key: cord-327324-4c4a4bfz authors: wilkinson, robert j title: tuberculosis and type 2 diabetes mellitus: an inflammatory danger signal in the time of covid-19 date: 2020-06-13 journal: clin infect dis doi: 10.1093/cid/ciaa747 sha: doc_id: 327324 cord_uid: 4c4a4bfz nan however it is increasingly appreciated dm itself is an inflammatory disorder that may therefore interact with infection in more complex ways [10] . transcriptomic profiling of whole blood is a technique that has been widely applied and proven insightful in many infectious and inflammatory disorders [11] . active tuberculosis has a transcriptomic signature dominated by a neutrophil-driven type 1 and 2 interferoninducible gene profile. the transcriptomic signature relates to disease extent and resolves during successful treatment [12] . the differentiation of active from latent tuberculosis and other conditions may be aided by transcriptomic profiling [13] . the exaggerated inflammation that characterizes hiv-tuberculosis-associated immune reconstitution inflammatory syndrome is triggered by toll-like receptor and inflammasome signalling [14] . transcriptomic signatures may predict progression of, or detect, subclinical tuberculosis [15] [16] [17] . in this issue of the journal eckold and colleagues present an analysis of the effect of dm the study has the advantage of power and of validation across populations. limitations are that hba1 c is a relatively insensitive test for dm. it is known that tuberculosis itself associates with transiently impaired glucose tolerance which may even be in the frank diabetic range, yet which may resolve during tuberculosis treatment [7] , but the proportion of cases of ih falling into this category was not investigated longitudinally. sex influences type 1 interferon and neutrophil transcript abundance and there were some very substantial gender differences between groups ranging from 18-90% males. this publication coincides with the world grappling with the covid-19 pandemic. are there inferences that can be made from this very detailed study of susceptibility to tuberculosis conferred by diabetes? male sex and diabetes have been identified in virtually every study as risk factors for severe covid-19 infection, associated in the largest study to date with an adjusted hazard ratio for in-hospital death of 1.99 (male sex) and 1.50 for controlled (hba1c < 58 mmol/mol) and 2.36 for uncontrolled dm [18] . the pulmonary innate immune response in covid-19 patients with severe disease implicates a macrophage subpopulation with an interferon-associated hyperinflammatory response analogous to that observed in tb patients [19] . type 1 and 2 interferon stimulation increases angiotensin converting enzyme 2 (ace2), the receptor for sars-cov2, expression on respiratory epithelial cells. in particular, lungs of patients with hiv-associated tb show a c c e p t e d m a n u s c r i p t 4 the highest expression of ace2 in epithelial cells, compared to lungs of patients with tb only and healthy lungs [20] . hiv-1 infection, prior to antiretroviral therapy (art) also induces a similar interferon signalling gene (isg) profile to that of tb patients, a pattern also implicated in the context of hiv-tb co-infection, even prior to tb symptom onset [16] . given these findings, it is plausible that individuals with hiv-1 and/or tb infection could be at increased risk of sars-cov2 infection not only via immunosuppression but also via increased ace2 expression, predisposing to enhanced viral uptake. this theory is diabetes mellitus increases the risk of active tuberculosis: a systematic review of 13 observational studies effect of glycemic control and type of diabetes treatment on unsuccessful tb treatment outcomes among people with tb-diabetes: a systematic review the impact of diabetes on tuberculosis treatment outcomes: a systematic review diabetes is a strong predictor of mortality during tuberculosis treatment: a prospective cohort study among tuberculosis patients from mwanza, tanzania hyperglycemia during tuberculosis treatment increases morbidity and mortality in a contemporary cohort of hiv-infected patients in rio de janeiro, brazil stress hyperglycaemia tuberculosis, hiv and the association with transient hyperglycaemia in peri-urban south africa consultation meeting on tuberculosis and diabetes mellitus: meeting summary and recommendations infections in patients with diabetes mellitus: a review of pathogenesis immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections uninfected african adults using whole blood rna expression signatures: a casecontrol study hiv-tuberculosis-associated immune reconstitution inflammatory syndrome is characterized by toll-like receptor and inflammasome signalling characterization of progressive hiv-associated tuberculosis using 2-deoxy-2-[18f]fluoro-d-glucose positron emission and computed tomography complement pathway gene activation and rising circulating immune complexes characterize early disease in hiv-associated tuberculosis a modular transcriptional signature identifies phenotypic heterogeneity of human tuberculosis infection opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients single-cell landscape of bronchoalveolar immune cells in patients with covid-19 sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is enriched in specific cell subsets across tissues rjw is supported by the francis crick institute which receives funding from wellcome (fc0010218), ukri (fc0010218) and cruk (fc0010218). he also receives support from wellcome (014803 and 203135), nih (r01ai145436) and edctp (ria2017t-2004) . the views in this article are the author's alone and do not reflect the views of these agencies. a c c e p t e d m a n u s c r i p t key: cord-309515-0pxl0sta authors: blanco, jose l; ambrosioni, juan; garcia, felipe; martínez, esteban; soriano, alex; mallolas, josep; miro, jose m title: covid-19 in patients with hiv: clinical case series date: 2020-04-15 journal: lancet hiv doi: 10.1016/s2352-3018(20)30111-9 sha: doc_id: 309515 cord_uid: 0pxl0sta nan as of march 24, 2020, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic has affected almost 400 000 people in 168 countries on five continents. older patients (>60 years) and those with comorbidities (eg, hypertension, diabetes, cardiovascular disease, lung disease, and chronic kidney disease) present with more severe infection and worse prognosis. 1 coronavirus disease 2019 (covid-19) has been described in only one patient with hiv in wuhan, china, 2 but case series in patients with hiv are lacking despite 37·9 million people having hiv globally. 3 here we describe, to our knowledge, the first single-centre experience of covid-19 in patients infected with hiv-1, including clinical characteristics, antiviral and antiretroviral treatment, and outcomes. all patients gave informed consent for publishing their clinical data. we used nasopharyngeal swab samples for all diagnoses, amplifying the betacoronavirus e gene and the specific sars-cov-2 rdrp gene by pcr. on we started all five patients on anti-sars-cov-2 treatment on the day of diagnosis. we gave all five patients boosted-protease inhibitor art. we explained to patients treated with art that we were making a transitional change in their regimen on the basis of the fact that hiv protease inhibitors might have activity against the have sex with men (msm). two patients had comorbid conditions. two patients were sex workers. four were virologically suppressed: two with protease-inhibitor (darunavir-boosted cobicistat) and two with integrase-inhibitor (dolutegravir)-based antiretroviral therapy (art). cd4 counts were above 400 cells per µl in all patients apart from patient 5, who was art naive and a very advanced late presenter. two patients had upperrespiratory tract infections, and three lopinavir-boosted ritonavir was given as 400 mg of ritonavir boosted with 100 mg of lopinavir twice a day for 14 days; azithromycin was given as 500 mg once a day, with a loading dose on the first day, and then 250 mg once a day for 4 days; hydroxychloroquine was given as 400 mg twice a day with a loading dose on the first day and then 200 mg twice a day for 4 days, and interferon beta-1b was given as 250 μg (8 million units) every 48 h. msm=men who have sex with men. nd=not done. *hepatitis c virus, hepatitis b virus, chronic obstructive pulmonary disease, asthma, chronic kidney failure, hypertension, cardiovascular disease, diabetes, solid organ transplantation, use of biologics, other types of immunosuppression. †tenofovir alafenamide, emtricitabine, and darunavir-boosted cobicistat was indicated before the information provided by janssen on march 18, 2020. ‡discharged with a supervised home-care programme. given remdesivir (only available through clinical trials, with restricted access at the time these patients were evaluated). we administered c o n c o m i t a n t a n t i b a c t e r i a l s in all three patients who had pneumonia (patients 2, 4, and 5), and corticosteroids in two patients (patients 4 and 5) and tocilizumab in one (patient 2). we have discharged four patients (80%); one remains in hospital in the intensive care unit (patient 2). our preliminary experience highlights several issues. first, patients with hiv accounted for almost 1% of patients with covid-19 who required admission to hospital in barcelona. we only observed the infection in people younger than 50 years, who identified as msm, and who have a covid-19 clinical pictures resembling the general population. none of these five patients has died, although we admitted two to intensive care, where one remains. more studies of covid-19 in patients with hiv are needed in the older msm population, drug users, and heterosexual men and women in middle-income and lower-income settings. second, two patients who were msm were sex workers, one reporting participating in a chemsex party 6 days before admission to hospital. during this pandemic, implementing health education programmes is very important to explain that such activities as these could cause clusters of sars-cov-2 transmission. third, we adapted art in all patients to a regimen based on protease inhibitors: three patients were given lopinavir-boosted ritonavir and two were given darunavirboosted cobici stat. in the past month, a clinical trial 4 found that lopinavirboosted ritonavir was ineffective as a monotherapy against severe pneumonia asso ciated with covid-19 in china. therefore, investigation of the efficacy of this treatment in patients with covid-19 in combined therapy in earlier stages of the disease is needed. additionally, janssen reported on march 18, 2020, that darunavir was ineffective against sars-cov-2 due to low affinity to coronavirus protease. fourth, we did not give our patients remdesivir, the most active in-vitro and in-vivo antiviral drug against coronavirus to date, 5 and is currently only available through clinical trials or for compassionate use. this drug has no pharmacokinetic interactions with any medication including art drugs. finally, in advanced patients (ie, late presenters), we must ensure differential diagnosis and initial antimicrobial treatment to address pulmonary opportunistic infections (eg, pneumocystis jirovecii, as seen in patient 5) presenting with similar clinical and radiological symptoms. this pandemic is a chall enge affecting everyone. by generating information such as we present here, the management and prognosis of patients co-infected with hiv and sars-cov-2 might be improved. hospital clinic, institut d'investigacions biomèdiques august pi i sunyer clinical characteristics of coronavirus disease 2019 in china co-infection of sars-cov-2 and hiv in a patient in wuhan city joint united nations programme on hiv/aids, 2020 a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov for more on covid-19 drug interactions see www.covid19-druginteractions.org for report from janssen on darunavir-based treatments for sars-cov-2 see see online for appendix key: cord-349104-p0egfpx9 authors: modi, anita r.; koval, christine e.; taege, alan j.; modaresi esfeh, jamak; eghtesad, bijan; narayanan menon, k. v.; quintini, cristiano; miller, charles title: coronavirus disease 2019 in an orthotopic liver transplant recipient living with human immunodeficiency virus date: 2020-06-17 journal: transpl infect dis doi: 10.1111/tid.13351 sha: doc_id: 349104 cord_uid: p0egfpx9 coronavirus disease 2019 (covid‐19), mediated by severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2), can manifest with flu‐like illness and severe pneumonia with acute respiratory distress syndrome (ards). immunocompromised patients merit particular attention as altered host immunity may influence both disease severity and duration of viral shedding as is described with several other ribonucleic acid respiratory viruses. yet immunocompromised status alone, in the absence of other comorbidities, may not necessarily predict severe illness presentations and poorer clinical outcomes as indicated by recent reports of covid‐19‐infected solid organ transplant recipients and people living with human immunodeficiency virus (hiv). such patients may even be spared the robust inflammatory response that precipitates ards associated with covid‐19, complicating the management of iatrogenic immunosuppression in this setting. we present a case of an orthotopic liver transplant recipient with well‐controlled hiv who successfully recovered from a mild, flu‐like illness attributed to sars‐cov‐2. ease severity and duration of viral shedding as is described with several other ribonucleic acid respiratory viruses. yet immunocompromised status alone, in the absence of other comorbidities, may not necessarily predict severe illness presentations and poorer clinical outcomes as indicated by recent reports of covid-19-infected solid organ transplant recipients and people living with human immunodeficiency virus (hiv). such patients may even be spared the robust inflammatory response that precipitates ards associated with covid-19, complicating the management of iatrogenic immunosuppression in this setting. we present a case of an orthotopic liver transplant recipient with well-controlled hiv who successfully recovered from a mild, flu-like illness attributed to sars-cov-2. covid-19, hiv, hydroxychloroquine, immunocompromised, orthotopic liver transplantation solid organ transplant recipients living with hiv uniquely demonstrate features of both immune suppression and immune activation, as evidenced by the increased rates of allograft rejection in such patients. 13 definitive guidance on management strategies for covid-19 in this specific population is lacking. we hope to contribute to the literature of covid-19 in immunocompromised patients by describing an orthotopic liver transplant (olt) recipient with well-controlled hiv who experienced a mild flu-like illness attributed to sars-cov-2. our patient is a 32-year-old african american man diagnosed with hiv 10 years prior to presentation with a peak viral load of 107 000 copies/ml and a cd4 + t-cell count of 477 cells/µl. efavirenz, emtricitabine, and tenofovir disoproxil fumarate were initiated at the time of diagnosis, and the patient rapidly achieved viral suppression. two years after diagnosis, he suffered progressive liver failure attributed to antiretroviral-induced hepatotoxicity and underwent olt in 2013. his maintenance immunosuppression consisted of mycophenolate mofetil (mmf), prednisone, and tacrolimus. his antiretroviral therapy (art) was changed to raltegravir, emtricitabine, and tenofovir disoproxil fumarate post-transplantation. he experienced two episodes of biopsy-proven acute cellular rejection diagnosed two years and four years after olt, treated with anti-thymocyte globulin. in march of 2020, five days after returning from a trip to new york city, he developed fatigue, fever, headache, and a dry cough. he presented to the emergency department and was found to have a temperature of 101 0 f. nucleic acid amplification testing for influenza a, influenza b, and respiratory syncytial virus (cepheid xpert xpress) performed on nasopharyngeal specimens was negative. nucleic acid amplification testing for sars-cov-2 (centers for disease control and prevention, usa) performed on nasopharyngeal and oropharyngeal specimens was positive. he had been working at a community health center since his return, but reported no overtly ill contacts in that setting. the patient was initially instructed to engage in supportive care measures at home; however, the development of chest tightness and shortness of breath prompted presentation to the hospital the following day. he complained of aggravating dry cough, but denied any abdominal symptoms. his vital signs were within normal limits. pertinent laboratory results are listed in table 1 . chest x-ray did not demonstrate any infiltrates. computerized tomography (ct) imaging was not obtained. given the patient's immunocompromised status and interleukin-6 (pg/ml) ta b l e 1 laboratory markers at baseline and throughout the clinical course of covid-19 infection concern for symptom progression, he was admitted to the hospital despite his mild illness presentation. mmf was discontinued; prednisone was maintained, and tacrolimus was dosed to target a lower trough of 5-9 ng/ml. hydroxychloroquine was administered outside of a clinical trial for five days. antiretroviral therapy was continued. the patient's respiratory symptoms gradually improved, and he never demonstrated fever or hypoxia. he was discharged home on the sixth day of admission and instructed to maintain isolation for 14 days. post-discharge laboratory testing demonstrated improved inflammatory markers and lymphopenia. figure 1 depicts his clinical timeline. short-term liver transplant recipients were exposed to augmented immunosuppression including lymphodepleting agents more recently than their long-term counterparts, the short-term liver transplant recipients were significantly younger and healthier overall. 11 our patient was transplanted seven years prior to diagnosis of covid-19 and last received a lymphodepleting agent for the treatment of acute cellular rejection three years previously. yet, his age and overall health more closely approximate those of the short-term liver transplant recipients included in this study. immunosuppressive agents administered to solid organ transplant recipients compromise t-cell immunity responsible for viral recognition and eradication, thus prompting particular concern for the severity of the early and localized pulmonary phases of well-controlled hiv, successfully recovered from covid-19. the impact of reduced immunosuppression and early hydroxychloroquine therapy in this setting has yet to be determined. the authors of this manuscript have no conflicts of interest to disclose. a novel coronavirus from patients with pneumonia in china clinical features of patients infected with 2019 novel coronavirus in wuhan characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention implications of emerging infections for transplantation severe acute respiratory syndrome (sars) in the liver transplant recipient and guidelines for donor sars screening mers cov infection in two renal transplant recipients: case report infectious diseases community of practice. rna respiratory viral infections in solid organ transplant recipients: guidelines from the american society of transplantation infectious diseases community of practice covid-19 in solid organ transplant recipients: initial report from the us epicenter covid-19 and kidney transplant patients covid-19 in long-term liver transplant patients: preliminary experience from an italian transplant center in lombardy covid-19 in patients with hiv: clinical case series outcomes of kidney transplantation in hiv-infected recipients covid-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) observational study of hydroxychloroquine in hospitalized patients with covid-19 coronavirus disease 2019 in an orthotopic liver transplant recipient living with human immunodeficiency virus key: cord-338438-q5fis2v8 authors: young, sean d.; schneider, john title: clinical care, research, and telehealth services in the era of social distancing to mitigate covid-19 date: 2020-05-21 journal: aids behav doi: 10.1007/s10461-020-02924-z sha: doc_id: 338438 cord_uid: q5fis2v8 nan the covid-19 pandemic and its related policies have the potential to fundamentally change how healthcare delivery and public health interventions are deployed. populations at high-risk for and/or living with hiv are especially impacted by covid-19 and related policies, making it important to understand and plan for ways that covid-19 might affect clinical care and research related to these populations. social distancing is the cornerstone of covid-19 prevention responses and follows long-standing and clear infection control principles. infection control has focused on decreasing exposure to respiratory droplets, and maintenance of a 6-foot buffer as the primary mechanism of containment. dramatic public health measures, including stay-at-home orders and closing of public spaces, have been implemented across the u.s. to facilitate social distancing. medical care systems, public health programs, and individual client care approaches are forced to rapidly adapt to these infection control measures, often without infrastructure or experience on how to do so. the perfect social distance is one where there is no chance of exposure or contact, as can be done using remote technologies. researchers who have studied and promised the potential of telehealth technologies therefore have an urgency to demonstrate how these tools might be quickly adapted for use in new clinical settings. in this note, we describe considerations for integrating technologies, such as telemedicine; social media, mobile applications (apps), and chatbots; and biosensors/wearables into clinical hiv care delivery and research, as well as case examples of current uses of these technologies in adapting to the changing clinical and research needs among populations at risk for and/living with hiv as a result of the covid-19 pandemic. telemedicine coverage policies are complex, dynamic and geographically specific. much of the current policies have limited the extent to which patients can be cared for remotely. many jurisdictions have strict requirements as to the type of provider, the location of the patient, and medium of communication. in addition, barriers to engaging patients where they are, are driven by early conceptions for telehealth. for example, hiv-related telemedicine was primarily developed as a way for hiv specialists or consultants to provide care to remote areas, including rural contexts, or to inaccessible patients, such as incarcerated persons. in such cases, there is local control on both ends of the connection with well-established and controlled systems. in illinois, for example, the provision of care required patients to be in a clinical setting. this system has recently been upended by the covid-19 pandemic in order to increase social distancing. on march 17th, 2020, the telehealth waiver in medicare under hr 6047 was implemented, allowing for an expanded fee for services provision [1, 2] . while the provision did not expand the allowable types of secure digital platforms or disciplines of care providers, this change importantly eliminated site limitations for patients, such that a patientcentered approach can be deployed allowing for patients to be seen where they wish. clearly, this is perfect social distancing in a covid-19 context. it ensures that providers and patients are kept as safe as possible, with the additional benefit of helping providers avoid clinical contexts that have additional coronavirus transmission risk, particularly in hyperepidemic locations such as new york city. as a case example of illinois, academic medical centers such as the university of chicago, and federally qualified health centers, such as howard brown health, rapidly transitioned scheduled patients to telemedicine, developed telemedicine protocols, billing algorithms, and hippaa compliant communication videoconferencing platforms. this occurred over days with clinical flows and patient acceptability still being examined. from an implementation science perspective, there are several important questions around telemedicine effectiveness, feasibility, acceptability and contextual variation [3] . in addition, while covid-19 presents a unique solution to social distancing, it is unclear how telemedicine will change as we move to the post-covid era and how hiv and routine prep care may be impacted. social media, chatbots, and mobile apps have been studied across a number of clinical and public health settings, including patient outreach, screening and monitoring; intervention delivery; remote vital sign assessment; as well for providing treatment recommendations and retaining patients in care. for example, social media discussions about hiv and syphilis-related risk behaviors can be modeled with artificial intelligence to monitor and predict future cases within regions, helping to inform interventions that can be digitally delivered and tailored to at-risk populations [4] . social media and mobile apps can also be used to engage and retain patients at risk for and living with hiv, helping to keep them connected to clinical care during times when non-essential in-person visits are discouraged. the already widespread use of these technologies makes them appropriate tools for keeping patients engaged in care. automatically-responding chatbots, can be also be integrated into health settings, such as by asking questions and routing patients to appropriate educational resources or next steps for treatment (e.g., setting up an appointment) based on responses. for example, specific to covid-19, the cdc has released a coronavirus self-checker tool to assess symptoms and provide recommendations for next steps based on screening results [5] . tools like this could be adapted for the special needs of hiv populations, including items on substance use-related risk behaviors and hiv medication adherence in order to assess comorbid risks between hiv, covid-19, substance, and mental health. biosensors and wearable (including non-wearing, remote sensing) devices can assist with clinical care, especially in settings where remote patient monitoring of behaviors, vital signs, and other clinical outcomes is needed [6] . in addition to the most common sensors being used that track sleep, activity, and heart rate, sensors are also available to track respiratory rate, sweat, temperature, breathing abnormalities/coughing and oxygen saturation, all important covid-19-related signs and symptoms. biosensors and wearables can be used to assist with clinical monitoring and care while maintaining social distancing and stay-at-home orders. for example, respiratory, oxygen saturation, and temperature sensors can be used to monitor vital signs among patients, such as for substance use/addiction medicine treatment monitoring, hypertensive medication adherence, and pulmonary care patients. biosensor technologies can also facilitate social distancing between patients, and between high-risk covid-19 patients and providers. newer approaches (e.g., digital contact tracing) are using bluetooth signals to identify whether and when people have been social/physical distancing by assessing distances between 2 or more phones/mobile devices. data from these devices can be used to not only inform social distancing efforts but also to inform hiv research efforts, as these methods of measuring distances between phones might be integrated into hiv studies to hypothesize whether and when people are engaging in sexual risk behaviors. similar to early studies on electronic health records, studies integrating telehealth technologies into hiv prevention interventions have had mixed results on health outcomes, for various practical and intervention design reasons. however, studies, including a report from the agency for health research and quality, largely support use of these tools to remotely monitor patients and keep them engaged, if used correctly [7] . findings suggest that these tools are especially impactful when delivered based on specific rather than general needs (e.g., keeping hiv patients engaged in sociallydistanced care), and on evidenced-based behavioral and social science frameworks. although this note has categorized and separated the technologies above to help distinguish their use, the most effective approaches would likely integrate several of these technologies because of their complementary features. a number of potential issues need to be addressed before implementing fully-integrated systems, however, including ethical/risk monitoring; patient engagement; costs; and staffing requirements. due to the rapid changes required related to covid-19, it is expected that these issues will be addressed quickly. covid-19 will continue to impact the way that technologies are integrated into hiv clinical care and research long after the removal of social distancing policies, making it important to begin investing in the knowledge, infrastructure, and implementation of these technologies now to be prepared for the future. funding this study was funded by the national institute of allergy and infectious diseases (7r01ai132030) and the national institute on drug abuse (1 u2c da050098 ). the funders played no role in the study planning, analysis, or outcomes. covid-19) in the u.s. centers for disease control and prevention using search engine data as a tool to predict syphilis covid-19). centers for disease control and prevention wearable sensors for remote health monitoring telehealth: mapping the evidence for patient outcomes from systematic reviews key: cord-346153-9162w7il authors: openshaw, p j title: crossing barriers: infections of the lung and the gut date: 2008-12-24 journal: mucosal immunol doi: 10.1038/mi.2008.79 sha: doc_id: 346153 cord_uid: 9162w7il although known as respiratory pathogens, severe acute respiratory syndrome (sars) and its sister coronaviruses frequently cause enteric symptoms. in addition, other classically non-enteric viruses (such as hiv and influenza) may also have enteric effects that are crucial in their pathogeneses. these effects can be due to direct infection of the gut mucosa, but can also be because of decreased antibacterial defenses, increased mucosal permeability, bacterial translocation, and systemic leak of endotoxin. supplementary information: the online version of this article (doi:10.1038/mi.2008.79) contains supplementary material, which is available to authorized users. when sars broke out in the winter of 2002 -2003, the world was gripped by a well-founded fear that it would become a lethal pandemic. e acronym resonated with the public, helping to focus attention not only on the virus but also on its transmissibility and global potential. e original animal reservoir of the sars coronavirus appears to be wild bats, although it probably adapted to infect nocturnal pine civet cats before moving in to man. 4 although sars was dubbed " respiratory ", the virus was clearly not just a lung pathogen -it also a ected the gut. e emerging epidemic came to the attention of virologists when it began to spread in hong kong in early march 2002. one of the rst major outbreaks was in a hospital, triggered by a " superspreading " event following the admission of a doctor who had acquired the infection while working with patients with atypical pneumonia in guangdong province of mainland china. in all, 70 hospital sta became infected in this one outbreak; this same index case apparently infected visitors to the hotel where the doctor stayed, one of whom ew to hanoi, was admitted to hospital and there led to an outbreak in which 63 hospital sta were infected. events in the same hotel probably led to the transmission of infection to toronto, where a major outbreak also occurred. the major community transmission in hong kong occurred in a tower block complex named amoy gardens. in this case, the evidence points to transmission from soil pipes and sewage which appears to have led to aerosolization of sars coronavirus, and to inhalation and transmission to approximately 331 new individuals. this alarming ability to swop between being a respiratory and a gastrointestinal pathogen was one of the features made sars so potentially devastating. molecular detection methods show that picornaviruses (rhinovirus and enterovirus) cause approximately 60 % of common colds in older children and adults. e next most common are the coronaviruses, causing about 15 % of colds. human coronaviruses are classi ed genetically into three groups. one of the group 2 viruses, oc43, shows remarkable antigenic and genetic similarities to a common bovine coronavirus that probably rst mutated and transmitted to man in the 1890s. 1 although now transmitted from person to person via the respiratory tract, oc43 causes gastrointestinal symptoms in up to 57 % of infected people, along with various combinations of rhinitis (36.6 % ), pharyngitis (30 % ), and bronchitis or bronchiolitis (26.6 % ). 2 therefore, gastrointestinal symptoms can be as prominent as respiratory symptoms in coronavirus colds, o en labeled " gastric u ". in veterinary practice, coronaviruses are also notorious for causing infection of either the gut or lung and for sometimes moving between sites. porcine transmissible gastroenteritis virus (tgev) is a coronavirus related to the 229e strain of coronavirus (another cause of common colds in man). tgev was a major cause of severe gastroenteritis in domesticated pigs, causing significant morbidity and mortality throughout worldwide. however, in 1984 spontaneous deletions caused a new strain to emerge transmitted via the respiratory route and causing predominately upper respiratory symptoms, and o en mild or inapparent infection. is new virus was su ciently antigenetically similar to tgev to cause cross-protection, 3 so that the new strain virtually wiped out the parental strain. erefore, the respiratory version of the coronavirus acted as a natural vaccine, eliminating tgev as a signi cant veterinary problem. although known as respiratory pathogens, severe acute respiratory syndrome (sars) and its sister coronaviruses frequently cause enteric symptoms. in addition, other classically non-enteric viruses (such as hiv and influenza) may also have enteric effects that are crucial in their pathogeneses. these effects can be due to direct infection of the gut mucosa, but can also be because of decreased antibacterial defenses, increased mucosal permeability, bacterial translocation, and systemic leak of endotoxin. nature publishing group commentaries sars was characterized by intense systemic symptoms and triggering of exuberant host immune responses. 5 detailed pathological studies showed that sars coronavirus could infect not only the respiratory epithelium, but also surface enterocytes in the small bowel. although infection caused di use alveolar damage, the changes in the gut are more subtle 6 and might include an increase in intestinal permeability to lipopolysaccharide (lps) and transmigration of intestinal bacteria ( figure 1c ) . remarkably, the onset of diarrhea in sars infected patients usually peaked on days 4 -9, by which time the fever had subsided. however, the coronavirus copy number in some studies showed an increase between day 5 and day 10, so that maximal infectivity followed the fever, 7 leading perhaps to a false sense of security amongst those caring for sars patients. although virus copy number in the pharyngeal aspirates dropped signi cantly between days 10 and 15, many patients still had sars coronavirus present in the stool at day 21, by which time under 50 % had virus present in the nasopharyngeal aspirate. erefore, late transmission by contact with stool was a particular unappreciated risk. infection with porcine respiratory coronavirus or porcine reproductive and respiratory syndrome virus has a synergistic e ect with bacterial products, demonstrated by enhanced in ammation induced by lps from escherichia coli . lps caused potentiation of disease, with enhanced production of tumor necrosis factor, interleukin-1, and interleukin-6. 8 it has been known for many years that healthy people o en carry pathogenic bacteria (such as neisseria meningitides or streptococcus pneumoniae ) in the upper respiratory tract, but may only develop invasive disease during coinfection with respiratory viruses. e reason for these interactions are incompletely understood, but intriguing recent study show that in uenza and respiratory syndrome virus are both capable of causing a persistent inhibition of the innate response to bacterial superinfection, and therefore to increased bacterial replication and disease. 9 although dual tropism for both the lung and gut may be an obvious and clear reason to consider the gut in respiratory infection, links can also be more subtle and complex. hiv targets cd4-expressing t cells and macrophages, thereby leading to immunosuppression. however, the symptoms of progressive hiv infection sometimes include profound weight loss ( " slim disease " ), and evidence of systemic immune activation. indeed, this immune activation may assist fresh infection by hiv of activated t cells, so enhancing the viral life cycle. e reasons for this immune activation seems to include the fact that hiv infects intestinal mucosal lymphocytes, including those in the peyer ' s patch and especially 17 cells that normally keep bacteria in check 10 ( figure 1b ) . this leads to enhanced gastrointestinal permeability to microbial products, causing increased levels of circulating lps, 11 further activating the innate and adaptive immune system. an additional intriguing possibility is that the distribution of foxp3 + regulatory t cells is a ected by hiv infection, 12 and that the balance between proin ammatory and antiviral effects is disturbed 13 thereby contributing to immune activation, malaise, and cachexia in hiv-infected patients. highly pathogenic strains of in uenza also cause intense systemic symptoms, sometimes associated with gastrointestinal disease. in waterfowl, in uenza is mostly an enteric pathogen, transmitted via the feces in the lakes and waterways, and e ciently transmitted to other birds feeding and breeding on the same water. when these viruses spread to man, viral replication can trigger hypercytokinaemia. 14 it therefore seems possible that the gut (known to be susceptible to infection with highly pathogenic strains of in uenza) 15 might also become permeable to intestinal lps in severe in uenza infections ( figure 1c ), or that bacterial translocation through the gut wall could contribute to systemic symptoms, cytokine release and circulatory collapse. in the era of integrative systems biology and holistic medicine, it is not only timely but also essential to view the gut and the lung as a continuous surface with distinct but overlapping susceptibilities. in addition, viral and bacterial infections should not be regarded as isolated events but viewed both in the context of intercurrent infection and of previous infection history. studies of single infections are certainly revealing, but in reality the secret to understanding variations in responses to infections must include an awareness of what else is present on a rich polymicrobial landscape. the author declared no conflict of interest. complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event detection of respiratory syncytial virus, parainfl uenzavirus 3, adenovirus and rhinovirus sequences in respiratory tract of infants by polymerase chain reaction and hybridization contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge evolutionary insights into the ecology of coronaviruses t cell responses to whole sars coronavirus in humans tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study in vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory infl uenza infection differential th17 cd4 tcell depletion in pathogenic and nonpathogenic lentiviral infections microbial translocation is a cause of systemic immune activation in chronic hiv infection preservation of foxp3+ regulatory t cells in the peripheral blood of human immunodefi ciency virus type 1-infected elite suppressors correlates with low cd4+ tcell activation coordination of early protective immunity to viral infection by regulatory t cells fatal outcome of human infl uenza a (h5n1) is associated with high viral load and hypercytokinemia infl uenza a h5n1 replication sites in humans key: cord-341323-mw352rr1 authors: logie, carmen h title: lessons learned from hiv can inform our approach to covid‐19 stigma date: 2020-05-04 journal: j int aids soc doi: 10.1002/jia2.25504 sha: doc_id: 341323 cord_uid: mw352rr1 nan stigma targeting people associated with covid-19, particularly persons of asian descent, has been reported in media spanning diverse global contexts [1] . the united nations described that "fear, rumours and stigma" are key challenges accompanying covid-19 [2] . the convergence of its framing as a "foreign virus" and an "infodemic" ignited this fear and stigma [2, 3] . this is not a new phenomenon. blaming epidemics on a foreign "other" is a recurring historic narrative [4, 5] . we can leverage our four decades of hiv research to understand and address covid-19 stigma. first, hiv reflects the health, moral and racial dimensions of stigma theorized by goffman [6] and aligned with historical patterns of disease attribution [4, 5] . stigma is produced in social processes of labelling that differentiate persons characterized as "normal" from the "abnormal" other. archetypes of the other include racial and religious minorities as well as people labelled as physically unhealthy or "immoral" [6] . in the early 1980s the hiv epidemicinitially coined gay-related immune deficiency (grid)was conceptualized as a plague that impacted "at risk" populations in the us known as the "4-h's" (haemophiliacs, heroin users, homosexuals, haitians) [7, 8] . this framing blamed racial (haitian) and "immoral" (e.g. gay men) others and positioned a foreign location (haiti) as the origin of hiv in the us. the world health organization deliberately named covid-19 to avoid conflation with a location of origin [3] , yet referrals to it as the "chinese" and "wuhan" virus persist [9] . the arrests of people for breaching covid-19 public health measures [10]and subsequent labelling as "intentional murderers" [11] and "super spreaders" [12] signal the creation of the "immoral" other. these arrests contradict unaids recommendations to avoid criminal repercussions for breaching covid-19 public health restrictions [13] . similar to hiv, we need to address several facets of covid-19 stigma to effectively reduce it. these include exposing and eliminating racism and xenophobia and recognizing the social processes of othering already experienced by persons blamed for covid-19 (including stigma and socio-economic exclusion experienced by immigrants [14] ). second, hiv has taught us about the complexity of stigma. we are moving away from siloed stigma research on individual health conditions (e.g. hiv, mental health), social identities (e.g. race, sexual orientation) and practices (e.g., sex work, drug use) [15] . instead, stigma is understood as intersectional, social ecological, and produced by drivers (e.g., misinformation) and facilitators (e.g., inequitable social norms) [15] [16] [17] [18] . intersecting stigmasuch as racism and povertyinteract with hiv-related stigma to harm health engagement and outcomes [16, 17] and may present analogous barriers to covid-19 testing and treatment [14] . stigma also operates across multiple, interacting dimensions of life. social ecological approaches to hiv remind us that stigma is intrapersonal (affecting our self-perception and mental health), interpersonal (altering our relationships), social (embedded in community norms and values) and structural (reproduced institutionally in health, legal, employment and other practices) [15, 16] . researchers can apply this lens to explore covid-19 stigma's effects on mental health, intimate relationships [18] , community cohesiveness, and interactions with police, employers, healthcare providers, among others. stigma experiences are shaped by intersecting social identities. researchers have called for a gender-based analysis of covid-19 [18, 19] . we urgently need to examine the gendered nature of covid-19 stigma, particularly in light of hivrelated stigma research that shows its associations with gender-based violence [e.g. 20]. age is another identity that may shape covid-19 stigma manifestations. there are complex associations between hiv-related stigma and age, whereby older persons living with hiv may experience reduced health effects of stigma [21, 22] . this could differ from covid-19, where the distressing choice of rationing intensive care hospital beds and ventilators has sparked debate over choosing who should live and who should die [23, 24] . the recommended utilitarian approach favours prioritizing treatment for young, severely ill persons [24] . what implications does this scarcity of covid-19 medical resources have on stigma towards older persons? understanding specific contexts of covid-19 stigma can inform tailored mitigation strategies. however, the great challenge remains that covid-19 is a moving target with continually changing dynamics. groups impacted by stigma may change as the pandemic evolves. while asian communities were initially blamed for covid-19 [1, 9] , will this liability shift to other marginalized communities, such as undocumented immigrants, homeless persons, and others who experience barriers to testing and care [14] ? we can also apply lessons from hiv-related stigma reduction interventions to covid-19. community-based approaches to reducing hiv-related stigma focus on generating solidarity and reclaiming identities [3, 13, 16, 25] . such covid-19 stigma resistance tactics have already emerged, evidenced with the twitter hashtags #iamnotavirus, #nosoyunvirus and #jenesuispasunvirus. there is a rich evidence-base of hiv-related stigma interventions for healthcare providers that provide hiv information, share how stigma affects communities, encourage reflection on personal biases and ensure institutional support for stigma mitigation [13, 26, 27] . other strategies include participatory learning through engaging activities such as discussions, games and role-play [26, 27] . the contact approach involves people who have experienced the stigma being targeted (e.g. persons living with hiv, persons experiencing covid-19 stigma) delivering the intervention to provide a face to the pandemic that in turn can foster empathy and reduce othering [26, 27] . moving forward we need not only focus on the stories of hardship in the midst of an epidemic [28] , but to also remember the complexity and fullness of people's lives. for instance, there are videos circulating on social media of people quarantined in italy for covid-19 singing to one another from their balconies. the hiv epidemic simultaneously produced stigma and created communities among affected persons [7, 8] . creating space for stories of covid-19 that reveal stigma and solidarity, of front-line healthcare workers' experiences, and of people living in quarantine, can reduce fear and spark empathy by helping us to see ourselves and our communities reflected in the pandemic [28] . understanding our shared humanity and the precarity of distinguishing the "sick" and "healthy" may be a step towards fostering solidarity. sontag [4] reminds us that we are interconnected in our vulnerability to sickness: illness is the night-side of life, a more onerous citizenship. everyone who is born holds dual citizenship, in the kingdom of the well and in the kingdom of the sick. although we all prefer to use only the good passport, sooner or later each of us is obliged, at least for a spell, to identify ourselves as citizens of that other place. (p. 3). factor-inwentash faculty of social work, university of toronto, toronto, canada a u t h o r s ' c o n t r i b u t i o n s chl conceptualized and wrote the manuscript. she read and approved the final manuscript. chl was supported during the writing of this manuscript by a brocher foundation residency and an eccles fellowship at the british library. she also receives support for her programme of research from canada research chairs, canada foundation for innovation, and the ontario ministry of research and innovation. funders played no role in writing this manuscript. the author also thanks the anonymous reviewers for their helpful feedback. as coronavirus spreads, so does xenophobia and anti-asian racism coronavirus covid-19 risk increased to 'very high' but containment still possible world health organization, international federation of red cross and red crescent societies, united nations children's fund. social stigma associated with covid-19. geneva: world health organization illness as metaphor history in a crisis -lessons for covid-19 stigma-notes on the management of spoiled identity aids and its metaphors aids: cultural analysis/cultural activism us push to include 'wuhan virus' language in g7 joint statement fractures alliance. cnn politics unaids.press statement: unaids condemns misuse and abuse of emergency powers to target marginalized and vulnerable populations intentional murder': careless covid-19 spreaders in italy could face homicide charges british broadcasting corporation (bbc) news. coronavirus: india 'super spreader' quarantines 40,000 people rights in the time of covid-19: lessons from hiv for an effective, community-led responses undocumented u.s. immigrants and covid-19 the health stigma and discrimination framework: a global, crosscutting framework to inform research, intervention development, and policy on health-related stigmas hiv, gender, race, sexual orientation, and sex work: a qualitative study of intersectional stigma experienced by hiv-positive women in ontario challenges and opportunities in examining and addressing intersectional stigma and health covid-19: what implications for sexual and reproductive health and rights globally? sex reprod health matters covid-19: the gendered impacts of the outbreak a longitudinal study of associations between hiv-related stigma, recent violence and depression among women living with hiv in a canadian cohort study age, stigma, adherence, and clinical indicators in hiv-infected women the impact of hiv-related stigma on older and younger adults living with hiv disease: does age matter? who should be saved first? experts offer ethical guidance fair allocation of scarce medical resources in the time of covid-19 community-based interventions that work to reduce hiv stigma and discrimination: results of an evaluation study in thailand stigma in health facilities: why it matters and how we can change it combating hiv stigma in health care settings: what works? the danger of stories in global health key: cord-351740-779g8tr1 authors: khaba, moshawa calvin; ngale, tshepo cletus; madala, nomandla title: covid-19 in an hiv-infected patient. lessons learned from an autopsy case date: 2020-09-25 journal: int j infect dis doi: 10.1016/j.ijid.2020.09.1435 sha: doc_id: 351740 cord_uid: 779g8tr1 despite measures put in places to curb the spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) across south africa, there has been a rapid spread which caused extensive morbidity and mortality. whilst there is currently increased covid-19 associated death, autopsies on covid positive individuals are not routinely performed. an autopsy was performed on a 19 years old african patient who was recently diagnosed with human immunodeficiency virus (hiv). he presented with clinical features of sars-cov-2 which subsequently tested positive for. important histopathological findings included diffuse alveolar damage and fibrin thrombi. no superimposed infections were noted. the cause of death was attributed to covid-19. we report the first autopsy case of hiv-infected individual with covid-19 as the cause of death. the first confirmed case of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection was reported in china in december 2019. this viral infection has since spread across the globe with the first case reported in south africa on march 20, 2020. coronaviruses are enveloped, non-segmented, positive-sense single-stranded rna viruses. the other two that are known to cause human diseases are beta coronaviruses-severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (1, 2) . the clinical features ranges from asymptomatic to mild symptoms such as cough, fever, dyspnoea and severe disease which leads to acute respiratory distress syndrome and death (1, 3) . older patients and individual with hypertension, diabetes and cancer are at increased risk of infection (3) . there is little knowledge on hiv/aids and its impact on the clinical outcomes in patients with . to the best of our knowledge, this manuscript represents the first published report of an autopsy performed on an hiv infected patient with cause of death attributed to covid-19. clinical features 19 years old male african patient who was referred from a local clinic with one-week history of generalised weakness, fatigue, cough and shortness of breath. he was recently diagnosed with human immunodeficiency virus (hiv) with cd4 t lymphocytes of 17 cell/ul and viral load of 1487946 copies/ml. he was not yet on anti-retroviral therapy (art). he had no other co-morbidities. on examination, his blood pressure was 95/33 mmhg, heart rate of 103 beats/minute, fever of 38,7 0 c and saturation of 95% on 40% oxygen. he was confused, pale and hypovalaemic with generalised lymphadenopathy. he had bilateral crackles on chest examination. abdominal examination revealed massive splenomegaly and hepatomegaly. he was given an intravenous stat dose of ceftriaxone and acetaminophen; and admitted to a patient under investigation (pui) ward for covid-19 suspects. the chest x-ray showed extensive bilateral infiltrates (fig. 1a) . the full blood count showed bicytopaenia with low haemoglobin and platelets of 100 x 10 9 /l; and transfused 3 units of red blood cells. the liver function tests were mildly deranged. c-reactive protein, ferritin and procalcitonin were raised. the renal function tests revealed pre-renal acute kidney injury most likely secondary to the hypovolaemia (see table 1 for investigations). despite sars-cov-2 infection, in view of the retroviral disease, pneumocystis pneumonia, bacterial pneumonia and tuberculosis could not be excluded. he was started on trimethoprim-sulfamethoxazole 1920mg 6hourly, hydrocortisone 200mg 8 hourly, azithromycin 500mg daily and enoxaparin 60mg daily. polymerase chain reaction of the nasopharyngeal swab detected severe acute respiratory syndrome coronavirus 2 (sars-cov-2). three days post admission, his confusion worsened; developed cardiorespiratory failure and died. a complete autopsy was performed in a negative pressure autopsy room with personal protective equipment (ppe), including n95 masks, eye protection, gloves and gowns. the patient's body mass index was 18.1 kg/m 2 . he was emaciated, hypovolaemic and pale with generalized lymphadenopathy. he had serosanguineous bilateral pleural effusion and ascites. the right and left lungs weighed 884g and 772g (n=360 -570g and 325 -480g) respectively. they were oedematous, firm with alternating pale and red areas (fig. 2b) . the spleen weighed 1310g (n=156) with haemorrhagic cut surface. the liver weighed 2400g (n= 1500 -1800) with pale, greasy and yellowish cut surface. the kidneys had smooth surface with good corticomedullary differentiation on cut section. the brain and heart were unremarkable. the bone marrow was pale and soft. microscopic features: sections of the lungs showed extensive oedema with bilateral diffuse alveolar damage (dad) evidenced by hyaline membrane formation (fig 1c-e) . adjacent small calibre blood vessels with fibrin thrombi were noted (fig 1f) . granulomatous inflammation or infectious pathogens were not seen. the spleen was poorly preserved; however, infectious pathogens or neoplastic infiltrate were not seen. sections of the liver showed microvesicular steatosis and lobular inflammation. the heart, brain and bone marrow were unremarkable. the kidney, lymph nodes and pancreas were poorly preserved. the well preserved glomeruli did not show features of hypertension, diabetes mellitus or hiv associated nephropathy. furthermore, fibrin thrombi were not seen within the glomerular capillaries. the final cause of death was sars-cov-2 (covid-19) infection in hiv infected patient. much as performing autopsies on covid-19 positive individuals is still not routinely perfomed due to safety reasons, there has been a recent increase in covid-19 autopsy. the spectrum of pathological findings has emerged from this. the most consistently described autopsy findings are diffuse alveolar damage with associated desquamation of pneumocytes, oedema and capillary congestion (1). increased intra-alveolar macrophage and enlarged, atypical pneumocytes have also been seen in advanced disease (4). vascular microthrombi are seen in areas of diffuse alveolar damage with diffuse endothelial damage. whilst this feature is not pathognomonic for covid-19, it has been postulated to be specific to covid-19 as this is not a normal finding in dad (4,5). luca carsana et al. found that fibrin thrombi of small vessels were observed in 87% of lung cases and high levels of d-dimers in the blood (1) elevated d-dimers is associated increased thrombin generation in covid-19 (6) . the existence of fibrin thrombi and high serum levels of d-dimers may explain the severe hypoxaemia that indicate acute respiratory distress syndrome in these patients (1) . d-dimer levels of >1 μg/ml is associated with increased mortality in covid-19 patients (6) . in accord to what is already published, the lung findings on the index patient showed early phase of diffuse alveolar damage with associated microthrombi which is seen in covid-19. the lung findings on this patient was not different from the ones reported on non-hiv infected patient. little is known of the interaction between hiv infection and sars-cov-2 pathogenesis. furthermore, there's little knowledge on the impact of hiv infection on the clinical outcomes of patients infected with sars-cov-2. whilst hiv infected people on treatment with normal cd4 count and low viral load may not be at a high risk of serious illness, the presence of other chronic conditions may increase their overall risk (7) the fact that sars-cov-2 can cause transient immune deficiency, it denotes that hiv and covid-19 interaction may have adverse immunological and clinical outcomes. therefore, defective cellular immunity in hiv infected patients may be paradoxically protective for severe cytokine dysregulation in patients with covid-19 (8) . the index patient's clinical emaciation suggests that the low cd4 count was pre-covid-19. shalev et al hypothesized that the absence of t-cell activation alleviates the severe immunopathological phenomena seen in covid-19. while his study had its limitation, he further suggested that sars-cov-2 does not act as an opportunistic pathogen in patients with uncontrolled hiv or aids (3). tuohy et al suggested that hiv status did not significantly impact clinical outcomes in patients with sars-cov-2 infection, albeit he detected trends suggestive of worse course outcome in hiv-positive patients (9) . although the index patient was hiv infected, he was young without comorbidities. he had lymphopaenia and was not yet on antiretroviral therapy. this is contrary to the few published cases which show that high mortality rate of covid-19 in hiv infected patients is usually associated with older patients (> 50 years) with diabetes, hypertension, etc. furthermore, in view of the index patient's low cd4 count, secondary or opportunistic infection such as tuberculosis, pneumocystis pneumonia or cryptoccocal infection would be expected. however, there was no superimposed infection identified on lung sections examined. this further favoured covid-19 as the sole cause of death in this patient. with its own limitation, this autopsy has not shown any distinct pathological findings specific to hiv infection in contrast to what is already described in non-hiv infected patients. in lieu of this, more studies regarding hiv and covid-19 association are warranted as typical clinicopathological findings may likely have important treatment implications for these patients. the autopsy was done seven days after he demised, awaiting a written informed consent from the family. hence some of the organs were autolysed. mck, tcn and nm conceptualised the report and wrote the manuscript. all authors have read and approved the submitted version of this manuscript. all materials and data described in this manuscript are available upon reasonable request to the corresponding author, and if complying with patients' privacy. written informed consent was obtained from the patients' parents for scientific publication of this case report. sefako makgatho health sciences university research ethics committee (smurec) approved the publication of these case series. smurec/m/215/2020 no funding was secured for this study. the authors declare that they have no conflicts of interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. legends fig 1: a , chest x-ray shows extensive bilateral infiltrates with left pleural effusion; b, firm lung with alternating red and pale areas, and oedema. no visible thrombus; c -e, diffuse alveolar damage with hyaline membrane formation ( ); f, small vessels with fibrin thrombi ( ). j o u r n a l p r e -p r o o f articles pulmonary post-mortem findings in a series of covid-19 cases from northern italy : a two-centre descriptive study clinical characteristics and outcomes in people living with human immunodeficiency virus hospitalized for coronavirus disease articles pulmonary and cardiac pathology in african american patients with covid-19 : an autopsy series from new orleans articles histopathology and ultrastructural findings of fatal covid-19 infections in washington state : a case series hematologic parameters in patients with covid-19 infection clinical features and outcomes of hiv patients with coronavirus disease 2019 covid -19 in people living with human immunodeficiency virus : a case series of 33 patients outcomes among hiv-positive patients hospitalized with covid-19 we thank ms b ngubeni and nhls management for motivation and support all research endeavours. j o u r n a l p r e -p r o o f key: cord-341304-jdvzpvdx authors: pata, rama kanth; ahmady, abolfazl; kiani, roudabeh title: human immunodeficiency virus: a dark cloud with silver lining during the covid-19 pandemic date: 2020-07-20 journal: cureus doi: 10.7759/cureus.9302 sha: doc_id: 341304 cord_uid: jdvzpvdx in december 2019, china reported a cluster of pneumonia patients infected by a new virus from the coronavirus family called severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the virus quickly spread around the world and infected millions of people, and the world health organization (who) declared coronavirus disease 2019 (covid-19) a pandemic on march 11, 2020. although some patients show only mild or even asymptomatic response to this infection, severe disease with rapid progression to acute respiratory distress and multiorgan failure is also commonly seen. in this report, we discuss three cases of hiv patients who survived covid-19. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a coronavirus with a positivesense single-stranded ribonucleic acid (rna). it is believed to have originated in bats because of the 96% genomic similarities it has with bat coronaviruses. the high incubation period (2-14 days) in conjunction with the high survivability of this virus and a high reproductive number (r0 ranging from 1.4 to 6.49, with a mean of 3.28 in one study and 2 to 3.5 in another) [1] [2] [3] have caused it to spread quickly throughout the world. on march 11, 2020 , the world health organization (who) declared the coronavirus disease 2019 (covid-19) outbreak a global pandemic. the covid-19 pandemic has expanded rapidly worldwide. the number of cases has skyrocketed as days passed, and mortality has rapidly increased globally. in this report, we present three cases of hiv patients who survived covid-19. on march 22, 2020, a 67-year-old female with a past medical history of asthma, coronary artery disease (status post-coronary artery bypass graft two years ago), hypertension, hyperlipidemia, and hiv on antiretroviral medications [bictegrav/emtricit/tenofov ala (biktarvy® 50-200-25 mg tablet, gilead sciences, foster city, ca) and darunavir/cobicistat (prezcobix® 800 mg-150 mg tablet, janssen pharmaceutica, beerse, belgium)] was brought in by emergency medical services (ems) for progressively worsening shortening of breath associated with weakness and two episodes of watery non-bloody diarrhea for one day. she had sought medical attention two days ago at an emergency department where she had been tested for covid-19 [reverse transcription-polymerase chain reaction (rt-pcr)]. she had been discharged on levofloxacin. she returned to the hospital for worsening of symptoms but denied any new symptoms including fever or cough. the covid-19 rt-pcr came back positive later. her chest ct scan showed multifocal patchy consolidations of the bilateral upper and lower lobes, and the electrocardiogram showed normal sinus rhythm with corrected qt interval (qtc) of 453 ms and t wave inversion in v2. two sets of blood and urine cultures were negative. other laboratory findings are presented in table 1 (column: case 1) . she was admitted for covid-19 pneumonia and placed on cardiac monitoring due to elevated troponin levels (0.04 ng/ml) and d-dimer (2,631 ng/ml). additionally, she was started on iv antibiotics (ceftriaxone and azithromycin) and iv fluids. ct scan of the chest on admission incidentally showed cholelithiasis without cholecystitis likely due to acute pancreatitis; amylase and lipase were 1,562 u/l and >900 u/l respectively, and toxicology was negative for alcohol. she was kept npo; a hepatobiliary iminodiacetic acid (hida) scan was performed after she refused a ct scan of the abdomen, which showed no scintigraphic evidence of biliary obstruction. in addition, heparin [5,000 units every 12 hours (q12h)] was started as standard deep vein thrombosis (dvt) prophylaxis. she had an acute drop in hemoglobin during her stay, and a fecal occult blood test was performed, which came back positive (march 23, 2020). therefore, heparin was discontinued, and she remained npo in view of concomitant pancreatitis and was started on an iv proton pump inhibitor. hemoglobin levels were monitored, and no further drop in hemoglobin level, hematemesis, or melena was observed. her clinical condition improved, hence diet was modified based on what she could tolerate. during the first four days of admission, she had fluctuating fever spikes ( figure 1) . additionally, throughout her stay, she had a few episodes of drops in her oxygen saturation ( figure 2) . consequently, she was placed on high flow oxygen cannula (2.0 l/min), and her oxygen saturation subsequently improved ( table 1 (column: case 2). he developed desaturation with increasing respiratory distress and altered mental status during his medical floor stay. arterial blood gas (abg) at 4 l nasal cannula showed hypoxia with pao 2 of 47 mmhg and o 2 saturation of 78.5% (figure 4) . consequently, he was upgraded to the intensive care unit due to the impeding respiratory arrest for close monitoring and further management; however, he refused intubation. during his stay, he began saturating well on a non-rebreather ( table 3) and was downgraded to the floor. he finished a course of ceftriaxone (1 gm daily ivpb for eight days) and azithromycin (500 mg ivpb daily for seven days) and received hydroxychloroquine (400 mg po daily) for 10 days while qtc interval and electrolytes level were monitored. additionally, he received hemodialysis as per his schedule. on april 3, 2020, sputum culture grew klebsiella pneumonia for which he received antibiotics (meropenem 500 mg daily for three days and ciprofloxacin 500 mg po daily for two days). his medical condition improved ( figure 5) , and he was downgraded to the floor after nine days of icu care. he was eventually discharged on april 11, 2020, with a prescription of 14 days of ciprofloxacin and instructions on social distancing and follow-up appointments. presented for feeling unwell for three days, specifically complaining of headaches, myalgia, dry cough, shortness of breath, and fever. his chest ct scan showed patchy infiltrates throughout both lungs with a ground-glass pattern, more prominent at the periphery and at the lung bases ( figure 6) . additionally, a covid-19 rt-pcr test was positive, and two blood cultures showed no growth. moreover, the electrocardiogram showed sinus rhythm with a corrected qt interval (qtc) of 418 ms. other blood test results are shown in table 1 (column: case 3). overall, he finished a course of ceftriaxone (1 gm daily for six days), tamiflu® (roche pharma, basel, switzerland, 75 mg daily for 10 days), hydroxychloroquine (400 mg for five days), and azithromycin (500 mg daily for four days) while qtc interval and electrolytes level were monitored. during his admission, he had spikes of fever; consequently, he was put on vancomycin and meropenem until he was afebrile for 72 hours. furthermore, he had a few episodes of the mild drop in his oxygen saturation for which he was put on 2 l nasal cannula, and his saturation subsequently improved ( table 4) . his symptoms improved gradually (figures 7, 8) , and he was discharged on april 6, 2020, and advised to practice social distancing. the leading cause of mortality in covid-19 patients is acute respiratory distress syndrome (ards). some covid-19 patients present with a cytokine profile resembling secondary haemophagocytic lymphohistiocytosis (shlh), a fulminant and fatal hypercytokinemia with multiorgan failure, which is most commonly triggered by a viral infection and most frequently presents with fever, cytopenias, and hyperferritinemia with the involvement of the lungs (ards) [4] [5] . systemic elevation in pyrogenic cytokines such as interleukin-6 (il-6), il-10, and tumor necrosis factor-α (tnf-α) has been observed, suggesting that covid-19 triggers hyper inflammation that initiates ards via a complex inter-relationship of cytokines and proinflammatory mediators involving humoral and cellular responses [3, 4, 6] . data suggest that the host immune status may influence the progression of covid-19. some studies have shown higher levels of type i interferon (ifn-i) in hiv patients, which may help clear the covid-19 infection. additionally, delayed antibody response to covid-19 in hiv patients has been reported, suggesting a possible influence of immune deficiency in hiv patients on clearance of covid-19 [7] . the impact of multiple anti-retroviral drugs such as lopinavir-boosted ritonavir, darunavir, and remdesivir on covid-19 has been studied. a case report by holshue et al. showed clinical improvement in the first case of covid-19 in the united states after the use of remdesivir [8] . currently, gilead sciences has initiated two phase-3 studies investigating the efficacy of remdesivir for the treatment of covid-19 [9] . clinical trials have shown varying levels of effectiveness of lopinavir-boosted ritonavir on reducing mortality rates in covid-19 patients. some reports suggest that the addition of lopinavir/ritonavir to the initial treatment reduces the overall death rate and intubation rate [10, 11] . additionally, janssen has announced that even though anecdotal instances of darunavir being used for the treatment of covid-19 have been reported, it is ineffective in treating covid-19 patients due to its low affinity to coronavirus protease [11] [12] . we propose that hiv could have modulated the immune system in these three patients in a way that led to a less severe immune response to covid-19. furthermore, the lower severity of covid-19 in these hiv patients could be explained by the effect of the anti-retroviral medications these patients were taking. in order to confirm the validity of these hypotheses and show that it was not just the heterogenicity playing a role in such less severe immune response to covid-19, extensive retrospective studies or prospective randomized control studies should be conducted to further evaluate these hypotheses. additionally, we recommend further studies on anti-retroviral medications and their effect on covid-19 patients. human subjects: consent was obtained by all participants in this study. in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. the reproductive number of covid-19 is higher compared to sars coronavirus unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures a comprehensive literature review on the clinical presentation, and management of the pandemic coronavirus disease 2019 (covid-19) covid-19: consider cytokine storm syndromes and immunosuppression the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease acute lung injury/acute respiratory distress syndrome (ali/ards): the mechanism, present strategies and future perspectives of therapies early virus clearance and delayed antibody response in a case of covid-19 with a history of co-infection with hiv-1 and hcv (epub ahead of print) first case of 2019 novel coronavirus in the united states gilead sciences initiates two phase 3 studies of investigational antiviral remdesivir for the treatment of covid-19 emerging pharmacotherapies for covid-19 covid-19 in patients with hiv: clinical case series lack of evidence to support use of darunavir-based treatments for sars-cov-2 we would like to acknowledge the hard work all the medical staff around the world, especially those in new york, did during the covid-19 pandemic. key: cord-355475-kdubhh73 authors: patton, lauren l. title: viral pandemics and oral health: lessons learned from hiv to sars-cov-2 date: 2020-11-05 journal: oral surg oral med oral pathol oral radiol doi: 10.1016/j.oooo.2020.10.022 sha: doc_id: 355475 cord_uid: kdubhh73 nan training was from james j. crawford, phd and i was memorably impacted by his "what if saliva were red" video. originally developed in the 1970s, this video graphically illustrated the widespread dissemination of the otherwise invisible saliva around the dental operatory during routine practice. from this dramatic demonstration, it became clear there was a need for improved cross-infection control practices to prevent infection with blood-borne pathogens. until around the discovery of hiv in 1985, dentists typically wore gloves only for surgical and some endodontic procedures, long sleeve gowns were nonexistent in restorative dental practice, handpieces were not routinely autoclave sterilized, and masks and protective eyewear was rarely used in general dental settings. and yes, as we somewhat painfully adapt today to the extra gear, particularly the n95 respirator and full-face shields, in the 1980's we mourned the loss of fine tactile sensations with routine use of gloves. in the early aids pandemic, enhanced sterilization and engineering designs helped keep us safer from sharps injuries, dental unit waterline contamination and suction equipment backflow, and handpiece cross-contamination, as today enhanced air handling systems, face shields, and new office designs help to keep us safer in our aerosol generating environment. we have now added plexiglass barriers as social distancing/sneeze-guard shields to protect our main office staff, as lessons from our early use of disposable plastic cover barriers on operatory light handles, dental chairs and operatory trays/handpiece setups taught us how to simplify maintaining clean surfaces prone to contamination with potentially infectious agents. we now are challenged with quickly introducing more safety measures for our work in an environment of droplets and aerosols from the mouth in this age of a respiratory pandemic. an early survey in may and june 2020 of practicing dentists in private practice and public health settings in the united states (u.s.), a short 2 months after the first covid-19 wave and national shortages of personal protective equipment caused offices to move to emergency only dental care, showed that 99.7% of offices had implemented enhanced infection control procedures. 1 these were most often more frequent disinfection, covid-19 screening procedures including temperature checks, social distancing, and providing face masks to staff and patients. 1 meeting the challenges of producing a safer office practice environment and educating the public and our dental team members on our risk mitigation strategies are helping overcome public fear of covid-19 spread in the dental office. the early days of the aids pandemic were filled with fear of spread of aids in the dental office. a common practice in the early days of the aids pandemic among practices that would accept aids patients was providing care only at the end of the day so the invisible virus would dry and die on surfaces overnight. uncertainty over many aspects of the new air-borne covid-19 disease and the dramatically increased demand on a limited supply of personal protective equipment caused a shut-down of dental practices to only emergency dental services and limitations on use of aerosol-generating dental equipment. today with practices reopened, a main concern is sars-cov-2 viral particles lingering in the air in the operatories, suggesting the need to allow time between patients for the room air to settle and/or implementation of high-efficiency particulate air filtration systems. fortunately, with today's wide-spread influence of the internet, transition to electronic health records and patient engagement in health, many patients have the capacity for audio/video visits with their healthcare providers. these technological advances helped to transform the nascent telehealth system into one that is a robust alternative for triage and dental visits not requiring hands-on examination and procedural-based care, helping to solve healthcare access challenges. as soon as vaccines became available against hbv, healthcare workers were immunized, and over a short time period vaccine technology moved from plasmaderived to recombinant dna technology. 2 despite globally wide-spread infant hbv vaccination programs implemented in the ensuing years, hbv has not been eradicated. while hiv has eluded the efforts to develop a preventive vaccine largely due to its global genetic diversity, 3 sars-cov-2 has appeared to be relatively genetically stable, thus creating hope for success in efforts toward creation of a sars-cov-2 vaccine. what would be most useful for prevention of future pandemics would be development of a vaccine that was widely effective against many alpha and beta human coronavirus strains. while hope for a covid-19 vaccine to quell transmission is widespread, we must not lose sight of the fact that diverse vaccine development technologies and novel drug discovery efforts made today will benefit our response to the next pandemic. given the length of time for drug and vaccine development and approval, we now need to commit to getting ahead of the curve with well-organized and funded collaborative efforts to add to the pipeline. molecular evidence of both viruses can be found in whole saliva fluids, although in lower concentration than in blood. evidence supports sars-cov-2 to be community spread via saliva droplets and aerosols through coughing, sneezing, speaking, singing and breathing. 4 one of the key reasons for hiv testing advocacy was the observation that hiv-infected individuals unaware of their infection were the most likely to spread hiv; this is also true for sars-cov-2 where 80% of transmission may be due to undocumented asymptomatic infection. 5 saliva based sars-cov-2 diagnosis of this enveloped, positive-sense, single-strand rna virus by rt-pcr or immunoglobulin/antigen detection has emerged as a promising testing option to the traditional nasopharyngeal swab test. 4 it is likely that the work that went into developing the more recent generation antibody/antigen tests for hiv helped accelerate the pace of development of testing modes for sars-cov-2. orasure technologies inc. (bethlehem, pa, usa) delved into saliva or oral-fluid based hiv antibody testing. their efforts that moved from lab to rapid point-of-care to at-home testing over the course of several years have likely paved the way for more rapid progress and acceptance of saliva-based rapid sars-cov-2 testing. to date, one study analyzing multiple immunoglobulin response to sars-cov-2 in various biological fluids, including self-collected saliva for rapid sars-cov-2 diagnosis, has been published as a protocol without results, 6 so additional results of this study and others are needed to support antibody testing for sars-cov-2 in saliva. the real hope is for saliva-based point-of-care rapid tests, as was created in oraquick advance® hiv-1/2 (orasure technologies, inc.) for diagnosis of hiv infection. there has been limited progress in developing a specific treatment for covid-19 at present despite advances in repurposing the nucleotide analog antiviral remdesivir® and exploiting passive immunity approaches with production of convalescent plasma from recovered patient therapies or multiple neutralizing monoclonal antibody cocktails (regeneron and eli lilly). it took considerable research investments and six years for approval of azidothymidine, the first antiretroviral drug in the fight against hiv, originally synthetized for use as a cancer treatment agent, and even longer until highly effective antiretroviral therapies were available. 7 we are just at the beginning of our human interaction with corona virus diseases and since cross-species transmission of viral pathogens has emerged as a threat to humans, 8 a sustained investment in antiviral research can better prepare us for the future. our repurposing of today's antiviral drug discoveries fuels hope of more rapid solutions to future viral pandemics that are inevitable. 10 of the different taste disorders, the most common was dysgeusia (38%), followed by 35% hypogeusia and 24% ageusia. 10 authors of this review report that the diversity of oral mucosal lesion presentations including irregular and aphthous-like ulcers, white and erythematous plaques, blisters, petechiae, and desquamative gingivitis, found on the lips, tongue, palate, buccal mucosa and gingiva, suggest coinfections and secondary manifestations of covid-19. patients. interestingly, the dermatology literature is also demonstrating that vesicular rashes appear early and may help diagnosis, while vascular rashes may be useful in predicting severe disease. 13 to determine co-occurrence of skin lesions (exanthems) and oral cavity lesions (enanthem) in patients with covid-19, oral cavities were examined in 21 patients with skin rashes and 6 (29%) had oral lesions, all on the palate, and described as macular and/or petechial, with no association with drug intake and laboratory studies suggested they were a stronger indicator of viral etiology than a drug reaction. 14 when the diversity of oral mucosal and salivary gland disorders were observed in hiv/aids patients, international collaborative groups such as the european community we learned from hiv disease management that the antiretroviral drugs can have acute and long-term toxicities including ulcers, xerostomia/parotid lipomatosis, taste disturbances, perioral paresthesia, erythema multiforme and facial fat wasting. 16 will there be oral toxicities or benefits of covid-19 treatments particularly as treatments target the cytokine storm, a procoagulant state, and local and systemic activation of inflammation? in this new global pandemic, we need to learn from early disease hotspots, and we have witnessed great strides in the early months of this pandemic when our u.s. national institutes of health created supplemental funding for existing extramural grants to add components related to the clinical impact of sars-cov-2 on patients' health outcomes. internationally, as was the case at my home academic institution, many researchers engaged in and supported for their hiv/aids work pivoted to work on covid-19. while many covid-19 studies could be conducted in laboratory biosafety level 2 (bsl-2) laboratories using standard precautions, those studies involving virus isolation needed bsl-3 laboratories and procedures. 19 as this is similar to the biosafety categorization for work with hiv, 20 many already established bsl-3 laboratories engaged in hiv research became rapidly repurposed as covid-19 research facilities. almost overnight, our hiv clinical researchers became covid-19 researchers, as critical new studies were designed. without hiv/aids, this research infrastructure needed for covid-19 work would likely not have existed. we now need better mobilization of scientific collaboration globally and continued support of federal and international agencies to sustain our work in prevention and treatment for covid-19 to prepare us for future outbreaks and sustain our global population. this is a global health crisis, needing a global response. the covid-19 pandemic has further revealed the fundamental place of dentistry in the health system as an essential health care service whose role is to assure eradication of disease and management of pain in the maxillofacial structures to preserve quantity and quality of life and to prevent decline in a person's systemic health. we have been effective at keeping patients with dental complaints out of our nation's hospital emergency departments during the early covid-19 shut-downs. we have adapted and will continue to adapt to the challenges ahead. the question arises as to whether we can now mobilize our oral health team to participate in viral disease testing and vaccination efforts in the days ahead. here's hoping we have many "wins" in 2021 for our further integration into the health system. estimating covid-19 prevalence and infection control practices among us dentists. jada. online ahead of print 15 hepatitis b: 50 years after the discovery of australia antigen global and regional molecular epidemiology of hiv-1, 1990-2015: a systematic review, global survey, and trend analysis covid-19 salivary signature: diagnostic and research opportunities substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2) detection of sars-cov-2 rna and antibodies in diverse samples: protocol to validate the sufficiency of provider-observed, home-collected blood, saliva, and oropharyngeal samples learning from history: do not flatten the curve of antiviral research! drug discov today viruses in bats and potential spillover to animals and humans ace2 & tmprss2 expressions in head & neck tissues: a systematic review oral manifestations in patients with covid-19: a living systematic review oral lesions in patients with sars-cov-2 infection: could the oral cavity be a target organ? oral surg oral med oral pathol oral radiol report of four cases. are they a true sign of covid-19 disease? spec care dentist cutaneous manifestations of covid-19: an evidence-based review classification and diagnostic criteria for oral lesions in hiv infection. ec-clearinghouse on oral problems related to hiv infection and who collaborating centre on oral manifestations of the immunodeficiency virus adverse effects of antiretroviral therapy: focus on orofacial effects world health organization. summary of the global hiv epidemic world health organization. coronavirus disease (covid-19) pandemic. numbers at a glance centers for disease control and prevention. interim laboratory biosafety guidelines for handling and processing specimens associated with coronavirus disease 2019 (covid-19). available at agent summary statement for human immunodeficiency viruses (hivs) including htlv-iii, lav, hiv-1, and hiv-2 key: cord-313617-hh7lccet authors: sigel, keith; swartz, talia; golden, eddye; paranjpe, ishan; somani, sulaiman; richter, felix; de freitas, jessica k; miotto, riccardo; zhao, shan; polak, paz; mutetwa, tinaye; factor, stephanie; mehandru, saurabh; mullen, michael; cossarini, francesca; bottinger, erwin; fayad, zahi; merad, miriam; gnjatic, sacha; aberg, judith; charney, alexander; nadkarni, girish; glicksberg, benjamin s title: covid-19 and people with hiv infection: outcomes for hospitalized patients in new york city date: 2020-06-28 journal: clin infect dis doi: 10.1093/cid/ciaa880 sha: doc_id: 313617 cord_uid: hh7lccet background: there have been limited data regarding the clinical impact of covid-19 disease on people with hiv (pwh). in this study we compared outcomes for pwh with covid-19 disease to a matched comparison group. design: we identified 88 pwh hospitalized with laboratory confirmed covid-19 in our hospital system in new york between march 12 and april 23, 2020. we collected data on baseline clinical characteristics, laboratory values, hiv infection status, covid-19 treatment, and outcomes from this group and matched comparators (one pwh to up to five patients by age, sex, race/ethnicity and calendar week of infection). we compared baseline clinical characteristics and outcomes (death, mechanical ventilation, hospital discharge) for these two groups, as well as cumulative incidence of death by hiv status. results: patients did not differ significantly by hiv status by age, sex or race/ethnicity due to the matching algorithm. pwh hospitalized with covid-19 had high proportions of hiv virologic control on antiretroviral therapy. pwh had greater proportions of smoking (p<0.001) and comorbid illness than demographically similar uninfected comparators. there was no difference in covid-19 severity on admission by hiv status (p=0.15). poor outcomes for hospitalized pwh were frequent but similar to proportions in comparators; 18% required mechanical ventilation and ultimately 21% died during follow-up (compared with 23% and 20% respectively). there was similar cumulative incidence of death over time by hiv status (p=0.94). interpretation: we found no differences in adverse outcomes associated with hiv infection for hospitalized covid-19 patients compared to a demographically similar patient group. a c c e p t e d m a n u s c r i p t background sars-cov-2, the causative agent of coronavirus disease-19 , has infected millions of people worldwide since its identification in december 2019, resulting in >400,000 deaths to-date. [1] patients with immunocompromise have overall poorer outcomes from most serious infections, but this may vary based on the type of immune deficiency and the specific pathogens. hiv infection is one of the most common causes of immunocompromise globally, with over 1 million people with hiv (pwh) in the united states alone. [2] despite the substantial reach of the covid19 in this study we assessed the outcomes of covid-19 disease in pwh, comparing a hospitalized cohort of pwh from our nyc health system and a demographically matched group of comparator patients. we then evaluated factors associated with mortality for pwh hospitalized with covid-19. a c c e p t e d m a n u s c r i p t we identified all hospitalized patients with laboratory confirmed sars-cov-2 infection at five hospitals in the mount sinai health system admitted between march 12 and april 23, 2020. from this cohort we identified 88 patients with hiv-related diagnostic codes or those being treated with antiretroviral medications identified from inpatient or outpatient medication orders. we then selected an hiv uninfected comparator group (from the larger laboratory confirmed sars-cov-2 cohort) using a similar methodology to that used by the veterans aging cohort study for comparisons by hiv status. [5] we identified 405 hiv uninfected patients, matching one pwh to up to five patients by age (+/-2.5 years), sex, race/ethnicity, and calendar week of infection (to account for temporally associated changes in covid-19 management and differences in follow-up time). electronic health record (ehr) data were then collected including demographics, all diagnostic codes and procedures, as well as clinical laboratory measurements and outcomes (death, mechanical ventilation, discharge). smoking status was ascertained from a structured ehr element. oxygen supplementation requirements on admission were collected, verified via chart review and used to categorize covid-19 severity using published criteria. a c c e p t e d m a n u s c r i p t we then compared baseline characteristics, treatments and outcomes, testing to assess significant differences between pwh and comparators using the wilcoxon test for continuous variables and the chi-square test for categorical variables. we also stratified baseline laboratory measures by covid-19 severity for pwh, testing for differences in measures by severity category using ordinal logistic regression. we then compared differences in time to death to assess disease trajectory for hospitalized patients by hiv status by fitting unadjusted cumulative incidence function curves with hospital discharge as a competing risk. curves were compared using the test of pepe and mori. [7] to compare cumulative incidence of death by hiv status accounting for potential confounding factors we then fit a multivariable survival model using fine-grey competing risk methods, including demographics, covid-19 severity, comorbid conditions and laboratory values that differed by hiv status. [8] finally, we compared characteristics of pwh who died during hospitalization to those who were discharged or still alive at the end of follow-up. we explored the independent association of significant factors associated with death for pwh from covid-19 by fitting separate multivariable competing risk models using demographic factors and significant univariate predictors (one model for each predictor). based on our sample size, our primary analysis of proportion of hospital deaths by hiv status had 80% power to detect a 15% increase in the absolute risk of death for pwh compared to uninfected persons. all analyses were conducted using stata version 15. this study was approved by our institutional review board. a c c e p t e d m a n u s c r i p t of the 4,402 patients hospitalized during the study period for covid-19, 88 (2%) were pwh. the median age of pwh hospitalized with covid-19 disease was 61 (table 1; intraquartile range [iqr] 54-67) and most pwh were black (40%) or hispanic/latino (30%). patients did not differ significantly by hiv status when comparing age, sex, or race/ethnicity due to the matching algorithm. consistent with trends noted in hiv cohort studies, [9] pwh also had greater proportions of smoking (55% versus 23%; p<0.001) and comorbid illnesses than demographically-similar uninfected comparators, most notably chronic obstructive pulmonary disease (copd; 10% vs. 3%; p<0.001), cirrhosis (6% vs. 2%; p=0.02) and a history of cancer diagnosis (17% vs. 6%; p=0.001). pwh and uninfected persons had similar covid-19 severity on admission as measured by oxygen supplementation requirements (p=0.15). all pwh admitted for covid-19 were prescribed antiretroviral therapy and most (78%) were receiving integrase inhibitor-based regimens. the majority (58%) of patients with cd4 measurements on hospital admission had counts >200 cells/mm 3 (table s2; p=0.005). the majority of pwh were treated with hydroxychloroquine and azithromycin; experimental or expanded-access agents were used less frequently. most hospitalized pwh were discharged from the hospital during the follow-up period (figure 1 ). there was no significant difference in intensive care use by hiv status in our cohort. poor outcomes for hospitalized pwh were still frequent but similar to proportions in comparators; 18% required a c c e p t e d m a n u s c r i p t mechanical ventilation and 21% died during follow-up compared with 23% and 20%, respectively. competing risks analysis showed similar cumulative incidence of death over time by hiv status with no significant difference in the curves (p=0.94; figure 2 ). hiv was not significantly associated with risk of death in multivariable competing risks analysis after adjusting for demographics, copd, smoking, baseline ferritin level and baseline white blood cell count (table s3) . comparisons of pwh who died to those who were still alive or discharged at the end of follow-up revealed few differences in patients who died compared to those who did not. we did find, however, differences in the proportions who were organ transplant recipients who died versus those who had not died ( among patients from five hospitals in a large health system during the peak of the spring 2020 nyc sars-cov-2 epidemic, we found that pwh hospitalized with covid-19 had a substantial burden of comorbid illness and smoking but had good hiv disease control. although mortality and other adverse outcomes were high among pwh in our cohort, these were not worse than a demographically and temporally matched group with similar covid-19 severity at presentation and fewer comorbidities. our study represents the largest and most diverse cohort of pwh with covid-19 that has been described to date and adds to existing limited evidence that hiv might not be associated with more severe covid-19 disease course. concern that covid-19 disease might be more severe in persons with immunodeficiency or immune dysregulation has been raised since the emergence of the earliest cases. [10] uncontrolled series of immunosuppressed persons such as kidney transplant recipients and cancer patients have shown a c c e p t e d m a n u s c r i p t high mortality rates. [11] outcomes analyses for pwh during the covid-19 pandemic have been limited however, consisting of small case reports or series. [12] a single center in spain reported outcomes for five male pwh hospitalized with covid-19. four of the five men had been discharged and the fifth was in intensive care at the time of their report. [13] larger uncontrolled series from new jersey (13 hospitalized patients), new york city (31 hospitalized patients) and madrid, spain (28 hospitalized patients) also subsequently demonstrated covid-19 outcomes for pwh similar to those described for the general population. [14] [15] [16] this is broadly consistent with our finding that respiratory failure requiring mechanical ventilation and death were not more frequent in pwh when compared to demographically similar persons with covid-19. the lack of outcome differences is even more striking when noting that copd, cancer and smoking, risk factors linked to worse covid-19 outcomes in several previous studies, were far more prevalent in pwh in our cohort than in comparator patients. [17, 18] immunomodulatory effects of sars-cov-2 have been associated with severe sequelae including a cytokine release syndrome. [4] several factors have supported theoretical risks of worse covid-19 disease in pwh including incomplete immune reconstitution and evidence of persistent immune activation in many patients prior to the pandemic. [19, 20] furthermore, increased il-6 and d-dimer measures have been independently associated with chronic hiv infection [21, 22] and have also been closely linked with covid-19 severity in data from mostly hiv uninfected persons. [23, 24] neither biomarker differed on presentation for patients by hiv status nor was either associated with covid-19 severity at presentation for pwh. among pwh, cd4 decline was noted in the majority of patients with available data, consistent with existing immunologic data on covid-19 natural history, but the decrease in cd4 percentage was not large. [25] we did not find evidence of associations between immunologic measures (either decreases from pre-covid-19 values or low values at the time of presentation) and adverse covid-19 outcomes for pwh. organ transplantation was associated with death for pwh in our study, however, suggesting that non-hiv causes of immunodeficiency may be more prominent risks for severe outcomes. our study benefited from data collected from diverse patient groups from five hospitals within a nyc large health system that is one of the largest hiv care providers in the united states. our sample size of pwh was limited but we were nonetheless able to identify a large, well-matched comparison group for comparison of outcomes. to maximize our comparison group size, we limited our matching strategy to demographic and temporal factors using a similar method to the largest american hiv cohort study although this yielded differences in comorbidity profiles for the two groups. [5, 29] this difference in comorbidity profiles may have also been influenced by more frequent medical care for pwh, leading to an imbalance in the ascertainment of comorbid diagnoses. however, the marked difference in smoking history for pwh versus hiv uninfected persons supports likely differences in the true prevalence of these comorbid diseases by hiv status in our cohort. in addition, some measures such as laboratory data were missing for substantial portions of the sample, in proportions too large for imputation. nonetheless, our key exposures and outcomes were manually verified and provide important information for this vulnerable population. in conclusion, we found no differences in adverse outcomes associated with hiv infection for a c c e p t e d m a n u s c r i p t m a n u s c r i p t anti-retroviral therapy (art) 88 (100) -integrase 69 (78) -covid-19 projections the time is now to end the hiv epidemic do biomarkers of inflammation, monocyte activation, and altered coagulation explain excess mortality between hiv infected and uninfected people? the trinity of covid-19: immunity, inflammation and intervention veterans aging cohort study (vacs): overview and description ultra-high-throughput clinical proteomics reveals classifiers of covid-19 infection marginal or conditional probability curves in summarizing competing risks failure time data practical methods for competing risks data: a review excess clinical comorbidity among hiv-infected patients accessing primary care in us community health centers the virus that changed spain: impact of covid-19 on people with hiv covid-19 and kidney transplantation co-infection of sars-cov-2 and hiv in a patient in wuhan city covid-19 in patients with hiv: clinical case series covid-19 pneumonia in patients with hiv -a case series clinical characteristics and outcomes in people living with hiv hospitalized for covid-19 description of covid-19 in hivinfected individuals: a single-centre, prospective cohort severity and mortality associated with copd and smoking in patients with covid-19: a rapid systematic review and meta-analysis cancer patients in sars-cov-2 infection: a nationwide analysis in china lack of mucosal immune reconstitution during prolonged treatment of acute and early hiv-1 infection hiv-associated chronic immune activation hiv status, burden of comorbid disease, and biomarkers of inflammation, altered coagulation, and monocyte activation markers of inflammation, coagulation, and renal function are elevated in adults with hiv infection risk factors for severity and mortality in adult covid-19 inpatients in wuhan clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study dysregulation of immune response in patients with covid-19 in wuhan, china a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 compounds with therapeutic potential against novel respiratory sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study aging and infectious diseases: do patterns of comorbidity vary by hiv status, age, and hiv severity? laboratory values -median (iqr) a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t >50 copies/ul 1 (9) 11 (21) a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-326744-eled2tgo authors: millett, gregorio a.; honermann, brian; jones, austin; lankiewicz, elise; sherwood, jennifer; blumenthal, susan; sayas, asal title: white counties stand apart: the primacy of residential segregation in covid-19 and hiv diagnoses date: 2020-10-01 journal: aids patient care stds doi: 10.1089/apc.2020.0155 sha: doc_id: 326744 cord_uid: eled2tgo emerging epidemiological data suggest that white americans have a lower risk of acquiring covid-19. although many studies have pointed to the role of systemic racism in covid-19 racial/ethnic disparities, few studies have examined the contribution of racial segregation. residential segregation is associated with differing health outcomes by race/ethnicity for various diseases, including hiv. this commentary documents differing hiv and covid-19 outcomes and service delivery by race/ethnicity and the crucial role of racial segregation. using publicly available census data, we divide us counties into quintiles by percentage of non-hispanic white residents and examine hiv diagnoses and covid-19 per 100,000 population. hiv diagnoses decrease as the proportion of white residents increase across us counties. covid-19 diagnoses follow a similar pattern: counties with the highest proportion of white residents have the fewest cases of covid-19 irrespective of geographic region or state political party inclination (i.e., red or blue states). moreover, comparatively fewer covid-19 diagnoses have occurred in primarily white counties throughout the duration of the us covid-19 pandemic. systemic drivers place racial minorities at greater risk for covid-19 and hiv. individual-level characteristics (e.g., underlying health conditions for covid-19 or risk behavior for hiv) do not fully explain excess disease burden in racial minority communities. corresponding interventions must use structuraland policy-level solutions to address racial and ethnic health disparities. t en weeks after the states began reopening in the wake of the white house's ''opening up america again'' guidelines, daily covid-19 cases have been increasing rapidly again in the united states. 1 sustained spikes in covid-19 cases in the south and the west have resulted in the doubling of us cases nationally in a matter of weeks. 2, 3 much of the corresponding press coverage has highlighted differing state and local public health responses based upon partisan divisions with differing observed trajectories of covid-19 in red and blue counties, 4 or the degree to which the surge in new infections is occurring among young adults. 5 mostly missing from the media narrative is the durability of racial disparities as covid-19 cases gain traction beyond the northeast. although an analysis recently linked the nationwide relaxation of public health restrictions to increases in covid-19 cases among latinx populations, 6 a more illuminating question is what is the trajectory of covid-19 cases nationally in overwhelmingly non-hispanic white counties since the reopening of the economy? some observers have been surprised by the rapid emergence of clear disparities in covid-19 acquisition and poor health outcomes along the lines of race/ethnicity for an infectious agent that can infect anyone. one approach to making sense of these disparities is to assess the impact of race and ethnicity in the context of hiv. like covid-19, hiv is an infectious disease that can infect anyone, yet for which large and persistent racial and ethnicity disparities exist. one important starting point is to acknowledge that race and ethnicity often not only determine where people live in the united states but their health status as well. redlining by the federal home owner's loan corporation in the 1930s not only codified racial segregation nationally by excluding specific communities from key financial and other resource investments, but also continues to determine current racial segregation patterns by census tract and subsequent health outcomes for various conditions, including covid-19 and hiv. 7, 8 a recent analysis reported an association between greater covid-19 mortality in primarily black neighborhoods and redlining practices in chicago. 9 racial segregation in another study explained 19% of hiv infection among black people who inject drugs (pwid) as opposed to 3% of hiv infections in white or latinx pwid; 10 and a new york city analysis found that 65% of black men diagnosed with hiv and 68% of new hiv diagnoses among black men occurred in specific zip codes. 11 using publicly available census data, we divided us counties into quintiles by percentage of non-hispanic white residents (quintile 1: <60.18%, quintile 2: 60.19-77.39%, quintile 3: 77.40-88.11%, quintile 4: 88.11-93.80%, and quintile 5: >93.80%). covid-19 diagnoses were standardized per 100,000 population, and graphs depicting the 7-day moving average of diagnoses through june 25, 2020, were generated. we hypothesized that counties with the greatest percentage of non-hispanic whites (hereafter ''white'') would have the least covid-19 cases before and after reopening the economy (most states partially reopened by may 15, 2020). 12 conversely, counties with the fewest white residents would have the greatest covid-19 cases. utilizing the cdc atlas hiv data per 100,000 population, we also hypothesized that hiv diagnoses data would follow a similar pattern as covid-19 cases as per county demographics. the same pattern is evident by region. irrespective of us region, covid-19 cases were lowest in counties with the most number of white residents (fig. 1 ). despite the increasing covid-19 pandemic across all quintiles in the south and west, infections remain lowest in counties with the highest proportion of white residents; whereas the declining covid-19 pandemic in the northeast and midwest was driven by the decline in cases among more racially diverse counties. remarkably, covid-19 diagnoses in northeastern and midwestern counties with primarily white residents have remained fairly low and stable throughout the entirety of the covid-19 crisis. surveys and studies have found differences by political parties in the acceptance of and adherence to covid-19 prevention measures such as social distancing 13 or wearing face masks, 14 as well as comfort in resuming daily activities as the nation opens. 15 it has been widely reported that there are increasing covid-19 cases in states that voted for donald trump in 2016, while cases are declining in states that voted for hillary clinton. 16 we find the same pattern, but the previous analysis misses two crucial details ( fig. 2) : first, irrespective of the 2016 presidential candidate preference, covid-19 cases remain lowest in the whitest counties in blue or red states relative to more diverse counties even after reopening. second, even though covid-19 cases have declined or remained stable in states that voted for hillary clinton, infections are increasing in the most diverse counties after reopening. lower infectious disease burden in primarily white communities is not confined to covid-19. we found the same stark differences in hiv diagnoses per 100,000 (fig. 3) , with the greatest prevalence in more diverse counties. as with covid-19 diagnoses, hiv diagnoses decrease progressively across counties nationally as the population of white residents increases. the burden of the covid-19 pandemic in the united states has not been borne equally. we published the first national study of covid-19 in black communities and found that both cases and deaths increased as the proportion of black residents increased across counties in the united states. 17 counties with a higher proportion of black americans (only 22% of counties nationwide and 35% of the us population) account for 47% of covid-19 cases and 54% of deaths nationally as of june 25, 2020. 18 these disparities are not confined to urban areas. we found the same racial disparity among counties in the small metropolitan and rural areas of the united states. communities other than black americans are at elevated risk for covid-19. 19 latinx households are more likely than other groups to report a myriad of symptoms associated with covid-19, 20 polls have found that more black and latinx americans than white americans know someone who has died of covid-19, 21 and in late may, the navajo nation surpassed new york and new jersey in per capita covid-19 cases. 22 there are multiple factors that contribute to the greater covid-19 burden in communities of color, but all stem from systemic racism. our published epidemiological analyses point to specific factors that exacerbate covid-19 diagnoses in black 17 and latino 23 communities, including poverty and living in densely occupied households, living in localities with greater air pollution, lack of health insurance, and being employed in jobs that increase exposure to sars-cov-2. our data also show that monolingual spanish speakers (an economically and politically vulnerable community) have an elevated risk of being diagnosed with covid-19, 23 possibly due to the same systemic inequities that place this same population at a higher risk for hiv opportunistic infections. 24 conversely, fewer white americans feel at risk for covid-19 as the demographics of those stricken by the pandemic have become clearer. a pew poll between april and june 2020 found increasing majorities of black and latinx respondents being very or somewhat concerned of being hospitalized for covid-19. only 43% of white respondents expressed the same concern in the june 2020 poll, which was an 8-point drop from the april poll. 25 this racial divide in covid-19 risk translated into diverging beliefs around reopening businesses during the pandemic. another poll conducted in march 2020 found that most black, latinx, and white americans believed that it was important to keep businesses closed for the sake of public safety (76-83%). 26 by june 2020, anxiety remained high among black and latinx americans (82% and 65%, respectively), but dropped to only 50% among white americans. many point to underlying health conditions (e.g., obesity, hypertension, diabetes, heart disease) as the source of covid-19 racial disparities. although underlying health conditions are greater in communities of color, our study and other studies show that these conditions are not driving covid-19 racial disparities. 27 we found that employment status, access to health insurance, and overcrowded homes were better predictors of covid-19 than any underlying condition. 17, 23 another recent analysis reported greater covid-19 cases among black americans despite the fact that social distancing behavior was more common among african americans. 28 attributing racial disparities to underlying conditions implicitly blames communities of color for covid-19 disparities due to poor health decisions; but there is ample literature showing how social determinants contribute to worse health outcomes in communities of color, 29 including well-cited hiv research studies of youth, gay men, and pwid, which show that hiv disparities persist in black communities despite similar or fewer behavioral risks than whites. [30] [31] [32] existing reports underline the contributions of segregation and privilege to race-specific covid-19 and hiv outcomes. studies in tennessee, 33 texas, 34 philadelphia, 35 and new york 35 have repeatedly found that covid-19 testing centers were initially located in white and wealthy rather than poorer or browner neighborhoods. similarly, an analysis found that whiter and wealthier neighborhoods in new york city witnessed a 40% decline in residents between march and may who fled the city to the safety of country homes, neighboring states, or other locales with fewer covid-19 diagnoses. 36 those left behind to weather the worst of the covid-19 crisis in nyc were disproportionately poor, less educated, black, latinx, or asian. another consequence of residential segregation is proximity to health care facilities. nationally representative surveys have found that racial minorities on average travel farther than whites to access health care and spend more time in a waiting room to see a health care provider. 37 moreover, the disproportionate number of black and brown workers who cannot work from home during the pandemic 38 and must take public transportation poses another risk for covid-19 infection after reopening the economy. 39 traveling further for care is associated with being uninsured among people living with hiv, 40 and distance to care is linked to poor hiv outcomes in communities of color. a cohort study of 3623 respondents (77% non-hispanic black and 6.2% latinx) receiving hiv care in the dc area outpatient clinics found that patients who had to travel 5 or miles had a 30% lower retention and care and viral load. 41 a separate study of people living with hiv in birmingham, alabama, reported that both lack of fuel and lack of a vehicle approached statistical significance in predicting late presentation to hiv care among african american clinic attendees. 42 hiv and covid-19 racial disparities are not a fait accompli. systemic inequities that have accrued over hundreds of years, and residential segregation in particular, are not subject to change overnight. this does not mean that these disparities are a fait accompli. there are lessons learned from the hiv literature in reducing health disparities, such as greater access to health care under medicaid expansion for communities of color, targeted testing, and the provision of wrap-around services. a higher proportion of blacks (45%) versus whites (35%) living with hiv receive care via medicaid nationally, 43 and states with expanded medicaid have lower rates of being uninsured as well as fewer hospitalizations among people living with hiv. 43, 44 most of the 12 states that have not expanded medicaid are in the southern united states, a region where 55% of the black community resides. the covid-19 crisis and the accompanying economic downturn are prompting referenda in various red states to consider the adoption of medicaid expansion, 45 which may increase health care access in communities of color and improve overall health outcomes. moreover, scaling up testing in communities of color can reduce disparities. a cdc demonstration project that scaled up hiv testing efforts in black and latino communities in the district of columbia dramatically reduced the proportion of concurrent aids diagnoses at first positive test; 46 and encouraging data from a recent study 35 show shifts in covid-19 testing from wealthier and white neighborhoods to poorer and more diverse neighborhoods in cities initially hit by the covid-19 pandemic. scaling up services that help blunt the social determinants of health are likewise useful. local and national studies of the ryan white program have found declining hiv-related disparities by race due to the provision of wraparound services, 47, 48 which may have implications for effectively addressing covid-19 disparities. equity in health care access and delivery can also reduce health-related disparities. although far from perfect, the us military has endeavored to meaningfully address institutional racism. 49 these efforts have also impacted health. a recent cdc study of active us service members living with hiv reported uniformly high rates of viral suppression irrespective of race or ethnicity, 50 and studies from the veterans administration (va) have found less pronounced health disparities by race in the va system compared with the us population overall. 51 similarly, a preprint of nearly 6000 veterans found greater covid-19 diagnoses among black and latinx veterans, but no difference by race in covid-19 deaths. 52 in other words, unlike what we have witnessed nationally, black and latinx covid-19 patients in the va's care are not experiencing excess deaths compared with whites. the premature reopening of our economy has only worsened the covid-19 crisis among communities of color irrespective of region or blue or red voting patterns. however, there are immediate steps that can be taken to achieve greater health equity and improve health outcomes among all americans. no competing financial interests exist. there was no funding provided for this article. reopening america: all 50 states have begun to reopen. see what that means for your state. nbc news the national. covid-19 cases rise in infections swamp the u.s. which recorded 42% of all its coronaviruses cases in july. new york times. 2020. available at the coronavirus is deadliest where democrats live as virus surges, younger people account for 'disturbing' number of cases many latinos couldn't stay home. now virus cases are soaring in their communities structural racism, historical redlining, and risk of preterm birth cancer stage at diagnosis, historical redlining, and current neighborhood characteristics: breast, cervical, lung, and colorectal cancers covid-19, race, and redlining risk environments, race/ethnicity, and hiv status in a large sample of people who inject drugs in the united states high hiv prevalence and diagnosis rates in new york city black men see how all 50 states are reopening (and closing again) trump campaign removed social-distancing stickers from seats at tulsa rally face masks make a political statement in era of coronavirus republicans, democrats move even further apart in coronavirus concerns the shift of the coronavirus to primarily red states is complete-but it's not that simple assessing differential impacts of covid-19 on black communities the foundation for aids research. covid-19 racial disparities in u.s. counties [database coronavirus is decimating racially diverse communities large and small latino homes report serious covid-19 symptoms nearly twice as often, survey of 1.6 million shows. usa today almost one-third of black americans know someone who died of covid-19, survey shows navajo nation surpasses new york state for the highest covid-19 infection rate in the us risk for covid-19 infection and death among latinos in the united states: examining heterogeneity in transmission dynamics factors in the delayed hiv presentation of immigrants in northern california: implications for voluntary counseling and testing programs to understand who's dying of covid-19, look to social factors like race more than preexisting diseases. stat news many white americans are ready to reopen the economy social sources of racial disparities in health social distancing across vulnerability, race, politics, and employment: how different americans changed behaviors before and after major covid-19 policy announcements the coronavirus doesn't discriminate, but u.s. health care showing familiar biases. national public radio sexual and drug behavior patterns and hiv/std racial disparities: the need for new directions comparisons of disparities and risks of hiv infection in black and other men who have sex with men in canada, uk, and usa: a meta-analysis racial differences in sexual behaviors related to aids in a nineteen city sample of street-recruited drug injectors texas cities experience disparities in access to the coronavirus testing positive tests higher in poorer neighborhoods sespite six times more testing in higher-income neighborhoods, researcher says. cbs philly spatial inequities in covid-19 outcomes in three us cities the richest neighborhoods emptied out most as coronavirus hit new york city disparities in time spent seeking medical care in the united states as states reopen, black workers are at greater risk for covid-19. cbs news coronavirus is putting public transportation riders at risk. usa today. 2020. available at travel distance to hiv medical care: a geographic analysis of weighted survey data from the medical monitoring project in philadelphia identifying spatial variation along the hiv care continuum: the role of distance to care on retention and viral suppression social determinants of late presentation to hiv care insurance coverage and viral suppression among people with hiv in the united states in four aca expansion states, the percentage of uninsured hospitalizations for people with hiv declined red state votes to expand medicaid-even as gop tries to kill obamacare amid pandemic. abc news expanded hiv testing and trends in diagnoses of hiv infection-district of columbia reducing hiv-related health disparities in the health resources and services administration's ryan white hiv/aids program improvement in the health of hiv-infected persons in care: reducing disparities diversity, inclusion, and equal opportunity in the armed services: background and issues for congress antiretroviral therapy and viral suppression among active duty service members with incident hiv infection-united states racial/ethnic disparities in mortality across the veterans health administration covid-19 by race and ethnicity: a national cohort study of 6 million united states veterans key: cord-355439-eqtk51q3 authors: lesko, catherine r; bengtson, angela m title: hiv and sars-cov-2: intersecting epidemics with many unknowns date: 2020-07-22 journal: am j epidemiol doi: 10.1093/aje/kwaa158 sha: doc_id: 355439 cord_uid: eqtk51q3 as of july 2020, approximately 6 months into the pandemic of novel coronavirus disease 2019 (covid-19), whether people living with hiv (plwh) are disproportionately affected remains an unanswered question. thus far, risk of covid-19 in people with and without hiv appears similar but data are sometimes contradictory. some uncertainty is due to the recency of the emergence of covid-19 and sparsity of data; some is due to imprecision about what it means for hiv to be a “risk factor” for covid-19. forthcoming studies on the risk of covid-19 to plwh should differentiate between 1) the unadjusted, excess burden of disease among plwh to inform surveillance efforts; and 2) any excess risk of covid-19 among plwh due to biological effects of hiv, independent of comorbidities that confound rather than mediate this effect. plwh bear a disproportionate burden of alcohol, other drug use, mental health disorders, and other structural vulnerabilities, which may increase their risk of covid-19. in addition to any direct effects of covid-19 on the health of plwh, we need to understand how physical distancing restrictions impact secondary health outcomes, and the need for, accessibility of, and impact of alternative modalities of providing ongoing medical, mental health, and substance use treatment that comply with physical distancing restrictions (e.g., telemedicine). cov-2 epidemic. it is critical that we understand these risks to modify ongoing hiv care accordingly, and to update future pandemic preparedness plans. herein, we outline several research questions to frame this research agenda, and we highlight existing data and future opportunities to answer these questions. we focus mainly on the intersecting epidemics of sars-cov-2 and hiv in the united states of america (usa), but many of the questions we pose apply to other settings as well. it is unclear whether or not plwh are at higher risk for infection with sars-cov-2 or for poor clinical outcomes subsequent to infection. there are reasons to hypothesize that plwh are a high-risk group: antibody responses to an immune system challenge are impaired in plwh, and plwh have high prevalence of risk factors for severe sars-cov-2 infection including hypertension, diabetes, cardiovascular disease, obesity, lung disease and smoking, male sex, and older age (1, 2) . alternatively, worse covid-19 outcomes may be due to immune (over)activation, and thus plwh might actually be at lower risk for poor outcomes following sars-cov-2 infection due to their reduced immune response (3) . however, there are not yet sufficient data to support or refute either of these hypotheses. early in the course of an epidemic of a novel pathogen, evidence is scarce and the most practical or indeed the only epidemiologic study design available to us is the case report or case series (4) (5) (6) (7) (8) . early case reports and case series of covid-19 in plwh told of an occasionally atypical, but not more severe, disease course, relative to people living without hiv (7) (8) (9) (10) (11) (12) (13) . some case series suggested that plwh with covid-19 may be younger than persons with covid-19 in the general population (11, 12). however, incidence and mortality rates for covid-19 will be a function of the age structure of the underlying populations of people with versus without hiv, thus it is difficult to compare rates without age-standardization. data on the incidence of covid-19 in plwh is slowly amassing from population-(i.e., surveillance) and clinic-based cohorts of plwh. while important, absolute risk estimates will illinois, usa (15%) was comparable to the positivity rate among people without hiv (19%) (16) . in contrast to these cohorts suggesting similar infection rates in people with and without hiv, unpublished surveillance data from south africa's western cape province through june 9, 2020 suggest that plwh were 2.3 times as likely to die from covid-19 as people without hiv, after age and sex standardization (17) . certainly, more information is needed. surveillance data, such as those available from south africa or wuhan, will provide the most complete picture of covid-19 risk among plwh (e.g., by not restricting to plwh who are in care and who are more likely to have wellcontrolled hiv disease); however clinical data, such as those from madrid, may provide the most depth (e.g., by allowing examination of the role of comorbidities, medications, and covid-19 treatments) as long as potential selection bias is considered. perhaps the most fruitful investigation would be one that merged clinical and surveillance data. strict initial guidelines for testing for sars-cov-2 infection that restricted testing to people with a history of travel to wuhan, and then to china, or to people with a known epidemiologic connection to a confirmed case limits our ability to accurately describe incidence of sars-cov-2 in plwh. even if testing were widely available, incidence estimates would be plagued by non-randomly missing data from people with poor access to health care, people who are avoiding healthcare settings for fear of contracting or transmitting sars-cov-2, and people who don't believe themselves to be infected. new serologic assays for past exposure to sars-cov-2 are rapidly becoming available (18) . sensitivity of serologic tests in plwh with compromised immune systems who may not mount a vigorous antibody response may be lower than the nominal sensitivity; unless test and patient characteristics are taken into account, serosurveys of plwh may underestimate the true burden of sars-cov-2 infection. as with estimation of incidence, attempts to estimate prevalence of past sars-cov-2 infection in plwh must take into account who is and is not included in any serosurvey. some states are randomly sampling residents for serosurveys (19) ; if sampling strategies considered groups of special interest, including plwh, these serosurveys may be an opportunity to get estimates of prior sars-cov-2 infection in plwh. useful epidemiologic investigations into the impact of covid-19 on plwh will need to carefully consider the research question of interest and how results will be used. there is justified concern about labeling hiv as an -independent risk factor‖ for poor covid-19 outcomes based on an arbitrary multivariable model as it may then be inappropriately used to ration care or guide treatment decisions. ambiguity about the meaning of the term -independent risk factor‖ make it highly likely that results will be misinterpreted and misapplied (20, 21) . if interest is in identifying groups that should be monitored more closely for sars-cov-2 infection, this is a descriptive epidemiology question and crude analyses (or perhaps age-and sex-adjusted analyses) may be sufficient (22) . while the most appropriate adjustment set for descriptive epidemiology is an unresolved question, associations from a multivariable model are matching factors included some confounders of the effect of hiv, such as admission date, age, gender, and tobacco history, but also included variables that might be considered mediators, such as body mass index, and history of chronic kidney disease, hypertension, asthma, chronic obstructive pulmonary disease, and heart failure (23) . in another matched cohort in new york, outcomes of people with and without hiv hospitalized for covid-19 were similar even without adjusting for higher prevalence of chronic obstructive pulmonary disease, prior cancer, cirrhosis, and current smoking in plwh (24) . in the data. certain antiretroviral medications, such as lopinavir-ritonavir (a protease inhibitor), were proposed and partially evaluated as treatments for other, similar coronaviruses (25) . however, a trial of 199 patients randomized to lopinavir-ritonavir versus standard of care found only small differences in time to clinical improvement (hazard ratio: 1.24, 95% confidence interval: 0.90, 1.72) and 28-day mortality (risk difference: -5.8%, 95% confidence interval: -17.3%, 5.7%). there was some hint that the impact of lopinavir-ritonavir on mortality was stronger if treatment was administered closer to symptom onset, although results were imprecise. results were reported as indicative of -no benefit‖ of lopinavir-ritonavir, although associations were suggestive of a potentially protective effect (26) . while these results do not support initiating treatment with lopinavir-ritonavir in patients with sars-cov-2, they might suggest some benefit to plwh on a lopinavir-ritonavir-containing antiretroviral therapy (art) regimen who continue on treatment while infected with sars-cov-2. darunavir (another protease inhibitor) has also been hypothesized to potentially have therapeutic action against sars-cov-2, however no trial results are yet available. thus far, in cohort studies of covid-19 among plwh, art regimen has not been consistently associated with disease incidence or severity. in a small cohort (n=88) of plwh hospitalized with covid-19 in new york city, new york, usa, being on a nucleoside reverse transcriptase inhibitor was protective against death (24) . in a cohort of over 77,000 plwh receiving art in spain, being on a regimen containing tenofovir/emtricitabine (a nucleotide reverse transcriptase inhibitor and a nucleoside reverse transcriptase inhibitor, respectively) was protective against covid-19 diagnosis and hospitalization (27) . data on the association between art regimen and covid-19 outcomes are still too limited as to support or exclude an effect of any particular regimen. engagement in ongoing care is essential to the health of plwh. hiv viral load and cd4 cell count should be monitored every 3-6 months (28) . in light of the risk of sars-cov-2 transmission associated with face-to-face contact, particularly in medical settings, many clinical encounters (for all people, including for plwh) were rapidly changed to telehealth visits starting in march 2020 as sars-cov-2 cases started increasing rapidly (29) . while telehealth visits eliminate the potential exposure to sars-cov-2 and thus may be necessary for some period, the costs and benefits associated with telemedicine need to be enumerated and weighed. prior to the sars-cov-2 outbreak, telehealth was studied as a potential intervention to increase access to care (30) particularly for plwh with transportation difficulties and those living in rural settings (31) . however, offering telehealth to persons who opt-in is a different intervention than requiring telehealth visits to all persons in the midst of a pandemic, and may result in different outcomes. there is, as yet, little data on the short and long-term impacts of the transition to telehealth on engagement in care and art adherence for plwh. in a narrative report, >90% of patients in a missouri hiv clinic (presumably among those who successfully completed a telehealth visit) reported their telehealth visit during covid-19 physical distancing restrictions was as good as or better than a traditional in-clinic visit (29) . not provided was the number of patients who failed to complete a telehealth visit. at a clinic in chicago, from late-march to mid-april, only 21% of scheduled visits were carried out virtually; 31% were rescheduled, 2% occurred in person, and 46% were not attended (16) . the impact of telehealth on high-need patients and new patients who have not yet established rapport with their providers has yet to be described (7). despite some good telehealth outcomes for some plwh, telehealth has the potential to exacerbate disparities in care for people with lower socio-economic status: lack of necessary technology and services, technology literacy, and safe, confidential surroundings to participate fully in telehealth may be barriers to engagement in care (32 distancing restrictions if they need to go outside their homes to access alcohol or other drugs, or critically, medication assisted treatments (such as methadone or buprenorphine). poor baseline mental health is likely to be exacerbated by physical distancing restrictions (42) . plwh, particularly older plwh, are already at high risk of social isolation (43, 44) , and social structures and creative outlets that have helped people cope in the past may be dismantled under physical distancing restrictions. breaking with physical distancing policy to seek out these coping outlets may be associated with additional stress due to fears of sars-cov-2 exposure or stigma. accurate estimates of the risk associated with such activities for plwh are critical to help individuals weigh the risk and benefits of participating in them, but are not currently available. people able to shelter in place in their homes, may face additional stressors at home, if they are alone in their home, if being at home imposes additional caregiving responsibilities, or if they live with someone who poses a physical or emotional threat. for persons with diagnosed mental health disorders, physical distancing restrictions and the transition to telehealth may lead to difficulty receiving or fully engaging in behavioral treatments for those disorders. indeed, while delivery of mental health counseling may be one of the medical services most amenable to delivery via video conferencing, it may also serve as a ‗canary in the coal mine' for emergent disparities due to access to technology and private, safe spaces to participate in counseling (30, 45) . for example, one hiv clinic in chicago, illinois, usa reported some patients who had been receiving mental health counseling prior to the institution of physical distancing measures temporarily discontinued services when they were offered via telehealth, but other patients engaged in tele-counseling for the first time. engagement in tele-counseling was universal among patients with stable income and housing, but entirely absent among patients who were unstably housed with no steady source of income; in lieu of tele-counseling, the latter group of patients received peer counseling, which was more flexible with respect to the time and locations in which it could occur (16) . in addition to exacerbated mental health symptoms as a result of physical distancing, persons with severe mental health symptoms may be at higher risk for sars-cov-2 infection if their understanding of public health messaging is impaired, and if they do not understand their risk and how to mitigate it (46) . the hiv epidemic has disproportionately impacted marginalized communities: people belonging to minority racial or ethnic groups, and in particular women of color, young men of color who have sex with men, people who inject drugs, transgender individuals, and people with a history of incarceration. the same structures that placed these groups at higher risk for hiv, including racism, stigmatization, limited economic opportunities, oppression, also place them at higher risk for sars-cov-2, such that the term syndemic has been used to describe these overlapping epidemics and vulnerabilities (47, 48) . less than 6 months into the covid-19 pandemic, we are already seeing staggering disparities in the proportion of confirmed sars-cov-2 infections and covid-19 deaths in black americans, and persons in homeless shelters and prisons (49) (50) (51) (52) . persons with limited income are likely to be able to take some precautions that require financial resources, such as driving in lieu of taking public transportation (53), stockpiling groceries, or paying for grocery delivery. indeed, even in the first two weeks of implementation of physical distancing regulations in alabama, usa, there was increased need for wrap-around social services such as provision of nutritional and personal care items (54) . there is likely to be increased need for services among plwh who were already receiving such services, and also increasing number of people in need of services. data that can help answer many of these questions are already being collected (or their collection is planned), but not yet available for analyses. because many cohorts of plwh prearound hiv provides many opportunities to answer some of these outstanding questions, as long as we adhere to good epidemiologic principles with regards to asking well-defined questions. leveraging these opportunities to inform public health practice, requires that the specific research question being addressed is clearly stated, and appropriate analyses for answering that questions are applied. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study risk factors of critical & mortal covid-19 cases: a systematic literature review and meta-analysis could hiv infection alter the clinical course of sars-cov-2 infection? when less is better co-infection of sars-cov-2 and hiv in a patient in wuhan city, china covid-19 in patients with hiv: clinical case series sars-cov-2 and hiv hiv/sars-cov-2 coinfected patients in istanbul, turkey a case of hiv and sars-cov-2 co-infection in singapore hiv and sars-cov-2 co-infection: a case report from uganda early virus clearance and delayed antibody response in a case of coronavirus disease 2019 (covid-19) with a history of coinfection with human immunodeficiency virus type 1 and hepatitis c virus clinical features and outcomes of hiv patients with coronavirus disease 2019 covid-19 in people living with human immunodeficiency virus: a case series of 33 patients prevalence, clinical characteristics and treatment outcomes of hiv and sars-cov-2 co-infection: a systematic review and meta-analysis a survey for covid-19 among hiv/aids patients in two districts of wuhan, china description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort outcomes among people living with hiv during the covid-19 pandemic people with hiv at greater risk of covid-19 death in south african study johns hopkins bloomberg school of public health center for health security blood tests show 2.2 percent of riers have coronavirus antibodies opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients the table 2 fallacy: presenting and interpreting confounder and modifier coefficients adjusted statistics, and maladjusted statistics outcomes among hiv-positive patients hospitalized with covid-19 covid-19 and people with hiv infection: outcomes for hospitalized patients coronaviruses -drug discovery and therapeutic options a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 incidence and severity of covid-19 in hiv-positive persons receiving antiretroviral therapy: a cohort study antiretroviral drugs for treatment and prevention of hiv infection in adults: 2016 recommendations of the international antiviral society-usa panel it is time to include telehealth in our measure of patient retention in hiv care overcoming technological challenges: lessons learned from a telehealth counseling study geographic access and use of infectious diseases specialty and general primary care services by veterans with hiv infection: implications for telehealth and shared care programs covid-19, telemedicine, and patient empowerment in hiv care and research general and health-related internet use among an urban, community-based sample of hiv-positive women: implications for intervention development mobile fact sheet a qualitative study investigating the use of a mobile phone short message service designed to improve hiv adherence and retention in care in canada (weltel bc1) digital divide persists even as lower-income americans make gains in tech adoption advantages and disadvantages for receiving internet-based hiv/aids interventions at home or at community-based organizations exploring the attitude of patients with hiv about using telehealth for hiv care epidemics: covid-19 and lack of health insurance rebalancing the ‗covid-19 effect' on alcohol sales. the nielsen company (us) reducing hiv risks in the places where people drink: prevention interventions in alcohol venues the mental health consequences of covid-19 and physical distancing: the need for prevention and early intervention mental health, psychosocial challenges and resilience in older adults living with hiv an examination of the social networks and social isolation in older and younger adults living with hiv/aids society of behavioral medicine calls for equitable healthcare during covid-19 pandemic patients with mental health disorders in the covid-19 epidemic the burden of covid-19 in people living with hiv: a syndemic perspective economic, mental health, hiv prevention and hiv treatment impacts of covid-19 and the covid-19 response on a global sample of cisgender gay men and other men who have sex with men epidemiology of covid-19 among people experiencing homelessness: early evidence from boston covid-19 outbreak among three affiliated homeless service sites covid-19 in prisons and jails in the united states flattening the curve for incarcerated populations -covid-19 in jails and prisons understanding socioeconomic disparities in travel behavior during the covid-19 pandemic from hiv to coronavirus: aids service organizations adaptative responses to covid-19 key: cord-322503-fynprt6f authors: thakur, aarzoo; tan, shawn pei feng; chan, james chun yip title: physiologically‐based pharmacokinetic modeling to predict the clinical efficacy of the coadministration of lopinavir and ritonavir against sars‐cov‐2 date: 2020-08-07 journal: clin pharmacol ther doi: 10.1002/cpt.2014 sha: doc_id: 322503 cord_uid: fynprt6f lopinavir/ritonavir, originally developed for treating the human immunodeficiency virus (hiv), is currently undergoing clinical studies for treating the severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2). although recent reports suggest that lopinavir exhibits in vitro efficacy against sars‐cov‐2, it is a highly protein bound drug and it remains unknown if it reaches adequate in vivo unbound (free) concentrations in lung tissue. we built a physiologically‐based pharmacokinetic (pbpk) model of lopinavir/ritonavir in caucasian and chinese populations. our aim was to perform pharmacokinetic/pharmacodynamic correlations by comparing simulated free plasma and lung concentration values achieved using different dosing regimens of lopinavir/ritonavir with ec(50,unbound) and ec(90,unbound) values of lopinavir against sars‐cov‐2. the model was validated against multiple observed clinical datasets for single and repeated dosing of lopinavir/ritonavir. predicted pharmacokinetic parameters such as the maximum plasma concentration, area under the plasma concentration‐time profile, oral clearance, half‐life and minimum plasma concentration at steady state were within two‐fold of clinical values for both populations. using the current lopinavir/ritonavir regimen of 400/100 mg twice daily, lopinavir does not achieve sufficient free lung concentrations for efficacy against sars‐cov‐2. although the chinese population reaches greater plasma and lung concentrations as compared to caucasians, our simulations suggest that a significant dose increase from the current clinically used dosing regimen is necessary to reach the ec(50,unbound) value for both populations. based on safety data, higher doses would likely lead to qt prolongation and gastrointestinal disorders (nausea, vomiting and diarrhea), thus, any dose adjustment must be carefully weighed alongside these safety concerns. on december 31 st , 2019, china notified the world health organization (who) regarding multiple cases of pneumonia in wuhan, china. today, this pneumonia is known as coronavirus disease 2019 (covid19) and has been found to be caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus leading to over 11.1 million positive cases and 528,000 deaths worldwide as of 5 th july 2020. 1 repurposing existing drugs with proven human safety profiles is expected to provide an expedited route to rapidly identify effective pharmacotherapy against sars-cov-2. one therapy currently of high interest is lopinavir/ritonavir, a protease inhibitor therapy initially approved for the treatment of human immunodeficiency virus (hiv). 2 this combination has attracted attention because of a long history of use, wide availability and evidence of efficacy against other coronaviruses such as mers-cov and sars-cov-1. 3, 4 lopinavir/ritonavir was tested in clinical trials globally through the solidarity trial conducted by who and there are many instances of off-label, compassionate use by physicians to treat covid-19. 5 despite widespread use, there is little evidence available in the literature to support its anti-viral activity against sars-cov-2. in particular, cao et al. reported that a recent randomized control trial on 199 critical patients with covid-19 did not demonstrate significant clinical improvement in those receiving 400/100 mg lopinavir/ritonavir twice daily for 14 days over standard of care. 6 we hypothesized that one possible explanation for the lack of efficacy reported by cao et al. could be insufficient unbound (free) concentrations of lopinavir in lung tissue (c u,lung ) achieved by the dosing regimen used in the study. to address this possibility, we utilized physiologically-based pharmacokinetic (pbpk) modeling to simulate the unbound lung concentration of lopinavir achieved by 400/100 mg twice daily dose of lopinavir/ritonavir in both caucasians and chinese populations. lopinavir (victim drug) exhibits complex drug-drug interactions with ritonavir (perpetrator drug), with cytochrome p450 (cyp450) mediated mechanism-based inactivation (mbi) dominating initially, followed by induction. 7 while a minimal pbpk model was previously reported for lopinavir/ritonavir, 8 it did not recapitulate the pharmacokinetic (pk) profile of the single dose administration of 400/100 mg lopinavir/ritonavir, as the model was optimized for a repeated dosing regimen. furthermore, the use of an empirical additional clearance component for the elimination of ritonavir, which in turn exerts a major influence on the systemic exposure of lopinavir, limited its mechanistic extrapolation to other populations (e.g. ethnic han chinese) with lower cyp450 enzyme expression levels. 9, 10 separately, a population pk model by aspiroz et al. 11 this article is protected by copyright. all rights reserved lopinavir clearance in order to simulate the effects of simultaneous inactivation and induction on steady state levels. this top-down approach would similarly limit mechanistic extrapolation to other populations. using over 40 sets of in vitro data, we built and rigorously validated a middle-out, full pbpk model for lopinavir alone, and lopinavir/ritonavir using the simcyp ® simulator. the model was used to predict c u,lung derived from human lung tissue-to-plasma partition coefficient (k p,lung ) values. as lopinavir is highly protein bound, the free fraction of lopinavir available to interact with pharmacological targets in human tissue and plasma is low. 13 such considerations influence the free fraction of lopinavir in in vitro assays, which in turn affects potency measurements. 14, 15 therefore, we derived unbound ec 50 (ec 50,unbound ) values against sars-cov-2 from various literature reports and compared it against the predicted c u,lung values to determine if clinically used doses of 400/100 mg twice a day would reach efficacious lung concentrations in caucasian and chinese populations. 16, 17 subsequently, we predicted the optimal dosing regimens required to reach the efficacious lung concentrations in both populations. this article is protected by copyright. all rights reserved middle-out pbpk models of lopinavir and ritonavir were built using population-based simcyp simulator version 19 (certara, simcyp division, sheffield, uk). in particular, a perfusion-limited full pbpk distribution model was implemented to permit prediction of tissue concentrations. for lopinavir, tissue k p values were predicted using the corrected poulin and thiel approach (method 1), except for human k p,lung which was calculated from experimental values of unbound lopinavir fraction in lung (f u,lung ) and plasma (f u,p ). 18 the hepatic metabolism of lopinavir by cyp3a4 was accounted for using microsomal kinetic parameters, 2 while the maximal inactivation rate constant (k inact ) for mbi of cyp3a4 by lopinavir was optimized according to wagner et al. 8 for ritonavir, extensive metabolic kinetic parameters from recombinant cyp enzymes were coupled with intersystem extrapolation factors and incorporated into the model to account for its hepatic metabolism. 19, 20 subsequently, lopinavir (substrate) and ritonavir (inhibitor) models were linked. experimental values for mbi and induction of cyp3a4 by ritonavir were used to define the complex time-dependent inhibition and induction of lopinavir metabolism by ritonavir 21 . finally, an active hepatic scalar was empirically fitted to achieve an optimal induction of lpv metabolism. all the drug specific parameters of lopinavir and ritonavir are listed in table s1 . simulations were performed for an oral administration of single dose of lopinavir 400 mg, single dose of lopinavir/ritonavir 400/100 mg and repeated dosing of 400/100 mg twice daily regimen for caucasian and chinese populations using the simcyp healthy volunteers and chinese healthy volunteers population database, respectively. simulated results were visually inspected by overlaying clinical plasma concentration-time profiles (extracted using webplotdigitizer, san francisco, california, usa) with model predictions. 2, [22] [23] [24] [25] [26] [27] [28] thereafter, the pbpk models were validated using a two-fold criterion to compare the clinically observed and model predicted pk parameters such as the maximum plasma concentration (c max ), area under the plasma concentration-time profile (auc 0-t ), time needed to reach c max (t max ), oral clearance (cl/f), halflife (t 1/2 ) and minimum plasma concentration at steady state (c min ). once the pbpk model was validated, it was first used to assess lopinavir's efficacy against hiv by comparing its unbound concentrations in white blood cells (c u,wbc ) with ec 50,unbound value against hiv-infected lymphocytes. this was done to verify that the current pbpk model is able to recapitulate the clinical efficacy of lopinavir against hiv. likewise, the pharmacodynamic (pd) this article is protected by copyright. all rights reserved assessment against sars-cov-2 was performed by predicting if lopinavir reaches therapeutic concentrations in the lung. c u,lung was compared with the ec 50,unbound and ec 90,unbound values of lopinavir against sars-cov-2-infected vero e6/tmprss2 cells, respectively. 16, 17 since lopinavir is a poor substrate of uptake or efflux transporters and its cellular entry occurs passively, 29, 30 it follows the free drug hypothesis. accordingly, at steady state, free concentration of lopinavir is same on both sides of any biomembrane. 31 therefore, c u,wbc and c u,lung were calculated using equation 1: where, c u,tissue represents the unbound tissue concentration; c plasma and c tissue are the total plasma and tissue concentrations, respectively; and f u,tissue is the unbound fraction in tissues. as the simcyp model does not possess a wbc compartment, c u,wbc was assumed to be the same as c u,plasma . c u,lung was calculated based on simulated total lung concentrations (c lung ) and experimental f u,lung measurements. the ec 50,unbound value against hiv was obtained from literature. 15 17 yamamoto et al. used 5% fcs in their culture media. 16 the model is defined by equation 2, where, k a is the association constant, f u,prot represents the fraction of unoccupied protein sites and p t is the total protein concentration. keeping the product of k a and f u,prot constant, equation 2 was analyzed using graphpad prism 8 (graphpad software, la jolla, ca, usa) to determine the f u,media in 2% fcs. the impact of protein binding on pk/pd assessments were then assessed by comparing the predicted total and unbound lung concentrations of 400/100 mg twice daily lopinavir/ritonavir with ec 50 and ec 50,unbound values of lopinavir against sars-cov-2 respectively. in both caucasian and chinese populations, simulations were performed with gradually increasing combinations of loading and maintenance doses in order to reach c u,lung values this article is protected by copyright. all rights reserved comparable with ec 50,unbound and ec 90,unbound values of lopinavir against sars-cov-2. the optimal dose was determined when the steady-state unbound lung c max reached the ec 50,unbound value. this article is protected by copyright. all rights reserved simulated plasma concentration-time profiles for single and repeated twice daily doses of 400/100 mg lopinavir/ritonavir in caucasian and chinese populations are shown in figure 1a -c. clinically observed and model predicted pk parameters were compared and the fold difference values were within the two-fold criterion ( the predicted lopinavir c u,wbc after twice daily dosing of 400/100 mg lopinavir/ritonavir in both caucasian and chinese populations were 100-fold higher than the ec 50,unbound value of lopinavir against hiv (0.00069 ± 0.00006 µg/ml). 15 the concentration-time profiles along with ec 50,unbound values are shown in figure 1d and e. the f u,media for lopinavir in 2% fcs was determined to be 0.355, which yielded an ec 50,unbound value of 0.386 µg/ml and ec 90,unbound value of 0.806 µg/ml against sars-cov-2 from ohashi et al., 17 while the corresponding f u,media in 5% fcs was reported by hickman et al. as 0.200, which yielded an ec 50,unbound value of 0.721 µg/ml from yamamoto et al. 16 using the same dosing regimen, a comparison of the c u,lung with both ec 50,unbound and ec 90,unbound values of lopinavir against sars-cov-2 showed that insufficient unbound lopinavir concentrations were achieved in lung tissue for both caucasian (unbound lung c max = 0.130 µg/ml) and chinese populations (unbound lung c max = 0.200 µg/ml) (figure 2a and b). as protein binding has a major impact on both unbound tissue concentrations and in vitro ec 50,unbound values, we compared the juxtaposition of both total and unbound values at steady state after twice daily dose of 400/100 mg lopinavir/ritonavir in caucasian populations (fig 2c) . the total lung c min ( this article is protected by copyright. all rights reserved values. c u,lung (a function of both plasma and tissue binding) was lower than the ec 50,unbound value of lopinavir (a function of binding to protein within the incubation media). different combinations of loading and maintenance doses of lopinavir/ritonavir were trialed to simulate c u,lung values, beginning with the 400/100 mg twice daily regimen currently tested in clinical trials ( figure 2d ). the predicted unbound lung pk parameters are listed in this article is protected by copyright. all rights reserved lopinavir is a perfusion-limited drug and is neither dependent on transporters for pulmonary entry nor undergoes elimination in the lung. additionally, it is a neutral compound and does not accumulate in the tissues due to ph-driven partitioning. therefore, the unbound plasma concentrations can be regarded to be equivalent to unbound tissue concentrations. based on these observations, we simulated the unbound concentrations of lopinavir in wbcs and lung tissue and juxtaposed these simulations against ec 50,unbound values for inhibition of hiv and this article is protected by copyright. all rights reserved measurements. 35, 36 current evidence indicates that sars-cov-2 utilises the tmprss2 protease for viral spike protein priming, which is necessary for sars-cov-2 viral entry. 37 furthermore, it has been found that the sars-cov-2 rna copies in the vero e6/tmprss2 cell culture supernatants were >100 times greater than those from vero e6 cells. 38 thus, the vero e6/tmprss2 cell system is more robust, sensitive and well-suited for performing in vitro efficacy measurements of pharmaceutical compounds against sars-cov-2, and here we utilized the ec 50 values obtained from such cell systems. understanding the free/unbound drug concentration at the target site is critical as it is the free drug that exerts its pharmacological action. 31 in caucasian populations, comparison of the predicted total and unbound lung concentration of lopinavir (on administration of twice daily 400/100 mg lopinavir/ritonavir) with its ec 50 and ec 50,unbound against sars-cov-2 yield contrasting results. even though the total lung concentration was greater than the ec 50 , when in vivo and in vitro protein binding were used to correct the corresponding lopinavir concentrations, the opposite trend is observed; where c u,lung is now lower than ec 50,unbound , indicating a lack of efficacy. this highlights the importance of accounting for the free fraction of a drug, both in vivo and in vitro, in performing pk/pd correlation and assessing efficacy particularly for highly protein bound drugs. our simulations show that none of the ec 50,unbound values are reached by the c u,lung when 400/100 mg twice daily dosing is administered to either caucasian or chinese populations. this article is protected by copyright. all rights reserved therefore, it is plausible that the failure of a 400/100 mg twice daily regimen for covid-19 reported by cao et al. 6 could be due to insufficient free lung concentrations. during the writing of this manuscript, the who announced their decision to discontinue the lopinavir/ritonavir treatment arm in hospitalised covid-19 patients taking part in the solidarity trials as well. 39 a dose adjustment may therefore be necessary to increase free lung concentrations of lopinavir. our were nausea, vomiting and diarrhoea. 6 moreover, sars-cov-2 can also adversely affect the heart, either directly by infecting cardiac cells and/or indirectly by inducing a cytokine storm. 42 finally, the elevated doses are highly likely to result in gi adverse effects which may be doselimiting. therefore, in the absence of efficacy at clinical doses and the presence of safety concerns at higher doses, the utility of lopinavir/ritonavir to treat covid-19 should be revisited. our study has several limitations. firstly, the f u,lung value used to predict k p,lung was determined using tissue homogenate and may not describe the intracellular drug binding accurately. 18 this article is protected by copyright. all rights reserved study highlights (150 words) recent reports indicate that lopinavir inhibits the in vitro replication of sars-cov-2 but it is uncertain if lopinavir exhibits clinical efficacy. we developed a physiologically-based pharmacokinetic (pbpk) model to predict unbound lung tissue concentrations of lopinavir when co-administered with ritonavir. we aimed to determine (1) if the recommended dose of 400/100 mg of lopinavir/ritonavir twice daily reaches effective unbound lung concentrations necessary for inhibition of sars-cov-2; (2) the optimal dosing regimens required in both caucasian and chinese populations; and (3) whether this optimal dosing regimen is clinically safe. the recommended dose for lopinavir/ritonavir does not reach therapeutic unbound lung concentrations against sars-cov-2. a significantly larger loading and maintenance dose is necessary but may pose safety concerns. our study exemplifies the utility of pbpk modeling to predict tissue concentrations for building pharmacokinetic/pharmacodynamic (pk/pd) correlations of drugs and the need to account for the free fraction of drugs when making pk/pd assessments. 1. table s1 accepted article this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved world health organization coronavirus disease centre for drug evaluation and research fda kaletra clinical pharmacology and biopharmaceutical review role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture solidarity" clinical trial for covid-19 treatments a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 complex drug interactions of hiv protease inhibitors 2: in vivo induction and in vitro to in vivo correlation of induction of cytochrome p450 1a2, 2b6, and 2c9 by ritonavir or nelfinavir physiologically based pharmacokinetic modeling for predicting the effect of intrinsic and extrinsic factors on darunavir or lopinavir exposure coadministered with ritonavir differences in cytochrome p450-mediated pharmacokinetics between chinese and caucasian populations predicted by mechanistic physiologically based pharmacokinetic modelling a review of clinical pharmacokinetic and pharmacodynamic profiles of select antiretrovirals: focus on differences among chinese patients population pharmacokinetics of lopinavir/ritonavir (kaletra) in hivinfected patients dosing will be a key success factor in repurposing antivirals for covid-19 metabolism and disposition of the hiv-1 protease inhibitor lopinavir (abt-378) given in combination with ritonavir in rats, dogs, and humans abt-378, a highly potent inhibitor of the human immunodeficiency virus protease estimation of serum-free 50-percent inhibitory concentrations for human immunodeficiency virus protease inhibitors lopinavir and ritonavir shutoku matsuyama; tyuji hoshino; naoki yamamoto nelfinavir inhibits replication of severe acute respiratory syndrome coronavirus 2 in vitro multidrug treatment with nelfinavir and cepharanthine against covid-19 evaluation of fraction unbound across 7 tissues of 5 species cytochrome p450-mediated metabolism of the hiv-1 protease inhibitor ritonavir (abt-538) in human liver microsomes investigating the contribution of cyp2j2 to ritonavir metabolism in vitro and in vivo time-dependent interaction of ritonavir in chronic use: the power balance between inhibition and induction of p-glycoprotein and cytochrome p450 3a pharmacokinetics of adjusted-dose lopinavir-ritonavir combined with rifampin in healthy volunteers steady-state pharmacokinetics of etravirine and lopinavir/ritonavir melt extrusion formulation, alone and in combination, in healthy hiv-negative volunteers pharmacokinetics of plasma lopinavir/ritonavir following the administration of 400/100 mg, 200/150 mg and 200/50 mg twice daily in hiv-negative volunteers evaluation of pharmacokinetic interactions between long-acting hiv-1 fusion inhibitor albuvirtide and lopinavir/ritonavir, in hiv-infected subjects, combined with clinical study and simulation results echinacea purpurea significantly induces cytochrome p450 3a activity but does not alter lopinavir-ritonavir exposure in healthy subjects once-daily versus twice-daily lopinavir/ritonavir in antiretroviral-naive hiv-positive patients: a 48-week randomized clinical trial pharmacokinetics of two generic co-formulations of lopinavir/ritonavir for hiv-infected children: a pilot study of paediatric lopimune versus the branded product in healthy adult volunteers protease inhibitors atazanavir, lopinavir and ritonavir are potent blockers, but poor substrates, of abc transporters in a broad panel of abc transporteroverexpressing cell lines interaction between hiv protease inhibitors (pis) and hepatic transporters in sandwich cultured human hepatocytes: implication for pi-based ddis the effect of plasma protein binding on in vivo efficacy: misconceptions in drug discovery measurement of antiretroviral drugs in the lungs of hiv-infected patients detection of intrapulmonary concentration of lopinavir in an hiv-infected patient intracellular drug concentrations and transporters: measurement, modeling, and implications for the liver identification of antiviral drug candidates against sars-cov-2 from fdaapproved drugs remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor enhanced isolation of sars-cov-2 by tmprss2-expressing cells world health organization (who) who discontinues hydroxychloroquine and the authors thank drs venkateshan srirangam prativadibhayankara and andrea kwa for helpful discussions on the clinical pharmacology of lopinavir/ritonavir. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-331879-w7008uyy authors: iversen, jenny; sabin, keith; chang, judy; morgan thomas, ruth; prestage, garrett; strathdee, steffanie a; maher, lisa title: covid‐19, hiv and key populations: cross‐cutting issues and the need for population‐specific responses date: 2020-10-01 journal: j int aids soc doi: 10.1002/jia2.25632 sha: doc_id: 331879 cord_uid: w7008uyy introduction: key populations at elevated risk to contract or transmit hiv may also be at higher risk of covid‐19 complications and adverse outcomes associated with public health prevention measures. however, the conditions faced by specific populations vary according to social, structural and environmental factors, including stigma and discrimination, criminalization, social and economic safety nets and the local epidemiology of hiv and covid‐19, which determine risk of exposure and vulnerability to adverse health outcomes, as well as the ability to comply with measures such as physical distancing. this commentary identifies common vulnerabilities and cross‐cutting themes in terms of the impacts of covid‐19 on key populations before addressing issues and concerns specific to particular populations. discussion: cross‐cutting themes include direct impacts such as disrupted access to essential medicines, commodities and services such as anti‐retroviral treatment, hiv pre‐exposure prophylaxis, opioid agonist treatment, viral load monitoring, hiv and sexually transmitted infections testing, condoms and syringes. indirect impacts include significant collateral damage arising from prevention measures which restrict human rights, increase or impose criminal penalties, and expand police powers to target vulnerable and criminalized populations. significant heterogeneity in the covid‐19 pandemic, the underlying hiv epidemic and the ability of key populations to protect themselves means that people who inject drugs and sex workers face particular challenges, including indirect impacts as a result of police targeting, loss of income and sometimes both. geographical variations mean that transgender people and men who have sex with men in regions like africa and the middle east remain criminalized, as well as stigmatized and discriminated against, increasing their vulnerability to adverse outcomes in relation to covid‐19. conclusions: disruptions to both licit and illicit supply chains, loss of income and livelihoods and changes in behaviour as a result of lockdowns and physical distancing have the potential to exacerbate the impacts of the covid‐19 pandemic on key populations. while these impacts will vary significantly, human‐rights approaches to covid‐19 emergency laws and public health prevention measures that are population‐specific and sensitive, will be key to reducing adverse health outcomes and ensuring that no one is left behind. the rapid spread of coronavirus disease (covid-19) has induced governments worldwide to introduce preventive measures, including physical distancing, bans on public gatherings, workplace and school closures and lockdowns designed to reduce contact and suppress transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov2), the virus that causes covid-19. in the absence of a vaccine or effective pharmaceutical treatments, modelling indicates the potential of non-pharmaceutical interventions to reduce covid-19related demand on health systems by two-thirds and to halve deaths [1] . people who inject drugs (pwid), sex workers, men who have sex with men (msm), transgender people, and people in prisons and closed settings, comprise key populations in the response to hiv [2] . in 2020, key populations and their sexual partners accounted for almost two-thirds of new hiv infections globally [3] . older people, men, and those with medical comorbidities including chronic pulmonary disease, cardiovascular disease, cerebrovascular disease, diabetes and compromised immunity, are at higher risk of covid-19 complications [4] . several risk factors for complications, including smoking and vaping drugs, are elevated among key populations [5] . in many settings, key populations also face stigma and discrimination, criminalization, homelessness and food insecurity, which may also exacerbate vulnerability to covid-19 complications. however, conditions faced by key populations vary according to social, structural and environmental factors, including the underlying epidemiology of both hiv and covid-19. as highlighted by sohn et al. [6] , the concentration of covid-19 risk is similar to hiv, where overlapping and intersecting individual, network and structural risks influence both acquisition and transmission and vulnerability to adverse health outcomes [7] , as well as the ability to comply with public health prevention measures. this commentary identifies common vulnerabilities and cross-cutting themes including access to prevention, treatment and care, and the need to respect health and human rights during the covid-19 pandemic, before addressing populationspecific issues and concerns. 2 | discussion 2.1 | common vulnerabilities and cross-cutting themes trust and respect for human rights have long been recognized as central to effective responses to hiv. four decades of the hiv epidemic have taught us that restrictive and stigmatizing measures drive people underground, perpetuate stigma, erode trust and respect for human rights and disproportionately impact vulnerable populations [8] . the potential for both the covid-19 pandemic, and the responses to it, to amplify existing inequalities underlines the need to address criminalization, stigma and discrimination, which are also structural drivers of the hiv epidemic. discriminatory law enforcement and overly restrictive lockdown orders may disproportionately impact key populations and undermine public health strategies and community trust in government [8] with increasing reports of sex workers, pwid, msm and transgender people being fined arrested or detained for breaching covid-19 related restrictions [9] . it is important to remember that the hiv epidemic is not over, with 38 million people living with hiv (plhiv) globally and 1.7 million new infections in 2019 [3]. covid-19-induced supply chain issues may also disproportionately impact key populations reliant on access to medications such as antiretroviral treatment (art), pre-exposure prophylaxis (prep) and opioid agonist treatment (oat), as well as services such as viral load monitoring, hiv and sti testing and condom and needle syringe distribution. in july 2020, the world health organization (who) reported that numerous countries had experienced disruptions to provision of art during the covid pandemic [10] . clinics around the world have reduced hours, reallocated staff or closed, leaving millions of plhiv with uncertain access to treatment [10] . south africa, with 20% of the world's plhiv and 3.5 of an estimated 7.7 million plhiv not virally suppressed [11] , has the highest covid-19 cases in africa and ranks in the top 20 globally for covid-19 deaths per 100,000 population [12] . in addition to disruptions in access to essential medicines, commodities and health services, some key populations are at increased risk of indirect impacts arising from responses to covid-19, particularly physical distancing measures. the negative consequences of these measures on general population health and well-being, such as mental health issues arising from isolation, loss of income and residential instability, will be exacerbated in vulnerable key populations who lack the resources to physically distance or who do not have access to social safety nets or the option of working from home [6] . in low-income countries characterized by a high burden of infectious diseases, including tuberculosis, cholera, typhoid, malaria and hiv/aids, structural and environmental conditions which impede adherence to physical distancing and hygiene measures will differentially impact key populations [13] . unaids has cautioned against the use of covid-19 emergency powers or public health justifications to restrict human rights and expand police powers to target vulnerable and criminalized groups [14, 15] and the united nations office of the high commissioner for human rights (ohchr) has expressed concerns about the use of imprisonment for noncompliance with public health measures [16] . there is a need for independent mechanisms to oversee the use of police powers and to ensure that police are accountable for their actions during the pandemic. to safeguard against the pandemic being used to introduce or expand laws, penalties and police powers that criminalize key populations, unaids recommends that covid-19 emergency laws and powers are necessary, proportionate, non-arbitrary, evidence-informed and lawful, as well as time-limited and renewable only through appropriate democratic mechanisms [17] . across all key populations, meaningful community participation by civil society will be essential to minimize the potential for collateral damage, maintain momentum towards global hiv targets and to ensure that the covid-19 response, or "cure, " is not worse than the disease itself. global networks, including the international network of people who use drugs (inpud), the global network of sex work projects (nswp), the global network of people living with hiv (gnp+) and mpact global action for gay men's health and rights have issued statements calling for urgent action to protect their communities and to address population-specific needs for prevention, care and treatment [9,18-20]. challenges faced by key populations and how networks and communities are responding vary; below we highlight some of these considerations and contexts in order to illustrate the heterogeneity of the covid-19 pandemic and its impacts. compared to the general population, pwid have a high burden of comorbid medical conditions [21] , exacerbated by criminalization and socio-economic disadvantage [22] , which place them at greater risk of infection and complications. restrictions on access to key services for pwid have the potential to increase overdose deaths and hiv and hcv transmission, undermining gains made by global elimination efforts [23] . these services, including needle syringe programs (nsp) and oat, have historically required frequent attendance and restricted access to takeaway oat [24] . covid-19 travel bans and border closures are also impacting illicit markets, with shortages of precursor chemicals, declining availability of opioids and increasing prices in some settings [25] potentially increasing demand for oat and naloxone [24] . countries such as nepal and morocco, with moderate hiv prevalence among pwid at 7 to 9% and limited covid-19 epidemics with less than one death per 100,000 population [12] , have responded by working to ensure supplies of oat for multimonth dispensing (mmd) and providing unsupervised dosing. canada, a country with 11% hiv prevalence among pwid, and a severe covid-19 epidemic with 21.3 deaths per 100,000 population [12] , has introduced biometric vending machines that dispense prescribed supplies of hydromorphone tablets to registered patients [26] . as government services have closed or reduced their hours, drug users and peer-run services, have stepped up to maintain and provide harm reduction services to pwid, as well as distributing oat, art and hcv medications to those in lockdown [27] . modifications have also been made to harm reduction programmes, including the distribution of supplies through outreach, pharmacies, vending machines and post and services previously rejected or under-scaled by policy makers, such as take-home dosing of methadone or hydromorphone. while policy changes to accommodate unsupervised oat demonstrate not only that flexibility in oat delivery is possible and can be done safely and effectively, oat continues to be unavailable in many settings including bahrain, belarus, brazil, cameroon, egypt, nigeria and russia [27]. in many settings sex workers remain vulnerable to hiv and other sexually transmitted infections (sti) due to multiple factors including criminalization, challenges negotiating consistent condom use, unsafe working environments, stigmatization, discrimination and violence [28, 29] . global covid-19 induced bans on sex work, including closures of brothels, mean that sex workers have faced loss of income and are unable to provide for themselves and their families [18] . furthermore, reduced numbers of clients and school closures may disproportionately impact women with responsibility for school-aged children. in most countries, national social protection schemes and emergency protection measures put in place for workers exclude sex workers, particularly where sex work is criminalized. this exclusion means that many sex workers are faced with putting their safety, health and lives at increased risk in order to survive. sex worker organizations have responded rapidly by providing resources, including getting started in online/non-contact work, working safely during the covid-19 pandemic and dealing with stress and emotional impacts, as well as implementing income support programmes. bangladesh, where hiv prevalence among sex workers is low at 0.2% and covid-19 related deaths are also low at less than one per 100 000 population [12] , is one of the few countries that has provided emergency income support for sex workers. in the absence of income support, sex work may be driven further underground with significant health and safety risks [30] . migrant sex workers and those who use drugs may be particularly vulnerable to exploitation by clients, including unsafe work practices and lower prices. sex workers have been targeted by police for physical distancing offences in several countries and covid-19-related policing of public health, including punitive crackdowns, raiding of homes, compulsory testing, arrests and threatened deportations of migrant sex workers [18], has the potential to undermine access to health services, as well as sex workers' ability to report crimes against them. social connectedness has been shown to promote health seeking and risk reduction, including access to hiv treatment and hiv prep among gay and bisexual men (gbm) [31] . lockdowns and physical distancing also threaten to undermine the centrality of peer support to optimizing health outcomes in this population. however, early research suggests that some gbm in high-income countries have adapted their sexual behaviour [32] . in australia where hiv prevalence in this population is 18% and covid-19 transmission has been limited to date, gbm dramatically reduced their sexual contacts following the introduction of physical distancing restrictions [33] . for many in the lesbian, gay, bisexual, transgender and intersex (lgbti) community, the family home may not be a safe place. in uganda where hiv prevalence among gbm is estimated at 85%, a raid on an lgbti community shelter resulted in 19 people arrested and detained without access to bail, legal representation or medication for allegedly violating physical distancing measures [14, 20] . their release was eventually secured after significant efforts by civil society and a court later awarded compensation for rights violations [8] . on any given day, <10 million people, including pre-trial detainees, are incarcerated worldwide, with an estimated 3.8% living with hiv, 15.1% with hcv, 4.8% with chronic hbv and 2.8% with active tuberculosis (tb) [17] , in conditions where physical distancing is impossible. many more are detained in compulsory drug detention, asylum seeker and immigration detention, and private drug treatment centres [9] . in the us, covid-19 outbreaks have been reported in prisons and jails, including in new york, illinois and ohio, with both staff and detainees infected [34, 35] . interim guidance by the united nations ohchr and the who has urged governments to reduce the number of people in detention by finding ways to release those at increased risk of covid-19, including older detainees and people with underlying health conditions, as well as children and those with low risk profiles and people incarcerated for minor offences [16]. estimates of the size of the transgender population vary by location, however population-based surveys report 0.5% to 1.0% of adults identify with a gender different to their sex assigned at birth [36] . similarly, hiv prevalence estimates among transgender populations are highly variable within and across geographic locations, although evidence suggests transgender populations are disproportionately impacted by hiv [37] . in some latin american countries, where hiv prevalence among transgender feminine people is typically >10% [36] , governments implemented gender-based lockdown policies, with designated days residents were permitted to leave the home based on their gender. these policies led to reports of discrimination and violence against transgender people who were away from home on a day that corresponded to their gender identity but did not match the gender listed on their identification documents [8] . in many settings, disruptions to both licit and illicit supply chains, loss of livelihoods, changes in behaviour as a result of lockdowns and physical distancing and discriminatory and coercive policing have the potential to inflict more damage on vulnerable communities than sars-cov2. unaids and who have called for a "people-centred approach" to ensure access to medication is maintained throughout the covid-19 pandemic [17] . expediting differentiated service delivery such as telehealth and expanded mmd for art for plhiv and oat and nsp for pwid [38] , and the roll-out of adaptive programmes like social protection support for sex workers, are essential to ensure that covid-19 is not used to disregard and further disenfranchise key populations. communities are uniting to find solutions and several countries, including african and south american nations, have developed and/or implemented community-based art distribution policies to reduce demands on health systems and to encourage people to stay at home [8] . covid-19 has the potential to reverse decreases in hiv, tb and viral hepatitis mortality, however, its impacts on key populations are likely to be uneven. pwid and sex workers face particular challenges in relation to physical distancing, including indirect impacts as a result of police targeting, loss of income and sometimes both. geographical variations mean that msm and transgender people in regions like africa and the middle east remain criminalized, as well as stigmatized and discriminated against, increasing their risk of adverse outcomes. while successful containment of sars-cov-2 in community settings also protects prisoners and detainees, this group remains vulnerable to the negative health consequences of social isolation. and under covid-19 pandemic conditions, plhiv in resource constrained settings with fragile health systems will be more likely than plhiv in the global north to experience art interruptions which compromise their health. research is also needed to guide public health responses. consistent with the "right to science"' [39] , interventions to prevent, diagnose and treat covid-19 need to be accessible and available to all, especially key populations for whom covid-19 is a pandemic on top of one or more epidemics. this has been reinforced by a recent call for a people's vaccine which guarantees covid-19 vaccines are available free of charge to everyone, everywhere with access prioritized for front-line workers, vulnerable people and low-and middle-income countries [40] . biomedical, socio-economic and behavioural data on the impacts of covid-19 on key populations are needed, including studies designed to assess how specific policy responses increase or decrease exposure to harmful consequences and to monitor the impact of changes to service delivery. existing surveillance mechanisms, with appropriate rights-based legal safeguards, must be adapted to assess the impacts of covid-19 on established epidemics, including hiv and viral hepatitis, in key populations [32] . understanding the impacts of lockdowns and physical distancing measures on key populations will be critical to monitoring trends in hiv and other infections. heterogeneity in the covid-19 pandemic and in the ability of key populations to protect themselves from covid-19 and its consequences necessitates rapid development and implementation of evidence-informed interventions that address the population determinants of transmission and local risks, while remaining sensitive to differences in the needs of key populations and the synergistic impacts of structural factors on particular communities. one of the key lessons from the hiv epidemic was that prevention responses are more effective when communities are empowered with knowledge about the virus and how to mitigate risk and are involved in, or lead, the process of developing inclusive responses. greater solidarity with, more guidance from, and the meaningful involvement of, key populations who are working to fill gaps in prevention, care and treatment, information and advocacy, is required to shape the covid-19 response. while covid-19 has exposed the moral and political barriers to implementing evidencebased public health responses [41] , the willingness to set aside ideological objections to services such as unsupervised oat in order to save lives may be short-lived. there is an urgent need for advocacy to ensure that the introduction or scale up of evidence-based interventions during covid-19 are sustained and integrated into routine service delivery. working together to understand how key populations experience, engage with and emerge from, covid-19 pandemic-induced change, developing responses that address populationspecific needs, and ensuring that no-one is left behind, will be vital to a post-covid-19 transition to a more sustainable, equitable and resilient society. impact of non-pharmaceutical interventions (npis) to reduce covid19 mortality and healthcare demand response team consolidated guidelines on hiv prevention, diagnosis, treatment and care for key populations joint united nations programme on hiv/aids (unaids) who guidance on covid-19 relevant to key populations know your epidemic, know your response: understanding and responding to the heterogeneity of the covid-19 epidemics across southeast asia global commission on hiv and the law. global commission on hiv and the law: risks, rights & health. supplement in the time of covid-19: civil society statement on covid-19 and people who use drugs what we know about hiv and covid-19 sars-cov-2 pandemic expanding in sub-saharan africa: considerations for covid-19 in people living with hiv mortality analysis: how does mortality differ across countries the right to health must guide responses to covid-19 unaids condemns misuse and abuse of emergency powers to target marginalized and vulnerable populations united nations office of the high commissioner for human rights (ohchr) and who. interim guidance covid-19: focus on persons deprived of their liberty hiv and the criminalisation of drug use among people who inject drugs: a systematic review global burden of hiv, viral hepatitis, and tuberculosis among prisoners and detainees association between rapid utilisation of direct hepatitis c antivirals and decline in the prevalence of viremia among people who inject drugs in australia dwindling drug supply on dtes drives prices up, leaves users desperate as covid-19 closes border challenges in maintaining treatment services for people who use drugs during the covid-19 pandemic international network of people who use drugs (inpud). inpud online survey on covid-19 & people who use drugs (pwud): data report the global response and unmet actions for hiv and sex workers a systematic review of the correlates of violence against sex workers conflicting rights: how the prohibition of human trafficking and sexual exploitation infringes the right to health of female sex workers in phnom penh, cambodia. health and human rights mental health, drug use and sexual risk behavior among gay and bisexual men? characterizing the impact of covid-19 on men who have sex with men across the united states in april physical distancing due to covid-19 disrupts sexual behaviors among gay and bisexual men in australia: implications for trends in hiv and other sexually transmissible infections covid-19 in prisons and jails in the united states covid-19 in correctional and detention facilities -united states global epidemiology of hiv infection and related syndemics affecting transgender people strategies for engaging transgender populations in hiv prevention and care the time is now: expedited hiv differentiated service delivery during the covid-19 pandemic science and economic, social and cultural rights art. 15.1.b, 15.2, 15.3 and 15.4 [cited preventing hiv outbreaks among people who inject drugs in the united states key: cord-312513-mad9xkz8 authors: iordanou, stelios; koukios, dimitris; matsentidou, chrystalla‐timiliotou; markoulaki, despina; raftopoulos, vasilios title: severe sars‐cov‐2 pneumonia in a 58‐year‐old patient with hiv: a clinical case report from the republic of cyprus date: 2020-05-25 journal: j med virol doi: 10.1002/jmv.26053 sha: doc_id: 312513 cord_uid: mad9xkz8 hiv and severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) co‐infection is a major challenge for the clinicians as it urged the importance of developing an optimal pharmaceutical scheme and patient's management. the reports that have been recently published regarding the course of sars‐cov‐2 in patients with hiv are sparse. in this brief report we describe, our first single‐centre experience from a 58‐year‐old caucasian male patient with hiv who developed a severe sars‐cov‐2 infection, including clinical characteristics, treatment, and outcomes. this article is protected by copyright. all rights reserved. minute. the patient was awake, alert, and fully oriented. the patient's medical history is notable for hiv infection since 1995, followed in an outpatient hiv clinic. his most recent (august 2019) cd4 cell count was 1,640 per μl, and viral load was undetectable under elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide fumarate. he had no comorbidities. the results of laboratory tests upon admission were unremarkable except for a mildly elevated crp (52mg per litter). specimens were collected in accordance with ecdc guidance 5 and included nasopharyngeal and oropharyngeal swab specimens for influenza a and b and sars-cov-2 (table 1 ). chest radiography was performed, which showed bilateral air space pacifications ( figure 1 ). the patient was started on levofloxacin (750mg once daily) and oseltamivir (standard dose, 75mg twice a day), pending results from pcr analyses. the first (hospital day 1) and a repeat (hospital day 3) upper respiratory specimen tested with reverse transcriptase real-time pcr (rt-pcr) for sars-cov-2 returned a negative result. covid-19 was finally confirmed from a third nasopharyngeal/oropharyngeal sample on hospital day 6. azithromycin (500 mg once daily) and chloroquine (500mg twice a day) was administered (table 1 ). influenza came out negative, and oseltamivir was subsequently stopped. the patient progressively developed severe acute respiratory distress syndrome (ards) (figure 1 ) with a po2/fio2 ratio of 55mmhg on hospital day 7; he was electively intubated and admitted to the icu (table 1) . given the changing clinical presentation and concern about hospital-acquired pneumonia, piperacillin-tazobactam (4.5gr four times a day), and vancomycin (1750mg loading dose followed by 1000mg three times a day) were initiated. nasal pcr testing for methicillin-resistant staphylococcus aureus was negative, as were all other obtained cultures. serial procalcitonin was tested negative. due to persistent fever, the antimicrobial treatment accepted article sedation and mechanical ventilation, the patient regained consciousness relatively quickly and remained oriented and cooperative during the entire stay. he was weaned off the ventilator on hospital day 29, and decannulation was performed on hospital day 31. the patient was discharged from the icu the following day and transferred to a clinic for rehabilitation. so far, he makes a quick and uneventful recovery. standard antimicrobial treatment was used in combination with chloroquine and azithromycin, based on studies that showed promising results. 10 the patient remained on his previous art (on tenofovir-containing regimen), 3, 4 given his excellent virologic and immunologic condition. remdesivir, lopinavir/ritonavir, tocilizumab, and corticosteroids were not used due to inconclusive data about their efficacy and safety. 11 covid-19 characteristics such as serial false-negative upper respiratory specimens, deterioration between days 7 and 10, near-normal lung compliance, hyperventilation, prolonged need for mechanical ventilation, and increased risk for thrombotic complications, were observed in our patient, in line with findings from other case studies. the absence of comorbidities, the virologic and immunologic condition, as well as the use of standard antimicrobial treatment in combination with chloroquine and azithromycin and the maintenance of previous art instead of adaptation art seem to have an impact on patient's recovery despite his age. the early initiation of antimicrobial treatment may be translated into a lower risk for opportunistic infections. situation update worldwide co-infection of sars-cov-2 and hiv in a patient in wuhan city covid-19 in patients with hiv: clinical case series key: cord-340703-vtuy806l authors: cascio, antonio; colomba, claudia; di carlo, paola; serra, nicola; lo re, giuseppe; gambino, angelo; lo casto, antonio; guglielmi, giuseppe; veronese, nicola; lagalla, roberto; sergi, consolato title: low bone mineral density in hiv-positive young italians and migrants date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0237984 sha: doc_id: 340703 cord_uid: vtuy806l background: human immunodeficiency virus (hiv) infected individuals may have osteoporosis. we aimed to evaluate the bone mineral density (bmd) in naïve antiretroviral (arv) treated hiv positive patients comparing native italian group (itg) to a migrants group (mig) upon arrival in italy. methods: we conducted a cross-sectional study on 83 hiv patients less than 50 years old. we used the dual-energy x-ray absorptiometry (dxa) within six months from the hiv diagnosis. participants were categorized as having low bmd if the femoral neck or total lumbar spine z-score was– 2 or less. results: mig showed low bmd more often than itg (37.5% vs.13.6%), especially for the female gender (16.7% vs. 0.0%). a low cd4 rate (<200 cells/μl) was most often detected in mig than itg. in particular, we found most often male italians with abnormal cd4 than male migrants (67.8% vs. 33.3%) and vice versa for females (30.5% vs. 66.7%). we found an abnormal bone mineral density at the lumbar site. low bmd at the lumbar site was more frequently observed in female migrants than female italians. both male and female migrants had a z-score value significantly lower than male and female italians, respectively. by logistic regression low vitamin-d level was positively correlated to low bmd in itg only. all data were verified and validated using a triple code identifier. conclusions: both dxa and vitamin-d evaluation should be offered after the diagnosis of hiv infection. lumbar site low bmd is an initial condition of bone loss in hiv young patients, especially in female migrants. vitamin d levels and supplementation may be considered after hiv diagnosis independently of age to improve bone health. highlights: this study evaluates the frequency of bone mineral density in hiv positive patients naive to antiretroviral therapy. it compares the density of the native italian population with that of hiv migrants upon arrival in italy. the results show that hiv positive migrants, even if younger than 50 years of age, are at risk for osteoporosis, especially if they are female. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 osteoporosis is a process characterized by increased bone resorption without reciprocal bone apposition. the decreased bone mass leads to an increased risk of bone fractures, which are particularly as the subject ages [1, 2] . currently, lifestyle factors, including alcohol, diet, hormones, physical activity, and smoking, are influencing the bone mass, and osteoporosis is affecting younger people than before [2] [3] [4] . the incidence of osteoporosis and osteoporosisrelated fractures varies across the european union/european economic area (eu/eea), and especially in the mediterranean area, it is increasing, as it has been evidenced in the literature with a clear link to nutrition [1, 2] . among human immunodeficiency virus (hiv)-related comorbidities, the bone disease has emerged as a significant question in this chronically infected population [3] [4] [5] [6] [7] [8] . several studies suggest that fracture rates are higher in communities with hiv than among matched uninfected controls despite anti-retroviral therapy (art) and gender [9] [10] [11] . the causes of a low bone mineral density (bmd) could be different in hiv-infected patients by considering the time of hiv infection [12] . previous studies conducted in our geographical area established that the prevalence of osteoporosis was significantly higher in hiv-infected than in uninfected subjects, which mirrors a result similar to previous meta-analyses [12] [13] [14] [15] . the prevalence of osteopenia and osteoporosis in hiv mono-infected patients in our geographical area was about 44.9%, and 20.9% in comparison with 18% reported in a healthy italian population [16] . a recent analysis of bone-healthy in the immigrant population conducted in sweden showed that women had low bmd for age according to the american and african-american referents [17] [18] [19] . moreover, a study conducted in german-turkish immigrants osteopenia was diagnosed in 32% and osteoporosis in 8% of young migrants [20] . in the migrant setting, it has been hypothesized that bmd is intriguingly associated with lifestyle, body mass index (bmi), and vitamin d levels. recently, low level of vitamin d were also reported in somali migrant women in sweden and refugees in canada using the calgary refugee health program, an urban family practice that serves newly arrived refugees in calgary, alberta [21] [22] [23] . although the number of migrants and refugees crossing the mediterranean sea has decreased in 2018-19 and 2020, during the covid-19 pandemic, this number has been unprecedented in the last ten years compared with the past twenty or thirty years [5] [6] [7] . topical italian data showed that the new diagnosis of hiv infection in 72% of migrants was late and less than six months before developing aids with half of the subjects coming from sub-saharan africa (ssa, 59.4%) and with an increasing percentage of fertile females [22] [23] [24] [25] [26] . further, the incidence of bmd loss and related fractures attributed to a specific eating pattern has also been targeted for guidelines and suggestions within the eu/eea [8] . our study aims to emphasize the burden of bone health in naïve arv hiv positive patients and compare the bone density of the native italian population group (itg) with that of hiv migrants (mig) upon arrival in italy. this investigation is a retrospective cross-sectional study. we gathered data from 83 arv naïve subjects consecutively admitted between january 2010 and may 2015 at the sicilian aids center of the university of palermo, italy. we retrospectively analyzed all patients who underwent dual-energy x-ray absorptiometry (dxa) within six months from hiv diagnosis independent of their bmi. in this study, we excluded all patients with the treatment of steroidinduced bone loss and subjects with active tuberculosis. dxa measures bmd utilizing two x-ray beams with different energy levels, which are aimed at the patient's bones (see below). of the 83 subjects, 59 were italian, and 24 were migrant patients living in italy for less than twelve months. table 1 shows the characteristics of enrolled patients. we collected the medical records and entered into an anonymous database, as previously reported [8, 9] . cd4+ t-cell count and plasma hiv-rna levels were assessed as previously reported [9] . 25-hydroxy-vitamin d (25(oh)d) was assayed as previously reported [21, 22, 25] . we considered a serum 25(oh)d concentration of 30 ng/ml as a threshold value for identifying low levels of vitamin d, as previously reported (standard value of our laboratory: 30-80 ng/ml) [13] . all subjects gave their informed consent for inclusion before they started participating in the study, which was conducted by the declaration of helsinki. the ethics committee of the university of palermo approved the study protocol. each participant was free to decline or withdraw from the study at any time, and this was explained in their original language or a language that the migrant knew. bmd was assessed by dxa, using a qdr discovery hologic dxa in the femoral neck and dxa in the lumbar spine by total body dxa. for each scan, bmd, z-scores were recorded as previously reported [9] [10] [11] . dxa measurements were performed in the femur (femoral neck and/or total hip) and lumbar spine in each patient. since the age of our patients ranged from 30 to 50 years, the use of z-scores (defined as an individuals' bmd in comparison to agematched normal individuals) was used for all the analyses, according to world health organisation (who) recommendation [27] . participants were categorized as having low bmd if the femoral neck or total lumbar spine z-score was-2 or less. to individualize sample sizes statistically significant in this study, we considered a bernoulli sampling for both italian and migrant groups [28] . for itg, the minimum sample size for this study was estimated equal to 41 patients affected by low bmd. it was obtained considering a statistical z-score at 95%, an error ε = 15% and hypothesizing a prevalence π, about 60% according to the studies of cavalli et al. [16] and tomažič et al. [29] . in this case, we estimated a prevalence range equal to 45%-75%. for mig, the minimum sample size for this study was estimated equal to 22 patients affected by low bmd. it was obtained considering a z-score at 95%, an error ε = 20%, and hypothesizing a prevalence π about 35%, according to varenna et al. [30] . in this way we estimated a prevalence range equal to 15%-55%. in this case, we considered an error ε greater to an error in itg because of the amount of information that could be gathered in migrants in italy. also, the sample size for itg and mig were increased to 59 and 24 patients, respectively considering the possibility of unexpected events and, consequently, the likelihood of patients' data loss. the statistical analysis was performed by matlab statistical toolbox version 2008 (math-works, natick, ma, usa). data are presented as number and percentage for categorical variables. numerical data are expressed as the mean ± standard deviation (sd) or median and confidence interval at 95% (ci). the χ 2 test and fisher's exact tests were performed to evaluate significant differences of proportions or percentages between two groups. the fisher's exact test was used where the χ 2 test was not appropriate. the mann-whitney test was used to test the difference between two independent samples (itg and mig group). it was the alternative for a t-test dealing with independent samples when the distribution of the samples is not normal. normal distributed independent samples were studied using the d'agostino-pearson test. linear correlation analysis was also performed, and the test on pearson's linear correlation coefficient r was performed with the t-student test under the null hypothesis of pearson's linear correlation coefficient r = 0. the logistic regression was performed to analyze the relationship between low bmd (dichotomous variable) and the independent variables: gender (dichotomous-m = 1, f = 0), previous fractures (dichotomous-yes = 1, no = 0), bmi (continuous), cd4 cells (continuous), and 25-hydroxy-vitamin d (continuous). finally, all tests with p-value < 0.05 were considered significant. the 83 patients, composed by 59.4% males and 40.6% females, with ages into range 30-50, mean 44.2 years old and standard deviation (s.d.) equal to 4.9 years old, was subdivided into two groups (table 1) in table 2 , we report the differences between itg and mig group and statistical tests. low bmd was significantly more frequent in mig group in comparison to itg group (37.5% > 13.6%, p = 0.0324), particularly it was significantly more frequently found in migrant females than in italian females (16.7% > 0.0%, p = 0.0058). significant differences were seen in low-bmd values. in fact, both female and male italians had a measure of bmd significantly greater than migrant females and males respectively (median: -1.0 > -2.3, p = 0.0272; 0.05 > -0.8, p = 0.0291). in addition, we found major presence of migrant females with abnormal lumbar value in comparison to italian females (16.7% > 0.0%, p = 0.0058). conversely, no significant differences between itg and mig were found considering the femoral bmd values. in examining the gender in mono-infected hiv patients, the percentages of hiv patients were significantly greater in italian males in comparison to migrant males (57.6% > 25%, p = 0.007). on the other hand, hiv was most frequently observed in female migrants in comparison to female italians (58.3% > 23.7%, p < 0.003). for co-infected patients, there were no significant differences in gender between itg and mig. also, for aids patients there was a significant difference between itg and mig (45.8% < 83.3%, p = 0.0017). in particular, aids was most frequent in female migrants in comparison to female italians (8.3%>8.5%, p<0.0001). we found a higher percentage of male italians with abnormal values of cd4 in comparison to male migrants (67.8% > 33.3%, p = 0.0039), and vice versa for the female gender ( in migrant patients, 25-oh-vitamin d mean values were significantly lower than in italian patients (14.8 > 11.8 ng/ml, p = 0.0466). also, male italians had mean value significantly figs 1 and 2 , the heat maps of lbd and fbd values for itg and mig groups are shown, respectively. every line represents the fbd and lbd values for each patient, and the graduation of the colors are representative of the z-score. in fig 3, we showed the scatter plots for itg and mig group between cd4 values with lbd and fbd (a-d). for every scatters plot, the red points on graphs are individuated by cd4 values and correspondent lumbar bmd and femoral bmd z-scores. the blue line is the best linear fit, and the red lines define the 95% prediction interval for the regression curve. for any given value of the independent variable (cd4), this interval represents the 95% probability for the values of the dependent variable (lbd or fbd). by linear correlation analysis, it results that for the itg group, there was a significant positive linear correlation between cd4 and femoral bmd (r = 0.34, p = 0.009) and between cd4 and lumbar bmd (r = 0.30, p = 0.020). in contrast, the mig group showed no significant correlation between cd4 and femoral bmd (r = 0.0, p = 1.0) and between cd4 and lumbar bmd (r = 0.23, p = 0.27). in other words, in italian patients, an increase/decrease of cd4 values implicate an increase/decrease of lumbar bmd or femoral bmd scores. we did not observe these correlations in the mig group. finally, in table 3 , we report the logistic regression analysis between low bmd variable (dichotomous) and the independent variables: gender (dichotomous), bmi (continuous), hydroxy-vitamin d (continuous), cd4 (continuous), and previous fractures (dichotomous) for the total sample, itg, and mig. for this scope, two models were considered. the null model: -2ln(l 0 ), where l 0 was the likelihood of obtaining the observations if the independent variables did not affect the outcome, and the full model: -2ln(l 0 ), where l 0 was the likelihood of obtaining the observations with all independent variables incorporated in the model. the difference between these two yields was estimated with the chi-square test to define how well the independent variables affect the outcome or dependent variable. if the chi-square test was positive (p < 0.05), then there was evidence that at least one of the independent variables contributes to the prediction of the outcome. by logistic regression, it resulted that only hydroxy-vitamin d was negatively correlated to low bmd both in total sample (odds ratio, or = 0.85 and p = 0.0063), and in itg (or = 0.84 and p = 0.0309). in other words, an increase (decrease) of hydroxy-vitamin d contribute to decreasing (increasing) of low bmd. in other words, a decrease of hydroxy-vitamin d contributes to increasing the probability of osteoporosis or osteopenia in the total sample, and particularly, in the italian patients. in the migrant group, we did not observe significant correlations by regression analysis, and the small sample size of migrants considered in our study may be the reason (see below). our study shows that "low bmd" was significantly more frequent in the mig group in comparison to the itg group (migrants 37.5% vs. 13.6%). in particular, it was significantly more frequently found in migrant females than in italian females. our previous reports [13, 14] on the prevalence of low-bmd in hiv mono-infected patients who underwent arv therapy showed higher percentage rates of osteopenia (44.9%) and osteoporosis (20.9%) than an agerelated healthy italian population (18%) [16] . our current study showed that the percentage of abnormal bmd was mostly restricted to the lumbar site. the low bmd of the lumbar site in female migrants was more evident than in female italians, and both males and female migrants had a z-score value significantly lower than males and females italians. studies conducted on hiv negative patients have, indeed, shown how osteophyte formation, bone sclerosis, disk space narrowing, and spondylolisthesis are positively correlated with lumbar spine bmd. at the same time, there was no association with femoral neck bmd [31] . in general, anatomical studies suggest that sex-differences in the lumbar spine appear to be supported by postural differences in sacral bone-orientation and morphological differences in the vertebral bodies of females [32] . moreover, emerging data show that pregnant and breastfeeding women are associated with early osteoporosis, especially in the thoracolumbar spine [12] . the involvement of the lumbar site is probably justified by the fact that our sample is younger than in other studies conducted on older populations of hiv negative subjects [13-15, 33, 34] . hcv is a risk factor for osteoporosis and fractures in the hiv population. in our studied population, hcv co-infection was found in 18.1% of the enrolled sample. recently, one of the authors identified a more lumbar than femoral "low bmd" in hiv/hcv coinfected patients [16] . the comparison of aids diagnosis between italian and migrants showed that aids was more frequent in migrants, especially in females. in general, we found more frequently low cd4 values (<200 cells/μl) in both male and female migrants in comparison with italian patients. these data are in agreement with the emerging late diagnosis of hiv infection in young italians comparing with age-matched female migrants [24, 25] . by linear correlation analysis, an increase/decrease of cd4 values implicates an increase/decrease of lumbar or femoral bmd scores only in italian patients. in contrast, we did not observe similar correlations in the mig group. this aspect is probably due to the element that all migrants had lower cd4 values than age-matched italians. indeed, no migrant had values of cd4 � 350. although the mean of bmi in migrants was lower than italian patients, the statistical analysis of this variable evidenced that low bmd was significant only in female migrants. on the other hand, the comparison of the percentage of patients with abnormal bmi was significant in male italians. the logistic regression showed a relationship between low hydroxy-vitamin d and low bmd only in the italian group. this data should be validated on a larger sample size. low consumption of dairy products, high consumption of soft drinks, moderate sun exposure, and a high prevalence of vitamin d deficiency have been identified among youth as risk factors for early osteoporosis. the nutrition factor is probably intrinsically associated with the geographical region and climate extremes (droughts and storms) may aggravate the bone mass loss of hiv-infected youth. as reported in other studies, we observed early natural menopause status both in italian than in migrant women. this finding was analogous to both groups. apart from hiv-related immunologic status (cd4 count and viral load) other socio-demographic variables (marital status, parity, education, or income) and religious clothing of the female migrant sample may have influenced the low bmd observed in our study. to the best of our knowledge, this study is the first dxa-based investigation to study and assess the bmd among the flow of migrants and refugees from countries with a high incidence of hiv infection across the mediterranean area. in this respect, the fact that our study includes only migrants, who were resident in italy for no more than one year, could offer the opportunity to carefully assess the risk factors, which stress the bone composition in the hiv infected population. a recent paper on osteoporosis among hiv positive patients highlighted how an assessment of baseline bone mass loss within the first two years from the start of arv is often lacking in longitudinal studies and claimed that there is some evidence concerning bone loss within the first year of hiv infection and art initiation [13, 17] . the findings of a high prevalence of low bmd in hiv infected migrants aged less than 50 years of age could suggest that the first screening for osteoporosis should be carried out as soon as possible and before the start of art. this aspect should help the clinician in arv management. planning hiv therapy to prevent bone loss is crucial. we suggest the use of tenofovir alafenamide (taf) molecule instead of tenofovir to reduce the risk of bone toxicity [35, 36] . a recent study enrolled young hiv positive with low bmd showed tenofovir as a key molecule in arv therapy [11] . taf is a hepatitis b virus (hbv) nucleotide reverse transcriptase inhibitor for the treatment of chronic hbv infection in adults with compensated liver disease and has been approved by the food and drug administration (fda) for the treatment of hiv-1 in november 2015. the altered bone remodeling in young hiv positive women may suggest some alertness about our current preventive health care services. most of the immigrants diagnosed with hiv are pregnant women who have undergone tests for sexually transmitted diseases. although this study is original and discloses essential steps to improve current management strategies because of new migrations from underdeveloped countries, there are some limitations. our research involves a small size of the groups, but the cross-sectional study design still maintains its validity. this study does not permit us to establish a cause-effect relationship from our results or to evaluate the impact of osteoporosis risk factors and/or lifestyle risk factors on the management of bone disease in the hiv population. thus, a multicentric and prospective study is warranted. in conclusion, our study raises the question of the need to plan preventive strategies in hiv migrants and refugees, also in the light of the great movement of people from southern mediterranean areas to northern european countries with low sun exposure [21, 22] . the female migrants must be protected from precocious low bmd with an additional layer of safety [17-19, 21, 22, 33] . the approach requires different and specific intervention strategies in the future. our data confirm that early screening for low bmd and other risk factors associated with bone loss in hiv patients is useful [2, 7, 15, [18] [19] [20] . in agreement with the osteoporosis foundation (http://share.iofbonehealth.org/wod/ compendium/2019-iof-compendium-of-osteoporosis-press.pdf) the authors stress the strategies for preventing osteoporosis. the role of nutrition in maintaining bone health associated with a personalized anti-hiv treatment and daily vitamin supplementation to reduce early bone loss as well as a baseline dxa are critical steps for hiv migrants and refugees after arrival in the host countries [13, 17, 20, 23, 36] . high-risk groups should be first identified, and osteoporosis investigations should be carried out earlier than at 50 years of age [37] [38] [39] . as indicated above, our results should be evaluated with caution due to the retrospective nature of the study. also, despite the estimated minimum sample sizes of itg and mig were correctly applied and the conditions for the sample size according to bernoulli are met, prospective and more extensive studies are needed to confirm our results. assessment of fracture risk and its application to screening for postmenopausal osteoporosis: report of a who study group world health organization dietary patterns, bone mineral density, and risk of fractures: a systematic review and meta-analysis pathological case of the month: classic rickets in a setting of significant psychosocial deprivation english disease": historical notes on rickets, the bone-lung link and child neglect issues strategy and action plan for refugee and migrant health in the who european region. copenhagen, denmark: world health organization nutrition, osteoarthritis and cartilage metabolism endocrinological aspects of hiv infection mapping a method for systematically reviewing the medical literature: a helpful checklist postmortem protocol of human immunodeficiency virus (hiv)-related pathology in childhood decreased apoptosis despite severe cd4 depletion in the thymus of a human immunodeficiency virus-1 infected child estimating minimum adult hiv prevalence: a cross-sectional study to assess the characteristics of people living with hiv in italy reduced bone mineral density among hiv infected patients on anti-retroviral therapy in blantyre, malawi: prevalence and associated factors reduced bone mineral density in human immunodeficiency virus-infected individuals: a meta-analysis of its prevalence and risk factors: supplementary presentation dairy calcium intake and lifestyle risk factors for bone loss in hiv-infected and uninfected mediterranean subjects associated factors and liver disease severity for decreased bone mineral density in hiv mono-and hiv/hcv co-infected patients vitamin d levels and il28b polymorphisms are related to rapid virological response to standard of care in genotype 1 chronic hepatitis c prevalence of osteoporosis in the italian population and main risk factors: results of bonetour campaign comorbidity and health-related quality of life in somali women living in sweden. scand j prim health care factors associated with bone mineral density in healthy african women site-specific differences in bone mineral density in black and white premenopausal south african women adult lactose intolerance, calcium intake, bone metabolism and bone density in german-turkish immigrants vitamin d status of refugees arriving in canada: findings from the calgary refugee health program high hiv-1 diversity in immigrants resident in italy impact of an 8-week exercise and sport intervention on post-traumatic stress disorder symptoms, mental health, and physical fitness among male refugees living in a greek refugee camp behavioral and clinical characteristics of people receiving medical care for hiv infection in an outpatient facility in sicily, italy. patient prefer adherence scientific group on the assessment of osteoporosis at primary health care level estimation of a population total under a "bernoulli sampling" procedure prevalence and risk factors for osteopenia/osteoporosis in an hiv-infected male population prevalence of osteoporosis and fractures in a migrant population from southern to northern italy: a cross-sectional, comparative study insulin/igf-1r, sirt1, and foxos pathways-an intriguing interaction platform for bone and osteosarcoma morphological and postural sexual dimorphism of the lumbar spine facilitates greater lordosis in females calcium and vitamin d in human health: hype or real? phalangeal quantitative ultrasound: cheaper methods for screening and follow-up of bone pathologies in hiv-infected women? optimizing antiretroviral therapy for women living with hiv planning hiv therapy to prevent future comorbidities: patient years for tenofovir alafenamide osteoporosis risk factors in hiv positive women with osteoporosis: a retrospective analysis vitamin d status and the relationship with bone fragility fractures in hiv-infected patients: a case control study subsequent fracture risk of women with pregnancy and lactation-associated osteoporosis after a median of 6 years of follow-up the authors ac, pdc, and ns, contributed equally to this paper. we are deeply grateful to the patients and their families for participating in the study. preliminary results of this study were presented at the hiv glasgow conference, oct. 23-26, 2016, glasgow, united kingdom. key: cord-338594-wft7yy6j authors: winkler, michael; gärtner, sabine; wrensch, florian; krawczak, michael; sauermann, ulrike; pöhlmann, stefan title: rhesus macaque ifitm3 gene polymorphisms and siv infection date: 2017-03-03 journal: plos one doi: 10.1371/journal.pone.0172847 sha: doc_id: 338594 cord_uid: wft7yy6j interferon-induced transmembrane proteins (ifitms) have been recognized as important antiviral effectors of the innate immune system, both in cell culture and in infected humans. in particular, polymorphisms of the human ifitm3 gene have been shown to affect disease severity and progression in influenza a virus (fluav) and human immunodeficiency virus (hiv) infection, respectively. rhesus macaques (macaca mulatta) are commonly used to model human infections and the experimental inoculation of these animals with simian immunodeficiency virus (siv) is one of the best models for hiv/aids in humans. however, information on the role of ifitm3 in siv infection of rhesus macaques is currently lacking. we show that rhesus macaque (rh) ifitm3 inhibits siv and fluav entry in cell culture, although with moderately reduced efficiency as compared to its human counterpart. we further report the identification of 16 polymorphisms in the rhifitm3 gene, three of which were exonic and synonymous while the remainder was located in non-coding regions. employing previously characterized samples from two cohorts of siv-infected rhesus macaques, we investigated the relationship between these rhifitm3 polymorphisms and both aids-free survival time and virus load. in cohort 1, several intronic polymorphisms were significantly associated with virus load or survival. however, an association with both parameters was not observed and significance was lost in most cases when animals were stratified for the presence of mhc allele mamu-a1*001. moreover, no significant genotype-phenotype associations were detected in cohort 2. these results suggest that, although ifitm3 can inhibit siv infection in cell culture, genetic variation in rhifitm3 might have only a minor impact on the course of siv infection in experimentally infected animals. interferon-induced transmembrane proteins are encoded by a gene family of diverse function in vertebrates [1] [2] [3] . a subgroup of immunity-related ifitms (ifitm1-3) has been recognized recently to inhibit multiplication of a broad range of viruses including, but not limited to, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 influenza a viruses (fluav), flaviviruses, coronaviruses, filoviruses and lentiviruses [4] [5] [6] [7] [8] [9] [10] [11] . ifitm proteins are type ii transmembrane proteins and consist of a variable cytoplasmic nterminus followed by a conserved intramembrane domain, an intracellular loop, a transmembrane domain and a short variable and luminal c-terminus [12] [13] [14] . it is well established that ifitms inhibit viral entry [11] . while the exact mechanism still has to be elucidated, ifitms seem to act through the arrest of virus-host cell membrane fusion at the hemifusion step by altering membrane fluidity [15, 16] . despite their broad antiviral activity, ifitms can also promote entry or assembly of selected viruses [17, 18] , indicating that they belong to a pathway that can positively and negatively regulate viral infection. all immunity-related ifitms (ifitm1-3) have been shown to affect early stages of human and simian immunodeficiency virus (hiv, siv) replication, with ifitm2 and ifitm3 inhibiting virus entry [8, 19, 20] . in addition, ifitms may target later stages of the viral replication cycle such as gag expression and proteolytic processing of env [8, 21, 22] . the antiviral activity of ifitm1 can restrict hiv-1 cell-to-cell spread, and mutations in env and vpu have been identified that rescue the virus from inhibition by ifitm1 at the cost of reduced viral fitness [21, 23] . moreover, ifitms are incorporated into lentiviral particles thereby reducing viral infectivity even though inhibition by ifitms and virion incorporation of ifitms were not found to be intimately related [21, 24, 25] . finally, unlike other antiviral effector proteins of innate immunity, ifitms do not seem to be antagonized by viral factors [22] . different lines of evidence suggest that ifitms can limit viral spread and reduce disease burden in the infected host. thus, absence of ifitm3 expression was found to be associated with reduced fluav spread and pathogenesis in experimentally inoculated mice [26, 27] . moreover, a polymorphism in the human ifitm3 gene (rs12252 t>c) was found in several studies to be associated with severe influenza [16, [27] [28] [29] , although one study reported an association with mild influenza only [30] . the rs12252 t>c polymorphism is believed to alter ifitm3 mrna splicing [27] and to result in the expression of an ifitm3 variant lacking the first 21 amino acids, which displays reduced antiviral activity against fluav upon directed expression in cell culture [31] . conversely, the truncated protein exerts increased anti-hiv-1 activity in transfected cells, due to preferential localization at the cell surface [32] , the site of hiv-1 entry and exit from target cells. however, an association between rs12252-c and more rapid progression to aids, but not risk of hiv-1 infection, has been demonstrated [33] , and the reason for the discrepancy between these results and the increased anti-hiv-1 activity of truncated ifitm3 in cell culture is at present unclear. finally, a recent study demonstrated that hiv-1 variants sexually transmitted between individuals are resistant against blockade by ifitm proteins indicative of a strong selection pressure exerted by ifitms [34, 35] . moreover, ifitm expression was shown to be a major contributor to the blockade of hiv-1 by treatment of cells with ifn [34] , suggesting that ifitms constitute important defenses against hiv-1 infection. non-human primates are an important animal model for infectious diseases in humans. in particular, experimental siv infection of rhesus macaques has contributed substantially to our current understanding of hiv/aids. polymorphisms in several immune-relevant gene loci such as mhc or kir are associated with the transmission and course of disease in siv infected rhesus macaques and hiv-1 infected humans [36, 37] . some primate ifitm gene products were recently shown to have antiviral activity against siv and hiv-1 [19, 20] . however, these analyses did not include rhesus macaque so that it is presently unknown whether rhesus macaque (rh) ifitm3 also inhibits siv infection in this species. moreover, no rhifitm3 gene polymorphisms have been described so far or their modulatory effect upon infection by siv or other viruses studied. therefore, we systematically searched for polymorphisms in rhifitm3 and assessed their association with viral load at set point and disease progression in the context of siv infection. human embryonal kidney (hek) 293t cells (atcc crl-3216) were cultivated in dulbeccos modified eagles medium (dmem), supplemented with 10% fetal calf serum (fcs), l-glutamine and penicillin/streptomycin in a humidified atmosphere containing 5% co 2 . the cells were obtained from collaborators and their identity was verified by short tandem repeat (str) analysis. plasmids encoding machupo virus glycoprotein (macv-gpc), murine leukemia virus envelope protein (mlv-env), fluav hemagglutinin (ha) and neuraminidase (na), vesicular stomatitis virus glycoprotein (vsv-g) and siv envelope protein (siv-env) were previously described [6, 7] . the mlv-based vector pqcxip (clontech, heidelberg, germany) encoding human and rhesus macaque ifitm3 proteins, chloramphenicol-acteyltransferase (cat) or firefly luciferase as well as the mlv gag-pol encoding plasmid were also reported before [6, 7] . for the production of mlv particles encoding ifitm proteins, we followed a published protocol [6, 7] . in brief, 293t cells were cotransfected with vector pqcxip harboring rhifitm3, huifitm3 or cat, an mlv gag-pol-encoding plasmid and a plasmid encoding vesicular stomatitis virus glycoprotein (vsv-g). for the production of reporter particles required for the analysis of ifitm3-mediated inhibition of viral entry, 293t cells were cotransfected with vector pqcxip encoding firefly luciferase, an mlv gag-pol encoding plasmid and a plasmid encoding the viral glycoprotein to be tested. medium was changed at 8 h and supernatants were harvested at 48 h post transfection, passed through a 0.45 μm filter, aliquoted and stored at -80˚c. the antiviral activity of rhifitm3 was analyzed as described previously [6] . in brief, 293t cells seeded at 10 4 cells per well in a 96-well plate were spin-oculated [38] at 4,000×g for 30 minutes with ifitm3 or cat encoding vectors. after incubation for 48h at 37˚c, the medium was replaced by 50μl of fresh culture medium followed by the addition of 50μl of mlv vectors harboring the viral glycoproteins to be analyzed and normalized for equal luciferase activity upon transduction of control cells. after incubation for 8h, medium was replaced by fresh culture medium and luciferase activities in cell lysates were analyzed at 72h post infection, employing a microplate reader, plate chameleon v (hidex, turku, finland), and a commercial luciferase assay system (promega, mannheim, germany) or beetle-juice (pjk, kleinblittersdorf, germany) kits. genetic and phenotypic data from two cohorts of siv-infected rhesus macaques (macaca mulatta) of indian origin, comprising a total of 94 individuals (19 female, 75 male), were screened retrospectively for rhifitm3 polymorphisms ( table 1) . samples of mhc-typed animals were selected that were infected intravenously, intrarectally or via tonsils through a single dose of sivmac239, sivmac251 or sivmac239/32h and which displayed the whole spectrum of disease progression, from rapid progression to long-term non progression, for each combination of virus and infection route. all macaques were either bred at the german primate centre and had known ancestry, or were from breeders in france, united kingdom or the united states. the definition of early aids-defining illness was based on clinical, necropsy and histopathological findings as described [39] . the 39 animals included in the first cohort were previously used as a 'screening cohort' to search for polymorphisms influencing aids-free survival time and viral load in siv infection [39] . it should be noted that no viral load data were available for some of the animals included in the survival analysis. the second cohort of 55 animals consisted of macaques more recently infected, and viral load data were available for all animals. the animals were housed and treated at the german primate centre under standard conditions which are in accordance with the german animal protection act and the european union guidelines on the use of non-human primates for biomedical research as described [40] . briefly, all experiments were approved by an external ethics committee authorized by the lower saxony state office for consumer protection and food safety (project licenses: 509.42502/08-02.95, 509.42502/08-13.98, 504.42502/08-03.90 (v+ä ), 33.9-42502-04-12/0820, 509-42502/08-04.03, 33.9.42502/04/017/07, 33.9.42502/04/72/08). according to §11 of the german animal welfare act, the dpz is permitted to breed and house non-human primates under license number 392001/7 issued by the local veterinary authorities. all macaques were under daily surveillance by veterinarians and animal caretakers. during quarantine, the animals were usually housed in groups of 2-3 animals per cage, with a minimum enclosure height of 180 cm and a cage volume of 3 to 4.5 m 3 . animals in experiments had to be housed singly (cage dimensions [in cm]: 190h × 90w × 90d), but with constant visual, olfactory and acoustic contact to their roommates. procedures for animal welfare and to minimize discomfort and suffering were undertaken in accordance with the recommendations of the weatherall report "the use of nonhuman primates in research". monkeys were fed with dry monkey biscuits supplemented with fresh fruit or vegetables twice daily and fresh water access ad libitum. for environmental enrichment, animals were offered feeding puzzles, varying toys and wood sticks for gnawing. in addition, music was played. for dna preparation, blood samples were drawn under anesthesia with 10 mg ketamine i.m. per kg body weight. animals were humanely euthanized by an overdose of pentobarbital-natrium (narcoren 1 , merial, hallbergmoos, germany) under anesthesia either for experimental reasons without exhibiting clinical symptoms or in cases of suffering predefined by a scoring system of termination criteria that was approved by the external ethics committee and corresponds to the iacuc endpoint guidelines as described [40] . for the purpose of this study, no monkey was sacrificed since archival samples were used. the rhifitm3 gene was amplified from genomic dna by pcr and screened for polymorphisms by re-sequencing. primers for amplification were based on the macaca mulatta chromosome 14 scaffold (mmul_051212) [41] . we used this scaffold before to successfully amplify cdnas of rhifitm genes [6] . amplification of genomic ifitm3 sequences (loc697829) was performed using primers mamuifitm3gen5-1fora (5'-tttgttccgccctcatctgg-3') and mamuifitm3gen5-1rev (5'-tctgagatccacgctcagga-3'). primers were designed using primer-blast [42] to specifically avoid amplification of rhifitm3(2) (loc697564) or rhifitm3 pseudogenes. reaction mixtures contained 100 ng genomic dna, 50 pmol of each primer, 100μm dntp mix, 10% betain, 1x hf synthesis buffer and 1 u phusion dna polymerase. pcr cycling started with an initial denaturation step at 95˚c for 5 min, followed by 40 cycles with denaturation at 95˚c for 30 s, annealing at 68˚c for 30s and elongation at 72˚c for 1:40 min followed by a post amplification step at 72˚c for 10 min. pcr products were gel purified and sequenced using abi big dye chemistry and primers mamuifitm3gen5-1fora, mamuifitm3gen5-1rev, if3gi-for (5'-gtgcccacgtc agtagcttta-3') or if3gi-for2 (5'-tgtgcccacgtcagtagcttt-3') and if3gi-rev (5'-cttcctcactcaggctcagac-3') or if3gi-rev2 (5'-caaactctgagcc agccagc-3'). amplicons were assembled on a scaffold sequence using vector nti contigexpress and polymorphisms identified by visual inspection. aids-free survival analysis, including a log-rank test for rhifitm3 genotype-related differences, was performed using the lifetest procedure of the sas software version 9.4 (sas institute inc., cary, nc). the association between rhifitm3 polymorphisms and virus load was assessed for statistical significance by means of either a kruskal-wallis test (three genotypes) or a mann-whitney test (two genotypes), as appropriate, using the graphpad prism software. all p-values were two-sided. the level of statistical significance was depicted as follows, ã , p<0.05; ãã , p<0.01; ããã , p<0.001. one ifitm1 gene and two ifitm3 genes have been identified in rhesus macaques. for the purpose of the present study, we will distinguish between the two rhifitm3 genes as rhifitm3 (loc697829) and rhifitm3(2) (loc697564) because rhifitm3(2) shows a slightly higher sequence homology to the human ifitm2 gene than rhifitm3. here, we investigated whether rhifitm3 inhibits viral entry in a way similar to human (hu) ifitm3, employing a pseudotyping approach. expression of huifitm3 in 293t cells did not appreciably inhibit entry driven by the mlv envelope protein (env) or the machupo virus glycoprotein (macv-gpc) whereas entry driven by the fluav-hemagglutinin (ha) was robustly inhibited (fig 1) , in keeping with published findings [4] [5] [6] . entry driven by siv-env was also reduced upon hui-fitm3 expression (fig 1) , again in agreement with published data [19, 20] . a similar tendency was observed for rhifitm3 even though inhibition of siv-env-and fluav-ha-driven entry was less robust than in the case of huifitm3 (fig 1) , despite comparable expression levels [6] . thus, rhifitm3 exerts antiviral activity but does so less efficiently than huifitm3. we next aimed to characterize polymorphisms in the rhifitm3 gene based upon a previously characterized cohort of 39 siv infected animals (cohort 1) (table 1) , where relationships between animals and confounding factors had been minimized [39] . to obtain results from a larger collection of animals, we randomly selected additional 55 animals for inclusion into a second cohort (cohort 2) ( table 1 ). the rhifitm3 gene has two exons leading to a mature mrna of 636 bp (xm_001085567.2) whereas the genomic region encoding the primary transcript comprises 1202 bp. we devised a pcr strategy to selectively amplify the genomic region of the rhifitm3 gene so as to yield a 1340 bp amplification product (fig 2) . the dna sequence was determined directly from purified pcr products, and sequence variations were identified at 16 positions (fig 3 and table 2 ). three polymorphisms were located in the coding region but did not change the amino acid sequence. twelve polymorphisms (including one single nucleotide deletion) were intronic and one polymorphism was located upstream of the coding sequence. seven polymorphisms had a low level of heterozygosity ( 10%) but, with few exceptions, the two cohorts were found to be characterized by similar major allele table 2 ). polymorphism g.488 c>a was only detected in the second cohort. polymorphisms g.279 g>a and g.678 g>t showed higher heterozygosity in cohort 2, while the opposite was true for g.693 dela, g.708 a>g, g.709 g>a and g.747 t>g. finally, a polymorphism in the 5´region of the coding sequence that corresponds to rs12252 t>c in the human ifitm3 gene was not identified in our sample. to assess whether the detected sequence variations had an influence on the course of siv infection, we first analyzed their effect on aids-free survival time (fig 4, table 3 ). polymorphisms g.270 c>t (genotype gt) and g.678 g>t (genotype gg) showed a nominally significant association with faster disease progression when all animals from both cohorts were considered. however, these associations were not observed when each cohort was analyzed separately and were not significant when animals with mhc allele mamu-a1 ã 001 were excluded from the analysis (not shown). polymorphism g.279 g>a showed a highly significant association of the aa genotype with faster disease progression (log-rank test < 0.0001) and this association was also detected when carriers of mamu-a1 ã 001 (log-rank test < 0.0001) were excluded (fig 4, table 3 ). however, this variant was only little polymorphic (maf: 0.95), and the significance of the association hinged heavily on a single aa animal in cohort 1. for a subset of 81 animals, virus load data were available that allowed the potential association between rhifitm3 polymorphism and siv amplification to be analysed. however, no statistically significant association with plasma viral rna copies at 20 weeks after infection or at time of death was noted for any of the polymorphisms (fig 5, table 4 ). only in cohort 1, we found a nominally significant association of polymorphisms g.693 dela (genotype -/-), g.708 a>g (genotype gg) and g.709 g>a (genotype aa) with increased viral load and of polymorphism g.678 g>t (genotype gt) with lower virus load (table 4 ). however, these findings were not replicated in cohort 2 and were not detected when the full set of animals was analyzed. immune-related ifitm proteins have been established as important antiviral effectors of the interferon response, and a polymorphism in the human ifitm3 gene has been found to be associated with disease severity and progression in fluav and hiv-1 infection [27, 33] . rhesus macaques are important animal models for human infections but it was hitherto unknown whether the rhifitm3 gene is polymorphic and whether possible polymorphisms would impact upon the course of siv infection. we demonstrated that rhifitm3 displays antiviral activity and identified 16 polymorphisms in rhifitm3. some of the polymorphisms were associated with either disease progression or viral load. however, none of the polymorphisms impacted on both parameters, and significant associations were not observed in cohort 2. moreover, most associations failed to attain statistical significance when mamu-a1 ã 001 animals were excluded from the analysis, arguing against a major impact of rhifitm3 polymorphisms on siv infection. recently, a comprehensive characterization of the antiviral activity of ifitm proteins of non-human primates revealed that these proteins, like their human counterparts, can block viral infection [20] . however, ifitm proteins from rhesus macaques were not included in these studies. our results demonstrate that rhifitm3 and huifitm3 target the same viral glycoproteins, but with somewhat different efficiency. thus, entry driven by fluav-ha and siv-env appears to be more efficiently inhibited by huifitm3 than by rhifitm3. these results are in agreement with our previous analysis demonstrating that huifitm3 inhibits entry driven by filovirus glycoproteins with greater efficiency than rhifitm3, despite comparable expression in cells [6] . in summary, rhifitm3 inhibits entry of siv in cell culture which raised the question whether rhifitm3 is polymorphic and whether possible polymorphisms would impact the course of siv infection. our sequence analysis identified variations at 16 positions present in at least two out of 94 animals. six of these polymorphisms were highly polymorphic (maf <75%) and homozygous animals could be detected for both alleles. three of the polymorphisms identified in our study were located within the coding sequences, but led to silent changes. this is noteworthy since ifitm3 polymorphisms in siv-infection the n-terminal residues of ifitm3 control intracellular localization and spectrum of antiviral activity [32, 43, 44] and were suggested to be shaped by selective pressure during primate evolution [32] . one polymorphism was located upstream of the coding sequence; all other polymorphisms were identified within the intron. we found no evidence for a polymorphism that would lead to the production of a truncated protein similar to rs12252-c in human ifitm3, a variant that is associated with the clinical severity of influenza disease [27] . our results were in keeping with a previous report suggesting that ifitm1 and ifitm3 in non-human primates probably underwent purifying selection [20] . thus, we did not find any non-synonymous polymorphisms, indicating that maintenance of the primary amino acid sequence is essential for the function of the protein in rhesus macaques. our analysis was based upon dna sequences derived from assembly mmul_051212 [41] . in the meantime, a new genomic framework (mmul_8.0.1) has been published [45] where the sequences at the 3' end of the coding regions of rhifitm3 (loc697829) and rhifitm3(2) (loc697564) have been exchanged in comparison to the previous assembly (mmul_051212). however, our previously reported cloning and sequencing of cdnas from both loci [6] and the analysis presented here show better agreement with the rhifitm3 locus as annotated in the older genomic assembly. therefore, we continued to use mmul_051212 throughout this study. a reconstruction of haplotypes using phase (v. 2.1) [46] based upon our results revealed 16 haplotypes for the rhifitm3 gene, all of which clustered together and were clearly distinct from rhifitm3(2) (data not shown). this reinsures us that all detected polymorphisms are true rhifitm3 polymorphisms. silent mutations may affect translational efficiency and protein folding [47] whereas polymorphisms in untranslated regions or introns may alter mrna stability or splicing [48] . in consequence, we analyzed the association between the polymorphisms and survival time and viral load in siv-infected rhesus macaques. for this, we used a sample that previously allowed detection of an association between tlr7 polymorphisms and survival time (cohort 1, [39] ) and a sample of randomly selected animals (cohort 2). when the full set of animals was considered, we identified three polymorphisms (g.270 c>t, g.279 g>a, g.678 g>t) with a nominally significant association between a genotype and aids-free survival, but not with virus ifitm3 polymorphisms in siv-infection load at set point (20 weeks post infection). however, for two of these polymorphisms the association was not significant if the two cohorts were analyzed separately or when animals with mhc allele mamu-a1 ã 001 were excluded from the analysis. the polymorphism g.279 g>a genotype aa showed a nominally significant association with aids progression even after adjustment for mhc alleles. however, this position is only modestly polymorphic and the association observed was dependent on a single animal with aa genotype. collectively, these results suggest that rhifitm3 polymorphism might not play a major role in siv infection. in summary, our study not only demonstrated that rhifitm3 displays antiviral activity, but also led to the identification of polymorphisms in the coding and non-coding region of the ifitm3 gene of rhesus macaques. notably, all polymorphism in the coding region were silent and strong evidence for an association of rhifitm3 polymorphisms with disease progression and viral load in siv infected animals was not obtained. we cannot exclude that the analysis of a substantially larger number of animals might reveal such associations. however, the frequency of potentially affected animals and/or the effect of the polymorphisms in terms of viral inhibition would be very low and are therefore not expected to significantly influence results obtained in the macaque model of aids. the dispanins: a novel gene family of ancient origin that contains 14 human members evolution of vertebrate interferon inducible transmembrane proteins evolutionary dynamics of the interferon-induced transmembrane gene family in vertebrates the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus comparative analysis of host cell entry of ebola virus from sierra leone, 2014, and zaire, 1976. the journal of infectious diseases ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms the ifitm proteins inhibit hiv-1 infection tmprss2 activates the human coronavirus 229e for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium the ifitms inhibit zika virus replication ifitm proteins-cellular inhibitors of viral entry interferon-induced transmembrane protein 3 is a type ii transmembrane protein a membrane topology model for human interferon inducible transmembrane protein 1 combined approaches of epr and nmr illustrate only one transmembrane helix in the human ifitm3 ifitm3 restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals interferon induction of ifitm proteins promotes infection by human coronavirus oc43 human cytomegalovirus exploits interferoninduced transmembrane proteins to facilitate morphogenesis of the virion assembly compartment primate lentiviruses are differentially inhibited by interferon-induced transmembrane proteins nonhuman primate ifitm proteins are potent inhibitors of hiv and siv characteristics of ifitm, the newly identified ifn-inducible anti-hiv-1 family proteins hiv-1 mutates to evade ifitm1 restriction ifitm proteins incorporated into hiv-1 virions impair viral fusion and spread ifitm proteins are incorporated onto hiv-1 virion particles and negatively imprint their infectivity ifitm3 limits the severity of acute influenza in mice. plos pathogens ifitm3 restricts the morbidity and mortality associated with influenza ifitm3 rs12252 t>c polymorphism is associated with the risk of severe influenza: a meta-analysis early hypercytokinemia is associated with interferoninduced transmembrane protein-3 dysfunction and predictive of fatal h7n9 infection ifitm3 and susceptibility to respiratory viral infections in the community. the journal of infectious diseases ifitm3 polymorphism rs12252-c restricts influenza a viruses natural mutations in ifitm3 modulate post-translational regulation and toggle antiviral specificity interferon-induced transmembrane protein-3 rs12252-c is associated with rapid progression of acute hiv-1 infection in chinese msm cohort resistance of transmitted founder hiv-1 to ifitm-mediated restriction the v3-loop of hiv-1 env determines viral susceptibility to ifitm3 impairment of viral infectivity making the animal model for aids research more precise: the impact of major histocompatibility complex (mhc) genes on pathogenesis and disease progression in siv-infected monkeys the role of nk cells in hiv-1 protection: autologous, allogeneic or both? human immunodeficiency virus type 1 spinoculation enhances infection through virus binding association of tlr7 variants with aids-like disease and aids vaccine efficacy in rhesus macaques noncytolytic cd8+ cell mediated antiviral response represents a strong element in the immune response of simian immunodeficiency virus-infected long-term non-progressing rhesus macaques evolutionary and biomedical insights from the rhesus macaque genome primer-blast: a tool to design target-specific primers for polymerase chain reaction the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication the interferon-induced transmembrane proteins, ifitm1, ifitm2, and ifitm3 inhibit hepatitis c virus entry a new rhesus macaque assembly and annotation for next-generation sequencing analyses a comparison of bayesian methods for haplotype reconstruction from population genotype data exposing synonymous mutations the dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding rna and synonymous mutations we thank heidi meyer for technical assistance and christiane stahl-hennig for the support of our work and for providing access to material and data that were generated under her supervision including the blood samples from the experimental animals. key: cord-333622-0ddutmdd authors: dyer, wayne b; zaunders, john j; yuan, fang fang; wang, bin; learmont, jennifer c; geczy, andrew f; saksena, nitin k; mcphee, dale a; gorry, paul r; sullivan, john s title: mechanisms of hiv non-progression; robust and sustained cd4+ t-cell proliferative responses to p24 antigen correlate with control of viraemia and lack of disease progression after long-term transfusion-acquired hiv-1 infection date: 2008-12-11 journal: retrovirology doi: 10.1186/1742-4690-5-112 sha: doc_id: 333622 cord_uid: 0ddutmdd background: elite non-progressors (plasma viral load <50 copies/ml while antiretroviral naive) constitute a tiny fraction of hiv-infected individuals. after 12 years follow-up of a cohort of 13 long-term non-progressors (ltnp) identified from 135 individuals with transfusion-acquired hiv infection, 5 remained ltnp after 23 to 26 years infection, but only 3 retained elite ltnp status. we examined the mechanisms that differentiated delayed progressors from ltnp in this cohort. results: a survival advantage was conferred on 12 of 13 subjects, who had at least one host genetic factor (hla, chemokine receptor or tlr polymorphisms) or viral attenuating factor (defective nef) associated with slow progression. however, antiviral immune responses differentiated the course of disease into and beyond the second decade of infection. a stable p24-specific proliferative response was associated with control of viraemia and retention of non-progressor status, but this p24 response was absent or declined in viraemic subjects. strong gag-dominant cytotoxic t lymphocyte (ctl) responses were identified in most ltnp, or pol dominant-ctl in those with nef-defective hiv infection. ctl were associated with control of viraemia when combined with p24 proliferative responses. however, ctl did not prevent late disease progression. individuals with sustained viral suppression had ctl recognising numerous gag epitopes, while strong but restricted responses to one or two immunodominant epitopes was effective for some time, but failed to contain viraemia over the course of this study. viral escape mutants at a hla b27-restricted gag-p24 epitope were detected in only 1 of 3 individuals, whereas declining or negative p24 proliferative responses occurred in all 3 concurrent with an increase in viraemia. conclusion: detectable viraemia at study entry was predictive of loss of ltnp status and/or disease progression in 6 of 8, and differentiated slow progressors from elite ltnp who retained potent virological control. sustained immunological suppression of viraemia was independently associated with preserved p24 proliferative responses, regardless of the strength and breadth of the ctl response. a decline in this protective p24 response preceded or correlated with loss of non-progressor status and/or signs of disease progression. declining or negative p24 proliferative responses occurred in all 3 concurrent with an increase in viraemia. conclusion: detectable viraemia at study entry was predictive of loss of ltnp status and/or disease progression in 6 of 8, and differentiated slow progressors from elite ltnp who retained potent virological control. sustained immunological suppression of viraemia was independently associated with preserved p24 proliferative responses, regardless of the strength and breadth of the ctl response. a decline in this protective p24 response preceded or correlated with loss of non-progressor status and/or signs of disease progression. a cohort of blood product recipients with transfusionacquired hiv (tahiv) infected between 1981 and 1984 was followed prospectively by the australian red cross blood service hiv lookback team since 1987. there are individuals in this cohort who have remained asymptomatic for 27 years since infection without antiretroviral therapy; some maintaining plasma hiv rna levels to below detectable levels and a stable cd4 t cell count, thus retaining elite non-progressor status. early natural history studies on this and other cohorts suggested that tahiv infection may result in a shorter time to aids than sexually-acquired (sa) hiv infection [1, 2] . this observed increase in the rate of disease progression in tahiv may be due to the higher inoculation volume of blood product compared with the much smaller blood or genital fluid exchange involved in sahiv infection [1] , as well as the known immunomodulatory effect of transfusion on immune function [3, 4] . age is also an independent predictor for an increased rate of hiv disease progression [5, 6] . the bias toward an aged population requiring transfusion is part of the composite disadvantage of transfusion as a route of hiv infection [1] . in addition to hiv infection, survival may be influenced by the underlying medical cause for transfusion. yet despite these disadvantages, we previously observed a high frequency of nonprogression in this tahiv cohort after 20 years of infection [7] . early studies on this cohort of tahiv patients led to the identification of the sydney blood bank cohort (sbbc) of long-term survivors [8] , and that an attenuated nef-deleted strain of hiv-1, transmitted from a single donor resulted in slow to non-progression in these individuals [9] . however, after prolonged infection, not all sbbc members maintained non-progressive disease [10] [11] [12] [13] . although hla type did not explain non-progression in this group [14] , we have observed differences in cd8 t cell responses that are associated with hla-dependent epitope recognition [15] , and we have detected increased preservation of helper t cell responses in non-progressors from this cohort [16, 17] . in addition to the well described host genetic factors which may prolong non-progression [7] , recent studies have suggested an influence from innate immune mechanisms, including polymorphisms that decrease tlr function thereby reducing immune activation upon exposure to infections diseases [18] , or the fcγriia polymorphism (r/r) which is strongly associated with progressive hiv disease as a result of impaired elimination of hiv immune complexes [19] . while host genetic factors may predispose an individual for delayed disease progression, there is substantial evidence that antiviral t cell responses are required to sustain non-progressor status. earlier studies have demonstrated an important role for gag-specific ctl in delaying disease progression [20, 21] . non-progressors that control viraemia in the absence of antiviral therapy also have strong cd4 t cell proliferative responses to the gag protein p24 [22] . importantly, for gag ctl to be efficient in killing hiv-infected cells and therefore protective in controlling viraemia, these must also be accompanied by p24-specific t cell proliferative responses [23] [24] [25] . appropriate t cell help is also required to achieve maturation and display of effector phenotypes on ctl associated with effective virological control [26] . to determine how these host genetic and immune factors combined to contribute to prolonged non-progression in our tahiv cohort, we report here on the current status of the elite non-progressors not on antiretroviral therapy (art), examining the factors that have influenced disease in the former non-progressors (now on therapy or deceased), and analyse potential mechanisms that have influenced non-progression in this cohort for up to 27 years. when this prospective study began in 1994, 13 ltnp were identified in the nsw tahiv cohort according to the original guidelines for classifying ltnp: at least 10 years infection, stable cd4 t cell counts >500 cells/μl, and no history of art [27, 28] . subsequently, loss of ltnp status was defined by any of the following events: a consistent decline in cd4 t cell counts below 500/μl, commencement of art, and after viral load testing became routine, plasma viraemia >5000 copies/ml. elite non-progressors were also defined by viraemia suppressed to <50 copies/ ml in addition to the above criteria. disease progression was defined by a cd4 t cell count of <200 and/or plasma viraemia >100,000 copies/ml. the two non-progressor groups in this study included the sbbc, consisting of 6 recipients of hiv-infected blood from a common donor, and the other (cohort 2) consisting of 7 recipients infected by blood from different donors. clinical data from these ltnp were collected prospectively since the late 1980s. t cell counts and viral load tests were performed as part of routine clinical care. blood samples and clinical histories were provided after informed consent was granted in accordance with guidelines from the arcbs institutional human research ethics committee. anti-hiv t cell function assays were performed as previously described [15, 29] . briefly, the proliferative response to hiv-1 p24 was determined by 6 day culture of pbmc (1 × 10 5 cells/well) in rpmi medium with 15% pooled human serum in round bottom microtitre plates, with 2 μg/ml hiv-1 sf2 p24 (chiron, emeryville, ca, usa), or medium alone for control. after 6 days, proliferative responses were determined by 3 h-thymidine incorporation during a further 6 hours culture, followed by cell harvest and reading in a liquid scintillation counter. results were expressed as stimulation index (si; mean counts antigen wells/mean counts control wells), and a si >3 was considered a positive response. the response of cd8+ t cells to hiv antigen was measured by ifnγ elispot, using pre-coated elispot kits according to the manufacture's protocol (mabtech, mosman, australia). firstly, the response to whole hiv proteins was determined in response to antigen presented by autologous b lymphoblastoid cell lines infected for 18 hours with 5 pfu/cell recombinant vaccinia expressing the hiv-1 iiib env, gag, pol, or nef genes (therion biologics, cambridge, ma, usa), or e. coli lacz as a control. gag responses were further characterised using overlapping gag peptides, firstly using a matrix of peptide pools, and then individual peptides for confirmation (full gag peptide set; kindly provided by the nih aids research and reference reagent program, division of aids, niaid, nih). dna from pbmc was isolated using a qiaamp dna mini kit (qiagen, valencia, ca) according to the manufacturer's protocol. a nested polymerase chain reaction (pcr) was used to amplify ~1.5 kb of the hiv gag gene using the following primers: rna was isolated from plasma using the qiaamp viral rna mini kit (qiagen, valencia, ca) according to the manufacturer's protocol. gag gene was amplified using the qiagen onestep rt-pcr kit using the outer primer pairs mentioned above. second round pcr reactions were performed using the inner primer pair under the same conditions. pcr products were purified using a millipore pcr purification plate (millipore, billerica, ma, usa) and sequenced by the abi prism bigdye terminator v3.1 ready reaction cycle sequencing kit (applied biosystems, foster city, ca, usa) on an abi 377 automated sequencer. multiple sequences derived from each patient were analysed using sequencher 3.11 software (gene codes corp., ann arbor, mi, usa). chromatograms derived from both forward and reverse primers were aligned with the reference strain hiv-1 hxb2. methods for hla and chemokine receptor polymorphisms [30] and toll-like receptor (tlr) and fcγriia polymorphisms [31] [32] [33] have been described elsewhere. the fishers exact test was used to associate genetic and immune factors with viraemia and non-progressor status. from all reported tahiv cases from the state of nsw, australia, a cohort of 13 (10%) remained asymptomatic after 10 years of infection. we now report that only 5 remain non-progressors after 23 to 26 years of hiv-1 infection. infection and treatment history for each subject is summarised in additional file 1. most of these individuals had a survival advantage, with 7 of 13 having at least one host genetic polymorphism associated with slow progression, and 6 of 13 were infected with the sbbc nefdefective hiv-1 strain [12] , and combined, 12 of 13 had at least one host or viral factor favouring slow progression. acting in opposition to these survival advantages, 5 of 8 former non-progressors had the fcγriia polymorphism (r/r). while this genotype was absent in current ltnp, the effect of the r/r genotype in promoting disease progression was not significant in this small study of 13 individuals. on balance, these competing survival factors along with antiviral immune responses enabled a nonprogressive disease course to be established early in infection. the loss of non-progressor status was based on increasing viraemia and/or decreasing cd4 counts in 5 of 8, and initiation of art in these individuals (additional file 1). patient c122 lost ltnp status due to gradually increasing viraemia, but died from unrelated causes before substantial t cell loss was observed. another two elderly individuals (c18 and c54; both sbbc members), each with low detectable viraemia, died before losing their non-progressor status [13, 17] . host and viral genetic factors may have played a role in delaying disease progression into the second decade of infection in these 13 individuals, but this study also demonstrates the importance of host immune responses in sustaining this non-progressive disease course into and beyond the second decade of infection. immune status and activity of hiv-specific cd4 t cells (proliferation) and cd8 t cells (ifn-γ response) is shown for the current non-progressors ( figure 1 ) compared with those that lost their non-progressor status or died ( figure 2 ). antiviral ctl responses were variable during the second decade of hiv infection, and did not always correlate with viremia for members of these cohorts. strong gag-specific ctl were detected in the cohort 2 non-progressors (c13, c53, c122, and c105 before art), but the predominant ctl response in the sbbc members was against pol antigens. these ctl appeared to be equally effective in containing viral replication, whether gag-specific as demonstrated in earlier time points in c122, or pol-specific in c18 ( figure 2 ). the main factor that differentiated ltnp from those that lost non-progressor status, was low or undetectable hiv viraemia (<100 copies/ml; p = 0.021), and low viraemia was associated with detectable p24 proliferative responses (p = 0.0047). loss of non-progressor status was strongly associated with undetectable or declining p24 responses (p = 0.0047). the combination of detectable p24 proliferative responses and strong (>500 sfc/10 6 pbmc) gag ctl responses was associated with low (<100 copies/ml) or undetectable viraemia (p = 0.032). illustrating the importance of these combined gag-specific t cell responses over time, low viraemia was intermittently detected at earlier time points in c122, with sharp increases in gag ctl temporally associated with control of transient viraemia at 17 years post infection. however, gag ctl later failed to contain viraemia in c122 beyond approximately 20 years, coinciding with weakening proliferative responses that gradually became negative. a similar correlation between anti viral immune responses and a spike in viral replication was demonstrated in sbbc member c18, shown in more detail in figure 3 . over the course of 12 months, in response to an increase in viraemia peaking at 3600 copies/ml, the p24 proliferative response increased, along with substantial expansions of pol-specific ctl in both precursor [15] and effector ctl populations. the durability of immune control in this individual was not determined as he died soon after from causes unrelated to hiv disease, aged 83. a decline in gag-specific t cell responses preceding detectable viraemia was demonstrated in c13. this decline up to year 16 was followed by a period of low detectable viraemia (50 -100 copies/ml) between years 19 -22. a rebound in these gag-specific t cell responses coincided with the first detectable viraemia at 19 years. these t cell responses may have helped contain viraemia to low levels over the following two years, but the sharp increase in viral rna at 22.7 years ( figure 1 ) coincided with a decline in gag-specific cd4 and cd8 t cell responses, whereas pol-specific ctl increased in response to rising viraemia. these examples demonstrate the influence of conserved gag-specific responses, particularly helper t cell responses, in reduced viral replication and delayed disease progression. while the decline in these responses preceded detectable viraemia in c13, sufficient patient specimens were not available to allow this critical observation to be made in others who progressed. to determine why strong gag ctl may have contained viral replication in some, but failed in others, we mapped the breadth of the gag ctl response over time in patients with at least moderate clt responses to whole gag antigens. pools of overlapping 15-mer gag peptides were used to test sequential pbmc spanning the study period by elispot. the composition of each peptide pool, and examples of responses to these are shown in figures 4 and 5 , indicating the relevant hla-specific epitopes contained in peptides at the intersection of positive pools. figure 4 demonstrates a broad strong response by c53's pbmc to multiple immunodominant epitopes, contrasted in figure 5 by the restricted response from c122 to only two immunodominant epitopes. the sequential analysis revealed relatively high stability in the repertoire of gag responses over the past 10 years in most subjects (additional file 2). relevant epitopes at intersecting positive peptide pools were then confirmed using individual peptides ( figure 6 ). this data demonstrates that retention of broadly reactive gag ctl was associated with ongoing non-progression (c49, c64, and c53), while restriction toward a narrow ctl specificity was observed in patients that eventually lost control of viraemia (c122 and possibly c13). the sbbc non-progressors c49 and c64 had responses to several gag epitopes, and although gag responses were moderate to weak in c64, this needs to be viewed in the context of pol ctl dominance in the sbbc. a strong but immunovirological status of the surviving non-progressors, showing t cell counts; viral rna copies/ml plasma (data generated from the roche amplicore standard assay, limit of detection 400, and ultrasensitive assay, limit of detection 50, plotted sepa-rately); t cell proliferative responses to recombinant hiv-1 p24 (stimulation index; significant responses >3, defined by the broken line); and ifnγ responses (elispot) by ctl against autologous bcl expressing hiv-1 antigens after infection with recombinant vaccinia figure 1 immunovirological status of the surviving non-progressors, showing t cell counts; viral rna copies/ml plasma (data generated from the roche amplicore standard assay, limit of detection 400, and ultrasensitive assay, limit of detection 50, plotted separately); t cell proliferative responses to recombinant hiv-1 p24 (stimulation index; significant responses >3, defined by the broken line); and ifnγ responses (elispot) by ctl against autologous bcl expressing hiv-1 antigens after infection with recombinant vaccinia. *sbbc member. immunovirological status of the former non-progressors (same parameters as in figure 1) figure 2 immunovirological status of the former non-progressors (same parameters as in figure 1 ). initiation of antiretroviral therapy is defined by an arrow in the viral load panels. other reasons for loss of non-progressor status are summarised in additional file 1. restricted gag response was also seen in c18, but these gag responses were likely to be secondary in controlling viraemia, as suggested by the kinetics of pol ctl in response to a spike in viraemia ( figure 3 ). pol ctl recognition was confirmed by subsequent analysis of responses to peptide pools derived from the full set of pol overlapping 15-mer peptides. moderate to strong responses to multiple pools containing epitopes in the reverse transcriptase protein were detected in sbbc members c49, c64, c18, c54, but weakly in c98 (data not shown). c18 also responded strongly to integrase peptides. a strong but narrow ctl response may eventually fail to control viral replication. restricted recognition of only one a3 and two b27 gag epitopes in c13 appeared sufficient to have contained viraemia for many years, but the most recent viral load result (figure 1 ) suggested that immune escape from these b27-restricted ctl may have occurred recently. similarly, the predominant response by c122 against an immunodominant b27 epitope ( figure 5 and 6) may have contained earlier spikes of increased viraemia, but ultimately failed to contain increasing viral replication in later years ( figure 2 ). to determine why immunodominant b27-restricted ctl initially contributed to reduced viral replication in c13 and c122, but not in c117, sequencing of plasma and pbmc derived virus spanning the period before and after signs of disease progression was carried out to determine if viral escape mutants had emerged in this region of gag (figure 7) . with the exception of one sample in 1996, a well characterised escape mutant [34] was detected from the earliest time point in c117. this escape mutant was not detected in c13 or c122, and hence was not the cause for the loss of control of viraemia in c122, nor was it detected in the latest time point from c13 when viraemia first increased above 1000 copies/ml. this suggests that immune escape at this b27 gag epitope was not a major cause of disease progression in very long term infected dynamics of immune responses during an episode of increased viral replication in sbbc patient c18 figure 3 dynamics of immune responses during an episode of increased viral replication in sbbc patient c18. individuals. the sole common factor was a decline in p24specific proliferative responses. non-progressors are considered to represent the tail end of the distribution curve of rates of disease progression, and although elite non-progressors are extending this curve even further, disease progression may be inevitable in this rare group of individuals. recent analyses of the sbbc may support this suggestion [13, 17] . however, death from other causes has prevented the establishment of definitive proof of disease progression in some individpool x1 x2 x3 x4 x5 x6 x7 x8 x9 x0 control sfc 505 155 0 100 185 3600 225 150 390 425 10 5 1 2 3 4 5 6 7 8 uals. two sbbc subjects that did not consent to prospective analysis died from unrelated causes in 1987 and 1994, and the sole sbbc recipient on therapy (c98) has since died from non-hiv causes. two other elderly subjects also died from non-hiv causes (c18 and c54), but control of viraemia at low levels along with normal cd4 t cell counts suggested there was no evidence for loss ltnp status before death. this leaves the three elite nonprogressors from the sbbc described in this study, and one is also advanced in age. one of the elderly cohort 2 ltnp (with wild-type hiv infection) also died recently from non-hiv causes aged 84 (c122). we may have an opportunity to determine the factors involved in disease progression in the other two cohort 2 non-progressors (c13 and c53). both had very low but detectable viraemia, but a recent inversion of cd4 : cd8 t cell ratio in c13 is evident of a change in hiv-induced immune activation. based on the decline in the proliferative response to p24 preceding this recent increase in viraemia, it is likely that these together signify a transitionary stage toward disease progression in c13. the p24 proliferative response was the single immune parameter that consistently defined control of viraemia and non-progression. the p24 proliferative response may also be specifically protective, as suggested by a study showing that responses to some pol antigens were associated with increased viraemia, whereas a protective association was always found in responses to gag antigens [35] . hiv-specific proliferative responses may also promote ctl proliferation. this was supported by the response to a spike in viraemia in patient c18, where the hiv-specific cd4 and cd8 memory t cell pool increased and were maintained throughout this period (both proliferation and ctl precursor assays measure proliferating antigenspecific memory t cells), whereas effector ctl (elispot-positive cells) peaked then declined as viraemia declined. these data suggest that effective helper t cell function involves proliferation followed by maturation into both effector and costimulatory cells that provide "help" for other lymphocyte functions. thus, antigen-specific cd8 t cell proliferation may be directly associated with cd4 proliferation to epitopes on the corresponding antigen. it is also possible that loss of protective cd4 and cd8 hivspecific proliferation may be mediated by a common immunological defect. the other distinguishing feature of most slow and nonprogressor subjects that we have studied is the predominance of gag ctl, but the sbbc non-progressors were exceptional in having a pol-dominant ctl response. this observation is unexpected, considering the extensively published role for gag ctl in controlling viraemia [20, 21, 23] . also peculiar to sbbc members, the strongest ctl responses were detected in those with detectable viraemia, and were weaker in subjects with undetectable viraemia [15] . in the non-attenuated hiv-infected nonprogressors, strong immunodominant ctl combined with detectable proliferative responses to p24 appears to have contributed to viraemia remaining <100 copies/ml after 23 years hiv-1 infection in patient c53. the individual with the strongest ctl response was c122, but ctl increased as viraemia increased in this patient, while proliferative responses to gag p24 declined. given the predominant ctl response in this subject was directed against an immunodominant hla b27 restricted p24 amino acid sequences of the hla b27 restricted gag epitope krwiilglnk) figure 7 amino acid sequences of the hla b27 restricted gag epitope krwiilglnk). epitope, and that there were no immune escape sequences detected at this epitope, it is likely that the decline in the p24-specific proliferative response was the key event that contributed to the failure of ctl to control viraemia, as it is understood that ctl have much reduced functional efficiency in containing viraemia in the absence of helper t cell responses [24] . another study of hla b57 positive individuals found no association between disease progression and the strength of ctl responses or the emergence of viral escape mutants at these epitopes, but it was found that viral replicative fitness influenced disease course [36] . the contribution of p24-specific proliferative responses was not investigated in that study. the neutralising antibody (nab) response is another immune mechanism that may contribute to long term control of viraemia [37] . we recently analysed viral replicative fitness and the strength of nab responses, and confirmed that nab titres in long-term infected subjects were inversely proportional to viral load. however, nab titres in sbbc members were comparatively weaker, and in parallel with ctl responses, were highest in those with detectable viraemia. the presence of strong nabs did not prevent some sbbc members from developing signs of disease progression [38] . hence, we suggest that while broad nabs might be generated due to a crippled infection they do not prevent disease progression, particularly in the absence of antiviral helper t cell responses. what is the key factor that sustains a non-progressive disease course, and what initiates the decline in protective immunity after many years of a non-progressive disease course. could a change in viral pathogenicity overcome this delicate balance between host and virus, or could progressive weakening of the cd4 t cell response by slow virus turnover gradually allow the virus to escape the combined effector mechanisms of the hiv-specific cell-mediated and humoral immune responses? escape mutants at the b27 epitope krwiilglnk have been observed under conditions of high viral load and evolutionary drift [34] , which was likely in c117, but not for both c13 and c122, who had prolonged periods of immune control in the presence of p24 proliferative responses, and very low viraemia up to or beyond the second decade of hiv infection. disease progression in one sbbc member (c98) and the sbbc infecting donor was associated with the emergence of divergent strains which preceded viral load increases and subsequent changes in immune responses [39, 40] . also, these individuals lacked protective p24 proliferative responses and had detectable viraemia before viral divergence occurred, providing further evidence that effective immune control over viral replication to levels below a putative threshold, may prevent the emergence of escape mutants or fitter variants. therefore, the common factor in all these observations is the decline or lack of p24 proliferative responses, suggesting that a lack of helper t cell responses may result in a reduced capacity to contain viral replication by other immune effector responses including ctl, independent of the presence of viral escape mutants. our studies have demonstrated that host and viral genetic factors can contribute to delayed disease progression, but the single immunological factor that functionally defined non-progression was gag-specific cd4 t cell proliferation. the maintenance of this p24-specific response does not require detectable viral replication for antigenic stimulation [41] . detectable p24-specific t cell proliferation defines the immunocompetent recall response to viral antigen, and when spikes of viral replication were detected in these individuals, these cells likely provided t cell help for maintaining functional antiviral effector responses by other cd4 and cd8 t cells, including noncytolytic antiviral mechanisms [41] , and may also provide t cell help for efficient generation of nab by hiv-specific b cells. we have demonstrated that a decline in this protective p24 response in slow progressors either preceded or coincided with classic signs of disease progression. the natural history of transfusion-associated infection with human immunodeficiency virus. factors influencing the rate of progression to disease the natural history of human immunodeficiency virus infection transfusion-induced immunomodulation and its clinical consequences immunomodulation and blood transfusion the influence of age on the latency period to aids in people infected by hiv through blood transfusion hiv infection in recipients of blood products from donors with known duration of infection high frequency of nonprogression 20 years after transfusion-transmitted human immunodeficiency virus type-1 infection long-term symptomless hiv-1 infection in recipients of blood products from a single donor genomic structure of an attenuated quasi species of hiv-1 from a blood transfusion donor and recipients sullivan js: immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of hiv-1. a report from the sydney blood bank cohort longitudinal analysis of nef/long terminal repeatdeleted hiv-1 in blood and cerebrospinal fluid of a long-term survivor who developed hiv-associated dementia longitudinal analysis of human immunodeficiency virus type 1 nef/long terminal repeat sequences in a cohort of long-term survivors infected from a single source replication-dependent pathogenicity of attenuated, nef-deleted hiv-1 in vivo update on long-term symptomless hiv type 1 infection in recipients of blood products from a single donor strong human immunodeficiency virus (hiv)-specific cytotoxic t-lymphocyte activity in the sydney blood bank cohort patients infected with nefdefective hiv type 1 lymphoproliferative immune function in the sydney blood bank cohort, infected with natural nef/long terminal repeat mutants, and in other long-term survivors of transfusion-acquired hiv-1 infection pathogenicity and immunogenicity of attenuated, nef-deleted hiv-1 strains in vivo influence of coinfecting pathogens on hiv expression: evidence for a role of toll-like receptors fcgammariia genotype predicts progression of hiv infection gag-specific cytotoxic responses to hiv type 1 are associated with a decreased risk of progression to aids-related complex or aids longitudinal analysis of human immunodeficiency virus type 1-specific cytotoxic t lymphocyte responses: a predominant gag-specific response is associated with nonprogressive infection vigorous hiv-1-specific cd4+ t cell responses associated with control of viremia cd8 t-cell recognition of multiple epitopes within specific gag regions is associated with maintenance of a low steady-state viremia in human immunodeficiency virus type 1-seropositive patients hiv type 1-specific helper t cells: a critical host defense loss of hiv-1-specific cd8+ t cell proliferation after acute hiv-1 infection and restoration by vaccine-induced hiv-1-specific cd4+ t cells hiv-1 specific cd8+ t cells with an effector phenotype and control of viral replication long-term hiv-1 infection without immunologic progression long-term survivors of hiv-1 infection: definitions and research challenges correlates of antiviral immune restoration in acute and chronic hiv-1 infection: sustained viral suppression and normalisation of t cell subsets hla and other host factors in transfusion-acquired hiv-1 infection association of toll-like receptor-4 (asp299gly and thr399ileu) gene polymorphisms with gastritis and precancerous lesions influence of fcgammariia and mbl polymorphisms on severe acute respiratory syndrome. tissue antigens clinical relevance of tlr2, tlr4, cd14 and fcgam-mariia gene polymorphisms in streptococcus pneumoniae infection clustered mutations in hiv-1 gag are consistently required for escape from hla-b27 restricted ctl responses cd4 responses to conserved hiv-1 t helper epitopes show both negative and positive associations with virus load in chronically infected subjects viral replication capacity as a correlate of hla b57/b5801-associated nonprogressive hiv-1 infection neutralizing antibody responses against autologous and heterologous viruses in acute versus chronic human immunodeficiency virus (hiv) infection: evidence for a constraint on the ability of hiv to completely evade neutralizing antibody responses viral phenotypes and antibody responses in long term survivors infected with attenuated human immunodeficiency virus type 1 containing deletions in the nef and long terminal repeat region phenotype and envelope gene diversity of nef-deleted hiv-1 isolated from long-term survivors infected from a single source macrophage tropism and cytopathicity of hiv-1 variants isolated sequentially from a long-term survivor infected with nef-deleted virus comprehensive analyses of a unique hiv-1-infected non-progressor reveal a complex association of immunobiological mechanisms in the context of replication incompetent infection we wish to thank the long-term dedicated participation of the study subjects. technical assistance was provided by jie liu, ingrid boehm and kirsi bourget (arcbs). anthony kelleher provided a critical review of the manuscript. this study was supported by the immunovirology research network, the australian centres for hiv and hcv virology research, and an nhmrc project grant (dam). prg is the recipient of an australian national health and medical research council r. douglas wright biomedical career development award. the authors declare that they have no competing interests. wbd wrote the manuscript and directed the immunology experimental work. bw and nks provided the sequence data, and jcl provided patient clinical details. ffy provided host genetic data. jjz and adk assisted with analysis of t cell immunity data, and dm and prg provided commentary on neutralising antibody and viral evolution and helped edit the manuscript. afg and jss contributed to the design of the ltnp studies. clinical status of the study subjects. ¶ ltnp cohort identified in 1994, consisting of the sbbc and "cohort 2" non-progressors. ‡ key: cord-342719-bdxb45us authors: yamamoto, shinya; saito, makoto; nagai, etsuko; toriuchi, keiko; nagai, hiroyuki; yotsuyanagi, hiroshi; nakagama, yu; kido, yasutoshi; adachi, eisuke title: antibody response to sars-cov-2 in people living with hiv date: 2020-10-02 journal: j microbiol immunol infect doi: 10.1016/j.jmii.2020.09.005 sha: doc_id: 342719 cord_uid: bdxb45us nan of the 83 covid-19 patients, five were plwh. the characteristics of them are summarized in table 1 . all of them received antiretroviral therapy and their cd4+ t-cell counts had been stable. three cases were men who have sex with men (msm), and two cases were transgender women. all five cases were classified mild to moderate severity of covid-19, and none required intubation. all cases were cured and discharged. all five patients received consecutive serological test, and four of five patients had seroconversion by one month after the symptom onset, which were similar to non-hiv-infected patients. 1 ample studies have demonstrated that plwh generally show poor serological response to other viruses or viral antigens such as hepatitis b vaccine, 2 especially for plwh with a high hiv viral load and decreased cd4+ t-cell. 2 one previous report described that an untreated hiv case had seroconversion of sars-cov-2 two months after symptoms appeared. 3 moreover, a fundamental research demonstrated b-cell dysfunction was caused by hiv-1 gp120 binds directly to primary b-cell in hiv viremic individuals. 4 therefore, uncontrolled hiv infection may be a factor to lower the rate of seroconversion, including false negative. art occurred similarly to that in covid-19 patients without hiv infection. absence of seroconversion, as was observed in our case 2, has been reported particularly in mild 1 or asymptomatic patients. 5 we highlighted that seroconversion of sars-cov-2 was similar between well-controlled plwh and patients without hiv. our findings showed no evidence of poor serological response to covid-19. further studies should be required to elucidate the serological mechanism with plwh, but coronavirus vaccine potentially could be suitable in plwh. antibody profiles in mild and severe cases of covid-19 undetectable plasma hiv rna load predicts success after hepatitis b vaccination in hiv-infected persons one case of coronavirus disease 2019 (covid-19) in a patient co-infected by hiv with a low cd4(+) t-cell count the hiv-1 envelope protein gp120 impairs b cell proliferation by inducing tgf-β1 production and fcrl4 expression antibody responses to sars-cov-2 at 8 weeks postinfection in asymptomatic patients. emerg infect dis key: cord-314560-rswa5zdn authors: manjunath, n.; kumar, priti; lee, sang kyung; shankar, premlata title: interfering antiviral immunity: application, subversion, hope? date: 2006-06-06 journal: trends immunol doi: 10.1016/j.it.2006.05.006 sha: doc_id: 314560 cord_uid: rswa5zdn rna interference (rnai), initially recognized as a natural antiviral mechanism in plants, has rapidly emerged as an invaluable tool to suppress gene expression in a sequence-specific manner in all organisms, including mammals. its potential to inhibit the replication of a variety of viruses has been demonstrated in vitro and in vivo in mouse and monkey models. these results have generated profound interest in the use of this technology as a potential treatment strategy for viral infections for which vaccines and drugs are unavailable or inadequate. in this review, we discuss the progress made within the past 2–3 years towards harnessing the potential of rnai for clinical application in viral infections and the hurdles that have yet to be overcome. conventionally, only specialized cells of the immune system and their secreted products are thought to be involved in protecting the body from foreign invaders such as viruses. however, in recent years, a new type of genomic immunity mediated by rna interference (rnai) has emerged and has sparked intense interest as a potential treatment strategy for a variety of diseases, including viral infections, cancer and degenerative diseases [1] [2] [3] [4] . rnai was first recognized as a naturally occurring anti-viral defense mechanism in plants. in rnai, long double-stranded (ds) rna generated during viral infection is cleaved by an enzyme termed dicer into short, 21-23 nucleotide (nt) dsrna molecules termed small interfering (si)rnas that mediate sequence-specific gene silencing [5, 6] . the sirna associates with a protein complex called the rna-induced silencing complex (risc) (figure 1 ), following which the sense strand is cleaved by the enzyme argonaute 2 (ago2) [7] . the antisense strand then guides the risc to the corresponding messenger (m)rna by sequence homology, and the ago2 nuclease cuts the mrna, resulting in specific gene silencing. in the context of rna viruses, sirnas can be designed to degrade not only viral mrna but also the negative sense viral genomic rna and the complimentary rna that serves as a template for new genomic rna synthesis [8] . although rnai is a natural phenomenon in plants and worms, long dsrna induces an interferon response in mammalian cells resulting in non-specific global suppression of protein synthesis and cell death [9] . a landmark development in the field occurred with the discovery that the introduction of 21-nt-long synthetic rna resembling the dicer-processed sirna into mammalian cells induces sequence-specific gene silencing without evoking the interferon response [10] . since then, rnai has been widely used as a quick reversegenetics approach for gene-function analysis and for ablating specific genes for therapeutic purposes. the exquisite sequence specificity and high potency of rnai makes it an attractive gene-silencing approach [6] . rnai was found to be 1000-fold more effective on a molar basis than antisense oligonucleotides [11] . this is probably owing to the autocatalytic effect of rnai whereby a single sirna molecule is reused for cleaving many target mrna molecules [10, 12] . rnai can be induced by the introduction of synthetic sirna or by intracellular generation from vector-driven expression of precursor short hairpin (sh) rnas (box 1). the optimal design of sirna or shrna is required for the potent induction of rnai (box 2). although rnai is an integral component of the innate immune response to viruses in plants, whether the same is true in mammals is unclear. however, the recently described virus-encoded counter-defense strategies, such as suppressors of rnai and micrornas, suggest a longstanding interaction of viruses with the rnai machinery (box 3). given the natural antiviral role of rnai in plants and its induction in mammals by introduced sirna, the phenomenon has generated great enthusiasm as a potential antiviral treatment strategy [1, 2] . several viruses with widely differing replication cycles have been inhibited in vitro by targeting viral and cellular genes involved in the viral life cycle ( figure 2 ). these include: the positive-stranded rna viruses, including polio, west nile, dengue and foot and mouth disease (fmd) viruses; the negative-stranded rna viruses, including respiratory syncytial virus (rsv) and influenza virus; the doublestranded rna rotavirus; hiv lentivirus; and dna viruses such as the polyoma virus, papilloma virus and herpes simplex virus (hsv). however, the replicative characteristics of certain viruses can protect them from rnai. for example, although sirnas can inhibit progeny virus production, the genomic rnas of rsv, hepatitis delta virus and rotavirus are resistant to rnai owing to tight figure 1 . rna interference. rna interference can be initiated in cells by the introduction of synthetic double stranded sirna or plasmid or viral vectors encoding shrna. the shrna is transcribed in the nucleus and exported to the cytoplasm, where it is processed into sirna by dicer or, possibly, another ribonuclease. in the cytoplasm, the sirna associates with the risc complex consisting of several proteins, which in human cells include dicer, argonaute 2 (ago-2), hiv-1 transactivating response rna-binding protein (trbp), protein activator of protein kinase r (pact) and, possibly, other proteins unidentified to date [50] . the sense (passenger) strand of the sirna is then cleaved by ago-2 within the active risc [7] . the passenger strand can also be removed, albeit at a slower rate, by a cleavage-independent 'bypass' mechanism used for microrna processing [7] . because the exact sequence of the molecular interactions involved in risc activation is unknown, the process is shown in a dotted box and the sirna guide strand is shown as curved to indicate its directional loading into the risc. the anti-sense (guide) strand associated with the mature risc guides the complex to the corresponding mrna because of sequence homology, and the same ago-2 nuclease then cuts the target mrna at a position corresponding to nt 10-11 from the 5 0 end of the anti-sense guide strand. the cleaved mrna is rapidly degraded resulting in gene silencing. rnai can be induced by synthetic sirna or by vector-driven expression of shrna. in the second method, sirna sequence followed by a w9nt loop and a reverse complement of the sirna sequence is cloned in dna or viral vectors to express endogenously the shrna, which is processed in the cytoplasm to sirna. whereas synthetic sirna is introduced into cells by transfection, shrna can be introduced by transfection of a dna vector or transduction through viral vectors. non-replicating, recombinant viral vectors are commonly used for shrna expression because of ease of delivery, particularly in difficult-to-transfect primary cells. adenoviruses and adeno-associated viruses have been used as vectors but lentiviral vectors are generally preferred because they infect actively dividing, and resting and differentiated cells such as stem cells, macrophages and neurons. because the viral dna is incorporated in the host genome, the main advantage of this method is the long-term expression of shrnas and gene silencing. in fact, knockdown persisted for at least six months in the mouse brain following transduction with a shrna-expressing vector [51] . although viral vectors deliver shrna efficiently, they have several disadvantages, including the vector-induced immune response and possible toxic effects of long-term rnai induction. moreover, retroviral integration into the host genome also enhances the risk of insertional mutagenesis, exemplified by the development of leukemia in patients undergoing retroviral-based gene therapy for severe combined immunodeficiency [49] . by contrast, synthetic sirna, similarly to drug treatment, provides a way to achieve transient gene silencing without the risks of insertional mutagenesis, immune response induction or the toxic effects of longterm rnai induction. the major challenge is its delivery to cells in vivo. also, because sirna becomes diluted by cell division, the silencing effect generally fades after 4-5 days in dividing cells. however, in non-dividing cells such as macrophages and neurons, sirna silencing has been observed for at least 3 weeks [38, 52] . thus, whereas sirna seems to be ideal for situations such as acute viral infection, shrna could be useful for treating chronic viral infections and cancer. shrna might also be useful to generate cells resistant to infection by transducing stem cells with shrnaencoding vectors. review trends in immunology vol. 27 no.7 july 2006 shielding by proteins or sequestration in membranous compartments [13, 14] . despite success in vitro, many hurdles need to be overcome before using rnai to counteract virus infections in vivo. one major bottleneck is the delivery of sirnas and shrnas to appropriate cell types in vivo but significant progress has been made in the past 2-3 years. another limitation is the lack of appropriate animal models for many human viruses. nevertheless, studies in mice have proved invaluable for testing the in vivo efficacy for some viruses. data obtained from these initial investigations provide a glimpse of the successes and challenges that can be expected in a clinical setting for specific viruses in terms of rnai design and delivery. in the next subsections, we describe some of the viral diseases for which substantial progress has been made in moving rnai towards therapy. although mice are not susceptible to hepatitis viruses, transfection with plasmids containing the viral genome recapitulates many steps in the viral life cycle, including dna replication and expression of the core and surface antigens. in one of the first demonstrations of rnai effectiveness in vivo, mccaffrey et al. injected the pthbv2 plasmid encoding the hepatitis b virus (hbv) genome alone or with a plasmid encoding anti-hbv shrnas by hydrodynamic intravenous injection (dna was rapidly injected intravenously in 10-15 seconds in a large volume of o1 ml) [15] . this resulted in a substantial knockdown of hbv transcription in the liver and also resulted in o90% reduction in serum hepatitis b surface antigen (hbsag) levels and viral core antigen expression by liver cells. hydrodynamic injection is not feasible in humans because it entails the injection of almost the whole blood volume. however, morrissey et al. showed that regular low-volume intravenous injection of a chemically modified sirna (containing a phosphorothioate backbone and 2 0fluoro and 2 0 -o-methyl substitutions to render the sirna nuclease-resistant) can also significantly reduce serum dna and hbsag levels in mice [16] . chemically modified sirnas have also been encapsulated in lipid nanoparticles for intravenous delivery [17] . this resulted in extending the serum half-life of sirna from w2 min to 6.5 h and reduced the sirna amounts required for a comparable reduction in viral dna and serum hbsag from 30 box 2. recent advances in sirna and shrna design until recently, the design of sirna was based on selecting a 19-bp unique sequence with w45-55% g:c content immediately following the nucleotides aa or na (n represents any nucleotide) within the target gene, and incorporating a 5 0 phosphate and 2-nt 3 0 overhangs [10] . the chances of making successful hits in this essentially trial-anderror process required testing multiple target sites for each gene. however, based on analyses of the biochemical properties of a large number of highly effective sirnas, more-stringent algorithms have now been developed for predicting functional sirnas [6] . the thermodynamic stability at the 5 0 end of the antisense strand has emerged as a crucial criterion for sirna effectiveness [53, 54] . a low internal stability at the 5 0 end enables proper directional loading of sirna to ensure stable incorporation of the antisense instead of the ineffective sense strand into the risc. this increases the potency of sirna almost a 100-fold, and, by reducing the amounts of sirna needed for silencing, it also minimizes off-target silencing. lower thermostability can also be ensured by incorporating mismatches in the sense strand at nucleotides complementary to positions 2-4 of the 5 0 end in the antisense strand. because the antisense strand is still unaltered in sequence, target specificity is not compromised. alternatively, guanosines can be replaced with inosines in the first 4 positions to give i:c base pairs that are similar in energy to a:u base pairs, to increase the propensity of this end to fray. low thermodynamic stability in the mrna cleavage region is also important to promote the release of the risc-antisense complex for multiple rounds of activity. using a 27-29 nt sirna instead of the conventional 21-nt sirna increases the potency by 10-100-fold without inducing an interferon response [55] . this could be because the longer sirna is processed by dicer to generate the optimal sirna endogenously. the pol iii promoters, such as u6, h1 and trna, are commonly used to drive shrna expression because they provide an efficient mechanism to generate small rna transcripts. however, it has recently been shown that inserting the shrna sequence into the backbone of a mirna (e.g. mir-30) at the stem increases the shrna potency enormously, even at the level of single-copy integration [56] . because the pol ii cytomegalovirus (cmv) promoter is used to drive this longer shrna, this system also enables multicistronic expression of multiple shrnas. rnai is used as a natural antiviral defense mechanism in plants, and plant viruses have also developed mechanisms to evade rnai [57] . although it is unknown whether rnai is induced naturally during viral infection in mammals, recent studies suggest that mammalian viruses can also suppress rnai [58, 59] . nodamura virus encodes a protein called b2 that interferes with dicer function and the incorporation of sirna into risc [60] . similarly, the e3l protein of vaccinia virus and the nonstructural protein ns1 of influenza virus can suppress rnai by sequestering dsrna [61, 62] . the nonstructural protein nss of la crosse bunyavirus and hiv-1 tat protein also suppress rnai [63, 64] . in addition to sirnas, another class of small rnas called mirnas also use the rnai pathway [65] . in contrast to sirnas, mirnas are cellular gene products and regulate endogenous gene expression in plants, worms and mammals. in fact, the altered development of t and b lymphocytes has been noted in specific mirna-knockout hematopoietic stem cells and in dicer-knockout mice [66, 67] . recently, viruses have also been found to encode their own mirna and manipulate host mirnas. epstein-barr virus encodes a mirna bart2, which has been predicted to target several cellular genes [68] . similarly, human cytomegalovirus encodes at least five mirnas, and the kaposi's sarcoma-associated herpesvirus expresses 11 mirnas that can target host genes [69] . the exact function of these viral mirnas has yet to be elucidated. however, the sv-40 encoded mirna downregulates sv-40 t antigens on infected tumor cells, making them less susceptible for cytotoxic t-cell recognition, providing the virus a way to evade host immunity [70] . viruses can also use different methods for subverting host mirnas for their own purposes. the liver-specific mirna mir-122 binds to the 5 0 end of the hepatitis c viral genome to greatly augment viral replication, and mir-122 inactivation abolishes viral replication [71] . by contrast, another host mirna, mir-32, targets the retrovirus primate foamy virus-1 genome to repress viral replication, and the viral tas protein suppresses the rnai pathway to overcome mir-32 action [72] . a mirna encoded within the hiv nef gene that can regulate viral transcription has also been documented [73] . review trends in immunology vol. 27 no.7 july 2006 to 3 mg/kg. moreover, the reduction in serum hbv dna could be sustained for 6 weeks simply by weekly sirna treatment, showing the feasibility of using sirna treatment for a chronic disease such as hbv infection. influenza virus (iav) is the major cause of respiratory infections worldwide. two groups have recently used rnai to suppress iav infection in mice. by targeting a conserved sequence in the viral nucleoprotein and acid polymerase genes, tompkins et al. prevented infection with multiple isolates of iav, including the virulent avian h5n1 strain [18] . for delivery to the lungs, they injected sirna by hydrodynamic intravenous injection combined with intranasal administration of sirna complexed with the transfection reagent oligofectamine. similarly, ge et al. achieved and polio), for which a single mrna is used to transcribe viral proteins, targeting any part of the coding sequence should result in degradation (scissors) of viral genomic rna and/or progeny mrna. by contrast, for (b) viruses containing segmented rna genomes for example, rotavirus or influenza, and for (c) dna viruses, for example, herpes simplex or papilloma viruses, for which many different rna molecules are used to generate viral proteins, it is necessary to target genes essential for the viral life cycle, such as the key viral enzymes or structural proteins involved in cell binding and fusion. (d) for retroviruses (e.g. hiv), although viral proteins are transcribed either from unspliced or multiple spliced viral rnas generated from the integrated proviral dna, the genomic rna is linear, thus, as for (a), targeting any part of the genome should suppress viral replication. in addition to viral genes, cellular genes involved in viral replication, such as the cellular receptors or co-receptors (e.g. cd4 and ccr-5 for hiv), can also be used as rnai targets for all viruses. abbreviation: rt, reverse transcriptase. review trends in immunology vol.27 no.7 july 2006 efficient lung delivery following regular intravenous injection of sirna-or shrna-encoding dna vector by complexing with the cationic polymer polyethyleneimine (pei) [19] . this treatment resulted in a 1-2 log reduction in viral titers even when administered 24 h post-infection. moreover, the intranasal administration of shrna vector with the surfactant infasurf r , or of sirna complexed with pei, were also effective, providing an intranasal approach to treat respiratory illnesses. respiratory syncytial virus rsv is another major respiratory pathogen that causes epidemics of respiratory illness with bronchiolitis and pneumonia. two studies have shown that sirnas can be used effectively for prophylaxis and treatment of rsv infection. the viral nonstructural protein ns1 suppresses type i interferon production in host cells, and ns1 deletion mutants are avirulent in vitro and in vivo. transfecting human dendritic cells with a plasmid encoding shrna to suppress rsv ns-1 resulted in the efficient induction of interferon and interferon-induced genes following rsv infection [20] . to test its antiviral effects in vivo, the vector was complexed in a chitosan polymeric nanoparticle. intranasal instillation of the nanoparticle was harmless in mice and substantially reduced viral titers and virusinduced pathology when administered 2 days before infection. importantly, it also reduced lung inflammation and viral titers by nearly 2 logs even when administered 2 days after infection. similar results were also obtained by bitko et al. [21] . in this report, the authors used a synthetic sirna targeting the viral p protein, an essential component of viral rna polymerase. when administered intranasally with the transit r transfection reagent, the sirna substantially suppressed virus replication and lung pathology. this effect was also seen, although to a lesser extent, when the sirna was administered 2-3 days after viral infection. interestingly, the intranasal application of naked sirna without a transfection reagent was also effective (80% activity). similarly, a sirna targeting the p protein of parainfluenza virus was also able to suppress viral replication and virus-induced pathology [21] . an epidemic caused by the recently emerged respiratory viral pathogen sars corona virus (scv) attracted worldwide attention because of the high degree of morbidity and mortality it carried. a rhesus macaque monkey model has been developed for this virus, in which intranasal instillation of the pumc01 strain of scv results in a disease that is similar to the human disease [22] . li et al. used this system to test sirna as a potential therapy for scv [22] . sirnas were used to target the scv genome at the spike protein coding region. because the lipid-based transfection reagents pei and transit tko r used as sirna carriers in previous studies might not be acceptable for human treatment owing to potential toxicity, the authors in this study used infasurf r or 5% d-glucose in water (d5w) for sirna delivery. infasurf r (a naturally occurring lung surfactant protein) and d5w are nontoxic and are currently in clinical use. d5w was 3-4 fold more effective than infasurf r in delivering sirna to the lungs. for testing the efficacy in monkeys, 10 mg/kg of sirna was intranasally instilled in 3 ml of d5w solution. this treatment substantially reduced clinical symptoms, lung pathology and viral burden. although the inhibitory effect was maximal when the sirna was administered 4h before or with viral challenge, disease mitigation was seen even when it was given 24h after viral challenge. thus, this study demonstrated for the first time the considerable antiviral potential of rnai in a non-human primate model using clinically acceptable carriers for sirna delivery. herpes simplex virus sirna has also been used as a potential topical microbiocide [23] . intravaginal application of an anti-greenfluorescent-protein (gfp) sirna mixed with oligofectamine in gfp-transgenic mice resulted in the loss of gfp expression throughout the vagina and cervix but not in distant organs, such as the liver. importantly, the topical application of sirnas targeting the essential hsv-2 genes ul27 (envelope glycoprotein b) and ul29 (a dna-binding protein) before viral infection reduced mortality by 60% and substantially reduced viral shedding from the vagina. combinations of sirnas were also effective when administered 3 and 6 h after infection. this study highlights the feasibility of using a similar approach for other sexually transmitted diseases, including hiv-1. japanese encephalitis (je) and west nile (wn) viruses can cause a devastating neurological illness. two studies have used rnai to suppress these viruses in mouse models. bai et al. injected a sirna targeting the viral envelope gene hydrodynamically 24h before an intraperitoneal wnv challenge and observed a 40% increase in survival [24] . kumar et al. targeted conserved regions in the viral envelope genes of jev and wnv and administered sirna through a lentiviral vector or as synthetic sirna complexed with the cationic lipid jetsi/dope to enable sirna delivery to neuronal cells [25] . a single intracranial treatment with lentiviral vector or synthetic sirna was sufficient to provide almost complete protection from fatality. sirna treatment given 18 h after infection was also effective but the treatment failed at later time points. significantly, by targeting a sequence that is highly conserved in jev and wnv, they achieved near-complete protection against fatal encephalitis induced by either jev or wnv. this offers the possibility of using sirna as a broad-spectrum antiviral agent to suppress related viruses across species. foot and mouth disease fmd is a highly contagious and economically devastating disease of domestic animals. chen et al. used a plasmid vector encoding a shrna targeting the viral vp-1 gene in a suckling mouse model [26] . the subcutaneous injection of 50 mg of plasmid dna 6h before fmd challenge resulted in 75% survival, compared with 100% mortality in control-dna-injected mice. however, dna vector injection at the time of viral challenge or increasing the challenge virus dose reduced the protection considerably. hiv interest in rnai as an alternative antiviral approach has been particularly strong for hiv-1 because of the problems of drug resistance, toxicity and the cost of highly active antiretroviral therapy (haart). several investigators have used rnai to suppress hiv in cell lines and primary cells [27] [28] [29] . however, given the specificity of rnai, the propensity of the virus to mutate can pose a serious challenge for therapeutic use. in vitro studies have documented the generation of rnai-escape mutants in long-term cultures [30, 31] . nevertheless, recent studies suggest that although the homology requirement for rnai is stringent for the crucial central residues, there is some tolerance for peripheral nucleotide changes [32] . moreover, because rnai requires only short stretches of homology, it is possible to target one or more regions which are highly conserved because of their essential structural and functional roles. several of these conserved target sites have been identified [33] [34] [35] [36] . by targeting a highly conserved vif sequence, lee et al. protected cd4 t cells from all hiv clades, including multiple isolates of clade b, which is prevalent in the west [33] . host genes important for hiv replication, including the viral receptor cd4 and the co-receptors ccr5 and cxcr4, have also been targeted to avoid viral escape. synthetic and lentivirally-expressed sirnas have been used to prevent hiv entry by silencing ccr5 [37, 38] . ccr5 is the favored target because a 32 base pair (bp) deletion of the gene is known to be harmless and confers resistance to hiv infection [39] . using a combination of sirnas targeting conserved viral sequences and host genes important in the viral life cycle might be the optimal therapeutic approach, akin to antiretroviral drug cocktails. in another study, rnai has been combined with other gene therapy approaches in a single lentiviral vector. the lentivirus was engineered to encode a shrna targeting hiv-1 rev/tat mrna, a short rna homologous to the viral transactivation response region (tar) to act as a decoy for tat binding, and a ribozyme targeting the host ccr5 gene. the approach might be a pragmatic way for achieving the stable long-term suppression of hiv replication because each of these therapeutic rnas targets a different gene product and blocks hiv replication by a distinct mechanism [40] . a novel approach has also been used for targeted sirna delivery to infected t cells. here, a chimeric recombinant protein consisting of a single-chain antibody to hiv-1 gp120 fused to the highly positively charged protamine was able to bind to sirna (by charge interaction) and deliver it specifically to hiv-infected cd4 c t cells [41] . moreover, when administered with bound anti-tumor sirnas, it specifically targeted and cleared gp120expressing experimental tumors in mice. another approach is to generate hiv-resistant progeny t cells and macrophages by transducing hematopoietic stem cells (hsc). the feasibility of this approach was shown in the severe combined immunodeficient (scid) mouse-human chimeric model by transplanting human cd34 c hematopoietic stem cells transduced with a lentivirus expressing an anti-hiv shrna [42, 43] . rnai has already entered phase i clinical trials for macular degeneration and rsv infection. however, there are several hurdles that must be overcome for routine therapeutic use. a significant limitation for its use in viral infections is the natural sequence differences that exist amongst the various serotypes and strains within a given viral species. sequence divergence can also occur because of the accumulation of mutations during viral replication or the active generation of escape mutants [2] . selecting conserved sequences where the virus is averse to mutate and using a combination of several viral and feasible cellular targets could overcome this limitation. even so, target sequences based on the data available for the few sequenced viruses might not ensure effectiveness against field strains of virus. thus, testing multiple field isolates in vivo might be necessary. although substantial progress has been made, more studies are required to achieve the effective and nontoxic delivery of sirna and shrna in vivo. toxicity can be a problem for in vivo use, particularly when carriers such as transfection reagents are used for delivery. for example, although pei has been used in one study in mice [19] , several reports suggest that it induces toxic effects that might result in the death of the animals [21, 22, 44] . thus, greater emphasis should be given to developing clinically acceptable carriers for systemic delivery. the encasement of sirna within nontoxic nanoparticles or liposomes might enhance stability and simultaneously avoid toxicity. combining a targeted delivery approach, using antibodies or receptor ligands, the introduction of chemical modifications in the sirna, and the encasement of sirna within nanoparticles or liposomes could be the ideal way to improve systemic delivery and reduce sirna requirements. delivery to the brain is still a challenge because of the blood-brain barrier. however, a recent study used transferrin receptor antibody-coated immunoliposomes to overcome this barrier [45] . another issue is the induction of interferon and the associated inflammatory effects. in fact, toll-like receptor (tlr) recognition of rna by plasmacytoid dendritic cells seems to be a major mechanism of interferon induction [46] , and the administration of naked sirna might induce interferon. however, certain motifs within the rna seem to be crucial for interferon induction; avoiding such motifs or encapsulating sirna within nanoparticles could reduce this risk [17, 47] . a better understanding of the immunostimulatory motifs within the rna that are needed for tlr activation might help to avoid non-specific immune activation. however, because most viral infections induce interferon and inflammation, this might not be a major limitation for antiviral therapy. the inadvertent targeting of host genes, observed in some in vitro studies [48] , is another issue that needs more-thorough investigation. although no grossly observable side effects have been reported in vivo, off-target effects might not necessarily manifest as symptoms in animals but could be unacceptable for human therapy. also, because the rnai machinery is used in mammalian cells to regulate cellular gene expression by micrornas (mirnas), the effect of the exogenously introduced sirna on mirna functioning review should be investigated. another concern is the effect of possible subversion of the rnai machinery by viral proteins or virally encoded micrornas (box 3). safety is an also an issue in using shrna delivery through viral vectors because of the possibility of insertional mutagenesis and malignant transformation [49] . another concern is that the effect of long-term sirna induction is unknown. inducible systems for the controlled expression of sirna from plasmid or viral vectors could offer hope in this regard. rnai has tremendous therapeutic potential in viral infections. the major hurdles of delivery to the appropriate cells necessary for its in vivo use as a therapeutic agent are being rapidly overcome and sirna therapy is already being tested in clinical trials. future studies should undoubtedly focus on further refining in vivo delivery methods, and minimizing off-target effects and the emergence of escape mutants in vivo. optimizing sirna design, delivery methods and delivery schedules to achieve sustained sirna levels in the target cells should be emphasized. with these investigations and further advancement in the understanding of the endogenous rnai mechanism, the next few years should prove to be an exciting time to discover whether rnai will become a viable approach to treat viral infections in humans, particularly after the appearance of clinical symptoms. the prospect of silencing disease using rna interference silencing viruses by rna interference rna interference in cancer silencing neurodegenerative disease: bringing rna interference to the clinic mechanisms of gene silencing by double-stranded rna unlocking the potential of the human genome with rna interference passenger-strand cleavage facilitates assembly of sirna into ago2-containing rnai enzyme complexes rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription a comprehensive view of regulation of gene expression by double-stranded rna-mediated cell signaling duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells comparison of antisense oligonucleotides and sirnas in cell culture and in vivo rna silencing: the genome's immune system rotavirus replication: plus-sense templates for double-stranded rna synthesis are made in viroplasms resistance of human hepatitis delta virus rnas to dicer activity rna interference in adult mice activity of stabilized short interfering rna in a mouse model of hepatitis b virus replication potent and persistent in vivo anti-hbv activity of chemically modified sirnas protection against lethal influenza virus challenge by rna interference in vivo inhibition of influenza virus production in virusinfected mice by rna interference inhibition of respiratory syncytial virus infection with intranasal sirna nanoparticles targeting the viral ns1 gene inhibition of respiratory viruses by nasally administered sirna using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque an sirna-based microbicide protects mice from lethal herpes simplex virus 2 infection use of rna interference to prevent lethal murine west nile virus infection a single sirna suppresses fatal encephalitis induced by two different flaviviruses rna interference targeting vp1 inhibits footand-mouth disease virus replication in bhk-21 cells and suckling mice hiv-1-specific rna interference rna interference as an antiviral approach: targeting hiv-1 control of hiv-1 replication by rna interference human immunodeficiency virus type 1 escape from rna interference human immunodeficiency virus type 1 escapes from rna interference-mediated inhibition risc is a 5 0 phosphomonoesterproducing rna endonuclease lentiviral delivery of short hairpin rnas protects cd4 t cells from multiple clades and primary isolates of hiv lentiviral sirnas targeting multiple highly conserved rna sequences of human immunodeficiency virus type 1 inhibition of human immunodeficiency virus type 1 replication by sirna targeted to the highly conserved primer binding site antiviral effects of human immunodeficiency virus type 1-specific small interfering rnas against targets conserved in select neurotropic viral strains inhibiting hiv-1 infection in human t cells by lentiviral-mediated delivery of small interfering rna against ccr5 sustained small interfering rna-mediated human immunodeficiency virus type 1 inhibition in primary macrophages the role of a mutant ccr5 allele in hiv-1 transmission and disease progression long-term inhibition of hiv-1 infection in primary hematopoietic cells by lentiviral vector delivery of a triple combination of anti-hiv shrna, anti-ccr5 ribozyme, and a nucleolar-localizing tar decoy antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors inhibition of hiv-1 by lentiviral vectortransduced sirnas in t lymphocytes differentiated in scid-hu mice and cd34 c progenitor cell-derived macrophages lentiviral vector delivery of sirna and shrna encoding genes into cultured and primary hematopoietic cells purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer intravenous rna interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer plasmacytoid dendritic cells in immunity sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna noise amidst the silence: offtarget effects of sirnas? gene therapy. second child in french trial is found to have leukemia the role of pact in the rna silencing pathway lentiviral vector-mediated delivery of short hairpin rna results in persistent knockdown of gene expression in mouse brain long-lasting rnai activity in mammalian neurons functional sirnas and mirnas exhibit strand bias asymmetry in the assembly of the rnai enzyme complex synthetic dsrna dicer substrates enhance rnai potency and efficacy a lentiviral microrna-based system for single-copy polymerase ii-regulated rna interference in mammalian cells induction and suppression of rna silencing: insights from viral infections micrornas and viral infection interaction of viruses with the mammalian rna interference pathway a virus-encoded inhibitor that blocks rna interference in mammalian cells induction and suppression of rna silencing by an animal virus three distinct suppressors of rna silencing encoded by a 20-kb viral rna genome la crosse virus nonstructural protein nss counteracts the effects of short interfering rna evidence that hiv-1 encodes an sirna and a suppressor of rna silencing silence from within: endogenous sirnas and mirnas micrornas modulate hematopoietic lineage differentiation aberrant t cell differentiation in the absence of dicer identification of virus-encoded micrornas identification of micrornas of the herpesvirus family sv40-encoded micrornas regulate viral gene expression and reduce susceptibility to cytotoxic t cells modulation of hepatitis c virus rna abundance by a liver-specific microrna a cellular microrna mediates antiviral defense in human cells regulation of human immunodeficiency virus 1 transcription by nef microrna this work was supported by nih grant u19 ai 056900 to m.n and p.s. key: cord-324056-cvvyf3cb authors: kelley, patrick w. title: global health: governance and policy development date: 2011-06-30 journal: infectious disease clinics of north america doi: 10.1016/j.idc.2011.02.014 sha: doc_id: 324056 cord_uid: cvvyf3cb global health policy is now being influenced by an ever-increasing number of nonstate and non-intergovernmental actors to include influential foundations, multinational corporations, multi-sectoral partnerships, and civil society organizations. this article reviews how globalization is a key driver for the ongoing evolution of global health governance. it describes the massive increases in bilateral and multilateral investments in global health and it highlights the current global and us architecture for performing global health programs. the article closes describing some of the challenges and prospects that characterize global health governance today. recent inspection rates it would cost the fda $3.16 billion and take more than 13 years to inspect the 189,000 foreign facilities involved with registered food production for export to the united states. 10 governance structures for building consensus and for collectively managing public health actions are necessary because the world's inhabitants cannot create societies and economies that are largely self-contained and insulated from outside threats. the world's inhabitants increasingly share the same air, water, exposure to infectious diseases, foods, pharmaceuticals, and health workforce. through climate change, the impact of human activities in a few countries can affect harvests, the epidemiology of infectious diseases, and the potential for global natural disasters. 11 even among countries with strong public health programs, harmonization of standards and processes is important for effective and efficient collective action. today's global health governance structures include a complex web of un agencies, public/private partnerships, donor and recipient governments, foundations, corporations, and civil society organizations. a recent report from the kaiser family foundation identified 50 multilateral international health treaties, commitments, partnerships, and other agreements, 26 of which are legally binding under international law. the united states is a party to 36 of these of agreements, 16 of which are legally binding. these agreements cover many topical areas, including specific diseases, environmental issues, trade and intellectual property, specific populations (eg, refugees or children), health security/preparedness, water, and food/nutrition. 12 in the aftermath of the nineteenth-century international sanitary conferences that focused on threats to ports and trade, the early twentieth century saw international $400,000 1 9 6 1 1 9 6 3 1 9 6 5 1 9 6 7 1 9 6 9 1 9 7 1 1 9 7 3 1 9 7 5 1 9 7 7 1 9 7 9 1 9 8 1 1 9 8 3 1 9 8 5 1 9 8 7 1 9 8 9 1 9 9 1 1 9 9 3 1 9 9 5 1 9 9 7 1 9 9 9 2 0 0 1 2 0 0 3 2 0 0 5 2 0 0 7 global health governance reflected in new institutions, such as the international sanitary bureau (isb) based in washington, dc, and the office internationale d'hygié ne publique in paris. the isb subsequently became the pan american health organization, which since 1948 has been one of 6 regional offices of the who. when the un system was conceived in 1945, brazil and china proposed the who. the who constitution contains several key principles, including: health is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity. the enjoyment of the highest attainable standard of health is one of the fundamental rights of every human being without distinction of race, religion, political belief, economic or social condition. the health of all peoples is fundamental to the attainment of peace and security and is dependent upon the fullest co-operation of individuals and states. 13 the broad mandate of the who has 6 core functions: the provision of collective health leadership, the shaping of research as well as the generation and dissemination of knowledge, the setting of norms and standards and the promotion and monitoring of their implementation, the production of ethical and evidence-based policy options, the provision of technical support and capacity-building, and the monitoring of health situations and trends. 14 the organization has a 6-point agenda: promote development; foster health security; strengthen health systems; harness research, information, and evidence; enhance partnerships; and improve performance. 15 this agenda is shaped at annual meetings in geneva by the 193 member countries that compose the world health assembly. an 8000-person secretariat based in geneva and at the 6 regional and 147 country offices performs who programs. the breadth of the programs undertaken is vast, although not always deep. some of the topics addressed include hiv/aids; malaria; tuberculosis; chronic noncommunicable diseases; mental disorders; violence; traffic safety; visual impairment; maternal and child health; aging; disasters; health equity; environmental health; nutrition and food safety; drug access; research; health systems strengthening; and tobacco, alcohol, and drug abuse, and other unsafe behaviors. when the who was envisioned, the concept of its operations was quite vertical and focused primarily on relationships with member states. few other actors were engaged in global health. since its founding, the who has celebrated major accomplishments but has also suffered from an erosion of its influence. beyond the eradication of smallpox (an event that has likely saved more than 20 million lives since 1977), who technical leadership has also played a role in the near eradication of polio; the containment of the deadly 2003 sars epidemic; the landmark 2003 who framework convention on tobacco control (the first treaty negotiated under the auspices of who); and the 2005 revision of the international health regulations (ihr). compared with the previous ihr that was focused on classical diseases of international importance, plague, yellow fever, and cholera, the 2005 revision is more powerful and practical in that it encompasses all acute public health risks of transnational significance. the new ihr defines broad reporting requirements and establishes the authority of the who to initiate disease-control actions, if necessary, through use of information sources other than country governments. the new ihr also requires countries to strengthen their existing surveillance capacities. many have hoped that the who would take a stronger role in the setting and enforcement of norms, given its extraordinary constitutional authorities and its quasi-legislative power to adopt binding regulations. the who has, however, leaned more toward providing technical advice than toward exerting bold, proactive leadership through binding norms and regulations. 16 kelley given its mandate, the who is handicapped by a modest budget and by its dependent fiscal relationships. the proposed budget for 2010 to 2011 was $4.94 billion before currency adjustments. because only about 18.8% of the who budget comes from the dues assessments of member countries, its flexibility in focusing its program based on the scientific priorities it determines is constrained. wealthier countries pay larger assessments and thus have more influence over the direction of who programming than poorer countries, which have the greater need for help. moreover, the remaining 81.2% of the budget is projected as "voluntary contributions," which are largely earmarked by donors for specific purposes. 17 this arrangement contributes to fragmented programming and perhaps to certain donor-influenced compromises. in this century, the who also faces growing challenges. although its members are sovereign governments, many other powerful actors (including nongovernmental entities, such as other un agencies, foundations, corporations, and civil society organizations) have entered the global-health arena. placating these competing interests is not a recipe for bold action. the who is the lead un agency for health. owing to the increasing recognition that health is fundamental to the broader un goals of fostering the international rule of law, global security, economic development, social progress, human rights, and world peace, health issues have now taken a more prominent place than they had in the united nation's first 50 years. in 2001, world leaders came together at the un headquarters to adopt the 8 millennium development goals (mdgs), 3 of which are focused on health. the leaders of 192 un member states agreed to partner to achieve these goals by 2015. the 3 health-related goals have directly provided high-level, consensus global health policy direction (box 1). another goal is related to hunger reduction. regrettably, the mdgs lack a target for chronic diseases, such as cardiovascular diseases and cancers, which now account for more than 50% of the global burden of disease. since 2000, progress toward the mdg targets has been mixed. for example, between 2003 and 2008, the number of individuals infected with hiv placed on antiretroviral drugs rose from 400,000 to 4 million (ie, 42% of the 8.8 million people in target 6c: have halted and begun to reverse the incidence of malaria and other major diseases by 2015 need). in developing countries, 37% of births in 2008 still took place without a trained birth attendant present, and maternal mortality remains shockingly high. 18 as a result of the hiv/aids pandemic and its immense scale and impact on human life, dignity, rights, social cohesion, and economic development, a un general assembly special session issued a formal declaration of commitment on hiv/aids in june 2001. 19 that declaration endorsed the establishment of the "global aids and health fund," which would pool contributions from countries and from the private sector to fund hiv prevention and treatment efforts. it also set out a policy approach to providing leadership; improving prevention, care, and treatment; preserving human rights; reducing the vulnerability of women and children at risk for or affected by hiv/aids; and mobilizing financial resources. a un special session on noncommunicable diseases and their prevention will convene in september 2011. as illustrated in box 2, the un has spawned a wide array of specialized agencies and other entities with direct or indirect interests in health. to some degree, these compete with the who for legitimacy and resources, and some have a significant role in global health governance. in world trade organization treatment, especially among migrants, women and girls, peacekeepers, travelers, and orphans; it unites the relevant un agencies, civil society organizations, governments, and the private sector; it advocates for rights, resources, and accountability; it empowers actors with strategic information; and it supports country leadership. created by the general assembly in 1946 for post-war relief, unicef is now focused on providing on-the-ground, long-term humanitarian and developmental assistance to children and mothers in developing countries. funded by governments and the private sector, the 2009 unicef expenditures totaled $3.3 billion. 20 based in rome, the food and agriculture organization of the united nations (fao) operates through regional offices and through more than 70 country offices around the world. with a biennial budget in 2008 to 2009 of $929.8 million, the fao leads un efforts to enhance food security and to eliminate hunger, the number-one mdg. the fao provides a neutral forum in which all countries can negotiate agreements and debate policy. its experts disseminate technical information and assist countries with disease outbreaks and in developing sound agriculture policies. where issues of human health and animal health intersect (eg, avian influenza or food safety emergencies), the fao plays a role. although not actually a un agency, the world organization for animal health (previously known as the office international des epizooties and still known as the oie) is also an intergovernmental organization; it seeks to perform global disease control for the animal population. based in paris and having regional and subregional offices worldwide, the oie is an important partner of the who, the fao, and the world trade organization (wto). like the who, it operates under the collective control and authority of an assembly of member countries. oie relevance to global health governance stems from the often-underappreciated connection between human health and animal health. this connection, embodied in the one-health concept, is most evident in the context of emerging zoonotic diseases and food safety. 21 in 2009, an institute of medicine (iom) committee concluded that the oie rules lacked critical provisions found in the 2005 who ihr, provisions that would be valuable for an organization involved in protecting animals capable of transmitting infections to humans. the committee recommended that the oie create legally binding obligations for members to develop and maintain core surveillance-and-response capabilities, that the oie be authorized to publically disseminate the animal-disease information received from nongovernmental sources when the member state does not do so in a timely and accurate way, and that the oie director general be empowered to declare animalhealth emergencies and related recommendations as appropriate. 22 the wto, a member of the un family since 1995, is primarily concerned with the rules of trade between nations. with globalization, however, trade and health are increasingly connected. the wto has several agreements related to health and health policies, including the agreements on technical barriers to trade; the sanitary and phytosanitary measures; the trade-related intellectual property rights (trips); and the trade in services. although trade can be restrained for scientifically valid reasons related to health, relevant interpretations can be controversial. some health issues relevant to wto agreements include food safety and protection from zoonotic diseases; trips patent protection for pharmaceuticals (to balance incentives for drug development vs ensuring affordable access to drugs); tobacco control; biotechnology; and the transnational migrations of health workers, patients, and investment in health services. 23 the world bank group, founded in 1944, is a critical source of financial and technical help for developing countries. headquartered in washington, dc, it employs more than 10,000 people worldwide. the bank provides low-interest loans, interest-free credits, and grants to developing countries for health and other purposes. the world bank consists primarily of 2 unique development institutions owned by 187 member countries: the international bank for reconstruction and development (ibrd) and the international development association (ida). the ibrd serves middle-income and creditworthy poorer countries; whereas, the ida mission is to serve the world's poorest countries. ibrd lending is primarily financed through aaa-rated bonds sold on the world's financial markets; the ida funds come from 40 donor countries and are replenished periodically. additional funds come from repayments of loan principal on longterm, no-interest loans. the ida accounts for more than 40% of world bank lending, and its loans are largely supervised and evaluated by the world bank country offices. ida loans have been used to improve sanitation and water supplies, support immunization programs, and combat the hiv/aids pandemic. in fiscal 2009, the world bank health, nutrition, and population program lent $2.9 billion, a 3-fold increase from 2008. since 1997, the bank group provided $17 billion in country-level project financing and $873 million in private health and pharmaceutical investments. in fiscal 2009, the bank disbursed $290 million for existing hiv projects and committed nearly $326 million to new hiv/aids efforts. it also committed funds in fiscal 2009 for pandemic preparedness. 24 achieving the mdgs will require an estimated $20 billion to $70 billion annually. 25 higher-income countries have made substantial policy commitments to support the lower-income countries that have insufficient resources to provide a basic package of essential health benefits (estimated at $34 per capita per year). funding for action by wealthier countries has often been coordinated through governance mechanisms, including the annual group of eight (g8) summits. g8 progress toward these commitments is summarized in table 1 . in 2002, the un millennium project estimated that to meet the 2015 mdgs, the total overseas development assistance (oda) from high-income countries would need to rise to 0.54% of gross national income (gni). in absolute terms, the us government has contributed great amounts to oda; however, as depicted in fig. 2 , the relative amount contributed in 2009 was only 0.20% of us gni, a figure less generous than the percentage contributed by most european countries, australia, and canada. the us government's global health program, compared with that of many other donor governments, cuts across many departments. it draws upon both the foreign-assistance structure but also the government's extensive public health capabilities. in addition to well-known actors, such as the us agency for international development and the us centers for disease control and prevention, executive branch agencies with a significant involvement in global health include the departments of state, defense, agriculture, homeland security, labor, and commerce, as well as the national institutes of health, the food and drug administration, the environmental protection agency, the peace corps, and the health resources and services administration. these agencies carry out programs in more than 100 countries and are overseen by more than 15 congressional committees. 26 the us financial commitment to global health has dramatically increased over the last decade, especially with the implementation of the president's emergency plan for aids relief (pepfar) and the president's malaria initiative (fig. 3) . originally authorized in 2003 at about $15 billion, pepfar focused on 15 countries, mostly in africa. over its first 5 years, pepfar sought to support antiretroviral treatment for 2 million people infected with hiv; prevent 7 million hiv infections; and provide care, including care for orphans and vulnerable children, for 10 million individuals. the program has been viewed as a major success for us global health policy. 27 with wide bipartisan support, pepfar was reauthorized in 2008 for another 5 years at $39 billion, with substantial increases in the prevention, treatment, and care targets. the legislation also authorized $4 billion for tuberculosis programming and $5 billion for malaria efforts. 28 although pepfar represents a landmark us achievement in global health, it also illustrates how policy can be skewed by nonscientific factors. fig. 4 shows that among i r e l a n d f i n l a n d u n i t e d k i n g d o m s w i t z e r l a n d s p a i n f r a n c e g e r m a n y a u s t r i a c a n a d a a u s t r a l i a n e w z e a l a n d p o r t u g a l u n i t e d s t a t e s g r e e c e j a p a n i t a l y health called for a rebalancing of this portfolio to better support initiatives against noncommunicable diseases, malnutrition, and injuries. 29 the signature global health program of the obama administration, the global health initiative (ghi), aims to provide $63 billion in health assistance between 2009 and 2014. although hiv/aids still dominates the ghi, increased investments are being made for neglected tropical diseases, malaria, maternal and child health, and family planning (box 3). the aims of the ghi are to implement a woman-centered and girlcentered approach; increase impact and efficiency through strategic coordination and integration; strengthen and leverage key partnerships, multilateral organizations, and private contributions; encourage country ownership and investing in country-led plans; improve metrics, monitoring, and evaluation; and promote research and innovation. 30 the us government's policy commitment to global health has been greatly lauded as a reflection of our vital health and economic self-interests, our humanitarian values, and "smart power." [31] [32] [33] in many ways, it is an important form of diplomacy; however, some aspects of it may also hinder diplomatic prerogatives. some forms of oda can be used as leverage in diplomatic negotiations, but once a patient infected with hiv is taken into a pepfar-funded treatment program, it would be unethical for the us government to threaten the loss of those lifesaving drugs for diplomatic advantage. 34 beyond un agencies and national governments, the global health enterprise is now a much more horizontal and networked endeavor than it was even 15 years ago. although this makes it harder for the who to lead, it also brings a wider array of talent and resources to bear on problems. fig. 5 illustrates an example of the partnership paradigm of global health governance today: the roll back malaria (rbm) partnership, a 500-member collaboration. even though the who was one of the founders of rbm and hosts the secretariat, it is not positioned as the dominant core of the effort. the rbm partnership not only works to facilitate policy coordination at the global level but also executes an operating framework with performance targets reflective of the mdg malaria goal; it articulates technical strategies and provides multidirectional accountability through its partners forum. a decision-making partnership board with 27 members (6 ex-officio) represents the 7 broad rbm constituencies and meets periodically. there are 21 voting members of the board representing foundations (1 seat), malaria-endemic countries (8 seats), multilateral institutions (4 seats), ngos (2 seats), organization for economic cooperation and development (oecd) donor countries (3 seats), private sector (2 seats), and research and academia (1 seat). the stop tb partnership (http://www.stoptb.org/) also has many similarities to rbm. founded in 2000, the global alliance for vaccines and immunizations (gavi) is another innovative partnership that links developing-world governments and donor governments; philanthropic foundations; the financial community; vaccine manufacturers from developed and developing countries; research and technical institutes; civil society organizations; and intergovernmental entities, such as who, unicef, and the world bank. immunizations funded by gavi have prevented an estimated 3.4 million deaths in developing countries. gavi also supports innovative financing box 3 the goals and targets of the us government global health initiative hiv/aids: the us president's emergency plan for aids relief will: (1) support the prevention of more than 12 million new hiv infections; (2) provide direct support for more than 4 million people on treatment; and (3) support care for more than 12 million people, including 5 million orphans and vulnerable children. malaria: the president's malaria initiative will reduce the burden of malaria by 50% for 450 million people, representing 70% of the at-risk population in africa, and expand malaria efforts into nigeria and the democratic republic of the congo. tuberculosis (tb): save approximately 1.3 million lives by reducing tb prevalence by 50%, which will involve treating 2.6 million new tb cases and 57,200 multidrug-resistant cases of tb. maternal health: save approximately 360,000 women's lives by reducing maternal mortality by 30% across assisted countries. child health: save approximately 3 million children's lives, including 1.5 million newborns, by reducing under-5 mortality rates by 35% across assisted countries. nutrition: reduce child undernutrition by 30% across assisted food-insecure countries, in conjunction with the president's feed the future initiative. family planning and reproductive health: prevent 54 million unintended pregnancies by meeting unmet need for modern contraception. contraceptive prevalence is expected to rise to 35% across assisted countries, reflecting an average annual increase of 2 percentage points. first births by women younger than 18 years should decline to 20%. neglected tropical diseases: reduce the prevalence of 7 neglected tropical diseases by 50% among 70% of the affected population, and eliminate onchocerciasis in latin america by 2016, lymphatic filariasis globally by 2017, and leprosy. data from available at: http://www.usaid.gov/ghi/factsheet.html. mechanisms, such as advanced market commitments to reduce the costs of immunizations needed by developing countries. the 2001 un declaration of commitment on hiv/aids called for the establishment of a global fund of pooled contributions to support hiv/aids needs. this global fund to fight aids, tuberculosis, and malaria (gfatm), unlike the previously mentioned partnerships, has as its sole focus mobilizing and distributing financial resources in a manner driven by technically sound national plans and priorities and principles of transparency and accountability. the gfatm is a partnership between governments, civil society, the private sector, and affected communities. more than 40 countries have pledged funds to the gfatm; other major donors include the bill and melinda gates foundation, unitaid, and chevron. about $21 billion has been pledged, of which more than $16 billion has already been paid. 35 with funding approved for more than 500 programs in nearly 150 countries, the gfatm is the source of onequarter of all international financing for aids globally as well as for two-thirds of that for tuberculosis and three-quarters of that for malaria. 36 an innovative fiscal contribution to solving the crisis of antimalarial drug resistance and the dwindling number of effective drugs has been the creation of the affordable medicines facility for malaria (amfm). conceived by the iom committee on the economics of antimalarial drugs, the amfm was designed to re-engineer market forces to favor the effective treatment of resistant malaria through the appropriate use of artemisinin-containing combination antimalarial drugs (acts). in 2004 the committee recommended the commitment of $300 to $500 million per year to subsidize the entire global act market to create a steady supply of affordable and desirably priced acts in all malarious areas. 37 several donors accepted this recommendation. the amfm was established by the gfatm and initially capitalized with more than $146 million. 35 although the greatest sources of funding, technical support, and leadership will continue to come from donor governments, recipient governments, and un agencies, contributions from the world of philanthropy have never been more significant. early in the twentieth century, rockefeller philanthropy supported efforts to eliminate hookworm, first domestically and then internationally. it has since taken on many other disease-control efforts directed toward conditions, including malaria, schistosomiasis, yellow fever, and vaccine-preventable childhood infections. in 1914, it created the china medical board to develop modern medicine in that country but arguably its most significant contributions to advancing global health governance were investments to establish the johns hopkins school of hygiene and public health as well as schools of public health at harvard and the university of michigan. reflecting the emergence of the new era in global health governance, in 1998 the rockefeller foundation established an initiative to create innovative new public-private partnerships, including the medicines for malaria venture, the global alliance for tb drug development, and the international partnership on microbicides. 38 over the years, many other foundations have made important contributions to facilitating global health action, including the ford foundation, atlantic philanthropies, the carnegie corporation, the bloomberg family foundation, the burroughs wellcome trust, the burroughs wellcome foundation, and the doris duke charitable foundation. the most noteworthy newcomer is the bill and melinda gates foundation. with assets of approximately $33 billion, the gates foundation is the largest private philanthropy in the world. its current annual disbursements are approximately $3 billion, much of which goes to global health. among foundations, its major commitments to gavi (at least $1.5 billion), the rotary international polio-eradication effort ($355 million), and the gfatm ($650 million) give it a uniquely powerful and sometimes controversial voice in global health governance. its investments in research, implementation, and advocacy encompass enteric and diarrheal diseases; hiv/aids; malaria, pneumonia, tuberculosis, and neglected and other infectious diseases; family planning; nutrition; maternal, neonatal and child health; tobacco control; and vaccine-preventable diseases. private funding now accounts for almost one-quarter of global health aid. 39 unlike the foundations previously mentioned, the william j. clinton foundation is not a grant-making organization. for nearly a decade, however, it has been a unique contributor to advancing global health. by capitalizing on the influence of former president bill clinton, this foundation has catalyzed many initiatives. among these initiatives have been tremendous gains in access to hiv/aids treatment through negotiations with suppliers of drugs and diagnostic tests. through successive agreements, suppliers to low-income countries have reduced the prices of first-line treatments by 50%, pediatric medicines by 90%, and second-line hiv/aids medicines by a cumulative reduction of 30%. 40 over the last decade, the corporate sector has also been making an increasing mark on global health. although corporate initiatives are too numerous to catalog in detail, their donations of drugs are especially noteworthy. since 1987, for example, merck has donated ivermectin for the control of onchocerciasis (river blindness) worldwide. in 1998, in partnership with glaxosmithkline, this commitment was expanded to include the elimination of lymphatic filariasis; ivermectin and glaxosmithkline's albendazole were coadministered in african countries and in yemen (countries where lymphatic filariasis and onchocerciasis are coendemic). over 21 years, more than 1 billion treatments for these infections have been provided though a large partnership. 41 johnson and johnson donates enough mebendazole each year to treat 25 million children for intestinal helminthes; boehringer ingelheim donates nevirapine to prevent mother-to-child transmission of hiv. pfizer has proved to be a valuable and innovative partner with its support for capacity-building activities, such as the pfizer global health fellows program. each year, this program deploys up to 50 talented employees to work on high-impact, capacity-building projects in developing countries. 42 similarly, bd strengthens capacity through a partnership with pepfar to improve laboratory systems in countries highly affected by hiv/aids and tb. 43 the emerging corporate role in global health is not limited to companies focused on the business of health. companies as diverse as exxonmobil, warner brothers, and nike have engaged in important partnerships focused on controlling malaria, hiv/ aids, and violence against girls. american cyanamid has donated millions of dollars of the larvicide temephos to support guinea worm eradication efforts. formal business coalitions have developed to take on issues of malaria, tuberculosis, and hiv/aids. 44 the role of civil society organizations in global health predates all of those entities previously named. the who lists 189 ngos with which it has an official relationship. 45 as the who notes: no longer the domain of medical specialists, health work now involves politicians, economists, lawyers, communicators, social scientists and ordinary people everywhere. the involvement of civil society has profoundly affected not only the concepts underpinning public health but the formulation and implementation of public health programs and policies as well. 46 global health: governance and policy development civil society organizations span a wide array of secular and faith-based entities. they include groups with a disease-specific orientation; groups with a professionalspecialty focus; charities that work on the ground; and global professional service organizations, such as rotary international. (rotary international is a key global partner in the who campaign for polio eradication. 47 ) perhaps the most exciting recent development in the united states has been the explosion in interest in global health education at universities. suffice it to say that if governance is the constellation of mechanisms a society uses to effect collective action toward common goals, then the catalyst of the many new us multidisciplinary university programs in global health education will initiate and energize an unprecedented level of collective action. 48 despite the vast inflow of resources for global health, the remaining policy challenges are significant. perhaps today's most acute global health challenge is achieving the 3 health-related mdgs. current trends indicate that none of these basic targets will be near achievement by 2015. overall access to care is still lacking or suboptimal for billions of people. access to clean water and essential medicines is uneven. modern pharmaceuticals are often unaffordable or unavailable. globalization has brought some health benefits to the world's poorest, but it has also fostered the transnational spread of infectious diseases, the brain drain of skilled health care workers from developing countries, and the trade in poor-quality food and pharmaceuticals. surveillance for human and animal diseases is of variable quality and the enforcement of the relevant regulatory regimes needs improvement. despite the challenges that remain in coordinating the many diverse players now engaged in global health, the vast increase in the commitment of both private and public wealth over the last decade is to be celebrated. commitments have gone far beyond money and have brought forth legions of individuals who choose to commit themselves in the global context to the universal value of health. research is steadily discovering and developing new technological interventions. new mechanisms of cooperation have been developed, and there is a growing interest in implementation science and in program evaluation to increase accountability and effectiveness. improved global health governance to better catalyze and coordinate collective action remains an essential underpinning to meeting the diverse challenges to saving lives in all parts of the globe. global governance in health -do historical experiences of industrialized countries teach any lessons? the emergence of new virus diseases: an overview learning from sars: preparing for the next disease outbreak (workshop summary) the domestic and international impacts of the 2009-h1n1 influenza a pandemic: global challenges, global solutions (workshop summary) infectious disease movement in a borderless world (workshop summary) convention on the prohibition of the development, production and stockpiling of bacteriological (biological) and toxin weapons and on their destruction top 15 u.s. agricultural import sources drug safety: preliminary findings suggest recent fda initiatives have potential, but do not fully address weaknesses in its foreign drug inspection program (testimony before the subcommittee on oversight and investigations, committee on energy and commerce, us house of representatives). washington, dc. the united states government accountability office global climate change and extreme weather events: understanding the contributions to infectious disease emergence (workshop summary) global health policy: u.s. participation in international health treaties, commitments, partnerships, and other agreements constitution of the world health organization the role of the who in public health global health governance report (commissioned paper). the u.s. commitment to global health: recommendations for the public and private sectors draft proposed programme budget united nations united nations general assembly special session on hiv the convention on the rights of the child. new york: unicef one health initiative task force: final report. one health -a new professional imperative sustaining global surveillance and response to emerging zoonotic diseases world trade organization/world health organization the world bank annual report 2009-year in review. the international bank for reconstruction and development/the world bank millennium development goals for health: what will it take to accelerate progress? global health policy: the u.s. government's global health policy architecture: structure, programs, and funding committee for the evaluation of the president's emergency plan for aids relief (pepfar) implementation. pepfar implementation: progress and promise. institute of medicine public law 110-293. united states global leadership against hiv/aids, tuberculosis, and malaria reauthorization act of the institute of medicine committee on the us commitment to global health. the u.s. commitment to global health: recommendations for the public and private sectors report of the csis commission on smart global health policy: a healthier, safer, and more prosperous world america's vital interest in global health: protecting our people, enhancing our economy, and advancing our international interests the institute of medicine committee on the u.s. commitment to global health. the u.s. commitment to global health: recommendations for the public and private sectors no good deed goes unpunished: the unintended consequences of washington's hiv/aids programs the global fund to fight aids, tuberculosis, and malaria -pledges about the global fund saving lives: buying time: economics of malaria drugs in an age of resistance rockefeller foundation-our history governing global health global health fellows: pfizer investments in health. available at: https:// globalhealthfellows.pfizer.com/login.asp?returnurl5home.asp. accessed september 12 bd's global health initiative global business coalition on hiv/aids, tuberculosis and malaria world health organization. list of ngos in official relations with who world health organization. civil society initiative (csi) rotary international/the rotary foundation the dramatic expansion of university engagement in global health: implications for us policy: a report of the csis global health policy center key: cord-341097-c96hm610 authors: mayer, craig s.; williams, nick; huser, vojtech title: analysis of data dictionary formats of hiv clinical trials date: 2020-10-05 journal: plos one doi: 10.1371/journal.pone.0240047 sha: doc_id: 341097 cord_uid: c96hm610 background: efforts to define research common data elements try to harmonize data collection across clinical studies. objective: our goal was to analyze the quality and usability of data dictionaries of hiv studies. methods: for the clinical domain of hiv, we searched data sharing platforms and acquired a set of 18 hiv related studies from which we analyzed 26 328 data elements. we identified existing standards for creating a data dictionary and reviewed their use. to facilitate aggregation across studies, we defined three types of data dictionary (data element, forms, and permissible values) and created a simple information model for each type. results: an average study had 427 data elements (ranging from 46 elements to 9 945 elements). in terms of data type, 48.6% of data elements were string, 47.8% were numeric, 3.0% were date and 0.6% were date-time. no study in our sample explicitly declared a data element as a categorical variable and rather considered them either strings or numeric. only for 61% of studies were we able to obtain permissible values. the majority of studies used csv files to share a data dictionary while 22% of the studies used a non-computable, pdf format. all studies grouped their data elements. the average number of groups or forms per study was 24 (ranging between 2 and 124 groups/forms). an accurate and well formatted data dictionary facilitates error-free secondary analysis and can help with data de-identification. conclusion: we saw features of data dictionaries that made them difficult to use and understand. this included multiple data dictionary files or non-machine-readable documents, data elements included in data but not in the dictionary or missing data types or descriptions. building on experience with aggregating data elements across a large set of studies, we created a set of recommendations (called consider statement) that can guide optimal data sharing of future studies. in recent years, efforts to define research common data elements (cdes) attempt to harmonize data collection across clinical studies [1] . sheehan at al. defined a cde as 'a combination of a precisely defined question paired with a specified set of responses to the question that is common to multiple datasets or used across different studies' [2] . cdes have been defined on both a general level applicable to a broad range of diseases and studies, as well as on a disease specific level that focuses on data elements applicable to a narrow context. an example of general data elements are those defined by phenx initiative, such as employment status, education attainment, or health insurance coverage (all with appropriate permissible values for such elements). an example of disease specific cdes are those defined by the therapeutic area user guide for hiv [3] published by the clinical data interchange standard consortium (cdisc). cdes are expected to deliver the following benefits: faster and cheaper study start-up, improved comparability and aggregation of data across studies, improved study data collection and study data quality, and improved data organization for re-use. another phenomenon that highlights the importance of cdes is the requirement to share de-identified individual participant data (ipd) of a completed observational study or interventional trial [4] . when data is shared, a data dictionary is typically provided to describe individual data elements used in a study. in recent decades, tens of new data sharing platforms with the aim of facilitating secondary research have emerged [5] . identification and standardization of cdes is, however, an ongoing challenge in the field of clinical research informatics [6] . some leaders have argued strongly for adoption of policies that require much stronger sponsor-enforced standardization [2, 7] , while others point to possible restrictions and additional cde burden on principal investigators of future studies [8, 9] . increased pressure for cde adoption can be seen in research efforts triggered by the covid-19 pandemic [10, 11] . many cde initiatives use expert consensus to achieve standardization. we refer to this method as a top down approach. if the number of data elements to standardize is large, expert consensus can be very time consuming. with increased availability of de-identified ipd data from completed studies, it is possible to arrive at cdes using a data-driven approach. most common data elements will simply appear in a high number of shared study datasets if a simple usage frequency approach is used. we refer to this method as a bottom up approach. a data-driven approach can also handle a large volume of common data elements. however, this data-driven approach depends on studies sharing their data elements in an analyzable format. these two general clinical research trends (effort to arrive at standardized cdes and increased sharing of completed studies) have also impacted the clinical domain of infectious diseases and hiv. for example, at clinicaltrials.gov, the number of studies that provide a link to de-identified ipd increased from 4.4% in 2014 (3 hiv studies have link to ipd out of 68 total hiv studies that completed in 2014) to 12.6% in 2019 (8 hiv studies have link to ipd out of 63 total hiv studies that completed in 2019). such numbers demonstrate a gradual shift towards increased data sharing. despite improvements, there are still opportunities for further enhancements in terms of format and the extent of data sharing in the hiv domain as well in clinical research in general). we present a study to analyze the quality and usability of data dictionaries of hiv studies. we focused on data dictionaries because they are an important common metadata artifact for data sharing. we thus search for hiv/aids interventional trials or observational studies using several approaches. in our article, we use the term study to refer to both interventional trials and observational studies. our goal is to find hiv studies that provide a data dictionary with a list of data elements used in a study and relevant additional metadata. we analyze the format and content of those data dictionaries. the study contribution lies in the generating of recommendations that improve data sharing of future hiv studies. our study is the first informatics study of data sharing format that analyzes a large body of hiv studies shared to date via various data sharing mechanisms. the presented study is limited to data dictionary analysis, although the motivation is to later analyze a large body of past hiv data elements to inform data-driven consensus on cdes. this study is part of a larger research project titled 'identification of research common data elements in hiv/aids using data science methods' [12] . in this section, we outline how we identified a set of analyzed hiv studies and define important terms for the analysis of data dictionaries. we also describe relevant standards for study data dictionaries. we searched clinical study data sharing platforms [5] for hiv studies that shared ipd. we defined hiv studies as any study relating to hiv, which includes any study with hiv positive participants, or any study relating to hiv infection (i.e. hiv prevention or vaccine trials). a data sharing platform is a web-based repository of completed clinical studies with study ipd and other study artifacts (such as protocol, study publications or case report forms) for each included study. the platforms we searched included national institute of drug abuse (nida) data share [13] , the national heart, lung and blood institute (nhlbi) biolincc, the national institute of child health and human development data and specimen hub (nichd dash), vivli, clinicalstudydatarequest.com, project data sphere, the national institute of mental health data archive (nda) and the yale open data access project. we also searched for hiv clinical trial networks using a web search engine and requested ipd from completed studies conducted by those networks. we excluded studies for which a data dictionary could not be obtained or inferred from ipd or studies that were not registered at clinical-trials.gov. in order to aggregate this data across many hiv studies, we obtained institutional review board approval at our local institution (or exemption if the data were de-identified). we filed appropriate data requests at the platforms where we found relevant hiv studies. for each hiv study included in our analysis, we organized the available data artifacts into the following categories: ipd data files, data dictionary files, study protocol documents and study de-identification notes. within the data dictionary category, we observed several different formats. some studies used single or multiple comma separated value files (csv), a widely accessible and machine-readable file format. some studies used portable document format (pdf) or a proprietary sas data catalog format ( � .sas7bcat) [14] . because we used computerized methods for aggregation, we did not include studies whose data dictionaries were not in a machine-readable format or could not be readily converted into such a format. this includes pdfs with scanned content. in our analysis of data dictionaries and data elements, we adopt the definitions developed by nih cde task force of the nih data council [15] . they clearly defined terms such as data element, common data element, form and permissible value [16] . in addition to using terminology defined by the nih cde task force, we have introduced three types of data dictionaries. we use the term element data dictionary (or element dictionary in shorter form) to describe a spreadsheet that enumerates individual data elements used in a study with fields such as data element name and description. we combined all data element data dictionaries to create an aggregate data elements file that contains data elements from a set of studies. due to the large number of data elements and to limit the project scope, we did not perform any semantic mapping of identical des. this aggregated file targeted the following attributes about each data element (referred to as target de model): (1) data element description, (2) data element id (sometimes called de name, de short label, variable name, or variable id), (3) data type (such as character, numeric, date, enumerated, or boolean), (4) length (provides information about the length of the character string or maximum value or range for a numeric data element), and (5) group id (sometimes called form name or form id; provides information on which form the de is being collected). to demonstrate some examples of study data elements, table 1 shows the element data dictionary from study nct00099359: 'trial of three neonatal antiretroviral regimens for prevention of intrapartum hiv transmission.' for brevity, the table shows only 10 selected data elements out of all 577 data elements used in that study. for each element data dictionary, we analyzed the accuracy and completeness of the dictionary. this included an analysis of the previously specified features, such as the presence and clarity of the data element descriptions and the prevalence, common usage, and accurate representation of the data type for each listed data element. we use the term forms data dictionary (or forms dictionary in shorter form) to refer to a data dictionary that provides a full list of titles and descriptions of all case report forms (crfs) used in the study (or other relevant metadata for data element grouping). table 2 shows an example of a forms dictionary from study nct01751646: 'vitamin d absorption in hiv infected young adults being treated with tenofovir containing cart.' we followed the same approach as with the element data dictionary and combined all form dictionaries across all analyzed studies. the intention was to see whether some crf titles appear more frequently. to eliminate similarly named forms, we manually mapped synonymous form titles to their preferred title and identified common crf names that appeared in at least two studies. for example, forms by the names 'f89' for nct01751646 and 'hxw0100' for nct01418014 both map to a common form name of 'family history'. the semantic mapping was much more feasible for forms (compared to data elements, where it was out of project scope) due to the relatively small total number of forms across all studies. for harmonizing data collection across studies, the issue of copyright protection on case report forms must be considered [17, 18] . to measure the impact of copyright, we analyzed whether any of the form (across all studies in our sample) were marked as copyrighted. finally, we use the term permissible values data dictionary (or permissible values dictionary in shorter form) to list permissible values that are possible for categorical data elements. each permissible value (on separate rows) is linked to the data element it provides the value for (the link is via data element id). for example, in study nct01772823 'an open label demonstration project and phase ii safety study of pre-exposure prophylaxis' for data element offtxr, which describes the reason for discontinuation, its permissible value dictionary has 6 permissible values defined: 1 (viral breakthrough), 2 (adverse reaction), 3 (subject's decision), 4 (clinician's decision), 5 (course completed), and 99 (other). each row represents a single possible value (organized under a respective data element). the permissible value information model has the following columns defined: (1) permissible value id (or permissible value short label; this column is optional and can be missing), (2) permissible value, (3) permissible value description (if the previous field does not sufficiently define the permissible value), and (4) data element id (for proper linking of this permissible value dictionary to the element dictionary). for the permissible value dictionaries, we assessed commonly used formats for permissible values, as well as the most commonly used values. for all analyzed studies, we also looked at primary and secondary outcomes [1] as defined for each study at clinicaltrials.gov. the assumption was that every outcome on clinicaltrials. gov would be linked to at least one study data element. during our review of the study's clini-caltrials.gov record we also determined whether the clinicaltrials.gov study record reflects the availability of the study results data, ipd data [19] or data dictionary. finally, during aggregation of data dictionaries across studies, we recorded positive features of dictionaries as well as challenges of dictionary formats that complicated the analysis. our goal was to create a set of recommendations for optimal data sharing for future hiv studies (presented in the discussion section). in addition to analyzing a set of real studies, we also investigated relevant standards for clinical study data dictionaries. our reason why we looked into such standards was to inform our efforts to combine multiple data dictionaries into a single data structure. there are two relevant data dictionary standards: cdisc define-xml and redcap. cdisc define-xml standard is used by the food and drug administration in the us [20] and pharmaceuticals and medical devices agency in japan. it was first released in 2005 and it uses extensible markup language (xml) to describe a data dictionary of a clinical study. redcap data dictionary format is another standard defined by the redcap electronic data capture system used by more than 3200 institutions world-wide [21] . redcap stands for research data capture. the redcap software was first released in 2004 and it uses a zip compressed spreadsheet file to represent a data dictionary. while it is not widely used by individual studies, it is of note that, for example, the phenx initiative provides their cdes in the redcap data dictionary format [22] . we also acknowledge that iso 11179 specification aims at describing data element metadata, however it does not clearly define an exchange format [23] . our platform search identified relevant hiv studies on three data sharing platforms: nhlbi biolincc (3 studies), nichd data and specimen hub (dash) (11 studies) and nida data archive (5 studies). we searched 5 additional data sharing platforms which did not yield any more results due to several reasons, including: (1) we did not find any hiv studies on those platforms, (2) the study data request process was still pending at the start of our dictionary analysis, or (3) we could not obtain the data. our web search identified 5 hiv/aids clinical trial networks. they were the hiv vaccine trials network (hvtn), the hiv prevention trials network (hptn), the aids clinical trial group (actg), the international maternal, pediatrics adolescent aids and the microbicide trials network (mtn). after filing official or email requests, we obtained studies from two networks: (1) hptn (9 studies were obtained), and (2) hvtn (2 studies were obtained). one trial network, mtn, was inactive and for the two remaining networks, actg and impaact, our data request was pending at the time of our data dictionary analysis. our web search for individual hiv studies (outside any network) identified one additional hiv study (multicenter aids cohort study; macs). our searches were conducted between october 1, 2018 and march 31, 2019. after pooling all possible study acquisition channels, we acquired a total of 31 hiv studies from 5 distinct sources. nida data share was the only source with a widely used standard as it used cdisc. both hptn and hvtn stated that newer trials in their networks would be standardizing to cdisc as well. all other data sharing platforms and studies allowed for custom formats to be used by the studies present on their platform. of our set of studies, one from hptn and the 5 acquired from the nida data archive used a cdisc format. studies that used cdisc format were excluded from the main analysis presented here (we have a separate research project that focuses solely on cdisc-formatted studies). for another 7 studies in our input set, the data dictionary was either (1) not convertible into a machine-readable format, (2) the dictionary was not included in the shared data package, or (3) the dictionary could not be readily inferred from the provided ipd data. this resulted in a total of 18 studies in our final sample of studies that we analyzed. table 3 provides a clinicaltrials.gov study identifier and the study titles for this final set. seven studies (38.9%) in our sample were observational studies while the remaining 11 (61.1%) were interventional trials. we identified a total of 26 328 data elements across the analyzed 18 studies. see supplemental file s1 at the project repository, https://github.com/lhncbc/cde/tree/master/hiv/ datadictionary, to see the aggregated data element file (complete list of data elements). the median number of data elements for the studies in our set was 427 elements. the number of elements ranged between a minimum of 46 elements (study nct02404311) and a maximum of 9 945 elements (study nct00046280). a total of 14 studies used only csv files to provide their data dictionaries. in most cases, the studies provided a single csv dictionary file for the entire study. a single dictionary file is the most user-friendly format for data re-using researchers. a minority of studies split the dictionary into multiple files. the most extreme case of a split dictionary was a study that provided 55 dictionary files (one for each of the 55 study data files generated). four studies (nct00000590, nct00005273, nct00005274, and nct01418014) used pdf files to share the element dictionary. this pdf format required a manual conversion into a csv machine readable format. one study (nct01233531) had a mixture of formats with some data elements provided in a pdf format and some in csv format (spread across 8 files). for some of the studies where we also obtained ipd data (10 studies), we saw data elements present in data, but missing and not defined in the data dictionary, making the data dictionary incomplete. to quantify this level of completeness, we generated data element dictionaries for half of the trials we had ipd for and calculated the percentage of data elements included in the data dictionary. table 4 presents the results of this dictionary completeness analysis and shows that completeness ranges from 45.4% to 100%. we observed missing or incorrect information about data types within our sample of studies. explicit declaration of data type for each data element is important for proper data interpretation and correct data analysis. in one psychology study, incorrect results were published when a categorical variable (code for country of birth) was incorrectly used as a numerical variable in the model [24] . thanks to data sharing and secondary analysis by a researcher (external to the original study team) the error was discovered and revised study results were generated. in terms of missing data type, 12 studies provided data type for all des. one study (nct01492842) had missing data type for 49% of its data elements. data type was completely missing in four studies (22% out of 18 studies). on an aggregate level, across all studies, a total of 10 755 des had missing data type (40.9% out of all 26 328 des). however, this was mainly due to a single study (the multicenter aids cohort study [macs]; nct00046280) with 9 945 des without data type which accounted for 92.5% of all des with missing data type. the breakdown of the number of data elements for a given data type can be found in table 5 . for the 15 573 data elements where data type was declared the most common data type was string with 48.6%, followed by numeric with 47.8%. in terms of incorrect data type, we observed that no studies used a categorical data type. use of categorical variables is common in research. for example, all 13 studies in the national sleep research resource [25] , which is a sharing platform that we consider exemplary in terms of data dictionary format, have at least one categorical variable. in addition, a special case of categorical data type is a boolean data type and it was also not present in any of the element dictionaries. both cdisc define.xml and redcap standards clearly distinguish categorical data elements and support enumeration of permissible values. for the purpose of data analysis and when data is loaded into a database, a categorical variable may still be implemented as a string or a number; however, a flag that indicates that only a set of permissible strings or numbers are expected as values represent good analytical practice. by inspecting the data element title and description and study data, we found numerous categorical data elements; however, their formally listed data dictionary data type was not categorical and there was no flag marking them as categorical. we consider string data type (sometimes also called character) to be a free-text entry with no restrictions. in other words, no set of permissible values is defined for a string data element. an example of a data element of type string (from study nct01751646) is the element titled table 4 . proportion of data elements found in ipd data that are also present in the data dictionary. 'reason missed vitamins-specify' (data element id: vtmrsp). it asks about the specific reasons why the patient missed taking vitamins. the study does not provide any list of permissible values for this question, and there are 107 distinct responses in the ipd data). to demonstrate incorrect data type, we provide two examples. the first example shows the imperfect use of string data type. the study nct00099359 contains a data element 'severity grade of a medical event' (data element id: grade). this categorical data element has five permissible values: 'mild', 'moderate', 'severe', 'life threatening' and 'death'. the ipd data shows 5 distinct values that are all subsumed by this permissible value set. an accurate data dictionary should adopt and use categorical data type as one of the valid data types. if, for some reason, this semantically-rich modelling approach for data dictionary is not used, it should at least use a flag or other mechanism to distinguish data as 'string-categorical' versus 'string-proper'. the second example shows the other variant of this misclassification problem when a categorical data element is in the data dictionary marked as numeric data type (with numbers representing codes for a particular permissible value). as demonstrated in the previously cited retracted analysis [24] , this misclassification can be even more error-prone. in our sample, an example of a numeric-categorical data element (a data element that is not formally designated as categorical in the data dictionary and not distinguished from numerical-proper by any flag) is from study nct00005273 with title 'pain severity' that asks about severity of pain. it has the following permissible values: 0 for none, 1 for mild, 2 for moderate, 3 for severe, and 9 for unsure. not interpreting this element as numerical value is crucial for correct data analysis. if the data dictionary does not model correctly categorical variables, the provision of a complete permissible value dictionary can still fully compensate for the lack of distinction between numeric-categorical and numeric-proper or string-categorical and string-proper. however, we found that many studies in our sample did not provide the permissible value dictionary, and the problem can thus still occur. for data re-using researchers, the ability to properly interpret each data element is crucial. for that purpose, having an unambiguous description for each data element (in addition to a data element id) facilitates this proper interpretation. if the data element definition or meaning cannot be fully understood (description is missing or is vague), the resulting analysis can misinterpret these data elements (leading to incorrect results) or exclude them from the analysis (leading to incomplete results). we found missing data element descriptions in three studies. the element description field was missing for 4 447 des (16.9% out of all 26 328 des). one study (nct00005274) with 4 249 des omitted descriptions for all its data elements, while two studies (nct00865566 and nct01233531) had missing descriptions for some des. the remaining 15 studies (83.3% out of 18 studies) provided descriptions for 100% of their des. we also do, however, acknowledge the fact that a well formulated data element name can be in some cases sufficient to fully define an element. in the aggregate de file, we observed 327 des where the de description was identical to the de name (1.9% out of all 17 014 elements with descriptions). we also found that some de descriptions were vague or difficult to understand. des with confusing description can sometimes be disambiguated by inspecting the ipd data. although, such inspection may require the submission (and approval) of a formal data request that may not be necessary for the data dictionary alone. review of the clinicaltrials.gov records showed that 7 studies (38.9% out of 18 studies) provided study results. no study included a hyperlink (or actual file) for the data dictionary. two studies (11.1%) referenced ipd availability on clinical-trials.gov. although it is theoretically possible for a study to not organize its des into groups, all 18 studies in our sample did group their elements and utilized a group id mechanism. the majority of studies (11 out of 18 studies) used the case report form as the organizing principle (group id is the form name). the remaining 7 studies organized their elements by study data table and used the table name as the group id. the median number of forms in a study (or distinct groups) was 27 (ranging from a minimum of 2 (nct02404311) to a maximum of 124 (nct00005274). a review of form names (or group ids) can provide data re-using researchers a highly pragmatic and quick overview of what data was collected in a given study. if a standardized data collection instrument was used in a study, it may be easiest to discover it via the review of the forms dictionary. if a previously defined and standardized form addresses well the study's data collection needs, use of such a standardized form allows for a very straightforward method of meta-analysis across studies. for example, cdisc clinical data acquisition standards harmonization standard (cdash) defines such common forms for 'protocol deviations', 'demographics', 'adverse event', 'end of study', or 'concomitant medications' [26] . we identified a total of 28 common form names. some form names, such as 'off study' (in 11 of 18 studies, 61.1.6%), 'eligibility' (in 10 on 18 studies, 55.6%) and 'visit report' (in 10 of 18 studies, 55.6%) were very common in our set of studies. we defined very common as forms being present in 50% or more studies out of a set of studies that had a forms dictionary. table 6 shows the very common form names and quantitative measures of their use (count of studies and percentage). less common form names (present in two studies) were body measurements, family history, medical history, missed visit, behavior, and skin test. our results for common form names are affected by the fact that 10 (out of 18 studies) were executed by the same research network (adolescent medicine trials network for hiv/aids interventions). similarly, to data elements, we have observed form names data inaccuracies in 11 studies within our sample. we observed form names (or group names) that were ambiguous, and instances of identical names used to refer to two clearly different forms (see table 2 for an example: two forms both titled 'specimen tracking form'). for data recipients, unambiguous and good data descriptions are important for proper analysis. no study in our sample marked any of the forms as copyrighted. copyright protection typically does not impact the use of the collected data in a secondary research analysis. however, for researchers obtaining common forms and data elements with the intent to use the most prominent ones in a future study (especially those used frequently in past studies), the copyright status is important. use of categorical data elements in research is extremely common and, as stated earlier, most studies would be expected to provide a permissible value dictionary. table 6 . very common form names and the number and percentage of studies their used in. we were able to extract permissible values for 11 studies. the aggregate file contained a total of 7 669 permissible values for 1 815 des. the mean number of permissible values per data element is 4.23. use of numerical ids is most common: 1 511 categorical des (83.3%) used numbers as permissible values. the most frequent permissible value description was 'no' (for 1 334 des in 10 distinct studies). in some cases, permissible values represent administered drugs (e.g., values 3tc, nfv, nvp and zdv were permissible values for data element 'drugnm' which represents the drug name in trial nct00099359). permissible values can also represent laboratory tests. for example, 'cd4 t cell percent' is a permissible value for data element 'testnm' in study nct01772823. some permissible values can be further linked to established healthcare terminologies, such as rxnorm terminology for drugs or logical observation identifiers names and codes (loinc) terminology for laboratory tests. both data dictionary standards (define-xml and redcap) allow for such annotation of permissible values by relevant external terminology codes. no study in our sample made any such annotations. six hiv studies we obtained for our analysis used a cdisc format. cdisc specifications mandate ipd data to be represented in an sdtm (study data tabulation model) format and a corresponding data dictionary in the define-xml format. table 7 lists the studies that used cdisc format (which are excluded from the main analysis). the sdtm format accommodates some data elements directly as a column in a standardized spreadsheet (we refer to those as sdtm-model-hardcoded des) and for other data elements it uses an entity-attribute-value (eav) approach with concepts for those entities defined in cdisc controlled terminology (we refer to those as sdtm eav des). cdisc controlled terminology (ct) thus represents yet a third, tightly linked and relevant cdisc standard. in fact, cdisc ct concepts are also used for coded permissible values. all six studies that used some cdisc standard used sdtm format and also provided a define-xml dictionary. we obtained a large number of hiv trials and analyzed the format and content of their data dictionaries. to our knowledge, our study is the first to aggregate hiv data elements from a large set of completed hiv studies that were later shared via a platform or other mechanism. the majority of studies used a custom, non-standardized format that required significant processing to make the data machine-readable. the lack of consensus to use a single standard should be viewed in light of the fact that data sharing of clinical study data is still a developing and evolving scientific challenge. moreover, the studies in our sample may have been initiated at a time when the emphasis on proper data sharing (by research enterprise in general) was smaller. only in recent years has the importance of study metadata become more prominent. in the clinical domain of hiv, we did not find any prior study that analyzed hiv-specific research data elements. cdisc hiv therapeutic area user guide, published in january 2019, is the only relevant hiv-specific data element effort [3] (in addition to base sdtm elements, it highlights lab codes for cd4 count, loinc codes for hiv viral load testing and mother-infant data linking among many other things). a study focused on data elements and data sharing for hiv registries was published by our team in 2019 [27] . there are, however, prior studies that are not specific to hiv and cover data elements and dictionaries for medicine in general. sharma et al. analyzed three data dictionaries and used archetype modeling language developed by the clinical information modeling initiative [28] . they also developed a platform (called d2refine) for data element harmonization and standardization. in their report they discuss impediments to comparison and interoperability caused by the lack of standardization of data dictionaries from clinical studies. this lack of standardization we observed in our analysis as well. due to the evolution of some of the data dictionary standards and sharing policies, it is difficult to recommend a single data dictionary standard. as outlined in section 2.3, there are essentially two data dictionary standards (cdisc define-xml and redcap) to consider. although, the redcap format is not backed up by any formal standard developing organization (sdo). a recommendation to adopt a cdisc define-xml triggers the requirement to also adopt other related cdisc standards (including cdisc controlled term terminology and possibly cdisc sdtm) and such adoption requires significant training and technical expertise. we consider it too significant a hurdle to be universally recommending such an adoption. with regards to the redcap format, we view it to be very similar to simply formulating a good set of best practices to create a single spreadsheet, compliant with fair principles [31, 32] , that captures all significant parameters of all study data elements (either unique to the study or formal cdes adopted by the study). in other words, if redcap format is not formally utilized, we also find it acceptable to use an ad-hoc single spreadsheet-based data dictionary. based on transformation of data dictionaries and the creation of the aggregated de file, we have designed a set of general recommendations for the optimal sharing of data element metadata for future studies in the hiv domain. we, however, do believe that these recommendations also apply beyond hiv and to other medical domains. we enumerate some of the most important recommendations as a list below. we developed a more detailed set of recommendations as part of consider statement (consolidated recommendations for sharing individual participant data from human clinical studies) to improve the practice of data sharing and reuse. it is available at w3id.org/consider and includes details defining the desired best data sharing practices, positive examples of studies following the recommendations, challenging examples of studies where recommendations are not followed and notes on how to evaluate adherence of a study to a given recommendation (consider checklist). • provide data dictionary documentation separate from de-identified individual participant data. since it contains no participant level data and it does not require local ethical approval as a condition of releasing the data dictionary (avoid a request wall for the data dictionary). • share a data dictionary as soon as possible. do not wait until the data collection is complete. • provide the data dictionary in a single, machine-readable file. • for each data element, provide a data type (such as numeric, date, string, categorical). • for categorical data elements, provide a list of permissible values and distinguish when a numerical code or a string code is a code for a permissible value (versus when it is an actual number or string) • provide a complete data dictionary (all elements in the data are listed in a dictionary) and all types of applicable dictionaries (date elements, forms [or groupings], and permissible values). utilize a description field, in addition to title, to fully describe a data element or form. • at study design time and when resources allow, adopt previously defined applicable common data elements (including adoption of grouped data elements). within the analyzed data dictionaries, we saw instances of closely related des. for example, hiv 1 rna viral load and hiv 1 rna viral load date. this split into multiple self-standing des that are closely related data elements created higher counts of distinct data elements. in recent decades, several common data models (cdms) for electronic health record (ehr) data have emerged that group related data elements into more complex structures. similarly, the cdisc sdtm standard provides higher level structures (lb [= laboratory] domain) that group data elements together into one row of data in a higher level structure. this grouping has been referred to as the ehr data convention [20] and is increasingly becoming the method of choice for modelling closely related des. sharing of clinical study data is inherently linked to de-identification. if a study collected sensitive information (patient's exact date of birth or other identifying data enumerated in regulations or various policies), such information is removed or obfuscated prior to data sharing. nine studies in our sample provided de-identification notes. maintaining a data element dictionary can greatly facilitate ipd de-identification. for example, all elements with date data type may need a relative time obfuscation (shifting all events for a given participant by some fixed number of days). annotation of data elements with common terminologies can help in finding data elements that need to be de-identified and it can help identify an appropriate deidentification method based on a knowledge base organized by cde created from prior instances of study de-identification. our findings are time dependent and based on studies shared at the time of our review (october 2019; 18 studies). ongoing studies or studies currently being planned may use a more complete data dictionary and employ better dictionary formats. thanks to requirements to use cdes included in new funding opportunity announcements [2] , newly initiated studies are more likely to adopt cdes during the study design stage. because we searched specifically for hiv studies, our findings may not generalize fully to other clinical domains. we analyzed 26 328 des from 18 hiv studies. all analyzed studies organized their des into groups. an average study, represented by the median of our set, had 427 data elements. the majority of studies used csv files to share a data dictionary while 22% of the studies used a non-computable, pdf format. string and numeric data types were the most frequent data types with many studies incorrectly representing categorical data elements (100%) and not providing a full list of permissible values (39%). only a minority of studies reported study results or linked ipd within their clinicaltrials.gov record. we also identified two relevant data dictionary standards that have many features that encourage proper data sharing. using our analysis and review, we designed a set of recommendations that provide best practices for data sharing for future studies. analyzing real-world use of research common data elements improving the value of clinical research through the use of common data elements hiv therapeutic area user guide (taug) area user guide (taug) data sharing and embedded research data sharing platforms for de-identified data from human clinical trials clinical research informatics: supporting the research study lifecycle guidance to encourage the use of cdes data sharing celebrating parasites big data and collaboration seek to fight covid-19 [internet]. the scientist magazine® data sharing in the era of covid-19. lancet digit health website for research project: identification of research common data elements in hiv/aids using data science methods data share platform using sas catalogs to develop and manage sas data step programs nih scientific data council committee nih common data element task force: glossary sub-group: definitions of terms reflection paper on copyright, patient-reported outcome instruments and their translations. health qual life outcomes questions of copyright. health qual life outcomes sharing of individual participant data from clinical trials: general comparison and hiv use case study data for submission to cder and cber | fda the redcap consortium: building an international community of software platform partners redcap instruments zip files one step away from technology but one step towards domain experts-mdrbridge: a template-based iso 11179-compliant metadata processing pipeline retraction notice to: the negative association between religiousness and children's altruism across the world the national sleep research resource: towards a sleep data commons evaluation of research accessibility and data elements of hiv registries standardized representation of clinical study data dictionaries with cimi archetypes a concept for a data dictionary system supporting for clinical research the fair guiding principles for scientific data management and stewardship. sci data problems in fairifying medical datasets. stud health technol inform key: cord-341503-3cvtoc2j authors: jaiswal, j.; loschiavo, c.; perlman, d. c. title: disinformation, misinformation and inequality-driven mistrust in the time of covid-19: lessons unlearned from aids denialism date: 2020-05-21 journal: aids behav doi: 10.1007/s10461-020-02925-y sha: doc_id: 341503 cord_uid: 3cvtoc2j nan during a pandemic about which too little is known, public health is facing a crisis on multiple levels, including regarding covid-19 related-health messaging. with the federal government's inadequate, inconsistent and largely nonevidence-based response [1] [2] [3] , and the far reach of social media "armchair experts" [4, 5] , tremendous uncertainty, fear, and anger has emerged with respect to the origins, treatments and prevention methods regarding covid-19. much of the evidence needed to fully inform clinical and public health responses is not yet available, making covid-19 uniquely vulnerable to a proliferation of disinformation, misinformation, and medical mistrust, including what are often called "conspiracy beliefs" [6, 7] . disinformation (strategically and deliberately spread false information), misinformation (false information, not necessarily with intent to mislead), and mistrust (more than the lack of trust; suspicion of ill intent) are multi-faceted phenomena, with heterogeneous underlying motivating factors. the purpose of this commentary is to suggest that understanding the etiologies of disinformation, misinformation, and medical mistrust must be an important component of the public health response to covid-19. this is especially critical when considering how the pandemic has affected communities of color, including asian communities who have been blamed for the introduction of sars-cov-2 to the u.s. [8, 9] and black communities who have been blamed for higher fatality rates among black populations [10] . we propose that two main forms of pushback against dominant scientific evidence have become prominent during covid-19: (1) disinformation propagated at the institutional/federal government level to preserve power and undermine already marginalized groups, and (2) inequality-driven mistrust among communities that have been made vulnerable by historical and ongoing structural inequities. while these two forms do not constitute a strict dichotomy, this distinction can help inform strategies to address erroneous information and mistrust and inform public health messaging. "conspiracy beliefs," characterized as "attempts to explain the ultimate cause of an event…as a secret plot by a covert alliance of powerful individuals or organizations, rather than as an overt activity or natural occurrence" [11] , feature prominently in disinformation, misinformation, and inequality-driven mistrust. it can be difficult to persuasively present evidence to refute these types of ideas, especially because experts are often seen as part of the conspiracy [12] , and new pieces of contrary evidence can be rationalized into an existing narrative [13] . for example, a pew research center survey conducted in march 2020 found that 29% of americans believed that sars-cov-2 was developed intentionally in a lab [14] , with many pointing to wuhan, china as the source [15] ; president trump has given this theory institutional legitimacy [16] , despite scientific consensus [17] [18] [19] [20] and the consensus of the u.s intelligence services that sars-cov-2 is not human-made [21] . this strategic disinformation has served several agendas: casting doubt on evidence presented by dr. anthony fauci, the director of the national institute of allergy and infectious diseases and member of the white house coronavirus task force, validating and reinforcing pre-existing xenophobia and racism [22] , and redirecting attention away from the white house's inadequate and delayed response to covid-19. state-sanctioned disinformation has proven disastrous in the past. in south africa during thabo mbeki's presidency (1999-2008), aids denialism was institutionalized at the highest levels of government. this disinformation delayed official recognition of hiv as the etiology of aids and the accessibility of life-saving anti-retroviral therapy, which directly contributed to more than 330,000 preventable deaths [23] [24] [25] [26] . these examples of disinformation share some commonalities: assertions and preservations of power, authoritarianism, fear mongering, scapegoating to deflect blame, and the creation or reinforcement of states of collective shock, which can be used to facilitate the implementation of political and economic agendas that are more difficult to achieve during periods of stability [27] . covid-19 disinformation appears to reflect agendas of white supremacy [28, 29] , anxiety over social and economic instability [30] , opportunistic unrestrained capitalism, and the cult of personality regarding the president [31, 32] . this is a contrast to the inequality-driven mistrust held by people who continue to experience disenfranchisement [33] . in the covid-19 pandemic, beliefs about deliberately withheld vaccines [34, 35] and the intentional human-made origins of sars-cov2 appear to be emerging in some black communities [36] . while disinformation and inequality-driven mistrust may result in similar or even shared fallacious beliefs, understanding their different origins is vital to delivering effective public health messaging. government officials promulgating disinformation about the origins of covid-19 or possible therapies-whether rooted in racism and xenophobia, or whether motivated by goals of deflecting responsibility and accountability-is fundamentally different from marginalized individuals or groups endorsing the same beliefs, for whom mistrust is rooted in ongoing trauma and direct memories of real betrayals [37] . medical mistrust is well documented among black people and other populations placed at risk for disproportionate harm [38] [39] [40] [41] [42] [43] [44] [45] . for example, endorsement of hiv-related "conspiracy beliefs" is associated with worse hiv-related outcomes among some black populations [46] [47] [48] . this manifestation of hiv-related mistrust can include the beliefs that the u.s. federal government was involved in creating or disseminating hiv as a form of genocide against people of color, that anti-retroviral therapies are harmful, and that a cure is available but is being secretly withheld by the government and pharmaceutical companies [46, 49] . referring to these ideas as "conspiracy beliefs" frames these beliefs as being irrational or even paranoid, yet for populations made socially and economically vulnerable by interlocking structures of inequality [50] , these ideas are often intergenerational in nature and can resonate strongly with ongoing stigmatizing and exclusionary experiences with healthcare, government, law enforcement, and criminal justice systems [37, [51] [52] [53] . similarly, inequality-driven mistrust around covid-19 may deter or prevent individuals from seeking covid-19-related medical care or adhering to evidencebased covid-19 prevention guidelines, such as physical distancing and self-quarantining. this is a critical consideration, as black, native, and latinx populations have been disproportionately affected by covid-19 infection, morbidity, and mortality [54] [55] [56] [57] [58] [59] 69] , are disproportionately arrested for physical distancing violations [3, 60] , and appear to be less likely to receive covid-19 testing [61] . more effective public health messaging is urgently needed to address these inequities. we suggest the following recommendations: the onus is on researchers and clinicians to better understand these beliefs and to more effectively bridge the mistrust gaps [62] . a social determinants of health framework [63] can help public health and medical professionals address the impact of population-level inequalities on health outcomes in addition to facilitate enhanced understandings of how social and economic conditions engender inequalitydriven mistrust. it is vital to consider how people, as individuals and as members of groups, experience and interpret social and economic inequality, and how those experiences affect their trust in or mistrust of evidence-based public health messaging, as well as their readiness to accept any promulgated misinformation or disinformation [64] . public health and medicine must address structural racism directly [65] . effective public health responses to the pandemic, and to its disproportionate impact on communities of color and other vulnerable populations, must recognize the complex dimensions of mistrust. this requires attention to the issues of structural racism and systematic discrimination which create, perpetuate, and sustain mistrust and influence people's acceptance or rejection of misinformation or disinformation. the failure to fully address differential risk at the community and structural level, and differential risk among various populations placed at greatest risk for harm, further drives mistrust, which then reinforces mistrust arising from people's daily lived experiences of racism, classism, and stigma [66] . anti-racism education as well as training on research with and care for marginalized populations, must be more fully integrated into public health and medical education [67] . avoiding terms like "conspiracy beliefs" may position us to better understand, and thus more effectively address disinformation, misinformation and inequality-driven mistrust that has emerged during this pandemic. referring to ideas as "conspiracy beliefs" risks obscuring and denying meaningful aspects of people's lived experiences, particularly regarding inequality-driven mistrust, and is an ethical and strategic mistake for public health [49, 68] . thus, we propose that public health abandon this term and instead endeavor to identify and distinguish the underpinnings of such beliefs, highlighting how false information may be driven either by agendas of power and racism, or instead driven by mistrust deriving from ongoing social and economic exclusion. this distinction will avoid placing blame on communities that are structurally placed at risk for disproportionate harm by elucidating the role of historical and ongoing social and economic inequalities, while recognizing the forces underlying propagated disinformation and holding accountable those with structural power. distinguishing between disinformation and inequalitydriven mistrust and shifting language away from "conspiracy beliefs" can help avoid pushing people further toward endorsing misinformation and disinformation. moreover, language without the negative connotations of "conspiracy beliefs" may leave rhetorical space for marginalized populations to express concerns shaped by historical and current trauma, and for public health to better understand why some people endorse such beliefs. public health and medical professionals have a responsibility to communicate science in an effective, accurate and accessible manner, without bias-and with the understanding that structural racism and other forms of oppression are root causes of inequality-driven mistrust. while erroneous beliefs may appear to similar in nature, the critical distinction is in the source of and paths to those beliefs. misinformation during a pandemic americans immersed in covid-19 news; most think media are doing fairly well covering it associated press. 9 out of 10 people arrested for coronavirus-related offenses in nyc have been black or hispanic the dangerous rise of covid-19 influencer and armchair epidemiologists please, let's stop the epidemic of armchair epidemiology why uncertainty about coronavirus breeds opportunity for misinformation. pbs news hour coronavirus misinformation and disinformation regarding coronavirus in social media asian americans are blamed by some for covid-19 outbreak. national public radio blame and discrimination attached to covid-19-an faq for us elected leaders and health officials stop blaming black people for dying of the coronavirus: new data from 29 states confirm the extent of the racial disparities the hidden impact of conspiracy theories: perceived and actual influence of theories surrounding the death of princess diana advances in conspiracy theor dead and alive: beliefs in contradictory conspiracy theories nearly three-in-ten americans believe covid-19 was made in a lab covid-19 conspiracy theories: was sars-cov-2 made in a lab? psychology today a timeline of the covid-19 wuhan lab origin theory. forbes genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event the proximal origin of sars-cov-2 statement in support of the scientists, public health professionals, and medical professionals of china combatting covid-19 office of the director of national intelligence. intelligence community statement on origins of covid-19. odni news the foreignness of germs: the persistent association of immigrants and disease in american society. milbank q aids and the scientific governance of medicine in post-apartheid south africa lessons from south africa's experience of hiv/aids there is no proof that hiv causes aids": aids denialism beliefs among people living with hiv/aids denying aids: conspiracy theories, pseudoscience, and human tragedy screen new deal. the intercept white supremacist groups are recruiting with help from coronavirus-and a popular messaging app coronavirus outbreak revives dangerous race myths and pseudoscience. nbc news coronavirus perceptions and economic anxiety the relation between media consumption and misinformation at the outset of the sars-cov-2 pandemic in the us all the president's lies about the coronavirus. the atlantic towards a more inclusive and dynamic understanding of medical mistrust informed by science nearly one-third of americans believe a coronavirus vaccine exists and is being withheld, survey finds. usa today tuskegee always looms in our minds": some fear black americans, hardest hit by coronavirus, may not get vaccine study: nearly a third of americans believe a conspiracy theory about the origins of the coronavirus medical apartheid: the dark history of medical experimentation on black americans from colonial times to the present ubiquitous yet unclear: a systematic review of medical mistrust medical mistrust and prep perceptions among transgender women: a cluster analysis the association of cultural and structural factors with perceived medical mistrust among young adult latinos in rural oregon medical mistrust, racism, and delays in preventive health screening among african-american men does discrimination breed mistrust? examining the role of mediated and non-mediated discrimination experiences in medical mistrust medical mistrust and less satisfaction with health care among native americans presenting for cancer treatment the effect of conspiracy beliefs and trust on hiv diagnosis, linkage, and retention in young msm with hiv the health and sociocultural correlates of aids genocidal beliefs and medical mistrust among african american msm conspiracy beliefs about hiv are related to antiretroviral treatment nonadherence among african american men with hiv the need for multi-level mitigation of medical mistrust among social network members contributing to antiretroviral treatment nonadherence in african americans living with hiv: comment on bogart medical mistrust is related to lower longitudinal medication adherence among african-american males with hiv hiv-related 'conspiracy beliefs': lived experiences of racism and socio-economic exclusion among people living with hiv mapping the margins: intersectionality, identity politics, and violence against women of color like i have no choice": a qualitative exploration of hiv diagnosis and medical care experiences while incarcerated and their effects the transgenerational consequences of discrimination on african-american health outcomes rumors and realities: making sense of hiv/aids conspiracy narratives and contemporary legends black americans face alarming rates of coronavirus infection in some states. the new york times questions of bias in covid-19 treatment add to the mourning for black families. the new york times age-adjusted rates of lab confirmed covid19 for native americans, covid-19 is "the worst of both worlds at the same time doctors without borders dispatches team to the navajo nation for latinos and covid-19, doctors are seeing an 'alarming' disparity. the new york times in-some-ofohi osmost-popul ous-areas -black -peopl e-were-at-least -4-times -as-likel y-to-be-charg edwit h-stay-at-home-viola tions -as-white s the coronavirus doesn't discriminate, but u.s. health care showing familiar biases. national public radio whose responsibility is it to dismantle medical mistrust? future directions for researchers and health care providers social determinants of health inequalities there's never been a more urgent moment to build black americans' trust in the medical system structural racism and supporting black lives-the role of health professionals prior experiences of racial discrimination and racial differences in health care system distrust a call for critical race theory in medical education dissecting the social body: social inequality through aids counternarratives racial capitalism: a fundamental cause of novel coronavirus (covid-19) pandemic inequities in the united states. health edu behav key: cord-321773-5fw9abzl authors: cheng, wenyu; chen, guohua; jia, huaijie; he, xiaobing; jing, zhizhong title: ddx5 rna helicases: emerging roles in viral infection date: 2018-04-09 journal: int j mol sci doi: 10.3390/ijms19041122 sha: doc_id: 321773 cord_uid: 5fw9abzl asp-glu-ala-asp (dead)-box polypeptide 5 (ddx5), also called p68, is a prototypical member of the large atp-dependent rna helicases family and is known to participate in all aspects of rna metabolism ranging from transcription to translation, rna decay, and mirna processing. the roles of ddx5 in cell cycle regulation, tumorigenesis, apoptosis, cancer development, adipogenesis, wnt-β-catenin signaling, and viral infection have been established. several rna viruses have been reported to hijack ddx5 to facilitate various steps of their replication cycles. furthermore, ddx5 can be bounded by the viral proteins of some viruses with unknown functions. interestingly, an antiviral function of ddx5 has been reported during hepatitis b virus and myxoma virus infection. thus, the precise roles of this apparently multifaceted protein remain largely obscure. here, we provide a rapid and critical overview of the structure and functions of ddx5 with a particular emphasis on its role during virus infection. dead-box polypeptide 5 (ddx5), also called p68, is a prototypical member of the large asp-glu-ala-asp (dead)-box atp-dependent rna helicases family, which was first identified in 1980 [1] . ddx5 has extensive amino acid sequence homology in various organisms from escherichia coli to humans [2] [3] [4] . the protein plays multifunctional roles involved in all aspects of rna metabolism, including translation, splicing, transcription regulation, ribosome biogenesis, mrna nuclear export, and micro rna (mirna) processing [5] [6] [7] [8] [9] [10] [11] . in addition, the functions in dna modification, cell cycle regulation, tumorigenesis, apoptosis, cancer development, adipogenesis, and nuclear translocation of the wnt-β-catenin protein through phosphorylation have been established [12] [13] [14] . in line with the function of the protein in transcription regulation and mrna splicing, ddx5 has been found to shuttle between the nucleus and the cytoplasm, albeit it predominately localizes in the cell nucleus [15] . given the crucial roles of ddx5 in rna biology, several rna viruses were found to interact with the protein to promote viral replication (table 1) , including severe acute respiratory syndrome (sars) coronavirus (cov) [16] , human immunodeficiency virus 1 (hiv-1) [17] , hepatitis c virus (hcv) [18] , japanese encephalitis virus (jev) [19] , porcine reproductive and respiratory syndrome virus (prrsv) [20] , and influenza virus [21] . additionally, it has been reported that ddx5 is inhibitory for viral replication for two dna viruses, hepatitis b virus (hbv) and myxoma virus (myxv) [22, 23] . in view of the importance of ddx5 in regulating virus infection, we here provide a brief and critical overview of the known functions of ddx5, with a particular emphasis on its role during the virus life cycle. rna or dna helicases are categorized based on the substrates they bind, and can be classified into three superfamilies and two families (named sf1 to sf5) based on the occurrences and characteristics of conserved motifs in their primary sequences [26, 27] . the dead-box helicase family is named for its conserved asp-glu-ala-asp motif and belongs to the sf2 group [26] [27] [28] , which shares nine conserved motifs shown to be involved in atpase and helicase activities [29] [30] [31] . ddx5 is a 68 kd dead-box helicase with 614 amino acids in its full length [2] . consistent with the members of the sf1 and sf2 helicases, ddx5 contains the motifs q, i, ia, ib, ii, iii, iv, v, and vi, which constitute the core region of its helicase function ( figure 1 ). the q motif is named for the conserved glutamine residue that is present in more than 99% of dead-box helicase sequences. the q motif has been found to be necessary for efficient binding of single-stranded rna (ssrna) as well as for the conformational changes that are driven by nucleotide binding and atp hydrolysis [31] . motif i has been found to be crucial for atpase and helicase activities through its interaction with motif ii and motif iii [26, 32] . motifs ia and ib participate in rna-binding and structural rearrangements that occur upon atp binding and hydrolysis [26, 33] . motifs ii and iii are necessary for atpase activity, and they interact with motifs i and vi to collectively form the atp binding pocket, which leads to the correct positioning of the residues necessary for hydrolysis [26, 30] . motif iv is believed to bind ssrna and functions as a functional connect ion between motifs iv and v [26] . motifs v and vi are both important for rna binding in association with motifs ia, ib, and iv [34] . ddx5 also contains a arg-gly-ser-arg-gly-gly (rgs-rgg) motif and an ile-gln (iq) motif, which locate in the c-terminal region. the rgs-rgg motif can function as an rna-binding site to interact with rna or ddx3 for modulating biological functions [35] . (q, i, ia, ib, ii, iii, iv, v, and vi) and the corresponding conserved amino acid sequences of motifs are presented. rgs-rgg: arg-gly-ser-arg-gly-gly. given the ubiquity of rna helicases in plants and animals and the highly conserved asp-glu-x-asp/his (dexd/h) atp-binding domain in all helicases, rna helicases are assumed to play essential roles in a broad array of biological processes, especially rna metabolism. growing evidence shows that ddx5 is implicated in the infection of several viruses. by exploiting the driving force of ddx5-mediated atp hydrolysis or assembling large ribonucleoprotein complexes, rna viruses can complete their life cycles. however, ddx5 exhibits an antiviral activity during hbv and myxv infection. severe acute respiratory syndrome coronavirus (sars-cov), the causative agent of sars, was responsible for a large outbreak associated with a 10% fatality rate in 2003 [36, 37] . this positive rna virus encodes a large replicase polyprotein made up of 16 nonstructural proteins (nsp1-16), among which nsp13 is thought to be essential for the virus life cycle [38, 39] . as the helicase of sars-cov, nsp13 characterizes the enzymatic activities that can unwind both rna and dna duplexes [40, 41] . although sars-cov carries its own rna helicases, host rna helicases may be hijacked by viruses to facilitate the replication of their viral genome; examples include hiv-1 and hcv [42] . using the sars-cov nsp13 gene as a probe for two-hybrid screening in yeast and mammalian cells, only the ddx5 gene was identified as interacting with the helicase [16] . further rna interference (rnai) assays confirmed that inhibition of ddx5 results in the suppression of viral replication [16] . through direct binding to the sars-cov helicase, ddx5 may act as a coactivator to enhance viral genome transcription and virus proliferation [16] . like ddx5, another dead-box rna helicase, ddx1, has been reported to be associated with sars-cov and coronavirus infectious bronchitis virus (ibv) nonstructural protein 14 (nsp14) [43] . ddx1 utilizes its c-terminal region, containing motifs v and vi, to interact with nsp14 via the latter's n-terminal portion. further manipulation of ddx1 expression by small interfering rna and overexpression confirmed that this interaction enhances coronavirus ibv replication [43, 44] . moreover, ddx1 has been identified as a member of the cellular interactomes for the ibv n protein, a nucleocapsid protein encoded by subgenomic mrnas of coronaviruses [44, 45] . recruitment of ddx1 to the phosphorylated-n-containing complex mediated by n phosphorylation can facilitate template readthrough and longer subgenomic mrna synthesis [44] . therefore, the main role of ddx1 and ddx5 when hijacked by coronavirus is to positively modulate viral genome transcription and virus proliferation. as the causative agent of acquired immune deficiency syndrome (aids), human immunodeficiency virus 1 (hiv-1) is a retrovirus of the lentivirus genus with an rna genome of 9 kb. nine polypeptides are encoded by the genome rna, containing three structural proteins env (envelope), gag (group-specific antigen), and pol (polymerase); four accessory proteins, nef, vif, vpu, and vpr; and the regulatory proteins, tat and rev [46, 47] . this "intelligent" pathogen utilizes many host cell factors for its replication. genome-wide screening technologies have been applied by many groups to clarify host factors that affect hiv-1 replication. more than 300 cellular factors have been identified that may be involved in this process [48] [49] [50] . among these factors, several members of the dead-box helicase family that act as co-factors to facilitate hiv-1 proliferation, such as rna helicase a (rha), ddx1, ddx3, ddx5, ddx10, ddx17, ddx21, ddx28, dhx36, ddx47, ddx52, and ddx56 [17, 49, [51] [52] [53] [54] [55] . research has revealed that host rna helicases rha and ddx3 act as cofactors of tat and enhance hiv-1 genes expression [56] [57] [58] . knockdown of ddx3 expression or depletion of ddx3 in cells effectively suppresses the translation of tat and rev as well as hiv-1 mrna transcription [57, 58] . ddx3 directly interacts with hiv-1 tat, which is partially targeted to cytoplasmic stress granules upon ddx3 overexpression or conditions of cell stress, suggesting a potential role of tat/ddx3 complex in translation. more findings have indicated that ddx3 is recruited to the trans-activation responsive (tar) hairpin by interaction with viral tat to facilitate hiv-1 mrna transcription and translation [57, 58] . recently, ddx5 was found to be a new co-factor of hiv-1 that interacted with hiv-1 rev through the rev response element (rre) axis, and thus affects rev function to enhance hiv-1 replication [17] . meta-analysis of host cell genes linked to hiv replication showed that ddx5 was involved in rev-associated complex, suggesting its specific links to hiv-1 replication [50] . ddx5 was confirmed in co-localization with rev in the nucleus, an interaction that is largely dependent on rna [17] . since the main function of rev is to bind with unspliced and partially spliced hiv-1 transcripts and shuttle them from the nucleus to the cytoplasm [59, 60] , ddx5 might be a co-factor to bind with rna and then affect splicing or export of rev/rre-dependent mrnas ( figure 2 ). findings made by lever's group showed that ddx5 and ddx17 exist as homo-or heterodimers to be used by hiv but function in different phases of the virus' lifecycle [55, 61] . ddx5 has a phenotype consistent with its involvement in viral transcriptional control, as mentioned above, while it is speculated that ddx17 acts at a later stage, after transcription [61] . similarly, ddx17 has also been shown to associate with hiv-1 rev, with helicase knockdown leading to reduced virus release from infected cells [53] . in addition to ddx3, ddx5, and ddx17, other rna helicases, including rha, ddx1, ddx21, and ddx56 also have been shown to associate with hiv-1 rev and to stimulate the rev function, suggesting distinct dead-box rna helicases could cross-talk to enhance the hiv-1 life cycle at multiple stages through modulating the hiv-1 rev function ( figure 2 ). compared with other etiological agents, the genome of viruses encodes fewer proteins. consequently, numerous host factors are harnessed by viruses to complete their life cycles, among which some are pro-viral host factors while others are anti-viral host factors. viral dna/rna replication may be the prime target for the development of efficient and safe novel vaccines or anti-viral therapeutics [62] [63] [64] . unlike sars-cov, hiv-1 does not encode its own rna helicase. taken together, host ddx5 could be a new potential molecular target for the development of anti-viral drugs. there are several studies that focus on specific inhibitors or drugs of the host dead-box helicase to inhibit virus replication or treat cancers [65] [66] [67] , but it remains to be determined whether small molecular inhibitors of the interaction between ddx5 and rev can be found. hepatitis c, a contagious liver disease, is caused by hcv, which belongs to a positive-stranded rna virus of the family flaviviridae. hcv infects several hundred million people worldwide and results in cirrhosis, steatosis, and hepatocellular carcinoma [68, 69] . at least 10 proteins are encoded by the positive single-stranded 9.6 kb rna genome of hcv, including three structural proteins (the core protein and envelope (e) proteins e1 and e2) and seven nonstructural (ns) proteins (p7, ns2, ns3, ns4a, ns4b, ns5a, and ns5b) [68, 69] . as the viral rna-dependent rna polymerase (rdrp), ns5b can transcribe the positive-and negative-strand rna genome of the virus; it has therefore become a common target for antiviral agents [70] . viral replication requires interaction between rna, viral, and host proteins [71] . ns5b has been shown to interact with a mass of host proteins through yeast two-hybrid screening, small interfering rna (sirna) screening, and transcriptome expression analysis, including t-plastin, eukaryotic initiation factor 4aii, peptidyl-prolyl isomerase pin1, and ddx5 [18, [72] [73] [74] [75] . using yeast-two-hybrid screening, ddx5 was identified as an interacting partner of hcv ns5b, one that affects hcv replication [18] . overexpression of ns5b causes the redistribution of endogenous ddx5 from the nucleus to the cytoplasm; deletion of the c terminal of ns5b abolishes this interaction, while the deletion of the n terminal of ns5b does not influence the distribution of ddx5 [18] . similarly, another study revealed that ddx5 is redirected from the nucleus to the cytoplasm in huh7 cells infected with cell culture-produced hcv (hcvcc) [76] . further experimentation has demonstrated that there are two independent ns5b-binding sites in ddx5: one located at the n-terminus and another at the c-terminus [77] . the first 51 residues of n-terminal fragment are variable, which could fold back to block one of the ns5b binding sites located between 61 and 305 residues in ddx5, suggesting the highly dynamic interaction between ddx5 and ns5b in infected cells [77] . previous studies have shown that knockdown of ddx5 by rnai in 293 cells reduced the synthesis of negative-strand hcv rna, suggesting that ddx5 may be beneficial to the synthesis of the hcv genome (hcv-s1 strain) ( table 2 ) [18] . similarly, hcv rna replication was decreased and the infectivity of hcv (hcv-jfh1 strain) was significantly suppressed in the ddx5 knockdown rsc cells [76] . in line with this observation, the knockdown of ddx5 in huh7.5 cells dramatically inhibits hcv replication as determined from the 5 -nontranslated region of the hcv genome [78] . however, contrary results have been shown in huh7.5 cells by knockdown of the ddx5 through rnai, in a study that revealed that the level of hcv rna in the ddx5 sirna treated cells was higher than in the cells treated with control sirna, suggesting that ddx5 has the ability to inhibit hcv rna replication [79] . further experiments using cell-free infectious virus particles to infect sirna treated cells showed, curiously, that the increased hcv rna in the ddx5 sirna treated cells did not cause increased production of cell-free infectious virus particles (table 2 ) [79] . this finding may indicate that ddx5 was involved in a regulatory role at a later stage of the hcv life cycle. ddx5 was also found to be interacted with the 3 -untranslated region (utr) of hcv, which acts as an enhancer of hcv ires (internal ribosome entry site) function in hepatic cell lines [80] . the knockdown of ddx5 results in reduction viral translation efficiency, but has no effects on hcv replicon, suggesting the essential roles of ddx5 in hcv ires translation [80] . so far, limited focus has been trained on the precise role of ddx5 in hcv replication. hence, further investigations and studies should aim to elucidate the precise role of ddx5 in hcv replication and determine if ddx5 can interact with other hcv proteins. japanese encephalitis virus (jev), a mosquito-borne neurotropic flavivirus, is one of the common causes for epidemic encephalitis in humans and animals. the resulting disease, je, affects large parts of asia and the pacific, where over 3 billion people at risk of exposure to the virus [81, 82] . jev possesses a single-strand positive-sense rna approximately 11 kb in length, encoding a single polyprotein composed of three structural proteins and seven non-structural (ns) proteins in the order 5 -c-prm-e-ns1-ns2a-ns2b-ns3-ns4a-ns4b-ns5-3 [83] . of these, the core (c), premembrane (prm), and envelope (e) proteins are components of extracellular mature virus particles [84] . the seven c-terminal non-structural proteins (ns1-ns5) are involved in multiple steps of viral life cycles such as rna replication, virus assembly, and innate immunity evasion [83] . recently, it has been shown that ddx5 can interact with jev core protein (c), ns3 and ns5, and can host ddx5 act as a positive regulator for jev replication [19] . both knockdown of ddx5 and overexpression of ddx5 mutants lacking helicase activity can decrease jev replication, suggesting that ddx5 and its helicase activity are required for jev replication [19] . ddx5 binds to the viral 3 -utr and colocalize with viral rna, which promotes virus rna replication but not in viral protein translation. as mentioned above, ddx5 has an interaction relationship with 3 -utr of hcv and plays a role in hcv ires translation. similarly, ddx5 might participate in the translation of jev ires. the interaction between ddx5 and the three viral proteins (c, ns3, and ns5) co-localized with viral rna in the cytoplasm during infection might facilitate the unwinding the intermediate rna duplexes [19] . the c protein of jev was found to localize in both the cytoplasm and the nucleus; the mature c protein, released from the endoplasmic reticulum (er) membrane, is believed to bind to the genomic rna [85] . kinetic study of viral rna and protein syntheses have shown that, following translation at the early step of infection, it was translocated into the nucleus, and then facilitated rna replication at the late phase of infection. ns3 functions as rna helicase, rna triphosphatase, and rna-stimulated nucleoside triphosphatase. ns5 works as rna-dependent rna polymerase and methyltransferase [86] . these two viral proteins (ns3 and ns5), together with ns2a, specifically bind the 3 -untranslated region and lead to the formation of replication complex (rc) [87] . host ddx5 is recruited by the viral proteins and binds to this rc, and then utilizes its helicase activity to regulate jev replication [19] . since viral rna replication is a quite dynamic process, ddx5 shuttles between nucleus and cytoplasm to increase the efficiency of rna separation [19] . in addition to ddx5, helicase ddx3 was also found to be involved in jev replication [88] . viral ns3 and ns5 may interact with ddx3, which binds to jev 5 -and 3 -untranslated regions to positively regulate viral rna translation. when using chemical compounds (cmp6 and cmp8) to inhibit the helicase activity of ddx3 in hiv and jev infected cells, viral rna replication was remarkably inhibited [65, 88] . however, no drugs or molecule inhibitors targeting ddx5 are available. therefore, ddx5 could be a novel target for the development of anti-viral drugs. besides hcv and jev, other members of the flaviviridae family were also found to hijack ddx5 during virus infection. the genus pestivirus, a group of small positive-stranded rna viruses, belongs to the family flavivirus, which cause economically important diseases of farm animals and includes bovine viral diarrhea virus (bvdv), classical swine fever virus (csfv), and border disease virus (bdv) [89] . the enveloped pestiviruses contain single-stranded rna genomes of approximately 12.3-12.5 kb that consists of a single open reading frame, encoding only 12 proteins. these are coand post-translationally processed from a single rna into four structural and eight nonstructural proteins in the order nh2-n pro -c-e rns -e1-e2-p7-ns2-ns3-ns4a-ns5b-cooh [90, 91] . n pro , named for the viral n-terminal protease, is a 168-amino-acid autoprotease, with cysteine protease activity in a glu22-his49-cys69 triad that acts to cleave itself from the nascent polypeptide [92] . early work using mass spectrometry showed that n pro binds to ddx5, and also to ddx3x and dhx9 [25] . the distribution of n pro in ribosomal and ribonucleoprotein particles, as well as its interactions with several rna helicases, suggest that n pro is involved with virus rna translation; ddx5 possibly contributes to this process. nevertheless, the function of ddx5 in modulation of viral replication remains to be determined. porcine reproductive and respiratory syndrome virus (prrsv), the causative agent of one of the most economically important global swine diseases, is classified in the genus arterivirus of the family arteriviridae [93] . prrsv contains a single-strand, positive-sense rna of approximately 15 kb in length, composed of at least 10 open reading frames (orfs), and a poly-a tail at the 3 -terminus [94] . orf1a and orf1b encode the replication-related polymerase proteins and are processed into at least 13 nonstructural proteins (nsps) by self-cleavage [95] . among the nsps encoded by the orf1b region of prrsv, nsp9 is considered to be involved in viral replication and genomic transcription [96] . this protein possesses a rna-dependent rna polymerase (rdrp) domain in its c-terminal portion that contributes to its polymerase activity and the virulence of prrsv [97] . using the yeast two-hybrid method, the host ddx5 was found to interact with the nsp9 of prrsv [20] . overexpression of ddx5 in marc-145 cells showed an enhancement effect on the replication of prrsv; meanwhile, silencing of ddx5 expression in marc-145 cells might significantly inhibit the replication of virus particles, suggesting that ddx5 might function as a cellular cofactor that positively regulates prrsv replication [20] . confocal immunofluorescence assay has revealed that ddx5 co-localizes with nsp9 in the cytoplasm with a perinuclear pattern in hek293 cells, marc-145 cells, and the pam cell line, suggesting that endogenous ddx5 might diffuse from nucleus to cytoplasm and help to unwind the viral rna through its helicase activity. this hypothesis was confirmed by co-immunoprecipitation (co-ip) assay, which showed that the dead and helicase domains (aa1-436) of ddx5 bind with the c-terminal portion of nsp9 [20] . interestingly, prrsv carries its own helicase, nsp10, with helicase activity; the recruitment of host ddx5 by nsp9 may function as a cofactor for rna unwinding, export, and/or translation of the prrsv during viral genomic replication and transcription [20, 96] . influenza a virus is a major public health problem, causing seasonal epidemics in humans and occasionally lethal global pandemics, as happened in 1918-1919 (h1n1), 1957 (h2n2), 1968 (h3n2), and 2009 (swine-origin h1n1) [98] . currently, the h5n1 subtype circulating in wild and domestic birds is capable of crossing the species barrier into humans and constitutes huge threats to both animals and public health [99] . as many as 500 million people a year are infected with influenza a virus worldwide, with more than 500,000 deaths [100] . characterized by its high potential for mutations and adaptations, influenza virus presents a significant challenge to disease control and the pharmaceutical industry [101] . it has been demonstrated that the trimeric viral polymerase (a complex consisting of the pb1, pb2, and pa subunits) and nucleoprotein (np) contribute to the pathogenicity of h5n1 viruses in humans and other species of mammals [102] . functional genomics and proteomics approaches have identified a number of human proteins that associate with viral polymerases, including ddx5 and ddx17 [21] . the required role of ddx5 and ddx17 in h5n1 virus replication has led to a significant reduction in the virus titer after knockdown of these proteins [21] . it has been observed that ddx5 colocalizes with viral nps in the nucleus during viral rna replication and transcription. likewise, ddx17 has been found to colocalize with h5n1 np. however, the predominant distribution of ddx17 in uninfected cells is in the nucleus, where ddx17 exhibits as punctated [21] , whereas, in h5n1 infected cells, ddx17 was shown to be relocated in the cytoplasm, a change that exhibits its essential roles in efficient h5n1 mrna and viral rna (vrna) synthesis in both human and avian cells. given the multiple functions of ddx5 and ddx17 in ribosomal pre-rrna (ribosomal rna) processing and rrna nuclear export [103] , we speculate that influenza viruses have evolved to hijack host ddx5 and/or ddx17 in order to facilitate their own proteins' nuclear export. epstein-barr virus (ebv), a type of oncogenic human herpesvirus, is ubiquitous, causing over 90% infection in the exposed human population [104] . infections cause no symptoms in young children but result in the development of infectious mononucleosis at adolescence or later [105] . as with other herpesvirus, ebv possesses a 170 kb double-stranded dna genome encoding different sets of viral proteins and micrornas during lytic and latent infection. epstein-barr virus nuclear antigen 2 (ebna2) is a latency type iii expressed protein that interacts with various types of host cell proteins to regulate cellular and viral gene transcription [106] . recent work using immunoprecipitation identified ddx5 as an interacting partner of ebna2 [24] . as both ebna2 and ddx5 contain rg-repeat elements and mono-methylated arginine (mma) residues [107] , it has been proposed that ddx5 probably serves as methylation substrate to facilitate gene expression activation by ebna2. however, the relevance of ebna2-ddx5 interaction and functions during virus infection still remains elusive, and more studies are needed. being a member of the leporipoxvirus genus, myxoma virus exhibits a specific rabbit-restricted host tropism but reveals a much broader cellular host range in cultured cells [108] . myxv has been shown to selectively infect a variety of human cancer cell lines derived from a diverse group of tissues, leading to study of myxv as a potential oncolytic virotherapeutic for various classes of human cancer [109] . recently, a sirna library screen targeting the 58 human dead-box rna helicases has shown that ddx5 knockdown enhances myxv replication. the knockdown of ddx5 in multiple human cell lines increased myxv replication and enhanced foci size and virus spread, suggesting its potential roles in antiviral responses or regulation of innate immune responses [22] . in addition, ddx5 has shown increased expression in human monocyte-derived dendritic cells infected with attenuated strains of vaccinia virus (vacv) [110] . however, the exact function of this molecule in vacv infection was not established in that study. unlike myxv, bunyavirus replication is unaffected by ddx5 silencing, so that the antiviral function of ddx5 remains undefined, as ddx5 depletion upregulated ddx17 expression, restricting bunyavirus infection in an interferon-independent manner [111] . additionally, ddx5 has a role in transcription of hbv minichromosome by a mechanism not yet determined [23] . hbv is also the causative agent of chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma (hcc) worldwide [112, 113] . although hbv is a dna virus, the replication of its dna genome depends on reverse transcription [112] . after entry, the hbv dna is transported into the nucleus and is converted into covalently closed circular (ccc) dna [114] , which can assume a minichromosome-like structure and acts as the template for transcription of four viral rnas [114] . recently, it was shown that ddx5, by regulating prc2 (polycomb repressive complex 2) stability and function, represses hbv minichromosome and the expression of specific host genes involved in hbv-mediated hepatocarcinogenesis [23] . ddx5 interacts with and stabilizes suppressor of zeste 12 homolog (suz12), which is an essential subunit of prc2 [23, 115] . as an expression of viral protein hbx in hbv-infected cells, ddx5 was downregulated and leads to the degradation of suz12, which results in the transcription viral cccdna-encoded genes [23] . moreover, ddx5 expression was found to be reduced in a group of hbv-associated hccs and this can be an important molecule in pathogenesis of poor prognosis hbv-mediated liver cancer [23] . in recent years, as the emerging roles of some rna helicases in host innate response have been established, studies have shown that viral nucleic acids are sensed by ddx1, dhx9, ddx21, dhx33, dhx36, ddx41, retinoic acid-inducible gene 1 (rig-i/ddx58), and melanoma differentiationassociated gene 5 (mda-5), leading to type i interferon production and an antiviral response [116] [117] [118] [119] [120] [121] . these helicases (rig-i and mda-5) are characterized by caspase activation and recruitment domains (card) at the n-terminal region, which interact with other card-containing proteins to transduce the signaling cascade [122] . alternatively, the functions in nucleic acids recognition of helicases-such as ddx1, dhx9, ddx21, dhx33, dhx36 and ddx41-which are lacking in card, have all been established in certain human plasmacytoid dendritic cells [116] [117] [118] [119] [120] . ddx5 lacks the card signaling domains, and no literature indicates the function of ddx5 in regulating the production of type i interferon. the issue of whether ddx5 contributes to antiviral responses or regulation of innate immune responses remains uncertain, and more studies are needed. as summarized in this review, the multifaceted protein ddx5 has been shown to be involved in rna metabolism and viral infection, especially for rna viruses. it is intriguing that ddx5 is the target of manipulation by different viruses. each rna virus seems to hijack host ddx5 in order to facilitate its own replication. with conserved atp binding motif and helicase motif, ddx5 utilizes the free energy to change of binding and hydrolyzing a nucleotide triphosphate to dissociate duplexes or displace bound proteins, which, on one hand, ddx5 may translocate along with viral dna/rna; on the other hand, ddx5 may unwind double-stranded regions to promote the expression of viral proteins [26] . however, ddx5 exhibits a role in antiviral responses during hbv and myxv infection. this might indicate that a requirement of ddx5 for viral replication is a more universal feature of rna viruses. the opposite roles of ddx5 between dna and rna infection likely reflect the different modes of the biosynthesis of rna and dna viruses. as mentioned, the using of ddx5 for viral replication could be a therapeutic target for development of antiviral drugs. even though emerging studies on ddx5 have supplemented our knowledge about the multiple functions of ddx5 in recent years, the overall picture still remains fragmentary. research is still needed to resolve seemingly contradictory data on the role(s) of ddx5 in response to different virus. as conserved motifs in the primary sequence, dexd/h helicases are highly conserved from viruses and bacteria to humans, and these proteins have overlapping functions. in addition, these proteins generally act as components of large multi-protein complexes, with each other or other factors, to regulate transcription. therefore, studies should also attempt to establish whether the effects of ddx5 on viral replication after knockdown are unique or whether other helicases supplement abolished function via small interfering rna. sv40 large t shares an antigenic determinant with a cellular protein of molecular weight 68,000 rna helicase activity associated with the human p68 protein identification of a putative rna helicase in e. coli 68 rna helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts a subfamily of rna-binding dead-box proteins acts as an estrogen receptor α coactivator through the n-terminal activation domain (af-1) with an rna coactivator rna helicase is an essential human splicing factor that acts at the u1 snrna-5 splice site duplex the p68 and p72 dead-box rna helicases interact with hdac1 and repress transcription in a promoter-specific manner atpase/helicase activities of p68 rna helicase are required for pre-mrna splicing but not for assembly of the spliceosome redundant role of dead-box proteins p68 (ddx5) and p72/p82 (ddx17) in ribosome biogenesis and cell proliferation the rna helicase ddx5/p68 is a key factor promoting c-fos expression at different levels from transcription to mrna export rna helicases ddx5 and ddx17 dynamically orchestrate transcription, mirna, and splicing programs in cell differentiation rna helicase ddx5 is a p53-independent target of arf that participates in ribosome biogenesis a chicken embryo protein related to the mammalian dead-box protein p68 is tightly associated with the highly purified protein-rna complex of 5-mec-dna glycosylase phosphorylations of dead-box p68 rna helicase are associated with cancer development and cell proliferation p68 rna helicase is a nucleocytoplasmic shuttling protein interaction between sars-cov helicase and a multifunctional cellular protein (ddx5) revealed by yeast and mammalian cell two-hybrid systems ddx5 facilitates hiv-1 replication as a cellular co-factor of rev cellular rna helicase p68 relocalization and interaction with the hepatitis c virus (hcv) ns5b protein and the potential role of p68 in hcv rna replication the dead-box rna helicase ddx5 acts as a positive regulator of japanese encephalitis virus replication by binding to viral 3 utr the dead-box rna helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral nsp9 in vitro host-and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins identification of host dead-box rna helicases that regulate cellular tropism of oncolytic myxoma virus in human cancer cells rna helicase dead-box protein 5 regulates polycomb repressive complex 2/hox transcript antisense intergenic rna function in hepatitis b virus infection and hepatocarcinogenesis antibodies against the mono-methylated arginine-glycine repeat (mma-rg) of the epstein-barr virus nuclear antigen 2 (ebna2) identify potential cellular proteins targeted in viral transformation host factors that interact with the pestivirus n-terminal protease, n pro , are components of the ribonucleoprotein complex the dead-box protein family of rna helicases happy birthday: 25 years of dead-box proteins helicase structure and mechanism the newly identified q motif of dead-box helicases is involved in adenine recognition dead-box proteins: the driving forces behind rna metabolism the newly discovered q motif of dead-box rna helicases regulates rna-binding and helicase activity dexd/h box rna helicases: from generic motors to specific dissociation functions rna helicase dynamics in pre-mrna splicing comparative structural analysis of human dead-box rna helicases the dead-box rna helicase ddx3 interacts with ddx5, co-localizes with it in the cytoplasm during the g2/m phase of the cycle, and affects its shuttling during mrnp export crystal structure and functional analysis of the sars-coronavirus rna cap 2 -o-methyltransferase nsp10/nsp16 complex rna 3 -end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex mechanisms and enzymes involved in sars coronavirus genome expression mechanism of nucleic acid unwinding by sars-cov helicase cooperative translocation enhances the unwinding of duplex dna by sars coronavirus helicase nsp13 sars-cov orf1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets viral and cellular rna helicases as antiviral targets the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology cell biology of hiv-1 infection of macrophages making sense of multifunctional proteins: human immunodeficiency virus type 1 accessory and regulatory proteins and connections to transcription a whole-genome association study of major determinants for host control of hiv-1 identification of host proteins required for hiv infection through a functional genomic screen host cell factors in hiv replication: meta-analysis of genome-wide studies a dead-box protein facilitates hiv-1 replication as a cellular co-factor of rev role of rna helicases in hiv-1 replication distinct ddx dead-box rna helicases cooperate to modulate the hiv-1 rev function multiple functions of ddx3 rna helicase in gene regulation, tumorigenesis, and viral infection identification of rna helicases in human immunodeficiency virus 1 (hiv-1) replication-a targeted small interfering rna library screen using pseudotyped and wt hiv-1 a role of rna helicase a in cis-acting transactivation response element-mediated transcriptional regulation of human immunodeficiency virus type 1 human ddx3 interacts with the hiv-1 tat protein to facilitate viral mrna translation ddx3 rna helicase is required for hiv-1 tat function hiv rev assembly on the rev response element (rre): a structural perspective the hiv-1 rev response element (rre) adopts alternative conformations that promote different rates of virus replication the roles of dead-box helicases in the life cycle of hiv-1 poxvirus dna replication host factor-targeted hepatitis b virus therapies recent strategies and progress in identifying host factors involved in virus replication discovery of the first small molecule inhibitor of human ddx3 specifically designed to target the rna binding site: towards the next generation hiv-1 inhibitors ketorolac salt is a newly discovered ddx3 inhibitor to treat oral cancer targeting ddx3 with a small molecule inhibitor for lung cancer therapy virus-specific mechanisms of carcinogenesis in hepatitis c virus associated liver cancer the core of hepatitis c virus pathogenesis nucleoside inhibitors of hepatitis c virus ns5b polymerase: a systematic review recent advances in the molecular biology of hepatitis c virus interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis c virus rna-dependent rna polymerase peptidyl-prolyl isomerase pin1 is a cellular factor required for hepatitis c virus propagation hepatitis c virus non-structural 5b protein interacts with cyclin a2 and regulates viral propagation chemical proteomic identification of t-plastin as a novel host cell response factor in hcv infection pml tumor suppressor protein is required for hcv production the variable n-terminal region of ddx5 contains structural elements and auto-inhibits its interaction with ns5b of hepatitis c virus identification of cellular factors associated with the 3 -nontranslated region of the hepatitis c virus genome understanding the interaction of hepatitis c virus with host dead-box rna helicases berzal-herranz, a. the cis-acting replication element of the hepatitis c virus genome recruits host factors that influence viral replication and translation japanese encephalitis: the virus and vaccines. hum. vaccines immunother japanese encephalitis surveillance and immunization-asia and western pacific regions regulation of flavivirus rna synthesis and replication capsid protein c of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence japanese encephalitis virus core protein inhibits stress granule formation through an interaction with caprin-1 and facilitates viral propagation an rna cap (nucleoside-2 -o-)-methyltransferase in the flavivirus rna polymerase ns5: crystal structure and functional characterization architecture of the flaviviral replication complex. protease, nuclease, and detergents reveal encasement within double-layered membrane compartments cellular ddx3 regulates japanese encephalitis virus replication by interacting with viral un-translated regions pestiviruses: how to outmaneuver your hosts the molecular biology of pestiviruses structures and functions of pestivirus glycoproteins: not simply surface matters autocatalytic activity and substrate specificity of the pestivirus n-terminal protease n pro prrsv structure, replication and recombination: origin of phenotype and genotype diversity overview: replication of porcine reproductive and respiratory syndrome virus the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins nsp9 and nsp10 contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china inhibition of porcine reproductive and respiratory syndrome virus by specific sirna targeting nsp9 gene h5 influenza, a global update a systematic review of the comparative epidemiology of avian and human influenza a h5n1 and h7n9-lessons and unanswered questions functional genomics reveals linkers critical for influenza polymerase a membrane-based purification process for cell culture-derived influenza a virus mutations in the pa protein of avian h5n1 influenza viruses affect polymerase activity and mouse virulence the dead-box proteins ddx5 (p68) and ddx17 (p72): multi-tasking transcriptional regulators epstein-barr virus lymphoproliferative disease after solid organ transplantation epstein-barr virus hijacks dna damage response transducers to orchestrate its life cycle proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest myxoma virus and the leporipoxviruses: an evolutionary paradigm oncolytic myxoma virus: the path to clinic distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors mva and nyvac stem-loop recognition by ddx17 facilitates mirna processing and antiviral defense hbv cure: why, how, when? hepatitis b virus infection and alcohol consumption molecular biology of hepatitis b virus infection hepatitis b virus-associated hepatocellular carcinoma and hepatic cancer stem cells ddx21, and dhx36 helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells aspartate-glutamate-alanine-histidine box motif (deah)/rna helicase a helicases sense microbial dna in human plasmacytoid dendritic cells the dhx33 rna helicase senses cytosolic rna and activates the nlrp3 inflammasome the helicase ddx41 senses intracellular dna mediated by the adaptor sting in dendritic cells is a novel antiviral factor promoting rig-i-like receptor-mediated signaling rig-i and mda-5 detection of viral rna-dependent rna polymerase activity restricts positive-strand rna virus replication emerging roles in viral replication and the host innate response this study was supported by grants from the natural science foundation of china (31402199) key: cord-355541-5sctqkwr authors: alcamí, josé; joseph munné, joan; muñoz-fernández, maría ángeles; esteban, mariano title: current situation in the development of a preventive hiv vaccine date: 2005-07-31 journal: enfermedades infecciosas y microbiología clínica doi: 10.1016/s0213-005x(05)75157-0 sha: doc_id: 355541 cord_uid: 5sctqkwr the uncontrolled progression of the aids epidemic has made the development of an efficacious human immunodeficiency virus (hiv) vaccine a major objective of scientific research. no effective preventive vaccine against hiv is currently available and sterilizing immunity has not yet been achieved in animal models. this review analyses the major challenges in developing an aids vaccine, in particular the mechanisms involved in viral escape from the immune response, and summarizes the results obtained with the different prototypes of therapeutic and preventive vaccines. finally, social, economic and healthcare aspects of research into hiv vaccines and current controversies regarding the development of clinical trials are discussed. in the year 2003, the aids pandemic was responsible for more than three million deaths, and five million people are calculated to have contracted the virus during this period. this takes the number of infected people throughout the world to 42 million, with 20 million deaths registered since the origin of the pandemic was identified 1 . the geographical and economic differences in this disease are obvious, with more than 95% of cases and deaths by aids occurring in the third world (70% in africa), especially among young adults, with a gradual increase in women. it is dramatic to see how, in sub-saharian africa, the epidemic continues to spread and, in many countries, the high percentage of people infected with aids has devastating effects on families and on the productive economy. the explosion of this epidemic in developing countries has raised the need to take urgent preventive measures and provide expanded access to antiretroviral therapy. however, in some parts of the world, these measures, although essential, are probably insufficient to curb the epidemic, so that the development of a vaccine is the only available possibility of controlling it. therefore, the development of an efficacious hiv vaccine is not only an area of aids research which has yet to be resolved but also an urgent need for developing countries. this awareness has led to a considerable increase in financing the search for an aids vaccine. to obtain an effective hiv vaccine represents a major challenge and priority scientific objective for public and private institutions, governments and ngos [2] [3] [4] (table 1) . this chapter analyses prototype vaccines being developed and the results obtained, the scientific difficulties in developing a vaccine, especially with regard to the mechanisms of viral escape from the immune response. furthermore, as this research is set in the social, economic and healthcare context of the aids pandemic, current controversies surrounding clinical trials with the different types of vaccine 5 are addressed in this review. situación actual en el desarrollo de una vacuna preventiva frente al vih el avance de la epidemia de sida ha convertido la obtención de una vacuna eficaz frente al virus de la inmunodeficiencia humana (vih) como un objetivo científico prioritario. en el momento actual no disponemos de una vacuna preventiva frente a la infección por el vih y en ningún modelo animal se ha conseguido la protección frente a la infección. en esta revisión se analizan las dificultades existentes en el desarrollo de una vacuna contra el sida, en especial los mecanismos de escape viral a la respuesta inmunitaria y se describen los prototipos de vacunas preventivas y terapéuticas en desarrollo y los resultados obtenidos. por otra parte se sitúa esta investigación en el contexto sanitario, económico y social de la pandemia de sida y se analizan las polémicas in order to develop a vaccine, it is necessary to know the genes of the pathogen involved in the induction of a specific immune response, and to use experimental models to test the efficacy of the virus. despite the fact that, in the past, there have been important advances in the control of infectious diseases by vaccination, many of the mechanisms used by the pathogen to take over cell machinery are unknown. the same applies to mechanisms of escape from the immune system and ways of boosting an immune response capable of eliminating the infected cell. in scientific terms, obtaining an efficacious vaccine capable of preventing hiv infection faces a series of challenges: the immune response begins with the recognition by cd4 lymphocytes via their receptor (tcr) of foreign antigens in the class-ii hla groove presented by cells specialized in antigen processing. antigen recognition activates the different effector mechanisms of the immune system: cytokine and chemokine synthesis, production of antibodies by b lymphocytes, activation of cd4 lymphocytes and generation of cytotoxic cd8 lymphocytes. these last events represent the main mechanism involved in killing of virus-infected cells and to initiate this process cytotoxic lymphocytes must recognize the antigenic determinants of the virus lodged in the class-i hla groove of infected cells 6 . as a consequence of the polymorphism of the hla system both in antigen presenting cells and target cells different peptides (that are anchored in class ii and class i hla molecules respectively) are selected according to individual hla haplotypes. in many viruses there are "major immunodominant determinants" (epitopes) which induce a potent response by the immune system. the efficacy of this response depends on two characteristics: first, they correspond to epitopes or domains of the viral proteins which are conserved among the different isolates, even in the context of highly variable viruses; second, these major determinants are capable of binding to the grooves of most hla haplotypes. the existence and identification of these major immunogenic determinants is crucial when developing a vaccine, since they make up viral targets par excellence by being "universal" in two senses: as epitopes conserved in the viral protein among different isolates and as epitopes susceptible to antigenic presentation by most subjects regardless of their hla haplotype. however, in the case of hiv, no similar dominant epitopes have been found to date, which represents a very important limitation when designing a vaccine. the presence of these major immunogenicity determinants in viruses with tremendous genetic variability, such as hiv-1, makes it practically impossible to define these epitopes empirically and experimentally. nevertheless, bioinformatics can provide us with predictive methods which make identification easier 7 . the objective of a vaccine is to induce an efficacious, memory-type immune response which allows the immune system to react against the infectious agent by preventing its spread. for this objective to be reached, it is essential to know which immunological effectors are efficacious in the control of the infection in order to define a series of surrogate immunological parameters which enable us to evaluate whether a vaccine preparation is efficient or not. the intense immune response of hiv-infected patients is reported to encompass practically all the effector mechanisms of the immune system. this response is relatively wide, since it is developed against numerous epitopes, and practically all the viral proteins, both structural and regulatory, are recognized as foreign antigens. however, controversy still surrounds the role of "protection" played by each of these components of the antiviral response. below, we describe the type of immune response generated against hiv infection. hiv infection induces an intense antibody response against practically all the regulatory and structural proteins of hiv 8 . some of these antibodies have neutralizing capacity in vitro 9 and in in vivo adoptive immunotherapy experiments 9, 10 . however, the production of antibodies with neutralizing capacity is scarce and viral escape from these antibodies is rapid 11 . furthermore, in the immunization models developed to date, high levels of neutralizing antibodies are not consistently obtained and their presence is not systematically associated with protection. these data raise severe concerns about the role of the humoral response in the control of hiv infection 12, 13 . nevertheless, almost all preventive vaccines induce neutralizing antibodies and their role as a surrogate protection marker is clearly demonstrated in other diseases. therefore, "a priori", a preventive hiv vaccine should induce broad-spectrum neutralizing antibodies 14 and this is one of the huge challenges currently facing the development of an aids vaccine. recent studies which define the location of neutralization epitopes, antibody structure and mechanism of action 15 represent an important advance to define the characteristics that a given vaccine must have to induce neutralizing antibodies. most studies agree that the combined response of cd4 and cd8 is probably the most important protective mechanism against hiv 16 . study of the cellular response has shown that in seropositive patients there is a clonal expansion of cd4 and cd8 lymphocytes which are active against hiv. this expansion is particularly intense in patients with primary infection and correlates with the control of viral replication 17, 18 . there are also reports showing an intense cd4 and cd8 response in some patients during immune reconstitution after antiretroviral therapy, especially in those with a good immunological status before starting therapy 19, 20 . the same phenomenon has been described in patients with structured treatment interruptions who spontaneously control viral replication 21 . although it is difficult to draw conclusions as to a cause-effect relationship between the appearance of a specific type of immune response and the control of viral replication, all the data seem to suggest that both helper and cytotoxic immune responses are essential to contain viral replication in the early stages of the disease, when the immune system is relatively undamaged. the most conclusive experimental data on the role of the cellular response in the control of viral replication come from studies in which selective depletion of cd8 lymphocytes in macaques infected with siv leads to a huge increase in viremia and accelerated evolution to aids 22 . hiv transmission occurs mainly via the mucosa. the large quantity of cd4+ lymphocytes in genito-rectal lymphoid tissue represent a major reservoir for the replication of hiv or siv, even when the infection is contracted intravenously. the gut-associated lymphoid tissue system (galt) is set up by activated b and t lymphocytes and dendritic cells which migrate through the lymphatic system and bloodstream to distant lymph nodes where they become resident. thus, the induction of a strong immune response in mucosa is probably a necessary requirement for a vaccine to be efficient against hiv. each family of viruses develops different escape mechanisms to avoid elimination by the immune system. a vaccine must face the same escape mechanisms and, to be successful, must induce a series of immune responses capable of overcoming them. the rate of variability of hiv is due to the high error rate of reverse transcriptase (one substitution per 10 3 -10 4 nucleotides and round of copy). in consequence there is wide intersubtype and intrasubtype variability but the immunological relevance for vaccine design of this genetic disparity is a matter of debate. several investigations have shown that genetic differences among hiv subtypes do not correlate with immunotypes. in fact, several genetic subtypes could share common protective epitopes and more than one immunotype can be found in the same genetic subtype. in general, neutralizing antibodies seem to be more strain-specific, whereas cellular immune responses have a broader spectrum. this lack of fidelity generates a high diversity in viral proteins which allows escape from the control of specific immune response. therefore, hiv shares with other rna viruses a common escape mechanism related to their high variability that allow the virus finding holes in the immune repertoire. together with the variability generated by the high error rate of reverse transcriptase other mechanisms such as genetic recombination, which produces new subtypes and "mosaic" viruses among different subtypes, are also involved in the generation of hiv variants. several molecular epidemiology studies have stressed the rapid dissemination of hiv variants and have described the distribution of several subtypes or recombinant viruses in different parts of the world. this could be an obstacle to the development of a universal vaccine 23 . one central aspect of hiv infection which is not totally understood is the reason why viral replication is not controlled despite the potent immune responses elicited in primary infection. although several explanations have been put forward, the most widely reported is viral escape through mutations in the viral epitopes recognized by the different effector mechanisms of the immune system 24 . escape from ctl response is due to ad hoc mutations of the viral epitopes which interact with the groove of the hla molecules. it has been shown that mutations in critical residues generate viral escape in both animal models and patients with primary infection and this event results in loss in the ctl response and parallel increase in viremia 25, 26 . however, in the chronic phase of infection, there is no clear correlation between the presence of specific ctl and the elimination or persistence of a given viral variant 27 . in addition to merely quantitative data, functional analysis have shown qualitative differences between ctl from progressors and non-progressors, such as the expression of perforins 28 , production of cytokines and chemokines, and reduced activity of the t-cell receptor against viral epitopes presented in the hla complex 23 . these data suggest that qualitative aspects of ctl may be also important in the control of viral replication. a general strategy for maximizing the efficacy of a vaccine would be to obtain a cytotoxic response against a large number of epitopes from several proteins. however, recent studies suggest that a more targeted approach can be more effective. thus, ctl against non-structural proteins (tat, nef) are more efficient in controlling infection but more prone to viral escape and do not last as long as ctl against the structural proteins gag and pol 29 . for a sterilizing vaccine, the objective would be to induce an intense ctl response against early proteins, whereas immunization against structural proteins would generate a response which would control viral replication thus attenuating infection. another problem which may be a serious genetic resistance barrier is the adaptation of the virus to the hla haplotype of the infected patient. in this situation peptides from viral mutants generated would reduce their affinity to hla thus decreasing recognition by tcr and generating a greater resistance to ctl response 30 . the structure of the viral envelope in its native form hides the domains of interaction with viral coreceptors due to the trimeric structure and folding of the protein (oligomeric exclusion and entropic masking) 31 . exposure of these conserved epitopes which are identified by neutralizing antibodies takes place at the moment of interaction between the viral and cellular membranes, a setting in which antibodies efficacy is lower given their low accessibility to neutralization epitopes. a second, more classic escape mechanism is epitopic mutation in the hypervariable regions found in the external domain of the viral envelope. nevertheless, recent studies show that escape from these antibodies does not always require mutation in amino acid residues but takes place by glycosylation of the residues and formation of carbohydrate structures on viral gp120 known as "glycan shields" that represent authentic barriers to the action of neutralizing antibodies 32 . one of the most spectacular studies published during the last year shows how, during evolution in a specific patient, the viral envelopes gradually become resistant to all types of neutralization by neutralizing antibodies via accumulation of the previously mentioned escape mechanisms 32 . both in animal models and in patients with primary infection through sexual contact the establishment of hiv infection is a very rapid process 33 . in a few hours, the lymphoid cells of the rectal and vaginal submucosa become infected and, in seven days, the infection spreads to systemic lymph nodes where it reaches viral and proviral loads similar to levels found in chronic infection 34 . the speed at which these reservoirs appear, before a specific immune response is triggered, represents a major obstacle to the control of viral replication since once established hiv infection will "persists" in lymphocytes despite immune response 35 . hiv can infect target cells in a latent form. in this situation no viral proteins are expressed on the membrane of infected cells thus allowing escape from immune surveillance. furthermore, reactivation-reinfection processes take place in lymphoid organs, which provide an ideal cellular microenvironment for the process of infection: dendritic cells express in their membrane a lectin (dc-sign) which interacts with the virions and lymphocytes and enhances hiv infection 36 . antigenic recognition by lymphocytes and the presence of cytokines in this microenvironment in turn increase infection of target cells and promote viral replication. as confirmation of these data, hiv-specific lymphocyte clones have been shown to be infected at higher proportion, which implies a preferential immunosuppression of the hiv-specific responses 37 . it must be stressed that the continuous generation of new, latently infected cells from the active viral replication compartment generates a "continuous archive" of changes produced in the virus throughout the disease, by including treatment-resistant mutated genomes and variants of the immune escape. the latent compartment is therefore not static and in some way hiv stores its "history" in latently infected cells 32 thus contributing to viral diversity as a mechanism of escape from antiretroviral therapy and vaccines. attenuated virus vaccines are without doubt the most efficacious because the germ carries out a limited series of replication cycles and simulates a low-level infection which induces the whole spectrum of antiviral response in a physiological setting. in the case of lentiviruses, one of the most spectacular findings was that which showed that a defective nef-deleted siv virus induced a protective response against the challenge with highly pathogenic aggressive viable viruses 38 . these experimental data had a natural correlate in the "sydney cohort", made up of 14 patients infected through blood transfusion from a seropositive donor and who, after 12 years of infection, had an excellent clinical and immunological status. the cloning and characterization of the virus in these patients and the donor showed that it presented deletions in the nef gene and in critical regulatory sequences of the ltr region 39 . these findings led to the proposal of attenuated hiv vaccines similar to defective siv mutants. however, it must be stressed that attenuated vaccines are usually used against viruses which do not persist or, alternatively, the attenuated virus used as vaccine is not capable of persisting in the host. this is not the case for nef-defective viruses which not only infect, but also replicate and persist in the host, with the risk of drifting towards more aggressive variants in the vaccinated subject. the first alarming data came from vaccination of newborn maca ques in which, in contrast with was found in adults, the innocuous virus rapidly induced aggressive infection and death by immunodeficiency 40 . furthermore, prolonged follow-up of patients from the sydney cohort enabled us to observe an immunological deterioration and blips of viremia in some subjects 41 . similarly, some adult maca ques vaccinated with the defective siv virus developed aids from the virus they had been vaccinated with, which suffered reversions of the mutant phenotype 42 . therefore, the use of vaccines from defective viruses has been ruled out, and this approach has been explicitly excluded in guidelines and recommendations of regulatory agencies. inactivated viruses have scarcely been used as preventive vaccines. on the contrary, this is the most widely used model in therapeutic vaccines of which remune ® is the prototype. these viral preparations are composed of complete virions or particles whose envelope has been eliminated, which are then inactivated using different chemical methods and administered in conjunction with freund's incomplete adjuvant 43 . the first hiv vaccines were based on the hepatitis b immunization model. they were composed of recombinant proteins gp 120 and gp 160 produced by genetic engineer-ing or using vaccinia virus as expression vectors. in preclinical studies and in phase i and phase ii clinical trials, the preparation was safe and induced antibody synthesis against the viral envelope 44 , but these antibodies were incapable of neutralizing wild-type variants "in vitro" 45 . in spite of the evidence against the efficacy of this prototype, phase iii trials were continued (see below). other trials have used the regulatory protein tat in toxoid form, which has provided good protection results in macaque studies, although its role remains controversial 46 . peptide vaccines have little immunogenic capacity, since, in many cases, the antibodies do not recognize the primary structure of the aminoacid sequence, but rather secondary and tertiary structures in the target proteins which are not simulated by the peptides. therefore, peptides are generally used in combination with other vaccine preparations such as viral vectors or dna in order to induce complementary immunization 47 . the advantages of these combinations are low toxicity, the possibility of preparing peptide "cocktails" which cover a wide range of viral isolates in proteins presenting high variability, and the use of "mixed peptides" which, by including t and b immunodominant epitopes induce cellular and humoral responses. these systems use viruses or bacteria into whose genome hiv genes are inserted in such a way that their proteins are expressed during the course of replication of the vectors in the host cell. the most developed models are those which use poxvirus (vaccinia, canarypox, modified ankara virus/mva) 48 and adenovirus 49 . other experimental approaches use bacteria (bcg, salmonella) 50 and rna viruses (coronavirus, vsv, sfv, reovirus, poliovirus, influenza) including also lentivirus 51 . some of these systems are limited by the risk that exogenous genetic information from the vector can be integrated in the host genome. the advantage of these viral and bacterial systems lies in the possibility of inserting several viral genes in their genomes and their capacity to express high levels of viral proteins. strong antigen expression can in turn induce a potent and prolonged immune stimulation, particularly of cellular immune responses, against these proteins. the vaccine prototypes currently being developed include the genes gag, pol, env and nef in different combinations 48, 49 , different priming-boosting strategies and vaccine doses. these types of preparation have failed as preventive vaccines in animal models, since they have not achieved protective immunity, probably due to the fact that the humoral response induced against hiv proteins is erratic and of reduced potency. they do, however, induce a potent cellular response which makes viral load stabilize at low levels 48, 49 . in the most optimistic scenario it has been suggested that this response could be enough to "attenuate" the infection and transforming vaccinated patients who become infected into "long-term survivors". new vectors, such as bcg, salmonella and poliovirus are expected to induce greater humoral and cellular immunity in the mucosa by means of oral administration, thus improving the efficacy of these vaccines. the observation that "naked dna" is capable of inducing an immune response against several viruses and in different animal models broke new ground in the development of vaccines 52 . in infection models with siv and shiv, it has been observed that, as with microbial vectors, immunization with dna is capable of inducing an immune response which, although it does not protect against infection, can often attenuate viral replication and clinical symptoms 53 . the main limitation of dna vectors is that the intensity of the immune response induced is low, therefore they are generally used in combination with viral vectors. a disadvantage of this type of vaccine is the potential long-term secondary effects owing to chromosomal integration processes. adjuvants are preparations which boost the immune response of vaccine antigens by different mechanisms. traditional adjuvants, such as freund's, are bacterial lysates which, by inducing a non-specific inflammatory response, "recruit" immune cells at the injection site. others, such as iscom or liposomes improve the presentation of antigens. recent studies have demonstrated the efficacy of interleukins, especially these activating th1 responses (interleukins 2 and 12) or chemokines, in boosting the response induced by attenuated-vectors or naked-dna vaccines 54 . successive inoculation with an interval of some weeks using two different vectors expressing the same hiv antigen (prime/booster) has been shown to induce a stronger cellular immune response against hiv antigens than when the same vector is administered in two doses. these procedures, which boost specific cd8 t cell induction, were developed in the murine malaria system by showing that this increase correlates with protection against the pathogen 55 . one of the best systems reported is based on recombinant poxviruses, especially the attenuated vaccinia virus ankara (mva). this vector must be administered at the second immunization (booster), whereas in the first inoculation (priming), dna, capsids and other viral protein-expressing vectors can be used indiscriminately. the most promising prime/booster combinations include dna/pox, sfv/pox, and adeno/pox. there is currently no available preventive hiv vaccine. in fact, previously described strategies have failed because no one single animal has been protected from infection in any experimental model. table 2 . this trial has raised controversy over whether it should be carried out, since both the experimental results and the immune response induced by these vaccine preparations have been very limited 58, 59 . the most advanced pre-clinical protocols of the new vaccine prototypes include that being developed by aventis pasteur in uganda, which uses a canarypox vector for the expression of viral structural proteins 60 . also in uganda, january 2003 saw the start of a phase i trial combining dna + mva (strain a) 61 . a similar phase i clinical trial sponsored by iavi and kavi is being carried out in kenya. unfortunately, it seems unlikely these assays go on due to the low proportion of individuals in which a relevant immune response has been elicited with the vaccine preparations tested. in europe, through the eurovacs initiative a clinical phase i trial using poxviral-based vaccine ny-vav has been completed by juin 2004. another phase i trial based on the combination dna/nyvac expressing gp120, gag, pol and nef of subtype c was launched also in juin 2004. a comparative assay between nyvac-c and mva-c is planned in 2005. this latter immunogen will be generated in spain at the national centre of biotechnology. complete and updated information on the situation of existing vaccines and clinical trials is available at: www. hvtn.org/trials. obtaining an aids vaccine has become a global enterprise 2-5 . this is very positive because it has increased social awareness of the disease and financial support. it is also important to note that priority given to vaccine research and the development of new approaches appears at a time when we know much more about the pathogenesis of aids than ten years ago. nevertheless, it must be remembered that demonstrating the usefulness of a vaccine is a long and expensive process. therefore, a critical aspect of the problem is to define strategies and criteria for the different phases in vaccine development: type of vaccine, objectives of the vaccine, experimental design, animal models, pre-clinical and laboratory analysis, phase i and ii trials and requirements and infrastructure for phase iii trials. given the healthcare, social and political priorities, this subject is sometimes affected by serious concerns outside the scientific world. these include the social pressure from international organizations and countries devastated by the epidemic and financial pressure from the pharmaceutical companies. although some of these motives are understandable, given the severity of the situation, these attitudes can also distort the scientific process. below, we summarize the key questions in the search for an "aids vaccine". some scientists doubt that an efficacious aids vaccine can be found 62 . the reason is the difficulty in obtaining what has been defined as "sterilizing immunity" against retroviruses. if we analyze the mechanisms of action of vaccines, in most cases they do not achieve "sterilizing im-munity", since they do not prevent infection but rather the persistence of the microorganism and development of disease: the germ infects, but the immune response prevents it from spreading and destroying new infected cells, thus helping to eradicate the infection. in the case of siv and hiv infection, we know that, after the first inoculum, infection takes place in a short period of time and an important reservoir of cells from the lymphoid system become infected. in some of these initially infected cells, the virus replicates actively, but in others it remains in a state of latency as an integrated provirus in the genome of the host cell. therefore, despite the immune response induced by vaccines, the virus can "persist" in the reservoirs from where it can replicate continuously. a particularly controversial area is the "final objective" of the vaccine: some people argue that if it is not possible to induce "sterilizing immunity" to prevent infection, will it be enough to have an immune response capable of controlling the level of viral replication to sufficiently low levels that allow the immune system to escape from huge destruction. the objective would not be so much to prevent infection but to attenuate it, in such a way that the infected patients become "long-term survivors" capable of living with the virus. another area of debate is the level of protection which must be "reached" by an aids vaccine. in contrast with the high efficacy of protection in most vaccines (above 90% of vaccinated patients) different authors propose that a partial protection rate of 25-40% should be considered as "sufficient". this reduction in the final objectives to be attained by an hiv vaccine is arguable. on one hand, it is doubtful that "attenuation of the infection" will be a definitive phenomenon in the medium-long term. both in animal models and in isolated cases in which an infection has been caused by a defective virus, this virus increases its virulence in the long-term. on the other hand, although it is true that the establishment of a low viral load after primary infection is a good prognostic factor in the medium term, this does not guarantee that patients who present low levels of viral load after vaccination will behave as long-term survivors. the fact that some scientists set the "sufficient" efficacy of a vaccine at 30-40% protection level can also be criticized. this could, perhaps, be considered a realistic stance and, even in specific prevalence rates in specific risk groups, a vaccine of this type could be efficacious, but we do not know its real impact on the evolution of the epidemic in the medium term. we must not forget that one of the mechanisms of vaccine efficacy is due to the "population or epidemiological impact" in the decrease in the prevalence of the infection and the consequent reduced possibility of transmitting the germ among the general population. this epidemiological impact of the vaccine would not exist with the proposed protection rates. some authors suggest that variability among subtypes represents an important obstacle for the development of a universal vaccine and that "ad hoc" vaccines should be manufactured based on the subtypes circulating in each region 23 . however, the new vaccine prototypes use other viral genes (env, nef, gag, pol, tat) as targets which have a much lower variability than the envelope. in fact, different studies show that the immune response induced by vaccination against a specific hiv subtype is capable of acting against other subtypes 63, 64 . how, when and where is the efficacy of the different vaccines to be evaluated? the efficacy of an aids vaccine must be evaluated in populations with a high rate of infection in order to obtain significant differences between the control group and the vaccinated group in the shortest time possible. this means that almost all the trials are carried out in africa and southeast asia, where annual seroconversion rate in the most affected areas is approximately 1% of the population 1 . carrying out trials in developing countries raises a series of ethical issues: 1. it is essential that the studies comply with all ethical requirements and that patients' rights are guaranteed 65 . 2. the vaccines tried must have satisfied the scientific and medical requisites of potency and safety which are necessary in any medicine tried on humans. 3. vaccine trials need a wide-ranging follow-up infrastructure which can guarantee patient follow-up. therefore, it is essential to develop healthcare structures and reference centers with the following objectives: recruitment and follow-up of volunteers, extraction, freeze and storage of blood according to standard procedures, and assessment of immunological parameters such as lymphoid populations, cytokine production and neutralizing antibodies. if this requisite is not met, the analysis of results could be skewed and/or incomplete, thus making it impossible to draw conclusions 66 . 4 . one demand by governments is that if a vaccine is efficacious, free access to the vaccine must be guaranteed to the country where the evaluation was carried out. 5. according to the ethical guidelines of unaids, lifelong antiretroviral therapy must be administered to any person infected during the clinical trial 67 . an important problem, now the center of social and scientific controversy, is to define the requirements a vaccine preparation must fulfill to start a phase iii clinical trial. the journal "science" has been the forum for a series of letters from prestigious scientists criticizing investment strategy in the development of an aids vaccine and the initiation of phase iii clinical trials 58, 59 . the strict scientific position defends that there are no consistent data on the efficacy of current vaccine prototypes to carry out phase iii clinical trials. consequently, such investment should be concentrated in basic research in order to get a better understanding on the mechanisms of protective immune responses and to develop new relevant animal models. faced with this stance, a more humanist position bases the start of phase iii trials on the catastrophic situation in developing countries and on the counterargument that, if there are no adequate animal models, it will be anyway necessary to carry out all the phases of the studies, including phase iii, in humans to obtain a definitive response. despite the reticence and pessimism of a large part of the scientific community, the general impression is that phase iii trials will be carried out. it is important to remember the cost and effort involved in these trials, which require the follow-up of 10,000 patients for at least five years to obtain conclusive results. therefore, with regard to aids vaccines, we are living in difficult times in which a huge economic investment will be necessary so that the scientific community can generate, develop and evaluate all the vaccine prototypes imaginable in animal models in order to find the holy grail of vaccines. as a reference, in case the european union decided to start a program of phase i and ii clinical trials with a reduced number of vaccine prototypes already generated in european laboratories an investment of 1.2 billions euros in the following 10 years should be required. with this objective in mind, the development of vaccine research centers has been proposed 68 . these centers would combine: (i) a critical mass of investigators, (ii) their sole dedication to the development of prototype hiv vaccines, (iii) a long-term commitment by academic, governmental and private institutions, (iv) sufficient resources and (v) continuous exchange of information and collaboration with the private sector. as a consequence of this policy the main leader organizations (nih, iavi, anrs, eu, gates foundation...) should finance vaccine development centers and would coordinate their work. the prototypes considered interesting would be prepared under the conditions of good manufacturing practice for use in humans and would enter a previously defined process of pre-clinical studies and phase i, ii and iii clinical trials. all the prototypes would meet the minimum requirements for clinical application, which would mean not only defining these criteria but also involving the regulatory authorities (fda, emea) in their development. the evaluation of prototypes also requires the definition of those immunological markers which must be used to evaluate their potential efficacy. this in turn would mean developing standardized and reproducible trials to evaluate the humoral and cellular responses to hiv and the approval of laboratories which would carry out these immunological determinations. lastly, the necessary healthcare structures should be set up to carry out the trials in clinical phases in developing countries, and the ethical criteria to be fulfilled in these trials should be defined. given the large number of current prototypes (table 2), the application of homogeneous evaluation criteria is the only way to reach consistent conclusions which can be extrapolated to all situations. nevertheless, it is important to be aware that this search is full of unanswered questions and that it can fail despite all the efforts made. as it may not be possible to develop a vaccine it may be time to convey this terrible possibility to society. the history of vaccines is defined by the words "empiricism" and "success". no intervention has saved as many lives throughout the history of medicine as vaccines. these successes were often the fruit of the most basic empiricism. however, at present, empiricism cannot serve as the basis of success in the scientific development of an aids vaccine. to conclude, in recent years, the development of an aids vaccine has changed radically due to different factors: the devastating growth of the epidemic, social awareness, fi-nancial investment and, in particular, a better understanding of the pathogenesis. all these new elements enable us to face this challenge rationally and with adequate resources. only scientific effort combined with unprecedented solidarity will allow us to decide whether it is possible to find a vaccine against hiv and whether its application will be sufficient to curb the current aids pandemic. genève: who the global alliance for vaccines and immunization: a millennial challenge accelerating the development and future availability of hiv-1 vaccines: why, when, where, and how? challenges and opportunities for development of an aids vaccines hiv vaccines 1983-2003 mhc-restricted cytotoxic t-cells: studies on the biological role of major transplantation antigens determining t-cell restriction-specificity, function and responsiveness genomewide conserved epitope profiles of hiv-1 predicted by biophysical properties of mhc binding peptides inmunidad humoral en la infección por el vih antibodies and resistance to natural hiv infection protection of macaques against vaginal transmission of a pathogenic hiv-1/siv chimeric virus by passive infusion of neutralizing antibodies rapid evolution of the neutralizing antibody response to hiv type 1 infection neutralizing antibody-independent containment of immunodeficiency virus challenges by dna priming and recombinant pox virus booster immunizations hiv-1 neutralizing antibodies: how full is the bottle? antibodies, viruses and vaccines hiv vaccine design and the neutralizing antibody problem cellular immune responses to hiv. nature vigorous hiv-1-specific cd4+ t cell responses associated with control of viremia quantitation of hiv-1-specific cytotoxic t lymphocytes and plasma load of viral rna immune control of hiv-1 after early treatment of acute infection hiv-1-specific cd4+ t cells are detectable in most individuals with active hiv-1 infection, but decline with prolonged viral suppression the challenge of immune control of immunodeficiency virus effects of in vivo cd8+ t cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine molecular epidemiology of hiv-1 genetic forms and its significance for vaccine development and therapy immunopathogenesis and immunotherapy in aids virus infections antiviral pressure exerted by hiv-1-specific cytotoxic t lymphocytes (ctls) during primary infection demonstrated by rapid selection of ctl escape virus acute phase citotoxic t lymphocyte escape is a hallmarg of simian immunodeficiency virus infection lack of strong immune selection pressure by the immunodominant, hla-a*0201-restricted cytotoxic t lymphocyte response in chronic human immunodeficiency virus-1 infection hiv-specific cd8+ t cell proliferation is coupled to perforin expression and is maintained in nonprogressors ctl ontogeny and viral escape: implications for hiv-1 vaccine design hiv-tailored to fit hiv-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites antibody neutralization and escape by hiv-1 rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus population biology of hiv-1 infection: viral and cd4+ t cell demographics and dynamics in lymphatic tissues the challenge of viral reservoirs in hiv-1 infection dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances trans-infection of t cells hiv preferentially infects hiv-specific cd4+ t cells protective effects of a live attenuated siv vaccine with a deletion in the nef gene genomic structure of an attenuated quasi species of hiv-1 from a blood transfusion donor and recipients live-attenuated, multiply deleted siv causes aids in infants and adult macaques declining cd4 t-cell counts in a person infected with nef-deleted hiv-1 pathogenic conversion of live attenuated siv vaccine is associated with expression of truncated evaluation of hiv-1 immunogen, an immunologic modifier, administered to patients infected whith hiv having 300 to 549 × 10 6 /l cd4 cell counts: a randomized controlled trial advancing aidsvax to phase 3. safety, immunogencity, and plans for phase 3 immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1 tat as one key to hiv-induced immune pathogenesis and tat toxoid as an important component of a vaccine neutralization of hiv-1 by secretory iga induced by oral immunization with a new macromolecular multicomponent peptide vaccine candidate immunization with a modified vaccinia virus expressing simian immunodeficiency virus (siv) gag-pol primes for an anamnestic gag-specific cytotoxic t-lymphocyte response and is associated with reduction of viremia after siv challenge replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity protective immune responses induced by secretion of a chimeric soluble protein from a recombinant mycobacterium bovis bacillus calmette-guérin vector candidate vaccine for human immunodeficiency virus type 1 in small animals lentiviral vectors: turning a deadly foe into a therapeutic agent hiv-1 dna vaccines and chemokines augmentation of immune responses to hiv-1 and simian immunodeficiency virus dna vaccines by il-2/ig plasmid administration in rhesus monkeys il-12 and il-18 act in synergy to clear vaccinia virus infection: involvement of innate and adaptive components of the immune system priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces cd8+ t-cell-mediated protective immunity against malaria preventive hiv vaccine development in thailand aidsvax flop leaves vaccine field unscathed public health. a sound rationale needed for phase iii hiv-1 vaccine trials policy rebuttal. hiv vaccine trial justified a canarypox vaccine expressing multiple human immunodeficiency virus type 1 genes given alone or with rgp120 elicits broad and durable cd8+ cytotoxic t lymphocyte responses in seronegative volunteers effective induction of simian immunodeficiency virus-specific cytotoxic t lymphocytes in macaques by using a multiepitope gene and dna primemodified vaccinia virus ankara boost vaccination regimen why an hiv vaccine is not currently within our grasp. xi croi clade b-based hiv-1 vaccines elicit cross-clade cytotoxic t lymphocyte reactivities in uninfected volunteers a human immunodeficiency virus prime-boost immunization regimen in humans induces antibodies that show interclade cross-reactivity and neutralize several x4-, r5-, and dualtropic clade b and c primary isolates the highest attainable standard: ethical issues in aids vaccines some important issues in the planning of phase iii hiv vaccine efficacy trials unaids. ethical considerations in hiv preventive vaccine research the needfor a global hiv vaccine enterprise our laboratories are funded by redes temáticas cooperativas de investigación en sida (ris g03/173) y de genética clínica y molecular (rig 03/07), "fundación para la investigación y la prevención del sida en españa" (fipse 36365/02;36344/02;12456/03;36259/01), "programa nacional de salud" (saf 2003-09209 y 0037-04-01) and comunidad de madrid. we are grateful to florencia etcheverry for her help in revising the bibliography of this manuscript. key: cord-337897-hkvll3xh authors: yang, zheng rong title: peptide bioinformaticspeptide classification using peptide machines date: 2009 journal: artificial neural networks doi: 10.1007/978-1-60327-101-1_9 sha: doc_id: 337897 cord_uid: hkvll3xh peptides scanned from whole protein sequences are the core information for many peptide bioinformatics research subjects, such as functional site prediction, protein structure identification, and protein function recognition. in these applications, we normally need to assign a peptide to one of the given categories using a computer model. they are therefore referred to as peptide classification applications. among various machine learning approaches, including neural networks, peptide machines have demonstrated excellent performance compared with various conventional machine learning approaches in many applications. this chapter discusses the basic concepts of peptide classification, commonly used feature extraction methods, three peptide machines, and some important issues in peptide classification. proteins are the major components for various cell activities, including gene transcription, cell proliferation, and cell differentiation. to implement these activities, proteins function only if they interact with each other. enzyme catalytic activity, acetylation, methylation, glycosylation, and phosphorylation are typical protein functions through binding. studying how to recognize functional sites is then a fundamental topic in bioinformatics research. as proteins function only when they are bound together, the binding sites (functional sites) and their surrounding residues in substrates are the basic components for functional recognition. studying consecutive residues around a functional site within a short region of a protein sequence for functional recognition is then a task of peptide classification that aims to find a proper model that maps these residues to functional status. a peptide classification model can be built using a properly selected learning algorithm with similar principles to those used in pattern classification systems. the earlier work was to investigate a set of experimentally determined (synthesized) functional peptides to find some conserved amino acids, referred in protease cleavage site prediction, we commonly use peptides with a fixed length. in some cases, we may deal with peptides with variable lengths. for instance, we may have peptides whose lengths vary from one residue to a couple of hundreds residues long when we try to identify disorder regions (segments) in proteins [ 5 ] . figure 9 .2 shows two cases, where the shaded regions indicate some disorder segments. the curves represent the probability of disorder for each residue. the dashed lines show 50% of the probability and indicate the boundary between order and disorder. it can be seen that the disorder segments have variable lengths. the importance of accurately identifying disorder segments is related to accurate protein structure analysis. if a sequence contains any disorder segments, x-ray crystallization may fail to give a structure for the sequence. it is then critically important to remove such disordered segments in a sequence before the sequence is submitted for x-ray crystallization experiments. the study of protein secondary structures also falls into the same category as disorder segment identification, where the length of peptides that are constructs for secondary structures varies [ 6 ] . because the basic components in peptides are amino acids, which are nonnumerical, we need a proper feature extraction method to convert peptides to numerical vectors. the second section of this chapter discusses some commonly used feature extraction methods. the third section introduces three peptide machines for peptide classification. the fourth section discusses some practical issues for peptide classification. the final section concludes peptide classification and gives some future research directions. currently, three types of feature extraction methods are commonly used for converting amino acids to numerical vectors: orthogonal vector, frequency estimation, and bio-basis function methods. the orthogonal vector method encodes each amino acid using a 20-bit long binary vector with 1 bit assigned a unity and the rest zeros [ 8 ] . denoted by s a peptide, a numerical vector generated using the orthogonal vector method is x î b 20×| s | , where b = {0, 1} and | s | is the length of s . the introduction of this method greatly eased the difficulty of peptide classification in the early days of applying various machine learning algorithms like neural networks to various peptide classification tasks. however, the method significantly expands the input variables, as each amino acid is represented by 20 inputs [ 9 , 10 ] . for an application with peptides ten residues long, 200 input variables are needed. figure 9 .3 shows such an application using the orthogonal vector method to convert amino acids to numerical inputs. it can be seen that data significance, expressed as a ratio of the number of data points over the number of model parameters, can be very low. meanwhile, the method may not be able to properly code biological content in peptides. the distance (dissimilarity) between any two binary vectors encoded from two different amino acids is always a constant (it is 2 if using the hamming distance or the square root of 2 if using the euclidean distance), while the similarity (mutation or substitution probability) between any two amino acids varies [ 11 -13 ] . the other limitation with this method is that it is unable to handle peptides of variable lengths. for instance, it is hard to imagine that this method could be used to implement a model for predicting disorder segments in proteins. an example of using the orthogonal vector for converting a peptide to numerical vector for using feedforward neural networks, where each residue is expanded to 20 inputs. adapted from [ 14 ] the frequency estimation is another commonly used method in peptide classification for feature extraction. when using single amino acid frequency values as features, we have the conversion as s ‫ۋ‬ x î ᑬ 20 , meaning that a converted vector always has 20 dimensions. however, it has been widely accepted that neighboring residues interact. in this case, di-peptide frequency values as features may be used, leading to the conversion as s ‫ۋ‬ x î ᑬ 420 . if tri-peptide frequency values are used, we then have the conversion as s ‫ۋ‬ x î ᑬ 8420 . this method has been used in dealing with peptides with variable lengths for peptide classification, for instance, secondary structure prediction [ 15 ] and disorder segment prediction [ 5 ] . one very important issue of this method is the difficulty of handling the large dimensionality. for any application, dealing with a data set with 8,420 features is no easy task. the third method, called the bio-basis function , was developed in 2001 [ 9 , 10 ] . the introduction of the bio-basis function was based on the understanding that nongapped pairwise homology alignment scores using a mutation matrix are able to quantify peptide similarity statistically . figure 9 .4 shows two contours where one functional peptide (sknypivq) and one nonfunctional peptide (sdgngmna) are selected as indicator peptides. we use the term indicator peptides , because the use of some indicator peptides transform a training peptide space to an indicator or feature space for modeling. we then calculate the nongapped pairwise homology alignment scores using the dayhoff mutation matrix [ 12 ] to calculate the similarities among all the functional/positive and these two indicator peptides as well as the similarities among all the nonfunctional/negative peptides and these two indicator peptides. it can be seen that positive peptides show larger similarities with the positive indicator peptide (sknypivq) from the left panel, while negative peptides show larger similarities with the negative indicator peptide (sdgngmna) from the right panel. we denote by s a query peptide and by r i the i th indicator peptide and each has d residue, the bio-basis function is defined as k(s, r ) = exp (s, r ) (r , r ) note that s j and r ij are the j th residues in the query and indicator peptides, respectively. the value of m ( s j , r ij ) can be found from a mutation matrix. figure 9 .5 shows the dayhoff mutation matrix [ 12 ] . from eq. 1 , we can see that k ( s , the equality occurs if and only if s = r i . the basic principle of the bio-basis function is normalizing nongapped pairwise homology alignment scores. as shown in fig. 9 .6 , a query peptide ( iprs ) is aligned with two indicator peptides ( kprt and ykae ) to produce two nongapped pairwise homology alignment scores a ( å 1 = 24 + 56 + 56 + 36 = 172) and b ( å 2 = 28 + 28 + 24 + 32 =112 ) respectively. because a > b , it is believed that the query peptide is more likely to have the same functional status (functional or nonfunctional) as the first indicator peptide. after the conversion using the bio-basis function, each peptide s is represented by a numerical vector x î ᑬ m or a point in an m -dimensional feature space, where m is the number of indicator peptides. note that m = 2 in fig. 9 .6 . the bio-basis function method has been successfully applied to various peptide classification tasks, for instance, the prediction of trypsin cleavage sites [ 9 ] , the prediction of hiv cleavage sites [ 10 ] , the prediction of hepatitis c virus protease cleavage sites [ 16 ] , the prediction of the disorder segments in proteins [ 7 , 17 ] , the prediction of protein phosphorylation sites [ 18 , 19 ] , the prediction of the o-linkage sites in glycoproteins [ 20 ] , the prediction of signal peptides [ 21 ] , the prediction of factor xa protease cleavage sites [ 22 ] , the analysis of mutation patterns of hiv-1 fig. 9 . 5 dayhoff matrix. each entry is a mutation probability from one amino acid (in rows) to another amino acid (in columns). reproduced from [ 14 ] with permission drug resistance [ 23 ] , the prediction of caspase cleavage sites [ 24 ] , the prediction of sars-cov protease cleavage sites, [ 25 ] and t-cell epitope prediction [ 26 ] . as one class of machine learning approaches, various vector machines are playing important roles in machine learning research and applications. three vector machines-the support vector machine [ 27 , 28 ] , relevance vector machine [ 29 ] , and orthogonal vector machine [ 30 ] -have already drawn the attention for peptide bioinformatics. each studies pattern classification with a specific focus. the support vector machine (svm) searches for data points located on or near the boundaries for maximizing the margin between two classes of data points. these found data points are referred to as the support vectors . the relevance vector machine (rvm), on the other hand, searches for data points as the representative or prototypic data points referred to as the relevance vectors . the orthogonal vector machine (ovm) searches for the orthogonal bases on which the data points become mutually independent from each other. the found data points are referred to as the orthogonal vectors . all these machines need numerical inputs. we then see how to embed the bio-basis function into these vector machines to derive peptide machines. the support peptide machine (spm) aims to find a mapping function between an input peptide s and the class membership (functional status). the model is defined as the bio-basis function. as iprs is more similar to kprt than ykae , its similarity with kprt is larger that that with ykae , see the right figure. reproduced from [ 14 ] with permission where w is the parameter vector, y = f ( s , w ) the mapping function, and y the output corresponding to the desired class membership t î {-1, 1}. note that -1 and 1 represent nonfunctional and functional status, respectively. with other classification algorithms like neural networks, the distance (error) between y and t is minimized to optimize w . this can lead to a biased hyperplane for discrimination. in fig. 9 .7 , there are two classes of peptides, a and b . the four open circles of class a and four filled circles of the class b are distributed in balance. with this set of peptides, the true hyperplane separating two classes of peptides, represented as circles, can be found as in fig. 9 .7(a) . suppose a shaded large circle belonging to class b as a noise peptide is included, as seen in fig. 9 .7(b) ; the hyperplane (the broken thick line) is biased because the error (distance) between the nine circles and the hyperplane has to be minimized. suppose a shaded circle belonging to class a as a noise peptide is included as seen in fig. 9 .7(c) ; the hyperplane (the broken thick line) also is biased. with theses biased hyperplanes, the novel peptides denoted by the triangles could be misclassified. in searching for the best hyperplane, the spm finds the set of peptides that are the most difficult training data points to classify. these peptides are referred to as support peptides . in constructing a spm classifier, the support peptides are closest to the hyperplane within the slat formed by two boundaries ( fig. 9 .7d ) and are located on the boundaries of the margin between two classes of peptides. the advantage of using an spm is that the hyperplane is searched through maximizing this margin. because of this, the spm classifier is robust; hence, it has better generalization performance than neural networks. in fig. 9 .7(d) , two open circles on the upper boundary and two filled circles on the lower boundary are selected as support peptides. the use of these four circles can form the boundaries of the maximum margin between two classes of peptides. the trained spm classifier is a linear fig. 9 .7 a hyperplane formed using conventional classification algorithms for peptides with a balanced distribution. b and c hyperplanes formed using conventional classification algorithms for peptides without balanced distribution. d hyperplane formed using spm for peptides without balanced distribution. the open circles represent class a , the filled circles class b , and the shaded circle class a or b . the thick lines represent the correct hyperplane for discrimination and the broken thick lines the biased hyperplanes. the thin lines represent the margin boundaries. the γ indicates the distance between the hyperplane and the boundary formed by support peptides. the margin is 2γ. reproduced from [ 14 ] with permission combination of the similarity between an input peptide and the found support peptides. the similarity between an input peptide and the support peptides is quantified by the bio-basis function. the decision is made using the following equation, y = sign {σ w i t i k ( s , r i )}, where t i is the class label of the i th support peptide and w i the positive parameter as a weight for the i th support peptide. the weights are determined by a spm algorithm [ 31 ] . the relevance peptide machine (rpm) was proposed based on automatic relevance determination (ard) theory [ 32 ] . in spm, the found support peptides are normally located near the boundary for discrimination between two classes of peptides. however, the relevance peptides found by rpm are prototypic peptides. this is a unique property of rpm, as the prototypic peptides found by a learning process are useful for exploring the biological inside. suppose the model output is defined as where t is a similarity vector and w = ( w 1 , w 2 , . . ., w n ) t . the model likelihood function is defined as follows: using ard prior to computation can prevent overfitting the coefficients by assuming each weight follows a gaussian where α = (α 1 , α 2 , . . . , α n ) t . the bayesian learning method gives the following posterior for the coefficients: where the parameters (covariance matrix σ , the mean vector u , and the hyperparameters for weights s ), which can be approximated: and the marginal likelihood can be obtained through integrating out the coefficients: in learning, α can be estimated in an iterative way: a formal learning process of the rpm is then conducted. the following condition is checked during learning: where θ is a threshold. if the condition is satisfied, the corresponding expansion coefficient is zeroed. the learning continues until either the change of the expansion coefficients is small or the maximum learning cycle is approached [ 33 ] . figure 9 .8 shows how the rpm selects prototypic peptides as the relevance peptides, compared with the spm, which selects boundary peptides as the support peptides. first, we define a linear model using the bio-basis function as y = kw (16) where k is defined in eq. 10 . the orthogonal least square algorithm is used as a forward selection procedure by the orthogonal peptide machine (opm). at each step, the incremental information content of a system is maximized. we can rewrite the design matrix k as a collection of column vectors ( k 1 , k 2 , . . . , k m ), where k i represents a vector of the similarities between all the training peptides and the i th the opm involves the transformation of the indicator peptides ( r i ) to the orthogonal peptides ( o i ) to reduce possible information redundancy. the feature matrix k is decomposed to an orthogonal matrix and an upper triangular matrix as follows: where the triangular matrix u has ones on the diagonal line: the orthogonal matrix satisfies where h is diagonal with the elements h ii as the space spanned by the set of orthogonal peptides is the same space spanned by the set of indicator peptides, and eq. 16 can be rewritten as we can define an error model as follows: suppose e ~ n (0, 1), the pseudoinverse method can be used to estimate a as follows: because h is diagonal, the element in a is then the quantities a and w satisfy the triangular system the gram-schmidt or the modified gram-schmidt methods are commonly used to find the orthogonal peptides then to estimate w [ 30 , 34 ] . hiv/aids is one of the most lethal and transmittable diseases, with a high mortality rate worldwide. the most effective prevention of hiv infection is to use a vaccine to block virus infection [ 34 ] . however, hiv vaccine is difficult to develop because of the expense and complexity in advancing new candidate vaccines. although some efficient models and integrated hiv vaccine research enterprises have been proposed [ 36 ] , there is little hope that an hiv vaccine would be developed before 2009 [ 37 ] . hiv is a type of retrovirus. it can enter uninfected cells to replicate itself. inhibiting viral maturation and viral reverse transcription are then the major methods so far for treating hiv-infected patients. two groups of inhibitors have since been developed. one group aims to stop protease cleavage activities and is referred to as the protease inhibitor . the other aims to stop reverse transcriptase cleavage activities and is referred to as the reverse transcriptase inhibitor . however, hiv often develops resistance to the drugs applied. drug-resistant mutants of hiv-1 protease limit the long-term effectiveness of current antiviral therapy [ 38 ] . the emergence of drug resistance remains one of the most critical issues in treating hiv-1-infected patients [ 39 ] . the genetic reason for drug resistance is the high mutation rate along with a very high replication rate of the virus. these two factors work together, leading to the evolution of drug-resistant variants and consequently resulting in therapy failure. the resistance to hiv-1 protease inhibitors can be analyzed at a molecular level with a genotypic or a phenotypic method [ 39 , 40 ] . the genotypic method is used to scan a viral genome for some mutation patterns for potential resistance. as an alternative, the viral activity can be measured in cell culture assays. yet another alternative, phenotypic testing is based on clinic observations, ithat is, directly measuring viral replication in the presence of increasing drug concentration. genotypic assays have been widely used as tools for determining hiv-1 drug resistance and for guiding treatment. the use of such tools is based on the principle that the complex impact of amino acid substitutions in hiv reverse transcriptase or protease on the phenotypic susceptibility or clinical response to the 18 available antiretroviral agents is observable [ 41 ] . genotypic assays are used to analyze mutations associated drug resistance or reduced drug susceptibility. however, this method is problematic, because various mutations and mutational patterns may lead to drug resistance [ 39 ] , and therefore the method occasionally fails to predict the effects of multiple mutations [ 42 ] . in addition, genotypic assays provide only indirect evidence of drug resistance [ 43 ] . although hiv-1 genotyping is widely accepted for monitoring antiretroviral therapy, how to interpret the mutation pattern associated with drug resistance to make accurate predictions of susceptibility to each antiretroviral drug is still challenging [ 44 ] . phenotypic assays directly measure drug resistance [ 43 ] , where drug resistance can be experimentally evaluated by measuring the ratio of free drug bound to hiv-1 protease molecules. however, this procedure is generally expensive and time consuming [ 39 , 42 ] . to deal with the difficulties encountered in genotypic assay analysis and phenotypic evaluation, a combination of inhibitor flexible docking and molecular dynamics simulations was used to calculate the protein-inhibitor binding energy. from this, an inhibitory constant is calculated for prediction [ 39 ] . later, some statistical models were established for predicting drug resistance. for instance, a statistical model was proposed to analyze the viral and immunologic dynamics of hiv infection, taking into account drug-resistant mutants and therapy outcomes [ 45 ] . the factors causing the increase in cd4 cell count and the decrease in viral load from baseline after six months of haart (highly active antiretroviral therapy) were analyzed. note that cd4 stands for cluster of differentiation 4, which is a molecule expressed on the surface of t helper cells. in addition to the baseline viral load and cd4 cell count, which are known to affect response to therapy, the baseline cd8 cell count and resistance characteristics of detectable strains are shown to improve prediction accuracy. note that cd8 stands for cluster of differentiation 8, which is a membrane glycoprotein found primarily on the surface of cytotoxic t cells. a logistic regression model was built for the prediction of the odds of achieving virology suppression after 24 weeks of haart in 656 antiretroviral-naive patients starting haart according to their week 4 viral load [ 46 ] . a regression model was built on a set of 650 matched genotype-phenotype pairs for the prediction of phenotypic drug resistance from genotypes [ 47 ] . as it was observed that the range of resistance factors varies considerably among drugs, two simple scoring functions were derived from different sets of predicted phenotypes. the scoring functions were then used for discrimination analysis [ 48 ] . in addition, machine learning algorithms were used. decision trees as a method for mimicking human intelligence were implemented to predict drug resistance [ 43 ] . a fuzzy prediction model was built based on a clinical data set of 231 patients failing highly active antiretroviral therapy (haart) and starting salvage therapy with baseline resistance genotyping and virological outcomes after three and six months [ 49 ] . in the model, a set of rules predicting genotypic resistance was initially derived from an expert and implemented using a fuzzy logic approach. the model was trained using the virological outcomes data set, that is, stanford's hiv drug-resistance database (stanford hivdb). expert systems were also used for this purpose [ 41 ] . neural networks as a type of powerful nonlinear modelers were utilized to predict hiv drug resistance for two protease inhibitors, indinavir and saquinavir [ 50 ] . other than using sequence information for prediction, a structure-based approach was proposed to predict drug resistance [ 42 ] . models of wt complexes were first produced from crystal structures. mutant complexes were then built by amino acid substitutions in the wt complexes with subsequent energy minimization of the ligand (a small molecule binding to a protein or receptor) and pr (protease receptor) binding site residues. a computational model was then built based on the calculated energies. the data set studied was obtained from an online relational database, the hiv rt and protease database ( http://hivdb.stanford.edu ) [ 51 ] . the susceptibility of the data to five protease inhibitors were determined, saquinavir (sqv), indinavir (idv), ritonavir (rtv), nelfinavir (nfv) and amprenavir (apv). we obtained 255 genotype-phenotype pairs for each of the protease inhibitor, except for apv, for which we obtained 112 pairs. phenotypic resistance testing measures in-vitro viral replication of the wild type and the viral sample in increasing drug concentrations [ 52 ] . the resistance factor, defined as the ratio of 50% inhibitory concentration (ic 50 ) of the respective viral sample to ic 50 of the nonresistant reference strain, reports the level of resistance as the fold change in susceptibility to the drug as compared to a fully susceptible reference strain. while genotypic resistance testing is done by scanning the viral genome for resistance-associated mutations. the major and minor mutations observed in a resistant hiv protease can be seen in figure 1 in [ 53 ] . hiv protease is an aspartyl protease with 99 residues. in this study, we considered major and minor mutation sites, obtaining a window size of 20 residues for each drug. we divided the sample set into two classes by attaching to each peptide a label 1 or -1 for resistant (functional) and susceptible (nonfunctional), respectively. this division depended on whether the resistance factor of a sample exceeded the cutoff value or not. based on previously published datasets and studies, we assumed the cutoff value for the resistant factor of each sample with respect to each protease inhibitor to be 3.5. we used matthews correlation coefficient [ 54 ] the larger the matthews correlation coefficient, the better the model fits the data. if the value of the matthews correlation coefficient is 1, it represents a complete correlation. if the value is 0, the prediction is completely random. if the value is negative, the prediction is on the opposite side of the target. the evaluation is carried out based on the simulation of fivefold cross-validation for each drug and each algorithm. figure 9 .9 shows the comparison between vector machines and peptide machines. it can be seen that the peptide machines outperformed the vector machines. we also constructed neural networks models for this task, their performance is far worse than vector machines (data are not shown). in building a computer model for a real application, there are two important issues for model validation in addition to model parameter estimation. the first is how to evaluate a model. the second is how to select a model based on the evaluation. there are always some hyperparameters for determination when using neural networks or machine learning algorithms. for instance, the number of hidden neurons in feedforward neural networks is such a hyperparameter, and the determination of the optimal number of hidden neurons is not an easy task. using the training data for the evaluation will not deliver meaningful result, as an optimization process can easily overfit the data, leading to poor generation capability. the generalization capability should measure how well a built model works with novel data. based on this requirement, we then need to consider how to use the available data for proper model validation. there are three commonly used methods for this purpose: cross-validation, resampling, and jackknife. all these methods use the same principle, that the validation data must not involve any process of model parameter estimation. this means that the available data must be divided into two parts. one is for model parameter estimation, which is commonly referred to as training . the other is for model evaluation (validation) and selection. the difference between these three methods is the strategy used for the division of a given data set. with the resampling method, we normally randomly sample a certain percentage of data for training and the rest for validation. such a process is commonly repeated many times. suppose there are n data points and we repeat the sampling process for m times. there will be m validation models, each of which has different training and validation data with some possible overlap. note that all these validation models use the same hyperparameters; for instance, they all use h hidden neurons. the parameters of the i th validation model are estimated using the i th training data set with k i < n data points. the i th validation model is validated on the i th validation data set with n -k i data points. because we use different training data sets each time, it is expected that the parameters of the i th validation model will be different from those of the j th validation model. the validation performance of the i th validation model is certainly different from that of the j th validation model as well. we denote by ω i the evaluated performance for the i th validation model. the evaluation statistic of the model with designed hyperparameters can follow to determine the proper values for the hyperparameters so as to select a proper model, we can vary the values assigned to the hyperparameters. if we have g hyperparameters for selection, the selection will be taken in a g -dimensional space, where each grid is a combination of hyperparameters. suppose we need to determine only the number of hidden neurons, we then have a series of m and s 2 . the best model can be selected through it should be noted that, for the resampling method, some data points may be used for multiple times in training or validation. in cross-validation, we normally randomly divide a data set into m folds. each fold contains distinctive data points. if we denote by w i as the set of data points in i th fold, we have w i ç w j = φ, meaning that two sets have no elements in common. every time, we select one fold as the validation set and the remaining m -1 folds are used as the training set for model parameters estimate. such a process is repeated for m times, until each fold has been used for validation once. this means that there are m validation models. again, all these validation models use the same hyperparameters. the i th validation model is trained using the folds except for the i th fold and validated on the i th fold. the parameters of the i th validation model also is different from those of the j th validation model, and the validation performance of different validation models will vary. note that each data point ise validated only once. we denote by when data size is not too large, one commonly prefers using the jackknife (often called leave-one-out cross-validation ) method. in using the jackknife method, we normally pick up one data point for validation and use the remaining data points for training. such a process is repeated for n times until each data point has been exhausted for validation. this means that there are n validation models for n data points. obviously, all these validation models use the same hyperparameters. the i th validation model is trained using all the data points except for the i th data point and validated on the i th data point. equation 31 can be used for evaluation. the best model can be selected through the use of eq. 30 . model validation can help us evaluate models and select models with a proper setting of hyperparameters. however, such evaluation values cannot be regarded as the final model evaluation, which can be delivered to users. the evaluated performance on validation data may not be able to reflect the true performance, because the validation data has been used to tune hyperparameters. as a consequence, the evaluated performance on validation data commonly overestimates the true model accuracy. based on this understanding, a proper peptide classification methodology must contain a blind test stage. this means that, after model evaluation and model selection, we may need to test the "best" model using another data set, which has never been used for estimating model parameters and tuning model hyperparameters or selecting models [ 7 ] . a very important issue must be discussed here, especially for peptide classification. many peptide classification tasks deal with functional site predictions. within each protein sequence, there might be a few functional sites, such as protease cleavage sites, protein interaction sites, or protein posttranslational modification sites. based on the substrate size, we can extract a peptide around one functional site. such a peptide is commonly regarded as a functional peptide. to conduct proper peptide classification, we have to have a set of nonfunctional peptides. each nonfunctional peptide has no functional site at the desired residue(s). we normally use a sliding window with a fixed length to scan a protein sequence from the n-terminal to the c-terminal, one residue by one residue to generate no-functional peptides. a commonly used method is to combine both functional and nonfunctional peptides to produce a data set. suppose we use the cross-validation method, the data set is then randomly divided into m folds for cross-validation. one then builds m validation models for model evaluation and model selection. now a question arises. when we use such kind of models for testing on novel whole protein sequences, the performance is commonly not as expected. this means that the model is some where overfitted. if model parameter estimation follows a proper procedure, the most probable problem is the data used for training and validation. we mentioned previously that we must maintain the independence of a validation data set from a training data set. when we check the method as described, we can clearly see two interesting facts. first, a training peptide and a validation peptide may come from the same protein. if one protein has conserved amino acids, the validation peptides generated this way may not be independent of the training peptides. second, a training peptide and a validation peptide may have many identical residues if they are extracted from neighboring sliding widows. based on this analysis, we proposed to use protein-oriented validation [ 24 ] . suppose we have n proteins, we may divide these n proteins into m folds. we then generate validation peptides using one fold and generate training peptides from the remaining folds. the validation models are constructed using the training peptides scanned from the sequences of the training proteins and verified on the validation peptides scanned from the sequences of the validation proteins. we always have an important issue when using machine learning approaches for peptide classification, that is, if the model is correct forever. the answer is no, as a peptide data set collected at a certain time can be far less than complete. based on this understanding, the models built using an incomplete set of peptides may not be able to generalize well forever. when new experimentally determined peptides have been collected, the exiting models must be corrected so that they can conduct classification tasks well. in this section, we investigate an interesting peptide classification topic, where we can use a model built on published peptides for peptide classification, and we can use a model built on both published peptides and newly submitted sequences in ncbi (www.ncbi.nih.gov) for peptide classification. we found there is a significant difference between the two. the case studied in this session is about hepatitis c virus (hcv), which is a member of the flaviviridae family [ 55 ] and is the major agent of parenterally transmitted non-a/non-b hepatitis [ 56 , 57 ] . the nomenclature of schechter and berger [ 1 ] is applied to designate the cleavage sites on the peptides, p 6 -p 5 -p 4 -p 3 -p 2 -p 1 -p 1 ' -p 2 ' -p 3 ' -p 4 ' , the asymmetric scissile bond being between p 1 and p 1 ' . two resources are available for modeling. first, research articles have published experimentally determined peptides, cleaved and noncleaved. second, some databank like ncbi has collected many new submissions. each submission contains a whole protein sequence with a number of experimentally determined cleavage sites. in terms of this, there are two strategies for modeling. if we believe that the published peptides are able to represent all the protease cleavage specificity, we can select indicator peptides from these published peptides for modeling. we refer to a model constructed this way as a type-i model . in fact, viruses mutate very rapidly; hence, the published peptides may not be able to represent the cleavage specificity in the recent submissions to ncbi. we can then select more indicator peptides, which show more viral mutation information from the new sequences downloaded from ncbi to build a more informative model referred to as a type-ii model. from published papers (data are not shown), we collected 215 experimentally determined peptides. they are referred to as published peptides in this study. among them, 168 are cleaved, while 47 are noncleaved. twenty-five whole protein sequences were downloaded from ncbi. they are aaa65789, aaa72945, aaa79971, aab27127, baa01583, baa02756, baa03581, baa03905, baa08372, baa09073, baa09075, baa88057, bab08107, caa03854, caa43793, cab46677, gnwvtc, jc5620, np_043570, np_671491, p26663, p26664, p27958, pc2219, and s40770. within these 25 whole protein peptides (data are not shown) are 123 cleavage sites. the cleavage sites with notes of potential, by similarity , or probable were removed. first, we built type-i models using the leave-one-out method. each model is built on 214 published peptides and tested on 1 remaining published peptide. the models are then evaluated in terms of the mean accuracy, which is calculated on 215 leaveone-out testes. the best model is selected and tested on 25 new sequences downloaded from ncbi, which are regarded as the independent testing data. the simulation shows that the noncleaved, cleaved, and total accuracies are 77%, 77%, and 77%, respectively. shown in fig. 9 .10 are the prediction results on five whole protein sequences, where the horizontal axis indicates the residues in whole protein sequences and the vertical axis the probability of positive (cleavage). if the probability at a residue is larger than 0.5, the corresponding residue is regarded as the cleavage site p 1 . the simulation shows that there were too many false positives. the average of false positive fraction was 27%, or 722 misclassified noncleavage sites. second, we built type-ii models. each model is built and validated using all the published peptides (215) plus the peptides scanned from 24 newly downloaded sequences. in this run, the protein-oriented leave-one-out method is used. with this method, 1 of 24 new sequences is removed for validation on a model built using 215 published peptides and the peptides scanned from the remaining 23 new sequences. this is repeated 24 times and the performance is estimated on the predictions on 24 new sequences. the best model is selected and tested on the peptides on the remaining new sequence. these peptides are regarded as the independent testing peptides. the noncleaved, cleaved, and total accuracies are 99%, 83%, and 99%, respectively. it can be seen that the prediction accuracy has been greatly improved compared with the type-i models. shown in fig. 9 .11 is a comparison between the type-i and the type-ii models. it shows that the type-ii models greatly outperformed the type-i models in terms of the performance for noncleaved peptides. more important, the standard deviation in the type-ii models is much smaller, demonstrating high robustness. shown in fig. 9 .12 are the prediction results on five protein sequences using the type-ii models, where the horizontal axis indicates the residues in whole protein tpf total type-i type-ii fig. 9 .11 a comparison between the type-i and type-ii models. it can be seen that the type-ii models performed much better than the type-i models in terms of the increase of specificity (truenegative fraction). therefore, the false-positive fraction (false alarm) has been significantly reduced. tnf and tpf stand for true-negative fraction (specificity) and true-positive fraction (sensitivity). reproduced from [ 58 ] with permission fig. 9.12 the probabilities of cleavage sites among the residues for five whole protein sequences for the type-ii models. the horizontal axes indicate the residues in whole sequences and the vertical axes the probabilities of positives (cleavage sites). the numbers above the percentages are the ncbi accession numbers and the percentages are the false positive fractions and the true positive fractions. the simulation shows that the type-ii models demonstrated very small falsepositive fractions compared with the type-i models. it can be seen that the probability of positives are very clean . reproduced from [ 58 ] with permission sequences and the vertical axis the probability of positive (cleavage). the simulation shows fewer false positives than the type-i models. the reason is that many of the 25 newly downloaded sequences were published after the reports with the published peptides. the published peptides may not contain complete information for all these 25 new sequences. this chapter introduced the state-of-the-art machines for peptide classification. through the discussion, we can see the difference between the peptide machines and other machine learning approaches. each peptide machine combines the feature extraction process and the model construction process to improve the efficiency. because peptide machines use the novel bio-basis function, they can have biologically well-coded inputs for building models. this is the reason that the peptide machines outperformed the other machine learning approaches. the chapter also discussed some issues related with peptide classification, such as model evaluation, protein-oriented validation, and model lifetime. each of these issues is important in terms of building correct, accurate, and robust peptide classification models. for instance, the blind test can be easily missed by some new bioinformatics researchers. protein-oriented validation has not yet been paid enough attention in bioinformatics. there is also little discussion on model lifetime. nevertheless, these important issues have been evidenced in this chapter. we should note that there is no a priori knowledge about which peptide machine should be used. research into the link between these three peptide machines may provide some important insights for building better machines for peptide classification. the core component of peptide machines is a mutation matrix. our earlier work shows that the model performance using various mutation matrices varies [ 10 ] . it is then interesting to see how we can devise a proper learning method to optimize the mutation probabilities between amino acids during model construction. on the active site of proteases, 3. mapping the active site of papain; specific peptide inhibitors of papain sequence and structure based prediction of eukaryotic protein phosphorylation sites the carbohydrates of glycoproteins machine learning algorithms for protein functional site recognition intrinsic protein disorder in complete genomes matching protein beta-sheet partners by feedforward and recurrent neural networks ronn: use of the bio-basis function neural network technique for the detection of natively disordered regions in proteins predicting the secondary structure of globular proteins using neural network models characterising proteolytic cleavage site activity using bio-basis function neural networks a novel neural network method in mining molecular sequence data basic local alignment search tool a model of evolutionary change in proteins. matrices for detecting distant relationships a structural basis for sequence comparisons-an evaluation of scoring methodologies application of support vector machines to biology linear optimization of predictors for secondary structure: application to transbilayer segments of membrane proteins reduced bio-basis function neural networks for protease cleavage site prediction predict disordered proteins using bio-basis function neural networks reduced bio basis function neural network for identification of protein phosphorylation sites: comparison with pattern recognition algorithms predicting the phosphorylation sites using hidden markov models and machine learning methods bio-basis function neural networks for the prediction of the olinkage sites in glyco-proteins predict signal peptides using bio-basis function neural networks a bio-basis function neural network for protein peptide cleavage activity characterisation bio-kernel self-organizing map for hiv drug resistance classification prediction of caspase cleavage sites using bayesian bio-basis function neural networks mining sars-cov protease cleavage data using decision trees, a novel method for decisive template searching predict t-cell epitopes using bio-support vector machines vapnik v (1995) the nature of statistical learning theory the kernel trick for distances sparse bayesian learning and the relevance vector machine orthogonal least squares learning algorithm for radial basis function networks bio-support vector machines for computational proteomics a practical bayesian framework for backpropagation networks orthogonal kernel machine in prediction of functional sites in proteins relevance peptide machine for hiv-1 drug resistance prediction how antibodies block hiv infection: paths to an aids vaccine the need for a global hiv vaccine enterprise hiv vaccine still out of our grasp molecular mechanics analysis of drug-resistant mutants of hiv protease prediction of hiv-1 protease inhibitor resistance using a protein-inhibitor flexible docking approach correlation between rules-based interpretation and virtual phenotype interpretation of hiv-1 genotypes for predicting drug resistance in hiv-infected individuals variable prediction of antiretroviral treatment outcome by different systems for interpreting genotypic human immunodeficiency virus type 1 drug resistance structure-based phenotyping predicts hiv-1 protease inhibitor resistance diversity and complexity of hiv-1 drug resistance: a bioinformatics approach to predicting phenotype from genotype comparison of nine resistance interpretation systems for hiv-1 genotyping the role of resistance characteristics of viral strains in the prediction of the response to antiretroviral therapy in hiv infection use of viral load measured after 4 weeks of highly active antiretroviral therapy to predict virologic putcome at 24 weeks for hiv-1-positive individuals geno2pheno: estimating phenotypic drug resistance from hiv-1 genotypes characterizing the relationship between hiv-1 genotype and phenotype: prediction-based classification construction, training and clinical validation of an interpretation system for genotypic hiv-1 drug resistance based on fuzzy rules revised by virological outcomes predicting hiv drug resistance with neural networks human immunodeficiency virus reverse transcriptase and protease sequence database rapid, phenotypic hiv-1 drug sensitivity assay for protease and reverse transcriptase inhibitors analysis of the protease sequences of hiv-1 infected individuals after indinavir monotherapy comparison of the predicted and observed secondary structure of t4 phage lysozyme classification and nomenclature of virus: fifth report of the international committee on taxonomy of viruses isolation of a cdna clone derived from a blood-borne non-a non-b viral hepatitis genome an assay for circulating antibodies to a major etiologic virus of human non-a non-b hepatitis predicting hepatitis c virus protease cleavage sites using generalised linear indicator regression models key: cord-317990-61is0hgm authors: quinn, katherine g. title: applying the popular opinion leader intervention for hiv to covid-19 date: 2020-06-25 journal: aids behav doi: 10.1007/s10461-020-02954-7 sha: doc_id: 317990 cord_uid: 61is0hgm nan the 2019 novel coronavirus disease (covid-19) pandemic has demanded an immediate public health response and community-wide adoption of various behavior change strategies. public health guidelines and recommendations emerged rapidly and have been modified as the pandemic has shifted. as a result, the public has received inconsistent and occasionally inaccurate information, contributing to misinformation and mistrust of public health recommendations [1] . as jaiswal et al. have recently noted, the spread of medical mistrust and public health misinformation evident in the current covid-19 pandemic mirrors long-standing challenges in the hiv epidemic [1] . accordingly, we can take lessons learned from the hiv epidemic about the spread of public health information and its effects on behavior change apply them to the current pandemic. this note focuses on social networks and the popular opinion leader model, which may be key in disseminating trusted information about covid-19 in a rapidly changing public health landscape. social networks are critical in norm formation and behavioral adoption, and have been shown to impact a variety of public health outcomes, including hiv [2] [3] [4] [5] . since the early years of the hiv epidemic, social network interventions have been used to disseminate trusted information to vulnerable communities [6, 7] . based on diffusion of innovation theory [8] , the popular opinion leader (pol) model posits that behavior change is achieved when new risk-reducing methods for hiv are disseminated by social network opinion leaders through their personal networks. as outlined by kelly, the pol model identifies socially influential members of a population who have sufficient large social networks. these individuals are then trained in how to effectively communicate risk reduction strategies to their peers during everyday conversations and are able to establish momentum as a community social movement [9, 10] . designed as a community-level intervention targeting gay and bisexual men, this intervention has demonstrated effectiveness at increasing hiv prevention knowledge and behaviors, facilitating pep interest and uptake, and reducing hiv-related stigma [10] [11] [12] [13] [14] . social network and pol interventions may be particularly applicable to other public health crises, including covid-19. similar to hiv, the early covid-19 pandemic has been characterized by stigma, misinformation, and medical mistrust [1, 15] . perceptions of blame, personal responsibility, and infection risk contribute to detrimental health outcomes and quality of life among affected individuals and those perceived to be at-risk. furthermore, stereotypes and judgments about disproportionately affected groups can generate stigma and inform the public response to disease [16] . for example, asymptomatic individuals who are infected with coronavirus have been referred to as "carriers" [17] reminiscent of the narrative surrounding "hiv carriers" evident in the early years of the disease. this type of terminology can contribute to fear, public perceptions about who might be a "carrier," and stigma about certain groups of people. the covid-19 outbreak began in wuhan, china, which sparked early reports of racism and xenophobia toward asian americans in the united states (us). since mid-march 2020, there have been over 1,700 reports of racism and xenophobia toward asian americans, including microaggressions and violent attacks [18] . in the months following, the concentration of diagnoses and deaths related to covid-19 in african american and latinx communities illuminated long-standing structural racism in the us [19, 20] , and led to blame of black individuals for high mortality rates within their communities [20] . emerging data show stark racial disparities in covid-19. for example, one analysis found that counties that had majority-black -populations had covid-19 infection rates three times that of majority-white counties [21] . additionally, a centers for disease control and prevention analysis found that of nearly 1500 hospitalizations due to covid-19, nearly one-third were among black people, despite accounting for 18% of the population in the areas studied [22] . similar to the disparities evident in hiv/aids, the stark racial disparities in covid-19 are rooted in systemic and contemporary racism and health and economic inequity that has made black americans more vulnerable to the virus. stigma resulting from xenophobia or racism can limit access to accurate public health information, alienate communities from needed prevention and treatment resources, and further contribute to disparities in disease outcomes [23] [24] [25] . similarly, misinformation about covid-19 has emerged as a result of medical mistrust, which has also plagued the public health response to the hiv epidemic [13, 26, 27] . medical mistrust is a primary driver of racial and ethnic health inequities [28] and can stem from historical and persistent racism, oppression, and disadvantage faced by people of color in the united states. similar to what has been documented in the hiv literature [25] [26] [27] 29] , medical mistrust may contribute to lack of covid-19 testing, delayed access to needed medical care, and misinformation about covid-19. yet, engaging trusted community leaders and social influencers to disseminate accurate public health information may help overcome these challenges to address inequities reduce covid-19 stigma, and strengthen norms that contribute to sustained behavior change (e.g. social distancing, mask wearing, hand washing). trusted influencers from within one's community who endorse covid-19 prevention steps can add trust and credibility to public health messaging, particularly if there is mistrust of official authorities (e.g. medical system, public health departments, and state and local government). our prior research has shown that peer endorsements and health advice from friends and members of one's social network are effective at facilitating population-level hiv behavior change [13, 30] . as we have demonstrated [13, 30] , most community members want to help friends in a time of crises. endorsements by popular opinion leaders within their social networks convey that health protective steps are possible, can reduce stigma, strengthen peer norms concerning the benefits of taking protective measures, and increase solidarity [5, 13, 30] . in racial and ethnic minority communities where medical mistrust is high [31, 32] , advice from personally known and trusted individuals establishes information credibility and can help overcome mistrust and misinformation [33] . given our experience implementing the pol model with populations at risk for hiv, we are currently applying this model to communities being affected by covid-19. as has been documented in many parts of the country, there are significant racial and ethnic disparities in covid-19 in milwaukee [34, 35] . although 6.7% of wisconsin's residents are black, they have comprised 21% of confirmed covid-cases and 29% of covid-related deaths. additionally, over 30% of covid cases are among individuals who identify as hispanic or latino, although they represent just 6.9% of the state's population [35] . with funding from the advancing a healthier wisconsin endowment, our project, 1000 hometown heroes, aims to engage 1000 local "heroes" in milwaukee from predominantly african american and latinx communities in an effort to address the racial and ethnic disparities in covid-19. these 'heroes' are social influencers and leaders on various social media platforms who will share accurate, culturally relevant, tailored information about covid-19 with their networks. we are recruiting pol who see themselves as change agents, want to help protect the health and psychosocial well-being of others in their communities, and are willing to spread the word about covid-19 prevention and reduce disease stigma. additionally, we are providing influencers information to connect community members to needed services, provide strategies to cope with stress, anxiety, and social distancing, and provide accurate, up-to-date information about the status of covid-19 in the milwaukee area. aligned with social distancing guidelines for covid-19 prevention, we are using online social networks as an efficient and effective way to disseminate accurate information and influence community norms and behaviors [36] . the pol model has been successfully adapted to online networks, allowing peer influencers to reach a large number of people in a cost-effective approach [37] . in many ways, this approach is a modernization of efforts implemented during the 1918 flu pandemic. postal workers, boy scouts, and teachers, as trusted community members, were asked to volunteer for health education campaigns and go door-to-door to educate families about public health practices [38] . with the advent of numerous social media platforms, we now have an opportunity for more efficient information spread from a larger segment of the population. over the next six months, we will be evaluating the program's reach and effectiveness, which may provide some early indication about how to quickly disseminate accurate, trusted information during public health crises, particularly to communities hardest hit by an epidemic. adapting the popular opinion model, which has demonstrated significant success in hiv prevention, to covid-19 is one example of how hiv prevention and intervention researchers can take lessons learned in hiv and apply them to current and future pandemics. while much of our current hiv research is on hold indefinitely, we have an opportunity to pivot and use what we have learned from nearly four decades of hiv research to assist in addressing the impact of covid-19. we have a wealth of knowledge about infectious disease, stigma, sustained behavior modification, and social and structural facilitators of health disparities that should be adapted and shared to address other emerging infectious diseases. disinformation, misinformation and inequality-driven mistrust in the time of covid-19: lessons unlearned from aids denialism network mixing and network influences most linked to hiv infection and risk behavior in the hiv epidemic among black men who have sex with men the spread of alcohol consumption behavior in a large social network social norms, social networks, and hiv risk behavior among injection drug users effects of a social network hiv/std prevention intervention for msm in russia and hungary: a randomized controlled trial randomised, controlled, community-level hivprevention intervention for sexual-risk behaviour among homosexual men in us cities social networks, sexual networks and hiv risk in men who have sex with men diffusion of innovations-fourth edition. rogers: everett m popular opinion leaders and hiv prevention peer education: resolving discrepant findings, and implications for the development of effective community programmes hiv risk behavior reduction following intervention with key opinion leaders of population: an experimental analysis impact of a community popular opinion leader intervention among african american adults in a southeastern united states community popular opinion leader intervention for hiv stigma reduction in health care settings the influence of peers on prep perceptions and use among young black gay, bisexual, and other men who have sex with men: a qualitative examination an hiv intervention tailored for black young men who have sex with men in the house ball community comparative stigma of hiv/aids, sars, and tuberculosis in hong kong no signs of coronavirus? here's why you could be carrying (and spreading) it stop aapi hate receives over 1700 incident reports of verbal harassment , shunning and physical assaults structural racism and health inequities in the usa: evidence and interventions stop blaming black people for dying fo the coronavirus the conronavirus is infecting and killing black americans at an alarmingly high rate hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019-covid-net, 14 states applying an intersectional framework to understand syndemic conditions among young black gay, bisexual, and other men who have sex with men how hiv stigma and racism affect perception of risk among young black men who have sex with men a gay man and a doctor are just like, a recipe for destruction": how racism and homonegativity in healthcare settings influence prep uptake among young black msm race-based medical mistrust, medication beliefs and hiv treatment adherence: test of a mediation model in people living with hiv/aids the health and sociocultural correlates of aids genocidal beliefs and medical mistrust among african american msm unequal treatment: institute of medicine (us) committee on understanding and eliminating racial and ethnic disparities in healthcare. unequal treat conspiracy beliefs about hiv are related to antiretroviral treatment nonadherence among african american men with hiv social network intervention to increase preexposure prophylaxis (prep) awareness, interest, and use among african american men who have sex with men race and sexual identity: perceptions about medical culture and healthcare among black men who have sex with men the role of stigma and medical mistrust in the routine health care engagement of black men who have sex with men prep4love: the role of messaging and prevention advocacy in prep attitudes, perceptions, and uptake among ymsm and transgender women milwaukee's coronavirus racial divide: a report on the early stages of covid-19 spread in milwaukee county covid-19 testing up but racial disparities still stark building trust while influencing online covid-19 conent in the social media world effects of internet popular opinion leaders (ipol) among internet-using men who have sex with men lessons learned from the 1918-1919 influenza pandemic in minneapolis acknowledgements special thanks to jeffrey a. kelly, phd for his direction and guidance on this work. funding for 1000 hometown heroes comes from the advancing a healthier wisconsin endowment, which allowed for a swift response from the research community in milwaukee to address the covid-19 pandemic. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-330465-16j5vm7h authors: marciniec, krzysztof; chrobak, elwira; dąbrowska, aleksandra; bębenek, ewa; kadela-tomanek, monika; pęcak, paweł; boryczka, stanisław title: phosphate derivatives of 3-carboxyacylbetulin: synthesis, in vitro anti-hiv and molecular docking study date: 2020-08-05 journal: biomolecules doi: 10.3390/biom10081148 sha: doc_id: 330465 cord_uid: 16j5vm7h lupane-type pentacyclic triterpenes such as betulin and betulinic acid play an important role in the search for new therapies that would be effective in controlling viral infections. the aim of this study was the synthesis and evaluation of in vitro anti-hiv-1 activity for phosphate derivatives of 3-carboxyacylbetulin 3–5 as well as an in silico study of new compounds as potential ligands of the c-terminal domain of the hiv-1 capsid–spacer peptide 1 (ca-ctd-sp1) as a molecular target of hiv-1 maturation inhibitors. in vitro studies showed that 28-diethoxyphosphoryl-3-o-(3′,3′-dimethylsuccinyl)betulin (compound 3), the phosphate analog of bevirimat (betulinic acid derivative, hiv-1 maturation inhibitor), has ic(50) (half maximal inhibitory concentration) equal to 0.02 μm. compound 3 inhibits viral replication at a level comparable to bevirimat and is also more selective (selectivity indices = 1250 and 967, respectively). molecular docking was used to examine the probable interaction between the phosphate derivatives of 3-carboxyacylbetulin and c-terminal domain (ctd) of the hiv-1 capsid (ca)–spacer peptide 1 (sp1) fragment of gag protein, designated as ctd-sp1. compared with interactions between bevirimat (bvm) and the protein, an increased number of strong interactions between ligand 3 and the protein, generated by the phosphate group, were observed. these compounds might have the potential to also inhibit sars-cov2 proteins, in as far as the intrinsically imprecise docking scores suggest. despite significant advances in medicine and continuous work on new pharmacotherapy methods, in addition to common preventive vaccination programs, in the first two decades of the 21st century, the world health organization (who) recorded many epidemics of viral diseases. among the great epidemics of this period were severe acute respiratory syndrome (sars) in the year 2003, the influenza h1n1 pandemic in 2009, middle east respiratory syndrome (mers) in 2012, and the zika virus in 2005, as well as the hiv/aids pandemic, which peaked in 2005-2012 [1] . the beginning of 2020 brought a new pandemic produced by the rapidly spreading virus that causes covid-19 disease. in the case of each new pathogen, the science world faces the difficult challenge of finding an effective therapy. chemical modification of natural substances is an important method used to obtain promising new therapeutic agents. pentacyclic triterpenes and their semi-synthetic derivatives are a large group of compounds known to demonstrate biological activity, including antitumor, antiviral, antimalarial, melting points of obtained compounds were measured in open capillary tubes on an electrothermal melting point apparatus without correction. 1 h nmr (600 mhz), 13 c nmr (150 mhz) and 31 p nmr (243 mhz) spectra were recorded on a bruker avance iii hd 600 spectrometer (bruker, billerica, ma, usa) in deuterated cdcl 3 , using the residual solvent signal as an internal standard. chemical shifts values were reported in parts per million (ppm). multiplicity was designated as singlet (s), doublet (d), doublet of doublets (dd) and multiplet (m). infrared spectra (pellets, kbr, merck, darmstadt, germany) were obtained using an iraffnity-1 ftir spectrometer (shimadzu, kyoto, japan). the measurement was recorded in the range of 4000-1000 cm -1 at 295k. high-resolution mass spectra have been measured with bruker impact ii (bruker, billerica, ma, usa). calculation of the theoretical molecular mass for compounds was performed using "the exact mass calculator, single isotope version" (http://www.sisweb.com/referenc/tools/exactmass.htm; (ringoes, nj, usa)). synthesis of bvm [3-o-(3',3'-dimethylsuccinyl)betulinic acid], used in the study as a reference, was performed as was previously described [12] . the final product was obtained at a yield of 70%. the melting point and spectroscopic characteristics of the compound were consistent with the literature data [20] . to the solution of 1 mmol of betulin 1 in 3 ml tetrahydrofuran (thf), pyridine (2.6 mmol, 0.24 ml) and n,n-dimethylaminopyridine (dmap; 0.1 mmol, 12 mg) was added. the obtained mixture was cooled in an ice-water bath to 0 • c, then diethylchlorophosphate (2 mmol, 0.29 ml) was added dropwise. the reaction was carried out under argon atmosphere for 9 h. then, the volatile components were evaporated on a vacuum evaporator. dichloromethane (15 ml) was added to the residue and washed with saturated sodium bicarbonate solution and water. the organic layer was dried with anhydrous sodium sulphate (vi), then concentrated until dry. the product was purified by column chromatography (sio 2 ; hexane/ethyl acetate ratio: 3:2, v/v) yielding compound 2. to the solution of 1 mmol 28-diethoxyphosphorylbetulin 2 in 2 ml pyridine, dmap (1.5 mmol, 0.19 g) and 5 mmol of appropriate acid anhydride (2,2-dimethylsuccinic anhydride or 3,3-dimethylglutaric anhydride or 2,2-dimethylglutaric anhydride) was added. the reaction vessel was placed in a microwave reactor and the reaction was carried out for 1.5 h at a temperature of 130 • c at a maximum wave power (300 w). after cooling, the mixture was diluted with 25 ml of ethyl acetate, and then washed with a 20% hydrochloric acid solution and with water. the organic layer was dried with anhydrous sodium sulphate (vi) and concentrated until dry in a vacuum evaporator. the crude product was purified by column chromatography (sio 2 ; chloroform/ethanol ratio: 15:1, v/v) to obtain phosphorus derivatives of 3-carboxyacyl-28-diethoxyphosphorylbetulin 3-5. for the determination of compounds' cytotoxicity, cem-t4 cells were obtained from the nih aids reagent program (nih, us) and were cultured in rpmi supplemented with 10% fcs (biochrom) and antibiotics at 37 • c in 5% co 2 on 96-well culture plates. the experiments were carried out in media containing tested compounds in concentrations of the appropriate range. cultures in a neat medium (rpmi, 10% fcs) were used as a control. viability of cells was determined after 7 days using the mtt assay [21] in which 10 µl of mtt solution (5 mg/ml) was added to each culture plate well, and cultures were incubated for 3 h at a temperature of 37 • c. after the centrifugation, the supernatant was removed, and dmso was added for lysis of the cells and to dissolve crystals of formazan. color intensity was measured with a plate reader at 560 nm. cem-t4 cells were preincubated (culture plates with 96 flat bottom wells) for 24 h under standard conditions (37 • c, 5% co 2 ) and in a standard medium (rpmi, fcs 10%) enriched with tested compounds in the concentration range from 0.02 to 10 µm. in each well, 20,000 cells were suspended in the solution of a tested compound (200 µl). for each concentration, cultures were run in triplicate. a wild-type hiv-1 was isolated from the hiv-positive patient in the laboratory of virology of the national medicines institute (warsaw, poland) and was used as a reference. a culture of cem-t4 lines in a standard neat medium (rpmu, fcs 10%) was used to produce viruses. after 24 h of incubation in a medium enriched with a tested compound, cells were inoculated with a known amount of hiv, and after 7 days, hiv replication was evaluated through the measurement of secreted viral protein p24 carried out with the enzyme-linked immunosorbent assay (elisa) technique [22] . for each tested compound and for each concentration, the measurements of p24 antigen were done in triplicate using the genscreen ultra hiv ag-ab kit (biorad, warszawa, poland) and following manufacturer's instructions. the 3d structures of studied compounds were generated in their low-energy conformation using the gaussian 16 (revision a.03) computer code [23] with the density functional theory (dft, b3lyp) and 63-11 + g(d,p) basis sets. target macromolecules for molecular docking studies were obtained from the protein data bank (https://www.rcsb.org/; research collaboratory for structural bioinformatics, usa). we used crystal structure of the ca-ctd-sp1 fragment of hiv-1 gag, sars-cov-2 main protein, sars-cov-2 rna-dependent rna polymerase, and sars-cov-2 spike ectodomain (pdb ids: 5i4t, 5r7z, 6m71, and 6vyb respectively). incomplete or missing side chains of ca-ctd-sp1 protein were restored with the swiss-model workspace (https://swissmodel.expasy.org/; swiss institute of bioinformatics, basel, switzerland) [24] . geometry optimization of the model was achieved by the optimization protocol in yasara energy minimization server (http://www.yasara. org/-minimizationserver.htm; yasara biosciences gmbh, vienna, austria). the sars-cov-2 main protease (m pro ) structure was obtained from pdb (pdb id: 5r7z). the native ligand for 5r7z is n-[2-(5-fluoro-1h-indol-3-yl)ethyl]ethanamide (pdb id: hwh). the region of interest used for vina docking was defined as all m pro residues within 40 × 40 × 40 å of the reference ligand. the homology model of the sars-cov-2 e protein catalytic domain was also generated in the swiss-model workspace [24] . based on maximum sequence identity (95%), structure with pdb id 5x29 is detected as the best homologous structure of human sars-cov-2 e protein. geometry optimization of model was done by the optimization protocol in yasara. for toxic/side effect study we used crystal structure of the pyruvate kinase (pdb id: 6nu1), aconitase (pdb id: 1c96), cytochrome c (pdb id: 1hrc), carbamoyl phosphate synthetase 1 (pdb id: 5dou), hypoxanthine-guanine phosphoribosyltransferase (pdb id: 6ar9), glutamate dehydrogenase (pdb id: 1bvg). in this study, autodock vina [25] tool compiled in pyrx [26] was employed to perform molecular docking. autodock vina incorporates limited flexibility in the receptor, and it combines an empirical free-energy force field with a lamarckian genetic algorithm, providing fast prediction of bound conformations with predicted free energies of association. the volume was set as 40 × 40 × 40 å. after calculations, only the 9 highest-scored poses were returned as a docking result for ligand-cavity configuration. all the obtained results were ranked according to their score values and presented in kcal/mol. molecular docking details were visualized using the biovia discovery studio virtual environment [27] . appropriate autodock vina output complexes were prepared for simulations using qwikmd [28] built in visual molecular dynamics (vmd https://www.ks.uiuc.edu/research/vmd/; theoretical and computational biophysics group, university of illinois at urbana-champaign, illinois, usa) software ver 1.9.3 [29] . simulation was carried out with namd (https://www.ks.uiuc.edu/research/namd/; theoretical and computational biophysics group, university of illinois at urbana-champaign, illinois, usa) ver. 2.13 [30] using charmm27 force field. periodic boundary conditions with an explicit solvent were employed. parameterization of ligands was conducted using a cgenff server [31, 32] . all protein and protein-ligand systems have been solvated with tip3p cubic water box at 15 å thickness. to neutralize the system, 0.15 mol/l of nacl salt was added. simulation protocol consists of 2000 steps of minimization, 144,000 steps of annealing, 500,000 steps of equilibration and 5,000,000 steps (10ns) of production md simulation. the system was heated to 300 k at rate of one kelvin degree per 1 ps. langevin dynamics were used to control the temperature. minimization, annealing and equilibration were restrained to backbone atoms of the protein, while the production run was unrestrained. timestep was set to 2 fs, all runs were performed in npt ensemble. vmd was used to analyze the results. the above-mentioned literature data and results of the anti-hiv activity test obtained for phosphate and phosphonate derivatives of betulinic acid [12] have become the starting point for the synthesis of betulin phosphate derivatives. the obtained compounds have a carboxyacyl group in position c3, whose presence is important for action against hiv-1, and a diethyl phosphate group in position c28 (scheme 1). this work attempts to answer the question of how the introduction of a phosphate substituent affects the activity of synthesized compounds, compared to substances with known potential-in this case, bvm. betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3 ,3 -dimethylsuccinic, 4 ,4 -dimethylglutaric, and 3 ,3 -dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc 50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic 50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . as can be seen, betulin 1 and betulin phosphate 2 did not demonstrate the ability to inhibit hiv replication in the tested range of concentrations. betulin derivatives containing a 3 ,3 -dimethylglutaric group that exhibit significant activity against hiv-1 are described in the literature [34, 35] . corresponding phosphorus derivative 4 demonstrated good activity and selectivity that were higher than obtained for 4 ,4 -dimethylglutaric derivative 5. on the other hand, compound 4 was more active than the previously described 3 ,3 -dimethylglutaric derivatives of betulinic acid, which in the c30 position contained a diethylphosphate or diethylphosphonate group (ic 50 = 0.6 µm and 0.9 µm, respectively) [12] . the results obtained for phosphate derivatives of 3-carboxyacylbetulin 3-5 indicate that the most active compound is derivative 3 with r = hoc(o)c(ch 3 ) 2 ch 2 c(o), which shows activity comparable to that of bvm (ic 50 = 0.02 and 0.03 µm, respectively). a chart of changes in hiv-1 inhibition of 3-carboxyacylbetulin phosphate and bvm in the tested concentration range is attached to the supplementary materials ( figure s1 ). however, compound 3 has a higher selectivity for action (therapeutic index (ti) = 1250 and 967, respectively). among the tested phosphate derivatives, compound 3 showed the lowest cytotoxicity, but it was at a level comparable to bvm. other phosphate derivatives were more toxic and also less active against hiv. as results from the data presented in the table 1 , compounds with a longer carbon chain in the carboxyacylic substituent are more cytotoxic. dimethylglutaric derivatives have an ic 50 figure s2 ). an important issue in the study of new chemical substances considered as potential drug candidates is the analysis of their physicochemical parameters. the in silico pharmacokinetic study is being used in the development of new drugs with good oral bioavailability, low toxicity and the least side effects. for phosphate derivatives 2-5 the adme (adsorption-distribution-metabolism-excretion) properties (supplementary materials table s1 ) such as number of hydrogen bond acceptors (nhba), number of hydrogen bond donors (nhbd), lipophilicity (log p), molecular weight m, number of rotatable bonds (nrotb) and topological polar surface area (tpsa) were evaluated through the pkcsm online server (http://biosig.unimelb.edu.au/pkcsm/; university of melbourne, australia) [36] . the hydrophobicity of compounds was determined on the basis of log p values. the log p parameter determines the strength of the drug and its distribution in the body after absorption stage. the obtained log p values (9.20-10.65) of triterpene derivatives indicate poor permeability across the cell membrane. the compounds shown in the table s1 having a number of rotational bonds in the range 8-13 show high conformational flexibility and good binding affinity with the binding pocket. topological polar surface area (tpsa) is important criteria for the determination of oral bioavailability. compounds that meet the criterion that tpsa ≤ 140 å may show oral bioavailability [37, 38] . the values of the parameters log ps, log bb and tpsa for the tested derivatives are characteristic for the cns-nonactive drugs [36] . the tested compounds can exist as two chemical species, carboxylic acids or carboxylate salts, depending on the ph of the aqueous solution. calculations performed with acd/percepta software [39] for bvm and compounds 3-5 show that in all cases the content of the carboxylate states at physiological ph is 100%. therefore, the carboxylate states of betulin derivatives were used in docking calculations. the three-dimensional (3d) structures of ligands required for docking studies were generated in their low-energy conformation using gaussian 16 computer code [24] . the last step of hiv-1 replication occurs after release of immature virion outside host cell. during maturation process, viral protease cleaves gag precursor protein to individual matrix (ma), spacer peptide (sp1), capsid (ca) and nucleocapsid (nc) peptides. this step is essential to produce mature and infectious virions. recent studies report that the immature ca-ctd-sp1 assembles to create a hexameric structure which forms a cone-shaped core built up with multiple hexameric proteins. protease can access the cleavage site only after the six-helix bundle unfolds [40] . based on atomic coordinates of the ctd of ca and sp1 of hiv-1 gag deposited in the protein data bank (pbd id 5i4t), one active site cavity was predicted [41] . this cavity was located inside the pore formed by the six-helix sp1 bundle (figure 1 ). we would like to point out that we used autodock ver.4.2.6 in our previous research on the antiviral activity of betulinic acid derivatives [12] . in the present study, we decided to use the program autodock vina (referred to as vina) for in silico research. vina uses a sophisticated gradient optimization method in its local optimization procedure. the calculation of the gradient effectively gives the optimization algorithm a "sense of direction" from a single evaluation. evaluation of the speed and accuracy of vina during flexible docking showed an improvement in speed of approximately two orders of magnitude, and a significantly higher accuracy of the binding mode prediction compared to autodock [25] . we also decided to redock the bvm molecule to hiv-1 ca-ctd-sp1 with the use of vina software. the betulin derivatives ranked by autodock vina are shown in table 2 . the lowest scores for binding energy (kcal/mol) of protein−ligand complexes correspond to a strong binding affinity, and the most probable ligand−protein system in vivo. results obtained with vina indicate that compound 3 exhibits a lower binding energy compared to the reference bvm ( table 2) . analysis of the bvm complex ( figure 2 ) included calculations, distance measurements, and pose geometries that determined salt bridge interaction of the ligand pose with lys227 residue of chain g and hydrogen bonding with lys158 in chain l. in addition, numerous hydrophobic interactions influence the increased stability of the complex. analysis of the compound 3 complex, the most potent compound in vitro (figure 3 ), determined salt hydrogen bonding between the carboxylate group of the ligand and lys158 residue of chain i, and hydrogen bonding with lys158 in chain l and the phosphate group. in addition, numerous hydrophobic interactions, including carbon−hydrogen bonding, are also visible. analysis of the compound 3 complex, the most potent compound in vitro (figure 3) , determined salt hydrogen bonding between the carboxylate group of the ligand and lys158 residue of chain i, and hydrogen bonding with lys158 in chain l and the phosphate group. in addition, numerous hydrophobic interactions, including carbon−hydrogen bonding, are also visible. analysis of the compound 3 complex, the most potent compound in vitro (figure 3) , determined salt hydrogen bonding between the carboxylate group of the ligand and lys158 residue of chain i, and hydrogen bonding with lys158 in chain l and the phosphate group. in addition, numerous hydrophobic interactions, including carbon−hydrogen bonding, are also visible. in order to validate docking results a molecular dynamics simulation (md) has been performed. after a 10-ns run, a root mean square deviation (rmsd) of the atomic positions of ligand-protein complexes have been calculated. low value of ligand rmsd followed by relatively constant value of protein backbone rmsd indicate complex stability and validate docking protocol. md simulation has been performed for compound 3, possessing the lowest score for binding energy. both ligand and protein showed low rmsd values below 2 å, proving stability of the ligand-protein system ( figure 6 ). the docking results are in line with cytotoxicity binding assay findings, which means that the tested compounds can interact with the ca-ctd-sp1 protein. in vitro studies in human cells, as well as preclinical studies, suggest that bevirimat should have low potential for cytotoxicity. there is no evidence of reproductive or developmental toxicity [42] . in order to check the potential toxic properties of the compounds 3-5, docking study of phosphate betulin derivatives to cellular proteins was carried out. it is known that deficiency and inhibition of some proteins important in normal cellular function may result in toxicity or side effects. examples of these proteins are those involved in key cellular metabolism processes such as glycolytic pathway, amino acid and nucleotide metabolism, urea cycle, citric acid cycle and oxidative phosphorylation in mitochondria [43] . table 3 gives a list of selected cellular proteins that are known to be associated with potential toxicity and side effects. information about the physiological function, site of action and effect of deficiency/inhibition for these proteins is also given in table 3 [44] [45] [46] [47] [48] [49] . table 3 . toxicity and side effect-causing protein target of drugs. physiological function site of action effect of deficiency/inhibition ref. in order to validate docking results a molecular dynamics simulation (md) has been performed. after a 10-ns run, a root mean square deviation (rmsd) of the atomic positions of ligand-protein complexes have been calculated. low value of ligand rmsd followed by relatively constant value of protein backbone rmsd indicate complex stability and validate docking protocol. md simulation has been performed for compound 3, possessing the lowest score for binding energy. both ligand and protein showed low rmsd values below 2 å, proving stability of the ligand-protein system ( figure 6 ). the docking results are in line with cytotoxicity binding assay findings, which means that the tested compounds can interact with the ca-ctd-sp1 protein. in vitro studies in human cells, as well as preclinical studies, suggest that bevirimat should have low potential for cytotoxicity. there is no evidence of reproductive or developmental toxicity [42] . in order to check the potential toxic properties of the compounds 3-5, docking study of phosphate betulin derivatives to cellular proteins was carried out. it is known that deficiency and inhibition of some proteins important in normal cellular function may result in toxicity or side effects. examples of these proteins are those involved in key cellular metabolism processes such as glycolytic pathway, amino acid and nucleotide metabolism, urea cycle, citric acid cycle and oxidative phosphorylation in mitochondria [43] . table 3 gives a list of selected cellular proteins that are known to be associated with potential toxicity and side effects. information about the physiological function, site of action and effect of deficiency/inhibition for these proteins is also given in table 3 the docking results are in line with cytotoxicity binding assay findings, which means that the tested compounds can interact with the ca-ctd-sp1 protein. in vitro studies in human cells, as well as preclinical studies, suggest that bevirimat should have low potential for cytotoxicity. there is no evidence of reproductive or developmental toxicity [42] . in order to check the potential toxic properties of the compounds 3-5, docking study of phosphate betulin derivatives to cellular proteins was carried out. it is known that deficiency and inhibition of some proteins important in normal cellular function may result in toxicity or side effects. examples of these proteins are those involved in key cellular metabolism processes such as glycolytic pathway, amino acid and nucleotide metabolism, urea cycle, citric acid cycle and oxidative phosphorylation in mitochondria [43] . table 3 gives a list of selected cellular proteins that are known to be associated with potential toxicity and side effects. information about the physiological function, site of action and effect of deficiency/inhibition for these proteins is also given in table 3 [44] [45] [46] [47] [48] [49] . [45] cytochrome c oxidative phosphorylation mitochondria increased sensitivity to cell heath signals triggered by tnf-α [46] carbamoyl phosphate synthetase i urea cycle mitochondria hyperammonemia [47] hypoxanthine-guanine phosphoribosyltransferase nucleotide biosynthesis mitochondria hyperuricemia [48] glutamate dehydrogenase amino acid degradation mitochondria nephrotoxicity [49] the results of docking experiments between bvm, compounds 3-5 and selected cellular proteins are shown in table 4 . according to the results of docking for the target proteins, all tested compounds showed a lower degree of fit to tested proteins compared to bvm. these results may indicate that modification of the bevirimat molecule by substitution of the carboxyl group by a phosphate substituent probably does not increase toxicity of phosphate derivatives compared to bvm. due to 2020's covid-19 pandemic, another group of rna viruses-coronaviruses-have become interesting to scientists. the majority of its large genome is transcribed and translated into polypeptide encoding proteins. these proteins are important for gene expression. the main protease (m pro ), and rna-dependent rna polymerase (rdrp) are key enzymes for coronavirus replication [50] . the m pro mediates the maturation of non-structural proteins (nsps), essential in the life cycle of the virus [51] . in research on various coronaviruses inhibitors, nsps and its rdrp domains have been used as a promising target for new drug candidates [52] . the sars-cov e protein is an integral membrane. most of the phases of the virus life cycle, such as envelope pathogenesis, formation, budding, and assembly are dependent on this protein [53, 54] . it has been suggested by several studies that the absence of the sars-cov-2 e protein may result in an "attenuated virus" [54] . spike (s) is the fundamental protein of the coronavirus, and forms a characteristic corolla structure on the membrane of the virion [55] . structural integrity of spike and cleavage activation play a key role in virus invasion and virulence. therefore, blocking coronavirus form entering host cell by targeting specific receptors on the host surface, such as s protein, might be therapeutic strategy of great value for the development of the anti-viral agents [56] . no specific medicine or treatment is currently available for sars-cov-2-related diseases. studies of remdesivir, phosphoramidate of an adenosine c-nucleoside analog, have brought attention to the possible application of this molecule as an anti-sars-cov-2 agent (figure 7 ) [57] . this rdrp inhibitor [18] can inhibit the virus by inhibiting synthesis of viral nucleic acid, and has been recently authorized for emergency use in acute covid-19 patients. the new betulin phosphate derivatives 2−5 reported in this paper were obtained from easily available material, botulin 1, via a brief two-step synthesis. compounds 3−5, with different carboxyacylic substituents at the c3 position, exhibited in vitro anti-hiv activity with ic50 values in the range of 0.02−0.22 μm. derivative 3, which has a 3',3'-dimethylsuccinyl moiety at the c3 position, exhibits the highest anti-hiv-1 activity (ic50 = 0.02 μm). for bvm, a known maturation inhibitor, the ic50 value determined in this study was comparable and equal to 0.03 μm. derivative 3 was characterized by slightly higher selectivity (ti values of 1250 and 967 for 3 and bvm, respectively). considering the literature reports on various potential mechanisms of anti-hiv activity of triterpenes, molecular modeling with ca-ctd-sp1 has been carried out. the optimal fit was demonstrated by compound 3. this effect suggests a potential molecular target that determines anti-hiv activity of the studied compounds. supplementary materials: the following are available online at www.mdpi.com/xxx/s1, characteristics of synthesized compounds, figure s1 : anti-hiv-1 activity of 3-carboxyacylbetulin phosphate and bvm in the tested concentration range, figure s2 : cytotoxicity of betulin phosphate 2-5 in the tested concentration range, table s1 : selected physicochemical properties of the compounds 2-5, table s2 : scoring functions of the tested compounds (sars-cov-2 proteins), figure s3 : the lowest-energy docking poses of sars-cov-2 mpro protein complexes with bvm (a) and betulinic acid (b), figure s4 : visualization of interaction between bvm (a) and compound 4 (b) with sars-cov-2 rdrp, figure s5 : docking pose of sars-cov-2 e protein complexes with bvm (a) and compound 6 (b), figure s6 : visualization of interaction between betulinic acid (a) and bvm (b) with sars-cov-2 spike protein monomer, figure s7 : rmsd for atoms of protein (mpro, rdrp, e) backbones (left) and ligands (right), figure s8 : rmsd for atoms of s protein backbones (left) and ligands (right), figure s9 : rmsd for atoms of e protein backbones without terminal residues, table s3 : interactions of tested compounds with sars-cov-2 proteins (references [59] [60] [61] are cited in the supplementary materials). on 31 january 2020, the new england journal of medicine reported the diagnosis and treatment of the first sars-cov-2 patient in the united states [58] , and remdesivir exhibited some potential in the treatment of this first patient. accordingly, we used remdesivir as a reference ligand. moreover, based on our previous study, we decided to dock to sars-cov-2 proteins betulinic acid and 30-diethoxyphosphoryl-3-o-(3 ,3 -dimethylsuccinyl)betulinic acid (compound 6, figure 7 ). compound 6 demonstrated in vitro anti-hiv-1 activity with an ic 50 value similar to that of bvm (ic 50 = 0.02 µm and 0.03 µm for 6 and bvm, respectively), and with a lower cytotoxicity (cc 50 = 68 µm and 29 µm for 6 and bvm, respectively) [12] . according to the results of docking (table s1 ) obtained from autodock vina, four potential sars-cov-2 inhibitors (bvm, betulinic acid, and compounds 4 and 6) were selected based on a lower negative dock energy value. detailed interactions between ligands and selected sars-cov-2 proteins are included in supplementary materials (figures s3-s6 and tables s2 and s3) . md simulation has been performed for compounds possessing the lowest score for binding energy (bvm, betulinic acid, and compounds 4 and 6). low fluctuations of rmsd of all proteins indicate that they reached stable conformation (supplementary materials figures s7-s9 ). the new betulin phosphate derivatives 2−5 reported in this paper were obtained from easily available material, botulin 1, via a brief two-step synthesis. compounds 3−5, with different carboxyacylic substituents at the c3 position, exhibited in vitro anti-hiv activity with ic 50 values in the range of 0.02−0.22 µm. derivative 3, which has a 3',3'-dimethylsuccinyl moiety at the c3 position, exhibits the highest anti-hiv-1 activity (ic 50 = 0.02 µm). for bvm, a known maturation inhibitor, the ic 50 value determined in this study was comparable and equal to 0.03 µm. derivative 3 was characterized by slightly higher selectivity (ti values of 1250 and 967 for 3 and bvm, respectively). considering the literature reports on various potential mechanisms of anti-hiv activity of triterpenes, molecular modeling with ca-ctd-sp1 has been carried out. the optimal fit was demonstrated by compound 3. this effect suggests a potential molecular target that determines anti-hiv activity of the studied compounds. managing epidemics: key facts about major deadly diseases. world health organization. license: cc by-nc-sa 3.0 igo pharmacological activities of natural triterpenoids and their therapeutic implications recent progress in the antiviral activity and mechanism study of pentacyclic triterpenoids and their derivatives hiv-1 gag polymorphisms determine treatment response to bevirimat (pa-457) pharmacological intervention of hiv-1 maturation antiviral drugs for cytomegalovirus diseases study of chemical stability of antivirally active 5-azacytosine acyclic nucleoside phosphonates using nmr spectroscopy intralesional cidofovir for the treatment of multiple and recalcitrant cutaneous viral warts jiangxi qingfeng pharmaceutical inc. lupane triterpenoid derivatives and pharmaceutcal use thereof new phosphorus analogs of bevirimat: synthesis, evaluation of anti-hiv-1 activity and molecular docking study biomolecules 2020 origin and evolution of pathogenic coronaviruses emerging threats from zoonotic coronaviruses-from sars and mers to 2019-ncov drug treatment options for the 2019-new coronavirus (2019-ncov) identification of novel compounds against three targets of sars cov-2 coronavirus by combined virtual screening and supervised machine learning in silico screening of chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus anti-aids agents-xxvii. synthesis and anti-hiv activity of betulinic acid and dihydrobetulinic acid derivatives rapid and automated tetrazolium-based colorimetric assay for the detection of anti-hiv compounds enzyme immunoassay for detection of human immunodeficiency virus antigens in cells cultures swiss-model: homology modelling of protein structures and complexes autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading small-molecule library screening by docking with pyrx discovery studio modeling environment qwikmd: integrative molecular dynamics toolkit for novices and experts vmd-visual molecular dynamics scalable molecular dynamics with namd general force field: a force field for drug-like molecules compatible with the charmm all-atom additive biological force field extension of the charmm general force field to sulfonyl-containing compounds and its utility in biomolecular simulations anti-hiv-1 phosphate derivatives of 3-carboxyacylbetulin, their preparation and their use anti-aids agents. 32. synthesis and anti-hiv activity of betulin derivatives alkyl amine bevirimat derivatives are potent and broadly active hiv-1 maturation inhibitors predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures synthesis, in vitro antimicrobial assessment, and computational investigation of pharmacokinetic and bioactivity properties of novel trifluoromethylated compounds using in silico adme and toxicity prediction tools computational prediction of pharmacokinetic, bioactivity and toxicity parameters of some selected anti-arrhythmic agents the sequence of the ca-sp1 junction accounts for the differential sensitivity of hiv-1 and siv to the small molecule maturation inhibitor 3-o-{3',3'-dimethylsuccinyl}-betulinic acid crystal structure of an hiv assembly and maturation switch bevirimat: a novel maturation inhibitor for the treatment of hiv-1 infectio prediction of potential toxicity and side effect protein targets of a small molecule by a ligand-protein inverse docking approach red cell pyruvate kinase deficiency: from genetics to clinical manifestations dependence of excitotoxic neurodegeneration on mitochondria aconitase inactivation role of cytochrome c in apoptosis: increased sensitivity to tumor necrosis factor alpha is associated with respiratory defects but not with lack of cytochrome c release fatal hyperammonemia and carbamoyl phosphate synthetase 1 (cps1) deficiency following high-dose chemotherapy and autologous hematopoietic stem cell transplantation hyperuricemia and gout due to deficiency of hypoxanthine-guanine phosphoribosyltransferase in female carriers: new insight to differential diagnosis. case rep chloroquine is a potent inhibitor of glutamate dehydrogenase in liver and kidney-cortex of rabbit the viruses and their replication substrate specificity profiling of sars-cov-2 m pro protease provides basis for anti-covid-19 drug design antiviral activity of nucleoside analogues against sars-coronavirus (sars-cov) insights into the recent 2019 novel coronavirus (sars-cov-2) in light of past human coronavirus outbreaks severe acute respiratory syndrome coronaviruses with mutations in the e protein are attenuated and promising vaccine candidates structural insights into coronavirus entry molecular docking study of receptor binding domain of sars-cov-2 spike glycoprotein with saikosaponin, a triterpenoid natural product remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro first case of 2019 novel coronavirus in the united states in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. the following are available online at http://www.mdpi.com/2218-273x/10/8/1148/s1, characteristics of synthesized compounds, figure s1 : anti-hiv-1 activity of 3-carboxyacylbetulin phosphate and bvm in the tested concentration range, figure s2 : cytotoxicity of betulin phosphate 2-5 in the tested concentration range, table s1 : selected physicochemical properties of the compounds 2-5, table s2 : scoring functions of the tested compounds (sars-cov-2 proteins), figure s3 : the lowest-energy docking poses of sars-cov-2 mpro protein complexes with bvm (a) and betulinic acid (b), figure s4 : visualization of interaction between bvm (a) and compound 4 (b) with sars-cov-2 rdrp, figure s5 : docking pose of sars-cov-2 e protein complexes with bvm (a) and compound 6 (b), figure s6 : visualization of interaction between betulinic acid (a) and bvm (b) with sars-cov-2 spike protein monomer, figure s7 : rmsd for atoms of protein (mpro, rdrp, e) backbones (left) and ligands (right), figure s8 : rmsd for atoms of s protein backbones (left) and ligands (right), figure s9 : rmsd for atoms of e protein backbones without terminal residues, table s3 : interactions of tested compounds with sars-cov-2 proteins (references [59, 60] are cited in the supplementary materials). key: cord-319354-jbain7n6 authors: gondim, ana c. s.; roberta da silva, suzete; mathys, leen; noppen, sam; liekens, sandra; holanda sampaio, alexandre; nagano, celso s.; renata costa rocha, cintia; nascimento, kyria s.; cavada, benildo s.; sadler, peter j.; balzarini, jan title: potent antiviral activity of carbohydrate-specific algal and leguminous lectins from the brazilian biodiversity date: 2019-01-14 journal: medchemcomm doi: 10.1039/c8md00508g sha: doc_id: 319354 cord_uid: jbain7n6 brazil has one of the largest biodiversities in the world. the search for new natural products extracted from the brazilian flora may lead to the discovery of novel drugs with potential to treat infectious and other diseases. here, we have investigated 9 lectins extracted and purified from the northeastern brazilian flora, from both leguminous species: canavalia brasiliensis (conbr), c. maritima (conm), dioclea lasiocarpa (dlasil) and d. sclerocarpa (dsclerl), and algae amansia multifida (aml), bryothamniom seaforthii (bsl), hypnea musciformis (hml), meristiella echinocarpa (mel) and solieria filiformis (sfl). they were exposed to a panel of 18 different viruses, including hiv and influenza viruses. several lectins showed highly potent antiviral activity, often within the low nanomolar range. dsclerl and dlasil exhibited ec(50) values (effective concentration of lectin required to inhibit virus-induced cytopathicity by 50%) of 9 nm to 46 nm for hiv-1 and respiratory syncytial virus (rsv), respectively, dlasil also inhibited feline corona virus at an ec(50) of 5 nm, and dsclerl, conbr and conm showed remarkably low ec(50) values ranging from 0.4 to 6 nm against influenza a virus strain h3n2 and influenza b virus. for hiv, evidence pointed to the blockage of entry of the virus into its target cells as the underlying mechanism of antiviral action of these lectins. overall, the most promising lectins based on their ec(50) values were dlasil, dsclerl, conbr, conm, sfl and hml. these novel findings indicate that lectins from the brazilian flora may provide novel antiviral compounds with therapeutic potential. for decades, lectins have been recognized as potential natural drugs capable of treating a range of diseases, and hence their biological activity has been studied intensely. [1] [2] [3] [4] [5] in this context, brazilian biodiversity can potentially provide a wealth of novel unknown lectins, which can be mined for novel bioactivity. lectins are found in a broad range of organisms, from land plants to marine algae, and are capable of discriminating between, and binding to specific carbohydrate structures. 6 the location of the carbohydrate binding sites on the lectin, sugar-binding epitopes and ligand disposition on its scaffold, give rise to a variety of types of interactions of lectins with their sugar ligands. 7 the avidity and specificity for binding to glycoconjugates relies on the carbohydrate recognition domains (crd) of the lectin on the one hand, and the density of the glycans, the glycan structure, and their multivalency, on the other. 8 recently the therapeutic potential of lectins from algae has been widely recognized. 9 algae are indeed a rich natural marine resource of lectins on the brazilian coast that may be endowed with important pharmacological activities. for example, lectins from bryothamnion triquetrum (btl) and bryothamnion seaforthii (bsl) have been used to differentiate human colon carcinoma cell variants, 10 while the algal (cyanobacterial) lectin cyanovirin-n (cv-n) shows not only potent antiviral activity against hiv-1 and hiv-2, but also against influenza virus and ebola virus. 7 lectins are proteins found in nature. this can have several disadvantages, including expensive production and scalingup, poor oral bioavailability, hemagglutination of human red blood cells, mitogenic activity, cellular toxicity and stimulation of differentiation/activation markers. 11, 12 the cyanobacterial lectin cyanovirin is a well-known example for several of these properties. 13 however, these potential compromising properties are highly dependent on the exact nature of the particular lectin. for example, many lectins agglutinate red blood cells derived from several animal species such as mice, rats, rabbits, but not of human origin. 11, 14 microvirin, an alphaij1,2)-mannose-specific lectin from microcystis aeruginosa has been shown to have comparable anti-hiv activity as cyanovirin, but a much higher safety profile. it proved 50-fold less cytotoxic than cyanovirin, and it did not increase the level of activation markers in cd4 + t lymphocytes. 15 also, the highly potent antiviral red algal lectin griffithsin was reported to have an outstanding safety and efficacy profile as a potential microbicidal drug candidate. 16 it induces only minimal changes in secretion of inflammatory cytokines and chemokines by epithelial cells and human pbmcs, does not markedly upregulate t cell activation markers and gene expression, and has no measurable effect on cell viability. even in the case of the mitogenic activity of a particular lectin such as the banana lectin banlec, it was recently shown that its mitogenic activity could be uncoupled from its antiviral activity by engineering this lectin through site-directed mutagenesis. thus, lectins can be modulated to remove a nondesirable activity (i.e. mitogenicity) while preserving a beneficial activity (i.e. antiviral activity). 17 moreover, since carbohydrate-binding agents such as lectins are prime candidate drugs for preventing sexually-transmitted viral infections (i.e. hiv, hbv, hcv, herpes viruses), oral bioavailability of such drugs is not required, and it can even be an advantage to have poor absorption through cell layers in order to avoid undesired systemic side effects. 18 evidence that lectins can realistically be used to treat pathogen infections in vivo has recently been provided by several investigators. scalable manufacture of cyanovirin 19 and griffithsin 20 and validation of the safety and antiviral efficacy of griffithsin in mice and rabbits as a topical microbicide has been reported by o'keefe et al., 21, 22 takebe et al., 23 kouokam et al., 24 farr zuend et al. 25 and girard et al. 26 interestingly, castillo-acosta et al. 27 recently demonstrated that pradimicin, a mannosespecific agent purified from actinomyces madura, can cure mice suffering from acute sleeping sickness caused by trypanosomes. currently, legume lectins are the most extensively studied, including lectins from seeds of plants belonging to the fabaceae family. 28 dolichos lablab lectin (dll) has been shown to weaken proangiogenic signals, specifically nuclear factor kappa b (nf-κb), matrix metalloproteinase (mmp-2 and 9) and vascular endothelial growth factor (vegf) in lectin-exposed mice. 29 lens culinaris agglutinin and pisum sativum agglutinin are capable of disrupting hiv infection by preventing the interaction of viral surface glycoprotein gp120 with the cellular cd4 receptor. 28 here, we show that several algal and leguminous lectins from the brazilian flora are highly potent inhibitors towards a variety of viruses, and discuss their target sites and possible mechanisms of action. the purified algal and leguminous lectins were initially investigated for their inhibitory activity against two different strains of hiv, hiv-1 (nl4.3) and hiv-2 (rod). for this assay, hiv-1 and hiv-2 infection was performed in human cd4 + t-lymphocyte (cem) cell cultures. the ec 50 values for legume lectins (conbr, conm, dlasil and dsclerl) were found to be in the nanomolar range and were generally somewhat lower (higher activity) for hiv-1 than hiv-2 (2-to 4-fold). interestingly, dlasil and dsclerl from diocleinae species were up to 3-fold more active than the lectins derived from canavalia species (table 1) . in a second series of anti-hiv assays, co-cultivation of uninfected supt1 and persistently hiv-1-infected hut-78 cells (expressing the viral surface glycoproteins gp120 and gp41) was performed. in these assays, giant cells (syncytia) derived from the fusion between the infected and the uninfected cells (by virtue of gp120/gp41-cd4 interaction) were abundantly formed within 20 hours of co-cultivation. the ec 50 values to prevent giant cell formation in the co-cultures in the presence of different concentrations of the lectins proved to be quite similar to the range of ec 50 values for hiv-1 infection in the previously mentioned set (table 1 ). these results indicate that leguminous lectins most likely block the adsorption/entry of the virus in the infection step, presumably by binding to the heavily glycosylated gp120/gp41 that is expressed on persistently hiv-1-infected hut78/hiv1 cells. the antiviral activity concentrations of the leguminous lectins were usually well below their toxicity threshold ( table 1) . the algal lectins (hml, bsl, aml, mel) were much less effective in blocking hiv-1 and hiv-2 activity than the leguminous lectins. for example, whereas hml was 3-fold less effective than the most active dsclerl lectin against hiv-1, sfl was 22-fold less inhibitory in comparison to the leguminous dsclerl lectin with ec 50 values of 440 and 304 nm against hiv-1 and hiv-2, respectively. these remarkable differences between algae and leguminous lectins illustrate the different selective recognition properties of the lectins, which should be further explored. given the potent anti-hiv activity of several lectins, and their pronounced effect on syncytium formation in supt1-hut-78/hiv-1 co-cultures, the binding of the lectins to the hiv-1 envelope glycoproteins gp120 and gp41 and the cellular cd4 receptor was investigated by surface plasmon resonance (spr) technology. these preliminary spr studies suggest that hiv-encoded surface glycoproteins are potential antiviral targets for the lectins. no attempt was made to determine binding constants (k d ) and related association (k a ) and dissociation (k d ) rates since the interpretation of data is complicated by the known auto-proteolysis of the leguminous lectins ( fig. s1 †) . the glycoproteins were bound on the sensor chip surface and binding of lectins flowing over the surface was recorded. the mannose-specific red algae-derived griffithsin lectin was used as a positive control for the assay. we selected only those lectins with promising ec 50 values against hiv-1 including dlasil, dsclerl, conbr, conm, sfl and hml. the leguminous lectins (dlasil, dsclerl, conbr, conm) appeared to show higher binding to the three glycoproteins than the algal (sfl, hml) lectins (table s1 †) . also, the lectins appeared to bind to a higher extent to gp120 and gp41. interestingly, there appears to be a positive correlation between the antiviral potential of the lectins in both primary infection and co-cultivation assays, and the binding of the lectins to gp120, gp41 and cd4. these preliminary spr studies suggest that hiv-encoded surface glycoproteins are potential antiviral targets for the lectins and that future detailed measurements on a low density ligand sensor chip to determine k d , k a and k d values would be worthwhile, if the problems of auto-proteolysis and glycation can be controlled. since, besides hiv, also influenza viruses contain a heavily glycosylated envelope, three influenza viruses, influenza a (h1n1 subtype), influenza a (h3n2 subtype) and influenza b were investigated using a cytopathic evaluation and a coloured dye-mts exposure assay in the presence of each of all 9 lectins. remarkably, the leguminous lectin dsclerl showed exquisitely potent antiviral activity against influenza a (h3n2) infection with an ec 50 as low as 400-1200 pm ( table 2) . it proved to be 2-to 4-fold more active than conbr. in contrast, much lower (if any) activity was observed towards the influenza a h1n1 strain (ec 50 >20 000 pm). the three leguminous lectins were also markedly active against influenza b virus (ec 50 in the higher picomolar/lower nanomolar range). surprisingly, dlasil, which was highly active against hiv, showed the poorest activity against all influenza virus strains. despite a generally lower activity noticed for the algal lectins, some of them (i.e. aml) also showed nanomolar activity, although highly dependent on the nature of the influenza virus strain. the algae-derived sfl did not show antiinfluenza virus activity at subtoxic (8-32 nm) concentrations. it proved to be markedly more cytotoxic than the other studied lectins. it is notable that the anti-influenza virus activity of the leguminous lectins was generally observed at concentrations that were markedly lower than their cytotoxic concentrations (40 to >200 nm). finally, the activity of nine lectins against another set of 12 different viruses was determined, including reovirus-1, sindbis virus, coxsackie b4 virus, punta toro virus, feline corona virus (fipv), feline herpes virus, vesicular stomatitis virus, respiratory syncytial virus, herpes simplex virus 1 (kos), herpes simplex virus 2 (g), herpes simplex virus 1 (tk-kos acv), vaccinia virus (table 3 ). in these assays, the lectins did not show any promising activity towards most viruses such as reovirus-1, sindbis virus, coxsackie b4 virus, parainfluenza-3 virus, vaccinia virus and punta toro virus. acquired immunodeficiency syndrome (aids) is caused by the human immunodeficiency virus (hiv), and the disease compromises the immune system, predominantly infecting t-cells, dendritic cells and macrophages. 30 the world health organization (who) predicts that in the next few years more than 37 million people will be infected by hiv. 31 hiv is an enveloped virus possessing a lipid bilayer protecting the viral rna and capsid. at the surface of the virus, the glycosylated glycoproteins gp120 and gp41 are imbedded in, and part of, the viral envelope at the start of the infection. glycoprotein gp120 binds to the cd4 receptor present on t-cells. cd4 binding induces changes in gp120 resulting in the exposure of envelope domains, which were previously hidden at the moment it binds to the host cells. such uncovered domains may then bind to the cxcr4 or ccr5 co-receptors on the cell membrane. it is difficult for antibodies to reach the initiallyuncovered surface protein domains efficiently, which also means that efficient vaccines are not available yet for hiv. 31 the hiv surface glycoprotein gp120 is one of the most heavily glycosylated proteins known so far, and its molecular weight consists of ∼50% of glycans. therefore, targeting these glycans by carbohydrate-binding agents (cbas) has been proposed earlier as a potential therapeutic approach to suppress hiv. 32 indeed, due to their high specificity for binding to carbohydrate chains on cell surfaces, lectins are widely studied in the glycobiology field, and participate in many important physiological processes. 31 several lectins have been reported as antiviral agents, but relatively few types of viruses have been investigated so far. 9 the best-studied cases of lectins with potent antiviral (i.e. hiv) activity have been the cyanobacterial cyanovirin n algal lectin and the red algal lectin griffithsin, which have picomolar potency against hiv-1. 33 interestingly, several lectins with anti-hiv activity show recognition of different carbohydrates. for example, polygonatum citronella and ophiopogon japonicus lectins have affinity for sialic acid. 31, 34 griffithsin binds to mannose/glucose structures, cyanovirin n predominantly binds to alphaij1,2)-mannose structures, galanthus nivalis agglutinin (gna) and hippeastrum hybrid agglutinin exhibit α(1,3)/α(1,6)-mannose-binding activity 33 and musa acuminata agglutinin (banlec) binds to high mannose carbohydrate structures. 35 the latter lectins showed different degrees of anti-viral activity in the high picomolar range (i.e. griffithsin) or low nanomolar range. 33 they block viral entry as the major mechanism of antiviral activity. thus, these and other lectins might act as antiviral compounds efficiently preventing viral entry into the host cells, which generally occurs through specific interactions of the lectins with glycans exposed on the gp120 (and gp41) glycoproteins (in the case of hiv) of the virus surface. 31 in fact, the first lectin discovered to have anti-hiv activity, cona, binds to glycoprotein structures, specifically at α-d-mannosyl and α-d-glucosyl groups found in the glycoproteins gp120 of hiv-1 and hiv-2, 31,36 and blocks the binding of hiv to its receptors on the host cells. 37 conbr, conm, dlasil and dsclerl reported here have the same specificity and high identity of amino acid sequence with cona (>97% for lectins from canavalia and up to 80% for dioclea lectins). differences in their amino acid sequence result in distinct biological activities, as noticed in our study. 38 our data for hiv show antiviral potency from 20 to 137 nm for the leguminous lectins, while algal lectins tested were generally less potent (table 1) . algae lectins are structurally classified into families and according to their binding profile. 39 in our study, aml, mel and sfl belong to the oaah lectin family that includes lectins from marine red algae eucheuma serra (esa) and kappaphycus alvarezii (kaa) and cyanobacteria oscillatoria agardhii (oaa). these lectins inhibited hiv infection through binding to high mannose (hm) oligosaccharides of enveloped glycoprotein gp120. 40 these carbohydrate profiles indicate that this family has anti-hiv activity preferring to link α1-3 man branched from the α1-6 man of the penta-saccharide core, showing high affinity to hm n-glycans. 40 oaa recognizes major high mannose sugars on hiv-1 showing similar ec 50 values supporting the similarities of the family. 40 this supports our findings for the algal lectins aml, mel and sfl, where ec 50 values showed minor differences that are associated with the number of sugar-binding epitope sites on the glycans that influence the anti-hiv activity. the reason why the leguminous lectins have superior antiviral potential compared to the algae-derived lectins studied here may not only be due to their different specificities for sugar structures but also due to their tetrameric structure compared to the monomeric conformation of the algaederived lectins ( table 4) . the tetrameric lectins (as also the case for hippeastrum hybrid (amaryllis) hha and galanthus nivalis (gna)) have multiple carbohydrate-binding sites allowing tighter binding to, and cross-linking of the glycans on the viral envelope. 52, 53 it would be of interest to obtain structural data on the molecular interactions of the most active lectins in complexes with their hiv gp120 target. generating nmr data, x-ray based crystallographic analysis and/or cryo-em data will be challenging, but would indeed add significantly to understanding the molecular interactions of the lectins with the glycans on hiv gp120 and will be important for the rational design of mutated lectins with improved interactions with their targets and eventual antiviral efficacy. indeed, it has been shown earlier that lectins with preferential alpha-1,2; 1,3 and 1,6-mannose specificity usually show potent antiviral (hiv) activity, 14, 18, 32, 54 due to the presence of a number of clustered high-mannose-type glycans on the surface glycoprotein of hiv. 32 also, it can be assumed that tetrameric lectins may have a higher degree of interaction with their target glycans than monomeric lectins. as a result, they may have a higher capacity to cross-link the glycans on hiv gp120/gp41 thereby "freezing" the glycans in a fixed conformational state and decreasing the overall flexibility of the surface glycoprotein. the lectins showed pronounced and comparable inhibition of both primary hiv infection (4 day assay) and giant cell formation in co-cultures of persistently hiv-1-infected and uninfected t-lymphocyte cells (∼20 h assay). these findings are highly suggestive for virus adsorption/entry as the underlying mechanism of antiviral action. the binding of the lectins to hiv-1 gp120 (and gp41), as measured by spr, and the marked positive correlation between binding of gp120 (and gp41) and antiviral activity strongly corroborate this hypothesis. the fact that the lectins also measurably bind to cd4 might explain their strong overall anti-hiv activity, but may make these lectins somewhat less selective than griffithsin (that strongly binds to gp120/gp41, but interacts poorly with cd4). further studies should reveal whether the additional binding of the lectins to cd4 eventually turns-out to be an advantage (in terms of antiviral efficacy), or a disadvantage (in terms of potential side-effects). another interesting set of viruses consists of the human influenza virus strains a and b. in particular the influenza a strains are very susceptible to antigenic drift and/or shift. they are subdivided based on the type of envelope substructures, e.g. influenza virus type a consisting of a well-defined hemagglutinin (ha) and neuraminidase (na), which can be subdivided into 18 (h1-h18) and 11 (n1-n11) types. 55 influenza virus binds to sialic acid structures present on the host cell surface, through the viral envelope. 6 according to the literature, humans and other vertebrates including wild birds, bats and pigs, can contract these type of viruses. their infection can become pandemic and has been responsible for more than fifty million deaths. they can also cause highly destructive loss in domestic poultry and pose risks for humans. 6, 55 lectin esa-2 from red alga eucheuma serra inhibits infection by influenza a h1n1, with an ec 50 of 12 nm, by recognizing high mannose n-glycans (hm) on the ha surface glycoprotein. 40 cona also binds to hm glycans and showed a somewhat weaker activity with an ec 50 of 41 nm, while lectins from aspergillus oryzae (aol) and aleuria aurantia (aal) that bind to fucose were less potent with ec 50 values of 50 to 100 nm. 40 our results are quite remarkable, since we identified a leguminous lectin dsclerl, with exquisitely high activity against influenza a (h3n2) infection with an ec 50 as low as 0.4-1.2 nm (table 2) . also, conm exhibited very low ec 50 values (high activity) of 0.2 to 1.5 nm against influenza a (h3n2) and influenza b viruses. beyond that, a high degree of selectivity for these lectins regarding their target influenza virus strain, was noticed, which, for conm, showed about 50-fold higher activity toward influenza a (h3n2) than influenza a (h1n1) ( table 2 ). these striking differences in antiviral potential between different strains of influenza virus may most likely be due to (often subtle) 56 which is about 10-fold less potent than our findings for dlasil against rsv (ec 50 = 9 nm). additionally, even the best activity against influenza b virus (ec 50 = 8 nm) and influenza a h3n2 (ec 50 = 51 nm) for ntl was also markedly lower than those found for dsclerl (h3n2, ec 50 = 0.4-1.2 nm) and conm (h3n2; b, ec 50 = 1.6-3.5 nm) in our current study. the investigation of 9 lectins isolated from the brazilian biodiversity flora against 18 different viruses that belong to a broad variety of viral families has revealed a remarkable panel of highly active lectins. interestingly, the most active lectins were of leguminous origin, with activity reaching the picomolar level. the pronounced antiviral potencies make them attractive as novel agents to be further investigated for their antiviral potential. they might be suitable for topical application. our findings encourage the further investigation of the potential of these lectins as antiviral agents. lectin purification. all the lectins used in this study have been well characterized by sds-page ( fig. s1 and s2 †), mass spectrometry (ms) and primary structure determination as described in the literature, and showed similar levels of purity as those reported. lectins conbr, conm, dlasil and dsclerl were purified by affinity chromatography on a sephadex g50 column using a reported procedure. 1, 4, 5 lectins from amansia multifida (aml), bryothamnion seaforthii (bsl), hypnea musciformis (hml), meristiella echinocarpa (mel) and solieria filiformis (sfl) were purified by combination of ammonium sulphate precipitation and ion exchange chromatography on a deae-sephacel column as previously described. 45 50 was based on the compound concentration required to prevent syncytium formation by 50%. all other antiviral assays (different from hiv) were based on inhibition of virus-induced cytopathicity in human embryonic lung (hel) (using herpes simplex virus type 1 (hsv-1), surface plasmon resonance measurements. the specific interactions of the lectins with immobilized hiv-1 gp120, hiv-1 gp41 and cellular cd4 receptor were investigated by surface plasmon resonance (spr) technology using the biosensor biacore t200 (ge healthcare, uppsala, sweden). the lectins dlasil, dsclerl, conbr, conm, sfl and hml were used at 100 nm in pbs (ph 7.4), except for dlasil and hml that were also used at 500 nm, and injected for 2 min onto the gp120-, gp41-, cd4-bound surface of a cm5 sensor chip at a flow rate of 5 μl min −1 (association phase). then, the change of the spr response was monitored at 25°c for another 8 min in the absence of the compound (dissociation phase). for immobilization, gp120, gp41 and cd4 were immobilized at 4690, 2300 and 5569 ru's in 10 mm sodium acetate buffer (ph 4.0). the authors declare no competing financial interests. the lectins properties, funtions and applications in biology and medicine we thank the coordenação de aperfeiçoamento de nível superior (capes) for providing phd fellowship to ana c. s. gondim, including one-year "sandwich fellowship" in the department of chemistry at warwick university, and support from the erc (grant no. 247450), epsrc (grant no. ep/ f034210/1 and ep/m027503/1) for pjs and (goa-ku leuven) (for jb, sl). we are grateful to dr eduardo sousa (federal university of ceará) for his helpful comments on the script. key: cord-342076-3a6aky7i authors: zhang, lei; fung chow, eric pui; zhang, jun; jing, jun; wilson, david p title: describing the chinese hiv surveillance system and the influences of political structures and social stigma date: 2012-09-07 journal: open aids j doi: 10.2174/1874613601206010163 sha: doc_id: 342076 cord_uid: 3a6aky7i china’s public health surveillance system for hiv was established in late 1980s and has evolved significantly during the past three decades. with the gradually changing mode of hiv transmission from sharing of intravenous injecting equipment to sexual exposure and the rapid spread of hiv infection among chinese homosexual men in recent years, an efficient and comprehensive population-level surveillance system for describing epidemics trends and risk behaviours associated with hiv acquisition are essential for effective public health interventions for hiv. the current review describes the overall strength of the chinese hiv surveillance system and its structural weaknesses from a political and social perspective. the hiv surveillance system in china has undergone substantial revamping leading to a comprehensive, timely and efficient reporting system. however, large data gaps and lack of quality control and sharing of information obstruct the full performance of the system. this is largely due to fragmented authoritarianism brought about by the underlying political structure. social stigma and discrimination in health institutes are also key barriers for further improvements of hiv diagnosis and surveillance in china. by the end of 2009 it was estimated that 740,000 (0.56 -0.92 million) people in china were living with hiv [1] . this estimate was obtained by using the workbook method recommended by unaids based on the numbers of newly diagnosed hiv cases and applying related mortality rates reported by the current hiv sentinel surveillance system. the overall national hiv prevalence is estimated to be 0.057% (0.042-0.071%) among the chinese population [2, 3] . however, data from the national sentinel surveillance for hiv/aids indicated magnitudes and trends in hiv prevalence vary substantially across different at-risk populations: e.g. 0.6% hiv prevalence among female sex workers (fsw) in 2009 [2] and increases from 1.4% in 2001 to 5.3% in 2009 among men who have sex with men (msm) [4, 5] , and from 5.9% in 2002 to 9.3% in 2009 among injecting drug users (idu) [2, 6] . the profile of hiv epidemics in china have also been gradually changing in mode of transmission. in the early 1990s, the main driver of hiv transmission was needle sharing among idu [6] [7] [8] [9] ; however, sexual transmission has now become the primary mode of transmission in recent years [2, [10] [11] [12] . the proportion of newly diagnosed hiv cases due to sexual transmission increased from 49.8% in 2005 to 74.7% in 2009 and newly diagnosed cases attributed to homosexual contact has substantially increased from 12.2% in 2007 to 32.5% in 2009 [1, 10] . effective public health interventions for hiv rely on accurate and timely information on the extent and patterns of *address correspondence to this author at the cfi building, corner of west and boundary streets, darlinghurst, sydney nsw 2010, australia; tel: +61 2 9385 0900; fax: +61 2 9385 0920; e-mail: lzhang@nchecr.unsw.edu.au spread of infection. as such, an efficient and comprehensive population-level surveillance system for describing epidemics trends and risk behaviours associated with hiv acquisition are essential [13] . previous studies have provided reviews of the current hiv surveillance system in china, mainly from a functional and epidemiological perspective [14] [15] [16] . in contrast, the current review aims to describe the overall strength of the chinese hiv surveillance system and its structural weaknesses from a political and social perspective. prior to 2004, newly diagnosed hiv/aids cases were reported through the national disease reporting system (ndrs) (see table 1 for a list of reportable items in china). the system consists of multiple level of reporting. villages, being the lowest level of administrative, collected hiv epidemiological data in their allocated areas and reported to prevention units in township hospitals. from the prevention units, hiv data were further sent through county health and epidemic-prevention stations (eps) to provincial centers and forwarded to the chinese academy of preventive medicine. with the support from the ministry of health and cooperation of provincial centers of health and epidemic prevention, a computerized system had worked effectively to network the monitoring of hiv epidemics at various levels in china and information transfer experienced no substantial changes for about 25 years. however, all communicable disease surveillance systems in china underwent significant upgrade in 2004. the severe acute respiratory syndrome (sars) outbreak in 2003-2004 exposed the great deficiencies in both timeliness and completeness of infectious disease reporting in ndrs [17] . this substantially changed the landscape of infectious diseases surveillance in china. just 6 months after the sars epidemic subsided, the china center for disease control (china cdc) launched the china information system for disease control and prevention (cisdcp) [18] . the system is an information platform constructed mainly with an infectious diseases orientation, aiming to improve timeliness of case reports, completeness and accuracy of surveillance data, ability for early detection of outbreaks and to provide direct information sharing among various levels of cdcs and health authorities, replacing the previous ndrs. the upgrade of overall infectious disease surveillance systems in china directly led to the construction of web-based hiv/aids case reporting system. under this reporting system, grassroot hospitals remain the primary source of hiv epidemiological information. all diagnosed and confirmed hiv-infected cases admitted to hospitals are obliged to report through the web-based reporting system to china cdc. improving from the previous step-wise hierarchical reporting framework, the new web-based reporting system provided the ability for direct reporting of diagnosed hiv cases from the grassroot hospitals to the centralised database in chinese moh through cisdcp (fig. 1) . that is, once disease cases are entered into the system, all levels of cdc can acquire the data 'live' according to their assigned access privileges [19] . this substantially reduced the average reporting period from 7-8 days to less than 2 days. in addition to their assumed responsibility for authenticating the hiv disease information in their administrative regions, municipal and provincial cdcs are required to report to their corresponding department of health (dohs) and bureau of health (bohs) and to form networks with local research bodies, universities and other health organisations. at the top of the hierarchy, the national center for hiv/aids prevention and control (ncaids) of china cdc is the overseeing organisation for assembling and analysing all hiv disease information and then presenting a final report to china moh. china moh remains the only legitimate office to disseminate hiv/aids-related information to the public, but provincial and municipal cdcs are also authorised to release information under the supervision and approval of china moh [20, 21] . china moh enacts health policies, designs prevention guidelines and intervention strategies based on the surveillance reports provided by ncaids, once a consensus agreement is established between moh and the chinese government, feedback recommendations and policies are issued to all lower levels of moh for execution. since the feedback information does not go through the webbased information pool, hospitals at grassroots level may have to wait for a considerable period of time to receive recommendations and guidelines from china moh. by the year 2009, 8563 laboratories (8273 screening laboratories, 254 confirmatory laboratories, 35 confirmatory central laboratories and 1 national aids reference laboratory) and 84 laboratories hospitals are capable of carrying out cd4 and hiv viral load testing in china [22] . confirmed diagnosed hiv cases were reported back to referring clinical sites then further integrated into the central database through cisdcp. the hospital-based surveillance in china has cumulatively diagnosed 326,000 people living aids and a total of 65,481 people are currently receiving art in 2009 [23] , which accounted for 62.4% of the aids cases [2] . the hospital-based surveillance relies on plhiv to attend hospital to be diagnosed and cases subsequently reported. complementing this passive reporting, active surveillance for hiv was adopted in 1995 and further expanded in 2003 to include second generation behavioral surveillance in china according to who recommendations [24, 25] . while the existing hospital-based diagnosis system provides hiv diagnosis and treatment data, sentinel surveillance collects information on hiv prevalence, incidence and the risk behaviours of at-risk populations. in china, hiv sentinel surveillance is collaboratively supervised by the chinese moh and who. in 2000, surveillance pilot sites targeting idu, fsw, male clients and the general female population were established in fujian, xinjiang, guangxi, shanxi, yunnan and sichuan. the approach utilised 21 surveillance points in these provinces and from every surveillance point 400 people are sampled twice each year to estimate the prevalence, incidence rate and risk behaviours of the targeted population [26] . the sampling and reporting for each round is completed within 4 weeks. in 2002, china issued "standards for hiv surveillance and hiv/aids" and "std comprehensive surveillance guideline" to provide guidelines to standardise the practice of hiv surveillance. in 2009, the number of sentinel sites in china increased to 1318, covering 14 at-risk population groups [2, 14, 27] . since the establishment of the web-based hiv reporting system in 2004, sentinel surveillance data is integrated with disease information reported from hospitals. this approach established a comprehensive database for data collection, analysis and sharing and enables high-level comparison and integration of epidemiological and behavioural surveillance data [14] . this further allows the conduct of evaluation and forecasting activities for guiding the formulation of public health intervention strategies. hiv sentinel surveillance has detected the changing trends and patterns of hiv epidemics in china, from transmission related to injecting practices to sexual transmission, especially homosexual transmission. further, surveillance data have revealed that the proportion of idu among all drug users has increased from 42% in 1994 [28, 29] to 53% in 2001 [29, 30] and 69% in 2009 [31] . in addition, during 1997-2009, the proportion of idu who report sharing needles increased from 25% to 38% [32] . among fsw, consistent condom use in commercial sexual contacts in the last month increased from 10% in 1995 to 62% in 2009, and the proportion who reported never using condoms decreased from 70.6% to 2.0% during the same period [14, 33] . the current hiv surveillance system in china appears to have improved in its timeliness and effectiveness for providing useful hiv disease information and regular behavioural monitoring of at-risk populations in china. however, under-utilisation and lack of transparency of information outside certain authorised channels are possibly the greatest hindrances to optimal use of the current system. since accessibility to health data in china is largely limited to a small group of authorised personnel at the top of the administrative hierarchy, it often requires extensive bargaining and bureaucratic procedures to obtain epidemiological and behavioural indicators that are commonly accessibly in other countries. hence, large amount of surveillance data remains in cisdcp and cannot be utilised widely for the purpose of hiv public health research, broader program evaluation and planning of control strategies. a transparent hiv surveillance system across all stakeholders, including policy makers, health officials, community health organisation leaders and hiv researchers, is beneficial for the entire hiv sector in the evaluation and implementation of hiv prevention and intervention strategies [34] . to understand barriers to optimal utilisation of available systems it is essential to first understand the political structure in china and its influences on surveillance. although hiv surveillance is considered as a purely public health issue in many countries, issues related to hiv in china are highly sensitive and politically motivated [13, 35, 36] . authoritarianism describes any political structure in which overall authority is concentrated in the hands of a single leader or a small group of elite. prior to economic reform in china, the ultimate political powers were mastered by the chairman of state, who is simultaneously the president of the party and chief commander of the army. political fig. (1) . schematic diagram of flow of information for hiv surveillance in china. decisions and national policies were implemented strictly "from top to bottom" through a pyramidal administrative structure (fig. 2a) . during the process of political decentralisation initiated by the economic reform in china, provincial governments and ministerial institutions were given high degrees of autonomy to stimulate economic growth. although the central government remains at the peak of the power hierarchy, opportunities for competition and bargaining between governments and institutions became possible. this phenomenon was coined "fragmented authoritarianism" by lampton in 1987 [37] . as a result of fragmented authoritarianism, government authority below the very peak of the beijing central government is fragmented and disjointed [37, 38] . as the highest administrative body, the central government does not generally dictate the execution of policies by provincial governments. between equal level governments or governmental institutions, and even between local and central governments, there exists a large space for competition and cooperation, which leads to extensive bargaining concerning policy execution and project development of common interests (fig. 2b) . fragmented authoritarianism in china can be viewed as the mixed product of traditional authoritarianism and demands for increase in political freedom triggered by the economic reform. the fragmented authoritarian political structure directly affects the hiv surveillance system in china. first, fragmented authoritarianism leads to the absence of accountability in vertical administration of cdcs and lack of cooperation among cdcs on the same level. in this structure, the central china moh cannot exercise direct supervision over the provincial mohs, which retain the authority of personnel appointment, organisation structure and budget distribution. conversely, policy immobility in inferior governments can only be overcome by the intervention of a superior government with the authority to resolve conflicting interests. in other words, lower-level governments tend to shift their policy overload to the upper levels in order to avoid assuming accountabilities [38] . consequently, a large number of responsibilities and agenda items accumulate at the upper end of the political hierarchy. therefore, china cdc, as a subordinate organisation of china moh, is caught in an uneasy position whereby it is unable to exercise its designated authority and simultaneously overloaded with responsibilities which it is not designated to bear. this is reflected by the large amount of collected but unanalysed hiv data accumulated in ncaids in central china cdc. as pointed out by sun, et al., the amount of hiv surveillance data from diagnoses, treatment, laboratory tests and behavioural surveillance overflows the organizational capacity of ncaids [14] . apart from key indicators that require to be distributed to moh and the general public, most of the surveillance data is under-utilised [14] . although the web-based hiv reporting system provides an advanced technology base for data reporting and collection, the dissemination of related summary and policy to direct hiv prevention and care strategies remain slow and inefficient. in addition, such a fragmented system makes it difficult to authenticate the quality of the data, as intermediate levels of cdcs do not assume accountability for the accuracy of reported data. an evaluation of hiv surveillance systems by loo et al., indicated that although the chinese hiv surveillance system obtained high scores for flexibility and timeliness across in its surveillance activities, representativeness and completeness of data are poor in its case reporting [34] . discrepancies are found between the hiv laboratory testing algorithm stated in protocols and the actual procedures implemented, which results in gaps in the data and reductions in quality [39] . second, a fragmented authoritarian political structure inevitably leads to a fragmented information system with little openness and systematic mechanisms for synthesis. as fig. (2) . change of political relationship between institutions at the same and different administrative levels in china prior to, and post, economic reform. previously analysed, lower-level cdcs can only access hiv information within its own jurisdiction, which represents only a subset of the information pool of the higher-level cdcs. as there is little information sharing, even between neighbour cdcs on the same level, these information subsets become isolated information islands which can only be integrated by their superiors. in this way, the central china cdc at the top of the administration pyramid has the ultimate authority of data collection, assembling, analysis and distribution. hence, information openness is strongly and solely dependent on the decision of central china cdc and there exists little monitoring mechanisms to ensure the accuracy of both the reported and published information. in the current political structure, all levels of government are only accountable to their upper-level administrations, but not to the general public. the absence of genuine engagement of civil society groups, including the media and the scientific communities, and the expectations and pressures from higher-level authorities, often results in a results-oriented policy implementation, such that local officials tend to manipulate nonscientific and arbitrary results to satisfy their superiors perfunctorily [40] . in this view, ensuring the accuracy of reported disease information should be a high priority in the future development of hiv surveillance in china. social stigma against plhiv is common in china. misconceptions about hiv transmission and prejudice towards plhiv are widespread among the general population. hiv-positive individuals are often denied employment, education and necessary medical services [41] and are thus more likely not to disclose their hiv status [42, 43] . a recent study by zhou in 2008 shows that the healthcare expenses for hiv-positive individuals were greatly elevated by social discrimination, and their available social resources were substantially reduced in the presence of social stigma [44] . the study further indicated that plhiv often perceive healthcare institutions as an indispensable source of social support and one of the few places where they may be willing to disclose their hiv-positive status [44] . however, a separate survey among health workers in hospitals demonstrated that hiv stigmatisation remains as the major health-seeking barriers for plhiv in china [45] . according to a unaids study in 2009, 15% of surveyed participants indicated that their hiv status was disclosed by health-care professionals to others without their consent [46] . according to the latest available statistics, there have been 326,000 cumulative hiv/aids cases in china, including 54,000 deaths, by the end of 2009 [23] . this accounts for only 44% of the estimated number of plhiv at that time. the percentage of undiagnosed cases cannot be improved upon if the barrier of stigma against plhiv from health institutions is not removed. in addition, plhiv that belongs to at-risk groups, especially msm, are subjected to greater social stigma due their identity and sexual orientation. recent study indicated that only 14-45% of chinese msm underwent hiv testing in the past 12 months during 2007-2009 [2, [47] [48] [49] [50] [51] [52] [53] . also, it is estimated that only 12-15% of hiv-positive msm are diagnosed based on an estimate among msm in yunnan province in 2009 [54] . due to the hidden nature of msm, sentinel surveillance targeting msm was also required to substantial scale-up. although the number of sentinel surveillance sites targeting msm has increased from 4 in 2006 to 25 sites in 2009 [1, 14] , both epidemic and behavioural surveillance for msm remains insufficient and limited in many parts of china. reducing social stigma and discrimination is necessary in improving hiv surveillance in china. in summary, the hiv surveillance system in china has undergone substantial revamping that leading to a comprehensive, timely and efficient reporting system. however, large data gaps and lack of quality control and sharing of information obstruct the full performance of the system. this is largely due to fragmented authoritarianism brought about by the underlying political structure. social stigma and discrimination in health institutes are also key barriers for further improvements of hiv diagnosis and surveillance in china. the insufficient biological and behavioural surveillance for chinese msm may result in underestimation of the actual prevalence and disease burden among this population. the study was funded by vice chancellor fellowship, the university of new south wales, 2010-2012. estimating the number of people living with hiv/aids in china: 2003-09 ministry of health of the people's republic of china hiv/aids epidemiology and prevention in china hiv prevalence is increasing among men who have sex with men in china: findings from a review and meta-analysis sexual behaviors and hiv/syphilis infection among men who have sex with men: a meta analysis of data collected between 2001 and 2006 in the chinese mainland risk of hiv/aids in china: subpopulations of special importance office of the state council working committee on aids china. progress on implementing ungass declaration of commitment in china comparing the cost-effectiveness of hiv prevention interventions injection drug use and hiv/aids in china: review of current situation, prevention and policy implications ministry of health of the people's republic of china sex, drugs, and hiv/aids in china the changing face of hiv in china china aids policy implementation: reversing the hiv/aids epidemic by 2015 the development of hiv/aids surveillance in china hiv/aids epidemic and the development of comprehensive surveillance system in china with challenges current challenge of aids epidemic surveillance in china overview on sars in asia and the world a sampling survey on syringe exchange and methadone maintenance treatment among drug abusers in yunnan province brief introduction of chinese infectious disease detection and reporting information system information release guidelines for infectious diseases epidemic and public health contigencies encyclopedia on the prc state secrets law. changchun: jilin people's publishers quality assurance in the hiv/aids laboratory network of china joint united nations programme on hiv/aids, world health organization geneva: world health organization and joint united nations programme on hiv regional differences in hiv prevalence among drug users in china: potential for future spread of hiv? group on hiv/aids in china. hiv/aids: china's titanic peril -2001 update of the aids situation and needs assessment report hiv prevalence among populations at risk, using sentinel surveillance data from 1995 to 2009 in china heterosexual transmission of hiv in china: a systematic review of behavioral studies in the past two decades national hiv/aids sentinel surveillance report in 2003. beijing: china cdc & national sentinel surveillance group injection drug use and hiv/aids transmission in china systematic review of hiv and hcv infection among drug users in china situations and trends of hiv and syphilis infections among drug users in china surveillance of aids among female sex workers in china improving hiv surveillance systems: country experiences and a proposal for evaluation framework quantitatively monitoring aids policy implementation in china living with hiv/aids in china policy implementation in post-mao china the sars epidemic and its aftermath in china: a political perspective evaluation of the guangdong hiv/aids surveillance system, china building a better infectious disease surveillance system for china: an evaluation from a political perspective discrimination against people with hiv persists in china disclosure of hiv status: cultural issues of asian patients disclosure of hiv status is a family matter: field notes from china help-seeking in a context of aids stigma: understanding the healthcare needs of people with hiv/aids in china hiv/std stigmatization fears as health-seeking barriers in china the china stigma index report evaluation of effect of community-based hiv/aids interventions among men who have sex with men in eighteen cities, china which chinese men who have sex with men miss out on hiv testing? investigation on the infection of hiv, hcv, syphilis and hbv among msm in dalian city in 2008 possible increase in hiv and syphilis prevalence among men who have sex with men in guangzhou, china: results from a respondent-driven sampling survey surveillance on the high risk behaviors among 239 men who have sex with men prevalence and correlates of hiv and syphilis infections among men who have sex with men in chongqing municipality survey of the knowledge, attitude, and practice on aids among msm population in urumchi the next era of hiv in china: rapidly spreading epidemics among men who have sex with men the authors confirm that this article content has no conflicts of interest. key: cord-341155-3d64mso0 authors: slots, jørgen; slots, henrik title: bacterial and viral pathogens in saliva: disease relationship and infectious risk date: 2010-12-07 journal: periodontol 2000 doi: 10.1111/j.1600-0757.2010.00361.x sha: doc_id: 341155 cord_uid: 3d64mso0 nan research on the infectious aspects of dental diseases has focused on the internal development and the pathogenicity of dental biofilms, and comparably little attention has been given to the source of the biofilm microorganisms. odontopathic bacteria exist in saliva before colonizing dental surfaces, and a better understanding of the acquisition of salivary pathogens may lead to new approaches for managing dental diseases. human viruses are also frequent inhabitants of the human mouth, and their presence in saliva may be caused by the direct transfer of saliva from infected individuals, a bloodborne infection of the salivary glands, infection of the oral mucosa, or serumal exudates from diseased periodontal sites. it has long been recognized that saliva can contain potential pathogens in quantities sufficient to infect other individuals (152) . the classic example of a serious infection contracted through saliva is epstein-barr virus-induced mononucleosis, which colloquially is termed the ôkissing diseaseõ. an example from the past is the ôno spittingõ signs to prevent inhalation of the tubercle bacillus from spit specimens on street pavements and public floors. dental clinics implement stringent infection-control measures to protect personnel and patients from pathogens in spatter, mists, aerosols, particulate matter or contaminated instruments (36) and, in the future, may adopt fully automated test systems to identify salivaborne pathogens of oral and medical diseases (72, 144) . the potential of salivary biomolecules to aid in the diagnosis of various conditions ⁄ diseases is a topic of current interest (209) . highly sensitive and specific molecular detection methods have greatly facilitated the search for salivary molecules of diagnostic value. polymerase chain reaction (pcr)-based assays can detect a large array of pathogens in saliva with no interference from pcr inhibitors (139) , and even more efficient identification techniques are rapidly emerging (97, 132) . a major advantage of salivary testing is the ease by which diagnostic samples can be collected by health professionals, by the individuals themselves, or by parents for young children. salivary sampling is painless and involves fewer health and safety issues than venepuncture, especially in patients with hemorrhagic diseases or virulent bloodborne pathogens. used as diagnostic aids, salivary biomolecules can identify a variety of cancers, illicit and prescription drug use, hereditary disorders and hormonal irregularities (87) . salivary testing can also screen for infection with the human immunodeficiency virus (hiv) (206) , herpesviruses (144, 172) , hepatitis viruses (144) , measles virus (70) and other pathogenic viruses and bacteria (discussed later). some biomarkers in saliva exhibit significant intrasubject fluctuations and may have limited diagnostic utility (189) . salivary microbial assays to assess the presence or the risk of dental diseases are premised on the idea that (i) whole saliva is the immediate source of oral biofilm bacteria, and saliva and dental biofilms tend to harbor similar relative levels of odontopathogens, and (ii) high salivary counts of odontopathic bacteria infer a high risk for dental disease and for pathogen transmission between individuals, and a decrease in the salivary count of pathogens can serve as an indicator of therapeutic effectiveness. periodontal disease severity may be ascertained by the salivary level of periodontal pathogens or host-response markers (67, 130, 146, 193, 214) , and the periodontopathic bacteria may be acquired from the infectious saliva of close family members (17) . caries risk is assessed by the levels of mutans streptococci and lactobacilli in stimulated saliva (94, 96) , and salivary transmission of cariogenic bacteria frequently occurs from the mother to her child (92, 100) . yeasts can be part of oral biofilms and cause candidosis and other oral diseases (157, 210) , especially in hiv-infected individuals, and candida albicans transmission between spouses can take place through saliva (26) . comparatively few parasites colonize the mouth, but systemic parasitic infections can affect the oral cavity (e.g. leishmaniasis), and oral protozoa may be more common than once thought (21) . this review article presents evidence that pathogenic bacteria and viruses can be present in saliva at levels that pose a disease risk for individuals with whom saliva is exchanged. emphasis is placed on the salivary route of transmission of periodontopathic bacteria and herpesviruses, and on the relationship between these infectious agents and periodontitis. the salivary presence of viral pathogens of rare, but serious, medical diseases is also reviewed. periodontitis and dental caries are infectious diseases, but the exact causes and their relative importance is still a matter of research. the search for etiological factors is closely connected to the question of how to avoid dental diseases. the consensus viewpoint of the scientific community is that specific bacteria cause both periodontitis and dental caries. this understanding has prompted the pursuit of microbiological methods to diagnose, prevent and treat dental infections. the periodontopathic microbiota has been studied for the purpose of developing more effective diagnostic tests and treatments (12, 165) . as periodontopathic bacteria also colonize the tongue dorsum and other nondental sites (43, 131) , and can be transferred via saliva to close family members (17) , periodontitis therapeutic measures ought to target periodontal pathogens in the whole mouth, not only in dental biofilms, and may even include entire family units in order to prevent cross-infection. umeda et al. (193) compared the presence of six species of periodontopathic bacteria in whole saliva and subgingival plaque from 202 subjects. each study subject contributed a whole saliva sample and a paper point sample pooled from the deepest perio-dontal pocket in each quadrant of the dentition, and the test bacteria were identified using a 16s ribosomal rna-based pcr assay (15) . a statistical relationship was found between the presence of porphyromonas gingivalis, prevotella intermedia, prevotella nigrescens and treponema denticola in whole saliva and in periodontal pocket samples, and in the event of disagreement, the organisms were more frequently present in whole saliva than in periodontal pockets (p < 0.01). the oral presence of aggregatibacter actinomycetemcomitans and tannerella forsythia was not reliably detected by sampling either whole saliva or periodontal pockets. other studies also found that a salivary sample alone did not identify all individuals infected with a. actinomycetemcomitans (176, 200) . taken together, a sample of whole saliva seems to be superior to a pooled periodontal pocket sample for detecting oral p. gingivalis, p. intermedia, p. nigrescens and t. denticola, but samples of both whole saliva and periodontal pockets may be needed in order to detect oral a. actinomycetemcomitans and t. forsythia with reasonably good accuracy. the reason for this is that a. actinomycetemcomitans and t. forsythia can persist in nondental sites, as best demonstrated in fully edentulous individuals (55, 194) . umeda et al. (192) also investigated risk factors for harboring a. actinomycetemcomitans, p. gingivalis, t. forsythia, p. intermedia, p. nigrescens and t. denticola in periodontal pockets, in whole saliva, or in both sites (i.e. orally). the study subjects included 49 african-americans, 48 asian-americans, 50 hispanics and 52 caucasians living in los angeles. periodontal probing depth was positively associated with all six study bacteria. african-americans were at increased risk (compared with caucasians) for harboring p. gingivalis in saliva [odds ratio (or) 2.95] and orally (or 2.66), and at reduced risk for harboring t. denticola orally (or 0.34). asian-americans showed an increased risk for harboring a. actinomycetemcomitans in periodontal pockets (or 6.63) and for harboring p. gingivalis in periodontal pockets (or 5.39), in saliva (or 5.74) and orally (or 5.81). hispanics demonstrated an increased risk for harboring a. actinomycetemcomitans in periodontal pockets (or 12.27) , and for harboring p. gingivalis in periodontal pockets (or 6.07), in saliva (or 8.72) and orally (or 7.98). age was positively associated with the prevalence of a. actinomycetemcomitans orally (or 1.18), and with p. gingivalis in saliva (or 1.20) and orally (or 1.20). the male gender was a risk factor for harboring p. intermedia in periodontal pockets (or 2.40), in saliva (or 3.31) and orally (or 4.25) , and for harboring p. nigrescens in saliva (or 2.85). the longer the subjects had resided in the usa, the greater the decrease in detection of a. actinomycetemcomitans orally (or 0.82). former smokers demonstrated a decreased risk for harboring a. actinomycetemcomitans in saliva (or 0.23), and current smokers displayed an increased risk for harboring t. denticola in periodontal pockets (or 4.61) . current and passive smokers revealed less salivary p. nigrescens than nonsmokers (127) . in sum, the study found a relationship between the presence of periodontopathic bacteria in whole saliva and in periodontal pockets, and pointed to the importance of genetic or environmental factors in the colonization of these pathogens. salivary tests for periodontitis may show increased accuracy if supplementing infectious disease variables with ethnic and social factors and with smoking habits (177) . studies have evaluated the salivary route of transmission of periodontopathic bacteria. transmission of periodontal pathogens from person to person depends on the salivary load of pathogens in the donor subject and various ecological factors in the recipient (16) . an early epidemiologic study found that members of the same family were infected with a. actinomycetemcomitans strains of the same biotype and serotype (213) . however, even in families with individuals heavily infected with a. actinomycetemcomitans, some family members did not harbor the organism, attesting to a relatively poor transmissibility of a. actinomycetemcomitans (213) . a study based on bacterial typing by means of the arbitrarily primed pcr method revealed an interspousal transmission of a. actinomycetemcomitans in 4 ⁄ 11 (36%) of married couples and of p. gingivalis in 2 ⁄ 10 (20%) of married couples (17) . parent-to-child transmission of a. actinomycetemcomitans took place in 6 ⁄ 19 (32%) families, whereas p. gingivalis was not transmitted from parent to child in any of the families studied (17) . similarities in the profile of periodontal bacteria have also been shown for 6-to 36-month-old children and their caregivers (186) . a review article described horizontal transmission between spouses to be 14-60% for a. actinomycetemcomitans and 30-75% for p. gingivalis, and vertical transmission to be 30-60% for a. actinomycetemcomitans and to occur only rarely for p. gingivalis (197) . the intrafamilial transmission of a. actinomycetemcomitans and p. gingivalis may in part explain the familial pattern of some types of periodontitis (13) . also, periodontal treatment and marked suppression of periodontopathic bacteria in members of a periodontitis-prone family may diminish the risk of transferring the pathogens and the disease to uninfected family members. the major cariogenic bacteria are mutans streptococci in incipient dental caries and lactobacilli in advanced caries lesions (95) , perhaps in combination with other bacteria of the dental biofilm (1, 142) . after adjusting for age and ethnicity, 6-to 36-month-old children with high levels of streptococcus mutans were found to be five times more likely to have dental caries than children with low levels of the bacterium (117) . recent large-scale microbiological studies have linked s. mutans to crown caries in children and adolescents (1, 42) and to root caries in elderly patients (142) . herpesviruses have been statistically associated with severe dental caries, but their role, if any, in the caries process remains obscure (38, 212) . an intrafamilial transfer of s. mutans was first suggested in the 1980s (23, 47) . transmission of cariogenic bacteria from the mother to the young child is particularly common, although the organisms also may be acquired from a spouse or from outside the family (98) . more recent studies have found a similar profile of cariogenic bacteria in young children and their caregivers (186) , and molecular typing studies have provided additional evidence of a transmission of mutans streptococci from mother to child (92, 100) . caries-free twins have a more similar oral microflora than twins that are caries-active, and hereditary factors seem to influence the colonization of oral bacterial species that protect against dental caries (41) . the finding of a relatively unique cariogenic microflora has a practical implication. routine testing for elevated caries risk, based on the salivary level of mutans streptococci (>1,000,000 per ml saliva) and lactobacilli (>100,000 per ml saliva), has been performed in sweden for more than 30 years (46, 94) . repeat swabbing of teeth of young children with 10% povidone-iodine can reduce the number of mutans streptococci (22) and the incidence of caries (106) . suppression of high levels of s. mutans in the mother may delay or prevent the establishment of the organism in her child (91) . a variety of bacterial pathogens of medical diseases can be present in the oral cavity and may be transmitted to individuals in close contact with the host (45) . medical pathogens are mostly detected in the mouth during the acute phase of the nonoral infection, but the organisms can also occur in the saliva of clinically healthy subjects. streptococcus pyogenes (beta-hemolytic group a streptococcus) is the cause of a variety of human diseases ranging from mild illnesses of the skin or throat (pharyngitis or ôstrep. throatõ) to severe invasive infections, including necrotizing fasciitis (flesheating disease), septicemia, toxic shock syndrome, erysipelas, cellulitis, acute postinfectious glomerulonephritis, rheumatic fever and scarlet fever (178) . s. pyogenes normally resides in the throat and is one of the most common medical pathogens in the saliva. an asymptomatic carriage stage of s. pyogenes was detected in approximately 10% of adults and 25% of children, and in as many as 60% of subjects during large outbreaks of streptococcal pharyngotonsillitis (178) . beta-hemolytic group a streptococci were found in 20% of pharyngeal samples and in 5% of saliva samples of young schoolchildren in new zealand, with a suggestion of a child-to-child transmission of the organism (185) . members in the same household of a patient with pharyngotonsillitis frequently harbor the same strain of beta-hemolytic group a streptococcus, indicating an intrafamilial transmission of the bacterium (58) . haemophilus influenzae can cause acute bronchitis and exacerbations of chronic obstructive pulmonary disease, as well as meningitis in children and other serious diseases (124) . despite the availability of highly effective vaccines since the early 1990s, 100,000s of unvaccinated children die every year from h. influenzae-related disease (208) . the organism resides in the pharynx and is rarely recovered from the saliva of healthy individuals (88) . it can reach quantities of 10 3 -10 8 ⁄ ml in the sputum of patients with lower respiratory tract infections and purulent sputum (61) . staphylococcus spp., pseudomonas spp. and acinetobacter spp. are also potential pathogens in respiratory (and other) diseases. these bacteria were detected in the oral cavity of 85% of hospitalized patients in brazil (216) and in subgingival sites of periodontitis patients in the usa (145, 174) . periodontal staphylococci occurred with highest proportions in younger individuals, and periodontal gram-negative bacilli were found mostly in older subjects (174) . staphylococci can also be prominent in the microbiota of failing dental implants (78) . gram-negative bacilli are frequent inhabitants of the oral cavity of individuals in developing countries, where the bacteria are probably acquired through contaminated potable water (8, 79, 175) . meningococcal invasive disease (septicemia and ⁄ or meningitis in association with hemorrhagic rash) is a life-threatening condition that primarily affects young children. meningococcal disease can also occur in teenagers, and is more common in collage ⁄ university students than in the general population (or 3.4) (190) . although neisseria meningitidis resides in the nasopharynx and in the tonsils, and is much less common in saliva (129), intimate kissing, especially with multiple partners, constitutes a risk factor for meningococcal disease (or 3.7) (190) . fortunately, the prevalence of meningitis caused by n. meningitidis, h. influenzae type b and streptococcus pneumoniae has decreased markedly after the introduction of vaccines against these bacteria (89) . neisseria gonorrhoeae (which causes gonorrhea) and treponema pallidum (which causes syphilis) can produce acute and chronic oral infections. gonorrhea is a widespread disease worldwide, with an estimated 600,000 new cases each year in the usa (103) . although oral gonorrhea is relatively rare, the literature describes more than 500 cases of oropharyngeal gonorrhea (20) . syphilis is re-emerging in many countries, especially in hiv-infected individuals and among men who have sex with men, and oral sex is often reported to be the route of t. pallidum transmission (37, 168, 198) . infants have contracted syphilis by the mouth-to-mouth transfer of prechewed food from actively infected relatives (215) . dentists can play an important role in the control of sexually transmitted diseases by identifying signs and symptoms of gonorrhea and syphilis and making appropriate referrals for treatment. tuberculosis remains a serious disease worldwide (68) . in 2005, there were an estimated 8.8 million new cases of tuberculosis, with 7.4 million occurring in asia and sub-saharan africa, and 1.6 million people died of tuberculosis, including 195,000 with hiv infection (114) . mycobacterium tuberculosis can be identified in the whole saliva of almost all tuberculosis patients (54) and of some nontuberculous individuals (101) , and has been recovered from alginate dental impressions (140) . the us centers for disease control has identified the personnel of a dental-care facility to be at increased risk for infection with m. tuberculosis (35) and has updated the tuberculosis infection control guidelines for dental clinics (40) . helicobacter pylori can cause gastritis, peptic ulcers and gastric adenocarcinoma (143) . the organism resides primarily in the human stomach and may colonize about 50% of the worldõs population. large quantities of h. pylori can be recovered from vomitus, and the bacterium can also be detected in saliva, especially in subjects suffering from gastric ulcer. however, published data on the occurrence of h. pylori in the mouth vary greatly (143) , perhaps because the oral carriage of h. pylori is population dependent or is only transient (50) . the transmission route is mainly from the mother, or an older sibling, to younger children. both gastro-to-oral and oral-tooral transmission are considered important. legionella pneumophila is the cause of legionellosis (legionnaireõs disease), a severe type of pneumonia with multisystem failure, and of pontiac fever, a self-limiting influenza-like illness (114) . the natural reservoir for l. pneumophila and other legionella species is aquatic habitats. legionellae have been isolated from sputum and other body fluids and sites (123) . l. pneumophila has also been recovered from dental unit water in england, germany and austria (184) , and from 8% of dental units in the usa (18) . however, no evidence exists to incriminate dental units as a significant source of legionellosis. herpesvirus species comprise the most prevalent viral family in human saliva and are important periodontopathic agents (173) . eight herpesvirus species, with distinct biological and clinical characteristics, can infect humans: herpes simplex virus-1 and -2, varicella-zoster virus, epstein-barr virus, human cytomegalovirus, human herpesvirus-6, human herpesvirus-7 and human herpesvirus-8 (kaposiõs sarcoma virus) (171) . herpesviruses establish a lifelong persistent infection, and some herpesvirus species infect as many as 90% of the adult population. the clinical outcome of a herpesvirus infection ranges from subclinical or mild disease to encephalitis, pneumonia and various types of cancer. herpesviral infections in the oral cavity may give rise to asymptomatic and unrecognized shedding of virions into saliva, or to diseases of the oral mucosa or the periodontium (171, 179) . a recent article reviewed acute herpesviral infections in the oral cavity of children (156) . herpesviruses exhibit a biphasic infection cycle involving a lytic, replicative (ôproductiveõ) phase and a latent, nonproductive phase (171) . the replicative phase involves expression of viral regulatory and structural proteins, and the formation of infectious virion particles (172) . the ability to switch between replicative and latent states ensures viral transmissibility between individuals as well as a permanent infection of the host. following the initial infection, herpesviruses preferentially exist in a state of latency in sensory ganglion cells (herpes simplex viruses and varicella-zoster virus), b-lymphocytes (epstein-barr virus, herpesvirus-8), or monocytes and t-lymphocytes (cytomegalovirus and herpesviruses-6 and -7). herpesvirus conversion from a latent form to lytic replication can occur spontaneously or be caused by environmental stimuli, chemical agents and physical and psychosocial stress events, as found in adults with an abusive early-childhood history, astronauts in space flight, students before important academic exams, elite athletes in intensive training and subjects with work-related fatigue ( table 1 ). reactivation of an oral herpesviral infection can be estimated by a rise in herpesvirus salivary counts or a significant increase in herpesvirus-specific salivary antibodies. immunocompetent individuals usually experience herpesvirus re-activation lasting for only a few hours or days (112) , which is probably too short a time period to initiate or exacerbate clinical disease. however, the egress of herpesvirus virions into saliva poses a risk for infecting individuals in intimate contact. by contrast, immunosuppressive conditions ⁄ diseases and long-term medications may result in the re-activation of oral herpesviruses that continues for an extended period of time and may pose a pathogenetic risk for the infected individual. the immune system of older persons may fail to control a latent varicella-zoster infection, resulting in herpes zoster outbreaks (29), or may not protect effectively against epstein-barr virus and cytomegalovirus re-activation (180) . the herpesvirus infection in such persons may be characterized as chronically re-activated instead of latent. the great majority of systemically healthy adults continually shed herpesvirus dna into saliva. herpes simplex virus-1 dna was detected in saliva in quantities up to 2.0-2.8 · 10 6 ⁄ ml (102, 118) . epstein-barr virus dna copies in saliva can reach levels of 10 8 ⁄ ml (76), 1.6 · 10 9 ⁄ ml (155), 7.1 · 10 5 ⁄ ml (181) and 2.2 · 10 6 per 0.5 lg of dna (202) . as the epstein-barr virus salivary count only decreased moderately after large-volume mouth gargles and rinses, or after normal swallowing every 2 min, a large quantity of the virus must constantly enter the saliva (76) . however, the salivary epstein-barr virus load can vary by as much as 4-5 logs over the course of several months, which complicates the categorizing of (76) . cytomegalovirus dna was detected in the saliva of 61% of immunocompetent and immunocompromised subjects (65) , and could reach salivary dna copy counts of 4.2 · 10 4 ⁄ ml (155) . herpesvirus-6 and herpesvirus-7 may occur in saliva, with prevalences exceeding 95% and in quantities of several million dna copies ⁄ ml (118) . salivary herpesvirus-8 dna, in quantities of 2.0-7.3 log 10 copies ⁄ ml, was detected in 61% of asymptomatic, immunocompetent men who have sex with men (32) , and in 37% of zimbabwean women with kaposiõs sarcoma, but not in women without the disease (99) . varicella-zoster virus dna is present at a low prevalence and in quantities of <1,100 copies ⁄ ml in the saliva of both healthy and hiv-infected individuals (205) . table 2 shows the association between salivary herpesviruses and periodontitis. a periodontal dual infection of herpesviruses and pathogenic bacteria gives rise to enhanced cytokine release and immune signaling dysregulation (27, 104, 187) , and tends to be associated with more severe periodontitis than a periodontal infection involving solely bacteria (172) . herpes simplex virus-1 may contribute to periodontitis in a subset of individuals (173) , and the virus was identified in whole saliva of 24% of patients with chronic periodontitis (71) . in the same group of patients, herpes simplex virus-1 dna was present in 16% of subgingival samples and in 8% of peripheral blood samples (71) . herpes simplex virus dna was found in the saliva of 84% of patients with overt herpetic lesions (144) . epstein-barr virus dna has been detected in whole saliva of 79% of periodontitis patients and 33% of gingivitis patients (155) , and in 49% of periodontitis patients and 15% of healthy individuals (82) . a correlation was found between salivary and subgingival levels of epstein-barr virus in one study (48) but not in another study (84) . as high quantities of salivary epstein-barr virus dna can be recovered from fully edentulous patients (155) , the occurrence of the virus in saliva may not be a reliable indicator of its subgingival level or of the periodontitis disease status. cytomegalovirus periodontal active infection is closely linked to aggressive periodontitis (173) . cytomegalovirus dna was detected in the saliva of 50% of periodontitis patients, but was not found in the saliva of gingivitis patients or complete denture wearers, suggesting that salivary cytomegalovirus originates mainly from periodontitis lesions (155) . also, cytomegalovirus dna from infected breast milk appeared in the saliva of infants at 4 months of age, peaked 4-10 months after birth, and thereafter decreased or became undetectable (122) . to sum up, a great proportion of salivary herpeviruses are shed from periodontal disease sites. as periodontal treatment can markedly reduce subgingival (73, 162) and salivary (82, 162) herpesvirus dna counts, the establishment of a healthy periodontium may diminish the risk of intersubject herpesvirus transmission and of herpesvirus-related diseases. the close relationship between some herpesvirus species and periodontitis also argues for examining the potential of using herpesvirus salivary counts to indicate periodontal disease risk. infectious mononucleosis is caused by a primary infection with epstein-barr virus, and predominantly by epstein-barr virus type 1 (44) . approximately 10% of mononucleosis-like disease is attributable to cytomegalovirus. the epstein-barr virus infects b-lymphocytes, which gives rise to the strong t-lymphocyte response that is characteristic of mononucleosis. clinical signs of infectious mononucleosis are long-lasting fever, tonsillopharyngitis, lymphadenopathy, fatigue, and occasionally splenomegaly, liver involvement and pericarditis (199) . oral signs are sore throat, palatal petechiae and enlarged lymph nodes in the throat and neck. the epstein-barr virus is transmitted through direct contact with virus-infected saliva, such as with kissing, and rarely via the air or blood. young adults with a primary epstein-barr virus infection can rapidly clear the virus from the blood but not from the oropharynx (19) . however, individuals who are already infected with the epstein-barr virus (and cytomegalovirus) are not at risk for infectious mononucleosis, even when exposed to individuals with the disease. other diseases have been linked to salivary herpesviruses (table 2) . relationships have been found between bellõs palsy (idiopathic peripheral facial paralysis) and an active herpes simplex virus-1 infection (3), between oropharyngeal lesions of the ramsay hunt syndrome and varicella-zoster virus (62, 144) , and between hiv infection and epstein-barr virus (74) and herpesvirus-8 (33) . young children with exanthem subitum acquired the disease from their mothers who excreted the causative herpesvirus-6 into saliva (121) . human immunodeficiency virus infection is a potent herpesvirus re-activator, as demonstrated by a strong correlation between decreasing cd4 cell counts in hiv-infected patients and increasing rates of herpesvirus re-activation (34) . an hiv infection is frequently associated with the salivary presence of several re-activated herpesvirus species (table 3 ). in the mode of synergism, herpesviruses (196) , p. gingivalis (83) and other periodontal bacteria (81) may resides in the buccal epithelial cells of hiv-infected subjects (134) , and can be transmitted horizontally from an hiv-infected mother to her young children (66, 111) but, despite the possibility of in utero infection (28) , vertical transmission of the virus is uncommon in infants born to an hiv-positive mother (111) . herpesvirus-8 can also be transmitted by oral sex. deep kissing was an independent risk factor (odds ratio of 5.4) for transmitting herpesvirus-8 from hiv-seropositive men to hiv-seronegative men, (134) . taken together, the saliva of hiv-infected persons is a risk factor for the transmission of several virulent herpesvirus species, and patients receiving haart cannot be assumed to be less infectious for herpesviruses than individuals not receiving haart. oral mucositis is an important complication of immunosuppressive radiotherapy, chemotherapy and radiochemotherapy (163) . the mucositis may involve herpesviruses, bacteria and yeasts, individually or in combination (163) . bone marrow and stem cell transplantation has been associated with oral cytomegalovirus re-activation (148) , and renal allograft transplantation has been associated with oral cytomegalovirus re-activation (128) and oral herpesvirus-8 re-activation (9) . also, although not studied in the oral cavity, corticosteroid immunosuppressive treatment may trigger the re-activation of herpesvirus species (14, 49, 164, 211) . viruses of serious medical diseases can be present in saliva at levels sufficient to be transmitted from person to person through close (within 2 meter) or intimate contact (table 4) . moreover, viral pathogens can be transferred to humans by animals or insects (table 4) , or from humans to animals and then later transferred back into humans (56) . viruses in saliva may infect the periodontium and exacerbate periodontal disease. human papillomaviruses are frequent inhabitants of the oral mucosa of normal adults (188) and have been found to occur in the saliva of 25% of healthy individuals (154) . papillomavirus dna was detected in 26% of gingival biopsies from periodontitis lesions (80) , and in as many as 92% of biopsies of cyclosporin-induced gingival hyperplasia from renal transplant recipients (30) . papillomavirus type 16 is associated with a subset of oropharyngeal squamous cell carcinomas (171) , and quantitative measurement of salivary papillomavirus-16 dna has shown promise for early detection of recurrence of head and neck squamous cell carcinoma (39) , and for surveillance of premalignant oral disorders (183) . papillomavirus dna was identified in the saliva of 10% (5) and 41% (154) of oral squamous cell carcinoma patients, and in the saliva of 35% of hiv-positive individuals (5) . a spouse had a 10-fold higher risk of acquiring a persistent oral papillomavirus infection if the other spouse had a persistent oral papillomavirus infection, a finding that is consistent with the oral route of papillomavirus transmission (149) . the likelihood of contracting an oral papillomavirus infection increases with increasing numbers of open-mouthed kissing partners and oral sex partners (52) , and papillomavirus-positive oral tumors are strongly linked to multiple oral sex partners (53) . the current prophylactic papillomavirus-6 ⁄ 11 ⁄ 16 ⁄ 18 vaccine, designed to prevent cervical cancer, generates an oral antibody response and will probably also reduce the incidence of papillomavirus-related diseases of the mouth (153) . human immunodeficiency virus is transmitted through sexual contact or by contaminated needles and blood, but only exceptionally rarely through saliva. a recent study provided compelling evidence that three infants acquired hiv ⁄ acquired immunedeficiency syndrome (aids) after receiving prechewed food (64) . the hiv-infected caregivers had bleeding gingiva while masticating food for the infants, and thus blood, not saliva, was probably the vehicle for hiv transmission in the three cases reported. in fact, submandibular ⁄ sublingual gland secretions contain mucin molecules that normally will prevent infection and transmission of hiv by the oral route (75) . thus, as is the case for hiv and for other viruses, saliva is not merely serving as a passive transport medium, but can significantly affect the efficiency of pathogen transmission and the course of disease. fortunately, anti-retroviral drugs have turned hiv infection into a manageable condition with a greatly reduced morbidity. the proviral dna of human t-cell lymphotropic virus type i, an oncogenic retrovirus, was detected in whole saliva of 77% of mashhadi-born iranian jews with viral myelopathy (4) . this finding may suggest the potential for a salivary transmission of human t-cell lymphotropic virus type i and may possibly help to explain the relatively high rate of myelopathy in the elderly mashhadi-jewish population. the human t-cell lymphotropic virus type i can also be present in the saliva of asymptomatic carriers of the virus (4). hepatitis viruses (designated a through g) cause the majority of cases of acute and chronic hepatitis and liver damage worldwide. hepatitis ranges pathologically from asymptomatic or mild disease to fulminant liver failure. hepatitis a and hepatitis e viruses are transmitted by water contaminated with feces (fecal-oral route), produce acute infections and do not induce a chronic carrier state. a high incidence of hepatitis a and hepatitis e viral infections occurs in countries with poor sanitary standards. hepatitis a virus rna was detected in the saliva of 50% of patients during a hepatitis a outbreak (11) . a study in cynomolgus monkeys found that the tonsils and salivary glands acted as extrahepatic sites for early hepatitis a virus replication and constituted potential sources for saliva-transmitted infection (10) . hepatitis b virus is parenterally transmitted and is frequently associated with chronic viremia. hepatitis b virus dna was found at concentrations of >10 5 copies ⁄ ml of saliva in 15% of patients with chronic hepatitis b (195) . that concentration may be sufficient to permit horizontal transmission of the virus, and perhaps some of the 20% of hepatitis b patients, who contract the disease without a known origin of the infection, may have acquired the hepatitis b virus by salivary transfer (195) . chronic hepatitis c affects more than 170 million people worldwide, and the hepatitis c virus persists in 80% of the infected individuals, where it can give rise to liver inflammation, liver cirrhosis and hepatocellular carcinoma (135) , and perhaps to periodontitis, sjögrenõs syndrome, oral lichen planus and sialadenitis (171) . hepatitis c virus rna was present in the saliva of 39-72% of subjects with chronic hepatitis (113, 133, 144, 204) , and was detected in 59% of gingival crevice fluid specimens from viremic patients (113) . the gingival crevice fluid was identified as the major source for salivary hepatitis c virus (113) . twenty-seven percent of spouses of individuals with chronic hepatitis c revealed antibodies against three hiv-positive infants (9-39 months old) were fed with premasticated food: two children by an hiv-infected mother with oral bleeding; and one child by an hiv-positive aunt (the mother was hiv-negative) the infants were not breastfed and perinatal transmission of hiv was previously ruled out. viruses of respiratory diseases are usually transmitted through coughs or sneezes that release large quantities of high-velocity droplets into the air, and the risk of cross-infection through salivary exchange is comparatively small. children with respiratory disease revealed respiratory viruses (respiratory syncytial virus, influenza virus, parainfluenza virus, adenovirus) in 74% of oral specimens and in 77% of nasopharyngeal specimens (150) , and respiratory syncytial virus rna in 76% of salivary samples (201) . although present in saliva (150) , influenza virions may not be infectious because of the anti-influenza virus activity of salivary glycoproteins (207) . the severe acute respiratory syndrome (sars) corona virus, the etiological agent of a highly lethal type of pneumonia, was detected in the saliva of each sars patient studied, and was present in quantities up to 6.38 · 10 8 copies ⁄ ml (203) . a dental clinic located in a sars-affected region must institute strict infectioncontrol measures in order to prevent cross-infection with the sars virus (158) . measles and rubella are rare diseases in vaccinated populations, but still occur in unvaccinated persons, commonly in developing countries. the causative viruses are spread through respiration, and can be present in saliva in high numbers during disease outbreaks (2, 126) . ebola is a viral hemorrhagic febrile disease that can cause death in 2-5 days. ebola virus was detected in the saliva of all 24 patients with a positive ebola diagnosis (60) , and transmission of the ebola virus through oral exposure has been demonstrated in nonhuman primates (85) . merkel cell carcinoma is a highly lethal neuroepithelial tumor of the skin, and at least some merkel cell carcinomas appear to be caused by a newly discovered polyomavirus. the merkel cell polyomavirus is found in relatively high numbers in respiratory secretions and in the saliva of patients with merkel cell carcinoma (107) , possibly exposing close individuals to a risk of infection. humans can contract serious viral diseases through zoonotic transfer ( table 4 ). the rabies virus resides in dogs, foxes, cats, vampire bats and other animals, and is transmitted to humans through the bite of a rabid animal. rabies virus rna was identified in 88% of salivary samples from humans with an ante-mortem diagnosis of rabies (125) . hantaviruses cause hemorrhagic fever with renal syndrome (in eurasia) or cardiopulmonary syndrome (in the americas), and rodent-to-human transmission usually occurs by the inhalation of aerozolized viruscontaminated rodent excreta. however, the andes hantavirus infects the secretory cells of human salivary glands and can be detected in the saliva after onset of disease symptoms, suggesting that the virus also may be transmitted by human-to-human contact (77, 137) . nipah virus, a paramyxovirus with a reservoir in fruit bats, can cause respiratory disease and severe encephalitis in humans. a study in bangladesh concluded that 50% of nipah patients acquired the virus through salivary transmission from person to person (108) . some viral diseases in tropical and subtropical parts of the world are acquired through insect bites. dengue fever, caused by a mosquito-borne flavivirus, afflicts more than 100 million subjects annually. patients with dengue fever revealed the dengue virus genome in saliva during the acute phase of the infection (120, 141) . crimean-congo hemorrhagic fever virus is transmitted by tick bites or by contact with the blood or tissues of infected patients and livestock. the genome of the crimean-congo hemorrhagic fever virus was detected in the saliva of five of six patients with confirmed disease (24) , increasing the likelihood of a human-to-human transmission. our knowledge of infectious agents in the human oral cavity has expanded greatly in recent years, mainly as a result of molecular techniques that can identify and quantify oral bacteria and viruses with great accuracy. several oral and medical pathogens occur in saliva at levels that are sufficient to infect close individuals, and contact with saliva may be a more important mode of pathogen transmission than previously realized. the rising awareness of the infectious potential of saliva raises challenging questions about the safety of intimate (ôdeepõ or ôopen mouthedõ) kissing contact. the risk of cross-infection by salivary transfer may not be trivial and needs to be studied further. the type of pathogenic agents that can retain infectiousness in saliva and that are efficiently spread by saliva needs to be identified and controlled. current knowledge of the oral ecology may form the basis for more efficient treatments of bacterial and viral infections around teeth and of the oral mucosa. the finding of major periodontopathic bacteria in nondental sites, especially on the tongue, argues for antimicrobial treatment of the entire oral cavity, not only of dental biofilms (151) . virtually all periodontal patients can benefit from treatment with antiseptics active against bacteria and herpesviruses, such as sodium hypochlorite and povidone-iodine (169) , and selective patients may benefit from treatment with systemic antibacterial (170) and antiviral (182) medications. effective periodontal therapy includes professional administration of a battery of well-tolerated antimicrobial agents, each exhibiting high activity against periodontal pathogens and delivered in ways that simultaneously affect pathogens residing in different oral ecological niches [i.e. chlorhexidine or dilute sodium hypochlorite (bleach) for general oral disinfection, povidone-iodine for subgingival irrigation, and systemic antibiotics to reach microorganisms within periodontal tissue and in difficult-to-reach subgingival and extra-dental sites]. the follow-up maintenance program should have a strong anti-infective emphasis, and may include patient-administered subgingival irrigation with dilute sodium hypochlorite and oral rinsing with sodium hypochlorite or chlorhexidine two to three times per week. full-mouth disinfection may also reduce the risk for cross-infection of oral pathogens between individuals in close contact. however, in the final analysis, most chronic infectious diseases such as periodontitis and dental caries will be defeated on a mass-scale only by employing effective, safe and inexpensive vaccines. vaccines may be prophylactic, therapeutic, or a combination of both. perhaps a vaccine that reduces the infectious load without actually eliminating the infectious agent is sufficient to arrest or prevent dental and other oral diseases. vaccination studies on herpesviruses and some oral bacteria have yielded occasional successes in animal models, but a number of human trials have failed to show adequate efficacy. vaccine development has been difficult because of the heterogeneity, variability and poor immunogenicity of the outer surface components of many infectious agents. nonetheless, despite the setbacks, vaccines against herpes zoster virus and oncogenic papillomaviruses were recently approved for clinical use by the us food and drug administration. effective and safe vaccines against oral infectious diseases constitute one of the most important needs in dentistry. bacteria of dental caries in primary and permanent teeth in children and young adults confirmation of rubella within 4 days of rash onset: comparison of rubella virus rna detection in oral fluid with immunoglobulin m detection in serum or oral fluid secretion and dynamics of herpes simplex virus in tears and saliva of patients with bellõs palsy detection of proviral human t-cell lymphotrophic virus type i dna in mouthwash samples of ham ⁄ tsp patients and htlv-i carriers hpv detection rate in saliva may depend on the immune system efficiency hepatitis c virus infection in spouses of patients with type c chronic liver disease prevalence and risk factors for intrafamilial transmission of hepatitis c virus in karachi, pakistan comparative detection frequency of 6 putative periodontal pathogens in sudanese and norwegian adult periodontitis patients extensive oral shedding of human herpesvirus 8 in a renal allograft recipient experimental hepatitis a virus (hav) infection in cynomolgus monkeys (macaca fascicularis): evidence of active extrahepatic site of hav replication comparison between serum and saliva for the detection of hepatitis a virus rna comparison of the microbiologic features of chronic and aggressive periodontitis comparison of the clinical features of chronic and aggressive periodontitis cytomegalovirus disease during severe drug eruptions: report of 2 cases and retrospective study of 18 patients with drug-induced hypersensitivity syndrome polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions oral ecology and person-to-person transmission of actinobacillus actinomycetemcomitans and porphyromonas gingivalis likelihood of transmitting actinobacillus actinomycetemcomitans and porphyromonas gingivalis in families with periodontitis legionella contamination of dental-unit waters a prospective clinical study of epstein-barr virus and host interactions during acute infectious mononucleosis gonococcal tonsillar infectiona case report and literature review parasitic infections affecting the oral cavity adjunctive chemotherapeutic suppression of mutans streptococci in the setting of severe early childhood caries: an exploratory study mouth-to-mouth transmission of the bacterium streptococcus mutans between mother and child detection of crimean-congo hemorrhagic fever virus genome in saliva and urine human polyoma viruses and disease with emphasis on clinical bk and jc distribution and hydrolytic enzyme characteristics of candida albicans strains isolated from diabetic patients and their non-diabetic consorts profiling of inflammatory cytokines produced by gingival fibroblasts after human cytomegalovirus infection postnatal human herpesvirus 8 and human immunodeficiency virus type 1 infection in mothers and infants from zambia varicella zoster virus: natural history and current therapies of varicella and herpes zoster. herpes human papillomavirus infection in cyclosporin-induced gingival overgrowth in renal allograft recipients spread of hepatitis c virus infection within families. investigators of an italian multicenter group frequent and asymptomatic oropharyngeal shedding of human herpesvirus 8 among immunocompetent men hiv infection and human herpesvirus-8 oral shedding among men who have sex with men the interaction between herpes simplex virus and human immunodeficiency virus guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilities. notice of final revisions to the ''guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilities transmission of primary and secondary syphilis by oral sex -chicago herpes simplex virus infection in patients affected by dental caries presence of hpv dna in convalescent salivary rinses is an adverse prognostic marker in head and neck squamous cell carcinoma tuberculosis epidemiology, diagnosis and infection control recommendations for dental settings: an update on the centers for disease control and prevention guidelines heritability of oral microbial species in caries-active and caries-free twins microbial risk indicators of early childhood caries detection of periodontal pathogens in oral mucous membranes of edentulous individuals a cohort study among university students: identification of risk factors for epstein-barr virus seroconversion and infectious mononucleosis bacterial infections of the oral mucosa multiple types of the bacterium streptococcus mutans in the human mouth and their intrafamily transmission salivary levels of epstein-barr virus dna correlate with subgingival levels, not severity of periodontitis cytomegalovirus retinitis following intravitreal injection of triamcinolone: report of two cases oral helicobacter pylori: can we stomach it? human herpesviruses 6 and 7 in salivary glands and shedding in saliva of healthy and human immunodeficiency virus positive individuals oral sexual behaviors associated with prevalent oral human papillomavirus infection case-control study of human papillomavirus and oropharyngeal cancer pcr method is essential for detecting mycobacterium tuberculosis in oral cavity samples bacterial colonization of oral implants from nondental sources the significant but understudied impact of pathogen transmission from humans to animals investigation of saliva, faeces, urine or semen samples for the presence of gbv-c rna the role of household contacts in the transmission of group a streptococci salivary cytomegalovirus (cmv) shedding, glycoprotein b genotype distribution, and cmv disease in human immunodeficiency virus-seropositive patients detection of ebola virus in oral fluid specimens during outbreaks of ebola virus hemorrhagic fever in the republic of congo ecology of haemophilus influenzae and haemophilus parainfluenzae in sputum and saliva and effects of antibiotics on their distribution in patients with lower respiratory tract infections varicellazoster virus dna level and facial paralysis in ramsay hunt syndrome quantitation of varicella-zoster virus dna in patients with ramsay hunt syndrome and zoster sine herpete practice of feeding premasticated food to infants: a potential risk factor for hiv transmission detection of human betaherpesvirinae in saliva and urine from immunocompromised and immunocompetent subjects human herpesvirus 8 primary infection occurs during childhood in cameroon, central africa saliva as a diagnostic tool for periodontal disease: current state and future directions global burden and epidemiology of tuberculosis epstein-barr virus reactivation and upper-respiratory illness in elite swimmers oral fluid, a substitute for serum to monitor measles igg antibody? herpes viruses in periodontal compromised sites: comparison between hiv-positive and -negative patients dentistsõ attitudes toward chairside screening for medical conditions detection of herpetic viruses in gingival crevicular fluid of patients suffering from periodontal diseases: prevalence and effect of treatment oral mucosal reactivation rates of herpesviruses among hiv-1 seropositive persons the role of crude human saliva and purified salivary muc5b and muc7 mucins in the inhibition of human immunodeficiency virus type 1 in an inhibition assay the dynamics of ebv shedding implicate a central role for epithelial cells in amplifying viral output sensitivity of andes hantavirus to antiviral effect of human saliva comparative biology of chronic and aggressive periodontitis vs. peri-implantitis subgingival microbial profiles in chronic periodontitis patients from chile, colombia and spain marginal periodontium as a potential reservoir of human papillomavirus in oral mucosa oral bacteria induce a differential activation of hiv-1 promoter in t cells, macrophages, and dendritic cells detection of epstein-barr virus in saliva by real-time pcr reactivation of latent hiv-1 infection by the periodontopathic bacterium porphyromonas gingivalis involves histone modification detection of epstein-barr virus and human cytomegalovirus in blood and oral samples: comparison of three sampling methods lethal experimental infection of rhesus monkeys with ebola-zaire (mayinga) virus by the oral and conjunctival route of exposure bk virus has tropism for human salivary gland cells in vitro: implications for transmission the diagnostic applications of saliva -a review haemophili and related bacteria in the human oral cavity acute bacterial meningitis in infants and children oral herpes simplex virus type 2 reactivation in hiv-positive and -negative men preventive measures in mothers influence the establishment of the bacterium streptococcus mutans in their infants longitudinal study of intrafamilial mutans streptococci ribotypes caries risk. a practical guide for assessment and control specific microorganisms and dental caries in children root surface caries: a problem for periodontally compromised patients genotyping to distinguish microbial pathogenicity in periodontitis an investigation into the use of restriction endonuclease analysis for the study of transmission of mutans streptococci detection of kaposiõs sarcoma-associated herpesvirus in oral and genital secretions of zimbabwean women demonstration of mother-to-child transmission of streptococcus mutans using multilocus sequence typing detection of hepatitis b virus and mycobacterium tuberculosis in korean dental patients asymptomatically shed recombinant herpes simplex virus type 1 strains detected in saliva gonorrhea: update. oral surg oral med oral pathol oral radiol endod cytokine responses against periodontal infection: protective and destructive roles hepatitis c-contamination of toothbrushes: myth or reality? topical antimicrobial therapy in the prevention of early childhood caries quantitative detection of merkel cell virus in human tissues and possible mode of transmission transmission of human infection with nipah virus epstein-barr virus (ebv) dna in saliva and ebv serology of hiv-1-infected persons with and without hairy leukoplakia shedding of cytomegalovirus and herpesviruses 6, 7 and 8 in saliva of human immunodeficiency virus type 1-infected patients and healthy controls evidence for horizontal and not vertical transmission of human herpesvirus 8 in children born to human immunodeficiency virus-infected mothers rapidly cleared episodes of herpes simplex virus reactivation in immunocompetent adults detection of hepatitis c virus rna from gingival crevicular fluid and its relation to virus presence in saliva respiratory tract infections and pneumonia stress-induced subclinical reactivation of varicella zoster virus in astronauts epstein-barr virus reactivation associated with diminished cell-mediated immunity in antarctic expeditioners dental caries and its relationship to bacterial infection, hypoplasia, diet, and oral hygiene in 6-to 36-month-old children effect of prophylactic valacyclovir on the presence of human herpesvirus dna in saliva of healthy individuals after dental treatment high prevalence of multiple human herpesviruses in saliva from human immunodeficiency virus-infected persons in the era of highly active antiretroviral therapy confirmation of dengue virus infection by detection of dengue virus type 1 genome in urine and saliva but not in plasma molecular epidemiological studies on human herpesvirus 6 in families quantitative detection of hcmv-dna in saliva from infants and breast milk on real-time polymerase chain reaction diagnosis of legionella infection nontypeable haemophilus influenzae as a pathogen in children ante mortem diagnosis of human rabies using saliva samples: comparison of real time and conventional rt-pcr techniques measles virus strains circulating in ethiopia in 1998-1999: molecular characterisation using oral fluid samples and identification of a new genotype association between involuntary smoking and salivary markers related to periodontitis: a 2-year longitudinal study human cytomegalovirus-associated periodontitis in renal transplant patients saliva and meningococcal transmission detection of multiple pathogenic species in saliva is associated with periodontal infection in adults the microbiota on different oral surfaces in healthy children molecular microbial diagnosis detection of hepatitis c virus-rna in saliva from chronically hcv-infected patients mucosal shedding of human herpesvirus 8 in men pathophysiology of hepatitis c virus infection and related liver disease incidence of epstein-barr virus in astronaut saliva during spaceflight hantavirus rna in saliva from patients with hemorrhagic fever with renal syndrome epstein-barr virus shedding by astronauts during space flight clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary epstein-barr virus infection in children incidence of viable mycobacteria tuberculosis on alginate impressions in patients with positive sputum detection of dengue virus in saliva and urine by real time rt-pcr bacterial profiles of root caries in elderly patients infections of the esophagus and the stomach reliable detection and quantitation of viral nucleic acids in oral fluid: liquid phase-based sample collection in conjunction with automated and standardized molecular assays staphylococci in human periodontal diseases giannobile wv. identification of pathogen and host-response markers correlated with periodontal disease quantitation of hepatitis c virus rna in saliva and serum of patients coinfected with hcv and human immunodeficiency virus quantitative determination of cmv-dna in saliva of patients with bone marrow and stem cell transplantation using taqman-pcr natural history of oral human papillomasvirus infections in female and male partners: a prospective finnish hpv family study use of throat swab or saliva specimens for detection of respiratory viruses in children the use of pvp-iodine as an adjunct to nonsurgical treatment of chronic periodontitis quantitative studies on the salivary flora antibody responses in oral fluid after administration of prophylactic human papillomavirus vaccines human papillomavirus in saliva of patients with oral squamous cell carcinoma periodontitis lesions are the main source of salivary cytomegalovirus oral viral infections of children oral mucosal fungal infections severe acute respiratory syndrome and dentistry: a retrospective view epstein-barr virus specific salivary antibodies as related to stress caused by examinations human cytomegalovirus salivary antibodies as related to stress academic stress, immunological reaction, and academic performance among students of nursing and physiotherapy periodontitis lesions are a source of salivary cytomegalovirus and epstein-barr virus oral mucositis: a challenging complication of radiotherapy, chemotherapy, and radiochemotherapy: part 1, pathogenesis and prophylaxis of mucositis reactivation of human herpesvirus (hhv) family members other than hhv-6 in drug-induced hypersensitivity syndrome microbial testing in periodontics: value, limitations and future directions investigating the concurrent presence of hcv in serum, oral fluid and urine samples from chronic hcv patients in faisalabad early childhood stress is associated with elevated antibody levels to herpes simplex virus type 1 the re-emergence of syphilis in the united kingdom: the new epidemic phases selection of antimicrobial agents in periodontal therapy systemic antibiotics in periodontics oral viral infections of adults herpesviral-bacterial interactions in periodontal diseases human viruses in periodontitis age and sex relationships of superinfecting microorganisms in periodontitis patients subgingival microflora of advanced periodontitis in the dominican republic actinobacillus actinomycetemcomitans in human periodontal disease: a crosssectional microbiological investigation genetic and environmental risk factors for chronic periodontitis and aggressive periodontitis oral herpetic infections (hsv 1-8) chronic herpesvirus reactivation occurs in aging relationship between porphyromonas gingivalis, epstein-barr virus infection and reactivation in periodontitis patient with severe periodontitis and subgingival epstein-barr virus treated with antiviral therapy progressive increase of human papillomavirus carriage rates in potentially malignant and malignant oral disorders with increasing malignant potential risk of exposure to legionella in dental practice a longitudinal study of lancefield group a streptococcus acquisitions by a group of young dunedin schoolchildren similarity of the oral microbiota of pre-school children with that of their caregivers in a population-based study cytokine regulation of immune responses to porphyromonas gingivalis high prevalence of human papillomaviruses in the normal oral cavity of adults within-subject variability in repeated measures of salivary analytes in healthy adults risk and protective factors for meningococcal disease in adolescents: matched cohort study cytokine secretion and latent herpes virus reactivation with 28 days of horizontal hypokinesia risk indicators for harboring periodontal pathogens the utility of whole saliva to detect the oral presence of periodontopathic bacteria do periodontopathogens disappear after full-mouth tooth extraction? paired, quantitative measurements of hepatitis b virus dna in saliva, urine and serum of chronic hepatitis b patients herpes simplex virus and hiv-1: deciphering viral synergy transmission of periodontal bacteria and models of infection syphilis epidemiology in sweden: re-emergence since 2000 primarily due to spread among men who have sex with men infectious mononucleosis and epstein-barr virus occurrence of aggregatibacter actinomycetemcomitans in brazilian indians from umutina reservation, mato grosso, brazil excretion patterns of human metapneumovirus and respiratory syncytial virus among young children multiple epstein-barr virus infections in healthy individuals detection of sars-associated coronavirus in throat wash and saliva in early diagnosis high serum hepatitis c virus (hcv) rna load predicts the presence of hcv rna in saliva from individuals with chronic and acute hcv infection low prevalence of varicella zoster virus and herpes simplex virus type 2 in saliva from human immunodeficiency virus-infected persons in the era of highly active antiretroviral therapy. oral surg oral med oral pathol oral radiol endod post-marketing surveillance team. post-marketing surveillance of oraquick whole blood and oral fluid rapid hiv testing multiple components contribute to ability of saliva to inhibit influenza viruses haemophilus influenzae type b (hib) salivary diagnostics powered by nanotechnologies, proteomics and genomics candida biofilms and oral candidosis: treatment and prevention disseminated varicella infection in a child receiving short-term steroids for asthma taqman real-time quantification of epstein-barr virus in severe early childhood caries actinobacillus actinomycetemcomitans in human periodontal disease. prevalence in patient groups and distribution of biotypes and serotypes within families the clinical value of salivary biomarkers for periodontal disease nonvenereal transmission of syphilis in infancy by mouth-to-mouth transfer of prechewed food prevalence of potential bacterial respiratory pathogens in the oral cavity of hospitalised individuals key: cord-345771-3v2avxiv authors: traub, ariana moriah; ifafore-calfee, temitayo; phelps, benjamin ryan title: multimonth dispensing of antiretroviral therapy protects the most vulnerable from 2 pandemics at once date: 2020-06-30 journal: glob health sci pract doi: 10.9745/ghsp-d-20-00160 sha: doc_id: 345771 cord_uid: 3v2avxiv we encourage governments in countries that have a high prevalence of people living with hiv to implement multimonth dispensing of antiretroviral therapy to safeguard both patients with hiv and health care workers from coronavirus disease 2019. s ince the emergence of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in december 2019, the virus has been identified in more than 183 countries. 1 its rapid spread is of major concern for those living in low-and middle-income countries and specifically people living with hiv (plhiv). with the dearth of studies on hiv-coronavirus disease 2019 (covid-19) coinfections, countries have no clear evidence on specific risks for plhiv. 2,3 however, as with other infectious diseases, it is expected that plhiv who are not virally suppressed and/or on antiretroviral therapy (art) might be at an increased risk of covid-19 infection, severe disease, and poor health outcomes. 2,3 thus, every precaution must be taken to avoid hiv-covid-19 coinfection and keep the number of covid-19 cases at a minimum in countries with high proportions of plhiv. to minimize the spread of covid-19, plhiv are encouraged to follow public health guidelines to limit in-person interactions and general mitigation recommendations, including social distancing and good hand hygiene. refilling lifesaving antiretroviral drugs requires interpersonal interaction and brings the potential of increasing the spread of sars-cov-2 and other transmissible diseases. thus, policy makers-including the u.s. president's emergency plan for aids relief-are encouraging countries to provide plhiv with a multimonth supply of art as a key strategy to safeguard plhiv and health care workers involved in providing hiv services. 4 multimonth dispensing (mmd) is an aspect of differentiated service delivery that provides patients with either 3 or 6 months of medication and eliminates the need for monthly clinic and/or community facility visits. from the patient's perspective, mmd has already been shown to reduce the cost of travel, reduce patient burden, and limit the hours of work or school lost. 5,6 importantly, mmd improves adherence and viral suppression. 5, 7 in the context of the covid-19 pandemic, this viral suppression strengthens the immune system and likely mitigates the risk of severe covid-19. 8 mmd, essential to caring for plhiv, is arguably even more critical during the covid-19 pandemic. currently, only 21 countries have formal 6-month dispensing policies. two countries allow clinicians to provide 6-month supply when they deem appropriate, and 8 countries have a 3-month dispensing policy in place. south africa, the country with the largest number of plhiv, only has a 2-month dispensing policy in place. to mitigate the damage caused by sars-cov-2 while protecting the health of plhiv, we encourage countries to rapidly implement 3-month if not 6-month dispensing. as a key strategy to safeguard patients and health care workers providing hiv services, mmd also reduces clinic visits, improves viral suppression, encourages social distancing, and potentially saves patients from the dual threat of sars-cov-2 and hiv. as sars-cov-2 spreads, patient caseloads are likely to increase across low-and middle-income countries where hiv burden is high and millions of people are stable on effective treatment. implementing mmd quickly will protect the time and health of existing health care professionals and ready our health systems for imminent threats as well as any that may follow. an interactive web-based dashboard to track covid-19 in real time interim guidance for covid-19 and persons with hiv president's emergency plan for aids relief. pepfar's hiv response in the context of coronavirus disease outcomes of community-based differentiated models of multimonth dispensing of antiretroviral medication among stable hivinfected patients in lesotho: a cluster randomised non-inferiority trial protocol cost effectiveness of a pharmacy-only refill program in a large urban hiv/aids clinic in uganda multi-month prescriptions, fasttrack refills, and community art groups: results from a process evaluation in malawi on using differentiated models of care to achieve national hiv treatment goals hiv infection key: cord-332093-iluqwwxs authors: lessler, justin; cummings, derek a. t. title: mechanistic models of infectious disease and their impact on public health date: 2016-02-17 journal: american journal of epidemiology doi: 10.1093/aje/kww021 sha: doc_id: 332093 cord_uid: iluqwwxs from the 1930s through the 1940s, lowell reed and wade hampton frost used mathematical models and mechanical epidemic simulators as research tools and to teach epidemic theory to students at the johns hopkins bloomberg school of public health (then the school of hygiene and public health). since that time, modeling has become an integral part of epidemiology and public health. models have been used for explanatory and inferential purposes, as well as in planning and implementing public health responses. in this article, we review a selection of developments in the history of modeling of infectious disease dynamics over the past 100 years. we also identify trends in model development and use and speculate as to the future use of models in infectious disease dynamics. from the 1930s through the 1940s, lowell reed and wade hampton frost used mathematical models and mechanical epidemic simulators as research tools and to teach epidemic theory to students at the johns hopkins bloomberg school of public health (then the school of hygiene and public health) (1, 2) . though never published by reed and frost (versions of the model were eventually published by their students (3, 4) ), their model was one of the first mechanistic models of infectious disease transmission, and at a time long before digital computing, they may have been the first to use simulation methods to understand the epidemic process. reed and frost were pioneers in the study of infectious disease dynamics using mechanistic models, a field of epidemiology that has developed in parallel with the associative statistical models and methods of causal inference that dominate much of epidemiologic research. over the past century, mechanistic models have played an essential role in shaping public health policy, the way we study interventions aimed at controlling infectious diseases, and the theory on which disease control is based. mechanistic models differ from traditional statistical models such as regression models because their structure makes explicit hypotheses about the biological mechanisms that drive infection dynamics. such hypotheses range from simple representations of the time it takes to complete some part of the disease process (e.g., sartwell's lognormal representation of the incubation period (5)) to complex agent-based models that attempt to explicitly represent social interactions of people in an entire country (6, 7) or even the world (8) . regardless of scale, approach, and complexity, these models have more of the flavor of models in physics than the statistical models that are used in other branches of epidemiology, and in many cases they can be used to predict the effectiveness of hypothetical interventions in controlling disease spread. perhaps the first mechanistic model of infectious disease transmission used in assessing intervention strategies was a mathematical model of malaria transmission developed and refined by ronald ross in a series of papers published between 1908 and 1921 (9) (10) (11) , pre-dating the work of reed and frost by decades. this model had a direct and powerful message for public health: malaria could be controlled and even eliminated through mosquito control, even if the vector could not be completely eliminated. ross used his theoretical framework to develop and advocate for multiple indices, including the prevalence rate and the entomological inoculation rate (12) , that could effectively characterize the intensity of transmission in an area and identify goals for control. in the wake of the founding of the global malaria eradication program by the world health organization, george macdonald (13) extended ross's work in order to justify the use of insecticide as a tool for global malaria eradication (14) . in particular, he showed that increasing daily mosquito mortality from 5% to 45% would be adequate to eliminate malaria even in locations with the highest transmission intensities in africa. mechanistic models continue to play an important role in the fight against malaria. the work of ross and macdonald looms large to this day, with a recent review finding that the majority of models published since 1940 depart from central hypotheses of the ross-macdonald model in only a few key assumptions, if any (15) . although there are numerous instances over the past century in which mechanistic models have contributed to the control of a single disease (see figure 1 for some examples), their larger contribution may be in our general understanding of disease control. the prime example is the concept of herd immunity and the critical vaccination threshold. herd immunity is the indirect protection offered to members of the population susceptible to the disease (i.e., not immune and with the potential to be infected) by the immunity of surrounding individuals, and the critical vaccination threshold is the percentage of the population that must be vaccinated in order for the introduction of an infectious case to not spark an epidemic (16) . to estimate the critical vaccination threshold, we must first understand one of the most critical concepts of infectious disease dynamics, the basic reproductive number, r 0 . r 0 is the number of cases that a single infectious individual is expected to cause in a fully susceptible population. this concept was first introduced in demography and underwent significant development by lotka while on a visit to the johns hopkins university school of hygiene and public health in 1925 (see heesterbeek (17) for a full history of the development of r 0 in infectious disease). although this value does vary by setting, for many pathogens it is remarkably consistent across contexts and serves as a rough quantification of pathogen transmissibility. based on dynamic models, it has been shown that if we vaccinate a proportion of the population equivalent to 1 − 1/r 0 , then the pathogen will fail to spread in that population. this is the critical vaccination threshold, and it has helped to set vaccination goals for a number of diseases, particularly when elimination is the goal. however, the dynamics of vaccines in real populations are complex, and mechanistic models have helped us to understand what to expect after changes in vaccination policy. for instance, immediately after the introduction of a vaccine or improvement in vaccination rates, a disease may appear to be eliminated from a population. however, this long honeymoon period may be followed by a large, resurgent, outbreak that bigger than the yearly epidemics seen before the introduction of vaccination (though the cumulative number of cases is still less than what would have been seen without vaccination) (18) . these results have helped public health officials to understand that initial apparent vaccine successes may not last, as well as what to expect after introducing a new vaccine. mechanistic models have also been used to understand the optimal age range for vaccination campaigns (19, 20) , how such campaigns should be timed (21) , and how best to use vaccines when supplies are limited (20, 22) . models have also been used to design active response strategies for vaccine use, including ring vaccination strategies such as those implemented in the smallpox eradication campaign (23) . models were also used to assess strategies to respond to a bioterrorist release of smallpox in the early part of the 21st century and were influential in setting policy for response (24) (25) (26) (27) . one counterintuitive prediction of mechanistic models is that in rare cases, increased population immunity from vaccination can actually increase the incidence of severe disease. the poster child for the phenomenon is congenital rubella syndrome (crs). for most people, rubella infection causes a relatively minor infection characterized by fever and rash; however, when pregnant women are infected during the first trimester of pregnancy, it cause crs, which results in severe complications of pregnancy including congenital disorders and death of the fetus (28) . because vaccination increases the average age of infection (by decreasing the hazard of infection), a vaccination program that does not achieve sufficient coverage can increase the number of pregnant women who are infected, thereby increasing incidence of crs (29) . this is not simply a theoretical concept; although there have been no sustained increases in the incidence of crs (in part due to the public health response), both costa rica and greece experienced transient increases in crs burden after rubella vaccination (30, 31) . in light of the threat of crs, mechanistic models have played an important role in setting world health organization recommendations for the introduction of a rubella vaccine. these recommendations encourage countries to wait to introduce the vaccine until measles vaccination rates (measles and rubella vaccines are usually given together) are high enough to guarantee a reduction in crs cases and to strongly consider vaccination campaigns in women of childbearing age before the vaccine is introduced (32) . vaccination is only 1 of a suite of control measures. another that is of particular importance in the control of macroparasite infections is mass drug administration. a key difference between microparasite and macroparasite dynamics is the huge variation in transmission potential of human hosts, with some individuals experiencing huge pathogen loads that contribute disproportionately to transmission within populations (33) . here, strategies have taken an eye toward reducing overall population burdens of macroparasites, including targeting those with the highest burdens. theoretical explorations of the impact of heterogeneity in transmissibility have helped inform interventions and aided in the development of theory exploring the impact of heterogeneities in microparasites (34) . (aids). ron brookmeyer (35) used the incubation period distribution of hiv to "back calculate" the number of hiv infections that must have occurred over the previous course of the epidemic and predict the number of future hiv/aids cases in those already infected with hiv. he thereby linked an observable quantity (the number of aids cases) with an unobservable one (the number of people living with hiv). longini et al. (36) then fit a more mechanistic model of disease progression to data from hiv-infected individuals in the united states army, achieving similar results by explicitly representing the biological process. when attempting to estimate global mortality from measles infection, simons et al. (37) used a state-space model (i.e., a hidden markov model) which linked an underlying model of measles epidemic dynamics (the process model) with nationally reported measles incidence via an observation model (38) . they thereby were able to estimate the extent to which national reports underestimated measles cases by reconciling these reports with what was likely given birth rates and a known epidemic process. planning for so called "black swans," which are unlikely but catastrophic events, is essential to ensuring security and population health. the prime example of an infectious disease black swan is the 1918 influenza pandemic, which is estimated to have killed 50-100 million people in 2 years (39) . governments and policy makers depend on simulations built on mechanistic models to decide the extent of these threats and what can be done to confront them. for the past decade and a half, there have been ongoing concerns that one of several strains of influenza a that have been known to infect humans from domestic poultry (h5n1, h9n2, etc.), might develop the ability to transmit efficiently in humans and cause a major pandemic. h5n1 strains are seen as particularly concerning because of their high case fatality rate and the substantial increase in the number of human cases (particularly in southeast asia) that started in 2003 (40) . independent teams of disease modeling experts developed sophisticated agent-based models of potential emergence events to determine whether effective antiviral agents could be used to contain an emerging influenza at the source (6, 41) . these models showed that under reasonable expectations of the transmissibility of an emerging influenza (i.e., r 0 in the 1.5-2.0 range), containment was possible, though perhaps not practical, as it would require the deployment of millions of courses of antiviral medication, very early detection of the disease, and rapid response. in parallel work, groups considered how the impact of a pandemic could be mitigated in the united states if the initial containment attempt was unsuccessful (42) (43) (44) . the efforts of independent groups showed that something more than social-distancing measures (e.g., school closure, case isolation) would be needed to control a pandemic and that effective antivirals could help. in part on the basis of this work, the united states and other countries decided to stockpile antivirals to combat a future pandemic, a decision that has since been criticized by some (45) . however, these criticisms have been focused on concerns about the efficacy of the stockpiled antiviral drugs (46) rather than the results of the modeling work itself. the question of the probability of h5n1 influenza evolving to become transmissible in humans has itself been the focus of mechanistic modeling (47) . after 2 research groups had identified 2 different sets of mutations to the h5n1 virus that would be sufficient to allow airborne transmission in a mammalian host (48, 49) , russell et al. (47) developed a mathematical model of the within host dynamics of influenza evolution. although the authors were unable to confidently estimate the probability of the emergence of a pandemic h5n1 strain because of uncertainties about the underlying biological processes involved, they were able to identify the biological factors on which this probability would most strongly depend and recommend studies (e.g., deep sequencing of viral samples from h5n1-infected hosts) that might help to develop more precise predictions. there has been considerable debate surrounding the ethics of gain-of-function experiments for h5n1 influenza (50) , but if such experiments are to be justified, they must provide us a way to have advanced warning of a coming pandemic, a task that may only be possible through mechanistic models. however, to be successful, these models will require substantial additional theoretical work on how viral evolution interacts with the distribution of immunity in the population. in the event that an outbreak of an emerging disease does occur, mechanistic models are one of the first tools used to characterize the threat and plan a response. when a pandemic influenza strain emerged in 2009, it was critical to quickly assess whether it had the potential to cause illness with high rates of fatality, like the virus that emerged in the pandemic of 1918, or was a more mild disease, akin to what was seen in the pandemics of 1957 and 1968. initial assessments relied heavily on dynamic models of a variety of types, including phylogenetic techniques paired with demographic models, models based on the probability of the observed number of introductions of pandemic h1n1 into populations outside of mexico, analysis of epidemic curves, and the results of detailed investigations of early outbreaks (51, 52) . analyses by a number of groups quickly showed that the emergent pandemic h1n1 virus was behaving very much like alreadycirculating strains, and although it was still potentially a significant public health threat, it was unlikely to have a qualitatively different impact on mortality or morbidity than circulating influenza strains. in addition to its role in the response to the 2009 influenza pandemic, mechanistic modeling has played a role in the response to most of the emerging disease threats of this century, from foot and mouth disease in the united kingdom (53), to severe acute respiratory syndrome coronavirus (54) , to middle east respiratory syndrome coronavirus in saudi arabia (55) , to ebola in west africa (56) . the last of these shows both the power of mechanistic approaches and the dangers of its misuse. in the summer and fall of 2014, the number of ebola cases in west africa was continuing to grow, and it was unclear how severe the epidemic would eventually become. to address this issue, as well as the threat of spread to other countries, a number of modeling exercises were conducted (e.g., gomes et al. (57) ). of particular note was a model released by the centers for disease control and prevention that predicted that, without further intervention, 1.4 million cases of ebola would occur in liberia and sierra leone by mid-january 2015 (58). this did not come to pass, and although the authors noted that such long-term projections were tenuous, the media and many in the public health community made much of this number. of course interventions and behavior change did occur, but the authors had also made tenuous assumptions about how the populations of liberia and sierra leone mix together, essentially treating each country as a homogenous entity. in contrast, the world health organization ebola response team, who also made projections based on an unconstrained epidemic, declined to forecast further than 2 months into the future (56), and though theirs was a moderate overestimate of total cases, they avoided publishing any panic-inducing overestimations (they projected approximately 20,000 cases by november 2, 2014; approximately 13,000 actually were reported by that point) (59) . forecasting the course of disease spread is difficult to do well, particularly in the context of an active response. it also may be the least of what mechanistic approaches to disease epidemiology have to offer. the aforementioned work, particularly that of the world health organization ebola response team, also characterized important aspects of ebola's natural history and epidemiology, including its basic reproductive number (r 0 ), the decline in r over the course of the epidemic, the incubation period, and the serial interval, properties of the disease that will be important to understand should it re-emerge. mechanistic and mathematical approaches aid not only in the response to particular diseases but also in illuminating basic epidemiologic principles and important parameters that dictate whether a novel (or existing) pathogen can be controlled. in a 2004 paper, fraser et al. (60) confronted the question of why severe acute respiratory syndrome coronavirus was successfully contained, whereas influenza, hiv, and numerous others were not. they were particularly interested in the effectiveness of the tools available when first confronting a novel pathogen: contact tracing, isolation, and quarantine. they presented evidence that a critical determinant of the controllability of a pathogen is the amount of transmission that occurs before symptom onset, expressed by their parameter θ. pathogens that had a low proportion of all transmission occurring before symptom onset are easier to control because symptomatic individuals can be targeted with isolation or pharmaceuticals before they transmit to others. although forecasting is difficult, particularly in the response to an emerging disease threat, it remains a major goal of the disease-modeling community. because disease reporting is often delayed, forecasting includes not only projections into the future but also "now casting" of incidence based on more readily available information. this has led to a number of approaches in which models have been used to either process a data stream that is a proxy of the data of interest but available more quickly (e.g., google flutrends) (61) or in analyses of ongoing outbreaks to assess (with available data) what might be the current situation given the limitations of the observation process and temporal lags in both reporting and outcomes being generated (e.g., calculating case fatality rates for the severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus outbreaks when many patients had yet to resolve) (55, 62) . at a larger time horizon, several efforts have attempted to forecast the impact of interventions on future incidence. one of the most successful was a project that forecasted the impact of respiratory syncytial virus immunization campaigns on the temporal pattern of incidence in the united states. using mechanistic transmission models, pitzer et al. (63, 64) made detailed predictions of the impact of vaccination on the multiannual dynamics of rotavirus, as well as the impact of the vaccine on genotype circulation. these forecasts of broad qualitative impacts of interventions are critical tests of models. detailed prospective predictions of changes that will occur with changes in health policy, which are then validated, will provide the best evidence of the utility of mechanistic models in the future. dependent happenings is the term coined by ronald ross (10) to capture the fact that for infectious diseases, an individual's risk of infection depends on the disease status of those around them (65) . this presents challenges for trial design and the interpretation of observational studies. cluster randomization and adjustment for intra-class correlation can be used to account for this effect in some cases (66) , but mechanistic models are often useful in trial design or in interpretation of results when cluster randomization is imperfect or impossible. under these conditions, simulations studies have been used to help in study design settings, including vaccine studies (67, 68) and combination approaches to hiv prevention (69) . mechanistic models have been particularly revealing for studies of vaccine effectiveness. for example, a naïve approach would be to consider that all vaccines acted in the same way, providing complete protection for some fraction of the population. however, in reality vaccines may be leaky and provide protection only in some dimensions (65) . vaccines may prevent infection all together (e.g., the measles vaccine) (70), offer protection against pathogenic disease but still allow individuals to become infected and transmit the disease (e.g., acellular pertussis vaccines (71)), or only prevent onward transmission of the disease (e.g., transmissionblocking vaccines for malaria (72) ). in order to anticipate and assess the impact of vaccines once scaled up to widespread use, the specific actions of the vaccine in reducing infection, onward transmission, and disease must be disentangled. these specific mechanisms will contribute differently to the direct, indirect, and total effects of a vaccine. these effects are increasingly targets of inference during trials (73) , and developments in infectious disease theory have driven development of both inference tools and study design to measure specific impacts (65) . in emerging outbreaks, simulation models have often been used as the framework to quickly quantitatively compare policy alternatives. the application of these models has yielded results ranging from broad information about the feasibility and potential impact of interventions to detailed recommendations about targeting of interventions. in the foot and mouth disease outbreak of 2001, models were used to determine optimal culling strategies that specified operational details of those strategies, including the timing and spatial extent of culling. even outside of public health crises, infectious disease models play an important role in setting public health policy. cost-effectiveness analyses are often built on mechanistic models of disease spread (74, 75) . models can help investigators choose between different intervention strategies, determine the potential of specific interventions, and compare investments across pathogens. infectious disease models play a critical role in incorporating indirect effects that can vary substantially across alternative programs. the design of immunization campaigns against human papillomavirus has to weigh the direct effects protecting women from human papillomavirus infection, as well as indirect protection resulting from immunization of both women and men. the tradeoffs of alternative programs in protecting individuals at risk of the most severe outcomes and those at little risk have been best evaluated in transmission models (64) . increasingly important is the marrying of mechanistic disease models with operations research by explicitly modeling the logistical constraints on public health intervention. this approach can be key when preparing for outbreaks or bioterrorism, as speed of deployment, hospital capacity, and other logistical factors can severely impact the efficiency of disease containment and its subsequent spread (25, 76) . likewise, a logistical analysis can assess the feasibility of novel diseasecontrol strategies, showing whether they are practical as well as efficacious; for instance, an analysis of the feasibility and potential effectiveness of passive immunotherapy in hong kong showed that this intervention could play an important role in controlling a mildly severe pandemic (77) . as the price of computation drops and we enter the era of "big data," the role of mechanistic models will only increase. a powerful new synergy is the combination of mechanistic models of disease spread with phylogenetic techniques outlining the evolutionary relationship between infecting pathogens. genetic sequence data present samples of pathogens taken from a large population of pathogens both within a host and among all hosts. understanding the impact of different selective pressures on pathogens is inherently a task of population genetics. models of the population dynamics of pathogens have been incorporated into models in order to explain the phylogenetic structure of pathogens. sequence data have been used to infer basic reproductive numbers of pathogens (51, 78) , harkening back to lotka's first use of the term to describe replication of organisms. in future work, we expect to see more direct integration of models with data at both population scales, as has been the tradition, and within models of infectious disease 419 host scales. traversing these scales will be a key challenge to the field. targeted funding and the relatively new paradigm (at least for epidemiology) of sanctioning competitions to identify the best methods of disease forecasting continue to in invigorate the field. in the united states, the models of infectious disease agent study and the recently completed research and policy for infectious disease dynamics program have led to well over 1,000 publications and continue to invigorate research and training in the field (79, 80) . similar initiatives in the united kingdom and other parts of europe, such as that from the medical research council's centre for outbreak analysis and modelling, have also been successful (81) . competitions such as the national oceanic and atmospheric administration's dengue forecasting project (82), the defense advanced research projects agency forecasting chikungunya challenge (83) , and the us center for disease control and prevention's predict the influenza season challenge (84) require researchers to assess and compare the performances of their models and stand by their predictions in the face of actual events. such initiative should serve to greatly improve the quality and number of models of infectious diseases, but this will only translate into improved public health if it is paired with greater engagement with policy and practice. in limited space, it is impossible to cover every important contribution that mechanistic models have made over the past century, and there is much important work that we have not covered. these contributions range from work showing the potential impact of test-and-treat strategies in hiv control (85) , to analyses of how to best use a limited supply of cholera vaccines to control disease (22, 86) , to fundamental work on the link between demographic characteristics and disease incidence (87) . these omissions should not be seen as a reflection of the quality of the work, but rather merely as the result of our need to select only a few of many good options. the use of mechanistic models in infectious disease epidemiology has shifted over the course of 100 years. the arc of their use spans beginnings as 1 of a group of statistical and mathematical tools used by epidemiologists to understand a multitude of phenomena, to use and development by an increasingly specialized group of researchers over the course of the 20th century, to more general use by a broader group of researchers. this arc still bends. at their core, these methods provide frameworks of analysis that can be treated in the same way as other statistical tools of analysis. refinement of methods has led to a theoretical base and application toolkit that allows nonspecialists to analyze and understand infectious disease dynamics with mechanistic models. this broader ecosystem of modelers, which includes methods-focused researchers and public health practitioners, has led to encouraging progress in tying models increasingly to data and to the most salient infectious disease problems facing global health. memoir on the reed-frost epidemic theory a commentary on the mechanical analogue to the reed-frost epidemic model an examination of the reed-frost theory of epidemics some mathematical developments on the epidemic theory formulated by reed and frost the distribution of incubation periods of infectious disease strategies for containing an emerging influenza pandemic in southeast asia modelling disease outbreaks in realistic urban social networks modeling the spatial spread of infectious diseases: the global epidemic and mobility computational model prevention of malaria in mauritius some quantitative studies in epidemiology the principle of repeated medication for curing infections revisiting the basic reproductive number for malaria and its implications for malaria control epidemiological basis of malaria control macdonald, and a theory for the dynamics and control of mosquito-transmitted pathogens a systematic review of mathematical models of mosquito-borne pathogen transmission: 1970-2010 herd immunity: history, theory, practice a brief history of r0 and a recipe for its calculation measles in developing countries. part ii. the predicted impact of mass vaccination impact of birth rate, seasonality and transmission rate on minimum levels of coverage needed for rubella vaccination optimizing the dose of prepandemic influenza vaccines to reduce the infection attack rate pulse mass measles vaccination across age cohorts reactive vaccination in the presence of disease hotspots smallpox and its eradication containing bioterrorist smallpox containing a large bioterrorist smallpox attack: a computer simulation approach smallpox bioterror response smallpox transmission and control: spatial dynamics in great britain the history and medical consequences of rubella vaccination against rubella and measles: quantitative investigations of different policies congenital rubella syndrome: progress and future challenges increase in congenital rubella occurrence after immunisation in greece: retrospective survey and systematic review: how does herd immunity work? world health organization. rubella vaccines: who position paper-recommendations heterogeneities in the transmission of infectious agents: implications for the design of control programs superspreading and the effect of individual variation on disease emergence reconstruction and future trends of the aids epidemic in the united states the dynamics of cd4+ t-lymphocyte decline in hiv-infected individuals: a markov modeling approach assessment of the 2010 global measles mortality reduction goal: results from a model of surveillance data tracking measles infection through non-linear state space models updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic avian influenza a (h5n1) infection in humans containing pandemic influenza at the source strategies for mitigating an influenza pandemic mitigation strategies for pandemic influenza in the united states modeling targeted layered containment of an influenza pandemic in the united states report disputes benefit of stockpiling tamiflu neuraminidase inhibitors for preventing and treating influenza in healthy adults: systematic review and meta-analysis the potential for respiratory droplet-transmissible a/h5n1 influenza virus to evolve in a mammalian host airborne transmission of influenza a/h5n1 virus between ferrets experimental adaptation of an influenza h5 haemagglutinin (ha) confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets ethical alternatives to experiments with novel potential pandemic pathogens pandemic potential of a strain of influenza a (h1n1): early findings outbreak of 2009 pandemic influenza a (h1n1) at a new york city school dynamics of the 2001 uk foot and mouth epidemic: stochastic dispersal in a heterogeneous landscape transmission dynamics and control of severe acute respiratory syndrome middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility who ebola response team. ebola virus disease in west africa-the first 9 months of the epidemic and forward projections assessing the international spreading risk associated with the 2014 west african ebola outbreak estimating the future number of cases in the ebola epidemic-liberia and sierra leone world health organization factors that make an infectious disease outbreak controllable detecting influenza epidemics using search engine query data methods for estimating the case fatality ratio for a novel, emerging infectious disease demographic variability, vaccination, and the spatiotemporal dynamics of rotavirus epidemics modeling rotavirus strain dynamics in developed countries to understand the potential impact of vaccination on genotype distributions design and analysis of vaccine studies statistics notes: the intracluster correlation coefficient in cluster randomisation modeling human papillomavirus vaccine effectiveness: quantifying the impact of parameter uncertainty design and evaluation of prophylactic interventions using infectious disease incidence data from close contact groups hptn 071 (popart): a cluster-randomized trial of the population impact of an hiv combination prevention intervention including universal testing and treatment: mathematical model acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model development of a transmission-blocking malaria vaccine: progress, challenges, and the path forward effects of vaccination on invasive pneumococcal disease in south africa a review of typhoid fever transmission dynamic models and economic evaluations of vaccination cost-effectiveness analyses of human papillomavirus vaccination emergency response to an anthrax attack logistical feasibility and potential benefits of a population-wide passive-immunotherapy program during an influenza pandemic evolutionary analysis of the dynamics of viral infectious disease models of infectious disease agent study (midas) about the centre for outbreak analysis and modelling national oceanic and atmospheric administration. dengue forecasting project chikungunya threat inspires new darpa challenge predict the influenza season challenge universal voluntary hiv testing with immediate antiretroviral therapy as a strategy for elimination of hiv transmission: a mathematical model the impact of a one-dose versus two-dose oral cholera vaccine regimen in outbreak settings: a modeling study a simple model for complex dynamical transitions in epidemics on the course of epidemics of some infectious diseases we thank c. jessica metcalf for suggestions and useful discussions.conflict of interest: none declared. key: cord-329890-wg23sa1u authors: quah, stella r. title: public image and governance of epidemics: comparing hiv/aids and sars date: 2007-02-28 journal: health policy doi: 10.1016/j.healthpol.2006.03.002 sha: doc_id: 329890 cord_uid: wg23sa1u abstract a comparative analysis of the 2002–2003 infectious disease outbreak, severe acute respiratory syndrome (sars), and the hiv/aids epidemic that has affected the world over the past two decades reveals the significant role of socio-cultural beliefs and attitudes in the shaping of people's lifestyles and approaches to the control and prevention of epidemics. the main research question is: what can we learn from the sars experience about effective prevention of hiv/aids? the sources of data include population figures on the development of these epidemics and findings from two sociological studies of representative samples of singapore's multi-ethnic population. the comparative study illustrates the impact of cultural beliefs and attitudes in shaping the public image of these two different infectious diseases; the relevance of public image of the disease for effective prevention and control of epidemics. traditionally, the human suffering inflicted by longterm epidemics have tended to find expression in literature and the fine arts thus becoming a visible part of the collective memory and shaping the public image of the disease. for example, the impact of the bubonic and pneumonic plague or "black death" had a major influence on painters of the gothic period [1] ; tuberculosis is featured in eugene g. o'neill's long day's journey into night; franz kafka's diaries; thomas mann's the magic mountain [2] ; victor hugo's les misérables; dickens' nicholas nickleby, brontë's wuthering heights; verdi's la traviata [3] . among e-mail address: socquahs@nus.edu.sg. epidemics in the past three decades (hiv/aids, sars, mad cow disease, and avian flu among others), only hiv/aids has lasted long enough to inspire artistic expressions in literature [4] [5] [6] [7] , theatre [8] , dance [9] , and film [10] mostly used as vehicles for hiv/aids preventive education programs particularly in africa [8] [9] [10] [11] [12] . one of the pioneer studies in prevention was published in 1939 by zinsser [13] . ever since, a community of experts worldwide has been dedicated to prevention [14] [15] [16] [17] [18] [19] [20] . however, despite the struggle to convey a more accurate and humane public image of aids in the past decade, the stigma attached to hiv/aids still persists as a formidable obstacle to prevention efforts [21] [22] [23] . figures on the spread of the disease suggest we are losing the battle against hiv/aids especially in developing countries [3, [24] [25] [26] [27] . in china alone, the estimated number of deaths due to aids as of december 2003 (latest figures available), ranged from 21,000 to 75,000 and 840,000 persons infected with hiv/aids. the figures for thailand, the second most affected east asian country, are 34,000-97,000 deaths, and 570,000 persons infected with hiv (appendix a, table a .1). in contrast to the dismal hiv/aids situation, the 2002-2003 outbreak of severe asymptomatic respiratory syndrome or sars, offers a completely different picture for the analysis of preventive efforts. although sars, like hiv/aids, was unknown in the medical world and hit unexpectedly, there are some significant differences, particularly in their etiology, epidemiology, natural history and clinical outcomes of the two diseases. hiv/aids is asymptomatic for 7-10 years after infection so that hiv-positive persons may continue to spread the disease unknowingly. the main mode of hiv/aids transmission is through direct contact with infected body fluids or blood (sexual intercourse, use of infected needles by drug users and receiving contaminated blood transfusions). sars is caused by the sars coronavirus and characterized by airborne transmission. sars develops very rapidly, with an average incubation period of 5 days or a range of 2-10 days after contact. within 1 week of the illness patients show typical influenza-like symptoms such as fever, malaise, and headache with cough and diarrhoea getting worst in the second week of infection. it has been determined that "transmission occurs mainly during the second week of illness". these external signs facilitate prompt action: exposed patients may be placed under fever surveillance twice a day "in an isolation facility or ward for at least 10 days after the last exposure to the source case(s)" [28] . in the span of 9 months sars infected 8096 persons, caused 774 deaths (appendix a, table a .2) and became a widespread visible threat through the serious disruption of normal daily activities of individuals and major sectors of the economy such as transportation, commerce, industrial production, and tourism [28] [29] [30] . the first probable sars case was reported in china on 16 november 2002 and the infection spread to 28 other countries around the world but the largest number of locally transmitted infections and deaths were reported in china, hong kong, taiwan, canada, and singapore (appendix a, table a .2). despite the fact that sars caught the world unprepared, hit at great speed, and it is very difficult to eradicate [31] , the outbreak was contained within 9 months, a relatively brief period of time (appendix a, table a .2). despite the medical differences (in etiology, epidemiology, natural development and clinical outcomes) between these two epidemics, i argue and attempt to demonstrate in this study that we may advance our knowledge on preventive strategies by conducting a systematic comparison of important social aspects of hiv/aids and sars. what can we learn from the sars experience about effective prevention of hiv/aids? more specifically, why were the efforts to contain and prevent the spread of a new epidemic like sars successful while it has taken 25 years so far to contain the spread of hiv/aids and no effective solution is yet in sight? social science research has identified over the past decades a complex array of factors and conditions associated with disease prevention in individuals (micro-level analysis) as well as collectivities (macro-level analysis) but the factors and conditions vary for different diseases and there may be many other factors yet to be identified. still, contrasting the two epidemics in terms of social attitudes and beliefs at the micro-and macro-levels, will help us to elucidate some of the major obstacles to hiv/aids prevention. therefore, this paper focuses on only three possible factors: the impact of perceived severity and susceptibility to infection and the public image of the epidemic (micro-level factors); and the governance of epidemics (macro-level factor). sociology and social psychology offer some interesting explanations of the sluggishness of preventive health behavior in individuals [32, 33] . among ten theories identified as the "most often used" today [34] , the top two explanatory models are the social cognitive theory (sct) and the health belief model (hbm) [34, 35] . both social theories are useful in the analysis of preventive action: they focus on the individual's capacity to make his/her own decisions, and the recognition that there are multiple and varied factors involved in a person's health-related actions. the sct explains people's health-related actions primarily in terms of their expectations of the outcome, their confidence in the success of their actions, and their ability to perform the action; but perhaps the most relevant aspect of the sct is its consideration of the individual's environment as one of the important determinants of his/her behavior [36] . the hbm proposes that the likelihood of a person taking preventive action increases if he/she believes in his/her personal susceptibility to the illness and in the severity of the illness; perceives the preventive action as beneficial; believes that there are no barriers to action or that barriers can be overcome; believes in the net gain -benefits exceed barriers or costs-of taking preventive action [33, 37, 38] . a comprehensive review of studies applying the hbm [37] found the variables perceived susceptibility and perceived severity to be closely correlated and to have significant influence upon a variety of preventive behaviors. the combination of both perceived severity and perceived susceptibility labeled "perceived threat" has been found to be a more significant explanatory variable than severity and susceptibility used separately [37] . the hbm variable influencing the perception of effective prevention of hiv/aids in three ethnic communities is perceived severity or seriousness of the disease, together with the perception of personal responsibility for contracting the disease [39] . i ascertained perceived severity of hiv/aids in terms of the respondents' subjective perception of the likelihood of death using the close-ended question "when you think about aids, how serious do you feel it is? this approach is basically the same as that of janz et al. [38] who define perceived severity as "one's belief of how serious a condition and its sequelae are". strecher et al. had offered earlier [37] a wider definition of perceived severity: "personal evaluations of the probable biomedical, financial and social consequences of contracting hiv and having aids". although there are slight variations in the wording of the question asked in interviews and in the definition of perceived severity, the general consensus in the literature is that this conceptual construct, often together with perceived susceptibility, is essential in the analysis of people's motivation to take preventive action. nevertheless, as preventive health behavior is influenced by a multiplicity of factors, the hbm, sct and all the other top eight theories [34] are limited as they offer only partial explanations of health-related behavior, but they are complementary. i include social constructs from the hbm and the sct and some relevant contextual social factors, to explore the research question "what can we learn from the sars experience about effective prevention of hiv/aids"? combining the analysis of individuals' responses with their collective implications, i attempt to demonstrate that perceived severity together with perceived susceptibility and the public image of the two epidemics help to explain some of the difference in prevention effectiveness. i test two related assumptions: (1) a higher perception of disease severity and personal susceptibility to sars as compared to hiv/aids, contributed to the higher effectiveness of sars prevention efforts; (2) the second assumption is two-fold: (a) in contrast to sars, the overall negative social 'image' of hiv/aids as a disease associated with particular types of individuals tends to weaken people's perception of susceptibility; (b) correspondingly, low perceived susceptibility tends to discourage public support for robust preventive efforts at the community level. these assumptions require elaboration. following the hbm, the first assumption to be tested is that a person's perception of the severity of the disease and his/her perceived susceptibility to that disease are likely to motivate him/her towards taking preventive action. all things being equal, such a commitment to prevention would weaken or be altogether absent when perceived severity and/or susceptibility are low or nil. the perceived severity of hiv/aids was ascertained through the question "when you think about aids, how serious do you feel it is? four alternative response categories were provided (see tables 1 and a.3) . for the logistic analysis, the responses were dichotomized into high perceived severity ((1) "very serious, causes death"), and low perceived severity ((0) "not very serious" or "not serious at all" and "don't know"). perceived susceptibility to hiv/aids was ascertained by the level of agreement to the statement "aids doesn't happen to people like me" (tables 1 and a. 3). in the study of sars, perceived susceptibility was ascertained through the question "how likely do you think it is for you to contract sars"? respondents expressed their belief in their personal susceptibility by indicating whether they saw the likelihood of contracting sars as "very likely", "likely", "not very likely", "not likely at all", or did not know ( table 2 ). the perception of severity of sars was measured by the question "if you have contracted sars, what is the likelihood of survival"? the four response categories were "very likely", "likely", "not very likely" and "not likely at all" ( table 2) . the second assumption is that in contrast to sars, the overall negative public 'image' of hiv/aids as a disease associated with particular types of individuals tends to weaken people's perception of susceptibility and, correspondingly, tends to discourage public support for robust preventive efforts at the community level. the individual's 'image' of the disease shapes his/her perception of seriousness and susceptibility and thus contributes to his/her motivation to take preventive action. that 'image' of the disease and of persons affected, however, is shaped to a large extent by prevailing values and normative beliefs in the community and it is subject to change over time. this process is suggested by many sociological theories including social networks and social support theory [40] , rational choice theory [41] , and the sct. the sct offers the concept "reciprocal determinism" [36] that proposes a dynamic interplay of "the person, behavior, and the environment". a eight preventive measures were considered as part of the respondents' "activities during the past 3 days": covering the mouth with paper tissue or handkerchief when sneezing or coughing; covering the mouth with bare hand when sneezing or coughing; washing hands after sneezing or coughing; using soap or liquid hand-wash when washing hands; wearing a mask over the mouth; using serving utensils (chopsticks or spoons) for shared food when joining others for meals; when touching objects that may possible carry the sars virus (e.g., door handles, buttons in lifts), taking preventive measures (e.g., pressing lift buttons with tissue paper); washing hands as soon as possible after touching objects that may possibly carry the sars virus (e.g., door handles, buttons in lifts). b the original response categories for perceived susceptibility (that is, the perceived likelihood of contracting sars) were: "very likely", "likely", "not very likely", "not likely at all" and "don't know". for the logistic regression analysis the latter group, 17.6% of respondents who had no idea on their susceptibility to sars, were contrasted with all other respondents who did have an assessment of their likelihood of getting infected. c the original response categories for perceived severity (that is, the likelihood of survival) were "very likely", "likely", "not very likely" and "not likely at all". for the logistic regression analysis, these responses were dichotomized into low perceived severity (survival "very likely/likely") and high perceived severity (survival "not very likely"/"not likely at all"). d the respondents' appraisal of the health authorities' crisis management was ascertained by their assessment of the distribution of information in terms of accuracy, clearness, sufficiency, timeliness, and trustworthiness in a scale from very negative (score 1) to very positive (score 6). the scale had high reliability (α = 0.813) and the mean score was 4.83 (s.d. = 0.617). in my view, the dynamic interaction of the individual's health-related behavior and the environment is better explained by the individual's subjective perception of the situation (for example a crisis or stressor) and the social context of the situation, as proposed by symbolic interaction and family stress theory [42] . thus, my assumption that a person's motivation to take preventive action may also be manifested in his or her cooperation with community-based preventive measures is explained clearly by the application of the conceptual premises on community responses to stressor events formulated by reiss and oliveri [43] . the public's perception of the scope of the problem was highlighted by these authors as part of their concept "community's punctuation of an event". they defined the community's punctuation of an event or perceived scope as "when the problem begins and when it ends and who is involved". they proposed that the community and its leaders would be more inclined to invest concerted efforts to solve a problem if three conditions are met: accountability, duty, and competence [43] . that is, the community and its policy-makers would be most inclined to mobilize assistance and preventive efforts when these three conditions are met: the persons affected are perceived as not being accountable for the problem; they are regarded as having the duty to request outside help; they are considered as lacking the competence to solve by themselves the problem affecting them. i suggest that these three conditions shape the public image of sars and hiv/aids and add to our understanding of the disparity in prevention effectiveness of these two epidemics at the community level. the public image of hiv/aids was ascertained through one open-ended question about persons living with hiv/aids: "what kind of people do you think are most likely to get aids"? the analysis of responses revealed three types of stereotypes or 'images': 'risktakers', 'deviants', and 'victims'. only a small group of respondents had not particular image of people living with hiv/aids ( table 1 ). the public image of sars may be ascertained indirectly through the respondents' perception of a sense of social responsibility and willingness to make some personal sacrifices in combating the disease. those perceptions reflect people's collective sense of accountability and duty and their recognition of the need for expertise to handle the crisis. referring the respondents to the measures implemented to prevent the spread of sars, they were asked their level of agreement (strongly agree to strongly disagree) with these statements: "people should be willing to make some personal sacrifices"; "people have mostly been socially responsible"; "if you did not develop symptoms of sars after having close contact with someone diagnosed with sars, would you agree to be quarantined for 10 days?"; "if you did not develop symptoms of sars after having non-close contact with someone diagnosed with sars, would you agree to be quarantined for 10 days"? ( table 2 ). the condition of "competence" identified by reiss and oliveri [43] as discussed above, has to do with expertise in handling the crisis, and thus brings in a final concept of relevance to this discussion: governance. governance is another socially significant aspect of hiv/aids and sars as a strong political will at the national level is needed to invest state resources, and to utilize knowledge and technology creatively for their detection and prevention. but these epidemics are also global problems that challenge national boundaries and the conventional idea of state sovereignty and governance, and test international cooperation, because of their mode of transmission and the increasing movement of people across countries for leisure, trade, and study among other activities. given the easiness and speed of their international transmission, these epidemics have unwittingly pushed forth a new phenomenon that fidler [30] calls "global health governance" that is, "the proposition that governance of public health issues must include not only state actors but also non-state actors". examining the international impact of sars, this international law expert concluded that "to govern an increasingly borderless world, requires, in essence, increasingly borderless governance" [30] . the analysis of the sars crisis management in singapore suggests that effective containment and prevention of infectious disease outbreaks requires dedicated and transparent governance at both levels, national and global. and to be successful, global governance requires timely and effective response and collaboration from sovereign nations. but as i shall discuss later, the governance approach to sars differs substantially from that applied to hiv/aids. first, a word of caution: i reiterate that this study is by necessity limited and exploratory because the two epidemics, hiv/aids and sars, are very different microbiologically and epidemiologically as indicated in the introduction; and there is a 10-year gap between the two surveys. the sars study was conducted as the outbreak progressed in may 2003, 10 years after the hiv/aids study. nonetheless, despite these methodological difficulties, i believe it is important to scrutinize available information on both epidemics in the hope of increasing our understanding of the dynamics of preventive action against hiv/aids. while the two epidemics are different in many respects, they are both serious public health threats that require a collective response [26, 30] as indicated earlier. two general types of data are discussed here: population figures and data from personal interviews. the international population figures on the impact and spread of hiv/aids and sars are taken from who's published reports [25, 28, 31, [44] [45] [46] . the analysis of behavior and attitudes of individuals is based on data from two separate studies i conducted in singapore as principal investigator. the data on hiv/aids are from a 1993 study of attitudes and preventive behavior regarding hiv/aids based on a survey of personal interviews with a representative stratified random sample of 660 adults aged 21 and older following a structured questionnaire. respondents were from the chinese, malay, and indian communities, the three largest ethnic groups in singapore. the sample characteristics and the details on measurement of the variables included in this analysis are described in tables 1 and a. 3. the data discussed in this paper are part of a larger study on preventive health behavior regarding cancer, heart disease, and hiv/aids supported by a research grant from the national university of singapore. further methodological details are provided elsewhere [39] . the new findings discussed in this paper were obtained through logistic regression analysis. the data on behavior and attitudes on sars are from a 2003 study based on telephone interviews with a representative stratified random sample of 1202 adults aged 21 and older, following a structured questionnaire. the three main ethnic groups in singapore (chinese, malays, and indians) were proportionally represented. the interviews were conducted within the span of 6 days, from 5 to 10 may, while the country was facing the sars epidemic. telephone interviews were the only data collection option because it was imperative at the time to follow the public health advice to restrict personal contact to home and the workplace, whenever possible. the main characteristics of the sample and the attitudinal measurements applied are presented in table 2 . further methodological details of this study are described elsewhere [29] . the new findings presented in this paper were obtained through logistic regression analysis. while the data refer to three ethnic communities in singapore the findings illustrate the impact of social attitudes upon the governance of epidemics in a high density global city. acknowledging the multiplicity of factors that may play a role in shaping the success or failure of illness prevention and containment of epidemics, this study deals only with a small number of variables. the analysis of data in both studies comprised two stages: an initial scrutiny of the main assumed correlations and attitudinal scales using partial correlation and factor analysis; logistic regression to explore the likelihood of occurrence of stereotypical images of people living with hiv/aids, the dependent variable in the hiv/aids study; and likelihood of public support of the sars crisis management, the dependent variable in the sars study. logistic regression is a very useful tool to explore the probability of occurrence of the dependent variable over the probability of it not occurring and the outcome is provided as odds ratios. the odds ratio is the odds of one variable occurring to the odds of another [47, 48] . the logistic regression analysis of the public image of hiv/aids comprised three sets of variables: sociodemographic variables (gender, age, ethnicity, marital status and religion); social class factors (occupation, personal monthly income, and educational level); attitudinal factors proposed by the hbm and the sct (tendency to worry about falling ill; future orientation; sense of personal control of one's life; life satisfaction; perceived severity of hiv/aids; perceived susceptibility to hiv/aids; belief in effective prevention of hiv/aids) and perception of hiv/aids. a description of these variables is provided in table a.3. five sets of variables were included in the logistic regression analysis of the public support of the sars crisis management: socio-demographic variables (gender; age; ethnicity; place of birth; marital status); social class (educational level and personal monthly income); health behavior variables (smoking, exercising regularly, and preventive measures against sars taken over the 3 days preceding the interview); attitudes suggested by the hbm including perceived susceptibility and perceived severity; and attitudes on sars crisis management. to meet requirements of the logistic regression analysis the response categories of the question on perceived susceptibility were dichotomized contrasting the respondents who expressed an estimation of their likelihood of infection on the one hand, with respon-dents who have no awareness of their susceptibility to sars, on the other hand. the response categories for perceived severity were dichotomized into "high" severity (survival not very likely or not likely at all) versus "low" severity (all other responses including "don't know"). the complete list and explanation of all the variables are presented in table 2 . the discussion follows the two related assumptions presented earlier. the first assumption to be tested is that a higher perception of disease severity and personal susceptibility to sars as compared to hiv/aids, contributed to the higher effectiveness of sars prevention efforts. show that the sars outbreak was contained within 9 months of its onset while the hiv/aids epidemic continues undefeated after nearly three decades. would people's sense of susceptibility to these diseases and their severity contribute to that difference? the survey data from singapore on the two epidemics provide a tentative yet useful indication of the differential perception of severity and susceptibility. only 31.5% of the respondents expressed high susceptibility to hiv/aids (table 1 ) compared to 82.4% of respondents in the case of sars ( table 2 ). the corresponding figures on the expression of high perceived severity are 85.6% of the respondents in the case of hiv/aids (table 1 ) and only 12.4% of the respondents in the case of sars ( table 2) . the findings from the analysis of the belief in effective hiv/aids prevention in the total sample (appendix a, table a.4) indicate that people who believe that hiv/aids is very serious and fatal (high perceived severity) are significantly more inclined than those with low perceived severity to believe there are effective ways of preventing the disease. this belief in effective prevention of hiv/aids is also found among people with high future orientation, those who are inclined to worry about falling ill; it is expressed by men more than women. the nagelkerke r 2 coefficient of 0.435 suggests that 43.5% of the overall variation in the belief in effective prevention of hiv/aids is predicted by the variables in the model. the model predicted correctly the belief in effective hiv/aids prevention 86.9% of the time. in the case of sars, preventive measures were being implemented as the outbreak progressed. the interviews took place in the midst of the crisis as the country and the region were coping with this completely new threat. no clear indication of effective prevention was in sight but through a steep learning process several effective preventive measures were being identified and the information transmitted from the experts to the public daily through various mass media including radio, newspapers, the internet, regular television, and a dedicated television channel set up specifically for that purpose. this special situation may explain the respondents' very low perceived severity of sars and their very high sense of susceptibility to it as ways of transmission encroached into people's daily life, for example: droplets from the sneezing or coughing of an infected person, and the touching of infected commonly used objects such as eating utensils, buttons in elevators, and door handles [29, 49] . the second assumption to be explored here is that in contrast to sars, the overall negative social 'image' of hiv/aids as a disease associated with particular types of individuals tends to weaken people's perception of susceptibility and, correspondingly, tends to discourage public support for robust preventive efforts at the community level. as suggested earlier, this assumption may be examined using reiss and oliveri's [43] concept "community's punctuation of an event" and the three conditions -accountability, duty, and competence -these authors identified as requirements for the community's positive response. in the case of hiv/aids and sars the focus is the community's endorsement of disease prevention and containment plans and their active collaboration in prevention efforts. when does the problem begin and when does it end and who is involved? the answers to these questions mark the community's punctuation of crises and show that the punctuation of the sars outbreak was rather different from that of hiv/aids. the punctuation of the sars outbreak as a crisis was very clear. sars was imported into singapore at the end of february, 2003, when an infected vacationer returned home from hong kong where she caught the infection while staying at the same hong kong hotel where a doctor from guangzhou, china who had treated sars patients there, was residing [50] . the unknown cause and nature of the disease deterred the assignment of blame or accountability. sars patients were not held accountable for their illness. nor were they assumed to have the expertise to solve the problem on their own although it soon became a duty for people with the publicized symptoms to seek immediate expert medical help [29, 51] . the findings from the sars study in table 2 show that 75.9% of the respondents made a positive appraisal of the health authorities' management and control of the sars crisis; 77.4% had the chance to express their opinions to the authorities; 91.3% were prepared to be quarantined for 10 days after close contact with an infected person and 71.6% would agree to be quarantined even if there was non-close contact. further indications of the public's willingness to collaborate in preventive efforts against sars were the very positive attitudes of the majority of respondents: although about one of every two agreed that preventive measures taken against sars "have affected my personal choice and freedom in life", most respondents (95.3%) agreed that "people should be willing to make some personal sacrifices" to contain the epidemic and 86% felt that "people have mostly been socially responsible". the findings from the logistic regression analysis of the sars study data confirm that this sense of social responsibility was a fundamental manifestation of the community's positive and compassionate 'image' of sars patients and it was significantly associated with their endorsement of the health authorities' management of the crisis in the total sample as well as among people with lower education, and ethnic minorities such as the singaporean malays (table 3) . among the 1202 respondents, the endorsement of the health authorities' crisis management was particularly supported by people who perceived the community as being socially responsible; those who believed that the crisis justified making some personal sacrifices (especially with regard to movement outside their homes, restricting or changing their travel patterns, and abiding by quarantine regulations); those felt that they had the chance to be part of the effort and express their personal opinions; people who had formed an opinion on their personal susceptibility to the infection (in contrast to those who had no or very little information on sars). the nagelkerke r 2 coefficient of 0.139 suggests the factors in the model explain 13.9% of the variation in endorsement of the crisis management in the total population. the analysis of the same factors was repeated among three specific subgroups that have shown less positive appraisal of crisis management: the senior cohort (respondents aged 60 and older), the lower educated (people with only primary or lower education), and malays. as illustrated in table 3 , even among the lower educated and the malay, the sense of social responsibility was significantly associated with their endorsement of preventive efforts (dependent variable). however, perceived susceptibility to sars did not influence significantly the appraisal of crisis management by the seniors and the less educated but it did among malays in the expected direction: persons who have no idea of their susceptibility (no awareness of it) were most likely to give a negative appraisal of crisis management. no significant impact of perceived severity upon people's appraisal of crisis management was detected in the total sample or any of the three subgroups. overall, the nagelkerke r 2 coefficients indicate that, compared to the total sample, variables in the model helped explain a larger proportion of the variance in the dependent variable among subgroups such as seniors (43.7%), the less educated (33.8%) and the malay community (29.0%) about 80% of the time. these figures point to the importance of variations the perception of and responses to crises among different segments of the population given their differences in life experiences, in knowledge and level of information on the problem, and in cultural values and beliefs, among other factors. as indicated earlier, from the contextual perspective proposed by reiss and oliveri [43] the public image of a crisis or stressor (e.g., an infectious disease epidemic) refers to the public's perception of its scope and its social acceptability. in terms of the scope of the problem -when it begins and when it ends -the quiet and prolonged way in which the hiv virus enters and destroys the immune system represents a major challenge for the mobilization of public interest and support of testing. visible signs of the disease tend to appear only in the late stages. the opposite occurred with respect to sars. two separate studies of the awareness of health threats in the united kingdom confirm these findings on impact of the public image of the problem and punctuation or scope of the event: british lay respondents and journalists were inclined to see aids as a "far-flung" risk of no immediate relevance to their lives [62, 63] . regarding social acceptability, if the stressor is seen by the community as the consequence of socially unacceptable behavior, one would expect collective apathy or reluctance or opposition to the investment of public funds and efforts to contain and solve the problem. the findings in tables 2, 3 and a.4 suggest that this appears to be the case with hiv/aids. the public image of hiv/aids tends to be shaped by normative expectations or stereotypes in the community. the large majority of the respondents (88.5%) associated a particular lifestyle with the contracting of the disease thus forming negative images of people living with hiv/aids. as shown in table 1 , over half of the respondents (57%) saw them as "risk-takers": people who engage in activities that put them at risk of infection such as having multiple sexual partners or procuring the services of commercial sex workers. another 26% of the respondents associated them with people who engage in 'deviant' activities such as commercial sex workers and injecting drug users who exchange infected needles. a small group (5.5%) considered them as "victims" of "fate" or "bad luck" or accidental infection. only 11.5% of the respondents did not have an opinion or image of people living with hiv/aids. practically all in this group had no information on hiv/aids. table 4 presents some of the factors that contribute to the formation of a particular 'image' of people living with hiv/aids. the odds of seeing them as 'victims' (column b) were significantly higher among older people, those who do not think hiv/aids is a serious and deadly disease; and those who do not believe there is an effective way of preventing the illness. interestingly, the same features are exhibited by the small group who did not put a label on people living with hiv/aids (column a): they tend to be older, unaware of the severity of the disease, and unaware of any effective preventive measures. the odds of perceiving hiv/aids sufferers as 'risk-takers' that is, associating a 'risk-taking' life style with hiv/aids infection (column c), decreased by 45% among men; increased significantly among people who worry about falling ill and those who believe that there are effective ways of preventing the disease. the most negative 'image' or lifestyle associated with hiv/aids infection is that labeled 'deviants' (column d). the odds of having this image of hiv/aids sufferers increase significantly among women in contrast to men; among younger people in contrast to people who are 60 or older; among those believe the disease is very severe, and among people who do not worry much about falling ill. the nagelkerke r 2 coefficients indicate that the variables in the model explain 45.6% of the overall variation in emphasis on the 'victim' image; 19.5% of the emphasis on the 'risk-takers' image; 16.9% of the emphasis on the 'deviant' image, and 58.1% of the variance on the absence of a stereotypical image of hiv/aids sufferers. this variable is explained correctly by the variables in the model 92.7% of the time (table 4 ). these findings fit the international pattern: the presence of stereotypical images of people who get infected with hiv/aids is not restricted to a particular country [23, 61] . state regulations on infectious diseases such as notification and surveillance systems have been in place for more than a century in many countries [30, 52] and today they are followed by all state members of the united nations including singapore [53] . but the official approach to the control and prevention of hiv/aids differs widely from that of sars and the difference has to do with the public image of the disease discussed in the preceding sections. the experience of sars in singapore provides an interesting illustration of the positive synergy between national governance and global health governance. the main features of the state's crisis management approach illustrate the situation well. those features were: (a) transparency; (b) public education; (c) multi-pronged approach; (d) legislation. in contrast to the situation in china and some other affected countries at the onset of the epidemic [54, 55] , the sars situation in singapore was characterized throughout by transparency on the part of the health authorities in their reporting and distribution of a continuous flow of information to the public on new infections and deaths, locations, and contact tracing efforts and approaches. it was believed that an informed public can collaborate better and participate more effectively in containing the spread of the disease than a public kept ignorant of the seriousness of the situation. news reports on the progress of the epidemic were transmitnotes: total sample size, 660. * statistically significant at p = 0.04-0.05. ** statistically significant at p = 0.01-0.03. *** statistically significant at p = 0.001-0.009. **** statistically significant at p = 0.0001 or lower. ted to the public through all printed media, radio and television in all the four official languages (mandarin, malay, tamil and english). exceptional measures were taken to reach as many people as possible throughout singapore: broadcasting was resumed temporarily in some of the chinese dialects that had not been used in tv for nearly two decades; a new dedicated tv channel was set up, sarstv. singapore was the only country affected by sars to set up this public service [56] . informing the public on the development of the epidemic went hand-in-hand with public health education whereby public information on the current state of knowledge on the disease was constantly updated at various levels of sophistication, from medical and epidemiological data in specialized publications [57, 58] to newspaper articles explaining how the coronavirus attacks a healthy cell, to cartoons illustrating the proper use of masks, of serving utensils, and to take one's temperature correctly with a thermometer, and how to wash hands thoroughly, among other things. it was evident to the authorities and the population that the sars epidemic affected the daily life activities of every citizen and demanded drastic changes in lifestyle. this realization led to the implementation of a multi-pronged approach to deal with the crisis. all relevant ministries, statutory boards and other organs of the state were mobilized and non-governmental organizations and the private sector joined the effort. this approach was in fact a typical response in singapore as "ministries and government agencies had honed emergency preparedness to a fine art" [49] . part of that preparedness was the use of legislation including quarantine laws and other preventive measures. for example, section 10 of the infectious diseases act was amended with effect from 27 april 2003 requiring "medical or dental practitioners to obtain information from their patients and transmit such information to the director [of medical services] to investigate the outbreak or prevent the spread of an infectious disease such as sars"; a new "patient declaration form" was used for this purpose during the outbreak [51] . while transparency, public education, the implementation of a multi-pronged approach, and the use of legislation were characteristics of the national government's response internally, there was also transparency and close collaboration of singapore with the who and other international organizations. in his global analysis of the epidemic, fidler highlights this feature: "singapore was initially scheduled to be removed from the [who's] list of sars-affected areas on 11 may; but, on that date, singapore reported a new case of sars to who, an indication of singapore's commitment to open reporting and cooperation with who" [28] . a sovereign state's abiding to international guidelines, even at the expense of its own economic interests, illustrates the importance of "global health governance" to deal with infectious disease epidemics and similar health threats in the 21st century. the control and prevention of hiv/aids has followed a different approach. fidler [30] correctly highlights "the conceptual and policy shifts" from standard procedures applied to infectious diseases by the who and public health experts. the international health regulations (ihr) "are the only set of international legal rules binding on who member states concerning the control of infectious diseases" [30] . yet, in fidler's view, one distinguishing feature of the official international approach to deal with the hiv/aids epidemic was that public health experts and the who did not follow the ihr's classical "westphalian" model -that emphasizes state sovereignty and non-intervention -to deal with infectious diseases "but rather turned to international human rights law to provide governance norms for the fight against this new plague" [30] . this conceptual shift was unique. the approach to hiv/aids was "the first time in history [that] preventing discrimination towards those affected by an epidemic became an integral part of a global strategy to prevent and control an epidemic of infectious disease" [59, 30] . danziger [26] suggests that this conceptual shift was promoted and supported by the "neoliberal democratic ideology" prevalent in western countries by the end of the 20th century and enthusiastic enough to lead some countries to abandon the compulsory surveillance methods and "placing protection of individual rights on a par with (or even above) the protection of the public health". apart from the ideological angle, the who's concern with human rights and particularly with the matter of discrimination of people living with hiv/aids has its roots in actual manifestations of social stigma associated with the presumed lifestyle of the first persons affected by the disease. in june and july, 1981, five young homosexual men were diagnosed with pneumocystis carinii pneumonia and 26 young homosexual men with kaposi's sarcoma "a rare form of cancer which had until then been associated with elderly americans"; added to the spectrum of aids features in the early 1980s was the confirmation that one of the main forms of transmission is sexual intercourse [26] . the spread of the epidemic has been so extensive geographically as well as socially, that people living with hiv/aids today come from all walks of life. but as the figures in appendix a, table a .1 indicate, the highest prevalence of hiv are still found among some distinct lifestyle groups including commercial sex workers and injecting drug users. the survey data discussed in the preceding sections suggest that the public image of hiv/aids reflects the prevalence figures among some specific groups; a large majority of respondents saw hiv/aids sufferers as following their lifestyle by personal choice. thus, their image remains negative despite public education efforts by the who, non-state organizations and non-governmental organizations. an additional aspect is the slow pace of development of the disease: people may be infected for many years without developing any symptoms and may thus continue unwittingly to infect others [26] . the global governance approach to protect people with hiv/aids from discrimination involves avoidance of disclosure of one's health condition and of routinely or compulsory name-linked testing and other features of standard infectious disease surveillance. danziger [26] sees this position as "possessive individualism" that demands testing to be done only if the person has consented freely and has received full information on the consequences through the process of informed consent. moreover, danziger points out that with the current stage of knowledge on hiv/aids, "there is little or no gain for a hiv-infected person to be tested and identified as hiv-positive" [26] . this reasoning creates a dilemma because experts agree that standard infectious disease surveillance is indispensable for the effective prevention and control of infectious diseases [3, [14] [15] [16] [17] [18] [19] [20] 30, 52] that is, the beneficiaries of effective control and prevention are the rest of the community. according to danziger [26] the dilemma has been sorted out internationally by an apparent consensus of stakeholders to consider hiv/aids as "a crisis of human rights" and not as a "public health crisis". however, with the epidemic proceeding unrelentingly, there are signs of concern. a recent development in singapore is illustrative of the range of opinions on this matter: the ministry of health announced on 15 july 2005 that "spouses of patients with hiv will be informed of their partner's illness, regardless of whether the infected person agrees" [60] . the newspaper report cited the case of four women who discovered they had hiv when they did their blood test during their pregnancies. the husband of one of them had been diagnosed with hiv since 2001. previously, patients were counseled and informed consent was required. now the doctor needs only to keep the patient "appropriately informed". the spouses will be informed "in a sensitive manner" by trained personnel of a new hiv prevention unit. the infectious diseases act will be invoked as is the case with all other infectious diseases [60] . the singapore shift towards a version closer to standard surveillance of hiv/aids is indicative of its concern for the rights of the infected person as well as the rights of the patient's partner, and of all other persons involved. the global governance of hiv/aids prevention needs to address the public image of the disease and the impact that such an image has upon efforts to mobilize the community in prevention efforts. for as long as hiv/aids is seen as a problem of particular lifestyles (that is, of specific types of people), prevention efforts such as condom use and testing are bound to have limited impact. the analysis was based on data from two separate studies of singapore residents' attitudes and behavior. given the differences in questions asked during the respective interviews, the 10-year difference between studies and the significant differences in the etiology, nature, and development of the two infectious diseases, the findings must be treated with caution. that said, the availability of data from the two studies offered a good opportunity for this exploratory comparison of attitudes towards and the public image of hiv/aids and sars in search of a better understanding of the social obstacles to effective prevention against hiv/aids. this exploratory comparison was guided by two main assumptions. the first assumption was that a higher perception of disease severity and personal susceptibility to sars as compared to the level of perceived susceptibility to and severity of hiv/aids, contributed to the difference in effectiveness of prevention efforts. the data from the singapore study support that assumption partially and revealed a new aspect of the problem. perceived susceptibility was high for sars but relatively low for hiv/aids. the opposite was found for perceived severity. the findings suggest that among the complex set of factors that motivate people to take preventive measures, perceived susceptibility to the disease (your subjective assessment of the likelihood of becoming infected) is more relevant than perceived severity of the disease. the data show a strong tendency for people to consider hiv/aids as peculiar to certain types of people different from themselves. thus their belief that their chances of being infected with the hiv virus are remote is not surprising. these findings on low perceived susceptibility and high perceived severity are also meaningful in the context of the second assumption tested. the second assumption explored in this study was two-fold: (a) that in contrast to sars, the overall negative social 'image' of hiv/aids as a disease associated with particular types of individuals tends to weaken people's perception of susceptibility; (b) that correspondingly, low perceived susceptibility tends to discourage public support for robust preventive efforts at the community level. the findings verify and clarify these assumptions. only 3 out of every 10 respondents expressed high perceived susceptibility to hiv/aids compared to 8 out of every 10 in the case of sars. but, as mentioned above, it was also found that the perceived severity of hiv/aids was significantly higher than the perceived severity of sars. more importantly, the labeling of people living with hiv/aids as 'risktakers' or 'deviants', was expressed by nine out of every 10 of the respondents who believed hiv/aids was a incurable disease (high perceived severity) and believed that the disease is mostly linked to one's lifestyle choice. the challenge for health authorities is to enhance the population's perceived susceptibility to hiv/aids. the first step in this direction is the widespread distribution of accurate, clear and consis-tent information on the 'silent' nature of the disease in its early stages. the second part of this assumption was on the mobilization of the community's endorsement and active participation in the control and prevention of the epidemics. i have discussed the elements involved including the community's punctuation of the crisis and the aspects of accountability, duty, and competence. in the case of epidemics, competence is typically found at the community level and this brings us to the question of governance: how does a government deal with the health crisis represented by an infectious disease epidemic? much has been learned over the centuries around the world but each new epidemic brings new dangers. the sars outbreak tested the state's level of emergency preparedness and commitment to transparency particularly in asian countries, but it also highlighted the need for global governance of health threats that cut across national boundaries. in contrast, the hiv/aids epidemic still represents a challenge in terms of public health, political ideology, human rights, and social discrimination. the lack of success in the control and prevention of the epidemic highlights the fact that after nearly three decades the global governance of hiv/aids is still a work-in-progress. the urgency of the problem is well recognized by most world leaders and specialists with some experts warning that "new threats to [world] stability and secu-rity may emerge as the pandemic escalates" because, among other reasons, large numbers among police and armed forces in many countries and un peacekeeping forces are getting infected [27] . the current global governance of hiv/aids requires critical scrutiny, as well as active exploration of solutions -among all types of stakeholders with differing ideological and social perspectives -to the slackness of preventive efforts against the two main behaviors that are sustaining the epidemic: "high-risk sexual activity and drug use" [27] . one aspect of that critical scrutiny is the systematic and comparative analysis of hiv/aids global governance with the global governance of other infectious disease epidemics that have been successful. in sum, the findings on the sars and hiv/aids experiences suggest that health authorities need to navigate effectively the local and global obstacles to prevention by: (a) enhancing the public's understanding of the etiology of hiv/aids, its modes of transmission and effective preventive measures; and (b) correcting the lay public's inaccurate perception of personal susceptibility and 'the punctuation of the event'. tables a.1-a.4. very serious because it can cause death and has no cure serious but has some partial cure only mildly serious because does not cause death not serious at all scores range from 4 (very serious) to 1 (not serious at all). for the logistic regression analysis these response categories were dichotomized: (1) "very serious" vs. (0) all other responses perceived susceptibility to hiv/aids "aids doesn't happen to people like me" mean, 0.315; s.d., 0.464; sample size, 660 this statement is part of a series involving cancer, heart disease and aids. respondents were asked to tell the interviewer if they strongly agree (sa), agree (a), disagree (d) or strongly disagree (sd) with each statement scores range from 1 (sa) to 5 (sd), with higher scores indicating higher perceived susceptibility for the logistic regression analysis these response categories were dichotomized: (1) "sa/a" vs. (0) all other responses belief in effective hiv/aids prevention "is there an effective way of protecting yourself from aids"? mean, 0.828; s.d., 0.376; sample size, 660 the responses were scored as "yes" (1) vs. "no" (0) dependent variable: perception of people living with hiv/aids "what kind of people do you think are most likely to get hiv/aids?" during the personal interviews this open-ended question was preceded by identical questions for cancer and heart disease. factor analysis of the responses revealed three categories or 'images': (a) "victims" of "fate" or "bad luck" or accidental infection; (b) "risk-takers": people who engage in activities that put them at risk of infection such as having multiple sexual partners or procuring the services of commercial sex workers; (c) people who engage in 'deviant' activities such as commercial sex workers and injecting drug users who exchange infected needles. a fourth category comprises a small group of respondents who did not label people living with hiv/aids. each of the four categories is examined using separate logistic regression analyses (see table 4 ) (1) 3.362 **** personal control: high (1) 1.583 life satisfaction: high (1) 1.224 perceived severity: high (1) 9.518 **** perceived susceptibility: high (1) 1.318 nagelkerke r 2 0.435 variance predicted correct (%) 86.9 a see table a .3 for the description of measurement of belief in effective prevention. total sample size: 660. ** statistically significant at p = 0.01-0.03. *** statistically significant at p = 0.001-0.009. **** statistically significant at p = 0.0001 or lower. dk publishing and national gallery of art illness as a metaphor and aids and its metaphors disease and the modern world. 1500 to the present day hiv stories: the archaeology of aids writing in france narrative in the time of aids: postcolonial kenyan women's literature the evil of the 20th century: poetry and aids on finding in a book of poems by norman dubie, a 25-year-old letter from the bookbinder to my cousin now dead of aids playing for life: performance in africa in the age of aids choreographer mark sieczkarek's 'living with aids' with the dance-factory in ghana nationalizing the gay body: aids and sentimental pedagogy in 'philadelphia'. american literary history narrative in times of crisis: aids stories in ghana playing for life: performance in africa in the age of rats, lice and history emerging issues in infectious disease epidemiology mapping the world of epidemiology. 1. the disease detectives disease detectives are turning to molecular techniques to uncover emerging microbes epidemiology: a science of patterns 21st century detectives-the laboratory professional in the prevention of infectious disease the making of a germ panic, then and now disease detectives celebrate 50 years of successful sleuthing a critical analysis of the brazilian response to hiv/aids: lessons learned for controlling and mitigating the epidemic in developing countries measuring stigma in older and younger adults with hiv/aids: an analysis of an hiv stigma scale and initial exploration of subscales aids-related discrimination in asia treat three million people living with hiv/aids by 2005. in: plenary address by director of hiv/aids, who at the seventh international congress on aids in asia and the pacific world health organization [who] position statement on condoms and hiv prevention the politics of emerging and resurgent infectious diseases. london: macmillan the lessons of hiv/aids world health organization. who guidelines for the global surveillance of severe acute respiratory syndrome (sars) crisis prevention and management during the sars outbreak sars, governance and the globalization of disease world health organization. who global conference on severe acute respiratory syndrome (sars)-where do we go from here? geneva: who health behavior. emerging research perspectives handbook of health behavior research health behavior and health education. theory, research and practice promoting health: intervention strategies from social and behavioral research how individuals, environments, and health behavior interact. social cognitive theory the health belief model and health behavior the health belief model hiv/aids prevention and public health education social networks and social support rational choice theory: advocacy and critique family stress management. a contextual approach family stress as community frame global atlas of infections -epidemiological fact sheet -2004 update world health organization. summary of probable sars cases with onset of illness from 1 summary table of areas that experienced local transmission of sars during the outbreak period from 1 ordinary least squares and logistic regression analysis medical statistics a defining moment. how singapore beat sars at the epicentre. hong kong and the sars outbreak infectious diseases law & sars. singapore: times editions the politics of emerging and resurging infectious diseases hiv in singapore-past, present and future the sars epidemic under china's media policy online news media as interactive community bulletin boards. coverage of sars in the greater china regions the sars channel in singapore severe acute respiratory syndrome (sars) human rights and aids: the future of the pandemic new rule overrides patient confidentiality in order to protect the vulnerable aids as a social problem. the creation of social pariahs in the management of an epidemic. in: ritzer g, editor. handbook of social problems. a comparative international perspective representations of far-flung illnesses: the case of ebola in britain representations of sars in the british newspapers key: cord-354974-bh2expef authors: peterson, ingrid; bar-zeev, naor; kennedy, neil; ho, antonia; newberry, laura; sanjoaquin, miguel a.; menyere, mavis; alaerts, maaike; mapurisa, gugulethu; chilombe, moses; mambule, ivan; lalloo, david g.; anderson, suzanne t.; katangwe, thembi; cunliffe, nigel; nagelkerke, nico; mcmorrow, meredith; widdowson, marc-allain; french, neil; everett, dean; heyderman, robert s. title: respiratory virus–associated severe acute respiratory illness and viral clustering in malawian children in a setting with a high prevalence of hiv infection, malaria, and malnutrition date: 2016-09-13 journal: journal of infectious diseases doi: 10.1093/infdis/jiw426 sha: doc_id: 354974 cord_uid: bh2expef background: we used data from 4 years of pediatric severe acute respiratory illness (sari) sentinel surveillance in blantyre, malawi, to identify factors associated with clinical severity and coviral clustering. methods: from january 2011 to december 2014, 2363 children aged 3 months to 14 years presenting to the hospital with sari were enrolled. nasopharyngeal aspirates were tested for influenza virus and other respiratory viruses. we assessed risk factors for clinical severity and conducted clustering analysis to identify viral clusters in children with viral codetection. results: hospital-attended influenza virus–positive sari incidence was 2.0 cases per 10 000 children annually; it was highest among children aged <1 year (6.3 cases per 10 000), and human immunodeficiency virus (hiv)–infected children aged 5–9 years (6.0 cases per 10 000). a total of 605 sari cases (26.8%) had warning signs, which were positively associated with hiv infection (adjusted risk ratio [arr], 2.4; 95% confidence interval [ci], 1.4–3.9), respiratory syncytial virus infection (arr, 1.9; 95% ci, 1.3–3.0) and rainy season (arr, 2.4; 95% ci, 1.6–3.8). we identified 6 coviral clusters; 1 cluster was associated with sari with warning signs. conclusions: influenza vaccination may benefit young children and hiv-infected children in this setting. viral clustering may be associated with sari severity; its assessment should be included in routine sari surveillance. it is estimated that, worldwide, the case-fatality rate of severe pneumonia in children aged <5 years is 8.9%, which, in 2011, amounted to 1.26 million deaths [1] . much of this burden falls on sub-saharan africa, where severe acute respiratory infection (sari), including pneumonia, is a leading cause of childhood hospital attendance and death [2] . although laboratory diagnostic facilities are rarely available in such settings, sentinel surveillance using multiplex molecular diagnostic assays has recently provided considerable insight into the true burden of disease and the complexity of sari etiology. respiratory syncytial virus (rsv), parainfluenza viruses, rhinoviruses, influenza viruses, and adenovirus have been commonly detected in sari surveillance across the african continent [3] [4] [5] [6] [7] [8] . while there are a few viruses for which detection in respiratory disease cases is likely causal (eg, influenza virus and rsv) [9, 10] , for other commonly identified viruses causality has been difficult to determine. use of multiplex assays has led to an increasing realization that children with sari commonly carry multiple viral pathogens that may potentially contribute to disease. in the context of a low-income population with multiple drivers of immune compromise (eg, human immunodeficiency virus [hiv] infection, malnutrition, and malaria) [11] , we conducted active surveillance at a large urban teaching hospital in malawi to estimate the incidence of childhood sari and explore the association of sari clinical severity with hiv infection and clustering of respiratory viral coinfection. while previous studies have focused on children aged <5 years, we included children aged 3 months to 14 years in our analysis, to better capture the total burden and identify age groups particularly at risk. it offers care free at the point of delivery. overall, 13% of children aged <5 years in malawi are moderately to severely underweight, and 4% are experiencing wasting; 80.9% of children aged 12-23 months have received all expanded program on immunization vaccinations [12] . there is no national routine influenza vaccination in malawi. in 2010, a monovalent vaccine campaign targeting 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09) achieved 74% coverage in pregnant women and 7% of the overall population [13] . an estimated 2.5% of children aged <15 years are hiv infected [14] ; the hiv prevalence in children aged <5 years on qech nonsurgical pediatric wards is estimated at 6%. blantyre has 2 distinct weather seasons, a rainy season (january-april) and a cool dry season (may-august). overall, 25.2% of paediatric accident and emergency unit (paeu) patients have a malaria parasitepositive blood slide; malaria presentations to the paeu peak from december to may. patients aged 3 months to 14 years presenting during surveillance hours (weekdays, from 8:00 am to 1:00 pm) from january 2011 through december 2014 were screened. consecutive patients fulfilling the sari case definition were recruited (maximum, 5 per day). demographic and clinical data were captured through an electronic data collection system [15] . nasopharyngeal aspirates (npas) were obtained and tested for influenza viruses; from 2011 to 2013, npas were also tested by multiplex assay for respiratory pathogens. thick blood films for detection of malaria parasites were performed for all children. sari was defined as (1) an acute illness with symptom onset <7 days and (2) a reported or recorded fever of ≥38°c (or hypothermia in children <6 months). additional criteria for sari varied by age. in children aged <6 months, additional criteria were (3) cough or apnea or (4) any respiratory symptom requiring hospitalization. in children aged 6-59 months, an additional criterion was (3) clinician-diagnosed lower respiratory infection. in children aged 6-14 years, additional criteria were (3) cough or sore throat and (4) shortness of breath or difficulty breathing. sari with warning signs was considered clinically more severe and defined as the occurrence of one of the following: admission to the hospital, chest recession, or blood oxygen saturation of ≤90%. in this resource-limited setting, some patients with severe illness requiring admission were sent home. thus, hospital attendance (not admission) was required for study enrollment. npas were stored at −80°c in universal transport medium (copan, brescia, italy) [16] and tested in batches for influenza viruses by real-time reverse transcription-polymerase chain reaction (rt-pcr). total nucleic acids were extracted from 300-µl aliquots of each specimen with the qiagen biorobot universal system, using the qiaamp one-for-all nucleic acid kit (qiagen, manchester, united kingdom). the quantity of nucleic acid used per reaction was 5 µl for the centers for disease control and prevention (cdc) human influenza real-time rt-pcr diagnostic panel (cdc influenza division), which detects influenza a and b viruses and influenza a subtypes h1, h3, 2009h1, and h5n1, and 10 µl for the ftd respiratory pathogens 33 kit (fast-track diagnostics, luxembourg). details on sample processing with by ftd real-time rt-pcr are provided in appendix 1. hiv serostatus was assessed by the rapid test (alere determine hiv-1/2 and trinity biotech uni-gold hiv) according to world health organization guidelines [17] . pcr for detection of hiv rna was performed in children aged 3-11 months who had a positive hiv rapid test. hiv infection was defined on the basis of positive results of an hiv rapid test (in the absence of an hiv-negative pcr); data were not collected on hiv exposure. ethics approval for this study was obtained from the liverpool school of tropical medicine research ethics committee (approval reth000790), the university of malawi college of medicine research ethics committee (comrec; approval 958), and the cdc through reliance on the comrec. informed consent was obtained from guardians of all study participants. numerators for minimum sari incidence estimates were generated by summing the number of cases resident in blantyre within strata of age category and hiv status. numerators were adjusted by multiplying by the reciprocal of the daily proportion of recruited cases among all sari cases attending the paeu. denominators for hiv and age strata were derived by applying age-specific hiv prevalence estimates to census figures for blantyre district's population aged 0-14 years [18] . the former were obtained by apportioning the total hiv prevalence among malawian children aged <15 years [14] according to the age distribution of pediatric hiv infections in mozambique, which borders malawi and has a similarly severe hiv epidemic [19, 20] . estimates of age-specific hiv prevalence were unavailable for malawi for the study period. the incidence was obtained by dividing numerators by denominators and multiplying by 10 000; hiv-associated incidence rate ratios (irrs) were calculated by dividing the incidence in hiv-infected strata by the incidence in hiv-uninfected strata; 95% confidence intervals (cis) of incidence and hiv-associated irrs were generated with 1000 bootstrap samples. data analysis was performed using sas 9.3 (sas institute, cary, north carolina). temporal trends in weekly sample counts for sari cases were assessed by plotting 5-week moving averages of sample counts by recruitment week. we developed 2 logistic regression models with a binary outcome factor for the child's clinical status. the first outcome represented sari with warning signs (ie, clinical markers of very severe illness) versus sari without warning signs. the second outcome represented influenza virus-positive sari versus influenza virus-negative sari. autoregressive correlation of residuals was removed by introducing a patient-level kernel weighted moving average of the prior probability of case status. parsimonious models were developed by stepwise logistic regression, retaining age, sex a priori, and explanatory factors with a 2-sided p value of <.05. adjusted relative risk ratios for factors associated with the outcomes were derived from these models. detection of multiple viruses in sari is common, with many possible combinations of viral carriage. conventional statistical techniques (eg, regression models, covariance matrices, and temporal plots) have limited capacity to quantify, characterize or identify factors associated with viral carriage groupings. to assess multiple virus carriage clusters in our setting, we performed nearest-neighbor discrete hierarchical cluster analysis in patients with viral codetection, using the gower distance [21] . distance was based on similarity of viral pathogens detected in the nasopharynx of patients with sari; each patient was a member of only 1 cluster. we defined clusters as those that increased the r 2 value by ≥0.05 (using the ward method); to improve precision, 10% of observations with the lowest densities were discarded. using univariate logistic regression, we identified factors associated with cluster membership. from table 2) . plots of weekly influenza virus-positive sari cases suggest both unimodal and bimodal (2 peaks per year) seasonality. weekly influenza virus-positive sari cases increased during the rainy figure 1 ). incidence estimates for sari and respiratory virus-associated sari sari incidence was 17.5 cases per 10 000 children annually, with the highest incidence in children aged 3-11 months table 5 ), as well as an increased incidence of sari, sari with warning signs, and influenza virus-positive sari (table 4) . hiv-associated irrs rose with increasing age. the hiv-associated irrs for sari with warning signs was 2.6 in children aged 3-11 months as compared to 37.7 in children aged 10-14 years. in children aged >5 years, the incidence of hospital-attended influenza virus-positive sari was at least 8-fold higher in hiv-infected children as compared to hiv-uninfected children. there was no difference in the incidence of rsv-positive sari between hiv-infected and hivuninfected children. in multivariable analysis controlling for etiology, patients with sari recruited during the rainy season (january-april) were more than twice as likely to have warning signs, compared with patients enrolled during september-december (arr, 2.4; 95% ci, 1.6-3.8; table 5 ). peaks in rsv and influenza virus activity corresponded to peaks in the occurrence of sari with warning signs (figure 1 ). detection of rsv in cases of sari warning signs was much higher during the rainy season (39.8%) as compared to other times of year (5.9%). the arr for a positive results of an influenza virus test in patients with sari increased with older age and rainy season of recruitment (table 3 ). after adjustment for age, sex, and hiv status, rainy season recruitment was significantly associated with sari with warning signs in influenza virus-positive patients with sari (arr, 3.42; 95% ci, 1.37-8.53; analysis not shown). in adjusted analysis, a(h1n1)pdm09 was associated with double the risk of sari with warning signs, compared with other influenza virus subtypes (arr, 2.10; 95% ci, .98-4.53; analysis not shown). detection of ≥2 viral pathogens by multiplex pcr occurred in 362 of 1835 sari cases (19.7%). viral codetection was highest in sari cases positive for coronavirus 229 (70.6%) and enterovirus (79.7%). viral codetection was least common in sari cases testing positive for a(h1n1)pdm09 (27.3%), influenza a(h3n2) virus (29.0%), and rsv (29.5%) ( table 2) . viral codetection per se was not associated with warning signs in sari (table 5) . we used discrete hierarchical cluster analysis based on similarity of viral pathogens detected by multiplex pcr assay in sari cases to explore whether particular groupings of viruses were associated with warning signs, host factors, or seasonal factors. we identified 6 clusters, which accounted for 48.3% of the total variation in viral pathogen test results in children with viral codetection. cluster size ranged from 23 to 96 members; smaller clusters had fewer viral pathogens and lower within-cluster heterogeneity. clusters were distinguishable by the type of viral pathogens detected. for example, 80% of influenza a(h3n2) viruses detected were found in cluster a; >65% of bocaviruses detected were found in cluster e (appendix 3). cluster membership was significantly associated with clinical and temporal factors (figure 2 ). among children with viral codetection, membership in cluster d (characterized by abbreviations: ci, confidence interval; irr, hiv-associated incidence rate ratio; rsv, respiratory syncytial virus. a inestimable. hospital-attended sari was common in this urban sub-saharan african setting, particularly in infants aged 3-11 months, in whom the incidence was 91.7 cases per 10 000 children annually. similar to studies from other settings, influenza viruses and rsv were important sari-associated pathogens [5-8, 22, 23] , with prevalence rates of 11% and 12%, respectively. as elsewhere, hiv infection increased the risk of sari and the presence of warning signs in sari cases [24] [25] [26] . among older children, hiv infection greatly increased the risk of influenza virus-positive sari, consistent with data from south africa [25] . viral coinfection occurred in almost 20% of sari cases, highlighting its potential impact in the development or clinical worsening of sari [27] . although viral codetection per se was not associated with clinical severity or season, we found 1 viral cluster, characterized by a high proportion of rsv and a(h1n1)pdm09 infection, which was significantly associated with clinical warning signs and rainy season recruitment. cluster members coinfected with rsv and a(h1n1)pdm09 had a higher rate of warning signs, but the number of coinfected individuals (within the cluster and the entire sample) was too small to formally test for interaction. it is unclear therefore whether clinical severity in this cluster resulted from biological interaction of pathogens, additive risks from each pathogen, or other underlying factors. clusters clearly mapped to peaks and troughs in individual pathogen activity. we suggest that this viral clustering, which was associated temporal dynamics of pathogen activity, may have arisen from complex virus-virus and host-virus pathogen interactions. clinical severity in sari demonstrated seasonal peaks, coinciding with rainy season peaks in rsv activity. rsv was detected in 40% of sari cases with warning signs recruited during the rainy season, compared with 6% recruited at other times of the year. thus, rsv may drive rainy season increases in clinical severity in pediatric sari in our setting, consistent with studies elsewhere in sub-saharan africa [28, 29] . nevertheless, the rainy season remained independently associated with an increased risk of warning signs in sari in figure 2 . dendrogram of coviral clusters. six coviral clusters (a-f) were identified in 362 pediatric sari cases, in whom >2 viral pathogens were detected in the nasopharynx. each severe acute respiratory infection (sari) case is a member of only one cluster; clusters membership is based on similarity of viral pathogens detected. as shown here, characteristics such as sari severity, number of viruses detected per child, and particular season and year of recruitment are more common in some clusters than others. green bars denote sari without warning signs, red bars denote sari with warning signs, bluish-gray bars denote detection of <3 viruses detected, orange bars denote detection of ≥3 viruses, lavender bars denote recruitment in the rainy season, yellow bars denote recruited outside of the rainy season, gray bars denote recruitment in 2011, blue bars denote recruitment in 2012, pink bars denote recruitment in 2013, and light green bars denote recruitment in 2014. multivariable analysis controlling for rsv, hiv, and other viral pathogens. therefore, the observed rainy season excess of clinical severity in sari is in part attributable to unmeasured factors. we speculate that these factors include other intervening illnesses and seasonal malnutrition (in malawi, the rainy season coincides with the so-called lean season after harvest [30] ). however, we cannot exclude seasonal differences in healthcare utilization. we acknowledge that our study has limitations. we did not recruit children aged <3 months, in whom the frequency of sari-related deaths is known to be elevated [31] . we were unable to determine the role of bacterial pathogens in sari, as we lacked laboratory data and systematic radiological data to identify probable infection in the context of a very high prevalence of bacterial carriage. our estimates of sari incidence by hiv strata were based on mozambican pediatric hiv prevalence rates because we lacked data from malawi. nevertheless, malawi and mozambique have similar rates of antenatal hiv prevalence [12, 32, 33] and similarly high rates of hiv-infected pregnant women accessing antiretroviral treatment [34] . we did not assess the impact of hiv exposure on sari risk in hiv-uninfected children. hiv exposure was associated with higher sari incidence and greater sari severity in hiv-uninfected south african children [35] . in conclusion, sari is common in this setting of high hiv prevalence, where influenza viruses, rhinoviruses, and rsv were the most prevalent viruses detected. hiv greatly increased the risk of influenza virus-associated sari in children, and therefore yearly influenza vaccination should be considered in routine pediatric hiv clinical care. influenza vaccination in hiv-infected children is safe, but it has low efficacy (<20%) and may only be immunogenic in older children and adolescents with virological suppression [36] [37] [38] . viral coinfection was common, with 1 coviral cluster associated with clinical severity in sari cases. in this context, there is considerable potential for the use of multiplex respiratory virus assays in tandem with cluster analysis to reveal multiplepathogen-associated outbreaks and disease burden. this approach may expose the potential for synergistic effects of vaccine strategies that disrupt viral clusters. vaccine probe studies to assess the impact of viral coinfection on clinical severity could clarify complex pathogen and host interrelationships and reveal the true burden of disease. global burden of childhood pneumonia and diarrhoea global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis identification of viral and bacterial pathogens from hospitalized children with severe acute respiratory illness in lusaka, zambia incidence of respiratory virus-associated pneumonia in urban poor young children of viral and bacterial causes of severe acute respiratory illness among children aged less than 5 years in a high malaria prevalence area of western kenya viral and bacterial etiology of severe acute respiratory illness among children < 5 years of age without influenza in niger influenza sentinel surveillance among patients with influenza-like-illness and severe acute respiratory illness within the framework of the national reference laboratory severe acute respiratory infection in children in a densely populated urban slum in kenya respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia viral etiology of severe pneumonia among kenyan infants and children do multiple concurrent infections in african children cause irreversible immunological damage? malawi demographic and health survey pandemic influenza a virus subtype h1n1 vaccination in africa-successes and challenges hiv and syphilis sero-survey and national hiv prevalence and aids estimates report for surveillance programme of inpatients and epidemiology (spine): implementation of an electronic data collection tool within a large hospital in malawi universal transport medium rapid hiv tests: guidelines for use in hiv testing and counselling services in resource-constrained settings malawi population and housing census inquérito nacional de prevalência, riscos comportamentais e informação sobre o hiv e sida em moçambique hiv prevalence estimates from the demographic and health surveys metric and euclidean properties of dissimilarity coefficients epidemiology of respiratory syncytial virus infection in rural and urban kenya results from the first six years of national sentinel surveillance for influenza in kenya increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodeficiency virus type-1 severe influenza-associated respiratory infection in high hiv prevalence setting, south africa epidemiology of severe acute respiratory illness (sari) among adults and children aged >/=5 years in a high hiv-prevalence setting mixed respiratory virus infections mortality associated with seasonal and pandemic influenza and respiratory syncytial virus among children <5 years of age in a high hiv prevalence setting-south africa global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis malawi: current issues and what the world food programme is doing global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis mapping hiv prevalence using population and antenatal sentinel-based hiv surveys: a multi-stage approach routine data from prevention of mother-to-child transmission (pmtct) hiv testing not yet ready for hiv surveillance in mozambique: a retrospective analysis of matched test results lessons learned from early implementation of option b+: the elizabeth glaser pediatric aids foundation experience in 11 african countries epidemiology of acute lower respiratory tract infection in hiv-exposed uninfected infants efficacy and immunogenicity of influenza vaccine in hiv-infected children: a randomized, double-blind, placebo controlled trial shedding of live vaccine virus, comparative safety, and influenza-specific antibody responses after administration of live attenuated and inactivated trivalent influenza vaccines to hiv-infected children hiv virological suppression influences response to the as03-adjuvanted monovalent pandemic influenza a h1n1 vaccine in hiv-infected children description of implementation of ftf multiplex assay ftd rrt-pcr assay was used in combination with the agpath one-step qrt-pcr reagents according to the manufacturer's instructions (applied biosystems, carlsad, california, usa) consolidated standards of reporting trials diagram of data analyses. abbreviations: ftd, ftd respiratory pathogens 33 kit; hiv, human immunodeficiency virus sari, severe acute respiratory infection disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention (cdc).financial support. this work was supported by with the cdc through a cooperative agreement (grant 5u01ck000146-04).potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-332396-nattdect authors: ejima, k.; koizumi, y.; yamamoto, n.; rosenberg, m.; ludema, c.; bento, a. i.; yoneoka, d.; ichikawa, s.; mizushima, d.; iwami, s. title: hiv testing by public health centers and municipalities, and new hiv cases during the covid-19 pandemic in japan date: 2020-10-18 journal: nan doi: 10.1101/2020.10.16.20213959 sha: doc_id: 332396 cord_uid: nattdect background: during the covid-19 outbreak, medical resources were primarily allocated to covid-19, which might have reduced facility capacity for hiv testing. further, people may have opted against hiv testing during this period to avoid covid-19 exposure. we investigate the influence of the covid-19 pandemic on hiv testing and its consequences in japan. methods: we analysed quarterly hiv/aids-related data from 2015 to the second quarter of 2020 using an anomaly detection approach. the data included the number of consultations that public health centers received, the number of hiv tests performed by public health centers or municipalities, and the number of newly reported hiv cases with and without aids diagnosis. as sensitivity analyses, we performed the same analysis for two subgroups: men who have sex with men (msm) and non-japanese. findings: the number of hiv tests (9,584 vs. 35,908 in the year-before period) and consultations (11,689 vs. 32,565) performed by public health centers significantly declined in the second quarter of 2020, while the proportion of hiv cases with aids diagnosis among all hiv cases (36.2% vs. 26.4%) significantly increased after removing the trend and seasonality effects. the number of hiv cases without aids diagnosis numerically decreased (166 vs. 217), although the reduction was not significant. we confirmed similar trend for the msm and non-japanese groups. interpretation: the current hiv testing system including public health centers misses more hiv cases at the early phase of the infection during the pandemic. given that the clear epidemiological picture of hiv incidence during the pandemic is still uncertain, continuously monitoring the situation as well as securing sufficient test resources using self-test is essential. evidence before this study 25 before this study, we searched pubmed, medline, and google scholar on oct 12, 2020, for articles 26 investigated the number of hiv test and hiv cases during the covid-19 pandemic in japan, using the 27 search terms "novel coronavirus" or "sars-cov-2", and "hiv" or "aids", and "japan", with no time 28 restrictions. we found no published work relevant to our study. 29 during the covid-19 pandemic in japan, the public health centers and municipalities temporarily 31 suspended facility-based hiv testing to concentrate their limited resources to covid-19 testing. we 32 investigated the impact of the covid-19 pandemic on the number of hiv tests in public health centers and 33 municipalities, and on the number of hiv cases with and without aids diagnosis. we confirmed that the 34 number of the test declined in the second quarter (april to june) of 2020, and the proportion of hiv with 35 aids diagnosis among all hiv cases increased during the same period. 36 providing sufficient hiv testing opportunities even during the pandemic, when facility-based testing is 38 challenging, is necessary for better clinical and public health outcomes. self-testing and home specimen 39 collection (e.g. dried blood spot or oral fluid test) could be a key to fill the gap between the need for hiv 40 testing and the constraints related to the covid-19 outbreak. 41 42 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 18, 2020 . . https://doi.org/10.1101 /2020 testing and its consequences in japan. 48 we analysed quarterly hiv/aids-related data from 2015 to the second quarter of 2020 using an anomaly 50 detection approach. the data included the number of consultations that public health centers received, the 51 number of hiv tests performed by public health centers or municipalities, and the number of newly reported 52 hiv cases with and without aids diagnosis. as sensitivity analyses, we performed the same analysis for 53 two subgroups: men who have sex with men (msm) and non-japanese. 54 the number of hiv tests (9,584 vs. 35,908 in the year-before period) and consultations (11,689 vs. 32,565) 56 performed by public health centers significantly declined in the second quarter of 2020, while the proportion 57 of hiv cases with aids diagnosis among all hiv cases (36·2% vs. 26·4%) significantly increased after 58 removing the trend and seasonality effects. the number of hiv cases without aids diagnosis numerically 59 decreased (166 vs. 217), although the reduction was not significant. we confirmed similar trend for the 60 msm and non-japanese groups. 61 the current hiv testing system including public health centers misses more hiv cases at the early phase of 63 the infection during the pandemic. given that the clear epidemiological picture of hiv incidence during the 64 pandemic is still uncertain, continuously monitoring the situation as well as securing sufficient test resources 65 using self-test is essential. 66 japan society for the promotion of science, japan science and technology agency, japan agency for 68 medical research and development. 69 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 18, 2020 . . https://doi.org/10.1101 /2020 introduction 72 the ongoing covid-19 pandemic has had broad influences on lifestyle and health issues beyond covid-73 19. there have been positive impacts, such as a curtailed 2019/2020 influenza season in japan (1), which 74 could be partially explained by measures taken to constrain the covid-19 outbreak. due to reduced 75 economic activity and human mobility during lockdown, air pollution, which is a risk factor of pulmonary 76 diseases, was temporally improved in china (2). however, there are many problems caused by risk 77 mitigating interventions for covid-19, many of which are under investigation. fear and anxiety about this 78 new disease and changed lifestyle have been stressful for many people, which has worsened mental health 79 (3) (4) (5) . 80 further, during this pandemic period, non-covid-19 diseases have been at a lower priority for treatment 81 because medical resources have been disproportionately allocated to treat covid-19 patients in many 82 health care facilities and the capacity to care for non-covid-19 disease has been limited. moreover, health 83 care facilities need to balance the benefits of providing care for non-critical conditions with minimizing the 84 risk of covid-19 infections for both health care professionals and patients (6). 85 people living with hiv infections have better outcomes with early diagnosis and connection to antiretroviral 86 treatment. early diagnosis is also key to preventing onward transmission because early infection is 87 characterized by high viral load and receiving an hiv diagnosis facilitates behavioural change. however, 88 lagat et al. reported a decline in hiv test volume in kenya due to many barriers related to covid-19, such 89 as lack of funds to visit clinics and fear of covid-19 infection at clinics (7). 90 to increase hiv testing opportunities, in the united states the cdc is recommending self-testing of hiv or 91 home specimen collection (e.g. dried blood spot or oral fluid test) to avoid covid-19 infection risk 92 associated with face-to-face testing service (8). in japan, the number of self-collection-based hiv tests has 93 increased, though the majority of tests are still conducted at public health centers and clinics (i.e., facility-94 based testing). however, during the pandemic, public health centers and clinics have been overwhelmed by 95 covid-19 pcr testing and administrative work and many have temporally suspended or limited hiv 96 testing. 97 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 18, 2020. . https://doi.org/10.1101/2020.10.16.20213959 doi: medrxiv preprint 6 aids is usually diagnosed by the specific symptoms and the average incubation period is 10 years (9, 10). 98 therefore, the impact of the pandemic on the number of reported hiv cases with aids diagnosis will not be 99 substantial in a short-term period (< 1 year). in contrast, the median time interval between hiv infection to 100 the diagnosis without aids symptoms is 1 ·0 years in tokyo, japan (11) without aids diagnosis in japan. we further extracted the data of hiv cases with and without aids 116 diagnosis for the following two subgroups: men who have sex with men (msm) and non-japanese, because 117 msm accounts for about 70% of the newly reported hiv cases (12), and foreigners are considered 118 vulnerable during disasters due to language barriers (13). the data on the number of tests and consultations 119 for these subgroups were not available. 120 note that hiv cases with aids diagnosis here represent new cases of hiv identified at late-stages, 121 compatible with an aids diagnosis. thus, this number does not include hiv cases with aids diagnosis that 122 were previously diagnosed and progressed to aids. to rephrase, the new hiv cases here are divided into 123 two categories: early diagnosis (hiv cases without aids diagnoses) and late diagnosis (hiv cases with 124 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 18, 2020. . https://doi.org/10.1101/2020.10.16.20213959 doi: medrxiv preprint 7 aids diagnoses). hiv tests at clinics/hospitals and self-testing are also available in japan. thus, the number 125 of hiv testing performed by public health centers or municipalities was used to examine the trend of 126 facility-based tests, rather than capturing the total number of hiv tests or examining the proportion of hiv 127 positive cases among all the hiv tests. 128 we applied an anomaly detection approach to those longitudinal data to identify the period when the number figure 1 shows the observed data points and the computed normal range (the shaded area). we observed a 144 significant downward trend in hiv cases with or without aids diagnosis (figure 1ab) , all the hiv cases 145 (figure 1c) , and the number of consultations (figure 1e ) over the period examined (from the first quarter 146 of 2015 to the second quarter of 2020). the trend in number of tests was not significant during the period 147 ( figure 1d) . the proportion of hiv cases with aids diagnosis among all the hiv cases showed a 148 significant downward trend during the period (figure 1f) , suggesting that more hiv cases have been 149 diagnosed in the early phase of infection recently. regarding anomaly by the last quarter of 2019 (i.e., 150 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 18, 2020. . https://doi.org/10.1101/2020.10.16.20213959 doi: medrxiv preprint 8 before the covid-19 pandemic), there are a couple of data points that significantly deviated from the 151 normal range. however, the magnitude of the deviation of those numbers was modest. 152 we did not observe deviation in any numbers in the first quarter of 2020. however, in the second quarter, 153 the numbers of hiv tests and hiv consultations significantly declined (figure 1de) , which is compatible 154 with a public health infrastructure overwhelmed by covid-19 related work. in contrast, the proportion of 155 hiv cases with aids diagnosis among all hiv cases significantly increased ( figure 1f) . intriguingly, the 156 number of hiv cases with aids diagnosis in the second quarter of 2020 showed the same trend as before 157 ( figure 1b) . the number of hiv cases without aids diagnosis numerically decreased in the second quarter 158 of 2020 (figure 1a) , however it was not significant. it is worth noting that the number of hiv cases without 159 aids diagnosis did not decline as much as the number of tests (the former dropped 23.5%, whereas the 160 latter dropped 73.3% compared with the year-before period). this might be because more hiv cases with 161 acute symptoms (i.e., fever) visited clinics for viral test for sars-cov-2 and hiv infection was 162 subsequently diagnosed. 163 as a sensitivity analysis, we examined the longitudinal data of hiv cases with and without aids diagnosis 164 of two subgroups: men who have sex with men (msm) and non-japanese. we confirmed the same 165 downward trend for the msm population as for the total population (figure 2a-c) in terms of newly 166 reported hiv cases with and without aids diagnosis. on contrary, the hiv cases without aids diagnosis 167 and all hiv cases among non-japanese were in upward trend ( figure 3ac ). the number of hiv cases 168 without aids diagnosis numerically decreased, whereas hiv cases with aids diagnosis numerically 169 increased or were in the same trend as before in these two populations in the second quarter of 2020 ( figure 170 2ab and 3ab). as a result, the proportion of hiv cases with aids diagnosis among all positive cases also 171 numerically increased in the second quarter of 2020 in these two subgroups (figure 2d and 3d) ; however, 172 they were not significant, which might be due to the small sample size. the proportion of hiv cases among 173 msm and non-japanese populations relative to total hiv cases was consistent over time including the 174 second quarter of 2020 (figure 2e and 3e) . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 18, 2020. we need to interpret the results carefully. first, we have analyzed the number of tests performed by public 188 health centers and municipalities. however, this type of test accounts for less than half of the tests 189 performed in japan. for example, self-collection-based tests (mostly using dried blood spot) are increasing, 190 and tests are available in clinics and hospitals; however, the count of those tests was not available. it is 191 probable that the number of different types of hiv test has increased during the same period as 192 compensation for the reduction in the number of tests performed by public health centers and municipalities. 193 second, we do not know the full picture of the impact of the pandemic on hiv epidemiology. we have used 194 the number of reported hiv cases; however, there is likely a substantial proportion of undiagnosed cases, 195 and it is uncertain whether the number and the proportion of undiagnosed cases changed during the 196 pandemic. it is possible that the incidence increased (or decreased) during the pandemic. therefore, even 197 though the number of new hiv cases did not change dramatically, it does not indicate that the number of 198 tests is sufficient to identify all hiv patients. indeed, the number of reported hiv positive cases without 199 progressed to aids in the second quarter of 2020 was the lowest in the last five years and the proportion of 200 hiv cases with aids diagnosis significantly increased. it raises a concern that the testing opportunities 201 might not be sufficient to fully capture the epidemiological situation of hiv/aids during the pandemic in 202 japan. because most of the hiv cases with aids diagnosis are identified in clinics or hospitals due to 203 specific symptoms, hiv cases with aids diagnosis are less prone to being affected by healthcare avoidance 204 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 18, 2020. . https://doi.org/10.1101/2020.10.16.20213959 doi: medrxiv preprint even under the pandemic. indeed, the number of hiv cases with aids diagnosis was in the same trend as 205 before the pandemic. we need to keep monitoring the situation as well as adapting testing strategies to work 206 in these unusual circumstances. 207 as the pandemic continues, we do not know how long the hiv testing opportunities provided by public 208 health centers could be disrupted and how it could affect hiv spread. given that early detection and 209 treatment initiation for hiv is lifesaving, providing sufficient hiv testing opportunities is important. 210 unaids has a proposed treatment target, "90-90-90" to control the hiv epidemic (17). a part of the target 211 is that "by 2020, 90% of all people living with hiv will know their hiv status." if the covid-19 212 pandemic continuously disrupts testing opportunities, diagnosing 90% of people living with hiv might be 213 difficult to achieve. self-testing and home specimen collection could be a key to fill the gap between the 214 need for hiv testing and the constraints related to the covid-19 outbreak. 215 the strength of this study is that we used the quarterly hiv/aids reports in japan, which include all the 216 diagnosed cases as physicians are required to report all diagnosed cases under the infectious diseases 217 control law(18). further, the cases reported as hiv cases with aids diagnosis include only the cases of 218 newly diagnosed hiv infection co-occurring with aids. therefore, we could quantify the proportion of 219 patients presenting with aids at the time of diagnosis, which captures late-presentation for diagnosis and is 220 more likely if testing resources are not accessible. 221 a limitation of this study is that we were not able to account for the heterogeneous epidemiology of hiv 222 between different regions. higher hiv incidence has been observed in urban areas such as tokyo and osaka 223 compared with rural areas. thus, public health centers in urban areas have allocated budgetary resources 224 towards hiv testing, whereas this has not been prioritized in rural areas. during the pandemic, the testing 225 opportunities in rural areas may have declined even further than urban areas. our study mainly focused on 226 hiv cases with and without aids diagnosis and testing regardless of the patients' characteristics. one of 227 the concerns in this pandemic is that more vulnerable populations, such as sex workers, low-income 228 populations, younger population, in addition to foreigners, may have experienced disproportionate harms. 229 further studies should focus on these populations and identify barriers to testing. we used only the number 230 of tests performed by public health centers and municipalities, thus self-tests and home specimen collection, 231 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 18, 2020. . https://doi.org/10. 1101 /2020 which are increasingly popular, and tests performed in clinics and hospitals were not counted. further 232 investigation is necessary to focus on these types of tests to fully understand the response of the hiv cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 18, 2020. range. the observed data outside of the normal rage is considered abnormal and red colored. 307 308 309 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 18, 2020 . . https://doi.org/10.1101 /2020 seasonal influenza activity during the sars-cov-2 outbreak in 259 japan the short-term impacts of covid-19 lockdown on urban air pollution in 261 china how mental health care 263 should change as a consequence of the covid-19 pandemic. the lancet psychiatry mental health and the covid-19 pandemic depression and loneliness during 266 covid-19 restrictions in the united states, and their associations with frequency of social and sexual 267 connections centers for disease control and prevention. hiv self testing guidance 2020 incubation period of aids in san francisco the incubation period of aids estimating hiv-1 280 incidence in japan from the proportion of recent infections disproportionate impact of the 284 covid-19 pandemic on immigrant communities in the united states stl: a seasonal-trend decomposition procedure 287 based on loess non parametric statistics for the behavioral sciences percentage points for a generalized esd many-outlier procedure overview of 295 infectious disease surveillance system in japan international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted the copyright holder for this preprint this version posted october 18, 2020. . https: //doi.org/10.1101//doi.org/10. /2020 the authors declare that they have no competing interests. 256 257 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review)the copyright holder for this preprint this version posted october 18, 2020 . . https://doi.org/10.1101 /2020 key: cord-328287-3qgzulgj authors: moni, mohammad ali; liò, pietro title: network-based analysis of comorbidities risk during an infection: sars and hiv case studies date: 2014-10-24 journal: bmc bioinformatics doi: 10.1186/1471-2105-15-333 sha: doc_id: 328287 cord_uid: 3qgzulgj background: infections are often associated to comorbidity that increases the risk of medical conditions which can lead to further morbidity and mortality. sars is a threat which is similar to mers virus, but the comorbidity is the key aspect to underline their different impacts. one uk doctor says "i’d rather have hiv than diabetes" as life expectancy among diabetes patients is lower than that of hiv. however, hiv has a comorbidity impact on the diabetes. results: we present a quantitative framework to compare and explore comorbidity between diseases. by using neighbourhood based benchmark and topological methods, we have built comorbidity relationships network based on the omim and our identified significant genes. then based on the gene expression, ppi and signalling pathways data, we investigate the comorbidity association of these 2 infective pathologies with other 7 diseases (heart failure, kidney disorder, breast cancer, neurodegenerative disorders, bone diseases, type 1 and type 2 diabetes). phenotypic association is measured by calculating both the relative risk as the quantified measures of comorbidity tendency of two disease pairs and the ϕ-correlation to measure the robustness of the comorbidity associations. the differential gene expression profiling strongly suggests that the response of sars affected patients seems to be mainly an innate inflammatory response and statistically dysregulates a large number of genes, pathways and ppis subnetworks in different pathologies such as chronic heart failure (21 genes), breast cancer (16 genes) and bone diseases (11 genes). hiv-1 induces comorbidities relationship with many other diseases, particularly strong correlation with the neurological, cancer, metabolic and immunological diseases. similar comorbidities risk is observed from the clinical information. moreover, sars and hiv infections dysregulate 4 genes (anxa3, gns, hist1h1c, rasa3) and 3 genes (hba1, tfrc, ghitm) respectively that affect the ageing process. it is notable that hiv and sars similarly dysregulated 11 genes and 3 pathways. only 4 significantly dysregulated genes are common between sars-cov and mers-cov, including nfkbia that is a key regulator of immune responsiveness implicated in susceptibility to infectious and inflammatory diseases. conclusions: our method presents a ripe opportunity to use data-driven approaches for advancing our current knowledge on disease mechanism and predicting disease comorbidities in a quantitative way. electronic supplementary material: the online version of this article (doi:10.1186/1471-2105-15-333) contains supplementary material, which is available to authorized users. the term "comorbidity" refers to the coexistence of multiple diseases or disorders in relation to a primary disease or disorder in an individual [1] . a comorbidity relationship between two diseases exists whenever they appear simultaneously in a patient more than chance alone [2] . it represents the co-occurrence of diseases or presence http://www.biomedcentral.com/1471-2105/ 15/333 to chronic obstructive pulmonary disease (copd) [6, 7] , obesity [8] , mental disorders [9] , immune-related diseases [10] , cancer [11] etc. comorbidity can be attributed to the disease connections on the molecular level, such as dysregulated genes, ppis (protein-protein interactions), and metabolic pathways as potential causes of comorbidity [1, 3, 12, 13] . from a genetic perspective, a pair of diseases is connected because they have both been associated with the same dysregulated genes [14, 15] , whereas from a proteomics perspective phenotypically similar diseases are related via biological modules such as ppis or molecular pathways [16, 17] . population-based disease association is important in conjunction with molecular and genetic data to uncover the molecular origins of diseases and disease comorbidities. patient medical records contain important clarification regarding the co-occurrences of diseases affecting the same patient [2] . during the last few years, several researchers have been conducted the disease comorbidity analysis to understand the origins of many diseases [1, 12, 18] . goh, cusick, valle, childs, vidal, barabasi et al. and feldman, rzhetsky, vitkup et al. built networks of gene-disease associations by connecting diseases that have been associated with the same genes [14, 15] , whereas lee, park, kay, christakis, oltvai and barabási et al. constructed a network in which two diseases are linked if metabolic reactions are associated between them [13] . disease association studies from proteomic point of view have been studied by rual, venkatesan, hao, hirozane-kishikawa, dricot, li, berriz, gibbons, dreze, ayivi-guedehoussou et al. and stelzl, worm, lalowski, haenig, brembeck, goehler, stroedicke, zenkner, schoenherr, koeppen et al. [19, 20] . rzhetsky, wajngurt, park and zheng et al. inferred the comorbidity links between 161 disorders from the disease history of 1.5 million patients [12] . however, all of these efforts have focused on the role of a single molecular or phenotypic measure to capture disease-disease relationships. in our work we have used disease-gene associations, ppis, molecular pathways and clinical information to obtain statistically significant associations and comorbidity risks among diseases. inflammation is a hallmark of many serious human infectious diseases associated to a wide variety of infections, such as hiv-1 [21] . uk doctor max pemberton says "i'd rather have hiv than diabetes" as life expectancy among diabetes patients is lower than that of hiv [22] . however, hiv has a comorbidity impact on the diabetes. also the flu can cause complications, including bacterial pneumonia, or the worsening of chronic health problems. asthma is the most common comorbidity in patients hospitalized for swine influenza (h1n1) infection [23] . dengue can cause myocardial impairment, arrhythmias and, occasionally, fulminant myocarditis [24] . chronic medical conditions, such as heart disease, lung disease, diabetes, renal disease, rheumatologic disease, dementia, and stroke are risk factors for influenza complications [25] . common chronic infections such as periodontitis or infection with helicobacter pylori may also increase stroke risk [26] . moreover, the severity of pneumonia in patients coinfected with influenza virus and bacteria is significantly higher than in those infected with bacteria alone. the incidence of flu is higher in children and younger adults than in older individuals, but influenzaassociated morbidity and mortality increase with age, especially for individuals with underlying medical conditions such as chronic cardiovascular diseases [27] . during the ageing process the immune system becomes compromised and it causes an increasing inflammation [28] . in particular, chronic inflammation (inflammageing) and metabolic function are strongly affected by the ageing process [29] . the ageing of populations is leading to an unprecedented increase different diseases like cancer and fatalities. it is reported that 80% of the elderly population has three or more chronic conditions [30] . on the other hand, respiratory viruses are an emerging threat to global health security and have led to worldwide epidemics with substantial morbidity and mortality [31] . coronaviruses (covs) cause respiratory and enteric diseases in human and other animals that induce fatal respiratory, gastrointestinal and neurological disease. severe acute respiratory syndrome (sars) is an epidemic human disease, is caused by a coronavirus (cov), called sarsassociated coronavirus (sars-cov) [32] . sars patients may present with a spectrum of disease severity ranging from flu-like symptoms and viral pneumonia to acute respiratory distress syndrome and death [33] . most of the deaths were attributed to complications related to sepsis, ards and multiorgan failure, which occurred commonly in the elderly for comorbidities [34] . age and comorbidity (e.g. diabetes mellitus, heart disease) were consistently found to be significant independent predictors of various adverse outcomes in sars [35] . children with sars have better prognosis than adults [34] . advanced age and comorbidities were significantly associated with increased risk of sars-cov related death, due to acute respiratory distress syndrome [35] . mild degree of anaemia is common in the sars infected patients and patients who have recovered from sars show symptoms of psychological trauma [34] . another novel coronavirus mers-cov, which is a new threat for public health, has similar clinical characteristics to sars-cov, but the comorbidity is the key aspect to underline their different impacts [36, 37] . mers-cov causes respiratory infections of varying severity and sometimes fatal infections in humans including kidney failure and severe acute pneumonia [38] . despite sharing some clinical similarities with sars (eg, fever, http://www.biomedcentral.com/1471-2105/15/333 cough, incubation period), there are also some important differences such as the rapid progression to respiratory failure, which we have studied on comorbidities point of view. infection with the human immunodeficiency virus-1 (hiv) and the resulting acquired immune deficiency syndrome (aids) affects cellular immune regulation [39] . hiv infection severely impacts on the immune system causing phenotypic changes in peripheral cells and dysregulates the innate immune system [40] . significant number of hiv-1 infected patients exhibits osteopenia and osteoporosis, leading to higher incidence to develop weak and fragile bones during the course of disease [41] . hiv has also been associated with an increased risk of developing both diabetes and cardiovascular disease [42] . infection with hiv weakens the immune system and reduces the body's ability to fight infections that may lead to cancer [43, 44] . people infected with human immunodeficiency virus (hiv) have a higher risk of some types of cancer (kaposi sarcoma, non-hodgkin lymphoma, cervical cancer, anal, liver, lung cancer, and hodgkin lymphoma) than uninfected people [45] . many people infected with hiv are also infected with other viruses that cause certain cancers [46, 47] . hiv infection even when controlled by highly active antiretroviral therapy (haart) is being linked to chronic inflammation [48] . people with hiv-1 infection appear to have a markedly higher rate of chronic kidney disease than the general public [49] . it is because some of the risk factors associated with hiv-1 acquisition are the same as those that lead to kidney disease because of the virus itself and some therapies (e.g. haart therapy). antiretroviral therapy for hiv may increase the risk of developing metabolic syndrome (abdominal obesity, hyperglycaemia, dyslipidaemia and hypertension) and thus predispose to type 2 diabetes and cardiovascular disease. many of the biologic factors thought to be causally associated with inflammation in hiv disease are also thought to be causally associated with the inflammation of ageing [50] . infections (acute and chronic conditions) are often associated to comorbidity that increases the risk of medical conditions which can lead to further morbidity and mortality. comorbidities related to flu have been recently investigated [51] . comorbidities for tuberculosis have also been studied recently [52, 53] . to understand the overall mechanism we have studied the comorbidity associations of sars and hiv infections. both hiv and sars are emerging infectious diseases in the modern world; each of these diseases has caused global societal and economic impact related to unexpected illnesses and deaths [54] . sras is a significant public health threat and hiv is a long term chronic infection. since these two infections are associated with high mortality rates and there are no clinically approved antiviral treatments or vaccines available for either of these infections, we have selected these two infections for our study. centred on the sars and hiv-1 infections we have investigated highly heterogeneous disease comorbidity networks using the disease-gene associations, ppi subnetwork, molecular pathways and clinical information. we have presented a systematic and quantitative approach to discover human disease comorbidities using different sources of available mrna expression, protein-protein interactions, signalling pathways, disease-gene associations, disease-disease associations and disease-drug associations data. it has been shown that sars coronavirus infects and replicates in a wide variety of host cells in susceptible animals and human beings [55, 56] . to understand the host response to this pathogen, we analysed the gene expression patterns of sars infected patients, compared to normal subjects using oligonucleotide microarrays from the ncbi geo (http://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse1739) [55] . we analysed the microarray gene expression data of over 8,700 genes from the pbmcs of 10 sars patients, and compared with healthy control samples. we found that 274 genes (p < 0.01, > 1.5 fold change) were differentially expressed as compared to healthy controls in which 120 genes were significantly up regulated and 154 genes were significantly down regulated (see additional file 1: table s1 ). on the other hand, monocytes are the key immune responsive cells whose function is adversely impacted by hiv-1. hiv-1 infection radically alters the monocyte phenotype, which is reflected in an hiv-1 induced gene expression analysis. monocyte gene expression microarray data were collected for control and hiv patients from the ncbi geo (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=gse18464) [57] . to find out the significant dysregulated genes during the hiv-1 infection, we have performed global gene expression analysis. we found that 186 genes (p < 0.01, > 1.5 fold change) were differentially expressed as compared to healthy controls in which 71 genes were up regulated and 115 genes were down regulated (see additional file 2: table s2 ). considering the significantly dysregulated genes of sars (274 genes) and hiv-1 (186 genes) infections, and gene-disease associations information, we have constructed two gene-disease associations networks (gdn), which are used to explore the shared genetic associations and disease comorbidity. starting from the bipartite graph we generated biologically relevant network projections and constructed multi-relational gene-disease network in which nodes are diseases or genes, and edges indicate association between gene and disease. this bipartite http://www.biomedcentral.com/1471-2105/15/333 graph consists of two disjoint sets of nodes, where one set corresponds to all known genetic disorders and the other set corresponds to all of our identified significant genes for sars and hiv-1 infections. the list of disorders, disease genes, and associations between them were obtained from the online mendelian inheritance in man (omim) [58] , a compendium of human disease genes and phenotypes (see details in the methods section). we classified each disorder into one of 21 disorder categories based on the physiological system affected as introduced in goh, cusick, valle, childs, vidal, barabasi et al. [14] . in the gdn, nodes represent diseases class or genes, and two disorders are connected to each other if they share at least one gene in which mutations are associated with both diseases groups (figures 1 and 2 ). the number of interlinked genes between sars infection and other diseases indicates that immunological, hematological, neurological, metabolic and dermatological diseases categories are strongly associated with the sars infection (see figure 1 and additional file 3: table s3 ). few genes are also shared between more than 2 categories of diseases i.e those disease groups are also associated through at least that genes. for an instance, the gene atm shared among sars infection, cancer and immunological diseases. therefore, cancer and immunological diseases are also interrelated through the gene atm. among all these disease classes immunological diseases class is tightly correlated with the sars infection due to the highest number of genes (12 genes) shared between them. on the other hand, the number of associated genes between hiv infection and other diseases indicates that neurological, metabolic, cancer and hematological diseases categories are strongly correlated with the hiv infection (see figure 2 and additional file 4: figure 1 the gene-disease association network centred on the sars infection is constructed based on the different categories of diseases that are connected and showed comorbidities with the sars infection through the different genes. red colour represents different categories of disorders and green colour represents different genes that are common with the other categories of disorders. the size of a disease node is proportional to the number of dysregulated genes shared between the infections/disorder groups. a link is placed between a disorder and a disease gene if mutations in that gene lead to the specific disorder. http://www.biomedcentral.com/1471-2105/15/333 figure 2 the gene-disease association network centred on the hiv infection is constructed based on the different categories of diseases that are connected and showed comorbidities with the hiv-1 infection through the different genes. red colour represents different categories of disorders and green colour represents different genes that are common with the other categories of disorders. the size of a disease node is proportional to the number of dysregulated genes shared between the infections/disorder groups. a link is placed between a disorder and a disease gene if mutations in that gene lead to the specific disorder. table s4 ). few hiv dysregulated genes are also shared between more than 2 categories of diseases such as the gene tgfb1 is shared among hiv infection, cancer and skeletal diseases. it is notable that 11 significant genes (4 upregulated and 7 downregulated) are similarly dysregulated in the both sars and hiv infections. to observe the association of sars and hiv infections with other 7 important diseases (chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, type 1 and type 2 diabetes), we have collected mrna microarray raw data associated with each disease from the gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo/) accession numbers are gse9006, gse9128, gse15072, gse7158, gse8977 and gse7621 [59] . after several steps of statistical analysis we have selected the most significant over and under expressed genes for each infection and disease. we also performed cross compare analysis to find the common significant genes between each disease and sars/ hiv-1 infection. we observed that sars infection shares 21, 12, 16, 5, 11, 11, 11 and 13 genes corresponding to the chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, hiv-1 infection, type 1 and type 2 diabetes. on the other hand, hiv-1 infection shares 11, 10, 17, 9, 7, 11, 9 and 7 genes corresponding to the chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, sars infection, type 1 and type 2 diabetes. then we built disease-disease relationships network for sars and hiv-1 infection with other diseases (see figures 3 (a) and (b) and additional file 5: table s5 and additional file 6: table s6 ). since genes do not function alone and they coordinate their activities in the form of complexes or molecular pathways. therefore two diseases are potentially inter-correlated to each other if they share at least one commonly associated pathway. for this http://www.biomedcentral.com/1471-2105/15/333 reason we have used reactome pathway database [60] and selected the pathways related to these 7 diseases as well as sars and hiv-1 infections. we have observed that diseases and infections shared pathways between them as shown in figures 3 (a) and (b) and additional file 5: table s5 and additional file 6: table s6 . dysregulation in a protein subnetwork may yield the dysfunction of multiple protein subnetworks. therefore, multiple diseases may be caused by the malfunction of a protein complex. so, two diseases are potentially related to each other if they share one or more commonly associated protein subnetwork. to identify the association between diseases based on the ppi subnetwork, we used significantly associated disease protein pairs data from the hprd data base [61] . to find statistically significant associations among diseases, we built disease networks centred on the sars and hiv infections in which two diseases are comorbid if there exists one or more protein subnetwork that are associated with both diseases. the disease similarity network and the protein-protein interaction network are integrated systematically and comprehensively in a simple and compact manner to formulate the disease comorbidity for the sars and hiv-1 infections as shown in figures 4 and 5. we showed that sars and hiv infections shared ppi subnetworks with the other 7 diseases or infections similar to the gene-disease and pathway-disease associations as shown in figures 4 and 5 . based on the gene expression, protein-protein interaction and molecular pathways data, we have found that both sars and hiv-1 infections have a strong association with other 8 diseases or infections (chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, hiv/sars infection, type 1 and type 2 diabetes). these diseases and infections are also strongly correlated among them. we present the correlation strength and distance between a pair of these diseases and infections in figure 6 . we show that some diseases (such as kidney disorders, breast cancer, osteoporosis and heart failure) are more associated with the sars infection (see figure 6 ). kidney disorder is also tightly connected with the hiv-1 infection. the probability of occurring comorbidities between the more tightly connected diseases is more than that of others. it is notable that the patient medical records contain important evidence regarding the co-occurrences of diseases affecting the same patient. so, we constructed a phenotypic disease comorbidity network using 32 million medical records of 13039018 patients data from medpar and analysed its structural properties to better understand the connections among diseases and infections. nodes are unique diseases and edges indicate co-morbidity of the diseases. we included edges between disease pairs for which the co-occurrence is significantly greater than the random expectation based on population prevalence of the diseases. as pointed out in [2] , the relative risk (rr ij ) overestimates relations involving rare infections and diseases, and underestimates relationships between very common disorders or infections. on the other hand, φ-correlation underestimates comorbidity between rare and frequent diseases, and discriminates associations between disorders of similar appearances. thus, we built a network by selecting only the statistically significant network edges having rr ij ≥ 20 and φ ij ≥ 0.06. figure 7 summarises the set of all comorbidity associations among all diseases expressed in the study population by constructing a phenotypic disease network (pdn). in the pdn, nodes are disease phenotypes identified by unique icd-9-cm (the international classification of diseases) disease codes, and links connect phenotypes that show significant comorbidity according to the relative risk rr ij ≥ 20 and the correlation φ ij ≥ 0.06. our phenotypic disease network consists of 336 unique diseases nodes and 1018 co-morbidity relationships. sars-associated coronavirus icd-9-cm diagnosis code is 079.82, which is under the group of "viral and chlamydial infection in conditions classified elsewhere and of unspecified site" and icd-9-cm diagnosis code 079. moreover, the icd-9-cm code 480.3 is for the pneumonia due to sars associated coronavirus. so we have considered both icd-9-cm codes 079.82 and 480.3 for our phenotypic sars comorbidity study. in our 3 digit code data we have considered 079 and for 5 digit code data we have considered 480.3. considering the relative risk rr ij ≥ 10 between the disease group 079 and other disorder categories, we have constructed the pdn as shown in figure 8 (a), and considering the relative risk rr ij ≥ 20 between the disease group 480.3 and other disorder categories, we have constructed the pdn as shown in figure 8 (b). we presented only the most significant relative risk associations (see additional file 7: table s7 and additional file 8: table s8 ). the icd-9-cm diagnosis code for the human immunodeficiency virus (hiv) infection is 042 to 044, which is under the group of "infectious and parasitic diseases" and icd-9-cm code (001-139). so we have considered both 3 digit and 5 digit icd-9-cm codes for our phenotypic comorbidity studies related to hiv infection. considering the relative risk rr ij ≥ 20 between the disease group 042 and other disorder categories, we have constructed the pdn as shown in figure 9 (a) and considering the relative risk rr ij ≥ 100 and φ-correlation φ ij ≥ 0.06 between the disease groups under the sub categories of 042 and other disorder categories, we have constructed the pdn as shown in figure 9 (b). only the most significant relative risk association is represented (see additional file 9: table s9 and additional file 10: table s10 ). to observe the trend of phenotypic relative risk corresponding to the number of shared genes between 2 http://www.biomedcentral.com/1471-2105/15/333 diseases, we have computed the number of shared genes between two diseases and their corresponding phenotypic relative risk of the occurrence of comorbidities as shown in figure 10 . we observed that with increasing number of shared biomarker genes between 2 diseases, the phenotypic relative risk is also increased. we may predict existing diseases of a patient and the prospective disease comorbidities through the identification of highly up and down dysregulated genes. so based on the available data we could predict the disease comorbidities and the level of the comorbidities using the regression model as figure 10 . it is notable that ageing is also a "disease", not a natural process, for which age-related diseases increase exponentially with chronological time. so, to understand the impact of ageing on the disease comorbidities for sars and hiv infections we have considered the ageing data from the genage database (http://genomics.senescence. info/genes/human.html) [62, 63] . after cross comparing http://www.biomedcentral.com/1471-2105/15/333 human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (sars-cov) [32] . thus, a comprehensive evaluation of the complex epithelial signalling to sars-cov is crucial to better understand sars pathogenesis. since both of the sars-cov and mers-cov infections cause severe lung pathology we compare and contrast the genes expression level of sars-cov infection and mers-cov infection. to compare between sars-cov and mers-cov infections, and the affect on the disease comorbidities, we have performed the time series microarray data analysis for the both types of infections on lung compared to controls. we have considered gene expression microarray data from the ncbi geo (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=gse45042) [64] . from the analysis of sars-cov vs mock-infected controls (treated the same way except without the virus) we have found 215 genes are highly significant and from the analysis of mers-cov vs mock we have found 234 gens are highly significant (see details in the additional file 11: table s11 and additional file 12: table s12 ). interestingly, only 4 genes (nfkbia, egr1, ddit31 and ifit2) are common between these two infections (see figure 13 ). however, only 2 genes (nfkbia and egr1) play an important role and differentially expressed among the both infections in lung and also in sars infected pbmcs. then from the hierarchical cluster analysis of the differentially expressed genes of the lung infection by sars-cov and mers-cov, we observed distinct groups of genes that were significantly changed over time (see additional file 13: figure s1 and additional file 14: figure s2 , and additional file 11: table s11 and additional file 12: table s12 ). the log fold changes of the common 4 genes (nfkbia, egr1, ddit31 and ifit2) expression level for the infection of mers-cov and sars-cov are presented in the figures 14 and 15 . we observed that the log fold changes of nfkbia genes expression level is sharply upregulated in the both types of infections corresponding to time point. so nfkbia is an important bio-marker for the both mers-cov and sars-cov infections. it is also observed that the inflammatory genes nfkbia is a highly over expressed in the both pbmcs and lung cells for the infection of sars and also for the infection of mers in the lung cells (see figure 16 ). indeed, the immune system plays a pivotal role in the outbreak of the inflammatory state. so in case of sars infection, the nfkbia gene plays an important role for the disease comorbidities. on the other hand, similar diseases share common genes and could be treated by the same drugs [17] , which may allow us to make predictions for new uses of existing drugs. for an instance, the anti-diabetic drug metformin plays a major protective effect against cancer development and increases significantly higher survival rate of the cancer patients [65] . the finding is that the earlier the metformin regimen was initiated, the greater the http://www.biomedcentral.com/1471-2105/15/333 preventive benefit for the cancer patient. there is an evidence that the antiviral medication, ribavirin, does not work in case of sars infection [66] . to this end, we used connectivity map (cmap), which is a database of more than 1,400 drug transcriptional signatures in several cell lines [67] . this database allows to identify of molecules that induce similar or opposite transcriptional changes relative to the query signature, based on their connectivity enrichment scores. as a query signature we used our 274 highly dysregulated genes for the sars infection. we generated the connectivity score value ranges between +1 and -1, where a highly positive score indicates that the drug induces changes similar to those induced by viral infection, while a highly negative score indicates that the drug reverses the expression of the sars signature. based on the connectivity score we have selected most potential positive and negative regulators of sars viral response (see details in the additional file 15: table s13 ). potential negative regulators indicate that drugs reverse the sars signature gene expression. among the negative potential regulator, the drug molecule tetracycline, zalcitabine, gibberellic acid, prestwick-642 and sulfaquinoxaline are more potential for the mcf7 cell line and vorinostat for the hl60 cell line. based on the data demonstrate the efficacy of different drug against sars virus can be predicted effective drug treatment for the emergent viruses. furthermore, immunomodulatory drugs that reduce the excessive host inflammatory response to respiratory viruses have therapeutic benefit to reduce the sars infection as well as disease comorbidities. we presented and analysed multi-relational disease comorbidity relationships of sars and hiv-1 infections with other diseases or infections based on the associations of genetics, proteomics, molecular signalling pathways and phenotypic disorders. the combination of molecular biology, genetics and clinical medicine has greatly facilitated understanding of how different diseases relate to each other. based on the combined genetics, ppis, pathways and clinical data, our disease networks can disclose potentially novel disease relationships that have not been captured by previous individual studies. the underlying hypothesis behind this line of research is that once we catalogue all disease-related genes, ppi complex and signalling pathways, if we do not consider environmental changes, we will be able to predict the susceptibility of each individual to future diseases using various molecular biomarkers and it will help us to enter an era of predictive medicine. our results indicate that such a combination of molecular and population-level data could help to build novel hypotheses about disease mechanisms. furthermore, if two or more diseases have associated comorbidity, the occurrence of one of them in a patient may increase the likelihood of developing the other diseases. we have also studied the differences between mers-cov and sars-cov in the host response. this enables rapid assessment of viral properties and the ability to anticipate possible differences in human clinical responses to mers-cov and sars-cov and their impact on comorbidities with respect to the general comorbidities conditions. we used this information to predict potential effective drugs against sars-cov, a method that could be more generally used to identify candidate therapeutics in future disease outbreaks. these investigation approach may also help to generate hypotheses and make rapid advancements in characterising the new viruses. we also found that patients' response of sars appears to be mainly an innate inflammatory response using nfk-bia, rather than any specific immune response against a viral infection such as hiv. however, hiv infection and highly active antiretroviral therapy (haart) also increase the immune reconstitution inflammatory syndrome (iris) and inflammation through the nf-κb pathways [68] . moreover we have studied before about the impact of hiv infection on bone diseases and infection (e. g. osteoporosis and osteomyelitis). we observed that genes (e.g. rankl) and pathways (e.g. nf-κb) that are dysregulated by hiv infection also impact on the bone remodelling and bone related diseases. it is also recognised that inflammation plays a role in cancer aetiology, and various studies have found that inflammation may causes iris, obesity and tumour-promoting effects [69] . moreover, inflammation is an important concomitant cause of many major age-associated pathologies such as cancer, neurodegeneration and diabetes [70] . our study provides important evidence to associate diseases with the ageing process at the system level and helps to understand more about the comorbidities of the complex diseases. the ageing process itself is accompanied by a chronic low-grade inflammation, which is termed "inflammageing". the combination of metabolic-driven and age-driven inflammatory pathways plays a pivotal role in disease progression. this observation suggests that inflammageing and meta-inflammation can share stimuli and pathogenic mechanisms for comorbidities. we suppose that what is happening for the comorbidities we investigate is similar to what found for prions [71, 72] . similar to most infectious agents, prion causes inflammatory responses by activating innate immunity through glial cells in the brain. the complete transcriptome of the prion brain at 10 different time points is observed during the 22-week period [71, 72] . at the beginning of the disease, both normal and diseased mouse networks were the same. although the disease started in the most unique network of prion accumulation and replication it is progressed to the other networks. based on this approach we may propose a pathway model for comorbidities how hubs genes dysregulate several other pathways to influence comorbidities. the number of dysregulated pathways could be proportional to the amount of dysregulation of hub genes. our pathway model may states that the hubs that are over turned on, may direct the signal to the different pathways creating comorbidities as shown in figure 17 . for the infection, one of the pathways related to the inflammation starts dysregulation. with increasing time, both confidence level of inflammation and the number of dysregulated pathways are increased. moreover, with the increasing of inflammation the number of diseases for the comorbidities may increase. so initially infection dysregulates one signalling pathway of any cells and that causes other pathways may be dysrupted. in this way disrupting pathways increase more diseases in the same patient and make multimorbidity. disease genes play a central role in the human interactomes. overlapping component genes serve as bridges across the relatively independent functional modules or pathways. so perturbation in one pathway, such as the nf-κb signalling pathway, could be propagated throughout the other relevant pathways. we found sars and hiv-1 infections share 11 significantly dysregulated genes as well as molecular pathways. both sars and hiv-1 viruses may infect and find an already existing comorbidity or generate a new comorbidity through the perturbation of the infected pathways. furthermore, it may provide us an opportunity to investigate the role of other http://www.biomedcentral.com/1471-2105/15/333 genes from the same pathway in the disease space. therefore, pathways could be used to represent the underlying biology of diseases and make prediction of disease comorbidities. in most of the cases, the correlativeness among genes, pathways and diseases are many-to-many, e.g. a disease is associated to many different genes and pathways; and a pathway is associated to many different diseases. this study suggests that a single pathway can be involved in many diseases whereas a disease may have dysregulation in many biological processes. hence, if a drug is already available to treat a disease through modulating the activity of a pathway, then it could potentially be used to treat other diseases that are strongly linked with the same pathway. on the other hand, when a disease shows dysregulation in multiple pathways, a pathwayguided combined drug may be employed in the treatment. moreover, the protein subnetwork-based approach to diseases may aid in drug discovery, in fact it can potentially be used to treat other diseases that are linked to the same protein complex. thus, our findings not only potentially help us to understand how different diseases are related based on their underlying molecular mechanisms but also provide insights into the design of novel, protein complex-guided therapeutic interventions for diseases. extending the concept of subclassifying patient cohorts to the single patient level refers to as personalised medicine. during the last few years, acceptance level of the personalised medicine is sharply increased as it has been apparent that standard treatment approaches are rarely efficient across the entire patient population. advances in highthroughput molecular assay technologies in the fields of genomics, proteomics and other "omics" is increasing the diagnostic and therapeutic strategies for personalised treatment. as a result, declining per-sample cost has given rise to numerous public repositories of biomolecular data. in particular, the availability of these data sets for many different diseases presents a ripe opportunity to use data-driven approaches to advance our current knowledge of disease relationships in a systematic way. the identified disease patterns can then be further investigated with regards to their diagnostic utility or help in predicting novel therapeutic targets. medicine will focus on each individual patient. it will become intrinsically proactive and will increasingly focus on wellness rather than disease. proactive and personalised medicine will bring fundamental changes to healthcare, taking carefully http://www.biomedcentral.com/1471-2105/15/333 targeted preventative or therapeutic action at the earliest indications of risk or disease comorbidities. we are entering into the genomic era of medicine, where a patient's genetic/genomic data is becoming important for clinical decision making, including disease risk assessment, disease diagnosis and subtyping, drug therapy and dose selection, risk assessment for adverse drug reaction, and family planning [73] . today multi-scale and complex biomedical data are gathered and analysed to uncover combinations of predictive disease profiles. our genome, as well as multiple proteomes, multiple transcriptomes, multiple metabolomes, and other personalised data sets obtained at different points in our lives, will be readily available at affordable prices for each individual. in the near future, clinicians will have to consider genetic/genomic implications to patient care throughout their clinical workflow, including electronic prescribing of medications. therefore, for the implementation of the personalised medicine system, a model could be developed that will take individual genetic data. dysregulated biomarker genes will be identified from this genetic data and disease will be identified from the gene-disease association database. based on the information of the existing disease, the model will predict disease comorbidities using the disease-disease associations database. this will provide us to detect many diseases at the earliest detectable phase, even weeks, months, or maybe years before the symptoms appear and it will afford crucial insights into optimizing of our wellness. thus, personalised medicine will give fundamental new insights into disease mechanisms, and hence will open new opportunities for diagnosis, therapy and prevention from the disease comorbidities. in this study, we have considered all available categories of omics and phenotypic data to quantify the sars and hiv-1 infections centred comorbidity associations. we have shown that the phenotype disease network (pdn) has a heterogeneous structure where some diseases are highly connected while others are hardly connected at all. our findings showed that disease progression can be represented and studied using network methods, offering the potential to enhance our understanding of the origin and evolution of human diseases. detecting comorbidity in a large population is of clinical interest due to the fact that it may reveal new information useful for cause of diseases as well as for new treatment strategies. this study demonstrates the value of an integrated approach in revealing disease relationships and new opportunities for therapeutic applications. so we can say that this kind of approach will be helpful for making evidence-based recommendations about disease comorbidities. moreover, considering environmental factors (such as physiological stress, diet), ethnic group and gender discriminations are important factors in the comorbidity analysis. our network approach could be extended as a comorbidity map by integrating diet, exercise and other factors as in [74] . the gene-disease associations data used in this study were collected from the online mendelian inheritance in man (omim) database (http://www.ncbi.nlm.nih.gov/ omim/). this omim database is the best-curated repository of all known disease genes and their associated http://www.biomedcentral.com/1471-2105/15/333 figure 17 progressive temporal activation of pathways. a schematic view of networks becoming disease comorbidities increased for the perturbation of the pathways dysregulation advances with time. the red circles indicate increased levels of dysregulated gene expression relative to control and the red linked indicate dysregulated pathways that have been increased from infection as compared with normal control. the green indicated transcripts that are the same in control and infection condition. the four panels represent the network with time intervals of the infection progression. with time the inflammation confidence level is increased which is indicated by confidence interval. disorders [75, 76] . genotype-phenotype relationships, as summarised in the omim database, contained more than 5000 human disease-genes associations involving 1500 diseases and 3000 disease associated genes. each entry of the omim is composed of four fields, the name of the disorder, the associated gene symbols, its corresponding omim id, and the chromosomal location. we selected the entries with the "(3)" tag, for which there is strong evidence that at least one mutation is cause of the disorder. omim initially focused on monogenic disorders but in recent years has expanded to include complex traits and the associated genetic mutations that confer susceptibility to these common disorders [58] . subsequently we classified each disorder into 21 primary disorder classes based on the physiological system affected as introduced in goh, cusick, valle, childs, vidal and barabasi et al. [14] . disorders having distinct multiple clinical features are assigned to the "multiple" class. this classification scheme reflects the phenotypic similarities among diseases in the same class and has been successfully used in the recent studies of systematic disease analysis [77] . the gene expression data used in this study was obtained from the ncbi gene expression omnibus (geo) (http://www.ncbi.nlm.nih.gov/geo/) [59] . we have considered 10 different data sets for our analysis (accession numbers are gse1739, gse45042, gse17400, gse9006, gse9128, gse15072, gse7158, gse8977, gse18464 and gse7621) [32, 55, 57, 64, [78] [79] [80] [81] [82] . these data sets contain data from the patients of different age and sex. after several rounds of filtering, normalization and statistical analysis, we had microarrays representing sars, mers, hiv-1 infections and 7 other human diseases (heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, type 1 and type 2 diabetes). http://www.biomedcentral.com/1471-2105/15/333 the protein-protein interaction (ppi) data for human was obtained from the human protein reference database (hprd) [61] . hprd contains the maximum number of ppi data among all publicly available literature-derived databases for human ppi [83] . we have used the reactome knowledge base of human biological pathways database for our pathways association analysis [60] . for the cross compare analysis between the sars and hiv infections, and ageing process we have download ageing data from the human ageing genomic resources (http://genomics.senescence.info/ download.html) [62, 63] . they have collected human ageing genes after an extensive review of the literature. these genes are commonly dysregulated during the ageing process. to test the validity of the proposed disease associations, we examined the disease co-occurrence information at the population level. we obtained statistically significant pairwise comorbidity associations reconstructed from over 32 million medical records in the us medicare claims database recorded in the icd-9-cm format (http://www. icd9data.com), which are frequently used for epidemiological and demographic studies and collected from [2] . we used medpar records from 1990 to 1993, where the dates and reasons for all hospitalizations were reported in icd-9-cm format and it contains the diagnoses of 13039018 elderly patients. each record consists of the date of visit, a primary diagnosis and up to 9 secondary diagnosis. all diagnoses are specified by icd9 codes of up to 5 digits. the first three digits specify the main disease category while the last two are used to give additional information about the disease. in total, the icd-9-cm classification consists of 657 different categories at the 3 digit level and 16,459 categories at the 5 digit level [2] . to determine whether some existing drug compounds can reverse the sars infection signature, we used the publicly available connectivity map (cmap) database [67] . cmap provides associations among genes, chemicals and disease or infection conditions. it is a collection of genome-wide transcriptional data from cultured human cells treated with 1,400 different compounds. the method of global gene expression analysis using oligonucleotide microarrays has proven to be a sensitive method to develop and refine the molecular determinants of human disorders [55] . using this technology, we compared the gene expression profiles of sars, hiv and other diseases. to avoid the problems of comparing microarray data of different platforms and experimental systems, we normalized the gene expression data in each microarray sample (disease state or control) using the z-score transformation (z ij ) for each disease gene expression matrix using z ij = where sd is the standard deviation, g ij represents the expression value of gene i in sample j. this transformation allows for the direct comparison of gene expression values across various microarray samples and diseases. to combined more than one data series or experiments for a given disease, we employed a linear regression approach to obtain a combined t-test statistic between two conditions. data were log 2 -transformed and we calculated expression level for each gene using a linear regression model : where y i is the gene expression value and x i is a disease state (disease or control). the coefficients β 0 and β 1 are the parameters of this model and were estimated by least squares. the t-test statistic, when estimating the value of β 1 , is the same as the standard t-test statistic between disease and control states. time series microarray gene expression data analysis was divide into two steps: pre-processing and identification of statistically significant points by t-test, anova and regression analysis to find differently expressed gene profiles in different time points. in the first step, we preprocessed the experimental data using different statistical methods and finally followed by post less normalization, recommended by the golden spike project [84] . in the second step, we have used a most suitable method "masigpro" (microarray significant profiles) to identify differentially expressed genes in time-course microarray experiments, which is a two step regression method successfully applied on more than one groups of time-series [85, 86] . this two steps regression strategy is used to find genes with significant temporal expression changes and significant differences between experimental groups. this procedure first adjusts this global model by the leastsquared technique to identify differentially expressed genes and selects significant genes applying false discovery rate control procedures. then stepwise regression is applied as a variable selection strategy to study differences between experimental groups and statistically significant profiles. after finding differentially gene expression profiles among the group of experiments, the next step is to cluster them according to their profile similarities. the hierarchical clustering and the median gene expression profiles of clusters are performed according to the "masigpro" package in r [85] . student's unpaired t-test was performed to identify genes that were differentially expressed in patients over normal samples and significant genes were selected. a threshold of at least 1.5 fold change and a p value for the t-tests of less than 0.01 were chosen. in addition, a two-way anova with bonferroni's post-hoc test was used to establish statistical significance between groups (< 0.01). pathways and functional categories were considered as over-represented when fisher's exact test p value was < 0.01. for presenting the signalling and http://www.biomedcentral.com/1471-2105/15/333 interaction pathways of the different significant genes, we used cytoscape for data integration and network visualization [87, 88] and reactome functional interaction (fi) cytoscape plugin for knowledge base of human biological pathways and network processes [60] . for the gene disease association, we have considered the neighbourhood based benchmark and topological methods, which are better suited to our networks [89] . in this case, topological refers to methods that rely only on the structure of the network to draw conclusions. we construct a gdn from gene-disease associations where the node in the network can be either a disease or a gene. this network can also be regarded as a bipartite graph. diseases are connected when the diseases share at least one significant dysregulated genes. let a particular set of human diseases d and a set of human genes g, genedisease associations attempt to find whether gene g ∈ g is associated with disease d ∈ d. if g i and g j , the sets of significant up and down dysregulated genes associated with diseases i and j respectively, then the number of shared dysregulated genes (n g ij ) associated with both diseases i and j is as follows: the co-occurrence refers to the number of shared genes in the gdn. the common neighbours is the based on the jaccard coefficient method, where the edge prediction score for the node pair is as: where e is the set of all edges. the number of shared pathways and protein subnetwork that links between diseases i and j are calculated using the equation 1 and the link prediction score is measured using the equation 2. to estimate the correlation starting from disease cooccurrence, we need to quantify the strength of disease association for comorbidities by dipicting a distance between two diseases. for the analysis of the phenotypic data, we used the relative risk (rr ij ) as the quantified measures of comorbidity tendency of two disease pairs and checked φ-correlation (φ ij ) to measure the robustness of the comorbidity associations. the rr ij is observing in a pair of diseases i and j affecting the same patient. when two diseases co-occur more frequently than expected by chance, we will get rr ij > 1 and φ ij > 0. however, rr ij and φ ij are not independent of each other and each carries unique biases that are complementary [1, 2] . so, we used both measures of comorbidity to ensure the robustness of our investigations. the rr ij allows us to quantify the co-occurrence of disease pairs compared with the random expectation which is calculated as: (3) where n is the total number of patients in the population, p i is the incidences/prevalences of disease i, p j is the incidence of disease j and c ij is the number of patients that have been diagnosed with both diseases i and j. for rr ij >= 1 comorbidity is larger than expected by chance and for rr ij < 1 comorbidity is smaller than expected by chance. to calculate the significance of the relative risk rr ij , we used the katz, baptista, azen and pike et al. method to estimate confidence intervals [90] . according to their estimation, the 99% confidence interval for the rr ij between two diseases i and j is calculated by: lower bounds of the confidence interval (lb) = rr ij * exp(−2.56 * σ ij ) and upper bounds of the confidence interval (ub) = rr ij * exp(2.56 * σ ij ), where σ ij is given by: disease pairs within the 99% confidence interval are only considered if the lb value is larger than 1 when rr ij is larger than 1, or if the ub value is smaller than 1 when rr ij is smaller than 1. relative risk measure is intrinsically biased towards overestimation of relationships between rare diseases and underestimates the co-morbidity of more frequent diseases [2] . this bias can be reduced by introduction of a φ-correlation measure. we can quantify the strength of comorbidities by calculating the correlation coefficient associated with a pair of diseases i and j as: where c ij is the number of patients affected by both diseases, n is the total number of patients in the studied population, and p i and p j are the morbidity or incidence of the i th and j th diseases respectively. the φ-correlation is the pearson's correlation for the variables which only take 0 or 1 values [91] . for φ ij > 0 comorbidity is larger than expected by chance and for φ ij < 0 comorbidity is smaller than expected by chance. we can determine the significance of φ = 0 by performing a t-test. this consists of calculating t according to the formula: t = φ √ n−2 √ 1−φ 2 , where n is the number of observations used to calculate φ. to predict the comorbidities considering the primary or index disease we have calculated the conditional relative risk (conditional rr ij ) as follows: for all possible disease pairs i and j, for the cases that one index disease (i) is present (k = true) or absent (k = false). (p = 0.01). we have weighted the edges using a mutual information metric which quantifies how much greater the edge relationship is with respect to co-occurrence. the mutual information weight between two diseases i and j is defined as w ij = c ij p i + p j − c ij (6) where c ij is the observed co-occurrence and p i and p j are the morbidity or prevalence of the i th and j th diseases respectively. to compare between sars-cov and mers-cov, a gene set enrichment analysis was undertaken using gsea [92] . to find out the correlation (similarities) and distance (dissimilarities) among the diseases from the integrated analysis of multidimensional data (gene expression and protein protein interaction), we have applied euclidian distance measurement and metric multi-dimensional scaling (mds) using majorization [93] . mds is a set of methods for discovering hidden structures in multidimensional data. based on a proximity matrix derived from variables measured on objects as input entity, these distances are mapped on a lower dimensional spatial representation. optimization problem is used to find mapping in target dimension of the data based on given pairwise proximity information while minimize the objective function. the particular objective function (or loss function) we used in this work is a sum of squares, commonly called stress. we used majorization to minimize stress and this mds solving strategy is known as smacof (scaling by majorizing a complicated function). stress majorization is an optimization strategy used in multidimensional scaling (mds) where, for a set of nm-dimensional data items, a configuration x of n points in r(<< m)-dimensional space is sought that minimizes the stress function σ (x). here r is 2 that means the (r × n) matrix x lists points in 2-dimensional euclidean space. we have applied the cost function σ to measures the squared differences between ideal (m-dimensional) distances and actual distances in r-dimensional space as follows: x 1 of dimension n 1 × p as the individual's or judge's configuration, and x 2 of dimension n 2 × p as the object's configuration matrix. the least squares metric multidimensional scaling or mds problem is the minimization of σ and over all m × p configurations x. here w ij are given non-negative weights and d ij are given non-negative dissimilarities. the d ij (x) are the euclidean distances between rows i and j of x. thus d ij (x1, x2) = p s=1 x 1is − x 2js 2 (8) where w ij ≥ 0 is a weight for the measurement between a pair of points (i, j), d ij (x) is the euclidean distance between i and j, and δ ij is the ideal distance between the points (their separation) in the m-dimensional data space. note that w ij is used to specify a degree of confidence in the similarity between points (e.g. 0 can be specified if there is no information for a particular pair). a configuration x which minimizes σ (x) gives a plot in which points that are close together correspond to points that are also close together in the original m-dimensional data space. programming scripts are freely available at www.cl.cam. ac.uk/~mam211/comor/. http://www.biomedcentral.com/1471-2105/15/333 information regarding each of the clusters and genes is described in additional file 12: table s12 . additional file 15: table s13 . connectivity map results of predicted drugs per instance (for each drug and cells line) to reverse sars-cov for early and sustained signature (drugs with negative enrichment scores). the impact of cellular networks on disease comorbidity a dynamic network approach for the study of human phenotypes comor: a software for disease comorbidity risk assessment comorbidity of cardiovascular disease, diabetes and chronic kidney disease in australia. canberra: australian institute of health and welfare mortality after incident cancer in people with and without type 2 diabetes impact of metformin on survival comorbidities in chronic obstructive pulmonary disease comorbidities of chronic obstructive pulmonary disease the incidence of co-morbidities related to obesity and overweight: a systematic review and meta-analysis comorbidity: a network perspective detecting shared pathogenesis from the shared genetics of immune-related diseases comorbidity and survival after early breast cancer. a review probing genetic overlap among complex human phenotypes the implications of human metabolic network topology for disease comorbidity the human disease network network properties of genes harboring inherited disease mutations a human phenome-interactome network of protein complexes implicated in genetic disorders network-based elucidation of human disease similarities reveals common functional modules enriched for pluripotent drug targets modelling osteomyelitis towards a proteome-scale map of the human protein-protein interaction network a human protein-protein interaction network: a resource for annotating the proteome origin and physiological roles of inflammation as a doctor, i'd rather have hiv than diabetes. the spectator magazine pandemic influenza a (h1n1) virus hospitalizations investigation team: hospitalized patients with 2009 h1n1 influenza in the united states cardiovascular manifestations of the emerging dengue pandemic complications of viral influenza association of symptoms of chronic bronchitis and frequent flu-like illnesses with stroke centers for disease control and prevention (cdc): recommendations of the advisory committee on immunization practices (acip) how ageing processes influence cancer the role of low-grade inflammation and metabolic flexibility in aging and nutritional modulation thereof: a systems biology approach prevalence of comorbidity of chronic diseases in australia early gene expression events in ferrets in response to sars coronavirus infection versus direct interferon-alpha2b stimulation dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection canadian sars research network, kelvin dj. interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome sars: prognosis, outcome and sequelae severe acute respiratory syndrome-coronavirus infection in aged nonhuman primates is associated with modulated pulmonary and systemic immune responses epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study severe respiratory illness caused by a novel coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia osteoimmunopathology in hiv/aids: a translational evidence-based perspective differential impacts of r5 vs. x4 hiv-1 on the transcriptome of primary cd4+ t cells hiv-1 triggers apoptosis in primary osteoblasts and hobit cells through tnfα activation a review of co-morbidity between infectious and chronic disease in sub saharan africa: tb and diabetes mellitus, hiv and metabolic syndrome, and the impact of globalization cancer risk in people infected with human immunodeficiency virus in the united states hepatocellular cancer in hiv-infected individuals: tomorrow's problem? vajdic cm: incidence of cancers in people with hiv/aids compared with immunosuppressed transplant recipients: a meta-analysis risk of human papillomavirus-associated cancers among persons with aids hiv infection and lymphoma control of antiviral immunity by pattern recognition and the microbiome safety and success of kidney transplantation and concomitant immunosuppression in hiv-positive patients hiv infection, inflammation, immunosenescence, and aging weakened immunity in aged hosts with comorbidities as a risk factor for the emergence of influenza a h7n9 mutants tuberculosis comorbidity with communicable and non-communicable diseases: integrating health services and control efforts heightened plasma levels of heme oxygenase-1 and tissue inhibitor of metalloproteinase-4 as well as elevated peripheral neutrophil counts are associated with tuberculosis-diabetes comorbidity emerging infectious diseases: threats to human health and global stability expression profile of immune response genes in patients with severe acute respiratory syndrome chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells interferon-α drives monocyte gene expression in chronic unsuppressed hiv-1 infection online mendelian inheritance in man (omim), a knowledgebase of human genes and genetic disorders ncbi geo: mining tens of millions of expression profiles database and tools update reactome: a knowledgebase of biological pathways human protein reference database-2009 update human ageing genomic resources: integrated databases and tools for the biology and genetics of ageing meta-analysis of age-related gene expression profiles identifies common signatures of aging cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus new users of metformin are at low risk of incident cancer a cohort study among people with type 2 diabetes severe acute respiratory syndrome (sars) applications of connectivity map in drug discovery and development developments in hiv neuropathogenesis infection, immunoregulation, and cancer charting the nf-κb pathway interactome map a systems approach to prion disease systems biology and p4 medicine: past, present, and future emerging landscape of genomics in the electronic health record for personalized medicine clinical assessment incorporating a personal genome mckusick's online mendelian inheritance in man (omim) a new face and new challenges for online mendelian inheritance in man (omim) evolutionary history of human disease genes reveals phenotypic connections and comorbidity among genetic diseases gene expression in peripheral blood mononuclear cells from children with diabetes gene expression profiles in peripheral blood mononuclear cells of chronic heart failure patients mitochondrial dysregulation and oxidative stress in patients with chronic kidney disease mesenchymal stem cells within tumour stroma promote breast cancer metastasis a genomic pathway approach to a complex disease: axon guidance and parkinson disease an evaluation of human protein-protein interaction data in the public domain preferred analysis methods for affymetrix genechips revealed by a wholly defined control dataset masigpro: a method to identify significantly differential expression profiles in time-course microarray experiments serial expression analysis: a web tool for the analysis of serial gene expression data cytoscape: a software environment for integrated models of biomolecular interaction networks cytoscape 2.8: new features for data integration and network visualization exploring and exploiting disease interactions from multi-relational gene and phenotype networks obtaining confidence intervals for the risk ratio in cohort studies applied multiple regression/correlation analysis for the behavioral sciences. routledge gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles a majorization algorithm for solving mds submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank fp7-health-f5-2012 for providing financial support, under grant agreement n 305280 (mimomics). additional file 1: table s1 . highly statistical significantly differential expressed genes between sars and control group in pbmcs. table s2 . highly statistical significantly differential expressed genes between hiv and control group. table s7 . phenotypic disease association for sars infection based on the icd9 codes at the 3-digit category level. only statistically significant links with high relative risk rr ij are considered. additional file 8: table s8 . phenotypic disease association for sars infection based on the icd9 codes at the 5-digit category level. only statistically significant links with high relative risk rr ij are considered.additional file 9: table s9 . phenotypic disease association for hiv infection based on the icd9 codes at the 3-digit category level. only statistically significant links with high relative risk rr ij are considered. additional file 10: table s10 . phenotypic disease association for hiv infection based on the icd9 codes at the 5-digit category level. only statistically significant links with high relative risk rr ij are considered.additional file 11: table s11 . highly statistical significant differentially expressed genes between sars-cov and reference group (mock) in lung epithelial cells.additional file 12: table s12 . highly statistical significant differentially expressed genes between mers-cov and reference group (mock) in lung epithelial cells.additional file 13: figure s1 . median expression profile of sars-cov vs mock using hierarchical clustering (ward method, pearson correlation) of 215 statistical significantly differential expressed genes (p < 0.001). the information regarding each of the clusters and genes is described in additional file 11: table s11 .additional file 14: figure s2 . median expression profile of mers-cov vs mock using hierarchical clustering (ward method, pearson correlation) of 234 statistical significantly differential expressed genes (p < 0.001). the the authors declare that they have no competing interests.authors' contributions mam and pl designed and mam implemented the analysis of the paper. mam and pl wrote the manuscript. both authors contributed to and approved the manuscript. key: cord-322256-mv9ll0h4 authors: edelman, e. jennifer; aoun-barakat, lydia; villanueva, merceditas; friedland, gerald title: confronting another pandemic: lessons from hiv can inform our covid-19 response date: 2020-05-12 journal: aids behav doi: 10.1007/s10461-020-02908-z sha: doc_id: 322256 cord_uid: mv9ll0h4 the novel coronavirus 2019 illness (covid-19) has completely transformed and uprooted lives across the globe. while different diseases, there are critical observations and lessons to be learned from the ongoing hiv epidemic to inform our response to covid-19. we reflect on how this relates to (1) testing, including contact tracing; (2) health system redesign; (3) telehealth; (4) health disparities; (5) political denial, with inadequate and uncoordinated governmental response; (6) occupational exposure; and (7) complex reactions among healthcare providers. decades of experiences with hiv provide an important framework for moving forward as we combat covid-19. in just a few months, the novel coronavirus 2019 illness (covid-19) has completely transformed and uprooted lives across the globe. as we fight locally, nationally, and globally to gain control of covid-19, there are critical observations and lessons to be learned from the ongoing hiv epidemic, which continues to claim over 1 million lives each year worldwide [1] . admittedly, hiv/aids and covid-19 are entirely different diseases with different modes of transmission and natural history. the former is a chronic disease spread by body fluids in specific risk behaviors; the latter is currently appreciated as an acute infection spread by respiratory droplets with potential for ubiquitous spread. nonetheless, given these inherent differences, both require confronting denial of their danger and similar and specific clinical and public health approaches. in both diseases, timely testing to identify those infected is crucial. as with hiv, individuals with covid-19 may be asymptomatic or pre-symptomatic, yet have high levels of transmissible virus. in both cases, insufficient testing leads to ongoing transmission and widespread dissemination and lack of critically needed epidemiologic information to focus control efforts. initially in hiv and now, even more dramatically in covid-19, there has been a tragic systemic delay in implementing widespread testing. in both, additional barriers to testing include inadequate access particularly in underserved populations; fear of adverse consequences, such as stigma; loss of job or housing; or lack of health literacy about potential clinical outcomes. efforts are needed to mitigate potential harms of a positive test and ensure that individuals who are less connected to the healthcare system know how and where to seek both testing and care for covid-19-related symptoms. in combating both diseases, adequate resources for sustained public health system preparedness are essential. experiences with hiv and partner services has taught us the critical role of public health collaboration to promote contact tracing to ensure that individuals who have been exposed to an infectious disease receive appropriate counseling, testing, and treatment [2] . in the current covid-19 context, prior to recognition of need for widespread testing there was an absence of focused contact tracing, particularly in the early phase of the pandemic, which could have provided a critical tool for containment. such efforts require well-developed and robust public health and disaster planning infrastructures with careful coordination and communication between testing sites, public health authorities, clinical facilities and individuals diagnosed with covid-19. efforts to scale-up contact tracing are now underway. as with hiv, covid-19 has mandated innovative efforts to optimize patient-centered care in a health care system illequipped to deal with a new highly infectious and rapidly fatal disease. in the case of hiv, the ryan white care act of 1990 resulted in a major restructuring of the healthcare system to meet the wide spectrum of needs of people with hiv to include integrated community-based prevention and care, comprehensive outpatient clinics, mental health and substance use treatment programs dedicated hospital wards, and non-medical needs (e.g., housing, food and transportation) [3] . on a more rapid time scale, covid-19 has dramatically led to health system redesign that currently focuses on increasing capacity to manage critical care needs of seriously ill patients with covid-19, such as expanding inpatient and critical care capacity, equipment (e.g., personal protective equipment [ppe], ventilators), and health care personnel; however, long-term needs, including creating systems for transition of care and outpatient follow-up, remain currently undefined and are quickly evolving. further, as we painfully learned with hiv, resources and infrastructure to expedite discovery and equitable availability of effective treatments and biomedical and behavioral prevention interventions for this new pathogen are desperately needed. novel telehealth-based models have been pioneered and developed in resource-limited settings to improve access for hiv prevention and treatment [4] . in the current covid-19 context, massive scale-up of telehealth has been launched to provide ongoing healthcare that is patient-centered and designed to protect healthcare workers. this may present a unique opportunity to evaluate patient preferences for telehealth, demonstrate its role in access to and impact on quality of care, and inform future models for providing patient-centered care and linkage to care for hard to reach populations. further, covid-19 care can be informed by extensive efforts that have been applied to enhance retention in lifelong hiv care and outreach efforts to identify and connect with patients vulnerable to effects of social isolation, worsening mental health, substance use, and intimate partner violence. unacceptable sociodemographic, racial and ethnic disparities observed with hiv, also characterize populations at greatest risk for covid-19, but have hitherto largely being ignored [5] . covid-19 disproportionally affects those who are challenged by poverty, inadequate housing, poor sanitation and crowding, and limited resources as well as increased numbers from minority populations with resultant higher rates of adverse outcomes and mortality. immediate efforts must broaden and target covid-19 testing in these populations at highest risk. in the longer term, sustained strategies to alleviate social and economic inequities are required to successfully reverse and control covid-19 and adequately prepare for subsequent pandemics. as with the hiv, political denial about the scope and consequences of, covid-19 has resulted in a wholly inadequate and uncoordinated governmental response. activism by community-based organizations like aids coalition to unleash power (act up) were critical for promoting broad recognition of and successful response to hiv/aids. similarly, forceful and energetic efforts by the health care and scientific professions and responsible media has been necessary to confront and accelerate the slow and uncoordinated governmental response in the united states. as communities begin moving towards liberalizing social distancing restrictions it will be essential to ensure that policies are grounded in science and fairness with close surveillance and accurate, responsible, and equitable delivery of information and resources to the public. with both hiv and covid-19, the risk of occupational exposure and the safety of health care workers has been a paramount concern. in hiv, rare but alarming occupational exposure resulting in hiv-seroconversions led to the establishment of universal precautions in health care settings, created innovative strategies to safeguard against needle stick injuries and other occupational disease exposures. the differences in routes of transmission render covid-19 many fold more dangerous than hiv in the health care setting and mandates the need for ensuring adequate ppe for healthcare workers and others providing care for individuals exposed by aerosols and contact with patients with covid-19 and cannot be overstated. the horrific delays in ensuring adequate ppe for healthcare workers has earned intense attention and must be rapidly rectified and never again arise. beyond the healthcare system, although quarantine and social distancing in community settings has gained needed traction, there remains an urgent need to enhance safety and protection for those providing other essential community-based services and those unable to remain home and who lack a strong coordinated voice in this current pandemic [6] . as with hiv, covid-19 is associated with many complex feelings among family and healthcare workers, including those of loss and grief. but, there is an additional painful difference between the two diseases as risk to health care workers and family from occupational exposure to covid-19 has resulted in clinicians being less able to provide the human and empathic support that remains at the core of their profession and essential for patients and their families. patients with covid-19 suffer and die in isolation, without their loved ones at the bedside. further, clinicians caring for patients with covid-19 must isolate themselves from their own families and can be stigmatized by neighbors, friends and families for fear of transmission and lack of understanding of their need and desire to be on the frontlines. physicians and other health care workers currently experience the uncertainty of lack of knowledge and inadequate clinical tools, such as effective medical treatments and an effective vaccine; a similar difficult scenario was experienced in the early decades of the hiv/aids epidemic and, although great success has been achieved, remains to the present. the availability of effective life prolonging antiretroviral treatment dramatically converted hiv/aids from an invariably fatal to a manageable chronic disease, although cure and effective vaccine still eludes us. in contrast, even as the painful toll of disease and social dislocation continues to rise, the comparative lower mortality rate from covid-19 and possibilities for future curative therapies provide hope for the future. even after we successfully address prevention and treatment of covid-19, as with hiv, the memory of patients' lives and resilience of health care workers and others' courage and dedication collectively provides comfort and strength during these uncertain times. while there are major differences in the timescale between the effects of hiv and covid-19, and much yet to learn about the course of covid-19, the decades of experiences with hiv can serve as a guidepost as we move onward to combat and mitigate the effects of this new global pandemic and prepare fully for subsequent ones. estimates of global, regional, and national incidence, prevalence, and mortality of hiv, 1980-2015: the global burden of disease study the effectiveness of hiv partner counseling and referral services in increasing identification of hiv-positive individuals a systematic review projecting the impact of aids on hospitals the current and future use of telemedicine in infectious diseases practice we're flying blind': african americans may be bearing the brunt of covid-19, but access to data are limited social distancing is a privilege new york times acknowledgements ej edelman received funding from the national institute of mental health (p30mh062294). the content is solely the responsibility of the authors and does not necessarily represent the official views of the center for interdisciplinary research on aids, the national institute of mental health, or the national institutes of health. key: cord-329482-haenltxn authors: small, eusebius; sharma, bonita b.; nikolova, silviya pavlova title: covid-19 and gender in lmics: potential lessons from hiv pandemic date: 2020-05-25 journal: aids behav doi: 10.1007/s10461-020-02932-z sha: doc_id: 329482 cord_uid: haenltxn nan the intersections between infectious diseases, including hiv and structural inequalities, cannot be overstated. hiv disproportionately impacts women [7] and is often concentrated in socially marginalized and disenfranchised communities [8] . covid-19 is affecting women disproportionately; they are "essential workers" taking the strain as food service industry workers, janitors, cashiers, and stockers. many live in densely populated areas that have no proper sanitation [7] . the social distancing and lockdown measures have impacted nearly 81% of the world's labor force, mostly women [7] . according to the world bank, almost 24 million fewer people will escape poverty in east asia and the pacific because of the financial impact of covid-19 in 2020 [9] . already half of the world population cannot access healthcare services, and large numbers of households are poor because of healthcare costs [9] . the emergence of covid-19 may widen the gender inequality gap to an unprecedented level and threaten women's health. for example, many hiv infected women will lack the essential antiretroviral therapy (art) drugs, or pre-exposure prophylaxis (prep) because of disruptions in the supply chain. as a consequence, many will develop an unsuppressed or uncontrolled viral load that will weaken the immune system making them susceptible to covid-19 [10] . covid-19 will exceedingly impact women in ssa, who already face food security challenges [11] . one in every five people in africa, nearly 250 million, did not have enough food before the covid-19 outbreak [12] . the long-term consequences are unknown; however, micronutrient deficiency and anemia are likely, which will worsen hiv/ aids disease for hiv patients. women are the caregivers and service providers who are 2.5 times more likely to do unpaid domestic work than men [13] . however, covid-19 responses have not been poverty or gender-sensitive to the needs of women [14, 15] . in lmics, women are the frontline workers, community health workers, and community engagement personnel; they are easily susceptible to covid-19 infections. without proper personal protective equipment (ppe), and as they work in environments with no water or sanitation, many of these workers could be exposed to covid-19 outbreaks [16] . although recommendations and obligatory hand hygiene have been emphasized as requisite to avoid covid-19 [16] , these recommendations are impervious to the social environments of the poor. millions of people in the world have no access to clean water or soap; asking them to wash their hands to prevent infections is insensitive to the plight of the poor. such recommendation parallel those made during the early days of hiv pertaining to safe sex practices. however, many populations could not afford condoms, and adequate guarantees were not put in place to acquire them [17] . the impact of covid-19 is undoubtedly felt unevenly across countries and regions [18] . among the lmics in sub-saharan africa, covid-19 could push these countries farther into a spiral of poverty, ravaging their already tenuous health systems [2, 5] . the vast global demand for ppes, for example, exerts substantial competition and demand for them. there is a troubling rich/poor divide, disadvantaging many lmics who lose out to the wealthier countries that outspend and outbid them, sometimes tripling the market price for ppes [19] . faced with such inequity gaps, many countries in latin america and africa are realizing unseen pre-existing inequities and cannot find enough materials and equipment to test for covid-19 and treat their populations [7] . the long-term impact on gdp, growth, and service delivery will be consequential as projections have already been revised downwards for most regions and countries [9] sexual and reproductive health sexual and reproductive health and rights (srhr) are critical to women's health [20] . evidence awash indicating that providing contraception to women is one of the simplest ways to reduce poverty [21] . during the hiv outbreak, a significant limited reproductive health care and family planning services were available to women. resources to address the disease were diverted elsewhere [6] . in the era of covid-19, disruptions in the manufacture and distribution of critical contraceptives might contribute to unwanted pregnancies [22] and sexually transmitted diseases like hiv. according to the united nations, an unrelated crisis impacting women worldwide are the spikes in domestic violence due to covid-19 lockdowns [7] . lockdowns and mandatory sheltering-in-place may be making violence in homes more frequent and severe as girls and women shelter in place with their abusers. domestic violence and sexual abuse are significant correlates of hiv risk for women as violence has been identified as an independent risk factor for hiv infection [23] . women who are abused may not ask their partner to use a condom, nor have the efficacy to say no to sex if their abusive partner does not want to use protection, which will put abused women at a higher risk for hiv. additionally, women who are transgender and are living with hiv are disproportionately impacted by intimate partner violence [24] , stay at home covid-19 orders could exacerbate their wellbeing. following covid-19, a renewed focus on reducing structural inequalities is needed to ensure that health is not a byproduct of privilege in any country or region. adapting existing practices and schemes to benefit women can simultaneously reduce the viral spread of the infection [25] . for example, cash benefits using e-payments and inkind transfers can improve economic security for women. also, policies and programs should ensure dignified work opportunities that promote sustainable economic growth, inclusive workspaces, and decent wages for women to ensure their full economic participation. particular attention should be paid to adolescent girls and young women and improve women's empowerment by challenging norms that increase their vulnerability in the community. the entrenched structural imbalance can dramatically weaken women's economic ability to recover from an epidemic, as evident with ebola quarantine measures [26] . women shoulder nearly 85% of austerity measures [27] . thus, involving women to assess and analyze policy decisions will ensure greater equity of impact. addressing the social determinants of health as a broader issue should incorporate women's lived experiences because of their unique perspectives on policymaking [28] . for example, cash-transfer programs (although not common in most lmics) should be sensitive to the women's lived reality. women are more likely to hold informal work and have child care obligations that may prevent them from accessing aid. in south asia, over 80% of individuals in non-agricultural jobs have informal employment; in ssa, this figure is 74%, yet social protection and consumer stimulus often depend on participation in the formal sector [29] . additionally, ensuring maternal and reproductive health in the form of insurance coverage for routine checks, especially in rural and marginalized communities, could reduce maternal mortality, reduce adolescent pregnancies and control hiv and sexually transmitted diseases. economic uncertainty, civil unrest, and disasters-including pandemics-are linked to increases in violence against women [30] . thus, anticipating a surge in vaw/c at the outset of an epidemic to promptly prepare is necessary. specifically, provide women (1) access to formal health/mental health services through increasing first responders, access to free mental health services, and free legal counsel. (2) increasing access to informal social support; (3) clear communication from governments on violence prevention/intervention, and steadily ease restrictions to help families reduce the financial stress that could trigger violent situations. icap at columbia university the social impact of covid-19 who. world health organization who | regional office for africa has covid-19 subverted global health the nih's slow response to the aids epidemic and the dismissal of pasteur's contributions: learning from history un secretary-general's policy brief: the impact of covid-19 on women | digital library: publications a pandemic of the poor: social disadvantage and the u.s. hiv epidemic world bank and who: half the world lacks access to essential health services, 100 million still pushed into extreme poverty because of health expenses maintaining hiv care during the covid-19 pandemic sofi 2019 -the state of food security and nutrition in the world coronavirus lockdowns are choking africa's food supply | time united nations development programme (undp) a gender-sensitive response is missing from the covid-19 crisis avenue hrw| 350 f, york 34th floor | new, t 1.212.290.4700 n 10118-3299 u| water, sanitation, hygiene, and waste management for the covid-19 virus: interim guidance rethinking gender, heterosexual men, and women's vulnerability to hiv/aids covid-19 situation reports accelerate progress-sexual and reproductive health and rights for all: report of the guttmacher-lancet commission do family planning programs decrease poverty? evidence from public census data covid-19 could lead to millions of unintended pregnancies, new un-backed data reveals gender-based violence increases risk of hiv/aids for women in sub-saharan africa -population reference bureau experiences of gender-based violence among female sex workers, men who have sex with men, and transgender women in latin america and the caribbean: a qualitative study to inform hiv programming uk. institute for fiscal studies warns impact of lockdown to hit women, you people and low earners hardest multisector impact assessment of gender dimensions of the ebola virus disease covid-19: women front and centre repor t/how-women s-parti cipat ion-confl ict-preve ntion -and-resol ution -advan ces-us-inter ests 29. women in informal economy infor mal-econo my 30. pandemics and violence against women and children key: cord-316789-nb4437qs authors: omel’yanchuk, l. v.; yudina, o. s. title: drosophila melanogaster as a model for studying the function of animal viral proteins date: 2011-07-16 journal: russ j genet doi: 10.1134/s1022795411040090 sha: doc_id: 316789 cord_uid: nb4437qs studies in which drosophila melanogaster individuals carrying transgenes of animal viruses were used to analyze the action of animal viral proteins on the cell are reviewed. the data presented suggest that host specificity of viruses is determined by their proteins responsible for the penetration of the virus into the cell, while viral proteins responsible for interactions with the host cell are much less host-specific. due to this, the model of drosophila with its developed system of searching for genetic interactions can be used to find intracellular targets for the action of viral proteins of the second group. drosophila melanogaster is a well studied model organism containing (according to the drosophila genome project) 10000-12000 genes [1] . many of these genes are similar to genes of other animals as well as to human genes. it was found that 70% of droso phila genes have human homologs [2] and 75% of human disease associated genes have homologs in drosophila [3] . irrespective of currently available large collections of mutant strains, it becomes more conve nient to study the function of drosophila genes with the use of rather elaborate transgenic constructs [4] . the system of ectopic expression of gal4 uas genes consists of two components: a driver (p element con taining the yeast gal4 protein coding region under the control of the genomic enhancer located close to the integrated element) and a target transgene under the control of the gal4 specific promoter uas (upstream activated system) [5] . drosophila has a set of drivers (hs gal4) able to activate uas transgenes in various tissues at different stages of development. there is also a possibility to activate the tissue specific transgene conditionally using an external stimulus and even to form a feedback loop for autoactivation of the trans gene (uas gal4). these possibilities, initially devel oped for genetic analysis of drosophila genes, can also be used to analyze the functions of foreign genes. viral genomes can maximally encode four to sev eral hundred proteins (the genome of mimivirus is 1.2 megabases in size and contains 1262 reading frames corresponding to 911 proteins [6] ), although most typical is the influenza virus encoding 11 proteins [7] . viral genomes encode viral envelope proteins responsible for interactions of the virus with the host cell at the stage of penetration into the cell. at the same time, it is commonly known that in addition to these proteins serving the structural function viral genomes contain genes responsible for finer aspects of interaction with host cells: cell proliferation (in fact, isolation of oncogenes was made on the basis of this property) and cell apoptosis. in this work, we review the results and methodical approaches of studies in which drosophila was used to analyze human viral infections. drosophila was successfully used to study viral genes and proteins inhibiting programmed death (apoptosis) of host cells. animal viruses are known to carry genes able to prevent apoptosis of host cells [8] [9] [10] [11] [12] [13] [14] [15] [16] . on this basis, hay et al. [17] cloned the gene p35 of the autographa californica baculovirus in the pgmr (glass multimer reporter) vector able to pro vide transgene overexpression in the eye. the effect of transgene overexpression in embryogenesis was exam ined in individuals containing an insertion of this vec tor and additionally carrying the hs glass transgene capable of activating the pgmr transgene in all tissues under the action of heat shock. heat shock induction in such individuals resulted in the lack of apoptosis in late embryos, while it is normally observed in wild type individuals [18] . to study apoptosis of eye cells, activation of the pgmr transgene was used. inhibition of apoptosis characteristic of normal development was demonstrated on the level of eye morphology of an adult individual and on the imaginal disc level. inhibi omel'yanchuk, yudina tion of apoptosis induced by x irradiation was also demonstrated. at present, constructs based on p35 serve as a commonly accepted test for classifying cell death as apoptosis. drosophila was successfully used to study hiv infec tion. the authors of the work [19] cloned the hiv tat gene in the pcasper vector containing the hs promoter capable of heat shock activation of transgene expression in all tissues. activation of the transgene in oogenesis resulted in an essential increase (from 1 to 10%) of the share of eggs with the phenotype of fused dorsal appendages. the authors found that the tat protein affects the localization of the gurken and kinesin pro teins, i. e., it alters both the dorsoventral and anteropos terior axes of embryos. activation of transgene expres sion in spermatogenesis showed colocalization of tat with alfa tubulin. furthermore, the authors established in an in vitro experiment that tat inhibits the assembly of microtubules. the study of the viral protein in accor dance to the commonly accepted scheme showed that it functions in the process of viral rna translation [20] . the study with the use of the drosophila model made it clear that in addition to this function the protein is also able to change the cell structure. to study the role of the hiv nef gene in hiv infec tion, lee et al. [21] cloned dna of this gene and dna of its mutant form (g2a substitution altering the myris tolation site of the protein) into the puast vector and activated the transgene with the use of drivers 1096 gal4 and ap gal4 in wing imaginal discs. expression of the wild type gene led to abnormalities in the wing shape, while no abnormalities were observed in the mutant. using brdu incorporation and detection of mitoses with the help of antibodies against the phosphorylated h3 his tone form, it was found that ectopic expression of hiv nef itself and its mutant form had no influence on wing cell proliferation. yet, ectopic expression increased the level of apoptosis, which was seen with the use of both acridinorange detection and antibodies against drosophila drice caspase. the authors found that genes involved in the jnk (june n terminal kinase) signaling pathway (bsk, hep) genetically interact with hiv nef (both uas con structs and point mutations) under conditions of overex pression, and the activity of the puc gene, which is a target for the action of bsk, is enhanced in the presence of an additional amount of the hiv nef gene product. since the activity of the jnk signaling pathway in drosophila is characterized by a complicated spatial regulation (for example, dorsal and thorax closures regulated by jnk are complicated events of morphologcal alterations of an embryo or a growing pupa), the authors also checked cel lular autonomy of the action of hiv nef with the help of the mosaic analysis. then the authors established that hiv nef, but not its mutant form, inhibits expression of the antimicrobial peptide dpt of drosophila. the protein relish (transcription factor nf kb) of drosophila, which controls dpt transcription, is translocated from the cyto plasm into the nucleus in the case of microbial infections. hiv nef expression inhibits this change in the localization of relish. the authors also studied the question as to whether inhibition of drosophila immunity is jnk dependent and showed that the effects of hiv nef on immunity and on jnk are independent of one another. thus, despite a small length (206 a.a.), this viral protein has two different targets in animal cells. leulier et al. [22] cloned cdna of the hiv vpu gene and its mutant form (ser52 and ser56 substituted by arg), which does not permit phosphorylation of the product, into the puast vector. the transgene was expressed with the use of the yolk gal4 driver activating expression in the fat body. in human cells, hiv vpu is found in the phosphorylated and nonphosphorylated forms. after treatment with alkaline phosphatase, the difference in the mobility of the proteins disappears. the authors showed that in drosophila cells the protein behaves analogously, i. e., it can be phosphorylated. this fact by itself is a strong argument in favor of using droso phila as a model object for studying viral infections. in drosophila, responsible for resistance to fungal infections is the toll signaling pathway controlling expression of the drs and mtk peptides [23] . induction of these peptides is essentially disturbed in individuals with hiv vpu expres sion, but is not disturbed in the nonphosphorylatable mutant. in support of this, the authors showed that the dominant tl 10b mutation of the toll signaling pathway, which causes connstitutive activation of drs and mtk, does not produce this effect against the background of vpu overexpression. at the same time, the authors found that the resistance to gram positive bacteria associated with the imd signaling activity does not change in the case of hiv vpu overexpression. the data on the ability of the drosophila model to reveal new aspects of hiv-host cells interactions were considered by spresser and carlson [24] . the authors proposed to study additionally genes ccr3, vpr, ca150, pol gamma, gl1, p tefb, and spt 4, 5, 6, since they have homologs in the drosophila genome and may well be of interest for studying hiv infections. drosophila was also used as a model object for studying infections caused by other viruses. to study the functional role of the 3a gene of the sars (severe acute respiratory syndrome) coronavirus encoding a 274 a.a. protein (expression of the protein was con firmed for patients infected by this virus), a transgenic drosophila strain containing a genomic insertion of the gene of this protein in the puast vector was gen erated in the work [25] . ectopic expression of the 3a protein in drosophila under the action of the eye driver gmr gal4 caused the dominant phenotype of the eyes. it was found that ectopic expression of 3a influ ences the vesicular cell traffic and induces cell apopto sis that can be modulated by the level of cellular cyto chrome c and by the activity of caspases destroying cellular structures during apoptosis. as shown by genetic screening, the phenotype resulting from ectopic expression is modified by heterozygous muta tions at 78 loci of drosophila. thus, the use of trans genic drosophila individuals was proved to be efficient for studying the function of viral genes. the epstein-barr virus causes infectious mononu cleosis, lymphomas, and nasopharyngeal carcinoma. the bzlf1 protein functions as a transcriptional acti vator of expression of early genes of the virus and con trols the transition from the latent form to the lytic form. bzlf1 also interferes with the cell cycle regula tion, intercellular signaling, and transcription. to study the function of bzlf1, adamson et al. cloned cdna of the gene into the pgmr vector [26] . the authors also cloned cdna of the mutant form of the a185l gene, which alters the dna binding domain of bzlf1 and leads to an experimentally recorded bind ing defect. several insertions differing in the level of expression of gmr bzlf1 and uas bzlf1 trans genes were obtained. the authors showed that insertions of the con structs obtained into the drosophila genome bring about morphological defects in the drosophila eye as a result of the disappearance of ommatidia. morpholog ical defects of the eye for normal and mutant bzlf1 did not differ, thus confirming that the viral protein acts not through transcription regulation, as was expected from the molecular function of the protein, but in some other manner [26] . the study of cell divi sions in transgenic individuals showed a reduction in the number of dividing cells in the second mitotic wave in the eye imaginal disc and an intensification of cell apoptosis. this is in good agreement with the data obtained for human cells that demonstrate a cell cycle arrest under the action of bzlf1. then adamson et al. [26] checked, using antibod ies against the drosophila eye protein elav, whether bzlf1 acts on differentiating photoreceptors of the eye. it turned out that the protein had no influence on the cells of the photoreceptors at this, later, stage. in addition, the authors activated the uas bzlf1 transgene with the use of the sev gal4 driver (it acts later than elav) and confirmed the lack of the eye phe notype. it was further found that in the case of bzlf1 overexpression the eye conic cells lose cut protein expression. the protein shaven (encoded by the sv gene) normally activates the expression of cut in these cells by binding with its promoter. using anti shaven antibodies, the authors demonstrated that bzlf1 expression affects the localization of shaven and the level of its expression. the authors assumed that the virul protein influences the process of cut transactiva tion with the help of shaven. by comparing the eye morphology in sv/+ and bzlf1/+; sv/+ individuals, the authors revealed genetic interactions between sv and bzlf1. on the basis of this finding, the authors proceeded to studying human homologs of shaven: proteins pax2, pax5, and pax8. since the epstein-barr virus infects b cells of the human immune sys tem, in which pax5 is functional, rather than the pax2 and pax8 homologs, the authors tried to elucidate whether the proteins bzlf1 and pax5 form a com plex. it was found that these proteins are able to form a complex and that the formation of a complex causes binding of bzlf1 with mitotic chromosomes. thus, the authors of this work succeeded in discovering and characterizing the human virul protein with the employment of the methods of molecular genetics of drosophila. the results obtained and the existence of homology between the human and drosophila proteins enabled the authors to return to studying the action of the human proteins. embryonic infection by the human cytomegalovirus (hcmv) leads to microencephalia, microgyria, and hydrocephalus [27] . transgenic constructs were obtained in the work [28] , of which one contained the major immediate early promoter (miep) of hcmv attached to the coding region of the yfp reporter. another con struct contained two viral genes, ie72 and ie86, under the control of the hs promoter of the pcasper vector. the promoter activity was detectable only in early embryos at the stage of blastoderm. this fact agrees well with the data on the expression of this promoter in mouse embryos and is a strong argument in favor of using droso phila as a model object for studying viral infections. moreover, the viral proteins under study were found to be localized in the nucleus. such a localization is expected for transcription factors. overexpression of the construct containing viral genes leads to the death of 20-30% of embryos with a phenotype called by the authors "frag mented embryo." normally, de cadherin in drosophila binds to the arm protein. both proteins are major com ponents of intercellular contacts (adherence junctions). while the localization of de cadherin in transgenic embryos overexpressing a cassette of viral transgenes remains unchanged, the pattern of arm expression rep resenting a system of bands changes up to becoming uni form. the authors concluded that the viral transgenes alter the composition of adherence junctions in embryos. the rabies virus, able to infect different mammals, is considered to have the widest range of hosts among ani mal viruses. at the same time, the action of many animal viruses is species specific [29] . the question arises as to whether the range of hosts is limited by genes determin ing the penetration of the virus into the cell or whether viral genes responsible for virus-host interactions also omel'yanchuk, yudina make a contribution to this limitation. the data consid ered in this review show that viral genes of the latter group can act on a broad diversity of organisms, including drosophila. in particular, the vpu protein of hiv can be phosphorylated in drosophila cells, and the cytomega lovirus promoter (miep) functions only in embryonic tissues of drosophila, which coincides with the phase of its action in mammals (see above). these facts cannot be accidental, and they indicate that the class of viral genes responsible for the interaction of the virus with host cells is similar to house keeping genes that perform the same functions in all animals. this means that host specificity is mainly a result of species specificity of proteins responsible for the penetration of the virus into the cell. the range of intracellular targets for genes of the second type is rather wide: cell apoptosis, assembly of microtubules, jnk signaling, cellular immunity, intercellular contacts, and vesicular traffic. it is hard to imagine that this class of viral proteins influencing all this diversity of cell functions occurs in viruses de novo. it is most likely that genes of this type are adopted by a persisting virus from the host genome, as can be often seen from homology of the primary struc tures. on the other hand, the study of the functions of viral proteins in drosophila can help in revealing the units of regulation of intracellular processes that have not yet been identified in direct studies. recall that adamson et al. [26] succeeded in discov ering and characterizing the human virul protein by the methods of molecular genetics of drosophila, and, pro ceeding from the results obtained and on the basis of homology of the human and drosophila proteins, they could return to studying the action of the virus on the human protein. thus, drosophila can be used as an inter mediate object for studying human viral infections. what tasks can be solved at this stage? first, ectopic expression of transgenes in drosophila is possible at any stage of development and in any tissue. this permits one to find a tissue and a developmental stage having targets for the action of viral proteins. of course, there are other objects more similar to humans (e. g., mouse) that can be used in such studies, but the generation of a transgenic mouse is incomparable in costs with the generation of a transgenic drosophila fly. second, drosophila has an elaborate sys tem of genetic screenings for discovering genetic interac tions. cases of application of this approach can be found in the works [21, 25] . genetic methods permit a more complete characterization of the spectrum of targets for the action of viral proteins. they also make it possible to discover weak interactions that can be revealed only by the modern methods detecting physical binding of mac romolecules. around the genomes: the drosophila genome project developmental control of cell cycle regulators: a fly's perspective a systematic analysis of human disease associated gene sequences in drosophila melanogaster transposable elements as tools for genomics and genetics in drosophila targeted gene expres sion as a means of altering cell fates and generating dominant phenotypes large scale sequencing of human influenza reveals the dynamic nature of viral genome evolution the y134. 5 gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteris tic of programmed cell death in neuronal cells prevention of apoptosis by a baculovirus gene during infection of insect cells an apopto sis inhibiting baculovirus gene with a zinc finger like motif induction of bcl 2 expression by epstein barr virus latent mem brane protein 1 protects infected b cells from pro grammed cell death epstein-barr virus coded bhrf1 protein, a viral homologue of bcl 2, protects human b cells from programmed cell death the 19 kilodalton adenovirus e1b transforming protein inhibits programmed cell death and prevents cytoly sis by tumor necrosis factor alpha a 14.700 mw protein from the e3 region of adenovirus inhibits cytolysis by tumor necrosis factor the 10.400 and 14.500 dalton proteins encoded by region e3 of adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor the e1b 19k protein of group c adenoviruses prevents cytolysis by tumor necrosis factor of human cells but not mouse cells expression of baculovirus p35 prevents cell death in drosophila programmed cell death during drosophila embryo genesis a drosophila model of hiv tat related pathogenicity mechanism of hiv 1 tat rna translation and its activation by the tat protein nef induces apoptosis by activating jnk signaling pathway and inhibits nf b dependent immune responses in droso phila directed expression of the hiv 1 accessory protein vpu in drosophila fat body cells inhibits toll dependent immune responses two distinct pathways can control expression of the drosophila antimicrobial peptide metchnikowin drosophila melano gaster as a complementary system for studying hiv 1 related genes and proteins in vivo func tional characterization of the sars coronavirus 3a protein in drosophila modeling early epstein-barr virus infection in droso phila melanogaster: the bzlf1 protein outcome of symptomatic congenital cytomegalovirus infection, results of long term longitudinal follow up human cytomegalovirus immedi ate early gene expression disrupts embryogenesis in transgenic drosophila understanding viruses key: cord-322915-zrjx31ev authors: demain, arnold l; sanchez, sergio title: microbial drug discovery: 80 years of progress date: 2009-01-09 journal: j antibiot (tokyo) doi: 10.1038/ja.2008.16 sha: doc_id: 322915 cord_uid: zrjx31ev microbes have made a phenomenal contribution to the health and well-being of people throughout the world. in addition to producing many primary metabolites, such as amino acids, vitamins and nucleotides, they are capable of making secondary metabolites, which constitute half of the pharmaceuticals on the market today and provide agriculture with many essential products. this review centers on these beneficial secondary metabolites, the discovery of which goes back 80 years to the time when penicillin was discovered by alexander fleming. back in 1928, alexander fleming 1 began the microbial drug era when he discovered in a petri dish seeded with staphylococcus aureus that a compound produced by a mold killed the bacteria. the mold, identified as penicillium notatum, produced an active agent that was named penicillin. later, penicillin was isolated as a yellow powder and used as a potent antibacterial compound during world war ii. by using fleming's method, other naturally occurring substances, such as chloramphenicol and streptomycin, were isolated. naturally occurring antibiotics are produced by fermentation, an old technique that can be traced back almost 8000 years, initially for beverages and food production. beer is one of the world's oldest beverages, produced from barley by fermentation, possibly dating back to the sixth millennium bc and recorded in the written history of ancient egypt and mesopotamia. another old fermentation, used to initiate the koji process, was that of rice by aspergillus oryzae. during the past 4000 years, penicillium roqueforti has been utilized for cheese production, and for the past 3000 years soy sauce in asia and bread in egypt has represented examples of traditional fermentations. 2 natural products with industrial applications can be produced from primary or secondary metabolism of living organisms (plants, animals or microorganisms). owing to technical improvements in screening programs, and separation and isolation techniques, the number of natural compounds discovered exceeds 1 million. 3 among them, 50-60% are produced by plants (alkaloids, flavonoids, terpenoids, steroids, carbohydrates, etc.) and 5% have a microbial origin. of all the reported natural products, approximately 20-25% show biological activity, and of these approximately 10% have been obtained from microbes. furthermore, from the 22 500 biologically active compounds that have been obtained so far from microbes, 45% are produced by actinomycetes, 38% by fungi and 17% by unicellular bacteria. 3 the increasing role of microorganisms in the production of antibiotics and other drugs for treatment of serious diseases has been dramatic. however, the development of resistance in microbes and tumor cells has become a major problem and requires much research effort to combat it. drugs of natural origin have been classified as (i) original natural products, (ii) products derived or chemically synthesized from natural products or (iii) synthetic products based on natural product structures. evidence of the importance of natural products in the discovery of leads for the development of drugs for the treatment of human diseases is provided by the fact that close to half of the best selling pharmaceuticals in 1991 were either natural products or their derivatives. 4 in this regard, of the 25 top-selling drugs reported in 1997, 42% were natural products or their derivatives and of these, 67% were antibiotics. today, the structures of around 140 000 secondary metabolites have been elucidated. it is important to understand that many chemically synthesized drugs owe their origin to natural sources. applications of chemically synthesized natural metabolites include the use of a natural product derived from plant salicyclic acid derivatives present in white willow, wintergreen and meadowsweet to relieve pain and suffering. concoctions of these plants were administered by hippocrates back in the year 500 bc, and even earlier in egypt and babylonia, for fever, pain and childbirth. synthetic salicylates were produced initially by bayer in 1874, and later in 1897, arthur eichengrun at bayer discovered that an acetyl derivative (aspirin), reduced acidity, bad taste and stomach irritation. these plant-based systems continue to play an essential role in health care, and it has been estimated by the world health organization (who) that approximately 80% of the world's inhabitants rely mainly on traditional medicines for their primary health care. 5 other synthesized compounds originating from natural products include a nonapeptide, designated teprotide, which was isolated from the venom of the brazilian pit viper bothrops jararaca. 6 this led to the design and synthesis of angiotensin-converting enzyme (ace) inhibitors such as captopril, which was the first marketed, orally active ace inhibitor. 7 enalapril, another ace inhibitor used in the treatment of cardiovascular disease, was approved for marketing by the food and drug administration (fda) in 1985. 6 the alkaloid quinine, the active constituent of the 'fever tree' cinchona succirubra, has been known for centuries by south american indians to control malaria. during the twentieth century, massive programs to synthesize quinoline derivatives, based on the quinine prototype, were carried out. the first of the new quinolones to be used clinically as an antibacterial agent was nalidixic acid, which emerged as part of a large chemical synthesis program developed at the sterling winthrop research institute. 8, 9 the program was begun when 7-chloro-1,4-dihydro-1-ethyl-4-oxoquinolone-3-carboxylic acid was obtained as a side product during purification of chloroquine and found to have antibacterial activity. the best compound found in the program was nalidixic acid, which had remarkable activity against gram-negative bacteria and was shown to be an inhibitor of dna gyrase. its discovery led to a whole series of synthetic quinolone and fluoroquinolone antibiotics (pefloxacin, norfloxacin, ciprofloxacin, levofloxacin, ofloxacin, lomefloxacin, sparfloxacin, etc.), which have been very successful in medicine and have achieved major commercial success (table 1) . it is important to appreciate that all quinolones, though synthetic, are based on the structure of the natural plant product quinine. secondary metabolites have exerted a major impact on the control of infectious diseases and other medical conditions, and the development of pharmaceutical industry. their use has contributed to an increase in the average life expectancy in the usa, which increased from 47 years in 1900 to 74 years (in men) and 80 years (in women) in 2000. 11 probably, the most important use of secondary metabolites has been as anti-infective drugs. in 2000, the market for such antiinfectives was us$55 billion (table 1 ) and in 2007 it was us$66 billion. table 1 shows that, among the anti-infective drugs, antivirals represent more than 20% of the market. two antivirals that are chemically synthesized today were originally isolated from marine organisms. they are acyclovir (active against the herpes virus by inhibition and inactivation of dna polymerase) and cytarabine (active against non-hodgkin's lymphoma). both compounds are nucleoside analog drugs, originally isolated from sponges. 12 other antiviral applications of natural compounds are related to human immunodeficiency virus (hiv) treatment. in the pathogenesis of this disease, hiv-1, similar to other retroviruses, depends on its stable integration into the host genome to facilitate efficient replication of the viral rna and maintenance of the infected state. therefore, de novo viral dna synthesized during reverse transcription is immediately integrated into the host cell dna (through the integration step), allowing for further transcription of viral rna. in the late phase of hiv viral replication, the large precursor polyprotein (gag-pol precursor, pr 160) must be appropriately cleaved by a viral protease. the cleavage of the gag precursor protein of hiv is critical for the maturation and infectivity of the viral particle. without the appropriate cleavage of the precursor polyproteins, non-infectious viral particles are generally produced. to confront this problem, a tremendous effort has been made at the us national cancer institute (nci), in search of natural metabolites capable of inhibiting hiv reverse transcriptase and hiv protease. chemically synthesized derivatives of these compounds are the main agents now used against hiv. furthermore, reports have been published on natural product inhibitors of hiv integrase obtained from among the marine ascidian alkaloids; that is, the lamellarins (produced by the mollusk lamellaria sp.), and from terrestrial plants (baccharis genistelloides and achyrocline satureioides). the most consistent anti-hiv activity was observed with extracts prepared from several baccharis species. 13 in addition, nci has been evaluating the hiv-1 inhibitory activity of pepstatin a, a small pentapeptide produced by several streptomyces species. it contains a unique hydroxyamino acid, statine, that sterically blocks the active site of hiv-1 protease. 14, 15 reasons for developing new antibiotics new antibiotics that are active against resistant bacteria are required. bacteria have lived on the earth for several billion years. during this time, they encountered in nature a wide range of naturally occurring antibiotics. to survive, bacteria developed antibiotic resistance mechanisms. therefore, it is not surprising that they have become resistant to most of the natural antimicrobial agents that have been developed over the past 50 years. 16 this resistance increasingly limits the effectiveness of current antimicrobial drugs. the problem is not just antibiotic resistance but also multidrug resistance. in 2004, more than 70% of pathogenic bacteria were estimated to be resistant to at least one of the currently available antibiotics. 17 the so-called 'superbugs' (organisms that are resistant to most of the clinically used antibiotics) are emerging at a rapid rate. s. aureus, which is resistant to methicillin, is responsible for many cases of infections each year. the incidence of multidrug-resistant pathogenic bacteria is increasing. the infectious disease society of america (idsa) reported in 2004 that in us hospitals alone, around 2 million people acquire bacterial infections each year (http://www.idsociety.org/content.aspx?id¼4682). s. aureus is responsible for half of the hospital-associated infections and takes the lives of approximately 100 000 patients each year in the usa alone. 18 the bacteria produce a biofilm in which they are encased and protected from the environment. biofilms can grow on wounds, scar tissues and medical implants or devices, such as joint prostheses, spinal instrumentations, catheters, vascular prosthetic grafts and heart valves. more than 70% of the bacterial species producing such biofilms are likely to be resistant to at least one of the drugs commonly used in anti-infectious therapy. 14 called 'nosocomial bacteria.' more than 60% of sepsis cases in hospitals are caused by gram-negative bacteria. 14 among them, pseudomonas aeruginosa accounts for almost 80% of these opportunistic infections. they represent a serious problem in patients hospitalized with cancer, cystic fibrosis and burns, causing death in 50% of cases. other infections caused by pseudomonas species include endocarditis, pneumonia and infections of the urinary tract, central nervous system, wounds, eyes, ears, skin and musculoskeletal system. this bacterium is another example of a natural multidrug-resistant microorganism. although many strains are susceptible to gentamicin, tobramycin and amikacin, resistant forms have also developed. these multidrug-resistant bacteria make hospitals ''dangerous places to be, especially if you are sick, but even if not.'' 19 although we are seeing a steady increase in resistance in almost every pathogen to most of the current antibiotics over time, not all the antibacterial agents show the same rate of resistance development. for example, antimicrobials such as rifampicin, which targets single enzymes, are most susceptible to the development of resistance, whereas agents that inactivate several targets irreversibly generate resistance more slowly. in addition to the antibiotic-resistance problem, new families of anti-infective compounds are needed to enter the marketplace at regular intervals to tackle the new diseases caused by evolving pathogens. at least 30 new diseases emerged in the 1980s and 1990s and they are growing in incidence. emerging infectious organisms often encounter hosts with no prior exposure to them and thus represent a novel challenge to the host's immune system. several viruses responsible for human epidemics have made a transition from animal host to humans and are now transmitted from human to human. hiv, responsible for the acquired immunodeficiency syndrome (aids) epidemic, is one example. although it has not been proven, it is suspected that severe acute respiratory syndrome (sars), caused by the sars coronavirus, also evolved from a different species. 20 in the early 1990s, after decades of decline, the incidence of tuberculosis began to increase. the epidemic took place owing to inadequate treatment regimens, a diminished public health system and the onset of the hiv/aids epidemic. the who has predicted that between 2000 and 2020, nearly 1 billion people will become infected with mycobacterium tuberculosis and that this disease will cost the lives of 35 million people. sexually transmitted diseases have also increased during these decades, especially in young people (aged 15-24 years). the human papillomavirus, chlamydia, genital herpes, gonorrhea and hiv/aids are examples. hiv/aids has infected more than 40 million people in the world. together with other diseases such as tuberculosis and malaria, hiv/aids accounts for over 300 million illnesses and more than 5 million deaths each year. additional evolving pathogens include the (i) ebola virus, which causes the viral hemorrhagic fever syndrome with a resultant mortality rate of 88%; (ii) the bacterium legionella pneumophila, a ubiquitous aquatic organism that lives in warm environments, which causes legionnaire's disease, a pulmonary infection; (iii) the hantavirus, which can infect humans with two serious illnesses: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome; (iv) at least three species of bacteria from the genus borrelia, which cause lyme disease, an emerging infection. in this case, the infection is acquired from the bite of ticks belonging to several species of the genus ixodes. borrelia burgdorferi is the predominant cause of lyme disease in the us, whereas borrelia afzelii and borrelia garinii are implicated in most european cases. the disease presentation varies widely, and may include a rash and flu-like symptoms in its initial stage, followed by musculoskeletal, arthritic, neurologic, psychiatric and cardiac manifestations. in the majority of cases, symptoms can be eliminated with antibiotics, especially if the treatment begins early in the course of illness. however, late or inadequate treatment can lead to 'late-stage' lyme disease that can be disabling and difficult to treat. 21 (v) other evolving pathogens include the escherichia coli 0157:h7 (enterohemorrhagic e. coli), a strain that causes colitis and bloody diarrhea by producing a toxin called shiga toxin, which damages the intestines. it is estimated that this bacterium causes infection in more than 70 000 patients a year in the usa. another example is (vi) cryptosporidium, an obligate intracellular parasite commonly found in lakes and rivers. cryptosporidium parvum is one of the common species affecting the digestive and respiratory organs. intestinal cryptosporidiosis is characterized by severe watery diarrhea. pulmonary and tracheal cryptosporidiosis in humans is associated with coughing and is frequently a low-grade fever. people with severely weakened immune systems are likely to have more severe and more persistent symptoms than healthy individuals. in the developing world, nearly 90% of the infectious disease deaths are caused by six diseases or disease processes: acute respiratory infections, diarrhea, tuberculosis, hiv, measles and malaria. in both the developing and developed nations, the leading cause of death by a wide margin is acute respiratory disease. in the developing world, acute respiratory infections are attributed primarily to seven bacteria: bordetella pertussis, streptococcus pneumoniae, haemophilus influenzae, staphylococcus aureus, mycoplasma pneumoniae, chlamydophila pneumoniae and chlamydia trachomatis. in addition, the major viral causes of respiratory infections include respiratory syncytial virus, human parainfluenza viruses 1 and 3, influenza viruses a and b, as well as some adenoviruses. these diseases are highly destructive in economic and social as well as in human terms and cause approximately 17 million deaths per year, and innumerable serious illnesses besides affecting the economic growth, development and prosperity of human societies. 22 morse 23 identified six general factors in the emergence of infectious diseases: ecological changes, human demographics and behavior, international travel, technology and industry, microbial adaptation and change, and breakdown in public health measures. 24 one additional reason for developing new antibiotics is related to their own toxicity. as with other therapeutic agents, the use of antibiotics may also cause side effects in patients. these include mild reactions such as upset stomach, vomiting and diarrhea (cephalosporins, macrolides, penicillins and tetracyclines), rash and other mild and severe allergic reactions (cephalosporins and penicillins), sensitivity to sunlight (tetracyclines), nervousness, tremors and seizures (quinolones). some side effects are more severe and, depending on the antibiotic, may disrupt the hearing function (aminoglycosides), kidneys (aminoglycosides and polypeptides) or liver (rifampin). during recent decades, we have seen an increasing number of reports on the progressive development of bacterial resistance to almost all available antimicrobial agents. in the 1970s, the major problem was the multidrug resistance of gram-negative bacteria, but later in the 1980s the gram-positive bacteria became important, including methicillin-resistant staphylococci, penicillin-resistant pneumococci and vancomycin-resistant enterococci. 25 in the past, the solution to the problem has depended primarily on the development of novel antimicrobial agents. however, the number of new classes of antimicrobial agents being developed has decreased dramatically in recent years. the advent of resistant gram-positive bacteria has been noticed by the pharmaceutical, biotechnology and academic communities. some of these groups are making concerted efforts to find novel antimicrobial agents to meet this need. a new glycopeptide antibiotic, teicoplanin, was developed against infections with resistant gram-positive bacteria, especially bacteria resistant to the glycopeptide vancomycin. in another instance, the approach involved the redesign of a mixture of two compounds, called streptogramin, into a new mixture, called pristinamycin, to allow administration of the drug parenterally and in higher doses than the earlier oral preparation. 26 the two components of streptogramin, quinupristin and dalfopristin, were chemically modified to allow intravenous administration. the new combination, pristinamycin, was approved by the fda for use against infections caused by vancomycin-resistant enterococcus faecium. additional moves against resistant microorganisms are the glycylcyclines developed to treat tetracycline-resistant bacteria. these modified tetracyclines show potent activity against a broad spectrum of gram-positive and gram-negative bacteria, including strains that carry the two major tetracycline-resistance determinants, involving efflux and ribosomal protection. two of the glycylcyline derivatives, dmg-mino and dmg-dmdot, have been tested against a large number of clinical pathogens isolated from various sources. the spectrum of activity of these compounds includes organisms with resistance to antibiotics other than tetracyclines; for example, methicillin-resistant staphylococci, penicillin-resistant s. pneumoniae and vancomycin-resistant enterococci. 27 tigecycline was approved by the fda in 2005 as an injectable antibiotic. 28 among the novel class of antimicrobial agents used in treating resistance to gram-positive infections, we can also mention the cyclic lipopeptide antibiotic daptomycin produced by streptomyces roseosporus. this compound was approved in 2003 by the fda for skin infections resulting from complications following surgery, diabetic foot ulcers and burns. it represents the first new natural antibiotic approved in many years. its mode of action is distinct from any other approved antibiotic: it rapidly kills gram-positive bacteria by disrupting multiple aspects of bacterial membrane function (by binding irreversibly to the bacterial cell membrane, causing membrane depolarization, destroying the ion concentration gradient and provoking the efflux of k + ). it acts against most clinically relevant gram-positive bacteria (staphylococcus aureus, streptococcus pyogenes, streptococcus agalactiae, streptococcus dysgalactiae subsp. equisimilis and enterococcus faecalis), and retains in vitro potency against isolates resistant to methicillin, vancomycin and linezolid. traditionally, these infections were treated with penicillin and cephalosporins, but resistance to these agents became widespread. [29] [30] [31] [32] daptomycin seems to have a favorable side effect profile, and it might be used to treat patients who cannot tolerate other antibiotics. telithromycin, a macrolide antibiotic, is the first orally active compound of a new family of antibacterials named the ketolides. it shows potent activity against pathogens implicated in communityacquired respiratory tract infections, irrespective of their b-lactam, macrolide or fluoroquinolone susceptibility. some of the microorganisms susceptible to this antibiotic are pneumococci, h. influenzae and moraxella catarrhalis, including b-lactamase-positive strains. in addition, telithromycin has a very low potential for selection of resistant isolates or induction of cross-resistance found with other macrolides. 33 clavulanic acid, first detected in streptomyces clavuligerus, contains a bicyclic b-lactam ring fused to an oxazolidine ring with an oxygen in place of a sulfur, a b-hydroxyethylidene substituent at c-2 and no acylamino group at c-6. it was first described in 1976 and shown to be a potent inhibitor of the b-lactamases produced by staphylococci and plasmid-mediated b-lactamases of e. coli, klebsiella, proteus, shigella, pseudomonas and haemophilus. although it is a broad-spectrum antibiotic, clavulanic acid possesses only very low antibacterial activity. therefore, the molecule has been combined, as a b-lactamase inhibitor, with a variety of broad-spectrum semisynthetic penicillins. for example, when administered with amoxicillin, it is used for the treatment of infections caused by b-lactamase-producing pathogenic bacteria. 34 it has world sales of over us$1 billion, and in 1995 it was the second largest selling antibacterial drug. clavulanic acid can also be combined with ticarcillin, which is a penicillin effective against organisms such as e. coli, proteus, salmonella, haemophilus, pseudomonas and s. aureus. it is normally used in hospitals for treating severe infections affecting blood or internal organs, bones and joints, upper or lower airways or skin and soft tissue. the combination extends ticarcillin antimicrobial activity by inhibiting the action of the b-lactamases produced by certain bacteria. mycosis is a condition in which fungi pass the resistance barriers of the human or animal body and establish infections. these organisms are harmless most of the time, but sometimes they can cause fungal infections. in most cases, these infections are not life threatening. however, when they are deeply invasive and disseminated, they lead to more serious infections, particularly in critically ill patients, elderly people and those who have conditions that affect the immune system (by disease or through the use of immunosuppressive agents). in addition, the use of antineoplastic and broad-spectrum antibiotics, prosthetic devices and grafts, and more aggressive surgery has increased invasive fungal infections. patients with burns, neutropenia, pancreatitis or after organ transplantation (40% of liver transplants, 15-35% of heart transplants and 5% of kidney transplants) are also predisposed to fungal infection. 35 approximately 40% of death from nosocomial infections are caused by fungi, and 80% of these are caused by candida and aspergillus, although cryptococcus spp., fusarium spp., scedosporium spp., penicillium spp. and zygomycetes are increasingly involved. 36 pulmonary aspergillosis is the main factor involved in the death of recipients of bone marrow transplants, and pneumocystis carinii is the leading cause of death in aids patients from europe and north america. 37 the rising incidence of invasive fungal infections and the emergence of broader fungal resistance have led to the need for novel antifungal agents. amphotericin b is the first-line therapy for systemic infection because of its broad spectrum and fungicidal activity. however, considerable side effects limit its clinical utility. echinocandins are large lipopeptide molecules that inhibit the synthesis of 1,3-b-dglucan, a key component of the fungal cell wall. three echinocandins (caspofungin, micafungin and anidulafungin) have reached the market. caspofungin is also known as pneumocandin or mk-0991. this compound was the first cell-wall-active antifungal approved as a new injectable antifungal; this was in 2000. 38 it irreversibly inhibits 1,3-b-d-glucan synthase, preventing the formation of glucan polymers and disrupting the integrity of fungal cell walls. 39 it is more active and less toxic than amphotericin b and shows a broad spectrum of activity against candida (including fluconozole resistance), aspergillus, histoplasma and p. carinii, the major cause of hiv death. micafungin is licensed for clinical use in asian countries and in the us. this compound exhibits extremely potent antifungal activity against clinically important fungi, including aspergillus and azole-resistant strains of candida. in animal studies, micafungin is as efficacious as amphotericin b with respect to improvement of survival rate. it is characterized by a linear pharmacokinetic profile and substantially fewer toxic effects. anidulafungin is currently licensed in the us. 40 although several new antifungal drugs have been developed in the past 6 years, some patients remain resistant to treatments. the main reasons for this include intrinsic or acquired antifungal resistance, organ dysfunction preventing the use of some agents and drug interactions. in addition, some drugs penetrate poorly into sanctuary sites, including the eye and urine, and others are associated with considerable adverse events. however, there has been some progress. posaconazole is a new member of the triazole class of antifungals. it has shown clinical efficacy in the treatment of oropharyngeal candidiasis and has potential as a salvage therapy for invasive aspergillosis, zygomycosis, cryptococcal meningitis and a variety of other fungal infections. it is available as an oral suspension and has a favorable toxicity profile. the wide spectrum of posaconazole activity in in vitro studies, animal models and preliminary clinical studies suggests that it represents an important addition to the antifungal armamentarium. 41 in addition to the screening programs for antibacterial activity, the pharmaceutical industry has extended these programs to other disease areas. 42, 43 microorganisms are a prolific source of structurally diverse bioactive metabolites and have yielded some of the most important products of the pharmaceutical industry. microbial secondary metabolites are now being used for applications other than antibacterial, antifungal and antiviral infections. for example, immunosuppressants have revolutionized medicine by facilitating organ transplantation. 44 other applications include antitumor drugs, enzyme inhibitors, gastrointestinal motor stimulator agents, hypocholesterolemic drugs, ruminant growth stimulants, insecticides, herbicides, coccidiostats, antiparasitics vs coccidia, helminths and other pharmacological activities. further applications are possible in various areas of pharmacology and agriculture, developments catalyzed by the use of simple enzyme assays for screening before testing in intact animals or in the field. in the year 2000, approximately 10 million new cases of cancer were diagnosed in the world, resulting in 6 million cancer-related deaths. the tumor types with the highest incidence were lung (12.3%), breast (10.4%) and colorectal (9.4%). 45 microbial metabolites are among the most important of the cancer chemotherapeutic agents. they started to appear around 1940 with the discovery of actinomycin and since then many compounds with anticancer properties have been isolated from natural sources. more than 60% of the current compounds with antineoplasic activity were originally isolated as natural products or are their derivatives. among the approved products deserving special attention are actinomycin d, anthracyclines (daunorubicin, doxorubicin, epirubicin, pirirubicin and valrubicin), bleomycin, mitosanes (mitomycin c), anthracenones (mithramycin, streptozotocin and pentostatin), enediynes (calcheamycin), taxol and epothilones. actinomycin d is the oldest microbial metabolite used in cancer therapy. its relative, actinomycin a, was the first antibiotic isolated from actinomycetes. the latter was obtained from actinomyces antibioticus (now streptomyces antibioticus) by waksman and woodruff. 46 as it binds dna at the transcription initiation complex, it prevents elongation by rna polymerase. this property, however, confers some human toxicity and it has been used primarily as an investigative tool in the development of molecular biology. despite the toxicity, however, it has served well against wilms tumor in children. the anthracyclines are some of the most effective antitumor compounds developed, and are effective against more types of cancer than any other class of chemotherapy agents. 47 they are used to treat a wide range of cancers, including leukemias, lymphomas, and breast, uterine, ovarian and lung cancers. anthracyclines act by intercalating dna strands, which result in a complex formation that inhibits the synthesis of dna and rna. it also triggers dna cleavage by topoisomerase ii, resulting in mechanisms that lead to cell death. in their cytotoxic effects, the binding to cell membranes and plasma proteins plays an important role. their main adverse effects are heart damage (cardiotoxicity), which considerably limits their usefulness, and vomiting. the first anthracycline discovered was daunorubicin (daunomycin) in 1966, which is produced naturally by streptomyces peucetius. doxorubicin (adriamycin) was developed in 1967. another anthracycline is epirubicin. this compound, approved by the fda in 1999, is favored over doxorubicin in some chemotherapy regimens as it appears to cause fewer side effects. epirubicin has a different spatial orientation of the hydroxyl group at the 4¢ carbon of the sugar, which may account for its faster elimination and reduced toxicity. epirubicin is primarily used against breast and ovarian cancer, gastric cancer, lung cancer and lymphomas. valrubicin is a semisynthetic analog of doxorubicin approved as a chemotherapeutic drug in 1999 and used to treat bladder cancer. bleomycin is a non-ribosomal glycopeptide microbial metabolite produced as a family of structurally related compounds by the bacterium streptomyces verticillus. first reported by umezawa et al. 48 in 1966, bleomycin obtained fda approval in 1973. when used as an anticancer agent (inducing dna strand breaks), the chemotherapeutic forms are primarily bleomycins a2 and b2. mitosanes are composed of several mitomycins that are formed during the cultivation of streptomyces caespitosus. although the mitosanes are excellent antitumor agents, they have limited utility owing to their toxicity. mitomycin c was approved by the fda in 1974, showing activity against several types of cancer (lung, breast, bladder, anal, colorectal, head and neck), including melanomas and gastric or pancreatic neoplasms. 49 recently, mitomycin dimers have been explored as potential alternatives for lowering toxicity and increasing efficiency. 50 mithramycin (plicamycin) is an antitumor aromatic polyketide produced by streptomyces argillaceous that shows antibacterial and antitumor activity. 51 it is one of the older chemotherapy drugs used in the treatment of testicular cancer, disseminated neoplasms and hypercalcemia. it binds to g-c-rich dna sequences, inhibiting the binding of transcription factors such as sp1, which is believed to affect neuronal survival/death pathways. it may also indirectly regulate gene transcription by altering histone methylation. with repeated use, organotoxicity (kidney, liver and hematopoietic system) can become a problem. streptozotocin is a microbial metabolite with antitumor properties, produced by streptomyces achromogenes. chemically, it is a glucosamine-nitroso-urea compound. as with other alkylating agents in the nitroso-urea class, it is toxic to cells by causing damage to dna, although other mechanisms may also contribute. the compound is selectively toxic to the b-cells of the pancreatic islets. it is similar enough to glucose to be transported into the cell by the glucose transport protein of these cells, but it is not recognized by the other glucose transporters. as b-cells have relatively high levels of glucose permease, the relative streptozotocin toxicity for these b-cells can be explained. 52 in 1982, fda granted approval for streptozotocin as a treatment for pancreatic islet cell cancer. pentostatin (deoxycoformycin) is an anticancer chemotherapeutic drug produced by s. antibioticus. it is classified as a purine analog, which mimics the nucleoside adenosine and thus tightly binds and inhibits adenosine deaminase (k i of 2.5â10 à12 m). it interferes with the cell's ability to process dna. 53 pentostatin is commonly used to treat hairy cell leukemia, acute lymphocytic leukemia, prolymphocytic leukemia (b-and t-cell origin), t-cell leukemia and lymphoma. however, it can cause kidney, liver, lung and neurological toxicity. 54 the fda granted approval for pentostatin in 1993. calicheamicins are highly potent antitumor microbial metabolites of the enediyne family produced by micromonospora echinospora. their antitumor activity is apparently due to the cleavage of double-stranded dna. 55 they are highly toxic, but it was possible to introduce one such compound into the clinic by attaching it to an antibody that delivered it to certain cancer types selectively. this ingenious idea of the wyeth laboratories avoided the side effects of calicheamicin. in this regard, gemtuzumab is effective against acute myelogenous leukemia (aml). calicheamicin is bound to a monoclonal antibody against a transmembrane receptor (cd33) expressed on cells of monocytic/myeloid lineage. cd33 is expressed in most leukemic blast cells, but in normal hematopoietic cells the intensity diminishes with maturation. it was approved by the fda for use in patients over the age of 60 years with relapsed aml who are not considered candidates for standard chemotherapy. 56 a successful non-actinomycete molecule is taxol (paclitaxel), which was first isolated from the pacific yew tree, taxus brevifolia, but is also produced by the endophytic fungi taxomyces andreanae and nodulisporium sylviforme. 57 this compound inhibits rapidly dividing mammalian cancer cells by promoting tubulin polymerization and interfering with normal microtubule breakdown during cell division. the drug also inhibits several fungi (pythium, phytophthora and aphanomyces) by the same mechanism. in 1992, taxol was approved for refractory ovarian cancer, and today it is used against breast and advanced forms of kaposi's sarcoma. 58 a new formulation is available in which paclitaxel is bound to albumin. taxol sales amounted to us$1.6 billion in 2006 for bristol myers-squibb, representing 10% of the company's pharmaceutical sales and its third largest selling product. currently, taxol production uses plant cell fermentation technology. the epothilones (a name derived from its molecular features: epoxide, thiazole and ketone) are macrolides originally isolated from the broth of the soil myxobacterium sorangium cellulosum as weak agents against rust fungi. 59 they were identified as microtubulestabilizing drugs, acting in a similar manner to taxol. 60,61 however, they are generally 5-25 times more potent than taxol in inhibiting cell growth in cultures. five analogs are now undergoing investigation as candidate anticancer drugs, and their preclinical studies have indicated a broad spectrum of antitumor activity, including taxol-resistant tumor cells. with the best currently available therapies, the median survival time for patients with metastatic breast cancer is only 2-3 years, and many patients develop resistance to taxanes or other chemotherapy drugs. one epothilone, ixabepilone, was approved in october 2007 by the fda for use in the treatment of aggressive metastatic or locally advanced breast cancer no longer responding to currently available chemotherapies. 62 in tumor cells, p-glycoprotein reduces intracellular antitumor drug concentrations, thereby limiting access of chemotherapeutic substrates to the site of action. the epothilones are attractive because they are active against p-glycoprotein-producing tumors and have good solubility. 62 epothilone b is a 16-membered polyketide macrolactone with a methylthiazole group connected to the macrocycle by an olefinic bond. testicular cancer is the most common cancer diagnosis in men between the ages of 15 and 35 years, with approximately 8000 cases detected in the united states annually. 63 the majority (95%) of testicular neoplasms are germ cell tumors, which are relatively uncommon carcinomas, accounting for only 1% of all male malignancies. remarkable progress has been made in the medical treatment of advanced testicular cancer, with a substantial increase in cure rates from approximately 5% in the early 1970s to almost 90% today. 64, 65 this cure rate is the highest of any solid tumor, and improved survival is primarily due to effective chemotherapy. a major advance in chemotherapy for testicular germ cell tumors was the introduction of cisplatin in the mid-1970s. two chemotherapy regimens are effective for patients with a good testicular germ cell tumor prognosis: four cycles of etoposide and cisplatin or three cycles of bleomycin, etoposide and cisplatin. 66 of the latter three agents, bleomycin and etoposide are natural products. enzyme inhibitors have received increasing attention as useful tools, not only for the study of enzyme structures and reaction mechanisms but also for potential utilization in medicine and agriculture. several enzyme inhibitors with various industrial uses have been isolated from microbes. 67 the most important are (1) clavulanic acid, the inhibitor of b-lactamases discussed above in the section 'moves against antibiotic resistance development in bacteria,' and the statins, hypocholesterolemic drugs presented below in the section 'hypocholesterolemic drugs.' some of the common targets for other inhibitors are glucosidases, amylases, lipases, proteases and xanthine oxidase (xo). acarbose is a pseudotetrasaccharide made by actinoplanes sp. se50. it contains an aminocyclitol moiety, valienamine, which inhibits intestinal a-glucosidase and sucrase. this results in a decrease in starch breakdown in the intestine, which is useful in combating diabetes in humans. 68 amylase inhibitors are useful for the control of carbohydratedependent diseases, such as diabetes, obesity and hyperlipemia. 69, 70 amylase inhibitors are also known as starch blockers because they contain substances that prevent dietary starches from being absorbed by the body. the inhibitors may also be useful for weight loss, as some versions of amylase inhibitors do show potential for reducing carbohydrate absorption in humans. 71, 72 the use of amylase inhibitors for the treatment of rumen acidosis has also been reported. 73 examples of microbial a-amylase inhibitors are paim, obtained from culture filtrates of streptomyces corchorushii, 74 and tai-a, tai-b, oligosaccharide compounds from streptomyces calvus tm-521. 75 lipstatin is a pancreatic lipase inhibitor produced by streptomyces toxytricini that is used to combat obesity and diabetes. it interferes with the gastrointestinal absorption of fat. 76 the commercial product is tetrahydrolipstatin, which is also known as orlistat. in the pathogenic processes of some diseases, such as emphysema, arthritis, pancreatitis, cancer and aids, protease inhibitors are potentially powerful tools for inactivating target proteases. examples of microbial products include antipain, produced by streptomyces yokosukaensis, leupeptin from streptomyces roseochromogenes and chymostatin from streptomyces hygroscopicus. 70 leupeptin is produced by more than 17 species of actinomycetes. 67 xo catalyzes the oxidation of hypoxanthine to uric acid through xanthine. an excessive accumulation of uric acid in the blood, called hyperuricemia, causes gout. 77 the inhibitors of xo decrease the uric acid levels, which result in an antihyperuricemic effect. a potent inhibitor of xo, hydroxyakalone, was purified from the fermentation broth of agrobacterium aurantiacum sp. nov., a marine bacterial strain. 78 fungal products are also used as enzyme inhibitors against cancer, diabetes, poisonings, alzheimer's disease, etc. the enzymes inhibited include acetylcholinesterase, protein kinase, tyrosine kinase, glycosidases and others. 79 immunosuppresants suppressor cells are critical in the regulation of the normal immune response. an individual's immune system is capable of distinguishing between native and foreign antigens and of mounting a response only against the latter. a major role has been established for suppressor t lymphocytes in this phenomenon. suppressor cells also play a role in regulating the magnitude and duration of the specific antibody response to an antigenic challenge. suppression of the immune response either by drugs or by radiation, to prevent the rejection of grafts or transplants or to control autoimmune diseases, is called immunosuppression. a number of microbial compounds capable of suppressing the immune response have been discovered. cyclosporin a was originally introduced as a narrow-spectrum antifungal peptide produced by the mold, tolypocladium nivenum (originally classified as trichoderma polysporum and later as tolypocladium inflatum), by aerobic fermentation. cyclosporins are a family of neutral, highly lipophilic, cyclic undecapeptides containing some unusual amino acids, synthesized by a non-ribosomal peptide synthetase, cyclosporin synthetase. discovery of the immunosuppressive activity led to its use in heart, liver and kidney transplants and to the overwhelming success of the organ transplant field. 80 cyclosporin was approved for use in 1983. it is thought to bind to the cytosolic protein cyclophilin (immunophilin) of immunocompetent lymphocytes, especially t lymphocytes. this complex of cyclosporin and cyclophilin inhibits calcineurin, which under normal circumstances is responsible for activating the transcription of interleukin-2. it also inhibits lymphokine production and interleukin release and therefore leads to a reduced function of effector t cells. sales of cyclosporin a have reached us$1.5 billion per year. other important transplant agents include sirolimus (rapamycin) and tacrolimus (fk506), which are produced by actinomycetes. rapamycin is especially useful in kidney transplants as it lacks the nephrotoxicity seen with cyclosporin a and tacrolimus. it is a macrolide, first discovered in 1975 as a product of s. hygroscopicus, and was initially proposed as an antifungal agent. however, this was abandoned when it was discovered that it had potent immunosuppressive and antiproliferative properties. this compound binds to the immunophilin fk506-binding protein (fkbp12), and this binary complex interacts with the rapamycin-binding domain and inactivates a serine-threonine kinase termed the mammalian target of rapamycin. the latter is known to control proteins that regulate mrna translation initiation and g1 progression. 81 the antiproliferative effect of rapamycin has also been used in conjunction with coronary stents to prevent restenosis, which usually occurs after the treatment of coronary artery disease by balloon angioplasty. rapamycin also shows promise in treating tuberous sclerosis complex (tsc), a congenital disorder that leaves sufferers prone to benign tumor growth in the brain, heart, kidneys, skin and other organs. in a study of rapamycin as a treatment for tsc, university of california, los angeles (ucla) researchers observed a major improvement in mice regarding retardation related to autism. 82 as rapamycin has poor aqueous solubility, some of its analogs, rad001 (everolimus), cci-799 (tensirolimus) and ap23573 (ariad), have been developed with improved pharmaceutical properties. everolimus is currently used as an immunosuppressant to prevent the rejection of organ transplants. although it does not have fda approval in the usa, it is approved for use in europe and australia, and phase iii trials are being conducted in the us. everolimus may have a role in heart transplantation as it has been shown to reduce chronic allograft vasculopathy in such transplants. 83 everolimus is also used in drug-eluting coronary stents as an immunosuppressant to prevent rejection. cci-779 is a rapamycin ester that can be converted to rapamycin in vivo. rad001 is a rapamycin analog currently being investigated in phase ii trials for recurrent endometrial cancer as a single agent, and in phase i/ii trials for the treatment of glioblastoma in combination with the inhibitor of certain epidermal growth factor receptor and vascular endothelial growth factor receptor family members. 84 ap23573 is a novel non-prodrug rapamycin analog with a nonlinear pharmacokinetic behavior that has demonstrated antiproliferative activity against several human tumor cell lines in vitro and against experimental tumors in vivo. 85 this agent is currently under evaluation in phase i-ii trials, including patients with different tumors. two additional small-molecule rapamycin analogs, ap23841 and ap23675, are currently in preclinical development for the treatment of bone metastases and primary bone cancer. 86 tacrolimus (fk506) was discovered in 1987 in japan. 87 it is produced by streptomyces tsukubaensis. however, its use was almost abandoned because of dose-associated toxicity. dr thomas starzl (university of pittsburgh) rescued it by using lower doses, realizing that it was approximately 100 times more active as an immunosuppressive than cyclosporin a. 88 it was introduced in japan in 1993, and in 1994 it was approved by the fda for use as an immunosuppressant in liver transplantation. furthermore, its use has been extended to include bone marrow, cornea, heart, intestines, kidney, lung, pancreas, trachea, small bowel, skin and limb transplants, and for the prevention of graft-vs-host disease. topically, it is also used against atopic dermatitis, a widespread skin disease. in the laboratory, tacrolimus inhibits the mixed lymphocyte reaction, the formation of interleukin-2 by t lymphocytes, and the formation of other soluble mediators, including interleukin-3 and interferon g. recently, it has been reported that tacrolimus inhibits tumor growth factor-b-induced signaling and collagen synthesis in human lung fibroblastic cells. this factor plays a pivotal role in tissue fibrosis, including pulmonary fibrosis. therefore, tacrolimus may be useful for the treatment of pulmonary fibrosis, although its use in the acute inflammatory phase may exacerbate lung injury. 89 hypocholesterolemic drugs atherosclerosis is generally viewed as a chronic, progressive disease characterized by the continuous accumulation of atheromatous plaque within the arterial wall. the past two decades have witnessed the introduction of a variety of anti-atherosclerotic therapies. the statins form a class of hypolipidemic drugs used to lower cholesterol by inhibiting the enzyme hmg-coa reductase, the rate-limiting enzyme of the mevalonate pathway of cholesterol biosynthesis. inhibition of this enzyme in the liver stimulates low-density lipoprotein (ldl) receptors, resulting in an increased clearance of ldl from the bloodstream and a decrease in blood cholesterol levels. through their cholesterol-lowering effect, they reduce the risk of cardiovascular disease, prevent stroke and reduce the development of peripheral vascular disease. 90 in addition, they are anti-thrombotic and antiinflammatory. currently there are a number of statins in clinical use. the entire group of statins reached an annual market of nearly us$30 billion before it became a generic pharmaceutical. the first member of the group (compactin; mevastatin) was isolated as an antibiotic product of penicillium brevicompactum and later from penicillium citrinum. although not of commercial importance, compactin's derivatives achieved overwhelming medical and commercial success. an ethylated form, known as lovastatin (monacolin k; mevinolin), was isolated in the 1970s in the broths of monascus ruber and aspergillus terreus. 91 lovastatin, the first commercially marketed statin, was approved by the fda in 1987. a semisynthetic derivative of lovastatin is simvastatin, a major hypocholesterolemic drug, selling for us$7 billion per year before becoming generic. another statin, pravastatin (us$3.6 billion per year), is made through different biotransformation processes from compactin by streptomyces carbophilus 92 and actinomadura sp. 93 other genera involved in the production of statins are doratomyces, eupenicillium, gymnoascus, hypomyces, paecilomyces, phoma, trichoderma and pleurotus. 94 a synthetic compound, modeled from the structure of the natural statins, is atorvastin, which has been the leading drug of the entire pharmaceutical industry in terms of market share (approximately us$14 billion per year) for many years. an insecticide is a pesticide used against insects in all developmental forms. they include ovicides and larvicides used against the eggs and larvae of insects, respectively. insecticides are used in agriculture, medicine, industry and households. the use of insecticides is believed to be one of the major factors behind the increase in agricultural productivity in the twentieth century. synthetic insecticides pose some hazards, whereas natural insecticides offer adequate levels of pest control and pose fewer hazards. microbially produced insecticides are especially valuable because their toxicity to non-target animals and humans is extremely low. compared with other commonly used insecticides, they are safe for both the pesticide users and consumers of treated crops. the action of microbial insecticides is often specific to a single group or species of insects, and this specificity means that most microbial insecticides do not naturally affect beneficial insects (including predators or parasites of pests) in treated areas. the spinosyns (a83543 group) are a group of natural products produced by saccharopolyspora spinosa that were discovered in 1989. the researchers isolated spinosyn a and d, as well as 21 minor analogs. they are active on a wide variety of insect pests, especially lepidopterans and dipterans, but do not have antibiotic activity. 95 the compounds attack the nervous system of insects by targeting two key neurotransmitter receptors, with no cross-resistance to other known insecticides. the spinosyns are a family of macrolides with 21 carbon atoms, containing four connected rings of carbon atoms at their core to which two deoxysugars (forosamine and 2,3,4, tri-o-methylrhamnose, which are required for bioactivity) are attached. novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of saccharopolyspora erythraea. 96 a mixture of spinosyn a (85%) and d (15%) (spinosad) is being produced through fermentation and was introduced to the market in 1997 for the control of chewing insects on a variety of crops. spinosyn formulations were recently approved for use on organic crops and for animal health applications. recently, a new naturally occurring series of insect-active compounds was discovered from a novel soil isolate, saccharopolyspora pogona nrrl30141. 97 the culture produced a unique family of over 30 new spinosyns. they have a butenyl substitution at the 21 position on the spinosyn lactone and are named butenyl-spinosyns or pogonins. herbicides are chemicals marketed to inhibit or interrupt normal plant growth and development. they are widely used in agriculture, industry and urban areas for weed management. approximately 30 000 kinds of weeds are widely distributed in the world; yield losses caused by 1800 kinds of weeds are approximately 9.7% of total crop production every year. 98 herbicides provide cost-effective weed control with a minimum of labor. most are used on crops planted in large acreages, such as soy, cotton, corn and canola. 99 there are numerous classes of herbicides with different modes of action, as well as different potentials for adverse effects on health and the environment. over the past century, chemical herbicides, used to control various weeds, may have caused many serious side effects, such as injured crops, threat to the applicator and others exposed to the chemicals, herbicide-resistant weed populations, reduction of soil and water quality, herbicide residues and detrimental effects on non-target organisms. 100 for example, alachlor and atrazine were reported to cause cancer in animal tests. with increasing global environmental consciousness, bioherbicides, which are highly effective for weed control and environmentally friendly as well, are very attractive both for research and for application. microbial herbicides can be divided into microbial preparations (microorganisms that control weeds) and microbially derived herbicides. the first microbial herbicide was independently discovered in germany and japan. in 1972, the zähner group in germany isolated phosphinothricin tripeptide, a peptide antibiotic consisting of two molecules of l-alanine and one molecule of the unusual amino acid l-phosphinothricin; that is, n(4[hydroxyl(methyl)phosphinoyl]homoalanyl)alanylalanine. they isolated it from streptomyces viridochromogenes as a broad-spectrum antibacterial including activity against botrytis cinerea. 101 in japan, it was discovered at the meiji seiki laboratories in 1973 from s. hygroscopicus and named bialaphos. 102 the bioactive l-phosphinothricin is a structural analog of glutamic acid, acting as a competitive inhibitor of glutamine synthetase, and has bactericidal (gram-positive and gram-negative bacteria), fungicidal (b. cinerea) and herbicidal properties. 103 glufosinate (dl-phosphinothricin) (without ala-ala) was developed as a herbicide. therefore, the agent acts as a herbicide with or without ala-ala. bialaphos has no influence on microorganisms in the soil and is easily degraded in the environment, having a half-life of only 2 h. this low level of environmental impact is of great interest to environmentalists. in 2006, the global animal health market was valued at us$16 billion, of which 29% was derived from parasiticides. parasites are organisms that inhabit the body and benefit from a prolonged, close association with the host. antiparasitics are compounds that inhibit the growth or reproduction of a parasite; some antiparasitics directly kill parasites. in general, parasites are much smaller than their hosts, show a high degree of specialization for their mode of life and reproduce more quickly and in greater numbers than their hosts. classic examples of parasitism include the interactions between vertebrate hosts and such diverse animals as tapeworms, flukes, plasmodium species and fleas. parasitic infections can cause potentially serious health problems and even kill the host. parasites mainly enter the body through the mouth, usually through ingestion of tainted food or drink. this is a very common problem in tropical areas, but is not limited to those regions. there are 3200 varieties of parasites in four major categories: protozoa, trematoda, cestoda and nematoda. the major groups include protozoans (organisms having only one cell) and parasitic worms (helminths). each of these can infect the digestive tract, and sometimes two or more can cause infection at the same time. the who reported that approximately 25% of the world's population is infected with roundworms. in addition, a major agricultural problem has been the infection of farm animals by worms. the predominant type of antiparasitic screening effort over the years was the testing of synthetic compounds against nematodes, and some commercial products did result. certain antibiotics were also shown to possess antihelmintic activity against nematodes or cestodes, but these failed to compete with the synthetic compounds. although merck had earlier developed a commercially useful synthetic product, thiabendazole, they had enough foresight to examine microbial broths for antihelmintic activity, and found a non-toxic fermentation broth that killed the intestinal nematode nematosporoides dubius in mice. the streptomyces avermitilis culture, isolated by ō mura and coworkers at the kitasato institute in japan, 104 produced a family of secondary metabolites (eight compounds) with both antihelmintic and insecticidal activities. these compounds, named 'avermectins,' are pentacyclic, 16-membered macrocyclic lactones, that harbor a disaccharide of the methylated sugar, oleandrose, with exceptional activity against parasites, especially nemathelminthes (nematodes) and arthropod parasites (10 times higher than any known synthetic antihelmintic agent). surprisingly, avermectins lack activity against bacteria and fungi, do not inhibit protein synthesis and are not ionophores. instead, they interfere with neurotransmission in many invertebrates, causing paralysis and death by neuromuscular attacks. 105 the annual market for avermectins surpasses us$1 billion. they are used against both nematode and arthropod parasites in sheep, cattle, dogs, horses and swine. a semisynthetic derivative, 22,23-dihydroavermectin b1 ('ivermectin') is 1000 times more active than thiabendazole and is a commercial veterinary product. the efficacy of ivermectin has made it a promising candidate for the control of human onchocerciasis and human strongyloidiasis. 106 another avermectin, called doramectin (or cyclohexyl avermectin b1), produced by 'mutational biosynthesis' was commercialized for use by food animals. 107 a semisynthetic monosaccharide derivative of doramectin called selamectin is the most recently commercialized avermectin, and is active against heartworms (dirofilaria immitis) and fleas in companion animals. although the macrocyclic backbone of each of these molecules (ivermectin, doramectin and selamectin) is identical, there are different substitutions at pharmacologically relevant sites such as c-5, c-13, c-22,23 and c-25. 108 the avermectins are closely related to the milbemycins, a group of non-glycosidated macrolides produced by s. hygroscopicus subsp. aureolacrimosus. 109 these compounds possess activity against worms and insects. coccidiostats are used for the prevention of coccidiosis in both extensively and intensively reared poultry. coccidiosis is the name given to a common intestinal disease caused by the invading protozoan parasites of the genus eimeria that affects several different animal species (cattle, dogs, cats, poultry, etc.). the major damage is caused by the rapid multiplication of the parasite in the intestinal wall and the subsequent rupture of the cells of the intestinal lining, leading to high mortality and severe loss of productivity. coccidia are obligate intracellular parasites that show host specificity; only cattle coccidia will cause disease in cattle; other species-specific coccidia will not. for many years, synthetic compounds were used to combat coccidiosis in poultry; however, resistance developed rapidly. a solution came on the scene with the discovery of the narrow-spectrum polyether antibiotic monensin, which had extreme potency against the coccidian. 110 made by streptomyces cinnamonensis, monensin led the way for additional microbial ionophoric antibiotics, such as lasalocid, narasin and salinomycin. all are produced by various streptomyces species. they form complexes with the polar cations k + , na + , ca 2+ and mg 2+ , severely affecting the osmotic balance in the parasitic cells and thus causing their death. 111 the widespread use of anticoccidials has revolutionized the poultry industry by reducing the mortality and production losses caused by coccidiosis. of great interest was another extremely valuable application of monensin; that is, growth promotion in ruminants. synthetic chemicals had been tested for years to inhibit wasteful methane production by cattle and sheep and increase fatty acid formation (especially propionate) to improve feed efficiency; however, they failed. the solution was monensin, which became a major success as a ruminant growth enhancer. 110 for more than 40 years, certain antibiotics have been used in foodanimal production to enhance feed utilization and weight gain. 112 from a production standpoint, feed antibiotics have been consistently shown to improve animal weight gain and feed efficiency, especially in younger animals. these responses are probably derived from an inhibitory effect on the normal microbiota, which can lead to reduced intestinal inflammation and improved nutrient utilization. 113 pigs in the usa are exposed to a great variety of antibiotics. these include b-lactam antibiotics (including penicillins), lincosamides and macrolides (including erythromycin and tetracyclines). all these groups have members that are used to treat infections in humans. in addition, bacitracin, flavophospholipol, pleuromutilins, quinoxalines and virginiamycin are utilized as growth stimulants. flavophospholipol and virginiamycin are also used as growth promoters in poultry. as described above, cattle are also exposed to ionophores such as monensin to promote growth. the animal health institute of america 114 has estimated that without the use of growth-promoting antibiotics, the usa would require an additional 452 million chickens, 23 million more cattle and 12 million more pigs to reach the levels of production attained by the current practices. considering that animal health research and the development of new anti-infective product discovery have decreased, the discovery of new antibiotics has decreased over the past 15 years, with few new drug approvals. 115 therefore, it will be incumbent on veterinary practitioners to use the existing products in a responsible manner to ensure their longevity. it remains to be seen what effects the dearth of new antibiotics for veterinary medicine will have on the future practice of veterinary medicine, production agriculture, food safety and public health. 116 since the 1999 eu decision to prohibit antibiotic use for foodanimal growth promotion, four antibiotic growth promoters have been banned, including the macrolide drugs tylosin and spiramycin. 117 although macrolides are no longer formally used as 'growth promoters,' their use under veterinary prescription has risen from 23 tons in 1998 to 55 tons in 2001, which suggests that more of them are being used now than before the prohibition. it is well known that the most effective route for feeding is via the gastrointestinal tract. many critically ill patients who accept early feeding improve their health. in some post-operative patients, gastric stasis and excessive volumes in the stomach increase the risk of aspiration and subsequent pneumonia. on account of the importance of achieving early and adequate nutritional intake, it is common practice in many intensive care units to use drugs to improve gastrointestinal motility. erythromycin is a macrolide antibiotic with a broad spectrum of activity. it is well recognized that when prescribed, either intravenously or orally, it causes side effects, such as diarrhea, nausea and vomiting. these side effects are, in part, due to the action of erythromycin at motilin receptors in the gut. this makes this antibiotic very attractive to be used in ill patients with gastrointestinal motility problems. there have been some developments on erythromycin analogs that lack antibiotic action but retain action at motilin receptors. these have been named 'motilides.' 118, 119 recently, an orally active erythromycinderived motilin receptor agonist (mitemcinal) has been tested in patients with idiopathic and diabetic gastroparesis. in both cases, an improvement of gastroparetic symptoms was observed. 120 the 80-year contribution of microorganisms to medicine and agriculture has been overwhelming. however, antibiotic resistance in microbes has created a dangerous situation and the need for new antibiotics is clear. unfortunately, most of the large pharmaceutical companies have abandoned the search for new antimicrobial compounds. owing to the economics, they have concluded that drugs directed against chronic diseases offer a better revenue stream than do antimicrobial agents, as for the latter the length of treatment is short and government restriction is likely. some small pharmaceutical and biotechnology companies are developing antibiotics, but most depend on venture capital rather than sales income, and with the present regulations, they face huge barriers to enter the market. these barriers were raised with the best intentions of ensuring public safety, but will have the opposite effect if they terminate antibiotic development while resistance continues to increase. 121 however, there are some bright possibilities. one of the most promising is the utilization of uncultivated microorganisms. considering that 99% of the bacteria and 95% of the fungi have not been cultivated in the laboratory, putting efforts into finding means to grow such microorganisms are proceeding and succeeding. 122 furthermore, researchers are now extracting bacterial dna from soil and marine habitats, cloning large fragments into, for example, bacterial artificial chromosomes, expressing in a host bacterium and screening the library for new antibiotics. this metagenomic effort is allowing access to a vast untapped reservoir of genetic and metabolic diversity, 123, 124 which could result in the discovery of new and useful natural products. 125 in addition to these two relatively new techniques, the chemical and biological modification of old antibiotics could still supply new and powerful drugs. these comments also apply to non-antibiotics such as antitumor agents and other microbial products. bioactive microbial metabolites. a personal view natural products in drug discovery and development natural products and design: interrelated approaches in drug discovery design of angiotensin converting enzyme inhibitors antibiotics: where did we go wrong? natural and synthetic substances related to human health the future of cephalosporins business infectious history oceans: medicine chests of the future? inhibitors of hiv-1 replication that inhibit hiv integrase medicinals for the millennia. the historical record natural products based anti-hiv drug discovery and development facilitated by the nci developmental therapeutics program the end of an era? where have all the antibiotic patents gone? barriers on the road to new antibiotics cycling may be bad for your health the microbial rosetta stone database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents sherris medical microbiology 4th edn health of nations: combating infectious diseases the public health threat of emerging viral disease the future challenges facing the development of new antimicrobial drugs jr problems with antimicrobial resistance in gram-positive cocci recent progress in the field of antibacterial pristinamycins recent developments in tetracycline antibiotics case studies in current drug development: 'glycylcyclines' development of daptomycin for gram-positive infections lipopeptides, focusing on daptomycin, for the treatment of gram-positive infections daptomycin-a novel antibiotic against gram-positive pathogens overcoming antimicrobial resistance: profile of a new ketolide antibacterial, telithromycin biosynthesis and molecular genetics of clavulanic acid invasive aspergillosis in organ transplant recipients: new issues in epidemiologic characteristics, diagnosis, and management invasive fungal infections: a review of epidemiology and management options antifungal resistance trends towards the year 2000. implications for therapy and new approaches caspofungin acetate: an antifungal agent antifungals targeted to cell wall: focus on b-1,3-glucan synthase role of micafungin in the antifungal armamentarium posaconazole: a new broad-spectrum antifungal agent signal-transduction cascades as targets for therapeutic intervention by natural products thiolactomycin and related analogues as novel anti-mycobacterial agents targeting kasa and kasb condensing enzymes in mycobacterium tuberculosis the combinatorial chemistry of nature anticancer drug discovery and development throughout the world actinomyces antibioticus, a new soil organism antagonistic to pathogenic and non-pathogenic bacteria anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity new antibiotics, bleomycin a and b the fam (5-fluorouracil, adriamycin, mitomycin c) and smf (streptozotocin, mitomycin c, 5-fluorouracil) chemotherapy regimens mitomycin dimers: polyfunctional cross-linkers of dna identification of two genes from streptomyces argillaceus encoding glycosyltransferases involved in transfer of a disaccharide during biosynthesis of the antitumor drug mithramycin glut2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice improved production of pentostatin and identification of fermentation cometabolites pentostatin in t-non-hodgkin's lymphomas: efficacy and effect on cd26+ t lymphocytes cleavage behavior of calicheamicin gamma 1 and calicheamicin t approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia study on the preparation and regeneration of protoplast from taxol-producing fungus nodulisporium sylviforme natural products as sources of new drugs over the last 25 years epothilons a and b: antifungal and cytotoxic compounds from sorangium cellulosum (myxobacteria): production, physico-chemical and biological properties epothilones, a new class of microtubulestabilizing agents with a taxol-like mechanism of action activities of the microtubule-stabilizing agents epothilones a and b with purified tubulin and in cells resistant to paclitaxel (taxol) epothilones: mechanism of action and biologic activity curing metastatic testicular cancer medical treatment of advanced testicular cancer refining the optimal chemotherapy regimen for good-risk metastatic nonseminomatous germ-cell tumors: a randomized trial of the genito-urinary group of the french federation of cancer centers (getug t93bp) enzyme inhibitors of microbial origin chemistry and biochemistry of microbial a-glucosidase inhibitors gastrointestinal and metabolic effects of amylase inhibition in diabetics enzyme inhibitors of marine microbial origin with pharmaceutical importance effect of an a-amylase inhibitor on body weight reduction in obese women effect of a purified amylase inhibitor on carbohydrate metabolism after a mixed meal in healthy humans treatment of rumen acidosis with alpha-amylase inhibitors european patent inhibitory properties of an a-amylase inhibitor, paim, from streptomyces corchorushii novel amylase inhibitors united states patent lipstatin, an inhibitor of pancreatic lipase, produced by streptomyces toxytricini progress towards the discovery of xanthine oxidase inhibitors agar plate method, a new assay for chitinase inhibitors using a chitin-degrading bacterium fungal enzyme inhibitors as pharmaceuticals, toxins and scourge of pcr history of the discovery of cyclosporin and of its early pharmacological development rapamycin, an mtor inhibitor, disrupts triglyceride metabolism in guinea pigs reversal of learning deficits in a tsc2 +/à mouse model of tuberous sclerosis everolimus for the prevention of allograft rejection and vasculopathy in cardiac-transplant recipients targeting mtor signaling for cancer therapy therapeutic targets: mtor and related pathways targeted mtor in human gynecologic cancers fk-506, a novel immunosuppressant isolated from a streptomyces. 1. fermentation, isolation, and physico-chemical and biological characteristics correlation of rejection episodes with fk 506 dosage, fk 506 level and steroid following primary orthotopic liver transplant use of tacrolimus, a potent antifibrotic agent, in bleomycin-induced lung fibrosis statins, high-density lipoprotein cholesterol, and regression of coronary atherosclerosis mevinolin, a highly potent competitive inhibitor of hydroxylmethylglutaryl-coenzyme a reductase and a cholesterol-lowering agent a two-component-type cytochrome p-450 monooxygenase system in a prokaryote that catalyzes hydroxylation of ml-236b to pravastatin, a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme a reductase a new hydroxylase system in actinomadura sp. cells converting compactin to pravastatin production and purification of statins from pleurotus ostreatus (basidiomycetes) strains evaluation and development of spinosyns to control ectoparasites on cattle and sheep engineered biosynthesis of novel spinosyns bearing altered deoxyhexose substituents butenyl-spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes research progress on microbial herbicides weed control for the preservation of biological diversity control of problem vegetation: a key to ecosystem management metabolic products of microorganisms. 98. phosphinothricin and phosphinothricyl-alanyl-analine studies on a new antibiotic, sf-1293. i. isolation and physicochemical and biological characterization of sf-1293 substances biosynthetic gene cluster of the herbicide phosphinothricin tripeptide from streptomyces viridochromogenes tu494 avermectins, antiparasitic lactones produced by streptomyces avermitilis isolated from a soil in japan avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in caenorhabditis elegans efficacy of ivermectin against strongyloides stercoralis in humans doramectin-a potent novel endectocide comparison of ivermectin, doramectin, selamectin, and eleven intermediates in a nematode larval development assay a new family of macrolide antibiotics. structure determination of milbemycins d polyether antibiotics: versatile carboxylic acid ionophores produced by streptomyces the determination of 5 anticoccidial drugs (nicarbazin, lasalocid, monensin, salinomycin and narasin) in animal livers and eggs by liquid chromatography linked with tandem mass spectrometry (lc-ms-ms) the role of enteric antibiotics in livestock production (national association for crop production and animal health antibiotics as growth promotants: mode of action antibiotic resistance back in the news antibiotic development pipeline runs dry the future of anti-infective products in animal health the european ban on growth-promoting antibiotics and emerging consequences for human and animal health bioactive metabolites of em574 and em523, erythromycin derivatives having strong gastrointestinal motor stimulating activity erythromycin as a gastrointestinal prokinetic agent clinical trial: effect of mitemcinal (a motilin agonist) on gastric emptying in patients with gastroparesis-a randomized, multicentre, placebocontrolled study the need for new antibiotics isolating 'uncultivable' microorganisms in pure culture in a simulated natural environment fulfilling the promise of biotechnology new antibiotics from bacterial natural products we thank beatriz ruiz and marco a ortiz for their assistance during the development of this review. key: cord-320832-q1oojklw authors: hanum, nadia; cambiano, valentina; sewell, janey; phillips, andrew n; rodger, alison j; speakman, andrew; nwokolo, nneka; asboe, david; gilson, richard; clarke, amanda; miltz, ada r; collins, simon; lampe, fiona c title: use of hiv pre-exposure prophylaxis among men who have sex with men in england: data from the aurah2 prospective study date: 2020-09-01 journal: lancet public health doi: 10.1016/s2468-2667(20)30186-9 sha: doc_id: 320832 cord_uid: q1oojklw background: since october, 2017 (and until october, 2020), pre-exposure prophylaxis (prep) has only been available in england, uk, through the prep impact trial, by purchasing it from some genitourinary medicine clinics, or via online sources. here we report changes from 2013 to 2018 in prep and postexposure prophylaxis (pep) awareness and use among hiv-negative gay, bisexual, and other men who have sex with men (msm) and assess predictors of prep initiation. methods: in the prospective cohort study attitudes to, and understanding of risk of acquisition of hiv 2 (aurah2), msm were recruited from three sexual health clinics in england: two in london and one in brighton, uk. men were eligible if they were aged 18 years or older and hiv-negative or of unknown hiv status. participants self-completed a baseline paper questionnaire at one of the three clinics between july 30, 2013, and april 30, 2016, and were subsequently able to complete 4-monthly and annual online questionnaires, which were available between march 1, 2015, and march 31, 2018, and collected information on sociodemographics, health and wellbeing, hiv status, and sexual behaviours. prep and pep use in the previous 12 months was obtained at baseline and in annual questionnaires. we assessed trends over calendar time in 3-month periods from first enrolment to the end of the study period (july–december, 2013, was counted as one period) in use of prep and pep using generalised estimating equation logistic models. we used age-adjusted poisson models to assess factors associated with prep initiation among participants who reported never having used prep at baseline. findings: 1162 men completed a baseline questionnaire, among whom the mean age was 34 years (sd 10·4), and of those with available data, 942 (82%) of 1150 were white, 1076 (94%) of 1150 were gay, and 857 (74%) of 1159 were university educated. 622 (54%) of 1162 men completed at least one follow-up online questionnaire, of whom 483 (78%) completed at least one annual questionnaire. overall, prep use in the past year increased from 0% (none of 28 respondents) in july to december, 2013, to 43% (23 of 53) in january to march, 2018. the corresponding increase in prep use among men who reported condomless sex with two or more partners was from 0% (none of 13 respondents) to 78% (21 of 27). pep use peaked in april to june, 2016, at 28% (41 of 147 respondents), but decreased thereafter to 8% (four of 53) in january to march, 2018. among 460 men who had never used prep at baseline, predictors of initiating prep included age 40–44 years (incidence rate ratio [irr] 4·25, 95% ci 1·14–15·79) and 45 years and older (3·59, 1·08–11·97) versus younger than 25 years; and after adjustment for age, recent hiv test (5·17, 1·89–14·08), condomless sex (5·01, 2·16–11·63), condomless sex with two or more partners (5·43, 2·99–9·86), group sex (1·69, 1·01–2·84), and non-injection chemsex-related drugs use (2·86, 1·67–4·91) in the past 3 months, pep use (4·69, 2·83–7·79) in the past 12 months, and calendar year (jan 1, 2017, to march 31, 2018 vs july 30, 2013, to dec 31, 2015: 21·19, 9·48–47·35). non-employment (0·35, 0·14–0·91) and unstable or no housing (vs homeowner 0·13, 0·02–0·95) were associated with reduced rates of prep initiation after adjustment for age. about half of prep was obtained via the internet, even after the prep impact trial had started (11 [48%] of 23 respondents in january to march, 2018). interpretation: prep awareness and use increased substantially from 2013 to 2018 among a cohort of msm in england. improving access to prep by routine commissioning by national health service england could increase prep use among all eligible msm, but should include public health strategies to target socioeconomic and demographic disparities in knowledge and use of prep. funding: national institute for health research. the proud study, an open-label randomised controlled trial carried out at 13 sites in england, uk, in 2015, reported that daily oral pre-exposure prophylaxis (prep) with tenofovir-emtricitabine resulted in an 86% reduction in hiv infection in gay, bisexual, and other men who have sex with men (hereafter referred to as men who have sex with men [msm]). 1 a subsequent modelling study has shown that the introduction of a prep programme for msm in the uk would be costeffective and possibly cost-saving in the long term. 2 in england, prep is freely available to people at risk of hiv only in the context of the prep impact trial by public health england that launched across england in october, 2017. all trial participants will get national health service (nhs) england funded prep until at least october, 2020 (the end of the trial). otherwise, people can legally purchase prep for their own use via the internet or from some genitourinary medicine clinics. a nationally commissioned prep programme for england has been agreed and should be operational by the end of the prep impact trial. among gay and bisexual men in the uk, modelling of available data suggests that the estimated annual number of new hiv infections has decreased by 71%, from 2800 in 2012, to 800 in 2018. 3 the annual number of hiv diagnoses recorded among gay and bisexual men in the uk has also decreased by 35%, from 3480 in 2014, to 2250 in 2018. 3 based on a cd4 cell count back-calculation model, the modelled number of incident infections among gay and bisexual in england has decreased by 65% since 2014, with the most rapid decrease occurring after 2016. 3 a combination of prep scale-up, a large increase in ever and repeat hiv testing, and rapid antiretroviral therapy initiation at diagnosis are most likely responsible for these steep decreases in new infections, largely among msm. 4 similar decreases in hiv diagnoses among msm have also been reported in san francisco, ca, and new york city, ny, in the usa, 5, 6 and new south wales, australia. 7 to date, little information exists on trends in prep awareness and uptake and predictors of prep initiation in england. such data would be helpful to further inform the unrestricted prep implementation programme in england in 2020, in which prep will be made routinely available after completion of the prep impact trial. 8 the attitudes to, and understanding of risk of acquisition of hiv 2 (aurah2) study is among the first prospective cohort studies of msm in england. we assessed changes in awareness of, and use of, prep and postexposure prophylaxis (pep), predictors of prep initiation, and factors associated with reporting the recent use of prep among initially hiv-negative msm between 2013 and 2018. the aurah2 study was a prospective cohort study that recruited msm who were hiv negative or of unknown hiv status from two large sexual health clinics in london (56 dean street and mortimer market centre) and one in brighton (claude nicol clinic), between july 30, 2013, and april 30, 2016. 9 additionally, participants were for more on the prep impact trial see https:// www.prepimpacttrial.org.uk/ for more on purchasing prep online see https://www.iwantprepnow.co. uk/buy-prep-now/ evidence before this study pre-exposure prophylaxis (prep) taken daily or on-demand has been shown to be highly effective for prevention of hiv infection among men who have sex with men (msm) in clinical trials and open-label studies. prep is recommended by who for hiv-negative individuals at substantial risk of sexually acquired hiv, including msm. we searched pubmed for longitudinal cohort studies in english that included msm in england, uk, published from database inception up to jan 31, 2020, using key search terms including "pre-exposure prophylaxis", "prep", "hiv", "msm", "homosexual", "men who have sex with men", "gay", "bisexual men", "longitudinal", "cohort", and "prospective". we identified 83 articles, which included articles of clinical trials, demonstration projects, and cohort studies mostly done in high-income countries. apart from reports on prep uptake and associated factors, among these articles were studies about sexual behaviours, sexually transmitted infections, and hiv incidence among prep users; prep awareness, acceptability, and willingness to use; prep retention, engagement, adherence, and discontinuation; and characteristics of prep users. we identified two studies run in england, two articles from the proud trial, and one article from our research group (the attitudes to, and understanding of risk of acquisition of hiv [aurah] and aurah2 study) that measured changes in the prevalence of sexual behaviours and prep use, by comparing data from the aurah cross-sectional study with baseline data from the aurah2 prospective cohort. to our knowledge, no data have been published from longitudinal cohort studies in england, excluding those from clinical trials or prep demonstration projects. this study provides the first estimates of trends in prep use and predictors of prep initiation among hiv-negative msm in england, using data from a prospective cohort, at a critical time in planning the roll-out of prep in england. we found that, despite free prep availability in england being only through the prep impact trial, both awareness and use of prep increased substantially from 2013 to 2018 among msm attending three sexual health clinics in south-east england. in 2018, prep use was almost 80% among men with multiple condomless sex partners. a substantial proportion of men accessing prep obtained it via the internet. prep use was lower among those with lower socioeconomic status than among those of higher socioeconomic status. the available evidence highly supports the addition of prep to the standard of care for msm who would benefit from the preventive prophylaxis. sociodemographic and economic barriers associated with prep use should be promptly addressed via routine commissioning of prep. eligible if they were aged 18 years or older and attending or had attended the study clinics for routine sexually transmitted infection (sti) or hiv testing. individuals who consented to participation in the study completed a confidential baseline paper questionnaire in the clinic. during the follow-up period, participants self-completed sub sequent 4-monthly and annual question naires that were available online from march, 2015, until march, 2018; hence, maximum follow-up was 3 years. the baseline and follow-up questionnaires gathered information on demographic and socio economic factors, health and wellbeing, knowledge and understanding of hiv, life style, sexual health, recent sexual behaviour, and prep and pep use. the study protocol has been described previously. 9 all participants provided written informed consent before taking part. all measures were self-reported from participant question n aires. information on prep and pep use was collected in the baseline and annual questionnaires, with information on prep and pep awareness collected at baseline only. the main outcomes of interest were prep and pep awareness and reported prep and pep use in the previous 12 months. study definitions and questions are shown in figure 1. to be classified as positive for having taken prep or pep in the past 12 months, at baseline or at the annual questionnaire, participants were required to have answered "yes" to the question on having ever taken prep or pep, and subsequently indicated use in the past year on the question on frequency of use. missing answers to prep and pep use questions were classified as no use. sociodemographic variables of interest included age group (<25, 25-29, 30-34, 35-39, 40-44, ≥45 years), country of birth and ethnicity (white uk born, other ethnicity uk born, white non-uk born, other ethnicity non-uk born), sexual identity (gay, bisexual or other), university education (yes, no), ongoing relationship (yes, no), employment status (employed, not employed), sufficient money for basic needs (all of the time, most of the time, sometimes or no), and housing status (homeowner, renting, unstable or other). we considered the following seven measures of hiv risk and related behaviours and activities (in the past 3 months, unless otherwise stated): condomless anal sex (condomless sex), condomless sex with two or more partners, group sex (sex involving more than two participants on the same occasion), recreational drug use classified into four groups (none; drug use but not injection or chemsexrelated; use of at least one chemsex-related drug [crystal metham phetamine, γ-hydroxybutyrate (ghb), γ-butyrolactone (gbl), or mephedrone] but no injection drug use; injection drug use), sti diagnosis (in the past 12 months at the baseline questionnaire, and in the past 3 months at the annual questionnaire), prep or pep use in the past 12 months, and having had a recent hiv test (in the past 6 months at the baseline questionnaire, and in the past 3 months at the annual questionnaire). mental health and lifestyle factors of interest included depressive symptoms (a score of ≥10 on the patient health questionnaire-9), 10 anxiety symptoms (a score of ≥10 on the generalised anxiety disorder-7 test), 11 and higher alcohol consumption (a score of ≥6 on the who alcohol screening tool audit, alcohol use disorders identification test-consumption [audit-c], questionnaire; first two questions only). 12 ethnicity, sexual identity, education, employment, financial status, and housing status were fixed variables derived from the baseline questionnaire, whereas age, depressive symp toms, anxiety symptoms, alcohol consumption, and hiv risk and related behaviours were time-varying variables derived from baseline and annual questionnaires. we treated missing values as "no" answers (these accounted for <5% for each variable), except for the few individuals with in preparation for the analyses, we considered calendar year 3-month periods from the first enrolment (july, 2013) to the end of the study period (march, 2018). information from each participant's questionnaires was ascribed to the 3-month period in which the questionnaire was completed. we combined data for the last two quarters of 2013 as one calendar period (july-december, 2013) because recruitment started on july 30, 2013, and so the third quarter of 2013 was less than 3 months; the number recruited by september, 2013, was too small to have a separate period. to describe the prevalence of prep and pep awareness at baseline, we used data from all available baseline questionnaires. we assessed trends over calendar time in the proportion of participants who indicated prep and pep awareness at baseline over the period from july, 2013, to april, 2016 (enrolment stage), and did a χ² test for linear trends in proportions. to examine trends in past 12-month prep and pep use over the entire study period, we used pooled data from all available baseline and annual questionnaires. we used univariate generalised estimation equation (gee) models with a logit link and robust ses to assess trends over calendar time during the period july, 2013, to march, 2018, in the proportion of questionnaires for which prep and pep use were reported, accounting for multiple questionnaire responses from individual parti cipants. calendar year was fitted as a continuous variable to obtain a test for linear trend. similarly, we investigated trends over calendar time in the proportion of men who reported condomless sex with two or more partners, and trends in prep use among these men specifically. we also assessed trends over time (in 6-month calendar periods) in source of prep, using questionnaires in which prep use was reported. we assessed, in a longitudinal analysis, factors associated with prep initiation during follow-up among those who reported not using prep at baseline and who had completed at least one annual questionnaire. prep initiation was defined as the first report of prep use in the past 12 months from an annual questionnaire; time to initiation was the time from baseline to the date of completion of the questionnaire in which prep was first reported, or from baseline to the end of follow-up if prep was not initiated. we considered each factor separately in age-adjusted poisson models (using age as a continuous variable) with robust ses. the predictors considered included sociodemographic factors, mental health, lifestyle factors, and sexual health and behaviours reported in past questionnaires associated with sub sequent prep initiation. we present these results as age-adjusted incidence rate ratios (irrs) with their corres ponding 95% cis. we did an additional cross-sectional analysis to examine factors associated with being on prep, using all available baseline and annual questionnaires. we used gee models with a logit link function, adjusted for age (as a continuous variable). also, separately among prep users, we analysed factors associated with reporting non-prescribed prep (ie, prep obtained via the internet, friends, or others sources) using all available annual questionnaires in which prep use was reported. we present these results as age-adjusted odds ratios (ors) with their corresponding 95% cis. p values below 0·05 were considered to be significant. we did all analyses using stata (version 15.1). the funder had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. 622 (54%) of 1162 men completed at least one online follow-up questionnaire, of whom 483 (78%) completed at least one annual questionnaire. a higher proportion of participants who were older, had greater financial security, and with more stable housing status continued on the study than did those who were younger, who only sometimes or did not have money to cover basic needs and who had unstable or no housing (table 1). the number of follow-up questionnaires (4-monthly and annual) completed by the end of the study period was 3277. to describe prep and pep awareness at enrolment by calendar period of baseline questionnaire, we used data from 1161 of 1162 participants who completed a baseline questionnaire (one questionnaire was excluded from the analysis due to missing data). overall, at baseline, 838 (72%) of 1161 participants were aware of prep and 1074 (93%) were aware of pep. the data are n/n (%), mean (sd), or median (iqr). data are from baseline paper questionnaire. condomless sex refers to condomless anal sex. data are missing from the baseline questionnaire as follows: age (n=9), money status (n=4), housing status (n=15); country of birth and ethnicity (n=12), sexual identity (n=12), university education (n=3), employment (n=3), relationship (n=3), hiv test (n=3), condomless sex in the past 3 months (n=3), condomless sex with ≥2 partners in the past 3 months (n=3), group sex in the past 3 months (n=3), pep use (n=3), prep use (n=3), recreational drug use (n=3), sti diagnoses (n=3), alcohol consumption (n=3), depressive symptoms (n=3), and anxiety symptoms (n=3). aurah2=attitudes to and understanding of risk of acquisition of hiv 2. prep=pre-exposure prophylaxis. pep=postexposure prophylaxis. sti=sexually transmitted infection. *other ethnicity includes black, asian, mixed, and other ethnic groups. †renting housing includes private renting and renting from council or housing association; unstable or other housing includes temporary accommodation, staying with friends or family, other accommodation, and homeless. ‡in the past 12 months at the baseline questionnaire and in the past 3 months at the annual questionnaire. §higher risk defined as a score of ≥6 on the who modified alcohol screening tool audit, alcohol use disorders identification test-consumption, questionnaire. ¶defined as a score of ≥10 on the patient health questionnaire-9. ||defined as a score of ≥10 on the generalised anxiety disorder-7. figure 2). by contrast, some fluctuation was seen in the trend of past 12-month use of pep. pep use was reported in 371 (18%) questionnaires. pep use was consistently higher than prep use until the period july to september, 2016, but after reaching a peak of 28% (41 of 147 respondents) in april to june, 2016, the data for condomless sex with ≥2 partners are from all baseline and annual questionnaires (ie, 1161 participants provided 2079 questionnaires; one questionnaire was excluded from the analysis due to missing data on year of enrolment). data on prep use in the past 12 months is from those with ≥2 condomless sex partners at baseline and in the annual questionnaires (n=755 questionnaires; one questionnaire was excluded from the analysis due to missing data on year of enrolment). condomless sex is condomless anal sex. p values are for linear trends. prep=pre-exposure prophylaxis. q=quarter. to examine factors associated with initiating prep, we restricted our analysis to the 460 participants who reported no previous prep use in the baseline questionnaire and who completed at least one annual follow-up questionnaire (data from 875 questionnaires). ageadjusted irrs for factors associated with initiating prep are shown in table 2. the prep initiation rate increased substantially from 2013 to 2018. when considering year as a continuous variable, the age-adjusted irr per calendar year was 5·91 (3·68-9·49; p<0·0001). compared with the category of younger than 25 years, rate of prep initiation was increased in the age categories of 40-44 years and age 45 years and older. in age-adjusted models, non-employment and unstable housing were significantly associated with a reduced rate of prep initiation compared with being a homeowner. behavioural factors associated with higher rates of prep initiation within the past 12 months were having a recent hiv test; reporting condomless sex; condomless sex with two or more partners, group sex, use of noninjected chemsex-related drugs, and use of pep in the past 12 months (table 2) . to assess factors associated with reporting prep use in the previous year, we used data from 2080 questionnaires representing 1162 respondents (baseline and follow-up; table 2). the predictors for prep use in the previous year were similar to those associated with initiating prep. in particular, older age and later calendar year were strongly associated with increased use of prep, while unstable or other housing was associated with less use of prep than being a homeowner. we found some evidence of a trend between money status and reporting prep use (p trend =0·037). behavioural factors associated with repor ting the use of prep were a recent hiv test, repor ting condomless sex, condomless sex with two or more partners, group sex, non-injection chemsex-related drugs use, and pep use. country of birth and ethnicity, sexual identity, education, ongoing relationship, higher alcohol use, sti diagnosis, and symptoms of depression and anxiety were not associated with initiation of prep or use of prep (table 2) . in an additional analysis we looked at the association between health and behavioural factors and source of prep amongst those reporting use (190 questionnaires from 128 participants). we found that past 3-month sti diagnosis (unadjusted or from gee-logistic model 2·00, 95% ci 1·09-3·69; p=0·025), condomless sex (unadjusted or 3·55, 95% ci 1·14-11·09; p=0·029), condomless sex with two or more partners (2·22, 1·01-4·85; p=0·045), group sex (2·32, 1·23-4·39; p=0·0090), use of noninjection chemsex-related drugs (3·69, 1·80-7·56; p<0·0001), and calendar year as a continuous variable (2·12, 1·43-3·15; p=0·028) were all associated with reported use of non-prescribed prep (data for other variables are not shown). to our knowledge, this article is the first prospective study of prep use among msm in england. in this study of msm attending sexual health clinics in london and brighton, uk, between 2013 and 2018, we found that use of prep increased substantially over the study period. the internet was the preferred source for obtaining prep, and online prep purchasing still continued even after the prep impact trial started. between january and march, 2018, about 48% of men who reported prep use obtained it from the internet, and paid for it using their own money. a substantial increase in prep use was also seen among men who self-reported engaging in condomless sex with two or more partners; in the period of january to march, 2018, the proportion was 78% compared with 0% in 2013. the increased level of awareness, use, and rates of initiation of prep in this study coincided first with the proud study (nov 29, 2012, to april 30, 2014), 1 and then the initiation of the prep impact implementation trial in england, and the availability of prep through nhs sexual health clinics in scotland and wales, and through a pilot in northern ireland. the prep impact trial started recruitment in 2017 and the rate of prep initiation among the men in our study, who at least at baseline had attended these study sites, increased by more than twenty times in 2017-18 compared with before 2015. the high proportion of men accessing prep online despite the prep impact trial opening for enrolment might have been because available places were rapidly filled and recruitment was closed temporarily. 13 as a result, men in need of prep were being turned away and had no choice but to purchase it via the internet (clarke a and nwokolo n, unpublished). substantial advocacy efforts from community-based organisations have also contributed to some men accessing prep online. in our analysis, older age was independently associated with being more likely to initiate prep, with the rate of initiation among men aged 40 years and older being four times higher than among those younger than 25 years. this finding was similar to that in a cohort in amsterdam in which the median age among men initiating prep was 40 years, 14 and a cohort in australia in which rates of prep initiation were highest among men aged 40 years and older. 15 we also found that indicators of socioeconomic disadvantage (eg, not being employed, having unstable housing status, and having less or no money for basic needs), were associated with a reduced rate of initiating prep or being on prep. previous research in the uk has shown that lower socioeconomic situation is associated with worse hiv treatment outcomes among individuals living with hiv. 16 efforts need to be made to ensure that socioeconomically disadvantaged individuals have equitable access to all effective hiv prevention strategies, including prep. high-risk sexual behaviours such as condomless sex, condomless sex with two or more partners, group sex, and using non-injection chemsex-related drugs were also associated with prep use, indicating appropriate use of prep by these men. similar to our findings, a recent national online prospective study in australia reported that younger age, less use of illicit party or sex drugs, and lower engagement in hiv sexual risk behaviours such as group sex or any condomless sex, were independently associated with non-uptake of prep. 15 the study also reported an increase in the uptake of prep from baseline (2014-15) to 24 months of follow-up. qualitative data from the proud study 17 showed that msm who were already having frequent condomless sex added prep as a prevention tool. msm with a high risk of contracting hiv through condomless sex should be offered prep as a matter of urgency. in our study, more than 22% of respondents in 2018 were not using prep when having condomless sex with multiple partners and so were still at risk. these data would support the national roll-out of prep in england. we did not find an association between sti diagnoses and taking prep, except among men who reported past 12-month non-prescribed prep use. a 2019 meta-analysis of 20 prep studies and trials among msm found high incidences of stis among msm taking prep, ranging from 33·0 per 100 person-years to 99·8 per 100 personyears. 18 however, whether prep use leads to increased rates of stis remains unknown. the meta-analysis generated estimates of sti incidence among msm who engaged in high-risk sexual behaviours, rather than comparing the rates among msm taking prep versus not taking prep. the proud study found extremely high levels of sti diagnoses, but detected no difference in the occurrence of stis between the immediate and deferred prep groups; 1 while, the prepx study in australia found the incidence of stis increased during prep use, but that this finding was partly explained by increased testing frequency. 19 additionally, in the prepx study, half of the participants were not diagnosed with an sti during follow-up and stis were highly concentrated among prep users with repeat stis, and associated with number of partners and group sex. regular sti testing should continue alongside prep use to ensure patients' good sexual health. although in our study we found no significant association between anxiety or depression and reporting recent prep use or prep, in a 2020 australian study, prep use was independently associated with lower levels of hiv-related anxiety among prep-eligible men (msm at high risk of hiv infection) than among prep ineligible men (msm at low risk). 20 alongside the increase in prep use, we found a substantial decreasing trend in pep use between 2013 and 2018, and that the use of pep was a predictor of future prep initiation. this finding suggests that transition from pep use to prep use occurred in these men. guidelines recommend transitioning msm who are at continuous risk of hiv from use of pep towards use of prep. 21 prep taken daily or on-demand before possible exposure is a highly effective strategy for reducing the risk of hiv acquisition among msm who are at high and ongoing risk of infection. 21 pep, on the other hand, is a short-term treatment and is to be used in emergency circumstances after recent hiv exposure (within 72 h). 22 both prep and pep should be a part of combination hiv prevention strategy. our study has some limitations. men in this cohort were recruited from three sexual health clinics where the proud study was run and might have been better informed about prep than the general msm population in england. therefore, prevalence of prep and pep use in this study might overestimate use in the msm population nationwide. men in this cohort were recruited from three sexual health clinics in urban areas in southeast england, and so the sample size was relatively small and these men might not be representative of the broader msm population in england and the uk. trends in use and predictors of prep initiation might also differ among msm who are not engaged with sexual health clinics. additionally, the sample comprised pre dominantly men who were highly educated, employed, in a stable economic situation, of white ethnicity, and with access to the internet (follow-up questionnaires were only available online, therefore to complete the questionnaires participants needed an internet connection), which might not allow generalisability to all msm living in england. however, the australian prospective cohort study that used a more diverse sample of msm reached similar findings as were observed in our study. 15 further research is needed to investigate prep use among msm who are more socioeconomically disadvantaged in england and the uk. recall bias and social desirability bias might be evident in these self-reported data; however, the study collected sensitive and personal data through an online follow-up questionnaire, which might have reduced such bias. 23 finally, the online retention of participants who initially registered in the study was lower than we hoped; however, more than 60% of participants who completed at least one online questionnaire (n=622) were followedup until the end of the study (n=400). 24 in summary, this study provides important data for prep implementation in england. prep use has increased substantially over the past 5 years, with a high proportion of prep being obtained via the internet. our data suggest that men engaging in sexual behaviour related to high hiv risk, who are older, and those of higher socioeconomic status are significantly more likely to use prep. a fully commissioned programme for prep in england has been agreed; however, implementation has been delayed (rodger aj, unpublished). due to the covid-19 pandemic, the programme might not be fully operational across england by the end of the prep impact trial in october, 2020. to transition participants of the prep impact trial onto the nationally commissioned programme, an interim supply of prep will be made available by the trial for participants with an ongoing need for prep and who attend services where the national programme has not yet commenced. the results of our study can inform the implementation of the national programme by highlighted patient groups who might be at increased risk of hiv infection but less likely to be aware of or using prep and who could benefit most from public health outreach and advice. improving access to prep via routine commissioning by nhs england could increase prep use among all eligible msm and reduce socioeconomic disparities, if it is accompanied by an understanding of these disparities and tailoring of public health message and services to address them. pre-exposure prophylaxis to prevent the acquisition of hiv-1 infection (proud): effectiveness results from the pilot phase of a pragmatic open-label randomised trial cost-effectiveness of pre-exposure prophylaxis for hiv prevention in men who have sex with men in the uk: a modelling study and health economic evaluation hiv in the united kingdom: towards zero hiv transmissions by 2030 rapidly declining hiv infection in msm in central london trends in the san francisco human immunodeficiency virus epidemic in the "getting to zero" era hiv surveillance annual report quarter 4 & annual 2018: data report. government of new south wales hiv drug prep to be available across england. london: uk government attitudes to and understanding of risk of acquisition of hiv over time: design and methods for an internet-based prospective cohort study among uk men who have sex with men (the aurah2 study) the phq-9: validity of a brief depression severity measure a brief measure for assessing generalized anxiety disorder: the gad-7 audit: the alcohol use disorders identification test: guidelines for use in primary care, second edition. department of metal health and substance dependence, world health organization prep trial updates pre-exposure prophylaxis among men who have sex with men in the amsterdam cohort studies: use, eligibility, and intention to use hiv pre-exposure prophylaxis (prep) uptake among gay and bisexual men in australia and factors associated with the nonuse of prep among eligible men: results from a prospective cohort study socioeconomic status and treatment outcomes for individuals with hiv on antiretroviral treatment in the uk: cross-sectional and longitudinal analyses the context of sexual risk behaviour among men who have sex with men seeking prep, and the impact of prep on sexual behaviour incidence of sexually transmitted infections in men who have sex with men and who are at substantial risk of hiv infection -a meta-analysis of data from trials and observational studies of hiv pre-exposure prophylaxis association of hiv preexposure prophylaxis with incidence of sexually transmitted infections among individuals at high risk of hiv infection use of hiv pre-exposure prophylaxis associated with lower hiv anxiety among gay and bisexual men in australia who are at high risk of hiv infection: results from the flux study bhiva/bashh guidelines on the use of hiv pre-exposure prophylaxis (prep) 2018 uk guideline for the use of hiv post-exposure prophylaxis following sexual exposure improving the validity of self-reported sexual behavior: no easy answers changes in chemsex and sexual behaviour over time, among a cohort of msm in london and brighton: findings from the aurah2 study ajr, anp, fcl, js, as, nn, da, rg, and ac conceived, designed, and managed the data collection in the aurah2 study. nh, vc, js, and arm conducted data cleaning and variable derivation. sc contributed to the conception of the analysis, interpretation of data, and final approval of the manuscript. nh did all analyses and drafted the manuscript. all authors contributed to the conception of this analysis and participated in interpretation of data, revising, and final approval of the manuscript. key: cord-340763-cxnu9g8y authors: grimm, sebastian k.; battles, michael b.; ackerman, margaret e. title: directed evolution of a yeast-displayed hiv-1 sosip gp140 spike protein toward improved expression and affinity for conformational antibodies date: 2015-02-17 journal: plos one doi: 10.1371/journal.pone.0117227 sha: doc_id: 340763 cord_uid: cxnu9g8y design of an envelope-based immunogen capable of inducing a broadly neutralizing antibody response is thought to be key to the development of a protective hiv-1 vaccine. however, the broad diversity of viral variants and a limited ability to produce native envelope have hampered such design efforts. here we describe adaptation of the yeast display system and use of a combinatorial protein engineering approach to permit directed evolution of hiv envelope variants. because the intrinsic instability and complexity of this trimeric glycoprotein has greatly impeded the development of immunogens that properly represent the structure of native envelope, this platform addresses an essential need for methodologies with the capacity to rapidly engineer hiv spike proteins towards improved homogeneity, stability, and presentation of neutralizing epitopes. we report for the first time the display of a designed sosip gp140 on yeast, and the in vitro evolution of derivatives with greatly improved expression and binding to conformation-dependent antibodies. these efforts represent an initial and critical step toward the ability to rapidly engineer hiv-1 envelope immunogens via directed evolution. broadly neutralizing antibodies (bnabs) are widely thought to represent the best means of preventing hiv infection, making development of an immunogen capable of raising bnabs a cornerstone and priority of vaccine development efforts [1, 2] . unfortunately, various factors relating both to the characteristics of known bnabs as well as the hiv virus itself have pointed toward substantial obstacles to the realization of this goal. the sequence diversity and instability of the trimer, its heavily glycosylated structure, low surface density on the viral particle, and limited access to functionally critical epitopes have confounded efforts to induce bnabs by vaccination [1, 3] . furthermore, cues from natural infection suggest that monoclonal bnabs are uncommon, arise after years of infection and high viral load, fail to control established infection, must have precisely oriented binding interactions, and often have unusual properties [4] [5] [6] [7] [8] [9] [10] [11] , indicating that without a fundamental breakthrough in immunogen design, the generation of such bnabs by vaccination is likely to remain a daunting challenge [12, 13] . even so, exciting progress has been achieved recently in characterizing the neutralizing capacity of antibodies generated in the course of natural infection [11, [14] [15] [16] [17] , as well as in identifying novel bnabs [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . these and other bnabs have greatly informed immunogen design, highlighting new regions of the envelope trimer, variable loops, envelope glycan, the membrane proximal region, novel quaternary epitopes, and receptor and co-receptor binding sites as epitopes with a combination of sufficient conservation and functional relevance to be key targets of an effective antibody response. it is anticipated that with the continued use of high-throughput b-cell screening methods, the set of bnabs with different fine-epitope specificities and viral coverage will continue to grow and provide a rich set of probes to reinvigorate and diversify immunogen design efforts. however, a high-throughput and adaptable platform is required to ensure these findings are efficiently translated into candidate immunogen development. in the context of natural infection, bnabs have tended to be isolated from individuals with high viral loads, persistent antigen exposure, and progressive disease. in the absence of replicating vectors, it is difficult to envision how similar levels of antigen exposure could be accomplished via vaccination. additionally, envelope diversity may be a key driver in the generation of neutralization breadth, posing another fundamental challenge. together, the antigen exposure associated with natural infection likely represents both orders of magnitude greater levels and diversity than can be achieved by current strategies, leading to the discouraging conclusion that a successful immunogen may need to possess an orders of magnitude improved capacity to elicit bnabs over natural envelope. with these technical and immunological gaps in mind, we sought to establish a yeast surface display (ysd) platform to apply directed molecular evolution principles to the development of hiv envelope variants with fundamentally improved biophysical properties. ysd allows the display of millions of sequence variants and selection based on flexible design criteria to allow efficient and deep coverage of the envelope structure:function landscape, representing a potentially enabling technology for the rapid translation of findings from basic studies to the development of novel candidate immunogens. as such, ysd has been established as a powerful method to engineer diverse proteins for a broad range of functional improvements, including stability, specificity, affinity, catalysis, and enantioselectivity [28] [29] [30] [31] . routinely, variants with million-fold improvements can be isolated from large libraries, and repeated cycling of mutagenesis and selection has resulted in evolution of some of the highest affinity synthetic interactions ever observed [32] . indeed, promising efforts aimed at the development of scaffolded epitopes and gp120 cores with desirable properties such as enhanced recognition of germline antibody families have routinely relied upon such combinatorial approaches and ysd-based directed evolution [33, 34] . however, yeast expression and display of complete gp120, much less gp140, has not been described. here we investigated yeast as a host for the display of hiv spike protein variants and report for the first time the display of full-length gp140 on s. cerevisiae. a jr-fl gp140 variant with the sosip mutations served as starting point because it has previously been engineered for stability and homogeneity, was well-expressed in mammalian cells [35] [36] [37] and because the sosip mutations proved key to enabling structural insights into the native hiv-1 envelope trimer [38] [39] [40] [41] . we have applied random mutagenesis and dna shuffling in combination with stringent vrc01 driven selections to isolate gp140 sosip variants with significantly enhanced expression and that bind to conformational hiv bnabs, suggesting proper antigenicity. the yeast-displayed gp140 sosip variants described here demonstrate the feasibility of production of hiv envelope in yeast, and the advantages of this robust platform in evolving variant envelopes. if trimeric presentation can be confirmed or further engineered, ysd may prove a robust platform for the rapid evolution and selection of promising vaccine immunogens. influence of n-glycosylation on binding of conformational hiv antibodies to yeast-produced gp120 since hiv spike proteins are highly glycosylated, proteolytically processed, and contain numerous disulfide bonds, only eukaryotic cells with appropriate protein processing machinery can serve as hosts for the display of functional protein. whereas mammalian cell lines are relatively slow growing, and achieving the single genotype-phenotype linkage requires viral transfections or other rather cumbersome and time-consuming methodologies, the yeast s. cerevisiae is a robust and well-described eukaryotic host for cell-surface display [42] . yeast are capable of displaying complex mammalian glycoproteins such as antibody fragments, full-length antibodies, peptide-mhc molecules, or growth factor receptors [42] [43] [44] [45] , as well as a growing number of viral envelope proteins of fragments thereof such as hemagglutinin, the gp120 core, dengue e protein, sars-cov, west nile virus envelope, and the hepatitis c virus envelope protein e2 [46] [47] [48] [49] [50] [51] , suggesting that the display of hiv envelope protein is feasible in principle. nevertheless, in s. cerevisiae, n-linked glycans are extended and mannose-rich [52] , though p. pastoris yeast strains with human or human-like n-linked glycosyation have been reported to overcome this limitation [53, 54] . to investigate whether heterologous n-glycosylation in yeast has a major effect on the recognition of gp120 by conformational antibodies, we compared hiv-1 jr-fl gp120 produced in either s. cerevisiae strain yvh10 or glycan-engineered p. pastoris [53] for binding to a panel of conformational hiv-1 bnabs. the mannose-rich n-glycosylation in yvh10 is trimmed to a more human-like 5-mannose stem in engineered pichia, which may promote proper protein folding and conformational ab recognition. however, we found that pichia-produced gp120 did not display a clearly improved binding profile across a panel of bnabs (s1 fig.) . furthermore, p. pastoris-produced gp120 appeared somewhat more fragmented on a western blot, suggesting more proteolytic degradation as compared to s. cerevisiae. therefore, and because of the well-established and characterized aga1/2-based display system, s. cerevisiae was chosen as host for hiv spike protein display. design of a codon-optimized gp140 for expression in s. cerevisiae since the secretion of a full-length jr-fl gp140 including the stabilizing sos and i559p substitutions (sosip jr-fl gp140 [36, 37] ) in s. cerevisiae yvh10 had failed, we fragmented the gene and compared the level of secreted protein between a gp120 stripped core construct described previously [47] , gp120 trimmed at n-and c-termini, full-length gp120, a full-length sosip gp140, as well as gp41, and gp41 lacking its hydrophobic fusion peptide (fig. 1a) . notably, proteins were not secreted as aga2 fusions in this study. firstly, both gp120 fragments and fulllength gp120 could be secreted at clearly detectable levels, but not full-length sosip gp140. secondly, the omission of the fusion peptide clearly enhanced secretion of gp41, suggesting that its omission or modification may also improve full-length gp140 secretion. thirdly, gp120 collapsed near its expected mw of 52 kda after treatment with n-glycosidase pngase f, suggesting extensive n-glycosylation ( fig. 1b and 1c ). based on these findings, we rationally designed a jr-fl sosip-based gp140 (designed, or d-sosip) with some modifications: codons were optimized for s. cerevisiae [55] ; hydrophobic amino acids within the fusion peptide were replaced by structurally related but more hydrophilic residues, which if possible were found among circulating viral sequences (fig. 1d) ; and lastly the furin protease cleavage site was substituted with a yeast kex2 recognition site, and a predicted internal kex2 site was removed. we then compared secreted protein content in cell culture supernatant and found that d-sosip, but not the initial jr-fl sosip gp140 could be detected at significant levels. the d-sosip variant was significantly n-glycosylated, and collapsed after pngase f-treatment (s1c fig.) . the rationally designed d-sosip variant and a mutant with disrupted cd4 binding site (cd4bs)-specific ab binding (d-sosip d368r) were displayed as aga2 fusion proteins on yeast ( fig. 2a ), and compared for display level and binding to a panel of hiv bnabs together with the well-characterized and folded yu2 gp120 core [47] and an unrelated viral envelope protein (e2) derived from hepatitis c virus (hcv e2) as positive and negative controls fig. 2) . notably, gp120 core epitopes are included in d-sosip, while the gp120 core lacks many of the epitopes of d-sosip, including variable loop and gp41-associated epitopes (fig. 1a) . induced yeast cells always included a population that did not display the heterologous protein, which is a feature of the aga2 display system [42] . the d-sosip constructs showed extremely low display levels as compared to the gp120 core (fig. 2b) , and only somewhat above background binding of the cd4bs-specific vrc01 ab (fig. 2c) . however, standardizing the binding of hiv-specific abs when variable display level was taken into account lead to detectable binding across a wide range of ab specificities, including conformational, linear, and glycan-dependent sites. yeast-displayed d-sosip bound well to the v3-specific ab 447-52d and to mper abs, as might be expected due to their ability to bind to unconstrained peptides. surprisingly, low but reproducible signal was observed, suggestive of recognition by glycan and trimer-preferring antibodies such as pgt121/126 [56] and pg9/16 [24] (fig. 2d and s2 table) . furthermore, the d-sosip d368r substitution was observed to reduce binding of cd4bs antibodies. nonetheless, the low signal relative to gp120 core for many of the antibodies tested clearly indicated significant room for improvement, which might be achieved by coupling the rational design modifications made thus far with directed evolution strategies. to select for properly folded sequence variants of d-sosip, a library of 2.0x10 7 members was prepared, with each library member containing an average of about 6.7 random amino acid mutations (table 1) . conformation-dependent mabs that specifically bind to discontinuous epitopes served as molecular probes for properly folded antigens. here, the d-sosip library was extensively screened for improved binding to the conformation-dependent cd4bs bnab vrc01, which mimics the natural ligand cd4 in its mode of binding [57] . the pool of clones selected was further diversified to up to 7.7x10 7 members during two additional cycles of error prone pcr and repeated facs selection for improved binding to either vrc01 (track 1), alternating enrichment based on binding to hiv cd4bs abs vrc01, ch31 or pgv04 (track 2), or against a pooled mix of the five cd4bs mabs vrc01, pgv04, ch31, b12 and b6 (track 3), as described in table 1 . these mabs were chosen because they all bind to the conformational, conserved and functionally critical cd4bs, while being different in sequence and paratope, so as to minimize the risk of selecting specific affinity-enhancing contact residues instead of general structurally stabilizing mutations. a comparative binding analysis of the initial d-sosip and the selected pools from cycle 2 onward showed enhanced binding not only to target bnab vrc01, but also to non-target cd4bs mab b6, suggesting a generally improved structural representation of the conformational cd4bs epitope (fig. 3b) , as vrc01 and b6 significantly differ in their epitope footprints within the cd4bs [58, 59] . dna sequencing revealed that two dominating and distinct clusters of sequences had been enriched (s3a fig.) . to select for combinatorial mutants with further improved binding properties among those clusters, and to remove structurally disruptive mutations that likely had been co-selected, d-sosip and the 3.3 pool variants were dna-shuffled [60] at a 1:1 ratio, and then subjected to an additional three rounds of facs following selection tracks 1, 2 and 3. the selected 4.3 population pools showed both greatly enhanced expression and binding to cd4bs mabs vrc01 and b6 as compared to the 3.3 pool selected before dna shuffling (fig. 3) , while there was no difference in binding observed between tracks 1, 2 and 3 (s4 fig.) . a total 16 clones from each selection track were evaluated for binding to vrc01 and absence of binding to two negative control mabs. using an aggregated ranking analysis, the 12 clones with the best vrc01 and least control mab binding were further screened for broad recognition of conformational cd4bs mabs pgv04 and b6, as well as the natural ligand cd4 and gp41 membrane-proximal extracellular region mab 2f5. f425 b4e8 and mper ab 2f5, as compared to the initial d-sosip. perhaps surprisingly, improved binding to natural ligand cd4-fc was not detected, but binding was maintained at a comparable level with d-sosip (fig. 4) . to determine whether the enhancements observed in displayed variants were generalizable to secreted protein, the clone 4.3.d01 was expressed solubly. the evolved variant was subsequently compared to yeast-secreted gp120 for binding to envelope-specific antibodies. notably, reference d-sosip could not be secreted at sufficient quantities in yeast for binding analyses, in line with the extremely low display level of d-sosip observed on yeast cells (figs. 2 and 3 ). beyond enabling expression, the 4.3.d01 clone exhibited improved binding to a number of mabs, notably nih45-46 g54w, relative to gp120. (s7 fig.) , suggesting that the improved binding properties observed on yeast cells correlate with improved binding properties of soluble gp140. mapping selected mutations onto the structure of bg505 sosip.664 mapping of the mutations on the crystal structure of bg505 sosip.664 gp140 [39] showed that the mutations found in 4.3.b01 and 4.3.d01 cluster in a relatively narrow region within the v1/v2 loop region of gp120, but proximal to the cd4bs (fig. 5a) . their side chains are buried within the protein or located in small cavities and do not appear to directly contact cd4bs mabs (fig. 5b) , suggesting that structurally stabilizing mutations have been selected. interestingly, mutated residues h66q and w112r may form hydrogen bonds stabilizing helices within the inner domain of gp120. similarly, i424n may form a novel hydrogen bond with y384 (fig. 5c) . mutation n156i disrupts an n-glycosylation motif located within the v1/v2 loop region. three additional gp120 mutations that were lost after shuffling (t50a, l122f, and s209t) were all surface located, suggesting that surface-located mutations may not be essential to improved protein folding in yeast. the mutations found within gp41 could not be mapped due to disordered electron density in this particular region of the sosip crystal structure. since 4.3.b01 and 4.3.d01 showed virtually identical binding profiles and expression levels (fig. 4) , these gp41 mutations do not appear to strongly contribute to either improved protein expression or ab recognition. a key attribute of most effective anti-viral vaccine responses is development of antibodies to viral envelope proteins which result in the ability to prevent or clear viral infection [61] . to this end, induction of neutralizing antibodies capable of blocking viral infection has been a cornerstone of hiv vaccine efforts. however, hiv has an arsenal of ways to evade antibody recognition, including multiple conformational states such as closed and open, cleaved or uncleaved trimer and various monomeric spike protein forms, which are thought to distract and dilute the adaptive immune response to the infectious "native" envelope trimer [11, [62] [63] [64] . immunogen design efforts aimed to circumvent these barriers stem back to the discovery of the hiv bnab b12, and began with the identification of epitope mimetic peptides that were capable of binding to b12 with high affinity. however, vaccination with mimetic peptides failed to induce abs that bound to gp120 [65] . other more recent approaches have been based on a subtractive principle, in which the immunogenic features of the antigen which lead to nonneutralizing but immunodominant antibody responses were removed or masked with glycans [58, 66, 67] , or even more dramatically, on the direct grafting of functionally relevant sites onto alternative scaffolds [68] [69] [70] , with the goal of focusing the immune response on functionally relevant epitopes. the subtraction of alternative epitopes, however, may not actively promote a response against the desired epitope, and may rather result in a blunted immune response, in which the titer of ab recognizing the selected epitope is not enhanced, but the total antibody response is decreased. while mixed success has recently been reported from these efforts in the setting of hiv [71] , the subtraction of epitopes may unnecessarily constrain the immune response-strongly limiting potentially protective recalled t cell responses, and non-productively decreasing the polyclonality of the antibody response, resulting in reduced recruitment of effector mechanisms reliant on avid interactions with antibody opsonized cells or virus. because these effector mechanisms have a demonstrated importance in preventing acquisition, decreasing viral burden, and delaying progression [72] , the possible immunologic outcome of vaccines with reduced epitopic coverage may be dividing rather than conquering. extensive studies utilizing soluble gp120 have generally failed to provide protective ab responses, and numerous full-length and trimeric gp140-based antigens that may better resemble the structure of the native spike have also been tested [73] [74] [75] , following the rationale that any antibody recognizing the native, trimeric spike should neutralize virus [41] . sosip trimers fall into this class, and a growing number of envelope sequences with sos and ip mutations have been individually constructed and tested, culminating with the bg505 sosip.664 gp140 trimer [39] . a complementary approach to the strictly rational development of stable, uniform and wellbehaved proteins is combinatorial protein engineering, involving screening of large libraries of protein mutants displayed on viruses or cells for improved properties such as affinity, stability or protein expression. toward this end, hiv virus particles have been exposed to temperature or chemical denaturant stress and screened for retained infectivity of mammalian cells. more homogeneous and stable viruses could be selected following this procedure [76] . however, cellular protein display methods, yeast display in particular, have standing as the gold-standard for evolution of complex and glycosylated proteins based on advantages in speed, library construction, scalable selections, and ease of use. our application of this system to a designed variant of jr-fl sosip gp140 represents the first use of a high-throughput platform for whole envelope evolution. the ease with which variants with improved expression and binding to conformational hiv antibodies could be evolved from repeated rounds of random mutagenesis and flow-cytometric selection points toward promising utility. while the mannose-rich glycosylation of s. cerevesiae in theory reduces enthusiasm about this host, major classes of bnabs, including glycan-specific abs, bound to yeast-produced gp140 variants, and loss of only one n-glycosylation motif (n156i) was observed after selection. while improved binding to cd4 was not observed, the selected variants exhibited similar cd4 binding as gp120 core, and showed broadly improved binding across multiple cd4bs mabs, suggesting a generally improved structural representation of this epitope. whether the selected d-sosip variants could elicit improved neutralizing immunity when applied as immunogens is unclear. because improved antigenicity does not equate to improved immunogenicity, immunization studies would be required to evaluate their ability to elicit neutralizing antibodies. differences in glycosylation can also have a large impact on antigenicity [17] , a factor that has not been optimized in our study. importantly, while yeast utilize the same glycosylation motif, the composition of the glycan incorporated differs significantly, and could be engineered. we rather consider the methodology described here and gp140 variants as an enabling platform for future investigations towards development of improved immunogens. a panel of diverse, stabilized gp140 variants is thought to likely be required to cover circulating virus diversity, minimize immune escape by mutation and glycan masking, and elicit broad neutralization activity [15] . the nine mutations within gp120 that contributed to the enhanced properties in yeast are located in a relatively narrow region, either in proximity of the cd4bs or within the v1/v2 loop domain, as determined by homology modeling. interestingly, three of the nine mutations (h66q, w112r and i424n) may form novel hydrogen bonds. the predominantly buried locations of the mutations identified and the predicted presence of additional, buried hydrogen bonds in selected clones suggest that the poor surface display level and limited binding of yeast-displayed gp140 to conformational abs observed initially were related to poor folding of gp140 in s. cerevisiae, and that improved gp120 core packing and hydrogen bonding within the core may have helped to stabilize the protein, enhancing folding and resulting in higher display levels. in fact, the display level of proteins fused to aga2p was shown previously to correlate with their thermal stability and soluble expression levels [77] , and yeast is known to contain a chaperone machinery that can retain and degrade misfolded protein [78] . further, the accumulation of mutations within the v1/v2 loop domain suggest that this region, in addition to the gp120 core itself, had a critical impact on obstructing proper folding of gp120. this finding is further supported by the notion that yeast produced gp120 core lacking this domain binds well to cd4bs conformational abs, while full-length gp120 containing v1/v2 did not. the more hydrophilic gp41 fusion peptide has provided a critical handle to rationally improve expression of d-sosip gp140 in s. cerevisiae, however no additional expression enhancing or apparently structurally stabilizing gp41 mutations were identified in the evolved d-sosip gp140 variants. evidence as to the utility of these rational mutations can be found in the absence of reversions observed among selected clones. notably, this study does not provide any evidence for the display of gp140 trimers on yeast, which from a steric perspective seems unlikely due to clashes of the aga2 cell wall anchoring fusion partners. further, the engineering of the fusion peptide towards reduced hydrophobicity may, while being as conservative as possible, have an effect on an overall trimer structure. to address this, in future studies, a secretion-capture-based display system could be developed [79] , allowing for the evolution of trimeric gp140 spike protein libraries that are non-covalently linked to aga2. the engineered fusion peptide could gradually be mutated back to wild-type if it affects trimer structure, or fusion peptide variants could be selected from yeast-displayed libraries that improve trimer-specific antibody recognition. in summary, the d-sosip gp140 variants evolved and the platform and approach described here provide a step forward toward the development of promising novel hiv immunogens, which could be rapidly and iteratively evaluated in immunization studies in animal models. such evaluation could help elucidate the value of whole envelope strategies relative to subtractive approaches focusing on a restricted set of epitopes. a less hydrophobic fusion peptide proved to be a critical feature to achieve an initial detectable expression level allowing for yeast display-based cell sorting. the shuffling of accumulated mutations and backcrossing to wild type greatly boosted binding to the target hiv mabs and potentially eliminated deleterious mutations. variants with further improved binding to certain hiv bnabs or their germline precursors, following the concept of b-cell-lineage immunogen design [80, 81] may be evolved relatively quickly using the approach described here. glyco-engineered yeast strains [82] could serve as hosts for future yeast-based display systems to select spike protein variants homogeneously decorated with human-like glycan that is involved in binding of many bnab-classes such as pg9/16 [24] or pgt121-123 [56] . such evolved yeast-produced spike protein derivatives may also be well suited for solving additional spike protein structures, facilitating rational vaccine design approaches, and further contributing to the immunogen pipeline. general pcr products were prepared using proofreading phusion dna polymerase (finnzymes) and oligonucleotide primers from integrated dna technologies, unless otherwise stated. dna restriction and modifying enzymes were from new england biolabs. pcr products were purified and gel-extracted using kits from either qiagen or geneaid. dna sequences of all constructs were verified using an applied biosystems model 3100 dna sequencer. e. coli strain dh5 alpha was used as host for molecular cloning. yeast strains s. cerevisiae yvh10 or p. pastoris superman5 (research corporation technologies) were used as hosts for soluble protein secretion and s. cerevisiae eby100 for cell surface display. plasmids pbgp1-jrfl-gp120, pbgp1-jrfl-gp41 and pbgp1-jrfl-gp41dfp for soluble protein secretion in p. pastoris strains were prepared as follows. hiv-1 jr-fl gp120, gp41 and gp41dfp were amplified from pppi4-jrfl-sosipopt_r6 gp140 [36] using primers jrfl-for2 and jrflgp120-rev2, jrflgp41-for2 and jrfl-rev3 or jrflgp41dfp-for and jrfl-rev3 and inserted between the ecori and xbai sites of pbgp1 [83] . plasmids prs-jrfl-gp140, prs-jrfl-gp120h and prs-jrfl-gp120 for soluble protein secretion in s. cerevisiae yvh10 were prepared by amplification of gp140, gp120h or gp120 using primers jrfl-for and jrfl-rev2, jrfl-for and jrflgp120h-rev or jrfl-for and jrflgp120-rev and insertion between eagi and bamhi sites of prs-4g [47] . for plasmid prs-d-sosip, a yeast codon-optimized and designed hiv-1 jrfl gp140, denoted d-sosip, based on jrfl-sosipopt_r6 gp140 [36] was constructed (dna2.0) and inserted between eagi and bamhi sites of prs-4g. the dna sequence of d-sosip is shown in s6 fig. plasmid pcha was obtained as a kind gift from jordi mata-fink [47] . plasmids pcha-d-sosip and pcha-jrfl-gp120 for s. cerevisiae cell surface display were prepared by amplification of gp140 or gp120 from prs-d-sosip using primers gp120co-for and gp140co-rev or gp120co-rev and insertion of pcr products between nhei and bamhi sites of pcha, resulting in an orientation of ha tag, d-sosip, c-myc tag, followed by aga2p from the n to c termini, respectively. prs-d-sosip-d368r was prepared using primers jrfld368r-for and jrfld368r-rev, following the quikchange site-directed mutagenesis procedure (agilent). pcr primers are listed in s1 table. protein production and purification for secretion of soluble hiv spike protein variants lacking any cell-wall anchoring domain from p. pastoris superman5, electro-competent yeast cells were prepared as described previously [84] , transformed with pbgp1 constructs, plated on ypd plates containing 100 î¼g/l zeocin (invivo gen) and incubated at 30â°c for 3 days to obtain single clones. typically 2 to 200 ml ypd cultures containing 100 î¼g/l zeocin were inoculated to an od600 of 0.1 from over-night pre-cultures and grown for 6 h at 30â°c followed by 5 days at 20â°c for constitutive protein expression. for secretion of soluble hiv spike protein variants lacking any cell-wall anchoring domain from s. cerevisiae yvh10, chemically competent yeast cells were prepared and transformed with prs constructs using the frozen-ez yeast transformation ii kit (zymo), plated onsd-caa plates supplemented with 40 mg/l l-tryptophan and incubated at 30â°c for 3 days to obtain single clones. typically, 2 to 50 ml sdcaa cultures supplemented with 40 mg/l l-tryptophan were inoculated from over-night pre-cultures to an od600 of 0.2 and grown for 6 h at 30â°c. for induction of protein production, cells were spun down, suspended in 200 ml sgcaa medium containing 40 mg/l l-tryptophan and grown for 5 days at 20â°c. media compositions have been described previously [84] . secreted spike protein variants were either detected directly in culture supernatant or imac-purified and buffer-exchanged to pbs prior to further analysis. yeast cell surface display and flow-cytometric analysis hiv spike protein variants were displayed on s. cerevisiae eby100 as decribed previously [47] . briefly, s. cerevisiae was transformed with pcha constructs using the frozen-ez yeast transformation ii kit (zymo) and clones were grown on sdcaa plates at 30â°c. typically 2 ml sdcaa medium was inoculated to an od600 of 0.2 from an over-night pre-culture, grown at 30â°c for 6 h, transferred to sgcaa medium and grown for 12 to 18 h at 30â°c. induced cells were analyzed for cell surface display on a macsquant analyzer (miltenyi biotec). typically 10 7 yeast cells were suspended in 200 î¼l pbs supplemented with 0.1% bsa (pbsf) in 96-well plates (greiner), spun down for 4 min at 2500 g and supernatant was removed by vacuum aspiration. cells were the suspended in 50 î¼l pbsf containing 1:200 dilutions of mouse monoclonal anti-ha tag antibody ha.11 clone 16b12 (covance) or chicken polyclonal anti-cmyc tag antibody (gallus immunotechnology) for detection of display levels, and human hiv antibody at various concentration and incubated for 1 h at ambient temperature while shaking at 1000 rpm. cells were then washed twice with 200 î¼l pbsf, suspended in 50 î¼l pbsf containing 1:200 dilutions of polyclonal goat anti-mouse or goat anti-chicken antibody and goat anti-human antibody conjugated to alexa488 or alexa647 (life sciences) and incubated for 20 min at ambient temperature while shaking. cells were again washed once, suspended in 200 î¼l pbsf, respectively, and kept cold for flow-cytometric analyses. routinely, cells were gated using forward-and side scatters and median fluorescence intensities (mfis) were determined from 10,000 scattergated events that were then gated for display signal. the gate for display level was set using a non-displaying control sample, assuring that non-displaying cells are excluded. mfi values associated with antibody binding were then normalized to display level by division with the mfi associated with expression tag (nmfis). all analyses were performed using macsquantify software (miltenyi). yeast-secreted and imac purified gp120 was denatured for 5 min at 95â°c, loaded on a nupage novex 3-8% tris-acetate gel (life scienes), and 150 v were applied for 1 h in mops buffer (life sciences). the gel was stained using gelcode blue stain reagent (pierce) and analyzed using a gel doc xr system (bio-rad). for western blotting analyses of hiv spike protein variants, typically 20 î¼l of denatured and reduced crude culture supernatant were loaded and the gel was blotted for 1.5 h at 30 v on a 0.45 î¼m pore-size pvdf membrane (life sciences). the membrane was washed twice for 10 min in pbs, blocked for 1 h in pbs supplemented with 5% bsa and 0.1% v/v tween 20, washed twice for 10 min in pbs supplemented with 0.05% v/v tween 20 (pbst) and once in pbs. the washed membrane was incubated with 1 ml pbs containing 3% bsa, 0.1% v/v tween 20 and 5 î¼l goat polyclonal anti-gp120 antibody hrp conjugate ab53840 (abcam) for gp120 detection or 1 î¼l mouse monoclonal anti-his antibody hrp conjugate 34460 (qiagen) for his-tag detection while covered by a transparency. the membrane was then washed as before and incubated for 1 min with enhanced chemiluminescence substrate (pierce) and analyzed using the gel doc xr system (bio-rad). jr-fl gp140 library insert was prepared based on a protocol by fromant et al. [85] , aiming for 5 amino acid mutations per gene. a 100 î¼l reaction containing 230 î¼m datp, 200 î¼m dctp, 420 î¼m dgtp, 2.9 mm dttp, 512 pm pcha-d-sosip template dna, 500 nm primer epyd-for2 and 500 nm primer epyd-rev2, 5 î¼g/ml bsa, 3.95 mm mgcl2, 500 î¼m mncl2, 5 u taq dna polymerase (neb) in 50 mm kcl and 10 mm tris-hcl ph 8.7 at 25â°c. thermo-cycling conditions were 94â°c for 5 min, followed by 16 cycles of 94â°c for 1 min, 65â°c for 1 min and 72â°c for 4 min, and finally 72â°c for 10 min. pcr products were column-purified and 200 to 800 ng were used as template for amplification in 1000 to 4000 î¼l of total pcr reaction volume using phusion dna polymerase and primers epyd-for3 and epyd-rev3. thermocycling conditions were 98â°c for 1 min, followed by 35 cycles of 98â°c for 10 s, 60â°c for 30 s and 72â°c for 1 min, and finally 72â°c for 10 min. library vector was prepared by cleaving 250 î¼g of pcha-sosip-jrfl-gp140-design5 with 400 u of bamhi and nhei, respectively, in 1 ml final volume for 8 h at 37â°c, followed by the addition of another 400 u of each enzyme, dtt to 1 mm and incubation over night at 37â°c. the reaction was heat-inactivated for 20 min at 65â°c. vector was gel-extracted using a dna extraction maxi kit (geneaid). both insert and vector preparations were ethanol-precipitated, washed and concentrated with pellet paint co-precipitant (millipore) and suspended in a final volume of approximately 200 î¼l (insert) and 80 î¼l (vector). a total of 1200 î¼l electrocompetent s. cerevisiae eby100 were prepared essentially as described previously [84] , mixed with all insert and vector, split in eight 2 mm cuvettes and transformed at 1.2 kv and 25 uf using a gene pulser xcell (bio-rad). transformed cells were grown in typically 500 ml sdcaa medium and passaged at least once prior to induction and facs. library size was determined by plating dilutions of the transformation on sdcaa plates and counting colonies. dna encoding the enriched and designed gp140 variants was shuffled based on a protocol by stemmer [86] . briefly, 2.5 î¼g of gp140 pcr products were incubated with 1 u dnasei in 50 î¼l for exactly 2 min at 24â°c, respectively, followed by immediate heat-inactivation at 75â°c for 10 min. reactions were separated on a 2% agarose gel and fragments between 25 bp and 1 kb were gel-extracted. approximately 80 ng of digested, selected gp140 pool was assembled with 80 ng of digested, initial d-sosip in 20 î¼l reactions using phusion dna polymerase and 30 s at 98â°c followed by 40 cycles of 10 s at 98â°c, 30 s at 55â°c, 45 s at 72â°c and finally 10 min at 72â°c. exactly 2 î¼l of assembly products were amplified in 50 î¼l reactions using primers epyd-for3, epyd-rev3 and phusion dna polymerase during 1 min at 98â°c, 30 cycles of 10 s at 98â°c, 30 s at 60â°c and 1 min at 72â°c and finally 5 min at 72â°c. the assembled pcr product was ethanol-precipitated, washed, concentrated and used for library preparation as described in the previous section. freshly grown yeast libraries were diluted to an od600 of 0.2 while oversampling the library size at least 10-fold and grown for at least 6 h at 30â°c while shaking at 250 rpm. cells were sedimented, suspended in sgcaa medium and induced for at least 12 h at 30â°c. induced cells were filtered using a 40 î¼m mesh (bd biosciences) and typically 2x10 8 cells were washed 3 times with 1 ml pbsf and incubated for 1 h at ambient temperature with varying concentrations of hiv antibodies (table 1 ) and optionally a 1:200 dilution of chicken polyclonal anti-cmyc tag antibody (gallus immunotechnology) in 500 î¼l pbsf. cells were washed twice with 1 ml pbsf and stained for 20 min on ice with 1:200 dilutions of goat anti-human polyclonal antibody conjugated to alexa647 (life sciences) and optionally goat anti-chicken polyclonal antibody conjugated to alexa488 (life sciences), while shielded from light. cells were washed twice, split in four aliquots and kept as pellets on ice and shielded from light until sorting. facs was performed using an icyt sy3200 cell sorter system (sony). briefly, aliquots of typically 4x10 7 stained yeast cells were suspended in 2 ml pbsf and sorted at around 10,000 events/s using 0.1% to 5% sorting gates in recovery mode during initial and ultra purity mode during later sort cycles. typical sorting gates for initial and later selection rounds are shown in s2 fig.. sorted cells were suspended in sdcaa medium and grown for 12 to 36 h for subsequent sorting rounds or analyses of selected pools. all binding analyses were performed at 25â°c using an octet red96 system (fortebio), 1000 rpm shaking speed and pbs containing 0.1% bsa, 0.02% v/v tween20 as sample buffer. protein a-coated biosensor tips were loaded for 10 min with 10 î¼g/ml igg, dipped in buffer and probed with 500 nm gp120 for 8 min, followed by a dissociation phase of 5 min in buffer. a trace obtained from igg probed with buffer only was subtracted as reference. the gp120 crystal structure used for modeling was taken from 4nco chain a. the cd4-binding site was determined using pymol interface residues script on gp120-cd4 co-crystal structure pdb id: 1gc1 and mapped to the corresponding side chains on 4nco. mutations of d-sosip picked up during selections were mapped onto the bg505 gp140.664 sosip structure via sequence alignment. side chain rotamers with minimal clashes were chosen. hydrogen bonds were designated at distance of <4 ã� and >2 ã�. mutation d180n and those in gp41 were not mapped due to lack of resolution in the crystal structure. toward an antibody-based hiv-1 vaccine rational antibody-based hiv-1 vaccine design: current approaches and future directions induction of immunity to human immunodeficiency virus type-1 by vaccination role of complex carbohydrates in human immunodeficiency virus type 1 infection and resistance to antibody neutralization a fusion-intermediate state of hiv-1 gp41 targeted by broadly neutralizing antibodies cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv-1 antibodies access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1 polyreactivity increases the apparent affinity of anti-hiv antibodies by heteroligation rapid evolution of the neutralizing antibody response to hiv type 1 infection challenges for structure-based hiv vaccine design antibody neutralization and escape by hiv-1 medicine. the need for a global hiv vaccine enterprise limitations to the structure-based design of hiv-1 vaccine immunogens co-evolution of a broadly neutralizing hiv-1 antibody and founder virus mapping the journey to an hiv vaccine developmental pathway for potent v1v2-directed hiv-neutralizing antibodies evolution of an hiv glycan-dependent broadly neutralizing antibody epitope through immune escape comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies efficient neutralization of primary isolates of hiv-1 by a recombinant human monoclonal antibody characteristics of the earliest cross-neutralizing antibody response to hiv-1 a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1 human monoclonal antibody 2g12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1 analysis of memory b cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from hiv-1-infected individuals broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1 structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact hiv-1 env trimers broadly neutralizing hiv antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers highly l and d enantioselective variants of horseradish peroxidase discovered by an ultrahigh-throughput selection method picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling yeast surface display for directed evolution of protein expression, affinity, and stability yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity engineering hiv envelope protein to activate germline b cell receptors of broadly neutralizing anti-cd4 binding site antibodies rational hiv immunogen design to target specific germline b cell receptors oligomeric and conformational properties of a proteolytically mature, disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1 a recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure cryo-em structure of a fully glycosylated soluble cleaved hiv-1 envelope trimer crystal structure of a soluble cleaved hiv-1 envelope trimer cleavage strongly influences whether soluble hiv-1 envelope glycoprotein trimers adopt a native-like conformation a next-generation cleaved, soluble hiv-1 env trimer, bg505 sosip.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies yeast surface display for screening combinatorial polypeptide libraries secretion-and-capture cell-surface display for selection of target-binding proteins yeast surface display of a noncovalent mhc class ii heterodimer complexed with antigenic peptide directed evolution of the epidermal growth factor receptor extracellular domain for expression in yeast fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic h5n1 influenza virus using yeast surface display rapid conformational epitope mapping of anti-gp120 antibodies with a designed mutant panel displayed on yeast genotype-specific neutralization and protection by antibodies against dengue virus type 3 antibody binding site mapping of sars-cov spike protein receptor-binding domain by a combination of yeast surface display and phage peptide library screening antibody recognition and neutralization determinants on domains i and ii of west nile virus envelope protein neutralizing monoclonal antibodies against hepatitis c virus e2 protein bind discontinuous epitopes and inhibit infection at a postattachment step engineering of glycosylation in yeast and other fungi: current state and perspectives engineering complex-type nglycosylation in pichia pastoris using glycoswitch technology production of complex human glycoproteins in yeast gene designer: a synthetic biology tool for constructing artificial dna segments broad neutralization coverage of hiv by multiple highly potent antibodies sequence and structural convergence of broad and potent hiv antibodies that mimic cd4 binding fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the cd4 binding site of human immunodeficiency virus type 1 gp120 focused evolution of hiv-1 neutralizing antibodies revealed by structures and deep sequencing dna shuffling of a family of genes from diverse species accelerates directed evolution correlates of protection induced by vaccination the hiv-1 envelope glycoproteins: fusogens, antigens, and immunogens structural basis of immune evasion at the site of cd4 attachment on hiv-1 gp120 few and far between: how hiv may be evading antibody avidity structure of a high-affinity "mimotope" peptide bound to hiv-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies improved design of an antigen with enhanced specificity for the broadly hiv-neutralizing antibody b12 hyperglycosylated resurfaced stabilized gp120 core as an immunogen elicits antibodies targeted at the cd4-binding site computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic hiv vaccine epitope elicitation of structure-specific antibodies by epitope scaffolds computation-guided backbone grafting of a discontinuous motif onto a protein scaffold an engineered mutant of hiv-1 gp120 formulated with adjuvant quil a promotes elicitation of antibody responses overlapping the cd4-binding site emerging concepts on the role of innate immunity in the prevention and control of hiv infection hiv-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120 human immunodeficiency virus type 1 env trimer immunization of macaques and impact of priming with viral vector or stabilized core protein comparative evaluation of trimeric envelope glycoproteins derived from subtype c and b hiv-1 r5 isolates increased functional stability and homogeneity of viral envelope spikes through directed evolution yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency protein folding and quality control in the endoplasmic reticulum: recent lessons from yeast and mammalian cell systems a flow cytometric assay for screening improved heterologous protein secretion in yeast b-cell-lineage immunogen design in vaccine development with hiv-1 as a case study vaccine design: emerging concepts and renewed optimism humanization of yeast to produce complex terminally sialylated glycoproteins an episomal expression vector for screening mutant gene libraries in pichia pastoris isolating and engineering human antibodies using yeast surface display direct random mutagenesis of gene-sized dna fragments using polymerase chain reaction dna shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution the authors thank dr. rogier sanders for providing the construct encoding for jr-fl sosip gp140, dr. jordi mata-fink and dr. dane wittrup for providing yeast strains and dna constructs, dr. laura walker and dr. dennis burton, and iavi for providing hiv mabs, bryan t. rogers, weijie lin, and morgan gilman for assisting experimentally, austin boesch for producing and providing target bnab vrc01, the nih aids reagent program for providing hiv mabs, dr. karl griswold and dr. sarah dostal for access to a cell sorter and technical assistance, dr. tom scanlon for valuable discussions and dr. benjamin hackel for providing the protocol for yeast library preparation. this study was supported by a grant from the national institute of health (nih), national institutes of allergy and infectious diseases (niaid) nih 1r01ai102691 to mea. key: cord-326642-kc85pev4 authors: cohen, adam l.; sahr, philip k.; treurnicht, florette; walaza, sibongile; groome, michelle j.; kahn, kathleen; dawood, halima; variava, ebrahim; tempia, stefano; pretorius, marthi; moyes, jocelyn; olorunju, steven a. s.; malope-kgokong, babatyi; kuonza, lazarus; wolter, nicole; von gottberg, anne; madhi, shabir a.; venter, marietjie; cohen, cheryl title: parainfluenza virus infection among human immunodeficiency virus (hiv)-infected and hiv-uninfected children and adults hospitalized for severe acute respiratory illness in south africa, 2009–2014 date: 2015-09-19 journal: open forum infect dis doi: 10.1093/ofid/ofv139 sha: doc_id: 326642 cord_uid: kc85pev4 background. parainfluenza virus (piv) is a common cause of acute respiratory tract infections, but little is known about piv infection in children and adults in africa, especially in settings where human immunodeficiency virus (hiv) prevalence is high. methods. we conducted active, prospective sentinel surveillance for children and adults hospitalized with severe acute respiratory illness (sari) from 2009 to 2014 in south africa. we enrolled controls (outpatients without febrile or respiratory illness) to calculate the attributable fraction for piv infection. respiratory specimens were tested by multiplex real-time reverse-transcription polymerase chain reaction assay for parainfluenza types 1, 2, and 3. results. of 18 282 sari cases enrolled, 1188 (6.5%) tested positive for any piv type: 230 (19.4%) were type 1; 168 (14.1%) were type 2; 762 (64.1%) were type 3; and 28 (2.4%) had coinfection with 2 piv types. after adjusting for age, hiv serostatus, and respiratory viral coinfection, the attributable fraction for piv was 65.6% (95% ci [confidence interval], 47.1–77.7); piv contributed to sari among hiv-infected and -uninfected children <5 years of age and among individuals infected with piv types 1 and 3. the observed overall incidence of piv-associated sari was 38 (95% ci, 36–39) cases per 100 000 population and was highest in children <1 year of age (925 [95% ci, 864–989] cases per 100 000 population). compared with persons without hiv, persons with hiv had an increased relative risk of piv hospitalization (9.4; 95% ci, 8.5–10.3). conclusions. parainfluenza virus causes substantial severe respiratory disease in south africa among children <5 years of age, especially those that are infected with hiv. parainfluenza virus (piv) is a paramyxovirus commonly detected in patients with respiratory illness, such as upper respiratory tract infections, laryngotracheobronchitis (croup), or pneumonia. there are 4 distinct types: 1, 2, 3, and 4. parainfluenza virus infection is most common in childhood, and serologic surveys have shown that nearly all children have antibodies to piv by 5 years of age [1, 2] . parainfluenza virus is associated with 3%-10% of hospitalized respiratory tract infections in children and adults [3] [4] [5] [6] [7] [8] [9] [10] . parainfluenza virus infections occur throughout the year, but there are recognized patterns of seasonality associated with different types [11] . the clinical significance of identifying piv infection among patients with respiratory illness does not necessarily imply causation because the virus may be found in individuals without respiratory symptoms [12] . in south africa, human immunodeficiency virus (hiv) is prevalent, which impacts respiratory disease burden [13] . in 2009, the national hiv prevalence estimate among children <5 years was 4.1% and decreased to 3.5% by 2013; the hiv prevalence among adults ≥18 years of age was approximately 16.0% during the same time [14] . a study from soweto prior to availability of antiretroviral therapy (art) found that hiv-infected children with piv-associated lower respiratory tract infection had greater morbidity and mortality than hivuninfected children [15] . a more recent study from cape town found that piv was associated with 12% of viral-associated pediatric intensive care unit admissions [16] . previous studies from our group have detected piv in 2.8% of adults and 9.4% of children hospitalized with severe acute respiratory illness [17] [18] [19] . little is known about the epidemiology of piv infection, including its association with illness and its clinical manifestation among both hiv-infected and -uninfected children and adults in south africa, especially in the era of art management. in this study, we aimed to describe the epidemiological and clinical characteristics of hiv-infected and -uninfected children and adults hospitalized with piv-associated pneumonia in south africa. in addition, we compared the prevalence of piv detection with controls for better interpretation of surveillance data. we conducted surveillance for children and adults hospitalized with pneumonia through the severe acute respiratory illness (sari) program, an active, prospective, sentinel hospitalbased surveillance system in 4 provinces in south africa, as described previously [13] . in february 2009, sari surveillance began in 3 of the 9 provinces of south africa (chris hani-baragwanath academic hospital [chbah] , an urban site in gauteng province; edendale hospital, a periurban site in kwazulu-natal province; and matikwana and mapulaneng hospitals, rural sites in mpumalanga province). in june 2010, an additional surveillance site was introduced at the klerksdorp-tshepong hospital complex, periurban sites in north west province. we stopped enrolling at chbah in december of 2013. in addition to sari cases, controls were enrolled from may 2012 at 2 outpatient clinics serving the same population as 2 of the sari sentinel sites: edendale hospital gateway clinic, kwazulu-natal province, and jouberton clinic, north west province. the case definitions used and enrollment procedures for both sari cases and controls are described fully in the supplementary material. this surveillance, including testing for influenza and hiv, received human subjects review and ethics approval by the universities of the witwatersrand, kwazulu-natal, and pretoria, all of south africa. the centers for disease control and prevention (atlanta, ga) deemed this a nonresearch, surveillance activity. nasopharyngeal aspirates from patients aged <5 years and nasopharyngeal and throat swabs from patients aged ≥5 years were placed in viral transport media, kept at 4-8°c, and sent to the national institute for communicable diseases (nicd) in johannesburg within 72 hours of collection. respiratory specimens were tested by multiplex real-time, reversetranscription polymerase chain reaction (pcr) assay for 10 respiratory viruses (piv types 1, 2, and 3; respiratory syncytial virus [rsv]; influenza a and b viruses; enterovirus; human metapneumovirus; adenovirus, and rhinovirus) [17] . we did not test for piv type 4 during the study period. we did not test for adenovirus from august to october 2009 because of unavailability of reagents. streptococcus pneumoniae was identified by quantitative real-time pcr detecting the lyta gene from whole blood specimens [20] . when available, hiv-infection status data were obtained through routine standard of care testing at the treating hospital. when not available, hiv testing was implemented at nicd through anonymized, linked dried blood spot specimen testing by hiv pcr assay for children aged <18 months and by enzyme-linked immunosorbent assay for those aged ≥18 months. we conducted 3 multivariable logistic regression models. in our first analysis, we implemented univariate and multivariable logistic regression models to determine the association of piv infection with sari (for all types together and for each viral type separately) compared with controls enrolled from may 2012 to december 2014 at the sites in kwazulu-natal and north west provinces. for the estimation of association with sari, we conducted an overall analysis adjusting for age, hiv serostatus, respiratory viral coinfection, and underlying illness and subanalyses stratifying by age and hiv serostatus and adjusting for respiratory viral coinfection and underlying illness. then, we estimated the attributable fraction (af) from the odds ratio (or) obtained from the multivariable model using the following formula: af = (or-1)/or × 100. in our second analysis, univariate and multivariable logistic regression was used to determine factors associated with hiv infection among patients with piv-associated sari from january 2009 to december 2014 at all sari sites. in our third analysis, we used multinomial regression to compare and contrast demographic and clinical characteristics and severity among patients infected with the 3 piv types. for the multinomial analysis, we used piv type 3 as the baseline category because type 3 is most common. the second and third analyses models were built using manual backward elimination in which nonsignificant variables were removed from the model one at a time starting with the variables with smallest magnitude of effect until all remaining variables had a p value of <.05. covariates with a p value of <.2 at univariate analysis were assessed for significance with multivariable analysis; statistical significance was assessed at p < .05 for all multivariable models. two-way interactions were assessed by inclusion of product terms for all variables remaining in the final additive models. for each univariate analysis, we used all available case information. for important variables in the hiv association and af analyses that had substantial missing data, namely hiv infection status, we multiply imputed that variable as well as any variables that were incomplete and associated with hiv using chained equation multiple imputation over 10 iterations. when adjusting for respiratory viral coinfection in our models, we evaluated coinfection with each virus separately and also as a combined variable of coinfection with any of the tested viruses. calculation of observed incidence and incidence adjusted for af of piv-associated sari hospitalizations was done for 1 study site (chbah) where population denominators were available, as described previously [13] . a complete description of the incidence calculation methods can be found in the supplementary material. . of the sari cases tested for piv, 1188 (6.5%) tested positive for any piv type: 230 (19.4%) were solely type 1, 168 (14.1%) were solely type 2, and 762 (64.1%) were solely type 3. coinfection with 2 types of piv occurred in 28 (2.4%) patients: 3 (0.3%) tested positive for both types 1 and 2, 13 (1.1%) for types 1 and 3, and 12 (1.0%) for both 2 and 3. children <5 years of age were more likely to test positive for piv (9.2%, 963 of 10 448) than individuals aged ≥5 years (2.8%, 214 of 7737, p < .001). males were slightly more likely to test positive for piv (7.1%, 637 of 9018) than females (5.9%, 540 of 9168, p = .001). the sari patients who died in hospital were less likely to test positive for piv (4.0%, 29 of 717) than those who did not (6.6%, 1142 of 17316, p = .007). there were no differences in percentage testing positive by race or surveillance site (data not shown). human immunodeficiency virus status was determined for 14 287 (76.3%) of sari cases, of which 5829 (40.8%) were infected with hiv. most of the patients with missing hiv status were children <5 years of age (30.6% [3290 of 10 754], compared with 13.3% [1038 of 7830] for individuals ≥5 years of age). on univariate analysis, sari patients that were hivinfected were less likely to test positive for piv (4.0%, 230 of 5794) than those that were hiv-uninfected (7.6%, 635 of 8365, p < .001). during the time period when controls were enrolled (may 2012-december 2014), 1472 sari cases were enrolled. for controls, 19 929 were screened and 1538 (7.7%) were enrolled. overall, 97 (6.6%) of the cases and 28 (1.8%) of the controls tested positive for piv ( table 1 ). the overall piv af was 65.6% (95% confidence interval [ci], 47.1-77.7), after adjusting for age, hiv serostatus, respiratory viral coinfection, and underlying illness, suggesting that more than two-thirds of pivassociated sari cases could be attributed to piv infection. a statistically significant af was seen among both hiv-infected (57.1%; 95% ci, 9.9-76.1) and hiv-uninfected individuals of any age (76.1%; 95% ci, 60.9-98.7); although the point estimates are different, the cis overlap. the af was highest among young children and older adults, although it was only statistically significant among children <5 years of age (for <1 year, 64.9%, 95% ci, 25.4-85.3; for 1-4 years, 77.3%, 95% ci, 46.2-90.3); this same association among children <5 years of age was seen among both hiv-infected and -uninfected individuals. we then calculated the af for piv types 1, 2, and 3 separately. the af was 73.1% (95% ci, 41.2-87.8) for piv type 1, 61.1% (95% ci, 0-92.9) for piv type 2, and 60.3% (95% ci, 33.3-76.5) for piv type 3. the age and hiv-status subgroup analysis results for piv types 1 and 3 separately were similar to those found for parainfluenza overall, except that some subgroup analyses involved smaller numbers of subjects and were not statistically significant (data not shown). we did not conduct the subgroup analysis for piv type 2 because it was not found to be statistically associated with disease. most (81.8%, 1043 of 1188) patients with piv-associated sari were <5 years of age, the group for which we found a statistically significant and substantial af. twelve percent (11.9%, 80 of 675) of children <5 years of age with piv-associated sari were infected with hiv ( tables 2 and 3 ). on multivariable analysis among individuals ≥5 years of age with piv-associated sari, hiv-infected individuals were more likely to be young adults 25-44 years of age (aor 5.2, 95% ci, 1.7-15.7), compared with children and young adults (5-24 years of age), and less likely to have an underlying illness other than hiv (aor 0.2, 95% ci, .1-.8; table 3 ). when we compared epidemiologic and clinical characteristics of the 3 piv types, we found statistically significant differences in age, year of infection, and coinfection with other respiratory viruses (supplementary table 1 ). on multivariable multinomial analysis, patients with piv type 3 infection were more likely to be <1 year of age and patients with piv type 2 infection were more likely to be 5-44 years of age, compared with patients with other piv types. compared with sari patients with piv type 3 infection, patients with either type 1 or 2 infection were more likely to be coinfected with rsv (for type 1, aor 2.2, 95% ci, 1.3-3.7; for type 2, aor 3.3, 95% ci, 1.9-5.7). in addition, patients with piv type 2 infection were more likely to be coinfected with rhinovirus (aor 2.0, 95% ci, 1.3-2.8) or enterovirus (aor 2.6, 95% ci, 1.5-4.4) than patients with type 3 infection. the overall observed incidence of piv-associated sari in soweto from 2009 to 2012 was 38 (95% ci, 36-39) cases per 100 000 population ( table 4 ). the observed incidence was highest in children <1 year of age (925 [95% ci, 864-989] cases per 100 000 population) and lowest in those 5-24 years of age (5 [95% ci, 4-6] cases per 100 000). in all age groups, the observed incidence was higher among hiv-infected compared with hivuninfected individuals. the subgroup with the highest incidence was hiv-infected infants <1 year of age (1361 [95% ci, 985-1834] cases per 100 000). the overall incidence adjusted for the af was 25 cases per 100 000 (95% ci, 24-26); the age-specific adjusted incidences followed the same trend as the observed incidences ( table 4 ). the age-adjusted relative risk of piv hospitalization by hiv status was 9.4 (95% ci, the 3 piv serotypes (1, 2, and 3) were found to cocirculate throughout the year and differed from year to year; seasonal peaks were observed for piv-3 between september and november, which is spring in south africa (figure 1 ). the percentage of specimens testing positive for any piv type varied by year, with a higher proportion of sari positive for piv in 2009 parainfluenza virus is associated with a significant amount of severe respiratory disease in south africa among children <5 years of age, especially those that are infected with hiv. the evidence for this is based on 2 of our findings: the af and the observed incidence. among children <5 years of age, a substantial amount of sari is attributable to piv, particularly types 1 and 3. the observed incidence of piv in soweto, south africa, is much higher among hiv-infected individuals and is similar to other respiratory viral pathogens such as influenza and human metapneumovirus [13, 17] . clinicians should recognize piv as a common cause of respiratory illness in children during the spring season, especially among children that are infected with hiv, and interventions should be developed to prevent and treat piv disease. as we found in south africa, studies from across the globe have found that piv is associated with up to 10% of inpatient respiratory illness, particularly among the very young. this has been found in bangladesh [21] , china [5, 22] , thailand [23] , the united states [3, 4] , and in multiple countries across the african continent. in kenya, 5.6% of individuals ≥5 years of age hospitalized with pneumonia had piv types 1-3 detected [6] . in ghana, 3.1% of children hospitalized with acute lower respiratory tract infection had piv types 1-3 [7] . in mozambique, 4.8% of infants <1 year and 5.1% of children <5 years of age hospitalized with acute respiratory illness tested positive for piv [8, 24] . it can be difficult to attribute respiratory disease to a specific pathogen. molecular tests may identify viruses in respiratory specimens that may not be causing illness, and coinfection with other potentially pathogenic respiratory viral infections was common in our population. we found that a majority of sari was attributable to piv types 1 and 3 in children, even when controlling for viral respiratory coinfection, but not all studies have found that piv is pathogenic. in fact, very few studies have adequately evaluated piv in the context of controls. two studies from kenya that compared patients with respiratory illness to controls did not find a statistically significant attributable risk for piv [6, 12] . however, a study comparing children <5 years of age from thailand hospitalized with pneumonia to controls found findings similar to ours, specifically that infection with piv types 1 or 3 was associated with disease [25] . the lower incidence and nonsignificant af from our data among individuals ≥5 years of age suggests that piv infection may not be associated with respiratory disease in older children and adults. we did not find a significant af for patients infected with piv type 2, but we may not have been powered to detect this because type 2 was the least common type. we found differences among patients infected with different types of piv and between patients infected and uninfected with hiv. in children, patients with piv-associated sari who were hiv-infected were more likely to be older and to be coinfected with pneumococcus; however, the af among adults was not statistically significant. the association between respiratory viral and pneumococcal infection, although not described previously for piv infection, is well described for other pathogens such as influenza [26] and rsv [27] . pneumococcal conjugate vaccine was introduced in the south africa childhood immunization system in 2009, so it was available and being used during the entire time of this study. the seasonality of piv in south africa is similar to that seen in other temperate countries such as the united states, where piv circulates during the northern hemisphere spring and winter seasons [28] . in contrast, in tropical and subtropical countries in africa, such as cameroon, ghana, and kenya, piv does not appear to have distinct seasonality [6, 7, 29] . this is one of few studies that describe the epidemiology of piv in both children and adults over a long period and in the context of controls. however, there were some limitations to our study. not all children were tested for hiv, and we did not analyze piv in cases of milder outpatient influenza-like illness, which includes common presentations of piv illness such as croup. although it is uncommon and uncommonly tested for, we did not test for piv type 4, so our incidence estimates would be a minimum estimate. we also did not test for all viral respiratory pathogens, such as coronavirus and bocavirus. the large number of patients from chbah (70%) may bias results toward the epidemiology at that one site. our calculation of incidence was for only 1 site and the calculation of af was conducted at 2, so these analyses may not be representative of all regions in south africa. the incidence may be an underestimate if patients did not seek medical care for their illness or sought care at a hospital other than chbah or died before reaching the hospital. the increased incidence of hospitalization among individuals infected with hiv may be due to a lower threshold for hospitalization compared with hiv-uninfected individuals. lastly, we did not have data on steroid use, which is a common treatment for croup in children, and for patients with hiv infection, we did not have information on art nor cd4 cell count. in conclusion, based on 6 years of respiratory disease surveillance, piv is a common cause of sari among children <5 years in south africa. among children, the observed incidence of piv is higher in the hiv-infected population than the hivuninfected population. a vaccine to protect against piv is not currently available, but vaccine candidates are in the clinical testing phase [30] . the findings of our study accentuate the need to target children for piv prevention strategies including vaccination, should a vaccine against piv become available. abbreviations: ci, confidence interval sari, severe acute respiratory illness preparedness and response to avian and pandemic influenza in south africa (cooperative agreement number: u51/ip000155-04), and the national institute for communicable diseases, south africa. potential conflict of interest. h. d. has received honoraria from msd-south africa and novartis-south africa, honoraria from pfizer-south africa for speaking engagements a study of the antibodies against parainfluenza viruses in childrens' sera half-life of human parainfluenza virus type 3 (hpiv3) maternal antibody and cumulative proportion of hpiv3 infection in young infants parainfluenza virus infection of young children: estimates of the population-based burden of hospitalization human parainfluenza virus-associated hospitalizations among children less than 6 years of age in the united states epidemiology and clinical presentation of the four human parainfluenza virus types etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana viral acute respiratory infections among infants visited in a rural hospital of southern mozambique community-acquired pneumonia among u.s. children community-acquired pneumonia requiring hospitalization among u.s. adults influenza and parainfluenza viral infections in children a preliminary study of pneumonia etiology among hospitalized children in kenya severe influenza-associated lower respiratory tract infection in a high hiv-prevalence setting-south africa severe lower respiratory tract infections associated with human parainfluenza viruses 1-3 in children infected and non-infected with hiv type 1 an investigation into the prevalence and outcome of patients admitted to a pediatric intensive care unit with viral respiratory tract infections in cape town, south africa respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa epidemiology of severe acute respiratory illness among adults and children aged ≥5 years in a high hiv-prevalence setting epidemiology of viral-associated acute lower respiratory tract infection among children <5 years of age in a high hiv prevalence setting evaluation and improvement of real-time pcr assays targeting lyta, ply, and psaa genes for detection of pneumococcal dna population-based incidence of severe acute respiratory virus infections among children aged <5 years in rural bangladesh viral etiologies of hospitalized acute lower respiratory infection patients in china hospitalization due to human parainfluenza virus-associated lower respiratory tract illness in rural thailand etiology and epidemiology of viral pneumonia among hospitalized children in rural mozambique: a malaria endemic area with high prevalence of human immunodeficiency virus incidence and etiology of acute lower respiratory tract infections in hospitalized children younger than 5 years in rural thailand interaction between influenza virus and streptococcus pneumoniae in severe pneumonia association between respiratory syncytial virus activity and pneumococcal disease in infants: a time series analysis of us hospitalization data seasonal trends of human parainfluenza viral infections: united states viral etiology of influenza-like illnesses in cameroon progress in the development of human parainfluenza virus vaccines we thank the surveillance officers, severe acute respiratory illness programme team, and especially the patients. we also acknowledge dorothy supplementary material is available online at open forum infectious diseases (http://openforuminfectiousdiseases.oxfordjournals.org/). key: cord-317277-rr9zue4l authors: cifuentes-munoz, nicolas; el najjar, farah; dutch, rebecca ellis title: viral cell-to-cell spread: conventional and non-conventional ways date: 2020-09-29 journal: adv virus res doi: 10.1016/bs.aivir.2020.09.002 sha: doc_id: 317277 cord_uid: rr9zue4l a critical step in the life cycle of a virus is spread to a new target cell, which generally involves the release of new viral particles from the infected cell which can then initiate infection in the next target cell. while cell-free viral particles released into the extracellular environment are necessary for long distance spread, there are disadvantages to this mechanism. these include the presence of immune system components, the low success rate of infection by single particles, and the relative fragility of viral particles in the environment. several mechanisms of direct cell-to-cell spread have been reported for animal viruses which would avoid the issues associated with cell-free particles. a number of viruses can utilize several different mechanisms of direct cell-to-cell spread, but our understanding of the differential usage by these pathogens is modest. although the mechanisms of cell-to-cell spread differ among viruses, there is a common exploitation of key pathways and components of the cellular cytoskeleton. remarkably, some of the viral mechanisms of cell-to-cell spread are surprisingly similar to those used by bacteria. here we summarize the current knowledge of the conventional and non-conventional mechanisms of viral spread, the common methods used to detect viral spread, and the impact that these mechanisms can have on viral pathogenesis. viruses are intracellular parasites that have coexisted with cells from ancient times. viruses hijack living cells and redirect many cellular processes to allow for efficient viral protein synthesis and replication. the success of the virus depends on the spread of some of these copies to new target cells to initiate a de novo infection. many animal viruses have evolved more than one mechanism of viral spread, revealing a previously unexpected heterogeneity. remarkably, some of these mechanisms allow the transfer of viral particles or components directly from cell-to-cell, without the need for virus release. cell-to-cell spread has important advantages over the well-studied mechanism of spread by diffusion of cell-free viral particles. to achieve direct cell-to-cell spread, some viruses induce a dramatic remodeling of cell architecture to reach uninfected cells and transfer viral particles or components. it is critical for our understanding of viral propagation to comprehend what circumstances promote the use of one mechanism of spread over others, and whether different mechanisms can operate simultaneously at different stages of the infectious cycle. in this review we have focused on the current knowledge of different mechanisms of cell-to-cell spread in animal viruses. release of viral particles into the extracellular space and the subsequent re-entry of these particles into new target cells is one of the best described and understood mechanisms of virus spread. the release of cell-free particles is critical for viral spread between distant cells, or for spread between hosts. virions released from a cell must interact with molecules at the cell surface of a new target cell as the first step to enter and initiate an infectious cycle. these molecules include receptors, co-receptors, and attachment factors. cell-free viral particles can be released into the extracellular space through different mechanisms, such as: (a) cell lysis induced by viral proteins, as is the case for many non-enveloped viruses such as reoviruses, rotaviruses, adenoviruses and picornaviruses (giorda and hebert, 2013; hu et al., 2012; nieva et al., 2012) ; (b) by budding directly from the plasma membrane, where virions acquire their envelope, as is the case of human immunodeficiency virus (hiv-1), influenza, paramyxoviruses, and pneumoviruses (lorizate and krausslich, 2011; votteler and sundquist, 2013; weissenhorn et al., 2013) ; (c) by exocytosis of intracellularly assembled viral particles, as is the case for bunyaviruses, flaviviruses and coronaviruses (cifuentes-munoz et al., 2014; lorizate and krausslich, 2011) . once released, different environmental and host factors affect the stability of viral particles and therefore their ability to initiate a new infection. many of the viruses that can be released as cell-free virions can use additional mechanisms of spread that are summarized below. vaccinia virus (vacv) replication occurs in cytosolic factories, and involves the formation of two forms of infectious virus, the intracellular mature virus (imv) and extracellular enveloped virus (eev) (fig. 1) . these two forms differ not only antigenically and structurally, but also in the way they exit the cell (smith and law, 2004) . the majority of imv particles are released from cells after lysis. late in infection, imv particles can also exit the cell through budding, but the significance of this mechanism is not completely understood. budding from the plasma membrane, together with exocytosis, are the proposed ways that eev exits cells. however, an important role of the actin cytoskeleton in vacv assembly was identified after early observations using high-voltage electron microscopy (stokes, 1976) . in infected cells, microvilli containing enveloped vacv particles at their tips were observed at the cell periphery (stokes, 1976) . the formation of the microvilli was shown to be sensitive to cytochalasin b but not to nocodazole, suggesting actin polymerization could play a role in their fig. 1 formation of actin tails. vaccinia virus (vacv) can spread from cell-to-cell through different mechanisms. in vacv infected cells, intracellular enveloped particles are transported to budding sites, where the outer viral membrane fuses with the plasma membrane (a). extracellular enveloped viral particles remain attached to the plasma membrane and several cellular factors are recruited to the site through a cascade of events initiated by phosphorylation of the a36r protein cytosolic tail (b). polymerization of f-actin underneath the plasma membrane occurs through activation of the cellular pathways n-wasp/arp2/3 and fhod1/rac1, leading to elongation of actin tails (c). viral particles can thus reach adjacent cells for rapid direct cell-tocell spread. stabilization. the term actin tails was proposed for vacv-induced microvilli, due to their resemblance to actin tails formed in bacterial infections with listeria, shigella or rickettsia (cudmore et al., 1995; welch and way, 2013) . actin tails are initially observed at the interior of the cell, but as the infection progresses they project from the cell surface up to 20 μm. enveloped virions located at the tip of actin tails were shown to project toward uninfected cells for direct cell-to-cell spread ( fig. 1) (cudmore et al., 1995) . soon after the discovery of actin tails, it was demonstrated that phosphorylation of the viral protein a36r by src and abl family kinases was essential for the actin-based motility of vaccinia (frischknecht et al., 1999a,b; newsome et al., 2006) . phosphorylated a36r recruits the adaptor proteins nck and grb2 and the downstream effector n-wasp (wiscott-aldrich syndrome protein) together with the wasp interacting protein (wip) (donnelly et al., 2013; frischknecht et al., 1999b; scaplehorn et al., 2002) . n-wasp can stimulate the actin-nucleating activity of the arp2/3 complex (taylor et al., 2011) . in addition, activation of the formin fhod1 by the small gtpase rac1 was shown to stimulate vaccinia virus-induced actin tail initiation and elongation, a mechanism independent of the n-wasp-arp2/3 pathway (alvarez and agaisse, 2013) (fig. 1) . two isoforms of cytoplasmic actin, β and γ-actin, are present in actin tails, but only β-actin was found to be involved in actin nucleation induced by vacv (marzook et al., 2017) . therefore, the signaling pathway initiated through phosphorylation of vacv a36r to induce actin tail formation for direct cell-to-cell spread was found to be an elegant mimic of cellular pathways. more recently, it was shown that two vacv proteins, a33 and a36, are necessary and sufficient to induce actin tail formation. the early expression of both proteins was shown to be crucial for rapid spread and repulsion of virions to neighboring uninfected cells (doceul et al., 2010) . additional cellular factors such as clathrin and the clathrin adaptor protein ap-2 enhance actin-based motility of vaccinia. clathrin and ap-2 are recruited by the extracellular virus during its egress to promote clustering of a36, thus potentiating actin-based motility and spread of the infection (humphries et al., 2012) . casein kinase 2 (ck2) also enhances actin tail formation of vaccinia virus, potentially through direct phosphorylation of a36 and the recruitment of phosphorylated src (alvarez and agaisse, 2012) . though actin tail formation has been explored in detail for vaccinia virus, the sequence homology of key viral proteins and additional evidence suggest that actin tail formation is a common strategy for orthopoxviruses (duncan et al., 2018; newsome and marzook, 2015; reeves et al., 2011; welch and way, 2013) . the fusion of membranes from adjacent cells results in multi-nucleated giant cells, also called syncytia. infection with different viruses including paramyxoviruses (takeuchi et al., 2003) , pneumoviruses (hamelin et al., 2004; neilson and yunis, 1990; vargas et al., 2004) , herpesviruses (cole and grose, 2003) and retroviruses (nardacci et al., 2005) results in syncytia formation in vitro and in vivo. cells infected with these viruses display a high concentration of viral fusion protein at the plasma membrane which can promote membrane fusion with neighboring cells. specific fusion proteins have different requirements for triggering the conformational changes needed to drive fusion, such as exposure to low ph or the presence of receptors which bind the fusion protein or an associated receptor binding protein (hernandez et al., 1996; kielian, 2014) . if the needed factors are present, the fusion protein can be activated to promote fusion between the membrane of the infected cell and the membrane of the target cell. the process of membrane fusion driven by viral fusion proteins involves several sequential steps, including: (a) activation of the fusion protein, which exposes a fusion peptide (fp); (b) insertion of the fp into the adjacent membrane; (c) clustering of fusion proteins at the site of fusion; (d) refolding of the fusion protein, which pushes together both adjacent membranes; (e) hemifusion, where only the outer leaflets of the membranes merge; (e) formation of a small fusion pore, and (f ) enlargement of the fusion pore until complete merging of both membranes (fig. 2) (hernandez et al., 1996; kielian, 2014; lorizate and krausslich, 2011) . iterations of this process generate massive syncytia containing numerous nuclei with the complete mixing of cytoplasmic material, which allows propagation of infection rapidly without the need for assembly of complete virions. large rearrangements of the actin cytoskeleton are induced during cell fusion and syncytia formation. these rearrangements are regulated by the action of rho gtpases cdc42, rac1 and rhoa (eitzen, 2003) . for example, it has been demonstrated that hiv-1 env-coreceptor interactions activate the gtpase rac1, which promotes actin rearrangements necessary for membrane fusion (pontow et al., 2004) . for the paramyxoviruses hendra virus and parainfluenza virus type 5 (piv5), constitutively active rac1 and cdc42 increased cell-to-cell fusion by their respective viral glycoproteins, as shown by quantitative reporter gene fusion assays. in contrast, constitutively active rhoa decreased hendra virus fusion protein (f)-mediated fusion and had minor effects on piv5 cell-to-cell fusion (schowalter et al., 2006) . cell-to-cell fusion mediated by the respiratory syncytial virus (rsv) fusion protein was shown to require rhoa signaling (gower et al., 2005) . interestingly, a recent report correlated a rsv fusion protein with a hyperfusogenic phenotype with increased pathogenesis in mice (hotard et al., 2015) . hypersyncytial viral strains (syn) have been extensively described for α-herpesviruses (ambrosini and enquist, 2015) . mutations associated with the syn phenotype have been mapped to viral genes encoding the glycoproteins, with a predominance in the gb and gk genes (ambrosini and enquist, 2015; bond et al., 1982) . early and recent reports have associated the hyperfusogenic strains of α-herpesviruses with increased electrical activity in infected neurons (mayer et al., 1985; mccarthy et al., 2009) . additionally, syncytia formed between neurons and satellite cells during varicella-zoster virus (vzv) infection have been suggested to contribute to neuropathogenesis (reichelt et al., 2008) . initially, the fusion protein is in an inactive prefusion state; (b) upon the proper stimulus (such as binding a receptor shown in purple on the target cell), the fusion protein is triggered, which induces conformational changes, insertion of a fusion peptide into the adjacent membrane, and conformational changes that bring both membranes, viral and cellular into close proximity, followed by a partial merging known as hemifusion (c). full merger of both membranes results in the opening of a fusion pore (d) that expands allowing the mixing of cytoplasmic material and spread of viral components between cells (e). iterations of the fusion process result in formation of large multinucleated syncytia. in contrast, syncytia formation by vzv mutant hyperfusogenic glycoproteins might be detrimental, rather than beneficial, for viral spread and pathogenesis in skin cells (yang et al., 2014) . whether syncytia formation contributes to pathogenicity of other viruses such as hiv-1 is still a matter of controversy (compton and schwartz, 2017) . remarkably, while viral fusion proteins are well studied in enveloped viruses, another type of viral fusogen that promotes syncytia formation has been described for the non-enveloped orthoreoviruses (duncan, 2019; shmulevitz and duncan, 2000) . this family of non-structural viral fusion proteins, named fusionassociated small transmembrane (fast) proteins, are unusual in that they do not mediate viral entry but rather fusion between cells using combinations of membrane effector motifs. cell-to-cell fusion mediated by the reptilian orthoreovirus p14 fusogen is accomplished by recruitment of the src kinase, the grb adaptor protein and n-wasp, thus initiating branched actin assembly (chan et al., 2020) . the factors recruited by the reptilian orthoreovirus p14 for cell-to-cell fusion are thus remarkably similar to those recruited by vaccinia virus to induce actin tail formation, as described above. despite their role in orthoreovirus cell-to-cell spread, syncytia formation is not essential for virus replication or progeny production of orthoreoviruses in vitro (duncan et al., 1996) . however, the maintenance of these proteins across reovirus species strongly suggests they provide a competitive advantage to the virus (duncan, 2019). cell junctions are structures composed by several different transmembrane proteins, whose main function is to form a seal between polarized epithelial cells. adhesion between epithelial cells is achieved by three main types of seals: tight junctions (tj), adherens junctions (aj) and desmosomes. these barriers cannot be penetrated by viruses unless there is substantial damage to the epithelia . however, epithelial cells can be infected from the apical or basolateral sides, with viral particles then transmitted to adjacent cells through specialized sites located in the vicinity of cellular junctions that are at least partially inaccessible from the extracellular environment. for example, in addition to cell-free spread, hepatitis c virus (hcv) can spread from cell-to-cell in a neutralizing antibodyindependent manner (brimacombe et al., 2011; timpe et al., 2008) . claudin 1 (cldn1) and occludin (ocln), components of tight junctions, are critical for cell-to-cell spread of hcv (brimacombe et al., 2011; timpe et al., 2008) . in addition, the cellular receptor cd81, scavenger receptor bi (sr-bi), apolipoprotein e, and low density lipoprotein receptor (ldlr) were shown to have a role in the cell-to-cell, but not cell-free, spread mechanism of hcv (brimacombe et al., 2011; fan et al., 2017; timpe et al., 2008) . direct transmission of assembled hcv particles was proposed to occur in cell-cell contacts across partially sealed cell junctions (brimacombe et al., 2011) . robust evidence shows the use of adherens junctions by herpesviruses to spread from cell-to-cell ( johnson and baines, 2011; johnson and huber, 2002) . herpes simplex virus type 1 (hsv-1) particles have been shown to be preferentially sorted towards cell junctions rather than to the apical surface of polarized epithelial cells ( johnson et al., 2001) . the transport of particles towards these sites is primarily directed by the glycoprotein complex gi/ge, with the cytosolic domain of ge having a critical role (dingwell et al., 1994; dingwell and johnson, 1998; farnsworth and johnson, 2006) . indeed, mutant viruses lacking gi/ge grow poorly in monolayers of human fibroblasts, and form plaques of small size (dingwell et al., 1994) . this phenotype was corroborated in vivo, as the △gi/ge mutant viruses produced small, punctate lesions in the corneal epithelium of rabbits and mice (dingwell et al., 1994) . the cell-to-cell spread mediated by gi/ge was found to be independent of neutralizing antibodies, suggesting that it occurs at sites of cellular junctions that are not accessible to these molecules (dingwell et al., 1994) . immunofluorescent staining showed that gi/ge colocalized with the adherens junction protein β-catenin, but not with zo-1, a component of tight junctions (dingwell and johnson, 1998) . in addition, nectin-1, a component of adherens junctions, has been reported as a receptor for the herpes simplex virus glycoprotein d (sakisaka et al., 2001; yoon and spear, 2002; zhang et al., 2011) . in this context, one accepted model is that gi/ge complexes accumulate at trans-golgi network (tgn) subdomains, from where nascent virions are sorted to basolateral cell junctions for cell-to-cell spread (farnsworth and johnson, 2006; johnson and baines, 2011) . at these sites, release of particles and their subsequent entry into the adjacent cell occur almost simultaneously. however, the critical function of gi/ge complexes extends beyond the spread between epithelial cells, and their role, together with pus9, in the anterograde transport (from cell body to axons) of hsv-1 particles has been extensively studied (howard et al., 2014; kratchmarov et al., 2013; mcgraw and friedman, 2009; snyder et al., 2008) . remarkably, the complex gi/ge together with pus9 promote anterograde transport of enveloped particles, nucleocapsids, and glycoproteins through kinesin-mediated vesicular transport using microtubules (diwaker et al., 2020; johnson and baines, 2011; kratchmarov et al., 2013) . the role of the gi/ge complex and us9 for cell-to-cell spread has additionally been reported for other herpesviruses including pseudorabies and vzv (cohen and nguyen, 1997; diwaker et al., 2020; johnson and huber, 2002; lyman et al., 2007; zsak et al., 1992) . the use of desmosomes for cell-to-cell spread has been studied less than spread at the other cell junctions described above. however, some reports describe an important role of keratin 1 in infection by lymphocytic choriomeningitis virus (lcmv) (labudova et al., 2009) (labudova et al., 2019) . keratin filaments are an important component of desmosomes, and the lcmv nucleoprotein was shown to bind and stabilize keratin 1 (labudova et al., 2009 ). this interaction resulted in increased desmosome formation and cell-cell adhesion capacity, facilitating cell-to-cell spread of lcmv (labudova et al., 2009 ). formation of virological synapses (vs) is a mechanism of cell-to-cell spread that has been shown to be used by retroviruses (alvarez et al., 2014; dupont and sattentau, 2020; . in the vs, several cellular factors, including cytoskeletal proteins, receptors, and adhesion molecules, are recruited to sites where virus particles can be transmitted quickly from an infected cell to a target cell. in lymphocytes infected with human t-cell leukemia virus type 1 (htlv-1), env is distributed evenly at the cell surface and clusters of gag are observed close to the plasma membrane (igakura et al., 2003) . upon contact with uninfected t-cells, a rapid recruitment of a large number of env, gag and viral rna molecules at cell-cell junctions is observed. subsequently, transfer of gag molecules and viral rna is detected from the infected t cell to the uninfected t cell, likely utilizing a mechanism in which viral particles are rapidly released by the infected cell and taken up by the target cell in the junction region (igakura et al., 2003) . the cellular adhesion molecule talin was frequently found at sites of cell-cell contact, and an important reorientation of the microtubule organizing center (mtoc) to cell-cell contact adjacent sites was shown to be critical for transport of gag molecules. the polarization of the mtoc towards sites of cell-cell contact by htlv-1 is triggered by the transcriptional transactivator viral protein tax (nejmeddine et al., 2005) . polarization of the mtoc/tax was shown to depend on intact microtubules, actin filaments, and the function of rac and cdc42 small gtpases (nejmeddine et al., 2005) . further evidence indicates that the engagement of the adhesion molecule icam-1 on the surface of htlv-1 infected cells is sufficient to trigger the intracellular polarization of the mtoc . analogous observations are seen in cells infected with hiv-1. dendritic cells (dc) infected with hiv-1 rapidly recruit virus to cell junctions formed between dcs and t lymphocytes (mcdonald et al., 2003) . remarkably, the un-infected t cells recruit receptor and co-receptor molecules including cd4, ccr5 and cxcr4 at the vs, which facilitates hiv transmission from cell-to-cell (mcdonald et al., 2003) . similar to htlv-1, in hiv-1-infected t cells (effector cells) env and gag are quickly recruited to the vs (do et al., 2014; . polarization of the mtoc and associated organelles towards the vs is induced in hiv-infected lymphocytes and dcs to hijack components of the secretory pathway (bayliss et al., 2020; jolly et al., 2011) . in adjacent uninfected t cd4 + cells, an actin-dependent recruitment of cd4, cxcr4 and lymphocyte function-associated antigen 1 (lfa-1) was found to facilitate spread of gag molecules between cells . quantitative 3d live microscopy revealed "buttons" of oligomerized hiv-1 gag at sites of cell-cell contact, which were shown to be the preferential route of infection between cd4-t cells (hubner et al., 2009) . thus, the proposed model of cell-to-cell infection involves budding of viral particles into the synaptic cleft and subsequent fusion of viral particles with the adjacent uninfected cell (hubner et al., 2009; miyauchi et al., 2009) . remarkably, it has been shown that hiv cell-to-cell spread can occur simultaneously from one infected lymphocyte to multiple adjacent recipient cells, a process named polysynapse formation (rudnicka et al., 2009 ). moreover, cell-to-cell transmission of retroviruses through the vs has been shown to play a critical role in viral dissemination in vivo (murooka et al., 2012; sewald et al., 2015) . in this context, studies that examined the accessibility of the vs to entry inhibitors have concluded that synapse-mediated spread is less sensitive than cell-free particle spread to neutralizing antibodies in vitro and vivo (abela et al., 2012; gombos et al., 2015; smith and derdeyn, 2017; zhong et al., 2013) . cell-to-cell contact sites have additionally been described as the main route of viral dissemination for the murine leukemia virus (mlv) by using live-cell imaging ( jin et al., 2009) . interestingly, a mechanism that resembles the vs was reported for spread of the nonlymphotropic hsv-1 (aubert et al., 2009) . infection of cd4 + and cd8 + t lymphocytes by hsv-1 was shown to occur in vivo. further, infection was more efficient by using cell-to-cell contacts than by cell-free virus, in a manner independent of the gi/ge glycoprotein complex (aubert et al., 2009 ). whether or not other non-lymphotropic viruses can establish vss warrants future research. htlv-1 cell-to-cell transmission does not only rely on the formation of vss. lymphocytes infected with htlv-1 have also been shown to store viral particles in an unconventional way, as clusters located on the surface of t cd4+ lymphocytes . these clusters correspond to viral assembly sites and are enriched in carbohydrates and extracellular matrix components including collagen and agrin, a heparan sulfate proteoglycan (pais-correia et al., 2010; thoulouze and alcover, 2010) . high-level expression of the host cell actin-bundling protein fascin is induced by htlv-1 tax in infected t-cells (kress et al., 2011) . fascin contributes to cell-to-cell htlv transmission, and has been suggested to be involved in transport and fitting gag into viral biofilms (gross et al., 2016) . remarkably, the clusters are transferred from infected cells to recipient cells during cell contact, and it was estimated that 80% of the infectious capacity of htlv-1 comes from these structures (pais-correia et al., 2010; thoulouze and alcover, 2010) . the mechanism shares several similarities with bacterial biofilms, and both improved spread and escape from the immune response might be advantages of this mechanism of viral cell-to-cell spread. tunneling nanotubes (tnts) were first described in 2004 as a new mechanism of cell-to-cell communication (rustom et al., 2004) . they are thin, elongated membrane-bound structures, between 50 and 200 nm in diameter, that connect two distant cells and allow the transfer of material including organelles, ions, proteins and mirnas. a very recent complete description of nanotube properties and their role in viral spread has been reported by jansens et al. ( jansens et al., 2020) . based on their diameter and composition, two classes of tnts are reported in macrophages: "thin tnts" that contain filamentous actin (f-actin) and "thick tnts" (greater than 0.7 μm) that contain f-actin and microtubules (onfelt et al., 2006) . organelles such as mitochondria, lysosomes and peroxisomes were detected in thick, but not in thin, tnts. interestingly, bacteria could "surf" along the surface of thin nanotubes using a constitutive flow (onfelt et al., 2006) . viruses can also exploit nanotubes for cell-to-cell spread, and this was first demonstrated for hiv (sowinski et al., 2008) . tunneling nanotubes containing f-actin were shown to connect distant t cells, to have a close-ended nature and not to be tethered to the substratum, in contrast to what is seen for filopodia. rapid spread of hiv particles through tnts was found to be receptor-dependent (sowinski et al., 2008) . the induction of tnts by hiv was additionally demonstrated in macrophages (eugenin et al., 2009) . interestingly, the authors identified three different processes in uninfected and hiv-infected macrophages: long tnts, short tnts and filopodia, based on intercellular communication and length. the authors reporter that tnts, but not filopodia, are open-ended and longer than filopodia, with lengths up to 150 μm (eugenin et al., 2009) . another retrovirus, htlv-1, was shown to form conduits in t cells that share characteristics of tnts (van prooyen et al., 2010) . induction of these conduits was shown to depend on the viral p8 protein and to be facilitated by the cellular factors icam-1 and lfa-1 (van prooyen et al., 2010) . further, the induction of tnts in infected cells was recently reported for influenza. influenza virus nucleocapsids were observed in long intercellular structures, likely tnts formed between lung epithelial cells (kumar et al., 2017; roberts et al., 2015) . direct transfer of influenza virus genomes and proteins was shown to require f-actin and actin dynamics. the process was also shown to be unaffected by the presence of neutralizing antibodies or oseltamivir, suggesting an open-ended nature of these tnts (kumar et al., 2017) . similar observations were made in cells infected with porcine reproductive and respiratory syndrome virus (prrsv) (guo et al., 2016) . infection by prrsv induces the formation of tnts that contain f-actin and myosin ii-a, a motor-binding protein. viral proteins and rna were observed through the length of tnts, and it was shown that viral proteins might interact directly or indirectly with myosin ii-a. the presence of neutralizing antibodies does not prevent cell-to-cell transmission of a fluorescently labeled prrsv and spread could occur in absence of cellular receptors, suggesting either a direct cytosolic connection between cells through tnts or transfer through sites of close-contact provided by the tnts that are inaccessible to antibodies (guo et al., 2016) . exploitation of tnts was additionally reported for pseudorabies virus ( jansens et al., 2017) . formation of tnts capable of intercellular spread of molecules was achieved by the expression of the viral protein us3 in the absence of other viral proteins (favoreel et al., 2005; jansens et al., 2017) . the tnts were associated with enhanced viral intercellular spread and were very stable. this stability was suggested to be the result of the presence of stabilized microtubules and an enrichment of the adherens junctions components β-catenin and e-cadherin at the site of contact between the tnt and the target cell. in this scenario, enveloped virions were shown to be transported by vesicles through the tnt and released by exocytosis from this structure at sites of contact between the tnt and the acceptor cells (favoreel et al., 2005; jansens et al., 2017) . recently, the induction of long intercellular connections has been reported upon infection by dna viruses, including vacv and the bovine herpesvirus 1 (bohv-1) (panasiuk et al., 2018; xiao et al., 2017) . the lack of adherence to substratum, their open-ended nature, length of up to 110 μm, and presence of f-actin and tubulin led to the proposal that the bohv-1-induced structures correspond to tnts. viral proteins were detected along the length of the tnts, and direct transfer of recombinant bohv-1 in a neutralizing antibody independent manner was observed, similar to what was reported for rna viruses (panasiuk et al., 2018) . filopodia are cellular structures involved in a wide array of functions including cell migration, wound healing, adhesion to the extracellular matrix and cell-cell interactions (faix and rottner, 2006; mattila and lappalainen, 2008) . their formation is induced through actin polymerization underneath the plasma membrane, which generates finger-like extensions that range between 0.1 and 0.3 μm in diameter. filopodia are filled with parallel bundles of f-actin (mattila and lappalainen, 2008) . several signaling pathways drive the protrusion of filopodia, including members of the family of rho gtpases like cdc42 (faix and rottner, 2006; mattila and lappalainen, 2008; nobes and hall, 1995) . because of the heterogeneous nature of the previously described tnts, it can be challenging to differentiate between tnts and filopodia. indeed, it was proposed that tnts arise from filopodium-like protrusions that connect to neighboring cells (rustom et al., 2004) . a more recent report suggests that two filopodial connections can interact with each other through n-cadherin, forming a bridge (chang et al., 2018) . rotation of the actin filaments inside these bridges by myosin proteins separates both filopodia, with one of them reaching a cell body and establishing a stable tnt-like connection via n-cadherin/β-catenin clustering (chang et al., 2018) . moreover, it has been shown that the cdc42/irsp53/vasp network that is upregulated during filopodia elongation acts as a negative regulator of tnt formation and vesicle transfer function (delage et al., 2016) . an interesting case of exploitation of filopodia during viral infection is reported for murine leukemia virus. after binding to receptors, mlv particles move along filopodia to reach the cell body and permit cell entry, a process dependent on actin and myosin that has been termed virus surfing (fig. 3a) (lehmann et al., 2005) . later in infection, it was shown that mlv particles move directly from infected to uninfected cells using filopodial bridges, named viral cytonemes, of about 5.8 μm in length (sherer et al., 2007) . movement of fluorescently labeled mlv particles was observed on the outer surface of cytonemes. the formation of cytonemes was shown to be dependent on env-receptor interactions, and antibodies targeting the extracellular domain of env could disrupt viral cytonemes. viral surfing through retrograde transport on the outer surface of filopodial structures has been also reported for dna viruses including hsv-1 and human papillomavirus types 16 and 31 (dixit et al., 2008; schelhaas et al., 2008; smith et al., 2008) . further evidence of animal virus infection inducing filopodia formation has been reported for asfarviruses ( jouvenet et al., 2006) . it was shown that african swine fever virus (asfv) induces filopodia formation at the plasma membrane with an average length of 10.5 μm. each projection contains the cellular proteins actin and fascin and single viral particles at their tips ( fig. 3b ) ( jouvenet et al., 2006) . similar observations were made for alphaviruses, as infection induces a dramatic remodeling of the cell architecture including formation of long intercellular projections (˃10 μm) (martinez et al., 2014; martinez and kielian, 2016) . as observed for other viruses, projections induced by alphaviruses contain actin and microtubules. however, they are different from those seen with asfv, where single virions are present at the tip of filopodia, as multiple alphavirus virions budding from the projections are observed (fig. 3c ). an interesting observation made for alphaviruses is that the filopodia-like extensions are not able to transfer cytosolic or plasma membrane components, suggesting they are not openended connections like tnts. instead, viral particles are hypothesized to bud into a protected space at the filopodial tip and then rapidly enter the target cell, preventing access of neutralizing antibodies. recent work has demonstrated that filopodia-like extensions are also a mechanism that respiratory viruses use for direct cell-to-cell spread. infection of human bronchial epithelial cells by human metapneumovirus (hmpv) resulted in a dramatic reorganization of the cell cytoskeleton (el najjar et al., 2016) . two major forms of projections were observed: long intercellular extensions of up to 18 μm, which resemble filopodia, and shorter abundant branching filaments (up to 6 μm) budding from the cell body and from the intercellular extensions (fig. 3d) . we speculate that the branching filaments correspond to filamentous forms of budding virus. as was seen for other viruses, f-actin and the rho gtpases cdc42 and rac1 were important for the formation of hmpv intercellular extensions. cdc42 is one of the main small gtpases involved in the formation of filopodia (nobes and hall, 1995) . up to 50% of cell-to-cell spread facilitated by hmpv intercellular extensions was shown to occur in the presence of neutralizing antibodies and in the absence of appropriate attachment factors, leading to the suggestion that direct intercellular transfer of viral components could occur. like hmpv, rsv induces formation of filopodial structures in lung epithelial cells which facilitate viral spread (mehedi et al., 2016) . clusters of filamentous structures were observed at the surface of rsv-induced filopodia, similar to branching filaments induced by hmpv (fig. 3d) . additionally, formation of filopodia was shown to confer increased motility to infected cells, thus facilitating cell-to-cell spread. filopodia induced by rsv were shown to be dependent on the rsv f protein and the cellular actin-related protein 2 (arp2), an important actin-nucleation factor (mehedi et al., 2016) . recently, a significant induction of filopodia containing budding particles was reported for the severe acute respiratory syndrome virus 2 (sars-cov-2) (bouhaddou et al., 2020) . filopodia induction was associated with an increase in signaling by casein kinase 2. indeed, ck2 was found in filopodia co-localizing with the sars-cov-2 nucleoprotein (bouhaddou et al., 2020) . analysis of measles virus (mev) infection in well-differentiated primary cultures of human airway epithelial (hae) cells revealed a previously unidentified mechanism of direct cell-to-cell spread that involves the opening of intercellular pores (singh et al., 2015) . a recombinant gfp-mev rapidly spreads in hae cell cultures to form infectious centers, but no syncytia were observed. these infectious centers are larger than those formed by other viruses such as rsv, sendai or piv5. cytoplasmic material from the infected cell was shown to suddenly flow into the adjacent cell through lateral pores of an estimated size of 250 nm (singh et al., 2015) . the formation of infectious centers by mev was dependent on an interaction between the actin filament binding protein afadin and nectin-4, the cellular receptor for mev entry (fig. 4) . recent work from the same group demonstrated that mev ribonucleoprotein complexes can spread to neighbor human airway epithelial cells by moving along the circumapical f-actin ring (fig. 4) (singh et al., 2019) . the circumapical f-actin ring encircles the uppermost portion of cells in hae cultures, close to and at the level of adherens junctions. further studies demonstrated that mev uses cellto-cell spread promoted by cell-cell contacts to move from infected immune it has been suggested that physical constraints due to the presence of the circumapical f-actin ring thwart expansion of the intercellular pores, in contrast to what is observed during syncytia formation. however, the pore has a size that permits direct spread of ribonucleoprotein complexes that can initiate infection in the adjacent cell. blue dots represent polymerase complexes bound to viral rna. cells to epithelial cells, rather than spread by cell-free virus (singh et al., 2016) . very recently, another mechanism of cell-to-cell spread exploited by mev has been reported. this mechanism involves the capture of membranes containing nectin-4 by cells expressing nectin-1, a process known as trans-endocytosis (generous et al., 2019) . cargo cytosolic material, including mev ribonucleocapsids, was shown to be transferred by trans-endocytosis and to remain functional in the recipient cell. this mechanism of spread was shown to occur between epithelial cells but also from infected epithelial cells to primary neurons. this type of spread does not require the assembly of complete virions, and demonstrates that transfer of ribonucleocapsids is sufficient to initiate infection in a target cell (generous et al., 2019) . a novel mechanism for transfer of multiple virus particles has been recently reported for enteroviruses. this virus family has historically been described as non-enveloped viruses that exit the cell by lysis. however, coxsackie virus b3 (cvb3) particles were shown to alternatively exit the cell enclosed within vesicles (robinson et al., 2014) . these extracellular microvesicles (emvs) were shown to contain autophagosomal membranes and to be infectious once added to recipient cells (robinson et al., 2014) . further evidence supporting extracellular vesicles as a mechanism for viral spread came from the demonstration of non-lytic release of poliovirus (pv) capsids (bird et al., 2014; chen et al., 2015) . large extracellular vesicles enriched with phosphatidyl serine (ps) of up to 500 nm in size were shown to contain multiple mature pv particles. the same observations were made for other enteroviruses including cvb3 and rhinovirus (chen et al., 2015) . the question of how these large pv-containing vesicles can enter a target cell was addressed, and the process was shown to be dependent on both the cellular receptor cd155 and the presence of ps on the membranes of vesicles. interestingly, it was shown that the pv particles contained within vesicles infect more efficiently than cell-free viral particles on a per-particle basis, potentially due to the synergistic effect of multiple particles entering the cell at the same time (chen et al., 2015) . non-lytic release of vesicles containing rotavirus from cultured cells was recently reported (santiana et al., 2018) . the biological relevance of this mechanism was strengthened by the observation of vesicles containing clusters of rotavirus in animal stool samples. on average, more than 15 rotavirus active particles were shown to be present in each vesicle, and these virus-containing vesicles were shed into stool. similar to what was found for pv-containing vesicles, rotavirus within vesicles was found to be more efficient in infection than cell-free viral particles (santiana et al., 2018) . norovirus particles were also shown to be released non-lytically within vesicles, but these vesicles were slightly smaller in size and had cellular markers of exosomes (santiana et al., 2018) . the first demonstrations of viral particles released in exosomal membranes came from studies with hcv and the non-enveloped hepatitis a (hav) virus (feng et al., 2013; ramakrishnaiah et al., 2013) . exosomes containing viral particles were shown to transmit hcv infection to recipient cells, a mechanism partly resistant to neutralizing antibodies (ramakrishnaiah et al., 2013) . similarly, enveloped vesicles with markers of exosomes were found to contain multiple hav particles and to mediate cell-to-cell transmission within the liver (feng et al., 2013) . these quasi-enveloped hav particles (ehav), are resistant to the action of neutralizing antibodies and can enter target cells through clathrin and dynamin-dependent endocytosis (feng et al., 2013; rivera-serrano et al., 2019) . endocytosed ehav are recognized by endolysosomal gangliosides that act as receptors, and the ehav is then trafficked to lysosomes, where the exosomal membrane is degraded and virus uncoated (das et al., 2020; rivera-serrano et al., 2019) . these novel mechanisms of spread represent an advantage for the virus, as neutralizing antibodies should not affect infectivity of vesicles the same way they neutralize cell-free particles since the particles are protected by the vesicular membrane. this was demonstrated for the human jc polyomavirus that also associates with extracellular vesicles (morris-love et al., 2019). furthermore, vesicles containing jc polyomavirus were shown to enter target cells in a manner that is independent of cellular receptors (morris-love et al., 2019). thus, production of extracellular vesicles containing infectious virus seems to be a mechanism shared by a number of rna and dna viruses, whether they are enveloped or not (altan-bonnet et al., 2019). while budding and release of cell-free virus particles enables dissemination from host-to-host and spread across long distances to infect distant tissues within a host, direct cell-to-cell spread and spread at specialized cell-cell contact sites have several potential advantages for viral spread and infection, and thus has implications for viral disease and pathogenesis. transmission of virus particles at cell-cell contact sites allows rapid viral dissemination within a tissue. in cell-to-cell spread, virus particles or virus genetic material are delivered right to target cells, making infection of target cells faster than with extracellular release and re-infection. cell-to-cell spread also overcomes the rate limiting fluid-phase virus diffusion step encountered by released particles. fitting with this, several studies have shown that the rate of hiv-1 spread through cell-to-cell transmission is faster than spread with cell-free virus (boulle et al., 2016; chen et al., 2007; dimitrov et al., 1993; sourisseau et al., 2007; spouge et al., 1996) . transfer of virus particles across cell-cell contacts can also facilitate receptor binding and entry. cell-cell contacts can specifically recruit receptors to the appropriate site, making the receptor attachment and entry steps more efficient (chen et al., 2007; sherer et al., 2007; vasiliver-shamis et al., 2009) . in some cases, like with measles virus, complete assembly of virus particles does not occur and direct spread is mediated by transfer of virus genetic material in the form of ribonucleoprotein (rnp) complexes to target cells (lawrence et al., 2000; singh et al., 2015 singh et al., , 2019 . intercellular spread is mediated by cytoskeletal forces and protein trafficking pathways, thus allowing faster transmission than that seen with particle diffusion. the actin comet tails induced by vaccinia virus act to propel virus particles quickly across long distances, allowing for faster spread (cudmore et al., 1995; doceul et al., 2010) . comparative analysis revealed that cell-to-cell spread can be significantly more efficient than cell-free spread for a number of viruses, as determined by higher infection rates that occur under conditions of cell-to-cell compared to lower infection by cell-free virus infection. hiv cell-to-cell transmission is 10-18,000â more efficient than extracellular spread (chen et al., 2007; kolodkin-gal et al., 2013; martin et al., 2010; sourisseau et al., 2007) . cell-to-cell spread of hsv and prv through intercellular extensions was shown to be essential for efficient spread in vitro (favoreel et al., 2005) . cell-to-cell transmission was initially identified for viruses that have low infectivity to particle ratio but show efficient transmission in cell culture (bangham, 2003; carr et al., 1999; dimitrov et al., 1993) . several studies have shown that spread of viruses at cell-cell contacts is associated with transfer of many virus particles or genomes from the donor to the target cell (del portillo et al., 2011; hubner et al., 2009; jin et al., 2009; jung et al., 2002; rager et al., 2002; russell et al., 2013; sherer et al., 2007; shirogane et al., 2012; sigal et al., 2011; zhong et al., 2013) . enhanced budding at cell-cell contact sites and transfer of multiple genomes provide high local multiplicity of infection (moi) (fig. 5) . this high moi makes cell-to-cell spread more efficient than cell-free transmission by increasing fig. 5 advantages of cell-to-cell spread. (a) cell-free viral particles released by budding can be affected by the presence of neutralizing antibodies in the extracellular environment. in contrast, viral particles that are disseminated from cell-to-cell at cell junction-specialized membrane contacts (as one example of cell-to-cell spread) are not accessible to neutralizing antibodies (b). once they enter a cell, individual viral particles can be targeted by intracellular restriction factors during uncoating. in contrast, multiple viral particles entering a target cell can saturate restriction factors available in the cell and proceed with infection. after successful uncoating, individual viral particles will initiate genome replication starting from single genomes (d), a highly inefficient process. in contrast, several viral genomes coming from particles will be more efficient in replication, leading to a rapid infection. alternatively, genomes can be transmitted from cell-to-cell directly through intercellular pores (as an example), which can bypass the uncoating step and inhibition by restriction factors, proceeding more efficiently with replication (c). the probability of successful infection through the introduction of multiple genomes into a single target cell, thus allowing spread even with low viral gene expression and poor particle release. it has been suggested that the transfer of multiple genomes during cell-to-cell spread has implications to virus fitness and evolution (sanjuan, 2017) . other advantages of high moi include trans-complementation of genetic defects, evasion of innate immune responses in the target cell, avoidance of the loss of viral components necessary for replication, alleviation of transmission bottlenecks and maintainance of genetic diversity (sanjuan, 2017; sanjuan and thoulouze, 2019) . although many virus particles or genomes can be transferred, only a limited number of genomes likely initiate infection in the target cell (miyashita et al., 2015) . it has been estimated that as many as 10 2 or 10 3 hiv-1 particles can be transmitted through a virological synapse (chen et al., 2007; hubner et al., 2009 ); however, hiv-1 infects with an average of 3.62 proviruses after cell-to-cell spread (del portillo et al., 2011) which is relevant in vivo where 3-4 genomes are detected in infected cells ( jung et al., 2002; law et al., 2016) . the alpha-herpesviruses hsv and prv infect with an average of 1.4 and 1.6 genomes, respectively, following cell-to-cell transmission although multiple virions were seen transported on distal axon sites (taylor et al., 2012a) . this suggests that although many virions or genomes can be transmitted at sites of cell-cell contacts, only a limited number establish infection which may be advantageous for the virus. further studies are needed to determine if this is true for other viruses that use cell-to-cell spread for transmission. spread of viruses by cell-to-cell transmission allows evasion of multiple barriers that exist for cell-free infection. one of the hallmarks of cell-to-cell spread is that it allows transmission in the presence of neutralizing antibodies (fig. 5) . spread at tight cell-cell contacts provides limited exposure time to neutralizing antibodies in the extracellular environment and can physically restrict access of neutralizing antibodies to the virus. neutralizingantibody resistant spread has been shown for viruses that use different modes of cell-to-cell spread. for example, transmission of viruses such as hcv, hiv-1 and hsv-1 across tight cell-cell contacts has been shown to shield the virus from neutralizing antibodies (brimacombe et al., 2011; favoreel et al., 2006) . viruses that use nanotubes, filopodia, or intercellular extensions, whether open-or close-ended, show a neutralizing antibody-resistant mode of spread. this includes the alphaherpesviruses prv and hsv-1 (favoreel et al., 2005; jansens et al., 2017; sarfo et al., 2017) , the alphavirus sindbis virus (martinez and kielian, 2016) , the respiratory viruses hmpv, influenza a virus, and piv5 (el najjar et al., 2016; kumar et al., 2017; roberts et al., 2015) , prrsv (guo et al., 2016 (guo et al., , 2018 , and coxsackievirus b3 (paloheimo et al., 2011) . antibody resistant cell-to-cell spread of enveloped vacv particles occurs in an actin-dependent or -independent manner likely mediated by protein a33 (krupovic et al., 2010; law et al., 2002) . the formation of viral biofilms by htlv-1 may also provide a way of physically protecting particles from circulating antibodies which recognize the env protein (pais-correia et al., 2010; thoulouze and alcover, 2010) . for hiv-1, several reports show conflicting results on the role of neutralizing antibodies in inhibiting cell-to-cell transmission, with some studies showing decreased neutralization during cellto-cell spread and others showing no inhibition of neutralizing antibodies on direct virus transfer. variations in the effects of the neutralizing antibodies depend on the epitope on env protein targeted by the antibody, the donor and target cell type, and virus strain used (abela et al., 2012; chen et al., 2007; ganesh et al., 2004; gupta et al., 1989; martin et al., 2010; massanella et al., 2009; pantaleo et al., 1995; van montfort et al., 2007; zhong et al., 2013) . in addition to evasion from neutralizing antibodies, cell-to-cell spread can play a role in overcoming intrinsic antiviral restriction factors. for retroviruses, restriction factors such as trim5α and tetherin can effectively inhibit cell-free virus spread, but are less effective, or fail completely, in preventing cell-to-cell transmission (celestino et al., 2012; jolly et al., 2010; richardson et al., 2008; zhong et al., 2013) . this may occur due to saturation of the restriction factors by accumulation of virus particles at sites of cell-cell contacts or because cell-associated virus is inaccessible to a restriction factor. it has been suggested that tetherin can promote accumulation of hiv-1 virus particles at the vs and even help to stabilize the synapse during cell-to-cell spread . cell-to-cell transmission may also act on overcoming the innate antiviral response. it has been suggested that transfer of influenza a virus ns protein through tunneling nanotubes may overcome the innate immune response in the target cell (kumar et al., 2017) . cell-to-cell spread has implications for the pathogenesis of viral infection as it contributes to viral persistence and latency, therapy failure/resistance, and immune evasion. the cell-to-cell mode of transmission is inherent to the pathogenesis of hsv-1; it is the main mode of spread in the epithelium and from epithelial cells to neurons during primary infection where it establishes latency and moves back through axons to epithelial cells during reactivation (dingwell et al., 1994; wang et al., 2010) . cell-to-cell transmission has also been implicated in persistence of hcv infection and establishment of chronic infection in the liver, in addition to resistance to direct-acting antiviral drugs and therapy failure (timpe et al., 2008; witteveldt et al., 2009; xiao et al., 2014; zeisel et al., 2013) . resistance to antivirals due to direct cell-to-cell spread has also been shown for influenza virus with resistance to oseltamivir treatment (kumar et al., 2017; mori et al., 2011; roberts et al., 2015) . the different modes of cell-to-cell transmission that hiv-1 utilizes contribute to the persistence of viral reservoirs (costiniuk and jenabian, 2014) and resistance to antiretroviral therapy (duncan et al., 2013; sigal et al., 2011) . understanding mechanisms of cell-to-cell spread is thus important for development of a deeper understanding of viral pathogenesis and for the development of potent antiviral therapies. cell-to-cell spread multiple approaches have been used in in-vitro cell culture models to study the different modes of virus transmission. experimental conditions were developed for blocking cell-free or cell-to-cell spread to determine the contribution of either in infection dynamics. addition of a viscous media such as methylcellulose or agarose to an infected cell culture suppresses diffusion of released virus particles and thus decreases spread by cell-free virus and not by direct cell-to-cell spread (el najjar et al., 2016; jin et al., 2009; martin et al., 2010; timpe et al., 2008) . another approach for blocking cell-free virus infection that provides more convincing experimental support is assaying spread in the presence of virus-neutralizing antibodies. while neutralizing antibodies are successful in completely, or almost completely, inhibiting cell-free infection, cell-to-cell transmission is generally resistant to neutralizing antibodies. spread in the presence of neutralizing antibodies has been used to demonstrate cell-to-cell transmission for several viruses including hcv-1, herpesviruses, alphaviruses, coxsackievirus b3, iav, hmpv and prrsv (brimacombe et al., 2011; favoreel et al., 2005 favoreel et al., , 2006 guo et al., 2016; jansens et al., 2017; martinez and kielian, 2016; roberts et al., 2015; sarfo et al., 2017) . in some cases however, such as hiv-1 and cmv, the effect of neutralizing antibodies can be inconsistent, as discussed above, and further validation of transmission modes requires additional investigation (dufloo et al., 2018; jacob et al., 2013) . in addition, some neutralizing antibodies can prevent cell-to-cell transmission by inhibiting establishment or stability of cell-cell contacts between an infected donor cell and an uninfected target cell. for instance, cell-to-cell transmission of hiv-1 depends on cell-cell contacts that form by the interaction of env protein in the donor cell with the cd4 receptor on the target cell; thus neutralizing antibodies against env protein can block cell-cell contact and cell-to-cell spread (chen et al., 2007; rudnicka et al., 2009) . it is therefore important to use neutralizing antibodies that do not interfere with the formation of cell-cell contacts in order to differentiate the susceptibility of cell-free and cell-to-cell transmission to neutralization. experimental assays to assess the role of direct contact between infected cells and neighboring cells in spreading infection include using a trans-well to physically separate donor and target cells and prevent cell migration, cell shaking to abolish the formation of cell-cell contacts or lowering cell density to prevent close cell contact. coculturing of donor infected cells with target cells is another widely used assay to identify cell-to-cell transmission. in this assay, infected target cells are harvested and cocultured with uninfected target cells under conditions that prevent infection by cell-free virus such as addition of neutralizing antibodies or blocking receptor binding (branscome et al., 2019; el najjar et al., 2016; martinez and kielian, 2016; xiao et al., 2014; zhong et al., 2013) . a fluorescent dye is usually used to differentiate target cells from donor cells, and quantification of cell spread is determined microscopically or by flow cytometry of a reporter virus or a labeled viral antigen. the cell coculture system has been used to identify viral and host factors that contribute to direct cell spread of several viruses. cell-to-cell transmission of hcv depends on viral envelope proteins and utilizes the same receptors used for cell-free infection including scavenger receptor bi, cd81, epidermal growth factor receptor (egfr) and the tight junction proteins claudin-1 and occludin (catanese et al., 2013; fofana et al., 2013; lupberger et al., 2011; witteveldt et al., 2009; zahid et al., 2013) . cell-to-cell spread for other viruses such as hmpv and sinv occurs in a manner that is independent, or partially independent, of the receptor binding which is needed for cell-free entry (el najjar et al., 2016; martinez and kielian, 2016) . for studying spread of herpesviruses, two cell culture systems are primarily used: a dissociated model where rat superior cervical ganglia (scg) are plated and form a network of axons; and a compartmentalized system that provides isolation of the neuron cell body from the distal axon termini (curanovic et al., 2009; ch'ng et al., 2005 ). the latter system has been used to study the number and diversity of viral genomes after anterograde direct spread of hsv-1 and prv particles, with results showing limited virus transmission from neurons to epithelial cells (taylor et al., 2012a) . experiments conducted in 2-dimensional cell culture models provide valuable information on the mechanisms of cell-to-cell spread of viruses; however, they do not take into account how the complexity and environment of the tissue impacts virus spread in vivo. to overcome some of these limitations, different 3-dimensional cell culture models have been utilized for studying spread for a number of viruses. a 3-d cell culture system using hiv-1-infected primary human cd4 t lymphocytes in a 3-d scaffold showed the importance of cell motility and density to hiv-1 spread (imle et al., 2019) . studying measles virus in a 3-d model of primary human differentiated airway epithelial cells revealed a novel mechanism of rnp horizontal spread along apical f-actin rings (singh et al., 2019) . a neutralizing antibody-resistant mode of spread was recently described for hmpv in a similar 3-d model of differentiated human bronchial epithelial cells (kinder et al., 2020) . further studies in 3-d tissue models are necessary to elucidate how viruses actually spread within the host and identify antivirals that affect one mode of virus transmission but not the other. imaging fixed cells by fluorescence and electron microcopy has been widely used for showing virus transfer between cells. detection of viral proteins in intercellular connections or at sites of cell-cell contact by immunofluorescence (if) is usually added to the line of evidence to demonstrate cellto-cell spread. a number of studies showed localization of viral proteins in tnts, filopodia, and other intercellular projections for viruses of different families that spread by direct cell-to-cell transmission, including the herpesvirus bohv-1, alphavirus sinv, prrsv and hmpv. localization of viral genomes within these cellular structures, as detected by fluorescence in situ hybridization (fish), indicated that not only viral antigens are localizing to intercellular connections but also genetic material, thus suggesting passage of virus particles or viral genomes across these structures (el najjar et al., 2016; favoreel et al., 2005; guo et al., 2016; martinez and kielian, 2016) . for hiv-1, a vs was initially described by if showing clustering of receptor on the target cell and env protein on the effector cell mcdonald et al., 2003) , and transmission electron microscopy provided cross sectional views of the synapse (wang et al., 2007) . advances in microscopic imaging facilitated by the generation of fluorescently labeled recombinant viruses helped to further deliver mechanistic insights underlying cell-to-cell spread of viruses. live cell imaging provides a powerful tool for demonstrating the dynamics of virus transport between cells. combining if and time lapse microscopy with correlative fluorescence and scanning microscopy revealed dynamics of movement of single mlv particles on the surface of cytonemes that form by interaction of env protein with the receptor mcat1 ( jin et al., 2009; sherer et al., 2007) . live cell 3-d microscopy and expansion microscopy of influenza virus infected cells, in addition to scanning electron microscopy (sem), showed that the ends of tnt are continuous with the cytoplasm of cells and that there are bundled fibers inside the tnts (kumar et al., 2017) . generation of fluorescently labeled alphaherpesviruses with fluorophore tagged capsid, tegument, or glycoproteins allowed examination of egress and spread mechanisms. live imaging of neurons infected with different labeled viruses indicated that both capsids and enveloped particles move across axons and that the us9 membrane protein is needed for anterograde transport of progeny virus particles (taylor et al., 2012b; wisner et al., 2011) . imaging alphaherpesviruses hsv-1 and prv, tagged with different fluorophores, allowed determination of the diversity and number of viral genomes following anterograde-directed spread from isolated neurites to epithelial cells in a compartmentalized cell culture model (taylor et al., 2012a) . imaging techniques have also advanced our understanding of measles virus spread. time lapse microscopy with 3-d z-stack imaging of gfp-tagged measles virus, or more recently of a recombinant virus containing gfp/p fusion protein, in a model of airway epithelial tissue was used to reveal rapid spread. infectious centers and rnp were shown along the apical f actin network stained with the lifeact marker as mentioned in more detail in section 2.9 (singh et al., 2015 (singh et al., , 2019 . more information has been gathered in recent years on the formation of the vs mediating hiv-1 spread. analysis using ion abrasion sem and electron tomography provided 3-d visualization of the vss with invaginations forming in the donor cell and a projection extending from the target cell (felts et al., 2010) . kinetics of virus clustering and transfer were determined by quantitative 3-d live imaging (hubner et al., 2009; real et al., 2018) . recently, live imaging using a gfp-tagged env mutant virus and cells stably expressing mcherry-gag revealed that the env protein is first transferred across the vs followed by redistribution of gag to cell-cell contact sites (wang et al., 2019) . super resolution microscopy revealed an organized localization of n and m proteins of hmpv in budding filaments with n protein on the inside surrounded by the m protein (el najjar et al., 2016) . combining functional and imaging approaches is thus essential for elucidating the mechanisms underlying cell-to-cell spread of different viruses. in addition, mathematical models have been employed in recent years to provide better quantitation of the contribution of the different modes of spread in infection dynamics. such models account for the role of space and effects of the immune response on viral spread and utilize experimental and clinical data to assess modes of virus spread in vivo (goyal and murray, 2016; graw et al., 2015; graw and perelson, 2016; kumberger et al., 2018) . research in the last several decades has highlighted both the variety of mechanisms that viruses utilize for spreading to new target cells and the surprising commonality of many mechanisms. recent work suggests that these mechanisms may be important for pathogenesis, providing a more efficient means of propagating infection under many circumstances. future work dissecting the role of cell-to-cell spread in animal models will be critical to understand the importance of these mechanisms in overall pathogenesis. in addition, dissection of these mechanisms may provide important new targets for antiviral treatment. cell-cell transmission enables hiv-1 to evade inhibition by potent cd4bs directed antibodies extracellular vesicles: vehicles of en bloc viral transmission casein kinase 2 regulates vaccinia virus actin tail formation the formin fhod1 and the small gtpase rac1 promote vaccinia virus actin-based motility unique features of hiv-1 spread through t cell virological synapses cell-fusion events induced by α-herpesviruses the virological synapse facilitates herpes simplex virus entry into t cells the immune control and cell-to-cell spread of human t-lymphotropic virus type 1 engagement of specific t-cell surface molecules regulates cytoskeletal polarization in htlv-1-infected lymphocytes identification of host trafficking genes required for hiv-1 virological synapse formation in dendritic cells nonlytic viral spread enhanced by autophagy components the isolation and characterization of mutants of herpes simplex virus type 1 that induce cell fusion the global phosphorylation landscape of sars-cov-2 infection hiv cell-to-cell spread results in earlier onset of viral gene expression by multiple infections per cell stem cell extracellular vesicles and their potential to contribute to the repair of damaged cns cells neutralizing antibody-resistant hepatitis c virus cell-to-cell transmission rapid and efficient cell-to-cell transmission of human immunodeficiency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes different requirements for scavenger receptor class b type i in hepatitis c virus cell-free versus cell-to-cell transmission feline tetherin is characterized by a short n-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein a viral fusogen hijacks the actin cytoskeleton to drive cell-cell fusion f-actin dynamics transform filopodial bridges into intercellular nanotubes capable of distant cell communication predominant mode of human immunodeficiency virus transfer between t cells is mediated by sustained envdependent neutralization-resistant virological synapses phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses culturing primary and transformed neuronal cells for studying pseudorabies virus infection hantavirus gn and gc envelope glycoproteins: key structural units for virus cell entry and virus assembly varicella-zoster virus glycoprotein i is essential for growth of virus in vero cells membrane fusion mediated by herpesvirus glycoproteins: the paradigm of varicella-zoster virus they might be giants: does syncytium formation sink or spread hiv infection? cell-to-cell transfer of hiv infection: implications for hiv viral persistence actin-based motility of vaccinia virus compartmented neuron cultures for directional infection by alpha herpesviruses gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis a virus multiploid inheritance of hiv-1 during cell-to-cell infection differential identity of filopodia and tunneling nanotubes revealed by the opposite functions of actin regulatory complexes quantitation of human immunodeficiency virus type 1 infection kinetics the herpes simplex virus ge-gi complex facilitates cell-to-cell spread and binds to components of cell junctions herpes simplex virus glycoproteins e and i facilitate cell-to-cell spread in vivo and across junctions of cultured cells deletion of the pseudorabies virus ge/gi-us9p complex disrupts kinesin kif1a and kif5c recruitment during egress, and alters the properties of microtubule-dependent transport in vitro herpes simplex virus type 1 induces filopodia in differentiated p19 neural cells to facilitate viral spread three-dimensional imaging of hiv-1 virological synapses reveals membrane architectures involved in virus transmission repulsion of superinfecting virions: a mechanism for rapid virus spread wip provides an essential link between nck and n-wasp during arp2/3-dependent actin polymerization hiv-1 cell-to-cell transmission and broadly neutralizing antibodies fusogenic reoviruses and their fusion-associated small transmembrane (fast) proteins avian reovirus-induced syncytium formation is independent of infectious progeny virus production and enhances the rate, but is not essential, for virus-induced cytopathology and virus egress high multiplicity hiv-1 cell-to-cell transmission from macrophages to cd4+ t cells limits antiretroviral efficacy loss of actin-based motility impairs ectromelia virus release in vitro but is not critical to spread in vivo macrophage cell-cell interactions promoting hiv-1 infection actin remodeling to facilitate membrane fusion human metapneumovirus induces reorganization of the actin cytoskeleton for direct cell-to-cell spread tunneling nanotubes (tnt) are induced by hiv-infection of macrophages: a potential mechanism for intercellular hiv trafficking the making of filopodia attachment and postattachment receptors important for hepatitis c virus infection and cell-to-cell transmission herpes simplex virus ge/gi must accumulate in the trans-golgi network at early times and then redistribute to cell junctions to promote cell-cell spread cytoskeletal rearrangements and cell extensions induced by the us3 kinase of an alphaherpesvirus are associated with enhanced spread herpesvirus interference with virus-specific antibodies: bridging antibodies, internalizing antibodies, and hiding from antibodies 3d visualization of hiv transfer at the virological synapse between dendritic cells and t cells a pathogenic picornavirus acquires an envelope by hijacking cellular membranes a novel monoclonal anti-cd81 antibody produced by genetic immunization efficiently inhibits hepatitis c virus cell-cell transmission tyrosine phosphorylation is required for actin-based motility of vaccinia but not listeria or shigella actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling infection of specific dendritic cells by ccr5-tropic human immunodeficiency virus type 1 promotes cell-mediated transmission of virus resistant to broadly neutralizing antibodies trans-endocytosis elicited by nectins transfers cytoplasmic cargo, including infectious material, between cells viroporins customize host cells for efficient viral propagation inhibitory effect of individual or combinations of broadly neutralizing antibodies and antiviral reagents against cell-free and cell-to-cell hiv-1 transmission rhoa signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology modelling the impact of cell-to-cell transmission in hepatitis b virus modeling viral spread quantification of hepatitis c virus cell-to-cell spread using a stochastic modeling approach the tax-inducible actin-bundling protein fascin is crucial for release and cell-to-cell transmission of human t-cell leukemia virus type 1 (htlv-1) porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread intercellular transfer of mitochondria rescues virusinduced cell death but facilitates cell-to-cell spreading of porcine reproductive and respiratory syndrome virus cell-to-cell transmission of human immunodeficiency virus type 1 in the presence of azidothymidine and neutralizing antibody human metapneumovirus: a new player among respiratory viruses virus-cell and cell-cell fusion identification of residues in the human respiratory syncytial virus fusion protein that modulate fusion activity and pathogenesis herpes simplex virus ge/gi extracellular domains promote axonal transport and spread from neurons to epithelial cells rotavirus nonstructural proteins: structure and function quantitative 3d video microscopy of hiv transfer across t cell virological synapses clathrin potentiates vaccinia-induced actin polymerization to facilitate viral spread spread of htlv-i between lymphocytes by virus-induced polarization of the cytoskeleton experimental and computational analyses reveal that environmental restrictions shape hiv-1 spread in 3d cultures neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus pseudorabies virus us3-induced tunneling nanotubes contain stabilized microtubules, interact with neighbouring cells via cadherins and allow intercellular molecular communication bridging the gap: virus long-distance spread via tunneling nanotubes assembly of the murine leukemia virus is directed towards sites of cell-cell contact herpesviruses remodel host membranes for virus egress directed egress of animal viruses promotes cell-to-cell spread herpes simplex virus ge/gi sorts nascent virions to epithelial cell junctions, promoting virus spread retroviral spread by induction of virological synapses hiv-1 cell to cell transfer across an env-induced, actin-dependent synapse cell-cell spread of human immunodeficiency virus type 1 overcomes tetherin/bst-2-mediated restriction in t cells the regulated secretory pathway in cd4(+) t cells contributes to human immunodeficiency virus type-1 cell-to-cell spread at the virological synapse african swine fever virus induces filopodia-like projections at the plasma membrane recombination: multiply infected spleen cells in hiv patients mechanisms of virus membrane fusion proteins respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) infections in 3-d human airway tissues expose an interesting dichotomy in viral replication, spread, and inhibition by neutralizing antibodies efficiency of cell-free and cell-associated virus in mucosal transmission of human immunodeficiency virus type 1 and simian immunodeficiency virus glycoproteins ge and gi are required for efficient kif1a-dependent anterograde axonal transport of alphaherpesvirus particles in neurons the tumor marker fascin is strongly induced by the tax oncoprotein of htlv-1 through nf-kappab signals protein a33 responsible for antibody-resistant spread of vaccinia virus is homologous to c-type lectin-like proteins influenza virus exploits tunneling nanotubes for cell-to-cell spread accounting for space-quantification of cell-to-cell transmission kinetics using virus dynamics models the nucleoprotein of lymphocytic choriomeningitis virus facilitates spread of persistent infection through stabilization of the keratin network absence of keratin 1 restricts the course of infection with lymphocytic choriomeningitis virus strain mx antibody-sensitive and antibody-resistant cellto-cell spread by vaccinia virus: role of the a33r protein in antibody-resistant spread in vivo hiv-1 cell-to-cell transmission promotes multicopy micro-compartmentalized infection measles virus spread between neurons requires cell contact but not cd46 expression, syncytium formation, or extracellular virus production actin-and myosin-driven movement of viruses along filopodia precedes their entry into cells role of lipids in virus replication egfr and epha2 are host factors for hepatitis c virus entry and possible targets for antiviral therapy pseudorabies virus us9 directs axonal sorting of viral capsids virological synapse-mediated spread of human immunodeficiency virus type 1 between t cells is sensitive to entry inhibition intercellular extensions are induced by the alphavirus structural proteins and mediate virus transmission imaging the alphavirus exit pathway divergent roles of beta-and gamma-actin isoforms during spread of vaccinia virus antigp41 antibodies fail to block early events of virological synapses but inhibit hiv spread between t cells connections matter-how viruses use cell-cell adhesion components filopodia: molecular architecture and cellular functions spontaneous electrical activity induced by herpes virus infection in rat sensory neuron cultures pseudorabies virus infection alters neuronal activity and connectivity in vitro recruitment of hiv and its receptors to dendritic cell-t cell junctions herpes simplex virus type 1 glycoprotein e mediates retrograde spread from epithelial cells to neurites actin-related protein 2 (arp2) and virus-induced filopodia facilitate human respiratory syncytial virus spread viruses roll the dice: the stochastic behavior of viral genome molecules accelerates viral adaptation at the cell and tissue levels hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes tamiflu-resistant but ha-mediated cell-to-cell transmission through apical membranes of cell-associated influenza viruses jc polyomavirus uses extracellular vesicles to infect target cells hiv-infected t cells are migratory vehicles for viral dissemination characterization of cell death pathways in human immunodeficiency virus-associated encephalitis demonstration of respiratory syncytial virus in an autopsy series human t-lymphotropic virus, type 1, tax protein triggers microtubule reorientation in the virological synapse viruses that ride on the coat-tails of actin nucleation abl collaborates with src family kinases to stimulate actin-based motility of vaccinia virus viroporins: structure and biological functions rho, rac, and cdc42 gtpases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria biofilm-like extracellular viral assemblies mediate htlv-1 cell-to-cell transmission at virological synapses coxsackievirus b3-induced cellular protrusions: structural characteristics and functional competence tunneling nanotubes as a novel route of cell-to-cell spread of herpesviruses effect of anti-v3 antibodies on cell-free and cell-to-cell human immunodeficiency virus transmission actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virusinduced cell fusion polyploid measles virus with hexameric genome length exosomemediated transmission of hepatitis c virus between human hepatoma huh7.5 cells live imaging of hiv-1 transfer across t cell virological synapse to epithelial cells that promotes stromal macrophage infection variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and src family tyrosine kinases mechanisms of varicella-zoster virus neuropathogenesis in human dorsal root ganglia mode of transmission affects the sensitivity of human immunodeficiency virus type 1 to restriction by rhesus trim5alpha cellular entry and uncoating of naked and quasi-enveloped human hepatoviruses influenza a virus uses intercellular connections to spread to neighboring cells simultaneous cell-tocell transmission of human immunodeficiency virus to multiple targets through polysynapses multiple proviral integration events after virological synapse-mediated hiv-1 spread nanotubular highways for intercellular organelle transport requirement of interaction of nectin-1alpha/hvec with afadin for efficient cell-cell spread of herpes simplex virus type 1 collective infectious units in viruses why viruses sometimes disperse in groups?(dagger) vesicle-cloaked virus clusters are optimal units for inter-organismal viral transmission the ul21 tegument protein of herpes simplex virus 1 is differentially required for the syncytial phenotype grb2 and nck act cooperatively to promote actin-based motility of vaccinia virus human papillomavirus type 16 entry: retrograde cell surface transport along actin-rich protrusions rho gtpase activity modulates paramyxovirus fusion proteinmediated cell-cell fusion retroviruses use cd169-mediated trans-infection of permissive lymphocytes to establish infection retroviruses can establish filopodial bridges for efficient cell-to-cell transmission cooperation between different rna virus genomes produces a new phenotype a new class of fusion-associated small transmembrane (fast) proteins encoded by the non-enveloped fusogenic reoviruses cell-to-cell spread of hiv permits ongoing replication despite antiretroviral therapy the nectin-4/afadin protein complex and intercellular membrane pores contribute to rapid spread of measles virus in primary human airway epithelia cell-to-cell contact and nectin-4 govern spread of measles virus from primary human myeloid cells to primary human airway epithelial cells measles virus ribonucleoprotein complexes rapidly spread across well-differentiated primary human airway epithelial cells along f-actin rings new connections: cell-to-cell hiv-1 transmission, resistance to broadly neutralizing antibodies, and an envelope sorting motif the exit of vaccinia virus from infected cells virus activated filopodia promote human papillomavirus type 31 uptake from the extracellular matrix herpes simplex virus ge/gi and us9 proteins promote transport of both capsids and virion glycoproteins in neuronal axons inefficient human immunodeficiency virus replication in mobile lymphocytes membrane nanotubes physically connect t cells over long distances presenting a novel route for hiv-1 transmission hiv-1 infection kinetics in tissue cultures high-voltage electron microscope study of the release of vaccinia virus from whole cells wild-type measles virus induces large syncytium formation in primary human small airway epithelial cells by a slam(cd150)-independent mechanism alphaherpesvirus axon-to-cell spread involves limited virion transmission visualization of an alphaherpesvirus membrane protein that is essential for anterograde axonal spread of infection in neurons subversion of the actin cytoskeleton during viral infection viral extracellular biofilms: a novel spreading trick of viruses? efficient capture of antibody neutralized hiv-1 by cells expressing dc-sign and transfer to cd4+ t lymphocytes human t-cell leukemia virus type 1 p8 protein increases cellular conduits and virus transmission pathology of human metapneumovirus infection: insights into the pathogenesis of a newly identified respiratory virus human immunodeficiency virus type 1 envelope gp120-induced partial t-cell receptor signaling creates an f-actin-depleted zone in the virological synapse virus budding and the escrt pathway functionally distinct transmission of human immunodeficiency virus type 1 mediated by immature and mature dendritic cells herpes simplex virus type 2 glycoprotein e is required for efficient virus spread from epithelial cells to neurons and for targeting viral proteins from the neuron cell body into axons sequential trafficking of env and gag to hiv-1 t cell virological synapses revealed by live imaging how to get out: ssrna enveloped viruses and membrane fission arp2/3-mediated actin-based motility: a tail of pathogen abuse anterograde transport of herpes simplex virus capsids in neurons by both separate and married mechanisms cd81 is dispensable for hepatitis c virus cell-to-cell transmission in hepatoma cells hepatitis c virus cell-cell transmission and resistance to direct-acting antiviral agents dynamic monitoring of membrane nanotubes formation induced by vaccinia virus on a high throughput microfluidic chip the cytoplasmic domain of varicella-zoster virus glycoprotein h regulates syncytia formation and skin pathogenesis disruption of adherens junctions liberates nectin-1 to serve as receptor for herpes simplex virus and pseudorabies virus entry the postbinding activity of scavenger receptor class b type i mediates initiation of hepatitis c virus infection and viral dissemination host-targeting agents for prevention and treatment of chronic hepatitis c-perspectives and challenges binding of herpes simplex virus glycoprotein d to nectin-1 exploits host cell adhesion cell-to-cell transmission can overcome multiple donor and target cell barriers imposed on cell-free hiv glycoprotein gi of pseudorabies virus promotes cell fusion and virus spread via direct cell-to-cell transmission this work was supported in part by nih grants ai051517 and ai140758 to r.e.d. and fondecyt inicio grant 11180269 to n.c.m. images were created using biorender.com. key: cord-343470-w215pzdc authors: tsai, kevin; cullen, bryan r. title: epigenetic and epitranscriptomic regulation of viral replication date: 2020-06-12 journal: nat rev microbiol doi: 10.1038/s41579-020-0382-3 sha: doc_id: 343470 cord_uid: w215pzdc eukaryotic gene expression is regulated not only by genomic enhancers and promoters, but also by covalent modifications added to both chromatin and rnas. whereas cellular gene expression may be either enhanced or inhibited by specific epigenetic modifications deposited on histones (in particular, histone h3), these epigenetic modifications can also repress viral gene expression, potentially functioning as a potent antiviral innate immune response in dna virus-infected cells. however, viruses have evolved countermeasures that prevent the epigenetic silencing of their genes during lytic replication, and they can also take advantage of epigenetic silencing to establish latent infections. by contrast, the various covalent modifications added to rnas, termed epitranscriptomic modifications, can positively regulate mrna translation and/or stability, and both dna and rna viruses have evolved to utilize epitranscriptomic modifications as a means to maximize viral gene expression. as a consequence, both chromatin and rna modifications could serve as novel targets for the development of antivirals. in this review, we discuss how host epigenetic and epitranscriptomic processes regulate viral gene expression at the levels of chromatin and rna function, respectively, and explore how viruses modify, avoid or utilize these processes in order to regulate viral gene expression. although all human cells in a given individual contain the same genome, they differ widely in the genes they express and, hence, in their biological properties. it is therefore well established that gene expression is not determined simply by the sequence information that is encoded into genomic dna, but rather is subject to multiple levels of control. this review discusses two complementary regulatory mechanisms that act, respectively, at the dna and rna levels. in the case of the host-cell genome, the level of expression of specific genes is regulated not only by flanking promoters and enhancers but also by how accessible the genes embedded within cellular chromatin are to transcription factors. this accessibility in turn is regulated by processes that include dna methylation, histone remodelling, alternative histone variant usage and the deposition of modifications on histone tails, collectively referred to as epigenetic gene regulation 1,2 (box 1). similarly, the function of the mrnas transcribed from a given gene is regulated not only by their nucleotide sequence but also by a range of covalent modifications that are added to individual nucleotides, the most prevalent of which is methylation of the n 6 position of adenosine (m 6 a) (refs 3,4 ). these rna modifications -which have been reported to regulate the stability, the translation and even the immunogenicity of rna molecules -are referred to as epitranscriptomic modifications. epigenetic and epitranscriptomic gene regulation likely initially evolved as means of regulating cell growth and differentiation. however, they are also relevant to several important diseases, including cancer, in which both processes have been reported to be dysregulated [5] [6] [7] . their importance for the regulation of viral gene expression has only recently begun to emerge. as obligate intracellular parasites, viruses misappropriate parts of the host-cell machinery in order to allow the expression of viral genes. this dependence on cellular gene regulation pathways can, however, also lead to viral gene expression being subject to host repression. indeed, epigenetic repression has been proposed to function as a form of antiviral restriction used by host cells as an innate immune defence against dna viruses, in a tug of war that is only revealed when viral countermeasures are experimentally removed 8 . alternatively, viruses may repurpose epigenetic repression in order to suppress their own gene expression, as a way to establish latent infections and prevent the production of immunogenic viral proteins 9 . by contrast, epitranscriptomic modifications generally enhance the function of viral mrnas, and both dna and rna viruses have therefore evolved to maximize the level of these modifications on their transcripts. in this review, we discuss epigenetic repression mechanisms, including histone tail modifications and chromatin a dna-protein complex consisting of genomic dna wrapped around organizing proteins called histones (see box 1) . the addition of a small chemical group called a methyl group (ch 3 ), found as a chemical modification on dna, rna and proteins (in particular, histones). a viral life-cycle stage in which the viral genomic material persists long-term in the host cell with minimal viral gene expression and replication. alternative histone variants that are loaded onto viral dna upon entry into host-cell nuclei and how viruses avoid or even utilize this repression to enhance viral replication or persistence. we then explore the epitranscriptomic rna modifications, including methylation and acetylation, found on viral transcripts, and how they enhance viral gene expression or help evade innate immune detection. we highlight the importance of non-encoded regulatory information found on viral chromatin and rna and discuss how the regulatory pathways that are influenced by this information could serve as novel targets for antiviral therapies. epigenetic repression and its avoidance incoming viral dna molecules are sensed by target cells and are rapidly loaded with histones bearing heterochromatic marks, thus inhibiting viral gene expression. how cells distinguish viral dna from their own remains undefined, though epigenetic repression of foreign dna seems mainly to be orchestrated by proteins associated with the pro-myelocytic leukaemia nuclear bodies (pml-nbs) and by the innate immune dna sensor interferon-inducible protein 16 (ifi16) 8,10-12 . here we discuss how herpesviruses, hepatitis b virus (hbv) and retroviruses can be epigenetically repressed and how these viruses can disrupt or avoid repression in order to initiate productive infections. we also discuss how epigenetic repression can be misappropriated to establish viral latent infections. epigenetic suppression of viral dna is frequently associated with nuclear protein aggregates called pml-nbs or nuclear domain 10 (nd10) ( fig. 1a ). pml-nbs consist of cage-like structures constructed of pml proteins, which contain other effector proteins that together appear as punctate foci when visualized by immunofluorescence microscopy [13] [14] [15] . several dna viruses, including simian virus 40, adenovirus type 5, herpes simplex virus 1 (hsv-1) and human cytomegalovirus (hcmv), localize to and establish viral replication sites adjacent to pml-nbs, suggesting that this structure is at least spatially, if not functionally, related to viral replication 16 . through the recruitment of cofactors, pml-nbs can be associated with many cellular functions, including antiviral defence and transcriptional repression. the expression of the pml-nb components sp100 and pml increases in response to interferon signalling, and pml-nb components including pml, sp100, daxx and atrx suppress viral replication 13, [17] [18] [19] [20] . daxx and sp100 also have transcriptional repressive functions, likely through recruiting co-repressors such as hp1 and hdac ii [21] [22] [23] [24] . moreover, daxx and atrx can load the histone variant h3.3 onto heterochromatic regions 25, 26 . all of the functions above implicate box 1 | relationship of chromatin architecture and histone modifications to gene expression the genomic dna of cells is bound by basic proteins called histones, with every ~147 bp of dna wrapped around a core octamer containing two molecules apiece of the histones h2a, h2b, h3 and h4, to form chromatin (see the figure) . although chromatin can protect genome integrity, histones can also hinder the accessibility of genomic dna for transcription. this accessibility is a function of the degree of compaction of the histones bound to that genomic dna, with open regions being referred to as euchromatin, whereas tightly compacted regions of dna are referred to as heterochromatin. chromatin compaction constitutes a form of gene regulation not directly encoded in the dna nucleotide sequence itself; this is termed epigenetic gene regulation, as it is the molecular mechanism underlying epigenetic inheritance 127 . epigenetic gene regulation can take the form of dna modifications, histone tail modifications, chromatin remodelling and alternative histone subunits (see the figure) . dna modifications mainly involve cpg methylation, in which a methyl group is added to deoxycytidine residues located in cg-rich genomic regions. cpg methylation is primarily associated with repressive heterochromatin. histone tail modifications -including methylations, acetylations, phosphorylations and ubiquitylations -are the best understood form of epigenetic regulation. examples have been found on all four canonical histones (h2a, h2b, h3 and h4). h3 and h4 acetylations are associated with active euchromatin, whereas methylations are more diverse in function. for example, h3 lysine 9 and lysine 27 tri-methylation (h3k9me3 and h3k27me3, respectively) mark heterochromatin, whereas h3k4me3 marks are found on actively transcribed euchromatin 128 . chromatin remodelling involves atp-dependent histone chaperones that may slide, evict or load histones, modulating the local density of histones on the chromatin. finally, chromatin accessibility can be modulated through the use of alternative histones, such as the h2a histone variant in place of h2, or the h3.3 histone variant as a replacement for h3.1 (ref. 129 ). the canonical h3 histone variants h3.1 and h3.2 are deposited by the histone chaperone caf-1 during dna replication. however, histones can be displaced by transcription, and they need to be replaced by the histone chaperone complex hira, which deposits the replacement histone h3 variant h3.3. thus, h3.3 previously was thought to mark transcriptionally active chromatin. however, h3.3 later was found also to be loaded by an alternative histone chaperone complex consisting of a heterodimer of daxx and atrx, which loads h3.3 onto repressed heterochromatic regions such as pericentromeric regions and telomeres, suggesting that different chaperones may load histone h3.3 with alternative histone tail marks onto either actively transcribed or repressed chromatin 25, 130, 131 . h3 the addition of a small chemical group called an acetyl group (ch 3 co), found on rna and proteins including histones. a family of highly basic proteins used to organize genomic dna (see box 1). epigenetic markers that mark a segment of chromatin for transcriptional repression, which usually coincide with 'closed chromatin' that is less accessible to transcription factors. microscopy assays in which subjects of interest are visualized though specific binding of antibodies labelled with fluorescent dyes. www.nature.com/nrmicro pml-nbs as subnuclear hubs of the epigenetic repression of viral dna. invading viral dna can also be detected by the innate immune dna sensor ifi16 ( fig. 1b ). ifi16 contains an oligomerizing pyrin domain (pyd) and two double-stranded dna (dsdna)-binding hin200 domains, and it oligomerizes on histone-free segments of dsdna longer than 70 bp 27 . ifi16 was initially characterized as a dna sensor that induces interferon-β through the signalling factors sting, tbk1 and irf3 (ref. 28 ), and it was shown to repress gene expression from transfected plasmids as well as from viruses including herpesviruses, papillomaviruses and hbv 11, [29] [30] [31] [32] . interestingly, ifi16 suppression of hsv-1 and kaposi sarcoma-associated herpesvirus (kshv) transcription involves the deposition of suppressive h3k9me3 marks in an interferon-independent manner, with ifi16 directly recruiting h3k9 methyltransferases to kshv dna 11, 29, 33 . although ifi16 has a key role in the epigenetic repression of viral dna, it should be noted that ifi16 function is likely closely connected to the inhibition by pml-nbs, as ifi16 is known to interact with pml-nb components 34 . herpesviruses. epigenetic repression of viruses is best understood for herpesviruses ( fig. 1 ). herpesvirus dna is packaged into virions 'naked' (that is, free of histones) [35] [36] [37] . upon release into the nucleus, viral dna is detected and then rapidly loaded with histones harbouring suppressive marks, as a form of innate immune response. upon infection of fibroblasts by the alphaherpes virus hsv-1, within 2 h viral dna is loaded with histones, mainly consisting of daxx-atrx and hira-loaded h3.3 with repressive h3k9me3 marks [38] [39] [40] [41] . hsv-1 disarms this repression in two stages. first, the viral protein vp16, which is packaged into the tegument layer of incoming virions, recruits host proteins, including host-cell factor 1 (hcf-1) and octamer-binding factor (oct-1), in order to form a complex that recruits the histone demethylases lysine-specific demethylase 1 (lsd1) and jumonji domain 2 (jmjd2) family members as a means to remove repressive h3k9 marks from viral immediate early promoters 42 upon entry into the cell nucleus, the dna of many viruses initiates the replication process adjacent to subnuclear structures called pro-myelocytic leukaemia nuclear bodies (pml-nbs). however, pml-nbs are an aggregation site for many heterochromatic repression proteins, which load repressive heterochromatin onto viral dna that shuts down viral transcription. in the absence of viral de-repression factors, viral episomal dna lacks active histone marks (shown as green histones with the active marks h3ac, h4ac and h3k4me3). a | several pml-nb components -including pml itself, sp100, smc5, smc6, daxx and atrx -are involved in the epigenetic repression of viruses, with the daxx-atrx complex having specifically been found to load the histone variant h3.3 bearing repressive marks onto viral dna, leading to the accumulation of heterochromatic marks and blocked transcription (dna is shown associated with red histones with the repressive marks h3k9me3 and h3k27me3). b | the innate immune dna sensor ifi16 drives an alternative mechanism of antiviral epigenetic repression, promoting repressive methylations on histone tail h3k9. c | specifically for the retrovirus murine leukaemia virus, np220 and the human silencing hub (hush) complex have also been shown to deposit heterochromatic marks on unintegrated viral dna. a family of proteins that are expressed by eukaryotic cells upon invasion by viruses, used to signal neighbouring cells to mount an antiviral response through the expression of a variety of interferon-stimulated genes. the space in viral particles between the outer membrane and the inner protein capsid shell; the term is most commonly used in the herpesvirus family, where proteins packaged in this space are termed tegument proteins. a small peptide that can be ligated onto other proteins, best known to mark proteins for degradation through the proteasome. h3k27me3 from the entire viral dna genome, ensuring a productive infection 45 . in parallel, icp0 has also been implicated in the degradation of ifi16 ( fig. 2c ). ifi16 suppresses gene expression from icp0-deficient mutants of hsv-1, with ifi16 directly binding viral dna, promoting the deposition of repressive h3k9me3 marks and the removal of active h3k4me3 marks 11, 29 . however, the role of icp0 in counteracting ifi16 is somewhat controversial, with one study reporting that inhibition of hsv-1 gene expression by ifi16 occurs only in the absence of icp0, whereas another study reported that ifi16 is also active against wild-type hsv-1 (refs 11,29 ). this discrepancy may be partially explained by the finding that icp0-mediated degradation of ifi16 is efficient in primary cells yet less so in transformed cell lines such as hela 46 57 . in this context, the structural maintenance of chromosome 5 and 6 (smc5/6) proteins, which colocalize with pml-nbs, act as restriction factors for hbv transcription. in a striking parallel to herpesviruses, the hbx protein recruits the cellular e3 ubiquitin ligase ddb1 in order to induce the ubiquitylation and degradation of smc5/6 (refs 58-60 ) ( fig. 2b ). importantly, artificial depletion of the pml-nb components pml and sp100 using rna interference not only results in the nuclear redistribution of smc6 but also allows the transcription of hbv cccdna in the absence of hbx. thus, hbv, like herpesviruses, disrupts pml-nbs as a means to circumvent the epigenetic silencing of viral dna. retroviruses. as we argued above, host cells can recognize incoming viral dna as targets for epigenetic silencing. whereas herpesviruses and hbv use viral proteins to disperse or degrade pml-nb components and so prevent silencing, retroviruses appear to avoid proviral dna silencing through the alternative strategy of chromosomal integration ( fig. 2d ). in the absence of integrase function, the potency of epigenetic repression becomes apparent, as unintegrated retroviral dna is poorly transcribed 61, 62 . retroviral particles contain rna genomes, which are reverse transcribed in the cytoplasm. like herpesviral dna, retroviral dna enters the nucleus 'naked' and is rapidly loaded with histones 63, 64 . specifically, unintegrated hiv-1 dna is loaded with the histone variant h3.3 bearing the repressive h3k9me3 mark and only low levels of the active mark h3ac. in the case of murine leukaemia virus (mlv), epigenetic repression requires the dna-binding protein np220, which recruits epigenetic repressors including the h3k9me3 methyltransferase setdb1, along with the human silencing hub (hush) complex 65 , which has previously been identified as a repressor of endogenous retrotransposons and has been proposed to have a role in the epigenetic silencing of latent hiv-1 proviruses 66 ( fig. 1c) . however, the previous study 65 also demonstrated that the hush complex has no role in silencing unintegrated hiv-1 proviruses; how these are targeted for epigenetic repression remains to be determined. it is also unclear how the repressive marks deposited on unintegrated proviral dna are removed after integration; however, hiv-1 proviruses are known to preferentially integrate into actively transcribed chromatin, which may allow active chromatin marks of the surrounding region to spread to the proviral dna 67, 68 . regardless, the ability of cells to effectively silence unintegrated retroviral dna means that integrase inhibitors are currently among the most clinically effective antiretroviral drugs. if unintegrated retroviral dna is indeed epigenetically silenced, this implies that a factor that induces the transcription of unintegrated retroviral dna should be able to rescue the replication of integrase-deficient (δin) retroviruses. indeed, this has recently been demonstrated. specifically, the tax protein encoded by human t cell leukaemia virus 1, which is a potent activator of the cellular nf-κb transcription factors rela and relb, was shown to rescue the robust replication of δin hiv-1 in t cells 69 . this rescue correlated with the effective recruitment of rela and relb to the two nf-κb binding sites located in the hiv-1 enhancer element as well as with the acquisition of active, and the loss of repressive, epigenetic marks on unintegrated hiv-1 dna. thus, in the presence of tax, δin hiv-1 replicates as transcriptionally active dna circles that are analogous to hbv cccdnas. upon infection of certain cell types, viruses may be epigenetically repressed, resulting in a latent infection. the successful repression of viral dna, while it prevents the killing of individual cells by lytic viral replication, does not necessarily reflect a clear victory for the host, as the establishment of latency can allow viruses to maintain long-term infections that avoid the host adaptive immune response yet revert to a lytic replication cycle at a later time. this is the case with latent infection of neurons by hsv-1, of b lymphocytes by ebv and even of resting t cells by hiv-1. each of these examples of latent infection is associated with the addition of repressive epigenetic marks to viral dna, including h3k9me3 and h3k27me3, and with low levels of active markers 70 . interestingly, latent infection by hsv-1 in neuronal cells may be partly explained by vp16 and its host cofactor hcf-1 being sequestered in the cytoplasm and thus unable to de-repress nuclear viral dna 8, 71 . the fact that hcf-1 is uniquely localized to the cytosol in neurons but not in other cell types suggests that hsv-1 may have evolved to utilize the cytosolic localization of hcf-1 to identify cell types in which latency is the preferred strategy. latency establishment is an intricately regulated process that allows viruses to temporarily adopt an alternative viral state. taking ebv as an example, upon infection of b lymphocytes, the virus first activates all eight latency genes to aid in the activation of naive b cells (type iii latency), and then certain genes are switched off (type i/ii latency), to minimize the number of viral proteins that are detectable by the immune system, with different latency types utilizing distinct viral promoters that are associated with active chromatin marks only when needed [72] [73] [74] . meanwhile, lytic viral genes remain transcriptionally silent and are associated with heterochromatic markers during latency 70, 75 . thus, herpesviruses appear to have subverted what may have originally evolved as an innate antiviral defence mechanism (the epigenetic silencing of viral dna) as a means to maintain viral latency by selectively silencing viral genes. in this way, herpesviruses can establish a long-lived viral reservoir that avoids detection by the host adaptive immune system. in conclusion, viral episomal dna can be epigenetically silenced by the host using mechanisms analogous to host heterochromatic gene repression, while dna viruses including herpesviruses and hbv package and/or encode viral proteins that can overcome the hbv genomic dna is packaged in the viral particle as a gapped, partially double-stranded dna with open dna ends. the ends are closed up upon infection of a host cell, resulting in a fully double-stranded circular dna genome, termed the covalently closed circular dna, which acts as a functional template for gene expression and replication. an essential retrovirus enzyme that catalyses the integration of reverse-transcribed viral dna into the host genomic dna. a viral life-cycle stage in which viruses undergo active replication in the host cell, in most cases resulting in the lysis of the host cell. nature reviews | microbiology this suppression. interestingly, the identified targets of viral de-repressor proteins have highlighted pml-nbs as a key structure in the epigenetic suppression of viral dna. although it remains enigmatic how pml-nbs differentiate between host and episomal viral dna, one hypothesis suggests that ifi16 may act as the dna sensor that recruits pml-nb components to viral dna, because ifi16 associates with pml-nb components and because both ifi16 and pml-nbs are recruited to incoming hsv-1 dna. by contrast, some reports suggest that ifi16 and pml-nbs represent distinct mechanisms 34, [76] [77] [78] . retroviruses appear to avoid epigenetic repression through integration of their dna into euchromatic regions of the host-cell genome, as the epigenetic repression of unintegrated retroviral dna closely mirrors that seen with mutant dna viruses that are unable to mount the necessary countermeasures. alternatively, when viruses invade host cells that are at least transiently non-permissive, viruses may utilize epigenetic suppression to enter a controlled, dormant state, thereby forming viral reservoirs that have so far proven impossible to eradicate. epitranscriptomic regulation rna transcripts are subject to a range of different covalent modifications at the single-nucleotide level, and over 100 such modifications have been identified, particularly on trnas and other non-coding rnas (ncrnas); several of these modifications have been shown to regulate ncrna function 3 . the repertoire of epitranscriptomic modifications (primarily but not exclusively methylations) found on eukaryotic mrnas is more limited than that found on ncrnas, and functional data exist for only a fraction of these mrna modifications 4 (table 1) . for the purposes of this review, we only discuss the four epitranscriptomic modifications that so far have been reported to affect viral gene expression -n 6 -methyladenosine (m 6 a), 5-methylcytidine (m 5 c), n 4 -acetylcytidine (ac 4 c) and 2′o-methylation of the ribose moiety of all four ribonucleosides (collectively nm) ( fig. 3 ). quantification of the levels of several different epitranscriptomic modifications on highly purified samples of the genomic rna of both hiv-1 and the model animal retrovirus mlv has revealed that these viral rnas contain far higher levels of m 6 a, m 5 c and nm residues than do cellular mrnas expressed in infected cells 79, 80 . specifically, m 5 c was detected at a level 14-30 times higher on these viral rnas than on cellular poly(a) + rna; nm levels were also 10-20 times higher, and m 6 a modifications were 2-10 times more prevalent. the level of ac 4 c was not examined in these two studies, but it was reported to be at a level comparable to that of m 6 a on hiv-1 transcripts in a third study that, despite having used virion rna samples of unknown purity, obtained m 6 a and m 5 c levels similar to those in the previous reports 81 . regardless, the very high prevalence of m 6 a, m 5 c, nm and likely ac 4 c modifications on these viral rnas indicates that viruses have evolved to maximize their addition to viral transcripts, which strongly suggests that these epitranscriptomic modifications are facilitating one or more steps in the viral replication cycle (fig. 3 ). n 6 -methyladenosine. the most common epitranscriptomic modification found on eukaryotic cellular mrnas is m 6 a, representing 0.2-0.4% of all adenosine residues 4,82,83 . as a result, m 6 a has been a major focus of epitranscriptomic research, resulting in the identification of methyltransferase-like 3 (mettl3) as the enzyme that adds m 6 a to mrnas (table 1) . mettl3, which is referred to as the m 6 a 'writer' , functions as part of a complex with several cofactors, including mettl14 and wtap, and uses s-adenosylmethionine (sam) as the methyl donor 4,82 ( fig. 3 ). this complex is predominantly nuclear, and the addition of m 6 a to mrnas has been reported to occur co-transcriptionally 84 . once deposited, m 6 a residues can be detected by five m 6 a 'readers' , including the predominantly nuclear protein ythdc1 and the cytoplasmic factors ythdc2, ythdf1, ythdf2 and ythdf3. all five of these proteins contain a yth domain, which directly binds m 6 a residues, and all five m 6 a readers appear to bind to all m 6 a sites equivalently. two m 6 a demethylases (also known as 'erasers'), called alkbh5 and fto, have also been reported, and it has been proposed that mrna modification by m 6 a may be dynamic and reversible, though this claim remains controversial 85, 86 . although the presence of m 6 a on viral transcripts was reported as long ago as the 1970s 87,88 , analysis of the effect of m 6 a residues on viral gene expression and replication only recently became technically feasible, with the development of techniques that allow epitranscriptomic modifications, including m 6 a, to be mapped to specific sites on rnas 83 (box 2). initially, we and others reported that m 6 a promotes hiv-1 gene expression and replication; knockdown of either mettl3 or the most highly expressed m 6 a reader, ythdf2, inhibited hiv-1 gene expression, whereas knockdown of the alkbh5 eraser or ythdf2 overexpression promoted hiv-1 gene expression 89, 90 . subsequent studies from our laboratory and others -using a combination of gene knockdown, knockout and overexpression strategies -demonstrated that m 6 a residues also promote the replication of a range of other viruses, including influenza a virus (iav), the picornavirus enterovirus 71, respiratory syncytial virus (rsv), human metapneumovirus (hmpv) and the polyomavirus sv40 (refs 91-95 ). in the case of iav, hmpv and rsv, viral variants carrying silent mutations that eliminated a subset cellular rnas that do not encode proteins. www.nature.com/nrmicro of the mapped m 6 a sites but that left the underlying open reading frames unaffected were found to be not only attenuated in culture but also substantially reduced in their pathogenic potential in rodents, suggesting that the elimination of m 6 a residues by mutagenesis might represent a novel strategy for the development of attenuated viruses that could potentially serve as vaccines 93 . although the addition of m 6 a residues to viral rnas clearly increases their expression 89, 91 , it has recently been reported that m 6 a addition to transcripts encoded by human hmpv also enables these viral rnas to escape recognition by the host innate immune rna sensor rig-i, thus avoiding the host antiviral response and promoting virus replication 95 . although the majority of studies have concluded that m 6 a promotes viral gene expression, some exceptions have been reported. for example, in the case of kshv, one group has reported that the addition of m 6 a to viral transcripts promotes lytic replication 96 , and another group has presented data arguing for the opposite conclusion: m 6 a can suppress lytic replication 97 . importantly, a third group has reported that m 6 a can both promote and inhibit kshv lytic replication, depending on the cell type being studied 98 . thus it appears that, in the context of the complex, temporally regulated replication cycles characteristic of large dna viruses such as kshv, m 6 a may exert different phenotypic effects depending on the cellular context. in addition, several flaviviruses associated with acute human infections -including zika virus, dengue virus, yellow fever virus and west nile virus -have been reported to contain conserved clusters of m 6 a residues, yet it was also reported that knockdown of the m 6 a writer mettl3 increased zika virus replication, while knockdown of the m 6 a eraser alkbh5 or fto exerted an inhibitory effect on viral replication 99, 100 . this result, which is opposite to what was seen with the other rna viruses discussed above, is difficult to understand, as it is not apparent why m 6 a sites would be evolutionarily conserved across several different flavivirus rna genomes if they act in cis to inhibit viral gene expression. recently, it was reported that the human m 6 a reader ythdf3 can inhibit hiv-1 replication, though the reported effect -a less than twofold inhibition in wild-type a3r5 t cells, when compared to ythdf3 knockout cells over an ~4-day infection period -was very modest 101 . ythdf3 was reported to be packaged into hiv-1 virions and to then reduce reverse transcription by ~30% 101 . the authors also reported that virion-associated ythdf3 was efficiently degraded by the hiv-1 protease, which they propose serves as a viral countermeasure. by contrast, we previously reported that ythdf2 overexpression in t cells increased hiv-1 replication, whereas ythdf2 knockout reduced hiv-1 replication 89 . one possibility that was not considered is that ythdf3 may act by competing with ythdf2 for binding to m 6 a sites on hiv-1 rna, thus reducing the positive effect on viral gene expression exerted by ythdf2. another relatively common epitranscriptomic mrna modification is m 5 c, which represents ~0.05-0.1% of all cytidine residues found in cellular mrnas, but up to ~1.4% of the cytidine residues in retroviral transcripts 79, 80, 83 . although cells encode at least eight cytidine methyltransferases that act on rna, all but one of which belong to the nsun family of proteins, current data indicate that nsun2 is the nuclear writer responsible for the large majority of, but not all, m 5 c residues added to mrnas, including viral mrnas 80, 102 (table 1, fig. 3 ). at present, no m 5 c readers are known. analysis of the effect of loss of nsun2 expression, and hence loss of m 5 c addition to mrnas, in hiv-1-infected t cells revealed a specific loss of translation efficiency for hiv-1 and for cellular transcripts that are normally highly m 5 c-modified 80 . by contrast, cellular mrnas that normally lack m 5 c, including several housekeeping genes, were unaffected by the loss of nsun2 expression. therefore, m 5 c seems to increase viral gene expression and replication by acting predominantly at the level of mrna translation. n 4 -acetylcytidine. recently, ac 4 c residues were detected on cellular mrnas at a level of ~1 ac 4 c residue per mrna, and the ac 4 c writer was identified as nuclear n-acetyltransferase 10 (nat10) 103, 104 (fig. 3) . however, no ac 4 c readers are currently known (table 1 ). in the case of cellular mrnas, ac 4 c was reported to enhance both the stability and the translation of cellular mrnas, in the latter case by acting in open reading frames to improve ribosomal decoding, especially when ac 4 c was present in the wobble position of codons 104 . this may relate to the fact that ac 4 c can form more stable base pairs with guanosine residues 105 . recently, we reported the mapping of ac 4 c residues in hiv-1 transcripts (box 2) and reported that the loss of nat10 expression results in a decline in hiv-1 gene expression in infected t cells 106 . however, unlike m 5 c, ac 4 c was found to increase hiv-1 gene expression by enhancing rna stability, while viral mrna translation was unaffected. in confirmation of this result, we also observed that the mutagenesis of ac 4 c clusters in the env gene of although high-performance liquid chromatography linked to tandem mass spectrometry (hplc-ms/ms) can identify and precisely quantify rna modifications, these methods do not provide location information. methods to map the location of modifications usually involve rna deep sequencing, which can be roughly separated into antibodydependent methods, modification-interacting protein pulldowns and chemical methods. the figure depicts the core mechanisms of modification identification used in various mapping techniques, with immunoprecipitationbased techniques on the left and chemical methods on the right. the simplest method used to map n 6 -methyladenosine (m 6 a) sites on mrna is m 6 a-seq (also known as methylated rna immunoprecipitation sequencing (merip-seq)), in which rna is typically fragmented by alkaline hydrolysis and then incubated with an m 6 a-specific antibody. the resulting rna-antibody complexes are then recovered and sequenced 132, 133 . however, the resolution of this mapping technique is limited by the average rna fragment size, which is generally around 125 nucleotides, resulting in fairly broad peaks (that is, it identifies broad regions that contain one or more m 6 a residues). one variation of this method, called m 6 a individual-nucleotide-resolution crosslinking and immunoprecipitation (miclip), uses ultraviolet (uv) light crosslinking (wavelength of 254 nm) of the antibody to the motif characteristic of m 6 a modification sites (minimally 5ʹ-rac-3ʹ, with r representing g or a), resulting in a cytidineto-thymidine conversion of the cytidine immediately 3ʹ to the m 6 a modification in the reverse-transcribed cdna library, allowing single-nucleotide resolution mapping 134 . another variation is photo-crosslinking-assisted m 6 a-seq (pa-m 6 a-seq), in which cells are first pulsed with the highly photoactive uridine analogue 4-thiouridine (4su). the 4su-containing rna is then isolated and uv-light-crosslinked (wavelength of 365 nm) in solution to an m 6 a antibody 135 . because this method utilizes rnase to degrade any rna not protected by the bound antibody, recovered bound rna fragments reflect the sequence protected by the antibody, which is ~32 nucleotides. in addition, uv-light crosslinking of proteins to 4su results in a thymidine-to-cytidine conversion during reverse transcription, which allows the removal of all background reads during final data analysis 136 . antibody-based methods can easily be adapted to mapping different modifications, as we have previously demonstrated by performing pa-m 6 a-seq with m 5 c and ac 4 c antibodies 80, 106 , whereas m 6 a-seq has also been adapted to ac 4 c 80, 104 . adapting miclip to other antibodies would require additional testing to discover the preferred mutation induced by uv-light crosslinking of the specific antibody. antibody-mapping results can be corroborated using the photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (par-clip) technique 136 to map the binding sites for modification-specific readers in living cells, if known. for example, the m 6 a reader ythdf2 can be utilized in this way 89 . in a method for mapping 5-methylcytidine (m 5 c), confusingly also named miclip, mutation of the m 5 c writer nsun2 at a key cysteine residue (c271a) causes nsun2 to covalently crosslink to the target cytidine residue on the substrate rna; thus, m 5 c modifications could be identified by immunoprecipitation of nsun2-c271a-bound rnas followed by rnase treatment, rna fragment recovery and deep sequencing 137 . for sites of 2′o-methylation (nm), no antibody is currently available, and mapping thus depends on chemical methods. ribomethseq exploits the fact that a methylation at the 2′o position can block alkaline hydrolysis at that position 138, 139 . in 2′ome-seq, reverse transcription stops at an nm site under low-dntp conditions 140 . nm-seq and ribooxiseq utilize the resistance of 3′-end nm to periodate cleavage (io 4 -) 141, 142 . all three of these chemical nm-mapping methods provide single-nucleotide resolution. however, because ribomethseq and 2′ome-seq depend on the absence of sequencing reads ending at the modification site, they require very high sequencing read depths. we note that the oxidation and elimination processes of nm-seq and ribooxiseq lead to the loss of most of the rna sample. thus, all current nm methods require large amounts of input rna. bisulfite sequencing has been used to map m 5 c residues on rnas, exploiting the fact that m 5 c residues are resistant to the cytidine-to-uridine conversion induced by bisulfite treatment 61 . however, we have observed that cytidine residues located in rna stems are also resistant to bisulfite treatment, potentially resulting in their incorrect identification as m 5 c residues. hiv-1, which forms part of the 3ʹ untranslated region of the viral gag mrna, nevertheless reduced gag mrna and protein levels equivalently. thus, in addition to rna methylation, acetylations in the form of ac 4 c can also be utilized to enhance viral gene expression, through the stabilization of viral rna transcripts. the fourth and final internal epitranscriptomic modification that has, so far, been reported to affect viral replication is 2ʹo-methylation of the ribose moiety of all four ribonucleosides (am, cm, gm and um, collectively known as nm). each of the four nm residues represents ~0.1% of the level of the relevant nucleoside found in cellular mrnas, yet this level was found to be up to 20 times higher when hiv-1 or mlv genomic rnas were analysed 79, 80 . the nm writer that acts on retroviral transcripts has been identified as the nucleolar protein ftsj3 (ref. 107 ), which was previously shown to function in pre-rrna processing 108 ( fig. 3 ). we note that ftsj3 was reported to be incapable of adding 2′o-methyl groups to cytidine residues 107 , which appears inconsistent with the high levels of cm detected on hiv-1 (1.02%) and mlv (0.74%) genomic rnas 79, 80 . moreover, preliminary data suggest that the yeast ftsj3 homologue (spb1) is able to methylate cytidine residues 109 . only one report has so far examined the phenotypic effect of nm residues on hiv-1 replication, and these researchers did not report any effect of nm residues on hiv-1 gene expression. instead, they found that hiv-1 virions produced in cells in which ftsj3 was knocked down by rna interference were potent activators of the cytoplasmic viral rna sensor mda5, a key component of the host antiviral immune response, when the virions were used to infect dendritic cells 107 . others have also reported that specific epitranscriptomic rna modifications, including not only nm but also pseudouridine, can attenuate cellular innate immune responses to transfected mrna molecules [110] [111] [112] [113] , whereas m 6 a was recently reported to prevent the activation of a second cytoplasmic rna sensor, rig-i 95 . clearly, it will be important to examine whether other viruses also use epitranscriptomic rna modifications, including but not limited to m 6 a and nm, to escape viral rna detection by host innate immunity factors. in conclusion, the current evidence suggests that several different epitranscriptomic rna modifications are able to promote viral replication, either directly, by increasing viral mrna stability or translation, or indirectly, by allowing viruses to elude the recognition of their transcripts as foreign by cytoplasmic viral rna sensors. it is therefore not surprising that viruses appear to have evolved so as to maximize the level of several epitranscriptomic modifications that are added to their mrnas. by contrast, mammalian cells have evolved the capacity to recognize viral dna as foreign and seek to silence that dna epigenetically as one form of innate immune response. viruses, in turn, have evolved mechanisms that allow them to avoid epigenetic silencing, either by the targeted destruction of relevant cellular factors, such as the components of pml-nbs, or by hiding in host-cell chromosomal dna, as is seen for retroviruses. in this review, we have argued that cells use epigenetic gene regulation as a potential mechanism to silence incoming viral dna molecules, whereas viruses have evolved to recruit the cellular epitranscriptomic modification factors in order to heavily modify viral transcripts, as a means to boost viral gene expression and/or replication. although the field of epigenetic gene regulation is fairly mature, the question of how cells distinguish between viral dna and host-cell dna remains unresolved. in particular, how unintegrated hiv-1 proviral dna is recognized and silenced remains unknown. by contrast, the field of epitranscriptomic gene regulation is still in its infancy. for example, how writers select specific bases for modification remains largely unknown, and even in the case of m 6 a -for which the consensus editing sequence 5ʹ-rac-3ʹ has been defined, where r is either g or a 3,4 -only a minority of sites with that sequence are, in fact, modified. moreover, although five m 6 a readers have been identified, the readers for all other rna modifications remain unknown, and even for m 6 a, how the readers exert their phenotypic effects remains largely undefined. it will be important to understand why m 6 a clearly promotes viral replication in most published studies, yet has also been suggested in other reports to inhibit viral replication, as discussed above. both epigenetic and epitranscriptomic regulatory pathways could in principle be targeted for antiviral drug development. in the case of epigenetic viral gene regulation, two strategies have emerged. in an approach termed 'shock-and-kill' , drugs that promote the formation of active chromatin, such as histone deacetylase (hdac) inhibitors, have been proposed as tools to activate latent viruses, including ebv [114] [115] [116] and hiv-1 (ref. 117 ). in the case of hiv-1, this strategy envisions using these drugs to reactivate latent proviruses, in individuals who are also on antiretrovirals, as a means of selectively eliminating latently infected cells. however, this strategy has yet to prove clinically useful. an alternative approach envisions the use of drugs that instead keep viral dna epigenetically suppressed, including drugs that inhibit the h3k9 demethylases lsd1 and jmjd2, which are recruited by hcf-1 to hsv-1 viral dna in order to remove repressive marks. inhibitors of lsd1 and jmjd2 have indeed been shown to suppress hsv-1 gene expression, replication and reactivation from latency, both in vitro and in vivo 43, 118, 119 . these drugs have also proved effective against other hcf-1-dependent herpesviruses, including hcmv and varicella zoster virus (vzv), and they might prove useful in the treatment of pathologies that result from the reactivation of latent herpesviruses, such as shingles. although in principle lsd1 and jmjd2 inhibitors could be used to entirely repress the reactivation of latent dna viruses, these drugs would then need to be taken long-term, and it seems unlikely that inhibition of host h3k9 demethylases for months or even years would be well-tolerated. inhibitors that block the epitranscriptomic modification of viral rnas clearly would be potentially even more interesting, as this should inhibit viral gene expression and/or promote antiviral immune responses. these drugs would presumably be targeted to the cellular a protein that normally localizes to the subnuclear structure called the nucleolus, which is where the rna components of the ribosome are produced. nature reviews | microbiology writers that add epitranscriptomic marks to mrnas (table 1) , and the emergence of drug-resistant viral mutants would therefore seem to be unlikely. conversely, as such drugs would also prevent the epitranscriptomic modification of cellular mrnas, toxicity might be an issue, and we therefore envision that such drugs would be used only briefly, during the 5-7-day acute phase of infection by viruses such as iav, rsv and possibly severe acute respiratory syndrome coronavirus 2, in order to reduce the peak viral load and limit viral pathogenicity until the adaptive immune system becomes effective. because the addition of m 6 a to mrnas has been implicated as a driver in some forms of cancer [120] [121] [122] , several biotech companies are already attempting to identify effective inhibitors of mettl3 function, and it would clearly be of interest to test these, once they have been identified, in animal models that support pathogenic infections by human viruses. in the interim, some data in fact already suggest that drugs that inhibit mrna methylation could prove to be effective pan-viral inhibitors. specifically, several drugs, including 3-deaza-adenosine (daa), are known to act as inhibitors of s-adenosylhomocysteine (sah) hydrolase and, as a result, to deplete cells of sam, the methyl donor used not only by mettl3 but also by nsun2, and likely also by the enzymes that add nm residues to mrnas. importantly, daa was shown to inhibit m 6 a addition to mrnas, while the formation of 7-methylguanosine, which forms part of the mrna 5ʹ cap, was largely unaffected 123 . daa acts as a potent pan-viral inhibitor in culture and is able to effectively inhibit a wide range of dna and rna viruses at doses that are >100-fold lower than the level found to exert a toxic effect on cultured primary cells 124 . in vivo, daa is also a remarkably effective broad-spectrum antiviral. for example, a single dose of daa given one or two days after infection reduced peak viraemia in ebolavirus-infected mice by >1,000 fold and resulted in the survival of almost all the treated animals, whereas untreated animals showed a 0% survival rate 125 . similarly, daa drastically reduced viral titers in cotton rats infected with rsv at doses of daa that were not detectably toxic 126 . although daa may not itself be a potentially useful drug, these studies do make the point that the epitranscriptomic modification of viral mrnas may represent a potential viral achilles heel, and that the identification of inhibitors of this process could therefore lead to the development of a novel class of potent, broad-spectrum antivirals. published online xx xx xxxx perceiving the epigenetic landscape through histone readers editing the epigenome: reshaping the genomic landscape the pivotal regulatory landscape of rna modifications dynamic rna modifications in gene expression regulation regulation of gene expression by n 6 -methyladenosine in cancer rna modifications regulating cell fate in cancer cancer epigenetics: moving forward nuclear sensing of viral dna, epigenetic regulation of herpes simplex virus infection, and innate immunity viral gene products actively promote latent infection by epigenetic silencing mechanisms viral dna sensors ifi16 and cyclic gmp-amp synthase possess distinct functions in regulating viral gene expression, immune defenses, and apoptotic responses during herpesvirus infection nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected dna regulation of alphaherpesvirus infections by the icp0 family of proteins pml and pml nuclear bodies: implications in antiviral defence structure, dynamics and functions of promyelocytic leukaemia nuclear bodies pml nuclear bodies are highly organised dna-protein structures with a function in heterochromatin remodelling at the g2 phase nuclear domain 10, the site of dna virus transcription and replication ifn enhance expression of sp100, an autoantigen in primary biliary cirrhosis the acute promyelocytic leukaemiaassociated pml gene is induced by interferon transcriptional induction of the pml growth suppressor gene by interferons is mediated through an isre and a gas element disruption of host antiviral resistances by gammaherpesvirus tegument proteins with homology to the fgarat purine biosynthesis enzyme sp100b, a repressor of gene expression preferentially binds to dna with unmethylated cpgs interaction of sp100 with hp1 proteins: a link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment rnai reveals anti-apoptotic and transcriptionally repressive activities of daxx daxx and histone deacetylase ii associate with chromatin through an interaction with core histones and the chromatin-associated protein dek daxx is an h3.3-specific histone chaperone and cooperates with atrx in replicationindependent chromatin assembly at telomeres the atrx syndrome protein forms a chromatin-remodeling complex with daxx and localizes in promyelocytic leukemia nuclear bodies the innate immune sensor ifi16 recognizes foreign dna in the nucleus by scanning along the duplex ifi16 is an innate immune sensor for intracellular dna this study reports the discovery of ifi16 as an antiviral dna sensor ifi16 restricts hsv-1 replication by accumulating on the hsv-1 genome, repressing hsv-1 gene expression, and directly or indirectly modulating histone modifications the intracellular dna sensor ifi16 gene acts as restriction factor for human cytomegalovirus replication the nuclear dna sensor ifi16 acts as a restriction factor for human papillomavirus replication through epigenetic modifications of the viral promoters nuclear sensor interferon-inducible protein 16 inhibits the function of hepatitis b virus covalently closed circular dna by integrating innate immune activation and epigenetic suppression ifi16, a nuclear innate immune dna sensor, mediates epigenetic silencing of herpesvirus genomes by its association with h3k9 methyltransferases suv39h1 and glp interactions of the antiviral factor interferon gamma-inducible protein 16 (ifi16) mediate immune signaling and herpes simplex virus-1 immunosuppression host and viral proteins in the virion of kaposi's sarcoma-associated herpesvirus proteins of purified epstein-barr virus identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome herpes simplex virus icp0 promotes both histone removal and acetylation on viral dna during lytic infection promyelocytic leukemia (pml) nuclear bodies (nbs) induce latent/quiescent hsv-1 genomes chromatinization through a pml nb/histone h3.3/h3.3 chaperone axis temporal association of the herpes simplex virus genome with histone proteins during a lytic infection the histone variant h3.3 regulates gene expression during lytic infection with herpes simplex virus type 1 inhibition of the histone demethylase lsd1 blocks alpha-herpesvirus lytic replication and reactivation from latency targeting the jmjd2 histone demethylases to epigenetically control herpesvirus infection and reactivation from latency the coactivator host cell factor-1 mediates set1 and mll1 h3k4 trimethylation at herpesvirus immediate early promoters for initiation of infection herpesviral icp0 protein promotes two waves of heterochromatin removal on an early viral promoter during lytic infection relative contributions of herpes simplex virus 1 icp0 and vhs to loss of cellular ifi16 vary in different human cell types the emerging role of nuclear viral dna sensors human cytomegalovirus (hcmv) ul82 gene product (pp71) relieves hdaxx-mediated repression of hcmv replication human cytomegalovirus protein pp71 displaces the chromatin-associated factor atrx from nuclear domain 10 at early stages of infection inactivating a cellular intrinsic immune defense mediated by daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate-early gene expression ebv tegument protein bnrf1 disrupts daxx-atrx to activate viral early gene transcription structural basis underlying viral hijacking of a histone chaperone complex viral reprogramming of the daxx histone h3.3 chaperone during early epstein-barr virus infection human cytomegalovirus pul83 stimulates activity of the viral immediate-early promoter through its interaction with the cellular ifi16 protein human cytomegalovirus tegument protein pul83 inhibits ifi16-mediated dna sensing for immune evasion innate nuclear sensor ifi16 translocates into the cytoplasm during the early stage of in vitro human cytomegalovirus infection and is entrapped in the egressing virions during the late stage epigenetic regulation of hepatitis b virus covalently closed circular dna: implications for epigenetic therapy against chronic hepatitis b hepatitis b virus x protein promotes degradation of smc5/6 to enhance hbv replication the smc5/6 complex restricts hbv when localized to nd10 without inducing an innate immune response and is counteracted by the hbv x protein shortly after infection this study reports the identification of smc5/6 as novel viral restriction factors active against hbv integration is essential for efficient gene expression of human immunodeficiency virus type 1 construction and analysis of deletion mutations in the pol gene of moloney murine leukemia virus: a new viral function required for productive infection unintegrated hiv-1 dnas are loaded with core and linker histones and transcriptionally silenced histones are rapidly loaded onto unintegrated retroviral dnas soon after nuclear entry np220 mediates silencing of unintegrated retroviral dna this study identifies the cellular factors that silence unintegrated murine leukaemia virus dna hush, a link between intrinsic immunity and hiv latency hiv-1 integration in the human genome favors active genes and local hotspots this study is the first to report the preference of the hiv-1 provirus for integration into open chromatin nuclear landscape of hiv-1 infection and integration reversal of epigenetic silencing allows robust hiv-1 replication in the absence of integrase function this article provides a useful review of the epigenetic regulation of viral latency nuclear localization of the c1 factor (host cell factor) in sensory neurons correlates with reactivation of herpes simplex virus from latency ctcf prevents the epigenetic drift of ebv latency promoter qp an atlas of the epstein-barr virus transcriptome and epigenome reveals host-virus regulatory interactions first days in the life of naive human b lymphocytes infected with epstein-barr virus epigenetic regulation of ebv persistence and oncogenesis dynamic response of ifi16 and promyelocytic leukemia nuclear body components to herpes simplex virus 1 infection mechanisms of host ifi16, pml, and daxx protein restriction of herpes simplex virus 1 replication the viral ubiquitin ligase icp0 is neither sufficient nor necessary for degradation of the cellular dna sensor ifi16 during herpes simplex virus 1 infection extensive epitranscriptomic methylation of a and c residues on murine leukemia virus transcripts enhances viral gene expression this study reports the comprehensive quantitative analysis of rna modifications on hiv-1 genomic rna, along with the first characterization of the role of the m 5 c rna positive-sense rna viruses reveal the complexity and dynamics of the cellular and viral epitranscriptomes during infection the dynamic epitranscriptome: n6-methyladenosine and gene expression control epitranscriptome sequencing technologies: decoding rna modifications m 6 a mrna modifications are deposited in nascent pre-mrna and are not required for splicing but do specify cytoplasmic turnover our views of dynamic n 6 -methyladenosine rna methylation jr pre-mrna processing includes n 6 methylation of adenosine residues that are retained in mrna exons and the fallacy of influenza viral mrna contains internal n6-methyladenosine and 5′-terminal 7-methylguanosine in cap structures methylated simian virus 40-specific rna from nuclei and cytoplasm of infected bsc-1 cells this study represents the first report of the methylation of internal (non-cap) residues on viral transcripts posttranscriptional m 6 a editing of hiv-1 mrnas enhances viral gene expression dynamics of the human and viral m6a rna methylomes during hiv-1 infection of t cells epitranscriptomic enhancement of influenza a virus gene expression and replication this study is the first to report that epitranscriptomic modification of viral rna enhances virus replication and pathogenesis in vivo addition of m6a to sv40 late mrnas enhances viral structural gene expression and replication viral n 6 -methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus n6-methyladenosine modification and mettl3 modulate enterovirus 71 replication this study reports that the m 6 a rna modification can prevent the detection of viral transcripts by the host innate immunity factor rig-i kaposi's sarcomaassociated herpesvirus utilizes and manipulates rna n6-adenosine methylation to promote lytic replication viral and cellular n 6 -methyladenosine and n 6 ,2′-o-dimethyladenosine epitranscriptomes in the kshv life cycle n6-methyladenosine modification and the ythdf2 reader protein play cell type specific roles in lytic viral gene expression during kaposi's sarcoma-associated herpesvirus infection dynamics of human and viral rna methylation during zika virus infection n6-methyladenosine in flaviviridae viral rna genomes regulates infection hiv protease cleaves the antiviral m6a reader protein ythdf3 in the viral particle 5-methylcytosine promotes mrna export-nsun2 as the methyltransferase and alyref as an m5c reader insights into rna biology from an atlas of mammalian mrna-binding proteins acetylation of cytidine in mrna promotes translation efficiency conformational preferences of modified nucleoside 5-taurinomethyluridine, taum 5 u occur at 'wobble' 34th position in the anticodon loop of trna acetylation of cytidine residues boosts hiv-1 gene expression by increasing viral rna stability ftsj3 is an rna 2′-o-methyltransferase recruited by hiv to avoid innate immune sensing the human nucleolar protein ftsj3 associates with nip7 and functions in pre-rrna processing conserved methyltransferase spb1 targets mrnas for regulated modification with 2′-o-methyl ribose 2′-o-methyl-modified rnas act as tlr7 antagonists suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna incorporation of pseudouridine into mrna enhances translation by diminishing pkr activation rnas containing modified nucleotides fail to trigger rig-i conformational changes for innate immune signaling virally targeted therapies for ebv-associated malignancies epstein-barr virus post-transplant lymphoproliferative disease and virus-specific therapy: pharmacological re-activation of viral target genes with arginine butyrate administration of vorinostat disrupts hiv-1 latency in patients on antiretroviral therapy emerging strategies to deplete the hiv reservoir a novel selective lsd1/kdm1a inhibitor epigenetically blocks herpes simplex virus lytic replication and reactivation from latency inhibition of lsd1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes n 6 -methyladenosine (m 6 a): a promising new molecular target in acute myeloid leukemia the critical role of rna m 6 a methylation in cancer emerging links between m 6 a and misregulated mrna methylation in cancer effects of the s-adenosylhomocysteine hydrolase inhibitors 3-deazaadenosine and 3-deazaaristeromycin on rna methylation and synthesis broad-spectrum antiviral activity of the carbocyclic analog of 3-deazaadenosine treatment of lethal ebola virus infection in mice with a single dose of an s-adenosyl-l-homocysteine hydrolase inhibitor evaluation of the toxicity and antiviral activity of carbocyclic 3-deazaadenosine against respiratory syncytial and parainfluenza type 3 viruses in tissue culture and in cotton rats transgenerational epigenetic inheritance: myths and mechanisms epigenetic modifications: basic mechanisms and role in cardiovascular disease the double face of the histone variant h3.3 the death-associated protein daxx is a novel histone chaperone involved in the replication-independent deposition of h3.3 distinct factors control histone variant h3.3 localization at specific genomic regions this early study uses merip-seq to characterize how the m 6 a modification regulates human mrna function comprehensive analysis of mrna methylation reveals enrichment in 3′ utrs and near stop codons single-nucleotide-resolution mapping of m6a and m6am throughout the transcriptome high-resolution n 6 -methyladenosine (m 6 a) map using photo-crosslinking-assisted m 6 a sequencing transcriptome-wide identification of rna-binding protein and microrna target sites by par-clip nsun2-mediated cytosine-5 methylation of vault noncoding rna determines its processing into regulatory small rnas illumina-based ribomethseq approach for mapping of 2′-o-me residues in rna profiling of ribose methylations in rna by high-throughput sequencing high-throughput single-base resolution mapping of rna 2-o-methylated residues nm-seq maps 2′-o-methylation sites in human mrna with base precision high-throughput and site-specific identification of 2′-o-methylation sites using ribose oxidation sequencing (riboxi-seq) this research was funded in part by nih grants r01-da046111 and u54-ai150470 to b.r.c., and by a duke university center for aids research (cfar, p30-ai064518) pilot award to k.t. the authors contributed equally to all aspects of the article. the authors declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-345342-04tvuj9f authors: kumar, rebecca n.; tanna, sajal d.; shetty, aneesha a.; stosor, valentina title: covid‐19 in an hiv‐positive kidney transplant recipient date: 2020-05-26 journal: transpl infect dis doi: 10.1111/tid.13338 sha: doc_id: 345342 cord_uid: 04tvuj9f we report a case of a 50‐year‐old male with a history of hiv and kidney transplant who presented with sars‐cov‐2. we also present a review of covid‐19 cases in kidney transplant recipients. since the first cases of an unusual pneumonia were reported in china in december 2019, coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has resulted in an ongoing pandemic 1 . by the beginning of may 2020, the centers for disease control and prevention reported 1,122,486 cases within the united states alone 2 . because this is a novel virus, outcomes associated with comorbidities, especially immunosuppressed or compromised states, are still being evaluated. this case describes the clinical course of a symptomatic kidney transplant recipient with hiv who tested positive for sars-cov-2. a 50-year-old hiv+ (cd4 395 cells/µl, cd4% 28%, hiv rna < 20 copies/ml) african-american male with deceased donor kidney transplantation 14 months earlier for end-stage renal disease secondary to hiv-associated nephropathy (hivan)/focal segmental glomerulosclerosis (fsgs) presented to the emergency department (ed) complaining of fevers for two days, with temperatures to 101°f, chills, nasal congestion, and mild cough. the past medical history also includes hypertension, asthma, steatohepatitis, and resolved hepatitis b infection. the patient denied shortness of breath, chest or abdominal pain, diarrhea, or vomiting. he did not have changes in his urine output, pain over the allograft, or dysuria. the patient reported known exposure to covid-19 at a family gathering seventeen days prior to symptom onset. his husband, who accompanied him to the event, tested positive for sars-cov-2 one week prior to onset of the patient's illness. the transplant history was notable for receipt of a phs-increased risk, hiv antibodypositive, hiv nat-negative deceased donor kidney allocated through the hiv organ policy equity (hope) act 3 . he received induction immunosuppression with basiliximab and steroid-sparing maintenance immunosuppression with tacrolimus (target trough 8-10 mcg/l) and mycophenolate mofetil (1250 mg twice daily orally initially followed by 1000 mg twice daily orally after six months post-transplant). post-transplant course was notable for postoperative perinephric seroma that required drainage. his renal function improved to a this article is protected by copyright. all rights reserved baseline serum creatinine of 1.9-2.2 mg/dl range post-transplant. the six-month and oneyear surveillance allograft biopsies were negative for acute rejection or recurrent hivan/fsgs, and he remained without development of donor-specific antibodies (dsa). two days prior to ed evaluation, when patient first reported fever and covid-19 exposure to the transplant department, the mycophenolate dose was reduced from 1000 mg twice daily to 250 mg twice daily orally due to suspicion for covid-19 as cause of the patient's illness. the patient was diagnosed with hiv infection in 1997, initiated antiretroviral therapy (art) at that time, and has had long-term viral suppression. at and since time of transplant, the art regimen consisted of dolutegravir, emtricitabine, and tenofovir alafenamide. he was also receiving maraviroc v. placebo as part of a randomized clinical trial (nct02741323). there have been no opportunistic infections. in the ed, the patient was hypertensive with blood pressure 172/95 mmhg and tachycardic with heart rate 108/minute, but he appeared well and had temperature 98.9°f and oxygen saturation 100% on room air. a nasopharyngeal swab was obtained, a respiratory viral panel (filmarray® respiratory panel assay) was negative, and sars-cov-2 real time pcr (northwestern memorial cdc covid-19 sars-cov-2 detection assay) was positive. no further lab work or imaging was performed. the patient enrolled in the covid home monitoring program through our medical center and was discharged to home. the patient had ongoing symptoms reported through the monitoring program including anosmia and ageusia one day after discharge, fatigue, and fevers. symptoms, including anosmia and ageusia, resolved ten days after illness onset. on a follow-up telehealth visit four weeks later, the patient reported his health was back to baseline. a subsequent laboratory evaluation was notable for white blood cell count 3.7 x 10 3 /µl and serum creatinine 1.93 mg/dl. the hiv rna remained < 20 copies/ml, and the cd4 count was 435 cells/µl, cd4% 31%, with a cd4/cd8 ratio 0.65 and absolute lymphocyte count of 1413 cells/µl. this article is protected by copyright. all rights reserved there have been reported cases of covid-19 in hiv-infected patients and cases of covid-19 in transplant recipients [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . however, this case is the first detailed report of an hivpositive kidney transplant recipient who developed and recovered from covid-19. unlike the case series from spain where hiv patients with covid-19 changed regimens to include a boosted protease inhibitor, no modifications were made to our patient's art regimen 6 . the current regimen was continued due to lack of demonstrated efficacy of lopinavir-ritonavir for covid-19 in a randomized control trial, as well as avoiding the interactions associated with ritonavir and tacrolimus 16, 17 overall, our patient did well without experimental antiviral or anti-inflammatory therapies and experienced no serious complications, including need for hospitalization or supplemental oxygenation. in fact, the patient appeared well enough that the ed did not order labs beyond respiratory virus and sars-cov-2 testing. immunosuppression was decreased within 48 hours of symptom onset, but it is unclear if this influenced clinical outcome. as the pandemic progresses and more data becomes available, the clinical spectrum of covid-19 infections in the transplant population will be better defined and will better inform the management of immunosuppression in this setting. concern for a potential immune reconstitution inflammatory syndrome (iris)-like reaction has made our center's transplant clinicians hesitant to discontinue immunosuppression altogether in the setting of acute infection; covid-19 increases this concern because of the potential for the heightened this article is protected by copyright. all rights reserved inflammatory state that occurs with critical illness within the non-immunosuppressed patient 19 . maraviroc is currently fda-approved for treatment of hiv in patients with r-5 virus, because it blocks the c-c chemokine receptor type 5 (ccr-5) receptor and prevents viral entry into cd4 cells. because of its importance in immune cell response, ccr5 blockade has been suggested as a potential way to reduce allograft loss recipients 20,21 . ccr-5 blockade with leronlimab is currently under investigation in a phase2b/3 for severely ill covid-19 patients 22 . in the case series from new york city, six severely ill patients received leronlimab on compassionate use basis; although there was improvement in interleukin-6 levels noted in five patients, only one patient did not require intubation 15 this article is protected by copyright. all rights reserved therapy. however, we would discourage the complete discontinuation of all immunosuppression to avoid an iris type reaction. consideration for remdesivir or therapies under investigation can be made on an individual basis for those patients who are critically ill or fail to clinically improve. as data emerges in the u.s., it is essential to systematically describe outcomes and identify unique features of patients with both hiv and organ transplants. these populations will also be important to include in clinical trials of covid therapies. cases this article is protected by copyright. all rights reserved accepted article this article is protected by copyright. all rights reserved 3. health r, services administration doh, human s. organ procurement and transplantation: implementation of the hiv case report: a kidney transplant patient with mild covid-19 covid-19 in long-term liver transplant patients: preliminary experience from an italian transplant centre in lombardy covid-19 in patients with hiv: clinical case series covid-19 in kidney transplant recipients sars cov2 infection in a renal transplanted patients. a case report early virus clearance and delayed antibody response in a case of covid-19 with a history of co-infection with hiv-1 and hcv case report of covid-19 in a kidney transplant recipient: does immunosuppression alter the clinical presentation? successful recovery of covid-19 pneumonia in a renal transplant recipient with long-term immunosuppression identification of kidney transplant recipients with coronavirus disease covid-19 in a kidney transplant patient first case of covid-19 in a kidney transplant recipient treated with belatacept covid-19 and kidney transplantation a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 american society of transplantation infectious diseases community of p. solid organ transplantation in the hiv-infected patient: guidelines from the american society of transplantation infectious diseases community of practice this article is protected by copyright. all rights reserved 46 12 key: cord-329361-0mpbau1b authors: bennasser, yamina; yeung, man lung; jeang, kuan-teh title: rnai therapy for hiv infection: principles and practicalities date: 2012-08-16 journal: biodrugs doi: 10.2165/00063030-200721010-00003 sha: doc_id: 329361 cord_uid: 0mpbau1b inside eukaryotic cells, small rna duplexes, called small interfering rnas (sirnas), activate a conserved rna interference (rnai) pathway which leads to specific degradation of complementary target mrnas through base-pairing recognition. as with other viruses, studies have shown that replication of the hiv-1 in cultured cells can be targeted and inhibited by synthetic sirnas. the relative ease of sirna design and the versatility of rnai to target a broad spectrum of mrnas have led to the promise that drug discovery in the rnai pathway could be effective against pathogens. this review discusses the current experimental principles that guide the application of rnai against hiv and describes challenges and limitations that need to be surmounted in order for sirnas to become practical antiviral drugs. the practical use of rnai therapy for hiv infection will depend on overcoming several challenges, including the ability to establish long-term expression of sirna without off-target effects and the capacity to counteract mutant escape viruses. rna interference (rnai) has emerged as a common in vitro the sirna duplex is loaded onto risc to serve as the guide strand while the second strand, the passenger rna, is degraded. [10] risc tool for silencing gene expression. [1, 2] the introduction of small uses its guide rna for base-pairing-mediated recognition of target rna duplexes of 19 to 21 nucleotides into cells can elicit specific messenger rna (mrna). once this specific hybridization is degradation of complementary gene sequences through base-pairachieved, the piwi domain of the ago2 protein within the risc ing. [3] it is thought that the rnai pathway serves as part of the complex degrades and silences the targeted substrate mrna. [11] innate immune defense of eukaryotes against invasion by exoge-because sirnas are easy to design and synthesize and because nous nucleic acids. [4] hence, in plants and drosophila, when a cell they have sequence specificity for silencing mrnas, these small is infected by a virus, an rnai response is triggered by the foreign rnas potentially represent a future class of antiviral drugs. double-stranded rna (dsrna) molecules that originate from the virus. [5] it has been shown recently that retroviruses such as human using small interfering rna (sirna) potentially be engaged into the rnai pathway of mammalian cells. [6, 7] in a relatively short period of time, rnai has been shown in an early step in the rnai response enlists an rnase iii enzyme cultured cells to be efficacious against various viruses including called dicer to bind and cleave long dsrnas into small duplexes respiratory syncytial virus (rsv), [12] influenza virus, [13] of 19 to 21 nucleotides, termed small interfering rna (sirna). poliovirus, [14] and hepatitis c virus (hcv). [15] for hiv, a variety dicer-sirna complex is then recognized by trans activation reof sirnas have also been reported to be effective in interrupting sponse region (tar) rna-binding protein (trbp) and protein viral infection. two sirna strategies have been considered for activator of pkr (pact), two cellular dsrna binding proteins. hiv; the first is to target essential viral genes and the second is to target cellular genes required by hiv for replication (table i) . the multi-protein entity is shuttled through interaction with the argonaute 2 (ago2) protein into an effector complex, the rna-to date, hiv sequences that have been targeted include the induced silencing complex (risc). [8, 9] one of the two strands of structural gag gene, [16] the infectivity factors vif and nef, [3] tat, rev, , a cellular protein involved in multivesicular body formation, [33] and targeting tumor susceptibility gene 101 [tsg101], also called vacuolar protein-sorting protein 23, involved in endocytic trafficking [36] ) 5. other cellular proteins used by hiv-1 for replication such as peptidylprolyl isomerase a (cyclophilin a), [34] the ras oncogene family member rab9, [35] and cdk2. [29] in many of the above examples, it should be pointed out that instead of directly introducing sirna duplexes into cells the effect of which is transient, in many cases, rnai was expressed using a dna plasmid expressing a short hairpin rna (shrna). it is recognized that the cell's dicer enzyme can remove the hairpin loop from shrna molecules, processing them to their sirna counterparts. currently, a myriad of systems are available for expressing shrnas (e.g. under the control of an rna polymerase iii u6 or h1 promoter or an rna polymerase ii cmv promoter) in several vector systems (e.g. adenoviral or lentiviral vector [17, 41] ), allowing for long-term silencing even in nondividing cells. hiv-1 is a notoriously mutable virus. to be effective, antiviral drugs based on sequence hybridization must take into account that hiv-1 mutates its sequence easily. indeed, hiv-1 can escape env, and tar rna. [21, 37, 38] in each instance, sequence-specific sirnas through nucleotide mutations. for example, das et al. [42] silencing of viral rna and transient suppression of hiv replicaobserved that an sirna targeted to nef rapidly elicited the emertion over a period of 3 to 4 days in single round infection of gence of sirna-resistant viruses with point mutations in the nef cultured cells have been achieved. however, it is important to note gene. alternatively, the deletation of an sirna recognition site that virion-associated hiv-1 genomic rna that infects cells apcan also allow the virus to evade restriction. [43] moreover, there is pears to be resistant to rnai-mediated degradation. [39] this findevidence that hiv-1 can undergo nucleotide substitutions that ing argues that if a goal is to prevent the genesis of integrated induce an alternative rna folding, thus shielding a previously provirus then, by using sirna, one should target cellular factors targeted sequence from access by the sirna. [42] required for early steps of hiv-1 replication rather than viral to minimize the mutational escape of hiv, one strategy is to sequences. simultaneously use multiple sirnas to target discrete sequences. sirna directed to several hiv-1 relevant cellular factors have the likelihood that a single viral rna molecule would mutate all targeted sequences becomes increasingly small as the number of indeed been tested for antiviral efficacy. these include sirna targets is escalated. [44] this multi-targeting strategy is aided by targeted to: computational modeling, which can predict how hiv-1 might 1. viral entry (e.g. sirna against hiv coreceptors chemokine evolve in cells that express multiple antiviral sirnas. [ [28, 30, 40] ) and cxcr4 have been reported. [46] in addition to evolving sirna-escape mutations, viruses can target effects can arise from as few as 7 nucleotides of fortuitous also encode suppressor factors that attenuate the cell's rnai base-pairing between sirna and mrna. [62] considered this way, response. [47, 48] while much remains to be elucidated, hiv-1 ap-a particular sirna could potentially suppress the expression of its pears to have multifaceted ways to suppress the cell's rnai intended target and several unintended mrnas. [63] this off-target machinery. [49] the viral tat protein appears to work as a protein phenomenon can be a significant drawback should sirnas be suppressor of rnai. [50] in addition, the viral rna, tar, [51] is also envisioned for in vivo use as human drugs. one anticipates that used by hiv-1 as a suppressor of rnai. [52, 53] if sirna-based methods and modifications that minimize off-target hybridization therapy of hiv-1 and other viruses is to be usefully contemplated, would need to be greatly improved to enable further sirna drug one needs to consider strategies that address virus-encoded rnai development. [64] suppressors. despite virally encoded suppressors, there is ample another unwelcome adverse effect of sirna use is the potenevidence for rnai-mediated inhibition of hiv-1 replication. it is tial to inappropriately elicit interferon and inflammatory possible that the suppressors are either synthesized too late or in cytokines. normally, rna duplexes shorter than 30 nucleotides in amounts too small to counter the effect of a pre-existing sirna or length would not be expected to trigger the interferon-linked shrna. protein kinase r pathway. however, recently it was found that some gu-rich sequences, such as 5′-ugugu-3′and 5′-guc-cuucaa-3′, which are toll-like receptor (tlr) immunostimulatory motifs, can trigger an interferon response [65] even when presented in duplexes shorter than 30 nucleotides. to minimize the sirna machinery used in eukaryotic cells is the same as nonspecific interferon responses, judicious care must be exercised that used to process a class of endogenous small rnas called in the sequence design of sirna drugs. micrornas (mirnas). mirnas are important regulators of at least 30% of cellular genes, including those involved in development, signal transduction, apoptosis, cell proliferation, and 4. development of antiviral sirna drugs: strategies tumorigenesis. [54] [55] [56] [57] to date, 474 human mirnas have been de-and limitations scribed, with more likely to be discovered (see mirna database [58] ). [59] recent optimism regarding the possibility of sirna drugs the mechanism of mirna action and the relevance of arises from studies that indicate that intravenous injections of an mirnas to hiv-1 infection have been reviewed elsewhere [47, 60] sirna directed against fas can protect mice from liver injury and and will not be elaborated further here. what needs to be empha-fibrosis in two models of autoimmune hepatitis [66] and from findsized is the fact that the shared factors for shrna, sirna, and ings that intranasal administrations of separate sirnas protected mirna processing are saturable entities. because mirnas are mice from rsv infection [67] and rhesus macaques from sars essential to cellular metabolism and viability, if the machinery for coronavirus infection. [68] sirna application to the mouse genital mirna genesis is diverted to handle exogenous sirna or tract has also been found to be protective against lethal herpes shrna, then a dearth of mirnas can potentially manifest as simplex virus type 2 infection. [69] these preliminary positive findcellular toxicity. this hitherto unexpected finding was indeed ings have prompted phase i studies to be initiated in humans. to demonstrated in a recent study of in vivo overexpression of shrna date, three clinical trials have been completed in humans and show in mice. [61] in that study, severe liver toxicity and the death of 150 no significant sirna toxicity. [70] sirnas are currently in clinical mice occurred within 2 weeks of shrna treatment. the explana-investigation for two diseases: age-related macular degeneration tion for the fatalities was the saturation by shrna of the pathway (amd) caused by an overexpression of the vascular endothelial normally used in the liver to process mirnas. hence, the high growth factor (vegf) and respiratory infection caused by the shrna expression in the mouse liver overwhelmed the export rsv. pathway (i.e. exportin-5) needed to transport precursor mirnas at this time, there is no sirna drug for hiv-1. as outlined from the nucleus into the cytoplasm. the interruption of proper above, there are several issues unique to the targeting of viral transport of mirna precursors led to reduced levels of mature genes which do not apply to the targeting of cellular genes. mirnas with resulting cellular toxicity. however, a question that is relevant to all sirna applications is apart from the problem of pathway saturation, off-target ef-how best to deliver sirna into virus-infected cells. in this regard, fects of sirnas raise additional concerns. on-target effects ema-several methods are being considered. for example, in hcv nate from perfect complementarity between guide-rna and target infections, a procedure that couples sirna to cholesterol [71] al-mrna (i.e. 19 of 19 matched nucleotide base-pairings), while off-lows for efficient targeting of hepatocytes, the cells preferentially infected by virus. elsewhere, a novel strategy using antibody-sette, which was shown to be effective in inhibiting hiv replicamediated sirna delivery has been proposed for hiv-1-infected tion [22] and a doxycycline-dependent lentivector. [73] going forcells. [18] whether these biochemical delivery techniques can dis-ward, a major challenge is to perfect vectors that are wholly silent tribute sirnas effectively to in vivo compartments awaits further for shrna expression unless specifically triggered by hiv-1 verification. infection. there are also virus-based approaches to deliver shrna; how-despite the optimized delivery of sirna and shrna into cells, ever, this type of gene therapy approach does raise issues related to the ability of viruses to mutate and escape from rnai is a biosafety. [72] a major focus for hiv-1-infected cells has been the significantly daunting problem. a multi-modal approach may use of lentiviral vectors, which have the ability to infect nondivid-have to be adopted in order to minimize viral escape. [44] for ing cells, such as resting t-cells, macrophages, and progenitor example, shrna targeted to rev can be used at the same time as a hematopoietic cells (cd34+ cells ogies has been proposed for hiv-1 therapy. [75] here, the strategy is inhibition of hiv-1 fusion with small interfering rnas targeting the chemokine coreceptor cxcr4 duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells inhibition of hiv-1 by lentiviral vector-411 (6836 modulation of hiv-1 replication by rna cd34+ progenitor cell-derived macrophages specific inhibition of hiv-1 replication by short induction and suppression of rna silencing: insights from viral hairpin rnas targeting human cyclin t1 without inducing apoptosis. febs infections innate immune defense through rna interfer-29 modulating hiv-1 replication by rna interferthe 3′ltr are alternatively spliced and polyadenylated the cellular hiv-1 rev cofactor hrip is type 1 ltr dna contains an intrinsic gene producing antisense rna and required for viral replication sam68 is absolutely required for rev complex to ago2 for microrna processing and gene silencing. nature 2005 function and hiv-1 production the role of lip5 and chmp5 in the role of pact in the rna silencing pathway. multivesicular body formation and hiv-1 budding in mammalian cells target cell cyclophilin a modulates human bly of sirna into ago2-containing rnai enzyme complexes ): replication of human immunodeficiency virus type 1, filoviruses, and measles 185-97 virus micrornas: genomics, biogenesis, mechanism, and function. cell protein sorting pathway are essential for hiv-1 budding inhibition of hiv-1 infection by small microrna.sanger.ac.uk/targets/v3 human microrna targets hiv-1-specific rna interference the virion-associated incoming hiv-1 fatality in mice due to oversaturation of rna genome is not targeted by rna interference principles of microrna-target recognitype 1 replication by rna interference directed against human transcription tion widespread sirna "off-target expression of small interfering rnas targeted silencing mediated by seed region sequence complementarity hiv-1 rev transcripts in human cells position-specific chemical modification of human immunodeficiency sirnas reduces "off-target" transcript silencing sequence-dependent stimulation of the by evolving an alternative structure in its rna genome. nucleic acids res mammalian innate immune response by synthetic sirna rna interference targeting fas protects mice from interference: a multiple shrna approach inhibition of respiratory viruses by strategies that resist human immunodeficiency virus escape using sirna in prophylactic and therapeutic cxcr4 and ccr5 shrna transgenic cd34+ cell derived regimens against sars coronavirus in rhesus macaque an sirna-based microbicide protects mirna and hiv: promises and mice from lethal herpes simplex virus 2 infection anti-viral rna silencing: do we look like plants? 70. behlke ma. progress towards in vivo use of sirnas gene by systemic administration of modified sirnas evidence that hiv-1 encodes an sirna and a suppressor of rna silencing retrovirus vectors: toward the plentivirus? detailed mutational analysis of tar rna: critical spacing between the bulge and loop recognition domains hiv-1 tar rna subverts rna interferencein tion transfected cells through sequestration of tar rna binding protein interference: viral diversion of a cellular pathway or evasion from antiviral 75 conserved seed pairing, often flanked by hiv shrna, anti-ccr5 ribozyme, and a nucleolar-localizing tar decoy. mol adenosines, indicates that thousands of human genes are microrna targets small rnas: classification, biogenesis, and function key: cord-341298-mqpovrms authors: morse, s.a.; meyer, r.f. title: viruses and bioterrorism date: 2016-10-31 journal: reference module in life sciences doi: 10.1016/b978-0-12-809633-8.11007-6 sha: doc_id: 341298 cord_uid: mqpovrms the target for a terrorist attack with a viral agent can range from humans to animals and plants. however, the use of a viral agent may pose a challenge due to problems associated with acquisition, cultivation, and dissemination. agricultural targets are of concern as they would require relatively little specialized expertise and technology and can have large economic consequences. viral agents are prone to genetic variation and mutation, and can be manipulated or created in the laboratory. unlike bacterial diseases, many of which are treatable with antimicrobials, there are fewer medical countermeasures to employ when dealing with viral infections. in 1775, during the revolutionary war, the british attempted to spread smallpox among the continental forces by inoculating (variolation) civilians fleeing boston. in the south, there is evidence that the british were going to distribute slaves who had escaped during hostilities, and were sick with smallpox, back to the rebel plantations in order to spread the disease (hopkins, 1983) . the use of viruses other than variola major is a more recent phenomenon and reflects an increased knowledge of how to grow and stabilize viruses for delivery purposes. allegations have been made by the government of cuba that the cia was responsible for the massive outbreaks of swine fever in 1971 and dengue fever in 1980 that ravaged the country. however, subsequent investigations have failed to find substantive proof of cia involvement in these outbreaks (zilinskas, 1999; leitenberg, 2001) . the aum shinrikyo, a religious cult responsible for the 1995 sarin gas release in the tokyo subway system, was also involved in biological warfare activity and sent a team of 16 cult doctors and nurses to zaire to acquire ebola virus (olson, 1999) . in 1997, unknown farmers in new zealand deliberately and illegally introduced rabbit hemorrhagic disease virus (a calicivirus) onto the south island as an animal control tool to kill feral rabbits (carus, 2002) . furthermore, some farmers further propagated the disease by mixing homogenized organs from infected rabbits with carrots and oats and spreading the infected bait in areas heavily infested with rabbits (carus, 2002) . for more than two decades, the human immunodeficiency virus (hiv) has been involved in a number of biocrimes. this most likely reflects the availability of hiv-contaminated blood as a source of this virus as well as widespread news reports concerning hiv and its association with the acquired immune deficiency syndrome (aids). for example, in 1990 graham farlow, an asymptomatic hiv-infected inmate at a prison in new south wales, australia, injected a guard with his hiv-contaminated blood. the guard became infected with hiv; farlow subsequently died of aids (carus, 2002) . in 1992, brian t. stewart, a phlebotomist at a st. louis, mo hospital, injected his 11-month-old son with hiv-contaminated blood during a fight over payment of child support (carus, 2002) . in 1993, iwan e. injected his former girlfriend with 2.5 ml of hiv-contaminated blood after she broke up with him (carus, 2002) . in 1994, dr. richard j. schmidt, a married louisiana gastroenterologist, injected his former lover with hiv-contaminated blood (carus, 2002) . molecular typing of the hiv strains demonstrated that she contracted the same strain of hiv as found in one of dr. schmidt's patients (metzker et al., 2002) . this case was the first time that phylogenetic evidence has been used as evidence in a united states criminal proceeding. in perhaps the most famous case, dr. david acer, a florida dentist infected with hiv, transmitted the disease to six of his patients between 1987 and 1990 (ou et al., 1992) . the mechanism by which hiv was transmitted to these patients was not unambiguously determined and intentional infection remains a possibility (ciesielski et al., 1994) . however, an independent reanalysis of the available sequence data strongly supported dental transmission of hiv (hillis and huelsenbeck, 1994) . in spite of these incidents and the public fear that hiv engendered, the virus was not included on lists of threat agents for public health bioterrorism preparedness (rotz et al., 2002) . however, others contend that hiv has great weapon potential if the goal is to destabilize a society (casadevall and pirofsky, 2004) . viruses have also been involved in suspected incidents or hoaxes. in 1999, an article appeared suggesting that the cia was investigating whether iraq was responsible for causing the outbreak of west nile fever in the new york city area (preston, 1999) . the story relied heavily on a previous story written by an iraqi defector, claiming that saddam hussein planned to use west nile virus strain sv 1417 to mount an attack. this was the first documented incidence of the west nile virus in the western hemisphere and raised concerns of an intentional introduction. however, an investigation revealed that the strain of west nile virus responsible for this outbreak was the same strain that had been circulating in the mediterranean region since 1998 (lanciotti et al., 1999) . a fictional "virus" was also responsible for one of the largest bioterrorism hoaxes in 2000. according to email messages widely circulated on the internet, an organization known as the klingerman foundation was mailing blue envelopes containing sponges sealed in plastic contaminated with a fictional pathogen called the "klingerman virus." according to the email alert, 23 people had been infected with the virus, including seven who died. this hoax was resurrected in the wake of the september 11, 2001 terrorist attacks on us and the subsequent anthrax attack, evidently because the specifics of the story resemble a germ warfare scenario. in 2011, a south african man was arrested for threatening to spread foot-and-mouth disease (fmd) virus in the us and great britain if he was not paid $4 million. if deployed, fmd virus would have caused the destruction of property and resulted in major economic losses. he was convicted of terrorist activity and money laundering even though he did not have fmd virus in his possession (keremidis et al., 2013) . advances in viral culture and virus stabilization made during the second half of the 20th century facilitated large-scale production of viral agents for aerosol dissemination. a report for the united nations (world health organization, 1970) on chemical and biological weapons and the effects of their possible use gave estimates on the numbers of casualties resulting from a hypothetical biological attack (table 1) . three viruses (rift valley fever virus, tick-borne encephalitis virus, and venezuelan equine encephalomyelitis (vee) virus) were evaluated in a scenario in which 50 kg of the agent was released by aircraft along a 2 km line upwind of a population center of 500,000. the viral agents caused significantly fewer casualties and impacted a smaller area when compared to the bacterial agents (francisella tularensis and b. anthracis) used in this hypothetical model. factors that may impact the effectiveness of the aerosol delivery of viruses include: type of viral nucleic acid (tseng and li, 2005) ; temperature and relative humidity (tang, 2009; lowen et al., 2007) ; and ultraviolet irradiation (tseng and li, 2005) . of note, smallpox was apparently not evaluated because it had not yet been eradicated and the level of vaccine-induced immunity in the population was high. viral agents were part of the biological weapons arsenal of both the soviet union and the united states (leitenberg, 2001 ; table 2 ). vee virus was stockpiled by both countries as an incapacitating agent; variola major and marburg viruses were stockpiled as lethal agents by the soviet union. the soviet union reportedly conducted a live field test of variola major virus on vozrozhdeniye island in the aral sea in the 1970s, in which 400 g of the virus was released into the atmosphere by explosion (shoham and wolfson, 2004; enserink, 2002) . unfortunately, a laboratory technician who was collecting plankton samples from an oceanographic research vessel 15 km from the island became infected. it was reported that after returning home to aralsk, she transmitted the infection to several people including children. all those infected died despite being immunized. it is unclear whether the strain of variola major released was india-1967 (enserink, 2002 or a highly virulent vaccine-resistant strain (shoham and wolfson, 2004) . a number of other viruses that infect humans (eg, ebola virus, lassa fever virus, enterovirus 70, yellow fever virus) or livestock (eg, foot and mouth disease virus, rinderpest, newcastle disease virus) have also been studied for their offensive capabilities or for the development of medical and veterinary countermeasures. today, with the increased level of concern, a number of viruses have been cited as possible weapons for use against humans or animals ( table 2) . requirements for an ideal biological warfare agent may include: availability; ease of production; stability after production; a susceptible population (human or animal); absence of specific treatment; ability to incapacitate or kill the host; appropriate particle size in aerosols so that the virus can be carried long distances by prevailing winds and inhaled deeply into the lungs of unsuspecting victims; ability to be disseminated via food or water; and, the availability of a vaccine to protect certain groups. many of the viruses listed in table 2 are biosafety level 4 (bsl-4) agents for which there is no available treatment (eg, ebola and marburg viruses). for those reasons, their production is likely to be restricted to national biological weapons programs. however, an agroterrorism attack would require relatively little expertise or the technology that is required to grow and disseminate many of the human viruses (wheelis et al., 2002) . terrorists can safely handle many of the animal viruses with no risk of becoming infected themselves. furthermore, terrorists do not necessarily have to acquire these agents from a laboratory and in theory, could acquire and use this kind of agent more easily than other biological agents that are pathogenic for humans (keremidis et al., 2013) . in addition, the risk of being caught in this kind of operation is low. furthermore, the increased importation of food, global food trading, and transportation of animals, have made us more vulnerable to terrorist attacks (polyak, 2004) . thus, other factors such as the economic impact of an attack on animal agriculture and the psychological impact on the population must also be considered. variola major ( table 2 ) is considered to be a major viral threat agent to civilian populations if used as a biological weapon (henderson et al., 1999) . thus, considerable effort has been expended toward preparing the public health and medical communities for the possibility that this virus will be employed by a terrorist. variola major is considered to be an ideal terrorist weapon because: it is highly transmissible by the aerosol route from infected to susceptible persons; the civilian populations of most countries contain a high proportion of susceptible persons; the disease is associated with a high morbidity and a mortality rate of 30% or more among unvaccinated persons and the absence of specific therapy; and initially, the diagnosis of a disease that has not been seen for more than 30 years would be difficult. in order to counteract the medical consequences of an attack with variola major, the united states has stockpiled enough vaccine for every american (300 million doses), medical countermeasures to address the needs of individuals for whom the vaccine is contraindicated, and about 2 million doses of a new fda approved antiviral, st246 (tecoviromat), against smallpox (smith et al., 2009) . alphaviruses ( table 2 ) are also of concern because they can be produced in large amounts using inexpensive and unsophisticated systems; they are relatively stable and aerosols are highly infectious for humans; and, strains are available that can produce note: these estimates are based on the following scenario: release of 50 kg of agent by aircraft along a 2 km line upwind of a population center of 500,000. classification of viral agents that are considered to be of concern for bioterrorism and biowarfare and those that have been weaponized or studied for offensive or defensive purposes as part of former or current national biological weapons programs incapacitating (eg, vee) or lethal infections (eee case fatality rates range from 50 to 75%) (sidwell and smee, 2003) . furthermore, the existence of multiple serotypes of vee and eee viruses, as well as the inherent difficulties of inducing efficient mucosal immunity, make defensive vaccine development difficult. veterinary vaccines utilizing inactivated alphavirus preparations are available and in routine use to control infection in endemic areas (barber et al., 1978) . unlicensed, investigational vaccines are also in use to protect at-risk laboratory workers (burke et al., 1977) . however, there are currently no vaccines licensed for general use in the united states for prevention or treatment of alphavirus infections, especially those acquired through the aerosol route (spurgers and glass, 2011) . the filoviruses and arenaviruses that cause hemorrhagic fever ( table 2 ) have also been considered as agents that might be used by terrorists because of their high virulence and capacity for causing fear and anxiety (rotz et al., 2002) . the filoviruses, ebola, and marburg, can also be highly infectious by the airborne route. humans are generally susceptible to infection with these viruses with fatality rates greater than 80%, and infection can be transmitted between humans through direct contact with virus-containing body fluids. there are five species of arenaviruses (lassa fever, junin, machupo, guanarito, and sabia) that can cause viral hemorrhagic fevers with a case fatality rate of about 20% (charrel and de lamballerie, 2003) . large quantities of these viruses can be produced by propagation in cell culture. infection occurs via the respiratory pathway suggesting that dissemination via aerosol might be used by a terrorist. human-to-human transmission has also been reported with aerosol transmission the most likely route for at least some of the secondary cases. the filoviruses and arenaviruses discussed above are bsl-4 agents and widespread diagnostic capabilities for infections caused by these viruses are limited. foot-and-mouth disease (fmd) is a severe, highly infectious viral disease of cattle, swine, sheep, goats, and other ruminant species; the virus is not a threat to human health. fmd is characterized by large blisters in the mouth, on the teats, and between the toes that burst to cause painful raw sores and even the loss of the hooves. animals cannot eat, drink, or walk, nor can they be milked. fmd is rarely fatal, but the recovered animal usually loses its productivity of milk or meat (breeze, 2004) . recovered animals may harbor and continue to shed infectious virus. fmd is the most infectious virus known; it is about 20-times more infectious than variola major. there are seven different types and 70 subtypes of fmd. thus, no single vaccine can be used to control this disease. about 15 different fmd vaccines manufactured throughout the world require the production and use of whole live virus. however, federal law prevents research or manufacture using live fmd virus in any part of the us mainland because of the risks of contagion. recently, a new molecular fmd vaccine was developed and granted conditional license for use in cattle by the usda. this vaccine can be manufactured on the us mainland because it does not use live fmd virus. the last fmd outbreak in the us was in 1929; currently fmd is not found in north and central america, the european union, australia, new zealand, or russia, but is present on a permanent or irregular basis in most of the rest of the world. countries that are free of fmd maintain restrictions on imports of live animals and animal products that might carry the virus. thus, the significance of fmd for the us is that our cattle, swine, sheep, and goats are totally susceptible to fmd and none have been vaccinated. these herds are the basis of a highly productive agribusiness, which is the largest in the world and a significant component of gdp and our foreign exports. the availability of fmd in endemic countries (eg, afganistan, pakistan, iraq, iran, and syria) and the ease with which an animal can become infected make this virus the primary agent of concern for agroterrorism (wheelis et al., 2002; breeze, 2004) . the economic loss from a fmd outbreak is considerable and is a major reason why this virus is important as an agent for agroterrorism. for example, in the 2001 fmd outbreak in the united kingdom, which lasted 8 months, cost more than $6.9 billion with more than 6 million animals destroyed and 120,300 farms depopulated in fmd affected areas. it took 18 months to regain normalization of trade following eradication of the disease. because the nucleic acid (dna or rna) of many viruses, including some that are currently not threats, can be manipulated in the laboratory, the potential for genetic engineering remains a serious threat. biotechnology, which has had a tremendous impact on the development of medicines, vaccines, and in the technologies needed to counter the threat of naturally occurring disease, can also be used to modify viruses with unintended consequences or even for the development of novel biological agents. several examples involving viruses are presented below. an australian research group (jackson et al., 2001) was investigating virally vectored immunocontraceptive vaccines based on ectromelia virus, the causative agent of the disease termed mousepox. the researchers created a recombinant virus, which expressed viruses and bioterrorism the mouse cytokine il-4 in order to enhance the antibody-mediated response to other recombinant antigens carried on the virus vector. instead, the ectromelia virus vector expressing il-4 altered the host's immune response to this virus resulting in lethal infections in normally genetically resistant mice (eg, c57bl/6). moreover, this virus also caused lethal infections in mice previously immunized with ectromelia virus. the creation of this "supermousepox" led to speculation that similar genetic engineering could be performed on another pox virus (ie, variola major) leading to a biological weapon that would be effective against an immunized population. the influenza pandemic of 1918-20, which followed world war i, was uniquely severe, causing an estimated 50 million deaths worldwide (johnson and mueller, 2002) . this pandemic happened before the advent of viral culture and very little was known about the virus until the development of polymerase chain reaction (pcr) technology. recently, the complete coding sequences of all eight viral rna segments has been determined by using reverse transcription-pcr (rt-pcr) to amplify fragments of viral rna retained in preserved tissues from individuals who died during this pandemic (reid et al., 1999; taubenberger et al., 1997 taubenberger et al., , 2005 . more recently, researchers reconstructed the 1918 spanish influenza pandemic virus using reverse genetics and observed that this reconstructed virus exhibited exceptional virulence in the model systems examined and that the 1918 hemagglutinin and polymerase genes were essential for optimal virulence (tumpey et al., 2005) . the milestone achievement of reconstructing an "extinct" pandemic virus raised a number of questions including whether it was necessary or wise to recreate by molecular means a naturally extinct virus that caused one of the deadliest pandemics in human history (taubenberger et al., 2012) . ultimately, senior us government scientists and officials at the department of health and human services concluded that research on this virus could play an important role in pandemic preparedness efforts as well as shed light on both the reasons for its extraordinary virulence and evolutionary origin. influenza pandemics in humans arise from animal influenza viruses, yet the molecular changes that are required for an animal virus to be transmitted efficiently between humans is poorly understood. the viral hemagglutinin (ha) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. highly pathogenic avian h5n1 influenza a viruses have circulated in poultry for more than 16 years. however, they only rarely cause human infections and do not transmit efficiently among humans. two controversial studies were published in 2012 that described mutations leading to the airborne transmission of h5n1 between ferrets, a mammalian model of influenza. in the first study, fouchier and his colleagues (herfst et al., 2012) used a combination of genetic engineering and serial infection of ferrets to create a mutant h5n1 virus that could spread among ferrets without direct contact. they used reverse genetics to introduce four amino acid substitutions in the ha protein that have been shown in previous research to make h5n1 viruses more human-like in that the ha would bind preferentially to alpha 2,6-linked sialic acid receptors instead of alpha 2,3-linked receptors. they also introduced a mutation in another viral gene, pb2, the polymerase complex protein, which was linked to the ability to grow in the human respiratory tract, which is cooler than the intestinal tract of birds, where the viruses usually reside. seeing that this mutant failed to achieve airborne transmission, the researchers serially passed this strain through a series of ferrets in an effort to force it to adapt to the mammalian respiratory tract. in the second study, kawaoka and his colleagues (imai et al., 2012) used a somewhat different approach in which they introduced four mutations in the ha gene from a h5n1 virus and then combined it with seven genes from a 2009 h1n1 virus, creating a hybrid virus that was able to spread by air between ferrets. on one hand, these studies provide further indication that a mammalian transmissible h5n1 influenza virus may arise naturally, and paves the way for improved influenza surveillance and pandemic preparedness. on the other hand, some scientists believe that the risks associated with these types of studies (particularly those by fouchier and colleagues) outweigh the benefits, particularly with respect to the consequences of an accidental release (roos, 2012) . a full-length poliovirus complementary dna (cdna) (c. 7500 bp) has been synthesized in the laboratory by assembling oligonucleotides of plus-and minus-strand polarity (cello et al., 2002) . the synthetic poliovirus cdna was transcribed by rna polymerase into viral rna, which was translated and replicated in a cytoplasmic extract of uninfected hela s3 cells, resulting in the de novo synthesis of infectious poliovirus. the publication of this research raised concerns that more complicated viruses (eg, variola major, ebola, or marburg) could be synthesized from scratch based on publically available sequences, or that viruses could be created that do not exist naturally. an effective defense requires a comprehensive approach that includes: prevention of access to viral stocks (morse and weirich, 2011) ; improved means of detecting deliberately induced disease outbreaks (elbers and knutsson, 2013; khan and pesik, 2011) ; rapid medical recognition of specific syndromes (eg, hemorrhagic fever syndrome); rapid laboratory identification of viruses in human or animal specimens (leijon and belak, 2013) ; prevention of human-to-human or animal-to-animal transmission (farsang et al., 2013) ; reliable decontamination procedures ; development of effective vaccines (spurgers and glass, 2011; lehrer and holbrook, 2011; marzi et al., 2011) ; and the development of effective antiviral therapies (smith et al., 2009; richardson et al., 2011) . rapid and accurate detection of biothreat agents is the basis of an effective public health response to bioterrorism. in order to address this issue, cdc in collaboration with other partners established an international network of laboratories called the laboratory response network (lrn) (morse et al., 2003) , which was provided with the tools to accomplish this mission. rapid assays utilizing advanced molecular and immunological technology for detection of agents such as variola virus, as well as emerging public health threats such as sars coronavirus and h5n1 influenza virus, were distributed to member laboratories. equipment, training, and proficiency testing are elements of the lrn and contribute to a uniform operational plan. the importance of high-quality standardized testing for detection of these agents is exemplified by the rapid need for medical countermeasures to protect or treat civilian populations. accurate laboratory analysis is a major element in the decision process for deployment of the federal government's strategic national stockpile (sns) of medical countermeasures (posid et al., 2011) . a similar system was developed by the us department of agriculture and its partners to protect us agriculture and the food supply from a bioterrorist attack (heintzelman, 2011) . as part of the effort to deter biological terrorism and strengthen the law enforcement investigative response to such an act, the us established a microbial forensic laboratory known as the national bioforensic analysis center (burans, 2011) that operates in partnership with the federal bureau of investigation. scientists are already developing methods for the forensic investigation of incidents involving viruses, including foot-and-mouth disease virus (carrillo, 2011) . for the terrorist, the use of a viral agent would pose a challenge due to problems associated with acquisition, cultivation, and dissemination. the target for an attack with a viral agent can range from humans to animals and plants. therefore, agricultural targets are a particular concern because a bioterrorist attack would require relatively little specialized expertise and technology, and can have very large economic consequences (wheelis et al., 2002; casagrande, 2002; breeze, 2004) . nature has provided many challenges to combating viral diseases. viral agents are much more prone to genetic variation and mutation, and can be manipulated or created in the laboratory to take on desired characteristics. with few exceptions, differentiating between natural and intentional viral disease outbreaks can be challenging. unlike bacterial diseases, many of which are treatable, there are fewer medical countermeasures to employ when dealing with viral infections. for many viruses, laboratory diagnostic methods and reagents must continuously be refined to account for genetic changes and variants. thus, the challenge of developing bioterrorism countermeasures is significant. fortunately, this effort contributes to combatting natural disease events more effectively, which has global benefits. efficacy of trivalent inactivated encephalomyelitis virus vaccine in horses agroterrorism: betting far more than the farm the national bioforensic analysis center persistence in humans of antibody to subtypes of venezuelan equine encephalomyelitis (vee) virus after immunizing with attenuated (tc-83) vee virus vaccine microbial forensics of rna viruses: foot-and-mouth disease virus bioterrorism and biocrimes. the illicit use of biological agents since 1900 the weapon potential of a microbe biological warfare targeted at livestock chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template arena viruses other than lassa virus the 1990 florida dental investigation agroterrorism targeting livestock: a review with a focus on early detection systems did bioweapons test cause a deadly smallpox outbreak? control of the deliberate spread of foot-and-mouth disease virus decontamination of high-risk animal and zoonotic pathogens biological and toxin weapons: research, development, and use from the middle ages to 1945 united states department of agriculture smallpox as a biological weapon airborne transmission of influenza a/h5n1 virus between ferrets princes and peasants: smallpox in history experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic historical perspective on agroterrorism: lessons learned from 1945 to 2012 forensic public health: epidemiological and microbiological investigations for biosecurity origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states tick-borne encephalitis vaccines review of a new molecular virus pathotyping method in the context of bioterrorism biological weapons in the twentieth century: a review and analysis influenza virus transmission is dependent on relative humidity and temperature vesicular stomatitis virus-based vaccines for prophylaxis and treatment of filovirus infections molecular evidence of hiv-1 transmission in a criminal case detecting biothreat agents: the laboratory response network select agent regulations aum shinrikyo: once and future threat? molecular epidemiology of hiv transmission in a dental practice the threat of agroterrorism: economics of bioterrorism center for disease control and prevention's bioterrorism preparedness program west nile mystery. the new yorker origin and evolution of the 1918 "spanish" influenza virus hemagglutinin gene evaluation of different strategies for post-exposure treatment of ebola virus infection in rodents fouchier study reveals changes enabling airborne spread of h5n1 public health assessment of potential biological terrorism agents the russian biological weapons program: vanished or disappeared? viruses of the bunya-and togaviridae families: potential as bioterrorism agents and means of control in vitro efficacy of st246 against smallpox and monkeypox vaccine development for biothreat alphaviruses the effect of environmental parameters on the survival of airborne infectious agents reconstruction of the 1918 influenza virus: unexpected rewards from the past initial genetic characterization of the 1918 characterization of the 1918 influenza virus polymerase genes risk factors for human disease emergence inactivation of virus-containing aerosols by ultraviolet germicidal irradiation characterization of the reconstructed 1918 spanish influenza pandemic virus world health organization, 1970. health aspects of chemical and biological weapons cuban allegations of biological warfare by the united states: assessing the evidence key: cord-337315-qv8ycdhe authors: miller, maureen; hagan, emily title: integrated biological–behavioural surveillance in pandemic-threat warning systems date: 2017-01-01 journal: bull world health organ doi: 10.2471/blt.16.175984 sha: doc_id: 337315 cord_uid: qv8ycdhe economically and politically disruptive disease outbreaks are a hallmark of the 21st century. although pandemics are driven by human behaviours, current surveillance systems for identifying pandemic threats are largely reliant on the monitoring of disease outcomes in clinical settings. standardized integrated biological–behavioural surveillance could, and should, be used in community settings to complement such clinical monitoring. the usefulness of such an approach has already been demonstrated in studies on human immunodeficiency virus, where integrated surveillance contributed to a biologically based and quantifiable understanding of the behavioural risk factors associated with the transmission dynamics of the virus. when designed according to strengthening the reporting of observational studies in epidemiology criteria, integrated surveillance requires that both behavioural risk factors – i.e. exposure variables – and disease-indicator outcome variables be measured in behavioural surveys. in the field of pandemic threats, biological outcome data could address the weaknesses of self-reported data collected in behavioural surveys. data from serosurveys of viruses with pandemic potential, collected under non-outbreak conditions, indicate that serosurveillance could be used to predict future outbreaks. when conducted together, behavioural surveys and serosurveys could warn of future pandemics, potentially before the disease appears in clinical settings. traditional disease-outcome surveillance must be frequent and ongoing to remain useful but behavioural surveillance remains informative even if conducted much less often, since behaviour change occurs slowly over time. only through knowledge of specific behavioural risk factors can interventions and policies that can prevent the next pandemic be developed. no other modern epidemic or pandemic mobilized the global health community to action like the 2013-2016 ebola virus disease outbreak in western africa. following the outbreak, calls for pandemic-threat warning systems came from both traditional public health policy-makers 1,2 and national governments. 3 as currently conceptualized, the first step in the identification of a pandemic threat requires an outbreak of sufficient size to come to the attention of medical personnel who are sufficiently influential and persistent to ensure action. 4 once an outbreak is verified, well-established protocols for disease investigation and control can be swiftly put in place -although it may be many months before the main risk factors and most effective control measures are identified. the ebola outbreak in western africa probably began in december 2013 5 but it took another year before traditional burial practices were found to be a leading cause of the rapid spread of the causative virus. 6 although human behaviours often increase the risk of acquiring an infectious disease, the systematic investigation of human risk behaviours is seldom included in disease surveillance strategies. 7 however, behavioural surveillance to improve the understanding of human immunodeficiency virus (hiv) and acquired immunodeficiency syndrome has been ongoing for decades. behavioural assessment was key to the identification of injecting drug users as a high-risk group for hiv infection in the early 1980s. 8 it was also crucial in documenting the risks posed by hiv to women. 9 subsequently, an innovative, practical method, which combines biological outcome data with behavioural risk factor data -i.e. exposure variables -was developed to document hiv transmission dynamics. such integrated biological-behavioural surveillance has since become well established and standardized and been frequently implemented globally. 10, 11 it has contributed extensively to a biologically-based and quantifiable understanding of the behavioural risk factors associated with the acquisition and transmission of hiv 12 and the early identification of subgroups of the population that may be more vulnerable to hiv infection. 9 more recently, data from integrated surveillance have been used to evaluate the impact of evidence-based interventions to prevent hiv infection and to monitor treatment uptake. 13 similar surveillance could help identify behavioural risk factors and high-risk subgroups for zoonotic infections such as ebola -potentially before diseases of pandemic potential are identified in clinical settings or major outbreaks occur in communities. approximately half of the emerging pandemic threats are zoonotic in origin. 14, 15 at the time of writing, the most lethal and costly pandemics of the 21st century, i.e. avian influenza, ebola, middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars), have all been caused by zoonotic viruses. [16] [17] [18] [19] little is known about either the risk factors that lead to the initial spillover of a zoonotic disease into human populations or the dynamics of any subsequent humanto-human transmission. 20 much more is known about (i) the locations of so-called hotspots where, many scientists believe, new pandemics of zoonotic disease are likely to emerge; 14 (ii) the kinds of ecological and environmental activities that have been associated with spillover and outbreaks of zoonotic disease in the past; 21 and (iii) the distinct spatial groupings of specific infectious diseases on a global scale, and the associ-abstract economically and politically disruptive disease outbreaks are a hallmark of the 21st century. although pandemics are driven by human behaviours, current surveillance systems for identifying pandemic threats are largely reliant on the monitoring of disease outcomes in clinical settings. standardized integrated biological-behavioural surveillance could, and should, be used in community settings to complement such clinical monitoring. the usefulness of such an approach has already been demonstrated in studies on human immunodeficiency virus, where integrated surveillance contributed to a biologically based and quantifiable understanding of the behavioural risk factors associated with the transmission dynamics of the virus. when designed according to strengthening the reporting of observational studies in epidemiology criteria, integrated surveillance requires that both behavioural risk factors -i.e. exposure variables -and disease-indicator outcome variables be measured in behavioural surveys. in the field of pandemic threats, biological outcome data could address the weaknesses of self-reported data collected in behavioural surveys. data from serosurveys of viruses with pandemic potential, collected under non-outbreak conditions, indicate that serosurveillance could be used to predict future outbreaks. when conducted together, behavioural surveys and serosurveys could warn of future pandemics, potentially before the disease appears in clinical settings. traditional disease-outcome surveillance must be frequent and ongoing to remain useful but behavioural surveillance remains informative even if conducted much less often, since behaviour change occurs slowly over time. only through knowledge of specific behavioural risk factors can interventions and policies that can prevent the next pandemic be developed. integrated surveillance for pandemic threats maureen miller & emily hagan ated ecological and virological barriers to the dispersal and establishment of those diseases. 22 in the development of pandemic-threat warning systems, integrated biological-behavioural surveillance can be tightly focused on specific viral families in the high-risk population subgroups that live in identified hotspots and are environmentally or occupationally exposed to animals. the remainder of this article presents an overview of issues relevant to the design of rigorous behavioural surveys to assess the spillover of emerging zoonotic disease and the associated transmission risk factors, which is the first step in designing effective integrated surveillance. we identified community-based serological surveys of viruses with pandemic potential as a possible source of useful biological outcome data. we summarize the results of such serosurveys, conducted in nonoutbreak settings in africa, and evaluate their usefulness -especially when used in combination with behavioural surveillance -in the prediction of future outbreaks. when designed according to strengthening the reporting of observational studies in epidemiology criteria, 23 integrated surveillance requires that both disease-indicator outcome variables and behavioural risk factors be measured in behavioural surveys. behavioural risk factors, i.e. exposure variables, simply represent the population prevalence of behaviours that may or may not increase the risk of disease. without the outcome variables, the exposure variables are of little use in elucidating the mechanisms of the spillover of zoonotic disease to humans or of subsequent human-to-human transmission. effective surveillance requires questions that assess a range of animal exposures, document experiences of unusual illness and measure contextual factors that can lead to an increase or decrease in the probabilities of behavioural risk factors and disease. in studies on zoonoses, the assessment of behavioural risk factors is complicated because different zoonotic diseases may be associated with different kinds of animal exposure. the spillover of zoonotic viruses from wildlife, the source of most emerging zoonoses, 15 has been difficult to document. behavioural risk may be either direct or indirect. direct contact with primate blood or bodily fluids has been associated with several zoonotic viruses found in humans, such as human t-lymphocyte virus, 24 simian foamy virus 25, 26 and, possibly, ebola. 5 indirect contact was responsible for the transmission of nipah virus to humans, which was mediated through date-palm sap contaminated with the urine of infected bats, 27 and hantavirus transmission is most frequently associated with inhalation of aerosolized virus from the excreta of infected rodents. 28 both general exposure to animals, e.g. when buying live animals at market, and more intimate exposure, e.g. during the slaughter of animals or as the result of animal bites, must therefore be assessed. syndromic surveillance is widely used to monitor illnesses of unknown etiology in clinical settings and can provide a useful referent in the identification of the outcome variables to be measured in behavioural surveys. several zoonotic diseases, such as avian influenza, 17 mers 16 and sars, 29 were identified via syndromic surveillance networks, from localized increases in the incidence of influenza-like illness or severe acute respiratory infection. by using standardized definitions, it should be easy to develop questions or symptom checklists for syndromic surveillance based on self-reported data. such definitions already exist for influenza-like illness, 30 severe acute respiratory infection 30 and other syndromes consistent with zoonotic infection, such as encephalitis of unknown origin, 31 fever of unknown origin 32 and haemorrhagic fever of unknown origin. 33 the risks posed to humans by exposure to animals may be modified by various biological, ecological, economic, political and sociocultural factors. 34 for example, poverty can place individuals and communities at the forefront of zoonotic disease risk through several mechanisms. exposure to dense or diverse rodent populations in urban or rural environments 35, 36 and the displacement of wildlife populations as land is cleared for crops are some mechanisms. 36 understanding the context within which spillover to humans can occur is an important component in the prevention of zoonotic outbreaks. the same behavioural risk factors may be risky in one context but not in another. for instance, the sharing of a water source with animals displaced by a change in land use may only have an adverse effect on human health if there is faecal-oral transmission of the zoonotic pathogen to humans. if such transmission requires contact with the animal blood, then the sharing of the water source should not increase the risks of either spillover or transmission. once human-to-human transmission of a zoonotic pathogen occurs, additional risks come into play, often as a consequence of the human infection, and these should also be measured pre-emptively. burial practices and health-care-seeking practices were associated with explosive increases in, respectively, the incidence of human infection with ebola virus in western africa 6 and mers in the republic of korea. 37 even in the absence of detailed biological outcome data, behavioural surveillance may be used to assess relationships between behavioural risk factors and self-reported experiences of unusual illness that are consistent with the symptoms of zoonotic disease. this could be done both rapidly and at a scale that facilitates epidemiologically relevant analyses. although not as conclusive as biological data, self-reported data could provide substantially more information than is currently available. in the field of pandemic threats, biological outcome data could address the weaknesses of any self-reported data collected in behavioural surveys. the ideal source of biological outcome data, i.e. data that provide the strongest evidence of zoonotic disease spillover, would be community-based screening for acute infection with zoonotic viruses. however, as such infection is rare under nonoutbreak conditions, many thousands of individuals would usually have to be screened for a meaningful analysis of the behavioural risk factors. serological assays, in which previous exposure to a virus is demonstrated by a positive result, can provide alternative biological outcome data. since many more individuals may have been exposed to a virus than are currently ill with the virus, serology can provide the larger number of individuals, with known viral exposure, required for powerful analyses of behavioural risk factors. we therefore investigated the results of serosurveys for their potential usefulness in the prediction of future outbreaks. we focused on studies conducted in communities under non-outbreak settings in africa. we collated results presented in peer-reviewed publications -in english, french or spanish -that we identified via google scholar and web of science searches that ended on 31 december 2015 (available from the corresponding author). we used "africa", "antibody", "serology", "serosurvey", "zoonoses" and "zoonotic disease" as search terms. we identified additional relevant results through the citations in the articles identified in the searches. serosurveys of zoonotic viruses have been conducted since the discovery of the ebola virus in the 1970s, mostly during or shortly after an outbreak of zoonotic disease. our searches revealed 38 serosurveys of zoonotic viruses in africa that had been conducted during non-outbreak conditions. 25, 26, [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] to identify any associations between population subgroup risk and seroprevalence, we divided the subjects of the serosurveys into three risk categories, based on the limited data from previous research on zoonotic disease spillover. for example, hunters have been consistently found to be a high-risk population subgroup, 25, 26, 42, 45, 48 followed by rural populations, who have been categorized as medium-risk because of their close and regular proximity to wildlife. 38, 39, 41, 43, 44, 46, 49 randomized or representative samples of general populations 40, 47 have been considered to be low-risk. serological assays for several different zoonotic pathogens were conducted as part of serosurveillance in each of the 14 studies included in our analysis. use of these assays led to the recording of a total of 38 unique zoonotic pathogen seroprevalences that ranged from 0% to 24%. of these 38 seroprevalences, nine were recorded for high-risk population subgroups, 19 for medium-risk and 10 for low-risk. evidence of previous exposure to a zoonotic pathogen, i.e. a seroprevalence of more than zero, was detected in eight (89%) of the high-risk population subgroups, 16 (84%) of the medium-risk and seven (70%) of the low-risk (available from the corresponding author). high seroprevalences, i.e. seroprevalences of at least 1%, represented the results for all eight of the high-risk subgroups with evidence of previous exposure to a zoonotic pathogen, 12 (75%) of the medium-risk subgroups with such evidence and three (48%) of the low-risk subgroups with such evidence (available from the corresponding author). exposure to wildlife therefore appeared to be associated both with any evidence of viral exposure and with high seroprevalence. since the first known outbreak in 1976, the united states centers for disease control and prevention have recorded 35 documented ebola outbreaks in africa. 50 more than 5% of the subjects included in serosurveys in gabon in 1981 43 and liberia in 1982 46 showed evidence of exposure to ebola virus, that is decades before an ebola outbreak was first reported in either of these countries. between 1.9% and 12.4% of the subjects included in ebola serosurveys in three countries that have never reported an ebola outbreak, the central african republic, 40, 44, 48 madagascar 47 and zimbabwe, 39 were also found to be seropositive. although serological assays exist for the coronaviruses that cause mers and sars, hantaan viruses and paramyxoviruses, most serosurveys of zoonotic viruses in africa have focused on haemorrhagic fevers. most of the serosurveys we reviewed had also been done before the widespread availability and use of viral detection tests. recently, population-based serosurveys have been increasingly adopted, in recognition of their utility in preparing health authorities for potential outbreaks or epidemics. 26, 38, 49 current serological assay methods tend to be labour-intensive and to suffer from cross-reactivity that prevents distinction between several antigenically related viruses. however, the last few years have witnessed major advances in the development of economically feasible methods for comprehensive serological profiling 51, 52 and these should facilitate the investigation of zoonotic spillover into human populations. we review the potential contributions that integrated biological-behavioural surveillance could make to pandemicthreat prediction, prevention and risk mitigation. if we are to mitigate the risk of a zoonotic disease outbreak, we need a better understanding of the mechanisms behind the spillover of zoonotic disease into human populations. by making such mechanisms the focus of integrated surveillance, we should be able to: (i) monitor the presence and prevalence of behavioural risk factors and the seroprevalence of specific zoonotic pathogens within particular population subgroups; (ii) deploy targeted control and mitigation strategies rapidly; and (iii) evaluate the efficacy of prevention policies and interventions. although traditional disease surveillance must be frequent and ongoing to remain useful, behavioural surveillance remains informative even if conducted much less often, since behaviour change occurs slowly over time. to be effective as a prevention tool, integrated biological-behavioural surveillance should be implemented as a baseline measure -to identify behavioural risk factors, determine the prevalence of those risk factors, especially in any population subgroups that are considered at higher risk of zoonotic spillover, and establish seroprevalence. should an outbreak occur, a database that documents local behaviours and practices, as well as the context within which such behaviours occur, can contribute to the development of appropriate and feasible strategies for disease control and mitigation. finally, data from integrated surveillance will be invaluable in both informing realistic and effective interventions and policies for the prevention of zoonotic spillover and transmission, and evaluating the impact of such interventions and policies efficiently. relative to the economic, social and political costs of epidemics, prevention will always be less expensive 3, 19 if the targets of prevention activities are well understood and acted upon. the fact that success can feel more like the status quo is a challenge unique to prevention. the political commitment for prevention activities will often be less than that for a reactive response elicited by the emergence of a terrifying new infectious disease. however, political support may be improved if surveillance is made to be, and appear, more cost-effective, by focusing on specific diseases 22 in population subgroups who live in ecologically fragile hotspots. 14, 21 there is substantial overlap between areas considered to be hotspots for zoonotic disease spillover and those considered hotspots for hiv, perhaps the best known zoonotic virus. 14, 53 this overlap opens a real possibility of merging attempts to detect zoonotic disease spillover with pre-existing population-based systems that have been used to investigate the hiv epidemic for several decades. for example, the demographic and health surveys program is implemented globally in settings without high-quality civil registration and has extensive experience in collecting integrated biological-behavioural surveillance data in community settings. 54 integrated surveillance will never be a viable alternative to traditional clinical disease surveillance for assessing active viral infections. rather, it can serve to complement virus detection efforts, by potentially identifying pandemic threats before the need for large-scale clinical intervention. as current behaviours may not reflect the behaviours that originally exposed the individuals who are found seropositive to the virus of interest, both current and lifetime behaviours need to be investigated. this is the strategy that has proved successful in identifying subtle exposure risks in the field of hiv, such as backloading of syringes with drug solution by injecting drug users. 55 in identifying specific behavioural risk factors, integrated biological-behavioural surveillance will be most effective when the reported syndromic symptoms are recent, e.g. occurring in the previous 12 months, and their probable association with a zoonotic virus can be confirmed by a positive serological test result. current pandemic-threat warning systems rely almost exclusively on disease surveillance in clinical settings. standardized biological-behavioural surveillance, in which both disease outcome data -self-reported and biological -and behavioural risk factors are measured, would complement traditional surveillance and greatly advance the understanding of behaviours and practices that could be targeted for risk mitigation and, ultimately, for prevention. the implementation of integrated biological-behavioural surveillance need not be frequent to be informative and useful in preventing the spillover of zoonotic agents with pandemic potential. ■ отличительной чертой 21-го века являются вспышки заболеваний, оказывающие разрушительный эффект на экономику и политику. хотя причины пандемий кроются в поведении людей, современные системы наблюдения, предназначенные для выявления угроз пандемии, в значительной мере полагаются на мониторинг исходов заболевания в клинических условиях. стандартизованное, комплексное, биологически поведенческое наблюдение можно и следует применять в условиях общин как дополнение к подобному клиническому мониторингу. целесообразность такого подхода уже была продемонстрирована в исследованиях вируса иммунодефицита человека, в рамках которых комплексное наблюдение способствовало биологически обоснованному и поддающемуся количественной оценке пониманию факторов поведенческого риска, связанных с динамикой передачи вируса. для комплексного наблюдения, разработанного в соответствии с критериями устранения недостатков предоставления информации в наблюдательных исследованиях по эпидемиологии, необходимо измерение фак торов поведенческого риска, т. е. переменных подверженности воздействию, и выходных переменных индикаторов заболеваемости в рамках исследований поведения. в сфере угроз пандемии с помощью информации о биологических исходах можно было бы устранить недостатки предоставленных респондентами данных, полученных в рамках поведенческих исследований. судя по данным серологических исследований вирусов, имеющих пандемический потенциал, которые были получены в условиях отсутствия вспышки, эпидемиологический надзор может быть использован для прогнозирования будущих вспышек. проведение поведенческих и серологических исследований вместе позволило бы спрогнозировать будущие пандемии теоретически до того, как заболевание возникнет в клинических условиях. польза традиционного наблюдения исходов заболеваний зависит от частоты и непрерывности его проведения, в то время как поведенческое наблюдение позволяет получить ценную информацию, даже если осуществляется гораздо реже, поскольку поведение со временем изменяется медленно. только зная конкретные факторы поведенческого риска, можно разработать мероприятия и политики, способные предотвращать будущие пандемии. los brotes de enfermedades perjudiciales para la economía y la política son una característica del siglo xxi. a pesar de que las pandemias se ven impulsadas por el comportamiento humano, los sistemas de vigilancia actuales para identificar amenazas de pandemia dependen enormemente del seguimiento de los resultados de las enfermedades en entornos clínicos. la vigilancia integrada y normalizada de datos biológicos y del comportamiento podría y debería utilizarse en comunidades como complemento de dicho seguimiento clínico. su utilidad ya se ha demostrado en varios estudios sobre el virus de la inmunodeficiencia humana, en los que la vigilancia integrada contribuyó a la comprensión cuantificable y biológica de los factores de riesgo del comportamiento asociados con la dinámica de la transmisión del virus. al estar diseñada según los criterios del fortalecimiento de la notificación de los estudios observacionales en epidemiología, la vigilancia integrada requiere tanto los factores de riesgo del comportamiento (es decir, las variables de exposición) como las variables de resultados del indicador de enfermedades se midan en encuestas sobre el comportamiento. en el campo de las amenazas de pandemia, los datos de los resultados biológicos podrían abordar la debilidad de los datos autodeclarados recopilados en las encuestas sobre el comportamiento. la información de las encuestas serológicas sobre virus con potencial pandémico, recopilada en condiciones en las que no se había reportado un brote, indica que podría utilizarse la vigilancia serológica para predecir futuros brotes. al realizarse juntas, integrated surveillance for pandemic threats maureen miller & emily hagan las encuestas serológicas y sobre el comportamiento podrían advertir sobre futuras pandemias, probablemente antes de que la enfermedad aparezca en entornos clínicos. la vigilancia tradicional de los resultados de las enfermedades debe ser constante y frecuente para que sea útil, aunque la vigilancia sobre el comportamiento sigue siendo meramente informativa, incluso si se realiza con menos asiduidad, dado que los cambios de comportamiento se producen lentamente con el paso del tiempo. únicamente a través del conocimiento de los factores de riesgo del comportamiento específicos se pueden desarrollar las intervenciones y las políticas capaces de evitar el desarrollo de la próxima pandemia. reforming the world health organization after ebola how to beat the next ebola a national blueprint for biodefense: leadership and major reforms needed to opitimize efforts report. bipartisan report of the blue ribbon study panel on biodefense integrating behavioral surveillance into emerging infectious disease prevention investigating the zoonotic origin of the west african ebola epidemic ebola transmission linked to a single traditional funeral ceremony from disease surveillance to the surveillance of risk factors an outbreak of community-acquired pneumocystis carinii pneumonia: initial manifestation of cellular immune dysfunction a different disease: hiv/aids and health care for women in poverty integrated biological and behavioral surveillance of key populations, toolkit for implementation. washington: national alliance of state & territorial aids directors (nastad) guidelines for surveys of populations at risk for hiv infection circumcision in hiv-infected men and its effect on hiv transmission to female partners in rakai second-generation hiv surveillance: better data for decision-making. bull world health organ risk factors for human disease emergence global trends in emerging infectious diseases mers: emergence of a novel human coronavirus human infection with a novel avian-origin influenza a (h7n9) virus isolation and characterization of viruses related to the sars coronavirus from animals in southern china economic optimization of a global strategy to address the pandemic threat origins of major human infectious diseases ecology of zoonoses: natural and unnatural histories global biogeography of human infectious diseases the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies bushmeat hunting, deforestation, and prediction of zoonoses emergence. emerg infect dis naturally acquired simian retrovirus infections in central african hunters cross-species transmission of simian foamy virus to humans in rural gabon, central africa transmission of human infection with nipah virus hantavirus pulmonary syndrome-united states: updated recommendations for risk reduction update: outbreak of severe acute respiratory syndrome-worldwide who surveillance case definitions for ili and sari world health organization international encephalitis consortium. case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium fever of unknown origin viral hemorrhagic fever (vhf) social epidemiology human-rodent contact and infection with lymphocytic choriomeningitis and seoul viruses in an inner-city population characteristics associated with contact with rodents in, around, and outside homes in khon kaen province, thailand middle east respiratory syndrome (mers) in the republic of korea; situation assessment high prevalence of both humoral and cellular immunity to zaire ebolavirus among rural populations in gabon viral haemorrhagic fever antibodies in zimbabwe schoolchildren antibody prevalence against haemorrhagic fever viruses in randomized representative central african populations african haemorrhagic fevers of viral origin. contribution to their study in central african republic ebola and marburg virus antibody prevalence in selected populations of the central african republic. microbes infect haemorrhagic fever in gabon. i. incidence of lassa, ebola and marburg viruses in haut-ogooué haemorrhagic fever virus activity in equatorial africa: distribution and prevalence of filovirus reactive antibody in the central african republic filovirus activity among selected ethnic groups inhabiting the tropical forest of equatorial africa a serological survey on viral hemorrhagic fevers in liberia antibodies to haemorrhagic fever viruses in madagascar populations veille microbiologique: les fièvres hémorragiques virales en république centrafricaine; données sérologiques actualisées chez l'homme evidence for henipavirus spillover into human populations in africa outbreaks chronology: ebola virus disease comprehensive serological profiling of human populations using a synthetic human virome determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics geneva: joint united nations programme on hiv/aids innovations in health and demographic surveillance systems to establish the causal impacts of hiv policies syringe-mediated drug-sharing (backloading): a new risk factor for hiv among injecting drug users key: cord-329396-cl28bjnd authors: carrico, adam w.; horvath, keith j.; grov, christian; moskowitz, judith t.; pahwa, savita; pallikkuth, suresh; hirshfield, sabina title: double jeopardy: methamphetamine use and hiv as risk factors for covid-19 date: 2020-04-07 journal: aids behav doi: 10.1007/s10461-020-02854-w sha: doc_id: 329396 cord_uid: cl28bjnd nan among men who have sex with men (msm), the co-occurrence of methamphetamine (meth) use and hiv could create a double jeopardy for coronavirus disease 2019 . co-occurring meth and hiv could amplify biological and behavioral risk for infection with the novel coronavirus (sars-cov-2), presenting unique challenges to halting community-level transmission. this confluence of biobehavioral risk factors related to meth and hiv may also synergistically enhance vulnerability to covid-19 progression. below we provide an overview of important areas for further research regarding the potential implications of the intertwining epidemics of meth use and hiv for the covid-19 pandemic in msm. the use of meth and other stimulants has consistently been identified as a potent driver of the hiv/aids epidemic in msm [1] [2] [3] . despite successful deployment of public health interventions that specifically targeted meth in msm [4] [5] [6] , there is a resurgent epidemic of meth use in the united states [7] . beginning in 2011, recent meth use doubled (from 4 to 9%) among msm in new york city [8] , and comparable increases in meth use were observed among msm in san diego [9] . this trend is corroborated by concomitant increases in meth use in other urban areas such that national rates are meeting or exceeding 2005 peaks [10] . there is also increasing recognition that stimulant use is prevalent in ethnic minority msm where hiv incidence is the highest [11] . for example, recent findings from our team indicate that one-in-five young black msm in texas reported stimulant use in the past 2 months [12] . there is an urgent need to deploy comprehensive, multi-level interventions targeting the intersection of stimulant use and hiv among msm [1, 13] . the resurgence of meth use in msm threatens to compromise biomedical approaches to hiv/aids prevention, such as treatment as prevention (tasp), and fuel the covid-19 pandemic. the prevalence of stimulant use is twofold greater among msm living with hiv [14] , and stimulant use undermines the clinical and public health benefits of tasp. our team and others have demonstrated that people living with hiv who use stimulants experience profound difficulties with navigating the hiv care continuum, including poorer anti-retroviral therapy (art) adherence and persistence, that lead to slower rates of viral suppression [15] [16] [17] [18] and faster mortality [19, 20] . even in the era of universal art, stimulant users in a clinical cohort comprised mostly of msm receiving hiv care at zuckerberg san francisco general hospital reached viral suppression more slowly [15] . it is plausible that the covid-19 pandemic will present new barriers to engagement along the hiv care continuum, which could disproportionally affect people who use stimulants. this could include difficulties with obtaining timely laboratory results or refilling art medications. the difficulties that people who use stimulants experience with achieving and maintaining viral suppression could enhance biological vulnerability to sars-cov-2 infection as well as lead to faster covid-19 progression. hiv is already damaging the immune system even when people are virally suppressed, a phenomenon referred to as residual immune dysregulation [21] , which meth use amplifies to create a double jeopardy for covid-19. there is increasing evidence from our team and others that meth use can induce gut-immune dysregulation, even among those with treated hiv infection [22] [23] [24] [25] [26] . in our prior bio-behavioral research conducted with msm with treated hiv infection who use meth, those who provided a urine sample that was reactive for stimulants (i.e., meth or cocaine) displayed differential expression of genes and 2-directional perturbation of pathways relevant to systemic immune activation and inflammation [25] . these perturbations in gene expression relevant to immune dysregulation among those providing a urine sample that was reactive for stimulants were paralleled by elevations in plasma tumor necrosis factor-alpha (tnf-α), an important marker of systemic inflammation. we also observed concomitant elevations in soluble cd14 (scd14), a clinically relevant plasma marker of monocyte activation [27] , in those providing a urine sample that was reactive for stimulants. this partially reflects stimulation of monocytes with lipopolysaccharide leaking from the gastrointestinal tract [26] . the clinical relevance of these findings is supported by the fact that markers of systemic immune activation and inflammation are implicated in multiple, chronic medical conditions such as cardiovascular disease in people living with hiv, which have been identified as key risk factors for covid-19 progression [28, 29] . meth use could also contribute local immune dysregulation as well as alter the expression of angiotensin-converting enzyme 2 (ace-2) receptors, a key site for sars-cov-2 binding in the lungs and small intestine [30] . smoking is a prevalent mode of administration for meth and crack-cocaine that could enhance local immune dysregulation in the lungs and modify the expression of ace-2 receptors to increase vulnerability to sars-cov-2 infection and covid-19 progression. there are also three plausible biological pathways for meth-associated exacerbation of microbial translocation (i.e., the leaky gut) that could serve as a primary driver of systemic immune dysregulation, particularly among those living with hiv. first, the meth decreases parasympathetic tone [31] , which could increase intestinal permeability. second, meth directly damages gut barrier integrity in selfadministering hiv-1 transgenic rats [32] . third, meth-associated indoleamine 2,3-dioxygenase upregulation in treated hiv infection damages gut barrier integrity [33] . these meth-induced alterations to the gastrointestinal tract may be partially responsible for systemic immune dysregulation in people living with hiv who use meth and further research is needed to determine if meth use alters ace-2 receptor expression in the small intestine [30] . taken together, pulmonary immune dysregulation, gut-immune dysregulation, and enhanced ace-2 receptor expression are biologically plausible pathways whereby co-occurring meth use and hiv could heighten vulnerability to sars-cov-2 infection and covd-19 progression. in early march of 2020, epidemiologic experts warned that as much as 70% of the population could become infected with sars-cov-2 without broad implementation of social distancing such as restrictions on interactions in large groups [34] . recent forecasting models and data strongly suggest that the number of covid-19 cases will grow exponentially in countries that do not achieve high rates of adherence to social distancing guidelines [35] . because meth use has been consistently linked to sexual risk taking behaviors among msm [1] , it is likely that people who use meth will experience greater difficulties with adhering to covid-19 social distancing guidelines. men will continue to seek out partners for substance use and sex, which will potentiate covid-19 clusters among msm and present a key challenge to halting community-level transmission. in fact, there are reports of meth-fueled sex parties among msm during the covid-19 lockdown in spain [36] and covid-19 clusters from a recent miami circuit party [37] . further research is needed to examine meth use and other behavioral correlates of adherence to social distancing guidelines. this represents a critical first step to informing targeted deployment of limited public health resources to "flatten the curve" of the covid-19 pandemic. it is also clear that the covid-19 pandemic represents a chronic, uncontrollable stressor that could exacerbate psychiatric disorders and increase risk for sars-cov-2 infection. those living with stimulant use disorders could experience new barriers to accessing and remaining engaged in substance use disorder treatment programs as well as 12-step self-help groups, increasing risk for relapse [38] . furthermore, the stress and social isolation individuals experience during social distancing could serve as a potent trigger for alcohol and other substance use. there is a clear need for scalable, mhealth interventions to address the psychiatric burden of the covid-19 pandemic. we have previously demonstrated the efficacy of a positive affect intervention for improving psychological adjustment and achieving durable reductions in viral load for people living with hiv [39] , including msm living with hiv who use methamphetamine [40, 41] . we have also demonstrated that it is feasible and acceptable to deliver this positive affect intervention to various populations in a self-guided, online format [42] [43] [44] [45] . furthermore, there is other evidence to support the potential benefits of text messaging for reducing condomless sex [46] and mhealth applications for improving art adherence in msm who use meth [47] . further clinical research is needed to characterize the psychiatric consequences of the covid-19 pandemic and test novel mhealth approaches to improve adherence to social distancing guidelines in msm who use meth and other stimulants. the covid-19 pandemic is rapidly evolving, leaving more questions than answers regarding how best to mitigate its devastating effects. at this stage, there clearly is an urgent need for research to examine the implications of co-occurring meth use and hiv for sars-cov-2 acquisition as well as covid-19 progression. identifying the biobehavioral mechanisms that could account for the potential double jeopardy experienced by those living with co-occurring meth use and hiv will inform efforts to halt community-level sars-cov-2 transmission and reduce risk for covid-19 progression. it is also clear that the covid-19 pandemic presents unique challenges to engagement along the hiv care continuum as well as engagement in substance use disorder treatment that warrant further study. finally, the covid-19 pandemic underscores the urgent need for mhealth approaches to reach high priority populations to "flatten the curve" of the covid-19 pandemic. efforts to stem the tide of the covid-19 pandemic will require a sustained, coordinated response that integrates behavioral and biomedical approaches much like we have witnessed in over three decades of the hiv/aids epidemic. amphetamine-group substances and hiv the methamphetamine epidemic: implications for hiv prevention and treatment not just the needle: the state of hivprevention science among substance users and future directions community-based harm reduction substance abuse treatment with methamphetamine-using men who have sex with men a public health response to the methamphetamine epidemic: the implementation of contingency management to treat methamphetamine dependence community reactions to campaigns addressing crystal methamphetamine use among gay and bisexual men in new york city the forgotten killer, is back. and it's everywhere hiv infection risk, prevention, and testing behaviors among men who have sex with men-national hiv behavioral surveillance clear links between starting methamphetamine and increasing sexual risk behavior: a cohort study among men who have sex with men walking the line: stimulant use during sex and hiv risk behavior among black urban msm spirituality/religiosity, substance use, and hiv testing among young black men who have sex with men critical review: when the party is over: a systematic review of behavioral interventions for substance-using men who have sex with men amphetamine-type stimulants and hiv infection among men who have sex with men: implications on hiv research and prevention from a systematic review and meta-analysis stimulant use and viral suppression in the era of universal antiretroviral therapy psychiatric risk factors for hiv disease progression: the role of inconsistent patterns of antiretroviral therapy utilization increased human immunodeficiency virus loads in active methamphetamine users are explained by reduced effectiveness of antiretroviral therapy engagement in hiv medical care and technology use among stimulant-using and nonstimulant-using men who have sex with men crack cocaine, disease progression, and mortality in a multicenter cohort of hiv-1 positive women stimulant use and progression to aids or mortality after the initiation of highly active antiretroviral therapy residual immune dysregulation syndrome in treated hiv infection drugs of abuse, immune modulation, and aids methamphetamine decreases cd4 t cell frequency and alters pro-inflammatory cytokine production in a model of drug abuse brief report: recent methamphetamine use is associated with increased rectal mucosal inflammatory cytokines, regardless of hiv-1 serostatus recent stimulant use and leukocyte gene expression in methamphetamine users with treated hiv infection substance-associated elevations in monocyte activation among methamphetamine users with treated hiv infection gut epithelial barrier dysfunction and innate immune activation predict mortality in treated hiv infection inflammation, immune activation, and cardiovascular disease in hiv clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus effect of methamphetamine dependence on heart rate variability colon dysregulation in methamphetamine self-administering hiv-1 transgenic rats tryptophan catabolism by indoleamine 2,3-dioxygenase 1 alters the balance of th17 to regulatory t cells in hiv disease coronavirus may infect up to 70% of world's population, expert warns day level forecasting for coronavirus disease (covid-19) spread: analysis, modeling and recommendations 8 men arrested for hosting drug-fuelled orgy during coronavirus lockdown in spain 3 more gay men who went to the winter party test positive for coronavirus & others are getting sick with meetings banned, millions struggle to stay sober on their own randomized controlled trial of a positive affect intervention for people newly diagnosed with hiv randomized controlled trial of a positive affect intervention for methamphetamine users randomized controlled trial of a positive affect intervention to reduce hiv viral load among sexual minority men who use methamphetamine the marigold study: feasibility and enhancement of an online intervention to improve emotion regulation in people with elevated depressive symptoms feasibility and acceptability of an online positive affect intervention for those living with comorbid hiv depression a randomized pilot trial of a positive affect skill intervention (lessons in linking affect and coping) for women with metastatic breast cancer an online positive affect skills intervention reduces depression in adults with type 2 diabetes theorybased text-messaging to reduce methamphetamine use and hiv sexual risk behaviors among men who have sex with men: automated unidirectional delivery outperforms bidirectional peer interactive delivery a pilot study of a mobile app to support hiv antiretroviral therapy adherence among men who have sex with men who use stimulants key: cord-331289-02411gfv authors: di minno, giovanni; perno, carlo federico; tiede, andreas; navarro, david; canaro, mariana; güertler, lutz; ironside, james w. title: current concepts in the prevention of pathogen transmission via blood/plasma-derived products for bleeding disorders() date: 2015-07-20 journal: blood rev doi: 10.1016/j.blre.2015.07.004 sha: doc_id: 331289 cord_uid: 02411gfv the pathogen safety of blood/plasma-derived products has historically been a subject of significant concern to the medical community. measures such as donor selection and blood screening have contributed to increase the safety of these products, but pathogen transmission does still occur. reasons for this include lack of sensitivity/specificity of current screening methods, lack of reliable screening tests for some pathogens (e.g. prions) and the fact that many potentially harmful infectious agents are not routinely screened for. methods for the purification/inactivation of blood/plasma-derived products have been developed in order to further reduce the residual risk, but low concentrations of pathogens do not necessarily imply a low level of risk for the patient and so the overall challenge of minimising risk remains. this review aims to discuss the variable level of pathogenic risk and describes the current screening methods used to prevent/detect the presence of pathogens in blood/plasma-derived products. acute bleeding episodes can arise either because of inherited bleeding disorders (e.g. haemophilia, von willebrand disease), acquired deficiency of haemostatic components (e.g. due to infection, malignancy or autoimmune disease), trauma, surgery or as a result of infection with an organism that causes haemorrhagic disease (e.g. ebola or marburg virus) [1] . various treatment options exist for preventing or treating acute bleeding episodes, including fresh-frozen plasma/cryoprecipitate, platelets and plasma-derived/recombinant clotting factor concentrates [2, 3] . the use of blood-derived and recombinant haemostatic products has increased markedly over recent years, as exemplified by the global use of factor viii products ( fig. 1) [4] . this increased use has been driven by improved availability of clotting factors, increased life expectancy of people with bleeding disorders [5, 6] , increased use of prophylaxis for severe bleeding disorders [7, 8] and decreased risk of transmission of infectious agents. historically, the risk of transmission of infectious agents via blood/ plasma-derived products has been of great concern to the medical community. this risk has reduced dramatically since the implementation of stricter donation screening/donor selection procedures and improved purification procedures, but cannot be fully eradicated. furthermore, the implementation of pathogen inactivation technology for blood/plasma-derived products has further reduced the risk of transmission of both known and emerging pathogens, although results can be variable according to the methods used [9, 10] . however, it is important to note that patient risk is highly dependent on the circumstances under which blood products are collected, handled and used. in general, clinicians assess the level of risk associated with the use of blood/ plasma-derived products by evaluating factors such as patient characteristics (e.g. age, immune status, geographical location, lifestyle) and the nature of the pathogen (e.g. physical characteristics, level of virulence, chronicity of infection, prevalence). the presence of a particular pathogen within blood/plasma-derived products may pose a significant threat to specific patient groups (e.g. the elderly or immunocompromised), while being of low risk to the general population (e.g. hev). while the clinical assessment of risk is based on a variety of factors, the virological assessment of risk is based solely on the presence or absence of pathogens. the presence of pathogens implies the possibility of infection, so only pathogen-free products can be described as entirely risk-free. adopting the virological approach (i.e. discarding all products a large number of pathogenic agents (including viruses, protozoan parasites and prions) can be transmitted via blood/plasma-derived products and are capable of causing disease in humans ( table 1) . the presence of viruses in plasma-derived products became a concern in the 1980s, when 60-70% of patients with severe haemophilia became infected with human immunodeficiency virus (hiv-1) [6] . this concern continued with the discovery that 80% of patients treated with plasmaderived products prior to 1992 had become infected with hepatitis c virus (hcv) [5] . current donor selection and screening practices have improved our ability to detect or reduce the presence of pathogens in blood/plasma-derived products; for example, the residual risk of transfusion-transmitted infection (tti) with hiv/hbv/hcv has fallen to near or less than 1 per million transfused units [14, 15] . despite this success, however, a residual risk still remains. the pathogenic agents shown in table 1 (and the supplementary appendix) do not form an exhaustive list. many microorganisms that are normally non-pathogenic have the potential to cause disease when responding to changes in the biological environment, or when transfused to an immunosuppressed patient. in addition, there is still a risk that new and emerging pathogens may enter the blood supply ( table 2) . the standard assays commonly used for blood screening are nucleic acid amplification technology (nat) and immunoassays for detection of antibody and/or antigen. immunoassays are frequently used for screening purposes as multiple samples can be processed together and they may yield semi-quantitative results. nat assays allow earlier pathogen detection than with immunoassays, but they are also more costly and complex. assay selection is generally determined by the level of accuracy/speed required, but factors such as the resources available (e.g. staff, infrastructure), assay complexity and cost considerations (e.g. consumables) must also be considered. most assays for blood donation screening are mandatory (particularly in europe and north america) and the world health organization (who) recommends that all whole blood (and blood which has been processed by apheresis) should undergo pathogen screening before it is used for clinical or manufacturing purposes. screening for hiv-1, hiv-2, hbv, hcv and treponema pallidum subspecies pallidum (t. pallidum; the causative agent of syphilis) is strongly advised. the who and world federation of hemophilia (wfh) suggest that countries should carry out individual routine screening for further pathogens based on epidemiological information for their region e.g. htlv-1 and trypanosoma cruzi [16] . the wfh also acknowledges the positive impact of hiv, hbv and hcv screening on global blood safety and recommends that these screening tests be implemented whenever possible [2] . details of the serological tests carried out on individual donor plasma and nat testing of plasma mini-pools are shown in tables 3 and 4 [17] . it is recommended that the minimum evaluated sensitivity/specificity level of any assay used for blood donation screening should be 99.5% or higher [16] . however, not all assays fulfil these criteria as sensitivity/ [196] . specificity levels vary according to assay type and type of microorganism being tested for (see table 1 for details). therefore, the general advice is to screen donated blood to as high a standard as possible [16] . in the early stages of infection with hiv, hbv or hcv, viraemia occurs in the host's bloodstream at variable levels. viral antigens may appear at the same time as dna/rna, but more often become detectable several weeks later. specific antibodies are measurable 2 to 6 weeks after infection; the time between initial infection and the appearance of detectable parameters of infection (e.g. viral nucleic acid/antigens/ antibodies) is known as the 'window period' [18] [19] [20] . 4.1.1.1. hiv. when screening blood for the presence of hiv-1/hiv-2, the use of a combined antigen/antibody assay is advised (combined hiv p24 ag and anti-hiv-1 + anti-hiv-2 antibodies) as it allows earlier detection of infection. the performance of nat testing is mandatory in many countries and further reduces the window period (from around 20 to 11 days) [20] [21] [22] . 4.1.1.2. hbv. the majority of diagnostic laboratories focus on the detection of hbsag, which is the first detectable serological marker of infection. however, there is a risk that the hbsag concentration may decline to undetectable levels during the course of infection, yielding a false negative result [23] . screening for antibodies to hbc is the most conservative approach for identifying potentially exposed donors, as this identifies all individuals who have ever immunologically experienced any type of hbv infection (either current, chronic or resolved) and who may experience viral reactivation during their lifetime (particularly under conditions of immunosuppression). however, assays to measure hbc antibodies are relatively non-specific and do not always correlate with the presence of hbv virus in plasma [18] . also, we cannot exclude the possibility that donors who test positive for anti-hbc do not have pulsed recurrences of virus replication, resulting in the presence of low levels of hbv-dna in plasma. for these reasons, national transfusion services do not always routinely screen donations for anti-hbc. nat assays can be carried out, but their use is restricted by potentially low levels of viral dna [16, 18] . the combined use of hbsag screening tests and nat assays has reduced the window period for detection of hbv infection from approximately 60 to 35.5 days [20, 24] . mutant hbv strains (escape variants) should also be considered, as they may occasionally escape serological detection (although most can be detected by nat assays) [24, 25] . these hbv-variants are rare, but therefore more likely to enter and contaminate the blood supply as they are more difficult to detect [25] . in summary, the screening of blood supplies for the presence of hbv is effective, but an optimal screening system has not yet been defined. 4.1.1.3. hcv. hcv (both recent and chronic infection) can be detected by screening blood for the presence of both hcv antigen and hcv antibody (anti-hcv). seroconversion occurs at approximately 6-8 weeks postinfection; however, steady improvements in screening technology (including the adoption of nat assays) have reduced the window period to approximately 1-3 weeks [20, 26] . as with hiv, nat assays are more useful for detecting early infection, although the issue of low viral rna concentration persists [27, 28] . htlv-1 and htlv-2 are endemic in some regions, but very rare in others, and therefore screenings are conducted on a geographical or at-risk basis. the presence of virus is mostly inferred by the detection of virus-specific antibodies, using sensitive immunoassays [16] . in this instance, screening involves detection of non-specific, nontreponemal or specific treponemal antibodies. nat assays are generally not used [29] . specific antibody tests identify all individuals who have ever been exposed to this bacterium (and may continue to yield positive results for more than ten years following initial infection), while the non-specific tests (e.g. vdrl or cardiolipin tests) are primarily of use in identifying donors who may have an active infection. since t. pallidum is heat-sensitive and cannot readily withstand extended storage at low temperatures, storage at 4°c for more than three days is sufficient to render the pathogen non-infectious. however, blood components (e.g. platelets) that are stored at temperatures of around 20°c do present a risk of t. pallidum persistence. therefore screening for antibodies of this organism is recommended [16] . blood can be screened for further pathogens as appropriate, according to geographical location, seasonal activity of the vector and also patient risk factors. a current viral pathogen of interest is the mosquitoborne flavivirus west nile virus (wnv), which was confirmed to have been transmitted via transfusion in 2002 [30] . an immediate screening policy was put in place in the usa in order to reduce the risk of further transmission. this policy included deferral of any individual displaying symptoms of infection, quarantine of plasma collected during periods of high mosquito activity (when wnv is most prevalent) and the rapid development/use of wnv-specific nat and serological assays. these measures were highly effective and caused a significant reduction in the number of confirmed cases of wnv transfusion-related transmission. however, wnv outbreaks still occur within the americas, indicating a potential need for seasonal blood screening for wnv [31] . wnv outbreaks have also occurred in europe (including italy and greece), prompting the implementation of seasonal blood screening procedures in the affected regions of those countries [32] . this is another mosquito-borne pathogen that could potentially pose a risk to transfusion safety, although to date reports of transfusionrelated transmission of this virus are rare [33] . a mutated form of the chikungunya virus has been responsible for several epidemics in the past decade, spreading to the reunion islands in the indian ocean (2005), italy (2007) and the caribbean area (2012/2013) [34] [35] [36] . the virus may be detected in blood donors by nat, which will help to reduce the level of transmission risk [35] . there is also concern about the possibility of parvovirus b19 in the blood supply. b19 is prevalent worldwide, with seroprevalence in blood donors varying from between 0.2-1.3% in the usa, europe and africa and 25-40% in asia [37] . the risk of parvovirus transmission is higher when units of blood are pooled (e.g. to create batches of clotting factor concentrates, albumin etc.) and so individuals with bleeding disorders are at a higher risk of infection. b19 dna was detected in 26% of clotting factor concentrates in a recent german study [38] , and another study found that populations receiving blood-derived products were 1.7 times more likely to display antibodies to b19 than populations who had not received blood products [39] . b19 lacks a lipid envelope, which renders it highly resistant to some methods of pathogen inactivation [40] . screening of blood donations for b19 dna is not currently routine, but many manufacturers only process plasma that has been screened for the absence of b19 dna in order to reduce the risk of transmission [41] . given the prevalence of b19 in different populations, it is difficult to define the residual tti risk of this virus. however, it is clear that a transmission risk does exist. trypanosoma brucei gambiense/rhodiense africa, 10% at risk [160] direct microscopic visualisation/antibody/nat nat: b100 trypanosomes/ml [161] nat: 1-10 trypanosomes/ml [162] antibody: catt -87-98% [161] nat: 88% [163] antibody: catt -95% [161] nat: 99.2% [163] (continued on next page) the potential presence of hepatitis e virus (hev) in blood or bloodderived products is relevant. currently there seems to be a discrepancy between the number of hev-rna positive blood donations in europe (ranging from 1 in 1240 in germany to 1 in 1761 donations in the netherlands) [42] , and the low number of confirmed cases of hepatitis e in blood transfusion recipients (one confirmed case in the uk [2006] , one in france [2007] and two in germany [2014]) [43] [44] [45] . this indicates that the subject of hev infectivity and pathogenicity needs to be investigated further [46] [47] [48] [49] . there is also a debate over the necessity of introducing screening blood for the presence of hev; the virus is not currently screened for in the uk and other european countries, although one study of english donors found hev to be widespread (1 in 2848 donations) within the donor population [50] . since infection with this virus can be harmful to immunocompromised individuals, the potential need for introducing hev screening should be considered [42] . the residual risk for tti of hbv, hcv, hiv and hev in selected countries is given in table 5 . despite recent advances in methods for the detection of prions, no single method has been developed as a screening test for blood, although several methods in animal models show great promise [51] [52] [53] . in humans, a protocol for the evaluation of a blood-based test for its suitability in the diagnosis of variant creutzfeldt-jakob disease (vcjd) has been established, but no test yet appears to satisfy the requirements of sensitivity and specificity [54] . the only report so far of a blood-based diagnostic test for vcjd claimed an assay sensitivity of 71.4% and a specificity of 100% in symptomatic patients and its potential applicability as a screening test to detect asymptomatic vcjd infection has recently been investigated [55] . there is currently no strategy for confirming a positive screening result, although the protein misfolding cyclical amplification technique has recently been shown to yield positive results in buffy coat/white blood cell samples from a small number of patients with vcjd [56] . extensive use of blood donor selection and testing does not always guarantee a safe product. if the test is insufficiently sensitive, then false negatives may occur. alternatively, tests which are not sufficiently specific (e.g. anti-hbc assay for hbv) may cause false positive results, leading to an unnecessary decrease in the number of clotting products available [16] . torque tenovirus (ttv) is an example where testing specificity has been an issue, as this virus exists in various divergent forms (23 distinct genotypes have been identified thus far) [57] . since ttv is often detected in healthy individuals and is not associated with any particular disease, routine screening for this virus is not considered to be necessary; even a test with excellent sensitivity/specificity would not contribute to increase the level of safety of blood/plasma-derived products with regard to ttv. insufficient assay sensitivity remains a rare but potential problem when blood donations are screened during the window period of initial hbv, hcv or hiv infection. an increase in testing sensitivity threshold is needed to prevent hbv transmission via blood/plasma-derived products and by blood transfusion, as extremely low concentrations of hbv (e.g. 1.6 copies/ml) is capable of viral transmission (see above section on anti-hbc positive patients) [58] . in the case of hiv and hcv, even nat testing may not always be sufficient to ensure sufficiently high levels of safety as virus transmission has previously occurred after transfusion of blood with undetectable levels of viraemia [59] . recent reports have highlighted concerns about the inability of nat assays to detect different variants of hiv. there have been at least four cases in which the presence of hiv-1 rna was undetected by nat assay screening, potentially putting transfusion recipients at risk [21] . two of these false-negative results occurred due to genetic mutation in the viral rna regions targeted by nat assay primers (although in in cases where no overall figures are available, specificity/sensitivity has been described as low, medium or high (as appropriate). catt, card agglutination test for trypanosomiasis; id-nat, individual donation nat; mp-nat, mini-pool nat; nat, nucleic acid testing; pcr, polymerase chain reaction; pfu, plaqueforming unit. the majority of cases serology yielded a positive result) [60, 61] . falsenegative results can be avoided by designing nat assays that target a minimum of two amplification regions; such testing will be mandatory in germany from 2015 [21, 62] . it is hoped that as the sensitivity and specificity of nat tests continue to improve, cases of undetected infection may become less of an issue in the future. a recent report suggests that it is not necessary to carry out serological screening for multiple hbv markers, and that nat based screening is preferred [58] . however, there is a risk that the practice of relying upon a single method of screening may lead to a higher incidence of false-negative results. in one case of transfusion-associated parvovirus b19 transmission, donated blood was screened for the presence of b19 antigen and deemed to be safe since no antigen was detected. since the recipient subsequently developed b19 infection, the donation was re-analysed and found to contain b19 dna [63] . reports such as this support the argument for multiple parameter testing. the localisation of pathogens within blood may also influence ease of detection. for example, wnv is present at 10-fold higher levels in whole blood than in plasma in viraemic seropositive donors. the situation is reversed in viraemic seronegative donors, who display higher wnv levels (4-fold) in plasma than in whole blood [64] . also, cellassociated viruses such as cmv and htlv-1 are less frequently transmitted with the use of leukoreduced products [65, 66] , indicating that these viruses are less likely to be found in blood/plasma-derived products. the minimum infective dose (mid: the lowest number of pathogenic particles required to successfully infect a host) of a particular pathogen is more likely to be reached in whole blood or blood-derived components, making the use of plasma-derived or recombinant clotting factors the safest option. to date, studies attempting to measure human mid values have generally determined the viral concentration needed to infect a particular percentage of the exposed population (e.g. 50%). this value (the human infective dose for 50% of the population) is referred to as hid 50 and is often described as the human mid [67] . the hid 50 value varies greatly between pathogens (even if they are physically similar) [68] and also varies depending upon the immune status of the recipient, as immunocompromised individuals, neonates and the elderly are at greater risk of infection than healthy individuals [67] . when this finding and the prevalence of immunocompromised patients receiving blood products are both taken into account, it implies a need for screening tests to have the highest sensitivity possible. the impact of transmission on morbidity and mortality is dependent on patient characteristics. for example, although the vast majority of cases of cmv infection are not clinically important, influencing factors such as genetic predisposition, malnutrition and pre-existing infection can lead to the development of severe disease [69] . national screening programmes do not currently screen for cmv as standard, but nat assays exist and may be carried out if required, e.g. if blood is specifically intended for vulnerable recipients, such as pregnant women or transplant patients [70] . it is the opinion of the authors that both serological assays and nat tests should be used in order to reach the highest level of safety possible, particularly when in the case of immunosuppressed patients. as well as blood donation testing, a range of other measures are used to increase the pathogen safety of blood-and plasma-derived products. these include donor selection and screening, recipient vaccination and the use of blood product purification/inactivation methods. the choice of inactivation method also impacts upon the level of risk. questionnaires are often used to attempt to assess donors' health status and their potential exposure to various risks. donors can be accepted or rejected on the basis of these answered questionnaires, or alternatively their blood may be put through additional screening tests as appropriate [16, 31] . patients with bleeding disorders should be vaccinated against hav and hbv. european studies have reported that universal hbv vaccination of blood donors could be cost-effective as this measure would reduce the risk of hbv transmission in general and might even remove the necessity for general hbv nat testing; however, this would not reduce the risk posed by hbv escape variants (as described earlier) [71, 72] . blood/plasma-derived products typically undergo various procedures designed to reduce the pathogen level, although these procedures are not effective against all pathogens. plasma donations undergo quarantine (approximately 4-6 months) prior to fractionation and when the donor is again screened negative ffp can then be subjected to chromatographic fractionation, solvent-detergent treatment, nanofiltration and/or heat inactivation [73, 74] . prolongation of product storage time can be effective in reducing the infectivity of temperature-sensitive pathogens (such as t. pallidum). production of recombinant products also follows strict protocols to remove and inactivate any viruses that might be present, even though the risk of viral presence is minimal. although current purification/inactivation techniques (such as solvent-detergent treatment, nanofiltration and heat activation) do reduce the risk of pathogen transmission, they are not always sufficient to render blood/plasma-derived products safe [75] . small nonenveloped viruses (e.g. hav, b19 and picornavirus) are often highly resistant to inactivation procedures and may still be infectious in some plasma-derived concentrates [75, 76] . the presence of prions is also a concern. attempts to remove prions from plasma-derived products have involved several techniques, including ion-exchange chromatography and nanofiltration [77, 78] . several problems exist with these approaches, in particular the use of exogenous "spikes" derived from prion-infected brain homogenates to measure prion clearance, which may result in an overestimation of the amount of prion removal, and the methods used for the estimation of the reduction in prion load, which ideally should involve bioassay to measure infectivity [79] . further developments in this field are required to address these issues. a recently proposed approach for the inactivation of infectious agents in blood is whole-blood treatment with ultraviolet (uv) light in combination with a photosensitiser such as riboflavin or amotosalen [80, 81] . the main disadvantage of this approach is that uv treatment has been linked to the formation of neoantigens, which may be generated via modification of the surface antigens of platelets. the presence of neoantigens may provoke a recipient immune response during transfusion of uv-treated platelets, causing them to be rapidly cleared from the circulation [81] . while this approach to pathogen inactivation is currently used for platelets and is effective, it needs further refinement for the inactivation of whole blood [81, 82] . the wfh strongly recommends the use of plasma-derived or recombinant products in preference to cryoprecipitate or fresh frozen plasma, as the infectious load of any infectious human pathogen is lower in plasma-derived products than in cryoprecipitate, and even lower in recombinant products [2] .in some european countries, recombinant products have almost completely replaced plasmaderived products [12] . the use of recombinant products, which have been manufactured and formulated with minimal addition of human/animal-derived materials, greatly reduces the risk of recipient exposure to plasma-derived infectious agents and after they have undergone virus removal/inactivation processes, recombinant products can be considered to be as safe as currently possible [83] . despite these benefits, the use of recombinant products may be limited by higher costs and perceived problems of inhibitor formation [83] . for indications where no recombinant factor concentrates are available, the use of inactivated plasma-derived concentrates is safer than fresh frozen plasma and will reduce the risk of other adverse effects such as hypervolemia, transfusion-related lung injury (trali) and hypersensitivity [84] . a minimal risk approach would ensure that patients receive effective treatment with the lowest possible risk, but this is difficult to achieve in practical terms. regulatory needs in different european countries are usually based on recommendations from the medical community, so in order to achieve minimal risk, it would be ideal for these regulations to be standardised and mandatory in all countries. directives issued by the european union commission describe the regulatory requirements for the safety of whole blood and plasma, stating that "all precautionary measures during their collection, processing, distribution and use need to be taken making appropriate use of scientific progress in the detection and inactivation and elimination of transfusion transmissible pathogenic agents" [85] . however, the relative safety of different screening tests, products and processing methods is not discussed and so individual countries may adopt different approaches towards minimising risk. although recombinant products are associated with the highest level of pathogen safety, higher costs for development and production may make them too expensive for some healthcare systems [86] . inhibitor formation also remains an important element of concern with both plasma-derived and recombinant products, particularly with regard to fviii in haemophilia a [87] . the risk of inhibitor formation was shown to be greater with recombinant versus plasma-derived factor viii concentrates in some cohort studies [88] , but similar in others [89] . in light of these considerations (availability, adverse reactions and cost), it appears that the issue of pathogen safety of blood/plasmaderived products is highly important but may not be the limiting factor with respect to overall patient safety. the benefits of treatment with a hypothetically 'unsafe' plasma-derived product may outweigh the risk of a negative outcome (e.g. bleeding, inhibitor formation), although we suggest that it may be clinically simpler to deal with inhibitor formation than to combat an infection from an unknown or untreatable pathogen. there are still significant knowledge gaps and areas of unmet need with respect to the pathogen safety of blood/plasma-derived and recombinant products. the incidence of hbv, hcv and hiv tti has fallen to near or below 1 per million transfused units in the industrialised world, indicating that current donor selection and blood screening strategies have had a positive impact on blood safety [14] . however, it is clear that screening both donors and donated blood cannot exclude all known pathogens or eliminate all risks from emerging pathogens [63, 90] . historical precedent indicates that the blood supply is always vulnerable to contamination by hitherto non-prevalent/unknown pathogens, and that this risk cannot be accurately gauged [91] . as we identify new infectious agents of concern and develop new tests for their detection, it will also be necessary to clearly define the infectious dose range for each agent and use appropriately sensitive tests for their identification. for example, in suspected cases of vcjd infection, considerable challenges remain in the development of screening and confirmatory tests that have sufficient sensitivity and specificity to be of use in both a clinical setting and within blood banks [92] . surveillance of people with haemophilia is required to monitor pathogen safety issues related to blood and plasma products. the european haemophilia safety surveillance system (euhass), which began in 2008, is a pharmacovigilance programme which spans 25 european countries and is designed to detect, monitor and investigate adverse drug reactions. reports of adverse events (such as acute/allergic events, ttis and inhibitors) are submitted to euhass by participating centres and cumulative patient and clotting factor data are recorded annually [93] . a formal coordinated risk-assessment and management action plan, in addition to a task force, should be developed to respond quickly to any emerging infections. such a plan should include long-term storage of samples from produced batches (for retesting in the case of outbreaks with known or recently emerged infectious agents) and guidance on responsibility for developing/performing tests for emerging pathogens (industry vs. regulatory agencies). guidance on approaching patients who have potentially been infected and surveillance strategies for patients at high risk would also be beneficial. the majority of evidence indicates that the concept of clinical safety of blood/plasma derivatives does not necessarily correspond to the concept of pathogen safety; blood can only be classed as microbially safe in reference to the infectious agents that are known and have been screened for. establishing whether the presence of undetected microbes in the blood is clinically relevant will require further long-term, detailed studies. it should also be noted that even though the risk of transmission of key detectable viruses (such as hiv, hbv and hcv) via transfusion has fallen significantly, transmission does still occur. in general, balancing safety, efficacy and practicalities is a difficult goal to achievepatient safety is typically the key driver, but striving for near-complete safety at the expense of the patient's health or quality of life may not be the best course of action for patients or clinicians. the lack of a cohesive international strategy for blood donation and screening is a pressing concern that needs to be addressed. furthermore, a formal coordinated continuous risk-assessment and management action plan needs to be developed to deal with the constant potential risk of emerging infections. establishing an international registry (or harmonising data collection in national registries) and dedicated task force may help to identify newly emerging pathogens more rapidly than in the past and to further improve pathogen safety of blood/plasma-derived products and the blood supply in general. • the use of blood/plasma-derived products for the treatment of bleeding disorders carries a risk of pathogen transmission. • blood donations are screened for key pathogens such as hbv, hcv, hiv and the causative agent of syphilis, but other screening tests should be conducted as required according to geographical location and patient risk factors. • screening tests for pathogens may lack sensitivity/specificity and so false negatives may occur, resulting in a residual pathogen risk to patients. • in terms of pathogen safety, recombinant products (products which have had minimal exposure to blood/plasma-derived proteins) are considered to pose the lowest level of risk to patients. research agenda • regional and international rates of transfusion-transmitted infection for key pathogens and emerging pathogens • safety and efficacy of blood/plasma-derived products and recombinant products for treatment of bleeding disorders. pathogenesis of the viral hemorrhagic fevers guidelines for the management of hemophilia plasma components: properties, differences, and uses a study of reported factor viii use around the world mortality and causes of death in patients with hemophilia, 1992-2001: a prospective cohort study past, present and future of hemophilia: a narrative review the management of hemophilia in elderly patients assessing emerging infectious threats to blood safety for the blood disorders community blood processing who technical report: guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products pathogen safety of long-term treatments for bleeding disorders: still relevant to current practice factor viii safety: plasma-derived versus recombinant products pathogen safety of long-term treatments for bleeding disorders: (un)predictable risks and evolving threats transfusion-transmitted emerging infectious diseases: 30 years of challenges and progress experience of german red cross blood donor services with nucleic acid testing: results of screening more than 30 million blood donations for human immunodeficiency virus-1, hepatitis c virus, and hepatitis b virus recommendations: screening donated blood for transfusion-transmissible infections registry of clotting factor concentrates medical virology of hepatitis b: how it began and where we are now a novel acute hiv infection staging system based on 4th generation immunoassay pathogen inactivation and removal methods for plasma-derived clotting factor concentrates how safe is safe: new human immunodeficiency virus type 1 variants missed by nucleic acid testing prevalence of human immunodeficiency virus rna and antibody in first-time, lapsed, and repeat blood donations across five international regions and relative efficacy of alternative screening scenarios recent advances in molecular diagnostics of hepatitis b virus surface antigen-negative hepatitis b virus infection in dutch blood donors potential threat of drug-resistant and vaccine-escape hbv mutants to public health hepatitis c virus: screening, diagnosis, and interpretation of laboratory assays the incidence/window period model and its use to assess the risk of transfusion-transmitted human immunodeficiency virus and hepatitis c virus infection comparative analysis of triplex nucleic acid test assays in united states blood donors novel treponema pallidum serologic tests: a paradigm shift in syphilis screening for the 21st century transmission of west nile virus through blood transfusion in the united states in 2002 emerging infectious agents and the nation's blood supply: responding to potential threats in the 21st century west nile virus in the transfusion setting with a special focus on italian preventive measures adopted in 2008-2012 and their impact on blood safety novel chikungunya virus variant in travelers returning from indian ocean islands the chikungunya epidemic in italy and its repercussion on the blood system chikungunya virus: new risk to transfusion safety in the americas a single mutation in chikungunya virus affects vector specificity and epidemic potential parvovirus transmission by blood products -a cause for concern? prevalence of nucleic acid sequences specific for human parvoviruses, hepatitis a and hepatitis e viruses in coagulation factor concentrates evidence for the continued transmission of parvovirus b19 in patients with bleeding disorders treated with plasma-derived factor concentrates human parvovirus b19 viral pathogens hepatitis e screening for blood donations: an urgent need? transfusiontransmitted hepatitis e in a 'nonhyperendemic' country transfusion-associated hepatitis e, france transfusion-transmitted hepatitis e in germany an assessment of hepatitis e virus (hev) in us blood donors and recipients: no detectable hev rna in 1939 donors tested and no evidence for hev transmission to 362 prospectively followed recipients autochthonous hepatitis e virus infections: a new transfusion-associated risk a nationwide survey for prevalence of hepatitis e virus antibody in qualified blood donors in japan hepatitis e virus antibodies in blood donors hepatitis e virus in blood components: a prevalence and transmission study in southeast england new generation quic assays for prion seeding activity protein misfolding cyclic amplification of infectious prions plasminogen-based capture combined with amplification technology for the detection of prp(tse) in the pre-clinical phase of infection evaluation of a test for its suitability in the diagnosis of variant creutzfeldt-jakob disease population screening for variant creutzfeldt-jakob disease using a novel blood test: diagnostic accuracy and feasibility study preclinical detection of variant cjd and bse prions in blood transfusion transmitted virus: a review on its molecular characteristics and role in medicine hepatitis b virus testing by minipool nucleic acid testing: does it improve blood safety? human immunodeficiency virus transfusion transmission despite nucleic acid testing first transmission of human immunodeficiency virus type 1 by a cellular blood product after mandatory nucleic acid screening in germany blood screening nucleic acid amplification tests for human immunodeficiency virus type 1 may require two different amplification targets instruction to implement measures for risk minimisation in using hiv-1 nat test systems erythroid crisis caused by parvovirus b19 transmitted via red blood cell transfusion relative distribution of west nile virus rna in blood compartments: implications for blood donor nucleic acid amplification technology screening epidemiology, treatment, and prevention of human t-cell leukemia virus type 1-associated diseases approaches to minimize infection risk in blood banking and transfusion practice minimum infective dose of the major human respiratory and enteric viruses transmitted through food and the environment mechanisms of pathogenesis, infective dose and virulence in human parasites cytomegalovirus: pathogen, paradigm, and puzzle seroprevalence of cytomegalovirus antibodies in blood donors in southern hepatitis b virus vaccination of blood donors-what costs may be expected a cost-benefit analysis of blood donor vaccination as an alternative to additional dna testing for reducing transfusion transmission of hepatitis b virus guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products quarantine plasma: quo vadis pathogen inactivation technology: cleansing the blood supply eis-hubinger am. contamination of coagulation factor concentrates with human parvovirus b19 genotype 1 and 2 removal of tse agent from plasma products manufactured in the united kingdom characterization of thrombate iii(r), a pasteurized and nanofiltered therapeutic human antithrombin concentrate comparison of nanofiltration efficacy in reducing infectivity of centrifuged versus ultracentrifuged 263 k scrapie-infected brain homogenates in "spiked" albumin solutions pathogen reduction technology treatment of platelets, plasma and whole blood using riboflavin and uv light uvc irradiation for pathogen reduction of platelet concentrates and plasma development of a riboflavin and ultraviolet light-based device to treat whole blood guideline on the selection and use of therapeutic products to treat haemophilia and other hereditary bleeding disorders. a united kingdom haemophilia center doctors' organisation (ukhcdo) guideline approved by the british committee for standards in haematology incidence of transfusion-related adverse reactions per patient reflects the potential risk of transfusion therapy in japan setting standards of quality and safety for the collection, testing, processing, storage and distribution of human blood and blood components and amending directive 2001/83/ec emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells is the incidence and prevalence of inhibitors greater with recombinant products? yes influence of the type of factor viii concentrate on the incidence of factor viii inhibitors in previously untreated patients with severe hemophilia a intensity of factor viii treatment and inhibitor development in children with severe hemophilia a: the rodin study clinical perspectives of emerging pathogens in bleeding disorders hemophilia therapy and blood-borne pathogen risk creutzfeldt-jakob disease: reflections on the risk from blood product therapy the european haemophilia safety surveillance system variant cjd infection in the spleen of a neurologically asymptomatic uk adult patient with haemophilia detection of prion infection in variant creutzfeldt-jakob disease: a blood-based assay world health organisation (who) development of a sensitive real-time reverse transcriptase pcr assay with an internal control to detect and quantify chikungunya virus development and evaluation of sybr green i-based one-step real-time rt-pcr assay for detection and quantification of chikungunya virus poor diagnostic accuracy of commercial antibody-based assays for the diagnosis of acute chikungunya infection cmv hhv6,7,8 r-gene® real-time pcr kit technicial specifications. 2015. available from detection of cytomegalovirus (cmv) dna in edta whole-blood samples: evaluation of the quantitative artus cmv lightcycler pcr kit in conjunction with automated sample preparation detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification simplexa™ dengue kit technical specifications development of multiplex real-time reverse transcriptase pcr assays for detecting eight medically important flaviviruses in mosquitoes evaluation of commercially available diagnostic tests for the detection of dengue virus ns1 antigen and anti-dengue virus igm antibody world health organisation (who) procleix® ultrio® assay hepatitis b virus serology: use and interpretation the reliability of hbv core antibody in serological screening for hepatitis b virus world health organisation (who) laboratory diagnostics for hepatitis c virus infection hepatitis c virus core antigen testing: role in diagnosis, disease monitoring and treatment hepatitis c diagnostics: clinical evaluation of the hcvcore antigen determination transfusion-transmitted hepatitis e: is screening warranted? comparison of real-time pcr and antigen assays for detection of hepatitis e virus in blood donors genotype 3 diversity and quantification of hepatitis e virus rna novel approach for detection of hepatitis e virus infection in german blood donors serologic assays specific to immunoglobulin m antibodies against hepatitis e virus: pangenotypic evaluation of performances transmission and disease association of kaposi's sarcoma-associated herpesvirus: recent developments quantitative real-time pcr detection of antibodies to kaposi's sarcoma-associated herpesvirus: a new approach using k8.1 elisa and a newly developed recombinant lana elisa global report: unaids report on the global aids epidemic comparison of hiv-1 viral load assay performance in immunological stable patients with low or undetectable viremia laboratory diagnostics for hiv infection molecular determinants of human t-lymphotropic virus type 1 transmission and spread abbott prism human t-lymphotrophic virus types i and ii assay development and validation of a multiplex real-time pcr for htlv-1/2 confirmatory diagnosis evaluation of a new screening assay for htlv-1 and -2 antibodies for large-scale use evaluation of the use of real-time pcr for human t cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors keeping pace with parvovirus b19 genetic variability: a multiplex genotype-specific qpcr assay quantitative real-time detection of parvovirus b19 dna in plasma detection of human parvovirus b19, human bocavirus and human parvovirus 4 infections in blood samples among 95 patients with liver disease in nanjing by nested pcr detection of parvovirus b19 igm by antibody capture enzyme immunoassay: receiver operating characteristic analysis placental transmission of human parvovirus 4 in newborns with hydrops serodiagnosis of primary infections with human parvovirus 4, finland human parvovirus 4 infection human parvovirus 4 viremia in young children novel parvovirus and related variant in human plasma detection of a novel dna virus (ttv) in blood donors and blood products validation of sybr green based quantification assay for the detection of human torque teno virus titers from plasma west nile virus: detection with serologic and real-time pcr assays diagnosis of west nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies second international diagnostic accuracy study for the serological detection of west nile virus infection global incidence and prevalence of selected curable sexually transmitted infections use of pcr in the diagnosis of early syphilis in the united kingdom evaluation of a pcr test for detection of treponema pallidum in swabs and blood the laboratory diagnosis of syphilis current advances in detection and treatment of babesiosis a new real-time pcr assay for improved detection of the parasite babesia microti multiplex assay detection of immunoglobulin g antibodies that recognize babesia microti antigens world health organisation (who) leishmaniasis: prevention, parasite detection and treatment evaluation of pcr procedures for detecting and quantifying leishmania donovani dna in large numbers of dried human blood samples from a visceral leishmaniasis focus in northern ethiopia comparison of pcr assays for diagnosis of cutaneous leishmaniasis world health organisation (who) emerging nucleic acid-based tests for point-ofcare detection of malaria serological detection of plasmodium vivax malaria using recombinant proteins corresponding to the 19-kda c-terminal region of the merozoite surface protein-1 factsheet no 259 -trypanosomiasis options for field diagnosis of human african trypanosomiasis nucleic acid sequence-based amplification with oligochromatography for detection of trypanosoma brucei in clinical samples diagnostic accuracy of pcr in gambiense sleeping sickness diagnosis, staging and post-treatment follow-up: a 2-year longitudinal study factsheet no 340 -chagas disease international study to evaluate pcr methods for detection of trypanosoma cruzi dna in blood samples from chagas disease patients evaluation of serological tests to identify trypanosoma cruzi infection in humans and determine cross-reactivity with trypanosoma rangeli and leishmania spp a novel rhabdovirus associated with acute hemorrhagic fever in central africa a novel bunyavirus in china hemorrhagic fever caused by a novel bunyavirus in china: pathogenesis and correlates of fatal outcome what we are watching-five top global infectious disease threats, 2012: a perspective from cdc's global disease detection operations center surveillance on a/h5n1 virus in domestic poultry and wild birds in egypt comparative epidemiology of human infections with avian influenza a h7n9 and h5n1 viruses in china: a population-based study of laboratory-confirmed cases viral lung infections: epidemiology, virology, clinical features, and management of avian influenza a(h7n9) genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa nosocomial outbreak of novel arenavirus infection, southern africa marseillevirus-like virus recovered from blood donated by asymptomatic humans marseillevirus prevalence in multitransfused patients suggests blood transmission transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk severe acute respiratory syndrome (sars) -frequently asked questions current status of severe acute respiratory syndrome in china confronting sars: a view from hong kong identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy no evidence of marseillevirus-like virus presence in blood donors and recipients of multiple blood transfusions the detection of acute hiv infection improving hiv-2 detection by a combination of serological and nucleic acid amplification test assays including source plasma, to reduce the risk of transmission of hepatitis b virus prevalence and incidence of hiv and hepatitis b among blood donors and estimated residual risk of transmission of hiv and hbv virus by blood transfusion. a study at the provincial general referee hospital bukavu, democratic republic of the congo risk factors for human immunodeficiency virus infection among brazilian blood donors: a multicentre case-control study using audio computer-assisted structured interviews the diversity of chronic hepatitis b virus infections within blood donors in england and north wales a method for estimating the residual risk of transfusion-transmitted hbv infection associated with occult hepatitis b virus infection in a donor population without universal anti-hbc screening hiv, hcv, hbv and syphilis surveillance among blood donors in evolution of residual risk for hiv, hcv and hbv hepatitis e virus in scottish blood donors occurrence of hepatitis e virus rna in plasma donations from sweden, germany and the united states world federation of hemophilia (wfh) annual global surveys we wish to thank professor hermann eichler for his appraisal of this manuscript and helpful suggestions. medical writing assistance was provided by hanna mourad-agha of inscience communications, springer healthcare. this assistance was funded by pfizer. a.t. has received grants and personal fees from bayer, grants and personal fees from baxter, grants and personal fees from biotest, grants and personal fees from csl behring, grants and personal fees from novo nordisk, grants and personal fees from pfizer, and grants and personal fees from octapharma, during the conduct of the study.c.f.p. has received grants as bureau speaker, consultant, or advisor, from gilead, merck sharp and dohme, roche, pfizer, abbott, bristol-myers squibb, viiv, and boehringer-ingelheim. none of these personal activities is in conflict with the opinions he expressed in this manuscript. d.n. has received honoraria for conferences from pfizer, roche pharma, roche diagnostics, abbott, msd, and astellas.g.d.m. has disclosed the following financial relationshipsspeaker or a member of a speaker bureau for: boehringer-ingelheim, sanofi-aventis, bayer, novo nordisk, pfizer, biotest, and grifols. consultant or ad hoc speaker/consultant for: boehringer-ingelheim, eli-lilly, sanofi-aventis, bayer, csl behring, novo nordisk, pfizer, biotest, and grifols.j.w.i. has received personal fees from piramal and grants from the department of health, uk, outside the submitted work.l.g. reports no potential conflicts of interest. m.c. has received research grants, lecture fees, and honoraria for consultancy from baxter, bayer, and pfizer. funding for medical writing assistance and two author meetings were provided by pfizer. pfizer had no editorial influence regarding the contents of this manuscript and did not comment on the outline or any drafts prior to journal submission. supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.blre.2015.07.004. key: cord-334133-61om170g authors: hollier, mark j.; dimmock, nigel j. title: the c-terminal tail of the gp41 transmembrane envelope glycoprotein of hiv-1 clades a, b, c, and d may exist in two conformations: an analysis of sequence, structure, and function date: 2005-07-05 journal: virology doi: 10.1016/j.virol.2005.04.015 sha: doc_id: 334133 cord_uid: 61om170g in addition to the major ectodomain, the gp41 transmembrane glycoprotein of hiv-1 is now known to have a minor ectodomain that is part of the long c-terminal tail. both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. however, data have so far been biologically based, and derived solely from t cell line-adapted (tcla), b clade viruses. here we have carried out sequence and theoretically based structural analyses of 357 gp41 c-terminal sequences of mainly primary isolates of hiv-1 clades a, b, c, and d. data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, β-sheet, membrane-spanning domains (msds). this means that the first (n-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. however, we suggest that only a minority of cell-associated gp41 molecules – those destined for incorporation into virions – has 3 msds and the minor ectodomain. most intracellular gp41 has the conventional single msd, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. the gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize hiv-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell. viruses of the lentivirus genus of the retroviridae have a single virus-encoded, envelope glycoprotein (env). this is a type i membrane protein with the n-terminus on the outside of the cell. env mrna is synthesized in the nucleus, and exported with the help of the viral rev protein to the cytoplasm where it is translated as a gp160 precursor. gp160 is then translocated through the rough endoplasmic reticulum (er) (dettenhofer and yu, 2001) , and folded with the assistance of the chaperone proteins, calreticulin, and calnexin (otteken et al., 1996) . glycans are added in the golgi, and subsequently trimmed (dash et al., 1994; fenouillet and jones, 1995) . gp160 oligomerizes in the er, forming a non-covalently linked trimer (earl et al., 1991; willey et al., 1988) . most gp160 is degraded, but about 5-15% is cleaved into gp120 (distal) and gp41 (membrane anchor) components. these remain associated non-covalently and are targeted to the cell surface (see below). gp120-gp41 trimers are incorporated into progeny virions. gp120 can be shed from the surface of cells and virions (willey et al., 1988 (willey et al., , 1991 . enzymatic cleavage of gp160 to gp120-gp41 is essential for env function. uncleaved gp160 cannot induce syncytium formation, and virions with uncleaved gp160 are not infectious. gp160 is rarely incorporated into virions, probably because so little reaches the cell surface (duensing et al., 1995; pal et al., 1991; pfeiffer et al., 1997; willey et al., 1988) . hiv-1 gp160 is cleaved between residues 518 r and 519 a by the subtilisin/kexin-like ca 2+ -dependent convertases such as furin, pace4, pc5/6-b, and pc1 (morikawa et al., 1993; moulard et al., 1994; vollenweider et al., 1996) . gp160 is probably cleaved by more than one cellular protease. gp160 cleavage occurs in the trans-golgi network (tgn) or after it exits the tgn (bultmann et al., 2000; pal et al., 1991; pfeiffer et al., 1997; stein and engelman, 1990) . brefeldin a, a1fn, monensin, and tunicamycin inhibit gp160 cleavage even when gp160 is allowed to accumulate in the tgn, suggesting that cleavage occurs in the late compartment of the golgi or after gp160 has exited from the golgi (dewar et al., 1989; kantanen et al., 1995; pal et al., 1991 pal et al., , 1988 . cleavage may occur in an acidic compartment, as it was inhibited by nh 4 cl and partially inhibited by chloroquine (courageot et al., 1999; willey et al., 1988) . however, these inhibitors may prevent gp160 from reaching the correct intracellular site for cleavage. methionine methyl ester failed to inhibit gp160 cleavage, indicating that cleavage may occur in a non-lysosomal compartment (willey et al., 1988) . only a small proportion of gp160 (5-40%) is cleaved into gp120-gp41, and this depends on the host cell (bird et al., 1990; hallenberger et al., 1993; jose et al., 1997; kimura et al., 1996; kozarsky et al., 1989; moulard et al., 1999; pfeiffer et al., 1997; willey et al., 1988 willey et al., , 1991 . for example, 10 -20% of gp160 is cleaved in peripheral blood lymphocytes (willey et al., 1988) . uncleaved, trimeric gp160 is degraded in the er and lysosomes. however, the proportion degraded in each location is not known. according to willey et al., most gp160 leaves the er and reaches the golgi, and the minority that remains in the er is degraded there (willey et al., 1988) . however, others find that most gp160 remains in the er and is degraded (bultmann et al., 2000; courageot et al., 1999; hallenberger et al., 1993; jabbar and nayak, 1990; pfeiffer et al., 1997) , or is rapidly sent to lysosomes and degraded (jabbar and nayak, 1990; pfeiffer et al., 1997) . whichever the location, degradation is relatively rapid, and only 10 -20% of gp160 remains after 8 h (willey et al., 1988) . when prevented from leaving the er, gp160 accumulates there and is degraded (courageot et al., 1999; pal et al., 1991; willey et al., 1991) . gp160 degradation has also been observed in proteosomes (bultmann et al., 2000) . the consensus view is that most gp160 is degraded in the er, and only a minority reaches the golgi. of the latter, most (85 -95%) is degraded in lysosomes, and the remainder is targeted as gp120-gp41 to the cell surface (willey et al., 1988) . gp120 is relatively stable, and 30 -50% can be detected as secreted or intracellular protein 24 h later (bird et al., 1990; willey et al., 1988 willey et al., , 1991 . the amount of gp160 secreted from the cell is less than 10% of the gp120 secreted (willey et al., 1988) , although the amount varies with the type of host cell (moulard et al., 1999) . tyrosine-dependent sorting signals (yxxb, where x represents any amino acid residue and b represents a bulky hydrophobic residue), and possibly di-leucine-sorting signals are found in the c-terminal tail of gp41, and have been implicated in transport of gp160 and gp120-gp41 (see results and discussion section and berlioz-torrent et al., 1999; boge et al., 1998; bu et al., 2004; deschambeault et al., 1999; egan et al., 1996; lodge et al., 1997; ohno et al., 1997; owens et al., 1991; rowell et al., 1995; west et al., 2002; wyss et al., 2001) . gp120 is the distal part of the envelope glycoprotein trimer, and recognizes the primary cd4 receptor, and coreceptors on the target cell. gp41 anchors the envelope glycoprotein in the cell or virion membrane, and mediates the virion fusion entry process that is activated by receptor binding. the n-terminal peptide of gp41 is inserted into the membrane of the target cell and leads to fusion of the virion and cell membranes, entry of the virus genome and associated proteins into the cell, and infection. gp41 comprises an ectodomain, a single membrane-spanning domain (msd), and a long c-terminal tail that in the past has been viewed as being entirely contained inside the cell or virion (gallaher, 1987; gallaher et al., 1989; gonzalez-scarano et al., 1987; levy, 1998; white, 1990) . however, there is abundant evidence that hiv-1 virions can be neutralized by antibodies directed to an epitope in the cterminal tail (buratti et al., 1998; chanh et al., 1986; cleveland et al., 2000a cleveland et al., ,b, 2003 dalgleish et al., 1988; durrani et al., 1998; ho et al., 1987; mcinerney et al., 1999; mclain et al., 1995 mclain et al., , 1996 mclain et al., , 2001 newton et al., 1995; reading et al., 2003) . since antibodies do not cross lipid bilayers, this means that part of the tail is exposed on the virion surface. we have called this exposed region of the gp41 tail the minor ectodomain, to distinguish it from the better known, and larger, major ectodomain. non-neutralizing antibodies specific for the minor ectodomain have also been shown to bind virions (cleveland et al., 2003; mclain et al., 2001) , providing further evidence of the externalization of the minor ectodomain. binding of antibody is abrogated by pre-treatment of virions with protease, again supporting the external location of this part of the tail (cleveland et al., 2003) . more recently, we demonstrated for the first time that neutralizing and non-neutralizing antibodies to the minor ectodomain bind to infected cells and that the neutralizing antibodies inhibit fusion of infected and non-infected cells (cheung et al., 2005; heap et al., 2005) . thus, part of the gp41 c-terminal tail is exposed on the surface of both infected cells and virions. until now, evidence for the exposure of part of the gp41 c-terminal tail has been based on the study of hiv-1 b clade, t cell line-adapted (tcla) viruses, and the generality of the existence of the minor ectodomain, and its structural basis, have not been explored. to remedy this, we have here analyzed 357 gp41 c-terminal tail database sequences from clades a, b, c, and d. this analysis shows that all these could potentially have a minor ectodomain of approximately 40 residues, supported by three msds, and an internal tail of approximately 100 residues. we shall further suggest that this 3-msd form of gp41 coexists with the accepted 1-msd form of gp41, and that the 3-msd form represents a minor population of intracellular gp41 that is destined for incorporation into virions. in contrast, 1-msd gp41 is the majority form of intracellular gp41, most of which is degraded. regions of conservation in the gp41 sequence 690 -793 of hiv-1 clades a to d are summarized in fig. 1 , with residues numbered according to ratner et al. (1985) . the only very highly conserved region throughout all clades is 713 nrvrqgysplsfq 725 , which contains the most n-terminal (first) tyrosine-dependent sorting signal ( 719 yspl 722 ). as expected, the region comprising the accepted msd, 690 kifimivggliglrivfavlsiv 712 , is highly conserved and forms part of a larger highly conserved sequence 690 kifimivggliglrivfavlsivnrvrq-gysplsfq 725 , which includes the region surrounding the first tyrosine-dependent sorting signal. however, there are conservative substitutions in individual clade consensus sequences of 700 i-v (clade b), 705 v-i (clade c), 711 i-l (clade d), and 712 v-i (clade a). the region 767 rslclfsyhrlr 779 containing the second potential tyrosine-dependent sorting signal ( 775 yhrl 778 ) is highly conserved. the highly conserved 790 ellg 793 contains a potential di-leucine signal, but the only di-leucine signal known to be functional in gp41 is 862 ll 863 (wyss et al., 2001) . the kennedy sequence ( 731 prgpdrpgrieeeg-geqdrdrs 752 ) in clade b viruses contains the neutralizing epitope core sequence 746 erdrd 750 , and two non-neutralizing epitopes ( 734 pdrpeg 739 and 740 ieee 743 ) (chanh et al., 1986; dalgleish et al., 1988; evans et al., 1989; ho et al., 1987; kennedy et al., 1986; niedrig et al., 1992; vella et al., 1993) . overall the kennedy sequence is only poorly to moderately conserved. specifically 731 prgpdrpgri 740 and 747 qdrdrs 752 are poorly to moderately conserved, but 741 eeegge 746 is moderately to highly conserved. 741 eeegge 746 is notable in having 67% acidic residues. 746 erdrd 750 is present only in clade b and the recombinant crf03 _ ab clade. clades a and c have 746 eqdrd 750 and clade d has 746 eqgrg 750 . the exchange of 747 r (basic, ionizable, hydrophilic, with a long side chain) for q (polar, hydrophilic, amidic, with a shorter side chain) might create a different epitope, and 746 erdrd 750 -specific neutralizing antibody may not recognize 746 eqdrd 750 . the gp41 tail reading frame overlaps the second exons of tat (+1 relative to env) and rev (+2). both of the latter start fig. 1 . summary of the conservation of gp41 c-terminal amino acid residues 690 -793 of hiv-1 clades a to d, using a 7-residue moving window. at the same position, equivalent to the codon for residue 725 of env. tat and rev stop at the equivalent of the codons for residues 740 and 816 of env, respectively. this is the only part of the env sequence where all three orfs are used, yet counter-intuitively, conservation of 725 -740 of env ranges from poor to high. it may be that the tat and/or rev sequences are conserved at the expense of env. all clades have a very highly conserved 745 g (100%), 758 g (99.9%), and 764 w (99.4%). the conservation of 745 g and 758 g may be a consequence of the reading frame shared with rev. the last two bases of a rev codon are the first two bases of the env codon. thus, if rev requires a tryptophan in its second exon, the overlapping codon of env has to be glycine, since tryptophan is only encoded by ugg, while the codon for glycine is ggx. the reason why 764 w is very highly conserved is not clear, but any change in its codon results in a different amino acid residue or a stop codon (uga or uag). regions of conservation for clades a to d consensus sequences of gp41 residues 690 -793, and an overall consensus sequence are summarized in fig. 2 . the consensus sequences of hiv-1 clades a to d derived above were analyzed individually (see materials and methods), but as the predicted structures were virtually identical, only clade a data are presented. according to kyte and doolittle (1982) , four regions (690 -699, 702-711, 758 -763, and 783-787) have hydropathy values of >1.6, indicating that they are potential msds (fig. 3a ). there are two single point peaks of 1.6 at residues 771 and 781, but the values of surrounding residues are too low for these to form an msd. the kennedy region (731 -752) is highly hydrophilic, with values of mainly à1, suggesting that it is exposed to solvent. essentially the same conclusions were reached (data not shown) using other hydropathy prediction algorithms (eisenberg et al., 1984; hopp and woods, 1981; sweet and eisenberg, 1983) . five regions (690 -694, 704 -711, 740 -747, 758 -766, and 779 -793) have a-helix potentials of >1.03 (fig. 3b) , and four regions (690 -719, 753-761, 770 -777, and 779-788) have h-sheet values of >1.05 (fig. 3c ). the most likely conformation is that with the highest predicted value (chou and fasman, 1978) . thus, 690 -719 (1.34) is likely to be hsheet. in this region there is a dip to 1.05 at residue 702, suggesting that 690-719 may comprise two discrete regions (690 -701 and 703-719). this is consistent with the two hydropathic regions predicted above. there are two regions with different conformations adjacent to each other at 753-766, with 753 -761 showing higher h-sheet potential and 761 -766 showing greater a-helix potential. 779-788 could fig. 2 . consensus sequences for gp41 c-terminal amino acid residues 690 -793 of hiv-1 clades a to d combined (top line), and for clades a to d individually. the potential tyrosine-dependent sorting signals are in red. a potential di-leucine signal is pink. residues common to the majority of sequences are in black, while residues that vary are in green. where two residues are equally represented between the consensus sequences of the four clades, the overall consensus sequence is based on the larger number of sequences analyzed. the proposed first, second, and third msds are overlined. underlined is the antigenically active kennedy sequence. be either a-helix or h-sheet. the shows no potential for h-sheet formation with values <0.95. however, 740 -747 of the kennedy region that in clade b tcla viruses contains the highly immunogenic and anti-genic epitope 740 ieee 743 (cleveland et al., 2000a ) may form an a-helix (>1.03). there are potential h-turns at 702, 721, 731, 733, 748, 768, and 778 (fig. 3d) . the h-turn at 702 is consistent with the hydropathy and h-sheet predictions that this links two discrete msds. residue 721 p is present in most of the first tyrosine-dependent sorting signals ( 719 yspl 722 ) and is discussed later. the kennedy region contains potential hturns at 731, 733, and 748, and with the prediction of only one short region of a-helix and no h-sheet, the region is probably unstructured. the potential h-turn at 749 r might be important for maintaining the complex conformational neutralizing epitope 746 erdrd 750 found in clade b virions (buratti et al., 1998; cheung et al., 2005; cleveland et al., 2000a cleveland et al., ,b, 2003 heap et al., 2005; mclain et al., 2001; reading et al., 2003; vella et al., 1993) . h-turns at 768 and 778 would occur after the putative third msd (see below). there are four highly non-polar regions (694 -699, 708 -711, 720-725, and 759-762) (fig. 3e) . regions 694 -699 and 708 -711 coincide with the msd 1 and 2 predicted above. the unequal polarity of these two domains adds to the suggestion that they are separate entities. residues 720 -725 contain the first tyrosine-dependent sorting signal, but the significance of its lack of polarity is not clear. residues 759 -762 coincide with the possible location of the third msd predicted above (754 -763). region 780 -793 is moderately polar and unlikely to form an msd. the kennedy region is the most polar region in the analyzed sequence, consistent with it being exposed to aqueous solvent and with its antigenic properties. regions 694 -712 (containing the predicted msd 1 and 2 at 691-700 and 703 -712) and 755 -763 are likely to be inaccessible to aqueous solvent (fig. 3f) . the dip at the center of 694 -712 (residue 703) supports the prediction that this region comprises two msds. region 755-763 coincides with the predicted third msd (754 -763). the kennedy region is accessible to solvent. taken together, these data suggest that the gp41 of hiv-1 clades a, b, c, and d all have the potential to have three msds in a h-sheet conformation. msd 1 and 2 are connected by a short h-turn ( 701 gl 702 ), and msd 2 and 3 support the highly antigenic kennedy sequence on the outside of the cell or virion. further the msds all have significant parallel and anti-parallel h-strand potential (lifson and sander, 1979 ; data not shown). the 1-and 3-msd forms of gp41 are shown schematically in figs. 4a and b. the significance of the new positioning of the potential tyrosine-sorting signals is discussed below. while msds are typically a-helical and approximately 20 residues in length (sabatini et al., 1982; singer, 1990) , h-sheets as short as 7 residues form msds as part of transmembrane h-barrel proteins of bacteria, choroplasts, and mitochondria (schultz, 2003) . short h-turns connect sequential msds and do not normally occur within msds (jahnig, 1990) , but the h-turn may intrude into the membrane, as suggested for herpes simplex virus glycoprotein b (pellett et al., 1985) . an arginine residue is unlikely to occur in the middle of an msd (singer, 1990) , and r703 of the 3-msd form of gp41 is predicted to be on the membrane surface, where the polar head groups of membrane lipids can neutralize its positive charge. however, we shall suggest below that the 3-msd gp41 is a minority form that is selectively incorporated into the plasma membrane and virions, and that most intracellular gp41 exists in the 1-msd conformation. finally preliminary sequence and structural analysis suggests that other primate lentiviruses (hiv-2, siv) have the potential to form a 3-msd structure (unpublished data). gp41 molecules of hiv-1 clades a to d all have the potential to form three short msds, which most likely have a h-sheet conformation. the position of the third msd places the first potential tyrosine-dependent sorting signal outside the membrane, where it is non-functional. all viruses have a hydrophilic, unstructured region of 41 residues supported by msds 2 and 3 that probably equates to the antigenically and biologically active minor ectodomain of hiv-1 clade b, tcla viruses. the definition and values of the peaks obtained in the above analysis make the data highly significant. the position and number of residues in the msds and other regions of interest are summarized in table 1 and 1999; egan et al., 1996; ohno et al., 1997; rowell et al., 1995; west et al., 2002) and basolateral sorting (deschambeault et al., 1999; lodge et al., 1997; owens et al., 1991) . the signal sequence and upstream residues are highly to very highly conserved in clades a to d: 716 r = 100%, 717 q = 96.4%, 718 g = 99.95%, 719 y = 99.7%, 720 s = 99.5%, 721 p = 99.8%, 722 l = 96.9%. the glycine immediately before a tyrosine signal signifies that it can function in the tgn to target the protein to lysosomes. also for signal functionality there is a strict requirement that the tyrosine residue is the 7th -11th residue from the membrane (rohrer et al., 1996) . gp41 is also involved in basolateral localization of envelope protein in the plasma membrane of polarized cells (owens et al., 1991) , a property that is lost when the tyrosine of the first signal is substituted (deschambeault et al., 1999; lodge et al., 1997) . in the 1-msd model of gp41, gallaher et al. (1989) have proposed that the msd ends at residue 712 v, and thus 719 y will be the 7th residue from the membrane, and . only a monomer is represented. there may be interactions between msd 1, 2, and 3, between the minor ectodomain and the gp41 major ectodomain, between the minor ectodomain and elements of gp120, or with the other gp41 monomers that form the trimer (arrows). the nine msds of the trimer could also interact with each other. the antigenically active kennedy sequence (731 -752) containing neutralizing and non-neutralizing epitopes is shown as the outer face of the minor ectodomain. table 1 predicted location of residues in the 3-msd conformation of the gp41 of hiv-1 clades a to d in the virion or cell membrane within the required distance for optimum function. much depends on knowing precisely which residue ends the msd. often a charged residue that acts as a stopper defines the end, and it is possible that the gp41 msd could end at 714 r. if so, 719 y would be 5 residues from the membrane, and this might compromise the endocytosis, basolateral sorting, and lysosomal targeting functions of the signal. the minimum distance of the tyrosine-signal from the membrane is required for both direct lysosomal targeting and endocytosis (collawn et al., 1990; pytowski et al., 1995; trowbridge and collawn, 1992; trowbridge et al., 1993) . however, only lysosomal targeting signals have a strict maximum distance from the membrane (collawn, 1990; rohrer et al., 1996; trowbridge and collawn, 1992; trowbridge et al., 1993) . the fact that the first tyrosine signal functions in endocytosis (as referenced above) argues that it is indeed at the required distance from the membrane, but at the time of writing there are no experimental data to show if the signal is active in directing gp41 to lysosomes. the 3-msd model, as stated above, puts the first tyrosine signal outside the membrane where it is non-functional for any transport function (fig. 5) . adaptor protein (ap) complexes interact with tyrosinesorting signals. ap-1 and ap-3 complexes are mainly found in the tgn and function in lysosomal targeting, while ap-2 is predominantly localized to the plasma membrane and functions in endocytosis. they have distinct preferences for specific residues or combinations of residues of the tyrosine signals, although there is overlap, particularly with ap-1 and ap-3 complexes (table 2) (boll et al., 1996; ohno et al., 1996 ohno et al., , 1998 . table 2 shows that the consensus sequence of the first tyrosine-sorting signal of the gp41 tail of clades a to d most closely matches the preferences of ap-1 and ap-3 complexes. the arginine residue at position y à 3 of the signal is 100% conserved, and there is almost complete conservation of the glycine at y à 1 (99.95%), the tyrosine itself (99.7%), and the proline at y + 2 (99.8%). the leucine residue at y + 3 is 96.9% conserved, but if leucine, isoleucine, and valine at y + 3 (all with similar properties and tolerated at this position of the tyrosine signal) are summed, conservation reaches 98.4%. thus, the first tyrosine-sorting signal ( 719 yspl 722 ) could interact with the ap-1 and ap-3 complexes in the tgn and target gp41 to the lysosomes. however, as stated above, this signal functions at the plasma membrane (berlioz-torrent et al., 1999; boge et al., 1998; deschambeault et al., 1999; egan et al., 1996; ohno et al., 1997; rowell et al., 1995; west et al., 2002) , and is not known to be active in the tgn. it may be that the amount of gp41 synthesized saturates the lysosomal targeting system in the tgn, allowing gp41 to reach the cell surface and the first tyrosine-sorting signal to function as an endocytosis signal. the possibility that the signal targets gp41 to the lysosomes is consistent with the observation that the majority of gp160 that reaches the golgi is degraded in the lysosomes (willey et al., 1988) . ap-2 complexes have the broadest specificity range and associate with the same signals as ap-1 and ap-3 complexes (ohno et al., 1998) . the endocytosis function of the first tyrosine-sorting signal may enable plasma membrane gp41 to be re-directed to the lysosomes if it escapes that route initially (ohno et al., 1998) . the second tyrosine-dependent sorting signal ( 775 yhrl 778 ) has no preceding glycine, and is non-functional in the context of the 1-msd model of gp41 (boge et al., 1998; rowell et al., 1995) . it is situated 63 and 12 residues from the membrane in the 1-msd and 3-msd models, respectively, the latter being close to the optimal 7-11 residue distance. the residues of the second signal are variably conserved: 772 l = 94.5%, 773 f = 96.8%, 774 s = 86.0%, 775 y = 99.7%, 776 h = 89.3%, 777 r = 86.5%, and 778 l = 99.8%. the f at position y à 2, r at y + 2, and l at y + 3 suggest that the signal interacts with the ap-2 complex ( table 2 ). the lack of an r at y à 3, glycine at y à 1, and proline at y + 2, and the fact that it is not within the favored distance from the membrane (rohrer et al., 1996) , indicate that this signal is not optimal for interacting with ap-1 or ap-3 complexes. ap-3 complexes disfavor serine at position y à 1 and makes this interaction less likely (ohno et al., , 1998 . as yet there is no evidence that this signal is functional in gp41 (boge et al., 1998; rowell et al., 1995) . a peptide containing the signal interacted table 2 comparison of the preferences of ap complexes for cellular tyrosine-dependent sorting signals a with the consensus sequence for the first and second potential tyrosine-dependent sorting signals of the gp41 of hiv-1 clades a to d position (relative to y) ap-1 preferences ap-2 preferences ap-3 preferences hiv-1 tyrosine-dependent sorting signal sequences first second np, no preference known at this position; x, any amino acid residue; b, a bulky hydrophobic residue. a boll et al. (1996) , heilker et al. (1999 ), hö ning et al. (1996 , ohno et al. (1995 ohno et al. ( , 1996 ohno et al. ( , 1998 , ooi et al. (1997) , owen and evans (1998) , simpson et al. (1997) , stephens and banting (1998) , stepp et al. (1997) . strongly with the medium subunit of ap-2 (boge et al., 1998; ohno et al., 1997) , but the presence of a major upstream sequence in the 1-msd model could alter its environment and its possible interaction with the ap-2 complex. all hiv-1 gp41 tail sequences have a potential n-terminal gyxxf-sorting signal of the type that would be expected to interact strongly with ap-1 and ap-3 complexes. thus, with the critical 7-residue spacing from the membrane, the signal is likely to be functionally important in targeting tgn gp41 to lysosomes. if the signal was required only for endocytosis, it is unlikely that the g at position y à 1, r at y à 3, and the 7-residue spacing from the membrane would be so highly conserved. however, this would not preclude it from functioning as an endocytosis signal at the cell surface. the second yxxf sequence is not so well conserved, is 63 residues from the membrane in the 1-msd model, and is unlikely to interact with ap-1 and ap-3 complexes, or to be involved in lysosomal targeting from the tgn. however, the y is almost completely conserved and the bulky hydrophobic residue is highly conserved. in the 3-msd structure of gp41, only the second signal is on the cytoplasmic side of the membrane, where it could be functional. in the infected cell there may be populations of gp41 with one msd and three msds until recently all primate lentivirus envelope protein anchor components were viewed as having a single msd (691 -712 in hiv-1; fig. 4a ) (gallaher, 1987 (gallaher, , 1989 gonzalez-scarano et al., 1987; levy, 1998; white, 1990) . however, published work and the discussion above suggest that the gp41 tail crosses the membranes of hiv-1 virions and infected cells three times (cheung et al., 2005; cleveland et al., 2003; mclain et al., 2001) . to resolve this apparent conflict, we shall argue here that the 1-and 3-msd forms of gp41 coexist in the infected cell, with the 1-msd version being the major form. however, we shall propose that only the minor 3-msd form is incorporated into virions. these proposals are consistent with, and rationalize the observed degradation of the majority (85 -95%) of the1-msd form of cellular gp160 (bultmann et al., 2000; courageot et al., 1999; jabbar and nayak, 1990; pfeiffer et al., 1997; willey et al., 1988) , the apparently contradictory evidence of a functioning first tyrosine-sorting signal (berlioz-torrent et al., 1999; boge et al., 1998; deschambeault et al., 1999; egan et al., 1996; ohno et al., 1997; rowell et al., 1995; west et al., 2002) and basolateral-sorting signal (deschambeault et al., 1999; lodge et al., 1997; owens et al., 1991) , with the immunogenic and antigenic properties of the kennedy region (chanh et al., 1986; dalgleish et al., 1988; evans et al., 1989; ho et al., 1987; kennedy et al., 1986; niedrig et al., 1992) . the c-terminal tail of the 1-msd form of hiv-1 gp41 starts at residue 713 (fig. 4a) . as already stated, the majority of this type of gp41 (85 -95%) is degraded intracellularly. the y residue of the first tyrosine-dependent sorting signal ( 718 gyspl 722 ) is situated precisely 7 residues from the membrane and conforms closely to the requirements for cellular gyxxf lysosomal targeting signals (see above). in the 1-msd form of gp41, the second tyrosinedependent sorting signal is apparently not needed, and we suggest may not be functional. the 3-msd form of hiv-1 gp41 has msds at 691-700, 703-712, and 754 -763, and its 718 gyspl 722 sequence is outside the virion. this model is supported by antigenic and other data showing that residues of the c-terminal tail are exposed on the surface of the virion (buratti et al., 1998; chanh et al., 1986; cleveland et al., 2000a cleveland et al., ,b,2003 dalgleish et al., 1988; durrani et al., 1998; ho et al., 1987; mcinerney et al., 1999; mclain et al., 1995 mclain et al., , 1996 mclain et al., , 2001 newton et al., 1995; reading et al., 2003) and the infected cell (cheung et al., 2005; heap et al., 2005) . furthermore, we propose that this form of gp41 represents the 5 -15% of the tgn gp160 that is directed to the cell surface. its gyxxf signal is outside the cell membrane and cannot function in lysosomal targeting or endocytosis, but the second potential tyrosine-dependent sorting signal ( 775 yhrl 778 ), which has none of the requirements for lysosomal targeting (see above), is 12 residues from the membrane, and well situated to function in endocytosis and recycling of cell membrane-inserted gp41. because of its location this gp41, in association with gp120, is the major gp41 form in the cell membrane, and the major gp41form incorporated into virions. co-existence of two forms of gp41 raises questions, which will require further work to answer. the mechanism by which two forms of gp41 arise is not known, although translocational pausing may be involved in formation of the multiple msds (dettenhofer and yu, 2001) . the 1-and 3-msd forms are proposed to be a-helix and h-sheet, respectively, and it is noted above that structure predications allow for either conformation. conformation may be determined by the length of unbroken msd sequence, as a long sequence of lower value a-helix, can take precedence over a higher value, shorter h-sheet region (chou and fasman, 1978) . the conventional 1-msd model still has the problem of 703 r being centrally located in the membrane with no counter charge. however, a recent suggestion that the 703 r equivalent in siv is situated in a position where it can react with the polar lipid head groups may provide a solution (west et al., 2001) . degradation of the 1-msd gp41 and recycling of the 3-msd form would both act to reduce the surface expression of gp120-gp41. this may be important, as high intracellular concentrations of gp41 can be cytotoxic (arroyo et al., 1995; chernomordik et al., 1994; comardelle et al., 1997; gawrisch et al., 1993; miller et al., 1993; zhang et al., 1996) , and provoke immune responses against the infected cell. it may be that such post-translational control measures, utilizing tyrosine-dependent sorting signals and the host cell's degradation pathways, have evolved as envelope expression is not easily controlled at a genetic level due to the overlap of the env, tat, and rev orfs. the suggestion above that a membrane-inserted viral protein can have different numbers of msds is not unique. for example, the gl envelope protein of equine arteritis virus is proposed to have 1 or 3 msds (snijder and meulenberg, 1998) , the m protein of transmissible gastroenteritis coronavirus and equine arteritis virus, and the s antigen of hepatitis b virus are proposed to have three or four msds (prange and streeck, 1995; risco et al., 1995; snijder and meulenberg, 1998) , and the herpes simplex virus glycoprotein b (pellett et al., 1985) , and the epstein -barr virus 58 kda latent protein hennessy et al., 1984) both have multiple msds. based on an analysis of their sequence and structure, we propose that the gp41 transmembrane region and c-terminal tail of all hiv-1 clades a to d can exist in two conformations, with either 1 msd (the conventional structure) or with 3 msds. we suggest that these are, respectively, the majority and minority forms of intracellular env. in the 3-msd form, msd 1 and msd 2 are separated by a highly conserved beta turn, while the msd 2 and msd 3 support an unstructured hydrophilic loop/minor ectodomain of 41 residues that in clade b strains is highly antibody-reactive and involved in fusion. all viruses have two potential tyrosine-dependent sorting signals within the region analyzed. in the 1-msd model it is likely that only the n-terminal signal is functional, and that this interacts with ap-1 and ap-3 to direct env from the tgn to degradation in the lysosomes. in the 3-msd version, the nterminal signal is situated outside the membrane and nonfunctional, thus allowing this form of gp41 to reach the cell membrane. thus, it seems that the 3-msd form is the majority species on the cell surface and hence in virions. we propose that the second signal is functional in the 3-msd gp41, and controls recycling of cell surface env. the 1-and 3-msd strategy can be seen as an evolutionary adaptation that allows hiv-1-infected cells to evade the immune system or to avoid gp41-induced cytotoxicity. hiv-1 gp41 sequences from infectious viruses, molecular clones, and pcr products from blood samples from infected individuals were obtained from http://hiv-web.lanl.gov/. we analyzed residues 690-793 of hiv-1 (numbering according to ratner et al., 1985) of the following number of sequences: clade a, n = 25; clade b, n = 245; clade c, n = 61; clade d, n = 26. the sequence analyzed comprises the msd and approximately two thirds of the c-terminal tail. conserved regions of sequence were aligned, with spaces as necessary to maintain alignment. the residue occupying each position was recorded as a percentage, and consensus sequences constructed for each clade. sequence conservation is defined here as poor (<80%), moderate (80 -89.9%), high (90 -96.4%), and very high (96.5 -100%). for structure predications we used the consensus sequences derived in this report for the gp41 msd and cterminal region of hiv-1 clades a to d. hydropathy values assigned to the amino acids are based on water vapor transfer free energies and the interior -exterior distribution of amino acid side chains (kyte and doolittle, 1982) . this system predicts that a region with a value >1.6 is likely to be an msd, and that a region with a value >1.09 is likely to be sequestered inside the protein. a-helices and h-sheets were predicted according to chou and fasman (1978) , where p a is the helix conformational parameter and p h is the h-sheet conformational parameter. any segment of !6 residues with ( p a ) !1.03 and ( p a ) > ( p h ) is predicted to be aàhelical, and any segment of three residues or longer in a native protein with ( p h ) !1.05 and ( p h ) > ( p a ) is predicted to be h-sheet. a segment containing overlapping aand hresidues is resolved through conformational boundary analysis so that ( p a ) > ( p h ) is a-helical, and ( p h ) > ( p a ) is h-sheet. a h-turn occurs where a polypeptide folds back on itself by nearly 180-and typically requires four consecutive residues (chou and fasman, 1978) . however, a h-turn of two residues occurs between two msds of herpes simplex virus glycoprotein b (pellett et al., 1985) . the lower cut-off value for h-turns of 0.75 was used here (chou and fasman, 1978) . polarity was determined according to zimmerman et al. (1968) . accessibility of a region of a protein to solvent was predicated using the percentage buried residues index (janin, 1979) . low values indicate a region that is likely to be accessible to solvent and hence surface exposed, and high values indicate regions not accessible to the solvent. predications used a moving window of 7 residues. membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adapter complexes modulate intracellular and cell surface expression of envelope glycoprotein expression of human immunodeficiency virus type 1 (hiv-1) envelope gene products transcribed from a heterologous promoter a membraneproximal signal mediates internalization of the hiv-1 envelope glycoprotein via interaction with the ap-2 clathrin adaptor sequence requirements for the recognition of tyrosine-based endocytic signals by clathrin ap-2 complexes enhancement of immunogenicity of an hiv env dna vaccine by mutation of the tyr-based endocytosis motif in the cytoplasmic tail ubiquitination of the human immunodeficiency virus type 1 env protein the neutralizing antibody response against a conserved region of hiv-1 gp41 (amino acid residues 731 -752) is uniquely directed against a conformational epitope induction of anti-hiv neutralizing antibodies by synthetic peptides an amphipathic peptide from the c-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes part of the c-terminal tail of the envelope gp41 transmembrane protein of human immunodeficiency virus type 1 (hiv-1) is exposed on the cell surface and is involved in virus-mediated cell-cell fusion prediction of the secondary structure of proteins from their amino acid sequence immunogenic and antigenic dominance of a non-neutralizing epitope over a highly conserved neutralizing epitope in the gp41 transmembrane envelope glycoprotein of hiv-1: its deletion leads to a strong neutralizing antibody response properties of a neutralizing antibody that recognises a conformational form of epitope erdrd in the c-terminal tail of human immunodeficiency virus type 1 a region of the c-terminal tail of the gp41 envelope glycoprotein of human immunodeficiency virus type 1 contains a neutralizing epitope: evidence for its exposure on the surface of the virion transferrin receptor internalization sequence yxrf implicates a tight turn as the structural recognition motif for endocytosis a synthetic peptide corresponding to the carboxyterminus of human immunodeficiency virus type 1 transmembrane protein induces alterations in the ionic permeability of xenopus laevis oocytes intracellular degradation of the hiv-1 envelope glycoprotein. evidence for, and some characteristics of, an endoplasmic reticulum pathway neutralization of diverse strains of hiv-1 by monoclonal antibodies raised against a gp41 synthetic peptide deletion of a single n-linked site from the transmembrane envelope protein of human immunodeficiency virus type 1 stops cleavage and transport of gp160 preventing env-mediated fusion polarized human immunodeficiency virus budded in lymphocytes involves a tyrosine-based signal and favors cellto-cell viral transmission characterization of the biosynthesis of human immunodeficiency virus type 1 env from infected t-cells and the effects of glucose trimming of env on virion infectivity biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport processing of the envelope protein gp160 in immunotoxin-resistant cell lines chronically infected with human immunodeficiency virus type 1 intranasal immunization with a plant virus expressing a peptide from hiv-1 gp41 stimulates better mucosal and systemic hiv-1-specific iga and igg than oral immunization folding, interaction with grp78-bip, assembly, and transport of the human immunodeficiency virus type 1 envelope protein human immunodeficiency virus type 1 envelope protein endocytosis mediated by a highly conserved intrinsic internalization signal in the cytoplasmic domain of gp41 is suppressed in the presence of the pr55gag precursor analysis of membrane and surface protein sequences with the hydrophobic moment plot an engineered poliovirus chimaera elicits broadly reactive hiv-1 neutralizing antibodies nucleotide sequence of an mrna transcribed in latent growth-transforming virus infection indicates that it may encode a membrane protein the glycosylation of human immunodeficiency virus type 1 transmembrane protein (gp41) is important for the efficient transport of the envelope precursor gp160 detection of fusion peptide sequence in the transmembrane peptide of human immunodeficiency virus a general model for the transmembrane proteins of hiv and other retroviruses interaction of peptide fragment 828 -848 of the envelope glycoprotein of human immunodeficiency virus type 1 with lipid bilayers sequence similarities between human immunodeficiency virus gp41 and paramyxovirus fusion proteins secretion of a truncated form of the human immunodeficiency virus type 1 envelope glycoprotein an antibody specific for the c-terminal tail of gp41 of hiv-1 mediates post-attachment neutralization probably through inhibiting virus-cell fusion recognition of sorting signals by clathrin adapters a membrane protein encoded by epstein -barr virus in latent growthtransforming infection human immunodeficiency virus virus-neutralizing antibodies recognise several conserved domains on the envelope glycoprotein the tyrosinebased lysosomal targeting signal in lamp-1 mediates sorting into golgiderived clathrin-coated vesicles prediction of protein antigenic determinants from amino acid sequences intracellular interaction of human immunodeficiency virus type (arv-2) envelope glycoprotein gp160 with cd4 blocks the movement and maturation of cd4 to the plasma membrane structure predictions of membrane proteins are not that bad surface and inside volumes in globular proteins megalomycin inhibits hiv-1 replication and interferes with gp160 processing endoproteolytic cleavage of hiv-1 gp160 envelope precursor occurs after exit from the trans-golgi network (tgn) antiserum to a synthetic peptide recognizes the htlv-iii envelope glycoprotein uncleaved env gp160 of human immunodeficiency virus type 1 is degraded within the golgi apparatus but not lysosomes in cos-1 cells glycosylation and processing of the human immunodeficiency virus type 1 envelope glycoprotein a simple method for displaying the hydropathic character of a protein hiv and the pathogenesis of aids antiparallel and parallel beta-strands differ in amino acid residue preferences the membraneproximal intracytoplasmic tyrosine residue of hiv-1 envelope glycoprotein is critical for basolateral targeting of viral budding in mdck cells analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying a hiv-1 peptide human immunodeficiency virus type 1 neutralizing antibodies raised to a gp41 peptide expressed on the surface of a plant virus stimulation of neutralizing antibodies to human immunodeficiency virus type 1 in three strains of mice immunized with a 22-mer amino acid peptide expressed on the surface of a plant virus different effects of a single amino acid substitution on three epitopes in the gp41 c-terminal loop of a neutralizing antibody escape mutant of human immunodeficiency virus type 1 alterations in cell membrane permeability by the lentiviral peptide (llp-1) of hiv-1 transmembrane protein legitimate and illegitimate cleavage of human immunodeficiency virus glycoproteins by furin kex2p: a model for cellular endopeptidase processing human immunodeficiency virus type 1 envelope glycoprotein precursor processing and routage of hiv glycoproteins by furin to the cell surface expression and immunogenicity of an 18-residue epitope of hiv-1 gp41 inserted in the flagellar protein of a salmonella live vaccine murine monoclonal antibodies directed against the transmembrane protein gp41 of human immunodeficiency virus type 1 enhance its infectivity interaction of tyrosine-based sorting signals with clathrin-associated proteins structural determinants of interaction of tyrosine-based sorting signals with the adaptor medium chains interaction of endocytic signals from the hiv-1 envelope glycoprotein complex with members of the adaptor medium chain family the medium subunits of adaptor complexes recognise distinct but overlapping sets of tyrosine-based sorting signals altered expression of a novel adaptin leads to a defective pigment granule biogenesis in the drosophila eye color mutant garnet folding, assembly, and intracellular trafficking of the human immunodeficiency virus type 1 envelope glycoprotein analyzed with monoclonal antibodies recognizing maturational intermediates a structural explanation for the recognition of tyrosine-based endocytotic signals human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells processing and secretion of envelope glycoprotein of human immunodeficiency virus type 1 in the presence of trimming glucosidase inhibitor deoxynojirimycin brefeldin a inhibits the processing and secretion of envelope glycoprotein of human immunodeficiency virus type 1 anatomy of the herpes simplex virus 1 strain f glycoprotein b gene: primary sequence and predicted protein structure of the wild type and of monoclonal antibody-resistant mutants transfer of endoplasmic reticulum and golgi retention signals to human immunodeficiency virus type 1 gp160 inhibits intracellular transport and proteolytic processing of viral glycoprotein but does not influence the cellular site of virus particle budding novel transmembrane topology of the hepatitis b envelope proteins an internalization motif is created in the first cytoplasmic domain of the transferrin receptor by substitution of a tyrosine at the first position of a predicted turn complete nucleotide sequence of the aids virus, htlv-iii a novel monoclonal antibody specific for the c-terminal tail of the gp41 envelope transmembrane protein of human immunodeficiency virus type 1 that preferentially neutralizes virus after it has attached to the target cell and inhibits the production of infectious progeny membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion the targeting signal of lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane endocytosis of endogenously synthesized hiv-1 envelope protein: mechanism and role in processing for association with class ii mhc mechanisms for the incorporation of proteins in membranes and organelles transmembrane h-barrel proteins characterization of the adaptor-related protein complex, ap-3 the structure and insertion of integral proteins in membrane the molecular biology of arteriviruses intracellular processing of the gp160 hiv-1 envelope precursor. endoproteolytic cleavage occurs in a cis or medial compartment of the golgi complex specificity of interaction between adaptor-complex medium chains and the tyrosine-based sorting signals of tgn38 and igp120 the yeast adaptor protein complex, ap-3, is essential for the efficient delivery of alkaline phosphatase by the alternate pathway to the vacuole correlation of sequence hydrophobicities measures similarity in three dimensional protein structure structural requirements for high efficiency endocytosis of the human transferrin receptor signal-dependent membrane protein trafficking in the endocytic pathway characterization and primary structure of a human immunodeficiency virus type 1 (hiv-1) neutralization domain as presented by a poliovirus type 1/hiv-1 chimera comparative cellular processing of the human immunodeficiency virus (hiv-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation and infectivity mutation of the dominant endocytosis motif in human immunodeficiency virus type 1 gp41 can complement matrix mutations without increasing env incorporation viral and cellular membrane fusion reactions biosynthesis, cleavage, and degradation of the human immunodeficiency virus type 1 envelope glycoprotein gp160 mutations with the human immunodeficiency virus type 1 gp160 glycoprotein alter its intracellular transport and processing the highly conserved c-terminal dileucine motif in the cytosolic domain of the human immunodeficiency virus type 1 envelope glycoprotein is critical for its association with the ap-1 clathrin adaptor amphipathic domains in the c terminus of the transmembrane protein (gp41) permeabilize hiv-1 virions: a molecular mechanism underlying natural endogenous reverse transcription the characterization of amino acids in proteins by statistical methods we thank richard compans for reading the manuscript and his advice and are pleased to acknowledge the financial support of avert, uk. key: cord-306111-wn1gxhk9 authors: dommett, r. m.; klein, n; turner, m. w. title: mannose‐binding lectin in innate immunity: past, present and future date: 2006-09-01 journal: tissue antigens doi: 10.1111/j.1399-0039.2006.00649.x sha: doc_id: 306111 cord_uid: wn1gxhk9 the human collectin, mannose‐binding lectin (mbl), is an important protein of the humoral innate immune system. with multiple carbohydrate‐recognition domains, it is able to bind to sugar groups displayed on the surfaces of a wide range of microorganisms and thereby provide first‐line defence. importantly, it also activates the complement system through a distinctive third pathway, independent of both antibody and the c1 complex. three single point mutations in exon 1 of the expressed human mbl‐2 gene appear to impair the generation of functional oligomers. such deficiencies of functional protein are common in certain populations, e.g. in sub‐saharan africa, but virtually absent in others, e.g. indigenous australians. mbl disease association studies have been a fruitful area of research and implicate a role for mbl in infective, inflammatory and autoimmune disease processes. overall, there appears to be a genetic balance in which individuals generally benefit from high levels of the protein. however, in certain situations, reduced levels of circulating mbl may be beneficial to the host and this may explain the persistence of the deleterious gene polymorphisms in many population groups. it is now 60 years since the australian nobel prize winner sir frank macfarlane burnet, together with john mccrea, identified three inhibitors in serum (called a, b and g), which were able to inactivate influenza virus (1) . we now know that the b inhibitor was, in fact, a protein called mannosebinding lectin (mbl), a component of the innate immune system (2) . during the past 30 years, our understanding of this protein has steadily increased as a result of extensive research activity in three main areas: (a) bio/immunochemistry (including molecular genetics), (b) microbiology and (c) immunodeficiency. work in these areas initially proceeded independently as evidence for both an inexplicable biological function and a clinical deficiency state emerged. the isolation and characterization of the protein were necessary in order to illuminate the observations of the so-called rarf bactericidal activity (3, 4) in the microbiology area and the opsonic deficiency reported in many paediatric populations. some of the main developments are summarized in table 1 . this review briefly addresses issues relating to the early history of mbl, its structure, function, genetics and disease associations. finally, future developments including the potential use of both plasma-derived and recombinant mbl are discussed. the existence of mammalian serum lectins was first predicted in 1975 by robinson et al. (5) , and the protein was first isolated in 1978 from cytosolic fractions of rabbit liver by kawasaki et al. (6) . subsequently, wild et al. (7) were able to isolate mbl from both human and rat liver. more recently, extrahepatic transcription of mbl has been reported and this may have implications regarding its role in localized host defence (8) . mbl belongs to a family of proteins called the collectins, which possess both collagenous regions and lectin domains. the other major human collectins, surfactant protein a and surfactant protein d, possess structural characteristics similar to those of mbl and are found predominantly in the lung and other mucosal sites (9) . plasma-associated phagocytic defect (28) 1975 existence of mammalian serum ôlectin-like proteins specific for mannoseõ predicted (5) 1976 association of opsonic defect with frequent infections in infancy, but deficiency also present in 5% of the general population (29) 1978 mbl isolated from rabbit liver (6) 1980 opsonic deficiency in infants with chronic diarrhoea (30) 1981 association of yeast opsonization defect with suboptimal c3b deposition (31) 1982 description of mouse rarf: a complement-activating bactericidal protein (3) 1983 human mbl isolated from liver (7); human serum mbl isolated (121) prospective study of opsonic deficiency in infancy (122) 1984 rarf activity present in vertebrate classes (4) 1985 bovine serum mbl described (123) opsonic defect linked to absence of an unidentified co-factor of the complement system (124) 1987 mbl activation of classical complement pathway (18) 1988 rat serum mbl a and c described (125) ; description of c-type crd (126) 1989 gene for human mbl cloned (33, 34) ; human mbl has bactericidal activity (127) ; opsonic nature of mbl demonstrated (35) mbl inhibits in vitro infection by hiv (85) correlation of opsonic defect with low serum mbl levels (32) 1990 bovine and mouse serum b inhibitors of influenza a virus identified as mbl (2) correlation of mbl levels with classical complement pathway activation at low serum concentrations (128) 1991 mouse mbl a and c described (129) opsonic deficiency and low mbl levels linked to single point mutation in codon 54 (variant b) (50) 1992 human mbl levels in acute-phase responses (59) ; crystallography of mbl crd (130) ; novel protease (masp-1) and complement activation by mbl (19) human rarf identical to mbl-masp (131) low mbl levels in africans linked to codon 57 (variant c) mutation in the mbl gene (51) 1994 third mbl mutation in codon 52 (variant d) described (52) 1995 polymorphisms found in promoter region of mbl gene (55) 1997 second masp found to activate complement (20) mbl mutations are an important risk factor for infections in children (132) 1998 reconstitution of opsonizing activity by infusion of purified mbl into mbl-deficient humans (112) 1999 truncated form of masp-2 -map19 (21) 2000 complement-activating complex of ficolins and masp (133) mbl shown to bind to clinically relevant organisms (15) structural aspects of mbl the protein structure of mbl has been studied extensively, and aspects are presented in figures 1 and 2 . the protein consists of multimers of an identical polypeptide chain of 32 kda. each chain comprises four distinct regions encoded by different exons of the mbl-2 gene, as will be discussed in more detail later. each chain has a c-terminal, calcium-dependent carbohydrate-recognition domain (crd); a short, a-helical, hydrophobic neck region (in the so-called coiled-coil configuration); a collagenous region containing 19 gly-xaa-xaa triplets and a cysteine-rich n-terminal region. three polypeptide chains form a triple helix within the collagenous region, stabilized by hydrophobic interactions and interchain disulphide bonds within the n-terminal cysteine-rich region. this is the basic building block of all circulating molecular forms of mbl. in serum, mbl consists of oligomers ranging from dimers to hexamers, and x-ray crystallographic studies/electron micrographs have revealed that these oligomers have a sertiform or a bouquetlike structure due to an interruption in the collagenous region, giving rise to a kink/hinge. the ability of the protein to bind effectively to microorganisms and activate complement appears to depend on the presence of higher order oligomers (tetramers and above). work by drickamer and colleagues (10, 11) and also by ezekowitz and colleagues (12) has provided an insight into the structure of the crd. each crd binds a calcium ion, enabling it to form co-ordination bonds with the 3-and 4-hydroxyl groups of specific sugars including mannose, n-acetyl-d-glucosamine, n-acetyl-mannosamine, fucose and glucose. the three crds in each structural subunit are separated by a constant 45-å distance (12) . clustering of the structural subunits provides a flat platform, permitting binding of mbl to the arrays of repeating sugar groups on microbial surfaces. although the binding affinity of each individual crd-sugar interaction is relatively low at 10 23 m (13), the formation of higher order oligomers provides multiple crds, which are able to bind simultaneously with high avidity. mbl is a major pattern-recognition molecule of the innate immune system. it primarily recognizes specific sugar groups (as above) on the surface of microorganisms, enabling it to distinguish self from non-self. it can also bind to phospholipids, nucleic acids (14) and non-glycosylated proteins. mbl has been shown to bind promiscuously to a wide range of bacteria, viruses, fungi and protozoa and some selected examples are listed in table 2 . neth et al. used flow cytometry to demonstrate mbl binding to clinically relevant bacterial isolates from immunocompromised children and noted differences in binding within some species such that one isolate might show strong binding, whereas another was much weaker (15) . the role of specific structural features of microorganisms (e.g. the capsule), which permit or prevent binding to mbl, has been explored in several studies. the earliest work was probably by kawakami et al. on the socalled rarf complex (which was later identified as mbl) and its interaction with salmonella enterica serovar typhimurium (3). this suggested that the structure and composition of lipopolysaccharide play a crucial role in mbl binding and function. other mechanisms that enable microorganisms to avoid recognition and killing by mbl include lipooligosaccharide sialyation (16, 17) . despite much progress in this area, many puzzles remain to be addressed, mostly related to the exact disposition of sugars on microbial surfaces. our understanding of mbl function has grown rapidly over the past three decades. it is now recognized to have a role in processes as diverse as complement activation, promotion of complement-independent opsonophagocytosis, modulation of inflammation, recognition of altered self-structures and apoptotic cell clearance. a role for mbl in host defence was first proposed in 1987 when ikeda et al. observed that the protein was able to activate the classical pathway of complement (18) . however, it is now clear that mbl activates a novel third pathway of complement, often termed the mbl pathway, in an antibody-and c1-independent fashion as illustrated in figure 3 . this functional activity reflects the fact that mbl circulates in association with a group of mbl-associated serine proteases (the so-called masps). in 1992, matsushita and fujita demonstrated the presence of a novel complement enzyme in serum, which was thought to generate the c3 convertase (c4bc2a), associated with classical pathway activation (19) . however, this activity was later found to be mediated by masp-2 (20) , and the original enzyme is now known as masp-1 and may activate c3 directly. subsequently, a small separately synthesized fragment of masp-2 termed smap or map19 was identified (21, 22) and a third masp (masp-3) with no known function was also described (23) . current understanding suggests that on binding to microorganisms, autoactivation of masp-2 occurs, permitting cleavage of c4 and c2 to form a c3 convertase, which is indistinguishable in specificity from the convertases found in the other two activation pathways of complement (24) . it should be noted that the so-called mbl pathway is also activated by another family of proteins called ficolins. the ficolins are structurally similar to collectins, with collagenous domains linked to fibrinogen-like domains having sugar-binding properties. l-and h-ficolins are humoral factors synthesized by hepatocytes, although h-ficolin has also been observed in bronchial/alveolar fluid and in bile (25) . in contrast, m-ficolin is found on peripheral blood mononuclear cells, polymorphonuclear cells and type ii lung epithelial cells (26) . ficolins are also found in complexes with the masps and are considered to have different binding specificities compared with mbl (27) . in 1968, miller et al. reported a plasma-associated defect of phagocytosis in a child with severe recurrent infections, failure to thrive and diarrhoea (28) . in vitro work revealed a failure of the childõs plasma to opsonize heat-killed bakers yeast (saccharomyces cerevisiae). this defect was later detected in the sera of children with recurrent unexplained infections (29) and chronic diarrhoea of infancy (30), but, interestingly, studies in the general population also revealed a relatively high frequency of the defect (5%). in 1981, studies linked this opsonic deficiency to the complement figure 3 complement activation pathway. the lectin pathway of complement is activated by mbl and ficolins. on binding to appropriate targets, mbl-masp-2 complexes cleave c4 and c2 to form c3 convertase (c4bc2a). mbl-masp-1 complexes may activate c3 directly. ficolins also work in combination with the masps. the classical and alternative pathways also generate c3 convertase enzymes, which cleave c3. the lytic pathway (c5-c9) is common to all three routes of c3 cleavage. mbl, mannose-binding lectin; masp, mbl-associated serine proteases; masp-1, mbl-associated serine protease-2; masp-2, mblassociated serine protease-2. system by demonstrating that sera with the deficiency deposited less c3b on yeast surfaces (31) . however, it was not until 1989 that the common opsonic defect was found to be associated with low levels of the mannose-binding protein, which we now refer to as mbl (32) . in that same year, the gene for mbl was cloned (33, 34) (genetics of human mbl). in a study of mbl-coated salmonella montevideo, kuhlman et al. reported that mbl was able to interact directly with cell surface receptors and promote opsonophagocytosis (35) . subsequently, a number of putative mbl-binding proteins/receptors have been proposed including cc1qr/ calreticulin (36), c1qrp (37) and cr1 (38, 39) . however, it is unclear whether mbl is acting as a direct opsonin or is merely enhancing other complement pathways and/or antibody-mediated phagocytosis. the role of mbl as a modulator of inflammation appears to be complex and, accordingly, its mechanism of action remains unexplained. one possible explanation is that mbl is able to trigger proinflammatory cytokine release from monocytes (40, 41) . this concept was addressed in studies by jack et al. using neisseria meningitidis incubated with increasing concentrations of mbl before being added to mbl-deficient whole blood. release of tumour necrosis factor a, interleukin (il)-1b and il-6 from monocytes was enhanced at mbl concentrations below 4 mg/ml but suppressed at higher concentrations (42) . clinical studies in this area are discussed later. the role of mbl in the recognition of altered self and apoptosis a role for mbl in the clearance of apoptotic cells was first proposed by ogden et al. in 2001 (43) . mbl was found to bind directly to apoptotic cells that expose terminal sugars of cytoskeletal proteins, thereby permitting their recognition and directly facilitating their phagocytosis by macrophages. defects in the clearance of apoptotic cells have been implicated in the pathogenesis of certain autoimmune conditions, although the precise role of mbl, if any, remains elusive. for example, in 2005, stuart et al. reported that although mbl-deficient mice displayed defective apoptotic cell clearance, they did not develop autoimmune diseases (44) . in animal studies, mbl has been implicated in the pathophysiology of ischaemia reperfusion injury due to its ability to recognize altered self-structures. stahl and colleagues have proposed the lectin pathway as a mediator of this process in certain organs, and the absence of mbl/masp pathway activation appears to afford protection in these disease models (45, 46) . however, the relevance of these findings to human health needs to be established. changes in cell surface structures during oncogenic transformation appear to promote binding of mbl to cancer cells (47) where the protein can mediate cytotoxic effects including mbl-dependent cell mediated cytotoxicity (48, 49) . the relative importance of such mechanisms in tumour immunology is, at present, unknown. there are two human mbl genes, but mbl-1 is a pseudogene and only mbl-2 encodes a protein product. the functional mbl-2 gene is located on chromosome 10 (q11.2-q21) and comprises four exons as illustrated in figure 1 . exon 1 encodes the signal peptide, a cysteine-rich region and part of the glycine-rich collagenous region. exon 2 encodes the remainder of the collagenous region and exon 3 encodes an a-helical coiled-coil structure, which is known as the ôneckõ region. exon 4 encodes the crd, which adopts a globular configuration. the promoter region of the mbl gene contains a number of regulatory elements, which affect transcription of the protein. in 1991, the complete nucleotide sequence of all four exons of the human mbl-2 gene was determined by sumiya et al. in two british children with recurrent infections and low mbl levels (50) . in both individuals, a point mutation was observed in codon 54, changing the codon sequence from ggc to gac and substituting aspartic acid for glycine in the translated protein. familial studies confirmed that the defect was inherited in an autosomal dominant fashion. in 1992, lipscombe et al. identified a second exon 1 mutation in codon 57 (gly / glu), when studying a sub-saharan african population (51) , and in 1994, madsen et al. reported a mutation in codon 52 (arg / cys) (52) . these point mutations are now commonly referred to as variants b, c and d respectively, with variant a indicating the wild type. the b variant mutation occurs at a gene frequency of approximately 25% in eurasian populations. in contrast, the c variant is rare in eurasians but is commonly seen in sub-saharan african populations, with frequencies of 50%-60%. population studies suggest that the b variant mutation may have arisen between 50,000 and 20,000 years ago (53) since no structural gene mutations have been identified in studies of indigenous australian populations who arrived on the continent approximately 50,000 years ago, whereas the b variant mutation was probably introduced into both north and south america at the time of the last glaciation approximately 20,000 years ago. the effect of these exon 1 mutations on the protein product continues to be the focus of study. they are believed to impair oligomerization and lead to a functional deficiency. the b and c mutations result in the replacement of critical axial glycines in the triple helix by dicarboxylic acids, resulting in distortion of this important part of the protein (50) . in contrast, the d mutation results in the replacement of arginine with cysteine. this extra cysteine has been proposed to cause formation of adventitious disulphide bonds that hinder higher oligomer formation (54) . several polymorphisms have also been reported in the promoter region of the gene. studies by madsen et al. investigating the large interindividual variation in serum mbl levels revealed three polymorphisms, h/l, x/y and p/q at positions 2550, 2221 and 14 of the mbl gene (55, 56) . subsequently, four common haplotypes were identified, namely lxp, lyp, lyq and hyp. of these, hyp, which is associated with medium to high levels of mbl and lxp, which is associated with low levels of the protein, appear to be most important. these promoter haplotypes are in strong linkage disequilibrium with the exon 1 mutations, resulting in seven common extended haplotypes, namely hypa, lypa, lyqa, lxpa, hypd, lypb and lyqc. other rare haplotypes have also been described (57) . figure 4 illustrates the frequency of these various haplotypes in selected populations and highlights the degree of ethnic variation. the combination of structural gene and promoter polymorphisms results in a dramatic variation in mbl concentration in apparently healthy individuals of up to 1000-fold (caucasian: range <20-10,000 ng/ml). in addition, ezekowitz and colleagues presented evidence in 1988 that mbl was an acute-phase reactant (58) . in these investigations, rna was isolated from a ônormalõ liver taken as part of a staging biopsy for hodgkins disease and was compared with rna isolated from a fresh post-mortem liver of a victim with severe trauma. the authors found that mbl messenger rna transcripts were barely detectable in normal liver but that induction was seen in liver exposed to acute stress. subsequent studies have shown that mbl levels can increase between 1.5 and threefold during the acute phase, but this response is variable between individuals (59) . it should also be noted that even during an acutephase response, individuals heterozygous or homozygous for mbl mutations appear unable to achieve the protein levels of those possessing a wild-type genotype. approximately one-third of the caucasian population possess genotypes conferring low levels of mbl, with approximately 5% having very low levels. no absolute level of mbl deficiency has been defined. genotype and phenotype show a relatively strong correlation and studies often use just one measure to infer deficiency. however, there is ôadded valueõ in performing both measures and we would strongly advocate this approach whenever possible. mbl occurs in two distinct forms in rodents and rhesus monkeys (60), but only one form is found in humans and chickens. as discussed previously, there are two human mbl genes, which are most likely due to a gene duplication event (61) . however, mbl-1 is a pseudogene and the potential mechanisms responsible for silencing the mbl-1 (63). such substitutions were also found in other higher primates including chimpanzees and gorillas but not in more distant primates such as the rhesus monkey. the authors concluded that both the mbl-1 and the mbl-2 genes have been selectively silenced by the same molecular mechanisms, but skewed in time resulting in overall downregulation of mbl levels in the present human population. the high frequency of variant alleles observed in certain populations was initially puzzling since it suggests that functional mbl deficiency may well be advantageous. similarities have been proposed between the mbl genetic system and the role of the sickle cell gene in protection against malaria as occurs in carriers of the sickle cell haemoglobin allele (64) . the argument runs as follows: certain intracellular parasites use c3 opsonization and c3 receptors on monocytes/macrophages to enter their host. therefore, any reduction in complement-activating function of the host may reduce the probability of parasitization. in support of this notion is a study on patients with visceral leishmaniasis, which revealed that such patients are more likely to have high mbl levels than uninfected controls (65) . a small study of ethiopian patients with lepromatous or borderline lepromatous leprosy also found that their mbl levels were significantly higher than those of healthy blood donors (66) . an alternative explanation of the unexpectedly high frequency of low mbl phenotype individuals found in many tropical regions is that excessive complement activation can result in immunopathologically mediated host damage; therefore, any mechanism that reduces complement activation may be beneficial (51) . the identification of mbl deficiency as the cause of the so-called common opsonic defect has been followed by a plethora of disease association studies aimed at defining the precise role of this protein. a number of the early studies concentrated on paediatric populations and mbl was suggested to provide substitute ôantibodyõ-like activity during the ôwindow of vulnerabilityõ (approximately 6-24 months), when maternal immunoglobulin g (igg) antibody levels have waned but the infantõs own adaptive immune response is still immature (32) . nevertheless, studies in adults suggested that there might be a role for mbl throughout life (67) . notwithstanding these reports, the majority of individuals possessing a variant mbl allele apparently suffer no ill effects and remain essentially healthy. in a study that apparently confirms this, dahl et al. monitored 9245 adults in a danish caucasian population and found no evidence for significant differences in infectious disease or mortality in mbl-deficient individuals compared with controls (68) . similar findings were reported by tacx et al. in unselected adults admitted to hospital with infections (69) . nevertheless, these studies should not be regarded as proof that mbl levels have no clinical relevance. many groups have undertaken case-control studies, which do indeed suggest that mbl is an important immunological modulator. in some cases, there is evidence that the significance of mbl deficiency is more readily appreciated when there is another co-existing defect (70), as we first proposed in 1991 (71). space does not permit a comprehensive review of all the mbl clinical studies that have been undertaken to date, and the topics covered below have been selected in order to illustrate examples of possible roles for mbl in a variety of clinical situations. most studies have explored the role of mbl in relation to the acquisition of an infectious organism (susceptibility) and the nature of the associated clinical course (severity). in clinical practice, this distinction can be difficult. however, for the purposes of this review, we will highlight examples of infections in which mbl appears to have an influence on one or other of these two aspects of infectious diseases. hamvas et al. have recently shown a role for mbl in mycoplasma infection (72) . they studied cases of infection in patients with primary antibody deficiencies (pad) that are known to be particularly susceptible to such organisms and compared them with a control population. more than two-thirds of pad patients with mycoplasma infections were mbl deficient (in possession of an exon 1 variant allele) compared with one-third of the control group. in the same study, they were able to demonstrate binding of mbl to three strains of mycoplasma using flow cytometry and proposed a role for mbl in prevention of invasive disease. in 2003, severe acute respiratory syndrome (sars) emerged as a highly infectious disease caused by a novel coronavirus (sars-cov). it provided a new challenge to previously unexposed individuals predominantly in asia. specific antibodies to sars-cov could be detected 10 days after the onset of symptoms, making sufferers reliant on innate immune mechanisms during the early phase of infection. since the structure of the virus was rapidly established (73, 74) , it also became clear that this novel infectious agent was rich in the sugars known to be targeted by mbl and it was hypothesized that this lectin might well be involved in first-line defence against this infection. subsequent studies found significant differences in the distribution of mbl-deficient genotypes in patients with sars compared with those in controls (75, 76) . these studies suggested that mbl plays a role in susceptibility to the infection but does not influence subsequent severity. in their investigations, ip et al. were also able to demonstrate binding of mbl to the virus and its ability to inhibit infection (75) . (77). the b variant allele was found more commonly in patients with symptomatic hepatitis b cirrhosis and in those with spontaneous bacterial peritonitis. it was also noted that mbl levels were lower in this patient cohort with chronic infection. screening for mbl mutations in such patients was suggested in order to enable identification of those at increased risk of complications who may benefit from prophylactic antibiotic treatment. in 2005, chong et al. also reported that mbl genotypes correlating with low protein levels were associated with the occurrence of cirrhosis and also hepatocellular carcinoma in hepatitis b carriers (78) . they also demonstrated that mbl is able to bind hepatitis b surface antigen. in the same year, thio et al. published the results of a nested case-control study of 527 patients who had either naturally recovered from hepatitis b (n ¼ 338) or had persistent infection (n ¼ 189). they found that mbl genotypes correlating with high serum levels were associated with recovery from infection, whereas those correlating with lower levels were associated with persistence of the virus (79) . it should be noted that approximately half of the subjects were also infected with human immunodeficiency virus (hiv), but the authors concluded that this did not influence the results obtained. matsushita et al. investigated the influence of mbl mutations in hepatitis c infection and found that sufferers who were homozygous for b variant alleles were less likely to respond to interferon treatment (80) . further work would be warranted in order to define the role of mbl in the pathogenesis of hepatitis infection. secondary immunodeficiencies due to disease or treatment have provided interesting patient populations within which to study the role of mbl. one such group comprises those receiving chemotherapy for malignancy. these patients are rendered neutropenic by their treatment (or underlying disease process) and are subsequently at increased risk of infectious complications. in 2001, two studies were published reporting an effect of mbl deficiency in such patients. neth et al. studied 100 children and measured mbl levels and genotype. children in possession of mbl variant alleles spent twice as many days in hospital with febrile neutropenia during the first 6 months of their treatment compared with wild-type individuals (81) . in the other study, peterslund et al. followed 54 adults undergoing chemotherapy for various haematological malignancies and found that those who developed ôsignificantõ infections (bacteraemia, pneumonia or both) in the 3-week periods post-treatment had significantly lower levels of mbl compared with those without significant infections (82) . subsequent studies have shown differing results, but drawing comparisons between them is inherently difficult. these patients are a highly heterogeneous population, with different underlying disease processes, undergoing treatment regimens of differing intensity, resulting in various degrees of immunosuppression. in one contrasting study, bergmann et al. followed 80 adults undergoing therapy for acute myeloid leukaemia, which involves intense highly myelosuppressive treatment. they found no effect of mbl deficiency on frequency, severity or duration of fever and suggested that the nature of the treatment overwhelmed any potential influence of mbl (83) . further clinical studies in such patients are required in order to delineate the exact role of mbl. an mbl double-knockout mouse model has been used to explore the above clinical conundrum. in 2004, shi et al. demonstrated that mbl null mice were highly susceptible to intravenous inoculation with staphylococcus aureus, all dying within 48 h, compared with 55% survival of mbl wild-type mice. however, when the mice were inoculated via the intraperitoneal route and rendered neutropenic (using cyclophosphamide), neutropenic mbl null mice were found to have higher accumulations of bacteria in the blood and organs compared with neutropenic wild-type mice. by day 8 post-infection, the neutropenic wild-type mice had cleared their blood, but the neutropenic mbl null mice had persistent bacteraemia. the authors were able to reverse the phenotype by treating the mbl null mice with recombinant mbl (84) . to date, nearly 40 million humans have been infected with hiv. the clinical consequences of viral exposure are variable. some individuals can be repeatedly exposed to the virus but remain free from infection. others can be infected but remain free from clinical disease. while numerous viral and host factors will determine the fate of an individual exposed to hiv, there are data to indicate that mbl can influence both susceptibility and severity of hiv infection. the likely target for hiv binding is the heavily glycosylated glycoprotein, gp120. while mbl can be readily demonstrated to bind to purified gp120 (85) , the capacity of mbl to neutralize primary hiv isolates is less convincing. recent data indicate the mbl can opsonize hiv but does not induce neutralization at the levels at which it is normally present in serum. however, binding and opsonization of hiv by mbl may alter virus trafficking and viral antigen presentation during hiv infection. mbl may influence uptake by dendritic cells (dc), which express a cell surface lectin called ôdc-specific intracellular adhesion molecule 3-grabbing non-integrinõ (dc-sign). dc-sign has been shown to mediate a type of infection called ôtransõ-infection, where dc bind hiv and efficiently transfer the virus to t cells. preincubation of hiv strains with mbl prevents dc-sign-mediated trans-infection of t cells and indicates that at least in vitro, mbl may inhibit dc-sign-mediated uptake and spread of hiv (86) . whatever the mechanism of mbl interactions with hiv, a number of clinical studies have suggested that deficiency of mbl is a risk factor for acquiring hiv infection. mbl deficiency appears to increase the acquisition of hiv infection by between three-and eightfold (87) (88) (89) (90) . there is also an increased risk of vertical transmission from infected mothers to their offspring (91) . however, these findings have not been replicated in all populations, with some studies failing to demonstrate a role for mbl in hiv infection (92) (93) (94) . there is even less clarity with regard to the role of mbl in hiv disease progression. garred et al. (87) demonstrated that men with mbl variant alleles had a shorter survival time following the onset of acquired immune deficiency syndrome (aids) than did patients with wild-type mbl alleles. however, in a well-characterized cohort of homosexual men, variant mbl alleles had an insignificant effect on survival following the diagnosis of aids (95) . in this latter study, there appeared to be a protective effect of mbl variant alleles, with a delay in the development of aids from the time of hiv seroconversion. patients with mbl variant alleles had lower cd4 counts at the time of developing aids, indicating that mbl deficiency may influence the onset of aids for any given cd4 count. furthermore, mbl mutations appeared to protect against the development of kaposi sarcoma, a finding that was difficult to explain (95) . in another study, prohaszka et al. (90) found that mbl levels were lower in asymptomatic hiv-positive individuals compared with hiv-negative controls. however, the protective effect of mbl was lost in patients with an aids diagnosis; patients with high mbl levels had significantly lower numbers of cd4 cells. a possible explanation is that enhanced proinflammatory cytokine production in advanced hiv disease acts to increase mbl synthesis (96) , elevating levels in patients with late-stage disease. indeed, a recent study has shown in vitro that mbl can enhance proinflammatory cytokine production and viral replication (97) . in the light of studies indicating a role for mbl in inflammatory modulation, it is tempting to suggest that under some circumstances, mbl may act to promote inflammatory cell activation, thereby accelerating the rate of cd41 t-cell depletion. few studies have assessed the impact of mbl in the context of effective antiviral therapy. however, one study has attempted to relate mbl status and hiv-infected longterm non-progressors (ltnps) (98) . mbl levels were consistent with a wild-type genotype in the six ltnps studied. amoroso and colleagues had also suggested such an effect in a study showing that children with rapidly progressing disease were more likely to have mbl variant alleles (codon 54) than slower progressors (99) . cystic fibrosis provides an example of a clinical condition where mbl appears to be exerting its role as an infection susceptibility gene and inflammatory modulator. garred et al. were the first group to report that patients with mbl variant alleles have significantly impaired lung function and decreased life expectancy in comparison with wild-type individuals (100) . the effect of mbl deficiency on the severity of lung disease was most apparent in patients with chronic pseudomonas aeruginosa infection and it was also found that burkholderia cepacia infection was more common in patients with mbl deficiency. in 2004, davies et al. reported that an effect of mbl was only seen in adults homozygous for mbl mutations. these patients had significantly reduced lung function, more frequent hospital admissions and raised systemic inflammatory markers. however, there was no evidence of increased susceptibility to burkholderia cepacia and pseudomonas aeruginosa (101) . whether mbl has an effect on early colonization with burkholderia cepacia and pseudomonas aeruginosa or subsequent secondary viral infections or whether there is an (anti)inflammatory effect on subsequent lung damage remains unclear. clinical studies of critically ill patients requiring intensive care management have shown that individuals who are mbl deficient are more likely to develop the systemic inflammatory response syndrome (sirs) ( figure 5 ) and progress to septic shock and death (102, 103) , findings which may well relate to the proinflammatory cytokine response. it should also be noted that chronic inflammation is now increasingly accepted to be a risk factor for myocardial infarction (mi), and a recent study by saevarsdottir et al. has found that patients with high mbl levels have a decreased likelihood of suffering a mi -again suggesting a potential role for mbl in modulating the inflammatory response (104). as a component of the complement system with similarities to c1q, but also as a player in infectious and inflammatory processes, the structure and function of mbl have prompted studies exploring a possible role in autoimmune conditions. systemic lupus erythematosus (sle) has been the focus of a number of mbl genotyping studies, but the results have been somewhat inconsistent. nevertheless, a recent meta-analysis has reviewed studies in this area and found that mbl variant alleles are indeed sle risk factors (105) . as with infectious disease, there is some evidence that the risk of pathology increases if there is another co-existing immune defect. for example, in a cohort of spanish patients, the odds ratio for developing sle was 2.4 for individuals with mbl deficiency, but this increased to 3.2 when there was also a co-existing partial c4 deficiency (106) . studies in patients with sle have reported that mbl deficiency also influences their risk of developing certain complications, which include arterial thromboses (107) and respiratory tract infections (108, 109) . a role for mbl in the pathogenesis of rheumatoid arthritis has also been suggested. malhotra et al. reported that changes in igg glycosylation secondary to the underlying disease results in mbl-associated complement activation (110) . such complement activation then contributes to chronic inflammation of the synovial membrane. however, graudal et al. found that patients with lower mbl levels experienced earlier, more severe, symptoms and had more rapid joint destruction as visualized radiologically (111) . several recent research publications suggest the directions in which future work on this collectin and its associated molecules may proceed. these include therapeutic interventions, functional assays and the evaluation of the importance of mbl in disease. these are considered briefly below. therapeutic potential of mbl mbl replacement was first attempted (without any knowledge of the deficiency) when fresh frozen plasma was given to patients and found to correct the opsonic defect (28, 29) . since then, affinity-purified, plasma-derived mbl has been safely given to many patients, resulting in normalization of enzyme-linked immunosorbent assay detectable mbl and complement-mediated opsonic activity (112) . a phase 1 study showed the half-life of the protein to range between 18 and 115 h (113) . the development of recombinant mbl is also at the phase 1 trial stage and such developments provide exciting prospects for the future exploration of the therapeutic potential of mbl. exactly who would benefit from replacement therapy is under debate and the importance of targeting well-defined patient groups will be vital to its success. the discovery of other components of the lectin pathway including the ficolins and the masps indicates that this limb of the immune system is complex and extends beyond mbl and masp-2 alone. this knowledge enables us to question the impact of these molecules either in isolation or in combination. functional assessment of the lectin pathway may be a far more accurate and clinically relevant measurement than mbl level and/or genotype alone. a number of different assays have been reported, which assess activity at different stages of the functional pathway; therefore, the results must be interpreted accordingly (114, 115) . the impact of deficiencies of the various adjunctive components is also the subject of much current research. in 2003, stengaard-pedersen et al. reported the first identified case of masp-2 deficiency (116) . functional analysis of the ability of mbl to activate the lectin pathway, estimating c4b deposition on a mannan surface, was performed on a group of patients with suspected immunodeficiency. one patient was found to have deficient pathway activity despite having sufficient mbl. no masp-2 or map19 was found in the plasma, and genetic analysis indicated that the patient was homozygous for a point mutation in exon 3 of the gene (d105g). clinically, the patient suffered from recurrent infections and autoimmune symptoms. subsequently, the frequency of this mutation has been assessed in a small number of populations and values range from 1.3% to 6.3% (117) . as discussed previously, the contribution of masp-1 and masp-3 in the pathway remains unexplained. the role of ficolins is now beginning to be addressed in clinical studies. like mbl, no absolute levels of deficiency have yet been defined. atkinson et al. studied more than 300 children with recurrent respiratory tract infections and measured l-ficolin levels (118) . an association with mbl deficiency in the same patient cohort had already been reported (119) . in this study, low levels of l-ficolin were more common in patients than in controls and most common in patients with co-existing atopic disorders, suggesting a role for l-ficolin in protection from microorganisms complicating allergic disease. polymorphisms in the ficolins have been identified, although their clinical significance is as yet unknown. mbl is an ancient molecule, which has probably been subject to a large number of evolutionary pressures. the last 50,000 years of human evolution have been associated with major changes as hominids moved from an essentially nomadic lifestyle to increasingly crowded living arrangements in large settled communities. associated with these changes, the spectrum of common infectious diseases would also have changed. more recently, the introduction of antibiotics, the emergence of novel infections and increasing use of immunosuppressive therapies have provided new challenges to our innate host defence system. despite all these changing evolutionary pressures, mbl gene polymorphisms persist at high frequencies, suggesting that they offer potential advantages to the host. thus, there exists a balance in which certain individuals benefit from the expression of high levels of the protein, whereas others (living in differing environments, eg. the tropics) may benefit from reduced levels of circulating mbl ( figure 6 ). mbl status may also be either advantageous or disadvantageous when considered from the viewpoint of the severity of a particular illness. thus, it is known that those with higher levels of mbl are better able to modulate inflammation, probably through an effect on cytokine responses. in contrast, those deficient in mbl appear to be at risk of sepsis and sirs. for these reasons, we believe that analyses of the relevance of mbl (120) should be extended beyond its role in infectious disease and include clinical areas such as autoimmunity and inflammatory disorders. inhibitory and inactivating action of normal ferret sera against an influenza virus strain bovine and mouse serum beta inhibitors of influenza a viruses are mannose-binding lectins properties of a new complement-dependent bactericidal factor specific for ra chemotype salmonella in sera of conventional and germ-free mice a group of bactericidal factors conserved by vertebrates for more than 300 million years affinity chromatography of human liver alpha-d-mannosidase isolation and characterization of a mannan-binding protein from rabbit liver isolation of mannosebinding proteins from human and rat liver extra-hepatic transcription of the human mannose-binding lectin gene (mbl2) and the mbl-associated serine protease 1-3 genes collections and ficolins: humoral lectins of the innate immune defense structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by mad phasing trimeric structure of a c-type mannose-binding protein human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil binding of sugar ligands to ca(21)-dependent animal lectins analysis of mannose binding by site-directed mutagenesis and nmr nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins a and d and mannose-binding lectin mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition activation of complement by mannose-binding lectin on isogenic mutants of neisseria meningitidis serogroup b the lipopolysaccharide structures of salmonella enterica serovar typhimurium and neisseria gonorrhoeae determine the attachment of human mannose-binding lectin to intact organisms serum lectin with known structure activates complement through the classical pathway activation of the classical complement pathway by mannose-binding protein in association with a novel c1s-like serine protease a second serine protease associated with mannan-binding lectin that activates complement a truncated form of mannose-binding lectin-associated serine protease (masp)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene masp-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway crystal structure of the cub1-egf-cub2 region of mannose-binding protein associated serine protease-2 hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile human m-ficolin is a secretory protein that activates the lectin complement pathway l-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of gram-positive bacteria, and activates the lectin pathway of complement a familial plasma-associated defect of phagocytosis defective opsonization. a common immunity deficiency yeast opsonisation in children with chronic diarrhoeal states a study of c3b deposition on yeast surfaces by sera of known opsonic potential association of low levels of mannan-binding protein with a common defect of opsonisation exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10 structure and evolutionary origin of the gene encoding a human serum mannose-binding protein the human mannose-binding protein functions as an opsonin human leukocyte c1q receptor binds other soluble proteins with collagen domains mannose binding protein (mbp) enhances mononuclear phagocyte function via a receptor that contains the 126,000 m(r) component of the c1q receptor complement receptor 1/cd35 is a receptor for mannan-binding lectin complement receptor type 1 (cr1, cd35) is a receptor for c1q activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by cd14 antigen, and mannan binding protein inhibits tnf-alpha release induction of tnf-alpha in human peripheral blood mononuclear cells by the mannoprotein of cryptococcus neoformans involves human mannose binding protein mannose-binding lectin regulates the inflammatory response of human professional phagocytes to neisseria meningitidis serogroup b c1q and mannose binding lectin engagement of cell surface calreticulin and cd91 initiates macropinocytosis and uptake of apoptotic cells mannose-binding lectin-deficient mice display defective apoptotic cell clearance but no autoimmune phenotype gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving c1q mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding proteindependent cell-mediated cytotoxicity antitumor activity of mannan-binding protein molecular basis of opsonic defect in immunodeficient children high frequencies in african and non-african populations of independent mutations in the mannose binding protein gene a new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein restricted polymorphism of the mannose-binding lectin gene of indigenous australians molecular determinants of oligomer formation and complement fixation in mannose-binding proteins interplay between promoter and structural gene variants control basal serum level of mannan-binding protein different molecular events result in low protein levels of mannan-binding lectin in populations from southeast africa and south america a new strategy for mannosebinding lectin gene haplotyping a human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins the concentration of the c-type lectin, mannan-binding protein, in human plasma increases during an acute phase response characterization of two mannose-binding protein cdnas from rhesus monkey (macaca mulatta): structure and evolutionary implications characterization of murine mannose-binding protein genes mbl1 and mbl2 reveals features common to other collectin genes the human ortholog of rhesus mannose-binding protein-a gene is an expressed pseudogene that localizes to chromosome 10 the ôinvolutionõ of mannose-binding lectin protection afforded by sickle-cell trait against subtertian malareal infection mannan-binding lectin enhances susceptibility to visceral leishmaniasis dual role of mannan-binding protein in infections: another case of heterosis? mannose binding protein gene mutations associated with unusual and severe infections in adults a population-based study of morbidity and mortality in mannose-binding lectin deficiency mannan binding lectin in febrile adults: no correlation with microbial infection and complement activation mannan binding lectin deficiency and concomitant immunodefects the molecular basis of a common defect of opsonization role for mannose binding lectin in the prevention of mycoplasma infection characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus mannose-binding lectin in severe acute respiratory syndrome coronavirus infection association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection mannose binding lectin gene mutations are associated with progression of liver disease in chronic hepatitis b infection mannose-binding lectin in chronic hepatitis b virus infection mannose binding lectin genotypes influence recovery from hepatitis b virus infection hepatitis c virus infection and mutations of mannose-binding lectin gene mbl deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study association between deficiency of mannose-binding lectin and severe infections after chemotherapy low levels of mannose-binding lectin do not affect occurrence of severe infections or duration of fever in acute myeloid leukaemia during remission induction therapy mannose-binding lectin-deficient mice are susceptible to infection with staphylococcus aureus a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose-binding lectin mannan-binding lectin in the sub-saharan hiv and tuberculosis epidemics the level of the serum opsonin, mannan-binding protein in hiv-1 antibody-positive patients mannan-binding lectin serum concentrations in hiv-infected patients are influenced by the stage of disease polymorphisms in the mbl2 promoter correlated with risk of hiv-1 vertical transmission and aids progression absence of association between mannose-binding lectin gene polymorphisms and hiv-1 infection in a colombian population mannose-binding protein in hiv-seropositive patients does not contribute to disease progression or bacterial infections circulating levels of mannose binding protein in human immunodeficiency virus infection presence of the variant mannose-binding lectin alleles associated with slower progression to aids human mannose-binding protein gene is regulated by interleukins, dexamethasone and heat shock modulatory effect of mannose-binding lectin on cytokine responses: possible roles in hiv infection low mannose-binding lectin serum concentrations in hiv long-term nonprogressors? polymorphism at codon 54 of mannose-binding protein gene influences aids progression but not hiv infection in exposed children association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis impaired pulmonary status in cystic fibrosis adults with two mutated mbl-2 alleles association of mannose-binding lectin polymorphisms with sepsis and fatal outcome, in patients with systemic inflammatory response syndrome increased incidence and severity of the systemic inflammatory response syndrome in patients deficient in mannose-binding lectin mannan binding lectin as an adjunct to risk assessment for myocardial infarction in individuals with enhanced risk the mannose-binding lectin gene polymorphisms and systemic lupus erythematosus: two case-control studies and a meta-analysis a dysfunctional allele of the mannose binding protein gene associates with systemic lupus erythematosus in a spanish population mannose-binding lectin variant alleles and the risk of arterial thrombosis in systemic lupus erythematosus association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients association of mannose binding lectin (mbl) gene polymorphism and serum mbl concentration with characteristics and progression of systemic lupus erythematosus glycosylation changes of igg associated with rheumatoid arthritis can activate complement via the mannose-binding protein mannan binding lectin in rheumatoid arthritis. a longitudinal study reconstitution of opsonizing activity by infusion of mannan-binding lectin (mbl) to mbl-deficient humans human plasma-derived mannose-binding lectin: a phase i safety and pharmacokinetic study an assay for the mannan-binding lectin pathway of complement activation functional analysis of the classical, alternative, and mbl pathways of the complement system: standardization and validation of a simple elisa inherited deficiency of mannan-binding lectin-associated serine protease 2 deficiency of the mannan-binding lectin pathway of complement and poor outcome in cystic fibrosis: bacterial colonization may be decisive for a relationship l-ficolin in children with recurrent respiratory infections mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system human mannose-binding lectin in immunity: friend, foe, or both? isolation and characterization of a mannan-binding protein from human serum a common congenital immunodeficiency predisposing to infection and atopy in infancy mannan-binding protein and conglutinin in bovine serum suboptimal c3b/c3bi deposition and defective yeast opsonization. i. evidence for the absence of essential co-factor activity isolation and characterization of two distinct mannan-binding proteins from rat serum two distinct classes of carbohydrate-recognition domains in animal lectins a serum lectin (mannan-binding protein) has complement-dependent bactericidal activity the level of mannan-binding protein regulates the binding of complement-derived opsonins to mannan and zymosan at low serum concentrations molecular characterization of the mouse mannose-binding proteins. the mannose-binding protein a but not c is an acute phase reactant structure of a c-type mannose-binding protein complexed with an oligosaccharide human mannose-binding protein is identical to a component of ra-reactive factor association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series cutting edge: complementactivating complex of ficolin and mannose-binding lectin-associated serine protease mannose-binding lectin accelerates complement activation and increases serum killing of neisseria meningitidis serogroup c activation of the lectin complement pathway by h-ficolin (hakata antigen) differential recognition of obligate anaerobic bacteria by human mannose-binding lectin differential binding of mannose-binding lectin to respiratory pathogens in cystic fibrosis human mannose-binding protein inhibits infection of hela cells by chlamydia trachomatis binding of mannan-binding protein to various bacterial pathogens of meningitis interaction of human mannose-binding protein with mycobacterium avium interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 high mannose glycans and sialic acid on gp120 regulate binding of mannose-binding lectin (mbl) to hiv type 1 mannose binding lectin (mbl) and hiv mannan-binding protein and bovine conglutinin mediate enhancement of herpes simplex virus type 2 infection in mice mannan-binding lectin modulates the response to hsv-2 infection mannose binding protein is involved in first-line host defence: evidence from transgenic mice binding of host collectins to the pathogenic yeast cryptococcus neoformans: human surfactant protein d acts as an agglutinin for acapsular yeast cells mannose-binding lectin is a component of innate mucosal defense against cryptosporidium parvum in aids recognition of plasmodium falciparum proteins by mannan-binding lectin, a component of the human innate immune system the major surface glycoprotein of trypanosoma cruzi amastigotes are ligands of the human serum mannose-binding protein novel masp2 variants detected among north african and sub-saharan individuals analysis of mannose-binding lectin 2 (mbl2) genotype and the serum protein levels in the korean population association of mannose-binding lectin gene haplotype lxpa and lypb with interferon-resistant hepatitis c virus infection in japanese patients key: cord-324034-6cmztvyf authors: ashare, rebecca l; bernstein, steven l; schnoll, robert; gross, robert; catz, sheryl l; cioe, patricia; crothers, kristina; hitsman, brian; marhefka, stephanie l; mcclure, jennifer b; pacek, lauren r; vidrine, damon j; vilardaga, roger; kaufman, annette; edelman, e jennifer title: the united states national cancer institute’s coordinated research effort on tobacco use as a major cause of morbidity and mortality among people with hiv date: 2020-08-17 journal: nicotine tob res doi: 10.1093/ntr/ntaa155 sha: doc_id: 324034 cord_uid: 6cmztvyf the use of antiretroviral therapy (art) for people with hiv (pwh) has improved life expectancy. however, pwh now lose more life-years to tobacco use than to hiv infection. unfortunately, pwh smoke at higher rates and have more difficulty maintaining abstinence than the general population, compounding their risk for chronic disease. in this commentary, we describe a united states national cancer institute (nci)-led initiative to address the relative lack of research focused on developing, testing, and implementing smoking cessation interventions for pwh. this initiative supports seven clinical trials designed to systematically test and/or develop and test adaptations of evidence-based smoking cessation interventions for pwh (e.g., combination of behavioral and pharmacological). we summarize each project, including setting/recruitment sites, inclusion/exclusion criteria, interventions being tested, and outcomes. this initiative provides critical opportunities for collaboration and data harmonization across projects. the knowledge gained will inform strategies to assist pwh to promote and maintain abstinence, and ensure that these efforts are adaptable and scalable, thereby addressing one of the major threats to the health of pwh. reducing smoking behavior may be particularly important during the covid-19 pandemic given that smokers who become infected with sars-cov-2 may be at risk for more severe disease. a c c e p t e d m a n u s c r i p t improved life expectancy for pwh, with some showing a life expectancy similar to the general population. 1 this public health achievement has increased the need to address modifiable health risk behaviors, most notably tobacco use. pwh now lose more life-years to tobacco use than to hiv 1 and almost one-quarter of all deaths among pwh on art is attributed to smoking. 2 although evidence is still emerging, tobacco use may be predictive of covid-19 disease progression, 3 making pwh who smoke particularly vulnerable to the current pandemic. unfortunately, the prevalence of tobacco use among pwh remains disproportionately higher than the general population. 4, 5 in 2014, the smoking rate among pwh in the united states was estimated to be 34% -two-times greater than the general population (17%). 6 global prevalence is also high: 27 low-and middle-income countries surveyed in 2015 indicated approximately 24% of pwh smoke. 7 moreover, smoking cessation rates among pwh are lowest among those with a higher prevalence of health disparities including women, non-hispanic blacks, those with lower educational attainment, and those living below poverty levels. a c c e p t e d m a n u s c r i p t while most pwh are interested in stopping smoking, 4-6 particularly when cessation treatment is integrated with hiv care, 9 there has been a relative lack of research focused on developing and testing smoking cessation interventions specifically for pwh. 4 reviews conclude that there are insufficient data to indicate that cessation interventions, which are efficacious in the general population, are similarly efficacious for pwh. 5, 10, 11 for example, while mobile behavioral treatments have demonstrated feasibility among pwh, abstinence rates for these interventions remained low at 3 months (10-12%). 12, 13 pilot studies of behavioral interventions that incorporated motivational interviewing, contingency management, and strategies to address negative affect 10, 14 have shown promise for pwh, but fully powered trials are lacking. most studies that have examined the impact of tobacco treatment medications in pwh have utilized nrt, although a few examined varenicline. 10, 14 recently, an algorithm-based approach for selecting smoking cessation medication demonstrated feasibility and reduced smoking behavior, but abstinence was not examined. 15 two randomized placebo-controlled trials conducted among pwh found that varenicline significantly increased quit rates, compared with placebo. 16, 17 however, cessation rates among varenicline-treated participants (28-29%) 16, 17 were lower than the general population (44% 18 ). the limited available research indicates that behavioral interventions and medications yield moderate effects on cessation, but abstinence rates among pwh are generally lower than in the general population. 5, 11 there is also a lack of robust literature regarding strategies for switching treatments when smokers are not responsive to the initial treatment. despite evidence that current treatments are better than no treatment, implementation of evidencebased treatments for smoking cessation among pwh remains low, and recommended treatments are inconsistently provided. 19 recent data from the veterans aging cohort study found that pwh were less a c c e p t e d m a n u s c r i p t likely to receive nrt than veterans without hiv. 19 moreover, less than 4% of pwh use varenicline, and only 20% of hiv clinicians recommend varenicline to their patients who smoke. 9 digital interventions have taken on greater significance during the covid-19 pandemic. however, a review of smoking cessation apps revealed that only two were developed for pwh. 20 thus, there is an urgent need to identify novel and scalable strategies that meet the needs of the patients and setting (i.e., community, clinic), including the need to deliver treatments remotely when face-to-face interactions pose health risks, such as during the covid-19 pandemic. there is also a need for simultaneous evaluation of the patient, clinician, and community/organizational level factors that may impact intervention delivery. importantly, the studies will be conducted in diverse geographic locations and will recruit pwh through various sites (e.g., va hospitals, community hiv clinics, academic medical centers, and social media). these studies will accumulate a large, diverse population that is more representative of the population of pwh who smoke than any single study could have achieved. in addition to enhancing reach, this will enable consideration of health disparities by racial/ethnic groups, socioeconomic status, gender, and sexual orientation. the sum of knowledge obtained from cross-project measures will also facilitate evaluation of effective interventions (vs control) and critically, which aspects are most valuable (e.g., pharmacotherapy vs behavioral; in-person vs remote; clinical vs community vs remote samples). lastly, another nci-led foa on tobacco use and hiv in low-and middle-income countries (https://grants.nih.gov/grants/guide/pa-files/par-18-023.html, https://grants.nih.gov/grants/guide/pafiles/par-18-022.html) will allow investigators to link parallel on-going and future research in geographic areas where hiv and tobacco use are major threats. because of the success of hiv treatment, we are faced with the need to address the major threat of tobacco dependence to the health of pwh. by supporting the rigorous evaluation of the adaptation and implementation of evidence-based treatments for pwh who smoke, this nci-led initiative will provide critical knowledge about how to promote abstinence and ensure that these efforts are scalable. these nci-supported studies will provide an opportunity to disseminate findings to key stakeholders to a c c e p t e d m a n u s c r i p t enhance reach and sustainability, thereby expanding the impact of the nih's investment. although additional research is needed, tobacco use likely exacerbates the effects of covid-19 infection. 3 physical distancing recommendations have made identifying creative strategies for delivering smoking cessation interventions without face-to-face interactions a priority. the current studies are well-positioned to provide critical information about the impact of covid-19 on pwh who smoke. stakeholders interested in tobacco cessation should be aware of this initiative and the rich evidence-base it will provide. smoking and life expectancy among hiv-infected individuals on antiretroviral therapy in europe and north america smoking-related health risks among persons with hiv in the strategies for management of antiretroviral therapy clinical trial impact of smoking status on disease severity and mortality of hospitalized patients with covid-19 infection: a systematic review and meta-analysis a review of the literature concerning hiv and cigarette smoking: morbidity and mortality, associations with individual-and social-level characteristics, and smoking cessation efforts tobacco use, use disorders, and smoking cessation interventions in persons living with hiv. curr hiv/aids rep trends in cigarette smoking among adults with hiv compared with the general adult population, united states -2009-2014 tobacco use among people living with hiv: analysis of data from demographic and health surveys from 28 low-income and middle-income countries. the lancet global health the impact of cigarette smoking on hiv/aids: urgent need for research and cessation treatment positive smoking cessation-related interactions with hiv care providers increase the likelihood of interest in cessation among hiv-positive cigarette smokers. aids care smoking cessation for people living with hiv/aids: a literature review and synthesis behavioral interventions for tobacco use in hiv-infected smokers: a meta-analysis long-term outcomes of a cell phone-delivered intervention for smokers living with hiv/aids a qualitative systematic review of cigarette smoking cessation interventions for persons living with hiv delivery and implementation of an algorithm for smoking cessation treatment for people living with hiv and aids. aids care placebo-controlled randomized clinical trial testing the efficacy and safety of varenicline for smokers with hiv. drug alcohol depend varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, vs sustained-release bupropion and placebo for smoking cessation: a randomized controlled trial receipt and predictors of smoking cessation pharmacotherapy among veterans with and without hiv mobile applications for the treatment of tobacco use and dependence harmonizing cigar survey data across tobacco centers of regulatory science, center for tobacco products, and population assessment of tobacco and health studies: the cigar collaborative research group a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-319263-g49jma8n authors: marziali, megan e.; card, kiffer g.; mclinden, taylor; wang, lu; trigg, jason; hogg, robert s. title: physical distancing in covid-19 may exacerbate experiences of social isolation among people living with hiv date: 2020-04-23 journal: aids behav doi: 10.1007/s10461-020-02872-8 sha: doc_id: 319263 cord_uid: g49jma8n nan since late 2019, pandemic coronavirus disease (covid19) has spread rapidly across the globe [1] . in response, the united states centers for disease control and prevention and other health organizations have advocated for both voluntary and enforced control measures. among basic public health recommendations, social distancing measures have been recommended, involving avoiding social gatherings with ten or more people, keeping a physical distance of at least 2 m, and cancelling non-essential in-person activities [2, 3] . more extreme measures have since been implemented, including statewide "shelter-in-place" or "stay-athome" orders [4, 5] . residents are allowed to leave their houses for essential services (e.g. grocery stores, pharmacies) but are otherwise expected to remain at home [4] . at the time of writing this note, nearly all u.s. states have some form of stay-at-home order in place [6] . the influence of these restrictions on physical connection, while necessary, may have an unforeseen impact on the well-being of the entire population. such quarantine measures have never before occurred at this scale, and many individuals may be finding themselves unprepared to cope with the circumstances. individuals may experience unfamiliar challenges in receiving social support, as they are not able to gather in person. it is thus likely that rates of social isolation and loneliness will increase dramatically during the covid-19 outbreak. social isolation, or disconnectedness at the individual level, can be quantified objectively by measuring level of engagement with peers [7, 8] . loneliness, on the other hand, is a subjective experience relating to one's perceived (versus actual) degree of social connectedness [9] . it is thus possible for someone to experience loneliness while also reporting high levels of social engagement. both social isolation and loneliness have been found to have notable impacts on health within the general population. specifically, previous research has highlighted that social isolation is comparable to wellestablished risk factors for mortality, such as smoking and high blood pressure [10] [11] [12] . we have discussed that people living with hiv (plhiv) are at greater risk of experiencing social isolation [13] . it has been suggested that this is primarily due to both experienced and perceived stigma inhibiting the formation of social networks [14, 15] . this has been shown to occur through mechanisms such as fear of rejection and concealment of hiv status, or the use of social isolation as a coping mechanism to avoid hiv disclosure [14, 16] . plhiv may have experienced the loss of social network members in the early years of the hiv epidemic [17] , further heightening potential vulnerability to experiencing isolation. as a matter of example, we have examined data from the longitudinal investigation into supportive and ancillary health services (lisa) study (2007-2010), regarding the extent of social isolation among plhiv [18, 19] . social isolation was measured using latent class analysis. other person, in a relationship, had someone reliable to count on for support and friendship, or reported being 'socially satisfied.' the mi group (n = 508, 54.3%) was composed of plhiv who reported: living alone, not engaged in any type of relationship, not socially satisfied, or not having someone reliable to count on for support and friendship. lastly, the si class (n = 88, 9.4%) included individuals likely to live alone, not in a relationship, not socially satisfied, or did not have someone to count on for support and friendship. when considering those included in both the minimally and socially isolated classes, 63.7% (n = 596) experienced some degree of social isolation in the lisa study. given the prevalence of social isolation that we identified among plhiv, it is particularly relevant to consider the impacts of the covid-19 outbreak within this population. as previously noted, older adults living with hiv have restricted social networks due to both hiv-related stigma and ageism [17] . the aging population is also most susceptible to severe health effects of covid-19 [2] . therefore, due to the various shelter-in-place and physical distancing measures, it is likely that this disease is resulting in more social isolation, and in greater severity, than previously experienced among plhiv. in addition, many aids service organizations and other community-based organizations, which can provide opportunities for socializing to combat isolation and loneliness among plhiv, have been required to limit non-essential programming [20] . while this is absolutely necessary for limiting covid-19 cases, it seems evident that actions taken to curb spread of this respiratory virus will, in fact, exacerbate isolation-related vulnerabilities among plhiv. however, it has yet to be seen how these vulnerabilities will play out. notably, our recent research has further highlighted the impact of loneliness on health status. among participants with poor self-rated physical health, of whom 42% are living with hiv, 87% experienced loneliness. this is in comparison to those who reported good physical health, of which 27% are living with hiv, and where 59% experienced loneliness [21] . this study provides further support for the notion that social isolation and loneliness can have a tangible impact on the health of an individual. social distancing measures are necessary to reduce the number of covid-19 cases. however, the long-term impacts of this and related measures must also be considered in terms of their effects on the health of plhiv. while many schools and companies have transitioned to functioning online, community based organizations may lack the capacity to do so. given that these organizations help support socializing needs of plhiv, it would be beneficial to provide increased funding at this time to support the establishment of online programming or telemedicine projects, for example. as previous research has shown that programs targeting loneliness are most beneficial when those affected are involved in the implementation and conceptualization of the projects [9] , it is imperative that plhiv are included when discussing needed programming. further, individuals marginalized by socio-structural inequities may not have access to materials required for online participation. therefore, it is necessary to also focus on evaluating mental health of plhiv, during and after the covid-19 control measures, to better understand and address loneliness in this population. early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia covid-19): how to protect yourself and others. centers for disease control and prevention coronavirus disease (covid-19) advice for the public [internet]. world health organization governor cuomo signs the 'new york state on pause' executive order governor gavin newsom issues stay at home order california state government see which states and cities have told residents to stay at home: new york times loneliness, social isolation, and behavioral and biological health indicators in older adults social disconnectedness, perceived isolation and health among older adults the cambridge handbook of personal relationships social connectedness is associated with fibrinogen level in a human social network social relationships and health social isolation: a predictor of mortality comparable to traditional clinical risk factors relationship between social isolation and mortality among people living with hiv in british columbia social relationships, stigma and adherence to antiretroviral therapy for hiv/aids internalized stigma and hiv status disclosure among hivpositive black men who have sex with men stress and coping in women living with hiv: a meta-analytic review the aging hiv/aids population: fragile social networks predictors of social isolation among people living with hiv in british columbia cohort profile: longitudinal investigations into supportive and ancillary health services response to covid-19 loneliness and self-rated physical health among gay, bisexual and other men who have sex with men in vancouver key: cord-340777-d1vwjqk6 authors: o’byrne, patrick; orser, lauren; vandyk, amanda title: immediate prep after pep: results from an observational nurse-led pep2prep study date: 2020-08-28 journal: j int assoc provid aids care doi: 10.1177/2325958220939763 sha: doc_id: 340777 cord_uid: d1vwjqk6 patients who use post-exposure prophylaxis (pep) are at ongoing risk for hiv acquisition after completing pep. while the centers for disease control and prevention recommends pre-exposure prophylaxis (prep) use immediately after pep, some practitioners are hesitant to offer pep-to-prep (pep2prep). we began offering pep2prep in the sexually transmitted infection clinic in ottawa, canada on august 5, 2018. during the first 16 months of pep2prep, 61 patients requested pep and 46 were initiated; 30 of these patients agreed to pep2prep and 26 followed through. none of our pep patients had confirmed hiv exposures; all fulfilled the initiation criterion of condomless anal sex with a male partner of unknown hiv-status. during the study, the number of pep requests and initiations was statistical unchanged, yet the seroconversion rate among patients who used pep decreased from 1.7% pre-pep2prep to 0% post-pep2prep. regarding follow-up, most discontinuations occurred between the prep intake and 1-month follow-up visit. persons who use post-exposure prophylaxis (pep), which is the use of antiretroviral medications after a potential hiv exposure to prevent seroconversion, are vulnerable to hiv acquisition in the past, present, and future: (1) in the past-this is why pep is used; (2) in the present-pep can fail, and did so at a rate of 19% in the only case-control study to evaluate its efficacy, 1 yet at rates of less than 5% to 10% in subsequent observation studies 2 ; and (3) in the future-because studies have found diagnosis rates of up to 13% among gay men within 12 months of pep use among patients who used this intervention. 3 this last point has led some guidelines to suggest that pep use more than once warrants preexposure prophylaxis (prep), which is the use antiretroviral medications before a potential hiv exposure to prevent seroconversion. 4, 5 the possibility of hiv acquisition in the near future (eg, less than 12-month period) after pep use has also led the united states centers for disease control and prevention 4 and the international antiviral association (usa panel) 6 to hiv prep is warranted after the use of hiv pep. immediate pep2prep corresponded with decreased hiv seroconversion rates among persons who used pep. what are your research's implications toward theory, practice, or policy? while more research is required to further show the safety and efficacy of pep2prep, our findings provide proof-ofconcept for this approach to hiv prevention and care. recommend immediate pep-to-prep (pep2prep) transitions, such that persons who use pep start prep without interruption in drug therapy. other authors, 7 however, caution against pep2-prep until patients are established as hiv-negative. this is because (1) persons use pep due to an hiv exposure, (2) pep is not guaranteed to prevent hiv acquisition, 4 and (3) prep initiation during hiv seroconversion can produce drug resistance, and did so in prep trials at rates of about 41% among participants with acute hiv infections, compared to 3% among those who became hiv-positive due to prep failure. 8 a more recent study 9 identified a risk ratio of 3.34 (95% ci: 1.11-10.06) for drug resistance among persons starting prep during acute hiv infection compared to those without acute infection. notably, despite this rate of occurrence for drug resistance, the iprex study 10 identified that most drug resistance waned by 24 weeks of prep discontinuation. the issue with the recommendations for and against pep2-prep is that little research supports both positions. compounding this is that the real-world pep delivery is often not clear-cut. because canadian 5 and united states 4 guidelines state that condomless anal sex between men of unknown hiv-status warrants pep, many patients initiate pep after unconfirmed exposures, of which an unknown subset would not actually have been bona fide exposures. while this point is important, in frontline clinical practices, such information often cannot be known. this is not to say that unconfirmed exposures do not warrant pep, but that, because an unknown subset of patients use pep when they did not actually have an hiv exposure, the concerns about prep after pep may be smaller than anticipated because no harms would be possible. compounding this situation is that, to then further delay prep in these cases could leave patients vulnerable to hiv acquisition until practitioners feel confident that patients do not have acute hiv infection, which may never occur if patients continue to engage in hiv practices that transmit hiv. as such, the decision to start pep2prep is a balance of stewardship (protecting against drug resistance) and ethics (withholding an intervention that prevents hiv seroconversion by 96%-99%). 4, 5 to inform this decision and address high seroconversion rates among pep patients, we began offering pep2prep to patients who (1) initiated pep at our sexually transmitted infection (sti) testing clinic, (2) reported good adherence to the pep, and (3) had no serologic or physical evidence of hiv infection. our objective was to explore the uptake rate of prep after pep, the relationships between specific patient characteristics and prep discontinuation of prep continuation, and the hiv seroconversion rate among our pep patients overall and pep2prep patients. we report here on the first 16 months of observational findings from a nurse-led prep clinic that is situated within the sti clinic in ottawa, canada. our sti clinic is located downtown ottawa, canada, which is a city of 1 million residents, with the greater region approaching 1.5 million residents. ottawa public health operates this clinic, which has over 20 000 patient visits per year, and offers full sti screening, pep, prep, and contraception services. this clinic is like most other publicly-funded public health sti clinics in canada. while nurses, physicians, and nurse practitioners work in this sti clinic, nurses operate the pep and prep projects under medical directives from nurse practitioners. at this clinic, we offer nurse-led pep and prep to patients who qualify based on guidelines and clinical judgement. 4, 11 for pep, we use once-daily oral emtricitabine-tenofovir df 200/ 300 mg plus raltegravir hd 1200 mg daily for 28 days. for prep, we use once daily oral emtricitabine-tenofovir df 200/ 300 mg. we do not offer intermittent prep use and refer any patient who requests this to an alternate prep clinic where this is available. for pep2prep, we offer patients an appointment to start prep at the point of pep initiation and, if declined at first offer, again after 2 weeks during routine nursing followup. patients who agree to prep are referred to our prep clinic for an initiation visit after approximately 3 weeks of pep (see figure 1 ). patients who decline pep2prep at both offers are informed they can request prep at any point. we also encourage these patients to do routine follow-up according to canadian guidelines, 5 and we follow these patients for up to 1 year after their pep use for routine hiv and sti screening. notably, in contrast to the canadian pep guidelines, 5 we offer prep to anyone who uses pep once. our rationale is that, before prep, approximately 13.1% of gay men who obtained pep from our sti clinic were diagnosed with hiv within 12 months of using pep. 3 we, therefore, feel that a single episode of pep use justifies prep use. at the first prep visit, we repeat hiv testing using the fourthgeneration abbott architect (which has an estimated 85% sensitivity to detect acute hiv infection in persons using prep), 12 do a thorough history and examination for signs/symptoms of hiv seroconversion, and collect blood for renal function testing. once results show the patient has no hiv antigen/antibodies and adequate renal function for prep, we send the patient a prescription to start prep the day after completing pep. we repeat these serologic and clinical assessments after 1 month of prep use and every 3 months thereafter. 13 we also perform indicated sti testing at those visits 13 ( figure 1 ). we follow-up by telephone with all prep patients who do not attend scheduled appointments, and immediately offer them a new appointment that is convenient for them. we then continue with appointments as scheduled for such patients. we proceed in the same way for patients who sporadically miss appointments. patients who miss 3 consecutive appointments, however, are informed that they can rebook for prep when it becomes more convenient for them and we offer immediate referrals to other prep clinics in the city. data for this study were extracted from our pep2prep patients' charts and input into excel by the authors. we initiated our pep2prep referrals august 5, 2018, and extracted data from the files of patients who accessed pep between that date and december 4, 2019. extracted data focused on demographics (age, gender, income), sexual and drug use practices (sex practices, gender of partners, substance use), mental health (depression, anxiety, using the patient health questionnaire 9 [phq9], and generalized anxiety 7 scale [gad7], respectively), reason for pep use and follow-up details (practices that warranted pep, symptoms, test results), and information on prep visits (attendance, symptoms, results). hiv diagnosis data were extracted from august 5, 2018, to march 4, 2020, with no subsequent data collection available due to covid-19. sociodemographic information was analyzed using descriptive statistics. the outcome of interest was prep discontinuation, defined as failing to present to clinical visit 3 (i.e., the 1 month prep recheck appointment). we were interested in exploring the relationships between specific patient characteristics and prep discontinuation. these variables included age (below versus above 25 years), income (high versus low), ethnicity (caucasian versus visible minority), anxiety (any symptoms versus none), depression (any symptoms versus none), whether the patient accepted pep2prep at the intake or follow-up visit, and previous sti (yes versus no). thus, for each variable we conducted a bivariate w 2 test to identify the presence of a statistically significant relationship (in any direction) between it and the outcome of interest at p < .1. it was our intention to explore the direction of these relationships and to include all variables deemed significant in the bivariate analyses into a multivariable model; however, our results were not amenable to this (ie, there were no significant relationships). spss version 26 was used for this part of the analysis. the ontario hiv treatment network funded this research, and the research ethics boards at the university of ottawa and at ottawa public health approved the project (ethics file numbers h-04-18-533 and 246-18, respectively). all participants in this study signed expressed consent for involvement in this project. notably, a patient's ability to obtain pep or prep was not contingent upon study enrollment. during the 16-months of data collection, 61 patients presented to our sti clinic requesting pep, 60.7% (n ¼ 37/61) were initiated and 81.1% (n ¼ 30/37) of those who used pep agreed to pep2prep. during the study period, 13.5% (n ¼ 5/37) patients accessed our clinic to initiate pep more than once, which is similar to the average of 12.7% (yearly range of 9.7-15.4%) between 2015 and 2019. of these patients, 31% (n ¼ 19/37) sought services at our clinic for the first time for pep. this rate of access and uptake, plus the client demographics of these patients, also resembles our published data on this clinical service. for more information on this pep program and its participants and outcomes, please see o'byrne et al. 3, 14 no patient initiated on pep, irrespective of prep use, was diagnosed with hiv since we implemented pep2prep. comparing pep requests and initiations pre-/post-pep2prep, 286 persons sought pep in the 60 months before we started pep2-prep for a rate of 4.76/month, and 61 sought pep in the 16 months after, for a rate of 3.81/month. for initiations, we gave pep to 207 patients in the 60 months preceding pep2prep, for a rate 3.45/month, compared to 46 initiations in the 16 months after, for a rate of 2.87/month. the observed changes in the rates of accessing and initiating pep were not significant pre-/ post-pep2prep (p ¼ .240 and p ¼ .281, respectively). the hiv seroconversion rate before pep2prep was 3.8% (n ¼ 11/286) and 0% after. of these 11 diagnoses, 6 occurred at intake when patients sought pep, and 5 occurred within 12 months of using pep; none of these 5 diagnoses were pep failures, as all had tested negative after using pep. the rate of hiv diagnosis after pep before we implemented pep2prep was thus 2.4% (n ¼ 5/207) for initiations, which, because we follow all pep patients for 1 year after pep use, equates to an hiv incidence of 5.4 per 100 person-years. using this pre-pep2prep seroconversion rate, from our 37 pep initiations who generated 24.5 person-years of follow-up as of march 4, 2020, we would have expected 1.3 diagnosis during the pep2-prep study period. patients who agreed to prep after pep use had a mean age of 30.3 years. they were 97% male (n ¼ 29/30), 61% caucasian (n ¼ 17/28), and all reported sex with male partners. as well, 39% screened positive for anxiety or depression on the phq-9 or gad-7-all of whom were already connected with mental health services. see table 1 for more details. all patients who agreed to prep after pep use started pep due to condomless anal sex with a male partner of unknown hiv-status, with 72% (n ¼ 21/29, 1 unknown) of these contacts being receptive anal sex. as such, there were no confirmed hiv exposures. of these patients, 93% (n ¼ 28/30) completed pep, with 1 lost to follow-up and 1 discontinuing. all had negative hiv test results at baseline and all but the 2 who did not follow-up had repeat negative test results at least 3 weeks later. of the 30 patients who initiated prep due to pep, 87% (n ¼ 26/30) did immediate pep2prep transitions; 13% (n ¼ 4/30) had completed pep for 4 to 16 weeks before prep. one patient accepted prep when offered at pep initiation but declined before starting. for the 26 patients who did immediate pep2prep transitions, 85% (n ¼ 22/26) attended their second visit, and 66% (n ¼ 14/21) of those eligible to do so attended their third visit (n ¼ 3 had not reached this visit yet and n ¼ 2 moved). of those eligible to attend the fourth visit, 79% (n ¼ 11/14) did so (n ¼ 2 had not reached this visit yet, n ¼ 1 was referred to another clinic). of those eligible to attend the fifth visit, 87.5% (n ¼ 7/ 8) did so (n ¼ 2 had not reached this visit yet). therefore, removing the 7 participants who had not yet progressed to visit 5, plus the 4 who continued at another clinic and the 2 who moved, we had 53.8% (n ¼ 7/13) of participants retained on prep by visit 5. table 2 and figure 2 show the pep2prep cascade. hiv tests at all visits were negative, and no patient reported symptoms of hiv seroconversion, although we diagnosed 16% (n ¼ 4) of the 25 patients we tested for stis with 5 stis: 2 cases of syphilis, 2 cases of rectal gonorrhea, and 1 case of oral gonorrhea. these diagnoses occurred at the 1-month visit (n ¼ 1 patient with rectal gonorrhea), the 4-month visit (n ¼ 1 patient with syphilis and rectal gonorrhea), and the 7month visit (n ¼ 1 patient with syphilis and n ¼ 1 patient with oral gonorrhea). moreover, no one discontinued prep due to renal dysfunction. chi-square tests found no significant differences between the observed and expected frequencies for attendance at visit 3 (which was the largest drop-out point for pep2prep) and the following items: age (below versus above 25 years), income (high versus low), ethnicity (caucasian versus visible minority), anxiety (any symptoms versus none), depression (any symptoms versus none), whether the patient accepted pep2-prep at the intake or follow-up visit, and previous sti (yes versus no). in this article, we reported on the findings of the first 16 months of our nurse-led prep clinic, focusing on the patients who agreed to pep2prep. from 46 eligible pep patients during the study period, 30 agreed to prep and 26 accepted immediate prep initiation after pep completion. notably, the rate of discontinuing prep was highest between visits two and three. for clinical outcomes, 16% of our pep2prep patients were diagnosed with an sti while using prep, but none was diagnosed with hiv in our clinic or elsewhere in ontario, although we do not have results for those who moved out of province or since the covid-19 shutdowns (as testing was restricted during that time). notably, nearly half of these participants reported mild or moderate anxiety and/or depression. these results raise a few points for discussion. first, these results contribute generally to the everexpanding literature on hiv prep and more specifically to the sparse literature on pep2prep. regarding the overall literature on prep, our findings add more data on the outcomes associated with nurse-led prep, showing its operational feasibility. [15] [16] [17] [18] [19] [20] while several studies confirm such findings about the functionality of nurse-led prep, [15] [16] [17] [18] [19] [20] much of this research has focused on evaluating if nurses can provide prep independently or in various collaborating roles and on the outcomes associated with nurses initiating and/or monitoring patients who use prep. also unique to our study is that we focused on pep2prep and the outcomes associated with an immediate transition from one intervention to another. while our sample is small, our results suggest that this intervention is reasonable. second, the hiv positivity rate among our patients who used pep was 0%, which decreased from the previous rate of 1.7% per year for the preceding 5 years. based on historical data, we would have expected at least 1 hiv diagnosis during the 16-month study period. while the patients who accessed pep before and after pep2prep are not the same persons, it is the same clinic and there were no other changes to the pep program during the study period. as well, that we diagnosed 16% of pep2prep patients with stis during follow-up signals ongoing sexual practices that can result in hiv acquisition, which aligns with published literature. 21 this lack of hiv diagnosis, however, could be coincidental or an artefact of missed diagnoses due to prep medication affecting diagnosis, although patients did seroconvert in the prep studies. 12 it is also reassuring that missed diagnoses are not likely because our patients reported good pep and prep adherence, had no signs/ symptoms of hiv seroconversion, and all had negative hiv test results at pep initiation, 1 week before starting prep, and 1 month after being on prep. however, longer-term follow-up may be required to better determine such outcomes. that there were no hiv diagnoses among our pep2prep patients suggests that this transition could be appropriate, which might help appease those who feel patients need to be established as hiv-negative through serologic testing after being off pep for the duration of the hiv testing window. seeing as 1.7% of our patients who used pep had historically become hiv-positive (with this rate rising to 10% among gay men), 3 delaying prep means depriving many highly vulnerable patients of an available intervention. reinforcing the importance of pep2prep is that other pep studies have similarly found high rates of seroconversion after pep use. 4, [22] [23] [24] our findings thus suggest that withholding pep2prep might not be ideal, although the lack of hiv diagnoses among our pep2-prep patients could be because no patient had a true hiv exposure; all sought pep due to condomless anal sex with same-sex male partners of unknown hiv-status. as noted above, the point here is not that pep was inappropriately initiated in such instances (it absolutely was warranted and appropriately provided); rather, our assertion is that, because an unknown subset of these instances of pep use were possibly not required, in these unknown cases, there would be absolutely no harms of associated prep drug resistance because the person did not have a true exposure. this point is further reassuring for this transition. third, despite the potential success of this program, there was a high loss-to-follow-up rate between the prep initiation and follow-up visit after 1 month of prep use. most who continued with prep after this visit continued thereafter, signaling a potential period to maintain retention in care. while patients may agree to initiate prep after pep use, a substantial number may not continue prep after this time, as was the case in our study where over 4 of 5 eligible participants agreed to prep after pep use, yet only 1 in 2 of participants who initiated prep were retained in care by visit 5. as was also found in other studies, [25] [26] [27] offering prep is insufficient; additional efforts to increase retention are required. without efforts to increase retention for prep use, the population-level success of this intervention will likely be limited. moreover, that there were no clear sociodemographic factors associated with loss-tofollow-up suggests that this discontinuation may relate to the intervention itself, rather than it being a group-specific factor that makes people less willing and/or able to engage in treatment. alternatively, the loss-to-follow-up in our study, which is higher than in most published prep studies, 15-20 may have arisen because our study was observational and involved routine clinical practices; in other words, our study was not a rigid clinical trial with study-funded retention strategies. as such, our findings might more accurately reflect real-world prep retention-and in fact do align with a published analysis of 16 907 participants from 95 studies, which identified that "by the end of 1 year, almost 40% of the participants who started treatment in these clinical trials had stopped taking the medication(s), despite the protocol-specific regimen of continuously maintained dosing." 28(p278) as such, our retention rate of 53.8% more closely aligns with this published rate of 60% continuation at 1 year, versus rates of 70% to 80% retention in the literature. [15] [16] [17] [18] [19] [20] fourth, that nearly half of the participants screened positive for mild-to-moderate anxiety or depression on the gad7 and phq9 29 emphasizes the need for clinicians to assess for these disorders among persons who seek pep and prep. other studies have similarly found elevated rates of mood disorders among patients who use pep and prep. [30] [31] [32] although no significant correlation was identified between anxiety or depression and prep discontinuation, previous studies have identified this relationship. 33 as such, mood disorders may be the reason prep is needed (in that sexual practices that can transmit hiv could be the manifestation of depression or anxiety) and might be reason it is not used in an ongoing fashion (in that these illnesses undermine retention in care). it is thus prudent for clinicians in pep and prep programs to screen for, and provide services or referrals related to, mental health. ignoring this may leave a driving force behind some sexual practices unaddressed and may undermine retention in care. our findings must be interpreted considering certain limitations. for one, our sample was small and decreased over time. whether these results would be maintained with a larger sample is unknown. it is also uncertain whether our results would translate to areas with different testing technologies and without nurses who are experts in hiv assessment-although the literature suggests that nurse-led prep is feasible. we also do not know whether patients who did not follow-up accessed prep elsewhere. lastly, our sample was well-educated and insured, with most being caucasian and identifying as gay. whether these findings would apply to other groups is unclear. our pep2prep study found that many patients who were eligible for prep declined this intervention and that many who started prep discontinued after 4 weeks. we also did not find any hiv diagnoses among prep2pep patients, and the diagnosis rate among pep patients decreased from 1.7% to 0% after starting pep2prep. this is a reassuring finding considering that we would have expected at least 1 hiv diagnosis during our follow-up period; however, this finding could have been an artefact of a small study sample and disruptions caused by covid-19. nonetheless, despite these limitations, our results provide a proof-of-concept for pep2prep as one strategy to reduce hiv transmission among a group with historically high rates of seroconversion. 3 now, larger scale studies need to determine if these results hold true for other populations. in the meantime, pep2prep appears to be an appropriate intervention, and our findings support the centers for disease control and prevention 4 guidelines to engage in this practice when clinicians can be reasonably certain that patients do not have acute hiv infection. perhaps such an approach would yield similar outcomes that we observed, resulting in decreased hiv transmission more broadly. a case-control study of hiv seroconversion in health care workers after percutaneous exposure the efficacy of post-exposure prophylaxis (pep) for hiv nurse-led pep program used by men at high risk for hiv seroconversion preexposure prophylaxis for the prevention of hiv infection in the united states-2017 update canadian guideline on preexposure prophylaxis and nonoccupational postexposure prophylaxis antiretroviral drugs for treatment and prevention of hiv infection in adults challenges of hiv diagnosis and management in the context of pre-exposure prophylaxis (prep), post-exposure prophylaxis (pep), and test and start and acute hiv infection: a scoping review should we fear resistance from tenofovir/emtricitabine prep? drug resistance during hiv pre-exposure prophylaxis hiv-1 drug resistance in the iprex preexposure prophylaxis trial responding to critiques of the canadian prep guidelines: increasing equitable access through a nurse-led active-offer prep service assessment of hiv screening tests for use in preexposure prophylaxis programs prep-rn: clinical considerations and protocols for nurse-led prep community-based, nurse-led post-exposure prophylaxis: results and implications evolution of a preexposure prophylaxis (prep) service in a community-located sexual health clinic: concise report of the prepxpress implementing hiv preexposure prophylaxis (prep): let's not get caught with our pants down nurse-led preexposure prophylaxis: a non-traditional model to provide hiv prevention in a resource-constrained, pragmatic clinical trial same-day hiv preexposure prophylaxis (prep) initiation during drop-in sexually transmitted diseases clinic appointments is a highly acceptable, feasible, and safe model that engages individuals at risk for hiv into prep care integration of prep services into routine antenatal and postnatal care: experiences from an implementation program in western kenya implementing pre-exposure prophylaxis for hiv prevention at an urban youth clinic global epidemiologic characteristics of sexually transmitted infections among individuals using preexposure prophylaxis for the prevention of hiv infection: a systematic review and meta-analysis high hiv incidence among msm prescribed postexposure prophylaxis, 2000-2009: indications for ongoing risk behaviour nonoccupational postexposure prophylaxis, subsequent risk behaviour and hiv incidence in a cohort of australian homosexual men a randomized noninferiority trial of standard versus enhanced risk reduction and adherence counselling for individuals receiving post-exposure prophylaxis following sexual exposures to hiv retention in care outcomes for hiv pre-exposure prophylaxis implementation programmes among men who have sex with men in three us cities patient disengagement from an hiv preexposure prophylaxis program in a sexually transmitted disease clinic. std hiv pre-exposure prophylaxis (prep) uptake and retention among men who have sex with men in a community-based sexual health clinic adherence to medications: insights arising from studies on the unreliable link between prescribed and actual drug dosing histories screening for depression: review of the patient health questionnaire-9 for nurse practitioners high levels of concomitant behavioural health disorders among patients presenting for hiv non-occupational post-exposure prophylaxis at a boston community health center between high prevalence of syndemic health problems in patients seeking post-exposure prophylaxis for sexual exposures to hiv high hiv risk and syndemic burden regardless of referral source among msm screening for a prep demonstration project in toronto mental health and hiv/aids: the need for an integrated response patrick o'byrne and lauren orser have contributed equally to the work. all authors have read and approved the final manuscript. patrick o'byrne contributed to conceptualization, data extraction, analysis, and writing. lauren orser contributed to data extraction, analysis, and writing. amanda vandyk contributed to data analysis and editing. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study was funded by the ontario hiv treatment network (ohtn). patrick o'byrne, rn-ec, phd https://orcid.org/0000-0002-0587-1409 key: cord-346557-s6c7d70y authors: brennan, david j.; card, kiffer g.; collict, david; jollimore, jody; lachowsky, nathan j. title: how might social distancing impact gay, bisexual, queer, trans and two-spirit men in canada? date: 2020-04-30 journal: aids behav doi: 10.1007/s10461-020-02891-5 sha: doc_id: 346557 cord_uid: s6c7d70y nan in response to the global covid-19 pandemic, canadians are being asked to engage in social distancing [1] . however, potential unintended consequences of self-imposed or legally-enforced social distancing in response to covid-19 are the potential to negative impacts on mental, social, sexual, and physical health [2] . this is especially true for marginalized populations, such as gay, bisexual, queer, trans, two-spirit, and other men who have sex with men (gbqt2+), who already have elevated rates of mental health challenges, such as anxiety, depression, and loneliness [3] [4] [5] . given that social support from friends, family, and partners is a known protective factor against negative mental health outcomes and sexual risk behavior among gbqt2+ [4] , social distancing may exacerbate negative mental health consequences. questions are also being raised about how social distancing will impact the sexual behavior of gbqt2+. social and sexual networking apps have long been used by gbqt2+ to seek social connections, either virtually or in-person. data from these applications suggest that individuals are seeking connection during the pandemic [6, 7] . for example, compared with the previous month, tinder has seen an increase of up to 15% on their apps in the united states and up to 25% in italy and spain. chat conversations are also up to 30% longer [8] . previous research on app use among gbqt2+ indicates that dating applications provide a means of engaging in romantic, sexual and interpersonal relationships, including casual sexual encounters [9] [10] [11] [12] [13] . as well, these apps are tools that help address issues of mental health, internalized homophobia and low self-esteem [14] . research is needed to understand the implications of social distancing on individuals and their sex-seeking behaviors, how individuals may be coping through dating application use, and whether individuals are continuing to engage in in-person sexual connections despite social distancing protocols. furthermore, it is unknown to what extent gbqt2+ may experience difficulty accessing health-related services and supplies (i.e., counselling, antiretroviral therapy,) during the covid-19 pandemic. indeed, services considered "non-essential" are closed or are being drastically restricted. for instance, the hassle free clinic, a toronto sti and hiv testing clinic provides services to gbqt2+ clients, has significantly reduced hours, removed clinic drop-in services, and stopped providing routine sti/hiv testing amid the pandemic [15] . additional community-based resources providing mental and sexual health-related services to gbqt2+ have significantly limited available resources or suspended programming, including sti/hiv testing, low-cost counselling services, mental health support groups, and walk-in health services [16] [17] [18] . across british columbia, as a part of cancelling all non-emergency surgeries, gender affirming surgeries have largely been cancelled or postponed indefinitely [19] . these reductions in the provision of services for gbqt2+ have the potential to cause significant harm among gbqt2+ as a group that is already marginalized. given that social distancing measures may yet last for several months [20] , and potentially even longer for older and immune-compromised gbqt2+ [21] , it is important to take this opportunity to study the impact of social distancing on marginalized populations-particularly if covid-19 we recruited participants using promotional materials in french and english shared by various partner organizations across the country through newsletters, listservs, and social media platforms (e.g., facebook, instagram, twitter). this included paid advertising and boosted posts on facebook and instagram for cbrc and partners who have a large social media presence. additionally, several community champions and local celebrity drag queens promoted the survey on their social media profiles and were provided with a small honorarium as compensation for their time. finally, cbrc purchased recruitment ads on popular sex seeking apps and websites (grindr, squirt, scruff, jack'd), porn sites (pornhub), and gbtq2-oriented media sites (xtra, fugues). the eligibility criteria restricted participation to individuals who identified as men or another gender other than women (e.g., non-binary, genderqueer, agender; inclusive of trans men and two-spirit people); identified as gay, bisexual, queer, asexual, or other non-heterosexual identity (inclusive of two-spirit) and/or have reported having had sex with another man in the last 5 years; were at least 15 years of age; lived in canada; provided informed consent; completed a questionnaire in either french or english; and did not already participate in this study cycle. participants were eligible to win a $500 gift card via random draw. among the many ways that social distancing can impact the health and vulnerability of gbqt2+, we are particularly concerned of how their social and mental health might be impacted. in fact, among 6198 gbqt2+ who participated in the 2019 sex now survey, over one-in-five gbqt2+ (21%) had patient health questionnaire-2 item [22; phq-2] depression scores greater than clinical screening thresholds and 29% self-reported having "fair" or "poor" mental health overall. further, a staggering 57% of respondents wanted help with a mental health problem they were facing. notably, of those who wanted help with a mental health problem, 19% wanted help dealing with suicidal thoughts. these statistics are alarming considering that we also see a dose-response relationship between the number of people gbqt2+ can turn to for support and the proportion who have depression symptoms and the proportion who want help dealing with suicidal thoughts. if social distancing prevents people from accessing these supports, it may contribute to worse mental health outcomes, potentially even suicide. in addition to the effects on social distancing, economic impacts may also harm gbqt2+ and their mental health. in sex now 2019, only one third (33%) of gbqt2+ reported having "extra" money and 10% reported not being able to "make ends meet." recent national research in canada highlighted that half of lgbtq2+ households (52%, compared with 39% of households overall) have experienced lay-offs or reduced employment as a result of the covid-19 pandemic [23] . the closures of bathhouses, gay bars, queer pop-up events, and lgbtq pride festival cancellations as a result of covid-19 control measures leaves connecting online as one of the few remaining options. as a baseline, 51% of sex now 2019 respondents reported meeting new sexual partners in the past three months, with two-thirds of these individuals having had sex with a new partner in the past 4 weeks (and 15% of all respondents reporting sex with a new partner in the week they completed the survey). while some people, including gbqt2+, will undoubtedly continue to connect in-person with sexual partners during the covid-19 pandemic, we know that the apps and websites are also used for online sexual acts. with 32% of gbqt2+ reporting camming or sexting online-it would suggest that at least some gbqt2+ may be able to find safe ways to fulfill their sexual needs while social distancing. it remains unclear whether these online encounters will fulfill the intimacy and connection needs of gbqt2+ men. social distancing is likely to have additional consequences with respect to antiretroviral medication adherence and implications as a result of changes in sexual health care. beyond issues of delayed or missed diagnoses of stis, especially asymptomatic ones, a lack of sti screening also means that clinical eligibility for prep [24] will be limited if rectal stis or syphilis are not diagnosed. regarding prep, 86% of hiv-negative gbqt2+ on prep reported daily use. fortunately, if access to prep is made difficult by increased burden on the healthcare system or if individuals are worried about accessing health services to get their prescription fulfilled these individuals might be able to switch to on demand regimens (only 11% were currently taking it on demand), but this may not provide adequate hiv prevention during sex if sexual frequency is very low. issues with antiretroviral adherence for people living with hiv may also be impacted by a lack of physical access to healthcare providers and peer navigators [25] . in sex now, there were already indications of sub-optimal adherence among people living with hiv: 33% reported having missed a dose in the past 4 weeks alone. disruptions to regular habits and schedules as well as potential challenges to medication access could compound these problems. control measures in response to the covid-19 pandemic has serious potential to impact the health and wellbeing of gbqt2+. studies conducted during this time should pay attention to impacts of social distancing on the mental, social, and sexual health of gbqt2+, as well as other marginalized populations. online surveys with comparable methodology to that of sex now might be particularly beneficial in order to allow comparisons to baseline indicators. supports to gbqt2+ organizations should be provided in order to help them adapt to the online provision of healthcare resources. community-based measures to mitigate the spread of coronavirus disease (covid-19) in canada centre for addiction and mental health. mental health and the covid-19 pandemic men's sexual orientation and health in canada number of psychosocial strengths predicts reduced hiv sexual risk behaviors above and beyond syndemic problems among gay and bisexual men sexual orientation and mental health how to date online in the age of covid-19. bloomberg dating app usage up thanks to covid-19, study suggests. toronto sun love in the time of coronavirus: covid-19 changes the game for online dating. deutsche welle the use of social networking applications of smartphone and associated sexual risks in lesbian, gay, bisexual, and transgender populations: a systematic review geosocial-networking app usage patterns of gay, bisexual, and other men who have sex with men: survey among users of grindr, a mobile dating app trends in internet use among men who have sex with men in the united states geosocial networking app use among men who have sex with men in serious romantic relationships breaking boundaries: the uses and gratifications of grindr use of the internet and mobile-based "apps" for sex-seeking among men who have sex with men in new york city hassle free clinic will be open by appointment only covid-19: the 519 information and updates covid-19, important updates covid-19 update: gender affirming surgeries impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand d-19?utm_sourc e=tw&utm_mediu m=socme d&utm_campa ign=03162 0&utm_conte nt=en the patient health questionnaire-2: validity of a two-item depression screener impact of covid-19: canada's lgbtqi2s community in focus canadian guideline on hiv pre-exposure prophylaxis and nonoccupational postexposure prophylaxis the hiv treatment cascade: patching the leaks to improve hiv prevention. the body pro key: cord-317213-vhprfb1o authors: tram, dai thien nhan; wang, hao; sugiarto, sigit; li, tao; ang, wee han; lee, chengkuo; pastorin, giorgia title: advances in nanomaterials and their applications in point of care (poc) devices for the diagnosis of infectious diseases date: 2016-09-26 journal: biotechnol adv doi: 10.1016/j.biotechadv.2016.09.003 sha: doc_id: 317213 cord_uid: vhprfb1o nanotechnology has gained much attention over the last decades, as it offers unique opportunities for the advancement of the next generation of sensing tools. point-of-care (poc) devices for the selective detection of biomolecules using engineered nanoparticles have become a main research thrust in the diagnostic field. this review presents an overview on how the poc-associated nanotechnology, currently applied for the identification of nucleic acids, proteins and antibodies, might be further exploited for the detection of infectious pathogens: although still premature, future integrations of nanoparticles with biological markers that target specific microorganisms will enable timely therapeutic intervention against life-threatening infectious diseases. according to the world health organization (who), in 2012 infectious diseases claimed 15 million lives worldwide (world health, 2013) . among them, human immunodeficiency virus (hiv) and tuberculosis were the leading causes of death at all age groups. in 2011, hiv claimed 1.3 million lives in sub-saharan africa alone (tarantola et al., 1993) . the extent of damage exerted by a particular infectious disease could reach well beyond the people directly plagued by the germs. a recent ebola outbreak caused so much trouble for the healthcare system in west africa that there were insufficient resources available for measles vaccination programs, thereby further adding to the death toll (takahashi et al., 2015) . an even more recent outbreak is represented by the zika virus, currently spreading in the americas and the pacific region. this has resulted in increased infections during pregnancy and microcephaly, as well as guillain-barré syndrome in adults. the transmission of pathogens is not limited to just humans. a number of transmissible microbes originated from animal vectors (e.g. birds, bats, ticks, etc.) could subsequently switch host to humans. severe acute respiratory syndrome (sars) virus, hantavirus, nipah virus and human immunodeficiency virus (hiv) are just a few of such examples (morse et al., 2012) . in the past few decades, the spread of once dreaded maladies such as smallpox and poliomyelitis have generally been kept under control, but these rigorous vaccination programs are far from being equally practiced across the globe (fonkwo, 2008) . in developing countries, a lack of proper sanitation, technologies, equipment, and human resources has been hampering efforts to provide timely treatments (batt, 2007) . identification of microorganisms by observing characteristic features of cultures has been in practice for decades. however, several limitations render this classical technique impractical for on-site diagnosis of infectious diseases, especially in resource-poor regions (kaittanis et al., 2010) . being time-consuming is one of the principal flaws of current diagnostic approaches. for preliminary results, each analysis takes 2-3 days. for more definite results, it might take up to 7-10 days. detection of salmonella typhimurium consumes 3-5 days before yielding results (he et al., 2013) , whereas diagnosis of tuberculosis via microbiological means may take weeks (dinnes et al., 2007) . an additional complication derives from the fact that, in order to procure meaningful observations, the initial serum samples must contain pathogen loads above a certain threshold level. this prerequisite might not be met if the patients are in early stages of infection. to worsen the situation, the life cycle of some bacterial strains includes a dormancy state, whereby organisms do not grow significantly in number when cultured. this could culminate in false negative results that critically undermine diagnoses. interferon gamma (inf-γ) release assay detects inf-γ produced by t-cells when the patient is exposed to mycobacterium tuberculosis antigen. however, a tuberculosis patient is usually affected by hiv at the same time. concurrent presence of hiv could readily impair the patient's immune systems. the resulting low t-cell count could mask a clinically relevant quantity of mycobacterium tuberculosis, hence leaving tuberculosis undetected (diel et al., 2011) . in the case of microbes more diminutive than bacteria (e.g. viruses, with average size of only about one-hundredth that of the average bacterium), an electron microscope is required for detailed visualization of the viral particles (i.e. virions). the growth of viral particles also necessitates a more sophisticated protocol than the one adopted for bacterial cultures (shinde et al., 2012) . technological advances have empowered medical professionals with a wide range of diagnostic tools. however, even state-of-the-art techniques are still far from being suitable for application in resourcepoor contexts, wherein infectious diseases have proven to be the most widespread. as of 2007, the gold standard for hiv diagnosis is an enzyme immunoassay which detects igm antibodies in the patient's serum, followed by western blot (branson, 2007) . two popular methods are enzymelinked immunosorbent assay (elisa) and nucleic acid test (nat) . in order to credibly detect a few virions in 100 μl of plasma sample, most commercially available methods require nucleic acid amplification (calmy et al., 2007 , fiscus et al., 2006 , rouet and rouzioux, 2007 . fourth-generation elisa, a combination assay capable of detecting both hiv igg/igm and the capsid protein p24, has a limit of detection (lod) of 4 pg/ml (speers et al., 2005) , thereby removing the need for nucleic acid amplification. the main downside is its high cost. amidst the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in 2003, real-time polymerase chain reaction (rt-pcr) (chan et al., 2004) was widely employed. however, sensitivity of the assay represented the main limitation. in specifics, it would appear below clinically established standards, were the patients infected fewer than six days before the sample extraction date (vasoo et al., 2009) . while a refinement of specimen extraction process does improve the sensitivity level, it leaves the cost issue unaddressed. another pathogen whose diagnosis utilizes rt-pcr as the standard test is the avian flu h1n1. commercially available immunochromatography-based strip for the diagnosis of h1n1 (welch and ginocchio, 2010) is not as costly, but low sensitivity and specificity limit its clinical utility (lee-lewandrowski and lewandrowski, 2001, posthuma-trumpie et al., 2009) . other than diagnosis, nat sees extensive use in screening of blood supply for common pathogens such as hiv, hepatitis b virus (hbv), and hepatitis c virus (hcv) (fiscus, cheng, 2006) . it is also employed to monitor patient progress throughout treatment courses. genexpert is the first fully integrated nat system. it could produce test outcomes in 2 h. despite the relatively shorter assay time, the problems of cost and energy consumption remain (meyer-rath et al., 2012) . according to the college of american pathologists, poc testing could be considered as on-site diagnostic tests carried out using mobile devices readily accessible to the patients and the in-charge physicians (lamb et al., 1995) . another more concise definition is 'testing done in the proximity of patient care' (kiechle et al., 1990) . the portable devices employed can be either hand-held or transported on a cart (urdea et al., 2006) . the acronym "assured" was coined by who to denote the fundamental criteria of poc testing: affordable, sensitive, specific, userfriendly, rapid and robust, equipment-free, and deliverable to end user (sista et al., 2008) . as mentioned above, there is an increasing demand for diagnosis of infectious diseases in resource-poor regions. a paucity of laboratory technicians with necessary know-hows is, among others, a major concern under such circumstances. hence, poc devices need to be userfriendly with easy-to-follow instructions. as such, routine tests may be performed by family members, or even the patients themselves. apart from mobility, short assaying time is crucial to poc testing. in cases where such time reduction does not hold much clinical significance, cutting down on the waiting time helps alleviate patients' discomfort (holland and kiechle, 2005) . conventionally, poc tests are classified into 4 main categories : 1) those whose speediness is the most valuable attribute, to reinforce decision on treatment regimen of lethal conditions (e.g. meningitis); 2) those whose short assaying time is also a crucial element for prompt measures to restrain an outbreak (e.g. mrsa in hospitals); 3) those simply for verification of the disease-causing microbes; 4) those for self-monitoring by patients who do not attend follow-ups (e.g. in the case of patients with sexually transmitted diseases). despite this classification, poc devices are still at their infant stage. by 2005, several poc systems had been investigated in clinical trials. however, none had been released for commercial uses (liao and huang, 2005) . in general terms, a poc device is designed to detect, either qualitatively or quantitatively, the presence of a specific biomarker characteristic of the malady at stake. at the moment, the analytes of interest range from nucleic acids of the microbes or proteins released by them during their time residing in the host, to antigens located on the surface of the microbes themselves. therefore, the review of the current pocs has been organized into 1) nucleic acids, 2) whole pathogens and 3) proteins & antibody detection systems. many diagnostic techniques have been revamped and adopted for detection of microorganisms' genetic materials: electrophoresis, spectrophotometry, rt-pcr, etc. (cagnin et al., 2009 ). however, adopting them not only for laboratory experiments, but also clinical applications at patient sites has proven to be a challenge. electrochemical (ecm) sensing was originally contrived for applications in laboratories, but recent advancements in technology have refined its suitability for the development of poc devices (liepold et al., 2005) . on its own, ecm already has desirable properties (lucarelli et al., 2009 , pohlmann et al., 2009 , wakai et al., 2004 , wei et al., 2010 . firstly, ecm sensors do not require much expertise to maneuver. the steps entailed in sample manipulation process are straightforward as well. in terms of resources, these systems can work with sample size smaller than usual, which ranges from a couple of microliters all the way to nanoliters, and do not consume much energy. all these fortes render ecm sensors suitable for poc assays. a typical ecm sensor consists of an electrode, a capture probe, and a reporter probe (fig. s1 (siangproh et al., 2011) and s2). capture probe is essentially an oligonucleotide whose sequence is complementary to that of the target nucleic acid. in most cases, the probe is conjugated to a surface, such as an electrode. after sample introduction, binding of target dna/rna to the capture probe generates a series of changes that eventually trigger the release of ecm signals by the reporter probe. even though a number of disposable electrodes such as glassy carbon (rivas et al., 2007) and pyrolytic carbon (stoner et al., 2014) have been in use for quite some time, non-disposable alternatives (e.g. indium tin oxide, pencil graphite, screen-printed carbon) are slowly but steadily taking over. after all, the latter are more economical and easier to produce (yeung et al., 2006) . for instance, indium tin oxide electrodes were exploited in a silicon-and glass-based microchamber for simultaneous diagnosis of escherichia coli and bacillus subtillis (xu et al., 2009) . capture probes were attached to the electrode surface by electrochemical copolymerization. nanoparticles (nps) have lately been used to complement electrodes in immobilizing probe (fig. 1 ). sun et al. employed gold nanoparticles (aunps) and multi-walled carbon nanotubes (mwcnts) to conjugate single-stranded dna probes for the detection of staphylococcus aureus dna . gold electrodes were also included in the set-up. rather than the common purpose of immobilizing capture probes, the electrode was instead used to concentrate the nano-sized anchors. in another example, each aunp served as the core for coconjugating a hairpin sequence of dna and a reporter dna. interestingly, the reporter dna only carried a sequence complementary to half of the target helicobacter pylori sequence. the other half was recognized by a capture probe anchored to a gold electrode (cui et al., 2015) . with a great surface-to-volume ratio, nps could potentially bind a greater density of capture probes. this indirectly magnifies the ecm signals ultimately generated (cui et al., 2015; sun et al., 2015) . at the same time, signal-to-noise (snr) ratio is improved. out of various kinds of nps, aunps are arguably the most extensively investigated as far as ecm sensing is concerned (table 1) . like most other nano-sized materials, aunps have inherently large surface area and surface free energy. these properties facilitate the adsorption of nucleic acid strands. nonetheless, there exist some challenges in terms of reliability, reproducibility, scalability in manufacturing aunp-based biosensing assays and long-term stability; these aspects have hampered the successful translation into clinical trial research. indeed, nanoparticles with different size ranges might show variable surface area, reactivity and orientation towards biosensing molecules. quite recently, aunps have been produced with very narrow size distribution (e.g. 12.7 ± 1 nm (lu et al. 2008) or even smaller, 1.6 ± 0.3 nm ) and further research efforts have resulted in samples with desirable polydispersity indices (b0.3). this process could be further enhanced via functional groups such as thiols and disulfides (galow et al., 1999, niemeyer and ceyhan, 2001) . since aunps are capable of forming table 1 examples of diagnostic poc systems whose target analytes are nucleic acids. key features ecm (yeung et al., 2006) analyte: escherichia coli dna or bacillus subtillis dna sample size: 1 μl sample pretreatment: • genome isolation using avidin-coated magnetic particles • amplification using pcr detection range: 10 2 -10 5 cells clinical/real-world samples tested? no performance: • capture probes were thermally stable throughout thermal cycling process • insignificant interference with quantification due to non-specific adsorption onto the electrodes • use of magnetic particles for dna isolation was compatible with pcr process (baeumner et al., 2002) analyte: dengue virus rna sample pretreatment: • amplification using isothermal nucleic acid sequence-based technique • mixing with liposomes assay time: 15 min (excluding rna amplification process) clinical/real-world samples tested? yes (human serum) performance: • sensitivity and specificity comparable to that of lab-based techniques • accurately detect dengue serotypes 1, 2 and 4 in clinical samples • minimal cross-reactivity with dengue serotype 3 (authier et al., 2001) analyte: 406-base pair hcmv dna sample pretreatment: • dna extraction from cell culture • amplification using pcr • denaturation in alkaline media at room temperature • 12-fold dilution with coating solution reproducibility: • ensured by slicing off a small segment at the end of the electrodes in between trials • enhanced by maintaining the screen-printed microband electrodes in a solution of (ferrocenylmethyl)trimethylammonium hexafluorophosphate • undermined with the use of manual screen-printer detection range: 5-500 pm clinical/real-world samples tested? no performance: • aunps label employed for hybridization assay proved to be more stable than radioisotopic or enzymatic labels • non-specific binding present in low level • selectivity demonstrated by testing against non-complementary human ets2 gene • lod better than that reported in an electrochemiluminescent hcmv dna method tested on 578-base pair hcmv dna (boom et al., 1999 ) analyte: staphylococcus aureus nuc gene sample size: 1 μl (after purification) sample pretreatment: • filtration of tap water samples through 22 μl membrane • inoculation with different amount of staphylococcus aureus • centrifugation at 10,000 rpm for 5 min • dna extraction using rapid boiling method • amplification using pcr • dilution with te buffer solution, followed by denaturation in boiling water bath detection range: • 1 fm-10 nm of nuc gene • 10-10 6 cfu ml −1 for real-world samples clinical/real-world samples tested? yes (tap water) performance: • achieved a lower lod (for tap water samples) than other reported methods such as aunp-based immunosensors (hejazi et al., 2008) or fluorescence-based assay using cdse quantum dots(yang and lai, 2011) optical (chen et al., 2014) analyte: detection of hev rna sample pretreatment: • reverse transcription of hev rna, followed by denaturation at 95°c • amplification using rt-lamp assay time: b3 min (excluding amplification step) detection range: n10 hev rna copies clinical/real-world samples tested? yes (human serum) performance: • selectivity demonstrated by testing against three other hepatitis strains hav, hbv, and hcv • results validated using agarose gel electrophoresis (phillips et al., 2008) analyte: escherichia coli dna sample size: 10 μl reproducibility: • reproducible fluorescence signals • extent varied depending on bacterial strains and species assay time: within min performance: • selectivity demonstrated by testing against twelve other species of bacteria (griffin et al., 2009) analyte: hcv rna detection range: 60-250 pm clinical/real-world samples tested? no performance: • no tagging is required • about two orders of magnitude more sensitive than some common colorimetric techniques • selectivity down to the level of single-base mismatch • quantitative signal intensity varied with the length of target rna sequence (nam et al., 2004) analyte: nucleotide sequence indicative of anthrax lethal factor sample size: 30 μl assay time: 3-4 h detection range: 500 zm-5 fm performance: • sensitivity on par with that of pcr-based methods, but did not require enzymatic amplification process • selectivity down to the level of single-base mismatch analyte: hiv subtypes (a, b, c, d, e, g, and panel) sample pretreatment: • system capable of effectively sequestrating separating viruses without the need for pretreatment reproducibility: strong covalent bonds with sulfhydryl groups, thiolation reaction could be readily achieved (daniel and astruc, 2004) . in contrast, nucleic acid strands with adenosyl phosphothiolate tails could be conjugated in a more direct manner (patolsky et al., 2006) . ease of functionalization and excellent biocompatibility (liu and ju, 2003) render aunps a great asset in both optical (cao et al., 2002) and electronic (park et al., 2002) dna detection methods. to incorporate aunps into ecm biosensors for poc devices, researchers have devised a handful of approaches for fully exploiting their potential . aunps, while being immobilized on genosensors, could be directly detected. in one experiment, target nucleic acid strands were first anchored onto au 67 quantum dots (qds) (pumera et al., 2005) . binding between target sequence and the capture probe, by then already conjugated to paramagnetic beads, led to the formation of a complex that enabled voltammetric detection of the gold qds. alternatively, it is possible to quantify au 3+ ions generated after aunps are exposed to a mixture of hydrogen bromide and bromine (i.e. acid dissolving step). such a strategy was employed in the detection of human cytomegalovirus (hcmv) dna sequence (authier et al., 2001) . since many au 3+ ions are released as each aunp is suspended in a medium, the ecm signal is enhanced. as a result, a limit of detection (lod) of 5 pm could be achieved. however, the mixture used for dissolving aunps is extremely harmful (hydrogen bromide is highly corrosive and irritating by inhalation). as such, this could limit the practicality of the technique (lucarelli et al., 2004) . for signal amplification, silver enhancement could be employed. cai et al. conjugated dna capture probe to aunps, and targeted dna to a glassy electrode (cai et al., 2002) . a silver enhancer solution was introduced to allow metallic silver to coat itself onto aunps. such coating process helped boost the voltammetric signals by more than 80 times. alternatively, signal amplification may be achieved by letting aunps act as carriers of electroactive labels. the incorporation of 6ferrocenylhexanethiol decreased the lod level to 2 pm for a sample size of 5 μl . aunps could be combined with other nano-size materials for application in ecm sensors. watanabe et al. recently combined the use of aunps and magnetic nps (mnps) in the detection of meca gene, a popular biomarker for mrsa (watanabe et al., 2015) . two dna probes were employed. one was anchored to mnps, whereas the other to aunps alongside ferrocene. the sequences of these probes were designed to be complementary to nearby regions located on meca gene. the co-binding of mnps permitted the isolation and enrichment of analyte complex prior to ecm measurement. even without the help of nucleic acid amplification via pcr, this system managed to detect as low as 10 pm of target dna. disposable biosensors are slowly but steadily assuming a larger role in poc technology, given the troubles commonly associated with nondisposable counterparts. a poc device that could be reused requires thorough cleaning after every assay to ensure no cross-assay contamination occurs, hence preserving the reliability of the assay. with that prerequisite, there is still the issue of how to do the washing without inadvertently damaging the integrity of the test reagents. this renders non-disposable poc devices unpractical in resource-poor settings. however, calibration and sterility problems have been reported for example in disposable clinical sensors, where the sensor-imbedded device required sensor calibration and/or validation by the clinician immediately prior to each use. some recent advances have enabled the production of pre-calibrated and pre-validated sensors (e.g. us 7857506 b2 patent), but they still require optimization in lowering the costs and increase performance before becoming suitable for a single-use sensor application. aunps have also been integrated into the design of disposable biosensors. they were used together with screen-printed electrodes for the diagnosis of respiratory pathogens such as mycoplasma pneumonia, streptococcus pneumonia, and chlamydophila pneumonia (bessede et al., 2010) . in another example of disposable biosensors, liposomes (another type of nps) were employed as carriers of dyes (ho et al., 2008) . this strategy was examined for the detection of serotype-specific rna fragments of dengue virus (baeumner et al., 2002) . a portable reflectometer was utilized for quantification of the nucleic acid materials. taking into account its many fortes (i.e. portable, inexpensive, user-friendly with easy-to-follow instructions, etc.), this system seems very promising. following isothermal nucleic acid sequence-based amplification, which requires only basic tools such as water baths, it only took another 15 min to produce results. the same research group has adopted a similar system for detection of viable escherichia coli in drinking water (baeumner et al., 2003) . thus far, we have discussed systems in which ecm labels play a major role in the quantification process. however, label-free sensors have also been investigated by numerous researchers. there are two main methods via which ecm sensing works without having to rely on electroactive labels (siangproh et al., 2011) . the first one is rather straightforward. nucleotide bases do have intrinsic redox properties. thus, they can generate ecm signals, which are indicative of the amount bound to the electrode. in the second approach, molecules which stably orient themselves into the groove of target dna duplex (e.g. methylene blue, daunomycin, aromatic amines, co(2,2'-bipyridyl) 3 3+ ) are employed. after the two dna strands get detached, these duplexintercalating entities are freed, hence generating ecm signals. the latter strategy has been adopted in the investigation of infectious pathogens such as escherichia coli, mycobacterium tuberculosis, hiv (haddache et al., 2014) , and hbv (meric et al., 2002) . however, a grave downside of this technique is the interaction between the chemicals and dna duplex, which makes them potentially mutagenic (watanabe et al., 2015) . depending on the nature of the pathogens (e.g. dna or rna viruses, according to the baltimore classification (baltimore, 1971) ), rna detection is another viable strategy that could benefit from the advent of nanotechnology into the development of poc systems. for instance, aunps were incorporated into a lateral flow test strip for the diagnosis of hiv through the quantification of viral rna in plasma samples (rohrman et al., 2012) . lateral flow nucleic acid test strips derived from the well-established immunochromatographic strips (mao et al., 2009) . at the moment, this kind of device has several limitations (carter and cary, 2007, corstjens et al., 2001) . before letting the sample flow through the devices, nucleic acid hybridization process is normally carried out in advance. this results in the addition of 10-30 min to the total assaying time. in general, quantification done with lateral flow assays necessitates the use of expensive equipment. the high cost, however, is not necessarily translated into great sensitivity of the assays (he et al., 2011) . there have been several attempts to augment the sensitivity of lateral flow assays. most of these ended up further complicating the protocol without really addressing the other issue (i.e. high cost) (rohrman et al., 2012 ). an example of such attempts to improve sensitivity exploited antigens and antibodies to detect hbv, hcv, and hiv viruses (dineva et al., 2005) . • surface chemistry of nps demonstrated to be reproducible to a considerable extent • analysis results reproducible for several hiv subtypes assay time: 1 h of capturing and 10 min of detection and analysis detection range: • varied between different subtypes • ranging from 98 ± 39 copies/ml (subtype d) to 120,159 ± 15,368 copies/ml (subtype e) clinical/real-world samples tested? yes (unprocessed whole blood) indeed, the specific detection hiv-1 rna is particularly challenging (rohrman et al., 2012) . it is known that the level of hiv genetic material present in the patients' bloodstream is naturally not very high. to be more precise, it is only a few copies per milliliter of blood. therefore, duplication of the nucleic acids prior to the assay is a vital prerequisite for an adequately sensitive test. to this end, rohrman et al. opted for isothermal nucleic acid sequence-based amplification, a popular technique mentioned above (baeumner et al., 2002) . this particular system has many laudable qualities (rohrman et al., 2012) , including a low cost (each strip costs no more than one us dollar) and simple production steps that involve commercially available reagents. from the very beginning to the completion of the assay, the user only needs to perform three steps. it takes in total only about 20 min, which is much shorter than the duration of any assay discussed thus far. in addition, simpleto-operate and relatively inexpensive instruments (e.g. heat block, scanner, camera, and pipette) are sufficient to perform the assay. in addition, when tested under different temperature conditions, the results produced by the assay remained essentially consistent. such a commendable level of robustness testifies to its suitability for use in africa and other places where a high temperature is a normal occurrence. consistent performance was also observed when the system was tested against varying storage periods. however, this technology is still far from ideal, as the use of heat blocks does consume a considerable amount of energy (labarre et al., 2011; liu et al., 2011) . it could be replaced by heater equipment that runs on battery, hence cutting down on costs. instead of imaging instruments, a color scale could be exploited to qualitatively simplify data interpretation process. up to this point, it should be fairly apparent that ecm sensing is one of the most extensively studied methods for the detection of pathogenic nucleic acid. another method that has also attracted much attention in the field of poc technology is optical sensing. in essence, presence of the biomarker of interest in the sample will trigger a chain of biochemical reactions. the end result is a change in optical properties of the system. such variation is designed to be proportional to the amount of the nucleic acid sequence to be analyzed (fig. 2) . one key advantage of optical sensing is that electroactive labels are not indispensable (shafiee et al., 2013) . once again, nanotechnology plays a huge role. metal nps especially show tremendous promises. among them, gold and silver nps are the better options, since they are less susceptible to oxidation than their copper counterparts (jain et al., 2008) . reportedly, nanorods (bi et al., 2015) and aunps were employed in a colorimetric assay for the diagnosis of hepatitis e virus (hev) (chen et al., 2014) . to ensure that the concentration of the biomarker fell within detectable range, real-time loopmediated isothermal amplification (rt-lamp) was employed as part of sample preparation process. streptavidin molecules were conjugated to aunps, which were then added to the sample that already underwent rt-lamp. if the sample was hev-positive, aunps would clump together as a response. this would in turn trigger a color change from red to purplish blue, hence permitting visual readout by naked eyes without any need for sophisticated instruments. on the contrary, had there been no hev genetic material in the test sample, the biotin added during rt-lamp process would help stabilize the aunps. as a result, the solution would remain red. a notable advantage demonstrated by this system was a remarkably short assay duration. not including rna amplification process, the test consumed only 3 min in total. surface plasmon resonance (spr)-based sensors have rapidly emerged as a popular type of optical biosensors (homola, 2008 , tokel et al., 2014 . they work by measuring changes in refractive index of metal-dielectric interface, which could occur following binding events between the analyte molecules and capture probes. surface plasmon bands absorbed by metal nps are closely related to the size of their aggregates, which come about as a consequence of nucleic acid hybridization (hazarika et al., 2004) . the more np aggregates there are, the greater the resulting red-shift becomes. an example of spr-based biosensor was used for the detection of hbv (chuang et al., 2012) . aunps were also employed in the said assay, which was economical and exhibited a lod of 2 fg/ml with just 17 min of assay time. nat has always been a valuable diagnostic tool of hbv infection. its utility is even more conspicuous during the 'window period' where other common techniques (e.g. immunoassays) are not practically reliable. the reason for this setback of immunoassays is the lack of antibodies against hbv in the body throughout the 'window period' (yildiz et al., 2015) . nat is also particularly useful in the diagnosis of occult hbv infection (ozsoz et al., 2003) . while dna of occult hbv are present in the bloodstream, there is no trace of their surface antigens. given its unique capability, nat is the preferred technique when it comes to scanning of blood transfusion sources (stramer et al., 2011) . fluorescence-based assays are categorized under optical sensing as well. storhoff investigated the use of this type of assay for the detection of meca gene of mrsa (storhoff et al., 2004) . the integration of nanosized materials (i.e. aunps) helped augment the sensitivity of the assay relative to that of similar methods. conveniently, nucleic acid amplification prior to quantification process was not necessary. a separate study adopted fluorescence-based sensors for the detection of escherichia coli dna (esteban-fernandez de avila et al., 2015) . in details, anionic molecules of poly(para-phenylenethynylene) (ppe) were immobilized onto aunps, which were already functionalized with ammonium groups. the resulting complex efficiently subdued fluorescent property of ppe. after sample introduction, if bacteria were present, there would be electrostatic interaction between their surface and the various positive charges lining along the surface of aunps. this triggered the release of ppe from the complex. once freed, ppe molecules regained their fluorescence, hence allowing quantitative measurements of the bacteria. as additional advantage, the assay is capable of identifying three distinct bacterial strains within min. fluorescence-based assays can also offer a final readout by naked eyes. zhang et al. reported a poc device that made use of microcapillaries for detection of two rna biomarkers that belong to different hiv strains . a simple uv-flashlight was sufficient to generate visible readouts of fluorescence signals from the indicator, calcein. in addition, the system is self-sufficient in the sense that it did not utilize any external source of electricity. more precisely, a pocket warmer was all it needed. the use of capillaries to introduce and hold samples permitted concurrent analysis of several samples. in brief, this system did manage to tackle some of the most troubling issues associated with diagnostic tools in resource-poor settings, namely time and energy consumption. these assays are generally based on immunoreactions between antibodies and antigens, which are characteristics of individual strains. relative to nat, immunological tests are generally capable of producing more robust results within a shorter span of time (shinde et al., 2012) . however, their level of specificity and sensitivity often pales in comparison. over the time, different kinds of antibodies (e.g. conventional, heavy chain, monoclonal, polyclonal, and recombinant antibodies) have been investigated in immunological tests. none of them have proven to be perfectly suitable for the role (o'kennedy et al., 2005 , shinde et al., 2012 . polyclonal antibodies could be produced in a more rapid and economical manner than monoclonal antibodies, but their intrinsically poor specificity represents a valid concern. the latter have their fair share of shortcomings though. the production of monoclonal antibodies requires more well-trained personnel, and more sophisticated machineries, whose cost is a setback. moreover, recombinant antibodies do not promise an acceptable level of sensitivity and affinity. to worsen the matter, they are rather vulnerable to interference from contaminants. monoclonal antibodies were utilized in the commercially available architect qualitative assay by abbott (lou et al., 2011) . this system was designed to detect hepatitis b surface antigens (hbsag) by conjugating anti-hbsag monoclonal antibodies to paramagnetic nps. recognition of the hbsag in plasma samples, now bound to paramagnetic nps, was handled by another set of acridinium-functionalized antibodies. upon the introduction of hydrogen peroxide and sodium hydroxide into the system, chemiluminescence signals indicative of the amount of hbsag were emitted. they were exploited by architect system optics for quantification purposes. in immunological tests, ecm sensing plays a significant role ( table 2 ). aunps served as the carriers for five different antibodies in an ecm immunosensor array that concurrently detects five separate strains of hbv (tang et al., 2010) . while its performance was comparable to that of conventional elisa, it was demonstrated to be more energy-efficient and able to produce results within 5 min (ye et al., 2003) . if aunps attract all the limelight in nat, a wide range of nps have found utility in the detection of pathogenic antigens. one such nanosized material is graphene. by virtue of its distinguished electron transfer quality, graphene film was used to construct electrodes for detecting rotavirus (liu et al., 2012) (fig. s3(a) ). antibodies specific to the viral particles were anchored onto the surface of the graphene-based electrodes. a wide range of graphene-based nanomaterials have been investigated for application in the field of nanomedicine. that alone is proof of the utility of graphene nps. however, development of graphenebased biosensors has been to some extent impeded by a lack of reproducibility and scalability of the manufacturing processes. graphene nps have also been exploited for other functions. for instance, graphene oxide nps were employed by chen et al. as fluorescence quenchers in an assay capable of simultaneously detecting both human enterovirus 71 (lods: 0.42 ng/ml) and coxsackievirus b3 (lod: 0.39 ng/ml) . this assay also made use of qds, another type of nps. for the detection of two unrelated species of viruses, two kinds of qds, which possessed distinct optical behaviors for selective quantification, were required to conjugate the two kinds of antibodies. both kinds of qds were in turn functionalized with graphene oxide, which efficiently suppressed fluorescent signals from qds. when either human enterovirus 71 or coxsackievirus b3 was present in the samples, the corresponding qds detached themselves from graphene oxide nps and emitted fluorescence. the signals could be picked up and quantified. in another study, graphene oxide nps were used together with silver nps (agnps) for simultaneous diagnosis of hbv, hiv and treponema pallidum (liu et al., 2013) . one major defect of graphene-based nanomaterials is their high hydrophobicity, which is responsible for formation of clumps in solution. these bulky aggregates indiscriminately bind biomolecules other than the desired targets, and could cause denaturation of the sample (yildiz et al., 2015) . mnps were employed in a sandwich-type immunoassay for the detection of salmonella typhimurium (gehring et al., 1996) . anti-s. typhimurium antibodies were conjugated onto superparamagnetic beads while being functionalized with alkaline phosphatase. phosphatase was the key element of ecm sensing mechanism in that investigation. after sample introduction, the inherent magnetism of mnps allowed to attract the complex towards disposable graphite ink electrodes. this step was included in the protocol to enhance the efficiency of ecm detection. a drawback of this particular assay was that it took in total 80 min, which made it much more time-consuming than many other poc systems. given the fact that gehring et al. reported this immunoassay more than a decade ago, the lack of efficiency in assay time was understandable. in an enzyme-free ecm immunosensor, silicon nanowires were functionalized with antibodies for the detection of influenza a virus (patolsky et al., 2004) (figure s3(b) ). a change in conductance was recorded once the complex was exposed to viral particles. such quantifiable change was observed when the sample contained paramyxovirus and adenovirus, but not influenza a virus. this helped establish that the assay possessed a clinically relevant level of selectivity. remarkably, samples containing single viruses could be detected without compromising the selectivity. this level of performance would compare favorably against the mainstream pcr-based techniques. moreover, the system was demonstrated to be capable of multiplexing. • excellent specificity (99.94%) when tested on 6482 specimens • performed better than other hbsag assays in terms of accurate detection • capable of detecting more substitution mutants than an earlier versions (tang et al., 2010) analyte: multiple types of hepatitis virus antigens (hav, hbv, hcv, hdv, hev) reproducibility: inter-assay imprecision level at 8.1% assay time: 5 min detection range: • lod slightly varied between antigen types, ranging from 0.8 ng/ml for hbv to 1.5 ng/ml for hcv and hev • same upper limit of linear range (350 ng/ml) clinical/real-world samples tested? yes (human serum) performance: • quality of results comparable with that of conventional elisa • some cross-reactivity between adjacent sites (≤7.5%) purpose: enterovirus 71 and coxsackievirus b3 assay time: shorter than equivalent methods (e.g. rt-pcr) detection range: • (perez et al., 2003) analyte: herpes simplex virus or adenovirus sample size: 10 μl sample pretreatment: minimal detection range: • 5 viral particles in 10 μl samples • 100 viral particles in 100 μl samples clinical/real-world samples tested? no performance: • superior than common pcr-based techniques • capable of analyzing complex turbid samples • more sensitive than elisa assays (lien et al., 2007) analyte: dengue virus serotype 2 sample size: 25 μl sample pretreatment: • the concentration of anchored antibody does influence performance of the assay (reducing the concentration from 4 mg/ml to 2 mg/ml lengthens the detection range) • selectivity demonstrated when tested against escherichia coli o157:h7 and liseria genus (zhao et al., 2004) analyte: escherichia coli o157:h7 sample pretreatment: • no amplification or enrichment required assay time: 20 min clinical/real-world samples tested? yes (spiked ground beef) performance: • could even detect a single bacterium in the samples (verified using two distinct quantitative techniques) as mentioned in the previous section, mnps have been integrated into ecm immunosensors before. nonetheless, they are more extensively applied in poc devices, which utilize magnetic resonance detection method ( fig. 3(a) ). a phage-based magnetoelastic biosensor was adopted to analyze salmonella typhimurium on fresh tomato surfaces (li et al., 2010) . use of filamentous e2 phages facilitated the binding of the analytes. the resonance frequency generated by the wireless biosensors could be quantified via magnetic fields. traditional magnetic beads typically used in biological separation have a diameter of about 1−5 μm. in contrast, mnps are much smaller (b10 nm in diameter). as such, they have a substantially larger surfaceto-volume ratio (josephson et al., 2002) . superparamagnetic iron oxide nps were used to anchor antibodies for the detection of herpesvirus or adenovirus in 10 μl of sample volume (perez et al., 2003) . the iron oxide nps were coated with dextran. this layer could be further functionalized with amino groups, thereby facilitating antibody conjugation (josephson et al., 1999) . existence of viral particles in the samples triggered self-aggregation of the mnps to form a complex with augmented magnetic properties. this change in structure then allowed for quantitative detection. it was observed that the percentage of serum of the samples did have an impact on the sensitivity of the assay. in 100% serum samples, the lod was 10 virions, but it dropped to as low as 5 virions when 25% serum samples were investigated. an immunosensor with that impressive lod is undoubtedly promising. while it can efficiently scan serum samples for viral infections, its application in the detection of bacteria seems far from being ideal (kaittanis et al., 2007) . upon being exposed to a low bacteria count, the mnps would clutter together on the surface of the pathogens. however, if the count was above a certain threshold value, the mnps would revert back to a dispersed state just like how they would behave under pathogen-free circumstances. therefore, the assay could potentially produce false negatives. with this serious flaw left unaddressed, applications of this system are confined to analysis of infectious diseases generally known to display a low serum pathogen count (e.g. mycobacterium avium spp. paratuberculosis (map)) (kaittanis et al., 2007) . a separate study investigated the use of dextran-coated iron oxide nps, this time in the form of nano-sized rods, also for the diagnosis of map (liao et al., 2009 ). an lod of 6cfu could be achieved after just 5 min. one principal shortcoming of magnetic resonance detection method is the requirement of machineries such as magnetic relaxometers or other instruments specialized to perform nuclear magnetic resonance (nmr). aside from being too costly, their operation demands a certain level of technical skill of the personnel. this ultimately undermines the suitability of this detection method for application in poc devices. magnetic properties of mnps have not only been exploited for magnetic resonance sensing, but also for deliberate isolation of the pathogens of interest from the sample (fig. 3 (b)(c)(d)). such maneuver allows for sample enrichment. lien et al. immobilized antibodies onto mnps to trap dengue virus from the sample (lien et al., 2007) . using a planar micro-sized coil, a magnetic field gradient was set up to attract the viral particles captured on mnps. the separation efficiency achieved was fairly high at 87%. as a result, pathogenic quantification could then be executed with a greater level of sensitivity. to be specific, the lod was brought down to 100 fu/ml. in another study, xia et al. employed mnps to bind and extract escherichia coli virions from the flow of a solution that simulated human blood samples (xia et al., 2006) . in place of a planar microcoil, a high-gradient magnetic field concentrator was used to generate the necessary magnetic field gradient. it was noticed that the separation efficiency of the system did not deteriorate with time. at flow rates from 25 to 40 μl/h, separation efficiency of mnp-bound bacterial cells ranged from 78% to over 90%. raising the cell density of input flow brought about a great increase in throughput rate. one common setback faced by both systems was a poor capacity for concentrating samples. nevertheless, it is not universally observed among experiments that perform magnetic separation using mnps. in a microfluidic chamber device, which employed a close-packed columns of polydispersed iron nps and a ndfeb permanent magnet, 0.5 ml of hiv-inflicted plasma samples was concentrated by 44 times. this remarkable concentrating power would significantly raise the sensitivity level of any quantification method subsequently employed for the diagnosis of hiv (chen et al., 2010a) . ecm sensing was one of the methods which have been coupled with magnetic separation to enhance sensitivity. escherichia coli o157:h7, an enterohemorrhagic serotype of escherichia coli, was subjected to diagnosis using this combination of techniques (gu et al., 2003) . setterington et al. took the modification a step further and bioconjugated the bacterial cells with polyaniline after they were magnetically separated (setterington and alocilja, 2011) . the electroactive label allowed quantification using cyclic voltammetry. this played a huge role in strengthening the ecm signals emitted. consequently, a lod of 70 cfu/ml was attained. notwithstanding the relatively lengthy sampling time of 70 min, this experimental poc system can be carried out using compact and mobile devices, hence confirming its suitability for a broad range of relevant applications. it has been demonstrated that magnetic separation of the pathogenic particles also allows for microscopic identification of the microorganisms. in one study, vancomycin was conjugated to mnps in order to trap vancomycin-resistant enterococci (gu et al., 2003) . with the help of an external magnetic field, the mnps-bound bacteria were accumulated into an area around 1 mm 2 large. biological separation was followed by observation using optical microscope, and finally verification with the help of electron micrograph. a lod as low as 10 cfu/ml was achieved. this kind of assays has several advantages (ghindilis et al., 1998 , lin and ju, 2005 , warsinke et al., 2000 . it has inherently excellent sensitivity relative to other kinds of diagnostic tests, could easily be adopted as poc technology, and is, above all else, cost-effective. nano-sized materials have been widely studied as add-ons to accompany transducers so as to facilitate electron transfer process, magnify the snr of the system, or to heighten the efficiency of antibody conjugation . in some cases, nps complex have also been explored as ecm labels, and anchor points for antibodies. it has been demonstrated that antibodies do retain their biological binding activity after being conjugated to nps (e.g. aunps) (liao et al., 2009) (table 3) . a range of nps have been investigated for application in proteinsensing ecm immunosensors ( fig. s4(a) ) (hansen et al., 2006 , liu et al., 2004 , yuan et al., 2015 . among them, qds are fairly popular in multiplexed assays for parallel detection of several biomarkers at the same time (fig. s4(b) ) (liu et al., 2004) . to this end, one set of antibodies is immobilized onto magnetic beadseach type of antibody belonging to the other set is conjugated to a distinct type of metal sulfide semiconductor. the examined qds included cds, zns, pbs, and cus, which all have comparable levels of sensitivity. the hydroxyl terminals of these nano-sized colloidal tracers enabled the antibody functionalization process via carbamate bonds. the use of 2 sets of antibodies served as the central elements of an ecm sandwich immunoassay. the antigens of interest, be it proteins or a human antibodies, were captured between magnetic beads and qds with the corresponding antibodies. each individual antibody-antigen binding event would produce a characteristic voltammetric peak. the position and magnitude of the peaks provide detailed information on how much of each table 3 examples of diagnostic poc systems whose target analytes are proteins and antibodies. author key features electrochemical (ambrosi et al., 2007) analyte: human igg sample size: 150 μl sample pretreatment: • mixing with antibody-coated magnetic beads • labeling with double codified aunps detection range: varied between the two quantification methods employed • spectrophotometry: lod of 52 pg/ml • ecm: lod of 260 pg/ml clinical/real-world samples tested? no performance: • more sensitive than conventional elisa techniques • selectivity demonstrated by testing against goat igg (yuan et al., 2015) analyte: c-reactive protein (hscrp) and soluble cd40 ligand (scd40l) sample size: 6 μl sample pretreatment: minimal reproducibility: • acceptable level of reproducibility • inter-assay standard derivations were 5.04% and 4.08% for hscrp and scd40l respectively detection range: 0.05-100 ng/ml • lod of hscrp: 16.7 pg/ml • lod of scd40l: 13.1 pg/ml clinical/real-world samples tested? yes (human serum) performance: • stability of system demonstrated by storing the immunosensor at 4°c for 30 days in between assays (91.63% and 90.02% of initial response achieved for hscrp and scd40l respectively) optical (zhu and yang, 2015) analyte: anti-rabbit human igg sample size: 18 μl sample pretreatment: minimal assay time: 25 min detection range: 1-10 μg/ml clinical/real-world samples tested? no performance: • reducing sample size to 6 μl helps reduce assay time to around 15 min, but at the same time compromises the sensitivity (higher lod of 5 μg/ml) • selectivity demonstrated by testing against bovine serum albumin (at much higher concentration than the target analyte) biomarker (e.g. β 2 -microglobulin, igg, bovine serum albumin, and creactive protein) was present in the sample. qds were accompanied by aunps and thiolated aptamers in an ecm immunesensor system, which reportedly yielded an lod of 20 ng/l (fig. s4(c) ) (hansen et al., 2006) . aptamers are synthetic nucleic acid ligands which have been studied as substitutes to antibodies. they possess several desirable qualities, out of which resistance to denaturation is perhaps the most laudable. in general, aptamer-based biosensors could achieve excellent sensitivity. this forte was apparent in this particular investigation, whereby trace amounts of proteins could be detected. being energy-efficient, easily miniaturized, and economical, the assay satisfied a handful of assured criteria. in another study, cadmium tellurite qds were conjugated to silica nps for magnification of ecm signals. the system was designed for detecting epstein-barr virus-derived latent membrane protein 1 (lmp-1) (chen et al., 2010b) . the especially large surface area of nano-sized carriers allowed for the immobilization of numerous qds. this served as the basis for the augmentation of ecm signals detected by square wave voltammetry. as a result, an lod of 1 pg/ml was achieved. the invariable efficiency of qds immobilization process also worked in our favor by ensuring excellent reproducibility of the assay. the flexibility of qds is apparent when we take into account the properties that make them excellent fluorescence emitters. by adjusting the size of these nano-sized semiconductors, it is possible to manipulate their emission wavelength range (pinaud et al., 2006) . ergo, a single absorption wavelength could be used to trigger a range of emission wavelength, given that qds of varying size are employed. this can be tapped on for potential development of fluorescence-based multiplexed assays (hare et al., 2015) . multiplexed assays can also be achieved with mnps. in one study, hybrid nps, which consisted of a nife 2 o 4 core enclosed within a sio 2 shell, were used to anchor different kinds of antibodies for concurrent detection of four distinct biomarkers (tang et al., 2007) . the extent of ecm signal interference between adjacent electrodes (each electrode designed to detect one biomarker) was minimal. ambrosi et al. reported an immunoassay compatible with two separate detection methods (ecm and optical) for quantifying human igg (ambrosi et al., 2007) . aunps were conjugated with antibodies specific to human igg. these antibodies were then bonded to horseradish peroxidase. detection step could be done spectrophotometrically by measuring the intensity of the solution's color emanated from aunps. alternatively, innate ecm behaviors of the aunps could be quantified with stripping voltammetry. the use of paramagnetic beads enabled magnetic separation of the labeled antibody complex. as a consequence, the sensitivity of the assay outperformed conventional elisa tests. the lod of optical and ecm detection methods was 52 pg/ml and 260 pg/ ml respectively. in addition, the use of mnps helped curtail incubation and washing time, which then contributed to a more desirable total assay time. just considering optical detection method alone, europium (iii) nps (eunps) have been contemplated as an excellent substitute for aunps. they were meant to help reduce the sophistication of the assays without compromising their sensitivity (tang et al., 2009 ). the optical properties of these nano-sized fluorophores render them suitable for immunosensors. after all, eunps are capable of producing robust and lasting fluorescence (hemmila et al., 1984) . in one investigation, they were encapsulated inside polystyrene nps for the detection of anthrax protective antigen (tang et al., 2009) . the system was considerably reliable, since no false negatives were observed. meanwhile, the assay attained a level of sensitivity 100 times greater than that of conventional elisa, whose lod was known to be around 1 ng/ml (moayeri et al., 2007) . poc devices which permit visible readouts are generally associated with a more affordable cost, since the need for advanced instruments for quantitative detection is eliminated. it is therefore a welcomed addition to poc technology, considering how it helps realize one key element of the assured criteria. one recent example microfluidic immunoassay with naked-eye readouts leveraged on the changing appearance of liquid crystals (zhu and yang, 2015) (fig. s5(a) ). binding events between antigens and immobilized antibodies triggered a shift of the lqc appearance from dark to bright. this phenomenon could be visualized without the need for sophisticated devices. the technique exhibited good robustness, a lod of 1 μg/ml. in addition, good specificity was demonstrated using bovine serum albumin as the non-target interference (10-fold concentration compared to the target analyte). another example is the volumetric bar-chart chip reported by song et al. for the detection of disease-specific proteins (song et al., 2012) (fig. s5(b) ). in this study, catalase and antibody molecules were both conjugated onto the surface of silica nps. if the target analyte was present in the sample, the enzyme would catalyze the decomposition of hydrogen peroxide in the solution to give out oxygen. the extent of pressure build-up inside the enclosed columns was proportional to the amount of oxygen gas produced. the rise in column pressure elevated the ink columns upwards. in a nutshell, the extent of elevation was designed to be indicative of the amount of the protein biomarkers in the sample. the presentation of quantitative results in the form of bar charts, as suggested by the name of the device, could be readily interpreted with just naked eyes. moreover, the oxygen-producing reaction facilitated by catalase occurred very rapidly, within seconds (george, 1947) . this was arguably the key feature of the system, which helped shorten the total assay time. duration was further decreased by virtue of this poc system's impressive multiplexing power, which permitted up to 50 concurrent tests. apart from the aforementioned strengths of this system, it did exhibit certain limitations (zhu et al., 2014) . the biocatalytic capability of the enzyme could possibly be impaired during the conjugation step. the fact that catalase enzyme itself is highly susceptible to hydrolysis further cast doubt on the reliability of the assay. more recently, zhu et al. developed an immunoassay based on similar concepts, but with certain alterations to expunge the existing drawbacks (zhu et al., 2014) . to tackle the problem at its root, the easily degraded catalase was replaced by hybrid nps comprised of a gold shell and a platinum core (au@ptnps). these au@ptnps were encapsulated inside aptamer-modified hydrogels. when the biomarker of interest was added, its interaction with the aptamers found on the exterior of the hydrogels would trigger their disintegration. au@ptnps would then come into contact with hydrogen peroxide molecules already present in the test solution. in this manner, the hybrid nps assumed the role of catalase. this led to the formation of visible bar-chart displays, as explained above in the study of zhu et al. (zhu and yang, 2015) . this novel system could be conveniently adopted for detecting numerous protein and antibody biomarkers. after all, a diverse selection of aptamers could be procured through different means (ellington and szostak, 1990, tuerk and gold, 1990) . subramaniam et al. demonstrated another system with visible readouts, referred to as metal-amplified density assay (subramaniam et al., 2015) . levitation of diamagnetic polystyrene beads was employed as the parameter for interpretation by naked eyes. in essence, antibodies specific to the biomarkers were conjugated onto these nano-sized beads. after sample introduction, successful immunoreactions would bring about a change in the density of the polystyrene beads. at first glance, such physical alteration proved to be too minute to be reliably detected. to address this issue, the authors revised the protocol by incorporating aunps into the poc system to amplify the change in density. visible readouts using floating height of small particles are much more attractive than those dependent on colorimetric interpretation (martinez, 2008) . the former have shown promises in parallel testing capacity by virtue of several kinds of colored beads. for instance, it was explored for simultaneous diagnosis of syphilis and hepatitis c (subramaniam et al., 2015) . however, its protocol required a handful of steps which could appear confusing to on-site test performers. by producing diagnostic test results within a short period of time, at low cost, and without the need for advanced instruments or welltrained technicians, poc devices are undoubtedly dream companions for medical professionals in resource-poor regions. sometimes, the availability of poc devices could very well make the difference between life and death, given their ability to produce timely test outcomes. for those mobile devices whose operational instructions have been sufficiently simplified, the task of carrying out the tests could be entrusted to the caretaker, or even the patients themselves if the need arises. notwithstanding its far-reaching medical applications, poc technology in general, and nano-sized materials in particular, are still in early phases of development. throughout this review, we have discussed several poc devices currently in experimental stage. they all excel in certain aspects, but at the same time fail to satisfy every single assured criterion. this makes a case for further improvements. one direction for improvement is to develop a platform which is able to deal with samples without the requirement of preprocessing (e.g. hiv in whole blood or pathogen on fresh tomato surface (li et al., 2010) ). this can be truly helpful to users for a one-step diagnosis. two feasible methods may be adopted for this purpose. one is further enhancement of sensitivity and another is having integration with microfluidic components for sorting and purification functions (bi et al. 2015) . advancements in the field have rendered the synthesis of inorganic nps (e.g. aunps (craig et al., 2012) , sio 2 nps (li and zhao, 2013) ) largely reproducible, with respect to physical parameters such as size distribution. attaining uniformity of organic nps used to be a real challenge, but progresses have been made in that aspect. recently reported syntheses achieved acceptable levels of reproducibility and desirable polydispersity indices (b0.3). since nps are primarily employed as carriers onto which biomolecules are conjugated, their size distribution is one of the key factors predisposing the performance of the poc systems. therefore, future researches into fine-tuning reproducibility of nps will further enhance the reliability of poc systems. it also helps that the developments in poc technology are easily adopted horizontally, as long as the biomarkers belong to the same class (e.g. nucleic acids). with the current amount of time and effort invested into researches on poc technologies, it is not much of an exaggeration to state that breakthroughs are bound to come in the near future. double-codified gold nanolabels for enhanced immunoanalysis gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus dna using disposable microband electrodes biosensor for dengue virus detection: sensitive, rapid, and serotype specific rna biosensor for the rapid detection of viable escherichia coli in drinking water expression of animal virus genomes materials science. food pathogen detection evaluation of the combination of the nuclisens easymag and the easyq applications for the detection of mycoplasma pneumoniae and chlamydia pneumoniae in respiratory tract specimens gene detection in complex biological media using semiconductor nanorods within an integrated microfluidic device a highly sensitive assay for detection and quantitation of human cytomegalovirus dna in serum and plasma by pcr and electrochemiluminescence state of the art for diagnosis of hiv infection overview of electrochemical dna biosensors: new approaches to detect the expression of life an electrochemical dna hybridization detection assay based on a silver nanoparticle label hiv viral load monitoring in resource-limited regions: optional or necessary? nanoparticles with raman spectroscopic fingerprints for dna and rna detection lateral flow microarrays: a novel platform for rapid nucleic acid detection based on miniaturized lateral flow chromatography. nucleic acids research electrochemical genosensors for biomedical applications based on gold nanoparticles detection of sars coronavirus in patients with suspected sars concentration and purification of human immunodeficiency virus type 1 virions by microfluidic separation of superparamagnetic nanoparticles sensitive detection of epstein-barr virusderived latent membrane protein 1 based on cdte quantum dots-capped silica nanoparticle labels simultaneous determination of human enterovirus 71 and coxsackievirus b3 by dual-color quantum dots and homogeneous immunoassay colorimetric detection of hepatitis e virus based on reverse transcription loop mediated isothermal amplification (rt-lamp) assay a polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity hbv loop-mediated isothermal amplification use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: a rapid, sensitive dna test to identify human papillomavirus type 16 infection cisplatin-tethered gold nanoparticles that exhibit enhanced reproducibility, drug loading, and stability: a step closer to pharmaceutical approval? hairpin dna as a biobarcode modified on gold nanoparticles for electrochemical dna detection gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology interferon-gamma release assays for the diagnosis of latent mycobacterium tuberculosis infection: a systematic review and meta-analysis simultaneous visual detection of multiple viral amplicons by dipstick assay a systematic review of rapid diagnostic tests for the detection of tuberculosis infection in vitro selection of rna molecules that bind specific ligands dual functional graphene derivative-based electrochemical platforms for detection of the tp53 gene with single nucleotide polymorphism selectivity in biological samples hiv-1 viral load assays for resource-limited settings pricing infectious disease. the economic and health implications of infectious diseases fluorocarbonylferrocene. a versatile intermediate for ferrocene esters and amides enzymelinked immunomagnetic electrochemical detection of salmonella typhimurium reaction between catalase and hydrogen peroxide immunosensors: electrochemical sensing and other engineering approaches sequencespecific hcv rna quantification using the size-dependent nonlinear optical properties of gold nanoparticles using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration labelfree photoelectrochemical detection of double-stranded hiv dna by means of a metallointercalator-functionalized electrogenerated polymer quantum-dot/ aptamer-based ultrasensitive multi-analyte electrochemical biosensor imaging metals in biology: balancing sensitivity, selectivity and spatial resolution reversible switching of dna-gold nanoparticle aggregation rapid and ultrasensitive salmonella typhimurium quantification using positive dielectrophoresis driven on-line enrichment and fluorescent nanoparticleslabel ultrasensitive nucleic acid biosensor based on enzyme-gold nanoparticle dual label and lateral flow strip biosensor construction, electrochemically biosensing and discrimination of recombinant plasmid (pethil-2) on the basis of interleukine-2 dna insert europium as a label in time-resolved immunofluorometric assays liposome-based immunostrip for the rapid detection of salmonella point-of-care molecular diagnostic systems-past, present and future surface plasmon resonance sensors for detection of chemical and biological species oligonucleotide-linked gold nanoparticle aggregates for enhanced sensitivity in lateral flow assays nanoplasmonic quantitative detection of intact viruses from unprocessed whole blood noble metals on the nanoscale: optical and photothermal properties and some applications in imaging, sensing, biology, and medicine high-efficiency intracellular magnetic labeling with novel superparamagnetic-tat peptide conjugates near-infrared fluorescent nanoparticles as combined mr/optical imaging probes one-step, nanoparticle-mediated bacterial detection with magnetic relaxation emerging nanotechnology-based strategies for the identification of microbial pathogenesis the effect of amino acids, monoamines and polyamines on pyruvate dehydrogenase activity in mitochondria from rat adipocytes preparation and characterization of 1-2 nm dendrimer-encapsulated gold nanoparticles having very narrow size distributions a simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings current nursing practice of pointof-care laboratory diagnostic testing in critical care units regulatory compliance for point-of-care testing. a perspective from the united states direct detection of salmonella typhimurium on fresh produce using phage-based magnetoelastic biosensors extension of the stober method to construct mesoporous sio 2 and tio 2 shells for uniform multifunctional core-shell structures femtomolar immunoassay based on coupling gold nanoparticle enlargement with square wave stripping voltammetry ultra-sensitive detection of mutated papillary thyroid carcinoma dna using square wave stripping voltammetry method and amplified gold nanoparticle biomarkers purification and enrichment of virus samples utilizing magnetic beads on a microfluidic system dna-arrays with electrical detection: a label-free low cost technology for routine use in life sciences and diagnostics electrochemical and chemiluminescent immunosensors for tumor markers a self-heating cartridge for molecular diagnostics nanomaterial labels in electrochemical immunosensors and immunoassays electrochemical coding for multiplexed immunoassays of proteins reagentless glucose biosensor based on direct electron transfer of glucose oxidase immobilized on colloidal gold modified carbon paste electrode graphene oxide/nucleic-acidstabilized silver nanoclusters: functional hybrid materials for optical aptamer sensing and multiplexed analysis of pathogenic dnas biological and chemical sensors based on graphene materials an improved abbott architect assay for the detection of hepatitis b virus surface antigen (hbsag) facile synthesis of gold nanoparticles with narrow size distribution by using aucl or aubr as the precursor carbon and gold electrodes as electrochemical transducers for dna hybridisation sensors split hybridisation probes for electrochemical typing of single-nucleotide polymorphisms disposable nucleic acid biosensors based on gold nanoparticle probes and lateral flow strip antibiotics and antibiotic resistance genes in natural environments electrochemical dna biosensor for the detection of tt and hepatitis b virus from pcr amplified real samples by using methylene blue the impact and cost of scaling up genexpert mtb/rif in south africa anthrax protective antigen cleavage and clearance from the blood of mice and rats prediction and prevention of the next pandemic zoonosis bio-bar-code-based dna detection with pcr-like sensitivity dna-directed functionalization of colloidal gold with proteins this work was supported by deutsche forschungsgemeinschaft and fonds der chemischen industrie. we thank prof. d. blohm for helpful discussions and generous support advances in biosensors for detection of pathogens in food and water electrochemical genosensor based on colloidal gold nanoparticles for the detection of factor v leiden mutation using disposable pencil graphite electrodes array-based electrical detection of dna with nanoparticle probes electrical detection of single viruses fabrication of silicon nanowire devices for ultrasensitive, label-free, real-time detection of biological and chemical species viral-induced selfassembly of magnetic nanoparticles allows the detection of viral particles in biological media rapid and efficient identification of bacteria using gold-nanoparticle-poly(para-phenyleneethynylene) constructs advances in fluorescence imaging with quantum dot bio-probes rapid, specific and sensitive electrochemical detection of foodborne bacteria lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. a literature survey magnetically trigged direct electrochemical detection of dna hybridization using au67 quantum dot as electrical tracer carbon nanotubes for electrochemical biosensing a lateral flow assay for quantitative detection of amplified hiv-1 rna hiv-1 viral load testing cost in developing countries: what's new? rapid electrochemical detection of polyanilinelabeled escherichia coli o157:h7 acute on-chip hiv detection through label-free electrical sensing of viral nano-lysate recent trends in in-vitro nanodiagnostics for detection of pathogens nanoparticle-based electrochemical detection in conventional and miniaturized systems and their bioanalytical applications: a review development of a digital microfluidic platform for point of care testing multiplexed volumetric bar-chart chip for point-of-care diagnostics combination assay detecting both human immunodeficiency virus (hiv) p24 antigen and anti-hiv antibodies opens a second diagnostic window selected topics on the synthesis, properties and applications of multiwalled carbon nanotubes homogeneous detection of unamplified genomic dna sequences based on colorimetric scatter of gold nanoparticle probes nucleic acid testing to detect hbv infection in blood donors metal-amplified density assays, (madas), including a density-linked immunosorbent assay (delisa) a highly selective and sensitive electrochemical cs-mwcnts/au-nps composite dna biosensor for staphylococcus aureus gene sequence detection reduced vaccination and the risk of measles and other childhood infections post-ebola magnetic control of an electrochemical microfluidic device with an arrayed immunosensor for simultaneous multiple immunoassays simultaneous determination of five-type hepatitis virus antigens in 5 min using an integrated automatic electrochemical immunosensor array detection of anthrax toxin by an ultrasensitive immunoassay using europium nanoparticles the hiv/aids pandemic and the global response advances in plasmonic technologies for point of care applications systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t4 dna polymerase requirements for high impact diagnostics in the developing world rapid antigen tests for diagnosis of pandemic (swine) influenza a/h1n1 a novel method of identifying genetic mutations using an electrochemical dna array amplified voltammetric detection of dna hybridization via oxidation of ferrocene caps on gold nanoparticle/ streptavidin conjugates electrochemical immunoassays a smart dna sensing system for detecting methicillin-resistant staphylococcus aureus using modified nanoparticle probes dna diagnostics: nanotechnology-enhanced electrochemical detection of nucleic acids role of rapid immunochromatographic antigen testing in diagnosis of influenza a virus 2009 h1n1 infection research priorities for the environment, agriculture and infectious diseases of poverty combined microfluidic-micromagnetic separation of living cells in continuous flow recent development of nano-materials used in dna biosensors effect of diluent chain length on the performance of the electrochemical dna sensor at elevated temperature electrochemical behavior and detection of hepatitis b virus dna pcr production at gold electrode a dna biochip for on-the-spot multiplexed pathogen identification recent advances in micro/nanotechnologies for global control of hepatitis b infection a simultaneous electrochemical multianalyte immunoassay of high sensitivity c-reactive protein and soluble cd40 ligand based on reduced graphene oxide-tetraethylene pentamine that directly adsorb metal ions as labels point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification a rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles microfluidic immunoassay with plug-in liquid crystal for optical detection of antibody au@pt nanoparticle encapsulated target-responsive hydrogel with volumetric bar-chart chip readout for quantitative point-of-care testing this research has been supported by the national university of singapore, department of pharmacy ((acrf) tier 1-frc grant r-148-000-164-112, r-148-000-213-112; nusage grant n-148-000-009-001), by moe of singapore (grant moe2009-t2-2-011, r-398-000-068-112) and by a-star-serc (r-148-000-222-305). leung kai fook grant (r-148-000-227-720). supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.biotechadv.2016.09.003. key: cord-327461-ohgkgvry authors: lu, ying; ni, yuxin; li, xiaofeng; he, xi; huang, shanzi; zhou, yi; dai, wencan; wu, dan; tucker, joseph d.; shen, guangquan; sha, yongjie; jiang, hongbo; huang, liqun; tang, weiming title: monetary incentives and peer referral in promoting digital network-based secondary distribution of hiv self-testing among men who have sex with men in china: study protocol for a three-arm randomized controlled trial date: 2020-06-12 journal: bmc public health doi: 10.1186/s12889-020-09048-y sha: doc_id: 327461 cord_uid: ohgkgvry background: human immunodeficiency virus (hiv) testing is a crucial strategy for hiv prevention. hiv testing rates remain low among men who have sex with men (msm) in china. digital network-based secondary distribution is considered as an effective model to enhance hiv self-testing (hivst) among key populations. digital platforms provide opportunities for testers to apply for hivst kits by themselves, and secondary distribution allows them to apply for multiple kits to deliver to their sexual partners or members within their social network. we describe a three-arm randomized controlled trial to examine the effect of monetary incentives and peer referral in promoting digital network-based secondary distribution of hivst among msm in china. methods: three hundred msm in china will be enrolled through a digital platform for data collection. the eligibility criteria include being biological male, 18 years of age or over, ever having had sex with another man, being able to apply for kits via the online platform, and being willing to provide personal telephone number for follow-up. eligible participants will be randomly allocated into one of the three arms: standard secondary distribution arm, secondary distribution with monetary incentives arm, and secondary distribution with monetary incentives plus peer referral arm. participants (defined as “index”) will distribute actual hiv self-test kits to members within their social network (defined as “alter”) or share referral links to encourage alters to apply hiv self-test kits by themselves. all index participants will be requested to complete a baseline survey and a 3-month follow-up survey. both indexes and alters will complete a survey upon returning the results by taking a photo of the used kits with the unique identification number. discussion: hiv testing rates remain suboptimal among msm in china. innovative interventions are needed to further expand the uptake of hiv testing among key populations. the findings of the trial can provide scientific evidence and experience on promoting secondary distribution of hivst to reach key populations who have not yet been covered by existing testing services. trial registration: the study was registered in the chinese clinical trial registry (chictr1900025433) on 26, august 2019, http://www.chictr.org.cn/showproj.aspx?proj=42001. prospectively registered. methods: three hundred msm in china will be enrolled through a digital platform for data collection. the eligibility criteria include being biological male, 18 years of age or over, ever having had sex with another man, being able to apply for kits via the online platform, and being willing to provide personal telephone number for follow-up. eligible participants will be randomly allocated into one of the three arms: standard secondary distribution arm, secondary distribution with monetary incentives arm, and secondary distribution with monetary incentives plus peer referral arm. participants (defined as "index") will distribute actual hiv self-test kits to members within their social network (defined as "alter") or share referral links to encourage alters to apply hiv self-test kits by themselves. all index participants will be requested to complete a baseline survey and a 3-month follow-up survey. both indexes and alters will complete a survey upon returning the results by taking a photo of the used kits with the unique identification number. discussion: hiv testing rates remain suboptimal among msm in china. innovative interventions are needed to further expand the uptake of hiv testing among key populations. the findings of the trial can provide scientific evidence and experience on promoting secondary distribution of hivst to reach key populations who have not yet been covered by existing testing services. trial registration: the study was registered in the chinese clinical trial registry (chictr1900025433) on 26, august 2019, http://www.chictr.org.cn/showproj.aspx?proj=42001. prospectively registered. keywords: hiv self-testing, monetary incentives, men who have sex with men, peer referral, secondary distribution background background and rationale men who have sex with men (msm) are one of the key populations affected by human immunodeficiency virus (hiv) [1] . compared to general populations, the risk of acquiring hiv is 22 times higher in msm [2] . in china, the hiv infection rate among msm was 6.9% by 2018 [3] . additionally, a large-scale systematic analysis illustrated that prevalence of hiv among msm in china increased substantially from 2001 to 2018 [4] . hiv testing is considered as a significant stage of the hiv care continuum [5] and the treat all strategy [6] , because serostatus awareness can link patients to timely treatment and prevent wider transmission of hiv [7] . thus, expanding hiv testing is crucial for hiv prevention and treatment. however, conventional hiv test services like healthcare facility-based tests fail to reach a wider hidden group of people, mainly due to the barriers including the stigma of hiv testing, the lack of confidentiality and privacy, low trust towards healthcare institutions, and inconvenience [8] [9] [10] [11] . to increase hiv testing among people who do not know their hiv status, the world health organization (who) recommends hiv self-testing (hivst) as an empowering and innovative way to reach those who have limited access to hiv testing and those who are at high risk of hiv infection [12] . with hivst, individuals can decide where and when to test while ensuring efficiency, privacy, and confidentiality. hivst may be an effective alternative for those who do not regularly attend healthcare facilities, which may also be a promising approach to increase the uptake of hiv testing in key populations such as msm [13, 14] . digital network-based secondary distribution of hivst could be an effective strategy for promoting hivst. this is a strategy that individuals (defined as indexes) to apply for multiple hivst kits and distribute them to their sexual partners or other members (defined as alters) within their social network [14, 15] . a cohort study conducted in kenya has proven the feasibility and acceptability of secondary distribution of hivst among female sex workers [14] . one observational study in china also indicated that secondary distribution of hivst successfully reached people who were not covered by traditional testing services and promoted case identification [16] . digital health is also considered to be an innovative strategy to deal with traditional health challenges, including challenges for hiv prevention [17] . for example, digital health has been used to promote safe sex, condom use, and awareness of hiv or sexually transmitted diseases among key populations [18, 19] . for msm, social networking or dating apps are widely used, especially among young msm, which provides a unique opportunity of leveraging digital health in improving health services among them. combining digital health and social network-based strategy for hiv testing (i.e., secondary distribution) promotion can help surpass traditional barriers and reach more people who have never been reached by the facility-based services and increase case finding. in this study, we intend to examine two modified secondary distribution approaches for hivst through a three-arm randomized controlled trial in zhuhai, china. the main purpose of this study is to compare the effectiveness of two modified secondary distribution approaches (monetary incentives, and monetary incentives plus peer referral) with the traditional secondary distribution approach, in order to determine whether these two approaches can increase the uptake of hivst among msm, especially the first-time testers among alters. our trial aims to enable more chinese msm to receive hiv self-testing, reach more first-time hiv testing alters, and identify more people with an hiv-positive (reactive) result by implementing the photo-verified hiv self-testing method under different scenarios, i.e., standard secondary distribution, secondary distribution with monetary incentives and secondary distribution with monetary incentives plus peer-referral links. hypothesis 1: compared with standard secondary distribution, secondary distribution with monetary incentives will promote index msm to distribute more hivst kits to people within their social network. hypothesis 2: compared with standard secondary distribution, secondary distribution with monetary incentives plus peer referral will promote index msm to distribute more hivst kits to people within their social network. hypothesis 3: compared with monetary alone secondary distribution, secondary distribution with monetary incentives plus peer referral will promote index msm to distribute more hivst kits to people within their social network. this is a three-arm randomized controlled trial among chinese msm. enrolled indexes will be randomly assigned to one of the three groups: standard secondary distribution, secondary distribution with monetary incentives arm, and secondary distribution with monetary incentives plus peer referral. further, indexes will be asked to complete a baseline survey at the beginning of the trial, and a three-month follow-up survey after their hivst kits applications. a flowchart of the trial is shown in fig. 1 . this trial, conducted in zhuhai, is a collaboration among the social entrepreneurship to spur health (sesh) research team, zhuhai center of diseases control (cdc), and zhuhai xutong voluntary services center (hereafter, xutong). zhuhai, located in southern china, has approximately 17,000 msm with an hiv prevalence rate of 7% [12] . xutong is a local gay community-based organization (cbo) founded in 2015 and has developed a digital network-based platform for individuals (all-over time) to apply for free hiv/syphilis self-testing kits provided by zhuhai cdc. with xutong's social impact within the gay community, its volunteers will help with study recruitment, and their online platform will be used to support our intervention implementation. study recruitment advertisement will be posted via xutong's official account on wechat, a popular social site in china similar to facebook and twitter. the recruitment information will also be advertised on blued, the largest social network app within the gay community in china. potential participants can join the trial via the study ads and sign up for xutong's online platform. an eligible index is required to meet the following criteria: 1) chinese born biologically male whose age is 18 years old or older; 2) ever had sex with another man; 3) willing to self-apply hivst kits via xutong's digital platform; 4) willing to provide personal contact information for future follow-up. all participants will need to sign an informed consent electronically before the study. randomization will be completed by a computer-generated program with a 1:1:1 allocation ratio. a consented participant will be allocated to one of three study groups, i.e., standard secondary distribution group, secondary distribution with monetary incentives group, and secondary distribution with monetary incentives plus peer referral group. standard secondary distribution arm/ control arm index msm in this arm will be eligible to apply for up to 5 hivst kits based on personal needs, and a 100 rmb (≈ 15 usd) deposit will be charged for each kit. the charged deposit will be refunded to the index if anyone (an index himself or alters to whom he distributed) return his/her testing result. in addition, the participants will need to provide contact information for kits shipping. all kits shipped to a participant will be packed with instructions in an unmarked box to protect privacy. each kit will be assigned with an identical confirmation code for future distribution tracking, and a unique "st" number for returned results tracking. after receiving kits, an index can choose to use the kits for themselves or distribute additional kits to alters such as sexual partners or friends. self-testing kit users can photograph and upload their results to the online platform anonymously by scanning the qr code attached on each kit box. figure 2 shows the hivst reporting qr code for testers to return results. further, alters will be asked to fill out a survey regarding their experience of and attitude toward hivst. after the survey is completed, the deposit will be returned to the matched index by tracking the confirmation code. index msm in this arm will follow the same application process as in the control arm. differently, a fixed 20 rmb (≈ 3 usd), designed as the financial incentives, will be offered to all self-testers who report their results. when an alter returns his/her results, his/her matched index msm will also receive an extra 20 rmb as incentives. index msm in this arm will first receive the same intervention as the participants received in the monetary incentives arm. in addition, apart from applying for up to five self-testing kits, each index msm in this arm will obtain a unique referral link, which can be shared with up to 5 individuals within their social network to apply for kits, and each alter can apply for only one kit through the link. similarly, indexes will be given 20 rmb for each matched alter who returns the result (whether through peer referral or direct distribution). all testers will receive a monetary incentive of 20 rmb once they complete uploading their results. the follow-up survey will be administered 3 months after indexes apply for hivst kits. this survey focuses on distribution history, the relationship between indexes and alters, and indexes' risky sexual behavior. the results from follow-up survey will be compared with baseline survey results to investigate whether the index msm has changed their behavior after hivst. xutong's volunteers will check returned self-test results. if there is any positive (reactive) result returned, volunteers will contact testers accordingly, and refer them to the local cdc to do confirmation tests. moreover, reactive testers will be encouraged to distribute hivst kits to their sexual partners or provide partners' contact information to volunteers for offering free testing service. the primary outcomes of this trial consist of three parts: 1) mean number of motivated alters who have photoverified self-testing per index in each arm; 2) proportion of first-time hiv testing among alters in each arm; 3) proportion of alters with a positive hiv testing result in each arm. the secondary outcomes of this study consist of three parts 1) risky sexual behavior among indexes and alters in each arm; 2) adverse events reported during secondary distribution among indexes and alters in each arm; 3) attitude towards and past experiences of hivst and sexual behavior among alters in each arm. in general, we aim to examine the effectiveness of our modified secondary distribution models. primary outcomes of this trial are the number of alters, first-time testing alters, and hiv-positive alters in each arm, therefore, all testers' information will be specifically clarified and recorded. all data will be collected through jin-shuju, a secure online platform where participants can apply for hivst kits, report testing results, and complete the baseline and follow-up questionnaires. all data collected from participants will be examined and deduplicated according to phone number and ip. in the baseline survey, we will collect the sociodemographic characteristics, sexual orientation, sexual behavior, hiv testing history, and social network. when alters return the results, they will also complete a questionnaire online. in this survey, except for basic information on socio-demographic characteristics, sexual orientation, sexual behavior, hiv testing history, and social network will be collected. we will also investigate the attitudes and experiences towards self-test. the follow-up survey for indexes will take place 3 months after the application completed, which focuses on experiences and attitudes on using and distributing self-test kits. the survey mainly inquires about the occurrence of intimate partner violence (ipv) or other adverse events, the relationship between the indexes and the alters, whether indexes have tested together with the alters, and whether indexes have guided the alters on how to perform hivst. specifically, in our surveys, socio-demographic characteristics include indexes' age, sex, marital status, the highest level of education completed, and monthly income level. sexual behavior within 3 months includes previous sex with males and females, role during sex with males, condom use, number of sex partners, and drug use. hiv testing history includes health care facility-based and online hiv testing experience. social network collects community engagements [7] , community connectedness [20] , identity fusion [21] , and social cohesion [22] . all blood samples will be analyzed using sd bioline hiv/syphilis duo test kits (sd bioline company, south korea). participants will collect fingertip blood samples by themselves according to the instruction. the trained staff of the cdc or cbo will check and read the photos of result and record them in jinshuju. the results are subject to the reading of the staff. participants will be involved at one to three stages: the baseline questionnaire, results return, and follow-up survey. however, there might be data missing in the primary and secondary outcomes. if there is < 20% of participants missing in the follow-up, a complete-case approach will be applied. if there is ≥ 20% of participants missing in the follow-up, we will investigate the missingness mechanism and use suitable imputation. sample size according to preliminary study results, on average, the number of alters motivated by an index man was 0.65 through standard secondary distribution, 1.0 through sd/monetary incentives intervention, and 1.4 through sd/monetary incentives plus peer referral intervention. we assumed that the variances of the three groups were equal with the same standard deviation of 0.5 (preliminary data, unpublished results). further, we estimated an effective sample size of 300 participants (100 in each group), with a power of 0.90, an alpha of 0.05, and a lost to follow-up rate of 0.20. all statistical analyses will follow the intention-to-treat principle. missing data will be handled by multiple imputations. categorical variables from the baseline survey will be aggregated in frequency distributions, and numerical variables will be summarized in mean and standard deviation. we will first calculate primary outcomes in each arm, i.e., mean number of alters that each index recruited, proportion of first-time hiv testers, and proportion of alter testers with an hiv-positive result. secondly, we will compare calculated means using two-sample t-test, and proportions using chi-square between sd/monetary incentives arm and control arm, sd/monetary incentives plus peer referral arm and control arm, and also between two intervention arms. results of the follow-up survey for indexes and the survey for alters will be used for measuring secondary outcomes, i.e. risky sexual behavior, adverse events during secondary distribution, and experience and attitude towards hiv testing. high-risk sexual behavior, defined as unprotected sex, substance use, or multiple sexual partners, are determined from survey questions such as "how often do you wear a condom during anal sex?", "in the past three months, how many sexual partners did you have?", "in the past three months, have you used drugs before sex?", etc. adverse events or ipv that happened on alters during distribution are determined from the question "have you experienced any of the following ipv when received the hivst kit from the index?". alters' experience and perception of hivst are determined from total 20 question items, e.g. "how difficult do you feel about completing hiv self-testing?", "how many men did you have anal sex with after hiv selftesting?", "which testing do you prefer, facility-based testing or hiv self-testing?", etc. we will use chi-square tests to compare each outcome of interests between sd/ monetary incentives group and control group, sd/monetary incentives and peer referral group and control group, and also between two intervention arms. despite global hiv control programs, hiv persists as a major public health threat among key populations such as msm [12] . therefore, the screening of hiv in msm plays a key role in furthering prevention and control. hivst was considered to be an alternative strategy to promote hiv testing. digital network-based secondary distribution takes advantage of digital network, which can be a promising approach to enhance hivst. we apply innovative strategies on the basis of secondary distribution model, adding monetary incentives and peer referral to explore a more effective secondary distribution model and promote hivst to wider populations. however, it is necessary to consider several limitations of this trial. first, due to the digital network strategy, access to this hivst service is limited so that the recruitment might overlook individuals who cannot access online social tools. however, the mobilization and promotion from the local cbo xutong, as well as the faceto-face distribution initiated by index msm can somehow resolve the problem. second, in the online surveys, behaviors of participants are self-reported, which may increase the possibility of social desirability bias. it can lead to the hawthorne effect that participants may have deviations in behavior reporting because of the awareness of the trial. however, the form of the online questionnaire and limiting collection of identifiers can reduce bias. third, from the perspective of implementation and promotion, each participant can only apply once due to the design of the trial, while there may be participants who have the habit of regular testing and request to apply for hivst kits multiple times. while in this project we cannot satisfy such demand, the future implementation model can expand access to multiple applications. this study generates a social innovation and policy implication that expands and improves the public service of hiv prevention and control, with the use of social network strategies. from the perspective of digital health, digital network-based secondary distribution of hivst is an innovative delivery approach that can reach hidden msm. beyond the geographical limitation, some msm, especially those in remote conservative areas, have limited access to facility-based services, while digital platforms might motivate them to perform hiv testing. furthermore, by taking advantage of digital network-based distribution, we aim to reduce fear of stigma associated with conventional hiv testing services in healthcare facilities. social network strategies, especially peer referral, taps into self-identity with a community and trust to provide services more effectively. empowering vulnerable individuals within a community by offering essential resources has both research and policy implementations for expansion of hiv services in other hardto-reach populations. upon completion of the study, we will provide the community with practical digital networkbased hivst interventions and scientific evidence on the feasibility and acceptability of the secondary distribution approaches. in addition, practical experience and knowledge gained from conducting the interventions can be considered and applied to further trials in enhancing hiv testing. if successful, the strategy has the potential to be implemented in similar regions. the study timeline was designed from 01, september 2019 to 31, may 2020. at the time of writing this draft protocol, study enrollment and data collection are ongoing. due to covid-19 pandemic, study recruitment expects to be delayed till 31, september 2020, and the follow-up will be finished by 31 december 2020. thus, we have further updated the trial registration status, and updated the study period as 01, september 2019 to 31, december 2020. ethics approval will be renewed annually. statistical analysis has not begun. the trial protocol conforms to the standard protocol items: recommendation for interventional trials (spirit) 2013 statement. global epidemiology of hiv infection in men who have sex with men unaids. global hiv & aids statistics -2019 fact sheet the prevalence of hiv among msm in china: a large-scale systematic analysis patching a leaky pipe: the cascade of hiv care an ambitious treatment target to help end the aids epidemic. geneva: unaids community engagement in sexual health and uptake of hiv testing and syphilis testing among msm in china: a cross-sectional online survey organizational characteristics of hiv/syphilis testing services for men who have sex with men in south china: a social entrepreneurship analysis and implications for creating sustainable service models hiv-testing behavior among young migrant men who have sex with men (msm) in beijing acceptability of hiv self-testing: a systematic literature review point-of-care testing for sexually transmitted infections: recent advances and implications for disease control status of hiv self-testing in national policies world health organization rapid hiv self-testing: long in coming but opportunities beckon promoting male partner testing and safer sexual decision-making through secondary distribution of hiv self-tests by hiv-uninfected female sex workers and women receiving antenatal and postpartum care in kenya: a cohort study promoting partner testing and couples testing through secondary distribution of hiv self-tests: a randomized clinical trial social-media based secondary distribution of hiv self-testing among chinese men who have sex with men: a pilot implementation program assessment [internet]. ias2019 digital health for sexually transmitted infection and hiv services: a global scoping review effects of internet popular opinion leaders (ipol) among internet-using men who have sex with men popular opinion leaders and hiv prevention peer education: resolving discrepant findings, and implications for the development of effective community programmes measuring community connectedness among diverse sexual minority populations identity fusion: the interplay of personal and social identities in extreme group behavior social cohesion, social participation and hiv testing among men who have sex with men in swaziland publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we appreciate the contributions from all study participants, cbo volunteers, and staff from zhuhai center for disease control and prevention, zhuhai xutong voluntary services center, and social entrepreneurship to spur health group. authors' contributions yl and yn drafted and finalized the paper with inputs from wt, dw and gs. jt provided critical revision of the paper. yz, dw, lh, and wt conceived the study. xh, xl, ys and sh assisted with recruitments. wt, yz, dw, jt, lh, wd, gs and hj provided oversight. wd and hj made insightful contributions to the study conception. all authors read and authorized the final version. this study received support from internal institute funding of zhuhai center for disease prevention and control, and the national institutes of health (nimh 1r34mh119963-01). the funding source had no role in the process of study design, data collection, and analysis, decision to publish, or preparation of the manuscript. data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.ethics approval and consent to participate ethical review of biomedical research has been obtained from the ethics committee of zhuhai center for disease control and prevention prior to study enrollment. all participants will be provided online consents and sign it electronically prior to taking part in the study. not applicable. the authors declare that they have no competing interests.author details key: cord-349358-leicos9j authors: ketzinel‐gilad, mali; shaul, yosef; galun, eithan title: rna interference for antiviral therapy date: 2006-06-16 journal: j gene med doi: 10.1002/jgm.929 sha: doc_id: 349358 cord_uid: leicos9j silencing gene expression through a process known as rna interference (rnai) has been known in the plant world for many years. in recent years, knowledge of the prevalence of rnai and the mechanism of gene silencing through rnai has started to unfold. it is now believed that rnai serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing rnai have been found in various viral pathogens. during the past few years, it has been demonstrated that rnai, induced by specifically designed double‐stranded rna (dsrna) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. however, many key questions and obstacles in the translation of rnai into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsrna that induces the system. it is expected that for the specific examples in which the delivery issue could be circumvented or resolved, rnai may hold promise for the development of gene‐specific therapeutics. copyright © 2006 john wiley & sons, ltd. the battle against viral infections is ferocious. since viruses are developing resistance to therapy, novel antiviral therapeutic modalities are in great demand. the currently approved antiviral therapies are based on the use of small molecular weight drugs, utilization of proteins simulating the innate immune response, and the adaptive immune system for both passive and active vaccination [1] . recently, an antisense drug against viral infection has also been approved, suggesting that newly developed approaches are acceptable. the first drug using antisense technology, fomivirsen (vitravene), developed by isis pharmaceuticals, was approved for the treatment of cytomegalovirus (cmv) retinitis. in general, small molecular weight drugs are mainly used today for chronic viral disease, although there are exceptions such as the anti-influenza compound, oseltamivir/tamiflu, which is used for acute infection. passive and active vaccination approaches are being developed for the prevention of severe acute diseases (in this case, there are also exceptions, such as life administration of anti-hepatitis b virus (hbv) antibodies to liver transplant patients at risk for hbv infection recurrence). the development and use of specific antiviral drugs and vaccines have been slower than expected and face major challenges. one particular example is the sluggish progress in generating effective drugs against the hepatitis c virus (hcv), even though the genomic sequence of the virus was unfolded over 15 years ago. hiv drug development also faces major drawbacks. although hiv replication is efficiently inhibited by the combination of highly active antiretroviral therapy (haart), long-term complications of haart include significant morbidity on the one hand, and the generation of multi-drug-resistant hiv strains on the other in a large proportion of patients. in spite of the comprehensive advancement in understanding the biology of the immune response, translating these findings into rational therapeutic platforms remains slower than expected. the need for preventive and effective vaccines remains as much a requisite today as it was in previous times in the history of mankind. indeed, at the beginning of the previous century, the world suffered devastating viral infection pandemics such as the one that occurred in 1918 where a quarter of the world population fell ill and the death toll reached over 25 million people from acute influenza infection. today, the achievement of peak vaccine efficacy to treat influenza stands at 75% among those under 65 years old and just 35% among the elderly. even today, the annual death toll from acute influenza infection in the usa tops 20,000 in spite of national vaccination programs. thus, different strategies are currently being suggested in an effort to be prepared for future pandemics [2] , and these include the development of escape mutants (antigenic drift) and reassortment of genetic segments of different quasispecies of the same virus or of different viruses (antigenic shift). presently, human viral pathogens are spreading worldwide, such as the much publicized menacing spread of the avian flu which is reported to have expanded to remote sites of russia and kazakhstan from the south-east, posing a major health threat to the entire world. in an effort to identify a novel class of efficient antiviral therapeutics, numerous technologies are currently being assessed for their antiviral potential. these include antisense oligonucleotides, antisense phosphorodiamidate morpholino oligonucleotides, ribozymes and, in recent times, rna interference (rnai). incidentally, we have been encouraged to learn that sirna (short interfering rna) against vascular endothelial growth factor (vegf) has recently been administered to patients in a clinical study without major side effects (asgt meeting, 2005) . in this review, we will summarize the recent developments in the use of rnai as an antiviral agent. rna interference (rnai) is a sequence-specific silencing of genes, induced by small molecules of double-stranded rna (dsrna). this phenomenon was first observed in plants in the late 1980s, but its molecular mechanism remained unclear until it was discovered in 1998 by fire et al., in the nematode c. elegans [3] . they showed that the presence of a very small quantity of dsrna led to almost a complete shut-off of the expression of the gene that was homologous to the dsrna. the interference process starts with cleavage of the dsrna that induced it into small rna duplexes, 21-23 nucleotides (nt) long, called sirnas (short interfering rnas) [4, 5] . these small dsrna duplexes have 2 nt overhang at their 3 end, a 5 -monophosphate and a 3 -hydroxyl group [6] . the enzyme responsible for that first step is dicer, a dsrna-specific nuclease that belongs to the rnaseiii family, and acts in an atp-dependent manner [7] . in the next step, the sirnas generated by the dicer are incorporated into the rna-induced silencing complex (risc), a multi-component enzymatic complex [5] . the risc unwinds the sirna in an atp-dependent manner, and uses its single-strand form to target the homologous transcript by base-pairing interactions. it then cleaves the mrna by its endonuclease component in a homology-dependent manner, only in the region corresponding to the sequence of the sirna. this process leads to degradation of the mrna [4, 8] . sirna-mediated gene silencing has also been found in lower organisms, such as plants, fungi, worms and flies [9] . it is a conserved mechanism of intracellular antiviral immunity that also protects the host genome from foreign genetic elements such as retroviruses, transposons and retrotransposons. these elements may have deleterious effects on the genomic dna of the host, and thus their mrna elimination may represent an earliest form of innate immunity. rnai was first suggested to evolve as a natural antiviral defense in plants, especially against rna viruses [10, 11] . in mammalians, rnai has also been reported to have gene-silencing properties. the rnai machinery was triggered experimentally by the introduction into the cells of artificially designed dsrna molecules, 21 nt in length, and the target gene was inhibited in a sequencespecific manner [8] . this effect has become very effective in silencing and knocking-down expression of specific genes in the cells. sirnas have become the method of choice for mammalian cell genetics as well as for potential sequence-specific therapeutic approaches. the inhibitory action of sirnas has been documented for numerous viruses. it works against rna viruses with negative-or positive-strand genome polarity, as well as against dna viruses. the sirna, as a therapeutic tool, can be targeted against the various phases of the viral life cycle of dna and rna viruses including replication, transcription, assembly of new virions, and budding out of the target cells ( figure 1 ). proteins for transcription. many rna viruses encode their own transcription factors. for example, the gag, pol and env genes of retroviruses are needed for an efficient transcription. indeed, sirnas directed against the gag and env genes of hiv-1 [12, 13] or the avian sarcoma leucosis virus [14] significantly reduce their overall transcription. another example is the dna human papilloma virus; sirna directed against the viral transcription factor e6 inhibits viral gene expression and growth in tissue culture [15, 16] . baculoviruses infect many different insect species. over many years, the autographa californica nucleopolyhedrosis virus has caused severe economic losses in the silk industry. inhibition of that virus was achieved by a pair of sirnas that target specifically the viral coded early transcription activator, and the major nucleocapsid [17] . one of the main steps in the process of viral infection is dna and rna genome replication of the virus. the genome of rna viruses, especially with plus polarity, serves both as mrna and as a replication template. many research groups applied the sirna method to inhibit replication of viruses in vitro and in vivo. in the absence of an efficient cell culture system for growing hcv, the sensitivity of hcv to rnai was shown in the replicon-based system. an hcv replicon is derived from an hcv consensus genome that was cloned from a viral rna isolated from an infected human and used to construct subgenomic selectable replicons. upon transfection of these subgenomic selectable constructs into a cell line, these rnas were found to replicate to high levels. in several studies, sirnas were directed against different targets in the virus genome [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . for example, sirna specific for the 5 untranslated region (utr) of the hcv genome, introduced into huh7 cells carrying the replicon system, inhibits hcv replication by up to 90% [20] , as measured by the expression level of the replicon luciferase reporter gene. sirnas targeting the viral polymerase ns5b region reduced expression of ns5b-luc chimera in mice [27] or in the replicon system in vitro. other studies that target other regions of the hcv genome reported a significant decline in the level of hcv proteins and the level of both the sense and antisense rna strands [25] . the sirna effect shown for hcv is ifn-and cell-cycleindependent [23] . in the hepatitis a virus (hav) replicon-based system, it has been reported that sirnas targeting the regions coding for the non-structural proteins of the virus give rise to partial inhibition of hav replication [28] . in that study, two sirnas specific for hav sequences increased rather than inhibited hav replication. this could be due to complex secondary structures of the target region that can limit and reduce the efficiency of the rnai process [29] . in another study, sirna targeted to various domains of the hav internal ribosomal entry site (ires) induced efficient and sustained suppression of viral genome translation and replication [30] . poliovirus is a highly cytopathic rna virus. sirnas specific to the poliovirus genome inhibited viral replication, as was demonstrated in a poliovirus replicon system. the sirna effect led to viral genome clearance from the infected cells, without destruction of the cells harboring the virus [31] . additional examples of inhibition of viral replication by sirna originated from the study of positive rna viruses such as dengue (denv), west nile (wnv), and severe acute respiratory syndrome (sars) [32] [33] [34] [35] [36] . sirnas targeting the 3 -utr sequence of denv, in a region that is conserved in all the dengue serotypes, reduced viral replication and infection in dendritic cells [32] . sirnas targeting the sars-cov rna polymerase gene inhibited viral rna replication, protein synthesis and reduced the viral cytopathic effects on vero cells [36] . likewise, expression vectors of sirnas specific for two different regions of the wnv genome protected 293t cells from wnv infection, and significantly reduced viral rna replication and virus production [35] . coxackievirus b3 (cvb3), a member of the picornaviridae family, is a major cause of many human diseases, such as meningioencephalitis and myocarditis. synthetic sirna targeted to the vp1 or to the viral polymerase showed antiviral effects in infected hela cells by inducing a significant reduction of viral replication [37] . the footand-mouth disease virus (fmdv) replication was inhibited in bhk-21 cells by sirnas targeting various conserved regions of the fmdv genome [38] . multiple sirnas have been used to target multiple conserved viral genes that are essential for virus replication, including a long 5non-coding region, a short 3 -non-coding region, the viral protein vpg, the viral polymerase, and the viral capsid protein vp4. the combination of those sirnas gave rise to a 10-1,000-fold inhibition in virus yield by specific inhibition of viral replication [38] . the antiviral properties of rnai have not been assessed in comparison for their effectiveness upon targeting the different intracellular stages of the viral life cycle. however, from current reports, we could surmise that targeting viral replication, similar to what has been described in several other types of antiviral methods, would probably be the suggested approach to suppress viral infection. replication of dna viruses can be inhibited by targeting their viral mrna, whereas replication of rna viruses can be inhibited by targeting either their mrnas or their viral rna, as was elegantly demonstrated for hiv [39] . in the later stages of the virus life cycle, the structural proteins are produced to assemble and form mature virions before egress. rotavirus causes severe diarrhea in infants and children worldwide. to combat this virus, dector et al., utilized sirna directed to the vp4, a viral structural protein that is essential for the attachment of the infecting virus to the cell surface. they showed a significant reduction in the number of viral particles produced in ma104, an infected monkey kidney cell line. moreover, most of the viral particles that were produced were poorly infectious [40] . however, there are only few reports assessing specifically the potential of the rnai effect on viral assembly [41] . the antiviral properties of rnai against viral assembly, a late stage in the intracellular viral life cycle, is expected to be less effective than rnai in the early steps of the viral life cycle. in many rna viruses, there is emergence of quasispecies that contain point mutations in the sirna's target sequences leading to evasion from inhibition by sirna. using a pool of sirnas to simultaneously target multiple sites in the viral genome can prevent the emergence of these resistant viruses [42, 43] . another approach that may partially solve this problem is targeting cellular factors rather than viral genes. during their life cycle, viruses apply cell membrane receptors for penetration, and cellular transcription factors for viral replication, harnessing very efficiently the cellular transcription and translation machinery for their life cycle. targeting those cellular genes may be another strategy for inhibition of viruses. and indeed, zhang et al., for example, succeeded in suppressing the replication of hcv in the replicon system by the expression of sirnas against cellular cofactors that are needed for viral replication, the polypyrimidine tract-binding protein (ptb) or eif2bg [44] . inhibition of the ptb alone by sirnas resulted in an efficient decrease in the levels of hcv proteins as well as hcv rna replication in huh7 cells harboring the hcv replicon [45] . in another study, sirna against cellular rna helicase p68 reduced hcv negative strand replication [46] . the e7 protein of human papillomaviruses (hpvs) contributes to oncogenesis. e7 was found to specifically activate the transcription of the cellular transcription factor e2f2 in an in vitro system of differentiating human keratinocytes. while suppression of e2f2 levels through the use of sirna decreased hpv replication, this loss did not affect cell proliferation. thus, e2f2 is a potential target for antiviral therapies [47] . the human cyclin t1 (hcyct1) is a cellular factor essential for transcription of messenger and genomic rnas from the long terminal repeat (ltr) promoter of the hiv-1 provirus. sirna directed against hcyct1 could effectively suppress hiv-1 replication without any induction of apoptotic cell death [48] . in previous studies, downregulation of other cellular factors, such as cd4, cxcr4, ccr5, nf-kb, p-tefb, cyclophilin a, dc-sign, spt-5 and parp-1, successfully inhibited hiv-1 replication [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] . however, since many of these molecules are essential for cellular processes (cd4, e.g., is a cell-surface molecule important for adaptive immune response), not all of them can serve as a practical target for hiv gene therapy. to conclude, sirnas can be used for inhibition of both rna and dna viral infections from the early stage of viral attachment to the cell to the late stage of viral assembly. sirnas can be targeted directly to the viral genes involved in the viral life cycle, or against cellular genes which are used by the viruses. in each case, the best strategy for viral inhibition needs to be assessed according to the virus type and its unique life cycle. interestingly, a combination therapy of two sirnas, targeting different viral sequences, each with inhibitory function, did not have an enhanced effect. this was also found by other groups, assessing the rnai effect in other human and non-human pathogens. these repeated observations could be related to the early saturation of the rnai cellular machinery. however, this issue will need further investigation, which could lead to improvement of the efficacy of rnai against viral infections. in some specific cases of acute viral infections, in particular those cases which could pose a major threat to the health care system, several hurdles remain to be overcome for the development of vaccines and specific small drugs. however, in most cases, the viral agent causing the acute severe endemic, possibly pandemic disease, could be rapidly identified and sequenced, as was the case during the recent outbreak of sars. in these cases, the development of sirnas targeted against various regions of the viral genome could lead to a quick development of a therapy against the acute viral infection. the production, delivery, dose, and modes of administration of sirnas could be tailor-designed for any group of targets. suffice to say, it is imperative that the timetable for the generation of a new sirna-based rna silencing drug could be shorter, so that a therapeutic platform against many specific infectious agents with pandemic potential could be forthcoming. obviously, it must be remembered that additional important factors need to be implemented for the development of an antiviral drug, such as in vitro and in vivo models, although these requirements are essential for any other type of therapeutic modality to be produced and tested. one recent report nicely exemplified the fact that synthetic sirna could be generated against a single (respiratory syncytial virus, rsv) or a double infection (with parainfluenza virus, piv), and rapidly tested in vitro, as a sufficiently predictive tool for an in vivo effect [65] . in this report, the sirnas against both rsv and piv were administered nasally with profound antiviral preventive and therapeutic effects without inducing interferon production. as mentioned in previous sections, sirna is very effective against other life-threatening pandemic threats such as influenza infections [66] . however, it must be remembered that we are probably just at the beginning of experimental assessments to determine the potential antiviral effects of sirna. the antiviral effects of short hairpin rnas (shrna) or sirnas were also assessed in other viral infections where there is practically no available therapy (shrnas are short hairpin rnas expressed by plasmid and viral vector systems and are subsequently processed to sirnas by the cellular machinery). sirna targeting the protease 2a region of the most common viral agent causing myocarditis, coxsackievirus b3, was found to be effective in inhibiting viral replication in vitro [67] . in addition, this same group also showed that the antisense sirna strand is critical for the rnai effect and that single nucleotide mutations at the central or 5 regions are detrimental for the antiviral effect. sirna was also effective against sars caused by the newly discovered coronavirus in a preventive model in vitro [68] . currently, since there is no available effective specific therapy against sars, rnai could be developed for this serious infection. in other cases of severe human infections by viral pathogens, there are fewer promising results than for the antiviral potential against viral replication by inducing rnai. sirna was also assessed against wnv infection. in one report, while the investigators failed to show an antiviral effect in active replicating cells [69] , they showed prevention of infection in a previous report. another group assessed the potential effect of sirna against wnv infection in vivo. again, only a preventive mode of therapy was found partially effective against viral replication and disease outcome in mice [70] . from the reports on the use of sirna against human viral pathogens causing acute disease, we could learn that for each specific pathogen infecting a specific cell lineage or tissue, we would probably need to perform an indepth assessment, with proper in vitro and in vivo models, and develop specific delivery systems. although the road towards rnai development could be visible for some of its destinations and the traveling speed could be changing, the target time remains unknown and unpredictable. an interesting new approach of preventing viral infection was reported by the group of judy lieberman [71] . in an effort to suggest a method of prevention of hsv2 sexual transmission, intravaginal installation of sirnas targeting two hsv-2 genes protected mice from a lethal hsv-2 challenge without inducing interferon-responsive genes. this encouraging result once again proves that combining a realistic method of gene delivery with a specific genetic drug payload for a specific disease could result in a beneficial gene therapy outcome. in most acute viral infections, the host overcomes the invading pathogen through a robust innate and adaptive immune response. however, in those cases where the virus causes severe disease as a result of a significant cytopathic effect, which could be related to a high multiplicity of infection (moi), low immunogenicity, high replication capacity or direct toxic effects, or a combination of all, could result in organ failure or even death. in these cases, rnai could significantly support, or even enhance, the antiviral effects for a short period of time and this could be achieved by the administration of sirnas. characterizing the specific viral pathogen, even in a pandemic situation, could enhance the rational design of a sequence-specific sirna that can be used as therapy. this situation is very different from cases of chronic viral infections of rna viruses, in that there could be quasispecies; in addition, the effect of the rnai should be prolonged and generated through an expression system rather than through a synthetic sirna administered once or only a few times. human chronic viral infections such as hbv, hcv and hiv are a worldwide threat. for hcv and hiv infections, there are no available vaccines, and, in addition, in both the prospect of vaccine development is not encouraging. furthermore, current therapeutics for both of these infections are suboptimal. for these viral infections, numerous gene-based approaches have been developed. although there are effective vaccines against hbv infection, chronic infection is still a major therapeutic challenge. the rnai was used to inhibit replication of dna viruses. hbv replication was inhibited in vitro and in vivo by rnai by us and by others [72] [73] [74] [75] [76] . sirnas targeted to different regions of the hbv surface antigen gene robustly inhibited viral gene expression and replication both in vitro and in vivo [72] . due to the overlapping gene structure of the hbv genome, targeting a region in the open reading frame (orf) of the x gene which is shared by all the viral transcripts resulted in a significant reduction of up to 90% in all viral transcripts and proteins and in a dramatic reduction of ∼95% in viral replication [75] . the x gene of hbv was also recently assessed as a target for rnai in vivo [77] . using two hbv mice models as a naked dna approach with the hydrodynamic method or expressed from an adenovector, the pol iii u6 promoter encoded short shrnas targeting conserved sequences of the hbx orf. the anti-hbv effect was significant without stimulating the interferon system. it is also known that hbx plays an important role in hepatocarcinogenesis. sirna against hbx was also used to test its effect against hepatocellular carcinoma cell lines which express hbx sequences [78] . this group demonstrated a significant reduction in cell proliferation, cell growth, anchorageindependent growth in soft agar, and tumor development in nude mice following the expression of sirna against hbx. a recent report suggested that the inhibitory effect of rnai on hbv expression is stronger than that of lamivudine in vitro [79] . we could further speculate that a combination of rnai and nucleoside analogs might encounter a synergistic effect, although this is yet to be determined. the most challenging part of rnai approaches for chronic viral infections is to design the best delivery method that would facilitate the targeting of the specific organ/cells with the appropriate expression system, for durable intracellular levels of gene-silencing effect. this also applies when designing an rnai approach for hcv infection, as well as for other chronic viral infections. the studies assessing the effect of rnai against hcv were mostly restricted to in vitro replicon systems, as discussed above. alternative in vivo systems were also adopted by some investigators, with reporting proteins used to assess the antiviral effect [80] . in early studies using the in vitro hcv replicon system, it was shown that a synthetic sirna targeting the 5 core region of hcv inhibited viral proteins and significantly suppressed viral replication for at least 8 days [21] . at about the same time, a different group showed, also in the replicon system, that sirna against the ns5b region (viral polymerase) is most effective in suppressing hcv replication [19] . this group had also transfected the huh7 replicon cells with a vector expressing complementary strands of sirna, again targeting the ns5b region, under the control of two separate h1 promoters (pcep4, invitrogen). in this case, the suppression effect on hcv replication lasted over 3 weeks. another group targeted similar hcv regions, ns3 and ns5b [26] ; they introduced shrnas targeted against these two genes into huh7-replicon cells. the delivery systems that were used in their study were plasmids or lentiviral vectors harboring shrnas against ns3 or ns5b, expressed from the u6 promoter. in both cases, they observed similar effects, i.e., suppression of hcv proteins and viral replication. however, shrna against the 5 -utr of hcv resulted in very low levels of inhibition of hcv replication. as mentioned earlier, viral replication is dependent on numerous cellular factors. targeting these viral replication/gene expression cofactors is a potential target for inhibition of viral replication. however, this approach should always be balanced against the potential of generating side effects which could overshadow the beneficial antiviral outcome. a recent study assessed this approach in vitro, targeting two hcv replication cofactors: proteasome α-subunit 7 and hu antigen r. shrna targeted against these two genes that were expressed from an expression vector transfected into huh7 hcv replicon cells showed a modest reduction of hcv transcription [81] . however, this modest inhibition on the one hand, and the potential role of both proteins in normal cellular function on the other, might suggest that it is advisable to abstain from such approaches, if possible. an interesting study in vivo targeting the hcv ires, translating a luciferase reporter protein, revealed that the in vitro synthesized shrna, administered systemically by the hydrodynamic method, encountered a sustained antiviral effect lasting over 4 days compared to synthetic sirna [80] . the use of rnai against hiv infection was reported by a number of groups (table 1) . although the results of these studies suggest that hiv could be targeted by rnai, there are major obstacles in translating this therapeutic approach into the clinical setting. most reports have used sequences from laboratory hiv strains. viruses with mutations at the rnai recognition site produced an escape mutant after a long-term rnai pressure. targeting relatively conserved hiv sequences could improve the efficacy of the rnai effect. a recent study looked at the protective effect of shrnas targeting the rev, gag, and vif sequences of a panel of hiv clades [82] . this study showed that targeting the vif hiv region had a significant inhibition effect on hiv replication. however, the long-term use of any specific sirna or shrna against hiv could probably induce the generation of escape mutants containing nucleotide substitutions at or near the target sites. furthermore, the escape from rnaimediated inhibition could also signify the emergence of mutations that change the hiv rna secondary structures [83] . all of these data emphasize the significance of hiv evolution during rnai pressure and its potential impact on the use of rnai against hiv. the virus also harbors a specific mechanism that evades the nucleicacid-based innate immunity of human cells against hiv. the genome of hiv contains a plethora of dsrna regions capable of being processed into sirnas targeting the viral genome to suppression [84] . however, the virus has evolved by a counter process, rendering itself resistant to rnai through the tat protein, altering the dicer effect on viral sequences, and abrogating the host cell innate immunity against hiv infection. interestingly, on the other hand, it is possible that hiv does apply the cellular rnai machinery for regulation of its own gene expression. the hiv nef region encodes a microrna, mir-n367, which can block nef expression [85] . later, it was shown by the same group that mir-n367 targets the hiv ltr promoter region, downregulating viral transcription; this might be a mechanism by which the virus regulates its own transcription [86] . single anti-hiv therapy is ineffective against viral replication and gene expression due to the high mutation rate of the virus. one option of overcoming this major obstacle is to generate therapeutics against highly conserved viral genomic regions. a recent report showed that it is possible to clone shrnas against the conserved regions of the hiv genome into hiv vectors, and to suppress hiv infection upon targeting the gag, pol, int and vpu sequences [87] . however, although this approach could be applied for prevention of infection, cessation of an ongoing hiv replication is prone to failure due to the high mutation rate of the virus. an alternative strategy could be to target essential cellular determinants for hiv infection. the early step of hiv infection would be attaching to the viral cellular receptor. during the progressive stage of the disease, most hiv isolates use the chemokine receptor cxcr4 for viral attachment and penetration into the host cells. patients with mutations of the cxcr4 receptor are less prone to hiv infection and are healthy by any significant measure. this finding was the rationale used to develop an anti-hiv approach by targeting cxcr4 expression and inhibiting viral fusion with the cellular membrane [54] . it is expected that such an approach will pose a major obstacle to viral evolution and prevent infection. other groups have also adopted this strategy to render cells resistant to hiv infection [52, 53, [58] [59] [60] . the hiv regulatory protein, rev, is essential for viral life cycle in a number of ways including splicing, translation and trans-activation. with regard to rev trans-activation properties, it needs to interact with the hypusine-containing protein eif-5a. the eif-5a rev cofactor is activated following a catalytic step performed by the human deoxyhypusine synthase (dhs). a recent study has suggested that rna interference inhibiting dhs blocked hiv replication [88] . again, as with other drugs in development against hiv infection, we are confronted with the following major questions: what are the potential side effects from such an approach, and what would be the therapeutic dose window? indeed, studies aimed at unfolding these issues are crucial before entering any clinical studies. other groups have also suggested the targeting of other cellular factors essential for the hiv life cycle, including parp-1 [55] , the elongation transcription factor p-tefb [56] , cyclophilin a, dc-sign [61, 63] , the human elongation transcription factor spt5 [64] , and human cyclin t1 [89] . the road towards the development of an efficient therapeutic modality using the anti-hiv rna interference strategy is bumpy, due to potential side effects; moreover, significant strides will have to be made towards harnessing clearcut approaches as described, as well as designing additional rational studies through wet and dry investigations [89] . reduction in cell-to-cell spread [170] the cellular level of dicer could be crucial for gene therapy approaches while utilizing the rnai machinery in targeted cells. recent data suggest that, although the expression of dicer and other proteins that participate in digesting long dsrnas into 21-25 nt, e.g., eif2c1 ∼ 4, decreases in differentiated cells, they retain a sufficient amount of enzymatic activity to induce rnai. however, when designing and planning any specific approach using sirna for gene therapy, it is advisable to assess the dicer activity in the targeted tissue if the expected sirna is unmet. since rna silencing acts as an antiviral defense mechanism in plants [91] , insects [92] and other eukaryotes, including mammalian cells [93] , it is not surprising that viruses have developed strategies to interfere with this effect. many plant dna and rna viruses have developed proteins that function as suppressors of rna silencing [94] [95] [96] [97] [98] . since the silencing suppression reduces the antiviral effects, the viruses can accumulate in their target cells and reach higher titers. comparing those suppressor genes did not reveal any sequence homology between them. the rna silencing suppressors can act upon the various sequences of the rnai machinery in several ways such as inhibition of sirna processing, inhibition of the incorporation step into the risc, or preventing the action of its effector molecules. although the mechanisms of inhibition of silencing are not fully understood, there are several suppressor genes that their targets have identified. the p19 protein from the tombusvirus was shown to bind specifically to the sirnas, and thus may inhibit the incorporation step of the sirnas into the silencing effector complexes [98] . the hc-pro protein, expressed by polyviruses, acts by targeting the risc [99, 100] . another example is the mosaic virus 2b protein (cmv2b). this nucleus-localized rnai suppressor protein inhibits the activity of the spreading signal of the rnai, and inhibits dna methylation processes in the nucleus that control the silencing pathways [97, 101] . the coat protein of the turnip crinkle virus (tcv) strongly suppresses the rna silencing process at an early initiation step, probably by interference with the function of the dicer cleavage reaction [96] . since the rnai machinery is conserved in mammals, it appears possible that, similar to plant viruses, viruses that infect invertebrates and vertebrates, including human viruses, have developed strategies to suppress rna silencing. and indeed, there is evidence that such inhibitors are not limited to plant viruses. in insects, the flock house virus (fhv) is a target of rna silencing. it has been shown that in drosophila and mosquito cultured cells, the fhv-encoded protein, b2, inhibited rna silencing, but the mechanism is still unknown. this protein also inhibited rnai in transgenic plants, suggesting that the rna silencing pathway is conserved in plants and animals [92, 102, 103] . recent evidence also indicates that human virus genes have the ability to inhibit the rnai pathway. both the influenza virus ns1 protein and the vaccinia virus e3l protein can inhibit rnai in drosophila s2 cultured cells [102] . the adenovirus encoded inhibitor of rnai is functional in mammalian cells. the human adenovirus inhibits rnai in the later stages of infection by suppressing the activity of dicer and the risc. the virus-associated rnas, va rnai and va rnaii, bind directly to dicer and function as competitive substrates, squelching it, and the resulting sirnas are incorporated into the active risc [104, 105] . the antiviral therapeutic potential of rnai would require identifying possible viral suppressors of the rnai machinery and designing strategies to inhibit their expression. one major drawback for most antiviral approaches is the development of resistance. this is most apparent in cases where the fidelity of the viral polymerase is low, especially in viruses with an rna genome. to overcome this hurdle, most antiviral therapeutic protocols harness a strategy that uses multiple drugs targeting different viral proteins or steps in its life cycle. one example where such resistance has been developed in vitro is in the case of rnai against the nef gene of hiv [106, 107] . to overcome this problem, it may be advisable to utilize a multi-targeted rnai approach possibly in combination with additional antiviral modalities. although targeting multiple genomic sites has probably no advantage with regard to the direct antiviral effect, it could repress the development of resistance. the initial steps towards focusing into this avenue have already been established. the group that initially described the sirna effect in vitro in the replicon system against hcv [19] have shown that, following several rounds of treatment with the same sirna against hcv, the replicon became resistant to that specific sirna, developing a point mutation at the target site. however, the replicon was still sensitive to a sirna targeting a different hcv region. in addition, the development of a resistant replicon was limited by the use of a combination of two sirnas targeting simultaneously different hcv genomic regions [108] . a similar approach has also been suggested by other groups [109] . the off-target effect of rnai is the silencing of nontargeted genes containing partial sequence identity to the sirna. in experiments conducted on specificity of sirna in cultured human cells, jackson et al., have demonstrated that sirna can cause direct downregulation of unintended targets containing as few as 11 contiguous bp complementarities [110] . to increase sirna specificity, there are many sirna design programs that employ various sequence alignment algorithms; however, maximum complementarity, by itself, is not enough for accurate prediction of off-targeting. a study done in dharmacon inc., identified a significant association between off-targeting and exact complementarity between the seed region of the sirna (bases 2-7) and their offtargeted genes. this pattern has been recognized in mirna-mediated gene silencing, thus suggesting that sirna off-targeting may operate by a mechanism similar to that of mirna targeting (a. birmingham, personal communication). until the off-target mechanism of sirna is understood, this issue can encounter deleterious side effects on the use of rnai. the role of interferon signaling in rnai has given rise to a series of conflicting reports. although most studies suggest that there is very little non-specific effect of sirna, others have shown that the jak-stat pathway is activated following sirna transfection. this effect is mediated by dsrna-dependent protein kinase (pkr) [111] . while we expect that this issue will foster additional debates, at this point, it would be important to impart cautious interpretations upon describing rnai effects. in addition, recent studies have suggested that although sirna does not activate the intracellular interferon machinery in mammalian cells upon entrance or in situ propagation inside the cells, if they are shorter than 30-nt dsrna, there is a non-specific innate immune response depicted by cytokine production [112] . furthermore, this effect is dependent on the toll-like receptor 3 (tlr3) that senses dsrna and serves as its receptor. tlr3 is located intracellularly on the endosome membrane and signals through nfκb nuclear translocation for the production of inflammatory cytokines. incubation of immune cells with sirna induces the activation of cells [113] . all of these effects could be dependent on the concentration of sirna. recent works identified putative immunostimulatory motifs within sirnas, and showed that even a slight change of these motifs did not significantly hamper the rnai process [114] . this research provides a basis for the design of synthetic sirnas that avoids activation of the innate immune response, and helps to minimize immunotoxicity. the group of reuven agami was the first to report a new vector system, called psuper, which directs the synthesis of sirnas in mammalian cells. they used the poly iii h1-rna gene promoter to express shrnas that specifically down-regulated gene expression, resulting in functional inactivation of the targeted genes [115] . recent reports showed that the expression of shrna from the h1 or the u6 pol iii promoters in a hiv-based vector induces the expression of interferon-stimulated genes (isgs). this effect is dependent on the presence of an aa dinucleotide near the transcription starting site. preserving a c/g sequence at positions −1/ + 1 prevents this effect [116] . in some cases, the expression from the u6 promoter is relatively low. the enhancer of the cmv immediateearly promoter enhances the u6 promoter activity [117] . others have also tested various promoters [118] reporting some beneficial effects on expression with the modified trna(met)-derived (mtd) promoter, upon expressing shrna against hiv-1 compared to u6 or h1 promoters [119] . it may happen that for each specific application, we would need to compare numerous regulatory elements to achieve the desired rnai effect. in some systems, it may be important to tightly control the expression of the shrna. although most controlled systems do not reach the desired stringency in vivo, some reports have suggested the use of specific systems. one such method is the tet-onoff expression technology [120] . again, each investigator should specifically assess the potential of this inducible system in a specific tissue culture or animal model. the teton-off systems were developed for naked dna transfection systems or incorporated into viral vectors like the lentiviral vector [121] . an additional long debate in the literature questions the choice of the loop structure to apply for the design of shrna. one specific strategy was to adopt the natural loop structure of microrna [118] . this was also used for shrna against hcv [80] . the major challenge in rnai gene therapy is to transform the in vitro robust effects of sirna into an in vivo genesilencing method. in other words, what would be the preferred delivery system to use in animals and later on in humans? as for gene therapy in general, and the specific aims of delivering rnai platforms, we need to tailor-design the tools to be used to the sought objective. this includes targeting the tissue, adjusting the desired level of expression (high level of sirna could induce nonspecific silencing [122] ), the longevity, and the specific maladies that we wish to treat. this is a complex situation, especially since our major barrier is the lack of a simple, non-immunogenic, targeted delivery system without side effects. however, in spite of all of these hurdles, we would like to discuss the potential available methods to deliver synthetic sirna. the most straightforward method of using sirna in vivo is by administering synthetic sirna. upon coinjecting sirna and its target being expressed from a plasmid vector, we could achieve knockdown of expression in the liver following a hydrodynamic injection. in our laboratory, we could show that hbv expression out of an hbv head-to-tail plasmid that supports viral replication in the liver of mice could effectively be silenced transiently with synthetic sirna [72] . this effect was dosedependent. however, the kinetics of this effect revealed that the silencing was transient as was also shown by other groups using a similar approach [27, [123] [124] [125] . the effect subsides after 48 to 72 h and is probably completely lost after 7 days. the silencing effect of sirna following systemic administration of duplex rna could have been hampered by various factors, thus imposing some logistic hurdles upon translating this approach into the clinical setting (additional examples of rnai against viral infections are depicted in table 1 ). although duplex rna is quite stable in serum [124] , and more stable than ssdna or ssrna, a high serum concentration could reduce stability. the introduction of phosphorothioate linkages could enhance stability in the serum [124] . others have used chemically modified sirna with the complete absence of 2 -oh residues on the sense and antisense strands of the dsrna, including 2 -fluoro, 2 -omethyl and 2 -deoxy sugars. these chemical modifications of sirna were produced in order to enhance its stability and effect [126] . not all modifications have resulted in beneficial silencing effects [127] [128] [129] ; a short review of these modifications was reported by paroo and corey [130] . one particular interesting report [131] shows that a specific sirna with a combined chemical modification had a significant and a relatively sustained effect in vitro and in vivo as compared to non-modified sirna. however, although sirna is relatively stable in the serum, there are disadvantages of using synthetic sirna: (1) the effect of synthetic sirna is transient; in order to impose a long-term effect, repeated administrations would be needed, and this might still be true for the chemically modified sirnas. (2) the production of synthetic sirna is expensive, making repeated administrations for longterm effect very costly. (3) it is very complicated to target synthetic sirna to a specific cell or tissue. however, in specific cases, the use of sirna directly administered into the target tissue could encounter a significant effect. in a recent report by dorn et al., they used sirna against the pain-related cation-channel p2x 3 , by intrathecal injection of phosphorothioated (ps) sirna in a rat model of neuropathic pain [132] . although they did not compare the non-ps-modified versus the ps-modified sirna, they have clearly shown a significant effect of sirna in relieving chronic pain. furthermore, the effect was superior to the comparable p2x 3 antisense oligonucleotides. one specific case where such an approach could be translated into an applicable clinical therapeutic modality is in post-herpetic neuralgia which could follow varicella-zoster infection. however, since chronic pain is a condition that is generally expected to last for months, this type of treatment would need to be readministered several times. a recent report took advantage of the knowledge generated regarding the stabilization of sirna by chemical modifications, and protection of the modified sirna with pegylated liposomes upon delivery. they assessed the antiviral effect of sirna against hbv in vitro and in vivo. interestingly, the modified sirna had induced less non-specific cytokine secretion combined with an effective and anti-hbv effect of up to 6 weeks after repeated weekly administrations [133] . in an effort to enhance and prolong the effect of sirna, various approaches have been undertaken using nonviral reagents. tailoring the specific delivery tactic and method is essential when designing a specific therapeutic strategy. one example is the use of sirna targeting the influenza virus genome. this malady can cause moderate to severe illness, can affect millions of people each year, and could be life-threatening. for the gene therapist, this means that the genetic therapy effect could be designed for a short window of time. in this case, the use of synthetic sirna with an enhanced transduction is an appropriate approach. in a study by ge et al., the systemic and intratracheal delivery of polyethylenimine (pei), a cationic polymer, promoting sirna delivery in mice, is beneficial for prophylaxis and therapy of the influenza virus infection [134] . pei was developed for in vitro and in vivo local and systemic gene delivery and pei-mediated gene delivery/transduction into the lung following systemic and intratracheal administrations. some investigators reported safety problems in specific cases upon the use of pei in animals. however, plasmid dna mixed with pei administered into the human bladder was safe (a. hochberg, personal communication). tompkins et al., also assessed the effect of sirna against the same pathogen, the influenza virus [135] . this group used a different therapeutic regimen. they used a preventive measure by administering naked sirna systemically, i.e., intravenously, before infection, and at the time of infection, they administered the sirna/oligofectamine  (a lipid carrier from invitrogen) intranasally. in this model, the undertaken therapeutic approach prevented death of animals. however, this study was limited to asking the general question of sirna effect against influenza virus infection rather than comparing different sirna delivery systems. thus, we cannot draw any conclusions that would suggest a preference for any specific delivery method in this disease model system. future animal studies would be required to determine the preferred delivery method. in addition to the lipid carriers, traditionally developed for in vitro and in vivo delivery of dna and now for sirna, alternative approaches have also been developed. one specific interesting report by minakuchi et al. describes the use of atelocollagen (at) [136] . at is prepared from type i collagen from calf dermis. this is a low immunogenic product that is already available in clinics for various indications like promoting wound healing. the same investigators showed that at enhanced dna delivery and supported prolonged expression. at mediated the delivery of sirna in vitro and was found in vivo to encounter a significant advantage over the sirna/liposome complex in inhibiting tumor growth in mice. recently, more sophisticated delivery methods of sirna have also been developed to treat brain tumors [137] . the obtained results could be important for those interested in developing sirna therapeutics for viral encephalitis. the model that they used in vivo to assess their delivery and rnai effects was an immunodeficient mouse with an inoculated intracranial human u87 glioma tumor that was dependent on egf signaling for growth. one of the major barriers for macromolecules to travel to diseased tissue in the brain is the blood-brain barrier (bbb). passing the bbb is a major challenge faced for the development of any pharmacological compound with high molecular weight. pegylated (polyethylene glycol) immunoliposomes (85 nm size liposomes designed with monoclonal antibodies over its outer surface) were able to support transvascular delivery of plasmid dna and to target and transduce specific cells in the brain. this group conjugated the pegylated liposomes with two monoclonal antibodies, one against the mouse transferrin receptor to enable bbb crossing, and the second against the insulin receptor to enhance cellular uptake. the generated pegylated immunoliposomes encapsidated a plasmid payload that was designed to express the sirna against the egf receptor in the transduced cell. the sirna complex of pegylated immunoliposomes showed an enhanced antitumor effect by prolonging survival of animals. the ability to treat brain tumors with a systemic approach rather than by stereotactic injection is a major achievement. recently, it has been reported that recombinant hbv capsids can be used as efficient vehicles for oligonucleotide delivery; they can encapsulate the oligonucleotides in vitro, and mediate their delivery into cells very efficiently. the process is not cell-type-specific. this method may be useful for in vivo systems for hbvinfected individuals or in other diseases provided that the immunogenicity of the viral capsids can be decreased; until then, it can be used in cell culture and in ex vivo systems [138] . the potential advantage of viral-mediated sirna delivery encouraged numerous groups to clone expression cassettes in transgenes and to encapsidate these into viral particles. each type of viral vector holds specific properties. these viral vector characteristics should be those that determine which viral delivery system needs to be applied for the specific therapeutic target. the adenovector [139] , and in particular the ad-gutless vector, hold major promise for liver-directed systemic delivery. in cases where short-term silencing effect is warranted, the non-gutless vector could then be applied; however, for prolonged silencing effects, the gutless vector might be more beneficial. in the gutless vector, there are practically no restrictions as to the size of the sequences to be incorporated and these could include marker genes, regulatory controlled cassettes or matrix-controlled regions for prolonged expression. controlling the expression of an sirna, specifically in tumor cells, could also be designed in a conditionally replicating adenovirus (crad). crads are designed to replicate and specifically kill tumor cells without harming normal cells. carette et al. [140] applied crad, which is dependent on rb deficiency for replication, to test its potential in silencing expression by shrnas, a marker gene in vitro in tumor cells. they showed that the silencing effect of a marker gene is dependent on crad replication. the combination of the crad antitumor effect with a sirna against a tumor-dependent growth gene should be assessed in the future; this possibility was not considered by this group, whether in vitro or in vivo. these issues would need further studies in an effort to assess and enhance their therapeutic potential. the major advantage of the retroviral delivery method is the potential to incorporate the payload transgene they 'carry' into the host cell genome. the integration site could not be specifically targeted and this could cause side effects. integration that occurs near cellular protooncogenes can lead to their aberrant expression from the viral ltr, or alternatively this could cause the disruption of a tumor suppresser gene expression. in the past 2 years, numerous groups have reported on various retroviral deliveries [141] , including lentiviral systems to express sirnas against viral pathogens and tumor cells. ralf bartenschlager and associates reported the use of the moloney murine leukemia virus (mo-mulv)-based vector (pbabe) as a delivery system for sirna targeting hcv [22] . in their publication, they also assessed a unique rnai approach against hcv infection. this was done in an effort to overcome the low fidelity of the viral polymerase, establishing a state of quasispecies by generating endoribonuclease-prepared sirna to simultaneously target multiple sites of the viral genome in order to prevent escape. as for the retroviral delivery approach, this group designed their sirna mainly against the viral ires sequences. their readout system to assess the silencing effect involved tissue culture cells transfected with the subgenomic hcv replicon. this replicon harbors the hcv ires upstream of the luciferase gene and the neomycin resistance region, and an additional non-hcv ires upstream of the viral non-structural sequences. the presence of the luciferase gene enables the determination of hcv ires activity in vitro. this system enables the assessment of the effect of sirna against the hcv ires as well as against the viral non-structural genome. the sirna in the retrovirus was expressed from the h1 promoter. they designed numerous sirnas in the vector and found that a specific region in the hcv ires, near the beginning of the viral coding nucleotides, was the most sensitive to rnai effect. although this is an interesting approach with clearcut results, significant developmental steps and modifications are required in order to translate this modality into the clinical setting. however, the results described in this report again suggest that the rnai encounters a therapeutic potential for chronic viral infection. similar observations were reported by other groups ( [18, 24] , see table 1 ). presently, most groups who use retroviral vectors to express sirna apply lentiviral vectors. however, it must be kept in mind that the clinical experience with this vector is very limited. numerous papers were recently published describing differently designed lentiviral vectors to meet copyright [121] or conditioning [142, 143] of sirna expression. veerle baekelandt and associates have designed a study to assess the potential use of lentiviruses in delivering sirna into brain tissue. in their recent report [144] , they constructed a lentiviral vector with sirna against the marker gene egfp. upon simultaneous administration into the brain tissue by stereotactic injection of the lentivirus expressing the egfp and the lentiviral vector expressing the sirna against the same gene, they were able to show almost complete knockdown of egfp expression. in two additional experiments in which the sirna lentivirus was administered before or after the marker gene, they were also able to show significant silencing of expression. interestingly, they claimed, albeit without showing the data, that this effect persisted for 6 months. however, the issue of delivery is again a major barrier. stereotactic administrations are possible, but alternative approaches of systemic delivery or intra-organ spreading of the vector would be beneficial. lentiviruses hold major promise in gene therapy. once the issue of integration and production is overcome, we expect that the lentiviralbased vector will be integrated into the clinical setting. the aav vector is currently perceived as a relatively safe vector since it supports long-term expression in most tissues from an episome. beverly davidson and associates reported an interesting study on the suppression of polyglutamine-induced neurodegeneration in a mouse model of spinocerebellar ataxia (sca1), a disease of the polyglutamine-expansion group which also includes huntington chorea [145] . they expressed the sirna under a modified cmv promoter due to its enhanced expression/silencing effect compared to the pol iii promoter, in their hands. in addition, they revealed that incorporating the mir23 loop (10-nt loop sequence) into the sirna expression cassette enhanced the silencing effect, resulting in improved suppression of the ataxin protein levels [118] . however, this effect was only apparent in the pol iii expression vector. how to select the best loop in any specific case is still an open question, and, presently, this is a matter of empiric assessment. in their mouse model, they injected the aav vector expressing the shrna against the mutant sca1 message directly into the brain tissue. this treatment showed longterm therapeutic effect on motor coordination as well as a histological improvement by reducing intranuclear inclusions. although this represents a step forward from previous studies with similar models that used antisense and ribozymes as therapeutic agents, we are still far from the clinic. the direct administration of a viral vector into brain tissue is a significant drawback for the current delivery systems. the potential side effects of aav administration into the brain may soon be revealed once results of clinical studies using the aav for direct brain administration in parkinson and alzheimer disease studies are available [146] . one specific point of importance should be mentioned on the issue of designing loop sequences stated previously. a number of investigators have assessed this matter as related to the effectiveness of silencing. we must bear in mind that for each specific case, there is a need to develop a specific structure to improve the silencing effect [147] . chemically modified oligonucleotides (oligos) vs. si/shrna as antiviral drugs the jury is still out as to when an antisense approach should be adopted against viral infections or when an si/shrna strategy should be applied. although antisense oligos were discovered more than 25 years ago [148] their role as antiviral tools in the clinic is still in progress. early studies with antisense oligos have shown promising antiviral potency. however, later reports determined that such approaches encounter significant problems. although comprehensive stringent comparison studies in vitro and in vivo between antisense oligos and sirna were not performed, we would like to stress a few practical points which characterize each group of compounds, in particular those which are important for those investigators interested in designing an antiviral strategy. antisense oligos with a high phosphorothioate content, which are early-generation antisense, interact directly and non-specifically with proteins, potentially interfering with their function [149] . however, recently developed 2nd and 3rd generation rnas -like oligos -have improved significantly the binding affinities of antisense oligos, as well as their nuclease and non-specific proteinbinding characteristics. the new generations include 2 -o-methyl (2om) and the additional version 2 -omethoxyethyl (2ome), both encountering improved nuclease resistance, binding affinity and reduced nonspecific binding affinity. recently, additional novel chemically modified versions of antisense oligos were developed including the locked nucleic acids (lna) [150] , anhydrohexitol nucleic acids (hnas), peptide nucleic acids (pnas), morpholino nucleic acids (mnas), and other uncharged oligos. all these novel chemicals have been tested and proven to hold significant antisense properties, with antiviral effects in models in vitro and in vivo. their effects are due to the high binding affinity and nuclease degradation resistance, without significant rnaseh activity. to overcome this hurdle, a chimeric version of oligos was generated, the gapmers, containing a core region of a phosphodiester/phosphorothioate flanked on both sides with modified oligo backbones. these gapmers also hold the rnaseh activity of traditional antisenses. when designing any specific experiment aimed to assess an antisense effect, it is important to select the best fitted oligo by testing 10 to 20 different sequences, and compare the selected oligo with appropriate controls containing scrambled, mismatched and irrelevant antisense oligos. although a significant amount of investigations would be needed to confirm which specific antiviral nucleic acid approaches would be best fitted for a specific disease therapy, by comparing antisense or ribozymes and sirna, some general guidelines can be phrased: (1) for acute viral infections, the preferred type of rna therapy could be a synthetic designed antisense or sirna. (2) for chronic viral disease targets, a preferred treatment would be such that continuously generates an antiviral drug. this could include an expression vector for any type of nucleicacid-based drug, including antisense, ribozyme or shrna. (3) for diseases of simple accessible organs, such as ocular cavity, oral cavity, vagina and epidermis, synthetic rnabased drugs might be beneficial. the need for repeated administration might be simple even in chronic disease states in cases of accessible organs. (4) for chronic multiorgan, or internal organ, involved in viral infections, e.g., chronic viral hepatitis b or c, intracellular continuous expression of high-level non-toxic, effective therapy is desirable. in this case, an expression system delivered into infected cells is warranted. designed shrna based on up-to-date criteria of shrna [151] [152] [153] might be the preferred gene therapy based treatment. (5) special attention must be given as to the specific type of virus to be targeted; whether a dna or a rna agent. furthermore, it is important to know where, inside the cellular compartments, should the rna-based drug be concentrated, e.g., cytoplasmatic vs. nuclear. for rna viruses, such as hcv, which replicate in the cytoplasm, an antisense approach could be suggested, although it should be kept in mind that the rnaseh effect is preferentially restricted to the nuclear compartment of the cell. however, for chronic hepatitis b virus infection, in which there is a nuclear reservoir of the virus in the form of super-coiled species, a vector which will express its shrna payload in the nuclear compartment is preferred. numerous new expression systems were recently suggested to encounter improved silencing properties [147] . most of these methods are waiting to be assessed as for their truly beneficial properties as antiviral reagents. in our modern world, viral infections still pose a major threat to mankind. since viruses are developing resistance to the current available therapies, there is an ongoing battle between the viruses and our ability to develop novel strategies to fight them. in vitro and in vivo experiments carried out so far conceivably demonstrate the effectiveness of rnai in inhibiting many viruses that cause severe health and economical problems. even though in vivo experiments in larger animals as well as developing efficient delivery methods have to be done before applying rnai in humans, this fascinating phenomenon will undoubtedly continue to provide new and exciting data regarding its mechanism of action and therapeutic applications in the years to come. strategies and mechanisms for host and pathogen survival in acute and persistent viral infections strategies for containing an emerging influenza pandemic in southeast asia potent and specific genetic interference by double-stranded rna in caenorhabditis elegans rnai: doublestranded rna directs the atp-dependent cleavage of mrna at 21 to 23 nucleotide intervals an rnadirected nuclease mediates post-transcriptional gene silencing in drosophila cells rna interference is mediated by 21-and 22-nucleotide rnas role for a bidentate ribonuclease in the initiation step of rna interference duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants initiation and maintenance of virus-induced gene silencing rna silencing in plants -defense and counterdefense prevention of hiv-1 infection in human peripheral blood mononuclear cells by specific rna interference inhibition of hiv-1 infection by small interfering rna-mediated rna interference rna interference against retroviruses in vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by e6 sirna selective silencing of viral gene e6 and e7 expression in hpv-positive human cervical carcinoma cells using small interfering rnas silencing structural and nonstructural genes in baculovirus by rna interference inhibition of intracellular hepatitis c virus replication by synthetic and vector-derived small interfering rnas rna interference blocks gene expression and rna synthesis from hepatitis c replicons propagated in human liver cells small interfering rna-mediated inhibition of hepatitis c virus replication in the human hepatoma cell line huh-7 clearance of replicating hepatitis c virus replicon rnas in cell culture by small interfering rnas alternative approaches for efficient inhibition of hepatitis c virus rna replication by small interfering rnas interference of hepatitis c virus rna replication by short interfering rnas inhibition of hepatitis c virus protein expression by rna interference interfering with hepatitis c virus rna replication suppression of hepatitis c virus replicon by rna interference directed against the ns3 and ns5b regions of the viral genome rna interference in adult mice interference of hepatitis a virus replication by small interfering rnas local rna target structure influences sirna efficacy: systematic analysis of intentionally designed binding regions suppression of hepatitis a virus genome translation and replication by sirnas targeting the internal ribosomal entry site short interfering rna confers intracellular antiviral immunity in human cells attenuation of dengue virus infection by adeno-associated virus-mediated sirna delivery rna silencing of dengue virus type 2 replication in transformed c6/36 mosquito cells transcribing an inverted-repeat rna derived from the virus genome rna interference, arthropod-borne viruses, and mosquitoes the utility of sirna transcripts produced by rna polymerase in down-regulating viral gene expression and replication of negative-and positivestrand rna viruses inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells a small interfering rna targeting coxsackievirus b3 protects permissive hela cells from viral challenge cross-inhibition to heterologous foot-and-mouth disease virus infection induced by rna interference targeting the conserved regions of viral genome modulation of hiv-1 replication by rna interference rotavirus gene silencing by small interfering rnas rotavirus replication: plus-sense templates for double-stranded rna synthesis are made in viroplasms sequence homology required by human immunodeficiency virus type 1 to escape from short interfering rnas poliovirus escape from rna interference: short interfering rna-target recognition and implications for therapeutic approaches down-regulation of viral replication by adenoviral-mediated expression of sirna against cellular cofactors for hepatitis c virus the polypyrimidine tract-binding protein (ptb) is required for efficient replication of hepatitis c virus (hcv) rna cellular rna helicase p68 relocalization and interaction with the hepatitis c virus (hcv) ns5b protein and the potential role of p68 in hcv rna replication hpv31 e7 facilitates replication by activating e2f2 transcription through its interaction with hdacs specific inhibition of hiv-1 replication by short hairpin rnas targeting human cyclin t1 without inducing apoptosis specific inhibition of hiv-1 gene expression by double-stranded rna sirna-directed inhibition of hiv-1 infection rna interference directed against viral and cellular targets inhibits human immunodeficiency virus type 1 replication bispecific short hairpin sirna constructs targeted to cd4, cxcr4, and ccr5 confer hiv-1 resistance inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using tat-or ccr5-specific small interfering rnas expressed from a lentivirus vector inhibition of hiv-1 fusion with small interfering rnas targeting the chemokine coreceptor cxcr4 rna interference directed against poly(adp-ribose) polymerase 1 efficiently suppresses human immunodeficiency virus type 1 replication in human cells inhibition of human immunodeficiency virus type 1 replication by rna interference directed against human transcription elongation factor p-tefb (cdk9/cyclint1) suppression of chemokine receptor expression by rna interference allows for inhibition of hiv-1 replication protection from hiv-1 infection of primary cd4 t cells by ccr5 silencing is effective for the full spectrum of ccr5 expression potent suppression of hiv type 1 infection by a short hairpin anti-cxcr4 sirna suppression of chemokine receptor expression by rna interference allows for inhibition of hiv-1 replication cyclophilin a retrotransposition into trim5 explains owl monkey resistance to hiv-1 rna interference of hiv replication lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells modulating hiv-1 replication by rna interference directed against human transcription elongation factor spt5 inhibition of respiratory viruses by nasally administered sirna the promise of sirnas for the treatment of influenza inhibition of coxsackievirus b3 replication by small interfering rnas requires perfect sequence match in the central region of the viral positive strand inhibition of sars-cov replication by sirna actively replicating west nile virus is resistant to cytoplasmic delivery of sirna use of rna interference to prevent lethal murine west nile virus infection an sirna-based microbicide protects mice from lethal herpes simplex virus 2 infection small interfering rna inhibits hepatitis b virus replication in mice short interfering rna-directed inhibition of hepatitis b virus replication inhibition of hbv replication by sirna in a stable hbv-producing cell line inhibition of hepatitis b virus expression and replication by rna interference inhibition of hepatitis b virus in mice by rna interference effective inhibition of hbv replication in vivo by anti-hbx short hairpin rnas knock-down of hepatitis b virus x protein reduces the tumorigenicity of hepatocellular carcinoma cells genomic analysis of anti-hepatitis b virus (hbv) activity by small interfering rna and lamivudine in stable hbv-producing cells small hairpin rnas efficiently inhibit hepatitis c ires-mediated gene expression in human tissue culture cells and a mouse model inhibition of hepatitis c virus translation and subgenomic replication by sirnas directed against highly conserved hcv sequence and cellular hcv cofactors lentiviral delivery of short hairpin rnas protects cd4 t cells from multiple clades and primary isolates of hiv hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome evidence that hiv-1 encodes an sirna and a suppressor of rna silencing hiv-1 nef suppression by virally encoded microrna regulation of human immunodeficiency virus 1 transcription by nef microrna lentiviral sirnas targeting multiple highly conserved rna sequences of human immunodeficiency virus type 1 identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy computational design of antiviral rna interference strategies that resist human immunodeficiency virus escape rnai induction and activation in mammalian muscle cells where dicer and eif2c translation initiation factors are barely expressed rna silencing as a plant immune system against viruses induction and suppression of rna silencing by an animal virus nucleic acid-based immune system: the antiviral potential of mammalian rna silencing adenosine kinase inhibition and suppression of rna silencing by geminivirus al2 and l2 proteins effects and side-effects of viral rna silencing suppressors on short rnas the coat protein of turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step a viral protein inhibits the long range signaling activity of the gene silencing signal a viral protein suppresses rna silencing and binds silencing-generated, 21-to 25-nucleotide double-stranded rnas virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway p1/hc-pro, a viral suppressor of rna silencing, interferes with arabidopsis development and mirna unction suppression of posttranscriptional gene silencing by a plant viral protein localized in the nucleus interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing a virus-encoded inhibitor that blocks rna interference in mammalian cells adenovirus va1 noncoding rna can inhibit small interfering rna and microrna biogenesis suppression of rna interference by adenovirus virus-associated rna human immunodeficiency virus type 1 escapes from rna interferencemediated inhibition human immunodeficiency virus type 1 escape from rna interference hepatitis c virus replicons escape rna interference induced by a short interfering rna directed against the ns5b coding region inhibition of hepatitis b virus gene expression by single and dual small interfering rna treatment expression profiling reveals off-target gene regulation by rnai activation of the interferon system by short-interfering rnas cationic liposome-mediated delivery of sirnas in adult mice small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3 sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna a system for stable expression of short interfering rnas in mammalian cells determinants of interferon-stimulated gene induction by rnai vectors an enhanced u6 promoter for synthesis of short hairpin rna short hairpin type of dsrnas that are controlled by trna(val) promoter significantly induce rnaimediated gene silencing in the cytoplasm of human cells promoter choice affects the potency of hiv-1 specific rna interference development and application of sirna expression vector conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible rna interference short-term cytotoxic effects and longterm instability of rnai delivered using lentiviral vectors efficient delivery of sirna for inhibition of gene expression in postnatal mice rna interference in mammalian cells by chemically-modified rna rna interference targeting fas protects mice from fulminant hepatitis in vivo activity of nuclease-resistant sirnas functional anatomy of sirnas for mediating efficient rnai in drosophila melanogaster embryo lysate sirna function in rnai: a chemical modification analysis functional anatomy of a dsrna trigger: differential requirement for the two trigger strands in rna interference challenges for rnai in vivo activity of stabilized short interfering rna in a mouse model of hepatitis b virus replication sirna relieves chronic neuropathic pain potent and persistent in vivo anti-hbv activity of chemically modified sirnas inhibition of influenza virus production in virus-infected mice by rna interference protection against lethal influenza virus challenge by rna interference in vivo atelocollagenmediated synthetic small interfering rna delivery for effective gene silencing in vitro and in vivo intravenous rna interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer recombinant viral capsids as an efficient vehicle of oligonucleotide delivery into cells adenovirus vectormediated doxycycline-inducible rna interference conditionally replicating adenoviruses expressing short hairpin rnas silence the expression of a target gene in cancer cells inducible, reversible, and stable rna interference in mammalian cells cre-lox-regulated conditional rna interference from transgenes cre recombinase-inducible rna interference mediated by lentiviral vectors lentiviral vector-mediated delivery of short hairpin rna results in persistent knockdown of gene expression in mouse brain rnai suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia first parkinson gene therapy trial launches optimization of an sirna-expression system with an improved hairpin and its significant suppressive effects in mammalian cells inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide antisense oligonucleotides: promise and reality lna: a versatile tool for therapeutics and genomics rational sirna design for rna interference asymmetry in the assembly of the rnai enzyme complex functional sirnas and mirnas exhibit strand bias attenuation of sars coronavirus by a short hairpin rna expression plasmid targeting rnadependent rna polymerase silencing sars-cov spike protein expression in cultured cells by rna interference using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque rna interference targeting vp1 inhibits foot-and-mouth disease virus replication in bhk-21 cells and suckling mice small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism susceptibility of human hepatitis delta virus rnas to small interfering rna action rna interference against enterovirus 71 infection inhibition of porcine endogenous retroviruses by rna interference: increasing the safety of xenotransplantation enhanced gene silencing of hiv-1 specific sirna using microrna designed hairpins expression of small hairpin rna by lentivirus-based vector confers efficient and stable gene-suppression of hiv-1 on human cells including primary non-dividing cells inhibition of hiv-1 by lentiviral vector-transduced sirnas in t lymphocytes differentiated in scid-hu mice and cd34+ progenitor cellderived macrophages efficient gene transfer of hiv-1-specific short hairpin rna into human lymphocytic cells using recombinant adeno-associated virus vectors inhibition of virus production in jc virus-infected cells by postinfection rna interference inhibition of the epstein-barr virus lytic cycle by zta-targeted rna interference selective inhibition of hepatitis b virus replication by rna interference clearance of hepatitis b virus from the liver of transgenic mice by short hairpin rnas short interfering rna-mediated inhibition of herpes simplex virus type 1 gene expression and function during infection of human keratinocytes the investigators are supported by the israeli ministry of science, through a grant to the national gene therapy knowledge center and through the ec grant lshb-ct-2004-512034. the investigators are also supported by the blum, grinspoon, horowitz & wolfson foundations and an isf grant. we would like to thank michal gropp and hilla giladi for carefully reading the review. key: cord-338804-nreqluol authors: heise, m.t. title: viral pathogenesis date: 2014-11-28 journal: reference module in biomedical sciences doi: 10.1016/b978-0-12-801238-3.00079-9 sha: doc_id: 338804 cord_uid: nreqluol viral pathogenesis describes the processes by which viral infections cause diseases and involves virus–host interactions at the cellular and systemic level that determine whether a virus will cause a disease, what form that disease takes, and how severe the disease will be. though the pathogenesis of each virus is unique, there are several common points in the virus life cycle that are shared between all pathogenic viruses, and by considering these common aspects of the virus-induced disease process, we can explore some general concepts in viral pathogenesis while illustrating some of the virus specific processes that shape disease outcomes. viral pathogenesis is a term that generally describes the processes by which viral infection results in a disease. however, viruses can range from small rna viruses (e.g., flaviviruses such as dengue virus) to large dna viruses (e.g., herpesviruses and poxviruses), all of which interact with the host in unique ways to drive the virus-induced disease process. these virus specific disease outcomes are driven by fundamental differences in viral replication cycles, modes of transmission, tissue tropism, interactions with the host immune response, as well as a multitude of other variables. furthermore, due to differences in a wide range of factors, including elements such as host genetic variation, host immune status, viral dose, or route of inoculation, infection with the same virus often results in varied disease outcomes in different individuals, where some individuals may develop no disease at all, while others are symptomatic and may develop serious or life threatening disease. therefore, it is difficult to provide a general overview of viral pathogenesis that accurately encompasses the full range of virus-induced disease processes. however, while the pathogenesis of each virus and its associated disease(s) has unique aspects, it is also true that there are several common stages in the viral life cycle/disease process that are shared between all pathogenic viruses, and consideration of these common processes can be used to illustrate several key concepts in viral pathogenesis. for example, since viruses are obligate intracellular pathogens that are not capable of reproducing themselves outside of a permissive host cell, a virus must successfully gain entry to a target cell and propagate itself to cause a disease. whether a virus can accomplish this task depends on interactions with key host molecules, such as cell surface receptors, which determine whether the virus can successfully infect and reproduce itself within its target cells. therefore, for the purposes of this overview, we will consider several common virus/host interactions, including: (1) factors that affect viral tissue tropism and host range, (2) viral immune evasion, and (3) viral effects on target cells or tissues. by focusing on these key steps in the viral life cycle/disease process, we can discuss some general concepts that illustrate how these interactions impact viral pathogenesis, while also emphasizing the virus specific aspects of these interactions that result in each virusinduced disease having unique attributes. as noted above, viruses cannot replicate outside of a permissive host cell, and gaining access to and being able to replicate within these cells represents a key part of any virus's life cycle. furthermore, the induction of disease is usually dependent upon the effects of the virus on cells or organ systems that it infects, such as direct killing of essential host cells (e.g., neurons) by the virus (see below). therefore, viruses that cannot gain access to and replicate within permissive host cells are generally not able to cause disease. furthermore, viruses that cannot gain access to the specific tissue that is associated with a disease, such as the central nervous system for viruses that cause encephalitis, are also less likely to cause severe diseases. though there are multiple virus-host interactions that determine whether a virus can successfully replicate within a target cell, for the purposes of brevity, we will consider specific examples of stages in the viral life cycle where interactions with the host determine whether the virus can successfully replicate in target cells, and how these interactions promote the virusinduced disease process figure 1 . viral entry into host cells usually involves interactions between molecules on the surface of the virus and specific cell surface receptors, which allow the virus to bind to the host cell and initiate the viral entry process. in some cases, viruses interact with a single cell surface molecule, which mediates viral binding to the cell and also facilitates viral cell entry. for example, several rhinoviruses bind to icam-1 (cd54), and these interactions promote viral infection of the cell (reviewed in rossmann et al., 2002) . in contrast other viruses, such as herpes simplex virus, interact with cell surface molecules such as heparin sulfate to facilitate viral attachment to the cell, and then engage specific host receptor proteins on the cell surface that mediate viral entry into the cell (reviewed in spear, 2004) . viral interactions with these receptors can have a significant impact upon several aspects of viral pathogenesis, including determining the cell or tissue tropism of a virus or even whether a virus can efficiently infect and cause disease in a specific host species. the importance of virus-receptor interactions in disease pathogenesis is nicely illustrated by interactions between human immunodeficiency virus (hiv) and two host molecules, cd4 and ccr5, which mediate hiv binding and entry. hiv infection requires interactions between the viral gp120 protein and cd4 (sweet et al., 1991) , a host protein expressed on a subset of t cells (cd4 positive helper t cells) and a limited number of other cell types, such as macrophages. since hiv infection is dependent on interactions with cd4, cd4 positive helper t cells are the major target of hiv replication, and viral replication in these cells results in the progressive loss of cd4 t cells over the course of the hiv infection. cd4 t cells play an essential role in regulating the immune response, and hiv-mediated killing of these cells ultimately leads to immune suppression that leaves hiv infected individuals susceptible to lethal opportunistic infections that would normally be controlled by persons with fully functional immune systems. therefore, viral interactions with cd4 and subsequent viral tropism for these cells, directly contributes to disease pathogenesis during hiv infection. in addition to the importance of cd4 in determining hiv tropism, a second hiv/receptor interaction further illustrates the importance of receptors in driving viral pathogenesis. following hiv gp120 binding to the cd4 molecule, the gp120 molecule undergoes a conformational change, which allows gp120 to interact with one of two coreceptor molecules, ccr5 or cxcr4, that then leads to viral fusion (alkhatib et al., 1996; feng et al., 1996) . the importance of these interactions is illustrated by the fact that a small subset of humans has a nonfunctional form of ccr5, and these individuals are highly resistant to hiv infection (liu et al., 1996; dean et al., 1996; huang et al., 1996) . in other words, if the virus cannot get into its appropriate target cells through interactions with ccr5, it cannot efficiently infect these individuals and cause disease. in addition to affecting disease pathogenesis by determining viral cell tropism, virus-receptor interactions can also broadly impact disease pathogenesis by determining whether a virus will efficiently infect a new host. this is particularly important for zoonotic viruses, which are viruses that naturally reside in animals, but which jump to humans and cause diseases. to successfully make the transition from its natural animal host to humans, a zoonotic virus must either interact with receptors that are highly conserved between species, or the virus must change (mutate) in a way that allows it to adapt to efficiently interact with receptors in the new host. an example of the first situation is provided by sindbis virus, a mosquitoborne virus that must efficiently infect both mosquitos and vertebrate hosts. recent studies have identified natural resistance-associated macrophage protein (nramp), as a receptor for sindbis virus in both mosquitoes and vertebrate cells (e.g., humans) (rose et al., 2011) . nramp is highly conserved between species, and it is likely that by interacting with this highly conserved receptor protein, sindbis virus is able to readily replicate in both mosquitoes and vertebrates. in contrast to situations where a virus interacts with a highly conserved receptor, there are also situations where the virus receptor is significantly different between species. therefore, for the virus to successfully make the transition between the original animal host and humans, it is likely that the virus will have to adapt to more efficient use of the receptor in the new species. an example of this type of interaction is provided by the sars coronavirus (sars-cov), which caused an outbreak of severe acute respiratory disease in 2002-03. sars-cov infects cells through interactions between the viral spike (s) protein and angiotensin converting enzyme-2 (ace2) (li et al., 2003) . however, sars-cov is thought to normally reside in bats (lau et al., 2005) and the bat-derived virus does not efficiently interact with human ace2. however, studies suggest that mutations within the receptor binding domain of sars led to more efficient interactions with the human ace2 molecule, and that viruses with these adaptive mutations were better able to infect human cells (reviewed in bolles et al., 2011; graham and baric, 2010) . this is supported by the finding that introducing the region of the s protein that binds to human ace2 into the bat sars virus allows that virus to bind to human ace2 and efficiently infect human cells (becker et al., 2008) . these results further reinforce the idea that virus receptor interactions play a crucial role in determining whether the virus can efficiently infect the host and ultimately cause disease. though receptor interactions represent a crucial component of virus-host interactions and viral pathogenesis, a number of other factors can also determine which tissues a virus infects. most viruses encode their own replication machinery, however they are still dependent upon the host cell for a number of functions, including processes that promote viral entry or the translation and assembly of viral proteins. recently, the field of virology has become very interested in identifying 'proviral' factors, which are host molecules that promote efficient viral replication. identification of these proviral factors not only enhances our knowledge of how viruses interact with the host cell, but also may identify host pathways that could be targeted to inhibit viral replication and develop new therapies. many proviral factors are components of generally important cellular processes, such as the host translation machinery or cellular protein transport pathways, which are likely to be important for broad classes of viral pathogens. however, there are instances where host factors interact with specific viruses to enhance viral replication in specific cell types, thereby affecting both viral cell tropism and disease pathogenesis. in the most extreme examples, viral replication, and hence the ability to cause disease, would be severely compromised by the absence of a specific proviral factor. an excellent example of a virusspecific proviral factor is provided by hepatitis c virus (hcv) interactions with mir-122. hcv is a positive stranded rna virus that infects the liver and causes chronic disease in a significant fraction of infected individuals, putting these individuals at risk for the development of chronic liver failure and the development of hepatocellular carcinoma (reviewed in cabibbo et al., 2012) . though hcv may be able to replicate in cell types such as b and t cells, the major site of hcv replication are hepatocytes within the liver. a key determinant of hcv's tropism for hepatocytes is the host microrna mir-122. micrornas are small (20-22) nucleotide host rnas, which regulate a number of biological processes within the host (reviewed in szabo et al., 2012) . in the context of hcv infection, mir-122 is expressed specifically within the liver and its expression enhances hcv rna levels in hepatocyte cell lines (jopling et al., 2005) . though the mechanisms of mir-122's actions on hcv are not completely understood, mir-122 interacts with rna structures in the 5 0 end of the viral genome, and these interactions are essential for mir-122's effects on hcv (jopling et al., 2005) . the importance of these interactions for hcv replication and disease pathogenesis is illustrated by recent studies in a chimpanzee model of hcv where administration of a mir-122 specific antagonist resulted in decreased viral loads within the liver and a reduction in hcv associated disease signs within the liver (lanford et al., 2010) . therefore, hcv interactions with mir-122 affect viral replication and disease pathogenesis within the liver, illustrating the potential importance of viral interactions with proviral host factors in promoting viral replication and driving the pathogenesis of virus-induced diseases. it is important to note that in addition to proviral factors, there are also classes of host proteins that can act as restriction factors that actively inhibit a virus's ability to replicate and cause disease. these types of host restriction factors have been extensively studied in the context of retro/lentivirus infection and an excellent example of this type of factor is provided by trim5a. trim5a is part of a class of tripartite motif containing host proteins, a large number of which have been shown to exhibit antiviral or immune regulatory functions (reviewed in mcnab et al., 2011) . studies looking at restriction factors that limit the ability of hiv to replicate in nonhuman primate cells found that the rhesus macaque trim5a molecule strongly interacts with the hiv capsid to block viral infection at an early stage in the viral replication process (stremlau et al., 2004) . in contrast, the human trim5a molecule, which interferes with retroviruses and lentiviruses such as equine infectious anemia virus, interacts less efficiently with hiv (stremlau et al., 2005) . efficient interactions between trim5 and hiv appear to protect macaques from hiv replication and disease, while less efficient interactions between human trim5a and hiv in part explain the enhanced susceptibility of humans to hiv infection. therefore, the presence or absence of appropriate restriction factors can have a major impact on host susceptibility to virusinduced disease, where the presence of a strong restriction factor would inhibit viral replication and prevent or limit virusinduced disease. in addition to trim5a, other factors that mediate host range restriction during lentivirus/retrovirus infection include apobec and tetherin (reviewed in luban, 2012) . the host innate and adaptive immune systems play a major role in protecting from virus-induced disease by limiting or preventing viral replication. the type i interferon system and other components of the innate immune response are rapidly activated in response to viral infection and play a crucial role in limiting viral replication and spread within the host. likewise, components of the adaptive immune system, such as cytotoxic cd8 positive t cells and antibody, are involved in clearing virus from infected tissues and can provide long-term immunity to prevent reinfection. the complexity of the immune systems and its interactions with each viral pathogen is such that a comprehensive overview of virus-host immune interactions is beyond the scope of this article. however, though there are aspects of the virus-host immune interaction that are unique to each pathogenic virus, every viral pathogen must successfully avoid or actively antagonize the host immune response to infect, replicate, and disseminate within the host. in fact, viruses with defects in blocking the host immune response are often attenuated in their ability to cause disease. therefore, immune evasion represents a common theme in viral pathogenesis that we will explore further. while different viruses employ a wide range of strategies to avoid/antagonize aspects of the innate and adaptive immune system, we will focus specifically on viral interactions with the innate immune system and the interferon response in particular to illustrate the importance of these interactions on viral pathogenesis. though multiple arms of the innate immune system contribute to viral control, including components such as the complement cascade, natural killer cells, and proinflammatory cytokines, several lines of evidence suggest that the type i interferon system plays an essential role in the pathogenesis of most viral pathogens. most, if not all of the pathogenic viruses affecting humans either avoid or actively antagonize some aspect of the type i interferon response, and viruses that are defective for avoiding/antagonizing the type i interferon system are often attenuated for replication and disease in immunocompetent animals (garcia-sastre et al., 1998; talon et al., 2000; leib et al., 1999; bouloy et al., 2001) . furthermore, animals lacking a functional type i interferon system exhibit enhanced sensitivity to a wide array of viral pathogens (leib et al., 1999; ryman et al., 2000; white et al., 2001; bouloy et al., 2001; schilte et al., 2010) , which suggests that type i interferon (ifn) plays an essential role in limiting viral replication and protecting from disease. the type i interferons are a group of cytokines consisting of a several related alpha interferon molecules and a single interferon beta protein, that are induced in response to stimuli associated with viral infections, such as double stranded rna, and which bind to a common type i interferon receptor complex. signaling via the type i interferon receptor leads to the induction of hundreds of interferon-stimulated genes (isgs). though the function of many of these isgs still needs to be elucidated, it is clear that many of these molecules have antiviral activity against one or more viral pathogens (schoggins and rice, 2011) , while some isg molecules modulate other aspects of the host immune response, including antigen presentation (schoggins and rice, 2011) . therefore, the type i interferon system limits viral replication and dissemination by inducing an antiviral state within host cells, and then promotes viral clearance by modulating other aspects of the host immune response, including host antibody and t cell responses. for the purposes of this overview, we will briefly summarize several key aspects of the type i interferon response to illustrate how viruses can avoid or antagonize this response. the production of type i interferon can be induced by signaling through several different pattern recognition molecules, including certain toll-like receptors and cytoplasmic nucleic acid sensors, such as rig-i. these pattern recognition molecules recognize pathogen associated molecular patterns (pamps), which are molecules that have signatures commonly associated with viral infection, such as double stranded rna. readers who are interested in more detailed discussion of the pattern recognition molecules that regulate the type i ifn response are directed to the following reviews (kawai and akira, 2006; nakhaei et al., 2009) . though simplified for this overview, following interactions with a viral pamp, each pattern recognition receptor will activate a signaling cascade that ultimately converges on a set of transcription factors (irf3 or irf7), which upon activation, transit to the nucleus to induce type i ifn transcription and subsequent production of type i interferon. type i interferons are secreted from the cell, and can then act in an autocrine (on the cell that produced the interferon) or a paracrine (affecting the surrounding cells) manner. the type i interferon receptor consists of two subunits ifnar1 and ifnar2, which upon interferon binding, dimerize at the cell surface. the receptors are associated with two protein tyrosine kinases, janus activated kinase 1 (jak1) and tyrosine kinase 2 (tyk2), that are brought together during receptor dimerization. jak1 and tyk2 then undergo auto-and/ or transphosphorylation (de weerd et al., 2007; de weerd and nguyen, 2012) , which leads to their activation, and they in turn phosphorylate tyrosine residues present on the receptor tails that serve as docking sites for signal transducers and activators of transcription (stat) factors. jak1 and tyk2 phosphorylate stat1 and stat2, which then form heterodimers and interact with interferon regulatory factor 9 (irf9), where this complex localizes to the nucleus and binds promoters containing ifn-stimulated response elements to drive expression of isgs, which mediate direct antiviral effector functions and modulate the host immune response (kawai and akira, 2006; nakhaei et al., 2009; deweerd, 2012) . importantly, though there are instances of viruses avoiding or antagonizing specific antiviral effector molecules (daffis et al., 2010) , the majority of known viral interferon evasion strategies are directed at either avoiding recognition by host pattern recognition molecules or targeting the signaling pathways associated with either type i interferon induction or interferon receptor signaling. therefore, we will briefly discuss a few examples of interferon antagonism to illustrate the importance of these interactions in viral pathogenesis. a number of viruses antagonize type i interferon induction either by targeting specific components of the interferon induction pathways or by broadly inhibiting de novo rna synthesis/protein translation in the infected cell, which effectively blocks the production of type i interferon. one strategy for avoiding type i interferon induction is to shield viral pamps (e.g., viral rna) from recognition by host rna sensors, such as rig-i and mda5. a number of viruses encode rna binding proteins that have been shown to inhibit type i ifn induction. for example, the ns1 protein of influenza a virus, which interferes with type i ifn induction at several stages in the induction process, exhibits potent antagonist activity against type i interferon induction, and this antagonism is in part due to rna binding activity by ns1 (donelan et al., 2003) . another example of this type of strategy is provided by the nucleocapsid protein (n) of the sars coronavirus, which also inhibits type i interferon induction at an early stage in the rna recognition process, and this inhibitory activity is dependent upon the rna binding activity of the n protein (lu et al., 2011) . in addition to blocking host recognition, a wide array of viruses encode proteins that block specific components of the type i interferon signaling cascade. for example, the influenza a virus ns1 protein, in addition to shielding the viral rna from recognition, prevents rig-i activation and interferon induction in response to influenza infection by blocking the function of trim25, a ubiquitin ligase that regulates the activation of the rna sensor, rig-i (gack et al., 2009) , while a second viral protein, pb1-f2 interferes with type i interferon induction through interactions with mavs, an adaptor molecule that is essential for rig-i-mediated type i interferon induction (conenello et al., 2011; varga et al., 2011) . the fact that influenza virus employs multiple strategies to block type i interferon induction argues that viral interactions with the type i interferon system are particularly important in regulating viral replication, and this is further supported by the fact that viruses with mutations in either ns1 or pb1-f2 induce higher levels of type i interferon and are subsequently attenuated in their ability to cause disease (conenello et al., 2011; donelan et al., 2003) . therefore, viruses that are defective in their ability to antagonize the host type i interferon system are often unable to replicate and spread efficiently within the host, illustrating the importance of viral immune evasion strategies in determining whether a virus will be pathogenic ( figure 2) . in addition to blocking type i interferon induction, a number of viruses also interfere with type i interferon receptor signaling, since this effectively blocks the induction of antiviral isgs. for example, the type i interferon signaling pathway is targeted at multiple stages by members of the flavivirus family, with multiple proteins from the same virus often targeting different steps of the pathway (reviewed in diamond, 2009) , again illustrating the importance of interferon antagonism for viral success. as is the case with antagonism of type i interferon induction, viral interactions with the type i interferon signaling pathways are likely to have a major impact on virus-induced disease. studies with reovirus have shown the induction of virus-induced myocarditis is associated with suppression of type i interferon receptor signaling, where the viral m1 gene from a myocarditic virus exhibits enhanced ability to interfere with the irf9 component of the isgf3 signaling complex (zurney et al., 2009) . these results suggest that viral antagonism on interferon receptor signaling can have a major impact on the pathogenesis of virus-induced disease, and that differences in the efficiency of interferon antagonism between related viruses can have significant impacts on what types of disease those viruses cause. as noted above, viruses are obligate intracellular pathogens that cannot reproduce themselves outside of host cells. therefore, the types of cells that a virus targets and what effect the virus infection has on a target cell plays a major role in determining whether a virus will induce disease and if so, what type of disease the virus causes. most simply, some viruses are cytopathic, in that the virus infection results in direct killing of the host cell, while other viruses are noncytopathic and do not directly kill the infected cell. however, while some viruses cause disease due to their targeting and killing of essential cell types, such as neurons, the mechanisms leading to virusinduced disease are often complex and we will discuss some examples of different interactions between viruses and host cells or the host immune response that affects disease outcomes (figure 3 ). immune killing of host cell transformation tumor development in the presence of a robust type i interferon response, and the absence of effective viral interferon antagonism, type i interferon will induce an antiviral state that limits viral replication and spread, thereby limiting virusinduced disease. (b) if the virus effectively interferes with the type i interferon response, interferon will be prevented from inducing a robust antiviral state within the host, and the virus is able to replicate to higher levels, will spread more efficiently, and may cause more severe disease. a number of viruses are directly cytotoxic and productive viral replication leads to direct cell killing. therefore, if the cell type that these viruses replicate in is essential, such as neurons, virus-induced killing of these cells can directly lead to disease. a number of viruses, including alphaviruses such as sindbis virus and venezuelan equine encephalitis virus (vee), cause direct killing of host cells. while the mechanisms leading to cell death are not completely worked out, it is generally accepted that these viruses induce apoptotic cell death in infected cells griffin et al., 1994) . since these viruses exhibit strong tropism for neurons, viral replication within the central nervous system leads to neuron death and degradation of neurologic function. though there is evidence that the host immune response exacerbates virus induced disease during both sindbis virus and vee infection (rowell and griffin, 2002; kimura and griffin, 2003; charles et al., 2001) , in the case of vee, mice lacking a functional adaptive immune system still succumb to virus-induced disease (charles et al., 2001) , suggesting the direct cell killing by the virus contributes to disease pathogenesis. the host immune systems plays a crucial role by protecting from virus-induced disease, however there are clear instances where an overactive or inappropriate host immune response contributes to the pathogenesis of virus-induced disease. this is perhaps most clearly illustrated in situations where a virus is noncytopathic and does not cause the direct death of infected host cells, yet viral infection still results in tissue destruction and disease. hepatitis b virus (hbv), which causes serious acute and chronic hepatitis in infected humans, nicely represents this situation and also illustrated both the importance of the host immune system in promoting viral clearance and the potential for ineffective or overly active immune responses to potentiate virus-induced disease. in most immunocompetent individuals, hbv infection causes acute hepatitis, where the host immune response plays an essential role in viral clearance, but in the process, also causes some liver injury (chisari et al., 2010) . hbv is noncytopathic for hepatocytes and during the early stages of hbv infection there are no signs of liver disease, even though the virus is replicating to high levels within the liver (chisari et al., 2010) . it is only after the host adaptive immune response has started to clear the virus from the liver that signs of liver injury are evidence, which suggests that though beneficial in clearing the virus, aspects of this immune response are pathologic. through the use of transgenic mouse models and hbv infection of chimpanzees, it has been shown the virus specific cd8 t cells are responsible for both the clearance of the virus from the liver and are the mediators of the acute liver injury associated with hbv infection (thimme et al., 2003) . immune interactions also play a major role in the pathogenesis of chronic hbv infection, where chronic disease is associated with a weak hbv specific cd8 t cell response that is unable to clear the infection (chisari et al., 2010) . furthermore, evidence from hbv transgenic mouse models suggests that suboptimal cd8 t cells responses that fail to clear hbv infection may be associated with chronic liver inflammation and damage that ultimately leads to the development of hepatocellular carcinoma (chisari et al., 2010) . immune pathology is not restricted to infection by noncytopathic viruses, and overactive or inappropriate immune responses are thought to contribute to disease even when the virus itself is capable of causing direct cell killing. as noted above, even though sindbis virus and vee are thought to directly contribute to virus-induced neurologic disease by killing neurons, it is also clear that components of the host adaptive immune response can exacerbate the disease process with these viruses (charles et al., 2001; rowell and griffin, 2002; kimura and griffin, 2003) . this concept is also illustrated by ross river virus (rrv), another alphavirus that is associated with severe arthralgia and myalgia in infected humans. studies in humans and mouse models have shown that rrv replicates to high levels in joint and muscle tissues and that viral replication within these tissues leads to the development of arthritis and myositis hazelton et al., 1985; morrison et al., 2006) . further analysis found that even though rrv is cytopathic, depletion of macrophages significantly reduced the severity of virus-induced disease, suggesting that components of the inflammatory response, rather than direct virus-induced killing, are responsible for virus-induced disease (lidbury et al., 2000) . furthermore, activation of specific components of the host complement cascade, including mannose binding lectin and the c3 component of complement, are associated with severe rrv-induced disease in humans and mice lacking either of these factors are highly resistant to virus-induced disease (morrison et al., 2007; gunn et al., 2012) . it is also important to remember that while the above examples focus on instances where components of the immune response directly contribute to cell killing in virally infected tissues, immune-mediated pathology may not always be restricted to direct effects on virally infected cells or within virally infected tissues. a number of chronic viral infections, including hepatitis c virus (hcv) infection, are associated with the development of immune complexes, where aggregates of virus and antibodies precipitate within small blood vessels and lead to the development of inflammation (vasculitis) (reviewed in lauletta et al., 2012) . therefore, while generally beneficial, there are instances where overactive or inappropriately activated components of the antiviral host immune response directly contribute to the pathogenesis of virusinduced disease. since there is significant person to person variation in immune function, variation in the magnitude and composition of the host immune response may play a major role in determining whether an individual mounts an immunopathologic immune response and thereby develops disease. not all pathologic interactions between viruses and their target cells/tissues involve direct cell killing and tissue damage, and virus-induced cancer is a serious consequence of some viral infections. while cancers associated with some viral infections are due to indirect effects such as immune suppression associated with hiv infection or chronic inflammation associated with hbv or hcv infection (see above), there are also examples of situations where viral infection directly promotes tumor development. several gamma herpes viruses, including the human gamma herpes viruses, epstein barr virus (ebv) and kaposi sarcoma-associated herpesvirus (kshv), are associated with lymphoproliferative disorders and cancer in humans. both of these viruses encode a number of proteins, such as ebv ebna2 and lmp1 or kshv lana-1 and v-cyclin that are associated with cellular transformation and cancer (reviewed in cesarman, 2011) . likewise, human papilloma viruses (hpv), which can cause several types of cancer, including cervical cancer, encode several proteins, including viral e6 and e7 proteins, that interfere with cell cycle progression checkpoints and promote cellular transformation (korzeniewski et al., 2011) . though infection with any of these viruses has the potential to cause cancer, as is the case with most aspects of viral pathogenesis, there are multiple additional factors that determine whether an individual is at risk for development of disease. in the case of ebv, since the viral proteins are recognized by the host immune response, individuals with healthy immune systems are at much lower risk of developing ebv associated cancers (reviewed in cesarman, 2011) . likewise, in the case of hpv infection, there are multiple hpv genotypes and the risk of developing hpv associated cervical carcinoma is much greater with certain high risk hpvs, such as hpv 16 or 18 (korzeniewski et al., 2011) . therefore, like other aspects of viral pathogenesis, a complex series of virus-host interactions determines whether infection with cancer associated viruses ultimately results in disease development. viral pathogenesis represents a complex series of interactions between viruses and the host that determine whether the virus will successfully establish infection within the host and if so, whether this infection will result in virus-induced disease. as discussed above, though the pathogenesis of each virus is unique, there are several stages in the viral life cycle that are shared by all pathogenic viruses which illustrate common themes in viral pathogenesis. however, it is important to remember, that within each of these common stages, there is tremendous variation in how each virus interacts with the host, and that these complex interactions are ultimately what determine whether a viral infection results in disease. isolation of ross river virus from epidemic polyarthritis patients in australia cc ckr5: a rantes, mip-1alpha, mip-1beta receptor as a fusion cofactor for macrophage-tropic hiv-1 synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice sars-cov and emergent coronaviruses: viral determinants of interspecies transmission genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein nss causes of and prevention strategies for hepatocellular carcinoma gammaherpesvirus and lymphoproliferative disorders in immunocompromised patients immunopathogenesis and immune modulation of venezuelan equine encephalitis virus-induced disease in the mouse pathogenesis of hepatitis b virus infection a single n66s mutation in the pb1-f2 protein of influenza a virus increases virulence by inhibiting the early interferon response in vivo 2'-o methylation of the viral mrna cap evades host restriction by ifit family members the interferons and their receptors -distribution and regulation type i interferon receptors: biochemistry and biological functions genetic restriction of hiv-1 infection and progression to aids by a deletion allele of the ckr5 structural gene. hemophilia growth and development study mechanisms of evasion of the type i interferon antiviral response by flaviviruses a recombinant influenza a virus expressing an rna-binding-defective ns1 protein induces high levels of beta interferon and is attenuated in mice hiv-1 entry cofactor: functional cdna cloning of a seven-transmembrane, g protein-coupled receptor influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i influenza a virus lacking the ns1 gene replicates in interferon-deficient systems recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission the effects of alphavirus infection on neurons mannose binding lectin is required for alphavirus-induced arthritis/myositis the inflammatory response in the synovium of a patient with ross river arbovirus infection the role of a mutant ccr5 allele in hiv-1 transmission and disease progression modulation of hepatitis c virus rna abundance by a liver-specific microrna tlr signaling extensive immune-mediated hippocampal damage in mice surviving infection with neuroadapted sindbis virus genomic instability and cancer: lessons learned from human papillomaviruses therapeutic silencing of microrna-122 in primates with chronic hepatitis c virus infection severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats hepatitis c virus infection and mixed cryoglobulinemia interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus macrophage-induced muscle pathology results in morbidity and mortality for ross river virus-infected mice homozygous defect in hiv-1 coreceptor accounts for resistance of some multiply-exposed individuals to hiv-1 infection sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism trim5 and the regulation of hiv-1 infectivity tripartite-motif proteins and innate immune regulation complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease characterization of ross river virus tropism and virus-induced inflammation in a mouse model of viral arthritis and myositis rig-i-like receptors: sensing and responding to rna virus infection natural resistanceassociated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts picornavirus-receptor interactions contribution of t cells to mortality in neurovirulent sindbis virus encephalomyelitis alpha/ beta interferon protects adult mice from fatal sindbis virus infection and is an important determinant of cell and tissue tropism type i ifn controls chikungunya virus via its action on nonhematopoietic cells interferon-stimulated genes and their antiviral effector functions herpes simplex virus: receptors and ligands for cell entry the cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys species-specific variation in the b30.2(spry) domain of trim5alpha determines the potency of human immunodeficiency virus restriction cd4: its structure, role in immune function and aids pathogenesis, and potential as a pharmacological target microrna silencing and the development of novel therapies for liver disease influenza a and b viruses expressing altered ns1 proteins: a vaccine approach cd8(þ) t cells mediate viral clearance and disease pathogenesis during acute hepatitis b virus infection neurovirulent strains of alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the e2 glycoprotein the influenza virus protein pb1-f2 inhibits the induction of type i interferon at the level of the mavs adaptor protein role of alpha/beta interferon in venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the 5' untranslated region reovirus mu2 protein inhibits interferon signaling through a novel mechanism involving nuclear accumulation of interferon regulatory factor 9 key: cord-351004-h6fde7vm authors: gudipati, smitha; brar, indira; murray, shannon; mckinnon, john e.; yared, nicholas; markowitz, norman title: descriptive analysis of patients living with hiv affected by covid-19 date: 2020-07-13 journal: j acquir immune defic syndr doi: 10.1097/qai.0000000000002450 sha: doc_id: 351004 cord_uid: h6fde7vm covid-19 disease has spread globally and was declared a pandemic on march 11, 2020, by the world health organization. on march 10, the state of michigan confirmed its first 2 cases of covid-19, and the number of confirmed cases has reached 47,182 as of may 11, 2020, with 4555 deaths. setting: currently, little is known if patients living with hiv (plwh) are at a higher risk of severe covid-19 or if their antiretrovirals are protective. this study presents epidemiologic and clinical features of covid-19 infected plwh in detroit, michigan. methods: this is a case series that included 14 plwh with laboratory-confirmed covid-19 infection who were evaluated at henry ford hospital in detroit, michigan, between march 20, 2020, and april 30, 2020. results: fourteen plwh were diagnosed with covid-19. twelve patients were men and 2 were women; 13 patients were virally suppressed. eight patients were hospitalized, and 6 patients were told to self-quarantine at home after their diagnoses. three patients who were admitted expired during their hospital stay. no patient required bilevel positive airway pressure or nebulizer use in the emergency department, and none developed acute respiratory distress syndrome, pulmonary embolism, deep venous thrombosis, or a cytokine storm while on therapy for covid-19. conclusion: although the clinical spectrum of covid-19 among plwh cannot be fully ascertained by this report, it adds to the data that suggest that hiv-positive patients with sars-cov-2 infection are not at a greater risk of severe disease or death as compared to hiv-negative patients. severe acute respiratory syndrome coronavirus-2 (sars-cov-2) is a novel coronavirus first detected in december 2019 in wuhan, hubei province, china. 1 many of the initial cases had a common exposure to the huanan wholesale seafood market that also traded live animals. 2 sars-cov-2 was then identified on january 7, 2020, by the chinese center for disease control and prevention, which then disclosed the genomic sequence on january 11, 2020. 3 the world health organization named the infection caused by sars-cov-2, covid-19. since the initial detection of covid-19, the disease has spread globally and was declared a pandemic on march 11, 2020, by the world health organization. 4 in the united states, detroit, michigan, had become a "hotspot" of covid-19 infected patients with the number of confirmed cases reaching 47,182 as of may 8, 2020, with 4555 deaths in michigan (fourth most deaths in the united states). 5 currently, little is known if patients living with hiv (plwh) are at a higher risk of severe covid-19 or if antiretroviral medications used to treat hiv are protective against severe covid-19. tenofovir has been shown in vitro to tightly bind to the sars-cov-2 rna-dependent rna polymerase. 6 alternatively, lopinavir-ritonavir has already been shown to have no benefit beyond standard care in a large randomized control trial. 7, 8 in addition, little is known if and how frequently plwh mount the intense cytokine response leading to cytokine storm and severe covid-19. we describe our single-center experience in detroit, michigan, of covid-19 in patients infected with hiv-1. we reviewed patients' demographics, clinical characteristics of both their hiv and covid-19 coinfections, the antiviral and antiretroviral treatments they received, and their clinical outcomes. this is a case series that included 14 plwh who were evaluated in the henry ford hospital (hfh) emergency department for laboratory-confirmed covid-19 infection, between march 20, 2020, and april 30, 2020. a confirmed case of covid-19 was defined by a positive result on a reverse transcriptase polymerase chain reaction assay of a nasopharyngeal swab sample. patients were identified, and clinical data were collected from electronic medical records. all laboratory tests and radiological assessments were performed at the discretion of the treating physician. categorical variables were analyzed using the x 2 test. approval from the henry ford health system institutional review board was obtained. from march 20, 2020, to april 30, 2020, 7372 people tested positive for covid-19 at hfh. of this group, 14 were plwh. twelve patients were men and 2 were women. twelve were african american and 2 were hispanic. six patients were discharged from the emergency department to self-quarantine. eight patients were admitted to the general practice unit, one of whom required supplemental oxygenation. two of the 8 patients were admitted to the intensive care unit and required invasive mechanical ventilation. the average duration of symptoms before admission was 12 days. the most common presenting complaints were fever (n = 7; 50%), shortness of breath (n = 7; 50%), cough (n = 10; 70%), diarrhea (n = 4; 29%), and loss of taste and smell (n = 4; 29%). one patient endorsed previous travel to texas and 2 patients endorsed exposure as health care workers. baseline characteristics of the patients are shown in table 1 . the most common comorbidities included obesity (n = 8; 57%), hypertension (n = 8; 57%), diabetes (n = 6; 43%), chronic kidney disease (n = 5; 36%), and end-stage renal disease requiring hemodialysis (n = 2; 14%). past or present smoking and alcohol use was noted in the medical histories of 7 patients. one patient was previously on an angiotensinconverting enzyme inhibitor, 2 patients were on an angiotensin ii receptor blocker, and 4 patients were on inhaled steroids for either chronic obstructive pulmonary disease or asthma. thirteen patients had suppressed hiv viral loads ( table 1 ) on combination antiretroviral therapy (cart) regimens. one patient was not on cart at presentation. all patients but one on cart had a regimen with a tenofovir component, and only 1 patient was on a protease inhibitor-based regimen with cobicistat-boosted darunavir. two patients had a history of an aids defining illness in the past. in the emergency department, antibiotics for community-acquired pneumonia were initiated for 4 of the 14 patients, intravenous fluids were given to 3 patients, and systemic corticosteroids were given to 3 patients. no patient required bilevel positive airway pressure or nebulizer use in the emergency department, and none developed acute respiratory distress syndrome, pulmonary embolism, deep venous thrombosis, or a cytokine storm while on therapy for covid-19. two patients were transferred to the intensive care unit during their admission and both expired; one from respiratory failure secondary to multifocal pneumonia, and the second one from cardiac arrest and had an extensive history of congestive heart failure. the third patient died while on the general practice unit from cardiac arrest. five patients were discharged home. all admitted patients received hydroxychloroquine 400 mg per os twice a day for 2 doses, followed by 200 mg per os twice a day for 4 days. the administration of hydroxychloroquine was consistent with our institution's treatment guidelines during that period. at 30 days after their covid-19 diagnoses, 11 of the 14 plwh were still alive. because of cardiac-associated mortality concern in the 3 patients who expired, a further medical record review was undertaken with a focus on covid-19 and hydroxychloroquine-associated causes. patient 13 died from worsening heart failure that was severe before a covid-19 diagnosis. the patient expired in the intensive care unit from pulseless electrical activity arrest with no documentation of torsades de pointes noted on cardiac monitoring. patient 9 expired on the general practice unit from a cardiac arrest. he was found pulseless and unresponsive by a nurse. this patient had multiple comorbidities, including metastatic cancer, heart although the clinical spectrum of covid-19 among plwh cannot be ascertained by this report, the course of infection among those described in this report is similar to what has been described in the literature. 1 a case series from germany presented 33 virally suppressed plwh infected with covid-19 with 76% having mild infection, 27% patients having severe infection, and 3% patients expiring. 9 these findings were also consistent with our results. an observational prospective study from madrid, spain, analyzed 51 plwh diagnosed with covid-19, of whom 69% required hospitalization, 63% had one comorbidity, and 12% were critically ill. the authors of this study noted that plwh should not be considered to be protected from covid-19 or to have a lower risk of severe disease. therefore, they should receive the same treatment approach applied to the general public. 10 plwh at hfh had similar comorbidities and hospital admissions compared with non-hiv patients admitted with covid-19. in a recent publication of 463 patients infected with covid-19, but without known hiv, the investigators from hfh found that the 3 most common comorbidities were hypertension (63.7%), obesity (57.6%), and diabetes (38.4%). patients in the hfh study also had a 16% overall 30-day mortality rate. 11 these comorbidities were also the 3 most common in our population (57%, 57%, and 43%, respectively) with a 21% overall 30day mortality rate. our case series, the current published literature on hiv and sars-cov-2, and the published data from hfh on covid-19 patients without known hiv supports the theory that there is not an excess morbidity and mortality among plwh affected by covid-19 compared with the general public. there are many hypotheses as to why plwh might have favorable outcomes to covid-19. one hypothesis is that most of these patients are on tenofovir-based regimens. tenofovir has been described as having the capacity to bind to sars-cov-2 rna-dependent rna polymerase. 6 this mechanism of action is similar to the antiviral drug remdesivir, which has been approved for treatment for covid-19 through emergency use by the united states food and drug administration. however, the affinity for the binding substrate was lowest in tenofovir, compared with galidesivir, remdesivir, and ribavirin. 6 tenofovir-emtricitabine was also recently shown to reduce virus titers in a highly susceptible ferret infection model. 12 in a spanish study, plwh receiving emtricitabine and tenofovir disoproxil fumarate were found to have a lower risk for covid-19 and related hospitalization than those receiving other nucleoside reverse transcriptase inhibitors. 13 whether the tenofovir-based regimen resulted in a reduction in the severity of response to covid-19 will need to be studied further, but this regimen did not protect our patients from acquiring the infection. early, cart can blunt the cytokine response in acute hiv infection. 14 in addition, tenofovir has been shown to have antiinflammatory effects in cells and tissue from hiv-uninfected donors. 15, 16 the impact on covid-19 of art, early therapy for sars-cov-2, or pre-existing immunodeficiency is unknown. however, none of these patients developed a clinical syndrome compatible with a cytokine storm. although it is of interest that cd4 and cd8 counts fall with sars-cov-2 infection, it is unknown whether this change exacerbates or dampens the cytokine response associated with covid-19 or leads to an increased risk of secondary infections. the absence of reports of covid-19 among immunosuppressed hosts is striking. the susceptibility, disease course, and outcome among plwh have not been described. given the role of the "cytokine storm" in the outcome of covid-19 infection, some immunodeficient states may be protective in terms of reducing the severity of infection. however, guan et al 17 in a review of 1590 patients found that any malignancy was associated with poor clinical outcomes regardless of an individual's hiv status. the covid-19 outbreak in detroit is at a plateau as of may 10, 2020, but the total number of plwh who have been diagnosed with covid-19 is unknown as universal testing is not consistently performed. 18 the michigan department of health and human services reported that as of june 30, 2020, 278 of the 17,093 (1.6%) known plwh in michigan tested positive for covid-19 with 8% expiring, 21% requiring hospitalization, and 5% requiring ventilation (table 2 ) (j. b. kent, personal communication, 2020). this is similar to the 1.7% prevalence of confirmed covid-19 in detroit. 5 of the approximately 1500 plwh followed in our hiv clinic in detroit, mi, 14 hiv-positive individuals were diagnosed with covid-19 in our laboratories. others have self-reported to have been diagnosed elsewhere, but because of a lack of our study had some limitations. we had a small sample size and it is unclear if our findings can be applied to a larger number of patients. all patients also received hydroxychloroquine that was consistent with our institution's guidelines during that period before the emergency use authorization being rescinded. however, data remain limited on coinfection with hiv and covid-19, and it is important to share experiences between health care professionals to enhance our knowledge about covid-19. in conclusion, although in our series all patients coinfected with hiv and sars-cov-2 were either african american or hispanic, they experienced a clinical course that was similar to that reported in the literature for individuals not infected with hiv. in this small group of individuals, we did not observe an unexpected increase in disease severity or mortality. it is possible that plwh are more likely to take precautions to prevent exposure, more likely to seek medical care, or more likely to be tested for covid-19, which may skew data to earlier and less symptomatic infection. nevertheless, we have seen no signals yet to suggest that care for patients coinfected with hiv and sars-cov-2 should be different from care for patients with covid-19 who are not infected with hiv. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study world healht organziation genomic epidemiology of hcov-19. gisaid database world health organization. who director-general's opening remarks at the media briefing on covid-19. world health organization ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 covid-19 in patients with hiv: clinical case series covid-19 in people living with human immunodeficiency virus: a case series of 33 patients description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort clinical characteristics and morbidity associated with coronavirus disease 2019 in a series of patients in metropolitan detroit antiviral efficacies of fda-approved drugs against sars-cov-2 infection in ferrets incidence and severity of covid-19 in hiv-positive persons receiving antiretroviral therapy: a cohort study association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute hiv infection tenofovir selectively regulates production of inflammatory cytokines and shifts the il-12/il-10 balance in human primary cells mucosal effects of tenofovir 1% gel comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis covid-19 in patients with hiv. the lancet hiv the authors thank our patients with hiv and our health care colleagues for providing care in our hospital system. key: cord-318591-ssnlfjap authors: pecego, ac; amâncio, rt; costa, dm; bozza, fa; siqueira, mm; oliveira, ml; cerbino-neto, j; japiassu, a title: etiology, clinical, and epidemiological characteristics of severe respiratory infection in people living with hiv date: 2020-01-22 journal: int j std aids doi: 10.1177/0956462419882587 sha: doc_id: 318591 cord_uid: ssnlfjap people living with hiv (plwh) are more prone to severe respiratory infections. we used the severe acute respiratory infection (sari) definition to describe the etiology, clinical, and epidemiological characteristics in this population. this was a prospective observational study including plwh hospitalized with fever and cough. those with symptom onset up to 10 days were classified as severe acute respiratory infection and 11–30 days as non-severe acute respiratory infection. blood, urine samples and nasopharyngeal swabs were collected. data were extracted from patient charts during their hospital stay. forty-nine patients were included, median cd4 cell count: 80 cells/mm(3), median time since hiv diagnosis and hospital admission: 84 months and 80% were antiretroviral therapy exposed. twenty-seven patients were classified as sari. etiology was identified in 69%, 47% were polymicrobial. respiratory virus (9 sari vs. 13 non-sari), bacteria (5 sari vs. 4 non-sari), mycobacterium tuberculosis (6 sari group vs. 7 non-sari group), pneumocystis jirovecii (4 sari vs. 1 non-sari), cryptococcus neoformans (1 sari vs. 3 non-sari), and influenza a (1 sari vs. 2 non-sari). dyspnea was statistically more prevalent in sari (78% vs. 36%, p = 0.011) but the risk of death was higher in the non-sari (4% vs. 36%, p = 0.0067). in the severely immunocompromised plwh, severe acute respiratory infection can be caused by multiple pathogens and codetection is a common feature. people living with hiv (plwh) are highly susceptible to respiratory infections. 1 even in the antiretroviral therapy (art) era, tuberculosis (tb) has fallen disproportionately among seropositive patients 2 and pneumonia remains five times more common among this population, despite achieving cd4 cell counts above 500 cells/mm 3 . 3 plwh are also at increased risk for poor influenza outcomes, which may be particularly risky for those living in countries with limited resources. [4] [5] [6] after the recent influenza a (h1n1, h5n1, and h7n9) and the middle east respiratory virus (mers-cov) outbreaks, the who is encouraging and supporting countries to strengthen surveillance on severe acute respiratory infections (sari) 7 but with limited information on plwh regarding etiology and prognosis, despite their increased risk for respiratory infections and adverse outcomes. [8] [9] [10] so, in this study, we described how sari is represented, according to clinical presentation, epidemiology and etiology in a population of plwh with respiratory infection residing in a high-prevalence tb area. a prospective observational study was conducted at the instituto nacional de infectologia evandro chagas (ini-fiocruz), rio de janeiro, brazil, from may 2012 to december 2013. ini-fiocruz is a national reference center for infectious diseases and has been a reference center for care, research, and training related to hiv/aids since 1986. the aids program at ini is one of the largest providers of primary, specialty, and tertiary care for plwh patients in rio de janeiro state with a cohort of about 4000 patients in active followup. we conducted surveillance of every hiv-positive patient hospitalized during the study period above. every monday, wednesday, and friday we carried out reviews of inpatient medical records and all plwh hospitalized with respiratory symptoms were screened by the principal investigator, a physician specialized in infectious diseases. in the presence of fever and/or cough and onset of symptoms for less than 30 days, the patient was invited to participate in the study, which involved the collection of clinical material as detailed below in the sample collection and processing section. patients were excluded if symptoms occurred more than 30 days ago or if no fever and/or no cough were reported by the patient. patients were classified as sari if the onset of symptoms was 10 days, according to who global standards for sari definition. 7 those with symptom onset greater than 10 days but up to 30 days prior were considered as non-sari patients ( figure 1 ). participants were screened by a physician regarding respiratory viral symptoms such as myalgia, diarrhea, arthralgia, coryza, and odynophagia. data on dyspnea, respiratory frequency, and peripheral oxygen saturation (spo 2 ) using pulse oximetry were collected by a respiratory therapist. patients were considered to have dyspnea if rated 5 on an ascending scale from 0 to 10. clinical and epidemiological data were collected during hospitalization using standardized forms, until hospital discharge or death ( table 1 ). variables of interest were age, gender, time since hiv diagnosis based on the first hiv-positive result, cd4 nadir cell count, and cd4 cell count at hospital presentation or up to 2 months before, having had a previous tb diagnosis (pulmonary or disseminated) or any other previous opportunistic infection. viral load was considered to be undetectable if less than 400 copies/ml, and we considered being art exposed if patient ever used art regardless of adherence. prophylaxis of respiratory infection was recorded, such as pneumocystis jirovecii pneumonia (pjp) prophylaxis and pneumococcal and influenza vaccines. data about the respiratory infection included leucocyte count, c-reactive protein, pulse oximetry, and crb-65 and the combined crb-65 þ o 2 score at admission. outcome variables of interest were: intensive care unit (icu) admission, noninvasive ventilation use beginning within 72 h of hospital admission and death. microbiologically-confirmed pneumonia was defined as fever (axillary temperature) 38 c, (new) pulmonary infiltrate on chest x-ray or computed tomography scan plus an etiological agent identified through the tests mentioned below. for every patient included we collected blood cultures for bacteria, fungi, and mycobacteria processed with the automated bactalert v r system (healthcare-bio merieux), urine samples for urinary streptococcus pneumoniae and legionella pneumophila serogroup 1 testing (binaxnow kit v r ) and triple respiratory swab (nasopharyngeal, one for each nostril and one for posterior pharynges). respiratory swabs were tested for respiratory syncytial virus (rsv), adenovirus (adeno), influenza a (flua), parainfluenza 1-3 (piv 1-3), human-metapneumovirus (hmpv), and rhinovirus (rv) using rt-pcr. probes were labeled at the 5 0 terminus with 6 carboxyfluorescein (fam) and with quencher blackhole-1 dt (biosearch technologies, inc., novato, ca). the tests were done following centers for disease control and prevention (cdc) guidance. sputum or induced sputum and bronchoalveolar fluid were tested for fungus and mycobacterium spp. under direct visualization followed by inoculation into sabouraud culture medium for fungi and bactec v r mgit tm 960 and lowenstein-jensen for mycobacterial culture. mycobacterium spp. identification was performed under conventional techniques (kent and kubica, 1985, cdc atlanta). all clinical samples were collected up to 72 h after hospital admission except for p. jirovecii detection which was done through staining and indirect immunofluorescence assay (ifl) on bronchoalveolar samples, which were performed only in mechanically-ventilated patients. table 1 . clinical and epidemiological characteristics of patients with less than 10 days since symptom onset (sari) vs patients with more than 10 days since symptom onset (non-sari). total we conducted a descriptive analysis of the convenience sample from the studied population. categorical variables were presented as proportion and continuous variables summarized as median and interquartile ranges for sari and non-sari patients. we compared the distribution of continuous variables by using u or mann-whitney test, and categorical variables by using the chi-squared or fisher's exact test when appropriate at univariate analysis. the analyses were processed using statistical package for social science (spss) and graphpad prism 3.0 for windows (graphpad software, san diego, ca, usa). statistical significance was considered whenever p value was less than 0.05. a total of 149 patients were screened and 74 patients met our inclusion criteria. among those, 8 patients refused to participate because of discomfort and pain during the nasal swab collection and 15 patients could not be included due to temporary lack of supplies. a total of 49 patients with fever and cough and symptoms lasting up to 30 days were included (figure 1 ), of which 27 (55%) met the sari case definition. a profound and prolonged immunosuppression is reflected by the median nadir lymphocyte cd4 cell count (58 cells/mm 3 ) and at hospital admission (80 cells/mm 3 ) and time since hiv diagnosis with a median of 84 months, except for five patients with recent hiv diagnosis (in the previous three months). this finding is supported by the fact that 38 (80%) patients were art exposed, but only 12 (25%) had an undetectable viral load. prevention of respiratory infection was observed in only 7 (14%) patients taking prophylactic sulfamethozaxole-trimethoprim for pjp, 7 patients (14%) who previously received s. pneumoniae vaccine and 9 (18%) received influenza seasonal vaccine. about half of the patients had a previous history of tb and 18 (37%) had a history of other opportunistic infections. pneumonia was confirmed in 46 (94%) patients and 31 (63%) had x-rays with multilobar infiltrates. in the remaining three patients, the chest x-ray was normal and ct scan could not be done (data not shown). pulse oximetry showed that 15 patients (30%) had spo 2 less than 90% and non-invasive ventilation was initiated in 67% of the patients in the first 72 h of admission. twenty (41%) patients were classified as crb-65 ¼ 0 points, 19 patients classified as crb-65 ¼ 1 (39%) point, 9 (18%) as crb-65 ¼ 2 points and 1 as crb-65 ¼ 3 points. although 39 patients had crb-65 0-1, the majority required non-invasive ventilation (niv) in the first 72 h of hospitalization and 25 had chest x-rays with multilobar infiltrates. the mortality risk for those 10 patients (20.4%) with crb-65 2-3 points was 4.5 higher (95% confidence interval [ci]: 0.94-21.92) compared to crb-65 0-1 points, p value ¼ 0.069, with sensitivity (s) and specificity (sp) of 85% and 44% and positive predictive value (ppv) and negative predictive value (npv) of 87% and 40%, respectively. when combined with pulse oximetry, 15 patients (30.6%) had crb65þo 2 of 2-4 points and a 10.5 higher risk of dying (95% ci: 1.87-59.1), p value ¼ 0.0051 with a s:75% and sp:78% and ppv: 94% and npv: 42% (data not shown). in total, nine patients (18.6%) died during hospitalization. the median time between symptom onset and hospital admission was 10 days (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) . according to sari classification, we saw that clinical and epidemiological characteristics were evenly distributed with no statistical difference between sari and non-sari patients, except for mortality rate, which was significantly more prevalent among non-sari patients (table 1) . symptoms related to respiratory viral infection were analyzed among sari and non-sari patients (figure 2 ). fever and cough were present in 100% of cases because they were used as inclusion criteria. dyspnea was the only symptom that was statistically more prevalent in the sari subgroup, p ¼ 0.011 ( figure 2) . arthralgia, conjunctivitis, odynophagia, and coryza were all infrequent in both subgroups. microbiological confirmation was possible in 34 (69%) patients and 16 (47%) patients had polymicrobial infections ( table 2 ). viral pathogens were the most common agents, occurring in 17 (35%) patients with 22 isolates. rv was the most common virus among sari (4; 15%) and non-sari (7; 32%) patients, followed by rsv in (5; 10%); the majority (4/5) in the non-sari group, influenza a in 3 (6%), hmpv in 2 (4%), and parainfluenza-2 in 1 (2%) patient. all three cases of influenza a happened in non-influenzavaccinated patients, but since the vaccination was infrequent, we could not explore its impact. influenza a (pdm09)h1n1 was confirmed in one patient classified as sari, and the other two influenza viruses were identified among patients whose symptom onset was greater than 10 days ago, both seasonal ah3n2 strains. the two isolates of hmpv and the only piv-2 were identified in patients with sari. mycobacterium tuberculosis (mtb) was the most common bacterial pathogen identified in 13 (27%) patients (table 2) . among the 24 patients (49%) who had been previously diagnosed with tb, 8/24 (33%) were readmitted with mtb infection the majority in the non-sari group (7/8 patients, 88%). s. pneumoniae was evenly distributed among sari (four isolates) and non-sari (three isolates), but pjp was more frequent in the sari group, while cryptococcus neoformans was more common in the non-sari group ( table 2) . among polymicrobial infections, 13 patients had 2 pathogens and 3 patients had 3 or more pathogens identified. polymicrobial infection was more frequent in the non-sari group, 9 (41%) vs. 7 (26%), but with no statistical difference. no single agent was significantly more prevalent according to this classification. the main agents associated with severity and icu admission were pjp in 80% (4/5); cryptococcus neoformans in 75% (3/4), s. pneumoniae in 43% (3/7), and pseudomonas aeruginosa in 100% (1/1) of the cases. infections were polymicrobial in 41% vs. 30% of the patients being admitted to the icu (p ¼ 0.52) and in 44% of the patients who died vs. 25% among survivors (p ¼ 0.25). we found that plwh hospitalized with fever and cough, with less than 30 days of symptoms were mostly young, with severe and prolonged immunosuppressed caused by hiv infection. there were no clinical or epidemiological aspects that could distinguish the classification of sari and non-sari. in terms of etiology, sari was represented by a broad range of infectious agents in this population and much of the lung infections were polymicrobial. in one-third of the cases, we found viruses in nasopharyngeal samples and two out of three influenza viruses were detected in non-sari patients. our findings highlight the importance of viral pathogens in community-acquired respiratory infections: viruses were implicated in 35% of respiratory symptoms in plwh and rv was the most common one. this may be due to the fact that the rv is present throughout the year regardless of specific seasonality. 11, 12 although being historically related to the common cold and to mild respiratory illness, [13] [14] [15] recent data suggest that rv can be related to more severe cases. 16 according to cohen et al. it was the principal agent of sari during a hospital surveillance period 2009-2012 in south africa, where 74% were plwh 9 and also an important agent in elderly hospitalized with sari in chile 17 as well as the causal agent of more severe cases requiring icu treatment, in codetection with s. pneumoniae. 18 most of our cases were found in co-detection with another agent suggesting that rv may contribute to more severe cases but the possibility of only being a bystander cannot be rule out in this context. in accordance to our findings, hmpv remains an infrequent cause of sari even in plwh. 19 apart from viral detection, s. pneumoniae is reemphasized as the main bacterial pathogen found in patients with cap who presented with sari, regardless of the cd4 lymphocyte count as shown by l opez-aldeguer et al. 20 another important aspect of our study was the fact this pyogenic bacterium was found in patients with late onset of symptoms (non-sari) and three out of seven patients presented with bacteremia and all three of them died, a feature often correlated with poor prognosis. 21 l. pneumophila remains an uncommon cause of community-acquired lung infection but we acknowledge that some cases might have been missed since the urinary antigen test can only detect type 1 infections and up to 14 days of onset. 20, 22 we found a high frequency of mtb in patients with few days of symptoms and in a population with a previous history of pulmonary or disseminated tb, suggesting that prolonged immunosuppression may reactivate the bacillus. [22] [23] [24] our findings are in agreement with coelho et al. who showed that tb was the most common opportunistic infection in the total cohort followed at our center. in high tb prevalence settings, such as the one observed in our context, this diagnosis should be pursued in plwh regardless of symptom duration. 25 another important aspect of our findings was to emphasize the polymicrobial aspect of respiratory infections in this population. co-detection of multiple pathogens can occur in the context of hiv infection 20, 25, 26 but also in the general adult population as recently demonstrated by karhu et al. (39% of severe acquired pneumonia patients) and in children with sari. [26] [27] [28] in our sample, coinfection could be associated with death (44% vs. 25%, p ¼ 0.25) but did not reach statistical significance, probably due to the small sample size. whether the presence of multiple pathogens leads to a more severe presentation with increased risk of poor outcomes is still unclear. 29 when looking at coinfection involving bacteria plus a respiratory virus, mortality seems to be higher in the bacterial coinfection subgroup, 30 32, 33 but these findings may not be applied to viral-viral coinfection. 34 since sari and non-sari were similar according to clinical and epidemiological aspects, the delay between symptom onset and the demand for care (non-sari group) could have contributed to the higher mortality observed in our findings, as shown previously by several authors. 6, 35, 36 although 39 patients had crb-65 of 0-1 points and could have been sent home with oral antibiotic therapy, the majority required niv in the first 72 h of hospital admission and 63% had x-rays with multilobar infiltrates, another predictor of unfavorable prognosis. similar findings were found by almeida et al. in 49 plwh admitted with pneumonia in an emergency department with a median crb65 score lower then hiv-negative patients but which were also associated with a higher risk of mortality. 37 in the paper of yone et al., 54 out 62 plwh admitted with community acquired pneumonia (cap) had crb65 of 0-1. 38 in our study, adding the pulse oximetry to crb-65 score, as suggested by the consensur ii, the latin america pneumonia working group, increased the ability to confidently predict the 30-day in-hospital mortality. 39 in the context of sari surveillance target at influenza, a possible explanation for our late detection relies on the fact that immunosuppressed and critically ill patients there seems to be prolonged viral shedding. 40 the median cd4 cell count in this study population was 80 (24-274) cells/mm 3 and the proportion of patients with undetectable viral load was 25% despite 80% being exposed to art. compared to the whole cohort followed at our center for the period 2012-2013, where 69.5% were male and the median age was 42.5 years, the median cd4 cell count was higher (542 cells/mm 3 ), and so was the proportion who had undetectable hiv viral load (69.5%) and ever being exposed to art (90.8%). 41 this shows that the population in this study is severely immunocompromised and found it harder to adhere to art at the time. we observed two cases of influenza in the non-sari group, respectively 20 and 30 days after symptom onset, and both patients developed respiratory failure that led to death (data not shown). this highlights the fact that immunosuppressed plwh with respiratory symptoms may not seek hospital attention in time to be captured by sari surveillance. finally, regarding symptom frequency, we found a low percentage of sore throat, runny nose and conjunctivitis and a high percentage of dyspnea, diarrhea, and myalgia. in fact, dyspnea was the only symptom more prevalent in the sari group (p < 0.05). although gastrointestinal involvement may have been influenced by the advanced immunodeficiency syndrome, other authors studying the symptoms of influenza in lessimmunocompromised plwh reached similar results. 42, 43 it is important to note that our study tried to address viral symptoms in a context of multiple pathogens. on one hand, it is difficult to assign a symptom to a specific virus or bacteria but on the other hand, it may reflect what actually happens in clinical practice, because as already highlighted above, a significant percentage of respiratory infections, especially in plwh are of mixed type. our study has limitations. the small sample size conducted at a single center does not allow generalization of the results and the detection of respiratory viruses from the lower respiratory tract would have been more representative of the etiology of respiratory infection. patients with advanced hiv infection may have multiple infections at the same time and an overlap of signs and symptoms is frequent, so the need for hospitalization may not be attributed exclusively to the respiratory infection per se. all cases in this study occurred in a 19-month period at a single institution, and viral epidemiology at this site and during this timeframe may not be representative of all seasons and locations. finally, due to the constraints of the respiratory viral kit used, we may not have identified all possible and important viral infections such as adenovirus, coronavirus and bocavirus and pcr technique may detect a virus that is representative of several conditions such as the true agent itself, a bystander or facilitating bacterial superinfection. 44 in severely immunocompromised plwh, sari can be represented by a wide variety of respiratory pathogens and the codetection of multiple pathogens is common. viruses must be considered as a common cause of respiratory infection either alone or in combination with other pathogens and in scenarios where tb is endemic, this agent should be pursued regardless of cough duration. hiv-1 and bacterial pneumonia in the era of antiretroviral therapy aidsrelated tuberculosis in rio de janeiro hospitalization for pneumonia among individuals with and without hiv infection, 1995-2007: a danish population-based, nationwide cohort study mortality associated with seasonal and pandemic influenza and respiratory syncytial virus among children <5 years of age in a high hiv prevalence setting-south africa deaths associated with respiratory syncytial and influenza viruses among persons 5 years of age in hiv-prevalent area mortality amongst patients with influenza-associated severe acute respiratory illness who|who global epidemiological surveillance standards for influenza surveillance for severe acute respiratory infections in southern arizona epidemiology of severe acute respiratory illness (sari) among adults and children aged 5 years in a high hiv-prevalence setting the role of influenza, rsv and other common respiratory viruses in severe acute respiratory infections and influenza-like illness in a population with a high hiv sero-prevalence viral etiology of influenza-like illnesses in cameroon the seasonality of rhinovirus infections and its implications for clinical recognition respiratory viruses in adults with community-acquired pneumonia etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods viral etiology of community-acquired pneumonia among adolescents and adults with mild or moderate severity and its relation to age and severity communityacquired pneumonia requiring hospitalization among u.s. adults clinical relevance of rhinovirus infections among adult hospitalized patients viral infection in patients with severe pneumonia requiring intensive care unit admission human metapneumovirus-associated severe acute respiratory illness hospitalisation in hiv-infected and hiv-uninfected south african children and adults outcomes in hiv-infected patients admitted due to pandemic influenza changing global epidemiology of pulmonary manifestations of hiv/ aids pyogenic bacterial lower respiratory tract infection in human immunodeficiency virus-infected patients hiv and tuberculosis: a deadly human syndemic tuberculosis and hiv co-infection prevalent tuberculosis at hiv diagnosis in rio de janeiro, brazil: the tb/hiv in rio (thrio) cohort respiratory viruses in hiv-infected patients with suspected respiratory opportunistic infection lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia the role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country does virus-bacteria coinfection increase the clinical severity of acute respiratory infection?: virus-bacteria coinfection and respiratory infection different pattern of viral infections and clinical outcomes in patient with acute exacerbation of chronic obstructive pulmonary disease and chronic obstructive pulmonary disease with pneumonia: respiratory viral infections in copd patients does viral co-infection influence the severity of acute respiratory infection in children? influenza virus infection is associated with increased risk of death amongst patients hospitalized with confirmed pulmonary tuberculosis in south africa epidemiology, co-infections, and outcomes of viral pneumonia in adults: an observational cohort study clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis severe 2009 pandemic influenza a (h1n1) infection and increased mortality in patients with late and advanced hiv disease outcomes of influenza a(h1n1)pdm09 virus infection: results from two international cohort studies curb-65 and other markers of illness severity in community-acquired pneumonia among hiv-positive patients influence of hiv infection on the clinical presentation and outcome of adults with acute community-acquired pneumonia in yaounde, cameroon: a retrospective hospitalbased study neumon ıa aguda adquirida en la comunidad en adultos: actualizaci on de los lineamientos para el tratamiento antimicrobiano inicial basado en la evidencia local del grupo de trabajo de sudame´rica influenza susceptibility, severity, and shedding in hiv-infected adults: a review of the literature hospitalization rates, length of stay and in-hospital mortality in a cohort of hiv infected patients from rio de janeiro, brazil influenza a h1n1 in hiv-infected adults*: influenza a h1n1 in hiv-positive adults hiv-infected hospitalized patients with 2009 pandemic influenza a (ph1n1)-united states, spring and summer editorial commentary: what is the real role of respiratory viruses in severe community-acquired pneumonia? we thank rogerio valls and jorge salluh for their valuable suggestions during the study period. authors' contribution acp collected data, performed the analysis, interpreted results, and wrote the manuscript. rta contributed to data management, performed the analysis and drafting the figures and tables. fab and aj participated in the conception of this study and revised the manuscript. aj and jcn participated in the interpretation of the results and writing. ms and mlo processed the respiratory samples. dcm participated in the data collection. all authors read and approved the final manuscript. de-identified data and materials are available on a case-bycase basis. please contact the corresponding author with requests. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the study was conducted in accordance with the declaration of helsinki. written informed consent was obtained from all patients prior to any study-related procedure. the national institute of infectious diseases review board approved study protocol (ini, caae 0014.0.009.000-08). participants who refused to participate or were excluded by exclusion criteria were not harmed by not participating in the study. the authors received no financial support for the research, authorship, and/or publication of this article. https://orcid.org/0000-0002-4131-1425 key: cord-354374-rtgjjglc authors: c.g. pollok, richard; j.g. farthing, michael title: enteric viruses in hiv-related diarrhoea date: 2000-12-01 journal: mol med today doi: 10.1016/s1357-4310(00)01816-5 sha: doc_id: 354374 cord_uid: rtgjjglc hiv-related diarrhoea is an important cause of morbidity and mortality in hiv infection. cytomegalovirus is a well-established cause of diarrhoea, but the role of other enteric viruses is less clear and will be discussed here. the clinical manifestations, disease mechanisms, diagnostic techniques and current treatments for the management of these infections are reviewed. cmv infection of the gi tract, in patients with aids have diminished greatly. following the initiation of haart, aids patients with cmv can successfully discontinue anticmv treatment without reactivation of the disease and with a parallel reduction in cmv viraemia. the pathogenic role of cmv infection is well established. although retinitis is the most common manifestation of infection, before the introduction of combination antiretroviral therapy, gi infection of the oesophagus, stomach, small bowel and colon occur in 5-15% of patients during the course of hiv infection. the incidence of cmv retinitis has declined following the introduction of haart, with a concomitant increase in survival 5 . this is also presumably true of gi cmv, although published data are limited. although cmv can infect any part of the gi tract, the most common site of infection is the colon 6 , and the most common manifestation of cmv colitis is chronic or intermittent diarrhoea in association with abdominal pain. the disease is also associated with mild or severe rectal bleeding or abdominal pain in the absence of diarrhoea and, in addition, fever is common at presentation. pain can precede the development of toxic megacolon, and intestinal perforation is rare but clearly life threatening. colonic cmv infection can occur in association with infection elsewhere in the gi tract including the oesophagus, which usually results in dysphagia and odynophagia, and the pancreatico-biliary tree, which results from aids-related cholangiopathy or pancreatitis and manifests as pain in the upper abdomen. gi infection can also herald cmv retinitis and careful retinal assessment is, therefore, essential in this group. the mechanisms by which cmv induces gi disease are poorly understood. cmv infection of the mucosa is associated with a marked local inflammatory response, which might have a role in inducing mucosal ulceration. wilcox and colleagues found that mrna levels of the proinflammatory cytokine tumour necrosis factor ␣ (tnf-␣) were elevated in oesophageal mucosa of patients with cmv oesophagitis, but returned to normal following treatment of cmv (ref. 7) . similarly, sharpstone et al. noted increased levels of tnf-␣ in faecal samples from patients with gi cmv (ref. 8) . the enteric ischaemia that results from cmvassociated vasculitis might conceivably play a role in gi cmv (ref. 9) . although this hypothesis remains unexplored in cmv enteritis, a similar mechanism has been invoked to explain the aetio-pathogenesis of the opportunistic viral enteritis is a potentially important gastrointestinal (gi) manifestation of hiv-related disease. however, with the exception of cytomegalovirus (cmv) and human herpes simplex virus (hsv), which are established aetiological agents of disease in the gi tract in patients with hiv, the role of other enteric viruses remains controversial. between 44% and 82% of hiv patients with chronic diarrhoea have a pathogen that is readily identifiable using a well-established diagnostic protocol that includes stool culture, microscopy and histological examination of biopsies obtained by endoscopy of the upper and lower gi tract [1] [2] [3] [4] . for example, using such a protocol, 82% of 155 cases of persistent diarrhoea were related to infection with identifiable bacteria, parasite or cmv, with cmv alone accounting for 18% of these cases 4 . the additional techniques of stool electron microscopy and specific immunological staining implicated other enteric viruses in a further 11% of cases. the role of these other viruses in so-called 'pathogen-negative' diarrhoea remains uncertain. the clinical importance of hiv enteropathy is probably limited. several viruses, including astrovirus, picobirnavirus, small round structured virus (srsv) and rotavirus, are implicated in hiv-related diarrhoea. in addition, adenovirus is associated with persistent diarrhoea in patients with characteristic adenovirus colitis. the evidence for a pathological role for these viruses will be discussed ( table 1) . it is well established that cmv can infect the gi tract and cause diarrhoea and methods of diagnosis and current treatments are reviewed. the spectrum of disease morbidity and mortality among hiv patients has altered dramatically since the widespread introduction of highly active antiretroviral therapy (haart). opportunistic infections, including a spectrum of both upper and lower gi manifestations of cmv infection have been described and a definitive diagnosis of cmv enterocolitis requires intestinal biopsy. in a prospective study, wilcox and colleagues evaluated 55 patients with hiv by sigmoidoscopy and colonoscopy. chronic diarrhoea and abdominal pain occurred in 80% and 50% of patients respectively and 9% presented with lower gi bleeding with a previous history of diarrhoea 12 . endoscopically, appearances were heterogeneous. three main categories were identified: (1) colitis associated with ulceration (39%); (2) ulceration alone (38%); or (3) colitis alone (20%). subepithelial haemorrhage was common in all groups. a total of 31 patients underwent complete endoscopy to the caecum. of these, four (9%) had disease that was proximal to the splenic flexure without distal involvement that was therefore inaccessible by flexible sigmoidoscopy. this contrasts with early reports suggesting higher rates of right-sided colitis 13 . the same group has assessed the varied endoscopic appearances of cmv infection of the oesphagus 14 . multiple ulcers, located in the middle or lower oesophagus, were identified in 58% of patients. these were usually less than 1 cm in diameter and superficial. the heterogeneous manifestation of cmv disease in both the colon and oesophagus makes biopsy essential for accurate diagnosis. a histologically based diagnosis of cmv enterocolitis depends largely on identification of characteristic cytomegalovirus inclusion bodies in samples usually obtained from the base of cmv ulcers. in addition, specific immunoperoxidase staining for cmv is also useful in identifying gi disease 15 ; positive staining is more likely from the edge of ulcers and some authors claim greater sensitivity compared to conventional histology, although it is unclear from these reports how rigorously inclusions bodies have been excluded 16, 17 . the value of a viral culture of intestinal biopsies is limited as this is a time-consuming procedure. furthermore, cmv viraemia can occur in the absence of mucosal disease, which can lead to a false-positive diagnosis if biopsies are contaminated with blood. although using in situ hybridization to assess gi cmv disease is both sensitive and specific, it probably offers little additional benefit over conventional histology 18 . using the pcr for the detection of cmv might also be more sensitive than using standard histological techniques; however, evidence suggests that this technique lacks specificity, with 28% of normal gi biopsies positive by pcr. furthermore, as with in situ hybridization, there is the risk of pcr giving a false-positive result in patients with viraemia. the sensitivity of conventional histology, viral culture, pcr and immunohistochemistry are summarized in table 2 (ref. 19 ). cotte and colleagues monitored cmv dna levels during treatment and levels broadly reflected clinical response 20 . newer techniques are now available to monitor the natural history and progression of cmv disease in the post-haart era. salmon-ceron and colleagues have compared three blood markers of cmv, levels of pp65 antigenaemia in viral culture, plasma levels of cmv dna and levels of late cmv mrna, in the assessment of cmv-disease progression in patients on haart. in a multivariate analysis, plasma cmv dna, raised pp65 antigenaemia or a cd4 count of 75 cells l ϫ1 were identified as independent risk factors in the development of cmv disease 21 . others have compared these techniques with retinal assessment in the evaluation of cmv retinitis. the digene hybrid capture cmv dna system was 85% as sensitive as retinal assessment, which is similar to the more cumbersome pp65 antigenaemia assay (80% as sensitive) 22 . table 3 glossary cholangiopathy -pathology relating to the biliary tree. crohn's disease -chronic inflammatory condition of the gastrointestinal tract, the cause of which remains unknown. enteric virus -virus with a tissue tropism for the intestinal tract. odynophagia -pain on swallowing. oesophagitis -inflammation of the oesophagus. pancreatitis -inflammation of the pancreas. retinitis -inflammation of the retina that can lead to visual impairment or blindness. toxic megacolon -pathological dilatation of colon associated with certain infections and inflammatory conditions of the bowel. viraemia -blood-borne carriage of virus. treatment of cmv enterocolitis requires parenteral therapy with either ganciclovir (5 mg kg ϫ1 twice daily) or foscarnet (90 mg kg ϫ1 twice daily), both of which can cause severe side effects. although these agents improve both endoscopic and symptomatic markers of cmv disease, the survival benefit is uncertain and needs to be re-evaluated following the introduction of haart (ref. 23) . ganciclovir can cause severe bone marrow depression with resultant anaemia and neutopaenia, and foscarnet can cause severe renal impairment (although a concomitant infusion of normal saline largely diminishes the risk of renal damage). in an open label, randomized study comparing a two-week course of foscarnet with ganciclovir, at the doses stated above, there was no significant difference in response of gi cmv disease between the two therapies 24 . approximately 75% of patients had good clinical and endoscopic responses with disappearance of inclusion bodies as determined histologically. relapse occurred within ten weeks in at least 50% of patients and survival without haart was ϸ20 weeks. surprisingly, blanshard and colleagues found that maintenance therapy did not increase the time taken for relapse of gi disease, although numbers were small and allocation of maintenance therapy was not randomized. the use of oral ganciclovir in maintenance therapy for retinitis is effective but has not been specifically evaluated for gi disease 25 . the efficacy of treatment with oral ganciclovir for primary prophylaxis of cmv disease is disputed and concerns about the development of viral resistance have been raised 26, 27 . it is hoped that newer agents, such as cidofovir, famiclovir, valganciclovir and formivirsen, will have a role in the management of cmv disease 28 . encouragingly, the advent of haart has led to a marked reduction in mortality and morbidity of hiv patients 29 . haart can result in remission of previously persistent opportunistic infections, including cmv infection 5 . however, the development of viral resistance and difficulties with compliance could lead to breakthrough hiv viraemia and the re-emergence or reacquisition of opportunistic gi infections. unfortunately, combination antiretroviral therapy is costly and, consequently, unavailable to the vast majority of hiv patients worldwide. patients on haart with stable cmv retinitis and cd4 counts of 150 cells l ϫ1 were able to successfully discontinue anticmv maintenance therapy without relapse of retinitis or development of extraocular disease over a mean follow-up period of 16 months 30 . importantly, immune reactivation retinitis occurred in 90% of patients started on haart before discontinuation of anticmv treatment, with substantial visual loss in the minority of patients. macdonald and colleagues report similar findings, although the follow-up period was considerably shorter, and they do not comment on immune reactivation retinitis 31 . others find that haart causes a significant and progressive decline in cmv viraemia in the absence of specific anticmv treatment 32 . it is intriguing to speculate about the possible occurrence of immune reactivation enteritis in patients with gi cmv; this has not been described to date. many questions remain about the host immune response to opportunistic infections following immune reconstitution with haart. what are the specific immune mechanisms leading to disease resolution following haart? does the t-cell repertoire expand following reconstitution to recognize 'new' cmv antigens? when can secondary prophylaxis be discontinued in patients that respond to haart? what are the immune consequences of retroviral rebound when haart fails? how can cmv indices be monitored to predict relapse in patients on haart? adenovirus is reported to cause infection in other immunosuppressed groups, including individuals with primary immunodeficiency and bone-marrow-transplant patients 33 . in hiv patients, dionisio and colleagues report increasing stool carriage of subgenus f type 40 adenovirus with increasing immunosuppression 34 . janoff and colleagues first described adenovirus colitis 35 . electron microscopy or culture of colonic biopsies from 67 hiv patients investigated for diarrhoea identified adenovirus in five patients. colonoscopy revealed mild inflammatory change in two of these patients. focal necrosis and amphophilic nuclear inclusions within degenerating epithelial cells were shown using light microscopy, and electron microscopy revealed characteristic hexagonal adenovirus particles within the inclusions (fig. 1) . maddox et al. have confirmed these characteristic features and found that specific immunostaining for adenovirus is both sensitive and specific for the identification of adenovirus inclusions 36 . the pathogenic role of adenovirus remains unclear as this group of patients is frequently co-infected with other known pathogens. thomas and co-workers report that adenovirus colitis is significantly more likely to be associated with chronic diarrhoea 37 , while schmidt and colleagues were able to detect adenovirus only in aids patients who are severely immunosuppressed 38 . although in the severely immunosuppressed group, adenovirus was detected more frequently in patients with diarrhoea than without (10% vs 3.3%), both positive and negative correlations between adenovirus and diarrhoea have been reported by other groups (table 1) [37] [38] [39] [40] [41] [42] [43] . most studies indicate a strong association between infection with adenovirus and co-infection with other pathogens, in particular cmv. it is difficult, therefore, to ascribe a pathogenic role to this virus with any certainty [37] [38] [39] . infection of enterocytes by hiv is well documented and is implicated as the cause of so-called 'hiv enteropathy', in which morphological and functional abnormalities of the gut are described in the absence of any other detectable pathogen 44 . the clinical importance of hiv enteropathy is probably limited, certainly 'pathogen-negative' diarrhoea is comparatively short lived and associated with a good prognosis 45 . several other enteric viruses are associated with hiv-related diarrhoea. grohman and colleagues examined stool specimens from patients with or without diarrhoea 39 . electron microscopy, polyacrylamide-gel electrophoresis and enzyme immunoassay were used to examine samples for rotavirus, adenovirus, calcivirus and picobirnavirus. paired sera were also analysed for antibodies to norwalk and picobirnavirus. overall, virus was detected in 35% of patients with diarrhoea and 12% without diarrhoea. astrovirus, picobirnavirus, calcivirus (including srsv) and adenovirus were identified significantly more often in patients with diarrhoea. unfortunately, co-infection with other known pathogens was not evaluated systematically and no information regarding the relative distribution of acute and chronic diarrhoea was provided. schmidt and colleagues detected virus in 17% of stool samples from 256 hiv-infected patients 38 . adenovirus and coronavirus were detected more frequently in patients with diarrhoea than without (10% vs 3.3% and 15% vs 6.6% respectively) and both were associated with severe immunosuppression. thea et al. found no association between enteric virus shedding and diarrhoea in a study performed in zaire 40 . overall, this group identified enteric virus, including rotavirus, srsv, coronavirus and adenovirus in 17% of samples analysed. they noted a trend towards increased shedding with greater immunosuppression, a finding in common with cunningham and colleagues 43 . an association of rotavirus with prolonged diarrhoea in hiv patients, as detected by enzyme immunoassay was also reported, although other groups do not support this finding 41 . data from selected studies that have evaluated enteric virus carriage in hiv patients are summarized in table 3 . in summary, data regarding the association of non-cmv enteric virus infection and diarrhoea is conflicting. the best evidence relates to adenovirus infection although its pathogenic role is far from certain. we speculate that many of these viruses cause only self-limiting acute diarrhoea or, as in the case of adenovirus, act as a cofactor in cmv infection. little is known about therapeutic options for these putative pathogens, although ribavarin might be effective in the treatment of adenovirus (r.c.g. pollok, unpublished). the impact of haart on non-cmv enteric virus infection has not yet been evaluated and warrants study. new developments in the diagnosis and treatment of cmv are being established. assays for cmv dna and the cmv pp65 antigen assay offer the prospect of cmv surveillance before the development of end-organ disease, which will allow the tailored introduction of prophylaxis. oral ganciclovir and valganciclovir (which, of the two, has greater bioavailability) are potentially useful prophylactic agents. second generation cmv treatments are becoming available and are being evaluated, largely in transplant patients. the advent of haart has dramatically reduced the occurrence of all opportunistic infections, including cmv. consequently, this has made evaluation of these newer agents difficult in patients with hiv. haart is unlikely to be widely available in the developing world in the immediate future and diarrhoeal disease continues to contribute to the death toll of hiv patients in these countries. the role of non-cmv enteric infection in hivrelated diarrhoea remains uncertain and unless further research is undertaken the importance of these viruses in hiv-related diarrhoea is unlikely to be established. • what are the specific immune mechanisms leading to disease resolution following haart? • what role do non-cmv enteric viruses, particularly adenovirus, have in hiv-related diarrhoea? • what is the significance of pathological change associated with 'adenovirus colitis'? • what role does haart play in the control of non-cmv enteric virus infection? prevalence of enteric pathogens in homosexual men with and without acquired immunodeficiency syndrome intestinal infections in patients with the acquired immunodeficiency syndrome (aids) chronic unexplained diarrhea in human immunodeficiency virus infection: determination of the best diagnostic approach investigation of chronic diarrhoea in acquired immunodeficiency syndrome. a prospective study of 155 patients changes in the natural history of cytomegalovirus retinitis following the introduction of highly active antiretroviral therapy cytomegalovirus colitis in hiv-1-infected patients: a prospective research in 55 patients high mucosal levels of tumor necrosis factor alpha messenger rna in aids-associated cytomegalovirus-induced esophagitis faecal tumour necrosis factor-alpha in individuals with hiv-related diarrhoea cytomegalovirus vasculitis. case reports and review of the literature isolation of cytomegalovirus-specific cytotoxic t-lymphocytes from gut-associated lymphoid tissue (galt) of hiv type 1-infected subjects progressive reduction of cmv-specific cd4ϩ t cells in hiv-1 infected individuals during antiretroviral therapy cytomegalovirus colitis in acquired immunodeficiency syndrome: a clinical and endoscopic study cytomegalovirus colitis in aids: presentation in 44 patients and a review of the literature cytomegalovirus esophagitis in patients with aids. a clinical, endoscopic, and pathologic correlation evaluation of diagnostic criteria for mucosal cytomegalic inclusion disease in the acquired immune deficiency syndrome cytomegalovirus esophagitis in aids: diagnosis by endoscopic biopsy cytomegalovirus infection in gastrointestinal tracts of patients infected with aids correlation of in situ hybridization with histology and viral culture in patients with acquired immunodeficiency syndrome with cytomegalovirus colitis frequency of positive tests for cytomegalovirus in aids patients: endoscopic lesions compared with normal mucosa cytomegalovirus dna level on biopsy specimens during treatment of cytomegalovirus gastrointestinal disease plasma cytomegalovirus dna, pp65 antigenaemia and a low cd4 cell count remain risk factors for cytomegalovirus disease in patients receiving highly active antiretroviral therapy comparison of three assays for cytomegalovirus detection in aids patients at risk for retinitis ganciclovir treatment of cytomegalovirus colitis in aids: a randomized, double-blind, placebo-controlled multicenter study treatment of aids-associated gastrointestinal cytomegalovirus infection with foscarnet and ganciclovir: a randomized comparison oral ganciclovir for cytomegalovirus retinitis in patients with aids: results of two randomized studies response of asymptomatic cytomegalovirus viraemia to oral ganciclovir 3 g/day or 6 g/day in hiv-infected patients a randomized, placebo-controlled trial of the safety and efficacy of oral ganciclovir for prophylaxis of cytomegalovirus disease in hiv-infected individuals. terry beirn community programs for recent advances in the therapy and prevention of cmv infections revision to the british hiv association guidelines for antiretroviral treatment of hiv seropositive individuals. bhiva guidelines writing committee discontinuation of anticytomegalovirus therapy in patients with hiv infection and cytomegalovirus retinitis lack of reactivation of cytomegalovirus (cmv) retinitis after stopping cmv maintenance therapy in aids patients with sustained elevations in cd4 t cells in response to highly active antiretroviral therapy decrease of cytomegalovirus replication in human immunodeficiency virus infected-patients after treatment with highly active antiretroviral therapy adenoviruses in the immunocompromised host chronic intestinal infection due to subgenus f type 40 adenovirus in a patient with adenovirus colitis in the acquired immunodeficiency syndrome adenovirus infection of the large bowel in hiv positive patients enteric viral infections as a cause of diarrhoea in the acquired immunodeficiency syndrome stool viruses, coinfections, and diarrhea in hiv-infected patients. berlin diarrhea/wasting syndrome study group enteric viruses and diarrhea in hiv-infected patients prevalence of enteric viruses among hospital patients with aids in kinshasa rotavirus antigen detection in patients with hiv infection and diarrhea prevalence of acute enteric viral pathogens in acquired immunodeficiency syndrome patients with diarrhea gastrointestinal viral infections in homosexual men who were symptomatic and seropositive for human immunodeficiency virus natural history and prognosis of diarrhoea of unknown cause in patients with acquired immunodeficiency syndrome (aids) key: cord-314753-xflhxb13 authors: manso, carmen f.; bibby, david f.; mbisa, jean l. title: efficient and unbiased metagenomic recovery of rna virus genomes from human plasma samples date: 2017-06-23 journal: sci rep doi: 10.1038/s41598-017-02239-5 sha: doc_id: 314753 cord_uid: xflhxb13 rna viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. new sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. through selective host rna depletion and compensatory protocol adjustments for ultra-low rna inputs, we are able to detect three major blood-borne rna viruses – hiv, hcv and hev. we recovered complete genomes and up to 43% of the genome from samples with viral loads of 10(4) and 10(3) iu/ml respectively. additionally, we demonstrated the utility of this method in detecting and characterising members of diverse rna virus families within a human plasma background, some present at very low levels. by applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of hcv genotype 6, and a co-infecting human pegivirus. this method builds upon earlier rna metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses. a complex host-enriched sample was prepared by diluting in negative human plasma (nhp, negative for each hiv, hcv, and hev) stored plasmas from four samples previously characterized by routine diagnostic testing to contain hcv (x2), hiv and hev (see table 1 for details). nhp was obtained by centrifuging negative human blood for 10 minutes at 500× g to remove cell debris. the final concentration of each virus in the primary panel sample was 10 6 iu/ml (copies/ml for hiv -implied by iu henceforth for convenience), and three serial tenfold dilutions in nhp were prepared from this stock. virus multiplex reference (vmr) panel. a reagent comprising a suspension in pbs of 18 rna viruses with different genomic and structural characteristics was provided by the national institute for biological standards and controls (nibsc, potters bar, uk). each viral component and its approximate relative concentrations is given in mee et al. 42 . prior to extraction, the panel was mixed 1:1 with nhp. duplicate 400 μl extractions were performed 44 , as was genotyping of the hcv 43 and hev 68 clinical samples of indeterminate hcv genotype. four plasma samples collected from a patient between 2014 and 2016 were submitted to public health england (phe) for metagenomic analysis as previous genotyping results had been inconsistent. the most recent such test employed ns5b sequencing 43 , and reported the presence of a virus belonging to genotype 6 but was unable to resolve the subtype with any further precision. rna extraction and quantification. before extraction, all samples were centrifuged for 10 min at 2,500 × g to remove cell debris. triplicate, duplicate and single extractions were performed on the diluted vmr panel samples (referred to as 'vmr panel a/b/pbs'), the bbv panel samples ('10 6-3 -a/b'), and the patient sample series, respectively. a negative control comprising 200 μl of the same plasma used to dilute the panels was also extracted. the split rna extraction kit (lexogen) was used to extract 200 μl of each sample input, according to the manufacturer's instructions. acidic phenol was used to preferentially recover the large rna fraction, which was eluted in 12 μl of nuclease-free water. rna eluates were quantified using qubit rna hs assay kit (thermo fisher scientific), which is accurate for concentrations between 250 pg/μl and 100 ng/μl. depletion of ribosomal rna and dna digestion. ribosomal rna depletion and dna digestion was achieved using the riboerase kit (kapa biosystems). as all sample extracts were below the detection limit of the qubit quantification system, the total rna input was less than the recommended 100 ng. the manufacturer's specifications were followed with the exception of using the entire 10 μl of the extract, and after the dna digestion reaction clean up, eluting the residual rna was in 10 μl of nuclease-free water. in the case of the bbv panel, two of the three extracts of each dilution were treated with the riboerase kit before rna library preparation. the third set of extracts remained untreated and was used to monitor the effect of the rrna depletion and dna digestion upon the subsequent library preparation and sequence analysis. in the case of the vmr panel (the two duplicates) and the negative control, rrna and dna depletion was performed on all extracts. in the case of the uncharacterized hcv strain, extracts from all four samples were treated with riboerase. an additional, untreated, extract of sample 4 was included, again to monitor the process. rna library preparation with ultra-low rna input. libraries were constructed from 10 μl of extracted rna or 10 μl of rrna-depleted dnase-digested rna, using the nebnext ultra directional rna library prep kit (new england biolabs). as the protocol is designed to use a minimum rna input of 10 ng, several modifications were made to adapt it to an ultralow rna input. these are listed in table 2 . libraries were analysed for size distribution using the high sensitivity dna kit (agilent) on a 2100 bioanalyser instrument, and were quantified using the kapa sybr fast universal qpcr kit for illumina libraries (kapa biosystems) on a 7500 real-time pcr system (applied biosystems). to determine the relative abundances of viral inserts, libraries constructed from the bbv panel were analysed by qpcrs with primers and probes targeting each of the three viral components (refs 44-46 and supplementary table s1 ). reactions were performed using the quantitect virus kit (qiagen) according to the manufacturer's instructions. libraries labelled with different indexes were diluted to 2 nm and pooled. sequencing was performed on an illumina miseq instrument using the miseq reagent kit v2 (300 cycles) (illumina) according to the manufacturer's guidelines, with the following minor modifications. the library pools were denatured with 0.2 n sodium hydroxide for 2 minutes rather than 5, diluted in kit reagent ht1 to produce a 20 pm solution and then these were further diluted to 11 pm. of this library pool dilution, 600 μl were loaded onto the miseq cartridge. data analysis. all paired end fastq files were processed with trimmomatic v0.30, removing the illumina adaptor sequences, then trimming leading and trailing bases with phred scores below 20. reads were discarded where the length of either trimmed end was below 50 bases. for the determination of genome sequences of blood-borne viruses, trimmed fastq sets were normalised using the normalise-by-median.py script in the khmer package (k = 31, c = 5) 47 and submitted to the spades de novo assembler 48 without error-correction, applying the default kmer sizes of 21, 33, and 55. output contigs that matched each virus were identified with the nhmmer function of the hmmer v3.1b2 package 49 using hidden markov models (hmms) built from alignments of each virus (detailed in supplementary table s2) . where necessary, the ends of contigs were trimmed to the whole genome alignment. bwa mem (v0.7.5a, default parameters) 50 was used to map the original trimmed fastqs to the genome sequence, and the sam files were converted to bam files using samtools v0.1.19 51 while discarding reads with either 0 × 04 and/or 0 × 08 flags set (i.e. retaining only fully-mapped paired-end reads). base frequencies at each nucleotide position within each component virus sequence were obtained from bam files using quasibam v2.2, an in-house c++ program that tabulates base frequencies at each nucleotide position within a reference and generates consensus sequences based upon user-defined depth and variant percentages 52 . mapping of trimmed paired-end fastq to one or more virus reference genomes was also performed using bwa mem 0.7.5a. in each case, two independent mappings were performed, using as a reference the viral sequences, supplemented firstly by the march 2009 'grch37' release of the human genome, and secondly by a set of human rrna sequences (nr_003286.1, nr_003287.1, v00589.1, nr_003285.2, gij251831106:648-1601, and gij251831106:1671-3229, as per malboeuf et al. 38 ). the second file was used solely to derive counts for reads mapping rrna which would otherwise be subsumed into the human genome mapping results. from the filtered sam files, the numbers of reads mapping to each reference sequence were counted. counts for each of the constituent sequences of the human genome and rrna were pooled into a "human" count and an "rrna" count. quasibam was used to derive nucleotide frequencies from which depth and coverage data were calculated. a minimum depth of 10 was required for inclusion in a derived consensus sequence for the bbv panel (1 for the vmr panel). bbv and vmr panel sequences. the members of the multi-fasta reference file for the bbv panel were obtained by submitting fastq sets from the rrna-depleted sample with the highest virus concentrations to the spades-hmmer-mapping approach described in the previous paragraph. vmr panel references were derived from sequences obtained from genbank using accession numbers from mee et al. 42 . additionally, the complete genome sequence of a human pegivirus (hpgv) present in the plasma diluent was discovered in the spades contigs file. a hmm profile was constructed from an alignment of genbank sequences (supplementary table s2 ). sample with uncharacterised hcv. to obtain full-length hcv genomes, each fastq set was submitted to the spades-hmmer-mapping process. where a complete genome was not obtained, hcv-matching contigs were aligned to the full-length genomes using mega5 53 . in addition, contigs with length > 5 kb that did not align to the hmm profile were submitted to blast 54 for identification. following this analysis, an additional pegivirus genome was derived in similar fashion to the hcv genomes, using the same hmm profile as above for the nhp pegivirus. when calculating the read percentages and coverage plots, both sample-derived full-length genome sequences (hcv and hpgv) were used as the reference sequence when mapping that sample's corresponding trimmed paired-end fastqs, as well where only incomplete hcv genomes were obtained. virus (bbv) panel was prepared, comprising two strains of hcv (genotypes 1a and 1b), and one each of hiv and hev diluted in nhp to 10 6 iu/ml. three tenfold serial dilutions in plasma were made from this original panel. step manufacturer's recommendations protocol modification riboerase rna input 0. 69 . ribosomal rna depletion was performed on two of each set of triplicate extractions prior to all three being subjected to the modified library preparation protocol. data from the most concentrated rrna-depleted samples were used to generate individual virus genome sequences for use in reference mapping. during this data analysis, an unexpected human pegivirus (hpgv) was found and traced to the nhp diluent. the full genome sequence of this hpgv was determined from the 10 3 -b data and included in the mapping references. table 3 gives the read counts, genome coverages and median depths for each virus-dilution combination, across each of the three samples per dilution (10 6-3 -untreated/a/b). each test sample yielded over 800,000 reads with the exception of 10 3 -a, which gave just over 140,000 reads. with the exception of the two 10 6 samples, in which only a very small volume of nhp was added to the clinical samples, the percentage of reads mapping to the hpgv remained relatively constant at 29-39%. the exception is 10 5 -b, in which the overall viral read percentage was lower than expected, with a corresponding elevation in reads mapping to the human genome suggesting possible incomplete dnase digestion during the rrna depletion step (supplementary table s3) . with increasing dilution, the total viral read percentages (excluding hpgv) decline from over 60% to 0.23%. complete and near-complete genome coverages with depths greater than 10 were achieved at 10 6 and 10 5 iu/ml for all four viruses. a few short regions in hiv had low coverages (<10) with 10 5 -b, reflecting the reduced overall viral reads in this dataset, but at a minimum depth of 1, 99.6% coverage was achieved with this sample, with only a 32-base sequence in pol having no coverage. at 10 4 iu/ml, hcv 1a and hev continued to give 98.1-99.7% coverage with median depths over 120. hcv 1b and hiv gave 91.2-93.5% coverage (82-95 median depth) and 55.0-90.2% coverage (12-83 median depth) respectively, and in 10 3 -b, despite only 0.23% of all reads mapping to the four viruses collectively, genome coverages of 18.1-72.5% were achieved, with median depths up to 29. figure 1 illustrates coverages and depths across target genome at each dilution, showing even distributions of reads across all four target genomes and hpgv. pooling duplicates consistently improved coverages (final column, table 3 ). this is most clearly seen at the lower viral loads, where at 10 4 iu/ml, three of the four viruses achieve combined coverages of >99.4% each, and 93.5% in hiv. at 10 3 iu/ml, the combined coverages for the four viruses are effectively what would be expected were the individual coverages independent, i.e. cov aub depletion of rrna substantially enhances the recovery of blood-borne virus sequences. the percentage of reads mapping to rna virus genomes in the rrna-depleted bbv panel samples was between 40 and 150-fold higher than in corresponding untreated controls. individual target virus ratios decreased as they became more dilute, from over 100-fold for hcv in 10 5 -a to 2.9-fold for hiv at the lowest dilution. concomitantly, the ratio for hpgv rose markedly, from 4.8-fold in 10 6 -a to 175 in 10 3 -b, reflecting an effectively constant viral load against decreasing quantities of panel viruses (table 3 and fig. 1 ). genome coverage and median depth values were also much higher in the treated samples than untreated comparators. at the two highest virus concentrations, median depths were between 47-and 274-fold higher in the treated versus the untreated samples. only short fragments of hev were recovered from the untreated 10 4 dilution, and almost no hiv or hcv sequences. by contrast, near complete genomes from all four target viruses were recovered from the treated comparators, with median depths of between 83 and 457 (as noted above, hiv in 10 4 -a was an exception at 54.0% coverage and a median depth of 13). recovery of partial and complete genomes of diverse virus types from human plasma. the ability of our method to recover genome sequences from a range of rna viruses in the context of human plasma was evaluated using a virus multiplex reference (vmr) panel, putatively containing 25 genomically and physicochemically diverse viruses. two plasma-diluted panels and one pbs-diluted panel were tested (table 4 ). no reads from either of the three samples mapped to either of the two norovirus genomes, coronavirus 229e or influenza b virus. by the panel distributor's qpcr 42 , the threshold cycle (c t ) of the coronavirus was >36 and the other three were not detected, hence these four targets were excluded from further analysis. notwithstanding influenza virus a h3n2 and parainfluenza virus type 3 also not being detected by the qpcr, we recovered reads from both, with genome coverages ranging from 2.7% to 21.6%. almost no reads belonging to the panel's dna viruses were found. sixty-nine percent of all reads obtained from the pbs-diluted panel mapped to vmr panel genomes, dropping to 41-44% for the plasma-diluted samples, although the distribution of reads between targets was very uneven. parechovirus and rotavirus accounted for 78.8-87.6% and 10.6-19.5% of all viral reads respectively, with the other viruses collectively accounting for 1.7-1.9%. depths and genome coverages showed some inverse correlation with the given c q values (fig. 2) . as with the bbv panel data, coverage plots of the samples diluted in plasma were largely unbiased, giving pooled genome coverages close to those expected by independent distributions of reads between replicates ( table 4 , final column). rotavirus and coxsackievirus were exceptions, where despite large numbers of mapped reads, almost identical patterns of read coverages and gaps were observed between their replicates, with minimal additive effect. the pbs-diluted sample gave larger read numbers, but their distribution was less even throughout the genomes, resulting in relatively lower coverages. characterisation of a new subtype belonging to hcv genotype 6 and discovery of a second virus in a patient sample series. four plasma samples from a patient with hcv were used as starting material. all extracts were subjected to riboerase treatment; a second extract of sample 4 remained untreated for comparison. de novo assembly analysis of fastq sets from samples 1, 3 and 4 each gave a full-length hcv genome sequence as a single contig. for sample 2, 6 partially-overlapping contigs were obtained, covering 66% of scientific reports | 7: 4173 | doi:10.1038/s41598-017-02239-5 the hcv sequence. additionally, in all four samples, a single contig was obtained that was determined by blast and subsequent hmmer analysis to comprise an hpgv genome. the hcv and hpgv full genome sequences were combined in a single file to carry out reference mapping and nucleotide frequency determination on the four sample fastq sets (table 5 ). samples 1, 3 and 4 had hcv read percentages ranging from 1.0 to 24.3%, and gave complete genomes with median depths greater than 700. sample 2 had the lowest viral load (2,000 iu/ml), had 0.3% of reads mapping to hcv giving a genome coverage of 87% at a minimum depth of 10 (96.5% at depth ≥1) and a median depth of 43. full coverage of the hpgv genome was obtained from all samples, with median depths over 8,700, and read percentages ranging from 34.2 to 63.3%. the depth plots in fig. 3 again show unbiased and even coverages across both genomes, and the percentages of reads mapping to viral targets was again much higher in the rrna-depleted sample than in the untreated comparator (61-fold and 85-fold for hcv and hpgv respectively). table 3 . detailed sequencing data from the bbv panel. for each of the three samples (untreated, a and b) at each dilution (10 6 -10 3 ), the number and percentage of reads mapping to each virus are given, together with the genome coverages (depth ≥10) and median depths. the final column gives these last two metrics from the combined data sets of both the a and b samples. included in the analysis are data for the hpgv discovered in the sample diluent. analysis of the hcv sequence showed it to belonging to a new subtype within genotype 6 of which the details are presented in a separate manuscript (in preparation). the hpgv clustered with genotype 1 strains, and is distinct from the nhp strain. including the nhp negative control were subjected to virus-specific qpcr for the detection and quantification of hcv, hiv and hev. all were detectable in the sample libraries, but were undetectable in the riboerase-treated negative control library (supplementary table s4 ). all samples were mapped against reference sequences that included human genome and human rrna sequences to evaluate the efficiency of riboerase treatment. the average ratio of the percentages of reads mapping to rrna in the untreated versus the treated samples was 32-fold with an approximate halving of the number of reads mapping to the human genome, across all panels (fig. 4) . with the exception of the expected human pegivirus, mapping of the negative control fastq set against the reference sequences of the four bbv panel viruses, the two pegiviruses, the vmr panel and the patient hcv gave table 5 . detailed sequencing data from the patient sample series. for each of the four samples 1-4, the number and percentage of reads mapping to both the hcv and hpgv genomes are given, genome coverages (depth ≥10) and median depths. the analysis of sample 4 extracted without host rrna depletion is in the untreated column. very low numbers of reads mapped to viral genomes and no consensus sequences could be derived. further data for this section are found in supplementary tables s3 and s5 . in light of the large and ever-increasing number of human rna virus pathogens, it is perhaps unsurprising that standard serological assays and nucleic acid tests suffer from a lack of sensitivity to diverse variants of target viruses, overlook the presence of new or unexpected viruses, and provide only limited information about those targets they do successfully detect. hence the three main aims of metagenomic virology are to detect & identify known agents irrespective of their diversity, to discover novel agents of disease, and to obtain complete sequence information of detected viruses. most existing protocols achieve a maximum of two of these aims, but difficulties in selectively isolating viral rna species and short read sequences from those of the super-abundant host nucleic acid have limited the utility of metagenomic approaches in diagnostic virology. this study has addressed these limitations by establishing a novel methodology suitable for the agnostic detection and characterization of blood-borne rna viruses in plasma samples. by depleting host-derived nucleic acids and making modifications to an existing library preparation protocol to account for ultra-low rna input quantities, we have been able to reconstruct effectively full-length genomes of hcv, hev and hiv from plasma samples with viral loads of 10 4 iu/ml (copies/ml for hiv) and substantial fractions of complete genomes at 10 3 iu/ml. when applied to a series of clinical samples, we could elucidate simultaneously the full genome sequences of both a novel subtype belonging to hcv genotype 6 and a hitherto-undetected human pegivirus. additionally, our system was able to recover viral sequences from a panel of diverse rna viruses diluted in human plasma, with a broad correlation between the genomic coverage and depth metrics and approximate concentration. although full genomes were not assembled in many cases, the independence of read distribution gave sufficient genome coverage for identification. the vast majority of rna molecules in a human plasma sample are host-derived, of which up to 80% comprises the six species of human rrna. their presence in our libraries was minimised by two key protocol steps in our modified protocol. firstly, we selected an extraction method that combined a phenol/chloroform step with a column format (lexogen split rna) which increased the amount of extracted viral rna by up to one log when compared to other extraction methods (data not shown). perhaps more importantly, by controlling the final precipitation step, small rna molecules below 150 nt such as 5 s rrna and trna are excluded from the eluates, as are the majority of molecules of human genomic dna. secondly, we employed dna probes complementary to human rrna such that hybridisation and subsequent digestion by rnase h dramatically reduced their frequency in the finished libraries. whilst this methodology has been successfully used in the detection and characterisation of two haemorrhagic fever viruses, the frequency of viral reads was often below 1% and an additional hybrid-capture step was employed to elevate read numbers 36 . methods that do not deplete rrna generally give poor recovery of viral reads, yielding viral genome fragments that necessitate further work 27, 32, 55, 56 , low read numbers even at viral loads over 10 4 iu/ml [20] [21] [22] 33 , or at best, requiring dilution of both host and virus in pbs in order to recover full hiv genomes at low copy numbers 38 . the resultant rrna-depleted sample extracts typically contain quantities of nucleic acid in the low picogram range. library preparation through hexamer-mediated reverse transcription followed by multiple displacement amplification constitutes an easy and effective means of amplifying very low amounts of dna 27, 38, 57 , but in several studies (and in the authors' laboratory), significant amplification biases have been observed, leading to gaps in target genome coverage 39, [58] [59] [60] . consequently, we adopted an approach using a standard rna library preparation kit, but with substantial modification to compensate for their minimum rna input requirements of at least 10 ng and optimally 100 ng-1 µg. we made key changes to the rna fragmentation and adaptor-ligation steps of the nebnext ultra directional rna library prep kit protocol. while prior rna fragmentation with heat and divalent cations improves sequence coverage, over-fragmentation of target genomes leads to the loss of material during the library preparation process 37 . lower amounts of rna thus require shorter optimum fragmentation times and we found that 1 minute at 94 °c was optimal in terms of breadth of genome coverage. under standard kit conditions, our ultra-low rna inputs dramatically skewed the ratio of cdna to adaptor. the resulting adaptor excess led to the preferential amplification of adaptor dimers during the pcr step, and despite increasing cycle number to amplify low rna inputs, we were generally unable to generate sufficient quantities of target-specific material. accurate quantification and consequent equimolar pooling of libraries was compromised, as was the miseq clustering efficiency. we found that a reduced final adaptor concentration of 1.4 nm was crucial in reducing the amount of adaptor dimers in libraries from rrna-depleted samples whilst simultaneously extending the pcr cycle number. in the present study, serial dilutions of the blood borne virus panel were prepared in negative human plasma, reducing both the absolute quantity and relative frequency of the viral rna targets while maintaining the complexity of the sample in terms of host nucleic acid, thus mimicking that of a clinical sample. with rrna depletion, the number and diversity of viral reads was consistently high, with over 35% of all reads mapping to constituent virus genomes. throughout the three sample series, we obtained relatively high genome coverages of low-frequency viral targets. co-infections with multiple blood-borne viruses are common 61 , so whilst we speculate that the depths and coverages of target viruses would be greater yet in these samples had it not been for the confounding effect of the unexpected human pegiviruses in both the plasma diluent and the patient sample series, it was reassuring to see the method performed well under such conditions. in our experiments using negative human plasma as sample diluent, we were able to recover levels of viral genomes comparable to previous work using pbs, both for bbv panel viruses 38 and for the vmr panel 41 , and we were able to recover from a patient sample a large percentage of the genome of a previously uncharacterised subtype of hcv genotype 6 when present at 2 × 10 3 iu/ml, a diagnosis not possible using existing genotyping assays. the presence of an undiagnosed pegivirus in this sample further demonstrated the utility of the method in metagenomic analysis of blood-borne virus co-infections where the relative abundances of each virus can be highly variable 22 . furthermore, in three of the four samples, depths greater than 1,000 were routinely obtained, which are likely to be sufficient to call minority variants for clinical resistance 62 . a full description of the patient series and the new hcv strain are provided in a separate manuscript (in preparation). our approach can therefore not only accurately characterise rare or novel variants of existing viruses, but also generates the same level of information regarding unexpected viruses present in the sample. by comparison, vidisca 32, 63, 64 and other random amplification-ngs techniques 30, 31 have detected novel viruses in diverse clinical samples, but all have required further techniques to achieve full genome sequences. together with the vmr panel results, we were able to recover identifying sequence from both enveloped viruses (hcv, hiv, hev, influenza, and several paramyxoviruses), and non-enveloped viruses (several enteroviruses, astrovirus, rotavirus, and sapovirus). for the majority of viruses in the vmr panel, whilst dilution in plasma reduced the total percentage of reads recovered when compared to the panel diluted in pbs, a greater breadth of genome coverage was achieved. in the absence of any host nucleic acid background, it is possible that the pbs extracts had such ultra-low quantities of rna that despite the adjustments made to the library preparation protocol, the rna was over-fragmented, leading to a smaller number of genome fragments that were individually amplified to a greater extent than the larger array of fragments surviving the plasma extraction. in developing a similar approach, kohl et al. were only able to recover a percentage of reads exceeding 6% at a viral load over 10 7 copies/ml. at an influenza a virus concentration of over 10 5 copies/ml, this dropped to just 0.5%, and at a reovirus concentration of 10 3 -10 4 copies/ml, no viral reads were detected 24 . with our method, whole genomes were obtained for those with the highest viral loads, and for minority viral targets, there was a correlation between ostensible quantity and coverage, including for two viruses undetectable by the panel distributor 42 , a result superior to that recently obtained from influenza in clinical respiratory samples 65 . again, the presence of high quantities of one or more target is likely to have inhibited the representation of the minority species such that if tested individually, superior depths and coverages would seem likely. with further reduction to the fragmentation time, or even its abolition, it may be possible to use this method to reconstruct genomes from old, partially degraded samples such as those recently used to re-evaluate the early hiv epidemic in the americas 66 . our negative control data suggest that the level of contamination is low, with most viral reads therein belonging to the most abundant vmr panel member. nelson et al. 67 identified a second source of contamination consisting of incorrect reads from other libraries that were sequenced during the same sequencing run due to truseq index misassignment (~0.06% of reads, 0.02% here). although cross-contamination between samples during the library preparation can be another source of contamination, the qpcr results suggest no bbv panel genomes were present after library preparation in the negative control sample. to conclude, by applying the three adaptations of selective large rna extraction, rrna depletion-dnase treatment, and the extensively modified library preparation in combination, ngs data sets can be produced from plasma samples that are rich in rna virus sequence data. complex bioinformatic processing has been employed to identify viruses within a metagenomic dataset 7, 25, 26, 32, 64, 65 , but here, only simple bioinformatic processing is needed for detection and identification of known viruses, and by applying only moderately more advanced tools, an agnostic approach to virus detection can be taken, together with characterisation of the full genome even at low viral loads. the role of mutational robustness in rna virus evolution rna virus quasispecies: significance for viral disease and epidemiology ecological origins of novel human pathogens isolation of a novel coronavirus from a man with pneumonia in saudi arabia zika virus in the americas-yet another arbovirus threat reappearance of chikungunya, formerly called dengue detecting the emergence of novel, zoonotic viruses pathogenic to humans human viruses: discovery and emergence estimates of global, regional, and national incidence, prevalence, and mortality of hiv, 1980-2015: the global burden of disease study global epidemiology and genotype distribution of the hepatitis c virus infection first genome characterization of a novel hepatitis c virus genotype 5 variant first ngs full genome characterization of a hepatitis c virus genotype 7 divergent subtype first evidence of transmission of an hiv-1 m/o intergroup recombinant virus rates of and reasons for failure of commercial human immunodeficiency virus type 1 viral load assays in brazil impact of hiv-1 genetic diversity on plasma hiv-1 rna quantification: usefulness of the agence nationale de recherches sur le sida second-generation long terminal repeat-based real-time reverse transcriptase polymerase chain reaction test blinded, multicenter comparison of methods to detect a drug-resistant mutant of human immunodeficiency virus type 1 at low frequency global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study comparison of real-time pcr and antigen assays for detection of hepatitis e virus in blood donors an introduction to the analysis of shotgun metagenomic data metagenomic profiling of the viromes of plasma collected from blood donors with elevated serum alanine aminotransferase levels viral metagenomics applied to blood donors and recipients at high risk for blood-borne infections utility of metagenomic next-generation sequencing for characterization of hiv and human pegivirus diversity comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood protocol for metagenomic virus detection in clinical specimens host-associated metagenomics: a guide to generating infectious rna viromes virome analysis for identification of novel mammalian viruses in bat species from chinese provinces evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery two novel parvoviruses in frugivorous new and old world bats identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections using next generation sequencing to identify yellow fever virus in uganda a new phlebovirus associated with severe febrile illness in missouri a sensitive assay for virus discovery in respiratory clinical samples a viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing sequence-specific cleavage of small-subunit (ssu) rrna with oligonucleotides and rnase h: a rapid and simple approach to ssu rrna-based quantitative detection of microorganisms evaluation of convenient pretreatment protocols for rna virus metagenomics in serum and tissue samples enhanced methods for unbiased deep sequencing of lassa and ebola rna viruses from clinical and biological samples library construction for next-generation sequencing: overviews and challenges complete viral rna genome sequencing of ultra-low copy samples by sequence-independent amplification multiple displacement amplification compromises quantitative analysis of metagenomes genomic dna amplification by the multiple displacement amplification (mda) method challenges of the unknown: clinical application of microbial metagenomics development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing determining hepatitis c genotype by analyzing the sequence of the ns5b region minor groove binder modification of widely used taqman probe for hepatitis e virus reduces risk of false negative real-time pcr results fast, reliable and low cost user-developed protocol for detection, quantification and genotyping of hepatitis c virus a novel internally controlled real-time reverse transcription-pcr assay for hiv-1 rna targeting the pol integrase genomic region the khmer software package: enabling efficient nucleotide sequence analysis spades: a new genome assembly algorithm and its applications to single-cell sequencing nhmmer: dna homology search with profile hmms fast and accurate short read alignment with burrows-wheeler transform the sequence alignment/map format and samtools assessment of the utility of whole genome sequencing of measles virus in the characterisation of outbreaks mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods blast+: architecture and applications comparative analysis of rna sequencing methods for degraded or low-input samples metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (ftls) in henan province, china: discovery of a new bunyavirus whole genome amplification: abundant supplies of dna from precious samples or clinical specimens environmental whole-genome amplification to access microbial populations in contaminated sediments sequencing genomes from single cells by polymerase cloning amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded dna viruses epidemiology and impact of hiv coinfection with hepatitis b and hepatitis c viruses in sub-saharan africa highly sensitive and specific detection of rare variants in mixed viral populations from massively parallel sequence data identification of a new human coronavirus identification of a contemporary human parechovirus type 1 by vidisca and characterisation of its full genome evaluation of unbiased next-generation sequencing of rna (rna-seq) as a diagnostic method in influenza virus-positive respiratory samples patient 0' hiv-1 genomes illuminate early hiv/aids history in north america analysis, optimization and verification of illumina-generated 16s rrna gene amplicon surveys a novel virus in swine is closely related to the human hepatitis e virus a modified rna-seq approach for whole genome sequencing of rna viruses from faecal and blood samples the authors would like to acknowledge the contribution of nibsc for kindly providing the viral multiplex reference panel and the blood borne virus unit of virus reference department, phe, for providing the hev sample. the genomic services unit at phe are acknowledged for running the miseqs. the authors are grateful to dr. brendan healy, dr. owen seddon and dr. nicola price from university hospital of wales for providing additional information regarding the patient sample series. this work was supported by funding from public health england. supplementary information accompanies this paper at doi:10.1038/s41598-017-02239-5competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-318587-ewvnkdr2 authors: steeds, kimberley; hall, yper; slack, gillian s.; longet, stephanie; strecker, thomas; fehling, sarah katharina; wright, edward; bore, joseph akoi; koundouno, fara raymond; konde, mandy kader; hewson, roger; hiscox, julian a.; pollakis, georgios; carroll, miles w. title: pseudotyping of vsv with ebola virus glycoprotein is superior to hiv-1 for the assessment of neutralising antibodies date: 2020-08-31 journal: sci rep doi: 10.1038/s41598-020-71225-1 sha: doc_id: 318587 cord_uid: ewvnkdr2 ebola virus (ebov) is an enveloped, single-stranded rna virus that can cause ebola virus disease (evd). it is thought that evd survivors are protected against subsequent infection with ebov and that neutralising antibodies to the viral surface glycoprotein (gp) are potential correlates of protection. serological studies are vital to assess neutralising antibodies targeted to ebov gp; however, handling of ebov is limited to containment level 4 laboratories. pseudotyped viruses can be used as alternatives to live viruses, which require high levels of bio-containment, in serological and viral entry assays. however, neutralisation capacity can differ among pseudotyped virus platforms. we evaluated the suitability of ebov gp pseudotyped human immunodeficiency virus type 1 (hiv-1) and vesicular stomatitis virus (vsv) to measure the neutralising ability of plasma from evd survivors, when compared to results from a live ebov neutralisation assay. the sensitivity, specificity and correlation with live ebov neutralisation were greater for the vsv-based pseudotyped virus system, which is particularly important when evaluating ebov vaccine responses and immuno-therapeutics. therefore, the ebov gp pseudotyped vsv neutralisation assay reported here could be used to provide a better understanding of the putative correlates of protection against ebov. www.nature.com/scientificreports/ and time required for plaque development, which can take approximately nine days, makes it time-consuming and restricts high-throughput sample processing. development of novel serological assays that utilise genetically modified recombinant or chimeric viruses with attenuated pathogenicity have enabled more widespread investigation of neutralising antibodies against highly pathogenic viruses including ebov 10, 11 . pseudotyped viruses are replication-defective chimeric virions that comprise the structural and enzymatic core of one virus, bearing the envelope protein or glycoprotein of another, and encode a quantifiable reporter gene. retroviruses, including lentiviruses and gammaretroviruses such as human immunodeficiency virus (hiv) and murine leukaemia virus (mlv), respectively, and rhabdoviruses, such as vesicular stomatitis virus (vsv), have been used extensively as cores for pseudotyped viruses 12, 13 , including for ebov 14, 15 . a number of ebov gp pseudotyped virus neutralisation assays have been developed to investigate immune responses to ebov infection and vaccination [16] [17] [18] , as well as for evaluation of monoclonal antibody (mab) therapies [19] [20] [21] . there are many factors that need to be considered when developing and optimising pseudotyped virus neutralisation assays, to assess experimental parameters that can affect assay performance and to ensure accuracy and reproducibility. these include, choice of core virus and reporter gene, determination of target cell line and amount of pseudotyped virus input, as well as correlation with live virus neutralisation 22 . the aim of this study was to assess the suitability of ebov gp pseudotyped hiv-1 and vsv systems to measure neutralisation by evd survivor plasma, in comparison with results from a live ebov neutralisation assay. cell tropism of ebov gp pseudotyped viruses. pseudotyped hiv-1 and vsv bearing the envelope gp from ebov (mayinga) were generated and quantified by measuring luminescence in a range of target cell lines, in order to determine the optimum cell line to use in neutralisation assays. cells only controls were used to determine background levels of luminescence ( supplementary fig. s1 ). reporter activity was detected in all cell lines infected with ebov gp pseudotyped hiv-1 and vsv, demonstrating the broad tissue range conferred by ebov gp, although differences in luminescence were observed (fig. 1a,b) . for ebov gp pseudotyped hiv-1, highest tcid 50 /ml values were observed in 293t/17 cells, followed by huh-7 cells (fig. 1c) . titres generated by infection of 293t/17 cells were approximately 3, 33 and 73 times greater than those produced by infection of huh-7, hela and vero e6 cells, respectively. for ebov gp pseudotyped vsv, highest titres were obtained in during the initial stages of assay development, it is important to evaluate neutralisation of pseudotyped viruses using well characterised antibodies in order to demonstrate the validity and accuracy of the assay. the ebov gp pseudotyped viruses were assessed for neutralisation by the human anti-ebov gp mab, kz52. kz52 is an antibody isolated from a human survivor of the 1995 outbreak in kikwit that neutralises ebov in vitro and recognises a conformational epitope at the base of the gp [23] [24] [25] . human anti-ebov gp mab, kz52 was unable to neutralise the ebov gp pseudotyped hiv-1 (fig. 2a) within the range tested, however it was able to neutralise the ebov gp pseudotyped vsv (fig. 2b) , suggesting that vsv-based pseudotyped viruses are more sensitive to neutralisation then lentiviral-based, possibly the density of ebov gp on the pseudotyped hiv-1 may differ from that on the pseudotyped vsv or live ebov. to determine the optimal pseudotyped virus input to use in the hiv-and vsv-based assays, neutralisation of different amounts of the ebov gp pseudotyped viruses by plasma from a guinean evd survivor donor or human anti-ebov gp mab kz52 was assessed. kz52 was selected as it is commercially available and there is accompanying information regarding its neutralisation activity against ebov gp pseudotyped vsv expressing luciferase. however, as the ebov gp pseudotyped hiv-1 was not neutralised by kz52 (fig. 2a) in the range tested, plasma from an evd survivor was used to assess the effect of pseudotyped hiv-1 input on neutralisation instead. survivor plasma sample cs001 was chosen as it displayed strong neutralising ability against live ebov neutralisation, with a geometric mean titre (gmt) of 1,218. percentage infectivity was determined relative to infectivity of cells by the ebov gp pseudotyped viruses alone (fig. 3a ,b) and 50% inhibitory concentration (ic 50 ) of pseudotyped virus neutralisation were estimated by model of nonlinear regression dose-response curves (fig. 3c,d) . plasma from evd survivor cs001 displayed neutralising activity against all amounts of ebov gp pseudotyped hiv-1 tested (fig. 3a) . lower pseudotyped virus input resulted in larger variability and less curve fitting. therefore, an ebov gp pseudotyped hiv-1 input of at least 8.6 × 10 4 rlu/well, with a target input of 2.0 × 10 5 rlu/well, was used in subsequent neutralisation assays. kz52 neutralised all dilutions of ebov gp pseudotyped vsv tested (fig. 3b) and ic 50 values decreased with decreasing amounts of pseudotyped virus input (fig. 3d , supplementary table s1 ). when using 3.9 × 10 4 rlu/well of ebov gp pseudotyped vsv, ic 50 of virus neutralisation (0.07 µg/ ml) was similar to that expected according to the manufacturer's product data sheet (0.06 µg/ml). therefore, a target input of approximately 3.7 × 10 4 rlu/well was used in subsequent ebov gp pseudotyped vsv neutralisation assays. table s2 ). neutralisation of ebov gp pseudotyped hiv-1 and vsv by positive (evd survivor) and negative (uk donor) control plasma was assessed in several independent assays ( supplementary fig. s2 ). the background level of neutralisation was determined using plasma from a uk negative control donor. for the hiv-1-based assay this was calculated as ic 50 6.28 reciprocal dilution. the negative control plasma displayed no neutralising activity against ebov gp pseudotyped vsv, and therefore the background level of neutralisation was assigned the lowwww.nature.com/scientificreports/ est dilution of sample tested in the assay (1/20). in the hiv-1-based assay, dose-response curves were unable to be fitted for three of the 30 evd survivor samples and six of the samples were deemed below the background level of neutralisation. in contrast, a dose-response curve was unable to be fitted for only one of the evd survivor samples tested in the vsv-based neutralisation assay. in the hiv-1-based assay, three of the 10 negative plasma samples tested were above the background level of neutralisation, whereas only one of the negative samples tested was above the background level of neutralisation in the vsv-based assay. although some differences in the discriminatory power of positive and negative samples between the assays were observed, a statistically significant difference in neutralisation titres was detected between the evd survivors and negative plasma samples in the hiv-1-based assay (mann-whitney, p = 0.0054) (fig. 4a ) and in the vsv-based assay (mann-whitney, p < 0.0001) (fig. 4b , supplementary table s3 ). remarkably, this difference was more significant and the separation of the positive and negative plasma was better in the vsv-based assay (fig. 4b) . the sum of these results clearly show that the vsv-based ebov gp neutralisation assay displayed better reliability, specificity and sensitivity compared to the hiv-1-based assay. correlation with live ebov neutralisation. the neutralising capacity of the individual plasma samples against authentic ebov was assessed by a live virus neutralisation assay (supplementary table s3 , supplementary fig. s3 ). when ic 50 values of ebov gp pseudotyped hiv-1 neutralisation of the 30 evd survivor and 10 negative plasma samples were compared with gmt values for the live ebov neutralisation assay, a positive correlation (r s = 0.54) was determined using the nonparametric spearman correlation coefficient (fig. 5a) and this was statistically significant (p = 0.0004). remarkably, a stronger statistically significant (p < 0.0001) positive correlation (r s = 0.86) was observed when ic 50 values of ebov gp pseudotyped vsv neutralisation were compared with gmt values for the live ebov neutralisation assay (fig. 5b) . the correlation coefficients for ebov gp hiv-1 and vsv ic 50 compared with live ebov gmt without the negative controls were 0.38 (p = 0.0375) and 0.69 (p < 0.0001), respectively. therefore, the vsv-based ebov gp pseudotyped virus neutralisation assay correlated better with live ebov neutralisation than the hiv-1-based neutralisation assay. pseudotyped viruses can be used as alternatives to infectious virus in serological assays to measure neutralising antibodies to viral envelope glycoproteins 11 . pseudotyped virus assays used to profile neutralising antibody responses against severe acute respiratory syndrome-associated coronavirus (sars-cov) 26 , influenza (h5n1 and h7n9) [27] [28] [29] , rabies 30,31 and chikungunya virus 32 , for example, found that results correlated well with those from replication-competent or live virus assays. a high degree of correlation has been demonstrated between ebov cl4 prnt and an ebov pseudotyped vsv cl2 fluorescence reduction neutralisation test (frnt) 33 . however, pseudotyped virus assays may not always accurately determine neutralisation 34, 35 . live ebov and ebov gp pseudotyped neutralisation assays have previously been shown to yield variable results 8, 36 , which could be due to differing experimental conditions and viral systems. it is therefore important to optimise pseudotyped virus neutralisation assays in context of the particular viral gp being studied in order to obtain reliable specificity and sensitivity. the aim of this study was to assess the suitability of ebov gp pseudotyped hiv-1 and vsv systems to measure the neutralising ability of plasma from evd survivors, when compared to live ebov neutralisation. reporter activity was detected in all cell lines (293t/17, huh-7, hela and vero e6) infected with ebov gp (mayinga) pseudotyped hiv-1 and vsv, demonstrating the broad tissue range conferred by ebov gp, although differences in luminescence were observed. this may reflect general defects in viral entry in different cells. a relatively lower level of ebov gp pseudotyped hiv-1 transduction was exhibited by vero e6 cells, which might be due to an intrinsic restriction factor, trim5α, which restricts retroviral infection by specifically recognising the hiv-1 capsid and promoting its rapid, premature disassembly 37 . highest tcid 50 values were obtained following ebov gp pseudotyped hiv-1 and vsv infection of 293t/17 and vero e6 cells, respectively. there seemed to be large variability of the luminescent measurement for the vsv-based platform, which may be caused by the www.nature.com/scientificreports/ sensitive nature of the luciferase signal detection. this highlights the importance of titrating each pseudotyped virus batch before use in neutralisation assays, and the inclusion of multiple replicates. the ebov gp pseudotyped viruses were used to assess the neutralising activity of a human anti-ebov gp mab, kz52. kz52 has been shown previously to neutralise ebov pseudotyped viruses 17, 19, 38 . however, within the range tested here, kz52 did not display neutralisation against ebov gp pseudotyped hiv-1, suggesting that the ebov gp on the pseudotyped hiv-1 might be at higher levels, thereby reducing assay sensitivity, and neutralisation may be observed using a higher concentration of kz52. in contrast, kz52 was able to neutralise the ebov gp pseudotyped vsv. to assess the effects of differing amounts of pseudotyped virus input on neutralisation, plasma from an evd survivor of the 2013-2016 ebov outbreak and kz52 were screened against different amounts of the ebov gp pseudotyped hiv-1 and vsv, respectively. decreasing quantities of pseudotyped hiv-1 led to more variable and unreliable results, and the kz52 ic 50 of pseudotyped virus neutralisation decreased with decreasing amounts of ebov gp pseudotyped vsv input. the variability in neutralisation observed between different amounts of pseudotyped virus input highlights the importance of including standards or reference material with a known activity or potency when comparing neutralising activity, allowing calibration of results 39 . both pseudotyped virus systems were able to measure neutralising antibodies in plasma from evd convalescent patients, and results correlated positively with a live ebov neutralisation assay. however, the discriminatory power of the hiv-1-based assay with regards to differing antibody titres appeared to be low. some of the samples tested, which showed neutralising activity against live ebov, did not display neutralisation against the pseudotyped virus and vice versa, therefore raising questions on the sensitivity and specificity of the pseudotyped hiv-1 assay. in the current study, human embryonic kidney (293t/17) cells were used for the pseudotyped hiv-1 neutralisation assays, whereas african green monkey kidney (vero) cells were used in the vsv-based assay and also the live ebov assay. therefore, this could account for some of the differences in results observed between the two assays and for the better performance of the vsv-based assay in relation to live ebov neutralisation. also, the hiv-1-and vsv-based pseudotyped virus systems assessed in the current study utilise different transfection methods, which could have implications on the composition of the pseudotyped viruses, density and/or glycosylation of the viral envelope protein on the surface, and consequently neutralisation results. this highlights the importance of assessing experimental conditions and methodology when developing and optimising pseudotyped virus neutralisation assays. a limitation to this study was that the level of ebov gp incorporation per pseudotyped virus type could not be assessed. also, for the vsv-based pseudotyped virus system, traces of vsv-g from the rvsv-δg-luc-vsv-g virus could be recycled into newly pseudotyped virions 40 . therefore, the use of anti-vsv-g hybridoma cell culture supernatant could give rise to pseudotyped virions covered by anti-vsv-g antibodies, but are still infectious due to ebola gp. this could potentially induce plasma specific reactivity of virions due to bound anti-vsv-g antibodies more than ebov gp specific reactivity. there are several differences between ebov gp pseudotyped and live ebov neutralisation assays that could affect their results 8 . due to their non-replicating nature, such pseudotype systems do not recapitulate all steps in the viral life cycle that may potentially be targeted by neutralising antibodies 41 . in addition, the round, spherical shape of ebov gp pseudotyped hiv-1 or bullet shape of ebov gp pseudotyped vsv compared to the filamentous shape of authentic ebov could affect their susceptibility to neutralisation. also, the density of gp on the surface of the pseudotyped virus may not be the same as that found on live ebov and may result in the loss or masking of quaternary epitopes 11, 42 . furthermore, gp maturation and assembly in live ebov could be different in the generation of an ebov pseudotyped virus and may result in different targets and/or conformational epitopes when using whole live ebov as opposed to ebov gp alone in a pseudotyped virus. the presence of shed gp or secreted gp (sgp) in the live ebov assay compared to absence in the ebov gp pseudotyped virus assays could also have an effect on neutralisation. in the live ebov assay, shed gp and sgp could reduce neutralisation of circulating virus by cross-reactive antibodies to surface gp. however, in the current study, weaker relative neutralisation was observed in the hiv-1 based pseudotyped virus assay. therefore, it is possible that cell debris or free gp generated during ebov gp pseudotyped hiv-1 production by polyethylenimine (pei) transfection could be interfering with neutralisation. finally, detection of infected cells via measurement of luminescence in the ebov gp pseudotyped virus neutralisation assay compared to plaque formation in the live ebov neutralisation assay could affect neutralisation readout. ebov gp pseudotyped virus neutralisation assays have value for vaccine evaluation and assessment of convalescent blood products and mabs for use as immunotherapeutics. however, pseudotyped virus assays may not always accurately determine neutralisation when compared with neutralisation against live virus. in this study, both ebov gp pseudotyped hiv-1 and vsv assays were able to detect neutralisation of plasma from evd survivors and correlated positively with live ebov neutralisation. however, the vsv-based assay performed better than the hiv-1-based assay in relation to specificity, sensitivity, and correlation with the live ebov neutralisation assay. this research has highlighted the importance of optimising pseudotyped virus neutralisation assays in context of the particular viral gp being studied, especially when evaluating vaccine responses and therapeutics, and could provide a better understanding of the correlates of protection against ebov. and from negative control blood donors in the uk and guinea, who were not knowingly exposed to persons with evd and did not attend high risk events such as funerals, were heat inactivated at 56 °c for 30 min. the samples were obtained from a pre-existing biobank, for which live ebov neutralisation 45 production of pseudotyped viruses. the generation of hiv-1 pseudotyped viruses was performed as detailed previously 44, 46, 47 . twenty-four hours prior to transfection, approximately 8 × 10 5 293t/17 cells were seeded into sterile, 6-well cell culture plates (corning, ewloe, uk) and incubated at 37 °c, 5% co 2 and 95% humidity until 60-80% confluence. the hiv gag-pol plasmid, p8.91, and the firefly luciferase reporter construct, pcsflw, were transfected simultaneously with the ebov (mayinga) gp expression vector at a ratio of 0.6:0.9:0.6 µg (core:reporter:envelope) using 10 µl of 1 µg/ml polyethylenimine (pei) (sigma-aldrich) per 1 µg dna in opti-mem medium (gibco). following overnight transfection, the cells were incubated with fresh medium and incubated at 37 °c, 5% co 2 . pseudotyped virus supernatants were harvested at 48 and 72 h posttransfection, passed through a 0.45 µm pore filter (millex, millipore, watford, uk) and stored at − 80 °c. ebov gp pseudotyped vsvs were prepared using recombinant vsv, in which the vsv-g gene had been deleted (rvsv-δg) and replaced with a luciferase reporter gene (rvsv-δg-luc) by a method similar to that described previously 48 . twenty-four hours prior to transfection, approximately 2.4 × 10 6 293t/17 cells were seeded into sterile, 100 mm cell culture dishes (corning) and incubated at 37 °c, 5% co 2 and 95% humidity until 60-80% confluence. the cells were transfected with the ebov gp expression vectors using transit-lt1 transfection reagent (mirus bio, madison, wisconsin (wi), usa) as per the manufacturer's instructions. following overnight transfection, the medium was removed and the cells were infected with rvsv-δg-luc that was pseudotyped with the vsv glycoprotein (rvsv-δg-luc-vsv-g) (masayuki saijo, national institute of infectious diseases, tokyo, japan) at a multiplicity of infection (moi) of 5 in opti-mem medium and incubated at 37 °c, 5% co 2 . after 2 h, the inoculum was removed, cells were washed twice with dulbecco's phosphate buffered saline (dpbs) (gibco) and fresh medium was added. pseudotyped virus supernatants were harvested at 18-24 h post-infection, clarified twice by centrifugation at 200xg for 5 min at 10 °c and stored at − 80 °c. prior to use, the pseudotyped viruses were incubated with anti-vsv-g hybridoma cell culture supernatant (masayuki saijo, national institute of infectious diseases, tokyo, japan) at a 1:125 dilution for 1 h at 37 °c to reduce background infection mediated by residual virus possessing vsv-g, which can be carried over during preparation 48 . all experiments involving pseudotyped viruses were performed in a cl2 facility at public health england (phe), porton down, uk. pseudotyped virus titration and neutralisation assays. titration and neutralisation assays were performed in 96-well solid white flat bottom polystyrene tc-treated microplates (corning) and were based upon previously described protocols 44, 46, 48 . for pseudotyped hiv-1 titration assays, five-fold serial dilutions of pseudotyped virus at a starting dilution of 1:5 were prepared in quadruplicate in opti-mem medium at a final volume of 100 µl/well. 100 µl of approximately 2 × 10 4 293t/17, huh-7 or vero e6 cells, or 1 × 10 4 hela cells were then added to each well and incubated at 37 °c, 5% co 2 for 48 h. the medium was removed and 50 µl of a 50:50 mix of bright-glo luciferase assay reagent (promega, southampton, uk):fresh medium was added to each well and incubated for at least 2 min at room temperature to allow complete cell lysis. luminescence was measured using a glomax-multi + detection system luminometer (promega) and relative luminescence units per ml (rlu/ml) were determined. the negative cut-off was set at 2.5 times the average rlus of the cells only control wells. 50% tissue culture infectious dose (tcid 50 )/ml values were determined using the reed-muench method 49 . for the pseudotyped hiv-1 neutralisation assay, two or threefold serial dilutions of plasma samples at a starting dilution of 1:5 or 1:10, respectively, were prepared in duplicate in opti-mem medium at a final volume of 50 µl/well and incubated with 50 µl of a standardised rlu per well of pseudotyped virus (as calculated from the titration assay), prepared in opti-mem medium, for 1 h at 37 °c. 100 µl of approximately 2 × 10 5 293t/17 cells were then added to each well and incubated for 48 h at 37 °c, 5% co 2 , prior to taking a chemiluminescent readout as described above. infectivity was calculated using the formula: percentage (%) infectivity = [(rlu with sample)/(rlu without sample)] × 100. www.nature.com/scientificreports/ for pseudotyped vsv titration assays, 24 h prior, approximately 2.5 × 10 4 293t/17 or 1 × 10 4 huh-7, hela cells or vero e6 cells were seeded in 96-well microplates and incubated at 37 °c, 5% co 2 and 95% humidity. the medium was removed and two-fold serial dilutions of pseudotyped virus in opti-mem medium, starting with neat pseudotyped virus were added to each well in quadruplicate at a final volume of 100 µl/well. after 24 h, a chemiluminescent readout was taken and tcid 50 /ml values were determined as described above. twenty-four hours prior to pseudotyped vsv neutralisation, approximately 1 × 10 4 vero e6 cells were seeded and incubated as for titration above. twofold serial dilutions of plasma samples at a starting dilution of 1:10 were prepared in duplicate in opti-mem medium at a final volume of 120 µl/well in 96-well microplates, and incubated with 120 µl of a standardised rlu per well of pseudotyped virus (as calculated from the titration assay), prepared in opti-mem medium, for 1 h at 37 °c. the medium was removed from the cells, 50 µl of the plasma-pseudotyped virus mixtures were added to each well in quadruplicate at incubated at 37 °c, 5% co 2 . after 1 h, 50 µl of fresh medium was added to each well. luminescence was measured after 24 h and infectivity was calculated as described above. statistical analysis. pseudotyped virus neutralisation assay raw data were normalised as percentage (%) infection relative to mean values for pseudotyped virus only controls (equivalent to 100% infection), then ic 50 of pseudotyped virus neutralisation were estimated by model of nonlinear regression fit with settings for log (inhibitor) vs. normalised response curves using graphpad prism v5 (san diego, california (ca), usa). statistical comparison between two unpaired groups was performed using the mann-whitney test (graph-pad prism v5). correlation between two variables was quantified using spearman nonparametric correlation (graphpad prism v5). www.nature.com/scientificreports/ open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/4.0/. ebola haemorrhagic fever emergence of zaire ebola virus disease in guinea world health organization. situation report -10 biochemical analysis of the secreted and virion glycoproteins of ebola virus a mutation in the ebola virus envelope glycoprotein restricts viral entry in a host species-and cell-type-specific manner characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines antibody-mediated neutralization of ebola virus can occur by two distinct mechanisms systematic analysis of monoclonal antibodies against ebola virus gp defines features that contribute to protection role of antibodies in protection against ebola virus in nonhuman primates immunized with three vaccine platforms the use of pseudotypes to study viruses, virus sero-epidemiology and vaccination current progress with serological assays for exotic emerging/re-emerging viruses construction and use of a human immunodeficiency virus vector for analysis of virus infectivity a system for functional analysis of ebola virus glycoprotein distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses immune protection of nonhuman primates against ebola virus with single low-dose adenovirus vectors encoding modified gps specific neutralizing response in plasma from convalescent patients of ebola virus disease against the west africa makona variant of ebola virus ebola virus neutralizing antibodies detectable in survivors of the yambuku, zaire outbreak 40 years after infection mechanism of binding to ebola virus glycoprotein by the zmapp, zmab, and mb-003 cocktail antibodies protective monotherapy against lethal ebola virus infection by a potently neutralizing antibody potent neutralizing monoclonal antibodies against ebola virus infection technical considerations for the generation of novel pseudotyped viruses ebola virus can be effectively neutralized by antibody produced in natural human infection pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody structure of the ebola virus glycoprotein bound to an antibody from a human survivor longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to h5n1 influenza viruses characterization of lentiviral pseudotypes with influenza h5n1 hemagglutinin and their performance in neutralization assays safe pseudovirus-based assay for neutralization antibodies against influenza a(h7n9) virus a robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in africa development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system development of a pseudotyped-lentiviral-vector-based neutralization assay for chikungunya virus infection high degree of correlation between ebola virus bsl-4 neutralization assays and pseudotyped vsv bsl-2 fluorescence reduction neutralization test human immunodeficiency virus type 1 env clones from acute and early subtype b infections for standardized assessments of vaccine-elicited neutralizing antibodies broadly neutralizing human monoclonal antibodies to the hepatitis c virus e2 glycoprotein comparison of platform technologies for assaying antibody to ebola virus specific recognition and accelerated uncoating of retroviral capsids by the trim5α restriction factor a shared structural solution for neutralizing ebola viruses ebola virus: pseudotypes, libraries and standards characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis c virus glycoproteins neutralizing antibodies inhibit chikungunya virus budding at the plasma membrane maturation of west nile virus modulates sensitivity to antibody-mediated neutralization multiply attenuated lentiviral vector achieves efficient gene delivery in vivo investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison phase 1 trials of rvsv ebola vaccine in africa and europe a sensitive retroviral pseudotype assay for influenza h5n1-neutralizing antibodies lyophilisation of influenza, rabies and marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation-assay based diagnostic kit generation of vsv pseudotypes using recombinant δg-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines a simple method of estimating fifty per cent endpoints the authors would like to thank masayuki saijo for providing the rvsv-δg-luc-vsv-g virus and anti-vsv-g hybridoma cell culture supernatant. we are grateful to eccac for providing vero e6 and hela cells, and to arvind patel for providing huh-7 cells. this work was funded by the u.s. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-71225 -1.correspondence and requests for materials should be addressed to m.w.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-334454-cqaado3u authors: leal, rodolfo oliveira; gil, solange title: the use of recombinant feline interferon omega therapy as an immune-modulator in cats naturally infected with feline immunodeficiency virus: new perspectives date: 2016-10-27 journal: vet sci doi: 10.3390/vetsci3040032 sha: doc_id: 334454 cord_uid: cqaado3u type i interferons (ifns) are well-known cytokines that, among their main functions, are key components of the host immune response against viral infections. due to its immune modulation properties, they are commonly used in the therapeutic approach of various retroviral infections, namely human immunodeficiency virus (hiv) and feline immunodeficiency virus (fiv). in hiv infection, it has been shown that ifn therapy limits early viral replication, particularly useful on post-exposure prophylaxis. in veterinary medicine, recombinant feline interferon omega (rfeifn-ω) was the first interferon licensed for use in cats. several studies have recently shown that this compound seems to stimulate the innate immunity, decreasing clinical signs and co-infections in naturally fiv-infected cats. more than summarizing the main conclusions about rfeifn-ω in cats, this review emphasizes the immune-modulation properties of ifn therapy, opening new perspectives for its use in retroviral infections. either in fiv-infected cats or in hiv individuals, type i ifns seem to induce an innate immune-modulation and should not be overlooked as a therapeutic option in retroviral infections. in clinical practice, the initial therapeutic approach in cats suspected of retroviral infections is always supportive and symptomatic. when the diagnosis of retroviral infection is established, antivirals and immune modulators can be considered on short-and long-term management. taking into account that most of these drugs are licensed for use in humans, there is a lack of well-controlled clinical trials in cats and their efficacy is not entirely clear [1, 2] . nowadays, there are no antivirals licensed for use in veterinary medicine with the exception of several immune modulators, which have concurrent antiviral properties. therefore, all the truly antiviral compounds used in dogs and cats are those licensed for use in humans, namely in hiv therapies [1] [2] [3] . on the other hand, interestingly, the administration of antivirals in cats has been frequently documented due to the fact that fiv-infected cats are commonly used as hiv experimental models. even if antivirals have shown a low efficacy in cats and can induce significant toxic effects, several of them are used on the management of retroviral infections [1, 2, 4] . among them, the rt inhibitors/nucleoside analogues are the most common. the majority are nucleoside analogues which, acting as anti-metabolites, are "false substrates" that bind to rt enzyme and block its activity [4] . other antivirals that can be used in retroviral infections are antagonists/homologous of receptors, namely byciclams such as plerixafor and amd 3100, which bind either to viruses (homologous) or to cell-receptors (antagonists), inhibiting the virus-cell interaction. with the exception of several drugs, these compounds are strongly selective for hiv, meaning that they are not used in veterinary medicine [4] . taking part in the group of "immune therapy", by definition, immune modulators are compounds that interfere with the immune system. they are commonly used in different clinical situations, particularly in canine and feline viral infections. it is believed that immune modulators restore several functions of the immune system, allowing a better management of opportunistic infections and a better clinical recovery. some of these compounds can even have a direct antiviral effect [4] . in between the well-known immune modulators, interferons (ifns) will be further discussed, mainly due to its current use in retroviral infections. interferons (ifns) are key components of the host immune system, being particularly relevant in viral infections [5] . the large family of ifns can be divided into different types. type i ifns are the most studied ones as they are commonly used for therapeutic purposes. among their major functions, type i ifns increase and sensitize the immune system towards the microbial recognition [6] , establishing an important link between innate and acquired immunity [7] . furthermore, they have different anti-viral properties, blocking viral replication and inducing apoptosis of infected cells [8] [9] [10] [11] . in human immunodeficiency virus (hiv), the therapeutic use of ifns has been assessed in various in vitro and in vivo studies. in vitro, it was proven that ifns are able to restrict hiv replication inducing an antiviral state and preventing viral infection [12, 13] . in vivo, despite the hiv's evasion mechanisms, ifns seem to play an important protective role against hiv infection in seronegative individuals exposed to the virus [14, 15] . moreover, its therapeutic administration showed a durable suppression of viral replication and decreased viral integration following anti-retroviral therapy in hiv individuals [16] . even if further studies are needed to investigate the therapeutic effects of ifn in hiv, its protective role on the early stages of infection aims to consider that ifn therapy seems particularly useful on post-exposure prophylaxis [15] . not only in humans but also in feline medicine, the use of type i ifns as immune modulation therapy is common, particularly in retroviral infections [17] [18] [19] . still used in several countries, huifn-α was the first interferon administered in cats, despite the fact that it is only licensed for human medicine [4] . among their major functions, it was shown that huifn-α inhibits oncogenic mutations induced by retroviruses, and it restrains viral nucleic acid synthesis and suppress protein production. due to these anti-viral and immune modulation properties, huifn-α is used with various human diseases such as myeloid leukemia, papilomatosis, and hiv [4] . for the same reasons, huifn-α has been also used in feline medicine, namely in fiv, felv, fhv-1, fcv, and fcov viral infections [20] [21] [22] [23] . in spite of its short-term effects, particularly on clinical improvement and the increase in survival time, the development of neutralizing antibodies several weeks after therapy makes huifn-α ineffective for long-term immune modulation therapy in cats [4, 19, 21] . this problem was bypassed by the more recent release of recombinant feline interferon omega (rfeifn-ω). rfeifn-ω is the first interferon compound licensed for use in veterinary medicine. specifically in cats, this product is approved for use in retroviral infections, namely feline immunodeficiency virus (fiv) and feline leukemia virus (felv). according to the manufacturer's instructions and license, it should be administered in three cycles of five daily subcutaneous injections of 1 mu/kg, beginning respectively on days 0, 14, and 60. despite the fact that it was licensed a few years ago, only a few studies supported its clinical benefits, particularly in retroviral infections. the first study, which described its clinical application dates from 2004 and reported that treated feline leukemia virus (felv) and fiv/felv co-infected cats, showed a significant clinical improvement and an increased survival time [18] . more recently, in 2011, another research group reinforced that rfeifn-ω improves clinical signs and hematologic parameters in retrovirally infected cats. however, according to this study, it does not change hypergammaglobulinemia, proviral load, or viremia, suggesting an overall effect mainly on the innate immune reaction rather than on the acquired immunity [17] . this means that rfeifn-ω does not seem to have an anti-viral effect in vivo, and the observed clinical improvement must be related to a potential immune modulation of the innate immune system [17] . moreover, in contrast to in vitro studies [24] , the anti-viral in vivo action of the rfeifn-ω licensed protocol towards fiv and felv was found to be negligible according to these authors [17] . these points highlight the need of further studies in order to clarify the mechanisms of action of rfeifn-ω. the authors tried to extend the current knowledge about this compound in order to support and clarify the evident clinical improvement previously described [17, 18] . experimental work relied on two distinct clinical trials [25] [26] [27] . one referred to 16 retrovirally infected cats (7 fiv, 6 felv, and 3 co-infected cats) treated with rfeifn-ω licensed subcutaneous protocol, while the other described an alternative oral rfeifn-ω protocol, administered to 11 fiv-infected cats [25, 26] . both protocols were compared in an immunological and virological perspective [25] [26] [27] . firstly, the effect of rfeifn-ω licensed protocol in cats living in an animal shelter was evaluated, assessing clinical improvement and monitoring concurrent viral excretion (namely herpesvirus, calicivirus, and coronavirus) [26] . a previous study in cats treated with huifn-α documented that the induced clinical improvement could be potentially related to a recovery of serious opportunistic infections [21] . in order to assess whether rfeifn-ω had the same properties, different hematology and biochemistry parameters were monitored, and a score-scale system that allowed the evaluation of clinical improvement in treated cats was validated. in agreement with the two previous referred studies [17, 18] , a significant clinical improvement in treated animals without relevant changes on hematology and biochemistry profiles was reported [26] . concerning these results, rfeifnω seems to be able to contribute to the management of this reality by improving clinical signs and decreasing concurrent viral excretion. therefore, rfeifnω must be considered as an effective immune-modulator therapy for use in shelter medicine where the prevalence of concomitant infections is higher. in further studies, the influence of rfeifn-ω in the acute phase reaction was investigated [28] . in human medicine, several studies have described its increase in hiv-positive patients [29, 30] , even after immunomodulation therapy with exogenous il-2 [31] . despite the similarity between hiv and fiv [2, 32] , the crp behavior in fiv-positive cats undergoing immune-modulation therapy remains unknown. following previous conclusions suggesting that this compound acts on the innate response rather than on the acquired immune system, its effect on acute phase proteins (apps) was evaluated [17] . taking into account that apps are one of the key components of the innate immune system, they seemed reasonable predictors of an innate immune-stimulation [33] . authors showed that apps significantly increased in cats treated with the licensed rfeifn-ω protocol [28] . despite the fact that apps usually increase in different situations such as chronic infection and severe inflammation [33, 34] , in this study, their increment was concomitant with the described clinical improvement and reduction in opportunistic infections. this work showed that the app increment might be beneficial in retrovirally infected cats, confirming that they can be a reasonable indicator of a potentiated innate immune response. being the first work documenting the effect of rfeifn-ω on app profiles, this study reinforces the action of this compound on the innate immune system, helping to clarify the mechanisms of action of the licensed protocol [28] . in order to explore in greater detail these conclusions, the evaluation of other innate immunity biomarkers (namely mx protein) would be useful. although its use in feline medicine is scarce, mx protein is a specific type i ifn biomarker [8, 35] ; hence, its production is directly related with the activation of a type i ifn signal transduction pathway. in this sense, from the authors' point of view, further studies are needed to strengthen these conclusions and to assess whether mx protein can be a reasonable biomarker of innate immunity in cats under rfeifn-ω therapy. recognizing that, in clinical practice, rfeifn-ω is very often a cost-limiting therapy, the use of an alternative oral protocol in fiv-infected cats has also been documented [25] . in fact, different authors reported the clinical benefits of oral low dose huifn-α protocols in viral infections such as fiv [19, 21] . after the release of rfeifn-ω, several clinical trials had also been conducted in specific-pathogen free cats and calicivirus-positive cats in order to study the immune-modulation properties of oral rfeifn-ω [8, 36] . however, no studies have documented its use in retrovirally infected cats, particularly in fiv-infected ones. in this sense, an oral rfeifn-ω protocol was developed and administered to 11 client-owned fiv-infected cats [25] . the oral dose was based on the referred previous studies and was decided according to the manufacturer's previous trials [8, 36] . the oral protocol was 10-fold lower that the licensed subcutaneous protocol, and it was given orally instead of subcutaneously and for a longer period of time (90 continuous days). following the same methodology of previous studies, the clinical improvement, concurrent viral excretion, app profiles, and different hematology and biochemistry parameters in fiv-infected cats treated with the oral protocol were assessed. similarly to what was observed for the licensed protocol, cats treated with oral rfeifn-ω showed a significant clinical improvement without remarkable changes in hematology and biochemistry profiles, which were within the reference range in the majority of the treated cats [25] . conversely, mainly due to the fact that client-owned cats were less prone to opportunistic infections, concurrent viral excretion was very low and did not change with the applied protocol. additionally, app profiles did not change in cats treated with oral rfeifn-ω, meaning that the two distinct protocols seem to act differently. taking apps as biomarkers of the innate immune response, it seems reasonable to say that innate immune reaction does not seem to be potentiated in the same way as observed in the licensed protocol. however, as interferons have pleotropic effects and apps are only a part of the innate immune response, it is overspeculative to assume that oral rfeifn-ω does not interfere with innate immunity. even if apps did not change, overall results showed an important clinical improvement and reinforced the potential extra-label use of rfeifn-ω in an oral continuous low-dose protocol. for a second time, the effect of both protocols of rfeifn-ω on cytokine profile, viremia, and proviral load was evaluated [27] . only one study had previously reported that the licensed protocol does not change viremia or proviral load in treated fiv-infected cats, suggesting that this compound may not act on acquired immunity [17] . in agreement with these results, authors concluded that viremia did not change in the group of fiv-infected cats treated with the licensed protocol [27] . however, in opposition to what was previously reported, a significant increase on proviral load was reported and correlated to a relative increase of lymphocytes cell count (even within the normal range) [27] . in this study, viremia and proviral load changes in fiv-infected cats treated with oral rfeifn-ω were also evaluated. as expected, no changes were obtained in either parameter, which reinforced the previous suggestion that, independently of the administered protocol, the rfeifn-ω's anti-viral effect in vivo is negligible. at this point, it was concluded that rfeifn-ω licensed protocol induced a decrease in concurrent viral infections, even if there were no true changes in fiv viral load. on the other hand, in the oral protocol, albeit no changes on the viral load and concurrent viral infections, there was a significant clinical improvement in treated cats. therefore, even if the antiviral properties against fiv seem negligible in both protocols, the fact that there was a decrease in concurrent viral infections in cats treated with the licensed protocol raises the question of whether there is a true antiviral effect towards other viruses or whether this is the result of an overall stimulation of the innate and acquired immune system. from the authors' point of view, it is overspeculative to state that rfeifn-ω does not act on the acquired immune system only based on viremia and proviral load. therefore, an evaluation of cytokine profiles in these animals seemed essential to fully understand the actions of rfeifn-ω. in order to reinforce the previous suggestions that rfeifn-ω does not affect the acquired immune system of fiv-infected cats, mrna expression and concurrent plasma levels of various cytokines were monitored using biological samples from the two groups of fiv-infected cats treated with either subcutaneous or oral rfeifn-ω protocols [27] . despite its pleiotropic effect, variations of th-1 and th-2 responses were assessed based on different measured cytokines. results showed that th-1 and th-2 responses did not significantly change in either protocol, which supported the previous suggestions that rfeifn-ω does not strongly affect the acquired immune system. among the measured cytokines, only il-6 (a pro-inflammatory cytokine involved in different immune pathways and particularly in the innate immune response) significantly changed in both groups. in fact, in cats treated with the licensed protocol, il-6 plasma levels were significantly reduced, whilst its respective mrna expression showed a decreasing tendency that was not statistically significant. on the other hand, in cats treated with oral rfeifn-ω, il-6 plasma levels did not change, but the concurrent mrna expression significantly decreased. all in all, it was documented that il-6 production is affected in both protocols, meaning that rfeifn-ω has anti-inflammatory properties. moreover, considering that plasma changes were only significant in cats treated with the licensed protocol, it seems reasonable to state that this higher pulsate therapeutic scheme is more efficient in reducing the pro-inflammatory stimuli than the continuous low dose therapy. these were the first studies exploring the effect of rfeifn-ω in different biomarkers of the immune system. however, there are still many points to investigate in order to explore the whole potential of this compound in retroviral infections. in fiv-infected cats, only two protocols were assessed. following the same research line, it would be interesting to reinforce these conclusions with pharmacokinetic and pharmacodynamic studies. in the subcutaneous protocol, it would be useful to document the effect of lower doses but in longer therapy cycles and vice-versa, in order to correlate the therapy requirements with clinical and virological conditions. for the oral group, it would also be interesting to assess the effect of intermediate higher doses for shorter periods and also to extend the protocol to felv-infected cats. in spite of the different physiopathology and the aggressive clinical portrait, it would be interesting to assess the effect of the suggested oral rfeifn-ω in these animals. according to the authors' point of view, it is unlikely that such a lower oral dose will have the same effect on clinical improvement of felv-infected cats since these animals are usually more symptomatic and in worse overall clinical condition than fiv-infected animals. consequently, the higher subcutaneous protocol will always be preferable in these cats. as previously stated, several studies have shown that ifns can be beneficial in the early stages of hiv infection [15] . recognizing that these results concern naturally infected cats in which the time of infection is unknown, these findings are in some way in agreement with these hiv studies as they indirectly reflect an innate immune-modulation and decrease in clinical signs and co-infections. compared with exploring the full potential of each route and dose, it would be interesting to study a combination of protocols to be used according to different clinical presentations. although there are no studies assessing a possible combination of both protocols, we think that they can be reasonably associated, as both therapies are well documented and do not show important adverse effects. for instance, in symptomatic animals, an initial subcutaneous protocol followed by an oral continuous lower-dose therapy can now be recommended. from the authors' point of view, this seems a reasonable approach that sooner or later can be considered in routine clinical practice. also regarding the follow-up and life span, studies are warranted. in fact, some authors have tried to perform monthly to trimester follow-ups after the end of therapy. in both groups, it was unsuccessful. in the animal shelter, clinical evaluations after therapy were unreasonable once the income and outcome of cats impaired correct conclusions. regarding client-owned cats, it was only correctly achieved in a small number of treated animals (unpublished data). in fact, the individual oral therapy for each cat was free of charge and comprised a total of 90 daily doses that were partially given to the owners at each evaluation time point. after the end of the therapy, the monitoring was unpractical once the majority of owners were unavailable to pursuit with monthly follow-ups. consequently, a structured prospective study would be interesting in order to evaluate not only the long-term benefits of therapy but also any effect on the mean life span of fiv-infected cats. these studies innovated in the extension of the main therapeutic properties of the licensed rfeifn-ω protocol in naturally retrovirally infected cats. it is now documented that it must be used not only in client-owned symptomatic cats but also in animals living in catteries or shelters where opportunistic infections are problematic. although without a truly effect on the cytokine profile, this compound induces a significant clinical improvement and seems to reduce the pro-inflammatory stimuli. particularly, for fiv-infected cats, this work presents a new oral rfeifn-ω protocol, which was successfully tested and validated. although it induces a significant clinical improvement, its overall action as an immune modulator is less than the subcutaneous protocol. in fact, it slightly decreases the pro-inflammatory stimuli without affecting the acquired immunity, or even other parameters of the innate response such as acute phase proteins. therefore, whilst the high pulsate subcutaneous protocol is strongly recommended for symptomatic fiv-infected cats, this lower continuous oral protocol is a good alternative for cats presenting mild clinical signs, for cases where cost limits the use of licensed protocol or even for cats, which previously received subcutaneous rfeifn-ω and require continuous immune modulation therapy. these studies did not contribute to a better understanding of rfeifn-ω as much as they explored its immune modulation properties and validated a new oral protocol, which can be included in future fiv-guidelines. in summary, this review reinforces the beneficial properties of ifn therapy opening new perspectives for its use in retroviral infections. even if rfeifn-ω is a species-specific compound, these conclusions can be extrapolated to a larger perspective. either in fiv-infected cats or in hiv individuals, type i ifns seem to induce an innate immune-modulation and should not be overlooked as a therapeutic option in retroviral infections. clinical aspects of feline retroviruses: a review clinical aspects of feline immunodeficiency and feline leukemia virus infection efficacy of antiviral chemotherapy for retrovirus-infected cats: what does the current literature tell us? chapter 2: antiviral and immmunomodulatory chemotherapy interferon-inducible antiviral effectors ifn-alpha regulates tlr-dependent gene expression of ifn-alpha, ifn-beta, il-28, and il-29 plasmacytoid dendritic cells in immunity activity of feline interferon-omega after ocular or oral administration in cats as indicated by mx protein expression in conjunctival and white blood cells interferons: cell signalling, immune modulation, antiviral response and virus countermeasures type i interferon responses in rhesus macaques prevent siv infection and slow disease progression hiv-1 and interferons: who's interfering with whom? apobec3g upregulation by alpha interferon restricts human immunodeficiency virus type 1 infection in human peripheral plasmacytoid dendritic cells hiv replication can be blocked by recombinant human interferon beta cohorts for the study of hiv-1-exposed but uninfected individuals: benefits and limitations interferon responses in hiv infection: from protection to disease pegylated interferon alfa-2a monotherapy results in suppression of hiv type 1 replication and decreased cell-associated hiv dna integration use of recombinant interferon omega in feline retrovirosis: from theory to practice therapeutic effects of recombinant feline interferon-omega on feline leukemia virus (felv)-infected and felv/feline immunodeficiency virus (fiv)-coinfected symptomatic cats immunomodulation and therapeutic effects of the oral use of interferon-alpha: mechanism of action susceptibility of feline herpesvirus 1 and a feline calicivirus to feline interferon and recombinant human leukocyte interferons low-dose interferon-alpha treatment for feline immunodeficiency virus infection inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro alpha interferon (2b) in combination with zidovudine for the treatment of presymptomatic feline leukemia virus-induced immunodeficiency syndrome antiviral potency of interferon omega against selected canine and feline viruses oral recombinant feline interferon-omega as an alternative immune modulation therapy in fiv positive cats: clinical and laboratory evaluation relevance of feline interferon omega for clinical improvement and reduction of concurrent viral excretion in retrovirus infected cats from a rescue shelter evaluation of viremia, proviral load and cytokine profile in naturally feline immunodeficiency virus infected cats treated with two different protocols of recombinant feline interferon omega monitoring acute phase proteins in retrovirus infected cats undergoing feline interferon-ω therapy the acute-phase protein response to human immunodeficiency virus infection in human subjects lipid and acute-phase protein alterations in hiv-1 infected patients in the early stages of infection: correlation with cd4+ lymphocytes. braz changes in the levels of some acute-phase proteins in human immunodeficiency virus-1 infected patients, following interleukin-2 treatment feline immunodeficiency. abcd guidelines on prevention and management acute phase proteins in dogs and cats: current knowledge and future perspectives the feline acute phase reaction the innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation comparative efficacy of a recombinant feline interferon omega in refractory cases of calicivirus-positive cats with caudal stomatitis: a randomised, multi-centre, controlled, double-blind study in 39 cats the research work that conducted to the mentioned conclusions was part of the phd project of rodolfo oliveira leal. this project was kindly sponsored by fct (fundação para a ciência e tecnologia-phd grant bd/62917/2009) and ciisa (centro de investigação interdisciplinar em sanidade animal) of the fmv-ulisboa and virbac company. author contributions: this review was entirely written by rodolfo oliveira leal and solange gil and was based on the recent conclusions of rfeifnomega studies. both authors contributed equally to the writing of this review. the authors declare no conflict of interest. virbac did not interfere with the presented conclusions. the company only sponsored the above mentioned research work in a practical manner by donating rfeifn-ω (virbagen) which was administered to fiv cats. key: cord-334104-mphz1aye authors: apisarnthanarak, anucha; mundy, linda m. title: etiology of community-acquired pneumonia date: 2005-03-28 journal: clin chest med doi: 10.1016/j.ccm.2004.10.016 sha: doc_id: 334104 cord_uid: mphz1aye community-acquired pneumonia (cap) is a serious lower respiratory tract infection associated with significant morbidity and mortality that is characterized by disputes over diagnostic evaluations and therapeutic decisions. with the widespread use of broad-spectrum antimicrobial agents and the increasing number of immunocompromised hosts, the etiology and the drug resistance patterns of pathogens responsible for cap have changed. streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis remain the leading causes of cap in immunocompetent patients. opportunistic infections with organisms such as pneumocystis jiroveci and mycobacterium tuberculosis and other opportunistic fungal pneumonias should also be considered in the differential diagnosis of cap in immunocompromised patients. this article examines the current peer-reviewed literature on etiology, risk factors, and outcomes of patients with cap. at least six important host defense mechanisms are important in the prevention of cap: aerodynamic filtration, the cough reflex, mucociliary transport, phagocytic cell function, immunologic function, and the clearance of pulmonary secretions. deficiencies in normal host defense mechanisms influence the epidemiology, risk factors, and outcomes for cap [5] . the etiology of cap can be defined broadly into typical pathogens (eg, s pneumoniae, h influenza, and moraxella catarrhalis), atypical pathogens (eg, legionella spp, m pneumoniae, c pneumoniae), viruses, aspiration, and other agents. the etiologic agents and risk factors of common cap pathogens for immunocompetent hosts are summarized in table 1 . in immunocompromised hosts, in addition to the usual pathogens, the etiology of cap also includes opportunistic infections, such as mycobacterium tuberculosis, pneumocystis jiroveci pneumonia (pcp), and other opportunistic fungal infections ( table 2 ). coexisting pulmonary pathogens should be considered when clinical isolates have been detected, when patients lack clinical improvement, and when patients have clinical deterioration despite seemingly appropriate treatment. epidemiology s pneumoniae is among the leading infectious causes of illness and death from cap worldwide for young children, for persons who have underlying 0272 chronic systemic conditions, and for the elderly. the pneumococcus had been identified in 5% to 11% of patients with cap treated on an ambulatory basis, in 5% to 43% of patients with cap who require hospitalization, and in 11% to 38% of patients with cap who require admission to an intensive care unit (icu) [4,6 -9] . in a meta-analysis of 122 reports of cap from 1966 through 1995, s pneumoniae accounted for the majority of over 7000 cases (66%) in which an etiologic diagnosis was made and for 66% of the lethal pneumonias [10] . in addition, the pneumococcus is the most common cause (60%) of bacteremic pneumonia [7] . specific risk factors for pneumococcal infection include dementia, seizure disorders, congestive heart failure, cerebrovascular disease, chronic obstructive pulmonary disease (copd), hiv, black race, overcrowding in institutions, and smoking [11 -12] . in the united states, nonsusceptibility to penicillin has increased significantly during the past decade [13] . frequently, rates of penicillin-resistant s pneumoniae (prsp) are in excess of 30% [14] . the minimal inhibitory concentrations (mics) for most strains with high-level resistance are 2 to 4 mg/ml. of concern, the increasing rate of penicillin resistance is associated with resistance to beta-lactams and other classes of antibiotics, such as macrolides, tetracycline, and trimethoprim-sulfamethoxazole [14] . with the increase in the rate of prsp, physicians should be aware of the changing epidemiology and microbiology of pneumococcal disease to provide appropriate empiric antimicrobial treatment. haemophilus influenzae, a fastidious gram-negative coccobacillus bacteria, is the third most common cause of cap identified in patients who require hospitalization [6] . h influenzae accounts for 2% to 12% of cap in patients treated on an ambulatory basis, 1% to 11% of cap in patients who require hospitalization, and 3% to 19% of pneumonic cases in nursing home residents [4,6 -9] . most h influenzae clinical isolates are nontypeable stains recovered from patients during the winter months [3] . in a metaanalysis of 33,148 patients from 127 studies, h influenzae was the cause in 844 (3%) patients [10] . most studies have shown a higher prevalence of h influenza pneumonia among patients with copd [15] . other risk factors include the use of antibiotics or oral steroids within the past 3 months [6] . currently, more than 30% of h influenzae isolates in canada have aminopenicillin resistance owing to b-lactamase production [16] . nearly all strains are susceptible to ceftriaxone and cefuroxime, yet more than 50% of b-lactamase -producing h influenza isolates display either intermediate or high-level or resistance to clarithromycin [17] . approximately 1% to 5% of healthy adults are colonized by m catarrhalis [18, 19] . adults with chronic lung disease have been reported to have higher rates of m catarrhalis respiratory tract colonization when compared with healthy adults [18] . a study of adult carrier rates of m catarrhalis showed differential rates by age, that is, 5% versus 27% for adults aged less and more than 60 years, respectively [19] . three separate but related clinical scenarios have been defined for m catarrhalis lower respiratory tract infections: (1) infection causing a copd exacerbation, (2) infection causing pneumonia, especially in an older adult, and (3) infection as a nosocomial respiratory tract pathogen [18] . m catarrhalis pneumonia occurs predominantly in the winter months and is responsible for 4% to 6% of nursing home -acquired pneumonia and 10% of cap in the elderly [3, 19] . most elderly patients who experience pneumonia owing to m catarrhalis have underlying cardiopulmonary disease, including copd, bronchiectasis, congestive heart failure, or predisposition to aspiration [20] . other predisposing conditions associated with m catarrhalis infection include corticosteroid therapy, diabetes mellitus and malignancies [18] . although m catarrhalis pneumonia causes a significant illness in elderly patients, fulminant pneumonia, pleural effusion, and empyema are uncommon [18] . most (90%) strains of m catarrhalis produce an inducible b-lactamase [21] . the b-lactamase is more active against penicillins than cephalosporins, and its activity is inhibited by b-lactamase inhibitors. these strains show an inoculum-dependent susceptibility to ampicillin; therefore, ampicillin should not be used for these strains regardless of the results of susceptibility testing. epidemiology pneumonia owing to s aureus accounts for 1% to 5% of patients with cap who require hospitalization and 2% to 26% of patients with nursing homeacquired pneumonia [4,6 -9] . clinical manifestations are similar to those with cap from other bacterial etiologies [22, 23] , although the mortality may be higher [23, 24] . radiologic patterns are protean, inclusive of cavity formations [23, 25] . methicillin-resistant s aureus (mrsa) remains more of a concern for hospital-acquired pneumonia (hap), although it has been reported as a cause in cap [23, 24] . the risk factors associated with s aureus pneumonia include advanced age, prolonged hospitalization, underlying lung disease, prior antibiotic therapy, and surgery or other invasive procedures [22, 24] . mrsa pneumonia tends to produce a significantly greater frequency of bacteremia and septic shock and may associated with higher mortality [22] . aerobic gram-negative pneumonia accounts for 1% to 7% of patients with cap who require hospitalization and 5% to 23% of hap and nursing home -acquired pneumonia [4,6 -9] . these pathogens also have been identified in severe cap; although rare, such cases often are rapidly progressive and may be fatal [26] . aerobic gram-negative pneumonia, especially with pseudomonas aeruginosa, is a prognostic indicator of mortality in patients with cap. most cases occur in patients with underlying diseases, such as malignancy, cystic fibrosis, aplastic anemia, and bronchiectasis [9] . environmental exposure to dusts containing metals such as iron have been associated with acinetobacter cap, whereas exposure to water aerosolization has been associated with p aeruginosa pneumonia [26] . the frequency of anaerobic infection among patients with cap is not known, because the methods required to obtain valid uncontaminated specimens for meaningful anaerobic culture have rarely been used. nonetheless, anaerobic bacteria are the most common etiologic agents of lung abscess and aspiration pneumonia, and these bacteria are relatively common isolates from empyemas [3, 27] . patients with anaerobic bacterial infection may also present with pneumonitis that is indistinguishable from other forms of bacterial pneumonia [28] . factors that predispose to anaerobic bacterial pneumonia include aspiration, infection of the gingival crevice (gingivitis), necrosis of tissue with abscess formation or bronchopulmonary fistula, infection complicating airway obstruction, and infection in a dependent pulmonary segment [27] . some studies have suggested that anaerobes may account for a substantial number of cases of cap that have these characteristic features [29] . epidemiology m pneumoniae is a common cause of respiratory tract infection in young adults, attributable for 17% to 37% of outpatient cap and 2% to 33% of patients with cap who require hospitalization [5 -9] . after an incubation period of 2 to 4 weeks, approximately 3% of patients have clinical and radiographic evidence of pneumonia. common symptoms include a prodromal period with fever, chills, headache, and sore throat followed by a dry nocturnal or productive cough of mucoid sputum that persists for 3 to 4 weeks [3] . extrapulmonary manifestations may include hemolytic anemia, nausea and vomiting, myocarditis, rash, and diverse neurologic syndromes. a possible clue to help diagnose m pneumoniae pneumonia is a history of contact with a person with a similar condition characterized by a long incubation period. currently, there is no reliable diagnostic test to detect m pneumoniae infection; therefore, macrolide or tetracycline-based therapy usually is empirical. the prevalence of pneumonia owing to c pneumoniae varies from year to year and within geographic settings. studies indicate that c pneumoniae is linked to 5% to 11% of patients with cap who are treated on an ambulatory basis, 5% to 18% of patients with cap who require hospitalization, and approximately 7% of patients with nursing homeacquired pneumonia [5 -9] . the clinical spectrum ranges from asymptomatic infection to life-threatening pneumonia. when seen in cap as one of two pathogens, the associated pathogen seems to influence the clinical course and outcome [30] . in c pneumoniae pneumonia, sore throat, hoarseness, and headache are important nonpneumonic symptoms; other findings include sinusitis, reactive airway disease, and empyema [3] . advanced age and several comorbidities are risk factors for hospitalization in patients with pneumonia owing to c pneumoniae [3] . the preferred diagnostic test is an assay of acute and convalescent specimens to detect a fourfold increase in antibody titers, with supporting evidence based on throat swab polymerase chain reaction or culture results [3, 31] . nevertheless, with the limitation and availability of diagnostic tests, treatment most often is empirical. legionella pneumophila and other legionella species legionella species are implicated in 2% to 6% of patients with cap who require hospitalization [7 -8] . although rare in immunocompetent adults younger than 30 years of age, legionellosis can be a major cause of lethal pneumonia, with mortality rates of 5% to 25% among immunocompetent hosts and substantially higher rates among immunosuppressed hosts [32] . clinical features of legionnaires' disease include high fever, hyponatremia, central nervous system manifestations, elevated lactate dehydrogenase levels, and the presence of severe disease [32] . epidemiologic risk factors for legionnaires' disease include recent travel with an overnight stay outside the home, recent repair of domestic plumbing, exposure to hot tub and whirlpool-type spas, renal or hepatic failure, diabetes, or systemic malignancy [33, 34] . in addition, increasing age, smoking, and compromised cell-mediated immunity are associated with legionnaires' disease [33] . diagnostic tests for legionnaires' disease include the urine antigen assay for l pneumophila serogroup 1 and culture on selective media, which detects all legionella strains but is technically demanding [3] . a wide variety of pathogens, such as m tuberculosis, fungi, viruses, nocardia, chlamydia psittaci, hantavirus, coxiella burnettii, p jiroveci, leptospira, and uncommon pathogens such as tularemia may be responsible for cap, depending on the patient's host defense system and exposures. physicians should consider epidemiologic risk factors in the diagnosis and treatment of cap, especially in patients who do not respond to a standard therapeutic regimen for cap (table 3) . immunocompromised patients, especially those with hiv infection, have increased risk for routine and unusual cap pathogens. the etiology of cap in immunocompromised patients, with a focus on hosts with hiv infection, has been summarized previously in table 2 . the etiology and epidemiology of cap in cancer and transplant patients is addressed elsewhere in this issue. bacterial pneumonia continues to be an important problem in patients with hiv infection. pneumonia remains the chief cause of hospitalization for patients with hiv/aids, even in the era of combination antiretroviral therapy (art) [35] . the incidence of bacterial pneumonia in hiv patients ranges from 2 to 19 cases per 100 patients/year [36 -38] . low cd4 cell counts, injection drug use, prior sinusitis, and prior lower respiratory tract bacterial infection are risk factors for bacterial pneumonia in patients with hiv infection [38, 39] , whereas trimethoprimsulfamethoxazole prophylaxis is associated with a lower risk of bacterial pneumonia [38, 40, 41] . several studies have confirmed bacterial pneumonia to be the most common pulmonary complication in patients with hiv/aids. s pneumoniae, s aureus, h influenzae, and m pneumoniae are the most frequent pathogens identified [5, 38, 42, 43] . in addition, there is a trend toward higher mortality and more frequent presentation of severe pneumonia caused by p aeruginosa, especially in patients with advanced hiv/aids [36, 44] . among the types of mycobacterial infections associated with hiv/aids, m tuberculosis is considered the most prevalent and important problem worldwide. the incidence of tuberculosis ranges from 1.4 to 2.2 cases per 100 person-years to 7.7 to 16.2 cases per 100 person-years, depending on the geographic location, prevalence rates of tuberculin test reactivity, and the demographic characteristics of the population [45, 46] . it is estimated that 6000 to 9000 new cases of tuberculosis occur annually in the united states in patients with hiv/aids [45, 47] . hiv-infected patients have markedly increased risks for primary infection, reactivation of tuberculosis, and second episodes of tuberculosis from exogenous reinfection [47] . most cases present as pulmonary infections, with case rates extraordinarily high among indigent patients and users of illicit drugs [47] . clinical studies have shown the detrimental effects of tuberculosis on the course of hiv infection, with a twofold higher mortality in dual-infected hosts when compared with hiv-infected patients without tuberculosis, independent of cd4 cell count [47] . the degree of immunosuppression is the most important predictor of survival in hiv-infected patients with tuberculosis [48] . notable nontuberculous mycobacterial infections in hiv-infected patients occur from m kansasii, m scrofulaceum, m terrae, m gordonae, m chelonae, m genavence, m xenopi, and m fortuitum. although rare, isolated pulmonary m avium complex (mac) infection has been reported in 20 patients with hiv infection [49] . with the advent of combination art, the incidence of opportunistic infections in hosts with hiv infection has substantially declined [50] . likewise, the incidence of opportunistic fungal infections is approximately 20% to 25% of the incidence seen in the mid-1990s. despite the decline in the incidence of opportunistic infections, fungal infections are still common in patients with advanced hiv disease. such infections occur in patients who are long-term nonpresenters, who are nonadherent to art, or who do not seek medical care [50, 51] . p jiroveci pneumonia (pcp) remains the most common opportunistic infection, whereas the incidence of pneumonia owing to cryptococcus neoformans, histoplasma capsulatum, coccidioides immitis, and penicillium marneffei varies greatly depending on the geographic location (see table 2 ) [50, 51] . as is true for tuberculous and nontuberculous mycobacterial infections, some cases of fungal pneumonias have been associated with the use of art and the subsequent immune restoration from aids [51] . because this syndrome may mask the clinical presentation of typical and atypical pneumonic infections, a high index of suspicion is necessary for early diagnosis. infections from cytomegalovirus, herpes simplex virus, respiratory syncytial virus, and influenza virus readily occur in patients with hiv/aids. the true incidence and prevalence of these pathogens as causes of cap in hiv-infected patients are unknown. in severely immunosuppressed hosts with pcp, coexisting pulmonary infection has been reported in as many as 40% of cases [52, 53] . in recent years, newer agents such as hantavirus, severe acute respiratory syndrome (sars), and avian influenza have been identified as causes of cap. although rare, an isolated case or outbreak from agents of bioterrorism is plausible (www.who.int and www.cdc.gov). pneumonic presentations of bioterrorist activity are likely attributed to bacillus anthracis, yersinia pestis, or francisella tularensis. any suspected case should be treated as an epidemiologic emergency. the local health department should be notified. because these agents may be transmittable from person to person, a high index of suspicion will lead to prompt diagnosis and proper treatment. initial assessment of all patients with cap should include a travel history, animal exposures, and occupational risk (see table 3 ). the growing list of etiologic agents associated with cap and the growing number of the at-risk population continue to challenge existing diagnostic modalities for this lower respiratory tract infection. physicians should be aware of unusual presentations of common causes of cap as well as common presentations of unusual causes of cap. empiric treatment remains the norm in patients with cap. community-acquired pneumonia the epidemiology of respiratory tract infections community-acquired pneumonia in adults: guidelines for management the role of atypical pathogens: mycoplasma pneumoniae, chlamydia pneumoniae, and legionella pneumophila in respiratory infection community-acquired pneumonia: impact of immune status canadian guidelines for the initial management of community acquired pneumonia: an evidence-based update by the canadian infectious diseases society and the canadian thoracic society community-acquired pneumonia requiring hospitalization: 5-year prospective study diagnosis of pneumonia by cultures, bacterial and viral antigen detection tests, and serology with special reference to antibodies against pneumococcal antigens severe community-acquired pneumonia: epidemiology and prognostic factors prognosis and outcomes of patients with community-acquired pneumonia an epidemic of pneumococcal disease in an overcrowded, inadequately ventilated jail cigarette smoking and invasive pneumococcal disease the prevalence of drug-resistant streptococcus pneumoniae in atlanta drug-resistant pathogens in community-and hospital-acquired pneumonia communityacquired pneumonia in chronic obstructive pulmonary disease: a spanish multicenter study comparison of oral cephalosporins and their activities against b-lactamase producing bacteria surveillance of antimicrobial resistant in streptococcus pneumoniae, haemophilus influenzae and moraxella catarrhalis in the united states in 1996 -1997 respiratory seasons lung infections. branhamella catarrhalis: epidemiological and clinical aspects of a human respiratory tract pathogen respiratory tract carrier rate of moraxella (branhamella) catarrhalis in adults and children and interpretations of the isolation of m catarrhalis from the sputum branhamella catarrhalis respiratory infections in vitro activity of 12 orally administered antimicrobial agents against four species of bacterial respiratory pathogens from us medical centers in 1992 and 1993 bacteremic pneumonia due to staphylococcus aureus: a comparison of disease caused by methicillin-resistant and methicillin-susceptible organisms gram-positive pneumonia clinical and epidemiologic investigations of nosocomial pulmonary infections caused by methicillin-resistant staphylococcus aureus pneumonia due to staphylococcus aureus infection pseudomonas aeruginosa community-acquired pneumonia in previously healthy adults: case report and review of the literature anaerobic bacterial infections of the lung and pleural space anaerobic bacterial pneumonitis diagnosis of bacterial pulmonary infections during quantitative protected catheter cultures obtained during bronchoscopy chlamydia pneumoniae (twar) diagnosis of chlamydia pneumoniae infection in patients with community-acquired pneumonia by polymerase chain reaction enzyme immunoassay surveillance for legionnaires' disease: risk factors for morbidity and mortality hot tub legionellosis: legionnaires' disease and pontiac fever after a point-source exposure to legionella pneumophila the impact of potent antiretroviral therapy on the characteristics of hospitalized patients with hiv infections communityacquired bacterial pneumonia in human immunodeficiency virus-infected patients community-acquired pneumonia in a cohort of former injection drug users with and without human immunodeficiency virus infection: incidence, etiologies and clinical aspects bacterial pneumonia in persons infected with the human immunodeficiency virus pyogenic bacterial pneumonia in human immunodeficiency virus-infected inpatients: a clinical, radiological, microbiological, and epidemiological study risk factors for community-acquired pneumonia among persons infected with human immunodeficiency virus a controlled trial of trimethoprim-sulfamethoxazole or aerosolized pentamidine for secondary prophylaxis of pneumocystis carinii pneumonia in patients with human immunodeficiency syndrome pulmonary complications of hiv infection: autopsy findings the etiology of community-acquired pneumonia at an urban public hospital: influence of human immunodeficiency virus infection and initial severity of illness pseudomonas spp complications in patients with hiv disease: an eight-year clinical and microbiology survey incidence of tuberculosis in the united states among hiv-infected persons: the pulmonary complications of hiv infection study group tuberculosis and hiv infection: a cohort study of incidence and susceptibility to tuberculosis drugs tuberculosis in patients with human immunodeficiency virus infection site of disease and opportunistic infection predict survival in hiv-associated tuberculosis isolated pulmonary mycobacterium avium complex infection in patients with human immunodeficiency virus infection: case reports and literature review epidemiology of human immunodeficiency virus-associated opportunistic infections in the united states in the era of highly active antiretroviral therapy treatment of acute cryptococcal disease dual pulmonary infection with mycobacterium tuberculosis and pneumocystis carinii in patients infected with human immunodeficiency virus trimetrexate in the treatment of pneumocystis carinii pneumonia in patients with acquired immunodeficiency syndrome key: cord-332610-t99l3zii authors: mayer, j.d. title: emerging diseases: overview date: 2008-08-26 journal: international encyclopedia of public health doi: 10.1016/b978-012373960-5.00453-6 sha: doc_id: 332610 cord_uid: t99l3zii emerging infectious diseases are diseases that are either new, are newly recognized, or are increasing in prevalence in new areas. resurgent diseases are also usually grouped in this category, as is antimicrobial resistance. these diseases have been given formal recognition in the past two decades, although a historical outlook demonstrates that the phenomenon has probably been persistent, although largely undetected, through recorded history. emergence has accelerated recently, driven by factors such as demographic change, land use change, increased rapidity and frequency of intercontinental transportation, and other mostly social trends. continued infectious disease emergence poses, and will continue to pose, significant challenges for public health and for basic science. emerging and re-emerging infectious diseases have been major features of contemporary societies. indeed, there is evidence that history has been characterized by the constant interplay of humans and pathogens (mcneill, 1977) . however, it is impossible to say when the terms 'emerging infection' or 'emerging infectious diseases' were first used to describe new infectious diseases, or diseases that meet the criteria that are described in this article. the belief in the 1960s that the threat of infectious diseases had been eliminated in developed countries was unfounded. a broader view of history would have demonstrated this. one possible reason for the optimism is that the 1960s was a decade of optimism in general. in the united states, social programs were instituted to address inequities; humankind had not only orbited the earth, but landed on the moon; the gains of science and technology were impressive; economic expansion was equally impressive; poliomyelitis had been all but eliminated in the united states; and the sense of 'control' was widespread. beyond the borders of the united states, however, in africa, asia, latin america, and elsewhere, malaria proved to be a huge challenge to life, although its prevalence was decreasing, and diarrheal diseases continued to take their toll, particularly among the young. transportation links created the potential for transmission of infection between tropical regions and developed countries such as the united states. the potential for new diseases to emerge in the united states was there, and it took just a few years until this happened, catching the medical and public health communities by surprise. in discussions of emergence, both 'emerging infections' and 'emerging infectious diseases' are commonly found. while the two are closely related, they are not synonymous. an infection does not necessarily represent a state of disease. 'infection' suggests that an agent (usually a microbe) has become resident in the host. usually that agent is replicating in the host. however, the host need not show any sign of disease, in the sense that it can conduct its normal activities without hindrance. 'disease' is a state in which the normal functioning of the host is impaired, and both signs and symptoms are present -indeed, they are what limit normal function. an infectious disease is therefore a disease that is due to a pathogen. appeared de novo, or are being experienced in a region with greater intensity, or for the first time. some authors have used a more specific definition of emerging to diseases and have specified five types of emerging diseases: (1) diseases that arise de novo, (2) diseases that are newly recognized, (3) diseases that have not previously existed in a specific area, (4) diseases that had not yet made a species jump to humans until the present, and (5) diseases that are increasing in prevalence. there are other definitions as well. the simplest definitions are frequently the most useful, and thus morse's definition will be used in this article. re-emerging infectious diseases are frequently thought of as being closely related phenomena to emerging infectious diseases. whereas emerging diseases denote diseases that are being experienced for the first time in a given location, re-emerging diseases are diseases that are reappearing in regions from which they have disappeared. usually eradication is due to deliberate efforts on the parts of government and public health agencies. for example, malaria control programs following the end of world war ii were instrumental in the elimination of malaria from some areas of the world, such as italy and spain. sometimes, malaria eradication was eliminated as part of multisector development programs. for example, the tennessee valley authority, created during the 1930s primarily for flood control, hydroelectric power, and economic development, also had an explicit aim of malaria control. this resulted in the drainage of most swamps, and the elimination of malaria from this part of the united states. just as malaria was disappearing from many regions in the 1950s, the next decade saw the resurgence of malaria, and the global prevalence of malaria has been increasing ever since. there are multiple reasons for this. these include anopheline spp. resistance to ddt, banning of ddt because of suspected environmental effects, and the development of resistance to chloroquine. malaria, then, is a re-emerging disease. another is tuberculosis. in many societies, tb had been nearly eliminated, but with the appearance of hiv/aids, immunocompromised individuals were much more susceptible to tb reactivation. tb, therefore, is also considered to be a re-emerging disease. the public and the medical and public health communities gradually came to realize that their complacency over the potential threat of infectious diseases was misplaced, and that new and emerging diseases constituted one foci of concern over health threats to the public. this change in attitude came gradually, and can be thought of as a series of historical 'moments,' each of which refocused attention on infectious diseases. while it is impossible to be exhaustive here, this section takes a roughly chronological approach in describing the events that led the public and professional communities to realize that infectious diseases had not been 'conquered.' the bicentennial of the united states was celebrated in 1976, and there were many gala events around the nation in july. one was the meeting of the pennsylvania chapter of the american legion. the events surrounding this meeting were the first to bring the attention of both the population and the broad scientific and medical communities to the argument that infectious diseases in the united states had been 'conquered,' and both alarmed the public and aroused the curiosity of the scientific and medical communities because this appeared to be a new disease. indeed, before legionellosis was identified and antimicrobial treatment identified, legionellosis was called a 'monster disease. ' over 220 members of the american legion who had attended the meeting developed an unusual respiratory illness, and it became clear that it was of bacterial etiology, although it was initially thought to be viral, due to its close clinical resemblance to influenza. approximately 34 people died as a result of this outbreak. however, two things remained unclear. first, the pathogen could not be identified with conventional methods, and second, no common source of exposure could be identified initially, although the fact that the number of incident cases followed a typical epidemic curve suggested very strongly that there was some sort of common exposure to the pathogen. the news media seized upon this medical 'mystery,' and the public knew that they were dealing with an unknown infectious disease. this constituted a historical moment in contemporary american history, because it had been decades since something like this had happened. six months later, the bacterium was finally identified. legionella was not a new bacterium. stored samples from outbreaks as early as 1943 tested positive for legionella spp. however, the bacterium had not been identified in these outbreaks because it had not yet been described and characterized. in retrospect, most renowned is an outbreak that occurred in pontiac, michigan in 1968, although the symptoms were milder than in the legionella outbreak in philadelphia. in fact, mild legionellosis with a nonpneumonic form is often called 'pontiac fever.' this is not the place to review the epidemiology, pathophysiology, and clinical aspects of legionellosis in depth. briefly, though, it usually has an acute onset, and is usually caused by legionella pneumophila, although other species are also pathogenic. in fact, there are 40 species of the genus, and numerous serotypes. epidemiologically, l. pneumophila is by far the dominant species in human disease. the major reservoirs are bodies of freshwater, and the main mode of transmission is through small droplets that are inhaled from the environment. in the philadelphia outbreak, the source was finally traced to the air conditioning system in the hotel in which most attendees were lodged; the attendees were inhaling small particles in certain parts of the building. dozens of subsequent outbreaks have been traced to similar mechanisms. these have been not only air conditioners but also shower heads, aerosolizers in sinks, and whirlpools. virtually anything that aerosolizes fresh water is a potential mechanism by which legionellosis may be transmitted. symptoms of classic legionnaires disease are nonspecific and include fever, malaise, headaches, and myalgias. frequently, rigors will develop, as will a productive cough (in about half the cases). dyspnea (shortness of breath) is almost invariably present, and chest pain is common, as is a relative bradycardia for the elevated temperature. there are a number of abnormalities in laboratory tests, and chest films are markedly abnormal. a urine antigen test is available for one serotype, so laboratory diagnosis must frequently rely on more complex and time-consuming laboratory methods such as dfa. sputum cultures or cultures from bronchoalveolar lavage have been the mainstay of laboratory diagnosis. since laboratory methods do not show a definitive diagnosis until a minimum of 3 days following onset, diagnosis is usually made on clinical grounds, and treatment is initiated based upon index of suspicion. erythromycin proved to be effective in 1976, and other macrolides (azithromycin, clarithromycin) are highly effective. tetracycline and doxycycline are frequently used, as are the fluoroquinolones, such as levofloxacin. in hosts who are not immunocompromised, the prognosis is generally positive. there is no doubt that legionellosis was an emerging disease when it was first identified. its particular significance lies in its historical context -in the fact that this was the first occurrence that began shaking the optimism of the 1960s and early 1970s that infectious diseases had been conquered, and also in the fact that the etiology of an obviously infectious syndrome with a reasonably high case fatality ratio remained unknown for a number of months. chronologically, the next event to bring infectious disease to the attention of the public was another emerging infectious syndrome. in late 1979 and 1980, a number of women in the united states became seriously ill with a syndrome characterized by high fever, shock, rash, hypotension, and capillary leak. this syndrome had been first described as such 2 years earlier, although in retrospect it had been noted in the medical literature in the 1920s. the 1978 paper identified toxic shock syndrome in males, females, and children -and the females were both menstruating and not menstruating. the 1980 outbreak was associated with menstruating women, many of whom were using superabsorbent tampons. although this was a major risk factor in the 1979-80 outbreak, much of the public and many physicians were under the erroneous impression that toxic shock syndrome (tss) was necessarily associated with menstruating women who were using superabsorbent tampons. although tss is not necessarily associated with menstruating women, this does remain a risk factor in the epidemiology of tss. as with legionnaires disease, tss was a rare disease, yet the public's perception of it was out of proportion to its true prevalence -the risk was exaggerated. this is something that social scientists have called the 'social amplification of risk' in the context of new events that are potentially dangerous, but that nonetheless carry with them a low risk. amplification takes place as a result of media coverage, and as a result of intrapsychic processes that tend to amplify the threat of novel threats when the locus of control over the event is external to the individual. during the outbreak of toxic shock syndrome, newspapers were full of stories about tss and the sometimes deadly consequences of developing the syndrome. these were frequently on 'page 1 above the fold' and necessarily caught the attention of the public. the same was true of television news. once this outbreak of tss appeared to be concentrated in one single group -menstruating women using superabsorbent tampons -the general public's fear of tss began to diminish, and the federal government mandated the withdrawal of those tampons from the market. the number of incident cases began a rapid decline, and was back to baseline of about 100 cases per year by 1985. some reports demonstrated that there was a decrease in the use of all tampons -not just superabsorbent tampons. it was already known in 1980 that toxic shock syndrome was caused by staphylococci (specifically, s. aureus). in these cases, treatment is threefold: removal of the tampon, indwelling tampon, or other hypothesized environmental cause; aggressive fluid resuscitation; and rapid use of antistaphylococcal antibiotics. other bacterial species can cause toxic shock syndrome. in rare cases, other staphylococcus species have been associated with toxic shock syndrome, and because they are coagulase-negative, they are difficult to treat. at this time, coagulase-negative staphylococci constitute the most common cause of hospital-acquired bacteremia. this sometimes results in endocarditis, and usually the only effective treatment is surgical valve replacement, particularly in the case of those who have had earlier valve replacement. aggressive antibiotic therapy is occasionally effective. should toxic shock syndrome be considered to be an emerging disease? it certainly was in 1980, when the public was so concerned with its appearance. now, in 2007, 29 years after it was first described, this label is more questionable. what was most significant about toxic shock syndrome, however, was its historical significance. it followed the outbreak of legionnaires disease so closely that it turned the public's attention, once again, to infectious diseases, and to infectious diseases that had been unknown. it also reminded the biomedical community that infectious diseases had not been conquered. the issue at the time was whether legionnaires disease and toxic shock syndrome were anomalies, whether the assumption of the conquest of infectious diseases had clearly been erroneous, or whether these two outbreaks were harbingers of a new stage in 'epidemiologic history'a historical period during which emerging infections would become common and would catch the attention of the public, the public health community, the medical community, and government agencies. the public health and medical communities were divided on this. it would soon become clear, however, that the latter would hold true -that emerging infectious diseases would come to the forefront of public health, epidemiology, and the medical community. in the cases of legionnaires disease and tss, the social amplification of risk exaggerated perceived threats. nonetheless, the public became more attentive to infection. two other phenomena would solidify this attention. one was the appearance of hiv/aids in the united states, and the other was public attention that was drawn to hemorrhagic fevers, mostly in africa. the details of hiv/aids are covered elsewhere in this encyclopedia, and there will be no attempt here to duplicate this material. rather, this discussion concentrates on the significance of hiv/aids. when hiv/aids first appeared in several urban areas in the united states in 1981, it appeared to be an anomalous syndrome. it was not called 'aids' until 1982, when the centers for disease control (cdc) gave the syndrome that label. in the same year, researchers at cdc also linked one of the pathways of transmission to blood and blood products, causing a great deal of public concernif it was possible to contract aids through a frequently used medical practice, it had the potential of affecting millions of people. until then, aids was thought to be restricted to the gay community. in 1983, blood banks were warned by the cdc that blood and blood products could definitely transmit aids, and surgeons and other medical personnel began rethinking the criteria necessary for transfusion. by 1983, it was clear that the exponential increase in the number of incident cases was a definite trend. in 1983 and 1984, two teams discovered that the pathogen causing aids was viral, and although it had a different nomenclature at first, there was a great deal of relief that the causal agent had been discovered. it is an interesting study in the sociology of science to analyze the competing claims by luc montagnier at the institut pasteur and robert gallo in the united states concerning their respective claims that they discovered hiv. it is now clear that montagnier discovered the virus. shortly after the virus was discovered and characterized, an antibody test was developed to detect hiv in vivo. this was quickly used to screen blood products as well as to detect hiv in individuals. whereas some people decried the slowness of the u.s. government's response to hiv, the time from the first presentation of a group of males with kaposi's sarcoma or oral thrush until the antibody test for a recently identified virus was only 3 years. granted, the president of the united states, ronald reagan, had not even mentioned aids, and funding was less impressive than it could have been, but the time was quite short. the real challenge with hiv has been to find an effective vaccine, or to find a 'cure,' although antivirals have been effective in suppressing viral load in the majority of cases since 1995-96. the prevalence and mortality data are well-known. the best estimates are that globally, over 40 million people are living with hiv/aids, and approximately 22 million have died of hiv/aids. currently, about 19-20 million of those living with hiv/aids are women, and in developing countries, particularly in sub-saharan africa, hiv/aids is becoming, increasingly, a disease of women. currently, approximately two-thirds of those living with hiv/aids are in sub-saharan africa, but the increasing prevalence and incidence of hiv/aids in asia -and particularly, in india and china -are making east asia and south asia regions of tremendous concern. this is because each country has over 1 billion people, and the prevalence rates do not have to be high to result in large numbers of infected people. the global significance of hiv/aids is that it, by itself, has altered demographic trends, and the political economy of nations and regions, not to mention the human suffering that this disease has exacted. in botswana and swaziland, for example, the gains in life expectancy during the 20th century have not only been completely reversed, but the life expectancy at birth is lower now than it was at the beginning of the 20th century. in the context of this article, hiv/aids is an emerging infectious disease par excellence. a generation ago, it was literally unheard of. now in all developed countries and in many developing countries, hiv/aids shapes many behaviors, is responsible for significant stigma, is feared, and causes a significant percentage of deaths. globally, hiv/aids is the fourth leading cause of death, although in many parts of africa, it is the leading cause of death. hiv/aids is an emerging infectious disease because of the historical rapidity with which it moved from an unknown localized zoonotic complex in west and central africa to the most prevalent infectious disease in the world. while the scientific evidence suggests that there were a number of species jumps of both hiv-1 and hiv-2 that occurred in africa, these were so localized and the societies isolated enough from the rest of the world that hiv went unnoticed. thus, it appeared as though the disease went from nonexistence to a major pandemic in a matter of a few years. and there is another major significant dimension. since hiv/aids appears to have originated in africa -'out there,' away from northern europe and north america -some have argued that hiv/aids acquired a certain nefariousness -a disease emerging from the dark, foreign, isolated jungle -the stereotypical cauldron of new diseases. viral hemorrhagic fevers have been in the public eye since 1969, when there was a major outbreak of a hemorrhagic fever in the jos plain of nigeria. the disease came to be called lassa fever, caused by an arenavirus (lassa) that seemed particularly undesirable to the public. the virus is named after the town in which this outbreak occurred. like all hemorrhagic fevers, including dengue in some cases, one of the characteristics of lassa fever is that it can disturb the clotting/coagulation mechanism, resulting in disseminated intravascular coagulation (dic) and diffuse hemorrhage. the 1969 outbreak was publicized in the united states through the news media, perhaps because it was an 'exotic' or newsworthy event, and once again, the social amplification of risk was responsible for exaggerated fears of 'what if it spreads here?' that this outbreak occurred in sub-saharan africa, which, in the eyes of the north american public, may have been thought to be all 'jungle' (the jos plain is not rain forest) probably also contributed to the amplification of risk. serologic tests demonstrate that exposure to lassa virus is common in west africa. for example, in parts of nigeria, seroprevalence is positive in 21% of those tested; in sierra leone, the figure varies from 8-52% depending on the region (richmond and baglole, 2003) . it is now known that humans are dead-end hosts, and that the rat species mastomys natalensi is the natural host. these rats are extremely common throughout sub-saharan africa. people become infected by inhaling aerosols from rat excreta, and risk is increased by eating them, which is a very common practice in west africa. modern modes of travel have allowed infected individuals who are either symptomatic or asymptomatic at time of entry to travel to other continents, where they require treatment for lassa fever. these cases have not been numerous, but cases have appeared in the united states and japan, as well as in several european countries. this has caught some clinicians unprepared, since they were not trained in tropical medicine and were unaware of how to diagnose or manage a viral hemorrhagic fever. the prevalence rate of lassa fever is much higher than was initially thought. in one series, lassa fever accounted for 30% of adult deaths in sierra leone, and as many as 16% of hospital admissions (richmond and baglole, 2003) . following the outbreak in 1969, it took some time to investigate adequate treatment protocols, but now, aggressive fluid replacement and the use of antiviralsparticularly ribavarin -are the treatments of choice. ebola hemorrhagic fever and closely related marburg virus are both single-stranded rna viruses, as are other viruses that cause hemorrhagic fevers. ebola and marburg are filoviruses; ebola virus is actually a genus and there are four species. it was first described in the sudan in 1976, and estimates are that mortality from this virus has now exceeded 1000 people. the case fatality ratio exceeds 50%, and may be as high as 90% in some cases. transmission is different than lassa fever. it is usually through direct contact with blood and bodily secretions from individuals who are ill with ebola fever, or from nonhuman primates who are also infected. evidence points to bats as the natural reservoir of ebola virus, but this is not certain. in several studies, however, bats have been shown to be infected by the virus (leroy et al., 2005) . this is highly suggestive, but it is not conclusive proof. like so many other viral hemorrhagic fevers, the symptomatology of ebola is very nonspecific and typical of viral syndromes in general. the clinician needs to have a high index of suspicion. at this point, the only certain treatment is supportive, and from a public health point of view, quarantine is of the utmost importance, since ebola fever is so contagious. this was well-documented by the news media in the outbreak in kikwit, democratic republic of the congo (drc, then zaire) in 1995. this was so well-documented that once again it led to exaggerated perceptions of risk, with overtones of the 'exotic disease' from sub-saharan africa and its possible spread to the united states. recent advances in understanding the pathogenesis of ebola and the role of proinflammatory cytokines has led to the use of some recombinant products that block the progression of the inflammatory cascade to dic in some animal models. nonetheless, this approach has not been used in humans as of 2007. there are three notable points that need to be mentioned concerning ebola. first is that it appears to be increasing in prevalence in africa. this may be because detection is better and the disease has been better described, both epidemiologically and pathophysiologically. second is that there is significant concern that ebola virus could be used as a biological weapon. it has thus been placed on the highest level (category a) of potential biological weapons by the cdc. finally, ebola, more than any other emerging infectious disease, typifies in the mind of the public the sort of dangerous, threatening disease risk that is associated with tropical areas, the 'jungle,' and the threats that are associated with a more interconnected world. bovine spongiform encephalopathy (bse), or 'mad cow disease' in nontechnical terms, is another infectious disease that focused public awareness on emerging infections. the pathogen in this case was unusual not only in the sense that it had not been described elsewhere, but also because the whole class of pathogens -prionshave been very rare. like another neurologic disease, kuru, bse turned out to be due to a prion. essentially, prions are very simple since they are just unusually folded and self-replicating proteins. they cannot even be described as organisms. the source of the prion is not known, although many speculate that it is somehow derived from sheep infected with scrapie. in 1986, an unusual disease seemed to be affecting cattle in the united kingdom, and by the end of the year, over 175 000 cattle had died because of spongiform encephalopathy. since it was apparent that the disease was contagious, over 4 million cattle were intentionally slaughtered to limit contagion and ensuing effects on the cattle industry. by the mid-1990s, there was a clear epidemiologic association between bse and a variant of a neurodegenerative disease in humans that had been described in the middle of the 20th century: creutzfeldt-jakob disease (cjd). however, there were some notable differences between cjd and the disease that was affecting humans in the 1990s. the median age of this new syndrome was much younger than in classical cjd; the median duration of survival from onset of symptoms was longer than in classical cjd; and pathological differences and differences on mri were apparent with this new variant. accordingly, the cjd associated with bse first was named 'new variant creutzfeldt-jakob disease' or 'nvcjd;' as time progressed, nvcjd was renamed 'variant cjd' or 'vcjd.' although there were very few cases of vcjd in the uk human population, the threat of this disease was great according to public perception. according to the world health organization (who), as of november 2002, there had been 129 cases of vcjd in the united kingdom, six in france, and one each in several other countries (who, 2002) . nearly all of those with vcjd died or would die within 3 years. because of the realistic fear of contagion, several steps have been taken to limit the spread of vcjd. feeding practices for cattle have changed so that it is no longer legal to feed animal protein that might contain any tissues proximal to the central nervous system to other cattle. in the united kingdom, there was a ban on cattle over 30 months old from entering the commercial food supply. in the united states, individuals who have lived in the united kingdom or who have spent more than 6 months in the united kingdom are banned from being blood donors on the assumption that they might have consumed infected beef during their stay(s) in the united kingdom. a ban was instituted on importing cattle and cattle feed from the united kingdom, and, occasionally, from canada, in an attempt to prevent bse from spreading to the united states (kuzma and ahl, 2006) . while the number of incident cases of vcjd and bse have decreased in a typical epidemic curve pattern, the effects of the bse 'scare' have been tremendous. the very credibility of the uk government was threatened. the whole cattle and meat industries were severely hurt. on the other hand, surveillance techniques and understanding of cattle food chains were vastly improved. severe acute respiratory syndrome (sars) proved to be of great import in both the public awareness of emerging infectious diseases and in the testing and real-time construction of both domestic and international systems of public health surveillance and response. it was particularly important in terms of public awareness because it spread very rapidly on the international and intercontinental scales. sars apparently began as a few cases of a viral pneumonia in guangdong province in southeastern china in late 1992. however, this was not immediately apparent to the global public health communities because it was not publicized by the chinese government. what catapulted sars to international attention in the media and in the public health community was the appearance and rapid increase of incident cases in guangdong in february 2003 (zhao, 2007) . sars spread rapidly to hong kong, where contact tracing eventually identified one night in a specific hotel where the index case stayed as being the epidemic focus. the index case infected at least 16 others who were in the hotel at one time or another during that night. sars spread from hong kong to other areas of hong kong and to singapore, vietnam, and canada (toronto, ontario). the spread of all these cases has been traced to airplane travel, followed by localized spread by an index case. a case definition was developed based upon clinical presentation, which typically consisted of fever, initially, followed by lower respiratory signs and symptoms, sometimes resulting in acute respiratory distress syndrome and respiratory distress typical of acute lung injury as a response to the inflammatory cascade. just over 8000 cases were identified worldwide, and 774 died, for a case fatality ratio just <10%. a disproportionate degree of contagion occurred in intensive care units and areas of hospitals in which hospital personnel were exposed to respiratory excretions; close proximity -within 1 m -to an infected patient who was undergoing endotracheal intubation was the single greatest risk factor for contracting sars. local measures to control the spread of sars consisted largely of quarantine and containment. in china, for example, separate quarters for sars patients were constructed very rapidly. in singapore, arriving and departing passengers were required to pass through automated temperature detectors, and anybody with a fever was required to undergo further medical evaluation. the same was true at most points of entry in most developed countries. since most cases were contracted in hospitals and health facilities, rigorous contact control procedures were instituted, and in some cases, hospitals were closed to visitors and new admissions. the identification of the pathogen causing sars constitutes a textbook example of how international cooperation in science and public health may occur when the willpower is there and the scientific capability exists. by mid-march 2003, many leading laboratories with advanced virologic capabilities had agreed to cooperate in a network that was coordinated by the world health organization. within 2 weeks, a pathogen was identified as a novel coronavirus, using a combination of methods: molecular polymerase chain reaction, culture, and electron microscopy, and shortly thereafter, the criteria of koch's postulates were met. thus, the evidence was quite clear that the new coronavirus was the pathogen. the virus was named the sars coronavirus, or, almost always, sars cov. the ecology of sars was not understood as quickly as the pathogen was identified. some features were identified within a number of months. first was the phenomenon of superspreaders, which is a concept that previously had received scant attention. in this case, it became apparent that a small number of individuals spread sars to a disproportionately large number of people. it is not clear whether this is because of behavioral factors, host-pathogen interaction, or environmental factors. what is fairly clear is that were it not for superspreaders, the epidemic would not have affected nearly as many people as it did. this is because the r 0 , or number of people who one individual could infect, was inflated by superspreaders. thus there was a domino effect of contagion. in 2007, bats were identified as the reservoir of sars cov. there had previously been some speculation about bats being the reservoir, but there was no solid evidence, and the reservoir had been a mystery. some had suggested that proximity of people to avian species could possibly be a factor in the pathogenesis of sars, because of the importance of this process in avian influenza. however, this turned out not to be the case with sars. sars is a prototype of an emerging infectious disease (berger et al., 2004) . there is no evidence that sars cov existed in the human population prior to the outbreak of late 2002-03. the specific syndrome surprised the public health and medical communities, yet its general features did not, and the emergence of new diseases had been a familiar concept since the u.s. institute of medicine report of 2002. at the same time, the rapidity of the appearance of sars and its very rapid spread at every scale fueled public apprehension, and even hysteria in some cases. evidence exists that history has been punctuated by relatively regular influenza epidemics and pandemics. the rapidity of epidemic spread, leading to pandemics, is largely determined by the velocity of the prevailing transportation modes. severe epidemics and pandemics are caused by genetic shift, whereby the viral genome expressing surface antigens (hemagglutinin and neuraminidase) undergoes relatively major change. relatively minor epidemics occur because of genetic shift, in which the surface antigens undergo minimal yet detectable changes in their configuration. following genetic shift, people have minimal immunity to the virus, and are susceptible. in one sense, each year influenza constitutes an emerging infection, because the precise genome of the influenza viruses and the surface antigens undergo change. similarly, whenever a pandemic occurs, influenza represents a more significant emerging infection. on the other hand, influenza represents a disease entity that is not new to the population. thus, it is a matter of semantics whether to consider influenza to be an emerging infection. avian influenza may constitute the next serious pandemic threat. it has been known for decades that genetic reassortment occurs in southeastern china because of the proximity of humans, avian species, and swine. an unusual number of influenza epidemics appear to arise there. however, the concern over avian influenza arises from a slightly different situation. it has been known for some time that no less than 24 influenza subtypes -different configurations of surface antigens -can infect aquatic bird species. it has been wellestablished that several of these subtypes can infect humans, although recent experience suggests that all subtypes that circulate in avian species may have the potential to infect humans. this is one of the reasons that has given rise to concern over the possibility of an avian influenza pandemic. this theoretical concern moved closer to reality in hong kong in 1997, when one influenza strain (h5n1) was transmitted directly from poultry to humans. this took place in 'wet markets' -markets in which live poultry are densely packed, and where people co-mingle with their intended purchases. the transmission in 1997 appears to have been limited: only 18 cases were confirmed. however, the case fatality ratio was high. six of the 18 people died. transmission also occurred with another strain h9n1 -in 1999, and in 2001 and 2002 there was widespread transmission and mortality among chickens in hong kong. because of a concern over possible transmission to humans, and because of the devastating economic potential in the poultry industry, containment of this epidemic in poultry was partly obtained by the slaughter of millions of chickens and other poultry. avian influenza viruses have shown some propensity, since 1997, for transmission to humans. so far, human cases of influenza that have been identified as avian strains have been limited to approximately 200, and these have all been in asia. human-to-human transmission has been implicated in only a few cases. if this is the case, what is the concern over avian influenza? because of the tendency for influenza viruses to mutate, many virologists and epidemiologists predict that there is a high likelihood that a mutation could occur that would facilitate human-to-human transmission of h5n1 and other avian subtypes that have been transmitted to humans. if this occurs, then there is little doubt that this strain would spread rapidly among the human population, and would spread locally, nationally, and between continents in a manner similar to sars. other epidemiologists and virologists are more circumspect in their predictions, and argue that the probability of a mutation that would increase the propensity of avian influenza to spread from human to human is unknown. a minority of authorities argue that the probability is low. thus, in assessing the overall threat of avian influenza, the crucial question is whether the virus will spread readily from human to human. at this point (mid-2007) , it is unknown whether this will occur. however, it is prudent public health policy to bolster surveillance systems, and governments are stockpiling neuraminidase inhibitors, which are medications that can moderate the course of influenza if taken early in the course of clinical disease, or sometimes prevent the onset of symptoms if taken prophylactically. similarly, there has been great emphasis on vaccine development and stockpiling. in response to growing public concern over emerging infectious diseases, both domestically and internationally, as well as to both interest and concern in the medical and public health communities, a major conference on emerging viruses was held at rockefeller university in 1989. the conference was cosponsored by several government agencies. the conference participants reached many conclusions, but two of them were that emerging infections had become a major focus for scientific research and that emerging infectious diseases had become and would remain a major public health challenge for the united states. accordingly, the institute of medicine of the national research council of the united states took a proactive role and sought funding for a major study of emerging infections. the study was funded by a number of government units, and in early 1991, a high-powered committee met in washington for the first time to: identify significant emergent infectious diseases, determine what might be done to deal with them, and recommend how similar future threats might be confronted to lessen their impact on public health. (institute of medicine, 1992: vi) the committee issued a report in 1992 that quickly became a standard scientific and policy reference on emerging infectious disease. emerging infections: microbial threats to health was the first major comprehensive discussion of how emerging infections arise, and how they might be addressed by the public health community. the committee also identified the six 'factors' or causes of emergence. briefly, the factors that this committee identified were the following: human demographics and behavior; technology and industry; economic development and land use; international travel and commerce; microbial adaptation and change; and the breakdown of public health measures. it is notable that five of these six factors are social factors that are consequences of changes in society. even microbial adaptation and change, such as the development of antimicrobial resistance as a response to selective pressure, has a large behavioral dimension. this is partly a response to a technical innovation -the development of antimicrobials -and partly a response to a behaviorthe prescribing of those antimicrobials. of course, one dimension of this factor is the nonselective and improper prescribing of antimicrobials. this has several dimensions: the prescription of antibiotics when none are needed, the prescription of broad-spectrum antibiotics when narrowspectrum antibiotics are sufficient, the free availability of antibiotics in many developing countries on the street and in pharmacies where no prescription is needed, and the free use of late-generation antibiotics in the food industry to promote the growth of cattle, chickens, and other animals intended for human consumption. so, in fact, all of the six factors of emergence are social and behavioral in nature. it is ironic that despite the fact that both institute of medicine reports concluded that the major causes of emergence have been social, there have been very few social analyses of emerging infections. for example, emerging infectious diseases, a new journal founded in 1995 in response to the growing importance of emerging infections, has an explicit aim of including a social understanding of emerging infections in its contents, yet there have been very few articles written by social scientists in this journal, and very few articles with any social content have been published. the main point is that the overwhelming understanding of emerging infections has been 'biomedical.' this is not a criticism of either the journal or of any field in public health or medicine. in large part, this is the result of the sociology of knowledge and science. for whatever reason, few social scientists have become involved in research on emerging infections, whereas the same cannot be said about chronic diseases. some researchers have asked the question of why emerging infectious diseases are emerging now and in the societies where they are emerging, and have sought a more contextual understanding of emerging infections. david bradley asks a very penetrating question: [a]ttaching a microbiological label to an outbreak. . .does not answer either the micro-scale questions such as ''why is there an outbreak here, now, of this size, affecting these people?'' nor does it answer the macro-questions such as ''why are there more (or fewer) outbreaks this decade than last?'' nor does it answer the question ''what drives the overall worldwide trends in such problems?'' (bradley, 1998: 1) for example, a number of individuals have argued that emerging infections may represent another stage in the epidemiologic transition. our understanding of emerging infections has not been totally devoid of social analysis. inequality and poverty have become a major focus for the social analysis of health and disease. the argument is that through a complicated series of pathways that are yet to be fully understood, both poverty and inequality result in poor health status. this has not been applied extensively to emerging infectious diseases, although paul farmer's (1999) insightful work has been applied to emerging infections. in his critical analysis of emerging infection, farmer asks, ''emerging for whom?'' in other words, the diseases that westerners might label as emerging may have been present or endemic in poorer societies for a long time: if certain populations have long been afflicted by these disorders, why are the diseases considered ''new'' or ''emerging''? is it simply because they have come to afflict more visible -read more ''valuable'' persons? this would seem to be an obvious question from the perspective of the haitian or african poor. (farmer, 1999: 39) in other words, farmer argues, the concept of emerging infectious diseases is one of epistemology -the theory of knowledge. how do emerging diseases come to be categorized as 'emerging'? by implication, many of these diseases have been present in poorer societies for a long time. the evidence affirms this. hiv was probably present in small foci in central africa for decades to centuries; ebola was similarly endemic in west africa for an unknown period, as was lassa fever. what is novel about the past few decades is greater interconnection between places, allowing diseases, and news of diseases, to spread; better methods of detection; and changing settlement geographies that have brought people into different forms of contact with animal reservoirs. the root cause of the infectious disease emergence is human action, both intentional and unintentional. most of this action is the result of cumulative individual acts on a mass scale. for example, the mass urbanization of society in poorer countries is the sum of millions of individuals who move from rural to urban areas. this is largely the result of the perceived economic opportunities in urban areas, and the 'push' factor of lack of opportunity in rural areas. yet, taken together, millions of individual moves result in urbanization, and this urbanization facilitates the spread of diseases by the respiratory route, the fecal-oral route, and many other modes of transmission. the institute of medicine committee also developed a set of policy recommendations. these concentrated in two areas: the need for vastly increased resources for interdisciplinary training in infectious diseases because of the depleted workforce resources in this area; and the need to develop new surveillance and public health response systems, since the committee had determined that emerging infections did, indeed, constitute a major public health threat to the united states. this report was issued with a great deal of publicity. the u.s. public's attention was already focused on emerging infectious diseases as a result of legionnaires disease, viral hemorrhagic fevers, and toxic shock syndrome. now there was a major quasi-governmental report by a group of the nation's leading scientists who issued the sobering conclusion that: even with unlimited funds, no guarantee can be offered that an emerging microbe will not spread disease and cause devastation. (institute of medicine, 1992: 169) part of the institute's report identified specific microbes and diseases that could possibly threaten public health in the future. three of these were e. coli 0157:h7, cryptosporidiosis, and hantavirus. the report was prescient, because within a few years there were serious outbreaks of all of these. in 1993, which was the year after the iom report was issued, there was a major outbreak of cryptosporidiosis on the south side of milwaukee, wisconsin. it caused diarrhea, ranging from mild to severe, in over 400 000 people. cryptosporidium parvum is a protozoan parasite; evidence in animal models is that ingestion of even one oocyst can result in severe gastrointestinal symptoms. in humans, as few as 12 oocysts can produce these effects (king and monis, 2007) . it is impervious to usual methods of water treatment, and only recently has an effective medication become available. the milwaukee outbreak was probably due to groundwater absorption of cattle feces, subsequent runoff due to both heavy rains and snow melting, transport of the oocysts to river tributaries, and movement of the oocysts into lake michigan, which serves as the water supply for the south side of milwaukee. the filtration plant for that water was ineffective in eliminating the oocysts. many of these events are putative, but together they constitute a logical chain. meanwhile, research is still proceeding on the ecology of cryptosporidiosis. understanding is progressing, but it is still incomplete. e. coli 0157:h7 was also mentioned in the iom report as being an emerging disease. in january 1993, the washington state department of health ascertained that an outbreak of 0157:h7 was occurring in the state, and this outbreak was associated with having eaten at jack in the box fast-food restaurants. subsequently, it became apparent that the epidemic was not limited to washington, but also included idaho and nevada. the epidemiologic investigation of this outbreak was intricate, and implicated a chain of events. first, because meat inspection in the united states was inadequate, one theory is that e. coli 0157:h7 from the bowels of cattle had gotten into meat that was sent to market when cattle were slaughtered, and the bowel was probably nicked or severed. another is that under stress, cattle defecate over one another, and fecal matter from one cow can contaminate the hides of other cattle. second, when this meat was ground into hamburger, it increased the surface area of the meat by several orders of magnitude, thereby allowing the pathogen a great deal of exposure. third, once this hamburger meat was shipped to jack in the box restaurants, it appears that hamburgers were being systematically undercooked, below industry standards. this allowed the e. coli to survive and enter the hosts' systems. the consequences of such infection can be severe, and were in 1993, with those who were symptomatic frequently suffering from bloody diarrhea, fever, cramps, and, in the worst case, hemolytic uremic syndrome. the pathogenesis of this disease was only partially understood in 1993, but understanding is more complete in 2007. the third disease that was mentioned in the iom report that occurred shortly after its publication was hantavirus. in may 1993, in the four corners area of arizona, new mexico, california, and utah, several males who were otherwise in good health developed a sudden serious respiratory disease that was thought to be a rapidly progressing acute respiratory distress syndrome, since this was the immediate cause of death. however, it was noted that these cases had formed a cluster, and investigators tried to find some sort of common source to explain a possible environmental exposure to explain this serious and sometimes fatal syndrome. though hantavirus had never been described in the united states, serologic tests in patients showed a surprising seropositivity to hantavirus. it was apparent that this was the pathogen that had caused the dozen deaths associated with the outbreak. the chain of events that led up to the outbreak is now fairly clear. winter 1993 was unusually warm in the four corners area as a result of el nino, and the spring was also unusually rainy. these two conditions led to the rapid and plentiful growth of pinon trees, which provided food for a number of rodents. there is consensus that the deer mouse (peromyscus maniculatus) population increased by an order of magnitude. testing demonstrated that about 30% of the mice that were trapped after this epidemic were infected with hantavirus, and studies demonstrate that households from which infected individuals came were far more likely to have heavy rodent infestations than were households of controls. more rigorous studies eventually showed that transmission occurred from rats to humans, and that many of the cases, in this instance, were associated with crawling under houses and other places in which rodent exposure was likely to occur. by 2000, many of the predictions of the first institute of medicine report (1992) had been realized, and understanding of emerging infectious diseases had improved. there was greater focus on globalization as a process of disease spread, and the attacks on the world trade center and pentagon on september 11, 2001 focused attention on terrorism. a new institute of medicine committee was formed to consider the nature of microbial threats and emerging diseases, and the report of this committee was issued in 2003 (institute of medicine, 2003) . this report represented a rethinking of the factors of emergence, and presented a more nuanced understanding of the causes of emerging diseases, most of which were still social at one level or another. bioterrorism ('intent to harm') was specifically mentioned as a factor of emergence, as was lack of political will. policy recommendations for surveillance, response, and training were more detailed than in the 1992 report, and there was a more urgent tone to the need to respond to emerging threats. in this report, the emphasis on biological and social interaction was strong: genetic and biological factors allow microbes to adapt and change, and can make humans more or less susceptible to infections. changes in the physical environment can impact on the ecology of vectors and animal reservoirs, the transmissibility of microbes, and the activities of humans that expose them to certain threats. human behavior, both individual and collective, is perhaps the most complex factor in the emergence of disease. emergence is especially complicated by social, political, and economic factors. . .which ensure that infectious diseases will continue to plague us. (institute of medicine, 2003: 2) increasing resistance to antibacterials, antivirals, and other antimicrobials is frequently grouped under the heading of 'emerging infections.' resistance is certainly a constantly growing and very major public health problem, but this is of importance to emerging infections only in the sense that diseases that were once highly treatable with first-and second-generation antimicrobials are no longer treatable by them. the selective pressures exerted by antimicrobials have made numerous pathogens resistant to even the newest antimicrobials due to mechanisms that are now understood. for example, many respiratory pathogens are no longer treatable by b-lactam antibiotics since their b-lactam rings are cleaved by b-lactamases. there are fluoroquinolone-resistant strains of neisseria gonorrhoeae, resistant strains of staphylococcus aureus, and so on. the problem is most severe in hospitals, where severe infections once responsive to vancomycin are now resistant to this glycopeptide. several new antimicrobials have been developed, in part to address vancomycin resistance, but resistance to these medications developed within a few years of their introduction. thus, antimicrobial resistance is both a community problem and a hospital problem. there is great concern over multiple drug-resistant tuberculosis, which is defined as tuberculosis that is resistant to two first-line medications, and extensively resistant tuberculosis, which has a more complex definition specifying several medications. there is not space in this article to explore antimicrobial resistance in greater depth. the relationship between people and pathogens has been an integral part of history, and will continue to be. the progress in the diagnosis, detection, and clinical management of infectious diseases has been substantial. indeed, fauci (2001) has gone so far as to argue that: the successful diagnosis, prevention, and treatment of a wide array of infectious diseases has altered the very fabric of society, providing important social, economic, and political benefits. nonetheless, infectious diseases, aggregated together, constitute the second leading cause of death worldwide, and in many regions, they account for the dominant cause. moreover, emerging diseases will continue to emerge, because of constantly changing social and demographic conditions, as well as selective pressures. the prototypical emerging infectious disease, hiv/aids, has an uncertain future in the long run. perhaps a vaccine will be developed that will be inexpensive, and perhaps distribution systems will be developed that will transport the vaccine to points of demand. perhaps antiretrovirals will become extremely inexpensive, and perhaps the failure rate for antimicrobials of 30% will be overcome. however, it is unlikely under present conditions that all of these improvements will occur. thus, the future of hiv/aids is more sobering. the same is true of antimicrobial resistance. in an age of optimism when antimicrobials were developed and used successfully -perhaps the first 30 years of antimicrobial use -concern over resistance was minimal. however, the fact that organisms adapt to changing environmental conditions and threats is something that has not been realized only recently. the inevitability of adaptation is undeniable, and the only way to meet the challenges of resistance is through a combination of appropriate antimicrobial use (including the use of narrow-spectrum antibiotics as soon as possible in the clinical course of an individual) and the development of new antimicrobials, as well as new understanding in the physiology and genetics of microorganisms, which might lead to the development of new technologies in addressing the pathogenic basis of disease. see also: aids, epidemiology and surveillance; antimicrobial resistance; severe acute respiratory syndrome (sars); transmissible spongiform encephalopathies; tuberculosis: overview; west nile disease. acromegaly a condition produced by overproduction of growth hormone, leading to excessive growth of the hands, feet, and jaw in postpubertal individuals and giantism in prepubertal children. adrenal glands two endocrine organs situated above the kidneys that make a series of hormones: cortisol (stress hormone), aldosterone (salt-retaining hormone), and catecholamines (stress hormones). autoimmunity a situation in which part of the body, often an endocrine organ, is recognized as 'foreign,' triggering an immune response that tends to lead to destruction of the endocrine gland. cushing syndrome excessive production of cortisol with loss of the normal circadian variation leading to weight gain, hypertension, and type 2 diabetes mellitus. g protein proteins within the cell that transfer the hormone message from the receptor to specific parts of the cell. graves disease a combination of thyroid overactivity due to an autoimmune disorder and eye problems. hypothalamus part of the brain containing control centers for appetite, thirst, and pituitary hormone secretion. pituitary major regulator of hormone production. secretion of hormones regulated by the hypothalamus. severe acute respiratory syndrome (sars): paradigm of an emerging viral infection new and resurgent infectious: prediction, detection, and management of tomorrow's epidemics infections and inequalities: the modern plagues infectious diseases: considerations for the 21st century emerging infections: microbial threats to health in the united states microbial threats to health critical processes affecting cryptosporidium oocyst survival in the environment living with bse fruit bats as reservoirs of ebola virus plagues and peoples factors in the emergence of infectious diseases lassa fever: epidemiology, clinical features, and social consequences variant creutzfeldt-jakob disease sars molecular epidemiology: a chinese fairy tale of controlling an emerging zoonotic disease in the genomics era the coming plague: newly emerging diseases in a world out of balance new and resurgent infections: prediction, detection, and management of tomorrow's epidemics the changing face of disease: implications for society the challenge of emerging and re-emerging infectious diseases an emptying quiver: antimicrobial drugs and resistance the politics of emerging and resurgent infectious diseases disease in evolution: global changes and emergence of infectious diseases endocrine diseases: overview p c hindmarsh key: cord-346314-o9fjpqaj authors: jarboui, mohamed ali; bidoia, carlo; woods, elena; roe, barbara; wynne, kieran; elia, giuliano; hall, william w.; gautier, virginie w. title: nucleolar protein trafficking in response to hiv-1 tat: rewiring the nucleolus date: 2012-11-15 journal: plos one doi: 10.1371/journal.pone.0048702 sha: doc_id: 346314 cord_uid: o9fjpqaj the trans-activator tat protein is a viral regulatory protein essential for hiv-1 replication. tat trafficks to the nucleoplasm and the nucleolus. the nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rrna and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. importantly, transient nucleolar trafficking of both tat and hiv-1 viral transcripts are critical in hiv-1 replication, however, the role(s) of the nucleolus in hiv-1 replication remains unclear. to better understand how the interaction of tat with the nucleolar machinery contributes to hiv-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of jurkat t-cells stably expressing hiv-1 tat fused to a tap tag. using an organellar proteomic approach based on mass spectrometry, coupled with stable isotope labelling in cell culture (silac), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in jurkat t-cell nucleolus upon tat expression. numerous proteins exhibiting a fold change were well characterised tat interactors and/or known to be critical for hiv-1 replication. this suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by tat provide an additional layer of control for regulating cellular machinery involved in hiv-1 pathogenesis. pathway analysis and network reconstruction revealed that tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, t-cell signaling pathways and genome integrity. we present here the first differential profiling of the nucleolar proteome of t-cells expressing hiv-1 tat. we discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust hiv-1 production. the nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (nors) formed by clusters of hundreds of rdna gene repeats organised in tandem head-to-tail repeat [1, 2] . a membrane-less organelle originally described as the ''ribosome factory'', the nucleolus is dedicated to rna-polymerase-i-directed rdna transcription, rrna processing mediated by small nucleolar ribonucleoproteins (sornps) and ribosome assembly. ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60s) and small (40s) ribosomal subunits, which are subsequently exported to the cytoplasm. this fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] . the nucleolus is the site of additional rna processing, including mrna export and degradation, the maturation of uridine-rich small nuclear rnps (u snrnps), which form the core of the spliceosome, biogenesis of t-rna and micrornas (mirnas) [4] . the nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] . the concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions. of importance, the nucleolus is also the target of viruses including hiv-1, hcmv, hsv and kshv, as part of their replication strategy [2, 17] . proteomics studies analysing the nucleoli of cells infected with human respiratory syncytial virus (hrsv), influenza a virus, avian coronavirus infectious bronchitis virus (ibv) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . interestingly, both hiv-1 regulatory proteins tat and rev localise to the nucleoplasm and nucleolus. both their sequences encompass a nucleolar localisation signal (nols) overlapping with their nuclear localisation signal (nls), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . furthermore, tat and rev interact with the nucleolar antigen b23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . nevertheless, a recent study described that in contrast to jurkat t-cells and other transformed cell lines where tat is associated with the nucleus and nucleolus, in primary t-cells tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . while the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing tat and rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . importantly, two major studies by machienzi et al. have revealed important functional links between hiv-1 replication and the nucleolus [49, 50] . first, they could inhibit hiv-1 replication and tat transactivation function employing a tar decoy specifically directed to the nucleolus. furthermore, using a similar approach, with an anti-hiv-1 hammerhead ribozyme fused to the u16 small nucleolar rna and therefore targeted to the nucleolus, they could dramatically suppress hiv-1 replication. collectively, these findings strongly suggest that hiv-1 transcripts and tat nucleolar trafficking are critical for hiv-1 replication. however the nature of these contributions remains to be elucidated. in this report, we systematically analysed the nucleolar proteome perturbations occurring in jurkat t-cells constitutively expressing hiv-1 tat, using a quantitative mass spectrometry approach. following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon tat expression, we focussed on the tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, dna replication and repair and metabolism and discuss their potential involvement in hiv-1 pathogenesis. in this study, we investigated the quantitative changes in the nucleolar proteome of jurkat t cells constitutively expressing hiv-1 tat (86aa) versus their tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (silac) technology, followed by esi tandem mass spectrometry and implemented the experimental approach described in figure 1a . first, using retroviral gene delivery, we transduced hiv-1 tat fused to a tandem affinity purification (tap) tag (consisting of two protein g and a streptavidin binding peptide) or tap tag alone (control vector) in jurkat leukemia t cell clone e6-1 and sorted the transduced cells (gfp positive) by facs. this resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( figure 1b) . the functionality of tap-tat was confirmed by transfecting jurkat tap-tat and tap cells with a luciferase reporter gene vector under the control of the hiv-1 ltr (pgl3-ltr) [36] . tap-tat up regulated gene expression from the hiv-1 ltr by up to 28 fold compared to control ( figure 1c ). to further address the functionality of tat fused to tap, we compared jurkat tap-tat with jurkat-tat, a cell line stably expressing untagged tat [51] . both cell line exhibited comparable hiv-1 ltr activity following transfection with pgl3-ltr ( figure s1 ). next, tat expression and subcellular localization was verified by subcellular fractionation followed by wb analysis ( figure 1e ). tap-tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. these observations were further validated by immunofluorescence microscopy ( figure 1e ). of note, jurkat-tat presented similar patterns for tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by wb analysis (figure s2 and s3). we next compared the growth rate and proliferation of the jurkat tap and tap-tat cell lines (materials and methods s1), which were equivalent ( figure s4a ). similarly, facs analysis confirmed that the relative populations in g1, s, and g2/m were similar for jurkat tap-tat and tap cells ( figure s4b ). we labeled jurkat tap-tat and jurkat tap cells with light (r0k0) and heavy (r6k6) isotope containing arginine and lysine, respectively. following five passages in their respective silac medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( figure 1a ) [52] . each step of the procedure was closely monitored by microscopic examination. to assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by western blot analysis ( figure 1d ). nucleolin (110 kda) and fibrillarin (fbl) (34 kda), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. this distribution pattern for nucleolin appears to be specific for jurkat t-cells as show previously [52, 53] . the nuclear protein parp-1 (poly adpribose polymerase 1) (113 kda) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. alpha-tubulin (50 kda) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination. subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. comparative quantitative proteomic analysis was performed using maxquant to analyse the ratios in isotopes for each peptide identified. a total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. the fully annotated list of the quantified nucleolar proteins is available in table s1 and the raw data from the mass spectrometry analysis was deposited in the tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. we annotated the quantified proteins using the toppgene suite tools [54] and extracted gene ontology (go) and interpro annotations [55] . the analysis of go biological processes ( figure 1f ) revealed that the best-represented biological processes included transcription (24%), rna processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of jurkat t-cell nucleolar proteome [52] . subcellular distribution analysis ( figure 1f ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. the subcellular distribution was similar to our previous analysis of the jurkat t-cell nucleolar proteome [52] . table s1 . the distribution of protein ratios are represented in figure 1g as log 2 (abundance change). the silac ratios indicate changes in protein abundance in the nucleolar fraction of jurkat tap-tat cells in comparison with jurkat tap cells. the distribution of the quantified proteins followed a gaussian distribution ( figure 1g ). a total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon tat expression (table 1) . of these, 30 proteins were enriched, whereas 19 proteins were depleted. cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (npm1/ b23), c23, fbl, nucleolar protein p120 (nol1), and nucleolar protein 5a (nol5a). the distinct ratios of protein changes upon tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus. we performed wb analysis to validate the silac-based results obtained by our quantitative proteomic approach ( figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon tat expression, including 9 enriched (hsp90b, stat3, prb, ck2a, ck2a', hsp90a, transportin, zap70, ddx3), and 3 depleted (ilf3, bop1, and ssrp1) proteins. in addition, we also tested by wb analysis, protein abundance not affected by tat expression (importin beta, fbl, b23, c23). these results highlight the concordance in the trend of the corresponding silac ratios, despite some differences in the quantitative ranges. of note, using wb, we could observe a change of intensity for protein with a silac fold change as low as 1.25-fold. of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. on the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in table 1 . alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. therefore, we next focused on both enriched or depleted individual proteins with activities associated with hiv-1 or tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes. we initially focused on signaling proteins interacting with tat and/or associated hiv-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by tat expression. phospho-protein phosphatases. phospho-protein phosphatase pp1 and pp2a are essential serine/threonine phosphatases [56, 57] . importantly, pp1 accounts for 80% of the ser/thr phosphatase activity within the nucleolus. in our study, pp1 was found to be potentially enriched by 1.52-fold in the nucleolus of jurkat cells expressing tat, which supports previous studies describing the nuclear and nucleolar targeting of pp1a by hiv-1 tat and how pp1 upregulates hiv-1 transcription [58, 59, 60, 61, 62] . pp1 c was also identified as part of the in vitro nuclear interactome [63] . similarly, ppp2ca, the pp2a catalytic subunit (1.29-fold) and its regulatory subunit pp2r1a (1.27-fold) were similarly enriched upon tat expression. interestingly, tat association with the pp2a subunit promoters results in the overexpression and up regulation of pp2a activity in lymphocytes [64, 65] . furthermore, pp2a contributes to the regulation of hiv-1 transcription and replication [61, 66] . retinoblastoma protein. the tumour suppressor gene prb protein displayed a 1.4-fold change in the nucleolus upon tat expression [67] . furthermore, wb analysis confirmed the distinct translocation of prb from the nucleoplasm to the nucleolus by tat ( figure 2 ). depending on the cell type, prb can be hyperphosphorylated or hypophosphorylated upon tat expression and can negatively or positively regulate tat-mediated transcription respectively [68, 69, 70] . interestingly, the hyperphosphorylation of prb triggers in its translocation into the nucleolus [71] . phosphorylation of prb is also associated with an increase in ribosomal biogenesis and cell growth [72] . stat3. the transcription factor signal transducer and activator of transcription 3 (stat3) was significantly enriched (1.86-fold) in the nucleolar fraction by tat constitutive expression. furthermore, wb analysis indicated that tat expression could promote the relocalisation of stat3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( figure 2) . interestingly, previous studies have demonstrated tat-mediated activation of stat3 signaling, as shown by its phosphorylation status [73] . interestingly, stat3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] . ybx1. ybx1, the dna/rna binding multifunctional protein was enriched by 1.38-fold in the nucleolus of jurkat cells upon tat expression. interestingly, ybx1 interacts with tat and tar and modulates hiv-1 gene expression [63, 75] . zap70. the protein tyrosine kinase zap70 (zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of jurkat cells expressing tat [76] . furthermore, wb analysis revealed that tat expression could promote the relocalisation of zap70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( figure 2 ). of note, zap70 is part of the in vitro nuclear tat interactome [63] . matrin 3. the inner nuclear matrix protein, matrin 3 (matr3), presented a 1.39-fold change in the nucleolus of jurkat cells expressing tat. it localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . matrin 3 has been identified as part of the in vitro hiv-1 tat nuclear interactome [63] . two recent studies have described matrin 3 as part of ribonucleoprotein complexes also including hiv-1 rev and (rev response element) rre-containing hiv-1 rna, and promoting hiv-1 post-transcriptional regulation [78, 79, 80] . casp10. the pro-apototic signaling molecule, caspase 10 (casp10), was significantly depleted from the nucleolus of jurkat-tat cells (0.82-fold) [81] . importantly, tat expression downregulates casp10 expression and activity in jurkat cells [82] . adar1. adenosine deaminase acting on rna (adar1), which converts adenosines to inosines in double-stranded rna, was significantly depleted from the nucleolus of jurkat-tat cells (0.78-fold). interestingly, adar1 over-expression up-regulates hiv-1 replication via an rna editing mechanism [83, 84, 85, 86, 87, 88] . furthermore, adar1 belongs to the in vitro hiv-1 tat nuclear interactome [63] . to underline the structural and functional relationships of the nucleolar proteins affected by hiv-1 tat, we constructed a network representation of our dataset. we employed cytoscape version 2.6.3 [89] and using the mimi plugin [90] to map previously characterised interactions, extracted from protein interaction databases (bind, dip, hprd, ccsb, reactome, intact and mint). this resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( figure 3a ). centrality analysis revealed a threshold of 23.7 interactions per protein. topology analysis using the centiscape plugin [91] showed that the node degree distribution follows a power law ( figure s5 ), characteristic of a scale-free network. importantly, when we analysed the clustering coefficient distribution ( figure s6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around hiv-1 tat. furthermore, node degree connection analysis of our network identified hiv-1 tat as the most connected protein ( figure s6 ). specifically, the topology analysis indicated that the values for tat centralities were the highest (node degree, stress, radiality, closeness, betweeness and centroid), characterising tat as the main hub protein of the nucleolar network. indeed, a total of 146 proteins have been previously described to interact with tat ( figure 3b , table s2 ). these proteins are involved in a wide range of cellular processes including chromosomal organization, dna and rna processing and cell cycle control. importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold). in parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( figure 4) . ribosomal biogenesis. we initially focused on ribosome biogenesis, the primary function of the nucleolus. we could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by tat expression (figure 4 ). while some ribosomal proteins remained unaffected, tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except rpl35a, for which tat expression caused a marked decrease at the nucleolar level (0.29-fold). similarly, several proteins involved in rrna processing exhibited an overall increase in nucleolar accumulation upon tat expression. these include human canonical members of the l7ae family together with members participating in box c/d, h/aca and u3 snornps ( figure 4) . conversely, bop1, a component of the pebow (pescadillo bop1 wdr12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of jurkat tap-tat cells (0.81-fold) and this was confirmed by wb analysis (figure 2 ) [93] . nevertheless, the other pebow complex components, pes1 (0.94-fold) and wdr12 (1.1fold), were not affected by tat expression. of note, we did not detect change in the abundance of protein participating in rdna transcription such as rnapoli, ubf. spliceosome. we identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . these proteins include the small nuclear ribonucleoproteins u1, u2 and u5, sm d1, d2, d3, f and b, and the heterogeneous nuclear ribonucleoproteins. our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of hiv-1 tat in jurkat t-cells (figure 3 and 4) . the only three proteins that were significantly depleted from the nucleolus upon expression of hiv-1 tat were rbmx (0.89-fold), hnrnpa2b1 (0.84-fold) and snrpa (0.81-fold). several investigations showed expression alteration in cellular splicing factors in hiv-1 infected cells [95, 96] . molecular chaperones. we have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of t-cells upon tat expression (figure 3 and 4) , many of which were previously characterised as part of the tat nuclear interactome [63] . several heat-shock proteins including dnajs, specific hsp90, hsp70 and hsp40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of jurkat cells expressing tat ( figure 4 ). as shown by wb, while hsp90a and b are mostly cytoplasmic, tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (figure 2) . similarly, heat-shock can cause the hsp90 and hsp70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . in a recent study, fassati's group has shown that hsp90 is present at the hiv-1 promoter and may directly regulate viral gene expression [102] . we also observed the coordinated increased abundance of class i (groel and groes) and class ii (chaperonin containing tcp-1 (ctt)) chaperonin molecules (figure 3 and 4) upon tat expression. ubiquitin-proteasome pathway. the ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . the 26s proteasome is composed of the 20s core particle (cp) and the 19s regulatory particle (rp). alternatively, cp can associate with the 11s rp to form the immunoproteasome. all the quantified proteins in our study are part of the 19s regulatory complex and include psmd2 (1.5-fold), psmd3 (1.32-fold), psmd11 (1.25-fold) and psmd13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of tat (figure 4) . interestingly, tat interacts with distinct subunits of the proteasome system, including the 19s, 20s and 11s subunits. the consequences of these interactions include the competition of tat with 11s rp or 19s rp for binding to the 20s cp, which resulted in the inhibition of the 20s peptidase activity [106, 107, 108, 109, 110, 111] . furthermore, tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic t-lymphocytes [112] . importantly, a recent study demonstrated that in the absence of tat, proteasome components are associated to the hiv-1 promoter and proteasome activity limits transcription [113] . addition of tat promoted the dissociation of the 19s subunit from the 20s proteasome, followed by the distinct enrichment of the 19s-like complex in nuclear extracts together with the tat-mediated recruitment of the 19s subunits to the hiv-1 promoter, which facilitated its transcriptional elongation [113] . we also quantified uba1 (1.36-fold), the e3 ubiquitin-protein ligase uhrf1 (1.13-fold), ubc (1-fold) and two ubiquitinspecific-peptidases, usp30 (1.28-fold) and usp20 (0.06-fold) (figure 4) . dna replication and repair. upon hiv-1 tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with dna replication and repairs pathways (figure 4) . tat induced the coordinated enrichment of the miniature chromosome maintenance mcm2-7 complex (from 1.23-to 3.30fold, respectively) [114] . mcm7, 6 and 3 were identified as part of the in vitro nuclear interactome of hiv-1 tat [63] . the structural maintenance of chromosomes 2, smc2, was enriched (1.35-fold) in the nucleolar fraction by tat expression. smc2 was identified as part of the in vitro nuclear interactome of hiv-1 tat [63] . while replication factor c1 (rfc1) and rfc2 (1.31-and 1.28-fold respectively) displayed an increased fold change and rfc5/3 were not affected, rfc4 was severely depleted (0.69-fold) from the nucleolar fraction upon tat expression [115] . rfc1 and rfc2 were identified as part of the in vitro nuclear interactome of hiv-1 tat [63] . tat induced the enrichment of xrcc6 (1.27-fold) and xrcc5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous dna end joining (nhej) [116] . xrcc6 associates with viral preintegration complexes containing hiv-1 integrase and also interact with tat and tar [117, 118, 119] . furthermore, in a ribozyme-based screen, xrcc5 (ku80) knockdown decreased both retroviral integration and tatmediated transcription [120] . as part of the base excision repair (ber), we have identified a major apurinic/apyrimidinic endonuclease 1 (apex1) (1.29-fold) . importantly, in a sirna screen targeting dna repair factors, apex1 knockdown was found to inhibit hiv-1 infection by more 60% [121] . the high mobility group (hmg) protein, hmga1 (1.30-fold), was enriched in the nucleolus following tat expression [122] . hmga1 interact with hiv-1 integrase and is part of the hiv-1 pre-integration complex [123, 124] . importantly, hmga1 has been identified in a proteomic screen, as a cellular cofactor interacting with the hiv-1 59leader [125] . metabolism. our proteomic data suggest that tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (figure 4 and figure s7 ). notably, in t cells expressing tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase b (ldhb) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (gapdh) (1.17-fold) and phosphoglyceric acid mutase (pgam1) (0.89-fold) ( figure 4 and figure s7 ). briefly, gpi catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. subsequently, pfkp catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. at the end of the glycolytic pathway, pkm2, in its tetrameric form, is known to generate atp and pyruvate, while ldhb diverts the majority of the pyruvate to lactate production and regeneration of nad+ in support to continued glycolysis, a phenomenon described for proliferative tcells [126] . of note, in highly proliferating cells, pkm2 can be found in its dimeric form and its activity is altered. this upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. as part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (g6pd) (2.11-fold), which branches of the glycolysis pathway to generate nadph, ribose-5phosphate an important precursor for the synthesis of nucleotides. consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. more specifically, impdh2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (prps2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (ctps) (1.74-fold) which catalyses the conversion of utp to ctp and the ribonucleotide reductase large subunit (rrm1) (1.56-fold). in parralel, we noted the increased abundance of the phosphoserine aminotransferase psat1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro. the host-virus interface is a fundamental aspect in defining the molecular pathogenesis of hiv-1 [127, 128, 129, 130, 131, 132, 133] . indeed, with its limited repertoire of viral proteins, hiv-1 relies extensively on the host cell machinery for its replication. several recent studies have capitalized on the recent advances in the ''omics'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . hiv-1 tat is essential for viral replication and orchestrates hiv-1 gene expression. the viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . we and others have employed system-level approaches to investigate tat interplay with the host cell machinery, which have characterised hiv-1 tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . here, we have investigated the nucleolar proteins trafficking in response to hiv-1 tat expression in t-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by tat in the fine-tuning of protein availability and function. we have developed for this study, a cellular model using jurkat t-cells stably expressing tat fused in its n-ternminal to tap-tag. jurkat t-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. importantly, they have been widely employed to evaluate tat-mediated pathogenesis using system-wide approaches and to analyse t-cell key cellular signaling pathways and functions [144, 150, 151, 152] . indeed, we have found them particularly suited for prolongued in vitro culture in silac medium and subsequent isolation of their nucleolus followed by ms analysis, which requires up to 85 millions of cells. we fused tat to the tap tag to enable future downstream applications such as tandem affinity purification or chromatin ip analysis. importantly, we have confirm that n-terminal tap-tag did not interfere with tat function nor its localisation in jurkat cells, when compared to untagged-tat. of note, tat subcellular distribution can vary according to the cell type employed. while tat is known to accumulate in the nucleus and nucleolus in jurkat cells and other transformed cell lines, in primary t-cells, tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . these differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by kuusisto et al. [39] . furthermore, stauber and pavlakis have suggested that tat nucleolar localisation could be the results of tat overexpression [31] . here, we have selected and employed a polyclonal population of jurkat t-cells expressing tat at different levels. we propose that this heterogeneity in tat expression levels might reflect tat stochastic expression described during viral replication [153] . using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. the extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with hiv-1 pathogenesis. hiv-1 tat is expressed early following hiv-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. in this context and based on our results, we propose that tat could participate in shaping the intracellular environment and metabolic profile of t cells to favor host biosynthetic activities supporting robust virions production. indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. with the notable exeption of rpl35a nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (nhp2l1, rlp14, rpl17, rpl27, rps2, rpl13). furthermore, this effect appears to be specific to hiv-1 tat since transcription inhibition by actinomycin d resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. in parralel, we observed the concordant modulation of pathways regulating protein homeostasis. we noted the significant nucleolar accumulation of multiple molecular chaperones including the hsps, the tcp-1 complex, and canx/calr molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . these observations further support previous studies describibing the modulation of the proteasomal activity by tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . we also observed the concomitant depletion of casp10 in the nucleolus of jurkat tap-tat. it has been suggested that casp10 could be targeted to the nucleolus to inhibit protein synthesis [154] . interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . as viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability. coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. similarly, these pathways are elevated in proliferative t-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the warburg effect [156, 157, 158, 159] . there, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the tca cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl coa and nadph for redox homeostasis. consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including imph2, a rate limiting enzyme known to control the pool of gtp. similarly, we noted the nucleolar enrichment of psat1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . collectively, we propose that by controlling protein homeostasis and metabolic pathways, tat could meet both the energetic and biosynthetic demand of hiv-1 productive infection. of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . furthermore glycolytic enzymes, such as pkm2, ldh, phosphoglycerate kinase, gapdh, and aldolase, also have been reported to display nuclear localization and bind to dna [162] . more specifically, pkm2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] . hiv-1 tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote tcr signaling [164] . we have observed the nucleolar enrichment by tat of key proximal or downstream components of t-cell signaling pathways, including zap70, ilf3 and stat3, which play crucial roles in t-cell development and activation. we had previously identified them as t-cell specific components of the nucleolus, and if studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . our results further support that tat could contribute to the dysregulation of tcr-derived signals and that the nucleolus could represent an important spatial link for tcr signaling molecules. we observed the coordinated nucleolar enrichment of key components of the dna replication, recombination and repair pathways by tat. these include xrcc5 and xrcc6, hmga1, apex1, mcm2-7, smc2, rfc1 and rfc2, while rfc4 was found to be significantly depleted. interestingly, these cofactors have been associated with the efficiency of retroviral dna integration into the host dna or the integrity of integrated provirus [166] . whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined. the mechanisms of tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with tat, since subcellular fractionation combined with wb analysis showed that the pattern and extent of subcellular redistribution between proteins varied. we could observe cases where tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (ddx3, tnpo1). alternatively, tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (prb). additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon tat expression (stat3, zap70 and hsp90). finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (ssrp1). it remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. remarkably, we identified several known hiv-1 tat partners involved in hiv-1 pathogenesis, which suggests that tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. this is exemplified by the following enriched proteins, prb, pp1 and stat3, for which phosphorylation is induced by tat. importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by tat. moreover, our data indicates that serine/threonine kinases (ck2 a') and phosphatases (pp1) were significantly enriched in the nucleolar fractions of jurkat tap-tat. these enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. in addition, tat significantly decreased the levels of sumo-2 in the nucleolus. similarly, sumo-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . given the potential importance of post-translational modifications, including phosphorylation in the tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach. the control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. ribosomal proteins are degraded by the ubiquitin-proteasome pathway to ensure their abundance matches up with rrna transcription levels. conversely, heat shock proteins hsp90s protect them from degradation. interestingly, our data showing that tat modulation the abundance proteins associated with the ubiquitin-proteasome and heat-shock pathway. this could contribute to the observed enrichment of ribosomal proteins by tat. nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of tat-mediated prevention of their export to the cytoplasm. interestingly, using a different cellular system, a drosophila melanogaster tat transgenic strain, ponti et al, analysed the effects of tat on ribosome biogenesis, following 3 days heat shock treatment to induce tat expression under the control of the hsp70 promoter [167] . following tat expression, they observed a defect in pre-rrna processing associated with a decrease in the level of 80s ribosomes [167] . nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours. while previous system-level studies have monitored the effects of hiv-1 tat expression on t cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to hiv-1 tat expression. using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to tat constitutive expression. our findings underscore that tat expressing t-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. importantly, we noted the functional relationship of nucleolar proteins of our dataset with hiv-1 pathogenesis and hiv-1 tat in particular. this further increases our confidence in our experimental strategy and suggests a role for tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. ultimatly, our study provides new insights on the importance of tat in the cross talk between nucleolar functions and viral pathogenesis. importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with hiv-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention. jurkat t-cells, clone e6.1 (atcc), jurkat ntap-tat and jurkat ntap were maintained in rpmi-1640 medium supplemented with 10% (v/v) foetal bovine serum (gibco, eu approved), and antibiotics. phoenix-gp cells (g.p. nolan; www.stanford.edu/ group/nolan/), were maintained in dmem medium supplemented with 10% (v/v) foetal bovine serum (gibco, eu approved). cells were counted using scepter tm 2.0 cell counter (millipore). the sequence of hiv-1 tat (hiv-1 hxb2, 86 amino acids) was sub-cloned into pentr 2b vector (invitrogen, a10463). using the gateway technology (invitrogen), we introduced the hiv-1 tat sequence into the plasmid pcemm-ntap(gs)-gw [168] . phoenix cells (g.p. nolan; www.stanford.edu/group/ nolan/), were transfected using fugene 6 (roche) with 5 mg of the plasmid ntap-tat or ntap and 3 mg of the pmdg-vsvg. viral supernatants were collected after 48 h, filtered and used to transduce the jurkat cell lines. the construct is termed ntap-tat, the empty vector was termed ntap. using retroviral gene delivery, we stably transduced jurkat cells (clone e6.1 (atcc)). the positive clones named jurkat ntap-tat and jurkat ntap were sorted to enrich the population of cells expressing gfp using the bc moflo xdp cell sorter (beckman coulter). sub-cellular fractions (10 mg) were resolved by sds-page and transferred onto biotrace pvdf membranes (pall corporation). the following primary antibodies were used: a-tubulin (sc 5286), c23 (sc 6013), and fibrillarin (sc 25397) were from santa cruz biotechnology, and parp (am30) from calbiochem, mouse anti-zap 70 (05-253, millipore), rabbit anti-stat3 (06-596, millipore), rabbit anti-ilf3 (ab92355, abcam), rabbit anti-hsp90 beta (ab32568, abcam), mouse anti-adar1 (ab88574, abcam), rabbit anti-hdac1 (ab19845, abcam), rabbit anti-ssrp1 (ab21584, abcam) rabbit anti-bop1 (ab86982, abcam), mouse anti-kpnb1 (ab10303, abcam), rabbit anti-hiv-1 tat (ab43014, abcam), rabbit anti-ck2a (ab10466, abcam), rabbit anti-ddx3x (ab37160, abcam), mouse anti-tnpo1 (ab2811, abcam), mouse anti-hsp90a (ca1023, merck), and rabbit-anti rb1 (sc-102, santa cruz).the following secondary antibodies were used ecl: anti-mouse igg and ecl anti-rabbit igg (ge healthcare), and donkey anti-goat igg (sc 2020) (santa cruz biotechnology). for silac analysis silac-rpmi r0k0 and silac-rpmi r6k6 (dundee cells) media supplemented with 10% dialyzed fbs (gibco, 26400-036) were used. the jurkat cells expressing ntap-tat and ntap were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. cells viability was checked with trypan blue (0.4% solution, sigma) and further confirmed using pi staining and facs analysis. cells were mixed to the ratio 1:1 to obtain 140610 6 cells. nucleoli were isolated from the mixed cell population as previously described in jarboui et al., [165] . nucleolar extracts (100 mg) were resuspended in 50 mm ammonium bicarbonate and in solution trypsin digested as previously described in jarboui et al. [165] . sample was run on a thermo scientific ltq orbitrap xl mass spectrometer connected to an eksigent nano lc.1dplus chromatography system incorporating an auto-sampler. sample was loaded onto a biobasic c18 picofrittm column (100 mm length, 75 mm id) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nl min-1. the mass spectrometer was operated in positive ion mode with a capillary temperature of 200uc, a capillary voltage of 46v, a tube lens voltage of 140v and with a potential of 1800 v applied to the frit. all data was acquired with the mass spectrometer operating in automatic data dependent switching mode. a high resolution ms scan was performed using the orbitrap to select the 5 most intense ions prior to ms/ms analysis using the ion trap. the incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''heavy'' medium as described in [169] . our analysis showed that we had an incorporation efficiency .95% (data not shown). the ms/ms spectra were searched for peptides identification and quantification using the maxquant software [170] (version 1.1.1.36), the human ipi database (version 3.83) and the andromeda search engine associated to maxquant [171] . standard settings were used for maxquant with the acetyl (protein n-term) as variable modification and carbamidomethyl (cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 da, respectively. each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. proteins were identified with the minimum of one peptide with a false discovery rate less than 1%. gene ontology, kegg pathway and pfam terms were extracted from uniprot entries using perseus, a software from the maxquant data analysis package (http://www.maxquant.org ), and the toppgene suite tools [54] . the jurkat ntap-tat and jurkat ntap were transfected using the amaxa electroporation system (amaxa biosystem) with the pgl3 (pgl3-ltr) (promega) as recommended by amaxa biosystem. dual-luciferase assays (promega) were performed according to the manufacturer's instructions. luciferase activity was measured and normalized against the total amount of proteins as quantified by the bca protein quantification kit (pierce, thermo scientific). to preserve their original shape, we performed immunostaining of jurkat cells in suspension. cells were fixed in 2% pfa for 10 min at rt, permeabilised in 0.5% triton x-100 for 15 min at rt and blocked with 5% fcs. cells were incubated with the rabbit hiv-1 tat antibody (ab43014, abcam) followed by the secondary antibody anti-rabbit alexa fluor 647 (a-21246, invitrogen). cells were allowed to attach to cell-tak (bd) coated silanised slides (daocytomation), and stained with dapi. images were captured with a carl zeiss confocal microscope equipped with a plan-apochromat 63x/1.4 oil dic objective. the proteomics raw data file from the mass spectrometry analysis was deposited to the tranche repository(https:// proteomecommons.org/tranche/) [172] . the file can be accessed and downloaded using the following hash key: (r3o5sv5z6hvwqrbndhp21txfetludwyxvwmifu-h6e1kmgaraucsq4dlncxeuvfohdezledcg4x5y8resb6-mua6wm1kiaaaaaaaab/w = = ). materials and methods s1 description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis. (docx) the epigenetics of rrna genes: from molecular to chromosome biology nucleolus: the fascinating nuclear body the nucleolus: a paradigm for cell proliferation and aging. brazilian journal of medical and biological research = revista brasileira de pesquisas medicas e biologicas/sociedade brasileira de biofisica in search of nonribosomal nucleolar protein function and regulation deciphering the human nucleolar proteome functional proteomic analysis of human nucleolus directed proteomic analysis of the human nucleolus nopdb: nucleolar proteome database-2008 update nucleolar proteome dynamics nopdb: nucleolar proteome database quantitative nucleolar proteomics reveals nuclear re-organization during stress-induced senescence in mouse fibroblast -dependent subcellular proteome localization following dna damage the nucleolus takes control of protein trafficking under cellular stress quantitative proteomics and dynamic imaging of the nucleolus reveals distinct responses to uv and ionizing radiation a quantitative proteomics analysis of subcellular proteome localization and changes induced by dna damage the nucleolus: reviewing oldies to have new understandings involvement of the nucleolus in replication of human viruses nucleolar proteomics and viral infection nucleophosmin phosphorylation by v-cyclin-cdk6 controls kshv latency elucidation of the avian nucleolar proteome by quantitative proteomics using silac and changes in cells infected with the coronavirus infectious bronchitis virus quantitative proteomics using silac coupled to lc-ms/ms reveals changes in the nucleolar proteome in influenza a virus-infected cells quantitative proteomic analysis of a549 cells infected with human respiratory syncytial virus quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals changes in the cytoplasmic, nuclear, and nucleolar proteomes in vero cells infected with the coronavirus infectious bronchitis virus proteomics analysis of the nucleolus in adenovirus-infected cells viral nucleolar localisation signals determine dynamic trafficking within the nucleolus protein b23 is an important human factor for the nucleolar localization of the human immunodeficiency virus protein tat spatial association of hiv-1 tat protein and the nucleolar transport protein b23 in stably transfected jurkat t-cells specific complex of human immunodeficiency virus type 1 rev and nucleolar b23 proteins: dissociation by the rev response element effects of a highly basic region of human immunodeficiency virus tat protein on nucleolar localization a region of basic amino-acid cluster in hiv-1 tat protein is essential for trans-acting activity and nucleolar localization intracellular trafficking and interactions of the hiv-1 tat protein phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of hiv-1 tat by infected t-cells the ins and outs of hiv-1 tat tuning the transport properties of hiv-1 tat arginine-rich motif in living cells in vivo study of hiv-1 tat arginine-rich motif unveils its transport properties direct interaction of the human i-mfa domain-containing protein tat results in cytoplasmic sequestration and control of tat activity the arginine-rich domains present in human immunodeficiency virus type 1 tat and rev function as direct importin betadependent nuclear localization signals the hiv-1 tat nuclear localization sequence confers novel nuclear import properties global enhancement of nuclear localization-dependent nuclear transport in transformed cells intermolecular masking of the hiv-1 rev nls by the cellular protein hic: novel insights into the regulation of rev nuclear import nuclear factor 90, a cellular dsrna binding protein inhibits the hiv rev-export function functional analysis of the interaction of the human immunodeficiency virus type 1 rev nuclear export signal with its cofactors the ins and outs of hiv rev the hiv-1 rev protein the specificity of the crm1-rev nuclear export signal interaction is mediated by rangtp requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function mechanisms of receptor-mediated nuclear import and nuclear export the nuclear pore component nup358 promotes transportin-dependent nuclear import a nucleolar tar decoy inhibitor of hiv-1 replication ribozyme-mediated inhibition of hiv 1 suggests nucleolar trafficking of hiv-1 rna constitutive expression of hiv-1 tat protein in human jurkat t cells using a bk virus vector gautier vw proteomic profiling of the human t-cell nucleolus nucleolin undergoes partial n-and o-glycosylations in the extranuclear cell compartment toppgene suite for gene list enrichment analysis and candidate gene prioritization interpro: the integrative protein signature database mitotic phosphatases: no longer silent partners emerging roles of nuclear protein phosphatases nuclear targeting of protein phosphatase-1 by hiv-1 tat protein expression of a protein phosphatase 1 inhibitor, cdnipp1, increases cdk9 threonine 186 phosphorylation and inhibits hiv-1 transcription regulation of hiv-1 transcription by protein phosphatase 1 dephosphorylation of cdk9 by protein phosphatase 2a and protein phosphatase-1 in tat-activated hiv-1 transcription nuclear protein phosphatase-1 regulates hiv-1 transcription vitro nuclear interactome of the hiv-1 tat protein association of tat with promoters of pten and pp2a subunits is key to transcriptional activation of apoptotic pathways in hiv-infected cd4+ t cells pp2a targeting by viral proteins: a widespread biological strategy from dna/rna tumor viruses to hiv-1 protein phosphatase 2a enhances activation of human immunodeficiency virus type 1 by phorbol myristate acetate rb1, development, and cancer human immunodeficiency virus type 1 tat protein modulates cell cycle and apoptosis in epstein-barr virus-immortalized b cells hiv-1 tat elongates the g1 phase and indirectly promotes hiv-1 gene expression in cells of glial origin retinoblastoma gene inhibits transactivation of hiv-ltr linked gene expression upon co-transfection in he la cells nucleolar size and activity are related to prb and p53 status in human breast cancer nucleolus, ribosomes, and cancer intracellular tat of human immunodeficiency virus type 1 activates lytic cycle replication of kaposi's sarcoma-associated herpesvirus: role of jak/stat signaling getting the message across, stat! design principles of a molecular signaling circuit interaction of yb-1 with human immunodeficiency virus type 1 tat and tar rna modulates viral promoter activity the structure, regulation, and function of zap-70 bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells matrin 3 and hiv rev regulation of mrna characterization of the hiv-1 rna associated proteome identifies matrin 3 as a nuclear cofactor of rev function matrin 3 is a co-factor for hiv-1 rev in regulating post-transcriptional viral gene expression playing the disc: turning on trail death receptor-mediated apoptosis in cancer hiv-1 tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-flip in lymphoid t cells: a potential molecular mechanism to escape trail cytotoxicity enhancement of replication of rna viruses by adar1 via rna editing and inhibition of rna-activated protein kinase multiple levels of pkr inhibition during hiv-1 replication adar2 editing enzyme is a novel human immunodeficiency virus-1 proviral factor alternate rrna secondary structures as regulators of translation adar1 interacts with pkr during human immunodeficiency virus infection of lymphocytes and contributes to viral replication double-stranded rna adenosine deaminases enhance expression of human immunodeficiency virus type 1 proteins cytoscape: a software environment for integrated models of biomolecular interaction networks michigan molecular interactions (mimi): putting the jigsaw puzzle together analyzing biological network parameters with centiscape network biology: understanding the cell's functional organization interdependence of pes1, bop1, and wdr12 controls nucleolar localization and assembly of the pebow complex required for maturation of the 60s ribosomal subunit comprehensive proteomic analysis of the human spliceosome the expression of the essential nuclear splicing factor sc35 is altered by human immunodeficiency virus infection hiv-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages nucleolar targeting of the chaperone hsc70 is regulated by stress, cell signaling, and a composite targeting signal which is controlled by autoinhibition stress inhibits nucleocytoplasmic shuttling of heat shock protein hsc70 heat-induced nuclear accumulation of hsc70s is regulated by phosphorylation and inhibited in confluent cells intracellular localization of the 90 kda heat shock protein (hsp90alpha) determined by expression of a egfp-hsp90alpha-fusion protein in unstressed and heat stressed 3t3 cells localization of heat shock proteins in mouse male germ cells: an immunoelectron microscopical study gyrase b inhibitor impairs hiv-1 replication by targeting hsp90 and the capsid protein ubiquitin-like and ubiquitin-associated domain proteins: significance in proteasomal degradation ubiquitin and ubiquitin-like proteins in the nucleolus: multitasking tools for a ribosome factory analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins the rtp site shared by the hiv-1 tat protein and the 11s regulator subunit alpha is crucial for their effects on proteasome function including antigen processing hiv-1 tat inhibits the 20 s proteasome and its 11 s regulator-mediated activation peptide sequencing identifies mss1, a modulator of hiv tat-mediated transactivation, as subunit 7 of the 26 s protease new human gene encoding a positive modulator of hiv tat-mediated transactivation a cdna for a protein that interacts with the human immunodeficiency virus tat transactivator human immunodeficiency virus-1 tat protein interacts with distinct proteasomal alpha and beta subunits hiv-1 tat protein modulates the generation of cytotoxic t cell epitopes by modifying proteasome composition and enzymatic activity the proteasome regulates hiv-1 transcription by both proteolytic and nonproteolytic mechanisms oncogenic activity of mcm7 transforming cluster unzipped and loaded: the role of dna helicases and rfc clamp-loading complexes in sister chromatid cohesion the xrcc genes: expanding roles in dna double-strand break repair role of the non-homologous dna end joining pathway in the early steps of retroviral infection lupus autoantigen ku protein binds hiv-1 tar rna in vitro effect of ku80 depletion on the preintegrative steps of hiv-1 replication in human cells identification of cellular cofactors for human immunodeficiency virus replication via a ribozyme-based genomics approach sirna screening of a targeted library of dna repair factors in hiv infection reveals a role for base excision repair in hiv integration the dynamics of hmg proteinchromatin interactions in living cells retroviral cdna integration: stimulation by hmg i family proteins hiv-1 cdna integration: requirement of hmg i(y) protein for function of preintegration complexes in vitro activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by ires trans-acting factors fuel feeds function: energy metabolism and the t-cell response human immunodeficiency virus type 1, human protein interaction database at ncbi patterns of hiv-1 protein interaction identify perturbed host-cellular subsystems the biological context of hiv-1 host interactions reveals subtle insights into a system hijack hivhost interactions: a map of viral perturbation of the host system cataloguing the hiv type 1 human protein interaction network retroviral proteomics and interactomes: intricate balances of cell survival and viral replication dual role of host cell factors in hiv-1 replication: restriction and enhancement of the viral cycle decoding the multifaceted hiv-1 virus-host interactome functions of tat: the versatile protein of human immunodeficiency virus type 1 chromatin dynamics associated with hiv-1 tat-activated transcription modifications in host cell cytoskeleton structure and function mediated by intracellular hiv-1 tat protein are greatly dependent on the second coding exon pathway analysis in hek 293t cells overexpressing hiv-1 tat and nucleocapsid expression profiles and pathway analysis in hek 293 t cells overexpressing hiv-1 tat and nucleocapsid using cdna microarray proliferative activity of extracellular hiv-1 tat protein in human epithelial cells: expression profile of pathogenetically relevant genes proteomics analysis of human astrocytes expressing the hiv protein tat hiv-1 tat reprograms immature dendritic cells to express chemoattractants for activated t cells and macrophages gene expression profile of hiv-1 tat expressing cells: a close interplay between proliferative and differentiation signals modifications in the human t cell proteome induced by intracellular hiv-1 tat protein expression cell-type-specific proteome and interactome: using hiv-1 tat as a test case differentially expressed genes in hiv-1 tat-expressing cd4(+) t-cell line hiv-1 tat assembles a multifunctional transcription elongation complex and stably associates with the 7sk snrnp the histone chaperone protein nucleosome assembly protein-1 (hnap-1) binds hiv-1 tat and promotes viral transcription genome-wide binding map of the hiv-1 tat protein to the human genome jurkat t cells and development of the t-cell receptor signalling paradigm global survey of human t leukemic cells by integrating proteomics and transcriptomics profiling a genome-wide short hairpin rna screening of jurkat t-cells for human proteins contributing to productive hiv-1 replication stochastic gene expression in a lentiviral positive-feedback loop: hiv-1 tat fluctuations drive phenotypic diversity dedd and dedd2 associate with caspase-8/10 and signal cell death chaperones and multitasking proteins in the nucleolus: networking together for survival? evidence for an alternative glycolytic pathway in rapidly proliferating cells glutamine uptake and metabolism are coordinately regulated by erk/mapk during t lymphocyte activation on the origin of cancer cells understanding the warburg effect: the metabolic requirements of cell proliferation characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of lserine biosynthesis proteomics profiling of nuclear proteins for kidney fibroblasts suggests hypoxia, meiosis, and cancer may meet in the nucleus glycolytic enzymes as dna binding proteins emerging roles of pkm2 in cell metabolism and cancer progression viral modulation of t-cell receptor signaling proteomic profiling of the human t-cell nucleolus the role of unintegrated dna in hiv infection the hiv tat protein affects processing of ribosomal rna precursor an efficient tandem affinity purification procedure for interaction proteomics in mammalian cells use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification andromeda: a peptide search engine integrated into the maxquant environment proteomecommons.org collaborative annotation and project management resource integrated with the tranche repository the authors wish to acknowledge access to and use of the ucd conway mass spectrometry resource instrumentation at the mass spectrometry resource (msr), conway institute for biomolecular and biomedical research, university college dublin is gratefully acknowledged. the authors wish to acknowledge prof. dimitri scholz director of biological imaging, conway institute, ucd, for his assistance with image acquisition and analysis. key: cord-347992-coby2m6e authors: marton, soledad; reyes-darias, josé a.; sánchez-luque, francisco j.; romero-lópez, cristina; berzal-herranz, alfredo title: in vitro and ex vivo selection procedures for identifying potentially therapeutic dna and rna molecules date: 2010-06-28 journal: molecules doi: 10.3390/molecules15074610 sha: doc_id: 347992 cord_uid: coby2m6e it was only relatively recently discovered that nucleic acids participate in a variety of biological functions, besides the storage and transmission of genetic information. quite apart from the nucleotide sequence, it is now clear that the structure of a nucleic acid plays an essential role in its functionality, enabling catalysis and specific binding reactions. in vitro selection and evolution strategies have been extremely useful in the analysis of functional rna and dna molecules, helping to expand our knowledge of their functional repertoire and to identify and optimize dna and rna molecules with potential therapeutic and diagnostic applications. the great progress made in this field has prompted the development of ex vivo methods for selecting functional nucleic acids in the cellular environment. this review summarizes the most important and most recent applications of in vitro and ex vivo selection strategies aimed at exploring the therapeutic potential of nucleic acids. nucleic acids, particularly rna, are extremely versatile molecules. apart from their role as carriers of genetic information they can also express a phenotype, e.g., they may show a catalytic activity, have a specific binding function, or have the capacity to recruit specific molecules. the appearance of sequence variation in a nucleic acid can, in some cases, provide an advantage to an organism: a key feature in natural evolution. over the last 20 years, advances in molecular biology and biotechnology have seen the development of methods that allow the effect of such naturally arising variation to be mimicked in the laboratory. it was in the 1960s when spiegelman's group first observed evolution in vitro [1] . these authors reported that changes in the rna genome of the qβ bacteriophage during replication led to the formation of rna molecules that were more efficiently copied by the viral replicase. these genomes lacked unnecessary sequences and were synthesized at a greater rate. however, these finding fell into oblivion until 1990, when the great potential of in vitro selection and evolution techniques was reported by three independent groups [2] [3] [4] [5] . since then, numerous authors have used these technologies to study the chemical and catalytic properties of nucleic acids (for further information see [6] [7] [8] [9] [10] [11] [12] [13] . they have also helped uphold the rna world hypothesis, and it seems likely that they may soon have applications in biomedicine. indeed, new selection methods -known as ex vivo selection procedures -are now being used to identify molecules that target viruses, subcellular fractions and even whole cells. these techniques overcome some of the limits imposed by in vitro technology and provide new environments and conditions in which to explore the properties and functions of nucleic acids. this review highlights the most recent advances in in vitro and ex vivo selection procedures for nucleic acids, and discusses their potential application in biomedicine. in vitro selection strategies have been used to select nucleic acids with a large variety of properties. although each strategy differs according to the feature or phenotype sought, all in vitro selection methods follow the same three-step pattern ( figure 1 ). genetic variability is introduced into the system (generally by chemical synthesis) to yield nucleic acid populations, the heterogeneity of which is determined by fixing the number of nucleotides to be mutagenized and the mutation rate per nucleotide. in most cases, completely random synthesis involving a fixed number of nucleotides yields a starting population of variant molecules that, a priori, differ only in the sequence and the structure of the variable region. constant sequences, or primer binding sites (pbs), flanking the variable region are incorporated during the design of the starting population to facilitate the amplification of desired molecules, although this limits the structural diversity of the rna populations to specific conformations [14] . different approaches have been tried to minimize the effect of pbss, e.g., the addition of customized primers or adapters by ligation before amplification, and their removal prior to the selection step [15] [16] [17] . the selection strategy needs to be specifically designed according to the phenotype sought. the initial pool of variants usually contains very few active molecules corresponding to the desired phenotype, and their enrichment can only be made possible by properly designing the selection step. the selection of inactive molecules may also be important since the analysis of these molecules can provide very valuable information on the sequence and structural requirements of active molecules [18] . in vitro selection strategies have been widely used for the selection of nucleic acids capable of catalyzing specific chemical reactions, i.e., dnazymes and ribozymes [19] . these strategies have also been successfully used to identify dna and rna molecules with affinity for a specific ligand (i.e., aptamers) [3] , being known as selex, which stands for systematic evolution of ligands by exponential enrichment [5] . sequence variants are separated into active, those that satisfy selection criteria, and inactive molecules. active molecules are selectively amplified by pcr resulting in the production of a new pool of molecules that can be the input of a new selection cycle. an additional reverse transcription and in vitro transcription steps are required, before and after pcr, respectively, when the selection is performed on rna molecules. alternatively, active and inactive resulting pools can be cloned and analyzed. variants that show the required phenotype need to be replicated to ensure their passage into the next generation and therefore their persistence in the population. specific primer binding sites are used to amplify the selected molecules. when necessary a rna polymerase promoter is incorporated at the 5' end of the pbs during the amplification step. in addition to these general steps, the amplified molecules may require additional manipulations prior to their introduction into the next round of selection when the selected molecule is ssdna, both dna strands must be separated and the positive one isolated, e.g., by incorporating a biotinylated residue into the unwanted strand [20, 21] via asymmetric pcr [22] . as a result of the described selection cycle, a population enriched in the sought-after molecules (but not composed of them entirely) is produced; a new selection cycle can then be undertaken. by iteratively executing the process of selection and amplification the complexity of the original population is reduced and enriched with candidates of interest. during this process, the stringency of selection can be increased to achieve the isolation of the molecules with the desired phenotype. the great advances made in in vitro selection procedures over recent decades have helped us improve our knowledge of the plasticity of nucleic acids and their potential applications as therapeutic tools. indeed, such have been the advances that selection strategies within living cells are now contemplated. these new methodologies are named according to the specific procedure employed in each method, e.g., ex vivo, in vivo, in cell selection, or cell-selex etc. these are here all referred to as ex vivo selection strategies. ex vivo selection methodologies follow the general pattern described above for in vitro techniques. these systems have mainly been used in the isolation of dna and rna molecules that interfere with the activity of a target molecule. when the expression of an oligonucleotide leads to a desired cell phenotype, it allows for the selection of these cells and the subsequent isolation of the oligonucleotide itself. following these principles a strategy was used to identify a transcriptional activator regulated by tmr (tetramethylrosamine) [23] . for this purpose, a chimera was constructed by tethering a tmr aptamer to the transcriptional activator for ms2 protein (also known as n40-26) [24] . variability was introduced by randomizing seven nucleotides within the linker region of the two rna domains. selection was performed in yeast cells containing a construct coding for the his3 and lacz genes under the control of the lexa operator, and expressing a lexa-ms2 coat protein fusion. this is a hybrid protein that binds to the operator and to the n40-26 ms2 rna hairpin present in the rna construct. only cells expressing an active transcriptional activator were capable of growth in the absence of histidine, and were the only ones capable of expressing β-galactosidase. the stringency of the selection process was increased by the presence of varying amounts of a competitive inhibitor of the his3p activity. analysis of the selected yeast clones revealed specific rna sequences that responded to tmr enhancing the transcription activator effect [23] . the selection of an artificial ribosome switched on or off via the external addition of a small molecule to the growth medium deserves special mention. the escherichia coli 16s ribosomal rna was fused to an aptazyme, a ribozyme capable of binding thiamine pyrophosphate (ttp) through an aptamer domain. the binding of ttp activates the ribozyme, reducing gene expression by cleaving the 16s ribosomal rna and thus working as a riboswitch. this allows for the identification and selection of specific e. coli colonies according to the phenotype expressed [25] . the ttp riboswitch was also included in the 5`utr region of a reporter gene, 30 nt upstream of the shine-dalgarno sequence. this linker between these elements was randomized and e. coli colonies selected depending on the expression of the reporter gene in response to ttp [26] . ex vivo selection methods in cells are limited by the number of sequences that can be studied, this number being determined by the number of available cells. another problem is the possibility of there being more than one sequence variant per cell, which could lead to many false positive. ellington's group developed a very interesting strategy that allowed the direct selection of active molecules instead of selecting cell clones [27] . the cleavage products of the autocatalytic ribozymes synthesized in the cell nucleus were extracted via hybridization to a biotinylated oligonucleotide, allowing the direct identification of active molecules. the selection of nucleic acids against whole cell targets has been successfully used to select nucleic acids that target cell surface receptors. this technology also known as cell-selex could have a role to play in the treatment of cancer. for most types of cancer cell there is a shortage of highly specific surface markers that can be used with diagnostic and therapeutic intent. aptamers generated from whole living cells are the optimal molecular probe for characterizing target cells at the molecular level. when bound to the membrane receptors of cell lines they provide an effective means of identifying disease markers. the pharmacokinetic and pharmacodynamic properties of a nucleic acid, and its resistance to nucleases, all condition its effectiveness as a therapeutic molecule. after selection, the most effective molecules can be modified to improve their nuclease resistance as well as their affinity for their targets, their cellular uptake and selectivity. a great diversity of post-selection modifications has been described. only those of interest for therapeutic purpose are discussed here. these include modifications of oligonucleotide size and sequence, and mainly certain chemical modifications (for a review see [28, 29] . advances in chemical synthesis have allowed the production of conjugates that combine an oligonucleotide sequence with compounds such as fluorophores, peptides, carbohydrates and lipids ( figure 2 ). these modified oligonucleotides show advantageous properties with respect to the native form. the ligands are usually linked to the 5' or 3' termini, which are the most accessible regions for chemical conjugation reactions; in addition, any disruption of the nucleic acid's folding and functional properties are minimized [30] . conjugations at the 2' position of ribose or involving the internucleotidic phosphodiester bonds are also possible. fluorophore conjugation is already used in clinical diagnosis, e.g., in fluorescence in situ hybridization (fish) and molecular beacons. fish can detect specific genes in cells, while molecular beacons act like switches, emitting fluorescent light when bound to their target sequence. cell uptake has been improved by the conjugation of oligonucleotides to peptides capable of translocating them across the cell membrane by a non-receptormediated endocytotic mechanism. some of the most frequently used peptides are residues 43-58 of the third helix of the antennapedia homeodomain (penetratin), the highly arginine/lysine rich region of the hiv-1 tat protein, the hydrophobic signal peptide, the nuclear localization sequence (nls), and transportan [31] . besides improving cellular uptake, peptide-oligonucleotide conjugates show increased stability to nucleases degradation and enhanced binding [32] . carbohydrate-oligonucleotide conjugates (cocs) have similar applications. in addition they confer cell or tissue specificity by their binding to sugar receptors (i.e., lectins) present at the cell surface capable of recognizing and internalizing (by endocytosis) glycoproteins bearing specific carbohydrates moieties [33] . lipophilic oligonucleotide conjugates (locs), such as cholesterol, reduce the hydrophobic character of the oligonucleotide, and some bind to blood lipoprotein carriers. lipophilic oligonucleotides are also used for designing supramolecular assemblages such as micelles, vesicles and liposome networks [34] . the ability of certain rna polymerases to incorporate modified nucleotides to the growing chain, such as the 2'-modified ribonucleosides, makes it possible to use the selection procedure with populations of chemically modified oligonucleotides [35, 36] . chemical modifications of the 2' hydroxyl group of rna, such as 2' fluoro, amino, methoxy and amido modifications, are noteworthy for their potential therapeutic applications since they increase rna stability, conferring greater resistance to nucleases. another important modification for therapeutic purposes is the use of locked nucleotides (lnas) in the nucleotidic chain. lnas contain a methylene link between the 2'-o and 4'-c of the ribose ring which locks the sugar moiety in the 3' endo conformation [37] . this generates the most stable hybrids ever characterized, with a δt m of +3 to +10 per lna residue upon binding to dna and rna respectively, thus conferring nuclease resistance [38, 39] . the introduction of lna modifications into in vitro selection techniques has so far been restricted to post selection incorporation (for a review see [40] ). nevertheless, it has recently been shown that locked nucleotides can be incorporated enzymatically into both dna and rna [41] [42] [43] . spiegelmers (spiegel = mirror in german), unnatural but biostable l-forms of d-aptamers, were developed to allow oligonucleotides to escape nuclease attack. naturally occurring proteins are lchiral, therefore natural nucleases digest d-oligonucleotides while l-nucleosides escape this fate. to obtain spiegelmers, natural oligonucleotides are used during the selection cycle against unnatural dproteins that mirror the natural structure of the l-target [44, 45] . the selection process yields l-form aptamer sequences that by virtue of the law of symmetry recognize their natural target. spiegelmers have been selected against peptide hormones such as gonadotropin-releasing hormone (gnrh) [46] , and ghrelin, an endogenous ligand for growth hormone secretagogue receptor 1a [47] . both have a neutralizing effect in vivo against these hormones after systemic administration. in vitro selection strategies have been extensively and successfully used to characterize known ribozymes and dnazymes, and to isolate new catalytic nucleic acids with unsuspected activities. in fact, the first observation of a dna molecule catalyzing a chemical reaction (dnazyme) was made when using an in vitro selection strategy [48] . although ribozymes and dnazymes have been extensively assayed as potential therapeutic agents, and different clinical trials have already tested their efficiency against various diseases [49] [50] [51] [52] , very few reports have described the direct application of in vitro selection strategies in the development of potentially therapeutic catalytic nucleic acids. ellington's group recently described a procedure aimed at identifying cleaving ribozymes active within the cell milieu, but this has not yet been used with therapeutic intent [27] . most of the work referred to herein describes the use of in vitro and ex vivo selection strategies for the identification of aptamers of therapeutic potential. assays with catalytic nucleic acids engineered by so-called 'rational design' are beyond the scope of this review. the idea that aptamers can modulate the activity of target proteins emerged from basic studies of viruses. in the 1980s, research on hiv and adenovirus led to the discovery that these viruses contain several structural rna domains that bind to viral or cellular proteins with high affinity and specificity. not surprisingly, functional analyses of these viral rna ligands demonstrated that the viruses had evolved these aptamers either to modulate the activity of proteins essential for their replication [53] or to inhibit the activity of proteins involved in cellular antiviral responses [54] . the first study performed to determine whether an rna aptamer could be used to inhibit the activity of a pathogenic protein was published in 1990. this work reported that the tar aptamer, evolved by hiv to recruit viral and cellular proteins to viral transcripts, could be turned against the virus to inhibit its replication [55] . the in vitro aptamer selection strategies developed during the 1990s prompted the idea of sullenger's group that therapeutic aptamers might be possible. several such aptamers have now completed various stages of preclinical development, and a number of others are currently being tested clinically (table 1) . indeed, one aptamer is already on the market as a therapeutic drug several modifications of the general selection process scheme have been described in order to achieve different goals. for example, the toggle-selex strategy is used for the selection of potentially therapeutic nucleic acids [56] . toggle-selex was designed for the isolation of aptamers with a broad range of specificities for closely related targets, such as the homologous proteins of different species. these aptamers were obtained by performing the selection procedure for related targets (i.e., homologous proteins) in alternating cycles. such alternation ensures that the rna or dna variants resulting from selection will bind to both proteins, most likely to domains conserved between them. sullenger's group described an in vitro selection strategy in which rna aptamers that bind both human and porcine thrombin were selected by "toggling" the protein target between the human and porcine forms in successive rounds of selection [56] . this yielded a family of aptamers, all of which bound both thrombin types with high affinity. toggle-25, a characteristic member, inhibited two of thrombin's most important functions: plasma clot formation and platelet activation [56] . this strategy could facilitate the isolation of ligands with properties required for gene therapy or other therapeutic or diagnostic applications. to date, the only aptamer approved by the fda [73] , known as pegaptanib or macugen, was approved in december 2004 for the treatment of wet type age-related macular degeneration (amd). this aptamer binds to vascular endothelial growth factor, vegf 165 [57, 74] , the main isoform of a family of growth factors involved in promoting blood vessel development and maintenance via tyrosine kinase receptor signaling. vegf 165 is also involved in several pathological processes such as amd, diabetic retinopathy and cancer [75] . a phase ii clinical trial to evaluate the use of this aptamer in the fight against diabetic retinopathy is currently underway [76] . the selection procedure involved a 2'-fluoro-pyrimidine (2'-fy)-modified rna pool. additional modifications were made after selection by adding 2'-o-methyl (2'-mr) to all purine residues of the aptamer except two, by adding a 3' cap, and by adding polyethylene glycol (peg; 40 kd) to the 5' end ( figure 3 ). the macugen aptamer binds to the heparin-binding domain of vegf 165 [75, 77] and efficiently inhibits the growth of blood vessels [57, 74] . this agent is of particular interest with respect to preventing tumor angiogenesis. different aptamers are currently being tested for the treatment of other degenerative diseases. niu's group attempted to interfere with the function of the glur2 ampa receptor associated with cerebral ischemia and amylotrophic lateral sclerosis [78] . a selected aptamer known as an58 acts as a glutamate antagonist preventing glutamate-induced activation of the cationic channel. interestingly, the aptamer adopts two mutually exclusive non-interchangeable isoforms that are both necessary for proper inhibition to occur [79] . recently, new rna aptamers have been isolated that bind to the nogo-66 neural receptor as antagonists of myelin-derived ligands. nogo-66 signaling blocks neurite outgrowth, but the binding of the aptamers allow axon growth in rat ganglion cells in vitro [80] . this aptamer is of special interest in the search for agents that aid neural repair, e.g., after spinal cord trauma. aptamers can also be targeted against disease-causing proteins such the scrapie isoform of the prion protein (prp sc ). a 2'-fy rna aptamer known as saf-93 [81] has been shown to prevent its aggregation in cell free systems, while a 2'-amino-2'-deoxypyrimidine-modified aptamer known as dp7 slows down its aggregation in neuroblastoma cells [35] . both aptamers target the same conserved region involved in prion interactions. neutrophil elastase (hne) is involved in the pathogenesis of inflammatory diseases such as acute respiratory distress syndrome (ards), septic shock, emphysema and arthritis, as well as ischemiareperfusion injuries [82] . a covalent inhibitor of hne, a diphenyl phosphate derivative of valine, has been coupled to an rna library to enhance its binding to hne [83] . after in vitro selection, an rna aptamer conjugated with dna:valp (rna10.11:dna:valp) was isolated that binds hne with high affinity. the bound molecule, unlike the aptamer rna 10.11 or dna:valp alone, also inhibits lung inflammation in an ex vivo rat model of ards [83] . more efficient inhibitors of hne were obtained from a valyl phosphonate:dna pool [84] . after selection, aptamer inhibitor ed45 inhibited hne formation two orders of magnitude greater than rna.10.11:dna:valp [83] . a truncated dna aptamer version named nx21909, composed of two annealed dna oligonucleotides, was tested in a rat model of lung inflammation and was found to inhibit neutrophil infiltration by 53% at a dose of 40 nmol [85] . immunoglobulin e (ige) plays an important role in protecting mammals from parasites [86] . however, its overproduction due to exposure to environmental antigens can result in allergies, atopic dermatitis and allergic asthma [87] . dna selection was performed against human ige to produce aptamers that bind it with high affinity [88] . these aptamers inhibited the binding of ige to its receptor fcεri, and also prevented ige-mediated cellular degranulation in the serum of patients with allergy to grass pollen. in patients exposed to grass pollen extract, the ic50s for the dna aptamers were 2-6 µm, but when triggered by anti-ige antibodies they reached 200-300 nm. these dna aptamers represent a novel class of ige inhibitors that may prove useful in the fight against allergic diseases. cancer has been one of the diseases most targeted by aptamers. alteration of the signaling pathways results in the escape of tumor cells from the control of cell division and apoptosis. the formation of metastases is also promoted. all of these processes have been the target of aptamers. the tyrosine kinase receptors (tkr) has been targeted by two aptamers: ret (rearranged during transfection) and her 3 (human epidermal growth factor receptor). the ret aptamer, d4, a 2'-fy aptamer, was isolated by ex vivo selection against the extracellular mutant ret c634y receptor [89] . briefly, an rna population was incubated against a parental pc12 cell line and variants expressing different ret mutants as a negative selection step. the unbound fraction of rna molecules was recovered and incubated against pc12 cells expressing the recombinant ret c634y receptor. the selected d4 aptamer inhibits neurite outgrowth and reverts the neoplastic phenotype of nih/men2a cells in vitro [89] and in three dimensional collagen gel matrix cultures [90] . the her-3 aptamer, a 2'-fy aptamer, known as a30, was isolated against the her-3 monomeric extracellular domain; this prevents her-2 signaling via her-3 heterodimerization in cell culture [91] . cell adhesion misregulation involved in metastasis has also been prevented by aptamers. the plasminogen activator inhibitor-1 (pai-1) is overexpressed in breast cancer cells and binds to vitronectin, leading to the loss of adhesion. a 2'-fy aptamer known as sm-30, specific for plasminogen activator inhibitor-1 (pai-1), restores cell adhesion to vitronectin-coated plates in vitro [92, 93] . another type of therapeutic signaling modulation is the targeting of nuclear factor κb (nf-κb) inside cells. maher's group isolated two rna aptamers, α-p50 and r1, by two independent in vitro selection procedures. these bind nf-κb p50 and p65 isoforms in vitro respectively [94, 95] . these aptamers underwent further ex vivo selection using the yeast three-hybrid system [96, 97] . the inhibition of nf-κb might be of therapeutic interest in different types of cancer, hiv-1 infection and inflammatory diseases. in fact, gene therapy with different anti-nf-κb aptamers administered via an adenoviral vector suppresses doxorubicin resistance in vivo in a lung tumor xenograft mouse model [98, 99] . the inhibition of nucleophosmin oligomerization by an aptamer promotes higher p53 levels and, consistently, sensitizes cells to dna-damaging-agent-induced apoptosis in cell culture [100] . the stimulation of the immune system can also be used in anti-cancer therapy. the activation of cd8 + t cells within a tumor would promote its cytotoxic involution. in this respect, the modulation of cytotoxic t-cell antigen-4 (ctla-4), 4-1bb and ox40 receptors by aptamers has been explored. multimeric aptamer forms frequently improve aptamer signaling properties and have proven especially importance in this area. it has been shown that an antagonist rna aptamer against ctla-4, a negative regulator of t-cell activation, inhibits its function [101] , while an agonistic aptamer against 4-1bb, a major co-stimulatory receptor, leads to the activation of t cells [102] . immunity against the tumor is induced in vivo in both cases. a specific aptamer for the dimeric murine ox40 combined with a dendritic cell-based tumor vaccine promotes tumor immunity in a xenograft melanoma model in mice [103] . aptamers have also been shown able to promote tumor cell death. when expressed in cells, an aptamer selected against nucleophosmin was shown to prevent the latter's oligomerization. higher p53 levels were therefore promoted that led to apoptosis. in addition, the sensitivity of cells to dnadamaging agents in cell culture was increased [100] . research into anti-angiogenesis aptamers has provided some very interesting results. sullenger's group reported the selection of specific rna aptamers against the ang1 and ang2 genes [36, 104] . aptamer ang9-4 binds to ang1 and inhibits its signaling pathway, leading to the reduced survival of huvec cells in vitro [36] . similarly, intraocularly administered aptamer 11-1.41 binds to ang2 and inhibits angiogenesis in rat corneal micropockets [104] . a 3' deoxythymidine cap protects this aptamer from rnases. the pegylated version of this molecule was shown to inhibit tumor angiogenesis and growth in an in vivo murine metastatic colon cancer model following systemic administration [105] . while the large majority of aptamers have been isolated by selex [106] , the anti-proliferative dna aptamer as-1411 was developed based on observations that guanosine-rich oligonucleotides have antiproliferative effects in tumor cells [107] . molecular studies have shown that this aptamer binds to the cell surface protein nucleolin and inhibits the activity of nf-kb, a ubiquitous transcription factor, through intracellular complex formation [108] . clinical studies of as-1411 have focused on patients with renal, pancreatic and other solid tumors. the aptamer was administered to patients as a continuous infusion for 4 or 7 days. selectins are a family of cell adhesion molecules expressed by leukocytes, endothelial cells and platelets [109] . they are involved in a number of inflammatory diseases as well as tissue injury and infection. dna selection against the l-selectin/igg fusion protein (ls-rg) was performed to find aptamers that could be tested in vivo [110] . aptamers ld201, ld174 and ld196 all bound with a kd of 1.8 nm at 37 ºc. truncated versions of these aptamers inhibited sl-rg binding to its ligand sialyl lewis x (slex) with an ic 50 of 3 nm. aptamer ld201t1 blocked l-selectin-mediated adhesion of human lymphocytes and neutrophils and inhibited human cell trafficking to peripheral and mesenteric lymph nodes in scid mice. platelet-derived growth factor (pdgf) is a ubiquitous mitogen and chemotactic growth factor in the form of three disulphide-linked dimers made of two homologous chains, a and b. it is involved in wound healing and is linked to the progression of numerous diseases, including atherosclerosis and glomerulonephritis [111, 112] . a hallmark of malignant transformation is the loss of dependence on exogenous mitogenic stimulation; many tumor cell lines are thought to produce and secrete pdgf for this reason [113] . dna selection against recombinant human pdgf-ab yielded dna specific aptamers of the pdgf b-chain that bound with subnanomolar affinity [114] . the consensus secondary structure motif for most of the high-affinity ligands is a three-way helix junction with a threenucleotide loop at the branch point. the pdgf aptamers inhibited the mitogenic effects of pdgf-bb in cells that expressed pdgf β receptors [114, 115] . peg-modified aptamers in a rat model of mesangioproliferative glomerulonephritis led to a 64% reduction in mitoses by day 6, and 78% by day 9. there was also a 95% reduction of proliferating mesangial cells by day 9 and a markedly reduced glomerular expression of the endogenous pdgf b-chain. aptamer-treated animals also showed a reduced influx of monocytes/macrophages and the overproduction of glomerular extracellular matrix on day 6 [109] . further studies revealed the inhibition of other disease mechanisms by the above aptamer in experimental glomerulonephritis [116, 117] . the expression of pdgf β receptors in tumors is associated with increased interstitial fluid pressure (ifp) in the dermis. this reduces the gradient between capillaries and the interstitium and impedes the exchange of solutes, such as anticancer agents, over the capillary membrane [118] . increasing this gradient may facilitate the transport of anticancer drugs to tumors [119] . to reduce the ifp, the pdgf-b aptamers [109] were tested in a rat tumor model. the treated animals had an ifp of 9.7 mm hg compared to 14.6 mm hg in scrambled-rna-treated animals [120] . another dna aptamer known as e1-0030 that targets the pdgf-b subtype is currently undergoing phase i clinical trials as an antivascular endothelial growth factor compound [121] . blood fluidity and blood vessel resistance are involved in numerous vascular diseases such as coronary and thrombotic syndromes and myocardial infarction. certainly, these factors must be carefully controlled during coronary surgery. the proliferation of cardiac and vascular cells is key in the development of vessel resistance in diseases such as cardiac intimal hyperplasia, cardiac hypertrophy and atherosclerosis, as well as in the development of malignancies [122, 123] . a recent study has reported the development of an rna aptamer able to specifically recognize members of the e2f transcription factors involved in cell proliferation. the binding of the 2'-fy aptamer 8-2 mainly to e2f3, reduces intimal hyperplasia and the pathological proliferation and migration of vascular smooth muscle cells (vsmcs) after bypass surgery in a mouse model [124] . this aptamer avoids the side effects derived from cross reactivity with other members of the e2f family. in a different approach, selex has been performed with the e2f1 protein to find in vitro selected rna aptamers that bind to and inhibit e2f activity. clone e1 rna was found to bind to e2f1 and blocked the latter's attachment to its dna binding site [125] . by impeding e2f activity, the e2f rna aptamer inhibited s-phase induction by 90% compared to controls. thus, both natural and in vitro selected aptamers appear able to limit cell proliferation. the coagulation signaling cascade offers several targets for the modulation of blood fluidity. the conversion of pro-thrombin to thrombin is delayed by a long half-life (15 h) 2'-fy aptamer targeting the catalyst factor viia in a dose dependent manner [126] . nevertheless, the rapid restoration of cascade integrity is needed to prevent the harmful effects of coagulation deficiency. with this aim, an rna-based aptamer-antidote system has been developed [127, 128] . the reg-1 rna aptamer targets factor xia. an antisense rna molecule was designed to specifically bind the 5'-half of the aptamer (the antidote). binding of the antidote abolishes the aptamer's binding to its target, thereby reversing the anticoagulant effect ( figure 4 ). neither antidote nor aptamer have been seen to cause any adverse effect in phase i clinical trials (ia and ib) when given to healthy people and patients with stable cardiovascular disease receiving antiplatelet therapy [61, 63] . the results of another phase i clinical trial (ic) indicate that the anticoagulation effect can be modulated by varying the dose of antidote rna [62] . in a recent phase iia trial, percutaneous coronary injection of the aptamer increased the activated clotting time (act) in patients immediately after its administration, reaching values close to those obtained with heparin. act values were restored 15 min after the administration of the antidote [129] . thrombin is a natural target for anticoagulation therapy and numerous aptamers have been generated with different capacities to inhibit its activity in vitro [56, 70] . the archemix corp., in collaboration with arca biopharma inc. (formerly nuvelo inc.), has tested nu-172, a dna aptamer against thrombin. in phase i clinical trials, nu-172 was administered intravenously as a continuous infusion to healthy volunteers. the results showed an increase in activated clotting time with a return to baseline when administration ceased (see archemix corp. website). a phase ii clinical study is currently underway with the goal of using nu-172 in coronary artery bypass graft surgery and percutaneous coronary intervention. figure 4 . mechanism of the apatamer-antidote pair for anticoagulant therapy.the intrinsic pathway of the blood coagulation cascade involves the activation of factor x. anticoagulation system reg1 consists of rb006 (drug), an injectable rna aptamer that specifically binds to activated factor ix (ixa) and prevents the proteolytic cleavage of factor x; and rb007 (antidote), a rna antisense oligonucleotide that neutralizes the anticoagulating effect of the aptamer rb006. in the presence of the antidote, the aptamer is released from factor ixa and clotting parameters return to normal. together with activated factor viii (viiia), factor ixa catalyzes the cleavage of factor x (pro-enzyme) to yield activated factor x (xa), which is required for the blood clotting cascade. a dna/rna aptamer conjugated to peg, known as arc-1779, generated against vwf (von willebrand factor), a central factor in the adhesion of platelets to the endothelial surface at vascular injury sites [130] , has been examined in a phase i trial in healthy volunteers. the aptamer increased platelet function in a whole-blood assay sensitive to vwf-mediated platelet inhibition. moreover, a slow intravenous bolus followed by 4 h of continuous infusion inhibited more than 95% of vwf function, which returned to baseline over 12-16 h after administration was suspended. nf-κb is involved in inflammation responses and modulates the synthesis of chemokines, interferons, major histocompatibility complex (mhc) proteins, growth factors, and the cell adhesion molecules that play a role in ischemia-reperfusion injuries seen in most myocardial infarctions [131] . a natural double stranded dna aptamer was found that binds to nf-κb with high affinity. in a rat cardiac ischemia-reperfusion model, this aptamer significantly reduced the expected injury [115] . in a rat cardioplegic arrest model, animals transfected with the nf-κb dna aptamer showed improved recovery of left ventricular function as well as coronary flow three days post-transfection compared to scrambled-dna controls (97% vs. 61%) [132] . the aptamer-treated group also showed a lower percentage of neutrophil adhesion to endothelial cells (38% vs. 81%) and a lower level of interleukin il-8 (109 vs. 210 ng/mg). the same aptamer was also studied in a murine model of nephritis, in which it abolished glomerular inflammation and the expression of inflammatory markers il-1α, il-1β, il-6, icam-2 (intracellular adhesion molecule 2), and vcam-1 (vascular cell adhesion molecule 1) [133] . a rat global brain ischemia model showed inhibition of tnf-α, il-1β and icam-1 expression in nf-κb aptamer-treated animals after 1 h of ischemia. moreover, 7 days after ischemia, neuronal damage was significantly attenuated in the nf-κb-aptamer-treated group compared to controls [134] . the proteins of different pathogens have also attracted the attention of researchers as targets for inhibitory nucleic acids. a recent study reported the use of a 2'-fy aptamer against the extracellular domain of the erythrocyte membrane protein 1 (pfemp1) of the parasitic protozoan plasmodium falciparum, achieving the efficient inhibition of erythrocyte rosseting in blood cultures [135] . rna viruses, especially hiv-1 and hcv, have been the main targets for therapeutic nucleic acids with catalytic activity. rossi's group designed a very interesting ex vivo selection procedure to identify anti-hiv hammerhead ribozymes. a pool of hammerhead catalytic domains containing randomized binding arm sequences was assayed against an hiv-1 chimera containing the thymidine kinase gene. after cell transfection of the ribozyme population expressed under the control of the u16snorna promoter, gancyclovir-resistant cells were selected. such antibiotic resistance suggests the presence of an active anti-hiv ribozyme [136] . banerjea's group designed a strategy to identify accessible cleavage sites within the hiv-1 gag rna and to pick out dnazymes effective against this target [137] . two dnazyme variant populations were synthesized. the specificity of the first was limited to all possible augs in the target rna, whereas the second population was designed to cleave any potential target site. these options were made possible by either fixing the nucleotides immediately preceding the catalytic motif to ca (for aug cleavage) or totally randomizing the seven bases on either side of the catalytic motif. dnazymes selected from both populations showed target-specific cleavage activities in the presence of mg 2+ , and significantly interfered with hiv-1-specific gene expression. this strategy could be used for the selection of effective target sites in any target rna [137] . viral proteins are favorite targets for the development of therapeutic aptamers. rna aptamers have been selected against different viral enzymes and proteins involved in host-cell interactions, such as hiv-1 reverse transcriptase (rt), hiv-1 glycoprotein 120, hcv rna-dependent rna polymerase (rdrp), sars coronavirus ntpase/helicase, and the hemagglutinin protein of human influenza virus b, all of which show efficient viral inhibition [138] [139] [140] [141] [142] [143] . human influenza b virus hemagglutinin protein has been inhibited in vitro by the rna pool resulting from 15 rounds of in vitro selection [143] . further studies are required for the identification of independent aptamers. recently, a dna aptamer (termed 93del) has been described that adopts a novel type of dimeric quadruplex folding and shows anti-hiv1 integrase activity in the nanomolar range in vitro by blocking several catalytic amino acid residues essential for integrase function. several other g-rich dna aptamers have been identified as remarkable hiv1-in inhibitors with ic50 values in the nanomolar range. t30695 is one such aptamer, and has been well studied in recent years, [144, 145] . astier-gi's group described the characterization of two dna aptamers (27v and 127v) that specifically bind to hepatitis c virus (hcv) rna polymerase (ns5b), inhibiting its activity in vitro [146] . aptamer 27v competed with the rna template for binding to the enzyme and blocked both the initiation and elongation phases of rna synthesis, whereas aptamer 127v exclusively inhibited initiation events. the authors also determined that, in addition to an in vitro inhibitory effect on rna synthesis, aptamer 27v interfered with the multiplication of hcv jfh1 in huh7 cells. the efficient cellular entry of these short dnas, and the inhibitory effect observed in human cells infected with hcv, indicate that aptamers are useful tools for the study of hcv rna synthesis; their therapeutic against hcv infection is also attractive [146] . an alternative and innovative potential therapeutic approach that has attracted much hope is the targeting of the structural domains of viral genomes which are frequently concentrated within untranslated terminal regions (utrs). the hiv-1 trans-activation response (tar) element is a polyfunctional rna domain mainly involved in the activation of transcription by its binding to the viral protein tat. aptamer r06 binds the tar element in vitro by a loop-loop interaction [147] . the intracellular expression of the aptamer by the nucleolar u16 rna promoter inhibits hiv-1 infection by more than 90%. this strategy takes advantage of hiv-1 rna nucleolar-trafficking to efficiently colocalize the aptamer and target [148] . the same r06 aptamer has been improved by the inclusion of extra binding rna domains targeting additional 5'-utr sites [149] . our own results show that presynthesized aptamers, or aptamers produced intracellularly via u6 rna promoter-driven expression, both with multiple binding sites targeting the whole hiv-1 5'-utr, efficiently inhibit hiv-1 replication in cell culture (sánchez-luque et al., unpublished). there have been different attempts to isolate rna aptamers against different domains of the hcv internal ribosome entry site (ires) located within the 5'-utr [150] [151] [152] . aptamer 3-07 binds domain iii-iv of hcv ires inhibiting its activity in cell-free systems [151] . this inhibition was further improved by conjugation of 3-07 with 2-02 in a chimeric molecule that targets domain ii [153] . rna aptamer ap30 isolated against the hcv (-) strand ires domain i partially inhibits hcv rna replicase-mediated (+) strand synthesis, probably by interfering with (-) strand binding [154] . a step forward was the design of an innovative selection approach to identify chimeric ribozyme/aptamer rna molecules against the entire hcv-ires. each selection cycle includes two consecutive selection steps for binding and cleavage of the viral rna [155] . after six selection rounds, seven families of inhibitor rnas were identified simultaneously targeting two sites within the hcv-ires, one for each inhibitory domain. these chimeric rna inhibitors promoted ires inhibition by up to 95% in cell extracts, identifying new targets for the development of anti-hcv agents. further characterization revealed up to 50% inhibition of viral translation and replication in a human liver cell line [156, 157] . the ability of selex to generate aptamers against any kind of target provides the possibility of their being used as therapeutic antidotes or palliatives. ricin is a toxic heterodimeric lectin from the castor bean plant ricinus communis. it disrupts protein synthesis by inactivation of the ribosomes. an aptamer isolated against the a chain partially restores protein synthesis levels in cell free translation systems and cell cultures [158] . a different approach has been explored for cocaine and anticonvulsant mk-801 alleviation. both compounds preferentially bind the open isoform of nicotine acetylcholine receptors (nachrs) found at neuromuscular junctions, autonomic ganglia and in the central nervous system. rna aptamers developed against nachrs with equal binding affinity for both open and closed sodium-potasium channel isoforms partially restore isoform equilibrium, alleviating drug channel inhibition in cells [159] [160] [161] . aptamers can distinguish between different isoforms of the same protein or different members of the same family. aptamer-sirna/toxin conjugates have been developed to deliver therapeutic agents within a specific target cell. ex vivo selection procedures performed against a specific cell type have yielded aptamers that are very efficiently taken up by those cells; they could therefore be used as a specific delivery tool. the tta1 aptamer of tenascin c, selected against the human glioma u251 cell line [110, 162] , was efficiently taken up by human tumor cells in a xenograft glioblastoma and breast tumor model [163] . aptamers targeting the prostate-specific membrane antigen (psma) are those most used as delivery tools. coffey's group reported two 2'-fy rna aptamers, a9 and a10, that bind specifically recombinant psma [164] . since psma is continually recycled from the cell surface, these aptamers are appropriate vehicles for delivering therapeutic compounds via the endosomal pathway. small interfering rnas against pro-survival factors over-expressed in prostate cancer cells, such as plk-1 and bcl-2 and eukaryotic elongation factor 2, have been delivered by the a10 aptamer [165] [166]. the aptamer-driven uptake of plk-1 bcl-2 sirnas leads to the death of psma-positive cells and tumor regression following intra-tumoral injection in mice xenograft models [165] . the improvement of the delivery vehicles were achieved in several ways and tumor regression through the almost complete loss of cell viability was observed when the eukaryotic elongation factor 2 sirna was delivered by a dimeric aptamer [166] . the delivery of pharmacological compounds by aptamers has also been studied by different approaches. doxorubicin (dox) is a chemotherapeutic intercalating agent used against cancer. dox intercalates between the aromatic rings of the gc pairs of the a10 aptamer, enhancing its cytotoxicity against psma-expressing cells and reducing the same in non-expressing cells [167] . the same results have been reported when using the a9-genolin conjugate, a toxin that blocks protein synthesis [168] . side toxicity of the chemotherapeutic compound can be further prevented by encapsulating it within nanoparticles or nanoconjugates [169] [170] [171] . aptamer b40, specific for hiv-1 gp120 [140] , has also been used to deliver sirna against infected cells. hiv-1 gp120 is found on the surface of infected cells and promotes cell fusion. consequently, besides neutralizing infective particles, the b40 aptamer can be used for anti-hiv-1 sirna delivery to infected cells. the first attempt involved a chimera of the b40 aptamer and sirna targeting the overlapping rev tat region [172] . the chimera was internalized in an aptamer-dependent manner, with inhibition dependent on the interference machinery. the aptamer-sirna linkage was further improved for the easy combination of sirnas to the aptamer and better sirna processing by the cellular machinery. this improved chimera resulted in the specific inhibition of hiv-1 replication and infectivity in pbmc and cultured cem t cells [173] . finally, aptamers can also be used to colocalize rna inhibitors with their specific target molecules at the subcellular level. aptamers can target rna domains, e.g., in viral rna genomes; they can therefore improve trans-cleaving ribozymes by anchoring them to their target. chimeric molecules composed of a hammerhead ribozyme and an aptamer both targeting the hcv ires have efficiently inhibited ires activity in human hepatocyte cell cultures [156, 157] . the procedures used to identify dna and rna molecules of interest in large populations of variant nucleic acid molecules have contributed significantly to the development of nucleic acid-based therapeutic drugs. aptamers show high specificity for their targets and have low toxicity and immunogenicity profiles. since the 1990s, the design and isolation of specific aptamers using selection and evolution techniques has been optimized and even automated. this has lead to great advances in our knowledge of aptamers as therapeutic agents and has expanded our bank of inhibitory nucleic acids and their possible targets, which now include cytokines, cell receptors, viruses and even whole cells. an aptamer drug is now on the market, and several mid and late clinical trials in progress that appears to confirm the great potential of these tools. with the improvement and optimization of selection strategies, and the ongoing discoveries made in this field, success in the development of nucleic acidbased therapeutics protocols might be predicted. an extracellular darwinian experiment with a selfduplicating nucleic acid molecule in vitro genetic analysis of the tetrahymena selfsplicing intron in vitro selection of rna molecules that bind specific ligands selection in vitro of an rna enzyme that specifically cleaves singlestranded dna systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t4 dna polymerase in vitro selection and evolution of rna: applications for catalytic rna, molecular recognition, and drug discovery in vitro selection of hairpin ribozymes in vitro selection of catalytic polynucleotides in vitro selection of functional nucleic acids in vitro selection of functional nucleic acid sequences in vitro selection procedures for identifying dna and rna aptamers targeted to nucleic acids and proteins berzal-herranz, a. rna selection and evolution in vitro: powerful techniques for the analysis and identification of new molecular tools aptamers: a new class of oligonucleotides in the drug discovery pipeline? in vitro rna random pools are not structurally diverse: a computational analysis in vitro selection using a dual rna library that allows primerless selection short bioactive spiegelmers to migraineassociated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-selex minimal primer and primer-free selex protocols for selection of aptamers from random dna libraries in vitro selection of active hairpin ribozymes by sequential rna-catalyzed cleavage and ligation reactions ribozyme catalysis of metabolism in the rna world selection-by-function: efficient enrichment of cathepsin e inhibitors from a dna library an allosteric synthetic dna engineering a ligand-dependent rna transcriptional activator in vivo evolution of an rna-based transcriptional activator aptazyme-mediated regulation of 16s ribosomal rna reengineering a natural riboswitch by dual genetic selection direct selection for ribozyme cleavage activity in cells chemical modification of sirnas for in vivo use therapeutic applications of dna and rna aptamers recent developments in oligonucleotide conjugation cell penetrating peptides: overview and applications to the delivery of oligonucleotides sequence-specific activity of antisense oligonucleotides conjugated to poly (l-lysine) carriers the cluster glycoside effect nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids prion-proteinspecific aptamer reduces prpsc formation a nuclease-resistant rna aptamer specifically inhibits angiopoietin-1-mediated tie2 activation and function the first analogues of lna (locked nucleic acids): phosphorothioate-lna and 2'-thio-lna structural studies of lna:rna duplexes by nmr: conformations and implications for rnase h activity the conformations of locked nucleic acids (lna) locked nucleic acids: promising nucleic acid analogs for therapeutic applications in vitro incorporation of lna nucleotides polymerase chain reaction and transcription using locked nucleic acid nucleotide triphosphates efficient enzymatic synthesis of lna-modified dna duplexes using kod dna polymerase mirror-image rna that binds dadenosine mirror-design of loligonucleotide ligands binding to l-arginine in vivo properties of an anti-gnrh spiegelmer: an example of an oligonucleotide-based therapeutic substance class inhibition of ghrelin action in vitro and in vivo by an rna-spiegelmer a general purpose rna-cleaving dna enzyme ribozyme structures and mechanisms gaining target access for deoxyribozymes dnazymes as potential therapeutic molecules inhibition of hiv-1 replication by rna-based strategies regulatory pathways governing hiv-1 replication identification and characterization of a hela nuclear protein that specifically binds to the trans-activation-response (tar) element of human immunodeficiency virus overexpression of tar sequences renders cells resistant to human immunodeficiency virus replication generation of species cross-reactive aptamers using "toggle pegaptanib, a targeted anti-vegf aptamer for ocular vascular disease antivascular endothelial growth factor therapy for neovascular agerelated macular degeneration inhibition of von willebrand factor-mediated platelet activation and thrombosis by the anti-von willebrand factor a1-domain aptamer arc1779 first-in-human evaluation of anti von willebrand factor therapeutic aptamer arc1779 in healthy volunteers first-in-human experience of an antidote-controlled anticoagulant using rna aptamer technology: a phase 1a pharmacodynamic evaluation of a drug-antidote pair for the controlled regulation of factor ixa activity a randomized, repeat-dose, pharmacodynamic and safety study of an antidote-controlled factor ixa inhibitor phase 1b randomized study of antidote-controlled modulation of factor ixa activity in patients with stable coronary artery disease discovery and development of the g-rich oligonucleotide as1411 as a novel treatment for cancer the nucleolin targeting aptamer as1411 destabilizes bcl-2 messenger rna in human breast cancer cells discovery and development of anticancer aptamers inhibition of platelet-derived growth factor b signaling enhances the efficacy of antivascular endothelial growth factor therapy in multiple models of ocular neovascularization sequential loss of tumor vessel pericytes and endothelial cells after inhibition of platelet-derived growth factor b by selective aptamer ax102 derivation of rna aptamer inhibitors of human complement c5 selection of single-stranded dna molecules that bind and inhibit human thrombin in vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits pilot study of the efficacy of a thrombin inhibitor for use during cardiopulmonary bypass pegaptanib sodium for ocular vascular disease 2'-fluoropyrimidine rna-based aptamers to the 165-amino acid form of vascular endothelial growth factor (vegf165). inhibition of receptor binding and vegf-induced vascular permeability through interactions requiring the exon 7-encoded domain the biology of vegf and its receptors a phase ii randomized doublemasked trial of pegaptanib, an anti-vascular endothelial growth factor aptamer, for diabetic macular edema inhibition of vegf blocks tgf-beta1 production through a pi3k/akt signalling pathway rna aptamers selected against the glur2 glutamate receptor channel one rna aptamer sequence, two structures: a collaborating pair that inhibits ampa receptors aptamer antagonists of myelin-derived inhibitors promote axon growth characterization of 2'-fluoro-rna aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion the role of neutrophil elastase in chronic inflammation in vitro selection of rna-based irreversible inhibitors of human neutrophil elastase highly potent irreversible inhibitors of neutrophil elastase generated by selection from a randomized dna-valine phosphonate library protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury high-affinity ige receptor on eosinophils is involved in defence against parasites the human ige network high-affinity oligonucleotide ligands to human ige inhibit binding to fc epsilon receptor i neutralizing aptamers from whole-cell selex inhibit the ret receptor tyrosine kinase distribution and bioactivity of the ret-specific d4 aptamer in three-dimensional collagen gel cultures inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3 antimetastatic potential of pai-1-specific rna aptamers rna aptamers as conformational probes and regulatory agents for plasminogen activator inhibitor-1 selection and characterization of an rna decoy for transcription factor nf-kappa b selection and characterization of anti-nf-kappab p65 rna aptamers yeast genetic selections to optimize rna decoys for transcription factor nf-kappa b characterization of anti-nf-kappab rna aptamer-binding specificity in vitro and in the yeast three-hybrid system h1 rna polymerase iii promoter-driven expression of an rna aptamer leads to high-level inhibition of intracellular protein activity rna aptamertargeted inhibition of nf-kappa b suppresses non-small cell lung cancer resistance to doxorubicin rna aptamers interfering with nucleophosmin oligomerization induce apoptosis of cancer cells multivalent rna aptamers that inhibit ctla-4 and enhance tumor immunity multivalent 4-1bb binding aptamers costimulate cd8+ t cells and inhibit tumor growth in mice assembling ox40 aptamers on a molecular scaffold to create a receptor-activating aptamer inhibition of rat corneal angiogenesis by a nuclease-resistant rna aptamer specific for angiopoietin-2 inhibition of in vivo tumor angiogenesis and growth via systemic delivery of an angiopoietin 2-specific rna aptamer aptamers: an emerging class of therapeutics antiproliferative activity of grich oligonucleotides correlates with protein binding agro100 inhibits activation of nuclear factor-kappab (nf-kappab) by forming a complex with nf-kappab essential modulator (nemo) and nucleolin novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers tenascin-c aptamers are generated using tumor cells and purified protein a subpopulation of smooth muscle cells in injured rat arteries expresses platelet-derived growth factor-b chain mrna platelet-derived growth factor (pdgf) and pdgf receptor are induced in mesangial proliferative nephritis in the rat structural and functional studies on platelet-derived growth factor inhibitory dna ligands to platelet-derived growth factor b-chain in vivo transfection of cis element "decoy" against nuclear factor-kappab binding site prevents myocardial infarction specific antagonism of pdgf prevents renal scarring in experimental glomerulonephritis the effects of platelet-derived growth factor antagonism in experimental glomerulonephritis are independent of the transforming growth factor-beta system transport of molecules in the tumor interstitium: a review delivery of molecular medicine to solid tumors inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors emerging pharmacologic therapies for wet age-related macular degeneration braking the cycle e2f: a link between the rb tumor suppressor protein and viral oncoproteins distinct roles of e2f proteins in vascular smooth muscle cell proliferation and intimal hyperplasia inhibition of cell proliferation by an rna ligand that selectively blocks e2f function blocking the initiation of coagulation by rna aptamers to factor viia rna aptamers as reversible antagonists of coagulation factor ixa antidote-mediated control of an anticoagulant aptamer in vivo nucleic acid aptamers as antithrombotic agents: opportunities in extracellular therapeutics von willebrand factor and the endothelium in vascular disease rel/nf-kappa b/i kappa b family: intimate tales of association and dissociation a novel strategy for myocardial protection using in vivo transfection of cis element 'decoy' against nfkappab binding site: evidence for a role of nfkappab in ischemia-reperfusion injury transcription factor decoy for nfkappab inhibits cytokine and adhesion molecule expressions in synovial cells derived from rheumatoid arthritis nuclear factor-kappa b decoy attenuates neuronal damage after global brain ischemia: a future strategy for brain protection during circulatory arrest in vitro selection of rna aptamers against a conserved region of the plasmodium falciparum erythrocyte membrane protein 1 use of a u16 snorna-containing ribozyme library to identify ribozyme targets in hiv-1 in vitro-selected rna cleaving dna enzymes from a combinatorial library are potent inhibitors of hiv-1 gene expression rna pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase bent pseudoknots and novel rna inhibitors of type 1 human immunodeficiency virus (hiv-1) reverse transcriptase neutralization of infectivity of diverse r5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2'f-rna aptamers in vitro selection of rna aptamers that bind the rna-dependent rna polymerase of hepatitis c virus: a possible role of gc-rich rna motifs in ns5b binding isolation of inhibitory rna aptamers against severe acute respiratory syndrome (sars) coronavirus ntpase/helicase an efficient rna aptamer against human influenza b virus hemagglutinin mechanism of inhibition of hiv-1 integrase by g-tetrad-forming oligonucleotides in vitro structure-activity of tetrad-forming oligonucleotides as a potent anti-hiv therapeutic drug astier-gin, t. inhibition of hepatitis c virus (hcv) rna polymerase by dna aptamers: mechanism of inhibition of in vitro rna synthesis and effect on hcv-infected cells in vitro selection identifies key determinants for loop-loop interactions: rna aptamers selective for the tar rna element of hiv-1 endogenous expression of an anti-tar aptamer reduces hiv-1 replication bimodal loop-loop interactions increase the affinity of rna aptamers for hiv-1 rna structures apical loop-internal loop interactions: a new rna-rna recognition motif identified through in vitro selection against rna hairpins of the hepatitis c virus mrna rna aptamers targeted to domain ii of hepatitis c virus ires that bind to its apical loop region a hepatitis c virus (hcv) internal ribosome entry site (ires) domain iii-iv-targeted aptamer inhibits translation by binding to an apical loop of domain iiid increased inhibitory ability of conjugated rna aptamers against the hcv ires isolation and characterization of rna aptamers specific for the hcv minus-ires domain i berzal-herranz, a. interfering with hepatitis c virus ires activity using rna molecules identified by a novel in vitro selection method berzal-herranz, a. inhibition of hepatitis c virus internal ribosome entry site-mediated translation by an rna targeting the conserved iiif domain berzal-herranz, a. inhibition of hepatitis c virus replication and internal ribosome entry site-dependent translation by an rna molecule protective effects of anti-ricin a-chain rna aptamer against ricin toxicity in vitro selection of rna molecules that displace cocaine from the membranebound nicotinic acetylcholine receptor mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and mk-801 minimal rna aptamer sequences that can inhibit or alleviate noncompetitive inhibition of the muscle-type nicotinic acetylcholine receptor a tenascin-c aptamer identified by tumor cell selex: systematic evolution of ligands by exponential enrichment tumor targeting by an aptamer identification and characterization of nucleasestabilized rna molecules that bind human prostate cancer cells via the prostate-specific membrane antigen cell type-specific delivery of sirnas with aptamer-sirna chimeras cell-specific induction of apoptosis by rationally designed bivalent aptamer-sirna transcripts silencing eukaryotic elongation factor 2. curr. cancer drug targets an aptamer-doxorubicin physical conjugate as a novel targeted drug-delivery platform aptamer:toxin conjugates that specifically target prostate tumor cells targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo formulation of functionalized plga-peg nanoparticles for in vivo targeted drug delivery targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized pt(iv) prodrug-plga-peg nanoparticles novel dual inhibitory function aptamer-sirna delivery system for hiv-1 therapy selection, characterization and application of new rna hiv gp 120 aptamers for facile delivery of dicer substrate sirnas into hiv infected cells the work of berzal-herranz's group is funded by grant bfu2009-08137 from the spanish ministry of science and innovation, grant cts-5077 from the junta de andalucía, and by feder funds from the e.u. key: cord-319609-y0gdjn64 authors: van duyne, rachel; guendel, irene; kehn-hall, kylene; easley, rebecca; klase, zachary; liu, chenglong; young, mary; kashanchi, fatah title: the identification of unique serum proteins of hiv-1 latently infected long-term non-progressor patients date: 2010-07-06 journal: aids res ther doi: 10.1186/1742-6405-7-21 sha: doc_id: 319609 cord_uid: y0gdjn64 background: the search for disease biomarkers within human peripheral fluids has become a favorable approach to preventative therapeutics throughout the past few years. the comparison of normal versus disease states can identify an overexpression or a suppression of critical proteins where illness has directly altered a patient's cellular homeostasis. in particular, the analysis of hiv-1 infected serum is an attractive medium with which to identify altered protein expression due to the ease and non-invasive methods of collecting samples as well as the corresponding insight into the in vivo interaction of the virus with infected cells/tissue. the utilization of proteomic techniques to globally identify differentially expressed serum proteins in response to hiv-1 infection is a significant undertaking that is complicated due to the innate protein profile of human serum. results: here, the depletion of 12 of the most abundant serum proteins, followed by two-dimensional gel electrophoresis coupled with identification of these proteins using matrix-assisted laser desorption/ionization time-of-flight (maldi-tof) mass spectrometry, has allowed for the identification of differentially expressed, low abundant serum proteins. we have analyzed and compared serum samples from hiv-1 infected subjects who are being treated using highly active antiretroviral therapy (haart) to those who are latently infected but have not progressed to aids despite the absence of treatment, i.e. long term non-progressors (ltnps). here we have identified unique serum proteins that are differentially expressed in ltnp hiv-1 patients and may contribute to the ability of these patients to combat hiv-1 infection in the absence of haart. we focused on the cdk4/6 cell cycle inhibitor p16(ink4a )and found that the treatment of hiv-1 latently infected cell lines with p16(ink4a )decreases viral production despite it not being expressed endogenously in these cells. conclusions: identification of these unique proteins may serve as an indication of altered viral states in response to infection as well as a natural phenotypic variability in response to hiv-1 infection in a given population. human serum is derived from the liquid plasma component of the blood with the fibrinogens, or clotting factors, removed and is composed of small molecules such as salts, lipids, amino acids, sugars and approximately 60-80 mg of proteins/ml [1] . serum is a readily obtainable peripheral bodily fluid from which the protein profile directly reflects the normal or disease state of the organism [2] [3] [4] . serum is a complex mixture of "classical" and "non-classical" proteins. classical serum proteins are involved in a number of processes including proteolysis, inhibition, binding, transport, coagulation, and immune response and are often secreted from the liver, through the intestines, and into the bloodstream [5] . "non-classical" proteins are proteins that are not directly tied to any known function within the serum and often originate from cellular leakage or shedding, and may utilize the bloodstream for transportation [5] . it is generally accepted that most of the significant changes in the serum will be found in these low abundant non-classical proteins, due to the hypothesis that the presence of these proteins should reflect changes in the diseased tissue. indeed, serum commonly contains upwards of 10,000 different proteins at any given time that are being actively produced and secreted by all cells and tissues, therefore, the proteomic profile of serum can give insight into the systemic reaction to a disease state and can serve as a pool of differentially expressed proteins [2, [6] [7] [8] [9] [10] [11] [12] [13] . recently, the interest in characterizing the human serum proteome has increased due to the determination of disease biomarkers for early detection, diagnosis, and drug targeting; however, due to the extensive dynamic range of protein concentration within the serum, the identification of low abundance proteins suitable for biomarker determination is often masked. the 22 highly abundant proteins contained within serum constitute approximately 99% of the total serum proteins, including albumin, igg, transferrin, haptoglobin, fibrinogen, etc. and interfere with the identification of low abundance proteins in the ng/ml concentration range. the presence of these highly abundant proteins necessitates the prefractionation of serum samples prior to analysis for low abundant proteins. due to the dynamic insight the analysis of the serum proteome can relate to a disease state, of particular interest is the identification of low abundant proteins that change in expression or abundance in response to a disease state. these low abundant proteins could potentially arise as an early diagnostic for a disease state, or a therapeutic target. serum proteomics has emerged as an integral biomarker identification and diagnostic tool, especially for infectious diseases and oncology. recently, novel serum biomarkers have been identified for liver fibrosis in hepatitis c virus (hcv) infected patients as well as unique protein signatures in sars coronavirus infections, and infant hepatitis syndrome induced by human cytomegalovirus (hcmv) infection [14] [15] [16] . characterization of the serum protein profile of these viral states helps provide insight into the expression changes associated with viral infection. in particular, hiv-1 infection, even at the acute phase, results in dramatic changes in both cellular and viral protein expression levels. as the hiv-1 viral tropism consists primarily of cd4+ t-cells, macrophages, and dendritic cells, the resulting protein changes can be seen systemically as infected cells travel throughout the body. additionally, the nature of this viral infection supports the secretion of altered proteins into the blood and subsequently the serum due to the propensity of the virus to stimulate apoptosis of infected cells, therefore emptying cellular contents into the serum. these characteristics of hiv-1 infection suggest that the analysis of the serum of infected patients is an appropriate reflection of a patients' altered protein expression state. due to innate genetic and phenotypic differences in the human population, significant variability exists in the sus-ceptibility to hiv-1 infection. amongst this diversity includes the well-studied ccr5δ32 inherited mutation, which prevents the binding of r5-tropic hiv-1 strains to the ccr5 chemokine receptor on the surface of cd4+ tcells, therefore preventing entry of the virus [17] . additionally, some individuals can be infected with hiv-1, however will not progress to aids even in the absence of therapy. these long term non-progressors (ltnps) are often characterized as being infected with hiv-1 but are also disease free and sustain a normal cd4 t-cell count and a low viral load. over the past 20 years, multiple studies have been aimed at determining the reason that these individuals are able to resist disease progression. there are studies that suggest that the virus infecting these cells could be deficient in some way, for example, nef deficient viruses and vpr r77q mutations are associated with ltnps [18] [19] [20] [21] [22] . a number of host factors have also been identified that may contribute to the observed resistance. ltnps have a higher prevalence of the ccr5δ32 allele [17, [23] [24] [25] . in addition, the presence of certain hla genes including hla-b27, hla-b*5701, hla-b*5401, and hla-b*1507 have been linked to ltnp [26] [27] [28] however, the identified alterations do not account for all cases of ltnp. therefore, the search for protective host factors is still an area of active investigation in hopes of obtaining information that could be of therapeutic value. here, we describe the detection of unique, low abundant serum proteins in latently infected hiv-1 ltnps as compared to serum from patients undergoing haart treatment, and those not infected with hiv-1. we attempted to characterize the underlying differences in ltnps that contribute to the ability of these patients to combat hiv-1 infections. we have depleted 12 of the most highly abundant serum proteins from three sets of serum samples (uninfected, infected on haart, ltnp) and identified differentially expressed proteins across the samples. in particular, we focus on the identified cellular protein p16 ink4a which is found preferentially in ltnp patient serum samples, but is not present in patients undergoing haart treatment. in vitro viral assays and viability studies confirm the loss of viral replication upon p16 ink4a treatment in latently infected cell lines and the non-toxic effect of the same treatment in corresponding uninfected cell lines. to begin the identification of unique serum proteins, we obtained 18 subject serum samples: six ltnp, six hiv-1 infected subjects receiving haart therapy (haart) and six hiv-uninfected individuals through the washington dc site of the women's interagency hiv study (wihs) georgetown site (table 1) . wihs is an nih multicenter study of the natural history of hiv-1 infection in women [29] . ltnps are defined by wihs as being hiv-1 infected, but disease free for at least five years, having a cd4 count of greater than 500 at all visits and having no history of anti-retroviral therapy. the difficulty associated with analyzing serum is the presence of a high abundance of proteins which mask potential low abundance biomarkers. to overcome this obstacle, we utilized the proteomelab igy serum depletion kit which removes 12 of the most abundant proteins in serum: albumin, igg, transferrin, fibrinogen, iga, α2-macroglobulin, igm, α1antitrypsin, haptoglobin, α1-acid glycoprotein, apolipoprotein a-i, and apolipoprotein a-ii. as can be observed in figure 1a , whole serum (lanes 2, 3) contains many proteins and is too complex to allow for confident identification of specific proteins. however, when the high abundant proteins (figure 1 , lanes 8, 9) are removed, lower abundant proteins that were originally masked ( figure 1 , lanes 4, 5) are able to be analyzed. along these lines, we found the proteomelab igy serum depletion kit to be the most appropriate and reproducible manner in which to fractionate our serum samples into high and low abundant fractions. we applied this depletion strategy to pooled patient samples, combining equal volumes of whole serum from each of the six patients per sample set (ltnp, haart, and negative), which were subsequently depleted into low and high abundance fractions. we began the analysis with pooled samples to assist in the identification of hiv-1 infection specific protein identification as opposed to identifying individual patient and serum variability. these pooled samples were separated based on 1d sds-page ( figure 1b ) and comparisons between ltnp, haart, and negative low abundant samples were carried out via in-gel trypsin digestion, peptide elution and desalting, followed by maldi-tof mass spectrometry as indicated by numbered arrows marking excised bands. the subsequent protein identifications served as a preliminary indication of differentially expressed proteins between the three patient types. these observations, as summarized in table 2 , provide an insight into the relevance of proteins identified in the context of the state of hiv-1 infection. of particular interest in table 2 is the identification of hiv-1 enhancer binding protein 1, (hivep1), ribonuclease iii, and heterochromatin protein 1 binding protein in the low abundance ltnp fraction. hivep1 is a member of the zas family of proteins which bind the promoter and enhancer regions of both cellular genes and infectious viruses, including hiv-1. also known as prdii-bf1 or mbp-1, this transcription factor binds to both the nf-κb and the tar transactivation response dna elements on the hiv-1 ltr in both the presence and absence of hiv-1 tat [30, 31] . it is not surprising that a transcription factor such as hivep1 would be present during hiv-1 infection; however, the identification of this protein is not necessarily a marker for a ltnp phenotype. ribonuclease iii, or drosha, is a cellular enzyme found in the nucleus which serves to cleave double-stranded rna hairpin transcripts as a key step in the production of mirnas in the rna interference pathway. interestingly, heterochromatin protein 1, or hp1 is a member of the chromatin remodeling family of proteins, which can bind histones at methylated lysine residues and can interact with many chromatin-associated nonhistone proteins. the hp1 family of proteins has been associated with promoting a heterochromatic cellular state, where latently hiv-1 infected cells can persist as a transcriptionally silent provirus [32, 33] . it may be of interest that an hp1 binding protein would be present in the serum of an hiv-1 infected patient as hp1, including its subtypes α, β, and γ, could be involved in the control of various stages of infection. it is possible that the association of this hp1 binding protein with varying subtype of hp1 could explain the differences in patient phenotypes, especially those that result in an altered susceptibility to viral infection. following the initial 1d separation and maldi-tof ms assisted identification, the pooled patient samples were subjected to 2d-gel electrophoresis (2dge); isoelectric focusing (ief) using ipg strips with a ph 3.0-10.0 range followed by sds-page using 4-20% tris-glycine criterion gels. the method of 2d-gel electrophoresis is much more sensitive than 1d-gel electrophoresis in that it provides separation of a complex mixture of proteins in two dimensions, therefore removing the complexity associated with overlapping proteins, or masking due to posttranslational modifications. 2dge is a more sensitive front-end purification approach to the isolation and identification of individual protein species by mass spectrometry. figure 2 depicts the ltnp, haart, and negative low abundance fractions in gels "a", "b", and "c", respectively, as well as the ltnp, haart, and negative high abundance fractions in gels "d", "e", and "f", respectively. indicated protein spots from all gels were excised based on a comparison of protein abundance and the presence of unique spots in a given patient set, were subjected to in-gel trypsin digestion, and were identified by maldi-tof mass spectrometry. it is important to note that although gels "d," "e," and "f" contain the majority of the high abundance proteins, unique small protein spots can still be visualized on these gels. this indicates that not only will these high abundant proteins mask proteins of interest; they can also interact with and seclude lower abundance proteins from being identified. peak lists from the collected mass spectra were processed via peptide mass fingerprinting (pmf) analysis using the mascot and profound databases, compared, and compiled into a nonexhaustive list of identified proteins as displayed in table 3 . of particular interest are those proteins identified from gel "a" indicating unique low abundance proteins in the serum of ltnp patients: tropomyosin 3, protein kinase 3, and cdk4/6 binding protein p16. tropomyosin interacts with actin filaments to provide stability and regulates other actin binding proteins. this family of proteins has been shown to be cleaved by hiv-1 protease in vitro, resulting in the dissociation of critical cytoskeletal elements, which may demonstrate the alteration of muscle structure in the presence of an hiv-1 infection [34] . protein kinase 3, or protein kinase c (pkc) is a member of the family of serine/threonine kinases that are integrally involved in key cellular signaling pathways and can phosphorylate a wide variety of substrates. not surprisingly, hiv-1 infection alters the pkc phosphorylation pathway to stimulate tnf-α production by monocytes as well as other cytokines and growth factors such as il-6, il-10, and mcp-1 [35] [36] [37] [38] [39] . pkc has also been shown to be necessary for hiv-1 tat-mediated transactivation as well as directly phosphorylating tat at serine 46 [40, 41] and plays an integral role in the signaling and secretion of cytokines in response to hiv-1 envelope proteins gp120, gp160, and gp41 [42, 43] . of particular interest in the low abundance, ltnp fraction is the presence of the cdk4/ cdk6 binding protein p16, or more specifically, p16 ink4a , a member of the inhibitor of kinase 4/alternative reading frame (ink4/arf) family of endogenous cdk (cyclindependent kinase) inhibitors [44] . dysregulation of the cell cycle, including the manipulation of cdks and their associated cyclins is often a hallmark of cancerous and infectious phenotypes. indeed hiv-1 and its associated proteins have been known to alter the phosphorylation state and activity of these kinases. p16 ink4a inhibits the phosphorylation of rb by competitively inhibiting the association of cdk4/cyclin d therefore inhibiting the release of rb-bound proteins, such as e2f, and the subsequent progression into the s phase of the cell cycle [44, 45] . this small molecular weight protein is an attractive candidate for a secreted, differentially expressed protein in response to hiv-1 infection. in addition to these low abundant protein identifications, the ltnp high abundant samples (gel "d") indicated the presence of the fgfr1 oncogenic partner and pctaire protein kinase 3 as well as an anti-hiv-1 gp120 igg 16 cκ light chain. fgfr1 oncogene partnered with the fibroblast growth factor receptor 1 (fgfr1) is thought to be associated with myeloproliferative disorders and as of yet is not associated with any hiv-1 protein interactions or associated disease phenotypes. pctaire protein kinase 3, however, is a member of the serine/threonine family of protein kinases and more specifically, the cdc2/cdkx subfamily that plays a role in broad signal transduction pathways. this serine/threonine kinase family member has also been associated with the essential regulation of cell cycle progression, as well as transcription and dna repair [46] . this protein identification again demonstrates the role that hiv-1 infection plays in the dysregulation of cellular kinases and specifically, cell cycle progression. the haart responder patient samples (gels "b" and "e") also contained unique protein candidates: serine/ threonine kinase 33, the kelch repeat domain containing protein 11, and the snw1 protein/apaf1 interacting protein in the low abundant fraction as well as the pre-bcell leukemia homeobox interacting protein 1 in the high abundant fraction. of functional interest is the general serine/threonine kinase 33 as we have already identified several cellular kinases of the same family. additionally, the snw1 protein is a transcriptional coactivator that induces the expression of vitamin d, retinoic acid, estrogen, and glucocorticoid associated genes. snw1/skip interacts with hiv-1 tat through the association with p-tefb (cdk9/cyclin t1) at the tar rna complex, stimulating hiv-1 transcription elongation [44] . interestingly, some of the protein spots identified the presence of serum albumin contamination (spots a2, d5, d7, and e2), which both served as an internal positive control for mass spectrometry and also indicated that the depletion columns are not completely efficient at removing contaminating high abundant proteins. in order to further confirm the presence of these proteins in the serum as identified by mass spectrometry, we performed western blots on the same low and high abundant pooled fractions ( figure 3a ). p16 ink4a is present in both the low and high abundant fractions of the pooled ltnps (lanes 3, 4) and is also observed in the high abundance fraction of uninfected patients ( figure 3a , lane 2). interestingly, this protein is not present in haart patient samples at all ( figure 3a , lane 5, 6). the presence of this protein in serum may be specific to individuals that confer resistance to chronic hiv-1 infection. as p16 ink4a is an inhibitor of cell cycle kinases, in particular cdk4 and cdk6, the levels of cdk4 in the serum samples was assayed and was shown to be ubiquitously expressed across all low and high abundance serum samples ( figure 3a , third panel from top). indeed, levels of cdk6 were not detectable in any of the patient serum samples as compared to a 293t whole cell extract positive control (data not shown). this implies that the presence of cdk4 in the serum is not dependent on the presence or absence of p16 ink4a and likewise, p16 ink4a does not affect the expression levels of cdk4 amongst the patient serum samples. the hp1 binding protein was initially identified in the 1d/mass spectrometry analysis in the ltnp low abundance fraction, therefore the serum levels of both hp1α and hp1γ subunits were assayed ( figure 3a ). the family of heterochromatin-associated proteins exist as three distinct isoforms, α, β, and γ and all act as regulators of heterochromatinmediated transcriptional silencing [47] . hp1α has been shown to directly interact with dna methyltransferases and histone methyltransferases to mediate transcriptional silencing [48, 49] and hp1γ, in particular, interacts with the histone methyltransferase suv39h1 to initiate a chromatin-mediated repressive state of the hiv-1 integrated virus [50] . hp1α was shown to be present in the low abundance fractions of all of the patient phenotypes whereas hp1γ was shown to be present in both the low and high abundant fractions across all patient types (fig-ure 3a ). hp1γ is observed in lower amounts in both the negative and haart high abundance fractions and all serum samples indicate the presence of a post-translational modification (i.e. a doublet band) as compared to the 293t whole cell extract positive control. this indicates that the hp1γ found in serum exists in both a modified and unmodified form. interestingly, pctaire was present in the highest abundance in the uninfected (negative), high abundance fraction ( figure 3a , lane 2), however low levels were also seen in both ltnp and haart high abundance fractions ( figure 3a , lane 4, 6). pctaire was identified initially by mass spectrometry in the high abundance ltnp sample and can be seen in the high abundance fractions of all three of the patient types biochemically, however it is present in lower amounts in the hiv-1 infected patients, indicating that this kinase may be differentially expressed upon infection though not necessarily a unique identifier for infection. p16 ink4a is the only protein identified from mass spectrometric analysis and confirmed biochemically that is specific for the 5) were collected as the flowthrough, the column was washed (lanes 6, 7) and the high abundant proteins eluted (lanes 8, 9) . briefly the observed high abundant proteins were compared to the known sizes of the expected proteins as indicated. b) equal volumes of serum from each of the six patients within each category (ltnp, haart, and negative) were pooled together to create a stock of each condition, independent of patient-to-patient variability. twenty microliters of each stock was subjected to depletion and equal concentration of low and high fraction were run on a 1d gel. lanes 2, 3 and 4 are the low abundance fractions of the pooled ltnp, haart, and negative patients, respectively. lanes 6, 8, and 10 are the high abundance fractions of the pooled ltnp, haart, and negative patients, respectively. the indicated arrows represent differentially expressed proteins that were excised, trypsinized, and identified using maldi-tof for preliminary protein screening. low abundance ltnp serum samples; although the protein is also identified in the uninfected and the high abundance ltnp fractions. these results are also interesting due to the involvement of p16 ink4a in alterations of cell cycle control, additionally, mutations in p16 ink4a are found in various cancers including pancreatic, lymphomas, and sarcomas, contributing to cancer progression [45] . these findings also indicate a difference in composition of serum proteins present in hiv-1 infected individuals undergoing haart treatment versus those that are naturally non-progressing. in order to address the concern that the protein signature of the pooled set of samples for each patient type may not be an accurate representation of the individual variability that could be present, we screened the low abundance fractions of the ltnps for the presence of both p16 ink4a and cdk4. results in figure 3b indicate the lack of detection of p16 ink4a in any of the low abundant ltnp samples, especially as compared to the pooled sample "a" (lane 2). in contrast, p16 ink4a was detectable in ltnp patients 1 and 2, as well as in the pooled sample "d" for the corresponding high abundance fractions (lanes 2, 3, 4) . due to the nature of the depletion step based on immuno-affinity, it is not surprising that p16 ink4a is detectable in individual high abundant samples, as it is probably coupled to a larger, more abundant protein and was not efficiently depleted. additionally, post-depletion, the low abundant fractions for each patient were very dilute and the protein levels were undetectable by traditional methods. cdk4 was detectable in the ltnp low abundant individual samples with variable abundance, correlating with the data in figure 3a . interestingly, cdk4 was not detectable in the ltnp high abundant individual samples, indicating that this protein was effectively isolated away from the high abundant proteins. although through a straight western blot, the levels of p16 ink4a were undetectable, figure 3c indicates that p16 ink4a can indeed be immunoprecipitated out of the individual low abundant ltnp samples and subsequently detected by western blot. lanes 1, 2, and 3 represent the pooled "a" sample and patient samples 2 and 3, respectively. the pooled "a" sample was incubated with α-igg, and patient samples 2 and 3 were incubated with α-p16. these three immunoprecipitations were subjected to very stringent wash conditions of tne 600 + 0.1% np-40, tne 300 + 0.1% np-40, and tne 50 + 0.1% np-40 in order to remove any non-specific proteins. as can be seen in figure 3c although we have identified p16 ink4a as differentially present in the serum of hiv-1 infected ltnps as compared to haart treated individuals, this may not directly correlate to viral pathogenesis or functionality of this protein. in order to gain insight into the reason why p16 ink4a may be present preferentially in the serum of ltnp patients, we treated latently infected hiv-1 cell lines (j1.1 and u1) with exogenous purified gst-p16 ink4a figure 4a depicts an rt assay which measures the viral reverse transcriptase activity of infected cells and is an indicator of functional particle production. in the presence of both 0.1 and 0.5 ug of gst-p16 ink4a , j1.1 latently infected t-cells exhibited a decrease in rt activity (cpm) whereas the higher concentration of gst-p16 ink4a was able to elicit a decrease in rt activity in the latently infected monocytes, u1, as compared to the gst treatment alone. this data suggests that the presence of p16 ink4a in serum may result in a decrease in viral replication, which may help to explain why the presence of p16 ink4a in the serum of ltnps could correlate with an overall lack of viral activity. to detect whether p16 ink4a had an effect on normal or uninfected cells, we performed an mtt assay to screen for the percentage of cells viable after p16 ink4a treatment. figure 4b depicts cem, jurkat, and h9 uninfected t-cell lines, as well as the uninfected monocytic u937 cell line treated with gst as well as gst-p16 ink4a . cem, h9, and u937 control cells showed no appreciable decrease in cellular viability upon 48 hours of treatment with any of the four conditions. interestingly, in the presence of 0.5 ug of gst-p16 ink4a , jurkat cells exhibit an almost 40% decrease in cellular viability. we next performed western blots on whole cell extracts from all four of these uninfected cell lines and observed only jurkat cells exhibiting an endogenous expression of p16 ink4a ( figure 4d, lane 2) . this suggests that the decrease in cellular viability seen in jurkat cells ( figure 4b ) treated with p16 ink4a can be correlated with the expression of exogenous p16 ink4a in these cells, resulting in an increase in cdk4,6/cyclin d inhibition and an increase in apoptosis. interestingly, no endogenous levels of p16 ink4a are detected in the infected j1.1 cells. p16 ink4a is a critical member of the rb tumor-suppressor pathway which acts to arrest the cell-cycle at g1/s by inhibiting the binding of cdk4/6 to cyclin d1 and subsequently inhibiting the phosphorylation of rb. in figure 4c , we investigate the levels of rb present in jurkat cells alone (lane 1) compared to jurkat cells treated with an excess of gst or gst-p16 ink4a (2.5 μg, lanes 2 and 3). interestingly, upon treatment of exogenous gst-p16 ink4a , we observed a decrease in cellular levels of rb; indeed there is also a decrease in rb with gst treatment alone the rb antibody used detects total rb levels in the cell, therefore we could not assume a loss of phosphorylation due to the inhibitory effect of p16 ink4a on cdk4/6. a recent paper has addressed the literature-wide discrepancies of rb dephosphorylation vs. degradation in response to drug treatment or cell senescence in various cell types [51] . it is possible that the increased amount of p16 ink4a present in these cells has induced a proteasomal degradation of rb that has not otherwise been characterized in t cells. the cell line panel in figure 4d was also screened for the presence of endogenous levels of rb in these cell lines, and interestingly there is a high degree of variability. the t cell lines cem, jurkat, and the hiv-1 infected j1.1 have the highest endogenous levels of rb. interestingly, the monocytic cell lines u937 and the hiv-1 infected u1 have the lowest amount of rb present, with almost completely undetectable levels in hiv-1 infected u1 cells. the variability supports the discrepancies seen in the literature about hypo-, hyper-phosphorylation of rb, as well as depletion or degradation of rb during cell cycle or cellular responses. in order to confirm that the effects seen by gst and gst-p16 ink4a treatment in figure 4a , b, and c, we checked to ensure that the purified proteins are actually entering the cell. jurkat, j1.1, u937, and u1 cell lines were treated with an excess (2.5 μg) of gst or gst-p16 ( figure 4e ). at 48 hours post treatment, the cells were harvested, washed extensively, lysed, and incubated with glutathione-sepharose beads overnight. the glutathione-sepharose beads were washed extensively to remove any non-specific proteins with buffers containing salts and detergents. the bound proteins were subjected to western blot for the presence of p16 ink4a as shown in figure 4e . jurkat whole cell extract served as the positive control (lanes1 in both blots) and a higher molecular weight band corresponding to gst-p16 ink4a was observed in the gst-p16 ink4a pulldown lanes for each of the cell lines (lanes 4 and 7 in both blots). the lack of detection in the untreated cell lysate incubated with beads alone indicates that the protein detected in lanes 5 and 8 are specifically the gst-bound proteins. these studies confirm that the gst proteins are indeed entering the cells when incubated in the extracellular environment. in order to confirm that the cellular effects shown in figure 4 are specific to the natural biological activity of p16 ink4a as a cdk4/6/cyclin d inhibitor, we attempted to mimic these studies with the small molecule compound inhibitor fascaplysin. fascaplysin (fasc) is a naturally derived molecule isolated from a marine sponge which specifically inhibits the interaction between cdk4/cyclin d at an ic 50 of approximately 0.35 μm, and to a lesser extent cdk6/cyclin d by binding the atp pocket of cdk4, resulting in cell cycle arrest at g1/s [52, 53] . again, we treated latently infected hiv-1 cell lines (j1.1 and u1) with three concentrations of fasc (100 nm, 500 nm, and 1 μm) and collected supernatants at 24, 48, and 72 hours post treatment. the rt activity of both j1.1 and u1 cells in the presence of fasc decreased over time with increasing concentration of the drug. this indicates that the presence of a general cdk4/cyclin d inhibitor is able correlating with the viability assay presented in figure 4b , approximately 50% of jurkat cells were killed due to additional cdk4/cyclin d inhibition by 1 μm of fasc treatment. none of the other cell lines exhibit appreciable cell death which indicates that the drug treatment itself is not toxic to the cells. in figure 5c , we investigate the levels of rb present in jurkat cells alone (lane 1) compared to jurkat cells that have been treated with three concentrations of fasc (lanes 3, 4, and 5). again, correlating with our exogenous p16 ink4a treatment data in figure 4 , we observed a decrease in total cellular rb levels at the highest concentration of fasc. this set of data confirms that the cellular effects we observed with exogenous p16 ink4a may be due to the specific cdk inhibitory activity of this molecule. it is interesting to note that these effects are seen with simple protein treatment of the cells with a purified molecule which may not have efficient entry as compared to transfection or drug treatment. this sug-gests that p16 ink4a in the serum may be able to enter and exit lymphocytes and exhibit its inhibitory effects during an hiv-1 infection. we previously showed that both p16 ink4a and fascaplysin treatment results in a loss of cellular viability in jurkat cells as well as a decrease in viral production in infected j1.1 and u1 cells. we were interested to detect the cell cycle pattern of jurkat, j1.1, u937, and u1 in response to fascaplysin treatment. cells were treated with three concentrations of fasc (100 nm, 500 nm, 1 μm) and were collected after 48 hours. cells were fixed and stained with propidium iodide and cell cycle analyzed using a facscalibur flow cytometer. in figure 6a the presence of an additional cdk4/6/cyclin d inhibitor, we observed the normal g1/s arrest effect. there were no major appreciable differences in cell cycle in the fasc treated u937 and u1, however, looking at the protein profile of endogenously expressed p16 ink4a and rb, it is not surprising that these monocytic cell lines would exhibit a different inhibitory pathway. taken together, the cell cycle data shown in figure 6 , supports the overall notion of cdk4/6/cyclin d inhibitory effect by p16 ink4a and fascaplysin. the global proteomic analysis of serum proteins is not without its challenges; however the presence or absence of proteins in such bodily fluids of patients is often the most accurate reflection of cellular leakage or secretion of proteins in response to a disease state. unfortunately, the classical serum proteome contains a large concentration of high abundance proteins that mask the individual proteins that are unique to a particular phenotype. the identification of such low abundance serum proteins is attractive as a method of early detection of a disease state or a response to infection. here, we demonstrate that the detection of unique proteins in the serum of hiv-1 infected long-term non-progressors may be indicative of a natural "immunity" to the progression of hiv-1 infection. specifically, we have identified p16 ink4a , a cdk4/6 inhibitor, as preferentially present in pooled serum of hiv-1 ltnp patients, as opposed to hiv-1 infected individuals responding to haart treatment. p16 ink4a is a member of the ink4/arf family of endogenous cdki's that serve to regulate cell cycle progression through the inhibition of specific cdk/cyclin interactions. p16 ink4a is a critical member of the rb/p16 tumor-suppressor pathway which inhibits the activation of cdk4/6, preventing the progression through the cell cycle. this tumor-suppressive pathway is mutated in close to 100% of human cancers and specific loss-of-function mutations are found within the rb gene or the cdkn2a gene encoding p16 ink4a [54] . in addition to the direct inactivation of these two proteins, cancer cells also override this regulatory pathway by overexpressing cdk/ cyclins as well as inducing the endogenous loss of expression of other cdk inhibitors. the loss of p16 ink4a results in the constitutive activation of cdk4/6 as well as prb hyperphosphorylation, therefore bypassing the antioncogenic senescence induced by this cdk inhibitor. in addition to necessitating an oncogenic state, the regulation and manipulation of cellular cdks and cyclins is also critical for an active hiv-1 viral infection and occurs mainly through the hiv-1 transactivator tat. tat interacts with the cdk2/cyclin e complex to phosphorylate cdk7 and assist in the phosphorylation of the c-terminal domain (ctd) of rna pol ii at the viral ltr (long-terminal repeat) to promote viral transcription [55] [56] [57] . cdk2 is also involved in the direct phosphorylation of tat at both serine 16 and 46, which are critical for efficient tat activity [56, 57] . hiv-1 transactivation is also heavily dependent on the recruitment of the cdk9/cyclin t1 complex to the viral tar element where it also assists in the phosphorylation of the ctd of rna pol ii as well as an autophosphorylation event which is important for the localization to the nucleus [58] . taking into consideration the recruitment of cellular kinases for hiv-1 transcription, it is not surprising that pharmacological cdk inhibitors have been developed (analogous to endogenous cdki's) as a template to specifically inhibit these kinases. therefore, the presence of the cdk inhibitor p16 ink4a in ltnp serum is suggestive of an existing defense mechanism in these patients that imparts a predisposition to a lack of hiv-1 disease progression. the development of pharmacological cdki's and peptide mimetics is a commonly used approach for both cancer and viral therapeutics. for instance, p16 ink4a peptides have previously been developed, which, when conjugated to localization proteins/peptides, were able to block cell cycle progression in breast cancer and colon cancer cell lines in vitro as well as reduce tumor size in an in vivo mouse model of pancreatic cancer [59] [60] [61] . these peptides have also been used for intracellular delivery to leukemia and lymphoma derived cells [59, 62] . these studies have demonstrated the ability of cdki-derived peptides to be used for both direct cell cycle arrest and the treatment of metastatic cancers. this type of therapy is an appropriate approach to cancerous states where the oncogenic mutation results in a loss of function or expression of the p16 ink4a in this study we also chose to reinforce the p16 ink4ainduced cdki effects seen on cellular viability and viral replication with a known cdk4/6/cyclin d inhibitor, fascaplysin. we show that treatment of jurkat cells with this inhibitor also results in a decrease in cellular viability, consistent with the effect seen by treatment with endogenous p16 ink4a . additionally, the treatment of both stably infected hiv-1 cell lines, j1.1 and u1, with fascaplysin resulted in a decrease in viral replication with a minimal decrease in cellular viability. the correlation of this pharmacological cdki inhibitor data with the functional-cdki p16 ink4a data suggests that the preferential inhibition of the cdk4/6/cyclin d interaction by ltnps may contribute to this phenotype. the high and low abundance fractions analyzed for each of the three patient types were a pooled representation of six individual patient samples that almost certainly have inherent variability. indeed, when assaying for p16 ink4a in high and low abundance serum fractions for each individual ltnp patient, p16 ink4a was present in only a subset of the patients analyzed. this indicates that although p16 ink4a may serve as a unique serum protein indicating a ltnp hiv-1 infected status, individual person-to-person changes will alter the serum proteome profile and needs to be taken into consideration. differences in expression of p16 ink4a can be due to either a host cellular response to the infection or from an influence of the infection itself. two different scenarios need to be finally, many complicating factors can contribute to the altered state of proteins present in the serum, not the least of which is the length of time in which the patient has been infected, age, gender, coinfections, and other preexisting conditions such as cancer or metabolic diseases. therefore, it is important to take into consideration some of these factors when applying global proteomic analyses to hiv-1 patient derived samples. 293t endothelial kidney cell line was harvested for whole cell extract and used as a positive control. 293ts were grown in dulbecco's modified eagle's medium (dmem) containing 10% fbs, 1% l-glutamine, and 1% streptomycin/penicillin (quality biological). latently infected hiv-1 cell lines j1.1 (t-cells) and u1 (monocytes) were used for rt assays and whole cell extracts were obtained for western blots. uninfected cem, jurkat, and h9 cell lines (t-cells) and uninfected u937 cell line (monocytes) were used for viability assays and whole cell extracts were obtained for western blots. uninfected cells were grown in rpmi-1640 media containing 10% fbs, 1% l-glutamine, and 1% streptomycin/penicillin (quality biological). all cells were incubated at 37°c and 5% co2. gst-p16 ink4a was a generous gift from dr. ming-daw tsai, institute of biological chemistry, academia sinica, nankang, taipei, taiwan eighteen subject serum samples (6 ltnp, 6 hiv infected subjects receiving haart therapy, and 6 uninfected individuals) were obtained through washington dc site of the women's interagency hiv study (wihs) ( table 1) . wihs is an nih multicenter study of the natural history of hiv-1 infection in women [29] . ltnps were defined by wihs as being hiv infected, but disease free for at least five years, a cd4 count of greater than 500 at all visits, and no history of anti-retroviral therapy. serum samples were subjected to depletion of the 12 most abundant serum proteins using the proteomelab igy-12 high capacity spin column proteome partitioning kit from this spin column consists of anti-human serum albumin, anti-igg, anti-fibrinogen, anti-transferrin, anti-iga, anti-igm, anti-hdl (anti-apo a-i and anti-apo a-ii) , anti-haptoglobin, anti-α1-antitrypsin, anti-α1-acid glycoprotein and anti-α2-macroglobulin conjugated to polymeric microbeads. twenty microliters of each serum sample was diluted 1:25 in dilution buffer and ran over the spin column. low abundant proteins were collected in the flowthrough and subsequent high abundant, bound proteins were retained and eluted with a low ph stripping buffer. protein concentrations of both low and high abundant fractions were calculated and pooled as indicated. prowl-cgi/profound.exe databases for peptide mass fingerprinting analysis. fifty microliters of pooled low abundance ltnp serum sample "a" and individual low abundance ltnp serum samples (1) (2) (3) (4) (5) (6) were incubated with either α-igg or α-p16 ink4a as indicated, volume was brought up to 500 μl with tne 50 western blots were performed to validate proteins identified from depleted serum samples. whole cell extracts were obtained from cell culture pellets washed twice with 25 ml of phosphate buffered saline (pbs) with ca2+ and mg2+ (quality biological) and centrifuged once more. cell pellets were resuspended in lysis buffer (50 mm tris-hcl, ph 7.5, 120 mm nacl, 5 mm edta, 0.5% np-40, 50 mm naf, 0.2 mm na3vo4, 1 mm dtt, one complete protease cocktail tablet/50 ml) and incubated on ice for 20 min, with a gently vortexing every 5 min. cell lysates were transferred to eppendorf tubes and were centrifuged at 10,000 rpm for 10 min. supernatants were transferred to a fresh tube where protein concentrations were determined using bio-rad protein assay (bio-rad, hercules, ca). antibodies against cdk4 (sc-749), p16 (sc-467), pctaire (sc-174), rb (sc-50), and actin (sc-1615) were purchased from santa cruz biotechnology (santa cruz, ca). antibodies against hp1α and hp1γ were purchased from cell signaling (danvers, ma). j1.1 and u1 cells (2 × 10 6 ) were treated with gst or gst-p16 (0.1 or 0.5 μg) or fascaplysin (100 nm, 500 nm, or 1 μm) and supernatants collected to test for the presence of virus 48 hours post treatment. jurkats were treated with gst or gst-p16 (2.5 μg) for 48 hours prior to hiv-1 infection with the dual tropic strain 89.6. supernatants were collected at various time points post infection to test for the presence of virus post treatment. viral supernatants (10 μl) were incubated in a 96-well plate with reverse transcriptase (rt) reaction mixture containing 1x rt buffer (50 mm tris-hcl, 1 mm dtt, 5 mm mgcl 2 , 20 mm kcl), 0.1% triton, poly(a) (1u/ml), pd(t) (1u/ml), and [3h]ttp. the mixture was incubated overnight at 37°c, and 5 μl of the reaction mix was spotted on a deae filtermat paper, washed four times with 5% na 2 hpo 4 , three times with water, and then dried completely. rt activity was measured in a betaplate counter (wallac, gaithersburg, md). five thousand cells were plated per well in a 96-well plate and the next day cells were treated with gst or gst-p16 (0.1 or 0.5 μg) or fascaplysin (100 nm, 500 nm, or 1 μm). forty-eight hours later, 10 μl mtt reagent (5 mg/ml) was added to each well and plates incubated at 37°c for 3 hours. next, 100 μl of dmso was added to each well to solubilize the violet crystals. the assay was read at 570 nm. cells were washed with pbs and fixed with 70% ethanol. following rehydration in pbs, cells were stained in pbs containing 25 ug/ml propidium iodide (sigma), 10 ug/ml rnase a (sigma) and 0.1% np-40. cells were analyzed on a bd facscalibur flow cytometer. cell cycle analysis and measurement of apoptosis was performed using cell-quest software. aggregates and debris were excluded by gating on the fl2w and fl2a parameters. apoptosis was considered to be the population of cells that were sub-g1. toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry a novel approach and protocol for discovering extremely low-abundance proteins in serum peptidomics-based approach reveals the secretion of the 29-residue cooh-terminal fragment of the putative tumor suppressor protein dmbt1 from pancreatic adenocarcinoma cell lines the role of proteomics in toxicology: identification of biomarkers of toxicity by protein expression analysis utilizing human blood plasma for proteomic biomarker discovery clinical potential of proteomics in the diagnosis of ovarian cancer progress and challenges in screening for early detection of ovarian cancer serum proteomics in cancer diagnosis and management therapeutic potential of the plasma proteome the human plasma proteome: history, character, and diagnostic prospects three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer current status of the molecular characterization of the ovarian cancer antigen ca125 and implications for its use in clinical screening the free/total prostate-specific antigen ratio (%fpsa) is the best predictor of tumor involvement in the radical prostatectomy specimen among men with an elevated psa serum proteomics with seldi-tof-ms in congenital human cytomegalovirus hepatitis serum proteomic fingerprints of adult patients with severe acute respiratory syndrome the hcv serum proteome: a search for fibrosis protein markers the role of a mutant ccr5 allele in hiv-1 transmission and disease progression comparison of proviral accessory genes between long-term nonprogressors and progressors of human immunodeficiency virus type 1 infection vpr and hiv-1 disease progression: r77q mutation is associated with long-term control of hiv-1 infection in different groups of patients badley ad: vpr r77q is associated with long-term nonprogressive hiv infection and impaired induction of apoptosis importance of the nef gene for maintenance of high virus loads and for development of aids genomic structure of an attenuated quasi species of hiv-1 from a blood transfusion donor and recipients increased frequency of ccr-5 delta 32 heterozygotes among long-term non-progressors with hiv-1 infection. the australian long-term non-progressor study group role of ccr5, ccr2 and sdf-1 gene polymorphisms in a population of hiv-1 infected individuals genetic restriction of hiv-1 infection and progression to aids by a deletion allele of the ckr5 structural gene. hemophilia growth and development study hla-b polymorphism in japanese hiv-1-infected long-term surviving hemophiliacs hla b*5701 is highly associated with restriction of virus replication in a subgroup of hiv-infected long term nonprogressors progression of hiv to aids: a protective role for hla-b27? the women's interagency hiv study transcription factor ap-2 activates gene expression of htlv-i transcription factor prdii-bf1 activates human immunodeficiency virus type 1 gene expression recruitment of chromatin-modifying enzymes by ctip2 promotes hiv-1 transcriptional silencing chromatin remodeling and modification during hiv-1 tat-activated transcription cleavage of human and mouse cytoskeletal and sarcomeric proteins by human immunodeficiency virus type 1 protease. actin, desmin, myosin, and tropomyosin il-16-and other cd4 ligand-induced migration is dependent upon protein kinase c hiv-1 tat protein induces il-10 production in monocytes by classical and alternative nf-kappab pathways human immunodeficiency virus type 1 tat protein induces an intracellular calcium increase in human monocytes that requires dhp receptors: involvement in tnf-alpha production signaling pathways triggered by hiv-1 tat in human monocytes to induce tnf-alpha hiv-1 tat protein induces interleukin-10 in human peripheral blood monocytes: involvement of protein kinase c-betaii and-delta trans-activation of hiv-1 ltrdirected gene expression by tat requires protein kinase c in vitro phosphorylation of human immunodeficiency virus type 1 tat protein by protein kinase c: evidence for the phosphorylation of amino acid residue serine-46 hiv envelope gp120 activates human arterial smooth muscle cells involvement of protein kinase c in hiv-1 gp120-induced apoptosis in primary endothelium mammalian cyclin-dependent kinases cyclin d-dependent kinases, ink4 inhibitors and cancer comparative genomics of cyclin-dependent kinases suggest co-evolution of the rnap ii c-terminal domain and ctddirected cdks heterochromatin formation in mammalian cells: interaction between histones and hp1 proteins functional cooperation between hp1 and dnmt1 mediates gene silencing the hp1alpha-caf1-setdb1-containing complex provides h3k9me1 for suv39-mediated k9me3 in pericentric heterochromatin suv39h1 and hp1gamma are responsible for chromatin-mediated hiv-1 transcriptional silencing and postintegration latency roninson ib: p21(waf1/cip1/sdi1) mediates retinoblastoma protein degradation inhibition of cyclin-dependent kinase 4 (cdk4) by fascaplysin, a marine natural product ca224, a non-planar analogue of fascaplysin, inhibits cdk4 but not cdk2 and arrests cells at g0/g1 inhibiting prb phosphorylation searching for selective cyclin-dependent kinase inhibitors to target the retinoblastoma/p16 cancer gene pathway phosphorylation of hiv-1 tat by cdk2 in hiv-1 transcription hiv-1 tat interaction with rna polymerase ii c-terminal domain (ctd) and a dynamic association with cdk2 induce ctd phosphorylation and transcription from hiv-1 promoter hiv-1 tat-associated rna polymerase c-terminal domain kinase, cdk2, phosphorylates cdk7 and stimulates tat-mediated transcription novel hiv-1 therapeutics through targeting altered host cell pathways therapeutic peptides for cancer therapy. part ii -cell cycle inhibitory peptides and apoptosisinducing peptides inhibition of prb phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16cdkn2/ink4a the p16(ink4a) tumour suppressor protein inhibits alphavbeta3 integrin-mediated cell spreading on vitronectin by blocking pkc-dependent localization of alphavbeta3 to focal contacts highly efficient delivery of p16 antitumor peptide into aggressive leukemia/lymphoma cells using a novel transporter system we would like to thank the members of the kashanchi lab for experiments and assistance with the manuscript. we thank dr. ming-daw tsai (institute of biological chemistry, academia sinica) for the gst-p16 ink4a plasmid constructs. most of the data on the current manuscript was generated using funds from nih grants ai078859, ai074410 and ai043894. data in this manuscript were the authors declare that they have no competing interests. rvd performed the serum depletion, 2d gel analysis, maldi preparation, and mass spectrometry analysis as well as the drafting of the manuscript. ig performed serum depletion, maldi preparation, and confirmatory western blots. kkh performed the rt and mtt assays. re performed the maldi preparation and analysis. zk provided support in drafting the manuscript and maldi analysis. cl established the patient definitions (ltnp, haart responders, etc.) and patient sample identification. my is the wihs contact and provided support and information on patient samples. fk provided overall direction and funding for the project. all authors have read and approved the final manuscript. key: cord-310867-78cx3o29 authors: mo, phoenix k. h.; ng, charlson t. y. title: stigmatization among people living with hiv in hong kong: a qualitative study date: 2017-02-14 journal: health expect doi: 10.1111/hex.12535 sha: doc_id: 310867 cord_uid: 78cx3o29 background: hiv/aids is one of the most stigmatized medical conditions across the world. self‐stigma is prevalent among people living with hiv (plhiv) and a major obstacle to hiv prevention and care. objective: this study aimed to describe the experiences of stigmatization and explore the possible factors that might be associated with stigmatization among plhiv in hong kong. design: qualitative in‐depth interviews were conducted. setting and participants: 15 plhiv were recruited from two local non‐governmental organizations on hiv prevention. main variables studied: participants were interviewed about their views and feelings towards oneself as a plhiv and contributing factors, experiences of discriminations, stigmatizing behaviours, issues about disclosure, social relationships and potential impact of hiv. results and conclusions: thematic analyses revealed three levels of factors which might be associated with stigmatization: (i) intrapersonal level (misconceptions about hiv, attribution of self‐responsibility, severe state of illness, side‐effects of medication), (ii) interpersonal level (discrimination, social rejection) and (iii) social level (mass media, public stereotypes). findings provide important insights into which interventions to reduce stigmatization of plhiv could be designed. hiv is a serious global epidemic causing heavy social and medical costs. although global hiv prevalence has levelled off, the total number of people living with hiv (plhiv) is increasing steadily due to the on-going acquisition of hiv infection, longer survival times of plhiv and a growing general population. 1 in hong kong, the hiv prevalence was low among the general population but high among some specific populations (e.g. 5.9% among men who have sex with men). 2 among the new hiv cases reported in the second quarter of hong kong, 86.7% were male, and 35.7%, 36.9% and 4.5% were infected through heterosexual, homosexual and bisexual contact, respectively. 3 the figures are comparable to those of the uk, which shows that out of those newly diagnosed with hiv in 2015, 75% were male, and 38.1% and 56.1% were infected through heterosexual and homosexual sex contact, respectively. 4 despite the promising efforts in counteracting hiv, hiv continues to be a major public health concern in hong kong. hiv has long been regarded as one of the most stigmatized medical conditions. 5 according to goffman, 6 stigma is defined as an attribute linking a person to a set of undesirable characteristics that may lead to prejudice and discrimination. while public stigma is the reaction that the general population has to the individuals who are considered different, self-stigma occurs when "people with devalued status who internalize the discriminatory beliefs and experience diminished selfesteem and self-efficacy". 7 corrigan suggested that both public stigma and self-stigma comprise three components: stereotype (negative belief about the group/self), prejudice (agreement with belief and/or negative emotional reaction) and discrimination (behaviour response to prejudice). 8 self-stigma has further been explained by the cognitiveaffective-behavioural model. 9 according to the model, the stigmatized individuals perceive their stigmatized identity as a burden and taint of their life, which lead to negative sense of self (self-stigmatizing cognitions). such conceptions might lead to a myriad of negative affective responses, encompassing feelings of anger, fear and shame (selfstigmatizing affect) which, in turn, resulting in identity concealment, social avoidance and self-denigration (self-stigmatizing behaviours). despite the continuous intervention efforts in combating hiv stigma, public stigma towards plhiv remains ubiquitous across the globe. in a meta-analysis of 21 studies, public stigma towards hiv/ aids is the greatest compared to genital herpes, hepatitis, drug abuse and cancer. 10 the high level of stigma towards plhiv can be due to several causes. first, a high level of self-responsibility is attached to hiv as the acquisition of hiv is due to behaviours that can be preventable, such as unsafe sex and needle sharing. 11, 12 some behaviours associated with hiv, such as same-sex behaviours, are also considered immoral and are thus condemned by the society. 5 misconceptions about hiv transmission routes and overestimation of the perceived contagiousness and risks through casual contact further evoke stigmatization towards hiv. 12 these cause an intensive level of blame and disapproval against plhiv, resulting in social rejection, discrimination and prejudice. 13, 14 it is contended that stigma against plhiv is even more intense in the chinese context, as the behaviours related to hiv infection, such as same-sex behaviours, multiple sex partnership and injecting drug use, are generally perceived as defying acceptable social norms and culture. [15] [16] [17] high level of ignorance, misconception and fear of hiv also contribute to the high level of stigma towards plhiv in the chinese context. 18, 19 furthermore, face concern, which refers to social image and social worth that are garnered based on one's performance in an interpersonal context, is particularly important in the chinese society. 20, 21 yang and kleinman 22 proposed that while the concept of "face" represents social power, capital and a person's value in society, plhiv experiences loss of face as a result of violation to cultural norms, which greatly affects their access to social capital and closely parallels how stigma works in chinese society. in hong kong, studies have documented a high level of unfavourable attitudes and stigmatizing behaviours towards plhiv. for example, a population-based study among hong kong adults reported that nearly half of them exhibited discriminatory attitudes towards plhiv. 23 a comparative study on the public's attitude towards different types of infectious diseases have shown that the level of public stigma towards hiv was the highest compared to other infectious diseases such as severe acute respiratory syndrome (sars) and tuberculosis (tb). 24 plhiv are also disadvantaged in terms of economic and social opportunities. for example, a local study reported that 20% of the studied companies would dismiss an employee if he or she was hiv+, and only a few companies indicated that they would provide counselling and support to an hiv+ employee. 25 comparative study on employer attitudes towards plhiv in beijing, hong kong and chicago revealed the trend of reluctance to hire plhiv are most pronounced among employers from beijing and hong kong. 26 study among plhiv in hong kong has also shown that over 50% felt that they were discriminated in different settings such as in the workplace and in social relationships. 27 hiv stigma poses a significant impediment to public health across the globe and a key obstacle to hiv treatment, prevention, care and support. 28, 29 it is noticeable that many plhiv have internalized the negative labels attached to them and experienced a high level of selfstigma. in one study among 322 plhiv in china, 78% of them had the feeling of negative worth and 58% of them were unwilling to disclose their hiv status. 30 it is also reported that the level of internalized stigma was similar between plhiv diagnosed less than 1 year ago and more than 1 year, indicating that level of self-stigma did not reduce across time. 31 self-stigma is significantly associated with various negative outcomes such as worse psychosocial well-being, 32 showed that attribution of perceived controllability of the disease, personal responsibility to the cause of the illness and self-blame was significantly associated with higher levels of self-stigma. 45, 46 however, one local study looking at factors associated with self-stigmatization using the attribution model for plhiv, including self-blame, responsibility for contacting hiv and internal controllability for contracting hiv, showed that although attributions of control predicted attributions of responsibility which, in turn, predicted self-blame, the linkage between self-blame and self-stigma was not significant. 47 other factors have also been proposed. recent studies in adults living with hiv have found that optimism predicts lower levels of hiv-related stigma indirectly through increasing psychological well-being 32 and also that personal meaning predicts lower level of hiv-related stigma indirectly through increasing social support. 48 these mechanisms have not been explored in the local context. furthermore, mass media has often been used to shape public attitudes and knowledge about hiv. it has been suggested that chinese individuals are more likely to obtain hiv information from mass media than from interpersonal sources. 49 a content analysis of articles about hiv published in chinese newspapers over a decade revealed that individuals who contracted hiv through socially unacceptable means were devalued as non-descript members of a deviant and dangerous group. 50 however, from our understanding, how mass media might lead to self-stigma among plhiv in the chinese context has not been examined. there is an urgent need to understand how internalization of stigma is formed and intensified among plhiv in the chinese context so that tailored services and intervention can be provided to this population. the purpose of this study was to describe and explore the possible factors that may contribute to internalization of stigma among plhiv in hong kong using a qualitative approach. participants were adults living with hiv in hong kong. inclusion criteria were (i) age of 18 or above, (ii) being diagnosed with hiv for more than 6 months, (iii) being able to speak in cantonese which is the native language of hong kong and (iv) mentally competent in taking part in a 1.5-hour in-depth interview. participants were recruited from two local non-governmental organizations (ngo) on hiv prevention using convenience sampling. staff of the participating ngo identified their members who fulfilled the inclusion criteria, briefed and referred them to contact the research staff who would make an appointment to meet with them. at the meeting, participants were briefed about the purpose and logistic of the study again. written informed consent was obtained before the in-depth interview was administered by the research staff in a private room, in the absence of any third person. data confidentiality was assured. a total of 15 eligible members were referred to the research team; all of them provided written consent to join the study and completed the in-depth interview. each interview lasted for about 1.5 hours. ethical approval was obtained from the chinese university of hong kong survey and behavioral research ethics committee. participants' socio-demographic characteristics, including age, gender, education level and employment status, were obtained. medical characteristics, including length of diagnosis, disease stage, and most recent cd4 count, were also obtained. interview questions were open-ended and broad to elicit a detailed description of participants' experiences. the interviews were audio-recorded and transcribed verbatim. responses characterizing the factors associated with internalization of stigma were noted and analysed inductively using thematic analysis. the coding process of thematic analysis involves recognizing patterns from the data and encoding them prior to the process of interpretation. 51, 52 the responses were read and reread several times, across both questions and respondents, to increase familiarity with the data. notes were made to reflect initial impressions and were progressively conceptualized into broader themes that best captured participants' viewpoints. 53 the coding framework and results were discussed between the authors, and any discrepancies were resolved to safeguard the reliability of the findings. findings from the interviews seem to suggest that various levels of factors contribute to self-stigma among plhiv. in particular, participants' responses to the open-ended questions were conceptualized in the following themes: misconceptions about hiv poor perception of self as a plhiv seems to build from such discriminatory experiences. when plhiv find that they do not receive any respect and support from others, they are more likely to conceal their identity and show negative feelings towards themselves. the negative perception might be further exacerbated when it comes to the clinic setting, as clinic staff is expected to show empathetic understanding to every patient. for most of the participants, hiv infection is considered as a shame to both plhiv and the groups they belong to, from the interview it was ubiquitous that many members were rejected from their social circle. findings suggest that participants seem to be rejected from their significant others, and these unpleasant responses tend to be the prime factor for their self-stigmatization. social rejection is particularly deleterious that plhiv might think that their identity incur shame to them and their families as well. it also appears to make them believe that being plhiv would only receive aversive reactions from their social network, leading to a negative perception towards themselves. findings also suggest that public stereotype about hiv appears to be the cause of their self-stigmatization. some respondents revealed that plhiv were perceived by the society as "lan gwan" (someone who always patronizes sex workers) and "dai sei"(someone who deserves to die), these stereotypes have further portrayed plhiv as "bad people," which jeopardizes their level of self-worth: these public stereotypes constitute a negative impression towards plhiv, constructing devaluated and marginalized social status on them. plhiv are highly aware of the stereotype attached on them, and they would then internalize those stereotypes and prejudices onto themselves. the stereotypes imbued by the society are that plhiv should be drugs users, homosexuals or promiscuous, which are bounded to be negative. the mass media, mainly the tv, advertisements and newspapers, serves as a tool to disseminate message and constructs a norm within the society. most of the respondents seem to blame the media for being the main source of creating unwelcoming and hostile climate towards plhiv. as one member explained: …media seemingly only focuses on the dark side of plhiv. for example, newspapers usually use an exaggerated title to describe a plhiv who has committed suicide. it has instilled the concept into general public that all plhiv will do the same thing as well. members claimed that the media was prone to cast a negative light on them by developing negative portrayal of plhiv: in some movies or tv programme, it was common to see that the actor would curse their enemy to die of hiv. although it seemed to be to a joke, it might defame us. the present study was the first attempt in understanding the stigmatizing experiences and the possible factors that may be related to self-stigmatization among plhiv in hong kong.. findings show that participants face strong stigmatization in various ways and suggest a number of factors that may be associated with their self-stigmatization. first, from the intrapersonal level, attribution of responsibility to self seems to be associated with self-stigmatization. our findings suggest that many participants have shown feelings of shame and guilt as they considered themselves responsible for the infection. as most of the participants acquired hiv through heterosexual contact with sex workers or homosexual contact with men, it is conceivable that participants demonstrated an internal attribution of the disease and thus reporting negative feelings of shame and guilt and a high level of self-stigmatization. findings are consistent with a local study which shows that the hong kong general public perceived plhiv as more responsible and blameworthy of their diseases than other conditions such as sars and tb. 24 findings also suggest that physical challenges, such as severe hiv symptoms or medication side-effects; tend to increase their level of self-stigmatization. participants described how they wanted to isolate themselves because of the physical symptoms, and how their physical symptoms made them felt negative about themselves. previous work has suggested that stigma associated with hiv increases as symptoms become more apparent to others. 11 findings are also consistent with previous studies that plhiv who greater severity of hiv symptoms experienced higher levels of self-stigma. 5 as hiv is a concealable condition, the appearance of physical symptoms might be one of the primary cues that someone is infected with hiv. the intense physical sufferings attached to hiv may cause plhiv distress and frustration. hiv is another factor leading to self-stigmatization. as suggested by the data, misconceptions about hiv, even among plhiv, were highly prevalent. findings corroborate with local studies that people in hong kong have poor knowledge about hiv. 23 it also supports previous studies that show the pathway between lack of knowledge about hiv to increased felt stigma and decreased intention to disclose one's hiv status. 54 despite the critics that education alone might not be effective in reducing stigma, 55 the present study suggests that providing plhiv knowledge of the disease is still the key and first step in stigma reduction. in the interpersonal level, findings also suggest that discrimination and social rejection emerged as a factor driving self-stigmatization. the present study indicates that plhiv experience discrimination and social rejection in various settings. in the study, most of the participants gave detailed accounts on their feelings of social isolation or experiences of being rejected by the family and society. some of them even reported facing discrimination in the health-care settings. indeed, stigma attached to hiv remains ubiquitous in hong kong. 23 study in china also revealed that people hold stigmatizing attitude to plhiv as they are blamed for their acquisition of the disease. 19 facing both social rejection and discrimination experiences, plhiv may endorse their stigmatizing attitudes and internalize negative feelings into themselves, which, in turn, develop poor sense of self and self-esteem. at the society level, the media also plays an important role in shaping the self-stigma of plhiv, mainly through delivering biased messages about hiv and developing a negative representation of plhiv. at the individual level, it is important to increase awareness of stigma and the benefits of reducing stigma among health workers. in the environmental level, there is a need to ensure that health workers have the information, supplies and equipment necessary to practice universal precautions and prevent transmission of hiv. it is also important to enact policies that protect the safety and health of patients and health-care professionals in order to prevent discrimination against plhiv. however, this might be difficult to achieve given the lack of policies to prevent discriminations against vulnerable groups in hong kong. the study also indicate that mass media is the main culprit of creating a biased image for plhiv, thus leading to their stigmatization. interventions to reduce hiv stigma should therefore extend beyond the individual level. previous study has reported that exposure to multiple sources of hiv information from mass media was significantly related to hiv knowledge and less stigmatizing attitude towards plhiv. 49 there are several limitations of the study that should be noted. first, a convenience sampling was used. therefore, results cannot be interventions to reduce stigma of plhiv are strongly warranted and should target not only the patients themselves, but also their family members, health-care professionals and the broader community. world health organization. global hiv prevalence has levelled off centre for health protection hiv surveillance and epidemiology press meeting presentation internalized stigma among people living with hiv-aids stigma: notes on the management of spoiled identity. harmondsworth: penguin understanding the impact of stigma on people with mental illness self-stigma and the "why try" effect: impact on life goals and evidence-based practices self-stigma across social minorities: conceptualization and unified measurement stigma associated with aids: a meta-analysis aids and stigma aids stigma and sexual prejudice can we measure hiv/aids-related stigma and discrimination? current knowledge about quantifying stigma in developing countries disentangling hiv and aids stigma in ethiopia, tanzania and zambia expressions of hiv-related stigma among rural-to-urban migrants in china homosexuality, seropositivity, and family obligations: perspectives of hiv-infected men who have sex with men in china prostitutes, prostitution and std/hiv transmission in mainland china stigma against hiv-infected persons among migrant women living in understanding hiv-related stigma and discrimination in a "blameless" population face concern: its role on stress-distress relationships among chinese americans understanding distress: the role of face concern among chinese americans, european americans, hong kong chinese, and mainland chinese face' and the embodiment of stigma in china: the cases of schizophrenia and aids discriminatory attitudes towards people living with hiv/aids and associated factors: a population based study in the chinese general population comparative stigma of hiv/aids, sars, and tuberculosis in hong kong aids-related discrimination in the workplace-the results of two evaluative surveys carried out during a three-year period in hong kong stigma in the workplace: employer attitudes about people with hiv in beijing, hong kong, and chicago needs assessment and social environment of people living with hiv/aids in hong kong stigma in the hiv/aids epidemic: a review of the literature and recommendations for the way forward hiv/aids stigma: an impediment to public health stigma reported by people living with hiv in south central china experiences of hiv-related stigma among young men who have sex with men optimism, wellbeing, and perceived stigma in individuals living with hiv perceived discrimination and mental health symptoms among black men with hiv. cult divers ethnic minor psychol disclosure of hiv status to sex partners and sexual risk behaviours among hiv-positive men and women stigma mediates the relationship between self-efficacy, medication adherence, and quality of life among people living with hiv/aids in china the effects of hiv stigma on health, disclosure of hiv status, and risk behavior of homeless and unstably housed persons living with hiv meta-analysis of health and demographic correlates of stigma towards people living with hiv a model of suicidal ideation in adults aging with hiv the association of stigma with self-reported access to medical care and antiretroviral therapy adherence in persons living with hiv/aids hiv stigma and missed medications in hiv-positive people in five african countries intentionality of medication non-adherence among individuals living with hiv/aids in hong kong impact of hiv-related stigma on treatment adherence: systematic review and meta-synthesis exploring hiv stigma and quality of life for persons living with hiv infection associations between perceived hiv stigma and quality of life at the dyadic lvel: the actor-partner interdependence model internalization of stigma for parents of children with autism spectrum disorder in hong kong cognitive insight and causal attribution in the development of self-stigma among individuals with schizophrenia examining attribution model of self-stigma on social support and psychological well-being among people with hiv+ personal meaning, social support, and perceived stigma in individuals receiving hiv mental health services mass media and hiv/aids in china chinese newspapers' coverage of hiv transmission over a decade (2000-2010): where hiv stigma arises a pragmatic view of thematic analysis transforming qualitative information: thematic analysis and code development competing paradigms in qualitative research understanding interrelationships among hiv-related stigma, concern about hiv infection, and intent to disclose hiv serostatus: a pretest-posttest study in a rural area of eastern china joint united nations programme on hiv/aids. key programmes to reduce stigma and discrimination and increase access to justice in national hiv responses self-stigma among concealable minorities: conceptualization and unified measurement interventions to reduce hiv/aids stigma: what have we learned? hiv interventions to reduce hiv/aids stigma: a systematic review a hiv stigma reduction intervention for people living with hiv and their families combating hiv stigma in health care settings: what works? we would like to express our heartfelt gratitude to mr. marco chau from heart to heart and ms. krystal yiu from hong kong aids foundation, who provided invaluable support with participant recruitment for this study. thanks are also given to all participants who participated in this study. all authors declare that they have no conflict of interests. key: cord-332569-af8oq2d6 authors: friedman, henry; ator, nancy; haigwood, nancy; newsome, william; allan, james s.; golos, thaddeus g.; kordower, jeff h.; shade, robert e.; goldberg, michael e.; bailey, matthew r.; bianchi, paul title: the critical role of nonhuman primates in medical research date: 2017-08-23 journal: pathog immun doi: 10.20411/pai.v2i3.186 sha: doc_id: 332569 cord_uid: af8oq2d6 the sponsors of this report endorse carefully regulated research with nonhuman primates. this research is essential to learning about the biology, treatment and prevention of diseases and conditions that cause human suffering. research with nonhuman primates (nhps) -monkeys for the most part -has led to critical health advances that have saved or improved millions of human lives. while nhps account for just one-half of one percent of animals in current medical research, it is no exaggeration to say they are essential to our ability to find cures for cancer, aids, alzheimer's, parkinson's, obesity/diabetes, and dozens of other diseases that cause human suffering and death. research with monkeys is critical to increasing our knowledge of how the human brain works and its role in cognitive, motor, and mental illnesses such as alzheimer's, parkinson's, and depression. this research is also fundamental to understanding how to prevent and treat emerging infectious diseases like zika and ebola. nhp research is uncovering critical information about the most common and costly metabolic disorder in the u.s. -type 2 diabetes -as well as the obesity that leads to most cases. without nhp research, we lose our ability to learn better ways to prevent negative pregnancy outcomes, including miscarriage, stillbirth, and premature birth. this research is also helping scientists to uncover information that makes human organ transplants easier and more accessible, literally giving new life to those whose kidneys, hearts, and lungs are failing. news headlines tout medical breakthroughs. breakthrough sounds dramatic, and to someone hearing about how the virus that causes polio is being used to put an aggressive form of brain cancer into remission, it is indeed. but as the scientists involved in that cancer research-and research into every other area of medicine-will tell you, breakthroughs might be dramatic, but they are never sudden. a well thought-out and structured process is behind virtually every medical breakthrough and the discovery process probably took decades or more. every step in the process was essential to the next, from basic research to human clinical trials. monkeys are often involved at the later stage of the processwhat is called translational or applied research. here all of the knowledge accumulated earlier is applied to specific medical questions such as: will this vaccine protect a pregnant woman (and her baby) from zika infection? and is the vaccine likely to be safe? research scientists are like detectives solving mysteries. we want to know why. more importantly, we want to save lives. but monkeys also play a vital role in basic science research that can come decades earlier. basic nhp research in the 1970s helped scientists understand the inner workings of the basal ganglia, the part of the brain that coordinates movement. those early findings led to the "breakthrough" 30 years later in which deep brain stimulation is used to reduce involuntary movements of parkinson's disease. see more breakthroughs linked to nhp research in appendix a. regardless of where it occurs in the scientific discovery process, research with monkeys is highly regulated (see appendix b). scientists use monkeys only when no other research model can provide the required information. while rodents are used extensively and are extremely helpful in answering many basic research questions, their usefulness is limited by differences from primates in their lack of sophisticated brain structures, less developed immune systems and motor skills, and differences in how their metabolism functions, among other traits. to cite an example, rodent brains are very different from human brains. the rodent lacks the prefrontal cortex specialization that is found in monkeys and humans. this difference limits the applicability of rodent studies in relation to studies of injury in the human brain. nhps are also the main animals that allow quick response and research into emerging viruses, like zika. what scientists learn about zika itself, as well as what they learn about the best use of monkeys in zika studies, they will apply to studies of future emergent diseases. and with recent history as a guide (zika, ebola, middle east respiratory syndrome [mers], sars, pandemic flu, etc.), we should expect more infectious disease outbreaks in the near future. modified poliovirus is being tested as a way to help the body's immune system see and destroy glioblastomas, the deadliest type of brain cancer. glioblastomas can double in size every two weeks and can be deadly within months of diagnosis. the new treatment has led to complete remission in two glioblastoma patients and investigations are ongoing. this type of research, called immunotherapy, uses the body's natural defense -the immune system -to destroy cancer cells. the harmless form of poliovirus is injected into glioblastoma tumors where it attaches to the cancer cells. the immune system recognizes the poliovirus as a dangerous invader and attacks -killing it and the cancer along with it. current studies in monkeys are helping to find ways to help wounded soldiers and stroke victims regain their independence after losing limbs or the ability to control them. this is the most promising therapy i've seen in my career. it was 18 years from the earliest research until the first study in humans. monkey research was essential in mapping out how to get the poliovirus through the brain and inside the cancerous tumors. the national institutes of health and the food and drug administration also mandated testing the treatment first in monkeys to be sure the modified poliovirus would be harmless to humans. doctors now are designing research to use this approach in treating many forms of cancer -including breast and prostate cancers -since the same receptor on the glioblastoma tumors that allows the poliovirus to attach itself also is found on virtually every cancerous tumor in humans. scientists are looking for vaccines that can prevent hiv infection and treatments leading to a cure. just 20 years ago medical advances changed the disease from a death sentence into a chronic, manageable disease. drugs that keep the virus in check now give millions of hiv-infected people hope for a long and productive life. drug therapies effectively prevent hiv disease, but scientific advances in hiv are still needed. people with well-treated hiv still face more health problems than those without hiv [1, 2] . they age faster, too. doctors estimate people with hiv are at least 5 to 14 years older than their chronological age [3, 4] . monkeys are crucial to ongoing hiv research because of the combination of their unique biology among animals and their longevity, which is key in hiv studies that take from months to years to complete. their similar biology helps scientists understand hiv disease, infection routes, the potential for vaccine-induced protection, and even an hiv cure. an experiment reported in early 2016 looked at preventing mother-tochild hiv transmission [5] . after being exposed to simian-human immunodeficiency virus (shiv), which is similar to hiv, infant monkeys with early stage infection were treated with human antibodies to block the infection. all of the monkeys in this experiment had no detectable virus in their blood or any of their tissues at the end of six months of observation. in another experiment reported this year, rhesus macaques infected with another hiv-like virus were treated with standard anti-hiv medications plus an experimental drug that stimulates the immune system [6] . at the end of the study, 90 days after both medications were stopped, two monkeys showed no detectable virus in their bloodstream. this immune system stimulator was tested earlier in nhps infected with chronic hepatitis b, leading to current research in humans with this potentially deadly infection. [7] the value of studying monkeys is scientifically proven. hiv is no longer an impending death sentence. several years ago, this young woman was diagnosed with a deadly brain tumor the size of a tennis ball. when conventional treatment didn't work her doctors suggested a new treatment developed with primate research. she agreed. today, she's cancer free. these studies hold promise for protecting babies from hiv infection and for finding a cure for those already infected, but much more research with monkeys will be needed to get there. in human clinical studies, a fundamental question is, "do the potential benefits of this treatment outweigh the potential risks?" this question takes on added meaning when the study is in pregnant women. researchers must not only consider the risks and benefits to the pregnant woman, but also to her developing fetus and ultimately to the child [8] . but how do researchers even begin to define these risks and benefits before human clinical trials? the answer is research with monkeys, since their fetal and placental development is uniquely similar to humans. researchers are working with macaque monkeys to understand the impact of zika, the latest virus to emerge as a global threat. zika infection in pregnant women can cause microcephaly, a condition where the child is born with a small head due to abnormal brain development. it also appears to cause stillbirth, miscarriages, and fetal growth restriction. these problems all appear to be rooted in how the zika virus affects the developing fetus and the placenta, which nourishes the baby in its mother's womb. the zika virus infects monkeys just as it does humans, and both experience the disease in the same way. researchers can study pregnant monkeys much as an obstetrician follows a woman's pregnancy -they can take blood, monitor fetal development through ultrasounds, and collect amniotic fluid. they can then test vaccines and drugs with the hope of protecting the fetus. no other animal model allows for this entire spectrum of study and application of the findings to pregnant women. more than 120,000 people in the u.s. are waiting for organ transplants and 22 of them die every day [9] . it is all the more tragic, then, when an organ transplant fails. this failure, or rejection, is caused when the recipient's immune system sees the new organ as "foreign" and attacks it. to reduce the chance of organ rejection, transplant patients receive drugs to suppress their immune system. but the drugs come with a risk of toxicity and increase the risk of other problems, including development of cancers and infections resulting from a weakened immune system. research with monkeys is focused on achieving transplant tolerancewhere the body's immune system does not see the new organ as foreign, thus eliminating the need for immunosuppressive drugs. while scientists have already made great strides in kidney transplant tolerance, they understand that tolerance is organ specific, so knowledge about the kidney primate research will help us learn new ways to prevent miscarriage, stillbirth, and premature babies. understanding all brain diseases relies on us understanding how the healthy brain works during normal functioning. may not transfer to the heart, lungs, liver, pancreas/pancreatic islets, or other types of transplants. transplant tolerance also differs by species. in other words, what works in a mouse may not work in a pig, and what works in a pig may not work in a monkey. scientists learned about kidney transplant tolerance by starting with mice and then working up through swine and eventually into monkeys and humans. the same process is underway now for many other types of transplants. how does the brain work? no question could be more important for understanding human behavior and mental health, and for acquiring new information about the triggers in the brain that cause psychiatric, movement and other neurological diseases. the u.s. national institutes of health-supported brain initiative has developed a plan for improving our knowledge in these areas and research with monkeys and other species is critical to its success [10] . scientists are mapping the activity of the billions of neurons deep inside the brain -the special cells that transmit the signals that drive thinking, mood, movement, and much more. by tracking neuron activity in monkeys while they are performing new tasks, scientists can actually see what parts of the brain are involved in sending the signals that take in, process, and store the newly acquired information. what is unique to -or at least greatly enhanced by -the use of monkeys in this research is the range of cognitive behaviors that can be studied, the amount and precision of the data that can be collected, and the relevance of that data to human behavior and mental activity. seeing what is happening in a healthy monkey brain helps scientists understand what has gone wrong when a human brain is no longer working as it should. this type of research has relevance to parkinson's disease and other movement disorders, all forms of dementia, including alzheimer's, and behavioral and psychiatric problems from alcoholism and attention-deficit disorder to bipolar disorder and autism. you see someone walking haltingly, dragging one leg behind him, or sitting with one arm draped listlessly on a table and immediately know he has had a stroke. scientists learned long ago that it's not the muscles that are at fault; it's the nerve impulses inside the brain that have been affected. alzheimer's and other dementias cost the u.s. $236 billion each year [11] . monkeys have certain traits and characteristics that make them essential and irreplaceable in medical research. they're the "bridge to the clinic." a combination of scientific breakthroughs in neuroscience, computer processing and robotics has led to development of "brain-machine interfaces" -devices that allow humans to interact with their environment with prosthetic arms when they have lost the use of their own. brain-machine interfaces translate signals in the brain into directions to move prosthetic arms. this area of research shows enormous promise for humans who are paralyzed, such as injured veterans, or those with brain damage and paralysis due to stroke. as nhps and humans have similarly developed brains and movements, experiments in monkeys have been vital to moving this field forward both conceptually and technically. nhps are essential to vaccine research. among research animals, they alone can reproduce the entire biological process of the infections being studied. they allow researchers to monitor for information that is vital in understanding human infectious diseases -such as how a virus or bacterium reproduces inside the body, what symptoms it causes, and how the body's immune system responds to attack the invader. among the viruses currently in vaccine research trials is respiratory syncytial virus, or rsv -the most common cause of lower respiratory tract infections in u.s. infants and small children [12] . there is no treatment for rsv, [13] which hospitalizes nearly 60,000 u.s. children under age 5 every year and sends 2.1 million more to the doctor [14] . vaccine research with monkeys is evaluating the safety of potential rsv vaccines in infants. other viruses under study include ebola and marburg, which can cause extreme bleeding that leads to death; the mosquito-borne dengue and zika viruses, capable of causing massive epidemics; and mers plus the dangerous h5 and h7 bird flu strains, all of which have very high death rates. lowering blood pressure is vitally important to individuals and our society. high blood pressure is a major factor in heart disease -the number one killer in the u.s. and the world [15] . and it's not just heart disease; high blood pressure leads to stroke, kidney damage, memory problems, and many other illnesses [16] . decades ago, researchers made a breakthrough discovery that longterm blood pressure regulation is nearly identical in humans, baboons, brain-machine interfaces can help paralyzed veterans interact with their environment. the top 3 benefits of our human/monkey research partnerships? safety. efficacy. and greater predictability. scientists recently discovered that baboons share another unique trait with humans -a characteristic in their red blood cells that can lead to salt-sensitivity and an inherited form of hypertension that is particularly difficult to treat. current research is looking for new targets to control this type of high blood pressure. research with monkeys provides another key benefit -lifespan. high blood pressure becomes more common as we age and researchers are able to work with older baboons to gain essential information about the mechanisms driving this age-based increase -vital to the health of our aging population. type 2 diabetes develops in monkeys just as it does in humans, even following the same age patterns, that is to say, more disease as we get older (one-fourth of u.s. seniors have diabetes) [17] . nhps with diabetes even develop the same complications that are common in humans: eye disease, kidney disease, nerve damage and pain, and blood vessel disease, among others [18] . nhps and humans have very similar systems that regulate blood sugar. for example, the structure and function of the group of cells in the monkey pancreas (called islets) that produce insulin are very similar to human islets. the islets in mice, rats, pigs, and other animals share some similarities with humans, but there are important differences, making monkeys a critical model for developing treatment and prevention methods, and for testing new therapies for people with diabetes. type 2 diabetes and the u.s. obesity epidemic are linked -obesity is a contributing factor to the condition. more than a third of u.s. adults are obese and another third are overweight [19] . as with diabetes research, monkeys provide a critically important study model for human obesity. monkeys that are fed a diet similar to the typical american diet respond like humans, gaining weight and later progressing to type 2 diabetes. researchers are examining the role of gastrointestinal proteins called glucagon-like peptides in the development of obesity in bonnet macaques. bonnet macaques are unique among nhps because they have a strong genetic predisposition to obesity. this research is looking for obesity treatments that will be as effective as invasive bariatric surgery, but with far less risk. in conclusion, because nhps are the most readily available models with the greatest psychological and genetic similarities to humans, they play an indispensable role in the process of medical research and development. nonhuman primates are the ideal model for testing new therapies for people with diabetes, including the artificial pancreas, drugs and devices. all of our ability to address diseases in applied research comes from efforts in basic science research. william newsome, phd, neurobiologist studying how â�¢ components of blood and plasma discovered. â�¢ ability to diagnose and treat typhoid fever. â�¢ modern anesthesia. â�¢ mumps virus discovered. â�¢ treatment of rheumatoid arthritis. â�¢ discovery of the rh factor, blood-typing knowledge critical for safe blood transfusions. â�¢ development of polio vaccine. â�¢ development of antipsychotic medication chlorpromazine and its tranquilizing derivatives. â�¢ cancer chemotherapy. â�¢ development of yellow fever vaccine. â�¢ mapping of the heart's connections to arteries. â�¢ development of german measles vaccine. â�¢ therapeutic use of cortisone for reducing inflammation and allergy symptoms. â�¢ corneal transplants. â�¢ development of treatment and prevention of radiation sickness. â�¢ development of measles, mumps, and rubella (mmr) vaccine. â�¢ discovery of the biochemical cause of depression. â�¢ transmissibility of human prion diseases, such as creutzfeldt-jacob disease, discovered. â�¢ treatment of leprosy. â�¢ procedures to restore blood supply in the brain. â�¢ interaction between tumor viruses and genetic material. â�¢ understanding of slow viruses, which linger in the nervous system. â�¢ understanding of the inner workings of the basal ganglia, the part of the brain that coordinates movement. â�¢ discovery of mechanisms of opiate withdrawal and the anti-withdrawal effects of clonidine. â�¢ development of cyclosporine and other anti-rejection drugs helpful for organ transplants. â�¢ processing of visual information by the brain. â�¢ identification of physiological and psychological co-factors in depression, anxiety and phobias. â�¢ treatment of malnutrition caused by food aversion following chemotherapy. â�¢ treatment of congenital cataracts and "lazy eye" in children. â�¢ first animal model for research on parkinson's disease, enabling doctors to more accurately research human parkinson's disease. â�¢ heart and lung transplant to treat cardiopulmonary hypertension. â�¢ first hepatitis b vaccine. â�¢ development of rhesus monkey model for hiv/aids. â�¢ addition of taurine to infant formulas. taurine is necessary for normal eye development. â�¢ first treatment of naturally diabetic nhps with a hormone-like insulin stimulus that is now in wide use both for diabetes and obesity treatment (glp-1 agonist). without continued nonhuman primate research, many important translational discoveries in the field of transplantation will never happen. these preclinical investigations are critical to the many patients who are currently waiting for transplantation and the many others who are facing the possibility of organ rejection after transplantation. â�¢ estrogen discovered to control an enzyme key to making serotonin, the brain chemical that regulates mood. represents first step to providing effective medications for depression at the end of the menstrual cycle, and postpartum and postmenopausal depression. â�¢ demonstration of the effectiveness of early administrationof azt to prevent or treat hiv infection. thanks to this, hiv-infected mothers can give birth to hiv-free babies. â�¢ demonstration in monkeys of the high efficacy of the hiv drug tenofovir to prevent or treat infection. â�¢ lead toxicity studies help u.s. fight childhood lead exposure. â�¢ ongoing development of a one-dose transplant drug to prevent organ rejection. â�¢ first controlled study to reveal that even moderate levels of alcohol are dangerous in pregnancy. â�¢ breakthroughs in understanding the mechanisms of puberty and disorders of puberty. â�¢ primate embryonic stem cells studied extensively for the first time, advancing efforts to better understand reproduction and genetic disorders. â�¢ control of intimal hyperplasia, a complication of coronary bypass surgery. â�¢ parent to child lung transplants for cystic fibrosis. â�¢ nhps shown to naturally develop diabetes, which is the same disease as in humans, thus opening the path to research for new treatments. â�¢ naturally regenerative mechanism discovered in the mature nhp brain, spurring new research toward curing alzheimer's and other degenerative brain disorders. â�¢ development of anthrax vaccine. â�¢ development of life-saving medications for lupus. â�¢ gene that boosts dopamine production and strengthens brain cells used to successfully treat monkeys showing symptoms of parkinson's disease. â�¢ monkey model developed to study the effects of malaria in pregnant women and their offspring. â�¢ nhps are prime model for development of hiv treatments and potential vaccines. â�¢ insulin-treated diabetic patients live longer, fuller lives. â�¢ the most common and debilitating complications of diabetes can now be studied in nhps. â�¢ high blood pressure is treated to prevent heart attack, stroke, and kidney failure. â�¢ patients can receive hip replacements and are no longer reliant on wheelchairs. â�¢ people with degenerative eye diseases are able to see more clearly. â�¢ better medications improve lives of people with severe depression, bipolar disorder, and other psychiatric illnesses. â�¢ better pre-and postnatal care protects children. â�¢ earlier diagnoses and better treatments help those with polycystic ovary syndrome, endometriosis, and breast cancer. â�¢ improved treatments help more men survive prostate cancer. â�¢ secondhand smoke shown to affect prenatal, neonatal and child lung development, cognitive function and brain development. â�¢ exposure to wildfire smoke adversely affects development of the immune system. â�¢ better understanding of the effects of bpa, a chemical found in plastic, on prenatal development improves health of children and adults. for research funded by any public health service entity, such as the national institutes of health (nih) or the national science foundation, the nih: g establishes public health service policy on humane care and use of laboratory animals. [20] g mandates use of the guide for the care and use of laboratory animals, which is issued by the national academy of science institute for laboratory animal research. this guide addresses day-to-day aspects of caring for laboratory animals. [21] g mandates that every institution appoints an institutional animal care and use committee (see below). the usda's animal plant and health inspection service enforces the animal welfare act with unannounced compliance inspections of all regulated entities using animals in research, testing or teaching at least yearly [22] . the association for the assessment and accreditation of laboratory animal care international is an independent non-government accrediting organization. while voluntary, this accreditation includes broader requirements than the regulations. this demonstrates research facilities want to go "above and beyond" in the care of animals [23] . every institution involved in nonhuman primate research is required by the animal welfare act as well as public health service policy to appoint and empower an institutional animal care and use committee [24] that reviews and approves the research. scientists must justify their use of primates and also explain why alternative forms of research (for example, studying cells or using computer simulations) are not able to achieve their scientific goals. they must also confirm that their research does not unnecessarily duplicate previous research [25] . the number of nonhuman primates used in research is less than 1%. but its impact on human health is enormous. all others geriatric syndromes in older hiv-infected adults the end of aids: hiv infection as a chronic disease methylome-wide analysis of chronic hiv infection reveals five-year increase in biological age and epigenetic targeting of hla acceleration of age-associated methylation patterns in hiv-1-infected adults early short-term treatment with neutralizing human monoclonal antibodies halts shiv infection in infant macaques gilead announces data from new preclinical study evaluating an investigational tlr7 agonist in siv-infected monkeys gs-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees ethical issues related to the inclusion of pregnant women in clinical trials (i) american transplant association. facts and myths national institutes of health. what is the brain initiative? available from alzheimer's disease facts and figures respiratory syncytial virus infection (rsv) american lung association. diagnosing and treating rsv the burden of respiratory syncytial virus infection in young children heart disease and stroke statistics-at-a-glance high blood pressure (hypertension) statistics about diabetes the rhesus monkey (macaca mulatta) manifests all features of human type 2 diabetes national institute of diabetes and digestive and kidney diseases public health service policy on humane care and use of laboratory animals. revised committee for the update of the guide for the care and use of laboratory animals; institute for laboratory animal research. guide for the care and use of laboratory animals animal welfare act inspections. at 1.usa.gov/1xcaq7m american association for the accreditation of laboratory animal care. the aaalac international accreditation program office of research integrity. how to work with your institutional animal care and use committee (iacuc) good laboratory practice for nonclinical laboratory studies the authors wish to acknowledge the following experts who participated in conference calls that served as the basis for the paper's content and/or who provided comments during drafting: key: cord-341838-lkz8ro90 authors: gervasoni, cristina; meraviglia, paola; riva, agostino; giacomelli, andrea; oreni, letizia; minisci, davide; atzori, chiara; ridolfo, annalisa; cattaneo, dario title: clinical features and outcomes of hiv patients with coronavirus disease 2019 date: 2020-05-14 journal: clin infect dis doi: 10.1093/cid/ciaa579 sha: doc_id: 341838 cord_uid: lkz8ro90 little is known about the clinical outcomes of hiv patients infected with sars-cov-2. we describe 47 patients referred to our hospital between 21 february and 16 april 2020 with proven/probable covid-19, 45 (96%) of whom fully recovered and two died. as of april 20, the pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection had affected 2.5 million people all over the world, and led to more than 165,000 deaths (update available at https://coronavirus.jhu.edu/map.html). initial information from china and evidence accumulated over recent weeks has allowed the identification of some of the risk factors associated with a negative prognosis, includingaging, male gender, hypertension, diabetes mellitus, and cardiovascular, lung and/or kidney diseases. [1] [2] [3] [4] however, little is known about the impact of hiv infection on the clinical outcomes of patients infected with sars-cov-2 because, to the best of our knowledge, only case reports or small case series have so far been published. [5] [6] [7] treated people living with hiv who have a normal cd4 t cell count and suppressed viral load may not be at increased risk of serious illness, but many also have other conditions that increase their overall risk: almost half of hiv patients are males, aged >50 years, and affected by chronic cardiovascular and lung diseases. the aim of this retrospective study was to describe the clinical characteristics and outcomes of hivinfected patients with a probable/proven diagnosis of sars-cov-2 infection who have been regularly followed up by our hospital. a c c e p t e d m a n u s c r i p t 4 we searched our database for hiv patients diagnosed as having probable or proven sars-cov-2 infection between 21 february and 20 april 2020. a diagnosis of probable sars-cov-2 infection was based on the presence of fever and respiratory symptoms (cough and dyspnea), epidemiological risk factors such as relatives or close colleagues with a proven diagnosis of covid-19, and/or a chest xray or ct diagnosis of interstitial pneumonia; proven sars-cov-2 infection required a throat swab positive for viral nucleic acid. we also recorded their main demographic data, pharmacological treatments and clinical outcomes. the database of our department of infectious diseases included nearly 6,000 hiv-positive patients (74% males; mean age 52±12 years); 90% had <20 copies/ml of hiv viral load and 76% had a cd4 cell count of >500 cells/mm 3 (3% had <200 cells/mm 3 ). during the observation period, 47 hiv patients with proven or probable sars-cov-2 infection were identified. they were mainly males (76%) and had a mean age of 51±11 years. as shown in table 1 , most of the patients showed suppressed hiv viremia and acceptable immune reconstitution (cd4 cell count >350 cells/mm 3 ); three (all males) had detectable hiv viral loads of 52, 72 and 134 copies/ml. nearly 64% had at least one co-morbidity (82% of the males and 58% of the females), mainly dyslipidemia (32%), arterial hypertension (30%) and hepatitis b or hepatitis c co-infections (11%). approximately 80% of the identified patients were receiving integrase inhibitor-based antiretroviral treatment and 11% a protease inhibitor-based regimen (11%); 42% were receiving a tenofovir-based regimen ( table 2) . twenty-eight patients (>50%) tested positive for sars-cov-2, including one female asymptomatic patient who was tested because she was a healthcare provider; the remainder were not tested mainly because they lived in the high-risk provinces of bergamo and brescia (lombardy) but were isolated at home and cared for by their general practitioners. the covid-19 diagnosis of the untested patients was based on their clinical symptoms and the presence of risk factors (13% were healthcare providers; 9% had been in close contact with sars-cov-2 positive working colleagues and 23% with sars-cov-2 positive relatives). interstitial pneumonia was diagnosed by means of an x-ray in three cases, and ground-glass opacity was identified by means of ct in one. thirteen of the 28 sars-cov-2 positive patients were hospitalised. six had severe lung disease (respiratory rate ≥30 breaths/min; resting percutaneous oxygen saturation ≤93% in room air), two of whom required mechanical ventilation: one recovered and was discharged and the other (a 47-year-old overweight man without other co-morbidities) died. another a c c e p t e d m a n u s c r i p t 6 patient with cardiovascular disease and a recent diagnosis of lung cancer died during hospitalisation. for comparative purposes, the crude mortality rate of the hiv-negative covid-19 patients in our hospital (n=502, 67% males, mean age 61±16 years) is currently forty-five of the patients recovered 14±8 days after symptom onset, with no significant difference between the females and males (11±7 versus 14±9 days; p=0.338). table 2 , fewer than 50% of the patients were given potential anti-sars-cov-2 treatments, specifically hydroxychloroquine (17%), azithromycin (15%), lopinavir/ritonavir (11%); one was treated with tocilizumab and remdesivir, and one with toxicizumab alone. this report describes our experience with hiv-positive patients regularly followed by our hospital (a reference hospital for the management of hiv infection in italy) who were infected with sars-cov-2. as in the general population, the large majority of our patients were males, but their mean age was nearly 10 years lower than that observed in hiv-negative covid-19 patients. [8] [9] [10] this is in line with the recent finding of blanco et al. who described five hiv-infected patients with covid-19 from spain who were aged 29-49 years. 5 the difference in age between hiv-positive and hiv-negative patients with covid-19 may possibly be explained by the general belief that hiv infection can add nearly 10 years to the chronological age. 11, 12 this is indirectly supported by the fact that, although they are about 50 years old, the large majority of our patients have at least one co-morbidity, a picture that is more frequently observed in patients aged >60 years in the general population. the risk of severe disease in our hiv-patients compared favourably with that observed in the general population of covid-19 patients. 13 likewise, the risk of death or admission to an intensive care unit was lower than that observed in the hiv-negative patients treated at our hospital and in another cohort of hiv-negative covid-19 patients of a similar mean age. 14 it is worth noting that these positive outcomes were achieved even though fewer than 50% of the patients were treated with drugs currently considered to be potential treatments for sars-cov-2 infection, and only two received remdesivir or tocilizumab. it could be argued that antiretroviral therapy may have played a role in the positive evolution of covid-19 in our hiv patients; indeed, lopinavir/ritonavir, darunavir/ritonavir and darunavir/cobicistat were all initially suggested as candidate treatments for sars-cov-2 infection. 15 however, we have recently shown that darunavir does not prevent sars-cov-2 infection in people living with hiv or protect against worsening respiratory function, at least not at a dose of 800 mg. 16 furthermore, the findings of this study document favourable outcomes in hiv patients treated mainly with integrase inhibitors (11% protease inhibitors), which apparently indicates that antiretroviral therapy does not play a key role, a c c e p t e d m a n u s c r i p t 8 although a potentially protective effect of tenofovir cannot be ruled out given its recently reported effect against sars-cov-2 rna-dependent rna polymerase. 17 another possible explanation of the more favourable clinical outcome observed in our hiv-positive patients is that, despite their effective antiretroviral therapy, they still had deficient immune systems (albeit to different degrees) and showed some persistent immune activation: consequently, the evolution of covid-19 may be milder and not progress to a severe cytokine storm. 18 finally, as all of our hiv-negative patients required hospitalisation, they may have been more susceptible to an unfavourable outcome. the main limitation of this retrospective observational study is that it also includes hiv patients with probable but not confirmed covid-19. however, given the paucity of the data published so far, we believe that our real-life experience can provide useful information concerning the management of hiv-infected patients with sars-cov-2 infection that may eventually be used for prospective investigations. in conclusion, our findings suggest that hiv-positive patients with sars-cov-2 infection are not at greater risk of severe disease or death than hiv-negative patients. however, the observed more favourable outcomes need to be confirmed in larger cohort studies. m a n u s c r i p t 15 risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study risk factors of healthcare workers with corona virus disease 2019: a retrospective cohort study in a designated hospital of wuhan in china risk factors associated with disease severity and length of hospital stay in covid-19 patients covid-19 in patients with hiv: clinical case series early virus clearance and delayed antibody response in a case of covid-19 with a history of co-infection with hiv-1 and hcv co-infection of sars-cov-2 and hiv in a patient in wuhan city review of the clinical characteristics of coronavirus disease 2019 (covid-19) clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region do people living with hiv experience greater age advancement than their hiv-negative counterparts? basic science and pathogenesis of ageing with hiv: potential mechanisms and biomarkers clinical characteristics of coronavirus disease 2019 in china clinical features and short-term outcomes of 102 patients with corona virus disease 2019 in wuhan, china. clin infect dis. 2020 therapeutic options for the 2019 novel coronavirus darunavir does not prevent sars-cov-2 infection in hiv patients ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study inflammation, immune activation, and antiretroviral therapy in hiv a c c e p t e d m a n u s c r i p t 10 a c c e p t e d m a n u s c r i p t 13 key: cord-315918-12rbbe8c authors: mukherjee, pulok k. title: antiviral evaluation of herbal drugs date: 2019-06-21 journal: quality control and evaluation of herbal drugs doi: 10.1016/b978-0-12-813374-3.00016-8 sha: doc_id: 315918 cord_uid: 12rbbe8c the viral infection and resistance to the existing antiviral drugs are alarming, which is a serious public health concern. medicinal plants are valuable resources for treatment of viral infections and can be used for the management of infections like herpes simplex virus (hsv), human immunodeficiency virus (hiv), influenza, etc. the antiviral screening of plant extracts should be highly selective, specific, and sensitive for bioactivity guided isolation of the active compounds from the plant extracts. the antiviral screening system should be validated for accuracy, reproducibility, simplicity, and cost effectiveness. this chapter highlights on various aspects for screening and evaluation of antiviral natural components including factors affecting antiviral in vivo studies, host cells, organisms, and culture media followed by different virus-specific assays for antiviral screening of natural products. although vaccines have been very successful in controlling many viral diseases, some diseases are likely to be controlled only by antiviral chemotherapy. the concept of antiviral drugs has only been accepted slowly, partly because of the toxicity of many of the earlier antiviral agents. in contrast to the development of antibiotics, attempts to develop antiviral drugs have indeed met a variety of problems. being strictly dependent on cellular metabolic processes, viruses possess only limited intrinsic enzyme systems and building blocks that may serve as specific targets for a drug. moreover, contrary to a bacteriostatic compound, an effective antiviral drug should not only display considerable specificity in its antiviral action, but should also irreversibly block viral synthesis in order to stop cell suicide due to the viral infection and restore normal cell synthesis (vanden berghe et al., 1986) . in addition to this inhibition, the antiviral agent must have a broad spectrum of activity, favorable pharmacodynamic properties, and not be immunosuppressive. in the ideal situation, the antiviral drug checks the infection while the immune system prepares to destroy the last virus particles (munro et al., 1987) . this point is critical for those immune-compromised by illness (aids, cancer) or drug therapy (transplants, cancer). a frequent cause of death in these instances is from viral infections, so that adjuvant antiviral chemotherapy is vital in these circumstances (shannon and schabel, 1980) . many viral infectious diseases still cause high mortality. although antiviral chemotherapy has shown outstanding progress, antiviral agents are still required. the emergence of drug-resistant viruses during treatment raises a potential problem for effective therapy. furthermore, new viral pathogens may be discovered. biologically active substances of plant origin have long been known as viral inhibitors. these antiviral compounds may be extracted from sources, such as higher plants, which have, for various reasons, been explored considerably less than the traditional ones. the first clinically useful antiviral drug was methylisatin-thiosemicarbazone (methisazone), which was active against pox viruses, especially smallpox virus. methisazone (25 mg/kg) was found to inhibit variola virus in mice and was later used successfully to treat cases of eczema vaccinatum and to treat and prevent smallpox. kaufman et al. (1962) , an ophthalmologist, successfully treated a herpes eye infection with an antineoplastic drug, idoxuridine. at the same time, a group of chemists at the du pont chemical company in the united states investigated the antiviral activity of a molecule called amantadine. it was active against influenza virus type a. in rapid succession, further nucleoside analogs, cytosine arabinoside, trifluorothymidine, and adenine arabinoside, were found to inhibit herpes virus. in the 1970s, the antiherpes activity of a new compound, 9-(2-hydroxyethoxymethyl) guanine (acyclovir), was detected by j. bauer at the wellcome research labs. within a decade, the same group was to discover azidothymidine (zidovudine), the first effective inhibitor of the newly emerged hiv. the alkaloid from the australian horse chestnut (castanospermine australe), isolated in the 1980s, was also found to be active against hiv. a research program to detect and isolate antiviral compounds from higher plants is best carried out by a multidisciplinary team, consisting of at least a pharmacognosist and a virologist. the antiviral screening system should meet all requirements of any good assay, including validity, lack of ambiguity, accuracy, reproducibility, simplicity, and reasonable cost. moreover, because we are dealing with plant extracts, the antiviral screen should be highly selective, specific, and sensitive; it is advisable to discriminate a true antiviral activity from a viricidal one at this stage. because most of the aforementioned requirements are better met by in vitro testing, we not only prefer in vitro screening of the plant extracts, but also the use of the same bioassay to guide the isolation of the antivirally active compounds from the plant extracts. the antiviral activity of the pure compounds then has to be confirmed in a later stage by in vivo assays (leven et al., 1982; vanden berghe et al., 1986) . one of the possible strategies for finding new antiinfective drugs may involve the search for compounds with chemotherapeutic activities supplementary to, and structures widely different from, those in current use. these compounds could be extracted from sources that have, for various reasons, been explored considerably less than the traditional microorganisms, including, among others, higher plants (mitscher and rao, 1984) . considering the enormous number and the amazing structural diversity of the currently available antimicrobially and antivirally active plant constituents, one might hope that promising systemic and/or locally acting antiinfective agents might be discovered in the plant kingdom (vanden berghe et al., 1986; vlientinck et al., 1988) . the increase of drug-resistant viruses in treatment raises an important issue for effective treatment. moreover, new viral pathogens might be found. along these lines, there is a strong requirement for promptly accessible antiviral medications at moderate costs with minimal side effects. henceforth, traditional medicines ought to be investigated as novel antiviral agents, as many of these ancient medicaments, containing different plant metabolites, have potent antiviral activities (chattopadhyay and khan, 2008) . the design of new antiviral drugs potentially focuses on the structural dynamics and replication cycles of the various infections causing viruses. a suitable example is the invention of acyclovir, which hinders certain proteins of herpes infections responsible for the triggering of disease. ethnomedicines are a vast repository of structural diversities and extensive bioactivities that can serve as a huge source of potential antiviral drugs. a significant number of medicinal plants from ayurveda and the traditional chinese system of medicine serve as potential remedies to decrease the severity of illness caused by viruses (chattopadhyay et al., 2009; khan et al., 2005; jadhav et al., 2012) . research on the antiviral potentials of plants was first started in 1952, and 12 out of 288 plants were found to be effective against influenza (chattopadhyay and naik, 2007) . numerous screening studies have been conducted in the last few years to determine the antiviral efficacy of medicinal plants using in vitro and in vivo assays. a few out of a 100 british colombian medicinal plants showed antiviral efficacy against respiratory syncytial virus (rsv), corona virus, influenza virus, and herpes simplex virus (hsv) (mccutcheon et al., 1995) , while the marine algae spirulina showed antimutagenic, immunomodulatory, and antiviral activities (chamorro et al., 1996) . interestingly, cyanovirin n, an 11-kda protein of blue green algae, inactivates hiv-1 by binding with its glycoprotein120 (clercq , while sulfated polysaccharides of seaweeds and algae showed anti-hiv and anti-hsv activities (schaeffer and krylov, 2000) . the plant kingdom is highly diverse and ranges from unicellular microscopic plants to long lived, huge trees. to screen each and every plant or their individual parts for the identification of antiviral components is a huge task. several examples of plants having antiviral properties and newly identified active compounds from them are reported in various journals. one of the examples that can be cited here is cyanovirin n (cv-n), an 11-kda protein isolated from the cyanobacterium nostoc ellipsosporum, which exhibits the property of inhibiting hiv-1 infection and also possesses broad-spectrum activity. the phytochemical, podophyllotoxin, isolated from the aqueous extract of podophyllum peltatum l., inhibited hsv type 1 (hsv-1) (bedows and hatfield, 1982) . the acetone extract of another plant, phyllanthus urinaria, also suppressed hsv-2 and hsv-1 (yang et al., 2007) . bessong et al. (2006) reported a comparison of various plants and their individual parts (stem, leaves, roots, and so forth.) in repressing viral reverse transcriptase (rt) and integrase, the two basic enzymes in hiv disease. after comparing all the extracts and fractions of the various plants, it was found that the n-butanol fraction of bridelia micrantha (hochst) exhibited the highest anti-rt activity. it has also been reported that the aqueous extract from the roots of carissa edulis (forssk.) vahl, a plant grown in kenya, displayed noteworthy activity against hsv for both wild type and resistant strains (tolo et al., 2006) . polyphenol-rich extract from the medicinal plant geranium sanguineum l. has been reported to show a strong antiinfluenza virus activity, as well as antioxidant and radical scavenging capacities (sokmen et al., 2005) hepatitis a, b, c, d, and e viruses are the leading causes for the prevalence of viral hepatitis and liver inflammation. despite the fact that presentation to any of these infections prompts intense disease, in any case, types b, c, and d are unique in causing chronic infection. plants belonging to the genus phyllanthus of the euphorbiaceae family were extensively used as a traditional remedy against these infections. clinical investigations were additionally intended to look at the inhibitory effects of different species of phyllanthus, that is, p. amarus (l.), p. niruri (l.), and p. urinaria (l.) (wang et al., 1995) . the screening of 56 different chinese medicinal herbs led to the identification of two potent plant extracts against duck hepatitis b virus, namely, ardisia chinensis and pithecellobium clypearia . similarly, this also led to the identification of oenanthe javanica (blume) dc flavones (ojf). they acted as a strong inhibitor of hbsag and hbeag secretion (involved in viral pathogenesis) in 2.2.15 cells and also reduced dhbv-dna levels in the hbv-infected duck model (wang et al., 2005) . because of the strong prevalence of hcv infection in poor countries, screening for the identification of anti-hcv potentials from medicinal plants are still ongoing. according to hussein et al. (2000) various plant extracts, such as methanol extracts acacia nilotica l. willd ex delile, boswellia carterii, embelia schimperi, quercus infectoria, trachyspermum ammi l., and aqueous extracts of piper cubeba l., q. infectoria, and syzygium aromaticum l., were found to possess significant inhibitory activity against hsv. combination therapy for treating diseases is an age-old practice of traditional medicine in which several plants are mixed together to develop an effective formulation for a particular disease. such combination therapies have also been tried for the inhibition of viral hepatitis. as an example, a chinese herbal medicine, prepared by liquid fermentation broth of ganoderma lucidum supplemented with an aqueous extract of radix sophorae flavescentis, was potent against hepatitis b virus activity in vitro and in vivo. viral infections are a matter of great concern for this planet. plants having broad-spectrum activity against many viruses could be evaluated for isolation and identification of molecules for treating viral infections. as an example, glycyrrhizin, a bioactive component of licorice (glycyrrhiza uralensis fisch), and lycorine, isolated from lycoris radiata l., showed strong anti-sars-cov activity, and was initially used for treating other indications (li et al., 2005a, b) . a variety of herbal preparations have shown potentials for inhibiting viruses that cause serious infections among humans, such as measles viruses (olila et al., 2002) , human rotaviruses (hrv) (husson et al., 1994; takahashi et al., 2001) , respiratory syncytial virus (rsv), human rhinoviruses (glatthaar-saalmuller et al., 2001) , the coxsackie group of viruses (evstropov et al., 2004; su et al., 2006) , neurotropic sindbis virus (nsv) (paredes et al., 2001) , and various strains of polio virus (andrighetti-frohner et al., 2005; melo et al., 2008) . one such illustration is the atomic investigation of the heated water concentrates of stevia rebaudiana l., which blocked a section of different irresistible serotypes of hrv into permissive cells by an anionic polysaccharide having a molecular weight of 9800 with uronic acid as a noteworthy sugar constituent (takahashi et al., 2001) . thus, an alkaloid concentrate of haemanthus albiflos globules repressed rna amalgamation of hrv spread in ma-104 cells (husson et al., 1994) . in contrast to the many publications on antibacterial and antifungal screening of plant extracts that have appeared in the last decades, far fewer antiviral screening studies of plant extracts have been reported. this is due chiefly to the complexity of the different techniques involved in such research, which consequently requires the know-how and dedication of a multidisciplinary team. nevertheless, many antiviral agents have been isolated from natural sources and partly or completely characterized. from these studies, several substances have emerged as true antivirals having a good chemotherapeutic index based on the viability of infected cells and on in vivo tests. thus, different 3-methoxy flavones and synthetic derivatives have shown to be promising leads for the development of antirhinovirus drugs (van hoof et al., 1984; vlientinck et al., 1988) , whereas the spanonin, glycyrrhizic acid, was found to be highly active in vitro against herpes simplex (pompei et al., 1979) , varizella-zoster (baba and shijeta, 1987) , and human immunodeficiency viruses (ito et al., 1987) . whether these compounds have any clinical potential, that is, in the therapy of the corresponding viral diseases, remains to be determined. there, in vivo bioavailability and other pharmacokinetic parameters are subjects of future study. because of the problems of drug resistance stated earlier and of side effects, the pharmaceutical industry is looking forward toward natural products (mainly medicinal plant extracts) as a source of possible antiviral agents. approximately 2500 medicinal plant species have been recorded globally to treat a myriad of inflictions and diseases. polyphenols, alkaloids, flavonoids, saponins, quinones, terpenes, proanthocyanidins, lignins, tannins, polysaccharides, steroids, thiosulfonates, and coumarins are prominent bioactive phytochemicals that have been observed to combat viral infections, as they are harmless to the systems of the human body. some selected anthraquinones and anthraquinone derivatives are noted for their dammarane saponins, ginsenoside rb1 (grb1), and chikusetsusaponin (chi-iii) have been found to possess antiviral activity against hsv-i using an in vitro plaque elimination assay. chi-iii prevented plaque formation at half the concentration of grb1 (fukushima et al., 1995 (hudson and towers, 1995) . the inhibitory effect of ferulic acid and isoferulic acid on murine interleukin-8 production in response to influenza virus infections in vitro has been reported (hirabayashi et al., 1995) and the effect of isoferulic acid was found to be greater than that of ferulic acid. hayashi et al. (1995) found a direct inhibitory activity of the steam distillate prepared from fresh plants of houttuynia cordata (saururaceae) against hsv-1, influenza virus, and hiv-1, without showing cytotoxicity, but not against poliovirus and coxsackie virus. montanha et al. (1995a) evaluated the action of a series of 19 aporphine alkaloids against hsv-1 in cell cultures. on the basis of viral titer reduction, six alkaloids were found to be active. ellagitannins isolated from phyllanthus myrtifolius and p. urinaria (euphorbiaceae) showed activity against epstein-barr virus dna polymerase. flavonoidal constituents, such as biflavonoids and robustaflavones, exhibited strong inhibitory effects against influenza a and b viruses. the antiviral potential of the flavonoids of chamaesyce thymifolia has been reported; they showed high cytotoxicity on hep-2 cells and moderate inhibitory activity against hsv-1 and bovine viral diarrhea virus (amaral et al., 1999) . sotanaphun et al. (1999) isolated sclerocarpic acid from the stem bark of glyptopetalum sclerocarpum, which exhibited antiviral activity against herpes simplex virus types 1 and 2. rhamnan sulfate, a natural sulfated polysaccharide isolated from monostroma latissimum, showed potent inhibitory effects on the virus replication of hsv-1, hcmv, and hiv-1 in vitro . matsuse et al. (1999) tested aqueous and methanolic extracts of 39 panamanian medicinal plants for anti-hiv effects. seven of these were found to be moderate inhibitors of hiv-protease enzyme. serkedjieva and ivancheva (1999) investigated the inhibitory effect of five extracts from the bulgarian medicinal plant g. sanguineum on herpes simplex virus types 1 and 2. yoosook et al. (1999) evaluated the anti-hsv-2 activities of barleria lupulina and clinacanthus nutans. the results suggested a therapeutic potential against hsv-2 for b. lupulina, but not for c. nutans. the antiviral activities of various water and methanol soluble substances isolated from g. lucidum against hsv types 1 and 2, influenza a virus, and vesicular stomatitis virus were studied using cytopathic effect inhibition assay and plaque reduction assay (eo et al., 1999) . kudi and myint (1999) investigated the antiviral activity of medicinal plant extracts against poliovirus, astrovirus, hsv, and parvovirus. most of the extracts showed activity against more than one virus at a dose rate of between 100 and 400 î¼g/100 î¼l (eo et al., 1999) . bourne et al. (1999) assessed 19 plant products in vitro by plaque reduction assay to determine their activity against a commonly transmitted pathogen, herpes simplex virus type 2. docherty et al. (1999) found that resveratrol, a phytoalexin, inhibited hsv-1 and hsv-2 replication in a dose-dependent reversible manner. anani et al. (2000) prepared methanol extracts from 19 medicinal plants of togo and analyzed them for antiviral and antibiotic activities. ten of the 19 showed significant antiviral activity against one or another of the test viruses (herpes simplex, sindbis, and poliovirus). hudson et al. (2000a, b) evaluated ethanolic extracts of 11 plants, endemic to madagascar, in order to determine the potential of malagasy plants as sources of antiviral activities. nine of the extracts had significant activity against hsv, whereas only four were active against the sindbis virus. a bioactive flavonoid, "baicalein," isolated from the chinese medicinal plant scutellaria baicalensis georgi showed antiviral properties using the high-speed countercurrent chromatography (hsccc) technique (li and chen, 2005) . many other substances, including flavonoids, phenolics, tannins, triterpenes, and alkaloids, interfere with host cell replication at their antivirally active concentrations or only exhibit extracellular viricidal activities. some of these viricides, including flavonoids and tannins present in foodstuffs, might be important, because they can inhibit virus replication of picorna-, rota-, and arena viruses in the gastrointestinal tract of humans and animals. artemisia capillaris was found to possess an active 6,7-dimethylesculetine having potent cytotoxic potential. in the fruits of schisandra chinensis (schizandraceae) used in oriental medicine, 22 lignans were identified, some of which, such as wuweizisu c and gomisine n, are very active. the same method threw light on the mechanism of the antihepatotoxic action of such well-known compounds as glycyrrhizin and its intestinal metabolites, which are protective against the first stages preceding hepatic lesions. other tests of this type are used to track down active substances. from taxus baccata, potier's group isolated new analogs of taxol, a terpenic compound displaying very good antileukemic and antitumor activities. taxol promotes the polymerization of tubulin into microtubules and inhibits their depolymerization. the choice among various fractions obtained by extraction was guided by the antitubulin activity in an in vitro test. many substances that are present in only trace quantities and are difficult to purify have been studied chemically; for example, the demonstration of xylose-bearing derivatives is new in this series of compounds. regarding structure activity relationships, in vitro cytotoxicity tests have shown that the activity is mainly related to the presence of a complex ester function in the compound. a list of plant extracts and their phytoconstituents that have antiviral potentials are listed in table 16 .1. in recent years, a lot of development has taken place regarding the identification of antiviral molecules from plant sources and the molecular mechanistic approach. compounds, such as spiroketalenol ether derivatives isolated from rhizome extract of tanacetum vulgare, have been reported to block virus entry and also arrest the synthesis of hsv-1 gc and hsv-2 gg glycoproteins (fernandes et al., 2012) . samarangenin b from the roots of limonium sinense exhibited inhibition of hsv-1î± gene expression (kuo et al., 2002) , whereas oxyresveratrol from artocarpus lakoocha plant was found to inhibit the early and late phases of viral replication of hsv-1 and hsv-2, respectively (chuanasa et al., 2008) . also, pterocarnin a compound isolated from pterocarya stenoptera inhibited hsv-2 from binding and penetrating to the host cells (cos et al., 2003) . the structures of some of the potential phytoconstituents having significant antiviral activity are depicted in fig. 16 .1. many of the antiviral drugs now known have been discovered by random search in the laboratory. most labs use a biological test system in which new molecules are added to tissue culture cells at a range of concentrations (e.g., 100-1000 î¼g/ ml); the drug-treated cells (and untreated cells as control) are then infected with a known multiplicity of infective virus particles. thousands of compounds can inhibit viral replication in a cell culture. in general, the more complex the regulatory mechanisms of a virus, the easier it is to find molecules that inhibit it. a broad estimate of the ratios of the activity of antiviral compounds in a cell culture, animal models, and humans is 1000:10:1. during the evaluation of antiviral agents, many test conditions, such as the cell culture system, virus strain, virus challenge dose, virus input multiplicity of infection, and time of harvesting, can affect or even alter the test results. to test the inhibitory activity of a new antiviral agent, it is first necessary to select the host cell system(s) in which the virus replication can be measured. viruses vary considerably in their ability to replicate in cultured cells. many viruses can cause cpe while some of them can form plaques. others can produce some specialized functions, such as hemagglutination, hemadsorption, or cell transformation. virus replication in cell cultures can also be detected by the presence of viral products, namely, viral dna, rna, or polypeptides. the antiviral tests selected may be based on: (a) inhibition of the virus-induced cytopathic effect (cpe) in which the 50% effective dose (ed 50 ) of the antiviral agent is expressed as the concentration that inhibits cpe in half of the quadruplicate cultures. (b) plaque reduction assay in which the dose of the drug required to reduce the plaque number by 50%, that is, ed 50 is calculated. (c) virus yield reduction assay in which the drug concentration required to reduce 90% (1 log 10 reduction), or 99% (2 log 10 reduction) of the virus yield as compared with the virus control (infected cultures without drug) are determined from the dose-response curves and are expressed as ed 90 or ed 99 of the antiviral agent. (d) assay systems based on the measurement of specialized functions and viral products; a number of viruses do not produce plaques nor do they cause cpe readily, but they may be quantified by certain specialized functions based on their unique properties, for example, hemagglutination and hemadsorption tests used to study the antiviral activity against myxoviruses and elisa, used to determine the extent of virus replication and, thus, obtain a measure of the inhibitory effect of various antiviral agents on virus replication, etc. (hu and hsiung, 1989 ). colorimetric assays quantify cell viability through enzyme-mediated biochemical reactions owing to ingress of certain dyes inside living cells. mosmann, borenfreund, and puerner first advocated the application of tetrazolium (mtt) and neutral red (nr) assay, respectively, to quantitate cell viability and the cytotoxicity to cells in vitro. parida et al. (1999) compared the efficacy of two colorimetric assays (mtt and neutral red) to determine viral infectivity in microculture virus titration employing polio virus type-3 and dengue virus type-4. mtt assay, also known as tetrazolium assay, has been exploited extensively to reveal the protective efficacy of therapeutic agents and plant extracts against cancer, hiv-1, hsv, polio virus type 3, den-4 virus, and to the determination of neutralizing antibody levels to hiv and respiratory syncytial virus. mtt assay using microculture virus titration (mcvt) was applied for the determination of infectivity titers of influenza viruses and was found to be compatible with the well-established procedure of egg infectivity assay. unlike mtt, neutral red dye uptake assay has not been substantially exploited in virologic research. nr dye assay was earlier performed for the study of the antiviral efficacy of basil leaves extract against polio virus type 3. mtt, a tetrazolium salt, is a yellow-colored dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] which gets cleared by mitochondrial succinate dehydrogenase enzyme into a blue-colored formazan in active cells. this product does not form crystals when it interacts with isopropyl alcohol and thus can be accurately measured. there is no need to harvest the viable cells, wherein the cell viability can be directly measured by a spectrophotometer (parida et al., 1999) . the assay procedure has been discussed in section 16.6.7. a colorimetric assay based on the cleavage of the tetrazolium salt wst-1 has been developed for human cytomegalovirus (hcmv) antiviral susceptibility testing and adapted to a microtiter plate format. bedard et al. (1999) developed a colorimetric assay based on the cleavage of the tetrazolium salt wst-1 for human cytomegalovirus antiviral susceptibility testing. the response of different cell cultures to a given antiviral agent, including the drug metabolism and toxicity, among other factors, may vary greatly. to perform antiviral testing against a particular virus infection, it is necessary to obtain a suitable host cell system for that virus infection. the same antiviral agent may behave very differently in different cell culture systems, although the same virus strain is used. (ii) virus strain and passage history the variability of sensitivity to a given antiviral agent has been noted among different strains of hsv and cmv. drugresistant strains have emerged to some antiviral agents, especially in the herpes virus group. the passage history of a virus strain may also affect its sensitivity to some antiviral agents. moi can influence substantially the evaluation of antiviral activity by the plaque reduction method or the virus yield reduction assay. high moi will decrease the sensitivity of the virus to an antiviral agent. this may also contribute to the disparity among antiviral evaluation results, even when the virus moi is kept constant (hu and hsiung, 1989) . ( the activities of several known antiviral phytochemicals are profoundly affected by the presence of serum components. for example, the terthiophene, î±-terthienyl (î±-t) was inhibited in a concentration-dependent manner by serum. in the case of the carboxylic acid derivative of î±-t, the compound appeared to have no antiviral activity at all in the presence of serum, yet in its absence this compound was as effective as î±-t. in contrast, the complex anthraquinone hypericin required a small amount of serum for maximal antiviral activity. the reactions were also strongly affected by the order of incubation of the components: virus, compound, serum, and light (hudson and towers, 1995) . the viruses to be selected for initial evaluation of plant extracts are obviously of major importance. they must be chosen to represent the different groups of viruses according to their morphology and various multiplication mechanisms and a range of virus diseases for which chemical control would be useful. besides the need for control, also the prevalence of the viral diseases and the resulting projection of the market potential, which are determined by the antigenic abundance of the causative viruses and the problems this represents for vaccine control, are important selection criteria (grunert, 1979) . in vitro methods are therefore more appropriate, because they allow simultaneous screening of a battery of viruses. in vivo screening of extracts against a broad array of viruses, in contrast, is not only very expensive but also extremely laborious. in vitro antiviral bioassays utilize thinly confluent monolayers of tissue culture cells with sufficient susceptibility to the infecting viruses that a visibly cytopathogenic effect (cpe) occurs, for example, rounding up. shrinking or detaching of cells from the monolayer can be produced and readily observed microscopically. a monolayer of cells consists of animal or human cells, such as chick embryo, rabbit, or green monkey kidney cells (vero cells), or human skin fibroblasts and carcinoma cells (hela cells), grown in a culture medium. such continuous cell lines used in virology are mostly "transformed" cells that can be maintained for an indefinite number of generations. the host cells require an appropriate tissue culture medium in which they can survive for at least 1 week without having to renew the medium. renewal of the medium causes changes in intra-and extracellular products and alters the virus concentration. the medium must have a stable ph during the whole incubation time and may contain only a small amount of serum, because blood products tend to absorb many compounds. mostly, a defined synthetic medium, supplemented by some type of serum (such as fetal bovine, calf, or horse), a buffer on sometimes bacterial and fungal inhibitory antibiotics, is used. according to experience, vero cells, which allow the growth of many human viruses with visible cpe, grown in the medium described by hronovosky et al. (1975) , and slightly modified by lowering the pyruvate concentration, are most suitable for antiviral screening of plant extracts (van den berghe et al., 1978) . many combinations of test viruses are possible, but a battery of six viruses seems to be quite acceptable. virus types and strains may vary in sensitivity, but have to be selected as a function of their ability to multiply in the same tissue culture when cell culture models are used as screening systems. in this way, an objective comparison of antiviral activities is possible, whereas toxicity tests are minimized. moreover, virus multiplication must cause a visible cpe within a reasonable period of time, preferably within a week after infection. which are expressed as the number of infectious units per volume. an infectious unit is defined as the smallest amount of virus capable of producing a reaction after virus inoculation and can be carried out by two generally applied methods, namely, the plaque test (pt) and the 50% endpoint titration technique (eptt). in the plaque test, monolayers of cells grown in plastic or glass petri dishes are inoculated with dilutions of a viral suspension. after adsorption of infectious virus particles to the host cell, the monolayers are overlaid with an agarosecontaining medium so that the newly formed virus particles are localized by the solid agar over layer. these newly formed infectious particles spread from the initially infected cell to the adjacent cells and develop well-circumscribed foci of cellular degeneration. these areas of dead cells are called "plaques" and are visualized by staining the cell monolayer with a vital dye, such as neutral red. the plaques may also be detected microscopically by determining the multinucleated cells (e.g., measles) or by immunofluorescence. the concentrations of viral suspensions measured by counting the plaques are expressed as plaque-forming units per ml (pfu ml â��1 ). in the endpoint titration technique, the concentration of infectivity is measured by determining the highest dilution of the suspension, which produces cpe in 50% of the cell cultures inoculated. this dilution is called the 50% tissue culture dose endpoint (tcd 50 ). dilutions are therefore made in a maintenance medium and a certain volume of each of them (0.05-0.1 ml) is added to four or more tube cultures. the proportion of positive cultures is registered for each dilution and the precise dilution at which 50% of the inoculated tube cultures are infected is calculated. at this dilution, the inoculum contains, on average, one tcd 50 or one tissue culture (infectious) dose for 50% of the tissue culture tubes. the influence of a plant extract on virus multiplication can be determined as a viricidal or an antiviral activity. the viricidal activity is measured by titration of the residual infectious virus after incubation of the plant extract with a virus suspension of at least 10 6 tcd 50 ml â��1 . on the other hand, the antiviral activity is determined by comparing the virus titers of infected cells, which have been cultured with a maintenance medium containing plant extracts or test substances and a maintenance medium without test material (colegate and molyneux, 1993) . in contrast with antibacterial screening, no solvents, other than physiological buffer solutions, should ideally be used in the in vitro antiviral screening of plant extracts because the samples have to be added to tissue culture cells. it has been observed that many nonpolar plant extracts, prepared and evaporated, are reasonably soluble in dimethyl sulfoxide (dmso), especially if little or no water is present in the sample and the dissolving sample is heated on a water bath. viricidal and antiviral determinations may then be carried out on test solutions containing not more than 10% and 1% dmso, respectively. therefore, dissolved samples of nonpolar plant extracts in dmso are added dropwise to the maintenance medium in a ratio of 1:10 or 1:100 under stirring. as already mentioned, the maintenance medium may contain antibiotics, such as penicillin g (20 î¼g ml â��1 ), neomycin (1 î¼g ml â��1 ), and amphotericin b (1 î¼g ml â��1 ), in order to avoid sterilization of the test solutions. any contamination by bacteria or fungi would indeed ruin the in vitro antiviral bioassay (colegate and molyneux, 1993) . there are various methods for validation of antiviral activity. the major techniques have been highlighted in the preceding sections and in fig. 16.2. most currently used antiseptics and disinfectants kill pathogenic bacteria and fungi at 25â°c within 5 min when present in a concentration of about 0.5% (3-log titer reduction). because it has been noticed that most of these preparations fail to kill all pathogenic viruses under these circumstances, a method has been developed for testing the in vitro viricidal effect of plant extracts, as will be described in the following section. thoroughly mix the preincubated (25â°c) plant extracts, dissolved in a physiological buffer, or their twofold dilutions (e.g., 1/2 to 1/16), with the same volume of a preincubated (25â°c) virus suspension of, for example, 10 6 pfu ml â��1 or tcd 50 ml â��1 in physiological buffer. incubate the mixture at 25â°c for 5 min. stop the incubation by the addition of a 10fold volume of ice-cold maintenance medium and filter the mixture immediately through a 0.22 î¼m filter to eliminate all possible precipitate. then, filter the ice-cold filtrate through a 0.01 î¼m filter to concentrate residual virus on the filter and separate the virus from possibly cytotoxic plant components, which pass the filter. remove the residual virus from the filter with maintenance medium, supplement with 5% serum (1 ml), sonicate in an ice-bath for 30 s, and titrate in 10-fold dilutions at 37â°c by plaque formation or in microtiter plates according to the eptt. carry out a virus control in a physiological buffer containing no plant extract simultaneously. an essential step of this methodology is the separation of all cytotoxic plant components from the residual virus, which has to be measured at 37â°c. cytotoxic substances have a greater influence on the activity of an extracellular virus at 37â°c than at 25â°c. this step, however, can be omitted when the plant extracts to be tested are not toxic to the host cells under the conditions of the evaluation procedure (colegate and molyneux, 1993) . the eptt technique is performed on preemptied confluent monolayers of vero or other cells, grown in the holes of microtiter plates, which are infected with serial 10-fold dilutions of a virus suspension (100 î¼l). starting with monolayers containing 10 4 cells per hole and a virus suspension of, for example, 10 7 tcid 50 ml â��1 or pfu ml â��1 , infect the first monolayers of cells with a multiplicity of infection (moi). the antiviral activity is expressed as the virus titer reduction at the maximal nontoxic dose (mntd) of the test substance, that is, the highest concentration (î¼g ml â��1 ) that does not affect the monolayers under the antiviral test condition. viral titer reduction factors, that is, the ratio of the viral titer reduction in the absence (virus control) and presence of the mntd of the test sample of 1 ã� 10 3 to 1 ã� 10 4 indicate a pronounced antiviral activity and are suitable as selection criteria for further investigation of plant extracts (colegate and molyneux, 1993) . the eptt is more suitable for testing complex samples, such as plant extracts, for many reasons. (i) first, the concentration of many compounds in the extract remains constant, and consequently the proportion of toxic versus active compounds does not change. (ii) second, the exact duration of the antiviral action can be determined by using the eptt, because the action starts from the moment the extract is added to the cells. (iii) third, the eptt using serial diluted extracts deals with a dynamic process, because the cells are subsequently infected with different moi. (iv) this system allows the correlation of all possible moi values in the same microtiter plate with decreasing amounts of plant extracts, so that the noncytotoxic concentration of plant extracts can be determined. at the same time, a correlation between extract toxicity and antiviral activity according to the corresponding moi can be determined in the same microtiter plate. (v) it can be stated as a general rule that the detected antiviral activity should be stable in at least two subsequent dilutions of nontoxic concentration of the extract; otherwise the activity is directly correlated with the toxicity of the extract or is only viricidal. moreover, a true antiviral product has to protect the cells, which have been infected with low virus dilutions (starting from 0.1 pfu per cell onward). (vi) finally, it should be stressed that all possible steps of virus manipulation are to be completed before the plant extracts are added. this means that an antiviral product tcd 50 ml â��1 ), under nontoxic conditions, must act on virus replication steps after uncoating. when the cells are completely protected only in the lower moi (0.1 tcd 50 ml â��1 ), replication processes before uncoating may be involved. an important aspect of the inhibition of viral cytopathic effect (cpe) is the determination of tcid 50 (50% tissue culture infective dose). after harvesting, dilute the virus stock 10-fold. add 0.1 ml of each dilution in 10 wells each of a 96-well microtiter plate. add 0.1 ml of cell suspension of 10,000 cells/well, incubate at 37â°c with 5% co 2 atmosphere, and observe for vial cpe on alternate days. take a final reading on the fifth day and calculate tcid 50 as per the method of reed and muench (1938) , from which tcid 50 can be further calculated from the log value. an antiviral drug will inhibit the cpe of a virus. therefore, for detecting an antiviral agent, the amount of inhibition of cpe of a virus can be observed microscopically (kenny et al., 1985) . for this purpose, trypsinise the monolayer and allow to seed in 96-well microtiter plates. after a 24-h incubation, wash the monolayer in each well and add different test drug dilutions and incubate. after 24 h, wash the cell culture and inoculate with 0.1 ml of 10 tcid 50 , 50 tcid 50 , and 100 tcid 50 of the virus suspensions in different wells. incubate them for 1 h at 37â°c in an incubator for the adsorption of the virus onto the cells. after incubation, remove excess virus suspension by washing with rpmi. add 0.1 ml of selected concentration of the test compound and keep both virus and cell control wells with 0.1 ml of rpmi containing 2% sheep serum. observe the plates under a microscope every 24 h until the virus control shows 100% cpe and tabulate the results (hu and hsiung, 1989) . trypsinize the cell monolayer, allow to seed in a 96-well microtiter plate and incubate for 24 h at 37â°c with 5% co 2 atmosphere. remove the medium, wash the cell monolayer with rpmi without serum, and add 0.1 ml of different virus suspensions in different wells containing the cell layer and incubate for 1 h for virus adsorption; wash off the excess virus suspension after adsorption. to each well, add the selected concentration of the test drug diluted in rpmi containing 2% sheep serum. to the virus control and cell control, rpmi is added (2% serum) and incubated for 24 h. freeze the plates at â��70â°c and thaw at room temperature a couple of times to liberate the associated virus. determine the virus titer by the end point dilution method and express as tcid 50 (cinatl et al., 1997) . trypsinize the monolayer culture and allow to seed in a 96-well microtiter plate. after a 24-h incubation, wash the monolayer in each well and add different test drug dilutions and incubate. after 24 h, wash the cell culture and inoculate with 0.1 ml of 10 tcid 50 , 50 tcid 50 , and 100 tcid 50 of the virus suspensions in different wells. incubate for 1 h at 37â°c in a co 2 incubator for adsorption of the virus onto the cell. after incubation, remove excess virus suspension by washing with rpmi without serum. add 0.1 ml of the selected concentrations of the test compound and keep both the virus and cell control wells with 0.1 ml of rpmi containing 2% sheep serum. incubate the plates at 37â°c for 72 h. after 72 h, discard the old media from the cell cultures and add 50 î¼l of 2 mg/ml of mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to each well and incubate for 3 h. after 3 h of incubation, discard the mtt and add 100 î¼l of isopropyl alcohol to each culture and set aside for 10 min. record the absorbance using an elisa plate reader at 540 nm. in this experiment, the effect of the test drug on mitochondrial synthesis due to viral infection is studied. the "plaque" is a confined region of contaminated cells formed by multiplying virus particles. the plaques are revealed either as regions of dead/decimated cells recognized by cell stains or as zones of polluted cells by immunostaining. the blended cell monolayer is infected with a log10 dilution of viral plaque-forming unit (pfu) in the absence or presence of the test drug and permitted to adsorb (1 h at 37â°c in 5% co 2 ); afterward, the cells should be washed twice with prewarmed tcid cpe at dilution next above cpe at dilution nex 50 50 50 % % % t t above cpe at dilution next below 50 50 % % % ⧠⩠ⷠminimum essential medium (mem). the overlaid drug dilutions are arranged in the overlay medium on the contaminated culture, without the test drug. then, 45 ml of carboxy methyl cellulose is added to 9 ml of the medium; the plates are incubated for 3-5 days, then settled with 10% formalin or 4% formaldehyde for 30 min. the cells are stained with methylene blue (1 ml/well) or 1% gem violet (w/v), washed, and dried to check the plaques (dark areas) by low-power amplification of a binocular microscope. the antiviral impact should be measured as the percentage inhibition of plaque formation: the concentration of the test drug required to exert 50% of virus inhibition (ic 50 or ec 50 ), as compared with the infection control, is evaluated from the graphical plot as dose-response curves by regression analysis . viruses, for example, influenza, can agglutinate rbcs due to surface ha proteins, which can be analyzed visually by blending viral dilutions with rbc. this can be utilized to inspect the inhibitory impact of any medication on ha. the 10-overlay serially diluted (1-1000x) test drug is used alongside the diluted viral stocks (1:4 to 1:128). this dilution (50 ml/well) is added to drug-containing wells. it should be preincubated for 45 min and mixed with rbc (1/20 in pbs) sample solution. here, up to a specific dilution, the viral particles possibly lose their capacity to agglutinate rbcs, which shows a linkage of the drug with the viral ha. known quantities of virus (moi 5-10) are used for infecting both untreated or drug-treated cells and allowed to adsorb for 45-60 min. the unabsorbed virus particles are washed and fresh media is added to incubate for 24-36 h. then, cells are washed with pbs, fixed with paraformaldehyde (3%-4%), and permeabilized with acetone or triton x-100. the cells are again washed and blocked with 1% bovine serum albumin (bsa) in pbs for 30 min. then, the cells are incubated with mouse or rabbit antibody against a specific viral protein for 1-4 h at 37â°c. after that, the cells are subjected to repeated washing and incubated with a fluorescent-tagged secondary antibody for 1 h and washed again. after washing, the cells are visualized under a fluorescent microscope and compared with the fluorescence of untreated and drug-treated cells. on the other hand, for quantitation, the cells are trypsinized after treatment and fixed with 4% paraformaldehyde. the cells are then washed, permeabilized, and labeled with a fluorescent-tagged antibody, followed by propidium iodide (pi: 50 mg/ml in pbs). the cells are then counted in a fluorescent-activated cell counter to quantitate the fluorescence percentage. known quantities of virus (moi 5-10) are used for infecting both untreated and drug-treated cells, adsorbed for 1 h, washed, and incubated (2-4 days) for evaluation of the inhibition of the virus-induced cytopathic effect (cpe). the virus stock is centrifuged after freeze thawing and diluted for elisa. each well of a plate coated with a virus-specific antibody is mixed with 100 ml of controls or test drug and incubated at room temperature (1 h) with horseradish peroxidase conjugate, alkaline phosphatase, or b-d-galactosidase-labeled virus-specific antibody. the wells are washed five times and the substrate (100 ml) is added and incubated in the dark for 10 min. the reaction is stopped by adding 5% h 2 so 4 solution and the absorbance is read at 450 nm. the 96-well plates are seeded with a quadruplicate cell monolayer having a log 10 dilution of the test drug followed by infection with the virus. after 16-20 h of incubation at 37â°c, the monolayers are fixed with 0.05% glutaraldehyde and examined for virus-specific protein(s) on the cell surface. the elisa should be performed with a monoclonal antibody to the specific protein of the corresponding virus and protein and the od (optical density) is measured at 450 nm. the concentration is calculated by 50% reduction in od values (ec 50 ) from extrapolating graphical plots followed by the determination of si (ratio of cc 50 :ec 50 ) in which the results are expressed as a percentage of virusinfected cells (virus control) . the test drug and the virus (10 4 pfu/ml) are mixed to incubate for 1 h at 37â°c. then, immediately dilute the virus-drug mixture to 100-fold (final inoculums 50 pfu/well) with media containing 2% fbs to get a subtherapeutic concentration of mean number of plaques in control mean number of plaques in sample m ean number of plaque in control the test drug. following that, mix the monolayer, with the virus inoculums seeded using a 12-well plate. alternately, add the virus-test drug mix diluted to 100-fold, with no incubation period, with the respective cells for infection. allow to adsorb for 1 h at 37â°c, discard the diluted inoculums, and wash the cells with pbs. pour an overlay medium (with 2% fbs), and incubate at 37â°c for 72 h, followed by plaque reduction assay. count the viral plaque numbers obtained from infections set in the presence of the test drug and compare it with the control. viral attachment to the cell surface is carried out at 4â°c, as it permits binding, but stops viral entry, by elisa. briefly, incubate susceptible cells (2 ã� 10 4 cells/well) in 96-well plates and allow growth overnight. the cell monolayers are cooled at 4â°c. the cells are infected with the virus (moi 5) using heparin in presence of the test drug for 3 h at 4â°c as a control. after washing the wells with ice-cold pbs, fix with prechilled 4% paraformaldehyde in pbs for 1 h on ice, blocked with 5% bsa at 4â°c. the samples are incubated at 37â°c for 1 h with a primary antibody in pbst pbs with 0.05% tween 20 along with 5% bsa. the wells are washed twice with pbst, 5% bsa, and again only with pbst twice, at 5-min intervals on a shaker. this is mixed with secondary antibody in pbst with 5% bsa and incubated at 37â°c for 1 h. the reaction is developed with a 3,3â�²,5,5â�²-tetramethyl-benzidine two component microwell peroxidase substrate for 20 min; the reaction is stopped with 1 m phosphoric acid. the absorbance is measured at 450 nm, and the values are expressed as the fold change of absorbance relative to the mock infection control (lin et al., 2011) . the cell monolayers are cooled and grown in 12-well plates at 4â°c for 1 h. subsequently, infect the prechilled cells with hsv-1 (100 pfu/well) and incubate for 3 h at 4â°c to allow for viral adsorption. incubate the infected cell monolayers in the presence of test drug or heparin (100 mg/ml) for an additional 20 min at 37â°c to facilitate hsv-1 penetration. the extracellular virus is inactivated by citrate buffer (ph 3.0) for 1 min, and the cells are washed with pbs followed by overlay with dmem containing 2% fbs. after 48 h of incubation at 37â°c, the viral plaques are stained and counted (lin et al., 2011) . add the plated cells (0.8 ã� 10 5 cells/well for a 12-well plate) grown overnight at 30% confluence (300 ml) with virus dilution and deae dextran at a final concentration of 20 mg/ml. after adsorption (2 h at 37â°c in co 2 incubator), place the plates in a rocker to prevent the cells from drying and add fresh medium (1-2 ml) containing the test drug to each well and incubate for 40-48 h in 5% co 2 at 37â°c for subconfluent growth. after removing the media, add fixing solution (1-2 ml) to each well and incubate for 5 min at room temperature (î²-galactosidase activity decreases dramatically if the fixing solution is left for >10 min). then, discard the fixing solution, wash the cells twice with pbs, stain, and incubate at 37â°c for 50 min. finally, stain the plates to count the number of blue syncytia and express the titration values as the number of stained cells multiplied by the viral dilution . this assay is applicable for hiv-1, simian immunodeficiency virus (siv), and simian-hiv and is carried out in tzm-bl cells as it reveal the reduction in tat-induced luciferase (luc) reporter gene expression after a single round of virus infection. place the tzm-bl cells (4 ã� 10 4 /well) in a 24-well plate and incubate overnight. in a separate vial, mix the hiv-1nl4.3 (moi 0.05) virion with the test drug or vehicle for 1 h at 37â°c, then add to tzm-bl cells and incubate for 4 h. wash the cells (with cold pbs), and add fresh media with the test drug to culture for 48 h, using untreated hiv-1-infected cells (negative) and azidothymidine (azt)-treated cells (positive) as controls. wash the cells twice with pbs, lyse with 1x lysis buffer, and add the supernatant with the substrate, which should then be analyzed for luciferase activity in an optiplate using a fluorimeter. the results are expressed as percentage inhibition: and the percent inhibition is calculated by subtracting the above value from 100 (wan et al., 2012) . luminescence in the experimental group test drug or azt lu / m minescence of infected cells without the drug u100 (b) cem-green fluorescent protein (gpt) cell-based assay cem-gfp is a stable t-cell line-containing a plasmid encoding gfp and is suitable for hiv-1nl4.3 (moi 0.05) culture. for postinfection, incubate the cells (2.0 ã� 10 5 /well) with the test drug up to 8 days, using azt and solvents (used to prepare the test drug) as control(s). lyse the virus-infected cells with 1x promega cell lysis buffer (150 ml), and transfer to a culture plate to read the absorbance at 485 nm (excitation) and 520 nm (emission) by a fluorimeter. the results can be expressed as percentage inhibition: with the he percent inhibition calculated by subtracting the above value from 100 . place hep ad38 cell suspension (6 ã� 10 5 viable cells/ml of hep ad38 seeding medium) in a 96-well microtiter plate, and incubate at 37â°c for 3 days. discard the medium and wash the cell monolayers with warm (37â°c) dpbs. to the proper wells, add 100 î¼l of hepad38 assay medium that contains either test or control compounds at the desired concentrations. also include wells with hep ad38 assay medium alone as "virus only" controls. incubate at 37â°c for 3 days. on day 3, wash the cells once with warm dpbs and add fresh medium containing the appropriate compound to the wells. after 24 h, transfer the supernatants to v-bottomed 96-well plates and remove cellular debris by centrifugation (15 min, 2500 rpm at 4â°c). transfer 90 î¼l of the clarified supernatants to new v-bottomed plates and store at â��70â°c for quantification of hbv dna. thaw the supernatants that were collected and add 90 î¼l of 2x denaturation solution to each well and mix. incubate at 37â°c for 20 min. cut the nylon membrane to size and prepare it for blotting by wetting it first with distilled water and then 20x hybridization buffer, ssc (saline sodium-citrate). dot-blot the denatured supernatants on to the nylon membrane as directed by the manufacturer. wash the blot with 200 î¼l of neutralization solution followed by 200 î¼l of 20x ssc. remove the blot, rinse it briefly in 2x ssc, and then crosslink the dna to the nylon filter by uv irradiation. prehybridize the blot at 42â°c for 1 h in 20 î¼l of hybridization solution. prepare a 32p-labeled probe by random priming using a portion of the hbv genome as a template. purify the probe through a clontech chroma spin column. denature the probe by boiling for 5 min and add it immediately to the hybridization solution. hybridize the nylon filter overnight at 42â°c. wash the nylon filters twice with 50 ml of 2x ssc, 0.1% sds (sodium dodecyl sulfate) at room temperature for 20 min and twice with 50 ml of 0.2x ssc, 0.1% sds at 65â°c for 20 min. expose the nylon filters to a molecular imager screen for 4 h and scan on a phosphor imager to obtain the data. to determine the percent inhibition of hbv replication, subtract the background value (counts of radiation detected from the nylon filter itself) from all control and experimental values. divide the average values of the experimental wells (cells treated with test compounds) by the average value for the "virus only" control (cells not treated with compound or tetracycline during the experiment) and multiply this number by 100 (king and ladner, 2000) . the above mentioned in vitro studies are listed in table 16 .2. drug development programs include preclinical screening of immense quantities of chemicals for specific and nonspecific cytotoxicity against numerous sorts of cells, which is imperative to show the potential therapeutic target and safety evaluation. the screening of plant extracts or pure compounds for investigating their antiviral properties can be more significant with cytotoxicity measures (meyer et al., 1982) . it is essential for an investigational item to establish the antiviral activity at concentrations that can be accomplished in vivo without inducing toxicity to cells. moreover, in a cell culture display, antiviral activity of an investigational item can be the aftereffect of host cell death after exposure to the item. cytotoxicity tests utilize a series of increasing concentrations of the antiviral product to determine what concentration results in the death of 50% of the host cells. this value is referred to as the median cellular cytotoxicity concentration (cc50 or ctc50 or ccic50). the relative effectiveness of the investigational product in inhibiting viral replication compared with inducing cell death is defined as the therapeutic or selectivity index (cc50 value/ec 50 value). it is desirable to have a high therapeutic index giving maximum antiviral activity with minimal cell toxicity. according to us fda guidelines, it is recommended to determine cc50 values in both stationary and dividing cells from multiple relevant human cell types and tissues to establish the potential for cell cycle, species, or tissue-specific toxicities. studies determining cytotoxicity and therapeutic indexes should be conducted before the initiation of phase 1 clinical studies. there are a number of advantages for in vitro testing using cell cultures, which include gfp fluorescence in the experimental group fluorescence in / i infected cells without the test drug ã�100 analysis of species specificity, feasibility of using only small amounts of test substances, and facility to do mechanistic studies (guidance for industry, 2006). after confirming the cytotoxic concentration, the drug concentrations are selected for antiviral studies based on the percentage viability of cells and are used to study the antiviral activity by cpe inhibition assay, virus yield assay, followed by mtt assay. any compound that is cytotoxic to cells inhibits the cell proliferation and kills the cells. trypan blue is a dye that is capable of penetrating dead cells; therefore, the dead cells take up the blue stain whereas the viable cells do not. this method gives an exact number of dead and viable cells (strober, 2001) . protein content is widely used for estimating total cellular material and can be used in growth experiments. the colorimetric method of estimating protein is more sensitive. the cell pellets are treated with 11% cold trichloroacetic acid to remove amino acid pools and dissolved in alkaline cupric sulfate and folin ciocalteau phenolic reagent. folin's reagent and cupric sulfate together react with amino acid to give a blue color and this color intensity is proportional to the protein concentration, which can be measured colorimetrically (maya et al., 1995) . to proceed with the same technique, the cells from the wells were trypsinized using 100 î¼l trypsin and transferred into eppendorf tubes and centrifuged at 5000 rpm for 10 min to obtain pellets. the cell pellets are dissolved in naoh and diluted , tzm-bl cell-based assay (hiv), cem-green fluorescent protein cell-based assay (hiv), hep ad38 assay (hbv), immunofluorescence assay, enzyme-linked immunosorbent assay (elisa) â�¢ reduction or inhibition of virus-specific polypeptides synthesis in infected cell cultures, e.g., viral nucleic acids, determination of the uptake of radioactive isotope labeled precursors or viral genome copy numbers other assays for validation of antiviral activity â�¢ virus inactivation assay, virus adsorption assay, virus attachment, and penetration assay to 0.1 n. the test drug is to be added to 200 î¼l of protein sample, mixed, and left for 10 min. to this, 100 î¼l of test reagent is added with constant mixing and left for 40 min in incubator. the absorbance was read at 655 nm using an elisa reader (bio-rad). the color development was correlated with the cell number as follows: the cytotoxic concentration found by dye exclusion techniques gives superficial data. the selected concentrations from a trypan blue dye exclusion study are used further for estimating proteins. mtt (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide) in live cells enters the cells and enzyme succinate dehydrogenase present in mitochondria reduces it to formazan blue product. the color intensity is directly proportional to the number of live cells. to perform this process, the plates were seeded with hep-2 cells at 10,000 cells/well. they are incubated for 24 h. after 24 h, the medium is discarded and drug concentrations were added and incubated for 72 h. then, 50 î¼l of 2 mg/ml of mtt is to be added and incubated for 3 h and then 100 î¼l of isopropyl alcohol is added and absorbance is read at 540 nm in an elisa plate reader (bio-rad). the results are tabulated and percentage growth inhibition is calculated using the following formula: the concentrations of the test drug used in the previous experiments can be further confirmed by studying the mitochondrial synthesis by mtt assay. the formazan blue color formation is directly proportional to the number of viable cells and therefore the absorbance is to be read at 540 nm. in this test, brine shrimp (artemia salina) eggs are hatched in artificial sea water (38 g/l of sea salt). the brine shrimp test (bst) bioassay experiment is performed according to the procedure described by meyer et al. (1982) . generally, samples of the test drugs for the experiment are prepared in methanol solution, which acts as control vehicle. after 48 h of incubation, 10 brine shrimps are transferred to each sample vial using a pasteur pipette and artificial sea water is added to make 5 ml. sample vials are previously prepared by dissolving specific concentrations of test drugs with different dilutions. the solvent is then evaporated overnight. survivors are counted after 24 h and the lc 50 values, with 95% confidence intervals are determined using probit analysis, as described by finney (1971) . control vials are prepared using methanol only. three replicates are prepared for each concentration of the test drugs. control disks are prepared using only methanol. replicates are prepared for each dose level. to begin the bioassay, brine shrimp eggs are hatched in a shallow rectangular dish (22 ã� 32 cm 2 ) under the same conditions described in the literature except that natural instead of artificial seawater is used. ten shrimps are selected and transferred into each sample vial by means of a 23-cm disposable pasteur pipette and the final volume in each vial is adjusted to 5 ml using natural seawater. a drop of dry yeast suspension (3 mg in 5 ml seawater) is added as food to each vial. the vials are maintained under illumination. survivors are counted with the aid of a stereomicroscope, after 6, 24, and 48 h, and the deaths at each dose level and control are determined. no deaths are usually observed to occur in the control after 48 h. the brine shrimp test (bst) represents a rapid, inexpensive, and simple bioassay for testing plant extract lethality, which, in most cases, correlates reasonably well with cytotoxic properties (mclaughlin, 1991) . following the procedure of meyer et al. (1982) , the lethality of the test drugs/plant extracts to brine shrimp is determined. the lethal concentrations of test drugs/plant extract resulting in 50% mortality of the brine shrimp (lc 50 ) and 95% confidence intervals are determined from the 24 and 48-h counts and the dose-response data are transformed into a straight line by means of a trend line fit linear regression analysis; the lc 50 is derived from the best-fit line obtained. caffeine (lc 50 = 306 î¼g/ml) (meyer et al., 1982) is used as a positive control and methanol (500 î¼l) as a solvent and a negative control in the bioassay experiments. the current scenario of viral diseases is lethal and there is an upsurge in new viral diseases and resistance to existing viral infections worldwide. the currently accessible antivirals, however effective, are exorbitant and past the reach of a majority of individuals. along these lines, the advancement of safe, effective, and low-cost antiviral medications, for example, rt inhibitors, is among the top priorities, as many viral infections are not yet treatable and have high death rates. for the past few years, substantial work has been carried out regarding the effectiveness of medicinal plants on hiv infection (premanathan et al., 1999; calabrese et al., 2000; asres et al., 2001) and an increasing popularity of over-the-counter plant products containing orthodox drugs has been observed. the main focus is to lower the adverse effects associated with viral infections and an inclination toward synergistic interactions of multiple molecules present in plant extracts. be that as it may, because mostly pharmacological mechanisms of the combinations are not studied, antagonistic impacts or remedial disappointments have been seen (chan et al., 2000) . a prerequisite that should be considered significant for medicinal plants is to identify and standardize the method of extract preparation, the suitable season for collecting plant material, and the details of its administration (chattopadhyay et al., 2006) . as a lot of plant extracts and subsequent formulations have shown significant outcomes, it seems to be rational to endorse the idea of the study of medicinal plants as a quest to find potential antivirals. antiviral investigation on the flavonoids of chamaesyce thymifolia investigation of medicinal plants of togo for antiviral and antimicrobial activities in vitro virucidal activity of selected anthraquinones and anthraquinone derivatives antiviral evaluation of plants from brazilian atlantic tropical forest antiviral activity against human immunodeficiency virus type 1 (hiv-1) and type 2 (hiv-2) of ethnobotanically selected ethiopian medicinal plants suppression of hepatitis c virus by the flavonoid quercetin is mediated by inhibition of ns3 protease activity anti-herpes virus activities of bioactive fraction and isolated pure constituent of mallotus peltatus: an ethnomedicine from andaman islands the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborine and the (n-acetylglucosamine) n -specific plant lactin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro 1-cinnamoyl-3,11-dihydroxymeliacarpin is a natural bioactive compound with antiviral and nuclear factor-kappa b modulating properties a high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors an investigation of the antiviral activity of podophyllum peltatum in vitro anti-hiv-1 activities of kaempferol and kaempferol-7-o-glucoside isolated from securigera securidaca further screening of venda medicinal plants for activity against hiv type 1 reverse transcriptase and integrase the natural compound silvestrolis a potent inhibitor of ebola virus replication plant products as topical microbicide candidates: assessment of in vitro and in vivo activity against herpes simplex virus type 2 a phase i trial of andrographolide in hiv positive patients and normal volunteers comparative study of propofol versus midazolam in the sedation of critically ill patients: results of a prospective, randomized, multicenter trial survey for the presence and distribution of human herpesvirus 8 in healthy brain ethnomedicines and ethnomedicinal phytophores against herpes viruses antivirals of ethnomedicinal origin: structure-activity relationship and scope dose dependent therapeutic antiinfectives from ethnomedicines of bay islands recent advancements for the evaluation of anti-viral activities of natural products validation of antiviral potential of herbal ethnomedicine inhibitory effects of quercetin 3-rhamnoside on influenza a virus replication anti-herpes simplex virus (hsv-1) activity of oxyresveratrol derived from thai medicinal plant: mechanism of action and therapeutic efficacy on cutaneous hsv-1 infection in mice chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and hiv infection antiviral effects of 6-diazo-5-oxo-1-nor-leucine on replication of herpes simplex type-1 novel compounds in preclinical/early clinical development for the treatment of hiv infections molyneux bioactive natural products plant substances as antiviral agents: an update potential antiviral lignans from the roots of saururus chinensis with activity against epstein-barr virus lytic replication drug screening for autophagy inhibitors based on the dissociation of beclin1-bcl2 complex using bifc technique and mechanism of eugenol on anti-influenza a virus activity structure and in vitro antiviral activity of triterpenoid saponins from calendula arvensis antiviral activity of chlorogenic acid against influenza a (h1n1/h3n2) virus and its inhibition of neuraminidase resveratrol inhibition of herpes simplex virus replication antiviral activities of various water and methanol soluble substances isolated from ganoderma lucidum sennoside a, derived from the traditional chinese medicine plant rheum l., is a new dual hiv-1 inhibitor effective on hiv-1 replication anti-enterovirus and immunostimulating activity of the polyphenol complex extracted from pethaphylloides fruticosa (l.) o. schwarz honokiol, a lignanbiphenol derived from the magnolia tree, inhibits dengue virus type 2 infection screening of brazilian plants for antiviral activity against animal herpes viruses probit analysis antiviral effects of glycyrrhiza species biotransformation of ursolic acid by syncephalastrum racemosum cgmcc 3.2500 and anti-hcv activity anti-aids agents 11. betulinic acid and platanic acid as anti-hiv principles from syzygium claviflorum, and the anti-hiv activity of structurally related triterpenoids antiviral activity of dammarane saponins against herpes simplex virus type 1 activity of melaleuca alternifolia (tea tree) oil on influenza virus a/pr8: study on the mechanism of action antiviral activity of an extract derived from roots of eleutherococcus senticosus search for antiviral agents antiviral product development-conducting and submitting virology studies to the agency. u.s. department of health and human services, food and drug administration michellamines d-f, new hiv-inhibitory dimeric naphthylisoquinoline alkaloids, and korupensamine e, a new antimalarial monomer, from ancistrocladus korupensis virucidal effects of the steam distillate from houttuynia cordata and its components on hsv-1, influenza virus, and hiv inhibitory effect of ferulic acid and isoferalic acid on murine interleukin-8 production in response to influenza virus infections in vitro and in vivo a modified plaque method for arboviruses on plastic panels evaluation of new antiviral agents: i, in vitro perspectives anti-viral effect of a compound isolated from liriope platyphylla against hepatitis b virus in vitro isolation of the anthropogenic compound fluoranthene in a screening of chinese medicinal plants for antiviral compounds further investigations on the antiviral activities of medicinal plants of togo antiviral activities in plants endemic to madagascar inhibitory effects of sudanese medicinal plant extracts on hepatitis c virus (hcv) protease study of antiviral action of total alkaloids from haemanthus albiflos inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus antiviral potential of selected indian medicinal (ayurvedic) plants against herpes simplex virus 1 and 2. n anti-hbv active constituents from piper longum the flavonoid ellagic acid from a medicinal herb inhibits host immune tolerance induced by the hepatitis b virus-e antigen comparison of specific antiviral agents in herpes simplex keratitis in vitro and in vivo anti picorna virus activity of some phenoxypyridine carbonitriles extracts and molecules from medicinal plants against herpes simplex viruses hep ad38 assay a high-throughput, cell-based screen for the evaluation of compounds against hepatitis b virus samarangenin b from limonium sinense suppress herpes simplex virus type 1 replication in vero cells by regulation of viral macromolecular synthesis antiviral activities against hsv-1, hcmv, and hiv-1 of rhamnan sulfate from monostroma latissimum antiviral effects of black raspberry (rubus coreanus) seed and its gallic acid against influenza virus infection in vitro antiviral activities of chinese medicinal herbs against duck hepatitis b virus plant antiviral agents iii. isolation of alkaloids from clivia miniata regel (amaryllidaceae) isolation and purification of baicalein, wogonin and oroxylin a from the medicinal plant scutellaria baicalensis by high-speed counter-current chromatography antiviral activity and mode of action of caffeoylquinic acids from schefflera heptaphylla (l) identification of natural compounds with antiviral activities against sarsassociated coronavirus mechanism of action of glycyrrhizic acid in inhibition of epstein-barr virus replication in vitro hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoproteine glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread saikosaponin b2 is a naturally occurring terpenoid that efficiently inhibits hepatitis c virus entry ethnopharmacology in overdrive: the remarkable anti-hiv activity of artemisia annua the alkaloids. vol antiviral potential of curcumin a search for anti-viral properties in panamian medicinal plants, the effects on hiv and its essential enzymes interaction of filacial proteins on growth regulation of normal lung epithelial cells in vitro antiviral screening of british columbian medicinal plants crown-gall tumors in potato discs and brine shrimp lethality: two simple bioassays for higher plant screening and fractionation the in vitro antivirial activity of an aliphatic nitro compound from heteropteris aphrodisiaca brine shrimp: a convenient general bioassay for active plant constituents natural products and drug development. munksgaard anti-herpes virus activity of apporphine alkaloids anti-herpes virus activities of achyranthes aspera: an indian ethnomedicine, and its triterpene acid bio organic marine chemistry pedilanthus tithymaloides inhibits hsv infection by modulating nf-îºb signalling screening extracts of zanthoxylum chalybeum and warburgia ugandensis for activity against measles virus (swartz and edmonston strains) in vitro biological activity of guatteria cardoniana fractions comparison of two colorimetric assays to determine viral infectivity in microculture virus titration characteristic of alkylated chalcones from angelica keiskei on influenza virus neuraminidase inhibition glycyrrhizic acid inhibits virus growth and inactivates virus particles in vitro anti-human immunodeficiency virus activity of polysaccharide from rhizophora mucronata poir maslinic acid, a natural triterpenoid compound from olea europaea, protects cortical neurons against oxygen-glucose deprivation induced injury in vitro study of the antiviral activity of some b-carboline alkaloids a simple method of estimating fifty percent endpoints anti-hiv activity of extracts and compounds from algae and cyanobacteria antiviral flavonoid from pterocaulon sphacelatum, an australian aboriginal medicine in vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus antiherpes virus activity of extracts from the medicinal plant geranium sanguineum l antiviral agents as adjuncts in cancer chemotherapy effects of phyllanthus plant extracts on duck hepatitis b virus in vitro and in vivo antiviral activity of carnosic acid against respiratory syncytial virus in vitro antioxidant activity of polyphenol extracts with antiviral properties from geranium sanguineum l a new antiviral and antimicrobial sesquiterpene from glyptopetalum sclerocarpum trypan blue exclusion test of cell viability antiviral activity and constituent of ardisia chinensis benth against coxsackie b3 virus analysis of anti-rotavirus activity of extract from stevia rebaudiana proanthocyanidin from blueberry leaves suppresses expression of subgenomic hepatitis c virus rna anti-viral activity of the extracts of a kenyan medicinal plant carissa edulis against herpes simplex virus screening of higher plants for biological activities. ii. antiviral activity plant products as potential antiviral agents plant antiviral agents: v. 3-methoxyflavones as potent inhibitors of viral-induced block of cell synthesis in vitro antiviral activity of plant extracts from asteraceae medicinal plants current organic chemistry, natural product chemistry issue plant flavonoids in biology and medicine ii: biochemical, cellular and medicinal properties inhibition of hepatitis c virus replication by chalepin and pseudane ix isolated from ruta angustifolia leaves fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing herbs of the genus phyllanthus in the treatment of chronic hepatitis b: observations with three preparations from different geographic sites effect of oenanthe javanica flavone on human and duck hepatitis b virus infection antiviral effect of cimicifugin from cimicifuga foetida against human respiratory syncytial virus three new secoiridoids, swermacrolactones a-c and anti-hepatitis b virus activity from swertia macrosperma lignans with antihepatitis b virus activities from phyllanthus niruri l antiviral activity of polymethoxylated flavones from guangchenpi, the edible and medicinal pericarps of citrus reticulata 'chachi the in vitro activity of geraniin and 1,3,4,6-tetra-o-galloyl-beta-d-glucose isolated from phyllanthus urinaria against herpes simplex virus type 1 and type 2 infection the protective effect of 3-deoxysappanchalcone on in vitro influenza virusinduced apoptosis and inflammation mechanism of inhibition of hiv-1 infection in vitro by purified extracts of prunella vulgaris evaluation of anti-hsv-2 activities of barleria lupulina and clinacanthus nutans novel antiviral activity of baicalein against dengue virus a dihydro-pyrido-indole potently inhibits hsv-1 infection by interfering the viral immediate early transcriptional events comparative inhibitory effects of suramin and other selected compounds on the infectivity and replication of human t-cell lymphotropic virus (htlv-iii)/lymphadenopathy-associated virus (lav) discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development emerging anti-hiv drugs agar diffusion method for detection and bioassay of antiviral antibiotics anti-herpes virus activity of aporphine alkaloids antiviral potentials of medicinal plants evaluation of anti-infective potential of a tribal folklore odina wodier roxb against some selected microbes and herpes simplex virus associated with skin infection sensitive and rapid assay on mt-4 cells for the detection of antiviral compounds against the aids virus a rapid and simple colorimetric test for the study of anti-hiv agents plant-derived leading compounds for chemotherapy of human immunodeficiency virus (hiv) infection resistance of human immunodeficiency virus type 1 to the high-mannose binding agents cyanovirin n and concanavalin a key: cord-355318-qm79gz8w authors: smit, albertus j.; fitchett, jennifer m.; engelbrecht, francois a.; scholes, robert j.; dzhivhuho, godfrey; sweijd, neville a. title: winter is coming: a southern hemisphere perspective of the environmental drivers of sars-cov-2 and the potential seasonality of covid-19 date: 2020-08-05 journal: int j environ res public health doi: 10.3390/ijerph17165634 sha: doc_id: 355318 cord_uid: qm79gz8w sars-cov-2 virus infections in humans were first reported in december 2019, the boreal winter. the resulting covid-19 pandemic was declared by the who in march 2020. by july 2020, covid-19 was present in 213 countries and territories, with over 12 million confirmed cases and over half a million attributed deaths. knowledge of other viral respiratory diseases suggests that the transmission of sars-cov-2 could be modulated by seasonally varying environmental factors such as temperature and humidity. many studies on the environmental sensitivity of covid-19 are appearing online, and some have been published in peer-reviewed journals. initially, these studies raised the hypothesis that climatic conditions would subdue the viral transmission rate in places entering the boreal summer, and that southern hemisphere countries would experience enhanced disease spread. for the latter, the covid-19 peak would coincide with the peak of the influenza season, increasing misdiagnosis and placing an additional burden on health systems. in this review, we assess the evidence that environmental drivers are a significant factor in the trajectory of the covid-19 pandemic, globally and regionally. we critically assessed 42 peer-reviewed and 80 preprint publications that met qualifying criteria. since the disease has been prevalent for only half a year in the northern, and one-quarter of a year in the southern hemisphere, datasets capturing a full seasonal cycle in one locality are not yet available. analyses based on space-for-time substitutions, i.e., using data from climatically distinct locations as a surrogate for seasonal progression, have been inconclusive. the reported studies present a strong northern bias. socio-economic conditions peculiar to the ‘global south’ have been omitted as confounding variables, thereby weakening evidence of environmental signals. we explore why research to date has failed to show convincing evidence for environmental modulation of covid-19, and discuss directions for future research. we conclude that the evidence thus far suggests a weak modulation effect, currently overwhelmed by the scale and rate of the spread of covid-19. seasonally modulated transmission, if it exists, will be more evident in 2021 and subsequent years. a novel coronavirus, thought to have made a zoonotic transition from bats, infected a human host in wuhan, hubei province, china [1] . by late january 2020, the virus, newly named sars-cov-2, and the disease it causes, covid-19, had spread to 18 other chinese provinces, and to japan, south korea, taiwan, thailand, and the usa. on the date of submission of this review (15 july 2020), there were 13,331,879 confirmed cases, in virtually every country worldwide (213 countries and territories, figure 1 ). at the time, it was reported that 577,825 people infected with the virus had died; both numbers have subsequently risen. the only comparable acute respiratory disease pandemic was spanish influenza (h1n1), transmitted from birds to people in 1918, which lasted until 1919 and killed an estimated 50 million people worldwide. in the current highly interconnected world, the impact of the covid-19 pandemic is likely to be felt for many years [2] [3] [4] . it is therefore crucial that all potential determinants of the rate and location of the pandemic spread receive careful consideration in order to make appropriate plans for its management. epidemiological models have been used worldwide to guide the imposition (or not) of policy and regulatory intervention [5, 6] . these models can be modified to include aspects of social characteristics of the infected populations; and they can also be adapted to reflect the modulating effect of environmental influences on the processes that determine transmission. many related respiratory diseases show a connection between climate variables and the dynamics of the disease. it is thus plausible that such a dependency could exist for sars-cov-2 (reviewed in section 4) . given that the covid-19 outbreak began in mid-winter in the northern hemisphere, where it was (at the time of writing) peaking toward the middle of the boreal summer, and that the opposite scenario seems to be playing out in many southern hemisphere countries, it is tempting to associate this pattern with climate seasonality, as many publications have suggested. however, it is also plausible that the association is spurious, related simply to coincidental spatial connectivity between countries. it is necessary to critically assess the evidence for environmental sensitivity, in both the virus and the disease, before arriving at conclusions that may have significant implications. in terms of a response to the pandemic, we need to understand whether and how environmental variables influence the infection rate. this knowledge provides clues for policy and practice to reduce the spread of the virus and potential for treatment options. for example, if analyses show that absolute humidity is strongly associated with reduced infection rates (e.g., influenza [7, 8] ), artificially epidemiological models have been used worldwide to guide the imposition (or not) of policy and regulatory intervention [5, 6] . these models can be modified to include aspects of social characteristics of the infected populations; and they can also be adapted to reflect the modulating effect of environmental influences on the processes that determine transmission. many related respiratory diseases show a connection between climate variables and the dynamics of the disease. it is thus plausible that such a dependency could exist for sars-cov-2 (reviewed in section 4) . given that the covid-19 outbreak began in mid-winter in the northern hemisphere, where it was (at the time of writing) peaking toward the middle of the boreal summer, and that the opposite scenario seems to be playing out in many southern hemisphere countries, it is tempting to associate this pattern with climate seasonality, as many publications have suggested. however, it is also plausible that the association is spurious, related simply to coincidental spatial connectivity between countries. it is necessary to critically assess the evidence for environmental sensitivity, in both the virus and the disease, before arriving at conclusions that may have significant implications. in terms of a response to the pandemic, we need to understand whether and how environmental variables influence the infection rate. this knowledge provides clues for policy and practice to reduce the spread of the virus and potential for treatment options. for example, if analyses show that absolute humidity is strongly associated with reduced infection rates (e.g., influenza [7, 8] ), artificially raising indoor absolute humidity during periods of low ambient humidity may be an effective intervention. second, if environmental variables do influence the trajectory of the pandemic, the seasonal progression of the disease will lead to different implications across the globe, varying by hemisphere, region, and climatic zone. in the extratropical northern hemisphere, there would be a real possibility of a second wave appearing during the next winter [9] . conversely, there is a danger that the initially slow pace of the epidemic in the southern hemisphere could be misinterpreted to mean that proactive management has supressed the disease spread. given that in the south, where the peak of covid-19 incidence is likely to coincide with the winter peak of other endemic respiratory illnesses, complicating diagnosis and placing additional strain on the health systems, missing the environmental drivers of covid-19, if they exist, would be profoundly damaging. as we will argue, many southern hemisphere countries are particularly vulnerable (they are in the developmental 'global south' as well as the geographical south). for these regions especially, clarifying the environmental sensitivity will assist the prioritisation of resources. third, for longer-term management of the disease, we need to understand whether the seasonal effect will manifest as it does in established or endemic respiratory viruses, in the absence of being able to predict in what period of time (in years) the virus will be eliminated [10] . in this review, we consider all the pertinent studies relating to the effect of a range of specific environmental and climatological variables on the biology of the virus and the epidemiology of the disease. in section 2, we develop our reasoning for why southern hemisphere countries can benefit from the lessons learnt in the north, if the application of that knowledge takes heed of particularly southern hemisphere issues. in section 3, we briefly present the main classes of epidemiological models, since key parameters revealing environmental modulation are derived from such analyses. in section 4, we explore environmental sensitivity in extant respiratory viral diseases and past epidemics in order to suggest why seasonally coupled environmental influences are also likely to exist for sars-cov-2. section 5 then critically reviews evidence for such signals in the literature that had accumulated to 15 july 2020. section 6 summarises our findings, and offers suggestions for future analyses of the seasonal modulation of covid-19. the situation regarding covid-19 in southern hemisphere is different from that in the north in three ways. first, while the northern hemisphere is moving out of winter at the time of their peak of infections, the southern hemisphere is moving into winter. second, a much larger proportion of countries in the southern hemisphere are developing countries, with significant resource limitations in their healthcare systems. third, many of the countries in the southern hemisphere, and on the african continent in particular, have a much higher incidence of pulmonary diseases such as tuberculosis, immunocompromising diseases such as hiv-aids, and a higher prevalence of diseases such as cholera and malaria, which may not be recognised as comorbidity risks in covid-19 but do place coinciding stressors on the health system. to their advantage, the delayed arrival of covid-19 in much of the southern hemisphere has allowed these countries the time to observe the efficacy of containment and treatment practices in the global north, and to adapt their healthcare and policy response accordingly. the initial outbreak of covid-19 in china, early epidemics in iran, italy, and later much of europe and the united states took place during the coldest months of their year, and were distributed within a narrow climatic band [11, 12] . during the early period of the outbreak in january and february 2020, few known cases had been recorded in the southern hemisphere, which was experiencing peak summer conditions. this could reflect a climate sensitivity, but could just as plausibly reflect dominant trade and human movement patterns [13] . thus, the initial relatively low rates of spread and mortality in southern africa, australia and some regions of south america may simply be a result of being at an earlier stage in these epidemics. however, in both the northern and southern hemisphere, influenza and other coronavirus diseases peak during their respective winter seasons [14] . thus, if climate factors do play a role in covid-19 infection rates, the concurrence of transition of southern hemisphere countries to their winter season with the mid-stages of the disease transmission trajectory is of concern, especially with respect to containment policy and health system resource allocation. the status of healthcare services in the global south is of concern even without a climatic component to covid-19. while australia and new zealand have healthcare services as good as any in the northern hemisphere [13] , much of south america and sub-saharan africa struggle with access to quality healthcare. this is associated with poverty and socio-economic inequalities and result in poor health outcomes and financial risk to the state and individuals [15] [16] [17] [18] . the healthcare sectors are understaffed, underresourced, and understocked under normal conditions, which were working at maximum capacity even before the covid-19 pandemic [19] , and will be severely challenged as covid-19 cases increase [20, 21] . early evidence from china shows a significant correlation between mortality and the healthcare burden in covid-19 cases [22] . efforts to model the preparedness of african countries have highlighted concerns relating to the staffing of testing centres, stock for testing, and the ability to implement effective quarantining both inside and outside of healthcare facilities [20] . the prevalence of pre-existing infectious diseases compounds this issue. in the period 2016-2018, 41 african countries have experienced at least one epidemic, while 21 have experienced at least one epidemic per year [23] . south america is currently struggling with outbreaks of measles in 14 countries, and a tripling of the incidence of dengue fever in four countries [24] . recent outbreaks of diphtheria, zika and chikungunya have further stretched the healthcare systems [24] . the most prevalent infectious diseases in sub-saharan africa include cholera, malaria, viral haemorrhagic fever, measles and malaria [19] . of particular concern in the global south is the possibility of comorbidity with hiv-aids and tuberculosis (tb). many tb cases are pulmonary in nature [25] , while patients with hiv are significantly immunocompromised [26] . there is considerable tb-hiv comorbidity [27] . corbett et al. [28] found a 38% incidence of hiv in tb-infected patients across africa, and for the countries with the highest hiv prevalence, up to 75% of tb patients also tested positive for hiv. comorbidity has decreased from 33% to 31.8% over the past decade, and over the period 1990-2017, tb incidence, tb mortality rate and hiv-associated tb have declined in a number of southern african countries [26] . south america has much lower cases of both hiv and tb, and a comorbidity of approximately 10% [29] . while results from spain suggest that hiv-positive patients currently on antiretroviral treatment have no higher risk of severe sars-cov-2-induced illness [30] , the comorbidity of those with a longer hiv history and tb comorbidity, with or without hiv, is unknown. there are further related concerns pertaining to continued hiv [31] and tb [32] care during covid-19, as social distancing requires people to stay indoors and hospitals are overstretched. finally, the relatively delayed spread of covid-19 to the southern hemisphere has allowed these countries to 'get ahead of the curve' through evidence-based management derived in the north [33] . recent experiences of two ebola epidemics have meant that many countries in sub-saharan africa implemented temperature screening at airports long before the first covid-19 cases were reported [20] , and contact tracing and epidemic management plans are in place [19] . south africa, kenya, uganda and zambia were reported as having all been particularly proactive in planning for their eventual covid-19 cases [19] . south america has arguably not been as prepared (rodriguez-morales et al. 2020). studies on modelling risk for the african continent are largely related to importation risk [20] , which has been capped due to lockdown in many countries. this form of response is important in delaying the peak and "flattening the curve", but is unlikely to completely avoid extreme pressure on already stressed healthcare systems [16, 22] . when assembling datasets from many different locations to test the effects of environment on covid-19 progression, it is essential that the criteria for determining the infection and mortality rates are consistent across sources. the data used to calibrate and validate epidemiological models (e.g., the covid-19 data repository, center for systems science and engineering (csse), johns hopkins university) consist of time series of infections, which often include only those with symptoms sufficiently severe that the patients sought medical assistance, and who subsequently tested positive using a pcr-based test for the presence of the sars-cov-2 virus [34] . this is known as the 'case rate'. as the number of tests increases and includes community-based testing, as opposed to testing only those displaying symptoms, the case rate will converge on a true infection rate. pcr testing is accurate (though reporting is often delayed by days to weeks [35] ), but if testing is mostly performed on those presenting symptoms and their close contacts, estimates of the true infection rate inevitably include large biases, especially given the high occurrence of asymptomatic or mild cases. compensating for this bias requires that the sample frame be weighted to be representative of the population as a whole. as antibody-based tests become more widely used, datasets that indicate post facto what fraction of the general population was exposed to the virus will emerge. antibody tests have variable accuracy, both in terms of false positives and false negatives [35] ; nevertheless, their overall accuracy is much better than the guesswork that otherwise goes into estimating the number infected from the medical case rate alone. it is suspected that mildly infected people and even asymptomatic cases can spread the disease [36] , but perhaps less effectively than severely ill individuals. it is likely that recovery from sars-cov-2 provides subsequent immunity, with initial indications that this may be persitent [37] . the models that predict mortality use a time series of recorded deaths. at a minimum, this includes the deaths recorded in hospitals for people being treated for sars-cov-2 at the time of death. more complete records are supplemented with data on people who died in the community or in nursing homes, and were inferred from the symptoms they displayed to have died from covid-19. for severely-affected areas, it is possible to estimate the anomalies between the covid-attributed death rate relative to the seasonally adjusted expected population death rate, and infer that these additional deaths ('excess deaths') were caused by the pandemic [38] . where this has been carried out, it suggests that the death rate is substantially higher than that initially reported; however, this approach conflates deaths directly caused by sars-cov-2, and those that may have resulted from overburdening the health system. making accurate estimates of transmission rates requires a sufficient number of cases. often the models are initiated only once 30 or 100 cases have occurred in a location [39] so that the effect of importation of cases due to travelling may be minimised. therefore, if the area selected for analysis is too small, the number of cases may be inadequate to support the more data-intensive approaches. in most countries, data are collected daily, but the daily data show a lot of noise, partly for stochastic reasons; also, for spurious reasons such as the effect of weekends, laboratory delays, or recoding the date of reporting rather than the date of testing or infection (section 5.1). smoothing the data over periods of a week helps to solve irregular daily data patterns [40, 41] , but this also means that the analyses are unresponsive to events at finer timescales. the need to match the time period for which infections and deaths are recorded and the period over which environmental drivers are integrated is widely accepted. similar considerations also apply to spatial resolution. covid-19 outbreaks are apparently highly clustered, often in small areas. environmental drivers are also spatially heterogeneous, some much more so than others. the resolution chosen for the environmental data needs to be appropriate for both the grain of the infection process and the grain of the environmental variable. several analytical typologies have been applied to epidemiological models, mostly based on what factors they take into account [42] . table 1 is a pragmatic classification of the types thus far predominantly used for covid-19 projections, based on the logic of their construction. most of these model types can be implemented either deterministically or stochastically for age-structured or non-age-structured populations; for a single, equally-exposed population or for a spatially disaggregated population with transfers between groups; and using frequentist or bayesian approaches. cuevas [49] simple extrapolation and phenomenological models are suitable for projections of less than one month into the future, whereas the somewhat mechanistic epidemiology models are more robust for projections months or years into the future. the various classes of models can in principle run at any spatial scale and over any time period, but in practice there are data-imposed constraints. environmental influences can be introduced into the basic model structures at a variety of points ( figure 2 ). where they are introduced and what the models are able to say about the relationship between the environmental influences and infection or mortality rates depend on the theoretical basis of the model ( table 1 ). models that best capture the functional relationship of confirmed daily cases across time are best suited for revealing environmental drivers. the phenomenological and compartmental models are the strongest contenders here. the raw time series of confirmed infections and deaths can be time aggregated, and time lagged with respect to the environmental factors, to find the best fits, as long as this is performed consistently, and considers the time lags already built into the model structure. one approach is to establish correlations, either over time or across space, between the infection rate at a given time and simultaneous metrics of environmental factors such as temperature, humidity and uv (see section 5.4). in seir and similar models, two metrics are available for this infection rate: r 0 , the basic reproductive number, and rt, the effective reproductive number. r 0 is defined as the expected number of secondary infectious cases generated by any single average infectious case in an entirely susceptible population. r 0 should be largely free from signals attributed to imposed factors that affect human behaviour. it is typically derived from the initial portion of the growth curve when the disease spreads in a population where everyone is susceptible, before control measures have been put in place (i.e., completely 'natural conditions' sensu shi et al. [50] ) or herd immunity had been attained. neher et al. [51] note that "r 0 is not a biological constant for a pathogen" (p. 1) but it is affected by factors such as the infectiousness of the virus, susceptibility of the hosts (e.g., due to age or an assortment of comorbidities), duration of the infectious period, density of susceptible people (also population density and the proportion of the population that is urbanised) or the contact rate with them (including aspects of mobility), and environmental influences (as shown in figure 2 ). these aspects are subject to localised idiosyncrasies across the globe and must be accounted for in regional or global analyses when calculating or comparing r 0 . one approach is to establish correlations, either over time or across space, between the infection rate at a given time and simultaneous metrics of environmental factors such as temperature, humidity and uv (see section 5.4). in seir and similar models, two metrics are available for this infection rate: r0, the basic reproductive number, and rt, the effective reproductive number. r0 is defined as the expected number of secondary infectious cases generated by any single average infectious case in an entirely susceptible population. r0 should be largely free from signals attributed to imposed factors that affect human behaviour. it is typically derived from the initial portion of the growth curve when the disease spreads in a population where everyone is susceptible, before control measures have been put in place (i.e., completely 'natural conditions' sensu shi et al. [50] ) or herd immunity had been attained. neher et al. [51] note that "r0 is not a biological constant for a pathogen" (p. 1) but it is affected by factors such as the infectiousness of the virus, susceptibility of the hosts (e.g., due to age or an assortment of comorbidities), duration of the infectious period, density of susceptible people (also population density and the proportion of the population that is urbanised) or the contact rate with them (including aspects of mobility), and environmental influences (as shown in figure 2 ). these aspects are subject to localised idiosyncrasies across the globe and must be accounted for in regional or global analyses when calculating or comparing r0. rt is a measure of observed disease transmissibility, defined as the average number of people a case infects at any time (t) once the epidemic is underway. rt incorporates changes in a society's behaviour (self-regulated responses and non-pharmaceutical interventions or npis [52] ) as the disease becomes widespread, and varies day to day. these effects are typically stronger than the environmental influences, and can easily mask them or generate spurious associations. it is not advised to base assessments of environmental effects on rt due to the 'noise' that the signal will contain, unless there is sufficient information that permits inclusion of the interventions as continuous, time-varying factors. for the compartment models, it is possible to derive the values of the key model parameters by model inversion, in near-real time, and from these, calculate r0. this needs at least one more observation than there are free parameters to be estimated. in practice, accurate estimates of r t is a measure of observed disease transmissibility, defined as the average number of people a case infects at any time (t) once the epidemic is underway. r t incorporates changes in a society's behaviour (self-regulated responses and non-pharmaceutical interventions or npis [52] ) as the disease becomes widespread, and varies day to day. these effects are typically stronger than the environmental influences, and can easily mask them or generate spurious associations. it is not advised to base assessments of environmental effects on r t due to the 'noise' that the signal will contain, unless there is sufficient information that permits inclusion of the interventions as continuous, time-varying factors. for the compartment models, it is possible to derive the values of the key model parameters by model inversion, in near-real time, and from these, calculate r 0 . this needs at least one more observation than there are free parameters to be estimated. in practice, accurate estimates of confidence limits require many more data points than parameters. the multiple observations can come from a single-population time series, but this would limit the degree to which changes over time can be resolved within the parameters themselves. if there are multiple time series from different populations, both temporal and spatial variation of the parameters can be obtained. phenomenological approaches typically use a variety of parametric regression models (see section 5.4). it is sometimes necessary to fit a piecewise model to accommodate the breakpoint that develops when country-specific npis are introduced. it is generally only possible to compare the parameters of the curves across locations (rather than within locations, over time) to determine whether there is a systematic pattern that relates to any environmental predictors. this is because fitting multi-parameter non-linear curves using data from only a part of the curve (in epidemics, usually just the initial part) is notoriously difficult and uncertain. if the effect of the environmental factors on the model parameters was known, they could be used to alter the curve parameters dynamically, and thus the projected outcomes; but the parameters typically have no intrinsic biological meaning. seasonality of prevalence is a common feature in most persistent and established or endemic respiratory infectious disease [53] [54] [55] [56] [57] [58] [59] [60] , as well as many other infectious diseases [61, 62] , in diseases (or endemic tolerated infections) of both humans and other animals. peaks incidence periods occur during the shoulder seasons or the winter, oscillating globally with the opposing boreal and austral climate. seasonally varying prevalence has a general latitudinal gradient and is accentuated in highly seasonal temperate and subtropical climates (with some rare exceptions) but is also observed in tropical regions [63] . seasonality is found in a wide range of viral respiratory diseases (vrds)-including influenza viruses, para-influenza virus (piv), human syncytial virus (rsv), rhinoviruses and human coronavirus strains (hcov) [55, 60] . for endemic viruses causing vrds in humans, seasonal peaks are usually quite predictable, but interannual variability in onset and duration of any season, and the virulence of respective seasonal strains, vary. it follows, therefore, that if vrd prevalence follows this climatological pattern, a mechanism(s) that connects and modulates the viral disease progression with seasonally varying climatological variables in individuals or populations must exist. this sensitivity must occur in at least one location of the seir model ( figure 2 ). in the case of novel viruses, the role of seasonality is more contentious, mainly because they have not existed for enough time for seasonality to be unambiguously established. the seasonal prevalence of pandemic strains of virus is often conflated with the so-called second wave, which may be coincidentally associated with the following winter season, suggesting that there is a climate-based modulating effect on its incidence [10, 64] . in the case of sars and mers, the attribution of resurgence to climatological drivers, as opposed to secondary circulation dynamics, remains unresolved [65, 66] . novel viruses are much less predictable than established viruses with respect to their persistence, re-emergence in the following years or seasons, and virulence in later outbreaks [64, 67] . until a novel virus becomes endemic and recycles (in its existing form or as mutated strains), its seasonal prevalence is difficult to assess [68] . the magnitude of the current sars-cov-2 pandemic is likely to result in an extended period of persistence [69] , and thus if seasonal prevalence exists, it should eventually be unambiguously apparent. in the generalised seir model shown in figure 2 , environmental modulation can primarily take place at two stages, namely susceptibility and exposure. environmental sensitivity insights can come from two basic sources. the first is observational data and laboratory studies and analyses of the environmental modulation on the sars-cov-2 virus biology and the incidence of the disease it causes (as in this review). second, we can examine data and information from published studies on respiratory viruses and vrds and related endemic and novel coronaviruses specifically (see [53, 55, 57, 59, 60, 67, 70] for general treatment of this topic). in this section, we examine three sets of hypothetical mechanisms which explain environmental modulation and seasonality of vrds other than covid-19: (i) physical environmental variable modulation, (iii) biological and host behavioural modulation, and (iii) viral molecular and biochemical modulation. physical environmental variable modulation hypotheses focus on the meteorological correlates of seasonality in the diseases [54, 58] and comprise the bulk of such studies. these all follow the basic tenet that selected environmental variables (such as temperature or humidity) vary in space and time with the progressing seasons, and if a mechanism that links them with a vrd can be demonstrated, this makes them a suitable candidate for explaining vrd seasonality. there is a lack of clarity in the literature regarding which definition of humidity is best applied as environmental moderator of respiratory viral epidemiology. studies employ relative humidity (rh), absolute humidity (ah), specific humidity (sh), vapour pressure or dew point (more in section 5 below). this renders comparisons and conclusions difficult to reach [7] . rh and sh have strong dependence on temperature, which further complicates studies that include both temperature and humidity as predictors. the postulated mechanisms are usually tested in laboratory studies which monitor the persistence of viable viruses in aerosol droplets and on surfaces [71] , perform experimental transmission studies in animal models [72] , or study the relationship between observed ambient or indoor environmental variability and infection rate, morbidity and mortality, with the assumption of causality (section 5). notably, results from temperate and tropical climate zones (or with ranging latitude) are often contradictory. this has led to a suggestion that different seasonality mechanisms are at play in different climate zones: humidity (aerosol droplet transmission) as the key driver in temperate regions, and precipitation driving behaviorally mediated contact transfections in the tropics [73] [74] [75] . the environmental determinants of virus transmission in aerosol liquid droplets have received substantial consideration. the premise is that, in winter, characterised by relatively lower humidity, pathogen-bearing aerosol droplets (pbads) are more persistent. pbads expelled by infected individuals often contain viruses or bacteria, in a mixture of mucus, saliva and dissolved salts, and can travel up 8 m from a simple sneeze [76] . upon leaving the airway with moisture saturation close to 100%, pbads are exposed to much drier air which results in evaporation. they can quickly lose up to 90% of their water mass and reduce in size. at an rh of 40-60%, the water loss greatly increases the salt concentration to levels that inactivate viruses. in contrast, for rh < 40%, the dissolved salts precipitate, resulting in a pbad with low salt concentration and a high number of infectious viruses [77] . pbads range in diameters 5-20 µm when the ambient rh is 30-60%, whereas below 30%, a pbad may immediately reduce its size below 0.5 µm, and become a droplet nucleus [78, 79] . thus, conditions of lower ambient rh result in the production of smaller, lighter (longer floating periods), and potentially more penetrative pbads, thereby elevating the exposure component of the seir model [80, 81] . the role of temperature in influencing the prevalence of vrds is more contested and complex. this is partly because temperature and ah together determine rh, which affects the rate of evaporation and thus pbad dynamics, as argued above [72, 82] ; and temperature could also have direct effects. several studies associate temperature with respiratory disease incidence, some by direct association [83, 84] and some focussed on the temperature changes (i.e., lowering temperatures rather than lower temperatures [85] ). temperature may also play a mediating role in other ways. the first set of hypotheses consider the direct effect of temperature on respiratory virus survival. there are very few such studies but they show that viruses in general are surprisingly tenacious, with survival periods of days at room temperature for sars-cov-1. effective inactivation occurs at temperatures of above 56 • c [86, 87] . another temperature-mediated mechanism with substantial literature involves the fomite viability of viruses [88, 89] , particularly in public spaces and hospitals, involving endemic coronaviruses and sars-cov-2 [90, 91] . some studies explore the role of temperature alone on specific surface types [88] , while others look at the combined role of temperature and humidity [90, 92] . respiratory viruses, including human coronaviruses, can remain viable as fomites on a range of surface types, indoors and in sheltered external environments, at room temperature and higher, for periods of hours to days and from days to weeks on refrigerated surfaces at 4 • c. persistence depends on both the surface type and the temperature and humidity range (see table 1 [91] for a recent summary). thus, the risk of infection from fomites (the exposure element of the seir model) increases as temperature decreases. the combination of temperature and humidity has been found important for fomite viability in the endemic human coronavirus hcov 229e (table 2 ). most studies aim to test sterilisation techniques and the efficacy of personal protective gear [91, [93] [94] [95] [96] . one hypothesis posits a predominance of surface contact transmission in the tropical climates, versus transmission through pbads in temperate climates [97] . a range of other physical environmental variables have been cited as moderators of respiratory viral epidemiology. they often co-vary with other causal variables. wind and wind speed are relatively neglected as physical environmental factors in infectious disease epidemiology. given that windy seasons occur in many climates zones, wind should not be discarded as a contributing variable [98] . for influenza, wind has been cited in some instances as a factor in transmission of infectious particles from remote locations, as promoting the extended local transmission of pbads [99, 100] , with a convincing account in one case of equine influenza [100] . barometric pressure has also been considered, for example in the case of respiratory syncytial virus, where it was found to have no statistically significant influence [101] . in other studies, it does have an influence, along with temperature [102] . guo et al. [103] found air pressure to be a predictor of the risk of influenza infection in children in guangzhou, china, with a differential effect by age. rainfall seasonality and disease incidence in general are well described [104] , but literature on the relationship between rainfall patterns and vrd epidemiology is restricted to tropical climates. most studies have considered rainfall either at a very local scale, or as part of a set of meteorological variables being tested. pica and bouvier [105] comprehensively review the literature on this rainfall and vrds, and conclude that for a range of respiratory viruses (primarily influenza and rsv), there are as many studies finding some association as there are studies finding no link. with attenuated intraseasonal temperature variation in the tropics, rainfall provides a key differentiator between seasons, possibly explaining the strong associations between rainfall and respiratory illness prevalence there. the mechanism of association is less clear. there is a suggestion that the tropical rainy season causes crowding, and thus increased exposure [106] , another suggesting that reduced sunlight is associated with pneumonia incidence [107] , and yet another citing diurnal temperature changes [108] . the improvement in air quality and reduction in allergen production following rainfall may be another mechanism [109] . solar ultraviolet radiation (solar uv) varies greatly with season everywhere and is thus an attractive candidate to explain seasonality of vrds. uv radiation in laboratory settings is a very effective means of deactivating viruses, and there are a plethora of studies of this effect on all kinds of pathogens (including coronaviruses sars-cov-1 and mers-cov), mainly targeting hygiene and outbreak management in public spaces and hospitals [110] [111] [112] . studies that consider the environmental effect of solar uv (a component of sunlight) without confounding effects of other variables are rare. sagripanti and lytle [113] state that, for influenza, "the correlation between low and high solar virucidal radiation and high and low disease prevalence, respectively, suggest that inactivation of viruses in the environment by solar uv radiation plays a role in the seasonal occurrence of influenza pandemics" but concede that there are a range of additional factors that need to be considered. despite uv being regarded by several authors as the "primary germicide in the environment", its independent effect as a seasonal driver of vrds remains uncertain (on this point, for influenza, see [114] ). a second set of hypotheses for explaining the seasonality of vrds consider behavioural and physiological responses to changing environmental variables such as temperature [54, 55, 60] , related mostly to the exposure but also the susceptibility component of the model in figure 2 . these include considering the consequences of confining people in sheltered and enclosed spaces during colder weather, with recirculating air and closer proximity to infected co-inhabitants, thus increasing the likelihood of exposure. they also include the idea that exposure to colder and drier air at the cellular level in the respiratory tract results in impaired physical or immune-system defences to infection, and hence increased susceptibility [60, 115] . large (<30 µm) and medium (<10 µm) inhaled pbads are normally captured in the upper nasal mucosa and upper respiratory tract, respectively, and are transported towards the mouth (and expelled) through a synchronised circular movement of cilia. the combination of the mucosal layer and cilia can effectively clear the particles [79] . however, low ambient rh has been demonstrated to reduce the effectiveness of both mucosal production and cilia action [60, 72] . a corroborating study demonstrates that dry air (low rh) impairs host defence against influenza infection in genetically engineered mice with human-like lung tissue, as well as slowing recovery [116] . the third set of hypotheses consider the biochemistry and molecular adaptation of the viral pathogens [55] . these take into account the temperature sensitivities of the various stages of the virus infection cycle, from binding to the host cell, replication of nucleic acids, the stability of secondary structures of viral proteins, and eventual ejection of the virus from the host cell [55] . given that there is a gradient of temperature within the respiratory tract, and that breathed air can greatly alter conditions in the upper respiratory tract, susceptibility can increase under cold conditions, especially to viruses which are adapted to be most efficiently infectious at temperatures slightly below normal body temperature [55, 115] . falling somewhere between the physical, physiological and biochemical hypotheses in explaining seasonality of respiratory viruses is the change in susceptibility with varying serological levels of vitamin d. vitamin d synthesis occurs when the skin is exposed to sunshine, which varies seasonally (confounded with uv, temperature and other variables). vitamin d has been suggested as an important form of defence against microbes, influenza and pneumonia in particular [117] [118] [119] [120] . shaman et al. [121] attempted to model this effect on influenza prevalence in the usa and concluded that seasonal variability in other factors such as humidity and even the school calendar were better at explaining their results. these considerations are incomplete, with a final abiotic aspect that must be included. air pollution refers to a wide range of harmful, primarily geogenic (naturally occurring) and anthropogenic particulate matter, chemicals or gasses that cause negative or dangerous physiological responses and effects in humans and biota. it is well known that poor air quality can have direct and indirect impacts on human health, and in particular on the susceptibility of humans to respiratory viral infections as well and a measurable effect on the severity and mortality rates [122] . gases such as nitrogen dioxide, ozone and especially particulates classified by size (pm10, pm2.5, and pm0.1) have different pathological mechanisms and effects but are all known to be associated with the increases in viral respiratory disease incidence, hospitalisation or attributed deaths, famously during the london fog of 1952 [123] and the 1918 spanish influenza pandemic [124] . clifford et al. [125] , for example, showed that pm10 inhalation exacerbates the response to influenza, and ye et al. [126] showed that 'haze' (a combination of air pollutants) was associated with the spread of respiratory syncytial virus in children. air pollution is also known to have a strong seasonality, driven by both seasonal economic production activity and also by ranging seasonal metrological conditions which can either concentrate and trap pollutants in surface air or conversely disperse pollutants and improve air quality [127] [128] [129] . therefore, it is a further consideration that seasonal variation in air quality and pollution is an additional factor for consideration as a contributor to the seasonality of respiratory viral infections that have been reported. it is most likely that each of these hypothesised mechanisms has some role, either in unison, or independently or that one mechanism dominates in particular conditions [60] . while the precise mechanism that explains the relationship between environmental factors and disease prevalence is important, particularly because it may reveal optimal management interventions (of transmission and for treatment), statistical attribution of a strong correlate may suffice for effective management [8] . evidence from the many studies on viruses not dissimilar from sars-cov-2 suggests that a seasonal and environmentally-mediated signal should be seen in the novel covid-19 epidemic. what do studies to date tell us? we comprehensively reviewed the preprint and peer-reviewed literature on the topic of environmental influences of sars-cov-2 transmission. we used the boolean search capability of google scholar to locate articles with the following keywords in the article title: "(covid-19 or sars-cov-2) and (pollution or humidity or temperature or uv or climate or weather or season or seasonality)". this returned 287 articles on 8 july 2020. on the same day, additional searches for these search terms were conducted in the title fields on pubmed and the title, abstract and subject fields on the who covid-19 literature database (https://search.bvsalud.org/global-literature-on-novel-coronavirus-2019-ncov/), returning 469 and 170 publications, respectively. all searches were constrained to the year 2020. we selected only those studies on infection rates or similar metrics, excluding studies based solely on mortality rates. the combined set, which contained many duplicates and triplicates due to the intersection of three sets of search results, was screened manually and papers suitable for inclusion in our review were retained. five reviews in preprint were excluded from our assessment, but we did verify that we included in our analysis all relevant papers cited in these reviews. since we a priori expected many preprint manuscripts, we did not embark on the review with the intention to be prisma compliant (as would be necessary for a meta-analysis and systematic reviews), and hence we did not count the number of duplicates and triplicates, the ineligible studies discarded, or the reasons why they were discarded. the result of our searches yielded 42 peer-reviewed publications and 80 preprint manuscripts (supplementary tables s1 and s2). the peer-reviewed publications were subject to normal review scrutiny, and form the main body of this section. we did not assess the outcomes of the preprint papers (i.e., they are not discussed in detail as part of section 5.5) in order to avoid erroneous conclusions based on unassessed data, results or interpretations; nor did we attempt to apply our own peer-review process. the peer-reviewed research conducted on the role of climatic variables in covid-19 transmission has been highly interdisciplinary, with authors spanning 25 broad academic backgrounds. the largest number of authors (27) currently work in disciplines of geography, earth and environmental sciences, which incorporate climate science. this is closely followed by the 26 authors working in public health, and 25 authors in disciplines of epidemiology, virology and disease control. a total of 40% of the authors are in fields directly relating to covid-19 and climate. there is, however, a notable spread of authors in more distal academic and medical fields. notably, the authorship of 18 papers included nobody with an explicitly medical background. of the multi-authored papers, only three were by researchers who all come from the same disciplinary background, and for two of these, the backgrounds were epidemiology and medical laboratories. collectively, the peer-reviewed studies provide only weak evidence that sars-cov-2 is more infectious under lower temperatures and lower levels of absolute humidity. similarly ambiguous relationships for air pollution, uv and wind are reported, with a smaller focus on these variables in the literature. there are considerable differences in the ways in which the relationships have been established, resulting from which co-varying variables were included or not; use of different metrics of viral transmission, and which statistical methods were applied. in many cases, insufficient information is provided on the methods and data used, making it impossible to replicate the analyses. this section is relevant because of the high dependence on spatial variance to provide information at this early stage of the pandemic. the geographical coverage of the literature on the environmental influences on sars-cov-2 is heavily weighted to the northern hemisphere. data from bolivia, ecuador, brazil and australia were included in only five studies, i.e., one-tenth of the total. most of the southern hemisphere studies are included in studies claiming to be near global in their sampling. only eight studies focus specifically on a country in the southern hemisphere, brazil [130] [131] [132] [133] [134] [135] [136] [137] , and none of them consider any african country. environmental variables considered in preprint and peer-reviewed publications as modulators of sars-cov-2 transmission rates include mean, minimum and/or maximum daily temperature, and diurnal temperature range; an undefined 'humidity' variable, relative humidity, specific humidity and absolute humidity; dew point temperature; rainfall; wind speed or wind power; air pressure; some metric of solar or uv radiation; and 'air quality' (supplementary tables s1 and s2 ). these choices are apparently strongly influenced by the literature on other viral respiratory diseases. which definition of 'humidity,' is selected is significant challenge for interpreting and comparing studies. humidity broadly refers to the amount of water vapour held by air (which effects on the viability of pathogens in exhaled aerosol droplets-see section 4). studies must account for the fact that atmospheric pressure and temperature modulate the amount of water that a volume of air is able to hold in a gaseous state. a relatively small amount of water vapour is able to saturate cold air, whereas more water vapour is required to bring warm air to saturation. the studies we reviewed that seek to establish whether humidity is a potential driver of covid-19 use absolute humidity, relative humidity or specific humidity. two studies use 'humidity' [138, 139] without qualifying whether it is relative, specific or absolute humidity. this ambiguous use of the term does not permit reproducibility or meta-analysis. absolute humidity is defined as the total amount of water vapour held by air, in units of g·m −3 . a temperature change will not necessarily change the moisture content; it simply changes the capacity of the volume of air to hold water. only if temperature drops to saturation point, will condensation occur and water vapour content (but not relative humidity) will drop. if temperature increases, water vapour content will only increase if a moisture source is available from where evaporation can take place, or if a moist air mass moves in to replace the drier one. relative humidity is the fraction of water vapour, expressed as a %, contained by air relative to the amount of water vapour required to result in saturation of air at a given temperature and pressure. specific humidity is the amount of water vapour per unit mass of dry air (g·g −1 ). the distinction between relative and absolute humidity matters less in situations when the seasonal thermal range is constrained to a narrow band, such as at some mid-latitude coastal locations and near the tropics. however, in space-for-time studies-such as are required for global syntheses of seasonality effects-the reliance on absolute humidity should allow the investigator to arrive at plausible conclusions about atmospheric water vapour's effect on viral transmissibility [140] [141] [142] . environmental data were obtained from various sources such as era interim [143] or local meteorological organisations, and maybe provided as daily data or aggregates on temporal scales from 10 days to months. some use 'seasonal climatologies', i.e., averaged long-term data. since symptoms first manifest 3 to 14 days after infection, analyses sometimes apply lags between the independent and dependent variables of up to 14 [40] or 21 days [41] . lags have been accommodated in the reviewed literature by applying moving average filters to the daily time series of environmental variables with a width of 7, 14 or 21 days [41] . another approach is to base the analysis on 10 day aggregates of environmental data [140] . it is uncertain how such discretised intervals can be aligned with case data that is typically daily, but yet contains various delays. some studies take the mean of the variable over the analytic time period; for example, jahangiri et al. [144] who ambiguously use either the mean temperature over the study period or over the year, or liu et al. [141] and sajadi et al. [12] who use the mean of the environmental variables over the period for which case incidence data were obtained. most studies, do not account for lag effects [138] , or if they do, fail to adequately explain how lags were accommodated [40] . which metric of sars-cov-2 transmission to use as dependent variable is critical in addressing the central question, "do environmental variables modulate the transmission of the virus?" we argue in section 3.3 that the basic reproductive number, r 0 , is the best parameter for this purpose since it excludes the effects spontaneous or imposed non-pharmacological control measures implemented to slow the spread of the disease, but which still incorporates the environmental influence of a particular place. the failure to adequately account for non-entrée influences is the achilles' heel of many of the studies reviewed. of the literature we assessed (supplementary tables s1 and s2) , only six studies base their assessment of the presence or magnitude of environmental influences on r 0 as the dependent variable [39, [145] [146] [147] [148] [149] . jebril [150] , luo et al. [151] , poirier et al. [142] , and wang et al. [152] used r t (see section 3.3) as the response variable. because r t is very context specific and sensitive to social factors and interventions, using this parameter to assess the presence and size of environmental influences will in most instances have a low signal: noise ratio. the usefulness of r t is that it demonstrates how effective npi measures are in controlling an epidemic, and provides information on how regulators must adapt these interventions over time, based on health and economic goals. the non-environmental 'noise' can be filtered out, but this requires a great deal of data regarding the nature of the specific interventions applied, movement patterns, precise knowledge about testing and reporting (which is not necessarily constant), and so forth. none of the r t based studies to date meet these preconditions, and are therefore not able to remove the non-climatic (social) influences from the rapidly fluctuating r t values. another approach that holds merit is to use the growth rate or doubling time estimated from the exponential increase in cases as dependent variable [148, [153] [154] [155] [156] [157] [158] [159] [160] . merow and urban [156] argue that these kinds of metric are robust even if the details of testing and reporting vary from place to place, as long as the detection probabilities at a place remain constant over the estimation period. this argument is equally valid for estimates of r 0 . another variation to this theme of estimating growth rate-related parameters as an indication of transmissibility is to take rates as time required to progress from the first reported case to 200 cases [161] , or to use the cumulative number of cases reached 28 days after the first reported case [162] . however, these approaches effectively fit a linear model to case vs. time data, which does not account for the accelerating rate of increase in number of cases. lolli et al. [163] use the daily icu case anomaly, but this of course entirely excludes all but the most severely ill patients and cannot be seen as being representative of disease transmissibility. other data-related considerations, particularly in relation to studies that use parameter estimates of the exponential relationship that daily new infections has with time, are that care must be taken to omit both (i) cases that result from the importation of infected individuals from the time series (i.e., new cases must be local transmissions only), and (ii) the case data obtained after the intervention period begins. requirement (i) can be affected by including only the portion of the time series after a certain minimum number of cases are present, as has been performed by caspi et al. [154] , merow and urban [156] , and notari [157] . requirement (ii) is met by ficetola and rubolini [155] , merow and urban [156] , notari [157] , and possibly for oliveiros et al. [158] , although it is uncertain how strictly this was implemented due to their statement that oliveiros et al. [158] "considered mainly the initial days of the time series" (p. 4). we will comment on the reproducibility of methods in section 6. requirement (ii) is implicit in the definition of r 0 , but the two requirements constrain the usable data to between 'not too early' and 'not too late'. the bulk of the studies in supplementary tables s1 and s2 used daily new or cumulative confirmed cases as response variables. this practice is not advised for largely the same reasons given for r t . such daily data are likely to carry too many other non-climatic signals to be generally used-unless, of course, analyses included a specific set of controls that would be difficult to extend to the global context. the studies in supplementary tables s1 and s2 employed the following statistical methods to evaluate relationships between environmental variables and the transmission rate of sars-cov-2: various linear, logistic, or exponential parametric models [39, 41, 50, 130, 134, 142, 146, 148, 151, 158, [164] [165] [166] [167] , sometimes with the inclusion of non-gaussian error structures as permitted by generalised linear models (glms) [141, 157, 162, 164, [168] [169] [170] [171] ; generalised additive models (gams) [41, 134, 167, 172, 173] ; distributed lag panel regression models [153] ; machine learning such as support vector machines and decision trees [147] ; local panel projection estimator within a country-level dynamic framework [174] ; loess smoothers/curves [142] ; bayesian methods [157] ; and pearson's, spearman's, and kendall's correlations [40, [138] [139] [140] 154, [175] [176] [177] . regression approaches allow functional relationships to be established between the driver (any of the environmental influences) and response variable (a metric of infection rate), allowing the magnitude of the environmental effect can be determined. robust implementation of a regression approach would include place as a random effect (i.e., as mixed models, also known as panel regressions; for example, [11, 153, 155, 178, 179] ). this allows the fact that the effect of the environment on viral transmission varies from place to place, for social and historical reasons. multiple regression allows the simultaneous evaluation of several predictor variables in terms of the influence they collectively or individually have on the outcome [39, 180, 181] . it is possible to establish which of the drivers, if any, has the greatest contribution to an effect seen in the outcome variable. for example, mollalo et al. [180] used multiple regression to evaluate the simultaneous contributions of environmental and socio-economic influences on usa county case counts. if parameterised properly, multiple regression can be used to rule out contributions of potentially confounding and multi-collinear variables. loess smoothers and correlation approaches, although useful for a qualitative assessment for the presence of environmental influences, cannot inform us about the relative importance of environmental modulators versus other location-specific or social influences. similar non-quantitative approaches that only hint at the presence of relationships include the simple visual mapping of the number of infections in relation to climate zones or latitudes [12, 150, [182] [183] [184] . these methods can at best raise an hypothesis that requires further testing. other approaches worth mentioning include the application of wavelet transforms [185] , multivariate analyses [130] , and ecological niche models [186, 187] . wavelet analysis, which requires a long time series, provides only a qualitative view of disease dynamics as modulated by weather or climate variables. ecological niche models are not suited for studies on covid-19 because disease dynamics are entirely different mechanistically from the principles that govern organisms and ecological systems (as reviewed by carlson et al. [188] ). multivariate methods are useful for examining environmental variable modulation of covid-19, since they provide many, if not all, of the benefits of multiple regressions, plus they have other features that confer flexibility and the ability to accommodate a range of data types. they are ideally suited for situations where there are many factors that might contribute simultaneously to the variation of one or many outcome variables. the application of a multivariate approach by auler et al. [130] uses data on the daily new confirmed cases (see critique above), and for this reason we do not consider the findings of this study further in our review. dynamic or mechanistic models (predominantly the compartment models of the seir family) are useful tools to explore how seasonality may impact on the evolution of the disease, and provide a way to discern the signature of seasonality in near real-time observational data. such an investigation recently reported on by baker et al. [189] concluded that under the high infection rates of covid-19, within the context of almost the entire population being susceptible at the onset of the disease, seasonality effects on the disease evolution will be limited initially. however it cannot be discounted at later stages, if for instance, the immunity gained by recovered patients is temporary, so that they become susceptible again in subsequent years or if herd immunity is not attained before managed abatement of the epidemic (as we are seeing in some countries experiencing resurgences). a similar study by neher et al. [51] came to similar conclusions. we will now discuss only the findings of those studies that have undergone peer review, have selected appropriate environmental data as influential variables, relied on suitable response variables (such as r 0 or parametric estimates) to estimate the local viral transmission rates in the absence of policy control measures, accounted for potential confounding influences, and applied appropriate statistical models. the only peer-reviewed paper that fulfils all of these criteria is that by yao et al. [39] , which undertakes an assessment of the effects that temperature, relative humidity, and uv radiation have on the r 0 . this study has a relatively narrow geographical focus: it includes 227 chinese cities. r 0 was calculated from data over the period 10 february to 9 march 2020. the authors assert that these data are for the "expected number of secondary cases generated by an initial infectious individual, in a completely susceptible population" [39] (p. 1). all daily environmental data were spatially matched as closely as possible to the cities they represent. given the large number of cities, each with its unique climate, this kind of study lends itself to a regression-type analysis if each of the daily observations per environmental variable are averaged over the study period duration before relating them to each locality's r 0 . this study did not find an influence due to any of the environmental variables studied on the rate of sars-cov-2 transmission. a weakness of the study was the failure to account in their multiple regression model for any of a large number of city-level confounding influences. a single published study does not provide robust support for the presence or absence of a climatic influence on sars-cov-2 transmission rates. the preprint studies [11, 147, 152, 153, 155, 156, 162, 174, 190] offer mixed statistical support (none, weak, or strong relationships) for the influence of environmental drivers. carlton et al. [153] show that that uv radiation affects covid-19 growth rates, but not temperature or humidity. merow and urban [156] offer comparable support for a uv radiation effect. according to ficetola and rubolini [155] and wan et al. [190] , covid-19 transmission is greatest at a temperature of 5 and 6.3 • c, respectively; the former authors further show that transmission peaks at a specific humidity,~4-6 g·m −3 (peaking implying optimum conditions above and below which transmission rates drop off). similarly, leung et al. [162] suggest support for the hypothesis that lower temperature and humidity enhance covid-19 transmission. similar responses are seen by lin et al. [165] and wilson [174] with regards to temperature, but they also suggest an interaction between temperature and relative humidity [165] and temperature and mobility [174] in terms of modulating infection rates. in contrast, gupta and gharehgozli [147] show that higher temperatures enhance the spread of the disease; they also show that viral transmission is enhanced under higher concentrations of pm2.5. this pandemic has rapidly mobilised scientists from diverse disciplines in a possibly unprecedented way. scientists have helpfully offered insights and analytical methods based on their own disciplines they did so efficiently and swiftly, particularly in those countries most heavily affected by the pandemic early on. the rush to contribute knowledge about the future spread of covid-19 resulted in a flood of papers appearing on preprint servers [191] , which will in due course be peer reviewed and some will be published. the pressure to speed up the peer-review process, in order to address the urgent challenge, may result in a compromise in the quality of both the review process and the science that is thereby published. in our screening process in section 5, we scrutinised 29 peer-reviewed publications and 23 preprint articles. of these, we found one published and potentially four preprint studies that offer credible insight into the climate-related sars-cov-2 and covid-19 dynamics and epidemiology with a reasonable degree of confidence and rigour. the general prevalence of climatologically-coupled seasonal signals and environmental variable modulation seen in the majority of other viral respiratory diseases creates the expectation for a similar effect on sars-cov-2 and in covid-19 epidemiology. however, this virus and disease have only been spreading for 8 months. observational evidence available to date has not yet been analysed sufficiently thoroughly to show that climate-related modulation is indeed a significant factor. the studies reviewed in section 5 have aimed to find signs for such a signal, but a variety of methodological problems render a definitive conclusion premature. the currently available time series do not capture a full annual cycle at any one location, or globally. the first studies appeared in late january on preprint servers (the majority of these are yet to be formally published as of mid-july 2020). as such, the initial reports looked for spatial variation in infectivity within a region and attempt to explain it in terms of associated variability in temperature, humidity or other environmental factors among these locations. later studies could have benefitted from the larger datasets and a wider range of variation in the environmental drivers, resulting from the global spread, but became increasingly confounded by co-varying differences among the countries' socio-economic conditions and pandemic responses. to date, the 'global' messages coming from the current body of covid-19 research in general, and in respect to the environmental drivers of the disease in particular, do not equitably address the specific dynamics and considerations pertaining to the 'global south'. this is in part likely due to the slightly later arrival of the disease in the southern hemisphere. thus, fewer southern hemisphere countries have suffered outbreaks of the same scale and severity (at the stage of assembling this manuscript) as the epidemics in the far east, europe and the united states. at the time of writing, the situation in some south american countries (such as brazil and peru) was deteriorating quickly. there is also a technical challenge in countries with relatively lower medical health research capacity, such as those in africa [192] . the upshot is a circumstantially driven bias in the current literature which needs to be corrected, for several reasons. neglecting the hemispheric disparities in knowledge regarding the role of environmental variables on sars-cov-2 and the modulation of the covid-19 epidemic influences the discussion on the attribution of the reductions in cases. northern countries are likely to move past peak daily infections coincidentally with the height of summer. it also neglects the urgent consideration of countries which are moving into winter. importantly, many of the countries in the global south have already-stressed healthcare systems, and accurate modelling is critical in determining policy interventions for control measures to protect the lives of some of the world's most vulnerable people. the collective global experience can provide a shortcut to knowledge and information regarding the role of environmental variables on sars-cov-2 biology and modulation of covid-19 epidemiology and seasonality, applicable anywhere, by exploiting the latitudinal phasing of seasons to conduct research in all climates zones simultaneously. this leads us to call for global collaboration on this topic. much of the work we reviewed failed to carefully consider the implications of the choice of available metrics for viral transmission. we deem r 0 to be best suited for the purpose of finding environmental sensitivity and seasonal climatic signals; some parametric estimates from regression models can also work, provided that care is taken to constrain the cases to those that result from local transmissions up to the time when npis come into play. r 0 is closely aligned with the sir-seir model family, and can be derived from the inversion of time series of case rate data using these models (see below). due to the effects of the incubation period, it may be important to use daily data (rather than data averaged over a several days) and a suitable lag period for both environmental and test-result data incorporated in the analysis. in the case of a highly infectious disease such as covid-19, manifesting in a densely populated location, the effect of daily weather variations on transmission mechanisms is likely to be overwhelmed by the sheer magnitude of exposure. it may be that environmental modulation is still an important factor in these circumstances, but may reflect in indoor environments rather than outdoor ambient conditions [193] . once the disease spread begins to approach an equilibrium (r t~1 ), the environmental effect may become more apparent. to date, studies that attempted to discern the effects of climate by comparing infection rates across regions with different climates have been compromised by the heterogeneities that exist across locations and times in terms of control measures applied [194] , and social, economic and cultural conditions that affect the practise of social distancing. most studies have omitted variables such as poverty, population size and demographics (particularly age frequencies of the populace), the density of the population and how much high-resolution clustering is present (such as in the informal settlements in many countries of the south), the degree of urbanisation, access to healthcare, mobility and migration, various types of comorbidities (e.g., tb, hiv, malnourishment), the effect of the bacillus calmette-guérin(bcg) vaccine [195] , and a plethora of additional influences which are still not well understood with regards to how they influence the unfolding of covid-19 across the globe. simple graphing of case numbers across time in relation to some of the potentially influential drivers (as for example permitted by the our world in data coronavirus pandemic data explorer) will help reveal which of the additional variables to admit into the analysis. an important obstacle to finding the seasonal signal in the global covid-19 data is to find a way to deal with the hemispheric disparity (gradient away from the equator) in out-of-phase climatic signals. comparing the evolution of covid-19 for northern hemisphere countries moving from winter to summer to its evolution in southern hemisphere countries moving from summer to winter provides a valuable opportunity to discern the signature of seasonality. however, such a comparison will remain compromised by short time series and can only fully fulfil its potential once both hemispheres have experienced a full annual seasonal cycle. we have concluded that due to high values of r 0 exhibited by sars-cov-2, seasonal climate modulation should not be relied on to significantly dampen the infection rate even in the midst of the northern hemisphere approaching summer. should the disease persist several years into the future, however, under the condition of an increasing fraction of the population of a given region having immunity, it is likely that the covid-19 will exhibit an increasingly clear seasonal cycle as evident in similar endemic human coronaviruses. such insights will only be apparent after the main pandemic surge in 2020. we suggest some avenues for progress in addressing the environmental sensitivity of the disease. in addition to regression and correlative empirical approaches (section 5.4), non-linear methods can also be applied. these may include the use of extended kalman filters and the inversion of compartment models. extended kalman filters are commonly used in data assimilation to infer parameters from high-dimensional input data sets. recently, pei et al. [196] applied an ensemble-adjusted kalman filter to infer the differential spatial distribution of covid-19 infection rates from empirical data collected across different counties in the usa, followed by their application in a seir model. it may be feasible to apply this technique to estimate the relative roles of non-pharmaceutical control measures and seasonality in determining the infection rate. inverse modelling, particularly using seir-type models, can infer infection rates from case and testing data, as demonstrated for the hubai province in china [46] . making use of large ensembles that ingest data from many locations and systematically explore various combinations of the forcings can potentially explore the relative sensitivities of infection rates to npi control measures and seasonality. we recommend the use of regression-type statistical analyses than can be adapted to accommodate many simultaneous driving variables, including both environmental and non-environmental factors, thereby removing confounding influences. these models also readily accept non-gaussian error terms and can account for autocorrelation in time series. lags between exposure and when an individual is confirmed as infected can be accommodated by distributed lag non-linear models [197, 198] . these techniques rely on generalised additive models (gams) for the flexible estimation of smooth responses and parametric terms. the recognition that disease dynamics may differ between locations for a multitude of reasons requires that 'location' be specified as random effect (notable examples involving covid-19 include carlton et al. [153] and wilson [174] ). such approaches can be accommodated by longitudinal models (called panel regressions by economists) (sensu gardiner et al. [199] ), which regress the dependant variable (plus covariates and constraints) as a function of time. care should be given to estimations of uncertainties around model predictions -such estimates of uncertainties are permitted by markov chain monte carlo (mcmc) approaches [42] . knowing the uncertainties is necessary in assessing projections from competing models in the public policy space. finally, multivariate approaches, such as redundancy analysis (rda) or constrained correspondence analysis (cca), will also accept a creative assignment of a host of response and influential variables simultaneously, and can be employed when research is faced with many potentially contributing factors, each of which might explain a portion of the overall variability. we noted a lamentable deficiency in the application of reproducible research practices in many of the publications we reviewed. clear, precise reporting of data sources and quality, data screening practices, listings of the ancillary data sources used, a detailed account of the data processing and statistical procedures and software used, and the exact reporting of all relevant diagnostic and supporting statistics, tables and figures is essential, particularly in this global emergency, where published data and information are used operationally, and where robust guidance is most likely to emerge from meta-analyses of many studies. lives, livelihoods, economies, and the public trust in science depend on rigour and reproducibility. it is thus incumbent upon global research organisations and agencies such as the world health organisation (who) and the world meteorological organisation (wmo) to provide leadership and guidance and to define best-practice protocols for the analysis of data and production of information. to this end, the who has produced a document entitled "a coordinated global research roadmap: 2019 novel coronavirus" [200] . its scope is broad, and thus does not specifically address some of the issues raised in our review. the authors are aware [201] that at the time of writing, the wmo has agreed to set up a task team which will focus on the environmental aspects of the covid-19 pandemic. datasets capturing even the first full seasonal cycle of covid-19 incidence in one locality, region or globally are not yet available and it is not possible at this stage to conclude that a definitive and unequivocal signal of environmental modulation is apparent from the reviewed literature. however, there is some evidence that environmental drivers played a role in transmission in some regions and at some (early) stages of the pandemic. under other circumstances, longer and denser datasets would be a minimum requirement to support a thorough statistical treatment to explore evidence of environmental modulation of the covid-19 pandemic and epidemiological dynamics. pressure for rapid answers and information has prompted impulsive and dubious forays into signal-finding missions, such as those that dominate the current body of literature that had accumulated to date (15 july 2020). analyses based on space-for-time substitutions have been inconclusive, primarily due to lack of care taken to account for the effects of strong confounding variables, such as socio-economic influences and effects of npis, which exist between jurisdictions. in terms of the outcomes of the published work, most studies are insensitive to the idiosyncratic conditions unique to many southern hemisphere countries, rendering it challenging to transfer findings from north to south. rigorous hypotheses, interrogation of assumptions, and careful selection and development of analytical approaches and statistical models are required to examine environmental signals in complex covid-19 incidence datasets, especially prior to longer and denser time series data being available. in the interim, there is merit in comparisons of signals among contrasting locations at different scales, and with due consideration paid to the implementation of npis and other sources of 'noise'. this outcome does not discount the role of environmental drivers in modulating the incidence or seasonality of person-to-person transfection mechanisms, or of the morbidity, severity and mortality associated with covid-19 infections. however, these may become unequivocally discernable only at later stages of the pandemic in 2020 or 2021, and globally coordinated efforts to test this robustly are essential. supplementary materials: the following are available online at http://www.mdpi.com/1660-4601/17/16/5634/s1, figure s1 : discipline backgrounds of authors whose publications were included in this review. table s1: studies that have aimed to establish links between sars-cov-2 infections and environmental variables, notably temperature and humidity. all studies have in common a finding that the transmission of the virus is enhanced under colder, dryer conditions. table s2: studies that have aimed to establish links between sars-cov-2 infections and environmental variables, notably temperature and humidity. all studies have in common a finding that the transmission of the virus is enhanced under colder, dryer conditions. author contributions: the conceptualisation of this study was by a.j.s.; he also performed the data extraction and wrote section 5 "critical assessment of studies of covid-19 climate susceptibility" and section 6 "discussion". data collection was undertaken by n.a.s., j.m.f. and a.j.s.; j.m.f. contributed section 2 "why the southern hemisphere is different". f.a.e. provided editorial input as required, and contributed his thinking around inverse modelling and some of the climatological considerations. r.j.s. provided the text under section 3 "monitoring and modelling the spread of covid-19", and also assisted with thorough language editing of the final document. n.s. added section 1 "introduction" and section 4 "implications for covid-19 of environmental sensitivity in other viral respiratory diseases", with g.d. contributing towards the latter section. funding for the covid-19 environmental reference group (cerg) endeavour was enabled by n.a.s. and he also provided overall project management for the group's efforts. all authors have read and agreed to the published version of the manuscript. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors, but the time of some of the authors of this paper was attributable to funding from the national research foundation and our respective home institutions. a novel coronavirus outbreak of global health concern the socio-economic implications of the coronavirus pandemic (covid-19): a review covid-19: how a virus is turning the world upside down the impact of covid-19 (coronavirus) on global poverty: why sub-saharan africa might be the region hardest hit covid-19: how doctors and healthcare systems are tackling coronavirus worldwide absolute humidity modulates influenza survival, transmission, and seasonality absolute humidity and the seasonal onset of influenza in the continental united states beware of the second wave of covid-19 global seasonal occurrence of middle east respiratory syndrome coronavirus (mers-cov) infection causal empirical estimates suggest covid-19 transmission rates are highly seasonal temperature, humidity, and latitude analysis to estimate potential spread and seasonality of coronavirus disease 2019 (covid-19) analysing health care systems performance: the story behind the statistics influenza seasonality in the tropics and subtropics-when to vaccinate? healthcare reform and poverty in latin america shortage of healthcare workers in developing countries-africa health-system reform and universal health coverage in latin america informal m-health: how are young people using mobile phones to bridge healthcare gaps in sub-saharan africa? is africa prepared for tackling the covid-19 (sars-cov-2) epidemic. lessons from past outbreaks, ongoing pan-african public health efforts, and implications for the future preparedness and vulnerability of african countries against importations of covid-19: a modelling study the covid-19 epidemic potential association between covid-19 mortality and health-care resource availability spatial and temporal distribution of infectious disease epidemics, disasters and other potential public health emergencies in the world health organisation africa region covid-19 in latin america: the implications of the first confirmed case in brazil xpert ® mtb/rif assay for pulmonary tuberculosis and rifampicin resistance in adults hiv prevalence among tuberculosis patients in sub-saharan africa: a systematic review and meta-analysis effect of routine isoniazid preventive therapy on tuberculosis incidence among hiv-infected men in south africa tuberculosis in sub-saharan africa: opportunities, challenges, and change in the era of antiretroviral treatment burden of hiv-associated histoplasmosis compared with tuberculosis in latin america: a modelling study covid-19 in hiv investigators covid-19 in patients with hiv: clinical case series maintaining hiv care during the covid-19 pandemic impact of covid-19 on tuberculosis control in china covid-19: getting ahead of the epidemic curve by early implementation of social distancing viral dynamics in mild and severe cases of covid-19 report from the american society for microbiology covid-19 international summit delivery of infection from asymptomatic carriers of covid-19 in a familial cluster targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals covid-19: a need for real-time monitoring of weekly excess deaths no association of covid-19 transmission with temperature or uv radiation in chinese cities impact of weather on covid-19 pandemic in turkey association between ambient temperature and covid-19 infection in 122 cities from china modeling infectious disease dynamics in the complex landscape of global health rt covid-19 renormalization group approach to pandemics: the covid-19 case. front. phys. 2020, 8 real-time forecasts of the covid-19 epidemic in china from data-based analysis, modelling and forecasting of the covid-19 outbreak covid-19 outbreak prediction with machine learning covid-19 pandemic prediction for hungary an agent-based model to evaluate the covid-19 transmission risks in facilities the impact of temperature and absolute humidity on the coronavirus disease 2019 (covid-19) outbreak-evidence from china potential impact of seasonal forcing on a sars-cov-2 pandemic estimating the number of infections and the impact of nonpharmaceutical interventions on covid-19 in 11 european countries influenza seasonality: underlying causes and modeling theories are meteorological parameters associated with acute respiratory tract infections? seasonality and selective trends in viral acute respiratory tract infections ecology, evolution and classification of bat coronaviruses in the aftermath of sars human coronavirus circulation in the united states association between viral seasonality and meteorological factors sick time seasonality of respiratory viral infections the calendar of epidemics: seasonal cycles of infectious diseases seasonality of viral infections: mechanisms and unknowns influenza in tropical regions reviewing the history of pandemic influenza: understanding patterns of emergence and transmission drivers of mers-cov transmission: what do we know? severe acute respiratory syndrome (sars): a year in review seasonality of infectious diseases and severe acute respiratory syndrome-what we don't know can hurt us projecting the transmission dynamics of sars-cov-2 through the postpandemic period part 1: the future of the covid-19 pandemic the cidrap viewpoint working group the effects of temperature and relative humidity on the viability of the sars coronavirus aerosol transmission of influenza a virus: a review of new studies influenza virus transmission is dependent on relative humidity and temperature transmission of influenza virus in temperate zones is predominantly by aerosol, in the tropics by contact: a hypothesis environmental predictors of seasonal influenza epidemics across temperate and tropical climates dynamic response of airborne infections to climate change: predictions for varicella relationship between humidity and influenza a viability in droplets and implications for influenza's seasonality characterization of infectious aerosols in health care facilities: an aid to effective engineering controls and preventive strategies who guidelines approved by the guidelines review committee. in natural ventilation for infection control in health-care settings evaporation and dispersion of respiratory droplets from coughing recognition of aerosol transmission of infectious agents: a commentary roles of humidity and temperature in shaping influenza seasonality using google trends and ambient temperature to predict seasonal influenza outbreaks excess mortality related to seasonal influenza and extreme temperatures in denmark decline in temperature and humidity increases the occurrence of influenza in cold climate stability and inactivation of sars coronavirus testing thermal resistance of viruses survival of influenza viruses on environmental surfaces significance of fomites in the spread of respiratory and enteric viral disease effects of air temperature and relative humidity on coronavirus survival on surfaces persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents role of absolute humidity in the inactivation of influenza viruses on stainless steel surfaces at elevated temperatures human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies survival of human coronaviruses 229e and oc43 in suspension and after drying onsurfaces: a possible source of hospital-acquired infections transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination how long do nosocomial pathogens persist on inanimate surfaces? a systematic review high temperature (30 • c) blocks aerosol but not contact transmission of influenza virus wind: a neglected factor in the spread of infectious diseases effects of absolute humidity, relative humidity, temperature, and wind speed on influenza the influence of meteorology on the spread of influenza: survival analysis of an equine influenza (a/h3n8) outbreak the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus meteorologic conditions and respiratory syncytial virus activity the effects of meteorological factors on influenza among children in guangzhou global precipitation: means, variations and trends during the satellite era (1979-2014) environmental factors affecting the transmission of respiratory viruses rainfall, household crowding, and acute respiratory infections in the tropics sunshine, rainfall, humidity and child pneumonia in the tropics: time-series analyses seasonal trends of viral respiratory tract infections in the tropics severe outbreaks of respiratory syndromes following autumn rainfall in khuzestan stability of sars coronavirus in human specimens and environment and its sensitivity to heating and uv irradiation inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov efficacy of an automated multiple emitter whole-room ultraviolet-c disinfection system against coronaviruses mhv and mers-cov inactivation of influenza virus by solar radiation inactivation of influenza a viruses in the environment and modes of transmission: a critical review an explanation for the seasonality of acute upper respiratory tract viral infections low ambient humidity impairs barrier function and innate resistance against influenza infection epidemic influenza and vitamin d toll-like receptor triggering of a vitamin d-mediated human antimicrobial response influenza, solar radiation and vitamin d vitamin d for treatment and prevention of infectious diseases: a systematic review of randomized controlled trials shortcomings of vitamin d-based model simulations of seasonal influenza air pollution and respiratory viral infection, inhalation. toxicology air pollution and daily mortality: a review and meta analysis pollution, infectious disease, and mortality: evidence from the 1918 spanish influenza pandemic geogenic pm10 exposure exacerbates responses to influenza infection haze is a risk factor contributing to the rapid spread of respiratory syncytial virus in children seasonal analyses of air pollution and mortality in 100 us cities dispersion of atmospheric air pollution in summer and winter season acute health impacts of the southeast asian transboundary haze problem-a review evidence that high temperatures and intermediate relative humidity might favor the spread of covid-19 in tropical climate: a case study for the most affected brazilian cities correlation between meteorological factors and covid-19 infection in the belem metropolitan region factors associated with the incidence and mortality from covid-19 in the autonomous communities of spain correlation between weather, population size and covid-19 pandemic: a study of brazilian capitals temperature significantly changes covid-19 transmission in (sub) tropical cities of brazil meteorological variables associations and the occurrence of covid-19 in the city of são paulo, brazil. revista ibero-americana de ciências ambientias 2020 gaussian approach for probability and correlation between the number of covid-19 cases and the air pollution in lima the role of climate during the covid-19 epidemic in new south wales correlation between climate indicators and covid-19 pandemic correlation between weather and covid-19 pandemic in jakarta effect of weather on covid-19 spread in the us: a prediction model for india in 2020 impact of meteorological factors on the covid-19 transmission: a multi-city study in china the role of environmental factors on transmission rates of the covid-19 outbreak: an initial assessment in two spatial scales the era-interim reanalysis: configuration and performance of the data assimilation system the sensitivity and specificity analyses of ambient temperature and population size on the transmission rate of the novel coronavirus (covid-19) in different provinces in iran non-linear modulation of covid-19 transmission by climate conditions transmissibility of covid-19 and its association with temperature and humidity developing a machine learning framework to determine the spread of covid-19. ssrn electron no evidence for temperature-dependence of the covid-19 epidemic no evidence for temperature-dependence of the covid-19 epidemic predict the transmission of covid-19 under the effect of air temperature and relative humidity over the year in baghdad the role of absolute humidity on transmission rates of the covid-19 outbreak temperature significant change covid-19 transmission in 429 cities ultraviolet radiation decreases covid-19 growth rates: global causal estimates and seasonal implications climate effect on covid-19 spread rate: an online surveillance tool climate affects global patterns of covid-19 early outbreak dynamics seasonality and uncertainty in covid-19 growth rates temperature dependence of covid-19 transmission. arxiv role of temperature and humidity in the modulation of the doubling time of covid-19 cases high temperature has no impact on the reproduction number and new cases of covid-19 in the association of uv with rates of covid-19 transmission and deaths in mexico: the possible mediating role of vitamin d high temperature slows coronavirus disease 2019 transmission rate: a within and among country analysis predictors of covid-19 incidence, mortality, and epidemic growth rate at the country level impact of meteorology and air pollution on covid-19 pandemic transmission in lombardy region roles of meteorological conditions in covid-19 transmission on a worldwide scale containing the spread of coronavirus disease 2019 (covid-19): meteorological factors and control strategies a retrospective analysis of influence of environmental/air temperature and relative humidity on sars-cov-2 outbreak effects of temperature and humidity on the daily new cases and new deaths of covid-19 in 166 countries preliminary evidence that higher temperatures are associated with lower incidence of covid-19, for cases reported globally up to 29th is temperature reducing the transmission of covid-19? high temperature and high humidity reduce the transmission of covid-19 possible environmental effects on the spread of covid-19 in china covid-19 transmission in mainland china is associated with temperature and humidity: a time-series analysis effect of temperature on the infectivity of covid-19 social distancing, and the spread of covid-19 investigation of effective climatology parameters on covid-19 outbreak in iran do latitude and ozone concentration predict covid-2019 cases in 34 countries? ssrn electron statistical investigation of relationship between spread of coronavirus disease (covid-19) and environmental factors based on study of four mostly affected places of china and five mostly affected places of italy. arxiv a spatio-temporal analysis for exploring the effect of temperature on covid-19 early evolution in spain association between climate variables and global transmission of sars-cov-2 gis-based spatial modelling of covid-19 incidence rate in the continental united states development of an assessment method for investigating the impact of climate and urban parameters in confirmed cases of covid-19: a new challenge in sustainable development does the pathogenesis of sars-cov-2 virus decrease at high-altitude? will coronavirus pandemic diminish by summer? ssrn electron on the global trends and spread of the covid-19 outbreak: preliminary assessment of the potential relation between location-specific temperature and uv index nexus between covid-19, temperature and exchange rate in wuhan city: new findings from partial and multiple wavelet coherence spread of sars-cov-2 coronavirus likely to be constrained by climate a global-scale ecological niche model to predict sars-cov-2 coronavirus infection rate species distribution models are inappropriate for covid-19 susceptible supply limits the role of climate in the early sars-cov-2 pandemic early transmission of covid-19 has an optimal temperature but late transmission decreases in warm climate how swamped preprint servers are blocking bad coronavirus research research capacity building-obligations for global health partners airborne transmission of sars-cov-2: the world should face the reality covid-19 health system response monitor barillas-mury, c. bcg vaccine protection from severe coronavirus disease 2019 (covid-19) differential effects of intervention timing on covid-19 spread in the united states mortality risk attributable to high and low ambient temperature: a multicountry observational study ambient particulate air pollution and daily mortality in 652 cities fixed effects, random effects and gee: what are the differences? novel coronavirus who/wmo climate and health joint office this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank the south african department of science and innovation for encouraging us to proceed with this work, and other members of the covid-19 environmental reference group (cerg) for providing valuable editorial input and discussion around the development of this review paper. the authors declare no conflict of interest. key: cord-319116-2ts6zpdb authors: ruggiero, emanuela; richter, sara n title: g-quadruplexes and g-quadruplex ligands: targets and tools in antiviral therapy date: 2018-04-20 journal: nucleic acids res doi: 10.1093/nar/gky187 sha: doc_id: 319116 cord_uid: 2ts6zpdb g-quadruplexes (g4s) are non-canonical nucleic acids secondary structures that form within guanine-rich strands of regulatory genomic regions. g4s have been extensively described in the human genome, especially in telomeres and oncogene promoters; in recent years the presence of g4s in viruses has attracted increasing interest. indeed, g4s have been reported in several viruses, including those involved in recent epidemics, such as the zika and ebola viruses. viral g4s are usually located in regulatory regions of the genome and implicated in the control of key viral processes; in some cases, they have been involved also in viral latency. in this context, g4 ligands have been developed and tested both as tools to study the complexity of g4-mediated mechanisms in the viral life cycle, and as therapeutic agents. in general, g4 ligands showed promising antiviral activity, with g4-mediated mechanisms of action both at the genome and transcript level. this review aims to provide an updated close-up of the literature on g4s in viruses. the current state of the art of g4 ligands in antiviral research is also reported, with particular focus on the structural and physicochemical requirements for optimal biological activity. the achievements and the to-dos in the field are discussed. g-quadruplexes (g4s) are nucleic acids secondary structures that can form within dna (1) or rna (2) guanine (g)-rich strands, when two or more g-tetrads stack on top of each other and coordinate monovalent cations, such as k + and na + . each tetrad is composed of four g residues that are linked by the sugar-phosphate backbone and connected through hoogsteen-type hydrogen bonds. g4s are highly polymorphic structures whose topology can be influenced by variations in strand stoichiometry and polarity, as well as by the nature and length of loops and their location in the sequence. g4s can fold intramolecularly from a single g-rich strand, or intermolecularly through dimerization or tetramerization of separate filaments: research of biologically relevant g4s has mainly focused on monomolecular g4s (3, 4) ; however, intermolecular g4s are gaining increasing attention (5) (6) (7) . strands orientation defines the parallel, antiparallel or mixed topology of g4s, which is directly correlated to the conformational state, anti or syn, of the glycosidic bond between the g base and the sugar (1). the anti conformation characterizes a parallel folding, while antiparallel g4s are found to adopt both syn and anti orientations (8) (figure 1 ). while rna g4s are mostly locked in a parallel conformation due to the 2 -hydroxyl group in the sugar which exclusively allows the anti orientation (2), dna g4s are in principle characterized by higher topological diversity, even though the majority of dna g4s examined so far adopt the parallel topology. computational analysis using different algorithms (9,10) indicated that 300 000 and up to around 3 000 000 potential g4-forming sequences may form in the human genome, correlated with specific gene functions (11) . these data have been corroborated by 'g4-seq' high-throughput sequencing method, which identified about 700 000 g4s (12) . however, mapping of g4s in chromatin by g4 chip-sequencing with an anti-g4 antibody (13) or footprinting (14) retrieved only about 10 000 g4s in highly transcribed regulatory nucleosome-depleted chromatin regions. these data indicate that g4s are mostly suppressed in chromatin and that, in turn, they may influence the occupancy and positioning of nucleosomes. in general, g4 sequences are non-randomly distributed but mainly clustered in pivotal genomic regions, namely telomeres, gene promoters and dna replication origins (15) . moreover, putative g4-forming sequences have been found in coding and non-coding regions of the human transcriptome, i.e. open reading frames and untranslated regions (utrs), and in the telomeric repeat-containing rna (2) .this evidence suggests that g4s are likely involved in the regulation of different biological pathways such as replication, transcription, translation and genome instability. in the past years, the resolution of g4 structures (16) (17) (18) and the employment of novel visualization approaches (19) (20) (21) helped researchers to validate the previous computational predictions, disclosing new aspects of the multifaceted g4s world, e.g. the effective occurrence of g4s within patient-derived cancer tissues (22) or the key role in the pathogenesis of two incurable neurodegenerative diseases, amyotrophic lateral sclerosis and frontotemporal dementia (23) . indeed, the presence of g4s in the human genome and their potential in diseases modulation have been extensively investigated, resulting in many good and exhaustive reviews focused on g4 structures (1, 8, 24, 25) and their biological role, particularly in telomeres (26) (27) (28) (29) and oncogene promoters (30) (31) (32) (33) (34) (35) . besides humans, putative g4-forming sequences have been found in other mammalian genomes (36) , yeasts (37), protozoa (38) , bacteria (39, 40) and viruses, therefore implicating g4s in many human infectious diseases. one review has been published in 2015 on the possible role of g4s in the antigenic variation systems of bacteria and protozoa and silencing of two viruses (41) . the possible role of g4s in viruses and the use of g4-forming oligonucleotides as antiviral agents have been discussed in 2014 (42) . the virus recognizes and binds the host cell surface receptors (step 1) to enter the cell (step 2). after penetration, the viral genome is uncoated (step 3) and its dna or rna nature determines where and how the genome is replicated (step 4): most dna viruses replicate in the cell nucleus, while the majority of rna viruses replicate in the cytoplasm of infected cells. after viral mrna production, viral proteins are expressed in the cytoplasm (steps 5-6). the newly synthesized viral genomes and proteins are then assembled into new virions (step 7), which are released outside the cell (step 8). since the number of reports describing the presence of g4s in virus genomes has boomed in the past 2 years and treatment with several g4 ligands has shown potentially interesting therapeutic activity, we here aim at presenting, organizing and discussing an up-to-date close-up of the literature on g4s in viruses and the classes of molecules that have shown antiviral activity by viral g4 targeting. in particular, we first focus on the presence and proposed function of g4s in virus genomes. next, we present the classes of g4 ligands that have reported successful antiviral activity, with special emphasis on the structural and physicochemical properties that characterize the viral g4/g4 ligand interaction. a general simplified virus life cycle is schematically depicted in figure 2 ; a summary of the viruses in which g4s have been reported and of the corresponding g4s is shown in figure 3 . since the use of g4-forming oligonucleotides as antiviral agents has been more recently addressed by musumeci et al. (43, 44) , this topic has not been considered in the present review. the human immunodeficiency virus (hiv) is the etiological agent of the acquired immune deficiency syndrome (aids), which to date affects more than 35 million people worldwide. albeit the current anti-retroviral therapy keeps the disease progression under control, people still die from hiv-related causes; thereby it is necessary to find alternative and effective antiviral targets. the hiv belongs to the retroviridae family; the single-stranded rna genome is processed by the viral retrotranscriptase and the newly formed double-stranded dna is integrated into the host cell chromosomes to form the proviral genome, from which viral mrnas and new genomes are transcribed. the research of g4s in the hiv-1 genome has been quite productive, concerning not only the two rna viral genome copies, but also the integrated proviral genome, specifically for each virus the following information is shown: virion structure and dimension, genome size and organization; schematic representation of the g4 (red dots) location in the viral genomes or in the mrna and g4 binding proteins; number of g4s assessed through bioinformatics analysis, according to the corresponding references; g4 ligands reported to date to display antiviral effect and corresponding references. in the long terminal promoter (ltr) region (45) (46) (47) and in the nef coding region (48) , as properly reviewed by metifiot et al. (42) . briefly, the ltr promoter is characterized by a highly conserved g-rich sequence in the u3 region, corresponding to sp1 and nf-b binding sites, where three mutually exclusive g4 structures can form, i.e. ltr-ii, ltr-iii and ltr-iv (46) . ltr-iv is a parallel g4 with a bulge at its 3end, as ascertained by nuclear magnetic resonance (nmr) characterization (49) . ltr-iii and ltr-iv exert opposite effects on ltr promoter activity, which is silenced when ltr-iii is folded and enhanced by ltr-iv stabilization (49) . in addition, the ltr g4 region is under the control of two nuclear proteins: nucleolin, which upon binding increases ltr g4 stability and thus silences transcription (50) and the human ribonucleoprotein (hnrnp) a2/b1, which unwinds the ltr region, decreasing its promoter activity (51) : these data suggest that the balance between g4s acts as a regulatory mechanism in hiv-1 promoter activity. interestingly, g4-forming sequences are present in the ltr promoter of all primate lentiviruses and display binding sites for transcription factors that are related to g4 regulation (52, 53) , supporting a role for g4s as crucial control elements for viral transcription, conserved throughout evolution (54) . g4s were also evidenced in the u3 region of the hiv-1 rna genome, where multiple highly stable parallel g4s can form (55) . rna sequences can dimerize through an intermolecular g4 interaction (56) , suggesting that the u3 region could represent an additional point of contact between the two viral genome copies. additionally, such rna g4s likely contribute to the observed increased genetic recombination rate in the u3 (57) . nef, a viral accessory protein, is an essential factor in proviral dna synthesis (58) and in the establishment of a persistent state of infection (59) . its coding region is located at the 3 -end of the viral genome and partially overlaps with the 3 -ltr. three g4 sequences have been identified in the most conserved region of the gene (48). g-rich sequences able to form g4s were reported in the hiv central dna flap overlapping positive-strand and were found to protect the pre-integrated genome from nuclease degradation (60) . stabilization of hiv g4s by small molecules showed antiviral effects at different levels: g4 ligand binding to dna ltr g4s decreased viral transcription, while binding to rna ltr g4s inhibited the reverse transcription process, leading in both cases to strong antiviral effects (46, 55, 61) . g4 ligand-mediated stabilization of the nef g4s induced nef-dependent antiviral activity (48) . very recently, g4 stabilizing agents were also employed in cells infected with latent hiv-1, where their activity resulted in a strong antiviral effect, especially in combination with a dna repair inhibitor, revealing new aspects of hiv-1 latent infection (62) . the specific molecules that were used as anti-hiv-1 agents are discussed in the 'antiviral g4 ligands' section of this review. herpesviridae is a large family of viruses with long linear double-stranded dna genomes. among the nine herpesvirus species that can infect humans, at least five are extremely widespread, i.e. herpes simplex virus 1 and 2 (hsv-1 and hsv-2), varicella zoster virus, epstein-barr virus (ebv) and cytomegalovirus, which cause orolabial and genital herpes (63) , chickenpox and shingles (64), mononucleosis (65) and some cancers (66) . more than 90% of adults have been infected with at least one of these (63) . herpesviruses also tend to display latent, recurring infections, with the virus remaining in some part of the infected organism and typically maintaining its genome as extrachromosomal nuclear episome (67) . recent genome-wide bioinformatics analysis revealed an impressively high density of putative g4-forming sequences in all herpesvirus species (68) . indeed, the presence of g4s has been experimentally reported for hsv-1, ebv, kaposi's sarcoma associated herpesvirus (kshv) and human herpesvirus 6 (hhv-6). hsv-1 establishes life-long persistent infections with a viral lifecycle that involves latency and reactivation/lytic replication. more than half of the world population suffers from hsv infections, the outcome of which may become severe in immunocompromised patients. anti-hsv-1 therapy can be very effective; however, the emergence of drugresistant viral strains urges the discovery of anti-herpetic drugs with innovative mechanisms of action. the hsv-1 genome, characterized by 68% gc-content, was found to contain numerous and highly stable g4-forming sequences that are mainly located in the repeated regions (69) . these hsv-1 g4s, visualized through a g4-specific antibody in infected cells at different time points post-infections, were shown to form in a virus cycle-dependent fashion: viral g4s form massively in the cell nucleus during viral replication, and localize in different cell compartments according to the viral genome movements (70) . ebv is associated not only with the well-known infectious mononucleosis, but also with a wider spectrum of illnesses, including several lymphoid malignancies. studies on the presence and role of g4s in ebv proved that the genome maintenance protein ebv-encoded nuclear antigen-1 (ebna1) stimulates viral dna replication by recruiting the cellular origin replication complex through an interaction with rna g4s (71) . the ebna1 mrna itself is rich in g clusters able to fold into parallel g4s, which behave as cis-acting regulators of viral mrna translation, producing ribosome dissociation. g4s in ebna1 mrna have been shown to modulate the endogenous presentation of ebna1-specific cd8 + t-cell epitopes, which are involved in persistent infections (72) . the cellular protein nucleolin counteracts this mechanism by interacting with ebna1 mrna g4s and thus downregulating ebna1 protein expression and antigen presentation (73, 74) . g4s can also be observed in the mrnas of other genome maintenance proteins that are known to regulate their selfsynthesis, suggesting that g4s are exploited as structural regulatory elements by the virus (75) . kshv is the etiological agent of all forms of kaposi's sarcoma and other numerous lymphoproliferative disorders, which mostly concern aids patients, and at the moment, no treatments for the lytic or latent infections are available (76) . the kshv genome is organized in a 137 kb long unique region, flanked by the terminal repeats, which are rich in g residues and able to form stable g4s, both in the forward and reverse strands (77) . hhv-6 is a ubiquitous virus that infects almost 100% of the human population. the diseases associated with hhv-6 include the febrile illness roseola infantum, also known as the sixth childhood eruptive disease (78) . reactivation of hhv-6 in immunosuppressed individuals is associated with adverse clinical outcomes, comprising life-threatening encephalitis or graft rejection in transplant patients (79) . the hhv-6 genome presents telomeric regions at its termini, which can integrate into the telomeres of human chromosomes: integration is considered one possible mode of latency (80) . since telomeres can fold into g4s, these structures may be involved in the mechanism of hhv-6 integration. indeed, stabilization of telomeric g4s by a g4 ligand inhibited hhv-6 chromosomal integration (81) . stabilization of herpesvirus g4s by g4 ligands led to antiviral activity. in hsv-1, inhibition of dna replication and reduction of late viral transcripts were observed (69, 82) . in ebv, a g4 ligand inhibited ebna1-dependent stimulation of viral dna replication (71) and ebna1 synthesis (75) . in contrast, another g4 ligand reduced nucleolin binding to ebna1 mrna (75) , which in turn resulted in enhanced ebna1 synthesis and antigen presentation (73, 74) . treatment of latently infected cells with g4 stabilizing compounds proved to negatively regulate viral replication, leading to a reduction in the kshv genome copies (77) . g4 ligands used against herpesviruses are discussed in the 'antiviral g4 ligands' section of this review. dna viruses. the human papillomavirus (hpv) is a double-stranded dna virus that can cause skin and genital warts and some types of cancer. its genome displays several g-rich sequences: stable g4s form in only eight out of 120 identified hpv types; however, the g4-forming hpvs include some of the most high risk hpv types, responsible for the majority of cases of cervical cancer (83, 84) . the hepatitis b virus (hbv) is a partially doublestranded dna virus, the best known member of the hepadnaviridae family. it causes the hepatitis b disease, which may lead to cirrhosis and hepatocellular carcinoma. a single putative g4-forming sequence was discovered in the promoter region of the pres2/s gene in hbv genotype b and was found to fold into an intramolecular hybrid g4 structure. surprisingly, the g4 acted as a positive regulator of hbv transcription, as revealed by luciferase reporter assays (85) . adeno-associated viruses (aav) are single-stranded dna viruses of the parvoviridae family. aav are not currently linked to human diseases and have been used as delivery vectors for gene therapy. a recent study reported the presence of g4s in the aav genome. the dna binding protein nucleophosmin (npm1), which is known to enhance aav infectivity, directly interacts with g4s: 18 putative g4s were identified, located within the inverted terminal repeat region (86) . amongst rna viruses, g4 putative sequences have been identified in three positive and singlestranded ones, namely the severe acute respiratory syndrome coronavirus (sars-cov), the hepatitis c virus (hcv) and the zika virus (zikv). the sars-cov belongs to the family of coronaviridae; its genome is about 29.7 kb, which is one of the largest among rna viruses. it has been identified after a massive outbreak in 2003 and is considered one of the most pathogenic coronaviruses in humans. within the nonstructural protein 3, the so-called sars unique domain (sud), which plays an essential role in viral replication and transcription, was found to preferentially bind g4-forming oligonucleotides (87, 88) . these may be found in the 3 -nontranslated regions of mrnas coding for host-cell proteins involved in apoptosis or signal transduction; therefore, it has been proposed that sud/g4 interaction may be involved in controlling the host cell's response to the viral infection. the hcv belongs to the flaviviridae family; it can cause both acute and chronic hepatitis, possibly leading to cirrhosis and liver cancer. bioinformatics and biophysical analysis demonstrated the existence of two highly conserved g4 sequences in the c gene of hcv (89) . the zikv is also included in the family of flaviviridae. it is transmitted to humans by mosquito bites; while in an adult it may cause mild symptoms or even be symptomless, it may be devastating in a pregnant woman as it causes microcephaly in the unborn child. several g4 sequences were discovered in the positive strand of the zikv genome: 7 of these are conserved within more than 50 flavivirus genomes, suggesting an important role in the life cycle of these viruses. furthermore, zikv presents an additional g4 in the unique 3 -utr region, crucial for initial viral replication of the negative-sense strand (90) . finally, g4s have been investigated in the ebola virus (ebov) and marburg virus (marv), two negative and single-stranded rna viruses belonging to the filoviridae family. these are deadly pathogens that cause haemorrhagic fever in humans and primates (91) . the presence of g4 sequences in the negative strand of ebov and marv was assessed by a fluorescent probe (92) . both zikv and ebolv went through massive outbreaks in the past three years, which makes them two of the most dangerous agents of viral epidemics of the current decade. in figure 3 , all the viruses in which g4s have been investigated are displayed. the stabilizing g4 ligands tested in some of these viruses are thoroughly described in the section below. in the past few years much effort has been directed toward the design of small molecules able to target g4s, leading to very promising potential therapeutics, especially against cancer. several updated reviews describe the use of g4 ligands that target telomeres and oncogenes to treat cancer (8, 34, (93) (94) (95) . despite the considerable achievements in antiviral research, viral infections still represent a major global threat nucleic acids research, 2018, vol. 46, no. 7 3275 for human health, causing significant morbidity and mortality. the recurrent onset of drug-resistant pathogens, combined with the fact that the majority of viruses still lack a specific vaccine, urges the development of novel therapeutic approaches for the management of viral diseases. to this end, g4 ligands provide both compounds with an innovative mechanism of action in antiviral treatment and valuable tools to better understand virus mechanisms. in the section below g4 ligands reported to exert antiviral activity have been grouped based on the chemical nature of their core. a description of their discovery, general g4 binding activity and biological effects in cells is initially provided. antiviral properties, activity and selectivity are then discussed. the n,n'-(9-((4-(dimethylamino)phenyl)amino)acridine-3,6-diyl)bis(3-(pyrrolidin-1-yl)propan-amide), labeled braco-19 (b19) (1, figure 4 ), is to date one of the most studied g4 ligands. it is the outcome of a complex and thorough medicinal chemistry investigation that started with the introduction of an acridine moiety as a new chromophore in the research of g4 binders. read and colleagues demonstrated that the acridine core was more active than the previously developed anthraquinone core (96, 97) , because of the presence of a nitrogen atom in the heterocyclic scaffold that could be protonated at physiological conditions. as a result, the electron deficiency in the chromophore was increased, with consequent enhancement of the g4 interaction (98) . in-depth structure-activity relationship (sar) analysis supported by molecular modeling techniques next led to the development of bi-and tri-substituted derivatives (99, 100) . these classes of compounds are characterized by a central planar pharmacophore that binds g-tetrads throughinteractions ( figure 5a ). additionally, two side chains functionalized with a tertiary amine moiety are needed to interact with the grooves: the amine group is crucial for activity since it is protonated at physiological ph, while it disrupts the g4 when substituted with bulky residues (101). the 3,6,9-trisubstituted acridines emerged as the most potent compounds among all the possible regioisomeric series that have been evaluated: they proved to act as g4mediated telomerase inhibitors. b19 showed telomerase inhibition at nanomolar concentration, with higher affinity for g4 with respect to duplex dna, and lower cytotoxicity when compared to first generation acridines. it induced long-term growth arrest and replicative senescence in the 21nt breast carcinoma cell line and was the first g4 ligand to prove anticancer activity in vivo, against human tumor xenograft models (102, 103) . the use of b19 in a viral environment was first analyzed in ebv, to investigate the functional and biochemical characteristics of ebna1. results showed that b19 stabilized the viral rna g4 and, during infection, was able to reduce ebv genome copy numbers in raji cells. it was also found to induce modest reduction of transcription levels of ebna2 and ebna3a and inhibition of ebna1dependent dna replication. these data indicate that g4-interacting molecules can block functions of ebna1 that are critical for viral dna replication (71) . in the ltr promoter region of the hiv-1 proviral genome, b19 was able to significantly stabilize the naturally occurring g4s, ltr-ii and ltr-iii, and to induce an additional g4, ltr-iv. in the presence of increasing concentration of b19, ltr promoter activity was decreased of almost 70% with respect to the untreated control, while no activity was detected in a mutated sequence unable to fold into g4s (46) . these results confirmed a g4-mediated mechanism of action. the anti-hiv-1 activity of b19 (ic 50 < 7.9 m) was tested in various cell lines, against different viral strains and was demonstrated to be g4 mediated. since g4 structures also formed in the pre-integration viral rna (55), a dual mode of action both at the pre-and post-integration level was proposed ( figure 6) . b19 antiviral activity was tested and confirmed in latent hiv-1 infected cells, where the acridine was able to reduce the viral titer to undetectable level, also in long-term treatment (62) . b19 exerted its g4 stabilizing activity also in the hsv-1 genome, where multiple g4s can form. treatment with b19 led to a significant antiviral effect (ic 50 = 8 m), with reduction in viral dna synthesis and late proteins production (69) . moreover, b19 was used in hhv-6a infected cells to evaluate the ability of g4 ligands to impair viral integration in the telomeric region, through stabilization of telomeric g4s. interestingly, in telomerase expressing cell lines, the frequency of chromosomal integration was reduced up to 50% upon treatment. however, effects of g4 ligands on hhv-6 replication and gene expression are yet to be discovered (81) . recently, b19 was employed in a luciferase reporter assay to analyze the role of g4s in hbv, where it enhanced promoter activity, suggesting a positive regulatory role of g4s in hbv transcription (85) . despite its good solubility in aqueous solutions and strong g4 binding, poor permeability across biological barriers, which characterizes most g4 ligands, restrains b19 pharmacological application (104) . nonetheless, b19 is still considered a reference compound in g4 research. the cationic porphyrin compound 5,10,15,20-tetrakis-(nmethyl-4-pyridyl)porphine (tmpyp4, 2, figure 4 ) was proposed as g4 binder because of its suitable physical properties, such as molecular size, planar core, positive charges and hydrophobicity, favorable for stacking with the g tetrads (105) ( figure 5b ). biophysical analysis demonstrated that tmpyp4 was actually able to stack and stabilize both parallel and antiparallel g4s, with mild selectivity for quadruplex over duplex dna (95) . since then, it has been widely employed as a tool to study g4s, especially because of the availability of a negative control compound, tmpyp2 (3, figure 4) , which is a structural isomer with n-methyl-2-pyridyl residues on the porphine core. intriguingly, tmpyp2 is sterically hindered from external stacking on the g4 with respect to tmpyp4, producing no biological effects (106, 107) . in biological assays, tmpyp4 was shown to inhibit human telomerase (ic 50 = 6.5 â± 1.4 m) (108) and downregulate the proto-oncogene c-myc expression as well as several c-myc-regulated genes containing g4-forming sequences. such modulation resulted in in vivo antitumor activity in different models where the porphyrin was able to decrease tumor growth and prolong survival (109) . in viruses, tmpyp4 was shown to stabilize g4s in the hiv-1 nef coding region and to induce their formation within the double-helix conformation. interestingly, in the tzm-bl reporter cell line, which supports nef-dependent hiv-1 replication, the porphyrin inhibited viral infectivity in a dose-dependent manner (48) . in addition, tmpyp4 administration was able to block viral replication in two different jurkat-derived t-cell lines with established hiv-1 latency. bambara's research group demonstrated that the antiviral activity was coupled with an increased rate of apoptosis/death when compared to untreated cells, and that this effect was enhanced by association with dna damage repair inhibitors (62) . in hcv, tmpyp4 was found to stabilize rna g4s and inhibit hcv c gene expression through a g4-mediated mechanism of action confirmed by an enhanced green fluorescent protein reporter gene system. in addition, in an infectious hcv culture system, administration of the porphyrin led to a dose-dependent decrease of viral rna levels (89) . tmpyp4 was also employed to investigate the role of g4s in ebov l gene. it exerted high stabilization of the target g4 rna in circular dichroism and rna stop assays. more importantly, after treatment with increasing concentrations of the compound, transcription of the l gene was gradually reduced. to confirm target selectivity, a mutant non-g4-forming sequence was used as a negative control, where tmpyp4 did not produce significant inhibition of transcription. in addition, the porphyrin was found to in-hibit replication of ebov mini-genome, a cell-based approach that uses firefly luciferase as reporter protein and thus can be used as an efficient antiviral screening system (91) . it is worth noting that the low selectivity of tmpyp4 towards g4 structures versus duplex dna (110) may suggest the antiviral activity to be ascribed to multiple mechanisms of action, limiting its biological and clinical application. perylenes represent a well-known family of g4 ligands, containing a differently substituted, large fused aromatic ring system: they are characterized by a hydrophobic heptacyclic central core, which is responsible for the binding to g quartets throughinteractions, and by up to four protonated side chains. accurate sar studies on this scaffold pointed out two crucial features for g4 binding: the basicity of the system, which prevents the compound from self-aggregation, and the distance between the aromatic central core and the quaternarized nitrogen residue in the side chain, which modulates ligand solubility and affects g4 recognition. the cationic amino moieties in the lateral substituents are thought to regulate specificity for g4 versus duplex dna (111) . piper, n,n'-bis[2-(1-piperidino) ethyl]-3,4,9,10-perylenetetracarboxylic diimide (4, figure 4 ) is the lead compound of this class; it was shown to induce and stabilize g4 structures in telomeres (112) , leading to telomere shortening, reduction of cell proliferation and tumorigenicity, and senescence (113) . in the effort to improve the physicochemical properties of the perylene scaffold, progressive surface reduction led to the more promising class of naphthalene diimide (ndi) derivatives. indeed, it was demonstrated that the dimensions of the planar core modulate the ability of this class of compounds to recognize different dna conformations. in particular, in the cyclic condensed system at least four rings are required to efficiently target g4s (114) . in addition, the ndi planar core can accommodate up to four side chains to enhance g4 affinity. these compounds were found to inhibit telomerase activity in the low micromolar range and to produce short-term cell growth inhibition against mcf-7 and a549 cancer cell lines (115) . to improve dna g4 alkylating properties, further modifications were introduced on the ndi scaffold, which include quinone methides precursors (116, 117) . these ligands revealed both reversible and irreversible binding properties toward telomeric dna, with promising duplex versus quadruplex selectivity (118) , and were found to impair the growth of different telomerase-positive cancer cell lines following telomerase activity inhibition (119) (120) (121) . crystallographic analyses of various ndi-telomere complexes provided a turning point for rational optimization of this class of compounds (122, 123) . neidle et al. reported that the tested ligands promoted a parallel g4 topology, forming a 1:1 complex with the oligonucleotide. this stoichiometry resulted from the combination of binding site affinity and direct groove interactions that are highly influenced by the protonated moiety in the side chains, which interacts with dna phosphates in the grooves. despite their high molecular weight, ndis are highly versatile structures, suitable for further medicinal chemistry modifications to improve their pharmacological profile (124, 125) . in the antiviral field, piper induced and stabilized g4 structures in the nef coding region of the hiv-1 genome (48) . however, the best results were obtained with coreextended ndi derivatives (c-exndis, 5, figure 4 ). this series of compounds, endowed with exceptional solubility properties, has been obtained by fusing the ndi core with a 1,4-dihydroquinoxaline heterocycle. interestingly, the newly developed ligands displayed greater in vitro binding and stabilization activity on viral hiv-1 ltr g4s than the human telomeric sequence, used as a cellular reference g4. most importantly, the c-exndis exhibited very promising antiviral activity in the low nanomolar range (ic 50 < 25 nm) against different strains of hiv-1, with very low cytotoxicity, yielding a wide and encouraging therapeutic window. the g4-related mechanism of action was proved combining time-related antiviral and reporter assays, using a non-g4-forming ltr-mutant sequence as control. it is reasonable that the higher antiviral activity depends on the selectivity toward the viral g4s, as, during the infection, ltr and telomeric g4s are likely the most abundant species in the cell (61) . the most active c-exndi was also analyzed in hsv-1 infection. in vitro cd and taq-polymerase stop assays indicated that the compound was able to bind and stabilize various g4-forming sequences of the hsv-1 genome. mass spectrometry competition analysis revealed a stronger preference for hiv-1 g4s over hsv-1, but generally, viral g4s were preferentially bound, when compared to the telomeric g4. indeed, c-exndi showed remarkable antiviral activity (ic 50 = 18.3 â± 1.4 nm). the anti-herpetic effect was ascribed to inhibition of viral dna replication, as gathered by time-of-addition assay and flow cytometry analysis using acyclovir as reference compound (82) . since c-exndi selectivity towards hsv-1 g4s in vitro resulted to be good but not outstanding, the marked anti-hsv-1 activity was likely due also to the massive presence of viral g4s in the cell nucleus, which was demonstrated to occur during hsv-1 replication (70) . pyridostatin (pds, 6, figure 4 ) has been rationally designed on the structural features shared by known g4-binding ligands, as it comprises a potentially planar electron-rich aromatic surface and the ability to participate in hydrogen bonding. moreover, the rotatable bonds provide a flexibility degree, which makes pds capable to adapt to the dynamism of g4s. pds strongly stabilized telomeric g4 with no effect on double-stranded dna: as a result, the shelterin complex integrity was altered, triggering a dna-damage response at telomeres (126) . numerous modifications have been introduced in the pds scaffold to further explore the role of this class in anticancer therapy. indeed, the obtained analogues showed remarkable growth-inhibitory effects in cancer cell lines and a complete arrest after long-term exposure to the drug. these results emphasize the high potential of these compounds to fine-tune their biological activity (127, 128) . in antiviral research, pds has been used to study the role of g4s in ebv ebna1 mrna, where it enhanced the stability of the g4-forming sequence, decreasing ebna1 synthesis level in a concentration-dependent fashion, both in vitro and in vivo. as a consequence, ebv-infected cells resulted less efficiently recognized by virus-specific t cells, albeit the mechanism of action still needs to be clarified (75) . in hbv, pds was used to unravel the positive regulatory role of g4s within the pres2/s gene promoter (85) . a pds analogue, namely pdp (7, figure 4) , was employed in hcv g4 research, along with tmpyp4. the pdp-induced stabilization of g4 structures located in the hcv rna downregulated c gene expression. in vivo, pdp inhibited intracellular replication of different hcv genotypes through a confirmed g4-related mechanism of action, resulting in antiviral activity in the low micromolar range (89) . bisquinolinium compounds are characterized by an aromatic nucleus substituted with two protonated quinoline moieties. the first reported compounds present a dicarboxamide-pyridine or -triazine ring as central core: the most promising of these ligands have shown to increase g4 stability in telomeres, with great selectivity over duplex dna (129) . these compounds are able to adopt an intramolecular syn-syn h-bond, which was proposed to be critical for g4 recognition, likely because the consequent rigidity of the compound promotes g-quartet overlap. on these bases, the central core was expanded without disrupting the h-bonds, leading to a new disubstituted-1,10phenanthroline series that displays exceptional selectivity for g4s (130) , due to the crescent-like shape which prevents such compounds to intercalate with duplex dna ( figure 5c ) (131, 132) . phendc3 (8, figure 4 ), the best representative of this class, is a potent telomeric g4 ligand able to reduce telomerase processivity (133) . phendc3 was used in kshv to evaluate its potential role in inhibiting latent viral replication. the ligand was found to elicit a stress response in infected bcbl-1 cells and to stall the replication machinery both in the leading and lagging strands of the kshv genome. furthermore, treatment with phendc3 resulted in the dramatic reduction (60%) of episome copy number, with no effect on cell growth and proliferation. these data represent the first use of g4 ligands in targeting latent viral infections (77) . phendc3 was also used in ebv, where it prevented binding of nucleolin to ebna1 mrna g4 and increased the endogenous ebna1 levels in ebv-infected b cells and in cells derived from a nasopharyngeal carcinoma. these results indicate that the nucleolin-ebna1 mrna interaction can also be targeted by antiviral g4-ligands (74) . a summary of g4 ligands and the viruses against which they have been tested is reported in figure 3 . in the last decades, research on the role of g4s in the human genome has been quite challenging and promising, leading to the awareness that these high-order structures nucleic acids research, 2018, vol. 46, no. 7 3279 play key regulatory roles in biological pathways such as transcription, replication, translation and telomere maintenance. the development of g4 binders with encouraging anti-cancer activity has prompted researchers to identify new ways to exploit g4 structures in human diseases, e.g. viral infections. because g4s are present both in cell and virus genomes, the challenge in developing antiviral g4 ligands reasonably consists in overcoming selectivity toward viral versus cellular g4s. a major limitation of the so far described g4 ligands is their large flat aromatic core that stacks on the g tetrad, which reduces the chances to discriminate among different g4s. moreover, they are generally characterized by high-molecular weights and protonated side chains, which are necessary for loops and grooves interaction, but, on the other hand, may affect cellular uptake. indeed, because of the low selectivity profile and poor drug-like properties, no g4 ligand has advanced beyond phase ii in the drug discovery pathway. quarfloxin, a fluoroquinolone derivative compound developed by hurley's research group (134) , is to date the only g4 ligand that has reached phase ii clinical trials but was withdrawn due to bioavailability related problems (35) . however, several data presented in the literature indicate that, in general, a certain degree of selectivity is achievable towards the viral g4 of interest in comparison to the telomeric g4, i.e. the most abundant cellular g4 (135) . in the case of hiv-1 g4s and c-exndi compounds, the higher affinity towards the viral structure is likely caused by the extension of the ndi core and thus by the interaction with the viral g4 loop region, which is unique for this g4 (61) . in general, loop and groove regions characterize each g4 and thus are amenable for selective recognition. structural studies on cellular g4/g4 ligand complexes indicated that most g4-binding molecules interact with g4s through quasi-external stacking, in which the heteroaromatic chromophore of the small molecules isstacked onto the face of an external g-quartet (136) ( figure 5 ) and onto the side chains positioned in the g4 grooves (94) . it is therefore conceivable that the reported antiviral activity of g4 ligands is mediated by an increased interaction, hence affinity, with the groove/loop moiety of the viral g4s. to date, only one viral g4 structure has been resolved through nmr spectroscopy (49), therefore future nmr and crystallographic resolutions of viral g4s and g4/g4 ligand complexes are necessary to define the viral g4s architecture. this could help researchers identifying possible unique g4 structures which could lead to the design and development of selective molecules. in other cases, g4 ligands did not show significant selectivity for the viral versus telomeric g4s, and the g4s present in oncogene promoters were usually strongly bound by the tested compounds (82) . nonetheless, the data so far presented on the antiviral use of g4 ligands have shown in general very promising activity against a wide range of virus species. one possible explanation is that the amount of the viral g4s in the infected cells largely surpasses that of the cellular g4s (70) . indeed, usually cells are exploited to function as factories in the production of new viral genomes that are eventually assembled into new mature virus particles (see figure 2 for the viral infection cycle). it is thus conceivable that the viral g4s become largely more abun-dant than the cellular g4s during virus replication. at least in one case this eventuality has been demonstrated: in hsv-1 there is a sharp increase in the number of viral g4s during viral dna replication (70) . combining the abundance of g4s per genome and the number of new genomes, the amount of viral g4s could outstand that of cellular g4s by several logs per cell. in addition, the so far identified viral g4s are usually key regulatory elements of the virus life cycle and their stabilization/unfolding by g4 ligands can likely explain the resulting massive virus inhibition. if this behaviour is demonstrated also in other viruses, it would be possible to exploit g4 ligands that are not strictly selective for the viral g4s. this scenario would highly and rapidly expand the research and pharmacological application of g4 ligands as antiviral agents. a further point to be addressed is the necessity to standardize methods to study the antiviral activity of the g4 ligands. one starting point should be the detection of the inhibitory activity of the ligand on the virus life cycle. if an effect is obtained, further investigation on the mechanism of action has to be performed. in this regard, the time of addition method (137) can be of assistance as it indicates the last viral step at which the compound is active and it thus narrows the possible molecular targets. however, because of the complexity and uniqueness of each virus, the investigation of the target and mechanism of action at the molecular level may not be straightforward. for example, pds inhibited ebna1 synthesis in vitro but not in cells, while phendc3 in cells led to the exact opposite effect, i.e. enhanced ebna1 synthesis (74, 75) . it is likely that multiple g4-mediated mechanisms are involved in the observed outcomes. finally, targeting g4s in the viral genomes leads to the exciting possibility of affecting viruses that undergo latency. these viruses, such as hiv, the herpes and papilloma virus families, comprise an initial acute infection and a subsequent latent infection. the latter is characterized by the maintenance of the virus genome in the human host for the entire life of the host. the latent virus may reactivate from time to time to produce new mature virus. current therapies that normally target viral proteins fail to remove the latent virus, i.e. the virus genome, from its host. selectively targeting the viral genome in a g4-mediated approach would allow removing not only the replicating virus but also the latent one, therefore eradicating so far incurable infective agents. in this picture it is worth considering the virus-induced manipulation of host chromatin. in recent years, studies about the role of chromatin in viral infections showed dynamic virus-host chromatin interactions and chromatin machinery modulation by virus encoded proteins (138) . for example, the hsv-1 epigenetic regulation of viral chromatin by viral gene products plays a key role in determining whether the virus develops a lytic or latent infection (139) . considering the recent evidences reported by hã¤nsel-hertsch et al. that g4 formation reflects the suppressive role of heterochromatin and that it occurs only in highly transcribed regulatory nucleosome-depleted chromatin regions (13) , it would be interesting to understand how the virus and its g4s affect and could be affected by such a complex mechanism. to conclude, all the data reported in this review indicate that: i) g4 structures are crucial elements in the regulation of viruses' life cycle, both in lytic and latent states; ii) g4 ligands efficiently act as antiviral agents. this should encourage researchers to continue investigating on g4-binding small molecules: as a matter of fact, albeit quarfloxin clinical evaluation did not progress, its success in phase i clinical trial, i.e. optimal toxicity profile (35) , suggests that improvements of g4 ligand pharmacological profiles will very likely lead to concrete clinical applications of these compounds. therefore, research in the next future will need to improve i) the understanding of g4 activity and regulation at the viral level, ii) the selectivity of g4 ligands toward the viral versus cellular g4s, iii) the drug-like properties of the antiviral g4 ligands to be employed in in vivo studies. g4-mediated antiviral drugs may represent a significant turning point in the management of viral infections, especially for people who cannot access immunization, like immunocompromised patients or elderly people. in addition, the g4-mediated antiviral effects reported in latent infections (62) may pave the way for cutting-edge therapeutic approaches in the treatment of human fatal malignancies related to latent viruses, such as aids, herpes-and hpvrelated cancer. quadruplex dna: sequence, topology and structure rna g-quadruplexes in biology: principles and molecular mechanisms g-quadruplexes: prediction, characterization, and biological application targeting unimolecular g-quadruplex nucleic acids: a new paradigm for the drug discovery? g-quadruplexes involving both strands of genomic dna are highly abundant and colocalize with functional sites in the human genome formation of dna:rna hybrid g-quadruplex in bacterial cells and its dominance over the intramolecular dna g-quadruplex in mediating transcription termination co-transcriptional formation of dna:rna hybrid g-quadruplex and potential function as constitutional cis element for transcription control four-stranded nucleic acids: structure, function and targeting of g-quadruplexes re-evaluation of g-quadruplex propensity with g4hunter prevalence of quadruplexes in the human genome gene function correlates with potential for g4 dna formation in the human genome high-throughput sequencing of dna g-quadruplex structures in the human genome g-quadruplex structures mark human regulatory chromatin permanganate/s1 nuclease footprinting reveals non-b dna structures with regulatory potential across a mammalian genome g-quadruplexes and their regulatory roles in biology high-resolution three-dimensional nmr structure of the kras proto-oncogene promoter reveals key features of a g-quadruplex involved in transcriptional regulation structure of two intramolecular g-quadruplexes formed by natural human telomere sequences in k+ solution crystal structure of parallel quadruplexes from human telomeric dna quantitative visualization of dna g-quadruplex structures in human cells detection of g-quadruplex dna in mammalian cells visualization of rna-quadruplexes in live cells elevated levels of g-quadruplex formation in human stomach and liver cancer tissues g-quadruplex-binding small molecules ameliorate c9orf72 ftd/als pathology in vitro and in vivo g-quadruplex structures and their interaction diversity with ligands structure, location and interactions of g-quadruplexes telomere g-quadruplex as a potential target to accelerate telomere shortening by expanding the incomplete end-replication of telomere dna telomeric g-quadruplex architecture and interactions with potential drugs human telomeric g-quadruplex: the current status of telomeric g-quadruplexes as therapeutic targets in human cancer cell cycle regulation of g-quadruplex dna structures at telomeres g-quadruplexes in human promoters: a challenge for therapeutic applications g4 dna in ras genes and its potential in cancer therapy small molecules targeting c-myc oncogene: promising anti-cancer therapeutics making sense of g-quadruplex and i-motif functions in oncogene promoters g-quadruplex structures in the human genome as novel therapeutic targets targeting g-quadruplexes in gene promoters: a novel anticancer strategy? genome-wide computational and expression analyses reveal g-quadruplex dna motifs as conserved cis-regulatory elements in human and related species genomic distribution and functional analyses of potential g-quadruplex-forming sequences in saccharomyces cerevisiae putative dna g-quadruplex formation within the promoters of plasmodium falciparum var genes genome-wide study predicts promoter-g4 dna motifs regulate selective functions in bacteria: radioresistance of d. radiodurans involves g4 dna-mediated regulation mapping and characterization of g-quadruplexes in mycobacterium tuberculosis gene promoter regions g-quadruplexes in pathogens: a common route to virulence control? g-quadruplexes in viruses: function and potential therapeutic applications g-quadruplex forming oligonucleotides as anti-hiv agents g-quadruplex-based aptamers against protein targets in therapy and diagnostics topology of a dna g-quadruplex structure formed in the hiv-1 promoter: a potential target for anti-hiv drug development a dynamic g-quadruplex region regulates the hiv-1 long terminal repeat promoter hiv-1 nucleocapsid protein increases strand transfer recombination by promoting dimeric g-quartet formation formation of a unique cluster of g-quadruplex structures in the hiv-1 nef coding region: implications for antiviral activity structure and possible function of a g-quadruplex in the long terminal repeat of the proviral hiv-1 genome nucleolin stabilizes g-quadruplex structures folded by the ltr promoter and silences hiv-1 viral transcription the cellular protein hnrnp a2/b1 enhances hiv-1 transcription by unfolding ltr promoter g-quadruplexes a non-canonical dna structure is a binding motif for the transcription factor sp1 in vitro the relationship of potential g-quadruplex sequences in cis-upstream regions of the human genome to sp1-binding elements conserved presence of g-quadruplex forming sequences in the long terminal repeat promoter of lentiviruses anti-hiv-1 activity of the g-quadruplex ligand braco-19 evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic rna mechanism of hiv-1 rna dimerization in the central region of the genome and significance for viral evolution nef stimulates human immunodeficiency virus type 1 proviral dna synthesis the human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages g-quartets assembly within a g-rich dna flap. a possible event at the center of the hiv-1 genome synthesis, binding and antiviral properties of potent core-extended naphthalene diimides targeting the hiv-1 long terminal repeat promoter g-quadruplexes deficiency in dna damage response, a new characteristic of cells infected with latent hiv-1 herpes simplex virus establishment, maintenance, and reactivation: in vitro modeling of latency diagnosis, antiviral therapy, and prophylaxis of varicella-zoster virus infections infectious mononucleosis herpesviruses and cancer genital herpes genome-wide analysis of g-quadruplexes in herpesvirus genomes the herpes simplex virus-1 genome contains multiple clusters of repeated g-quadruplex: implications for the antiviral activity of a g-quadruplex ligand visualization of dna g-quadruplexes in herpes simplex virus 1-infected cells role for g-quadruplex rna binding by epstein-barr virus nuclear antigen 1 in dna replication and metaphase chromosome attachment mrna structural constraints on ebna1 synthesis impact on in vivo antigen presentation and early priming of cd8+ t cells a yeast model for the mechanism of the epstein-barr virus immune evasion identifies a new therapeutic target to interfere with the virus stealthiness nucleolin directly mediates epstein-barr virus immune evasion through binding to g-quadruplexes of ebna1 mrna g-quadruplexes regulate epstein-barr virus-encoded nuclear antigen 1 mrna translation kaposi sarcoma herpesvirus-associated cancers and related diseases g-quadruplex-interacting compounds alter latent dna replication and episomal persistence of kshv clinical impact of primary infection with roseoloviruses roseoloviruses in transplant recipients: clinical consequences and prospects for treatment and prevention trials the latent human herpesvirus-6a genome specifically integrates in telomeres of human chromosomes in vivo and in vitro stabilization of telomere g-quadruplexes interferes with human herpesvirus 6a chromosomal integration a core extended naphtalene diimide g-quadruplex ligand potently inhibits herpes simplex virus 1 replication the effect of single nucleotide polymorphisms in g-rich regions of high-risk human papillomaviruses on structural diversity of dna human papillomavirus g-quadruplexes a g-quadruplex motif in an envelope gene promoter regulates transcription and virion secretion in hbv genotype b the function of dna binding protein nucleophosmin in aav replication a g-quadruplex-binding macrodomain within the "sars-unique domain" is essential for the activity of the sars-coronavirus replication-transcription complex the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes a highly conserved g-rich consensus sequence in hepatitis c virus core gene represents a new anti-hepatitis c target zika virus genomic rna possesses conserved g-quadruplexes characteristic of the flaviviridae family chemical targeting of a g-quadruplex rna in the ebola virus l gene ebola virus derived g-quadruplexes: thiazole orange interaction recent advances in targeting the telomeric g-quadruplex dna sequence with small molecules as a strategy for anticancer therapies quadruplex nucleic acids as targets for anticancer therapeutics g-quadruplexes: targets in anticancer drug design human telomerase inhibition by regioisomeric disubstituted amidoanthracene-9,10-diones inhibition of human telomerase by a g-quadruplex-interactive compound molecular modeling studies on g-quadruplex complexes of telomerase inhibitors: structure-activity relationships human telomerase inhibition by substituted acridine derivatives structure-based design of selective and potent g quadruplex-mediated telomerase inhibitors trisubstituted acridine derivatives as potent and selective telomerase inhibitors the g-quadruplex-interactive molecule braco-19 inhibits tumor growth, consistent with telomere targeting and interference with telomerase function a g-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumor activity biopharmaceutical characterization of the telomerase inhibitor braco19 structural basis for binding of porphyrin to human telomeres interactions of tmpyp4 and tmpyp2 with quadruplex dna. structural basis for the differential effects on telomerase inhibition cationic porphyrins as telomerase inhibitors: the interaction of tetra-(n-methyl-4-pyridyl)porphine with quadruplex dna effects of cationic porphyrins as g-quadruplex interactive agents in human tumor cells the cationic porphyrin tmpyp4 down-regulates c-myc and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo shedding light on the interaction between tmpyp4 and human telomeric quadruplexes synthesis of a non-cationic, water-soluble perylenetetracarboxylic diimide and its interactions with g-quadruplex-forming dna nmr-based model of a telomerase-inhibiting compound bound to g-quadruplex dna telomere shortening and cell senescence induced by perylene derivatives in a549 human lung cancer cells tri-, tetra-and heptacyclic perylene analogues as new potential antineoplastic agents based on dna telomerase inhibition tri-and tetra-substituted naphthalene diimides as potent g-quadruplex ligands binol-amino acid conjugates as triggerable carriers of dna-targeted potent photocytotoxic agents binol quinone methides as bisalkylating and dna cross-linking agents quinone methides tethered to naphthalene diimides as selective g-quadruplex alkylating agents hybrid ligand-alkylating agents targeting telomeric g-quadruplex structures targeting loop adenines in g-quadruplex by a selective oxirane naphthalene diimide scaffolds with dual reversible and covalent interaction properties towards g-quadruplex structural basis for telomeric g-quadruplex targeting by naphthalene diimide ligands structure-based design and evaluation of naphthalene diimide g-quadruplex ligands as telomere targeting agents in pancreatic cancer cells targeting multiple effector pathways in pancreatic ductal adenocarcinoma with a g-quadruplex-binding small molecule a g-quadruplex-binding compound showing anti-tumour activity in an in vivo model for pancreatic cancer a novel small molecule that alters shelterin integrity and triggers a dna-damage response at telomeres small-molecule-mediated g-quadruplex isolation from human cells pyridostatin analogues promote telomere dysfunction and long-term growth inhibition in human cancer cells cell senescence and telomere shortening induced by a new series of specific g-quadruplex dna ligands highly efficient g-quadruplex recognition by bisquinolinium compounds quadruplex nucleic acids as novel therapeutic targets solution structure of a g-quadruplex bound to the bisquinolinium compound phen-dc(3) reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action design and synthesis of fluoroquinophenoxazines that interact with human telomeric g-quadruplexes and their biological effects how shelterin protects mammalian telomeres structural basis of dna quadruplex recognition by an acridine drug a time-of-drug addition approach to target identification of antiviral compounds snapshots: chromatin control of viral infection chromatin control of herpes simplex virus lytic and latent infection key: cord-319002-xmsfkaoc authors: brown, james; lipman, marc title: community-acquired pneumonia in hiv-infected individuals date: 2014-02-22 journal: curr infect dis rep doi: 10.1007/s11908-014-0397-x sha: doc_id: 319002 cord_uid: xmsfkaoc community-acquired pneumonia continues to be an important complication of hiv infection. rates of pneumonia decrease with the use of antiretroviral therapy but continue to be higher than in hiv uninfected individuals. risk factors for pneumonia include low blood cd4+ count, unsuppressed plasma hiv load, smoking, injection drug use and renal impairment. immunization against streptococcus pneumoniae and smoking cessation can reduce this risk. it is unclear whether newly reported viral respiratory pathogens (such as the middle east respiratory syndrome coronavirus, will be more of a problem in hiv-infected individuals than the general population. community-acquired pneumonia (cap) remains an important cause of morbidity and mortality in hiv-positive individuals [1] . although the incidence has decreased with the use of antiretroviral therapy (art), rates continue to be elevated compared to hiv-uninfected individuals in both developing and developed societies [2] . the clinical presentation, range of typical pathogens and outcomes of cap are similar to those without hiv infection, but important differential diagnoses and implications for future health merit particular attention in hiv-infected individuals [3] . this review considers the evidence concerning cap in hiv-infected individuals, with particular emphasis on studies reported in the last year. we consider the role of bacterial and viral causes of pneumonia and other conditions that can mimic pneumonia. the management of hospital-acquired pneumonia and infections caused by opportunistic pathogens such as pneumocystis jirovecii, cmv, fungal and mycobacterial organisms are covered elsewhere and are not discussed here, beyond stressing that they can all present as an acute pneumonic illness, and hence will often need to be considered as part of the differential diagnosis. prior to the development of effective art, respiratory tract infection was the most common complication of hiv infection [1] . the advent of art has been associated with a substantial decline in the rates of opportunistic infections and as the frequency of pulmonary infections such as p. jirovecii pneumonia have fallen, bacterial pneumonias have become increasingly prominent in the care of hiv-infected people [4•, 5•] . although there has been debate as to the increased rate of pneumonia in hiv-infected persons following art, recent data from several large observational cohorts have significantly increased our current knowledge on this subject. the patient cohorts in the multicentre aids cohort study (macs) and the womens interagency hiv study (wihs) have been followed since 1984 and 1994, respectively, and allow analysis of rates of pneumonia in the art era. in the macs cohort the adjusted odds ratio for bacterial pneumonia compared with hiv-uninfected individuals has declined from 21.8 in the pre-art period to 4.14 following the introduction of art [6••] . the wihs cohort of hiv-infected women have also shown an elevated incidence of pneumonia despite art use with an adjusted odds ratio of 9.55 compared with hiv-uninfected controls. similarly, in the veterans aging cohort study (which involved a virtual cohort of 33,420 hiv-infected individuals using data from the us national va health information system from the date of hiv diagnosis until 2007) a rate of 28 per 1,000 person-years was found in hiv-infected individuals compared with 5.8 per 1,000 person-years in hivnegative controls [7••] . a lower incidence has been seen in the eurosida study of more than 18,000 patients in 34 countries studied between 2006 and 2011, in which an incidence of pneumonia of 5.36/1000 person-years was found [8••] . this may reflect the better immune status of participants in the latter era, who had a mean blood cd4+ count of 457 cells/μl, as very low levels of severe bacterial infections were demonstrated in the eurosida study in those with cd4 counts above 500 cells/μl (a level generally regarded as near normal). studies in populations other than in europe and the us have confirmed the importance of bacterial pneumonia in hiv-infected individuals, with recent work in taiwan showing this to be the most common respiratory complication of hiv infection in those with cd4 counts above 200 cells/μl [9] . however, the general improvement in incidence rates has not been found in all populations, as surveillance data from soweto, south africa, indicate persisting high rates of invasive pneumococcal disease, with no decrease since the introduction of art [10] . this may be due to the high levels of immunocompromise in this population despite the availability of art, although an increase in invasive pneumococcal disease was found amongst women in that study, suggesting that general uptake of the childhood pneumococcal conjugate vaccination (pcv; which now forms part of the childhood immunization schedule in south africa) may be particularly effective at reducing rates of invasive pneumococcal disease amongst hiv-infected adults in this community. specific risk factors for cap can be identified in the cohorts studied. most have studies have shown that pneumonia is more common in hiv-infected people at any level of immunosuppression, and rates of pneumonia increase with increasing immunosuppression [8••] . cigarette smoking is consistently reported to increase the risk of pneumonia, with a hazard ratio of 0.48 in former smokers compared to current smokers in a cohort study from france [11•] . injecting drug use increases the risk of pneumonia, and although this may be due to confounding diseases, poor adherence to art or socioeconomic factors, some recent evidence suggests that direct effects of opiates or their withdrawal may play a role [12] . ethnic differences in pneumonia rates, which may be reflective of socioeconomic factors, have been found in some cohorts: rates of pneumonia in african-americans in california are significantly higher than in other ethnic groups [13] , aboriginal ethnicity is a risk factor for invasive pneumococcal disease in canada [14••] , and asian ethnicity was associated with reduced risk in the evaluation of subcutaneous interleukin-2 (esprit) trial [15] . being a care-giver to young children (who have high rates of pneumococcal carriage) may predispose to invasive pneumococcal disease, and a recent study from south africa confirms significant child-to-mother transmission of pneumococcal strains [16•] . the eurosida study showed that a reduced estimated glomerular filtration rate (egfr) is a significant risk factor for severe bacterial infections including pneumonia, with an incidence rate ratio of 5.07 for those with egfr <60 ml/min/1.73 m 2 compared to those with egfr >90 ml/min/1.73 m 2 [8••] . it is not clear if this is a direct effect on immune function caused by renal impairment, or the result of other comorbid conditions or lifestyle factors. art is an important protective factor against pneumonia. several cohorts have demonstrated that suppressed hiv loads are associated with a lower incidence of pneumonia, most recently the icona study in italy, which showed an overall incidence of 5.66 per 1,000 person-years with increased risk in people with low nadir cd4+ count, low current cd4 count or high viral load [17•] . this trial followed 4,942 individuals for a median of 63.7 months between 1996 and 2011; 15 % had an aids diagnosis prior to art initiation and 46 % had a nadir cd4 count below 200 cells/μl. a low nadir cd4 count was associated with a hazard ratio for an episode of bacterial pneumonia of 0.86 (per 100 cells/μl higher). low current blood cd4 was associated with a hazard ratio of 0.88 (per 100 cells/μl higher) and an unsuppressed plasma viral load was associated with a hazard ratio of 1.29. the strategies for management of antiretroviral therapy (smart) trial demonstrated reductions in the incidence of pneumonia in those on continuous compared to those on intermittent art, as well as a lower incidence in those with an undetectable plasma hiv load [18•] . in this trial (which recruited patients with a cd4 count of >350 cells/μl between 2002 and 2006) intermittent rather than continuous art was associated with a 1.55-fold increase in pneumonia incidence. this finding is of particular importance as the randomized trial methodology of intermittent versus continuous art allows confidence that the observed differences in pneumonia incidence are the result of art rather than unmeasured confounding factors. the esprit trial (which evaluated the effect of subcutaneous il-2 in hiv-infected patients on or starting art with a cd4 count >300 cells/μl) also found that a higher hiv plasma viral load was associated with increased risk of pneumonia with a (hazard ratio for a 1 log 10 higher viral load of 1.28) [15] . a reduction in pneumonia incidence has also been reported in hiv-infected children in the us, this has been attributed to the use of art and pneumococcal vaccination. an observational cohort study of 736 hiv-infected children observed between 2000 and 2005 showed lower cd4 counts in those with pneumonia than in those without pneumonia (668 cells/ μl, or 23 %. in a multivariate analysis a hiv viral load of >100,000 copies/ml was associated with a hazard ratio for pneumonia of 3.98 [19] . work in hiv-uninfected populations suggests that the use of statins (hmg coenzyme a reductase inhibitors) may reduce pneumonia-related mortality, possibly by attenuating the associated inflammatory response [20] . however, a recent study in the netherlands using population-based data found there to be neither a reduction in the frequency of pneumonia nor a worse outcome, when it did occur, in hiv-infected individuals taking regular statin therapy [21] . it should be remembered that the higher incidence of pneumonia in hiv-infected individuals means that presentation with cap should prompt consideration of hiv testing in patients not known to be hiv-infected. it is estimated that around 24 % of the hiv-infected population in the uk and 18 % of the hiv-infected population in the us are unaware of their serostatus [22, 23] . surveys from sub-saharan africa undertaken by usaid have shown that in many countries the majority of hiv-infected individuals do not know their serostatus [24] . bacterial pneumonia should be regarded as an indicator condition that must prompt discussion of hiv testing. british hiv association guidelines recommend that all patients admitted with bacterial pneumonia should be offered an hiv test, and analysis of primary care data confirms that bacterial pneumonia is one of the strongest indicator conditions for hiv infection [25••] . invasive pneumococcal disease is particularly strongly associated with hiv infection, with recent uk population-based data showing that 2.4 % of patients with invasive pneumococcal disease have undiagnosed hiv infection [26••] . recent studies have contributed to our understanding of the pathogenesis of pneumonia in hiv-infected individuals. although depletion of the cd4+ t cell is the hallmark of hiv infection, it is recognized that impairment of innate immune responses plays an important role in the increased susceptibility to pneumonia [27] . reduced cd4+ t-cell responses to respiratory antigens have been demonstrated in bronchoalveolar cd4+ cells when compared with those in hiv-uninfected controls, and a disruption in the t-cell response to pneumococcus appears to precede cd4+ t-cell depletion [28, 29•] . hiv causes chronic activation of dendritic cells of the innate immune system, triggering immune dysregulation and apoptosis of cd4+ and cd8+ t cells [30, 31] . impairment of cd8+ t-cell responses may also be important, as cd8 levels have been found to be predictive of pneumonia in the hiv epidemiologic research study (hers) of hiv-infected women in the us after adjustment for age, cd4+ cell count, viral load and antiretroviral use [32•] . impairment of immune responses within the lung of hivinfected individuals results in changes to the oral and airway microbiota with increased numbers and diversity of microorganisms, including potentially pathogenic species found in hiv-infected patients with pneumonia compared to hivnegative controls [33] . hiv-infected individuals appear to be particularly susceptible to pneumococcal disease [34] . rates of carriage of streptococcus pneumoniae were found to be higher in hiv-infected individuals in a prospective cohort of hiv-infected and hiv-uninfected mothers in zambia, although it should be noted that the cd4 counts in these subjects were not known [35] . smoking appears to increase rates of pneumococcal carriage [36] as does a lower nadir cd4 count and an aids diagnoses [37] . there may be an interaction between respiratory viral pathogens and rates of invasive pneumococcal disease, as influenza infection has been reported to be associated with increased blood pneumococcal load [38] . viral pathogens are responsible for a large proportion of respiratory tract infections in hiv-infected individuals, although there are limited data available on the role of viral pathogens. a prospective study in montreal found that among 50 hiv-positive patients with fever and symptoms consistent with respiratory tract infection, viruses accounted for 64 % of these illnesses [39••] . in this study, 90 % of patients were on art and had a median cd4+ t cell count of 325 cells/μl, and a median viral load of <50 copies/ml. influenza was the most important viral cause of respiratory tract infections with 22 of 34 identified viral infections due to influenza, with equal numbers of influenza a and b. other viruses isolated were human metapneumovirus types a and b, respiratory syncytial virus, parainfluenza types 2 and 3 and coronavirus. these pathogens also cause respiratory tract infections in hivuninfected individuals, and it is not known if the incidence or severity of these infections is different in hiv-infected individuals, although respiratory syncytial virus has been reported to be a cause of severe cap in hiv-positive individuals [40, 41] . although it is often suggested that hiv-infected individuals may be more susceptible to viral respiratory tract infections, or suffer more severe disease associated with viral pathogens, the evidence to support this is mixed. studies prior to the availability of art suggested that hiv-infected individuals have more severe disease due to influenza infection [42] . cohen et al. have reported an excess mortality amongst hiv-infected persons during influenza epidemic periods in south africa and the us, with reductions in this excess mortality since the widespread use of art in the us [43] . however, it is not clear if this excess mortality arises directly from influenza infectionfor example, an observational study in barcelona found no difference in the severity of influenza in patients with and without hiv infection admitted with influenza h 1 n 1 [44] . the excess deaths in influenza epidemic periods found by cohen et al. may instead be the result of other conditions which are more prevalent at these times, including pneumococcal pneumonia. in general, data from the h 1 n 1 pandemic of 2009 do not support the suggestion that hiv-infected individuals experience more severe disease [45, 46] . emerging respiratory pathogens such as the middle east respiratory syndrome coronavirus (mers cov) may pose a threat to everybody. no data are yet available on the relative susceptibility of hivinfected individuals to this condition [47] . it is possible that antiretroviral medication may modify the presentation of influenza infection as protease inhibitors have been shown to block influenza viral replication, although the clinical impact of this observation remains unclear [48] . hiv-infected individuals with pneumonia present with typical clinical features of an acute illness characterized by fever, pleuritic chest pain, breathlessness and hypoxaemia [2] . the use of established severity scores has been found to be valid in hiv infection, with the pneumonia severity index (psi) being the best-studied scoring system to assess severity and predict the likelihood of mortality, as confirmed by a recent study from california [49] . although the management of cap in hiv-infected persons is specifically excluded from pneumonia guidelines from the us and europe [50] [51] [52] . treatment should follow similar principles to those in patients without hiv infection. patients with high curb-65 or psi scores have a significant risk of death and should be managed in critical care areas where possible. typical bacterial pathogens are similar to those in hiv-negative populations, with s. pneumoniae consistently found to be the most common pathogen. other significant pathogens include haemophilus influenza, klebsiella pneumoniae, staphylococcus aureus, moraxella catarrhalis and pseudomonas aeruginosa [3] . "atypical" organisms such as legionella pneumophila, mycoplasma spp. and chlamydia pneumoniae represent less than 5 % of cases, although the incidence of these infections in hiv-infected people has not been systematically evaluated. it should be remembered that opportunistic pathogens such as p. jirovecii and cryptococcus neoformans can present with acute pneumonic illnesses and these should be considered in the differential diagnosis of pneumonia in immunocompromised individuals. the incidence of mycobacterium tuberculosis infection is greatly increased in hiv-infected individuals and can present with clinical and radiological features identical to those of bacterial pneumonias (fig. 1) . the choice of empirical antibiotic treatment will differ between populations depending on local antibacterial drug resistance. in 2011, rates of penicillin resistance in s. pneumoniae in europe ranged from <1 % to 61 % [53] . some data suggest that antibiotic resistance in respiratory pathogens may be higher in hiv-infected individuals, a finding that may be related to the use of cotrimoxazole for pneumocystis pneumonia prophylaxis, or to differences in serotypes infecting hiv-positive individuals [54, 55] . mortality rates in cap differ among published series, but most fall in the range 10-15 % of cases [3] . although this has been the subject of some controversy, mortality and length of hospital stay in hiv-infected persons with pneumonia do not seem to differ from those in patients without hiv infection [56] . it should be noted that most data arise from the us and europe, and may not be applicable to resource-limited settings. recent studies have added to the evidence that blood cd4 counts do not predict mortality and should not be used to guide therapy or decisions regarding admission to intensive care units [57••] . changes in the demographic composition of hiv-infected populations may lead to changes in outcome as hiv-positive populations age. for example, information derived from hospital claims databases in the us suggest a higher case fatality rate for pneumonia and influenza in hiv-infected elderly individuals compared to those without hiv infection [58••] . pneumonia is an important cause of morbidity and mortality in hiv-infected persons and several interventions can reduce this risk. in particular smoking cessation and immunization against pneumococcus and influenza offer the opportunity to modify a patient's risk of pneumonia. hiv-infected populations continue to have high rates of exposure to cigarette smoke [59] [60] [61] . rates of pneumonia are higher in smokers than nonsmokers, and smoking cessation can reduce this [62••] . a recent systematic review of the literature by de et al. found that current smokers have an increased risk of bacterial pneumonia compared to nonsmokers (hazard ratio 1.73, 95 % confidence interval 1.44 -2.06) and that this risk is reduced by smoking cessation. in a prospective study in france, a hazard ratio of 0.48 was found in former smokers compared to current smokers (p=0.02) [11•] . the use of medication as an adjunct to smoking cessation has been extensively investigated in hiv-uninfected smokers, but there are few data from hiv-infected people. a small study of the safety and tolerability of varenicline tartrate in 36 hiv-positive individuals found a high frequency of adverse events, most commonly nausea, but excellent quit rates with a 42 % abstinence rate at 12 weeks. there were no adverse changes in hiv viral loads detected [63] . given the high incidence of invasive pneumococcal disease in hiv-infected persons, and the efficacy of pneumococcal vaccination in other at-risk groups, there has been considerable interest in immunization against pneumococcus. two forms of pneumococcal vaccination, the 23-valent pneumococcal polysaccharide vaccine (ppv-23) and pcv have been developed. ppv-23 has been studied in one randomized trial and 15 observational studies, which are the subject of a recent systematic review [64••] . this concluded that the current evidence base lends only moderate support for the efficacy of the ppv immunization. the only randomized trial was carried out in africa in a population without widespread access to art and with a high mortality rate, and found an increased rate of pneumonia in the immunized group. however, these results may not be applicable to a population with access to art and lower background rates of pneumococcal pneumonia, and most observational studies have shown that ppv-23 immunization is associated with a reduced rate of pneumococcal disease, although in many studies confounding factors that could have influenced this observation were not completely accounted for. ppv-23 suffers from relatively poor immunogenicity, particularly in those with low cd4 counts, and one study of 103 hiv-infected individuals found that antibody responses declined to pre-immunization levels after 12 months [65] . in an observational cohort study in canada, 74 % of pneumococcal serotypes isolated were from ppv-23, despite the fact that 73 % of this population had received this immunization, and vaccine failure was associated with low cd4 counts [14••] . pcv has theoretical advantages in that it induces t celldependent immune responses and has been demonstrated to be immunogenic in hiv-infected adults in several studies [66] . these have recently been reviewed by nunes and madhi who concluded that pcv is effective at reducing rates of invasive pneumococcal disease, and produces more effective and durable antibody responses in individuals on art than in those who are art-naive [67] . it should be noted that immune responses to pneumococcal vaccination are significantly better in those with a higher cd4 count [68] . apart from the possible direct benefits of pneumococcal immunization, vaccination of young children with pcv-7 (which offers protection against seven common serotypes) has been associated with indirect benefits in hiv-infected individuals. in the us, widespread use of the pcv-7 as part of the childhood immunization schedule has been associated with a 91 % reduction in invasive pneumococcal disease caused by vaccine serotypes in hiv-infected adults. these changes in pneumococcal epidemiology following routine childhood immunization have also been observed in other populations, with an 81 % reduction in invasive pneumococcal disease caused by vaccine serotypes in spain [69] . however, this study also noted an increase in severity and need for mechanical ventilation for pneumococcal pneumonia. work in south africa has also demonstrated reduced levels of pneumococcal carriage following the introduction of childhood pcv, including reductions amongst unvaccinated adults. there appeared to be reductions in nonvaccine pneumococcal serotypes which were not restricted to hiv-infected individuals [70] . analysis of data from england and wales since the introduction of childhood pcv-7 immunization suggests that there has been a 54 % reduction in invasive pneumococcal disease caused by the serotypes targeted by the vaccine [26••] . this study found that 61 % of invasive pneumococcal disease in hiv-infected patients was caused by the 13 serotypes that would be covered by the new 13-valent pcv (pcv-13). at present, guidelines from the british hivassociation (2008) recommend pneumococcal polysaccharide vaccination in hivinfected individuals with a cd4 count above 200 cells/μl whilst stating that this should be considered in individuals with lower cd4 counts and noting that efficacy may be poorer in those with low cd4 counts who are not on art [71] . the infectious disease society of america recommends the combination of pcv-13 and pneumococcal polysaccharide (ppv-23) immunizations with one dose of pcv-13 followed by ppv-23 after 8 weeks, although they suggest that this may be delayed until blood cd4 counts are above 200 cells/μl, when on art [72] . a health-economic analysis by rozenbaum et al. concluded that the addition of pcv-13 for hiv-infected adults in england would not be cost effective [73• ]. smith et al. have explored the current recommendations in the us and conclude that a single dose of pcv may be more cost-effective than the current dual immunization strategy, whilst cho et al. have report that the current recommendations would still be cost-saving [74, 75] . it should be noted that these studies of cost-effectiveness are very sensitive to the assumptions made regarding vaccine effectiveness. however, these estimates of the cost-effectiveness of pneumococcal immunization are limited by the poor uptake of the pneumococcal immunization amongst hiv-positive adults which appears to be below that of other immunizations [76] . clinical guidelines in the us and uk advise immunization of hiv-infected individuals with the influenza vaccine. a metaanalysis by beck et al. of published studies suggests that influenza vaccination is effective at reducing the incidence of influenza and is well tolerated [77] . it should be noted that respiratory comorbidities such as copd are common in hivinfected individuals and immunization of these individuals against influenza may be particularly important. recent work suggests that the immunogenicity of influenza immunization in hiv-infected persons may be improved by high-dose (four times the standard trivalent dose) vaccination [78] . whilst the increased dose was well tolerated, it was not clear whether the enhanced antibody response translates into better clinical, cost-effective protection. cap is a major cause of morbidity and mortality amongst hiv-infected individuals. although a wealth of evidence attests to the increased rates of pneumonia in immunocompromised individuals prior to the use of art, there have been clear reductions over recent years. hiv-infected populations have high rates of smoking and comorbid respiratory pathologies but current data suggest that hiv infection remains an independent risk factor for cap. at present it is not known whether hiv-infected individuals will have an increased susceptibility to new respiratory pathogens such as mers-cov. the management and outcome of pneumonia in hivinfected individuals does not appear to differ from those in hiv-uninfected persons. several interventions can be made that have been shown to reduce this risk; these include: the use of art and achievement of an undetectable plasma hiv load, smoking cessation, and the uptake of the pneumococcal and influenza immunizations, which international guidelines recommend for hiv-infected individuals. despite an increasing knowledge base there continues to be uncertainty regarding the degree of increased risk of pneumonia in hiv-infected individuals and the best option to reduce this. smoking cessation is of importance, and evidence is required as to the best immunization strategy for this population. conflict of interest marc lipman and james brown have no conflicts. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by the author. american thoracic society committee on hiv pulmonary disease. an official ats workshop report: emerging issues and current controversies in hiv-associated pulmonary diseases hiv-associated bacterial pneumonia pulmonary infections in hiv-infected patients: an update in the 21st century declining incidence of aids-defining opportunistic illnesses: results from 16 years of population-based aids surveillance declining incidences of opportunistic infections based on us aids surveillance data all-cause mortality in hospitalized hiv-infected patients at an acute tertiary care hospital with a comprehensive outpatient hiv care program in new york city in the era of highly active antiretroviral therapy (haart) the impact of haart on the respiratory complications of hiv infection: longitudinal trends in the macs and wihs cohorts hiv infection and risk for incident pulmonary diseases in the combination antiretroviral therapy era severe bacterial non-aids infections in hiv-positive persons: incidence rates and risk factors etiology of pulmonary complications of human immunodeficiency virus-1-infected patients in taiwan in the era of combination antiretroviral therapy: a prospective observational study persistent high burden of invasive pneumococcal disease in south african hiv-infected adults in the era of an antiretroviral treatment program groupe d'epidémiologie clinique du sida en aquitaine. bacterial pneumonia among hiv-infected patients: decreased risk after tobacco smoking cessation opioid drug abuse and modulation of immune function: consequences in the susceptibility to opportunistic infections epidemiologic relation between hiv and invasive pneumococcal disease in san francisco county :314. important study of the epidemiology of invasive pneumococcal disease amongst hivinfected people in canada insight-esprit study group. predictors of bacterial pneumonia in evaluation of subcutaneous interleukin-2 in a randomized international trial (esprit) dynamics of pneumococcal transmission in vaccine-naive children and their hiv-infected or hiv-uninfected mothers during the first 2 years of life icona foundation study group. incidence, timing, and determinants of bacterial pneumonia among hiv-infected patients: data from the icona foundation cohort pneumonia in hiv-infected persons: increased risk with cigarette smoking and treatment interruption incidence of and risk factors for community acquired pneumonia in us hiv-infected children the immunomodulatory effects of statins in community-acquired pneumonia: a systematic review the effect of statin therapy on pneumonia in an hivinfected population in the netherlands health protection agency. hiv in the united kingdom: 2012 report monitoring selected national hiv prevention and care objectives by using hiv surveillance data -united states and 6 u.s. dependent areas -2010 demographic patterns of hiv testing uptake in sub-saharan africa. dhs comparative reports no. 30. calverton: icf international analysis from the thin database of primary care records comparing records of 939 cases of hiv infection with 2576 controls demonstrating importance of hiv invasive pneumococcal disease among hiv-positive individuals monocyte and macrophage dysfunction as a cause of hiv-1 induced dysfunction of innate immunity bronchoalveolar cd4+ t cell responses to respiratory antigens are impaired in hiv-infected adults impaired cd4 t cell memory response to streptococcus pneumoniae precedes cd4 t cell depletion in hiv-infected malawian adults dendritic cell dysregulation during hiv-1 infection cd4-and dynamin-dependent endocytosis of hiv-1 into plasmacytoid dendritic cells cd8+ t cells and risk for bacterial pneumonia and all-cause mortality among hiv-infected women oral and airway microbiota in hiv-infected pneumonia patients invasive pneumococcal disease in patients infected with hiv: still a threat in the era of highly active antiretroviral therapy impact of human immunodeficiency virus infection on streptococcus pneumoniae colonization and seroepidemiology among zambian women streptococcus pneumoniae colonization among patients with human immunodeficiency virus-1 who had received 23-valent polysaccharide pneumococcal vaccine risk factors for pneumococcal nasopharyngeal colonization before and after pneumococcal conjugate vaccination in persons with hiv: brief report hiv and influenza virus infections are associated with increased blood pneumococcal load: a prospective, hospital-based observational study in south africa viral pathogens including human metapneumovirus are the primary cause of febrile respiratory illness in hiv-infected adults receiving antiretroviral therapy respiratory syncytial virus (rsv) community-acquired pneumonia (cap) in a hospitalized adult with human immunodeficiency virus (hiv) mimicking influenza a and pneumocystis (carinii) jiroveci pneumonia (pcp) a case of respiratory syncytial virus infection in an hiv-positive adult. case rep infect dis influenza susceptibility, severity, and shedding in hiv-infected adults: a review of the literature influenza-related mortality among adults aged 25-54 years with aids in south africa and the united states of america influenza a h1n1 in hiv-infected adults influenza and hiv: lessons from the 2009 h1n1 influenza pandemic pandemic h1n12009 influenza and hiv: a review of natural history, management and vaccine immunogenicity epidemiological, demographic and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hiv-protease inhibitors block the replication of both vesicular stomatitis and influenza viruses at an early post-entry replication step predictors of pneumonia severity in hiv-infected adults admitted to an urban public hospital pneumonia guidelines committee of the bts standards of care committee. bts guidelines for the management of community acquired pneumonia in adults: update thoracic society consensus guidelines on the management of community-acquired pneumonia in adults joint taskforce of the european respiratory society and european society for clinical microbiology and infectious diseases. guidelines for the management of adult lower respiratory tract infections risk factors for multidrug-resistant invasive pneumococcal disease in south africa, a setting with high hiv prevalence invasive pneumococcal disease in hiv-infected adults in france from 2000 to 2011: antimicrobial susceptibility and implication of serotypes for vaccination community-acquired pneumonia organization investigators. hiv infection does not influence clinical outcomes in hospitalized patients with bacterial community-acquired pneumonia: results from the capo international cohort study cd4+ cell counts and hiv-rna levels do not predict outcomes of community-acquired pneumonia in hospitalized hivinfected patients pneumonia and influenza hospitalization in hiv-positive seniors factors associated with smoking in hiv-infected patients and potential barriers to cessation smoking behaviors in a communitybased cohort of hiv-infected indigent adults the prevalence of smoking and the knowledge of smoking hazards and smoking cessation strategies among hiv-positive patients in systematic review and metaanalysis: influence of smoking cessation on incidence of pneumonia in hiv safety and tolerability of varenicline tartrate chantix(®)) for smoking cessation in hiv-infected subjects: a pilot open-label study the effectiveness of pneumococcal polysaccharide vaccination in hiv-infected adults: a systematic review rapid loss of specific antibodies after pneumococcal vaccination in patients with human immunodeficiency virus-1 infection serologic response to primary vaccination with 7-valent pneumococcal conjugate vaccine is better than with 23-valent pneumococcal polysaccharide vaccine in hiv-infected patients in the era of combination antiretroviral therapy safety, immunogenicity and efficacy of pneumococcal conjugate vaccine in hiv-infected individuals immunization against pneumococcal disease in hiv-infected patients: conjugate versus polysaccharide vaccine before or after reconstitution of the immune system (ctn-147) invasive pneumococcal disease in hiv-infected adults: clinical changes after the introduction of the pneumococcal conjugate vaccine in children temporal changes in pneumococcal colonization in a rural african community with high hiv prevalence following routine infant pneumococcal immunization british hiv association guidelines for immunization of hiv-infected adults guidelines for the prevention and treatment of opportunistic infections in hiv-infected adults and adolescents vaccination of risk groups in england using the 13 valent pneumococcal conjugate vaccine: economic analysis costeffectiveness of pneumococcal conjugate vaccination in immunocompromised adults costeffectiveness of administering 13-valent pneumococcal conjugate vaccine in addition to 23-valent pneumococcal polysaccharide vaccine to adults with immunocompromising conditions an audit of pneumococcal and hepatitis vaccination in an outpatient hiv clinic university of nottingham influenza and the immunocompromised (uniic) study group. influenza vaccination for immunocompromised patients: systematic review and meta-analysis by etiology improved immunogenicity with high-dose seasonal influenza vaccine in hiv-infected persons: a single-center, parallel, randomized trial key: cord-353895-tgn1kk07 authors: kavanagh, matthew m; katz, ingrid t; holmes, charles b title: reckoning with mortality: global health, hiv, and the politics of data date: 2020-07-03 journal: lancet doi: 10.1016/s0140-6736(20)31046-1 sha: doc_id: 353895 cord_uid: tgn1kk07 nan example, the institute for health metrics and evaluation's global burden of disease studies. 6 these estimates allow aid agencies, such as the us president's emergency plan for aids relief (pepfar) and the global fund, to justify their financing, and national politicians in both donor governments and implementing governments to take (often deservedly) credit for any progress. however, these estimates do little to help local leaders, programme managers, and clinicians, all of whom exercise far less power over budgets, to assess the effectiveness of hiv services. mature hiv programmes face the complex task of accelerating enrolment while simultaneously improving the quality of services and addressing longterm retention of people who start treatment when still asymptomatic. monitoring systems could track actual deaths and their causes, feeding these data back for use throughout the health system, but these systems have not been prioritised. the existing priorities perpetuate the long-standing challenge in global health of missing mortality data for programmatic and research purposes. 7, 8 for example, south africa's success in establishing a system for registering deaths makes it the only country in sub-saharan africa with a classification higher than very low in vital registration capacity. 9 the absence of data on actual mortality at the programme and clinic level, whether all-cause mortality or aids-related mortality, has undermined the aids response on several fronts. most fundamentally, it has led to death being undervalued as an outcome in hiv programmes. many clinics and programmes track in this context, a broad set of patients have for years been categorised as lost to follow-up, conflating all individuals who transfer to another clinic or disengage from care and stop treatment with individuals who have actually died. this amalgamation of different groups is a problem not just in hiv but in multiple efforts to fight disease, from tuberculosis to cardiovascular disease and diabetes. 10, 11 one study that traced a sample of people lost to followup in uganda, kenya, and tanzania found that 27% of these individuals had actually died, three times more than clinic-level data suggested. 12 in another sample of people who initiated antiretroviral therapy in 22 african countries, about 15% had died within 5 years, 2·5 times the rate shown in clinic records. 13 although distinguishing aids mortality from non-aids mortality is difficult in lowresource settings, tracking all-cause mortality among people living with hiv at a granular level will yield actionable insights, even as greater diagnostic capacity is built. these measures can also support integration efforts by increasing awareness of the numerous so-called silent deaths due to non-hiv causes, such as cardiovascular disease. one of the most important implications of the paucity of data of local mortality is that programmes do not sufficiently prioritise interventions for people with advanced disease. studies in south africa, kenya, zambia, and the democratic republic of the congo have shown that most patients with hiv admitted to hospital have already been on antiretroviral therapy (often for years) but they either stop treatment or are on a treatment regimen that is not effectively suppressing the virus. 14-16 a high proportion of patients die because of hiv-related illnesses that could have been prevented. a set of evidence-based clinical interven tions has been recommen ded by who to prevent this mortality, including point-of-care cryptococcal screening and tuberculosis urine lipoarabinomannan screening, along with prophylactic and preventive treatment. however, uptake has been slow and insufficient in a context of scarce resources. 17 data on localised and specific mortality could provide a basis for programme managers to target resources to clinics, regions, or populations where these interventions are most needed. without granular mortality data, comparisons between different sites, regions, and subpopulations cannot be made. a study across four provinces in zambia, for example, found that some clinics had mortality rates greater than ten times those of the best performing clinics; a degree of heterogeneity that could be related to clinical, structural, or other factors. 18 health leaders who have access to reliable mortality information can identify and learn from successful programmes and apply these findings to programmes that underperform. generating information to be used at the low levels of the health-care system, by clinic managers, district-level public health officials, and local community groups, has not been a political priority. generating this kind of information is also not simple. medical record systems in low-income and middle-income countries remain weak. many deaths among people living with hiv occur outside the health-care system. yet action is even more urgent in situations where deaths are concentrated among the individuals who are hardest to reach. 1 we see at least three opportunities to address these challenges and enable timely mortality tracking, alongside efforts to prevent unnecessary deaths. first, progress in countries as diverse as south africa, malaysia, nicaragua, and fiji shows that developing more robust vital registries is possible within a few years and with relatively small amounts of funding. in south africa, in particular, tracking the mortality of young people using systems at the local level helped monitor the effectiveness of hiv programmes. low-cost efforts to create sample vital registration systems with verbal autopsy also show promise. 8 importantly, these efforts have the advantage of supporting all health programmes. hiv funders, including pepfar and the global fund, should partner with national governments and health funders, such as the uk department for international development and world bank, to develop and strengthen vital registries, building on momentum from who, unicef, and others. 19 second, robust patient-tracing activities should be incorporated as core objectives of all hiv programmes and funded accordingly. actual mortality rates should be key programme indicators, made easier by investments in vital registration. pepfar took the bold step, in 2019, of requiring the programmes it funds to report on hiv mortality. 20 hopefully, this step will improve patient outcomes by incentivising effective interventions for advanced hiv disease and support for people who have stopped treatment to re-enter care. 17 third, we can move towards a variety of outcomeoriented global health programmes beyond hiv, for which measures of success move from the number of patients receiving services to explicit reductions in mortality rates. some maternal health efforts have shown it is possible to uncover and address the causes of maternal deaths, but many have struggled to have a widespread effect. these approaches face similar challenges to hiv with regard to data limitations, adequate scaling, sufficient resources, and a high number of deaths outside facilities. 21 other compelling examples, such as the public-private saving mothers giving life programme, show how focusing on maternal mortality rates and leveraging systems developed for the hiv response could improve facilities and increase demand. initiated by the obama administration, the programme helped decrease maternal mortality by 41% in regions of uganda, and 44% in regions of zambia before it was discontinued by the trump administration. 22 these programmes should be revived and expanded. broader global health efforts can learn from the hiv experience. mortality estimates based on aggregate national mod elling can be influential in drawing political attention to these issues, and in encouraging collective action and making high-level decisions on the allocation of resources. however, as effective programmes are scaled up, mortality is likely to become more localised, heterogeneous, and less susceptible to single interventions. from emerging infectious diseases to cancer and mental health diseases, this path is likely to be similar. as efforts to address non-communicable diseases in low-income and middle-income countries gain momentum, they are likely to include provision of long-term biomedical interventions such as anti-hypertensives for high blood pressure and oral hypoglycaemic medi cations for diabetes. tracking actual mortality and building the necessary capacity to identify the causes of death will be key for programmes to fully account for the progress that is made and motivate filling gaps in the quality of services. making the shift to reporting on and addressing actual mortality will require a political reorientation by funders towards prioritising information that might be less useful to them than high-level modelled estimates but essential for the people designing and implementing frontline programmes. the hiv response could lead this change by committing resources as part of an effort to regain the momentum against preventable deaths. this commitment will require a collective effort to mobilise the political will needed to improve and empower decisionmaking and a renewed focus on what ultimately matters most to the individuals in the pandemic's path-avoiding unnecessary deaths. global aids update: communities at the centre institute for health metrics and evaluation. global burden of disease compare beyond precision: embracing the politics of global health numbers generation of political priority for global health initiatives: a framework and case study of maternal mortality metric partnerships: global burden of disease estimates within the world bank, the world health organisation, and the institute for health metrics and evaluation counting the dead and what they died from: an assessment of the global status of cause of death data reliable direct measurement of causes of death in low-and middle-income countries a global assessment of civil registration and vital statistics systems: monitoring data quality and progress improved retention rates with low-cost interventions in hypertension and diabetes management in a rural african environment of nurse-led care: a cluster-randomised trial loss-to-follow-up on multidrug resistant tuberculosis treatment in gujarat, india: the when and who of it estimation of mortality among hiv-infected people on antiretroviral treatment in east africa: a sampling based approach in an observational, multisite, cohort study retention and mortality on antiretroviral therapy in sub-saharan africa: collaborative analyses of hiv treatment programmes high proportions of patients with advanced hiv are antiretroviral therapy experienced: hospitalization outcomes from 2 sub-saharan african sites hiv-related medical admissions to a south african district hospital remain frequent despite effective antiretroviral therapy scale-up care continuum and postdischarge outcomes among hiv-infected adults admitted to the hospital in zambia guidelines for managing advanced hiv disease and rapid initiation of antiretroviral therapy. geneva: world health organization estimated mortality on hiv treatment among active patients and patients lost to follow-up in 4 provinces of zambia: findings from a multistage samplingbased survey the future for women and children: unicef and who joint statement on strengthening civil registration and vital statistics (crvs) president's emergency plan for aids relief. monitoring, evaluation, and reporting indicator reference guide maternal death surveillance and response: a tall order for effectiveness in resource-poor settings impact of the saving mothers, giving life approach on decreasing maternal and perinatal deaths in uganda and zambia all authors contributed to conceptualising, drafting, and editing the manuscript. mmk received a grant for a joint policy mapping project with unaids. all other authors declare no competing interests. key: cord-347356-uc9dqhyq authors: cooper, tj; woodward, bl; alom, s; harky, a title: coronavirus disease 2019 (covid‐19) outcomes in hiv/aids patients: a systematic review date: 2020-07-15 journal: hiv med doi: 10.1111/hiv.12911 sha: doc_id: 347356 cord_uid: uc9dqhyq objectives: the aim of the study was to systematically review current studies reporting on clinical outcomes in people living with hiv (plhiv) infected with severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2). methods: we conducted a systematic review using the preferred reporting items for systematic reviews and meta‐analysis (prisma) guidelines. a comprehensive literature search was conducted in global health, scopus, medline and embase using pertinent key words and medical subject headings (mesh) terms relating to coronavirus disease 2019 (covid‐19) and hiv. a narrative synthesis was undertaken. articles are summarized in relevant sections. results: two hundred and eighty‐five articles were identified after duplicates had been removed. after screening, eight studies were analysed, totalling 70 hiv‐infected patients (57 without aids and 13 with aids). three themes were identified: (1) controlled hiv infection does not appear to result in poorer covid‐19 outcomes, (2) more data are needed to determine covid‐19 outcomes in patients with aids and (3) hiv‐infected patients presenting with covid‐19 symptoms should be investigated for superinfections. conclusions: our findings suggest that plhiv with well‐controlled disease are not at risk of poorer covid‐19 disease outcomes than the general population. it is not clear whether those with poorly controlled hiv disease and aids have poorer outcomes. superimposed bacterial pneumonia may be a risk factor for more severe covid‐19 but further research is urgently needed to elucidate whether plhiv are more at risk than the general population. in december 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), emerged in the city of wuhan, china. sars-cov-2 causes coronavirus disease 2019 (covid19) , which has resulted in the most catastrophic pandemic in modern history [1] . presentation can be asymptomatic or consist of mild symptoms, from cough and fever to severe and lifethreatening acute respiratory distress syndrome (ards), sepsis, multi-organ failure and death [2] . there is no current specific treatment for covid-19 but rather organ support is provided, and severe cases require admission to hospital for supportive management including mechanical ventilation. evidence is emerging that suggests that increasing age, hypertension and diabetes are risk factors that correlate with worse outcomes [3, 4] . however, it is not clear if people living with hiv (plhiv) are at greater risk than the general population [5] . left untreated, hiv infection results in a reduced number of cd4 t cells, leading to aids. aids is defined as a cd4 t-cell count < 200 cells/ ll [3] or the presence of an aids-defining illness [6] . in 2018, it was estimated 37.9 million people worldwide have hiv infection, 23.3 million of whom are on treatment with antiretroviral therapy (art) [7] . eighty-six per cent of those on treatment have successful viral suppression, resulting in undetectable viral load and untransmissible disease, known as u = u [8] [9] [10] . if art is maintained and adhered to, plhiv are not immunocompromised. [11] . despite this, plhiv may be at risk of severe covid-19, especially in areas where hiv infection is poorly controlled. limited evidence is available on the impact of hiv on sars-cov-2 infection and on whether it has any effect on covid-19 outcomes [12] . there is a need to understand whether plhiv are at greater risk of severe illness so that adequate preventative measures can be put in place. the aim of this systematic review was to identify studies that discuss plhiv who have been infected with sars-cov-2 and that report whether coinfection results in a greater risk of adverse outcomes and, furthermore, whether controlled hiv infection vs. uncontrolled hiv infection or aids results in different covid-19 disease outcomes. we define controlled hiv infection as an undetectable viral load and a cd4 count ≥ 200 cells/ll. a comprehensive literature search was carried out in global health, scopus, medline and embase to identify articles that discussed hiv-positive patients and the clinical implications of hiv infection in covid-19 in accordance with the preferred reporting items for systematic reviews and meta-analysis (prisma) guidelines [13] . keywords were deconstructed into two categories (table 1) . pertinent keywords and medical suject headings (mesh) terms related to these categories were used to maximize the output from the literature search. all relevant articles were identified and screened by two authors; the results are summarized in a narrative manner in each relevant section within the text of this review. inclusion and exclusion criteria are outlined in table 2 . studies were included if they discussed the correlation between confirmed hiv infection and the diagnosis or prediction of severity of covid-19. all articles were screened by two authors and any disagreement was resolved by consensus or the involvement of a third author. data were extracted by two authors and validated by a third author. the quality of each publication was evaluated by two independent reviewers according to a predefined scoring system, using the national heart lung and blood institute (nih) quality assessment tool for case series and casecontrol studies, as appropriate (table 3 ) [14] . it was not possible to conduct an appropriate meta-analysis because there were not enough research data in the studies on this subject. a prisma flow chart for the literature search is shown in figure 1 . a total of 445 articles were found. after removal of duplicates, a total of 285 articles were used for full-text screening and, finally, only eight studies were included in our analysis. table 3 summarizes the study characteristics and the data extracted. a narrative "coronavirus" or "ncov*" or "2019-ncov" or "covid*" or "sars-cov*" hiv "hiv" or "human immunodeficiency virus*" or "aids" or "acquired immunodeficiency syndrome" • median was higher than the median age of wuhan; however this was consistent with the epidemic in wuhan. 8 and required intubation for 4 days. • the patient made a full recovery synthesis was conducted as a consequence of the qualitative nature of the studies analysed. the themes that emerged were (1) controlled hiv infection does not appear to result in poorer covid-19 outcomes than those found in the general population, (2) more data are needed for covid-19 outcomes in aids patients and (3) hiv-infected patients presenting with covid-19 symptoms should be investigated for superinfections. these themes are explored in a narrative manner in the sections below. of the studies analysed, one case report reported a difference between the symptoms of covid-19 in plhiv and the symptoms of covid-19 as defined by the who [15] . haddad et al., reported encephalopathy and seizure activity in a patient with controlled hiv on day 8 of their symptoms with covid-19. this was not seen in any other studies, and the patient made a full recovery [16] . the other studies included in our review all reported that symptoms of covid-19, such as cough, fever, malaise and breathlessness, in plhiv were not dissimilar to the normal population [15] . this indicates that co-infection hiv and sar-cov-2 does not appear to cause a different presentation. two studies reported that one patient in wuhan was asymptomatic despite a positive rt-pcr test for sar-cov-2 rna from a nasopharyngeal swab [17, 18] . no other study tested asymptomatic plhiv and therefore it is not clear what proportion of plhiv experience asymptomatic infection and how this compares to the general population. the rate of asymptomatic infection in plhiv is likely to be underestimated. blanco et al. [19] , studied five patients in barcelona; they report four patients with controlled hiv, three recovered from covid-19, one patient did not need treatment and recovered well, whilst another patient had developed severe covid-19 and required mechanical ventilation in icu. this patient was 49 with hypothyroidism. all patients were on art before they were admitted. h€ arter et al. [20] , investigated the outcomes of 32 patients coinfected with hiv and sars-cov-2 in a retrospective case series in germany. ninety percent of the cohort made a full recovery with 76% experiencing mild symptoms, while the reported mortality was 9%. mortality, hospitalisation and critical case rate was higher than covid-19 (six via nasopharyngeal swab and two via ct chest). this represented 0.68% of patients with hiv who were surveyed, slight but not significantly higher than the percentage of people diagnosed with covid-19 in wuhan (0.5%). outcomes for were that six recovered fully after mild illness, one experienced severe illness but recovered and one patient died. all patients had hiv controlled with art. neither study was controlled or matched for hiv negative patients, however the mortality rates do not appear to be different to those in the general population in wuhan [21] . zhao et al. [21] , also highlighted a case study of a hiv patient with sars-cov2 co-infection, diagnosis of viral pneumonia was made on clinical examination and chest ct findings. three nasopharyngeal swabs sars-cov-2 were negative. sars-cov-2 infection was confirmed with antibody testing at a later date. the patient made a full recovery without the need for admission to icu or ventilation. furthermore, karmen-tuohy et al. [22] showed no statistical difference in outcomes of covid-19 between plhiv and the general population. twenty-one patients with hiv and sars-cov-2 co-infection were matched to 42 hiv negative patients with sars-cov-2, with no difference in demographics or comorbidities. twenty of the 21 patients with hiv had normal cd4 t-cell counts and were virally suppressed. these patients are not representative of aids patients. blanco et al. [19] , reported one case of sars-cov-2 in a patient who was art na€ ıve and a late diagnosis of hiv. the patient presented with a cd4 t-cell count of 13 cells/mm 3 and had superimposed pneumocystis jirovecii pneumonia (an aids defining organism) but no other comorbidities [23] . the patient was admitted to icu and required non-invasive ventilation (niv), responded well to treatment with oxygen, antibiotics, corticosteroids and hydroxychloroquine and was discharged after 12 days. h€ arter et al. [20] , reported that one patient out of three who died had a low cd4 t-cell count of 69 cells/mm 3 with no other stated comorbidities. the study also included three other patients with low cd4 t-cell counts who recovered from covid-19, but no further information regarding treatment, disease was given. yang et al. [18] , presented a case of a patient with a low cd4 t-cell count (21 cells/mm 3 ) and kaposi's sarcoma; this patient was also discussed by guo et al. [17] the patient tested positive for sars-cov-2 and remained asymptomatic until two negative tests, with no abnormal findings on ct chest. in contrast, wang et al., reported a patient with 34 cd4 t cells/mm 3 who experienced a 2-month disease course, far exceeding the typical natural history of covid-19 [24] . viral pneumonia was confirmed via ct chest but sars-cov-2 rt-pcr was negative on four occasions. one positive test for orf1ab (but not the n-gene) confirmed sars-cov-2. the patient tested negative twice for sars-cov-2 antibodies, but later tested positive for igm antibodies, indicating a delayed immune response which may explain the prolonged disease course. it is not clear if there is an increased risk of worse outcomes of covid-19 for aids patients. outcomes in included studies have ranged from asymptomatic infection to death. until more good quality, case-controlled studies are produced, conclusions cannot be drawn on this point. we found reports of superimposed bacterial pneumonia with covid-19. these patients generally had poorer outcomes irrespective of controlled hiv or aids. blanco et al. [19] , described a case of pneumocystis jirovecii in a patient with aids and covid-19 which responded well to antibiotics, as discussed earlier in this review. they raised the issue of ensuring that pulmonary opportunistic infections are considered in the differential diagnoses of sars-cov-2 and hiv co-infection, particularly in aids patients. this was supported by karmen-tuohy et al. [22] , who found three patients with hiv and covid-19 developed superimposed non-aids related bacterial pneumonia, compared to only one hiv negative patient with covid-19. all of the patients who developed a superimposed bacterial pneumonia died in this study, despite receiving antibiotic treatment. whilst this is a small cohort of patients, it highlights potentially worse outcomes for patients who have a superimposed bacterial pneumonia with covid-19. patients with hiv show a higher incidence of bacterial pneumonia, which is inversely proportional to cd4 t-cell count, when compared to the general population. thus, superimposed bacterial pneumonia with covid-19 is a significant consideration in plhiv [25] [26] [27] [28] [29] . at present, the covid-19 guidelines for well-controlled hiv infection state that it is unlikely that plhiv are at any greater risk of contracting covid-19 or experiencing more severe disease than the general population [12, 13] . our findings support this conclusion, but the data are currently very limited. the recommendations in both the uk and usa are in line with the infection control measures provided to the general public [30] . specific recommendations include ensuring at least a 30-day supply of art and advising up-to-date vaccinations including pneumococcal and influenza vaccines [12] [13] [14] . clinicians should reassure plhiv that, if the hiv infection is controlled, their risk of serious complications of covid-19 and therefore poor outcomes is likely to be low. however, the presence of known covid-19 risk factors may put them at greater risk of worse outcomes. the same cannot be said for poorly controlled hiv infection or aids. in previous outbreaks of sars, h1n1 and middle east respiratory syndrome (mers), hiv infection was not associated with increased disease severity. in the sars and mers outbreaks, there were only a few reports of mild disease among plhiv [31, 32] . the risk factors for these outbreaks were similar to those being observed for sars-cov-2. h1n1 outcomes were similar to outcomes for sars-cov-2, whereby clinical outcomes of those with well-controlled hiv infection were similar to those of the general population [33, 34] . a literature review by cooper [35] further highlighted that hiv-infected patients and patients who were immunocompromised for other reasons recovered from h1n1 infection without complications. sars-cov-2 cannot be presumed to be the same as these viruses. we do not know enough about its mechanisms of action to be sure of the impact of hiv infection in those coinfected with sars-cov-2 and hiv. however, data from previous outbreaks are reassuring at least in that well-controlled hiv infection did not pose a greater risk of more severe disease compared with the general population. we identified a small number of studies specifically looking at plhiv and covid-19. we identified two patients who were diagnosed with hiv infection at the same time as sars-cov-2 infection who were art na€ ıve [19, 24] . both patients had low cd4 t-cell counts. in 2018, an estimated 8.1 million cases of hiv infection globally were undiagnosed and 28% of patients known to be infected were not on treatment [7] . we recommend that, if a clinical picture suggests viral pneumonia, such as symptoms of covid-19 or typical viral pneumonitis on a computed tomography (ct) scan (including mild and severe cases of , patients should be offered testing for hiv to rule out undetected hiv infection. we found a disparity in methods of covid-19 diagnosis between studies. in six studies, sars-cov-2 was confirmed by reverse transcriptase-polymerase chain reaction (rt-pcr) [16] [17] [18] [19] [20] 22] , and in two studies covid-19 was diagnosed using ct thorax findings [17, 21] .in order to ensure that the diagnosis of covid-19 is correct and other differential diagnosis are ruled out , we suggest that future studies reporting detection of the virus should use a consistent method of diagnosising covid-19. also, other causes of pneumonia should be screened for and ruled out to ensure that data are accurate both to confirm the causative agent and to identify any coinfection that may exacerbate symptoms and severity of covid-19. viral pneumonia is generally more severe if a concurrent bacterial pneumonia is present, in both the normal population and plhiv. this is a plausible reason why some patients included in this review had more severe covid-19 [25] [26] [27] [28] [29] . we recommend that sputum and blood cultures should be taken early for detection of superimposed bacterial pneumonia and the presence of other causative agents. the continuation of out-patient care for plhiv presents unique challenges as hospitals deal with covid-19 patients. plhiv could be deterred from accessing hiv care as a consequence of the perceived risks of attending hospitals, and also the risk that out-patient services may be affected by the re-deployment of health care professionals to other areas of the hospital. this presents an opportunity for the advancement of telemedicine, the remote diagnosis and treatment of a patient using technology. young et al. [36] and ohl et al. [37] both found that, where telemedicine was available to patients, greater viral suppression was achieved. rogers et al. [38] have described an effective implementation of telemedicine in a clinic in the usa, to ensure continuity of care during the covid-19 pandemic. clinics who implemented telemedicine also reported fewer missed appointments and higher patient engagement. furthermore, studies showed that text message services resulted in significant improvements in art adherence and rates of viral suppression in sub-saharan africa [39, 40] . telemedicine can therefore be effectively used to manage patients with hiv infection, without the need for them to attend clinic, allowing patients to avoid unnecessary journeys to hospitals and clinics and to maintain social distancing. having said this. as stressed by mgbako et al. [41] , this must be led by a patient-centred approach, ensuring good communication as well as a good professional-patient relationship. the disruption in the continuity of care for plhiv, increased social isolation and the psychological stress of living through a pandemic are all factors that could worsen the mental health problems that plhiv are at a higher risk of experiencing [42] . plhiv are more likely to experience social isolation; however, as a consequence of measures to prevent the spread of covid-19, this experience may be amplified, which may have a negative effect on mental health [43] . furthermore, covid-19 will potentially exacerbate other psychological stressors such as food insecurity and increased societal stigma [42, 44, 45] . these psychosocial stressors further may increase the risk of adverse health outcomes among plhiv as a consequence of reduced medication adherence, leading to failure to achieve adequate hiv management [42] . whilst further high-quality studies would be beneficial to confirm that there is no difference in outcomes of covid-19 between plhiv who have controlled hiv infection and the general population, as a consequence of the gap in knowledge about outcomes in aids patients with covid-19, there needs to be an urgent focus on high-quality research looking at outcomes for patients with aids and covid-19. furthermore, more research into the impact and incidence of superimposed bacterial pneumonia is needed in both the general population and plhiv. this would help to influence management and provide a good basis for developing treatment pathways that may reduce the trend towards higher mortality. the limitations of this review were the small number of studies included in our analysis that were relevant to our outcomes. the studies included were all case series or case reports with small sample sizes, and confounding variables were not accounted for in the reporting of such data. we acknowledge that, in view of the nature of the study designs included in our analysis, our interpretations and conclusions should be treated with caution. our findings indicate that currently plhiv are not likely to be at increased risk of poorer outcomes of covid-19 disease than the general population if they have an undetectable viral load and an adequate cd4 count. this suggests that well-managed hiv infection is not a risk factor for more severe covid-19. however, it is unknown if poorly controlled hiv infection and aids put people at a greater risk of severe covid-19. bacterial superinfection appears to be a potential risk factor for poorer outcomes in plhiv. therefore, our findings highlight the need for further investigation to elucidate the impact of hiv infection in covid-19. conflicts of interest: none to be declared. financial disclosure: none to be declared. european centre for disease prevention and control. event background covid-19 covid-19 and multiorgan response covid-19: risk factors for severe disease and death risk factors of critical & mortal covid-19 cases: a systematic literature review and meta-analysis rethinking high-risk groups in covid-19 undetectable = untransmittable and your health: the personal benefits of early and continuous therapy for hiv infection global statistics partners of people on art -a new evaluation of the risks (the partner study): design and methods multicentre, prospective, observational study art in hiv-positive persons with low pretreatment viremia: results from the start trial sars-cov-2 and hiv interim guidance for covid-19 and persons with hiv covid-19 and persons with hiv preferred reporting items for systematic reviews and meta-analyses: the prisma statement study quality assessment tools encephalopathy and seizure activity in a covid-19 well controlled hiv patient the reflection on an aids patient with asymptomatic covid-19 -abstract -europe pmc covid-19 in patients with hiv: clinical case series covid-19 in people living with human immunodeficiency virus: a case series of 33 patients early virus clearance and delayed antibody response in a case of coronavirus disease 2019 (covid-19) with a history of coinfection with human immunodeficiency virus type 1 and hepatitis c virus outcomes among hiv-positive patients hospitalized with covid-19 hiv-associated pneumocystis pneumonia case report: one case of coronavirus desease 2019(covid-19) in patient co-nfected by hiv with a low cd4+ t cell count community-acquired bacterial pneumonia in adult hivinfected patients global strategies to prevent bacterial pneumonia in adults with hiv disease severe bacterial nonaids infections in hiv-positive persons: incidence rates and risk factors bacterial pneumonia, hiv therapy, and disease progression among hiv-infected women in the hiv epidemiologic research (her) study mortality and causes of death in people diagnosed with hiv in the era of highly active antiretroviral therapy compared with the general population: an analysis of a national observational cohort covid-19) and hiv -update from the british hiv association (bhiva) mers coronavirus outbreak: implications for emerging viral infections network-based analysis of comorbidities risk during an infection: sars and hiv case studies potential impact of sars-cov-2 infection in hiv-positive patients in south africa influenza and hiv: lessons from the 2009 h1n1 influenza pandemic pandemic h1n12009 influenza and hiv: a review of natural history, management and vaccine immunogenicity improved virologic suppression with hiv subspecialty care in a large prison system using telemedicine: an observational study with historical controls impact of availability of telehealth programs on documented hiv viral suppression: a cluster-randomized program evaluation in the veterans health administration development of telemedicine infrastructure at an lgbtq+clinic to support hiv prevention and care in response to covid-19 effect of mobile phone reminders on follow-up medical care of children exposed to or infected with hiv in cameroon (more care): a multicenter, single-blind, factorial, randomized controlled trial effects of a mobile phone short message service on antiretroviral treatment adherence in kenya (weltel kenya1): a randomized trial covid-19, telemedicine, and patient empowerment in hiv care and research the burden of covid-19 in people living with hiv: a syndemic perspective physical distancing in covid-19 may exacerbate experiences of social isolation among people living with hiv the lancet hiv. the syndemic threat of food insecurity and hiv challenges to hiv care and psychological health during the covid-19 pandemic among people living with hiv in china key: cord-318363-1mv5j4w2 authors: zvolensky, michael j.; garey, lorra; rogers, andrew h.; schmidt, norman b.; vujanovic, anka a.; storch, eric a.; buckner, julia d.; paulus, daniel j.; alfano, candice; smits, jasper a.j.; o'cleirigh, conall title: psychological, addictive, and health behavior implications of the covid-19 pandemic date: 2020-08-27 journal: behav res ther doi: 10.1016/j.brat.2020.103715 sha: doc_id: 318363 cord_uid: 1mv5j4w2 • the public health impact of covid-19 on psychological symptoms and disorders, addiction, and health behavior is substantial and ongoing. • an integrative covid-19 stress-based model could be used to guide research focused on the stress-related burden of the pandemic. • this work could provide a theoretical and empirical knowledge base for future pandemics. around some of the most clinically important psychological disorders, addictive behaviors, and health behaviors for well-being. in the first section, we describe the covid-19 implications for mental health focusing on (a) anxiety/stress and mood disturbance, (b) obsessive compulsive symptoms and disorders, and (c) posttraumatic stress. such mental health problems, although certainly not exhaustive of the scope of psychological disorders impacted by covid-19, are some of the most common mental health issues in the general population and are frequently comorbid with chronic illness. in the second section, we focus on addictive behaviors, including (d) tobacco (combustible and electronic), (e) alcohol use and misuse, and (e) cannabis. these forms of drug use represent the most prevalent types of substance use and are frequently associated with chronic illness and premature death. in the third section, we spotlight health behavior and chronic illness by discussing the role of (f) sleep health and behavior, (g) chronic illness using the example of hiv/aids as an illustrative model, and (h) physical activity. health behaviors represent vital targets for the mitigation of covid-related disease and may play a key role in psychological adjustment and recovery. in the final section, we highlight sociocultural factors (e.g., race/ethnicity, economic adversity), developmental considerations, and the role of individual difference factors for psychological, addictive, and health behavior and chronic illness. we conclude by offering an integrative covid-19 stress-based model that could be used to guide research focused on the stress-related burden of the pandemic. fear is an adaptive defense mechanism that is fundamental for survival and involves several psychological and biological processes of preparation for a response to potentially j o u r n a l p r e -p r o o f threatening events. covid-19 represents a true threat, with many unknowns. if you are infected, there is a chance you could die, regardless of your current age, sex, or health status. as such, fear is a natural and adaptive response to this pandemic. on the other hand, every year tens of thousands die from influenza as well as many other preventable or unexpected causes. this raises the key question regarding the degree to which we should be anxious and fearful of . how much anxiety is reasonable? since even basic knowledge about covid-19 is undeveloped, it will be difficult to clearly discriminate between normal, adaptive fear responses and less adaptive responses. that said, such an overarching true threat and the concomitant stressors such as social isolation, economic uncertainty and so forth could in fact recalibrate what is considered a normal level of anxiety in the general population. research has demonstrated that trait levels of anxiety have increased in the us in recent decades, though the cause of such increases is unknown (twenge, 2000) . the covid-19 pandemic is likely to contribute to these basic levels of trait anxiety, thus creating a "new normal" level of anxiety. if we consider the likely general increase in anxiety and stress in the context of diathesisstress conceptualizations of mental illness, we expect that such a salient and broad reaching stressor to increase the incidence of pathological anxiety. anxiety conditions are already highly prevalent (bandelow & michaelis, 2015) , and we may see an increased incidence of anxiety psychopathology if the pandemic serves to push vulnerable individuals toward the expression of maladaptive levels of anxiety. moreover, those with preexisting conditions are likely to have their symptoms intensify. one could further speculate that forms of pathological anxiety will increase. first responders and hospital personnel, particularly in affected areas are already showing troubling signs of stress and psychopathology (joob & wiwanitkit, 2020) . it is highly j o u r n a l p r e -p r o o f likely that we will see increased rates of generalized anxiety and posttraumatic stress related to the pandemic and its sequelae. beyond the somewhat vague notion of covid-19 acting as a stressor to increase both normal and pathological anxiety, it is interesting to consider the specific mechanisms that play a role in this process. there are several well-established parameters that relate to the genesis and maintenance of anxiety that seem highly relevant to the current situation. these processes include perceptions relating to predictability/certainty and controllability of threat (barlow, 2004) . coming across a shark while swimming is quite different from viewing the same shark in an aquarium since a potential threat in the wild is far less predictable or controllable than one in an enclosure. historically, epidemics and pandemics were considered divine punishments that were essentially uncontrollable. although medical understanding of pathogens has advanced, globalization now facilitates the spread of pathological agents, which diminishes the degree to which we can control them. similarly, naturally occurring mutations and adaptation of viruses ensure that novel pathogens like covid-19 will emerge and spread. these conditions leave us in a state of uncertainty, except that we can be certain that covid-19 and other infectious agents will persist. thus, covid-19 affects many of the core anxiety generating mechanisms since it leads to a sense of diminished predictability and controllability along with increased uncertainty relating to a true threat. ultimately, the covid-19 pandemic creates an ideal environment for the onset, maintenance, and exacerbation of anxiety symptoms and syndromes. the dsm-5 posttraumatic stress disorder (ptsd) criterion a (american psychiatric association [apa] , 2013, p.271) defines trauma as "exposure to actual or threatened death." individuals who are closer to that exposure --providing healthcare to those infected, witnessing j o u r n a l p r e -p r o o f the deleterious and perhaps deadly effects of the virus on patients or loved ones, enduring losses of patients, family, or friends --might experience the crisis as potentially traumatic. people on the frontlines of the pandemic, including healthcare personnel, first responders, grocery store clerks, and other essential workers, encounter the threat of possible exposure to the virus regularly and on an ongoing basis. similarly, incarcerated populations and those who might feel compelled, financially or otherwise, to work in close quarters without adequate personal protective equipment (e.g., factory workers) may be exposed to the covid-19 virus for extended periods without perceived or actual recourse and suffer negative mental health repercussions as a result. covid-19 survivors, particularly those who might have struggled through various medical procedures and prolonged hospitalizations, may emerge with unique or shared constellations of mental health reactions from risk to resilience. additional high-risk groups include healthcare professionals or first responders who may have experienced significant moral injuries (jinkerson, 2016; joannou, besemann, & kriellaars, 2017; williamson, stevelink, & greenberg, 2018 ) as a result of making unfathomable decisions on the job (e.g., providing admission or ventilator access to one patient at the sacrifice of another). yet, in addition to considering direct impacts of the novel covid-19 virus on our population, it is imperative to understand the secondary potentially traumatic effects of the pandemic on individuals and communities. the combination of prolonged stress, close quarters, and self-isolation guidelines has increased risk of domestic violence, child abuse, and substance use (abramson, 2020; national institute on drug abuse, 2020; santhanam, 2020; taub, 2020) . indeed, physical and sexual violence may escalate without the regular societal checks provided by employers, schools, and loved ones. furthermore, such violence may stem from and/or intensify more unbridled substance use (carter et al., 2020) emerging from a context where j o u r n a l p r e -p r o o f uncertainty and unpredictability are high, practical stressors (e.g., unemployment, financial stress, food insecurity) may be difficult to problem-solve, and social supports may be distant. furthermore, in this pandemic, issues of grief and loss are inevitably interwoven with those of potential trauma. spiritual and emotional grief processes to honor and emotionally mourn the losses of loved ones may be interrupted by this pandemic, potentially exacerbating or prolonging grief, traumatic bereavement, or ptsd reactions. to understand the effects of covid-19 on the mental health of those who experience it as potentially traumatic, we need to recognize first that the impacts of trauma may not be fully determined nor completely recognizable until after the traumatic stressor has concluded. the covid-19 crisis is going to have a long, yet undetermined course, and thus our ongoing reactions to it are dynamic but indicative of peri-traumatic rather than post-traumatic coping (bell, boden, horwood, & mulder, 2017; lapid pickman, greene, & gelkopf, 2017) . based upon decades of research, we can expect the majority of the population, regardless of level of proximity to or interaction with covid-19, to demonstrate resilience and to recover psychologically in the aftermath of the pandemic (alisic et al., 2014; kilpatrick et al., 2013) . a relative minority, the proportions of which are unknown, may emerge from the crisis with clinical or subclinical ptsd or with exacerbation in pre-existing ptsd symptoms and related mental health conditions (e.g., depression, substance use disorder). women are at heightened risk of ptsd following potentially traumatic events (gaffey et al., 2019; rattel et al., 2019) and racial/ethnic minority populations may be especially impacted due to socioeconomic inequities and health-related disparities with regard to financial security and access to healthcare and treatment (asnaani & hall-clark, 2017; cross et al., 2018; sibrava et al., 2019) . the intersections of trauma and the covid-19 pandemic are complex. many constellations of interweaving risk and protective factors, learning histories, and life circumstances can affect how trauma histories and potentially traumatic experiences during the covid-19 crisis can affect individual journeys of recovery. for example, more unbalanced, negative individual interpretations of the covid-19 crisis and related changes in beliefs about oneself, others, or the world may have lasting deleterious effects (e.g., "i am damaged"; "people cannot be trusted"; "the world is dangerous and unsafe"; beierl, böllinghaus, clark, glucksman, & ehlers, 2019; bernardi & jobson, 2019; köhler, goebel, & pedersen, 2019; losavio, dillon, & resick, 2017; scher, suvak, & resick, 2017) . similarly, avoidance of thoughts or emotions related to the covid-19 crisis may increase the risk of developing ptsd symptoms and/or exacerbating or maintaining pre-existing trauma-related symptoms (e.g., orcutt, reffi, & ellis, 2020) . additional risk factors for the development or exacerbation of ptsd symptoms include a prior history of trauma or mental health disturbances, depressed or anxious mood, significant concurrent life stressors (e.g., financial problems, job loss, relationship stress), low social connectedness or support, sleep disturbance, substance use, and emotional numbing or detachment (colvonen, straus, acheson, & gehrman, 2019; cusack et al., 2019; germain, mckeon, & campbell, 2017; hancock & bryant, 2018; shalev et al., 2019; steenkamp et al., 2017; vujanovic & back, 2019) . navigating the covid-19 crisis requires a tolerance of uncertainty that is challenging for all, but especially trauma survivors who may have endured, sometimes over months or years (e.g., combat, childhood abuse), unfathomable circumstances that were, by definition, unpredictable and uncontrollable (e.g., raines, oglesby, walton, true, & franklin, 2019; vujanovic & zegel, 2020) . undoubtedly, social connection and a sense of community and collectivism, hope, psychological awareness, and healthy coping will j o u r n a l p r e -p r o o f differentiate risk versus resilience trajectories during and after this crisis (bernardi & jobson, 2019; long & gallagher, 2018; thompson, fiorillo, rothbaum, ressler, & michopoulos, 2018) . learning who suffers long-term negative effects of the covid-19 pandemic, why, and under what circumstances will help us to understand how to intervene most effectively to psychologically support trauma survivors in the aftermath of this and future societal crises. indeed, reactions of trauma survivors to the covid-19 crisis are also likely to be as diverse as the traumas and individuals themselves with the possibility of emergent themes. theoretically, individuals with histories of being directly impacted by natural disasters, people recovering from severe medical conditions, and those with histories of imprisonment or captivity may feel especially emotionally reactive to the large community-level impact, the social distancing and quarantining aspects of weathering covid-19, and the continual perceived health threat inherent to the pandemic. individuals with interpersonal trauma histories may experience a solidification or exacerbation of maladaptive beliefs relevant to trust, safety, or power. others may feel increased social detachment or engage in increased harmful, self-injurious, or suicidal behaviors, particularly those with mood or substance use disorders. for some trauma survivors, following social distancing and self-quarantine guidelines may lead to less frequent exposure to trauma-related reminders in the outside world and/or a lower perceived interpersonal threat due to social-isolation, but increased trauma-related avoidance during the covid-19 crisis in turn may exacerbate ptsd symptoms in the long-term. a high-risk subset may emerge who are slow or reluctant to heed public health guidelines due to a reaction against efforts to control, an increased risk-taking propensity, all-or-none thinking, or helplessness resulting from a history punctuated by traumatic, uncontrollable events. this may lead to incessant attempts, by some, to attain perceived control via closely monitoring news, stockpiling food, or supplies, and maintaining constant vigilance. for those affected by trauma prior to and/or during the covid-19 crisis, the current, chronically stressful global atmosphere where uncertainty reigns may feel especially overwhelming. for others, this crisis may foster growth and resilience as they endure and overcome a crisis of epic and unimaginable proportions. obsessive-compulsive disorder (ocd) is a common (1-2% incidence; (nestadt, bienvenu, cai, samuels, & eaton, 1998; ruscio, stein, chiu, & kessler, 2010) , disabling mental health condition characterized by presence of obsessions and/or compulsions (american psychiatric association, 2013; markarian et al., 2010) . symptoms present in a heterogeneous fashion across a number of dimensions, including contamination/cleaning, taboo obsessions (i.e., sexual, aggressive content), symmetry/repeating/ordering, and checking (mckay et al., 2006) . childhood onset occurs in over 50% of cases and symptoms run a chronic course without adequate intervention (pinto, mancebo, eisen, pagano, & rasmussen, 2006) . clinical presentation is further characterized by frequent comorbidity (stein et al., 2019) and variable degrees of insight (hamblin, park, wu, & storch, 2017) . the covid-19 pandemic is likely to have a number of effects on those with ocd, as well as those at risk. this includes the potential for symptom exacerbation and increased incidence of ocd cases, as well as having implications for assessment and treatment post-covid-19. patients with ocd commonly present with contamination obsessions and associated cleaning compulsions (mataix-cols, do rosario-campos, & leckman, 2005; pinto et al., 2006) . some individuals with contamination related ocd have reported that their symptoms have worsened in light of public health recommendations for increased cleaning behaviors (e.g., washing, wearing masks) and other safety behaviors (e.g., social distancing, wearing masks), j o u r n a l p r e -p r o o f which may be difficult for some patients to maintain within recommended guidelines. covid-19 has become a feared outcome for many patients with contamination-related ocd similar to other what has been observed with other infectious diseases (e.g., hiv). outside of contamination-focused symptomology, other obsessive-compulsive symptoms may be affected such as harm obsessions whereby someone fears that they may have unintentionally spread covid-19. stress has an established relationship with worsened obsessive-compulsive symptoms (adams et al., 2018; brander, perez-vigil, larsson, & mataix-cols, 2016) , and availability of coping strategies is taxed for many; this may further impact ocd symptom presentation as well as comorbidity patterns. although systematic data have not been presented, clinical accounts support symptom worsening for some affected individuals while, on balance, many others have not experienced negative symptomatic change. beyond worsening of symptoms in those with ocd, there is the possibility that there will be increased cases in the near future. this may involve those with subclinical symptoms or other risk factors experiencing onset or worsening of symptoms. the behavioral cycle of ocd/anxiety highlights the role of negative reinforcement in which rituals/avoidance are reinforced by distress reduction and creating a cognitive sense of control (i.e., not getting covid-19 is due to compulsions; rector, wilde, & richter, 2017) . in this scenario, a person with or at risk for ocd may engage in rituals/safety behaviors in response to obsessional distress which in turn reduces anxiety and is perceived as reducing the risk. reduction in distress may motivate further safety behaviors which, for some at risk, could begin to exceed recommended guidelines. while ordinary levels of risk have risen requiring increased hygiene, it remains to be seen what happens when risk levels decline. that is, do cleaning behaviors likewise decline or remain at elevated states thereby impacting diagnosis rates? assessment approaches should continue to capture j o u r n a l p r e -p r o o f obsessive-compulsive symptoms that are impairing, distressing and excessive relative to current risk levels and not count symptoms that reflect behaviors consistent with accepted public health standards. there are also treatment implications. the gold standard psychological treatment for adult and childhood ocd is cognitive behavioral therapy with exposure and response prevention (erp; mcguire et al., 2015; olatunji, davis, powers, & smits, 2013) . this treatment involves gradual exposure to triggers that evoke obsessive-compulsive symptoms while refraining from completing rituals or other avoidance behaviors. a core element to this treatment is that exposure to triggers involves exposure to 'ordinary' levels of risk. covid-19 understandably has shaken what is perceived as ordinary; fortunately, adept therapists have shifted their practice to utilize exposures that reflect this new normal such as relying on imaginal exposures or exposures targeting rituals in excess of public health agency recommendations. at the same time, some clinicians have negative attitudes towards exposure (meyer, farrell, kemp, blakey, & deacon, 2014) which is related to reduced practice of this core therapeutic technique (farrell, deacon, kemp, dixon, & sy, 2013) . it will be critical to provide guidelines established by expert erp clinicians for how providers integrate realistic covid-19 concerns into their ongoing practice, as well as that in the future. a concerning possibility is that erp treatment post-covid-19 is diluted by virtue of therapists not practicing exposures to the actual level of risk. cigarette smoking remains the leading cause of preventable death and disability globally. smoking may confer worse covid-19 outcomes given extensive evidence for the negative impact of smoking on lung health and respiratory function (tonnesen, marott, nordestgaard, j o u r n a l p r e -p r o o f bojesen, & lange, 2019). indeed, emerging evidence has identified smoking as a possible risk factor for adverse covid-19 prognosis and disease progression (patanavanich & glantz, 2020; vardavas & nikitara, 2020) . in the largest study of covid-19 patients, 16.9% of severely affected patience were current smokers relative to 11.8% of non-severe patients (guan et al., 2020 ). an inverse pattern emerged with non-smokers such that a greater proportion of nonsevere patients identified as a non-smoker relative to severe patients. moreover, 25.5% of covid-19 patients who either needed mechanical ventilation, were admitted to an intensive care unit, or died from complications related to the disease were current smokers relative to 11.8% of those not experiencing these outcomes. similar disparities in covid-19 severity across smoking status have been observed in other samples (w. j. zhang et al., 2020) . thus, these data, albeit preliminary and limited by sample size, indicate that smoking is a risk factor for covid-19 progression (w. . taking a biological perspective to understand why smokers are more susceptible to severe covid-19 symptoms, recent research has proposed that smoking and covid-19 susceptibility and symptom severity may be related to an upregulation of the angiotensin-converting enzyme-2 (ace2) receptor (brake et al., 2020) . ace2, a membrane-bound aminopeptidase that plays a vital role in cardiovascular and immune systems, is highly expressed in the heart and the lungs (turner, hiscox, & hooper, 2004; wang, luo, chen, chen, & li, 2020) . studies have established that ace2 is a receptor for the covid-19 virus (j. , and greater ace2 gene expression has been observed in smokers compared to non-smokers (brake et al., 2020; cai, 2020; emami, javanmardi, pirbonyeh, & akbari, 2020; tian et al., 2020; wan, shang, graham, baric, & li, 2020; zhao et al., 2020; . the upregulation in ace2 creates an environment that allows greater potential for covid-19 to j o u r n a l p r e -p r o o f infect human cells among smokers through more opportunity to bind to this receptor (olds & kabbani, 2020; zuluaga, montoya-giraldo, & buendia, 2020) . in part, this biological mechanism may help explain observed sex differences in covid-19. specifically, covid-19 symptom severity and mortality rates in china indicate worse outcomes for men than for women, where 52.1% of men and 2.7% of women are current smokers (parascandola & xiao, 2019; sun et al., 2020) . it is possible that the elevated smoking rates among men in china, and therefore greater upregulation in ace2, contributed to significant gender difference in covid-19 incidence and severity (j. . in addition to combustible cigarette smoking, there also is growing concern for the impact of electronic cigarette (e-cigarette) use on covid-19 infection and disease progression (lewis, 2020) . although it is believed that the worldwide distribution and adoption of ecigarettes has the potential to increase population-level vulnerability to respiratory infecting diseases (olds & kabbani, 2020) , such as covid-19, no studies have assessed e-cigarette use among covid-19 patients (farsalinos, barbouni, & niaura, 2020) . given evidence for the impact of various e-cigarette formulations on lung health and functioning (viswam, trotter, burge, & walters, 2018) as well as the fact that most e-cigarette users are former or current combustible cigarette users (mirbolouk et al., 2018) , it is possible that product use will critically impact the course of covid-19 among users. additionally, similar to combustible cigarette use, it has been theorized that e-cigarette use may engage an upregulation in ace2 that parallels that of combustible cigarette use and increases the likelihood of covid-19 infection (brake et al., 2020) . further research on these products and their influence on covid-19 outcomes is urgently needed. a final point to consider is the effect that the covid-19 pandemic itself has on smoking. one of the leading reasons for smoking is stress management (baker, piper, mccarthy, majeskie, & fiore, 2004; garey et al., in press) . the psychological effect of the current global environment, characterized by feelings of fear, uncertainty, isolation, and stress (mertens, gerritsen, salemink, & engelhard, 2020) , coupled with limited availability of adaptive coping tools due to regulations and consequences of covid-19 (i.e., social distancing, financial hardship) likely increases the risk for smoking onset, increased intensity, and relapse (patwardhan, 2020; stubbs et al., 2017) . smoking initiation and severity, in turn, increase susceptibility for covid-19 and worse disease-related outcomes. behavioral scientists must engage in targeted efforts to support current smokers and former smokers in achieving and maintaining cessation during this particularly challenging time. there are promising initial findings from smoking cessation programs implemented in smokers managing other infectious disease that may help guide some of these initiatives . as more is learned about covid-19, it is imperative that health care providers assess smoking (and e-cigarette) use status as well as relapse potential among former users and provide appropriate education and intervention to help mitigate the potential risk of this health behavior on disease infection and course. the (mis)use of alcohol is a leading risk factor for global disease burden and preventable death (degenhardt et al., 2018; organization, 2019) . alarmingly, alcohol use, high-risk drinking, alcohol use disorder (aud), and alcohol-related deaths were increasing before the covid-19 pandemic (grant et al., 2017; white, castle, hingson, & powell, 2020) . despite the widespread belief that moderate alcohol consumption may confer health benefits (diaz et al., 2002; j o u r n a l p r e -p r o o f et al., 2007) , more recent work suggests that any alcohol consumption is associated with health risks (griswold et al., 2018) . in fact, given the immunosuppressing effects of alcohol both generally and in the respiratory system specifically (molina, happel, zhang, kolls, & nelson, 2010; szabo & mandrekar, 2009) , it is germane to consider the role that alcohol consumption, whether chronic or in acute response to the ongoing crisis, may have on contraction of the covid-19 virus. in addition to the direct physiological impact of alcohol consumption on the body, the disinhibiting properties of alcohol (kumar et al., 2009; oscar-berman & marinković, 2007) may put individuals at risk for other risky/poor decisions (george, rogers, & duka, 2005) . for example, those under the influence of alcohol may be more likely to violate social distance protocols, exhibit poor hand washing procedures, or refuse/forget to wear a face covering in public, leading to potential exposure to and/or spreading of the virus. importantly, impulsivity has reciprocal relationships with alcohol such that consumption increases impulsive behaviors and individuals with greater trait impulsivity (mis)use alcohol to a greater extent (dick et al., 2010) . moreover, the effects of impulsivity on alcohol (mis)use can be amplified by other factors, such as stress, to confer greater risk for alcohol (mis)use (fox, bergquist, gu, & sinha, 2010) . it is well-documented that stress, both acute and chronic, is a trigger for alcohol (mis)use (becker, lopez, & doremus-fitzwater, 2011; blaine & sinha, 2017) . the covid-19 pandemic has brought about both acute (e.g., work displacement, limited availability of cleaning supplies) and chronic stress (e.g., financial difficulty, isolation) that likely will contribute to alcohol (mis)use for coping. it also is reasonable to expect that alcohol (mis)use will worsen during the crisis in response to the stress and uncertainty. for example, during the 2008-2009 economic recession, although there was a decrease in prevalence of alcohol use overall (i.e., increase in j o u r n a l p r e -p r o o f abstainers), there was an increase in prevalence of binge drinking (bor, basu, coutts, mckee, & stuckler, 2013) . this suggests that there may be a realignment/concentration of problematic drinking such that a greater segment of those who do consume alcohol may be doing so in a maladaptive or harmful way. although sales to restaurants and events have reduced markedly during the pandemic, sales of online and to-go alcohol have skyrocketed (nielsen, 2020) . given shelter in place orders and limits on socializing, it is possible that greater amounts of alcohol are being consumed at home/solitarily relative to social contexts. solitary drinking can, in some circumstances, lead to greater alcohol consumption than social drinking (kuendig & kuntsche, 2012) and is associated with greater alcohol-related consequences overall (christiansen, vik, & jarchow, 2002) . for many, the covid-19 pandemic has led to significant social isolation with in-person socializing virtually eliminated and many working from home (if at all). these conditions may also exacerbate a common reason for alcohol-related relapse: boredom (levy, 2008) . without other adaptive ways to manage stress, socialize, or simply occupy one's mind, it is possible that craving for alcohol may intensify. finally, there are important treatment implications for alcohol (mis)use during covid-19. individuals already report numerous barriers to seeking drug/alcohol treatment (mcgovern, xie, segal, siembab, & drake, 2006) . in the wake of the pandemic additional barriers may arise such as the perception that one's treatment is not a priority during a 'life or death' pandemic or not worth the risk of leaving one's home. alternatively, for those seeking treatment, there may simply not be local resources available or treatment facilities may have waitlists. although the use of telehealth services are growing in general (dorsey & topol, 2016) , there is more work to be done, with specific considerations for low-income individuals (e.g. recently unemployed) who j o u r n a l p r e -p r o o f may be reluctant to spend money on treatment, perceive treatment to be a luxury, or not have technological resources or a private location to engage in telehealth. affordable computer-based treatments without the need for a provider that focus on stress and alcohol use (paulus, gallagher, neighbors, & zvolensky, 2020) could be particularly pertinent during this pandemic. administration center for behavioral health statistics and quality, 2019) presumably due at least in part to legalization of recreational and/or medical marijuana at the state level (johnston, o'malley, miech, bachman, & schulenberg, 2015) . notably, cannabis users report using more cannabis during times of heightened distress following national disasters such as the september 11, 2001 terrorist attacks, a pattern that was especially prominent among individuals who experienced post-traumatic stress disorder and depression (vlahov et al., 2002) . it therefore follows that cannabis use and associated problems may increase during the covid-19 pandemic. cannabis use increases during times of distress to manage negative affect. in support of this contention, cannabis users report relaxation and tension relief as one of the most common reasons for use (copeland, swift, & rees, 2001; hathaway, 2003; reilly, didcott, swift, & hall, j o u r n a l p r e -p r o o f 1998). data from experimental studies support these self-reports. to illustrate, current cannabis users were randomly assigned to an anxiety-induction or non-anxious control condition and cannabis craving increased from before to during the task among participants in the anxiety condition, but not among those in the control condition (buckner, ecker, & vinci, 2013) . these data indicate that cannabis users were especially vulnerable to wanting to use cannabis during an anxiety-provoking situation, which has direct implications for the covid-19 pandemic characterized by heightened stress. notably, this effect was specific to cannabis craving and was not observed for craving for alcohol or cigarettes in this sample of cannabis users. coping motives are the most common reasons cited for wanting to use during laboratory-induced anxiety (buckner, zvolensky, ecker, & jeffries, 2016) . prospective data collected via ecological momentary assessment also confirm that anxiety is positively, significantly related to cannabis craving at the momentary level, and is related to greater subsequent craving (buckner, crosby, silgado, wonderlich, & schmidt, 2012) . further, although positive and negative affect were greater immediately prior to cannabis use compared to non-use episode, negative affect increased at a significant rate prior to cannabis use, and decreased at a significant rate following cannabis use; changes in positive affect were not significantly related to use (buckner et al., 2015) . further, the stress associated with the covid-19 pandemic may serve as trigger for lapse and/or relapse among individuals undergoing a cannabis quit attempt. in a qualitative interview following cannabis quit attempts, situations involving negative affect and exposure to others smoking cannabis were among the most difficult situations individuals reported in which to abstain (hughes, peters, callas, budney, & livingstone, 2008) . among cannabis users undoing a self-guided quit attempt, data from ecological momentary analysis indicated that although positive and negative affect were significantly higher during cannabis lapse episodes compared j o u r n a l p r e -p r o o f to non-use episodes, when negative and positive affect were analyzed simultaneously, negative affect, but not positive affect, remained significantly related to lapse (buckner, zvolensky, & ecker, 2013) . again, the most common reason for use cited during lapse episodes was to cope with negative affect. not only could covid-19 increase cannabis use, but cannabis use may exacerbate covid-19 symptoms given that smoking cannabis damages the lungs. respiratory toxins (including carcinogens) in cannabis smoke are similar to that of tobacco smoke but notably the smoking topography for cannabis leads to higher per-puff exposures to inhaled tar and gases (tashkin & roth, 2019) . further, respiratory symptoms such as chronic cough, sputum, and airway mucosal inflammation are also similar between cannabis smokers and tobacco smokers. the impact on respiratory functioning of cannabis smoke has led for the consideration of cannabis use as a pre-exiting condition that could increase the likelihood of more severe complications should one contract covid-19 (national institute on drug abuse, 2020). sleep is a fundamentally restorative process, but it is also highly responsive to stress (irwin, 2015) . during times of increased stress, sleep, quite paradoxically, serves both as a major line of defense and as a source of heightened vulnerability. these relationships derive from the fact that sleep and immunological functioning are reciprocally related: sleep promotes healthy immune responses and healthy immune responses (e.g., to infectious agents) promote deeper, more restorative sleep (opp, 2005) . precise mechanisms are of course complex, but several specific links are noteworthy. immune-signaling proteins called cytokines, such as tumor necrosis factor (tnf) and interleukin-1 (il-1) directly target infection and inflammation but are j o u r n a l p r e -p r o o f also known to promote sleepiness and non-rapid eye movement (nrem) sleep (jewett & krueger, 2012) . the hormone melatonin, which provides an endogenous marker of circadian phase peaks during the nocturnal sleep period but also has important immunomodulatory effects. conversely, the hypothalamus-pituitary-adrenal (hpa) axis and the sympathetic nervous system (sns), two primary stress response systems, are down-regulated during sleep, decreasing immune-regulating cortisol levels (besedovsky, lange, & born, 2012) . however, when sleep is inadequate or disrupted, alteration in these systems is readily observable. experimental sleep research provides overwhelming evidence for the detrimental effects of chronic sleep disruption on immune responses including increases in multiple inflammatory markers such as c-reactive protein, diminished immune response to vaccination, and enhanced susceptibility to bacteria and toxins (besedovsky et al., 2012) . rather than representing enhanced immunity, elevated levels of inflammation are associated with a range of health risks including cardio-pulmonary disease (libby, 2006) . sleep's inextricable role in human immunological functioning clearly place it at the forefront of critical behaviors during a pandemic. unfortunately, multiple aspects of the covid-19 pandemic threaten healthy sleep patterns which in turn endanger both physical and mental health. widespread uncertainty, 24-hour media coverage (including misinformation), fear for one's own health and the health of loved ones, and potential loss of employment/wages are but a few of the significant sources of stress present during these unprecedented times. heighted psychological and physiological arousal elicited by such stress falls in direct odds with a calm, quiescent state necessary for sleep onset and maintenance. further, common behaviors aimed at managing increased stress and anxiety such as smoking, alcohol consumption, and decreased physical activity can give rise to or worsen sleep disruption via known negative effects on sleep j o u r n a l p r e -p r o o f duration and quality (irish, kline, gunn, buysse, & hall, 2015) . moreover, sleep deprivation can amplify inflammatory responses (bollinger, bollinger, oster, & solbach, 2010) , increasing the risk for poor outcomes in covid-19 as unrestrained inflammation is implicated in the pathophysiology of the disease (gamaldo, shaikh, & mcarthur, 2012) . although predisposing (e.g., genetics) and precipitating (e.g., trauma) factors play a role, stress is considered a primary cause of insomnia (morin, rodrigue, & ivers, 2003 ) and among insomniacs, perceived inability to sleep often becomes a major source of stress in its own right. studies that have systematically examined incidence and severity of insomnia symptoms during a global pandemic are unavailable despite ubiquitous anecdotal reports and cautions from health professional regarding the immunosuppressive effects of poor sleep. however, in a recentlypublished study conducted between january 29 and february 3, 2020, c. zhang et al. (2020) surveyed medical staff responding to the covid-19 pandemic in china using the insomnia severity index (isi; morin, belleville, bélanger, & ivers, 2011) . more than a third of workers (36.1%) endorsed symptoms indicative of clinical insomnia and those with insomnia reported elevated levels of depression. insomnia is well-known to herald the onset of depression both acutely and years later even among those who have never been depressed (baglioni et al., 2011) . studies directed at uncovering precise mechanisms of affective risk during the covid-19 pandemic must therefore consider the presence and severity of insomnia symptoms. the covid-19 pandemic also has upended daily routines and associated 'cues' that serve to maintain regular sleep schedules. working from home, altered mealtimes, increased sedentary behavior, social distancing, and increased "screen time" are only some of the changes that hold potential to disrupt circadian rhythms that govern sleep-wake patterns. other factors such as social activities also can affect sleep-wake patterns. the human internal circadian clock j o u r n a l p r e -p r o o f runs slightly longer than 24 hours and therefore needs to be 'entrained' to the 24-hour day via internal and external cues (czeisler et al., 1999) . sunlight is the most potent exogenous cue that aligns our internal rhythm to the external environment, but quarantine measures and greater time spent indoors means that many individuals are receiving inadequate dosages of light exposure. although public health guidelines center on sufficient sleep duration (watson et al., 2015) , sleep timing is equally critical for overall health and well-being. misalignment of the sleep period with the body's 'biological night' is routinely linked with a host of serious risks, including anxiety, depression, suicide, cardiac events, and several forms of cancer (baron & reid, 2014) . healthcare workers who are working long hours and night shifts during the covid-19 pandemic are therefore a particularly high-risk group for circadian shifts and associated comorbidities. considering sleep's role in immunological function, this represents an area of priority for future research. the intersection of covid-19 with pre-existing chronic medical illness (e.g., cardiovascular disease, diabetes, hiv) raises additional challenges to the patient for managing multiple treatment cascades. these challenges are exacerbated by the poorer survival and disease course for patients with underlying medical conditions (emami et al., 2020) which in turn seems to be driving, in part, the alarming covid-19 racial disparity (laurencin & mcclinton, 2020) . the overlapping epidemic of covid-19 with hiv, for example, presents unique challenges for hiv access to care, hiv treatment engagement, and prevention. infection or if it exacerbates the likelihood of poor covid-19 outcomes. however, people living with hiv may have other comorbidities, such as cardiovascular disease and chronic lung disease, j o u r n a l p r e -p r o o f that increase the risk for a more severe course of covid-19 illness (guaraldi et al., 2011; guo et al., 2020) . there is also a concern that individuals who are immunocompromised, such as those with hiv, may be at greater risk for severe covid-19 symptoms (cdc, 2020a; duffau et al., 2018) . in the u.s., most people living with hiv (plwh) are tested, linked to hiv care, well engaged in antiretroviral treatment, and achieve hiv viral suppression thus ensuring their optimal health and protecting the public health by containing onward transmission (cdc, 2020b). however, structural and individual barriers to treatment and prevention create enduring inequalities and significantly increase the risk of infection, reduce access to, and engagement in, hiv care, and compromise participation in hiv biobehavioral prevention among particular risk groups. gay and bisexual men (particularly hispanic and african american men) are most impacted by hiv and account for nearly 70% of new hiv cases. hiv incidence rates in the u.s. are also significantly higher for those who are homeless or living in poverty (denning & dinenno, 2020) . with respect to individual barriers to care, plwh are disproportionally affected by traumatic life experiences, anxiety, depression, and substance use (brandt et al., 2017; nanni, caruso, mitchell, meggiolaro, & grassi, 2015; c. o'cleirigh, magidson, skeer, mayer, & safren, 2015) . each of these also have been associated with poorer engagement in hiv care, worse antiretroviral medication adherence, and poorer hiv disease course. their co-occurrence and interaction significantly increases both the risk for hiv infection (mimiaga et al., 2015) and poorer hiv disease management among those already infected (harkness et al., 2018; pantalone, valentine, woodward, & o'cleirigh, 2018) . these mental health barriers to full engagement in hiv care may well be exacerbated by increased levels of covid-19 specific anxieties and j o u r n a l p r e -p r o o f increases in general health-related anxieties. the requirements of social distancing also may contribute to feelings of isolation and loneliness which may in turn contribute to increased depression or depression-related withdrawal. both anxiety-related avoidance and depressive related withdrawal will likely have negative consequences for self-care generally and for hiv care specifically. these increases in distress will occur at a time when access to behavioral health services is already severely restricted. some plwh who become co-infected with covid-19 will already be struggling with hiv disease management (e.g., missed medical appointments, sub-optimal medication adherence) and may require additional supports to manage care and treatment at a time when many routine supports may not be available due social distancing and lack of routine medical services. protecting access to care and treatment among those already struggling with the complexities of the hiv care cascade who must now manage the additional burdens of the covid-19 illness is a robust clinical concern. here, we underline the importance of community (carrico et al., 2020) and health worker based approaches (operario, king, & gamarel, 2020) to hiv treatment and protecting access to care through innovative and virtual care models. many of those at risk for being lost to care during this covid-19 pandemic also may be vulnerable to perceived stigma (krier, bozich, pompa, & friedman, 2020; logie, 2020 ). many will have multiple stigmatized identities with respect to hiv status, covid-19 status, substance use, sexual or gender minority status, and others. keeping our community members and peers involved in our service delivery will help ensure our treatments are delivered in stigma-free contexts. empirical support for integrated treatment platforms that address mental health (ironson et al., 2013; safren, o'cleirigh, skeer, elsesser, & mayer, 2013) and substance use issues (mimiaga et al., 2019; safren et al., 2012) to protect engagement in hiv treatment and j o u r n a l p r e -p r o o f prevention (mayer et al., 2017; conall o'cleirigh et al., 2019) are available to guide these initiatives. in addition, protecting access and supporting engagement (virtual or otherwise), to mental health and substance use treatment will be critically important. these approaches may be particularly key for protecting access to hiv prevention services (i.e., hiv testing, access to preexposure prophylaxis [prep]) for those at risk for hiv. access to these services may be particularly important for those whose behavioral risk profiles and risk appraisals may be disturbed because of the impact of social distancing on usual patterns of substance use or sexual behavior. although much remains unknown about covid-19 and the mental health consequences of the pandemic, it is likely that regular physical activity offers protective effects. regular physical activity reduces risk of and helps manage conditions that appear to increase risk of adverse outcomes of covid-19 (e.g., obesity, cardiovascular disease, diabetes; lee et al., 2012) , and improves immune function (nieman & wentz, 2019) which likely positively affects the progression of covid-19. it also buffers the effect of stressors and (in part thereby) can prevent the onset of mental health conditions (harvey et al., 2018; jacquart et al., 2019) . further, diminished physical activity can disrupt sleep quality (buman & king, 2010; youngstedt & kline, 2006) , which increases susceptibility to infection and mental and physical illness (see sleep section). hence, establishing or maintaining a regular physical activity habit has the potential to mitigate the impact of the pandemic both at a personal and societal level. establishing and maintaining a regular physical activity habit has proven to be challenging. indeed, only 24% of adults meet the guidelines set forth by the department of health and human services (whitfield et al., 2019) . the covid-19 pandemic has impacted j o u r n a l p r e -p r o o f several factors, including a change in the daily routine and increased stress and anxiety, that can affect the intent of or ability to engage in behavior change. it is important to acknowledge the relationship between factors such as stress or changes in routine and physical activity participation can vary in strength or direction (i.e., negative or positive) depending on the individual and their context. for example, for some routine changes have created barriers for exercise participation, while for others changes to the daily structure have opened opportunities to engage in regular exercise. similarly, stress and anxiety at the "right" level can be motivating for some make exercise part of their daily routine, but when stress and anxiety become overwhelming, automated emotion action tendencies often cause people to move away from healthy (coping) behaviors such as exercise (otto et al., 2016) . importantly, such relationships may further vary within and across individuals depending on other individual difference variables (e.g., risk factors, protective factors, [mental] health diagnosis) and contextual factors (e.g., job loss, financial stress, isolation). research aimed at understanding the relationship between covid-19 and physical activity mostly likely will benefit from considering the importance of individual differences and the influence of contextual factors. comprehensive assessment batteries and statistical models that include the testing of these complex moderation effects are key. this perspective that acknowledges nuance in the relationship between covid-19 (pandemic) and physical activity also will aid efforts to develop or fine-tune intervention programs for physical activity uptake. the covid-19 pandemic, although still ongoing and presently under investigated from a behavioral health perspective, is apt to impart acute and potentially chronic exacerbations in psychological symptoms and disorders, addictive behavior, and health behavior and chronic j o u r n a l p r e -p r o o f illness. across various phenotypes overviewed in the current essay, previous scientific work and theoretical models predict covid-19, regardless of acquisition of the virus, has and will continue to have a strong negative psychological impact on negative mood states, various forms of substance use, and sleep, chronic illness, and physical activity. although many of these relations would be expected, theoretically, to be negative, select subgroups will certainly adaptively respond to covid-19 related stress (e.g., improve their physical fitness, improve self-care routines, quit/reduce maladaptive behaviors that place them at risk). in this final section of the paper, we discuss sociocultural considerations, developmental issues, and the role of individual difference factors for covid-19-related psychological, addictive, health behavior and chronic illness. we conclude by offering an integrative covid-19 model that could be used to guide research focused on the stress-related burden of the pandemic. certain subpopulations and contextual factors (e.g., loss of work) are likely to signify a vulnerability gradient for covid-19 in terms of mental health, addictive behavior, and health behavior. although there are numerous possible sociocultural factors that could be relevant, we highlight first responders and medical professionals, economic adversity, and racial/ethnic factors as three prototypical factors of public health importance. of all the sectors of the population, first responders and front-line healthcare professionals are arguably at the greatest risk for at least acute disruptions in anxiety, stress, and negative mood. first responders and healthcare professionals at the front line of the covid-19 pandemic have at their core mission to protect and preserve life (prati & pietrantoni, 2010) . these groups, although engaging in a diverse range of specific occupational activities (e.g., direct medical care, transport, public safety j o u r n a l p r e -p r o o f enforcement), share in common that they are among the first to respond to the covid-19 crisis and take primary responsibility for attending to covid-19 related health issues. first responders and healthcare professionals are undoubtedly experiencing emotionally challenging and unpredictable situations that can place their lives in danger. the acute emotional effects of managing covid-19 cases is likely to be amplified by heavy work schedules and reduced access to and isolation from social support systems (e.g., self-isolation after finishing a shift). it is likely that first responders and healthcare professionals working with covid-19 cases in hospitals will be exposed to potentially traumatic events, the greater-than-usual experience of life-threatening situations, working with emotional strain related to isolation of patients from their families (e.g., compassion stress in the form of offering emotional support to patients in a manner that family or caregiver of patients would typically offer), and exposure to the struggle to life and death. these experiences are apt to challenge the coping resources of even the most seasoned professionals, which can result in higher degrees of anxiety, stress, and depressed mood (lafauci schutt & marotta, 2011) . such elevated stress levels are likely to be related to changes in cognition and physical health, including emotional exhaustion, fatigue, sleep dysfunction, and problems with interpersonal relationships (kronenberg et al., 2008; lane, lating, lowry, & martino, 2010) . cognitive-based beliefs about personal safety and health can be altered and memories of potentially traumatic events engrained (setti & argentero, 2014) . collectively, the covid-19 related stress burden, as discussed in several sections of the current essay, will have a high likelihood of being related to increased risk of anxiety and depression for first responders and medical professionals working at the front line. moreover, consistent with past literature of these populations, the regulation of affect will be associated with addictive and health behavior to modulate such affect (e.g., physical activity, substance j o u r n a l p r e -p r o o f use). although some regulatory behavior will be adaptive (e.g., increasing sleep where possible to aid in recovery, engaging in regular physical exercise), others may be less adaptive (e.g., smoking to reduce stress) and promote the risk for other health problems (e.g., physical illness). economic adversity. economic hardship related to covid-19 is already evident at numerous levels of analysis, including job loss, reduced earnings, higher debt relative to assets ratio, inability to pay mortgage and bills, meeting governmental guidelines for poverty status, and worry about financials resources going forward due to the turbulent nature of the economy. past work has shown that economic hardship is related to behavioral health problems, including psychological disorders, addictive behavior, physical health problems, and interpersonal dysfunction in adults and children (k. j. conger et al., 2012; sareen, afifi, mcmillan, & asmundson, 2011) . for instance, economic adversity has been linked to reduced social competence and elevated physiological markers of stress (k. e. bolger, patterson, thompson, & kupersmidt, 1995; evans & english, 2002) . further, economic hardship is related to selfregulation capacity and the corresponding difficulty in dealing with additional responsibilities. for example, past work has found limited socioeconomic resources are related to harsher parenting behavior and greater substance use (r. d. conger & donnellan, 2007) . the negative effects may be particularly profound when economic hardship is severe or chronic (dearing, mccartney, & taylor, 2001; magnuson & duncan, 2002) . the totality of worsening economic conditions for individuals and families in the larger context of an uncertain economic future are apt to be related to elevations in anxiety, stress, and depression as well as other negative emotional states (e.g., anger, frustration, fatigue; newland, crnic, cox, & mills-koonce, 2013) . such emotional symptoms and problems are likely to be related to elevations in substance use and other maladaptive behavior (e.g., less supportive interpersonal behavior, less affection) and j o u r n a l p r e -p r o o f may exacerbate chronic health conditions. other work has found that these processes also disrupt social interconnections (scaramella, sohr-preston, callahan, & mirabile, 2008) . primary care givers who have children home from school, are unlikely to be able to work at their full capacity even with added flexibility in schedules. although certain occupations have decreased activity, many have not. therefore, it could be expected that for individuals with added responsibilities of educating their children at home occupational stress may be greater compared to those without such responsibilities. further, it is possible that the accumulation of occupational responsibilities that are not addressed for persons with additional educational responsibilities will accumulate and make it more challenging to recover when going back to 'normal,' resulting in a greater degree of occupational stress. grappling with lower socioeconomic states related to covid-19 will, for certain segments of the population, offer an additional psychological challenge. indeed, past work has repeatedly documented that lower socioeconomic status is related to adverse health outcomes for chronic illness and mortality rates (adler et al., 1994; adler, boyce, chesney, folkman, & syme, 1993) . moreover, harms faced by people who cannot afford not to work in dangerous settings can exacerbate the psychological and health risk associated with coid-19. further essential workers are more apt to be persons of color (handerson, mccullough, & treuhaft, 2020) . certain groups will be more likely to recover than others, which past work indicates is related to poorer health outcomes even at higher socioeconomic levels (kraus, borhani, & franti, 1980) . moreover, research has found that lower socioeconomic persons experience more chronic stress and negative life events (stansfeld, north, white, & marmot, 1995) . additionally, lower socioeconomic status is related to cognitive biases for threat (chen & matthews, 2001) , which engender greater degrees of interpersonal conflict and heightened negative emotional states j o u r n a l p r e -p r o o f (matthews et al., 2000; stansfeld, head, & marmot, 1997) . it would be expected that such negative emotional experiences will be related to maintained direct relations with poorer health behavior and health outcomes (mcewen & stellar, 1993) . in fact, research has consistently found that lower socioeconomic status is related to greater degrees of anxiety, stress, and depression when compared to those higher in socioeconomic status (mcleod & kessler, 1990) . this heightened stress reactivity may be at least in part attributable to having fewer resources. consequently, those struggling with a lower socioeconomic status due to covd-19 may be more contexts in which they must utilize their emotional resources and be less likely to be in a sociocultural context wherein such resources can be replenished (holahan, moos, holahan, & cronkite, 1999) . this perspective is in line with past work that has found that when persons are exposed to chronic stress, emotional resources are challenged, and there is a greater risk for future emotional distress (n. bolger & zuckerman, 1995; ensel & lin, 1991) . there is broad band evidence that significant health disparities exist for persons of racial/ethnic minority in the u.s. and beyond prior to covid-19 for psychological, addictive behavior, and health behavior as well as chronic illness. for example, african american/black individuals experience a disproportionate burden in disease morbidity, mortality, disability, and injury (mechanic, 2005; mensah, mokdad, ford, greenlund, & croft, 2005) . indeed, african american/black individuals remain significantly and consistently more at risk for early death than do similar non-latinx white individuals (williams, neighbors, & jackson, 2003; williams, yu, jackson, & anderson, 1997) ; overall early death rates of african american/black individuals are comparable to those observed among non-latinx whites in the u.s. decades ago (levine et al., 2016; williams & jackson, 2005) . differences in prevalence and rate of growth of chronic illness are not accounted for solely by j o u r n a l p r e -p r o o f exposure to lower income environments (franks, muennig, lubetkin, & jia, 2006) . indeed, social determinants of health (e.g., racism; krieger & sidney, 1996) , addictive behavior (e.g., tobacco use; sakuma et al., 2015) , and stress represent robust and consistent factors related to health inequalities among african american/black individuals and those from other underrepresented racial/ethnic groups. the covid-19 pandemic has appeared to strike racial and ethnic minority populations (e.g., african american/black) hard and with possible longerterm consequences. for example, less access to health care services for chronic illness, addictive behavior, and mental illness could exacerbate covid-19 related symptoms or promote a greater degree of stress-related burden associated with the pandemic (e.g., worry that loved ones, if infected, cannot access care). consequently, addictive behaviors (e.g., smoking, alcohol misuse) and health behaviors (e.g., disrupted sleep, emotional eating) may be used in the short-term to cope with such covid-19 related stress, increasing the longer-term risk for more severe negative emotional symptoms and health complaints (e.g., pain) and chronic health problems (e.g., obesity). additionally, situations characterized by mass fear and confusion, such as the current pandemic, also can elicit a human instinct to resolve the confusion and mitigate the fear by identifying a culprit for the introduction or spread of the disease (bard, verger, & hubert, 1997; bromet, 2011) . asian american persons are one group that has been singled out as responsible for the covid-19. the misdirection of fear and/or anger related to covid-19 toward a racial or ethnic group instead of the disease, however, can perpetuate fear and contribute to racism and stigma. several reports have already documented the rise in violent crimes and discrimination experienced by asian american persons related to covid-19 beliefs (e. liu, 2020) . covid-19 specific language, such as referring to covid-19 as 'the chinese virus,' has created a platform j o u r n a l p r e -p r o o f to propagate stigma and discrimination towards asian americans. it is likely that stigma and discrimination experienced by asian americans in response to covid-19 will increase emotional distress, coping-oriented addictive behavior, and may alter health behavior or exacerbate chronic illness. it would also be remiss to not call explicit attention to the fact that societies marked by greater economic and social inequality experience far more medical, psychological, and social pathology than do societies where such wealth inequalities are less pronounced (wilkinson & pickett, 2006 . further, such adverse effects occur across social classes, not merely among the most disadvantaged. yet, the adverse effects of economic (and thus social) inequality hurt everyone, although the poorest or most marginalized are affected the most (pickett, kelly, brunner, lobstein, & wilkinson, 2005; wilkinson & pickett, 2008) . there are far-reaching implications for psychological health, addictive behavior, and health behavior from a developmental perspective. for children, despite covid-19 appearing to have less severe symptoms and lower mortality rates than other age groups, are among the highest risk groups (sinha et al., 2020; zimmermann & curtis, 2020) . estimates suggests that there are over 1 billion children not in school (cluver et al., 2020) . the economic impact of covid-19 will likely be related to greater risk for children to be utilized to offset such financial hardship (e.g., selling merchandise on the street, forced begging for food and goods) and be a more likely to be abused (campbell, 2020) . for example, it is possible that children will be more likely to be used for child labor and be exploited for sexual behavioral and experience corresponding risk for sexual disease and pregnancy as well as serious psychological distress. interpersonal violence and child abuse will affect children at a significant rate, especially under j o u r n a l p r e -p r o o f conditions wherein there is no oversight from educational systems due to quarantine. world health organizations are already predicting an increase in children who will be orphaned and exposed to abuse and neglect (cluver et al., 2020) . child abuse is less likely to be detected during the covid-19 pandemic because the reduction or lack of child protection agencies monitoring cases, and teachers less able to detect signs of abuse. further, children who received meals at school through government programs such as the national school lunch program may now no longer have access to nutritious food, which can negatively impact their development. the lack of structure from schooling and missed education will have a lasting impact on well-being and apt to be related to increased anxiety, depression, and stress about educational attainment and progress going forward (van lancker & parolin, 2020) . although on-line school may help offset some of these challenges, disparities will exist for those who are most vulnerable, including those who lack internet access or cannot afford technology. older children and young adults may be more likely to drop out of school to help offset family needs. children and youth also may be engaging in more on-line behavior in general or due to emotional distress (e.g., loneliness due to social isolation) and be increasing the chance for solicitation from others who prey on their emotional vulnerabilities (peterman et al., 2020) . lacking access to physical activity due to quarantine protocols may reduce fitness levels and immunological response as well as decrease psychological wellbeing (rundle, park, herbstman, kinsey, & wang, 2020) . children and youth in juvenile systems, such as orphanages, already were exposed to high density living conditions and often lack access to proper medical or psychological care. the covid-19 pandemic is likely to place pressure on such systems (e.g., more children) and the physical environments of these settings may be amenable to the spread of infection. likewise, refugee or otherwise displaced children and youth often live-in high-density environments j o u r n a l p r e -p r o o f wherein social distancing is challenging if not impossible. further, lack of access in these settings to cleaning supplies and water can catalyze the spread of covid-19 or even the basic fear of acquiring the virus. to the extent the covid-19 challenges the medical system, it is possible other forms of medical care necessary for child welfare (e.g., routine exams, immunizations) will be reduced, as was the case during other pandemics such as ebola (mupere, kaducu, & yoti, 2001) . collectively, covid-19 places an enormous stress on children and youth, placing them at an increased risk for psychological disturbances and physical health vulnerability (j. j. liu, bao, huang, shi, & lu, 2020) . covid-19 also will affect ranges of the lifespan, including adults and older adults. the well-publicized health risks for older adults place an obvious psychological and health pressure on this group. older adults are among the most likely to have a chronic illness (e.g., diabetes, cancer, cardiovascular disease) and consequently they maintain an increased vulnerability to deteriorating health and death from covid-19. however, even in the absence of exposure to the virus, the fear and worry about contracting the disease is apt to be significant for this group, especially when in homecare facilities such as nursing homes or hospitals (armitage & nellums, 2020) . this group also is at significant risk for lacking transportation for food, which could challenge the quality of nutrition and have a negative effect in immunological function. similarly, older adults are among the least physical active groups, which again, will have the potential for decreasing psychological wellbeing and immunity. although not specific to older adults, the potential for disruption in grief and loss of others also is a significant psychological stressor. during the pandemic, regular methods of grieving such as funerals have been limited if not all together impossible. the inability to grieve with others or as traditionally done may spur escalation in psychological distress (e.g., sadness, j o u r n a l p r e -p r o o f depression) and complicate the grief process (wallace, wladkowski, gibson, & white, 2020) . to the extent that grief is impaired, individuals may engage in maladaptive addictive behaviors (e.g., alcohol misuse) to cope with the aversive experiences. similar types of emotional reactions may occur when parents are separated from their children due to quarantine protocols and disruptions in travel (e.g., cannot travel to see children located in another region). there are several individual difference factors at a psychological level of analysis that will place people at an increased or decreased risk for psychological problems, addiction, and poor health behavior, and chronic illness during and after the pandemic. research over the past few decades has theorized and found consistent empirical support for emotional symptoms and disorders as well as addictive behavior being explained by individual differences in transdiagnostic processes (sauer-zavala et al., 2012) . transdiagnostic factors may contribute to onset, maintenance, and exacerbation of emotional symptomatology and addictive and health behavior. a core aspect of transdiagnostic models is that they seek to identify basic processes underlying multiple, usually comorbid, psychopathologies or addictive behavior. one set of transdiagnostic factors relevant to covid-19 may be those that are "reactive" vulnerabilities; that is, individual differences that reflect a heightened emotional response to stressful stimuli. such vulnerabilities influence emotion experience by enhancing or diminishing the normative response to emotion stimuli and states, resulting in an excess or deficit, respectively, beyond typical emotional functioning; or altering the type of response to emotion stimuli and states (gratz & roemer, 2004; reiss, 1991; zvolensky, bernstein, & vujanovic, 2011) . in both instances, such reactive processes may be maladaptive because they serve to j o u r n a l p r e -p r o o f reinforce the intensity and frequency of future emotional symptoms. for example, when faced with negative emotion states, individuals with an emotional vulnerability factor that limits their capacity to handle distress may be more apt to execute behaviors that preclude habituation to negative emotion states, which could ultimately increase the intensity of future negative affect and solidify beliefs and learned responses that interfere capacity to adaptively respond to distress. to illustrate, a transdiagnostic factor that may be especially relevant to covid-19 related stress responsivity, substance use, and physical health is anxiety sensitivity (taylor, 2014) . anxiety sensitivity is a malleable, cognitive-affective factor reflecting the tendency to respond to interoceptive distress with anxiety (mcnally, 1989) . anxiety sensitivity is related to, yet distinct from, negative affectivity and trait anxiety (keough, riccardi, timpano, mitchell, & schmidt, 2010) . anxiety sensitivity has demonstrated racial/ethnic, gender, age, and time invariance (ebesutani, mcleish, luberto, young, & maack, 2014; farris et al., 2015; jardin et al., 2018) . given covid-19 can produce physical sensations and even when not infected, covid-related stress can elicit a range of interoceptive sensations, persons higher in anxiety sensitivity may be more be emotional reactive to such stimuli and engage in behavior to dampen stress symptoms (e.g., using tobacco, alcohol). for example, persons may interpret the onset of aversive bodily sensations (e.g., runny nose, cough, fever) as intolerable or catastrophic, exacerbating the experience of such interoceptive symptoms. further, interoceptive symptoms might be particularly salient to persons with higher anxiety sensitivity who are prone to health inequalities (e.g., racial/ethnic minorities, persons in financial stress), as they may be more apt to perceive these internal sensations as uncontrollable because resources to regulate symptoms (i.e., adaptive cognitive and behavioral skills) are likely diminished due to chronic stress exposure j o u r n a l p r e -p r o o f (e.g., low socioeconomic status, discrimination). in turn, persons higher in anxiety sensitivity may be motivated to use substances to reduce emotional and interoceptive distress, elevating their chance for physical illness and compromised immune system function. although this illustrative example represents only one of many possible transdiagnostic amplifying factors, it draws attention to the fact that individual differences in psychological processes are apt to play a central role in the relation between covid-19 related stress and mental health, addictive behavior, health behavior, and chronic illness. individual difference factors also may play roles in offering resilience to covid-19 related stress. that is, individual differences may contribute to the likelihood of a resilient response to covid-19 in the short and long term. thus, in addition to the many situational and contextual factors, individual difference factors will likely shape the level of resiliency to covid-19 pandemic. here, it is likely individual difference factors that de-amplify stress responses will play a central role in offsetting relative risk for psychological, addictive, and health behaviors problems and exacerbation of chronic illness (pidgeon & keye, 2014) . as with affect amplifying factors, such as anxiety sensitivity, there most certainly is a range of factors of potential importance, including flexible coping repertoires, mindfulness, self-efficacy, selfcompassion, and proneness to experience positive affect. to illustrate, individual difference in the capacity to accept difficult covid-19 related stress may offset the potential escalation of anxiety, stress, and depression and mitigate the need for addictive or unhealthy behaviors (e.g., emotional eating) to delimit aversive internal experiences (ranzijn & luszcz, 1999) . consequently, the corresponding risk for health complaints or worsening of chronic health conditions can be offset. indeed, there is a large theoretical and empirical literature that suggests the capacity to accept difficult emotions experiences is related to psychological well-being and j o u r n a l p r e -p r o o f adaptation. for example, one of the reasons meditative practice is related to decreased stress is via change emotional acceptance (teasdale et al., 2002) . this type of work has robust implications in efforts to intervene on covid-19 related stress in the immediate context and for those that struggle to regain stability and growth in the future in terms of mental health, addictive behavior, and health behavior. despite the present lack of systematic empirical work on covid-19 in terms of behavioral health problems, there is good theoretical basis from past scientific work to hypothesize that covid-19 related stress burden, due to a myriad of sources, may play a major vulnerability role in terms of mental health, addictive disorders, and health behaviors as well as chronic illness. for some, the stress-related burden of covid-19 may elicit fundamental changes in risk potential and serve as a fertile basis for future behavioral health problems. for others, the ability to adapt to covd-19 will offer a different course; one that is characterized by greater stability, speed of recovery, and growth. further, it is important to recognize that the adaptation process to covid-19 related stress is apt to be non-linear in many instances. that is, contextual factors (e.g., future life stressors, access to social support) can influence the degree of risk for future problems. research described in this essay provides a basis to develop a theoretical model that could be used to evaluate covid-19 related stress burden on psychological, addictive, and health behaviour problems. we therefore begin this section by briefly outlining a general model that can be used as a heuristic for understanding the complex issues at hand. see figure 1 for a graphical depiction of the model. in general, we predict individual differences in affect amplifying and de-amplifying factors will predict the course of psychological, addictive behavior j o u r n a l p r e -p r o o f and health behavior and chronic illness even when considering differences in exposure to covid-19 experiences (e.g., time of quarantine, acquisition of virus). we would predict, based on past work that transdiagnostic affect amplifying factors will influence addictive and health behaviour, which in turn, will increase (or decrease if de-amplifying) the risk of chronic illness and psychological problems and their comorbidity. further, we can expect that this type of perspective will be moderated by daily stress in the future and access to stress-dampening resources (e.g., social support). accordingly, certain subgroups more prone to greater and more chronic stress, such as first responders and racial/ethnic minorities and orphaned children, may be particularly vulnerable. this conceptual model predicts that the associations which exist between are reciprocal and dynamic. although the model offered here is purposively general and is offered only as a heuristic, it is presumed that there is, in fact, specificity between specific affect amplifying and deamplifying factors, moderators, mediators, and various forms of psychological and chronic illness. that is, a specific type of individual difference factor like anxiety sensitivity is linked to a particular type of problem (e.g., anxiety disorder, worsening of a chronic respiratory illness, severity of hazardous drinking) via a specified mediating process (e.g., smoking, sleep disruption) in the context of certain moderating variables (e.g., higher levels of covid-19 stress burden). the core idea being that the underlying mechanism in this hypothetical example may be quite different from that explaining other problems. the above theoretical model requires empirical testing, and if it is confirmed, one next logical step would be to intervene in it to reduce the burden of mental health, addictive disorders, poor health behaviours, and chronic health conditions related to covid-19 stress burden. ideally, this type of intervention approach would target the root of the pathway, including affect j o u r n a l p r e -p r o o f amplifying (i.e., decreasing levels) and de-amplifying (i.e., promoting growth). however, intervention efforts sit in the fact that the healthcare system will continue to shift and adapt to treatment delivery, including the uptake of digital health technologies. digital health, including mobile health (mhealth), telemedicine/telehealth, and health information technology (e.g., mobile phones, wearable sensors), can be used to develop scalable interventions to promote adherence public health guidelines for mitigating the spread of covid-19. they also can be combined with greater attention to affect amplifying (i.e., decreasing levels) and de-amplifying (i.e., promoting growth) factors that govern many psychological, addictive, and health behaviour processes. here, there is great opportunity for growth of digital health interventions to offer standalone clinical grade therapeutic tactics and as an adjunct to face-to-face interventions. this type of work can close the gap in access to care and offer evidence-based interventions to large segment of society. for example, digital interventions can be used to combat resistance to public health measures at the level of individuals and institutions with a consideration of individual difference factors that affect emotional and behavioral self-regulation. indeed, the public's response to public health measures is itself a potential risk and protective factor for many of the psychological, addictive, and health behavior problems reviewed in this essay. the public health impact of covid-19 on psychological symptoms and disorders, addiction, and health behavior is substantial and ongoing. there is a need for financial and social investment in research to better understand how covid-19 affects the onset, maintenance, and relapse potential for some of the most common, costly, and chronic behavioral health conditions in the general population. further, there is a need for the study of the role of psychological processes, addictive behavior, and health behavior in terms of the onset and maintenance of j o u r n a l p r e -p r o o f covid-19 infection and stress burden. there most certainly will be a demand for preventative and intervention efforts for managing the impact of covid-19 among individuals with elevated negative mood symptoms and disorders, addictive behavior, and certain health behaviors (e.g., sleep disorders) and chronic illness. this work is important to offset the current and projected burden to personal, system, and societal entities, and for providing a theoretical and empirical knowledge base for future pandemics. we presented a heuristic model, which posits that covid-19 related stress and mood, addictive, and health behavior may, in fact, exacerbate each other via several distinct mechanisms. future research in this emerging area has the potential to refine both theory and application with respect to covid-19 and its relation to affect, addiction, and health behavior as well as chronic disease. j o u r n a l p r e -p r o o f • the public health impact of covid-19 on psychological symptoms and disorders, addiction, and health behavior is substantial and ongoing • an integrative covid-19 stress-based model could be used to guide research focused on the stress-related burden of the pandemic • this work could provide a theoretical and empirical knowledge base for future pandemics j o u r n a l p r e -p r o o f how covid-19 may increase domestic violence and child abuse. paper presented at the american psychological association forum the role of stress in the pathogenesis and maintenance of obsessive-compulsive disorder socioeconomic status and health: the challenge of the gradient socioeconomic inequalities in health: no easy solution rates of post-traumatic stress disorder in trauma-exposed children and adolescents: meta-analysis diagnositic and statistical manual of mental disorders covid-19 and the consequences of isolating the elderly. the lancet public health recent developments in understanding ethnocultural and race differences in trauma exposure and ptsd. current opinion in psychology diagnostic and statistical manual of mental disorders: dsm-5 insomnia as a predictor of depression: a meta-analytic evaluation of longitudinal epidemiological studies addiction motivation reformulated: an affective processing model of negative reinforcement epidemiology of anxiety disorders in the 21st century chernobyl, 10 years after: health consequences anxiety and its disorders: the nature and treatment of anxiety and panic circadian misalignment and health effects of stress on alcohol drinking: a review of animal studies cognitive paths from trauma to posttraumatic stress disorder: a prospective study of ehlers and clark's model in survivors of assaults or road traffic collisions the role of peri-traumatic stress and disruption distress in predicting symptoms of major depression following investigating the moderating role of culture on the relationship between appraisals and symptoms of posttraumatic stress disorder sleep and immune function covid-19: undocumented migrants are probably at greatest risk. bmj alcohol, stress, and glucocorticoids: from risk to dependence and relapse in alcohol use disorders psychosocial adjustment among children experiencing persistent and intermittent family economic hardship a framework for studying personality in the stress process sleep, immunity, and circadian clocks: a mechanistic model alcohol use during the great recession of smoking upregulates angiotensin-converting enzyme-2 receptor: a potential adhesion site for novel coronavirus sars-cov-2 (covid-19) systematic review of environmental risk factors for obsessive-compulsive disorder: a proposed roadmap from association to causation anxiety symptoms and disorders among adults living with hiv and aids: a critical review and integrative synthesis of the empirical literature immediate antecedents of marijuana use: an analysis from ecological momentary assessment cannabis use vulnerability among socially anxious users: cannabis craving during a social interaction antecedents and consequences of cannabis use among racially diverse cannabis users: an analysis from ecological momentary assessment cannabis use during a voluntary quit attempt: an analysis from ecological momentary assessment cannabis craving in response to laboratory-induced social stress among racially diverse cannabis users: the impact of social anxiety disorder exercise as a treatment to enhance sleep tobacco-use disparity in gene expression of ace2, the receptor of 2019-ncov an increasing risk of family violence during the covid-19 pandemic: strengthening community collaborations to save lives double jeopardy: methamphetamine use and hiv as risk factors for covid-19 daily patterns of substance use and violence among a highrisk urban emerging adult sample: results from the flint youth injury study cognitive appraisal biases: an approach to understanding the relation between socioeconomic status and cardiovascular reactivity in children college student heavy drinking in social contexts versus alone parenting in a time of covid-19 a review of the relationship between emotional learning and memory, sleep, and ptsd economic hardship and its consequences across generations an interactionist perspective on the socioeconomic context of human development clinical profile of participants in a brief intervention program for cannabis use disorder trauma exposure, ptsd, and parenting in a community sample of low-income, predominantly african american mothers and children prevalence and predictors of ptsd among a college sample stability, precision, and near-24-hour period of the human circadian pacemaker change in family income-to-needs matters more for children with less the global burden of disease attributable to alcohol and drug use in 195 countries and territories, 1990-2016: a systematic analysis for the global burden of disease study communities in crisis: is there a generalized hiv epidemic in impoverished urban areas of the united states? influence of alcohol consumption on immunological status: a review understanding the construct of impulsivity and its relationship to alcohol use disorders state of telehealth multimorbidity, age-related comorbidities and mortality: association of activation, senescence and inflammation markers in hiv adults a bifactor model of anxiety sensitivity: analysis of the anxiety sensitivity index-3 prevalence of underlying diseases in hospitalized patients with covid-19: a systematic review and meta-analysis. archives of academic emergency medicine the life stress paradigm and psychological distress the environment of poverty: multiple stressor exposure, psychophysiological stress, and socioemotional adjustment do negative beliefs about exposure therapy cause its suboptimal delivery? an experimental investigation evaluation of the anxiety sensitivity index-3 among treatment-seeking smokers smoking, vaping and hospitalization for covid-19 interactive effects of cumulative stress and impulsivity on alcohol consumption the burden of disease associated with being african-american in the united states and the contribution of socio-economic status race, psychosocial vulnerability and social support differences in inner-city women's symptoms of posttraumatic stress disorder the sleep-immunity relationship the role of anxiety in smoking onset, severity, and cessation-related outcomes: a review of recent literature the acute effect of alcohol on decision making in social drinkers sleep in ptsd: conceptual model and novel directions in brain-based research and interventions. current opinion in psychology prevalence of 12-month alcohol use, high-risk drinking, and dsm-iv alcohol use disorder in the united states multidimensional assessment of emotion regulation and dysregulation: development, factor structure, and initial validation of the difficulties in emotion regulation scale alcohol use and burden for 195 countries and territories, 1990-2016: a systematic analysis for the global burden of disease study clinical characteristics of coronavirus disease 2019 in china premature age-related comorbidities among hiv-infected persons compared with the general population cardiovascular implications of fatal outcomes of patients with coronavirus disease ethnic and racial differences in the smoking-related risk of lung cancer variation in government responses to covid-19. blavatnik school of government working paper variable insight in ocd perceived control and avoidance in posttraumatic stress a profile of frontline workers in the bay area. bay area equity atlas longitudinal effects of syndemics on art non-adherence among sexual minority men exercise and the prevention of depression: results of the hunt cohort study cannabis effects and dependency concerns in long-term frequent users: a missing piece of the public health puzzle resource loss, resource gain, and depressive symptoms: a 10-year model cancer facts & figures for african americans attempts to stop or reduce marijuana use in non-treatment seekers the role of sleep hygiene in promoting public health: a review of empirical evidence gender-specific effects of an augmented written emotional disclosure intervention on posttraumatic, depressive, and hiv-disease-related outcomes: a randomized, controlled trial why sleep is important for health: a psychoneuroimmunology perspective the effects of exercise on transdiagnostic treatment targets: a meta-analytic review towards a greater understanding of anxiety sensitivity across groups: the construct validity of the anxiety sensitivity index-3 humoral sleep regulation; interleukin-1 and tumor necrosis factor vitamins & hormones maintaining hiv care during the covid-19 pandemic defining and assessing moral injury: a syndrome perspective project trauma support: addressing moral injury in first responders monitoring the future national survey results on drug use traumatization in medical staff helping with covid-19 control anxiety symptomatology: the association with distress tolerance and anxiety sensitivity national estimates of exposure to traumatic events and ptsd prevalence using dsm-iv and dsm-5 criteria ptsd severity among emergency personnel: an investigation based on the ehlers and clark cognitive model socioeconomic status, ethnicity, and risk of coronary heart disease racial discrimination and blood pressure: the cardia study of young black and white adults assessing hiv-related stigma in healthcare settings in the era of the covid-19 pandemic first responder culture: implications for mental health professionals providing services following a natural disaster solitary versus social drinking: an experimental study on effects of social exposures on in situ alcohol consumption the role of gaba a receptors in the acute and chronic effects of ethanol: a decade of progress personal and environmental predictors of posttraumatic stress in emergency management professionals differences in compassion fatigue, symptoms of posttraumatic stress disorder and relationship satisfaction, including sexual desire and functioning, between male and female detectives who investigate sexual offenses against children: a pilot study sense of threat as a mediator of peritraumatic stress symptom development during wartime: an experience sampling study the covid-19 pandemic: a call to action to identify and effect of physical inactivity on major non-communicable diseases worldwide: an analysis of burden of disease and life expectancy black-white inequalities in mortality and life expectancy, 1933-1999: implications for healthy people listening to our clients: the prevention of relapse smoking or vaping may increase the risk of severe coronavirus infection inflammation and cardiovascular disease mechanisms covid-19 has inflamed racism against asian-americans. here's how to fight back mental health considerations for children quarantined because of covid-19. the lancet child & adolescent health analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease lessons learned from hiv can inform our approach to covid-19 stigma hope and posttraumatic stress disorder the oxford handbook of hope cognitive factors in the development, maintenance, and treatment of post-traumatic stress disorder. current opinion in psychology parents in poverty multiple pathways to functional impairment in obsessive-compulsive disorder a multidimensional model of obsessive-compulsive disorder do the daily experiences of healthy men and women vary according to occupational prestige and work strain? psychosomatic medicine optimizing pre-exposure antiretroviral prophylaxis adherence in men who have sex with men: results of a pilot randomized controlled trial of "life-steps for prep stress and the individual: mechanisms leading to disease addiction treatment services and co-occurring disorders: prevalence estimates, treatment practices, and barriers a meta-analysis of cognitive behavior therapy and medication for child obsessive-compulsive disorder: moderators of treatment efficacy, response, and remission the structure of childhood obsessions and compulsions: dimensions in an outpatient sample. behavior, research, and therapy socioeconomic status differences in vulnerability to undesirable life events is anxiety sensitivity distinguishable from trait anxiety? reply to lilienfeld policy challenges in addressing racial disparities and improving population health state of disparities in cardiovascular health in the united states fear of the coronavirus (covid-19): predictors in an online study why do clinicians exclude anxious clients from exposure therapy? behavior, research, and therapy the effect of psychosocial syndemic production on 4-year hiv incidence and risk behavior in a large cohort of sexually active men who have sex with men an initial randomized controlled trial of behavioral activation for treatment of concurrent crystal methamphetamine dependence and sexual risk for hiv acquisition among men who have sex with men prevalence and distribution of e-cigarette use among u.s. adults: behavioral risk factor surveillance system focus on: alcohol and the immune system the insomnia severity index: psychometric indicators to detect insomnia cases and evaluate treatment response role of stress, arousal, and coping skills in primary insomnia ebola haemorrhagic fever among hospitalised children and adolescents in nothern uganda: epidemiologic and clinical observations covid-19: potential implications for individuals with substance use disorders the family model stress and maternal psychological symptoms: mediated pathways from economic hardship to parenting rebalancing the 'covid-19 effect' on alcohol sales the compelling link between physical activity and the body's defense system integrated treatment for smoking cessation, anxiety, and depressed mood in people living with hiv: a randomized controlled trial prevalence of psychiatric and substance abuse symptomatology among hiv-infected gay and bisexual men in hiv primary care cognitive behavioral therapy for trauma and self-care (cbt-tsc) in men who have sex with men with a history of childhood sexual abuse: a randomized controlled trial change in perceived risk associated with marijuana use in the united states from cognitive-behavioral therapy for obsessive-compulsive disorder: a meta-analysis of treatment outcome and moderators is nicotine exposure linked to cardiopulmonary vulnerability to covid-19 in the general population? prioritizing community partners and community hiv workers in the covid-19 pandemic cytokines and sleep experiential avoidance and ptsd emotion in posttraumatic stress disorder global status report on alcohol and health 2018: world health organization alcohol: effects on neurobehavioral functions and the brain anxiety sensitivity and working memory capacity: risk factors and targets for health behavior promotion syndemic indicators predict poor medication adherence and increased healthcare utilization for urban hiv-positive men who have sex with men tobacco and the lung cancer epidemic in china covid-19: risk of increase in smoking rates among england's 6 million smokers and relapse among england's 11 million ex-smokers computer-delivered personalized feedback intervention for hazardous drinkers with elevated anxiety sensitivity: study protocol for a randomized controlled trial pandemics and violence against women and children wider income gaps, wider waistbands? an ecological study of obesity and income inequality relationship between resilience, mindfulness, and pyschological well-being in university students the brown longitudinal obsessive compulsive study: clinical features and symptoms of the sample at intake the relation of perceived and received social support to mental health among first responders: a meta-analytic review intolerance of uncertainty and dsm-5 ptsd symptoms: associations among a treatment seeking veteran sample acceptance: a key to wellbeing in older adults? peritraumatic unconditioned and conditioned responding explains sex differences in intrusions after analogue trauma obsessive compulsive disorder and comorbidity long-term cannabis use: characteristics of users in an australian rural area expectancy model of fear, anxiety, and panic covid-19: disproportionate impact on ethnic minority healthcare workers will be explored by government moderate alcohol consumption and the immune system: a review covid-19 related school closings and risk of weight gain among children the epidemiology of obsessive-compulsive disorder in the national comorbidity survey replication cognitive behavioral therapy for adherence and depression (cbt-ad) in hivinfected injection drug users: a randomized controlled trial project enhance: a randomized controlled trial of an individualized hiv prevention intervention for hiv-infected men who have sex with men conducted in a primary care setting smoking trends and disparities among black and non-hispanic whites in california why child welfare experts fear a spike of abuse during covid-19 relationship between household income and mental disorders: findings from a population-based longitudinal study the role of negative affectivity and negative reactivity to emotions in predicting outcomes in the unified protocol for the transdiagnostic treatment of emotional disorders a test of the family stress model on toddler-aged children's adjustment among hurricane katrina impacted and nonimpacted low-income families trauma cognitions are related to symptoms up to 10 years after cognitive behavioral treatment for posttraumatic stress disorder the role of mindfulness in protecting firefighters from psychosomatic malaise estimating the risk of ptsd in recent trauma survivors: results of the international consortium to predict ptsd (icpp) the burden of covid-19 in people living with hiv: a syndemic perspective posttraumatic stress disorder in african american and latinx adults: clinical course and the role of racial and ethnic discrimination covid-19 infection in children. the lancet explaining social class differences in depression and well-being. social psychiatry and psychiatric epidemiology work characteristics and psychiatric disorder in civil servants in london predictors of ptsd 40 years after combat: findings from the national vietnam veterans longitudinal study obsessive-compulsive disorder -w substance abuse and mental health services administration center for behavioral health statistics and quality early epidemiological analysis of the coronavirus disease 2019 outbreak based on crowdsourced data: a population-level observational study. the lancet digital health a recent perspective on alcohol, immunity, and host defense pulmonary effects of inhaled cannabis smoke a new covid-19 crisis: domestic abuse rises worldwide anxiety sensitivity: theory, research, and treatment of the fear of anxiety: routledge metacognitive awareness and prevention of relapse in depression: empirical evidence coping strategies as mediators in relation to resilience and posttraumatic stress disorder potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody. emerging microbes & infections secular trends in smoking in relation to prevalent and incident smoking-related disease: a prospective population-based study ace2: from vasopeptidase to sars virus receptor the age of anxiety? the birth cohort change in anxiety and neuroticism covid-19, school closures, and child poverty: a social crisis in the making covid-19 and smoking: a systematic review of the evidence. tobacco induced diseases respiratory failure caused by lipoid pneumonia from vaping e-cigarettes increased use of cigarettes, alcohol, and marijuana among manhattan posttraumatic stress and substance use disorders: a comprehensive clinical handbook distress tolerance and posttraumatic stress grief during the covid-19 pandemic: considerations for palliative care providers receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china susceptibility analysis of covid-19 in smokers based on ace2 recommended amount of sleep for a healthy adult: a joint consensus statement of the american academy of sleep medicine and sleep research society using death certificates to explore changes in alcohol-related mortality in the united states trends in meeting physical activity guidelines among urban and rural dwelling adults-united states income inequality and population health: a review and explanation of the evidence the problems of relative deprivation: why some societies do better than others. social science & medicine income inequality and socioeconomic gradients in mortality social sources of racial disparities in health racial/ethnic discrimination and health: findings from community studies racial differences in physical and mental health: socio-economic status, stress and discrimination occupational moral injury and mental health: systematic review and meta-analysis evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission covid-19 and african americans patients with mental health disorders in the covid-19 epidemic epidemiology of exercise and sleep survey of insomnia and related social psychological factors among medical staff novel coronavirus disease outbreak clinical characteristics of 140 patients infected with sars-cov-2 in wuhan single-cell rna expression profiling of ace2, the receptor of sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin coronavirus infections in children including covid-19: an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children. the pediatric infectious disease journal are the high smoking rates related to covid-19 outbreaks? distress tolerance: theory, research, and clinical applications we wish to draw the attention of the editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributions to this work zvolensky receives personal fees from elsevier, guilford press, and is supported by grants from nih he receives research support from nih, texas higher education coordinating board, rebuild texas and greater houston community fund. he receives travel support and honorarium from iocdf for training in ocd treatment schmidt is supported by the military suicide research consortium (msrc), department of defense, and visn 19 mental illness research, education, and clinical center buckner receives funding from the u.s. department of health & human services' graduate psychology education (gpe) program (grant d40hp33350) smits reports grants from cancer prevention and research institute of texas; personal fees from big health, ltd., personal fees from aptinyx, inc., personal fees from elsevier vujanovic receives book royalties from routledge press and is supported, in part cleirigh is supported by grants from the nih and the centers for disease control and prevention we confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed he/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. we confirm that we have provided a current, correct email address which is accessible by the corresponding author and which has been configured to accept email from mjzvolen@central key: cord-324829-0nz0qioh authors: carabineiro, sónia alexandra correia title: applications of gold nanoparticles in nanomedicine: recent advances in vaccines † date: 2017-05-22 journal: molecules doi: 10.3390/molecules22050857 sha: doc_id: 324829 cord_uid: 0nz0qioh nowadays, gold is used in (nano-)medicine, usually in the form of nanoparticles, due to the solid proofs given of its therapeutic effects on several diseases. gold also plays an important role in the vaccine field as an adjuvant and a carrier, reducing toxicity, enhancing immunogenic activity, and providing stability in storage. an even brighter golden future is expected for gold applications in this area. it is well known that gold is inert in the bulk stable and is only active as a catalyst in the form of nanoparticles [1] [2] [3] [4] [5] [6] [7] . as the name suggests, nanoparticles have small (nano) size and corresponding large surface/volume ratio. examples are shown in figure 1 . due to these characteristics, au nanoparticles can have an important role, not only in catalysis, but also in nanomedicine, suitable for several biological applications. in fact, it has been argued that au nanoparticles could be used in almost all medical applications, from diagnostics to therapy, prevention, and hygiene [8] [9] [10] [11] [12] [13] [14] . gold nanoparticles have remarkable properties and can also play an important role in vaccine research for several diseases, as showed in several recent reviews [8, 9, [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . vaccines have been a significant advancement in public health, preventing the death of many millions of people every year [31] . the main purpose of vaccination is to introduce an antigen from a vaccines have been a significant advancement in public health, preventing the death of many millions of people every year [31] . the main purpose of vaccination is to introduce an antigen from usually nanoparticles are synthesized in colloidal solution by reduction of chloroauric acid (haucl4) by variations of the so-called turkevich method [46] . their characterization is carried out by ultraviolet-visible spectroscopy, their diameters determined by transmission electron microscopy. then, they can be further functionalized with the desired molecules, according to their specific needs. one example is given in scheme 1, showing a procedure carried out by saha et al. [47] . gold nanoparticles were synthesized in aqueous medium by an ultra-sound assisted green method using black pepper extract and chitosan as reducing agent and a stabilizing agent, respectively. the biopolymer-inspired biogenic method (scheme 1) was found to be stable and with enhanced bioactivity. the developed nanomaterial boosts the production of reactive oxygen species and misbalances the antioxidant parameters of detoxification enzymes of filarial parasites, such as gst, which besides its immune prophylactic potency, is also a promising candidate vaccine as shown in s. cervi and w. bancrofti [47] . gold nanoparticles, in general, have remarkably high surface-to-volume ratio, are biocompatible and inert, and can be easily functionalized with several molecules; thus, they can also play an important role in the vaccine field as adjuvants, reducing toxicity, enhancing immunogenic activity, and providing stability of vaccine in storage, and have great potential as carriers for the development of a great diversity of fully synthetic vaccines [8] [9] [10] [11] [12] 15, 20, 21, 26, [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] . their shape and size can affect immunological responses in vivo and in vitro [51, [62] [63] [64] [65] . moreover, they are able to penetrate blood vessels and tissue barriers and to be targeted to a specific cell by means of specifically functionalized molecules [59] . moreover, gold nanoparticles can be packaged inside virus-like particles generated by heterologous expression of viral structural genes that are powerful tools in vaccine development [66] . anionic gold nanoparticles increased the half-life of a green fluorescent protein expressing adenovirus from similar to 48 h to 21 days at 37 °c (from 7 to >30 days at room temperature) [61] . promising cd8 + t cell results were obtained with polyelectrolyte multilayers assembled on gold nanoparticles [67, 68] . a positively charged antigen and a negatively charged immuno-adjuvant on gold nanoparticles resulted in a new vaccine platform. usually nanoparticles are synthesized in colloidal solution by reduction of chloroauric acid (haucl 4 ) by variations of the so-called turkevich method [46] . their characterization is carried out by ultraviolet-visible spectroscopy, their diameters determined by transmission electron microscopy. then, they can be further functionalized with the desired molecules, according to their specific needs. one example is given in scheme 1, showing a procedure carried out by saha et al. [47] . gold nanoparticles were synthesized in aqueous medium by an ultra-sound assisted green method using black pepper extract and chitosan as reducing agent and a stabilizing agent, respectively. the biopolymer-inspired biogenic method (scheme 1) was found to be stable and with enhanced bioactivity. the developed nanomaterial boosts the production of reactive oxygen species and misbalances the antioxidant parameters of detoxification enzymes of filarial parasites, such as gst, which besides its immune prophylactic potency, is also a promising candidate vaccine as shown in s. cervi and w. bancrofti [47] . gold nanoparticles, in general, have remarkably high surface-to-volume ratio, are biocompatible and inert, and can be easily functionalized with several molecules; thus, they can also play an important role in the vaccine field as adjuvants, reducing toxicity, enhancing immunogenic activity, and providing stability of vaccine in storage, and have great potential as carriers for the development of a great diversity of fully synthetic vaccines [8] [9] [10] [11] [12] 15, 20, 21, 26, [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] . their shape and size can affect immunological responses in vivo and in vitro [51, [62] [63] [64] [65] . moreover, they are able to penetrate blood vessels and tissue barriers and to be targeted to a specific cell by means of specifically functionalized molecules [59] . moreover, gold nanoparticles can be packaged inside virus-like particles generated by heterologous expression of viral structural genes that are powerful tools in vaccine development [66] . anionic gold nanoparticles increased the half-life of a green fluorescent protein expressing adenovirus from similar to 48 h to 21 days at 37 • c (from 7 to >30 days at room temperature) [61] . promising cd8 + t cell results were obtained with polyelectrolyte multilayers assembled on gold [47] ). an immunostimulatory nanocomposite (cpg-au@hbc vlp), designed by self-assembling engineered virus-like particles encapsulating cpg-gold nanoparticle conjugates through electrostatic interactions, showed an increase in cd4 + and cd8 + t cell numbers, inducing important cellular and humoral immune response [69] . ultrasmall graphene oxide-supported gold nanoparticles (usgo-au) were obtained from reduction of chloroauric acid using usgo and then decorated with ovalbumin antigen (ova) through physical adsorption to obtain usgo-au@ova and used immune adjuvants [70] . it was shown that usgo-au@ova can efficiently stimulate raw264.7 cells to secrete tumor necrosis factor (tnf-), a mediator for cellular immune response. usgo-au@ova can also promote robust ova specific antibody response, cd8 + t cells proliferation, and secretion of different cytokines. fluorescent au nanoclusters were synthesized using ovalbumin-cpg oligodeoxynucleotides (odns) conjugates as templates [71] . the nanoclusters can act as self-vaccines to assist in generation of high immunostimulatory activity. gold-based nanovaccines were synthesized using a self-assembling conjugation method [53] . dendritic cells uptake gold nanovaccines with minimal toxicity and are able to process the vaccine peptides on the particles to stimulate cytotoxic t lymphocytes (ctls). these high-peptide density au nanovaccines can stimulate ctls better than free peptides and have great potential as carriers for various vaccine types [53] . hydrophilic gold nanodots were used to control lipopolysaccharide assembly to ease the formation of stable endotoxin nanovesicles, which are stable precursors of cubosomes and hexosomes with specific immunological effects that might be useful in vaccine development [72] . needle-free vaccine delivery systems efficiently transport powdered or particulate dna and protein vaccines into the epidermal tissue [73] [74] [75] [76] . it can be used to directly transfect antigen presenting cells by formulating dna or protein vaccines onto 1-3 µm gold particles (particle-mediated immunization). a solid-in-oil dispersion of gold nanorods can also enhance transdermal protein delivery and skin vaccination [77] . plasmid dna (pdna)-coated gold nanoparticles were successfully delivered into ex vivo murine and porcine skin at low inlet pressures using parallel arrays of microchannels [78] . it was shown that full-length pdna was preserved after each particle preparation and jetting procedures. below, several examples are given on the role of gold nanoparticles in the attempts of producing vaccines for several diseases. cancer is one of the main causes of death worldwide, that can affect people at all ages, even small children and foetuses, the risk usually increasing with age [32, 79] . the present treatments consist of surgery, chemotherapy, or radiotherapy, which can have adverse side effects, due to a lack of specificity for tumors [80] . an ideal treatment should aim only at the target tumor cells and have limited detrimental effects on normal cells [32, 79] . [47] ). an immunostimulatory nanocomposite (cpg-au@hbc vlp), designed by self-assembling engineered virus-like particles encapsulating cpg-gold nanoparticle conjugates through electrostatic interactions, showed an increase in cd4 + and cd8 + t cell numbers, inducing important cellular and humoral immune response [69] . ultrasmall graphene oxide-supported gold nanoparticles (usgo-au) were obtained from reduction of chloroauric acid using usgo and then decorated with ovalbumin antigen (ova) through physical adsorption to obtain usgo-au@ova and used immune adjuvants [70] . it was shown that usgo-au@ova can efficiently stimulate raw264.7 cells to secrete tumor necrosis factor (tnf-), a mediator for cellular immune response. usgo-au@ova can also promote robust ova specific antibody response, cd8 + t cells proliferation, and secretion of different cytokines. fluorescent au nanoclusters were synthesized using ovalbumin-cpg oligodeoxynucleotides (odns) conjugates as templates [71] . the nanoclusters can act as self-vaccines to assist in generation of high immunostimulatory activity. gold-based nanovaccines were synthesized using a self-assembling conjugation method [53] . dendritic cells uptake gold nanovaccines with minimal toxicity and are able to process the vaccine peptides on the particles to stimulate cytotoxic t lymphocytes (ctls). these high-peptide density au nanovaccines can stimulate ctls better than free peptides and have great potential as carriers for various vaccine types [53] . hydrophilic gold nanodots were used to control lipopolysaccharide assembly to ease the formation of stable endotoxin nanovesicles, which are stable precursors of cubosomes and hexosomes with specific immunological effects that might be useful in vaccine development [72] . needle-free vaccine delivery systems efficiently transport powdered or particulate dna and protein vaccines into the epidermal tissue [73] [74] [75] [76] . it can be used to directly transfect antigen presenting cells by formulating dna or protein vaccines onto 1-3 µm gold particles (particle-mediated immunization). a solid-in-oil dispersion of gold nanorods can also enhance transdermal protein delivery and skin vaccination [77] . plasmid dna (pdna)-coated gold nanoparticles were successfully delivered into ex vivo murine and porcine skin at low inlet pressures using parallel arrays of microchannels [78] . it was shown that full-length pdna was preserved after each particle preparation and jetting procedures. below, several examples are given on the role of gold nanoparticles in the attempts of producing vaccines for several diseases. cancer is one of the main causes of death worldwide, that can affect people at all ages, even small children and foetuses, the risk usually increasing with age [32, 79] . the present treatments consist of surgery, chemotherapy, or radiotherapy, which can have adverse side effects, due to a lack of specificity for tumors [80] . an ideal treatment should aim only at the target tumor cells and have limited detrimental effects on normal cells [32, 79] . it was first postulated by coley in 1928 that the immune system was able to recognize and set a response against tumors [81] . later, it was shown that immunization of mice with mutated tumor cells could induce a protective anti-tumor immune response against non-immunogenic tumor [32] . this led to cancer immunotherapy research and to the development of cancer vaccines capable of generating an active tumor-specific immune response. cancer vaccines have high specificity for tumor cells and long-lasting immunological memory that may safeguard against recurrences, and can be used to either prevent (prophylactic) or treat established cancer (therapeutic) [32, 79] . therapeutic cancer vaccines (also called active immunotherapy) work to enable the immune system of a patient to eradicate cancer cells [82] . identification of tumor-associated antigens (taas) and tumor-specific antigens (tsas) has led to increased efforts to develop vaccination strategies. vaccines may be composed of whole cells or cell extracts, genetically modified tumor cells, dendritic cells (dcs) loaded with taas, immunization with soluble proteins or synthetic peptides, recombinant viruses or bacteria encoding tumor-associated antigens, and plasmid dna-encoding tsas or taas in conjunction with appropriate immunomodulators [32, 79] . all of these vaccination approaches aim to induce specific immunological responses and are localized into taas, destroying only the tumor cells and leaving the majority of other cells of the body undamaged. an efficient delivery to cells is needed and gold nanoparticles have proved to be excellent carriers [83] [84] [85] [86] [87] [88] [89] . gold nanoparticles enabled efficient antigen delivery to dendritic cells and then activated the cells to facilitate cross-presentation, inducing antigen-specific cytotoxic t-lymphocyte responses for effective cancer therapy [88] . moreover, they show several advantages over other nanoparticulate-based carriers, such as the ease with which they control their size for different applications, the variety of antigens and adjuvants that can be easily linked to and displayed on their surface, the fact that they can be detected using non-invasive imaging techniques, providing clinicians with information on where or whether the vaccines have been delivered, which helps to predict or evaluate therapeutic efficacy, and the biocompatibility and non-toxicity of gold [86, 87, 90] . au nanoparticle immunotherapies are well suited for synergistic combination therapy with existing cancer therapies such as photothermal ablation [59, 87] . all these features suggest that gold nanoparticle-based antigen delivery systems may be a useful vaccine technology that is able to prevent and/or treat cancer. the several functionalities of gold nanoparticles make them promising vehicles for immune therapies, especially for combinatorial treatment approaches that target multiple immune pathways (figure 3) . a polymerizable version of the tn-antigen glycan was synthesized and the polymers were conjugated to gold nanoparticles, producing nanoscale glycoconjugates [91] . these nanomaterials generated a strong and long-lasting production of antibodies, selective to the tn-antigen glycan and cross-reactive toward mucin proteins displaying tn, and thus represent a simple approach to the synthesis of anticancer vaccines. gold nanoparticles obtained by a modified turkevich method were functionalized with mucin-1 (muc-1) glycoprotein using clealand's reagent [82] . (muc-1 is involved in fundamental biological processes that can be found over-expressed and with a clearly changed glycan pattern on epithelial tumor cells, and thus is a promising target structure in the search for effective carbohydrate-based cancer vaccines and immunotherapeutics [58] ). the obtained au-muc-1 nano-construction has been shown to be an important macrophage activator, leading to the release of cytokines such as tnf-alpha, il-6, il-10, and il-12 on peritoneal macrophages isolated from mice [82] . au nanoparticle immunotherapies are well suited for synergistic combination therapy with existing cancer therapies such as photothermal ablation [59, 87] . all these features suggest that gold nanoparticle-based antigen delivery systems may be a useful vaccine technology that is able to prevent and/or treat cancer. the several functionalities of gold nanoparticles make them promising vehicles for immune therapies, especially for combinatorial treatment approaches that target multiple immune pathways (figure 3) . a polymerizable version of the tn-antigen glycan was synthesized and the polymers were conjugated to gold nanoparticles, producing nanoscale glycoconjugates [91] . these nanomaterials generated a strong and long-lasting production of antibodies, selective to the tn-antigen glycan and cross-reactive toward mucin proteins displaying tn, and thus represent a simple approach to the synthesis of anticancer vaccines. gold nanoparticles obtained by a modified turkevich method were functionalized with mucin-1 (muc-1) glycoprotein using clealand's reagent [82] . other authors also reported on the synthesis of muc1-glycopeptide antigens and their coupling to gold nanoparticles of different sizes and developed a new dot-blot immunoassay to test the potential antigen-antibody binding [58] . a glycopeptide sequence derived from muc-1 glycoprotein and the t-cell epitope p30 sequence were immobilized gold nanoparticles attached to polyethylene glycol chains [92] . after immunization, mice showed significant mhc-ii mediated immune responses, and their antisera were able to recognize human mcf-7 breast cancer cells. gold nanoparticles designed this way have the potential to be used in the development of anticancer vaccines. gold nanoparticles proved to be efficient in facilitating the delivery of the ovalbumin (ova) peptide antigen and the cpg adjuvant and enhance their therapeutic effect in a b16-ova tumor model. gold nanoparticle-mediated ova peptide delivery can have important therapeutic benefits without the need of an adjuvant, showing that gold nanoparticles are effective peptide vaccine carriers, having the potential to allow the use of lower and safer adjuvant doses during vaccination [93] . the use of gold nanoparticles in hiv vaccine development has been recently reviewed by several authors [94] . after more than 30 years since the discovery of hiv in 1983, no effective vaccine is yet available [24, [95] [96] [97] . some histocompatibility complex molecules expressed on the surface of hiv are potential targets for neutralizing antibodies [98] , as shown in figure 4 . among the few broadly neutralizing hiv monoclonal antibodies, 2g12 is the only carbohydrate-directed that is able to recognize a cluster of high-mannose glycans on the viral envelope glycoprotein gp120 [95] . this type of glycan is thus envisaged as a target to develop an hiv vaccine capable of eliciting 2g12-like antibodies. gold nanoparticles coated with self-assembled monolayers of synthetic oligomannosides, which are present in gp120, are able to bind 2g12 with high affinity and to interfere with 2g12/gp120 binding [95] . thiol-terminated oligosaccharides have been attached on gold nanoparticles and have been used in attempts to develop an hiv vaccine [99] . two nanometer gold glyconanoparticles were coated with synthetic partial structures of several mannosides [100] . the assembly of the antennas of the gp120 high-mannose type glycan on gold glyconanoparticles provided superior binding to the anti-hiv antibody 2g12, which could help in the design of a carbohydrate-based vaccine against hiv. it was shown that conjugation to negatively charged gold glyconanoparticles could stabilize either the alpha-helix or beta-strand conformation of the third variable region (v3 peptide) of the hiv-1 gp120 [101] . the peptide on the nanoparticles shows more stability toward peptidase degradation compared to the free peptide. v3 beta-glyconanoparticles produce antibodies in rabbits that recognize a recombinant gp120, and the serum showed consistent neutralizing activity. these results potentially allow for the design of new fully synthetic hiv vaccine candidates [101] . gold nanorods modified with poly(diallydimethylammonium chloride) or polyethyleneimine can significantly promote cellular and humoral immunity, as well as t-cells proliferation, through activating antigen-presenting cells, when compared to naked hiv envelope plasmid dna treatment in vivo [102] . this makes gold nanorods promising dna vaccine adjuvants for hiv treatment. the use of gold nanoparticles in hiv vaccine development has been recently reviewed by several authors [94] . after more than 30 years since the discovery of hiv in 1983, no effective vaccine is yet available [24, [95] [96] [97] . some histocompatibility complex molecules expressed on the surface of hiv are potential targets for neutralizing antibodies [98] , as shown in figure 4 . among the few broadly neutralizing hiv monoclonal antibodies, 2g12 is the only carbohydrate-directed that is able to recognize a cluster of high-mannose glycans on the viral envelope glycoprotein gp120 [95] . this type of glycan is thus envisaged as a target to develop an hiv vaccine capable of eliciting 2g12-like antibodies. gold nanoparticles coated with self-assembled monolayers of synthetic oligomannosides, which are present in gp120, are able to bind 2g12 with high affinity and to interfere with 2g12/gp120 binding [95] . thiol-terminated oligosaccharides have been attached on gold nanoparticles and have been used in attempts to develop an hiv vaccine [99] . two nanometer gold glyconanoparticles were coated with synthetic partial structures of several mannosides [100] . the assembly of the antennas of the gp120 high-mannose type glycan on gold glyconanoparticles provided superior binding to the anti-hiv antibody 2g12, which could help in the design of a carbohydrate-based vaccine against hiv. it was shown that conjugation to negatively charged gold glyconanoparticles could stabilize either the alpha-helix or beta-strand conformation of the third variable region (v3 peptide) of the hiv-1 gp120 [101] . the peptide on the nanoparticles shows more stability toward peptidase degradation the use of gold nanoparticles in designing vaccines against tick-borne encephalitis (tbe) was proposed for the first time by demenev et al. [103] . the vaccine was prepared by conjugating colloid gold with a soluble tbe antigen. in animals immunized with the experimental vaccine, the protection coefficient and mean survival time were, respectively, 1.3-1.5 times and 10-30% higher than in mice immunized with a commercial vaccine. moreover, the mean survival time was 1.2-1.7 times longer in animals injected with antibodies from mice immunized with the experimental vaccine, compared with the commercial one. in a more recent study, colloidal gold particles were used as carriers of protein antigen of the capsid of the tbe virus in the antiviral vaccine [104] . after two inoculations, the mice in that study were found to resist challenge with 100,000 times the 50% lethal dose (ld50) of jev (beijing-1 strain), even when immunized with a relatively small dose of 0.5 mug of plasmid dna. japanese b encephalitis vaccine is an important vaccine to prevent this serious mosquito-borne disease caused by the virus [105, 106] . there are some semiquantitative methods to determine this vaccine, such as plaque forming unit and the animal testing, but they have low sensitivity, are time-consuming, and have a high cost. a label-free amperometric immunosensor for fast and sensitive assay of japanese b encephalitis vaccine was reported with a specific response in the range 1.1 × 10 −8 to 1.9 × 10 −6 lg·pfu/ml (pfu/ml is plaque forming unit and lg is common logarithm) with a detection limit of 6 × 10 −9 lg·pfu/ml [105] . an immunosensor based on the immobilization of antiserum of japanese b encephalitis on a gold electrode modified by [nano-au/co(bipyridine) 3 3+ ] 2 /nano-au/l-cysteine has also been reported [106] . it exhibited fast potentiometric high sensitivity and long-term stability. these works are promising test methods for biological products. hepatitis is inflammation of the liver that may originate a yellow color in the skin and eyes and abdominal pain, among other symptoms that can be caused by viruses or from the abuse of alcohol and certain medications. there are several types of viral hepatitis: types a, b, c, d, and e. hepatitis a and e are often spread by contaminated food and water, while hepatitis b is mainly sexually transmitted but may pass from mother to child during pregnancy. types b and c are usually spread through figure 5 shows a comparison between the viruses. hepatitis is inflammation of the liver that may originate a yellow color in the skin and eyes and abdominal pain, among other symptoms that can be caused by viruses or from the abuse of alcohol and certain medications. there are several types of viral hepatitis: types a, b, c, d, and e. hepatitis a and e are often spread by contaminated food and water, while hepatitis b is mainly sexually transmitted but may pass from mother to child during pregnancy. types b and c are usually spread through infected blood. hepatitis d can only infect people already infected with hepatitis b. figure 5 shows a comparison between the viruses. concerning hepatitis b vaccine research, hbsag-dna can be introduced into the host by intramuscular or intradermal route using a needle with syringe. an alternative is using gene-gun, biolistic ® , powderjecttm, accell ® , or particle-mediated dna delivery, when dna-coated microscopic gold particles are transported using a needle-free device directly into the epidermic cells [75] . micron-size gold particles were used as particulate adjuvants and coinjected intradermally with plasmid dna encoding the hbsag into mice [107, 108] . the presence of gold particles accelerated the antibody response significantly, increased the percentage of responding animals, and shortened the time taken to reach maximal antibody titers by two weeks. these immunizations were effective in protecting mice against tumor challenge with cancer cells, expressing hbsag as a surrogate cancer antigen. good results were also obtained with minipigs [109] . electrically activated plasmonic au nanoparticles can drive vibrational and dipole-like oscillations that are able to disrupt nearby cell membranes, allowing enhanced cell poration and facilitating the uptake of a model hepatitis c virus dna vaccine was recently reported [110] . immunized mice showed up to 100-fold higher gene expression compared to control treatments (without nanoparticles) and exhibited significantly increased levels of both antibody and cellular immune responses. a new method of preparation of a vaccine for hepatitis e (heva) using in situ easy growth of gold clusters in the vaccine was recently reported [111] . the prepared heva/au enhances its immune responses in vivo and reduces the potential toxicity of heva. the fluorescence of gold clusters enables the heva to be traceable, which may open a way to track the dynamic behavior of vaccines and further help to optimize an individual therapeutic regimen for immunotherapy [111] . gold has been used in several other vaccines. some examples are reported below. gold nanoparticles were conjugated to a synthetic peptide of the vp1 capsid protein of the foot-and-mouth disease virus, which causes an acute, highly contagious infection of domestic and wild animals, transmittable to humans, for which the existing vaccines are not much effective [112] . the resulting conjugate (with or without the use of complete freund's adjuvant) was compared to a commercial vaccine and to the native peptide, in immunization of guinea pigs. the titer and sensitivity of the antibodies were maximal for the combination comprising the nanoparticle-peptide conjugate and complete freund's adjuvant. gold nanoparticles were evaluated as vaccine carriers for enhancing the antibody response against a resembling foot-and-mouth disease virus peptide [62] . particles with 8-17 nm in diameter stimulated the highest antibody levels and accumulated at the highest numbers in the spleen of mice. recently, the potential of chitosan functionalized gold nanoparticles (csaunps) for the transmucosal delivery of tetanus toxoid vaccine was shown [63, 113, 114] . excellent stability was found for the formulation at recommended storage conditions. csaunps were also used as a carrier for the tetanus toxoid (tt) antigen along with the immunostimulant quillaja saponaria extract (qs) [115] . the synthesized csaunps were spherical, around 40 nm in size and conferred protection to tt against gastric hydrolysis. tt-qs-csaunps induced up to 28-fold immune responses compared to tt and tt-qs controls, after oral administration in mice. the delivery of tt and qs with functionalized csaunps might be a good approach for oral vaccine delivery [115] . gold glyconanoparticles proved to be good carriers for a synthetic streptococcus pneumoniae type 14 conjugate vaccine [50] . a gold nanorod construct that displayed the major protective antigen of the respiratory syncytial virus (which causes pneumonia and wheezing in children and the elderly), the fusion protein (f) was reported, which can be a candidate vaccine preparation by the covalent attachment of viral protein using a layer-by-layer approach [116] . gold nanoparticles of 12 nm were conjugated to the matrix 2 protein (m2e) of influenza a virus m2e through thiol-gold interactions [117] . this virus ( figure 6 ) is often responsible for seasonal epidemics and is also a high risk for pandemics [118] . vaccination of mice with m2e-au conjugates induced m2e-specific igg serum antibodies, which significantly improved by addition of cpg as adjuvant, as mice immunized that way were fully protected against lethal pr8 [117] . the same authors also showed that the inclusion of excess soluble m2e antigen along with m2e immobilized on au nanoparticles is vital for inducing high levels of antibody response, and in providing complete protection [118] . also showed that the inclusion of excess soluble m2e antigen along with m2e immobilized on au nanoparticles is vital for inducing high levels of antibody response, and in providing complete protection [118] . a recent study showed that animals immunized with a transmissible gastroenteritis virus, conjugated with colloidal gold nanoparticles, produced antibodies with a higher titer than those produced in response to the native virus [119] . a gold nanoparticle antigen delivery approach was used together with a novel polysaccharide nanoparticulate adjuvant, and an effective t-cell vaccine was developed providing protection in animal models of listeriosis (a fatal infection for fetuses and newborns that causes meningitis and cutaneous lesions) [120] . this antigen delivery approach against listeriosis, using gold nanoparticles and bacterial peptides, elicits protective cellular immunity, independent of the adjuvant used, either as a carbohydrate such as advax [121] or a tlr-4 agonist such as dio-1 [120] . gold glyconanoparticles loaded with listeriolysin peptide 91-99 (llo91-99) or glyceraldehyde-3-phosphate dehydrogenase 1-22 peptide (gapdh ) were successfully administrated to pregnant female mice as a vaccination for this disease [122] . neonates from vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice showed clear brain affections and cutaneous diminishment of melanocytes. amphiphilic surface ligand-coated au nanoparticles able to target myeloid dendritic cells in lymph nodes were used as a peptide antigen carrier, increasing the efficacy of a model vaccine in b16-ova melanoma mouse models [123] . gold nanoparticles were coated with the f1-antigen of yersinia pestis (bacterium responsible for the plague), using n-hydroxysuccinimide and n-(3-dimethylaminopropyl)-n′-ethylcarbodiimide a recent study showed that animals immunized with a transmissible gastroenteritis virus, conjugated with colloidal gold nanoparticles, produced antibodies with a higher titer than those produced in response to the native virus [119] . a gold nanoparticle antigen delivery approach was used together with a novel polysaccharide nanoparticulate adjuvant, and an effective t-cell vaccine was developed providing protection in animal models of listeriosis (a fatal infection for fetuses and newborns that causes meningitis and cutaneous lesions) [120] . this antigen delivery approach against listeriosis, using gold nanoparticles and bacterial peptides, elicits protective cellular immunity, independent of the adjuvant used, either as a carbohydrate such as advax [121] or a tlr-4 agonist such as dio-1 [120] . gold glyconanoparticles loaded with listeriolysin peptide 91-99 (llo91-99) or glyceraldehyde-3-phosphate dehydrogenase 1-22 peptide (gapdh(1-22)) were successfully administrated to pregnant female mice as a vaccination for this disease [122] . neonates from vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice showed clear brain affections and cutaneous diminishment of melanocytes. amphiphilic surface ligand-coated au nanoparticles able to target myeloid dendritic cells in lymph nodes were used as a peptide antigen carrier, increasing the efficacy of a model vaccine in b16-ova melanoma mouse models [123] . gold nanoparticles were coated with the f1-antigen of yersinia pestis (bacterium responsible for the plague), using n-hydroxysuccinimide and n-(3-dimethylaminopropyl)-n -ethylcarbodiimide hydrochloride coupling chemistry, and administrated to mice, triggering an igg antibody response to the f1-antigen [124] . the sera raised against f1-antigen coupled to au nanoparticles were able to competitively bind to recombinant f1-antigen, displacing protective macaque sera. target antigens identified in plasmodium falciparum, the parasite responsible for malaria, include surface proteins pfs230 and pfs48/45 in male and female gametocytes and pfs25 expressed in zygotes and ookinetes. the codon-harmonized recombinant pfs25 in escherichia coil (chrpfs25) is able to produce malaria transmission-blocking antibodies in mice. chrpfs25 was also investigated, with gold nanoparticles of different shapes, size and physicochemical properties as adjuvants for induction of transmission blocking immunity, causing transmission blocking antibodies [125] . these results show that gold nanoparticle-based formulations can be developed as nanovaccines to enhance the immunogenicity of vaccine antigens. gold nanoparticles were conjugated to n-terminal domains of pseudomonas aeruginosa flagellin recombinant protein through direct interaction of thiol molecules of the cysteines with gold and formation of au-s bond [126] . mice that received aunp-flagellin((1-161)) with freund's adjuvant produced high titers of anti-flagellin((1-161)) antibodies that effectively recognized the native flagellin on the bacteria. synthetic virus-like particles (svlps) were prepared by incubating 100 nm gold nanoparticles in a solution containing an avian coronavirus spike protein [127] . after removing the free proteins, antigen-laden particles were recovered and showed morphological semblance to natural viral particles. vaccination with the svlps showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic t-cell response, and reduced infection-associated symptoms, and provided superior antiviral protection when compared to a commercial whole inactivated virus vaccine [127] . burkholderia mallei are bacteria responsible for the glanders disease, which is recently classified as a tier 1 agent, since they can be weaponized for aerosol release, cause high mortality rates, and show multi-drug resistance, and for which there is so far no vaccine available [128] . gold nanoparticles were covalently coupled with one of three different protein carriers (tethc, hcp1, and flic) followed by conjugation to lipopolysaccharide (lps) generated significantly higher antibody titers compared with lps alone. in another study, a gold nanoparticle glycoconjugate composed of burkholderia thailandensis lps conjugated to flic was evaluated for the first time as a candidate vaccine for glanders on rhesus macaques (macaca mulatta) [57] . it has recently been shown that the pichia pastoris yeast expression system was adequate for the production of recombinant-truncated proteins, and their apparent bioactivity suggests that torf25, torf25c, and torf25d are potential candidate vaccines against cyprinid herpes virus 2 infection. found in farmed gibel carp carassius gibelio, it is an infectious disease that recently emerged in china that has been troubling the aquaculture industry [129] . colloidal gold-labelled immunoglobulin was used to confirm rv1268c protein localization on mycobacterium tuberculosis [130] . these results, along with those obtained for other proteins, might lead to the discovery of select peptides that could have the potential to be included in a subunit-based vaccine for tuberculosis, an infectious disease that remains lethal around the world. biocompatible gold nanoparticles were complexed with anti-dengue virus small interfering rnas (sirna), and were able to enhance the sirna delivery and stability, becoming a novel strategy against a virus that causes flu-like symptoms, haemorrhagic fever, and death [131] . escherichia coli as a model pathogen for the design of an antibacterial vaccine [132] . the bacterial outer membrane vesicles were collected and successfully coated onto gold nanoparticles with a 30 nm diameter. the resulting bacterial membrane-coated gold nanoparticles, when injected subcutaneously, induced rapid activation and maturation of dendritic cells in the lymph nodes of the vaccinated mice, generated durable antibody responses, and induced a high production of interferon gamma and interleukin-17, but not interleukin-4, indicating its capability of generating strong th1-and th17-biased cell responses against the used bacteria. a monoclonal antibody raised against human cytomegalovirus (cmv, a herpes virus) surface glycoprotein (gb) was chemically conjugated with gold nanoparticles [133] . the gb-gold nanoparticles blocked viral replication, virus-induced cytopathogenic effects, and virus spread in cell culture without inducing cytotoxicity, and cells treated with them gained resistance to cmv infection. as discussed above, research on gold has indicated its potential for several applications in nanomedicine and its (bio)medical applications, including vaccines, and, as shown in this review, has been increasing. the overall conclusion is that the potential of gold for stimulating research in vaccines is considerable. the results will certainly lead to more practical and commercial applications, the full extent of which has yet to be envisaged. certainly, the use of gold carries added costs. as newer and more expensive vaccines are introduced and attempted to reach people of different ages and in new settings, the logistics systems must be strengthened and optimized, as recently highlighted and reviewed by zaffran et al. [134] . catalysis by gold nanoparticles when gold is not noble: catalysis by nanoparticles gold catalysis catalysis by gold catalytic applications for gold nanotechnology anisotropic gold nanoparticles: preparation and applications in catalysis gold nanoparticles in biology and medicine: recent advances and prospects gold nanoparticles in biomedical applications: recent advances and perspectives biological applications of gold nanoparticles particulate inorganic adjuvants: recent developments and future outlook gold nanoparticles and their applications in biomedicine applications of nanotechnology in the agriculture, food, and pharmaceuticals use of nanoparticles to deliver immunomodulatory oligonucleotides current status of multiple antigen-presenting peptide vaccine systems: application of organic and inorganic nanoparticles vaccine delivery using nanoparticles applications of nanomaterials as vaccine adjuvants. hum. vaccines immunother oral delivery of nanoparticle-based vaccines synthetic nanoparticles for vaccines and immunotherapy gold nanoparticles and vaccine development gold nanocluster-based vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides gold nanoparticles (aunps): a new frontier in vaccine delivery design of nanomaterial based systems for novel vaccine development recent advances in synthetic carbohydrate-based human immunodeficiency virus vaccines advanced nanobiomaterials: vaccines, diagnosis and treatment of infectious diseases recent advances in subunit vaccine carriers nanomaterial-based vaccine adjuvants understanding the immunogenicity and antigenicity of nanomaterials: past, present and future immunological properties of gold nanoparticles role of metallic nanoparticles in vaccinology: implications for infectious disease vaccine development vaccine technologies: from whole organisms to rationally designed protein assemblies review: cancer therapy and vaccination biomedicine: new insights from plant viruses nanovaccines and their mode of action glycodendrimers: versatile tools for nanotechnology. braz functional nanomaterials can optimize the efficacy of vaccines nanomedicine in the management of microbial infection-overview and perspectives nanomedicine scale-up technologies: feasibilities and challenges nanoparticle based tailoring of adjuvant function: the role in vaccine development the potential of nanoparticles for the immunization against viral infections learning from vaccines and progressing to antigen-specific immunotherapies advances in multifunctional glycosylated nanomaterials: preparation and applications in glycoscience nanoparticle-based modulation of the immune system real-time assessment of nanoparticle-mediated antigen delivery and cell response a study of the nucleation and growth processes in the synthesis of colloidal gold development of chitosan based gold nanomaterial as an efficient antifilarial agent: a mechanistic approach gold colloidal solution is used as an adjuvant, provides reducing toxicity of vaccines, to enhance their immunogenic activity and to provide stability of vaccine in storage. patent ru2218937-c2 new adjuvant composition comprising colloidal gold, used for therapeutic vaccine or for inducing immune response gold nanoparticles as carriers for a synthetic streptococcus pneumoniae type 14 conjugate vaccine gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo injectable nanomaterials for drug delivery: carriers, targeting moieties, and therapeutics high-density sub-100-nm peptide-gold nanoparticle complexes improve vaccine presentation by dendritic cells in vitro gold nanoparticle conjugates: recent advances toward clinical applications polymeric nanoparticles potent vectors for vaccine delivery targeting cancer and infectious diseases tracking targeted bimodal nanovaccines: immune responses and routing in cells, tissue, and whole organism protection of non-human primates against glanders with a gold nanoparticle glycoconjugate vaccine synthesis of tumor-associated muc1-glycopeptides and their multivalent presentation by functionalized gold colloids metal based frameworks for drug delivery systems gold nanoparticles: recent advances in the biomedical applications additives for vaccine storage to improve thermal stability of adenoviruses from hours to months assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide structure function attributes of gold nanoparticle vaccine association: effect of particle size and association temperature intracellular accumulation and immunological properties of fluorescent gold nanoclusters in human dendritic cells different-sized gold nanoparticle activator/antigen increases dendritic cells accumulation in liver-draining lymph nodes and cd8+t cell responses yeast-expressed bacteriophage-like particles for the packaging of nanomaterials polyelectrolyte multilayers assembled entirely from immune signals on gold nanoparticle templates promote antigen-specific t cell response impact of dose, route, and composition on the immunogenicity of immune polyelectrolyte multilayers delivered on gold templates construction and immunological evaluation of cpg-au@hbc virus-like nanoparticles as a potential vaccine ultrasmall graphene oxide supported gold nanoparticles as adjuvants improve humoral and cellular immunity in mice engineered cpg-antigen conjugates protected gold nanoclusters as smart self-vaccines for enhanced immune response and cell imaging endotoxin nanovesicles: hydrophilic gold nanodots control supramolecular lipopolysaccharide assembly for modulating immunological responses epidermal powder immunization induces both cytotoxic t-lymphocyte and antibody responses to protein antigens of influenza and hepatitis b viruses powder and particle-mediated approaches for delivery of dna and protein vaccines into the epidermis recent advances in hepatitis b vaccination nanoparticulate mediated transcutaneous immunization: myth or reality a solid-in-oil dispersion of gold nanorods can enhance transdermal protein delivery and skin vaccination a microarray mems device for biolistic delivery of vaccine and drug powders cancer: facts, causes, symptoms and research application of nanostructured drug delivery systems in immunotherapy of cancer: a review end results in hodgkin's disease and lymphosarcoma treated by the mixed toxins of erysipelas and bacillus prodigiosus, alone or combined with radiation in vitro administration of gold nanoparticles functionalized with muc-1 protein fragment generates anticancer vaccine response via macrophage activation and polarization mechanism glycopolymer-functionalized gold nanoparticles: a new strategy toward synthetic anticancer vaccines development of a novel cancer vaccine based on multivalent presentation of tumor-associated carbohydrate antigens on gold nanoparticle scaffolds design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines imageable antigen-presenting gold nanoparticle vaccines for effective cancer immunotherapy in vivo gold nanoparticle mediated cancer immunotherapy gold nanoparticles displaying tumor-associated self-antigens as a potential vaccine for cancer immunotherapy current status and future directions of nanoparticulate strategy for cancer immunotherapy gold nanoparticles as an antigen and as an adjuvant multicopy multivalent' glycopolymer-stabilized gold nanoparticles as potential synthetic cancer vaccines glycopeptide-functionalized gold nanoparticles for antibody induction against the tumor associated mucin-1 glycoprotein in vivo gold nanoparticle delivery of peptide vaccine induces anti-tumor immune response in prophylactic and therapeutic tumor models role of nanotechnology in hiv/aids vaccine development gold nanoparticles coated with oligomannosides of hiv-1 glycoprotein gp120 mimic the carbohydrate epitope of antibody 2g12 gold manno-glyconanoparticles for intervening in hiv gp120 carbohydrate-mediated processes enhancing dendritic cell activation and hiv vaccine effectiveness through nanoparticle vaccination immunization with recombinant hla classes i and ii, hiv-1 gp140, and siv p27 elicits protection against heterologous shiv infection in rhesus macaques design and synthesis of thiol-terminated oligosaccharides for attachment on gold nanoparticles: toward the development of an hiv vaccine assembling different antennas of the gp120 high mannose-type glycans on gold nanoparticles provides superior binding to the anti-hiv antibody 2g12 than the individual antennas negatively charged glyconanoparticles modulate and stabilize the secondary structures of a gp120 v3 loop peptide: toward fully synthetic hiv vaccine candidates surface-engineered gold nanorods: promising dna vaccine adjuvant for hiv-1 treatment perfection of methodical approaches to designing vaccines against tick-borne encephalitis inoculation of plasmids encoding japanese encephalitis virus prm-e proteins with colloidal gold elicits a protective immune response in balb/c mice a label-free amperometric immunosenor based on multi-layer assembly of polymerized o-phenylenediamine and gold nanoparticles for determination of japanese b encephalitis vaccine layer-by-layer self-assembly of films of nano-au and co(bpy)(3)(3+) for the determination of japanese b encephalitis vaccine accelerated immune response to dna vaccines enhancement of the effectiveness of electroporation-augmented cutaneous dna vaccination by a particulate adjuvant the assessment of local tolerance, acute toxicity, and dna biodistribution following particle-mediated delivery of a dna vaccine to minipigs electrically oscillating plasmonic nanoparticles for enhanced dna vaccination against hepatitis c virus assembly of hepatitis e vaccine by 'in situ' growth of gold clusters as nano-adjuvants: an efficient way to enhance the immune responses of vaccination use of a synthetic foot-and-mouth disease virus peptide conjugated to gold nanoparticles for enhancing immunological response gold nanoparticles as a potential carrier for transmucosal vaccine delivery enhanced mucosal immune responses against tetanus toxoid using novel delivery system comprised of chitosan-functionalized gold nanoparticles and botanical adjuvant: characterization, immunogenicity, and stability assessment quillaja saponaria extract as mucosal adjuvant with chitosan functionalized gold nanoparticles for mucosal vaccine delivery: stability and immunoefficiency studies gold nanorod vaccine for respiratory syncytial virus gold nanoparticle-m2e conjugate coformulated with cpg induces protective immunity against influenza a virus m2e-immobilized gold nanoparticles as influenza a vaccine: role of soluble m2e and longevity of protection immunostimulatory effect of gold nanoparticles conjugated with transmissible gastroenteritis virus novel nanoparticle vaccines for listeriosis. hum. vaccines immunotherapeut a gold glyco-nanoparticle carrying a listeriolysin o peptide and formulated with advax™ delta inulin adjuvant induces robust t-cell protection against listeria infection pregnancy vaccination with gold glyco-nanoparticles carrying listeria monocytogenes peptides protects against listeriosis and brainand cutaneous-associated morbidities high-throughput quantitation of inorganic nanoparticle biodistribution at the single-cell level using mass cytometry conjugation of y. pestis f1-antigen to gold nanoparticles improves immunogenicity nanovaccines for malaria using plasmodium falciparum antigen pfs25 attached gold nanoparticles induction of humoral immune response against pseudomonas aeruginosa flagellin(1-161) using gold nanoparticles as an adjuvant synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection a gold nanoparticle-linked glycoconjugate vaccine against burkholderia mallei protective immunity in gibel carp, carassius gibelio of the truncated proteins of cyprinid herpesvirus 2 expressed in pichia pastoris rv1268c protein peptide inhibiting mycobacterium tuberculosis h37rv entry to target cells delivery of antiviral small interfering rna with gold nanoparticles inhibits dengue virus infection in vitro modulating antibacterial immunity via bacterial membrane-coated nanoparticles inhibition of cytomegalovirus infection and photothermolysis of infected cells using bioconjugated gold nanoparticles the imperative for stronger vaccine supply and logistics systems key: cord-348409-oxjd263z authors: stern, zachariah; stylianou, dora c.; kostrikis, leondios g. title: the development of inovirus-associated vector vaccines using phage-display technologies date: 2019-09-08 journal: expert rev vaccines doi: 10.1080/14760584.2019.1651649 sha: doc_id: 348409 cord_uid: oxjd263z introduction: inovirus-associated vectors (iavs) are derived from bacterial filamentous viruses (phages). as vaccine carriers, they have elicited both cellular and humoral responses against a variety of pathogens causing infectious diseases and other non-infectious diseases. by displaying specific antigen epitopes or proteins on their coat proteins, iavs have merited much study, as their unique abilities are exploited for widespread vaccine development. areas covered: the architectural traits of filamentous viruses and their derivatives, iavs, facilitate the display of specific antigenic peptides which induce antibody production to prevent or curtail infection. inoviruses provide a foundation for cost-efficient large-scale specific phage display. in this paper, the development of different applications of inovirus-based phage display vaccines across a broad range of pathogens and hosts is reviewed. the references cited in this review were selected from established databases based on the authors’ knowledge of the study subject. expert commentary: the importance of phage-display technology has been recently highlighted by the nobel prize in chemistry 2018 awarded to george p. smith and sir gregory p. winter. furthermore, the symbiotic nature of filamentous viruses infecting intestinal f(+) e. coli strains offers an attractive platform for the development of novel vaccines that stimulate mucosal immunity research regarding the potential to use viruses to combat disease has been ongoing for over 100 years [1, 2] . while viruses have often been considered pernicious as they co-opt a host for their own survival, often killing the host in the process, new work with viruses that can be exploited as bacteriophages but without the harmful effects has arisen [3] . these viruses, which can be easily manipulated and employed for phage display ability, are being increasingly used for a variety of potent biomedical tools [4] . these filamentous bacterial viruses, which make up the genus inovirus in the family inoviridae, are thread-like viruses containing single-stranded dna genomes know as filamentous bacteriophages [5] [6] [7] . over 50 different species of filamentous viruses are known, of which a majority can infect gram-negative bacteria. although inoviruses are now being used for their phage display capabilities, these filamentous viruses have a relationship with the cell that they infect that is more similar to symbiotic non-pathogenic animal viruses than classical phages. unlike phages, which term comes from the greek word φάγος for destroyer, inoviruses do not kill their host and only slightly affect cell growth despite yielding titers of up to 10 13 virions per milliliter of liquid culture. progeny virions are assembled in the host cell's membrane where single-stranded dna binding proteins are replaced by major capsid protein subunits before being released into the cell, resulting in opaque plaques on bacterial lawns [8, 9] . receptor organelles in the bacterial host that are encoded by transmissible plasmids facilitate the interactions between inovirus and cell [5, 10] . the functional architecture of inoviruses provides the foundation for their application in vaccine-related projects since inoviruses do not cause harm. a great number of inovirus species across the world have been isolated and characterized [5] . despite variation by species, they have the same general physical characteristics. the virions are flexible, thin cylindrical filaments [6, 7] under 10 nm in diameter and approximately 1000 nm in length (see figure 1 (a) for details). most of a single virion is composed of several thousand major capsid or coat protein subunits. these surround a circular single-stranded dna molecule. at the proximal end of the virion there are a few minor proteins which attach to the cell to initiate infection. at the distal end, there other minor proteins which are used for nucleation and assembly on the host membrane. the structures and life cycles are conserved across different species of inoviruses, resulting in similar functional applications. research into inovirus structure and application has been dominated by studies of ff [11] , which infect male (f + ) strains of e. coli. the most used and best understood of the ff inoviruses are the closely related fd, f1, and m13 types (for reviews see [9] [10] [11] ) for which extensive information regarding their life cycle and genetics is known. these nearly identical viruses almost perfectly share all structural motifs, including dna and protein sequences and gene organization [12] [13] [14] , although there are slight variations between the genomes. there are 10 genes and a non-coding intergenic region which are conserved across the ff species [15] [16] [17] as well as dna replication and transcriptional machinery (for a recent review see [10] ). proteins are encoded by 5 genes in the virion (g3, g6, g7, g8, and g9). gene 8 proteins (gp8), the major coat protein, make up the vast majority of the virion, occurring in fivefold axial symmetry. gene proteins 3 and 6 (gp3 and gp6) are located on the proximal end of the virion and are involved in stabilization and infecting the host cell. gene proteins 7 and 9 (gp7 and gp9) are on the distal end of the virion and perform initiation assembly [18] [19] [20] . as shown in the end-to-end model in figure 1 (a), minor coat protein subunits have fivefold axial symmetry. while much research has been done into the structure of the ff virus over the past 50 years, a conclusive structure has not been determined since the viruses cannot be crystallized. x-ray fiber diffraction studies and physiochemical measurements have revealed the five-star helical symmetry (5fold rotation axis) of the gp8 subunit and are referred to as class i [21] [22] [23] . however, the structure of the ssdna and its relationship to the protein sheath is poorly understood, due to the small amount of dna in individual virions. however, the architecture is sufficiently understood to take advantage of the structures and capabilities of the virus throughout its life cycle. the life cycle of ff filamentous viruses begins when an adsorption structure on the proximal end of the virus is adsorbed to the tip of the f + specific pilus of e. coli. following binding between the virus and the bacterial cell (for a recent review, see [10] ), the major coat proteins of the article highlights • inoviruses are easily manipulated viruses which coexist with their host while causing minimal harm and are highly reproducible. • simple genetic manipulation makes the insertion of random or specific oligonucleotides into the inovirus genome. these genetically modified inoviruses can display corresponding oligopeptides as fusion proteins on their surface and are referred to as inovirusassociated vectors (iavs). • iavs displaying randomly generated oligopeptides are used to identify the epitopes of specific antibodies through biopanning. by isolating recombinant inoviruses displaying mimotypes resembling an antibody discontinuous target epitope, the dna and amino acid sequence of the oligopeptides of interest can be determined. • iavs have bene used to do create vaccines against a variety of infectious and non-infectious diseases, being used both to the screen for immunogenic peptides and directly present immunogenic peptides to the organism to elicit proper antibody production. • iavs are highly immunogenic, can stimulate both humoral and cellular responses in a variety of diseases, pose no known health risks, and are cost-efficient to produce. this box summarizes key points contained in the article. virus become associated with the inner membrane of the cell [24] [25] [26] . the virion's circular single-stranded dna (cssdna) is then ejected into the cytoplasm where it is converted to a parental double-stranded replicative form (rf). using a rollingcircle mechanism, ff inoviruses replicate their genome. the new virion is assembled through a complex set of interactions that binds the protein subunits to the ssdna [15] [16] [17] and embeds newly synthesized coat proteins into the bacterial membrane [27] [28] [29] . ssdna is passed through the mature coat protein, spanning the bacterial membrane, and additional coat proteins are added on the internal edge of the membrane. additional proteins are used to package the inovirus and release it into the cell [23, 30] . inovirus assembly on the inner membrane of the bacteria is a harmonized sequential process with both viral-encoded and host proteins playing important roles. for more extensive reviews on inovirus life cycle and replication, see reviews [10, 31] . importantly, this process, which requires both virus and host to complete, does not kill the host, making inovirus replication a sustainable process. inoviruses are useful for vaccine development because it is possible to insert random oligonucleotides into their genome. this easy genetic manipulation is the basis for inovirus display (phage display) technology [32, 33] . inoviruses that have been genetically modified to display these oligopeptides as fusion proteins on their surface are referred to as inovirus-associated vectors (iavs). oligopeptides can be displayed on any capsid protein (gp3, gp6, gp7, gp8, and gp9) following genetic modification. a specific oligonucleotide sequence can be inserted into the viral genome to display the desired oligopeptide as a fusion with capsid proteins gp3, gp7, gp8, or gp9. this results in the oligopeptide's display on every copy of the target capsid protein. however, mosaic inovirus particles can be created where the specific capsid proteins display a mix of wild type and recombinant proteins with the desired oligopeptide [34] . this is done using a phagemid vector which has an extra copy of a capsid protein fused to the specific oligonucleotide. a host exposed to both the phagemid vector and a wild type capsid protein from a deficient helper phage produces mosaic iavs displaying both wild type and oligopeptide fused capsid proteins. work using the capsid protein gp6 has resulted only in the production of mosaic iavs (for reviews see [35] [36] [37] ) while both mosaic and non-mosaic iavs have been produced using gp3 and gp8 (for a review see [10] ) and gp7 and gp9 (for reviews see [38] [39] [40] ). as a result of the different locations, structures, and abundances of the capsid proteins in iavs, they have differential abilities to display different oligopeptides. figure 1(b) shows how each of the five capsid proteins of an iav can display antigens, as have been demonstrated in published literature. the capsid protein that can display the most copies of the desired oligopeptide is gp8 due to the large amount of gp8 in each inovirus. a non-mosaic iav can display a peptide on each of the approximately 2,700 copies of gp8. however, doing so with large peptides distorts the virus; only peptides of up to 6 amino acids can be displayed in such great quantity without affecting the structure of the inovirus. however, producing a mosaic iav with oligopeptides on far fewer gp8 units will not cause distortion [33] . while it is theoretically possible to display an entire protein on gp3 [41] , presenting on all 5 copies per virion, studies have demonstrated that at most one copy of gp3 will display the desired protein [34] . the ability of iavs to display different random oligopeptides on their surface has many applications, and is especially useful in for vaccine development. the creation of random peptide libraries (rpl), where random oligopeptides are fused to major capsid proteins (gp3 or gp8) and displayed on individual inovirus clones creating a random variety of iavs which can be used for vaccine design via epitope mapping using monoclonal or polyclonal antibodies. these random iavs, whose oligopeptide diversity increases with increased peptide length, can be used to identify the epitopes of specific antibodies through a process called biopanning. this allows for the isolation of iavs displaying mimotopes which mimic the antibody discontinuous target epitope. by isolating the recombinant inoviruses bearing mimotopes, the dna and amino acid sequence of the oligopeptides of interest can be determined. through this breakthrough technology which was the subject matter of the nobel prize in chemistry 2018 (see 'expert commentary' below), inovriuses displaying oligopeptides mimicking antigens (or specific epitopes of an antigen) can be used to vaccinate hosts thus inducing the desired antibody production. vaccines of this sort have been developed in a variety of organisms against many different diseases (see tables 1 and 2 in [31] for a list of inovirus-based vaccines), as the introduction of the inoviruses can induce the production of specific antibodies. while there have already been various successes, this method can potentially open the door to the development of many novel vaccines and vaccine methodologies. iavs have been successfully used as vaccine carriers for a variety of species and diseases across a range of vaccine studies (as shown in [31] ). these vaccines have been effective against infections agents such as viral, protozoan, and worm parasites as well as non-infectious diseases including alzheimer's and a variety of cancers. inovirus display technology has been used in two different ways to develop vaccines. the first method employs inovirus display technology to screen rpls with monoclonal antibodies to determine which peptides can be used. these immunogenic peptides are used as vaccines either with carrier proteins or in their soluble forms to elicit an immune response [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] . the second method not only employs inoviruses for epitope mapping, but also utilizes inoviruses to serve as the carrier for the isolated immunogenic peptide . directly using iavs to present the peptide has been more consistently successful in producing the production of the proper antibody than the introduction of soluble peptides. inovirus-bound peptides (on iavs) consistently retain their 3d structure and are more stable than soluble peptides, which do not always reflect the desired antigen epitope [84] . iavs are structurally simple, which allows the immune system to respond to the fused peptides rather than the viral coat. combined with ability to display many copies of the desired peptide, iavs are highly immunogenic, creating effective vaccines [85] . because the inoviruses replicate in e. coli cultures, the production of large numbers of vaccines is cost-efficient. iav's structural simplicity, high immunogenicity, and economical production make them an efficient and attainable system for creating a variety of effective vaccines. much of the research involving inovirus-based vaccines has been to target infectious diseases in animals. studies have shown that iavs have been constructed to protect vaccinated animals against a variety of infectious diseases by eliciting both humoral [82] and cellular [86] immune responses. to test the efficacy of iav vaccines against target pathogens, studies were conducted in which animals were challenged with a specific pathogen following iav vaccination. in these studies, iavs were shown to mitigate or prevent infection from viruses and parasites. in one study, mice were completely vaccinated against human respiratory syncytial virus (rsv) by binding a 15-mer linear epitope to gp3, which induced a humoral response [56] . monoclonal antibodies against various viral diseases have also been successfully screened against rpls to produce recombinant inovirus vaccines against herpes simplex virus type 2 (hsv-2) using a 15-mer peptide [57] and against neurotropic murine coronavirus sing a 13mer peptide [58] . these inovirus vaccines induced humoral responses in mice, greatly reducing mortality and providing protection relative to the dose of the vaccine [57] . iav vaccines have also been developed against fungal parasites in animals, providing both individual protection and large scale vaccination success. [59, 60] . not only did vaccination lower the burden of infection, but it also increased the lifespan in the infected mice [59] . in 2004, manoutsarian et al. used iavs to vaccinate pigs against taenia solium, a parasitic worm which causes neurocysticercosis in humans but uses pigs as intermediate hosts. unlike previous studies, which used a single specific peptide fused to a inovirus, four different antigenic peptides were displayed by inoviruses in a cocktail of recombinant iavs. the induction of a cellular response completely vaccinated 1/3 of the pigs in the study and reduced the number of cysticerci in all other pigs [61] . following the success of this study, a large-scale vaccination of 1047 pigs in mexico was undertaken in 2008. the pigs in a natural environment were successfully immunized, reducing parasite presence in the vaccinated pigs, and providing a more costefficient alternative to vaccination with synthetic peptides [87] . many additional vaccines have been developed that induced both humoral and cellular responses against parasites in animals providing partial protection against the infection and reducing infection burden. many of these studies were done by screening monoclonal and polyclonal antibodies against large rpl (for a review of more inovirus vaccine studies see [31] ). in addition to their success against infectious diseases, inovirus display technology has been successfully used to design vaccines which prevent or mitigate the progression of non-infectious diseases. iavs have been used to display antigen epitopes which elicit immune responses against tumors and other non-infectious diseases. an epitope of the melanoma antigen a1 (mage a1) displayed on gp8 induced a cellular immune response against the melanoma tumor in vaccinated mice. not only did vaccination inhibit tumor growth, but it reduced mortality in the targeted mice [88] . induction of a cellular response, decreased tumor growth, and higher survival rate was observed in mice vaccinated against murine mastocytoma p815 in a later study [89] . the highly immunogenic characteristics of inovirus display technology has also been used to activate immune responses against colorectal cancer tumors, which often evade antitumor immune responses, and to reduce tumor growth [90] . inovirus-based vaccines have also been used against diseases such as alzheimer's. the same methods have been used to induct antibodies against β-amyloid plaques by displaying a specific 4 amino acid antigenic epitope on the surface of the inovirus. multiple studies in mice using recombinant inoviruses have induced a humoral response and reduced βamyloid plaque burden [91] [92] [93] [94] . while not used preventatively as has been done with infectious diseases, iavs can vaccinate animals against further disease progression in non-infectious diseases. these findings suggest that there will be future development of inovirus-based vaccines against non-infectious diseases that mitigate the damage done to both animals and humans. despite the advances in the use of iavs to combat disease, the consistent development of inovirus-based vaccines still faces challenges. even when inoviruses have been screened with specific antibodies to bear the desired peptide mimotopes, immunization using iavs does not always produce the expected immune response. keller et al. in 1993 were unable to induce the production of broadly neutralizing antibodies against human immunodeficiency virus type-1 (hiv-1) in rabbits despite having screened for the proper mimotopes [66] . dorgham et al. in 2005 screened a 15-mer rpl using a broadly neutralizing antibody and, using the selected inoviruses, were able to induce antibodies in vaccinated mice. however, the induced antibodies did not exhibit neutralizing activity against hiv-1 [68] . other rpls screened against monoclonal and broadly neutralizing antibodies have produced antibodies that do not have neutralizing capabilities [55, 70] . in one such case, crystallization of the specific mimotope revealed that the oligopeptide borne by the inovirus was structurally different that the natural antibody epitope [69] . this structural issue is likely at the foundation of many of the issues involving ineffectual inovirus-based vaccines. when iavs fail, it is probable that the chosen mimotopes poorly resemble the original antibody epitopes and cannot function in the desired fashion, either failing to induce antibodies or inducing of antibodies without neutralizing capabilities. despite the efficacy of these inovirus-based vaccines against a variety of infectious and non-infectious diseases, there are still many diseases for which effective vaccines have not been developed using phage display or other methods. for example as discussed above, despite extensive work with inoviruses beginning in 1993 by keller et al. [66] , there has been very little success developing a (hiv-1) vaccine [95] . while four major epitopes on the hiv-1 envelope gp41 and gp120 glycoproteins have been identified [96] [97] [98] , inducing the production of effective antibodies has not been successful [70] . while current techniques have not succeeded, both humoral and cellular responses to hiv-1 through inovirus-based vaccines are being researched [99, 100] . however, the groundwork for future work involving hiv-1 and other diseases has been laid out, paving the way for a multitude of various new inovirus-based vaccines. while there have been many vaccine studies targeting hiv-1 using iavs, none have been completely successful. studies involving inovirus based-vaccines targeting hiv-1 initially only used the broadly neutralizing monoclonal antibodies 2f5, 2g12 and b12 [55, [66] [67] [68] [69] [70] . however, as discussed above, these studies have either failed to induce antibodies or induced non-neutralizing antibodies, despite often inducing a humoral response. in other studies, polyclonal sera from hiv-1-infected individuals have been used to screen rpls to search for new broadly neutralizing monoclonal anti-hiv-1 antibodies [71] [72] [73] [74] [75] 101] . these studies have used iavs to induce the protection of neutralizing antibodies in mice [73] and macaques (against simian-human immunodeficiency virus) [75] . inovirusbased vaccines targeting hiv-1 have been able to control viral load in a variety of non-human mammals after a challenge by hiv-1 or induce neutralizing antibodies, but not inhibit novel infection or induce broadly neutralizing antibodies. additionally, inovirus-based hiv-1 vaccines have often failed due to the autoreactivity of the monoclonal antibodies that can hopefully be avoided with the new 'next generation' broadly neutralizing antibodies [102] [103] [104] [105] [106] . inoviral vectors have been used extensively in the development of vaccines against a variety of infectious and noninfectious diseases over the past two decades. as shown above, the iavs have successfully induced a humoral or cellular response, or both. in the studies, the vaccines could provide partial or complete protection against pathogens causing infectious disease or merely lower the burden of infection. against non-infectious diseases, inovirus-based vaccines have been shown to be effective at mitigating disease development by initiating productive immune responses. there have already been many applications of vaccines against infectious and non-infectious diseases using inoviral vectors and iavs have distinct characteristics that make them more applicable for the development of new vaccines than other viral vectors. inoviruses and by extent iavs are highly immunogenic and do not require adjuvants, facilitating the ease and simplicity of usage as vaccines. they can display multiple (from few to thousands) copies of a peptide on their surface while still maintaining the desired structure and conformation as well as infectivity with their bacterial hosts. iavs, by displaying only particular peptides, allow the immune system to interact with a specific epitope rather than a larger, more complex protein, yielding a more targeted response. they are capable of stimulating humoral and cellular immune responses and do not pose known health risks to animals or humans. iavs can be administered safely in high doses through multiple routes, enhancing their usage in a variety of contexts [107] . with their cost-efficient production, structural simplicity, and record of success in a variety of contexts, iavs have the potential to be exploited for many new vaccines in humans and animals against a variety of diseases. advances in inovirus research and phage display technology are making the development of new vaccines for a variety of diseases feasible in the near future. currently, there are many diseases without vaccines or for which treatment is highly invasive. further development involving iavs will be likely to introduce more, better, and increasingly cost-effective vaccines. as the structure and tools for genetic manipulation of inoviruses are being better developed, iavs will likely begin to be more extensively introduced into animal and human use. the current research is only revealing the tip of the iceberg of the extensive ways in which inovirus-based vaccines will be applied in future use. accordingly, there are aspects of the genetic engineering of phage-display that need to be further expanded. specifically, further studies should be conducted to evaluate the maximum antigenic load capacity of the gp8 coat protein without jeopardizing the architectural integrity and infectivity of the iav (ff.g8, see figure 1 ). furthermore, additional research about the potential of using the other capsid proteins (gp3, gp6, gp7, and gp9) or combinations of any of the capsid proteins for antigen display is needed. additionally, future studies could elucidate the nuances of the uses of individual capsid proteins for specialized applications such as antigen display, penetration, targeting, etc. an additional aspect of iavs that needs to be further explored is how antigenic display on the surface of iavs induces immunogenicity. this predictive work needs to be undertaken by experimental and computational (in silico) research. further research will involve the development of new vaccines and improving the efficacy of current vaccines. additional undertakings should be done into identifying the structure of the viruses, since this will better be able to inform how the phage-display technology will best be applied as more complex work is being done using monoclonal and polyclonal antibodies to create more nuanced vaccines. the future has a dual focus: the development of vaccines for infectious diseases and the induction of immune responses in non-infectious diseases to limit the harm that they do (or eliminate them). the underlying principle of future iav-based vaccine development could be based on the unique natural symbiosis, which is known to exist between humans, non-pathogenic bacteria such as e. coli, and inoviruses. it is well established that humans or other warm-blooded animals acquire e.coli, including f+ strains, during their first few days in life or even before birth and that they are never thereafter without it. fspecific inoviruses (ff inoviruses) cannot infect humans, but they propagate at high viral loads in specific strains of e. coli including non-pathogenic strains which are symbiotic to humans. future iav-based vaccines within the next five to ten years could take advantage of the triple symbiotic nature between humans, enteric f + e. coli strains and f+-specific inoviruses to target the induction of mucosal immunity. this approach will be particularly effective against sexually transmitted pathogens such as hiv-1. much future study lies the further development of iavs and how they can induce immune responses on a broad scale. research regarding the development and advantages of phage display technology was recently recognized by the nobel committee. george p. smith and gregory p. winter were awarded the nobel prize in chemistry 2018 for the 'elegant method known as phage display, where a bacteriophagea virus that infects bacteriacan be used to evolve new proteins … [this] revolution … is bringing and will bring the greatest benefit to humankind' [108] . the development of more and better inovirus-based vaccines will continue to advance preventative treatment against diseases which have not been able to be combatted. papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers an investigation on the nature of ultra-microscopic viruses sur un microbe invisible antagoniste des bacilles dysentériques phage display as a promising approach for vaccine development beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold • a review of novel future applications of phage-display technology mahy bwj, regenmortel mhvv structure and assembly of filamentous bacteriophages dna packing in filamentous bacteriophages replication of coliphage m-13. i. effects on host cells after synchronized infection filamentous bacterial viruses filamentous bacteriophage: biology, phage display and nanotechnology applications a recent comprehensive review about the molecular biology and life cycle of ff viruses (f + -specific filamentous viruses) filamentous phage: structure and biology nucleotide sequence of bacteriophage fd dna nucleotide sequence of bacteriophage f1 dna nucleotide sequence of the filamentous bacteriophage m13 dna genome: comparison with phage fd genetic analysis of the filamentous bacteriophage packaging signal and of the proteins that interact with it functional analysis of bacteriophage f1 intergenic region filamentous phage morphogenetic signal sequence and orientation of dna in the virion and gene-v protein complex a specific dna orientation in the filamentous bacteriophage fd as probed by psoralen crosslinking and electron microscopy morphogenesis of filamentous bacteriophage f1: orientation of extrusion and production of polyphage orientation of the dna in the filamentous bacteriophage f1 the symmetries of filamentous phage particles helical viruses three-dimensional structure of a cloning vector. x-ray diffraction studies of filamentous bacteriophage m13 at 7 a resolution chemical modification of the coat protein in bacteriophage fd and orientation of the virion during assembly and disassembly bacteriophage f1 infection: fate of the parental major coat protein the fate of the protein component of bacteriophage fd during infection detection of prokaryotic signal peptidase in an escherichia coli membrane fraction: endoproteolytic cleavage of nascent f1 pre-coat protein isolation of mutants in m13 coat protein that affect its synthesis, processing, and assembly into phage conserved residues of the leader peptide are essential for cleavage by leader peptidase filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short c-terminal fragment of piii architectural insight into inovirus-associated vectors (iavs) and development of iav-based vaccines inducing humoral and cellular responses: implications in hiv-1 vaccines a detailed review of the architecture of inoviruses and major inovirus-associated vectors (iavs) and related vaccines libraries of peptides and proteins displayed on filamentous phage phage display • an early overview of the phage-display and its applications eliminating helper phage from phage display identification of peroxisomal proteins by using m13 phage protein vi phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-coa reductase phage display of cdna repertoires: the pvi display system and its applications for the selection of immunogenic ligands surface expression and ligand-based selection of cdnas fused to filamentous phage gene vi multivalent display system on filamentous bacteriophage pvii minor coat protein membrane insertion and assembly of epitopetagged gp9 at the tip of the m13 phage next generation phage display by use of pvii and pix as display scaffolds phage display: concept, innovations, applications and future vaccination with cetuximab mimotopes and biological properties of induced anti-epidermal growth factor receptor antibodies identification and characterization of protective epitope of trichinella spiralis paramyosin mimotopes selected with neutralizing antibodies against multiple subtypes of influenza a induction of humoral immune response against plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library high-molecular-weight melanoma-associated antigen mimotope immunizations induce antibodies recognizing melanoma cells differential immunogenicity of two peptides isolated by high molecular weight-melanoma-associated antigen-specific monoclonal antibodies with different affinities vaccination with a human high molecular weight melanoma-associated antigen mimotope induces a humoral response inhibiting melanoma cell growth in vitro targeting melanoma cells with human high molecular weight-melanoma associated antigen-specific antibodies elicited by a peptide mimotope: functional effects specificity of mimotopeinduced anti-high molecular weight-melanoma associated antigen (hmw-maa) antibodies does not ensure biological activity a mimotope peptide-based anti-cancer vaccine selected by bat monoclonal antibody peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-egfr immune response mimotope vaccination for epitope-specific induction of anti-vegf antibodies peptide mimics of the group b meningococcal capsule induce bactericidal and protective antibodies after immunization peptides selected from a phage display library with an hiv-neutralizing antibody elicit antibodies to hiv gp120 in rabbits, but not to the same epitope protective immune responses induced by the immunization of mice with a recombinant bacteriophage displaying an epitope of the human respiratory syncytial virus immunisation with phage displaying peptides representing single epitopes of the glycoprotein g can give rise to partial protective immunity to hsv-2 characterization of murine coronavirus neutralization epitopes with phage-displayed peptides prophylactic vaccination with phagedisplayed epitope of c. albicans elicits protective immune responses against systemic candidiasis in c57bl/6 mice protective immune responses against systemic candidiasis mediated by phage-displayed specific epitope of candida albicans heat shock protein 90 in c57bl/6j mice recombinant bacteriophage-based multiepitope vaccine against taenia solium pig cysticercosis schistosoma japonicum: isolation and identification of peptides mimicking ferritin epitopes from phage display library protective immunity induced by phage displayed mitochondrial related peptides of schistosoma japonicum induction of immunity in sheep to fasciola hepatica with mimotopes of cathepsin l selected from a phage display library trichinella spiralis: characterization of phagedisplayed specific epitopes and their protective immunity in balb/ c mice identification of hiv vaccine candidate peptides by screening random phage epitope libraries identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12 immunogenicity of hiv type 1 gp120 cd4 binding site phage mimotopes a peptide inhibitor of hiv-1 neutralizing antibody 2g12 is not a structural mimic of the natural carbohydrate epitope on gp120 constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-hiv-1 mab 2f5 selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv-1-positive sera collection of phage-peptide probes for hiv-1 immunodominant loop-epitope mimotopes selected with antibodies from hiv-1-neutralizing long-term non-progressor plasma hiv-1 v3 loop crown epitope-focused mimotope selection by patient serum from random phage display libraries: implications for the epitope structural features protection of rhesus macaques against disease progression from pathogenic shiv-89.6pd by vaccination with phage-displayed hiv-1 epitopes variable epitope library-based vaccines: shooting moving targets variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response a general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera induction of hepatitis b virus-specific cytotoxic t lymphocytes response in vivo by filamentous phage display vaccine phage display for sitespecific immunization and characterization of high-risk human papillomavirus specific e7 monoclonal antibodies peptide mimotopes of rabies virus glycoprotein with immunogenic activity immunogenicity and epitope mapping of foreign sequences via genetically engineered filamentous phage multiple display of foreign peptides on a filamentous bacteriophage. peptides from plasmodium falciparum circumsporozoite protein as antigens random-peptide libraries and antigenfragment libraries for epitope mapping and the development of vaccines and diagnostics developing strategies to enhance and focus humoral immune responses using filamentous phage as a model antigen phage display of peptide epitopes from hiv-1 elicits strong cytolytic responses inexpensive anticysticercosis vaccine: s3pvac expressed in heat inactivated m13 filamentous phage proves effective against naturally acquired taenia solium porcine cysticercosis the potential of phage display virions expressing malignant tumor specific antigen mage-a1 epitope in murine model phage display particles expressing tumor-specific antigens induce preventive and therapeutic antitumor immunity in murine p815 model a filamentous bacteriophage targeted to carcinoembryonic antigen induces tumor regression in mouse models of colorectal cancer active immunization against alzheimer's beta-amyloid peptide using phage display technology immunization against alzheimer's beta -amyloid plaques via efrh phage administration reduction of betaamyloid plaques in brain of transgenic mouse model of alzheimer's disease by efrh-phage immunization efrh-phage immunization of alzheimer's disease animal model improves behavioral performance in morris water maze trials hiv-1 vaccine strategies utilizing viral vectors including antigen-displayed inoviral vectors hiv-1 neutralizing antibodies: understanding nature's pathways antibodies in hiv-1 vaccine development and therapy broadly neutralizing antibodies and the search for an hiv-1 vaccine: the end of the beginning profound early control of highly pathogenic siv by an effector memory t-cell vaccine immune clearance of highly pathogenic siv infection inducing cross-clade neutralizing antibodies against hiv-1 by immunofocusing cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv-1 antibodies the role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-hiv-1 envelope human monoclonal antibodies 2f5 and 4e10 to glycoprotein 41 membrane proximal envelope epitopes specific phospholipid recognition by human immunodeficiency virus type-1 neutralizing anti-gp41 2f5 antibody the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2g12 recognizes a cluster of alpha1->2 mannose residues on the outer face of gp120 antibody polyspecificity and neutralization of hiv-1: a hypothesis immunocontraception: filamentous bacteriophage as a platform for vaccine development the nobel prize in chemistry we thank the university of cyprus for its support. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. key: cord-338572-5ifc2lx6 authors: nagarakanti, sandhya r.; okoh, alexis k; grinberg, sagy; bishburg, eliahu title: clinical outcomes of patients with covid‐19 and hiv coinfection date: 2020-09-19 journal: j med virol doi: 10.1002/jmv.26533 sha: doc_id: 338572 cord_uid: 5ifc2lx6 background: patients with human immune deficiency virus (hiv) infection may be at an increased risk for morbidity and mortality from the coronavirus disease‐2019 (covid‐19). we present the clinical outcomes of hiv patients hospitalized for covid‐19 in a matched comparison with historical controls. methods: we conducted retrospective cohort study of hiv patients who were admitted for covid‐19 between march 2020 and april 2020 to newark beth israel medical center. data on baseline clinical characteristics and hospital course was documented and compared with that of a matched control group of covid‐19 patients who had no history of hiv. kaplan meier survival curves and the log‐rank tests were used to estimate and compare in‐hospital survival between both unmatched and matched groups. results: twenty‐three patients with hiv were hospitalized with covid‐19. median age was 59 years. the rates of in‐hospital death, the need for mechanical ventilation and intensive care unit admission were 13% (n=3), 9% (n=2) and 9% (n=2) respectively. the hiv infection was well controlled in all patients except for 3 patients who had presented with acquired immune deficiency syndrome (aids). all aids patients were discharged home uneventfully. a one‐to‐one propensity matching identified 23 covid‐19 patients who served as a control group. in both pre‐ and post‐match cohorts, survival between hiv and control groups were comparable. conclusions: in our cohort of hiv infected patients hospitalized for covid‐19, there was no difference in mortality, icu admission and the need for mechanical ventilation when compared to a matched control of covid ‐19 patients with hiv. this article is protected by copyright. all rights reserved. since the declaration of the coronavirus disease 19 (covid-19) as a pandemic by the world health organization (who), the centers for disease control and prevention (cdc) have cautioned that, compared to the general population, this article is protected by copyright. all rights reserved. people living with human immunodeficiency virus syndrome (hiv) may be at a higher risk for complications and death associated with covid-19. 1 disease severity in covid-19 caused by the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has been described to vary in different demographic populations based on their age, body mass index, traditional cardiovascular risk factors and underlying co-existing conditions. the elderly and people with chronic conditions are more likely to experience worse outcomes from covid-19. 2 patients living with hiv may be at an increased risk for covid-19 related complications due to (i) a higher rate of co-existing conditions than the general population (ii) side effects of anti-retroviral therapy (art), and (iii) traditional cardiovascular risk factors such as obesity, alcohol, and tobacco use disorder. 3 an early report suggested that, hiv-related lymphopenia may have a protective role from severe disease in hiv patients who are susceptible to moreover, the role of art in the outcomes of hiv patients who present with covid-19, has been controversial, with some in-vitro data showing activity of art against sars coronavirus and sars-cov-2. [5] [6] to the best of our knowledge, data on covid 19 patients with underlying immunodeficiency such as hiv has been limited to few cases and clinical outcomes of hospitalized patients has not been well characterized. in this report, we sought to describe our experience with hiv patients who were hospitalized for covid 19. we evaluated their clinical outcomes and compared them to that of a well-matched control group of patients with no hiv. this study is a retrospective review of a prospectively maintained institutional review board (irb) approved covid-19 database in newark beth israel medical center, in newark, new jersey. the database includes all patients with laboratory-confirmed covid-19 infection, which was defined as a positive result accepted article on a reverse-transcriptase-polymerase reaction (rt-pcr) assay (abbott m2000 real time system, chicago, il) of a specimen collected on a nasopharyngeal swab. we queried the database for, (a) adults 18-years of age or older (b) with a history of hiv (confirmed with architect hiv1/2 antigen/antibody combination fourth generation testing) and hospitalized between march 10, 2020 and may 10, 2020 for covid-19 infection with follow up through may 30, 2020. there were no clear strict guidelines used for admission. hospitalization decision was mainly based on the individual admitting physician's discretion. some of the criterial used for admission were (a) signs of sepsis or septic shock defined by the 2016 third international consensus definition for sepsis and septic shock 7 (b) dyspnea requiring a step up in oxygenation therapy to maintain oxygen saturation between 88% -94%. we collected data on demographic, laboratory, and clinical outcomes from electronic medical records using a standardized data collection protocol. these data points included hiv-associated characteristics such as most recent cd4+ t cells, cd4/cd8 ratio, ( obtained by flow cytometry clinical outcomes of hospitalized patients were compared to that of a propensity matched cohort of covid-19 patients who had no history of hiv infection. inhospital survival was compared between both unmatched and matched groups. all data were summarized by using descriptive statistics, presenting continuous variables as median, interquartile range and categorical variables as proportions or percentages. we used the mann-whitney u test, chi-square, or fisher's exact tests to compare differences when appropriate. to estimate the effect of hiv on clinical outcomes, we accounted for covariates that predicted the probability of presenting with covid. propensity scores from a this article is protected by copyright. all rights reserved. we used kaplan maier survival curves and the log-rank and stratified log rank tests to estimate the survival and compare their differences between study groups. statistical significance was considered when a two-sided alpha of less than 0.05 was reached. all analyses were performed using the jmp version 14.0.2 statistical software (sas institute inc., cary, nc, usa). during the study period, 23 hiv patients were admitted for covid-19. the most common presenting symptoms were cough (n=20), fever (n=18) and dyspnea (n=17) over a median duration of 3 days ( figure 1 ). shown in table 1 undetectable viral load except for three patients who had advanced aids. one patient had a cd4 count of 10 cells/µl and hiv rna viral load 26,900copies/ml, the other one had cd4 count of 116 cells /µl and an hiv rna viral load of > 2 million copies ml and the other one had cd 4 of 179µl. the viral load in the last patient with aids was unavailable. all patients were discharged home alive after medical management which included anti-microbial therapy, administration of steroids, and respiratory support. given in table 2 are the demographic, clinical and outcomes comparison between hiv and non-hiv infected patients before and after 1:1 propensity score matching. a cohort of 254 non-hiv patients who were hospitalized was compared to 23 hiv patients who were also hospitalized during the same period. may not be generalizable to other centers and may also be limited by its small sample size. a larger study from multiple centers will be needed to verify findings from this study. we attempted to account for the differences in clinical characteristics by using a propensity score method to identify the control group and to reduce selection bias. also, we did not have full data on the immunological profile of all study subjects. a direct association between immunological profile and outcomes can therefore not be inferred from the presented dataset. nevertheless, the hundred percent survival rates of the 3 patients who presented with aids may suggest a paradoxical association between a worse immunological profile and improved outcomes in covid-19. this article is protected by copyright. all rights reserved. in this cohort of hiv patients hospitalized for covid-19, we found a similarity in morbidity and mortality to that of historical controls. in-hospital survival was 87% and 9% required icu admission. all patients had well controlled hiv infection except for 3 who had presented with aids. as the science regarding the management of covid-19 evolves, larger scale studies are needed to better understand the prognosis of hiv patients who present with covid. the data that support the findings of this study are available on request from the corresponding author. the data are not publicly available due to privacy or ethical restrictions. this article is protected by copyright. all rights reserved. vital signs at presentation (14) 44 ( covid-19) in people with hiv clinical features of patients infected with 2019 novel coronavirus in wuhan, china aging, inflammation, and hiv infection could hiv infection alter the clinical course of sars-cov-2 infection? when less is better immunosuppression drug-related and clinical manifestation of coronavirus disease 2019: a therapeutical hypothesis in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds the third international consensus definitions for sepsis and septic shock (sepsis-3) co-infection of sars-cov-2 and hiv in a patient in wuhan city a survey for covid-19 among hiv/aids patients in two districts of, china wuhan could hiv infection alter the clinical course of sars-cov-2 infection? when less is better description of covid-19 in hiv -infected individuals a single -center, prospective cohort covid-19 in people living with human immunodeficiency virus: a case series of 33 patients covid-19 in patients with hiv: clinical case series one case of coronavirus disease 2019 (covid-19) in a patient co-infected by hiv with a low cd4+ t-cell count coronavirus disease 2019 (covid-19) outcomes in hiv/aids patients: a systematic key: cord-317533-xpfqdeqv authors: smuts, heidi title: human coronavirus nl63 infections in infants hospitalised with acute respiratory tract infections in south africa date: 2008-07-24 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2008.00049.x sha: doc_id: 317533 cord_uid: xpfqdeqv background human coronavirus nl63 (hcov‐nl63) is a novel respiratory virus which is associated with respiratory tract infections in children. objective to determine the role of hcov‐nl63 in infants and young children hospitalised with acute respiratory tract infections (ari) in cape town, south africa. methods respiratory specimens were collected from 1055 infants and young children hospitalised with ari in 2003–2004. samples were screened by rt‐pcr to detect hcov‐nl63 and human metapneumovirus (hmpv). standard shell vial culture and immunofluoresence was used to detect the common respiratory viruses including rsv, influenza a and b viruses, parainfluenza viruses 1, 2, 3, adenovirus and cmv. results a respiratory virus was found in 401/1055 (38·0%) samples. hcov‐nl63 was detected in 9/1055 (0·85%) with peak activity during autumn (67%). most patients had a diagnosis of pneumonia or lower respiratory tract infection (6/9; 67%). conclusions this is the first report of hcov‐nl63 infections in hospitalised children in africa. during the 2‐year period hcov‐nl63 played a minor role in ari in children. a number of respiratory viruses including influenza viruses, respiratory syncytial virus (rsv), parainfluenza viruses, adenovirus and the recently described human metapneumovirus (hmpv) play an important role in acute respiratory tract infections (ari) in children. infections with these viruses may often lead to hospitalisation. however, in a substantial portion of respiratory infections the aetiological agent is not known. there has been renewed interest in human coronaviruses (hcov) as a cause of some of these infections. coronaviruses are large enveloped single-stranded rna viruses that can infect both humans and a variety of domestic animals causing respiratory and enteric illness. until recently human coronavirus (hcov) 229e and oc43, identified in the 1960s, 1 were the only known coronaviruses to infect humans. although primarily responsible for mild infections including the common cold 2 reports of more severe upper and lower respiratory tract infections associated with hcov-229e and hcov-oc43 have been documented. 3, 4 the identification of a coronavirus, sars-cov, as the causative agent of severe acute respiratory syndrome in 2003 5 has resulted in an increased interest in this group of viruses. subsequently two new human coronaviruses, hcov-nl63 6,7 and hcov-hku1, 8 have been described. both infect young children, the elderly and immunocompromised and can lead to severe respiratory tract infections requiring hospitalisation. the prevalence and clinical importance of hcov-nl63 in the south african hospital setting is not known. in this retrospective study 1055 nasopharyngeal, tracheal aspirate and bronchoalveoloar lavage samples were taken from children (age 13 days to 5 years) hospitalised with respiratory tract infections in 2003 and 2004 in the red cross war memorial children's hospital in cape town. croup as a specific diagnosis was not reported for any of these children. samples had previously been screened by using an indirect immunofluorecence assay (light diagnostics, chemicon international, temecula, ca, usa) for the common respiratory viruses including rsv, influenza viruses a and b, parainfluenza viruses 1, 2, 3, adenovirus and cmv. hmpv was also tested for using reverse-transcription-pcr (rt-pcr). in the south african setting, where the prevalence of hiv is high, all infant respiratory samples are routinely screened for cmv as in our setting this virus is a major cause of pneumonia in hiv-infected children. for the detection of hcov-nl63, rna was extracted from 200 ll of sample using the seek viral rna kit according to the manufacturer's instructions (talent, trieste, italy). ten microlitres rna was reverse transcribed into cdna using random primers (roche diagnostics gmbh, penzberg, germany) and the iscript kit (bio-rad, hercules, ca, usa). pcr amplification of a region of the 1b gene of hcov-nl63 was used for screening and positive samples were confirmed by amplification of a portion of the 1a gene. briefly 10 ll of cdna was added to a 50 ll of pcr mix containing 2 iu supertherm polymerase (jmr holdings, kent, uk), 1ae5 mm mgcl 2 , 200 lm each dntp and 0ae2 lm primers. pcr was performed with two sets of outer primers, one set to the 1b gene and one targeting the 1a gene from the study of van der hoek et al. 6 to improve sensitivity a nested pcr was performed using 2ae5 ll outer product and inner primers described by smuts et al. 9 the 1b and 1a pcr products were 169 and 520 bp respectively. the 1a amplicons were sequenced directly and the nucleotide sequences were deposited into genbank (eu477762-eu477770). a respiratory virus was detected in 401 ⁄ 1055 (38ae0%) samples collected over the 2-year period from 2003 to 2004. the detection rate was higher in 2004, 248 ⁄ 559 (44ae4%), compared with 2003, 153 ⁄ 496 (30ae8%). cmv was most frequently found (158 ⁄ 1055; 15ae0%) followed by adenovirus (n = 65; 6ae2%), rsv (42; 4ae0%), parainfluenza 3 (n = 32; 3ae0%), hmpv (n = 28; 2ae6%), influenza virus a (n = 8; 0ae76%), parainfluenza virus 1 (n = 6; 0ae57%), parainfluenza virus 2 (n = 4; 0ae38%) and influenza virus b (n = 1; 0ae09%). in 48 (4ae5%) samples a known respiratory virus was grown in shell vial culture but could not be further identified. of cmv-positive respiratory samples 44ae9% (71 ⁄ 158) were from hiv-infected children, 24ae0% (38 ⁄ 158) from hiv-negative children and for the remainder the hiv status was unknown. in both hiv-positive and hiv-negative groups the rates of co-infection with another known respiratory virus were similar, 12 ⁄ 71 (16ae9%) and 7 ⁄ 38 (18ae4%) respectively. hcov-nl63 was detected in 4 ⁄ 496 (0ae81%) and 5 ⁄ 559 (0ae89%) samples from 2003 and 2004 respectively. all hcov-nl63-infected children, with the exception of one aged 30 months, were under 2 years ( table 1 ). the majority, 6 ⁄ 9 (66ae7%), were <6 months. the hiv status of only those children from 2004 was known and 4 ⁄ 5 hcov-nl63-infected children from this year were hiv-positive. in two instances a co-pathogen was identified, hmpv and adenovirus. in 2003 3 ⁄ 4 positive samples were collected in march (autumn) while in 2004 hcov-nl63-positive samples were found in march, may, august and september (table 1) . a diagnosis of pneumonia or lower respiratory tract infection was made in six (67%) children. two hcov-nl63-positive infants aged 50 and 71 days respectively, required admission to the intensive care unit; both were hiv-positive. to determine the role hcov-nl63 plays in respiratory illness in infants, respiratory samples that had previously been screened for rsv, influenza viruses a and b, parainfluenza viruses 1, 2, 3, adenovirus, hmpv and cmv were also tested by rt-pcr for hcov-nl63. due to limited resources, screening for other viruses such as enteroviruses, rhinoviruses and the other hcovs was not undertaken. this may be considered a limitation of the study as there is accumulating evidence that these viruses, in particular rhinoviruses, may play a more significant role in lower respiratory tract infections than previously recognised. 10 the role and clinical significance of the recently identified human bocavirus 11 and polyomaviruses 12, 13 in respiratory disease is still under investigation. ideally comprehensive screening of respiratory samples for most if not all respiratory viruses and relevant respiratory bacteria should be undertaken in order to obtain greater insight into the epidemiological significance of these pathogens in respiratory disease in the local setting. further this would be beneficial to the clinical management of patients, including the administration of appropriate antiviral drugs and antibiotics. cmv was the most prevalent virus detected in the study samples. cmv-pneumonia is an important life-threatening complication in hiv-infected infants in south africa. a post-mortem study of hiv-infected children in kwazulu-natal, south africa showed frequent (52%) cmv detection in lung tissue compared with uninfected controls (4%). 14 the significance of cmv detection in respiratory samples from uninfected children is not known but probably represents viral shedding from either a recently acquired primary infection or reactivation. this study is the first to report the presence of hcov-nl63 in children hospitalised with respiratory illness in africa. hcov-nl63 was found to circulate in infants and young children in both 2003 and 2004 with similar low prevalence rates of 0ae8%. this finding is lower than that reported in previous studies where detection of hcov-nl63 ranged from 1% to 7ae3% 6,7,15-20 although higher prevalences of 8ae8% and 9ae3% have also been reported. 21, 22 most hcov-nl63-positive children (75%) were under 6 months, indicating that this younger age group may be more susceptible to severe infections requiring hospitalisation; a finding supported by other studies where 38 ⁄ 76 (50%), 4 ⁄ 5 (80%) and 11 ⁄ 12 (92%) of hcov-nl63 infections occurred in this age group respectively. 7, 19, 21 further, immunosuppression may also contribute to increased susceptibility to severe hcov-nl63 infection. in this study 4 ⁄ 5 hcov-nl63-infected whose hiv status was known, were hiv infected. all four were <4 months of age and two required admission to icu. it is not known whether maternal antibodies, if present, would provide protection or reduce the severity of infection. this protection is likely to be most effective in infants under 2 months. in this study very few infected infants were under this age, indicating a possible protective advantage. this observation is supported by the findings of other studies. 7, [15] [16] [17] in a very recent study by dijkman et al., hcov-nl63 specific maternal antibodies were found in all newborns studied. these antibodies disappeared within 3 months providing further evidence of their possible protective role in early life. in a recently published study 9 of ambulatory children presenting with acute wheezing at the outpatient's department of the same hospital in 2004, 3ae6% (3 ⁄ 83) showed evidence of hcov-nl63 infection. this is significantly different (p = 0ae0186) from the prevalence in the hospitalised group. this indicates that the virus is circulating in the community, probably causing mild symptoms which may trigger a wheezing episode requiring medical attention. in contrast the lower rate of hcov-nl63 infection identified in the hospitalised children suggests that the virus rarely causes severe lower respiratory tract infections. however, all hcov-infected children in the ambulatory group were over 12 months of age supporting the possibility that younger children are more susceptible to severe infections requiring hospitalisation. hcov-nl63 infections appear to be seasonal; 67% of infections occurred during autumn. no hcov-nl63 infection was detected during the winter months in either 2003 and 2004, a finding also noted in the ambulatory population 9 this pattern differs from that previously reported where hcov-nl63 infection was predominantly found during the winter season. 6, [15] [16] [17] [18] [19] 21, 22 detection during early spring and summer indicates that hcov-nl63 may circulate at low levels throughout the year. in this study the clinical symptoms of hcov-nl63infected infants were similar to those previously reported with lower respiratory tract infections including pneumonia predominating in this group. 6, 7, 15, 17, 20, 21 in this study only specimens from hospitalised children were examined resulting in a bias towards children with more severe respiratory illness. mild hcov-nl63 symptoms have also been reported 9, 16, 23 indicating that hcov-nl63 infections are probably more severe in the very young and immunocompromised. the role of hcov-nl63 in enteric disease has not been established but reports of gastroenteritis associated with coronavirus infection have been documented with frequencies ranging from 6% to 33%. 15, 17, 20, 22 in this study one child had a history of gastroenteritis and vomiting. a significant association with hcov-nl63 infection and croup has been made 23 but in this study it could not be determined if any of the samples were from children with croup. in conclusion these findings suggest that although hcov-nl63 is circulating in the community it plays a minor role in severe respiratory tract infections in young children who require hospitalisation. ethical approval (018 ⁄ 2004) was granted by the research ethics committee of the faculty of health sciences, university of cape town, south africa. coronaviridae: the viruses and their replication isolation of rhinoviruses and coronaviruses from 38 colds in adults frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infections by use of a novel real-time reverse-transcription polymerase chain reaction coronavirus hku1 and other coronavirus infections in hong kong a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans characterisation and complete genome sequence of a novel coronavirus hku1 from patients with pneumonia role of human metapneumovirus, human coronavirus nl63 and human bocavirus in infants and young children with acute wheezing rhinovirus-associated hospitalisations in young children cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third polyomavirus identification of a novel polyomavirus from patients with acute respiratory tract infections pneumocystis carnii and cmv infections in severely ill hiv-infected african children new human coronavirus, hcov-nl63, associated with severe lower tract disease in australia human coronavirus nl-63 infections in children: a 1-year study a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl63 in children hospitalised with respiratory tract infections in belgium human coronavirus nl63 and 229e seroconversion in children detection of human coronavirus nl63, human metapneumovirus and respiratory syncytial virus in children with respiratory tract infections in southwest sweden clinical disease in children associated with newly described coronavirus subtypes evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children human coronavirus nl63 croup is associated with the novel coronavirus nl63 the author thanks the staff of the virology diagnostic laboratory for performing the shell vial culture and immunofluorescence and dr diana hardie (division medical virology ⁄ national health laboratory service, university of cape town) for critical reading of the manuscript. this study was funded by the poliomyelitis research foundation (grant number 05 ⁄ 09). key: cord-342936-43u7afl3 authors: balzarini, jan title: targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy date: 2007 journal: nat rev microbiol doi: 10.1038/nrmicro1707 sha: doc_id: 342936 cord_uid: 43u7afl3 several chronic viral infections (such as hiv and hepatitis c virus) are highly prevalent and are a serious health risk. the adaptation of animal viruses to the human host, as recently exemplified by influenza viruses and the severe acute respiratory syndrome coronavirus, is also a continuous threat. there is a high demand, therefore, for new antiviral lead compounds and novel therapeutic concepts. in this review, an original therapeutic concept for suppressing enveloped viruses is presented that is based on a specific interaction of carbohydrate-binding agents (cbas) with the glycans present on viral-envelope glycoproteins. this approach may also be extended to other pathogens, including parasites, bacteria and fungi. any attempts to develop an efficient vaccine against chronic viruses, such as hiv and human hepatitis c virus (hcv), have so far failed. this is mainly due to the inventive immunological escape mechanisms of these viruses 1,2 , as well as a lack of efficient long-term protective vaccines that can be directed against conserved epitopes of viruses that are involved in acute infections (such as the influenza virus) 3 . instead, a broad range of chemotherapeutic agents is available for the treatment of various viral infections, in particular for hiv 4 . however, the appearance of long-term side effects and, in particular, the eventual emergence of viral resistance under drug pressure, often weakens the therapy and makes the drugs useless and even harmful in the long run. the targets of the currently available antiviral agents are essential virus-encoded enzymes, virus-specific structural proteins or cellular proteins (that is, viral receptors) 4 , but the sometimes-abundant presence of glycans on viral-envelope glycoproteins has never been seriously envisaged as a therapeutic target. glycans on the viral envelope often have a crucial role in enabling an efficient transmission of the pathogen and/or entry into its susceptible target cells. moreover, it has been shown that the presence of glycans on the envelope of viruses, such as hiv and hcv, is also of crucial importance for the evasion of the immunological surveillance of the host. agents that interact with the viral-envelope glycans may, therefore, compromise the efficient entry of the virus into its susceptible target cells. such agents do not interfere with the glycosylation enzymes from the cell, but rather act by directly binding to the intact glycans on the viral envelope. perhaps more importantly, such carbohydrate-binding agents (cbas) may force the virus to delete at least part of its glycan shield to escape drug pressure 5 ; this might result in the initiation of an immune response against uncovered immunogenic envelope epitopes. cbas may become the first chemotherapeutics with a dual mechanism of antiviral action: first, through direct antiviral activity, by binding to the glycans of the viral envelope and subsequently blocking virus entry, and second, through indirect (additional) antiviral action resulting from the progressive creation of deletions in the envelope glycan shield, thereby triggering the immune system to act against previously hidden immunogenic epitopes of the viral envelope (fig. 1) . in the broader perspective, apart from viruses, other pathogens such as mycobacterium tuberculosis, helicobacter pylori and some parasites may also be susceptible to this novel therapeutic approach. this review will focus on cbas and the molecular mechanism of their antiviral activity (fig. 1) . the escape mechanisms of hiv in response to cba pressure and how these escape mechanisms might involve the immune system to further combat the viral infection will also be discussed. the interference of cbas with the dendritic cell (dc)-specific intercellular adhesion molecule 3 (icam-3)-grabbing non-integrin (dc-sign)-directed capture and transmission of hiv and other pathogens will also be highlighted, and the unique features of the cba therapeutic concept and its potential pitfalls will be discussed. mannose-binding lectins (mbls) . a superfamily of strictly mannose-specific lectins, all of which consist of subunits with a similar sequence and overall threedimensional structure. glycosylation is a highly diverse co-and post-translational protein-modification reaction that can be broadly divided into two categories; o-linked and n-linked glycosylation. in o-linked glycosylation, which probably occurs in the golgi apparatus, the carbohydrate moiety is covalently linked to the hydroxyl oxygen of serine and threonine, but it can also be bound to tyrosine, or to 5-hydroxylysine and 4-hydroxyproline 6 . o-glycosylation has various functions, such as providing ligands for selectins, resistance to proteolysis of stem regions of membrane proteins and creating specific recognition phenomena 6 . it can also help to mask immunogenic epitopes on the protein. o-linked glycosylation usually has n-acetylgalactosamine (galnac) as the binding sugar but can also involve other sugars, such as fucose, glucose and n-acetylglucosamine (glcnac). galactose and/or sialic acid are also often found in o-glycans. the covalently n-linked glycans (fig. 2) are added co-translationally to native polypeptides in the endoplasmic reticulum (er) as blocks of fourteen sugars (glc 3 man 9 glcnac 2 ). these glycans are then subject to extensive modification during their transport through the er and the golgi complex before reaching their final destinations inside or outside the cell 7 . in the er and the early secretory pathway, the oligosaccharide repertoire is still small. in the golgi complex, however, the glycans acquire complex and highly diverse structures by terminal glycosylation, which results in a tremendous heterogeneity. such diversity differs between cell types, tissues and species, and helps to further increase microheterogeneity in the presence of an identical genetic polypeptide background. this results in the creation of new functionalities and specificities 8, 9 . the n-glycans may also have an important role in proper protein folding and degradation 10 , and solubility, by avoiding the precipitation that is caused by lipophylic aminoacid stretches in the nascent polypeptide 11 . they also control proper peptidic oligomerization and the sorting of the peptides, as well as peptide transport and trafficking (by acting as universal 'tags' for specific cellular lectins and modifying enzymes) 7, 9, 12 . the presence of a glycan shield on the peptides also enables the efficient protection of the glycoproteins against degradation by proteases. the interactions of carbohydrates with cellular lectins are also of crucial importance for the efficient operation of the innate immune system. examples include the mannose-binding lectin (mbl) 13 , dc-sign 14 , defensins 15 and macrophage mannose receptors 16 . leukocyte interactions with endothelial cells represent a well-characterized example of a cell-adhesion event that depends on glycan-receptor interactions 17 . cell-surface glycoproteins can, therefore, mediate cell adhesion and signalling events, as well as intercellular communication. as well as mammalian cells, many different pathogens, including viruses, bacteria, fungi and parasites, also use glycoproteins extensively for diverse functions, in part similar to eukaryotic cells. however, as the glycans on the pathogen (in particular, viral-derived glycoproteins) are produced by the cellular machinery, they are often recognized as 'self ' by the immune system. therefore, the glycans on pathogen glycoproteins in the viral envelope or bacterial cell wall help to escape recognition by the immune system, and the subsequent destruction or neutralization of the pathogen. classes of carbohydrate-binding agents arbitrarily, two different categories of cbas can be distinguished: lectins, which are proteins that specifically recognize carbohydrate (glycan) structures, and non-peptidic small-size agents that may have a good and often specific affinity for monosaccharide and/or oligosaccharide structures. exposure of cbas to the virus in cell culture has also been shown to force the virus to delete part of the protective glycan shield that is present on its envelope glycoprotein gp120. it is assumed that such glycan deletions trigger an enhanced neutralizing antibody response to the previously hidden immunogenic epitopes of gp120 and possibly also a cellular immune response. parts c and d reproduced with permission from ref. 122 © (2006) university of amsterdam. ccr5, chemokine receptor 5; lfa-1, lymphocyte function-associated antigen 1. ∼asn-x-ser/thr∼ c syncytium a multinucleated giant cell that is formed following the fusion of infected cells expressing hiv-encoded envelope glycoproteins and uninfected cells expressing the cd4 co-receptor. the resulting syncytium subsequently undergoes apoptosis. (table 1) . the most well-studied cba is undoubtedly cyanovirin-n (cv-n), an 11-kda protein (composed of 101 amino acids consisting of two sequence repeats) originally purified from extracts of the cyanobacterium nostoc ellipsosporum 18 . the elucidation of cv-n crystal structures revealed the existence of a domain-swapped dimer, with two primary carbohydrate-binding sites and two secondary carbohydrate-binding sites on opposite ends of the dimer [19] [20] [21] . the carbohydrate-recognition sites have a binding geometry of high-mannose glycans, in particular α(1,2)-linked mannose oligomers 22, 23 . a monomeric 13-kda protein isolated from the unicellular freshwater bloom-forming cyanobacterium microcystis viridis nies-102 strain (microcystis viridis lectin (mvl)) 24 was also shown to be composed of two tandemly repeated homologous domains, with specificity for α(1,6)and possibly α(1,3)-mannose oligomers. its smallest target is a man 2 glcnac 2 tetrasaccharide core 25 . scytovirin (svn), a 9.7-kda peptide, with 95 amino acids, has most recently been isolated from the cyanobacterium scytonema varium 26 and was shown to have a pronounced affinity for α(1,2)-α(1,6)-mannose trisaccharide units 27 . both cv-n and svn inhibit hiv infection in cell culture, at 50% effective concentrations of 0.1 and 0.3 nm, respectively. mvl, however, is less inhibitory against hiv 28 . a cba derived from the sea coral gerardia savaglia (gsa) was one of the first lectins isolated from a primitive eukaryotic organism 29 . this d-mannose-specific cba is a dimer, with each monomer being 14.8 kda, and requiring calcium to preserve full carbohydratebinding activity. actinohivin 30 , derived from the actinomycete longisporum alba (a 12.5-kda protein, with 114 amino acids), and griffithsin (grft) 31 , isolated from the red alga griffithsin spp. (a 13-kda protein, with 121 amino acids), were also recently shown to recognize mannose-type glycans. interestingly, the calcium-independent grft, a dimeric protein with four α(1,2)-mannose carbohydrate-binding domains (cbds) separated by short linker sequences, has no homology to any other primary amino-acid sequence that has been found so far 31 . gsa showed complete suppression of hiv-1 infection in the h9 cell line at a concentration of 0.2 µm. at the same concentration, syncytia formation between h9 and hiv-1-persistently infected jurkat cells was blocked 32 . actinohivin inhibits both t-cell and macrophage infection by hiv-1 at 60 to 700 nm concentrations in cell culture 33 ; grft is exquisitely active against cxc-chemokine receptor 4 (cxcr4)-tropic hiv-1 (x4 hiv-1) and cc-chemokine receptor 5 (ccr5)-tropic hiv-1 (r5 hiv-1) isolates, with ec 50 s ranging from 0.04 to 0.63 nm 31 . cbas that have a broad array of carbohydrate specificities, including mannose, glucose, galactose, fucose, sialic acid, glcnac and galnac oligomers, are prevalent in many plant families. monomer and dimer forms of plant lectins predominate, but trimer, tetramer and even octamer plant lectins exist that lead to quaternary protein complexes that have relatively high molecular weights (for an overview, see refs 34, 35) . the crystal structures of a number of plant lectins in complex with carbohydrate oligomers have been determined, such as the mannose-specific lectin from galanthus nivalis (gna) 36 examples of the structural composition of high-mannose-type n-glycans. a | tri-antennary complex-type n-glycans. b | hybrid-type n-glycans. c | high-mannose-type n-glycans that are abundantly present on the envelope glycoprotein gp120 of hiv, but are rare on mammalian glycoproteins. besides high-mannose-type n-glycans, the complex-type and hybrid-type n-glycans are also present on gp120. asn, asparagine; fuc, fucose; gal, galactose; glcnac, n-acetylglucosamine; man, mannose; sa, sialic acid; ser, serine; thr, threonine; x, any amino acid except proline. cbas have also been isolated from invertebrates, such as cvl from chaetopterus variapedatus 42 or mermaid from laxus oneistus 43 . whereas cvl has β-galactose specificity, mermaid is a calcium-dependent mannose-specific cba. interestingly, mermaid was reported to have a strong structural resemblance to mammalian dc-sign 43 . the antiviral activity of cvl against hiv is in the range of 0.004-0.06 µm 42 ; the anti-hiv activity of mermaid has not yet been reported. mammalians also have several types of cba. mbl is a calcium-dependent multimeric cba that is found in serum 13, 44 and contains subunits of approximately 31 kda. besides mannose, it also binds glcnac and fucose. mbl is part of the innate immune system and binds pathogens as the initiating step of the lectin pathway in order to opsonize the pathogen 44, 45 . dc-sign is another example of a vertebrate mannose-specific lectin that is predominantly present on immature dcs 46 . it functions in dc recognition and the uptake of pathogens (such as hiv), leading to antigen presentation to t cells 47 . various other cbas, apart from mbl or dc-sign, are also part of the innate and/or adaptive immune system. mammalian defensins (α, β and cyclic φ), a family of soluble glycan-binding proteins, are probably the best-studied lectins of our immune system 15 . galectins also have a role in cell-cell recognition and the triggering of intracellular signalling cascades that lead to apoptosis 48 . finally, the monoclonal antibody 2g12 is one of the few broadly neutralizing anti-hiv antibodies. it is directed against an epitope on the hiv envelope glycoprotein gp120, that lies around the c4-v4 region 49, 50 . this epitope contains high-mannose-type glycans, which are present at several highly conserved n-glycosylation sites (specifically n295, n332 and n392 in gp120) 49, 51 . the predominant interaction sites of 2g12 with gp120 are probably the terminal α(1,2)mannose oligomers of the high-mannose glycans. it should be noted, however, that 2g12 specifically recognizes hiv gp120 glycans, but does not specifically interact with peptide moieties near the glycan structures on gp120 (refs 49-53). the 2g12 antibody was found to be inhibitory to hiv-1(iii b ) in different cell types at an ec 50 of 0.02 to 0.2 µg per ml. however, it should be noted that 2g12 activity can vary depending on the nature of the hiv-1 subtype isolates that are evaluated 54 . in the course of screening for new antibiotics that are active against fungi, the actinomycete strain actinomadura hibisca was found to produce pradimicin a (prm-a) 55 . this antibiotic has a unique non-peptidic structure that contains the amino acid d-ala and the carbohydrates d-xylose and 4,6-dideoxy-4-methylamino-d-galactose attached to a substituted 5,6-dihydrobenzo[a]naphtacenequinone (fig. 3a) . prm-a binds to terminal d-mannose pyranoside and calcium to yield a ternary complex that consists of two molecules of prm-a, four molecules of mannose and one calcium atom 56 . benanomicin a (bnm-a) (fig. 3b) , a closely related antibiotic with mannose specificity that is similar to prm-a, has also been isolated from the actinomycete actinomadura spadix and studied for antifungal activity 57, 58 . prm-a, bnm-a and semisynthetic analogues of these compounds are the only antibiotics that are formally known to have well-defined carbohydrate-binding properties and for which the antiviral activity in cell culture has been reported (the 50% effective concentration against hiv-1 ranks in the lower micromolar range) [58] [59] [60] . several research groups focus on the synthesis and characterization of synthetic cbas that bind specific oligosaccharide structures. binuclear copper (ii) complexes 61 , acyclic pyridine-and pyrimidine-based compounds 62 , and tetrapyrrole (porfyrin) derivatives have all been reported to have carbohydrate-binding properties. these compounds may have potential benefit as both antiviral and diagnostic agents. however, for these small-size cbas, few, if any, antiviral data are available, and their potential for cytotoxicity has not been carefully addressed so far. it would be interesting, therefore, to explore the antiviral properties of such compounds to identify novel synthetic low-molecular-weight cba lead compounds that have chemotherapeutic potential. a broad range of proteins bind high-mannose-type glycans of hiv gp120. several binding modes can be distinguished 63 . one group of lectins interact as c-type lectins via a calcium ion. two of the best-known examples of such calcium-dependent carbohydrate-binding lectins of the innate immune system are dc-sign and serum or liver mbls. another group of lectins interacts with single-terminal carbohydrates or have more intimate interactions with multiple sugar rings, without the need of a metal ion. for a third group of lectins, the interactions have not yet been resolved. the carbohydrate specificity of mbl is broad. the mbls recognize d-mannose, glcnac and l-fucose. a common motif among these sugars is defined by figure 3 | low-size non-peptidic carbohydrate-binding antigens (cbas). structural formulae of the calciumdependent mannose-binding pradimicin a (a) and benanomicin a (b) antibiotic cbas that are produced by actinomadura hibiscus and actinomadura spadix, respectively. red represents the d-alanine moiety; black represents the dihydrobenzonaphtacenequinone core; green represents the carbohydrate part of the molecule. another example of a c-type lectin is dc-sign that has been crystallized in complex with man 3 glcnac 2 (ref. 65) (protein data bank (pdb) id lk9i) (fig. 4b) . interestingly, the internal α(1-3)-mannose (man)-linked core carbohydrate binds to the principal calcium site. the equatorial c-3 and c-4 hydroxyls coordinate the calcium ion and form hydrogen bonds with the amino acids that coordinate the calcium. uda is an example of a calcium-independent cba. a bound glcnac 3 molecule is sandwiched between the binding sites of the high-affinity cbd of a first uda molecule and the low-affinity cbd of a second uda molecule. the high-affinity cbd is formed by the residues ser19, trp21, trp23 and tyr30. the low-affinity calcium is shown as a green sphere, whereas the carbohydrate is shown in a yellow stick representation. the calcium coordination is shown in detail, with the hydrogen bonds to important coordinating residues shown as dotted spheres. the gray sphere represents a calcium site of another carbohydrate-recognition domain. c | hydrogen bonding, van der waals interactions and aromatic ring stacking of glcnac 3 , with the glcnac-specific lectin from urtica dioica (uda) 37 (pdb id 1ehh). two isolectin vi molecules are shown in blue and green. dotted lines are hydrogen-bonding contacts between uda and glcnac 3 . sugar residues are labelled with a, b, and c from the non-reducing end. d | hydrogen-bonding pattern and van der waals contacts of the narcissus psuedonarcissus lectin 7 (npl7) carbohydrate-binding domain complexed to manα cbd is formed by the residues ser65, his67, trp69 and tyr76. of most importance for sugar recognition are the multiple hydrogen bonds, van der waals interactions and aromatic-ring stacking of glcnac 3 with the two uda molecules 37 (pdb id 1ehh) (fig. 4c) . indeed, at least five hydrogen bonds are formed in the carbohydrate-lectin complex, as well as several ring stackings between the individual glcnac entities and the trp21, trp23, trp69 and his67 residues of the uda molecules. the molecular interaction of the narcissus pseudonarcissus lectin with manα1-3man has also been revealed 66 (pdb id 1npl) (fig. 4d) . the narcissus psuedonarcissus lectin 7 (npl7) isolectin contains three cbds in one monomer. each cbd in npl7 seems to have equal affinity for the particular carbohydrates and are equally occupied. in addition to the three conserved cbds, a unique fourth (low-affinity) carbohydrate-binding site has been observed near the tryptophan cluster. three residues (asn30, asp28 and gln26) create a polar patch in the cbd pocket that restricts the carbohydate ligand to an axial hydroxyl group at c-2. the multi-alignment of various monocot mannosebinding lectins revealed a striking sequence identity among narcissus pneudonarcissus agglutinin (npa), narcissus hybrid cultivar agglutinin (nha), galanthus nivalis agglutinin (gna), lycoris radiata agglutinin (lra), lycoris aurea agglutinin (laa) and zephyranthus grandiflora agglutinin (zga), all of which contain three mannose-binding sites represented by the conserved qxdxnxvxy motif. this motif binds mannose through a network of four hydrogen bonds that interconnects the hydroxyls of c2, c3 and c4 of mannose to the four (qdny) amino-acid residues 36,66-68 . a valine present within this motif also interacts with c3 and c4 of mannose through van der waals interactions. interestingly, as also described for npa, a fourth mannose-binding site has been found in laa near the tryptophane cluster 68 . investigation of the complex molecular interactions of cbas with their carbohydrate ligands should enable the rational design of small-size molecules, including peptidomimetics. it is obvious that such low-molecularweight cbas will, by necessity, have fewer interactions with the individual carbohydrates of the gp120 glycans, and thus may display a lower affinity towards the carbohydrate oligomers. however, the non-peptidic small-size antibiotics prm-a and bnm-a (fig. 3) have shown that their affinity and specificity for mannose oligomers are sufficiently high to enable an efficient antiviral activity. unfortunately, no crystal structures have yet been obtained for prm-a that allow visualization of the molecular interactions of the antibiotic with its target α(1,2)-mannose glycan. if such information became available, further optimization of such cbas may be possible. numerous studies have shown that cbas can prevent hiv infection of cell cultures. this has been shown for many virus strains (including laboratory hiv-1 and hiv-2 strains, members of group o and different hiv-1 clades) that infect many different target cells (including laboratory cell lines, peripheral-blood mononuclear cells (pbmcs), macrophages and dcs). inhibition of hiv infection by cbas occurs at 50% effective concentrations that rank between the lower nanomolar and micromolar ranges. the antiviral activity will also depend on the nature of the cba and the virus strains that are investigated 18, 26, [30] [31] [32] [38] [39] [40] [41] 58, 60, 63, 69, 72 . although most studies have been performed with laboratory virus strains that are adapted to growth in immortalized cell lines (such as mt-4, cem and molt), several other studies have shown the pronounced activity of cbas against different virus clade isolates in pbmc cultures or against the r5-hiv-1 strain ba-l in primary macrophage cell cultures 41, 60, 69, 71 . the size of the cbas and their carbohydrate specificity seem to have an important role in their eventual potency and antiviral efficacy. in general, whereas most mannose-specific cbas are inhibitory to hiv, uda is the only example of an hiv-1-active glcnac-specific lectin, and poor, if any, anti-hiv activity has been reported for lectins with other carbohydrate specificities 72 . interestingly, cbas that are assumed to have a well-defined glycan specificity may still differ considerably in antiviral potency, although this phenomenon is not well understood. it is clear, however, that subtle differences in their three-dimensional conformation, as well as the proper steric availability of the glycans that efficiently interact with cbas and the presence, or lack, of well-defined glycan conformations on the viral envelope, have an important role in the eventual antiviral efficacy of cbas. the formation of syncytial giant cells between cells persistently infected with hiv-1 and uninfected cells can also be efficiently prevented by cbas 40 . inhibition of giant-cell formation in hiv-1-infected and non-infected t-cell co-cultures usually requires cba concentrations that are five to tenfold higher than those needed for inhibition of cell-free virus infection of the target cells. some cbas (such as the non-peptidic prm-a), however, are equally effective in their inhibitory potential for both modes of viral transmission 60 . recently, it was shown that cbas inhibit hiv-1 infection of dcs and dc-directed hiv-1 transfer 71 . it has also been demonstrated that cbas have a pronounced inhibitory effect on virus capture by cells that express dc-sign and on the subsequent transmission of the virus to uninfected t cells 73 . dc-sign is a natural c-type lectin that has a pivotal role in innate immune defence 46 (box1; fig. 1) . to eliminate the pathogen from the infected host, dc-sign captures pathogens such as hiv through its envelope glycans, and subsequently presents the pathogen or pathogen fragments to appropriate t cells 74 . these observations are potentially important from a microbicide viewpoint, as microbicides aim to prevent virus infection that may occur after the exposure of an individual to the virus through sexual intercourse. indeed, dc-signmediated virus capture and transmission to t cells is believed to be among the first events that occur during the viral infection process through sexual contact 75, 76 (box 1). however, its exact role in hiv transmission remains controversial -boggiano et al. 77 and wang et al. 78 agents that efficiently prevent the first steps of virus propagation may qualify as promising microbicidal agents and have the potential to prevent persistent establishment of infection. the observation that cbas might compete directly with dc-sign to capture hiv deserves further investigation in terms of their impact on the efficiency of virus capture and the subsequent transmission of the virus to t cells. interestingly, it was recently reported that langerin, a c-type lectin that is present on epithelial langerhans cells (lcs), prevents hiv-1 transmission by lcs by internalization of captured hiv particles and subsequent intracellular degradation 79 . although such a mechanism may be efficient for a first-line inactivation of hiv, cba-exposed hiv strains may decrease the efficiency of lcs to eliminate hiv, but at the same time may compromise the ability of the virus to be efficiently transmitted by dcs. the interactions of several cbas have been extensively investigated, including: the prokaryotic cv-n and actinohivin; a variety of plant lectins, including hippeastrum hybrid agglutinin (hha) and uda; the non-peptidic low-molecular-weight antibiotic prm-a; and the monoclonal antibody 2g12 with the hiv-envelope gp120 and/or several glycan structures. interestingly, deletion of the n-glycans at amino-acid positions 295, 392 and/or 406 rendered the 2g12 monoclonal antibody completely incapable of efficiently neutralizing the virus particle 54 . the asparagines at positions 295, 332 and 392 were previously shown in crystallographic gp120-2g12 complexes to be indispensable for an efficient interaction with the 2g12 monoclonal antibody 80 . the interaction of the monoclonal antibody 2g12 with the glycans on a well-defined gp120 epitope has also been revealed in several studies 49, 51, 52 . alteration of the glycosylation pattern of hiv gp120 in the er by inhibiting the glycosidases i and ii prevents proper folding of gp120. consequently, the interaction of gp120 with the er lectin calnexin is inhibited 81 and, as a result, fewer infectious virus particles are released by the drug-treated cells. in addition, virus particles from which the glycans had been stripped became markedly less infectious. interestingly, combining cbas with the mannosidase inhibitor 1-deoxymannojirimycin (dmj) led to the expression of synergistic antiviral activity 82 . exposure of glycandeleted mutant hiv-1 strains to dmj results in an enhanced suppression of mutant virus-induced cytopathicity in cell culture 82 . therefore, the presence of an intact glycan shield on gp120 has also been proven to be indispensable for optimal infectivity and for an efficient interaction of hiv with its target cells. studies determining the direct interaction of cbas with fixed gp120 molecules have revealed the strong binding affinity of cv-n, hha, prm-a and actinohivin with gp120. however, for certain cbas (such as cv-n and hha), a low dissociation rate was observed 22, 60 . these results indicate that some of the cbas exert a strong and virtually irreversible binding to gp120. this is in agreement with cellular experiments, which show that a short pre-incubation of cell-free virus particles with cbas before infection of t cells markedly increases the antiviral activity of the cbas 18, 40 . the binding of the cba to gp120 does not prevent the initial virus-cell interaction. indeed, it was found that, in the presence of cbas, the virus could still efficiently bind cd4 + t cells 38 . however, one of the next steps during the entry process (which is binding to the co-receptor(s) and/or the subsequent exposure of gp41 to the target-cell membrane) becomes blocked in the presence of the cba. the exact molecular mechanism of the blockade of virus entry and/or fusion has not yet been determined. however, it can be assumed that the attachment of the cba to the glycans of gp120 hinders or prevents the conformational changes and flexibility of gp120 that are required to properly interact with the cell-membrane receptor, before or during the fusion process. in a number of cases, it has even been observed that the binding of virus particles to their target cells can be enhanced in the presence of some cbas 60 . this phenomenon may indicate that some sort of crosslinking between the virus particles and the cell-membrane glycoproteins occurs. alternatively, cba-induced conformational changes in gp120 may allow a more optimal interaction of the cd4-binding site on gp120 with the cellular receptor. it should be mentioned that the cd4-binding site on gp120 does not contain n-glycans, and deletion of n-glycans in the vicinity of the cd4-binding site on gp120 (of simian immunodeficiency virus strain sivmac239) has been reported to increase the binding efficiency of the mutant gp120 with cd4; it was concluded that most pathogens that bind to dc-sign (dendritic cell (dc)-specific intercellular adhesion molecule 3 (icam-3)-grabbing non-integrin) cause long-lasting and chronic infections, and often induce tolerance or immune evasion 74 . dc-sign is a c-type (calcium-dependent) lectin that is predominantly expressed on dcs, but also to some extent on macrophages, activated b cells, lymphoid tissues, skin dermis, placenta and the intestinal and genital mucosa. it functions as a tetramer, and consists of a cytoplasmic domain, a transmembrane domain, an exogenous (hepta) repeat domain that allows multimerization, and a terminal carbohydrate (high-mannose)-recognition domain 120 . dcs that are present at the sites of pathogen entry (for example, at the mucosal barrier underneath the vaginal epithelia) recognize and trap the pathogen. the activated dcs then migrate to draining lymph nodes, where naive t cells are primed to eradicate the pathogen (fig. 1) . one of the roles of dc-sign is to grab icam-3 that is present on t cells to enable close contact and allow antigen presentation to the t cells. dcs can efficiently capture hiv-1 particles through their c-type lectin (dc-sign) receptors. following dc contact with t cells, virus particles can be observed at the cell-cell junctions, which possibly creates an infectious synapse in which the passage of virions between the two cell types is facilitated. such a synapse depends on dc-sign expression and strong cell-cell adhesion mediated by the icam-1-lfa-1 (lymphocyte function-associated antigen 1) interaction. it has been shown that, in the case of hiv, the infectious synapse leads to an efficient transfer of the pathogen to t cells 120 . certain glycans might be particularly important for the shielding of the cd4-binding site from antibody recognition 83 . the exposure of hiv to any antiviral drug will eventually result in the appearance of phenotypic resistance, both in cell culture and in hiv-infected individuals. antiviral drug resistance is usually due to aminoacid mutations and/or deletions in the viral target with which the particular drug directly or indirectly interacts. in many cases, one amino-acid change is sufficient to provoke a marked degree of drug resistance, although sometimes several mutations are required before pronounced phenotypic resistance becomes evident. viral fitness or infectivity is often compromised in the presence of such resistance mutations. however, compensatory mutations may also occur. these aminoacid mutations do not contribute to drug resistance, but rather are meant to restore the fitness of the virus. for hiv, exposure to cbas invariably results in the appearance and accumulation of amino-acid mutations, predominantly in the putative n-glycosylation motifs of gp120 (refs 41, 54, 60, 69, (84) (85) (86) . either an asparagine, or a serine or threonine, are mutated, leading to the annihilation of the glycosylation site. no glycan deletions were observed, however, in the hiv-1 glycoprotein gp41 of such mutant virus strains. the hiv-1 gp120 consists of approximately 30-40% high-mannose-type glycans (approximately 10 out of 24 glycans in hiv-1/iii b ) 87, 88 (fig. 5a) , which are the preferential sites for glycan deletions that occur under cba pressure (fig. 5b) . indeed, up to 80% of high-mannose-type glycan sites are affected in hiv-1 gp120 under prolonged cba pressure. interestingly, such a phenomenon consistently occurs regardless of the nature of the cba, and has been observed in the presence of the α(1,2)-man-specific cv-n 69 and prm-a 60 , the α(1,3)-and/or α(1,6)-manspecific plant lectins (hha and gna) 84, 85 , the glcnacspecific plant lectin uda 41 and the n-glycan-recognizing monoclonal antibody 2g12 (ref. 54 ). there is a close correlation between the number of glycan deletions in the envelope of a particular hiv strain and the degree of phenotypic drug resistance and, in general, the greater the number of glycan deletions in hiv gp120, the greater the degree of cba resistance 41, 84, 85 . however, when the first glycosylation-site deletions occur under cba pressure in hiv-infected cell cultures, phenotypic resistance is rarely observed. for example, mutant virus strains have been isolated that contain at least three or more glycosylation-site mutations in gp120 without the appearance of visible phenotypic resistance to the glcnac-specific uda 41, 85 . therefore, there seems to be a threshold for the number of glycan deletions below which no significant phenotypic cba resistance is evident. so far, many mutant hiv-1 strains containing up to nine glycan deletions in gp120 have been isolated under escalating cba pressure 41 and such mutant virus strains can be up to 100-fold less sensitive to some cbas. interestingly, several of these mutant virus strains are less infective, resulting in a lower viral fitness compared with the wild-type virus 60 . however, in a few cases, mutant virus strains have been isolated that contain 4 to 5 glycan deletions in gp120, yet have an increased infectivity and fitness 85 . it is unclear what structural requirements the mutant gp120 must fulfill to have an increased infectivity. however, such virus strains have never been observed to emerge when more than five glycans were concomitantly affected in gp120. in fact, among more than fifty independent mutant virus isolates that emerged under cba pressure, only three were demonstrated to have an increased infectivity. there is much evidence indicating that the glycan shield of hiv-1 prevents the immune system from efficiently neutralizing the virus. hiv-1 strains lacking the highly conserved n-linked glycan at position 306 (designated as 301 in fig. 5 owing to a different numbering of the amino acids) within the v3 loop of gp120 are highly sensitive to neutralization. bolmstedt and co-workers 89 also showed that glycosylation at this amino-acid position shields hiv-1 from neutralizing antibodies. kang and colleagues 90 recently reported that hiv env-encoded proteins, with deleted glycans in the gp120 domains surrounding the cd4 binding site, or in the gp120 variable loop, expose immunogenic epitopes at much higher levels than wild-type virus does, which may provide a tool for novel vaccine immunogens. specific n-linked glycosylation modifications in the envelope v1 domain of siv or in a siv-hiv hybrid variant have also been shown to evolve in the host and alter recognition by neutralizing antibodies 91, 92 . studies with sivmac239, which is highly resistant to neutralization by polyclonal antisera or monoclonal antibodies, have shown that elimination of n-glycan attachment sites in the envelope gp120 results in a dramatically increased sensitivity to neutralization by monoclonal antibodies 93 . importantly, removal of specific n-glycans from v1 and v2 led to an increase in sensitivity to neutralization by antibodies recognizing epitopes from both within and outside the v1-v2 sequence. indeed, mutations in v1 not only resulted in an increased antibody recognition to epitopes in v1, but also in a redirection of antibody responses to the v3 loop, which is distant in the linear polypeptide sequence 94 . when rhesus monkeys were infected with mutant siv strains that were lacking in combinations of the two n-glycosylation sites in gp120, a marked increase in antibody binding to specific peptides derived from the glycan-deleted regions was observed, which resulted in an increased neutralizing activity. these results convincingly demonstrated that the presence of n-glycans limits the neutralizing antibody response to siv, and helps shield the virus from immune recognition 95 . these results also illustrate that deletion of as few as two glycosylation sites in the viral env gene is sufficient to trigger a significant neutralizing antibody response. blay et al. 96 showed that a significant divergence in the env proteins occurs over time in macaques infected with the siv strain shiv-89.6p (containing hiv env subtype b in a siv background). importantly, the total number of potential n-glycosylation sites did not increase over time, and there was a remarkable degree of conservation in patterns of change in env glycans. these findings suggest that the configuration of the glycan shield is under considerable constraints, which is in agreement with the findings of poon et al. 97 , who showed that negative (exclusive) interactions occur more often between co-localized glycans, whereas positive (inclusive) interactions are restricted to more distant glycans. these data imply that the adaptive repertoire of alternative configurations in the hiv-1 glycan shield is limited by functional interactions between the n-glycans 97 . therefore, it seems likely that cba exposure to siv or hiv strains would seriously compromise these constraints, by forcing the aids, acquired immunodeficiency syndrome; dc-sign, dendritic-cell-specific intercellular adhesion molecule 3-grabbing non-integrin; hcv, human hepatitis c virus; l-sign, liver/lymph node-sign ; sars, severe acute respiratory syndrome. virus to progressively delete envelope glycans and allowing the immune system to become actively involved in inhibiting the virus infection. in conclusion, much data are currently available to show that glycan deletions in the viral envelope uncover immunogenic epitopes that result in an increased neutralization of the mutant virus. pathogen susceptibility to cba therapy it has been shown that dc-sign can recognize and internalize numerous other viruses, bacteria and protozoa in addition to hiv 98 (table 2) . in this way, dc-sign can be considered to be a universal pathogen receptor. indeed, the recent identification of the carbohydrate specificity of the sign molecules for high-mannose-and/or fucose-containing glycans has led to the identification of various pathogens that are recognized by these receptors (table 1) . dc-sign binds and internalizes dengue virus 99 , human cytomegalovirus 100 , hcv 101 and ebola virus 102 to allow efficient trans infection of the target cells. it is also well documented that hcv closely interacts with both dc-sign and liver/lymph node (l)-sign, a close homologue of dc-sign that is expressed on specialized liver and lymph-node endothelial cells that have antigen-presenting capacity 98 . the capture of hcv by the sign-positive cells, through the highly glycosylated (high-mannose type) envelope glycoprotein e2, facilitates hcv transmission to proximal hepatocytes 102 . dc-sign and l-sign were also shown to enhance infection mediated by the marburg virus glycoprotein gp and the s protein of severe acute respiratory syndrome coronavirus (sars-cov) through ph-dependent endocytosis, and might promote virus dissemination 103 . although m. tuberculosis primarily infects macrophages, it also binds to dcs through the interaction of its cell-wall component manlam (mannosylated lipoarabinomannan) with dc-sign 104, 105 . the binding of manlam to dc-sign on dcs blocks dc maturation and induces the expression of immunosuppressive interleukin-10 (il-10) 104 . as a result, m. tuberculosis enables the suppression of immune activation signals, which allows immune escape. probiotic bacteria, such as lactobacillus spp., also exert immune suppression through dc-sign by the induction of il-10-producing regulatory t cells 106 . it has recently been shown that serotypes 3 and 14 of streptococcus pneumoniae specifically interact with dc-sign through the capsular polysaccharide, but the immunological consequences are unclear 107 . h. pylori and the parasite (protozoa) schistosoma mansoni were shown to bind to dc-sign through non-sialylated lewis antigens that are expressed on lipopolysaccharides of the bacterium and the cell-wall glycolipids of the parasite, respectively 108, 109 . this results in immune regulation that is to the advantage of the pathogen 110, 111 . leishmania mexicana has also been shown to express glycoconjugates that are recognized by dc-sign 108 . it is clear that the often-indispensable interactions of various pathogens with dc-sign are required to allow efficient pathogen transmission to its eventual target cells and/or immune escape and successful persistence in the host. it is therefore likely that cbas, by binding to the pathogens' glycoconjugates, directly compete with the lectins of the innate immune system. the cba may prevent efficient capture and transmission of the pathogen and/or suppression of an efficient immune response against the pathogen. therefore, it can be predicted that cbas may have a more general role in abrogating successful pathogen infection and persistence. cbas should, therefore, be put in a broader microbial therapeutic context, and not be explored solely for hiv therapy. unique features of the cba concept it has been unambiguously shown that cbas efficiently inhibit virus entry by inhibiting the fusion of cell-free hiv particles with susceptible cells, and forming syncytia between persistently infected and uninfected cells 38 . cbas also prevent the capture of virus particles by dc-sign, and the subsequent transmission of the virus to t cells 73 . cba treatment of virus-infected cells provokes drug pressure on the virus, resulting in predominant deletions of n-glycans in the hiv gp120 envelope 121 . such glycan deletions uncover previously hidden immunogenic epitopes on gp120, which may give the immune system the opportunity to produce a humoral and/or cellular response (fig. 1) . what are the unique features of the cba concept that differentiate this therapeutic approach from those that are currently available (box 2)? • the direct interaction of carbohydrate-binding agents (cbas) with the glycans of the viral envelope glycoproteins. • cba pressure forces hiv to delete n-glycans in the envelope glycoprotein gp120. • multiple cbas bind to one envelope gp120 target molecule. • a high genetic barrier. • no cross-resistance of cba-resistant mutant virus strains to other antivirals. • altered interaction of mutant (glycan-deleted) viruses with target cells. • the cba-induced glycan deletions compromise the protective role of the intact glycan shield on hiv gp120. • the eventual antiviral cba activity may combine a direct drug-mediated virus suppression and an indirect (delayed) induction of a specific antiviral immune response against the mutant gp120 envelope. • the cba concept may also apply to other chronic enveloped virus infections, such as the human hepatitis c virus. • other pathogens such as dendritic-cell-specific intercellular adhesion molecule 3-grabbing non-integrin (dc-sign)-recognizing bacteria, fungi or parasites may also be susceptible to the cba approach. in contrast to the existing drugs for the treatment of hiv, which interact with specific amino-acid configurations on their target proteins, cbas directly interact with the glycans that are present on the envelope gp120 of hiv. it is important to realize that cbas do not need to be taken up by the virus-infected cell in order to exert their antiviral activity and do not interfere with the synthesis of the glycans on glycoproteins per se. in this respect, this concept differs entirely from inhibitors of cellular glycosylation, such as dmj and castanospermine, which aim to disturb the glycan formation in viral glycoproteins, but at the same time may also disturb the formation of glycans on cellular glycoproteins. cba pressure progressively forces the virus to delete n-glycans in gp120. this mutational pattern is unique and does not consistently occur in the presence of other anti-hiv drugs, nor in any other known antiviral. a relatively high number of n-glycans are present on each hiv-1 gp120 molecule (approximately 20 to 30 glycans, depending on the nature of the virus clade and the individual virus strain) 87 . it can be reasonably assumed that multiple individual cbas simultaneously bind to every hiv-1 gp120 molecule. by contrast, hivhiv-1 inhibitors other than cbas stoichiometrically bind to their target -one drug molecule interacts with one target protein molecule. the multiple bindings of cbas to single gp120 molecules results in the cbas having a high genetic barrier. this means that several mutations (owing to glycan deletions) need to accumulate in gp120 before significant phenotypic drug resistance becomes evident. with the 'traditional' current drugs, the appearance of a single mutation, or at least two or three mutations in the target protein, is usually sufficient to produce a significant drop in sensitivity of the virus to the particular drug. as cbas selectively target n-glycans on gp120, cba-mutated virus strains are not likely to show crossresistance to drugs that act against other targets (such as protease, integrase and reverse transcriptase). the exception to this rule may be drugs that target the hiv-1 transmembrane gp41. indeed, preliminary findings indicate that some mutant virus strains that contain various n-glycan deletions in gp120 show some diminished sensitivity to the fusion gp41 inhibitor enfuvirtide (also known as t20 or fuzeon). it remains to be seen, however, whether this is a consistent behaviour of these mutant virus strains or whether the particular glycan-deleted virus strains have an intrinsically low sensitivity to enfuvirtide that is unrelated to the absence of some of the n-glycans of gp120. certain glycans may have an instrumental role in the correct folding of the protein immediately after the native peptide has been formed on the ribosomes of the er 7 . therefore, it can be expected that correct folding can become hampered if glycans are lacking on the viral envelope after amino-acid mutation of the glycosylation motif. such compromised (or altered) gp120 folding may affect the efficient interaction of the mutated gp120 envelope with the co-receptor molecules, virus fusion efficacy and the eventual fitness of the mutant virus strains. as the transmission of hiv is believed to be mediated by carbohydrate-recognizing dc-sign-expressing cells (that is, immature dcs) that are present in the vaginal-uterine mucosa-epithelial border 46 , it can be assumed that the mode of interaction of cells with mutated glycan-deficient hiv particles is altered owing to a changed glycan landscape on the gp120 envelope. the protective role of the glycan shield in hiding immunogenic epitopes on gp120 from the immune system may get lost, or at least compromised, on deletion of the particular glycans 112 . such glycan deletions may trigger the production of neutralizing antibodies that are specific for those gp120 peptide epitopes that were previously shielded by the glycans. whether this phenomenon will occur in cba-treated hiv-1-infected individuals remains to be determined, but it seems likely. it is also currently unknown how strong and efficient the neutralizing antibody response will be on the mutant virus strains that emerge under cba pressure and whether the virus can use other immunological subversions to circumvent cba drug pressure. it would also be interesting to see whether, and to what extent, a cellular immune response would be triggered under such conditions, and what contribution a provoked cellular immune response might make in the eventual inhibition of the virus infection in cba-treated individuals. none of the existing antiviral chemotherapeutics has been shown to have the potential to act in concert with the immune system to further increase the therapeutic pressure on the mutated virus. therefore, treatment of hiv with cbas may become the first strategy to combine drug-mediated virus suppression and induction of a specific antiviral immunological response 5 . such a phenomenon may result in 'self-vaccination' of the cba-exposed hiv-infected individuals by means of a chemotherapeutic agent. if this principle of a combined concerted action between chemotherapy and the immune response proves valid, it may also be applied to other chronic infections by viruses with a highly glycosylated envelope, such as hcv 113, 114 . also, more acute virus infections (for example, influenza virus, sars-cov and ebola virus) may be highly sensitive to the inhibitory action of cbas, as has been shown for certain cbas against feline and human coronaviruses 115, 116 and for cv-n against ebola virus 117 in cell culture. besides viruses, other pathogens, such as the dc-sign-recognizing m. tuberculosis and h. pylori or fungi that contain a glycan-rich cell wall (such as aspergillus spp., cryptococcus spp. and candida spp.), and even parasites, may become ideal candidate microorganisms to explore their susceptibility to the inhibitory action of cbas. therefore, the potential of cbas to selectively target some enveloped viruses may also be extended to other pathogens of an entirely different nature. if the glycans on these pathogens are sufficiently different from those of the host, an acceptable therapeutic window may be achieved. considering cbas in the larger context, beyond that of the therapeutic field of virus infections, may reveal an unprecedented therapeutic potential, and should trigger extensive efforts by both chemists and microbiologists to explore this novel therapeutic avenue in the broadest possible sense. potential pitfalls of the cba concept virtually all known cbas that are inhibitory to hiv infection (with the exception of the low-molecular-weight non-peptidic antibiotic prm-a and bmn-a analogues (fig. 3) ) are proteins. such agents are expensive to produce, scale-up and purify. there may also be storage and stability problems, although some plant lectins are remarkably temperature-and ph-stable 34, 40 . bioavailability is also expected to be low for peptidic cbas and, therefore, their pharmacokinetics and pharmacodynamics could be unfavourable, particularly for chronic therapeutic administration. besides a sometimes pronounced mitogenic and red-blood-cell-agglutinating activity, lectins might also be endowed with inflammatory activity and cellular toxicity 34 . also, lectins such as cv-n have the capacity to stimulate various differentiation markers (such as cd25, cd69 and human leukocyte antigen (hla-dr)) 69 . again, it must be emphasized that, although these properties are unfavourable and undesirable from a therapeutic viewpoint, the number and intensity of biological side effects is highly dependent on the nature of the cba. for example, whereas cv-n was shown to display a broad variety of side effects 69 , other cbas such as gna and hha showed much fewer, if any, side effects 118 . moreover, some of the side effects that were observed for cv-n were shown to be independent of its carbohydrate-binding properties 69 . therefore, the proper selection of cbas that have a high selectivity for viral glycans and minimal cellular side effects must be an achievable goal. given the proteinaceous nature of lectins, it could be assumed that repeated systemic administration of cbas will eventually elicit a specific antibody response. such a reaction by the immune system may hamper and attenuate the activity of the cba against the viral carbohydrates (making them less antivirally active). it may also provoke hyperreactivity of the immune system, which would necessitate premature abrogation of the continued administration of the cba. obviously, lowmolecular-weight non-peptidic cbas will not suffer from this potential drawback. the greatest concern, for both protein and non-peptidic cbas, is the degree of selectivity they may eventually show for the viral glycoproteins -that is, their potential to discriminate between pathogen (non-self) glycoproteins and cellular (self) glycoproteins. however, the hiv envelope gp120 carries a higher proportion of high-mannosetype glycans than do mammalian glycoproteins 87 . the three-dimensional configuration of the glycans that are displayed on the glycoproteins of the pathogen has been shown to be important, which may help the cba to distinguish between 'non-self ' glycans of the pathogen and 'self ' glycans of the host. high-mannose-type glycans contain terminal α(1,2)-mannose oligomers that are rare on glycans of mammalian glycoproteins. in fact, mbl and dc-sign can distinguish between pathogen-derived glycoproteins and cellular glycoproteins, enabling a selective elimination of the pathogen through specific interaction with its carbohydrate configuration 65, 119 . it is also important that the cba can discriminate between the glycans present on commensal bacteria and the glycans that must be targeted on the viral-envelope glycoproteins. it will be a challenging goal, but one, i believe, which is achievable, to discover or design cbas that show a marked degree of discriminating selectivity between pathogen glycans and the glycans of the host, including the glycans of commensal bacteria. although most cbas are proteins (such as prokaryotic and plant-lectin cbas), the demonstration that small-size nonpeptidic cbas (that is, those with a molecular weight of less than 1-2 kda) (fig. 3) can efficiently suppress viral and fungal infections makes this class of compounds a feasible and realistic tool for pathogen inhibition in the clinical setting. however, much work still has to be done, and more synthetic low-molecular-weight compounds need to be designed or discovered to enable the efficient exploration of this novel functional class of antivirals. this is the only way to enable a careful and rational selection of cbas that have a high specificity and selectivity for the pathogen and few, if any, side effects in the host, especially when included in long-term treatment modalities. several synthetic cba lead compounds are already available 61, 62 , which should trigger the synthesis of structurally related compounds, by organic and medicinal chemists, to allow extensive structure-activity relationship studies. such investigations could indicate which cbas are likely to be the most potent and selective candidates for further pre-clinical investigations. glycans on the envelope or cell wall of pathogens often seem to have similar functional roles, such as escape from recognition by the immune system or recognition by lectins from the innate immune system, that allow efficient transmission of the pathogen. the concept of interfering with and/or abrogating these protective mechanisms should, therefore, be put into a broader context than solely antiviral therapeutic intervention. many different microorganisms other than viruses (such as certain bacteria, fungi, yeasts and parasites) should be thoroughly investigated for their potential interactions with cbas. however, care should be taken to ensure that those microorganisms that have a pivotal role in maintaining the homeostasis of human functions, such as non-pathogenic lactobacilli in the vaginal environment or commensal bacteria in the intestine, are not negatively affected by the cba. including such bacterial strains in the screening of cbas may allow selection of the highest-possible pathogen-selective and specific cbas in the early stages of drug development. cbas should be considered to be valuable agents in their own right, directly suppressing or preventing pathogen infection in the host (fig. 1) . however, as they can force the pathogen to mutate (or delete) its protective glycan shield to escape cba pressure this adds an exciting new dimension to cbas as a novel conceptual class of antimicrobial agents. they may indeed represent the first agents that combine direct chemotherapeutic activity and an indirect, active involvement of the immune system by triggering a humoral response (by producing neutralizing antibodies) and/or a cellular response (t-cell-based immunity). although the triggering of a cellular immune response by cbas is still to be confirmed in vivo, indirect information suggests that an immune response is to be expected after prolonged treatment of the pathogen with cbas. immunology of hepatitis b virus and hepatitis c virus infection progress and obstacles in the development of an aids vaccine viral escape by selection of cytotoxic t-cell-resistant variants in influenza a virus pneumonia antivirals and antiviral strategies the first extensive description of the new concept that cbas may have a dual mechanism of antiviral action database analysis of o-glycosylation sites in proteins intracellular functions of n-linked glycans glycosylation: heterogeneity and the 3d structure of proteins biological importance of glycosylation role of n-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation hydration of a glycoprotein: relative water affinity of peptide and glycan moieties n-glycan processing in er quality control role of the mannose-binding lectin in innate immunity specificity of dc-sign for mannose-and fucose-containing glycans defensins: natural anti-hiv peptides carbohydrate recognition systems in autoimmunity adhesion molecules in leukocyte endothelial interaction discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development solution structure of cyanovirin-n, a potent hiv-inactivating protein crystal structure of cyanovirin-n, a potent hiv-inactivating protein, shows unexpected domain swapping structures of the complexes of a potent anti-hiv protein cyanovirin-n and high mannose oligosaccharides solution structure of a cyanovirin-n:man α1-2manα complex: structural basis for high-affinity carbohydrate-mediated binding to gp120 cyanovirin-n defines a new class of antiviral agent targeting n-linked, highmannose glycans in an oligosaccharide-specific manner isolation and characterization of a mannan-binding lectin from the freshwater cyanobacterium (blue-green algae) microcystis viridis crystal structures of the hiv-1 inhibitory cyanobacterial protein mvl free and bound to man 3 glcnac 2 : structural basis for specificity and highaffinity binding to the core pentasaccharide from n-linked oligomannoside a potent novel anti-hiv protein from the cultured cyanobacterium scytonema varium oligosaccharide and glycoprotein microassays as tools in hiv glycobiology: glycandependent gp120/protein interactions structural studies of algal lectins with anti-hiv activity a d-mannose-specific lectin from gerardia savaglia that inhibits nucleocytoplasmic transport of mrna molecular cloning of actinohivin, a novel anti-hiv protein from an actinomycete, and its expression in escherichia coli isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia spp the d-mannose-specific lectin from gerardia savaglia blocks binding of human immunodeficiency virus type 1 to h9 cells and human lymphocytes in vitro actinohivin, a novel anti-hiv protein from an actinomycete that inhibits syncytium formation: isolation, characterization, and biological activities handbook of plant lectins: properties and biomedical applications lectins 2nd edn the mannose-specific bulb lectin from galanthus nivalis (snowdrop) binds monoand dimannosides at distinct sites. structure analysis of refined complexes at 2.3 å and 3.0 å resolution crystal structures of urtica dioica agglutinin and its complex with triacetylchitotriose -3)-and α-(1-6)-d-mannosespecific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro the mannose-specific plant lectins from cymbidium hybrid and epi pactis helleborine and the (n-acetylglucosamine) n -specific plant lectin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro mannose-specific plant lectins from the amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp120. a new therapeutic concept to hit the achilles heel of hiv a β-galactose-specific lectin isolated from the marine worm chaetopterus variopedatus possesses anti-hiv-1 activity a new c-type lectin similar to the human immunoreceptor dc-sign mediates symbiont acquisition by a marine nematode mannose-binding lectin (mbl) and hiv mannose-binding lectin: do we need it? identification of dc-sign, a novel dendritic cell-specific icam-3 receptor that supports primary immune responses dc-sign, a dendritic cellspecific hiv-1-binding protein that enhances transinfection of t cells targeting diversity the broadly neutralizing antihuman immunodeficiency virus type 1 antibody 2g12 recognizes a cluster of α1→2 mannose residues on the outer face of gp120 human monoclonal antibody 2g12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1 dissection of the carbohydrate specificity of the broadly neutralizing anti-hivhiv-1 antibody 2g12 the mannose-dependent epitope for neutralizing antibody 2g12 on human immunodeficiency virus type 1 glycoprotein gp120 antibody domain exchange is an immunological solution to carbohydrate cluster recognition resistance of hiv-1 to the broadly hiv-1-neutralizing, anti-carbohydrate antibody 2g12 pradimicin, a novel class of potent antifungal antibiotics studies on the mode of antifungal action of pradimicin antibiotics. iii. spectrophotometric sequence analysis of the ternary complex formation of bmy-28864 with d-mannopyranoside and calcium new antifungal antibiotics, benanomicins a and b from an actinomycete new antifungal antibiotics, benanomicins a and b inhibit infection of t-cell with human immunodeficiency virus (hiv) and syncytium formation by hiv pradimicin a inhibition of human immunodeficiency virus: attenuation by mannan pradimicin a, a carbohydratebinding nonpeptidic lead compound for treatment of infections with viruses with highly glycosylated envelopes, such as human immunodeficiency virus a sugar discriminating binuclear copper (ii) complex molecular recognition of carbohydrates with artificial receptors: mimicking the binding motifs found in the crystal structures of protein-carbohydrate complexes orientation of bound ligands in mannose-binding proteins. implications for multivalent ligand recognition an extensive and detailed overview of carbohydrate-binding proteins that interact with the hiv structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr insights into carbohydrate recognition by narcissus pseudonarcissus lectin: the crystal structure at 2 å resolution in complex with α1-3 mannobiose strucure of mannose-specific snowdrop (galanthus nivalis) lectin is representative of a new plant lectin family a novel tetrameric lectin from lycoris aurea with four mannose binding sites per monomer mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type 1 strains with n-glycan deletions in their gp120 envelopes role of envelope glycoprotein carbohydrate in human immunodeficiency virus (hiv-1) infectivity and virus-induced cell fusion sugar-binding proteins potently inhibit dendritic cell human immunodeficiency virus type 1 (hiv-1) infection and dendritic-cell-directed hiv-1 transfer inhibition of hiv entry by carbohydratebinding proteins carbohydrate-binding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign)-directed hivhiv-1-1 dc-sign: escape mechanism for pathogens anti-human immunodeficiency virus (hiv-1) microbicide drug candidates to prevent hiv infection maturation of blood derived dendritic cells enhances hiv-1 capture and transmission dendritic cell-mediated trans-enhancement of human immunodeficiency virus type 1 infectivity is independent of dc-sign cd4 co-expression regulates dc-signmediated transmission of human immunodeficiency virus type 1 langerin is a natural barrier to hiv-1 transmission by langerhans cells antibody domain exchange is an immunological solution to carbohydrate cluster recognition glycobiology at oxford. a personal view the α(1, 2)-mannosidase i inhibitor 1-deoxymannojirimycin potentiates the antiviral activity of carbohydrate-binding agents against wild-type and mutant hiv-1 strains containing glycan deletions in gp120 identification of two n-linked glycosylation sites within the core of the simian immunodeficiency virus glycoprotein whose removal enhances sensitivity to soluble cd4 profile of resistance of human immunodeficiency virus to mannose-specific plant lectins marked depletion of glycosylation sites in hiv-1 gp120 under selection pressure by the mannose-specific plant lectins of hippeastrum hybrid and galanthus nivalis resistance of human immunodeficiency virus type 1 to the high-mannose binding agents cyanovirin n and concanavalin a assignment of intrachain disulphide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in chinese hamster ovary cells a general model for the surface glycoproteins of hiv-1 and other retroviruses enhanced immunogenicity of a human immunodeficiency virus type 1 env dna vaccine by manipulating n-glycosylation signals. effects of elimination of the v3 306 glycan modified hiv envelope proteins with enhanced binding to neutralizing monoclonal antibodies specific n-linked and o-linked glycosylation modifications in the envelope vi domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies selection for neutralization resistance of the simian human immunodeficiency virus shivsf33a variant in vivo by virtue of sequence changes in the extracellular envelope glycoprotein that modify n-linked glycosylation assorted mutations in the envelope gene of simian immunodeficiency virus lead to loss of neutralization resistance against antibodies representing a broad spectrum of specificities removal of n-linked glycosylation sites in the v1 region of simian immunodeficiency virus gp120 results in redirection of b-cell responses to v3 a role for carbohydrates in immune evasion in aids consistent patterns of change during the divergence of human immunodeficiency virus type 1 envelope from that of the inoculated virus in simian/human immunodeficiency virusinfected macaques evolutionary interactions between n-linked glycosylation sites in the hiv-1 envelope distinct functions of dc-sign and its homologues l-sign (dc-signr) and msignr1 in pathogen recognition and immune regulation dc-sign (cd209) mediates dengue virus infection of human dendritic cells human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection c-type lectins l-sign and dc-sign capture and transmit infectious hepatitis c virus pseudotype particles c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus mycobacteria target dc-sign to suppress dendritic cell function dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells selective probiotic bacteria induce il-10-producing regulatory t cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin dc-sign specifically recognizes streptococcus pneumoniae serotypes 3 and 14 references 46 and 47 provide the first identification of the important role of the natural c-type lectin dc-sign in innate pathogen recognition, capture and antigen presentation to the immune system the dendritic cell-specific c-type lectin dc-sign is a receptor for schistosoma mansoni egg antigens and recognizes the glycan antigen lewis x helicobacter pylori modulates the t helper cell 1/t helper cell 2 balance through phase-variable interaction between lipopolysaccharide and dc-sign dc-sign mediates binding of dendritic cells to authentic pseudo-lewisy glycolipids of schistosoma mansoni cercariae, the first parasitespecific ligand of dc-sign demonstration of the crucial importance of how a continuously evolving glycan shield on the hiv envelope gp120 escapes immune surveillance in hiv cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans the inhibition of infection and entry of the human immunodeficiency virus (hivhiv-1) and hepatitis c virus (hcv) closely correlates for carbohydrate-binding agents (cba), but not for polyanions. virology (in the press) plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle cyanovirin-n binds to the viral surface glycoprotein, gp1, 2 and inhibits infectivity of ebola virus safety concerns for the potential use of cyanovirin as a microbicidal anti-hiv agent in abstracts of the 14th conference on retroviruses and opportunistic infections glycosylation and the immune system dendritic-cell interactions with hiv: infection and viral dissemination carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses? dendritic cell-mediated hiv-1 transmission. thesis structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody hepatitis c virus targets dc-sign and l-sign to escape lysosomal degradation west nile virus discriminates between dc-sign and dc-signr for cellular attachment and infection the role of dc-sign and dc-signr in hiv and ebola virus infection: can potential therapeutics block virus transmission and dissemination? dc-sign is a receptor for human herpes virus 8 on dendritic cells and macrophages measles virus targets dc-sign to enhance dendritic cell infection dc-sign interacts with mycobacterium leprae but sequence variation in this lectin is not associated with leprosy in the pakistani population role of the c-type lectins dc-sign and l-sign in leishmania interaction with host phagocytes dc-sign mediates the binding of aspergillus fumigatus and keratinophylic fungi by human dendritic cells the c-type lectin dc-sign (cd209) is an antigen-uptake receptor for candida albicans on dendritic cells the research of the author has been supported by grants from the rega center of excellence at the katholieke universiteit leuven, the geconcerteerde onderzoeksacties and the european commission. d. schols and s. liekens, rega institute, leuven, belgium, are acknowledged for the critical reading of the manuscript and valuable discussions. i thank c. callebaut and c. biernaux for their dedicated editorial help. the authors declare no competing financial interests. key: cord-330581-g5r2b043 authors: marini, elena; tiberio, laura; caracciolo, sonia; tosti, giorgio; guzman, carlos a.; schiaffonati, luisa; fiorentini, simona; caruso, arnaldo title: hiv‐1 matrix protein p17 binds to monocytes and selectively stimulates mcp‐1 secretion: role of transcriptional factor ap‐1 date: 2007-10-26 journal: cell microbiol doi: 10.1111/j.1462-5822.2007.01073.x sha: doc_id: 330581 cord_uid: g5r2b043 hiv‐1 matrix protein p17 activates a variety of cell responses which play a critical role in viral replication and infection. its activity depends on the expression of p17 receptors (p17r) on the surface of target cells. whether p17 also plays a role in stimulating human monocytes, a major hiv‐1 reservoir, is not known. here we show that human monocytes constitutively express p17rs and that p17 selectively triggers these cells to produce mcp‐1. the effect of p17 on mcp‐1 expression was observed at the transcriptional level and was primarily dependent on the activation of the transcription factor ap‐1. p17 increased the binding activity of ap‐1 complexes in a time‐ and dose‐dependent manner. deletion of the ap‐1 binding sites in the mcp‐1 promoter resulted in the lack of p17‐induced mcp‐1 transcription. in particular, the p3 binding site located between −69 and −63 position seems to be essential to mcp‐1 mrna induction in p17‐treated monocytes. an ever increasing amount of evidences shows a tight link between biologically dysregulated monocytes, ap‐1 activation, mcp‐1 release and hiv‐1 pathogenesis. overall our results suggest that p17 may play a critical role in the monocyte‐mediated inflammatory processes, which are suspected to be major precipitating events in aids‐defining diseases. blood monocytes are major in vivo cell targets of hiv-1 infection. they are known to be less susceptible to viral cytopathic effects and more resistant to hiv-1-induced apoptosis than cd4 + t lymphocytes (herbein et al., 2002; kedzierska et al., 2003) . due to their ability to survive hiv-1 infection, and because of a chronic low-level viral infection, monocytes serve as a major virus reservoir (ho et al., 1994) . activation of monocytes by infectious and/or inflammatory stimuli increases plasma viral load (donovan et al., 1996; goletti et al., 1996) combined with increased numbers of hiv-1-positive cells. in addition, activated monocytes can traffic and invade tissues and organs, homing to sites of infection and inflammation (fischer-smith et al., 2001) . productively infected monocytes may therefore ultimately serve as 'trojan horses' for viral dissemination to different organs. furthermore, a monocyte-dependent mechanism of viral amplification at local sites of initial infection appears to be a prerequisite for efficient dissemination of infection (hirsch et al., 1998) . the analysis of infected monocytes reveals evidence of signalling cascades leading to upregulation of specific inflammation-related molecules, which include mcp-1 (wahl et al., 2003) . mcp-1 plays an important role in sustaining both inflammation and hiv-1 replication (fantuzzi et al., 2003) and is also involved in mass recruitment of such hiv-1 targets as dendritic cells, monocytes, macrophages and t lymphocytes, into localized sites of infection (cinque et al., 1998; fantuzzi et al., 2000; alfano and poli, 2001) . the pro-hiv-1 role of mcp-1 has been confirmed by the recent finding that hiv-1 infection and disease progression are influenced by a mutant mcp-1 allele linked to increased monocyte infiltration of tissues and mcp-1 levels (gonzalez et al., 2002) . considerable attention should therefore be given to the mechanisms triggered by hiv-1 to induce enhanced mcp-1 production by monocytes. the hiv-1 matrix protein p17 is a structural protein that plays an important part in the life cycle of the retrovirus (fiorentini et al., 2006) . it is released by infected cells (fiorentini et al., 2006) , and has recently been shown to accumulates in lymph nodes in the absence of any detectable hiv-1 replication (popovic et al., 2005) . hiv-1 p17, as a recombinant protein, exerts a cytokine-like activity after binding to a specific cellular receptor (p17r) (de francesco et al., 2002) . it is able to modulate the activation and proliferation status of t and nk cells (de francesco et al., 1998; vitale et al., 2003) and to enhance hiv-1 replication (de francesco et al., 1998) . moreover, it increases the release of such proinflammatory molecules as tnf-a and ifn-g, while counteracting the anti-inflammatory activity of il-4 (de francesco et al., 2002) . it is likely that, through the release of pro-inflammatory molecules, p17 may induce the activation of monocytes and modulate their functions. however, the potential direct effect of p17 on monocytes has not yet been investigated. in this study, we show that human monocytes constitutively express p17rs on their surface and that p17 is capable of triggering monocytes to selectively produce mcp-1. furthermore, we show that the p17-mediated induction of mcp-1 production is dependent on the activation of the cellular transcriptional factor ap-1. biotin-conjugated p17 was allowed to react with viable peripheral blood mononuclear cells (pbmcs) obtained from healthy donors. as shown in fig. 1a (left), cell staining with monoclonal antibody (mab) to cd14 revealed that almost all monocytes constitutively expressed p17rs on their surface. the presence of p17rs on human monocytes was confirmed on cells purified by magnetic sorting using anti-cd14-coated paramagnetic beads as a specific reagent (fig. 1a , right). the specificity of the binding was confirmed by displacement of p17/p17r interaction by the anti-p17 mab mbs-3 (data not shown). the stability of the p17r + phenotype was then evaluated in purified monocyte preparations. almost 100% of monocytes showed a p17r + phenotype after 72 h of culture, with a mean fluorescence intensity (mfi) similar to or even higher than that observed on cells soon after their purification (fig. 1a , right). at the same time, we observed a consistent increase in the mfi of p17rs expressed on cells stimulated for 16 h with ifn-g or lps. as shown in fig. 1b (top) , ifn-g increased the expression of p17rs on purified monocytes already at a dose as low as 0.2 ng ml -1 . a peak of p17r expression was reached at an ifn-g dose of 25 ng ml -1 . lps was less efficient than ifn-g in upregulating p17rs expression. a peak in p17r expression was observed at an lps dose of 10 ng ml -1 . a kinetic analysis of p17r expression on 4, 8, 16, 24 and 48 h) showed that p17r expression was enhanced on monocytes within 8-24 h ( fig. 1b, bottom) . the slow surface induction of p17rs is therefore indicative of the de novo synthesis of p17rs following monocyte activation. it is not yet known how p17r surface expression is regulated. therefore, we examined the baseline turnover of p17rs in purified monocytes. cells were cultured in the presence or absence of the protein synthesis inhibitor biotin-conjugated p17 was allowed to react with pbmcs or with purified monocytes. detection of p17 on the cell surface was performed using apc-conjugated streptavidin as a specific reagent. a. left. cells were also stained with fitc-conjugated anti-cd14 mab. data are displayed as bivariate dot plot and show that approximately all monocytes express p17rs. right. the expression of p17rs on purified monocytes is displayed as hystograms. histograms: bold, p17r + cd14 + cells within freshly purified monocytes; dotted: cd14 + cells within purified monocytes stained with an unrelated biotinilated protein; solid, p17r + cells within purified monocytes after 72 h of culture in complete medium. b. expression of p17rs on purified activated monocytes. cells were cultured in the presence of different doses of ifn-g or lps and assayed for p17r expression after 16 h of stimulation (top). the kinetics of p17r upregulation on cells stimulated up to 48 h with optimal doses of ifn-g (25 ng ml -1 ) or lps (10 ng ml -1 ) is shown in the bottom panel. these results are representative of three independent experiments. cycloheximide and the kinetics of p17r expression was examined over a 24 h time-course (i.e. 6, 12 and 24 h). another cell surface receptor, namely hla-dr, was included for comparison because of its high stability (lanzavecchia et al., 1992) and known recycling kinetics in allophycocyanin (apc) (tulp et al., 1994) . as shown in fig. 2a , cells treated with cycloheximide showed a downregulation of p17r surface expression to the background levels after 24 h of culture. in comparison with the hla-dr molecule, whose expression on the surface of monocytes was not affected, as expected, by cycloheximide, p17r clearly showed an accelerated turnover which suggests that a de novo synthesis is required to maintain its normal expression level. treatment of monocytes with trypsin for 15 min at 37°c reduced p17r expression almost to background levels (fig. 2b) . the direct involvement of trypsin in p17r degradation was confirmed by the lack of activity in trypsin inactivated by soybean trypsin inhibitor. moreover, the effect of trypsin was not due to cell death, as there was no significant increase in the percentage of cells stained with propidium iodide after trypsin treatment, as assessed by flow cytometry (data not shown). to assess whether the loss of p17rs on trypsinized cells was transient, trypsin-treated monocytes were allowed to recover for different periods of time at 37°c before p17 binding analysis. restoration of p17r expression started approximately after 24 h of culture, and involved 68 ϯ 13.2% of all monocytes, whereas a total recovery of p17r expression was reached only after 48 h of culture (fig. 2b ). the slow kinetic suggests that protein synthesis may be required. in order to completely exclude the presence of pre-formed intracellular p17rs, cells were cultured soon after trypsin treatment in medium containing or not cycloheximide. re-expression of p17rs was completely blocked by cycloheximide, which shows that it is most probably due to protein synthesis rather than to mobilization of a pre-formed intracellular pool. to characterize the effect of p17 on purified monocytes, we investigated the level of different cytokines and chemokines (i.e. il-1a, il-1b, il-6, il-8, il-12, tnf-a, mcp-1, mip-1a, mip-1b and rantes) in the medium of p17-treated or -untreated cells 48 h after the beginning of culture. untreated monocytes constitutively released il-8 in a range of 0.5-2.2 ng per 10 6 cells and, occasionally, also traces of il-6 and tnf-a. the addition of p17 at the beginning of culture induced, in a dose-dependent manner, the production of mcp-1 in the cells' supernatant ( fig. 3a) , whereas it did not affect the levels of the other cytokines or chemokines tested (data not shown). to assess whether the increase in mcp-1 protein levels induced by p17 treatment was paralleled by an increase in a. purified monocytes were stained for p17r and hla-dr expression, as described in experimental procedures. at the beginning of culture (0 h) almost 100% of cells were p17r + and hla-dr + (bold lines). controls (dotted lines) represent cells stained with isotype igg (versus hla-dr) or with an unrelated biotinylated protein (versus p17r). the cells were then cultured, for 6, 12 and 24 h in the presence (solid lines) or in the absence (bold lines) of cycloheximide. b. treatment of monocytes with trypsin for 15 min (0 h) reduced p17r expression (bold line) to almost background levels (dotted line). the cells were then allowed to recover in complete medium in the presence (solid line) or absence (bold line) of cycloheximide. monocytes were collected 24 h and 48 h after trypsin treatment for p17r staining. these results are representative of three independent experiments. relative mrna, we analysed the kinetics of mcp-1 mrna appearance by quantitative real-time pcr taqman assay (rtpcr). monocytes were treated or not with p17 and total rna was extracted from cell lysates at 1, 2, 3, 4 and 6 h after the beginning of culture. mcp-1 messenger levels were quantified according to b-actin mrna expression. as shown in fig. 3b , an enhancement of mcp-1 mrna expression (2.4-fold increase) started 4 h after p17 treatment reaching approximately an eightfold increase 6-8 h after the beginning of treatment. activation of mcp-1 gene transcription in different cell systems is known to be under the control of the cellular transcription factors nf-kb and/or ap-1 (shyy et al., 1993) . to assess the mechanism(s) whereby p17 induces an increase in mcp-1 gene expression, we verified whether p17 treatment increases nf-kb and ap-1 dna-binding activity. preliminary experiments were run using the transsignal array, a protein/dna array that includes, among others, specific probes for these two transcriptional factors. as shown in fig. 4a , nuclear extracts from untreated monocytes showed a basal level of ap-1 dnafig. 3 . induction of mcp-1 production by p17-treated monocytes. a. purified monocytes obtained from eight healthy donors were treated or not with p17 at a concentration of 0.1 or 1 mg ml -1 . culture supernatants were collected 48 h after the beginning of culture and analysed for the presence of mcp-1 by a standard quantitative elisa. bars represent the mean value of all samples and p-values were calculated by the wilcoxon test. b. cells treated or not with p17 (1 mg ml -1 ) were harvested at the specified times, and mcp-1 and b-actin levels were analysed by taqman rtpcr. mcp-1 mrna levels, normalized to b-actin levels in the same samples, are expressed as fold induction of the levels detected in p17-untreated cells at the same time points. bars represent the mean ϯ sd of triplicate samples. fig. 4 . ap-1 activation in p17-treated human monocytes. nuclear protein extracts obtained from monocytes collected after p17 treatment (1 mg ml -1 ) were analysed for their binding activity to oligonucleotides specific for different transcription factors. a. representative fluorograms of protein/dna array analysis of ap-1, nf-kb and oct-1 dna-binding activity in nuclear extracts (10 mg per sample) from human monocytes collected 1 h after p17 treatment. each transcription factor was evaluated in duplicate at 1¥ concentration (1¥) and at 0.1¥ concentration (0.1¥) of the specific oligonucleotides. b. representative emsa autoradiograms using nuclear extracts (10 mg per sample) from human monocytes collected at the specified times after p17 treatment, and ap-1-specific radiolabelled oligonucleotides. c: p17-untreated cells. protein-dna complex specificity was confirmed by competition with an excess of unlabelled oligonucleotide probe. dna-binding activity to oct-1 was used as loading control. the right panel shows a densitometric analysis of changes in the dna-binding activity of ap-1 relative to oct-1, expressed as fold of induction over control untreated cells. c. ap-1 protein family profiling for dna-binding activity of an elisa-based transcription factor assay kit using nuclear extracts (4 mg per sample) from human monocytes cultured for 1 h in the presence or absence of p17-and ap-1-specific oligonucleotides immobilized to a 96-well plate. levels of fos or jun family members in the active ap-1 complex of p17-treated or -untreated monocytes are expressed as percentage of relative activity where 100% is referred to 0 h values. statistical analysis was performed by one-way anova test. *p < 0.05, statistically different compared with 0 h values. bars represent the mean ϯ sd of triplicate samples. representative data from one of three experiments are shown. binding activity that was enhanced after p17 treatment for 1 h at 37°c. conversely, treatment of monocytes with p17 did not modify nf-kb dna-binding activity which, however, is present at low levels under basal conditions. data obtained by protein/dna array analysis were confirmed by electrophoretic mobility shift assay (emsa). preliminary experiments using p17 at concentrations ranging from 0.1 to 1 mg ml -1 showed that p17 activated ap-1 dna-binding activity in a dose-dependent manner with a peak at the 0.4-0.5 mg ml -1 dose (data not shown). time-course analysis using 1 mg ml -1 p17 showed that the activation of ap-1-binding activity is also time-dependent and peaks (approximately a threefold increase) at 1 h after the beginning of p17 treatment, after which it declines (fig. 4b) . the specificity of protein-dna complexes was confirmed by competition experiments using an excess of specific unlabelled oligonucleotide. as ap-1 proteins bind to dna as homo-or heterodimers of the jun (c-jun, jund and junb) and fos (c-fos, fra1, fra2 and fosb) proto-oncogene families, experiments were performed to characterize the proteins involved in p17induced ap-1 protein-dna complexes. data obtained by transam™ ap-1 family transcription factor assay showed that c-fos, fosb and junb were the main components of the p17-inducible ap-1 complexes (fig. 4c) . due to the large number of primary monocytes required for the analysis of transcription factor dna-binding activity in protein nuclear extracts, we further investigated the involvement of ap-1 transcription factor in p17-induced mcp-1 transcriptional events using the monocytic cell line thp-1, which constitutively expresses p17rs on its surface (data not shown). results from emsa experiments showed that treatment of thp-1 cells for 4 h at 37°c with p17 (1 mg ml -1 ) induced ap-1 activation, with a peak (5.2-fold increase) at 1 h after treatment, but did not induce any nf-kb activation (fig. 5a) . emsa experiments showed that pre-treatment of exogenous p17 protein with the neutralizing anti-p17 mab mbs-3 blocked ap-1 activation (data not shown). further analyses were performed to characterize the specificity and protein composition of ap-1 complexes induced by p17 treatment in thp-1 cells. transam™ analysis showed a preferential involvement of c-fos and junb in the active ap-1 complexes (fig. 5b) . altogether, our results confirm a similar ap-1 activation pattern by p17 in human primary monocytes and thp-1 cells, which suggests that this cell line may be used as a model in future studies of the biological activity of p17 in the monocyte compartment. thp-1 cells were also used as a target to assess the stability of p17 to heat or enzymatic degradation. we therefore treated exogenous p17 with 1% trypsin for 1 h at 37°c or boiled it for 15 min at 100°c. both treatments completely abolished p17induced ap-1 activity as assessed by emsa (fig. 5c ), providing evidences that the viral protein is sensitive to protease and heat denaturation. 5 . ap-1 activation in p17-treated thp-1 cells. nuclear protein extracts from thp-1 cells collected at different times after p17 treatment (1 mg ml -1 ) were analysed for binding activity to oligonucleotides specific for different transcription factors. a. representative autoradiograms of emsa using nuclear extracts (10 mg per sample) from thp-1 cells at the specified times after p17 treatment and radiolabelled oligonucleotides specific for nf-kb, ap-1 or oct-1. c: untreated cells; lps: lps-treated cells (1 mg ml -1 lps for 0.5 h), used as positive control for nf-kb activation. protein-dna complex specificity was confirmed by competition with an excess of unlabelled oligonucleotide probe. the right panel represents a densitometric analysis of changes in dna-binding activity of ap-1 relative to oct-1, used as loading control. data are expressed as fold of induction over control untreated cells. b. ap-1 family profiling for dna binding activation from an elisa-based kit using nuclear extracts (4 mg per sample) from thp-1 cells cultured for 1 h in the presence or absence of p17and ap-1-specific oligonucleotides immobilized to a 96-well plate. levels of fos or jun family members in the active ap-1 complex of p17-treated or -untreated monocytes are expressed as percentage of relative activity where 100% is referred to 0 h values. statistical analysis was performed by one-way anova test. *p < 0.05, statistically different compared with 0 h values. bars represent the mean ϯ sd of three separate experiments in triplicate. c. representative emsa autoradiograms using nuclear extracts from thp-1 cells collected at the specified times after treatment with boiled or trypsinized p17 and oligonucleotides specific for ap-1 and for oct-1, as loading control. the human mcp-1 gene contains one ap-1 binding site in the enhancer region (p1) and two ap-1 binding sites in the promoter region (p2 and p3). to investigate which regions are involved in the transcriptional activation of mcp-1 by p17, primary monocytes were transfected with different plasmids containing the reporter luciferase gene cloned downstream to the mcp-1 promoter. mcp-1 promoter luciferase reporter plasmids used included: pglm-prm containing the proximal promoter region of the mcp-1 gene, pglm-enh, which contains both the proximal promoter and the distal enhancer regions, pglm-prm-p2mut mutated into the p2 ap-1 binding site, and phmcp128, a plasmid containing a mcp-1 proximal promoter region whose ap-1 binding sites (p2 and p3) have been deleted (fig. 6a) . flow cytometric analysis of cells transfected with a control gfp-encoding plasmid (pmax-gfp) showed a high transfection efficiency of primary monocyte with over 40% of the viable cells expressing gfp as early as 8 h after pmax-gfp plasmid transfection (fig. 6b) . soon after transfection, cells were left untreated or treated with p17 (1 mg ml -1 ). pma (20 ng ml -1 ) or tnf-a (10 ng ml -1 ) was used as controls of nf-kb and ap-1 activation. pma and tnf-a resulted in a significant transcriptional activation (3.9-and 2.2-fold respectively) in cells transfected with the pglm-enh plasmid, in which both the mcp-1 promoter and enhancer regions are present (not shown). in p17-treated monocytes the luciferase activity of constructs pglm-enh and pglm-prm was induced approximately twofold (fig. 6c) . the lack of difference between the promoter and the enhancer regions in response to p17, despite the potential ap-1 binding site in the enhancer region, indicates a critical role for ap-1 binding sites in the promoter region. indeed, deletion of both ap-1 binding sites (phmcp128) resulted in complete loss of p17-induced activation. to further assess whether p17-induced mcp-1 expression was dependent on the p2 or p3 ap-1 binding site, we mutated the p2 region in the pglm-prm plasmid (pglm-prm-p2mut) and tested for p17 activation. treatment with p17 resulted in luciferase expression in both cells transfected with the pgml-prm or pgml-prm-p2mut plasmids. this finding suggests that the p3 ap-1 binding site in the promoter region is necessary for optimal p17 stimulation. monocyte-mediated inflammatory processes and mcp-1 production are likely to be major precipitating events in fig. 6 . analysis of p17-induced mcp-1 transcriptional activation. a. schematic representation of the four human mcp-1 reporter constructs used to perform our experiments. pgl-enh contains both the proximal promoter (between -107 and +60) and distal enhancer (between -2742 and -2513) regions of mcp-1. this construct has three ap-1 binding sites identified by open boxes: p1 is located in the enhancer region while p2 and p3 are placed in the promoter region. pgl-prm contains the proximal promoter region only, whereas pgl-prm-p2 mut contains the proximal promoter region but has a mutated p2 ap-1 binding site. phmcp-128 is a construct with deletion of both p2 and p3 ap-1 binding sites. b. the transfection efficiency of purified human monocytes was assessed by flow cytometry using a gfp-expressing plasmid as a tracer. in the left panel, gfp + monocytes are displayed in bold in the histogram, as opposed to untransfected cells (thin line). the percentage of gfp + cells is also reported. transfection by nucleofection did not impair p17r expression on the surface of monocytes (right). cells transfected with the pmax-gfp plasmid were stained with biotin-conjugated p17 and apc-conjugated streptavidin to detect p17r expression, and with pi to mark dead cells. viable monocytes (pi -) were gated on fsc/pi dot plot (not shown) and an analysis was performed of gfp versus p17r dot plot. quadrant statistics are shown. c. purified human monocytes were transiently co-transfected with the prl-tk control vector together with one of the plasmids described in (a). soon after transfection, cells were treated or not with p17 (1 mg ml -1 ). renilla and firefly luciferase activities were determined 6 h after p17 treatment. relative activity is expressed as fold stimulation as described in experimental procedures and represent the mean ϯ sd of quadruplicate samples from three independent experiments. aids pathogenesis. an increasing amount of evidence shows a tight link between activated monocytes recruitment, mcp-1 release and hiv-1 pathogenesis, especially in aids patients suffering from hiv-1-associated dementia (had). the selective accumulation of mcp-1 in the cerebrospinal fluid of aids patients with had, together with the finding that mcp-1 levels correlate with the degree of dementia (cinque et al., 1998; conant et al., 1998; kelder et al., 1998) , substantiates a possible role of mcp-1 in neuropathogenesis. it is worth noting that the presence of activated monocytes in the brain is a better correlate of had than the extent of viral infection (glass et al., 1995) . a chronic inflammatory state of circulating monocytes emerges over time also in hiv-1 patients receiving highly active anti-retroviral therapy (haart). in particular, these elements evolve to a chronic inflammatory state characterized by an increase in chemotactic macrophage markers and a more mature (monocyte/ macrophage hybrid) profile (pulliam et al., 1997; . considerable interest is focused therefore on the mechanism by which hiv-1 makes active to induce dysregulation of monocyte activity. understanding this mechanism will contribute to further identify the linkage between hiv-1 infection and monocyte activation and, ultimately, to achieve improved control over the replication and spread of the virus, and of the prevention and treatment of immune disorders. highly active anti-retroviral therapy usually leads to prolonged control of hiv-1 replication. yet the blocking of hiv-1 replication is not linked to a complete recovery from immune dysfunction, thus suggesting that mechanisms other than active viral replication are involved in chronic immune perturbation. structural hiv-1 proteins have been associated with immune activation and loss of functional competence by different immune cells (quaranta et al., 2004) . among these, the hiv-1 matrix protein p17 has been recently identified as a critical determinant in aids pathogenesis as it binds to a cellular receptor expressed on immune cells and enhances viral replication and infectivity (de francesco et al., 1998) , through a combination of different effector functions (de francesco et al., 2002; vitale et al., 2003) . this structural viral protein accumulates and persists in the lymph nodes of patients under haart (popovic et al., 2005) . moreover, amounts of the hiv-1 protein did not noticeably differ in lymph nodes specimens either before initiation of therapy or during treatment where the virus burden significantly decreased. this finding strongly suggests that p17 persists in patients under haart for a long time in the absence of detectable virus replication making conceivable that their chronic activity on immune cells can lead to immune dysfunction. furthermore, we have recently shown that p17 is released by infected cells (fiorentini et al., 2006) and can be detected in sera obtained from hiv-1-infected patients using a newly developed p17 capture immunoassay (s. fiorentini et al., in preparation) . the pro-inflammatory role for p17 is suggested by its ability to induce tnf-a and ifn-g expression on t cells and to counteract the antiinflammatory activity of il-4 (de francesco et al., 2002) . in the present study we have described the ability of the hiv-1 matrix protein p17 to perturb the biological activity of primary monocytes, by selectively inducing increased mcp-1 production and release. this process is mediated by the expression of p17rs on the monocyte surface. the biological activity of p17 is exerted after binding to a specific receptor that is absent on resting t cells but expressed on activated t cells. accordingly, p17 activity is observed only after pre-activation of t cells with mitogens, which per se exert a biological effect. the in vitro biological activity of p17 has therefore been described so far as a synergistic effect that cannot be completely separated from that triggered by the mitogen itself. this study shows that circulating monocytes constitutively express p17rs on their surface and for the first time furthers our understanding of signalling triggered by p17/p17r interaction. trypsin-sensitive membrane proteins are shown to be involved in p17 binding to human monocytes, while the re-expression of p17r on monocytes after trypsin treatment required several hours and was impaired by cycloheximide. this suggests that de novo protein synthesis is needed to maintain p17r expression on monocytes rather than p17rs mobilization from intracellular stores. interestingly, p17r expression -in terms of mean fluorescence intensity -was strongly up-modulated on the surface of these cells upon activation of monocytes with ifn-g or lps. the finding that p17r expression is increased by two known monocyte stimulatory molecules warrants the definition of this molecule as a new marker attesting for the monocyte activation status. it is not yet clear, however, why p17r expression is specifically sustained on monocytes but not on t cells. several signalling pathways have been hypothesized for the transcriptional regulation of mcp-1. in the human mcp-1 gene, tre and kb enhancer elements exist in the 5′-flanking region of mcp-1 gene, thus suggesting a role for ap-1 and nf-kb (shyy et al., 1993) . we therefore decided to use the primary human monocyte model to assess p17 signal transduction pathways involved in mcp-1 gene expression. emsa revealed that p17 does not cause significant changes to the dna-binding activity of nf-kb, whereas it increases ap-1 binding in a timeand dose-dependent manner. induction of ap-1 in both primary human monocytes and thp-1 cells was associated with increased levels of c-fos and junb proteins in the active ap-1 complexes. these results indicate that the p17-induced ap-1 complexes responsible for the mcp-1 transcriptional activity are likely to be c-fos/junb heterodimers. deletion of ap-1 binding sites in the hiv-1 p17 induces mcp-1 by ap-1 activation 661 enhancer region of human mcp-1 (p1) or mutation of the p2 ap-1 binding site in the mcp-1 proximal promoter region had no effect on the transcriptional activity in response to p17. instead, deletion of both ap-1 binding sites (p2 and p3) in the promoter region resulted in reduced p17-induced mcp-1 gene transcription levels, close to basal transcription values. such evidence suggests that the upregulation of mcp-1 transcription in human monocytes by p17 may depend on ap-1 activation and binding to the p3 site in the mcp-1 proximal promoter region. however, a direct evidence for the involvement of the p3 ap-1 binding site in p17-triggered mcp-1 transcription is still lacking, as all our efforts to obtain a mcp-1 promoter-luciferase construct containing a mutated p3 ap-1 binding site were unsuccessful. the finding suggesting that in primary human monocytes ap-1 could be a major regulator of mcp-1 transcription, independently of nf-kb activation, is in line with previous studies (hanazawa et al., 1993; wang et al., 1999; lee et al., 2001; cho et al., 2002) . moreover, cho et al. (2002) and lim and garzino-demo (1999) have clearly shown that the p3 ap-1 binding site abolished inducible mcp-1 promoter activity in human endothelial cells and human astrocytoma cells (u-87) respectively. taken together, these results suggest that, at least in certain cells, ap-1 activation can circumvent the nf-kb pathway by means of different signal transduction pathways leading to the induction of the mcp-1 gene. interestingly, our experiments show a peak of ap-1 activation at 1 h after p17 stimulation and persists, at lower levels, up to 4 h. at the same time, mcp-1 transcripts increase at 4 h after p17 treatment reaching a plateau at 6 h of stimulation. the kinetics of ap-1 activation does not apparently well correlate with induction of mcp-1 gene. this finding may reflect the capability of p17 to trigger an unusual pathway that induce ap-1 binding to the mcp-1 promoter leading to the production of mcp-1 transcripts but also to their stabilization, ultimately allowing their accumulation. indeed, it is well known that regulation of mcp-1 transcription is due to both gene transcription activation and mrna post-transcriptional stabilization. different studies have demonstrated that, upon different exogenous stimuli, mcp-1 mrna half-life increases to 3 h while anti-inflammatory molecules may reduce it to 15 min (poon et al., 1999; dhawan et al., 2007) . on the other hand, we cannot completely rule out the possibility that p17 may activate other transcription factors that cooperate with ap-1 to determine the prolonged activation of mcp-1 transcription. it is important to note that several viruses have been reported to modulate ap-1 activity (sadowska et al., 2003; kwon et al., 2004; xie et al., 2005; holloway and coulson, 2006) . in some cases, the viral protein responsible for ap-1 activation has been identified, e.g. the hepatitis b virus x protein (tanaka et al., 2006) , the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein (he et al., 2003) , the papilloma virus e2 protein (behren et al., 2005) , the epstein-barr virus latent protein 1 and the parvovirus non-structural protein (ns) 1 (kieser et al., 1997) . the common exploitation of the ap-1 pathway points to the wider role of this signalling event and of its downstream gene targets in viral infections. in the case of hiv-1, ap-1 activation is a redundant phenomenon, being performed by such viral proteins as tat (kumar et al., 1998) , nef (biggs et al., 1999) , gp120 (gibellini et al., 1999) , vpr (varin et al., 2005) and, as shown here, by the matrix protein p17. in the case of hiv-1, ap-1 activation may not only enhance viral replication via trans-activation of the hiv-1-ltr (rabbi et al., 1997) but, as demonstrated above for p17, it may also modulate the expression of cellular genes. these effects could not only promote hiv-1 infection but also lead to functional defects and pathogenetic events, as demonstrated for mcp-1 and its contribution to the pathogenesis of aids dementia. our results suggest that it is necessary to block the biological activity of the hiv-1 proteins (including p17) continuously released by infected cells despite the successful blocking of viral replication obtained by haart. at the same time, the knowledge that p17 induces monocytes to produce mcp-1 by activating the transcriptional factor ap-1 should be followed up by new anti-hiv-1 therapeutic strategies capable of neutralizing the functions of molecules induced by the hiv-1 proteins which are known contributors to virulence and pathogenesis. purified, endotoxin-free recombinant hiv-1 matrix protein p17 was produced as described previously (de francesco et al., 2002) . an aliquot of the purified p17 preparation was biotinylated by using ah-nhs-biotin (biospa, milan italy) according to the manufacturer's instructions. recombinant human ifn-g and tnf-a were obtained from r&d system (minneapolis, mn, usa). lps from escherichia coli, phorbol 12-myristate 13-acetate (pma) and cycloheximide were supplied by sigma-aldrich (milan, italy). mbs-3, an anti-p17 mab that recognizes the p17 epitope responsible for p17r binding and neutralizes the p17/p17r interaction (de francesco et al., 2002) , was produced and purified in our laboratory. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation from the blood of healthy hiv-1seronegative donors, who gave informed consent to this research under the helsinki declaration. cells were then plated onto tissue culture dishes in rpmi-1640 supplemented with 10% (v/v) fetal calf serum (fcs) (invitrogen, milan, italy), 100 u ml -1 penicillin, 100 mg ml -1 streptomycin, 2 mm l-glutamine (complete medium). monocytes were purified from pbmcs using a cd14 microbeads kit (miltenyi biotec, bologna, italy) and automacs (miltenyi biotec), according to the manufacturer's instructions. the purity of monocytes was assessed after overnight incubation at 37°c in complete medium by cd14 staining and flow cytometry, and found to be greater than 95%. the thp-1 human monocytic cell line was a gift from dr maura ferrari (istituto zooprofilattico sperimentale, brescia, italy). cells were grown and maintained in culture in complete medium. staining of cells for p17r expression was performed as already described previously (de francesco et al., 2002) . briefly, the cells were incubated for 30 min on ice with biotin-conjugated recombinant p17 at a concentration of 400 ng ml -1 . after washing with pbs containing 1% fcs, they were incubated for 30 min on ice with apc-conjugated streptavidin (bd biosciences, milan, italy) . for experiments performed on pbmcs, cells were stained for 30 min on ice with fitc-conjugated cd14 mab to identify p17r + monocytes. all data obtained were analysed with cellquest software (bd biosciences). in some experiments cells were also counterstained with pe-conjugated anti-hla-dr mab (bd biosciences). purified monocytes (1 ¥ 10 6 ) were treated with 1 ml of trypsin solution (sigma-aldrich) for 15 min at 37°c. protease activity was stopped by the addition of ice-cold complete medium or soybean trypsin inhibitor (sbti, type ii-s; sigma-aldrich). control cells were treated with buffer only or using a combination of trypsin and sbti was added simultaneously. the protease-treated cells were washed twice in rpmi-1640 and subsequently assayed for p17r expression, as described above. the viability of cells was > 85% as determined by propidium iodide (2.5 mg ml -1 ) staining and flow cytometric analysis. for experiments measuring the recovery of p17r expression after trypsin treatment, cells were re-suspended at a concentration of 1 ¥ 10 6 cells ml -1 in complete medium and incubated for 48 h at 37°c. in some experiments, monocytes were cultured in the presence of cycloheximide (1 mg ml -1 ) (sigma-aldrich). mcp-1, rantes, mip-1a and mip-1b mrna levels were determined by quantitative rtpcr. total rna was extracted from p17treated or -untreated monocytes using the trizol ® rna isolation kit (invitrogen), according to the manufacturer's instructions. after reverse transcription using improm reverse transcriptase (promega, milan, italy), the relative amounts of mcp-1, rantes, mip-1a, mip-1b and b-actin mrnas were determined by rtpcr. amplification was carried out in a 25 ml reaction mix containing 12.5 ml of 2¥ taqman universal pcr master mix (applied biosystems, monza, italy), and 1.25 ml of primer and fam dye-labelled probe (gene expression assays, applied biosystems). rtpcr assays were performed in triplicate on a 7500 rtpcr system (applied biosystems) with the following pro-gramme: 40 cycles at 95°c for 20 s and at 60°c for 1 min. analysis of rtpcr data was performed with the 2 -ddct method using relative quantitation study software (applied biosystems). quantification of each chemokine cdna was normalized in each reaction according to the internal b-actin control. the levels of each chemokine mrna were expressed as fold difference in p17-treated cells in comparison with untreated cells (calibrator sample). cytokines and chemokines, namely il-1a, il-1b, il-6, il-8, il-12, tnf-a, mcp-1, mip-1a, mip-1b and rantes, were evaluated in the supernatant of monocytes cultured in the presence or absence of p17 -at a concentration ranging from 0.1 to 1 mg ml -1 -by searchlight chemiluminescent arrays (endogen, rockford, il). in some experiments, mcp-1 quantification in the supernatant of p17-treated or -untreated monocytes was performed using a quantitative elisa kit (endogen). for each treatment condition, nuclear extracts of thp-1 cells or primary blood monocytes were prepared from 1 or 2 ¥ 10 7 cells respectively. the cells were washed with ice-cold pbs and lysed on ice for 10 min in 5¥ packed cell volume of lysis buffer containing 10 mm hepes ph 7.9, 1.5 mm mgcl2, 10 mm kcl, 0.1 mm egta, 1 mm dtt, 0.2% nonidet p-40 and protease inhibitor cocktail (sigma-aldrich). the nuclei were then pelleted by centrifugation at 3000 r.p.m. at 4°c for 10 min and re-suspended in 3¥ packed nuclear volume of high-salt buffer containing 20 mm hepes ph 7.9, 1 mm edta, 1 mm egta, 420 mm nacl, 20% glycerol, 1 mm dtt and a protease inhibitor cocktail. the suspension was rocked for 20 min at 4°c, gently vortexed and subsequently centrifuged at 10 000 r.p.m. for 30 min at 4°c. the supernatant/nuclear extracts were collected and stored in aliquots at -80°c. the protein concentration was determined by using the bio-rad protein assay kit (bio-rad, hercules, ca). nuclear extracts (10 mg of protein per sample) prepared as described above were incubated with biotinylated dna oligonucleotides of 54 selected transcription factor binding element sequences (transsignal array; panomics, redwood city, ca). after isolation of protein-dna complexes, the samples were denaturated and retained binding elements hybridized to membranes containing complementary sequences. hybridized biotinylated oligonucleotides were visualized using horseradish peroxidase (hrp)-conjugated streptavidin and ecl reagents (panomics). each transcription factor was quantified in duplicate at 1¥ concentration, and in duplicate at 0.1¥ oligonucleotide concentration under each condition. with p17 (1 mg ml -1 ), were incubated with 32 p-labelled ap-1 or nf-kb dna probe (tiberio et al., 2007) , and the mobility of dnaprotein complexes (emsa) was analysed as described previously (tiberio et al., 2006) . in some experiments recombinant p17 was boiled (20 min at 100°c) or treated with 1% trypsin for 15 min at 37°c. protease activity was stopped by the addition of ice-cold complete medium. for competition experiments, a 100-fold excess of specific unlabelled probe was incubated with nuclear extracts before addition of the radiolabelled probe. for loading controls, the nuclear extracts were analysed also for the dna-binding activity of octamer-1, whose site is present in many housekeeping genes (tiberio et al., 2007) . autoradiographic signals were quantified by molecular dynamics phosphoimager (mdp) analysis (typhoon 8600; molecular dynamics, sunnyvale, ca). in order to quantify ap-1 activation, and to assess the nature of dimeric ap-1 complexes induced by p17, we used an elisabased kit (transam™; active motif, carlsbad, ca) consisting of an oligonucleotide that contains a tissue-type plasminogen activator (t-pa)-responsive element (tre) immobilized on solid phase. the assay was run according to the manufacturer's instructions. briefly, nuclear extracts obtained from both monocytes and thp-1 cells treated or not with p17 for 1 h at 37°c were allowed to interact with tre-coated wells (4 mg of nuclear extract per well). then, ap-1 dimers contained in nuclear cell extracts were detected using antibodies directed against c-fos, fosb, fra-1, fra-2, phospho c-jun, junb or jund. antibody binding to the ap-1/tre complex was detected by adding an hrpconjugated secondary antibody. mcp-1 promoter-luciferase constructs containing the proximal promoter (between -107 and +60) (pglm-prm) and distal enhancer (between -2742 to -2513) (pglm-enh) regions of mcp-1 (kindly provided by dr t. yoshimura, laboratory of immunobiology, national cancer institute, frederick, md) have already been described (ueda et al., 1997) . site-directed mutation was introduced in the ap-1 binding p2 site of the promoter region with a quick-change site-directed mutagenesis kit (stratagene, la jolla, ca). briefly, 15 ng of pgl-prm and 125 ng of oligonucleotides: (5′-cag ccc act tat gac aga tgg aag atc cct cc-3′/5′-gga ggg atc ttc cat ctg tca taa gtg ggc tg-3′) were incubated with 2.5 u of pfu dna polymerase and cycled 12 times under the following conditions: denaturation at 95°c for 30 s, annealing at 55°c for 1 min and extension at 68°c for 6 min. the reaction mix was cooled on ice for 2 min and digested with 10 u of dpni at 37°c for 1 h; 2 ml of the reaction mix was transformed into e. coli xl1-blue supercompetent cells. plasmids were isolated from colonies grown overnight at 37°c on ampicillin agar plates, and sequencing was carried out to identify those containing the target mutated sequence of interest (pglm-prm-p2mut). the plasmid phmcp128 containing deletion of both p2 and p3 ap-1 binding sites (kindly provided by dr a. garzino-demo, institute of human virology, university of maryland, baltimore, md) has been previously described (lim and garzino-demo, 1999) . purified monocytes were co-transfected by nucleofection using the human monocyte kit (amaxa ag, cologne, germany) with the individual luciferase (firefly) test plasmids (2 mg ml -1 ) and a renilla luciferase expression plasmid (prl-tk as an internal control to signal the total cellular transcription level (promega). cells were then plated into a 24-well plate (10 6 cells per well) and exposed or not to p17 (1 mg ml -1 ) in complete medium. after 6 h, they were harvested and luciferase expression was quantified using a dual-luciferase reporter assay system, according to the manufacturer's instructions (promega). test values were corrected for the luciferase activity value of the internal control plasmid prl-tk. the results for the p17 exposed cells are expressed as fold induction of luciferase activity by the same construct under control conditions, taking the control (no p17 added) value as 1. cytokine and chemokine based control of hiv infection and replication papillomavirus e2 protein induces expression of the matrix metalloproteinase-9 via the extracellular signal-regulated kinase/activator protein-1 pathway induction of activator protein 1 (ap-1) in macrophages by human immunodeficiency virus type-1 nef is a cell-type-specific response that requires both hck and mapk signalling events induction of the gene encoding macrophage chemoattractant protein 1 by orientia tsutsugamushi in human endothelial cells involves activation of transcription factor activator protein 1 elevated cerebrospinal fluid levels of monocyte chemotactic protein-1 correlate with hiv-1 encephalitis and local viral replication induction of monocyte chemoattractant protein-1 in hiv-1 tat-stimulated astrocytes and elevation in aids dementia hiv-1 matrix protein p17 increases the production of proinflammatory cytokines and couteracts il-4 activity by binding to a cellular receptor hiv p17 enhances lymphocyte proliferation and hiv-1 replication after binding to a human serum factor a novel role for the glucocorticoid receptor in the regulation of monocyte chemoattractant protein-1 mrna stability changes in viral load markers during aids-associated opportunistic diseases in human immunodeficiency virus-infected persons regulation of chemokine/cytokine network during in vitro differentiation and hiv-1 infection of human monocytes: possible importance in the pathogenesis of aids endogenous ccl2 (monocyte chemotactic protein-1) modulates human immunodeficiency virus type-1 replication and affects cytoskeleton organization in human monocyte-derived macrophages functions of the hiv-1 matrix protein p17 cns invasion by cd14 + /cd16 + peripheral blood-derived monocytes in hiv dementia: perivascular accumulation and reservoir of hiv infection hiv-1 gp120 induces the activation of both c-fos and c-jun immediate-early genes in hel megakaryocytic cells immunocytochemical quantitation of human immunodeficiency virus in the brain: correlation with dementia effect of mycobacterium tuberculosis on hiv replication. role of immune activation hiv-1 infection and aids dementia are influenced by a mutant mcp-1 allele linked to increased monocyte infiltration of tissues and mcp-1 levels tumor necrosis factor-a induced expression of monocyte chemoattractant je via fos and jun genes in clonal osteoblastic mc3t3-e1 cells activation of ap-1 signal transduction pathway by sars coronavirus nucleocapsid protein macrophage activation and hiv infection: can the trojan horse turn into a fortress? vpx is required for dissemination and pathogenesis of siv(sm) pbj: evidence of macrophage-dependent viral amplification the macrophage and hiv-1 rotavirus activates jnk and p38 signaling pathways in intestinal cells, leading to ap-1-driven transcriptional responses and enhanced virus replication the influence of cytokines, chemokines and their receptors on hiv-1 replication in monocytes and macrophages beta-chemokines mcp-1 and rantes are selectively increased in cerebrospinal fluid of patients with human immunodeficiency virus-associated dementia epstein-barr virus latent membrane protein-1 triggers ap-1 activity via the c-jun n-terminal kinase cascade hiv-tat protein activates c-jun n-terminal kinase and activator protein-1 induction of chemokines in human astrocytes by picornavirus infection requires activation of both ap-1 and nf-kappa b irreversible association of peptides with class ii mhc molecules in living cells high glucose induces mcp-1 expression partly via tyrosine kinase-ap-1 pathway in peritoneal mesothelial cells the human immunodeficiency virus type 1 tat protein up-regulates the promoter activity of the beta-chemokine monocyte chemoattractant protein 1 in the human astrocytoma cell line u-87 mg: role of sp-1, ap-1 and nf-kb consensus sites identification of a novel dexamethasone-sensitive rna-destabilizing region on rat monocyte chemoattractant protein 1 mrna persistence of hiv-1 structural proteins and glycoproteins in lymph nodes of patients under highly active antiretroviral therapy unique monocyte subset in patients with aids dementia invasive chronic inflammatory monocyte phenotype in subjects with high hiv-1 viral load hiv-1 nef equipe dendritic cells to reduce survival and function of cd8 + t cells: a mechanism of immune evasion u5 region of the human immunodeficiency virus type 1 long terminal repeat contains trelike camp-responsive elements that bind both ap-1 and creb/atf proteins regulation of human polyomavirus jc virus gene transcription by ap-1 in glial cells activation of mcp-1 gene expression is mediated through multiple signalling pathways the hepatitis b virus x protein enhances ap-1 activation through interaction with jab1 mechanisms of interleukin-6 protection against ischemia-reperfusion injury in rat liver interleukin-6 sustains hepatic regeneration in cirrhotic rat isolation and characterization of the intracellular mhc class ii compartment transcriptional regulation of the human monocyte chemoattractant protein-1 gene. cooperation of two nf-kappa b sites and nf-kappa b/rel subunit specificity synthetic vpr protein activates activator protein-1, c-jun n-terminal kinase, and nf-kb and stimulates hiv-1 transcription in promonocytic cells and primary macrophages hiv-1 matrix protein p17 enhances the proliferative activity of natural killer cells and increases their ability to secrete proinflammatory cytokines viral and host cofactors facilitate hiv-1 replication in macrophages adenovirus-mediated overexpression of c-jun and c-fos induces intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human endothelial cells kaposi's sarcoma-associated herpesvirus induction of ap-1 and interleukin 6 during primary infection mediated by multiple mitogen-activated protein kinase pathways this work was supported by a grant from the istituto superiore di sanità aids projects (grant 40g.16) and by ministero dell'università e della ricerca, prin projects. key: cord-323261-1of5ertf authors: lo, catherine yuk-ping title: securitizing hiv/aids: a game changer in state-societal relations in china? date: 2018-05-16 journal: global health doi: 10.1186/s12992-018-0364-7 sha: doc_id: 323261 cord_uid: 1of5ertf background: china has experienced unprecedented economic growth since the 1980s. despite this impressive economic development, this growth exists side by side with the human immunodeficiency virus/acquired immune deficiency syndrome (hiv/aids) and severe acute respiratory syndrome (sars) crises and the persisting deficiencies in public health provision in china. acknowledging the prevailing health problems, the chinese government has encouraged the development of health non-governmental organizations (ngos) to respond to the health challenges and address the gaps in public health provision of the government. hiv/aids-focused ngos have been perceived as the most outstanding civil society group developed in china. considering the low priority of health policies since the economic reform, the limitation of the “third sector” activity permitted in authoritarian china, together with the political sensitivity of the hiv/aids problem in the country, this article aims to explain the proliferation of hiv/aids-focused ngos in china with the usage of the securitization framework in the field of international relations (ir). methods: the research that underpins this article is based on a desk-based literature review as well as in-depth field interviews with individuals working in hiv/aids-focused ngos in china. face-to-face interviews for this research were conducted between january and may in 2011, and between december 2016 and january 2017, in china. discourse analysis was in particular employed in the study of the security-threat framing process (securitization) of hiv/aids in china. results: this article argues that the proliferation of hiv/aids-related ngos in china is largely attributed to the normative and technical effects of hiv/aids securitization ushered in by the united nations security council (unsc) and supported by the global fund to fight aids, tuberculosis, and malaria (hereinafter global fund) observed in china. despite depicting a positive scenario, the development of hiv/aids-focused ngos in china generated by the international securitization efforts is largely limited. an internal and external factor was identified to verify the argument, namely (1) the reduction of international financial commitments, as well as (2) the fragmentation of hiv/aids-focused ngo community in china. conclusions: this article shows that international securitization weakened with the rise of chinese commitment on hiv/aids interventions. in other words, hiv/aids-related responses delivered by the national government are no longer checked by the global mechanism of hiv/aids; thus it is unclear whether these ngos would remain of interest as partners for the government. the fragmentation of the hiv/aids community would further hinder the development, preventing from ngos with the same interest forming alliances to call for changes in current political environment. such restriction on the concerted efforts of hiv/aids-related ngos in china would make achievement of the sustainable development goals (sdgs) to foster stronger partnerships between the government and civil society difficult, which in turn hindering the realization of ending hiv/aids in the world by 2030. china has experienced remarkable economic growth since the initiation of the economic reform in the 1980s. the average gdp growth in china was 9.6% annually during the first two decades of the economic reform [1] . the rising nation became the world's leading manufacturing power in 2011, and it surpassed the united states as the world's largest trading country [2] . jakovljievic further predicted that the economic growth in china would continue at least up to the middle of the 21st century [3] . despite such impressive economic performance, the provision of public health and the control of infectious diseases have been marginalized. between the late 1980s and 2003, the chinese national government public health spending as a proportion of total health spending plunged from 30% to just over 15% [4] , despite the fact that china has outperformed other developing countries, such as other brics members, regarding the public health spending in nominal terms [5] . as argued by yip and mahal, chinese political leaders in the abovementioned period of time simply perceived health as "a consumption activity rather than a productive good and therefore was given lower priority in government funding" [4] . in this regard, the chinese authorities deliberately curtailed the level of public health expenditures so that economic development could be pursued and the legitimacy of the one-party authoritarian regime bolstered. acknowledging the economic success in the past decades, hiv/aids nevertheless emerged as one of the "negative externalities" of public health threats brought about by the 1980s economic reform. hiv/aids was first discovered in china during the mid-1980s, the same period when the economic reform was just beginning. the epidemic was exacerbated in china in the 1990s mainly because of government-supported blood plasma selling, which did not follow adequate sterilization procedures. this lack of regulation resulted in the emergence of so-called aids villages (aizibing cun) in henan and its outlying provinces, where a large percentage of rural residents were infected with hiv/aids [6] . having little benefit from the economic reform in ways that coastal provinces did during the 1980s, many residents in the central plains had little choice but to engage in the blood-plasma industry to improve their standard of living. since 2000, hiv/aids has become a politically sensitive issue in china because of the international condemnation of the henan blood scandal. according to the latest figures released by the joint united nations program on hiv/aids (unaids), 660,000 people were estimated to have hiv/aids in china in 2016 [7] . the country has the second largest hiv/aids population in asia after india. the chinese government started addressing the hiv/ aids problem after years of denial and underestimation of the hiv/aids prevalence in the country, especially among the most-at-risk populations (marps) including injecting drug users (idus), female sex workers (fsw), and men who have sex with men (msm) [8] . however, national hiv/aids policies in the early years were very much driven in a top-down manner with government institutions (i.e. mainly the chinese centers for disease control and prevention [china cdc hereinafter] ) playing a leading role and with limited engagement of the "third sector" [9, 10] . given the association of the disease with behaviors that are legally or socially unacceptable in the country (i.e., commercial sex work, injecting drug use, and men having sex with men), hiv/aids high-risk individuals refrain from using the related services provided by the government agencies because they wish to avoid being seized or humiliated by public security officials or by thugs hired by local officials [11] . in this regard, gåsemyr claimed that the emergence of hiv/aids-focused non-governmental organizations (ngos) 1 in china during the early 1990s could fill the capacity gap of the government by implementing national hiv/aids policies and providing the related services for those marps that are hard to reach by the authorities [12] . considering the low priority of health policies since the economic reform, the limitation of the "third sector" activity permitted in authoritarian china, together with the political sensitivity of the hiv/aids problem in the country, it is believed that the involvement of hiv/aids-related ngos in china would be largely limited. this is however not the case. over the last 15 years there has been a dramatic increase in the number of hiv/aids-focused ngos operating in china. while gåsemyr argued that the growth of hiv/ aids-related ngos is mainly attributed to the devastating health crisis of sars in china and the new chinese leadership in 2003 [12] , the previous work cannot explain why a high degree of political recognition was observed in solely hiv/aids ngos, instead of other health ngos, such as tb or cancer ngos in china. this article aims to fill this research gap with regard to the proliferation of hiv/aids-focused ngos in china with the use of the securitization framework in the field of ir. the research that underpins this article is based on desk-based literature review as well as in-depth field interviews with individuals working in hiv/aids-focused ngos in china. discourse analysis was in particular employed to study the securitization of hiv/aids in china. potter argued that "discourse analysis has an analytic commitment to studying discourses as texts and talks in social practice…the focus is…on language as… the medium for interaction…one theme that is particularly emphasized here is the rhetorical or augmentative organization of talk and texts" [13] . silverman pointed out that the intellectual ancestor of discourse analysis is john langshaw austin, the original inventor of the idea of speech acts that later became the core element in securitization theory [14] . concerning discourse analysis and speech acts share the austinian concern with the rhetorical structure of talk and texts, the founding scholars of securitization theory, buzan, waever and wilde explicitly stated, "the obvious method [to study securitization] is discourse analysis, since we are interested in when and how something is established by whom as a security threat" [15] . the authors also noted that the technique of employing discourse analysis to securitization studies is to "read[ing], looking for arguments that take the rhetorical and logical form defined here as security" [15] . the meaning of security is constructed when the political leaders (securitizing actors) frames an issue as an existential threat to the referent objects, 2 claiming the issue as having an absolute priority on the government agenda, thereby invoking a right to handle such an issue with extraordinary/emergency measures to ensure the survival of the referent objects [16] . in this research, public speeches (in written form) related to hiv/aids problems, which were delivered by the top-level political leaders, were examined. a specific rhetorical structure had to be located in their discourses, consisting of either one or all of the following elements: (1) the existence of an existential threat; and/or (2) the perceived threat is challenging the survival of the states or people in the states (referent objects), and/or which it requires to (3) be urgently dealt with by new political measures of the respective country. an array of documents ranging from official documents, reports, newspaper articles was initially identified via the search engines of google scholar and proquest with the keywords of "hiv/aids", "security threat", and "china". nine key articles were consequently singled out to identify the textual discourses related to hiv/aids threat/ security nexus in china, published or released between 1987 and 2011 (table 1) . a total of 47 semi-structured interviews were conducted along with the discourse analysis. apart from the background information of the respective ngos, the interview questions used in this study are categorized into three main dimensions: (1) the priority of hiv/aids on the government agenda; (2) the perceptions on the changing international and national financial commitment on hiv/aids interventions; and (3) civil society involvement and coordination in national hiv/aids policies. the first dimension aimed to ensure that the ngos respondents were knowledgeable about the changing national government perceptions and policy priority towards hiv/aids in china, therefore, the researcher could rule out those comments given by unknowledgeable respondents so as to enhance the internal validity of the data collected. the second dimension was included to study their perceptions on the level of hiv/aids funding and effect (if any) on the service provision. the third dimension aimed to discover the actual level of involvement of ngos in national hiv/aids interventions as well as the level of coordination and cooperation between different ngos working on hiv/aids. two types of non-probability sampling-purposive sampling, including "theoretical sampling" and snowball sampling-were employed in the selection process of key informants. for purposive sampling method, samples were selected based on the "logic" or "commonsense" of the researcher in terms of the target population, its elements, and the purpose of the study. in this study, the target population consisted of the individuals working in hiv/aids-related ngos at global, national, and grassroots levels. in the course of the data collection, "theoretical sampling" was also applied to understand the subjects from a theoretical perspective. hence, academic scholars who have conducted research on hiv/aids policies in china were also included in the interviewee list. interviewees were also recruited via snowball sampling. the researcher collected data on the few members, and each respondent was asked to suggest people whom the respondent believed to be the most influential for interviewing. interviews for this research were undertaken between january and may in 2011, and between december 2016 and january 2017 with leaders or senior officers working on hiv/aids in china, including 15 international ngos, nine self-help groups, four gongos, five advocacy groups, two national ngo, one independent consultant, two academia, and two networks (see table 2 ). twenty-six respondents were pinpointed based on the civil society (minjian shehui) 3 was not considered as part of the political system in the pre-modern era; the emergence of a relatively independent civil society is a product of modern china. the changing socio-economic relations brought about by "open door policy" resulted in the recognition and growth of civil society for the first time in the chinese history [17] . despite the early emergence of civil society, the 1999 falung gong incident showcased the destructive power of civil society in the eyes of the chinese government, worrying a robust, well-organized, and out-of-control civil society could overthrow and replace the ccp ruling position. meanwhile, the authorities realized the supplementary role ngos could perform in policy implementation. the chinese government has thus resolved such dilemma by adopting a "state-led" approach to manage civil society in china. based on frolic's concept of "state-led civil society", civil society is created by and belong to the state, thus the independence and autonomy of civil society are at all time bounded by the state [18] . the state has the role of legitimating social organizations, demanding a disciplined partnership. accordingly, antagonism inherited in the western concept of civil society is not allowed to exist in the state-society relations in chinese authoritarian regime; any alternative force to the state is considered as an attempt to curtail or overturn its political legitimacy and power. accordingly, the state apparatus has managed the numbers and restricted the growth of both international and grassroots ngos via various legislative means, thereby allowing ngos to operate openly but at the same time keep the organization growth in check [19, 20] 4 despite these regulations, the chinese authorities had relatively given more autonomy to ingos than grassroots ngos operating in its territory, since the government could access international expertise and tap foreign money to tackle with the emerging social problems in china [22] . such degree of autonomy is nevertheless problematic after the promulgation of foreign ngos management law (jingwai fei zhengfu zuzhi guanli fa) in april 2016. the 2016 law stipulates that ingos operating in china must register with public security officials, and must not engage in political or religious activities that damage "china's national interests" or "ethnic unity". international community and western governments perceived the new law as a reinforcement of restriction of the numbers and scopes of foreign entities operating in the authoritarian regime. grassroots ngos are regulated by the regulation on registration and administration of social organizations (shehui tuanti dengji guanli tiaoli). according to the 1998 regulation (amended in february 2016), a ngo must possess a minimum asset of 100,000 yuan and have a "professional management unit" (zhuguan danwei) that acts as a supervisory body to the organization in order to register under the ministry of civic affairs (moca). 5 fulfilling these two requirements is very formidable for grassroots ngos. the financial situation is problematic in many grassroots ngos because of the limited source of funding. on the one hand, most grassroots ngos receive limited financial support from the government, as they usually do not have political ties with the government. grassroots ngos also seldom receive donations from local communities because (1) newly developed ngos do not have good track records that enable them to win the trust of the locals, and (2) donors cannot receive tax breaks for their donation to unregistered ngos [23, 24] . in addition, most government departments simply reject their applications due to fear of consequences of taking responsibilities for grassroots ngos. many grassroots ngos are hence often unregistered or registered as business entities with the ministry of industry and commerce (moic) [25] . 6 in addition to the abovementioned restrictions, the regulation also bans "similar organizations" coexisting at the various administrative levels [21] , facilitating the management and even control the legal status of grassroots ngos in china. 7 having official registration, to certain extent, is sine qua non to the survival of organizations as the legal status entitles ngos as official recognized entities to receive legal and financial supports from the government; unregistered ngos are officially perceived as illegal that would be subjected to prosecution and coercion by the state apparatus. 8 considering a restrictive engagement of grassroots ngos in china, instead of grassroots ngos, early hiv/ aids-related responses at societal level were largely conducted by gongos [26] , 9 [27] . considering the low priority of health policies since the economic reform, the limitation of the "third sector" activity permitted in authoritarian china, together with the political sensitivity of the hiv/aids problem in the country, it is intriguing to uncover the reasons for the growth of hiv/aids-focused ngos in china since 2003. gåsemyr argued that the growth of hiv/aids-related ngos is mainly attributed to the devastating health crisis of sars in china and the new chinese leadership in 2003 [12] . having said that the chinese government realized the impacts health problems or infectious diseases could have on its economic and social development, the previous work cannot explain why a high degree of political recognition was observed solely in hiv/ aids ngos, instead of other health ngos, such as tb or cancer ngos in china. considering the regime type of the government, allowing the growth of local ngos would in turn potentially attenuate the supremacy of the chinese communist party (ccp) in ruling the country or erode the autonomy of the state, why the chinese government tolerated, allowed, or even encouraged the growth of ngos in fixing hiv/aids problems, especially the epidemic has been viewed as a politically sensitive issue in china? how can one account for and understand the phenomenon? answering the key question with an ir perspective, this article argues that the rapid development of hiv/ aids-related ngos in china is attributed to the effects of hiv/aids securitization at the international level since 2000. considering one of the most influential theories developed in the field of ir, securitization describes the process of defining an issue as a security threat (speech acts) and the corresponding political responses to block the adverse development of the perceived threat (emergency measures). this article particularly pinpoints the unsc and the global fund, which provide normative and technical supports, to study this endeavor. normative influences in this article refer to the effects of security-threat discourses, primarily resolution 1308 of the unsc, on the national government's perception on hiv/aids problems, whereas technical influences refer to the effects of global emergency measures, in particular the global fund, on the funding and management of hiv/aids-related policies at the state level [28] . this article aims to demonstrate the ways in which international hiv/aids securitization has served as a game changer in altering state-society relations in china. drawing on literature reviews and fieldwork materials, this article further argues that the proliferation of hiv/aids-focused ngos in china generated by the international securitization efforts is nevertheless largely limited owing to the reduction of international financial commitments. the fragmentation of hiv/aids-focused ngo community is another factor hindering the development of a full-fledged third sector in the country. securitization in the ir discipline is a model outlining the process of framing an issue as a security threat and the corresponding political reactions to block the adverse development of the perceived threat. employing the theory, one is required to look for three criteria to determine whether an issue is a threat to the country and its people, including 1) discourses uttered by toplevel political leader (securitizing actor) declaring a particular referent objects as an existential threat to security; 2) acceptance by the targeted audience convinced of its potential to be an existential threat (audience acceptance); and also 3) redirections of existing financial resources, new policies, or practices (emergency measures) are implemented to address the issue following the security-threat claim [15] . in other words, the resolution of the "existential threat" posed by "x" takes precedence over other problems [29] . numbers of security studies scholars believed that hiv/aids is the first infectious disease being securitized by the international institutions and national governments as the abovementioned criteria were all fulfilled in the case of hiv/aids [9, [30] [31] [32] . speech acts concerning hiv/aids as a security threat were first identified in a unsc meeting in january 2000. 10 in july of the same year, the unsc passed resolution 1308, acknowledging the urgent need to address hiv/aids, which threatens the stability and security if left unchecked [33] . it is perceived that the particular security framing of hiv/aids gained acceptance by the national governments (audience of the hiv/aids security-threat claim) since 189 countries unanimously adopted and signed the declaration of commitment on hiv/aids in 2001. the urgency of addressing hiv/aids problems was reinforced in the special united nations general assembly special session (ungass) devoted to hiv/aids that has taken place every 5 years since 2001 [34] [35] [36] [37] . the abovementioned events are significant because they represented the first time that the unsc general assembly devoted an entire session to a single disease, and the first time that the international political authorities framed hiv/ aids as a global health threat to national and international security instead of solely as a developmental or public health problem [38] . 11 the 2000 rhetorical act was backed by operational emergency responses in terms of the augmentation of the global hiv/aids spending since 2000. the amount was exponentially surged from about us$900 million in 1999 to us$16 billion by 2009; and to an estimated us$19 billion in 2013 [39] . several hiv/aids-related funding agencies and health programs formulated after 2000, also referred to global health initiatives (ghis), brought in additional monetary resources for hiv/aids [40] . 12 perceiving as the most prominent ghis, the global fund pledges to accelerate the end of the three epidemics, [hiv/]aids, tuberculosis, and malaria by providing financial support (us$4 billion annually) for low-income countries to respond to the three diseases [41] . undoubtedly, the introduction of the abovementioned ghis has brought about unprecedented levels of funding for diseases, in particular hiv/aids, in countries that have insufficient resources, whether because of poor socio-economic development or lack of political will, to orchestrate appropriate responses to the intractable disease. acknowledging international securitizing efforts on hiv/aids, this article illustrates the normative effects of resolution 1308 of the unsc and the technical influences of the global fund on national-level hiv/aids responses. china is selected as a case study to investigate this endeavor. china's response to the hiv/aids problem changed from an ignorance of "global evidencebased policy recommendations and international advice" into "one of our success stories [in global hiv/aids fight] during this past 15 years," as claimed by michel sidibe, executive director of the unaids [42, 43] . the subsequent sections demonstrate the ways in which the securitization effort in the international level acted as a game changer for hiv/aids-focused ngos in participating in national hiv/aids undertakings in china. chinese case study discourse analysis: normative influences and the role of the unsc in china, many of the first cases of hiv/aids since 1985 occurred among foreigners and homosexuals. therefore, the official discourses at that time promoted the idea that hiv/aids only affected non-local chinese people and foreigners, asserting "drug taking, alcoholism, robbery, homicide, suicide, divorce, prostitution, homosexuality, syphilis, aids…these kinds of western social ills come from their ideology", and also claiming "china had no sources of hiv/aids" [44, 45] . the rhetorical acknowledgement of hiv/aids as a health security threat to the chinese population was largely absent during the early period of time. ramiah also pinpointed that the chinese leadership was extremely reluctant to acknowledge hiv/aids as a problem and securitize hiv/aids in china prior to the 2000s [46]. one probable reason for the resistance to hiv/aids securitization is economic: the period of early hiv/aids outbreak coincided with the initial years of open door policy and economic reform (gaige kaifang) of china to the outside world. the thirst for economic growth and performance preceded the urgency of disease management: local authorities feared their respected jurisdictions would lose external or foreign investment if the full extent of the problem were known, or that they would be punished by superiors for failing to prevent the hiv/aids spread. the "cover-up" practice also ocin response to the designated targets, the securitythreat framing of hiv/aids has been fully adopted in the official discourses of chinese leaders and in official documents since 2003. previously viewing hiv/aids as a "western disease" or "non-chinese disease", executive vice minister of health gao qiang stated in the hiv/ aids high-level meeting of the un general assembly that "hiv/aids is a common enemy of the whole mankind as it seriously threatens public health and safety. the chinese government has attached great importance to hiv/aids prevention and treatment and has treated it as a strategic issue for social stability, economic development, national prosperity and security, making it a first priority of the government work" [emphasis added] [47] . the chinese president hu jintao once claimed in a 2003 public speech that "hiv/aids prevention, care and treatment is a major issue pertinent to the quality and prosperity of the chinese nation"; whereas premier wen likewise asserted that "dealing with hiv/aids as an urgent and major issue is related to the fundamental interests of the whole chinese nation" [emphasis added] [47] . the ideas in the speeches were restated in the official document entitled state council notice on strengthening hiv/aids prevention and control (guowuyuan guanyu qieshi jiangqiang aizibing fangzhi gongzuo de tongzi). this 2004 document explicitly demonstrated the determination of the chinese authorities in grappling with the disease and stated that "hiv/aids prevention and control is linked to economic development, social stability, and national security and prosperity. long-term commitment to respond to hiv/aids is hence necessary" [emphasis added] [48] . china's hiv/ aids prevention and control policies were further strengthened in the state council notice on further strengthening hiv/aids prevention and control (guowuyuan guanyu jinyibu jiangqiang aizibing fangzhi gongzuo de tongzi) in late 2010, claiming that "the prevention and control of hiv/aids is related to the people's physical health and social and economic development, as well as to national security and the rise and fall of the nation. the party central committee and the state council have always attached great importance to the prevention and treatment of hiv/aids" [emphasis added] [49] . based on the discourse analysis of the official documents and newspaper articles, it is argued that chinese national leaders followed suit the international move (i.e. unsc resolution 1308) to securitize hiv/aids in the country, framing hiv/ aids as a threat with social, political, economic, and security implications. such an alteration in perception toward the epidemic brought about the political recognition of the hiv/ aids-focused ngos, which has also been highlighted by multiple statements delivered by various top-level government officials since 2003. in 2004, vice premier wu yi stated, "we should mobilize all the partners in society to participate in the fight against hiv/aids. we need to improve our policies and strategies to build a better environment for the society to participate in the response, and try our best to facilitate the involvement of all sectors" [49] . in 2011, vice minister of health yin li publicly proclaimed "civil societies play an indispensable role in effective hiv/aids interventions led by the government" [50] . premier li keqiang even promoted tax break for ngos specializing in hiv/aids prevention, claiming the role of ngos in hiv/aids prevention "is an irreplaceable and unique force" [51] . to a certain extent, these aforementioned statements demonstrate the state's recognition of ngos' works and its willingness to engage with ngos in national hiv/aids interventions, although the political space of involvement is by and large bounded by the state [52] . the previous section illustrates the change in the perception of the chinese government toward the hiv/ aids problems: from denial to the acceptance of the threats that could be posed by the disease on the country and its people. such threat recognition also brought about the acknowledgement that the government alone could not resolve the hiv/aids problems because of the complicated nature of the epidemic; the involvement of civil society groups was a globally recognized recommendation leading to the successful containment of the epidemic. nevertheless, verbal recognition is not sufficient to enhance the engagement and development of hiv/aids-focused ngos, especially in the preexisting authoritarian government structure; the governance and funding mechanism for hiv/aids responses requires alternation in china. as illustrated in the following section, this was the mechanism of the global fund that enhanced the funding for hiv/aids-focused ngos, facilitating the involvement of civil society groups in translating the rhetorical acts into emergency responses of hiv/aids in china. among the international funding agencies emerging after the hiv/aids securitization launched by the unsc, the global fund was the most renowned major contributor to the hiv/aids interventions in china. the global fund finally accepted the first proposal submitted by the chinese government on hiv/aids interventions in late 2003, after the previous proposals being turned down twice in 2002 [52] . between 2003 and 2012, the chinese government has received over us$8 billion in grants from the global fund; approximately us$324 million were disbursed to hiv/aids intervention programs [41] . further information regarding the global fund on hiv/aids in china is illustrated in table 3 . intervention of the global fund's funding mechanism was deemed to transform the primary hiv/aids governance in china [53] . the global fund required the governments to set up a country-level country coordination mechanism (ccm) to construct the grant proposals in line with the national hiv/aids-related strategies, manage the use and distribution of grants, and also oversee the implementation of successful applications. as per the requirement of the global fund, ccm should consist of a broad representation from the government, ngos, multilateral and bilateral agencies, and private sector [54] . in the existing government structure in china, grassroots ngos have limited channels to participate in policymaking and implementation processes. thus, the multi-sectoral structure of ccm contributed to enhancing the government's commitment to involve ngos in national hiv/aids prevention and control measures [55] . two among the 22 seats in chinese ccm were reserved for the representatives of hiv/ aids-related grassroots ngos and people living with hiv/aids are a case in point [56] . considering the existing government structure does not allow the full involvement of ngos in china, the ccm provided valuable opportunities for civil society groups to engage in hiv/aids-related policymaking. the involvement of ngos in national hiv/aids programs was notably highlighted in round 6 of the global fund in china, since the theme of the grant proposal submitted by the ccm was directly linked to the development of civil society groups (refer to table 3 ). in the course of the interview, a director of a hong kongbased ngo commented on the profound influence of the round 6 on the ngo development: "round 6 of the global fund acts as a pushing force for the government to involve more grassroots ngos in hiv/aids interventions" (national ngo-1). in line with the respondent's opinion, kaufman argued the round 6 in particular "was seen by many as a further mechanism to institutionalize hiv/[aids] ngos roles in china's [hiv]/aids response [42] ." in addition to the increase in participation, financial support for ngos working on hiv/aids-related programs surged through the global fund. as the principal recipient (pr) of the country, the chinese government (i.e., china cdc or the nhfpc) was required to disburse a designated portion of the grants to hiv/aids-related ngos (in the form of sub-recipients) operating in the country [41] . following the instruction, 20% of the total grant was allocated to support ngos in round 3 and 4, whereas 50% of its grant was distributed to ngos in round 5 of the global fund [56] . in line with the theme of round 6 entitled "mobilizing civil society to scale up hiv/aids control efforts in china" proposed by the chinese ccm, the proportion granted to hv/aids-related ngos reached 100% [52] . owing to the constructive political atmosphere and promising financial resources in the country, a burgeoning number of individual-organized hiv/aids-focused ngos was established in china; over 43% of the number of grassroots ngos increased in china between 2003 and 2004 [57] . the suspension of the global fund in 2011 demonstrated that the funding mechanism to some extent held the chinese government accountable for the financial supports of hiv/aids-focused ngos. the global fund once suspended its funding to chinese hiv/aids programs in early 2011 because the chinese ccm failed to allocate 35% of the us$283 million hiv/aids grant to grassroots ngos as pledged; less than 11% was distributed to hiv/aids-focused ngos [58] . a program officer of a ngo mentioned the failure of ccm to allocate the right portion of the global fund grant to grassroots ngos: "government officials and representatives from gongos dominate the ccm. only a few representatives are from grassroots ngos and civil society" (advocacy group-1). the improper management and use of the global fund was even admitted by a senior government official in the shanghai cdc in times of the interview: first, the global fund required at least 25 percent to 30 percent of the grant should be given to grassroots ngos. however, the government failed to achieve this requirement. second, the management fee in some provinces was too high. third, one of the elements in the rcc program was related to budget aggregation (i.e., monetary contribution by both the national government and the global fund). however, the chinese government did not allocate enough money for the aggregated budget (ccdc/nhfpc-1). the determination of the chinese government to resolve the suspension of the global fund was observed in the course of fieldwork research. during a face-to-face interview inside the office building of the ministry of health (renamed national health and family planning commission in 2013) in may 2011, the respondent told the investigator "the representatives of the chinese government and the global fund representatives are meeting upstairs to negotiate for the resumption of the 2011 grants" (ccdc/nhfpc-2). the dispute was eventually resolved; the chinese government promised to allocate at least 25% of the global fund grant to ngo work [59] . in this regard, the global fund mechanism contributed to the political and financial supports of ngos, thereby resulting in the proliferation of hiv/aids-specific grassroots ngos in china, and also the proactive involvement of ngos in national hiv/aids undertakings. in other words, international securitization altered the hiv/aids policy in china, allowing numerous grassroots-level ngos working on hiv/aids to grow in a prevailing political system with "bounded autonomy". an injecting drug use self-help group leader believed the upsurge in the involvement of civil society also resulted in the improvement of state-societal relations, stating, "in the past, the relationship with the government was not good, but it becomes much better now. the government sent officials to visit our center to learn the management strategy of our organization" (self-help group-1). another ngo interviewee in beijing also claimed that "an increase in the acceptance of the government, especially the ministry of health, on grassroots ngos, [is observed in china] . the ministry of health does open the opportunity for ngos to cooperate with the government" (advocacy group-2). having said that the improvement of state-societal relations, it is noted that the chinese state is not a monolithic actor, but a conglomeration of departments and officials with competing and sometimes conflicting agendas in the national and sub-national levels [22] . a beijing ngo leader pinpointed this phenomenon, stating, "the ministry of health is fine to cooperate with those ngos that can help, regardless of whom. however, state security bureau usually holds a relatively hostile view on the existence of grassroots ngos since the bureau is concerned about social stability" (advocacy group-2). considering hiv/aids as a health threat to the country and its people, the recognition and involvement by the central government and the nhfpc and other related government agencies is of particular vital to the proliferation of hiv/aids-focused ngos in a statedominated society in the current political regime. despite depicting a positive scenario, this article further illustrates that the development of hiv/aids-focused ngos in china generated by the international securitization efforts is largely limited. in other words, the growth of the number of ngos working on hiv/ aids in china would be ceased or even decreased in long term, and also the political space for grassroots to involve in hiv/aids policy making and implementation would be further restricted. an internal and external factor was identified to verify the argument, namely (1) the reduction of international financial commitment, as well as (2) the fragmentation of hiv/aids-focused ngo community in china. external factor: the reduction of international financial commitment china resembles some developing countries in that it has faced the problem of decreasing international hiv/ aids financial commitments in supporting its national hiv/aids responses [60] . given the 2008 global financial crisis and the continuous economic boom in china, international and bilateral funding agencies argue that china should be the donor instead of the recipient of international hiv/aids-specific grants and loans [52] . owing to a lack of international and national hiv/ aids funding, the number of ngos working on hiv/ aids was drastically reduced; many ngos and cbos were simply closed down, only a few of them could barely survive with the support by other funding sources (national ngo-2). the retreat of the global fund has subsequently created a policy vacuum for the chinese government in taking the lead in managing the development of hiv/ aids-related ngos in china. mimicking the funding mechanism of the global fund, the state council launched the china aids fund for non-governmental organizations (cafngo) (shehui zuzhi canyu aizibing fangzhi jijin), providing financial resources for hiv/ aids-related ngos via the submission of grant proposal starting from 2017 [61] . the reduction of the chinese government's reliance on the global fund implies that the hiv/aids-related responses delivered by the national government are no longer checked by the external mechanism. the authoritarian regime is probably the only agency defining/restricting the political space that ngos could work in. explaining the interaction between the chinese authorities and ngos, a university professor in beijing suggested: "in normal circumstances, local cdc staff hand over treatment delivery work to grassroots ngos. nonetheless, cdc officials intend to keep these ngos small, giving them just enough money to run the programs, so that they will not grow too strong to pose potential threats to the government" (academic-1). along with the weakening of international securitization efforts and the rise of chinese government's involvement in managing ngos in the post-global fund era, the continuous proliferation of ngos is further complicated by the fragmented nature of hiv/aids-focused civil society groups in china. it is argued that the existing ngo registration system, as previously mentioned, has brought about a fragmented ngo community in china. apparently, governmentorganized non-government organization (gongo) and registered ngos working on hiv/aids are included in hiv/aids national policy implementation processes, whereas excluding unregistered grassroots ngos that are perceived as illegal or "anti-government" organizations. based on the nature of services that individual hiv/ aids-focused ngos provided, service providers are preferred by the state, whereas ngos serving as advocates of human rights and agencies providing legal services for hiv/aids-infected people would be subjected to prosecution and coercion. a hiv/aids specialist working in an international ngo in beijing stated, "during a 2011 hiv/aids ngo meeting, the chinese government clearly claimed that the authorities would like to work with ngos/cbos conducting service delivery, but not with those working on hiv/aids-related human rights or gender issues" (ingo-1). to further illustrate this point, the chinese political leaders publicly praised a guangxi-based ngo named aids care china, recognizing the work the organization had been done in hiv/aids prevention, meanwhile stifling numbers of hiv/aids activists through imprisonment, house arrest, or assault. dr. wang yanhai and dr. gao yaojie were two of the prominent chinese hiv/aids activists that fled to the united states in 2009 and 2010, respectively, due to the recurrent pressure and disturbance exerted by chinese authorities owing to their advocacy work. in june 2015, a hiv/aids-related legal aid ngo, beijing yirenping, was raided, and two of its activists were detained. in this regard, the continuous inclusion/exclusion selection process would weaken unregistered or advocacy groups that conceived as threats to the legitimacy of the authoritarian regime [62] , while preserving ngos that are willing to under control by the state apparatus and prepared to accept the regime's policy measures. for the sake of reaching a modus vivendi, these ngos usually display self-limiting behaviors, avoiding actions that would be interpreted as threats to the regime [20] , such as having close relationship or cooperation with ngos being "excluded" by the state apparatus. while a formal local network for hiv/aids-related ngos or civil society groups has been established in many countries in asia, such coordination mechanism between non-state actors is largely absent in china. 13 the fragmentation of hiv/aids ngo community is likewise observed in the course of interview sessions. amongst the interviewees, the majority of the ngo respondents believed that cooperation dramatically increased between the government and ngos working on hiv/aids, especially in the areas of treatment, care, and prevention. only a few of them nevertheless stated that communication and coordination occurred amongst ngos in policy implementation. a national-wide ngo respondent claimed that "we seldom cooperate with other ngos or ingos; we have our own teams to do the work" (national ngo-3). some grassroots ngo respondents in shanghai and kunming expressed their unwillingness to cooperate with so-called "unauthentic" ngos or ngos having so-called "government background": few ngos have cooperative relationship. there are lots of "fake" ngos in china. the cdc found some patients to set up grassroots ngos and then through the name of such ngo to apply for the global fund. after they received the money, cdc would become the one who controlled the funding (self-help group-2). eighty-five percent of the hiv/aids-focused ngos in yunnan have government background…for those ngos that are funded by the global fund or china-bill gates projects, these are organizations with the government background…or if you read their introduction saying "with the help and support of government", you will know these are ngos with the government background (self-help group-3). a "we-they" distinction has apparently prevailed amongst the hiv/aids ngos community in china, reinforcing the atomization of hiv/aids-focused ngos, discouraging the proliferation of civil society in china. horizontal relationship amongst ngos working on hiv/aids is relatively weak and ill-organized, thereby avoiding these organizations joining hand to call for favorable political environment or monetary supports to continue their operation in china. owing to the reduction of international financial commitments, as well as the fragmentation of hiv/aids-focused ngo community in china, the proliferation of hiv/aids-focused ngos in china generated by the international securitization efforts is nevertheless largely limited in long run. such restriction on the concerted efforts of hiv/aids-related ngos will inevitably pose challenges to china to fulfill the pledge of the 2030 agenda to end hiv/aids as one of the targets of the sustainable development goals (sdgs) [63] . hiv/aids was officially announced as a security issue in 2000 by the unsc resolution 1308, which claimed that the pandemic could pose security threats if left unchecked. the rhetorical security-threat claim resulted in the burgeoning number of ghis organized by the international and bilateral organizations operating at the state level. this article illustrates the ways in which the unsc resolution 1308 and the global fund influencing the hiv/aids governance in china. rhetorically acknowledged the hiv/aids problems and the irreplaceable role of hiv/aids-focused ngos in combating the disease by the chinese government, the monetary and technical supports by the global fund enabled hiv/aids-focused ngos to participate in the national policymaking process, facilitating the proliferation and development of civil society in china. this success nevertheless does come with concerns regarding sustainability. this article shows that international securitization weakened with the rise of chinese commitment on hiv/aids interventions. in other words, hiv/aids-related responses delivered by the national government are no longer checked by the global mechanism of hiv/aids; thus it is unclear whether these ngos would remain of interest as partners for the government [64] . the fragmentation of the hiv/aids community would further hinder the development, preventing from ngos with the same interest forming alliances to call for changes in current political environment. such restriction on the concerted efforts of hiv/ aids-related ngos in china would make achievement of the sdgs to foster stronger partnerships between the government and civil society (goal 17) difficult, which in turn hindering the realization of ending hiv/aids in the world by 2030 (goal 3). this research has several limitations. firstly, there is a lack of available information regarding the operation of local or grassroots hiv/aids-specific ngos that are in particular helping marps in china, owing to the political sensitivity of hiv/aids, the unregistered status of ngos working on hiv/aids, the increasing restrictive environment for ngos operating in china especially under the xi administration, together with the social stigma attached to their service objects as well as the ngos themselves as the service providers. secondly, it is difficult to conduct longitudinal study on the development of hiv/aids-specific ngos in china. the researcher realized that many of the ngos that had been interviewed in 2011 were no longer under operation in the follow-up fieldwork in 2017, either because these ngos could not secure sustainable funding, or they were cracked down by local authorities for advocating hiv/aids-related human rights or gender equality, or having very close links to foreign (western) organizations. further research therefore needs to be conducted to remedy these technical weaknesses. it is suggested that comparative research could be conducted on the effects (if any) of the regime types, such as china and thailand, and the level of socio-economic development of the country, such as china and singapore, on the effects of international hiv/aids securitization in the national levels. endnotes 1 here ngos does not include government-organized non-governmental organizations (gongos). 2 referent objects refer to "things that are seen to be existentially threatened and that have a legitimate claim to survival." see buzan b et al., security: a new framework for analysis, p. 36. 3 "civil society" is often translated as "minjian shehui", "shiming shehui" and "gongming shehui" in chinese. in fact, the three different chinese terms do not have the same meaning. many scholars actually use these different chinese terms of civil society simultaneously. "minjian shehui" is used in this article because the term is widely used in the academic research in china's modern civil organizations. "civil society" in this article refers to a sphere that is conceptually separable from the state and government. 4 at the same time the state council abolished the jijin guanli banfa stipulated in 1988. 5 the chinese and english versions of the revised regulation (2016) can be found in chinalawinfo. "regulation on registration and administration of social organizations (2016) (shehui tuanti dengji guanli tiaoli). 6 nevertheless, grassroots ngos registered under the moic also encounter problems with regard to the amount and source of funding. as registered commercial entities, grassroots ngos are required to pay a 5 % tax on any revenue, even for funding received for non-profit purposes. 7 in recent years, the abovementioned legislative hurdle in ngo registration has been relaxed in some parts of china, first in shenzhen and then in beijing. the local government in these two cities relaxed its ngo registration rule, and allowed ngos in the fields of business, charity, welfare, and social services to register directly with the moca. such a move can also be observed at the national level. in early 2011, the national government started planning to revise the regulations on ngo registration and management. if the revised version is passed by the state council, the individual-organized or grassroots ngos can directly register with the moca without seeking a supervisory body prior to the registration. 8 it is noted that the author is fully aware of the arguments put forward by timothy hildebrandt, stating chinese ngos could be favored or cracked down by the state apparatus irrespective of registration status. the author is argued that unregistered ngos are comparatively easier to be targeted and cracked down by the government officials in the name of "unlawful organizations". for hildebrandt's argument, see hildebrandt, t. the political economy of social organization registration in china. the china quarterly. 2011;208:970-89. 9 gongos are composed of three types of organizations existed in the 1950s and 1960s: (1) private organizations from the "old china"; (2) friendship associations for the promotion of trade, and cultural exchange agencies; and (3) people's organizations and mass organizations. 10 in the course of the meeting, us vice president al gore claimed that hiv/aids was a security threat because "it threatens not just individual citizens, but the very institutions that define and defend the character of a society. the disease weakens workforces and saps economic strength. hiv/aids strikes at teachers, and denies education to their students. it strikes at the military, and subverts the forces of order and peacekeeping". 11 mcinnes and rushton (2010) argued that the hiv/ aids securitization in 2000 was not a successful or complete securitization. however, this article does not intend to examine whether the international hiv/aids securitization has been fully accepted by the target audience. it intends to examine the influences of the hiv/ aids international securitization on the national and sub-national levels in china. 12 in 2003, the bush administration launched the president's plan for emergency aids relief (pepfar), pledging us$15 billion from 2003 to 2008 to "turn the tide against hiv/aids." the who launched the "3 × 5" campaign that targets getting 3 million people on antiretroviral treatment by 2005, while the multi-country hiv/aids program of the world bank provided more than us$1.3 billion for grants and concessional loans to help governments respond to hiv/aids issues. 13 examples included the indian network of people living with hiv/aids, thai network of people living with hiv/aids, asia pacific council of aids service organization in malaysia, and also action for aids singapore, economic reform and growth in china routledge handbook of chinese security. london: routledge bric's growing share of global health spending and their diverging pathways the health care systems of china and india: performance and future challenges evolving health expenditure landscape of the brics nations and projections to 2025 rural china, a steep price of poverty: dying of aids: new york times unaids data china's pragmatic approach to aids hiv/aids in china and india: governing health security geopolitics in health: confronting obesity, aids, tuberculosis in the emerging brics economies unleash civil society in china to save lives. human rights watch twenty years of mobilizing around aids in china: the main actors and influences behind organizational growth discourse analysis as a way of analyzing naturally-occurring talk interpreting qualitative data: methods for analyzing talk, text and interaction security: a new framework for analysis. usa: lynne rienner publishers security, insecurity and asecurity in the west european non-war community civil society, corporatism and capitalism in china the civil society in china social organizations and the authoritarian state in china ngos in china: an overview. international community foundation is china's new overseas ngo management law sounding the death knell for civil society? maybe not china's evolving civil society: from environment to health china's rapidly growing non-governmental organizations. eai background brief no. 514 from spectators to implementers: civil society organizations involved in aids programs in china defining chinese nongovernmental organizations. volunt int j volunt nonprofit org globalization and its methodological discontents: contextualizing globalization through the study of hiv how is health a security issue? politics, responses and issues securitizing infectious disease should hiv/aids be securitized? the ethical dilemmas of linking hiv/ aids and security where are the links? council on foreign relations on the responsibility of the security council in the maintenance of international peace and security: hiv/aids and international peace-keeping operations. united nation security council united nations general assembly united nations general assembly political declaration on hiv/aids: intensifying our efforts to eliminate hiv/ aids. united nations general assembly united nations general assembly aids and security: where are we now? hiv/aids and securitization theory the global fund to fight aids, tuberculosis, and malaria china's evolving aids policy: the influence of global norms and transnational non-governmental organizations china among success stories in global hiv/aids fight: unaids chief. xinhua. 2015 peking daily cautions against western threats of aids, drugs: the associated press leexisnexis newspapers 46. ramiah i. securitizing the aids issue in asia gao qiang, at the hiv/aids high-level meeting of the un general assembly. permanent mission of the people's republic of china to the un state council notice on strengthening hiv/aids prevention and control. ncaids, china cdc 国务院关于进一步加强艾滋病防治工作的通知 [state council notice on further strengthening hiv/aids prevention and control.] 国发48号 [state council document no red ribbon forum redoubles aids fighting bid. china daily keqiang wants tax break for ngos specializing aids/hiv work. south china morning post china after the global fund investment in hiv/ aids programs: does it help strengthen health systems in developing countries? glob health the global fund: managing great expectations china country coordinating mechanism secretariats. the global fund to fight aids, tuberculosis, and malaria people's republic of china: national center for aids/std control and prevention aids funds frozen for china in grant dispute global fund lifts china grant freeze. the body china aids fund for non-governmental organizations making turkey's transformation possible: claiming "securityspeak"-not desecuritization! southeast european and black sea studies estimates of global, regional, and national incidence, prevalence, and mortality of hiv, 1980-2015: the global burden of disease study the political economy of social organization registration in china this work is supported by a hong kong university research council general research fund grant #144913. the author would like to thank 2 anonymous reviewers for their useful suggestions. the author would also like to express the appreciation to dr. nicholas thomas for his comments on an earlier version of the manuscript. all errors and omissions are the author's responsibility alone. the author declares that she has no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. the author read and approved the final manuscript. key: cord-339796-gccnvh0z authors: zhang, si min; jejcic, alenka; tam, james p.; vahlne, anders title: membrane-active sequences within gp41 membrane proximal external region (mper) modulate mper-containing peptidyl fusion inhibitor activity and the biosynthesis of hiv-1 structural proteins date: 2015-07-31 journal: plos one doi: 10.1371/journal.pone.0134851 sha: doc_id: 339796 cord_uid: gccnvh0z the membrane proximal external region (mper) is a highly conserved membrane-active region located at the juxtamembrane positions within class i viral fusion glycoproteins and essential for membrane fusion events during viral entry. the mper in the human immunodeficiency virus type i (hiv-1) envelope protein (env) interacts with the lipid bilayers through a cluster of tryptophan (trp) residues and a c-terminal cholesterol-interacting motif. the inclusion of the mper n-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-hiv peptidyl fusion inhibitors t20 and t1249. as a type i transmembrane protein, env also interacts with the cellular membranes during its biosynthesis and trafficking. here we investigated the roles of mper membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. we found that elimination of the membrane-active elements in mper peptides, namely, penta trp→alanine (ala) substitutions and the disruption of the c-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped hiv-1. furthermore, as compared to c-terminal dimerization, n-terminal dimerization of mper peptides and n-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. the secondary structure study revealed that the penta-trp→ala substitutions also increased the helical content in the mper sequence, which prompted us to study the biological relevance of such mutations in pre-fusion env. we observed that ala mutations of trp664, trp668 and trp670 in mper moderately lowered the intracellular and intraviral contents of env while significantly elevating the content of another viral structural protein, p55/gag and its derivative p24/capsid. the data suggest a role of the gp41 mper in the membrane-reactive events during both viral entry and budding, and provide insights into the future development of anti-viral therapeutics. the envelope protein (env) of human immunodeficiency virus type i (hiv-1) is a class i fusion glycoprotein [1] . it protrudes out of the viral envelope as homotrimers composed of non-covalently-linked gp120/gp41 heterodimers [2] [3] [4] . recognition of the viral receptor and co-receptor by the surface gp120 subunit activates the fusion machinery in the transmembrane (tm) gp41 subunit (fig 1) [5] [6] [7] [8] , resulting in the insertion of gp41 n-terminal fusion peptide region (fp) into the target cell membrane. this pre-fusion intermediate conformation of gp41 connects the cellular membrane and the viral envelope, exposing and extending the two heptad repeat (hr) regions, hr1 and hr2 [9] [10] [11] . the intermediate conformation quickly resolves into a stable six-helix bundle (6-hb) conformation, after hr2 folds back onto the central hr1 to form a coiled-coil trimer-of-dimers [12, 13] . this predisposes the opposing membranes into sufficient proximity for subsequent envelope fusion with the plasma membrane and viral content delivery [14] . the post-6-hb-formation lipid mixing and subsequent membrane fusion is mediated by the membrane proximal external region (mper) in gp41 (fig 1) , a hydrophobic region between hr2 and the tm domain [16, 17] . mper induces fusion-required membrane perturbation through a direct interaction with the membranes [18, 19] . sequential alignments have revealed a high conservation of mper among different groups of hiv-1 (fig 1) [15] . in particular, it contains two conserved sequence elements that contribute to its membrane perturbation function. one is an enrichment of aromatic amino acids, in particular trp (fig 1) , and the other is its cholesterol-interacting c-terminus. previous studies have shown that ala substitutions of the five trp residues abrogated the ability of mper-containing peptides to partition into and destabilize liposomal membranes [15] . sequences of the anti-hiv-1 first and second generation fusion inhibitor, t20 and t1498, respectively, are shown together with the mper-containing peptides tested in this study, ek37, el30, qk26, qt19, lk21 and lk21-5w5a, and all are aligned with the mper sequence. the mper sequence is highlighted in bold with its conserved residues shaded. peptide lk21-5w5a have all five tryptophan residues in mper sequence substituted by ala. [18, 19] . in the context of gp41, the trp!ala substitutions in mper inhibit membrane fusion events, such as the fusion pore expansion, during viral entry. [17] . furthermore, the mper cterminus (lwyik) shows sequence characteristics of the cholesterol recognition/interaction amino acid consensus (crac) motif,-l/v-(x)(1-5)-y-(x)(1-5)-r/k[20, 21] . cholesterol is enriched in viral envelopes and also in the cell membrane lipid rafts where viral receptors are enriched during viral entry, making the membranes rigid and counteracting viral entry [22, 23] . the c terminus of mper can facilitate the induction of membrane destabilization and subsequent fusion in the cholesterol-enriched liposomal membranes [24] [25] [26] . the membrane destabilizing ability of mper sequences has implication in designing viral fusion inhibitors. t20 and t1249, two of the first fusion inhibitors, are synthetic peptides containing sequences from both the hr2 and the n-terminus of mper (fig 1) [27, 28] . hr2 sequences enable these inhibitors to bind to the exposed hr1 of gp41 in the pre-fusion intermediate conformation, and thereby halt the formation of the 6-hb [9] . in addition, mper sequences act as inhibitors at a later stage of the viral entry, possibly through anchoring the peptide into the cellular membrane through their last four residues, wnwf [29] [30] [31] . the membrane anchoring abilities of the two fusion inhibitors correlate with their antiviral activity [32] . still, peptide fusion inhibitors, such as t20 and t1249, only included partial mper sequences without the c-terminal sequence [33] . due to the high variation and mutation rate of hiv-1 env protein, the fusion inhibitors were constantly challenged by drug resistance issues. the mper is a conserved, exposed and accessible region, and therefore it could be an additional target for the design of potential fusion inhibitors. apart from its role in viral entry, env also interacts with host cellular membranes during its biosynthesis and trafficking to the viral budding site. as a type i transmembrane protein, env is co-translationally translocated into the rough endoplasmic reticulum (er) and further transported to the golgi complex for maturation into gp160 and subsequent proteolytic processing [34, 35] . the resulting gp120/gp41 trimers are then transported to the cholesterol-rich plasma membrane regions (e.g. lipid rafts), following the secretory pathway [36] . studying sars-cov (severe respiratory syndrome associated corona virus) we observed that the trp residues in mper modulate the selective incorporation of the spike protein into lipid rafts (unpublished observations, see s1 and s2 figs). however, salzwedel and colleagues found that neither deletion nor trp substitution mutations in the hiv-1 mper affected env maturation, or steadystate levels, but had an effect on its incorporation into virus particles [17] . here we describe the roles of the trp residues in the membrane-active mper sequence in anti-hiv fusion inhibitor design and a surprising role in the biosynthesis of viral structural proteins. six peptides ranging from 19 to 37 amino acids (a.a.) were designed to contain the mper sequence in its full length, with c-terminal truncation, or with penta trp!ala substitution (fig 1) . their anti-viral activities were tested in a single-round infectivity assay using pseudovirus. dimerization of anti-viral peptides has been shown to enhance both their structural stability and the number of interaction sites and thus their anti-viral efficacy [37] [38] [39] [40] . therefore, we also tested our peptides as n-or c-terminal dimers. hiv-1 env with mutations of trp residues at the mper region were also constructed to examine the roles of the trp residues in the biosynthesis, maturation, trafficking, and viral incorporation of the viral structural proteins. hiv-1 gp41-derived monomeric peptides were custom synthesized by synpeptide co ltd (shanghai, china) and the dimeric peptides by pepscan presto bv (amsterdam, netherlands). the antibody to gp160/41 (chessie 8) and vif (#319) were obtained through the nih aids research and reference reagent program [41] . polyclonal rabbit anti-nef antibody was obtained from thermo fisher scientific, inc. monoclonal mouse anti-β-actin antibody was obtained from sigma. the antibody to p24 (ef7), has previously been described [42] . the secondary antibodies, hrp-conjugated polyclonal goat anti-rabbit immunoglobulins and hrpconjugated polyclonal rabbit anti-mouse immunoglobulins, were obtained from dako. hltat (cat. #1293) and tzm-bl (cat. #8129) cell lines were obtained through nih aids research and reference reagent program [43] [44] [45] [46] [47] and cultured in dulbecco's modified eagle medium (life technologies) supplemented with 10% fetal bovine serum (life technologies) and 1% penicillin-streptomycin (life technologies). the expression plasmid for env, nef (negative regulator factor) and vpu (virion protein u) from the hiv-1 strain nl4-3 (pnl1.5eu+) was kindly provided by dr. stefan schwartz (lund university, lund, sweden) [48] . pnlhivxδuδss contains a vpu-deficient hiv-1 genome with the signal sequence of env truncated and was constructed from pnl4-3 with quick change ii xl (stratagene) [49] . specifically, the fragment ecori-bamhi from pnl4-3 was cloned into puc18 to create puc18envxδu that has the vpu start codon mutated to ata and contains a xma i cleavage site immediately upstream of vpu, using the primer pair b(xδu): (forward) 5'-gcagtaagtagtacccggga tacaacctataatagtagcaatag-3', (reverse) 5'-ctattgctactattataggttg tatcccggtactacttactgc-3'. the same mutation was introduced into δnss-gp160 [50] , encoding vpu and a gp160 with a signal peptide of 11 a.a. length, using the primer pair a (xδu): (forward) 5'-gaagcgcgcacggagtacccgggatacaacctataatagtagc-3', (reverse) 5'-gctactattataggttgtatcccgggtactccgtgcgcgcttc-3'. the xmai-nhei fragment of the constructed mutant δnss-gp160 was then directionally inserted into pucenvxδu to create pucenvxδuss11, in which site-directed mutagenesis was carried out with primer pair(δss): (forward) 5'-ggatattgataatctgtagtgctatggaaaaattgtgg tc-3', (reverse) 5'-gacccacaatttttccatagcactacagattatcaatatcc-3' to introduce gp160 signal sequence mutation. the ecori-nhei fragment of the resulted mutant construct was then subcloned into p83-10 [51] , of which the saii-ncoi fragment was directionally transferred into pnl4-3, creating pnlhivxδuδss. pnl1.5eu+w5a, pnl1.5eu+w3a and pnl1.5+w2a were constructed from pnl1.5eu+, using quick change ii (agilent technologies). pnl1.5eu+w2a was constructed using the primer pair a(w676.678): (forward) 5'-gcaagtttgtggaattggtttaacataacaaa tgcgctggcgtatataaaattattcataatgatagtaggagg-3', (reverse) 5'-cc tcctactatcattatgaataattttatatacgccagcgcatttgttatgttaaacc aattccacaaacttgc -3'. pnl1.5eu+w3a was constructed using the primer pair b (w664.668.670): (forward) 5'-gaatgaacaagaattattggaattagataaagcggca agtttggcgaatgcgtttaacataacaaattggctgtggta -3', (reverse) 5'-tacc acaccacatttgttatgttaaacgcattcgccaaacttgccgctttatctaattc caataattcttgttcattc -3'. pnl1.5eu+w5a was constructed from pnl1.5eu+w3a using primer pair a (w676.678). the mutated pnl1.5eu+ plasmids were sequenced between nucleotide 7988 to 8533 with the sequencing primer (7988f-ctcctggggatttggggttg and 8533r-gtctctcaagcggtggtagc). hltat cells (5 x 10 5 ) were co-transfected with the hiv-1 proviral clone pnlhivxδuδss, and with the env-and vpu-expressing plasmid pnl1.5eu+, using fugenehd (roche). for the production of mutant pseudotyped hiv-1, we used pnl1.5eu+w5a, pnl1.5eu+w3a or pnl1.5eu+w2a during transfection, instead of pnl1.5eu+. the cell culture supernatant was collected 48 hours (h) post-transfection, clarified by centrifugation and 0.45-μm filtration, used for virion precipitation or stored at -80°c until further use. viral particles were precipitated from the clarified and 0.45μm-filtered cell culture supernatant at 4°c for 48h in 1:6 (vol:vol) polyethylene glycol 6000 containing 0.667m nacl. the precipitated particles were further concentrated by centrifugation at 16,000 times g for 20 min at 4°c. the virus pellets were dissolved in radio-immunoprecipitation assay (ripa) buffer containing 50 mm tris-hcl (ph = 7.4), 1% triton x-100, 1% deoxycholate, 150 mm nacl, 1 mm edta, and 0.1% sodium dodecyl sulfate (sds) and supplemented with complete protease inhibitor cocktail (roche). cell-free pseudotyped hiv-1 virus of 100tcid50 was either pre-incubated with peptides of various concentrations at 37°c for 1 h, or applied directly to infect 10 4 cd4+, ccr5+ tzm-bl cells. tzm-bl cells contain integrated copies of an ltr (long terminal repeats)-driven luciferase reporter gene. seventy-two h post-infection, the infectivity of pseudovirus was assessed as luciferase activity, using the one-glo luciferase assay system (promega). the tcid50 of pseudotyped hiv-1 was calculated based on the luciferase activity of the infected tzm-bl cells, using the reed-muench method and the cut-off value set at 3 times of the background signal [52] . effective concentrations of peptides inhibiting 50% (ic 50 ) and 80% (ic 80 ) of viral infectivity were estimated with graphpad prism. the ic50 and ic80 values were estimated from the dose-response curves that were curve-fitted with the sigmoidal dose-response non-linear regression model on prism graphpad software, using the percentage of inhibition data and the log values of peptide concentrations. the cytotoxicity effect of the peptides on tzm-bl cells were determined by prestoblue cell viability assay (lifetechnologies, singapore), according to the manufacturer's protocol. briefly, peptides of different concentrations were added to 10,000 tzm-bl cells seeded in 96-well plates. upon 24 h of incubation, prestoblue cell viability reagent was added to the cells and incubated for 30 min at 37°c. resulting absorbance values were recorded at 570nm and 600nm (baseline). final spectrum was obtained by normalizing the 570 nm values to the 600 nm values. circular dichroism spectroscopy analysis was performed to study the secondary structure of the monomeric peptides in trifluoroethanol (tfe). tfe was used to mimic the hydrophobic environment at the membrane fusion junction. the measurements were made on chirascan circular dichroism spectrometer (applied photophysics). fifty μm peptide was dissolved in 10%, 20%, or 40% tfe and subjected to the measurement with three repeats in a cell of 0.1mm pathlength (hellma uk ltd.) at 25°c. samples were measured between 190nm and 260nm, with a 0.5nm step resolution, a measurement speed of 60nm/min and a 1nm bandwidth. the baseline was measured with 10% tfe, with three repeats. the final spectrum was generated by subtracting the averaged sample spectrum with the baseline spectrum, followed by smoothing with a savitsky-golay filter. the secondary structures of the peptides were estimated from deconvoluting the respective circular dichroism spectra using the cdsstr deconvolution algorithm on dichroweb, with a cut-off nrmsd value set at 0.15 [53] [54] [55] . hltat cells (5 x 10 5 ) expressing wt or mutant pseudotyped hiv-1 were lysed in ripa buffer on ice, centrifuged and mixed with laemmli reducing buffer. precipitated pseudotyped hiv-1 viral particles dissolved in ripa buffer were also prepared in laemmli reducing buffer. cell lysates or virus lysates were resolved by sds-polyacrylamide gel electrophoresis (page), transferred to nitro-cellulous membranes and immunoblotted with anti-gp41 antisera and followed by hrp-conjugated anti-rabbit secondary antibody for env expression, or with anti-p24 antisera and followed by hrp-conjugated anti-rabbit secondary antibody for p24 and p55/gag expression, or with anti-nef antibody and followed by hrp-conjugated anti-rabbit secondary antibody for nef expression, or with anti-vif and followed by hrp-conjugated anti-mouse secondary antibody for vif expression, or with anti-β-actin and followed by hrp-conjugated anti-mouse secondary antibody for β-actin expression. the membranes were either exposed to film or analyzed with g:box chemi xx6 (syngene). the band intensities were quantified by imagej software. p24 levels in the cell-free lysate of the virus-producing hltat cells or the cell culture supernatants were quantified by the automated system architect (abbott). a standard curve was generated using p24 of known concentration and curve-fitted with the linear non-regression model on prism graphpad software. prior to each measurement, the samples were diluted to the concentrations within the linear range of the standard curve. six mper-containing peptides (fig 1) ranging from 19 to 39 a.a were prepared with acetylated n-termini and amidated c-termini and were tested as fusion/entry inhibitors against pseudotyped hiv-1(nl4-3). to ensure study the early events of the hiv-1 viral replication cycle a hiv-1 pseudovirus system allowing only a single replication cycle was employed. pseudovirus particles were produced by the co-transfection of the env-and vpu-expression-deficient proviral vector pnlhivδuδss and the env-, vpu-and nef-expressing vector pnl1.5eu+ [48] , which generates viral particles capable of entering and infecting target cells but not capable of giving rise to infectious second-generation viral particles. the pseudotyped hiv-1 (nl4-3) particles were pre-incubated with each of the six mper-containing peptides for 1 h and then added to the target cells, tzm-bl cells, which stably express a tat-responsive luciferase reporter gene allowing for the monitoring of successful hiv-1 entry and viral protein production. the inhibition of infection was monitored by measuring the luciferase expression at 72 h post-infection. peptide lk21, which contained the entire and exclusively the mper sequence, inhibited 50% and 80% of viral entry and infection at 8.0 μm and 12.3 μm, respectively ( fig 2a) . inclusion of five hr2 residues to lk21 n-terminus, generating the 26-a.a. peptide qk26, decreased the ic 50 and ic 80 values to 3.9 μm and 8.8 μm, respectively. however, further n-terminal extension with the addition of nine and sixteen hr2 residues to lk21, resulting in peptides ek30 and ek37, did not lead to any substantial increase in their anti-viral potency. however, adding the hr2 hydrophilic residues greatly enhanced the solubility and structural stability of the mper-containing peptides qk26, ek30 and ek37, allowing further application and design, such as peptide dimerization that will be elaborated on in section 2. concentrations of the peptides yielding a 50% and 80% reduction in luciferase activity were estimated with graphpad prism. results shown were summarized from three independent experiments with serial dilutions of peptides in replicates of two. b. no cytotoxicity effect was observed for the mper-derived peptides at 50 μm in tzm-bl cells. fifty μm of the peptides were incubated with 10,000 vero cells for 24 h. prestoblue cell viability reagent was subsequently added to the cells, and cytotoxicity effects were monitored as absorbance values (od) at 570 nm and 600 nm (baseline). c. circular dichroism spectra and the estimated secondary structure contents of the peptide lk21, qk26, ek30, ek37 and qt19 in 10% tfe. fifty μm of the peptides were dissolved in h 2 o supplemented with 10% tfe and were subjected to circular dichroism spectroscopy measurement. d. circular dichroism spectra and the estimated secondary structure contents of peptide lk21 in increasing concentrations of tfe. fifty μm of the peptides were dissolved in h 2 o supplemented with 10%, 20% or 40% tfe and were subjected to circular dichroism measurement. e. circular dichroism spectra and the estimated secondary structure contents of peptide lk21-5w5a in increasing concentrations of tfe, measured as described in c. to determine the necessity of the conserved mper c-terminal cholesterol-interacting motif (lwyik) for the antiviral effect of the peptides, the mper c-terminal sequence, nwlwyik, was deleted from the most active peptide, qk26, creating the peptide qt19. this deletion abrogated the antiviral activity, as qt19 failed to inhibit viral infection at concentration up to 33.3μm (fig 2a) . in addition, the importance of the enriched aromatic residue trp in the mper region was examined by mutating all the trp to ala in the exclusively mper-containing peptide, lk21, resulting in the peptide lk21-5w5a. interestingly, the substitutions of all the trp did not just abolish the antiviral activity of the peptide, but even enhanced viral infectivity in a dose-dependent manner (fig 2a) . at 26.6 μm, the lk21-5w5a increased the viral infectivity by 50%. the inhibitions of viral infectivity by the mper-derived peptides were not due to cytotoxicity, as incubating tzm-bl cells with 50 μm of the peptides for 24 h did not result in any statistical difference in cell viability between the control (dmso-treated) and the peptide-treated cells (fig 2b) . to investigate the structure-function relationship of mper-containing peptides in inhibiting the entry of pseudo-hiv-1, the secondary structures of the peptides were determined by circular dichroism (fig 2c) . the hr2 region of another class i viral fusion glycoprotein, the spike (s) protein of the severe acute respiratory syndrome associated coronavirus (sars-cov), has previously been shown to be largely α-helical and a synthetic peptide (hr2-s) derived from this region served as a control peptide in the following cd study [56] . the peptides were prepared in 10% tfe that mimics the lipidic environment at the juxtamembrane junction. the n-terminal extension of lk21 with hr2-derived residues generally increased the helicity in the mper-containing peptides. deleting the c-terminal sequence, nwlwyik, from qk26 did not change the secondary structure drastically (fig 2c) . to mimic the increasingly lipidic environment transition, which the mper undergoes during membrane fusion, the tfe concentration was increased from 10%, 20% and to 40%. with increasing tfe concentrations, lk21 gradually exhibited a more alpha-helical conformation, with the first minimum of its spectra shifted from 212 nm to 209 nm, and then to 208 nm, and the estimated α-helical content increased from 41% to 52% and then to 71% (fig 2d) . in contrast, lk21-5w5a, the peptide with all the trp replaced by ala, exhibited canonical alpha-helical spectra and a high α-helical content (84%) starting from 20% tfe (fig 2e) , which indicates the importance of trp residues in maintaining the structural plasticity of the mper sequence. we next investigated if the antiviral effect of our peptides could be enhanced by dimerization at either the n-or c-terminus. we hypothesized that n-terminally dimerized mper-containing peptides would mimic the fusion-active oligomerization state of gp41 mper, thus having an enhanced binding affinity with their interaction partners (e.g. fp), and thereby possess an improved viral inhibitory effect. dimeric peptides were constructed with parallel peptide chains with either two carboxylic termini or two amino termini using chemoselective ligation strategy [57] . to obtain the nterminal linked dimers (-dn), monomeric peptides were synthesized with an additional n-terminal cys residue, which was further ligated via a thiazolidine linkage to a linker molecule consisting of two ser branching from lys (fig 3) . the c-terminal peptide dimers (-dc) were synthesized on mbha resins, and ligated c-terminally to the linker via the amino functional groups on the linker (fig 3) . n-and c-terminal dimers were synthesized from the peptide ek30 and ek37. c-terminally linked qk26 dimer was also synthesized, but due to synthetic difficulties, its n-terminal dimer was not obtained. the dimeric peptides were tested for their ability to inhibit viral entry using the single-round infectivity assay with tzm-bl cells as described above for the monomeric peptides (fig 4a-4d) . the n-terminal dimerization of ek37 and ek30 enhanced their anti-viral potencies (fig 4b and 4c) . the ek37-dn and ek30-dn have ic 50 values of 1.2 μm and 1.1 μm, an increase of the potency by 5.2-and 1.9-fold compared to the monomeric ek30 and ek37, respectively (fig 4d) . in contrast, the c-terminal dimerization decreased the potency of ek37 and qk26, with ic 50 values of ek37-dc and qk26-dc elevated by 1.7 and 1.3 fold, respectively ( fig 4d) . meanwhile, the c-terminal dimerization had inconsistent effect on the antiviral potency of the ek30-dc, its ic50 value decreased and ic80 value significantly increased with respect to its monomeric form (fig 4d) . in summary these data indicate that the anti-viral activity is benefited by n-terminal dimerization of mper-containing peptides. no cytotoxicity effect of the monomeric nor dimeric peptides was observed in vitro in tzm-bl cells at concentrations up to 100 μm, as determined by the prestoblue cell viability assay (fig 4e) . our circular dichroism data suggest that the penta-trp!ala substitutions induces the mper peptide to commit to a predominantly helical structure regardless of the environmental lipidity fig 2e) . this poses the possibility that the same mutations may also affect the secondary structure of the mper sequence within the hiv-1 precursor env glycoprotein, gp160, which in turn may disturb its proper folding in the er, or affect its biosynthesis in other ways and eventually lead to viral defect. to investigate this, site-directed mutagenesis was performed on envexpressing plasmid pnl1.5eu+ [48] and generated three gp160 mutants, where all five trp, the three n-terminal trp (w664, w668 and w670), or the two c-terminal trp (w676 and w678) in the mper sequence were substituted with ala. the resulting constructs were termed w5a, w3a, and w2a, respectively. to examine the effect of the trp substitutions on env expression and viral maturation in the context of virus-producing cells and budding viral particles, hltat cells were co-transfected with the env-and vpu-expression-deficient proviral vector pnlhivxδuδss (described above) and with the wt or mutant pnl1.5eu+ to produce pseudo-typed hiv-1 (nl4-3) viral particles containing wt, w5a, w3a or w2a env. accordingly, the expressed pseudo-hiv-1 particles were termed hiv(wt), hiv(w5a), hiv(w3a) or hiv(w2a). at 48 h post-transfection, it was found that the env levels in cell lysates containing hiv(w5a), hiv(w3a) or hiv (w2a) were lower than that in lysate containing the hiv(wt) (fig 5b) , with the steady-state intracellular gp160 levels in cells producing hiv(w5a), hiv(w3a), hiv(w2a) were approximately 60% of that in hiv(wt) (p = 0.038 for w5a, p = 0.020 for w3a, p = 0.026 for w2a, n = 5) (fig 5c) . more striking effects of trp mutations were observed in the other viral structural proteins, specifically p55/gag and its derivative proteins. the hiv-1 genome encodes three structural proteins, which are the precursor protein env, p55/gag, and gag-pol. while env is translated into the er and is transported to the site of viral assembly at the plasma membrane via the secretory pathway, the p55/gag and gag-pol are expressed in the cytosol. the two viral cytosolic precursor structural proteins meet up with env at the plasma membrane where p55/gag directly or indirectly interacts with cytosolic tail of env to recruit it into the assembling viral particles [36] . during or after viral budding, the viral protease within gag-pol is activated leading to processing of gag-pol and p55/gag. among the p55/gag-derived proteins is the capsid protein, p24, which is commonly used to detect viral particle production [58, 59] . analysis of the p24 levels in cells producing wt or mutant pseudo-hiv-1 revealed that at 48 h post-transfection p24 in cells producing hiv(w3a) was approximately 45% (p<0.0001, n = 7) higher as compared to p24 in cells producing hiv(wt) (fig 5a) . however, the other two mutants, hiv(w5a) and hiv(w2a), showed no increased levels of intracellular p24 (fig 5a) . the analysis was performed using the automated system archiect (abbot) accreditated for detection of p24 in human serum. we also performed in-house validation experiments with hiv-1 infected cells in the presence of the protease inhibitor indinavir to confirm that the system did not detect the p24 within p55/gag but only the mature p24 once processed from its precursor protein. the corresponding cell lysates were therefore subjected to analysis by western blot and the findings clearly confirmed the elevated intracellular levels of p24 in the hiv(w3a) as compared to the hiv(wt) (fig 5b) . two other viral proteins, vif and nef were blotted and served as control for the transfection efficiency to which p55/gag and p24 expression was standardized. the results further indicated that the intracellular elevation of p24 by 93% (p = 0.01, n = 5) was a result of increased expression of p55/gag, as the p55/gag levels were 330% in the hiv(w3a) mutant as compared to that in the hiv(wt) (p = 0.007, n = 5) (fig 5b and 5c) . the extracellular p24 levels in the hiv(w3a)-expressing cultures, analyzed by the automated system archiect (abbot), were further found to be 67% higher than those of the hiv(wt) (p<0.0001, n = 7), consistent with the increased intracellular p24 expression levels from this mutant (fig 5d) . a smaller increase by 10% of extracellular p24 levels were detected in the hiv(w5a) (p = 0.0378, n = 7) expressing cultures (fig 5d) . in addition, the viral particles were isolated from equal volumes of the respective cell culture supernatants and subjected to analysis for env, p55/gag and p24 by western blot followed by densitometric analysis. as compared to that in hiv(wt), there was moderate decrease in env level among the mutant viral particles. in contrast, p55/gag and p24 contents in isolated hiv(w3a) virus particle were elevated by approximately 200% and 310%, respectively (fig 5e and 5f ), which is in line with elevated p24 levels detected in both the cell culture lysates and supernatants by the automated system, architect. it further indicates an increased hiv(w3a) viral particle production as were produced by co-transfecting hltat cells with pnlhivxδuδss and pnl1.5eu+, pnl1.5eu+w5a, pnl1.5eu+w3a, or pnl1.5eu+w2a, respectively. the p24 levels (ng) in cell lysates were quantified by the automated system architect (abbott). ***p < 0.001 as compared to wt by the unpaired student's t test. b. steady-state intracellular levels of viral proteins in pseudovirus-producing hltat cells. hltat cells from a were harvested 48 h post-transfection and the lysates were resolved by sds-page and immunoblotted with antibodies against gp41, p24, vif, nef and β-actin. vif and nef expression served as the transfection control. un-transfected hltat cells served as negative control. c. densitometric analysis of protein bands in blots from two independent experiments as described in in b was performed in imagej and presented as means ± sd, with gp160, p55/gag, and p24 levels in hiv(wt) standardized to 100%. *p < 0.05; **p < 0.01 as compared to wt by the unpaired student's t test. d. p24 levels (ng) in the culture supernatants of pseudovirus-producing hltat cells. p24 levels in the culture supernatant of hltat cells in a was quantified by the automated system architect (abbott). ****p < 0.0001 as compared to wt by the unpaired student's t test. e. env gp41, p55/gag and p24 levels in precipitated hiv(wt), hiv (w5a), hiv(w3a) and hiv(w2a). viral particles from the cell culture supernatant from a were precipitated, lysed, separated by sds-page and immunoblotted with antibodies against gp41 and p24. f densitometric analysis of the blot in e was performed in imagej and presented as means, with gp41, p55/gag and p24 a result of increased intracellular p55/gag expression. similar increase of p55/gag and p24 contents was observed in the hiv(w2a) particle isolate (fig 5e and 5f ). the influence of the trp substitutions to ala in the mper on viral entry and infectivity was also tested by adding equal volumes of the respective cell free culture supernatant to the tzmbl cells. forty-eight hours post-infection, the tat-activated expression of luciferase in the tzm-bl cells were measured and showed that in contrast to the hiv(wt), all the mutant particles hiv(w5a), hiv(w3a) and hiv(w2a) lost their ability to infect the tzm-bl cells (fig 5g) . these data collectively suggest that, while substitutions of the trp to ala in the mper sequence of gp160 decreased its intracellular expression levels and consequently moderately reduced the env incorporation into the viral particles, the mutations significantly influenced the intracellular expression levels of p55/gag. in particular, the substitutions of the trp664, trp668 and trp670 in the mper significantly elevated the intracellular p55/gag expression and subsequently the viral particle production. furthermore, the substitutions of the trp to ala rendered the viral particles non-infectious, in accordance with the previous literature [17] . the gp41 mper (hiv-1 nl4-3 : ldkwaslwnwfnitnwlwyik) induces membranefusion-required membrane perturbation in the viral envelope and cellular membranes, through its two conserved membrane-active sequence elements; the enrichment of aromatic residues (e.g. trp) and a c-terminal cholesterol interacting motif (lwyik) [17] . here we show that the membrane active sequential elements of gp41 mper are vital for mper-containing short peptidyl fusion inhibitors, as the omission of the c-terminal motif (lwyik) and the penta-trp!ala substitution abrogated their anti-hiv-1 activity. the peptide anti-viral activity could be enhanced through n-dimerization, but not c-dimerization. in this study, peptide lk21, containing the entire mper sequence, inhibited pseudotyped hiv-1(nl4-3) entry and infection with the ic 50 values at 8.0 μm. n-terminal extension of lk21 with five amphipathic residues derived from hr1-biding region of hr2 (628 a.a.-666 a. a.) enhanced the anti-viral effect and reduced the ic 50 by half. further n-terminal extension of lk21 did not enhance its anti-viral effect substantially, probably because this sequence does not include enough residues to mediate a stable interaction between the peptide and the viral hr1 as 6-hb formation. instead, the enhancement of the anti-viral efficacy through addition of five amphipathic residues correlated with the subtle increase in the helicity of the resulting peptides while maintaining the general structural profiles, as estimated from the respective circular dichroism spectrum. in the context of gp41, this amphipathic sequence upstream to the mper serves as an extension into hr2 and induces the n-terminus of mper in fusogenic gp41 to transform from an extended conformation to a helix upon increases in local lipidity [60] . this suggests that the addition of five residues of the n-terminal amphipathic sequence confers the peptides a more stable conformation and a capacity to interact with hr1, which lead to their enhanced anti-viral efficacy. the formation of 6-hb has been suggested to induce not only a secondary structural transformation in the mper n-terminus, but also to result in its quaternary structural rearrangement and oligomerization [60, 61] . meanwhile, the mper c-termini of the neighboring gp41 hiv-1 env mper in anti-viral design and viral life cycle stay monomeric and assume an extended platform to destabilize the cholesterol-enriched lipid bilayers, likely through its crac motif (lwyik) [24] [25] [26] . our data show that deletion of the c-terminal sequence including the conserved crac motif in the mper-containing peptide qk26 abrogated its anti-viral effect, suggesting that this membrane-active sequence plays an essential role in the anti-viral mechanism of the peptide. the peptide dimerization data further support that the inclusion of free c-termini for membrane interaction in the mper peptides are important for their inhibition of hiv-1 entry, as constraining the peptide c-termini through c-terminal resulted in the anti-viral effects of peptide ek37 and qk26 unenhanced. a previous study by nomura et.al also observed that the c-terminal trimerization of t20 failed to enhance its anti-viral efficacy significantly, presumably because the trimerization constrains its membrane-active mper sequence, offsetting the activity enhancement due to the potential cooperative interaction between its hr2 sequences and the viral gp41 hr1 [38, 62] . the enrichment of trp residues in mper is a second important membrane-active characteristic exhibited by class i fusion glycoproteins [63] . trp contains a large indole-ring side-chain that is preferred by the juxtamembrane interface of proteins, facilitating protein-membrane interaction and stabilizing protein structure [64] [65] [66] . in this study, we examined the influence of trp residues in the design of fusion inhibitors by substituting the indole-ring side-chains of the five trp residues in lk21 with the alkyl moieties of ala. surprisingly, the resulted lk21-w5a peptide promoted viral infectivity rather than inhibiting it, a phenotype that has been also observed when ala-substituting the trp residues of the sars-cov mper peptide [67] . the data indicate the significance of membrane-active elements in mper-containing peptides, both the crac motif and trp residues, for the inhibition of hiv-1 entry. this is in agreement with previous findings that increased membrane reactivity owing to inclusion of the mper n-terminal sequence (ldkwaslwnwf) as in t20 and t1249, correlated with an enhanced anti-viral effect of the peptides [29] [30] [31] [32] . of note, our data further demonstrate the involvement of the cterminal crac motif (lwyik) in facilitating viral inhibition for short mper-containing peptides with minimal inclusion of hr2 sequences. aside from its involvement in membrane fusion events, trp residues have also been shown to modulate the interaction between mper and other viral domain(s) during viral entry. specifically, gp41 mper interacts with fp to form a continuous hydrophobic track along with the 6-hb and promote membrane juxta-positioning [68, 69] . shortly before the interaction stabilizes and during the transition from the pre-fusion to post-fusion conformation, the mper and/or fp could be temporarily exposed and vulnerable to dominant-negative binding by peptidyl fusion inhibitors, such as lk21 of this study. we have recently shown that mper in the sars-cov spike protein interact with the internal fp (ifp) in a trp-dependent manner [67] . in the same study, trp!ala substitutions also resulted in mper-containing peptides to lose the dose-dependent inhibition of coronavirus entry, correlating with the disruption of the mper-ifp interaction. in gp41, trp670 has previously been shown to mediate the mper-fp interaction [70] . its ala substitution in the peptide lk21-5w5a could lead to a diminished affinity between the peptide and the fp in gp41, and hence contribute to the loss of the antiviral effect of the peptide. furthermore, penta-trp!ala substitutions in lk21 prematurely predisposes the peptide to the final helical conformation, losing the lipidity-induced structural plasticity. it suggests that the potential interactions between mper and other viral regions (such as fp) could also be conformation-dependent, and that the transition of mper from an extended to helical conformation could result in an intermediate species for such interaction to take place. whilst the c-terminal dimerization failed to enhance the anti-viral efficacy of peptides studied here, n-terminal dimerization lowered their ic 50 and ic 80 values up to 5-fold. our data would suggest that n-terminally dimerized mper-containing peptides mimic the fusion-active oligomerization state of gp41 mper, thus enhancing binding affinity with their interaction partners (e.g. fp and membrane), thereby having an improved anti-viral effect. the optimal length of the linker between the monomers could be explored to enhance the flexibility of the unit peptide, which may further enhance the cooperative interactions between the peptide multimer and gp41. the differential effects of n-and c-dimerization on the anti-viral effects of mper-containing peptides have been previously observed in our group, with sars-cov as the model virus ( s3 fig). n-and c-terminally dimerized peptides containing the s protein mper sequence were prepared as described in this paper, and enhanced and inhibited, respectively, the anti-viral effects against pseudotyped sars-cov compared to the monomeric peptide. the hiv-1 mper contains neighboring epitopes for broadly neutralizing antibodies, including 662-dkwa-665 for antibody 2f5 and 669-nwfnit-674 for antibody 4e10 [71, 72] . both antibodies have been shown to neutralize different strains of primary isolates of hiv-1 when administrated in cocktails in animal models [73] . despite being proven difficult, a tetramer peptide mimetic containing the mper sequence has elicited broadly neutralizing antibodies 4e10 and 2f5 in guinea pigs [74] . this rises the potential immunogenicity problem with the mper-derived anti-viral peptides. however, any such immune responses is not expected to induce major adverse effects in host, as any elicited anti-hiv-mper antibodies could probably be immunologically tolerated by the host, and may even further help to control the hiv replication. the direct evidence of the host immunological tolerance of 2f5 and 4e10 has been provided their recognition of two autoantigens, human kynureninase and splicing factor 3b subunit 3 [75] . furthermore, t20 which contains the epitopes for 2f5 and 4e10 has not only been clinically proven to be safe and effective in the presence of cross-reactive antibodies [76] , it has been shown to act synergistically with 4e10 in inhibiting viral infectivity [72] . in addition, the neutralizing capacities of the antibodies 2f5 and 4e10 require functional env trimers and probably would not neutralize the mper-derived peptides' anti-viral effect [73] . nevertheless, at the prospect of developing mper-derived peptidyl entry inhibitors, any potential elicited immunological responses should be examined and investigated [77] . finally, the peptides should be tested with different subtypes of hiv-1 to confirm the antiviral activity. env interacts with host membranes during both fusion/entry, and its biosynthesis and trafficking during viral budding. the substitution of all five trp to ala in the mper antiviral peptide resulted in it predominantly adopting a helical structure regardless of the environmental lipidity. this suggest that the trp residues could be equally vital for the secondary structure of the mper region within gp160, and consequently their substitution could hamper a proper folding and function of gp160. we further investigated the biological relevance of the mper trp residues in the biosynthesis of env and other viral proteins. previously, salzwedel et al. found that trp mutations in the mper affected incorporation of env into virions but no effect of the mutations was seen on the env levels in the cell lysate or on the plasma membranes [17] . here we observed that, in the transfected hltat cells expressing pseudotyped hiv-1, ala substitutions of all five trp, the n-terminal three trp (w664, w668, w670), and the c-terminal two trp (w676 and w678) residues, although moderately, lowered the steady-state intracellular gp160 and intraviral gp41 levels without affecting the migration patterns as compared to wt. the discrepancy between their and our findings may be due to differences in the assay used to quantify env, immunoprecipitation using patient serum versus immunoblotting. more interestingly, we observed that the mutations in env up-regulated the expression of the capsid protein p24 through up-regulating its precursor protein p55/gag, despite differential biosynthesis pathways between p55/gag and env. the results generally agreed with the previous understandings that env expression inhibits the steady-state intracellular level of p55/gag. it has been shown that the downregulation of p55/gag expression by env could be executed at both protein level through the env cytoplasmic tail, or at rna level through actions via the rev response element within the env gene [78, 79] . in this study, the upregulation of gag/p55 protein intracellular and intraviral levels were not proportional to the reduction of env protein levels and interestingly the pseudo-hiv-1 in which the n-terminal three trp or the c-terminal two trp were substituted gave a much higher increase of p55/gag than the pseudo-hiv-1 with all five trp replaced with an ala. hence it remains an open question if there is another distinctive mechanism through which the trp!ala mutations in env mper upregulated the gag expression, besides through lowering the intracellular env levels. while env is responsible for receptor/co-receptor recognition, membrane fusion and viral entry; p55/gag can independently induce the assembly and budding of virus-like particles in living cells and in vitro. its derivative protein, p24, dictates the proper maturation, size and morphology of the budding virions, which are essential for viral infectivity [80] . our data indicate that the trp residues in env mper are important for the biosynthesis of env and another major viral structural proteins p55/gag, which could collectively affect the viral fitness and be an additional factor, besides the absence of membrane-active trp indole ring sidechain, for the failed viral entry, as observed in a previous study [17] and confirmed in this study. in summary, our findings suggest active participation of membrane-active elements within mper (e.g. trp) in events that require protein-membrane interactions during both viral entry and assembly. these results indicate the importance of the five trp residues and c-terminal sequence (nwlwyik) in mper for the design of future mper-based fusion inhibitors and offer further insights into the understanding of viral structural proteins biosynthesis. the role of gp41 mper trp residues in modulating the viral contents of gag proteins might guide the discovery of potential therapeutic targets against hiv-1 infection. supporting information s1 fig. trafficking of the sars-cov spike protein to the lipid rafts. 293t cells were transiently transfected to express wild type spike protein (swt). twenty-four h post-transfection, cells were harvested and lysed on ice in 1% triton x-100 tne lysis buffer, and the cell postnuclear extracts were fractionated by 5%-30% sucrose gradient ultracentrifugation. eleven fractions were collected from top to bottom after centrifugation. samples were resolved by sds-page and western blot, with or without pngase f treatment. caveolin-1 serves as a positive marker for lipid raft. were detected in the lipid-raft-containing interfacial section between 5% sucrose and 30% sucrose, co-localizing with the lipid raft marker caveolin-1. both constructs contain two protein species with different sizes of 180 kda (mature) and 170 kda (immature), due to different glycosylation and maturation stages [81] . for both swt and s3w3a, n-deglycosylation via pngase f confirmed the gp180 and gp170 species originated from a common precursor but differed in glycosylation stage. the majority of swt gp180 was directed to lipid-raft containing fractions, while swt gp170 was predominantly retained in the bottom fractions. triple trp!ala substitutions resulted in an altered trafficking pattern of the mature form of the s protein. in s3w3a, both s3w3a gp180 and s3w3a gp170 were found in the upper and bottom fractions at equal amounts, suggesting that a lower percentage of mature s3w3a was recruited to the lipid raft. the data suggest that the trp residues function to fine-tune the clustering of fully mature s protein into lipid rafts during budding. (tif) s3 fig. effects of nand c-dimerization on the anti-viral effects of peptides containing sars-cov spike mper. peptide m sars, a peptide containing the sars-cov s protein mper sequence (kyeqyikwpwyvwlgf) and its n-and c-terminal dimers, n-m sars and c-m sars , were tested as fusion inhibitors against pseudotyped sars-cov. pseudotyped sars-cov was prepared by co-transfecting 293t cells using calcium phosphate transfection method with pnl4-3luc+env-vpr-and pcdna3.1-opt9-s mutant plasmids. pnl4-3luc +env-vpr-was kindly provided by prof. zhang linqi (aaron diamond aids research center, rockefeller university, new york 10016). peptides were incubated with the virus for 1 h under 5% co 2 at 37°c, prior to being added to vero e6 cells and incubated for another 72 h. inhibitory activities of the peptides were calculated from the luciferase activities of the vero e6 cells, determined by a td-20/20 luminometer (tuner designs viral membrane fusion a trimeric structural domain of the hiv-1 transmembrane glycoprotein electron tomography analysis of envelope glycoprotein trimers on hiv and simian immunodeficiency virus virions inhibition of furin-mediated cleavage activation of hiv-1 glycoprotein gp160 the t4 gene encodes the aids virus receptor and is expressed in the immune system and the brain hiv-1 entry cofactor: functional cdna cloning of a seventransmembrane, g protein-coupled receptor chemokine receptors as hiv-1 coreceptors: roles in viral entry, tropism, and disease capture of an early fusion-active conformation of hiv-1 gp41 dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates conformational changes in hiv-1 gp41 in the course of hiv-1 envelope glycoprotein-mediated fusion and inactivation atomic structure of the ectodomain from hiv-1 gp41 atomic structure of a thermostable subdomain of hiv-1 gp41 hiv-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation comparative study of adaptive molecular evolution in different human immunodeficiency virus groups and subtypes role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion a conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity the pre-transmembrane region of the human immunodeficiency virus type-1 glycoprotein: a novel fusogenic sequence membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion identification of a conserved domain of the hiv-1 transmembrane protein gp41 which interacts with cholesteryl groups peripheral-type benzodiazepine receptor function in cholesterol transport. identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern hiv-1 assembly: viral glycoproteins segregate quantally to lipid rafts that associate individually with hiv-1 capsids and virions evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts the c-and the n-terminal regions of glycoprotein 41 ectodomain fuse membranes enriched and not enriched with cholesterol, respectively the tryptophan-rich region of hiv gp41 and the promotion of cholesterol-rich domains cholesterol-dependent membrane fusion induced by the gp41 membrane-proximal external region-transmembrane domain connection suggests a mechanism for broad hiv-1 neutralization peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection short-term safety and antiretroviral activity of t-1249, a second-generation fusion inhibitor of hiv hiv gp41 c-terminal heptad repeat contains multifunctional domains. relation to mechanisms of action of anti-hiv peptides mode of action of an antiviral peptide from hiv-1. inhibition at a post-lipid mixing stage different from the hiv fusion inhibitor c34, the anti-hiv drug fuzeon (t-20) inhibits hiv-1 entry by targeting multiple sites in gp41 and gp120 hiv fusion inhibitor peptide t-1249 is able to insert or adsorb to lipidic bilayers. putative correlation with improved efficiency updating the use of synthetic peptides as inhibitors of hiv-1 entry control of expression, glycosylation, and secretion of hiv-1 gp120 by homologous and heterologous signal sequences endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus hiv-1 envelope glycoprotein biosynthesis, trafficking, and incorporation increase of anti-hiv activity of c-peptide fusion inhibitors using a bivalent drug design approach multimerized chr-derived peptides as hiv-1 fusion inhibitors modular assembly of dimeric hiv fusion inhibitor peptides with enhanced antiviral potency covalent stabilization of coiled coils of the hiv gp41 n region yields extremely potent and broad inhibitors of viral infection epitope mapping and topology of baculovirus-expressed hiv-1 gp160 determined with a panel of murine monoclonal antibodies mapping of b-cell epitopes in rabbits immunised with various gag antigens for the production of hiv-1 gag capture elisa reagents sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor t-20 is modulated by coreceptor specificity defined by hiv-1 env mper in anti-viral design and viral life cycle emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (t-20) monotherapy. antimicrob agents chemother identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research evidence that ecotropic murine leukemia virus contamination in tzm-bl cells does not affect the outcome of neutralizing antibody assays with human immunodeficiency virus type 1 cloning and functional analysis of multiply spliced mrna species of human immunodeficiency virus type 1 env and vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mrnas production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone small molecule targets env for endoplasmic reticulum-associated protein degradation and inhibits human immunodeficiency virus type 1 propagation construction and in vitro properties of hiv-1 mutants with deletions in "nonessential" genes estimation of protein secondary structure from circular dichroism spectra: comparison of contin, selcon, and cdsstr methods with an expanded reference set dichroweb, an online server for protein secondary structure analyses from circular dichroism spectroscopic data protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases sars-cov heptad repeat 2 is a trimer of parallel helices a facile ligation approach to prepare three-helix bundles of hiv fusion-state protein mimetics initial cleavage of the human immunodeficiency virus type 1 gagpol precursor by its activated protease occurs by an intramolecular mechanism sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual gag polyprotein cleavage sites structural and functional roles of hiv-1 gp41 pretransmembrane sequence segmentation structure of an hiv-1-neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer a synthetic c34 trimer of hiv-1 gp41 shows significant increase in inhibition potency interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission non-random distribution of amino acids in the transmembrane segments of human type i single span membrane proteins membrane proteins: from sequence to structure. annual review of biophysics and biomolecular structure the preference of tryptophan for membrane interfaces tryptophan-dependent membrane interaction and heteromerization with the internal fusion peptide by the membrane proximal external region of sars-cov spike protein the membrane proximal external region of the hiv-1 envelope glycoprotein gp41 contributes to the stabilization of the six-helix bundle formed with a matching n' peptide functional links between the fusion peptide-proximal polar segment and membrane-proximal region of human immunodeficiency virus gp41 in distinct phases of membrane fusion crystal structure of hiv-1 gp41 including both fusion peptide and membrane proximal external regions a conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1 anti-human immunodeficiency virus type 1 (hiv-1) antibodies 2f5 and 4e10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize hiv-1 the membrane-proximal external region of hiv-1 gp41: a vaccine target worth exploring multiple antigen peptide mimetics containing gp41 membrane-proximal external region elicit broad neutralizing antibodies against human immunodeficiency virus type 1 in guinea pigs identification of autoantigens recognized by the 2f5 and 4e10 broadly neutralizing hiv-1 antibodies. the journal of experimental medicine t-20) cross-reactive glycoprotein 41 antibody does not impair the efficacy or safety of enfuvirtide an ancestral hiv-2/simian immunodeficiency virus peptide with potent hiv-1 and hiv-2 fusion inhibitor activity downregulation of human immunodeficiency virus type 1 gag expression by a gp41 cytoplasmic domain fusion protein rre-dependent hiv-1 env rna effects on gag protein expression, assembly and release the retroviral capsid domain dictates virion size, morphology, and coassembly of gag into virus-like particles we thank dr. y. liao and dr. t. l. neo for the valuable discussions and technical support in this study. we also thank the original donors and the nih aids research and reference reagent program, division of aids, niaid for the cell lines hltat iii from barbara k. felber and dr. george n. pavlakis, and tzm-bl from john c. kabbes, xiaoyun wu, and transzyme, inc. we are grateful for the anti-gp41 antibody (chessie 8) from dr. george lweis and the plasmid pnl4-3 from dr. malcolm martin. we also thank jorma hinkula for kindly providing the anti-p24 (ef7) antibody. key: cord-312332-rwmuucsp authors: dicker, kate; järvelin, aino i.; garcia-moreno, manuel; castello, alfredo title: the importance of virion-incorporated cellular rna-binding proteins in viral particle assembly and infectivity date: 2020-09-10 journal: semin cell dev biol doi: 10.1016/j.semcdb.2020.08.002 sha: doc_id: 312332 cord_uid: rwmuucsp rna is a central molecule in rna virus biology due to its dual function as messenger and genome. however, the small number of proteins encoded by viral genomes is insufficient to enable virus infection. hence, viruses hijack cellular rna-binding proteins (rbps) to aid replication and spread. in this review we discuss the ‘knowns’ and ‘unknowns’ regarding the contribution of host rbps to the formation of viral particles and the initial steps of infection in the newly infected cell. through comparison of the virion proteomes of ten different human rna viruses, we confirm that a pool of cellular rbps are typically incorporated into viral particles. we describe here illustrative examples supporting the important functions of these rbps in viral particle formation and infectivity and we propose that the role of host rbps in these steps can be broader than previously anticipated. understanding how cellular rbps regulate virus infection can lead to the discovery of novel therapeutic targets against viruses. viruses are obligate intracellular pathogens that represent a threat to human health and the economy of countries. viral genomes are typically small, encoding only a few proteins. to circumvent this limitation, viruses hijack host resources to complete their infection cycle. rna is a central molecule in rna virus biology since it functions not only as a messenger for protein synthesis (i.e. mrna), but also as a genome. since viruses cannot encode all the proteins necessary for viral (v)rna replication/transcription, translation and packaging, viruses usurp cellular rna-binding proteins (rbps) [1] [2] [3] [4] [5] [6] [7] [8] . on the other hand, cellular rbps are also central to the antiviral defences [9, 10] . the host cell senses viruses through specialised rbps that recognise unusual signatures present in vrnas, known as pathogen-associated molecular patterns (pamps), leading to the stimulation of interferon production and the activation of mechanisms to inhibit protein synthesis [11] . cellular rbps are thus fundamental components of the virus-host cell battlefield, either sustaining or restricting virus infection. understanding how viruses interact with these cellular proteins can thus be instrumental for the development of novel antiviral therapies. rbps interact with rna forming dynamic complexes known as ribonucleoproteins (rnps), which are critical for mediating gene expression [12] . recently, the repertoire of human cellular rbps was increased dramatically to over 1500 proteins by the development of a proteome-wide approach known as rna interactome capture (ric) [13] [14] [15] . ric employs ultraviolet (uv) crosslinking of 'zero' distance (< 2 å) protein-rna interactions, followed by cell lysis under denaturing conditions, isolation of rbps crosslinked to poly(a) rna via oligo(dt) capture and quantitative mass spectrometry. additionally, new methods based on organic-aqueous phase separation to detect also proteins bound to non-poly(a) rnas have contributed to greatly extend the number of rbps in the cell [16] [17] [18] . while a substantial proportion of these proteins interact with rna using a defined set of well-characterised rna-binding domains (rbds), such as rna-recognition motifs (rrms), many rbps lack them, suggesting the existence of unconventional modes of rna binding [13] [14] [15] . indeed, dozens of novel rbds were later discovered using proteomic-based methods. these domains included enzymatic cores, protein-protein interaction surfaces and dna-binding domains [19, 20] , as well as intrinsically disordered regions that emerged as a prominent mode of rna binding [19, 21] . importantly, both classical and unconventional rbps have been linked to virus infection and immunity, as reviewed before [5] . moreover, analysis by comparative ric of the 'rna-binding proteome' of cells infected with the rna virus sindbis (sinv), revealed that the complement of cellular rbps is pervasively remodelled upon infection [7] . strikingly, many rbps activated by sinv either sustain or repress infection, highlighting the critical roles of cellular rbps as regulators of virus infection. because sinv rna is polyadenylated, ric isolates both host and viral rna. indeed, many of the proteins enhanced by sinv infection were shown by orthogonal approaches to accumulate within the viral replication factories, suggesting a direct interplay with vrna. however, ric will also discover rbps with differential rna-binding activity that interact with cellular rna instead of vrna. several approaches have been developed to elucidate the composition of viral ribonucleoproteins (vrnps). initially, studies on influenza a virus (iav) and vesicular stomatitis virus (vsv) focused on isolating viral rna polymerase complexes, as these should reflect the composition of replicating vrnps to some extent [22] [23] [24] . while useful, these datasets were biased towards direct protein-protein interactions. more recently, different groups have employed diverse approaches to capture vrna together with its interacting proteins, which are then detected by mass spectrometry. all these methods start by 'freezing' protein-vrna complexes. this can be achieved by uv protein-rna crosslinking exploiting the excitability of natural bases upon irradiation with uv light at 254 nm [2] . alternatively, photoactivatable nucleotides, such as 4thiouridine (4su), can be incorporated into nascent vrna, and protein-rna crosslinking is achieved by irradiation with 365 nm uv light [1, 3] . uv crosslinking only promotes covalent bonds between proteins and rnas placed at 'zero' distances, thus displaying high specificity with the cost of low efficiency. another approach typically used to immobilise protein-rna interactions is formaldehyde crosslinking [4, 6, 8] . while more efficient than uv, formaldehyde also induces covalent bonds between proteins, which promotes the isolation of indirect binders through protein-protein bridges. once protein-rna complexes are 'frozen', the second step is to isolate the vrna together with its covalently bound proteins. typically, vrna is captured with antisense dna probe(s). this approach substantially enriches for vrna [2, 4, 8] , although can still co-purify rnas in a non-specific fashion through the formation of partial hybrids with non-target sequences. if vrna is labelled with 4su (see above), it can alternatively be isolated by biotinylation of the sulfhydryl group in the 4su base, coupled with streptavidin pull down [6] . since exposed cysteines on protein surfaces can also be biotinylated, it is not surprising that this approach identifies a large proportion of cellular proteins lacking rna-binding activity. collectively, these methods have been applied to several virus models including dengue virus (denv) [2, 3, 8] , zika virus (zikv) [8] , sinv [6] , poliovirus [1] and human immunodeficiency virus 1 (hiv-1) [4] , representing important advances towards understanding vrnp composition. the interactomes between host proteins and incoming viral particles have been recently unravelled using a novel technique named vir-clasp [25] . vir-clasp uses 4su to label the genomic rna in infected cells, which is later incorporated into viral particles. viruses released to the supernatant harbouring 4su-labelled genomes are then used in very high multiplicity to infect new cells. infection is followed by crosslinking with uv light at 365 nm, solid-phase capture of protein-rna complexes and protein identification by mass spectrometry. vir-clasp has been successfully applied to chikungunya virus or iav and have revealed important regulators of the initial steps of the infection cycles of these viruses [25] . despite their strengths and limitations, all these studies agree that both classical and unconventional rbps engage with vrna and play critical roles in infection. in general, infection cycle of rna viruses consist of three main phases ( fig. 1) : (1) attachment of the viral particle to the cell and entry; (2) viral genome replication and expression; and (3) assembly and egress of the virus progeny [26] . the viral cycle begins when a virus binds to specific surface receptors and enters a host cell. once inside the cell, the genomic rna of positive-stranded viruses engages directly with the host protein synthesis machinery to produce the viral proteins required for replication. conversely, negative-stranded rna viruses typically carry the transcription machinery with them into the newly infected cell. retroviral rna must be reverse transcribed into dna, which is then imported into the nucleus and integrated into the host chromosome to be transcribed by the cellular rna polymerase ii. the next stage involves the synthesis of the components (i.e. viral structural proteins and genomes) required to assemble the progeny viral particles that leave the producer cell to infect a new one. virtually all these stages can be regulated by cellular rbps (fig. 1) , although most work so far has focused on viral replication and protein synthesis [27] [28] [29] [30] [31] [32] . however, new discoveries have highlighted the importance of cellular rbps in the assembly of viral particles as well as in the very initial steps of infection, when the rna genome is delivered into a newly infected cell. these steps are the focus of the present review. one of the main open questions is how vrna is recognised specifically by viral proteins with limited, if not non-existent, specificity for vrna during the assembly of viral particles [33, 34] , and whether highspecificity cellular rbps may cooperate with viral proteins to recognise the vrna. moreover, vrna is generally highly structured [35, 36] . thus, it is plausible that cellular rbps with helicase and chaperone activities facilitate the binding of capsid/nucleocapsid proteins across the vrna to enable rna packaging into virions. immediately upon entry into the cell, vrna must either be translated (positive-stranded rna viruses), transcribed and replicated (negative-stranded rna viruses) or reverse transcribed (retroviruses) without alerting the host immune defences, especially in the initial steps of infection where the virus is more vulnerable. how viruses achieve this remains largely unknown; however, recent evidence supports the idea that in certain cases such as hiv-1, some of the lifecycle of the vrna takes place inside the 'protective walls' of the capsid shell [37] [38] [39] . another interesting idea is whether vrnas carry the necessary cellular components from the producer cell to maximise the efficiency of the subsequent initial steps of infection. different proteomic studies have identified hundreds of cellular factors within the particles of several rna viruses [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] , many of which are rbps. here, we discuss the 'knowns' and 'unknowns' of the roles that virion-incorporated cellular rbps could play in the assembly of viral particles and the early steps of infection in the new host cell. in the last two decades, the composition of the particles of several rna viruses has been elucidated by proteomics. however, no work has currently focused on identifying the scope of cellular rbps that are present in virions. to gain insights into this question, we compiled the proteomes of particles from ten different human rna viruses (fig. 2 ) [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] . upon building the superset of host proteins present in virions (table s1a) , we cross-referenced it to the complement of experimentally determined human rbps [15] as well as gene ontology (go) terms and protein domains related to rna binding. the resulting in virion rbps (ivrbps) are listed in table s1b , including the dataset in which each protein was reported. we discover that virions contain many rbps (fig. s1a ), 58 % of which harbour known rbds, while the other 42 % were just recently classified as rbps by ric studies and their modes of rna binding remain unknown [15] . analysis of go biological process terms (fig. s1b ) and proteinprotein interaction networks (fig. s2 ) indicate that ivrbps can potentially participate in a wide variety of processes, e.g. transport, exocytosis, response to stress, immune system pathways, cytoskeleton organization, translation, rna processing, rna stability or protein folding. however, the main limitation of the cited proteomic analyses is that budding of the viral particles results in the uptake of a portion of the cytoplasm and plasma membrane. hence, it is challenging to discriminate between abundant proteins randomly incorporated into virions and with no function in virus biology, from those with an active regulatory role. we reasoned that host proteins with a true function in infection are likely to be identified in multiple datasets and across different virus species and cell lines (fig. s3) . strikingly, we noticed that 14 ivrbps are commonly detected in the particles of different viruses (at least 5 viruses out of 10, table s1b ). for example, cofilin 1 (cfl1) was identified in 8 of the 10 viruses studied here. cfl1 is a regulator of actin cytoskeleton reorganisation and has been shown to play a crucial role in measles virus (mev) rnp formation for vrna synthesis [56] , iav assembly and budding [57] and the production of infectious respiratory syncytial virus (rsv) [58] . however, whether the rna-binding activity of cfl1 plays a role in infection requires further investigation. moreover, the importance of cfl1 incorporation into particles should also be further tested functionally, since it could also be due to passive uptake with actin, which is often present in viral particles. many ivrbps such as annexins, heat shock family proteins (hsp), peptidylprolyl isomerase a (ppia -also cyclophilin a), eukaryotic translation elongation factors (eef), heterogeneous nuclear ribonucleoproteins (hnrnp) or poly(rc) binding protein 1 (pcbp1), have been linked to infection in multiple ways (fig. s2) , and here we show that they are incorporated in the particles of several viruses (table s1b) . whether these proteins are incorporated passively due to high abundance or through convergent, virus-driven active mechanisms such as specific interactions with viral proteins or rna, requires further investigation. however, ppia illustrates well why the second option should not be underestimated, as it interacts specifically with the capsid of hiv and this interaction is critical for capsid core stability [59, 60] . whether ppia rna-binding activity plays a role on this recruitment remains unknown. many ribosomal proteins are also present in the superset of ivrbps, together with rna helicases that aid the unwinding of rna (fig. s2 ). interestingly, several ivrbps are restricted to the virions of a certain superclass of viruses. for example, heat shock protein 90 alpha (hsp90aa1), transgelin 2 (tagln2) and hnrnpd are found exclusively in negative-stranded rna viruses. nevertheless, it is important to note that all these virion proteomes have been generated using different i) virion isolation methods, ii) proteomic approaches and iii) data analyses. these divergences are noticeable when comparing datasets generated in closely related viruses or even the same virus (fig. s3 ). for example, each of the six hiv-1 virion proteomes that we examined identified a distinct number of proteins ( fig. 2 ), reflecting differences in virion isolation stringency, proteomic depth and/or statistical thresholds. the analysis of virion proteomes is complicated by potential contamination with cellular proteins, either within extracellular vesicles that copurify with viral particles or via nonspecific interactions with the virus exterior. most of the viruses used in our metanalysis were purified on sucrose cushions or density gradients, and some lack additional purification steps [41, 43, 47, 48, 50, 52] . few of the latter studies showed that the vast majority of their samples were intact viral particles using transmission electron microscopy. interestingly, several studies incorporated further purification strategies: (1) treatment of viral particles with proteases such as subtilisin [40, 44] or proteinase k [45, 46] to remove non-specific binders and vesicles due to an alteration in their density; (2) cd45 immunoaffinity depletion as cd45 is present on microvesicles but not in hiv particles [42] ; (3) solubilisation of lipid bilayers for isolation of hiv cores by including a detergent layer on the sucrose density gradient [49] ; (4) haemadsorption to and elution from red blood cells to select for iav particles with receptor binding and cleavage activity, provided by haemagglutinin and neuraminidase [51] ; (5) sequential affinity purification with a heparin column to select for hcv particles containing the viral e2 protein, followed by antibody-or tag-mediated capture of envelopecontaining particles [53] . by focusing on proteins identified in multiple datasets, we expect to minimise technical differences between datasets, as well as biological factors, such as the incidence of randomly incorporated proteins (see above). however, passive incorporation due to abundance cannot be excluded a priori, and we recommend interested readers to test the abundance of their candidates in proteomic analyses of whole cell lysates, if available. table s1 highlights many of these robustly identified ivrbps and we discuss below several ivrbps with well-known roles in infection, and others that remain poorly characterised. k. dicker, et al. seminars in cell and developmental biology xxx (xxxx) xxx-xxx 2. do cellular rbps participate in viral particle assembly? the cytoplasm of the cell is a hostile environment for vrna due to the presence of antiviral sensors. these cytoplasmic immune surveillance factors are mostly specialised rbps that detect viral doublestranded (ds)rna, under-methylated cap structures or triphosphate 5′ ends, which are common signatures present in vrna [61] [62] [63] [64] . the recognition of vrna as 'foreign' nucleic acid can trigger the antiviral response and direct the rna decay machinery towards the vrna [65, 66] . hence, it is crucial for vrnas to effectively engage with cellular rbps to increase their stability and translatability [67] . upon accumulation of viral rna and proteins, these components must travel to the virus assembly areas and, in some instances, vrna trafficking has been proposed to be mediated by the cytoskeleton [68] [69] [70] [71] [72] [73] . the role of protein chaperones in the trafficking of vrnps is becoming increasingly evident. many cellular chaperones and peptidylprolyl cis/trans isomerases (immunophilins) are hijacked by viruses to enable viral replication and translation by folding viral proteins [74, 75] . interestingly, new proteome-wide approaches have revealed that both chaperones and immunophilins interact with rna in vivo and exert critical functions in rna metabolism, including regulatory roles within the spliceosome, control of transcription and mrna stability and ribonucleoprotein remodeling [13] [14] [15] . moreover, their rna-binding surfaces have recently been revealed, suggesting that their ability to act on proteins and bind to rna is interconnected [19] . ric analysis of sinv-infected cells revealed that the rna-binding activity of several protein chaperones and immunophilins is enhanced upon infection and that their functional perturbation severely impairs viral fitness [7] . the importance of host chaperones in infection is well illustrated by hiv-1 rnps. viral genomic rna interacts with staufen double-stranded rnabinding protein 1 (stau1) in the cytoplasm forming hiv-1-dependent stau1-rnps [76] [77] [78] . compositional characterisation of these rnp complexes revealed the presence of the chaperones hspd1 (hsp60), hspa1 (hsp70) and hspa8 (hsc70) 78]. it has been proposed that the stress-inducible chaperone hsp70 and its constitutive form, hsc70, interact with nascent hiv-1 gag-vrna complexes and hold them in an assembly-competent conformation during their transport towards the plasma membrane [79] . strikingly, these proteins are also present within hiv-1 virions (fig. s2c) , suggesting that they associate with vrna at different stages of the infection cycle 78]. the participation of hsc70 in viral particle assembly is also illustrated by hepatitis c virus (hcv) infection. hsc70 interacts with hcv rna and colocalises with the hcv capsid and e2 proteins at assembly sites [80] . knockdown of hsc70 [80] or abrogation of protein activity with allosteric inhibitors [81] decreases viral particle production, but has no effect on viral replication. whether direct binding of hsc70 to the hcv rna genome contributes to the formation of infective virions is still unclear. the roles of cellular rna-binding chaperones, therefore, may span replication, translation, trafficking and virion assembly (fig. s2) . cellular rbps are also thought to contribute to capture the vrna, amongst all the cellular rnas in the cytoplasm, to be packaged into the virion. several viruses, such as the bacteriophage ms2, encode capsid proteins with high specificity and affinity for stem loops present within the vrna [82] . however, other viruses, such as hiv-1, do not encode for any protein with high specificity and affinity for the vrna. hence, how these viruses select and package their vrna into virions against cellular counterparts remains poorly understood. for example, it was believed that the hiv-1 nucleocapsid subunit of the viral polyprotein gag recognises vrna from the pool of cellular rnas by a unique binding event at a cis-acting packaging element in the 5′ leader of the vrna [83] . however, a recent study using clip-seq (crosslinking and immunoprecipitation followed by sequencing) discovered that, in addition to the binding site in the 5′ leader, gag interacts in the cytoplasm with other discrete sites across the hiv-1 rna [34] , placed at the rev recognition element (rre) and 3′ utr. however, when the vrna reaches the plasma membrane, this binding pattern changes dramatically, and gag covers virtually the whole vrna. how gag switches from selective to non-selective binding is not understood. since many cellular rbps interact with hiv-1 rna and gag [4, 78, 84] , it is tempting to speculate that they might cooperate with hiv-1 nucleocapsid to recognise hiv-1 genome in the cytoplasm of the infected cell. in agreement, it has been proposed that stau1 assists gag interaction with hiv-1 rna [85] . the resulting rnp is transported through the cytoplasm to the plasma membrane where stau1 facilitates the multimerization of gag on the vrna [86] . this multimerization step enables the formation of immature virus particles, where the hiv-1 genomic rna acts as a nucleation site for assembly [87, 88] . interestingly, stau1 only modulates gag assembly once the tail of gag has anchored into the plasma membrane suggesting that stau1 may play a role in ensuring the rnp is delivered to the membrane [86] . stau2 has also been shown to promote the activity of hiv-1 rev protein in exporting vrna from the nucleus, along with dexh-box helicase 9 (dhx9) and arfgap with fg repeats 1 (agfg1) [89] [90] [91] . together, these studies suggest that staufen proteins play crucial roles in the transport of hiv-1 rnps from the nucleus to the plasma membrane for viral particle formation. additional studies should be carried out to determine if other cellular rbps cooperate with viral capsids and nucleocapsids to recognise the vrna and to enable vrnp trafficking. the cellular endosomal sorting complex required for transport (escrt) is a large multi-component machinery comprised of five complexes, escrt-0, escrt-i, escrt-ii, escrt-iii and vps4, which assemble sequentially in a multi-step manner following ubiquitination of escrt-0 [92] . the escrt machinery is hijacked for viral particle release across most types of viruses including retroviruses, filoviruses, arenaviruses, paramyxoviruses, rhabdoviruses, flaviviruses, reoviruses and picornaviruses [93] . viruses can either hijack escrt for assembly and budding at the plasma membrane or for budding at endosomal membranes into the cytoplasm. escrt is typically involved in the budding of vesicles from the endosomal membrane into the endosomal compartment. however, escrt proteins can also perform a "reverse topology" membrane fission. escrt is the only cellular machinery with such ability and this is perhaps why so many viruses have evolved to exploit it [93] . escrt-i/ii essentially function to recruit the escrt-iii protein chmp4 (charged multivesicular body protein 4) to membranes where the formation of escrt-iii filaments is then promoted. the formation of escrt-iii filaments drives the budding of the membrane, but it is not understood exactly how this is achieved. programmed cell death 6 interacting protein (pdcd6ip or alix) recruits chmp4 and the escrt-iii complex to the plasma membrane. several studies have shown that many viral structural proteins initiate escrt driven budding from the membrane by recruiting alix. examples include gag of hiv-1 [94] , the accessory c protein of human parainfluenza type 1 [95] , ebola virus vp40 [96] , px of hepatitis a virus [97] and the ns3 protein of denv [98] and yellow fever virus [99] . although alix has largely been studied from a proteo-centric perspective, ric studies have revealed that it is endowed with rnabinding activity [15] . in addition, alix has recently been implicated in the recruitment of cellular rnas to extracellular vesicles [100] . importantly, the interaction of hiv-1 gag with alix relies on the presence of vrna. the n-terminal bro1 domain and the central v-shaped domains of alix interact with hiv-1 nucleocapsid and p6 domains of gag respectively [101, 102] . interestingly, the interaction between alix bro1 domain and hiv-1 nucleocapsid is disrupted by rnase treatment, suggesting that the vrna molecule forms a bridge between the two proteins. both alix bro1 and nucleocapsid are highly positively charged and are believed to establish electrostatic interactions with the phosphate backbone of the vrna. the recruitment of alix by vrna raises the question of whether alix is recruited to the hiv-1 rnps in the cytoplasm or in the plasma membrane as the virus assembles 102]. additionally, proteomic-based compositional analysis of the zikv and denv rnps revealed that alix also interacts with these vrnas [8] , although the exact role of these interactions remain unexplored. another class of unconventional rbps implicated in virus release is the annexin protein family. annexins are calcium-dependent membrane binding proteins that carry out a diverse range of functions. they can reversibly associate with components of the cytoskeleton or with regulatory proteins and rnas that mediate stress-induced intra-and inter-cellular signalling [103] . annexin a2 (anxa2) is a multifunctional, ubiquitously expressed protein with roles in membrane domain organisation, membrane fusion, vesicle aggregation and exocytosis [104] . it has been shown to play a role in cell attachment and entry or replication of enterovirus, rsv, hcv and iav, amongst other viruses [104] . however, we still lack a molecular understanding of the relationship between anxa2 rna-binding activity and its regulatory roles in infection. anxa2 was shown to be involved in the formation of the hcv replication complex and can bind both hcv rna, in a sequence-specific manner, and non-structural protein ns5b forming a ternary complex [105] . the silencing of anxa2 has no effect on vrna levels suggesting that it does not influence replication. instead it significantly reduces the number of produced viral particles indicating that anxa2 plays a role in hcv virion formation or release, although the mechanism by which this is achieved remains unknown [106] . anxa2 has also been shown to be involved in virion assembly of mev through recruitment of the viral matrix protein to the plasma membrane [107] . anxa2 and other members of the annexin family have been identified within the viral particles of ebola and marburg virus [48] , hiv-1 [42] , iav [44] , vsv [45] , rift valley fever virus [52] and rsv [47] (table s1b ). together, these data suggest that anxa proteins may be involved in the formation and release of particles of a broad range of viruses. however, the mechanisms of action of anxa proteins and their rna-binding activity in infection remains largely uncharacterised. the compositional analysis of different virions has also revealed the presence of some members of the adp ribosylation factor (arf) gtpase family, suggesting a role in viral particle formation and/or release [55] . this is a ras-related subfamily fundamental for the regulation of vesicle formation, trafficking and docking at target membranes, and has been implicated in the infection cycles of many pathogens [108] . arfs have been involved in the recruitment of hiv-1 rnps to the plasma membrane and the release of hiv-1 particles [109] , and arf1 regulates hiv-1 trafficking to the virological synapse [110] . additionally, arf1 is necessary for hcv rna replication and production of infectious particles [111] . the possibility that the rna-binding activity of arf1 facilitates the localisation of vrnps at the sites of viral particle assembly and egress calls further investigation. after entry into the host cell, most positive-stranded rna viruses release the genomic mrna into the cytoplasm to be immediately translated into the viral replicase complex by the cellular translation machinery (fig. 1) . this is the case for sinv, a representative member of the alphavirus genus [112] . it was recently described that sinv infection of mammalian cells produce two subpopulations of infectious viral particles. one of them, known as 'heavy' viral particles, exhibits an enhanced translation of the vrna once inside the newly infected cell [113] . only one homogeneous population of virions is released from the other natural host of sinv, mosquito, with an infectivity that matches that of 'heavy' viral particles. authors showed that virion-incorporated host-derived factors, including the ribosomal components rps14, rps18 and 18s ribosomal rna, and the cellular rna binding motif protein 3 (rbm3), are responsible for vrna superior translation. rbm3 has been shown to promote translation in different contexts [114] and its incorporation into sinv particles was recently confirmed [54] , together with other ribosomal proteins and many cellular rbps (table s1c) . interestingly, while sinv particles with increased infectivity can be produced in either mammalian or mosquito cells, the enhanced vrna translation only occurs in a newly infected mammalian cell, but not in a mosquito cell [113] . this striking phenomenon is recapitulated in animal models and in other alphaviruses [115] . this suggests that rbps pre-loaded in the viral particle may interact specifically with mammalian proteins controlling translation initiation. the identity of these mammalian proteins remains unknown and uncovering them will be a challenge. however, recent work has shown that the mammalian ribosome establishes interactions with hundreds of cellular rbps, which represent potential regulators of translation [116] . as many of these ribosome interactors are indeed found inside virions (table s1), we anticipate that they may accompany the vrna into the newly infected cell. once the vrna is released into the cytoplasm, these proteins may facilitate translation initiation by interacting with the ribosome. to what extent virion composition determines translation of incoming viral genomes awaits mechanistic characterisation. moreover, whether this phenomenon can be extended to other rna viruses beyond sinv remains to be explored. particles of negative-stranded rna viruses contain a viral rna-dependent rna polymerase and accessory proteins to transcribe the vrna into mrna upon entry into the host cell (fig. 1) . later, this 'transcriptase' complex is modified to form a 'replicase' complex that synthesizes an intermediary positive-stranded rna that serves as a template for producing new copies of the negative vrna genome [117] . thus, viral transcription and replication are two separate processes controlled by distinct vrnp complexes. a representative example is vsv that forms two complexes composed of the viral rna polymerase (l) and different viral and host proteins [22] . the vsv transcriptase complex contains the viral proteins l and p and the host proteins rna guanylyltransferase and 5′phosphatase (rngtt) capping enzyme, eef1a and hsp60 [22, 118] . the last two proteins are important for the rna polymerase activity of l and can be found within virions (table s1b ). in addition, ppia is also bound to the vrnp complex inside purified vsv particles and is used for post-entry primary transcription [119] . interestingly, the mentioned host rbps are absent in the vsv replicase complex, which contains n, l and p viral proteins and synthesizes the plus strand 'antigenome' vrna used as template for copying the viral genome [22] . this suggests that differential association of the rna polymerase complex with host proteins may regulate the switching from transcription to replication. the cellular rbp hnrnpu interacts with the vsv leader rna that is required for vrna replication and inhibition of cellular transcription [120] . this protein is also packaged into vsv particles, but the potential association of virion-incorporated hnrnpu with the transcriptase or replicase complexes and its activity in the early steps of vsv infection is not well understood. in iav, the viral polymerase consists of three subunits: pb1, pb2 and pa. the cellular chaperone hsp90 is involved in the transport of pb1 and pb2 to the nucleus and modulates the interaction of pa with pb1 [121] . after binding of pa to pb1-pb2, hsp90 dissociates from the complex. in this way, authors suggested that hsp90 regulates the assembly of the mature trimeric polymerase complex. on the contrary, hsp90 was found to be associated with the trimeric polymerase complex in a different study [23] . hsp90 relocates to the nucleus after iav infection [121, 122] , and this could reflect its interaction with newly synthesized polymerase subunits. interestingly, hsp90 has been found inside purified iav particles (as well as in other viruses, table s1b), but k. dicker, et al. seminars in cell and developmental biology xxx (xxxx) xxx-xxx the possibility that virion-incorporated hsp90 participates in transport of the incoming vrnp to the nucleus and/or the initial viral transcription has not been explored so far. in vitro, hsp90 stimulates iav rna synthesis by interacting with pb2 122] , and binding of hsp90 to pb2 is increased during rna synthesis [121] . authors suggested that hsp90 may participate in the early steps of transcription elongation by dissociating the polymerase from the vrnp and stabilizing the different subunits during their transfer between rna templates [121] . however, further investigation in the context of iav-infected cells is required to elucidate the role of hsp90 and its recently described rna-binding activity [19] in early iav transcription. it was believed that the capsid shell protecting hiv-1 rna was disassembled upon entry into the host cell, allowing viral rna and proteins to associate with host rbps in the cytoplasm of the newly infected cell to initiate reverse transcription. however, more than a decade of intensive work has challenged this model by discovering that reverse transcription takes place inside the capsid core [37] [38] [39] [123] [124] [125] , protected from the hostile intracellular environment, and uncoating occurs at the nuclear pore complex [126, 127] or even inside the nucleus [128] (fig. 1) . recently, it was shown that the presence of pores in the capsid hexamers allows deoxynucleotides to traverse the capsid shell to feed reverse transcription [38] . however, at only 8 å wide, these pores are too small to allow proteins to pass through. hence, host rbps participating in reverse transcription must be present inside the capsid core prior to cell entry, likely being incorporated in the producer cell during the assembly of viral particles. the idea of bringing up key proteins from the producer cell is well characterised for negative stranded rna viruses, which incorporate viral polymerase complexes including host factors within the viral particles to enable replication upon entry in the host cell (see above). similarly, hiv-1 particles contain reverse transcriptase and integrase, two viral enzymes that are critical in both reverse transcription and proviral dna integration into the host chromosome. recently, integrase was shown to bind not only dna but also specific structures in hiv-1 rna within virions, and these interactions are critical for the correct localization of the vrnp inside mature virions [129] . several studies have revealed that specific cellular ivrbps either promote (e.g. upf1 rna helicase and atpase [upf1] [130] , y-box binding protein 1 [ybx1] [131] , dhx9 [132] , eef1a [133, 134] , ppia [135] and aminoacyl-trna synthetases [136] ) or inhibit (e.g. mov10 risc complex rna helicase [mov10] [137] , pyruvate kinase m1/2 [pkm] [138] , enolase 1 [eno1] [139] ) reverse transcription (table s1e) . nonetheless, the scope of cellular rbps hijacked into hiv-1 particles and whether they play critical roles in these initial steps of infection inside the virion is not well defined yet. as example, upf1 is a cytosolic rna helicase that plays crucial roles in hiv-1 infection and is incorporated into virions (table s1e) . strikingly, reverse transcription fails if virions are generated in cells depleted of upf1 or expressing an atpase-defective upf1 mutant [130] . authors suggested that upf1 could mediate the remodelling the vrnp to facilitate reverse transcription or, alternatively, promote the annealing of trna lys3 primer to the viral genome, as shown in other helicases such as dhx9 [130, 140] . conversely, recent evidence suggested that dhx9 participates in the elongation phase of reverse transcription but not in the annealing of trna lys3 to the vrna [132] . further research is required to elucidate the exact mechanism of action of upf1, dhx9 and other cellular rbps during hiv-1 reverse transcription. interestingly, cellular rbps have also been found in hepatitis b virus (hbv) nucleocapsids. hbv is a 'gapped' dna virus but initially a singlestranded rna pre-genome (pgrna) is packaged into viral particles. the pgrna is reverse transcribed to a circular dsdna within the viral capsid before the virus matures and is secreted from the cell [141] . the pgrna is packaged through recognition of epsilon (ε) stem-loop structure in the 5′ terminus by hbv polymerase (pol). it was shown that eukaryotic translation initiation factor (eif)4e enables this recognition by forming an eif4e-pol-ε rnp complex that is incorporated into the nucleocapsid [142] . reverse transcription of the pgrna then requires hsp90 [143] , which forms a rnp together with other cellular rbps including hsc70 and dnaja1 (hsp40) [144, 145] to enable chaperone-mediated specific binding of hbv reverse transcriptase to pgrna. how the rna-binding activity of these proteins contribute to viral capsid assembly and/or reverse transcription remains poorly understood. finally, other cellular ivrbps such as dead-box helicase 3 x-linked (ddx3x) [145] , apolipoprotein b mrna editing enzyme catalytic subunit 3 g (apobec3 g) [146, 147] and mov10 [148] negatively regulate the very early steps of hbv reverse transcription. ddx3x interacts with hbv polymerase and requires atpase but not helicase activity for minus-strand dna synthesis inhibition [145] ; apobec3 g acts via an unknown deaminationindependent mechanism [147] that may involve direct binding to reverse transcriptase [146] , while mov10 binds directly to hbv rna but not the viral polymerase [148] . however, the precise mechanisms by which these proteins affect hbv dna synthesis still remain unclear. it is an evocative idea that cellular antiviral factors may be also packaged with the vrna into virions in order to interfere with the initial steps of infection. well-known antiviral rbps such as zinc finger ccch-type containing antiviral 1 (zc3hav1), apobec3c and apobec3f are detected in the proteome of purified sinv particles (table s1c) . however, studies exploring their role in the context of the viral particle or the early phase of sinv infection are non-existent to our knowledge. in addition, antiviral rbps might be under-represented in the different virion proteomes analysed here (table s1 ). this is most likely due to 1) the existence of sophisticated mechanisms to avoid the presence of restriction proteins inside virions, or 2) because the viral particles used in these proteomic studies were often produced in highly permissive cell lines with damaged interferon pathway. the first possibility has been widely studied in hiv-1, which excludes antiviral proteins from virions by different mechanisms. for example, serine incorporator 5 (serinc5) and serinc3 are relocalized from cell surface to endosomes by the accessory viral protein nef [149, 150] ; yth n 6 -methyladenosine rna binding protein 3 (ythdf3) is cleaved by hiv-1 protease inside the viral particle [151] , and apobec3 proteins are targeted for degradation by the viral protein vif [152] . ythdf3 has a strong affinity for n 6 -methyladenosine (m 6 a), a posttranscriptional rna modification that has recently emerged as a key regulator of vrna fate [153] . m 6 a can be found in the rna of iav, denv, zikv, hcv, yellow fever virus, west nile virus, enterovirus 71 and hiv-1. in the case of hiv-1, m 6 a modification can influence different steps of the virus infection cycle including reverse transcription. recently, ythdf3 was found to be encapsidated into hiv-1 particles by interacting with nucleocapsid and negatively affected reverse transcription once the capsid core is delivered into the newly infected cell [151] . interestingly, hiv-1 protease cleaves ythdf3 into smaller fragments inside the virion, thus counteracting its antiviral activity. other 'readers' of m 6 a rna can be detected inside the viral particles of different viruses, including eif3, insulin like growth factor 2 mrna binding protein 1 (igf2bp1) and hnrnpa2b1 (table s1b) . further work is required to discover the potential role of m 6 a-modified vrna and ivrbps in the early steps of infection. one of the best studied cases of antiviral rbp acting inside virions is the apobec3 proteins [152] . these are cellular cytidine deaminases that catalyse the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine. to exert their antiviral effects in hiv-1, these proteins are recruited into viral particles through binding to the vrna and nucleocapsid. apobec3f, g and h have preference for g-rich and a-rich sequences which resemble the rna-binding pattern of the hiv-1 nucleocapsid domain in gag polypeptide, as determined by clip-seq in cells and purified virions [34, 154] . apobec3 proteins bind to a large proportion of cellular mrnas in infected cells due to their low specificity. however, the g/a-rich sequence bias ensures that a proportion of apobec3 molecules interact with hiv-1 rna [155] as well as with other retroviruses [156] . once inside the viral particle, apobec3 proteins induce cytidine deamination of the reverse-transcribed dna strand. changes from c to u cause complementary g to a conversion during second strand synthesis and this hypermutation inhibits hiv-1 replication [152] . in addition, apobec3 proteins also restrict hiv-1 by a deamination-independent mechanism that consist of altering reverse transcription template switching frequency [157] . apobec3 g has also been shown to hinder replication of mev, mumps virus and rsv [158] . however, it is unclear whether the deaminase activity is responsible for the inhibition of replication of these viruses. apobec3 proteins, including apobec3c, f and h, also restrict replication of the human coronavirus nl63 via two mechanisms: cytidine deamination and binding to nucleocapsid protein [159] . apo-bec3c restricts zikv infection in a non-editing-dependent manner and is partially counteracted by a small subgenomic flavivirus rna that sequesters it [160] . while apobec3s suppress the infection of a wide range of viruses by different, poorly understood mechanisms, it remains unknown if they are incorporated into these viral particles and exert their antiviral activity in the early steps of infection. this possibility deserves further investigation. in summary, many cellular rbps are hijacked by viruses to sustain infection and are involved in virtually all stages of their infection cycle. we have highlighted here many previously known and novel rbps that are found inside virions from different human rna viruses and depicted a clear gap in our understanding of their function in infection. cellular ivrbps may facilitate trafficking and selection of vrna into assembling viral particles, protect the vrna from the hostile cellular environment, or streamline the initial processes of vrna metabolism upon entrance in a new host cell. alternatively, some of these ivrbps may function as part of the immune surveillance system of the cell and are incorporated into viral particles to interfere with infectivity. whilst we have discussed several roles for ivrbps in this review, much of these remain poorly characterised and mechanistic data is still missing. future research must focus on deciphering the roles of ivrbps and their newly described rna-binding activities in virus infection. by broadening our understanding on these proteins, it will be possible to identify new targets with potential for host-based antiviral therapies. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.semcdb.2020.08.002. thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification identification of proteins bound to dengue viral rna in vivo reveals new host proteins important for virus replication identification of rna binding proteins associated with dengue virus rna in infected cells reveals temporally distinct host factor requirements elucidating the in vivo interactome of hiv-1 rna by hybridization capture and mass spectrometry unconventional rna-binding proteins step into the virus-host battlefront identification and characterization of sindbis virus rna-host protein interactions system-wide profiling of rna-binding proteins uncovers key regulators of virus infection an rna-centric dissection of host complexes controlling flavivirus infection viruses seen by our cells: the role of viral rna sensors intracellular sensing of viral genomes and viral evasion to translate, or not to translate: viral and host mrna regulation by interferon-stimulated genes the expanding universe of ribonucleoproteins: of novel rna-binding proteins and unconventional interactions the mrna-bound proteome and its global occupancy profile on protein-coding transcripts insights into rna biology from an atlas of mammalian mrnabinding proteins a brave new world of rnabinding proteins comprehensive identification of rna-protein interactions in any organism using orthogonal organic phase separation (oops) the human rna-binding proteome and its dynamics during translational arrest purification of cross-linked rna-protein complexes by phenol-toluol extraction comprehensive identification of rna-binding domains in human cells high-resolution mapping of rna-binding regions in the nuclear proteome of embryonic stem cells the new (dis)order in rna regulation two rna polymerase complexes from vesicular stomatitis virus-infected cells that carry out transcription and replication of genome rna identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches comprehensive proteomic analysis of influenza virus polymerase complex reveals a novel association with mitochondrial proteins and rna polymerase accessory factors discovery of widespread host protein interactions with the prereplicated genome of chikv using vir-clasp fields virology diverse roles of host rna binding proteins in rna virus replication the dependence of viral rna replication on co-opted host factors viral subversion of the host protein synthesis machinery host factors in the replication of positive-strand rna viruses tinkering with translation: protein synthesis in virus-infected cells regulation mechanisms of viral ires-driven translation a protein ballet around the viral genome orchestrated by hiv-1 reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed rna to vpr-bridged dna global changes in the rna binding specificity of hiv-1 gag regulate virion genesis physical and functional analysis of viral rna genomes by shape exploring the architecture of viral rna genomes hiv-1 capsid uncoating initiates after the first strand transfer of reverse transcription hiv-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated dna synthesis reverse transcription mechanically initiates hiv-1 capsid disassembly actin-binding cellular proteins inside human immunodeficiency virus type 1 proteomic profiling of cellular proteins interacting with the hepatitis c virus core protein proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins cellular proteins in influenza virus particles analysis of virion associated host proteins in vesicular stomatitis virus using a proteomics approach quantitative proteomic analysis of lentiviral vectors using 2-de protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein 90 in virus particle assembly identification of essential filovirion-associated host factors by serial proteomic analysis and rnai screen virus-producing cells determine the host protein profiles of hiv-1 virion cores comparative proteomic analysis of hiv-1 particles reveals a role for ezrin and ehd4 in the nef-dependent increase of virus infectivity conserved and host-specific features of influenza virion architecture multi-faceted proteomic characterization of host protein complement of rift valley fever virus virions and identification of specific heat shock proteins, including hsp90, as important viral host factors proteomics of hcv virions reveals an essential role for the nucleoporin nup98 in virus morphogenesis comparative characterization of the sindbis virus proteome from mammalian and invertebrate hosts identifies nsp2 as a component of the virion and sorting nexin 5 as a significant host factor for alphavirus replication host proteins identified in extracellular viral particles as targets for broad-spectrum antiviral inhibitors actin-modulating protein cofilin is involved in the formation of measles virus ribonucleoprotein complex at the perinuclear region cofilin-1 is involved in regulation of actin reorganization during influenza a virus assembly and budding new host factors important for respiratory syncytial virus (rsv) replication revealed by a novel microfluidics screen for interactors of matrix (m) protein the host proteins transportin sr2/tnpo3 and cyclophilin a exert opposing effects on hiv-1 uncoating cyclophilin a stabilizes the hiv-1 capsid through a novel non-canonical binding site the dsrna protein kinase pkr: virus and cell control rig-i detects viral genomic rna during negative-strand rna virus infection ifits: emerging roles as key antiviral proteins cytosolic innate immune sensing and signaling upon infection rna degradation in antiviral immunity and autoimmunity attacked from all sides: rna decay in antiviral defense strategies for viral rna stability: live long and prosper the taking of the cytoskeleton one two three: how viruses utilize the cytoskeleton during egress hiv trafficking in host cells: motors wanted! host cytoskeleton in respiratory syncytial virus assembly and budding microtubule regulation and function during virus infection friend or foe: the role of the cytoskeleton in influenza a virus assembly the role of host cytoskeleton in flavivirus infection role of rna chaperones in virus replication emerging picture of host chaperone and cyclophilin roles in rna virus replication identification of staufen in the human immunodeficiency virus type 1 gag ribonucleoprotein complex and a role in generating infectious viral particles novel staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of hiv-1 genomic rna characterization of staufen1 ribonucleoproteins by mass spectrometry and biochemical analyses reveal the presence of diverse host proteins associated with human immunodeficiency virus type 1 specific incorporation of heat shock protein 70 family members into primate lentiviral virions chaperones in hepatitis c virus infection allosteric heat shock protein 70 inhibitors block hepatitis c virus assembly bacteriophage ms2 genomic rna encodes an assembly instruction manual for its capsid life of psi: how full-length hiv-1 rnas become packaged genomes in the viral particles proteome analysis of the hiv-1 gag interactome the double-stranded rna-binding protein staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic rna encapsidation the host protein staufen1 participates in human immunodeficiency virus type 1 assembly in live cells by influencing pr55gag multimerization dimeric rna recognition regulates hiv-1 genome packaging hiv-1 rna genome dimerizes on the plasma membrane in the presence of gag protein hrip, a cellular cofactor for rev function, promotes release of hiv rnas from the perinuclear region the cellular hiv-1 rev cofactor hrip is required for viral replication human protein staufen-2 promotes hiv-1 proliferation by positively regulating rna export activity of viral protein rev teis, escrt and membrane protein ubiquitination virus budding and the escrt pathway escrt-ii's involvement in hiv-1 genomic rna trafficking and assembly alix serves as an adaptor that allows human parainfluenza virus type 1 to interact with the host cell escrt system alix rescues budding of a double ptap/ppey l-domain deletion mutant of ebola vp40: a role for alix in ebola virus egress hepatitis a virus structural protein px interacts with alix and promotes the secretion of virions and foreign proteins through exosome-like vesicles assembly and release of dengue virus: role of alix protein interaction between the yellow fever virus nonstructural protein ns3 and the host protein alix contributes to the release of infectious particles role of alix in mirna packaging during extracellular vesicle biogenesis human immunodeficiency virus type 1 gag engages the bro1 domain of alix/aip1 through the nucleocapsid identification of the hiv-1 nc binding interface in alix bro1 reveals a role for rna functional association between regulatory rnas and the annexins annexin a2 in virus infection characterization of interactions between hepatitis c virus ns5b polymerase, annexin a2 and rnaeffects on ns5b catalysis and allosteric inhibition role of annexin a2 in the production of infectious hepatitis c virus particles annexin a2 mediates the localization of measles virus matrix protein at the plasma membrane arfs at a glance gga and arf proteins modulate retrovirus assembly and release identification of host trafficking genes required for hiv-1 virological synapse formation in dendritic cells role for adp ribosylation factor 1 in the regulation of hepatitis c virus replication the regulation of translation in alphavirus-infected cells encapsidation of host-derived factors correlates with enhanced infectivity of sindbis virus cold-inducible proteins cirp and rbm3, a unique couple with activities far beyond the cold encapsidated host factors in alphavirus particles influence midgut infection of aedes aegypti the mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity the rna synthesis machinery of negative-stranded rna viruses rna polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 alphabetagamma for its activity requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype specific interaction of heterogeneous nuclear ribonucleoprotein particle u with the leader rna sequence of vesicular stomatitis virus involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits identification of hsp90 as a stimulatory host factor involved in influenza virus rna synthesis hiv-1 dna flap formation promotes uncoating of the pre-integration complex at the nuclear pore isolated hiv-1 core is active for reverse transcription ip6 is an hiv pocket factor that prevents capsid collapse and promotes dna synthesis hiv-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein nup358 single hiv-1 imaging reveals progression of infection through ca-dependent steps of docking at the nuclear pore, uncoating, and nuclear transport hiv-1 uncoats in the nucleus near sites of integration hiv-1 integrase binds the viral rna genome and is essential during virion morphogenesis upf1 is crucial for the infectivity of human immunodeficiency virus type 1 progeny virions y-box-binding protein 1 supports the early and late steps of hiv replication virion-associated, host-derived dhx9/rna helicase a enhances the processivity of hiv-1 reverse transcriptase on genomic rna hiv-1 uncoating and reverse transcription require eef1a binding to surface-exposed acidic residues of the reverse transcriptase thumb domain specific interaction between eef1a and hiv rt is critical for hiv-1 reverse transcription and a potential anti-hiv target cyclophilin a promotes hiv-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cdna role of host trnas and aminoacyl-trna synthetases in retroviral replication p body-associated protein mov10 inhibits hiv-1 replication at multiple stages virion-packaged pyruvate kinase muscle type 2 affects reverse transcription efficiency of human immunodeficiency virus type 1 by blocking virion recruitment of trna(lys3) virion-incorporated alpha-enolase suppresses the early stage of hiv-1 reverse transcription coordinate roles of gag and rna helicase a in promoting the annealing of formula to hiv-1 rna hepatitis b viruses: reverse transcription a different way incorporation of eukaryotic translation initiation k. dicker, et al. seminars in cell and developmental biology xxx (xxxx) xxx-xxx factor eif4e into viral nucleocapsids via interaction with hepatitis b virus polymerase hsp90 is required for the activity of a hepatitis b virus reverse transcriptase hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids ddx3 dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids reverse transcriptase-and rna packaging signal-dependent incorporation of apobec3g into hepatitis b virus nucleocapsids deamination-independent inhibition of hepatitis b virus reverse transcription by apobec3g the mov10 helicase restricts hepatitis b virus replication by inhibiting viral reverse transcription hiv-1 nef promotes infection by excluding serinc5 from virion incorporation serinc3 and serinc5 restrict hiv-1 infectivity and are counteracted by nef hiv protease cleaves the antiviral m6a reader protein ythdf3 in the viral particle apobecs and virus restriction n(6)-methyladenosine and viral infection the rna binding specificity of human apobec3 proteins resembles that of hiv-1 nucleocapsid promiscuous rna binding ensures effective encapsidation of apobec3 proteins by hiv-1 broad antiretroviral defence by human apobec3g through lethal editing of nascent reverse transcripts apobec3 host restriction factors of hiv-1 can change the template switching frequency of reverse transcriptase the innate antiviral factor apobec3g targets replication of measles, mumps and respiratory syncytial viruses apobec3-mediated restriction of rna virus replication zika virus noncoding sfrnas sequester multiple host-derived rna-binding proteins and modulate mrna decay and splicing during infection key: cord-339341-c2o42b5j authors: matibag, gino c.; igarashi, manabu; la porte, ron e.; tamashiro, hiko title: advocacy, promotion and e-learning: supercourse for zoonosis date: 2005-09-01 journal: environmental health and preventive medicine doi: 10.1007/bf02897702 sha: doc_id: 339341 cord_uid: c2o42b5j this paper discusses the history of emerging infectious diseases, risk communication and perception, and the supercourse lectures as means to strengthen the concepts and definition of risk management and global governance of zoonosis. the paper begins by outlining some of the key themes and issues in infectious diseases, highlighting the way which historical analysis challenges ideas of the ‘newness’ of some of these developments. it then discusses the role of risk communication to public accountability. the bulk of the paper presents an overview of developments of the internet-based learning system through the supercourse lectures that may prove to be a strong arm for the promotion of the latest medical information particularly to developing countries. we live in a dangerous world. yet it is also a world far safer in many ways than it has ever been. diseases that only recently were mass killers have been all but eradicated. advances in public health, medicine, environmental regulation, food safety, and worker protection have dramatically reduced many of the major risks we faced just a few decades ago. governance refers to how societies structure responses to the challenges they face. analyses of emerging and re-emerging infectious diseases (eids) have made it clear that national and international societies are confronting increased microbial threats (1) (2) (3) . whether the focus is bioterrorism, hiv/aids, severe acute respiratory syndrome (sars), or avian influenza, germs increasingly pose dangers to human societies. germ governance concerns how societies, both within and beyond national borders, structure their responses to pathogenic challenges (4) . the global nature of the microbial threat requires that governance address the borderless challenges presented by infectious diseases. the emergence of sars is a major global public health threat that requires a coordinated global response in terms of continued and improved surveillance and of research into a number of important public health issues. while much has been learnt about sars since it was brought to international attention in march 2003, there remain many unanswered questions about where it came from, how it spreads, and the effectiveness of public health and other measures employed to control the disease. the overall goal of the "supercourse for zoonosis" is to show the most recent development in the knowledge of sars and other zoonotic diseases such as avian influenza and bovine spongiform encephalopathy (bse), inter alia, which have significant global impact not only on health but also on the economy. the specific objectives of "supercourse for zoonosis" are to develop a set of educational materials for the control of zoonotic diseases, to disseminate them effectively via the internet, to facilitate their use in the prevention and control of the diseases, and to promote human health while minimizing their economic impact. in the light of all these advances, it is most appropriate that all countries remain vigilant, not only with sars, but also to all zoonotic diseases that may toll the productivity of human, animal, ecological, and economical sectors of our daily lives. emerging infections (eis) can be defined as infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range (5) . re-emerging and resurging infections are those that existed in the past but are now rapidly increasing either in incidence or in geographical or human host range (2) . the term deliberately emerging refers to both naturally occurring microbial agents such as anthrax (3) , and to bioengineered microorganisms such as those created by the insertion of genetic virulence factors that produce or exacerbate disease. about 15 million (more than 25%) of 57 million annual deaths worldwide are estimated to be related directly to infectious diseases; this figure does not include the additional millions of deaths that occur as a consequence of past infections (for example, streptococcal rheumatic heart disease), or because of complications associated with chronic infections, such as liver failure and hepatocellular carcinoma in people infected with hepatitis b or c viruses (6) . the burden of morbidity (ill health) and mortality associated with infectious diseases falls most heavily on people in developing countries (7), and particularly on infants and children (about three million children die each year from malaria and diarrheal diseases alone (6)). in developed nations, infectious disease mortality disproportionately affects indigenous and disadvantaged minorities (8) . bacteria and viruses existed long before humans evolved, and bacterial diseases probably co-evolved with each species. many bacterial diseases that we see today have been around for as long as we have, others may have developed later. many examples can be cited in addition to the black death and the 1918 influenza pandemic, such as certain biblical pharaonic plagues and the unidentified plague of athens, which heralded the end of greece's golden age (9) . importation of smallpox into mexico caused 10-15 million deaths in 1520-1521, effectively ending aztec civilization (10, 11) . with the beginning of microbiology, pathogens became apparent. the establishment of the germ theory and the identification of specific microbes as the causative agents of a wide variety of infectious diseases (12) (13) (14) led to enormous progress, notably in the development of vaccines and ultimately of antimicrobials (14) . by the 1950s, which had witnessed the widespread use of penicillin, the development of polio vaccines and the discovery of drugs for tuberculosis, complacency had set in (15) , and in 1967, the us surgeon general stated that the war against infectious diseases has been won (16) . some experts remained skeptical, aware of the current lessons from history. they were less persuaded by successes than alarmed by failures, such as the lack of progress against infections in the developing world and the global spread of antimicrobial resistance. the emergence of aids led to renewed appreciation of the inevitability and consequences of the emergence of infectious diseases (17) (18) (19) (20) (21) (22) (23) . in the past 25 years, some of the factors that resulted in aids have also led to the re-emergence of historically important diseases such as cholera, diphtheria, trench fever and plague. many re-emergences have been catalyzed by wars, loss of cohesion, and natural disasters such as earthquakes and floods, indicating the importance not only of microbial and viral factors, but also of social and environmental determinants (17) (18) (19) (20) (21) (22) (23) . these infections are those that have not previously been recognized in man. many diverse factors contribute to their emergences (table 1) . numerous microbial, host and environmental factors interact to create opportunities for infectious agents to evolve into new ecological niches, reach and adapt to new hosts, and spread more easily between them. at the end of 2004, an estimated 39.4 million (range 35.9-44.3 million) people around the world were living with hiv, including the 4.9 million (range 4.3-6.4 million) people who acquired hiv in 2004. the epidemic claimed an estimated 3.1 million (range 2.8-3.5 million) lives in 2004. sub-saharan africa remains the most affected region and is home to about 65% of the total number of people living with hiv worldwide (18) . before jumping to humans an estimated 60-70 years ago (24) , perhaps through consumption of 'bush meat' from nonhuman primates, hiv-1 and hiv-2 had ample opportunity to evolve in hosts that were genetically similar to man (the chimpanzee, pan troglodytes, and the sooty mangabey, cercocebus atys). but hiv/aids may never have emerged had it not been for disruptions in the economic and social infrastructure in postcolonial sub-saharan africa. increased travel, the movement of rural populations to large cities, urban poverty and a weakening of family structure, all these promoted sexual practices such as promiscuity and prostitution that facilitate hiv transmission (24) (25) (26) (27) . infections in animals that are transmitted to humans (zoonoses), and those transmitted from one vertebrate to another by an arthropod vector (vector-borne diseases), have repeatedly been identified as ranking among the most important eis (17, 18) . viruses in these groups have co-evolved with specific rodent species whose contact with humans has increased as a result of modern environmental and human behavioral factors. farming, the keeping of domestic pets, hunting and camping, deforestation and other types of habitat destruction all create new opportunities for such infectious agents to invade human hosts (17) (18) (19) (20) (21) (22) (23) . examples include the arenavirus hemorrhagic fevers (argentine, bolivian, venezuelan and lassa hemorrhagic fevers) and hantavirus pulmonary syndrome (hps). variant creutzfeldt-jakob disease (vcjd) is another example of a zoonotic disease emerging in humans. it is caused by the human-adapted form of the prion associated with the emerging epizootic (large-scale animal outbreak) of bovine spongiform encephalopathy (bse), commonly known as mad cow disease. the new bse prion has become uncharacteristically promiscuous: unlike most known prions, it readily infects multiple species in addition to humans. this suggests the possibility of further emerging diseases associated with prions with currently unknown transmissibility to humans (28) . infectious agents indirectly transmitted to or between humans by way of human-modified environments account for other emerging zoonoses. legionnaire's disease, first identified in 1976, is caused by legionella pneumophilia, whose emergence as a human pathogen might not have occurred were it not for the environmental niche provided by air-conditioning systems (18) . campylobacter jejuni and escherichia coli infect agricultural animals, gaining access to humans through food, milk, water or direct animal contact. other enteric pathogens, such as the vibrios which cause classic cholera and the zoonotic protozoa cryptosporidium parvum and cyclospora cayetanensis (18), seem to have come from environmental or animal organisms that have adapted to human-to-human 'fecal-oral' transmission through water. some eis come from microorganisms that once caused familiar diseases, but which now cause new or previously uncommon diseases. streptococcus pyogenes caused a fatal pandemic of scarlet and puerperal fevers between 1830 and 1900 (29) . scarlet fever, then the leading cause of death in children, is now rare, but has largely been supplemented by other streptococcal complications such as streptococcal toxic shock syndrome, necrotizing fasciitis and re-emergent rheumatic fever (31) . streptococcus pyogenes has been studied more extensively, but the basis of severe disease emergence seems to be more complex. many factors associated with streptococcal virulence have been identified in strains bearing the m1 surface protein as well as in other m protein strains, among them bacteriophage-encoded superantigen toxins and a protein known as sic (streptococcal inhibitor of complement), which seems to be strongly selected by human host mucosal factors. several lines of evidence suggest that changes in streptococcal virulence reflect genetic changes associated with phage integration, large-scale chromosomal rearrangements and possibly the shuffling of virulence cassettes (clusters of genes responsible for pathogenicity), followed by rapid human spread and immune selection (31, 32) . infectious agents that are associated with chronic diseases are one of the most challenging categories of newly emerging (or at least newly appreciated) infections. examples include the associations of hepatitis b and c with chronic liver damage and hepatocellular carcinoma, of certain genotypes of human papillomaviruses with cancer of the uterine cervix, of epstein-barr virus with burkitt's lymphoma (largely in africa) and nasopharyngeal carcinoma (in china), of human herpesvirus 8 with kaposi sarcoma, and helicobacter pylori with gastric ulcers and gastric cancer (33) (34) (35) . some data even suggest infectious etiologies for cardiovascular disease and diabetes mellitus (36) , major causes of death and disability worldwide. other associations between infectious agents and idiopathic chronic diseases will inevitably be found. re-emergence is caused by some of the factors that allow for newly emerging infectious diseases, factors such as microbial evolutionary vigor, zoonotic encounters and environmental encroachment. re-emergences or at least cyclical resurgences of some diseases may also be climate-related-for example, the el niño/southern oscillation (enso) phenomenon is associated with resurgences of cholera and malaria (37) . travel has an important role in bringing people into contact with infectious agents (38) . an increase in travelassociated importations of diseases was anticipated as early as 1933, when commercial air travel was still in its infancy (39) . this has since been demonstrated dramatically by an international airline hub-to-hub pandemic spread of acute hemorrhagic conjunctivitis in 1981 (40), by epidemics of meningococcal meningitis associated with the hajj, and more recently by the exportation of epidemic sars (a newly emerging disease) from guangdong province, china, to hong kong, and from there to beijing, hanoi, singapore, toronto and elsewhere (41) . plasmodium falciparum malaria was neglected for several decades, but is now among the most important re-emerging diseases worldwide. years of effective use of dichlorodiphenyltrichloroethane (ddt) had led to the abandonment of other mosquito-control programs, but the insecticide fell into disuse because of mosquito resistance and concerns about the insecticide's potentially harmful effects on humans and wildlife. consequently, malaria has re-emerged, and the situation has been worsened by the development of drug resistance to chloroquine and mefloquine (42) . research efforts focus on the development of vaccines and new drugs, and on re-establishing public health measures such as the use of bed nets (43) . the remarkable re-emergence of tuberculosis was fuelled by the immune deficiencies of people with hiv infection, which greatly increases the risk of latent mycobacterium tuberculosis infections progressing to active disease, and being transmitted to others. inadequate courses of anti-tuberculosis therapy compound the problem, leading to the emergence and spread of drug resistant and multidrug-resistant strains, and a need for more extensive treatment strategies such as directly observed therapy (44) . it has been known for over a century that tuberculosis is a disease of poverty, associated with crowding and inadequate hygiene. the continuing expansion of global populations living in poverty makes tuberculosis more difficult to control. drug resistance, another factor causing microbial and viral re-emergence, may result from mutation or from bacterial acquisition of extraneous genes through transformation or infection with plasmids. sequential emergences of staphylococcus aureus that are resistant to sulpha drugs (1940s), penicillin (1950s), methicillin (1980s) and to vancomycin in 2002 (45)-a last line of antibiotic defense for some multiply drug-resistant bacteria-are troubling. nosocomial enterococcus faecalis became fully resistant to vancomycin by 1988, and then apparently transferred vana resistance genes to co-infecting staphylococci (46) . methicillin-resistant staphylococci are now being isolated from livestock that have been fed with growthpromoting antibiotics (45) , possibly contributing to resistance problems in humans. immune deficiency associated with hiv infection, and with chemotherapy for cancer, immune-mediated diseases and transplantation, has contributed to an enormous global increase in the numbers of immunosuppressed people over the past few decades (probably more than 1% of the world's population), setting the stage for the re-emergence of many opportunistic infections. hiv, which has infected more than 60 million people globally (47) , is the largest single cause of human immune deficiency and markedly increases vulnerability to a wide range of opportunistic pathogens, including pneumocystis carinii, various fungi, tuberculosis, protozoa and herpesvirus (48) . the simultaneous 1999 emergences of encephalitis due to west nile virus (wnv) in the united states and russia (49, 50) reflect abundances of eclectic vector mosquitoes and avian hosts in these locations. both were probably connected to endemic sites by virus carriage in migratory birds and travelers. the remarkable geographical spread of wnv in the five years since its introduction into the western hemisphere reflects an unfortunate confluence of viral promiscuity and ecological diversity (51) . although humans are dead-end hosts for wnv, the risk of infection is greatly increased by marked zoonotic viral amplification and persistence in the environment. although wnv is now a major epidemiological concern in the developed world, dengue remains the most significant and widespread flavivirus disease to have emerged globally (52) . usually transmitted by aedes aegypti mosquitoes, dengue has recently been transmitted by aedes albopticus-a vector switch of potential significance with respect to dengue reemergence (52) . dengue re-emergence is further complicated by disturbing increases in a serious and formerly rare form of the disease, dengue hemorrhagic fever (dengue shock syndrome being its highly fatal form). these severe complications are thought to result from the evolution of dengue viruses to escape high population immunity, seen in increased viral virulence and human immunopathogenesis due to antibody-dependent enhancement of viral infection (53) . cholera is also of interest, not only as an important cause of mortality, but also because of the complexity of factors that determine its re-emergence. both virulent and avirulent strains of these zoonotic bacteria are maintained in the environment and are rapidly evolving in association with phyto-and zooplankton, algae and crustaceans. such environmental strains seem to act as reservoirs for human virulence genes and to undergo gene transfer events that lead to new strains containing further virulence gene combinations (54) . thus, although cholera has appeared to be clinically and epidemiologically stable at least since the third pandemic (in the 1840s), modern evidence suggests that such apparent stability masks aggressive bacterial evolution in complex natural environments. influenza a viruses, which are endemic gastrointestinal viruses of wild waterfowl, have evolved elaborate mechanisms to jump species into domestic fowl, farm animals and humans. periodic gene segment reassortments between human and animal viruses produce important antigenic changes, referred to as 'shifts'. these can lead to deadly pandemics, as occurred in 1888, 1918, 1957 and 1968 (55, 56) . in intervening years, shifted viruses undergo continual but less dramatic antigenic changes called 'drifts,' which allow them partially to escape human immunity raised by previously circulating influenza viruses. influenza drift is an evolutionary success story for the virus. influenza a has a seemingly inexhaustible repertoire of mutational possibilities at several critical epitopes surrounding the viral hemagglutinin site that attaches to human cells. deliberately emerging microbes are those that have been developed by humans, usually for nefarious use. they include microorganisms or toxins produced in a form that would cause maximal harm because of ease of dissemination, enhanced infectivity or heightened pathogenicity (57) . two modern attacks have been well documented. in 1984, an oregon religious cult spiked restaurant salad bars with salmonellae in an attempt to sway a local election (58) . a 2001 anthrax attack (59) , in which a terrorist mailed anthrax-sporefilled letters to prominent figures, including two us senators, resulted in illness in at least 18 people and the death of five of these individuals. the united states, the united kingdom, the russian federation and other nations once had sophisticated offensive bioweapons programs that included the production of weaponized anthrax spores (57) . in japan, the doomsday sect, aum supreme truth, carried out a nerve-gas attack on the tokyo subway in 1995, and made a trial run on an anthrax weapon, using harmless vaccine bacteria as a test (60) . bioterror agents have been grouped into three categories (a, b and c) according to risk (61) . the six category a agents (anthrax, smallpox, plague, tularaemia, viral hemorrhagic fevers and clostridial botulinum toxin) are given top priority because they are highly lethal and readily deployed as weapons. category b and c agents include food-borne and water-borne organisms that incapacitate but usually do not kill. risk is the probability that exposure to a hazard will lead to a negative consequence (62) . risk communication is an interactive exchange of information and opinion on risk among risk assessors, risk managers, and other interested parties (63) . humans tend to fear similar things, for similar reasons. scientists studying human behavior have discovered psychological patterns in the subconscious ways we "decide" what to be afraid of and how afraid we should be. any given risk has a set of identifiable characteristics that help predict what emotional responses that risk will trigger. people's perceptions of the magnitude of risk are influenced by factors other than numerical data (64) . the factors influencing risk perception are summarized in table 2 . merely disseminating information without regard for communicating the complexities and uncertainties of risk does not necessarily ensure effective risk communication. wellmanaged efforts will help ensure that messages are constructively formulated, transmitted, and received and that they result in meaningful actions. the seven cardinal rules of risk communication are demonstrated in table 3 . the fundamental goal of risk communication is to provide meaningful, relevant and accurate information, in clear and understandable terms, targeted to a specific audience (63) . the goals of risk communication are summarized in table 4 . it may not resolve all differences between interested parties, but may working in earthquake-prone areas hormone replacement therapy 4. risks perceived to be fairly distributed are more accepted than risks to be unfairly distributed. gun shots (in japan) traffic accidents 5. risks perceived to be natural are more accepted than risks perceived to be manmade. radiation from mobile phones radiation from the sun 6. risks perceived to be statistical are more accepted than risks perceived to be catastrophic. eaten by a shark (catastrophe) heart disease (statistics) 7. risks perceived to be generated by a well-known source are more accepted than risks perceived to be generated by a less known source. private industry government 8. risks perceived to be familiar are more accepted than risks perceived to be exotic. 9. risks perceived to affect adults are more accepted than risks perceived to affect children. asbestos exposure for children asbestos exposure in workplace 10. risks perceived with less uncertainty are more accepted than risks with high uncertainty. new technology conventional technology 11 . risks perceived that could directly affect others are more accepted than risks that could affect oneself. table 3 seven cardinal rules of risk communication* 1. accept and involve the public as a partner. the goal is to produce an informed public, not to defuse public concerns or replace actions. different goals, audiences, and media require different actions. 3. listen to the public's specific concerns. people often care more about trust, credibility, competence, fairness, and empathy than about statistics and details. 4. be honest, frank, and open. trust and credibility are difficult to obtain; once lost, they are almost impossible to regain. 5. work with other credible sources. conflicts and disagreements among organizations make communication with the public much more difficult. 6. meet the needs of the media. the media are usually more interested in politics than risk, simplicity than complexity, danger than safety. 7. speak clearly and with compassion. never let efforts prevent acknowledging the tragedy of an illness, injury, or death. people can understand risk information, but they may still not agree; some people will not be satisfied. * from: covello v and allen f. 1988. (reference no. 67) lead to a better understanding of those differences. it may also lead to more widely understood and accepted risk management decisions. effective risk communication should have goals that build and maintain trust and confidence. it should facilitate a higher degree of consensus and support by all interested parties for the risk management options being proposed. many considerations for effective risk communication, especially those involving the public, can be grouped in a sequence following the systematic approach of the risk communication process. this starts with gathering background and needed information, followed by the preparation and assembly of the message and its dissemination and distribution, with a follow-up review and evaluation of its impact (63) . the general considerations for effective risk communication are demonstrated in table 5 . risk communication efforts and programs need to be evaluated both regularly and systematically to determine their effectiveness and to provide for change when needed. communication aims and objectives need to be clearly stated if an evaluation is to be effective. this could include the proportion of at-risk population to be reached, adoption of appropriate risk reduction practices, and the extent of resolution of the crisis. it is important to learn from both positive and negative risk communication experiences, in order to adjust and improve ongoing communication activities. only through systematic evaluations, which are performed throughout the communication process, can that process be strengthened (63) . globalization of disease prevention lectures, through the supercourse prevention project, is funded by the us national institutes of health (nih). the supercourse is an internet library of lectures on prevention, shared for free by 10,000 table 4 goals of risk communication* 1. promote awareness and understanding of these specific issues under consideration during the risk analysis process, by all participants; 2. promote consistency and transparency in arriving at and implementing risk management decisions; 3. provide a sound basis for understanding the risk management decisions proposed or implemented; 4. improve the overall effectiveness and efficiency of the risk analysis process; 5. contribute to the development and delivery of effective information and education programs, when they are selected as risk management options; 6. foster public trust and confidence in the safety of the food supply; 7. strengthen the working relationships and mutual respect among all participants; 8. promote the appropriate involvement of all interested parties in the risk communication process; and, 9. exchange information on the knowledge, attitudes, values, practices and perceptions of interested parties concerning risks associated with food and related topics. table 6 . japan has associated with the global health network through supercourse japan (66) where a series of lectures in health, environment and sustainable development, epidemiology for decision-making, and zoonosis have so far been developed. the supercourse on health, environment and sustainable development was designed to provide an overview on health and environment in the context of sustainable development for public health students around the world, as well as decision makers, community leaders, scientists and professionals in government and non-governmental organizations, who are interested in health and environmental linkages in sustainable development (66) . the supercourse on epidemiology for decision-making provides a learning resource for students of environmental and occupational epidemiology, and its main purpose is to promote the understanding and application of epidemiology in the prevention of environmental and occupational disease and the promotion of health (66) . the supercourse on zoonosis aims to disseminate rapidly the latest information on animal diseases transmittable to humans. severe acute respiratory syndrome (sars) and bovine spongiform encephalopathy (bse) are some of the lectures discussed which will help in information sharing to developing countries (66) . a joint action plan through the 'the pacific islands forum' was created in 2003. its five priority policy targets are: 1) enhancement of security in the pacific region, 2) creation of a safer and more sustainable environment, 3) improvement in education and human resources development, 4) improvement in health, and 5) promotion of more vigorous and continued trade and economic growth. the countries in the pacific region face a contradictory dilemma: some of the poorest countries have the most expensive telecommunications, yet information and telecommunication technology (ict) need to be extensively utilized. in these circumstances telemedicine, such as for remote clinical and pathological diagnosis, is too expensive and technically demanding to be widely used. since information in the future will be technology-and network-based, it is imperative for "supercourse asia" to fully exploit the potential of ict for health education and health service development in the region. the advantages of this approach are to use the most appropriate, inexpensive, opensource, and low-band ict, and to operate it through active national networking. the supercourse asia network (scan) is an offshoot of this initiative and aims that all children and adults will have equal access to health and education of sufficient quality to empower them to break the poverty cycle, to improve their quality of life (qol), and to participate effectively in national development. the mission is to alleviate poverty and improve health and qol of developing countries in the pacific region through advances in a cost-effective ict-based health educational system. the internet is the most inexpensive and speed efficient means to penetrate the remote places such as the pacific islands. japan, a leader of modern technology in the region, is in the best position to provide these technological and educational tools to contribute to the well-being of people in the region. the scan is a group of primarily academics, united by a common belief: the internet is the best way to disseminate knowledge, especially knowledge about health promotion and prevention. they are working towards facilitating the dissemination of health-related information over the internet and improving teaching in the field of education, social/preventive medicine, public health and epidemiology. the members are voluntary professionals in academia, healthcare, telecommunications, the government, ngos, and other public and private sector organizations who will work together towards developing "supercourse asia." they are not only active members of the scan but also the main contributors of "supercourse asia" lectures, reviewers, translators, and its major user group. the challenges for the scan are 1) how to effectively utilize the national networks among the participating countries and within each participating country that may be geographically remote, 2) how to evaluate the effects of the scan on the advancement of health and qol, and the alleviation of poverty though "supercourse asia" in the region, and 3) how to sustain the network activities for many years to come. currently, the public has become concerned with information and safety, however, gaps between the understanding of safety and assurance are widening. health advocacy and promotion through e-learning are becoming more important in the framework of risk communication for the community. the supercourse lectures will serve as an international platform for sharing information, lectures, and ideas. understand the public perception of the risk through such means as risk surveys, interviews and focus groups expect different people to see the risk differently. preparation/assembly 1. avoid comparisons between familiar risks and new risks, as they may seem flippant and insincere unless presented properly. 2. recognize and respond to the emotional aspects of risk perceptions. speak with sympathy and never use logic alone to convince an audience characterized by emotion express risk in several different ways, making sure not to evade the risk question explain the uncertainty factors which are used in risk assessment and standard setting maintain an openness, flexibility, and recognition of public responsibilities in all communication activities accept and involve the public as a legitimate partner by describing risk/benefit information and control measures in an understandable way. 2. share the public's concern rather than deny it as not legitimate or as unimportant. be prepared to give people's concerns as much emphasis as the risk statistics be honest, frank, and open in discussing all issues if explaining statistics derived from risk assessment, explain the risk assessment process before presenting the numbers coordinate and collaborate with other credible sources evaluate the effectiveness of risk messages and communication channels. 2. emphasize action to monitor, manage, and reduce risk plan carefully and evaluate efforts it is based on the open source model, which allows free redistribution, shows the source code, and allows modifications and derived works this is especially useful for developing countries and minority populations with limited access to resources it aims to overcome the digital divide by improving access in places with low bandwidth internet connection it is a teaching support system -differing from a traditional distance education system it provides timely information for action -one of the greatest advantages of having a regional faculty base with varied areas of expertise which are very important in the field of public health (e.g., lectures on sars from the members in china and singapore, where it is most rampant) it is a "hyper-text comic book format hypertext links from text, images, tables, and pictures take the reader to other relevant supercourse lectures, images, or other websites on the internet oaks sc editors. emerging infections; microbial threats in the united states world health organization. removing obstacles to healthy development global defence against the infectious disease threat. world health organization globalisation of prevention education: a golden lecture factors in the emergence of infectious diseases threats to global health and survival; the growing crises of tropical infectious diseasesan unfinished agenda emerging infectious diseases among indigenous peoples epidemiology of the plague of athens the columbian exchange: biological and cultural consequences of 1492. greenwood, westport: connecticut princes and peasants. smallpox in history. chicago: univ untersuchungen über bacterien. v. die aetiologie der milzbrand-krankheit, begründet auf die entwicklungsgeschichte des bacillus anthracis spreading germs: diseases, theories, and medical practice in britain the greatest benefit to mankind: a medical history of humanity from antiquity to the present the evolution and eradication of infectious diseases infectious diseases: considerations for the 21st century oaks sc editors. emerg infect. microbial threats to health in the united states lederberg j editors. microbial threats to health in the united states: emergence, detection and response emerging and re-emerging infectious diseases: a multidisciplinary perspective emerging and re-emerging infectious diseases emerging infectious diseases in the 21st century emerging and re-emerging infectious diseases the origins of acquired immune deficiency viruses: where and when population migration and the spread of types 1 and 2 human immunodeficiency viruses the aids knowledge base slowing heterosexual hiv transmission variant creutzfeldt-jakob disease and the acquired and transmissible spongiform encephalopathies severe streptococcal infections in historical perspective emerging infections group a streptococcus: allelic variation, population genetics, and hostpathogen interactions genome sequence of a serotype m3 strain of group a streptococcus: phage-encoded toxins, the highvirulence phenotype, and clone emergence identification if herpesvirus-like dna sequences in aids-associated kaposi's sarcoma microbes and malignancy: infection as a cause of human cancers helicobacter pylori-associated diseases current clinical topics in infectious diseases el niño and health island epidemics epidemiology in relation to air travel acute hemorrhagic conjunctivitis: dealing with a newly emerging disease the severe acute respiratory syndrome two worlds of malaria research toward vaccines against malaria the global situation of mdr-tb methicillin (oxacillin)-resistant staphylococcus aureus strains isolated from major food animals and their potential transmission to humans centers for disease control prevention. staphylococcus aureus resistant to vancomycin-united states joint united nations programme on hiv/aids. aids epidemic update surveillance for aids-defining opportunistic illnesses, 1992-1997. mmwr 48. cdc surveillance summary no. ss-2 the outbreak of west nile virus infection in the new york city area in 1999 outbreak of west nile virus infection west nile virus: epidemiology and ecology in north america dengue and dengue hemorrhagic fever antibody-dependent enhancement of infection and the pathogenesis of viral disease molecular ecology of toxigenic vibrio cholerae the next influenza pandemic: lessons from hong kong a molecular whodunit the chilling true story of the largest covert biological weapons program in the world-told from the inside by the man who ran it a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars investigation of bioterrorism-related anthrax, united states, 2001: epidemiologic findings japanese sect was close to bioterrorism journal says: live anthrax found at office threats in bioterrorism: i. cdc category a agents risk: a practical guide for deciding what's really safe and what's really dangerous in the world around you world health organization. the application of risk communication to food standards and safety matters, a joint fao/ who expert consultation supercourse: epidemiology, the internet, and global health seven cardinal rules of risk communication key: cord-333730-qsx0m68e authors: tsai, y. c.; tsai, t. f. title: oral disease-modifying antirheumatic drugs and immunosuppressants with antiviral potential, including sars-cov-2 infection: a review date: 2020-09-03 journal: ther adv musculoskelet dis doi: 10.1177/1759720x20947296 sha: doc_id: 333730 cord_uid: qsx0m68e there have been several episodes of viral infection evolving into epidemics in recent decades, and severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the latest example. its high infectivity and moderate mortality have resulted in an urgent need to find an effective treatment modality. although the category of immunosuppressive drugs usually poses a risk of infection due to interference of the immune system, some of them have been found to exert antiviral properties and are already used in daily practice. recently, hydroxychloroquine and baricitinib have been proposed as potential drugs for sars-cov-2. in fact, there are other immunosuppressants known with antiviral activities, including cyclosporine a, hydroxyurea, minocycline, mycophenolic acid, mycophenolate mofetil, leflunomide, tofacitinib, and thalidomide. the inherent antiviral activity could be a treatment choice for patients with coexisting rheumatological disorders and infections. clinical evidence, their possible mode of actions and spectrum of antiviral activities are included in this review article. lay summary: immunosuppressants often raise the concern of infection risks, especially for patients with underlying immune disorders. however, some disease-modifying antirheumatic drugs (dmards) with inherent antiviral activity would be a reasonable choice in the situation of concomitant viral infections and flare up of autoimmune diseases. this review covers dmards of treatment potential for sars-cov-2 in part i, and antiviral mechanisms plus trial evidence for viruses other than sars-cov-2 in part ii. infections are a common concern of immunosuppressive drugs. however, some immunosuppressants or disease-modifying antirheumatic drugs (dmards) show antiviral activity and may be safely used or even beneficial in patients with selected concomitant viral infections. certain dmards may even be considered as an alternative treatment for recalcitrant infections. moreover, the concomitant use of immunosuppressants and antiviral agents was proved to be more effective than antiviral agent monotherapy in some reports. 1 the antiviral property of immunosuppressants may act through (a) direct virucidal activity, (b) blockage of receptors, (c) inhibition of necessary molecules for viral replication in the hosts, or (d) amelioration of inflammatory symptoms. also, control of inflammation may decrease the susceptibility or enhance host ability to defend against viral infection. the are indicated to treat and prevent malaria. they are also used as dmards for rheumatoid arthritis, lupus erythematosus, and porphyria cutanea tarda. in addition, the application for viral infections in off-label use has recently been investigated vigorously. the antiviral activity is through blocking the virus/cell fusion via increasing endosomal ph and hindering the glycosylation of cellular receptors ( figure 2 ). 4 in vitro cq revealed low half-maximal effective concentration (ec50) and high half-cytotoxic concentration (cc50) for covid-19. 5 a preliminary study conducted in china showed benefits in pneumonia image, shortening of disease course, and promoting a virus-negative conversion compared with control group. 6 then, four completed clinical studies demonstrated favorable outcomes in clinical and radiologic amelioration, while another two randomized controlled trials (rcts) illustrated no statistically significant change compared with control arms. [7] [8] [9] [10] [11] [12] based on the inhibitory effect of azithromycin against ebola and zika viruses in vitro, and the possibility of preventing from progressing to severe respiratory tract infections, two french trials which combined the use of azithromycin and hcq revealed better efficacy. 7, 9 however, further studies are still needed to draw conclusions because most of these studies bear limitations including selection bias, allocation bias, or insufficient case numbers. several multicenter, double-blind, and well-designed controlled trials are already underway to assess the efficacy and safety of cq or hcq in the treatment of covid-19 pneumonia. in the absence of other confirmed effective therapy specific to sars-cov-2, both drugs are currently still listed in the treatment guidelines (table 1) . baricitinib, blocking janus kinase (jak)1 and jak2, is approved for rheumatoid arthritis and has been investigated in atopic dermatitis. sars-cov-2 binds on the angiotensin-converting enzyme 2 (ace2) receptors and enters lung cells through receptor-mediated endocytosis. some of the numb-associated kinase (nak) family members, ap2-associated protein kinase 1 (aak1) and cyclin g-associated kinase (gak), are hypothesized to regulate the ace2-mediated endocytosis. baricitinib demonstrated high affinity to aak1 and in one controlled open-label study (n = 24), patients were given either baricitinib 4 mg/day plus lopinavir-ritonavir or antiretroviral plus hydroxychloroquine (control group) for 2 weeks. significant improvement of symptoms and laboratory results, no intensive care unit transfer (versus 33% transfer in control cases), and 58% discharge from wards (versus 8% in control) was shown among the baricitinib-treated individuals. 14 in addition to antiviral property, baricitinib has been suggested as an approach for a cytokine storm syndrome, which features hypercytokinemia and multi-organ failure. elevated ferritin and il-6 in covid-19 cases were predictive of a high mortality rate according to a china retrospective study. 17 baricitinib inhibits cytokines including il-2, il-6, il-10, interferon gamma (ifn-γ), and granulocytecolony-stimulating factor (g-csf) 13, 18 and may bring the benefit of immune reconstruction which could be used in rapidly progressive diseases. however, there are competing ideas about the interference of jak inhibitors with ifn-mediated antiviral activities. ifns prohibit viral spreading in the early phase of infections. in animal models of sars and middle east respiratory syndrome (mers), ifn-α and ifn-β showed benefit at the early stage but were harmful at the late phase. patients with severe sars who died of hypoxemia revealed high ifn-α, -γ, while those discharged dmards, disease-modifying anti-rheumatic drugs. journals.sagepub.com/home/tab 5 from hospital had low ifn-α, -γ. therefore, some experts suggested baricitinib's use in the situation of hyperinflammation and cytokine syndrome, rather than in those with mild diseases. in fact, clinical trials have commenced to evaluate the optimal timing, duration, and safety of baricitinib in viral infections, including sars-cov-2. [19] [20] [21] cyclosporine a cyclosporine a (csa) is indicated for rheumatoid arthritis, psoriasis, organ transplants to prevent injection, and keratoconjunctivitis sicca. it is also used in severe atopic dermatitis, chronic urticaria, pyoderma gangrenosum kimura disease, acute systemic mastocytosis, and ulcerative colitis. csa inhibits lymphocyte function, mainly t cells, by forming a complex with cyclophilin. cyclophilin-csa complex binds on the calcineurin, which blocks the dephosphorylation of nuclear factor of activated t cells (nf-at). this interferes with entry of nf-at into the t-cell nucleus and further suppresses cytokine production such as il-2. severe covid-19. experimentally, csa targets cyclophilin d to inhibit mitochondrial permeability transition pore (mptp) opening and rescues mitochondria from apoptosis. [22] [23] [24] moreover, melanoma-differentiation-activated protein 5 (mda5), an rlr helicase and putative cytoplasmic receptor of sars-cov-2, is also the target antigen of clinically amyopathic dermatomyositis (cadm). patients with mda5 plus cadm have higher risks of developing rapidly progressive interstitial lung diseases and respiratory failure, while this could be reversed by calcineurin inhibitors. based on these hypothetical functions, csa was proposed as a modulator for cytokine storm syndrome in covid-19 infections. 25 mycophenolic acid (mpa), an active metabolite of mycophenolate mofetil (mmf), inhibits inosine monophosphate dehydrogenase (impdh), an essential enzyme in the de novo purine synthesis pathway. impdh inhibition especially influences t and b lymphocytes because they use almost a de novo pathway to synthesize (minimally use a salvage pathway). mmf and mpa are utilized in organ transplantation, crohn's disease, and as steroid-sparing agents for conditions such as pemphigus, behçet's disease, and lupus erythematosus. although they were associated with higher risk of opportunistic infections including herpes zoster, cytomegalovirus (cmv), and bk virus (bkv) nephropathy, literature also revealed its possible benefit for hiv and influenza virus. 26, 27 in vitro: mmf showed low ec50 (0.47 μmol/l) in sars-cov-2-infected vero e6 cells, while the ec50 of remdesivir, as a positive control, was 0.77 μmol/l. besides, mmf probably inhibited sars-cov-2 through impdh and especially dihydroorotate dehydrogenase (dhodh). dhodh is another essential enzyme for pyrimidine synthesis, and mmf might control viral infection by depleting the intracellular pyrimidine pools. 28 thalidomide thalidomide, a derivative of glutamic acid, is approved for erythema nodosum leprosum and is also used in many conditions such as prurigo nodularis, pyoderma gangrenosum, bechet's disease, lupus erythematosus and erythema multiforme. it exerts anti-inflammatory effect through cereblon e3 ubiquitin ligase as the primary target and thus inhibits chemotaxis of leukocytes, monocytes as well as the production of tumor necrosis factor (tnf)-alpha, il-8, and il-12. case report: a 45-year-old woman with critical symptoms of covid-19 was treated by thalidomide 100 mg every 24 h. after the first day use of thalidomide, clinical conditions including oxygen index improved. cytokines such as il-6, il-10, ifn-γ all decreased to normal range. 15 proposed mechanisms are as follows: thalidomide inhibits nf-κb, which further suppresses the production of pro-inflammatory cytokines such as tumor necrosis factor alpha (tnf-α) and il-8, and prevents the cytokine surge. it also regulates immune function by activating t cells and t-cell receptors. moreover, the sedative and antiemetic property of thalidomide helps anxious patients calm down, which reduces oxygen consumption. 15 now, at least one clinical trial has been conducted to investigate the efficacy and safety of thalidomide as an adjuvant therapy for covid-19 pneumonia. 29 part ii: dmards with antiviral potential other than sars-cov-2 many oral dmards have inherent antiviral activity and could be the treatment of choice for patients with coexisting immune-based diseases and infections. especially when the infection is still in progression, choosing dmards with anti-microbial evidence would bring double benefits for better infection control without sacrificing underlying disease management. the antimicrobial mechanisms of dmards are often distinct from their immunomodulatory pathway, and the efficacy is different in viral species (tables 2, 3 leflunomide is approved for rheumatoid arthritis and psoriatic arthritis (not in the united states). leflunomide inhibits the synthesis of pyrimidine via acting on the mitochondrial enzyme dhodh; therefore, rapidly dividing cells, especially lymphocytes, are suppressed. on the other hand, leflunomide showed antiviral activity at least for cmv, bkv, and hiv. it works by teriflunomide, the active metabolite of leflunomide, which disrupts nucleocapsid tegumentation, and thus prevents virion assembling, rather than influences the de novo pyrimidine synthesis pathway. 97 herpes simplex virus a case report with perianal hsv-2 lesions in an acyclovir-resistant hiv patient significantly improved with leflunomide 40 mg, twice a day. 98 another hiv patient with hsv-1/hsv-2 pseudo-tumors on the perineum and scrotum only slightly improved with valacyclovir, foscarnet, and imiquimod. after 9 months of leflunomide, complete regression of the lesions was noted. leflunomide has both immunomodulation and antiviral activities in the hsv pseudotumors because pseudotumor formation is an immune reconstruction phenomenon in hiv patients. 99 human immunodeficiency virus an rct (n = 18) demonstrated leflunomide decreased the activation and cycling of cd4+ t cells. the expression of hiv co-receptors ccr5 and cxcr4 was also reduced compared with placebo. 102 three patients with atopic dermatitis treated with azathioprine developed multiple verrucae and molluscum contagiosum. due to treatment resistance, azathioprine was switched to leflunomide (100 mg loading 3 days, then 20 mg/day). all the lesions subsided in three patients within 2 months of leflunomide treatment. 103 multiple recalcitrant verrucae in three and molluscum in one of renal allograft recipients cleared after switching from mmf to leflunomide. 104 leflunomide can serve as a potential option for patients with skin warts or molluscum concomitant with immune conditions that require immunosuppressants. leflunomide was regarded as an add-on treatment for multi-drug-resistance cmv infection. in vitro anti-cmv properties of leflunomide were not through blocking the replication of viral dna, so it is effective even in patients with direct antiviral drug-resistance history. 105 disseminates to the urinary-tract system and lives there persistently. a sudden increase of bk-virusassociated nephropathy is related to the administration of potent immunosuppressants such as mmf and tacrolimus. leflunomide is now generally accepted as a second choice after reduction of immunosuppressive agents. however, in a phase ii rct (n = 46), viremia was decreased in the group of leflunomide active metabolites, but no significant improvement of renal function was noted. 108 treatment options for rsv are limited to supportive care or ribavirin with only marginal effectiveness. leflunomide showed a potent, dose-dependent anti-rsv activity in cell cultures. 92 also, pulmonary viral loads were prominently reduced in cotton rats, even if there was a 3-day delay of leflunomide administration after viral inoculation. 109 leflunomide offers a dual benefit of both viral-load reduction and anti-inflammatory effects that attenuate the destruction of cytokine-related diseases. tofacitinib, a jak1 and jak3 inhibitor, is indicated in rheumatoid arthritis, ulcerative colitis, and psoriatic arthritis. it is also used off label for vitiligo and alopecia areata. tofacitinib treats inflammatory diseases by interfering with the activation of the jak/signal transducers and activators of transcription (stat) pathway, which inhibits gene transcription, and cytokine production is thereby reduced. htlv-1, a retrovirus, has been linked to diseases such as adult t-cell lymphoma/leukemia (atll), htlv-1-associated myelopathy (ham), and uveitis. ex vivo and animal studies revealed positive results of tofacitinib for atll and ham. 110 htlv-1-encoded tax protein activates il-2, -9, -15, which further trigger jak3-stat5 pathway. accumulating data demonstrated a major role of jak3 in the pathophysiology of atll. 114 as a result, tofacitinib targeting jak3 has been suggested as a therapeutic strategy in future studies. hydroxyurea, a deoxyribonucleic acid (dna)synthesis inhibitor, belongs to the antineoplastic medications. however, it may be used as a second-line drug for psoriasis and palmoplantar pustulosis 115 based on the ability to slow down the rapid division of keratinocytes. bone marrow suppression is the major and common adverse effect of hydroxyurea. hydroxyurea demonstrated promising results in reducing hiv rna viral loads in five placebocontrolled clinical trials. among all the trials, hydroxyurea was combined with didanosine, a nucleoside analog reverse-transcriptase inhibitor (nrti). however, one should be reminded that decreased cd4 counts were noted in some studies. therefore, close follow up of hematologic change is required in daily practice. [65] [66] [67] [68] [69] in vitro studies demonstrated the antiviral modes of hydroxyurea. first, hydroxyurea depletes deoxynucleoside triphosphate (dntp) pools, which impedes dna synthesis and in turn slows down the production of viral dna. second, hydroxyurea enhances nrti phosphorylation and reduces resistance to nrtis. this may partially explain the benefits of adding hydroxyurea to nrti for viral control. finally, cytotoxic effect of hydroxyurea makes cellular division of cd4+ t cells decline. this enables hydroxyurea to block hiv proliferation, because hiv could only replicate in dividing cd4+ t cells. 64 although the mode of action of hydroxyurea for hcv, hbv, hsv, and b19v is unknown, viral replications were inhibited by hydroxyurea in in vitro studies. 70, [74] [75] [76] small-scaled clinical trials showed significant reduction of hcv rna levels and hbv viral loads in chronic hcv and hbv carriers, respectively. 71, 72 however, there is a case report of an elderly patient with essential thrombocythemia experiencing reactivation of hbv during treatment with hydroxyurea. 73 a retrospective review of children with sickle cell anemia demonstrated decreased requirement of blood transfusion and attenuation of clinical symptoms when using hydroxyurea in patients with b19v infection. 77 minocycline, a second-generation of tetracyclines, is frequently used for bacterial infections, acne, and rheumatoid arthritis. the small size and lipophilic nature facilitate its penetration into blood-brain barrier easily. the neuroprotection and anti-inflammation effects 116, 117 brought interest in the treatment of virus-induced encephalitis such as hiv, japanese encephalitis virus (jev), and reovirus. the antiviral property is not clearly known but seems to be diverse, including neuroprotective, antiapoptotic, interference of viral protein expression, and anti-inflammatory effects. in microglial cell culture, minocycline reduced viral replication by 71-96%. 78 in vivo, macaque monkeys treated with minocycline showed less destruction of axons and less replication of viruses in the central nervous system. the experiment suggested that the antiviral effect of minocycline was through reducing the activation of monocytes and hence, viral replication was blocked. 79 nevertheless, two double-blind, randomized, placebo-controlled human studies revealed that under minocycline 100 mg twice daily, there was no difference in cognitive function compared with placebo. 80, 81 japanese encephalitis virus minocycline showed high efficacy in animal models and in vitro studies for the treatment of jev. 82 33, 34 four clinical trials showed positive results of hiv control, either in the reduction of immune activation or lowering the vertical transmission. [35] [36] [37] [38] however, another two trials (one rct, one single-arm) revealed no efficacy. 39, 40 as for dengue, 41-44 chikungunya, [45] [46] [47] influenza a viruses, [48] [49] [50] [51] and hcv, 52,53 paradoxical outcomes were found in the literature. therefore, the utilization of cq in these viral diseases still needs further investigation. the major concern of zika virus infection is that it can transmit from placenta to fetus and cause microcephaly or congenital defects. cq prevented zika virus internalization in cell cultures and reduced morbidity or mortality in mice. in addition, it prevented fetal mice from microcephaly. 54 therefore, cq might be a potential treatment waiting for clinical verification. hcq had been reported to downregulate the expression of ifn genes and reduce the production of type i ifns. this phenomenon was noted in vitro 118 and in human studies of autoimmune diseases. 119 since ifns are crucial in innate immunity to defend viral infections, the usage of hcq may raise concerns about the counter effects in viral control. nevertheless, opposite results were also presented: hcq activated ifnβ signaling pathways in cell studies of dengue virus. 120 betaretrovirus is regarded as one of the environmental factors triggering the recurrence of primary biliary cirrhosis (pbc) after liver transplantation. earlier and more severe recurrence of pbc occurred with tacrolimus compared with csa as an immunosuppressant. this may be partially explained by the antiviral activity of csa. according to an in vitro study, it was suggested that csa interrupted viral replication through inhibiting viral protein synthesis, gag and envelope assembly, and particle budding. 63 the combination of mmf and highly active antiretroviral therapy improved the control of viral replication and delayed viral-load rebound in a randomized pilot study (n = 17 the effectiveness of thalidomide for ks might be related to anti-angiogenesis, and experts hypothesized the modulation of the immune system to trigger an antiviral action. the treatment of immune-based diseases has been revolutionized by the introduction of target therapy, mainly biologics. compared with biologics, conventional synthetic dmards exert broad-spectrum functionality. dmards work through immunosuppressive and antiinflammatory effects with the possibility of higher infection risk. however, many none-biologic dmards demonstrate antiviral activities instead. although in most instances, the antiviral activity of dmards is based on in vitro or small-scale controlled studies, this property would be useful in the choice of dmards for patients with concomitant viral infections. also, the combinational use of antiviral drugs and dmards has been shown to be more effective and less resistant in the control of some viral infections. furthermore, in the face of novel viral infection, such as sars-cov-2, screening of existing chemicals, including dmards, may prove to be fruitful. dr tsen-fang tsai has conducted clinical trials or received honoraria for serving as a consultant for abbvie, boehringer ingelheim, bristol-myers squibb, celgene, elililly, galderma, gsk-stiefel, janssen-cilag, leo-pharma, merck, novartis, pfizer inc., and ucb pharma. dr ya-chu tsai has delivered speeches held by abbvie, elililly, janssen-cilag, leo-pharma, novartis, pfizer. the authors received no financial support for the research, authorship, and/or publication of this article. efficacy of combination therapy of antiviral and immunosuppressive drugs for the treatment of severe acute exacerbation of chronic hepatitis b antiviral, immunosuppressive and antitumour effects of ribavirin analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with moderate covid-19 clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial. medrxiv. epub ahead of print 2020 treating covid-19 with chloroquine hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial mechanism of baricitinib supports artificial intelligence-predicted testing in covid-19 patients baricitinib therapy in covid-19: a pilot study on safety and clinical impact thalidomide combined with low-dose short-term glucocorticoid in the treatment of critical coronvirus disease baricitinib as potential treatment for 2019-ncov acute respiratory disease covid-19: consider cytokine storm syndromes and immunosuppression jak1/2 inhibition with baricitinib in the treatment of autoinflammatory interferonopathies baricitinib for covid-19: a suitable treatment? janus kinase inhibitor baricitinib is not an ideal option for management of covid-19 baricitinib for covid-19: a suitable treatment?-authors' reply why choose cyclosporin a as first-line therapy in covid-19 pneumonia. reumatol clin. epub ahead of print 16 a novel in vitro cypd-mediated p53 aggregation assay suggests a model for mitochondrial permeability transition by chaperone systems loss of cyclophilin d reveals a critical role for mitochondrial permeability transition in cell death chloroquine is a potent inhibitor of sars coronavirus infection and spread anti-hiv effects of chloroquine: mechanisms of inhibition and spectrum of activity the additive in vitro anti-hiv-1 effect of chloroquine, when combined with zidovudine and hydroxyurea hydroxychloroquine treatment of patients with human immunodeficiency virus type 1 effect of chloroquine on human immunodeficiency virus (hiv) vertical transmission reduction of immune activation with chloroquine therapy during chronic hiv infection hydroxychloroquine drastically reduces immune activation in hiv-infected, antiretroviral therapy-treated immunologic nonresponders effects of hydroxychloroquine on immune activation and disease progression among hiv-infected patients not receiving antiretroviral therapy: a randomized controlled trial assessment of chloroquine as a modulator of immune activation to improve cd4 recovery in immune nonresponding hiv-infected patients receiving antiretroviral therapy chloroquine inhibits dengue virus type 2 replication in vero cells but not in c6/36 cells antiviral activity of chloroquine against dengue virus type 2 replication in aotus monkeys chloroquine use improves dengue-related symptoms a randomized controlled trial of chloroquine for the treatment of dengue in vietnamese adults chikungunya virus: an update on antiviral development and challenges chloroquine phosphate treatment of chronic chikungunya arthritis. an open pilot study on chikungunya acute infection and chloroquine treatment confronting an influenza pandemic with inexpensive generic agents: can it be done? anti-malaria drug chloroquine is highly effective in treating avian influenza a h5n1 virus infection in an animal model influenza a/h1n1 vaccination of patients with sle: can antimalarial drugs restore diminished response under immunosuppressive therapy? chloroquine for influenza prevention: a randomised, doubleblind, placebo controlled trial lysosomotropic agents as hcv entry inhibitors porphyria cutanea tarda in an hcv-positive liver transplant patient: a case report fdaapproved drug, prevents zika virus infection and its associated congenital microcephaly in mice functional association of cyclophilin a with hiv-1 virions long-term follow-up of hiv positive asymptomatic patients having received cyclosporin a placebo-controlled trial of cyclosporin-a in hiv-1 disease: implications for solid organ transplantation critical role of cyclophilin a and its prolylpeptidyl isomerase activity in the structure and function of the hepatitis c virus replication complex cyclophilin b is a functional regulator of hepatitis c virus rna polymerase combined interferon alpha2b and cyclosporin a in the treatment of chronic hepatitis c: controlled trial cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral ns5 protein a screen of fda-approved drugs for inhibitors of zika virus infection cyclosporine a inhibits in vitro replication of betaretrovirus associated with primary biliary cirrhosis rationale for the use of hydroxyurea as an anti-human immunodeficiency virus drug combination of a drug targeting the cell with a drug targeting the virus controls human immunodeficiency virus type 1 resistance a placebo-controlled trial of didanosine plus stavudine, with and without hydroxyurea, for hiv infection. the swiss hiv cohort study the hydile trial: efficacy and tolerance of a quadruple combination of reverse transcriptase inhibitors versus the same regimen plus hydroxyurea or hydroxyurea and interleukin-2 in hiv-infected patients failing protease inhibitorbased combinations activity, safety, and immunological effects of hydroxyurea added to didanosine in antiretroviral-naive and experienced hiv type 1-infected subjects: a randomized, placebo-controlled trial, actg 307 a randomized trial to investigate the recycling of stavudine and didanosine with and without hydroxyurea in salvage therapy (restart) hydroxyurea as an inhibitor of hepatitis c virus rna replication hydroxyurea suppresses hcv replication in humans: a phase i trial of oral hydroxyurea in chronic hepatitis c patients amazing results with hydroxyurea therapy in chronic hepatitis b: a preliminary report reactivation of hepatitis b virus during treatment with hydroxyurea in an elderly patient with essential thrombocythemia reversible inhibition of herpes simplex virus replication by hydroxyurea hydroxyurea enhances the activity of acyclovir and cidofovir against herpes simplex virus type 1 resistant strains harboring mutations in the thymidine kinase and/or the dna polymerase genes hydroxyurea inhibits parvovirus b19 replication in erythroid progenitor cells original research: parvovirus b19 infection in children with sickle cell disease in the hydroxyurea era a novel action of minocycline inhibition of human immunodeficiency virus type 1 infection in microglia neuroprotective and anti-human immunodeficiency virus activity of minocycline minocycline treatment for hiv-associated 20 journals.sagepub.com/home/tab cognitive impairment: results from a randomized trial randomized trial of minocycline in the treatment of hiv-associated cognitive impairment minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following japanese encephalitis minocycline differentially modulates viral infection and persistence in an experimental model of japanese encephalitis minocycline trial in japanese encephalitis: a double blind, randomized placebo study role of oral minocycline in acute encephalitis syndrome in india: a randomized controlled trial drug repurposing of minocycline against dengue virus infection antibiotic minocycline prevents respiratory syncytial virus infection antiinflammatory and antiviral effects of minocycline in enterovirus 71 infections transcriptomic characterization of the novel avian-origin influenza a (h7n9) virus: specific host response and responses intermediate between avian (h5n1 and h7n7) and human (h3n2) viruses and implications for treatment options minocycline inhibits west nile virus replication and apoptosis in human neuronal cells minocycline delays disease onset and mortality in reovirus encephalitis therapy with minocycline aggravates experimental rabies in mice effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo mycophenolic mofetil, an alternative antiviral and immunomodulator for the highly pathogenic avian influenza h5n1 virus infection broadspectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset inhibition of herpes simplex virus type 1 by the experimental immunosuppressive agent leflunomide successful treatment of acyclovirresistant herpes simplex virus type 2 proctitis with leflunomide in an hiv-infected man leflunomide in the treatment of a pseudotumoral genital herpes simplex virus infection in an hiv patient inhibition of hiv replication by a77 1726, the active metabolite of leflunomide, in combination with pyrimidine nucleoside reverse transcriptase inhibitors anti-hiv-1 activity of leflunomide: a comparison with mycophenolic acid and hydroxyurea the effect of leflunomide on cycling and activation of t-cells in hiv-1-infected participants leflunomide: an immune modulating drug that may have a role in controlling secondary infections with review of its mechanisms of action conversion from tacrolimus/mycophenolic acid to tacrolimus/leflunomide to treat cutaneous of four pediatric renal allograft recipients leflunomide for cytomegalovirus: bench to bedside concurrent antiviral and immunosuppressive activities of leflunomide in vivo leflunomide as part of the treatment for multidrug-resistant cytomegalovirus disease after lung transplantation: case report and review of the literature assessment of efficacy and safety of fk778 in comparison with standard care in renal transplant recipients with untreated bk nephropathy inhibition of respiratory syncytial virus in vitro and in vivo by the immunosuppressive agent leflunomide cp-690,550, a therapeutic agent, inhibits cytokine-mediated jak3 activation and proliferation of t cells from patients with atl and ham/tsp activity of thalidomide in aids-related kaposi's sarcoma and correlation with hhv8 titre activity of thalidomide in aids-related kaposi's sarcoma a prospective evaluation of leflunomide therapy for cytomegalovirus disease in renal transplant recipients ferm domain mutations induce gain of function in jak3 in adult t-cell leukemia/ lymphoma pustulosis palmaris et plantaris treated with hydroxyurea inhibition of autoimmune encephalomyelitis by a tetracycline minocycline protects pc12 cells from ischemic-like injury and inhibits 5-lipoxygenase activation hydroxychloroquine is associated with impaired interferon-alpha and tumor necrosis factor-alpha production by plasmacytoid dendritic cells in systemic lupus erythematosus hydroxychloroquine treatment downregulates systemic interferon activation in primary sjögren's syndrome in the joquer randomized trial hydroxychloroquine-inhibited dengue virus is associated with host defense machinery predictors of major infections in systemic lupus erythematosus previous antimalarial therapy in patients diagnosed with lupus nephritis: influence on outcomes and survival infection risk in systemic lupus erythematosus patients: susceptibility factors and preventive strategies thalidomide in the treatment of kaposi's sarcoma visit sage journals online journals.sagepub.com/ home/tab key: cord-326833-boxgt4kb authors: marimuthu, janakiram; kumar, bubby s; aravind gandhi, p title: hiv and sars cov‐2 co‐infection: a retrospective, record based, case series from south india date: 2020-07-07 journal: j med virol doi: 10.1002/jmv.26271 sha: doc_id: 326833 cord_uid: boxgt4kb hiv prevalence in india is about 0.22%, with the total number of people living with hiv/aids (plha) is estimated at 21.40 lakhs, constituting third largest epidemic in world. however, no study on hiv‐covid‐19 co‐infection has been reported from india. we conducted a retrospective, record based case series including three males, 2 females and 1 transgender plha co‐infected with sars cov‐2 in the indian state of tamil nadu. fever (5), followed by cough (2) and sore throat (1), were the presenting symptoms. latest median cd4 count among our patients was 535 cells/ mm3. one of the patients was not under clinical hiv control, with an opportunistic infection two among our patients were having hypertension. the mainstay of treatment given for the patients consisted of multi‐vitamins in addition to the arv drugs, anti‐pyretics and anti‐tussives. one of the patient was on low dose ritonavir boosted haart regimen. all patients had stable vitals at room conditions, did not have any complications during their entire stay in health care facility for covid‐19, treated and discharged. this article is protected by copyright. all rights reserved. with co-morbidities like diabetes mellitus, hypertension etc also needs to be comprehensively managed. through literature search in pubmed and world health organization' (who) database on covid-19, we obtained 5 case reports, and 5 case series on plha infected with severe acute respiratory syndrome coronavirus 2 (sars cov-2). the 5 case series were, one from germany (32 patients), one from spain (5 cases), two from united states of america [usa] (9 cases and 4 cases) and one from turkey (4 cases). [3] [4] [5] [6] [7] india, the second most populous country in the world, reported its first covid-19 case on january 30, 2020, and presently crossed 548, 318 cases. 1 hiv prevalence in india is about 0.22%, 8 with the total number of plha estimated at 21.40 lakhs, constituting third largest epidemic in world. however, no study on hiv-covid-19 co-infection could be found from india. hence, we conducted a retrospective, record based case series on the plha who were infected with sars cov-2, in the indian state of tamil nadu. the data of the hiv-covid19 patients, treated and discharged, till june 10 th , 2020, was collected using a data extraction sheet from the records of the covid-19 designated hospitals, across the state. all data has been anonymized. ethical permission for the study was obtained (ref no. 961/aids/art/c2/tansacs/2018) from the research review committee of tamil nadu aids control society (tansacs). six plha co-infected with sars cov-2 were identified during the study period: three males, two females and one transgender patient. the median age of the patients was 38 years (22 -55 years). one of the patients was not under clinical hiv control, with an opportunistic infection. five (83.3%) of our patients had mild illness with symptoms, while one of the patients was asymptomatic. fever (5), followed by cough (2) and sore throat (1), were the presenting symptoms in our patients. five (83.3%) of our patients were symptomatic contacts of known covid-19 positive cases, and one (16.7%) had a travel history from mumbai. five patients were on highly active anti-retroviral therapy (haart), one patient was art naïve. all patients had stable vitals at room conditions, did not have any complications during their entire stay in health care facility for covid-19, treated and discharged, according to the government of india guidelines. other characteristics of the patients are tabulated in table 1 . latest median cd4 count among our patients was 535 cells/ mm 3 (129-1047). there was no difference in course of illness and the outcome of the patients with varying cd4 counts in our study, thus indicating cd4 may have no role in covid-19 prognosis. this is in contrast to the studies which observed that lower cd4 counts could actually protect against severe form of covid-19. 4, 7 it is based on the hypothesis that immune system activation may actually increase the injury caused by covid-19. 9 however, suwanwongse et al observed that low cd4+ count in the hiv infected patients may adversely affect the covid-19 outcomes . 6 one of our patients presented with orooesophageal candidiasis. it might be an hiv opportunistic infection due to poor art adherence or treatment failure, which must be probed further. it might also be a secondary infection to sars cov-2. though reports on fungal infection in sars cov-2 are inadequate, candida infection has been reported among sars patients in 2003. 10 blanco et al reported a concurrent penumocytis jiroveci infection in one of their cases. 4 two among our patients had hypertension as co-morbidity. co-morbidities of hypertension, diabetes mellitus 2, chronic obstructive pulmonary disease, hyperlipidemia were reported among the hiv-covid 19 co-infected as well as exclusively covid-19 infected patients, across the globe. [3] [4] [5] [6] [7] the mainstay of treatment given for our patients consisted of multi-vitamins, art drugs, paracetamol and anti-tussives. vitamins, especially a and d, have shown to improve the immune system and may have potential benefit against viral infections. 11 anti-retro viral(arv) drugs such as darunavir, lopinavir/ritonavir and remdesivir by virtue of their anti-viral properties may have a prophylactic and protective role in keeping the covid-19 infection as mild in plhas. but clinical evidence against the lopinavir/ritonavir and other hiv protease inhibitors efficacy in covid 19 management is emerging. 12 molecular dynamics (md) simulation studies have identified zidovudine as the next potential candidate among the arvs for clinical trials in covid 19 treatment. 13 among our patients, one was on low dose ritonavir boosted haart and two were on zidovudine containing regimen. but the art naïve patient too had mild infection similar to the ones on haart, thus giving an inconclusive picture on the effect of haart on covid 19 disease course in our study. recovery rate was 100% among our patients, same as that of benkovic et al. 3 in contrast, suwanwongse et al and harter et al reported a higher case fatality rate (cfr) of 78%, 6 and 9% 5 , respectively. however, harter et al included only symptomatic covid-19 plha and observed that they might have overestimated the morbidity and mortality than the general population. 5 our study reports that the clinical features of covid-19 co-infection among plha in south india is mild, and the clinical outcomes are favorable. co-morbidities were present in our patients, but did not impact the outcome. however, the number of cases included was low and all cases were from a single state. further studies including greater number of patients should be done, to better understand the epidemiology, clinical outcomes and appropriate treatment modalities in the hiv-covid 19 co-infection. this article is protected by copyright. all rights reserved. funding: none 10 zhou p, liu z, chen y, xiao y, huang x, fan xg. bacterial and fungal infections in covid-19 patients: a matter of concern. infect. control hosp. epidemiol. 2020;: 1. world health organization. coronavirus disease (covid-19) situation report-161 4 cases: hiv and sars-cov-2 co-inf ection in patients from long island covid-19 in patients with hiv: clinical case series covid-19 in people living with human immunodeficiency virus: a case series of 33 patients clinical features and outcome of hiv/sars-cov-2 coinfected patients in the b ronx, new york city sars-cov-2 co-infected patients in istanbul, turkey hiv facts & figures | national aids control organization | mohfw | goi could hiv infection alter the clinical course of sars-cov-2 infection? when less is better we would like to thank mr deepak jacob ias, project director, tansacs for his valuable support in writing the case series and dr manimaran deputy director (targeted interventions) tansacs for his guidance. we acknowledge the art medical officers and other health care workers who managed the hiv-covid 19 patients across the state, and aided us in collating the data for the case series. key: cord-330970-6kkqoh7f authors: weiss, robin a title: apes, lice and prehistory date: 2009-02-10 journal: j biol doi: 10.1186/jbiol114 sha: doc_id: 330970 cord_uid: 6kkqoh7f although most epidemic human infectious diseases are caused by recently introduced pathogens, cospeciation of parasite and host is commonplace for endemic infections. occasional host infidelity, however, provides the endemic parasite with an opportunity to survive the potential extinction of its host. such infidelity may account for the survival of certain types of human lice, and it is currently exemplified by viruses such as hiv. hans zinsser's rats, lice and history [1] is a classic in microbiology. written in 1934 and subtitled the biography of a bacillus, it tells the tale of that dreaded disease typhus, its reservoir in rats and its transmission among humans by lice. here, i discuss how we may in the course of prehistory have acquired the lice, and how other infections may, like the typhus bacillus, come to be shared by us and the animal species with which we are in close contact. it is a tale of infidelity that i shall begin with the recent research on lice of david reed and colleagues [2, 3] and of mark stoneking's group [4] who, on the basis of phylogenetic analysis, have speculated that we may have acquired a clade of head lice from another hominid species and pubic lice from gorillas; they have also suggested that lice might help determine the date when humans adopted clothing. i shall examine this unfolding story in the context of what we know about microbial infections, and will look at the promiscuity of viruses through the lens of modern molecular technology; and i will add my own speculation on why naked apes have pubic hair. lice are small, wingless insects that cannot live independently from their hosts ( figure 1 ). they are frequent parasites of birds and mammals, each host species having its own type of louse. humans harbor three kinds of these ectoparasites: head lice, body lice and pubic lice. for much of human history and prehistory blood-sucking lice have been so prevalent that they became part of our everyday language. we speak about feeling lousy and admonish our friends for nit-picking. nits are the eggs of lice, expertly cemented onto hair shafts, as many parents know from painstakingly combing them out of their children's hair. grooming among monkeys and apes is not only a means of social bonding but also a useful way of controlling nits and lice ( figure 2 ). f fa am mi il ly y h he ei ir rl lo oo om ms s a an nd d n ne ew w a ac cq qu ui is si it ti io on ns s writing on infections of humankind, tony mcmichael and i [5] have called those that cospeciated with their hosts 'family heirlooms' and those that crossed over from other hosts in recent evolutionary time 'new acquisitions'. the new acquisitions were initially derived from zoonotic infections but have flourished as self-sustaining infections in the human population. they have diverged from their progenitors in the original host: for example, measles is now distinct from rindepest. the majority of zoonoses, however, remain in their animal reservoirs and, so far as their sojourn in humans goes -even with limited human-tohuman transfer (as with ebola or severe acute respiratory syndrome (sars)) -we can regard them as 'temporary exhibits'. human dna might show 98% similarity to that of chimps, but we share less than 50% of our microbes and parasites with them. ashford [6] argues that the great apes became more specialized forest dwellers at the same time that early hominids explored the savannah, and that human gut parasites resemble those of omnivorous baboons more than those of chimps because humans, like baboons but unlike chimps, are omnivorous. further opportunities for horizontal crossover of microbes and parasites from animals to humans arose when humans spread out of africa. when we domesticated ruminants, and animals such as dogs, cats and rats 'domesticated' us for the rich pickings around human habitation, we acquired many infections from our new neighbors [5, 7] . thus a shared habitat, rather than a shared ancestry, is important for the acquisition of many infections. most human pandemic infections were acquired horizontally very recently on the evolutionary timescale, even though diseases such as typhus, measles and smallpox first occurred in prehistoric times. these new acquisitions originate from evolutionarily distant host species. the influenza pandemic of 1918-1919 came from birds, and the 1968 influenza strain could be an avian-porcine recombinant. today's novel viral infections are more likely to originate from exotic species than from animals that were domesticated long ago [5] . the market for 'bushmeat' has led to ebola virus outbreaks in africa from butchering primates, and to the sars outbreak in china from eating small carnivores, such as civet cats. the primary reservoirs of the ebola filovirus, the sars coronavirus and the nipah paramyxovirus, however, seem to be in fruit bats (flying foxes). lice and nits have been found in textiles, hair and combs excavated from archaeological sites [1, 8] . given that the closest relative of the human head and body louse, pediculus humanus, is p. schaeffi, which infests chimpanzees, one might assume that human and chimp lice have cospeciated with their hosts as family heirlooms ever since they diverged from a common ancestor. this requires, however, that the divergence among hosts and parasites approximates to the same timescale. after all, the closest relative to human immunodeficiency virus type 1 (hiv-1) is the simian immunodeficiency virus of chimpanzees (sivcpz), but it would be facile to suggest that hiv-1 co-evolved with humans, because molecular clock estimates place the most recent common ancestor of the pandemic form of hiv-1 at 75-100 years ago [9, 10] , and this is likely to be close to the time of the species crossover event. hiv-1 has invaded humans at least three times (groups m, n and o), and such is the power of modern forensic dna virology that the precise location in cameroon has been mapped for the chimpanzees carrying the sivcpz most closely related to group m [11, 12] . louse molecular clock. what is more remarkable, however, is that reed et al. [2] found that human lice split into two quite distinct clades, a and b, about 1.18 mya. there is a worldwide clade (which includes both head and body lice) and a new world clade (exclusively head lice). so how can humans harbor two clades of louse that diverged from each other over one million years ago, when that separation is tenfold older than the emergence of homo sapiens? the answer, reed et al. [2] suggested, is that the separation took place around the time of divergence of the ancestors of modern humans from homo erectus. these two hominid lineages then co-existed for about one million years until the demise of h. erectus. when modern humans radiated across asia they might have had contact with h. erectus, just as in more recent millennia h. sapiens met h. neanderthalensis in europe, as dramatized by the nobel laureate william golding [14] . there is no evidence that different human species interbred, but they may well have exchanged ectoparasites. thus, the new world clade of head louse may have crossed horizontally from h. erectus to h. sapiens within the last 100,000 years. zinsser [1] noted that the hair of ancient peruvian mummies and the scalps of pre-columbian native americans contained nits or lice. recent dna analysis of lice from similar remains indicates that they belong to the worldwide clade a, so this clade must have been present in pre-columbian american populations [15] . a third clade of head lice has been delineated in ethiopia and nepal and this clade, c, diverged from clades a and b about 2 mya [16] . if reed et al. [2] were correct to postulate that clade b came from h. erectus, one must wonder in which hominid population might clade c lice have maintained their separate identity. head and body lice used to be designated pediculus capitis and p. corporis but they are now known to belong to the same species, p. humanus [16, 17] . fifty years ago levene and dobzhansky [18] showed that head lice could be trained or adapted to become the rather larger body lice by attaching them to the body in small pill boxes. as we celebrate the 150th anniversary of darwin's origin of species we might recall that it was theodosius dobzhansky, an eminent evolutionary biologist and a russian orthodox christian, who in 1973 famously challenged creationists by declaring that "nothing in biology makes sense except in the light of evolution." his research on lice was no exception. kittler et al. [4] initially reckoned that body lice diverged from head lice approximately 70,000 years ago, but they f fi ig gu ur re e 2 2 nit-picking is an ancient habit, as seen in ( (a a) ) apes (photograph by eric c matthews, reproduced with permission) and in humans as shown in ( (b b) ) a painting by jan siberechts, cour de ferme, 1662. musée des beaux-arts, brussels, belgium. later increased this estimate to 107,000 years ago by correcting an error concerning the original outlier sequence. they postulated that this date of about 100,000 years ago coincided with or followed soon after the origin of clothing, because the naked human body is an inhospitable place for lice to breed. head lice feed on the scalp and breed in hair, whereas body lice feed on the skin but breed in clothing. more extensive phylogenetic analyses [16, 17] indicate that body lice evolved from head lice several times within the worldwide clade a, as they are found in many branches of the cladisitic tree. multiple derivations of body lice from head lice had already been considered by zinsser [1] , and it makes good sense if one considers that clothing was not a single invention. wearing animal pelts fur-side next to the skin would have provided a suitable place for lice to breed before fabrics were developed with the inventions of spinning and weaving. in 17th and 18th century europe, most of the aristocracy and gentry shaved their hair and wore wigs. had this custom arisen to protect them from lice as zinsser [1] suggests? not according to samuel pepys' diary, as he complained more than once about his wig being infested: "thence to my barbers, to have my periwig cleared of its nits." i wonder if they were head or body lice -is a wig hair or clothing? l li ic ce e a an nd d n nu ud di it ty y why naked apes are naked and when we 'lost' our hair has long been disputed, as discussed by desmond morris in the naked ape [19] . rantala [20] suggested that nakedness could have had a selective advantage to rid the body of lice and other ectoparasites, a view also championed by pagel and bodmer [21] , who added that being seen to be free of lice would be a fitness indicator and a good mating strategy. i am rather drawn to the theory first postulated by alister hardy [22] that humans evolved through an aquatic stage, although most anthropologists disparage this hypothesis. yet ashford [6] points out that several parasites specific to humans, such as three schistosoma species, depend for their transmission on our entering water, which again distinguishes us from the great apes. pubic lice are commonly called crabs because of their appearance (figure 1c) (figure 3 ). thus, it seems clear that humans acquired pubic lice horizontally, possibly at the time of the pthirus species' split and probably directly from gorillas. because they were already adapted to the coarse body hair of the gorilla, crabs would have found a suitable niche in human pubic hair. indeed, the diameter of the hair is most likely the key rather than the pubic region, because pediatric infestation of p. pubis is well documented: the crab is then found on the eyelashes of the infant. reed et al. [3] suggest that the most recent common ancestor of the genera pediculus and pthirus was about 12.5 mya, which is earlier than the estimated divergence of gorillas and the chimpanzee-human lineage. so was there duplication and separation of lice in the african anthropoid ape lineage, where they could have occupied separate ecological niches, rather as human head lice and pubic lice do today? although this is an intriguing hypothesis, i was having difficulty envisioning a clear separation of habitats between the groin and other parts of our ancient common ancestor. [23] , all the species of apes, old world monkeys and new world monkeys seem to be less hairy in the pubic region than elsewhere; fur is present but it is short and fine. why do adult humans sport a thick bush of wiry pubic hair, uniquely among primates? it must surely be because we are otherwise naked. it probably serves both a visual and an odorous function, because hair aids the distribution of apocrine scent secretion, like our less visually stunning axillary hair. unlike a beard, pubic hair is not sexually dimorphic, yet it is a feature of sexual maturation. no wonder that pubic lice are said to be the most contagious of all sexually acquired infections. which came first, nakedness or pubic hair? i would postulate that the development of pubic hair was a consequence of the visible nakedness elsewhere on the body. perhaps the acquisition of p. pubis 3.3 mya provides the clue to when hominids developed thick pubic hair, rather as the evolution of body lice is thought to be broadly contemporaneous to the development of clothing. it is noteworthy that the prevalence of infestation by pubic lice seems to be decreasing among women and men who remove their pubic hair using 'bikini wax', rather as the renaissance painters discreetly depilated classical female nudes. a study from the general infirmary, leeds, uk, records the increasing predilection among attendants at a clinic for sexually transmitted infections to undertake extensive pubic hair waxing known as the 'brazilian', leaving only a thin strip of hair. armstrong and wilson [24] noted a significant fall in the incidence of pubic lice among patients, although gonorrhea and chlamydia increased over the same period. thus, there may be a health benefit to this emerging sexual lifestyle, and this finding would also lend support to the notion that nakedness protects humans from ectoparasites. h hu um ma an ns s a as s r re ep po os si it to or ri ie es s o of f a an nc ci ie en nt t i in nf fe ec ct ti io on ns s parasites that have tightly cospeciated with their hosts are, of course, in grave danger of extinction when that host becomes an endangered species. but the 'smart' parasite would gain a whole new lease of evolutionary opportunity if it engaged in occasional 'infidelity', analogous to mutation in dna replication. a dna lineage that does not mutate would have an extraordinarily slow rate of evolution, whereas a super-mutator without repair would provide little opportunity for natural selection before the genotype changed further. thus, a low mutation rate within broad fidelity of dna replication allows for both inheritance and evolution. likewise, total cospeciation dooms the parasite to the fate of the host, whereas the ability to move horizontally to closely related hosts would provide flexibility. parasite jumping between related hosts might occur more frequently than realized because it might easily be overlooked. by this reckoning, the new world clade of head lice formerly faithful to the h. erectus lineage [2] adopted modern humans in the nick of time. hiv-1 might well be another successful case of jumping off a sinking ship, because its former host is not likely to survive for many generations longer in the wild. now that hiv-1 has adopted a new host species, it is enjoying a most successful adaptive radiation and has already colonized around 60 million humans (25 million of whom have died from aids). such crossover events, however, are relatively rare, and only one of the three ape-to-human transfers of hiv-1 has taken off to cause the aids pandemic [11] . it pays the host to place barriers known as restriction factors in the path of potential pathogens. if a species barrier is not recognized by the new invader or is successfully circumvented, the infection can be more virulent in the new host. regarding the intestinal parasites and ectoparasites that specialize in human infestation, ashford [6] pointed out that we not only house two kinds of closely related lice (he meant pediculus head and body lice), but also two species of cimex bedbugs, two of demodex mites and two of taenia tapeworms. he therefore asked if we were once two separate populations that rejoined after a long separation, but ashford did not have dna sequences and molecular clock estimates available to him. can we now view this phenomenon in the same way as the lice, as one of the pair of parasites cospeciating with h. sapiens and the other jumping from non-ancestral archaic humans to the modern human lineage? it would be intriguing to conduct similar molecular phylogenies of ashford's other pairs. ashford [6] finished by stating: "over to the microbiologists: what do the bacteria, viruses and fungi tell us?" there are ample examples both of cospeciation and horizontal transmission. the malaria parasite plasmodium falciparum exemplifies a complex cospeciation between the parasite, its human host and its mosquito vector. anopheles gambiae has also coevolved to be a specialist feeder on humans; by contrast, the other human malaria parasite, p. vivax, has an origin in south east asian monkeys and is transmitted by the more promiscuous culex species. might vivax malaria have first adapted to h. erectus as an intermediate between monkeys and humans? as a virologist, i have felt stimulated [23] to take up ashford's challenge to consider pairs of related human viruses. hiv-1 and hiv-2 are very recent 20th century arrivals, from chimpanzees and sooty mangabey monkeys, respectively. with human t-cell lymphotropic viruses types 1 and 2 (htlv-1 and htlv-2), there have been multiple introductions of htlv-1 from african and asian monkeys and apes [25] . the provenance of htlv-2 is puzzling, as its reservoir is in indigenous american populations and in african pygmies, which are as far apart in h. sapiens as one can find. humans have five distinct polyoma viruses and multiple genital papilloma virus types. the different clades of hepatitis c virus (hcv) have deep roots but there are no known animal relatives to provide an anchor or time calibration. it would be fascinating to learn whether archaic humans harbored hcv variants, but as hcv has an rna genome there is not much hope of gaining direct evidence by sequence amplification from ancient specimens. all members of the herpesvirus family are thought to have strictly cospeciated with their hosts, though i have my doubts. the closest relatives to human herpes simplex virus (an α herpesvirus), cytomegalovirus (β) and epstein-barr virus (γ), seem to be those in the chimpanzee. several simian species have a pair of distinct rhadinoviruses, whereas so far humans are only known to harbor one, kaposi's sarcoma herpesvirus. on the other hand, humans have two herpes simplex viruses (hsvs), types 1 and 2. phylogenetic analysis [26] indicates that hsv-1 and hsv-2 are further apart from each other than hsv-1 is from its chimp ortholog [27] . so where did hsv-2 come from? from its estimated age of divergence from the chimp-human hsv-1 lineage, i would place a bet on horizontal transmission from gorillas or possibly from orang-utans [23] . is it a coincidence that three human parasites acquired horizontally from great apes, namely pubic lice, hiv-1 and speculatively hsv-2, are sexually transmitted? now, before one conjures up a king kong scenario, it should be noted that predators can pick up parasites from their prey. the close contact involved in human ancestors butchering gorillas could have enabled pthirus to jump hosts, rather as bushmeat slaughter practices probably led sivcpz and other retroviruses to invade humans from chimpanzees in modern times [11, 25] . h hu um ma an ns s a as s a a s so ou ur rc ce e o of f i in nf fe ec ct ti io on ns s with the advent of globalization, previously isolated human populations lost nine-tenths of their number to the infections introduced by intrepid migrants and invaders [7] . hernan cortes conquered the mighty aztec empire thanks to smallpox and measles, which the invaders inadvertently introduced to the disease-naive new world peoples [5] . the subsequent population crash was severe; so few indigenous people survived that the lucrative african slave trade was established in order to provide labor in the plantations. this pattern of export of old world diseases was repeated in south america, australia and oceania. as charles darwin remarked in his diary on the voyage of the beagle, "wherever the european has trod death seems to pursue the aboriginal." it is plausible, then, that modern humans could have transmitted lethal infectious diseases to archaic human species. h. erectus might have given us a clade of head lice, but h. sapiens may have conveyed more deadly infections to h. erectus and later to h. neanderthalensis. hard evidence is lacking, and prehistoric legends seldom tell the tale from the point of view of the vanquished, although golding did through the eyes of the neanderthals [14] . to paraphrase darwin, wherever modern humans trod during the pleistocene era, death seemed to pursue archaic humans. there is also a danger today that the surviving great apes may be subjected to a coup de grace from human infections transmitted through jungle safaris and ecotourism. cross-species virulence is well known. myxamotosis resident in american cotton-tail rabbits devastated the european rabbit, and the disappearance of red squirrels in britain wherever american gray squirrels occur is probably due to a pox virus rather than direct competition for habitat. similarly, the α-herpesviruses that have cospeciated with indian and african elephants cause nothing more severe than cold sores in their natural host but each seems to be lethal to the other when the two species are unnaturally housed together in zoos [28] . sivcpz has little effect on chimpanzees but hiv-1 causes aids in humans. thus, it might pay the host to carry a fairly harmless parasite if that infection is lethal to the host's competitors. body lice, and occasionally head and pubic lice, transmit bacterial diseases: typhus (rickettsia prowazekii) [29] , trench fever (bartonella quintana) and relapsing fever (borellia recurrentis). the lice themselves succumb to typhus infection but pass the rickettsia in their feces, which the human then scratches into the skin. typhus is known as 'war fever' and 'jail fever' because it appears in conditions where lice thrive. at the end of rats, lice and history zinsser [1] wrote "typhus is not dead. it will live on for centuries and will break into the open whenever human stupidity and brutality give it a chance." sadly, zinsser's words were prophetic. within a few years, typhus became the major slayer in the nazi concentration camps; typhus broke out among rwandan refugees in burundi in 1994, in bosnia in 1995 and most recently in goma. human brutality is another feature shared with chimpanzees that has survived in the human lineage [30] . it is too bad that we are not closer to the pygmy chimp (bonobo), which evolved a means of conflict resolution between troupes through alpha females engaging in lesbian sex. but that is another story. a ac ck kn no ow wl le ed dg ge em me en nt ts s i am grateful to tim harrison and david reed for commenting on a draft of this paper. r re ef fe er re en nc ce es s zinsser h: rats, lice and history ge en ne et ti ic c a an na al ly ys si is s o of f l li ic ce e s su up pp po or rt ts s d di ir re ec ct t c co on nt ta ac ct t b be et tw we ee en n m mo od de er rn n a an nd d a ar rc ch ha ai ic c h hu um ma an ns s p pa ai pa ar ra a--s si it te es s r re eg ga ai in ne ed d s so oc ci ia al l a an nd d e en nv vi ir ro on nm me en nt ta al l r ri is sk k f fa ac ct to or rs s i in n t th he e e em me er rg ge en nc ce e o of f i in nf fe ec ct ti io ou us s d di is se ea as se es s germs and steel: a short history of everybody for the last 13,000 years b bo od dy y l lo ou us se e r re em ma ai in ns s f fo ou un nd d i in n t te ex xt ti il le es s e ex xc ca av va at te ed d a at t m ma as sa ad da a, i is sr ra ae el l t ti im mi in ng g t th he e a an nc ce es st to or r o of f t th he e h hi iv v--1 1 p pa an nd de em mi ic c s st tr ra ai in ns s d di ir re ec ct t e ev vi id de en nc ce e o of f e ex xt te en n--s si iv ve e d di iv ve er rs si it ty y o of f h hi iv v--1 1 i in n k ki in ns sh ha as sa a b by y 1 19 96 60 0 c ch hi im mp pa an nz ze ee e r re es se er rv vo oi ir rs s o of ve er rs si it ty y a an nd d p ph hy yl lo og ge eo og gr ra ap ph hi ic c c cl lu us st te er ri in ng g o of f s si iv vc cp pz zp pt tt t i in n w wi il ld d c ch hi im mp pa an nz ze ee es s i in n c ca am me er ro oo on n ge en no om mi ic c r re el la at ti io on ns sh hi ip ps s a an nd d s sp pe ec ci ia at ti io on n t ti im me es s o of f h hu um ma an n, c ch hi im mp pa an nz ze ee e, a an nd d g go or ri il ll la a i in nf fe er rr the inheritors. london: faber and faber m mo ol le ec cu ul la ar r i id de en nt ti if fi ic ca at ti io on n o of f l li ic ce e f fr ro om m p pr re e--c co ol lu um mb bi ia an n m mu um mm mi ie es s the naked ape: a zoologist's study of the human animal hu um ma an n n na ak ke ed dn ne es ss s: : a ad da ap pt ta at ti io on n a ag ga ai in ns st t e ec ct to op pa ar ra as si it te es s? ? l le es ss so on ns s f fr ro om m n na ak ke ed d a ap pe es s a an nd d t th he ei ir r i in nf fe ec ct ti io on ns s b br ra az zi il li ia an n t to op pi ic cs s i in n h he er rp pe es sv vi ir ru us s g ge en no om mi ic cs s a an nd d e ev vo ol lu ut ti io on n i is so ol la at ti io on n a an nd d c ch ha ar ra ac ct te er ri iz za at ti io on n o of f a a c ch hi im mp pa an nz ze ee e a al lp ph ha ah he er rp pe es sv vi ir ru us s pr ri im ma at te e t t--l ly ym mp ph ho ot tr ro op pi ic c v vi ir ru us se es s a am mo on ng g c ce en nt tr ra al l a af fr ri ic ca an n b bu us sh hm me ea at t n no ov ve el l e en nd do ot th he el li io ot tr ro op pi ic c h he er rp pe es sv vi ir ru us se es s f fa at ta al l f fo or r a as si ia an n a an nd d a af fr ri ic ca an n e el le ep ph ha an nt ts s e em me er rg gi in ng g a an nd d r re e--e em me er rg gi in ng g r ri ic ck ke et tt ts si io os se es s: : e en nd do ot th he el li ia al l c ce el ll l i in nf fe ec ct ti io on n a an nd d e ea ar rl ly y d di is se ea as se e e ev ve en nt ts s demonic males: apes and the origins of human violence key: cord-305602-yzc4bosn authors: llano, manuel; peña-hernandez, mario a. title: chapter seven defining pharmacological targets by analysis of virus–host protein interactions date: 2018-12-31 journal: advances in protein chemistry and structural biology doi: 10.1016/bs.apcsb.2017.11.001 sha: doc_id: 305602 cord_uid: yzc4bosn abstract viruses are obligate parasites that depend on cellular factors for replication. pharmacological inhibition of essential viral proteins, mostly enzymes, is an effective therapeutic alternative in the absence of effective vaccines. however, this strategy commonly encounters drug resistance mechanisms that allow these pathogens to evade control. due to the dependency on host factors for viral replication, pharmacological disruption of the host-pathogen protein–protein interactions (ppis) is an important therapeutic alternative to block viral replication. in this review we discuss salient aspects of ppis implicated in viral replication and advances in the development of small molecules that inhibit viral replication through antagonism of these interactions. chromatographic resolution of human cell extracts subsequently analyzed by quantitative tandem mass spectrometry resulted in the identification of 13,993 physical interactions established by 3006 individual proteins. computational analysis of the physical interactions led to the mapping of 622 complexes, suggesting an average of 4 proteins per complex (havugimana et al., 2012) . these estimates are also supported by other analysis indicating that most of the mammalian complexes are formed by the association of three or four different proteins. importantly, some host proteins are found in more than one complex at a time (wong et al., 2008) . similarly, a study in human cells combining large-scale immunoprecipitation of 331 bait proteins followed by high-throughput mass spectrometry analysis led to the discovery of 6463 interactions between 2235 unique proteins, suggesting an average of 3 proteins per complex (ewing et al., 2007) . protein complexes are stable assemblies that carry out most of the biochemical activities in the cell. corum, a public database of mammalian protein complexes, compiles complexes reported in nonhighthroughput, physical interaction experiments. of the proteins annotated in corum, 78% belong to one protein complex. fig. 1 represents the frequency of annotated complexes in the corum database. interestingly, protein complexes implicated in subcellular localization processes are notoriously overrepresented in this database, whereas others implicated in energy production, protein synthesis, and tissue differentiation are underrepresented. in these collection of experimentally characterized protein complexes, approximately 16% of all predicted human open reading frames are organized in 1815 protein complexes (ruepp et al., 2010) . however, this number is expected to be only a minor fraction of the human complexome considering that in yeast 45% of the encoded proteins reside in complexes (ruepp et al., 2010) . hypothesis-driven experiments conducted at small scale have notably contributed to our understanding of protein-protein interactions (ppis) implicated in viral replication. in addition, high-throughput technologies analyzing ppis, gene loss of function, or transcriptomic profiles have been utilized to define cellular complexes implicated in different steps of the viral life cycle. large-scale physical analysis of ppi is conducted by yeast two-hybrid (y2h) screens (calderwood et al., 2007; de chassey et al., 2008; fossum et al., 2009; lee et al., 2011; rajagopala, casjens, & uetz, 2011; shapira et al., 2009; trigg et al., 2017; uetz et al., 2006) and tandem affinity purification followed by mass spectrometry (tap-ms) (germain et al., 2014; pichlmair et al., 2012; ramage et al., 2015; rozenblatt-rosen et al., 2012) . y2h screens can analyze only binary interactions, whereas tap-ms allows for the identification of more complex interactors. then, the physical interactions identified with these methods are subjected to organization in functional networks by computational approaches. despite of their rate of success in ppis identification, both methodologies interrogating physical interactions of proteins fail to detect multiple types of ppis. for example, interactions involving transmembrane proteins, or transient/ weak interactions escape detection. similar to the physical methods, computational systems biology approaches successfully predict exploitation of biological processes by viruses (kitano, 2002a (kitano, , 2002b navratil, de chassey, combe, & lotteau, 2011; zak, tam, & aderem, 2014) . these methods use gene expression and gene loss-of-function systemic analysis to identify potential host factor networks implicated in viral replication (hirsch, 2010) . the main problem that the high-throughput functional approaches discussed earlier has is the poor data overlap resulting from these analyses. this is even though these methods have a high degree of similarity or complementary. therefore, validation of the discovered ppis is required (rozenblatt-rosen et al., 2012; shapira et al., 2009) . this validation involves the demonstration of the interaction by coimmunoprecipitation analysis, particularly during infection. prioritization analysis of ppis can be implemented by combining the findings from the direct interaction approaches with comprehensive gene loss-of-function screenings. an example of the effectiveness of this multifactorial approach for the identification of ppis is a study aimed to identify host-influenza ppis (shapira et al., 2009) . in this study, 35% of the 1745 genes initially found to be involved in viral protein interactions were demonstrated to influence viral replication by subsequent rnai screening. this high rate of positive validation may have been the result of the selection of the final candidate genes by a combined analysis of the results obtained in y2h screens using viral and host proteins (direct interactors), in transcriptional profiling experiments to define genes regulated by viral infection, and in in silico predictions of genes implicated in the protein networks found in the first two experimental approaches. comparison of the cellular complexes exploited by different pathogens indicates an significant overlap, leading to the definition of cellular processes implicated in infection (brander & walker, 2000; davis et al., 2015; dyer, murali, & sobral, 2008; hirsch, 2010; hiscott, nguyen, arguello, nakhaei, & paz, 2006; pichlmair et al., 2012; rozenblatt-rosen et al., 2012; shapira et al., 2009) . therefore, shared cellular complexes could constitute broad-spectrum therapeutic targets potentially impairing more than one pathogen. an example of this strategy is discussed later when we consider the implication of protein translation in the mechanism of replication of multiple viruses. the overlap in the utilization of cellular complexes between different pathogens also brings support to the hypothesis that the innate immune system has evolved the ability of detecting patterns of pathogenicity. these patterns are generated by the activation of similar biological processes by different pathogens, indicating the existence of pathogen-induced processes (dyer et al., 2008; pichlmair et al., 2012; vance, isberg, & portnoy, 2009) . the viral manipulation of the activity of type i interferon (ifn)-stimulated genes (isg) illustrates this concept. protein kinase, rna-activated (pkr) is an isg that upon activation phosphorylates the α-subunit of the translation initiation factor eif-2, inhibiting protein synthesis. viral dsrna is the main pkr activator in virus-infected cells, whereas the cellular protein pact (protein activator of pkr), that heterodimerizes with pkr, is an important activator of the kinase in stressed, noninfected cells in the absence of dsrna (li et al., 2006; patel & sen, 1998) . hiv-1 evades the robust type i ifn response mounted by plasmacytoid dendritic cells in infected individuals by rewiring the pact/pkr complex. in infected cells, hiv-1 tar nucleates a complex with the viral protein tat and the cellular proteins pact and adar1 (adenosine deaminase acting on rna 1). in this complex, adar1 inhibits pact pkr activatory function by direct interaction with pact (clerzius et al., 2013) . another example of pathogeninduced host complex rewiring will be discussed later in relation to the role of hiv-1 accessory proteins in the targeting of the ubiquitin/proteasome system to degrade different restriction factors. importantly (chukwurah, handy, & patel, 2017) . therefore, the pact-adar1-pkr axis is a cellular pathway commonly manipulated by different viruses that could constitute a therapeutic target with broad antiviral activity or pathogen-induced processes detected by the innate immune system. the hijacking of similar cellular pathways by different viruses also allows for the identification of broader biomarkers of viral infection. generally viral pathogens target cell cycle regulation, nuclear transport, and immune response processes (dyer et al., 2008; shapira et al., 2009) . ssrna(à) viral proteins tend to interact with processes implicated in the protection of rna from degradation and rna processing, whereas dsrna viruses are more connected to protein degradation processes. dna viruses, however, target proteins connecting the cell cycle with other processes such as chromosomal and transcriptional homeostasis (pichlmair et al., 2012; shapira et al., 2009 ). rnai-based screens have also been useful in identifying host factors implicated in viral replication. interestingly, the number of host factors identified for the different viruses analyzed in these screens outnumber by a factor of 10 the proteins encoded by these viruses (hirsch, 2010) . this disproportion could illustrate the multifunctional character of viral proteins. in this case viral proteins are expected to establish multiple independent binary interactions with independent host proteins. alternatively, viral proteins could interact with host proteins organized in complexes. therefore, rnai-mediated downregulation of the subunits of these protein complexes will produce similar phenotypes. correspondingly, y2h screenings have found that viral proteins establish a disproportionate number of binary interactions with the human proteome. these numbers of interactions registered for viral proteins exceed the predicted number of interactions estimated from the analysis of the human interaction network (shapira et al., 2009) . for example, assessment of the interaction of each of the 10 viral proteins encoded by influenza with 12,000 human orf through y2h screening found 135 interactions with 87 human proteins (shapira et al., 2009) . these evidences support a model indicating that viral proteins evolve multiple direct interactions. furthermore, computational analysis of host factors implicated in these pairwise interactions indicated that the host proteins occupy central positions, hub proteins, within the cellular interactome (shapira et al., 2009) . this higher than expected connectivity suggests that the direct interactions of viral proteins with host factors allow the access of the virus to cellular complexes. computational analysis of the interaction of human proteins binding to different viral proteins encoded by 35 different viruses showed that approximately 97% ae 9.1% of the 1396 unique targeted human proteins interact at least with one other human protein (dyer et al., 2008) . these data indicate that the majority of the host factors implicated in viral replication are in complexes. these conclusions are further supported by tap-ms and functional genomic studies. tap-ms studies of the virus-host interactome indicate that viral proteins tend to interact with proteins that have multiple interacting partners and participate in more cellular pathways (protein hubs), and also with proteins that are central to many pathways and, therefore, occupy a more central position within these networks (protein bottlenecks) (dyer et al., 2008; pichlmair et al., 2012; shapira et al., 2009) . for example, in a tap-ms experiment were mapped 3787 complex associations between 54 viral proteins from different viruses and 1079 host proteins (rozenblatt-rosen et al., 2012) , highlighting the high degree of connectivity of the interacting proteins. as discussed above, some of the host factors predicted, by the combined transcriptional profiling and in silico analyses, to interact with host proteins implicated in direct binary contacts with influenza proteins (y2h interactors) were demonstrated to influence viral replication in functional screenings (shapira et al., 2009 ). these findings demonstrate that influenza, and potentially other viruses, hijacks cellular networks by combining physical and regulatory (transcriptional level) interactions with these pathways. this multilayer regulation offers further alternatives of blocking physical or regulatory interactions aiming to impair viral replication. interestingly some of the host factors found in the networks of protein interacting with viral proteins are induced at the transcriptional level by infection in a type i ifnindependent manner (shapira et al., 2009 ). this indicates that viruses modulate, at the transcriptional levels, the abundance of proteins that will engage in physical binary or multiprotein complex interactions. therefore, computational analysis of the pathways discovered by functional genomics could lead to the identification of protein networks implicated in physical interactions with viral proteins. protein complexes established by viruses seem to determine the disease associated to the infection. for example, analysis of the interactome of e6 proteins from hpv types differing in their oncogenic potential associates with different subset of host proteins (rozenblatt-rosen et al., 2012) . therefore, disruption of these complexes could also prevent viral pathogenesis. almost 78% of the human-pathogen ppis reported belong to hiv-1 (dyer et al., 2008; fahey et al., 2011; ruepp et al., 2010) . generally host factors interacting with viral proteins tend to preserve the host protein interactions mapped in noninfected cells (rozenblatt-rosen et al., 2012; yu et al., 2011) , indicating that the normal host protein complexes rather than new virus-induced protein complexes are implicated in viral replication. this opens the opportunity to utilize the current body of knowledge on the human interactome to define the host complex hijacked by viruses. however, examples of virus-specific complex variants also occur. among them, one of the best characterized is the complex between hiv-1 vif and the transcriptional and protein degradation cellular machineries. vif removes apobec3 family restriction factors by targeting the ubiquitin ligase complex cul5 to this restriction factor, and inducing its proteasomemediated degradation. other hiv-1 proteins, vpu and vpr, also exploit cullin-ring e3 ligase to degrade other restriction factors. vif directly interacts with the substrate, the transcription factor cbf-β, the n-terminus of cul5 (cullin-ring e3 ligase), and the heterodimeric substrate adaptor elob/eloc. in turn, the c-terminus of cul5 binds rbx2 and recruits an e2 ubiquitin conjugation enzyme that mediates the transfer of polyubiquitin to the substrate proteins (jager, kim, et al., 2011; zhang, du, evans, yu, & yu, 2011) . cbf-β is required in this complex for the assembly of the vif-cul5 e3-ubiquitin-ligase complex, but not for the binding of vif to the substrate . cbf-β normally heterodimerizes with the runx family of transcription factors preventing its degradation and vif competes with runx1 for binding to cbf-β (guo et al., 2014) . the surface area buried at the interface of the interaction of vif and cbf-β is large ($4800 a 2 ) suggesting that is undruggable (salter, morales, & smith, 2014) , as we will discuss later for other surfaces of ppis. however, alanine scanning indicated that aa 5-11 of vif are a hot spot (defined in a later section of this review) in the interaction with cbf-β. furthermore, residues phe68 and ile55 of cbf-β establish important interactions with trp5 in vif through hydrophobic interactions (desimmie, smith, matsuo, hu, & pathak, 2017) , highlighting further opportunities for disruption of this complex with small molecules. therefore, these structural characteristics of the vif-cbf-β surface of interaction do not exclude its amenability to small-molecule interference, as we will show later for similar ppis. interfaces of ppi were considered undruggable for sometime, mainly because these surfaces of interactions are bigger than classical druggable protein surfaces, such as allosteric and catalytic sites in enzymes. the total area buried by the interactors in the binding site in ppis is in average 1600 (ae400) a 2 , with some proteins extending to 2000-4660 a 2 . in contrast, surfaces of interaction between proteins and small molecules are approximately from 300 to 1000 a 2 (arkin, tang, & wells, 2014; lo conte, chothia, & janin, 1999) . in addition, the surface of protein-protein interaction, although very variable in characteristics, was generally considered large patches of segmented complementary surfaces lacking small grooves or pockets (hopkins & groom, 2002; jones & thornton, 1996 lo conte et al., 1999) . furthermore, attempts to find small molecules interfering with ppis showed a very high failure rate ($60%) (brown & superti-furga, 2003) , therefore reinforcing the concept that surface of ppis is not druggable. in correlation with this, estimates using very stringent concepts for drugs indicate that only approximately 10% of the human proteome could be targeted by oral, drug-like small molecule. these predictions also indicate that of the druggable gene products only between 5% and 50% were implicated in diseases (hopkins & groom, 2002) . our understanding of the rules driving the ppis importantly changed with the discovery that the different residues buried in the large surfaces of interactions contribute differentially to the binding strength of the interacting proteins (clackson & wells, 1995) . this characteristic reduced considerably the area required to be targeted by small molecules. interrogation of the surfaces of binding in ppis by mutagenesis of individual residues (alanine scanning) indicated that mutation of a few residues implicated in these binding surfaces was sufficient to disrupt the ppis. residues importantly contributing to the binding free energy were defined as hot spots (clackson & wells, 1995) . these residues tend to cluster in tightly packed regions in the center of the surfaces of interactions and are called hot regions (clackson & wells, 1995; keskin, ma, & nussinov, 2005) . hot spots can be amenable to drug targeting due to their small surface area and important contribution to the total binding energy of the complex. however, not always hot spots are vulnerable to drugs, as additional topological constraints for drug binding limit the number of druggable hot spots (zerbe, hall, vajda, whitty, & kozakov, 2012) . in addition, in silico analysis indicated that interfaces could contain more than one hot region, and in some ppis the establishment of the complex depends on the cooperative interaction of different hot spots within the region (keskin et al., 2005) . by 2011, more than 40 ppis have been reported to be targeted by small molecules (morelli, bourgeas, & roche, 2011) . twenty-seven proteinprotein complexes were included in the 2016 release of the small-molecule orthosteric modulators of ppis database 2p2idb. in this site are listed only those ppis in which there is structural information of the protein-protein and protein-inhibitor complexes (basse, betzi, morelli, & roche, 2016) . the majority of the most successful ppis inhibitors target hot-spot residues that cluster in small binding pockets (250-900 a 2 ), and the surface of interaction of the binding proteins has short primary sequences (arkin et al., 2014) . structural analysis of protein-inhibitor complexes indicated that in some cases small molecules interrupting ppis bind to cavities not present in the surface of interaction of the protein-protein complex or in the interactor proteins when they are in isolation. these findings indicate some degree of adaptability in the surfaces of interaction initially considered lacking of druggable groves or pockets. according to in silico simulations the opening of some of these binding pockets is transient (wells & mcclendon, 2007) . most of the small molecules interfering with ppis bind directly to the implicated surfaces of interactions (orthosteric modulators) by targeting hot spot residues or by molecular mimicry of elements of secondary structures (arkin et al., 2014; basse et al., 2016; fry, 2006; wells & mcclendon, 2007; yin & hamilton, 2005) . however, several successful examples of small molecules disrupting ppis act through their binding to allosteric sites (crump et al., 2004; mcmillan et al., 2000; oswald et al., 2016; roche et al., 2016; szilagyi, nussinov, & csermely, 2013) . a well-characterized model of small-molecule disruption of a host ppi, deposited in the 2p2idb database, is the mdm2/p53 interaction. mdm2 binds to p53, impairing its transcriptional activity and stability. the interaction surfaces are formed by a hydrophobic pocket (aa 25-109) in the n-terminus of mdm2 that is occupied by the hydrophobic side of an amphipathic α-helix in p53 (aa 19-26) . mutations at any of the four residues within the mdm2-binding pocket or of three residues within the p53 α-helix block this ppi (moll & petrenko, 2003) . small molecules interrupting this interaction bind to the p53-binding pocket in mdm2 (vassilev et al., 2004) . most of the data on small molecules targeting ppis in the 2p2idb database correspond to host-host ppis and only two are relevant to viruses. that is the case of the interaction between the hpv proteins e2 and e1, and of allosteric inhibitors of hiv-1 integrase activity (allinis) (engelman, kessl, & kvaratskhelia, 2013) . hpv e2 and e1 bind cooperatively to the viral origin of dna replication and are required for initiation of dna replication (berg & stenlund, 1997) . indandione derivatives that bind to the e2 transactivation domain block this interaction. one of these small molecules was shown to contact 7 of the 20 residues implicated in this interaction. allinis bind to the dimer interface of integrase to the same residues of the host protein ledgf/p75 (engelman et al., 2013) . ledgf/p75 is a chromatin-bound protein required for efficient hiv-1 cdna integration (llano et al., 2006; shun et al., 2007) , an essential step in the viral life cycle. ledgf/p75 binds to the dimer interface of the catalytic core domain of integrase. two residues in the integrase-binding domain of ledgf/p75 are essential in this interaction. asp366 in an interhelical loop of the host protein contacts, via hydrogen bonds, with residues glu170 and his171 in one integrase monomer, whereas ledgf/p75 residue ile365 interacts with a hydrophobic pocket (leu102, ala128, trp132) in the other integrase subunit (cherepanov, ambrosio, rahman, ellenberger, & engelman, 2005; cherepanov, sun, et al., 2005) . allinis bind to the viral integrase at the ledgf/p75-binding site, preventing the binding of ledgf/p75 and affecting integrase catalytic core domain dimerization. this disrupts integrase assembly with viral dna and allosterically inhibits its activity (engelman et al., 2013) . 2-(quinolin-3-yl) acetic acid derivatives (ledgins), and in particular compound 6, showed potent inhibitory activity of hiv-1 replication (christ et al., 2010) . this compound establishes hydrogen bonds with integrase residues glu170, his171, and thr173 that are hot spots in the ledgf/p75-binding site in integrase (christ et al., 2010) . as allinis were more potent against hiv-1 in cells lacking ledgf/p75, the contribution of ledgf/ p75-binding inhibition to their mechanism of action is not clear (wang et al., 2012) . another successful strategy in the design of small molecules interfering viral replication has been the targeting of multimeric viral rna polymerases. influenza virus and vaccinia virus encode rna polymerases formed by the essential interaction of several subunits. in influenza virus each of the three viral subunits of the polymerase, pb1, pb2, and pa, is required to assemble in a complex for the polymerase activity. therefore, small molecules that interrupt the binding of pb1 into a hydrophobic groove in the c-terminus of pa (he et al., 2008) block replication of influenza a and b viruses (muratore et al., 2012) . similarly, vaccinia virus depends for replication on the formation of a trimeric complex between the dna polymerase e9, the uracil dna glycosylase d4, and the viral protein a20 (stanitsa, arps, & traktman, 2006) . small molecules disrupting this complex by impairing the interaction d4-a20 inhibit replication of vaccinia virus and cowpox virus (schormann et al., 2011) . there are two types of physical interactions that can be targeted to interrupt the utilization of cellular complexes by the virus: the virus-host interface or the host-host interface of complexes hijacked by the virus. targeting protein interaction surfaces containing viral proteins is always associated with the selection of mutant viruses that escape control of the inhibitor (de clercq, 2007; strasfeld & chou, 2010) . this is the result of the highly mutagenic rate of viruses. however, targeting host complexes utilized by viruses at the host-host interface is expected to do not exhibit mechanisms of resistance, since host proteins are genetically more stable than their viral counterparts. therefore, disruption of the host complexes exploited by viruses rather than interference of the virus-host protein interface is an attractive alternative to avoid the selection of escape mutants resistant to the inhibitors. a major disadvantage of this strategy is the potential toxicity associated to the disruption of cellular complexes. potentially, this could be minimized if the interruption of the complex is transient. viral replication is a fast process that occurs in a few hours, and disruption of these complexes for a few hours could greatly affect the ability of the virus to replicate without affecting cellular viability. we will later discuss experimental findings supporting this view. in addition, the high degree of functional overlap in the human proteome, that is absent in viruses because of their relative smaller genomes, could also ameliorate the toxicity caused by the disruption of host-host ppis implicated in viral replication. the differences in functional redundancy suggest that the disruption of cellular complex will be more tolerable for the host than the pathogen. in support of this view, shrna-based studies have showed that many host factors required for viral replication do not carry essential cellular functions. for example, out of 54,509 transcripts permanently targeted with lentivirus-encoded shrnas, 17% were dispensable for the cell and deficient stable cell lines were developed but a fraction of them were required for efficient hiv-1 replication (yeung, houzet, yedavalli, & jeang, 2009 ). finally, targeting host complexes relevant for different viruses could allow the development of broad-spectrum antivirals. protein translation is essential for the virus and the cell. however, transient interference with protein synthesis at the initiation step has been demonstrated to be more detrimental for the pathogen than the host at a cellular level. both cellular mrna and mrna from different viruses carry a 7-methylguanosine cap (cap) on their 5 0 terminal nucleotide. therefore, cap-dependent cellular translation is exploited by many different viruses. critical to this translation mechanism is the eukaryotic initiation factor 4f complex (eif4f). this complex is required for the efficient recruitment of ribosomes to capped mrnas. the eif4f complex includes a capbinding protein (eif4e), an rna helicase (eif4a), and a large scaffolding subunit (eif4g). eif4e protein binds to cap-mrna and then recruits eif4g that binds eif4a. therefore, inhibitors of eif4e-eif4g interaction (i.e., 4e2rcat ) or the helicase activity (hippuristanol) drastically impair translation. 4e2rcat completely blocks the replication of the human alphacoronavirus 229e (cencic, desforges, et al., 2011) or murine norovirus 1 (royall et al., 2015) at doses that impair approximately 40% of the cellular protein synthesis (cencic, desforges, et al., 2011) or do not alter at all cell viability. at these doses 4e2rcat affects eif4e-eif4g interaction as determined by coimmunoprecipitation (royall et al., 2015) . these results indicate that coronaviruses are more dependable on eif4f for ribosome recruitment mrnas than the host. hippuristanol is a natural product that inhibits the activity of eif4a by binding to its c-terminal domain. this compound impairs eif4a rnabinding, atpase, and helicase activities. hippuristanol specifically impaired replication of poliovirus , caliciviruses (chaudhry et al., 2006) , human cytomegalovirus (lenarcic, ziehr, de leon, mitchell, & moorman, 2014) , and junin virus (linero, thomas, boccaccio, & scolaro, 2011) . despite the cell toxicity of hippuristanol at the doses and length of the treatment used in these reports, viral replication was affected specifically since this compound did not alter cellular viability. for example, 6-h treatment of raw 264.7 cells with hippuristanol reduced cell viability by 10%, but murine norovirus production was reduced by over 3000-fold (chaudhry et al., 2006) . as described earlier, eif4e interacts with eif4g and assembles into the eif4f complex together with eif4a on the capped mrna. eif4f complex formation leads to eif4e phosphorylation at ser209 by the eif4g-associated kinase mnk1. inhibition of mnk1 by cgp57380 specifically impairs protein synthesis in a variety of large dna viruses (walsh, mathews, & mohr, 2013) . for example, this compound reduced by 10 2 -fold the replication of hsv-1 in quiescent primary fibroblasts without affecting cell viability (walsh & mohr, 2004) . in addition, cgp57380 inhibits reactivation of kaposi's sarcoma-associated herpesvirus (walsh et al., 2013) . because mnk1 is not essential for cell growth or development, it is a potential therapeutic target (ueda, watanabe-fukunaga, fukuyama, nagata, & fukunaga, 2004) . there are other examples of small molecules inhibiting ppis in host complex that are not essential for the cell. some compounds targeting host-virus ppis implicated in the earlier steps of the hiv-1 life cycle are in this category. for example, the hiv-1 entry inhibitor maraviroc, that reached clinical use, acts through an allosteric mechanism. this drug binds to a hydrophobic pocket located in the transmembrane domain of ccr5, altering the conformation of the extracellular domain of ccr5 and preventing in this manner the binding of env . in addition, the small molecule pf74, that inhibits hiv-1 replication by triggering early uncoating (shi, zhou, shah, aiken, & whitby, 2011) , binds to a pocket encompassing the n-terminal domain-c-terminal domain interface of ca in the assembled capsid (bhattacharya et al., 2014) . cleavage and polyadenylation-specific factor 6 (cpsf6) also binds to the same pocket in the capsid during hiv trafficking to the nucleus. this ppi delays uncoating, allowing hiv-1 to evade the innate immune activation through cytoplasmic nucleic acid sensor signaling (rasaiyaah et al., 2013) . we have presented literature evidences indicating that viruses interact for replication with cellular complexes. within these complexes, viral proteins bind to cellular proteins that are central (bottleneck proteins) in the interacting networks and establish multiple interactions in the human interactome (hub proteins). these ppis offer different opportunities for the development of small molecules to block viral replication. virusvirus, virus-host, and host-host ppis are amenable for pharmacological disruption. different viruses utilize similar host protein complexes during infection, showing overlap in the cellular processes hijacked. targeting these shared cellular processes opens the possibility to generate broad-spectrum antivirals and general biomarkers of viral infection. potentially, the innate immune system recognizes these shared pathogen-induced processes to counteract infection. a better understanding of this mechanism could greatly impact the development of stronger vaccine adjuvants. small-molecule inhibitors of protein-protein interactions: progressing toward the reality 2p2idb v2: update of a structural database dedicated to orthosteric modulation of protein-protein interactions functional interactions between papillomavirus e1 and e2 proteins structural basis of hiv-1 capsid recognition by pf74 and cpsf6 functional characterization of ireses by an inhibitor of the rna helicase eif4a modulation of host immune responses by clinically relevant human dna and rna viruses rediscovering the sweet spot in drug discovery epstein-barr virus and virus human protein interaction maps blocking eif4e-eif4g interaction as a strategy to impair coronavirus replication reversing chemoresistance by small molecule inhibition of the translation initiation complex eif4f caliciviruses differ in their functional requirements for eif4f components structural basis for the recognition between hiv-1 integrase and transcriptional coactivator p75 solution structure of the hiv-1 integrase-binding domain in ledgf/p75 rational design of small-molecule inhibitors of the ledgf/p75-integrase interaction and hiv replication adar1 and pact contribute to efficient translation of transcripts containing hiv-1 trans-activating response (tar) element a hot spot of binding energy in a hormone-receptor interface the pkr activator, pact, becomes a pkr inhibitor during hiv-1 replication structure of an allosteric inhibitor of lfa-1 bound to the i-domain studied by crystallography, nmr, and calorimetry global mapping of herpesvirus-host protein complexes reveals a transcription strategy for late genes hepatitis c virus infection protein network the design of drugs for hiv and hcv identification of a tripartite interaction between the n-terminus of hiv-1 vif and cbfbeta that is critical for vif function the landscape of human proteins interacting with viruses and other pathogens allosteric inhibition of hiv-1 integrase activity large-scale mapping of human protein-protein interactions by mass spectrometry gps-prot: a web-based visualization platform for integrating host-pathogen interaction data evolutionarily conserved herpesviral protein interaction networks protein-protein interactions as targets for small molecule drug discovery elucidating novel hepatitis c virus-host interactions using combined mass spectrometry and functional genomics approaches structural basis for hijacking cbf-beta and cul5 e3 ligase complex by hiv-1 vif a census of human soluble protein complexes crystal structure of the polymerase pa(c)-pb1(n) complex from an avian influenza h5n1 virus the use of rnai-based screens to identify host proteins involved in viral replication manipulation of the nuclear factor-kappab pathway and the innate immune response by viruses the druggable genome global landscape of hiv-human protein complexes purification and characterization of hiv-human protein complexes vif hijacks cbf-beta to degrade apobec3g and promote hiv-1 infection principles of protein-protein interactions analysis of protein-protein interaction sites using surface patches hot regions in protein-protein interactions: the organization and contribution of structurally conserved hot spot residues computational systems biology systems biology: a brief overview an integrated approach to elucidate the intra-viral and viral-cellular protein interaction networks of a gamma-herpesvirus differential role for host translation factors in host and viral protein synthesis during human cytomegalovirus infection molecular basis for pkr activation by pact or dsrna junin virus infection impairs stress-granule formation in vero cells treated with arsenite via inhibition of eif2alpha phosphorylation an essential role for ledgf/p75 in hiv integration the atomic structure of protein-protein recognition sites allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry the mdm2-p53 interaction chemical and structural lessons from recent successes in protein-protein interaction inhibition (2p2i) small molecule inhibitors of influenza a and b viruses that act by disrupting subunit interactions of the viral polymerase when the human viral infectome and diseasome networks collide: towards a systems biology platform for the aetiology of human diseases intracellular allosteric antagonism of the ccr9 receptor pact, a protein activator of the interferon-induced protein kinase viral immune modulators perturb the human molecular network by common and unique strategies the protein interaction map of bacteriophage lambda a combined proteomics/genomics approach links hepatitis c virus infection with nonsense-mediated mrna decay hiv-1 evades innate immune recognition through specific cofactor recruitment molecular gymnastics: mechanisms of hiv-1 resistance to ccr5 antagonists and impact on virus phenotypes murine norovirus 1 (mnv1) replication induces translational control of the host by regulating eif4e activity during infection interpreting cancer genomes using systematic host network perturbations by tumour virus proteins corum: the comprehensive resource of mammalian protein complexes-2009 structural insights for hiv-1 therapeutic strategies targeting vif identification of protein-protein interaction inhibitors targeting vaccinia virus processivity factor for development of antiviral agents a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection small-molecule inhibition of human immunodeficiency virus type 1 infection by virus capsid destabilization ledgf/p75 functions downstream from preintegration complex formation to effect gene-specific hiv-1 integration vaccinia virus uracil dna glycosylase interacts with the a20 protein to form a heterodimeric processivity factor for the viral dna polymerase antiviral drug resistance: mechanisms and clinical implications allo-network drugs: extension of the allosteric drug concept to protein-protein interaction and signaling networks cry2h-seq: a massively multiplexed assay for deep-coverage interactome mapping mnk2 and mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4e but not for cell growth or development herpesviral protein networks and their interaction with the human proteome patterns of pathogenesis: discrimination of pathogenic and nonpathogenic microbes by the innate immune system in vivo activation of the p53 pathway by small-molecule antagonists of mdm2 tinkering with translation: protein synthesis in virus-infected cells phosphorylation of eif4e by mnk-1 enhances hsv-1 translation and replication in quiescent cells hrp2 determines the efficiency and specificity of hiv-1 integration in ledgf/p75 knockout cells but does not contribute to the antiviral activity of a potent ledgf/p75-binding site integrase inhibitor reaching for high-hanging fruit in drug discovery at protein-protein interfaces an evolutionary and structural characterization of mammalian protein complex organization a genome-wide short hairpin rna screening of jurkat t-cells for human proteins contributing to productive hiv-1 replication strategies for targeting protein-protein interactions with synthetic agents nextgeneration sequencing to generate interactome datasets systems-level analysis of innate immunity relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces t-cell differentiation factor cbf-beta regulates hiv-1 vif-mediated evasion of host restriction we thank zachary s. martinez and angelica lopez, university of texas at el paso, for reviewing the manuscript. key: cord-344084-z4t2wkgk authors: ellwanger, joel henrique; kulmann-leal, bruna; kaminski, valéria de lima; rodrigues, andressa gonçalves; de souza bragatte, marcelo alves; chies, josé artur bogo title: beyond hiv infection: neglected and varied impacts of ccr5 and ccr5δ32 on viral diseases date: 2020-05-30 journal: virus res doi: 10.1016/j.virusres.2020.198040 sha: doc_id: 344084 cord_uid: z4t2wkgk the interactions between chemokine receptors and their ligands may affect susceptibility to infectious diseases as well as their clinical manifestations. these interactions mediate both the traffic of inflammatory cells and virus-associated immune responses. in the context of viral infections, the human c-c chemokine receptor type 5 (ccr5) receives great attention from the scientific community due to its role as an hiv-1 co-receptor. the genetic variant ccr5δ32 (32 base-pair deletion in ccr5 gene) impairs ccr5 expression on the cell surface and is associated with protection against hiv infection in homozygous individuals. also, the genetic variant ccr5δ32 modifies the ccr5-mediated inflammatory responses in various conditions, such as inflammatory and infectious diseases. ccr5 antagonists mimic, at least in part, the natural effects of the ccr5δ32 in humans, which explains the growing interest in the potential benefits of using ccr5 modulators for the treatment of different diseases. nevertheless, beyond hiv infection, understanding the effects of the ccr5δ32 variant in multiple viral infections is essential to shed light on the potential effects of the ccr5 modulators from a broader perspective. in this context, this review discusses the involvement of ccr5 and the effects of the ccr5δ32 in human infections caused by the following pathogens: west nile virus, influenza virus, human papillomavirus, hepatitis b virus, hepatitis c virus, poliovirus, dengue virus, human cytomegalovirus, crimean-congo hemorrhagic fever virus, enterovirus, japanese encephalitis virus, and hantavirus. subsequently, this review addresses the impacts of ccr5 gene editing and ccr5 modulation on health and viral diseases. also, this article connects recent findings regarding extracellular vesicles (e.g., exosomes), viruses, and ccr5. neglected and emerging topics in “ccr5 research” are briefly described, with focus on rocio virus, zika virus, epstein-barr virus, and rhinovirus. finally, the potential influence of ccr5 on the immune responses to coronaviruses is discussed. inflammatory cells play a crucial role in protecting the host from viral infections. leukocyte migration is a fundamental step of the inflammatory response to viruses, a process regulated by the interaction between chemokines and their receptors. therefore, dysregulations in the chemokine-mediated inflammatory process may contribute to viral pathogenesis (glass et al., 2003) . the c-c chemokine receptor type 5 (ccr5) interacts primarily with the chemokines ccl3 (mip-1α), ccl4 (mip-1β), and ccl5 j o u r n a l p r e -p r o o f (rantes) , which act as ccr5 agonists by stimulating cell migration and mediating inflammatory responses. on the other hand, the chemokine mcp-3/ccl7 is the main ccr5 antagonist ligand (blanpain et al., 1999; zlotnik and yoshie, 2000; glass et al., 2003; alkhatib, 2009 ). in addition to regulating the migration of non-specific leukocytes during inflammatory responses, ccr5 controls the action of specific cell types, including natural killer (nk) cells (khan et al., 2006; weiss et al., 2011) and regulatory t (treg) cells (wysocki et al., 2005; tan et al., 2009; dobaczewski et al., 2010) . ccr5 is also expressed by tissue-resident memory t cells. these ccr5 + cells support barrier immunity (davis et al., 2019) . the ccr5 is a g-protein-coupled receptor (gpcr), containing seven transmembrane α-helices, three extracellular loops, and three intracellular loops (tan et al., 2013) . figure 1 shows the structure of ccr5, highlighting its transmembrane domains and extra and intracellular loops. the specificity of interaction between ccr5 and chemokines is mediated by the second extracellular loop (samson et al., 1997) . helices 2 and 3 have a fundamental role in chemokine-induced ccr5 activation (govaerts et al., 2003) . the steps required from ligand binding culminating in cell migration encompass a series of intracellular interactions, including the g-protein heterotrimer and downstream effectors (lacalle et al., 2017) . after stimulation by chemokines or natural reactive antibodies and subsequent triggering of chemotaxis, ccr5 is phosphorylated and internalized in the cytoplasm (signoret et al., 2000; venuti et al., 2015; venuti et al., 2016; lacalle et al., 2017; venuti et al., 2017; venuti et al., 2018) . the number of available receptors on the cell surface is related to the rate of internalization and recycling of ccr5, which affects the activation of ccr5 and consequent signaling of specific pathways that culminate in chemotaxis processes (mueller et al., 2002) . of note, intracellular pools of ccr5 can be detected in the cells. these pools are probably formed by internalized or immature/precursor forms of ccr5 molecules (mirzabekov et al., 1999; kohlmeier et al., 2008; achour et al., 2009; guglielmi et al., 2011; shirvani et al., 2011) that can be rapidly expressed on the cell surface in response to viral stimuli and inflammatory responses (kohlmeier et al., 2008) . in other words, ccr5 molecules are recycled by cells. specifically, ccr5 recycling can be mediated by degradation followed by de novo synthesis (in response to stimulation by natural antibodies) or occur in the classic short-term system without de novo synthesis (in response to stimulation by ccl5, for example) (venuti et al., 2015; venuti et al., 2016) . the traffic of ccr5 between the plasma membrane and the intracellular medium is mediated by different molecules, including clathrins, β-arrestin 2, and extracellular signal-regulated kinase (erk) 1 (venuti et al., 2015; venuti et al., 2016; venuti et al., 2018) . also, intracellular cd4 regulates the expression of ccr5 on the cell surface (achour et al., 2009 ). the human ccr5 protein (352 residues) is encoded by the ccr5 gene [chromosome 3 (3p.21.31)], which is very polymorphic (blanpain et al., 2000; hoover, 2018) . among polymorphisms of the ccr5, the ccr5δ32 (rs333) has been intensively studied in different human populations. the frequency of the ccr5δ32 is quite variable. in general, the δ32 allele frequency is high in european-derived populations (for example, 16% in norway and 11% in germany) and low or absent in african and asian populations j o u r n a l p r e -p r o o f (solloch et al., 2017) . however, although the δ32 allele is more frequent in european populations, there are exceptions due to migratory events. for example, the frequency of the δ32 allele is high in south africa (13%) and chile (12%) (solloch et al., 2017) . also, the frequency of the δ32 allele can be quite variable within the same country. in brazil, the frequency of the allele in the general population is around 4-5% (silva-carvalho et al., 2016; solloch et al., 2017) . in the southern region of the country, the frequency can reach up to 9% due to the past migration of european populations to this region (boldt et al., 2009; pena et al., 2011; schauren et al., 2013) . figure 2 summarizes basic aspects of ccr5 and shows the frequency of the δ32 allele in various countries. the ccr5δ32 is the most studied genetic variant of the ccr5 gene because of its strong protective effect against hiv infection (considering susceptibility to ccr5-tropic strains). hiv entry into cd4 + t cells is mediated by the interaction of the virus with cd4 and with a co-receptor, usually ccr5. the ccr5δ32 variant is a 32 base-pair deletion in the ccr5 coding region, which causes a frameshift, resulting in a truncated protein that is not directed to the cell surface. ccr5δ32 in heterozygosis promotes a decrease in the expression of functional ccr5 on the cell surface compared to ccr5 wild-type cells. therefore, individuals with heterozygous genotype for ccr5δ32, if infected with hiv, have a small protection against disease progression due to the reduced expression of ccr5 on the surface of cd4 + t cells (reduced hiv-ccr5 interaction). in ccr5δ32 homozygous cells, no ccr5 is expressed in the plasmatic membrane. therefore, homozygous individuals for this polymorphism (δ32/δ32) show virtually total protection against hiv type 1 infection, since no ccr5 expression is verified on cell surface (no hiv-ccr5 interaction at cell surface is possible) (deng et al., 1996; dragic et al., 1996; huang et al., 1996; samson et al., 1996; wu et al., 1997; proudfoot, 2002; venkatesan et al., 2002; picton et al., 2012) . figure 3 illustrates the phenotypic effects of ccr5δ32 in human cells. the main results involving the triad "ccr5, hiv, and ccr5δ32" were published in 1996 in nature, cell and science papers by different groups (parmentier, 2015) . since then, the research involving ccr5 has explored the role of the ccr5 protein and ccr5δ32 polymorphism in different diseases, as well as the therapeutic potentials of ccr5 blockade. currently, the physical interaction of ccr5 with hiv is known in detail (shaik et al., 2019) ryst, 2015; latinovic et al., 2019) . also, a recent study has shown that molecules that inhibit ccr5 trafficking to the plasma membrane also have a therapeutic potential against hiv infection (boncompain et al., 2019) . research involving ccr5 has also brought other important advances in combating hiv infection. of note, there are already two cases of sustained remission of hiv infection following stem-cell transplantation using ccr5δ32 homozygous donor, the "berlin patient" (hütter et al., 2009) and the "london patient" (gupta et al., 2019; gupta et al., 2020) . articles that evaluated the involvement of ccr5 or ccr5δ32 in the infections caused by the mentioned viruses were analyzed. subsequently, the google scholar (https://scholar.google.com.br/) was consulted to detect relevant papers that were not indexed in pubmed, using the terms "ccr5" in association with the name of each of the viruses covered in the review. the authors of this review tried to include the largest number of original articles that addressed the topic covered in this work, intending to write a broad and complete article. however, some papers were not included because it was not possible to obtain clear conclusions from the studies. the reference lists of the consulted articles were also used to complement the selection of articles for this review. as previously mentioned in the introduction, a discussion addressing the involvement of ccr5 in tbev infection was not included in this work. articles addressing the involvement of ccr5 and ccr5δ32 in hiv infection were included only to present to the reader some basic and historical aspects related to such topics, cited mainly in the introduction section and figures, but not included as a major section. considering the importance of the coronavirus disease 19 (covid-19) pandemic, a section addressing the potential influence of ccr5 on the immune responses to coronaviruses was included in the article. review articles were also selected in pubmed and google scholar for writing the sections and paragraphs that address the basic aspects of viruses, exosomes, and diseases (e.g., epidemiological, molecular, clinical aspects). exceptionally, review articles with outstanding discussions regarding the role of ccr5 in viral infections were also cited in this work. regarding tables, it is important to note that the data available in the "population" columns are limited and often represent only general characteristics of the evaluated population. in many studies, a population can be composed of individuals from various ethnic groups. this limitation must be taken into account when evaluating the results of the studies cited in the tables. finally, also in "population" columns, "information not available" was used when this information was not clearly described in the cited article. west nile virus (wnv) is a neurotropic positive-sense single-stranded rna flavivirus endemic in various parts of the world. wnv transmission to humans occurs through the bite of infected mosquitoes, especially species of the culex genus (suthar et al., 2013) . although different animals can participate in the wnv transmission cycle, birds are the classic amplifier hosts. humans, horses and other mammals are deadend hosts (kramer et al., 2008; cdc, 2018) . among humans, blood transfusion, organ transplantation, and breast milk can also transmit the virus. vertical transmission may also occur. however, compared to transmission by mosquito bites, these routes of transmission are rare (kramer et al., 2008) . about 25% of wnv-infected individuals develop west nile fever, a clinical condition with variable symptoms and severity. in less than 1% of the infected individuals, wnv invades the central nervous system (cns), causing neurological manifestations (neuroinvasive disease), including meningitis, encephalitis, and acute flaccid paralysis. of note, west nile neuroinvasive disease shows a 10% to 20% fatality rate. severe illness is associated with older age and other factors, including genetic traces (petersen et al., 2013; sejvar, 2016) . wnv infection is considered the leading cause of arboviral encephalitis in the world (ciota, 2017) . the treatment of wnv infection is supportive (petersen et al., 2013) . it is known that the ccr5 protein interferes in the clinical course of wnv infection (glass et al., 2005; diamond, 2009; michlmayr and lim, 2014) , but the effects of ccr5δ32 on the susceptibility to this infection and disease progression are different. according to lim et al. (2010) and danial-farran et al. (2015) , ccr5δ32 has no important effect on susceptibility to wnv infection. in accordance, loeb et al. (2011) found no association between ccr5δ32 and wnv infection. conversely, there is robust populationbased data showing a strong association between the ccr5δ32 homozygous genotype and increased risk of developing symptomatic wnv infection lim et al., 2008; lim et al., 2010) . also, bigham et al. (2011) showed that the ccr5δ32 variant was associated with symptomatic wnv infection when the dominant model of inheritance was considered in the analysis. a recent meta-analysis confirmed the role of the ccr5 gene in wnv infection, specifically the association between the ccr5δ32 with severe disease (cahill et al., 2018) . table i summarizes the results of the studies that evaluated the ccr5δ32 genetic variant in the context of human wnv infection. in agreement with studies showing that ccr5δ32 homozygous genotype is a risk factor for symptomatic wnv infection in humans, ccr5-/-wnv-infected mice showed a reduced capacity of viral control, increased disease severity, impaired leukocyte trafficking towards the brain, and high mortality rates than ccr5 wild-type mice. these findings reinforce that ccr5 is a key molecule in the immune response against wnv (glass et al., 2005; durrant et al., 2015) . taking together, these pieces of evidence robustly support the role of ccr5 as a protective molecule on wnv pathogenesis. specifically, ccr5+ leukocytes play a fundamental role in combating wnv in the brain (glass et al., 2005; lim et al., 2006; michlmayr and lim, 2014; durrant et al., 2015) . in this sense, ccr5 plays a specific and non-redundant role in controlling wnv infection (lim and murphy, 2011; durrant et al., 2015; ellwanger et al., 2020a) . based on the data mentioned above, the lack of ccr5 expression linked to ccr5δ32 homozygosis is an important risk factor for increased severity of wnv-associated disease. the opposite effects of the ccr5δ32 genetic variant on both hiv and wnv infections are summarized in figure 5 . hereupon, individuals homozygous for ccr5δ32 and living in endemic areas of the wnv should take additional care to prevent wnv infection (e.g., use of repellents, mosquito nets). also, the use of ccr5 blockers to treat hiv infection may have a negative impact on populations living in wnv-endemic areas. to avoid this negative impact, hiv-infected individuals who live in such areas and who use ccr5 blockers must apply robust measures against mosquito bites lim et al., 2006; lim and murphy, 2011). j o u r n a l p r e -p r o o f influenza infection affects humans seasonally, causing recurrent epidemics and even pandemics in some years. in humans, the infection is caused basically by influenza a and influenza b, both enveloped negative-sense single-stranded rna viruses belonging to orthomyxoviridae family (krammer et al., 2018; petrova and russell, 2018) . influenza a is a zoonotic disease, and influenza b circulates primarily in humans. influenza infection affects mainly the respiratory tract, which can cause mild to severe disease depending on viral and host characteristics. secondary bacterial infection may also occur (krammer et al., 2018) . human co-infection with multiple influenza types is an important neglected problem (gregianini et al., 2019) . influenza is a prevalent infection worldwide, and new vaccines are produced annually, based on strains circulating each year in the northern and southern hemispheres (krammer et al., 2018; petrova and russell, 2018) . antivirals can be used in the treatment of influenza infection (krammer et al., 2018) . investments in new vaccines, antiviral drugs, and surveillance systems are needed to reduce the global burden associated with influenza infection (petrova and russell, 2018) . the severity of influenza infection is related to the intensity of proinflammatory responses and the predominant profile of cytokine production by the host . a body of evidence has shown that both ccl5 and ccr5 participate in the modulation of the immune response to influenza virus infection (matsukura et al., 1998; tyner et al., 2005; sládková and kostolanský, 2006; kohlmeier et al., 2008; oslund and baumgarth, 2011) . of note, ccr5 mediates the recruitment of nk cells to the lungs in influenza a infection (carlin et al., 2018) and participates in neutrophil action in the lungs during influenza pneumonia (rudd et al., 2019) . although flow cytometry data did not indicate significant changes regarding ccr5 expression on the surface of human monocytes after experimental influenza a infection (salentin et al., 2003) , various studies have shown that, in mice, the lack of ccr5 expression is associated with a higher risk of death by influenza infection (dawson et al., 2000; tyner et al., 2005; fadel et al., 2008; tavares et al., 2020) . based on these findings, it was postulated that pharmacological ccr5 blockade may have some undesirable effect on the immune response against the influenza virus in humans (fadel et al., 2008) . importantly, more research on this aspect is needed since this data suggests that, in humans, the ccr5 absence due to the ccr5δ32 polymorphism could affect pathogenesis and the lethality rate of influenza infection. falcon et al. (2015) evaluated the frequency of the ccr5δ32 in pandemic h1n1-infected spanish individuals and revealed an association between the polymorphism with fatal outcome. the ccr5δ32 was also associated with increased disease severity in other studies (keynan et al., 2010; rodriguez et al., 2013) . however, these results should be interpreted with caution since both studies were based on very small sample sizes (keynan et al., 2010; rodriguez et al., 2013) . importantly, other studies addressing humans reported no association between ccr5δ32 and severity of influenza infection (sironi et al., 2014; maestri et al., 2015; matos et al., 2019) . taking together, the body of evidence suggests that ccr5δ32 has little j o u r n a l p r e -p r o o f influence on severity of influenza infection (table 2) . results described by falcon et al. (2015) seem to be specific to that studied population, composed of individuals from 13 regions of spain. human papillomavirus (hpv) is a double-stranded dna virus belonging to the papillomavirus family. hpv is transmitted by direct contact (for example, through sexual intercourse). the hpv infection is quite common worldwide, and is usually controlled by the immune system. viral clearance occurs in most cases within 1-2 years after infection. however, if the infection is not controlled, hpv can cause noncancerous mucosal lesions or different types of malignant lesions such as anal cancer, penile cancer, vulvar cancer, head cancer, neck cancer, and especially cervical cancer. hpv is an oncogenic pathogen because it inhibits the activity of p53 and prb (retinoblastoma protein) tumor suppressor molecules through the action of e6 and e7 viral proteins, respectively. these proteins also exhibit other oncogenic mechanisms. worldwide, between 5-10% of all cancers in women are due to hpv infection (schiffman et al., 2016; sanjosé et al., 2018) . there are several hpv genotypes (>200), and those most associated with cancer are: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and probably 68 (schiffman et al., 2016) . of note, the genotype 16 (hpv16) has a prominent role in cancer development. vaccination is one of the most effective ways to prevent hpv infection, having important positive impacts on multiple aspects of population health. hpv vaccine protects the vaccinated individual from infection per se and from hpv-related cancer (schiffman et al., 2016; sanjosé et al., 2018) . the expression of ccr5 is increased in cervical cancer tissues (sales et al., 2014; che et al., 2016) . also, in vitro growth, proliferation, and invasive capacity of cervical cancer cells can be inhibited through ccr5 downregulation (che et al., 2016) . these findings suggest the involvement of ccr5 in the development of cervical lesions. in a study performed with indian individuals (singh et al., 2008) , the genotype and allele frequencies of ccr5δ32 were not different between cervical cancer patients and controls. however, when the patients were stratified by cancer stage (stages 1b to 4), the ccr5 heterozygous genotype was associated with stage 1b of cervical cancer. the hpv positivity rate among the evaluated patients was not described (singh et al., 2008) . the relationship between ccr5δ32, hpv infection, and cervical lesions is addressed in other studies (table 3 ). ccr5δ32 homozygous genotype was associated with increased susceptibility to hpv infection in a study performed with swedish individuals (zheng et al., 2006) . mangieri et al. (2019) investigated the potential influence of ccr5δ32 on susceptibility to hpv infection and cervical lesions in brazilian women. however, the genetic variant was not significantly associated with susceptibility to hpv infection (considering allele frequency, codominant model and dominant model) or hpv-associated lesions (mangieri et al., 2019) . in accordance, previous studies performed with brazilian women did not find an association between ccr5δ32 and susceptibility to hpv infection (suzuki et al., 2008) or between the polymorphism and hpv-related cervical lesions (suzuki et al., 2008; santos et al., 2016) . lastly, ccr5δ32 j o u r n a l p r e -p r o o f did not affect susceptibility to hpv infection in lithuanian individuals with laryngeal cancer (stumbrytė-kaminskienė et al., 2020) . in conclusion, although tissue analysis and evidence obtained in vitro suggest that the ccr5 is potentially involved in the pathogenesis of hpv, most studies point to a lack of involvement of ccr5δ32 in susceptibility to hpv infection or hpv-associated diseases. the the ccr5 and its ligands regulate the action of t cells and other leukocytes in the liver. thus, ccr5 regulates liver inflammation and participates in the local immune response against viruses (ajuebor et al., 2006; sanchooli et al., 2014) . in mice models of hepatitis, ccr5 deficiency was associated with increased liver inflammation, tissue injury, and liver failure (ajuebor et al., 2005; moreno et al., 2005; stevens et al., 2019) . deficiency of ccr5 expression is generally associated with reduced migration of inflammatory cells, which would translate into less inflammation. this reasoning is correct and applies to different situations and tissues (braunersreuther et al., 2007; muntinghe et al., 2009; kaminski et al., 2019a) . however, ccr5 is an immunoregulatory molecule (doodes et al., 2009; dobaczewski et al., 2010; christmann et al., 2011; hütter et al., 2011) and therefore its deficiency can also cause deregulation in the action of various immune cell types (e.g., nk and treg cells), increasing the inflammatory status in some tissues. in humans, multiple evidence has shown the involvement of ccr5 (protein and gene) in distinct aspects of hbv infection (ahn et al., 2006; trehanpati et al., 2009; ahmadabadi et al., 2013; yang et al., 2018) . interestingly, ccr5δ32 and other host genetic factors can affect the immunogenicity of the hbv vaccine (ganczak et al., 2017; ellwanger and chies, 2019b) . considering these aspects, the influence of the ccr5δ32 on susceptibility/resistance to hbv and disease severity is quite plausible. several studies investigated the role of the ccr5δ32 polymorphism in hbv infection. some of them found no association between those variables (arababadi et al., 2010; khorramdelazad et al., 2013; safari et al., 2017; zhang et al., 2018; moudi et al., 2019) . in other studies, the absence of ccr5δ32 allele in the sample, due to ethnic features of the evaluated population, precluded the analysis concerning the potential impact of this genetic variant on hbv infection (ahn et al., 2006; li et al., 2011) . some authors have reported significant influences of ccr5δ32 on hbv infection. suneetha et al. (2006) reported an association between the ccr5δ32 heterozygous genotype and chronic hbv infection. in contrast, thio et al. (2007) found an association between the ccr5δ32 allele and infection recovery in a study that analyzed individuals with persistent hbv infection and individuals who recovered from the infection. subsequently, thio et al. (2008) associated the hbv infection recovery with an epistatic effect between the ccr5δ32 and the rantes-403a promoter polymorphisms. finally, abdolmohammadi et al. (2016) found a protective effect of the ccr5δ32 genetic variant against hbv infection, once the δ32 allele was more frequent in controls compared to hbv-infected individuals. recently, we investigated the frequency of ccr5δ32 in hbv mono-infected and hbv/hiv coinfected brazilian individuals (ellwanger et al., 2020b) . a control group and hiv mono-infected individuals were also evaluated in our study. a total of 1113 individuals were studied, which represents the largest study involving ccr5δ32 and hbv infection to date (see table 4 for comparisons with other studies). we found a significant protective effect of ccr5δ32 on hbv/hiv co-infection, a result probably due to the partial protective effect of ccr5δ32 against hiv infection since no important impact of ccr5δ32 on susceptibility to hbv mono-infection was observed (ellwanger et al., 2020b) . the hepatitis c virus (hcv) was formally described in 1989 (choo et al., 1989) and, currently, hcv infection is one of the most important infectious diseases in terms of global public health burden. hcv is an enveloped single-stranded positive-sense rna virus, has seven genotypes, and belongs to the flaviviridae family, hepacivirus genus (pietschmann and brown, 2019) . it is estimated that 71 million individuals are chronically infected by hcv worldwide (viganò et al., 2019) . similar to the hbv infection, hcv-infected individuals can eliminate the virus naturally or develop chronic infection, which occurs in 55-85% of the cases. chronic infection can cause liver inflammation, cirrhosis, and hepatocarcinoma (lingala and ghany, 2015) . in addition to liver damage, hcv causes a series of immune-mediated extrahepatic manifestations, including rheumatologic, dermatologic, ophthalmologic, renal, pulmonary, neuropsychiatric, cardiovascular, and hematologic manifestations, especially mixed cryoglobulinemia (romano et al., 2018) . hcv therapy using direct-acting antivirals (daas) shows cure rates over 95% (pietschmann and brown, 2019) . early treatment of infected patients decreases death rates from hcv-associated liver disease, reduces disease transmission, and alleviates extrahepatic health problems. focusing the efforts on hcv treatment is extremely important because there is no effective hcv vaccine (viganò et al., 2019) . hcv seropositivity is an important risk factor for hiv infection (zwolińska et al., 2013) . like hiv, hcv is primarily transmitted through blood transfusion and sexual intercourse, and hcv/hiv co-infection is a major problem worldwide. depression of the immune system due to uncontrolled hiv infection may contribute to hcv progression (schlabe and rockstroh, 2018) . both susceptibility to hcv infection and j o u r n a l p r e -p r o o f disease progression are affected by viral and environmental factors and physio-metabolic, immune, and genetic components of the host (ellwanger et al., 2018a) . chemokines and chemokine receptors participate in the recruitment and activity of inflammatory cells in the liver, acting on anti-hcv immune responses and ultimately modifying the rate of inflammation and other histological manifestations observed during infection. based on this rationale, the ccr5 molecule was postulated as having an impact on hcv-induced liver injury, susceptibility to hcv infection, and modulation of the possibilities of viral clearance. the downregulation of ccr5 due to ccr5δ32 may interfere in these processes (ahlenstiel et al., 2004; coenen and nattermann, 2010) . in agreement, several polymorphisms in other immune system genes [especially human leukocyte antigen (hla), mannosebinding lectin (mbl), toll-like receptor (tlr), interleukins (il), and interferon (ifn) gene families] indeed modify both susceptibility to hcv infection and disease progression (ellwanger et al., 2018a) . especially the focus of this review, clinical response to hcv therapy is influenced by ccr5 gene polymorphisms (konishi et al., 2004; omran et al., 2013) . also, there is evidence showing that ccr5 haplotypes can affect susceptibility to hcv infection (huik et al., 2013) . a recent in vitro study suggested that ccr5 blockage could have a beneficial effect on the treatment of hcv infection since ccr5 antagonists (maraviroc and cenicriviroc) inhibit hcv replication (blackard et al., 2019) . the use of ccr5 antagonists in humans is safe (fätkenheuer et al., 2010; gulick et al., 2014; giaquinto et al., 2018) and these drugs have the potential to treat a number of diseases in which ccr5 is involved, including hcv-associated liver disease. although the use of ccr5 antagonists on hcv monoinfection is not yet approved, it can be useful specifically for co-infected hiv/hcv patients, where ccr5 blocking (maraviroc) is already recommended (haïm-boukobza et al., 2013; blackard et al., 2019) . however, the detailed patterns of ccr5 expression in different tissues and at various points in the clinical course of hcv infection are still poorly understood. according to a recent study, the expression of ccr5 in cd8+ t cells is increased in the liver of chronic hcv-infected patients (pirozyan et al., 2019) , but other studies have found mixed results regarding ccr5 expression on t cells in the context of hcv infection (lichterfeld et al., 2002; vincent et al., 2005; larrubia et al., 2007; zahran et al., 2020) . therefore, considering that the role of ccr5 in hcv infection is still uncertain, the potential use of ccr5 blockers to treat hcv mono-infection should be cautious. the influence of the ccr5δ32 variant on hcv infection susceptibility was investigated by several studies. woitas et al. (2002) found a significantly higher frequency of the ccr5δ32 homozygous genotype in hcv-infected individuals compared to controls, hiv-infected and hcv/hiv co-infected individuals, suggesting the ccr5δ32 as a risk factor for hcv infection. in agreement, the δ32 allele was a significant risk factor for infection when the authors compared the hcv-infected group to both controls and hivinfected individuals. moreover, the ccr5δ32 homozygous genotype was associated with increased hcv loads. in their study, it was observed an important deviation from the hardy-weinberg equilibrium in data from hcv-infected individuals; and a high portion of the individuals included in the study was hemophiliac j o u r n a l p r e -p r o o f (woitas et al., 2002) . hemophiliac individuals were at high risk of exposure to hcv and hiv until the mid-1980s (promrat et al., 2003; zhang et al., 2003) . considering that the ccr5δ32 homozygous genotype provides protection against hiv infection, a high frequency of this genotype in an hcv-infected group may be due to hiv resistance, but not to hcv, among individuals highly exposed to both viruses. due to those and other reasons, the results of woitas et al. (2002) were criticized by different authors (klein, 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003) . in this sense, no influence of ccr5δ32 on susceptibility to hcv infection were reported in studies performed with various populations (glas et al., 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003; ruiz-ferrer et al., 2004; wald et al., 2004; wasmuth et al., 2004; thoelen et al., 2005; goyal et al., 2006) . reinforcing the observations of those different studies, our group found no association between the ccr5δ32 and susceptibility to hcv infection or hcv/hiv co-infection in a study that evaluated a large number of brazilian individuals (ellwanger et al., 2018b) . bineshian et al. (2018) did not detect the ccr5δ32 allele in any iranian hcv-infected individual and controls included in their study, preventing any conclusion in terms of susceptibility in that population. finally, it is important to mention that in addition to the study by woitas et al. (2002) , a study performed in 2013 also found an association between the ccr5δ32 homozygous genotype with chronic hcv infection in europeans, but the authors of the study mentioned that specific factors regarding selection bias (e.g., co-exposure to hiv) may have influenced their results (suppiah et al., 2013) . taken together, the above-mentioned results called attention for the importance of performing genetic variant studies in different populations, exposed to different social and environmental factors and presenting distinct ethnic backgrounds. considering multiple clinical and histological parameters, two main different results were obtained when the ccr5δ32 genetic variant was evaluated in the context of hcv-related diseases: reduced liver inflammation in δ32 allele carriers (hellier et al., 2003; wald et al., 2004; goulding et al., 2005) ; and no association between the ccr5δ32 and clinical variables (glas et al., 2003; mangia et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; ruiz-ferrer et al., 2004; morard et al., 2014; ellwanger et al., 2018b) . in the context of persistence/resolution of hcv infection and viral control, in the one hand, studies described association of the ccr5δ32 allele with reduced rates of spontaneous viral clearance (nattermann et al., 2011; morard et al, 2014) , higher viral load (yilmaz et al., 2014) , and reduced anti-hcv immune response (ahlenstiel et al., 2009 ). on the other hand, studies have reported association between the ccr5δ32 variant and increased rates of spontaneous viral clearance (goulding et al., 2005; el-moamly et al., 2013) . no significant effect of the ccr5δ32 on viral clearance was reported by other authors (mascheretti et al., 2004) . the potential impact of the ccr5δ32 polymorphism on response to hcv therapy was also evaluated by some authors. ahlenstiel et al. (2003) found an association between the ccr5δ32 allele and reduced response rates to interferon-α monotherapy, but the polymorphism did not affect the response to the combined interferon/ribavirin therapy. this finding shows that the use of more robust therapeutic regimens j o u r n a l p r e -p r o o f compensates the undesirable effects of ccr5δ32 on hcv therapy with interferon-α monotherapy. of note, the effect of ccr5δ32 may be negligible in the context of modern hcv therapies (ahlenstiel et al., 2003) . other studies did not report significant effects of the polymorphism on response to hcv therapy (glas et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; suppiah et al., 2013; morard et al., 2014) . again, it is important to mention that the ethnic distribution of the ccr5δ32 allele could interfere with study results. konishi et al. (2004) , for example, did not detect the ccr5δ32 allele in any japanese individual included in their study focused on host genetic factors involved in the response to interferon therapy. lastly, the polymorphism also does not appear to be associated with any specific hcv genotype (glas et al., 2003; wasmuth et al., 2004; goyal et al., 2006) . ahlenstiel et al. (2004) highlighted that the impact of ccr5 on hcv infection was controversial. in 2020, many controversies remain, although some points are better defined. table 5 summarizes the main findings of the studies that evaluated ccr5δ32 on hcv infection. although some studies indicate an influence of the polymorphism on susceptibility to infection, most studies indicate that the δ32 allele has little (or no) influence on hcv susceptibility. the impact of the ccr5δ32 on hcv-related liver disease is quite variable and context-dependent. finally, available data suggest some benefit of ccr5 antagonists for the treatment of hcv mono-infection. however, these data are still limited and further studies evaluating this topic are needed. poliovirus (pv) is a single-stranded rna enterovirus of the family picornaviridae. there are three types of pv: wild pv type 1 (wpv1), type 2 (wpv2), and type 3 (wpv3). the pv replicates in the tonsils and intestinal tract. in few infection cases (~1%), the virus invades the cns and can cause poliomyelitis resulting in paralysis. poliomyelitis is a condition characterized by inflammation of the gray matter of the spinal cord and muscle paralysis unleashed by pv replication in motor neurons (racaniello, 2006; kew and pallansch, 2018; keohane et al., 2020) . polioencephalitis can also occur and is characterized by the pv outbreak occurred in that country (table 6 ). the authors found no statistically significant effect of the ccr5δ32 on pv infection; only a trend of association between the δ32 allele and increased risk of pv infection was observed (rosenberg et al., 2013) . however, this study had a very small sample size (only seven cases of severe pv infection were evaluated) and therefore the results were not conclusive. in addition, due to the declining number of pv infection cases in the world, the effect of ccr5δ32 will be increasingly difficult to be assessed in population-based studies. dengue virus (denv) is a single-stranded rna virus that belongs to the flaviviridae family, flavivirus genus. there are four denv serotypes, being all transmitted to humans by aedes mosquitoes (aedes aegypti and aedes albopictus) (guzman et al., 2016) . denv infection is a global health problem with huge impacts on public health systems, especially in tropical countries (bhatt et al., 2013) , being considered the most common arbovirosis in the world (stanaway et al., 2016) . globally, the incidence of symptomatic denv infection is within the range of 50 to 100 million cases per year, resulting in ~10,000 deaths each year (stanaway et al., 2016) . clinically, dengue illness is divided into three basic phases: acute febrile phase, critical phase, and recovery (convalescent) phase. dengue disease occurs with/without warning signs or severe dengue. warning signs (suggestive signs or symptoms of important fluid loss, capillary leakage, and shock, such as severe abdominal pain and mucosal bleeding; observed at the end of the febrile phase) allow the rapid identification of patients who need more clinical attention and supportive therapy, in an attempt to avoid severe dengue. when severe disease occurs, this condition can lead to serious organ involvement, shock, and hemorrhage, among other signals and symptoms (guzman et al., 2016) . infection with a denv serotype triggers long-term immunity to that specific serotype (homotypic denv). immunity to heterotypic denv also occurs, but it is transitory. therefore, an individual can have dengue disease more than once. severe dengue occurs more frequently in recurrent infection with a different viral serotype (murphy and whitehead, 2011; st john and rathore, 2019 ). an immune response mediates denv clearance and the resolution of dengue diseases, but it is also involved in the disease pathogenesis (murphy and whitehead, 2011) . some evidence suggests the participation of ccl5/ccr5 axis in the protection against denv (sierra et al., 2014) , as well as in the pathogenesis of dengue disease (islam et al., 2019) . indeed, denv infection is associated with increased frequency of human ccr5+ t cells (de-oliveira-pinto et al., 2012; badolato-corrêa et al., 2018) . in a study performed by marques et al. (2015) , lower viral replication was found in macrophages treated with ccr5 blockers. in the same study, ccr5-/-mice were protected from denv infection. these findings suggest that ccr5δ32 could be a protective factor against denv infection. however, xavier-carvalho et al. (2013) found no statistical difference in the frequency of ccr5δ32 polymorphism between brazilian children with j o u r n a l p r e -p r o o f severe denv infection and healthy controls. in the same direction, no effect of ccr5δ32 on susceptibility to denv infection was found in a small sample-size study performed with individuals from western australia (brestovac et al., 2014) and recent data suggested no important involvement of ccr5 gene or ccr5 polymorphisms in denv infection (cahill et al., 2018; ornelas et al., 2019) . finally, the ccr5δ32 allele was not identified in a small group of indian denv-infected individuals (islam et al., 2019) . table 6 shows some details of the studies involving ccr5δ32 and denv infection. taking together, the data mentioned above indicate that the effects of ccr5 on denv infection are very different between humans and rodents. however, it should be noted that the approach of each mentioned study is quite particular, and we cannot exclude some potential effects of ccr5 and ccr5δ32 on denv infection in humans. cmv is also linked to cancer development, once several cmv proteins (e.g., pul122, pul123, pus28, pul83, pul111a) activate pro-oncogenic pathways, including angiogenesis, escape of immune control and tumor suppressors, tumoral inflammation, invasion and metastasis, genome instability, and increased cell survival and proliferation (herbein, 2018) . poor socioeconomic condition favors cmv infection. antibodies indicating past cmv infection are found in ~60% of adults from high-income countries. in low-income countries, the rate of past infections can reach 100% (griffiths et al., 2015) . cmv can manipulate the immune system producing virokines (virus-encoded cytokine/chemokine homologs) and viroceptors (virus-encoded cytokine/chemokine receptor homologs), molecules that enable the virus to evade host immune defenses. such molecules can also facilitate viral replication (lucas et al., 2001; froberg, 2004; vomaske et al., 2012) . importantly, cmv-encoded proteins can interact with ccr5, (johnson et al., 2015) . however, mixed results were reported regarding the effect of cmv on ccr5 expression since there is evidence indicating that cmv infection may reduce ccr5 expression in various cell types (lecointe at al., 2002; varani et al., 2005) . interestingly, these mixed results may not be contradictory. cmv-infected cells may indeed exhibit decreased ccr5 expression, limiting hiv infection in these cells. however, cmv-infected cells release cmv-associated soluble factors that increase ccr5 expression in non-infected bystander cells, then facilitating hiv replication in such cells and, consequently, contributing to hiv pathogenesis (king et al., 2006) . there is evidence showing that variants in the ccr5 gene can influence multiple aspects of cmv infection (loeffler et al., 2006; sezgin et al., 2011) . for example, the ccr5 promoter polymorphism rs1800023 affects cmv replication (bravo et al., 2014; corrales et al., 2015) . in a study evaluating children, kasztelewicz et al. (2017) found no influence of ccr5δ32 on susceptibility to congenital cmv infection, severity of congenital cmv disease, or cmv-related sensorineural hearing loss at birth. as an individual genetic factor, ccr5δ32 was not statistically associated with the progression of cmv retinitis, a condition that cmv can cause in immunocompromised individuals (sezgin et al., 2011) (table 6 ). bunyaviridae. cchfv circulates in africa, europe, middle east, and asia countries, and can infect a variety of domestic animals and wild species, but without causing symptomatic illness. humans are accidental hosts of cchfv, for which the virus is transmitted mainly by tick-bites (especially ticks of the genus hyalomma), although other routes of transmission also exist, such as exposure to blood of infected animals. most cchfv-infected individuals have no symptoms or have mild nonspecific febrile syndrome. however, in some individuals, the infection can cause the crimean-congo hemorrhagic fever, a severe disease characterized by fever, myalgia, hemorrhage, among other manifestations. neurological complications can also occur, being the spectrum and intensity of the disease quite variable (ergönül, 2006; bente et al., 2013; garrison et al., 2019) . hemorrhagic fever (ergönül, 2006; saksida et al., 2010; bente et al., 2013; garrison et al., 2019) . in a study performed by arasli et al. (2015) , expression of the ccr5 ligands ccl2, ccl3, and ccl4 was increased in cchfv-infected adults compared to controls. considering these same chemokines in cchfv-infected children, only ccl4 was significantly increased compared to pediatric controls (arasli et al., 2015) . engin et al. (2009) evaluated the ccr5δ32 in 15 turkish cchfv-infected individuals and observed the wild-type homozygous genotype in all cases. in a subsequent study evaluating the turkish population, rustemoglu et al. (2017) found a protective effect of ccr5δ32 heterozygous genotype and δ32 allele on cchfv infection, since the genotype and allele frequencies were higher in controls than in cchfv-infected individuals. conversely, the wild-type genotype (normal ccr5 expression) was prevalent among infected j o u r n a l p r e -p r o o f individuals. these findings suggest that ccr5 contributes to susceptibility to cchfv infection and that ccr5 down-regulation due to ccr5δ32 results in some protection against the infection. however, further studies are needed to explain the mechanisms by which ccr5 participates in cchfv infection. in the same study, the ccr5δ32 was not significantly associated with disease severity, clinical parameters, or mortality rate (rustemoglu et al., 2017) . together, these findings indicate that the effect of ccr5δ32 is given specifically on resistance against cchfv infection, without affecting the pathogenesis/outcome of crimean-congo hemorrhagic fever. the genus enterovirus is composed of non-enveloped, positive-stranded rna viruses, belonging to the picornaviridae family. enteroviruses (ev) can infect the gastrointestinal tract, cns, and other organs, including heart (tapparel et al., 2013) . cardiomyopathy is a common consequence of ev infection in the heart. ev infection is associated with myocardial inflammation (myocarditis) and other damages to heart tissues (badorff et al., 2000; cooper, 2009; sagar et al., 2012; tapparel et al., 2013; weintraub et al., 2017) . damage heart tissues. viral persistence in individuals with enteroviral cardiomyopathy is associated with an increased mortality rates (kühl et al., 2005; lassner et al., 2018) . some studies suggest that ccr5 influences different aspects of the pathogenesis of viruses belonging to the picornaviridae family, including encephalomyocarditis virus (christmann et al., 2011; shaheen et al., 2015) , coxsackievirus b3 (valaperti et al., 2013) , and rhinovirus (muehling et al., 2017) , once ccr5 participates in the regulation of the host immune response during infection by these viruses. considering the effects of the genetic variant ccr5δ32 on ccr5 expression and ccr5-related immune responses, it is possible that ccr5δ32 also shows some impact on ev-related diseases. in a german study that evaluated patients with enteroviral (chronic/inflammatory) cardiomyopathy (lassner et al., 2018) , the ccr5δ32 was strongly associated with spontaneous viral clearance and better clinical outcome (reduced mortality rate) ( table 6 ). these findings indicate a critical involvement of the ccr5 molecule in the pathogenesis of ev cardiomyopathy. it was suggested that the ccr5δ32 genotyping could be used to assist in the prediction of the clinical progression of enteroviral cardiomyopathy: the δ32 allele as a predictor of a better prognosis, without the need of antiviral interferon-β (ifn-β) therapy; and the wild-type genotype as a predictor of a worse prognosis and immediate need of antiviral ifn-β therapy (lassner et al., 2018) . the clinical use of ifn-β is effective to eliminate the virus, avoid irreversible cardiac injury, and reduce mortality rates, but it is also associated to numerous adverse effects (kühl et al., 2012; lassner et al., 2018) . considering the prognostic value of the ccr5δ32 on the clinical course of enteroviral cardiomyopathy, it is necessary to evaluate the relationship between the ccr5δ32 and the disease in different human populations, mainly through genetic association studies. if this association is confirmed in other populations, the ccr5δ32 genotyping will be a broad-spectrum clinical tool, enhancing and driving the treatment of enteroviral cardiomyopathy. jev is the etiological agent of most cases of viral encephalitis in many countries, reaching ~30% mortality rate (van den hurk et al., 2009; tiwari et al., 2012; le flohic et al., 2013) . some evidence obtained in laboratory conditions (in vitro analysis and murine models) showed that jev infection induces the up-regulation of ccr5 gene (gupta and roa, 2011; pereek et al., 2014; zhang et al., 2019) . also, increased infiltration of ccr5 + cd8 + t cells was observed in the brains of jev-infected mice (zhang et al., 2019) . in this context, using a mouse model of japanese encephalitis, larena et al. (2012) showed that ccr5 protects the host against jev infection in the cns, being essential for disease recovery. ccr5-deficient mice showed higher viral loads in the brain and spinal cord as well as increased mortality rate, as compared to control mice (larena et al., 2012) . also using a mouse model of japanese encephalitis, kim et al. (2016) demonstrated that ccr5 controls the infiltration of cd4 + foxp3 + t regulatory cells (treg) in the cns, contributing to protection against japanese encephalitis. the infiltration and action of inflammatory cells in the brain are important to limit neuroinvasive infections, processes that are regulated by multiple factors, especially cytokines, chemokines and their receptors, including ccr5. however, the uncontrolled action of inflammatory cells can cause damage to the cns. of note, cd4 + foxp3 + treg cells regulate the immune responses, avoiding undesirable or excessive action of inflammatory cells (bardina and lim, 2012; veiga-parga et al., 2013; simonetta and bourgeois, 2013; campbell, 2015; kim et al., 2016) . according to these pieces of evidence, ccr5-mediated action of treg cells is critical for the control of japanese encephalitis. however, the role of ccr5 in jev infection may be more complex than it appears at first. liu et al. (2018) demonstrated that the use of the ccr5 antagonist maraviroc reduces the jev-induced inflammation in mice brain, increasing survival rate. this result suggests that potential deleterious effects of ccr5-mediated inflammation in the brain may override the effects of ccr5-mediated action of treg cells and, therefore, the use of ccr5 antagonists to treat japanese encephalitis may be beneficial. based on these complex and contradictory results, it can be concluded that ccr5 indeed has an important influence on japanese encephalitis, but it is not yet possible to state its specific roles, once they are varied and appear to be context-dependent. also, the results obtained in animal models may not fully represent the disease course in humans. indeed, some evidences suggest the involvement of ccr5 in the pathogenesis of japanese encephalitis in humans. in indian individuals with mild cases of japanese encephalitis, a significant dowregulation of the ccr5 gene was observed as compared to healthy controls (chowdhury and khan, 2019) . however, the ccr5δ32 does not have a relevant influence on the disease. deval et al. (2019) investigated the effect of various genetic variants, including ccr5δ32, on the development of japanese encephalitis in individuals from north india (table 6 ). no statistically significant difference was found when the ccr5δ32 (as an individual factor) was compared between cases and controls (deval et al., 2019) . considering that both studies mentioned above were performed in the indian population, studies evaluating the potential effects of ccr5 and ccr5δ32 on japanese encephalitis in other populations are necessary. puumala virus (puv) infection is a zoonosis endemic in europe. puv is an enveloped singlestranded rna virus, belonging to the bunyaviridae family, genus hantaviridae. most puv infections are mild or subclinical. in some infected individuals, puv is responsible for the development of nephropathia epidemica, a milder form of hemorrhagic fever with renal syndrome, a condition typically caused by other hantaviruses (settergren, 2000; mustonen et al., 2013; lebecque and dupont, 2019 ). an in vitro study found increased ccr5 gene expression in primary monocytes infected by hantaviruses (hantaan virus, sin nombre virus and andes virus) (markotić et al., 2007) . in this sense, host genetic factors and the immune responses affect different aspects of puv infection and progression of nephropathia epidemica (mustonen et al., 2013) , although the role of ccr5 in this disease is little known. (table 6 ). of note, in their study, the authors stated hantavirus infection as the cause of nephropathia epidemica, not specifying the puv detection. no statistical difference was observed in ccr5δ32 genotype frequencies between cases and controls, indicating that ccr5δ32 does not influenced the susceptibility to hantavirus infection in the studied population. considering only nephropathia epidemica cases, the study revealed an association between the wild-type homozygous genotype (functional ccr5) and increased severity of the disease. conversely, the heterozygous genotype was considered a protective factor against increased disease severity ( in 2019, the multiple roles of ccr5 in physiological and pathological conditions gained the attention of the scientific community and lay public due to ccr5 gene editing in human embryos, the "crispr babies", performed by a chinese biophysicist. the researcher claimed to have used crispr gene editing technology to edit the ccr5 of germline cells to mimic the effects of ccr5δ32, aiming to generate babies resistant to hiv infection. the ethical, safety, and legal aspects related to this procedure have caused an intense and broad discussion in the media and scientific literature (cohen, 2019; daley et al., 2019; greely, 2019; rosenbaum, 2019) , and this case culminated in a three years prison sentence for the biophysicist responsible for the procedure (cyranoski, 2020). also, the consequences of the ccr5 absence have brought many concerns to light, once the ccr5 protein participates in tissue development processes, control of immune responses, and several other physiological processes. considering these concerns, our group and others described some undesirable effects associated to natural ccr5 absence (due to the ccr5δ32 homozygous genotype) and ccr5 editing (ellwanger et al., 2019; wang and yang, 2019; xie et al., 2019) . besides gene editing techniques, the expression of ccr5 can be artificially modulated through the use of pharmacological antagonists (e.g. maraviroc), chemokine ligands, and monoclonal antibodies (fantuzzi et al., 2019) . the use of ccr5 antagonists is well established for the treatment of hiv infection and is currently being tested for the treatment of many other diseases, such as cancer (pervaiz et al., 2019; huang et al., 2020a) , stroke (joy et al., 2019), and cocaine addiction-related disorders (hall et al., 2020; nayak et al., 2020) . taking together, it is increasingly clear that the specific inhibition of ccr5 expression through different techniques is gaining pace in different clinical contexts. the pharmacological ccr5 blockade has many benefits in different clinical situations, particularly in the treatment of hiv infection. also, the potential of gene editing (especially in somatic cells) for the treatment of genetic diseases is very promising. however, considering viral infections, two aspects must be considered, as follows: i) the non-redundant characteristics of the ccr5 should be taken into consideration when studying the ccr5 protein and the effects of ccr5δ32 on viral infections: the traditional concepts of redundancy and robustness of the chemokine system consider that the absence of a specific chemokine or chemokine receptor is adequately compensated by other molecules. although these concepts are generally correct for some chemokines/receptors and for some physiological conditions, the lack of ccr5 expression may affect the protection against some specific infectious diseases once the ccr5 absence is not perfectly compensated for by other receptors (ellwanger et al., 2020a) . the nonredundancy of ccr5 is relevant especially for infections by neuroinvasive flaviviruses (bradina and lim, 2012) . for example, the absence of ccr5 due to ccr5δ32 is considered deleterious in wnv infection lim et al., 2008) and in some aspects of tbev infection (ellwanger and chies, 2019a) . the non-redundant role of ccr5 is also likely in jev infection (larena et al., 2012) . it is possible that those individuals using ccr5 antagonists (e.g., for the treatment of hiv infection) and living in areas endemic for neuroinvasive viruses, especially wnv and tbev, may be at increased risk of j o u r n a l p r e -p r o o f developing viral encephalitis-related problems. although, it is still necessary that studies consistently evaluate this assumption, since the available evidence does not support risks for the use of ccr5 antagonists in endemic areas of flaviviruses (martin-blondel et al., 2016) . as mentioned in the topic addressing wnv in this review, it is prudent to recommend to individuals using ccr5 antagonists to adopt measures to minimize the risk of neuroinvasive infections, such as the use of mosquito repellents and mosquito nets (considering the risk of wnv infection), and to limit their exposure to tick-infested areas (considering the risk of tbev infection). concerns regarding the use of ccr5 antagonists in areas of jev circulation have also been raised by other authors (kim et al., 2016; larena et al., 2012) . if the connection between the use of ccr5 antagonists and increased risk of neuroinvasive infections is confirmed in methodologically wellcontrolled studies, these recommendations should be considered of essential importance for users of ccr5 antagonists. "extracellular vesicles" (evs) is a general term used to designate different membranous vesicles released by various cell types. evs include microparticles, microvesicles, nanovesicles, nanoparticles, ectosomes, exosomes, exovesicles, exosome-like vesicles, oncosomes, among other vesicle types. evs act in the transport of different molecules (e.g. micrornas, mrnas, proteins) between cells and tissues in a regulated and precise manner. evs promote the maintenance of physiological processes, also participating in the pathogenesis of various diseases, such as cancer and inflammatory diseases (colombo et al., 2014; théry et al., 2018) . besides, the regulation of multiple aspects of the immune system is strongly influenced by evs (o'neill and quah, 2008; colombo et al., 2014; ellwanger et al., 2016) . the multiple roles of evs in viral infections began to be studied more intensively in recent years, representing an emerging topic in the field of infectious diseases. currently, the relationship between evs and viral infections has already been explored (to a greater or lesser extent) in the context of the following . evs participate in immune evasion processes, ultimately allowing viruses to bypass host defenses (kaminski et al., 2019b) . for example, hiv is likely to usurp the exosome biogenesis pathway in such a way that enhances its infectivity, while increasing its evading strategies from the host immune defenses (ellwanger et al., 2017a) . regarding tbev, exosomes were indicated as important participants in both viral infection and pathogenesis; in this case, tick-derived exosomes facilitate tbev transmission to the host. also, exosomes derived from tbev-infected host neurons can facilitate the spread of the virus in the cns . exosomes have shown multi-dimensional roles in denv life cycle. they can modulate negatively or positively denv pathogenesis, where they are suggested as instrumental for dengue j o u r n a l p r e -p r o o f hemorrhagic fever development in reason of the transportation of specific micrornas (mishra et al., 2019) . since hcv can be found inside exosomes (liu et al., 2014) , it is not suprising that blood-derived exosomes from hcv-infected patients can transmit the virus to other cells (bukong et al., 2014) . these findings suggest that exosomes and other evs assist in the transport of hcv particles/components between cells, ultimately facilitating viral infection. of note, evs can transport multiple viral components or molecules from virus-infected cells that end up facilitating the spread of the virus in the host. on the other hand, evs can act in favor of the host, transporting molecules that assist host defenses in the elimination or control of infections (kaminski et al., 2019b) . evs can transport a range of cytokines and chemokines, such as il-1, il-2, ifn-α, ifn-β, ccl2, ccl3, ccl4, and cxcl10 (chen et al., 2011; konadu et al., 2015; hosseinkhani et al., 2018; kodidela et al., 2018; gao et al., 2019a; gao et al., 2019b; aiello et al., 2020; chiantore et al., 2020) . besides, evs and microparticles also transport chemokine receptors (shen et al., 2016; liang et al., 2018) , including ccr5 tokarz et al., 2019) . interestingly, evs from ccr5 wild type cells can deliver ccr5 molecules to ccr5 δ32/δ32 cells, making them susceptible to hiv infection . a similar phenomenon has been reported involving microparticles, hiv, and cxcr4 (rozmyslowicz et al., 2003) . ccr5 expression is also influenced by the release of evs, specifically microparticles (renovato-martins et al., 2017) . therefore, evs have the potential to attribute greater complexity to ccr5 roles in viral infections. it is possible that the presence of ccr5 in cells is not only dependent on mechanisms of membrane expression and internalization of the receptor. it can also be dependent on ccr5 delivery mediated by evs. however, the actual impact of evs on ccr5-mediated immune response in infections remains to be determined and deserves further investigation. finally, a truncated ccr5 soluble form (tsimanis et al., 2005) , as well as soluble cxcr4, have also been reported in the plasma of humans (malvoisin et al., 2011) . of note, the truncated ccr5 soluble form has ~22 kda (half the size of the ccr5 found on cell membranes) and is truncated at the end of the second extracellular loop (the third extracellular loop and the subsequent three transmembrane regions are absent) (tsimanis et al., 2005) . however, the existence of cell-free soluble ccr5 is still controversial and does not represent a consensus in the scientific community. such doubts occur mainly in consideration of the structure of the receptor, which is highly characteristic of a cell membrane-associated molecule. we believe that evscontaining ccr5 can explain potential (misleading) detections of soluble ccr5. the importance of circulating evs-containing ccr5 on viral infections represents an additional and interesting open question to be investigated. chávez et al. (2013) (ellwanger et al., 2017b) . specifically, using a mouse model of rocv-associated encephalitis, ccr5-deficient mice showed increased survival rate and reduced levels of brain inflammation compared to mice expressing ccr5, indicating the participation of ccr5 in rocv-associated encephalitis (chávez et al., 2013) . although other studies discussed in this review have shown an important involvement of ccr5 in infection by flaviviruses [especially as an important molecule for the resolution of neuroinfection, in opposition to results of chávez et al., (2013) ], the role of the ccr5 in rocv infection represents a neglected topic. this knowledge gap should be addressed in further studies since the reemergence of rocv in brazil is a concern in terms of public health (figueiredo, 2007; ellwanger et al., 2017b) . although no other rocv outbreaks have been reported in brazil after the 1980s, there is evidence of rocv circulation in animals (casseb et al., 2014; pauvolid-correa et al., 2014; silva et al., 2014) . ccr5 expression on t cells. of note, zikv is another mosquito-borne flavivirus that recently reemerged in many countries, affecting brazil in particular. zikv infection is associated with microcephaly and other human development problems (baud et al., 2017) . zachova et al. (2019) showed that epstein-barr virus (ebv)-infected b cells have increased ccr5 expression compared to ebv-non-infected cells. also, recent evidence points to a pivotal role of the ccr5 in the attenuation of rhinovirus-associated inflammation, by controlling the activity of cd4 + foxp3 + treg cells (hossain et al., 2020) . altered levels of cytokines and chemokines are associated with several aspects of zikv (barros et al., 2018; naveca et al., 2018; khaiboullina et al., 2019; lima et al., 2019) , ebv (piovan et al., 2005; ehlin-henriksson et al., 2009; cohen et al., 2017) , and rhinovirus infections (rajan et al., 2014; shelfoon et al., 2016; hansel et al., 2017) . however, the potential role of the chemokine receptor ccr5 in the pathogenesis of zikv, ebv, and rhinovirus represents open questions in the field of ccr5 research. coronaviruses are positive-sense rna viruses that belong to the coronaviridae family (li et al., 2020) . coronaviruses infect a wide range of species, including humans. until 2019, humans have faced two major outbreaks of high pathogenic coronaviruses, the severe acute respiratory syndrome coronavirus (sars-cov) outbreak and the middle east respiratory syndrome coronavirus (mers-cov) outbreak (fung and liu, 2019) . in 2019, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan (hubei province, china) and rapidly spread to more than 180 countries/regions (interactive dashboard of global covid-19 cases -johns hopkins university: https://arcg.is/0fhmtx ; dong et al., 2020) . of note, sars-cov-2 infection causes the "coronavirus disease 2019" and therefore is also called "covid-19". considering the ongoing sars-cov-2 pandemic and the clinical variability observed in the affected individuals, ranging from mild to severe respiratory disease j o u r n a l p r e -p r o o f 2020b), the following question arose: does ccr5 have any clinically relevant influence on sars-cov-2 or other coronaviruses infections? recent data indicated that sars-cov-2 binds to the ace2 receptor (andersen et al., 2020) , using this receptor for entry into human cells. therefore, it is unlikely that ccr5 or ccr5δ32 have some significant effect on the susceptibility or resistance to sars-cov-2 infection. however, ccr5 may have some impact on the clinical course of sars-cov-2 infection. the pattern and intensity of the human immune responses regulate the clinical progression and even the outcome of infections by coronaviruses (li et al., 2020) , including sars-cov-2 (qin et al., 2020; shi et al., 2020) . sars-cov-infected mice showed increased expression of ccr5 mrna in lungs along with the production of ccl2, ccl3 and ccl5 chemokines, the major ccr5 natural agonists (chen et al., 2010) . intracranial infection of mice with mouse hepatitis virus (mhv, a murine coronavirus) induces an increased ccr5 expression, which contributed to mhv-induced demyelination through ccr5-mediated macrophage recruitment to the cns (glass et al., 2001) . subsequent studies using mhv-infected mice showed that ccr5 participates in the regulation of cd4 + and cd8 + t cell activities against the virus in the cns (glass and lane, 2003a; glass and lane, 2003b) . also, sars-cov-infected human monocyte-derived dendritic cells showed increased ccr5 expression in vitro (law et al., 2009) . taking together, the findings mentioned above suggest that ccr5 could have some influence on the clinical course of sars-cov-2 infection. however, future studies addressing humans are needed to clarify this suggestion. in this sense, we emphasize that so far, there is no evidence showing a clear involvement of ccr5 in sars-cov-2 human infection. the ccr5δ32 is a highly penetrating polymorphism, and exerts a robust phenotypic effect on ccr5. however, the expression of ccr5 is regulated by other polymorphisms in addition to ccr5δ32 (mehlotra, 2019) . also, the ccr5 expression is influenced by non-coding genetic elements. the effect of the genetic backround of the host can also be exacerbated or diminished depending on the environmental conditions to which the individual is exposed (ellwanger et al., 2018c; kulkarni et al., 2019) . taking together, these factors help to explain why many of the effects exerted by ccr5 on a given disease are specific to a certain population, ethnic group, or individual. research involving ccr5 and hiv began in the mid-1990s. since then, many achievements in the field of ccr5/hiv research have been made, such as the identification of ccr5δ32 as a strong protective factor against hiv infection and the development of ccr5 antagonists for the treatment of hiv infection. currently, these drugs are being tested for the treatment of other inflammatory and infectious diseases. in this context, the use of ccr5 antagonists has raised some concerns, since the blockade of ccr5 can affect j o u r n a l p r e -p r o o f or even weaken the host defenses against certain infections, especially neuroinvasive infections by flaviviruses. however, to date, there is no strong evidence indicating that the use of ccr5 antagonists has increased the susceptibility of individuals to problematic flaviviruses infections. the frequency of ccr5δ32 is quite varied among human populations, and therefore the effects of the allele in a particular population may not apply to another population. moreover, the effects of ccr5 and ccr5δ32 are disease-specific. for instance, the ccr5δ32 does not significantly affect susceptibility to wnv infection. however, the effect of the ccr5 and ccr5δ32 on wnv disease progression is very robust. some animal-derived findings suggest the involvement of the ccr5 in the pathogenesis of denv infection. however, the effects of ccr5 on denv infection are very different between humans and rodents. of note, ccr5δ32 does not significantly affect susceptibility to denv infection or disease progression. moreover, the effect of ccr5δ32 on hbv-related disease is population-specific. interestingly, cmv release virokines, which are molecules that can manipulate the host immune response and affect the ccr5 function. the examples cited above highlight the varied impacts of ccr5 and ccr5δ32 on viral infections. the few studies involving cchfv, ev, and hantavirus infection have indicated important effects of ccr5δ32 on these conditions. based on these findings, further studies should investigate the role of ccr5δ32 and ccr5 protein in such infections, considering populations with distinct genetic backgrounds. some evidence suggested the participation of ccr5 in infections by zikv, ebv, and rhinovirus. also, a mouse model of rocv-associated encephalitis suggested an important role for ccr5 in host immune responses against the virus. however, the roles of ccr5 and ccr5δ32 in infections by zikv, ebv, rhinovirus, and rocv are still poorly understood and need to be investigated in future studies. the role of evs in the transport of ccr5 between cells indicates that the expression of ccr5 on the cell surface may also depend on the release of evs containing ccr5. also, the transport of host molecules and viruses through evs adds complexity to the topics covered in this review and should be taken into account in future studies that investigate the role of ccr5 in viral infections. gene editing technologies have the potential to be used to treat different diseases, mainly when applied to somatic cells. however, ccr5 gene editing in human embryos presents a number of ethical problems. besides, the absence of ccr5 can have deleterious effects in certain conditions, such as increased susceptibility to symptomatic wnv infection. finally, this article showed that the participation of ccr5 in different viral infections is complex and varied and, therefore, cannot be generalized. this article also pointed out neglected gaps in knowledge involving ccr5 that should be addressed in future studies. the authors declare no conflicts of interest. ccr5 polymorphism as a protective factor for hepatocellular carcinoma in hepatitis b virus-infected iranian patients cd4-ccr5 interaction in intracellular compartments contributes to receptor expression at the cell surface effects of the ccr5-δ32 mutation on antiviral treatment in chronic hepatitis c effects of the ccr5-δ32 mutation on hepatitis c virus-specific immune responses in patients with haemophilia cc-chemokine receptor 5 (ccr5) in hepatitis c--at the crossroads of the antiviral immune response? downregulation of ccr5 expression on the peripheral blood + t cells of southeastern iranian patients with chronic hepatitis b infection association of genetic variations in ccr5 and its ligand, rantes with clearance of hepatitis b virus in korea an emerging interplay between extracellular vesicles and cytokines lack of chemokine receptor ccr5 promotes murine fulminant liver failure by preventing the apoptosis of activated cd1d-restricted nkt cells ccr5 in t cell-mediated liver diseases: what's going on? the biology of ccr5 and cxcr4 gene editing of hiv-1 co-receptors to prevent and/or cure virus infection the proximal origin of sars-cov-2 host genetics, innate immune responses, and cellular death pathways in poliomyelitis patients peripheral blood cd8 + t cells ccr5 expression and its δ32 mutation in iranian patients with occult hepatitis b infections elevated chemokine levels during adult but not pediatric crimean-congo hemorrhagic fever human t cell responses to dengue and zika virus infection compared to dengue/zika coinfection enteroviral cardiomyopathy: bad news for the dystrophinglycoprotein complex ccr5 proinflammatory allele in prostate cancer risk: a pilot study in patients and centenarians from sicily the role of chemokines in the pathogenesis of neurotropic flaviviruses acute zika virus infection in an endemic area shows modest proinflammatory systemic immunoactivation and cytokine-symptom associations an update on zika virus infection crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity the global distribution and burden of dengue host genetic risk factors for west nile virus infection and disease progression a study on the association between ccrδ32 mutation and hcv infection in iranian patients ccr5 receptor antagonism inhibits hepatitis c virus (hcv) replication in vitro multiple nonfunctional alleles of ccr5 are frequent in various human populations ccr5 binds multiple cc-chemokines: mcp-3 acts as a natural antagonist analysis of the ccr5 gene coding region diversity in five south american populations reveals two new non-synonymous alleles in amerindians and high ccr5*d32 frequency in euro-brazilians targeting ccr5 trafficking to inhibit hiv-1 infection ccr5 but not ccr1 deficiency reduces development of dietinduced atherosclerosis in mice looking for biological factors to predict the risk of active cytomegalovirus infection in non-immunosuppressed critically ill patients ccr5 revisited: how mechanisms of hiv entry govern aids pathogenesis primary acute dengue and the deletion in chemokine receptor 5 (ccr5δ32) exosomes from hepatitis c infected patients transmit hcv infection and contain replication competent viral rna in complex with ago2-mir122-hsp90 identification of genetic variants associated with dengue or west nile virus disease: a systematic review and meta-analysis control of regulatory t cell migration, function, and homeostasis natural killer cell recruitment to the lung during influenza a virus infection is dependent on cxcr3, ccr5, and virus exposure dose seroprevalence of flaviviruses antibodies in water buffaloes (bubalus bubalis) in brazilian amazon division of vector-borne diseases (dvbd) mip-1 α axis in the pathogenesis of rocio virus encephalitis in a mouse model downregulation of ccr5 inhibits the proliferation and invasion of cervical cancer cells and is regulated by microrna-107 cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4 + t cells are important in control of sars-cov infection epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type i t cells infiltration into central nervous system chemokine-containing exosomes are released from heatstressed tumor cells via lipid raft-dependent pathway and act as efficient tumor vaccine human papillomavirus and carcinogenesis: novel mechanisms of cell communication involving extracellular vesicles sickle cell disease: a chronic inflammatory condition isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome differential expression levels of inflammatory chemokines and tlrs in patients suffering from mild and severe japanese encephalitis ccr5 regulates inflammatory gene expression in response to encephalomyocarditis virus infection west nile virus and its vectors regulation of inflammatory chemokine receptors on blood t cells associated to the circulating versus liver chemokines in dengue fever identification of a major co-receptor for primary isolates of hiv-1 association of single nucleotide polymorphisms in tnfa and ccr5 genes with japanese encephalitis: a study from an endemic region of north india virus and host determinants of west nile virus pathogenesis ccr5 signaling suppresses inflammation and reduces adverse remodeling of the infarcted heart, mediating recruitment of regulatory t cells an interactive web-based dashboard to track covid-19 in real time ccr5 is involved in resolution of inflammation in proteoglycan-induced arthritis hiv-1 entry into cd4 + cells is mediated by the chemokine receptor cc-ckr-5 ccr5 limits cortical viral loads during west nile virus infection of the central nervous system changes in chemokines and chemokine receptor expression on tonsillar b cells upon epstein-barr virus infection host immunogenetics in tick-borne encephalitis virus infection-the ccr5 crossroad host genetic factors can impact vaccine immunogenicity and effectiveness exosomes are possibly used as a tool of immune regulation during the dendritic cell-based immune therapy against hiv-i rocio virus: an overview ccr5 gene editing -revisiting pros and cons of ccr5 absence what we say and what we mean when we say redundancy and robustness of the chemokine system -how ccr5 challenges these concepts immunogenetic studies of the hepatitis c virus infection in an era of pan-genotype antiviral therapies -effective treatment is coming role of the genetic variant ccr5δ32 in hbv infection and hbv/hiv co-infection ccr5δ32 in hcv infection, hcv/hiv co-infection, and hcv-related diseases exosomes in hiv infection: a review and critical look microrna-related polymorphisms in infectious diseases-tiny changes with a huge impact on viral infections and potential clinical applications role of ccr5δ32 mutation in protecting patients with schistosoma mansoni infection against hepatitis c viral infection or progression cytochrome p450 2d6 and mdr1 gene mutation in relation to mortality in patients with crimean-congo hemorrhagic fever: a preliminary study crimean-congo haemorrhagic fever cxcr3-deficiency protects influenza-infected ccr5-deficient mice from mortality ccr5 deficiency predisposes to fatal outcome in influenza virus infection dual ccr5/ccr2 targeting: opportunities for the cure of complex disorders short-term administration of the ccr5 antagonist vicriviroc to patients with hiv and hcv coinfection is safe and tolerable emergent arboviruses in brazil human coronavirus: host-pathogen interaction possible impact of 190g > a ccr2 and δ32 ccr5 mutations on decrease of the hbv vaccine immunogenicity-a preliminary report exosomes derived from septic mouse serum modulate immune responses via exosome-associated cytokines association between cytokines and exosomes in synovial fluid of individuals with knee osteoarthritis animal models for crimean-congo hemorrhagic fever human disease pharmacokinetics, safety and efficacy of maraviroc in treatment-experienced pediatric patients infected with ccr5-tropic hiv-1 the δ32 mutation of the chemokine-receptor 5 gene neither is correlated with chronic hepatitis c nor does it predict response to therapy with interferon-α and ribavirin functional expression of chemokine receptor ccr5 on cd4 + t cells during virus-induced central nervous system disease functional analysis of the cc chemokine receptor 5 (ccr5) on virus-specific + t cells following coronavirus infection of the central nervous system chemokine receptor ccr5 promotes leukocyte trafficking to the brain and survival in west nile virus infection reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor ccr5 following infection with a neurotropic coronavirus ccr5 deficiency increases risk of symptomatic west nile virus infection chemokine regulation of inflammation during acute viral infection the ccr5-δ32 mutation: impact on disease outcome in individuals with hepatitis c infection from a single source activation of ccr5 by chemokines involves an aromatic cluster between transmembrane helices 2 and 3 ccr5δ32 mutation does not influence the susceptibility to hcv infection, severity of liver disease and response to therapy in patients with chronic hepatitis c controls for lung dendritic cell maturation and migration during respiratory viral infection crispr'd babies: human germline genome editing in the 'he jiankui affair dual and triple infections with influenza a and b viruses: a case-control study in southern brazil the pathogenesis of human cytomegalovirus circulating human cd4 + t cells have intracellular pools of ccr5 molecules five-year safety evaluation of maraviroc in hiv-1-infected treatment-experienced patients transcriptomic profile of host response in japanese encephalitis virus infection hiv-1 remission following ccr5δ32/δ32 haematopoietic stem-cell transplantation evidence for hiv-1 cure after ccr5δ32/δ32 allogeneic haemopoietic stem-cell transplantation 30 months post analytical treatment interruption: a case report dengue infection blockade of ccr5 to protect the liver graft in hiv/hcv co-infected patients ccr5 and responses to cocaine: addiction is not just about the brain a comprehensive evaluation of nasal and bronchial cytokines and chemokines following experimental rhinovirus infection in allergic asthma: increased interferons (ifn-γ and ifn-λ) and type 2 inflammation (il-5 and il-13) association of genetic variants of the chemokine receptor ccr5 and its ligands, rantes and mcp-2, with outcome of hcv infection the human cytomegalovirus, from oncomodulation to oncogenesis intragenus (homo) variation in a chemokine receptor gene (ccr5) ccr5 attenuates neutrophilic airway inflammation exacerbated by infection with rhinovirus extracellular vesicles work as a functional inflammatory mediator between vascular endothelial cells and immune cells clinical features of patients infected with 2019 novel coronavirus in wuhan the ccr5 antagonist maraviroc causes remission of pancreatic cancer liver metastasis in nude rats based on cell cycle inhibition and apoptosis induction the role of a mutant ccr5 allele in hiv-1 transmission and disease progression ccr5 haplotypes influence hcv serostatus in caucasian intravenous drug users the effect of the ccr5-delta32 deletion on global gene expression considering immune response and inflammation long-term control of hiv by ccr5 delta32/delta32 stemcell transplantation significance of rantes-ccr5 axis and linked downstream immunomodulation in dengue pathogenesis: a study from guwahati, india human cytomegalovirus enhances placental susceptibility and replication of human immunodeficiency virus type 1 (hiv-1), which may facilitate in utero hiv-1 transmission cytomegalovirus upregulates expression of ccr5 in central memory cord blood mononuclear cells, which may facilitate in utero hiv type 1 transmission immunological cure of hbv infection the chemokine receptor ccr5, a therapeutic target for hiv/aids antagonists, is critical for recovery in a mouse model of japanese encephalitis the role of ccr5/cxcr3 expressing cd8+ cells in liver damage and viral control during persistent hepatitis c virus infection ccr5del32 genotype in human enteroviral cardiomyopathy leads to spontaneous virus clearance and improved outcome compared to wildtype ccr5 ccr5 inhibitors and hiv-1 infection toll-like receptors, chemokine receptors and death receptor ligands responses in sars coronavirus infected human monocyte derived dendritic cells puumala hantavirus: an imaging review human cytomegalovirus infection reduces surface ccr5 expression in human microglial cells, astrocytes and monocyte-derived macrophages updates on chronic hbv: current challenges and future goals review of climate, landscape, and viral genetics as drivers of the japanese encephalitis virus ecology coronavirus infections and immune responses polymorphisms of ccl3l1/ccr5 genes and recurrence of hepatitis b in liver transplant recipients horizontal transfer of exosomal cxcr4 promotes murine hepatocarcinoma cell migration, invasion and lymphangiogenesis the transcriptional and protein profile from human infected neuroprogenitor cells is strongly correlated to zika virus microcephaly cytokines phenotype evidencing a persistent inflammation in the cns the chemokine receptor ccr1 is identified in mast cell-derived exosomes reduced cc chemokine receptor (ccr) 1 and ccr5 surface expression on peripheral blood t lymphocytes from patients with chronic hepatitis c infection ccr5: no longer a 'good for nothing' gene--chemokine control of west nile virus infection genetic deficiency of chemokine receptor ccr5 is a strong risk factor for symptomatic west nile virus infection: a meta-analysis of 4 cohorts in the us epidemic ccr5 deficiency is a risk factor for early clinical manifestations of west nile virus infection but not for viral transmission chemokine control of west nile virus infection natural history of hepatitis c chemokine receptor antagonist block inflammation and therapy japanese encephalitis virus infection in mouse model the cytokine storm of severe influenza and development of immunomodulatory therapy exosome-associated hepatitis c virus in cell cultures and patient plasma genetic variants and susceptibility to neurological complications following west nile virus infection polymorphisms in the genes encoding chemokine receptor 5, interleukin-10, and monocyte chemoattractant protein 1 contribute to cytomegalovirus reactivation and disease after allogeneic stem cell transplantation viral escape mechanisms -escapology taught by viruses transfer of the chemokine receptor ccr5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection the ccr5δ32 (rs333) polymorphism is not a predisposing factor for severe pandemic influenza in the brazilian admixed population soluble chemokine receptor cxcr4 is present in human sera hcv chronic infection and ccr5-δ32/δ32 ccr5 genetic variants and epidemiological determinants for hpv infection and cervical premalignant lesions pathogenic hantaviruses elicit different immunoreactions in thp-1 cells and primary monocytes and induce differentiation of human monocytes to dendritic-like cells dengue virus requires the ccchemokine receptor ccr5 for replication and infection development ccr5 blockade for neuroinflammatory diseases-beyond control of hiv genetic variants in the ccr gene cluster and spontaneous viral elimination in hepatitis c-infected patients expression of rantes by normal airway epithelial cells after influenza virus a infection ccr5 promoter polymorphism -2459g > a: forgotten or ignored? cells chemokine receptors as important regulators of pathogenesis during arboviral encephalitis enhanced expression, native purification, and characterization of ccr5, a principal hiv-1 coreceptor dengue haemorrhagic fever: a job done via exosomes? clinical significance of the ccr5delta32 allele in hepatitis c ccr5 deficiency exacerbates t-cell-mediated hepatitis in mice ccr5, mcp-1 and vdr gene polymorphisms are associated with the susceptibility to hbv infection pathways for internalization and recycling of the chemokine receptor ccr5 single-cell tracking reveals a role for pre-existing ccr5 + memory th1 cells in the control of rhinovirus-a39 after experimental challenge in humans ccr5δ32 genotype leads to a th2 type directed immune response in esrd patients ccr5 deletion protects against inflammation-associated mortality in dialysis patients immune response to dengue virus and prospects for a vaccine the pathogenesis of nephropathia epidemica: new knowledge and unanswered questions the predictive value of il28b gene polymorphism for spontaneous clearance in a single source outbreak cohort is limited in patients carrying the ccr5δ32 mutation analysis of the immunological biomarker profile during acute zika virus infection reveals the overexpression of cxcl10, a chemokine linked to neuronal damage chemokine ccr5 and cocaine interactions in the brain: cocaine enhances mesolimbic ccr5 mrna levels and produces place preference and locomotor activation that are reduced by a ccr5 antagonist why and where an hiv cure is needed and how it might be achieved a study of cc-chemokine receptor 5 (ccr5) polymorphism on the outcome of hcv therapy in egyptian patients exosomes secreted by bacterially infected macrophages are proinflammatory association between mbl2 haplotypes and dengue severity in children from rio de janeiro, brazil influenza-induced innate immunity: regulators of viral replication, respiratory tract pathology & adaptive immunity mir-155 induction in microglial cells suppresses japanese encephalitis virus replication and negatively modulates innate immune responses ccr5 and hiv infection serological evidence of widespread circulation of west nile virus and other flaviviruses in equines of the pantanal, brazil the genomic ancestry of individuals from different geographical regions of brazil is more uniform than expected ccr5 blockage by maraviroc: a potential therapeutic option for metastatic breast cancer west nile virus: review of the literature the evolution of seasonal influenza viruses marked differences in ccr5 expression and activation levels in two south african populations hepatitis c virus chemokine-regulated recruitment of antigen-specific t-cell subpopulations to the liver in acute and chronic hepatitis c infection chemokine receptor expression in ebv-associated lymphoproliferation in hu/scid mice: implications for cxcl12/cxcr4 axis in lymphoma generation frequency of the 32-base pair deletion in the chemokine receptor ccr5 gene is not increased in hepatitis c patients associations of chemokine system polymorphisms with clinical outcomes and treatment responses of chronic hepatitis c chemokine receptors: multifaceted therapeutic targets dysregulation of immune response in patients with covid-19 in wuhan, china one hundred years of poliovirus pathogenesis human rhinovirus induced cytokine/chemokine responses in human airway epithelial and immune cells metabolomics as an approach to characterise the contrasting roles of ccr5 in the presence and absence of disease microparticles derived from obese adipose tissue elicit a pro-inflammatory phenotype of cd16 + , ccr5 + and tlr8 + monocytes characterization in vitro and in vivo of a pandemic h1n1 influenza virus from a fatal case uncommon immune-mediated extrahepatic manifestations of hcv infection the future of gene editing -toward scientific and social consensus ccr5 deficiency and severe polio infection in the 1984 outbreak in finland platelet-and megakaryocyte-derived microparticles transfer cxcr4 receptor to cxcr4-null cells and make them susceptible to infection by x4-hiv neutrophils induce a novel chemokine receptors repertoire during influenza pneumonia analysis of ccr5-δ32 and ccr2-v64i polymorphisms in a cohort of spanish hcv patients using real-time polymerase chain reaction and fluorescence resonance energy transfer technologies the possible role of ccr5δ32 mutation in crimean-congo hemorrhagic fever infection the ccr5-δ32 mutation: impact on disease outcome in individuals with hepatitis b infection in the southern khorasan population (east of iran) interacting roles of immune mechanisms and viral load in the pathogenesis of crimean-congo hemorrhagic fever chemokine receptor expression and chemotactic responsiveness of human monocytes after influenza a virus infection ccr5 expression is elevated in cervical cancer cells and is up-regulated by seminal plasma host genetic risk factors for community-acquired pneumonia the second extracellular loop of ccr5 is the major determinant of ligand specificity resistance to hiv-1 infection in caucasian individuals bearing mutant alleles of the ccr-5 chemokine receptor gene ccr5 plays important roles in hepatitis b infection the natural history of human papillomavirus infection ccr2 and ccr5 genes polymorphisms in women with cervical lesions from pernambuco, northeast region of brazil: a case-control study ccr5delta32 in systemic lupus erythematosus: implications for disease susceptibility and outcome in a brazilian population carcinogenic human papillomavirus infection west nile virus infection structural basis of coreceptor recognition by hiv-1 envelope spike clinical aspects of nephropathia epidemica (puumala virus infection) in europe: a review association of host genetic risk factors with the course of cytomegalovirus retinitis in patients infected with human immunodeficiency virus ccr5-dependent activation of mtorc1 regulates translation of inducible no synthase and cox-2 during encephalomyocarditis virus infection chemokine release from human rhinovirus-infected airway epithelial cells promotes fibroblast migration ccr2 positive exosome released by mesenchymal stem cells suppresses macrophage functions and alleviates ischemia/reperfusion-induced renal injury covid-19 infection: the perspectives on immune responses evidence for internal stores of ccr5 in blood cells role of cc chemokine receptor 1 and two of its ligands in human dengue infection. three approaches under the cuban situation endocytosis and recycling of the hiv coreceptor ccr5 a saint louis encephalitis and rocio virus serosurvey in brazilian horses frequency of the ccr5-delta32 allele in brazilian populations: a systematic literature review and meta-analysis cd4+foxp3+ regulatory t-cell subsets in human immunodeficiency virus infection ccr5-δ32 polymorphism and susceptibility to cervical cancer: association with early stage of cervical cancer the ccr5δ32 allele is not a major predisposing factor for severe h1n1pdm09 infection the role of cytokines in the immune response to influenza a virus infection frequencies of gene variant ccr5-δ32 in 87 countries based on next-generation sequencing of 1.3 million individuals sampled from 3 national dkms donor centers adaptive immune responses to primary and secondary dengue virus infections the global burden of dengue: an analysis from the global burden of disease study advances in the treatment of hiv/hcv coinfection in adults ccr5 deficiency enhances hepatic innate immune cell recruitment and inflammation in a murine model of acute hepatitis b infection combined effect of hpv and several gene snps in laryngeal cancer association between vitamin d receptor, ccr5, tnf-α and tnf-β gene polymorphisms and hbv infection and severity of liver disease interaction between rantes promoter variant and ccr5δ32 favors recovery from hepatitis b frequency of the ccr5-δ32 mutant allele is not increased in belgian hepatitis c virus-infected patients japanese encephalitis: a review of the indian perspective retinopathy severity correlates with rantes concentrations and ccr 5-positive microvesicles in diabetes soluble chemokine ccr5 receptor is present in human plasma ccl5-ccr5 interaction provides antiapoptotic signals for macrophage survival during viral infection ccr5δ32 mutation, mycoplasma pneumoniae infection, and asthma innate immune interleukin-1 receptor-associated kinase 4 exacerbates viral myocarditis by reducing ccr5 + cd11b + monocyte migration and impairing interferon production ecology and geographical expansion of japanese encephalitis virus maraviroc -a ccr5 antagonist for the treatment of hiv-1 infection human cytomegalovirus inhibits the migration of immature dendritic cells by down-regulating cell-surface ccr1 and ccr5 role of regulatory t cells during virus infection reduced cell surface expression of ccr5 in ccr5δ32 heterozygotes is mediated by gene dosage, rather than by receptor sequestration the role of natural antibodies to cc chemokine receptor 5 in hiv infection class b β-arrestin2-dependent ccr5 signalosome retention with natural antibodies to ccr5 the abrogation of phosphorylation plays a relevant role in the ccr5 signalosome formation with natural antibodies to ccr5 erk1-based pathway as a new selective mechanism to modulate ccr5 with natural antibodies real life experiences in hcv management t-cell surface ccr5 density is not correlated with hepatitis severity in hepatitis c virus/hivcoinfected individuals: implications for the therapeutic use of ccr5 antagonists cytomegalovirus cc chemokine promotes immune cell migration the ccr5δ32 allele is associated with reduced liver inflammation in hepatitis c virus infection gene-edited babies: what went wrong and what could go wrong cc chemokine receptor 5 δ32 polymorphism in two independent cohorts of hepatitis c virus infected patients without hemophilia dilated cardiomyopathy ccr5 deficiency regulates the proliferation and trafficking of natural killer cells under physiological conditions frequency of the hiv-protective cc chemokine receptor 5-δ32/δ32 genotype is increased in hepatitis c a new coronavirus associated with human respiratory disease in china ccr5 levels and expression pattern correlate with infectability by macrophagetropic hiv-1, in vitro critical role for ccr5 in the function of donor cd4 + cd25 + regulatory t cells during acute graftversus-host disease single nucleotide polymorphisms in candidate genes and dengue severity in children: a case-control, functional and meta-analysis study the potential risks of c-c chemokine receptor 5-edited babies in bone development bioinformatic identification of key genes and pathways that may be involved in the pathogenesis of hbv-associated acute liver failure effects of the chemokine receptor 5 (ccr5)-delta32 mutation on hepatitis c virus-specific immune responses and liver tissue pathology in hcv infected patients hepatitis b virus infection multiparametric flow cytometry analysis of peripheral blood b cell trafficking differences among epstein-barr virus infected and uninfected subpopulations differential expression of tim-3, pd-1, and ccr5 on peripheral t and b lymphocytes in hepatitis c virus-related hepatocellular carcinoma and their impact on treatment outcomes correlations between polymorphisms in the uridine diphosphate-glucuronosyltransferase 1a and c-c motif chemokine receptor 5 genes and infection with the hepatitis b virus in three ethnic groups in china pd1 + ccr2 + cd8 + t cells infiltrate the central nervous system during acute japanese encephalitis virus infection high frequency of ccr5-δ32 homozygosity in hcv-infected, hiv-1-uninfected hemophiliacs results from resistance to hiv-1 genetic polymorphism of chemokine receptors ccr2 and ccr5 in swedish cervical cancer patients host micrornas and exosomes that modulate influenza virus infection structure of cc chemokine receptor 5 with a potent chemokine antagonist reveals mechanisms of chemokine recognition and molecular mimicry by hiv exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral rna and proteins to the vertebrate neuronal cells chemokines: a new classification system and their role in immunity protective effect of ccr5-δ32 against hiv infection by the heterosexual mode of transmission in a polish population american 395 symptomatic wnv+ individuals; 145 symptomatic but noninfected individuals; 1318 controls (blood donors)the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection glass et al. (2006) american 224 symptomatic wnv+ individuals; 1318 controls (blood donors)corroborating data from glass et al. (2006) , the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection lim et al. (2008) american 634 wnv+ individuals; 422 non-infected individuals ccr5δ32 homozygous genotype was associated with clinical symptoms of wnv infection; no association between ccr5δ32 and susceptibility to wnv infection lim et al. (2010) american, canadian 385 symptomatic wnv+ individuals; 328 asymptomatic wnv+ individuals; 1318 controls [blood donors from glass et al. (2006)] no association of ccr5δ32 and wnv infection considering symptomatic and asymptomatic wnv+ individuals; considering a dominant model of inheritance of ccr5δ32 and using controls from glass et al. (2006) , the ccr5δ32 was associated with symptomatic wnv infection and infection risk bigham et al. (2011) key: cord-306972-alyyju5x authors: james, peter bai; wardle, jonathan; steel, amie; adams, jon title: an assessment of ebola-related stigma and its association with informal healthcare utilisation among ebola survivors in sierra leone: a cross-sectional study date: 2020-02-05 journal: bmc public health doi: 10.1186/s12889-020-8279-7 sha: doc_id: 306972 cord_uid: alyyju5x background: we examined the magnitude and correlates of ebola virus disease (evd)-related stigma among evd survivors in sierra leone since their return to their communities. in addition, we determined whether evd-related stigma is a predictor of informal health care use among evd survivors. methods: we conducted a cross-sectional study among 358 evd survivors in five districts across all four geographic regions (western area, northern province, eastern province and southern province) of sierra leone. ebola-related stigma was measured by adapting the validated hiv related stigma for people living with hiv/aids instrument. we also measured traditional and complementary medicine (t&cm) use (as a measure of informal healthcare use). data were analysed using descriptive statistics and regression analysis. results: evd survivors report higher levels of internalised stigma (0.92 ± 0.77) compared to total enacted stigma (0.71 ± 0.61). social isolation (0.96 ± 0.88) was the highest reported enacted stigma subscale. ebola survivors who identified as christians [aor = 2.51, 95%ci: 1.15–5.49, p = 0.021], who perceived their health to be fair/poor [aor = 2.58, 95%ci: 1.39–4.77. p = 0.003] and who reside in the northern region of sierra leone [aor = 2.80, 95%ci: 1.29–6.07, p = 0.009] were more likely to experience internalised stigma. verbal abuse [aor = 1.95, 95%ci: 1.09–3.49, p = 0.025] and healthcare neglect [aor = 2.35, 95%ci: 1.37–4.02, p = 0.002] were independent predictors of t&cm use among evd survivors. conclusion: our findings suggest evd-related stigma (internalised and enacted) is prevalent among evd survivors since their return to their communities. religiosity, perceived health status and region were identified as independent predictors of internalised stigma. verbal abuse and healthcare neglect predict informal healthcare use. evd survivor-centred and community-driven anti-stigma programs are needed to promote evd survivors’ recovery and community re-integration. the 2014-2016 ebola virus disease (evd) outbreak in west africa is considered the largest and unprecedented public health emergency in the history of the disease [1] . as at the 30th march 2016, the morbidity and mortality figures due to evd were estimated at 28, 646 and 11, 322 respectively [2] . the west african ebola outbreak also recorded the highest number of survivors, and current estimates put the number of evd survivors at more than 10,000 [3] . many evd survivors are known to be suffering from short and long-term physical symptoms and mental complications as a result of surviving evd [4] [5] [6] . psychosocial consequences of evd survivorship can be traumatic, due to the adverse psychological experiences of individuals with evd had to grapple with during infection, treatment and post-discharge. these adverse experiences includes various forms of psychosocial challenges such as depression, anxiety and grief due to loss of loved ones and stigma [4] . stigma constitutes negative attitudes and beliefs that discredit an individual or group of individuals leading to prejudice and societal exclusion [7] . stigma can lead to experiences and feelings of blame, shame, worthlessness, loneliness, isolation, social exclusion and discrimination in accessing social amenities and healthcare services [8, 9] . socially undesirable manifestations (prejudice and discrimination) expressed against those with the stigmatizing attributes are known as enacted stigma whereas the feeling of shame, guilt or worthlessness experienced as a result of having the stigmatising attribute is referred to as internalised stigma [10] . evd-related stigma is largely based on community fear that evd survivors are still contagious [11] . such fear is profound if evd survivors experience post-ebola sequelae [12, 13] or are aware that the ebola virus can be present in certain immune-protective parts of the body after convalescence (for example, the semen, breast milk, ocular (eye) fluid, and spinal column fluid) [14, 15] . evd-related stigma has led to evd survivors being mocked by their communities [16, 17] , being evicted from their homes by their property owners [13, 17] , losing their former jobs [11] and being divorced by their spouses [12, 13] . some evd survivors have been prevented from visiting public places such as public toilets and have experienced difficulty in trading commodities at their local market due to a community reluctance to touch their items or money [12, 13] . evd-related stigma has been reported by evd survivors and their communities in dr congo (35%), guinea (26%) and liberia (3%) [18] [19] [20] , and may be more common among female rather than male evd survivors [12] . other factors, which have been reported as predictors of evd-related stigma, are age, level of education, and having accessed medical care [21] . liberian research also suggests evd survivors are reported to be more likely to experience stigma compared to their close contacts who were not infected with evd virus [22] however the degree of evd-related stigma may decline among survivors over time [21, 23] . in sierra leone, stigmatisation is reported in approximately one third of evd survivors [24, 25] . stigma associated with infectious disease has been linked to poor adherence to conventional treatment and the utilization of informal or non-integrated forms of health care such as traditional and complementary medicine (t&cm) [26, 27] . t&cm refers to a number of health systems, products and practices considered to be predominantly outside conventional medical practice and the medical curriculum [28, 29] . in sub-saharan africa, an average of 58% of the general population is estimated to use t&cm products and 29% consult t&cm practitioners [30] . the key reasons for t&cm use in africa have been attributed to its low cost, easy accessibility, the alignment between t&cm philosophy and local cultural and religious values, perceived safety and efficacy, and dissatisfaction with conventional medicine [30] . in sierra leone, t&cm utilisation is common especially among hypertensive, pregnant women, infertile women, and lactating mothers and in the management of malaria and diarrhoea [31] [32] [33] [34] [35] [36] . studies have reported individuals with hiv/aids or mental health diagnoses that experience stigma are more likely to access t&cm services [37, 38] . this pattern of use is reportedly due to the users' perception of t&cm as less stigmatizing than conventional medicine, partly justified by the view that these t&cm approaches are deeply rooted in the local cultural and traditional practices [37, 38] . among sars survivors, t&cm was reported to be useful in overcoming sars-related stigmas by creating new social support networks and counteracting potential future stigmatization and discrimination [39] . most studies on stigma among evd survivors have focussed on its magnitude and nature both immediately following and over a number of years after discharge from an ebola treatment centre [11-13, 17-21, 23, 25, 40] . although recent studies have reported the use of informal healthcare services among evd survivors [41, 42] , globally, no study to date has reported whether evd-related stigma is associated with t&cm utilisation among evd survivors. in addition, none of the published studies in sierra leone on evd survivors has explored the sociodemographic and health-related factors associated with evdrelated stigma. such associations are important, as they will inform the design and implementation of future antistigma interventions. therefore, we examined the magnitude and the sociodemographic and health related correlates of enacted and internalised stigma among evd survivors in sierra leone since their return to their communities. in addition, our study determined whether enacted and internalised stigma are possible predictors of informal healthcare service utilisation (t&cm use) among evd survivors in sierra leone. we conducted a cross-sectional questionnaire study between january and august 2018 among evd survivors across all four geographic regions (western area, northern province, eastern province and southern province) of sierra leone. participants in this study were adult evd survivors aged 18 years and older experiencing post-ebola sequelae. we excluded evd survivors whose physical and psychological health limited them from providing information, such as those survivors with memory loss, hearing loss, high fever and bleeding or those experiencing acute emotional distress. a sample of 351 evd survivors was determined using a sample size formula for cross-sectional studies (n = z2 pq/ d 2 ). we increased our sample to 400 to make up nonresponses. multistage sampling method was used to recruit participants across the country. data was collected from the four geographic regions of sierra leone (western area, northern province, southern province and eastern province). five districts were purposefully selected to cover all four geographic regions of the country. the location of the five districts in sierra leone are shown in fig. 1 . the five districts are western area urban and western area rural districts (both in the western area), bo district (southern province), kenema district (eastern province) and bombali district (northern province). these five districts were chosen based on the epidemiological profile of the total confirmed ebola cases and because they are host to the highest number of ebola survivors in sierra leone. we randomly sampled the required number of evd survivors in all five districts based on proportional representation using the national list of registered ebola survivors obtained from the sierra leone association of ebola survivors (slaes). survivors who were randomly chosen were invited to participate in the study via telephone. the survey instrument measures evd demographics such age, sex, marital status, educational status, religious affiliation, employment status, financial status, place of residence (urban/rural), geographical region (north, south, east regions and western area) and time (months) since post-discharge. perceived health status was measured using a five-point likert scale that ranged from "excellent" to "poor". evd survivors were asked if they had been diagnosed with any chronic condition prior to being infected with evd virus. the ebola-related stigma instrument was adapted from the hiv-related stigma for people living with hiv/aids (hasi-p). the hasi-p is a validated 33-item scale that measures stigma among hiv/aids patients in the past 3 months [43] . this instrument was validated among hiv/aids patients in five african countries: lesotho, malawi, south africa, swaziland and tanzania. it consists of the following subscales and this includes verbal abuse (eight items, α = 0.886); healthcare neglect (seven items, α = 0.832); social isolation (five items, α = 0.890); fear of contagion (six items, α = 0.795); and workplace stigma (two items, α = 0.758) all of which measures enacted stigma. the final subscale called negative selfperception (five items, α = 0.906) measures internalised stigma [43] . we decided to use hiv/aids related stigma scale (hasi-p) because hiv/aids patients share similar psychosocial challenges with evd survivors in terms of social isolation, fear of contagion and family and community stigma and discrimination [44] . in addition, there is widespread misinformation about hiv/aids and evd. for instance, evd and hiv/aids only affects certain groups of people in society (the poor for evd and promiscuous adults or homosexuals for hiv/aids) and the unfounded community fear of being infected with the virus through means that have not being scientifically proven [44] . to adapt to our setting, the hasi-p was reviewed by two experts in sociology and evd as well as piloted among 10 evd survivors. based on their feedback, we decided to remove the two items that measure workplace stigma since the majority of evd survivors did not have any paid job before or after evd. we also removed the statement "at the hospital, i was left in soiled bed" from the healthcare neglect subscale since majority of survivors were not admitted at the clinic/hospital. in addition, the wording of some statements were changed to fit the local evd survivorship context. further, we decided to assess stigma experienced by evd survivors since their discharged from ebola treatment centre instead of the past 3 months, as was the case when the instrument was validated among hiv/aids patients [43] . the final adapted hasi-p instrument used in our study is attached as an additional file 1. evd survivors were asked about their health care utilisation, including whether they have used t&cm treatment (products and practitioners) since their discharge from the etc. the common t&cm modalities considered in our study were informed by studies undertaken previously in sierra leone [31-33, 35, 45-47] and across africa [30] . we considered t&cm in our study to include biological based therapy (herbal medicine and animal extract), spiritual therapy (prayer/faith healing), alternative medicine systems (chinese herbal medicine, and acupuncture), and physical therapy/body manipulations (massage therapy, traditional bone setting). trained data collectors obtained the relevant information from evd survivors using self-administered or interviewer-administered (for illiterate participants) formats. the university of technology sydney human research ethics committee (uts-hrec-eth17-2080) and the sierra leone ethics and scientific review committee granted ethical clearance. a participant information sheet, explaining the purpose and scope of the study, as well as the option to opt out, was given or read (illiterate) to evd survivors before seeking their consent to participate. survivors signing or thumb printing the consent form was interpreted as their willingness to participate. survivors who signed or thumbed printed (for illiterate participants) the consent form were then given the questionnaire to fill or to be interviewed(for illiterate participants).three hundred and fifty eight evd survivors consented and completely filled the questionnaire and were included in the data analysis. we collected our data between may and august 2018 and it was done either at the regional office of evd survivors or their homes or the village courtyard. we used ibm spss statistics version 25 to perform all analyses. each of the 30 stigma items was assigned a score of 0 to 3 (0 = never, 1 = once or twice, 2 = several times and 3 = most of the time). for each participant, we summed the scores and divided by the number of items to get the mean score for each of the factors/subscales. to obtain the overall total stigma mean score, we summed up the mean scores of each of the factors and divided by 30. stigma was analysed as a binary variable (yes/no). mean stigma score of zero means that none of the items (experiences) in each of the subscales (internalised stigma, verbal abuse, healthcare neglect, fear of contagion and social isolation) occurred since discharged from the etc. a mean stigma score greater than zero indicated that at least one of the items in each of the subscales occurred once or twice or several times or most of the time. as a binary variable, mean score of zero was taken as the absence of stigma and greater than zero was taken present of stigma. we employed chi-square and fischer exact two tailed tests to determine the association between stigma subscales and sociodemographic and health related variables. we conducted a backward stepwise regressions analysis to establish the most parsimonious model that determines the sociodemographic and health related predictors of internal and enacted stigma. we also used backward stepwise regressions analysis to establish the most parsimonious model that predicts whether internal and enacted stigma is an independent predictor of informal healthcare use (t&cm use). to determine the independent association between evd -related stigma and t&cm use, all of the sociodemographic (age, sex, marital status, religious affiliation, employment status residence etc.) and health related (perceived health status, duration(years) since discharged from etc, known chronic disease) variables were taken as potential cofounders and were adjusted for in the regression analysis. probability less than 0.05 was as statistically significant for all inferential statistical analyses. out of the 400 survivors invited to participate in the study, 377 of them agreed to take part in the study. however, 19 failed to completely fill the questionnaire. thus, complete data on 358 evd survivors were analysed. table 1 gives a summary of evd survivors' sociodemographic and healthrelated characteristics. more than half (n = 194, 54.2%) of survivors were within the ages of 18-33 years and close to two-thirds (n = 223, 62.3%) were females. also close to three -fourths (n = 262, 73.2%) of survivors perceived their health to be fair/poor. based on the calculated mean scores, evd survivors reported higher levels of internalised stigma (0.92 ± 0.77) compared to enacted stigma (0.71 ± 0.61). among the enacted stigma subscale, social isolation (0.96 ± 0.88) and healthcare neglect (0.46 ± 0.53) were the highest and least respectively. we categorised stigma scores into (yes /no) as there was little variability in stigma scores. in general, majority of ebola survivors endorsed at least one item exploring internalised stigma (n = 298, 83.2%) and any of the three subscales measuring enacted stigma (n = 333, 93%). verbal abuse (n = 276, 77.1%) and fear of contagion (n = 225, 62.8%) were the highest and least reported enacted stigma subscales respectively (see table 2 ). association between stigma and sociodemographic and health related variables among ebola survivors table 3 summarises the comparison of internalized and enacted stigma with sociodemographic and health related variables among ebola survivors. religious affiliation (p = 0.038) and perceived health status (p = 0.004) were associated with internalised stigma. none of the sociodemographic and health related variables was associated with table 4 ). no sociodemographic and health related variables predicted total enacted stigma. association between t&cm use and internalised and enacted stigma this is the first nationally representative study to determine the prevalence of stigma, its sociodemographic correlates and association with informal and nonintegrated forms of health care such as t&cm use among evd survivors in sierra leone. one key finding from our study is that evd survivors report high levels of internalised and enacted stigma since discharge from an ebola treatment centre which is in line with findings from a longitudinal liberian study that reported high levels of stigma at baseline but lower levels at subsequent follow-up visits [21, 23] . our finding also resonates with similar short term and smaller sample size cross-sectional studies in sierra leone [24, 25, 48] , liberia [20] , guinea [49] , and dr congo [19, 40] ,which reported that evd survivors experience several forms of internalised and enacted stigma. our result identifies higher occurrence of internalised stigma when compared with the occurrence of total enacted stigma experienced by evd survivors. our result contrasts to findings reported in a liberian longitudinal cohort study that employed a different stigma instrument [23] but is in line with a south african study that employed the same stigma tool to measure stigma among hiv/aids patients to that employed in our study [50] . the higher frequency of internalised stigma (negative self-perception) among evd survivors in our study is a cause for concern and warrants further research attention as it can lead to low self-esteem, low self-efficacy, loss of hope for the future and can interfere with life goal achievement [51] . the findings for evd studies appear to be similar to some other infectious diseases. for example, similar sequelae have been reported among hiv/aids patients in hong kong [52] and uganda [53] , in which hiv/ aids patients reported to feel less worthy of themselves, guilt, shame and self-blame for having hiv/aids. the common types of enacted stigma faced by evd survivors in our study were social isolation, verbal abuse and fear of contagion, all of which are congruent with the common forms of stigma reported by evd survivors in the wider literature [6] . these findings may be applicable to other emerging infectious disease survivors more generally, as similar forms of stigma from the public and healthcare staff have also been reported among sars survivors in hong kong [54] . social isolation, verbal abuse and fear of contagion can lead to increased levels of psychological distress, delayed access to medical care, low adherence to medical therapy and reduced quality of life as it has also been reported among hiv/aids and mental health patients [55, 56] . drawing from lessons learnt from hiv/aids-related stigma, several evd survivor-centred and community-driven strategies have been suggested that could contribute to evd survivors' recovery and community re-integration. these include community long-term psychosocial counselling for evd survivors to enhance their coping skills, community education and social support programs for evd survivors, recruitment and training of trusted opinion leaders that can spread accurate de-stigmatising messages within communities, minimising social isolation and promoting economic empowerment of evd survivors and evd affected communities [44, 57] . the mental health impact of surviving ebola is enormous, and previous studies have reported that psychological distress, anxiety and depression are widespread among ebola survivors [4, 6] . although the impact of ebola -related stigma on mental illness among ebola survivors is not well understood, stigma induced psychological distress and anxiety have been found to be associated with adverse mental health outcome among hiv/ aids patients [58] . since hiv/aids and ebola virus disease share similar stigmatizing attributes [44] , it is possible that ebola -related stigma maybe contributing to the mental health complications among ebola survivors. thus, it likely that stigma reduction strategies will help reduce the mental health burden among evd survivors. evd survivors in our study who identified as christians and reside in the northern region were more likely to experience internalised stigma. the reasons for the high levels of internalised stigma among christians remain unclear. going forward, an in-depth ethnography study would be required to explain the high levels of internalised stigma amongst christians compared to muslims that was observed in our study. our study findings also reveal that evd survivors who perceive their health to be fair/poor are more likely to experience internalised stigma than those who perceive their health to be good. in hiv/aids patients, the link between stigma and perceived poor health status is postulated to be because stigma is known to promote poor adherence to treatment, lowers emotional coping and social support networks and reduces access to and usage of health and social services leading to poor health outcomes [26, 38] . the similarity of our findings suggest that similar concerns may be present for evd survivors. further studies are needed to explore the link between internalised stigma and religiosity as well as perceived poor health status among evd survivors in sierra leone. nonetheless, our results have revealed that religiosity, perceived health status and spatial location are potential predictors of internalised stigma among evd survivors and, that healthcare provider and social workers should consider these characteristics as possible risk factors for internalised stigma among evd survivors in sierra leone. further analysis of the enacted stigma subscales revealed verbal abuse was more likely to occur among evd survivors residing in urban locations when compared to those living in rural areas. our finding may be explained by the fact that adherence to local bylaws to prevent stigma and discrimination by the community was more prevalent in rural areas compared to urban areas [59] . also, previously identified urban-rural community differences in knowledge and perception of, and attitude towards, evd may also explain our finding [60] . our study also revealed that evd survivors who are unemployed were more likely to be socially isolated by their communities than their counterparts who were employed. such a finding maybe explained given that unemployed evd survivors are likely to be economically and socially dependent on their families and their communities for their wellbeing and, as such are more likely to experience stigma in the form of isolation from their families and communities compared to employed evd survivors [17, 61] . evd survivors who experienced healthcare neglect in conventional healthcare settings in our study were more likely to use t&cm. our finding is not surprising given that healthcare neglect (negative attitude of healthcare providers, long waiting time or being the last person to be seen by the doctor) leads to patient's dissatisfaction with conventional healthcare -a key driver for t&cm use in the general and sub-health populations in africa [30] . thus, it is important for policy makers and health providers to bear in mind that, like other sub-health populations, evd survivors will likely seek informal healthcare options if they feel neglected by the conventional health system. at policy level, laws are needed that allow evd survivors to receive appropriate care in a safe environment without being stigmatised or discriminated. in addition, educational interventions to change the negative attitude towards evd survivors among health providers are required. however, there were also positive attributes identified for t&cm use. the high rate of t&cm use among evd survivors who experienced enacted stigma (healthcare neglect and verbal abuse) maybe related to the notion that t&cm may serve as a stigma reduction strategy. for instance, t&cm has been used by patients to resist the terminal understanding of hiv/aids and believing that hiv/ aids is chronic rather than a terminal illness [27] . also, hiv/aids patients and sars survivors have used t&cm practices such as yoga and tai chi to create social support groups as people in such settings are less likely to act differently to each other since they share similar health status and experiences [27, 39] . drawing from the experiences of hiv/aids patients and sars survivors in using t&cm in managing stigma, it is possible that evd survivors will be using t&cm not only to address their physical health needs but also to as a coping mechanism against the stigma they are experiencing in their communities and at healthcare facilities. as such, there may be a role for integration of some t&cm where appropriateto help improve conventional health options for evd survivors. going forward, welldesigned qualitative research is required to have a deeper understanding of the meanings of t&cm practice in the everyday lives of evd survivors. the following limitations must be considered when interpreting our findings. first, our study may suffer from recall bias as we relied entirely on self-reported data. second, our study employed a cross-sectional design and, therefore we cannot infer causality between independent and outcomes variables. third, we adapted the hiv/aids-related stigma scale (hasi-p) [43] to measure evd related stigma among evd survivors, as there is no detailed or validated tool exist for evd related stigma. we decided to use hiv/aids related stigma scale (hasi-p) because hiv/aids share similar characteristics with evd in terms of social isolation, fear of contagion and family and community stigma and discrimination [44] . finally, our findings are only applicable to evd survivors in sierra leone and may not be representative of evd survivors in other neighbouring evd affected countries. nevertheless, the national nature of this survey represents one of the most representative samples of stigma in evd survivors. the majority of evd survivors in sierra leone experience both internalised and enacted ebola-related stigma although internalised stigma was the most common in terms of occurrence. to reduce evd related stigma, and the impacts of such stigma on evd survivors' health and wellbeing, evd outbreak responses should include evd survivor-centred and community-driven interventions that can help contribute to evd survivors' recovery and community re-integration. evd survivors appear drawn to informal and non-integrated care (t&cm) via both push (i.e. dissatisfaction with conventional care) and pull (i.e. empowerment and social commitments from t&cm). future research is needed to have a deeper insight of the meanings of t&cm practice in the everyday lives of evd survivors. supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-8279-7. additional file 1. ebolarelated stigma questionnaire. abbreviations etc: ebola treatment centre; evd: ebola virus disease; t&cm: traditional and complementary medicine we want to extend our thanks and appreciation to the ebola survivors who consented to take part in this study. we also want to extend our appreciation to the staff of the sierra leone ebola survivors association and staff of the ebola clinic at 34 military hospital wilberforce freetown as well as all data collectors for their support during data collection.. we also extend our thanks to the faculty health, university of technology sydney to help fund the field work for this study. in addition, we extend our thanks and appreciation to dean and staff of the faculty of pharmaceutical sciences, college of medicine and allied health sciences, university of sierra leone. the authors thank mr. john alimamy kabba for helping in creating the map used in this manuscript. authors' contributions pbj and jw conceived of the study while, pbj, jw, as & ja contributed in designing the study. pbj analysed the data and wrote the initial draft of the manuscript. jw, as and ja supervised the process and contributed to the intellectual content of the manuscript. all authors read and approved the final version of the manuscipt. the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. availability of data and materials due to confidentiality and privacy concerns, and given the sensitivity surrounding stigma and discrimination among ebola survivors, our study did not receive approval from the university of technology sydney human research ethics committee and the sierra leone ethics and scientific review committee to publicly share the raw data. also, ebola survivors consented to participate in the study on the basis that their data would not be shared with anyone except members of the research team (my supervisors and i). the raw data informing the findings of this study are stored privately at the university of technology sydney data storage platform called cloudstor. however, upon reasonable request, the anonymised raw data underlying the findings of this study can be made available through the following persons not applicable. the authors declare that they have no competing interest. exposure patterns driving ebola transmission in west africa: a retrospective observational study ebola situation report who: clinical care for survivors of ebola virus disease. interim guidance neuropsychological longterm sequelae of ebola virus disease survivors -a systematic review sequelae of ebola virus disease: the emergency within the emergency post-ebola psychosocial experiences and coping mechanisms among ebola survivors: a systematic review stigma: introduction and overview social stigma and its consequences for the socially stigmatized the impact of stigma in healthcare on people living with chronic illnesses re-framing stigma: felt and enacted stigma and challenges to the sociology of chronic and disabling conditions groupe d'etude p. ebola in guinea: experience of stigma among health professional survivors cultural contexts of ebola in northern uganda psychological distress among ebola survivors discharged from an ebola treatment unit in monrovia, liberia -a qualitative study ebola rna persistence in semen of ebola virus disease survivors-preliminary report persistence of ebola virus in ocular fluid during convalescence experiences of ebola survivors: causes of distress and sources of resilience once there is life, there is hope' ebola survivors' experiences, behaviours and attitudes in sierra leone multidisciplinary assessment of post-ebola sequelae in guinea (postebogui): an observational cohort study survey among survivors of the 1995 ebola epidemic in kikwit, democratic republic of congo: their feelings and experiences care of ebola survivors and factors associated with clinical sequelae ebola virus disease-related stigma among survivors declined in liberia over an 18-month, post-outbreak period: an observational cohort study diminished quality of life among women affected by ebola stigma and ebola survivorship in liberia: results from a longitudinal cohort study mobile health clinic for the medical management of clinical sequelae experienced by survivors of the 2013-2016 ebola virus disease outbreak in sierra leone, west africa posttraumatic stress reactions in ebola virus disease survivors in sierra leone examining the associations between hiv-related stigma and health outcomes in people living with hiv/aids: a series of meta-analyses the meaning of complementary and alternative medicine practices among people with hiv in the united states: strategies for managing everyday life world health organization traditional, complementary and integrative medicine: an international reader: macmillan international higher education traditional, complementary and alternative medicine use in sub-saharan africa: a systematic review herbal medicine use among hypertensive patients attending public and private health facilities in freetown sierra leone herbal medicines use during pregnancy in sierra leone: an exploratory cross-sectional study prevalence and correlates of herbal medicine use among women seeking care for infertility in freetown healthcare seeking for diarrhoea, malaria and pneumonia among children in four poor rural districts in sierra leone in the context of free health care: results of a cross-sectional survey herbs and herbal combinations used to treat suspected malaria in herbal medicine use during breastfeeding: a cross-sectional study among mothers visiting public health facilities in the western area of sierra leone adaptation and translation of mental health interventions in middle eastern arab countries: a systematic review of barriers to and strategies for effective treatment implementation use of traditional complementary and alternative medicine for hiv coping with future epidemics: tai chi practice as an overcoming strategy used by survivors of severe acute respiratory syndrome (sars) in post-sars hong kong neurological, cognitive, and psychological findings among survivors of ebola virus disease from the 1995 ebola outbreak in kikwit, democratic republic of congo: a cross-sectional study pattern of health care utilization and traditional and complementary medicine use among ebola survivors in sierra leone utilisation of and attitude towards traditional and complementary medicine among ebola survivors in sierra leone validation of the hiv/aids stigma instrument -plwa (hasi-p) addressing ebola-related stigma: lessons learned from hiv/aids the role of traditional treatment on health care seeking by caregivers for sick children in sierra leone: results of a baseline survey awareness, use, attitude and perceived need for complementary and alternative medicine (cam) education among undergraduate pharmacy students in sierra leone: a descriptive crosssectional survey exploring self-use, attitude and interest to study complementary and alternative medicine (cam) among final year undergraduate medical, pharmacy and nursing students in sierra leone: a comparative study sequelae of ebola virus disease study of ebola virus disease survivors in guinea the prevalence and predictors of stigma amongst people living with hiv/aids in the western province on the self-stigma of mental illness: stages, disclosure, and strategies for change stigmatization among people living with hiv in hong kong: a qualitative study factors associated with perceived stigma among people living with hiv/aids in post-conflict northern uganda the sars-associated stigma of sars victims in the post-sars era of hong kong correlates and consequences of internalized stigma for people living with mental illness: a systematic review and meta-analysis how does stigma affect people living with hiv? the mediating roles of internalized and anticipated hiv stigma in the effects of perceived community stigma on health and psychosocial outcomes reducing hiv-related stigma and discrimination in healthcare settings: a systematic review of guidelines, tools, standards of practice, best practices, consensus statements and systematic reviews behavior: from conceptualizing to measuring hiv stigma: a review of hiv stigma mechanism measures samba tt: 'when ebola enters a home, a family, a community': a qualitative study of population perspectives on ebola control measures in rural and urban areas of sierra leone national survey of ebola-related knowledge, attitudes and practices before the outbreak peak in sierra leone profile and reintegration experience of ebola survivors in guinea: a cross-sectional study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-350540-s6is9ndm authors: pinto, rogério m.; park, sunggeun title: covid-19 pandemic disrupts hiv continuum of care and prevention: implications for research and practice concerning community-based organizations and frontline providers date: 2020-04-28 journal: aids behav doi: 10.1007/s10461-020-02893-3 sha: doc_id: 350540 cord_uid: s6is9ndm nan the united states department of health and human services has proposed a reduction in the number of hiv infections in the united states by 90% within the current decade [1] . the world health organization's 90/90/90 goal also aims to contain the hiv pandemic by 2030, at which time 90% of all people living with hiv (plwh) are projected to know their hiv status; 90% with diagnosed hiv infection are projected to receive antiretroviral therapy; and 90% of those receiving antiretroviral therapy are projected to show viral suppression. ending the hiv pandemic requires that we gather local knowledge on which to base sustainable action. it requires leveraging local efforts led by community-based organizations helping those at high risk for hiv navigate complex diffusion systems and promote health equity-a national priority to improve health access for all, regardless of geographic boundaries, race/ethnicity, and sexual orientation [2, 3]. community-based organizations (cbos) employ frontline service providers-social workers, health educators, navigators-to help (1) individuals of unknown hiv status access testing; (2) those at high-risk for hiv but who test negative to access physicians who can prescribe prep; (3) those who test positive for hiv to access primary care [4] ; and all at-risk clients to access support services to help them stay on the hiv continuum of care and prevention ("care continuum") [5] [6] [7] [8] . nonetheless, community-engaged research suggests that, prior to the covid-19 pandemic, these frontline providers had not been consistent in how often or in how they linked clients to care continuum services. using cross-sectional data from nearly 300 frontline providers in new york city, an epicenter of both the hiv and covid-19 pandemics, our research shows that, in the six months prior to data collection, 50% of provider participants had linked fewer than five clients to hiv testing; 48% linked fewer than five clients to primary care; and 48% had not provided any client with prep education [9] . these numbers are worrisome, but longitudinal data show a brighter picture. we detected increased involvement in at least some service linkage from 76% providers at baseline to 81% over two years [10] . nearly half (47%) of providers offered prep education at both of these time points; 19% started offering prep education since baseline; while 5% stopped offering education at follow-up [11] . furthermore, evidence suggests that frontline providers who more frequently link clients to hiv services tend to do so as part of face-to-face client meetings, at which the provider might ask the client, while still in their presence, to contact the referral, or the provider might make the contact and hand the client written information [12] . this level of proximity is a challenge under the shelter-in-place and physical distancing orders. before we crafted this note from the field, our cbo partners (i.e. managers and providers) informed us that the covid-19 pandemic had already disrupted all care continuum services. physical distancing between providers and clients will likely have a negative impact on how often and how frontline providers will link clients to services. this opens up a field of inquiry needed to develop best practices for referral-making. the covid-19 pandemic quickly exposed medical (e.g. difficult access to services, lack of testing and insurance), and structural (e.g. unemployment, food insecurity, geographic isolation) vulnerabilities which have also historically undermined individual-and system-level hiv prevention [13] . cbos are following stay-in-shelter orders and using online platforms and telephones to communicate with clients. some of these platforms are unreliable, and clients and providers alike may encounter difficulties navigating them; many do not have access to high-speed internet or have enough of a data allowance on their cellular phone plans. to offer basic information about hiv prevention, cbos are turning to social media: teaching clients about hiv testing and prep; reminding clients of the importance of physical distancing; and providing social support for newly diagnosed individuals. since access to in-person hiv tests has been hampered, one cbo partner mentioned about their network, "as far as i know, no one is doing any hiv testing right now." another cbo offers at-home hiv test kits to high-need clients, but the number of kits available is limited. frontline providers (e.g., prep navigators) are not allowed (confidential hipaa compliant) to take client information from their offices, thus they have stopped making referrals to physicians who can prescribe prep. providers have cut back on or stopped altogether making referrals to primary care. they are nonetheless providing limited harm reduction and support services, with all parties adhering to physical distancing-for example, clean syringes passed through mobile unit windows and food and other items dropped off in front of clients' homes. meanwhile, cbo managers are working on safety protocols and are purchasing protective materials in preparation for resuming face-to-face consultation when the stay-in-shelter order is lifted. the covid-19 pandemic presents multifaceted challenges to hiv service cbos, including but not limited to resource shortages, low staff morale, and disruption to patient-centered service provision. suspending services creates budgetary shortfalls for cbos that heavily rely on program revenues [14] . given the skyrocketing numbers of unemployment claims in the last two weeks of march and two first weeks of april [15] , many vulnerable clients may not be able to make co-payments for services, even after cbo doors are open again. cbos expect significant declines in private donations, as also observed in the 2008 recession [16] . now and during the stay-in-shelter period, cbos are likely to rely on small business and individual donations and government programs. they are likely to strengthen relationships with private and public partners (e.g., other cbos) in their communities in order to advocate for more effective government responses such as those initiated in response to the great depression of the 1930s [17] . the pandemic has created a substantial decline in provider morale. many have and will continue to lose colleagues and clients to covid-19. with limited resources and capacities, they are likely to be forced to make difficult choices as to which cases to prioritize for services [18, 19] . cbo staff will likely face layoffs and/or reduced paychecks as organizations struggle to stay open. these factors will continue to impact negatively frontline providers' sprit, motivation, and mental health. providers having day-to-day interactions with clients in primary care, outpatient, and prevention settings are poised to help plwh and vulnerable individuals overcome hiv-related stigma, prep stigma, inadequate health insurance, and can help improve hiv testing rates [20] [21] [22] [23] [24] [25] . provider engagement of clients in referral-making processes seems to improve client access to hiv testing, prep, and primary care, even when provider caseloads are high, clients may lack insurance, and cbos may fear losing clients and revenue to other cbos [5, 26, 27] . however, in the face of covid-19, such engaged, face-to-face interactions and referrals might not be feasible. community-focused research can help track the degree to which covid-19 is disrupting cbo operations, provider behaviors, and client experiences and outcomes. the disruptions caused by covid-19 allow us to see how the hiv care continuum has been undermined routinely by insufficient concrete and human organizational resources, and by failures to follow up and track provider referrals to hiv services. to demonstrate how structural failures, highlighted by the covid-19 pandemic, prevent providers from keeping their clients on the care continuum, we must study how cbos' organizational supports incorporate client perspectives. covid-19 exposed the need for research to understand how high volumes of incomplete referrals-i.e. clients not accessing services to which they are referred-waste time allotted for services, increases costs, lengthen waitlists, and jeopardize health outcomes (e.g., retention in care, viral suppression) [6, 28] . providers' active referral-making (including subsequent coordination and tracking efforts) can facilitate clients' timely access to needed services and reduce waste of organizational resources (e.g., staff hours, social capital) [29] . despite the growing emphasis on person-centered care [30] [31] [32] [33] , few empirical studies have investigated the implications of person-centered care for organizations offering hiv services [34] [35] [36] . we also need to look at current literature and identify not only gaps but the limitations of past research (e.g. lack of large-scale qualitative evidence and limited involvement of clients and providers). doing so should help researchers address the limited evidence on referral-making and linkage practices that could help clients access the hiv services to which they are referred ("referral completion"), and, ultimately, end the hiv pandemic within this decade. ending the hiv epidemic: a plan for the united states disparities in health and health care: five key questions and answers. henry j kais fam found recommendations for hiv screening of gay, bisexual, and other men who have sex with men -united states dc: danya international interventions to improve outpatient referrals from primary care to secondary care. cochrane database syst rev brief strengths-based case management promotes entry into hiv medical care: results of the antiretroviral treatment access study-ii recommendations to increase testing and identification of hiv-positive individuals through partner counseling and referral services interprofessional collaboration and on-the-job training improve access to hiv testing, hiv primary care, and pre-exposure prophylaxis (prep) interprofessional collaboration improves linkages to primary care: a longitudinal analysis interprofessional collaboration improves the odds of educating patients about prep over time preparation) more is better: active and passive referral-making increases linkages to hiv primary care covid-19). centers for disease control and prevention nonprofits for hire: the welfare state in the age of contracting this thing is going to come for us all nonprofit growth and decline during economic uncertainty a history of social welfare and social work in the united states present, and future the hardest questions doctors may face: who will be saved? who won't? the new york times perspectives of linkage to care among people diagnosed with hiv hiv testing, care referral and linkage to care intervals affect time to engagement in care for newly diagnosed hiv-infected adolescents in fifteen adolescent medicine clinics in the united states challenges and successes in linking hiv-infected women to care in the united states barriers and facilitators of linkage to hiv primary care in new york city barriers to hiv care and treatment among participants in a public health hiv care relinkage program why physicians don't ask: interpersonal and intrapersonal barriers to hiv testing-making a case for a patient-initiated campaign a client-centered relational framework on barriers to the integration of hiv and substance use services: a systematic review factors that influence linkages to hiv continuum of care services: implications for multi-level interventions unpredictable work timing in retail jobs: implications for employee work-life conflict limited effectiveness of antiviral treatment for hepatitis c in an urban hiv clinic the values and value of patient-centered care beyond patient-centred care: a conceptual framework of co-production mechanisms with vulnerable groups in health and social service settings crossing the quality chasm: a new health system for the 21st century what 'patient-centered' should mean: confessions of an extremist retention in hiv care among participants in the patient-centered hiv care model: a collaboration between community-based pharmacists and primary medical providers is the quality of the patient-provider relationship associated with better adherence and health outcomes for patients with hiv? co-production in the treatment of substance use disorder and its relationship to clinic's service output patterns publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we wish to convey our enormous appreciation for our community partners, richard yancy, hiv prevention specialist (matrix human services) and leon golson, director of prevention programs (unified -hiv health and beyond), who contributed critical information and support for the writing of this piece. key: cord-333405-ji58jbct authors: morens, david m.; folkers, gregory k.; fauci, anthony s. title: the challenge of emerging and re-emerging infectious diseases date: 2004-07-08 journal: nature doi: 10.1038/nature02759 sha: doc_id: 333405 cord_uid: ji58jbct infectious diseases have for centuries ranked with wars and famine as major challenges to human progress and survival. they remain among the leading causes of death and disability worldwide. against a constant background of established infections, epidemics of new and old infectious diseases periodically emerge, greatly magnifying the global burden of infections. studies of these emerging infections reveal the evolutionary properties of pathogenic microorganisms and the dynamic relationships between microorganisms, their hosts and the environment. merging infections (eis) can be defined as "infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range" 1 . eis have shaped the course of human history and have caused incalculable misery and death. in 1981, a new disease -acquired immune deficiency syndrome (aids) -was first recognized. as a global killer, aids now threatens to surpass the black death of the fourteenth century and the 1918-1920 influenza pandemic, each of which killed at least 50 million people 2, 3 . of the 'newly emerging' and 're-emerging/resurging' diseases that have followed the appearance of aids (fig. 1) , some have been minor curiosities, such as the 2003 cases of monkeypox imported into the united states 4 , whereas others, such as severe acute respiratory syndrome (sars), which emerged in the same year 5 , have had a worldwide impact. the 2001 anthrax bioterrorist attack in the united states 6 falls into a third category: 'deliberately emerging' diseases. eis can be expected to remain a considerable challenge for the foreseeable future. here we examine the nature and scope of emerging and reemerging microbial threats, and consider methods for their control. we emphasize that emergence results from dynamic interactions between rapidly evolving infectious agents and changes in the environment and in host behaviour that provide such agents with favourable new ecological niches. about 15 million (>25%) of 57 million annual deaths worldwide are estimated to be related directly to infectious diseases; this figure does not include the additional millions of deaths that occur as a consequence of past infections (for example, streptococcal rheumatic heart disease), or because of complications associated with chronic infections, such as liver failure and hepatocellular carcinoma in people infected with hepatitis b or c viruses 7 (fig. 2) . the burden of morbidity (ill health) and mortality associated with infectious diseases falls most heavily on people in developing countries 8 , and particularly on infants and children (about three million children die each year from malaria and diarrhoeal diseases alone 7 ). in developed nations, infectious disease mortality disproportionately affects indigenous and disadvantaged minorities 9 . eis have been familiar threats since ancient times. they were once identified by terms such as ȕșȓȗș ȝ (loimos) 10 , and later as 'pestilences' , 'pestes' , 'pests' and 'plagues' . many examples can be cited in addition to the black death and the 1918 influenza pandemic, such as certain biblical pharaonic plagues and the unidentified plague of athens, which heralded the end of greece's golden age 11 . the age of discovery, starting in the fifteenth century, was a particularly disastrous period with regard to the spread of infectious diseases. importation of smallpox into mexico caused 10-15 million deaths in 1520-1521, effectively ending aztec civilization 12, 13 . other amerindian and pacific civilizations were destroyed by imported smallpox and measles [13] [14] [15] [16] [17] . historians have referred to these events as apocalypses 16 and even as genocide 15 . for centuries, mankind seemed helpless against these sudden epidemics. but the establishment of the germ theory and the identification of specific microbes as the causative agents of a wide variety of infectious diseases [18] [19] [20] led to enormous progress, notably the development of vaccines and ultimately of antimicrobials 20 . in fact, the era of the identification of microbes had barely begun 18 when optimists at the end of the nineteenth century predicted the eradication of infectious diseases 21 . by the 1950s, which witnessed the widespread use of penicillin, the development of polio vaccines and the discovery of drugs for tuberculosis, complacency had set in 22 , and in 1967, the us surgeon general stated that "the war against infectious diseases has been won" 23 . some experts remained sceptical, aware of recurrent lessons from history. they were less persuaded by successes than alarmed by failures such as the lack of progress against infections in the developing world and the global spread of antimicrobial resistance. richard krause, then the director of the us national institute of allergy and infectious diseases, warned in 1981 (ref. 24) that microbial diversity and evolutionary vigour were still dynamic forces threatening mankind. as krause was completing his book the restless tide 24 , aids -one of history's most devastating pandemics -was already insidiously emerging. the emergence of aids led to renewed appreciation of the inevitability and consequences of the emergence of infectious diseases [25] [26] [27] [28] [29] [30] [31] . in the past 25 years, some of the factors that resulted in aids have also led to re-emergences of historically important diseases such as cholera, diphtheria, trench fever and plague. many re-emergences have been catalysed by wars, loss of social cohesion, and natural disasters such as earthquakes and floods, indicating the importance not only of microbial and viral factors, but also of social and environmental determinants [25] [26] [27] [28] [29] [30] [31] . the challenge of emerging and re-emerging infectious diseases the classification of eis as 'newly emerging' , 're-emerging/resurging' or 'deliberately emerging' is useful because the underlying causes of emergence and the optimal prevention or control responses frequently differ between the groups. newly emerging infections are those that have not previously been recognized in man. many diverse factors contribute to their emergences (see box 1); these include microbial genetic mutation and viral genetic recombination or reassortment, changes in populations of reservoir hosts or intermediate insect vectors, microbial switching from animal to human hosts, human behavioural changes (notably human movement and urbanization), and environmental factors. these numerous microbial, host and environmental factors interact to create opportunities for infectious agents to evolve into new ecological niches, reach and adapt to new hosts, and spread more easily between them. any discussion of recent eis must begin with the human immunodeficiency virus (hiv) that causes aids. hiv has so far infected more than 60 million people worldwide 33 . before jumping to humans an estimated 60-70 years ago 34 , perhaps as a consequence of the consumption of 'bush meat' from non-human primates, hiv-1 and hiv-2 had ample opportunity to evolve in hosts that were genetically similar to man (the chimpanzee, pan troglodytes, and the sooty mangabey, cercocebus atys). but hiv/aids might never have emerged had it not been for disruptions in the economic and social infrastructure in post-colonial sub-saharan africa. increased travel, the movement of rural populations to large cities, urban poverty and a weakening of family structure all promoted sexual practices, such as promiscuity and prostitution, that facilitate hiv transmission [34] [35] [36] [37] . such complex interactions between infectious agents, hosts and the environment are not unique to the epidemiology of hiv/aids. the examples cited below further illustrate how changes in population density, human movements and the environment interact to create ecological niches that facilitate microbial or viral adaptation. some infectious agents that have adapted to non-human hosts can jump to humans but, unlike hiv, are not generally transmitted from person to person, achieving only 'dead end' transmission. infections in animals that are transmitted to humans (zoonoses), and those transmitted from one vertebrate to another by an arthropod vector (vector-borne diseases), have repeatedly been identified as ranking among the most important eis 25, 26 . examples include the arenavirus haemorrhagic fevers (argentine, bolivian, venezuelan and lassa haemorrhagic fevers) and hantavirus pulmonary syndrome (hps). viruses in these groups have co-evolved with specific rodent species whose contact with humans has increased as a result of modern environmental and human behavioural factors. farming, keeping domestic pets, hunting and camping, deforestation and other types of habitat destruction all create new opportunities for such infectious agents to invade human hosts [25] [26] [27] [28] [29] [30] [31] . the first epidemic of hps, detected in the southwestern region of the united states in 1993 (ref. 38) , resulted from population booms of the deer mouse peromyscus maniculatis, in turn caused by climate-related and recurrent proliferation of rodent food sources. increased rodent populations and eventual shortages of food drove expanded deer mouse populations into homes, exposing people to virus-containing droppings. the 1998-1999 malaysian nipah virus epidemic 39 further illustrates the influence of human behaviours and environmental perturbations on newly emerging human infections. pigs crammed together in pens located in or near orchards attracted fruit bats whose normal habitats had been destroyed by deforestation and whose droppings contained the then-unknown paramyxovirus. virus aerosolization caused infection of pigs, with overcrowding leading to explosive transmission rates and ultimately to infections in pig handlers. variant creutzfeldt-jakob disease (vcjd) is another example of a zoonotic disease emerging in humans. vcjd is caused by the humanadapted form of the prion associated with the emerging epizootic (large-scale animal outbreak) of bovine spongiform encephalopathy (bse) 40 figure 1 global examples of emerging and re-emerging infectious diseases, some of which are discussed in the main text. red represents newly emerging diseases; blue, re-emerging/resurging diseases; black, a 'deliberately emerging' disease. adapted, with permission, from ref. 23. epizootic/vcjd epidemic, primarily affecting great britain, probably resulted from the now-abandoned practice of supplementing cattle feed with the pulverized meat and bones of previously slaughtered cattle. bse itself is suspected to have emerged because of even earlier use of cattle feed containing the agent of sheep scrapie, a prion disease recognized by farmers more than 250 years ago 41 . alarmingly, the new bse prion has become uncharacteristically promiscuous: unlike most known prions, it readily infects multiple species in addition to humans. this suggests the possibility of further emerging diseases associated with prions with currently unknown transmissibility to humans 40 . the recent reports of variant strains of the bse prion 42 suggest that the bse agent could be a more serious threat than other animal prions. infectious agents indirectly transmitted to or between humans by way of human-modified environments account for other emerging zoonoses, as well as certain non-zoonotic diseases, which are discussed below. for example, legionnaires' disease, first identified in 1976, is caused by legionella pneumophila, whose emergence as a human pathogen might not have occurred were it not for the environmental niche provided by air-conditioning systems 26 . campylobacter jejuni and shiga-toxin-producing escherichia coli (e. coli o157:h7 and other agents of haemolytic-uraemic syndrome) infect agricultural animals, gaining access to humans through food, milk, water or direct animal contact. other enteric pathogens, such as the vibrios causing classical cholera (re-emerging; see below) and serogroup o139 cholera, and the zoonotic protozoa cryptosporidium parvum and cyclospora cayetanensis 26 , seem to have come from environmental or animal organisms that have adapted to human-to-human 'faecal-oral' transmission through water. some eis come from microorganisms that once caused familiar diseases, but which now cause new or previously uncommon diseases. streptococcus pyogenes caused a fatal pandemic of scarlet and puerperal fevers between 1830 and 1900 (ref. 44) . scarlet fever, then the leading cause of death in children, is now rare, but has been largely supplemented by other streptococcal complications such as streptococcal toxic shock syndrome, necrotizing fasciitis and re-emergent rheumatic fever 45 . when new microbes are discovered, their emergences as disease-causing pathogens may be delayed. for example, in 1883, robert koch was unable to show that the newly discovered koch-weeks bacillus caused serious disease. more than a century later, a fatal ei dubbed brazilian purpuric fever was linked to virulent clonal variants of haemophilus influenzae biogroup aegyptius (the koch-weeks bacillus) 46 . although the bases of emergences of new and more severe diseases caused by s. pyogenes and h. influenzae biogroup aegyptius are not fully known, in both cases complex microbial genetic events are suspected. the distinctive clonal variants associated with severe h. influenzae biogroup aegyptius disease have been shown by pcr (polymerase chain reaction)-based subtractive genome hybridization to contain not only a unique plasmid, but also unique chromosomal regions, some of which are encoded by bacteriophages 47 . this research has narrowed the search for virulence determinants to unique proteins, some of which may have been acquired from other organisms by horizontal gene transfer. streptococcus pyogenes has been studied more extensively, but the basis of severe disease emergence seems to be more complex than for h. influenzae biogroup aegyptius. many factors associated with streptococcal virulence have been identified in strains bearing the m1 surface protein as well as in other m protein strains, among them bacteriophage-encoded superantigen toxins and a protein known as sic (streptococcal inhibitor of complement), which seems to be strongly selected by human host mucosal factors. several lines of evidence suggest that changes in streptococcal virulence reflect genetic changes associated with phage integration, large-scale chromosomal rearrangements and possibly the shuffling of virulence cassettes (clusters of genes responsible for pathogenicity), followed by rapid human spread and immune selection 48, 49 . infectious agents that are associated with chronic diseases are one of the most challenging categories of newly emerging (or at least newly appreciated) infections. examples include the associations of hepatitis b and c with chronic liver damage and hepatocellular carcinoma, of certain genotypes of human papillomaviruses with cancer of the uterine cervix, of epstein-barr virus with burkitt's lymphoma (largely in africa) and nasopharyngeal carcinoma (in china), of human herpesvirus 8 with kaposi sarcoma, and of helicobacter pylori with gastric ulcers and gastric cancer [50] [51] [52] . some data even suggest infectious aetiologies for cardiovascular disease and diabetes mellitus 53 , major causes of death and disability worldwide. other associations between infectious agents and idiopathic chronic diseases will inevitably be found. re-emerging and resurging infections are those that existed in the past but are now rapidly increasing either in incidence or in geographical or human host range. re-emergence is caused by some of the factors that cause newly emerging infectious diseases, such as microbial evolutionary vigour, zoonotic encounters and environmental encroachment. re-emergences or at least cyclical resurgences of some diseases may also be climate-related -for example, the el niño/southern oscillation (enso) phenomenon is associated with resurgences of cholera and malaria 54 . the impact of both new and re-emerging infectious diseases on human populations is affected by the rate and degree to which they spread across geographical areas, depending on the movement of human hosts or of the vectors or reservoirs of infections. travel has an important role in bringing people into contact with infectious agents 55 . an increase in travel-associated importations of diseases was anticipated as early as 1933, when commercial air travel was still in its infancy 56 . this has since been demonstrated dramatically by an international airline hub-to-hub pandemic spread of acute haemorrhagic conjunctivitis in 1981 (ref. 57) , by epidemics of meningococcal meningitis associated with the hajj, and more recently by the exportation of epidemic sars (a newly emerging disease) from guangdong province, china, to hong kong, and from there to beijing, hanoi, singapore, toronto and elsewhere 5 (fig. 3) . the persistent spread of hiv along air, trucking, drug-trafficking and troop-deployment routes is a deadly variation on this theme [35] [36] [37] . plasmodium falciparum malaria was neglected for several decades, but is now among the most important re-emerging diseases worldwide (fig. 2) . years of effective use of dichlorodiphenyltrichloroethane (ddt) led to the abandonment of other mosquito-control programmes, but the insecticide fell into disuse because of mosquito resistance and concerns about the insecticide's potentially harmful effects on humans and wildlife. consequently, malaria has re-emerged, and the situation has been worsened by the development of drug resistance to chloroquine and mefloquine 58 . research efforts focus on the development of vaccines 59 and new drugs, and on re-establishing public health measures such as the use of bed nets. tuberculosis is one of the most deadly re-emerging diseases (fig. 2) . the discovery of isoniazid and other drugs initially led to effective tuberculosis cures, empty sanitoria and the dismantling of public health control systems in developed nations. consequently, by the 1980s, when tuberculosis had re-emerged in the era of hiv/aids, local and state health departments in the united states lacked field, laboratory and clinical staff and so had to reinvent tuberculosiscontrol programmes 25 . the remarkable re-emergence of tuberculosis was fuelled by the immune deficiencies of people with aids, which greatly increases the risk of latent mycobacterium tuberculosis infections progressing to active disease, and being transmitted to others. inadequate courses of anti-tuberculosis therapy compound the problem, leading to the emergence and spread of drug-resistant and multidrug-resistant strains 60 , and a need for more expensive treatment strategies such as directly observed therapy. it has been known for over a century that tuberculosis is a disease of poverty, associated with crowding and inadequate hygiene. the continuing expansion of global populations living in poverty makes tuberculosis more difficult to control. 63 . immune deficiency associated with aids, and with chemotherapy for cancer, immune-mediated diseases and transplantation, has contributed to an enormous global increase in the numbers of immunosuppressed people over the past few decades (probably more than 1% of the world's population), setting the stage for the re-emergence of many opportunistic infections. hiv, which has infected more than 60 million people globally 33 , is the largest single cause of human immune deficiency and markedly increases vulnerability to a wide range of opportunistic pathogens, including pneumocystis carinii, various fungi, tuberculosis, protozoa and herpesviruses 64 . breakthroughs in cancer therapy and in immunosuppressive therapies used to treat immune-mediated diseases and for transplantation 65, 66 can also leave patients susceptible to opportunistic infections. human organ transplantation adds a further risk of infection with undetected pathogens in donor tissues, and transplantation of animal organs introduces the risk of transmission to humans of animal microbes 67 . the emergence of zoonotic and vector-borne diseases can also be associated with human behaviours and environmental perturbation. although west nile virus is now a major epidemiological concern in the developed world, dengue remains the most significant and widespread flavivirus disease to have emerged globally 71 . a 2001-2002 epidemic in hawaii -fortunately without fatalities -is a reminder that dengue has also re-emerged in locations once considered to be dengue-free. usually transmitted by aedes aegypti mosquitoes, dengue has recently been transmitted by aedes albopictus -a vector switch of potential significance with respect to dengue re-emergence 71 . in the americas, including many us southern states, a. albopictus has been spreading into areas where a. aegypti mosquitoes are not found, and persisting for longer seasonal periods, putting tens of millions more people at risk of dengue infection. dengue re-emergence is further complicated by disturbing increases in a serious and formerly rare form of the disease, dengue haemorrhagic fever (dengue shock syndrome being its highly fatal form). these severe complications are thought to result from the evolution of dengue viruses to escape high insight review articles 246 nature | vol 430 | 8 july 2004 | www.nature.com/nature population immunity, seen in increased viral virulence and human immunopathogenesis due to antibody-dependent enhancement of viral infection 72 . cholera is also of interest, not only as an important cause of mortality, but also because of the complexity of factors that determine its re-emergence. both virulent and avirulent strains of these zoonotic bacteria are maintained in the environment and are rapidly evolving in association with phyto-and zooplankton, algae and crustaceans. such environmental strains seem to act as reservoirs for human virulence genes (for example, genes for the phage-encoded cholera toxin and the toxin-coregulated pilus (tcp) factor associated with attachment), and to undergo gene transfer events that lead to new strains containing further virulence gene combinations. these result in periodic cholera emergences that cause epidemics and pandemics 73 . thus, although the disease we know as cholera has appeared to be clinically and epidemiologically stable at least since the third pandemic (in the 1840s), modern evidence suggests that such apparent stability masks aggressive bacterial evolution in complex natural environments. influenza a viruses, which are endemic gastrointestinal viruses of wild waterfowl, have evolved elaborate mechanisms to jump species into domestic fowl, farm animals and humans. periodic gene segment reassortments between human and animal viruses produce important antigenic changes, referred to as 'shifts' . these can lead to deadly pandemics, as occurred in 1888, 1918, 1957 and 1968 (refs 74, 75) . in intervening years, shifted viruses undergo continual but less dramatic antigenic changes called 'drifts' , which allow them partially to escape human immunity raised by previously circulating influenza viruses. influenza drift is an evolutionary success story for the virus. influenza a has a seemingly inexhaustible repertoire of mutational possibilities at several critical epitopes surrounding the viral haemagglutinin site that attaches to human cells. it remains something of a mystery how zoonotic influenza viruses mix with each other and with human strains to acquire the additional properties of human virulence and human-to-human transmissibility. before 1997, mild cases of human disease associated with avian influenza viruses were occasionally reported 76 . these events have become more frequent, sometimes resulting in severe cases of disease and death. avian influenza has recently made dead-end jumps to humans -for example, the 1997 hong kong outbreak of the newly emergent h5n1 influenza, the 2003 h7n7 epidemic in the netherlands, the 2003-2004 h5n1 and h7n3 epizootics in asia and elsewhere, and occasional cases of h9n2 disease (fig. 4) . meanwhile, back-switches of human h3n2 viruses have emerged in pigs, from which both doubly mixed (pig-human) and triply mixed (pig-human-avian) viruses 74, 75 have been isolated. such enzootic/zoonotic mixing is suspected to have occurred in the influenza pandemic of 1918-1920, which was caused by an h1n1 virus with an avian-like receptor-binding site 77 . the predicted virulence genes of this virus are now being sought from 85-year-old pathology specimens and from frozen corpses 78 . the implications of interspecies genetic mixing for future influenza pandemics are troubling. although much remains speculative about how influenza viruses emerge and spread, it seems clear that the process is driven by prolific and complex viral evolution (genetic reassortment and mutational 'drift'), interspecies mixing and adaptation, and ecological factors that bring humans into contact with animals and each other. by whatever means new influenza virus pandemic strains emerge, they eventually reach a critical threshold of human transmission beyond which epidemic and pandemic spread follows mathematically predictable patterns. the dynamics and determinants of such epidemic development have been studied since the nineteenth century for several infectious diseases. for influenza, both historical and prospective epidemics have been described or predicted using deterministic and stochastic mathematical models, often with surprising accuracy when compared with actual epidemic data. more complicated mathematical models that describe how diseases spread by means other than personto-person aerosol transmission have generally been less successful in describing and predicting epidemics, but have nonetheless been helpful in planning public health responses to epidemics caused by hiv 79 , vcjd 80 and other diseases. mathematical modelling is also used to determine the impact of emerging epidemics. for example, it has been difficult to estimate overall influenza mortality because fatal infections are often neither diagnosed nor accurately recorded in hospital records and death certificates, especially in the elderly. recent epidemiological attempts to obtain improved influenza mortality estimates from seasonal excess mortality data 81 have indicated that influenza mortality may be insight review articles nature | vol 430 | 8 july 2004 | www.nature.com/nature 247 greater than was previously suspected, because influenza deaths are frequently coded under seemingly unrelated categories such as cardiovascular diseases. the same approaches also show that other influenza-like deaths may actually be due to other agents, such as respiratory syncytial virus (rsv), a common childhood virus that in the past decade has emerged as a major cause of adult mortality 81 . deliberately emerging microbes are those that have been developed by man, usually for nefarious use. the term 'deliberately emerging' refers to both naturally occurring microbial agents such as anthrax 6 , and to bioengineered microorganisms such as those created by the insertion of genetic virulence factors that produce or exacerbate disease. deliberately emerging microbes include microorganisms or toxins produced in a form that would cause maximal harm because of ease of dissemination, enhanced infectivity or heightened pathogenicity 82 . as concepts, bioterrorism and biowarfare are probably not new. the alleged catapulting of plague-ridden corpses over enemy walls in the 1346 siege of caffa (the modern crimean port of feodosia, ukraine) and the dispatch of smallpox-impregnated blankets to indians by british officers in the seven years war (1754-1763) have frequently been cited as examples of bioterrorism or biowarfare 83, 84 . two modern attacks have been well documented. in 1984, an oregon religious cult spiked restaurant salad bars with salmonellae in an attempt to sway a local election 85 . a 2001 anthrax attack 6 , in which a terrorist mailed anthrax-spore-filled letters to prominent figures, including two us senators, resulted in illness in at least 18 people and the death of five of these individuals. public alarm was elevated by the knowledge that bacillus anthracis is a common and easily obtainable enzootic and soil organism found in laboratories worldwide, and that scientific technology had increased its lethality: the spores had been weaponized by being concentrated, finely milled and packed with a dispersal agent to increase their capacity to disseminate 82 . the united states, the united kingdom, the soviet union and other nations once had sophisticated offensive bioweapons programmes that included the production of weaponized anthrax spores 82 . soviet scientists continued to produce large quantities of organisms adapted for biowarfare and bioterror -among them the agents of smallpox, plague, tularaemia and marburg virus -for several years after their signing of the bioweapons and toxins treaty convention in 1972, which forbade such activities 82 . by 1987, the soviet programme was annually producing 5,000 tonnes of weaponized anthrax spores, packing them into warheads and other delivery devices 82 . before the 2001 anthrax attacks 6 , the us scientific community had for several years been bolstering its biodefence research capacity. the anthrax attacks greatly accelerated this expansion as part of a national defence plan, which includes efforts to provide a knowledge base for the development of effective countermeasures against agents of bioterror, such as diagnostics, therapeutics and vaccines, and to translate this knowledge into the production and delivery of such measures 86 . bioterror agents have been grouped into three categories of risk 87 . the six category a agents (anthrax, smallpox, plague, tularaemia, viral haemorrhagic fevers and clostridial botulinum toxin) are given top priority because they are highly lethal and readily deployed as weapons. category b and c agents include food-borne and water-borne organisms that incapacitate but usually do not kill. infectious diseases will continue to emerge and re-emerge, leading to unpredictable epidemics and difficult challenges to public health and to microbiology and allied sciences. surveillance and response, the key elements in controlling eis, be they naturally occurring or deliberately engineered, depend on rapid clinical diagnosis and detection and containment in populations and 89 , which along with state and local health departments and other agencies have been making significant strides in national surveillance-response capacity. the enormous influx of us government-funded research resources (largely through the national institutes of health) and public health resources (mainly through the cdc, and state and local public health agencies) in response to the increased threat of a bioterrorist attack 86 will fortify the response capabilities related to all eis. however, it is clear that surveillance and other activities that traditionally fall within the domain of public health are not in themselves sufficient to adequately address the problem of eis. of critical importance are basic, translational and applied research efforts to develop advanced countermeasures such as surveillance tools, diagnostic tests, vaccines and therapeutics 86 . genomics, proteomics and advances in nanotechnology 90 are increasingly being exploited in diagnostic, therapeutic and microbial research applications, and in rational drug and vaccine design. direct and computational structural determination 91 , prediction of protein-protein interactions between microorganisms and drugs, and sophisticated bioinformatics techniques support research in all of the above areas. these technologies have led to numerous advances in real-world utility against eis, most notably in the development of more than 20 antiretroviral drugs that can effectively suppress hiv replication. where they are available and properly used in hiv-infected individuals, these medications have dramatically reduced hiv morbidity and mortality 92 . gene-and protein-based microarrays can be used to detect pathogen signals, to monitor resistance to anti-infective agents, to characterize host gene responses to recent infections, and to facilitate the development of new drugs and vaccines 93 . basic and applied research together have provided promising new vaccine platforms, such as recombinant proteins, immunogenic peptides, naked dna vaccines, viral vectors of extraneous genes encoding immunogenic proteins (including chimaeras), replicons and pseudovirions 94 . many novel vaccine candidates are now being developed against eis such as hiv, ebola virus, west nile virus, dengue, the sars coronavirus, tuberculosis and malaria. of particular note are novel tuberculosis vaccines that recently entered clinical trials -the first time in more than 60 years that new approaches to vaccination for tuberculosis have been assessed in humans 95 . chimaeric flavivirus vaccines for west nile virus, dengue and japanese encephalitis virus are effective in animal models and are in various stages of clinical testing 95 . our growing understanding of the human immune system is also helping to accelerate vaccine development. this is especially true in the case of innate immune responses, which are evolutionarily older, less specific and faster-acting than the adaptive responses that have been the traditional targets of vaccines 96 . as we learn more about innate immunity and its relationship with the adaptive immune system, opportunities to create more effective vaccine adjuvants will emerge. for example, synthetic dna sequences that contain repeated cpg motifs mimic the stimulatory activity that bacterial dna fragments exert on the innate immune system. these sequences show promise as vaccine adjuvants that accelerate and augment immune responses 97 . we can anticipate more progress of this kind as we continue to delineate the complex interactions between innate and adaptive immune responses. the sequencing of the human genome, the genomes of six other animals, including the mouse, and those of microbial vectors and microbes themselves (for example, p. falciparum and its mosquito vector, anopheles gambiae), have elevated microbiology to a wholegenome level. the ability to sequence microbial genomes in a few days 98 or less, and to examine host-vector-microbe interactions at both the genome level and at the tertiary protein structural level, will help us to understand the molecular mechanisms that underlie the pathogenesis of infectious disease and host defences, including resistance and immune evasion. these advances will facilitate the development of new countermeasures. other fertile areas of research include the use of geographical information systems 99 and satellite imaging to support field study and epidemic prevention (for example, predicting hps and rift valley fever epidemics in indigenous areas by satellite imagery of water and vegetation related to animal reservoir and vector prevalence). underlying disease emergence are evolutionary conflicts between rapidly evolving and adapting infectious agents and their slowly evolving hosts. these are fought out in the context of accelerating environmental and human behavioural alterations that provide new ecological niches into which evolving microbes can readily fit. it is essential that broadly based prevention strategies, as well as new and improved countermeasures (that is, surveillance tools, diagnostics, therapeutics and vaccines), be continually tested, refined and upgraded, requiring a strengthened relationship between public health and basic and clinical sciences. the challenge presented by the ongoing conflict between pathogenic microorganisms and man has been well summarized by a noted champion of the war on eis, joshua lederberg: "the future of microbes and mankind will probably unfold as episodes of a suspense thriller that could be entitled our wits versus their genes" 29 . the global scientific and public health communities must confront this reality not only with wit, but also with vision and sustained commitment to meet a perpetual challenge. in figure 2 of this review, the pie chart was drawn incorrectly, with the wedge sizes not in proportion to the total size. the correct figure is shown below. asthma and chronic obstructive pulmonary disease 3.0 million factors in the emergence of infectious diseases the wordsworth encyclopedia of plague and pestilence updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic centers for disease control and prevention. multistate outbreak of monkeypox -illinois the severe acute respiratory syndrome investigation of bioterrorism-related anthrax, united states, 2001: epidemiologic findings threats to global health and survival: the growing crises of tropical infectious diseases -an "unfinished" agenda emerging infectious diseases among indigenous peoples ǽșȓȗșȕșȍȓȋ -sive, pestis nuperae apud populum londinensem grassantis narratio historica epidemiology of the plague of athens the columbian exchange: biological and cultural consequences of 1492 princes and peasants. smallpox in history ch island populations of the pacific the gifts of civilization. germs and genocide in hawai'i agents of apocalypse: epidemic disease in the colonial philippines measles in fiji, 1875: thoughts on the history of emerging infectious diseases die aetiologie der milzbrand-krankheit, begründet auf die entwicklungsgeschichte des bacillus anthracis. beiträge zur biologie der pflanzen spreading germs: diseases, theories, and medical practice in britain the greatest benefit to mankind: a medical history of humanity from antiquity to the present the extermination of infectious diseases the evolution and eradication of infectious diseases infectious diseases: considerations for the 21st century the restless tide: the persistent challenge of the microbial world (national foundation for infectious diseases committee on emerging microbial threats to health. emerging infections. microbial threats to health in the united states committee on emerging microbial threats to health in the 21st century. microbial threats to health in the united states: emergence, detection and response emerging and re-emerging infectious diseases: a multidisciplinary perspective emerging and re-emerging infectious diseases infectious history emerging infectious diseases in the 21st century emerging and re-emerging infectious diseases human frontiers, environments and disease the origins of acquired immune deficiency viruses: where and when? phil population migration and the spread of types 1 and 2 human immunodeficiency viruses the aids knowledge base slowing heterosexual hiv transmission a novel hantavirus associated with an outbreak of fatal respiratory disease in the southwestern united states: evolutionary relationships to known hantaviruses nipah virus: a recently emergent deadly paramyxovirus variant creutzfeldt-jakob disease and the acquired and transmissible spongiform encephalopathies nützliche und auf die erfahrung gegründete einleitung zu der landwirthschaft identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic creutzfeldt-jakob disease scrapie and chronic wasting disease severe streptococcal infections in historical perspective emerging infections brazilian purpuric fever: evolutionary genetic relationships of the case clone of haemophilus influenzae biogroup aegyptius to encapsulated strains of haemophilus influenzae identification and characterization of genomic loci unique to the brazilian purpuric fever clonal group of h. influenzae biogroup aegyptius: functionality explored using meningococcal homology group a streptococcus: allelic variation, population genetics, and host-pathogen interactions genome sequence of a serotype m3 strain of group a streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma microbes and malignancy: infection as a cause of human cancers helicobacter pylori-associated diseases current clinical topics in infectious diseases vol el niño and health epidemiology in relation to air travel acute haemorrhagic conjunctivitis: dealing with a newly emerging disease two worlds of malaria research toward vaccines against malaria the global situation of mdr-tb staphylococcus aureus resistant to vancomycin -united states methicillin (oxacillin)-resistant staphylococcus aureus strains isolated from major food animals and their potential transmission to humans the crisis in antibiotic resistance surveillance for aids-defining opportunistic illnesses, 1992-1997. mmwr 48 (cdc surveillance summary no. ss-2) infections in patients with cancer undergoing chemotherapy: aetiology, prevention, and treatment impact of current transplantation practices on the changing epidemiology of infections in transplant recipients xenotransplantation: public health risks -patient vs. society in an emerging field the outbreak of west nile virus infection in the new york city area in 1999 outbreak of west nile infection west nile virus: epidemiology and ecology in north america dengue and dengue haemorrhagic fever antibody-dependent enhancement of infection and the pathogenesis of viral disease molecular ecology of toxigenic vibrio cholerae the next influenza pandemic: lessons from hong kong a molecular whodunit avian influenza viruses infecting humans structure of the uncleaved human h1 haemagglutinin from the extinct 1918 influenza virus the origin of the 1918 pandemic influenza virus: a continuing enigma potential impact of low-efficacy hiv-1 vaccines in populations with high rates of infection short-term projections for variant creutzfeldt-jakob disease onsets mortality associated with influenza and respiratory syncytial virus in the united states the chilling true story of the largest covert biological weapons program in the world -told from the inside by the man who ran it (random house document zur geschichte des schwarzen todes. mitgetheilt und eingeleitet smallpox and the indians in the american colonies a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars biodefence on the research agenda: the world needs new and creative ways to counter bioterrorism threats in bioterrorism: i. cdc category a agents world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) preventing emerging infectious diseases: a strategy for the 21st century (department of health and human services protein structure prediction and structural genomics guidelines for using antiretroviral agents among hiv-infected adults and adolescents new technology platforms in the development of vaccines for the future the jordan report: 20th anniversary adjuvants of immunity: harnessing innate immunity to promote adaptive immunity recent advances in the discovery and delivery of vaccine adjuvants the value of complete microbial genome sequencing (you get what you pay for) the authors thank r. m. krause for helpful discussions, and j. weddle for graphic design. the authors declare that they have no competing financial interests.insight review articles key: cord-349790-dezauioa authors: johnson, stephanie; parker, michael title: ethical challenges in pathogen sequencing: a systematic scoping review date: 2020-06-03 journal: wellcome open res doi: 10.12688/wellcomeopenres.15806.1 sha: doc_id: 349790 cord_uid: dezauioa background: going forward, the routine implementation of genomic surveillance activities and outbreak investigation is to be expected. we sought to systematically identify the emerging ethical challenges; and to systematically assess the gaps in ethical frameworks or thinking and identify where further work is needed to solve practical challenges. methods: we systematically searched indexed academic literature from pubmed, google scholar, and web of science from 2000 to april 2019 for peer-reviewed articles that substantively engaged in discussion of ethical issues in the use of pathogen genome sequencing technologies for diagnostic, surveillance and outbreak investigation. results: 28 articles were identified; nine united states, five united kingdom, five the netherlands, three canada, two switzerland, one australia, two south africa, and one italy. eight articles were specifically about the use of sequencing in hiv. eleven were not specific to a particular disease. results were organized into four themes: tensions between public and private interests; difficulties with translation from research to clinical and public health practice; the importance of community trust and support; equity and global partnerships; and the importance of context. conclusion: while pathogen sequencing has the potential to be transformative for public health, there are a number of key ethical issues that must be addressed, particularly around the conditions of use for pathogen sequence data. ethical standards should be informed by public values, and further empirical work investigating stakeholders’ views are required. development in the field should also be under-pinned by a strong commitment to values of justice, in particular global health equity. genetic information derived from pathogens is an increasingly essential input for infectious disease control, public health and research 1 . although routine sequencing of pathogens was, until recently, unthinkable, the centers for disease control (cdc), food and drug administration (fda), state, and global public health laboratories now routinely sequence more than 200 foodborne bacterial isolates a day and more than 6,000 influenza virus genomes a year 2,3 . in the united kingdom, public health england now engages in routine clinical genomic diagnostics and drug sensitivity testing for mycobacterium tuberculosis 4 . in the research setting, phylogenetic analysis (the study of evolutionary relationships among pathogens) is being used to track and understand factors associated with the spread of infections such as hiv 5 and to monitor the global spread of drug-resistant infections 6 . mobile genomic sequencing technology is also being applied to disease outbreak investigation, most publicly in the case of the ebola outbreak in west africa 7-9 . going forward, the routine implementation of genomic surveillance activities and outbreak investigation is to be expected. while the technical developments of sequencing technology are being implemented at a rapid pace, the non-technical aspects of implementing this technology are still being broadly discussed between the different stakeholders involved 1 . the successful implementation of this rapidly developing technology will, for example, require sharing of samples and metadata, interdisciplinary global collaborative partnerships, and will need to offer useful evidence for public health decision-making. importantly, the successful and appropriate response to these challenges will also require the systematic identification, analysis and addressing of a number of complex ethical, legal and social issues. a number of factors will contribute to the types of ethical issues that arise in different instances. these are likely to include characteristics of the disease, the environmental, political and geographical context, existing laws and policies, public attitudes, and cultural differences 10 . in the work reported in this paper, taking these and other ethical issues as our focus, we sought to systematically examine the available literature to: identify the emerging ethical challenges and proposed solutions; and to systematically assess the gaps in ethical frameworks or thinking and identify where further work is needed to solve practical challenges. scoping reviews seek to identify literature relevant to a research objective and may include a variety of research formats and conceptual literature [11] [12] [13] . this study sought to review published literature on ethical aspects of pathogen sequencing. inclusion criteria for the study encompassed a broad range of article types, including empirical studies, news articles, opinion pieces, features, editorials, reports of practice, and theoretical articles. we systematically searched indexed academic literature from pubmed, google scholar, and web of science from 2000 to april 2019 for peer-reviewed articles that substantively engaged in discussion of ethical issues in the use of pathogen genome sequencing technologies for diagnostic, surveillance and outbreak investigation. the search was then updated in january 2020. the initial search strategies were developed through an iterative process and used a combination of controlled vocabulary (mesh terms) and free text words. an example medline search strategy is provided in table 1 . reference lists of included articles were searched for relevant articles and further database searchers were conducted using the names of researchers commonly publishing in this field. finally, we also reviewed relevant international research and clinical practice guidance for relevant guidelines e.g. website of the world health organization. we sought to maximize the literature included in the review by reviewing guidelines, frameworks, commentaries and original research reviews related to pathogen sequencing. we also included studies on molecular typing where enough accuracy could be included to include transmission tracking, as this was thought to provide useful insights into the ethical challenges pathogen sequencing technologies may pose. we excluded studies considering genomics outside of infectious disease or focusing on host response studies as these were not deemed relevant or specific enough to the topic under investigation. duplicates were removed. sj undertook title and abstract screening to remove obviously irrelevant studies, borderline cases were discussed with mp and a decision reached by consensus. data was then abstracted by sj and cross-checked for accuracy by mp. names of study authors, institutions, journals of publication and results were non-blinded. analysis sj initially inductively analysed all studies, recording the aims and main findings in microsoft excel 2016 (16.0.4699.1000), and developing descriptive codes to chart the broad themes describing the literature base. similar findings were then grouped according to topic area and a preliminary list of themes was developed in collaboration with mp. both authors engaged in iterative discussions about organization of findings, after which the final themes were decided. sj subsequently re-examined each study and extracted data using a standard format (design, results and recommendations). the search produced 466 articles after duplicates were removed. all articles were initially screened by title and abstract and thirty-nine full text articles were assessed for eligibility. twenty eight articles were included in the final analysis; three ethical guidelines or frameworks 14-17 ; seven empirical research studies 1,18-23 ; eight reviews of the ethics 2,17-21 ; and ten publications that contained a section on the ethical aspects of pathogen sequencing 24-33 . the literature largely originated from the us and other high-income countries (hics, country determined by lead author institution): nine from the united states (us), five from the united kingdom (uk), five from the netherlands, three from canada, two from switzerland, one from australia, and one from italy. only two publications, by the same author, originated from the global south (south africa). eight articles were specifically about the use of sequencing in hiv 14, 15, 17, 20, 22, 28, 34, 35 . eleven were not specific to a particular disease. table 2 presents a summary of studies. results were organized into four themes: tensions between public and private interests; difficulties with translation from research to clinical and public health practice; the importance of community trust and support; equity and global partnerships; and the importance of context. when considering the implications of collecting, using and sharing pathogen genome sequence data, the interests and rights of individuals were universally acknowledged. in particular, the literature pointed to the importance of considering and/or protecting individual rights to autonomy 19,21,32,36 , and to privacy 18,21,22,28 . many pointed out that the potential of sequencing techniques to detect the origin and routes of transmission of an outbreak may result in negative consequences for the individuals involved 25 . consequences may include stigmatization, penalties, economic risks, problems with interpersonal relationships (e.g. inadvertent disclosure of infidelity), emotional distress and the capacity for discrimination 19, 21, 22, 24, 25, 27, 30, 32, [37] [38] [39] . there was also concern that sequencing could lead to serious legal consequences, particularly with regards to the criminalization of hiv transmission 14, 15, 17, 22, 24, 28, 34, 35 . it was also acknowledged that individuals may have an interest in avoiding the use of information about them for purposes they do not endorse, such as to support anti-gay sentiment or as part of a criminal investigation 20, 24 . somewere concerned about forced testing either of certain groups such as gay men 28 or healthcare workers 19,21,38-40 . there was acknowledgment of individual professional interests of researchersand practitioners to ownership and use of data 1, 16, 32 . sequencing was also seen to carry risks for communities and groups. many authors noted that certain groups can be placed at risk through characterization as high risk or likely to transmit virus (hiv), including geographically defined groups, sexual or gender minorities, or those defined by ethnicity, nationality, or migration status 14 . similarly, data regarding transmission patterns of multidrug-resistant tuberculosis (tb) could be used for discrimination based on ethnicity, and possible challenges to immigration 20 . institutions could be subject to increasing numbers of legal claims, or companies could suffer reputational or economic damage 39 . it was also noted that some communities may be particularly at risk of being exploited by research, especially during emergency outbreak situations 14,32 . on the other hand, it was acknowledged that widespread availability and use of sequence data contributes important benefits to the clinical and research communities 17 . in particular, the rapid sharing of data can help identify etiological factors, predict disease spread, evaluate existing and novel treatments, symptomatic care and preventive measures, and guide the deployment of there was strong support for the permissibility of conducting sequencing studies, as long as potential risks were thoughtfully mitigated 14,18,19,21,22 . in one empirical study, patients and healthcare workers were asked if the benefits of hiv molecular epidemiology outweigh the risks; all said yes 22 . threequarters of respondents answered with an unqualified, yes, and one quarter gave a positive answer with qualifications, such as 'it's very necessary, just as long as parameters are set in place and they're kept', or 'with proper protections in place, the benefits outweigh the risks' 22 . in another study, expert delphi panelists held that the protection of the public was of overriding importance, but that most of the potential harms could be managed 19 . there were differences across the literature in the priority afforded to different conditions of use, and to the types of risk or amount of risk deemed acceptable. it was broadly agreed that any research should have a favourable risk benefit ratio 14,20 , and that maximizing the utility of data must be weighed against concerns over interests of individuals and that policies on data collection and release should seek to align the interests of different parties 36 . there was, however, disagreement as to whether privacy concerns or public interest should take precedence 24,25,31 and some noted that the balance between the public health benefit and personal privacy risk for individuals whose genetic data (personal or pathogen) are included is difficult to delineate, since neither the true benefit nor the actual risk to participants has been adequately defined 27,35 . below we set out the key recommendations from the literature on the conditions of use for pathogen sequence data. box 1 summarises recommendations from the literature for future research focus and study design. it was clear that the release of information of relevance to public health should not be delayed by publication timelines or concerns over academic ownership of data 16,26,32 . recommendations to address such conflicts of interests included: that medical journals should update their policies to support pre-publication sharing of pathogen sequence data related to outbreaks 16 ; publication disclaimers prohibiting use of sequence data for publication without permission 16,32 ; acknowledgment of data sharing contributions and the inclusion of such criteria to the assessment of academic research credit 1 ; establishment of governance structures and dispute resolution mechanisms that can mediate where disagreements arise 16 . much of the literature suggested that traditional methods of de-identification or anonymization of data are insufficient to meet their purpose in the context of pathogen sequencing 2,14,17,23,28,33,39 . existing approaches to minimize the risk of privacy loss to participants are based on de-identification of data by removal of a predefined set of identifiers 17 . however, this has three key limitations in the context of pathogen genomics. first, sharing of corresponding sample metadata (minimally time and place of collection, ideally with demographic, laboratory, and clinical data) is essential to enhance the interpretation and the value of genomic data 16 , therefore removal of key identifiers such as geographic location may severely limit the utility of genomic data 39 . second, removing predefined identifiers may be ineffective at protecting privacy and confidentiality 17,19,22,39 . for example, one study demonstrated how sample collection dates associated with microbiological testing at a large tertiary hospital were highly correlated with patient admission date (protected health information), meaning data is re-identifiable 23 . small study populations may also mean that individuals who are part of a transmission chain may be able to identify others during the course of routine contact tracing (e.g. sexual partners) 17 . third, anonymization of data does little to mitigate potential risk to communities and groups 14,17,39 . perhaps a consequence there was clear support for re-visioning of existing privacy standards, and for privacy policies specific to the context of sequencing studies. there was debate in the literature around the importance of consent to the use of sequence data and associated meta-data in epidemiological investigation. in the research setting, coltart et al. 14 state that research participants and patients whose samples are being used for phylogenetic analysis should ideally have consented to such use, but suggest that when using data from previous studies, where only broad consent for hiv-related research might have been obtained, waivers of specific consent are allowable when samples are no longer linked to identifiers, or when broad consent for sample collection for research and storage in future studies was given 14 . in the public health and clinical setting, an australian study reported that one of the key differences amongst participants in a modified delphi social and behavioural research into conceptual and normative aspects should be backed up by empirical research 35 . 5. new inter-disciplinary collaborations including microbiologists, engineers and bioethicists 30 . 6. as real-time and other intervention strategies that build on hiv phylogenetic information continue to emerge, it will be critical to address questions of efficacy for cluster growth interventions to ensure that the benefits outweigh potential risks. implementation science research may also inform best practices for discussing the meaning and limitations of sequence data and cluster membership with community members and help to identify acceptable and evidence-based approaches that impose the least risk to persons within specific contexts. these might involve partnerships with providers for non-intrusive patient follow-up related to clusters, more detailed consent procedures for future follow-up related to hiv test results or partner services referrals, and specific guidelines and education to mitigate criminalization risks 28 . 7. communication methods that increase the understanding of phylogenetic studies need to be designed and evaluated. these must emphasise potential harms, thoughtful mitigation of harms to risk groups, processes for monitoring risk, and clear protection procedures to minimise risks 14 . 8. collaboration between stakeholders is necessary, with an active exchange of experiences and best practices. the first step, should be sought in creating awareness and consensus within sectors on the causal factors of barriers to sharing of sequence data 1 . ethical conduct of studies 9. need to pre-define exceptional circumstances where un-validated techniques might be used in emergency situations 27 . 10. to ensure scientific validity, researchers and their associates should be competent to implement the proposed study design. in order to maximize scientific validity, the researchers should ensure that they have all necessary resources, that the community accepts the protocol and that a competent and independent research ethics committee (rec) or institutional review board (irb) reviews and approves the protocol 35 . 11. the scientific objectives of research should guide the choice of participants and determine the inclusion criteria and appropriate recruitment strategies. it is unethical to use privilege, convenience and/or vulnerability as criteria for selecting participants. exclusion of certain population sub-groups or communities in a research study without appropriate scientific justification is also considered unethical 35 . 12. risk mitigation strategies must also provide for redress mechanisms in cases of abuse or misuse of phylogenetic data. these strategies might require the establishment of ties with local legal services, organisations working to protect people with hiv, and criminalised or stigmatised populations, to ensure that they have access to the means to protect their rights 14 . study included the necessity for consent before testing and data-linkage. no panelists agreed with the statement "under no conditions should a study be conducted without prior consent", although only ten of thirty agreed that consent is not required under any conditions 21 . in a dutch study, outbreak managers thought intervention without seeking explicit consent of all individuals involved is justified when there is at least be a substantial public health threat, realistic expectation that deploying the techniques will help to mitigate the outbreak, and that source and contact tracing would most likely not be successful without the use of molecular typing techniques 21 . there was a strong commitment to rapid and open data-sharing, particularly in emergency or outbreak situations 16,27,32 and in such conditions for incentives and safeguards to encourage rapid and unrestricted access to data release 16,27,32 . the world health organization (who) recommended that in emergency outbreak situations "the first set of sequences providing crucial information on the pathogen, genotype, lineage, and strain(s) causing the outbreak should be generated and shared as rapidly as possible. sharing of corresponding anonymised sample metadata (minimally time and place of collection, ideally with demographic, laboratory, and clinical data) is essential to enhance the interpretation and the value of genomic data" 16 . however, access to data gathered as part of clinical care was seen as ethically more contentious as "publicly accessible databases are not an appropriate storage location for the level of metadata required to enable clinical and epidemiological analysis for the purposes of providing patient and population care" 39 . suggestions were made for a tiered approach to data release, whereby a separate database governed by appropriate public health authorities would collate and store metadata in a location to which access to data could be limited to users with a legitimate clinical or public health need to use it, and data that cannot be released into public domains but is needed by authorised healthcare and public health professionals for service delivery remains within a suitable secured access database 39 . a public survey on tb explored questions related to database access and the potential benefits and risks associated with it. most felt that medical professionals and the research community should have access to such a database; and a significant proportion thought that other agencies, such as the police (10%) and immigration officials (13%), should also have access to the genomic database. experts, however, were clear that they felt transmission data should not be used in litigation; this was partially because it was deemed too unreliable 24,27 and also because of the potential for 'abuse' of data 14,15,24 . overall, there was broad support for further work in defining the conditions for collection use and storage of data and samples 2,27,30,32,37 and for policy and legal clarity to aid the ethical implementation of these technologies. this will require more work to carefully assess and understand risks 39 ; research to decide how much individual privacy might be risk in the name of public health 31 ; consideration of alternative strategies required to mitigate this risk, such as suppression of data in the public domain where it may cause serious harm; and adjustments to communication plans 14 . effective phylogenetic work often occurs at the interface between research and public health practice because the same data can be used for both purposes 14 . in this regard, pathogen sequencing was described as 'straddling the boundary between research and clinical use' 27 . the hybrid nature of sequencing activities imposes important ethical challenges. clinical implementation of metagenomics sequencing (un-targeted testing) has the potential to detect unexpected or incidental findings that may include infections with hepatitis or hiv 39 . incidental findings of a different type may occur if non-germline samples (such as faecal samples) are contaminated with germline cells, which could potentially reveal predictive information about developing inherited disease 39 . furthermore, informed consent for phylogenetic studies that are difficult complex and difficult to understand, and in which the benefits and risk may not be fully determined, may also be difficult to achieve 35 . mutenherwa suggests that where sequence data are generated for routine clinical management, its subsequent use for research and surveillance may be underestimated by patients 20 . others suggested that the right to withdraw from research activities-a key indicator of voluntary participation in research-was overlooked by expert stakeholders 20 . understanding and interpreting phylogenetic data requires significant expertise 25,27 and presents a challenge to established professional boundaries. expertise in phylogenetic studies creates new obligations for researchers, such as deciding whether or not to participate in forensic investigations and potential prosecutions of individuals 24 , and to consider the down-stream uses and misuses of data 14 . the routine implementation of pathogen sequencing studies may create new responsibilities for clinical microbiologists (related to public health) 2 , and require major changes in culture such that diagnostic interpretation, therapeutic management decisions and antimicrobial treatment regimes are delegated to physicians instead of microbiologists 31 . many noted that there are important reasons to ensure that the public and individuals understand the uses of data collected as part of a sequencing studies, and the potential risks. first, this was seen to have some intrinsic value in that it supports patient autonomy and truth telling is a respected moral virtue 14,15,19,21 . second, truth-telling was seen to be important because it may lead to better outcomes in research and public health practice. this is both because this was deemed to promote trust in research and therefore lead to increased participation, and because it promotes disclosure, which is helpful from a public health perspective. third, promoting understanding of uses and risks of data was also seen as a way of avoiding harm and exploitation of vulnerable individuals and communities, by enhancing understanding of risks that may be specific to that them. in some cases, this was balanced by a number of practical challenges to telling people the truth, such as: risk of fear mongering 19 ; information needed for legal proceedings in public interest 14 ; and the fact that it may be difficult to adequately inform the public and/or ensure full understanding 20 . none-the-less, there was a clear recommendation in the literature to raise public awareness and understanding of these techniques 19,22,30 , and for early and meaningful community engagement 15,22,32,34 prior to conducting sequencing studies. this was seen as particularly important when working with vulnerable groups or when the risks of participation are high. the notion of justice appeared to be a widely recognized ethical principle in the field 1 . stakeholders pointed to the importance of equitable access to data 27 , and to benefit-sharing obligations 16,26 . this included an ethical imperative that outbreak related research and countermeasures, such as diagnostics and vaccines, should be accessible to all affected countries 26 , and towards reciprocal arrangements such that countries that participate in sequencing activities should derive some corresponding local benefit 27 . collaboration between researchers from africa and hics was raised as an important ethical consideration 20 . in one empirical study, interviewees were concerned that african researchers were not meaningfully engaged in the scientific research process in health research in general and phylogenetic research in particular, and that for equitable and mutually beneficial collaborative research partnerships to be realized, local researchers were encouraged to take leading and active roles throughout the research process 20 . this type of collaborative research practice was supported elsewhere in the literature 14,27,37 . for some, this was to enhance equity as well as to help maximize the utility of data and lead to better public health outcomes. it was argued that local researchers were more likely to understand their health care and research systems and study results were more likely to be easily translated into policy 20 , and that context specific responses to particular outbreaks were likely to be required. recommendations were made to conduct studies exploring the nature of existing collaborative partnerships between researchers from low-and middle-income countries (lmics) and hics to explore team composition and distribution of roles, including contribution to intellectual property 20 . outbreak related research and countermeasures, such as diagnostics and vaccines, must be accessible to all affected countries not only as a legal obligation, but also as an ethical imperative 26 . it was also noted that global and interdisciplinary partnerships are a necessary component of an effective genomic informed response to infectious disease. this was because of the vast range of stakeholders and varying interests involved in control of infectious disease outbreaks, and because issues may resist simple resolution and span multiple jurisdictions 27 . for example, conflict may result from governments wishing to keep an outbreak quiet and/or from the tension between lmics with few resources for generating and using data and the researchers or response teams from better-resourced settings 1,37 . ownership of samples and data was seen as an important barrier to global cooperation. the nagoya protocol (np), for example, was developed to facilitate access to genetic resources and the fair and equitable sharing of benefits arising from their utilization 1 . nevertheless, despite the importance of reinforcing sovereignty rights of states over genetic resources in their territory, uncertainties about intellectual property rights and the resulting disputes hamper access to samples 1,32 . ribeiro et al. 1 explain that: "the real or perceived possibilities for the commercial valorization of microbial genetic resources (mgr) has enforced their appropriation for further use in research, innovation and product development. the problem for public health surveillance occurs when such appropriation is triggered at initial (upstream) phases of the research and innovation cycle, such as sampling and sequencing of microorganisms, instead of later stages, such as the actual product development (in this case drugs, diagnostics and vaccines). as such, stakeholders are reluctant to share their (intangible) assets even in early phases of the innovation process, decreasing the scope of innovation efforts due to the lack of access to upstream research inputs." the same authors suggest that standardized and simplified sharing agreements 26 , and collaboration between stakeholders with an active exchange of experiences and best practices 1 are required. in general, recommendations were made for a global approach to ethics, policy and legal frameworks 16,27,29,32,37 . for example, it was suggested global data sharing arrangements should include "a global data governance or ethical framework, supplemented by local memoranda of understanding that take into account the local context" 29 ; or to investigate how the global alliance for genomics and health (ga4gh) framework for responsible data-sharing could be adapted for digital pathogen surveillance 27 . lastly, it was clear that the types of ethical issues likely to arise are in part dependent upon the contexts in which studies are conducted, as well as the nature of the pathogen under study. chiefly, information that may impact on interpersonal relationships was viewed as particularly sensitive and therefore, worthy of additional ethical reflection. examples included: sexually transmitted infections 14,20 ; consent requirements to use isolates collected from dead neonates for the purposes of epidemiological research 19 ; and disclosure of family members as the source of infection 38 . it was suggested that the balance of risks to patients and public health benefits is likely to be affected by the characteristics of the pathogen, in terms of likely morbidity and mortality: infectivity; treatability and drug resistance 39 . stakeholders also suggested that the ethical permissibility of sharing data about, particularly with regards to the source of transmission, may be different in professional contexts, where healthcare providers or companies are seen to carry a responsibility to control risk, as opposed to outside of professional contexts where protecting individuals from 'naming and shaming' may be of greater concern 21 . it was also noted that the legal and regulatory structures in which studies are conducted may also influence the implementation and ethics of conducting pathogen sequencing studies. in particular, use of phylogenetic analyses in criminal convictions was raised as an ethical risk 14,17,24 . although quality assessment of all included materials is desirable in systematic reviews, it was not possible in this case due to the inclusion of a diverse range of research formats and literature, such as commentaries and ethics guidelines. a second limitation of this review is that a large proportion of the literature included related to phylogenetic and hiv specifically (8 out of 28), meaning that the issues relevant to this context may be over-represented. this review highlights that while pathogen sequencing has the potential to be transformative for public health and clinical practice and to bring about important health benefits, there are a number of key ethical issues that must be addressed. in particular, there was clear support in the literature for innovative and critical thinking around the conditions of use for pathogen sequence data. this includes context specific standards of practice for consent, data collection, use and sharing. these practices should be informed by public values, and further empirical work investigating stakeholders' views are required. this should include experts in pathogen sequencing, patients and the general public, as well as end users such as public health professionals and clinicians. lastly, it is both a scientific and an ethical imperative that development in the field is under-pinned by a strong commitment to values of justice, in particular global health equity. all data underlying the results are available as part of the article and no additional source data are required. this manuscript presents the results of a scoping review of the literature on the topic of the ethical issues in the use of pathogen genome sequencing technologies. the authors make excellent use of the scoping review methodology which they implemented clearly (good use of tables!) and rigorously. there are minor formatting issues and typos: p. 4 under theme 1: somewere concerned about forced testing either of certain groups such as gay men 28 or healthcare workers 19,21,38-40 . there was acknowledgment of individual professional interests of researchersand practitioners to ownership and use of data. p. 9 under theme 3: 'that may be specific to that them' the main substantial issue is the substantial focus of the manuscript on the hiv context (8 articles out of 28) which the authors acknowledge. hiv is a particularly stigmatized, serious condition, with lifelong consequences for patients which is not the case for many other communicable diseases. the problem is that in the past few months, covid-19 has been a game changer in the field. covid-19 is both much more contagious but less stigmatizing and, for most people, less dangerous than hiv. considering that the last update to this scoping review was made in january 2020, none of the covid-19 emerging literature was considered. the result, which is not the fault of the authors, is that the article will only have a limited relevance to the current global pandemic. given the importance of covid to the field and beyond, it could be worth it for the author to take the time to update their research accounting for very recent developments before publishing. otherwise, the publication maybe perceived as already outdated and not garner much attention from readers. a second issue of the manuscript is that while it acknowledges the tension and blurry demarcation between the research and the public health context, it doesn't really provide any solution in this regard. for example, in the context of pathogen genome sequencing for outbreak surveillance during a public health emergency, informed consent is often not required. however, the lack of consent can create issue later for data sharing with the research community. such a scenario is not really discussed in the manuscript. similarly, the impact of the public health vs. research situation on the potential requirement for ethics review is not discussed. perhaps this was not touched upon in the literature, but it is certainly a preoccupation of researchers in the covid context. is the study design appropriate and is the work technically sound? yes are all the source data underlying the results available to ensure full reproducibility? yes are the conclusions drawn adequately supported by the results? yes in the paper in the review appear to be appropriate and informative. some additional concepts that weren't included, possibly because the article presents a review of already published ideas, are the following: the potential for civil legal consequences from publication of genomic data. the article discusses at a few different points the potential for criminal prosecution, but publication of data could also open up participants of a study to civil penalties as well. how ownership rights over microorganisms affect infectious disease control and innovation: a rootcause analysis of barriers to data sharing as experienced by key stakeholders pubmed abstract | publisher full text | free full text societal implications of the internet of pathogens reviewer expertise: ethical, legal and social issues of genomic research, medical law, bioethics. reviewer report 06 july 2020 https://doi.org/10.21956/wellcomeopenres.17333.r39365 © 2020 armstrong g. this is an open access peer review report distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. office of advanced molecular detection, national center for emerging and zoonotic infectious diseases, centers for disease control and prevention, atlanta, ga, usa the article is generally well written, although it would be stronger if it had a more concise, more focused set of recommendations for readers to act on. the focus of the paper is mostly on the use of microbial genomic data for research but also touches a little on the issues around public health use of the data. this reviewer is not an expert in ethics; that said, the ethical concepts brought out ○ the potential for violation of a privacy when cases are rare. for example, early in an outbreak, as is highlighted in the article, there is an urgency to making sequence data from the pathogen in question public. however, it's common early in an outbreak for the media to discover and publish the names of early cases. in that situation (which is relatively common), there's a risk that sequence data made public could be linked to a specific person. ○ one concern that is often under-appreciated is that, under certain circumstances, a researcher or public health agency might be legally compelled to release the identity of someone. in general, public health laws are quite strong in shielding public health data, but that may not be the case with research data. if there is uncertainty about whether such data are protected, any participants in research should be notified. ○ gh4ge is mentioned in the manuscript, but there's no mention of pha4ge ( https://pha4ge.github.io/), which is much more applicable here. ○ issues around the nagoya protocol are addressed, but the tension between gisaid and insdc is not. gisaid provides protections for intellectual property rights that the insdc members do not, and such protections are key for participation of lmics. however, where public funds are used to obtain data, there is a strong argument that the data should be made publicly available (i.e., through insdc) without restriction as long as privacy and confidentiality are not placed at risk. the tension between the two models has been particularly strong with the advent of covid-19, and the very assertive push by gisaid to prevent researchers from submitting to insdc or from citing it. ○ some (minor) specific issues that need to be addressed: i would remove the reference to microsoft excel. there's a strong argument for including information about statistical software, but which spreadsheet is used is not particularly relevant.○ "theme 1: …": there are typos in the first paragraph.○ box 1, item 5: this is not a complete, declarative sentence like the others.○ reference 29 appears to be the incorrect reference. also, the quote from it (pages 9-10) appears to be a quote from a separate document (that should be cited separately, if still available). is the study design appropriate and is the work technically sound? if applicable, is the statistical analysis and its interpretation appropriate? are all the source data underlying the results available to ensure full reproducibility?no source data required competing interests: no competing interests were disclosed.reviewer expertise: pathogen genomics to support public health.i confirm that i have read this submission and believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. key: cord-325300-wawui0fd authors: tulchinsky, theodore h.; varavikova, elena a. title: 4 communicable diseases date: 2000-12-31 journal: the new public health doi: 10.1016/b978-012703350-1/50006-1 sha: doc_id: 325300 cord_uid: wawui0fd publisher summary in a world of rapid international transport and contact between populations, systems are needed to monitor the potential explosive spread of pathogens that may be transferred from their normal habitat. the potential for the international spread of new or reinvigorated infectious diseases constitute threat to mankind akin to ecological and other man-made disasters. public health has addressed the issues of communicable disease as one of its key issues in protecting individual and population health. methods of intervention include classic public health through sanitation, immunization, and well beyond that into nutrition, education, case finding, and treatment, and changing human behavior. the knowledge, attitudes, beliefs, and practices of policy makers, health care providers, and parents is as important in the success of communicable disease control as are the technology available and methods of financing health systems. together, these encompass the broad programmatic approach of the new public health to control of communicable diseases. important for all health providers and public health personnel so as to be able to cope with the scale of these problems and to absorb new technologies as they emerge from scientific advances and experience, and their successful application. lived. it was to be observed, indeed, that it did not come straight on toward us; for the city, that is to say within the walls, was indifferently healthy still; nor was it got over the water into southwark; for though there died that week 1,268 of all distempers, whereof it might be supposed above 900 died of the plague, yet there was but 28 in southwark, lambeth parish included; whereas in the parishes of st. giles the agent-host-environment triad, discussed in chapter 2, is fundamental to the success of understanding transmission of infectious diseases and their control, including those well known, those changing their patterns, and those newly emerging or escaping current methods of control. infection occurs when the organism successfully invades the host body, where it multiplies and produces an illness. a host is a person or other living animal, including birds and arthropods, who provides a place for growth and sustenance to an infectious agent under natural, as opposed to experimental, conditions. some organisms, such as protozoa or helminths, may pass successive stages of their life cycle in different hosts, but the primary or definitive host is the one in which the organism passes its sexual stage. the secondary or intermediate host is where the parasite passes the larval or asexual stage. a transport host is a carrier in which the organism remains alive, but does not develop. an agent of an infectious disease is necessary, but not always sufficient to cause a disease or disorder. the infective dose is the quantity of the organism needed to cause clinical disease. a disease may have a single agent as a cause, or it may occur as a result of the agent in company with contributory factors, whose presence is also essential for the development of the disease. a disease may be present in an infected person in a dormant form such as tuberculosis, or a subclinical form, such as poliomyelitis or hiv. the virulence or pathogenicity of an infective agent is the capacity of an infectious agent to enter the host, replicate, damage tissue, and cause disease in an exposed and susceptible host. virulence is indicated by the severity of clinical disease and case fatality rates. the environment provides a reservoir for the organism, and the mode of transmission, by which the organism reaches a new host. the reservoir is the natural habitat where an infectious agent lives and multiplies, from which it can be transmitted directly or indirectly to a new host. the reservoir refers to the natural habitat of the organism, which may be in people, animals, arthropods, plants, soil, or substances in which an organism normally lives and multiplies, and on which it depends for survival or in which it survives in a dormant form. contacts are persons or animals who have been in association with an infected person, animal, or contaminated inanimate object, or environment that might provide an opportunity for acquiring the infective agent. persons or animals that harbor a specific infectious agent, often in the absence of discernible clinical disease, and who serve as a source of infection or contamination of food, water, or other materials, are carriers. a carrier may have an inapparent infection (a healthy cartier) or may be in the incubation or convalescent stage of the infection. communicable diseases may be classified by a variety of methods: by organism, by mode of transmission, by methods of prevention (e.g., vaccine preventable, vector controllable), or by major organism classification, that is, viral, bacterial, and parasitic disease. a virus is a nucleic acid molecule (rna or dna) encapsulated in a protein coat or capsid. the virus is not a complete cell and can only replicate inside a complete cell. the capsid may have a protective envelope of a lipid containing membrane. the capsid and membrane facilitate attachment and penetration of a host cell. inside the host cell, the nucleic molecule may cause the cell's chromosomes to be changed in its own genetic material or so that there is cellular manufacture and virus replication. viroids are smaller rna structures without capsids which can cause plant disease. prions are recently discovered (stanley prusiner, nobel prize, 1997) variants of viruses or viroids which are the infective agents cause of scrapie in sheep, and similar degenerative central nervous system diseases in cattle and in man (mad cow disease or creutzfeld-jakob disease in humans). bacteria are unicellular organisms that reproduce sexually or asexually, grow on cell-free media, and can exist in an environment with oxygen (aerobic) or in one lacking oxygen (anaerobic). some may enter a dormant state and form spores where they are protected from the environment and may remain viable for years. bacteria include a nucleus of chromosomal dna material within a membrane surrounded by cytoplasm, itself enclosed by the cellular membrane. bacteria are often characterized by their coloration under gram's stain, as gram-negative or gram-positive, as well as by their microscopic morphology, colony patterns on growth media, by the diseases they may cause, as well as by antibody and molecular (dna) marking techniques. bacteria include both indigenous flora (normal resident) bacteria and pathogenic (disease causing) bacteria. pathogenic bacteria cause disease by invading, overcoming natural or acquired resistance, and multiplying in the body. bacteria may produce a toxin or poison that can affect a body site distant from where the bacterial replication occurs, such as in tetanus. bacteria may also initiate an excessive immune response, producing damage to other body tissues away from the site of infection, e.g;, acute rheumatic fever and glomerulonephritis. parasitology studies protozoa, helminths, and arthropods that live within, on, or at the expense of a host. these include oxygen-producing, flagellate, unicellular organisms such as giardia and trichomonas, and amoebas such as entamoeba important in enteric and gynecologic disorders. sporozoa are parasites with complex life cycles in different hosts, such as cryptosporidium or malarial parasites. parasitic disease usually refers to infestation, with fungi, molds, and yeasts that can affect humans. helminths are worms that infest humans especially in poor sanitation and tropical areas. transmission of diseases is by the spread of an infectious agent from a source or reservoir to a person (table 4 .1). direct transmission from one host to another occurs during touching, biting, kissing, sexual intercourse, and projection via droplets, as in sneezing, coughing, or spitting, or by entry through the skin. indirect transmission includes via aerosols of long-lasting suspended particles in air, fecal-oral transmission such as food and waterborne as well as by poor hygenic conditions with inanimate materials, such as soiled clothes, handkerchiefs, toys, or other objects. vector-borne diseases are transmitted via crawling or flying insects, in some cases with multiplication, and development of the organism in the vector, as in malaria. the subsequent transmission to humans is by injection of salivary gland fluid during biting, e.g., congenital syphilis, or by deposition of feces, urine or other material capable of penetrating the skin through a bite wound or other trauma. transmission may occur with insects as a transport mechanism, as in salmonella on the legs of a housefly. airborne transmission occurs inderectly via infective organisms in small aerosols that may remain suspended for long periods of time and which easily enter the respiratory tract. small particles of dust may spread organisms from soil, clothing, or bedding. vertical transmission occurs from one generation to another, or from one stage of the insect life cycle to another stage. maternal-infant transmission occurs during pregnancy (transplacental), delivery, as in gonorrhoea, breast-feeding, e.g., hiv, with transfer of infectious agents from mother to fetus or newborn. resistance to infectious diseases is related to many host and environmental factors, including age, sex, pregnancy, nutrition, trauma, fatigue, living and socioeconomic conditions, and emotional status. good nutritional status has a protective effect against the results of an infection. vitamin a supplements reduce complication rates of measles and enteric infections. tuberculosis may be present in an individual whose resistance is sufficient to prevent clinical disease, but the infected person is a cartier of an organism which can be transmitted to another or cause clinical disease if the person's susceptibility is reduced. immunity is resistance to infection resulting from presence of antibodies or cells that specifically act on the microorganism associated with a specific disease or toxin. immunity to a specific organism can be acquired by having the disease, that is, natural immunity, or by immunization, active or passive, or by protection box 4.3 vaccines and prevention "the greeks had two gods of health, aesculapius and hygeia, therapy and prevention, respectively. medicine in the twentieth century retains those two concepts, and vaccination is a powerful means of prevention. what follows is information on the vaccines that together with sanitation, make modem society possible, and that if wisely used will continue to bestow on mankind the gift of prevention, which according to proverb is worth far more than cure." source: plotkin, s. a., mortimer, e. a. 1994 . vaccines. second edition. philadelphia: wb saunders (with permission). infectious agent: a pathogenic organism (e.g., virus, bacteria, rickettsia, fungus, protozoa, or helminth) capable of producing infection or an infectious disease. infection: the process of entry, development, and proliferation of an infectious agent in the body tissue of a living organism (human, animal, or plant) overcoming body defense mechanisms, resulting in an inapparent or clinically manifest disease. antigen: a substance (e.g., protein, polysaccharide) capable of inducing specific response mechanisms in the body. an antigen may be introduced into the body by invasion of an infectious agent, by immunization, inhalation, ingestion, or through the skin, wounds, or via transplantation. antibody: a protein molecule formed by the body in response to a foreign substance (an antigen) or acquired by passive transfer. antibodies bind to the specific antigen that elicits its production, causing the infective agent to be susceptible to immune defense mechanisms against infections e.g., humoral and cellular. immunoglobulins: antibodies that meet different types of antigenic challenges. they are present in blood or other body fluids, and can cross from a mother to fetus in utero, providing protection during part of the first year of life. there are five major classes (igg, igm, iga, igd, and ige) and subclasses based on molecular weight. anfisera or antitoxin: materials prepared in animals for use in passive immunization against infection or toxins. source: jawetz, melrick, and adelberg, medical microbiology, 1998. through elimination of circulation of the organism in the community. immunity may be by antibodies produced by the host body or transferred from externally produced antibodies. the body also reacts to infective antigens by cellular responses, including those that directly defend against invading organisms and other cells which produce antibodies. the immune response is the resistance of a body to specific infectious organisms or their toxins provided by a complex interaction of antibodies and cells including a. b cells (bone marrow and spleen) produce antibodies which circulate in the blood, i.e., humoral immunity; b. t cell-mediated immunity is provided by sensitization of lymphocytes of thymus origin to mature into cytotoxic cells capable of destroying virusinfected or foreign cells; c. complement, a humoral response which causes lysis of foreign cells; d. phagocytosis, a cellular mechanism which ingests foreign microorganisms (macrophages and leukocytes). surveillance of disease is the continuous scrutiny of all aspects of occurrence and spread of disease pertinent to effective control of that disease. maintaining ongoing surveillance is one of the basic duties of a public health system, and is vital to the control of communicable disease, providing the essential data for tracking of disease, planning interventions, and responding to future disease challenges. surveillance of infectious disease incidence relies on reports of notifiable diseases by physicians, supplemented by individual and summary reports of public health laboratories. such a system must concern itself with the completeness and quality of reporting and potential errors and artifacts. quality is maintained by seeking clinical and laboratory support to confirm first reports. completeness, rapidity, and quality of reporting by physicians and laboratories should be emphasized in undergraduate and postgraduate medical education. enforcement of legal sanctions may be needed where standards are not met. surveillance of infectious diseases includes the following: 1. morbidity reports from clinics to public health offices; 2. mortality reports from attending doctors to vital records; 3. reports from selected sentinel centers; 4. special field investigations of epidemics or individual cases; 5. laboratory monitoring of infectious agents in population samples; 6. data on supply, use, and side effects of vaccines, toxoids, immune globulins; 7. data on vector control activities such as insecticides use; 8. immunity levels in samples of the population at risk; 9. review of current literature on the disease; 10. epidemiologic and clinical reports from other jurisdictions. epidemiologic monitoring based on individual and aggregated reports of infectious diseases provide data vital to planning interventions at the community level or for the individually exposed patient and his contacts, along with other information sources such as hospital discharge data and monitoring of sentinel centers. these may be specific medical or community sites that are representative of the population and are able to provide good levels of reporting to monitor an area or population group. a sentinel center can be a pediatric practice site, a hospital emergency room, or other location which will provide a "finger on the pulse" to assess the degree and kind of morbidity occurring in the community. it can also include monitoring in a location previously known for disease transmission, such as hong kong in relation to influenza. epidemiologic analysis provided by government public health agencies should be published weekly, monthly, and annually and distributed to a wide audience of public health and health-related professionals throughout the country. feedback to those in the field on whose initial reports the data are based is vital in order to promote involvement and improved quality of data, as well as to allow evaluation of the local situation in comparison to other areas. in a federal system of government, national agencies report regularly on all state or provincial health patterns. state or provincial health authorities provide data to the counties and cities in their jurisdictions. such data should also be readily available to researchers in other government agencies, universities, and other academic settings for further research and analysis both on internet and hard-copy publications. notifiable diseases are those which a physician is legally required to report to state or local public health officials, by reason of their contagiousness, severity, frequency, or other public health importance (table 4 .2). public health laboratory services provide validation of clinical and epidemiologic reports. they also provide day-to-day supervision of public health conditions, and can monitor communicable disease and vaccine efficacy and coverage. in addition, they support standards of clinical laboratories in biochemistry, microbiology, and genetic screening. nosocomial or hospital-acquired infections constitute a major health hazard associated with care in institutions. in the united states, they occur in 5-10% of hospital admissions and are the cause of lengthening of hospital stay and an estimated 30,000 deaths per year. in developing countries, nosocomial infection rates may occur in up to 65% of hospitalizations. this category of infectious disease most commonly includes infections of the urinary tract, surgical wounds, lower respiratory tract (pneumonias), and blood poisoning or septicemias. in the united states, up to 60% of hospital-acquired infections are caused by multidrug resistant organisms. staphylococcus infections resistant to many current antibiotics, for example, methicillin and vancomycin, are a notable cause of prolongation of hospitalization or even death. the increasing number of immunodeficient patients has increased the importance of prevention of nosocomial infections. where standards of infection control are lacking, in both developed and developing countries, hospital staff are vulnerable to serious infection. in developing countries, deadly new viruses, such as ebola and marburg viruses mainly affect nursing, medical, and other staff as secondary cases. surveillance and control measures are important elements of hospital management. hospital epidemiologists and infection control staff are part of modem hospital staffing. the cost to the health system of nosocomial infections is a major consideration in planning health budgets. reducing the risk of acquiring such infections in hospital justifies substantial expenditures for hospital epidemiology and infection control activities. with diagnostic related group payment for hospital care (by diagnosis rather than by days of stay) the good manager has a major incentive to ensure that the risk of nosocomial infections is minimized, since they can greatly prolong hospital stays, raising patient dissatisfaction and health care costs. an endemic disease is the constant usual presence of a disease or infectious agent in a given geographic area or population group. hyperendemic is a state of persistence of high levels of incidence of the disease. holoendemic means that the disease appears early in life and affects most of the population, as in malaria or hepatitis a and b in some regions. an epidemic is the occurrence in a community or region of a number of cases of an illness in excess of the usual or expected number of cases. the number of cases constituting an epidemic varies with the disease, and factors such as previous epidemiological patterns of the disease, time and place of the occurrence, and the population involved must be taken into account. a single case of a disease long absent from an area, such as polio, constitutes an epidemic, and therefore a public health emergency because a clinical case may represent a hundred carriers with nonparalytic or subclinical poliomyelitis. in the 1990s, two to three or more cases of measles linked in time and place may be considered sufficient evidence of transmission and presumed to be an epidemic. a pandemic is occurrence of a disease over a very wide area, crossing international boundaries, affecting a large proportion of the population. each epidemic should be regarded as a unique natural experiment. the investigation of an epidemic requires preparation and field investigation in conjunction with local health and other relevant authorities. verification of cases and the scope of the epidemic will require case definition and laboratory confirmation. tabulation of known cases according to time, place, and person are important for immediate control measures and formulation of the hypothesis as to the nature of the epidemic. an epidemic curve is a graphic plotting of the distribution of cases by the time of onset or reporting, which gives a picture of the timing, spread, and extent of the disease from the time of the initial index cases and the secondary spread. epidemic investigation requires a series of steps. this starts with confirmation of the initial report and preliminary investigation, defining who is affected, determining the nature of the illness and confirming the clinical diagnosis, and recording when and where the first (index) and follow-up (secondary) cases occurred, and how the disease was transmitted. samples are taken from index case patients (e.g., blood, feces, throat swabs) as well as from possible vectors (e.g., food, water, sewage, environment). a working hypothesis is established based on the first findings, taking into account all plausible explanations. the epidemic pattern is studied, establishing common source or risk factors, such as food, water, contact, environment, and drawing a time line of cases to define the epidemic curve. how many are ill (the numerator) and what is the population at risk (the denominator) establish the attack rate, namely, the percentage of sick among those exposed to the common factor. what is a reasonable explanation of the occurrence; is there a previous pattern, with the present episode a recurrence or new event? consultation with colleagues and the literature helps to establish both a biological and epidemiologic plausibility. what steps are needed to prevent spread and recurrence of the disease? coordination with relevant health and other officials and providers is required to establish surveillance and control systems, document and distribute reports, and respond to the public's fight to know. the first reports of excess cases may come from a medical clinic or hospital. the initial (sentinel or index) cases provide the first clues that may point to a common source. investigation of an epidemic is designed to quickly elucidate the cause and points of potential intervention to stop its continuation. this requires skilled investigation and interpretation. epidemiologic investigations have defined many public health problems. rubella syndrome, legionnaire's disease, aids, and lyme and hantavirus diseases were first identified clinically when unusually large numbers of cases appeared with common features. the suspicions that were raised led to a search for causes and the identification of control methods. a working hypothesis of the nature of an epidemic is developed based on the initial assessment, the type of presentation, the condition involved, and previous local, regional, national, and international experience. the hypothesis provides the basis for further investigation, control measures, and planning additional clinical and laboratory studies. surveillance will then monitor the effectiveness of control measures. communication of findings to local, regional, national, and international health reporting systems is important for sharing the knowledge with other potential support groups or other areas where similar epidemics may occur. the centers for disease control and prevention (cdc), originally organized in 1942 as the office for malaria control in war areas, is part of the u.s. public health service. as of 1993, the cdc had a budget of $1.5 billion, and 7300 employees include epidemiologists, microbiologists, and many other professionals. the cdc includes national centers for environmental health and injury control, chronic disease prevention and health promotion, infectious diseases, prevention services, health statistics, occupational safety and health, and international health. the epidemic intelligence service (eis) of the cdc in the united states is an excellent model for the organization of the national control of communicable diseases. young clinicians are trained to carry out epidemiologic investigations as part of training to become public health professionals. eis officers are assigned to state health departments, other public health units, and research centers as part of their training, carrying out epidemic investigation and special tasks in disease control. the cdc, in cooperation with the who, has developed and offers free of charge, a personal computer program to support field epidemiology, including epidemic investigations (epi-info), which can be accessed and down-loaded from the worldwide web. this program should be adopted widely in order to improve field investigations, to encourage reporting in real time, and to develop high standards in this discipline. 1 cdc's morbidity and mortality weekly report (mmwr) is a weekly publication of the cdc's epidemiologic data, also available free on the internet. it includes special summaries of reportable infectious diseases as well as noncom although an infectious disease is an event affecting an individual, it is communicable to others, and therefore its control requires both individual and community measures of protection. control of the disease is a reduction in its incidence, prevalence, morbidity, and mortality. elimination of a disease in a specified geographic area may be achieved as a result of intervention programs such as individual protection against tetanus; elimination of infections such as measles requires stoppage of circulation of the organism. eradication is success in reduction to zero of incidence of the disease and presence in nature of the organism, such as with smallpox. extinction means that a specific organism no longer exists in nature or in laboratories. public health applies a wide variety of tools for the prevention of infectious diseases and their transmission. it includes activities ranging from filtration and disinfection of community drinking water to environmental vector control, pasteurization of milk, and immunization programs (see table 4 .3). no less important are organized programs to promote self protection, case finding, and effective treatment of infections to stop their spread to other susceptible persons (e.g., hiv, sexually transmitted diseases, tuberculosis, malaria). planning measures to control and eradicate specific communicable diseases is one of the principal activities of public health and remains so for the twenty-first century. treating an infection once it has occurred is vital to the control of a communicable disease. each person infected may become a vector and continue the chain of transmission. successful treatment of the infected person reduces the potential for an uninfected contact person to acquire the infection. bacteriostatic agents or drugs such as sulfonamides inhibit growth or stop replication of the organism, allowing normal body defenses to overcome the organism. bacteriocidal drugs such as penicillin act to kill pathogenic organisms. traditional medical emphasis on single antibiotics has changed to use of multiple drug combinations for tuberculosis and more recently for hospital-acquired infections. antibiotics have made enormous contributions to clinical medicine and public health. however, pathogenic organisms are able to adapt or mutate and develop resistance to antibiotics, resulting in drug resistance. wide-scale use of antibiotics has led to increasing incidence of resistant organisms. multidrug resistance constitutes one of the major public health challenges at the end of the twentieth century. antiviral agents (e.g., ribovarin) are important additions to medical treatment potential, as are "cocktails" of antiviral agents for management of hiv infection. antibiotic use is a health problem requiting attention of clinicians and their teachers as well as the public health community and health care managers, representing the interaction of health issues across the entire spectrum of services. organized public health services are responsible for advocating legislation and for regulating and monitoring programs to prevent infectious disease occurrence and/or spread. they function to educate the population in measures to reduce or prevent the spread of disease. health promotion is one of the most essential instruments of infectious disease control. it promotes compliance and community support of preventive measures. these include personal hygiene and safe handling of water, milk, and food supplies. in sexually transmitted diseases, health education is the major method of prevention. each of the infectious diseases or groups of infectious diseases have one or more preventive or control approaches (table 4 .3). these may involve the coordinated intervention of different disciplines and modalities, including epidemiologic monitoring, laboratory confirmation, environmental measures, immunization, and health education. this requires teamwork and organized collaboration. very great progress has been made in infectious disease control by clinical, public health, and societal means since 1900 in the industrialized countries and since the 1970s in the developing world. this is attributable to a variety of factors, including organized public health services; the rapid development and wide use of new and improved vaccines and antibiotics; better access to health care; and improved sanitation, living conditions, and nutrition. triumphs have been achieved in the eradication of smallpox and in the increasing control of other vaccine-preventable diseases. however, there remain serious problems with tb, stds, malaria, and new infections such as hiv, and an increase in multiple drug-resistant organisms. vaccines are one of the most important tools of public health in the control of infectious diseases, especially for child health. vaccine-preventable diseases ta b l e 4.4 annual incidence of selected vaccine-preventable infectious diseases in rates per 100,000 population selected years, united states, 1950 -1996 disease 1950 1960 1970 1980 1985 1996 the body responds to invasion of disease-causing organisms by antigenantibody reactions and cellular responses. together, these act to restrain or destroy the disease-causing potential. strengthening this defense mechanism through im-box 4.5 definitions of immunizing agents and processes vaccines: a suspension of live or killed microorganisms or antigenic portion of those agents presented to a potential host to induce immunity to prevent the specific disease caused by that organism. preparation of vaccines may be from: a. live attenuated organisms which have been passed repeatedly in tissue culture or chick embryos so that they have lost their capacity to cause disease but retain an ability to induce antibody response, such as polio-sabin, measles, rubella, mumps, yellow fever, bcg, typhoid, and plague. b. inactivated or killed organisms which have been killed by heat or chemicals but retain an ability to induce antibody response; they are generally safe but less efficacious than live vaccines and require multiple doses, such as polio-salk, influenza, rabies, and japanese encephalitis. c. cellular fractions usually of a polysaccharide fraction of the cell wall of a disease-causing organisms, such as pneumococcal pneumonia or meningococcal meningitis. d. recombinant vaccines produced by recombinant dna methods in which specific dna sequences are inserted by molecular engineering techniques, such as dna sequences spliced to vaccinia virus grown in cell culture to produce influenza and hepatitis b vaccines. toxoids or antisera: modified toxins are made nontoxic to stimulate formation of an antitoxin, such as tetanus, diphtheria, botulism, gas gangrene, and snake and scorpion venom. immune globulin: an antibody-containing solution derived from immunized animals or human blood plasma, used primarily for short-term passive immunization, e.g., rabies, for immunocompromised persons. antitoxin: an antibody derived from serum of animals after stimulation with specific antigens and used to provide passive immunity, e.g., tetanus. munization is one of the outstanding achievements of public health, as treatment of infectious diseases by antimicrobials is a major element of clinical medicine. immunization (vaccination) is a process used to increase host resistance to specific microorganisms to prevent them from causing disease. it induces primary and secondary responses in the human or animal body: a. primary response occurs on first exposure to an antigen. after a lag or latent period of 3-14 days (depending on the antigen) specific antibodies appear in the blood. antibody production ceases after several weeks but memory cells that can recognize the antigen and respond to it remain ready to respond to a further challenge by the same antigen. b. secondary (booster) response is the response to a second and subsequent exposure to an antigen. the lag period is shorter than the primary response, the peak is higher and lasts longer. the antibodies produced have a higher affinity for the antigen, and a much smaller dose of the antigen is required to initiate a response. c. immunologic memory exists even when circulating antibodies are insufficient to protect against the antigen. when the body is exposed to the same antigen again, it responds by rapidly producing high levels of antibody to destroy the antigen before it can replicate and cause disease. immunization protects susceptible individuals from communicable disease by administration of a living modified agent, or subunit of the agent, a suspension of killed organisms or an inactivated toxin (see table 4 .5) to stimulate development of antibodies to that agent. in disease control, individual immunity may also protect another individual. herd immunity occurs when sufficient persons are protected (naturally or by immunization) against a specific infectious disease reducing circulation of the organism, thereby lowering the chance of an unprotected person to become infected. each pathogen has different characteristics of infectivity, and therefore different levels of herd immunity are required to protect the nonimmune individual. the critical proportion of a population that must be immunized in order to interrupt local circulation of the organism varies from disease to disease. eradication of smallpox was achieved with approximately 80% world coverage, followed by concentration on new case findings and immunization of contacts and surrounding communities. for highly infectious diseases, such as measles, immunization coverage of over 95% is needed to achieve local eradication. immunization coverage in a community must be monitored in order to gauge the extent of protection and need for program modification to achieve targets of disease control. immunization coverage is expressed as a proportion in which the numerator is the number of persons in the target group immunized at a specific age, and the denominator is the number of persons in the target cohort who should have been immunized according to the accepted standard: vaccine coverage = no. persons immunized in specific age group â�¢ 100 no. persons in the age group during that year immunization coverage in the united states is regularly monitered by the national immunization survey by a household survey in all 50 states, as well as selected urban areas considered to be at high risk for undervaccination. an initial telephone survey is followed by confirmation, where possible, from documentation from the parents or health care providers. the survey for july 1994-june 1995 examined children born between august 1991 and november 1993 (i.e., aged 19-35 months, median age 27 months). the results show improving coverage, with 95% having received three or more doses of dpt (diphtheria, pertussis, and tetanus), 88% with three or more doses of opv (oral polio vaccine), 92% with three or more doses of haemophilus influenzae, type b (hib), but only 62% with three or more doses of hepatitis b. however, only 75% had received all recommended vaccines at the recommended ages. eases that still cause millions of deaths globally each year. other important infectious diseases are still not subject to vaccine control because of difficulties in their development. in some cases, a microorganism can mutate with changes. viruses can undergo antigenic shifts in the molecular structure in the organism, producing completely new subtypes of the organism. hosts previously exposed to other strains may have little or no immunity to the new strains. antigenic drift refers to relatively minor antigenic changes which occur in viruses. this is responsible for frequent epidemics. antigenic shift is believed to explain the occurrence of new strains of influenza virus necessitating, for example, annual reformulation of the influenza vaccine associated with large scale epidemics and pandemics. new variants of poliovirus strains are similar enough to the three main types so that immunity to one strain is carded over to the new strain. molecular epidemiology is a powerful new technique used to specify the geographic origin of organisms such as poliomyelitis and measles viruses, permiting tracking of the source of the virus and epidemic. combinations of more than one vaccine is now common practice with a trend to enlarging the cocktail of vaccines in order to minimize the number of injections, and visits required. this reduces the number of visits to carry out routine immunization saving staff time and costs, as well as increasing compliance. there are virtually no contraindications to use of multiple antigens simultaneously. examples of vaccine cocktails include dpt (diphtheria, pertussis, and tetanus) in combination with haemophilus influenzae b, poliomyelitis, and varicella, or mmr (measles, mumps, and rubella) vaccines. interventions in the form of effective vaccines save millions of lives each year and contribute to improved health of countless children and adults throughout the world. vaccination is now accepted as one of the most cost-effective health interventions currently available. continuous policy review is needed regarding allocation of adequate resources, logistical organization, and continued scientific effort to seek effective, safe, and inexpensive vaccines for other important diseases such as malaria and hiv. new technology of recombinant vaccines, such as that of hepatitis b, holds promise for important vaccine breakthroughs in the decades ahead. internationally, much progress was made in the 1980s in the control of vaccinepreventable diseases. at the end of the 1970s, fewer than 10% of the world's children were being immunized. who, unicef, and other international organizations mobilized to promote an expanded programme on immunization (epi) with a target of reaching 80% coverage by 1990. immunization coverage increased in the developing countries, preventing some 3 million child deaths annually. bacillus calmette-gu6rin (bcg) coverage rose from 31 to 85%; poliomyelitis with opv (three doses) from 24 to 80%, and tetanus toxoid for pregnant women from 14 to 54%. since 1991, there has been a decline in coverage in some parts of the world, mainly in sub-saharan africa. the challenge remains to achieve control or eradication of vaccine-preventable diseases, thus saving millions of more lives. part of the hfa stresses the epi approach, which includes immunization against diphtheria, pertussis, tetanus, po-liomyelitis, measles, and tuberculosis. an extended form of this is the epi plus program which combines epi with immunization against hepatitis b and yellow fever and, where appropriate, supplementation with vitamin a and iodine. the success in international eradication of smallpox is now being followed by a campaign to eradicate poliomyelitis and other important infectious diseases. diphtheria. diphtheria is an acute bacterial disease of the tonsils, nasopharynx, and larynx caused by the organism corynebacterium diphtheriae. it occurs in colder months in temperate climates where the organism is present in human hosts and is spread by contact with patients or carriers. it has an incubation period of 2-5 days. in the past, this was primarily an infection of children and was a major contributor to child mortality in the prevaccine and preantibiotic eras. diphtheria has been virtually eliminated in countries with well-established immunization programs. in the 1980s, an outbreak of diphtheria occurred in the countries of the former soviet union among people over age 15. it reached epidemic proportions in the 1990s, with 140,000 cases (1991 -1995) with 1100 deaths in 1994 in russia alone. this indicates a failure of the vaccination program in several respects: it used only three doses of dpt in infancy; no boosters were given at school age or subsequently; the efficacy of diphtheria vaccine may have been low, and coverage was below 80%. efforts to control the present epidemic include mass vaccination campaigns for persons over 3 years of age with a single dose of dt (diphtheria and tetanus) and increasing coverage of routine dpt vaccines to four doses by age 2 years. the epidemic and its control measures have led to improved coverage with dt for those over 18 years, and 93% coverage among children aged 12-23 months. who recommends three doses of dpt in the first year of life and a booster at school entry. this is considered by many to be insufficient to produce long-lasting immunity. the united states and other industrialized countries use a four-dose schedule and recommend periodic boosters for adults with dt. pertussis. pertussis is an acute bacterial disease of the respiratory tract caused by the bacillus bordetella pertussis. after an initial coldlike (catarrhal) stage, the patient develops a severe cough which comes in spasms (paroxysms). the disease can last 1-2 months. the paroxysms can become violent and may be followed by a characteristic crowing or high pitched inspiratory whooping sound, followed by expulsion of a tenacious clear sputum, often followed by vomiting. in poorly immunized populations and those with malnutrition, pneumonia often follows and death is common. pertussis declined dramatically in the industrialized countries as a result of widespread coverage with dpt. however, because the pertussis component of the vaccine caused some reactions, many physicians avoided its use, using dt alone. during the 1970s in the united kingdom, many physicians recommended against vaccination with dpt. as a result, pertussis incidence increased with substantial mortality rates. this led to a reappraisal of the immunization program, with insti-tution of incentive payments to general practitioners for completion of vaccination schedules. as a result of these measures, vaccination coverage, with resulting pertussis control, improved dramatically in the united kingdom. pertussis continues to be a public health threat and recurs wherever there is inadequate immunization in infancy. a new acellular vaccine is ready for widespread use and will be safer with fewer and less severe reactions in infants, increasing the potential for improved confidence and support for routine vaccination. use of the new vaccine is spreading in the united states and forms part of the u.s. recommended vaccination schedule. tetanus. tetanus is an acute disease caused by an exotoxin of the tetanus bacillus (clostridium tetani) which grows anaerobically at the site of an injury. the bacillus is universally present in the environment and enters the human body via penetrating injuries. following an incubation period of 3-21 days, it causes an acute condition of painful muscular contractions. unless there is modem medical care available, patients are at risk of high case fatality rates of 30-90% (highest in infants and the elderly). antitetanus serum (ats) was discovered in 1890 and during world war i, ats contributed to saving the lives of many thousands of wounded soldiers. tetanus toxoid was developed in 1993. the organism, because of its universal presence in the environment, cannot be eradicated. however, the disease can be controlled by effective immunization of every child during infancy and school age. adults should receive routine boosters of tetanus toxoid once very decade. newborns are infected by tetanus spores (tetanus neonatorum) where unsanitary conditions or practices are present. it can occur when traditional birth attendants at home deliveries use unclean instruments to sever the umbilical cord, or dress the severed cord with contaminated material. tetanus neonatorum remains a serious public health problem in developing countries. immunization of pregnant women and women of childbearing age is reducing the problem by conferring passive immunity to the newborn. the training of traditional birth attendants in hygienic practice and the use of medically supervised birth centers for delivery also decreases the incidence of tetanus neonastorum. elimination of tetanus neonatorum by the year 2000 was made a health target by the world summit of children in 1990. in that year, the number of deaths from neonatal tetanus was reported by unicef as 700,000 infants worldwide, declining to 600,000 in 1993. immunization of pregnant women increased from under 20% in 1984 to 52% in 1995-1997 . despite progress, coverage is still too low to achieve the target of elimination. poliomyelitis. polio virus infection may be asymptomatic or cause an acute nonspecific febrile illness. it may reach more severe forms of aseptic meningitis and acute flaccid paralysis with long-term residual paralysis or death during the acute phase. poliomyelitis is transmitted mainly by direct person-to-person contact, but also via sewage contamination. large-scale epidemics of disease, with attendant paralysis and death, occurred in industrialized countries in the 1940s and 1950s, engendering widespread fear and panic and thousands of clinical cases of "infantile paralysis". growth of the poliovirus by john enders and colleagues in tissue culture in 1949 led to development of the first inactivated polio vaccine by jonas salk in the mid-1950s and gave hope and considerable success in the control of the disease. the development of the live attenuated oral poliomyelitis vaccine by albert sabin, licensed in 1960, added a new dimension to its control because of the effectiveness, low cost, and ease of administration of the vaccine. the two vaccines in their more modern forms, enhanced strength inactivated polio vaccine (eipv), and triple oral polio vaccine (topv), have been used in different settings with great success. oral polio vaccine (opv) induces both humoral and cellular, including intestinal, immunity. the presence of opv in the environment by contact with immunized infants and via excreta of immunized persons in the sewage gives a booster effect in the community. immunization using opv, in both routine and national immunization days (nids) has proven effective in dramatically reducing poliomyelitis and circulation of the wild virus in many parts of the world. use of the enhanced strength ipv (eipv) produces early and high levels of circulating antibodies, as well as protecting against the vaccine-associated disease. in rare cases opv can cause vaccine-associated paralytic poliomyelitis (vapp), with a risk of 1 case per 520,000 with initial doses, and 1 case per over 12 million with subsequent doses. approximately eight to ten cases of vapp occur annually in the united states, with clinical, ethical, and legal implications. use of ipv as initial protection eliminates this problem. experience in gaza and the west bank in the 1970s and 1980s, and later in israel, showed that a combination of ipv and opv is effective in overcoming endemic and imported poliovirus. opv requires multiple doses to achieve protective antibody levels. where there are many enteroviruses in the environment, as is the case in most developing countries, interference in the uptake of opv may result in cases of paralytic poliomeylitis among persons who have received 3 or even 4 doses of adequate opv. controversy as to the relative advantages of each vaccine continues. the opv program of mass repeated vaccination in control of poliomyelitis in the americas established the primacy of opv in practical public health, and the momentum to eradicate poliomyelitis is building. a combined schedule of ipv and opv would eliminate the wild virus and protect against vaccine-associated disease. the sequential use of ipv and opv was adopted as part of the routine infant immunization program in the united states in 1997, but ipv alone was adopted in 1999. there are concerns that exclusive use of either vaccine alone will not lead to the desired goal of eradication of polyomyelitis. progress in global eradication of polio has been impressive. global coverage of infants with three doses of opv reached 81% in 1996 as compared to 83% in 1995. the african region of who had an increase in opv coverage from 58% in 1995 to 60% in 1996. national immunization days (nids) were conducted in 62 countries in 1995 and 82 in 1996, covering 419 million children in 1996. mopping up operations to reinforce coverage of children in still endemic areas is proceeding along with increased emphasis on acute flaccid paralysis (afp) monitoring. confirmed polio cases reported continued at 5-6,000 per year in 1997-1998. with continued national and international emphasis, and support of who, rotary international, unicef, donor countries, and others, there is a real prospect of a world without polio, if not by the year 2000, then or shortly thereafter. measles is an acute disease caused by a virus of the paramyxovirus family. it is highly infectious with a very high ratio of clinical to subclinical case ratio (99/1). measles has a characteristic clinical presentation with fever, white spots (koplik spots) on the membranes of the mouth, and a red blotchy rash appearing on the 3rd-7th day lasting 4-7 days. mortality rates are high in young children with compromised nutritional status, especially vitamin a deficiency. the measles virus evolved from a virus disease of cattle (rinderpest) some 3000-5000 years ago, becoming an important disease of humans with high mortality rates in debilitated, poorly nourished children, and significant mortality and morbidity even in industrialized countries. in the prevaccine era, measles was endemic worldwide, and even in the late 1990s it remains one of the major childhood infectious diseases. it is one of the commonest causes of death for school age children worldwide. despite earlier predictions that measles deaths would be halved to 500,000 by 1996, who reported 1.1 million measles deaths in that year and over 1 million in 1997. eradication in the first decade of the next century is a feasible goal, provided that there is an adequate international effort. measles immunization increased from under 40% worldwide in 1985 to 79% in 1995-1996, but 56% in sub-saharan africa. single-dose immunization failed to meet control or eradication requirements even in the most developed parts of the world. a live vaccine, licensed in 1963, was later replace by a more effective and heat stable vaccine, but still with a primary vaccination failure rate (i.e., fails to produce protective antibodies) of 4-8%, and secondary failure rate (i.e., produces antibodies but protection is lost over time) of 4%. a two-dose policy incorporates a booster dose, usually at school-age, in addition to maximum feasible infant coverage of children in the 9-15 month period (timing varies in different countries). catch-up campaigns among schoolage children should be carried out until the routine two-dose policy has time to take full effect. nearly universal primary education in developing countries, offers an opportunity for mass coverage of school age children with a second dose of measles and a resulting increase of herd immunity to reduce the transmission of the virus. the two-dose policy adopted in many countries, should be supplemented with catch-up campaigns in schools to provide the booster effect for those previously immunized and to cover those previously unimmunized, especially in developing countries. the cdc considers that domestic transmission in the united states has been interrupted and that most localized outbreaks were traceable to imported cases. south america and the caribbean countries are now considered free of indigenous measles, based on their successful use of nids, although a large epidemic occurred in 1999 in brazil. it now appears that eradication has become a feasible target during the early part of the next century, with a strategy of levels of coverage in in-fancy with a two-dose policy, supplemented by catch-up campaigns to older children and young adults, and outbreak control. mumps. mumps is an acute viral disease characterized by fever, swelling, and tenderness usually of the parotid glands, but also other glands. the incubation period ranges between 12 and 25 days. orchitis, or inflammation of the testicles, occurs in 20-30% of postpubertal males and oophoritis, or inflammation of the ovaries, in 5% of postpubertal females. sterility is an extremely rare result of mumps. central nervous system involvement can occur in the form of aseptic meningitis, almost always without sequelae. encephalitis is reported in 1-2 per 10,000 cases with an overall case fatality rate of 0.01%. pancreatitis, neuritis, nerve deafness, mastiffs, nephritis, thyroiditis, and pericarditis, although rare, may occur. most persons born before 1957 are immune to the disease, because of the nearly universal exposure to the disease before that time. the live attenuated vaccine introduced in the united states in 1967 is available as a single vaccine or in combination with measles and rubella as the measlesmumps-rubella (mmr) vaccine. it provides long-lasting immunity in 95% of cases. mumps vaccine is now recommended in a two-dose policy with the first dose of mmr given between 12 and 15 months of age and a second dose given either at school entry or in early adolescence. mmr in two doses is now standard policy in the united sates, sweden, canada, israel, the united kingdom, and other countries. the incidence of mumps has consequently declined rapidly. local eradication of this disease is worthwhile and should be part of a basic international immunization program. rubella. rubella (german measles) is generally a mild viral disease with lymphadenopathy and a diffuse, raised red rash. low grade fever, malaise, coryza, and lymphadenopathy characterize the prodromal period. the incubation period is usually 16-18 days. differentiation from scarlet fever, measles, or other febrile diseases with rash may require laboratory testing and recovery of the virus from nasopharyngeal, blood, stool, and urine specimens. in 1942, norman gregg, an australian ophthalmologist, noted an epidemic of cases of congenital cataract in newborns associated with a history of rubella in the mother during the first trimester. subsequent investigation demonstrated that intrauterine death, spontaneous abortion, and congenital anomalies occur commonly when rubella occurs early in pregnancy. congenital rubella syndrome (crs) occurs with single or multiple congenital anomalies including deafness, cataracts, microophthalmia, congenital glaucoma, microcephaly, meningoencephalitis, congenital heart defects, and others. moderate and severe cases are recognizable at birth, but mild cases may not be detected for months or years after birth. insulin-dependent diabetes is suspected as a late sequela of congenital rubella. each case of crs is estimated to cost some $250,000 in health care costs during the patient's lifetime. prior to availability of the attenuated live rubella vaccine in 1969, the disease was universally endemic, with epidemics or peak incidence every 6-9 years. in unvaccinated populations, rubella is primarily a disease of childhood. in areas where children are well vaccinated, adolescent and young adult infection is more apparent, with epidemics in institutions, colleges, and among military personnel. a sharp reduction of rubella cases was seen in the united states following introduction of the vaccine in 1970, but increased in 1978, following rubella epidemics in 1976-1978. a further reduction in cases was followed by a sharp upswing of rubella and crs in [1988] [1989] [1990] . an outbreak of rubella among the amish in the united states, who refuse immunization on religious grounds, resulted in 7 cases of crs in 1991. it is now thought that vaccination of sufficient numbers in the united states reduced circulation of the virus and protected most vulnerable groups in the population. in the past, immunization policy in some countries was to vaccinate school girls aged 12 to protect them for the period of fertility. the current approach is to give a routine dose of mmr in early childhood, followed by a second dose in early school age to reduce the pool of susceptible persons. women of reproductive age should be tested to confirm immunity before pregnancy and immunized if not already immune. should a woman become infected during pregnancy, termination of pregnancy previously recommended is now managed with hyperimmune globulin. the infection of pregnant women during their first trimester of pregnancy is the primary public health implication of rubella. the emotional and financial burden of crs, including the cost of treatment of its congenital defects, makes this vaccination program cost-effective. its inclusion in a modem immunization program is fully justified. elimination of crs syndrome should be one of the primary goals of a program for prevention of vaccine-preventable disease in developed and developing countries. adoption of mmr and the two-dose policy will gradually lead to eradication of rubella and rubella syndrome. viral hepatitis. viral hepatitis is a group of diseases of increasing public health importance due to their large scale worldwide prevalence, their serious consequences, and our increasing ability to take preventive action. viral hepatic infectious diseases each have specific etiologic, clinical, epidemiologic, serologic, and pathologic characteristics. they have important short-and long-term sequelae. vaccine development is of high priority for control and ultimate eradication. hepatitis a. hepatitis a (hav) was previously known as infectious hepatitis or epidemic jaundice. hav is mainly transmitted by the fecal-oral route. clinical severity varies from a mild illness of 1-2 weeks to a debilitating illness lasting several months. the norm is complete recovery within 9 weeks, but a fulminating or even fatal hepatitis can occur. severity of the disease worsens with increasing age. hav is sporadic/endemic worldwide. improving sanitation raises the age of exposure, with accompanying complications. it now occurs particularly in persons from industrialized countries when exposed to situations of poor hygiene, or among young adults when traveling to areas where the disease is en-demic. common source outbreaks occur in school-aged children and young adults from case contact, or from food contaminated by infected handlers. hepatitis a may be a serious public health problem in a disaster situation. prevention involves improving personal and community hygiene, with safe chlorinated water and proper food handling. hepatitis a vaccine has been recently licensed for use in the united states, and will probably soon be recommended for routine vaccination programs, as well as for persons traveling to endemic areas. hepatitis b. hepatitis b (hbv) once called serum jaundice, was thought to be transmitted only by injections of blood or blood products. it is now known to be present in all body fluids and easily transmissible by household and sexual contact, perinatal spread from mother to newborn, and between toddlers. however, it is not spread by the oral-fecal route. hepatitis b virus is endemic worldwide and is especially prevalent in developing countries. carrier status with persistent viremia varies from < 1% of adults in north america to 20% in some parts of the world. carders have detectable levels of hbsag, the surface antigen (i.e., australian antigen), in their blood. high risk groups in developed countries include intravenous drug users, homosexual men, persons with high numbers of sexual partners, those receiving tattoos, body piercing or acupuncture treatments, and residents or staff of institutions such as group homes and prisons. immunocompromised and hemodialysis patients are commonly carders of hbv. hbv may also be spread in a health system by use of inadequately sterilized reusable syringes, as in china and the former soviet union. transmission is reduced by screening blood and blood products for hbsag and strict technique for handling blood and body fluids in health settings. hbv is clinically recognizable in less than 10% of infected children but is apparent in 30-50% of infected adults. clinically hbv has an insidious onset with anorexia, abdominal discomfort, nausea, vomiting, and jaundice. the disease can vary in severity from subclinical, very mild to fulminating liver necrosis, and death. it is a major cause of primary liver cancer, chronic liver disease, and liver failure, all devastating to health and expensive to treat. hepatitis b virus is considered to be the cause of 60% of primary cancer of the liver in the world and the most common carcinogen after cigarette smoking. the who estimates that more than 2 billion people alive today have been infected with hbv. it is also estimated that 350 million persons are chronic carriers of hbv, with an estimated 1-1.5 million deaths per year from cirrhosis or primary liver cancer. this makes hepatitis b control a vital issue in the revision of health priorities in many countries. strict discipline in blood banks and testing of all blood donations for hbv, as well as hiv, and hepatitis c, is mandatory, with destruction of those with positive tests. contacts should be immunized following exposure with hbv immunoglobulin and hbv vaccine. the inexpensive recombinant hbv vaccine should be adopted by all countries and included in routine vaccination of infants. catch-up immunization for older children is also desirable. immunization programs should include those exposed at work, such as health, prison, or sex workers and adults in group settings. hbv immunization has been included in who's epi-plus expanded program of immunization. hepatitis c. first identified in 1989, and previously known as non-a, non-b hepatitis, hepatitis c (hcv) has an insidious onset with jaundice, fatigue, abdominal pain, nausea, and vomiting. it may cause mild to moderate illness, but chronicity is common going on to cirrhosis and liver failure. the cdc estimates that 4 million americans are chronically infected with hcv, with 8000-10,000 resulting deaths per annum, and the main cause of liver transplants. hcv is transmitted most commonly in blood products, but also among injecting drug users (90% of intravenous drug users were hcv positive in a vancouver study in 1998), and is also a risk for health workers. the disease may also occur in dialysis centers and other medical situations. person-to-person spread is unclear. prevention of transmission includes routine testing of blood donations, antiviral treatment of blood products, needle exchange programs, and hygiene. the who in 1998 has declared hepatitis prevention as a major public health crisis, with an estimated 170 million persons infected worldwide (1996) , stressing that this "silent epidemic" is being neglected and that screening of blood products is vital to reduce transmission of this disease as for hiu hcv is a major cause of chronic cirrhosis and liver cancer. no vaccine is available at present, but an experimental vaccine is undergoing field trials. interferon and ribavirin treatment is reportedly effective in 40% of cases. hepatitis d. hepatitis d virus (hdv) also known as delta hepatitis, may be self-limiting or progress to chronic hepatitis. it is caused by a viruslike particle which infects cells along with hbv as a coinfection or in chronic carriers of hbv. hdv occurs worldwide in the same groups at risk for hbv. it also occurs in epidemics and is endemic in south america, africa, and among drug users. prevention is by measures similar to those for hbv. management for hdv is by passive immunity with immunoglobulin for contacts and high risk groups, and should include hbv vaccination as the diseases often coincide. there is currently no vaccine for hdv. hepatitis e. hepatitis e virus has an epidemiological and clinical course similar to that of hav. there is no evidence of a chronic form of hev. one striking characteristic of hev is its high mortality rate among pregnant women. the disease is caused by a viruslike particle with an incubation period of 15-64 days and is most common in young adults. sporadic cases as well as epidemics have been identified in india, pakistan, burma, china, russia, mexico, and north africa. hev results from waterborne epidemics or as sporadic cases in areas with poor hygiene, spread via the oral-fecal route. it is a hazard in disaster situations with crowding and poor sanitary conditions. prevention is by safe management of water supplies and sanitation. disease management is supportive care; passive immunization is not helpful and no vaccine is currently available. teria which causes meningitis and other serious infections in children under 18 months of age. before the introduction of effective vaccines, as many as 1 in 200 children developed invasive hib infection. two-thirds of these had hib meningitis, with a case fatality rate of 2-5%. long-term sequelae such as hearing impairment and neurological deficits occurred in 15-30% of survivors. the first hib vaccine was licensed in 1985, based on capsular material from the bacteria. extensive clinical trials in finland demonstrated a high degree of efficacy, but less impressive results were in seen in postmarketing efficacy studies. by 1989, a conjugate vaccine based on an additional protein cell capsular factor capable of enhancing the immunologic response was introduced. several conjugate vaccines are now available. the conjugate vaccines are now combined with dpt as their schedule is simultaneous with that of the dpt. although the hib vaccine has been found to be cost-effective, despite initially being as costly as all the basic vaccines combined (i.e., dpt, opv, mmr, and hbv). for this reason, its use thus far has been limited to industrialized countries. the vaccine is a valuable addition to the immunologic armamentarium. it showed dramatic results in local eradication of this serious early childhood infection in a number of european countries and a sharp reduction in the united states. impressive field trials in the gambia showed a sharp reduction in mortality from invasive streptococcal diseases. the price of the vaccine has also fallen dramatically since the mid 1990s. as a result, in 1997, the world health organization recommended inclusion of hib vaccine in routine immunization programs in developing countries. influenza. influenza is an acute viral respiratory illness characterized by fever, headache, myalgia, prostration, and cough. transmission is rapid by close contact with infected individuals and by airborne particles with an incubation period of 1-5 days. it is generally mild and self-limited with recovery in 2-7 days. however, in certain population groups, such as the elderly and chronically ill, infection can lead to severe sequelae. gastrointestinal symptoms commonly occur in children. during epidemics, mortality rates from respiratory diseases increase because of the large numbers of persons affected, although the case fatality rates are generally low. over the past century, influenza pandemics have occurred in 1889, 1918, 1957, and 1968 , while epidemics are annual events. the influenza pandemic of 1918 caused millions of deaths among young adults, by some estimates killing more than had died in world war i. it was the fear of recurrence of this pandemic which led the cdc to launch a massive immunization program in the united states in 1976 to prevent swine flu (the virus was a strain antigenically similar to that of the 1918 pandemic influenza) from spreading from an isolated outbreak in an army camp. the effort was stopped after millions of persons were immunized with an urgently produced vaccine when serious reactions occurred (guillain-barre syndrome, (i.e., a type of paralysis), and when no further cases of swine flu were seen. this demonstrated the difficulty of extrapolating scenarios from a historical experience. each year, epidemiologic services of the who and collaborating centers such as the cdc recommend which strains should be used in vaccine preparation for use among susceptible population groups. these vaccines are prepared with the current anticipated epidemic strains. the three main types of influenza (a, b, and c) have different epidemiological characteristics. type a and its subtypes, which are subject to antigenic shift, are associated with widespread epidemics and pandemics. type b undergoes antigenic drift and is associated with less widespread epidemics. influenza type c is even more localized. active immunization against the prevailing wild strain of influenza virus produces a 70-80% level of protection in high risk groups. the benefits of annual immunization outweigh the costs, and it has proven to be effective in reducing cases of influenza and its secondary complications such as pneumonia and death from respiratory complications in high-risk groups. pneumococcal disease. pneumococcal diseases, which are caused by streptococcus pneumoniae, include pneumonia, meningitis, and otitis media. the 23 capsular types of pneumococci selected out of 83 known types of the organism for the vaccine are those responsible for 88% of pneumococcal pneumonia cases and 10-25% of all pneumonia cases in the united states, and are responsible for some 40,000 deaths per year. this vaccine has been found to be cost-effective for high risk groups, including persons with chronic disease, hiv carriers, patients whose spleens were removed, the elderly, and those with immunosuppressive conditions. it should be included in preventive-oriented health programs, especially for long-term care of the chronically ill. because pneumococci cause bacterial meningitis, pneumococcal vaccine may be a future candidate for use in routine immunization programs for children (over age 2). varicella is an acute, generalized virus disease caused by the varicella zoster virus (vzv). despite its reputation as an innocuous disease of childhood, varicella patients can be quite ill. a mild fever and characteristic generalized red rash lasts for a few hours, followed by vesicles occurring in successive crops over various areas of the body. affected areas may include the membranes of the eyes, mouth, and respiratory tract. the disease may be so mild as to escape observation or may be quite severe, especially in adults. death can occur from viral pneumonia in adults and sepsis or encephalitis in children. neonates whose mothers develop the disease within 2 days of delivery are at increased risk with a case fatality rate of up to 30%. long-term sequelae include herpes zoster or shingles with a severely painful, vesicular rash along the distribution of sensory nerves, which can last for months. its occurrence increases with age and it is primarily seen in the elderly. it can, however, occur in immunocompromised children (especially those on cancer chemotherapy), aids patients, and others. some 15% of a population will experience herpes zoster during their lifetimes. reye's syndrome is an increasingly rare but serious complication from varicella or influenza b. it occurs in children and affects the liver and central nervous system. congenital varicella syndrome with birth defects similar to congenital rubella syndrome has been identified recently. varicella vaccine is now recommended for routine immunization at age 12-18 months in the united states, with catch-up for children up to age 13 years and for occupationally exposed persons in health or child care settings. varicella vaccine is also recommended for nonpregnant women of child bearing years. cost-benefit studies indicate a 1:5 ratio if both direct and indirect costs are included (see chapter 11). varicella vaccine is likely to be added to a "cocktail vaccine" containing dpt, polio (ipv), and hib. meningococcal meningitis. meningococcal meningitis, caused by the bacterium neisseria meningitides, is characterized by headache, fever, neck stiffness, delirium, coma, and/or convulsions. the incubation period is 2-10 days. it has a case fatality rate of 5-15% if treated early and adequately, but rises up to 50% in the absence of treatment. there are several important strains (a, b, c, x, y, and z). serogroups a and c are the main causes of epidemics, with b causing sporadic cases and local outbreaks. transmission is by direct contact and droplet spread. meningitis (group a) is common in sub-saharan african countries, but epidemics have occurred worldwide. during epidemics, children, teenagers, and young adults are the most severely affected. in developed countries, outbreaks occur most frequently in military and student populations. in 1997, meningococcal meningitis spread widely in the "meningitis belt" in central africa. epidemic control is achieved by mass chemoprophylaxis with antibiotics (e.g., rifampin or sulfa drugs) among case contacts, although the emergence of resistant strains is a concern. vaccines against serotypes a and c (bivalent) or a, c, w, and y are available. their use is effective in epidemic control and prevention institutions and military recruits, especially for a and c serogroups. vaccination is one of the key modalities of primary prevention. immunization is cost-effective and prevents wide-scale disease and death, with high levels of safety. despite the general consensus in public health regarding the central role of vaccination, there are many areas of controversy and unfulfilled expectations. a vaccination program should aim at 95% coverage at appropriate times, including infants, school children, and adults. immunization policy should be adapted from current international standards applying the best available program to national circumstances and financial capacities (table 4 .6). public health personnel with expertise in vaccine-preventable disease control are needed to advise ministries of health and the practicing pediatric community on current issues in vaccination and to monitor implementation and evolution of control programs. controversies and changing views are common to immunization policy, so that discussions must be conducted on a continuing basis. policy should be under continuing review by a ministerially appointed national immunization advisory committee, including professionals from public health, academia, immunology, laboratory sciences, economics, and relevant clinical fields. bduring 1999, the recommendation for polio virus was changed to 3 doses of ipv in infancy. vaccine supply should be adequate and continuous. supplies should be ordered from known manufacturers meeting international standards of good manufacturing practice. all batches should be tested for safety and efficacy prior to release for use. there should be an adequate and continuously monitored cold chain to protect against high temperatures for heat labile vaccines, sera, and other active biological preparations. the cold chain should include all stages of storage, transport, and maintenance at the site of usage. only disposable syringes should be used in vaccination programs to prevent any possible transmission of blood-borne infection. a vaccination program depends on a readily available service with no barriers or unnecessary prerequisites, free to parents or with a minimum fee, to administer vaccines in disposable syringes by properly trained individuals using patientoriented and community-oriented approaches. ongoing education and training on current immunization practices are needed. incentive payments by insuring agency or managed care systems promote complete, on-time coverage. all clinical encounters should be used to screen, immunize, and educate parents/guardians. contraindications to vaccination are very few; vaccines may be given even during mild illness with or without fever, during antibiotic therapy, during convalescence from illness, following recent exposure to an infectious disease, and to persons having a history of mild/moderate local reactions, convulsions, or family history of sudden infant death syndrome (sids). simultaneous administration of vaccines and vaccine "cocktails" reduces the number of visits and thereby improves coverage; there are no known interferences between vaccine antigens. accurate and complete recording with computerization of records with automatic reminders helps promote compliance, as does co-scheduling of immunization appointments with other services. adverse events should be reported promptly, accurately, and completely. a tracking system should operate with reminders of upcoming or overdue immunizations; use mail, telephone, and home visits, especially for high risk families, with semiannual audits to assess coverage and review patient records in the population served to determine the percentage of children covered by second birthday. tracking should identify children needing completion of the immunization schedule and assess the quality of documentation. it is important to maintain up-to-date, easily retrievable medical protocols where vaccines are administered, noting vaccine dosage, contraindications, and management of adverse events. all health care providers and managers should be trained in education, promotion, and management of immunization policy. health education should target parents as well as the general public. monitoring of vaccines used and children immunized, individually and by category of vaccination can be facilitated by computerization of immunization records, or regular manual review of child care records. where immunization is done by physicians in private practice, as in the united states, determination of coverage is by periodic surveys. inspection of vaccines for safety, purity, potency, and standards is part of the regulatory function. vaccines are defined as biological products and are therefore subject to regulation by national health authorities. in the united states, this comes under the legislative authority of the public health service act, as well as the food, drug and cosmetics act, with applicable regulations in the code of federal regulations. the federal agency empowered to carry out this regulatory function is the center for drugs and biologics of the federal food and drug administration. litigation regarding adverse effects of vaccines led to inflation of legal costs and efforts to limit court settlements. the u.s. federal government enacted the child vaccine injury act of 1988. this legislation requires providers to document vaccines given and to report on complications or reactions. it was intended to pay benefits to persons injured by vaccines faster and by means of a less expensive procedure than a civil suit for resolving claims. using this no-fault system, petitioners do not need to prove that manufacturers or vaccine givers were at fault. they must only prove that the vaccine is related to the injury in order to receive compensation. the vaccines covered by this legislation include dtp, mmr, opv, and ipv. development of vaccines from jenner in eighteenth century to the advent of recombinant hepatitis b vaccine in 1987, and of vaccines for acellular pertussis, varicella, hepatitis a, and rotavirus in the 1990s, has provided one of the pillars of public health and led to enormous savings of human life. vaccines for viral in-fections in humans for hiv, respiratory syncytial virus, papilloma, epstein-barr virus, dengue fever, and hantavirus are under intense research with genetic approaches using recombinant techniques. the potential for the future of vaccines will be greatly influenced by scientific advances in genetic engineering, with potential for development of vaccines attached to bacteria or protein in plants, which may be given in combination for an increasing range of organisms or their harmful products. recombinant dna technology has revolutionized basic and biomedical research since the 1970s. the industry of biotechnology has produced important diagnostic tests, such as for hiv, with great potential for vaccine development. traditional whole organism vaccines, alive or killed, may contain toxic products that may cause mild to severe reactions. subunit vaccines are prepared from components of a whole organism. this avoids the use of live organisms that can cause the disease or create toxic products which cause reactions. subunit vaccines traditionally prepared by inactivation of partially purified toxins are costly, difficult to prepare, and weakly immunogenic. recombinant techniques are an important development for production of new whole cell or subunit vaccines that are safe, inexpensive, and more productive of antibodies than other approaches. their potential contribution to the future of immunology is enormous. molecular biology and genetic engineering have made it feasible to create new, improved, and less costly vaccines. new vaccines should be inexpensive, easily administered, capable of being stored and transported without refrigeration, and given orally. the search for inexpensive and effective vaccines for groups of viruses causing diarrheal diseases led to development of the rotavirus vaccine. some "edible" research focuses on the genetic programming of plants to produce vaccines and dna. vaccine manufacturers, who spend huge sums of money and years of research on new products, tend to work on those which will bring great financial rewards for the company and are critical to the local health care community. this has led to less effort being made in developing vaccines for diseases such as malaria. yet industry plays a crucial role for continued progress in the field. since the eradication of smallpox, much attention has focused on the possibility of similarly eradicating other diseases, and a list of potential candidates has emerged. some of these have been abandoned because of practical difficulties with current technology. diseases that have been under discussion for eradication have included measles, tb, and some tropical diseases, such as malaria and dracunculiasis. eradication is defined as the achievement of a situation whereby no further cases of a disease occur anywhere and continued control measures are unnecessary. reducing epidemics of infectious diseases, through control and eradication in selected areas or target groups, can in certain instances achieve eradication of the disease. local eradication can be achieved where domestic circulation of an organism is interrupted with cases occurring from importation only. this requires a strong, sustained immunization program with adaptation to meet needs of importation of carriers and changing epidemiologic patterns. smallpox was one of the major pandemic diseases of the middle ages and its recorded history goes back to antiquity. prevention of smallpox was discussed in ancient china by ho kung (circe ao 320), and inoculation against the disease was practiced there from the eleventh century ad. prevention was carried out by nasal inhalation of powdered dried smallpox scabs. exposure of children to smallpox when the mortality rate was lowest assumed a weakened form of the disease, and it was observed that a person could only have smallpox once in a lifetime. isolation and quarantine were widely practiced in europe during the sixteenth and seventeenth centuries. variolation was the practice of inoculating youngsters with material from scabs of pustules from mild cases of smallpox in the hope that they would develop a mild form of the disease. although this practice was associated with substantial mortality, it was widely adopted because mortality from variolation was well below that of smallpox acquired during epidemics. introduced into england in 1721 (see chapter 1) it was commonly practiced as a lucrative medical specialty during the eighteenth century. in the 1720s, variolation was also introduced into the american colonies, russia, and subsequently into sweden and denmark. despite all efforts, in the early eighteenth century smallpox was a leading cause of death in all age groups. toward the end of the eighteenth century an estimated 400,000 persons died annually from smallpox in europe. vaccination, or the use of cowpox vaccinia virus to protect against smallpox, was initiated late in the eighteenth century. in 1774, a cattle breeder in yorkshire, england, inoculated his wife and two children with cowpox to protect them during a smallpox epidemic. in 1796, edward jenner, an english country general practitioner, experimented with inoculation from a milkmaid's cowpox pustule to a healthy youngster, who subsequently proved resistant to smallpox by variolation (see chapter 1). vaccination, the deliberate inoculation of cowpox material, was slow to be adopted universally, but by 1801, over 100,000 persons in england were vaccinated. vaccination gathered support in the nineteenth century in military establishments, and in some countries which adopted it universally. opposition to vaccination remained strong for nearly a century based on religious grounds, observed failures of vaccination to give lifelong immunity, and because it was seen as an infringement of the state on the rights of the individual. often the protest was led by medical variolationists whose medical practice and large incomes were threatened by the mass movement to vaccination. resistance was also offered by "sanitarians" who opposed the germ theory and thought cleanliness was the best method of prevention. universal vaccination was increasingly adopted in europe and america in the early nineteenth century and eradication of smallpox in developed countries was achieved by the mid twentieth century. in 1958, the soviet union proposed to the world health assembly a program to eradicate smallpox internationally and subsequently donated 140 million doses of vaccine per year as part of the 250 million needed to promote vaccination of at least 80% of the world population. in 1967, who adopted a target for the eradication of smallpox. a program was developed which included a massive increase in coverage to reduce the circulation of the virus through person-to-person contact. where smallpox was endemic, with a substantial number of unvaccinated persons, the aim of the mass vaccination phase was 80% coverage. increasing vaccination coverage in developing countries reduced the disease to periodic and increasingly localized outbreaks. in 1967, 33 countries were considered endemic for smallpox, and another 11 experienced importation of cases. by 1970, the number of endemic countries was down to 17, and by 1973 only 6 countries were still endemic, including india, pakistan, bangladesh, and nepal. in these countries, a new strategy was needed, based on a search for cases and vaccination of all contacts, working with a case incidence below 5 per 100,000. the program then moved into the consolidation phase, with emphasis on vaccination of newborns and new arrivals. surveillance and case detection were improved with case contact or risk group vaccination. the maintenance phase began when surveillance and reporting were switched to the national or regional health service with intensive follow-up of any suspect case. the mass epidemic era had been controlled by mass vaccination, reducing the total burden of the disease, but eradication required the isolation of individual cases with vaccination of potential contacts. technical innovations greatly eased the problems associated with mass vaccination worldwide. during the 1920s, there was wide variation in sources of smallpox vaccine. in the 1930s, efforts to standardize and further attenuate the strains used reduced complication rates from vaccinations. the development of lyophilization (freeze-drying) of the vaccine in england in the 1950s made a heat-stable vaccine that could be effective in tropical field conditions in developing countries. the invention of the bifurcated needle (bernard rubin 1961) allowed for easier and more widespread vaccination by lesser trained personnel in remote areas. the net result of these innovations was increased world coverage and a reduction in the spread of the disease. smallpox became more and more confined by increasing herd immunity, thus allowing transition to the phase of monitoring and isolation of individual cases. in 1977 the last case of smallpox was identified in somalia, and in 1980 the who declared the disease eradicated. no subsequent cases have been found except for several associated with a laboratory accident in the united kingdom in 1978. the who recommends that the last stores of smallpox virus should be destroyed in 1999. the cost of the eradication program was $112 million or $8 million per year. worldwide savings are estimated at $1 billion annually. this monumental public health achievement set the precedent for eradication of other infectious diseases. the world health assembly decided to destroy the last two remaining stocks of the smallpox virus in atlanta and moscow in 1999. destruction of the remaining stock was delayed in 1999 to 2002 because of concern that illegal stocks may be held by some states or potential bioterrorists for potential use in weapons of mass destruction, concern regarding the appearance of monkeypox and a wish to use the virus for further research. in 1988, the who established a target of eradication of poliomyelitis by the year 2000. global immunization coverage with three doses of opv increased from some 45% in 1984 to over 80% in 1990, with a slight decline in the period 1991-1993. support from member countries and international agencies such as unicef and rotary international has led to widescale increases in immunization coverage throughout many parts of the world. the world health organization promotes use of opv only as part of routine infant immunization or national immunization days (nids). this strategy has been successful in the americas and in china, but india and the middle east remain problematic. eradication of wild poliomyelitis by the year 2000 will require flexibility in vaccination strategies and may require the combined approach, using opv and ipv, as adopted in the united states in 1997 to prevent vaccine-associated clinical cases. the combination of opv and ipv may be needed where enteric disease is common and leads to interference in opv uptake, especially in tropical areas where endemic poliovirus and diarrheal diseases are still found. the world bank estimated that achievement of global eradication would save $300 million annually in the united states alone. since the eradication of smallpox, discussion has focused on the possibility of similarly eradicating other diseases, and a list of potential candidates has emerged. some of these have been abandoned because of practical difficulties with current technology. diseases that have been under discussion for eradication have included measles, tb, and tropical diseases such as malaria and dracunculiasis. eradication of malaria was thought to be possible in the 1950s when major gains were seen in malaria control by aggressive case environmental control, case finding, and management. however, lack of sustained vector control and an effective vaccine has prevented global eradication. malaria control suffered serious setbacks because of failure in political resolve and capacity to continue support needed for necessary programs. in the 1960s and 1970s, control efforts were not sustained in many countries, and a dreadful comeback of the disease occurred in africa and asia in the 1980s. the emergence of mosquitoes resistant to insecticides, and malarial strains resistant to antimalarial drugs, have made malaria control even more difficult and expensive. renewed effort in malaria control may require new approaches. use of community health workers (chws) in small villages in highly endemic regions of colombia resulted in a major drop in malaria mortality during the 1990s. the chws investigate suspect cases by taking clinical histories and blood smears. 1. scientific feasibility a. epidemiologic vulnerability; lack of nonhuman reservoir, ease of spread, no natural immunity, relapse potential; b. effective practical intervention available; vaccine or other primary preventive or curative treatment, or vectoricide that is safe, inexpensive, long lasting, and easily used in the field; c. demonstrated feasibility of elimination in specific locations, such as an island or other geographic unit. 2. political will/popular support a. they examine smears for malaria parasites and a diagnosis is made. therapy is instituted and the patient is followed. quality control monitoring shows high levels of accuracy in reading of slides compared to professional laboratories. in the late 1970s, there was widespread discussion in the literature of the potential for eradication of measles and tb. measles eradication was set back as breakthrough epidemics occurred in the united states, canada, and many other countries during the 1980s and early 1990s, but regional eradication was achieved combining the two-dose policy with catch-up campaigns for older children or in national immunization days, as in the caribbean countries. tuberculosis has also increased in the united states and several european countries for the first time in many decades. unrealistic expectations can lead to inappropriate assessments and policy when confounding factors alter the epidemiologic course of events. such is the case with tb, where control and eradication have receded from the picture. this deadly disease has returned to developed countries, partly in association with the hiv infection and multiple-drug-resistant strains, as well as homelessness, rising prison populations, poverty, and other deleterious social conditions. directly observed therapy is an important recent breakthrough, more effective in use of available technology and will play a major role in tb control in the twenty-first century. a decade after the eradication of smallpox was achieved, the international task force for disease eradication (itfde) was established to systematically evaluate the potential for global eradicability of candidate diseases. its goals were to identify specific barriers to the eradication of these diseases that might be surmountable and to promote eradication efforts. the subject of eradication versus control of infectious diseases if of central public health importance as technology expands the armamentarium of immunization and vector control into the twenty-first century. the control of epidemics, followed by interruption of transmission and ultimately eradication, will save countless lives and prevent serious damage to children throughout the world. the smallpox achievement, momentous in itself, points to the potential for the eradication of other deadly diseases. the skillful use of existing and new technology is an important priority in the new public health. flexibility and adaptability are as vital as resources and personnel. selecting diseases for eradication is not purely a professional issue of resources such as vaccines and manpower, organization and financing. it is also a matter of political will and perception of the burden of disease. there will be many controversies. the selection of polio for eradication while deferring measles when polio kills few and measles kills many may be questioned. the cdc published criteria for selection of disease for eradication are shown in box 4.8. the who, in a 1998 review of health targets in the field of infectious disease control for the twenty-first century, selected the following targets: eradication of chagas' disease by 2010; eradication of neonatal tetanus by 2010; eradication of leprosy by 2010; eradication of measles by 2020; eradication of trachoma by 2020; reversing the current trend of increasing tuberculosis and hiv/aids. in 1998, a conference in atlanta, georgia, reviewed the subject, which is still very much in a state of flux. table 4 .7 summarizes the selection of diseases which are presently seen as controllable and those considered to be potentially eradicable. the subject will be under review in the years ahead. mycobacterium tuberculosis in humans and m. bovis in cattle. the disease is primarily found in humans, but it is also a disease of cattle and occasionally other primates in certain regions of the world. it is transmitted via airborne droplet nuclei from persons with pulmonary or laryngeal tb during coughing, sneezing, talking, or singing. the initial infection may go unnoticed, but tuberculin sensitivity appears within a few weeks. about 95% of those infected enter a latent phase with a lifelong risk of reactivation. approximately 5 % go from initial infection to pulmonary tb. less commonly, the infection develops as extrapulmonary tb, involving meninges, lymph nodes, pleura, pericardium, bones, kidneys, or other organs. untreated, about half of the patients with active tb will die of the disease within 2 years, but modern chemotherapy almost always results in a cure. pulmonary tb symptoms include cough and weight loss, with clinical findings on chest examination and confirmation by findings of tubercle bacilli in stained smears of sputum and, if possible, growth of the organism on culture media, and changes in the chest x-ray. tuberculosis affects people in their adult working years, with 80-90% of cases in persons between the ages of 15 and 49. its devastating effects on the work force and economic development contribute to a high cost-effectiveness for tb control. the tubercle bacillus infects approximately 1.7 billion people in the world today, causing over 7 million cases and nearly 3 million deaths in 1997. during 1995, new cases of tb included 2.8 million (40%) in southeast asia and the western pacific regions of who, with 2.3 million cases in india, and 0.5 million in indonesia. by 2005, the incidence of tb may increase to 11.9 million new cases per year, a 58% increase over 1990. between 1990 and 1999, who estimates there were 88 million new cases of tb, of which 8 million cases were in association with hiv infection. during the 1990s, an estimated 30 million persons died of tb, including 2.9 million with hiv infection. a new and dangerous period for tb resurgence has resulted from parallel epidemiologic events: first, the advent of hiv infection and second, the occurrence of multiple drug resistant tb (mdrtb), that is, organisms resistant at least to both isoniazid (inh) and rifampicin, two mainstays of tb treatment. mdrtb can have a case fatality rate as high as 70%. hiv reduces cellular immunity so that people with latent tb have a high risk of activation of the disease. it is estimated that hiv negative persons have a 5-10% lifetime risk of tb; hiv positive people have a risk of 10% per year of developing clinical tuberculosis. drug resistance, the long period of treatment, and the socioeconomic profile of most tb patients combine to require a new approach to therapy. directly observed treatment, short-course (dots), has shown itself to be highly effective with patients in poor self-care settings, such as the homeless, drug users, and those with aids. the strategy of dots uses community health workers to visit the patient and observes him or her taking the various medications, providing both incentive, support, and moral coercion to complete the needed 6 to 8 month therapy. dots has been shown to cure up to 95% of cases, at a cost of as little as $11 per patient. it is one of the few hopes of containing the tb pandemic. in 1994, who released a new strategy for control of tuberculosis over the next decade. the plan calls for new guidelines for control, new aid funds for developing countries, and enlistment of ngos to assist in the fight. the new guidelines stress short-term chemotherapy in well-managed programs of dots, stressing strict compliance with therapy for infectious cases with a goal of an 85 % cure rate. even under adverse conditions, dots produces excellent results. it is one of the most cost-effective health interventions combining public health and clinical medical approaches. tuberculosis incidence in the united states decreased steadily until 1985, increased in 1990, and has declined again since. from 1986 to 1992, there was an excess of 51,600 cases over the expected rate if the previous decline in case incidence had continued. this rise was largely due to the hiv/aids epidemic and the emergence of mdrtb, but also greater incidence among immigrants from areas of higher tb incidence, drug abusers, the homeless, and those with limited access to health care. this is particularly true in new york city, where mdrtb has appeared in outbreaks among prison inmates and hospital staff. from 1992 to 1997, tb incidence in the united states declined by 26% and in some states, including new york, by 50% or more. this turnaround was due to stronger tb control programs that promptly identified persons with tb and initiated and ensured completion of appropriate therapy. aggressive staff training, outreach, and case management approaches were vital to this success. concern over rising rates among recent immigrants and the continued challenge of hiv/aids and coincidental transmission of hepatitis a, b, and c among drug users and marginal population groups show that continued support for tb control is needed. bacillus calmette-gurrin (bcg) is an attenuated strain of the tubercle bacillus used widely as a vaccination to prevent tb, especially in high incidence areas. it induces tuberculin sensitivity or an antigen-antibody reaction in which antibodies produced may be somewhat protective against the tubercle bacillus in 90% of vaccinees. although the support for its general use is contradictory, there is evidence from case-control and contact studies of positive protection against tb meningitis and disseminated tb in children under the age of 5. in some developed, low-incidence countries, it is not used routinely but selectively. it may also be used in asymptomatic hiv-positive persons or other high risk groups. the bcg vaccine for tuberculosis remains controversial. while used widely internationally, in the united states and other industrialized countries, it is thought to hinder rather than help in the fight against tb. this concern is based on the usefulness of tuberculin testing for diagnosis of the disease. where bcg has been administered, the diagnostic value of tuberculin testing is reduced, especially in the period soon after the bcg is used. studies showing equivocal benefit of bcg in preventing tuberculosis have added to the controversy. while those in the field in the united states continue to oppose the use of bcg, internationally it is still felt to be of benefit in preventing tb, primarily in children. a 1994 metaanalysis of the literature of bcg carried out by the technology assessment group at harvard school of public health concluded: on average, bcg vaccine significantly reduces the risk of tb by 50%. protection is observed across many populations, study designs, and forms of tb. age at vaccination did not enhance predictiveness of bcg efficacy. protection against tuberculous death, meningitis, and disseminated disease is higher than for total tb cases, although this result may reflect reduced error in disease classification rather than greater bcg efficacy. [colditz et al., jama, 1994.] box 4.9 control of tuberculosis 1. identifying persons with clinically active tb; 2. diagnostic methods--clinical suspicion, sputum smear for bacteriologic examination, tuberculin skin testing, chest radiograph; 3. case finding and investigation programs in high risk groups; 4. contact investigation; 5. isolation techniques during initial therapy; 6. treatment, mainly ambulatory, of persons with clinically active tb; 7. treatment of contacts; 8. directly observed treatment, short-course (dots), where compliance suspect; 9. environmental control in treatment settings to reduce droplet infection; 10. educate health care providers on suspicion of tb and investigation of suspects. currently, the who recommends use of bcg as close to birth as possible as part of the expanded programme of immunization (epi). tuberculosis control remains feasible with current medical and public health methods. deterioration in its control should not lead to despair and passivity. the recent trend to successful control by dots despite the growing problem of mdrtb suggest that control and gradual reduction can be achieved by an activist, community outreach approach. the who in 1999 made tb control one of its major priorities, expressing grave concern that the mdr organism, now widely spread in countries of asia, eastern europe, and the former soviet union, may spread the disease much more widely. the disease constitutes one of the great challenges to public health at the start of the new century. acute infectious diseases caused by group a streptococci include streptococcal sore throat, scarlet fever, puerperal fever, septicemia, ersypelas, cellulitis, mastoiditis, otitis media, pneumonia, peritonsillitis (quinsy), wound infections, toxic shock syndrome, and fasciitis, the "flesh eating bacteria." streptococcus pyogenes group a include some 80 serologically distinct types which vary in geographic location and clinical significance. transmission is by droplet, person-to-person direct contact, or by food infected by carriers. important complications from a public health point of view include acute rheumatic fever and acute glomerulonephritis, but also skin infections and pneumonia. acute rheumatic fever is a complication of strep a infection that has virtually disappeared from industrialized countries as a result of improved standards of living and antibiotic therapy. however, outbreaks were recorded in the united states in 1985, and an increasing number of cases have been seen since 1990. in developing countries, rheumatic fever remains a serious public health problem affecting school age children, particularly those in crowded living arrangements. longterm sequelae include disease of the mitral and aortic heart valves, which require cardiac care and surgery for repair or replacement with artificial valves. acute glomerulonephritis is a reaction to toxins of the streptococcal infection in the kidney tissue. this can result in long-term kidney failure and the need for dialysis or kidney transplantation. this disease has become far less common in the industrialized countries, but remains a public health problem in developing countries. the streptococcal diseases are controllable by early diagnosis and treatment with antibiotics. this is a major function of primary care systems. recent increases in rheumatic fever may herald a return of the problem, perhaps due to inadequate access to primary care in the united states for large sectors of the population, along with increased social hygiene problems. where access to primary care services is limited, infections with streptococci can result in a heavy burden of chronic heart and kidney disease with substantial health, emotional, and financial tolls. measures to improve access to care and pub-lic information are needed to assure rapid and effective care to prevent chronic and costly conditions. zoonoses are infectious diseases transmissible from vetebrate animals to humans. common examples of zoonoses of public health importance in nonindustrialized countries include brucellosis and rabies. in industrialized countries, salmonellosis, "mad cow disease" and influenza have reinforced the importance of relationships of animal and human health. strong cooperation between public health and veterinary public health authorities are required to monitor and to prevent such diseases. brucellosis is a disease occurring in cattle (brucella abortus), in dogs (br. cahis), in goats and sheep (br. melitensis), and in pigs (br. suis). humans are affected mainly through ingestion of contaminated milk products, by contact, or inhalation. brucellosis (also known as relapsing, undulant, malta, or mediterranean fever) is a systemic bacterial disease of acute or insidious onset characterized by fever, headache, weakness, sweating, chills, arthralgia, depression, weight loss, and generalized malaise. spread is by contact with tissues, blood, urine, vaginal discharges, but mainly by ingestion of raw milk and dairy products from infected animals. the disease may last from a few days to a year or more. complications include osteoarthritis and relapses. case fatality is under 2%, but disability is common and can be pronounced. the disease is primarily seen in mediterranean countries, the middle east, india, central asia, and in central and south america. brucellosis occurs primarily as an occupational disease of persons working with and in contact with tissues, blood, and urine of infected animals, especially goats and sheep. it is an occupational hazard for veterinarians, packinghouse workers, butchers, tanners, and laboratory workers. it is also transmitted to consumers of unpasteurized milk from infected animals. animal vectors include wild animals, so that eradication is virtually impossible. diagnosis is confirmed by laboratory findings of the organism in blood or other tissue samples, or with rising antibody titers in the blood, with confirmation by blood cultures. clinical cases are treated with antibiotics. epidemiologic investigation may help track down contaminated animal flocks. routine immunization of animals, monitoring of animals in high risk areas, quarantining sick animals, destroying infected animals, and pasteurizing milk and milk products prevents spread of the disease. control measures include educating farmers and the public not to use unpasteurized milk. individuals who work with animals (cattle, swine, goats, sheep, dogs, coyotes) should take special precautions when handling animal carcasses and materials. testing animals, destroying carriers, and enforcing mandatory pasteurization will restrict the spread of the disease. this is an economic as well as public health problem, requiring full cooperation between ministries of health and of agriculture. rabies is primarily a disease of animals, with a variety of wild animals serving as a reservoir for this disease, including foxes, wolves, bats, skunks, and raccoons, who may infect domestic animals such as dogs, cats, and farm animals. animal bites break the skin or mucous membrane, allowing entry of the virus from the infected saliva into the bloodstream. the incubation period of the virus is 2-8 weeks; it can be as long as several years or as short as 5 days, so that postexposure preventive treatment is a public health emergency. the clinical disease often begins with a feeling of apprehension, headache, pyrexia, followed by muscle spasms, acute encephalitis, and death. fear of water ("hydrophobia") or fear of swallowing is a characteristic of the disease. rabies is almost always fatal within a week of onset of symptoms. the disease is estimated to cause 30,000 deaths annually, primarily in developing countries. it is uncommon in developed countries. rabies control focuses on prevention in humans, domestic animals, and wildlife. prevention in humans is based on preexposure prophylaxis for groups at risk (e.g., veterinarians, zoo workers) and postexposure immunization for persons bitten by potentially rabid animals. because reducing exposure of pets to wild animals is difficult, immunization of domestic animals is one of the most important preventive measures. prevention in domestic animals is by mandatory immunization of household pets. all domestic animals should be immunized at age 3 months and revaccinated according to veterinary instructions. prevention in wild animals to reduce the reservoir is successful in achieving local eradication in settings where reentry from neighboring settings is limited. since 1978, the use of oral rabies immunization has been successful in reducing the population of wild animals infected by the rabies virus. rabies eradication efforts, using aerial distribution of baits containing fox rabies vaccine in affected areas of belgium, france, germany, italy, and luxembourg, have been underway since 1989. the number of rabies cases in these affected areas has declined by some 70%. switzerland is now virtually rabies-free because of this vaccination program. the potential exists for focal eradication, especially on islands or in partially restricted areas with limited possibilities of wild animal entry. livestock need not be routinely immunized against rabies, except in high risk areas. where bats are major reservoirs of the disease, as in the united states, eradication is not presently feasible. salmonella, discussed later in this chapter under diarrheal diseases, is one of the commonest of all infectious diseases among animals and is easily spread to humans via poultry, meat, eggs, and dairy products. specific antigenic types are associated with food-borne transmission to humans, causing generalized illness and gastroenteritis. severity of the disease varies widely, but the diseases can be devastating among vulnerable population groups, such as young children, the elderly, and the immunocompromised. epidemiologic investigation of common food source outbreaks may uncover hazardous food handling practices. laboratory confirmation or serotypes helps in monitoring the disease. prevention is by maintaining high standards of food hygiene in processing, inspection and regulation, food handling practices, and hygiene education. bacillus anthracis causes a bacterial infection in herbivore animals. its spores contaminate soil, worldwide. it affects humans exposed in occupational settings. transmission is cutaneous by contact, gastrointestinal by ingestion, or respiratory by inhalation. it has gained recent attention (iraq, 1997) as a highly potent agent for germ warfare or terrorism. limited supplies of vaccine are available. creutzfeld-jakob disease is a degenerative disease of the central nervous system linked to consumption of beef from cattle infected with bovine spongiform encephalopathy. it is transmitted by prions in animal feed prepared from contaminated animal material and in transplanted organs. this disease was identified in the united kingdom linked to infected cattle leading to a 1997 ban on british beef in many parts of the world and slaughter of large numbers of potentially contaminated animals. the tapeworm causing diphyllobothriasis (diphyllobothrium latum) is widespread in north american freshwater fish, passing from crustacean to fish to humans by eating raw freshwater fish. it is especially common among inuit peoples and may be asymptomatic or cause severe general and abdominal disorder. food hygiene (freezing and cooking of meat) is recommended; treatment is by anthelminthics. leptospiroses are a group of zoonotic bacterial diseases found worldwide in rats, raccoons, and domestic animals. it affects farmers, sewer workers, dairy and abattoir workers, veterinarians, military personnel, and miners with transmission by exposure to or ingestion of urine-contaminated water or tissues of infected animals. it is often asymptomatic or mild, but may cause generalized illness like influenza, meningitis, or encephalitis. prevention requires education of the public in self protection and immunization of workers in hazardous occupations, along with immunization and segregation of domestic animals and control of wild animals. vector-borne diseases are a group of diseases in which the infectious agent is transmitted to humans by crawling or flying insects. the vector is the intermediary between the reservoir and the host. both the vector and the host may be affected by climatic condition; mosquitoes thrive in warm, wet weather and are suppressed by cold weather; humans may wear less protective clothing in warm weather. the only important reservoir of malaria is humans. its mode of transmission is from person to person via the bite of an infected female anopheles mosquito (ronald ross, nobel prize, 1902) . the causative organism is a single cell parasite with four species: plasmodium vivax, p malariae, p falciparum, and p ovale. clinical symptoms are produced by the parasite invading and destroying red blood cells. the incubation period of approximately 12-30 days, depending on the specific plasmodium involved. some strains of p vivax may have a protracted incubation period of 8-10 months and even longer for p ovale. the disease can also be transmitted through infected blood transfusions. confirmation of diagnosis is by demonstrating malaria parasites on blood smears. falciparum malaria, the most serious form, presents with fever, chills, sweats, and headache. it may progress to jaundice, bleeding disorders, shock, renal or liver failure, encephalopathy, coma, and death. prompt treatment is essential. case fatality rates in untreated children and adults are above 10%. an untreated attack may last 18 months. other forms of malaria may present as a nonspecific fever. relapse of the p ovale may occur up to 5 years after initial infection; malaria may persist in chronic form for up to 50 years. malaria control advanced during the 1940s-1960s through improved chlovaquine treatment and use of ddt for vector control with optimism for eradication of the disease. however, control regressed in many developing countries as allocations for environmental control and case findings/treatment were reduced. there has also been an increase in drug resistance, so that this disease is now an extremely serious public health problem in many parts of the world. the need for a vaccine for malaria control is now more apparent than ever. the world health organization estimated that, in 1997, sub-saharan africa (ssa) had 270 million new malaria cases, with 5% of children up to age 5. over 1 million deaths occur annually from malaria more than two-thirds of them in ssa. large areas, particularly in forest or savannah regions with high rainfall, are holoendemic. in higher altitudes, endemicity is lower, but epidemics do occur. chloroquine-resistant p. falciparum has spread throughout africa, accompanied by an increasing incidence of severe clinical forms of the disease. the world bank estimates that 11% of all disability-adjusted life years (dalys) lost per year in ssa are from malaria, which places a heavy economic burden on the health systems. in the americas, the number of cases detected has risen every year since 1974, and the who estimates there to have been 2.2-2.5 million cases in 1991. the nine most endemic countries in the americas achieved a 60% reduction in malaria mortality between 1994 and 1997. southeast asian region reports some 3.4 million cases of malaria in 1996 and 8000 deaths from tb. this accounts for more than one-third of all non-african malaria cases. there is an increase in resistant strains to the major available drugs and of the mosquitoes to insecticides in use. vector control, case finding, and treatment remain the mainstay of control. use of insecticide-impregnated bed nets and curtains, and residual house spraying, and strengthened vector control activities are important, as are early diagnosis and carefully monitored treatment with monitoring for resistance. control of malaria will ultimately depend on a safe, effective, and inexpensive vaccine. attempts to develop a malaria vaccine have been unsuccessful to date due to the large number of genetic types of p. falciparum even in localized areas. a colombian-developed vaccine is being field-tested with partial effectiveness. research in vaccines for malaria has also been hampered by the fact that it is a relatively low priority for vaccine manufacturers because of the minimal potential for financial benefit. research on malaria concentrates on the pharmacological aspects of the disease because of increasing drug resistance. in 1998, who has initiated a new campaign to "roll back malaria" and maintain the dream of eradication in the future. effective low technology interventions include community-based case finding, early treatment of good quality, insecticide use, and vector control. the use of community health workers in endemic areas, has shown promising results. local control and even eradication can be achieved with currently available technology. this requires an integration of public health and clinical approaches with strong political commitment. the rickettsia are obligate parasites, i.e., they can only replicate in living cells, but otherwise they have characteristics of bacteria. this is a group of clinically similar diseases, usually characterized by severe headache, fever, myalgia, rash, and capillary bleeding causing damage to brain, lungs, kidneys, and heart. identification is by serological testing for antibodies, but the organisms can also be cultured in laboratory animals, embryonic eggs, or in cell cultures. the organisms are transmitted by arthropod vectors such as lice, fleas, ticks, and mites. the diseases caused millions of deaths during war and famine periods prior to the advent of antibiotics. these diseases appear in nature in ways that make them impossible to eradicate, but clinical diagnosis, host protection, and vector control can help reduce the burden of disease and deal with outbreaks that may occur. public education regarding self-protection, appropriate clothing, tick removal, and localized control measures such as spraying and habitat modification are useful. epidemic typhus, first identified in 1836, is due to rickettsia prowazekii. spread primarily by the body louse, typhus was the cause of an estimated 3 million deaths, i.e., during war and famine, in poland and the soviet union from 1915-1922. untreated, the fatality rate is 5-40%. typhus responds well to antibiotics. it is currently largely confined to endemic foci in central africa, central asia, eastern europe, and south america. it is preventable by hygiene and pediculicides such as ddt and lindane. a vaccine is available for exposed laboratory personnel. murine typhus is a mild form of typhus due to rickettsia typhi, which is found worldwide and spread in rodent reservoirs. scrub typhus, also known as tsutsugamushi or japanese river fever, is located throughout the far east and the pacific islands, and was a serious health problem for u.s. armed forces in the pacific during world war ii. it is spread by the rickettsia tsutsugamushi and has a wide variation in case fatality according to region, organism, and age of patient. rocky mountain spotted fever is a well-known and severe form of tick-borne typhus due to rickettsia rickettsii, occurring in western north america, europe, and asia. q. fever is a tick-borne disease caused by coxiella burnetii and is worldwide in distribution, usually associated with farm workers, in both acute and chronic forms. regular anti-tick spraying of sheep, cows, and goats helps protect exposed workers. protective clothing and regular removal of body ticks help protect exposed persons. arthropod-borne viral diseases are caused by a diverse group of viruses which are transmitted between vertebrate animals (often farm animals or small rodents) and people by the bite of blood-feeding vectors such as mosquitoes, ticks, and sandflies and by direct contact with infected animal carcasses. usually the viruses have the capacity to multiply in the salivary glands of the vector, but some are carried mechanically in their mouthparts. these viruses cause acute central nervous system infections (meningoencephalitis), myocarditis, or undifferentiated viral illnesses with polyarthritis and rashes, or severe hemorrhagic febrile illnesses. arbovirus diseases are often asymptomatic in vertebrates but may be severe in humans. over 250 antigenetically distinct arboviruses are associated with disease in humans, varying from benign fevers of short duration to severe hemmorhagic fevers. each has a specific geographic location, vector, clinical, and virologic characteristics. they are of international public health importance because of the potential for spread via natural phenomena and modem rapid transportation of vectors and persons incubating the disease or ill with it, with potential for further spreading at the point of destination. arboviruses are responsible for a large number of encephalitic diseases characterized by mode of transmission and geographic area. mosquito-borne arboviruses causing encephalitis include eastern and western equine, venezuelan, japanese, and murray hill encephalitides. japanese encephalitis is caused by a mosquito-borne arbovirus found in asia and is associated with rice-growing areas. it is characterized by headache, fever, convulsions, and paralysis, with fatality rates in severe cases as high as 60%. a currently available vaccine is used routinely in endemic areas (japan, korea, thailand, india, and taiwan) and for persons traveling to infected areas. tick-borne arboviruses causing encephalitis include the powassan virus, which occurs sporadically in the united states and canada. tickborne encephalitis is endemic in eastern europe, scandinavia, and the former soviet union. an epidemic of mosquito-borne encephalitis in new york city in 1999 included 54 cases and 6 deaths, due to the west nile fever virus, never before found in the united states. other insect vectors. it affects animals and humans who are in direct contact with the meat or blood of affected animals. the virus causes a generalized illness in humans with encephalitis, hemorrhages, retinitis and retinal hemorrhage leading to partial or total blindness, and death (1-2%). it also causes universal abortion in ewes and a high percentage of death in lambs. the normal habitat is in the rift valley of eastern africa (the great syrian-african rift), often spreading to southern africa, depending on climactic conditions. the primary reservoir and vector is the aedes mosquito, and affected animals serve to multiply the virus which is transmitted by other vectors and direct contact with animal fluids to humans. an unusual spread of rvf northward to the sudan and along the aswan dam reservoir to egypt in 1977-1978 caused hundreds of thousands of animal deaths, with 18,000 human cases and 598 deaths. rvf appeared again in egypt in 1993. this disease is suspected to be one of the ten plagues of egypt leading to the exodus of the children of israel from egypt during pharaonic-biblical times. in 1997, an outbreak of rvf in kenya, initially thought to be anthrax, with hundreds of cases and dozens of deaths, was related to abnormal rainy season and vector conditions. satellite monitoring of rainfall and vegetation is being used to predict epidemics in kenya and surrounding countries. animal immunization, monitoring, vector control, and reduced contact with infected animals can limit the spread of this disease. arboviruses can also cause hemorrhagic fevers. these are acute febrile illnesses, with extensive hemorrhagic phenomena (internal and external), liver damage, shock, and often high mortality rates. the potential for international transmission is high. yellow fever. yellow fever is an acute viral disease of short duration and varying severity with jaundice. it can progress to liver disease and severe intestinal bleeding. the case fatality rate is <5% in endemic areas, but may be as high as 50% in nonendemic areas and in epidemics. it caused major epidemics in the americas in the past, but was controlled by elimination of the vector, aedes aegypti. a live attenuated vaccine is used in routine immunization endemic areas and recommended for travelers to infected areas. determining the mode of transmission and vector control of yellow fever played a major role in the development of public health (see chapter 1). in 1997, the who reported 200,000 cases and 30,000 deaths from yellow fever globally. dengue hemorrhagic fever. dengue hemorrhagic fever is an acute sudden onset viral disease, with 3-5 days of fever, intense headache, myalgia, arthralgia, box 4.10 dengue fever and dengue hemorrhagic fever, 1996 dengue fever, a severe influenza-like illness, and dengue hemorrhagic fever are closely related conditions caused by four distinct viruses transmitted by aedes aegypti mosquitos. dengue is the world's most important mosquito-borne virus disease. a total of 2,500 million people worldwide are at risk of infection. an estimated 20 million cases occur each year, of whom 500,000 need to be hospitalized. this is a spreading problem, especially in cities in tropical and subtropical areas. major outbreaks were reported in colombia, cuba, and many other locations in 1997. source: world health organization. 1998 . world health report 1998 gastrointestinal disturbance, and rash. hemorrhagic phenomena can cause case fatality rates of up to 50%. epidemics can be explosive, but adequate treatment can greatly reduce the number of deaths. dengue occurs in southeast asia, the pacific islands, australia, west africa, the caribbean, and central and south america. an epidemic in cuba in 1981 included more than 500,000 cases, and 158 deaths. vector control of the a. aegypti mosquito resulted in control of the disease during the 1950s-1970s, but reinfestation of mosquitoes led to incresased transmission and epidemics in the pacific islands, caribbean, central and south america in the 1980s and 1990s. outbreaks in vietnam included 370,000 cases in 1987, another 116,000 cases in 1990, and a similar sized outbreak in 1997. indonesia had over 13,000 cases in 1997 with 240 deaths, and in 1998 over 19,000 cases (january-may) with at least 531 deaths. in 1998, epidemics of dengue were reported in fiji, the cook islands, new caledonia, and northern australia. the who estimates 140,000 deaths and 3.1 million cases worldwide in 1997. monkeys are the main reservoir, and the vector is the a. aegypti mosquito. no vaccine is currently available, and management is by vector control. lassa fever. lassa fever was first isolated in lassa, nigeria, in 1969 and is widely distributed in west africa, with 200,000-400,000 cases and 5000 deaths annually. it is spread by direct contact with blood, urine, or secretions of infected rodents and by direct person-to-person contact in hospital settings. the disease is characterized by a persistent or spiking fever for 2-4 weeks, and may include severe hypotension, shock, and hemorrhaging. the case fatality rate is 15%. marburg disease. marburg disease is a viral disease with sudden onset of generalized illness, malaise, fever, myalgia, headache, diarrhea, vomiting, rash, and hemorrhages. it was first seen in marburg, germany, in 1967, following ex-posure to green monkeys. person-to-person spread occurs via blood, secretions, organs, and semen. case fatality rates can be over 50%. ebola fever. ebola fever is a viral disease with sudden onset of generalized illness, malaise, fever, myalgia, headache, diarrhea, vomiting, rash, and hemorrhages. it was first found in zaire and sudan in 1976 in outbreaks which killed more than 400 persons. it is spread from person to person by the blood, vomitus, urine, stools, and other secretions of sick patients, with a short incubation period. the disease has case fatality rates of up to 90%. an outbreak of ebola among laboratory monkeys in a medical laboratory near washington, d.c., was contained with no human cases. the reservoir for the virus is thought to be rodents. an outbreak of ebola in may 1995 in the town of kikwit, zaire, killed 245 persons out of 316 cases (78% case fatality rate). this outbreak caused international concern that the disease could spread, but it remained localized. another outbreak of ebola virus occurred in gabon in early 1996, with 37 cases, 21 of whom had direct exposure to an infected monkey, the remainder by human-to-human contact, or not established; 21 of the cases died (57%). this disease is considered highly dangerous unless outbreaks are effectively controlled. in zaire, lack of basic sanitary supplies, such as surgical gloves for hospitals, almost ensures that this disease will spread when it recurs. lyme disease is characterized by the presence of a rash, musculoskeletal, neurologic, and cardiovascular symptoms. confirmation is by laboratory investigation. it is the most common vector-borne disease in the united states, with 33,000 cases reported between 1993 and 1995. it primarily affects children in the 5-14 age group and adults aged 30-49. lyme disease is preventable by avoiding contact with ticks, by applying insect repellant, wearing long pants and long sleeves in infected areas, and by the early removal of attached ticks. several u.s. manufacturers produced vaccines which are approved for animal and human use. in the mid 1970s, a mother of two young boys who were recently diagnosed with arthritis in the town of lyme, connecticut, conducted a private investigation among other town residents. she mapped each of the six arthritis cases in the town, cases which had occurred in a short time span among boys living in close proximity. this suggested that this syndrome of "juvenile rheumatoid arthritis" was perhaps connected with the boys playing in the woods. she presented her data to the head of rheumatology at yale medical school in new haven, who investigated this "cluster of a new disease entity." some parents reported that their sons had experienced tick bites and a rash before onset of the arthritis. a tick-borne, spiral shaped bacterium, a spirochete, borrelia burgdorferi, was identified as the organism, and ticks shown to be the vector. cases repond well to antibiotic therapy. in 1996 over 16,000 cases (6.2 per 100,000) were reported from 45 states, an increase from 11,000 in 1965 and 13,000 in 1994. cases were mainly located in the northeast, north central, and mid-atlantic regions. the disease accounts for over 90% of vector-borne disease in the united states and was the ninth leading reported infection in 1995. lyme disease has been identified in many parts of north america, europe, the former soviet union, china, and japan. a newly licensed vaccine is effective for people exposed to ticks but not general usage. personal hygiene for protection from ticks and environmental modification are important to limit spread of the disease. source: cdc, 1996, mmwr, 45:481-484; and cdc, 1997, mmwr, 46, no. 23 . lyme disease website http://www.cdc.gov/ncidad/disease/lyme/lyme.htm medically important parasites are animals that live, take nourishment, and thrive in the body of a host, which may or may not harm the host, but never brings benefit. they include those caused by unicellular organisms such as protozoa, which include amoebas (malaria, schistosomiasis, amebiasis, and cryptosporidium), and helminths (worms), which are categorized as nematodes, cestodes, and trematodes. public health continues to face the problems of parasitic diseases in the developing world. increasingly, parasitic diseases are being recognized in industrialized countries. giardiasis and cryptosporidium infections in waterborne and other outbreaks have occurred in the united states. parasitic diseases are among the most common causes of illness and death in the world, e.g., malaria. milder illnesses such as giardiasis and trichomoniasis cause widespread morbidity. intestinal infestations with worms may cause of severe complications, although they commonly cause chronic low-grade symptomatology and iron deficiency anemia. echinococcosis (hydatid cyst disease) is infection with echinococcus granulosus, a small dog tapeworm. the tapeworm forms unilocular (single, noncompartmental) cysts in the host, primarily in the liver and lungs, but they can also grow in the kidney, spleen, central nervous system, or in bones. cysts, which may grow up to 10 cm in size, may be asymptomatic or, if untreated, may cause severe symptoms and even death. this parasite is common where dogs are used with herd grazing animals and also have intimate contact with humans. the middle east, greece, sardinia, north africa, and south america are endemic areas, as are a few areas in the united states and canada. the human dis-ease has been eliminated in cyprus and australia. while the dog is the major host, intermediate hosts include sheep, cattle, pigs, horses, moose, and wolves. preventive measures include education in food and animal contact hygiene, destroying wild and stray dogs, and keeping dogs from the viscera of slaughtered animals. a similar, but multilocular, cystic hydatid disease is widely found in wild animal hosts in areas of the northern hemisphere, including central europe, the former soviet union, japan, alaska, canada, and the north-central united states. another echinococcal disease (echinococcus vogeli) is found in south america, where its natural host is the bush dog and its intermediate host is the rat. the domestic dog also serves as a source of human infection. surgical resection is not always successful, and long-term medical treatment may be required. control is through awareness and hygiene as well as the control of wild animals that come in contact with humans and domestic animals. control may require cooperation between neighboring countries. tapeworm infestation (taeniasis) is common in tropical countries where hygienic standards are low. beef (taenia saginata) and pork (t. solium) tapeworms are common where animals are fed with water or food exposed to human feces. freezing or cooking meat will destroy the tapeworm. fish tapeworm (diphyllobothrium latum) is common in populations living primarily on uncooked fish, such as inuit people. these tapeworms are usually associated with northern climates. toddlers are especially susceptible to dog tapeworm (dipylidium caninum), which is present worldwide, and domestic pets are often the source of oral-fecal transmission of the eggs. the disease is usually asymptomatic. similarly, dwarf tapeworm (hymenolepis nana) is transmitted through oral-fecal contamination from person to person, or via contaminated food or water. rat tapeworm (hymenolepis diminuta) also mostly affects young children. onchocerciasis (fiver blindness) is a disease caused by a parasitic worm, which produces millions of larvae that move through the body causing intense itching, debilitation, and eventually blindness. the disease is spread by a blackfly that transmits the larva from infected to uninfected people. it is primarily located in sub-saharan africa and in latin america, with over 120 million persons at risk. control is by a combination of activities including environmental control by larvicidal sprays to reduce the vector population, protection of potential hosts by protective clothing and insect repellents, and case treatment. a who-initiated program for onchocerciasis control started in 1974 is sponsored by four international agencies: the food and agriculture organization (fao), the united nations development program (undp), the world bank, and who. it covers 11 countries in sub-saharan africa, focusing on control of the blackfly by destoying its larvae, mainly via insecticides sprayed from the air. prevalence in 1997 was reported by who as over 17 million persons. the program has been successful in protecting some 30 million persons and helping 1.5 million infected persons to recover from this disease. who estimates that the program will have prevented 500,000 cases of blindness by the year 2000 and has freed 25 million hectares of land for resettlement and cultivation. the program cost $570 million. this investment is considered by the world bank to have a return of 16-28% in terms of large scale land reuse and improved output of the population. a who program, the african program for onchocerciasis control (apoc), started in 1996, uses a new drug (ivermectin) and selective vector control efforts by spraying. this involves 30 countries in africa, and 6 in a similar program in south america. see website http://www/who.int/ocp and is financed by many donor countries, internation organizations, merck & company, and ngos. dracunculiasis (guinea worm disease) is a parasitic disease of great public health importance in india, pakistan, and central and west africa. it is an infection of the subcutaneous and deeper tissues caused by a large (60 cm) nematode, usually affecting the lower extremities and causing pain and disability. the nematode causes a burning blister on the skin when it is ready to release its eggs. after the blister ruptures, the worm discharges larvae whenever the extremity is in water. the eggs are ingested in contaminated water and the larva released migrate through the viscera to locate as adults in the subcutaneous tissue of the leg. incubation is about 12 months. the larva released in water are ingested by minute crustaceans and remain infective for as long as a month. prevention is based on improving the safety of water supplies and by preventing contamination by infected persons. education of persons in endemic areas to stay out of water sources and to filter drinking water reduces transmission. insecticides remove the crustaceans. chlorine also kills the larvae and the crustaceans which prologue larval infectivity. there is no vaccine. treatment is helpful, but not definitive. dracunculiasis was traditionally endemic in a belt from west africa through the middle east to india and central asia. it was successfully eliminated from central asia and iran and has disappeared from the middle east and from some african countries (gambia and guinea). the world health organization has promoted the eradication of dracunculiasis. major progress has been made in this direction. worldwide prevalence is reported to have been reduced from 12 million cases in 1980 to 3 million in 1990, 152,814 in 1996, and 77,863 cases in 1997. eradication was anticipated for the year 2000, and in 1995 the who established a commission to monitor and certify eradication in formerly endemic areas. india's reported cases fell from 17,000 in 1987 to 900 in 1992, and the country was free of transmission in 1997. in 1997, formerly high prevalence countries such as kenya reported no cases in 1997, while chad, senegal, cameroons, yemen, and the central african republic less than 30 cases each. eradication of this disease appears to be imminent. the who eradication program was developed successfully as an independent program with its own direction and field staff, but further progress will require the integration of this program with other basic primary care programs in order to be self-sustaining as an integral part of community health. community-based surveillance systems for this disease are being converted to work for monitoring of other health conditions in the community. schistosomiasis (snail fever or bilharziasis) is a parasitic infection caused by the trematode (blood fluke) and transmitted from person to person via an intermediate host, the snail. it is endemic in 74 countries in africa, south america, the caribbean, and asia. there are an estimated 200 million persons infected worldwide and more than 600 million at risk for the disease. the clinical symptoms include fever, nausea, vomiting, abdominal pain, diarrhea, and hematuria. the organisms schistosoma mansoni and s. japonicum cause intestinal and hepatic symptoms, including diarrhea and abdominal pain. schistosoma haematobium affects the genitourinary tract, causing chronic cystitis, pyelonephritis, with high risk for bladder cancer the ninth most common cause of cancer deaths globally. infection is acquired by skin contact with freshwater containing contaminated snails. the cercariae of the organism penetrate the skin, and in the human host it matures into an adult worm that mates and produces eggs. the eggs are disseminated to other parts of the body from the worm's location in the veins surrounding the bladder or the intestines, and may result in neurological symptoms. eggs may be detected under microscopic examination of urine and stools. sensitive serologic tests are also available. treatment is effective against all three major species of schistosomiasis. eradication of the disease can be achieved with the use of irrigation canals, prevention of contamination of water sources by urine and feces of infected persons, treatment of infected persons, destruction of snails, and health education in affected areas. persons exposed to freshwater lakes, streams, and rivers in endemic areas should be warned of the danger of infection. mass chemotherapy in communities at risk and improved water and sanitation facilities are resulting in improved control of this disease. leishmaniasis causes both cutaneous and visceral disease. the cutaneous form is a chronic ulcer of the skin, called by various names, e.g., rose of jericho, oriental sore, and aleppo boil. it is caused by leishmania tropica, l. brasiliensis, l. mexicana, or the l. donovani complex. this chronic ulcer may last from weeks to more than a year. diagnosis is by biopsy, culture, and serologic tests. the organism multiplies in the gut of sandflies (phlebotomus and lutzomi) and is transmitted to humans, dogs, and rodents through bites. the parasites may remain in the untreated lesion for 5-24 months, and the lesion does not heal until the parasites are eliminated. prevention is through limiting exposure to the phlebotomines and reducing the sandfly population by environmental control measures. insecticide use near breeding places and homes has been successful in destroying the vector sandflies in their breeding places. case detection and treatment reduce the incidence of new cases. there is no vaccine, and treatment is with specific antimonials and antibiotics. visceral leishmaniasis (kala azar) is a chronic systemic disease in which the parasite multiplies in the cells of the host's visceral organs. the disease is characterized by fever, the enlargement of the liver and spleen, lymphadenopathy, anemia, leukopenia, and progressive weakness and emaciation. diagnosis is by culture of the organism from biopsy or aspirated material, or by demonstration of intracellular (leishman-donovan) bodies in stained smears from bone marrow, spleen, liver, or blood. kala azar is a rural disease occurring in the indian subcontinent, china, the southern republics of the former u.s.s.r., the middle east, latin america, and sub-saharan africa. it usually occurs as scattered cases among infants, children, and adolescents. transmission is by the bite of the infected sandfly with an incubation period of 2-4 months. there is no vaccine, but specific treatment is effective and environmental control measures reduce the disease prevalence. this includes the use of antimalarial insecticides. in localities where the dog population has been reduced, the disease is less prevalent. sleeping sickness. sleeping sickness a disease caused by trypanosoma brucei, transmitted but the tsetse fly, primarily in the african savannahs, affecting cattle and humans. some 55 million persons are at risk in sub-saharan africa. who reported 200,000 new cases, a total prevalence of 300,000 cases, and 150,000 deaths from this disease in 1996. prevention depends on vector control, and effective treatment of human cases. chagas disease is a chronic and incurable vector and blood transfusion borne parasitic disease (trypanosoma cruzi) which causes disability and death. it affects some 17 million persons mainly in latin america, with some 300,000 new cases and 45,000 deaths occurring annually. about 30% of affected persons develop severe heart disease. brazil, which accounts for 40% of the cases prevalent in latin america, achieved elimination of transmission in 1998, after uruguay (1996) and venezuela (1997) and followed by argentina (1999) . elimination of transmission is projected by who by the year 2010. control is difficult, but control measures include reducing the animal host and vector insect population in its habitat by ecological and insectiside measures, education of the population in prevention by clothing, bednets, and repellents, and with chemotherapy for case management. amebiasis. amebiasis is an infection with a protozoan parasite (entamoeba histolytica) which exists as an infective cyst. infestation may be asymptomatic or cause acute, severe diarrhea with blood and mucus, alternating with constipation. amebic colitis can be confused with ulcerative colitis. diagnosis is by microscopic examination of fresh fecal specimens showing trophozoites or cysts. transmission is generally via ingestion of fecal-contaminated food or water containing cysts, or by oral-anal sexual practices. amebiasis is found worldwide. sand filtration of community water supplies removes nearly all cysts. suspect water should be boiled. education regarding hygienic practices with safe food and water handling and disposal of human feces are the basis for control. ascariasis. ascariasis is infestation of the small intestine with the roundworm ascaris lumbricoides, which may appear in the stool, occasionally the nose or mouth, or may be coughed up from lung infestation. the roundworm is very common in tropical countries, where infestation may reach or exceed 50% of the population. children aged 3-8 years are especially susceptible. infestation can cause pulmonary symptoms and frequently contributes to malnutrition, especially iron deficiency anemia. transmission is by ingestion of infective eggs, common among children playing in contaminated areas, or via the ingestion of uncooked products of infected soil. eggs may remain viable in the soil for years. vermox and other treatments are effective. prevention is through education, adequate sanitary facilities for excretion, and improved hygienic practices, especially with food. use of human feces for fertilizer, even after partial treatment, may spread the infestation. mass treatment is indicated in high prevalence communities. pinworm disease or enterobiasis. pinworm disease (oxyuriasis) is common worldwide in all socioeconomic classes; however, it is more widespread when crowded and unsanitary living conditions exist. the enterobius vermicularis infestation of the intestine may be symptomless or may cause severe perianal itching or vulvovaginitis. it primarily affects schoolchildren and preschoolers. more severe complications may occur. adult worms may be seen visually or identified by microscopic examination of stool specimens or perianal swabs. transmission is by the oral-fecal ingestion of eggs. the larvae grow in the small intestine and upper colon. prevention is by educating the public regarding hygiene and adequate sanitary facilities, as well as by treating cases and investigating contacts. treatment is the same as for ascariasis. mass treatment is indicated in high prevalence communities. ectoparasites. ectoparasites include scabies (sarcoptes scabiei), the common bed bug (cimex lectularius), fleas, and lice, including the body louse (pediculus humanis), pubic louse (phthirius pubis), and the head louse (pediculus humanus capitis). their severity ranges from nuisance value to serious public health hazard. head lice are common in schoolchildren worldwide and are mainly a distressing nuisance. the body louse serves as a vector for epidemic typhus, trench fever, and louse-borne relapsing fever. in disaster situations, disinfection and hygienic practices may be essential to prevent epidemic typhus. the flea plays an important role in the spread of the plague by transmitting the organism from the rat to humans. control of rats has reduced the flea population, but during war and disasters, rat and flea populations may thrive. scabies, which is caused by a mite, is common worldwide and is transmitted from person to person. the mite burrows under the skin and causes intense itching. all of these ectoparasites are preventable by proper hygiene and the treatment of cases. the spread of these diseases is rapid and therefore warrants attention in school health and public health policy. legionnelae, a gram-negative group of bacilli, with 35 species and many serogroups. the first documented case was reported in the united states in 1947, and the first disease outbreak was reported in the united states in 1976 among participants of a war veterans convention. general malaise, anorexia, myalgia, and headache are followed by fever, cough, abdominal pain, and diarrhea. pneumonia followed by respiratory failure may follow. the case fatality rate can be as high as 40% of hospitalized cases. a milder, nonpneumonic form of the disease (pontiac fever) is associated with virtually no mortality. the organism is found in water reservoirs and is transmitted through heating, cooling, and air conditioning systems, as well as from tap water, showers, saunas, and jaccuzzi baths. the disease has been reported in australia, canada, south america, europe, israel, and on cruise ships. prevention requires the cleaning of water towers and cooling systems, including whirlpool spas. hyperchlorination of water systems and the replacement of filters is required where cases and/or organisms have been identified. antibiotic treatment with erythromycin is effective. leprosy (hansen's disease) was widely prevalent in europe and mediterranean countries for many centuries, with some 19,000 leprosaria in the year 1300. leprosy was largely wiped out during the black death in the fourteenth century, but continued in endemic form until the twentieth century. leprosy is a chronic bacterial infection of the skin, peripheral nerves, and upper airway. in the lepromatous form, there is diffuse infiltration of the skin nodules and macules, usually bilateral and extensive. the tuberculoid form of the disease is characterized by clearly demarcated skin lesions with peripheral nerve involvement. diagnosis is based on clinical examination of the skin and signs of peripheral nerve damage, skin scrapings, and skin biopsy. transmission of the mycobacterium leprae organism is by close contact from person to person, with incubation periods of between 9 months and 20 years (average of 4-8 years). rifampicin and other medications make the patient noninfectious in a short time, so that ambulatory treatment is possible. multidrug therapy (mdt) has been shown to be highly effective in combating the disease, with a very low relapse rate. treatment with mdt ensures that the bacillus does not develop drug resistance. mdt is covering 91% of known cases in 1996, according to who reports, as compared to only 55% in 1994. the increase has been associated with improved case finding. bcg may be useful in reducing tuberculoid leprosy among contacts. investigation of contacts over 5 years is recommended. the disease is still highly endemic primarily in five countries, india, brazil, indonesia, myanmar, and bangladesh, and is still present in some 80 countries in southeast asia, including the philippines and burma, sub-saharan africa, the middle east (sudan, egypt, iran), and in some parts of latin america (mexico, colombia) with isolated cases in the united states. world prevalence has declined from 10.5 million cases in 1980, 5.5 million in 1990, to less than 1 million cases in 1995. the world health organization expects to eliminate leprosy as a public health problem by the year 2000, defined as prevalence of less than 1 per 10,000 population, or less than 300,000 cases. trachoma is currently responsible for 6 million blind persons or 15% of total blindness in the world. the causative organism, chlamydia trachomatis, is a bacteria which can survive only within a cell. it is spread through contact with eye discharges, usually by flies, or household items (e.g., handkerchiefs, washcloths). trachoma is common in poor rural areas of central america, brazil, africa, parts of asia, and some countries in the eastern mediterranean. the resulting infection leads to conjuncfival scarring and if untreated, to blindness. who estimates there are 148 million cases of active disease in 46 endemic countries. hygiene, vector control, and treatment with antibiotic eye ointments or simple surgery for scarring of eyelids and inturned eyelashes prevent the blindness. a new drug, azithromycin, is effective in curing the disease. the who is promoting a program for the global elimination of trachoma using azithromycin and hygiene education in endemic areas. chlamydia (chlamydia pneumonia) is suspected of playing a role in coronary artery disease by intraarterial infection, with plaque formation and occlusion of the artery by thrombi consisting mainly of platelets. if borne out, this will provide potential for low cost intervention to reduce the burden of the leading worldwide cause of death. sexually transmitted diseases (stds) are widespread internationally with an estimated 330 million new cases per year, with 5.8 million new cases, over 30 million total cases, and 2.3 million deaths (1997), aids has captured world attention over the past decade. the global burden of stds is enormous (table 4 .8), and the public health and social consequences are devastating in many countries. sexually transmitted diseases, especially in women, may be asymptomatic, so that severe sequelae may occur before patients seek care. infection by one std increases risk of infection by other diseases in this group. syphilis is caused by the spirochete treponema pallidum. after an incubation period of 10-90 days (mean -21), primary syphilis develops as a painless ulcer or chancre on the penis, cervix, nose, mouth, or anus, lasting 4-6 weeks. the patient may first present with secondary syphilis 6-8 weeks (up to 12 weeks) after infection with a general rash and malaise, fever, hair loss, arthritis, and jaundice. these symptoms spontaneously disappear within weeks or up to 12 months later. tertiary syphilis may appear 5-20 years after initial infection. complications of tertiary syphilis include catastrophic cardiovascular and central nervous system conditions. early antibiotic treatment is highly effective when given in a large initial dose, but longer term therapy may be needed if treatment is delayed. gonorrhea (gc) is caused by the bacterium neisseria gonorrhoeae. the incubation period is 1-14 days. gonorrhea is often associated with concurrent chlamydia infection. in women, gc may be asymptomatic or it may cause vaginal discharge, pain on urination, bleeding on intercourse, or lower abdominal pain. untreated, it can lead to sterility. in men, gc causes urethral discharge and painful urination. treatment with antibiotics ends infectivity, but untreated cases can be infectious for months. drug resistance to penicillin and tetracycline has increased in many countries so that more expensive and often unavailable drugs are necessary for treatment. prevention of gonococcal eye infection in newborns is based on routine use of antibiotic ointments in the eyes of newborns. chancroid. chancroid is caused by haemophilus ducreyi. in women chancroids may cause a painful, irregular ulcer near the vagina, resulting in pain on in-tercourse, urination, and defection, but it may be asymptomatic. in men it causes a painful, irregular ulcer on the penis. the incubation period is usually 3-5 days, but may be up to 14 days. an individual is infectious as long as there are ulcers, usually 1-3 months. treatment is by erythromycin or azithromycin. herpes simplex. herpes simplex is caused by herpes simplex virus types 1 and 2 and has an incubation period of 2-12 days. genital herpes causes painful blisters around the mouth, vagina, penis, or anus. the genital lesions are infectious for 7-12 days. herpes may lead to central nervous system meningoencephalitis infection. it can be transmitted to newborns during vaginal delivery, causing infection, encephalitis, and death. cesarian delivery is therefore necessary when a mother is infected. anti-viral drugs are used in treatment, orally, topically, or intravenously. chlamydia. chlamydia is caused by chlamydia trachomatis. in women, it is usually asymptomatic but may cause vaginal discharge, spotting, pain on urination, lower abdominal pain, and pelvic inflammatory disease (pid). in newborns, chlamydia may cause eye and respiratory infections. in men, chlamydia causes urethral discharge and pain on urination. the incubation period is 7-21 days and the infectious period is unknown. treatment for chlamydia is doxycycline, azithromycin, or erythromycin. chlamydia infection, not necessarily venereal in transmission, may be transmitted to newborns of infected mothers. chlamydia pneumoniae, presently under investigation as a possible cause or contributor to coronary heart disease, and is widespread in poor hygenic conditions. trichomoniasis. trichomoniasis is caused by trichomonas vaginalis. the incubation period is 4-20 days (mean = 7). in women, trichomoniasis may be asymptomatic or may cause a frothy vaginal discharge with foul odor, and painful urination and intercourse. in men, the disease is usually mild, causing pain on urination. treatment is by metronidazole taken orally. without treatment, the disease may persist and remain infectious for years. (hpv). it is a sporadic disease which may be associated with cervical neoplasia and cancer of the cervix. hpv includes many types associated with a variety of conditons. the search for a hpv vaccine to prevent cancer of the cervix looks promising. in areas where a full range of diagnostic services is lacking, a "syndromic approach" is recommended for the control of stds. the diagnosis is based on a group of symptoms and treatment on a protocol addressing all the diseases that could possibly cause those symptoms, without expensive laboratory tests and repeated visits. early treatment without laboratory confirmation helps to cure persons who might not return for follow-up, or may place them in a noninfective stage so that even without follow-up they will not transmit the disease. std incidence between 1950 and 1996 is shown in table 4 .9, with decline overall except around 1990, with subsequent further fall in incidence. screening in prenatal and family planning clinics, prison medical services, and selected years 1950 -1996 disease 1950 1960 1970 1980 1985 1996 syphilis ( [1985] [1986] [1987] [1988] [1989] [1990] and subsequent decline by more than 50% in reported cases includes all three stages of the disease as well as congential syphilis. rates are cases per 100,000 population, rounded. in clinics serving prostitutes, homosexuals, or other potential risk groups will detect subclinical cases of various stds. treatment can be carried out cheaply and immediately. for instance, the screening test for syphilis costs $0.10 and the treatment with benzathine penicillin injection costs about $0.40 in 1998. partner notification is a controversial issue, but may be needed to identify contacts who may be the source of transmission to others. control of stds through a syndrome approaach based on primary care providers is being promoted by who. health education directed at high risk target groups is essential. providing easy and cost-free access to acceptable, nonthreatening treatment is vital in promoting the early treatment of cases and thereby reducing the risk of transmission. promoting prevention through the use of condoms and/or monogamy requires long-term educational efforts that are now fostered by the hiv/aids pandemic. increased use of condoms for hiv prevention is associated with reduced risk of other stds. training medical care providers in std awareness should be stressed in undergraduate and continuing educational efforts including personal protection as care givers. human immunodeficiency virus (hiv) is a retrovirus that infects various cells of the immune system, and also affects the central nervous system. two types have been identified: hiv 1, worldwide in distribution, and the less pathogenic hiv2, found mainly in west africa. hiv is transmitted by sexual contact, exposure to blood and blood products, perinatally, and via breast milk. the period of communicability is unknown, but studies indicate that infectiousness is high, both during the initial period after infection and later in the disease. antibodies to hiv usually appear within 1-3 months. within several weeks to months of the infection, many persons develop an acute self-limited flulike syndrome. they may then be free of any signs or symptoms for months to more than 10 years. onset of illness is usually insidious with nonspecific symptoms, including sweats, diarrhea, weight loss, and fatigue. aids represents the later clinical stage of hiv infection. according to the revised cdc case definition (1993), aids involves any one or more of the following: low cd4 count, severe systematic symptoms, opportunistic infections such as pneumocystis pneumonia or tb, aggressive cancers such as kaposi's sarcoma or lymphoma, and/or neurological manifestations, including dementia and neuropathy. the who case definition is more clinically oriented, relying less on often unavailable laboratory diagnoses for indicator diseases. aids was first recognized clinically in 1981 in los angeles and new york. by mid-1982 it was considered an epidemic in those and other u.s. cities. it was primarily seen among homosexual men and recipients of blood products. after initial errors, testing of blood and blood products became standard and has subsequently closed off this method of transmission. transmission has changed markedly since the initial onslaught of the disease, with needle sharing among intravenous drug users, heterosexual, and maternal-fetal transmission becoming major factors. comorbidity with other stds apparently increases hiv infectivity and may have helped to convert the epidemiology to a greater degree of heterosexual transmission. the disease grew exponentially in the united states (table 4 .10), but incidence of new cases nas declined since 1993. aids has become a major public health problem in most developed and developing countries, reaching catastrophic proportions in some sub-saharan african countries affecting up to 30% of the population. hiv-related deaths were the eighth leading cause of all deaths in 1993 in the u.s., the leading cause among men aged 25-44 years of age, and the fourth leading cause for women in this age group. by 1996, aids had been diagnosed in 548,000 persons and 343,000 had died. it is estimated that up to 1 million persons are hiv infected in the united states. globally, deaths from aids totalled 2.3 million in 1997, with an estimated 11.7 million person having died from this pandemic up to 1997. in 1998, an estimated 3.1 million person were hiv infected with 5.8 million new infection in 1997. the declining incidence of new cases in the industrialized countries may be the result of greater awareness of the disease and methods of prevention of transmission. improving early diagnosis and access to care, especially the combined therapy programs that are very effective in delaying onset of symptoms, are important parts of public health management of the aids crisis. until an effective vaccine is available, preventive reliance will continue to be on behavior risk-reduction and other prevention strategies such as needle and condom distribution among high risk population groups. throughout the world, hiv continues to spread rapidly, especially in poor countries in africa, asia, and south and central america. the united nations reports that 21 million persons are living with hiv/aids, 90% of them in developing countries, where transmission is 85% by heterosexual contact. every day, more than 8500 persons are infected, including 1000 children. in thailand, 1 person in 50 is now infected. in sub-saharan africa 1 person in 40 is infected, and in some cities as many as 1 person in 3 carries the virus. estimations of new infections per year in sub-saharan africa range from 1 to 2 million persons, while in asia the range is from 1.2 to 3.5 million new infected persons per year. lessons are still being learned from the aids pandemic. the explosive spread of this infection, from an estimated 100,000 people in 1980 to an anticipated 40 million persons hiv infected, shows that the world is still vulnerable to pandemics of "new" infectious diseases. enormous movements of tourists, business people, truck drivers, migrants, soldiers, and refugees promote the spread of such diseases. widespread sexual exchange, traffic in blood products, and illicit drug use all promote the international potential for pandemics. war and massive refugee situations promote rape and prostitution, worsening the aids situation in some settings in africa. hiv has arrived in almost every country. however, there is the somewhat hopeful indication that the rate of increase, has slowed in the united states. this may be an indication either of higher levels of self-protective behavior, or that the most susceptible population groups have already been affected and the spread into the general population is at a slower rate. it is also possible that this may yet prove to be only a lull in the storm, as heterosexual contact becomes a more important mode of transmission. the eleventh international conference on aids, held in vancouver, canada, in july 1996, reported signs that combinations of several drugs from among a number of antiretroviral medications are showing promise to suppress the aids virus in infected people. at a current annual price of $10,000-15,000 per patient, these sums well beyond the capacity of most developing countries. development of methods of measuring the hiv viral load have allowed for better evaluation of potential therapies and monitoring of patients receiving therapy. in developed countries, transmission by blood products has been largely controlled by screening tests; transmission among homosexuals has been reduced by safe sex practices; transmission to newborns has been reduced by recent therapeutic advances. safe sex practices and condom use may have helped in reducing heterosexual transmission. further advances in therapy and prevention with a vaccine are expected over the next decade. the hiv/aids pandemic is one of the great challenges to public health for the 21st century due to its complexity, its international spread, its sexual and other modes of transmission, its devastating and costly clinical effects, and its impact on parallel diseases such as tuberculosis, respiratory infections, and cancer. the cost of care for the aids patient can be very high. needed programs include home care and community health workers to improve nutrition and self-care, and mutual help among hiv carriers and aids patients. the ethical issues associated with aids are also complex regarding screening of pregnant women, newborns, partner notification, reporting, and contact tracing, as well as financing the cost of care. diarrheal diseases are caused by a wide variety of bacteria, parasites, and viruses (table 4 .11) infecting the intestinal tract and causing secretion of fluids and dissolved salts into the gut with mild to severe or fatal complications. in developing countries, diarrheal diseases account for half of all morbidity and a quarter of all mortality. diarrhea itself does not cause death, but the dehydration resulting from fluid and electrolyte loss is one of the most common causes of death in children worldwide. deaths from dehydration can be prevented by use of oral rehydration therapy (ort), an inexpensive and simple method of intervention easily used by a nonmedical primary care worker and by the mother of the child as a home intervention. in 1983, diarrheal diseases were the cause of almost 4 million child deaths, but by 1996 this had declined to 2.4 million, largely under the impact of increased use of ort. diarrheal diseases are transmitted by water, food, and directly from person to person via oral-fecal contamination. diarrheal diseases occur in epidemics in situations of food poisoning or contaminated water sources, but can also be present at high levels when common source contamination is not found. contamination of drinking water by sewage and poor management of water supplies are also major causes of diarrheal disease. the use of sewage for the irrigation of vegetables is a common cause of diarrheal disease in many areas. salmonella are a group of bacterial organisms causing acute gastroenteritis, associated with generalized illness including headache, fever, abdominal pains, and dehydration. there are over 2000 serotypes of salmonella, many of which are pathogenic in humans, the most common of which are salmonella typhimurium, s. enteritidis, and s. typhi. transmission is by ingestion of the organisms in food, derived from fecal material from animal or human contamination. common sources include raw or uncooked eggs, raw milk, meat, poultry and its products, as well as pet turtles or chicks. fecal-oral transmission from person to person is common. prevention is in safe animal and food handling, refrigeration, sanitary preparation and storage, protection against rodent and insect contamination, and the use of sterile techniques during patient care. antibiotics may not eliminate the carrier state and may produce resistant strains. shigella are a group of bacteria that are pathogenic in man, with four groups: type a = shigella dysenteriae, type b = s. flexneri, type c = s. boydii, and type d = s. sonnei. types a, b, and c are each further divided into a total of 40 serotypes. shigella are transmitted by direct or indirect fecal-oral methods from a patient or carrier, and illness follows ingestion of even a few organisms. water and milk transmission occurs as a result of contamination. flies can transmit the organism, and in nonrefrigerated foods the organism may multiply to an infectious dose. control is in hygienic practices and in the safe handling of water and food. escheria eoli e. coli are common fecal contaminants of inadequately prepared and cooked food. particularly virulent strains such as o 157:h17 can cause explosive outbreaks of severe (enterohemmorhagic) diarrhoeal disease with a hemolytic-uremic syndrome and death, as occurred in japan in 1998 with cases and deaths due to a foodborne epidemic. other milder strains cause travellers diarrhoea and nursery infections. inadequately cooked hamburger, unpasturized milk, and other food vectors are discussed under food safety in chapter 8. cholera is an acute bacterial enteric disease caused by vibrio cholerae, with sudden onset, profuse painless watery stools, occasional vomiting, and, if untreated, rapid dehydration, and circulatory collapse, and death. asymptomatic infection or carrier status, and mild cases are common. in severe, untreated cases, mortality is over 50%, but with adequate treatment, mortality is under 1%. diagnosis is based on clinical signs, epidemiologic, serologic and bacteriologic confirmation by culture. the two types of cholera are the classic and el tor (with inaba and ogawa serotypes). in 1991, a large scale epidemic of cholera spread through much of south america. it was imported via a chinese freighter, whose sewage contaminated shellfish in lima harbor in peru (box 4.12). the south american cholera epidemic has caused hundreds of thousands of cases and thousands of deaths since 1991. prevention requires sanitation, particularly the chlorination of drinking water, prohibiting the use of raw sewage for the irrigation of vegetable crops, and high standards of community, food, and personal hygiene. treatment is prompt fluid therapy with electrolytes in large volume to replace all fluid loss. oral rehydration should be accomplished using standard ort. tetracycline shortens the duration of the disease, and chemoprophylaxis for contacts following stool samples may help in reducing its spread. a vaccine is available but is of no value in the prevention of outbreaks. viral gastroenteritis can occur in sporadic or epidemic forms, in infants, children, or adults. some viruses, such as the rotaviruses and enteric adenoviruses, afin the 1980s, peruvian officials stopped the chlorination of community water supplies because of concern over possible carcinogenic effects of trihalomethanes, a view encouraged by officials of the u.s. environmental protection agency (epa) and the u.s. public health service. in january 1991, a chinese freighter arrived in lima, peru, and dumped bilge (sewage) in the harbor, apparently contaminating local shellfish. consumption of raw shellfish is a popular local delicacy (ceviche) and associated with cases of cholera seen in local hospitals. contamination of local water supplies from sewage resulted in the geometric increase in cases, and by the end of 1992 the pan american health organization (paho) reported an epidemic of 391,000 cases and 4002 deaths. the epidemic spread to 21 countries, and in 1992 there were a further 339,000 cases and 2321 deaths spreading over much of south america, continuing in 1999. in the united states, 102 cases of cholera were reported in 1992; of these, 75 cases and 1 death were among passengers of an airplane flying from south america to los angeles in which contaminated seafood was served. in 1993, 91 cases of cholera were reported in the united states which were unrelated to international travel. these occurred mostly among persons consuming shellfish from the gulf coast with a strain of cholera similar to the south american strain, also possibly introduced in ship ballast. cholera organisms are reported in harbor waters in other parts of the united states (promed, 1999 , 1991 promed, 1999. fect mainly infants and young children, and may be severe enough to cause hospitalization for dehydration. others such as norwalk and norwalk-like viruses affect older children and adults in self-limited acute gastroenteritis in family, institution, or community outbreaks. rotaviruses cause acute gastroenteritis in infants and young children, with fever and vomiting, followed by watery diarrhea and occasionally severe dehydration and death if not adequately treated. diagnosis is by examination of stool or rectal swabs with commercial immunologic kits. in both developed and developing countries, rotavirus is the cause of about one-third of all hospitalized cases for diarrheal diseases in infants and children up to age 5. most children in developing countries experience this disease by the age of 4 years, with the majority of cases between 6 and 24 months. in developing countries, rotaviruses are estimated to cause over 800,000 deaths per year. the virus is found in temperate climates in the cooler months and in tropical countries throughout the year. breastfeeding does not prevent the disease but may reduce its severity. oral rehydration therapy is the key treatment. a live attenuated vaccine was approved by the fda in 1998 and adopted in the 1999 u.s. recommended routine vaccination programs for infants. adenoviruses. adenoviruses, norwalk, and a variety of other viruses (including astrovirus, calcivirus, and other groups) cause sporadic acute gastroenteritis worldwide, mostly in outbreaks. spread is by the oral-fecal route, often in hospital or other communal settings, with secondary spread among family contacts. food-borne and waterborne transmission are both likely. these can be a serious problem in disaster situations. no vaccines are available. management is with fluid replacement and hygienic measures to prevent secondary spread. giardiasis. giardiasis (caused by giardia lamblia) is a protozoan parasitic infection of the upper small intestine, usually asymptomatic, but sometimes associated with chronic diarrhea, abdominal cramps, bloating, frequent loose greasy stools, fatigue, and weight loss. malabsorption of fats and vitamins may lead to malnutrition. diagnosis is by the presence of cysts or other forms of the organism in stools, duodenal fluid, or in intestinal mucosa from a biopsy. this disease is prevalent worldwide and affects mostly children. it is spread in areas of poor sanitation and in preschool settings and swimming pools, and is of increasing importance as a secondary infection among immunocompromised patients, especially those with aids. waterborne giardia was recognized as a serious problem in the united states in the 1980s and 1990s, since the protozoa is not readily inactivated by chlorine, but requires adequate filtration before chlorination. person-to-person transmission in day-care centers is common, as is transmission by unfiltered stream or lake water where contamination by human or animal feces is to be expected. an asymptomatic carrier state is common. prevention relies on careful hygiene in settings such as day-care centers, filtration of public water supplies and the boiling of water in emergency situations. cryptosporidium. cryptosporidium parvum is a parasitic infection of the gastrointestinal tract in man, small and large mammals and vertebrates. infection may be asymptomatic or cause a profuse, watery diarrhea, abdominal cramps, general malaise, fever, anorexia, nausea, and vomiting. in immunosuppressed patients, such as persons with aids, it can be a serious problem. the disease is most common in children under 2 years of age and those in close contact with them, as well as in homosexual men. diagnosis is by identification of the cryptosporidium or-ganism cysts in stools. the disease is present worldwide. in europe and the united states, the organism has been found in <1 to 4.5% of individuals sampled. spread is common by person-to-person contact by fecal-oral contamination, especially in such settings as day-care centers. raw milk and waterborne outbreaks have also been identified in recent years. a large waterborne disease outbreak due to cryptosporidium occurred in milwaukee in 1986 described in chapter 9. management is by rehydration and prevention is by careful hygiene in food and water safety. helicobacter pylori. helicobacter pylori, first identified in 1986, is a bacterium causally linked to duodenal ulcers and gastritis, contributing to high rates of gastric cancer (chapter 5). it is an important example of the link between infection and chronic disease. this has enormous implications for prevention of cancer of the stomach, chronic peptic ulcers and large-scale use of hospitals and other medical resources (see chapter 5). the control of diarrheal diseases requires a comprehensive program involving a wide range of activities, including good management of food and water supplies, education in hygiene, and, particularly where morbidity and mortality are high, education in the use of oral rehydration therapy (ort). oral rehydration therapy (ort) is considered by unicef and who to have resulted in the saving of 1 million lives each year in the 1990s. proper management of an episode of diarrhea by ort (table 4 .12), along with continued feeding, not only saves the child from dehydration and immediate death, but also contributes to early restoration of nutritional adequacy, sparing the child the prolonged effects of malnutrition. the world summit for children (wsc) in 1990 called for a reduction in child deaths from diarrheal diseases by one-third and malnutrition by one-half, with emphasis on the widest possible availability, education for, and use of ort. this requires a programmatic approach. public health leadership must train primary care doctors, pediatricians, pharmacists, drug manufacturers, and primary care health workers of all kinds in ort principles and usage. they must be backed by the widest possible publicity to raise awareness among parents. oral rehydration therapy is an important public health modality in developed countries as well as in developing countries. diarrhoeal disease may not cause death as frequently in developed countries, but it is still a significant factor in infant and child health and, even under the most optimal conditions, can cause setbacks in the nutritional state and physical development of a child. use of ort does not prevent the disease (i.e., it is not a primary prevention), but it is excellent in secondary prevention, by preventing complications from diarrhoea, and should be available in every home for symptomatic treatment of diarrheal diseases. an adaptation of ort has found its place in popular culture in the united states. a form of ort, marketed as "sports drinks," is used in sports where athletes lose large quantifies of water and salts in sweat and insensible loss from the respiratory tract. the wider application of the principles of ort for use in adults in dry hot climates and in adults under severe physical exertion with inadequate fluid/salt intake situations requires further exploration. management of diarrheal diseases should be part of a wider approach to child nutrition. the child who goes through an episode of diarrheal disease may have a faltering in growth and development. supportive measures may be needed following the episode as well as during it. this involves providing primary care services that are attuned to monitoring individual infant and child growth. growth monitoring surveillance is important to assess the health status of the individual child and the child population. supplementation of infant feeding with vitamins a and d, and iron to prevent anemia are important for routine infant and child care, and more so for conditions affecting total nutrition such as a diarrheal disease. in the developing world, respiratory infections account for over one-quarter of all deaths and illnesses in children. as diarrheal disease deaths are reduced, the major cause of death among infants in developing countries is becoming acute respiratory infections (aris). in industrialized countries, aris are important for their potentially devastating effects on the elderly and chronically ill. they are also the major cause of morbidity in infants in developed countries, causing much anxiety to parents even in areas with good living conditions. cigarette smoking, chronic bronchitis, poorly controlled diabetes or congestive heart failure, and chronic liver and kidney disease increase susceptibility to aris. aris place a heavy burden on health care systems and individual families. improved methods of management of such chronic diseases are needed to reduce the associated toll of morbidity, mortality, and the considerable expenses of health care. acute respiratory infections are due to a broad range of viral and, to a lesser extent, bacterial infections. it is the latter which can progress to pneumonia with mortality rates of 10-20%. acute viral respiratory diseases include those affecting the upper respiratory tract, such as acute viral rhinitis, pharyngitis, and laryngitis, as well as those affecting the lower respiratory tract, tracheobronchitis, bronchitis, bronchiolitis, and pneumonia. aris are frequently associated with vaccine-preventable diseases, including measles, varicella, and influenza. they are caused by a large number of viruses, producing a wide spectrum of acute respiratory illness. some organisms affect any part of the respiratory tract, while others affect specific parts and all predispose to bacterial secondary infection. while children and the elderly are especially susceptible to morbidity and mortality from acute respiratory disease, the vast numbers of respiratory illnesses among adults cause large-scale economic loss from work absence. bacterial agents causing upper respiratory tract infection include group a streptococcus, mycoplasma pneumonia, pertussis, and parapertussis. pneumonia or acute bacterial infection of the lower respiratory tract and lung tissue may be due to pneumococcal infection with streptococcus pneumoniae. there are 83 known types of this organism, distinguished by capsule characteristics; 23 account for 88% of pneumococcal infections in the united states. an excellent polyvalent vaccine based on these types is available for high risk groups such as the elderly, immunodeficient patients, and persons with chronic heart, lung, liver, blood disorders, or diabetes. opportunistic infections attack the chronically ill, especially those with compromised immune suystems, often with life-threatening aris. mycoplasma (primary atypical pneumonia) is a lower respiratory tract infection which sometimes progresses to pneumonia. tb and pneumonocytis carynia are especially problematic for aids patients. other organisms causing pneumonias include chlamydia pneumoniae, h. influenza, klebsiella pneumonia, escherichia coli, staphylococcus, rickettsia (q fever), and legionella. parasitic infestation of lungs may occur with nematodes (e.g., ascariasis). fungal infections of the lung may be caused by aspergillosis, histoplasmosis, and coccidiomycosis, often as a complication of antibiotic therapy. access to primary care and early institution of treatment are vital to control excess mortality from aris. in developed countries, aris as contributors to infant deaths are largely a problem in minority and deprived population groups. because these groups contribute disproportionately to childhood mortality, infant mortality reduction has been slower in countries such as the united states and russia than in other industrialized countries. the continuing gap in mortality rates between white and black children in the united states can, to a large extent, be attributed to aris and less access to organized primary care. children are brought to emergency rooms for care when the disease process is already advanced and more dangerous than had it been attended to professionally earlier in the process. many field trials of ari prevention programs have been proved successful involving parent education and training of primary care workers in early assessment and, if necessary, initiation of treatment. this needs field testing in multiple settings. reliance on vaccines to prevent respiratory infectious diseases is not currently feasible. aris are caused by a very wide spectrum of viruses, and the development of vaccines in this field has been slow and limited. the vaccine for pneumococcal pneumonia has been an important breakthrough, but it is still inadequately utilized by the chronically ill because of its limitations, costs, and lack of sufficient awareness, and it is too expensive for developing countries. improvements in bacterial and viral vaccine development will potentially help to reduce the burden of aris. a programmatic approach with clinical guidelines and education of family and care givers is currently the only feasible way to reduce the still enormous morbidity and mortality from aris on the young and the elderly. the success of sanitation vaccines and antibiotics led many to assume that all infectious diseases would sooner or later succumb to public health and medical technology. unfortunately, this is a premature and even dangerous assumption. despite the longstanding availability of an effective and inexpensive vaccine, the persistence of measles as a major killer of 1 million children per year represents a failure in effective use of both the vaccine and the health system. the resurgence of tb and malaria have led to new strategies, such as managed or directly observed care, with community health workers to assure compliance needed to render the patient noninfectious to others and to reduce the pool of carriers of the disease. current successes in reducing poliomyelitis, dracunculiasis, onchocerciasis, and other diseases to the point of eradication has raised hopes for similar success in other fields. but there are many infectious diseases of importance in developed and developing countries where existing technologies are not fully utilized. oral rehydration therapy (ort) is one of the most cost-effective methods of preventing excess mortality from ordinary diarrheal diseases, and yet is not used on sufficient scale. biases in the financing and management of medical insurance programs can result in underutilization of available effective vaccines. hospital-based infections cause large-scale increases in lengths of stay and expenditures, although application of epidemiologic investigation and improved quality in hospital practices could reduce this burden. control of the spread of aids using combined medical therapies is not financially or logistically possible in many countries, but education for "safe sex" is effective. community health worker programs can greatly enhance tuberculosis, malaria, and std control, or in aids care, promote prevention and appropriate treatment. in the industrialized and mid-level developing countries, epidemiologic and demographic shifts have created new challenges in infectious disease control. prevention and early treatment of infectious disease among the chronically ill and the elderly is not only a medical issue, it is also an economic one. patients with chronic obstructive lung disease (copd), chronic liver or kidney disease, or congestive heart failure are at high risk of developing an infectious disease followed by prolonged hospitalization. public health has addressed, and will continue to stress the issues of communicable disease as one of its key issues in protecting individual and population health. methods of intervention include classic public health through sanitation, immunization, and well beyond that into nutrition, education, case finding, and treatment, and changing human behavior. the knowledge, attitudes, beliefs, and practices of policy makers, health care providers, and parents is as important in the success of communicable disease control as are the technology available and methods of financing health systems. together, these encompass the broad programmatic approach of the new public health to control of communicable diseases. in a world of rapid international transport and contact between populations, systems are needed to monitor the potential explosive spread of pathogens that may be transferred from their normal habitat. the potential for the international spread of new or reinvigorated infectious diseases constitute threat to mankind akin to ecological and other man-made disasters. the eradication of smallpox paved the way for the eradication of poliomyelitis, and perhaps measles, in the foreseeable future. new vaccines are showing the capacity to reduce important morbidity from rubella syndrome, mumps, meningitis, and hepatitis. other new vaccines on the horizon will continue the immunologic revolution into the twenty-first century. as the triumphs of control or elimination of infectious diseases of children continue, the scourge of hiv infection continues with distressingly slow progess an effective vaccine or cure for the disease it engenders. partly as a result of the hiv/ aids, tb staged a comeback in many countries where it was thought to be merely a residual problem. at the same time an old/new method of intervention using directly observed short-term therapy has shown great success in controlling the tb epidemic. the resurgence of tb is more dangerous in that mdrtb has become a widespread problem. this issue highlights the difficulty of keeping ahead of drug resistance in the search for new generations of antibiotics, posing a difficult challenge for the pharmaceutical industry, basic scientists as well as public health workers. the burden of infectious diseases has receded as the predominant public health problem in the developed countries but remains large in the developing countries. with increases in longevity and increased importance of chronic disease in the health status of the industrial and mid-level developing nations, the effects of infectious disease on the care of the elderly and chronically ill is of great importance in the new public health. long-term management of chronic disease needs to address the care of vulnerable groups, promoting the use of existing vaccines and antibiotics. most important is the development of health systems that provide close monitoring of groups at special risk for infectious disease, especially patients with chronic diseases, the immunocompromised, and the elderly. the combination of traditional public health with direct medical care needed for effective control and eradication of communicable diseases is an essential element of the new public health. the challenge is to apply a comprehensive approach and management of resources to define and reach achievable targets in communicable disease control. access to e-mail and the internet are vital to current practice of public health and nowhere is this more important than in communicable diseases. there are many such information sites and these will undoubtedly expand in the coming years. several sites are given as examples. the internet has great practical implications for keeping up to date with rapidly occurring events in this field. outstanding encyclopedia database on infectious diseases (available via mdcassoc@ix.netcom.com at reduced price for promed users, and free to sub-saharan african sites) promed is an excellent, free report on current events in communicable diseases internationally; join via owner-promed @usa recommended readings centers for disease control. 1992. update: international task force for disease eradication addressing emerging infectious disease threats: a prevention strategy for the united states. executive summary update: trends in aids incidence--united states one thousand days until the target date for global poliomyelitis eradication tuberculosis morbidity--united states measles--united states, 1997. morbidity and mortality weekly report national adult immunization awareness week--october 11-17, recommended readings 1998; and influenza and pneumococcal vaccination levels among adults aged ---65 years impact of the sequential ipv/opv schedule on vaccination cover-agemunited states advances in global measles control and elimination: summary of the 1997 international meeting recommended childhood immunization schedulemunited states impact of vaccines universally recommended for childrenmunited states progress toward global poliomyelitis eradication global disease elimination and eradication as public health strategies childhood immunizations rotavirus vaccines: who position paper. weekly epidemiologic record infectious diseases of humans: dynamic and control vaccines and world health: science, policy, and practice control of communicable diseases manual jawetz, melnick and adelberg's medical microbiology, twenty-first edition preventive medicine and public health, second edition efficacy of bcg vaccine in the prevention of tuberculosis. meta-analysis of the published literature manson's tropical diseases vaccination and world health principles and practice oflnfectious diseases immunization of adolescents: recommendations of the advisory committee on immunization practices, the american academy of pediatrics, the american academy of family physicians and the combination vaccines for childhood immunization: recommendations of the advisory committee on immunization practices, the american academy of pediatrics, the american academy of family physicians and the poliomyelitis prevention: revised recommendations for use of inactivated and live oral poliovirus vaccines diphtheria outbreakmrussian federation rubella and congenital rubella syndrome~united states compendium of animal rabies control, 1996: national association of state public health veterinarians progress toward elimination of haemophilus influenzae type b disease among infants and children in the united states tetanus surveillance~united states, 1991-1994 recommendations and reports--vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of measles: recommendations of the advisory committee on immunization practices national, state and urban area vaccination coverage levels among children aged 19-35 months~united sates varicella related deaths among children--united states progress toward global poliomyelitis eradication ten great public health achievements--united states a ten-year experience in control of poliomyelitis through a combination of live and killed vaccines in two developing areas measles control in developing and developed countries: the case for a two-dose policy integration of vitamin a supplementation with immunization. weekly epidemiological record update cholera--western hemisphere, 1992. morbidity and mortality weekly report isolation of vibrio cholerae o 1 from oystersmmobile bay, 1991-1992 estimates of future global tuberculosis morbidity and mortality arbovirus disease--united states ~:~50 other communicable diseases update: outbreak of legionnaire's disease associated with a cruise ship rift valley fever--egypt the role of bcg vaccine in the prevention and control of tuberculosis in the united states: a joint statement by the advisory council for the elimination of tuberculosis and the advisory committee on immunization practices update: trends in aids incidence--united states case definition for infectious conditions under public health surveillance guidelines for treatment of sexually transmitted diseases primary and secondary syphilis--united states global tuberculosis incidence and mortality during the 20th century pandemic: need for surveillance and research escherichia coli o157:h7 diarrhoea in the united states: clinical and epidemiologic features the state of the world's children the rational use of drugs in the management of acute diarrhoea in children world health organization. 1994. the malaria situation in 1991 aids: images of the epidemic. geneva: who. world health organization progress toward the elimination of leprosy as a public health problem the world health report 1996: fighting disease, fostering development the world health report health for all in the twenty-first century. eb 101/8. geneva: who. world health organization. 1998. the world health report 1998: life in the twenty-first century: a vision for all world health organization. 1999. the world health report 1999: making a difference key: cord-340879-gu91cact authors: li, miao; liu, qin; cui, yajuan; li, dong; wang, hexiang; ng, tzi bun title: isolation and characterization of a phaseolus vulgaris trypsin inhibitor with antiproliferative activity on leukemia and lymphoma cells date: 2017-01-23 journal: molecules doi: 10.3390/molecules22010187 sha: doc_id: 340879 cord_uid: gu91cact a 17.5-kda trypsin inhibitor was purified from phaseolus vulgaris cv. “gold bean” with an isolation protocol including ion exchange chromatography on deae-cellulose (diethylaminoethyl-cellulose), affinity chromatography on affi-gel blue gel, ion exchange chromatography on sp-sepharose (sulfopropyl-sepharose), and gel filtration by fplc (fast protein liquid chromatography) on superdex 75. it dose-dependently inhibited trypsin with an ic(50) value of 0.4 μm, and this activity was reduced in the presence of dithiothreitol in a doseand time-dependent manner, signifying the importance of the disulfide linkage to the activity. it inhibited [methyl-(3)h] thymidine incorporation by leukemia l1210 cells and lymphoma mbl2 cells with an ic(50) value of 2.3 μm and 2.5 μm, respectively. the inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 μm. protease inhibitors have been isolated from the seeds of different monocots and dicots, including maize [1] , wheat [2] , wampee [3] , bitter gourd [4] , momordica cochinchinensis [5] , and legumes [6] [7] [8] . some of them display a variety of biological activities including antifungal [9] , immunomodulatory [5] and antitumor/antiproliferative [8, 10] activities. thus, they have drawn the attention of many researchers. one class of protease inhibitors is trypsin inhibitors. trypsin inhibitors are divided into kunitz type [11] , bowman-birk type [12, 13] , and squash type [4] . the three types have a molecular mass of about 20 kda, 8 kda, and 3 kda, respectively. the soybean produces both kunitz and bowman-birk inhibitors [6] , while melons belonging to family cucurbitaceal produce squash-type inhibitors [4] . leguminous seeds are a rich source of proteins, including protease inhibitors, lectins [14] , antifungal proteins [15] , ribosome inactivating proteins [16] , and arcelins [17] . some of the proteins have interesting biological activities, such as insecticidal [14] , immunomodulatory [5] , antifungal [18] , and antitumor [8, 10] activities. the intent of the present study was to isolate a trypsin inhibitor from the gold bean and to test it for inhibitory action on tumor cells, viral enzymes, and fungal growth. upon ion exchange chromatography on deae-cellulose, the bean extract was separated into three fractions of approximately equal size, an unadsorbed fraction d1 and two adsorbed fractions d2 and d3. trypsin inhibitory activity was located in fraction d2. when fraction d2 was subjected to affinity chromatography on affi-gel blue gel, the activity was recovered in the larger unadsorbed fraction b1 (data not shown). upon ion exchange chromatography on sp-sepharose, fraction b1 was resolved into a small unadsorbed fraction s1 and two large adsorbed fractions (s2 and s3) of approximately equal size. activity resided in fraction s2. further purification of s2 on superdex 75 yielded two fractions, su1 and su2. only fraction su2 exhibited trypsin inhibitory activity. su2 was the purified trypsin inhibitor (gbti). the yields of the various chromatographic fractions are presented in table 1 . the n-terminal sequence of the trypsin inhibitor is shown in table 2 . gbti exhibited a molecular mass of 17.5 kda in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and gel filtration ( figure 1 ). its trypsin inhibitor activity remained unchanged in the temperature range 20-80 • c, was reduced by 60% at 90 • c, and, was totally abolished after exposure to 100 • c. upon ion exchange chromatography on deae-cellulose, the bean extract was separated into three fractions of approximately equal size, an unadsorbed fraction d1 and two adsorbed fractions d2 and d3. trypsin inhibitory activity was located in fraction d2. when fraction d2 was subjected to affinity chromatography on affi-gel blue gel, the activity was recovered in the larger unadsorbed fraction b1 (data not shown). upon ion exchange chromatography on sp-sepharose, fraction b1 was resolved into a small unadsorbed fraction s1 and two large adsorbed fractions (s2 and s3) of approximately equal size. activity resided in fraction s2. further purification of s2 on superdex 75 yielded two fractions, su1 and su2. only fraction su2 exhibited trypsin inhibitory activity. su2 was the purified trypsin inhibitor (gbti). the yields of the various chromatographic fractions are presented in table 1 . the n-terminal sequence of the trypsin inhibitor is shown in table 2 . gbti exhibited a molecular mass of 17.5 kda in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and gel filtration ( figure 1 ). its trypsin inhibitor activity remained unchanged in the temperature range 20-80 °c, was reduced by 60% at 90 °c, and, was totally abolished after exposure to 100 °c. trypsin inhibitor-enriched fractions are highlighted in boldface. results represent mean ± sd, n = 2. trypsin inhibitor-enriched fractions are highlighted in boldface. results represent mean ± sd, n = 2. gbti inhibited trypsin with an ic 50 of 0.4 µm ( figure 2 ). dithiothreitol (dtt) treatment lowered the trypsin inhibitory activity in a dose-and time-dependent manner ( table 3 ). the ic 50 values of the inhibitory effects of gbti on l1210 cells and mbl2 cells were 2.3 µm and 2.5 µm, respectively (table 4) . there was no inhibition on hiv-1 reverse transcriptase when it was tested at various concentrations up to 100 µm ( table 5 ). the inhibitor had no effect on the activities of hiv-1 integrase (table 5 ) and sars coronavirus proteinase at 100 µm. there was no inhibitory action on fungal growth at 100 µm (table 6) . table 3 . inhibition rate (%) of dithiothreitol (dtt) on the activity of gbti and soybean trypsin inhibitor after incubation at 37 • c for different durations. results are presented as mean ± sd (n = 3). different letters (e.g., a,b and c ) indicate statistically significant differences (p < 0.05) when (i) data at the same time point and different dtt concentrations or (ii) data at the same dtt concentration, but different time points, were analyzed by analysis of variance followed by duncan's multiple range test. inhibition rate (%) of purified or soybean trypsin inhibitor at x mm dtt = (trypsin inhibitory activity of purified or soybean trypsin inhibitor−trypsin inhibitory activity of purified or soybean trypsin inhibitor in presence of x mm dtt)/trypsin inhibitory activity of purified or soybean trypsin inhibitor × 100%. results are presented as mean ± sd (n = 3). different letters (e.g., a,b,c and d ) indicate statistically-significant differences (p < 0.05) when data were analyzed by analysis of variance followed by duncan's multiple range test. gold bean trypsin inhibitor (gbti) is unadsorbed on affi-gel blue gel, but adsorbed on deae-cellulose. this chromatographic behavior is distinctly different from that of other leguminous antifungal proteins which are unadsorbed on the ion exchanger and adsorbed on the affinity chromatography media [15, 19] . hence, the purification procedure described herein provides a convenient means to separate trypsin inhibitors from antifungal proteins which may be present in the same legume. the trypsin inhibitor from gold beans demonstrate some sequence resemblance to other leguminous trypsin inhibitors including those of cowpea, mung bean, and garden pea. it is noteworthy that it exerts a potent antiproliferative action on both l1210 cells and mbl2 cells. some of the legume trypsin inhibitors, for instance, field bean trypsin inhibitors, inhibit in vitro proliferation of cancer cells and metastasis in vivo [20, 21] . gold bean trypsin inhibitor is dissimilar from broad bean trypsin inhibitor [9] in that the former lacks hiv-1 reverse transcriptase inhibitory activity. the lack of hiv-1 reverse transcriptase inhibitory activity in gold bean trypsin inhibitor is similar to the findings on lily bulb trypsin inhibitor [22] . gold bean trypsin inhibitor is also devoid of any inhibitory effect on hiv-1 integrase and sars coronavirus proteinase. the absence of antifungal activity in gold bean trypsin inhibitor is also in agreement with the observation on some trypsin inhibitors, such as lily bulb trypsin inhibitor [22] . the results demonstrate that hiv-1 reverse transcriptase inhibitory and antifungal activities of trypsin inhibitors have structural requirements different from those of trypsin inhibitory activity. the importance of the disulfide linkage in gold bean trypsin inhibitor to its trypsin inhibitory activity is revealed by the ability of the reducing agent dithiothreitol to reduce the activity. this is reminiscent of the results on trypsin-chymotrypsin inhibitor from vigna mungo seeds [23] . gold bean trypsin inhibitor (gbti) is unadsorbed on affi-gel blue gel, but adsorbed on deae-cellulose. this chromatographic behavior is distinctly different from that of other leguminous antifungal proteins which are unadsorbed on the ion exchanger and adsorbed on the affinity chromatography media [15, 19] . hence, the purification procedure described herein provides a convenient means to separate trypsin inhibitors from antifungal proteins which may be present in the same legume. the trypsin inhibitor from gold beans demonstrate some sequence resemblance to other leguminous trypsin inhibitors including those of cowpea, mung bean, and garden pea. it is noteworthy that it exerts a potent antiproliferative action on both l1210 cells and mbl2 cells. some of the legume trypsin inhibitors, for instance, field bean trypsin inhibitors, inhibit in vitro proliferation of cancer cells and metastasis in vivo [20, 21] . gold bean trypsin inhibitor is dissimilar from broad bean trypsin inhibitor [9] in that the former lacks hiv-1 reverse transcriptase inhibitory activity. the lack of hiv-1 reverse transcriptase inhibitory activity in gold bean trypsin inhibitor is similar to the findings on lily bulb trypsin inhibitor [22] . gold bean trypsin inhibitor is also devoid of any inhibitory effect on hiv-1 integrase and sars coronavirus proteinase. the absence of antifungal activity in gold bean trypsin inhibitor is also in agreement with the observation on some trypsin inhibitors, such as lily bulb trypsin inhibitor [22] . the results demonstrate that hiv-1 reverse transcriptase inhibitory and antifungal activities of trypsin inhibitors have structural requirements different from those of trypsin inhibitory activity. the importance of the disulfide linkage in gold bean trypsin inhibitor to its trypsin inhibitory activity is revealed by the ability of the reducing agent dithiothreitol to reduce the activity. this is reminiscent of the results on trypsin-chymotrypsin inhibitor from vigna mungo seeds [23] . a lectin, an antifungal protein and a trypsin inhibitor can be isolated from the gold bean [24, 25] . all three proteins can be regarded as defense proteins that protect the plant from pathogenic and predatory organisms. previously, a trypsin inhibitors have been isolated from phaseolus vulgaris. however, they have not been examined for biological activities other than trypsin inhibitory activity [26] [27] [28] [29] [30] [31] [32] . in conclusion, a new trypsin inhibitor was purified from gold bean. it dose-dependently inhibited trypsin, and this activity was reduced in the presence of dithiothreitol in a dose-and time-dependent manner, signifying the importance of the disulfide linkage to the activity. it inhibited [methyl-3h] thymidine incorporation by leukemia l1210 cells and lymphoma mbl2 cells. a water extract of gold beans phaseolus vulgaris cv. "gold bean" from mainland china (260 g) was made by homogenizing them in distilled water (6 ml/g). the homogenate was then centrifuged 4) . the unadsorbed proteins (fraction b1) were dialyzed against 10 mm nh 4 ac buffer (ph 5) and applied on a 2.5 × 20 cm column of sp-sepharose (ge healthcare, uppsala, sweden). after elution of unadsorbed proteins (fraction s1), the column was eluted with a 0-1 m nacl concentration gradient in the nh 4 ac buffer. the first adsorbed fraction (s2) was then subjected to gel filtration on a superdex 75 hr 10/30 column (ge healthcare) in 0.2 m nh 4 hco 3 buffer (ph 8.5). the second absorbance peak (su2) represented purified trypsin inhibitor (gbti). the assay for trypsin inhibitory activity was carried out by addition of test sample (20 µl) to 160 µl of a 1% casein solution in 0.1 m tris-hcl buffer (ph 7.4). trypsin (20 µl of a 0.5 mg/ml solution) was then added and the mixture was incubated at 37 • c for 15 min before 0.4 ml 5% trichloroacetic acid was added to terminate the reaction. after centrifugation, the absorbance of the supernatant, which reflects the amount of casein fragments, was measured at 280 nm [22] . the purified trypsin inhibitor was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page, woodinville, wa, usa) for molecular mass determination in accordance with the procedure of laemmli and favre [33] . gel filtration on an fplc-superdex 75 column (ge healthcare, uppsala, sweden), which had been calibrated with molecular mass markers including phosphorylase b (94 kda), bovine serum albumin (67 kda), ovalbumin (43 kda), carbonic anhydrase (30 kda), soybean trypsin inhibitor (20 kda) and α-lactalbumin (14.4 kda) (ge healthcare), was conducted to determine the molecular mass of the protein. the n-terminal sequence of the trypsin inhibitor was determined by using a hewlett-packard hp g1000a edman degradation unit (hewlett packard company, palo alto, ca, usa) and an hp 1000 hplc system (hewlett packard company, palo alto, ca, usa). the purified trypsin inhibitor was incubated in a water bath at different temperatures from 20 • c to 100 • c for 15 min, then immediately chilled on ice. the reaction mixture was then subjected to the assay of trypsin inhibitory activity. the purified trypsin inhibitor (2.5 µm) was incubated with dithiothreitol (dtt) at the final concentration of 2.5, 10, and 40 mm for 5, 20, and 80 min at 37 • c, respectively. for comparison, soybean trypsin inhibitor purchased from sigma chemical co., (st. louis, mi, usa) (2.5 µm) was similarly treated. the reaction was terminated by adding iodoacetamide at twice the amount of thiol functions at each dtt concentration. the remaining trypsin inhibitor activity was measured at ph 7.4, as described above. the highest iodoacetamide concentration used in the test was devoid of any effect on the activity of trypsin and the trypsin inhibitory activity of purified trypsin inhibitor and soybean trypsin inhibitor [34] . the antiproliferative activity of the purified protein was determined as follows. the cell lines l1210 (leukemia) and mbl2 (lymphoma) were purchased from american type culture collection. the cell line was maintained in dulbecco modified eagles' medium (dmem) supplemented with 10% fetal bovine serum (fbs) and 100 mg/l streptomycin and 100 iu/ml penicillin at 37 • c in a humidified atmosphere of 5% co 2 . cells (1 × 10 4 ) in their exponential growth phase were seeded into each well of a 96-well culture plate (nunc, roskilde, denmark) and incubated for 3 h before addition of the trypsin inhibitor. incubation was carried out for another 48 h. radioactive precursor, 1 µci ([ 3 h-methyl]-thymidine, from ge healthcare), was then added to each well and incubated for 6 hrs. the cultures were then harvested by a cell harvester. the incorporated radioactivity was determined by liquid scintillation counting [15] . the assay for hiv reverse transcriptase inhibitory activity was carried out in view of the report that trypsin inhibitors manifest this activity [35, 36] . it was conducted according to instructions supplied with the assay kit from boehringer mannheim (basel, switzerland). the assay takes advantage of the ability of reverse transcriptase to synthesize dna, starting from the template/primer hybrid poly (a) oligo (dt) 15. the digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into one of the same dna molecule, which is freshly synthesized by the reverse transcriptase (rt). the detection and quantification of synthesized dna as a parameter for rt activity follows a sandwich elisa protocol. biotin-labeled dna binds to the surface of microtiter plate modules that have been pre-coated with strepatavidin. in the next step, an antibody to digoxigenin, conjugated to peroxidase enzyme, binds to the digoxigenin-labeled dna. in the final step, the peroxidase substrate is added. the peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product. the absorbance of the sample at 405 nm can be determined using a microtiter plate (elisa) reader and is directly correlated to the level of rt activity. a fixed amount (4-6 ng) of recombinant hiv-1 reverse transcriptase was used. the inhibitory activity of the protein was calculated as percent inhibition compared to a control without the protein [15] . the positive control used was kale antifungal peptide. the plasmid that expressed his-tagged wild-type hiv-1 integrase, pt7-7-his (y|tx)-hiv-1-in, was a generous gift from professor s.a. chow (school of medicine, ucla). to express the protein, a 1-liter culture of e. coli bl21 (de3) cells containing the expression plasmid was grown at 37 • c until od600 reached 0.7-0.8. cells were induced by addition of 0.8 mm iptg (isopropyl-β-d-thiogalactopyranoside) and harvested, after 4 h of incubation, by centrifugation at 6000× g for 10 min at 4 • c cells were suspended at a concentration of 0.1 g/ml wet cell paste in 20 mm tris-hcl buffer (ph 8.0) containing 0.1 mm edta (ethylenediamine tetraacetic acid), 2 mm β-mercaptoethanol, 0.5 m nacl and 5 mm imidazole. lysozyme was added to a concentration of 0.2 mg/ml. after incubation at 4 • c for 1 h, the lysate was sonicated and centrifuged at 40,000× g at 4 • c for 20 min. the pellet was homogenized in 50 ml buffer a (20 mm tris-hcl, ph 8.0, 2 m nacl, 2 mm β-mercaptoethanol) containing 5 mm imidazole. the suspension was rotated at 4 • c for 1 h, and cleared by centrifugation at 40,000× g at 4 • c for 20 min. the supernatant was loaded onto a 1-ml chelating sepharose column (ge healthcare) charged with 50 mm niso 4 and was equilibrated with buffer a containing 50 mm imidazole. the column was washed with five column volumes of buffer a containing 5 mm imidazole, and the protein was eluted with three column volumes of buffer a containing 200 and 400 mm imidazole, respectively. protein containing fractions were pooled, and edta was added to a final concentration of 5 mm. the protein was dialyzed against buffer b (20 mm hepes, ph 7.5, 1 mm edta, 1 m nacl, 20% glycerol) containing 2 mm β-mercaptoethanol, and then against buffer b containing 1 mm dithiothreitol. aliquots of the protein were stored at −70 • c [19] . a non-radioactive elisa-based hiv-1 integrase assay was performed according to the dna-coated plate method. in this study, 1 µg of smal-linearized pbluescript sk was coated onto each well in the presence of 2 m nacl as target dna. the donor dna was prepared by annealing vu5br (5'-biotin-gtgtggaaaatctctagcagt-3') and vu5 (5'-actgctagagattttccacac-3') in 10 mm tris-hc1, ph 8.0, 1 mm edta and 0.1 m nacl at 80 • c, followed by 30 min at room temperature. integrase reaction was performed in 20 mm hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (ph 7.5) containing 10 mm mncl 2 , 30 mm nacl, 10 mm dithiothreitol and 0.05% nonidet-p40 (sigma). after the integrase reaction, the biotinylated dna immobilized on the wells was detected by incubation with streptavidin-conjugated alkaline phosphatase (boehringer-mannheim, mannheim, germany), followed by colorimetric detection with 1 mg/ml p-nitrophenyl phosphate in 10% diethanolamine buffer (ph 9.8) containing 0.5 mm mgcl 2 . the absorbance due to the alkaline phosphatase reaction was measured at 415 nm. the ribosome inactivating protein luffin was used as a positive control [16] . the activity of sars coronavirus (cov) protease was indicated by a cleavage of designed substrate which was composed of two proteins linked by a cleavage site for sars cov protease. the reaction was performed in a mixture containing 5 µm sars cov protease, 5 µm sample, 20 µm substrate, and buffer [20 mm tris-hcl (ph 7.5), 20 mm nacl and 10 mm beta-mercaptoethanol] for 40 min at 37 • c. after 40 min, the reaction was stopped by heating at 100 • c for 2 min. then the reaction mixture was analysed by sds-page. if sars cov protease is inhibited by the test sample, there is only one band, which is the intact substrate, shown in sds-page [19] . this assay was conducted in view of the report on antifungal activity of some trypsin inhibitors [9] . the assay for antifungal activity toward mycosphaerella arachidicola and fusarium oxysporum was carried out in 100 mm × 15 mm petri dishes containing 10 ml of potato dextrose agar. after the mycelial colony had developed, sterile blank paper disks (0.625 cm in diameter) were placed at a distance of 0.5 cm away from the rim of the mycelial colony. an aliquot (15 µl) of the purified trypsin inhibitor was added to a disk. the plates were incubated at 23 • c for 72 h until mycelial growth had enveloped the disks containing the control and had formed crescents of inhibition around the disks containing samples with antifungal activity [15] . the positive control used was kale antifungal peptide. a 17.5-kda trypsin inhibitor was purified from phaseolus vulgaris cv. "gold bean". it was adsorbed on deae-cellulose, unadsorbed on affi-gel blue gel, and adsorbed on sp-sepharose. it dose-dependently inhibited trypsin with an ic 50 value of 0.4 µm. about 80% and all of the trypsin inhibitory activity were abolished after treatment with 2.5 mm dithiothrietol for 5 min and 20 min of incubation, respectively. [methyl-3h] thymidine incorporation by leukemia l1210 cells and lymphoma mbl2 cells was inhibited with an ic 50 value of about 2 µm. mycelial growth in fusarium oxysporum and mycosphaerella arachidicola were unaffected. the activities of hiv-1 reverse transcriptase, hiv-1 integrase and sars coronavirus proteinase were unaltered after exposure to the trypsin inhibitor. amino acid sequence and secondary structural analysis of the corn inhibitor of trypsin and activated hageman factor primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin a homodimeric sporamin-type trypsin inhibitor with antiproliferative, hiv reverse transcriptase-inhibitory and antifungal activities from wampee (clausena lansium) seeds bitter gourd proteinase inhibitors: potential growth inhibitors of helicoverpa armigera and spodoptera litura isolation of a trypsin inhibitor with deletion of n-terminal pentapeptide from the seeds of momordica cochinchinensis, the chinese drug mubiezhi protein proteinase inhibitors in legume seed-overview trypsin inhibitor from poecilanthe parviflora seeds: purification, characterization, and activity against pest proteases effects of the medicago scutellata trypsin inhibitor (msti) on cisplatin-induced cytotoxicity in human breast and cervical cancer cells a bowman-birk-type trypsin-chymotrypsin inhibitor from broad beans wolfenstein-todel, c. a novel trypsin inhibitor from peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis characterization and pharmacological properties of a novel multifunctional kunitz inhibitor from erythrina velutina seeds formation of bowman-birk inhibitors during the germination of horsegram (dolichos biflorus) reductive unfolding and oxidative refolding of a bowman-birk inhibitor from horsegram seeds (dolichos biflorus): evidence for "hyperreactive" disulfide bonds and rate-limiting nature of disulfide isomerization in folding a comparison of the short and long term effects of insecticidal lectins on the activities of soluble and brush border enzymes of tomato moth larvae (lacanobia oleracea) a mitogenic defensin from white cloud beans (phaseolus vulgaris) purification and characterization of novel ribosome inactivating proteins, alpha-and beta-pisavins, from seeds of the garden pea pisum sativum molecular characterization of a new arcelin-5 gene distribution of proteinases and carbohydrates in the midgut of the larvae of the sweet potato weevil cyclas formicarius and response of proteinase to inhibitors from sweet potato concurrent purification of two defense proteins from french bean seeds: a defensin-like antifungal peptide and a hemagglutinin treatment with field bean protease inhibitor can effectively repress ethylnitrosourea (enu)-induced neoplasms of the nervous system in sprague-dawley rats the field bean protease inhibitor has the potential to suppress b16f10 melanoma cell lung metastasis in mice isolation and characterization of a novel trypsin inhibitor from fresh lily bulbs trypsin-chymotrypsin inhibitors from vigna mungo seeds an antifungal peptide from phaseolus vulgaris cv. brown kidney bean purification and characterization of a lectin from phaseolus vulgaris cv. (anasazi beans) characterization of trypsin inhibitors from tora-mame seeds, one of the japanese cultivars of phaseolus vulgaris trypsin isoinhibitors from garden beans (phaseolus vulgaris) isolation and characterization of some proteinase inhibitors from phaseolus vulgaris var. nanus natural plant enzyme inhibitors: isolation and properties of a trypsin/chymotrypsin inhibitor from kidney bean (phaseolus vulgaris) the isolation and characterization of a trypsin inhibitor from kintoki bean (phaseolus vulgaris) the isolation of two proteins, glycoprotein i and a trypsin inhibitor, from the seeds of kidney bean (phaseolus vulgaris) enzyme inhibitory activities of two leguminosae: phaseolus vulgaris l. pods and vicia faba l. hulls. trypsin inhibitory activity maturation of the head of bacteriophage t4. i. dna packaging events baeyens-volant, d. the papaya kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein a novel lectin from pseudostellaria heterophylla roots with sequence simularity to kunitz-type soybean trypsin inhibitor marmorin, a new ribosome inactivating protein with antiproliferative and hiv-1 reverse transcriptase inhibitory activities from the mushroom hypsizigus marmoreus the authors declare no conflict of interest.molecules 2017, 22, 187 key: cord-325936-rwxg187r authors: eyal, nir; halkitis, perry n. title: aids activism and coronavirus vaccine challenge trials date: 2020-06-26 journal: aids behav doi: 10.1007/s10461-020-02953-8 sha: doc_id: 325936 cord_uid: rwxg187r nan the who working group has articulated important criteria for assessing a challenge study, but we believe that they left out the most important one: until there is an approved treatment, a challenge trial with a potentially fatal and as-yet untreatable pathogen is unacceptable [2] . most sars-cov-2 vaccine candidates are not ready for efficacy testing yet. challenge trials will also need to wait sars-cov-2 culture and viral dose confirmation. by then, therapeutics for treating covid (beyond remdesivir) might prove efficacious. but we shall argue that even now, the risk, which is real in sars-cov-2 challenge trials, is already tolerable, for four reasons that should especially resonate with hiv/aids advocates and activists. therefore, the who working group (in which neither of us participated) was right to consider sars-cov-2 vaccine challenge trials permissible when conducted ethically. the ideas that follow build upon the pioneering effort undertaken throughout the course of the aids epidemic, especially in the first 2 decades when few options were available for people living with hiv (plwh), as well as in recent attempts to reach a sterilizing cure or controlled drug-free remission for hiv. throughout the aids epidemic, risky scientific efforts helped transform the disease and advance the well-being of plwh. similar ingenuity is required in the current pandemic. below we weigh the benefits of enacting sars-cov-2 vaccine challenge trials expeditiously in relation to four domains: relative risk, personal autonomy, indirect medical benefits and social value. to minimize risk to participants, live sars-cov-2 vaccine challenge trials would need to recruit participants who, in the-likely-event of infection, would remain at relatively low fatality risk. that means, young people without any major risk factors for severe clinical cases of covid following sars-cov-2 infection [1, [3] [4] [5] . based on concurrent evidence [6] , the who working group assessed sars-cov-2 infection fatality rate for people in their twenties at 0.03% [1] . that equals the fatality rate following live kidney donation [5, 7] , a widely-supported practice given the informed consent of the donor and the expected benefit to a single recipient. in the case of sars-cov-2 vaccine trials, the expected societal benefit is far greater than from a single transplantation. indeed, since the who working group published its guidance, new data suggest a lower infection fatality rate for people in their twenties-0.007% [8] , less than a quarter the fatality rate following kidney donation. and these figures cover both healthy and unhealthy people in their twenties, so if recruitment focuses, as it should [1, [3] [4] [5] , only on those without known major risk factors for severe covid following infection, risk of death will surely go down further. the avac and tag statement overlooks the targeted nature of recruitment to challenge trials. at one point, it warns about "a live challenge for a significant number of those at risk in a disease with a currently estimated case fatality rate (cfr) greater than 1 percent." [2] but in assessing the challenge, what matters is the risk of fatality among the low-risk subpopulation that it would recruit, and not the risk of fatality in the general population, or those at special risk. enacted with healthy volunteers, phase 1 studies (including studies for new vaccines) also have higher mortality rates than the latest figure we mentioned above [9] , as do some of the interventions recently deployed in cure-related hiv research, enacted with participants who were stable on art [10] . the ethical justification for all these risky studies is, in part, the free and informed consent of volunteers [11] [12] [13] . just like these other risky studies, sars-cov-2 challenge trials will not enlist war prisoners, incarcerated people, children, or adults with compromised decisional ability. rather, they would be preceded by thoroughgoing procedures to verify the comprehension of risks, benefits, and alternatives among non-coerced cognitively-able adults, as delineated e.g. in the belmont report [14] , which guides all us human subjects research. willingness to contribute to risky experimentation has been a hallmark of the hiv-affected community for decades. long-term survivors of hiv who were infected prior to the development and implementation of haart have recounted how any medical approach (chinese cucumbers included) would have been undertaken at the time in order to survive [15] . we owe treatments to thousands who were willing to engage in early trials toward them. indeed, millions around the world continue to dose art daily with scarce indication of its long-term effects, e.g. on older adults living with hiv [16] . surely, volunteers' consent to participate in at least some of these trials, and patients' consent to use these relatively novel technologies, were sometimes valid, notwithstanding risks and uncertainties. shortly after hiv sterilizing cure trials transplanted allogenic stem cell in participants, with well over a thousand times the fatal risk of sars-cov-2 infection in healthy young participants [17, 18] , aids activist david evans interviewed the participants of these risky trials and concluded, "we should recognize their great capacity to understand the risks they may confront as research participants and, after a careful ethical and scientific review, respect the motivations of those who decide that the benefits of knowing that their contributions may help others outweighs the risks" [19] . still, when it comes to sars-cov-2 challenge trials, the avac and tag statement summarily questions the possibility of informed consent: in recent times, live pathogen challenge trials have been conducted in diseases where a safe, effective approved treatment is available, or for which patho-genesis and risks are reasonably well characterized. that is not the case for covid-19, which means that adequately communicating about and assessing potential risks and benefits of participating in a challenge study and ensuring appropriate informed consent may be impossible. it is true that scientists have only limited understanding of sars-cov-2 and covid-19 risks, but informed consent can be 100% valid when scientists' understanding is limited. if ample scientific understanding were required to keep consent valid, then whenever older science became obsolete, we would have to condemn earlier studies for alleged invalid consent-an absurdity. first-in-human trials, including any testing of new vaccines, would always be wrongful-another absurdity. what informed consent requires is that scientists communicate their best concurrent understanding of relevant features of the study to participants, who then consent [20, 21] . if anything, scientists' limited understanding may make informed consent simpler; instead of communicating a large body of knowledge, scientists need only communicate something like "uncertainty runs high-take it or leave it." alluding to people who have declared their initial interest on a website in participating in challenge trials (nearly 27,000 so far), [22] the avac and tag statement adds, "we do not believe that individuals' expressed willingness to participate in such a trial is an adequate or appropriate measure of informed consent" [2] . as a closer look at the website and forms for volunteering would reveal, this initial and entirely revocable expression of interest is not made out to constitute the actual informed consent process, which would need to be very thorough [1, [3] [4] [5] . some of these volunteers are highly educated [22, 23] , and may comprehend complex risks after proper disclosure. the statement paternalistically questions the agency of thousands of individuals, and assumes unlikely knowledge of their decision making processes. challenge trials should, with some limitations, recruit in populations where many infections are expected [3, 5] . that would reduce somewhat the incremental risk from the infection in the trial. incremental risk of medical interventions, namely, the direct risk they introduce minus any risk that they remove, arguably matters more in their assessment than their sheer direct risk. in effect, the appropriate guaranteed access to life support and to any therapeutics proven by then for challenge participants could provide an important indirect medical benefit if those are scarce in high-transmission background communities where demand surges [3] . some calculations of challenge studies' overall effect on participants strangely ignore the incremental risks and indirect medical benefits and focus exclusively or myopically on the direct risks to them [5] . but the overall calculation must heed these factors [3, 24] , as hiv cure-related research powerfully demonstrates. in studies of hiv sterilizing cure strategies, far riskier allogenic stem cell transplantation was justified primarily by indirect medical benefits given high background risk-specifically, the crucial benefit for patients who, in addition to hiv infection, had a far more serious terminal cancer [17] . the level of risk that is tolerable in a study with moderate social value is nothing like the one that is tolerable in a study of immense social value [25] . in risky hiv curerelated research, another part of the justification would be the global health value of a cure [12] , yet the scalability and cost-effectiveness of several cure-related strategies being investigated remains unclear [26, 27] . by contrast, the supreme global health value of rolling out earlier a proven sars-cov-2 vaccine is beyond doubt [24, 28] . not only is enacting timely and potentially high-impact vaccine trials important for humanity at large. it is especially important for plwh, who may develop more severe covid complications [29] . many live with additional morbidities and heightened psychosocial stressors [30, 31] , primarily in countries with weakened health systems [32, 33] , all of which may exacerbate risk for severe covid outcomes. while there is speculation that art may provide protection from covid (or that immune dysregulation itself may help) [34] , 1 in 5 plwh are unaware of their serostatus [35] . the pandemic is everywhere disrupting hiv care, and extinguishing the hope to end hiv in this decade [36] [37] [38] [39] . statements such as those of avac and tag are extremely well intentioned, but who guidance is correct, and sars-cov-2 vaccine efficacy trials must be accelerated. as scientists we know all too well that the development of interventions cannot assure 100% safety, especially given evolving knowledge of emerging infections. without risk, science would never advance. one of us witnessed the ravages of aids on his own social circle prior to 1996, and remembers all too well the desperation for identifying treatments so as to save lives. today, we should all feel that same urgency. funding ne's work was funded by niaid (ai114617-01a1). pnh's work was funded by nida (da025537) and niaid (132020). who working group for guidance on human challenge studies in covid-19. key criteria for the ethical acceptability of covid-19 human challenge studies avac and tag statement on ethical conduct of sars-cov-2 human challenge studies to accelerate coronavirus vaccine licensure extraordinary diseases require extraordinary solutions ethics of controlled human infection to study covid-19 estimates of the severity of coronavirus disease 2019: a model-based analysis risks of living kidney donation: current state of knowledge on outcomes important to donors. evid-based nephrol estimating the burden of sars-cov-2 in france quantifying the risks of non-oncology phase i research in healthy volunteers: meta-analysis of phase i studies international aids society global scientific strategy: towards an hiv cure 2016 the benefit/risk ratio challenge in clinical research, and the case of hiv cure: an introduction how to keep high-risk studies ethical: classifying candidate solutions ethical guidelines for deliberately infecting volunteers with covid-19 the belmont report: ethical principles and guidelines for the protection of human subjects of research the aids generation: stories of survival and resilience mental health, psychosocial challenges and resilience in older adults living with hiv hematopoietic stem cell transplantation for hiv cure death after hematopoietic stem cell transplantation: changes over calendar year time, infections and associated factors an activist's argument that participant values should guide risk-benefit ratio calculations in hiv cure research disclosure and consent to medical research participation stanford encyclopedia of philosophy the covid challenge. 2020; www.theco vidch allen ge.org coronavirus: would you volunteer to be exposed? these stanford grads did covid-19 human challenge studies: ethical issues why we (probably) must deliberately infect the social value of candidate hiv cures: actualism versus possibilism maximizing the global health impact of future hiv cure-related interventions through advance planning first-wave covid-19 transmissibility and severity in china outside hubei after control measures, and second-wave scenario planning: a modelling impact assessment covid-19 in patients with hiv: clinical case series covid-19, hiv, and migrant workers: the double burden of the two viruses the burden of covid-19 in people living with hiv: a syndemic perspective covid-19 threatens health systems in sub-saharan africa: the eye of the crocodile three lessons for the covid-19 response from pandemic hiv why aren't people living with hiv at higher risk for developing severe coronavirus disease 2019 (covid-19)? unaids. global hiv & aids statistics-2019 fact sheet hiv care continuum and covid-19 outcomes among people living with hiv during the covid-19 pandemic challenges to hiv care and psychological health during the covid-19 pandemic among people living with hiv in china covid-19 pandemic disrupts hiv continuum of care and prevention: implications for research and practice concerning community-based organizations and frontline providers the potential impact of the covid-19 epidemic on hiv, tb and malaria in low-and middle-income countries key: cord-305394-wwabxlgr authors: venter, w d francois; nel, jeremy title: covid-19: first data from africa date: 2020-08-31 journal: clin infect dis doi: 10.1093/cid/ciaa1293 sha: doc_id: 305394 cord_uid: wwabxlgr nan m a n u s c r i p t africa is no stranger to infectious disease pandemics prior to sars-cov-2, with recent major outbreaks spanning ebola to listeria and measles, and ongoing high background incidence of malaria, hiv and tb. most countries on the continent have severe health resource constraints, and the focus of many far richer countries hit by the sars-cov-2 epidemic -pcr-guided contact tracing to prevent spread, reliance on intensive-care and ventilation to deal with those who became severely ill -struck fear into african public health practitioners, health workers and the public, where sophisticated laboratory infrastructure and high-tech intensive care is often severely rationed (1) . there was speculation that the continent may be relatively protected from the worst of covid by its younger population age structure (and fewer associated chronic comorbidities linked to covid-19 mortality), limited public transport, possibly greater circulating seasonal coronaviruses, and even antiretroviral therapy programs with anti-coronavirus activity. however, the possibility of hiv and tb being risk factors for covid severe events, both relatively unusual in the richer countries, could mean worse consequences in these populations (2) . dr mary-anne davies presents important data from the western cape province, the epicenter for south africa's initial wave of infections, in this edition of cid (3) . south africa as a major business and tourism hub was always likely to be one of the earliest african countries struck. initial severe lockdown measures were announced in march as the first internal cases in international travelers were reported. community seeding, especially in poorer and more densely populated communities, was delayed, but infections rapidly accelerated, and at the time of writing (late august 2020), the country has the fifth largest epidemic in the world (4). this data is from an ongoing surveillance cohort that has previously generated rich data on disease patterns in the western cape, and currently continues to provide near real-time updates on the impact of pcr-confirmed sars-cov-2 on factors ranging from death to oxygen consumption within hospitals. data from electronic clinical information systems is synthesized with laboratory, pharmacy and administrative data, providing a powerful population level dataset. reported data from other national surveillance systems from across the country confirms much of davies' findings, although her dataset is remarkable in its detail. key strengths of the paper include a dataset covering over 3 million healthcare users in the western cape province, and the use of both hospitalized and nonhospitalized cases and deaths davies' data shows similar mortality risk factors, including age, sex, diabetes (especially uncontrolled diabetes), hypertension and renal disease to other cohorts from richer countries. the data do provide vital information on south africa's other two continuing pandemics, with hiv and past or current tb all giving a roughly two-fold increased risk of death. earlier reports from europe and north america had not found a clear association with mortality, but they were limited by small sample sizes and selection biases. davies' cohort had about 4000 hiv positive patients with covid in it -about 40 times more than all the other published case series combined to date (5) . the dataset shows an association between hiv infection and increased mortality, with a hazard ratio after adjusting for age, sex and other comorbidities of 2.14 (95% ci 1.7-2.7). the possibility of residual confounding exists, but davies makes a plausible case for hiv being an independent risk factor for covid-19-related death. interestingly, davies' dataset does suggest a higher mortality in hiv patients with virological failure and/or pre-covid cd4 counts under 200 cells/µl compared with those who were virally suppressed, though there is substantial overlap in the confidence intervals, m a n u s c r i p t and may be confounded by social factors associated with non-adherence, as well as covid-19 mortality. whether antiretroviral therapy somehow mitigates covid outcomes has been widely speculated in the hiv scientific literature, with in-vitro data suggesting tenofovir has activity against coronaviruses (6) . intriguingly, in the multivariate analysis davies found that patients receiving tenofovir disoproxil fumarate as part of their antiretroviral therapy saw a statistically significant reduction in mortality (ahr 0.41, p=0.007). this supports observational data from a spanish cohort that similarly suggested better outcomes for those on tenofovir-based antiretroviral therapy (5) . however, extreme care should be taken interpreting the tenofovir data, as the association is at high risk of confounding and channeling bias. patients not on tenofovir in south africa are very likely to have underlying renal dysfunction, or be on second-line therapy, which is often again associated with coexisting social issues that may make them more vulnerable to covid-19 consequences. davies' data again makes a substantial addition to the covid-19 literature with respect to tuberculosis, a previously unreported topic. both active tuberculosis and past tuberculosis were associated with an elevated risk of mortality in the study, the latter presumably on the basis of residual lung damage and consequent poor respiratory reserve. what does this data mean for the rest of africa? we should be wary of extrapolating too far -africa has enormous diversity, and western cape demographics differ markedly even from the rest of south africa. however, the data is broadly similar to other regions in the world, and so the immediate priority for african health systems remains to rapidly and practically design systems to protect the elderly and those with chronic diseases or tb. for south africa, a sigh of relief at a relatively small increase in mortality in hiv and tb should be quickly tempered; diabetes was the second commonest cause of death in the country pre-covid-19, and most patients in the country have poor glucose control, a major risk factor from davies' data (7) . in addition, obesity is an independent risk factor for covid-19 mortality in other cohorts, and while this was not collected in this dataset (weight and height was not available for this cohort), the country's obesity epidemic, especially among women, is well documented. among hiv patients, again more among women, the large-scale introduction of dolutegravir in late 2019 may markedly aggravate obesity and covid mortality in the near-6 million south african patients on antiretrovirals (8) . sadly, south africa has not learnt from other african countries and their epidemics (2) . when ebola struck in west africa, the consequence of locking down the society and suspending vaccine programs extracted a mortality from measles alone estimated to be similar to that from ebola (9) . initially lauded by the who for the speed and severity of covid-19 lockdown measures, the south african response became increasingly militarised (over 200 000 people charged or incarcerated since the start), characterized by bizarre decisions on commerce, an incoherent pcr-based testing and tracing program, poor food support programs, corruption around procurement of personal protective equipment, and chaotic re-opening of schools, with a temporary decimation of vaccination and hiv and tb programs (10) . the country will face years of continuing infectious and other diseases well beyond this sars-cov-2 era, that may well lead to a far greater mortality than covid-19. low-and-middle income countries will need to ponder future pandemic responses that pit any current epidemic against hard-won public health interventions. m a n u s c r i p t finally, the value of having integrated surveillance systems such as the western cape's that can rapidly inform urgent public health responses in real time are demonstrated in this paper. african governments and donors should invest more in these systems, as a major adjunct to public health programs. fv reports research grants from bill and melinda gates foundation, unitaid, usaid, samrc, and viiv healthcare; drug donations from gilead sciences and viiv healthcare; and honoraria from gilead, viiv, mylan, merk, adcock-ingram, aspen, abbott, roche, and j&j, all outside the submitted work. j.n. has no potential conflicts to disclose. covid-19: shining the light on africa tackling covid-19: can the african continent play the long game? j glob health risk factors for covid-19 death in a population cohort study from the western cape province who coronavirus disease (covid-19) dashboard coinfection: case reports, retrospective cohorts and outcomes triphosphates of the two components in descovy and truvada are inhibitors of the sars-cov-2 polymerase (preprint) mortality and causes of death in south africa: findings from death notification weight gain and integrase inhibitors reduced vaccination and the risk of measles and other childhood infections post-ebola covid-19 lockdowns in low-and middle-income countries: success against covid-19 at the price of greater costs a c c e p t e d m a n u s c r i p t key: cord-317988-1buh1wm0 authors: kalichman, seth c.; eaton, lisa a.; berman, marcie; kalichman, moira o.; katner, harold; sam, soya s.; caliendo, angela m. title: intersecting pandemics: impact of sars-cov-2 (covid-19) protective behaviors on people living with hiv, atlanta, georgia date: 2020-06-05 journal: j acquir immune defic syndr doi: 10.1097/qai.0000000000002414 sha: doc_id: 317988 cord_uid: 1buh1wm0 covid-19 and its social responses threaten the health of people living with hiv. we conducted a rapid-response interview to assess covid-19 protective behaviors of people living with hiv and the impact of their responses on hiv-related health care. method: men and women living with hiv (n = 162) aged 20–37 years participating in a longitudinal study of hiv treatment and care completed routine study measures and an assessment of covid-19–related experiences. results: at baseline, most participants demonstrated hiv viremia, markers indicative of renal disorders, and biologically confirmed substance use. at follow-up, in the first month of responding to covid-19, engaging in more social distancing behaviors was related to difficulty accessing food and medications and increased cancelation of health care appointments, both by self and providers. we observed antiretroviral therapy adherence had improved during the initial month of covid-19 response. conclusions: factors that may pose added risk for covid-19 severity were prevalent among people living with hiv, and those with greater risk factors did not practice more covid-19 protective behaviors. social distancing and other practices intended to mitigate the spread of covid-19 interfered with hiv care, and impeded access to food and medications, although an immediate adverse impact on medication adherence was not evident. these results suggest social responses to covid-19 adversely impacted the health care of people living with hiv, supporting continued monitoring to determine the long-term effects of co-occurring hiv and covid-19 pandemics. the sars-cov-2 pandemic has rapidly emerged as a significant threat to public health, with the greatest degree of morbidity and mortality occurring among the elderly and individuals with underlying chronic health conditions, including people with compromised immune systems. [1] [2] [3] although the factors and combinations of underlying conditions that determine the severity of the sars-cov-2 disease (covid19) are under investigation, immune system dysfunction and other co-occurring chronic conditions raise concerns for covid-19 severity. 4 for people living with hiv, unsuppressed virus is the hallmark of hiv infection progression and may increase covid-19 severity. in addition, indicators of renal disease that are commonly observed with hiv infection can lead to more severe covid-19 outcomes. 5 liu et al, 6 for example, found that covid-19 severity is significantly greater in patients with urinary glucose and protein markers. furthermore, conditions of poverty, particularly poor nutrition resulting from food insecurity, can impede immune functioning. 7 substance use also raises concerns for increased covid-19 severity. tobacco use, particularly cigarette smoking, is associated with covid-19 severity, 8 and tobacco use may interact with other substances to further compromise the immune system. 9, 10 alcohol and other drug use also suppresses immune responses, [11] [12] [13] particularly among people living with hiv. 14 along with underlying health conditions, substance use raises concerns for covid-19 morbidity and mortality in people living with hiv. 15 high prevalence of substance use and co-occurring underlying health conditions have the potential to amplify the severity of covid-19 in people living with hiv. 16, 17 the increased vulnerability for covid-19 severity in people living with hiv shines a light on the necessity of adopting covid-19 protective behaviors, avoiding public gatherings, reducing social contacts, and physical distancing. covid-19 protective behaviors were recommended in early march 2020 as some states (eg, california, washington, and new york) responded to the unfolding health crisis. by contrast and in the absence of a national covid-19 strategy, the state of georgia was late in response. on march 13, 2020, the us government declared a national state of emergency, and on march 15, 2020, the centers for disease control and prevention issued recommendations to avoid social gatherings. 18 initial reports from hiv clinical settings indicated the potential for interruptions in essential hiv care services. 19 concerns have also been raised that stay-at-home orders and physical distancing could exacerbate what is already a high prevalence of food insecurity among people living with hiv. 20 here, we report the results of a rapid-response interview with men and women living with hiv in atlanta, ga, conducted at the start of the us covid-19 epidemic. there were 2397 diagnosed cases and 12 deaths in the state of ga on march 17, 2020, and 20,058 diagnosed cases and 749 deaths 1 month later. 21 during the final week of data collection for the current study, the 7-day average number of covid-19 diagnoses in the state of georgia was greater than 600, and the average daily deaths was greater than 25. more than 40% of cases and 40% of deaths in georgia occurred in the atlanta metro area. the city of atlanta acted to issue orders for protective policies before the state government. on march 16, 2020, the mayor of atlanta issued a state of emergency, and on march 19, 2020, all nonessential businesses closed, 22 which remained in effect throughout the data collection period. we examined social responses to covid-19 including physical distancing and reducing social contacts in response to the earliest public alerts. we specifically tested the association between covid-19 protective actions and their impact on hiv-related care and treatment. participants in the current study were men and women living with hiv in atlanta, ga, who were between the ages of 20 and 37 years and screened positive for active substance use. the current sample was actively participating in an ongoing antiretroviral therapy (art) adherence study at the time of the covid-19 outbreak. participants were taking part in an 18-month longitudinal hiv treatment engagement and adherence study. men and women living with hiv were recruited through social media websites, targeted online ads, and a participant-driven adaptation of snowball-sampling techniques. specifically, participants were encouraged to refer their hiv-positive friends to the study and were offered a modest incentive for their efforts. after informed consent, participants completed measures of demographic and health characteristics, including audio-computer-assisted self-interviews, blood samples for hiv viral load testing, and urine samples for substance use testing and urinary health markers. after the baseline assessment, participants were contacted monthly to complete health care engagement and health behavior interviews. all interviews were conducted by telephone as part of the original study protocol using methods consistent with best practices for conducting remote research. 23 during a 1-month period, median 9 months from baseline, partic-ipants due for their routine telephone interview completed questions regarding their experience with covid-19. the university of connecticut institutional review board approved all study procedures. participants were asked their self-identified gender, race, age, years of education, the stability of their current housing, income, and employment status. we also administered the centers for epidemiological studies depression scale to assess symptoms of depression (alpha = 0.87) and the 3-item consumption subscale of the alcohol use disorders identification test (audit-c). 24, 25 to assess food insecurity, we used items adapted from the us food security scale that have been validated in the past research and used by the us census bureau. 26 we constructed an unweighted index of 6 underlying health indicators for increased risk of covid-19 morbidity and mortality. the markers include hiv viremia, renal health markers, tobacco, alcohol, cannabis, and other drug use that were collected at the baseline assessment. to determine hiv rna concentrations (viral load), participants provided 80 ml of fingerstick blood for dried blood spots collected in hemaspot hf devices that were frozen before laboratory delivery. hiv-1 viral load testing was conducted using the abbott realtime hiv-1 assay, a reverse transcription pcr assay performed on the automated abbott m2000 platform (abbott molecular inc, des plaines, il). 27 the target sequence for the assay is the highly conserved pol/integrase region of the hiv-1 genome. the limit of detection of the assay is 2.92 log copies/ml, and it can quantify up to 7.0 log copies/ml. 28 all samples required an upfront processing to improve assay sensitivity before subjecting to rna extraction. forty-nine (30%) participants were unable or unwilling to provide a blood sample and were given the option to provide a recent viral load test result obtained within 90 days of the assessment from a health care provider. because hiv viremia indicates greater immune suppression, a detectable viral load was coded as an indicator of covid-19 severity risk. urine specimens were collected and tested on site for 4 health markers: glucose, an indicator of diabetes or renal disease; leukocytes, an indicator of urinary system inflammation 29, 30 ; protein as indicative of kidney disease; and blood as an indicator of multiple disease processes. 31 we used any positive urine health indicator as a marker for a potential underlying renal condition. because any substance use is a known immune suppressant, we conducted a multipanel urine dip test to detect common drug use. the test strip uses a lateral flow chromatographic immunoassay for the qualitative detection of 12 drugs and drug metabolites (redwood toxicology labs-reditest-12). these tests are fda approved and are reliable and valid for initial screening of drug use in the previous 72-96 hours. we considered thc as a separate marker because it is prevalent and its administration is typically smoked, posing specific risks for compromised lung function. 32 for alcohol use, we used the 500 ng/ml cutoff for ethyl glucuronide from a separate dip test with detection of ethyl glucuronide up to 80 hours after drinking. 33 finally, participants reported smoked tobacco products with items adapted from the alcohol, smoking and substance involvement screening test (assist). 34 telephone interviews assessed current hiv care engagement, treatment status, and art adherence. measures of health care engagement were adapted from the hiv cost and services utilization study consortium 35 and include attending and not attending scheduled care appointments. to assess art adherence, we used the 3-item self-report instrument for retrospective adherence (ira) developed and validated by wilson et al. 36 the items represent the number of days medications were taken over the previous 7 days, the frequency of taking medications as directed, and a selfperception rating of how well medications were taken over the previous week. we used methods suggested by wilson et al to convert scores for each item on a scale of 0-100 using linear transformations and calculating the mean to a single adherence score with a range from 0 to 100, interpreted as percent adherence over the past week (alpha = 0.73). wilson et al 36 found that the ira correlates 0.74 with electronically monitored art adherence. the ira was administered during the phone assessment, as well as the phone assessment conducted the previous month. participants reported whether they heard of the novel coronavirus/covid-19, how much they had heard about it, whether they had been tested for covid-19, and whether they had been diagnosed with covid-19. we also asked participants whether they had engaged in 5 recommended actions to mitigate their risk for contracting covid-19. all behaviors assessed were recommended by the us federal government and the state of ga to mitigate the spread of sars-cov-2 at the time the study commenced. the specific actions are shown in the results. we created an index of covid-19 protective actions by summing the use/nonuse of the 5 behaviors. we also assessed participant concerns that they may contract covid-19 using a 100-point rating scale in response to the question: "from 0 to 100, how concerned are you about catching the new coronavirus, with 0 = not at all concerned and 100 = extremely concerned." we asked participants whether they had been unable to get the food they need, get to the pharmacy, and get their medicines in relation to their response to covid-19. participants were also asked whether hiv care providers and other service providers had canceled any of their care appointments because of covid-19. we conducted descriptive analyses for participants identifying as male and female on demographic, health, and covid-19 protective behaviors using contingency table x 2 tests for categorical variables and independent t tests for continuous measures. we also formed 2 groups based on relatively fewer (#2, n = 65) and relatively greater ($3, n = 97) covid-19 protective behaviors and examined differences between groups using contingency table x 2 tests for categorical variables and independent t tests for continuous measures. poisson regression was used to test a multivariable model predicting the number of covid-19 protective behaviors from participant characteristics and the history of covid-19 severity risks. art adherence for the assessment period and the previous month was compared using a dependent (paired) t test. all statistical tests defined significance by p , 0.05. table 1 shows the participant demographic and health characteristics. most participants demonstrated a history of underlying health risks for severe covid-19. as shown in table 1 , half of participants did not know their cd4 cell count, an indicator of not being fully engaged in hiv care. among those participants who did know their most recent cd4 cell count, more than 1 in 4 indicated cd4 cell counts under 350 cells/mm 3 . in addition, 1 in 4 participants were not hiv suppressed and 1 in 3 tested positive for at least 1 underlying renal condition. most participants were actively using substances, including 40% tobacco, 19% alcohol, 65% cannabis (thc), and 29% testing positive for other drugs. overall, 90% of our participants had at least 1 indicator of immune suppression beyond their hiv status. nearly all participants reported staying indoors and away from public places to avoid contracting covid-19. table 2 shows the frequency of covid-19 protective behaviors in the early days of the pandemic. most participants had canceled plans, asked others to stay away, and avoided public transportation to mitigate their risk for contracting covid-19. on average, participants had taken multiple steps to reduce their risks. however, there were gender differences in covid-19 protective actions, with women more likely to stay indoors and away from public places than men. consistent with this pattern, women also rated greater concern about contracting covid-19 than men. poisson regression indicated that participant gender, wald x 2 = 8.9, p , 0.01, and level of concern for contracting covid-19, wald x 2 = 31.2, p , 0.001, predicted the number of covid-19 protective behaviors practiced; women engaged in more protective behaviors than men (b = 20.255, se = 0.085, 95% confidence interval: 20.422 to 20.088), and greater concern was related to engaging in more protective behaviors (b = 0.006, se = 0.001, 95% confidence interval: 0.004 to 0.008). the remaining factors in the model were not significantly associated with covid-19 protective behaviors: years of education, wald x 2 = 2.1, ns, years since testing hiv positive, wald x 2 = 0.1, ns, and number of risks for covid-19 severity, wald x 2 = 0.6, ns ( table 2) . results showed that 40% of participants reported being unable to access food because of the covid-19 outbreak. participants who engaged in greater protective behaviors also experienced more difficulty accessing food compared with persons who engaged in less protective behaviors. among participants who reported being unable to access food due to covid-19, 22% were not food insecure at baseline (table 3) . nearly 1 in 5 participants (n = 31, 19%) indicated that they had missed a scheduled hiv care appointment in the previous 30 days. when asked the reason for their missed appointment, 14 (45%) spontaneously stated the reason was directly related to covid-19. in addition to their missing a scheduled medical appointment, 45% of participants reported that a medical provider canceled an appointment, and 40% reported a nonmedical service provider contacted them to cancel an appointment due to covid-19. participants who engaged in greater covid-19 protective behaviors also experienced more nonmedical service cancelations than participants engaging in fewer covid-19 protective behaviors (table 3) . practicing greater covid-19 protective behaviors was also related to an inability to get to the pharmacy and an inability to access medications. there were no associations between covid-19 protective actions and art adherence in the month before, or the month during, the covid-19 assessment. however, adherence improved significantly in the month since the onset of covid-19 protective actions, t (159) = 17.2, p ,0.01. covid-19 adds to what is already a complex matrix of co-occurring epidemics and health disparities facing people living with hiv. 37 participants in the current study presented multiple challenges in managing their hiv infection before the emergence of covid-19, including substance use, mental health problems, history of comorbidities, and food insecurity. most participants were polysubstance users, demonstrated clinical indications of depression, and experienced food insecurity as severe as hunger. we found that all participants were aware of covid-19 at the earliest days of response by their city officials. nearly all participants were taking some steps to mitigate their risk for covid-19. their immediate response, however, exacerbated food insecurity, with more than 1 in 3 participants reporting difficulty accessing food, including individuals who were not previously food insecure reporting an inability to access food. covid-19 protective behaviors also impeded their health care, created barriers to accessing medications, and disrupted social services. similar impacts of covid-19 on hiv care have been reported by others 1, 38 and have implications for widening what are already entrenched hiv-related health disparities. 39 we also observed an unexpected significant increase in art adherence over the 1 month of response to covid-19. participants may have improved their art adherence out of health concerns or because they were home more, or possibly other reasons. 40, 41 this positive change occurred despite interruption in accessing medications. it is possible that this increase in adherence is a blip and will subside, especially if barriers in accessing medications are not resolved. nevertheless, the heightened and uncontrollable health concerns brought by covid-19 may have motivated health behaviors for which individuals do have control, such as taking their medications. monitoring art adherence over the course of covid-19 should be a priority in ongoing studies. the current findings should be interpreted in light of their methodological limitations. the sample for this study was one of convenience and cannot be considered representative of people living with hiv. although large enough for the number of variables tested, the sample was relatively small. in addition, the sample was largely male and african american and therefore limited in its generalizability. in addition, we assessed substance use with biological testing and could not discern whether some drugs, particularly methamphetamine and cocaine, we smoked, with greater implications for complicating covid-19. the study was undertaken in immediate response to the covid-19 public health crisis, and our findings may be transient and specific to people living with hiv may be at increased risk for a more severe clinical course of covid-19, and our sample demonstrated a history of multiple factors that would likely contribute further to their risks for greater severity of covid-19. of particular concern were high rates of hiv viremia, smoking, 8 other substance use, 32 and depression symptoms. 42 we found that at baseline 1-in-3 participants had evidence of a potential underlying renal condition. of particular concern are 10% and 15% of participants testing positive for urinary glucose and protein, respectively, both of which are associated with covid-19 severity. 6 the accumulation of risk factors for severe covid-19 among people living with hiv, who are already immune compromised, signals an urgent need to mitigate their exposure to sars-cov-2. we found that women engaged in more protective behaviors than men and that protective behaviors were associated with how concerned participants were about contracting covid-19. however, there was no association between the number of underlying risks for severe covid-19 and the number of protective actions taken. at the time of this study, public health messaging focused on increased covid-19 risks for the elderly and people with cardiovascular disease, diabetes, and immune suppression. 43 although people with hiv will recognize their increased risks due to an immune suppressive condition, the added burden of smoking and other substance use, as well as underlying conditions common to hiv infection, have not been included in centers for disease control and prevention reports of severe case outcomes and have not been included in public health messaging. 44 patients should be fully informed of their risks for severe covid-19 to understand the importance of long-term mitigation. maintaining hiv care during the covid-19 pandemic covid-19 in patients with hiv: clinical case series tuberculosis and hiv responses threatened by covid-19 epidemiological and clinical features of 125 hospitalized patients with covid-19 in clinical manifestation, diagnosis, prevention and control of sars-cov-2 (covid-19) during the outbreak period the value of urine biochemical parameters in the prediction of the severity of coronavirus disease 2019 role of nutrition in hiv infection: review of evidence for more effective programming in resource-limited settings the impact of copd and smoking history on the severity of covid-19: a systemic review and meta-analysis investigating the relationships between alcohol consumption, cannabis use, and circulating cytokines: a preliminary analysis cumulative exposure to stimulants and immune function outcomes among hiv-positive and hiv-negative men in the multicenter aids cohort study opposing effects of alcohol on the immune system double jeopardy: methamphetamine use and hiv as risk factors for covid-19 drug use is associated with anti-cd4 igg-mediated cd4+ t cell death and poor cd4+ t cell recovery in viralsuppressive hiv-infected individuals under antiretroviral therapy immune activation and neuroinflammation in alcohol use and hiv infection: evidence for shared mechanisms alcohol abuse and smoking alter inflammatory mediator production by pulmonary and systemic immune cells sars-cov-2 pandemic expanding in sub-saharan africa: considerations for covid-19 in people living with hiv symptoms, stress, and hiv-related care among older people living with hiv during the covid-19 pandemic three months in: a timeline of how covid-19 has unfolded in the us 2020 hiv care continuum and covid-19 outcomes among people living with hiv during the covid-19 pandemic hiv and food insecurity: a syndemic amid the covid-19 pandemic georgia department of public health covid-19 daily status report 2020 city of atlanta coronavirus disease 2019 (covid-19) response 2020 achieve research continuity during social distancing by rapidly implementing individual and group videoconferencing with participants: key considerations, best practices, and protocols development of the alcohol use disorders identification test (audit): who collaborative project on early detection of persons with harmful alcohol consumption ii an empirical investigation of the factor structure of the audit food security, poverty, and human development in the united states il: abbott laboratories hiv-1 viral load measurement in venous blood and fingerprick blood using abbott realtime hiv-1 dbs assay genital tract leukocytes and shedding of genital hiv type 1 rna developing an adherence support intervention for patients on antiretroviral therapy in the context of the recent idu-driven hiv/aids epidemic in estonia a new test-strip for demonstrating erythrocytes and haemoglobin in urine (author's transl) collision of the covid-19 and addiction epidemics quantification of etg in hair, etg and ets in urine and peth species in capillary dried blood spots to assess the alcohol consumption in driver's licence regranting cases validation of the alcohol, smoking and substance involvement screening test (assist) prevalence and predictors of highly active antiretroviral therapy use in patients with hiv infection in the united states. hcsus consortium. hiv cost and services utilization cognitive and field testing of a new set of medication adherence self-report items for hiv care the burden of covid-19 in people living with hiv: a syndemic perspective from hiv to coronavirus: aids service organizations adaptative responses to covid-19 responding to pandemics: what we've learned from hiv/aids medication management and adherence during the covid-19 pandemic: perspectives and experiences from low-and middle-income countries telemedicine and support groups in order to improve the adherence to treatment and health related quality of life in patients affected by inflammatory skin conditions during covid-19 emergency editorial: covid-19 and anxiety and depression in 2020 severe outcomes among patients with coronavirus disease 2019 (covid-19)-united states hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019-covid-net, 14 states key: cord-319043-hczwgf6o authors: ashkenazi, avraham; viard, mathias; unger, linor; blumenthal, robert; shai, yechiel title: sphingopeptides: dihydrosphingosine-based fusion inhibitors against wild-type and enfuvirtide-resistant hiv-1 date: 2012-08-07 journal: the faseb journal doi: 10.1096/fj.12-215111 sha: doc_id: 319043 cord_uid: hczwgf6o understanding the structural organization of lipids in the cell and viral membranes is essential for elucidating mechanisms of viral fusion that lead to entry of enveloped viruses into their host cells. the hiv lipidome shows a remarkable enrichment in dihydrosphingomyelin, an unusual sphingolipid formed by a dihydrosphingosine backbone. here we investigated the ability of dihydrosphingosine to incorporate into the site of membrane fusion mediated by the hiv envelope (env) protein. dihydrosphingosine as well as cholesterol, fatty acid, and tocopherol was conjugated to highly conserved, short hiv-1 env-derived peptides with no antiviral activity otherwise. we showed that dihydrosphingosine exclusively endowed nanomolar antiviral activity to the peptides (ic(50) as low as 120 nm) in hiv-1 infection on tzm-bl cells and on jurkat t cells, as well as in the cell-cell fusion assay. these sphingopeptides were active against enfuvirtide-resistant and wild-type cxcr4 and ccr5 tropic hiv strains. the anti-hiv activity was determined by both the peptides and their dihydrosphingosine conjugate. moreover, their mode of action involved accumulation in the cells and viruses and binding to membranes enriched in sphingomyelin and cholesterol. the data suggest that sphingopeptides are recruited to the hiv membrane fusion site and provide a general concept in developing inhibitors of sphingolipid-mediated biological systems.—ashkenazi, a., viard, m., unger, l., blumenthal, r., shai, y. sphingopeptides: dihydrosphingosine-based fusion inhibitors against wild-type and enfuvirtide-resistant hiv-1. the first step in the life cycle of enveloped viruses is entry into their host cells by membrane fusion (1) . to mediate fusion, the viral envelope (env) protein needs to assist in overcoming the energy barriers to fusion imposed by its surrounding lipids (2) . emerging studies on hiv and other viruses, such as influenza, west nile, and murine coronavirus, suggest membrane-ordered domains as a lipid platform for virus release and entry (3) (4) (5) (6) (7) (8) (9) . this results in viral envelopes with elevated levels of ordered lipid domains (10) . ordered lipid domains are assemblies in cell membranes that are enriched in cholesterol and sphingolipids (11) . the formation of these domains and the partitioning of proteins and lipids into them are dynamic processes (12) . therefore, it is of great importance to identify lipids that can be incorporated into the fusion site during the dynamics of the membrane fusion events. the hiv env is composed of 2 noncovalently associated subunits: the gp120 subunit enables binding of cell receptors and coreceptors, whereas the gp41 transmembrane subunit mediates the physical membrane fusion reaction (13) (14) (15) . binding of gp120 to cd4 and a coreceptor involves conformational changes in both gp120 and gp41, resulting in gp41 prehairpin conformation (14, 16, 17) . this conformation is sensitive to gp41-derived peptide fusion inhibitors that capture it in an intermediate state (18 -22) . as a result, the folding of the protein into the hairpin conformation is prevented, leading to inhibition of viral fusion. the hairpin conformation (23) comprises a trimeric central coiled-coil that is created by 3 n-terminal heptad repeat (nhr) regions, into which three c-terminal heptad repeat (chr) regions are packed in an antiparallel manner (24, 25) . the structure is usually referred to as the "6-helix bundle" or "core" structure, which is required for complete membrane fusion. similar bundles are created in other viral fusion proteins and in intracellular vesicle fusion by soluble n-ethylmaleimidesensitive factor accessory protein receptor proteins, demonstrating a common mechanism in diverse systems (26) . the hiv lipidome shows a remarkable enrichment in dihydrosphingomyelin (10) . this is an unusual sphingolipid in which the backbone is formed by dihydrosphingosine (sphinganine; refs. 27, 28) . in this study, we investigated the ability of sphinganine to incorporate into the site of the hiv env-mediated fusion reaction. for this purpose, sphinganine and other lipids from different classes were conjugated to highly conserved short fragments from the env core that could capture the env in its intermediate state and inhibit fusion when they are in proximity (fig. 1) . we hypothesized that by minimizing the affinity of the peptides to the viral fusion site (using short fragments from the core); we would mainly identify the contribution of the conjugated lipid moiety to antiviral activity. we showed that sphinganine endowed significant antiviral activity to otherwise poorly and nonactive, short c and n peptides derived from the core in enfuvirtideresistant and wild-type hiv-1. we address its plausible mode of action in the context of the lipid-protein rearrangements underlying virus-mediated cell fusion. peptides were synthesized on rink amide mbha resin (novabiochem ag, laufelfingen, switzerland) by using the f-moc strategy as described previously (29) . several peptides contain a lysine residue at their c terminus with an 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2h-tetrazolium bromide side chain protecting group (novabiochem) that requires a special deprotection step under mild acidic conditions [two 1-min washes of 5% trifluoroacetic acid (tfa) in dichloromethane (dcm) and 30 min of 1% tfa in dcm]. this enables the conjugation of a lipid moiety or a fluorescent probe to the c terminus. conjugation of hexadecanoic (palmitic) acid (c16) or ␣-tocopherol succinate (sigma-aldrich, rehovot, israel) to the n terminus of selected peptides was performed using standard f-moc chemistry. conjugation of cholesterol to the n terminus of a peptide was performed by adding 10 equivalents of cholesteryl chloroformate (alfa aesar, ward hill, ma, usa) dissolved in dcm, together with 3 equivalents of triethylamine to the peptide resin. conjugation of d-erythrodihydrosphingosine (d-erythro-sphinganine; matreya, lcc, state college, pa, usa) to the n or c terminus of a peptide was performed as follows: first, 10 equivalents of n,n=-disuccinimidyl carbonate (chem-impex international, wood dale, il, usa) and 20 equivalents of n,n-diisopropylethylamine (diea) were added to the resin for 2 h in dimethylformamide (dmf). then, 2 equivalents of sphinganine and 2 equivalents of diea were added for overnight incubation in dmf anhydrous. addition of nbd-f, a fluoride [4-fluoro-7-nitrobenzofurazan (nbd)] fluorescent probe (biotium, hayward, ca, usa), to the n or c terminus of selected peptides was performed in dmf for 1 h. all peptides were cleaved from the resin by a tfa/double-distilled water/n-tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid (93.1:4.9:2, v/v) mixture and purified by reverse-phase hplc to ͼ95% homogeneity. the molecular weight of the peptides was confirmed by platform electrospray mass spectrometry. for hydrophobicity tests, the peptides were eluted with a flow rate of 0.6 ml/min using a 2-step linear gradient from ch 3 nucleosil analytical c2 column (7-m particle size, pore size 100 å; macherey-nagel, düren, germany). a stock of fully infectious hiv-1 hxb2 concentrated virus was a kind gift from the aids vaccine program (saic-frederick, figure 1 . model for hiv membrane fusion and the structure of lipids investigated in this study. a) hiv gp41 transmembrane protein has at least 3 major conformations during membrane fusion: the native nonfusogenic conformation, the prehairpin conformation, and the hairpin conformation. b) side view of the recently determined hairpin structure of gp41 (protein data bank identification number 2x7r; ref. 56) . the trimeric inner coiled-coil of the n helices is shown in red; the 3 packing c helices are in blue. c, carboxyl terminus; n, amino terminus. investigated short domains within the central core of the protein are indicated: the 17-mer conserved pocket sequence, termed n17; and the 19-mer n-helix binding sequence, termed dp19. c) chemical structures of the hydrophobic conjugates investigated: the cellular moieties consisted of dihydrosphingosine (sphinganine), palmitic acid, and cholesterol; the noncellular moiety consisted of tocopherol. inc., national cancer institute-frederick, frederick, md, usa). the infectivity of hiv-1 hxb2 was determined using the tzm-bl cell line as a reporter. cells were added (2ϫ10 4 cells/well) to a 96-well clear-bottomed microtiter plate with 10% serum-supplemented dmem. plates were incubated at 37°c for 18-24 h to allow the cells to adhere. the mediam was then aspirated from each well and replaced with serum-free dmem containing 40 g/ml diethylaminoethyl (deae)dextran. stock dilutions of each peptide were prepared in dmso so that each final concentration was achieved with 1% dilution. on addition of the peptides, the virus was added to the cells diluted in serum-free dmem containing 40 g/ml deae-dextran. the plate was then incubated at 37°c for 18 h to allow the infection to occur. luciferase activity was analyzed using the steady-glo luciferase assay kit (promega, madison, wi, usa). sometimes cell-washing experiments were performed, as follows: peptides were preincubated with tzm-bl cells at 37°c for 1 h, followed by 3 washes with culture medium to remove unbound peptides and the addition of hxb2 viruses to start the infection. after 18 h, the antiviral activities of the remaining peptides that survive the washing were determined by measuring luciferase activity as described previously. for the virus treatment experiment, the virus was diluted in 100 l of serum-free dmem, and sphingopeptides were added in dmso to a final concentration of 2 m. after a 10-min incubation at room temperature, 400 l of pbs was added. the sample was then centrifuged using amicon centrifugal filters (emd millipore, billerica, ma, usa) with a 30-kda cutoff for 15 min at 10,000 g. the viruses were recovered (40 l) , and their infectivity was tested using the tzm-bl cell line as described above. fitting of the data points was performed according to eq. 1, derived from the hill equation, as described previously (30): in brief, in this equation, b is the maximum value; therefore, it equals 100% fusion, a is the value of an inhibitory concentration at 50% viral infectivity (ic 50 ), and c represents the hill coefficient. for the fitting, we uploaded the x and y values of the data into a nonlinear least-squares regression (curve fitter) program that provided the ic 50 value (parameter a). cotransfection of 293t cells with the pnlluc plasmid and hiv lai env, vesicular stomatitis virus g (vsv-g) env [obtained from the u.s. national institutes of health (nih) aids research and reference reagent program], and hiv ad8 env (a gift from dr. eric freed, national cancer institute, bethesda, md, usa) plasmids was performed. after 48 h of transfection, the viral supernatant was harvested and titrated with the tzm-bl cells. the pseudoviruses thus generated have the same core and differ only by the fusion protein present on their surface. infectivity assays were performed as described for the hxb2 assay. to assess viral infection with jurkat clone e6-1 t cells, lai-pseudotyped viruses were used. the jurkat cells were used at 1.5 ϫ 10 4 cells/well, and the infection was performed for 3 d. virus stocks of the wild-type lai and v38e mutant were prepared with the use of infectious molecular clones as described previously (31) . molecular clones of hiv-1 lai and nl4-3 were obtained from the nih aids research and reference reagent program. ecori-xhoi fragments from the lai molecular clone were cloned into pcdna3.1 expression vector (invitrogen, carlsbad, ca, usa), making the wild-type lai env construct. the fragment contains open reading frames of env, tat, and rev genes. point mutation (v38e) was introduced in the wild-type env construct using the quikchange site-directed mutagenesis kit (stratagene, la jolla, ca, usa). the mutant was numbered based on the env sequence of the hxb2 reference strain. the mutant env region was also introduced into the nl4-3 molecular clone using the ecori-xhoi fragments to generate infectious molecular clones containing mutant lai env. next, 293t cells were transfected with the infectious molecular clones. the virus supernatant was collected 48 h post-transfection, cleared of cellular debris by centrifugation, portioned into aliquots, and stored at ϫ70°c. infection with those viruses was performed as described previously for the hxb2 virus. effector cells were the env-expressing cells, hl2-3, a heladerived cell line, which constitutively expresses the hxb2 strain of the hiv-1 env glycoprotein along the tat protein, and as target cells, tzm-bl cells were used. the fusion of hl2-3 cells with tzm-bl cells was assessed through luciferase expression. the tzm-bl cells were seeded at 2 ϫ 10 4 cells/ well overnight in a 96-well plates. the medium was then aspirated from each well and replaced with serum-free dmem containing 40 g/ml deae-dextran. stock dilutions of each peptide were prepared in dmso so that each final concentration was achieved with 1% dilution. on addition of the peptides, the hl2-3 cells were added to the tzm-bl cells in serum-free dmem containing 40 g/ml deae-dextran at a 1:1 cell ratio. the cells were cocultured at 37°c for 6 h to allow the fusion to occur. luciferase activity was analyzed using the steady-glo luciferase assay kit. large unilamellar vesicles (luvs) were prepared as described previously (32) from egg phosphatidylcholine, cholesterol, and egg yolk sphingomyelin (sigma-aldrich). a dried film of lipids containing a total of 2 mg of phosphatidylcholine/ sphingomyelin/cholesterol (1:1:1) or 2 mg of phosphatidylcholine/cholesterol (9:1) was suspended in pbs and vortexed for 1.5 min. the lipid suspension underwent 5 cycles of freezing-thawing and then extrusion through polycarbonate membranes with 1-and 0.1-m diameter pores for 25 times. the degree of peptide association with lipid vesicles was measured by addition of lipid vesicles to 0.1 m fluorescent nbd-labeled peptides at room temperature, as described previously (33) . the fluorescence intensity was measured as a function of the lipid/peptide molar ratio, with excitation set at 467 nm (10-nm slit) and emission set at 530 nm (10-nm slit). to determine the extent of the contribution of the lipid to any given signal, the readings after the addition of lipid vesicles were subtracted as background from the recorded fluorescence intensity. the affinity constants were then determined by a steady-state affinity model using nonlinear least squares. the nonlinear least squares fitting was done using eq. 2: where x is the lipid concentration, y(x) is the fluorescence emission, f max is the maximal difference in the emission of nbd-labeled peptide before and after the addition of lipids (it represents the maximum peptide bound to lipid), and k a is the affinity constant. circular dichroism measurements were performed by using a spectropolarimeter (applied photophysics, leatherhead, uk). the spectra were scanned using a thermostatic quartz cuvette with a pathlength of 1 mm. wavelength scans were performed at 25°c; the average recording time was 15 s, in 1-nm steps, in the wavelength range of 190-260 nm. each peptide concentration was 10 m in hepes buffer (5 mm, ph 7.4). fractional helicities were calculated as described previously (34) . the 17-mer sequence, termed the pocket, is a conserved domain within the hiv-1 gp41 protein core. this is a deep cavity on the surface of the grooves of the nhr trimer that is important for stabilizing the trimer; it interacts with the chr to maintain the stability of the gp41 core (35, 36) however, a synthetic n peptide derived from the pocket domain (termed n17) was not active in inhibiting viral infectivity up to 4 m, the maximal concentration tested ( table 1) . we conjugated to n17 the following hydrophobic moieties (fig. 1b, c) : dihydrosphingosine (sphinganine), which forms the backbone of dihydrosphingomyelin (27, 28) ; cholesterol; and palmitic acid, which is a building block in many lipids, including phospholipids. for a comparison, we also conjugated to n17 tocopherol, a hydrophobic noncellular compound. the hybrid compounds were tested for their ability to inhibit viral infectivity. notably, the inhibitory activity of n17 depends on the nature of the lipid moiety (table 1 and fig. 2) . sphinganine was the most prominent one and potentiated the antiviral activity of n17 into the nanomolar concentration range. this exclusive inhibitory activity of sphinganine-based peptides (sphingopeptides) was further observed in a wider spectrum of viral entry systems, consisting of different hiv strains (wildtype hxb2 and lai) and different target cells (tzm-bl reporter cells and jurkat t cells), as well as in cell-cell fusion assay (fig. 2) . increased antiviral activity of short peptides is sometimes attributed to increased hydrophobicity (30, 37) or increased ␣-helical content of the compound via conjugation to a specific moiety (38) . therefore, we checked the apparent hydrophobicity of n17 and its lipid conjugates ( table 1 ). the most hydrophobic compounds were the tocopherol and cholesterol conjugates. sphinganine and palmitic acid conjugates exhibited similar medium hydrophobicity, and n17 alone was the least hydrophobic. clearly, there was no correlation between the extent of hydrophobicity and antiviral activity in this case. in addition, there was no increase in the ␣-helical content of n17 on conjugation (table 1) . we further investigated the interplay between sphinganine and its conjugated peptide in inhibiting viral infectivity. viruses were constructed to have the same hiv core with three different envs: lai (cxcr4 tropic), ad8 (ccr5 tropic), and vsv-g. the three constructed viruses 50 for each compound was calculated as described in materials and methods. results are means ϯ sd); n ϭ 3. b hydrophobicity was analyzed by rp-hplc. retention time of each compound is presented. c percentage of ␣-helical structure was determined as described in materials and methods. were allowed to infect tzm-bl cells in the presence of increasing concentrations of sphingopeptides (fig. 3a) . antiviral activity was observed for lai and ad8 but not for vsv-g. the importance of peptide interactions to the overall activity of the molecules was examined using sequence mutagenesis. mutated sphingopeptides were prepared by replacing n17 peptides residues in positions a and d of the helical wheel, termed n17m(a,d), thus knocking out their ability to self-assemble (fig. 3b, c) . the rationale followed the report by bewley et al. (39) for n36 peptides, in which these mutations completely abrogated n-peptide antiviral activity. alternatively, we randomly reorganized the n17 sequence to give scrambled peptides (fig. 3c) . none of the mutant n17 peptides or their sphinganine conjugates showed antiviral activity (fig. 3d) . in addition, a 17-mer n peptide was synthesized from the same region as n17 but shifted in its sequence from the gp41 pocket (the peptide termed shn17 ; fig. 4a ). sphingopeptides comprising the shn17 sequence exhibited antiviral activity that was 7.3-fold lower than those with the pocket ic 50 values are presented as means ϯ sd, n ն 3. *p ͻ 0.05, **p ͻ 0.02. sequence (fig. 4b ). the addition of sphinganine alone, up to 1 m (a concentration at which sphinganine-n17 completely blocks fusion), did not affect viral infectivity (fig. 4b) . changing the orientation of sphinganine toward n17 was achieved by addition of lysine to its c terminus, enabling c-terminal lipid conjugation. the addition of the lysine did not alter the antiviral activity of the compound according to its inhibitory concentration at 50% infectivity (ic 50 ) of 121 ϯ 36 and 116 nm ϯ 12 nm to sphinganine-n17 and sphinganine-n17k, respectively (fig. 4b, c) . the inhibitory potentiation of sphinganine was still powerful via c-terminal conjugation, exhibiting an ic 50 value of 287 ϯ 143 nm to n17ksphinganine (fig. 4c) . we then investigated whether the ability of sphinganine to potentiate the activity of short n peptides could be exploited to other peptides from the gp41 core. therefore, a short 19-mer c peptide from the chr, termed dp19, was synthesized (figs. 1b and 4a). dp19k-sphinganine was significantly more potent than dp19k, exhibiting an ic 50 value of 350 ϯ 60 nm (fig. 4d) . accordingly, this amplification of fusion inhibition is not restricted only to n peptides. it can be implemented in other short fragments from different regions within the gp41 core. the giv sequence within the nhr of gp41 (fig. 4a ) is well established as a site for escape mutations of the virus against the fusion inhibitor drug, enfuvirtide, which leads to viral resistance (40) . we introduced to the hiv construct a mutation in the giv sequence (v38e) that considerably weakened the antiviral activity of enfuvirtide, whereas sphinganine-n17 preserved its potent antiviral activity (fig. 4e ). the ability of sphingopeptides to bind and accumulate in the cell membrane was examined by preincubating sphinganine-n17 with target cells, followed by washing before addition of the virus to initiate infection. we calculated the ic 50 of the peptide with or without washing the cells (ic 50 1 or 2, respectively) to evaluate the ability of residual peptides to sustain inhibitory potency (fig. 5a) . surprisingly, the ic 50 value of sphinganine-n17 increased only by 5-fold after cell washing. we compared this with gp41 peptide fusion inhibitors with different membrane-binding properties: c34, which poorly binds membranes, and enfuvirtide, which has an intrinsic membrane-binding ability (41, 42) . the ic 50 of c34 dramatically increased by 305-fold, whereas a 36-fold increase was observed for the ic 50 of enfuvirtide after cell washing. we then directly treated the viruses with sphinganine-n17, purified the viruses, and added them to the cells. a remarkable reduction in infectivity was observed for the treated viruses (fig. 5b) . furthermore, the sphingopeptides were added to target cells before or after addition of the virus. in the second procedure, both viral and cell membranes are presented at the time of addition, and the sphingopeptides could theoretically bind to any of them. if binding to the viral membrane has a more pronounced effect, we might expect differences in the viral infectivity curves of the compound, depending on the time that the virus was added. importantly, no difference between the viral infectivity curves was observed for sphinganine-n17 (fig. 5c ). the degree of compound association with lipid vesicles was estimated by titrating nbd-labeled compound with e n f u v ir t id e figure 5 . sphingopeptides strongly bind and accumulate in the cell membrane and in the viral membrane. a) sphingopeptides are strongly anchored to the membrane and sustain a potent inhibitory effect on washing. c34, enfuvirtide, and sphing-n17 were preincubated with tzm-bl cells, followed by washing to remove unbound peptides and the addition of fully infectious viruses to start the infection. ic 50 of the peptide with or without washing the cells (ic 50 1 or 2, respectively) was calculated. results are presented as the mean ϯ sd ratio of ic 50 1/2 (nն2), which reflects the fold increase in ic 50 after cell washing. b) infectivity of purified viruses that were pretreated with sphing-n17 or dmso. infectivity of the purified viruses was measured by luciferase activity in tzm-bl cells and is expressed in relative light units. c) antiviral activity of sphing-n17 when it is preincubated with tzm-bl cells before the addition of fully infectious virus or when it is added to a mixture of the virus with cells. luvs. the membrane-binding k a values derived from eq. 2 are presented in table 2 . the data include sphinganine-n17, c16-n17, and enfuvirtide associated with vesicles enriched in sphingomyelin and cholesterol and with vesicles comprising only phosphatidylcholine and cholesterol. the backbone of both palmitic acid and sphinganine is based on a linear saturated carbon chain, and the lipids exhibited similar hydrophobicity. however, palmitic acid-conjugated peptides showed no antiviral activity (table 1) . thus, the fatty acid conjugates were used as controls for the binding assay of sphingopeptides. sphinganine-n17 and c16-n17 had higher binding affinities to both types of lipids compared with enfuvirtide. interestingly, sphinganine-n17 preferably binds lipid vesicles enriched in sphingomyelin and cholesterol ( table 2 ). enfuvirtide also exhibits some preference toward these vesicles but to a lesser extent, and c16-n17 retains binding affinity similar to that of both types of vesicles. these observations will be further discussed in the context of the plausible antiviral mode of action of sphingopeptides. the structural organization of lipids in the membrane is a key factor in cell physiology and is exploited by pathogens to infect cells (12) . hiv is believed to take advantage of cell membrane-ordered domains to facilitate its entry and budding. these were mainly explored by cholesterol depletion from viral and host cell membranes, together with altering the cellular sphingolipid metabolism before virus-cell fusion (43) (44) (45) (46) (47) (48) . much less, however, is known about the recruitment of specific lipid moieties to the site of fusion during the dynamic hiv fusion reaction. a substantial enrichment of dihydrosphingomyelin was observed in hiv particles that bud from their host cells (10) . thus, we hypothesized that its dihydrosphingosine (sphinganine) backbone may penetrate into the site of membrane fusion mediated by the hiv env. to investigate this, we conjugated sphinganine as well as other lipids to short, otherwise inert, peptides from the hiv-1 env core and their antiviral activities were investigated in several types of hiv-1 infection assays. the only lipid moiety that endowed potent antiviral activity to the peptides was sphinganine (a summary of the inhibitory ic 50 is presented in supplemental table s1 ). the analysis of the interplay between the lipid backbone and its conjugated hiv-1 peptide revealed that the anti-hiv effect is determined both by a specific peptide sequence and by a specific lipid moiety (i.e., sphinganine). this is based on the following findings: first, sphingopeptides exhibited antiviral activity against hiv-1 and not against vsv-g (fig. 3a) . second, mutagenesis analysis of the peptide interactions by knocking out their ability to self-assemble or by randomizing the sequence completely abrogated the antiviral activity of sphingopeptides (fig. 3b-d) . third, sphinganine alone did not alter hiv-1 infection at a concentration at which sphinganine-n17 completely blocked infection (fig. 4b) , and fourth, changing the sequence of the peptide, while keeping the same lipid backbone yielded different inhibitory levels (fig. 4b, d) . the viral membrane has a different lipid composition than does the cell membrane (10) . this parameter may affect the types of lipids that are bound to the cell and the viral membranes during fusion. we showed that sphingopeptides could inhibit when bound to the cell membrane or to the viral membrane by their fusion inhibitory activity on cell-cell fusion assay; the ability of sphinganine to preserve sufficient amounts of bound peptides within the target cells after washing, which prolonged their antiviral potencies; and the reduced infectivity of viruses pretreated with sphingopeptides. membrane binding affinity analysis revealed that sphinganine-n17 preferably binds lipid vesicles enriched in sphingomyelin and cholesterol (table 2) . several lines of evidence support the interaction of dihydrosphingomyelin with membrane-ordered domains. in model membranes, cholesterol interacts with dihydrosphingomyelin to form more condensed domains than the ones formed with sphingomyelin (49) . studies in bilayers that resemble the ocular lens membranes show that the amount of cholesterol crystallite formation is much higher with dihydrosphingomyelin than with sphingomyelin (50) . in addition, the polar dihydrosphingosine base could favor its interaction with the sphingolipid backbone chains through hydrogen bonds (27) . sphingolipids play an auxiliary function in hiv entry by several proposed models (44) . sphingolipids may compound association with lipid vesicles was estimated by titration of nbd-labeled compound with large unilamellar vesicles. mole ratio between phosphatidylcholine (pc), sphingomyelin (sm), and cholesterol (chol) is shown for each vesicle. membrane-binding constants (k a ) were calculated from eq. 2 in materials and methods. results represent means ϯ sd of the fitting curve; n ϭ 3. enhance virus attachment by direct interaction with the hiv-1 envelope (6, 51) . in addition, they are involved in clustering of sufficient receptors to the fusion site (52, 53) . in intestinal epithelial cells, both gp120 and gp41 bind to galactosyl ceramide, located in membrane-ordered domains. thus, they allow the transcytosis of the virus across the epithelial barrier (54) . resistant strains emerge from treatment with gp41 c-peptide fusion inhibitors. mutations in a contiguous 3-aa sequence within the nhr region, giv, are often related to the acquisition of viral resistance to enfuvirtide (40) . the alterations in that specific region were also observed during phase i of the clinical trial with enfuvirtide (55) . we show that sphinganine endows antiviral activities to short conserved n peptides to overcome enfuvirtide-resistant viruses. hence, there may be implications for the use of derivatives of n peptides as potential microbicides. possible future applications for sphingopeptides are to extend the halflife time of fusion inhibitors in vivo and to act as topical blockers of viral transmission during sexual intercourse. in summary, the data suggest that sphingopeptides are recruited to the hiv membrane fusion site with enhanced membrane-binding affinity that prolonged their inhibitory potencies. the anti-hiv activity is determined by both the peptides and their sphinganine conjugate. this unique characteristic makes sphingopeptides the shortest lipid-based hiv peptides that potently inhibit viral fusion. because sphingolipids are involved in many cellular processes such as signaling, viral infection, and membrane trafficking, the present data suggest a general concept of developing inhibitors to sphingolipid-mediated biological systems. viral and cellular membrane fusion proteins mechanics of membrane fusion evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts plasma membrane rafts play a critical role in hiv-1 assembly and release role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells identification of a common sphingolipidbinding domain in alzheimer, prion, and hiv-1 proteins influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release west nile virus entry requires cholesterol-rich membrane microdomains and is independent of ␣v␤3 integrin the hiv lipidome: a raft with an unusual composition functional rafts in cell membranes revitalizing membrane rafts: new tools and insights chemokine receptors as hiv-1 coreceptors: roles in viral entry, tropism, and disease mechanisms of viral membrane fusion and its inhibition the hiv env-mediated fusion reaction the function of coreceptor as a basis for the kinetic dissection of hiv type 1 envelope protein-mediated cell fusion topological layers in the hiv-1 gp120 inner domain regulate gp41 interaction and cd4-triggered conformational transitions hiv-1 inhibition by a peptide capture of an early fusion-active conformation of hiv-1 gp41 hiv entry and its inhibition membrane-anchored inhibitory peptides capture human immunodeficiency virus type 1 gp41 conformations that engage the target membrane prior to fusion dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41 the structural biology of type i viral membrane fusion core structure of gp41 from the hiv envelope glycoprotein atomic structure of the ectodomain from hiv-1 gp41 intracellular and viral membrane fusion: a uniting mechanism biophysics of sphingolipids i. membrane properties of sphingosine, ceramides and other simple sphingolipids the ins and outs of sphingolipid synthesis synthesis of the antibacterial peptide cecropin a (1-33) virus-cell and cell-cell fusion mediated by the hiv-1 envelope glycoprotein is inhibited by short gp41 n-terminal membrane-anchored peptides lacking the critical pocket domain site-specific mutations in hiv-1 gp41 reveal a correlation between hiv-1-mediated bystander apoptosis and fusion/hemifusion fatty acids can substitute the hiv fusion peptide in lipid merging and fusion: an analogy between viral and palmitoylated eukaryotic fusion proteins androctonin, a hydrophilic disulphide-bridged nonhaemolytic anti-microbial peptide: a plausible mode of action spectroscopic and functional characterization of the putative transmembrane segment of the mink potassium channel amino acid conservation in the gp41 transmembrane protein and natural polymorphisms associated with enfuvirtide resistance across hiv-1 variants evidence that a prominent cavity in the coiled coil of hiv type 1 gp41 is an attractive drug target membrane-anchored hiv-1 n-heptad repeat peptides are highly potent cell fusion inhibitors via an altered mode of action design of potent inhibitors of hiv-1 entry from the gp41 n-peptide region design of a novel peptide inhibitor of hiv fusion that disrupts the internal trimeric coiled-coil of gp41 emergence of a drugdependent human immunodeficiency virus type 1 variant during therapy with the t20 fusion inhibitor demonstrating the c-terminal boundary of the hiv 1 fusion conformation in a dynamic ongoing fusion process and implication for fusion inhibition different from the hiv fusion inhibitor c34, the anti-hiv drug fuzeon (t-20) inhibits hiv-1 entry by targeting multiple sites in gp41 and gp120 lipid rafts and hiv pathogenesis: host membrane cholesterol is required for infection by hiv type 1 sphingolipids: modulators of hiv-1 infection and pathogenesis the role of cholesterol and sphingolipids in chemokine receptor function and hiv-1 envelope glycoprotein-mediated fusion the role of glycosphingolipids in hiv signaling, entry and pathogenesis ceramide, a target for antiretroviral therapy dihydrosphingomyelin impairs hiv-1 infection by rigidifying liquid-ordered membrane domains membrane properties of d-erythro-n-acyl sphingomyelins and their corresponding dihydro species cholesterol in bilayers of sphingomyelin or dihydrosphingomyelin at concentrations found in ocular lens membranes specific interaction of hiv-1 and hiv-2 surface envelope glycoproteins with monolayers of galactosylceramide and ganglioside gm3 sphingomyelinase restricts the lateral diffusion of cd4 and inhibits human immunodeficiency virus fusion sphingolipids, cholesterol, and hiv-1: a paradigm in viral fusion secretory iga specific for a conserved epitope on gp41 envelope glycoprotein inhibits epithelial transcytosis of hiv-1 emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (t-20) monotherapy crystal structure of hiv-1 gp41 including both fusion peptide and membrane proximal external regions the authors thank batya zarmi for her valuable help with peptide purification and winfried weissenhorn for providing the recent 3-dimensional structure of gp41. this study was supported by the israel science foundation (to y.s.) and in part by federal funds (to r.b.) from the frederick national laboratory, national cancer institute, u.s. national institutes of health (contract hhsn26120080001e). the content of this publication does not necessarily reflect the views or policies of the u.s. department of health and human services, nor does mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. y.s. is the incumbent of the harold s. and harriet b. brady professorial chair in cancer research. key: cord-324137-nau83mjv authors: saranathan, nandhini; vivekanandan, perumal title: g-quadruplexes: more than just a kink in microbial genomes date: 2018-09-14 journal: trends microbiol doi: 10.1016/j.tim.2018.08.011 sha: doc_id: 324137 cord_uid: nau83mjv g-quadruplexes (g4s) are noncanonical nucleic acid secondary structures formed by guanine-rich dna and rna sequences. in this review we aim to provide an overview of the biological roles of g4s in microbial genomes with emphasis on recent discoveries. g4s are enriched and conserved in the regulatory regions of microbes, including bacteria, fungi, and viruses. importantly, g4s in hepatitis b virus (hbv) and hepatitis c virus (hcv) genomes modulate genes crucial for virus replication. recent studies on epstein–barr virus (ebv) shed light on the role of g4s within the microbial transcripts as cis-acting regulatory signals that modulate translation and facilitate immune evasion. furthermore, g4s in microbial genomes have been linked to radioresistance, antigenic variation, recombination, and latency. g4s in microbial genomes represent novel therapeutic targets for antimicrobial therapy. the transient formation of g4s under thermodynamically favorable conditions has important regulatory roles dictated by the genomic location. g4s are ubiquitously found in the telomeres of eukaryotes [4] . the formation of g4s by the g-rich telomeric repeats inhibits extension of telomeres by telomerase; thus stabilization of g4s in telomeres with ligands represents a potential anticancer strategy [5] . nearly 50% of human genes have a g4 motif in their promoter region [6] . importantly, oncogenes like c-myc, vegf [7] , and kras [8] are negatively regulated by their promoter-borne g4s [5] . g4 structures can also form in rna. quadruplexes formed in the 5 0 utr of the mrna inhibit cap-dependent translation (e.g., nras and bcl-2) and enhance ires-mediated cap-independent translation (e.g., hveg-f and fgf2) [9, 10] . besides, g4s also influence other molecular mechanisms in rna biology such as splicing, ribosomal frameshifting, mrna localization, repeat-associated non-aug (ran) translation, and maturation of mirnas (reviewed in [10] ). furthermore, formation of an intermolecular hybrid quadruplex (hq) between nontemplate dna and nascent mrna acts as a transcription-termination signal [10] . in addition to gene expression, the spatial association of quadruplex motifs with the recombination hotspots in the human genome implicates quadruplexes in recombination [11] . quadruplexes are increasingly being recognized as novel therapeutic targets in microbes. several reports convincingly demonstrate antimicrobial activity of quadruplex-binding ligands against clinically challenging pathogens, including hiv-1, hcv, ebola virus, plasmodium falciparum, and mycobacterium tuberculosis. the repertoire of cellular proteins binding g4s is both structurally and functionally diverse; it comprises a number of zinc-finger transcription factors (sp1, maz, parp, cnbp), splicing factors (u2af), proteins of the shelterin complex, rna-binding proteins such as hnrnps and rhau, and rgg-box-containing multifunctional proteins, including nucleolin and fmrp [12] [13] [14] . persistence of g4 structures can dysregulate the cellular activities they control and also compromise genomic integrity [15, 16] . helicases, including fancj, pif1, dhx36 [17] , blm, and wrn, can unwind the g4s in eukaryotic genomes. extensive research on g4s has led to the identification of g4 ligands which are compounds that can specifically bind to these nucleic acid secondary structures [18] . not much was known about g4s in microbial genomes about a decade ago. in the last few years, the roles of g4s in microbes have been increasingly recognized ( figure 2 ). in this review, we aim to provide insights on the biological role of quadruplexes in microbial genomes with an emphasis on recent findings. in this section, we discuss three virulence-related microbiological features regulated by g4s. virulence factors are biomolecules produced by the pathogen that enable them to successfully establish infection and multiply in the host. they include adherence factors, immunomodulators, drug efflux pumps, and toxins. depending on the tissue environment, pathogens have specific surface adaptations, including pili, fimbriae (in bacteria) and host cell receptor-binding glycoproteins (in viruses and parasites), all of which enable entry into the host. the surface-exposed proteins are at the interface of host-microbe interaction and are highly antigenic. the surface proteins of some pathogens are continuously altered by antigenic and phase variation to overcome host adaptive immune responses. besides sequence mutation and natural competence to dna transformation [19] , the molecular basis of antigenic variation (av) also involves recombination of genomic segments leading to the production of altered surface proteins [20] . interestingly, g4 motifs have been identified at the recombination sites associated with av in bacteria and parasites [21, 22] . indicate the types of microbe affected for each biological process (see the legend beside the graph). the specific microorganism under each type is denoted either by an abbreviation (for viruses) or a three-letter notation (for bacteria and parasites) in the vertical bars in the innermost circle (see the legend beside the graph). the vertical bars in the innermost circle are colour-coded for bacteria (green), viruses (red), and parasites (black). the curved lines connect a given microbe with multiple quadruplexregulated biological processes. red curved lines connect viruses, and blue curved lines connect parasites. intramolecular quadruplexes have been identified to play potential roles in the av of (i) pile in neisseria gonorrhoeae, the bacterium that causes gonorrhea, (ii) vlse in the lyme disease agent borrelia burgdorferi, and (iii) tprk in [ 3 1 6 _ t d $ d i f f ] treponema pallidum [23] [24] [25] . conventionally, av by gene conversion involves the unidirectional transfer of genetic segments from the donor loci, a tandem array of silent alleles of the surface protein, to a downstream recipient locus that actively expresses the gene encoding the surface protein; this process is assisted by recombinases. pili are hair-like appendages made up of pilin proteins which are present on the bacterial cell surface and exhibit av. in n. gonorrhoeae, it has been demonstrated experimentally that the g4 formed near the pile, a pilin-expression locus, binds reca and provides a topological advantage for the nicking process essential to initiate recombination [26] . deletion of this quadruplex motif suppresses av in neisseria. lyme disease is caused by a tick-borne bacterium belonging to the genus borrelia. the vls locus in b. burgdorferi is associated with av. the coding strand of the vls locus has over a 100fold enrichment of guanine (g)-runs of at least three nucleotides or more despite the preference for at-rich codons [24] . these g-rich sequences form g4s and are suggested to play a role in recombination-mediated av in borrelia species. t. pallidum is a spirochete that causes syphilis. tprk is a surface protein that undergoes av in t. pallidum. g4-forming sequences were identified proximal to the tprk gene, indicating a possible role for these dna secondary structures in av among treponemes [25] . however, the proposed functional roles for quadruplexes identified in the av loci of the two spirochetes are not supported by experimental evidence. [ 3 1 7 _ t d $ d i f f ] the family of erythrocyte membrane proteins-1 (pfemp-1) is an important virulence factor of the malarial parasite, plasmodium falciparum. symptoms of malaria appear in about a week after exposure when the parasite enters red blood cells (rbcs) and digests hemoglobin [27] . [ 3 1 8 _ t d $ d i f f ] proteins of the pfemp-1 family are expressed on the surface of infected erythrocytes during the asexual life cycle in man and are encoded by var, a family of 60 genes which are predominantly present in the subtelomeric region of chromosomes [28] . the var genes undergo recombination (indels and translocations) to facilitate sequence variation in pfemp1 and immune evasion. interestingly, about a quarter of all putative quadruplex motifs in the p. falciparum genome are associated with the promoters of the var genes [29] . stanton et al. identified a close association between the recombination breakpoints and g4 motifs in p. falciparum [30] . breakpoints were found to occur proximal to quadruplexes, especially in subtelomeic regions, indicating that g4s have a role in var-associated recombination. although recombination in p. falciparum genomes occurred proximal to quadruplex motifs, the median distance of a g4 from a breakpoint was about 16 kb. the specific mechanisms underlying g4assisted var gene recombination are not fully understood; nonetheless, a potential role for dna repair has been speculated. in addition to contributing to antigenic diversity in microbes (discussed above), g4s facilitate generation of genetic heterogeneity and evolution of hiv-1, the causative agent of aids. the recombination rate of hiv-1 stems from the ability of the reverse transcriptase (rt) to switch between rna templates and generate chimeric proviral dna. recombinant hiv-1 strains have been associated with increased transmission efficiency and resistance to anti-hiv therapy [31] . the two positive-sense rna strands of hiv-1 are held together by hairpin loops in the dimerization site (dis) at the 5 0 end of each of the rnas [32] . this allows for strand transfer by rt, making the region a recombination hotspot. similar to the hairpin loops, intermolecular g4s tether the recombining segments, thus bringing them into each other's proximity to promote initiation of recombination. independent studies identified intermolecular g4 motifs in three regions of the hiv-1 rna (i) a 130-nt region comprising the dis and 5 0 portion of the gag, (ii) central polypurine tract (cppt), and (iii) the u3 region on either termini of rna [33] [34] [35] [36] . under in vitro conditions, synthetic rna oligonucleotides corresponding to these g4s caused pausing of rt in the presence of potassium ions. moreover, in an in vitro strand transfer assay, efficient switching over of rt between templates was observed under conditions that promote quadruplex formation (presence of potassium ions). these studies suggest that quadruplexes allow for dimerization of the hiv-1 genome at multiple loci along its length, thus contributing to recombinogenicity and rapid evolution of hiv-1. in addition to hiv-1 evolution, other functional aspects of g4s discussed in this review, and the selective retention or exclusion of g4s from specific genomic loci in bacteria and yeast, indicate that g4s play a role in the evolution of microbes ( figure 3 ) [30, [37] [38] [39] [40] [41] [42] . gene expression and packaging of virions long terminal repeats (ltrs) present on either termini of the hiv-1 genome enable integration of the hiv-1 provirus into the host genome and contain within them the necessary genomic elements for expression and control of hiv-1 genes. three overlapping quadruplex motifs were identified in the u3 promoter region of the ltr between positions à105 and à48 [43] . the motifs encompass the binding sites of the two transcription factors, nf-kb and sp1. these g4s negatively regulate the activity of the ltr promoter and hence the replication of hiv-1. interestingly, the presence of the quadruplex in the ltr promoter is not restricted to hiv-1 but is evolutionarily conserved among the primate lentiviruses [41] . human herpesviruses (hhvs) are large double-stranded dna (ds-dna) viruses that infect a variety of tissues. in hhvs, putative promoter regions have higher g4 densities than the coding regions, suggesting a regulatory role for g4s in gene expression [44] . g4s in the promoters of ul2, ul24, and k18, all of which have previously established roles in virulence, were found to be negative regulators of promoter activity ( figure 4a ) [45] [46] [47] [48] . herpesvirus genes are divided into immediate early (ie), early (e), and late (l) based on the time at which they are expressed in the replication cycle. ie genes act as trans-activators or trans-repressors of the e and l genes. ie genes are expressed within a few hours of virus entry into the host cells. interestingly, the regulatory regions of ie genes were particularly enriched for g4 motifs [44] . besides transcription, a recent report also implicates g4s in the packaging of herpesvirus genomes [49] . an earlier study reports that, following concatemeric replication of hhv-1, the cleavage of unit length genomes and their encapsidation is achieved by the binding of virus proteins to a dna secondary structure formed by a dna packaging sequence (pac-1) [50] . it has now been identified that the dna secondary structure formed by pac-1 is a g4 ( figure 4b ) [49] . in fact, the pac-1 sequences of all the eight human herpesviruses contain a highly conserved g4 motif that predominantly forms intermolecular quadruplexes. human papilloma virus (hpv) is a dna virus that causes warts and cervical cancer. tluckova et al. identified the presence of three-tetrad g4 motifs in the long control region (lcr) and in the coding regions of e1, e2/e4, and l2 proteins of eight hpv types [51] . interestingly, two-tetrad quadruplexes were identified in the same genomic regions (i.e., e1, e2/e4 and lcr) of manatee papilloma viruses [52] . in papilloma viruses, the lcr contains a number of cis-acting regulatory elements for virus replication and transcription that play a role in determining tissue tropism [53] . the early proteins (e1-e7) are nonstructural proteins and have pivotal roles in the modulation of the host regulatory network while the late proteins (l1, l2) are required for virion assembly [54] . the specific biological roles for the g4s in papilloma viruses remain to be discovered; however, potential functional roles in gene expression have been speculated for these dna secondary structures based on their key genomic locations in certain hpv types. severe acute respiratory syndrome-coronavirus (sars-cov) is an enveloped virus with a positive-sense single-strand rna genome. nonstructural protein (nsp3) is a multidomain protein that is a part of the replication/transcription complex (rtc) of the virus [55] . the sars-unique domain (sud), exclusively present in the nsp3 of sars-cov, is believed to contribute to the higher pathogenicity of sars-cov as compared to other human coronaviruses [55] . interestingly, sud was identified to bind g-runs and the more ordered g4s, in both dna and rna [56] . the g4-binding property is mapped to the m domain nested within the sud and is indispensable for the replication and transcription of the virus [57] . putative g-rich targets of sud include host mrnas encoding proteins that regulate key cellular processes such as apoptosis and cell proliferation. therefore, g[ 3 2 0 _ t d $ d i f f ] 4-binding microbial proteins may potentially play a role in the modulation of key cellular proteins and signaling pathways in the host [55, 56] . the latency programme of viruses allows them to survive inside the host and protects them against the immune surveillance of the host. the expression of latency-associated genes leads to heterochromatinization of the virus genome and the inhibition of proteins necessary for virus replication [58, 59] . figure 4c ). interestingly, even upon removal of the ligand, the number of kshv genome copies was lower as compared to that in cells without the ligand, indicating reduction of virus episomes. integration into the host genome is another strategy employed by herpesviruses for latent survival. human herpesvirus 6a, the causative agent of roseola infantum, undergoes stable integration at the telomeric ends of the host chromosomes by homologous recombination. about 1% of the human population has the congenital presence of hhv-6a due to integration of the virus in germline cells [61] . the formation of g4s by the telomeric repeats (ttaggg) is well documented [4] . a recent study demonstrated that the stabilization of telomeric g4s by braco-19 (n-[9-[4-(dimethylamino)anilino]-6-(3-pyrrolidin-1-ylpropanoylamino)acridin-3-yl]-3-pyrrolidin-1-ylpropanamide) significantly reduced the integration frequency of hhv-6a in telomerase-expressing cells [ 3 2 3 _ t d $ d i f f ] [61] . the authors argue that stabilization of the telomeric g4s interferes with telomerase activity, resulting in reduced chromosomal integration of hhv-6a. epstein-barr virus (ebv) is an oncogenic herpesvirus that is associated with b cell lymphoma and nasopharyngeal carcinoma. during latency, ebna1, a genome-maintenance protein (gmp), tethers the circular ebv episome to cellular chromatin and ensures its transmission to daughter cells on completion of each cell cycle [62] . ebna-1 also regulates host and viral transcription. it is imperative that the synthesis of the latency proteins is tightly controlled lest they are processed and presented to mhcs as antigens, defeating the primary biological function of this group of proteins. murat et al. identified putative quadruplex motifs in ebna1 and gmps encoded by other gamma herpesviruses [63] . furthermore, they also describe quadruplex-mediated repression of ebna1 protein levels. the translation of several oncogenic proteins in humans is known to be controlled by quadruplexes in the 5 0 utr or coding region [9] . ebna-1 is a key player in ebv-induced oncogenesis [64, 65] . interestingly, the level of the ebv oncogene, ebna-1, is maintained by the folding and unfolding dynamics of its mrna-borne quadruplex. the segment of ebna1 mrna that encodes its glycine-alanine repeat (gar) domain was found to be rich in g4 motifs. polysome distribution profiling, in vitro translation, and cell culture experiments identified that formation of quadruplexes obstructs the progress of ribosome machinery, resulting in low levels of ebna1 and a concomitant decrease in presentation to t cells ( figure 4d ). in addition, nucleolin was identified to bind the g4 formed in the gar domain of ebna-1 mrna [66] . this interaction limits the synthesis of ebna-1 to levels that allow persistence of the virus and evasion of the host immune system. mutation of the ebna-1 mrna quadruplex or absence of nucleolin resulted in alleviation of translation inhibition and increased presentation of ebna-1. besides regulating synthesis, g4s also come into play in the functional aspects of ebna-1. as a gmp, ebna-1 is involved in episomal replication and attachment to metaphase chromosomes. these functions are carried out by the linking regions lr1 and lr2 present in ebna-1. lr1 and lr2 bind cellular rna-quadruplexes to recruit origin recognition complex (orc) to orip, the origin of episomal replication in ebv [67] . braco-19 outcompetes ebna-1 in binding to the intermediary rna quadruplex, thus inhibiting the replication of ebv in latently infected cells. consequently, a reduction in the ebv copy number and its attachment to metaphase chromosomes was observed by q-pcr and flow cytometry, respectively. viruses exploit the molecular machinery of the host for successful infection. they utilize the quadruplex-binding abilities of host proteins to regulate the dynamics of quadruplex formation in their genome and the downstream effects thereof. the quadruplex in the ltr promoter of hiv-1 provirus binds the host protein nucleolin [68] . nucleolin stabilizes the quadruplex and represses the transcriptional activity of the ltr promoter, allowing the virus to enter latency. interestingly, heterogeneous nuclear ribonucleoprotein a2/b1 (hnrnpa2/b1), a host protein, binds and unfolds the quadruplex in the ltr promoter of hiv-1 provirus, leading to enhanced transcription [69] . taken together, these results suggest that g4s in virus genomes may interact with host proteins not only to facilitate virus latency but also to revoke viruses from latency. these studies on g4-protein interactions highlight how g4s contribute to the molecular milieu of host-pathogen interaction. the emergence of antimicrobial resistance is a major limiting factor in the management of infectious diseases such as aids and tuberculosis. indiscriminate use of antimicrobial agents and patient noncompliance contribute to the emergence of antimicrobial resistance [70] . the need to develop or identify novel therapies as well as novel therapeutic targets to tackle antimicrobial resistance has been increasingly recognized. the identification of g4s in microbial genomes as targets of antimicrobial therapy has led to the identification of novel antimicrobial agents. g4s have been shown to inhibit the transcription or translation of structural and nonstructural proteins in viruses, deleteriously affecting the virus loads and their pathogenicity; the stabilization of these quadruplexes with ligands has been investigated as a potential mechanism for targeting viruses. for example, the hiv-1 nef gene contains quadruplex motifs that inhibit synthesis of the nef protein [71] . the addition of tmpyp4, a quadruplex-binding ligand, further lowered the expression of this protein. the nef protein is required for efficient viral entry, integration of provirus into host genome, and replication in the host cells [72] . it also modulates a number of cellular immunity factors like cd4 and mhc i to enhance the survival of the virus [73] . defects in the nef gene or its deletion from the virus genome affect the infectivity of the virus and delay the progression to aids [74, 75] . hcv is an enveloped positive-sense rna virus. chronic hcv infection is a major cause of hepatocellular carcinoma (hcc). a quadruplex motif in the core gene inhibits the synthesis of core (capsid) protein and replication of hcv [76] . stabilization of this quadruplex in the hcv core gene with ligands results in stalling of the viral rna-dependent rna polymerase (rdrp) at the g4 motif, resulting in decreased hcv core protein levels. ebola virus, a negative-sense rna virus, causes hemorrhagic fevers and represents one of the well studied zoonotic filoviruses. a 27-nt long g4 motif was identified in the l gene of ebola virus that encodes the rdrp [77] . stabilization of this g4 motif in the l gene with a quadruplexbinding ligand led to reduced transcription of the l gene. the rdrp encoded by the l gene is indispensable for the life cycle of ebola virus. the stabilization of the g4 motif in the l gene with ligands reduces the replication competence of ebola virus. furthermore, the authors report g4 ligands as more potent antiviral agents as compared to ribavirin. negative regulation of virus transcription, translation or replication by quadruplex motifs in virus genomes forms the basis of using g4-binding ligands as antiviral agents. considering that the g4s that negatively regulate virus replication are retained in virus genomes during evolution, it is likely that viruses may stand to benefit from these g4s in their genomes. further research in this area may help us better understand this conundrum. in the last few years, the antimicrobial activity of quadruplex-binding ligands has been demonstrated in bacteria and parasites in addition to viruses (table 1) . although g4-binding ligands appear to be promising as potential antimicrobial agents, an important but often ignored aspect is specificity. it is very likely that g4 ligands will bind several host g4 motifs, which outnumber the microbial quadruplex motifs. studies investigating the undesired interaction of g4-binding ligands with g-quartets in the host genome may help us to better understand the therapeutic potential of this class of drugs. across different types of microbes, the modulation of transcription by g4s and its cascading effect on specific microbial phenotypes appears to be a common theme ( figure 5 ). inhibition of hsv-1 dna replication ( figure 4e ) [78, 79] hiv-1 inhibition of reverse transcription and transcription by binding to g4 in the u3 region of rna and proviral dna, respectively [80] tmpyp4 inhibition of nef-dependent hiv replication [71] c-exndi-2 negative regulation of hiv-1 transcription by binding to the g4 in the ltr [81] mycobacterium tuberculosis braco-19, c-exndi inhibition of bacterial growth (no specific mechanism is elucidated) [82] plasmodium falciparum quarfloxin deregulated expression of g4associated genes and inhibition of ring-stage parasites [83] ebola virus tmpyp4 inhibition of the l (polymerase) gene expression [77] hepatitis c virus pdp and tmpyp4 inhibition of core gene expression [76] a some of these require experimental validation. hbv is an enveloped hepatotropic dna virus that replicates with an rna intermediate. persistent infection with hbv can cause serious liver damage leading to cirrhosis and hcc. the hbv genome exhibits a high degree of genetic variability owing to the lack of proof-reading ability in the hbv reverse transcriptase. as a result, hbv is classified into 10 hbv genotypes (a through j) with an intergenotypic sequence variation of at least 8% [84] . the hbv genotypes differ in transmissibility, virus loads, response to antiviral therapy, and ability to cause liver disease [84, 85] . however, genotype-specific regulatory mechanisms in hbv remain elusive. we had recently identified a g4 motif as a genotype-specific regulator of hbv replication [40] . a conserved three-tetrad g4 motif, 190 bp upstream of the transcription start site, was identified in the pres2/s promoter of hbv genotype b. this motif was virtually absent in the rest of the hbv genotypes. this quadruplex specifically enhanced the transcription of the pres2/s transcript and the production of hbv surface antigen (hbsag). point mutations disrupting the g4 motif in the pres2/s promoter of hbv genotype b led to a reduction in hbsag production resulting in a fivefold reduction in virion secretion [26] . virion secre on in hbv replica on of hiv-1, hcv, ebov replica on of plasmodium parasite in the erythrocy c stages nitrate metabolism in p. denitrificans figure 5 . phenotypic effects of the modulation of transcription by g-quadruplexes (g4s). g4-mediated control of microbial transcription appears to be a common theme leading to tangible differences in the phenotype of microbes. g4s in microbial genomes may regulate microbial transcription either negatively (pink circles) or positively (green circle). d. radiodurans, deinococcus radiodurans; p. denitrificans, paracoccus denitrificans; ebov, ebola virus. deinococcus radiodurans is an extremophilic bacterium tolerant to ionizing radiations such as gamma rays and uv rays. beaume et al. analyzed the promoters of d. radiodurans and found that g4 motifs were particularly enriched within the 200 bp upstream region in genes that confer radiation resistance; these include reca, recf, reco, recr, recq, and mutl, all of which are involved in recombinational dna repair [86, 87] . interestingly, the addition of an intracellular g4-binding ligand led to a marked reduction in the expression of genes associated with radiation resistance, thus rendering d. radiodurans sensitive to radiation. the ability of g4 motifs to modulate radioresistance in d. radiodurans sheds light on how these dna secondary structures contribute to microbial tolerance to environmental pressures by regulating the transcriptional machinery. paracoccus denitrificans is a facultative anaerobe capable of metabolizing nitrogen, nitrate, and ammonia. reduction of nitrate or nitrite to dinitrogen, a cellular process known as denitrification, is associated with the nasabghc gene cluster in p. denitrificans [88] . this nitrateassimilatory system (nas) is regulated by a two-component nass-nast system. nast is an effector molecule that positively regulates transcription of nas genes by acting as an antitermination signal [88] . the gc-rich genome of p. denitrificans contains a three-tetrad quadruplex motif 150 nucleotides upstream of the nast gene [89] . stabilization of the g4 by ligands (tmpyp4 and a benzophenoxazine ligand) or by cations (kcl) inhibited the transcription of the nast gene. similarly, the presence of g4-stabilizing ligands inhibited the growth of p. denitrificans in media containing nitrate as the sole source of nitrogen. this work on p. denitrificans highlights a role for g4-linked transcriptional control in modulating specific metabolic pathways. studies investigating bacterial and yeast genomes found an enrichment of g4s in promoters of genes involved in carbohydrate, amino acid, and nucleotide metabolism [37, 86, 90] . although the functional significance of the g4s in the promoters of bacterial and yeast genes involved in metabolism remains to be demonstrated, it may not be too speculative to suggest a possible role for these dna secondary structures in regulating the synthesis of macromolecules in bacteria and yeast by modulation of key metabolic pathways. trypanosoma brucei is a parasitic kinetoplastid that causes african sleeping sickness in humans. mitochondrial transcripts of kinetoplastid organisms undergo extensive editing post-transcription; this mrna editing involves deletion or insertion of 'u' residues at multiple locations specified by the anchoring of guide rnas (grnas) encoded by the mitochondrial genome [91] . the nucleotide composition of the pre-mrnas may be potentially altered by up to 50% as a result of editing, which is referred to as pan-editing [92] . matthias-leeder et al. analyzed nine mrnas of t. brucei and found that the guanosine (g) content is lowered to about 19% from about 34% during pan-editing [93] . importantly, the authors used computational methods to demonstrate the progressive decrease in g4 content during pan-editing. therefore, pan-editing in african trypanosomes has been suggested as a g4-resolving process that leads to the generation of g4-free translatable orfs. the authors also propose the formation of dna/rna hybrid g4s (hq) between the nontemplate dna strand and pre-edited transcripts. furthermore, it is speculated that the formation of hqs is involved in the termination of transcription and the initiation of mitochondrial replication. thus, quadruplexes may play a crucial role in switching between the two mutually exclusive processes of mitochondrial replication and transcription in trypanosomes. among the microorganisms that contain a g4 in their genome, the over-representation of viruses associated with cancer, namely, kshv, ebv, hcv, hbv, and hpv, is noteworthy [40, 51, 60, 63, 76] . the existence of these secondary structures in zoonotic agents such as ebola virus and vector-borne pathogens such as zika virus, plasmodium spp., b. burgdorferi, and t. brucei, is particularly interesting [24, 42, 93, 94] . from an evolutionary perspective, it may be of interest to identify g4-influenced adaptations, if any, that facilitate the survival of these microbes in different hosts. repeat regions in herpesviruses contain important regulatory elements for replication, packaging, latency, and reactivation [95, 96] . the existence of rgqms amplifies the g4 load of the genomes of hhvs manifold [44] . such g4-forming iterative g-rich units also comprise the simple sequence repeats (ssrs) present in the noncoding regions of nostoc sp. and xanthomonad spp. [97] . bacterial ssrs are known to be implicated in antigenic and phase variation. given the functional significance of repeat sequences in microbial genomes it may be interesting to investigate the link between the tandem array of g4s and molecular processes related to microbial pathogenesis. recent reports on g4 motifs in viruses infecting nonhuman hosts shed light on how g4s have been exploited by viruses for virulence and genome regulation throughout evolution [52, 98] . the identification of g4s in microbial genomes has opened up new avenues for therapeutics; additional studies on the specificity of g4-binding ligands and their undesired effects may help us to better understand the therapeutic potential of this novel group of antimicrobial agents. host protein-microbial g4 interaction or the host g4-microbial protein interactions at the molecular interface of the host and microbe during infection are fascinating and merit further investigation [55, 56, [66] [67] [68] [69] . it would be interesting to understand if such interactions defend the host or demonstrate yet another mechanism of microbial pathogenesis. intuitively, the threshold to transcend the thin line between these two opposing outcomes may be subject to complex regulation which may be important for an understanding of the therapeutic potential of targeting this host-microbe interaction. the nucleotide sequences complementary to g4 motifs are cytosine-rich and may form i-motifs which are higher-order nucleic acid structures formed in near-neutral or acidic ph [99] . recently, i-motifs were visualized in human cells [100] . it may be particularly interesting to study the molecular dynamics of g4s and i-motifs and its impact on microbial pathogenesis and evolution. supplemental information associated with this article can be found do g4-unwinding helicases encoded by the host interact with g4s in pathogens during infection? if so, are such host-microbe interactions doubleedged swords that help the host to defend some infections and prove to be detrimental in others? it may also be interesting to study if pathogens modulate the expression profile of hostencoded g4 helicases during infections. do g4s represent structural elements for conservation of energy in bacterial metabolism? is g4-mediated control of gene expression exercised only in the early stages of central dogma to conserve energy? are the prokaryotic orfs devoid of g4s to cut down on the energy cost of resolution of g4 for translation? does the presence of g4s as an additional level of coordinated gene control in operons also signify the same? as in d. radiodurans, are the unique adaptations of other extremophiles regulated by g4s? the primary sequence of viruses coevolves with that of their hosts. nonetheless, it is not known whether nucleic acid secondary structures, such as g4s, in virus genomes coevolve with host genomes. as in nostoc spp. and xanthomonas spp., are the plasmids of other bacteria also devoid of g4s? given that plasmids harbor important bacterial genes needed for virulence, antibiotic resistance etc., and the ability of g4s to pause replication, are they selectively eliminated from extrachromosomal elements to minimize interference, if any, in vertical transmission? do the pathogenicity islands in bacterial genomes have unique g4 profiles? are the differences in the density and distribution of g4s in pathogens influenced by the ecological relationship they share with the host? in other words, are there differences in the genomic densities of g4s among parasites, symbionts, commensals, and mutualists? stability and kinetics of g-quadruplex structures high-throughput sequencing of dna g-quadruplex structures in the human genome how long is too long? effects of loop size on g-quadruplex stability stability of telomeric g-quadruplexes human telomere, oncogenic promoter and 5 0 utr g-quadruplexes: diverse higher order dna and rna targets for cancer therapeutics g-quadruplexes and their regulatory roles in biology evidence of the formation of g-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene g-quadruplex formation within the promoter of the kras proto-oncogene and its effect on transcription 0 -utr rna gquadruplexes: translation regulation and targeting rna g-quadruplexes in biology: principles and molecular mechanisms genome-wide analyses of recombination prone regions predict role of dna structural motif in recombination dna and rna quadruplex-binding proteins zinc-finger transcription factors are associated with guanine quadruplex motifs in human, chimpanzee, mouse and rat promoters genome-wide on characterizing the interactions between proteins and guanine quadruplex structures of nucleic acids g-quadruplex unwinding helicases and their function in vivo g4-interacting dna helicases and polymerases: potential therapeutic targets structural basis of g-quadruplex unfolding by the deah/rha helicase dhx36 g-quadruplexes and gquadruplex ligands: targets and tools in antiviral therapy genetic mechanisms of bacterial antigenic variation shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections. microbiol g-quadruplexes in pathogens: a common route to virulence control? above and beyond watson and crick: guanine quadruplex structures and microbes an alternative dna structure is necessary for pilin antigenic variation in neisseria gonorrhoeae suggested role for g4 dna in recombinational switching at the antigenic variation locus of the lyme disease spirochete comparative investigation of the genomic regions involved in antigenic variation of the tprk antigen among treponemal species, subspecies, and strains reca-binding pile g4 sequence essential for pilin antigenic variation forms monomeric and 5 0 endstacked dimeric parallel g-quadruplexes the pathogenesis of malaria: a new perspective pfemp1: an antigen that plays a key role in the pathogenicity and immune evasion of the malaria parasite plasmodium falciparum putative dna g-quadruplex formation within the promoters of plasmodium falciparum var genes recombination events among virulence genes in malaria parasites are associated with g-quadruplexforming dna motifs recombination in hiv: an important viral evolutionary strategy hiv-1 rna dimerization: it takes two to tango a recombination hot spot in hiv-1 contains guanosine runs that can form a g-quartet structure and promote strand transfer in vitro evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic rna mechanism of hiv-1 rna dimerization in the central region of the genome and significance for viral evolution u3 region in the hiv-1 genome adopts a g-quadruplex structure in its rna and dna sequence genome-wide prediction of g4 dna as regulatory motifs: role in escherichia coli global regulation g-quadruplex dna sequences are evolutionarily conserved and associated with distinct genomic features in saccharomyces cerevisiae rna g-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria a g-quadruplex motif in an envelope gene promoter regulates transcription and virion secretion in hbv genotype b conserved presence of g-quadruplex forming sequences in the long terminal repeat promoter of lentiviruses zika virus genomic rna possesses conserved g-quadruplexes characteristic of the flaviviridae family a dynamic g-quadruplex region regulates the hiv-1 long terminal repeat promoter genome-wide analysis of g-quadruplexes in herpes virus genomes mutation of ul24 impedes the dissemination of acute herpes simplex virus 1 infection from the cornea to neurons of trigeminal ganglia importance of the herpes simplex virus ul24 gene for productive ganglionic infection in mice modulation of host gene expression by the k15 protein of kaposi's sarcoma-associated herpesvirus evidence that the herpes simplex virus type 1 uracil dna glycosylase is required for efficient viral replication and latency in the murine nervous system pac1 signals of human herpesviruses contain a highly conserved g-quadruplex motif herpes simplex virus dna packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery human papillomavirus g-quadruplexes identification of g-quadruplex forming sequences in three manatee papillomaviruses regulatory elements in the viral genome human papillomavirus: gene expression, regulation and prospects for novel diagnostic methods and antiviral therapies the 'sars-unique domain' (sud) of sars coronavirus is an oligo(g)-binding protein the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes a g-quadruplex-binding macrodomain within the 'sars-unique domain' is essential for the activity of the sars-coronavirus replication-transcription complex molecular mechanisms of hiv latency the molecular basis of herpes simplex virus latency g-quadruplex-interacting compounds alter latent dna replication and episomal persistence of kshv stabilization of telomere g-quadruplexes interferes with human herpes virus 6a chromosomal integration epstein-barr virus latent genes g-quadruplexes regulate epstein-barr virus-encoded nuclear antigen 1 mrna translation is the epstein-barr virus ebna-1 protein an oncogen? epstein-barr virus-encoded ebna1 regulates cellular gene transcription and modulates the stat1 and tgfbeta signaling pathways nucleolin directly mediates epstein-barr virus immune evasion through binding to g-quadruplexes of ebna1 mrna role for g-quadruplex rna binding by epstein-barr virus nuclear antigen 1 in dna replication and metaphase chromosome attachment nucleolin stabilizes g-quadruplex structures folded by the ltr promoter and silences hiv-1 viral transcription the cellular protein hnrnp a2/b1 enhances hiv-1 transcription by unfolding ltr promoter gquadruplexes the antimicrobial resistance crisis: causes, consequences, and management. front formation of a unique cluster of gquadruplex structures in the hiv-1 nef coding region: implications for antiviral activity the activity of nef on hiv-1 infectivity serine phosphorylationindependent downregulation of cell-surface cd4 by nef characterization of three nef-defective human immunodeficiency virus type 1 strains associated with long-term nonprogression. australian long-term nonprogressor study group the human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages a highly conserved g-rich consensus sequence in hepatitis c virus core gene represents a new antihepatitis c target chemical targeting of a g-quadruplex rna in the ebola virus l gene the herpes simplex virus-1 genome contains multiple clusters of repeated g-quadruplex: implications for the antiviral activity of a g-quadruplex ligand a core extended naphtalene diimide g-quadruplex ligand potently inhibits herpes simplex virus 1 replication anti-hiv-1 activity of the g-quadruplex ligand braco-19 synthesis, binding and antiviral properties of potent core-extended naphthalene diimides targeting the hiv-1 long terminal repeat promoter g-quadruplexes mapping and characterization of gquadruplexes in mycobacterium tuberculosis gene promoter regions g-quadruplex dna motifs in the malaria parasite plasmodium falciparum and their potential as novel antimalarial drug targets hepatitis b virus genotypes and variants. cold spring harb hepatitis b virus genotypes: global distribution and clinical importance genome-wide study predicts promoter-g4 dna motifs regulate selective functions in bacteria: radioresistance of d. radiodurans involves g4 dna-mediated regulation g-quadruplex forming structural motifs in the genome of deinococcus radiodurans and their regulatory roles in promoter functions transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier paracoccus denitrificans control of bacterial nitrate assimilation by stabilization of g-quadruplex dna genomic distribution and functional analyses of potential g-quadruplex-forming sequences in saccharomyces cerevisiae rna editing in trypanosomes the rna chaperone activity of the trypanosoma brucei editosome raises the dynamic of bound pre-mrnas multiple g-quartet structures in preedited mrnas suggest evolutionary driving force for rna editing in trypanosomes genome-wide regulatory dynamics of g-quadruplexes in human malaria parasite plasmodium falciparum mutagenesis of the herpesvirus saimiri terminal repeat region reveals important elements for virus production timeless-dependent dna replicationcoupled recombination promotes kaposi's sarcoma-associated herpesvirus episome maintenance and terminal repeat stability investigation of a quadruplex-forming repeat sequence highly enriched in xanthomonas and nostoc sp structure of genes coding the envelope proteins of the avian influenza a virus and bovine leucosis virus ) i-motif dna: structure, stability and targeting with ligands i-motif dna structures are formed in the nuclei of human cells key: cord-306050-y8i8759c authors: garcía, juan ignacio; meléndez, johanna; álvarez, rosa; mejía-chew, carlos; kelley, holden v.; sidiki, sabeen; castillo, alejandra; mazariegos, claudia; lópez-téllez, cesar; forno, diana; ayala, nancy; balada-llasat, joan-miquel; mejía-villatoro, carlos rodolfo; wang, shu-hua; torrelles, jordi b.; ikeda, janet title: accuracy of the tuberculosis point-of-care alere determine lipoarabinomannan antigen diagnostic test using α-mannosidase treated and untreated urine in a cohort of people living with hiv in guatemala date: 2020-10-19 journal: aids res ther doi: 10.1186/s12981-020-00318-8 sha: doc_id: 306050 cord_uid: y8i8759c background: improved point-of-care diagnostic tests for tuberculosis (tb) in severe immune suppressed people living with hiv (plwh) are needed to decrease morbidity and mortality outcomes. the aim of the study is to evaluate the performance of the lipoarabinomannan antigen test (lam-test) with and without α-mannosidase pre-treated urine in a cohort of plwh in primary care clinics in guatemala. we further determined tb incidence, and mortality rates and its risk factors in plwh with tb symptoms. methods: prospective longitudinal study of plwh with tb symptoms. urine samples were collected at 2 hiv sites to test the sensitivity of the lam-test in urine with and without α-mannosidase pre-treatment. a composite reference standard of either a positive mycobacterium tuberculosis complex culture and/or genexpert(®) mtb/rif (xpert, cepheid, sunnyvale, ca, usa) results was used in the lam-test diagnostic accuracy studies. cox proportional hazards regression was used to study mortality predictors. results: the overall sensitivity of the lam-test was of 56.1% with 95% ci of (43.3–68.3). there were no differences in the lam-test sensitivity neither by hospital nor by cd4 t cell values. lam-test sensitivity in plwh with < 200 cd4 t cells/µl was of 62.2% (95% ci 46.5–76.2). there were no significant differences in sensitivity when comparing lam-test results obtained from untreated vs. α-mannosidase treated urine [55.2% (95% ci 42.6–67.4) vs. 56.9% (95% ci 44–69.2), respectively]. tb incidence in our cohort was of 21.4/100 person years (pys) (95% ci 16.6–27.6), and mortality rate was of 11.1/100 pys (95% ci 8.2–15.0). importantly, plwh with a positive lam-test result had an adjusted hazard ratio (ahr) of death of 1.98 (1.0–3.8) with a significant p value of 0.044 when compared to plwh with a negative lam-test result. conclusions: in this study, α-mannosidase treatment of urine did not significantly increase the lam-test performance, however; this needs to be further evaluated in a large-scale study due to our study limitations. importantly, high rates of tb incidence and mortality were found, and a positive lam-test result predicted mortality in plwh with tb clinical symptoms. mycobacterium tuberculosis culture is the reference standard for tuberculosis (tb) diagnosis worldwide; however, it is not routinely used in low-income countries due to its high cost, lack of laboratory capacity and long turnaround time (2 to 6 weeks). consequently, in high tb burden settings with limited resources, sputum smear microscopy detecting acid fast bacilli (afb) remains the main laboratory tb diagnostic test for the majority of rural health care centers [1, 2] . in 2010, the world health organization (who) endorsed the genexpert ® mtb/rif assay (xpert, cepheid, sunnyvale, ca, usa) as the initial diagnostic test for people living with hiv (plwh) with presumptive pulmonary tb (ptb). the xpert rapidly (2-h) detects 99% of smear-positive and 70% of smear-negative in people with ptb [3] . conversely, severely immune suppressed plwh with presumptive extrapulmonary or disseminated tb fail in getting a positive xpert result, mainly because they are too ill to produce a quality sputum sample, or their sputum contains very few afb. for these individuals, currently the xpert is unlikely to be useful and thus, the need of alternative tb diagnostic tests with increased sensitivity to provide early treatment and care and reduce mortality in plwh having extrapulmonary or disseminated tb [4] . lipoarabinomannan (lam) is a m. tuberculosis cell envelope lipoglycan that is being explored as a biomarker for active tb disease. lam is a heterogeneous, stable, immunogenic, and virulent factor thought to be released into the milieu by active or degrading bacilli [5, 6] . m. tuberculosis lam is a tripartite structure composed of a lipid moiety [glycosyl-phosphatidyl-myo-inositol (gpi) anchor] with different degree of acylation by different fatty acids. it also contains d-mannan and d-arabinan domains, and in its non-reducing end has mannose-caps, defining the typical m. tuberculosis complex manlam [7] . lam heterogeneity resides at the different degrees of acylation in its gpi-anchor, mannose-capping, and branching in both d-mannan and d-arabinan domains. once released into the bloodstream, lam is filtered by the kidneys and detected in the urine [8, 9] . in this context, alere determine tb lam ag test (lam-test) performed in urine has great expectations for its potential to improve the diagnosis of tb in plwh with low cd4 cell counts, and who are at greatest risk of death if tb remains undiagnosed. there are several studies that performed lam-test tb diagnosis accuracy assays in tb high burden areas [10] . lawn et al. described that among plwh eligible to start antiretroviral treatment in south africa, the urine lam-test has highest sensitivity for the detection of culture positive ptb at low cd4 cell counts. however, in this study, the estimates precision of the lam-test was poor due to wide and overlapping confidence intervals, with 66.7% sensitivity (95% ci 41.0-86.7) in plwh with cd4 < 50 cells/ µl, 51.7% (32.5-70.6) at cd4 < 100 cells/µl, and 39.0% (26.5-52.6 ) at cd4 < 200 cells/µl [11] . notably, this study identified a subgroup of plwh with tb disease with poor prognosis and high mortality. conversely, peter et al. observed a higher sensitivity in those with cd4 < 200 cells/ml (72%, 95% ci 61-80) compared to cd4 > 200 cells/ml (54%, 95% ci 36-71) [12] . this variability observed is thought to be related to: (i) the failure to contain the infection in the lungs resulting in disseminated tb with renal involvement and presence of lam in urine; (ii) dysfunctional humoral immunity results in abundant free lam, which can pass into the urine through the glomerular filtration, whereas immune complexes of antibody-bound lam do not; (iii) dysfunctional cellular immunity drives host susceptibility to less virulent mycobacteria, which may have different lam structures; and/or iv) different geographically distributed strains presenting diverse lam structures [13] . to date, no studies have evaluated the lam-test in central america. herein, we focused on the lam-test to address its efficacy in detecting m. tuberculosis in a cohort of plwh with tb symptoms in guatemala. we also determined if a 30 min α-mannosidase pre-treatment of urine using an enzyme that removes the mannose caps of lam could improve the sensitivity of the lam-test as we have shown in the laboratory settings [14] , and further correlated lam-test results with tb incidence, mortality rates, and risk factors in our cohort of plwh. the ethical review committee of the guatemalan ministry of health and social welfare approved this study protocol with approval number . the institutional review board at the ohio state university approved this study protocol with approval number 2013h0251. a prospective cohort study of plwh recruited from april 2015 to december 2017. follow up period ended on december 31st, 2017. all plwh with clinical (4-symptoms who active tb screening: i.e., cough, weight loss, night sweats, and fever [15] ) and/or radiological abnormalities concerning for ptb or extrapulmonary tb were offered to participate in this study. a total of 361 plwh with tb symptoms were enrolled and followed up during the study period. after tb/hiv counselling, participants provided their written inform consent and were recruited following our human subjects irb approved protocols. to estimate tb incidence and death rates, person-years of follow up (pys) were calculated using participants' entry date to the study and censoring occurred at either tb date, lost to follow up, death, or study recruitment ending date (december 31st 2017). the last study participant was recruited in december 6th 2017. this study took place in two uai clinics ('unidad de atención integral') in guatemala. uai clinics function as a one stop tb/hiv service delivery model [16] for integral management of the tb/hiv syndemic [17] . the 'dr. isaac cohen alcahé' uai (henceforth uai 1) is located within the regional, respiratory infection and tb specialty hospital 'rodolfo robles' in the rural highland region of quetzaltenango, guatemala. it attends mostly to indigenous individuals, who come from distant communities in western guatemala and the southern part of chiapas, mexico. these communities have limited resources and low educational levels, and an increasing number of lesbian, gay, bisexual, and transgender (lgbt) populations [18, 19] . the other uai involved in this study is the 'dr. carlos rodolfo mejía-villatoro" clinic (henceforth uai 2) the largest hiv clinic in guatemala, located within the roosevelt hospital in guatemala city, one of the two national reference hospitals in guatemala. uai 2 attends individuals that have been referred from other smaller uais and from patients from all over the country. participants in this study were both, ambulatory and hospitalized patients admitted from emergency services, external consultations within hospitals and waiting rooms in uais. site 1 includes uai 1 and hospital rodolfo robles and site 2 includes uai 2 and roosevelt hospital. enrolled plwh initiated anti-tb treatment in accordance with the national tb control program of guatemala guidelines [20] ; urine samples were collected from all enrolled subjects prior they started their anti-tb treatment. all subjects were recruited from inpatient and outpatient setting and screened for who-defined four tb clinical symptoms [21] . rapid antibody hiv tests were offered to all individuals with tb clinical symptoms. positive rapid tests were confirmed with fourth generation elisa/p24 tests. the study team (infectious disease specialist, a nurse, and a microbiologist) explained risks and benefits of participating in the study and obtained informed consent. potential participants were screened for inclusion criteria. the gold standard for tb diagnosis was a composite reference standard defined as either a m. tuberculosis positive culture or a positive xpert result. samples obtained included smear sputum for acid-fast bacilli (afb) and xpert, and urine for xpert and lam-test. in some instances, abscesses, biopsies or other body fluids were taken. both uai 1 and uai 2 followed the combined antiretroviral therapy (cart) protocol for early cart initiation (2 weeks after the onset of anti-tb treatment [20] ). ambulatory plwh were referred to the health center nearest to their residence to initiate anti-tb direct observed treatment (dot). for mycobacteria culture, clinical specimens (sputum, cerebrospinal fluid, pleural fluid, biopsy lysates) were first decontaminated using the bd bbl mycoprep ™ system. once decontaminated, 5 drops (50 μl/drop) of the decontaminated sample were added into two tubes with löwenstein-jensen (lj) medium and in uai 2, 500 μl were added into the bd bactec ™ mgit (automated liquid mycobacterial growth indicator tube) system for mycobacteria growth detection. lj tubes were incubated at 37 °c and taken out for manual reading twice a week for 8 weeks. all solid (lj) and liquid (mgit) cultures suggestive of m. tuberculosis growth were further tested with the sd bioline tb ag mpt64 immunochromatographic test to confirm m. tuberculosis complex strains. mpt64 test negative isolates were verified later as possible nontuberculous mycobacteria (ntm). the final identification of all m. tuberculosis complex strains was confirmed using hain lifescience genotype mycobacterium cm and genotype mycobacterium as identification kits. drug susceptibility of the m. tuberculosis confirmed strains was performed using the hain (lifescience genotype mtbdrplus and genotype mtbdrsl) kits. in uai 1, all solid (lj) cultures suggestive of m. tuberculosis growth were further tested for drug susceptibility at the national reference laboratory. the xpert test was performed from decontaminated clinical specimens. following the manufacturer's instructions, 1 ml of the specimen was mixed with 2 ml of the kit reagent. the mixture was pipetted into the cartridge and inserted into the xpert instrument for processing. all urine samples were collected at recruitment, and stored at − 20 °c before use. for each individual, the same urine sample was used to perform the lam-test in both, untreated and α-mannosidase treated urine. briefly, for the lam-test, 60 μl of urine were directly pipetted on the lam-test strip. after 25 min, results were read and interpreted following the manufacturer's instructions [3] . for the α-mannosidase treatment of urine, a urine-sodium bicarbonate mixture (1:1, v/v, 100 μl final volume) was treated with 0.1 iu of α-mannosidase (sigma-aldrich, san louis, mo) and incubated at 37 °c for 15 min. the lam-test was performed using 60 μl of this mixture according to the manufacturer's instructions. a lam-test was read as positive using an intensity band grade scale from 1 to 4. two independent clinicians agreed on readings for both, lam-test and lam-test after α-mannosidase treatment of urine; however, lamtest band intensity readings were not recorded. combinational antiretroviral therapy (cart) regimens are defined in table 1 . diabetic plwh were defined as those with percentage of haemoglobin a1c (% hba1c) measurements above 6.1 [22] . body mass index (bmi) was re-categorized as binary defining ≥ 17 kg/m 2 and < 17 kg/m 2 . who hiv stage was binary defined as 1 or 2 and 3 or 4. in the descriptive analysis, proportions were compared using the chi square test. u mann-whitney was used to compare medians in different groups as appropriate. the significance for all comparison tests was set at p < 0.05. sensitivity and specificity were compared between the two participating uais using the fischer exact test. cox proportional hazard regression was used to calculate unadjusted (uhr) and adjusted hazard (ahr) ratios to explore predictors of mortality in the cohort of plwh with tb symptoms. the following confounding factors were used for adjustment: age, sex, cd4 values, cart, viral load, and who hiv stage. data analyses were conducted using stata, ver. 14 (stata corporation, college station, tx, usa). plwh flow charts for the diagnostic accuracy analysis of the lam-test vs. our composite referenced standard, and for the lam-test performed in untreated vs. α-mannosidase treated urine are depicted in fig. 1 . briefly, 292 out of 361 (80.9%) eligible plwh had valid results for xpert, culture and both lam-tests (untreated vs. α-mannosidase treated urine) and thus, these were used in the diagnostic accuracy analysis. ptb disease was diagnosed in 59/292 (20.2%) plwh with tb clinical symptom/radiological abnormality. finally, there were 343 subjects with tb symptoms with information on mortality outcomes that were used to study predictors of death by cox regression analysis. enrolled plwh characteristics at baseline are shown in table 1 . no differences were observed in age, sex and bmi at both study sites, except for clinical symptoms of weight loss and fever. interestingly, site 2 had higher proportion of second line cart regimens (65.3% vs. 10.6%) and higher mortality risk (27.34% vs. 11.2%) with p values < 0.001 when compared to site 1. we further determined the sensitivity and specificity of the lam-test in α-mannosidase treated vs. untreated urine when compared to the defined gold standard composite. results were stratified by site and cd4 t cell values (table 2 ). no differences were observed in the performance of the lam-test with and without α-mannosidase treatment of urine, neither when stratified by site nor by cd4 values. there were 3 plwh that had a positive lam-test result only when their urine was α-mannosidase treated. only 1 out of these 3 plwh also had a positive culture. conversely, there were 5 plwh that their lam-test result converted from a positive to a negative when their urine was α-mannosidase treated. only 1 out of these 5 plwh also had a confirmed tb positive culture, the others presented negative cultures. tb incidence and mortality rates in our cohort of plwh with tb clinical symptoms were calculated using person-years (pys). overall, 59 plwh were diagnosed with tb by the lam-test, and 53 of those died. tb incidence was of 21.4/100 pys with 95% ci of (16.6-27.6) for results evaluating risk factors, as predictors for mortality and mortality outcomes for the 343 plwh enrolled in this study, are shown in table 3 currently, this is the first reported diagnostic accuracy analysis of the urine lam-test performed in central america in plwh. it is also the first time that an innovative procedure using α-mannosidase pre-treatment of urine was implemented in clinical settings to evaluate its diagnosis accuracy [14] . importantly, a positive lamtest result confirmed mortality prediction in our plwh cohort with tb clinical symptoms as previously reported [23] [24] [25] [26] [27] [28] . our published laboratory data indicate that α-mannosidase treatment of lam-spiked human urine using purified lam from different m. tuberculosis strains, can increase tenfold the detection of lam molecules in urine by the lam-test [14] . our field validation analysis of the lam-test performed in α-mannosidase treated vs. untreated urine was run in two different clinical settings. on one hand, plwh attending site 1 used less second line cart, had higher cd4 t cell numbers and lower viral load values, and lower mortality rates compared to plwh attending site 2. most of plwh attending site 1 were recruited as ambulatory patients, compared to a higher proportion of hospitalized patients recruited in site 2, which includes the largest hiv clinic of the country located in guatemala city. differences in baseline characteristics of plwh in these two sites might explain some of the differences in diagnostic accuracy of the lam-test. however, the lam-test when compared to the composite reference gold standard performed equally in both sites, regardless of the severity of who clinical stage, cart status, viral load, and cd4 t cell numbers. in addition, recent analysis show similar hiv cascades of care in both site1 and site 2 [29] . in this study, the lam-test sensitivity in plwh with < 50 cd4 t cells/µl was similar to plwh with < 200 cd4 t cells/µl. a recent meta-analyses [26] showed a median pooled lam-test sensitivity for tb diagnosis in plwh of 45% with 95% credible interval of (29 to 63%), which is similar to our results in this study [(56.1% with 95% ci (43.3-68.3)] but with a smaller range of interval overlaps. contrary to our study in the laboratory setting, where α-mannosidase treatment of lam spiked human urine significantly increased the efficiency of the lam-test [14] , we did not find any sensitivity and specificity differences comparing the lam-test results in α-mannosidase treated vs. untreated urine using our composite gold referenced standard. this could be explained by several factors, including the viability and storage conditions of the α-mannosidase enzyme being used in the iaus, as well as the use of a twofold diluted urine in sodium bicarbonate when performing the lam-test in the α-mannosidase treated urine. this was performed with the rationale behind that sodium bicarbonate impairs micelle formation of lam molecules in aqueous buffer [30, 31] , but at the same time, the tested urine was diluted twofold, which could affect the sensitivity of the lam-test. another potential reason is the non-use of low-binding protein tubes during the test, as well as inconsistency in the incubation periods and temperature, showing in the lab setting to be optimal at 37 °c for 15 min [32] . conversely, lam in the infected host could be metabolized prior reaching the urine and thus, different epitopes could be exposed on lam molecules making the α-mannosidase treatment less relevant than in the laboratory in vitro setting. however, in this regard ongoing studies show that this later possibility may not be plausible, as mannose-monocapped lam motifs maybe detected in urine isolated from active tb patients [33] . indeed, we found 3 plwh that had an α-mannosidase lam-test positive result, which were negative without this treatment. of these, only 1 had a positive culture. interestingly, there were 5 plwh that had a lam-test negative result only after α-mannosidase treatment, that otherwise were positive by the lam-test using untreated urine. of these, only 1 had a tb culture confirmed, indicating that α-mannosidase treatment of urine could potentially help in reducing false positive lam-test results and detect true tb positives that otherwise could be missed. this is the first time that a modification of the lam-test to increase its sensitivity is performed in real hospital-based settings worldwide showing promising results in its feasibility; however, further careful largescale implementation and comparison analyses are necessary to verify its true usefulness in the field, especially in rural areas with limited tb diagnosis testing accessibility. the impact of the current coronavirus disease 2019 (covid-19) pandemic on the number of new tb and hiv cases is unknown. thus, in the years to come, improving tb diagnosis in plwh is a key target to achieve towards the end tb strategy by 2030 [34] [35] [36] . tb/hiv services integration is highly recommended to improve tb and hiv treatment outcomes, as well as to establish tb/hiv programmatic indicators for surveillance, and monitoring and evaluation purposes [37, 38] . in this regard, tb incidence and mortality rates in plwh with tb symptoms found in our plwh cohort showed that even in hiv clinics with a "one stop service delivery model" for tb and hiv care, tb incidence and mortality are still a serious concern. our tb incidence results of 17.9/100 pys and 95% ci of (10.4-30.8) in plwh with < 100 cd4 t cells/µl, are similar to those of 25.5/100 pys and 95% ci of (21.6-30.3) found in a plwh cohort with similar cd4 t cell values in south africa [39] . indeed, our tb incidence rates of 22.5/100 pys with 95% ci of (11.2-45.0) in plwh with < 50 cd4 t cells/µl, are far higher than those of 4.2/100 pys with 95% ci (1.4-12.7) found in a cohort of plwh in nigeria with similar cd4 t cell values [40] . several studies have emphasized different factors behind high proportions of aids defining illnesses such as late presentation, tb diagnostic delay, and attrition in plwh [27] . social forces are key determinants in explaining tb morbidity and mortality outcomes in the central american region that need to be further explored. our data also indicate that a positive lam-test result in plwh with tb clinical symptoms predicts mortality outcomes [ahr of 1.98 (1.0-3.8) p value = 0.044]. this was previously suggested in recent sub-saharan africa studies involving hospital inpatients [4, 41] , as well as in plwh receiving treatment for hiv-associated active tb in sub-saharan africa and thailand [42, 43] , and in newly diagnosed plwh screened for tb in south-africa [44] . this pilot study was originally focused as a feasibility study and a proper sample size calculation is missing. lam-test results using two independent readers in diagnostic accuracy analysis of the composite gold standard vs. the lam-test (urine untreated vs. α-mannosidase treated) were not properly recorded. thus, kappa concordance agreement studies were not performed. lamtest after urine α-mannosidase treatment was performed in twofold diluted urine in sodium bicarbonate in place of being directly performed in undiluted urine. however, lam-test using α-mannosidase diluted samples performed equally to lam-test alone with undiluted samples finally, lam-test band intensity was not properly registered and thus, results were only ascertained as positive or negative using the lam-tests grade 1 to 4 independently of the intensity of the band visualized, which limited to know if α-mannosidase treatment, a part of uncovering true positives, was able to allow better detection of lam molecules in urine by the lam-test, and further correlate the bacterial burden (culture) with the intensity of the lam-test band (i.e., a direct correlation between more bacteria producing more lam molecules resulting in a high intensity band). in this study, α-mannosidase treatment of urine did not significantly increase the lam-test performance; however, the performance of the lam-test after α-mannosidase treatment of urine needs to be carefully revaluated in the field at larger scale and proper controls. this simple α-mannosidase treatment of urine could increase the sensitivity of the lam-test (and other urine-based test such the new fujilam [45] ), when compared to untreated urine in field settings using large cohorts of hiv-uninfected and plwh clinically suspicious of active tb disease. in this regard, it will be important to evaluate in the field, if α-mannosidase treatment of urine can certainly increase the detection of lam molecules and correlate these results with bacterial burden. finally, high rates of tb incidence and mortality were found within established "one stop" services, and a positive lam-test result could predict mortality in plwh with tb disease clinical symptoms. tb diagnostic capacity in sub-saharan african hiv care settings smear positivity in paediatric and adult tuberculosis: systematic review and meta-analysis lateral flow urine lipoarabinomannan assay (lf-lam) for the diagnosis of active tuberculosis in people living with hiv. policy update 2019. who. world health organization risk score for predicting mortality including urine lipoarabinomannan detection in hospital inpatients with hiv-associated tuberculosis in sub-saharan africa: derivation and external validation cohort study mycobacterial extracellular vesicles and host pathogen interactions mycobacterium tuberculosis membrane vesicles inhibit t cell activation mannose-capped lipoarabinomannan in mycobacterium tuberculosis pathogenesis detection of lipoarabinomannan (lam) in urine is indicative of disseminated tb with renal involvement in patients living with hiv and advanced immunodeficiency: evidence and implications lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in hiv-positive adults point-of-care detection of lipoarabinomannan (lam) in urine for diagnosis of hiv-associated tuberculosis: a state of the art review diagnostic accuracy of a lowcost, urine antigen, point-of-care screening assay for hiv-associated pulmonary tuberculosis before antiretroviral therapy: a descriptive study diagnostic accuracy of a urine lipoarabinomannan strip-test for tb detection in hiv-infected hospitalised patients mannose-capped lipoarabinomannan in mycobacterium tuberculosis pathogenesis improved alere determine lipoarabinomannan antigen detection test for the diagnosis of human and bovine tuberculosis by manipulating urine and milk guidelines for intensified tuberculosis case-finding and isoniazid preventive therapy for people living with hiv in resource-constrained settings delivering tb/hiv services in ghana: a comparative study of service delivery models hiv-associated tb syndemic: a growing clinical challenge worldwide risk behaviors and perceptions among self-identified men who have sex with men (msm), bisexuals, transvestites, and transgender women in western guatemala impact of integrating hiv and tb care and treatment in a regional tuberculosis hospital in rural guatemala manual de tratamiento antiretroviral y de infecciones oportunistas en guatemala guidelines for intensified tuberculosis case-finding and isoniazid preventative therapy for people living with hiv in resource-constrained settings. department of hiv/aids a comparison of hba1c and fasting blood sugar tests in general population hiv-associated tuberculosis: relationship between disease severity and the sensitivity of new sputumbased and urine-based diagnostic assays disseminated tuberculosis among hospitalised hiv patients in south africa: a common condition that can be rapidly diagnosed using urine-based assays performance of urine lipoarabinomannan assays for paediatric tuberculosis in tanzania lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in hiv-positive adults rapid microbiological screening for tuberculosis in hiv-positive patients on the first day of acute hospital admission by systematic testing of urine samples using xpert mtb/rif: a prospective cohort in south africa lateral flow urine lipoarabinomannan assay (lf-lam) for the diagnosis of active tuberculosis in people living with hiv programa nacional de prevención y control de its vih y sida (pns) informe nacional de la cascada del continuo de atención en vih isolation of a distinct mycobacterium tuberculosis mannose-capped lipoarabinomannan isoform responsible for recognition by cd1b-restricted t cells fine discrimination in the recognition of individual species of phosphatidyl-myo-inositol mannosides from mycobacterium tuberculosis by c-type lectin pattern recognition receptors les bacilles de la tuberculose bovine: une évolution aux dépens de la transmissibilité chez l'homme comparative structural study of terminal ends of lipoarabinomannan from mice infected lung tissues and urine of a tuberculosis positive patient world health orgnaization (who) the who's new end tb strategy in the post-2015 era of the sustainable development goals global tuberculosis targets and milestones set for 2016-2035: definition and rationale world health organization. a guide to monitoring and evaluation for collaborative tb/hiv activities. geneva: who. world health organization world health organization. who | who policy on collaborative tb/hiv activities. world health organization: who tuberculosis among hivhiv-infected population: incidence and risk factors in rural tanzania incidence and predictors of tuberculosis among hiv-infected adults after initiation of antiretroviral therapy in nigeria effect on mortality of point-of-care, urine-based lipoarabinomannan testing to guide tuberculosis treatment initiation in hiv-positive hospital inpatients: a pragmatic, parallel-group, multicountry, open-label, randomised controlled trial detection of lipoarabinomannan (lam) in urine is an independent predictor of mortality risk in patients receiving treatment for hiv-associated tuberculosis in • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research ready to submit your research ? choose bmc and benefit from: sub-saharan africa: a systematic review and meta-analysis utility of urine lipoarabinomannan (lam) in diagnosing tuberculosis and predicting mortality with and without hiv: prospective tb cohort from the thailand big city tb research network urinary lam grade, culture positivity, and mortality among hiv-infected south african out-patients novel lipoarabinomannan point-of-care tuberculosis test for people with hiv: a diagnostic accuracy study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations dedicated to the memory of dr. carlos rodolfo mejía-villatoro, who devoted his entire life to improve the medical care for guatemalans, a co-author of this study, a colleague, and a friend that suddenly passed away during this study. we thank the plwh participants that provided samples for this study. 1 authors' contributions jig, jm, ra, hvk, ss, ac, cmc, cm, df, na, jmbll, clt, crmv, shw, jbt, and ji made substantial contributions to the conception, experimental design, performance of the experiments, analysis, and/or interpretation of data; jig and jbt wrote the manuscript; shw and ji edited the manuscript and/or revised it critically for important intellectual content; shw, jbt and ji provided funding for this study. this study was partially supported by probitas foundation (spain) to shw, jbt, ji; and the ohio state university public health preparedness for infectious diseases (osu-phpid) funds to shw and jbt. jig was partially supported by the cowles foundation fellowship. jbt was partially supported by the the robert j. kleberg, jr. and helen c. kleberg foundation. due to the ethically sensitive nature of this research, datasets generated during the current study are not publicly available but available to researchers only without personal identifiers. the ethical review committee of the guatemalan ministry of health and social welfare approved this study protocol with approval number 45-2014. the institutional review board at the ohio state university approved this study protocol with approval number 2013h0251. all authors consented and agreed to publish this research study. the authors declare that they have no competing interests. key: cord-308916-6p2qutc5 authors: le roux, david m.; zar, heather j. title: community-acquired pneumonia in children — a changing spectrum of disease date: 2017-09-21 journal: pediatr radiol doi: 10.1007/s00247-017-3827-8 sha: doc_id: 308916 cord_uid: 6p2qutc5 pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. new conjugate vaccines against haemophilus influenzae type b and streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. the importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. better access to effective preventative and management strategies is needed in lowand middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. pneumonia has been the leading cause of death in children younger than 5 years for decades. although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. however in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. in this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. the overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . this is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the global burden of disease study [1] . pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . these data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. the incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . on average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . in addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . childhood pneumonia can also lead to significant morbidity and chronic disease. early life pneumonia can impair longterm lung health by decreasing lung function [6] . severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . a meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . the risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. in 2005, to aid in defining outcomes of pneumococcal vaccine studies, the world health organization's (who) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . the term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). widespread use of pneumococcal conjugate vaccination and haemophilus influenzae type b conjugate vaccination has decreased the incidence of radiologic pneumonia. in a review of four randomized controlled trials and two case-control studies of haemophilus influenzae type b conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in california between 1995 and 1998 [14] . in vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the gambia [15] , 25% in south africa [16] and 26% in the philippines [17] . the who radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. however, despite widespread field utilization, there are concerns regarding inter-observer repeatability. there has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . in addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . a revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with who-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum c-reactive protein of at least 40 mg/l [21, 22] . this definition has been shown to have greater sensitivity than the original who radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in south america [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (hiv) in south africa [21] . thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. empyema is a rare complication of pneumonia. an increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19a [24] . in the united states, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . in australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . in scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . these trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. data from the united states suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the united kingdom and scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . several national guidelines from high-income countries, as well as the who recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. however, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . in addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . an analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in canada, italy, australia, poland and the united states showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . in the united states after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . a systematic review of etiology studies prior to availability of new conjugate vaccines confirmed s. pneumoniae and h. influenzae type b as the most important bacterial causes of pneumonia, with staphylococcus aureus and klebsiella pneumoniae associated with some severe cases. respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza a and b, parainfluenza, human metapneumovirus and adenovirus [37] . more recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. severe disease in particular is often caused by multiple pathogens. with high coverage of pneumococcal conjugate vaccination and haemophilus influenzae type b conjugate vaccination, viral pathogens increasingly predominate [38] . in recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in south africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in sweden [40] . in a large multi-center study in the united states, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . a meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . however there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . another etiology is pertussis. in the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. their infants might therefore be born with low transplacental anti-pertussis immunoglobulin g levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . in 2014, more than 40,000 pertussis cases were reported to the centers for disease control and prevention in the united states; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . in contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . however recent evidence from south africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. postmortem studies of children dying from acute respiratory illness have commonly reported m. tuberculosis [48, 49] . a recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . because of the effectiveness of pneumococcal conjugate vaccination and haemophilus influenzae type b conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . it is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . the single strongest risk factor for pneumonia is hiv infection, which is especially prevalent in children in sub-saharan africa. hiv-infected children have 6 times increased odds of developing severe pneumonia or of death compared to hiv-uninfected children [52] . since the effective prevention of mother-to-child transmission of hiv, there is a growing population of hiv-exposed children who are uninfected; their excess risk of pneumonia, compared to hiv unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . the pneumococcal conjugate vaccination and haemophilus influenzae type b conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . however, equitable coverage and access to vaccines remains sub-optimal. by the end of 2015, haemophilus influenzae type b conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. however, inequities are still apparent among regions: in the americas coverage is estimated at 90%, while in the western pacific it is only 25%. by 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . to address this issue, the who's global vaccine access plan initiative was launched to make life-saving vaccines more equitably available. in addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in bangladesh [63] and 50.4% in south africa [64] ). however as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. the who integrated global action plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . prevention of pediatric hiv is possible by providing interventions to prevent mother-to-child transmission [72] . early infant hiv testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among hiv-infected children [73] . community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . if these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. all children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. when implemented in highburden areas before the availability of conjugate vaccines, case management as part of integrated management of childhood illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . however the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . in two studies, performed in denmark and in india, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . in the third study, in pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . furthermore, because who-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . this has been reflected in british [33] and spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. the united states' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . however, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised world health organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the who pneumonia case definitions [80] . use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . however, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . much progress has been made in decreasing deaths caused by childhood pneumonia. improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. stronger strategies to prevent and manage hiv have reduced hiv-associated pneumonia deaths. however, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia. global and national burden of diseases and injuries among children and adolescents between 1990 and 2013: findings from the global burden of disease 2013 study world population prospects: the 2015 revision, key findings and advance tables epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries vaccines to prevent pneumonia in children -a developing country perspective global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis lung function in african infants in the drakenstein child health study: impact of lower respiratory tract illness risk factors for chronic obstructive pulmonary disease in a european cohort of young adults early life origins of chronic obstructive pulmonary disease long term sequelae from childhood pneumonia; systematic review and meta-analysis global, regional, and national disabilityadjusted life-years (dalys) for 315 diseases and injuries and healthy life expectancy (hale), 1990-2015: a systematic analysis for the global burden of disease study a systematic review on the diagnosis of pediatric bacterial pneumonia: when gold is bronze standardized interpretation of paediatric chest radiographs for the diagnosis of pneumonia in epidemiological studies the effect of haemophilus influenzae type b and pneumococcal conjugate vaccines on childhood pneumonia incidence, severe morbidity and mortality effectiveness of heptavalent pneumococcal conjugate vaccine in children younger than 5 years of age for prevention of pneumonia: updated analysis using world health organization standardized interpretation of chest radiographs efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in the gambia: randomised, double-blind, placebo-controlled trial a trial of a 9-valent pneumococcal conjugate vaccine in children with and those without hiv infection efficacy of an 11-valent pneumococcal conjugate vaccine against radiologically confirmed pneumonia among children less than 2 years of age in the philippines: a randomized, double-blind, placebo-controlled trial accuracy of the interpretation of chest radiographs for the diagnosis of paediatric pneumonia variability in the interpretation of chest radiographs for the diagnosis of pneumonia in children chest x-rayconfirmed pneumonia in children in fiji effectiveness of pneumococcal conjugate vaccine against presumed bacterial pneumonia hospitalisation in hiv-uninfected south african children: a casecontrol study efficacy of pneumococcal nontypable haemophilus influenzae protein d conjugate vaccine (phid-cv) in young latin american children: a double-blind randomized controlled trial usefulness of creactive protein to define pneumococcal conjugate vaccine efficacy in the prevention of pneumonia five-fold increase in pediatric parapneumonic empyema since introduction of pneumococcal conjugate vaccine emergence of parapneumonic empyema in the usa increased paediatric hospitalizations for empyema in australia after introduction of the 7-valent pneumococcal conjugate vaccine trends in pneumonia and empyema in scottish children in the past 25 years effect of 13-valent pneumococcal conjugate vaccine on admissions to hospital 2 years after its introduction in the usa: a time series analysis has the incidence of empyema in scottish children continued to increase beyond additive impact of pneumococcal conjugate vaccines on pneumonia and empyema hospital admissions in england international guidelines on tackling community-acquired pneumonia show major discrepancies between developed and developing countries the management of community-acquired pneumonia in infants and children older than 3 months of age: clinical practice guidelines by the pediatric infectious diseases society and the infectious diseases society of america british thoracic society guidelines for the management of community acquired pneumonia in children: update should ultrasound be included in the initial assessment of respiratory patients? a role for streptococcus pneumoniae in virus-associated pneumonia the worldwide impact of the seven-valent pneumococcal conjugate vaccine epidemiology and etiology of childhood pneumonia childhood pneumonia: the role of viruses aetiology of childhood pneumonia in a well vaccinated south african birth cohort: a nested case-control study of the drakenstein child health study respiratory viruses associated with community-acquired pneumonia in children: matched case-control study community-acquired pneumonia requiring hospitalization among u.s. children aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis specimen collection for the diagnosis of pediatric pneumonia pertussis epidemic -california the resurgence of mumps and pertussis the pertussis enigma: reconciling epidemiology, immunology and evolution incidence and diagnosis of pertussis in south african children hospitalized with lower respiratory tract infection burden of respiratory tract infections at post mortem in zambian children lung diseases at necropsy in african children dying from respiratory illnesses: a descriptive necropsy study tuberculosis as a cause or comorbidity of childhood pneumonia in tuberculosisendemic areas: a systematic review risk factors for severe acute lower respiratory infections in children: a systematic review and meta-analysis global, regional, and national estimates of pneumonia burden in hiv-infected children in 2010: a meta-analysis and modelling study epidemiology of acute lower respiratory tract infection in hiv-exposed uninfected infants incidence and severity of childhood pneumonia in the first year of life in a south african birth cohort: the drakenstein child health study clinical outcomes of hiv-exposed, hiv-uninfected children in sub-saharan africa infant morbidity, mortality, and breast milk immunologic profiles among breastfeeding hiv-infected and hiv-uninfected women in botswana risk factors for presumed bacterial pneumonia among hiv-uninfected children hospitalized in soweto interventions to address deaths from childhood pneumonia and diarrhoea equitably: what works and at what cost? impacts of the 13-valent pneumococcal conjugate vaccine in children keeping children healthy: the vaccine alliance progress report 2015. gavi, the vaccine alliance world health organization perspectives on the contribution of the global alliance for vaccines and immunization on reducing child mortality maternal immunization effectiveness of maternal influenza immunization in mothers and infants influenza vaccination of pregnant women and protection of their infants duration of infant protection against influenza illness conferred by maternal immunization: secondary analysis of a randomized clinical trial vaccination against respiratory syncytial virus in pregnancy: a suitable tool to combat global infant morbidity and mortality? lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics ending preventable child deaths from pneumonia and diarrhoea by 2025. development of the integrated global action plan for the prevention and control of pneumonia and diarrhoea interventions to improve breastfeeding outcomes: a systematic review and metaanalysis population-wide preventive interventions for reducing the burden of chronic respiratory disease respiratory effects of air pollution on children consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection: recommendations for a public health approach. world health organization community-acquired pneumonia in hivinfected children: a global perspective effect of pneumonia case management on mortality in neonates, infants, and preschool children: a metaanalysis of community-based trials does 3-day course of oral amoxicillin benefit children of non-severe pneumonia with wheeze: a multicentric randomised controlled trial antibiotic treatment of pneumonia and bronchiolitis. a prospective randomised study comparison of oral amoxicillin with placebo for the treatment of world health organizationdefined nonsevere pneumonia in children aged 2-59 months: a multicenter, double-blind, randomized, placebo-controlled trial in pakistan a double blind communitybased randomized trial of amoxicillin versus placebo for fast breathing pneumonia in children aged 2-59 months in karachi, pakistan (retapp) antibiotics for bronchiolitis in children under two years of age integrated management of childhood illness: chart booklet. world health organization an evaluation of oxygen systems for treatment of childhood pneumonia setting research priorities to reduce global mortality from childhood pneumonia by acknowledgments h.j.z. acknowledges grants for studies on childhood pneumonia from the bill and melinda gates foundation (opp 1017641); from the national institutes of health, united states (h3africa 1u01ai110466-01a1); from the mrc south africa; and from the national research foundation, south africa. key: cord-309489-ubf55eux authors: carvalho, john j. title: our common enemy: combatting the world's deadliest viruses to ensure equity health care in developing nations date: 2009-02-19 journal: zygon doi: 10.1111/j.1467-9744.2009.00985.x sha: doc_id: 309489 cord_uid: ubf55eux in a previous issue of zygon (carvalho 2007), i explored the role of scientists—especially those engaging the science‐religion dialogue—within the arena of global equity health, world poverty, and human rights. i contended that experimental biologists, who might have reduced agency because of their professional workload or lack of individual resources, can still unite into collective forces with other scientists as well as human rights organizations, medical doctors, and political and civic leaders to foster progressive change in our world. in this article, i present some recent findings from research on three emerging viruses—hiv, dengue, and rotavirus—to explore the factors that lead to the geographical expansion of these viruses and the increase in frequency of the infectious diseases they cause. i show how these viruses are generating problems for geopolitical stability, human rights, and equity health care for developing nations that are already experiencing a growing poverty crisis. i suggest some avenues of future research for the scientific community for the movement toward resolution of these problems and indicate where the science‐religion field can be of additional aid. the beginning of the twenty-first century has witnessed economic instability in war-torn areas such as the middle east, the advent of more advanced terrorist activity, and global warming. within this new era, the emergence of viruses and the infectious diseases they cause is becoming a worldwide threat of the utmost importance. indeed, unlike the migration of peoples, which can be curbed by national and continental boundaries, viruses have no boundaries that constrict their spread. they are free to expand their geographical habitats irrespective of governmental restrictions, economic factors, or other forces that normally affect the movements of populations. as a result, many developing countries are experiencing larger outbreaks of viral diseases, including diseases such as polio thought to be eradicable by vaccine programs. and not only the developing nations are suffering from emerging viruses. even in developed countries such as the united states, certain viruses have expanded their geographical range and become more frequent in first-world hospitals. of the emerging viruses, five have particular importance for what scientists and world leaders can learn concerning their impact on geopolitical stability, human rights, and equity health care for the underprivileged in both developed and developing nations. they are (1) human immunodeficiency virus (hiv), the causative agent of acquired immune deficiency syndrome (aids), (2) dengue virus, the causative agent of dengue fever and dengue shock syndrome, (3) rotavirus, the causative agent of viralinduced gastroenteritis, (4) influenza virus, and, surprisingly, (5) the coronavirus that causes severe acute respiratory syndrome (sars). this last one is included not because of the number of illnesses it has caused but rather because of the economic impact and societal panic it has been responsible for recently (gostin, bayer, and fairchild 2003; zhong and zeng 2006) -manifestations that can tell us much about what could transpire if more deadly viruses or deadlier versions of the sars coronavirus surfaced. for the sake of this synopsis, i focus on hiv, dengue, and rotavirus. by far, the greatest emerging threat internationally is hiv because developing nations, especially those in africa, are experiencing the enormous influence of this virus. since its discovery in 1981, hiv has confounded scientists with its enigmatic biology and, consequently, has caused a worldwide pandemic. globally, estimates of hiv infections range from 40 to 65 million, with 22.5 million infections in sub-saharan africa alone (white and fenner 1994; flint et al. 2000; fauci 2007; kuritzkes and walker 2007) . to make matters worse, the distress caused by hiv/aids is somewhat of an underestimate, because persons presently infected will eventually die if a cure is not discovered-creating further instability at the geopolitical, economic, and social levels and reversing progress in many societies for decades while simultaneously contributing to the geographical expansion of the virus (see below). some estimates suggest that the workforce in fifteen countries will be 24 million fewer by 2020 and that some nations, including south africa, will experience a 17 percent lower gdp by the beginning of the next decade because of the impact of hiv (piot et al. 2001) . many scholarly journals consider the effect of geopolitical stability on public health, but rarely do they consider the effects of public health on geopolitical stability. consequently, the contribution of hiv/aids to such stability is underappreciated. increase in hiv/aids cases is linked to the breakdown of country boundaries, a rise in the number of ill refugees migrating across borders and to different areas within their host countries, an increase in military personnel or world peacekeepers becoming infected with the virus, and the deterioration of national infrastructures. unaids estimates that hiv rates among armed forces in some developing nations is higher than that of the general population and that hiv is responsible for causing a 50 percent mortality in some militaries, to say nothing of the financial burden these militaries must face to combat the virus (feldbaum, lee, and patel 2006) . furthermore, the impact of the virus on some civilian populations such as in sub-saharan africa raises the alarming possibility of a "state meltdown" (feldbaum, lee, and patel 2006) . that hiv is causing problems in certain strategically important nations such as russia, china, and india is of much concern given that estimates suggest these regions could witness 193-259 million hiv cases by 2025 (eberstadt 2002) . the effect of hiv on the geopolitical order of these strategic nations, as well as developing nations that require robust militaries or the presence of peacekeeping forces in order to maintain national stability, has great importance for world peace (feldbaum, lee, and patel 2006) . furthermore, in many of these hardest hit nations hiv/aids is disproportionately affecting younger people, so that the coming decades may reveal a population inversion, with individuals in their 60s and 70s outnumbering those in their 40s and 50s (piot et al. 2001) . the expansion of hiv/aids is not just an economic and geopolitical crisis; it is also a human-rights problem. in a previous article in zygon i mentioned that the united nations' universal declaration on human rights affirms that all persons should have the right to adequate medical care and social services in order for the war on global poverty to be successful (carvalho 2007, 292) . unfortunately, as the hiv/aids pandemic spreads, it is largely the "poorest of the poor" who suffer from inadequate health care and lack of equity social services (reviewed extensively in farmer 2005) . indeed, hiv/aids exacerbates and prolongs poverty in every respect. it has a devastating impact on the stability of households in developing nations. in africa, 13.2 million children have been orphaned because of aids, and this phenomenon is causing serious strain on national health infrastructures and human relief agencies (piot et al. 2001) . similarly, education-a primary means of breaking the poverty cycle-in many nations is being detrimentally affected because sick teachers are dying, children are dropping out of schools because of personal illness or the illness of parents or guardians, and families cannot pay for school supplies because of the economic burden of hiv/aids on household incomes (piot et al. 2001) . hiv/aids also contributes to problems in the agricultural industries of these nations as manpower dwindles because people become sick with the virus and livestock need to be sold off to handle family funeral expenses (piot et al. 2001) . the impact on food supply further exacerbates the poverty-and therefore human rights-crisis already exhibited in these regions. furthermore, the progression from infection to full-blown aids appears to occur faster in patients living in lower-income countries because of malnutrition and the lack of equity health care (pinching 1988; piot et al. 2001; farmer 2005; sanchez and swaminathan 2005; wanke 2005; cohen 2006) . in summary, the hiv/aids pandemic has emerged into an economic, geopolitical, and human-rights crisis that feeds off poverty and the lack of equity health care in developing nations while further intensifying poverty and the lack of equity health care. these factors lead to the inability to control hiv and thus promote the geographical expansion of the virus. arguably, hiv/aids is a model case by which to measure how viruses can expand their presence in the world by collaborating with and strengthening the factors that allow them to perpetuate in the human population. although hiv/aids is considered the most pressing worldwide concern for scientists, dengue virus is also alarming. dengue is a member of the flaviviridae family of viruses, which includes yellow fever, japanese encephalitis, and west nile viruses, among others (reviewed in gubler, kuno, and markoff 2007; lindenbach, thiel, and rice 2007) . it is the causative agent of two major diseases, dengue hemorrhagic fever and dengue shock syndrome (deen et al. 2006) . early epidemics of dengue were documented in jakarta, cairo, and philadelphia in the late 1700s (mairuhu et al. 2004 ). since that time, epidemics have occurred on all continents every ten to thirty years, but the disease was relatively benign, showing just fever and severe bone and back pain in patients (mairuhu et al. 2004) . after 1945 the disease became more fatal, displaying signs of hemorrhage in subjects from the philippines in 1953 and shock in those from southeast asia (mairuhu et al. 2004) . during the 1980s dengue spread into india, pakistan, china, sri lanka, and the maldives. it has since become a significant health problem in south and central america and the caribbean, where a recent outbreak was documented in puerto rico (mairuhu et al. 2004; torres and castro 2007; morens and fauci 2008; usa today 2007; msnbc 2007) . the disease is presently confined to tropical and subtropical regions and is transmitted to humans by the bite of infected female mosquitoes of the genus aedes (aedes aegypti, aedes albopticus, and aedes polynesiensis) (gubler, kuno, and markoff 2007) . according to the world health organization, nearly half the world's population is now living in dengue endemic areas, and it is estimated that annually there are 50-100 million dengue fever cases, 500,000 of them the dengue hemorrhagic fever variety (deen et al. 2006; gubler, kuno, and markoff 2007, 1172) . given the possibility for severe disease in infected individuals and the ease by which dengue is transmitted by mosquitoes, the spread of dengue virus could be considered one of the most dangerous emerging threats facing the world. a number of factors of human origin contribute to the geographical expansion of dengue virus and increased incidence of severe disease. these factors exacerbate one other to amplify both the spread of the virus and the negative impact of each factor on suffering human populations. for example, the failure to control the spread of aedes mosquito populations is critical because these mosquitoes are the vectors that carry the virus. mosquito programs have met with repeated failure in certain countries and have been linked to a resurgence of the virus in the americas in the 1970s (gubler 1989) . much of this failure came from insecticide resistance and reduction in program support in poorer nations (mairuhu et al. 2004 ). in addition, as resolutions to control global warming were neglected by governments over the past few decades, climate changes have resulted in large areas of standing water following major monsoons that have acted as ideal breeding grounds for mosquitoes (torres and castro 2007) . concurrently, haphazard deforestation has compounded the emergence of vector-borne diseases like dengue (torres and castro 2007) . the above factors are coupled with unprecedented population growth and uncontrolled urbanization, both of which have greatly expanded the geographical range of the mosquitoes and the viruses they harbor-in essence providing hyperendemic areas where multiple serotypes of dengue virus can circulate in human and aedes populations simultaneously. for example, in latin america, population growth and uncontrolled migration from the countryside to the cities have resulted in poor housing conditions, inappropriate disposal of waste, and lack of adequate food, clean water and health care-all of which are concurrent with an increase in infected mosquitoes carrying different versions of dengue virus (torres and castro 2007) . the biology of the virus itself compounds the problem, with new, more pathogenic dengue serotypes emerging that cause the more severe hemorrhagic and shock forms of the disease, such as in cuba in 1981 when 344,000 infections were reported (mairuhu et al. 2004; guzman 2005) . the multiple circulating serotypes can contribute to more severe forms of disease upon secondary infections (mairuhu et al. 2004 ). one immunological theory postulates that some dengue-specific antibodies from prior infections can cross-react with viruses from secondary infections without neutralizing the virus but acting as a mediator for increased uptake of the virus into target immune cells (such as monocytes and macrophages) where the virus can replicate and then be secreted from these cells (clyde, kyle, and harris 2006) . as indicated, these factors feed off each other. by far, social inequalities that stem from poverty are the biggest concern. educational scarcity on dengue virus and the diseases it causes, poor sanitation, lack of quality public health surveillance, and delayed clinical diagnosis are dominantly seen in more underprivileged areas (morse 1995; ligon 2004; torres and castro 2007; khun and manderson 2007; 2008) . spread of the virus in these areas further aggravates the lack of equity health care-a human rights issue-to the most disadvantaged. much of this is a result of the economic impact of dengue on poor nations. estimates of total costs from epidemics in puerto rico, cuba, and nicaragua are as high as $15.6 million, $103 million, and $2.7 million, respectively (torres and castro 2007) . endemic dengue contributes even more to the economic burden (torres and castro 2007) . because most families in endemic areas such as latin america and the caribbean earn us $10,000/y ear, the economic load of dengue is harshest for the poorest families in society, where even a brief hospital stay takes a significant toll on household income. in turn, the missing workdays by sick patients results in reduced revenue for the already cash-strapped governments these citizens belong to. essentially, lack of financial resources contributes to the deterioration of the established public health infrastructure, a breakdown that in turn jeopardizes the maintaining of mosquitocontrol, entomological-surveillance, and prevention programs and reduces the likelihood of rapid diagnoses in hospitals and clinics because of poorly trained staff or lack of medical resources including available doctors. as with the hiv pandemic, it is the poorest of the poor who suffer from the inadequate health care in dengue endemic regions, such as latin america and cambodia (torres and castro 2007; khun and manderson 2007; 2008) . and when poor families do not have access to health care, there is the further possibility of infected individuals not seeking treatment, which in turn leads to underreporting of disease cases, which further hampers governmental effort on resource allocation to combat the disease (torres and castro 2007; khun and manderson 2007; 2008) . the lack of equity health care in endemic areas becomes a human rights issue rather than just a medical one-an issue that has great importance for the medical community's attempts to stop the geographical expansion of dengue virus. rotavirus, a member of the reoviridae family of viruses, is the major causative agent of viral-induced gastroenteritis (reviewed in estes and kapikian 2007) . there are approximately 2.1 million rotavirus infections and 660,000 rotavirus-induced deaths per year, affecting mostly children in developing nations, with 80 percent of deaths occurring in south asia and sub-saharan africa (bresee et al. 2005; parashar et al. 2006; estes and kapikian 2007) . rotavirus is spread by fecal-oral contamination and via airborne viruses or contaminated respiratory droplets (estes and kapikian 2007) . new rotavirus strains can emerge from nucleic acid "reassortment," whereby simultaneous or asynchronous infection of organisms with human and animal rotaviruses leads to new, more pathogenic viruses being produced with mixed genetic material from both humans and animals (cook et al. 2004) . the geographical expansion of rotavirus, like hiv and dengue, is caused by a variety of factors: the ability of the virus to infect many different types of organisms, contaminated water supplies, poor hygiene and inadequate sanitation, lack of education, lack of prevention/treatment strategies and the supplies needed to carry out prevention/treatment, and the cycle of poverty in rotavirus endemic regions (levine et al. 1986; cook et al. 2004; estes and kapikian 2007) . as with hiv and dengue, poverty and rotavirus infections feed off each other. viral infections add to the economic burden on suffering nations. sick children and adults may be absent from school or work, impeding gdp (gray et al. 2008) . rotavirus outbreaks may deter travelers willing to visit exotic countries, which leads to loss of the tourism dollars that may be the foundation of the economies for such countries (diemert 2006) . medical costs for treatment of infected individuals can be enormous. studies of eight latin american and caribbean countries (argentina, brazil, chile, dominican republic, honduras, mexico, panama, and venezuela) reveal that for every 1,000 children born in 2003, us $7,971 was expended in direct medical costs during their first five years of life (rheingans et al. 2007 ). indirect costs likely increase these numbers, further indicating that rotavirus gastroenteritis results in substantial economic burden for poorer families in these nations. in poland, each nosocomial infection can cost as much as us $2,500 for treatment, with yearly impact amounting to us $4.5 million (chandran et al. 2006) . overall, the cost of treatment in the united states is around $1 billion, due partly to increased hospital visits from rotavirus infections (estes and kapikian 2007) . the economic burden of rotavirus exacerbates difficulties in providing equity health care, with poorer populations suffering the most during epidemics. the data in the literature on the effects of rotavirus on geopolitical instability are underappreciated. nevertheless, if the hiv pandemic teaches us anything, it is that it is highly likely that the lack of proper health care and economic difficulty in the poorest rotavirus endemic regions contribute to problems in geopolitical stability and that the continued geographical expansion of rotavirus will likely intensify these problems in the future. to make matters more worrisome, the zoonotic potential of rotavirus can have a detrimental impact on agricultural safety, with poorer nations lacking adequate protections. global warming has been a cause of increased monsoons, with greater potential for contaminated cattle excreta to run off into fresh water supplies such as rivers and lakes (cook et al. 2004) . contaminated water supplies can further contaminate crops. already, outbreaks of rotavirus infections from contaminated food have been seen around the world (cook et al. 2004) . the zoonotic potential of rotavirus becomes further important because more dangerous viruses could emerge and because rotavirus can be repeatedly introduced into the human population from a variety of sources (cook et al. 2004 ). scholars have written on the link between human rights, world poverty, and the epidemiology of disease (sen 1999; farmer 2005) . i myself have suggested how the theoretical discussions in the science-religion dialogue could contribute to concerns for equity health care in the developing world (carvalho 2007 ) and how such discussions can expand the science-religion audience (carvalho 2008) . i and others also have suggested that scientists and science-religion scholars-who understand the impact of technology, philosophy, religion, and culture on societies-should engage the wider political and social realm to tackle present, practical problems (carvalho 2007; 2008; deane-drummond 2007; polkinghorne 2007; wildman 2007) . continuing with these themes, it is clear that the geographical expansion of three viruses (hiv, dengue, and rotavirus), the increase in frequency of the infectious diseases they cause, and the relationship between these viruses and geopolitical stability, human rights, and equity health care for developing nations are problems of great concern promoted not only by biological and technological factors but also by social, religious, and cultural ones. consequently, the issues must be tackled from many different angles: clinical intervention, biomedical research, philosophical analysis of social/cultural/religious issues, and public policy. in my view, scholars in the biomedical research and public policy arenas have a number of avenues to pursue to combat the geographical expansion of hiv, dengue, and rotavirus and the increase in the infectious diseases they cause. for example, unlike with hiv, most of the data on the economic and societal impact for dengue and rotavirus are derived from epidemic as opposed to endemic cases. this lack of sufficient data is relevant for how governments will allocate resources for research, prevention, and more aggressive control activities. this is an area for immediate attention by the scholarly community. similarly, better epidemiological and entomological surveillance must be established, and the sources of the vectors for the viruses-such as water reservoirs that act as breeding grounds for dengue-infected mosquitoes-must be reduced, partly through curbing global warming. there should be additional research on the control of the zoonotic potential of rotaviruses given the large number of animals able to be infected with these viruses and the recurrent infection of the human population. better and more rapid clinical diagnosis of patients, especially underprivileged individuals, is a must. delays in diagnosis in poorer communities continue to contribute to the geographical expansion of hiv, dengue, and rotavirus. in order to achieve this end, the health funding structures in developing nations need to be evaluated. indeed, the creation of appropriate funding mechanisms (such as the availability of equity health insurance) is crucial to ensure access to health care for poorer families. otherwise sick individuals who do not have insurance and who cannot present cash for clinical care will not seek treatment, which invariably leads to delayed diagnosis and inappropriate surveillance and, additionally, detrimentally influences how funds are allocated to combat the viruses. at the public policy level, there needs to be enforcement of international human rights standards to protect the most underprivileged, such as refugees who may be infected with hiv (see unhcr 2006) . in terms of the science-religion dialogue, i would argue that public policies should contain insights from science-religion scholars or biologists who maintain a human rights perspective, because human rights and equity health care are inherently linked to the epidemiology of hiv, dengue, and rotavirus. science-religion scholars could act as resources for education on the moral dimension of viral infections. hiv, for example, has become a major epidemic in certain developing nations-such as those in latin america and the caribbean-because people in these regions possess an erroneous theological view that hiv infection is a "moral curse" that is a "punishment" for unethical behavior. (we have even seen this position in the united states.) societies in these areas therefore impose a great stigma on hiv-infected patients that leads to widespread discrimination in all forms, including unwillingness from physicians to even treat the disease (ramirez 2008) . such discrimination promotes the lack of equity health care for the neediest individuals and can be witnessed in dengue and rotavirus cases as well. given that science-religion scholars are likely to promote more correct theological insights into morality while simultaneously possessing a quality understanding of science, they could be powerful proponents in educating societies that viral diseases result largely from causes that are not based on incorrect presuppositions about morality or social taboos. educational campaigns dealing with hiv/aids could have benefited greatly in the earlier years of the epidemics if erroneous theology had been corrected by insights from the science-religion field. this can be seen especially with the early epidemics in the united states and mexico (cohen 2006) . such insights could prevent future mistakes in handling disease epidemics. it cannot go unnoticed that activism from religious organizations that accept advice from science-religion scholars or the human rights field could be pivotal in curbing the geographical expansion of viruses in certain underprivileged communities. cases in point are mexico and honduras (cohen 2006) . additionally, the burden caused by these viruses to underprivileged families-financial, psychological, social, religious, and cultural-needs to be better defined, as does how this burden relates to human rights and equity health care. science-religion scholars are more likely to understand the complicated dimensions of many societies, so once again they would be helpful in educating the scholarly community on the religious and cultural parameters of viral epidemics. for example, during the hiv/aids epidemic in africa some ritual practices involving blood may have resulted in hiv infections. similarly, male circumcision has been suggested to reduce hiv infections (morris 2007) , but circumcision practices that reuse unsterilized objects in certain communities could be problematical (see unaids 2006) . cultural insights from science-religion scholars on the religious traditions in these communities could have resulted in an educational campaign to modify cultural and religious ceremonies to make them safer. to illustrate with another case, it greatly surprised the medical community when polio vaccine distribution in the muslim world met fierce resistance due to mistrust of western medicine (roberts 2004) . knowing this is important for vaccine distribution for other viruses like rotavirus. again, an educational campaign with religious and cultural insights from science-religion experts on islam could be helpful in future public policy and procedures for vaccine distribution in muslim communities. apart from these concerns, the issue of equity needs to be further explored and the problems resolved. lack of equity health care is not the only crisis exacerbated by viruses; equity in education (such as access to educational materials and scientific data in developing nations) is also a problem. for example, a proper theological perspective on the dignity and rights of the human person could have thwarted epidemics within certain communities, such as the mayan community in guatemala, where individuals are treated as second-class citizens and are not afforded access to health care or even educational materials on hiv (cohen 2006) . scholars within the science-religion field have suggested that addressing the health crisis in poorer areas of the world could help eliminate superstitious beliefs (budenholzer 2003, 143-45) . i would claim that some of these beliefs clearly have a negative impact on equity. i have argued that biologists should address global health in numerous ways (carvalho 2007) . i also have articulated that professional societies should devote sections of their journals to philosophical analysis and public policy suggestions that attempt to resolve world health issues (carvalho 2007) . interdisciplinary collaboration has been successful in other cases, and it is my expectation that continued interdisciplinary collaboration should keep biomedical research focused and relevant. science-religion scholars could provide a "moral compass," encouraging the biomedical community to focus not only on discovering drugs helpful to already developed nations (and therefore profitable to drug companies) but also on research into antiviral medications that would have a greater impact on developing countries and therefore be more likely to slow the geographical expansion of viruses from their niches into first-world communities. in addition, science-religion scholars with expertise in bioethics and/or human rights could be helpful as interdisciplinary collaborators for public policy initiatives such as the implementation of new medicines or vaccines. research on vaccines is always ongoing in the biomedical community, but it is important to determine proper cost/benefit information whenever a new vaccine is made, and such cost/benefit information needs to take into account the wider impact of vaccine distribution. for example, the removal of one rotavirus vaccine from the international market in 1999 because of a side effect that occurred in very few cases and that could have been resolved by a simple medical procedure may have thwarted an additional 5 million rotavirus-induced deaths in the developing world (strauss and strauss 2008, 203-5) . cost/benefit analysis is based on financial feasibility and the goal of causing no harm in any patient. much of this analysis presently is based on the effects that could occur to already developed nations. science-religion scholars could have provided additional bioethical insights into cost/benefit analysis for such vaccine distribution in developing nations. by educating public health agencies and governments on the burdens (financial, social, cultural) of rotavirus epidemics in these poorer countries, science-religion scholars could have presented a better case for how the vaccine would have slowed the geographical expansion of rotavirus in the hardest hit endemic areas and how it would have aided poorer communities at many levels beyond just the medical. it is doubtful that previous cost/benefit analysis took into consideration how the additional rotavirus deaths would have also impacted family structure and family relationships to the wider community, or the added economic burdens and burdens on national health infrastructure resulting from the new deaths because of lack of an adequate vaccine. in conclusion, the battle against infectious diseases must involve a multifaceted approach that takes into account the social, religious, and cultural dimensions of disease epidemics. the interdisciplinary background of scholars within the science-religion dialogue could be extremely valuable in identifying what these dimensions are and how they can be approached for resolution. rotavirus in asia: the value of surveillance for informing decisions about the introduction of new vaccines sars and superstition the scientist as statesman: biologists and third world health a biologist's perspective on the future of the science-religion dialogue in the twenty-first century nosocomial rotavirus infections: a systematic review recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis hiv/aids in latin america and caribbean experiencing wonder and seeking wisdom the who dengue classification and case definitions: a time for a reassessment prevention and treatment of traveler's diarrhea the future of aids rotaviruses pathologies of power: health, human rights, and the new war on the poor. berkeley: univ 25 years of hiv/aids science: reaching the poor with research advances the national security implications of hiv/aids principles of virology: molecular biology, pathogenesis, and control ethical and legal challenges posed by severe acute respiratory syndrome rotavirus aedes agypti and aedes agypti-borne disease control in the 1990s: top down or bottom up flaviviruses deciphering dengue: the cuban experience health seeking and access to care for children with suspected dengue in cambodia: an ethnographic study poverty, user fees and ability to pay for health care for children with suspected dengue in rural cambodia hiv-1: pathogenesis, clinical manifestations, and treatment pediatric diarrhea: the challenge of prevention emerging and re-emerging infectious diseases: review of general contributing factors and of west nile virus flaviviridae: the viruses and their replication dengue: an arthropod-borne disease of global importance dengue and hemorrhagic fever: a potential threat to public health in the united states why circumcision is a biomedical imperative for the 21st century factors in the emergence of infectious diseases dengue fever surging in puerto rico rotavirus and severe childhood diarrhea factors affecting the natural history of human immunodeficiency virus infection the global impact of hiv/aids science and religion: bottom-up style, interfaith context hiv/aids in latin america economic and health burden of rotavirus gastroenteritis for the 2003 birth cohort in eight latin american and caribbean countries polio: the final assault? cutting world hunger in half development as freedom viruses and human disease the health and economic impact of dengue in latin america male circumcision and hiv note on hiv/aids and the protection of refugees, idps and other persons of concern puerto rico warns of dengue fever outbreak nutrition and hiv in the international setting retroviridae from grand dreaming to problem solving what we have learnt from the sars epidemics in china key: cord-350443-ca5avyjf authors: zhang, lei; wilson, david p. title: trends in notifiable infectious diseases in china: implications for surveillance and population health policy date: 2012-02-16 journal: plos one doi: 10.1371/journal.pone.0031076 sha: doc_id: 350443 cord_uid: ca5avyjf this study aimed to analyse trends in notifiable infectious diseases in china, in their historical context. both english and chinese literature was searched and diseases were categorised according to the type of disease or transmission route. temporal trends of morbidity and mortality rates were calculated for eight major infectious diseases types. strong government commitment to public health responses and improvements in quality of life has led to the eradication or containment of a wide range of infectious diseases in china. the overall infectious diseases burden experienced a dramatic drop during 1975–1995, but since then, it reverted and maintained a gradual upward trend to date. most notifiable diseases are contained at a low endemic level; however, local small-scale outbreaks remain common. tuberculosis, as a bacterial infection, has re-emerged since the 1990s and has become prevalent in the country. sexually transmitted infections are in a rapid, exponential growth phase, spreading from core groups to the general population. together human immunodeficiency virus (hiv), they account for 39% of all death cases due to infectious diseases in china in 2008. zoonotic infections, such as severe acute respiratory syndrome (sars), rabies and influenza, pose constant threats to chinese residents and remain the most deadly disease type among the infected individuals. therefore, second-generation surveillance of behavioural risks or vectors associated with pathogen transmission should be scaled up. it is necessary to implement public health interventions that target hiv and relevant coinfections, address transmission associated with highly mobile populations, and reduce the risk of cross-species transmission of zoonotic pathogens. china has experienced a large decline in the spread and burden of infectious diseases since the early 1960s, associated with effective and large-scale public health interventions and large populationbased vaccination programmes. china successfully eliminated 11 infectious diseases -including smallpox from the general chinese population in early 1960s [1] , 19 years before its global eradication -and another 10 infectious diseases, including poliomyelitis, have more recently been eliminated [2] . a further 13 diseases, including measles, are thought to be contained well at low endemic levels [3] . surveillance systems for infectious diseases in china are mainly hospital based. the latest available statistics (from 2006) indicate that china has 18,703 county hospitals, 40,907 township hospitals and 201,562 medical clinics [4, 5] . hospitals at the prefecture level or above are usually equipped with reference laboratories that are capable of carrying out molecular surveillance for cases. together with laboratories from academic institutions, they form the front line of surveillance for outbreak detection and notification of infectious diseases in china. all hospitals and clinics are obliged to report both suspected and confirmed cases of notifiable infectious disease to their nominated county centre for disease control (cdc). after recording the details of the reported cases (including their geographical location, demographicl information and infection status), county cdcs send the information to the country's central cdc, through the national infectious diseases monitoring information system database, which was established in 2004 [6] . this reporting mechanism bypasses the previous stepwise hierarchical reporting framework, thus allowing information to flow directly from grassroots cdcs to china's central disease database. however, regional, provincial and national cdcs do not have equal access to all of the data in the database: their access rights are limited to their own administrative regions, and only national cdcs has the full access to all data [7] . in addition to their responsibility for verifying the disease information from their administrative regions, municipal and provincial cdcs are required to report to the appropriate level of the bureaus of health or department of health and to form networks with local research bodies, universities and other health organisations. at the top of the hierarchy, china's central cdc is the overseeing organisation with responsibility for assembling and analysing diagnostic data for all diseases and then presenting a final report to the ministry of health, which is the main public health policymaker in china. the central cdc is also the only legitimate office for disseminating infectious disease information to the public, but provincial and municipal cdcs are also authorised to publicise the information to people in their jurisdiction, under the supervision of the ministry of health [8] . in contrast to disease surveillance systems in europe, the chinese surveillance system uses a multilayer administrative mechanism that enables rapid and efficient upward flow of epidemic information. china is a populous country of 1.3 billion people [9] . over the last 30 years of economic reform in the country, there have been environmental, demographic, social and behavioural changes in the population. as china's ties with the rest of world become increasingly strong, infectious diseases in china no longer remain a domestic issue. a thorough review of infectious disease surveillance in china is timely and important for the country's disease prevention and control strategies. currently, 39 infectious diseases are classified as notifiable in china. this study reviews trends in notifiable infectious diseases in china, in their historical context, discusses the current epidemiological state of these infections and their implications for disease surveillance and public health interventions. the review is structured by category of disease or transmission route. currently, 39 infectious diseases are notifiable in china, classified as a, b or c according to their epidemic levels and potential population threats. groups a and b (total 28 diseases) represent categories of diseases with high risk of outbreaks or that are likely to result in rapid spread once an outbreak occurs. mortality and morbidity related to group a and b diseases are reported and published by the chinese ministry of health on a monthly basis. group c diseases are less infectious and, when outbreaks occur, are epidemiologically less severe. they are required to be reported only when outbreaks occur. in this review, we searched published peer-reviewed research articles as well as online reports and grey literature from 1985 to 2010 relevant to disease surveillance in china in the following databases: pubmed, chinese scientific journals fulltext database (cqvip), china national knowledge infrastructure (cnki) and wanfang data. keywords used in the database search included ['chinese' or 'china'] and ['infectious diseases surveillance' or 'surveillance system' or 'infectious diseases monitoring' or 'infectious diseases information' or the type of infectious disease or the name of individual infectious diseases]. we also searched governmental reports, reports of non-governmental organisations and other grey literature from online sources. we then collated data on notified cases and mortality related to these diseases, using the latest available information from the ministry of health [10] . notifiable infectious disease data, including morbidity and mortality rates, was summarised by chinese ministry of health and published annually in chinese health yearbook and online accessible through cnki database. notably, this dataset does not contain data on sars and influenza a(h5n1) virus infection. hence we did not include them in our statistical analysis, but describe them in the text. the selected diseases were categorised into eight major types according to their diseases characteristics and origins. the morbidity and mortality for each disease type were calculated as the sum of the corresponding rates of individual diseases. the total number of diagnosed and death cases were estimated by multiplying morbidity and mortality rates by the overall chinese population in the study years. case fatality rate was defined as the percentage of persons diagnosed with the disease who die as a result of the illness during the calendar year, and was estimated by dividing mortality rate by morbidity rate of the diseases. further, for each individual disease, we calculate the disease-specific mortality rates among the chinese population in five-year intervals (1999-2003 and 2004-2008) . for each disease, the mean annual increase or decrease during 1999-2008, with 95% confidence intervals, is estimated, based on linear regression of logarithmic values of the number of annual death cases. trends in infectious diseases in china, 1999 china, -2008 morbidity of notifiable infectious diseases in china, represented by estimated numbers of new cases, declined substantially (.90%) from 22,000 cases per million in 1975 to a nadir level of 1,800 cases per million in 1995 (figure 1a ). since then, the rate of new infectious disease cases gradually reverted and maintained an upward trend. in 2008, the estimated rate of infectious diseases among the general chinese population reached over 3000 cases per million population. the composition of diagnosed diseases cases also changed substantially, in 1975, the three most reported diseases were gastrointestinal diseases (41.9%), vector-borne diseases (30.8%) and vaccine-preventable diseases (21.1%), corresponding to a total of 93.8% of all diagnosed cases ( figure 1a ). additionally, these three types of diseases account for 35.5, 21.7 and 18.4 million reported cases respectively during the period 1975-1979 (table 1 ). in contrast, in 1995, although gastrointestinal diseases remained the dominating disease type (41.6%), the proportion of all cases that were due to viral hepatitis quickly rose to 35.7%. sexually transmitted diseases re-emerged, and together with hiv, consisted of a substantial 6.3% of all reported cases. in 2008, the three most frequently reported disease types included viral hepatitis (38.3%), bacterial infections (33.3%) and stis and hiv (9.8%), which account for 5.4, 4.8 and 1.4 million diagnosed cases respectively during the period 2005-2008 (table 1) . rapid declines in infectious diseases mortality and its similar saddle pattern were also observed in the past 35 years. the overall mortality rate in china decreased from 66 cases per million in 1975 to 5 cases per million in 1995, then it gradually reverted to 10 cases per million in 2008 ( figure 1b ). vaccine-preventable diseases, bacterial infections and gastrointestinal diseases were the greatest causes of death, accounting for 30.0%, 24.0% and 19.5% of reported infectious diseases death cases among chinese population in 1975 ( figure 1b) . however, the rank of composition shifted to zoonoses (22%), viral hepatitis (17.3%) and quarantinable diseases (15.3%) in 1995. since then, the proportion of deaths caused by stis and hiv, and bacterial infections rapidly increased. by 2008, stis and hiv (39.5%) has become the mostly deadly infectious disease, followed by bacterial infections (24.7%) and zoonoses (20.0%). during the period 2005-2008, these three types of diseases have led to approximately 12,500, 13,300 and 11,400 deaths in china, respectively (table 1) . case fatality rate measures the percentage of deaths among people who contracted a disease. during 1975-2008, the disease type with the highest fatality rate is zoonoses, consistently causing 5-15% of deaths among the infected population. following that, quarantinable diseases killed 1.4-5.6% of its infected population during the same period. in comparison, fatality rates in vaccinepreventable (0. (table 1) . plague, cholera and epidemic haemorrhagic fever (which is caused by hantaviruses and mainly transmitted by rodents) have had diverse impacts on the chinese population at different stages of recent history. during 1944 to 1949, there were 179 notified outbreaks of plague across china, which resulted in an estimated 2.4 million deaths. from 1950 to 1954, the mean number of annual notifications of plague infection was 1,373 cases per year [11, 12] . this was then substantially reduced to 20 cases per year over the following 30 to 40 years. since 1990, the number of annual reported plague cases has increased to approximately 52, which is equivalent to 0.004 per 100,000 population per year [11, 12] . there is no observed trend in the table 1 . estimated number of diagnosed cases, death cases and case fatality rate of eight major infectious disease types in china in 5-year intervals. period 1975 period -1979 period 1980 period -1984 period 1985 period -1989 period 1990 period -1994 period 1995 period -1999 period 2000 period -2004 period 2005 period -2008 quarantinable diseases mean annual mortality due to plague during 1999-2008 (table 2) . cholera has caused two major outbreaks in china in recent decades: the first started in 1973, leading to a large peak in the number of cases in 1980, with an annual notification rate of four cases per 100,000 population ( figure 2a ). the rate then gradually subsided but there was a second major outbreak in the early 1990s. during 2004 to 2008, cholera cases were reported at relatively low incidence levels: the disease is well controlled, with a mean annual decline in mortality due to the disease of 48% (95% ci, 12-70%, table 2 ). despite a 13-fold decrease in the epidemic haemorrhagic fever notification rate since 1987 and continuous decline in the diseasespecific mortality rate, china is still the country most severely affected by this disease, with 90% of the global cases of haemorrhagic fever with renal syndrome (hfrs) occurring in china [13, 14] . hfrs remains an important public health issue in china, as an estimated 20,000-50,000 new cases are reported each year [15] . it is important to establish effective strategies to reduce the incidence of hfrs. the mean annual decline in mortality due to this disease is 12% (95% ci, 3-20%, table 2 ). persistent public health campaigns have been effective in reducing the number of rodents, which has been largely responsible for the decreasing trend in plague and epidemic haemorrhagic fever notifications. improved hygiene and sanitary conditions have also led to the decline in incidence and mortality due to cholera. overall, these quarantinable diseases persist at low endemic levels, but still pose potential threats. in the early 1960s, a nationwide vaccination programme was implemented to eradicate smallpox, measles, poliomyelitis, tuberculosis (tb), pertussis, diphtheria and tetanus. measles vaccine (one-dose schedule) was introduced in the country in 1965, and from 1978 it has been administered routinely to all infants through the country's expanded programme on immunisation [16] . measles mortality and morbidity has declined continuously and substantially since 1978. during 1995 to 2007, the incidence was less than one per 100,000 population (figure 2b), with fewer than 250 deaths due to measles reported annually [17] . however, limited small-scale outbreaks continue to occur among susceptible children in rural areas with low routine immunisation coverage and among susceptible children of 'floating immigrants' (the relatively large population of internal chinese immigrants who leave their rural hometown and move to urban areas to seek better employment opportunities) [18] . in addition, although a two-dose measles vaccination schedule has been recommended in china since 1986, the second dose is widely perceived as a nonmandatory booster. coverage of the second dose is not reported, but is thought to be low [19] . a global effort to eradicate polio began in 1988 [20] ), wild-type poliovirus was eradicated in china in 1994 [21, 22] ; extensive surveillance for acute flaccid paralysis is thought to have strongly contributed to polio eradication in the country [23] . vaccination against diphtheria, pertussis and tetanus was also made mandatory for all infants under the country's expanded immunisation programme since 1960s. in 2006, vaccination coverage of the vaccine against the three diseases reached 99.0%, comparable with polio vaccination (99.0%) and measles vaccination (98.6%) [24] . since 2006, the total annual incidence of reported cases of diphtheria, pertussis and tetanus decreased to below 0.5 cases per 100,000 population ( figure 2b ). widespread vaccination against these diseases is one of the important contributors to improvements in child health in china: infant mortality rate dropped from 20% in 1950 to 1.7% in 2006 [24] . mandatory vaccination of children throughout the past decades has been demonstrated to be successful in containing the spread of these diseases in china. it is estimated that polio vaccination alone has saved a total of 1,128,000 children in china from disabling disease and suffering [24] . mortality due to pertussis and tetanus experienced significant annual decays at rates of 44% (95% ci, 23-59%) and 12% (95 ci%, 30-65%) ( table 2) . trends in tb are discussed in the section on bacterial infections. the number of reported cases of bacillary and amoebic dysentery has declined rapidly since 1975 in china (figure 2c ). an initial large decline occurred in the mid-1970s, largely due to substantial improvements in sanitary conditions and quality of drinking water. however, approximately 84 million diarrhoeal episodes were still reported in china each year at the end of the 1990s: 25% of the episodes were in children under five years of age [25] . shigella bacilli and entamoeba histolytica have been found to be the main aetiological organisms [25] . a live oral shigella vaccine was then developed in 1997 in china, which provided 60-70% protection against s. flexneri serotype 2a and s. sonnei infection [26] . between 1999 and 2008, reported deaths due to dysentery decreased by a mean of 10% (95 ci%, 1-18%) per year ( table 2 ). the number of notified cases of typhoid and paratyphoid fevers is very low in china, with an annual rate of 1.2 notifications per 100,000 population in 2008 (figure 2c ). the number of reported deaths due to these infections has decreased at a mean rate of 22% (95% ci, 2-39%) per year (table 2) . however, these infections remain endemic in some rural areas of southern china, where there is a lack of adequate sewage disposal and safe water supplies [27] . a large, localised outbreak occurred during 1996 to 1998 in xing-an county in the southern province of guangxi, which resulted in an annual incidence rate of 39 to 103 cases per 100,000 population [28] . subsequently 61,030 doses of vi polysaccharide vaccine were administered in 1999 and the epidemic quickly subsided. no further typhoid fever outbreaks were reported in china. national public health campaigns since 1949 in china effectively improved hygiene and sanitary conditions, which substantially reduced most of vector-borne infectious diseases. in 1955, a total of 5,970,000 malaria cases were reported nationally, accounting for 68% of the total number of reported infectious disease cases and an incidence of 1,028 per 100,000 population. malaria disproportionately affected some regions, with incidence rates as high as 10,360 per 100,000 population in hainan province [29] . in 1960 and 1970, there were major outbreaks of the disease, leading to a national incidence of 1,554 and 2,961, respectively, per 100,000 population [29] . it took considerable time for the incidence to fall: the number of cases did not decrease to 1955 levels until 1982, but it has since continued to decrease. by 1998, the rate of notifications decreased to 31.3 per 100,000 population, corresponding to more than a 99% reduction and accounting for only 1.3% of the total number of reported cases of notifiable conditions [29] . data for 2004 to 2008 indicate that the mean annual mortality rate due to malaria was reduced to 0.004 per 100,000 population (table 2) , corresponding to only 52 reported deaths per year countrywide. malaria no longer represents a major threat to the chinese population. the ongoing governmentmobilised mass movement for vector control is probably an effective method for eliminating mosquitoes and their habitats and consequently reducing the disease burden due to malaria in the country. the universal healthcare system provided an effective base for the implementation of countrywide interventions [6] . as a result of the national public health campaigns to eliminate infectious disease vectors, forest encephalitis (also named russian spring-summer encephalitis), tick-borne relapsing fever, tsutsuga-mushi disease (also known as scrub typhus) and epidemic typhus were reduced to very low levels by the late 1980s and were then no longer notifiable. the number of visceral leishmaniasis (also known as kala-azar), japanese encephalitis, malaria and dengue fever epidemics has also decreased, with few cases per year (figure 2d) . however, there are signs that climate change may have led to local outbreaks of some these vector-borne diseases in the past decade in china [30] . rabies is one of the zoonotic diseases on the rise in china. during a 58-year period between 1950 and 2007, a total of 117,530 human rabies cases were reported, corresponding to an incidence rate of 9.1 cases per 100,000 population. three major epidemics were observed [31, 32] : the first occurred in the mid-1950s when the annual number of reported cases reached a peak of about 2,000. the epidemic subsided in the 1960s and then started to surge again in the early 1970s. after reaching a sustained peak from 1982 until the end of the decade, at a level of 5,000 to 6,000 cases per year [33] , in 1996 the number of cases was at its lowest, with 159 reported cases [33] . as the disease in humans is closely associated with transmission of rabies virus by infected dogs, in the past epidemics, strict policies for vaccination of owned dogs and elimination of stray dogs were put in place, resulting in effective control of the epidemic. however, since 2000, due to a new wave of rapid expansion of the pet dog population in urban china, there has been a third outbreak, in which the number of cases has increased rapidly: in 2007, the number of notified cases climbed to 3,250 [34, 35] . the rabies-related mortality rate increased during 1999 to 2008 by a mean of 26% (95% ci, 14-37%) per year ( table 2 ). this should serve as a call for action to prevent the further spread of rabies in china. severe acute respiratory syndrome (sars), caused by a coronavirus that was transmitted to humans through close contact with civet cats, spread from guangdong, china, quickly leading to a worldwide epidemic in 2003. china reported 5,327 (66%) of the 8,071 sars cases globally and 349 sars-related deaths during the epidemic, which lasted for approximately a year [36] . the first human case of highly pathogenic avian influenza a (h5n1) virus infection was reported in hong kong in 1997. by 2009, mainland china had 88 avian influenza outbreaks among birds in 23 provinces and a total of 38 human cases and 25 deaths had been reported [37] . in 2010, the epidemic had spread to 15 countries and resulted in 296 deaths, out of 500 cases [38] . in comparison, the 2009 influenza a(h1n1) pandemic caused more than 154,000 human cases and 842 deaths in china alone [39] , despite the virus being much less virulent than influenza a(h5n1) virus. china, with concentrated human activity in its fast-growing major cities that may lead to effective transmission of human-tohuman transmission of h1n1, is very vulnerable to the transmission of respiratory infectious diseases of zoonotic origin. during the mid-to-late stages of the sars outbreak and, more apparently throughout the course of the 2009 influenza pandemic, the chinese government responded swiftly with openness and transparency to control both diseases and prevent further episodes. in both cases, the epidemics caused widespread social panic. instead of the ministry of health, the state councilsrepresenting the highest administrative authority of the chinese government -assumed direct leadership in combating the epidemics. case notifications for leptospirosis, anthrax and brucellosis have remained at low levels since 2000. there was an increase in the number of notified case of brucellosis from 0.17 per 100,000 population in 2000 to 1.50 per 100,000 population in 2007, but this was probably due to improved surveillance and is believed not to reflect an increase in incidence [40] . the annual number of notified cases of leptospriosis and anthrax dropped below 0.01 per 100,000 population in 2008. the mortality rate due to brucellosis remains very low and stable, whereas the rates due to leptospriosis and anthrax have dropped significantly, by 20% and 31%, respectively, during 1999 to 2008 (table 2) . china has the second largest tb epidemic in the world (after india) [41] . in 2008, some 4,500,000 patients were living with pulmonary tb in china, corresponding to an incidence of 111 per 100,000 population, and resulting in 130,000 deaths from tb in the same year [42] . since 2005, an estimated 20,000 additional tb reactivation cases have arisen per annum in china due to human immunodeficiency (hiv) coinfection [42] . the tb epidemic has spread unevenly in china: in 2005, china reported a national tb prevalence of 0.2% [41] , whereas the southern province of guangxi, which borders vietnam, reported a much higher prevalence of 0.65% [43] . in 1991, in adopting dots -the internationally recommended tb control strategy -china launched a 10-year infectious and endemic disease control project in an effort to curb its expanding tb epidemic in 13 of its 31 provinces [44, 45] . it is estimated that the number of chinese people infected with tb between 1990 and 2000 decreased by 30% and that 300,000 tb deaths were averted. with a success rate greater than 90% [45] , dots has led to the avoidance of an estimated 1.5 million tb cases [44, 45] . however, since 2000, multidrug-resistant (mdr) tb and extensively drug-resistant (xdr) tb have emerged as a severe public health issue in the country. in 2004, china reported approximately 140,000 mdr tb cases, accounting for about one third of the estimated global burden of mdr tb [46] . in some provinces, the proportion of mdr tb among newly notified tb cases and previously treated cases was found to exceed 10% and 30%, respectively [47] . the latest study indicates that the prevalence of mdr tb among tb patients is as high as 19.4%, and 14.9% of mdr tb cases were xdr during 2007 to 2009 [48] . the mortality due to tb increased at a rate of 28% (95% ci, 14-43%) per year. hydroelectric projects that led to the formation of a large number of dams in china, as well as climate change, have substantially increased the risk of schistosomiasis occurring and its spread to non-endemic areas of the country, leading to small-scale outbreaks and increases in rates of case notifications near the affected areas [49, 50] . the incidence of schistosomiasis has increased from 0.10 cases per 100,000 population in 2004 to 0.26 per 100,000 population in 2008 (figure 2f ). since the founding of china in 1949, laws have been enacted to make commercial sex illegal. brothels were closed down and sex workers were sent to camps to be 're-educated'. special institutions for treatment of sexually transmitted infections (stis), at the time mostly syphilis, gonorrhoea and chlamydia, were established for the training of medical personnel, who were then sent to areas previously associated with large sex industries to treat stis at beginning of 1950s. during 1950s, a universal healthcare system was established that provides treatment for sti patients free of charge. designated laboratories for stis were built for diagnosis and research [51] . more importantly, population-based health education campaigns about stis were carried out, and detection and treatment of stis were portrayed as patriotic actions [52] . as a result, the number of cases of notifiable stis decreased from 10 million in 1950 to reportedly eliminated from 1964 [51, 53, 54] . however, notifiable stis have re-emerged, leading to a fastspreading epidemic in the country. in 1987, the incidence for stis was 6.64 per 100,000 population and in 1996, the rate increased to 34.6 per 100,000 population, which corresponded to 1.8 million new sti cases [52] . the annual growth rate of the number of sti cases in china exceeds 20% [52] . the rise of sti incidence is mainly due to unprotected sexual acts and other high-risk behaviours [55, 56] . since early 1980s, underground prostitution has again become rampant in major cities in china [57] . the number of chinese men, increased purchasing power of urban residents and trends away from traditional chinese family values of fidelity all led to an increased number of male clients for commercial sexual services [58] . in addition, lack of sexual health knowledge among commercial sex workers and their inability to effectively negotiate condom use during sexual acts substantially increased the risk of sti transmission [57, 59] . the previously hidden population of men who have sex men (msm) is becoming more overt due to increasing acceptance in society. high-risk sexual behaviours, including a low percentage of condom use, in this population is also contributing to the fast transmission of stis [60] . for example, in 1998, the incidence of syphilis in china was 0.17 per 100,000 population: it increased 20-fold to 4.31 cases per 100,000 population in 2002 and further to 15.88 cases per 100,000 population in 2007 [10, 51] (figure 2g ). in particular, msm, injecting drug users (idus) and female sex workers have the highest syphilis prevalence in china, of 10-20% [61, 62] , 5.4% [63] and 7-12% [64] , respectively. the first case of acquired immunodeficiency syndrome (aids) in china was reported in 1985, but a local hiv epidemic was not detected until 1989 when a cluster of infections was diagnosed among idus in yunnan province [65] . before 2000, china had fewer than 20,000 reported hiv/aids cases, but in 2007, the number rose to 200,000. the latest estimates are that 700,000 people were living with hiv/aids in china at the end of 2007, with 70,000 new infections added every year since then [66] and 20,000-30,000 annual aids-related deaths [67] . although the hiv/aids epidemic first began and predominated among idus, it rapidly spread to other population groups through both heterosexual and homosexual transmission. the latest figures demonstrate that heterosexual transmission has accounted for approximately 38-50% of new hiv infections since 2005 [68] , which may level at or exceed the percentage (30-49%) caused by transmission due to the sharing of injection equipment among idus [69, 70] . notably, hiv infection has become more prevalent among msm, as male homosexual activities account for approximately 10% of new infections [71] . aids mortality rate increased from 0.10 in 1999 to 1.01 cases per million in 2005, then to 4.58 cases per million in 2008, corresponding to an annual growth rate of 44% (95% ci, 30-58%) the rate is expected to increase sharply if the epidemic remains uncontrolled and there is no large procurement of antiretroviral drugs. cumulatively, over 9000 infants were infected through mother-to-child transmission by 2005 and it was estimated that about 500 million chinese were carriers of mycobacterium tuberculosis in 2003 [72] : coinfection of hiv and m. tuberculosis could therefore have a large epidemiological impact, in the absence of appropriate public health interventions. viral hepatitis is prevalent in china, but the prevalence differs according to the type of virus. before the 1990s, hepatitis a virus (hav) was the most prevalent. in the early 1990s, the annual number of reported hav diagnoses was more than 50 per 100,000 population. this dropped by 90% to 5 per 100,000 population in 2005 to 2006 [73] (figure 2h ). the mean annual death rate due to hav decreased at a rate of 21% (95% ci, 8-32%) during 1999 to 2008 ( table 2 ). the decrease is probably a result of mass vaccination efforts in introducing the hav vaccine (h2 strain) in the 1990s and improvement of hygiene and sanitary conditions that broke the cycle of food and water contamination [73] . according to 2008 data [74] , hav persists at low, endemic levels ( figure 2h ). in comparison, infections of hepatitis b virus (hbv), which is transmitted mainly through body fluid from mother to child, and hepatitis c virus (hcv), transmitted predominantly by intravenous injections or blood transfusion, are becoming alarmingly widespread in china. the annual number of new hbv cases increased rapidly from 20 per 100,000 population in 1990 and reached 100 per 100,000 population in 2008, despite an increasing vaccination coverage of newborns from 30% in 1992 to 93.4% in 2005 [75] . hcv infections followed a similar trend and in 2007, the annual notification rate was about 8.8 per 100,000 population. the 2007 hcv and hbv prevalence among the general population was 0.5% and 6%, respectively [74] . mortality due to hbv and hcv increased at annual rates 9% (95% ci, 1-20%) and 30% (16-46%) respectively. in contrast to hav infection, which always causes acute hepatitis but never develops into a chronic condition, hcv and hbv infections often cause chronic hepatitis and may develop into cirrhosis and hepatocellular carcinoma [76] . coinfection of hbv or hcv with other pathogens causing chronic infections, such as hiv, results in substantial increases in the disease burden on individuals, accelerates disease progression and complicates treatment [77, 78] . since hbv and hcv can both be transmitted through exchange of body fluids, coinfection of these hepatitis viruses with hiv is common in at-risk groups in china. approximately 67-71% of the 700,000 registered idus in china are infected with hcv [79, 80, 81] and 17-26% of them are infected with hbv [79, 82] . the prevalence of hiv/hcv coinfection is 6.45% in idus [80] . among blood donors, the prevalence of hiv/hcv coinfection is 5.8-6.5% [80, 83] , whereas the figure for hiv/hbv coinfection is 3.7% [84] . in comparison, coinfection of hiv and hcv among the general population remains unknown as there is currently no reports on its prevalence. however, coinfection is expected to be increasingly noticeable as the hiv epidemic transforms into a generalised epidemic in china [6] . overall, this study investigated the temporal trends of major types of notifiable infectious diseases in china. our analysis indicated that while the morbidity and mortality of most infectious diseases reduced substantially during 1975-1995, there is an increasing trend of re-emergence of previously prevalent diseases and emergence of new infectious diseases since 1995, in particular, stis and hiv, viral hepatitis and zoonoses diseases. these diseases accounted for the majority of deaths associated with infectious diseases in china since 2005. consistent with this, analysis of case fatality rates indicated a higher percentage of deaths among zoonotically infected people (5-15%) and an elevated proportion (0.894%) among stis and hiv-infected individuals. our study has important implications for the surveillance and control of infectious diseases in china. first, the persistent small outbreaks of particular infectious diseases across the country indicate that any decrease in prevention efforts will probably trigger re-emergence of the diseases. surveillance activities are therefore essential to inform and prioritise public health responses. although efficient and well-developed disease surveillance systems have been implemented in many urban areas, hygiene conditions, health services and monitoring of patterns of spread and disease burden are largely lacking in rural china [6] . second, the rapid rise in the number of notified cases of stis, especially hiv infection, and viral hepatitis in china is associated with growth of the sex industry, increasingly frequent risky sexual behaviours and an increasing number of sexual partners in the general chinese population. the newly implemented internetbased national infectious diseases monitoring information system database not only provides a platform for integrating epidemiological data on hiv infection and other stis, but also initiated second-generation behavioural surveillance of the at-risk populations [70] . our analysis indicates that both epidemiological and behavioural surveillance of hiv infection and other stis is essential to understand and forecast the trends in these epidemics. extending surveillance efforts into population groups that were previously considered to be less at risk may further improve the quality and reliability of surveillance data. third, coinfections, especially those involving hiv, are likely to become major public health issues in the future. we demonstrate that tb/hiv, hbv/hiv and hcv/hiv coinfections have become increasingly prevalent in china and the trend is likely to continue in the absence of strong public health interventions. thus, inclusion of testing for tb and viral hepatitis coinfections as part of hiv voluntary counselling and testing could be useful among at-risk populations. scaling up of free-of-charge antiretroviral therapy should also include treatment for these coinfections. fourth, surveillance of zoonotic infections becomes increasingly important due to the interactions between humans and animals in china. china has established an efficient surveillance system for human infectious diseases [6, 85] : a parallel national centralised system for surveillance of disease in animals may be useful for reducing the risk of animal-to-human transmission. one of the potential limitations in our investigation may be poor data quality in the passive notification-based surveillance system. it is widely perceived that underreporting exists in the chinese infectious diseases surveillance system, but little is known about the extent of underreporting as systematic evaluations have never been conducted [6] . this issue requires further consideration in the future. an overview of prevalence studies would be useful for comparison with numbers of notified cases in order to estimate numbers of undiagnosed cases. a comprehensive evaluation would also involve assessments of the efficiency and effectiveness of the surveillance system, both quantitative and qualitative. understanding changes in testing rates in various population groups will also be useful for interpreting trends in notification data. but such investigations are beyond the scope of this review. infectious disease surveillance and interventions in china face several major challenges. the country's major economic reform in 1979 has created a large economic disparity between rural and urban areas, resulting in a large population of floating immigrants (also known as 'peasant workers'). this accounts for 10% of the total chinese population in 2005 [86] . the vast majority of these immigrants are from rural environments but work in urban areas, away from their families, and hence regularly travel between the two sites. in addition to the natural population growth of major cities, the inflow of internal immigrants substantially increases the population size and density in urban areas. these immigrants often live in overcrowded residential areas with compromised hygiene conditions. female immigrants often become targets of those seeking to recruit them for commercial sex work [57, 87] . these floating immigrants are often more disadvantageous than the local residents. they tend to be less educated compared with others in urban areas who have permanent resident status and less likely to seek medical treatment when sick due to financial constraints or lack of health insurance [6] . not surprisingly, the population of floating immigrants has been found to be more vulnerable to infectious diseases, especially stis, hiv and viral hepatitis [6] . additionally, their high-risk behaviours and mobility promotes transmission of infectious disease agents and creates a major challenge for disease detection and control. given the characteristics, behaviours and the increasing population size of floating immigrants, it becomes apparent that the capacity of current chinese healthcare system cannot meet the needs of both the surveillance activities and health problems of these migrant workers. the infectious disease surveillance system needs to be tailored for its high mobility, and a dramatic expansion of the private health sector is required to address the health needs of this population. transmission of zoonotic infectious agents relies on close interaction between infected animals and humans. as a result of the increasing social trend of keeping house pet dogs, it is estimated that the number of pet dogs in china reached 80-200 million in 2004 [88] . however, only 30% of dog owners register their animals with government authorities and only 2-8% of dogs are vaccinated against rabies [31, 89] -coverage of greater than 70% is needed to sufficiently control rabies, as determined by the world health organization (who) [90] . the high rabies prevalence (6.4%) among dogs and the expanding number of pet dogs pose a continual and increasing threat of transmission of rabies virus from dogs to humans in china [89] . in addition, the consumption of game meat, especially in southern china, is one of the important sources of zoonotic diseases [91] . markets in guangdong provinces sell live poultry, fish, reptiles and mammals, including dogs and civet cats, for food [92] : the housing of these live animals, often in packed conditions, together with human activities, makes such markets the ideal hub for cross-species transmission of zoonotic agents [91] . since there is no systematic surveillance of these live markets, it is difficult to estimate the number of such markets and the amount of game meat consumed by residents in southern china. the sars epidemic in 2003 demonstrates that transmission of pathogens originally associated with animals can cause worldwide outbreaks in human populations. the current absence of both epidemiological and behavioural data, including their frequency of contacts and means of animal handling, on animal infectious diseases is a strong barrier to effective surveillance and control. environmental changes facilitate the emergence and transmission of bacterial infections. the formation of a large number of dams in china, as a result of the increasing implementation of hydroelectric projects, have substantially increased the risk of schistosomiasis emergence and its spread to non-endemic areas of the country. further, the dense population conditions in urban china and the high mobility of its floating migrants substantially promote the rapid transmission of tuberculosis [93] . strengthening infectious diseases surveillance for bacterial infections among these specific affected populations should be a priority for the chinese government. conceived and designed the experiments: lz dw. analyzed the data: lz. wrote the paper: lz dw. establishment and amelioration of planned immunization in china disease control and prevention in china in the 20th century and prospects for the new millennium study on the strategy for shanghai disease control and prevention web-based infectious disease reporting using xml forms yearbook of chinese health statistics. beijing: chinese academy of medical sciences and peking union building a better infectious disease surveillance system for china: an evaluation from a political perspective the functionality and developmental strategy of pla cdc in current situation china cdc information release guidelines for infectious diseases epidemic and public health contigencies yearbook of chinese health statistics beijing: ministry of health plague and the control strategies in china the current plague epidemic in china investigating the effects of climatic variables and reservoir on the incidence of hemorrhagic fever with renal syndrome in huludao city, china: a 17-year data analysis based on structure equation model spatiotemporal dynamics of hemorrhagic fever with renal syndrome, beijing, people's republic of china spatial analysis of hemorrhagic fever with renal syndrome in china expanded program on immunization molecular epidemiology of measles viruses in china genetic characterization of measles viruses in china progress in accelerated measles control in the people's republic of china fine pe polio: measuring the protection that matters most status of the eradication of indigenous wild poliomyelitis in the people's republic of china eradication of poliomyelitis: progress in the people's republic of china active surveillance for acute flaccid paralysis in poliomyelitis high-risk areas in southern china chinese child health care in 60 years survey on the prevention and treatment of diarrhea in parts of china double-blind field trial of oral live f 2a-sonnie (fs) dysentery vaccine efficacy trial of vi polysaccharide vaccine against typhoid fever in south-western china an outbreak of typhoid fever, xing-an county, people's republic of china, 1999: estimation of the field effectiveness of vi polysaccharide typhoid vaccine prevention and control of malaria in china, in last 50 years el nino-southern oscillation and vector-borne diseases in anhui human rabies in china molecular characterization of rabies virus isolates in china during a molecular epidemiological study targeting the glycoprotein gene of rabies virus isolates from china rabies trend in china (1990-2007) and post-exposure prophylaxis in the guangdong province rabies in china: an update. vector borne zoonotic dis overview on sars in asia and the world cumulative number of confi rmed human cases of avian influenza a/(h5n1) reported to who the influenza a(h5n1) epidemic at six and a half years: 500 notified human cases and more to come world lab-verified swine flu infections epidemiology and control of brucellosis in china global tuberculosis control -surveillance, planning, financing. geneva: the organization a cross-sectional study of hiv and tuberculosis coinfection cases in mainland china basic situation and influence factor of implementation on tuberculosis dots strategy in guangxi the effect of tuberculosis control in china the dots strategy in china: results and lessons after 10 years global incidence of multidrug-resistant tuberculosis epidemiology of antituberculosis drug resistance (the global project on antituberculosis drug resistance surveillance): an updated analysis prevalence of multidrug and extensively drug-resistant tuberculosis in beijing, china: a hospital-based retrospective study potential impact of climate change on schistosomiasis transmission in china a potential impact of climate change and water resource development on the transmission of schistosoma japonicum in china epidemiologic trends of sexually transmitted diseases in china the past experience of control of sexually transmitted diseases and causes of re-emergency in china sexually transmitted diseases in the people's republic of china in y2k: back to the future prevention and treatment of infectious diseases in new millenium in china syphilis among female sex workers in southwestern china: potential for hiv transmission hidden epidemic of sexually transmitted diseases in china: crisis and opportunity female sex workers in china: vectors of disease? surplus men, sex work, and the spread of hiv in china understanding interrelationships among hiv-related stigma, concern about hiv infection, and intent to disclose hiv serostatus: a pretest-posttest study in a rural area of eastern china survey of high risk behaviors related to aids among men who have sex with their regular male sex partners the influence of social and sexual networks in the spread of hiv and syphilis among men who have sex with men in shanghai relationship between syphilis and hiv infections among men who have sex with men in beijing strategies for implementing health-promoting schools in a province in china effect of foliar application of zinc, selenium, and iron fertilizers on nutrients concentration and yield of rice grain in china epidemic of hiv infection and prevention research in yunnan, 1989-98 state council aids working committee office and un themem group on hiv/ aids in china prevalence of infection with herpes simplex virus types 1 and 2 in australia: a nationwide population based survey high prevalence of risk behavior concurrent with links to other high risk populations: a potentially explosive hiv epidemic among men who have sex with men in guangzhou update on the hiv/aids epidemic and response in china regional differences in hiv prevalence among drug users in china: potential for future spread of hiv? state council aids working committee office and un themem group on hiv/ aids in china (2004) a joint assessment of hiv/aids prevention, treatment and care in china the spread of hiv/aids and strategies for prevetion and control decline in the risk of hepatitis a virus infection in china, a country with booming economy and changing lifestyles general epidemiological parameters of viral hepatitis a, b, c, and e in six regions of china: a crosssectional study in evaluation of the impact of hepatitis b vaccination among children born during 1992-2005 in china hepatitis viruses: changing patterns of human disease opportunistic disease and mortality in patients coinfected with hepatitis b or c virus in the strategic management of antiretroviral therapy (smart) study management of chronic hepatitis b and c in hiv-coinfected patients incidence of hiv, hepatitis c and hepatitis b viruses among injection drug users in southwestern china: a 3-year follow-up study systematic review of hiv and hcv infection among drug users in china review of hiv and hcv infection among drug users in china preliminary research on the co-infection of human immunodeficiency virus and hepatitis virus in intravenous drug users coinfection with hiv and hepatitis c virus in former plasma/blood donors: challenge for patient care in rural china epidemiology, clinical and laboratory characteristics of currently alive hiv-1 infected former blood donors naive to antiretroviral therapy in anhui province, china emergence and control of infectious diseases in china infectious diseases in floating population: prevalent features and control migration and health in china: problems, obstacles and solutions. singapore: asian metacentre for population and substainable development analysis pivotal role of dogs in rabies transmission inferior rabies vaccine quality and low immunization coverage in dogs (canis familiaris) in china world health organization (1996) world health organization expert consultation on rabies the role of the legal and illegal trade of live birds and avian products in the spread of avian influenza severe acute respiratory syndrome analysis of factors affecting the epidemiology of tuberculosis in china key: cord-322581-v96k4yxg authors: mockiene, vida; suominen, tarja; välimäki, maritta; razbadauskas, arturas; caplinskas, saulius; martinkenas, arvydas title: nurses' willingness to take care of people living with human immunodeficiency virus/acquired immunodeficiency syndrome (hiv/aids) — does a teaching intervention make a difference? date: 2011-08-31 journal: nurse education today doi: 10.1016/j.nedt.2010.10.021 sha: doc_id: 322581 cord_uid: v96k4yxg summary the aim of this study is to describe the impact of an education intervention programme on nurses' willingness to care for hiv-positive people in lithuania. methods the study utilizes a randomized controlled trial design (rct). the total sample comprises 185 nurses working in medical, surgical and gynaecological units, and primary health care centres from the same hospital areas in three lithuanian hospitals. the data were analyzed using spss 12.0 and descriptive statistics. findings our educational intervention did not have an impact on the nurses' willingness to take care of people living with hiv (plhiv), as their level of willingness was high already before the education intervention. conclusions further research on this issue is needed to try to understand the forces acting on our nursing staff in order to ensure appropriate care for plhiv. diseases such as acquired immune deficiency syndrome (aids), severe acute respiratory syndrome (sars) and the avian influenza seem to raise new challenges for society as well as for nursing research. these new diseases certainly indicate that diseases nowadays are global phenomena and that new problems may be transported around the world rapidly (hallberg, 2006) . since the first cases were recorded in 1981, aids and its causative agent, hiv (human immunodeficiency virus), have taken an enormous toll around the world. according to the world health organization (2009) every day more than 6800 people become infected with hiv and more than 5700 die, mostly because they have no access to hiv prevention, treatment and care services. despite progress made in scaling up the response over the last decade, the hiv pandemic remains the most serious infectious disease challenge to global public health. at the front line of the war against hiv, health service providers are positioned to respond with needed services and care (li et al., 2007) . it is acknowledged that personal factors, health care systems and cultures may have an impact upon the willingness of nurses to work with hiv-positive people (ives et al., 2009) . while general societal attitudes towards plhiv may be less favourable, these negative attitudes may also be seen among health care personnel (tyer-viola, 2007) . in lithuania, the aids centre conducted an anonymous survey in 1990 to clarify the attitude of medical staff towards those living with hiv. the results showed that up to 90% of the medical staff were not willing to care for patients with hiv. although the situation changed over the years (from 1990 to 1999) and the proportion of staff unwilling to care for patients with hiv was reduced to 52.9%, doctors and nurses, in particular those not working in major cities, still feel a certain fear and tend to separate people living with hiv (lithuanian country report, 2007). for example, 78% of lithuanian health care workers would not allow their child to a kindergarten group, which is visited by a hiv-positive child (lithuania national aids program evaluation, 2005) . these examples show that broad-scale education and extensive information to reduce these barriers are still required. however, little research has focused on adapting the education interventions to target new behaviours associated with hiv, such as increasing nurses' engagement in hiv health care and supportive services. the medline, cochrane library, eric databases, and lithuanian aids centre were searched for relevant english-language citations between 2000 and 2010 using the following search terms: education intervention, hiv, lithuania, nurse, and willingness to take care. the keywords were used both alone and interchangeably. willingness to care is defined as the caregiver's attitude towards providing emotional, physical and instrumental support for plhiv. when willingness to care is assessed in the context of an existing relationship, the primary concerns are whether the relationship can be sustained over time and what issues or perceptions may need to be addressed to make it mutually functional for caregiver and care recipient (abell, 2001) . it has been debated whether it is ethically permissible to refuse to treat those with hiv. ehrenstein et al. (2006) found that 28% of german healthcare workers may abandon work in favour of protecting themselves and family. another study (qureshi et al., 2005) found that the most significant barrier to us healthcare workers' willingness to work with plhiv was fear for their own and their family's health. most recent studies (gurung and sangchart, 2008; williams et al., 2006) reported that the majority of nurses are willing to care for and treat plhiv, but some other studies (oyeyemi et al., 2008; cai et al., 2007) showed that nurses are reluctant to deal with these patients. different background factors have been found to have an impact on whether nurses are willing to provide care or not: cultural values (oyeyemi et al., 2006) , age (välimäki et al., 2008) , gender (oyeyemi et al., 2008; cai et al., 2007) , whether nurses have met (tyer-viola, 2007) , taken care of (oyeyemi et al., 2008; pisal et al., 2007; williams et al., 2006; peate et al., 2002) plhiv or worked with colleagues with hiv (kiragu et al., 2004) . the number of working years has been shown to be negatively associated with willingness to treat plhiv (e.g. worthington et al., 2008) . furthermore, nurses' willingness to take care of plhiv has been found to be related to whether the hiv-positive people were homosexuals, intravenous drug users or prostitutes (tyer-viola, 2007) . nurses' continuing education may be a way to support nurses' willingness to care for plhiv. slaten et al. (2000) , for example, reported after seven training workshop sessions during a five-month period a positive impact on nurses' willingness to take care of plhiv. buskin et al. (2002) found after two lectures that additional education could help eliminate a substantial amount of unwillingness to be in proximity of a person with hiv. it is not clear, however, what educational methods would be best to make an impact. in most intervention studies educational programmes have included workshops (williams et al., 2006; ezedinachi et al., 2002; slaten et al., 2000) or lectures (buskin et al., 2002) . it has been assumed that the educational programmes related to hiv should consist of various different teaching methods to allow debating and discussion about willingness to take care of plhiv (wu et al., 2002; uwakwe, 2000) . models of education that show most promise are those that use experiential styles of learning. it has been shown that it is possible to increase nurses' empathic ability (e.g. brunero et al., 2010) . furthermore, uwakwe (2000) found that an indicator of the degree of success of the programme is the increased willingness of the participating nurses subsequently to work with and treat plhiv. the aim of the study is to explore the impact of an intervention programme on nurses' willingness to take care of hiv-positive people in lithuania. in lithuania, aids is a late arrival (caplinskas, 2004) . while the first hiv-positive case was reported in 1988, today there are 1506 positive cases in the country (population about 3.5 million). (lithuania aids centre, 2009 ). the study population was made up of registered nurses in lithuania who work in the surgical, medical, or gynaecological wards of the hospital and in primary health care centres attached to the hospitals. these nurses were selected because they worked in both hospitals and primary health care areas, and were thus at the front line of hiv prevention, care and advocacy. all nurses in the selected wards of three lithuania hospitals (approximately 300 from each) and primary health care centres attached to the hospital areas constituted the selected population. firstly, nine biggest lithuanian hospitals were chosen for the study. three hospital areas were randomly selected from these. a total of 240 randomly selected nurses from these hospital areas were invited to participate in the study. the recruitment occurred at the same time for all groups. the study utilized a randomized controlled trial design with pre-test evaluation and a three-month repeated follow-up evaluation. randomisation was made by a statistician together with the researchers. to determine the number of participants needed in the study, we performed a power analysis for a one-way anova. the minimum required number of participants was 55 per group. however, we decided to increase the sample size because of possible dropouts. a total sample of 240 participants was recruited: the first educational intervention (two-day workshop and written material) group consisted of 80 participants (eg1), the second educational intervention (written material) group consisted of 80 participants (eg2), as did the control group (cg). the participants were selected by the cluster random sampling method using the spss 12.0 statistical analysis software. nurses from one hospital were selected for the first intervention group, nurses from the second hospital were chosen for another intervention group and nurses from the third randomly selected hospital were used as a control group. the data collection was conducted between december 2008 and march 2009. the first data collection sample included 206 participants. the response rate was 86.3% (n = 69) in eg1, 87.5% (n = 70) in eg2 and 83.8% (n = 67) in cg in december 2008. the follow-up data collection consisted of 185 participants. the response rate after one reminder letter (in march 2009) was for the first group 79% (n = 63), for the second group 79% (n = 63) and for the control group 74% (n = 59). there were two different educational interventions in this study. the first intervention included a two-day workshop (13 h) and the distribution of written material (20 pages). the content areas covered were: hiv and aids related epidemiology and history, prevention, transmission, hiv treatment, and counselling hiv-positive patients and ethical considerations. the intervention included lectures, group discussions, conversations with a hiv-infected person, watching a film about hiv and the distribution of written materials. the lectures were delivered by a physician-nurse pair. also an hiv-positive person participated in the group discussions. in addition to the lecture handouts, the participants were asked to review lithuanian academic journal articles (20 pages) of the content areas mentioned above. the second intervention consisted of the articles (20 pages) that were provided to the eg1 nurses and two pages about new statistics of the hiv situation in lithuania and in the world from eg1 were provided to eg2. in total, eg2 participants received 22 pages of written materials. cg nurses received no lectures or written materials. additionally, the participants from both intervention groups received continuing education credits as participation incentive from the continuing educational centre. the pre-test was done at the beginning of the first day of the twoday workshop among the members of the first education intervention group (eg1) in december 2008. the other groups (eg2 and cg) received the questionnaires at the same time by post. reminder letters (both pre-test and post-test) were sent to all participants one week later. the post-test was repeated for all groups after three months by post. the questionnaire used consisted of two parts: the background questions and the nursing willingness questionnaire (nwq). the background questions asked for demographic information (i.e. age, gender, mother tongue, marital status, number of children, education, and work experience) while the general questions were related to taking care of hiv positive (i.e. if they were willing to care for plhiv). additionally, the respondents were asked to answer dichotomous questions (yes/no answers). they were also asked about their general willingness to take care of plhiv. the nurses' willingness to care for plhiv was evaluated using the nwq scale developed by dubbert et al. (1994) . previous studies suggest that the nwq is a reliable and valid instrument for evaluating a construct of current concern to nursing administrators and educators (dubbert et al., 1994) . the original self-report instrument used a 370-word vignette, whereas the present modified version was reduced to a vignette of 13 english words to describe a person who has progressed to aids (called henes), whose health was deteriorating. the patient's symptoms were: diarrhoea, high temperature, double incontinence, vomiting and mental confusion. using these items, the nurses' willingness to carry out certain nursing activities was explored by 13 items (1 = strongly agree, 2 = agree, 3 = undecided, 4 = disagree, and 5 = strongly disagree). the translation into lithuanian was achieved by using the backtranslation technique (burns & grove, 2001) . the modified version has previously been used and found to be valid and reliable in finland, estonia and lithuania; for the whole data set among the three countries the cronbach's alpha value was 0.93 and in lithuania 0.95 (välimäki et al., 2008 ). in the current study, the cronbach's alpha values for the total data set were 0.83 (in the three groups before the intervention) and 0.81 (in the three groups after the intervention) which varied before and after the intervention among the groups as follows: for the first group 0.74/0.79, second group 0.84/0.74 and control group 0.81/0.87. permission to conduct the study was obtained from institution authorities and approved by the ethics committees of the hospitals and one university. each participant was given a unique identification (id) code which was used during the research and evaluation process to ensure nurses' confidentiality. individual consent was sought from the study participants before starting the study. the consent forms and the letters informing about the study were mailed to the possible participants. the letter contained relevant information on the study and a statement of voluntary participation, as well as assurances of confidentiality and honesty in the reporting of the results. the nurses (eg2 and cg) were provided with information on respondent anonymity, and all nurses were free to withdraw without prejudice at any stage of the research. additionally, the researchers were working at universities, and study participants in hospitals or public health care centres. statistical analysis of the data was performed by using the spss 12.0 software package. nurses' demographic variables and items concerning their perceptions related to their willingness to care for plhiv were examined using descriptive analysis. after testing for normality, we used parametric and nonparametric criteria, the student's t, anova, and mann-whitney and kruskal-wallis tests to compare two or more groups. cronbach's alpha was used to evaluate the internal consistency of the scales. p-values less than 0.05 were interpreted as statistically significant. in the following text only statistically significant results are reported. all participating nurses were female (n= 206, 100%). the nurses ranged in age from 23 to 67 years, their mean age being 43.1 (sd = 8.8). the nurses in the first group were the youngest, with the difference between the groups being statistically significant (p= 0.016). nurses in the first group had less children (p= 0.46), and more university-level qualifications (p = 0.001). also the length of the nurses' work experience at the present workplace varied in the different groups as follows: eg1 0.5-38 (mean 19.7, sd 8.8); eg2 0.8-46 (mean 23.5, sd 10.00) and cg 2.0-40 (mean 22.2, sd 9.11). the nurses from the first group had the least work experience (p= 0.051). other demographic differences are presented in table 1 . our educational interventions, meaning the workshop and the written materials distributed to the nurses (eg1), or the written materials distributed alone (eg2), did not have an impact on the nurses' willingness to take care of plhiv. however, the nurses from all groups were willing to care for plhiv even before the education intervention (nwq scores: 1.27-1.41), 1 meaning the most positive and 5 the most negative score. changes in willingness were found only in eg1, among those who had received the workshop and the written materials (mean score 1.37, sd 0.66), but the result was not statistically significant (table 2) . when comparing the nurses' willingness in the 13 specific nursing activities, it was found that, because of the intervention, nurses in eg1 were more willing to take an hiv-patient's vital signs, clean supplies, complete catheter care, shave, empty the urinary draining bags, start intravenous fluids and administer a blood transfusion, that is, do more "dirty" nursing activities than before the intervention (table 3) . before the intervention, nurses from eg2 were the most willing to care for plhiv (mean score 1.27, sd 0.59) while those from the cg were the least willing to provide care (mean score 1.52, sd 0.77). there were no statistically significant changes in the nurses' willingness to take care of plhiv in eg2 nor in cg after the intervention. in eg2, participants were asked to answer just one general background question on whether they were willing to take care of plhiv or not. after the intervention, the nurses tended to be more willing to take care of plhiv (mean score 1.11, sd 0.15 versus mean 1.21, sd 0.33, p = 0.066). as a background factor, all participants were asked to answer one general question concerning their willingness to take care of a patient with hiv. we found that after the intervention, nurses from all three groups: eg1 68%, n = 43; eg2 89%, n = 56; cg 93%, n = 55, reported unwillingness to take care of a patient with hiv. no adverse events occurred during the intervention study. we should point out that there could be some limitations in using the three intervention arms because of the demanding practical arrangements and/or scenario used in this teaching programme. however, at the same time it was valuable to see the impact of two different programmes in a country with limited funding possibilities. the mean difference is significant at the 0.05 level (mann-whitney test). eg1intervention group (2-day workshop and written material); eg2intervention group (written material); and cgcontrol group. the aim of this study was to ascertain what kind of impact the intervention has on nurses' willingness to take care of hiv-positive people or those with aids in lithuania. the ultimate goal was to create a realistic teaching programme with limited funding in order to establish an ongoing continuing education programme for lithuanian nurses. our study revealed that, in general, nurses from all three groups (eg1, eg2 and cg) were willing to perform required nursing activities for a fictional person who has progressed to aids (see dubbert et al., 1994) . as health professionals become part of the social space of people living with these infections, they become part of the patient's social surroundings as empathic figures, people who know "how it feels" (worthington et al., 2008) . hiv-related stigma is reduced if plhiv are not seen as "different". however, when using just one general background question on whether nurses were willing to take care of plhiv, up to 93% of the control group nurses reported unwillingness to care for plhiv. keeping in mind that drug use is the main route of transmission in lithuania, this may be a potential cause of the lack of willingness to provide care. drug use generally is a stigmatised activity, which is why people may be unwilling to care for drug users. although our first teaching intervention showed a positive impact on nurses' knowledge level (mockiene et al., 2011) only minor changes could be observed in the willingness to care. after the educational intervention, nurses in eg1 were more willing, for example, to carry out daily nursing activities such as to clean up faeces or vomit only using gloves and feed dinner. similar results were obtained by wu et al. (2002) and williams et al. (2006) . however, the fact that nurses in the first intervention group were younger, had more university-level qualifications and less work experience at the present ward than in the other groups, may have had have a positive impact on the results (see also välimäki et al., 2008 , worthington et al., 2008 . the distribution of written materials only (eg2) did not have a positive impact on increasing nurses' willingness to take care of plhiv. uwakwe (2000) indicated that the mere production of hiv information materials and dissemination with minimal personalized contact does not always yield optimum results in health behaviour modification and written materials, no matter how good they are, may not always be read by the target group. our study showed that the nurses who had more work experience were more willing to care for plhiv than nurses whose work history was short. this result differed from the results of another study which showed that the number of years in professional practice was negatively associated with willingness to treat plhiv (worthington et al., 2008) . it is likely that the preparation of nurses to take care of hiv-positive patients is influenced by different educational backgrounds, whereas the limitations of the education systems in lithuania, for example, can be problematic in themselves. according to williams et al. (2006) , nurses need to have the knowledge and confidence to protect themselves from performing the mean difference is significant at the 0.05 level (mann-whitney test). eg1intervention group (2-day workshop and written material); eg2intervention group (written material); and cgcontrol group. this work effectively, and must be well-informed about the clinical course of hiv and about effective strategies for its prevention and treatment. besides, they must be prepared to care for patients from a variety of cultural and social backgrounds whose experiences and values may differ from their own, and recognize that patients with hiv should be given the same care as hiv-negative patients. the present study was the first attempt to conduct a rct in the field of nursing research in lithuania. we emphasize the importance of such studies in all east-european countries in order to develop evidence-based nursing. even though it is problematic to carry out research on interventions (also internationally), and usually the conclusions cannot explain causality, such studies are needed to advance nursing knowledge (hallberg, 2009) . this is also true for research on implementing evidence for education. several variables may affect empathy education that need to be accounted for in future studies such as gender, cultural values and clinical speciality experience. models of education that show most promise are those that use experiential styles of learning. studies have demonstrated that it is possible to increase nurses' empathic ability (brunero et al., 2010) . the fact that lithuania was attributed to the low-prevalence countries after the first hiv cases, had a negative impact of hiv awareness. this might have been influenced by the attitude towards continuing studies of nurses. however, having reviewed the plans of the institutions providing services in lithuania, it was noted that the training in this area is the thing that nurses miss most. it is therefore important to focus on improving nurse education, and basic educational support should be a priority for those working with plhiv. a more organized educational structure, targeted at all health care professionals in the form of health talks/ seminars, in-service training, continuing medical education and nursing curricula, would improve the knowledge level related to hiv for health care providers most efficiently and effectively. consequently, educational programmes based on research evidence must play a leading role in educational strategies to help nurses understand hiv-positive people and change their attitudes towards taking care of them. in the future, we need more outcome research and intervention studies to assess their effects systematically as stated by hallberg (2006) . assessing willingness to care for persons with aids: validation of a new measure a review of empathy education in nursing the practice of nursing research: conduct, critique and utilization hiv/aids knowledge and attitudes in chinese medical professionals and students before and after an informational lecture on hiv/aids inequality and unwillingness to care for people living with hiv/aids: a survey of medical professionals in southeast china epidemiology of hiv/aids in lithuania in 1988-2001: review of present situation and prognosis of hiv transmission trends development of a measure of willingness to provide nursing care to aids patients influenza pandemic and professional duty: family or patient's first? a qualitative survey of hospital employees the impact of an intervention to change health workers' hiv/aids attitudes and knowledge in nigeria: a controlled trial nurses knowledge, attitude and willingness to take care for hiv/aids patients in bhutan challenges for future research: providing evidence for health-care education moving nursing research forward towards a stronger impact on health care practice? healthcare workers' attitudes to working during pandemic influenza: a qualitative study colleagues with hiv/ aids: perspectives from health workers in zambia using case vignettes to measure hiv-related stigma among health professionals in china the impact of an intervention to change nurses' knowledge and hiv/aids related attitudes in lithuania: a randomized controlled trial caring for patients living with aids: knowledge, attitude and global level of comfort knowledge, attitude and willingness of nigerian physiotherapists to provide care for patients living with acquired immunodeficiency syndrome hiv/aids and its impact on student nurses nurses health education program in india increases hiv knowledge and reduces fear healthcare workers' ability and willingness to report to duty during catastrophic disasters training mental health professionals on ethical issues and hiv obstetric nurses' attitudes and nursing care intentions regarding care of hiv-positive pregnant women systematized hiv/aids education for student nurses at the university of ibadan, nigeria: impact on knowledge, attitudes and compliance with universal precautions willingness to care for patients with hiv/aids effectiveness of an hiv/ aids educational programme for chinese nurses priority interventions hiv/aids: prevention, treatment and care in the health sector. world health organization, hiv/aids department rehabilitation professionals and human immunodeficiency virus care: results of a national canadian survey diffusion of hiv/aids knowledge, positive attitudes, and behaviours through training of health professionals in china the researchers would like to thank the finnish nursing education foundation for financial support.we would also like to thank the nurses in lithuania for their contribution in this study. key: cord-316534-ep7ezoko authors: gamble, lena j; matthews, qiana l title: current progress in the development of a prophylactic vaccine for hiv-1 date: 2010-12-22 journal: drug des devel ther doi: 10.2147/dddt.s6959 sha: doc_id: 316534 cord_uid: ep7ezoko since its discovery and characterization in the early 1980s as a virus that attacks the immune system, there has been some success for the treatment of human immunodeficiency virus-1 (hiv-1) infection. however, due to the overwhelming public health impact of this virus, a vaccine is needed urgently. despite the tireless efforts of scientist and clinicians, there is still no safe and effective vaccine that provides sterilizing immunity. a vaccine that provides sterilizing immunity against hiv infection remains elusive in part due to the following reasons: 1) degree of diversity of the virus, 2) ability of the virus to evade the hosts’ immunity, and 3) lack of appropriate animal models in which to test vaccine candidates. there have been several attempts to stimulate the immune system to provide protection against hiv-infection. here, we will discuss attempts that have been made to induce sterilizing immunity, including traditional vaccination attempts, induction of broadly neutralizing antibody production, dna vaccines, and use of viral vectors. some of these attempts show promise pending continued research efforts. since its discovery and characterization in the early 1980s as a virus that attacks the immune system, leaving patients unable to fight off opportunistic infections, there has been an ebb and flow of effective treatments and hope as scientists continue to search for ways to eradicate human immunodeficiency virus-1 (hiv-1) from the human population similar to what has been accomplished in the case of smallpox. the majority of the effort and nearly all of the success has come in the area of patient treatment rather than inhibition of contraction or spread of the virus. a class of treatments, antiretroviral therapies (arts) and later highly active antiretroviral therapies (haarts), has been the mainstay of disease control during the last 15 years. notwithstanding the increased life span of patients, increased time to full-blown aids, and decreased contraction of opportunistic infections and aids-related diseases (ie, non-hodgkin's lymphoma, kaposi's sarcoma, etc) by patients treated with haart, there are several reasons why development of an hiv-1 vaccine is still warranted. five of these reasons are as follows: 1) nearly two-thirds of the patients who contract hiv-1 live in underdeveloped countries and cannot afford the expensive haart regimen, 1 2) both the art and haart regimen are complex and are disruptive to patients' lives and diets, making long-term compliance an issue, 2 3) the potential side effects of art/haart treatments negatively affect the long-term health of patients and include diabetes, cardiovascular disease, fractures, etc, 3-5 4) development of haart drug resistance, and 5) the presence of latent hiv-1 reservoirs harboring viral strains that were produced through mutation throughout the duration of the infection of the host also play a role in the failure of haart. 6 these reasons, as well as many others, underscore the need for a prophylactic hiv-1 vaccine. possibly, the strongest argument for development of a prophylactic vaccine may be the need for control of the virus spread worldwide. every day, 7500 patients worldwide are infected with hiv-1. 1 production of a vaccine that could inhibit infection, reduce spread, or both would aid in the reduction of the burden of aids and aids-related diseases. the expenses incurred by the aids epidemic can hardly be calculated. they range from tens of thousands of dollars per patient for the haart regimen, to millions of dollars required for building of orphanages by governments for children whose parents have succumbed to the disease, to the unknown cost of educational materials and condoms in the effort to prevent further spread of the disease. this public health challenge has not gone unnoticed and has been addressed by scientists' ongoing efforts to develop a safe and effective hiv-1 vaccine. a prophylactic hiv-1 vaccine would offer sterilizing immunity to patients, preventing infection upon presentation of the virus. a prophylactic vaccine must also be effective at all possible portals of hiv-1 entry, especially the mucosa. 7 for this to occur, the vaccine must offer broad and durable immunity. several consortia have worked diligently to produce a vaccine that will induce broadly reactive neutralizing antibodies (nabs). these consortia include major international efforts as well as efforts of individual countries, regions, and institutions including, but not limited to: the international aids vaccine initiative neutralizing antibody consortium, 8 the center for hiv-aids vaccine immunology, the hiv vaccine trials network, us military hiv research program, the collaboration for aids vaccine discovery, and the vaccine research center at the national institutes of allergy and infectious diseases of the national institutes of health. to date, however, no hiv-1 candidate vaccine has induced broadly reactive nabs. 8 in the absence of a vaccine that can prevent infection of hiv-1, there are still many benefits to be realized from production of a therapeutic vaccine. a therapeutic vaccine would be supremely valuable if it were able to increase the titer of virus necessary for infection, increase the time to clinical manifestation of virus, control viral load after infection, and reduce secondary transmission. [9] [10] [11] [12] [13] a vaccine that could induce this type of response would invariably decrease contagiousness, decrease the need for costly and potentially dangerous art/haart, and decrease the number of opportunistic infections of patients. while the effect of controlling the normal hiv-1 pathology with therapeutic vaccines will be favorable for the individual patient as well as society, the effect of preventing hiv-1 infections in humans with a prophylactic vaccine is also broadly appealing. this potential for eradicating the hiv-1 virus from human hosts drives scientists to continue to find ways to circumvent the challenges presented by this unique virus in order to induce production of the nabs that are critical for sterilizing immunity. this review, therefore, will focus on the specific challenges presented by hiv-1 and strides that have been made toward creating a prophylactic vaccine, including past efforts that have failed and lessons that have been learned from those failures. we will also discuss novel vaccine options and some of the promising trials that are currently underway. while several problems face scientists who are attempting to create an hiv-1 vaccine, three problems in particular have posed extremely daunting challenges. these three problems are 1) degree of diversity of the virus, 2) ability of the virus to evade the hosts' immunity, and 3) lack of appropriate animal models in which to test vaccine candidates. these three major problems will be discussed in more detail below. traditionally, prophylactic vaccines have been made by exposing some part of a pathogen's structure as an antigen to the host's immune system, and eliciting an immune response, resulting in the production of long-term memory lymphocytes that are capable of mounting a strong immune response upon later infection with the pathogen. the premise upon which this manipulation of the immune system is based is the ability of the immune system to make long-lasting antibodies to conserved structures on exposed proteins that are native to the pathogen. ideally, both humoral and cell-mediated immunity would be induced creating long-lasting immunity. traditional attempts to recreate this process using live attenuated simian immunodeficiency virus-1 (siv-1) viruses in an effort to vaccinate macaques against siv-1 have been proven safe and effective in macaques that were subsequently challenged with siv-1. 14, 15 however, an incidental study of the effect of liveattenuated hiv-1 (containing deletions of the nef gene and the long terminal repeat) was proven pathogenic in humans when three out of six treated patients developed late-onset immunosuppression. [16] [17] [18] killed viruses have also been tested as a potential vaccine approach, but safety concerns have halted their use. these safety concerns include incomplete inactivation of the virus leading to potential residual infectivity during the vaccine preparation. 19 due to the ineffectiveness of traditional vaccine approaches to date, scientists have attempted to use recombinant hiv-1 proteins to stimulate the production of nabs. these attempts failed due to their inability to induce a lasting, broad range of nabs that would inhibit infection in humans. [20] [21] [22] [23] perhaps these failures are a result of the inherent diversity of hiv-1. this diversity has presented a major roadblock to development of a prophylactic vaccine. there are three main groups of hiv-1 (m, o, and n) 24 as well as a recently discovered group, p. 25 each group consists of several subtypes, clades. the various clades display biological differences with respect to transmission, 26 replication, 27 and disease progression. 28, 29 these differences result in an inability to produce a generalizable vaccine that would induce the breadth of nabs necessary to counter an infection by a wide range of hiv-1 clades that may be encountered in a natural setting. 30 the degree of diversity seen in hiv-1 is greater than that of any other virus observed. 31, 32 this problem is being addressed by development of multiclade (multiple env and/ or subtype b gag, pol, nef) 33, 34 and mosaic vaccines which incorporate sets of 10 immunogenic proteins from 4 different clades or bivalent proteins from clades b and c. [35] [36] [37] there are proof of principle studies that illustrate immunological protection against hiv-1 in nonhuman primates that were passively treated with broadly reactive nabs. [38] [39] [40] these studies show that protection against infection with hiv-1 can be conferred by the presence of broadly reactive nabs. the next step toward production of a prophylactic vaccine would involve induction of production of these or similar broadly reactive nabs by the host's immune system. the rate at which the hiv-1 virus mutates, due to the nature of the reverse transcriptase enzyme responsible for transcribing its rna, ensures that nearly every daughter virion will have a different genome than its parent. 41 when these changes occur in the hiv-1 env protein that is needed for antibody recognition, they inhibit the immune system's ability to mount a sufficient response. one attempt to circumvent this problem has been to induce the production of nabs to the conserved regions of hiv-1 proteins. a major problem with this approach is that the conserved regions of hiv-1 proteins are often shielded from exposure to nabs within the hiv-1 envelope. the native structure of the envelope protein, reportedly the only hiv-1 protein susceptible to nabs, 31 shields it from the immune system as a glycosylated trimer of heterodimers. the glycosylation of the envelope protein allows for the carbohydrates to masquerade as 'self' thereby forming an immunologically silent face and protects neighboring epitopes via an 'evolving glycan shield'. [42] [43] [44] additionally, the gp41 coreceptor binding site, another conserved site, is not presented until primary binding to cd4 + has occurred. 45 an attempt to create antibodies to the cd4-binding region of the gp120 protein was made in rhesus macaques in 2007 and results indicated that vaccinated hosts were able to withstand challenge with shiv. 46 other attempts to create an hiv-1 vaccine have focused on overcoming the ability of hiv-1 to escape immune surveillance through use of antibodies that are able to neutralize diverse isolates of hiv-1. these antibodies include pg9, pg16, 47 2f5, 2g12, 4e10, b12, [48] [49] [50] [51] and most recently scd4-17b 52 and others. 53 identification of these antibodies gives hope that their induction or the induction of other such broadly reactive nabs may provide the basis for a prophylactic vaccine in the future. the use of animal models for development of therapeutics offers the benefit of thorough testing and validation prior to introduction of a vaccine in humans. in the past, vaccines were made by observing and then mimicking the immune response mounted by individuals who had recovered from a particular disease. to date, however, there are no known cases of individuals who have recovered from hiv-1 infection. however, data can be gathered from long-term nonprogressors -patients who have been infected with hiv-1 for at least 7 years and do not display any hiv-1-related symptoms. 54, 55 another option that may be critical to the development of a prophylactic vaccine is the use of relevant animal models. such models will allow for analysis of the effect of a potential vaccine on an intact host prior to use in humans. one particular challenge with the use of animal models for development of a prophylactic hiv-1 vaccine is that there are very few naturally occurring disease models of hiv-1. only a few nonhuman primates are susceptible to infection with hiv-1 and infected animals do not progress to aids. 56 therefore, it is important to use other disease models that mimic the hiv-aids pathologic progression. 57 one such potential model is feline immunodeficiency virus (fiv). fiv was discovered in 1986 and is known to cause an aids-like disease in domestic cats and mimics hiv-related dementia in humans. 58 a vaccine for fiv was approved by the fda in 2002. 59 while the fiv model is potentially informative, its use is not sufficient as a basis for development of a prophylactic hiv-1 vaccine. an ideal animal model would display a pathological response to infection with hiv-1 that is very similar to the one that occurs in humans. unfortunately, hiv-1 does not cause pathology leading to the development of aids in any host other than humans. [60] [61] [62] [63] however, animal models have been developed and used that allow partial understanding of the pathology of hiv-1, the natural immunological response to infection, and the response of the host to novel therapeutics. one of these models involves the simian immunodeficiency virus mac (siv mac ) that replicates and causes an aids-like disease in baboons, cynomolgus, and pigtailed macaques. while the similarities of siv mac to hiv-1 have allowed for insight into pathology, transmission, and immunological response of the infected host to the virus, the differences between siv mac and hiv-1 are still too great to be able to draw conclusions regarding potential human responses to an hiv-1 prophylactic vaccine. 63 therefore, to broaden the scope of animal model usage, a chimeric shiv virus was engineered to incorporate both siv and hiv-1 proteins or genes. 64 while macaques infected with shiv do go on to develop aids, the time to progression is much different from the time to progression to aids of hiv-1-infected humans. infection of macaques with siv mac 251 strain mimics hiv-1 infection in humans by leading to chronic, slow disease progression. route and dose required for infection, viral tropism, replicative capacity of the viruses, and pathology of siv/shiv-infected monkeys are all very different than these parameters in humans. 65, 66 this distinction has been well characterized by the recent phase iib step trial, which involved 3000 healthy, uninfected volunteers. the result of this trial was termination at its first scheduled efficacy assessment due to its failure to suppress viral load in subsequently infected individuals and then-suspected increased hiv-1 infection due to interaction of the immune system with vaccine components. 67 the vaccine, a recombinant adenovirus serotype 5 (ad5) virus incorporating the gag, pol, and nef genes from hiv-1, had been previously tested in an shiv model in macaques and the results of that experiment were not suggestive of the results of the human trial. 68 this disparity underscores the need for animal models that more closely reflect the pathology seen in human infection with hiv-1 as well as identification of immunological correlates of protection that reflect control of hiv-1 viral load in human subjects. therefore, the search for an appropriate animal model or the appropriate use of current animal models in the search for a prophylactic hiv-1 vaccine continues. until a model can be derived that will allow for observation of each stage of infection, progression of disease, and response of the immune system in a way that is comparable to this process in humans, we will not be able to logically predict which vaccine candidates should be moved forward to clinical trials. several attempts to stimulate the immune system to provide protection against hiv infection have been attempted so far (table 1) . hope for creating a prophylactic vaccine lies in the ability of the scientific community to identify and induce a broad neutralizing antibody response that would offer sterilizing immunity to vaccinated patients. to this end, several novel approaches are being studied. as mentioned in the previous section, there are several daunting problems facing scientists who are attempting to create an hiv-1 vaccine. in hopes of creating a vaccine which elicits sterilizing immunity to hiv-1, researchers have focused their efforts on (1) the use of plasmid dna vaccines, (2) live recombinant vectors for vaccine development (expressing or presenting hiv antigens), and (3) mucosal immunity. these critical topics will be discussed in more detail below. vaccines should elicit a robust immune response that is long lasting and is able to provide protection against various strains of a pathogen. plasmid dna vaccinations can induce a strong humoral and t-cell response. dna-based vaccination has been used as a powerful tool to fight against parasitic, fungal, bacterial, and viral infections. [115] [116] [117] [118] [119] there are multiple advantages for using plasmid dna for vaccination: they are generally safe, nontoxic, and through the delivery of a gene encoding important immunogenic epitopes, the dnabased vaccine exploits biosynthetic machinery of the host cell. one such example was in 1990, whereby wolff and colleagues illustrated protein expression after intramuscular (im) injection of plasmid dna into myocytes. 120 despite these promising results, there had been speculation regarding dna vaccination strategies. for example, it was shown that protein production in response to dna plasmids that contained hiv inserts elicited substantial cellular response in mice and nonhuman primates. however, these products were poorly immunogenic in humans. one strategy to improve immune response of the plasmid dna vaccine strategy is by coadministration of dna plasmids coding for cytokines (eg, inf-g, il-2, il-12, il-18, and il-15). [121] [122] [123] [124] a second strategy which has been utilized to improve plasmid dna vaccination has been the administration of plasmid dna with adjuvants (eg, cpg oligodeoxynucleotides), or the use of dna-delivery systems (eg, microparticles, cochleates, and linear polyenimines). [125] [126] [127] [128] a third strategy to improve vaccine efficacy involves the coadministration of plasmid dna in combination with viral vectors. for instance, research performed by harari and colleagues in 2008 demonstrated that vaccination by means of an hiv-1 clade c dna prime in combination with a pox vector (nyvac) boost induces a reliable polyfunctional and longlasting anti-hiv t-cell response in human participants. 129 along these same lines, work recently published by jaoko and group demonstrated safety and immunogenicity of a multiclade hiv-1 ad-based vaccine alone or in combination with a multiclade hiv-1 dna vaccine in africa. these results also demonstrated that dna priming increased the frequency and magnitude of cellular and humoral responses; however, there was no effect of recombinant ad5 dosage on immunogenicity endpoints. 130 the previously mentioned dna-delivery strategies have been used in combination with viral vectors or alone by means of a variety of immunization routes (eg, im, intravenous [iv], intradermal [id], intranasal [in] , oral, rectal, or vaginal). in the majority of reported studies, dna vaccines have been administered by the im and/or id routes. however, as it relates to hiv vaccination, mucosal immunity could potentially be an important factor to consider, with mucosal immunity being achieved optimally by in or oral routes of administration. the topic of mucosal immunity will be discussed in more detail in a later section within this review. after immunization, it is assumed that the dna vaccination immunogen is produced in the skeletal muscles, dendritic cells, and macrophages at the site of immunization. however, in adults, the skeletal muscles are not involved in a high level of protein synthesis as compared to the liver. therefore, the delivery of dna to cells, which are capable of high protein synthesis, such as hepatocytes, epithelia cells of the intestines, or salivary pancreas, may result in high levels of protein expression. the hepatocytes express enzymes involved in the formation of intrachain and interchain disulfide bonds required for proper folding and assembly of proteins. in addition, the liver expresses glycosyltranferases, which are essential for synthesis of both n-and o-linked glycan side chains; this may not be the case for other cell types, 131,132 the significance of this point being the fact that broadly crossclade nabs such as 2g12 recognize glycan moieties on the heavily glycosylated hiv-1 envelope antigens. 44, 133, 134 another advantage of protein expression within the liver is that significantly lower amounts of dna are needed for protein expression of a particular antigen in the hepatocytes vs another cell type. for the immunization of humans, milligram quantities of dna are necessary to achieve adequate levels of immune response. 119 any method whereby there would be a reduction in dna quantity needed to vaccinate humans would provide significant economic advantages. based on the previously mentioned reasons, it is not a surprise that the liver has been exploited extensively as a site for gene delivery due to its ability to produce proteins and glycoproteins. [135] [136] [137] [138] hydrodynamic delivery is the application of controlled hydrodynamic pressure in capillaries to enhance endothelial and parenchymal cell permeability; this methodology had its inception in the late 1990s with investigations into intravascular injection of plasmid dna solution for gene delivery in whole animals. [139] [140] [141] [142] hydrodynamic plasmid dna delivery is well tolerated in mice. in 2008, raska and colleagues demonstrated in mice that iv hydrodynamic vaccination with hiv-1 envelope dna injections resulted in high levels of expression of hiv antigen in the liver. in mice, immunological data illustrated that hydrodynamic administration of hiv-1 plasmid dna was superior to vaccination with dna by in, id, im, and intrasplenic routes. further results illustrated that after boosting, hydrodynamic vaccination yielded levels of hiv-1-specific antibodies that were 40-fold higher than those elicited by other routes tested. 132 however, this delivery scheme is not feasible in large animals and humans. as an alternative, receptor-mediated dna binding to hepatocytes could be a viable approach. molecules with terminal galactose residues covalently linked to dna are recognized by the hepatocyte-expressed galactosespecific asialoglycoprotein 143 receptor for internalization. 144 this alternative would avoid delivery through the hepatic system and the need for expansion of the blood volume. in addition, galactose-linked dna packaged in delivery vehicles such as liposomes, choleates, or microspheres can be given by oral administration, which would be absorbed by the intestine and ultimately delivered to the hepatic vein. as an additional alternative to hydrodynamic delivery in humans, it might be possible to express hiv antigens in the liver by means of plasmid dna delivery via viral vectors such as the ad. ads have been shown to transduce the liver efficiently in vivo by means of the hexon proteins. 145, 146 in this regard, production of translation of hiv-1 proteins primarily in the liver might allow for the production of heavily glycosylated hiv-1 envelope antigens and thus the production of nabs. prophylactic vaccine for hiv-1 live recombinant vectors for vaccine development viral vectors are potent inducers of cellular and humoral response. viral vectors can express proteins from bacteria or viral pathogens to vaccinate against infectious diseases. there are several viral vaccine vectors that have been used successfully in models for vaccination. these vectors include alphaviruses, human rhinoviruses (hrvs), ads, picornaviruses, poxviruses, measles viruses, influenza, and vaccinia viruses. 30, 129, [147] [148] [149] [150] [151] [152] [153] [154] [155] [156] each of these vectors has its respective disadvantages and advantages with respect to vaccine development. some advantages of a few of these vectors include their ability to naturally infect a wide variety of cell types and tissues of interest. [157] [158] [159] [160] [161] [162] each respective vector has its own set of disadvantages. for instance, one disadvantage of using the poliovirus or the hrv as a vaccine vector is the insert size limit restriction of these vectors as compared to the large insert size (∼8 kb) accommodation of ad vectors. the most common disadvantage of the majority of viral vaccine vectors is reduced vaccine efficacy due to vector preexisting immunity (pei). [163] [164] [165] [166] [167] various strategies have been employed to circumvent the problems associated with vector pei. specifically, as it relates to ad vectors, pei is a tremendous problem. of the identified serotypes of ad vectors, human serotypes 5 (ad5) and 2 (ad2) have been the most extensively used for gene therapy protocols. ad5 has been used for hiv-1 vaccination protocols, most recently in the step study. as it relates to ad2 and ad5, pei to these vectors may be found in up to 50% of the american population and up to 95% of the population of other countries. this ad pei can limit the effectiveness of ad-based vaccinations. [168] [169] [170] to circumvent ad2 or ad5 pei, researchers have employed the use of vector chimeras, 166,171 use of alternative serotypes, [172] [173] [174] [175] [176] [177] [178] and the use of nonhuman ads, 151 such as chimpanzee ad. the chimpanzee ad virus was demonstrated to not be significantly neutralized by human sera, which gives chimpanzee ad an advantage for human vaccine development. [179] [180] [181] other strategies have been used to reduce the immune response against ad vectors such as the use of helperdependent ad (hd-ads) vectors, 182-187 the use of ad delivery in combination with biochemical modifications such as pegylation, [188] [189] [190] [191] [192] [193] [194] and the use of vector delivery by means of cell vehicles. 195, 196 with respect to the hd-ads, these vectors were produced to further increase the safety and cloning capacity of first-generation ad vectors. hd-ads lack ad genes and contain only the packaging signals and end terminal repeats. these vectors were designed to avoid cellular immunity and diminish liver toxicity, thus promoting long-term transgene expression. [197] [198] [199] [200] the reduced immune response against hd-ads has allowed for transgene expression in mice and baboons for years. 182, 183, 185, 200 this long-term transgene expression could be helpful for antigen production for an hiv vaccine, thus producing an opportunity to have increased protection against hiv, with reduced frequency of vaccinations. although nabs to ad5 may reduce the immunogenicity of ad5-based vectors in animal model systems, their effect on the immunity in subjects with previous ad5 exposure is still largely unknown. as previously mentioned, the step trial, which tested a merck recombinant ad5 (rad5) vaccine (encoding hiv-1 gag, pol, and ne1 genes), failed to yield protection, either by lowering viral load or by decreasing acquisition of infection. 13 analysis of data from this study aroused speculation that subjects with pre-existing nabs from wild-type ad5 infection had an increased risk of hiv infection after vaccination. one recent study has shown that there was no causative role for ad5-specific cd4 + t cells in increasing hiv-1 susceptibility in the merck trial. 201 in this regard, there are multiple studies ongoing to elucidate a concrete finding with respect to the role of ad5 pei and increased activation of cd4 + t cells in the mucosal milieu. 202, 203 recently, there was a report by cheng and colleagues that attempted to characterize the specificity of rad5 nabs in ad5immune subjects and determine the impact of ad exposure on immune responses elicited by ad5-based vaccinations. cheng and colleagues reported that rad5 nabs were directed toward different components of the ad virion, depending on whether the ad5 infection was natural or from ad-based hiv vaccine trials. for example, ad nabs generated by natural infection are directed primarily to fiber components, while vector exposure elicits responses primarily to capsid proteins other than fiber. nabs elicited by natural infection significantly reduced the cd8 + and cd4 + cell responses to hiv gag after dna/rad5 vaccination. this report concluded that ad5 nabs differ based on the route of exposure and that previous ad5 exposure compromises ad5 vaccine-induced immunity to weak immunogens, such as hiv-1 gag. 204 these results have a tremendous impact on hiv-1 vaccine trials and the design of next generation viral vaccine vectors. viral vectors such as ad, influenza, and polio have been used as vaccine vectors for many reasons. one important advantage of these vectors, which makes them attractive, is that they can provide mucosal immunity because they can easily infect the mucosal surfaces as well as act to induce cytokine and chemokine production at the mucosal entry sites. ad, influenza, and polio also have the advantage of being able to be delivered orally, without the use of needles. this is an important fact in developing countries where needle cost is prohibitive to vaccine administration. as it relates to hiv vaccine development, mucosal immunity is a debatable factor to consider. when deciding upon a vaccine agent, the importance of considering if the ultimate goal is to induce systemic immunity, mucosal immunity, or both is worth careful consideration. [205] [206] [207] it is believed that 80% of hiv-1 i nfection will occur from heterosexual viral transmission and most of the rest will occur from homosexual or perinatal transmission. 152 although the biology of sexual transmission is poorly understood, it is clear that the essential first step in the infection pathway is the transfer of infectious virus or hiv-infected cells through the mucosal surfaces. after hiv has entered a new host, the hiv or hiv-infected cells will soon encounter susceptible host target cells at the mucosal point of entry where the virus replicates and then invades local lymphatic tissues, initiating systemic hiv infection. on this basis, strong immunity is required to provide a protective immunological barrier at the most common point of entry, the mucosal surfaces of the reproductive tract. due to the compartmentalization of the secretory and systemic immune systems, parenterally administered antigens do not consistently stimulate mucosal immunity. 152 therefore, it is important to consider a vaccine regime that induces mucosal immunity. since cd4 + ccr5 + memory t cells are the primary target of hiv infection in the gut and mucosa and rapid depletion of this subset occurs early after infection, 208, 209 several studies have investigated the role of hiv mucosal immunity. previous studies have demonstrated the importance of a mucosal siv/hiv vaccine producing both strong mucosal antibody and cd8 + response capable of blocking the escape of virus from the intestinal mucosa into systemic lymphoid organs. 207, [210] [211] [212] [213] [214] however, in other instances, the necessity of exclusive mucosal hiv immunity will be further debated based on the promising results found in a heterologous prime/boost regimen using dna/89.6-expressing siv and hiv-1 transcripts 215,216 and modified vaccinia virus ankara (mva/89.6)-expressing siv and hiv-1 transcripts under the control of vaccinia virus early/late promoter. in this case, either id or im dna/mva vaccination was able to provide protection against a intrarectal shiv-89.6 challenge. 153 along these same lines, recently, promising results were found by hessell and colleagues in 2010. hessell and colleagues demonstrated that after an iv administration of monoclonal antibodies 2f5 or 4e10 to six monkeys followed by a shiv ba-l challenge, five out of six monkeys from either group showed complete protection and sterilizing immunity. a low level of viral replication could not be ruled out for the six monkeys in either group. 217 replicative ad yields a robust immune response at the mucosal sites partly because ad is known to infect and replicate in epithelial cells. [218] [219] [220] [221] various strategies have been used to achieve mucosal immunity via the oral route. one such strategy embodies the development of replication-defective recombinant ad serotype 41 (ad41) vector. 222 serotype 41 vectors are being currently used because ad41 has a natural tropism for the gut and causes no pathological disease outside of the gastrointestinal tract. 223 ad41 vectors are likely to have a preferential tropism for the gut because ad41 appears to have a resistance to acidic ph 224 and the capsid configuration of long and short fibers allows the ad41 virus to preferentially infect the gut. 177, 225 live recombinant vectors for vaccine development engineered to express/present hiv-1 antigens as previously mentioned, viral vectors are potent inducers of cellular and humoral responses. of note, viral vectors have been practically used for human applications and have progressed treating a variety of disease contexts such as cancer and infectious diseases. [226] [227] [228] [229] traditional viral vector immunization embodies the concept that the vector uses the host cell machinery to express antigens, which are encoded as transgenes within the viral vector. cellular and humoral immune responses are generated against these antigens. over the last 20 years, several viral vectors have been derived to express hiv-1 antigens for vaccine purposes. some researchers have taken an alternative approach to conventional transgene expression of antigens by means of viral vectors; this alternative approach embodies the capsid incorporation of antigens. this innovative paradigm is based upon the vector presenting the antigen as a component of the capsid rather than an encoded transgene. incorporation of immunogenic peptides into the vector capsid offers potential advantages. in this regard, the processing of the capsidincorporated antigen via the exogenous pathway should result in a strong humoral response similar to the response provoked by native ad capsid proteins. in this arrangement, potentially, hiv peptide antigens accrue the potent immunostimulatory effects of the native ad vector capsid proteins, which effectively perform an adjuvant function. on this basis, the immune response directed against vector capsid proteins with repetitive vector administration should achieve a booster effect against the incorporated antigen. 230 most importantly, as it relates to hiv infection, this strategy yields the potential of generating antibodies to hiv proteins. recent crystallographic, cryo-electron tomography, and molecular modeling studies have provided valuable insight to molecular surfaces recognized by antibodies as well as assisted in rationale vaccine design of immunogens. [231] [232] [233] [234] [235] these structural technologies can also potentially improve the abilities of scientists to advance the antigen capsidincorporation strategy. if the antigen capsid incorporation is effective, it can provide a way forward with respect to inducing sterilizing immunity. 68, 236, 237 the antigen capsid-incorporation strategy has been used for ad-based vaccines in the context of many diseases. 230, [238] [239] [240] [241] [242] one of the first examples where the antigen capsid-incorporation strategy was used was with research performed by crompton in 1994. 242 crompton and colleagues inserted an eight-amino acid sequence of the vp1 capsid protein of poliovirus type 3 into two regions of the ad2 serotype hexon. one of the chimeric vectors produced grew well in tissue culture. in addition, antiserum raised against the ad with the polio insert specifically recognized the vp1 capsid of polio type 3. as it relates to ad5 serotype, wu and group demonstrated that his 6 epitopes could be incorporated into ad hexon hypervariable regions (hvrs) 1-7 (now reclassified as 1-9) without perturbing viral viability and any major biological characteristics such as replication, thermostability, or native infectivity. this study by wu and colleagues demonstrated that his 6 appeared to be surface exposed at these regions. 243 with respect to peptide incorporation within ad5 hexon, hvr2 and hvr5 appear to be the most promising locales for peptide/antigen incorporation based on x-ray and peptide analyses along with molecular studies. 244 our laboratory and others have focused on incorporations at hvr5 or single-site incorporations (such as fiber and pix). 230,238-241-,243,245,246 however, we recognized that the ability to place antigen within multiple sites of the ad capsid protein would hold important potential for presenting multiple epitopes/ antigens or several copies of the same epitope within a single ad vector-based vaccine. in an effort to create multivalent hiv vaccine vectors, our 2008 study explored the use of ad5 hvr2 and hvr5 in hopes of creating vectors which contained hiv antigenic epitopes at both locales. to compare the flexibility and capacities of ad5 hvr2 and hvr5, we genetically incorporated identical epitopes of incrementally increasing size within hvr2 or hvr5 of ad5 hexon. we incorporated identical epitopes ranging from 33 to 83 amino acids within the ad5 hexon hvr2 or hvr5 region. viable viruses were produced with incorporations of 33 amino acids plus a 12-amino acid linker at hvr2 or hvr5. in addition, viable viruses were produced with incorporations of up to 53 amino acids plus a 12-amino acid linker at hvr5. with respect to identical antigen incorporations at ad5 hvr2 or hvr5, hvr5 was more permissive allowing an epitope incorporation of 65 amino acids in total. these model antigens were surface exposed via elisa analysis. in vivo immunization with these vectors illustrated an antigen-specific immune response. 240 along these same lines, abe and colleagues evaluated the ability of ad5-based vectors expressing an hiv transgene to induce antigen-specific immune responses under ad5 preimmune conditions. to overcome limitations that are generally experienced as a result of pei to ad5, they constructed vectors that have a modification in the hvr5. their study characterized various immunological parameters generated by these vectors such as vector neutralization, acquisition of adaptive immune response, and comparison of protective immunity. first, in order to evaluate the utility of the modified ad vector, they measured the neutralizing activity of sera by a modified ad vector. they administered ad-luc (luciferase protein expressed as a transgene in the ad e1 region), ad-hisluc (his 6 epitope presented in hvr5 region and luciferase protein expressed as a transgene), or ad-end/ aaaluc vector (containing three amino acid mutations in hvr5 and expressing luciferase protein) to mice im. after administration of these vectors, neutralizing activity against ad5 was observed for 0-8 weeks. the hexon-modified vector (ad-hisluc) generated the lowest ad5-specific neutralizing activity, which was significantly lower than what was generated by ad-luc at weeks 6 and 8, and by ad-end/aaaluc vector at week 8. the individual neutralizing activity of ad-hisluc immunization was significantly lower than that of ad-luc immunization. additional studies performed by abe and colleagues support the concept that modified hexon thwarts ad5 nabs and promotes cellular immune responses. 247 studies performed by this research group indicate that a change in the immunogenic epitope is necessary to avoid neutralization by pre-existing nabs. our recently published work exploits the antigen capsidincorporation strategy for hiv vaccination. our novel vectors were constructed in hopes of moving toward the goal of creating vectors that will provide cellular and humoral hiv immunity. our study is the first of its kind to genetically incorporate an hiv antigen within the ad5 hexon hvr2 alone or in combination with genomic incorporation of a gag transgene (ad5/hvr2-mper-l15(gag)). in this study, we successfully incorporated a 24-amino acid epitope of hiv-1 within hvr2. the hiv-1 region selected for hvr2 incorporation was the membrane proximal ectodomain region (mper) derived from hiv-1 glycoprotein 41 (gp41). our rationale for choosing a portion of the mper (eknekel-leldkwaslwnwfditn) derived from gp41 was based on the fact that the gp41 envelope protein ectodomain is a target of three broadly neutralizing anti-hiv-1 antibodies. 248 when the mper was incorporated into hvr2 in combination with transgene expression, we observed growth kinetics and thermostability changes similar to those of other capsid-incorporated vectors generated in other studies, 249, 250 indicating that incorporation of the mper epitope within hvr2 was not dramatically detrimental to virological characteristics. 250, 251 in addition, we demonstrated that the mper epitope is surface exposed within hvr2. most importantly, we observed a humoral anti-hiv response in mice vaccinated with the hexon-modified vector. the mper-modified vector allows boosting compared to adcmvgag, possibly because the ad5/hvr2-mper-l15(gag) ad elicits less anti-ad5 immune response. it is possible that the mper epitope reduced the immunogenicity of the ad5 vector. this finding is noteworthy because hvr2 has not been fully explored for antigen capsid-incorporation strategies. 252 these vectors are currently being analyzed by cryo-electron microscopic analysis to determine the critical correlates related to antigen placement/configuration and immune response. in addition, with respect to hiv-1 vaccination, the antigen capsid-incorporation strategy has been evaluated within the context of hrv. research groups have constructed human rhinovirus:hiv-1 chimeras in an effort to stimulate immunity against hiv-1. 148, 253 in an effort to develop hiv-1 vaccines, researchers within this same group generated combinatorial libraries of hrv capsid-incorporated hiv-1 gp41 epitope. their results indicated that they were successful in eliciting antibodies whose activity can mimic the nab effect. 149 commercial and clinical ad development of hiv-1 vaccines have progressed preferentially more than vector systems such as hrv because the flexibility of ad generally exceeds current rhinovirus systems. for example, because hrv is a relatively small rna virus, the hrv platform can display an array limited to 60 copies of a single hiv-1 epitope. 148, 253 in contrast, the ad vector platform allows incorporation of the hiv-1 mper epitope into three structurally distinct locales, including hvr2, hvr5, 247 and protein ix (our unpublished data). in comparison, the ad mper antigen capsid-incorporation display platform could present an array of 720 hiv-1 epitope copies within ad hexon and 240 hiv-1 epitope copies within pix. if a multivalent ad vector is generated with hiv-1 epitopes within the hexon and the pix locales, this would represent 960 hiv epitopes within one ad vector. another significant difference between the ad and hrv platforms is in the number of locales that have been successfully used for heterologous epitope insertion. finally, in contrast to the rhinovirus that lacks this capacity, the ad platform has sufficient coding capacity allowing for hiv-1 transgene expression in combination with presenting the same or a different antigen on the viral capsid surface. this latter finding is important because it provides the basis for constructing vectors that will provide cellular and humoral hiv-1 immunity. vectors which provide both cellular and humoral immunity may be the way forward with respect to prophylactic hiv vaccine development. recently, there have been encouraging developments regarding hiv vaccination. in the 1980s, in thailand, there was a substantial increase in the prevalence of infection with hiv-1. [254] [255] [256] by first observation, these groups consisted of intravenous-drug users and commercial sex workers; this infected group then expanded to the general population. 101 by the mid 1990s, the overall seroprevalence of hiv-1 reached a peak of 3.7% among members of the royal thai army and of 12.5% among people from northern thailand. 255, 257 the thai ministry of public health acted by starting an effective hiv-prevention campaign. with this effort, the number of new hiv-1 infections per year decreased from an estimated 143,000 in 1990 to 14,000 in 2007. 255, [258] [259] [260] although this decrease was promising, there was still a desire to do more to prevent hiv infection. to achieve this goal, an hiv phase iii study was begun. the thai phase iii hiv vaccine study, also known as rv144, opened in the fall of 2003. the placebo-controlled trial tested the safety and effectiveness of a prime-boost regimen of two vaccines: alvac-hiv vaccine (the prime), a modified canarypox vaccine, and aidsvax b/e vaccine (booster), a gp 120 vaccine. the vaccines were based on the subtype e and b hiv-1 strains that commonly circulate in thailand. the subtype b hiv-1 strain is the most commonly found strain in the united states. the trial, conducted in the chonburi and rayong provinces of thailand, enrolled 16,402 women and men aged 18-30 years at various levels of risk for hiv infection. study participants received the placebo or alvac hiv vaccine at enrollment and again after 1, 3, prophylactic vaccine for hiv-1 and 6 months. the placebo or aidsvax b/e vaccine was given to participants at 3 and 6 months. participants were tested for hiv-1 infection every 6 months for 3 years. during each clinic visit, study participants were counseled on how to prevent hiv-1 infection. the results showed that 74 of 8198 placebo recipients became infected with hiv-1 compared with 51 of 8197 participants who received the vaccine. this level of effectiveness in preventing hiv-1 infection was found to be statistically significant. the vaccine strategy had no effect, however, on the amount of virus in the blood of volunteers who acquired hiv-1 infection during the study. based on the final analysis of the study, the surgeon general of the us army, the trial sponsor, announced that the prime-boost investigational vaccine regimen was safe and 31% effective in preventing hiv-1 infection. with respect to an hiv-1 vaccine that can provide sterilizing hiv immunity, this is the best result in humans to date. however, the modest protection effect appeared limited to low-risk individuals, and there were data which suggest that this effect was confined to the first year following administration of the vaccine. efforts must continue to focus on evaluating the immune response induced by the vaccine to establish potential correlates of protection. over the last three decades, the world has been faced with the emergence and subsequent epidemic of hiv/aids. there has been much progress with respect to diagnosis and prevention. on the treatment front, there have been several significant advances with respect to drug development (ie, art/haart). however, there is a desperate need for an effective and safe vaccine. there has been tremendous difficulty with regard to developing a vaccine that provides sterilizing immunity. this has been the case due to some of the factors mentioned in this review such as hiv diversity, immune evasion, and lack of appropriate animal models. due to these obstacles, many researchers assumed that the control of hiv-1 viremia by vaccination would be a more realistic goal than the development of sterilizing immunity. the road to a safe and effective hiv-1 vaccine received a serious setback in the fall of 2007 with the premature termination of the merck-hiv-1 vaccine step trial due to the lack of efficacy and early speculation that the vaccine might have increased the risk of hiv infection in some populations of vaccinees. in late 2009, promising results came in from thailand in response to their efforts to create a safe and effective vaccine against hiv-1. a community-based, randomized, multicenter, double-blinded, placebo-controlled efficacy trial using a prime-boost combination showed 31% effectiveness in preventing hiv-1 infection. these results lend promise to the hope of producing an hiv-1 vaccine vector that yields sterilizing hiv-1 immunity. in the future, research scientists must work together to increase hiv-1 vaccine effectiveness beyond 31%. realization of this goal may be accomplished by some of the techniques mentioned in this review, such as acquisition of hiv mucosal immunity, development of effective prime-boost strategies, development of better animal models, better molecular antigen modeling and presentation, avoidance of pei (by the means of using novel vector serotypes in combination with pegylation), and/or induction of nabs (by means of capsid incorporation of hiv antigens within viral vectors). these are just a few considerations that scientists and clinicians must consider with respect to the development of an effective and safe hiv-1 vaccine. scientists and clinicians must also consider that one vector or scheme may not be sufficient with respect to providing effective hiv-1 immunity and some combination of the above-mentioned potential strategies may offer the most promising method of producing an effective hiv-1 prophylactic vaccine. submit your manuscript | www.dovepress.com dovepress dovepress report on the global aids epidemic adherence to antiretroviral therapy: a survey of factors associated with medication usage lipodystrophy, metabolic disorders, and human immunodeficiency virus infection: aquitaine cohort, france, 1999. groupe d'epidemiologie clinique du syndrome d'immunodeficience acquise en aquitaine a syndrome of lipoatrophy, lactic acidaemia and liver dysfunction associated with hiv nucleoside analogue therapy: contribution to protease inhibitor-related lipodystrophy syndrome longitudinal evolution of bone mineral density and bone markers in human immunodeficiency virus-infected individuals the latent hiv-1 reservoir in patients undergoing haart: an archive of pre-haart drug resistance mucosal immunity to hiv: a review of recent literature hiv vaccine design and the neutralizing antibody problem aids/hiv. developing an aids vaccine: need, uncertainty, hope hiv-1 vaccine development: progress and prospects t cell vaccines for microbial infections the failed hiv merck vaccine study: a step back or a launching point for future vaccine development? protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus long-term symptomless hiv-1 infection in recipients of blood products from a single donor genetic instability of live, attenuated human immunodeficiency virus type 1 vaccine strains update on long-term symptomless hiv type 1 infection in recipients of blood products from a single donor hiv vaccines: new frontiers in vaccine development placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent hiv-1 infection correlation between immunologic responses to a recombinant glycoprotein 120 vaccine and incidence of hiv-1 infection in a phase 3 hiv-1 preventive vaccine trial lessons from failure -preparing for future hiv-1 vaccine efficacy trials randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 hiv-1 vaccine among injection drug users in global and regional distribution of hiv-1 genetic subtypes and recombinants in 2004 a new human immunodeficiency virus derived from gorillas preferential in-utero transmission of hiv-1 subtype c as compared to hiv-1 subtype a or d cell reservoirs in lymph nodes infected with hiv-1 subtype e differ from subtype b: identification by combined in situ polymerase chain reaction and immunohistochemistry different rates of disease progression of hiv type 1 infection in tanzania based on infecting subtype relation between chemokine receptor use, disease stage, and hiv-1 subtypes a and d: results from a rural ugandan cohort escape artist par excellence genetic subtypes, humoral immunity, and human immunodeficiency virus type 1 vaccine development viral sequence diversity: challenges for aids vaccine designs standardized assessment of nab responses elicited in rhesus monkeys immunized with single-or multi-clade hiv-1 envelope immunogens a phase 1/2 study of a multiclade hiv-1 dna plasmid prime and recombinant adenovirus serotype 5 boost vaccine in hiv-uninfected east africans (rv 172) mosaic vaccines elicit cd8(+) t lymphocyte responses that confer enhanced immune coverage of diverse hiv strains in monkeys hiv vaccines: mosaic approach to virus diversity mosaic hiv-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys antibody protects macaques against vaginal challenge with a pathogenic r5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro human neutralizing monoclonal antibodies of the igg1 subtype protect against mucosal simian-human immunodeficiency virus infection protection of macaques against vaginal transmission of a pathogenic hiv-1/siv chimeric virus by passive infusion of neutralizing antibodies evolutionary and immunological implications of contemporary hiv-1 variation the antigenic structure of the hiv gp120 envelope glycoprotein structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody antibody neutralization and escape by hiv-1 cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus antibodies to cd4-induced sites in hiv gp120 correlate with the control of shiv challenge in macaques vaccinated with subunit immunogens broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target efficient neutralization of primary isolates of hiv-1 by a recombinant human monoclonal antibody. science a conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1 a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1 cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric cd4-igg scd4-17b bifunctional protein: extremely broad and potent neutralization of hiv-1 env pseudotyped viruses from genetically diverse primary isolates analysis of memory b cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from hiv-1-infected individuals long-term nonprogression in hiv infection: methodological issues and scientific priorities the immunology of hiv-infected long-term nonprogressors-a current view long-term observations of human immunodeficiency virus-infected chimpanzees simian and feline immunodeficiency viruses: animal lentivirus models for evaluation of aids vaccines and antiviral agents the feline model of neuroaids: understanding the progression towards aids dementia feline immunodeficiency virus model for designing hiv/aids vaccines human immunodeficiency virus infection of human-pbl-scid mice suppression of hiv infection in azt-treated scid-hu mice disseminated and sustained hiv infection in cd34+ cord blood cell-transplanted rag2-/-gamma c-/-mice animal models of aids infection of cynomolgus monkeys with a chimeric hiv-1/sivmac virus that expresses the hiv-1 envelope glycoproteins a chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an aids-like disease after in vivo passage in rhesus monkeys highly pathogenic shivs and sivs target different cd4+ t cell subsets in rhesus monkeys, explaining their divergent clinical courses efficacy assessment of a cell-mediated immunity hiv-1 vaccine (the step study): a doubleblind, randomised, placebo-controlled, test-of-concept trial replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity importance of the nef gene for maintenance of high virus loads and for development of aids protective effects of a live attenuated siv vaccine with a deletion in the nef gene molecular and biological characterization of simian immunodeficiency virus macaque strain 32h proviral clones containing nef size variants macaques infected with live attenuated sivmac are protected against superinfection via the rectal mucosa immunization with a live, attenuated simian immunodeficiency virus (siv) prevents early disease but not infection in rhesus macaques challenged with pathogenic siv live, attenuated simian immunodeficiency virus sivmac-m4, with point mutations in the env transmembrane protein intracytoplasmic domain, provides partial protection from mucosal challenge with pathogenic sivmac251 protection of macaques against simian immunodeficiency virus infection with inactivated vaccines: comparison of adjuvants, doses and challenge viruses. the european concerted action on 'macaque models for aids research' whole inactivated siv vaccine grown on human cells fails to protect against homologous siv grown on simian cells protection of monkeys by a split vaccine against sivmac depends upon biological properties of the challenge virus dual-subtype vaccine (fel-o-vax fiv) protects cats against contact challenge with heterologous subtype b fiv infected cats dual-subtype fiv vaccine protects cats against in vivo swarms of both homologous and heterologous subtype fiv isolates effect of dual-subtype vaccine against feline immunodeficiency virus infection dual-subtype fiv vaccine (fel-o-vax fiv) protection against a heterologous subtype b fiv isolate efficacy testing of recombinant human immunodeficiency virus (hiv) gp160 as a therapeutic vaccine in early-stage hiv-1-infected volunteers. rgp160 hase ii vaccine investigators randomised trial of mnrgp120 hiv-1 vaccine in symptomless hiv-1 infection phase ii controlled trial of post-exposure immunization with recombinant gp160 versus antiretroviral therapy in asymptomatic hiv-1-infected adults. vaxsyn protocol team repeated immunization with recombinant gp160 human immunodeficiency virus (hiv) envelope protein in early hiv-1 infection: evaluation of the t cell proliferative response a phase i evaluation of the safety and immunogenicity of vaccination with recombinant gp160 in patients with early human immunodeficiency virus infection. military medical consortium for applied retroviral research two double-blinded, randomized, comparative trials of 4 human immunodeficiency virus type 1 (hiv-1) envelope vaccines in hiv-1-infected individuals across a spectrum of disease severity: aids clinical trials groups 209 a nd 214 a randomized, placebocontrolled study of the immunogenicity of human immunodeficiency virus (hiv) rgp160 vaccine in hiv-infected subjects with . or = 400/ mm3 cd4 t lymphocytes improved cell-mediated immune responses in hiv-1-infected asymptomatic individuals after immunization with envelope glycoprotein gp160 immunization with envelope mn rgp120 vaccine in human immunodeficiency virus-infected pregnant women immunogenicity of the yeast recombinant p17/p24:ty virus-like particles (p24-vlp) in healthy volunteers immunization with recombinant p17/p24:ty virus-like particles in human immunodeficiency virus-infected persons safety and immunogenicity of a candidate therapeutic vaccine, p24 virus-like particle, combined with zidovudine, in asymptomatic subjects therapeutic vaccination with p24-vlp and zidovudine augments hiv-specific cytotoxic t lymphocyte activity in asymptomatic hiv-infected individuals induction of potent humoral and cell-mediated immune responses following direct injection of dna encoding the hiv type 1 env and rev gene products retroviral vectors for vaccine development: induction of hiv-1-specific humoral and cellular immune responses in rhesus macaques using a novel mlv(hiv-1) pseudotype vector a single administration of lentiviral vectors expressing either full-length human immunodeficiency virus 1 (hiv-1)(hxb2) rev/env or codon-optimized hiv-1(jr-fl) gp120 generates durable immune responses in mice rabies virus-based vaccines elicit neutralizing antibodies, poly-functional cd8+ t cell, and protect rhesus macaques from aids-like disease after siv(mac251) challenge highly attenuated rabies virus-based vaccine vectors expressing simian-human immunodeficiency virus89.6p env and simian immunodeficiency virusmac239 gag are safe in rhesus macaques and protect from an aids-like disease protection of rhesus monkeys against infection with minimally pathogenic simian-human immunodeficiency virus: correlations with neutralizing antibodies and cytotoxic t cells vaccination with alvac and aidsvax to prevent hiv-1 infection in thailand vector-mediated gene transfer engenders long-lived neutralizing activity and protection against siv infection in monkeys enhancement of humoral immunity to sivenv following simultaneous inoculation of mice by three recombinant adenoviruses encoding sivenv/poliovirus chimeras, tat and rev replication-defective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in nonhuman primates adenovirus vectored vaccines replication-deficient recombinant adenoviruses expressing the human immunodeficiency virus env antigen can induce both humoral and ctl immune responses in mice multi-envelope hiv-1 vaccine devoid of siv components controls disease in macaques challenged with heterologous pathogenic shiv cytotoxic t lymphocyte and antibody responses generated in rhesus monkeys immunized with retroviral vector-transduced fibroblasts expressing human immunodeficiency virus type-1 iiib env/rev proteins evaluation of a self-inactivating lentiviral vector expressing simian immunodeficiency virus gag for induction of specific immune responses in vitro and in vivo chimaeric hiv-1 subtype c gag molecules with large in-frame c-terminal polypeptide fusions form virus-like particles optimization of chimeric hiv-1 virus-like particle production in a baculovirus-insect cell expression system increased incorporation of chimeric human immunodeficiency virus type 1 gp120 proteins into pr55 gag virus-like particles by an epstein-barr virus gp220/350-derived transmembrane domain recombinant human immunodeficiency pr55 gag virus-like particles presenting chimeric envelope glycoproteins induce cytotoxic t-cells and neutralizing antibodies development of hiv/aids vaccine using chimeric gag-env virus-like particles dna vaccines induction of gp120-specific protective immune responses by genetic vaccination with linear polyethylenimine-plasmid complex dna vaccines: a review comparison of protective effect of protein and dna vaccines hsp90 in murine model of systemic candidiasis dna vaccines against human immunodeficiency virus type 1 in the past decade direct gene transfer into mouse muscle in vivo enhancement of human immunodeficiency virus type 1-dna vaccine potency through incorporation of t-helper 1 molecular adjuvants modulation of cellular and humoral immune responses to a multiepitopic hiv-1 dna vaccine by interleukin-18 dna immunization/ viral protein boost evaluation in macaques of hiv-1 dna vaccines containing primate cpg motifs and fowlpoxvirus vaccines co-expressing ifngamma or il-12 augmentation and suppression of immune responses to an hiv-1 dna vaccine by plasmid cytokine/ig administration cell-specific delivery of genes with glycosylated carriers organspecific gene expression in the rhesus monkey eye following intravenous non-viral gene transfer systemic linear polyethylenimine (l-pei)-mediated gene delivery in the mouse targeted gene delivery to cancer cells: directed assembly of nanometric dna particles coated with folic acid an hiv-1 clade c dna prime, nyvac boost vaccine regimen induces reliable, polyfunctional, and long-lasting t cell responses safety and immunogenicity study of multiclade hiv-1 adenoviral vector vaccine alone or as boost following a multiclade hiv-1 dna vaccine in africa differential effect of lentil feeding on proteosynthesis rates in the large intestine, liver and muscle of rats delivery of dna hiv-1 vaccine to the liver induces high and long-lasting humoral immune responses immunopathogenesis and immunotherapy in aids virus infections a role for carbohydrates in immune evasion in aids electrically mediated plasmid dna delivery to hepatocellular carcinomas in vivo tail-vein injection of mannan-binding lectin dna leads to high expression levels of multimeric protein in liver mechanism of plasmid delivery by hydrodynamic tail vein injection. ii. morphological studies mechanism of plasmid delivery by hydrodynamic tail vein injection. i. hepatocyte uptake of various molecules the efficient expression of intravascularly delivered dna in rat muscle hydrodynamics-based transfection in animals by systemic administration of plasmid dna high levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid dna hydrodynamic gene delivery: its principles and applications the asialoglycoprotein receptor: relationships between structure, function, and expression synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting adenovirus serotype 5 hexon mediates liver gene transfer adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo viral vectors for malaria vaccine development human rhinovirus type 14:human immunodeficiency virus type 1 (hiv-1) v3 loop chimeras from a combinatorial library induce potent neutralizing antibody responses against hiv-1 broad neutralization of human immunodeficiency virus type 1 (hiv-1) elicited from human rhinoviruses that display the hiv-1 gp41 eldkwa epitope use of adenovirus in vaccines for hiv development of nonhuman adenoviruses as vaccine vectors poliovirus vaccine strains as mucosal vaccine vectors and their potential use to develop an aids vaccine control of a mucosal challenge and prevention of aids by a multiprotein dna/mva vaccine recombinant poxviruses as mucosal vaccine vectors induction of neutralizing antibody responses to anthrax protective antigen by using influenza virus vectors: implications for disparate immune system priming pathways subcutaneous administration of a recombinant vaccinia virus vaccine expressing multiple envelopes of hiv-1 an adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism targeted adenoviral vectors viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics live attenuated measles vaccine as a potential multivalent pediatric vaccination vector infection of monocytes during measles vaccinology, immunology, and comparative pathogenesis of measles in the quest for a preventative against aids preexisting immunity to poliovirus does not impair the efficacy of recombinant poliovirus vaccine vectors strategies to overcome host immunity to adenovirus vectors in vaccine development neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein construction and characterization of adenovirus serotype 5 packaged by serotype 3 hexon hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity immune responses to adenovirus and adeno-associated virus in humans the impact of developmental stage, route of administration and the immune system on adenovirus-mediated gene transfer circumventing the immune response to adenovirus-mediated gene therapy hexon gene switch strategy for the generation of chimeric recombinant adenovirus sero-switch' adenovirusmediated in vivo gene transfer: circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype circumvention of vectorspecific neutralizing antibody response by alternating use of human and non-human adenoviruses: implications in gene therapy generation of a novel replication-incompetent adenoviral vector derived from human adenovirus type 49: manufacture on per.c6 cells, tropism and immunogenicity dependence of adenovirus infectivity on length of the fiber shaft domain adenovirus type 40 virions contain two distinct fibers human adenovirus type 41 contains two fibers structure-based identification of a major neutralizing site in an adenovirus hexon characterization of a family of chimpanzee adenoviruses and development of molecular clones for gene transfer vectors replication-defective vector based on a chimpanzee adenovirus chimpanzee adenovirus vaccine protects against zaire ebola virus efficient flpe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination high-capacity, helperdependent, 'gutless' adenoviral vectors for gene transfer into brain regulatable gutless adenovirus vectors sustain inducible transgene expression in the brain in the presence of an immune response against adenoviruses immunization against the transgene but not the teton switch reduces expression from gutless adenoviral vectors in the brain comparison of replicationcompetent, first generation, and helper-dependent adenoviral vaccines protection against mucosal shiv challenge by peptide and helper-dependent adenovirus vaccines effects of shielding adenoviral vectors with polyethylene glycol (peg) on vector-specific and vaccine-mediated immune responses pegylated adenovirus for targeted gene therapy development of pegylated adenovirus vector with targeting ligand neutralizing antibody evasion ability of adenovirus vector induced by the bioconjugation of methoxypolyethylene glycol succinimidyl propionate (mpeg-spa) pegylated adenovirus vectors containing rgd peptides on the tip of peg show high transduction efficiency and antibody evasion ability evaluation of polyethylene glycol modification of first-generation and helper-dependent adenoviral vectors to reduce innate immune responses pegylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile vaccination against hepatitis c virus with dendritic cells transduced with an adenovirus encoding ns3 protein cell vehicle targeting strategies current strategies and future directions for eluding adenoviral vector immunity factors influencing therapeutic efficacy and the host immune response to helper-dependent adenoviral gene therapy in hemophilia a mice elimination of innate immune responses and liver inflammation by pegylation of adenoviral vectors and methylprednisolone administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons baseline ad5 serostatus does not predict ad5 hiv vaccine-induced expansion of adenovirusspecific cd4+ t cells translational mini-review series on vaccines for hiv: t lymphocyte trafficking and vaccine-elicited mucosal immunity route of adenovirus-based hiv-1 vaccine delivery impacts the phenotype and trafficking of vaccine-elicited cd8+ t lymphocytes differential specificity and immunogenicity of adenovirus type 5 neutralizing antibodies elicited by natural infection or immunization distribution of poliovirus antibody in serum, nasopharynx and alimentary tract following segmental immunization of lower alimentary tract with poliovaccine local antibody response to experimental poliovirus infection in the central nervous system of rhesus monkeys strategies for optimizing targeting and delivery of mucosal hiv vaccines primary hiv-1 infection is associated with preferential depletion of cd4+ t lymphocytes from effector sites in the gastrointestinal tract cd4+ t cell depletion during all stages of hiv disease occurs predominantly in the gastrointestinal tract mucosal aids vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques impact of vaccineinduced mucosal high-avidity cd8+ ctls in delay of aids viral dissemination from mucosa a novel functional ctl avidity/activity compartmentalization to the site of mucosal immunization contributes to protection of macaques against simian/human immunodeficiency viral depletion of mucosal cd4+ t cells intranasal immunization is superior to vaginal, gastric, or rectal immunization for the induction of systemic and mucosal anti-hiv antibody responses prophylactic vaccine for hiv-1 comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women nucleocapsid protein zinc-finger mutants of simian immunodeficiency virus strain mne produce virions that are replication defective in vitro and in vivo an internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface broadly neutralizing monoclonal antibodies 2f5 and 4e10 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus shivba-l immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees long-term protection of chimpanzees against high-dose hiv-1 challenge induced by immunization protection against mucosal simian immunodeficiency virus siv(mac251) challenge by using replicating adenovirus-siv multigene vaccine priming and subunit boosting evaluation of combination dna/replication-competent ad-siv recombinant immunization regimens in rhesus macaques novel adenovirus vaccine vectors based on the enteric-tropic serotype 41 human viral gastroenteritis unique physicochemical properties of human enteric ad41 responsible for its survival and replication in the gastrointestinal tract human enteric adenovirus type 41 (tak) contains a second fiber protein gene adenovirus-based primeboost immunization for rapid vaccination against anthrax development of a preventive vaccine for ebola virus infection in primates adenoviral expression of a truncated s1 subunit of sars-cov spike protein results in specific humoral immune responses against sars-cov in rats adenoviruses as vectors for hiv vaccines characterization of a permissive epitope insertion site in adenovirus hexon challenges for structure-based hiv vaccine design designing immunogens to elicit broadly neutralizing antibodies to the hiv-1 envelope glycoprotein rational antibody-based hiv-1 vaccine design: current approaches and future directions molecular architecture of native hiv-1 gp120 trimers structural definition of a conserved neutralization epitope on hiv-1 gp120 one step forwards, one step back prospects for vaccine protection against hiv-1 infection and aids modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses protection against p. aeruginosa with an adenovirus vector containing an oprf epitope in the capsid optimization of capsid-incorporated antigens for a novel adenovirus vaccine approach epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity expression of a foreign epitope on the surface of the adenovirus hexon identification of sites in adenovirus hexon for foreign peptide incorporation structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods protective immunity to pseudomonas aeruginosa induced with a capsid-modified adenovirus expressing p. aeruginosa oprf rgd inclusion in the hexon monomer provides adenovirus type 5-based vectors with a fiber knob-independent pathway for infection adenovirus type 5 with modified hexons induces robust transgene-specific immune responses in mice with pre-existing immunity against adenovirus type 5 structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2f5 in complex with its gp41 epitope genetic incorporation of a herpes simplex virus type 1 thymidine kinase and firefly luciferase fusion into the adenovirus protein ix for functional display on the virion derivation of a triple mosaic adenovirus based on modification of the minor capsid protein ix genetic incorporation of hsv-1 thymidine kinase into the adenovirus protein ix for functional display on the virion hiv antigen incorporation within adenovirus hexon hypervariable 2 for a novel hiv vaccine approach use of random systematic mutagenesis to generate viable human rhinovirus 14 chimeras displaying human immunodeficiency virus type 1 v3 loop sequences drug design, development and therapy the asian epidemic model: a process model for exploring hiv policy and programme alternatives in asia hiv/aids prevention in thailand: success and challenges the epidemiology of hiv infection and aids in thailand preventive hiv vaccine development in thailand impact of thailand's hiv-control programme as indicated by the decline of sexually transmitted diseases risk factors for hiv infection among young adult men in northern thailand hiv/aids preventive vaccine 'prime-boost' phase iii trial: foundations and initial lessons learned from thailand grant support was provided by national institutes of health grants 1r33ai076096-01, 2p30ai027767-21a1, and 3p30ca013148-38s9. we thank erin e thacker for her thoughtful insight. the authors report no conflicts of interest in this work. submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal drug design, development and therapy is an international, peerreviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are a feature of the journal, which has also been accepted for indexing on pubmed central. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord-318272-spt0oea0 authors: bhardwaj, prateek; bhatia, eshant; sharma, shivam; ahamad, nadim; banerjee, rinti title: advancements in prophylactic and therapeutic nanovaccines date: 2020-04-05 journal: acta biomater doi: 10.1016/j.actbio.2020.03.020 sha: doc_id: 318272 cord_uid: spt0oea0 vaccines activate suitable immune responses to fight against diseases but can possess limitations such as compromised efficacy and immunogenic responses, poor stability, and requirement of adherence to multiple doses. ‘nanovaccines’ have been explored to elicit a strong immune response with the advantages of nano-sized range, high antigen loading, enhanced immunogenicity, controlled antigen presentation, more retention in lymph nodes and promote patient compliance by a lower frequency of dosing. various types of nanoparticles with diverse pathogenic or foreign antigens can help to overcome immunotolerance and alleviate the need of booster doses as required with conventional vaccines. nanovaccines have the potential to induce both cell-mediated and antibody-mediated immunity and can render long-lasting immunogenic memory. with such properties, nanovaccines have shown high potential for the prevention of infectious diseases such as acquired immunodeficiency syndrome (aids), malaria, tuberculosis, influenza, and cancer. their therapeutic potential has also been explored in the treatment of cancer. the various kinds of nanomaterials used for vaccine development and their effects on immune system activation have been discussed with special relevance to their implications in various pathological conditions. statement of significance: interaction of nanoparticles with the immune system has opened multiple avenues to combat a variety of infectious and non-infectious pathological conditions. limitations of conventional vaccines have paved the path for nanomedicine associated benefits with a hope of producing effective nanovaccines. this review highlights the role of different types of nanovaccines and the role of nanoparticles in modulating the immune response of vaccines. the applications of nanovaccines in infectious and non-infectious diseases like malaria, tuberculosis, aids, influenza, and cancers have been discussed. it will help the readers develop an understanding of mechanisms of immune activation by nanovaccines and design appropriate strategies for novel nanovaccines. since the advent of the first vaccine centuries ago, researchers have been trying to find the answer of most common pandemic diseases through immunotherapy. vaccination has successfully managed to eradicate a lot of prevalent diseases, but associated off-target responses, lack of prolonged protection across variable pathogenic strains and allergy, limits its horizon for the prevention or treatment of other globally prevalent diseases like tuberculosis, aids, malaria, influenza and cancer. though prophylactic vaccines use live-attenuated or inactivated pathogens, lack of response and protection against diverse pathogenic strains [2] . in addition, nanoparticles have the ability to impart more stability with high antigen loading and less proteasomal degradation of antigenic subunits. small and more specific subunits often tend to be less immunogenic, which can be overcome by the use of adjuvants that produce co-immunostimulatory or immunomodulatory signals. side effects associated with such conventional adjuvants with individual-specific response, immunotolerance to the target antigen and unwanted reactions towards self-antigens limit their use [3] . therefore, biocompatible nanoparticles can also be directly used as adjuvants and may mitigate the need of strong conventional adjuvants (e.g., alum). another reason associated with low subunit immunogenicity is their low cellular internalization and rapid clearance from the body. nanoparticles with tunable physicochemical properties can overcome this limitation by enhancing circulation time, bio-accumulation in lymphoid organs and efficient targeting of immune cells. they can also increase crosspresentation by antigen-presenting cells (apcs) and activate the immune system at much lower doses. for instance, poly (lactic-coglycolic acid) (plga)-based nanoparticles enhanced the cross presentation of ovalbumin through major histocompatibility complex-i (mhc-i) in bone marrow derived primary dendritic cells due to which t cells were stimulated at 10 0 0-fold lower concentrations as compared to soluble antigen [4] . different kinds of nanoparticles (lipid-based, polymeric, inorganic and protein/peptide-based) have been extensively used as adjuvants, immunogens and antigen delivery vehicles for immune activation [5] . most of the synthetic nanoparticles interact with apcs structurally to increase internalization rather than functionally as they lack specific binding locations for cell receptors. on the contrary, protein-based nanoparticles can interact both structurally and functionally as they can carry antigens as well as interact with the pattern associated receptors on apcs [6] . nanovaccines have the ability to induce both the components of human adaptive immunity i.e. cell-mediated as well as antibody-mediated immunity with induction of memory response. prolonged release of antigens from nanoparticle depots can cause enhanced stimulation for a long period of time, alleviating the need for frequent booster doses and therefore, nanovaccines can be used as both prophylactic or therapeutic which involves its administration before or after the occurrence of the disease respectively. however, the safety, biodistribution and residence of the nanoparticles must be optimized to obtain preferred immune responses towards nanoparticle-based vaccines. in this review, the potential and pros and cons of nanoparticlebased vaccines are explored. the role of different nanovaccines in activating various arms of immunity with an intent to abate the use of frequent booster doses as vaccines for tuberculosis, malaria, hiv (human immunodeficiency virus), influenza, and cancer are discussed. the applications and advantages of nanovaccines against various infectious and non-infectious diseases are also further delineated in this article. the ultimate goal for vaccination is to generate safe and efficient primary as well as secondary immune responses in the body. in a simplified way, primary immune responses protect the body from potential damages that might occur on first exposure of the pathogen or antigen. on the other hand, secondary immune responses, which are comparatively rapid and stronger, are elicited depending on the immunological memory generated during first exposure and protect the body from future encounters of the same epitope [7] . nanoparticle-based vaccine delivery emerged as an appealing platform for boosting both primary as well as secondary immune responses in the body [8 , 9] . there are multiple mechanisms by which nanovaccines help enhancing immune responses. nanovaccines facilitate uptake of antigens in dendritic cells (dcs), which can further be enhanced by decorating the particle surface with ligands that selectively recognize receptors on dcs (refer fig. 1 ). for example, ligands including fc fragment, mannose and anti-dec-205 (mab) have been widely explored which selectively binds fc receptors, mannose receptors and dec-205 receptors respectively on dcs [10 , 11] . in addition to efficient internalization, ability to co-deliver multiple-antigens and adjuvants, inherent adjuvant property, rapid lymph node drainage and efficient antigen presentation on dc's surface are other predominant mechanisms by which nanovaccines help increase the duration as well as the magnitude of the immune responses in immunized animals [12 , 13] . interestingly, these properties of nanoparticles have been substantially engineered to achieve desirable immune response for developing either prophylactic or therapeutic vaccines [9] . for designing a safe and effective nanovaccine, it is imperative to understand the mechanism by which the nanovaccine activates both innate and adaptive immunity in the body [14] . prophylactic nanovaccines are generally administered subcutaneously, intramuscularly, or intranasally whereas therapeutic vaccines (e.g. for cancer treatment) are injected intravenously. depending on the route of immunization, nanovaccines first encounter immune cells including neutrophils, macrophages, dcs and natural killer (nk) cells which quickly recognize the nanovaccine (foreign particle) based on the pathogen associated molecular patterns (pamps) associated with them [15] . pamps (e.g. bacterial lps, teichoic acid, lectins, oligonucleotides or material from the carrier nanoparticle etc.) serve as the ligands for prrs [e.g. toll-like receptors (tlrs), c-type lectin receptors (clrs), retinoic acid-inducible gene-i -like receptors (rlrs) etc.] abundantly present on these cells [16 , 17] . thus, interaction of pamps with prr trigger endocytosis of larger particles (usually > 500 nm) preferentially by macrophages and smaller particles by dcs. engulfment of nanovaccines by macrophages results in gradual degradation of nanovaccine particles and the encapsulated antigens. to prevent this, nanovaccine particles have been engineered to survive the attack by macrophages and facilitate direct delivery to apcs [18 , 19] . additionally, depending on the type of pamps, neutrophils and macrophage secrete a variety of cytokines and chemokines which further activate apcs. besides pamps, there are several unknown mechanisms which can activate innate immunity for example, induction of immune responses (potential adjuvant effect) by glycolic acid component of plga co-polymer [20] or by cationic liposomes [21] . eventually, the secreted cytokines and chemokines stimulate maturation and activation of apcs (mainly dcs) which initiate strong adaptive immune response (refer fig. 1 ). therapeutic nanovaccines developed for treatment of metastatic cancer directly target dcs for strengthening adaptive immune responses in body [22] . activated dcs serve as the link between innate and adaptive immunity. adaptive immunity generally has two arms; cell-mediated and antibody-mediated immunity. adaptive immune responses are generated within few days to several weeks and last longer. essentially, both types of immunity are necessary for a protective and prolonged immune response. however, their requirement is different in both prophylactic and therapeutic nanovaccines. for instance, strong cell-mediated cytotoxic responses are more desirable in case of therapeutic nanovaccine in order to effectively kill the metastatic cancer cells [23] . vaccination provokes cell-mediated immunity generated by b cells and t cells which serve to neutralize the pathogen/antigen and simultaneously stimulating the body for developing immunological memory [24] . the cmi responses are triggered by migration of activated dcs (following uptake of nanovaccine particle) to lymphatic organs (spleen and lymph noted). activated dcs present their antigens via mhc class i receptor to cd8 + cytotoxic t lymphocytes (ctls) (refer fig. 1 ). activated ctls elicit strong cmi and selectively kill target cells (infected altered cells or cancerous cell) by inducing apoptosis in them [20] . killing target cells is a complex immunological process that attracts several compliment systems (cs) proteins and co-stimulatory molecules. therapeutic cancer nanovaccines are preferentially designed to strengthen ctls responses by ex vivo educating dcs via adoptive transfer of antigen (for example, sipuleucel-t, a therapeutic vaccine approved for treatment of metastatic contraction resistant prostate cancer) [25] . on other hand, activated dcs also present antigens to cd4 + t-helper cells (th cells) via mhc-ii receptor. subsequently, effector th cells undergo activation and maturation leading to secretion of variety of cytokines signals (refer fig. 1 ). depending on the type of cytokine secreted, th populations are divided into th1 cells and th2 cells subsets [7] . th1 subset majorly secretes proinflammatory cytokines (e.g. inf-γ , tnf-α, il-1) which stimulates proliferation of ctls and strengthen the function of cmi. th2 subset secrete class of cytokines (e.g. il-4, il-5, il-6, il-9, il-10 and il-13) which stimulate b cell proliferation during antibody-mediated immune responses [7 , 26] . a subtle balance between th1/th2 responses determines overall therapeutic or prophylactic vaccine potential of candidate nanovaccine [27] . in some cases, nanovaccines also work by suppressing t-regulatory (treg) cells, which naturally suppress activation and proliferation of effector t cells in the body [28] . depending on the potential of nanovaccines to generate strong th2 cytokine response, naïve b cells located in spleen and lymph nodes get stimulated and bind to soluble antigen via b cell receptors (bcrs). activated b cell population undergoes proliferation in the germinal center. further, by somatic hyper mutation, b cells becomes specific to particular epitope of an antigen and then only epitope-specific b cells are selectively (clonal selection) proliferated. at this stage, activated b cells either differentiate into antibody-secreting plasma cells and secrete soluble antibodies against target antigen or remain dormant as memory cells for a future encounter of the same antigen (refer fig. 1 ) [29] . plasma cells are short-lived and their number gradually declines over time, as do the corresponding antibody titers [30] . in that case, memory cells (b cells and t cells) which were generated and stored in the lymphatic organs or bone marrow for several months, come into play and protect the body from reinfection of the same antigen. in such situations, memory b cells quickly proliferate and turn to antibody-secreting (mainly igg) plasma cells to neutralize the antigen. on the other hand, memory t cells (both types cd4 + and cd8 + ) serve to produce more cytokine and chemokine signals to stimulate enhanced cmi and antibody-mediated responses. however, in case the structure of the antigen (epitope) has undergone significant changes due to cleavage, aggregation or re-folding, even memory cells cannot provide the required protection [31] . although, in some cases, primary immunization can offer up to 90% protection, however, as even the rest 10% can be disastrous, therefore booster doses are scheduled to increase protection up to 100% [32] . booster doses promote generating of more population and a variety of memory cells. generally, booster doses are scheduled after the antibody titer from plasma cells has dramatically declined to allow more competitive binding of memory cells to the injected antigen [33 , 34] . in a re-lated context, the use of nanoparticles help recalling more robust immunological memory even at a low dose [35] . nanoparticles (e.g. plga based nanovaccine) facilitate prolonged availability of intact antigen in blood due to sustained release that leads to a higher proliferation of b cells and hence more memory cells [36 , 37] . also due to small size, nanoparticles can travel through the narrow lymphatic system to lymph nodes inducing differentiation of more memory cells [38] . for example, demento et al., reported that over alum-based (free antigen) or liposome-based (burst release) carriers, mice immunized with ova-expressing listeria monocytogenes (lm-ova) encapsulated in plga particles (sustained-release) showed the highest protection against a lethal dose of l. monocytogenes even after 13 weeks post immunization. they observed that mice immunized with antigen in plga nanoparticles had a robust population of cd8 + t memory cells [36] . similarly, kanchan et al., showed that encapsulation of tetanus toxoid (tt) inside poly(lactic acid) (pla) particles generated long lasting primary antibody response in mice and interestingly even after 18 months re-exposing the animal using very low antigen dose induced heightened and robust b cell responses [39] . nanomaterials can be efficiently used for designing nanovaccines for the enhancement of immune-modulation. based on their potential use, they can be employed as adjuvants, immunogens or nanocarriers for enhanced and prolonged antigenic delivery. these properties of a nanoparticle are strongly governed by its size, shape, composition, surface chemistry and route of administration. it plays an important role in deciding its capability to induce inflammatory response or expression of specific defenserelated genes. different types of nanoparticles like protein, lipid, polymer, or inorganic particles (such as gold, iron, carbon and silica nanoparticles) can induce immune stimulation and have been discussed below [40] . adjuvants are used with vaccines to enhance their potency by combination of mechanisms. adjuvants activate innate immune responses that shape and trigger the adaptive immune responses to vaccines [41] . traditionally used adjuvants such as alum, complete freund's adjuvant and as04 (combination of alum and monophosphoryl lipid a) are known to form local depots that induce secretion of cytokines, chemokines and recruitment of immune cells. absorption of antigen on surface of adjuvants enhances antigen uptake and presentation, thus promoting antigen transport to draining lymph nodes [42] . activation of tlrs has been potentially used for designing vaccine adjuvants. tlr ligands or agonists have served as potential immunostimulatory molecules as tlrs activate innate immune responses by identifying pamps and down signaling the transcription factors responsible for immune responses such as nf-κb, irfs and lymphocyte activation [43] . the co-delivery of antigen along with tlr ligands promotes antigen presentation to cd4 + t cells via mhc-ii signaling and crosspresentation to activate cd8 + t cells [44] . tlr agonist such as cpg up-regulates expression of cd40, cd54, cd80, cd86, and mhc class ii molecules thus enhancing antigen processing and presentation in plasmacytoid dcs [45] . nanovaccines generate amplified, specific and robust responses as compared to traditional vaccines despite having similar mechanism of immune activation. the exaggerated immune response generated by nanoparticle associated antigen is due to their ability to specifically target dendritic cells with enhanced cellular uptake and antigen presentation. moreover, nanoparticles can be used as adjuvants with specific antigens or as immunogen themselves. vaccine antigens are either encapsulated inside or surface decorated over nanoparticles to get delivered efficiently (refer fig. 2 ). there are chances that free antigens may degrade and elicit local immune responses at the site of administration, therefore, encapsulating free antigen inside nanoparticles prevents their degradation and provides prolonged and controlled release at the target site. the controlled release of antigen prevents burst responses and eliminates the need of booster doses. surface decoration of nanoparticles with antigen also confers the advantage of presenting the antigen to cells in a similar way as presented by pathogens [46] . polyanhydride-based nanoparticles encapsulating f1-v antigen when administered intranasally induced an immune response that persisted for 23 weeks and elicited a high anti-f1-v igg1 antibody response post-vaccination and conferred long-lived protective immunity against yersinia pestis infections compared to recombinant f1-v antigen [47] . similar nanoparticles were functionalized with di-mannose targeted macrophage mannose receptor (mmr). these carbohydrate functionalized nanoparticles enabled sustained release of immunogen for 30 days and enhanced the uptake of antigen by dendritic cells simultaneously upregulating the co-stimulatory molecule clr, cd206, and cd40 in cells as compared to non-functionalized nanoparticles [48] . apart from encapsulation, several other strategies have been exploited for the co-delivery of antigens and nanoparticle such as surface absorption, conjugation, and mixture (refer fig. 2 ). nevagi et al., observed enhanced immune activation against gas (group a streptococcus ) antigen using tri-methyl chitosan (tmc) nanoparticle conjugated with b cell and t cell epitope as compared to antigen mixed with tmc [49] . nanoparticles have also shown potential to be used as adjuvants because of their immunomodulating activities such as inflammasome activation, complement system activation and recruitment of immune cells [50] . due to above-mentioned effects, nanoparticles act as a better adjuvant in comparison to complete freund's adjuvant and conventional aluminum containing salts by inducing higher immunogenic responses at low antigen doses [51] . also, their small size facilitates internalization by dendritic cells and induces adaptive immune responses. studies by swaminathan et al., have proved enhanced immune responses by lipid nanoparticle (lnp) adjuvant formulations against ovalbumin (ova) and hepatitis b virus surface antigen (hbsag) in balb/c and c57bl/6 mice injected with hbsag along with lnps as compared to hbsag alone [52] . co-administration of quarternized chitosan nanoparticles as adjuvants along with ovalbumin intranasally in female balb/c mice induced activation of apcs followed by enhanced lymphocyte proliferation and differentiation as compared to ova mixed with aluminum hydroxide gel [53] . asgary et al., also reported that silver nanoparticles showed increased antibodymediated responses against cvs-11 rabies and reduced toxicity as compared to conventionally used adjuvants such as alum [54] . although nanoparticles possess great adjuvant properties, their codelivery along with generally used adjuvants such as tlr agonists can provide very robust immune protection. the use of tlr agonists is restricted because of their instability, particularly while using rna as tlr agonists. nugyen et al., have designed lipidoids to effectively deliver immunostimulatory rna to activate innate and adaptive immune responses. these lipid-rna nanoparticles enhanced ifn-γ responses and anti-ova cell-mediated and antibodymediated responses. these lipidoids were superior to the conventionally used n-[1-(2,3-dioleoyloxy) propyl]-n,n,n-tri-methyl ammonium methyl sulfate-rna delivery system [55] . similarly unmethylated cpg-rich oligodeoxynucleotides (cpg), a tlr agonist. these nanoparticles induced pro inflammatory responses and antigen-specific antibody and cell-mediated immunity. colocalization of mpla and cpg also induced functional memory cd8 + cells. these findings clearly demonstrated the ability of bacterial elements and tlr agonists towards developing a more effective vaccine design [56] . in addition, tlr agonists have been used in designing disease-specific nanovaccines, which are discussed later in this review. layer by layer protein nanoclusters synthesized from recombinant trimeric hemagglutinin were immunogenic by themselves and induced protective antibody-mediated responses without the use of adjuvants. such high immunogenic potential of peptide nanoclusters is because of the high antigen density available on the surface of nanoparticles [57] . various types of nanoparticles have been used for nanovaccine formulation and each type serves a different function, which has been elaborated in the next section. protein-based nanovaccine can serve both structural as well as functional purpose by carrying antigens and engaging pattern recognition receptors of apcs. the protein-based nanovaccine can be categorized as biomimetic (virus-like particles, cages, vaults), rationally designed (nanofibrils, self-assembled protein nanoparticles, nanoclusters) and micelles. biomimetic protein-based nanovaccines : biomimetic proteinbased nanovaccines are of viral origin with self-assembled viral capsid proteins devoid of genetic material and are categorized as virus-like particles (vlps) (refer fig. 3 (e) ). on the other hand, those of prokaryotic or eukaryotic origin are called protein cages and vaults. cages and vaults are generally used for biocatalysis or molecular transport. they share common properties with vlps like nanoscale, symmetry and container like geometries. all these structures can be modified chemically or biologically with antigens for immune activation [6] . vlps (60-80 nm) of salmonella typhimurium bacteriophage p22 were engineered to encapsulate two respiratory syncytial virus (rsv) protein antigens, matrix (m) and matrix 2 (m2). mice vaccination and intranasal boosting showed antigen-specific cd4 + and cd8 + t cell responses. upon subsequent rsv challenge, 10 0 0-fold reduction in viral load and increased antigen-specific cd8 + t cells were observed in the lungs of vaccinated mice as compared to empty p22 vaccinated and unvaccinated animals [58] . cages like 25 nm e2 cage derived from the pyruvate dehydrogenase complex of bacillus stearothermophilus [59] , 13 nm cage of human heavy chain ferritin (an iron storage protein) [60] , ~24 nm encapsulins (class of icosahedral cage structures) of bacteria and archaea are used for the presentation of different antigens to elicit immune response after immunization [61] . a recent study on nonviral e2 protein-based biomimetic nanoparticle-containing acidlabile cpg (dc activating molecule) and siinfekl peptide epitope conjugate has shown the potential to enhance the cpg mediated dc activation by 25-fold in vitro as compared to unbound cpg. interestingly, co-delivery of siinfekl peptide conjugated with cpg has shown elevated cross-presentation and cd8 + t cell activation by inducing a 3-fold greater display of siinfekl on mhc-i by dcs as compared to unbound peptide or peptide bound directly to e2 protein. this shows the ability of biomimetic protein-based nanoparticles to facilitate enhanced immune response by dc activation and cross-presentation [62] . vaults are eukaryotic ribonucleoproteins assembly of a cage-like barrel-shaped structure (approximately 70 nm × 40 nm × 40 nm) made from the major vault protein (mvp) [63] . it has been shown that increased antigen-specific cd4 + t cell response with a reduction in genital bacterial load and inflammation was observed after chlamydia muridarum polymorphic membrane protein g-1(pmpg) encapsulating vaults were administered intranasally [64] . rationally designed protein-based nanovaccines: nanofibrils are generally made of engineered β-sheets or α-helical coiled coils. β-sheet nanofibrils can be formed with peptide sequences like q11 (qqkfqfqfeqq) and kfe8 (fkfefkfe) appended to c terminus of a peptide antigen with a short linker sequence (refer fig. 3 (d)). similarly, α-helical coiled-coil can also be formed into fibrils from α-helical peptide-like coil29 [65] . such peptide sequences undergo assembly into fibrils with the antigenic epitope sticking outward from the fibril surface [6] . these fibrils have been assembled with various epitopes of plasmodium falciparum [66] , mycobacterium tuberculosis [67] , streptococcus aureus [68] , t-helper cells epitope padre [68] and ova epitopes [69] . conjugation of e2ep3 immunogenic epitope of chikungunya virus e2 glycoprotein with β-sheet self-adjuvanted amyloid assemblies showed cytocompatibility with enhanced macrophage uptake and robust igg response against e2ep3 epitope [70] . self-assembling protein nanoparticles (sapns) are 20-30 nm icosahedrons containing pentameric and trimeric helical coiledcoils conjugated with (glycine) 2 linker (refer fig. 3 (f)). antigens and adjuvants can be inserted depending on the expected structure from in silico modeling [71] . subunit malaria [72] , hiv [73] , toxoplasma [74] and severe acute respiratory syndrome [75] . wahome et al., encapsulated two hiv protein epitopes (4e10 and 2f5) onto sapn surface and observed enhanced production of epitope-specific neutralizing antibodies. this indicates the potential of sapn as nanovaccine to activate immune response against hiv [73] . nanoclusters have also emerged as a prominent vaccine platform prepared from desolvation of proteins and peptides. they are generally made up of antigens and crosslinkers with an intent to increase antigen loading and eliminate off-target sequences. intranasal vaccination of influenza matrix protein 2 (m2e) containing self-assembled protein nanoclusters elicited enhanced immune response and complete protection after lethal challenge with hetero and homo-subtypic virus [76] . since the preparation of nanoclusters requires organic solvents, it may alter the secondary or quaternary structure of full protein antigens [57] . micelles: antigenic molecules can be conjugated to micelles in two different ways. either they can be covalently linked onto the micellar surface or can be conjugated to the variety of alkyl tails to form amphiphilic peptide micelles (pams). micelles of different sizes and shapes affect their immune activation potential [77] . studies have shown that cylindrical or spherical pams are efficiently able to reach lymph nodes and carry the required amount of antigen and adjuvant [78] . studies have reported pam mediated increased immunogenicity of t cells [79] and b cells epitopes [80] . such peptide lipid conjugates also form nano-disc vaccines (refer fig. 3 (g)). subcutaneous vaccination with neo-antigen adpgk nanodiscs showed enhanced cd8 + cellular response, apc uptake and increase survival after b16f10 melanoma challenge as compared to intramuscular vaccination [81] . peptide-polymeric micelles have also been explored as a vaccine platform by conjugating hydrophilic peptide antigen with dendrimer polymers for immunotherapy [24] . the most common lipid-based nanovaccines getting explored from the past few decades are liposomal nanovaccines (refer fig. 3 (b) ). they offer a distinct advantage over other nanovaccine systems in terms of being biodegradable, tolerable, less immunogenic and having tunable physicochemical properties. they provide a wide repertoire to load antigens of different philicity inside and outside of liposomes with easy surface conjugation [82] . different physicochemical properties (like size [83] , surface charge [84] , surface modifications (peg) [85] , bilayer composition and fluidity [86] ) affect the immunological outcome of liposomal nanovaccines. size-dependent differential activation of immune response indicated the activation of th2 response by 100 nm sized liposomes as compared to th1 response by liposomes ≥400 nm [83] . transdermal immunization of mice with dissolving microneedles loaded with opposite charged transferosomes indicated the enhancement of th1 immune response by cationic transferosomes as compared to anionic counterparts by exhibiting strong endocytic escape property with fecilitating antigen processing via mhc-i pathway and larger accumulation in lymph nodes [87] . kaur et al., showed the effect of bilayer fluidity on immune activation by varying cholesterol concentrations. relatively lesser in vivo igg production and ifn-γ was observed by liposomal formulations containing high cholesterol [85] . similarly, the effect of another important factor affecting bilayer fluidity on liposomal mediated immune activation was observed by preparing liposomes with phospholipids having different transition temperatures (tm). high tm phospholipids containing liposomes stimulated th1 immune response as compared to low tm phospholipids that induced th2 response in mice immunized with leishmaniasis rgp63 antigen [86] . numerous clinical studies showed the use of liposomes as adjuvants in therapeutic vaccines. liposome encapsulated d. pteronyssinus vaccination reduced allergen bronchial provocation induced inflammatory changes and improved condition of asthma patient after sustained mite exposure [89] . a phase iii study of tecetomide vaccine, containing blp lipopeptide antigen incorporated into dmpg:dppc:chol:mpl-liposomes was performed for stage iii non-small cell lung cancer after subcutaneous immunization (nct01015443) [90] . however, the study was terminated due to no effect on survival and primary or secondary endpoint in a phase ii study [91] . a phase i trial (nct00922363) showed safety and immunogenicity of caf01 (liposomal adjuvant system) given with a subunit vaccine against m. tuberculosis antigen h1. this adjuvanted vaccine showed long-lasting t cell response with no antibody response observed in healthy adults [92] . viral liposomes called "virosomes" have also been studied in the literature. virosome exhibits viral capsid proteins onto the liposomal surface for effective fusing with the target cell membrane. recently, virosomes and liposomes mediated immune response was investigated on human respiratory tract triple culture model. both virosomes and liposomes were prepared from the same neutral phospholipids, dopc (1,2-dioleoyl-sn-glycero-3-phosphocholine) and pope (1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine), but virosomes were also fused with influenza membrane protein. results showed enhanced internalization of virosomes in epithelial cells of triple culture as compared to liposomes of similar sizes [93] . a recent study has shown the use of lipid-based 40-60 nm particles (iscomatrix particles) as adjuvant with chimera peptide vaccine containing gp21, tax, gp46 and gag epitopes of human t cell lymphotropic virus type 1 when given subcutaneously in mice. enhanced production of mucosal iga and igg2a antibody titers along with ifn-γ and il-10 cytokines were observed as compared to vaccine formulation [94] . another class of lipid-based nanocarrier includes 'hexosomes' and 'cubosomes' that can provide a high membrane surface area for antigen and membrane protein loading. hexosomes are generally made up of lipids known to form non-lamellar structure in hydrated systems with inverse hexagonal liquid crystalline phase. comparative adjuvant activity of mono mycoloyl glycerol-1 (mmg-1) immunopotentiator containing hexosomes and cationic liposomes (caf04) loaded with chlamydia trachomatis major outer membrane protein (momp) antigen in cb6/f1 mice after vaccination was studied by rodrigues et al. mmg-1 hexosomes made up of lipid phytantriol induced a strong momp-specific antibodymediated immune response whereas mmg-1 cationic liposomes elicited much stronger momp-specific cell-mediated response. it indicated the immune activation by two lipid-based adjuvants via different mechanisms [95] . in addition, cubosomes are generally made up of lipid bilayers forming continuous periodic cubic lattice structures with interwoven water channels and a stabilizing polymeric outer corona [96] . phytantriol, pluronic f127 and propylene glycol-based cubosome formulation containing (imiquimod and monophosphoryl lipid a (mpl)) adjuvants and model ova antigen elicited robust ifn-γ production and cd4 + and cd8 + t cell proliferation as compared to similar adjuvant containing liposomal formulation and alum [97] . such lipid-based self-assembled structures can provide high antigen and adjuvant loading with enhanced immunoactivation in comparison to conventional lipidbased carriers like liposomes. polymeric particles showed a great possibility to be used as nanovaccines due to their ease of preparation, biocompatibility and biodegradability, fine-tuning of surface properties and controlled release kinetics (refer fig. 3 (a) ) [98] . the most commonly used polymeric nanoparticle for antigen delivery is poly (lactic-coglycolic acid) plga or poly (lactic acid) pla that has been used to deliver a broad range of antigens like hepatitis b virus antigen [99] , tetanus toxoid [100] , bacillus anthracis antigen [101] , mycobacterium tuberculosis antigen [102] and ovalbumin [36] . other natural polymers like chitosan [103] , alginate [104] , pullulans [105] , and inulin [106] have also been used as adjuvants and antigen carriers. chitosan alginate-based nanovaccines for hepatitis b virus [103] and dna-based chitosan vaccines for mycobacterium tuberculosis ( mtb h37rv ) showed a marked increase in immunologic and protective efficacy in immunized mice [107] . hyperbranched polyglycerol (hbpg) globular polymer can be a potential candidate for inert dendrimer based multi-valent vaccine coupled with b cell epitope for tumor-associated antigen (muc1) and t cell epitope for tetanus toxin p2. it showed enhanced immune response and igg antibodies against breast cancer cells. hence polymeric nanoparticles can be used for potential multivalent vaccines [108] . several inorganic nanoparticles (gold, iron, carbon, silica nanoparticles) are being explored for vaccine delivery in different disease models (refer fig. 3 (c) ). ease of antigen functionalization onto easily accessible surface groups, robust properties with reproducibility outweighs their low biodegradability and gives them an edge to be used as nanovaccines. they also increase antigen stability by preventing premature proteolytic degradation. however, there are reports showing dose and size-dependent clinical toxicities of these nanoparticles [109] . amongst all the inorganic nanoparticles, gold nanoparticles have gained maximum attention in vaccine delivery. these particles have been used for the epitope delivery against hiv [110] , influenza [111] , malaria [112] and tumor [113] . effect of shape of gold nanovaccine on antibody production against wnv envelope e protein showed that rods induced only 50% of the antibody response as compared to spherical nanoparticles [114] . multicopy multivalent nano-glycoconjugates of gold nanoparticles decorated with tn-antigen glycan generated strong long-lasting antibodies against breast cancer expressing aberrant mucin glycan [115] . inorganic carbon spherical nanoparticles and nanotubes were used as adjuvants to increase immunogenicity and act as delivery vehicles for peptides and protein against various viral infections [116 , 117] . oral immunization of bovine serum albumin (bsa) loaded carbon nanoparticles showed effective stimulation of mucosal iga antibodies in intestinal, vaginal and salivary mucosa along with th1 and th2 responses [116] . abundance of silanol groups over silica nanoparticle surface provides an advantage for easy conjugation of targeting moiety for various viral infections [118] . silica vesicles adsorbing e2 glycoprotein of bovine viral diarrhea virus (bvdv) showed 6-month antibody-mediated and cellmediated immune response with no histopathological changes at the site of infection [119] . the use of magnetic iron oxide nanoparticles for tumor-associated carbohydrate antigen (taca) through phospholipid functionalization of taca glycopeptide showed enhanced antibody titers against both human and mouse tumor cells expressing that glycopeptide [120] . calcium nanoparticles have also been reported for their superior adjuvant property with dna vaccines and to confer mucosal immunity. they can also induce enhanced antibody production against viral antigens as compared to aluminum adjuvants [121] . since different types of nanoparticles impart different properties to the developed nanovaccine, their vast use in immune activation against a variety of antigens have been observed. though clear demarcation or direct comparison across nanocarriers has not been studied but there are unique properties and advantages associated with each nanocarrier that gives them an edge over each other. biomimetic carriers like vlps are expected to possess inherent immunogenicity that can elicit antibody reaction against carrier antigens itself. this could lead to the competition with the target antigen and alter antibody repertoire or memory response against it. reactive toxicities or neutralization upon repeated dosing can also limit their multiple use in a patient [122] . such problems can be avoided by the use of nanoclusters that are solely made up of single type of antigen. these protein-based biomimetic and rationally designed nanocarriers are most suitable for the incorporation of a highly hydrophobic antigens of varied sizes with an intent to preserve its native structure, whereas hydrophilic peptides can be well delivered using amphiphilic micelles [6] . carrier specific immune response has also been observed for certain polymeric and lipid-based carriers but it can be avoided using natural or biomimetic components (e.g., cellular lipid isolates or extracellular matrix polymers) [6] . control over spatiotempotable 1 . different type of nanovaccines can escalate the immunogenic potential of antigens by boosting the immunogenic responses and memory generated against antigens at low doses. immunogenic responses generated against certain antigens can be used in two different scenarios by varying the use of vaccines as prophylactic or therapeutic (refer fig. 4 ) . prophylactic vaccines aim to develop protective immunity and are administered before the onset of disease. the goal of prophylactic vaccines is to develop immunogenic memory against certain infections. major challenge of prophylaxis is obtaining sufficiently long-lasting immunogenic memory. for instance, single-dose immunization of mice with polyanhydride nanoparticle containing pneumococcal surface protein a (pspa), a virulence factor of s. pneumonia activated protective immunity and enhanced the survival of animals upon challenge even at 25-fold reduction in dose. pspa based nanovaccine formulation performed comparable to protein adjuvanted with alum, with much less tissue reactivity at the site of immunization [123] . prophylactic nanovaccines confer protective immunity mostly against infectious conditions at low doses of immunostimulating antigen and reduced need for adjuvants, thus mitigating associated toxicities. therapeutic vaccines are administered after the onset of diseases to alter the course of disease by encouraging the immune system to fight harder against the prevailing conditions. unique immune responses are generated against disease-specific antigens. generally, therapeutic vaccines are used for cancer treatment, but there are compelling evidences suggesting their potential use in autoimmune disorders also. the advantage of using therapeutic vaccines instead of conventional pharmaceutical treatment lies in their specificity [124] . mono-specific, diseasephase ii [170] relevant pmhc complexes when coated on nanoparticle surfaces triggered the selective expansion of memory-like autoregulatory cd8 + t cells that arise only in affected individuals. such engineered nanoparticles have the potential to become suitable vaccines with the capacity of resolving organ and disease-specific autoimmune responses. systemic delivery of type-i diabetes relevant peptide-mhc-nanoparticle complex triggered expansion of memory autoregulatory t cells and suppressed the autoimmune attack against insulin-producing beta cells, thus restoring glucose balance [125] . in certain cases, nanovaccines can be formulated to achieve both prophylactic and therapeutic responses. poly ( γ -glutamic acid) based synthetic vaccine nanoparticles (svnps) loaded with ovalbumin and toll-like receptor 3 agonist (poly (i:c)) were synthesized by kim et al., both of them enhanced the uptake of antigens by apcs and enhanced the secretion of inflammatory cytokines (tnf-α, il-6) and type i interferon (ifn-α, ifn-β). simultaneous injection of both svnp-ova and svnp-ic conferred both the protective as well as therapeutic immunities by inhibiting tumor growth in eg7-ova tumor-bearing mice [126] . it is clear from the above-mentioned examples that nanovaccines modulate the immune system either for providing prophylaxis or therapeutic responses against pathological conditions. majorly prophylactic vaccines are used to prevent infections ( table 1 ) and therapeutic vaccines have been used for cancer ( table 2 ) treatment. infections that can be prevented by vaccination still contribute to approximately half of the child morbidity each year [127] .treatment and immunization of such infectious conditions is a greater challenge due to the rapid emergence of new pathogenic variants. in spite of advancements in vaccine development over the years, many vaccines are associated with the risk of regaining their pathogenicity under certain immuno-compromised conditions. so, there is an imperative need for the development of risk-free and effective vaccines that confers desired antibody-mediated and cellmediated immunity against infectious diseases [128] . subunit nanovaccine against enterohemorrhagic escherichia coli (ehec) was developed using gold nanoparticle conjugated with ehec antigens and induced high titers of serum igg upon subcutaneous administration in mice. it has also prevented colonization of ehec in both the cecum and large intestine at 3 days post-immunization [129] . mice immunized against anthrax with a single dose of pad4-np (plga-based protective antigen domain, 4 nanoparticles) elicited robust antibody-mediated and cell-mediated responses with mixed igg1/igg2a subtypes and th1/th2 response. the survival rate of mice immunized with pad4-np was more than for pad4 immunized mice after a lethal challenge with bacillus anthracis spores [101] . nanovaccines have also shown great prospects in mitigating parasitic invasion. chimeric peptides containing leishmania infantum epitopes (histone h1, kinetoplastid membrane protein 11 and cysteine peptidase a) were encapsulated in plga nanoparticles. stimulation with the peptide-based nanovaccine induced maturation of dcs and il-12 production subsequently promoting allogeneic t cell proliferation and production of ifn-γ by cd4 + and cd8 + t cell. on immunization of transgenic mice with this peptide-based nanovaccine induced peptide-specific ifn-γ producing cd8 + t cells and conferred protection against l. infantum infection [130] . multiple self-assembling peptide-based nanovaccines containing epitopes of toxoplasma gondii specific antigens have been designed. immunization with self-assembled protein nanoparticles activated cd8 + t cells to produce ifn-γ and protected transgenic mice against subsequent challenge with type ii parasites [131 , 132] . some more example of nanovaccines for highly prevalent infectious diseases have been discussed below in detail. tuberculosis being the deadliest infectious disease, is the greatest cause of mortality worldwide. 10% of infected individuals develop the disease within one or two years of infection and the rest develop the disease in later stages of life when immune functions are compromised. co-infection of hiv patients with mtb ( mycobacterium tuberculosis ), further complicates the situation with 33% mortality each year [133] . bcg (bacille calmette-guerin) is a widely accepted vaccine against tb (tuberculosis), but its protective efficacy is limited by age due to the absence of consensus genomic loci that are present in most of the virulent strains of mtb. it induces short term and variable immune responses from 0% to 80% [134 , 135] . there is an urgent requirement of booster vaccines that augment t cell immunity in the lungs of bcg-vaccinated individuals. to address this, ansari et al., have synthesized archeosomes encapsulating a t cell antigen rv3619c, that upon immunization elicited type-1 cytokine response in mice. also, it increased the production of igg2a antibodies, antigen-specific t lymphocytes and enhanced jid: actbio [m5g; april 15, 2020; 3:1 ] expression of co-stimulatory molecules on the surface of apcs. by the virtue of all these properties, vaccination with archeosomes containing antigen reduced mycobacterial burden in the lungs and spleen of animals upon challenging with mtb [135] . similarly, selfassembled peptide nanofibers, when loaded with t cell epitope, also showed similar responses by inducing corresponding effector memory t cells and increasing cytotoxic t cell population in the lungs upon intranasal immunization. mice vaccinated with coassembly of nanofibers containing cd8 + t cell epitopes and tlr2 agonists in the lungs increased the expansion of multi-functional cytotoxic t cell population. on further challenge with mtb, significant reduction in lung bacterial load was observed in comparison to bcg primed mice [67] . in another study, mtb lipid-based nanovaccines were used wherein chitosan nanoparticles bound with mtb lipids induced cell-mediated and antibody-mediated immunity by secretion of immunoglobulins (igg, igm) and prominent th1 and th2 cytokines in lymph node and spleen cells [136] . the higher potency of nanoparticle encapsulated antigen preparation is because of their ability to form depots with a slow and steady release in the surrounding milieu and enhanced uptake of antigen by antigen-presenting cells [135] [136] [137] . as shown in a study by diogo et al., liposome-encapsulated antigens elicited more profound antibody-mediated and cell-mediated responses than free antigens. mice were immunized with free antigens and liposomeencapsulated antigen, further challenging mice 16 weeks post immunization with mtb, it was observed that mtb burden in the lungs and spleen was significantly less in mice immunized with liposome-encapsulated antigen as compared to free bcg vaccine [137] . the potential of nanoparticles in augmenting the prophylactic activity of antigens against tb was well understood and human trials were initiated. first-in-man trial on novel liposome caf01 as an adjuvant along with the tuberculosis vaccine ag85b-esat-6 (h1) is reported in 2014 [46] . human volunteers were vaccinated with escalating doses of caf01at 0 and 8 weeks. after immunization strong antigen-specific t cell responses persisted for 150 weeks and did not cause local or systemic adverse effects [92] . above discussed preclinical and clinical assessment of various antigen-loaded nanoparticles indicates the potential of nanovaccines in inducing robust and long-lasting cellular immunity against mtb and shows hope for the development of a different and more effective prophylactic regime against tb. it is clear that with the advent of nanotechnology, adjuvant and delivery vehicle associated problems have been reduced, but the effect of the host environment and lack of consensus genetic loci still remains an inevitable challenge while developing efficient vaccines against tb. malaria is known to affect almost 200 million people annually, with half a million deaths worldwide [138] . fighting malaria is challenging because of its multistage life cycle. various efforts have been made in designing vaccines against the pre-erythrocytic and blood stage of malaria [139 , 140] . similarly, nanovaccines have been used to target multiple stages of malaria. iron oxide (io) nanoparticles have been used for the delivery of merozoite surface proteins (rmsp1). immunization of mice with rmsp-1 loaded nanoparticles induced parasite inhibitory antibodies with high titers. macrophages and dendritic cells showed more than 90% internalization of io nanoparticles and enhanced the expression of cytokines and chemokines [141] . immune responses generated against sexual stage antigens of malaria impairs its transmission and reduces the disease burden. pfs25 is one such malaria transmission-blocking vaccine antigen, but its immunogenicity is limited in humans. hence, pfs25 has been used along with nanoformulations to achieve desired im-mune responses. for example, codon harmonized pfs25 (chrpfs25) has been used with gold nanoparticles as adjuvants for the induction of immunity against transmission. strong transmissionblocking antibodies elicited by simultaneous delivery of chrpfs25 with gold nanoparticles could be due to the co-ingestion of nanoparticles and antigen by immune cells [112] . similarly, recombinant polyhistidine-tagged (his-tagged) pfs25 when mixed with preformed co-porphyrin containing liposomes at the time of immunization resulted in spontaneous nanoliposome antigen particularization (snap). immunization of mice and rabbits with snap resulted in higher functional antibody generation as compared with other 'mix-and-inject' adjuvants. they have also been used for developing multi-antigen vaccines (apical membrane antigen-1, pfg27, pfs230 and the nanp peptide), targeting multiple stages of the plasmodium life cycle using liposomes [142] . ovalbumin loaded carboxylated polystyrene nanoparticles acted as an adjuvant and induced il-10 and granulocyte colonystimulating factor, that affects the migratory and homing ability of dendritic cells. mice challenged with malaria two weeks after immunization with nanoparticles produced anti-malaria antibodies and create the state of immune readiness to a subsequent infectious challenge [143] . stable nanomimic, which are polymersomes with receptors for parasite attachment were synthesized. they bind to host cells and block re-invasion of the parasite after their release from host cells in vitro . this strategy can be used in the future to modulate immune responses against malaria and in the efficient designing of the vaccine [144] . thus, the increased role of nanovaccines in targeting multiple life cycle stages of malaria shows new avenues in building a robust vaccination regime for prevention against malaria infection. three types of influenza viruses a, b and c have different viral nucleoproteins and matrix proteins giving rise to variable antigenic differences among them. influenza virus a and b cause epidemics attacking both adults and children, causing around 5 million infections annually [145] . presently, two types of vaccines are used against influenza based on strain a and strain b that target and produce antibody-mediated protective responses against hemagglutinin and neuraminidase. these antigens are highly prone to antigenic shift and drift due to error-prone rna dependent rna replication in influenza [146 , 147] . due to environmental selection and antigenic variations, chances of influenza epidemic still persists [148] . although animal influenza virus is distinct from the human virus, zoonotic animal viruses can still occasionally infect humans through direct/indirect contact. avian and swine influenza viruses have been known to infect humans in some countries. thus, there is an unmet need for producing more efficient vaccines that can provide both antibody-mediated and cell-mediated immunity to confront homologous and heterologous variants [149] . association of various influenza antigens with nanoparticles have shown promising preliminary results in providing enhanced immune responses against variable influenza antigens. as the antigenic variability limits the use of a single vaccine against influenza a virus (iav), there are effort s to develop nanovaccines conferring protection against more than one serotype by using most conserved ectodomain of influenza matrix protein 2 (m2e). nanoparticles containing self-assembling recombinant cage of human heavy chain ferritin presented with 3-sequential repeats of m2e protein was observed to elicit elevated levels of m2especific igg and mucosal secretory iga antibodies with enhanced t cell response against h1n1 and h5n1 lethal infection after intranasal administration in mice [60] . nanovaccines against the conserved ectodomains can confer protection against inter-species viral infection. self-assembled virus-like particles (vlps) containing multiple copies of m2e protein inserted into capsid (cap) protein of porcine circovirus type 2 (pcv2) have shown dual protection against iav and pcv2 lethal challenge in mice and pigs after generating m2e specific and pcv2 neutralizing antibodies [150] . hemagglutinin trimer has also been exploited as a potential antigen for inducing protective immunity against influenza virus. pham et al., immunized balb/c mice with nanodiamond conjugated trimeric h7 and observed a significant higher h7 specific igg production as compared to immunization with h7 trimeric antigen alone [151] . similarly, ross et al., have shown that intranasal or subcutaneous immunization of balb/c mice with polyanhydride nanoparticles encapsulated h53 induced high titer neutralizing antibodies. also, significantly higher cd4 + t cell expansion was observed in vaccinated mice as compared to non-vaccinated mice, thus providing cell-mediated and antibody-mediated protective immunity to mice. mice were further challenged intranasally with a/h5n1 vnh5n1-pr8cdc-rg influenza virus at 63 days post immunization and viral load was significantly reduced as compared to saline-administered mice [152] . it has also been demonstrated that the polyanhydride nanoparticles give equivalent responses at 64-fold lower doses as compared to free antigen [153] . also, encapsulation of antigen in polyanhydride nanovaccines exhibited stability of antigen for one year at room temperature, thus providing a major benefit for maintaining stocks of pandemic vaccines [154] . as discussed above, the nanovaccines have been designed by various research groups as combinations of nanomaterials and different antigens, but they provide protection from a particular strain of the virus. due to antigenic variations, multiple strains of influenza virus have emerged over the years posing serious public threats, indicating the need for a universal vaccine, which includes broad cross-presentation against multiple strains of influenza to reduce community threats. insights into the structural properties of antigens or peptides can be further explored for the rational designing of broad-spectrum nanovaccines using multiple antigens. on similar lines, to increase the breadth and potency of nanovaccines, deng et al., have designed double-layered protein nanoparticles. the core of the protein nanoparticles (4mtg) contains four types of m2e from human (hum2e), swine (swnm2e), avian (avim2e), and domestic fowl (fwlm2e) viral consensus sequences coated with ha variants hrh1 and hrh3 (refer fig. 5 ). mice were immunized intramuscularly with uni4mc (pnp), uni4c1 (hrh1-coated double-layer pnp) and uni4c3 (hrh3-coated doublelayer pnp) and uni4c13 (cocktail of uni4c1 and uni4c3). robust antibody-mediated responses were generated and induced m2e antibodies were strongly cross-reactive to diverse antigens. also, uni4c13 group produced broad cellular responses by significantly inducing more ifn-γ secreting splenocytes after stimulation with diverse antigen peptides. naïve mice were vaccinated with sera from pre-immunized mice with dpbs, uni4mc, or uni4c13 and then challenged intranasally with pr8 or aic after 24 hours. sera from uni4c13 immunized mice prevented the mice from a viral infection, indicating the role of serum antibodies. also, antibodies specific to human m2e, pr8, and aic were viable for four months after the immunization in mice and implied long-lasting protection [155] . from the examples discussed above, it is evident that with the help of structurally important antigens, smartly designed protein nanomaterials will have the potential of inducing long-lasting immunity against broad spectrum of antigens, thus providing platforms for producing efficient nanovaccines to combat pandemic viruses. hiv is the sixth leading cause of death worldwide. hiv infection causes systemic depletion of cd4 + t cells, hence compromising the activity of the immune system and the development of aids. immune functions can be preserved in the early stages of hiv by using antiretroviral therapy (art). it targets the hiv life cycle, but the major challenge is strict adherence to complex art regimens. this necessitates the urgent need for the development of a prophylactic vaccine against hiv. the most recent and promising hiv vaccine rv144 is in the phase iii trial [156] . igg antibodies raised against v1v2 loop were inversely correlated with the risk of infection in the clinical trial of rv144 [157] . sapn-based vaccine reduces the need for essential glycosylation of the hiv-1 env v1v2 loop to form a native-like structure to act as potential immunogen [157] . although self-assembling vlps as vectors have shown great potential in the development of immunity against hiv, anti-vector immunity is a significant concern. here, synthetic nanoparticles offer versatile platform technologies that can induce strong adaptive immune responses while avoiding anti vector immunity and toxicity issues [156] . plga based nanoparticles have been used for co-utilization of tlr agonist and the antigen. intradermal immunization of mice with hiv-1p24-nef/flic/plga (antigen/agonist/nanoparticle) enhanced igg production even at 4-fold lesser dose of antigen. nanovaccines shifted the immune response towards th1 polarization and enhanced the th1 cytokine pattern [158] . recently, protease cleavage sites (pcs) has been utilized as a strategy to hamper the maturation and infectivity of hiv. due to low immunogenicity of peptide antigens, they have been crosslinked with chitosan and hyaluronic based nano-formulation. nanoparticles containing pcs showed enhanced activation of apcs and the production of anti-pcs antibody. also, these nanoparticles generated memory t cells at longer time points after the last booster dose, indicating their capacity to elicit a good immune response upon infection [159] . silver nanoparticles coated with amantadine triggered the production of hiv specific ctls in the spleen of mice with 8-fold stronger tnf-α production in vivo . the co-culture of these hiv specific ctls with hiv infected cells in vitro enhanced the death of infected cells and reduced the production of hiv [160] . similarly, hiv-1 peptide and oligosaccharide loaded aunps enhanced antigen presentation to isolated t cells from hiv-1 patients. treatment with aunp formulated vaccine increased proliferation of hiv-specific cd4 + and cd8 + t cells with increased secretion of pro-th1 cytokines and pro-inflammatory cytokines in vitro [161] . malik et al., have shown the prophylactic potential of intravaginally delivered nanogold loaded formulation. poloxomer and hyaluronic based thermogels were loaded with gold niosomes and mannosylated gold niosomes along with efavirenz (efv). efv and gnps inhibited viral dissemination in pbmcs (peripheral blood mononuclear cells) host cells, but their individual potential in inhibiting pre-interaction viral dissemination was lower than that of the combination. a substantial 42.6% increase in activity was seen when efv was given with gnps. both the moieties had a dual-faced attack wherein gnps inhibited hiv entry into the cell by causing denaturation of gp120 glycoprotein and efv inhibited transcriptase enzyme. the mannosylated efv-gnps (man(egnz)) showed further potential activity in inhibiting viral dissemination when the host was pre-exposed to them before viral infection. thermogels containing efv-gnp and man(egnz) showed 87.6 ± 2.4% and 98.1 ± 0.2% inhibition respectively in p24 antigen during anti-hiv prophylactic challenged study [162] . various such formulations have been developed over the years that enhanced the prophylactic action of raltegravir and efavir [163] , phthalate and efavirenz [164] or efavirenz and saquinavir [165] . bayon et al., have also shown the capacity of nano-lipid complexes (nlc) to induce p24 specific immune responses against hiv in non-human primates (nhp). four intradermal immunization over a period of 7 months showed that nlc-loaded p-24 antigen elicited significantly higher p24-specific antibodies as compared to the combination of free p24 antigen and cpg adjuvant [166] . there have been advancements in understanding the role of nanovaccines in fighting hiv and development of nanovaccine loaded formulations for prophylaxis of hiv. with the emerging use of nanotechnology development of an efficient vaccine system for hiv prevention is not that far. despite constant efforts to develop effective cancer vaccines, their efficacy in clinics has always been limited. nonetheless, two prophylactic vaccines for hepatitis b virus-associated liver cancer and human papillomavirus virus-associated cervical cancer have been approved by us fda till now [50] . the prophylactic vaccine development for cancer eradication is a challenge due to inefficient identification of tumor-specific antigens (neoantigens), rapid clearance with less accumulation in the lymphoid organs and generation of weak memory immune response. therefore, the use of nanoparticles to overcome these limitations would be an advantageous strategy. prolonged and sustained delivery of antigen would be extremely important for the generation of immunologic memory and booster dose reduction. immunization of mice with protein nanoparticles conjugated to melanoma-associated epitope gp100 and cpg adjuvant enhanced the production of melanoma-specific cd8 + t cells. pre-immunization with nanoparticles delayed onset of tumor growth and increased the survival rate to 40%, showing prophylactic potential of protein nanoparticles [59] . luo et al., showed strong cytotoxic t cell response by a physical mixture of pc7a polymeric nanoparticle and ova antigen. this was achieved by enhanced cytosolic delivery and antigen presentation in apcs of draining lymph node by pc7a nps and activation of type-i interferon stimulating genes. nanovaccines showed tumor growth inhibition in melanoma, human papilloma virus e6/e7 tumor and colon cancer models. the combination of anti-pd1 with pc7a nps showed synergism with 100% survival in a tc-1 model over 60 days. further, re-challenging of tumor-free mice with tc-1 cells after immunization with pc7a nanoparticles and anti-pd1 showed complete tumor growth inhibition. this indicates the prophylactic response of nanovaccine by generating long-term antitumor memory [46] . liu et al., developed a unique prophylactic nanovaccines using metallo-organic nanoparticles (mof) coated with fused membrane (fm) of dendritic cells and cancer cells. presence of whole tumor antigens and immunological co-stimulatory molecules on its cytomembranes impart them apc like properties for cancer specific t cell immuno-activation. since, fusion of dc and cancer membrane is accompanied by dc maturation, mof@fm nanovaccines possessed lymph node homing molecules. therefore, enhanced retention and homing of mof@fm in lymph nodes and spleen as compared to only cancer membrane fused (mof@cm) and dendritic cell membrane fused (mof@dm) nanovaccines was observed. pre-immunization of mice with mof@fm has significantly prevented the development of 4t1 tumors by promoting differentiation of cd8 + t cell precursors into cytotoxic t cells with enhanced generation of ifn-ɣ and il-6 immuno-stimulatory cytokines (refer fig. 6 ) [77] . tumor heterogeneity with differential expression of antigens makes it challenging to develop a prophylactic treatment strategy. therefore, most of the nanovaccines being developed for cancer are therapeutic. the detailed description of the same is given in the next section. unlike conventional therapeutics, therapeutic nanovaccines offer the prospect of eliciting antigen-specific responses without any additional non-specific responses. therapeutic vaccines have been used against autoimmune disorders by antigen-specific targeting of t-regulatory cells [193] . clinical studies revealed the therapeutic efficacy of vlp in allergen-induced asthma. bacteriophage derived vlps were packed with cpg and tlr 9 and induced th1 response in immune system. a total of 63 asthmatic patients were treated with 7 injections of vlps as a part of double-blind randomized trial and it was observed that the asthma was well-controlled in patients treated with vlps as compared to the placebo group [170] . unlike cancer, nanovaccines showed promising results in pathogenic infections because of the presence of extremely potent foreign non-self antigens. therefore, vaccine-based therapy is still limited in cancer treatment but can provide an alternative to, or complement conventional cancer treatments. till now, cancer vaccines have failed to make a mark clinically as compared to other immunotherapies like t cell therapy and checkpoint blockade. the only us fda approved therapeutic cancer vaccine, provenge (sipuleucel-t), has shown a moderate increase in clinical efficacy in prostate cancer [25] . the major problem in successfully developing a cancer vaccine is the selection of tumor-specific antigens called "neoantigens". conventionally, cancer vaccines were developed by targeting tumor-associated antigens (taas), which are overexpressed specifically in a particular tumor type across patients. however, taas are also present in normal tissues; therefore vaccines specific for them can induce peripheral or central immunotolerance leading to autoimmunity or low vaccination efficiency [88] . neoantigens are tumor-specific antigens derived from random somatic mutations but absent in normal cells. hence, vaccines targeting such neoantigens using nanoparticles may enhance the outcome of cancer immunotherapy. cancer nanovaccines can present unique and highly immunogenic tumor-specific antigens with increased antigen loading and efficient delivery, controlled antigen presentation, and retention in lymphoid organs [194] . a recent study showed the targeting of bone marrow and splenic dcs with peptide ty using mesoporous silica nanoparticles conjugated with ova/cpg for immune activation (msn-ty/ova/cpg). therapeutic application of (msn-ty/ova/cpg) in naïve c57bl/6j b16-ova mice enhanced dc activation and tumor elimination by eliciting tumor-specific cd8 + t cell response, with prolonged survival and less systemic toxicity [195] . to overcome inadequate loading efficiency, weak immune response, and complex preparation process, dong et al., prepared antigen nanoparticles (ovalbumin nps) containing cpg adjuvant (onps-cpg) with high antigen and adjuvant loading for cancer immunotherapy. the in vitro and in vivo results showed enhanced immune response including dc maturation, t cell activation and ifn-γ production. onps-cpg induced remarkable anti-tumor efficacy in mouse lymphoma model [121] . biomimetic-approach for developing cancer nanovaccines: overcoming cancer heterogeneity by using patient-derived cell membranes for the development of smart nanovaccines can be a promising biomimetic approach for personalized therapy. various strategies have been explored in the form of developing artificial antigen-presenting cells (aapcs) based nanovaccines, use of different cell membrane fused nanovaccines and membrane coated viruses for enhanced cross-presentation and immune stimulation (refer fig. 7 ) . recently, kuai et al., have shown the anti-tumor efficacy of high-density lipoprotein (hdl) nanodiscs loaded with neoantigenic peptide and cpg t cell receptor agonist in b16f10 and mc-38 colon carcinoma model. nanodiscs have delivered the neoantigen to draining lymph nodes and enhanced antigen presentation by apcs. it causes enhanced stimulation of cytotoxic t cell response that potentially inhibited tumor growth. delivery of nanodiscs with checkpoint blockade (anti-pd1 and anti-ctla-4) therapy also eradicated established b16f10 and mc-38 colon carcinomas. this strategy showed a powerful therapy to generate personalized cancer medicine [196] . zhang et al., developed a magnetic artificial antigen-presenting cell (aapc) for enhancing cd8 + t cell-mediated tumorigenic response. they developed fe 3 o 4 magnetic nanoclusters coated with leucocyte membrane antigens decorated with mhc-i loaded peptide (siinfekl) and anti-cd28 co-stimulatory ligand for eg-7 tumors. these biomimetic aapcs stimulated ot-1 cd8 + t cells and visually guided them to tumor magnetically, causing reduced tumor burden [197] . this offers a great platform for t cell-based immunotherapy. the fusion of biological membranes with nanovaccines has emerged as an interesting concept towards biomimetic nanovaccines. a recent study showed biomimetic cancer-derived magnetosome with enhanced retention in lymph nodes after subcutaneous administration. toll-like receptor (tlr) agonist (cpg) loaded fe 3 o 4 magnetic nanoclusters camouflaged with cancer membrane and was decorated with anti-cd205 for preferential recognition by cd8 + dc population for cross-presentation to cd8 + t cells via mhc-i. enhanced stimulation of tumor-specific cd8 + t cells due to prolonged retention of magnetically guided nanocluster in lymph nodes showed anti-tumor efficacy with improved survival in different mice models [191] . similarly, guo et al., developed an erythrocyte membrane coated plga nanoparticle-containing hgp10025-33 antigenic peptide and monophosphoryl lipid-a (mpl-a) tlr-4 agonist for enhanced anti-tumor immunity. intradermal injection in c57bl/6 had shown reduced tumor burden along with metastasis and prolonged tumor occurring time after re-challenge in metastatic models [198] . studies exploiting cancer cell membranes as an envelope to impart a wide repertoire of cancer-specific antigens can offer an advantage to develop various personalized vaccines towards multiple tumor types. another interesting strategy for developing personalized biomimetic cancer nanovaccines is the use of cancer cell membrane coated virus for increased adjuvanticity, infectivity and oncolytic activities to generate a strong anti-tumor immune response. a recent study showed the prophylactic and therapeutic potential of a 117.7 ±0.8 nm biohybrid artificially enveloped virus extracrad (extra conditionally replicating adenovirus). oncolytic adenovirus serotype 5 containing a cpg island with a 24 base pair deletion was encapsulated into b16.ova cancer cell line membrane. coating of the cancer membrane was found to enhance the in vitro cytotoxicity of extracrad as compared to the naked virus indicating the enhanced infectivity of viral vaccine as compared to the naked virus in multiple cell lines. intra-tumoral injection of extracrad in c57bl/6 mice containing b16.ova, b16f10 and ll/2 induced models showed in vivo therapeutic vaccine efficacy in facilitating reduced tumor proliferation as compared to the naked virus, individual vaccine component controls and virus membrane mixture because of enhanced cross-presentation and t cell activation. the prophylactic potential was explored by vaccinating mice 3 times with extracrad.cmt64.ova and extracrad.b16f10 vaccine before cmt64.ova and b16f10 cells re-challenge, respectively. prolonged overall survival and enhanced inhibition of tumor proliferation in homologous membrane enveloped viral vaccinated groups as compared to the naked virus and heterologous membrane enveloped group indicated the strong potential of viral-based vaccine imparting wide repertoire of cancer-specific antigens as preventive vaccines [199] . another viral protein-coated dendrimer nanovaccine containing e6 and e7 peptide epitope of hpv showed enhanced cytotoxic t cell stimulation followed by hpv infected cells eradication. tumor elimination was observed in 50% of treated mice with no recurrence up to 3 months post initial challenge [24] . therefore, to address the problems associated with cancer vaccine development, special emphasis has been given on biomimetic personalized nanovaccines. the use of an apcs and nanoparticles coated with the patient's own cancer cell membranes can provide effective antigenic diversity to overcome tumor heterogeneity and tumor-specific immune response. this provides a key strategy to train one's own immune system with appropriate antigens. research using such a biomimetic personalized approach can significantly improve the outcome of current treatment modalities. nanovaccines have shown potential in augmenting the therapeutic and prophylactic efficacy of cancer vaccines. in the past decade, research on vaccines has taken a big leap forward, exploring their vast potential to combat numerous diseases. specifically, strong interactions of nanoparticles with the immune system and the associated benefits have attracted attention with the hope of producing less toxic and effective nanovaccines. physicochemical variations in nanoparticles are one of the prime factors affecting their fate as "nanovaccines" inside the body in terms of their clearance, specific bioaccumulation, adjuvanticity, antigenicity and toxicity. potent antigenicity with prolonged retention and release can cause nanovaccines to elicit both cellmediated and antibody-mediated antibody along with memory effector response, thus alleviating the use of frequent booster doses. although nanovaccines are still in their infancy because of its limited clinical trials but new generation nanovaccines possess enormous potential both as prophylactic or therapeutic. hence, exploring new avenues to increase nanovaccine safety and efficacy should be the driving force for further research in this domain of bionanotechnology. nanomedicine toxicity, scaling-up processes and lack of regulatory guidelines can be considered as the limiting factors in the production of nanovaccines. the nano-scale size can work as a double edge sword as pre-clinical and clinical reports showed their dosedependent acute and chronic toxicities with preferential bioaccu-mulation depending on the route of administration. few classes of nanoparticles possess inherent toxicity after prolonged exposure like inorganic nanoparticles (e.g., metallic nanoparticles). moreover, iscoms are being used in a number of animal vaccines, but toxicity associated with saponin-based adjuvants prohibited their use in humans. therefore, concerns over nanoparticle usage for vaccines are still in existence. scaling up is another major problem that has been minimized up to an extent because of technological advancements, but scale-up in a sterile environment is still a significant challenge [213] . though, the conventional treatments in this era of modern medicine is saving countless lives. the steady rise in antibiotic resistance and lack of curative cancer therapy because of continuous causative strain variations and cancer heterogeneity respectively drives the focus towards personalized therapeutics. hence, on similar lines, vaccines against bacterial infections and tumors could benefit significantly, but the current vaccine's antigenic breadth and lack of potency are the limiting factors. therefore, researchers have recently shifted towards biomimetic nanotechnology to develop biomimetic nanovaccines as personalized medicines. such nanovaccines can be inherently immuno-stimulatory and multiantigenic. coating of bacterial outer membrane vesicles (omvs) jid: actbio [m5g; april 15, 2020; 3:1 ] onto nanoparticles for anti-virulence vaccination to deliver and neutralize bacterial toxins outplays the pathogen by utilizing its own survival mechanism. this can prevent bacterial colonization effectively and reduce direct selective pressure to develop antibiotic resistance. a large number of such nano-toxoid formulations can be developed by varying the outer membrane coating of these biomimetic nanovaccines [213] . despite the advancements in the field of cancer nano-based immunotherapy, the major challenge is the toxicological assessments of these nanovaccines. prior evaluation of autoimmune side effects generated from nanovaccine induced immune activation needs to be done. over-activation of antigen presenting cells by nanovaccine can prove to be detrimental as it may cause the death of dendritic cells [47] . last but not least, increased complexity, cost of manufacturing, and commercialization hurdle related to nanovaccine therapy requires strategies to minimize these limitations for the successful translation of nano-based immunotherapy. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. a brief review of computer-assisted approaches to rational design of peptide vaccines peptide vaccine: progress and challenges principles of vaccine design-lessons from nature enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles advances and opportunities in nanoparticle-and nanomaterial-based vaccines against bacterial infections protein and peptide biomaterials for engineered subunit vaccines and immunotherapeutic applications immunological mechanisms of vaccination micro and nanoparticle-based delivery systems for vaccine immunotherapy: an immunological and materials perspective towards tailored vaccine delivery: needs, challenges and perspectives cd40-targeted dendritic cell delivery of plga-nanoparticle vaccines induce potent anti-tumor responses dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting induction of anti-tumor cytotoxic t cell responses through plga-nanoparticle mediated antigen delivery innate and adaptive immune cells in the tumor microenvironment trained innate immunity as underlying mechanism for the long-term, nonspecific effects of vaccines pathogen-associated molecular patterns on biomaterials: a paradigm for engineering new vaccines vaccine delivery: a matter of size, geometry, kinetics and molecular patterns engineering nanoparticles to overcome barriers to immunotherapy poly(lactide-co-glycolide)-rifampicin nanoparticles efficiently clear mycobacterium bovis bcg infection in macrophages and remain membrane-bound in phago-lysosomes a therapeutic microparticle-based tumor lysate vaccine reduces spontaneous metastases in murine breast cancer enantiospecific adjuvant activity of cationic lipid dotap in cancer vaccine dendritic cell-based nanovaccines for cancer immunotherapy adoptive immunotherapy for cancer: harnessing the t cell response skwarczynski , multiantigenic peptide-polymer conjugates as therapeutic vaccines against cervical cancer provenge (sipuleucel-t) in prostate cancer: the first fda-approved therapeutic cancer vaccine dendritic cells and other innate determinants of t helper cell polarisation rosenberg , cancer immunotherapy based on mutation-specific cd4 + t cells in a patient with epithelial cancer how regulatory t cells work human memory b cells originate from three distinct germinal center-dependent and -independent maturation pathways rituximab specifically depletes short-lived autoreactive plasma cells in a mouse model of inflammatory arthritis the cell biology of antigen processing quantitative review of antibody response to inactivated seasonal influenza vaccines, influenza other respir single-injection vaccines: progress, challenges, and opportunities switched memory b cells maintain specific memory independently of serum antibodies: the hepatitis b example nanoparticle conjugation of cpg enhances adjuvancy for cellular immunity and memory recall at low dose role of sustained antigen release from nanoparticle vaccines in shaping the t cell memory phenotype polymeric particles in vaccine delivery exploiting lymphatic transport and complement activation in nanoparticle vaccines memory antibody response from antigen loaded polymer particles and the effect of antigen release kinetics applications of nanotechnology for immunology vaccine adjuvants: mode of action mechanisms of action of adjuvants toll-like receptor signaling pathways unleashing the potential of nod-and toll-like agonists as vaccine adjuvants activation with cpg-a and cpg-b oligonucleotides reveals two distinct regulatory pathways of type i ifn synthesis in human plasmacytoid dendritic cells vaccine delivery using nanoparticles design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases carbohydrate-functionalized nanovaccines preserve hiv-1 antigen stability and activate antigen presenting cells polyglutamic acid-trimethyl chitosan-based intranasal peptide nano-vaccine induces potent immune responses against group a streptococcus applications of nanomaterials as vaccine adjuvants nanovaccines and their mode of action a novel lipid nanoparticle adjuvant significantly enhances b cell and t cell responses to sub-unit vaccine antigens curdlan sulfate-o-linked quaternized chitosan nanoparticles: potential adjuvants to improve the immunogenicity of exogenous antigens via intranasal vaccination green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine lipid-derived nanoparticles for immunostimulatory rna adjuvant delivery artificial bacterial biomimetic nanoparticles synergize pathogen-associated molecular patterns for vaccine efficacy coated protein nanoclusters from influenza h7n9 ha are highly immunogenic and induce robust protective immunity viruslike particles encapsidating respiratory syncytial virus m and m2 proteins induce robust t cell responses viral-mimicking protein nanoparticle vaccine for eliciting anti-tumor responses intranasal nanovaccine confers homo-and hetero-subtypic influenza protection encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science biomimetic protein nanoparticles facilitate enhanced dendritic cell activation and cross-presentation vault particles: a new generation of delivery nanodevices a protective vaccine against chlamydia genital infection using vault nanoparticles without an added adjuvant a supramolecular vaccine platform based on alpha-helical peptide nanofibers self-assembled peptide nanofibers raising durable antibody responses against a malaria epitope nanoscale peptide self-assemblies boost bcg-primed cellular immunity against mycobacterium tuberculosis titrating t-cell epitopes within self-assembled vaccines optimizes cd4 + helper t cell and antibody outputs intranasal delivery of adjuvant-free peptide nanofibers elicits resident cd8( + ) t cell responses synergistic sting activation by pc7a nanovaccine and ionizing radiation improves cancer immunotherapy design and optimization of peptide nanoparticles protective antibody and cd8 + t-cell responses to the plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine conformation-specific display of 4e10 and 2f5 epitopes on self-assembling protein nanoparticles as a potential hiv vaccine protein nanovaccine confers robust immunity against toxoplasma peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine nanoclusters self-assembled from conformation-stabilized influenza m2e as broadly cross-protective influenza vaccines cytomembrane nanovaccines show therapeutic effects by mimicking tumor cells and antigen presenting cells a sting-activating nanovaccine for cancer immunotherapy self-assembled peptide amphiphile micelles containing a cytotoxic t-cell epitope promote a protective immune response in vivo peptide amphiphile micelles self-adjuvant group a streptococcal vaccination subcutaneous nanodisc vaccination with neoantigens for combination cancer immunotherapy comparison of the immune response against polio peptides covalently-surface-linked to and internally-entrapped in liposomes, asian pac the role of liposome size on the type of immune response induced in balb/c mice against leishmaniasis: rgp63 as a model antigen liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a cmi response manipulation of the surface pegylation in combination with reduced vesicle size of cationic liposomal adjuvants modifies their clearance kinetics from the injection site, and the rate and type of t cell response enhancement of immune response and protection in balb/c mice immunized with liposomal recombinant major surface glycoprotein of leishmania (rgp63): the role of bilayer composition a surface charge dependent enhanced th1 antigen-specific immune response in lymph nodes by transfersome-based nanovaccine-loaded dissolving microneedle-assisted transdermal immunisation antigen-specific vaccines for cancer treatment liposome-entrapped , pteronyssinus vaccination in mild asthma patients: effect of 1-year double-blind, placebo-controlled trial on inflammation, bronchial hyperresponsiveness and immediate and late bronchial responses to the allergen inspire: a phase iii study of the blp25 liposome vaccine (l-blp25) in asian patients with unresectable stage iii non-small cell lung cancer some-based adjuvants for subunit vaccines: formulation strategies for subunit antigens and immunostimulators a novel liposomal adjuvant system, caf01, promotes long-lived mycobacterium tuberculosis-specific t-cell responses in human a triple co-culture model of the human respiratory tract to study immune-modulatory effects of liposomes and virosomes the novel immunogenic chimeric peptide vaccine to elicit potent cellular and mucosal immune responses against htlv-1 immune responses induced by nano-self-assembled lipid adjuvants based on a monomycoloyl glycerol analogue after vaccination with the chlamydia trachomatis major outer membrane protein cubosomes: the next generation of smart lipid nanoparticles? cubosomes containing the adjuvants imiquimod and monophosphoryl lipid a stimulate robust cellular and humoral immune responses in vitro protein release and degradation of poly-dl-lactide-poly(ethylene glycol) microspheres with entrapped human serum albumin: quantitative evaluation of the factors involved in protein release phases aerosolized pla and plga nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis b vaccine enhancement of immune responses by co-delivery of a cpg oligodeoxynucleotide and tetanus toxoid in biodegradable nanospheres a single-dose plga encapsulated protective antigen domain 4 nanoformulation protects mice against bacillus anthracis spore challenge role of trehalose dimycolate in recruitment of cells and modulation of production of cytokines and no in tuberculosis immune response by nasal delivery of hepatitis b surface antigen and codelivery of a cpg odn in alginate coated chitosan nanoparticles bioreducible alginate-poly(ethylenimine) nanogels as an antigen-delivery system robustly enhance vaccine-elicited humoral and cellular immune responses in vitro stimulation of cd8 and cd4 t cells by dendritic cells loaded with a complex of cholesterol-bearing hydrophobized pullulan and ny-eso-1 protein: identification of a new hla-dr15-binding cd4 t-cell epitope a novel hepatitis b vaccine containing advax, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing enhanced immune response and protective effects of nano-chitosan-based dna vaccine encoding t cell epitopes of esat-6 and fl against mycobacterium tuberculosis infection a fully synthetic glycopeptide antitumor vaccine based on multiple antigen presentation on a hyperbranched polymer toxicologic effects of gold nanoparticles in vivo by different administration routes assembling different antennas of the gp120 high mannose-type glycans on gold nanoparticles provides superior binding to the anti-hiv antibody 2g12 than the individual antennas m2e-immobilized gold nanoparticles as influenza a vaccine: role of soluble m2e and longevity of protection nanovaccines for malaria using plasmodium falciparum antigen pfs25 attached gold nanoparticles in vivo gold nanoparticle delivery of peptide vaccine induces anti-tumor immune response in prophylactic and therapeutic tumor models gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo multicopy multivalent' glycopolymer-stabilized gold nanoparticles as potential synthetic cancer vaccines synthesis of a novel kind of carbon nanoparticle with large mesopores and macropores and its application as an oral vaccine adjuvant single-walled carbon nanotubes deliver peptide antigen into dendritic cells and enhance igg responses to tumor-associated antigens immunisation of mice by hollow mesoporous silica nanoparticles as carriers of porcine circovirus type 2 orf2 protein silica vesicle nanovaccine formulations stimulate long-term immune responses to the bovine viral diarrhoea virus e2 protein lipopeptide-coated iron oxide nanoparticles as potential glycoconjugate-based synthetic anticancer vaccines a visible codelivery nanovaccine of antigen and adjuvant with self-carrier for cancer immunotherapy effect of preexisting neutralizing antibodies on the anti-tumor immune response induced by chimeric human papillomavirus virus-like particle vaccines room temperature stable pspa-based nanovaccine induces protective immunity reversal of autoimmunity by boosting memory-like autoregulatory t cells peptide-mhc-based nanovaccines for the treatment of autoimmunity: a "one size fits all" approach? synthetic vaccine nanoparticles target to lymph node triggering enhanced innate and adaptive antitumor immunity the contribution of vaccination to global health: past, present and future nanoparticle vaccines against infectious diseases development of a gold nanoparticle vaccine against enterohemorrhagic escherichia coli o157:h7 a poly(lactic-co-glycolic) acid nanovaccine based on chimeric peptides from different leishmania infantum proteins induces dendritic cells maturation and promotes peptide-specific ifngamma-producing cd8( + ) t cells essential for the protection against experimental visceral leishmaniasis effectiveness of a novel immunogenic nanoparticle platform for toxoplasma peptide vaccine in hla transgenic mice adjuvanted multi-epitope vaccines protect hla-a * 11:01 transgenic mice against toxoplasma gondii multidrug and extensively drug-resistant tb (m/xdr-tb) rd antigen based nanovaccine imparts long term protection by inducing memory response against experimental murine tuberculosis vaccine delivery system for tuberculosis based on nano-sized hepatitis b virus core protein particles chitosan nanoparticles as an efficient delivery vehicle for mycobacterium tuberculosis lipids to induce potent cytokines and antibody response through activation of gammadelta t cells in mice immunisation with mycobacterium tuberculosis antigens encapsulated in phosphatidylserine liposomes improves protection afforded by bcg pre-erythrocytic malaria vaccines: identifying the targets progress and prospects for blood-stage malaria vaccines iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood-stage human malaria vaccine a malaria vaccine adjuvant based on recombinant antigen binding to liposomes plebanski , nanoparticles modify dendritic cell homeostasis and induce non-specific effects on immunity to malaria nanomimics of host cell membranes block invasion and expose invasive malaria parasites influenza vaccines: challenges and solutions influenza virus antigenic variation, host antibody production and new approach to control epidemics changing selective pressure during antigenic changes in human influenza h3 cellular immunity and memory to respiratory virus infections nanovaccine confers dual protection against influenza a virus and porcine circovirus type 2 nanodiamond enhances immune responses in mice against recombinant ha/h7n9 protein hemagglutinin-based polyanhydride nanovaccines against h5n1 influenza elicit protective virus neutralizing titers and cell-mediated immunity single immunisation with a suboptimal antigen dose encapsulated into polyanhydride microparticles promotes high titer and avid antibody responses amphiphilic polyanhydride nanoparticles stabilize bacillus anthracis protective antigen double-layered protein nanoparticles induce broad protection against divergent influenza a viruses vaccine nanoparticles for protection against hiv infection glycosylation of the hiv-1 env v1v2 loop to form a native-like structure may not be essential with a nanoparticle vaccine co-utilization of a tlr5 agonist and nano-formulation of hiv-1 vaccine candidate leads to increased vaccine immunogenicity and decreased immunogenic dose: a preliminary study polysaccharide nanoparticles can efficiently modulate the immune response against an hiv peptide antigen amantadine surface-modified silver nanorods improves immunotherapy of hiv vaccine against hiv-infected cells loading dendritic cells with gold nanoparticles (gnps) bearing hiv-peptides and mannosides enhance hiv-specific t cell responses efaverinz and nano-gold-loaded mannosylated niosomes: a host cell-targeted topical hiv-1 prophylaxis via thermogel system development and evaluation of a thermosensitive vaginal gel containing raltegravir + efavirenz loaded nanoparticles for hiv prophylaxis thermosensitive gel containing cellulose acetate phthalate-efavirenz combination nanoparticles for prevention of hiv-1 infection drug synergy of tenofovir and nanoparticle-based antiretrovirals for hiv prophylaxis overcoming immunogenicity issues of hiv p24 antigen by the use of innovative nanostructured lipid carriers as delivery systems: evidences in mice and non-human primates a virus-like particle vaccine candidate for influenza a virus based on multiple conserved antigens presented on hepatitis b tandem core particles a novel vaccine using nanoparticle platform to present immunogenic m2e against avian influenza infection vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge the novel tlr-9 agonist qbg10 shows clinical efficacy in persistent allergic asthma gold nanoparticle-m2e conjugate coformulated with cpg induces protective immunity against influenza a virus simultaneous surface display and cargo loading of encapsulin nanocompartments and their use for rational vaccine design development of a self-assembling protein nanoparticle vaccine targeting plasmodium falciparum circumsporozoite protein delivered in three army liposome formulation adjuvants a randomized trial assessing the safety and immunogenicity of as01 and as02 adjuvanted rts,s malaria vaccine candidates in children in gabon randomized, double-blind, phase 2a trial of falciparum malaria vaccines rts,s/as01b and rts,s/as02a in malaria-naive adults: safety, efficacy, and immunologic associates of protection evaluation of rts,s/as02a and rts,s/as01b in adults in a high malaria transmission area mechanisms of protective immune responses induced by the plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine surface-engineered gold nanorods: promising dna vaccine adjuvant for hiv-1 treatment a randomized, controlled dose-finding phase ii study of the m72/as01 candidate tuberculosis vaccine in healthy ppd-positive adults teyton , t cells control the generation of nanomolar-affinity anti-glycan antibodies immunogenicity and efficacy of flagellin-envelope fusion dengue vaccines in mice and monkeys encapsulation of an ep67-conjugated ctl peptide vaccine in nanoscale biodegradable particles increases the efficacy of respiratory immunisation and affects the magnitude and memory subsets of vaccine-generated mucosal and systemic cd8( + ) t cells in a diameter-dependent manner chitosan-based nanoparticles for improving immunisation against hepatitis b infection bivalent mucosal peptide vaccines administered using the lcp carrier system stimulate protective immune responses against streptococcus pyogenes infection lipid core peptide/poly(lactic-co-glycolic acid) as a highly potent intranasal vaccine delivery system against group a streptococcus phase i study of a neisseria meningitidis liposomal vaccine containing purified outer membrane proteins and detoxified lipooligosaccharide preparation and efficacy of a live newcastle disease virus vaccine encapsulated in chitosan nanoparticles enhanced mucosal immune responses against tetanus toxoid using novel delivery system comprised of chitosan-functionalized gold nanoparticles and botanical adjuvant: characterization, immunogenicity, and stability assessment assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide immunisation with tumor neoantigens displayed on t7 phage nanoparticles elicits plasma antibody and vaccine-draining lymph node b cell responses engineering magnetosomes for high-performance cancer vaccination induction of immune response to the 17 kda ompa burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an omp nanoemulsion-based vaccine treg inducing adjuvants for therapeutic vaccination against chronic inflammatory diseases, front. immunol cancer immunotherapy: a treatment for the masses dendritic cell targeting peptide-based nanovaccines for enhanced cancer immunotherapy designer vaccine nanodiscs for personalized cancer immunotherapy biomimetic magnetosomes as versatile artificial antigen-presenting cells to potentiate t-cell-based anticancer therapy erythrocyte membrane-enveloped polymeric nanoparticles as nanovaccine for induction of antitumor immunity against melanoma artificially cloaked viral nanovaccine for cancer immunotherapy therapeutic vaccination using cationic liposome-adjuvanted hiv type 1 peptides representing hla-supertype-restricted subdominant t cell epitopes: safety, immunogenicity, and feasibility in guinea-bissau ) fusion peptide and gm-csf dna elicits potent and prolonged cd8( + ) t cell-dependent anti-tumor immunity in mice de berardinis , vectorized delivery of alpha-galactosylceramide and tumor antigen on filamentous bacteriophage fd induces protective immunity by enhancing tumor-specific t cell response targeting mutated plus germline epitopes confers pre-clinical efficacy of an instantly formulated cancer nano-vaccine heat shock protein-peptide and hsp-based immunotherapies for the treatment of cancer pre-clinical development of listeria-based nanovaccines as immunotherapies for solid tumours: insights from melanoma antitumor humoral and t cell responses by mucin-1 conjugates of bacteriophage qbeta in wild-type mice intradermal delivery of vaccine nanoparticles using hollow microneedle array generates enhanced and balanced immune response effect of tlr ligands co-encapsulated with multiepitopic antigen in nanoliposomes targeted to human dcs via fc receptor for cancer vaccines tumor growth inhibition by msteap peptide nanovaccine inducing augmented cd8( + ) t cell immune responses, drug deliv heat-shock protein peptide complex-96 vaccination for recurrent glioblastoma: a phase ii, single-arm trial phase 1 clinical trial of her2-specific immunotherapy with concomitant her2 kinase inhibition enhanced intratumoral delivery of sn38 as a tocopherol oxyacetate prodrug using nanoparticles in a neuroblastoma xenograft model biomimetic nanotechnology toward personalized vaccines authors would like to thank iit bombay, ministry of human resource and development, government of india, for their research fellowships. key: cord-354972-nc496v6s authors: margolin, emmanuel; burgers, wendy a.; sturrock, edward d.; mendelson, marc; chapman, rosamund; douglass, nicola; williamson, anna-lise; rybicki, edward p. title: prospects for sars-cov-2 diagnostics, therapeutics and vaccines in africa date: 2020-09-10 journal: nat rev microbiol doi: 10.1038/s41579-020-00441-3 sha: doc_id: 354972 cord_uid: nc496v6s the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has resulted in a global pandemic, prompting unprecedented efforts to contain the virus. many developed countries have implemented widespread testing and have rapidly mobilized research programmes to develop vaccines and therapeutics. however, these approaches may be impractical in africa, where the infrastructure for testing is poorly developed and owing to the limited manufacturing capacity to produce pharmaceuticals. furthermore, a large burden of hiv-1 and tuberculosis in africa could exacerbate the severity of infection and may affect vaccine immunogenicity. this review discusses global efforts to develop diagnostics, therapeutics and vaccines, with these considerations in mind. we also highlight vaccine and diagnostic production platforms that are being developed in africa and that could be translated into clinical development through appropriate partnerships for manufacture. coronaviruses are ubiquitous rna viruses that are responsible for endemic infections in humans and other animals, and sporadic outbreaks of potentially fatal respiratory disease in humans. four human coronaviruses, namely hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1, circulate in the human population, causing the common cold, with some causing potentially life-threatening disease in infants, young children, older individuals and individuals who are immunocompromised 1 . in the recent past, two additional coronaviruses have crossed the species barrier from other animals to infect humans. these are severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), which emerged in 2002 and 2012, respectively 2,3 . in december 2019, a novel betacoronavirus, subsequently named sars-cov-2, was implicated in an outbreak of respiratory disease in wuhan, china 4 . the first cases to be reported presented as atypical pneumonia and were traced to the huanan seafood wholesale market, although cases without any association with the market, and predating the putative index cases, were subsequently recognized. following these first reports, community transmission rapidly ensued, culminating in a global pandemic 5, 6 . the virus is speculated to have originated in bats and possibly to have passed through another host before infecting humans, but an intermediate host or intermediate hosts have yet to be defined. this remains the subject of considerable debate, and recent work suggests that the host receptor-binding motif of sars-cov-2 was acquired through recombination with a pangolin coronavirus [7] [8] [9] , but further work is needed to establish the origin of the virus. infection with sars-cov-2 in humans manifests as coronavirus disease-2019 (covid19) , a spectrum of disease that ranges from asymptomatic infection to acute respiratory distress syndrome with multisystem involvement. older individuals and individuals with co-morbidities are at greatest risk 10 . in individuals who are symptomatic, fever and cough are most commonly reported, although sore throat, shortness of breath, fatigue, anosmia, dysgeusia and gastrointestinal involvement are also frequently observed 5, 11 . extrapulmonary manifestations of covid-19 are being increasingly recognized. among adults with preexisting diabetes mellitus, diabetic ketoacidosis may be a common complication and is associated with a poor prognosis 12, 13 . according to the international diabetes federation, africa has an estimated 19.4 million adults aged between 20 and 79 years living with diabetes and is the region with the highest proportion of undiagnosed diabetes 14 . neurological and neuropsychiatric complications have also been recognized as presenting or complicating factors 15 . children generally have a milder course of disease and are more likely to be asymptomatic, although recent reports have described hyperinflammatory shock in children who were previously asymptomatic that seems similar to kawasaki disease [16] [17] [18] . further studies are required to determine the prevalence of this phenomenon and to define the immunological the first documented cases in a disease outbreak. a rare condition associated with inflammation in blood vessels that most commonly presents in children under 5 years of age. drivers of the illness. however, the contrasting presentation of covid-19 in children and adults suggests that the immune responses of children and adults to sars-cov-2 may be different. the virus continued to spread globally, prompting the implementation of radical travel restrictions and social distancing measures 19 . at the time of writing this article, the virus has resulted in over 25 million confirmed infections and has claimed the lives of over 850,000 people 6 . as of 8 august 2020, there have been over 1.2 million confirmed cases of covid-19 in africa, with 29,833 deaths reported (africa cdc) there is concern that the pandemic may pose an even greater risk to countries in africa owing to their weak health-care infrastructure, large burden of co-infections, including hiv-1 and tuberculosis, and ongoing outbreaks of emerging and re-emerging infections such as ebola virus (democratic republic of congo) and lassa haemorrhagic fever (nigeria) that will divert much-needed resources away from the fight against covid-19 (ref. 20 ) ( fig. 1 ). differences in global population demographics and health status are also likely to affect the severity of the pandemic in different regions and are a major concern in africa ( fig. 2 ). in addition to the health-care infrastructure, the general infrastructure throughout africa is also highly variable, and thus access to appropriate medical care is an important determinant of covid-19 disease outcome. the number of hospital beds in a population of 10,000 individuals varies from as low as 1 in mali to 32 in libya. however, libya is an exception for the region, and many central and west african countries are at the lower end of this range and generally report fewer than 10 beds per 10,000 individuals. this is in stark contrast to other developed countries such as germany and the usa, where 80 and 28.7 beds per 10,000 individuals are available, respectively. a similar trend is also seen for the number of doctors per 10,000 individuals. in more than 10 african countries, less than 1 doctor is available (per 10,000 individuals), whereas germany and the usa report 42 and 26 doctors (per 10,000 individuals), respectively 21 . concerns have been raised regarding the impact of the pandemic on other diseases and access to essential medicines 22 . for example, according to a newspaper article, the ministry of health in zimbabwe reported a 45% increase in malaria infections compared with 2019 (ref. 23 ). many african countries lack the capacity to implement widespread testing, including the identification of asymptomatic and mild infections that are major drivers of the pandemic 24 . although it is difficult to determine the number of tests conducted in many africa countries, the publicly available data clearly highlight the limited testing capacity on the continent. south africa is currently conducting the largest number of tests per 10,000 individuals (0.5/10,000 individuals), whereas many other countries, including ethiopia, nigeria, zimbabwe, tunisia, senegal and rwanda, perform fewer than 0.1 tests/10,000 individuals. this is markedly less than in the usa (1.54/10,000 individuals), the uk (0.9/10,000 individuals), italy (0.91/10,000 individuals) or germany (0.55/10,000 individuals) 25 . similar infrastructure limitations constrain the development of prophylactic vaccines and therapeutic interventions, which results in a concerning reliance on developed countries. another important consideration in the response to the pandemic in africa will be to limit the impact of the virus on vulnerable economies where prolonged lockdowns may not be feasible. the first case of covid-19 in africa was reported in egypt on 14 february 2020; subsequently, infections -19) pandemic. this is worsened by the high burden of infectious diseases, which may worsen disease outcome and compete for the available resources. a further challenge is the dire economic consequences of prolonged lockdowns in countries with weak economies. www.nature.com/nrmicro have been documented in 44 other african countries, with south africa reporting the highest number of cases 6 . interestingly, in spite of the obvious challenges in combatting the growing pandemic, african countries have observed a delay in the exponential growth trajectory that has been described by countries in the developed world 26 . this may be partly attributable to lower testing capacity in the region and the impact of implementing lockdowns in the early phase of the pandemic. the warmer climate has also been proposed to influence the spread of covid-19, which could explain the delayed pandemic in africa compared with the rest of the world, although this is largely speculative (box 1). the transition into winter in southern africa has been accompanied by an increase in sars-cov-2 infections, further complicated by seasonal influenza and limited influenza vaccine availability. in this review, we discuss the global efforts to develop diagnostic tests and therapeutic options to treat covid-19, as well as the vaccine platforms for immunization, with a focus on the opportunities and challenges for africa. the diagnosis of sars-cov-2 poses a major challenge owing to the prevalence of asymptomatic infections, pre-symptomatic infections with high viral loads in the upper airways (probably at peak infectivity) and the range of non-specific symptoms that manifest in individuals who are symptomatic 5, 24 . widespread testing is therefore critical to identify infected individuals who are asymptomatic, pre-symptomatic and symptomatic, and to enable contact tracing and isolation 27 . whereas this has been highly successful in countries such as germany and south korea, it is not generally possible in most african countries where the infrastructure is weak. indeed, in countries such as south africa, where widespread community testing was attempted, this has resulted in a very large backlog of tests and delays of weeks for returning test results, which are then rendered meaningless for quarantining of cases and containment 28 . in many african countries, testing is only available for severe cases of presumed covid-19, and self-isolation is recommended for less severe cases. therefore, reported cases and true prevalence do not equate. accordingly, the capacity provided by academic laboratories and pharmaceutical companies is being leveraged to increase testing capacity further, as has been necessary even in developed countries 29 . diagnosis of acute infection is by pcr with reverse transcription of respiratory tract specimens, which is generally performed in central laboratories with specialized equipment 30 . scale-up of testing is a major challenge for countries in africa, owing to laboratory infrastructure, costs and availability of test reagents that are largely imported and currently stretched global supply chains. a recently launched, continent-wide initiative, population demographics and prevalence of known co-morbidities for each of the six world health organization regions. although africa reports a lower average age compared with other regions, the burden of infectious disease is disproportionately high. both hiv and tuberculosis are associated with an increase in coronavirus disease-2019 (covid-19) disease severity, and their prevalence in africa will increase the risk of fatal infection for a large number of people. there is also a large proportion of individuals in africa with raised blood pressure, which is a known risk factor for severe disease. other known co-morbidities, including raised cholesterol, raised glucose and obesity, are less prevalent in africa compared with the other reported regions. raised blood pressure (systolic blood pressure ≥140 mm/hg or diastolic blood pressure ≥90 mmhg), raised fasting blood glucose levels (≥7 mmol/l or taking medication), raised total cholesterol levels (≥5 mmol/l) and body mass index (bmi) >25 are reflected as age-standardized estimates. all data shown reflect the latest available data from the world health data platform (global health observatory). the number of people living with hiv-1/aids (in millions) reflects the population of individuals who were infected in 2018, tuberculosis cases shown reflect the number of incident cases in 2018 and malaria cases reflect the estimated number of cases in 2017. nature reviews | microbiology the africa medical supplies platform, seeks to leverage collective purchasing for procurement of testing supplies, personal protective equipment, medical equipment and even, potentially, future vaccines 31 . in addition, repurposing of rapid, automated molecular diagnostics platforms such as genexpert® (cepheid), which is widely used for the diagnosis of tuberculosis in south africa, has the potential to decentralize and accelerate testing in certain countries, including using mobile testing centres, although test kits are also in limited supply. however, this has to be understood in the light of the potential for unintended consequences on the management of tuberculosis, with fewer diagnostic platforms being available as a result of increased sars-cov-2 testing. testing for tuberculosis in south africa has reportedly decreased by 50% during the lockdown period and, concurrently, the weekly average of microbiologically confirmed tuberculosis cases decreased by 33% (ref. 32 ). recently, a rapid method of heating samples prior to quantitative pcr with reverse transcription has shown promise to improve the turnaround time for testing and bypasses the need to order rna extraction reagents or kits 33 . furthermore, a rapid crispr-cas12-based test has also been developed to diagnose infection from respiratory sample-derived rna 34 . the test yields a result within 1 h and is less reliant on sophisticated laboratory infrastructure and test reagents that are in limited supply. implementing this test in africa could be a useful way of expanding the current testing capacity and could offer a faster turnaround time for high-priority cases. serology-based testing approaches have been proposed, but the delay between infection and the development of detectable antibodies (within 19 days) renders this approach impractical for the diagnosis of acute infection 35, 36 . nonetheless, these tests are critical for seroprevalence studies and to identify appropriate donors for convalescent sera, and potentially for the isolation of monoclonal antibodies that can be developed as therapeutics. serology studies are also crucial for understanding the longevity of the antibody response after infection, with the key caveat that it is not known whether humoral responses are a correlate of immunity against the virus. in addition, preliminary data suggest that not all individuals who are infected may seroconvert 37 , and early evidence is emerging that antibody levels may wane rapidly during the convalescent phase 38 . several serological assays have already been developed, and binding antibodies against the spike and nucleocapsid proteins are both indicative of past sars-cov-2 infection 39, 40 . many of these assays are also commercially available, but their specificity and sensitivities seem to be variable 41 . a major outstanding question is which antigen, or region of the antigen, is most appropriate for serology testing. most assays have favoured the spike glycoprotein for the detection of an immune response against the virus, although it is worth noting that the nucleocapsid is the most abundant viral antigen 42 . recent work has suggested that the receptor-binding domain alone may be sufficient to detect antibody responses to sars-cov-2, and given that it is not conserved between coronaviruses, its use may limit cross-reactivity arising from other coronavirus infections 43 . nonetheless, a nucleocapsid-based elisa (enzyme-linked immunosorbent assay) may be the easiest to implement in an african context as the antigen could easily be produced locally at low cost. nucleocapsid could be produced in escherichia coli, pichia pastoris or even in plants, as has been reported for the nucleocapsid proteins of three bunyaviruses, two of which were used successfully in validated assays [44] [45] [46] . moreover, the biovac institute in south africa has the capacity for bacterial fermentation and the required infrastructure for downstream processing, and a new plant-based production facility (cape bio pharms) is currently generating s1 protein derivatives as reagents. although the spike glycoprotein is heavily glycosylated and needs to be expressed in a more complex expression host to ensure appropriate post-translational modifications, both mammalian cells and plants would be suitable to produce both spike and nucleocapsid, and novel approaches to enhance recombinant glycoprotein production in plants have also been developed in south africa 47 . given the optimistic development timeline of 12-18 months before any vaccines could be available for widespread use, it is clear that these efforts will not box 1 | potential impact of climate on sars-cov-2 dissemination the comparatively low incidence of coronavirus disease-2019 (covid19) in africa has raised the possibility that climate could influence the spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). there is some circumstantial evidence describing a possible association between higher temperatures and lower severity of covid-19 to support this hypothesis; however, outbreaks in malaysia, hong kong, australia and south africa seem to be inconsistent with this theory as large numbers of infections have been reported despite higher temperatures [130] [131] [132] . the influence of climate could potentially account for the severity of the pandemic in central china and northern italy, where winter may have been particularly conducive to the spread of the virus 131 . these cold conditions are reminiscent of the environment in which sars-cov first emerged in china in november 2002 (ref. 133 ). although these observations are compelling, it is noteworthy that many of these studies have yet to undergo formal peer review, and the accuracy of species distribution models is constrained by variability in global testing capacity 134 . for example, infections in many african countries are expected to be an underestimate that reflects the lower number of tests conducted. it is also acknowledged that numerous other variables could influence the spread of the virus and may confound interpretations of the impact of climate. these variables may include variation in population density and age distribution, timely lockdown measures, adherence to social distancing protocols or even childhood vaccination with mycobacterium bovis bacille calmette-guérin as examples 135 . the impact of differing behaviour, with increased social mixing, in the winter months also cannot be discounted 136 . as with many respiratory pathogens, both middle east respiratory syndrome (mers-cov) and sars-cov exhibit decreased viability in the laboratory following exposure to increasing temperature and humidity 137, 138 . similar observations have also been reported for influenza virus and respiratory syncytial virus, for which the incidence of infection is highest under cold and dry conditions, which results in seasonal cycles of infection 139, 140 . a similar seasonality has also been observed for other endemic human coronaviruses, which led to the speculation that sars-cov-2 may also conform to a seasonal cycle of infection 136 . however, although all four endemic human coronaviruses (hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1) exhibit a marked winter seasonality 141 , the pathogenic human coronaviruses (mers-cov and sars-cov) do not conform to such a defined infection cycle. for example, mers-cov generally occurs mostly during summer months in the middle east despite temperatures often exceeding 50 °c 142 . by contrast, the highest incidence of sars-cov was reported during the winter months, although the outbreak continued to spread throughout spring in hong kong 143, 144 . therefore, more research is needed to define the impact of climate on the spread of sars-cov-2. a diagnostic test that measures the presence of antibodies in blood to determine exposure to pathogens or to diagnosis autoimmune diseases. sera obtained from individuals who have recovered from an infectious disease and contain antibodies against the pathogen. www.nature.com/nrmicro affect the first wave of the pandemic 48 . more importantly, the lack of manufacturing capacity in africa and the global demand for immunization against the virus will further delay the availability of vaccines in the region. repurposing existing drugs presents a feasible short-term strategy to manage the pandemic, especially given that some of the drug candidates are already available and have an established safety profile in humans 49 . these drugs would face lower regulatory barriers for approval and, in addition to being used for treating active infections, may have potential to be used as prophylactics for individuals at high risk, such as health-care workers or those who have been in contact with documented cases of infection. currently, two treatments have been shown to have an effect on the outcome of covid-19. the broad-spectrum antiviral drug remdesivir has been shown to shorten the recovery time in adults admitted to hospital with severe covid-19 in a publication of preliminary results from a double-blind, randomized, placebo-controlled trial in the usa 50 . however, remdesivir did not reduce mortality. by contrast, initial data from the recent recovery trial in the uk suggest that daily oral or intravenous doses of dexamethasone (10 mg for 10 days) reduced mortality by one-fifth in hospitalized patients with proven covid-19 requiring oxygen therapy, and that mortality was reduced by one-third in patients who needed mechanical ventilation 51 . it had no effect on patients hospitalized with covid-19 who were not requiring oxygen. the reductions in mortality were seen in patients whose symptoms started >7 days before receipt of the drug. the fact that a commonly used corticosteroid could reduce mortality in this trial is promising, as numerous other corticosteroids such as prednisolone and hydrocortisone (which were options in the recovery trial in pregnant women) are equally available, and some of them are manufactured in africa, which means that access may be less of an issue than for other more novel medicines. more commonly available medicines have been, and some continue to be, used in investigational treatments for covid-19. the commonly available antimalarials chloroquine and hydroxychloroquine were among the first to be investigated. initial studies were small and underpowered, and some combined hydroxychloroquine with azithromycin 52 and some proved highly controversial in relation to their conduct, leading to retraction 53 . one of the arms of the recovery trial included hydroxychloroquine, and on 4 june 2020 the independent data monitoring committee review of the data concluded that there was no beneficial effect of hydroxychloroquine in patients hospitalized with covid-19 (ref. 54 ). shortly after, the world health organization (who) announced that recruitment for the hydroxychloroquine arm of the solidarity trial was being stopped 55, 56 . all experimental treatments should either be introduced into properly conducted clinical trials or, if a country decides to use such a medicine outside a trial, then it should be controlled according to the who's monitored emergency use of unregistered interventions (meuri) framework, whereby it can be ethically appropriate to offer individuals investigational interventions on an emergency basis, in the context of an outbreak characterized by high mortality 57 . large-scale adaptive studies such as the recovery and solidarity trials continue, and such trials will reduce the time taken for randomized clinical trials 58 . several african countries, including south africa, burkina faso and senegal, are in the process of joining the solidarity study. similarly, small studies are ongoing in several countries, looking at the utility of convalescent plasma from patients who recently recovered from covid-19 as potential prophylaxis or treatment 59 . the need for randomized control trials using this treatment modality has been stressed 60 . unlike other investigational medicines, convalescent plasma can be readily produced, even in low and middle-income countries, through the national blood transfusion service, making it an attractive option for study. however, scaling production for use is the rate-limiting step for this intervention. preliminary studies identified monoclonal antibodies with the ability to neutralize sars-cov-2, which may also be important candidates for both treatment and prophylaxis, although similar issues with manufacture are a challenge 61, 62 . there is increasing recognition that pathophysiology of severe covid-19 includes an appreciable component of hyperactivation of inflammatory responses, manifesting as a cytokine storm and secondary haemophagocytic lymphocytic histiocytosis. in addition to the findings relating to dexamethasone detailed above, various immune-modulating drugs have been proposed as treatment options for covid-19. the il-6 inhibitors tocilizumab (actemra; roche) and sarilumab (kevzara; sanofi and regeneron), which are used to treat arthritis, are already being used in patients with covid-19 (nct04327388) 63 . their mechanism of action involves the prevention and the inhibition of the overactive inflammatory responses in the lungs. both drugs have entered phase iii clinical trials for sars-cov-2. a late-stage clinical trial with another il-6 inhibitor, siltuximab (sylvant; eusa), started in italy in mid-march (nct04322188). anti-inflammatory drugs used in combination with an antiviral drug such as remdesivir may increase the potential of the drug to improve disease outcome 64 . genentech has recently initiated a phase iii trial (remdecta) to study the efficacy and safety of tocilizumab and remdesivir in patients hospitalized with severe covid-19 pneumonia (nct04409262). additionally, the covacta study (nct04320615) will evaluate tocilizumab and standard of care versus standard of care alone in a similar cohort 65 . patients who have chronic medical conditions may be at higher risk for serious illness from covid-19, including those with pulmonary fibrosis 66 . the antifibrotic drug pirfenidone (genentech) has already entered a study to evaluate its efficacy and safety (nct04282902). recombinant angiotensin-converting enzyme 2 (ace2; apn01) that lacks the transmembrane region of the protein was developed by apeiron biologics for the treatment of acute lung injury and pulmonary artery hypertension. the soluble ace2 has the potential to reduce lung injury by activating the anti-fibrotic and a disproportionately large cytokine response that promotes inflammation and is harmful to the host. nature reviews | microbiology anti-inflammatory angiotensin (1-7)-mas receptor axis of the renin-angiotensin-aldosterone system, and by acting as a decoy and preventing infection by binding to the sars-cov-2 virus and inactivating it. apn01 is being tested in a phase i trial in china, and approval has been secured to carry out phase ii trials in austria, germany and denmark (nct04335136). currently, there are no targeted therapies for covid-19. however, numerous drug discovery programmes are in progress, and a recent study reported a structure-based drug design strategy, as well as virtual and high-throughput screening to identify lead compounds that bind to the main protease of the virus (m pro ; also known as 3cl pro ) 67 . the active site of the protease is highly conserved among coronaviruses, making a strong case for pursuing an m pro -targeting drug. the organoselenium drug ebselen, which is an anti-inflammatory and antioxidant, showed high affinity for m pro and showed promising antiviral activity (concentration that gives half-maximal response ec 50 = 4.67 μm). thus, the presented approach may greatly accelerate the discovery of drug leads with potential in the clinic. the drug discovery and development centre (h3d) based at the university of cape town is the only fully integrated drug discovery centre in africa that has taken a drug into a phase ii clinical trial. the centre has very strong collaborations with the pharmaceutical industry and mmv, a leading product development partnership, as well as the infrastructure and expertise to find potential therapies against covid-19. h3d has assembled chemical libraries for its malaria and tuberculosis projects that could be screened to identify possible drug leads against sars-cov-2; however, this will require additional resources and funding because the centre is contractually focused on antimalarial and anti-tuberculosis drug development. the infrastructure for large-scale, high-volume vaccine manufacturing is largely absent in africa, and the rapidly escalating covid-19 pandemic highlights the urgent need for capital investment in the region to lessen reliance on developed countries. the few facilities that are available are specialized, and are not well-suited to produce vaccines for sars-cov-2 (table 1) . it is also anticipated that it would take a minimum of 18 months to build a suitable manufacturing plant under ideal conditions, and therefore to contribute to the global covid-19 vaccine initiative, african developers will need to outsource large-scale manufacturing in the short term. the african vaccine manufacturing initiative, which aims to develop local manufacturing capacity in africa, has established a working group and is actively engaged with key stakeholders to meet the local need for a vaccine. innovative biotech (nigeria) has already partnered with medigen (usa) and merck (germany) to apply their insect cell production platform to producing virus-like particles with the intention of initiating a clinical trial in nigeria. similarly, the ethiopian public health institute (ephi) is planning to partner with techinvention (india) to produce the sars-cov-2 spike protein in a yeast-based fermentation system, although limited details are available (personal communication, s. agwale, ceo of innovative biotech). last, biovac (south africa) has modern facilities at a modest scale and has initiated a feasibility study for a large-scale facility with an annual minimum production capacity of 100 million vaccine doses for covid-19 and future pandemic vaccines, as well as vaccines for routine immunization use (personal communication, p. tippoo, head of science and innovation, biovac). given the global demand for a covid-19 vaccine, it is likely that even when a suitable candidate is approved for human use, there will be a considerable delay before it is available in africa. this is not unprecedentedduring the 2009 h1n1 influenza pandemic, a global shortage of influenza vaccines resulted in limited supplies being provided for countries in the region, and, in fact, the vaccines only became generally available after 2010 (ref. 68 ). this unfortunate, but entirely plausible, scenario may necessitate prioritizing high-risk groups, such as health-care workers and older individuals, to receive the first sars-cov-2 vaccines to reach africa. more than 100 vaccine candidates are currently in preclinical development around the world, and 15 vaccines are already being tested in clinical trials 69, 70 (table 2) . these vaccines are mostly focused on eliciting immunity against the spike glycoprotein, although other viral antigens may also have a role in vaccine-mediated protection (box 2). the speed of clinical deployment of these vaccines is unprecedented, but there are concerns regarding the longevity of immune responses and the potential although the rapid progress to clinical testing is encouraging, it is still too early to determine whether they will confer immunity against sars-cov-2 infection or whether they will ameliorate the disease course following infection. the only peer-reviewed report of a sars-cov-2 vaccine in clinical trial to date is for cansino biologics' ad5-ncov vaccine, which recently completed phase i testing. encouragingly, the vaccine elicited both binding antibodies and antigen-specific t cells, although, disappointingly, only 50% of volunteers developed neutralizing antibodies in the low (5 × 10 10 viral particles) and medium (1 × 10 11 viral particles) dose regimens. however, 75% of the high-dose group (1.5 × 10 11 viral particles) developed neutralizing antibodies. perhaps unsurprisingly, the high-dose group also reported a higher incidence of adverse effects following vaccination and only the low and intermediate doses will be pursued in phase ii trials 71 . despite the absence of suitable facilities for current good manufacturing practice (cgmp)-compliant vaccine or therapeutics manufacturing in most of africa, considerable expertise in preclinical vaccine development is also available in academic institutes, and vaccines could be manufactured on contract for clinical trials as was the case for the south african aids vaccine initiative 72 . accordingly, groups at the university of cape town (south africa), the national research centre (egypt) and the kenya aids vaccine initiative (kavi) have all confirmed that early-stage research on sars-cov-2 vaccine development is underway -although further details have not been disclosed 73 . important considerations for these vaccines will be the cost, their safety in individuals who box 2 | sars-cov-2 virus structure and targets for vaccine development severe acute respiratory syndrome coronavirus 2 (sars-cov-2) comprises pleomorphic virions, ranging from 60 to 140 nm in diameter, with prominent glycoprotein spike proteins projecting from the virus surface 4 . the virion also contains the membrane, envelope and nucleocapsid proteins, which encapsulate the viral genome and accessory proteins (see the figure, left). the spike protein is a glycosylated type 1 fusion protein that mediates infection by binding the host membrane-anchored angiotensin-converting enzyme 2 (ace2) 145 . the glycoprotein is organized into extracellular (s1) and membrane-spanning (s2) subunits, which mediate receptor binding and membrane fusion, respectively (not shown). binding of the spike protein to ace2 results in a conformational change that enables the dissociation of the s1 subunit and the insertion of the fusion peptide into the host membrane 146 . the spike glycoprotein is the primary target of vaccine development, based on the premise that neutralizing antibodies against spike will prevent viral entry into susceptible cells (see the figure, right). this is supported by preclinical immunogenicity studies, for the related middle east respiratory syndrome coronavirus (mers-cov) and sars-cov, for which immunization with spike-based vaccines elicited protective antibody responses 147, 148 . more recently, neutralizing antibodies against the sars-cov-2 spike have been reported in natural infection; these are readily elicited and frequently target the receptor-binding domain in s1 (ref. 35 ). the potential role of cell-mediated immunity in coronavirus vaccines generally has not been as well explored. it is reasonable to expect that cellular immune responses would contribute to viral clearance and ameliorate the severity of the disease, as well as support the development of antibody responses. accordingly, robust and durable cellular responses have been observed against the spike, membrane, envelope and nucleocapsid proteins in patients who recovered from sars coronavirus infection [149] [150] [151] . ultimately, both cell-mediated and humoral responses are desirable in a vaccine, especially given the observation that cellular responses are longer lived than antibodies following infection with sars coronaviruses 152, 153 . mhc, major histocompatibility complex. humoral immunity: • neutralizing antibodies prevent interaction of spike with ace2 • antibody effector functions can contribute to viral clearance • b cell memory for durable immunity genetic immunization with plasmid dna is perhaps the easiest vaccine modality to develop for clinical trials as the manufacturing process is well established, the incumbent costs are low compared with other platforms and multiple clinical trials have shown their safety. technological advances have also substantially reduced the time from identifying the viral sequence to initiating immunizations in humans 74 . accordingly, dna vaccines have been advanced into the clinic in response to several emerging pathogens, including mers-cov, and inovio pharmaceuticals (usa) have already completed recruiting participants to initiate a phase i trial with a candidate dna vaccine against sars-cov-2 (nct04336410) 75 . recent preclinical data demonstrated that the vaccine elicited neutralizing antibodies in both mice and guinea pigs, and an unrelated study reported that immunization with a dna vaccine protected against viral challenge in macaques 76, 77 . genetic immunization is well-suited to clinical development for africa, and candidate vaccines could be manufactured to cgmp standards using one of the contract manufacturers offering this service. however, there are no licensed human vaccines based on this platform, and the current delivery methods are not suitable for large-scale immunization. host-restricted viral vectors are another promising vector platform for immunization in africa 78 . replication-deficient chimpanzee adenovirus-based vaccines have shown promise for several emerging viruses, and given their simian origin, they circumvent concerns for vector-specific immunity as was observed when using human adenoviral vectors for immunization 79 . a single dose of a mers-cov-2 vaccine using this platform was reported to elicit protective immunity in non-human primates 80 . more recently, a single immunization with chadox1 encoding the sars-cov-2 spike protected against pneumonia and lowered viral loads in both bronchoalveolar lavage and respiratory tract samples in macaques following challenge 81 . this effect was observed in the absence of high titres of neutralizing antibodies and the impact of the vaccine was to ameliorate severe disease rather than to prevent infection. although it is disappointing that the vaccine did not confer sterilizing immunity in monkeys, it is noteworthy that the monkeys only received a single immunization and that the inoculum used for challenge was high. it should be noted that the high-challenge inoculum was conceived to determine whether immunization resulted in vaccine-mediated enhancement of infection, and that there was no evidence to suggest that this would be a concern 81 . this is the vaccine being pursued by the university of oxford in collaboration with astrazeneca that is now in phase ii testing. a clinical trial for this vaccine has recently been initiated in johannesburg (south africa), and this is the first vaccine for sars-cov-2 to be tested in africa. the manufacturing cost of chadox1 would be far less than for a subunit vaccine and, moreover, no adjuvant is needed for immunization. poxvirus-based vectors are similarly attractive: they elicit strong humoral and cellular immune responses, can be manufactured at low cost and are stable in the absence of a sustained cold chain 78, 82 . in addition, they can accommodate larger genetic insertions, which could be exploited to encode multiple sars-cov-2 genes (such as the spike, nucleocapsid, membrane and envelope antigens) and could potentially produce virus-like particles. suitable examples of candidate poxvirus vectors include the attenuated orthopoxviruses modified vaccinia ankara (mva) 83 and nyvac 84 , the avipoxviruses canarypox virus (alvac) 85 and fowlpox virus (fwpv) 86 , and the capripoxvirus lumpy skin disease virus (lsdv) 87 . mva is the most widely explored of these vectors. having been attenuated by more than 570 passages in chick embryo fibroblast cells, mva has a well established safety record, including in individuals who are immunocompromised, and has recently been approved as a vaccine against smallpox 88, 89 . nyvac was engineered by the purposeful deletion of 18 genes involved in host range and pathogenicity; it causes no disseminated disease in immunodeficient mice, like mva, and is unable to replicate in humans 90 . several mva-vectored vaccines of particular relevance to africa have shown promise in clinical trials, usually in prime-boost regimens together with other vectors such as dna or adenovirus. these include vaccines against hiv-1 (ref. 91 ), mycobacterium tuberculosis 92 and box 3 | immunological challenges for sars-cov-2 vaccine development two concerns have been raised that could undermine the vaccines against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in clinical testing: the longevity of immunity, and the potential for adverse effects following sars-cov-2 infection in immunized volunteers. the durability of antibody responses has implications for vaccine development, as immunization may need to induce stronger immunity than natural infection. this concern is partly due to observations of waning neutralizing antibody titres after sars coronavirus infection, and a lack of knowledge regarding the potential for sars-cov-2 re-infection [154] [155] [156] . encouragingly, preliminary data suggest that rhesus macaques may be resistant to challenge with sars-cov-2 after clearing the primary infection 157 . the duration of this protection remains unclear, as do the correlates of immunity. low neutralizing antibody titres were recently reported in 30% of patients who recovered from mild infection with sars-cov-2, which suggests that cellular responses may have an important role in viral clearance. however, it is plausible that neutralizing antibody titres correlate with disease severity and merely reflect the extent of antigenic stimulation 61 . another concern is vaccine-induced enhancement of infection. this can manifest as either antibody-dependent enhancement or cell-mediated inflammatory responses that result in pathology following exposure to the virus. accordingly, type 2 t helper cell-mediated lung pathology with eosinophilic infiltrates has been observed in vaccinated and challenged animals for both middle east respiratory syndrome coronavirus (mers-cov) and sars-cov [158] [159] [160] . the potential impact of antibodydependent enhancement in the context of coronavirus vaccines has not been as well defined, although the phenomenon has been described for a monoclonal antibody targeting the mers coronavirus spike glycoprotein 161 . preliminary data suggest that antibody-dependent enhancement may account for the severity of covid-19 in some cases, where previous exposure to other coronaviruses may have elicited responses that enhanced infection, although this remains to be determined 162 . www.nature.com/nrmicro ebola virus 93 . lsd, a notifiable disease of cattle worldwide, is prevalent in most african countries, and the live-attenuated neethling vaccine strain is widely used to control the disease on the continent 94 . lsdv is being developed both as a multivalent cattle vaccine vector 87, 95 and as a host-restricted hiv-1 vaccine vector 96 . it has been shown to have no adverse effects in immunodeficient mice, and although this vector could not be used in countries free of lsdv, it has potential as a human vaccine in sub-saharan africa 96 . together with mva and nyvac, the avipoxvirus vectors alvac (attenuated canarypox virus) and fwpv are probably more realistic targets for rapid clinical development, as they have also undergone testing in humans, and alvac is already licensed for several veterinary applications 97 plant-based vaccine protein production is an emerging technology that is well-suited to resource-limited areas given the capacity of the system for rapidly scalable production, the low manufacturing costs and the less sophisticated infrastructure requirements than mammalian expression systems 98 . the platform is well established to produce diverse classes of recombinant proteins, and recent advances in expression technologies and molecular engineering have also enabled improvements in glycoprotein production in plants 47, 99 . encouragingly, a preliminary pilot study suggests that these appro aches can be applied to produce the sars-cov-2 spike in nicotiana benthamiana plants, warranting further testing of the recombinant antigen in preclinical vaccine immunogenicity models 100 . three leading plant biotechnology companies, medicago inc. (canada), ibio inc. (usa) and kentucky bioprocessing inc. (usa), have already announced the successful production of candidate virus-like particle vaccines against sars-cov-2. although plant-based manufacturing of recombinant protein antigens may be the most suitable solution for africa, it may also pose a challenge for manufacturing. the major advantages of plant-based vaccine production for sars-cov-2 in africa are the lower costs and the potential for rapid production scale-up to accommodate the large demand for a vaccine. this is best demonstrated in the context of influenza vaccine development, as a fully formulated virus-like particle vaccine was produced within 3 weeks following release of the viral sequence 101 . this rapid development timeline supported the production of 10 million doses of the vaccine within 1 month 102 . however, despite the costs to establish a gmp-compliant plant-based manufacturing facility being considerably less than those for the equivalent mammalian platform (for example, us$80-100 million versus us$250-350 million, respectively), they are not insignificant, and the capital investment required has been prohibitive for africa 98 . furthermore, there are few suitable contract manufacturing organizations worldwide, and these are already invested in their own sars-cov-2 vaccine development programmes. several recent preliminary data have suggested a possible correlation between bacille calmette-guérin (bcg) vaccination and lower prevalence and mortality due to covid-19 (refs [103] [104] [105] [106] ). the bcg vaccine is one of the most widely used vaccines worldwide and has been used to vaccinate against tuberculosis for nearly 100 years. the vaccine comprises a live, attenuated form of mycobacterium bovis, which provides protection against disseminated forms of tuberculosis in infants but gives variable protection against pulmonary tuberculosis in adults 107, 108 . non-specific cross-protection against other pathogens, including those causing respiratory tract infections, has also been documented 109 . this effect may be attributable to altered expression of host cytokines and pattern-recognition receptors, as well as the reprogramming of different cellular metabolic pathways that, in turn, increases the innate immune response to other pathogens 109, 110 . however, potential correlation between bcg vaccination and covid-19 severity should be interpreted with caution. first, it is unlikely that bcg vaccination at birth will still provide non-specific cross-protection against viral pathogens in older individuals. second, the correlation could be influenced by numerous unknown confounding factors, including variation in testing between countries, which leads to differences in the recorded case numbers; differences in average population age, ethnic and genetic backgrounds; the stage of the pandemic in each country; and different approaches to mitigating the spread of the disease in different countries. numerous clinical trials are presently underway to determine whether bcg vaccination reduces the incidence and severity of covid-19 in health-care workers and older individuals (supplementary table) . a trial has also started in egypt (nct04347876), where disease severity and mortality in patients with covid-19 will be compared between those with positive and negative tuberculin tests. in brazil, the bcg vaccine will be given to patients with covid-19 as a therapeutic vaccine to evaluate the impact on the rate of elimination of sars-cov-2, the clinical evolution of covid-19 and the seroconversion rate and titres of anti-sars-cov-2 antibodies. as the bcg vaccine has been administered to most neonates in south africa and france until recently, these trials will also investigate the effect of revaccination with the bcg vaccine. in addition, a new modified version of bcg, namely vpm1002, which expresses listeriolysin instead of urease c, will be tested in health-care workers and older individuals in germany 111 . securing a reliable supply of bcg vaccine doses could be a challenge in africa if re-immunization shows promise, as there is limited manufacturing capacity for the vaccine on the continent. historically, shortages of the vaccines were documented in 41% of countries on the continent between 2005 and 2015 (ref. 112 ). this was largely due tuberculosis diagnostic tests that involve the intradermal injection of bacterial antigens to determine whether the recipient mounts an immune response at the site of injection. nature reviews | microbiology to lack of supply, but the limited availability of financing, procurement shortcomings and ineffective vaccine management also contributed to the shortage. the low price for a bcg vaccine and limited investment has also reduced the incentive for manufacturers to redesign and improve production processes in the region. from 1988 to 2001, the 172-tokyo bcg strain was produced at the state vaccine institute in cape town, south africa; however, this was discontinued as the cost of importing the vaccine was lower than that of local manufacture. the potential impact of co-infections africa shoulders a considerable burden of co-infections. although hiv-1 and tuberculosis may be the most important infections when considering potentially enhanced covid-19 disease severity, the high incidence of malaria and helminth infections as well as multiple ongoing outbreaks of ebola virus disease, lassa fever, cholera, measles, yellow fever, hepatitis e and chikungunya virus 113 all represent infections with unknown interactions with sars-cov-2. the high prevalence of hiv-1 and tuberculosis in sub-saharan africa presents an important but largely unknown challenge for the continent with regard to covid-19. the urgent question that needs answering is whether individuals with hiv-1, or those with past or current tuberculosis, have a higher risk of infection or greater morbidity and mortality from covid-19. of the 37.9 million people living with hiv-1 globally, 25.6 million live in sub-saharan africa, and it is estimated that 64% are accessing antiretroviral therapy and 54% are virally suppressed 114 . although individuals who are immunocompetent with well-controlled hiv-1 infections may be at no greater risk for covid-19, there remains a considerable number of individuals with low cd4 counts and uncontrolled hiv-1 viraemia who may be at risk of severe disease. to date, there have been two published reports of concurrent covid-19 and hiv-1 infection 115, 116 . although the cohort was an extremely limited group of patients predominantly established on antiretroviral therapy, the pattern of clinical disease did not differ from that observed in the general population, but more research is needed to confirm this result. the severity of other respiratory infections concomitant with hiv-1 may provide some clues: although the immunopathogenesis of sars-cov-2 is probably distinct in several aspects from influenza viruses, there are some shared clinical features. hiv-1 infection is associated with a greater susceptibility to influenza virus infection, increased severity of influenza-related disease and poorer prognosis in patients who are severely immunocompromised 117 . a large south african study observed an eightfold higher incidence of influenza virus infection and a fourfold greater risk of death in the case of hiv-1 co-infection 118 . paradoxically, there is also evidence that lower inflammatory responses in individuals who are immunocompetent and infected with hiv-1 may lead to milder influenza-related disease 117 . in addition to altering the clinical course of disease, hiv-1 infections may result in poorer antibody responses that may lead to prolonged viral shedding, thereby influencing disease transmission 119 . tuberculosis, a disease that causes chronic lung damage, may also present a challenge in the covid-19 era. there were approximately 2.4 million new cases of tuberculosis in africa in 2018 (ref. 120 ). in a south african study of patients who were hospitalized for severe respiratory illness, those with influenza virus infection together with laboratory-confirmed tuberculosis had a 4.5-fold greater risk of death 121 . hiv-1 largely drives the tuberculosis epidemic in sub-saharan africa, and the 'triple-hit' of hiv-1, tuberculosis and sars-cov-2 infection is consequently of considerable concern. a preliminary study suggests that hiv-1 infection increases the risk of mortality from covid-19 by 2.39-fold, and this increased risk seemed to be independent of suppressed hiv-1 viral load due to antiretroviral therapy. individuals with current tuberculosis had a 2.7-fold greater risk of death 122 . these figures represent a modest increased risk compared with older age and co-morbidities such as diabetes in the same population, which suggests that hiv-1 and tuberculosis may not be considered major risk factors for covid-19. although this would be considered good news, further studies are awaited to confirm these initial observations. the two main potential issues for using sars-cov-2 vaccines in individuals infected with hiv-1 are safety and efficacy. however, potential safety issues are likely to be restricted to use of certain vaccine modalities, such as live-attenuated or replicating vaccines, in individuals who are highly immunosuppressed. when considering vaccine efficacy, the magnitude and durability of immunity in individuals infected with hiv-1 for both vaccination against and natural infection with sars-cov-2 is unknown. to date, there are no reports describing immune responses to sars-cov-2 in individuals infected with hiv-1. it is possible that individuals with hiv-1 may have incomplete immune reconstitution and impaired immunity that may influence vaccine safety and efficacy, even if they are receiving antiretroviral therapy, owing to persistent immune activation and incomplete recovery of t cell and b cell immunity 123, 124 . suboptimal neutralizing antibody responses have been described following immunization against influenza virus or other pathogens in individuals infected with hiv-1 (ref. 125 ). weaker antibody responses and lower influenza virus-specific memory b cell responses in individuals infected with hiv-1 were directly related to cd4 counts 126 . it will be important to test candidate vaccines for their ability to generate immune responses in a range of high-risk groups, including patients with hiv-1. several strategies may improve the magnitude and durability of vaccine responses in individuals infected with hiv-1, such as higher doses, booster immunizations and/or the use of adjuvants 127 . substantive data on the clinical and immunological interaction of hiv-1, tuberculosis and covid-19 will emerge from africa in time for improved strategies to guide clinical management of patients who are co-infected and the vaccine regimens. finally, an important additional point to note is the indirect effects of covid-19 on health in africa within the setting of a high burden of infectious diseases. the who estimates that the disruption in vaccination due to www.nature.com/nrmicro disruption in supply could put 80 million infants at risk of contracting vaccine-preventable diseases 128 . several countries have reported reduced uptake of tuberculosis testing, and patients failing to collect tuberculosis medication or antiretroviral therapy owing to overwhelmed health-care systems, lockdown interventions and public fear of contracting covid-19 (ref. 129 ). mitigating these interruptions in prevention, diagnosis and treatment, and ensuring that essential health services continue, will ultimately lower the overall impact of the covid-19 pandemic in africa. the ongoing covid-19 pandemic presents an unprecedented global humanitarian and medical challenge. although this has prompted unparalleled progress in the development of vaccines and therapeutics in many countries, it has also highlighted the vulnerability of resource-limited countries in africa. not only do these countries have limited testing capacity but the infrastructure to manufacture tests, vaccines and therapeutic drugs is largely absent, and few clinical trials are underway on the continent to combat sars-cov-2. clearly, there is an urgent need for capacity development and the available resources should focus on solutions that are specific to the needs of the continent. for example, there is an urgent need to inexpensively manufacture viral antigens for serological testing: this will determine the seroprevalence of the virus where pcr-based testing is not available for mild infections. therapeutics development should focus on repurposing existing drugs, or using convalescent plasma that can rapidly be used to treat infection and could be prioritized for individuals who are at high risk. appropriate manufacturing partnerships need to be established to produce vaccines that could be tested and licensed on the continent, to limit reliance on global initiatives that may be overwhelmed by the global demand for a vaccine. in fact, this may present an opportunity for governments to finally invest in much-needed cgmp-compliant vaccine manufacturing facilities. although the situation is unquestionably dire, africa has an important role in the global fight against covid-19, and the resilience and resourcefulness of the people are not to be underestimated. published online xx xx xxxx epidemiology, genetic recombination, and pathogenesis of coronaviruses identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus from patients with pneumonia in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study an interactive web-based dashboard to track covid-19 in real time a pneumonia outbreak associated with a new coronavirus of probable bat origin probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak emergence of sars-cov-2 through recombination and strong purifying selection clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of coronavirus disease 2019 in china covid-19 infection may cause ketosis and ketoacidosis diabetic ketoacidosis in covid-19: unique concerns and considerations neurological and neuropsychiatric complications of covid-19 in 153 patients: a uk-wide surveillance study hyperinflammatory shock in children during covid-19 pandemic kawasaki-like disease: emerging complication during the covid-19 pandemic sars-cov-2 infection in children the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak looming threat of covid-19 infection in africa: act collectively, and fast hospital beds (per 10 000 population covid-19: keep essential malaria services going during pandemic, urges who zimbabwe under strain as malaria cases surge during covid-19 fight substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) coronavirus (covid-19) testing. global change data lab covid-19 in europe: the italian lesson feasibility of controlling covid-19 outbreaks by isolation of cases and contacts south africa's coronavirus testing strategy is broken and not fit for purpose: it's time for a change let africa into the market for covid-19 diagnostics detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr african countries unite to create 'one stop shop' to lower cost of covid-19 tests and ppe covid-19 lockdowns in low-and middle-income countries: success against covid-19 at the price of greater costs an alternative workflow for molecular detection of sars-cov-2 -escape from the na extraction kit-shortage crispr-cas12-based detection of sars-cov-2 antibody responses to sars-cov-2 in patients with covid-19 antibody tests for identification of current and past infection with sars-cov-2 dynamics of igg seroconversion and pathophysiology of covid-19 infections rapid decay of anti-sars-cov-2 antibodies in persons with mild covid-19 sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients evaluation of nine commercial sars-cov-2 immunoassays developing antibody tests for sars-cov-2 the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients plant-produced crimean-congo haemorrhagic fever virus nucleoprotein for use in indirect elisa expression of rift valley fever virus n-protein in nicotiana benthamiana for use as a diagnostic antigen the expression of sars-cov m gene in p. pastoris and the diagnostic utility of the expression product co-expression of human calreticulin significantly improves the production of hiv gp140 and other viral glycoproteins in plants vaccines: status report a sars-cov-2 protein interaction map reveals targets for drug repurposing remdesivir for the treatment of covid-19 -preliminary report dexamethasone in hospitalized patients with covid-19 -preliminary report hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid-19: a multinational registry analysis no clinical benefit from use of hydroxychloroquine in hospitalised patients with covid-19. nuffield department of population health solidarity" clinical trial for covid-19 treatments hydroxychloroquine use against sars-cov-2 infection in non-human primates world health organization guidance for managing ethical issues in infectious disease outbreaks race to find covid-19 treatments accelerates effectiveness of convalescent plasma therapy in severe covid-19 patients deployment of convalescent plasma for the prevention and treatment of covid-19 neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications human neutralizing antibodies elicited by sars-cov-2 infection effective treatment of severe covid-19 patients with tocilizumab covid-19: combining antiviral and anti-inflammatory treatments phase iii trial to study combination of genentech's actemra, gilead's remdesivir versus severe covid-19 pulmonary fibrosis and covid-19: the potential role for antifibrotic therapy structure of m pro from covid-19 virus and discovery of its inhibitors setting up a platform for plant-based influenza virus vaccine production in south africa the covid-19 vaccine development landscape covid-19 vaccine development pipeline gears up safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial subtype c gp140 vaccine boosts immune responses primed by the south african aids vaccine initiative dna-c2 and mva-c hiv vaccines after more than a 2-year gap african nations missing from coronavirus trials novel vaccine technologies: essential components of an adequate response to emerging viral diseases new vaccine technologies to combat outbreak situations immunogenicity of a dna vaccine candidate for covid-19 dna vaccine protection against sars-cov-2 in rhesus macaques the evolution of poxvirus vaccines chimpanzee adenoviral vectors as vaccines for outbreak pathogens a single dose of chadox1 mers provides protective immunity in rhesus macaques chadox1 ncov-19 vaccine prevents sars-cov-2 pneumonia in rhesus macaques vaccinia-based vaccines to biothreat and emerging viruses the smallpox vaccination strain mva: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defence mechanism the poxvirus vectors mva and nyvac as gene delivery systems for vaccination against infectious diseases and cancer applications of canarypox (alvac) vectors in human and veterinary vaccination fowlpox virus as a recombinant vaccine vector for use in mammals and poultry immunogenicity of a recombinant lumpy skin disease virus (neethling vaccine strain) expressing the rabies virus glycoprotein in cattle vaccination against pox diseases under immunosuppressive conditions phase 3 efficacy trial of modified vaccinia ankara as a vaccine against smallpox nyvac: a highly attenuated strain of vaccinia virus aiming for protective t cell responses: a focus on the first generation conserved-region hiv consv vaccines in preventive and therapeutic clinical trials a phase iia trial of the new tuberculosis vaccine, mva85a, in hiv-and/or mycobacterium tuberculosis-infected adults safety and immunogenicity of a 2-dose heterologous vaccine regimen with ad26.zebov and mva-bn-filo ebola vaccines: 12-month data from a phase 1 randomized clinical trial in nairobi, kenya review: capripoxvirus diseases: current status and opportunities for control evaluation of lumpy skin disease virus, a capripoxvirus, as a replication-deficient vaccine vector a novel candidate hiv vaccine vector based on the replication deficient capripoxvirus, lumpy skin disease virus (lsdv) development and registration of recombinant veterinary vaccines. the example of the canarypox vector platform molecular pharming for low and middle income countries when plant virology met agrobacterium: the rise of the deconstructed clones calreticulin co-expression supports high level production of a recombinant sars-cov-2 spike mimetic in nicotiana benthamiana the production of hemagglutininbased virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza darpa's blue angel -pentagon prepares millions of vaccines against future global flu correlation between universal bcg vaccination policy and reduced morbidity and mortality for covid-19: an epidemiological study connecting bcg vaccination and covid-19: additional data differential covid-19-attributable mortality and bcg vaccine use in countries association of bcg vaccination policy with prevalence and mortality of covid-19 systematic review and meta-analysis of the current evidence on the duration of protection by bacillus calmette-guérin vaccination against tuberculosis the efficacy of bacillus calmette-guérin vaccination of newborns and infants in the prevention of tuberculosis: meta-analyses of the published literature non-specific effects of bcg vaccine on viral infections bcg-induced cross-protection and development of trained immunity: implication for vaccine design safety and immunogenicity of the recombinant mycobacterium bovis bcg vaccine vpm1002 in hiv-unexposed newborn infants in south africa bacillus calmette-guérin (bcg) vaccine: a global assessment of demand and supply balance outbreaks and emergencies bulletin covid-19 in patients with hiv: clinical case series co-infection of sars-cov-2 and hiv in a patient in wuhan city impact of hiv on the severity of influenza severe influenza-associated respiratory infection in high hiv prevalence setting influenza viral shedding in a prospective cohort of hiv-infected and uninfected children and adults in 2 provinces of south africa who. global tuberculosis report the impact of influenza and tuberculosis interaction on mortality among individuals aged ≥15 years hospitalized with severe www.nature.com/nrmicro respiratory illness in south africa hiv and risk of covid-19 death: a population cohort study from the western cape province, south africa effect of antiretroviral therapy on the memory and activation profiles of b cells in hiv-infected african women residual t cell activation and skewed cd8 + t cell memory differentiation despite antiretroviral therapy-induced hiv suppression immunization for hiv-positive individuals compromised b cell responses to influenza vaccination in hiv-infected individuals vaccination in hiv-infected adults at least 80 million children under one at risk of diseases such as diphtheria, measles and polio as covid-19 disrupts routine vaccination efforts, warn gavi, who and unicef potential impact of the covid-19 pandemic on hiv, tuberculosis, and malaria in low-income and middle-income countries: a modelling study evidence that higher temperatures are associated with lower incidence of covid-19 in pandemic state, cumulative cases reported up to distribution of the sars-cov-2 pandemic and its monthly forecast based on seasonal climate patterns role of meteorological temperature and relative humidity in the coronavirus as a possible cause of severe acute respiratory syndrome species distribution models are inappropriate for covid-19 spatial modeling cannot currently differentiate sars-cov-2 coronavirus and human distributions on the basis of climate in the united states seasonality of respiratory viral infections stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions the effects of temperature and relative humidity on the viability of the sars coronavirus absolute humidity and the seasonal onset of influenza in the continental united states epidemic dynamics of respiratory syncytial virus in current and future climates epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study molecular evolution of the sars coronavirus during the course of the sars epidemic in china epidemiology, transmission dynamics and control of sars: the 2002-2003 epidemic cryo-em structure of the 2019-ncov spike in the prefusion conformation tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion a dna vaccine induces sars coronavirus neutralization and protective immunity in mice evaluation of candidate vaccine approaches for mers-cov persistent memory cd4 + and cd8 + t cell responses in recovered severe acute respiratory syndrome (sars) patients to sars coronavirus m antigen human memory t cell responses to sars-cov e protein long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients the host immune response in respiratory virus infection: balancing virus clearance and immunopathology virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection disappearance of antibodies to sars-associated coronavirus after recovery two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome positive rt-pcr test results in patients recovered from covid-19 primary exposure to sars-cov-2 protects against reinfection in rhesus macaques immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge molecular mechanism for antibodydependent enhancement of coronavirus entry medical countermeasures analysis of 2019-ncov and vaccine risks for antibody-dependent enhancement (ade) the authors acknowledge support from the south african medical research council with funds received from the south african department of science and technology, core funding from the wellcome trust (203135/z/16/z) and funding from the south african research chairs initiative of the department of science and technology and national research foundation (grant number 64815). the authors contributed equally to all aspects of the article. the authors declare no competing interests. nature reviews microbiology thanks m. baylis, g. dougan, s. jiang and l. f. p. ng for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. supplementary information is available for this paper at https://doi.org/10.1038/s41579-020-00441-3. key: cord-326725-0jgw083h authors: klamroth, robert; gröner, albrecht; simon, toby l. title: pathogen inactivation and removal methods for plasma‐derived clotting factor concentrates date: 2013-09-30 journal: transfusion doi: 10.1111/trf.12423 sha: doc_id: 326725 cord_uid: 0jgw083h pathogen safety is crucial for plasma‐derived clotting factor concentrates used in the treatment of bleeding disorders. plasma, the starting material for these products, is collected by plasmapheresis (source plasma) or derived from whole blood donations (recovered plasma). the primary measures regarding pathogen safety are selection of healthy donors donating in centers with appropriate epidemiologic data for the main blood‐transmissible viruses, screening donations for the absence of relevant infectious blood‐borne viruses, and release of plasma pools for further processing only if they are nonreactive for serologic markers and nucleic acids for these viruses. despite this testing, pathogen inactivation and/or removal during the manufacturing process of plasma‐derived clotting factor concentrates is required to ensure prevention of transmission of infectious agents. historically, hepatitis viruses and human immunodeficiency virus have posed the greatest threat to patients receiving plasma‐derived therapy for treatment of hemophilia or von willebrand disease. over the past 30 years, dedicated virus inactivation and removal steps have been integrated into factor concentrate production processes, essentially eliminating transmission of these viruses. manufacturing steps used in the purification of factor concentrates have also proved to be successful in reducing potential prion infectivity. in this review, current techniques for inactivation and removal of pathogens from factor concentrates are discussed. ideally, production processes should involve a combination of complementary steps for pathogen inactivation and/or removal to ensure product safety. finally, potential batch‐to‐batch contamination is avoided by stringent cleaning and sanitization methods as part of the manufacturing process. h uman plasma is a source of clotting factor concentrates used for treatment of bleeding disorders caused by coagulation factor deficiencies. such disorders include hemophilia a (factor [f]viii), hemophilia b (f ix), and von willebrand disease (von willebrand factor [vwf]); rarer disorders such as fibrinogen, fxiii, and fx deficiencies; and a number of acquired bleeding disorders. for von willebrand disease, some rare bleeding disorders, and acquired bleeding, plasma-derived clotting factor concentrates, but not recombinant concentrates, are available. a four-factor prothrombin complex concentrate (kcentra [beriplex p/n in europe] , csl behring, marburg, germany) has been recently approved in the united states for reversal of warfarin. 1 efforts to prevent transmission of infectious agents were accelerated after the recognition of human immunodeficiency virus (hiv) transmission to hemophilia patients in 1983 and the identification of hepatitis c virus (hcv) thereafter. by the 1990s, there was substantial clinical evidence of a lack of transmission of lipid membrane-enveloped viruses (hepatitis b [hbv] , hcv, and hiv) using solvent/detergent (s/d) treatment. 2 nonenveloped viruses, including hepatitis a virus (hav) and parvovirus b19 (b19v), presented additional challenges, as they are not inactivated by this technique. [3] [4] [5] in germany, pasteurization (heating at 60°c for 10 hours in a stabilized aqueous solution) was developed in the late 1970s for a clotting factor concentrate as a dedicated virus reduction method; this fviii and vwf concentrate was licensed in 1981 and no proven cases of virus transmissions were reported. 6 more recently, prion transmission has become a concern. 7-9 manufacturing processes have evolved to include multiple steps to prevent transmission of both known and emerging blood-borne pathogens. 10 the objective of this review is to examine current methods to prevent pathogen transmission via plasma-derived clotting factor concentrates by state-of-the-art selection of plasma, and effective pathogen inactivation and removal by the manufacturing process. clotting factor concentrates are derived from pools consisting of large numbers of donations. 2 the fractionation pool size varies depending on the size of the vessel used to pool the plasma. a batch of final product can include several fractionation pools. donor screening and testing of donations and fractionation pools are the first steps in product safety, followed by pathogen inactivation and/or removal (i.e., reduction) during manufacturing. source plasma from apheresis commonly comes from compensated donors, while recovered plasma is from whole blood donors not compensated in cash. 11, 12 the manufacturing process of clotting factor concentrates from both starting materials and their safety records are virtually identical. 11, 12 the european medicines agency (ema) published a guideline focusing on the safety of plasma-derived products; 10 the fourth version details measures necessary to prevent transmission of viruses to recipients of plasmaderived products. these measures include selection of donors, screening of donations and plasma pools for markers of infection with known viruses, and a manufacturing process with a high capacity to inactivate and/or remove viruses by selected steps validated for their virus reduction capacity. the guidelines state that "effective steps for the inactivation/removal of a wide range of viruses of diverse physicochemical characteristics" are required during manufacture. both the ema and the world health organization (who) recommend that two distinct steps with complementary modes of action be used, 10, 13 helping to ensure that any virus surviving step 1 is inactivated and/or removed in step 2. at least one step should inactivate or remove nonenveloped viruses. the ema guideline further states that if a manufacturing process contains a step that reliably inactivates and/or removes a wide range of enveloped and nonenveloped viruses and additional manufacturing steps reliably contribute to inactivation and/or removal of viruses, the second step might not be required. 10 in the united states, the manufacture of plasma-derived products must include at least one effective step for removal and/or inactivation of viruses. 14 further, careful validation is required for each inactivation or removal step. various methods of virus inactivation and removal used in concentrates available in many countries are presented in table 1 . [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] to estimate the potential of specific steps for reducing prion infectivity, the ema recommends evaluation studies for prions. 7, 9 established processes for the production of clotting factor concentrates have been shown to reduce prion infectivity. 7, 33, 34 biologic products, including recombinant clotting factor products, are required to undergo complementary pathogen reduction steps analogous to those of plasmaderived clotting factor concentrates. 35, 36 validation requirements are also similar for recombinant proteins. 35, 36 the safety focus in plasma-derived products has received greater attention because of the history of virus transmissions before 1990. however, the potential for virus contamination is an issue for all biologic products. adherence to strict donor selection criteria and screening of donations effectively reduces the virus load to be inactivated and/or removed from the plasma pool for fractionation. currently, no screening tests are able to detect prion infectivity in donors, [37] [38] [39] although an experimental blood test for prion infection was recently developed. 40 as a precaution, the ema, the us food and drug administration (fda), and who recommend deferral of donors at risk of developing creutzfeldt-jakob disease (cjd) and its variant (vcjd) using a series of questions to exclude donations at risk from pooling and further processing. [7] [8] [9] although screening for viral markers by serology and virus nucleic acid by nucleic acid testing (nat) ensures that nearly all plasma units entering production are free of hbv, hcv, and hiv, inactivation and removal steps are necessary to reduce any viruses that may enter the plasma pool during a "window period" before markers can be detected. 6 in 1999, it was estimated that this window for serologic testing could result in the identification of up to 1 in 10 plasma pools containing an hiv-infected unit and up to one in five containing an hbv-or hcv-infected unit. 2 for a particular nat assay and pool configuration, nat has been shown to decrease the diagnostic window from 22 to 11 days for hiv-1, 59 to 34 days for hbv, and 82 to 23 days for hcv, thereby decreasing the amount of virus entering the plasma pool for fractionation. 6 this reduces the residual risk by decreasing both the number of potentially infected units in the pool and the concentration of virus. however, screening does not sufficiently ensure the safety of products, as tests have limits of detection and are target specific. 13 in addition, the size of the minipool for nat impacts its effectiveness. the plasma industry developed two sets of standards through the plasma protein therapeutics association. 41 the international quality plasma program focuses on donor and center management. the second standard, known as quality standards of excellence, assurance, and leadership (qseal), addresses manufacturing. 41 part of qseal is the inventory hold standard, which was introduced to address the "window period" described previously; this standard allows removal of previously donated units from donors who subsequently test positive for a virus (i.e., removal of window donations). this has been highly effective in reducing the risk of donations from infected donors who are nonreactive in serology (and nat) at the time of donation from entering a manufacturing pool. 42 the inventory hold is not a quarantine, as it would not interdict an infectious unit in the nat window period if the donor did not return. the effectiveness of the hold is related to the large number of donors who donate frequently. all source plasma donations used for manufacturing are equivalent to donations for transfusion in terms of pathogen safety. in addition to manufacturing standards, qseal includes qualified donor and virus marker standards identical to those in international quality plasma program. 41 validation of production steps is necessary to estimate pathogen inactivation and/or removal capacity and provides confidence in the safety of a plasma-derived clotting factor product. 6, 43 the virus reduction capacity of a manufacturing process is tested in a dedicated laboratory using a validated scaled-down version of the manufacturing process by employing process intermediates spiked with high titers of an appropriate virus and then determining the virus reduction factor achieved. 6 viruses selected for validation should closely resemble those that pose the greatest threat of contamination, such as hepatitis viruses and hiv. for some viruses, no practical cell culture system is available; thus, specific model viruses are used. the selected viruses should include different genome, size, and envelope characteristics to represent the wide range of the physicochemical properties of viruses. this allows manufacturers to fully test the virus reduction capacity of the manufacturing process and to provide indirect evidence that it reduces viruses in general. 13, 14, 43 reduction factors greater than 4 log are generally considered effective. 43, 44 some virus reduction factors achieved by individual inactivation and/or removal steps are presented in table 2 . 17, 19, 32, [45] [46] [47] [48] removal of prions from plasma-derived products is evaluated similarly to virus validation studies, based on a valid scaled-down version of the manufacturing process. 33, 49, 50 for prion detection, the quantitative animal bioassay is considered most appropriate, although the in vitro methods of western blot assay, enzyme-linked immunosorbent assay, and conformation-dependent immunoassay are also used for the detection of misfolded prion protein. pasteurization by heating the protein in aqueous stabilized solution at 60°c for 10 to 11 hours inactivates both lipid membrane-enveloped and a range of nonenveloped viruses (table 3) . 13 coagulation factors are heat sensitive; therefore, stabilizers (usually sugars, amino acids, or acetate) are added to preserve protein integrity and are removed after pathogen inactivation. homogeneity of temperature throughout pasteurization must be validated by temperature mapping techniques. studies on inactivation of hiv, hav, and b19v by pasteurization in a fviii/ vwf concentrate have demonstrated virus reduction factors of at least 6.4, 4.2, and at least 3.9 log, respectively. 6, 32 table 4 lipid membranes of enveloped viruses are disrupted by s/d mixtures, thereby preventing binding to and infection of cells by these viruses. 51 organic solvents (e.g., tri(nbutyl)phosphate), combined with a nonionic detergent (polyethylene glycol octylphenyl ether [triton x-100] or polysorbate 80), are typically used for treatment; s/d compounds are removed by chromatography or oil extraction. 51 product solutions are filtered to remove viruses entrapped in particles and thus protected from exposure to s/d. 13 homogeneous mixing and maintenance of a consistent temperature throughout incubation must be validated. plasma protein therapeutics association member companies have compiled data on effective virus inactivation by s/d treatment for fviii, f ix, intravenous immunoglobulin, and intramuscular immunoglobulin. 52 virus inactivation was evaluated in 308 studies reflecting production conditions, as well as variables significantly beyond process specification; the results demonstrated that product class, process temperature, protein concentration, and ph were not substantially involved in virus inactivation. in contrast, the concentration of s/d was critical when it was significantly below that specified for the process. 52 for a fviii and vwf concentrate, s/d treatment studies demonstrated reduction factors of more than 7.5 log for hiv, sindbis virus (sinv), and prv; 46 for a f ix concentrate, reduction factors were greater than 4.5 log for hiv, sinv, vesicular stomatitis virus, bvdv, and prv. 47 an important limitation of s/d treatment is specificity for lipid-enveloped viruses; 4,53 there have been reports of patients with hemophilia with hav or b19v infections after receiving s/d-treated fviii concentrates. 3 therefore, a second virus reduction step is required in the manufacture of such plasma-derived products to close this gap in virus safety. most plasma-derived concentrates are lyophilized and, especially those treated with s/d, subsequently treated with dry heat to inactivate nonenveloped viruses that resist s/d treatment. lyophilization confers a certain degree of virus inactivation; 2,57-59 the moisture content of lyophilized products undergoing dry heat treatment should be kept low (typically <2%), as residual moisture may affect product stability, 45 although higher levels may enhance inactivation of some viruses. 60 dry heat treatment of lyophilized products has demonstrated favorable results for inactivation of relevant or model viruses of hav, hbv, hcv, and hiv. 45, 46, 61, 62 dry heating of a lyophilized fviii product at 80°c for 72 hours inactivated a wide range of viruses, 60 and dry heating of fviii or f ix concentrates reduced the risk of hcv transmission. 61 an additional study demonstrated that terminal dry heating of a lyophilized fviii concentrate at 100°c for 30 minutes inactivated hav and hiv to below detectable levels within 10 minutes, while retaining approximately 95% of fviii activity, 45 whereas heating a lyophilized fviii and vwf concentrate at 100°c for 120 minutes reduced a wide array of viruses (hiv, sinv, prv, reovirus type-3, hav, and b19v) by more than 4 log. 46 although b19v was reduced by dry heat in validation studies, the reduction factor may not be sufficient for complete inactivation of the virus load in the final product; asymptomatic b19v infection was detected in a patient who received fviii concentrate treated at 80°c for 72 hours. 63 one drawback to lyophilization is that in addition to stabilizing coagulation factors, the removal of water can also stabilize potential viruses in the product. 62 by adding water vapor to lyophilized products before heating, higher levels of virus inactivation can be achieved at equivalent temperatures. 13 vapor heating of lyophilized products targets enveloped and nonenveloped viruses and has demonstrated inactivation of hepatitis viruses and hiv. 19 lyophilization and vapor heat at 60°c inactivated approximately 6 log of hav in spiked fviii concentrates within 8 to 10 hours and similarly reduced hav titers in f ix concentrates within 3 hours. 64 among 20 patients with hemophilia who received vapor-heated f ix infusions, none developed markers for infection with hcv or hiv during 6 to 15 months of follow-up, suggesting that vapor heattreated concentrates may be associated with a low risk of viral infection. 65 however, vapor heat at 60°c may be insufficient to inactivate hbv completely, depending on the virus load in the starting material (batch related) and the amount of product given to patients not vaccinated against hbv; there are reports of four hemophilia patients not vaccinated against hbv who became infected with hbv after receiving vapor-heated fviii concentrate. 66 in addition to virus inactivation, viruses can also be physically removed from plasma-derived products by fractionation (precipitation or chromatography) and filtration (table 5) . before the 1980s, plasma fractionation was mainly considered a step in protein purification; no dedicated virus reduction step (except for pasteurization of human albumin) was implemented during the manufacturing process. later virus validation studies showed that fractionation reliably contributes to the overall virus reduction capacity for plasma-derived proteins. 47, 68, 69 although ethanol precipitation is the most widely used plasma fractionation method worldwide, 13 it is not used for the production of coagulation factor concentrates, as this treatment denatures the desired proteins. after initial cryoprecipitation, many clotting factor concentrates are purified chromatographically. in a study by roberts and colleagues, 69 purification of f ix concentrate using metal chelate affinity chromatography reduced poliovirus and sinv by reduction factors of 4.0 and 6.5 log, respectively. some products are further purified using affinity chromatography mediated by an antibody against the protein of interest. 44 affinity chromatography has been used to purify fviii concentrates, resulting in reduction factors of 8.3 log for encephalomyocarditis virus, 68 4.3 log for sinv, 68 4.2 log for porcine parvovirus (ppv; a model virus for b19v), 21 and 5.3 log for hav. 21 purification of a fviii concentrate using monoclonal antibody (moab) affinity chromatography contributed to overall virus reduction capabilities of 5 to 6 log for hiv, sinv, vesicular stomatitis virus, and prv before pasteurization. 23 for a f ix concentrate, moab affinity chromatography alone resulted in reduction factors of at least 5.8 log for enveloped viruses hiv, bvdv, prv, and wnv and reduction factors of 3.3 and 6.6 log for nonenveloped viruses hav and parvovirus, respectively. 24 an important consideration exists in the reuse of chromatography columns; 13 viruses adhere to resins and cannot be completely washed out. therefore, equipment and material must be sanitized with validated chemical and/or physical treatments to inactivate and remove viruses to ensure no cross-contamination of subsequent batches of product. 13 viruses may be removed from clotting factor concentrates via filtration employing retentive filters with pores smaller than the virus diameter (virus filtration, also called nanofiltration). 67 filtration may not be feasible for products containing proteins of a comparable or larger size to the viruses or pore size of the filter; thus, filtration is mainly limited to smaller-molecular-weight coagulation factors (e.g., f ix or fviii). however, a vwf concentrate filtered through 35-nm pore membranes resulted in reduction factors of at least 5.0 log of enveloped viruses hiv, bvdv, and prv, although filtration removed large vwf multimers as well. 70 filtration of a fviii concentrate using 35-and 15-nm sequential filtration resulted in removal of enveloped (hiv, bvdv, and prv) and nonenveloped (hav and ppv) viruses. 71 virus filtration alone resulted in virus reduction factors of at least 3.6 log, while combination of s/d, chromatography, and virus filtration yielded reduction factors of at least 5.1 log for all tested viruses. virus filtration of a fxiii concentrate using a 20-nm filter resulted in a reduction of enveloped (hiv, bvdv, prv) and nonenveloped (hav) viruses by at least 5.5 log and a reduction of a nonenveloped parvovirus by 3.4 log. 72 table 6 is an example of virus reduction achieved through the addition of a filtration step. in a safety study on f ix concentrate, a final virus filtration step was added following fractionation, chromatography, and s/d treatment. 47 the 35-to 15-nm sequential filtration step resulted in a reduction factor of at least 6.8 log for hav and at least 6.6 log for bvdv. 47 filtration of another f ix concentrate using two filters (20-nm mean pore size) in series very effectively removed enveloped and nonenveloped viruses. 24 an appropriate margin of safety with respect to bloodborne viruses is achieved when the overall virus reduction factor clearly exceeds the amount of virus potentially entering the manufacturing pool. in europe, quantitative risk assessments are required. 10 an estimate of virus particle content of the finished product is determined based on the worst case scenario of virus concentration in the starting material and the amount of plasma required to produce one vial, divided by the virus reduction factor a high margin of safety is documented when fewer than one virus particle per million doses is expected; this is based on the sterility assurance level with respect to bacteria, molds, and yeasts by pharmacopoeias worldwide. as viruses cannot replicate in a cell-free medium such as the dissolved final product, application of the sterility assurance level for viruses is conservative. transmissible spongiform encephalopathies are a group of prion-associated, fatal neurodegenerative diseases. three possible cases of prion transmission resulting in vcjd and one probable case of asymptomatic infection have been reported in patients in the united kingdom who received non-leukoreduced red blood cells from asymptomatic infected individuals. 73 to date, no recipients of pooled plasma-derived products have developed vcjd; 39 however, there is one report of asymptomatic prion detection at autopsy in a hemophilia patient, 74 probably due to prion-contaminated fviii concentrate. 74 as prion screening tests are currently unavailable, donor selection and manufacturing processes for prion removal are important. 39 as requested by the who, ema, and fda, restrictions on plasma donation have been imposed in many countries. [6] [7] [8] [9] 33 plasma-derived proteins are leukoreduced by plasma preparation, decreasing the risk for prion transmission. additionally, manufacturing steps for plasma fractionation and virus removal, including precipitation, chromatography, and filtration (table 4) , have also proven efficacy in evaluation studies. 6, 34, 75 inactivation of prions in plasma-derived clotting factor concentrates is not possible, as the needed techniques denature coagulation factors. 33 appropriate cleaning and sanitization methods for material and equipment used in the pro-duction of plasma-derived products inactivate and remove prion proteins; thus, batch-to-batch segregation can be achieved and batch-to-batch contamination excluded. [76] [77] [78] [79] emerging microbial pathogens or parasites are less of a threat because of sterile filtration, which, in combination with other steps before preparation of the finished product, removes these pathogens. however, "new emerging viruses" must be assessed very carefully with regard to potential impact on the safety of the plasma pool for fractionation and the final products. new emerging viruses may be novel, zoonotic viruses that are encountered as humans enter new geographic areas with previously undiscovered animal viruses that cross the species barrier to enter the human disease chain (e.g., hiv, yellow fever virus, severe acute respiratory syndrome coronavirus, menangle virus, hendra virus, nipah virus, hantaviruses, monkeypox virus). hepatitis e virus has been detected in different regions of the world and is considered a zoonosis; in particular, genotype 3, mainly detected in domestic and wild pigs, has been found in humans in developed countries. 80, 81 human parvovirus 4 (parv4) and related parvoviruses have been identified in cows, pigs, primates, and humans. 82-86 furthermore, a "new" virus can emerge and/or reemerge in new geographic regions; for example, wnv was known in africa and the middle east but emerged in north america in 1999. 87 improved diagnostic methods have resulted in detection of previously unknown viruses (which should not be considered as emerging), such as b19v in 1975, 88 hcv in 1989, 89 human herpesvirus type 8 in 1996, 90 and tt virus in 1997. 91 to meet this challenge, the epidemiology of emerging pathogens in the donor population and the potential infectious virus load in a donation during asymptomatic incubation must be addressed. diligent surveillance of available information may result in a (temporal) deferral of donors based on geographic risk. parv4 can reach a high load in an infected person, 92 but the prevalence of the virus is low in many regions. 93, 94 contamination of plasma pools for fractionation with parv4 has been reported, 95 resulting in final products with parv4 dna. however, parv4 dna in current products is undetectable 96 or lower than in products manufactured between 1970 and 1980, 97,98 possibly because of thorough screening of donors and testing of donations for pathogens that may be associated with parv4. some manufacturing processes for coagulation factor concentrates remove and/or inactivate low levels of parv4. 98 dedicated virus reduction steps in the manufacture of coagulation factor concentrates also reduced arthropodtransmitted viruses such as wnv, 99 other flaviviruses (dengue virus), 100 and togaviruses (chikungunya virus); 101 reduction of these viruses during manufacture of clotting factor concentrates was also demonstrated using model viruses for validation. these data demonstrate that the measures currently taken are also relevant for emerging viruses. the combination of inactivation and removal methods, in addition to donation screening, has essentially eliminated transmission of viruses of principal concern, namely hav, hbv, hcv, hiv, and b19v. as shown in validation studies, a strategy involving different steps for inactivation and removal is optimal for targeting viruses with different physicochemical properties. for instance, although s/d is highly effective against enveloped viruses, it is ineffective against nonenveloped viruses (e.g., hav and b19v); thus, a second step, often dry heating of the lyophilized product, is implemented. procedures for purification and concentration of the desired protein are usually less effective in virus removal than dedicated reduction steps, such as inactivation by heat and s/d treatment or removal by virus filtration. as diagnostic techniques advance, emerging pathogens may be identified, 92 and their genomes may be detected in the final product (e.g., parv4), depending on the manufacturing process. 96, 98 the methods employed make it extremely likely that any new pathogen would be inactivated or removed to a high degree, but diligence must be maintained. immune globulin and other plasma products, such as α1-proteinase inhibitor and c1 esterase inhibitor, are subject to similar pathogen reduction measures. in addition, low ph can be utilized for immune globulin products to inactivate certain viruses. thus, the same high safety record has been observed with these products in recent years. albumin has always been pasteurized to prevent pathogen transmission. a product similar to fresh-frozen plasma (ffp), but pooled and treated with s/d, has been available in europe and was recently licensed in the united states (octaplas). 102 enveloped viruses are effectively inactivated by s/d treatment, and chromatographic methods reduce prion transmission potential. this product relies on testing of the donations for protection against nonenveloped virus, specifically hav, b19v, and more recently, hepatitis e virus. improved virus safety compared with ffp is based solely on the reductions observed with s/d methods for blood products. 103 over the past 20 years, fears of potential infection and the introduction of recombinant factor products have fueled a shift toward recombinant products as the first choice for treatment of patients with hemophilia. however, opinions on the role of recombinant and plasma-derived concentrates for treatment vary. the united kingdom hemophilia center directors organiza-tion recommends recombinant products for all patients with hemophilia, whereas other european countries recommend them as the first choice for children only. in contrast, in germany and austria, there are no clear recommendations on use of recombinant factor concentrates in patients with hemophilia. furthermore, patients with other factor deficiencies for which no recombinant product is currently available (e.g., von willebrand disease) must use plasma-derived concentrates. physicians currently have fewer safety concerns using plasma-derived products than in the past; in fact, 45% of fviii consumption and approximately 65% of f ix consumption in germany are plasma-derived. for patients with hemophilia a and inhibitors against fviii, plasmaderived fviii products containing vwf are widely used as second-line treatment for immune tolerance induction. due to the improved and comprehensive pathogen reduction steps, physicians and patients should have confidence in the safety of plasma-derived products for the treatment of bleeding disorders. csl behring gmbh the epidemiology of virus transmission by plasma derivatives: clinical studies verifying the lack of transmission of hepatitis b and c viruses and hiv type 1 transmission of hepatitis a to patients with hemophilia by factor viii concentrates treated with organic solvent and detergent to inactivate viruses. the italian collaborative group clinical evaluation of viral safety of coagulation factor viii and ix concentrates human parvovirus b19 infection in hemophiliacs first infused with two highpurity, virally attenuated factor viii concentrates pathogen safety of plasma-derived products-haemate-p/humate-p chmp position statement on creutzfeldt-jakob disease and plasma-derived and urine-derived medicinal products guidance for industry: revised preventive measures to reduce the possible risk of transmission of creutzfeldt-jakob disease (cjd) and variant creutzfeldt-jakob disease (vcjd) by blood and blood products world health organization. who guidelines on transmissible spongiform encephalopathies in relation to biological and pharmaceutical products guideline on plasmaderived medicinal products payment, compensation and replacement-the ethics and motivation of blood and plasma donation monetary compensation for plasma donors: a record of safety guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products guide to inspections of viral clearance process for plasma derivatives clinical uses of plasma and plasma fractions: plasma-derived products for hemophilias a and b, and for von willebrand disease composition, quality control, and labeling of plasma-derived products for the treatment of von willebrand disease antihemophilic factor/von willebrand factor complex coagulation factor viii/von willebrand factor complex factor (human) antihemophilic factor (human) factor ix complex) prothrombin complex concentrates: an update cryoprecipitate versus commercial fibrinogen concentrate in patients who occasionally require a therapeutic supply of fibrinogen: risk comparison in the case of an emerging transfusiontransmitted infection human prothrombin complex) antihemophilic factor/von willebrand factor complex clearance of prions during plasma protein manufacture prion removal capacity of plasma protein manufacturing processes: a data collection from ppta member companies guidance for industry: q5a viral safety evaluation of biotechnology products derived from cell lines of human or animal origin note for guidance on quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin overall blood supply strategy with regard to variant creutzfeldt-jakob disease (vcjd)": statement on the development and implementation of test systems suitable for the screening of blood donors for vcjd-dated variant creutzfeldt-jakob disease: an update an update on the assessment and management of the risk of transmission of variant creutzfeldt-jakob disease by blood and plasma products detection of prion infection in variant creutzfeldt-jakob disease: a blood-based assay plasma protein therapeutics association the residual risk of a window period (wp) unit entering a pool of source plasma note for guidance on virus validation studies: the design, contribution and interpretation of studies validating the inactivation and removal of viruses the use of chromatography to manufacture purer and safer plasma products dry-heat treatment process for enhancing viral safety of an antihemophilic factor viii concentrate prepared from human plasma characterisation of a novel high-purity, double virus inactivated von willebrand factor and factor viii concentrate (wilate) inactivation and clearance of viruses during the manufacture of high purity factor ix inactivation of parvovirus b19 during stim-4 vapor heat treatment of three coagulation factor concentrates guideline on the investigation of manufacturing processes for plasma-derived medicinal products with regard to vcjd risk purity of spiking agent affects partitioning of prions in plasma protein purification pathogen reduction of blood components robustness of solvent/detergent treatment of plasma derivatives: a data collection from plasma protein therapeutics association member companies effectiveness of alternative treatments for reducing potential viral contaminants from plasma-derived products the outbreak of hepatitis a in italian patients with hemophilia: facts and fancies a new cluster of hepatitis a infection in hemophiliacs traced to a contaminated plasma pool detection of hepatitis a virus from clotting factors implicated as a source of hav infection among haemophilia patients in korea pharmacokinetics and acute tolerance of a double virus inactivated plasma derived factor viii concentrate protein modification during anti-viral heat-treatment bioprocessing of factor viii concentrates, factor ix concentrates, and model proteins in the presence of sucrose virus inactivation during the freeze-drying processes as used for the manufacture of plasma-derived medicinal products effect of manufacturing process parameters on virus inactivation by dry heat treatment at 80 degrees c in factor viii effect of dry-heating of coagulation factor concentrates at 80 degrees c for 72 hours on transmission of non-a, non-b hepatitis. study group of the uk haemophilia centre directors on surveillance of virus transmission by concentrates in vitro" and in animal model studies on a double virus-inactivated factor viii concentrate parvovirus b19 transmission by heat-treated clotting factor concentrates inactivation of hepatitis a virus in plasma products by vapor heating low risk of viral infection after administration of vapor-heated factor vii concentrate or factor ix complex in first-time recipients of blood components. international factor safety study group prospective study of hepatitis after factor viii concentrate exposed to hot vapour nanofiltration of plasmaderived biopharmaceutical products affinity chromatography to remove viruses during preparation of plasma derivatives removal and inactivation of enveloped and non-enveloped viruses during the purification of a high-purity factor ix by metal chelate affinity chromatography in vitro study of a triple-secured von willebrand factor concentrate a solvent/detergenttreated and 15-nm filtered factor viii: a new safety standard for plasma-derived coagulation factor concentrates high margin of pathogen safety of a plasma-derived fxiii concentrate variant creutzfeldt-jakob disease variant cjd infection in the spleen of a neurologically asymptomatic uk adult patient with haemophilia managing the risk of transmission of variant creutzfeldt jakob disease by blood products transmission of scrapie by steel-surface-bound prions sodium hydroxide renders the prion protein prpsc sensitive to proteinase k critical factors influencing prion inactivation by sodium hydroxide evaluation of a cleaning procedure for its capacity to remove prions from equipment used in the production of plasma derivatives genetic variability and evolution of hepatitis e virus seroprevalence and incidence of hepatitis e virus infection in german blood donors high prevalence of porcine hokovirus in german wild boar populations diversity of parvovirus 4-like viruses in humans, chimpanzees, and monkeys in hunter-prey relationships new dna viruses identified in patients with acute viral infection syndrome identification of novel porcine and bovine parvoviruses closely related to human parvovirus 4 widespread infection with homologues of human parvoviruses b19, parv4, and human bocavirus of chimpanzees and gorillas in the wild the west nile virus outbreak of 1999 in new york: the flushing hospital experience parvovirus-like particles in human sera isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome primary characterization of a herpesvirus agent associated with kaposi's sarcomae a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology virologic and clinical features of primary infection with human parvovirus 4 in subjects with hemophilia: frequent transmission by virally inactivated clotting factor concentrates absence of detection of novel human parvoviruses in german plasma donations parvoviruses in blood donors and transplant patients novel parvovirus and related variant in human plasma prevalence of nucleic acid sequences specific for human parvoviruses, hepatitis a and hepatitis e viruses in coagulation factor concentrates human parvovirus parv4 in clotting factor viii concentrates frequency of contamination of coagulation factor concentrates with novel human parvovirus parv4 west nile virus and the safety of plasma derivatives: verification of high safety margins, and the validity of predictions based on model virus data clearance of dengue virus in the plasma-derived therapeutic proteins chikungunya virus and the safety of plasma products octapharma pharmazeutika produktionsges m.b.h viral safety of solvent-detergent treated blood products rk has acted as a consultant and a paid speaker for csl behring, bayer, baxter, pfizer, novo nordisk, and sobi. ag and tls are employees of csl behring. key: cord-302928-nnly9ju8 authors: adachi, akio title: grand challenge in human/animal virology: unseen, smallest replicative entities shape the whole globe date: 2020-03-18 journal: front microbiol doi: 10.3389/fmicb.2020.00431 sha: doc_id: 302928 cord_uid: nnly9ju8 nan by far the most abundant species in nature is the virus that cannot replicate by itself. viruses, parasitic entities, are found in virtually all unicellular and multicellular creatures. viruses are everywhere. while smallest in size among all species, they can ingeniously replicate, persist, and survive in their individual hosts and/or host populations, and are transmittable among hosts. viruses are sometimes inflicting or fatal for host species and are sometimes inter-species replicons. they keep interacting with their hosts in numerous different manners. unseen viruses thus can reshuffle the whole world through accumulations of their subtle biological effects. in other words, viruses can shape the entire environment around us and are able to directly and/or indirectly influence us by their biologic activities. virology is a multidisciplinary research field and, as an academic discipline of the biology, it extensively analyzes all aspects of viruses derived from every living species by scientific systems/methodologies currently available to us as exemplified and fully described in a series of frontiers special issues designated "research topic" (rt) in the virology section of frontiers in microbiology adachi, 2010, 2017; miyazaki et al., 2012; nomaguchi et al., 2012; berkhout and coombs, 2013; sato et al., 2013; adachi and miura, 2014; dutilh et al., 2017; sanfaçon, 2017; yamamoto et al., 2017) . i wish to emphasize here again that virology is a branch of biological sciences studying the fundamental attributes of a wide variety of unique characteristic viruses. virology concerns biological issues in a broad sense. in the past decade, as many as of 1,500 articles approximately have been published and almost 80 rts for specific subjects have been issued or are on the way in the virology section. during this period, we have seen constant and vast increases both in virology-specific, in a narrow sense, articles, and in those giving more conceptual and general knowledge of the biology. notably, the observed advances frequently have been accompanied by remarkable development of novel innovative technologies. in the next decade, concomitant with successful proceeding of the powerful and sharp analyzing systems and/or methods such as computational biology, structural biology, bioinformatics, multi-omics, next-generation sequencing, genome editing, single-cell methodology, and organoid technology (bai et al., 2015; angermueller et al., 2016; chen et al., 2017; dutta et al., 2017; hasin et al., 2017; adli, 2018; artegiani and clevers, 2018; cheng, 2018; ibrahim et al., 2018; rossi et al., 2018; sbalzarini and greber, 2018; van dijk et al., 2018; pickar-oliver and gersbach, 2019) , virology will certainly continue to contribute much to the progress in all areas of the basic biology from molecular and structural biology to environmental science. in addition, virology will surely represent one of major driving forces to solve a wide variety of practical issues, which are directly or indirectly related to the virus issue itself, i.e., nanotechnology, viral vectors, antiviral drugs, vaccines, gene therapy, interferon therapy, immune therapy, and so on (de clercq, 2007; guimarães et al., 2015; szunerits et al., 2015; de clercq and li, 2016; athanasopoulos et al., 2017; grimm and büning, 2017; singh et al., 2017; snell et al., 2017; chambers et al., 2018; colino et al., 2018; kaufmann et al., 2018; ono et al., 2018; sulczewski et al., 2018; vetter et al., 2018; dionne, 2019; graham et al., 2019) . it is well-expected from our plentiful experience in the past decade that the virology section, as an outstanding communication platform, plays a critical and central role in various activities of basic and applied sciences. human and animal virology investigates viruses that infect human and all species of animals. upon replication, some viruses are damaging for their hosts to various degrees whereas others rather peacefully co-exist with the hosts. among these viruses, those that cause serious infectious diseases in human and/or animals, are medically, socially, and economically of particular importance, as a matter of course for scientific significance. in fact, our virology section has published numerous articles related to this kind of human and animal viruses and has edited relevant rts in the past decade. on one hand, more general subjects covering various pathogenic viruses and related research areas have been targeted as well. of socially important pathogenic viruses, some are solely tropic for humans, hiv-1 as an example (hatziioannou et al., 2006 (hatziioannou et al., , 2009 kamada et al., 2006; nomaguchi et al., 2008 nomaguchi et al., , 2013b , and some like human norovirus are known not to replicate in cultured cells (duizer et al., 2004; herbst-kralovetz et al., 2013; ettayebi et al., 2016; murakami et al., 2020) . indeed, these viral properties hamper and limit our experimental strategies, and are representing major study projects to overcome in the virology. alternative practical experimental systems, irrespective of being commonly useful and applicable to numbers of viruses or specifically to certain viruses, to circumvent the observed difficulties are definitely required. expert researchers on the viruses have been making every effort to achieve the primary purposes (kamada et al., 2006; nomaguchi et al., 2011 nomaguchi et al., , 2013b soll et al., 2013; hatziioannou et al., 2014; ettayebi et al., 2016; doi et al., 2018; schmidt et al., 2019; murakami et al., 2020) . the trend described above is understandable and wellrecognized by published articles and rts in the human and animal virus field in the virology section. most cited articles and most viewed rts in the human/animal virus field of the section (top 5, as of february 3, 2020) are as follows, respectively: articles, "epidemiological aspects and world distribution of htlv-1 infection" (gessain and cassar, 2012) , "pathology of asthma" (kudo et al., 2013) , "challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data" (beerenwinkel et al., 2012) , "er stress, autophagy, and rna viruses" (jheng et al., 2014) , and "zika virus: the latest newcomer" (saiz et al., 2016) ; rts, "highly mutable animal rna viruses: adaptation and evolution" , "virus discovery by metagenomics: the (im)possibilities" (dutilh et al., 2017) , "zika virus research" (bueno-marí et al., 2018), "pathophysiology and epidemiology of virus-induced asthma" (kimura and ryo, 2014) , and "forefront studies on htlv-1 oncogenesis" (mahieux and watanabe, 2013) . the results described above may indicate the research field in which our peers are mostly interested. another point worth mentioning here is the emerging pathogenic viruses such as hemorrhagic ebola virus and pneumonia-causing new coronavirus designated sars-cov-2. these viruses are becoming more and more important under today's world environment, as is the case for the influenza virus, a continuous global health threat, that potentially can induce an awful pandemic. it is more and more probable that humans may encounter emerging zoonotic viral pathogens and also re-emerging viruses through extensive inroads into untouched areas. we of course do not know much about the biology, including the ecology, origin, mutation, adaptation, etc., of these new viruses as yet. once transmitted to humans, they readily replicate in and spread among human populations without effective immunity against them. care should be taken not to be infected with the viruses at individual and population levels. infected individuals must be treated properly. in this regard, we virologists always have to prepare the ground for sudden onsets of new emerging diseases. routine collaborative research activities among basic researchers, clinical doctors, and relevant staffs of various expertise underpin the basis for swift responses against them. valid anti-viral strategies can be generated by such well-organized medical teams. viruses can cause acute or chronic infections in human and animal hosts in many mechanistically different ways. while causative viruses for the former do not persist long in individual hosts in general, the latter viruses generate persistent infection and may give rise to latent infection. it is scientifically and practically important and of great interest to determine the underlying mechanisms. elucidating biological and molecular bases for the conflict between viruses and their hosts is therefore one of major missions of current virology. especially, for some pathogenic viruses that have incredibly lengthy interacting period with hosts, or that are prone to readily alter in nature, it is critically important to dynamically investigate their mutations, adaptation, and evolution. hiv-1, a representative virus for such category, readily mutates and adapts itself in the course of infection as experimentally revealed by a series of analytical studies (nomaguchi et al., 2008 (nomaguchi et al., , 2013a (nomaguchi et al., ,c, 2014 (nomaguchi et al., , 2018 saito et al., 2011; yokoyama et al., 2016; doi et al., 2019) . here, following seven rts in our virology section are cited as examples of various cases for viral interactions with their hosts: "codon usage and dinucleotide composition of virus genomes: from the virus-host interaction to the development of vaccines, " "host and pathogen mechanisms underpinning viral ecology and emerging infections, " "revealing hiv hiding places: identification and characterization of cellular and tissue reservoirs, " "the interplay between innate immunity and herpesviruses, " "hiv-1 genetic diversity, " "highly mutable animal rna viruses: adaptation and evolution" , and "the past and the future of human immunity under viral evolutionary pressure" (hurst and magiorkinis, 2019) . as can be realized by the brief descriptions for the rts and articles published in the rts, there are indeed numerous distinct virus-host interactions to be noted. lastly, regardless of their medical or economic effects on human society, all rna and dna viruses are equally valid targets for extensive scientific studies in today's virology. we aim to publish important articles resulting from those studies that significantly advance our understanding of the viruses and related issues in our virology section. the most noticeable topic in the field of human and animal virology in the next decade would depend upon what happens in the virological world around us. it could be a deadly virus that causes global infection, or, could be some discovery or invention that changes our concept. attention should be paid to endogenized viral elements that affect the hosts in a biologically significant way. the relevant research field is rapidly growing as described recently in our journal (staege and emmer, 2018; turnbull and douville, 2018; flynn and moreau, 2019; moelling and broecker, 2019) . as for the correct answer to the question above, it is of course unpredictable at the present time but the virological information and biological finding of the first magnitude that move the academic and public communities will surely determine the result. based on enormous accumulations of scientific and statistical data of numerous kinds in various fields, together with orthodox and innovative analytical systems, it is quite expected that future virology can further deepen studies on virus populations and predictive science as well as those on a specific individual virus. from this point of view, i would like to enthusiastically encourage researchers outside the virology field, in addition to those who inside, to submit their representative works to our virology section, if there is a little relevance. virology is multidisciplinary in its marked and essential characteristic as stated above. nevertheless, the bottommost role for current virology in the era of "big data" is to functionally/biologically characterize individual viruses for the future by all available tools. by focusing on viruses, we can study and analyze living replicating creatures in all directions. we the virology section of frontiers in microbiology most welcome the collaborative and integrative studies that impact and stimulate the whole world. join us and share the fruits of virology. aa is a sole contributor to this manuscript and approved its submission. animal model studies on viral infections the crispr tool kit for genome editing and beyond deep learning for computational biology use and application of 3d-organoid technology nonintegrating gene therapy vectors how cryo-em is revolutionizing structural biology challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data quantitative omics and its application to study virus-host interactions-a new frontier editorial: zika virus research overview of the baculovirus expression system protein bioinformatics databases and resources membrane protein structural biology in the era of single particle cryo-em nanoparticles for signaling in biodiagnosis and treatment of infectious diseases the design of drugs for hiv and hcv approved antiviral drugs over the past 50 years key principles of antiretroviral pharmacology hiv-1 clones are both tractable to grow in rhesus macaques concomitant enhancement of hiv-1 replication potential and neutralizationresistance in concert with three adaptive mutations in env v1/c2/c4 domains laboratory efforts to cultivate noroviruses editorial: virus discovery by metagenomics: the (im)possibilities disease modeling in stem cell-derived 3d organoid systems replication of human noroviruses in stem cellderived human enteroids assessing the diversity of endogenous viruses throughout ant genomes epidemiological aspects and world distribution of htlv-1 infection structurebased vaccine antigen design small but increasingly mighty: latest advances in aav vector research, design, and evolution vaccines, adjuvants and autoimmunity multi-omics approaches to disease a macaque model of hiv-1 infection hiv-1-induced aids in monkeys generation of simian-tropic hiv-1 by restriction factor evasion lack of norovirus replication and histo-blood group antigen expression in 3-dimensional intestinal epithelial cells editorial: the past and the future of human immunity under viral evolutionary pressure a new era of virus bioinformatics er stress, autophagy, and rna viruses generation of hiv-1 derivatives that productively infect macaque monkey lymphoid cells host-directed therapies for bacterial and viral infections pathophysiology and epidemiology of virus-induced asthma forefront studies on htlv-1 oncogenesis structural biology for virus research viruses and evolution -viruses first? a personal perspective bile acids and ceramide overcome the entry restriction for gii. 3 human norovirus replication in human intestinal enteroids virology as biosystematics: towards understanding the viral infection biology editorial: highly mutable animal rna viruses: adaptation and evolution macaquetropic hiv-1 derivatives: a novel experimental approach to understand viral replication and evolution in vivo systemic biological analysis of the mutations in two distinct hiv-1mt genomes occurred during replication in macaque cells species barrier of hiv-1 and its jumping by virus engineering hiv-1 mutates to adapt in fluxing environments natural single-nucleotide variations in the hiv-1 genomic sa1prox region can alter viral replication ability by regulating vif expression levels production of hiv-1 vif mrna is modulated by natural nucleotide variations and slsa1 rna structure in sa1d2prox genomic region natural single-nucleotide polymorphisms in the 3' region of the hiv-1 pol gene modulate viral replication ability generation of rhesus macaque-tropic hiv-1 clones that are resistant to major anti-hiv-1 restriction factors gag-ca q110d mutation elicits trim5-independent enhancement of hiv-1mt replication in macaque cells baculovirus as a tool for gene delivery and gene therapy the next generation of crispr-cas technologies and applications progress and potential in organoid research improved capacity of a monkey-tropic hiv-1 derivative to replicate in cynomolgus monkeys with minimal modifications zika virus: the latest newcomer grand challenge in plant virology: understanding the impact of plant viruses in model plants, in agricultural crops, and in complex ecosystems genomics and computational science for virus research how computational models enable mechanistic insights into virus infection derivation of simian tropic hiv-1 infectious clone reveals virus adaptation to a new host the role of nanotechnology in the treatment of viral infections type i interferon in chronic virus infection and cancer assisted evolution enables hiv-1 to overcome a high trim5α-imposed genetic barrier to rhesus macaque tropism editorial: endogenous viral elementslinks between autoimmunity and cancer? nanoparticle vaccines against viral infections nanostructures for the inhibition of viral infections related endogenous retrovirus-k elements harbor distinct protease active site motifs the third revolution in sequencing technology understanding modern-day vaccines: what you need to know editorial: perspectives for the next generation of virus research: spearheading the use of innovative technologies and methodologies in silico analysis of hiv-1 env-gp120 reveals structural bases for viral adaptation in growth-restrictive cells i would like to thank prof. masako nomaguchi (tokushima university, tokushima, japan) for continuous hot/extensive discussions about virology. i also appreciate ms. fumie nishina (kansai medical university, osaka, japan) and ms. kazuko yoshida (tokushima university) for excellent editorial assistance. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 adachi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-304873-ppb9k3zu authors: kang, hunseung title: direct structural evidence for formation of a stem-loop structure involved in ribosomal frameshifting in human immunodeficiency virus type 1 1 kumho life and environmental science laboratory publication no. 8. 1 date: 1998-04-01 journal: biochimica et biophysica acta (bba) gene structure and expression doi: 10.1016/s0167-4781(98)00004-9 sha: doc_id: 304873 cord_uid: ppb9k3zu abstract programmed ribosomal frameshifting in viral messenger rna occurs in response to neighboring sequence elements consisting of: a frameshift site, a spacer, and a downstream enhancer sequence. in human immunodeficiency virus type 1 (hiv-1) mrna, this sequence element has a potential to form either a stem-loop or a pseudoknot structure. based on many mutational studies, the stem-loop structure has been proposed for the downstream enhancer region of the hiv-1 mrna. this stimulatory stem-loop structure is separated from the shift site by a spacer of seven nucleotides. in contrast, a recent report has proposed an alternative model in which the bases in the spacer sequence form a pseudoknot structure as the downstream enhancer sequence [du et al., biochemistry 35 (1996) 4187–4198.]. using uv melting and enzymatic mapping analyses, we have investigated the conformation of the sequence region involved in ribosomal frameshifting in hiv-1. our s1, v1, and t1 endonuclease mappings, together with uv melting analysis, clearly indicate that this sequence element of the hiv-1 mrna frameshift site forms a stem-loop structure, not a pseudoknot structure. this finding further supports the stem-loop structure proposed by many mutational studies for the downstream enhancer sequence of the hiv-1 mrna. ž . human immunodeficiency virus hiv is a retrovirus that employs y1 ribosomal frameshifting to synthesize essential viral enzymes, including reverse transcriptase, integrase, and protease, as fusion prow x teins with gag proteins 1-3 . the signals for this ) fax: q82-62-972-5085; e-mail: hskang@ksc.kumho.co.kr 1 kumho life and environmental science laboratory publication no. 8. frameshifting process consist of a heptanucleotide shift site and a downstream enhancer sequence in the viral rna. these two important signals are separated by a spacer with a nearly constant number of nucleotides. the mechanism of ribosomal frameshifting is not well understood, although the simultaneous slippage model is the most plausible. the pre-translocation slippage model proposes that when the ribosome ž . enters the frameshift region, the peptidyl p and ž . aminoacyl a sites of the ribosome slip backward simultaneously into the y1 frame at the shift site, 0167-4781r98r$ 19 .00 q 1998 elsevier science b.v. all rights reserved. ž . pii s0167-4781 98 00004-9 ( ) and that translation continues in the new reading w x frame 4,5 . in addition to this pre-translocation slipw x page model, a report by horsfield et al. 6 proposes an erp site post-translocation slippage model in e. coli containing hiv-1 gag-pol frameshift sequence. the enhancer secondary rna structure, either a stem-loop or a pseudoknot, downstream of the shift site induces pausing of the ribosome and stimulates w x slippage at the shift sequence 4-7 . enhancement of ribosomal frameshifting by rna secondary structures has been studied by many mutational analyses. an rna pseudoknot is a common žw x enhancer for highly efficient frameshifting 8 and . references therein . however, in hiv-1 and human ž . t-cell leukaemia virus type ii htlv-ii , frameshifting occurs at a frequency of 1-5% both in vivo and in vitro, and a simple stem-loop structure has been w x implicated for the enhancer region 9,10 . this downstream enhancer structure is separated from the shift site by a spacer sequence of typical length. the importance of the proper distance between this two w x signals was evidenced by a mutational study 11 . w x however, recently du et al. 12 proposed an alternative model involving a pseudoknot for the frameshifting region of the hiv-1 mrna. in this pseudoknot model, the bases in the spacer region are part of stem 1 of the pseudoknot, thus leaving no nucleotides between the shift site and the downstream enhancer ž . fig. 1 structure. direct probing of the rna secondary structure by single-strand and double-strand specific enzymes can distinguish between these two models. here, we determine the secondary structure of the hiv-1 mrna frameshifting sequence elements including the shift site, the spacer, and the downstream enhancer sequence by using ultraviolet melting and enzymatic mapping analysis. the rna sequence element has a potential to form either a stem-loop or a pseudoknot structure, as illustrated in fig. 1 . we show that the downstream enhancer sequence adopts a simple stem-loop structure. the spacer region is not involved in the formation of the stem of a pseudoknot w x as predicted by du et al. 12 , but is in a singlestranded conformation. our results agree well with the mutational studies showing a simple stem-loop structure for the downstream enhancer sequence of the frameshifting region of the hiv-1 mrna. oligoribonucleotides were synthesized in vitro from dna templates by using t7 rna polymerase w x 13,14 . dna templates were synthesized on an applied biosystem 381a and purified by 20% polyacrylamide gel electrophoresis under denaturing con-ž . ditions 7 m urea . rna oligonucleotides were purified by denaturing 20% polyacrylamide gel electrophoresis. the rna transcripts were visualized by uv shadowing, were cut from the gel, were eluted ž from the gel by electroelution schleicher and . schuell , and were precipitated with ethanol. the purified rna oligonucleotides were dialyzed first against 10 mm sodium phosphate, ph 7.0, 100 mm nacl, 5 mm edta, next against 10 mm sodium phosphate, ph 6.4, 100 mm nacl, and finally against 10 mm sodium phosphate, ph 6.4, 50 mm nacl, 5 mm mgcl . 2 all rna melting experiments were done with a gilford model 250 spectrophotometer interfaced to a personal computer. rna samples were in 10 mm ( ) sodium phosphate, ph 6.4, 50 mm nacl, 5 mm mgcl . melting curves were obtained at rna con-2 centrations from 2 mm to 1 mm. rna samples were heated to 908c for 1 min, slowly cooled, and equilibrated to 08c for 20 min. data were recorded at either 260 or 280 nm with the temperature increased from 0 to 958c at a rate of 0.58c min y1 by a gilford model 2527 thermoprogrammer. rna oligonucleotides were dephosphorylated using calf intestinal phosphatase and were 5 x -end labeled using g-32 p-atp and bacteriophage t4 ž . polynucleotide kinase usb . labeled rna was purified by denaturing 20% polyacrylamide gel electrophoresis. the secondary structures of 5 x -32 plabeled rna were probed by using nuclease s and 1 w x rnase v 15 . rnase t digestions were performed 1 1 in 10 mm tris-hcl, ph 7.0, 100 mm kcl, 10 mm mgcl on ice for 10 min. the reactions were stopped 2 ž . by adding 9 m urea, 0.05% wrv xylene cyanol in ž tbe 50 mm tris-hcl, ph 8.1, 50 mm boric acid, 1 . mm edta . the digestion products of each reaction were analyzed on the denaturing 20% polyacrylamide gel. optical melting studies were done for two rna ž . fragments fig. 1 , b and d in 10 mm sodium phosphate, ph 6.4, 50 mm nacl, 5 mm mgcl . the uv 2 absorbance profiles and their first derivatives as a function of temperature are presented in fig. 2 . as expected for a unimolecular melting, the melting temperature was found to be independent of the rna concentration from 2 mm to 1 mm. after heating the rna to 958c, the rna oligonucleotides were refolded by cooling to 08c, and the melting experiments were repeated. the same melting profiles and melting temperatures were recorded, indicating that the melting profiles were not perturbed by any hydrolysis of the rna. the melting temperatures of the ž hiv-1 rna frameshifting-enhancer sequence fig. . 2a and of the truncated rna with the spacer region ž . deleted fig. 2b are identical. this indicates that the spacer region is not involved in the secondary structure of the sequence. it is possible that the stem-loop and the pseudoknot shown in fig. 1 have a same melting temperature. however, considering the truncated rna oligonucleotide without the spacer region can form only a stem-loop conformation, the nearly identical uv melting profiles and melting temperatures for the two rna oligonucleotides indicate indirectly that the rna containing the spacer and the enhancer sequences adopts a stem-loop structure, but not a pseudoknot structure. in order to obtain structural information for the frameshifting sequence elements, enzymatic mapping of the rna was done. before probing the secondary structure of the rna oligonucleotides using enzymatic mapping analysis, we tested for the presence of a single conformation in the samples by a native polyacrylamide gel electrophoresis at 48c. only one ž . band was observed on the native gel data not shown , indicating that the rna oligonucleotides in 10 mm sodium phosphate, ph 6.4, 50 mm nacl, 5 mm mgcl adopt a single conformation. rnase t cleavage patterns of the frameshifting se-1 quence elements consisting of the spacer and the ž enhancer of hiv-1 mrna the sequence shown in . fig. 1a ,b . nucleotides from g1 to g5 were strongly cleaved by single-strand specific nuclease s , but 1 were protected from cleavage by double-strand specific rnase v . however, at a high enzyme concen-1 tration, some of the nucleotides in the region of g1 to g10 was cleaved by rnase v , suggesting that 1 residues 1 to 10 are partially stacked. guanine residues g5-g7, and g9, which are located in the stem region of the proposed pseudoknot structure ž . fig. 1c , were strongly attacked by rnase t that 1 cleaves the g in single-stranded region. these results indicate that residues 1-10 are in the single-stranded conformation. nucleotides a24-a27 were cleaved this cleavage pattern, as summarized in fig. 4 , indicates that in the context of the frameshifting sequence elements of the hiv-1 rna consisting of the spacer and the enhancer, the enhancer sequence adopts a stem-loop structure separated from the shift site by a spacer sequence. it is not consistent with the formation of a pseudoknot. this stem-loop structure agrees with the absorbance melting results and is consistent with the calculated secondary structure from the zuker algorithm. the spacer and the enhancer is solely folded into the stem-loop structure in fig. 1b with a free energy of y21.1 kcal mol y1 . the same free energy of folding is also calculated for the stem-loop of the truncated rna with the spacer ž . region deleted fig. 1d . the requirement of a downstream rna structure to enhance ribosomal frameshifting in hiv has been verified by many mutational studies. by expressing the hiv-1 gag-pol domain in cultured vertebrate w x cells, parkin et al. 9 showed that a stem-loop structure downstream of the hiv-1 shift site is important for wild-type levels of frameshifting. in a cell culture analysis for the sequences and distance between two cis-acting signals in hiv-1 and htlv-2, kollmus et w x al. 11 observed that the slippery sequence alone is sufficient for basal level of frameshifting, but that the ( ) frameshifting efficiency is enhanced by a downstream stem-loop structure. a similar stimulating role of the stem-loop sequence in hiv-1 mrna to enhance frameshifting was also observed in mammalian w x w x cell culture 16 . honda et al. 17 used an in vitro luciferase assay system for hiv-1 translation frameshifting and noted that the stem-loop is not essential for basal-level frameshifting, but it does influence the efficiency of ribosomal frameshifting. . ž t specific for single-strands , and for v specific for double1 1 . strands and stacked bases at a high concentration are consistent with a stem-loop. fig. 4 . summary of the v , s , and t mapping analysis. 1 1 1 nuclease s and ribonuclease t at 48c strongly cleave the 1 1 nucleotides in the spacer sequence, indicating that the spacer sequence is in a single-stranded conformation. the simultaneous slippage model of ribosomal frameshifting proposes that an rna secondary structure downstream of the shift site may slow or stall ribosomes during translation, thus increase the probability of the ribosome to slip backward into the y1 frame at the slippery site. such a model predicts that the correct spacing must be maintained between the shift site and the downstream rna structure. in analysis of the rna sequences responsible for frameshifting in the avian corona virus infectious ž . w x bronchitis virus ibv , brierley et al. 18 observed that alteration of the relative position of the shift sequence by as little as three nucleotides with the respect to the downstream pseudoknot causes a dramatic reduction in frameshifting efficiency. morikawa w x and bishop 19 also demonstrated for the gag-pol ribosomal frameshift site of feline immunodeficiency ž . virus fiv that mutations which alter the distance between the frameshift signal and the downstream pseudoknot structure reduces frameshifting efficiency. in a systematic analysis of the effect of the sequence and the distance between two signals on frameshifting efficiency in htlv-ii, kollmus et al. w x x 11 demonstrated that the base composition 3 to the slippery sequence does not have a significant influence on frameshifting, but that the spacing between the slippery sequence and the 5 x end of the stem-loop is stringently restricted to an optimal distance of seven nucleotides. all of these mutational results imply that the downstream rna structure should be located at a precise position relative to the shift site to enhance frameshifting. in the viruses reported so far, the ( ) relative spacing distance between the stimulator and the slippery sequence varies between five and eight w x nucleotides 8 . of course it is possible that the distance from the more efficient slippery sequence to the downstream rna structure needs not be so tightly constrained. since hiv-1 contains the highly slippery sequence uuuuuua, the distance between two signals needs not be so stringently maintained, and the spacing may vary from three to nine nucleotides to promote different level of frameshifting; the maximum frameshifting efficiency is observed in the context of relative spacing between two signals separated w x by seven nucleotides 11 . although a pseudoknot is the usual enhancer structure in retroviral frameshifting, there is no support for the proposed pseudoknot w x structure for hiv-1 by du et al. 12 . in a recent w x paper, du and hoffman 20 mentioned that the possible pseudoknot structure for hiv-1 is speculative, since it lacks a spacing sequence between the frameshift site and the pseudoknot. the mutation data and our direct structural studies all agree that a stem-loop structure separated from the slippery sequence by about seven nucleotides is necessary for efficient ribosomal frameshifting in hiv-1. proc. natl. acad. sci. ž i thank professor ignacio tinoco, jr. at the department of chemistry, university of california, berkeley for the helpful advice and discussion, and mr. david koh for synthesizing dna templates. the uv melting experiments were done at professor i. tinoco's laboratory. key: cord-310430-7eww1oet authors: singh, ram sarup; thakur, shivani rani; bansal, parveen title: algal lectins as promising biomolecules for biomedical research date: 2013-07-16 journal: crit rev microbiol doi: 10.3109/1040841x.2013.798780 sha: doc_id: 310430 cord_uid: 7eww1oet lectins are natural bioactive ubiquitous proteins or glycoproteins of non-immune response that bind reversibly to glycans of glycoproteins, glycolipids and polysaccharides possessing at least one non-catalytic domain causing agglutination. some of them consist of several carbohydrate-binding domains which endow them with the properties of cell agglutination or precipitation of glycoconjugates. lectins are rampant in nature from plants, animals and microorganisms. among microorganisms, algae are the potent source of lectins with unique properties specifically from red algae. the demand of peculiar and neoteric biologically active substances has intensified the developments on isolation and biomedical applications of new algal lectins. comprehensively, algal lectins are used in biomedical research for antiviral, antinociceptive, anti-inflammatory, anti-tumor activities, etc. and in pharmaceutics for the fabrication of cost-effective protein expression systems and nutraceutics. in this review, an attempt has been made to collate the information on various biomedical applications of algal lectins. lectins are proteins/glycoproteins of non-immune origin that bind non-covalently and reversibly to aposing cells bearing specific sugars culminating their agglutination (singh et al., 2011a) . stillmark (1888) enunciated first lectin (then called hemagglutinin) from seeds of ricinus communis. after that thousands of lectins have been isolated from different sources including plant seeds and roots, bacteria, algae, fungi, body fluid of invertebrates, lower vertebrates and mammalian cell membranes (singh et al., 1999) . they are type cast with respect to carbohydrate-binding specificity, molecular structure, biochemical and biomedical properties. among microbes, occurrence of lectins has been widely reported from algae and mushrooms (singh et al., 2010) . algae are amidst the most diverse organisms in the plant kingdom. they are photosynthetic, mainly aquatic organisms, devoid of vascular tissues, true roots, stems, leaves and possess simple reproductive structures. according to the latest system of classification based on ultra structure of the plastid, algae are classified into four groups which are further subdivided into eight divisions (lee, 1999) : group 1prokaryotic algae containing division (1) cyanophyta; group 2 -eucaryotic algae containing divisions (2) glaucophyta, (3) rhodophyta, (4) chlorophyta; group 3 -eucaryotic algae containing divisions (5) euglenophyta and (6) dinophyta; group 4 -eucaryotic algae containing division (7) heterokontophyta and (8) pyrmnesiophyta. the presence of agglutinins in marine algae was firstly reported by boyd et al. (1966) . later on, lectins have been reported from a large number of algae. algal lectins are generally referred to as phycolectins (matsubara et al., 1996; rogers et al., 1977) and they differ from plant lectins in a variety of physico-chemical characteristics. in general, marine algal lectins are monomeric, low molecular weight proteins, exhibiting high content of acidic amino acids, with isoelectric point (pi) in the range of 4-6, do not require metal ions for their biological activities and most of them show specificity for glycoproteins than monosaccharides rogers & hori, 1993; shiomi et al., 1981) . based on the binding properties to glycoproteins, algal lectins are categorized into three major categories, complex type n-glycan specific lectins, high mannose (hm) type n-glycan specific lectins and lectins with specificity to both the above types of n-glycans . mannose binding lectins are considered essential as they interplay with cell-surface glycoconjugates. due to their small size and presence of disulphide linkages, algal lectins are antigenic and highly stable. furthermore, the peculiar small structure of algal lectins makes them more expedient for use as specific molecular diagnostic probes against the cell surface carbohydrates and in drug targeting (nascimento et al., 2006) . recently, algal lectins have received greater attention due to their robust oligosaccharide-binding specificity (okuyama et al., 2009) . lectins are the most versatile group of proteins used in biological and biomedical research. they posses enormous potential as they play a major role in cell-cell recognition (singh et al., 2011b) and are widely used in drug delivery (singh et al., 1999) . algal lectins have various biomedical properties such as anti-tumor, anti-hiv, anti-inflammatory, anti-fungal, anti-microbial, etc. swamy, 2011; teixeira et al., 2012) . the occurrence of biomedically important lectins among various divisions of algae is represented in figure 1 . lectins have the property of adherence to sugars on cellmembranes, thereby reforming the physiology of membrane which leads to agglutination and other biochemical changes in cells (neves et al., 2007) . algal lectins have been detected using animal erythrocytes as well as human blood type erythrocytes. the susceptibility of erythrocytes to certain lectins increases upon mild treatment with proteolytic enzymes which leads to exposure of cryptic residues present on erythrocytes surface (sharon & lis, 1972) . the biological action spectrum of biomedically important algal lectins is summarized in table 1 . animal erythrocytes especially from sheep and rabbit have been reported to be more suitable for lectin detection in marine algae than human erythrocytes (freitas et al., 1997) . the extracts of microcystis viridis induced agglutination in hen, rabbit and horse erythrocytes, but no agglutination has been reported with human erythrocytes (yamaguchi et al., 1999) . hori et al. (1988) screened a plethora of marine algae for hemagglutinins. they concluded that marine algal agglutinins are most sensitive to protease treated sheep erythrocytes followed by native rabbit and sheep erythrocytes, but not to human and chicken red blood cells. caulerpa cupressoides lectin agglutinated trypsin treated sheep, rabbit and chicken erythrocytes (ainouz & sampaio, 1991) . lectin activity increased significantly when rabbit red blood cells were treated with trypsin, bromelain, papain and subtilisin, but chicken erythrocytes treated with only bromelain showed agglutination (freitas et al., 1997) . serraticardia maxima lectin has been reported to agglutinate native, trypsin and papain treated erythrocytes of horse, cow, sheep, rabbit, guinea pig, mouse and chicken. non-treated horse erythrocytes were most agglutinated, while non-treated cow erythrocytes were least agglutinated (shiomi et al., 1980) . lectin from ulva rigida promoted agglutination of sheep and rabbit erythrocytes (bird et al., 1993) , whereas lectin from ulva pertusa specifically agglutinated rabbit erythrocytes (wang et al., 2004) . bryothamnion seaforthii lectin has been found to agglutinate both native and trypsin treated rabbit as well as trypsin treated chicken and cow erythrocytes (ainouz & sampaio 1991; ainouz et al., 1995; vieira et al., 2004) . lectin from bryothamnion triquetrum has been shown to agglutinate enzyme treated erythrocytes from rabbit, chicken, goat and pig (ainouz et al., 1992) . sheep and rabbit erythrocytes were found sensitive to gracilaria sp. lectin (bird et al., 1993; chiles & bird, 1990) , while extracts from eucheuma serra agglutinated both native and trypsin treated sheep erythrocytes as well as trypsin treated rabbit erythrocytes (kawakubo et al., 1997) . trypsin treated rabbit and chicken erythrocytes were sensitive to crude extract of red algae gracilaria ornata, whereas no agglutination has been reported against native and trypsin treated human erythrocytes (leite et al., 2005) . the extract of oscillatoria agardhii agglutinated both trypsin treated red blood cells of rabbit and pronase treated erythrocytes of sheep (sato et al., 2000; sato & hori, 2009) . the agglutination of blood type a, b and o erythrocytes occurs due to robust binding of lectins to the n-acetyl-dgalactosamine, d-galactose and l-fucose moieties, respectively present on their surface (khan et al., 2002) . the extracts of chlorella sp. i, chlorella sp. 21 and chlorella sp. w have been reported to agglutinate blood type a, b and o erythrocytes (chu et al., 2007) . when native erythrocytes were used, both the crude extract and pure lectin of ptilota plumosa was found to be specific towards human blood group b erythrocytes. after papain treatment, only the pure lectin showed blood type b specificity, whereas the crude extract also showed low agglutination with blood group a and o erythrocytes (sampaio et al., 2002) . human a-type specific agglutinating activity has been reported from bryopsis plumosa . native, trypsin and bromelain treated erythrocytes from mouse, chicken and humans were used to determine the blood specificity of bryopsis hypnoides lectin. the lectin exhibited a preference for trypsin treated human blood group o and chicken erythrocytes (niu et al., 2009) . enzyme treated erythrocytes of human abo blood type were agglutinated by b. triquetrum (ainouz et al., 1992) . sato et al. (2000) green algae boodlea coacta nd h b nd nd nd nd nd nd nd nd nd nd nd hori et al. (1986) bird et al. (1993) red algae haemagglutination activity also with guinea pig erythrocytes. c haemagglutination activity also with carp erythrocytes. the binding specificity of lectins is established by the shape of binding site and the amino-acid residues to which the carbohydrate is linked. alterations in the binding site can essentially change their specificity. algal lectins display a considerable repertoire of carbohydrate specificities and physico-chemical characteristics. the carbohydrate specificity and characteristics profile of biomedically important algal lectins is summarized in table 2 . most of the red algal lectins have high content of acidic and hydroxyl amino acids, but lower levels of basic amino acids. they have low molecular weight, show high affinity to glycoproteins and do not require divalent cations for their biological activity okamoto et al., 1990; rogers & hori, 1993; sampaio et al., 1999) . lectin (oaa) from oscillatoria agardhii belongs to a new lectin family nies-204 and arrayed high binding specificity for high-mannose n-glycans and gp120 (envelope protein of hiv) in picomolar range (sato & hori, 2009 ). lectin (mvl) isolated from m. viridis shown inhibition activity with yeast mannan, whereas lectin (svn) from scytonema varium shown the specificity for man 8 glcnac 2 /man 9 glcnac 2 (li et al., 2008; yamaguchi et al., 1999) . cyanovirin-n (cv-n) lectin of 11 kda from nostoc ellipsosporum showed specificity towards man 9 glcnac 2 (ziolkowska & wlodawer, 2006) . the structural integrity of cv-n lectin has been reported to be highly resistant to degradation upon treatment with detergents, organic solvents, freezing and heating up to 100 c (boyd et al., 1997) . green algae lectin isolated from bryopsis hypnoides shown specificity for n-acetyl glucosamine, n-acetyl galactosamine and bovine submaxillary mucin and was of 27 kda having pi 5-6 (niu et al., 2009) . the lectin was stable in a ph range of 4-8 and does not require metal ions for hemagglutination activity. the lectin from u. pertusa exhibited carbohydrate content of 1.2% with molecular weight of 23 kda, thermal stability up to 70 c for 30 min and carbohydrate specificities for n-acetyl-d-glucosamine and bovine thyroglobulin (wang et al., 2004) . c. cupressoides lectin (ccl) displayed specificity for simple sugars like raffinose, lactose, galactose and fructose, derivatives of galactose and porcine stomach mucin. the molecular weight of the lectin was 44.7 kda and it had carbohydrate content of 11.05% (benevides et al., 2001; freitas et al., 1997) . the three isolectins isolated from kappaphycus alvarezii revealed their monomeric nature, having molecular weight of 28 kda and moreover, displayed affinity for glycoproteins bearing high-mannose n-glycans (hung et al., 2011) . lectin kaa-2 shared physico-chemical properties with esa-2 lectin from e. serra (sato et al., 2011a) . small-sized (9 kda) isolectins (hypnin a1, a2, a3) from hypnea japonica belonged to a new lectin family and showed no affinity for monosaccharides. these isolectins have been reported to bind only to core (a1-6) fucosylated n-gycans which makes them a valuable tool for cancer diagnosis and quality control of medicinal antibodies (okuyama et al., 2009) . amansia lectin of 26.9 kda isolated from amansia multifida contained 2.9% neutral carbohydrates and showed specificity for avidin (costa et al., 1999; neves et al., 2007) . tichocarpus crinitus lectin (tcl) is an acidic glycoprotein with pi 4.93, containing 6.9% carbohydrate content and its amino acid content revealed the presence of aspartic acid and glutamic acid residues (molchanova et al., 2010) . thermostable fetuin, avidin and mucin specific lectins have been reported from b. seaforthii and b. triquetrum with molecular weight of 4.5 kda and 3.5 kda, respectively (ainouz et al., 1995) . the sugar inhibition studies on lectins having molecular weight 41 kda and 25 kda purified from s. maxima and gracilaria verrucosa, respectively, revealed that both are not inhibited by simple sugars (shiomi et al., 1980; 1981) . several bioactivities have been attributed to algal lectins which include anti-inflammatory, anti-adhesion, anti-hiv, antinociceptive, antibiotic, mitogenic and human platelet aggregation inhibition activities (harnedy & fitzgerald, 2011) . the ability of these lectins to stimulate lymphocytes as well as other cells has made them important tools for experiments and diagnostics. biomedical potential of various algal lectins is depicted in figure 2 . the most abeyant biomedical applications of algal lectins are summarized in table 3 . a wide variety of mephitic stimuli are known to bring about powerful inhibition of pain sensation at a remote region of body; nociceptors are sensitized by tissue injury and inflammation. kurihara et al. (2003) reported that primary nociceptor which is known as hyperalgesia in humans and nociception in animal models, which is common for all inflammatory pain types. currently, opioids and non-steroidal anti-inflammatory drugs are used as analgesic. but due to their side-effects and low potency there is a need for an alternative. therefore, the hunt for new compounds for controlling pain and inflammation with low side effects has switched to marine algae. specific binding of lectins with carbohydrates acts an integral part of host defense system. this has opened up a new component of the immune system with both fundamental and practical implications (ahmadiani et al., 1998; vanderlei et al., 2010) . antinociceptive effect of lectins from marine alga a. multifida, b. seaforthii and b. triquetrum has been reported both at central and peripheral levels of the nervous system (neves et al., 2007; viana et al., 2002) . a. multifida lecin (amansin/lec) has also been indicated as a potential analgesic drug (neves et al., 2007) . agglutinin from hypnea cervicornis (hca) showed antinociceptive activity via interaction of the lectin carbohydrate-binding site. lectin hca was able to reduce writhings which suggests inhibition of the release of mediators in response to acetic acid. but formalin-induced nociception suggested that inflammatory pain is mainly responsible for antinociceptive effect; however, the hot plate test postulated peripheral acting mechanism of antinociception (bitencourt et al., 2008) . significant antinociceptive effects have also been demonstrated by chlorella stigmatophora and phaeodactylum tricornutum lectins which reduce neutrophil migration to peritoneal cavity (guzman et al., 2001) . lectin from green algae c. cupressoides produces antinociceptive and anti-inflammatory response in models of nociception in mice and inflammation in rats which attributes peripheral antinociception action against the release of mediators in response to acetic acid (vanderlei et al., 2010) . inflammation is a body's defense reaction caused by injury or damage, which is characterized by rubor (redness), tumor (swelling), calor (heat) and dolar (pain). the first phase of inflammation and edema is marked by the release of histamine and serotonin, second phase involves the release anti-inflammatory 18% anti-nociceptive 21% others 21% anti-viral anti-inflammatory anti-nociceptive anti-cancer others tichocarpus crinitus tcl stimulate synthesis of pro-inflammatory cytokinies tnf-a, ifn-g, il-6 by human whole-blood cells. of cytokines followed by prostaglandins (vanderlei et al., 2010) . hca lectin isolated from h. cervicornis induces antiinflammatory effects in models of paw edema and peritonitis which is elicited by a reduction in leukocyte migration to the peritoneal cavity of the animals. thus, the anti-inflammatory effects occur via competition of mucins of cell glycoproteins with selectins which results in neutrophil reduction and blockade of leukocyte adhesion to the endothelium (neves et al., 2007) . anti-inflammatory effects have also been demonstrated by lectins from c. cupressoides, c. stigmatophora, p. tricornutum and a. multifida which results in neutrophil migration to peritoneal cavity and carrageenaninduced paw-edema of rats (guzman et al., 2001; neves et al., 2007; vanderlei et al., 2010) . lectins in oncology can be used as diagnostic probes and biological response modifiers. due to their small size and several disulphide bridges, marine algal lectins possess greater stability and specificity for complex carbohydrates and glycoconjugates. therefore, they are thought to induce immunogenicity. many algal lectins are reported to possess anti-tumor activity against human cancer cells (karasaki et al., 2001; timoshenko et al., 2001; wang et al., 2000) . tumorspecific ''active targeting'' is often practiced which is achieved by immobilizing tumor-specific ligands (antibodies, peptides or saccharides) onto drug-carrier systems (forssen & wills, 1998; peer et al., 2007; trochilin, 2005) . e. serra agglutinin (esa) induced cell-death of colon cancer colo201 cells and cervix cancer hela cells (sugahara et al., 2001) . esa lectin had strong mitogenic activity against human and mouse lymphocytes due to affinity for glycoproteins bearing highmannose type n-glycans. when immobilized onto span 80 (sorbitan monooleate) vesicles, esa drastically decreased the viability of colo201cancer cells in vitro, whereas normal cells remained unaffected. the vesicles also manifested inhibition of cancer cell growth in nude mice and diminished tumor growth after intravenous administration to nude mice having an implanted colo201 tumor (kawakubo et al., 1997) . lectins bsl and btl from b. seaforthii and b. triquetrum, respectively, were capable of differentiating human colon carcinoma cell variants with respect to their cell membrane glycoreceptors and could be used for structural modifications of cell membrane glycoconjugates in cancer cell systems (pinto et al., 2009) . the binding of both lectins to carcinoma cells results in internalization which could be used for drug delivery. lectins derived from marine algae are a rich source of antiviral products (triveleka et al., 2003) . antiviral activity depends on the ability to bind mannose-containing oligosaccharides present on surface of viral envelope glycoproteins. lectins from cyanobacteria and other marine macro-algae are specific for high-mannose which makes them promising candidates for the prevention of transmission of various enveloped viruses such as human immunodeficiency virus (hiv), influenza virus, hepatitis c virus (hcv), ebola virus and severe acute respiratory syndrome coronavirus (sars-cov) (ziolkowska & wlodawer, 2006) . the specific interaction of algal lectins with target glycans on virus surfaces suppresses virus infection (balzarini, 2007) . boodlea coacta lectin (bca) has been reported to be the first hiv-and influenza virus-inhibiting protein from green algae. bca revealed potent antiviral activity against most of the influenza virus strains tested by binding to the envelope ha (a trimeric glycoprotein expressed on influenza virus membrane) including a clinical isolate of pandemic h1n1-2009 virus (sato et al., 2011b) . lectin isolated from k. alvarezii (kaa-2) exhibited strong antiviral activity against broad range of influenza virus strains including swine-origin h1n1 influenza virus; regardless of the virus subtype and strain. inhibition of influenza virus propagation occurred due to the blocking of viral entry into the host cell by binding to hm glycans on the surface envelope glycoprotein ha. this clearly indicates that kaa-2 completely inhibits yeast mannan bearing hm glycans and binds strongly to ha via hm glycans. the strain-independent inhibition by kaa-2 might be more effective than antibody-based medicines that are more prone to antigenic shift/drift. kaa-2 can be used as novel antiviral agent (sato et al., 2011a) . in a recent groundbreaking study, griffithsin from griffithsia sp. has been reported to be a potent inhibitor of the life-cycle of the coronavirus which is responsible for sars. the antiviral potency of griffithsin is due to presence of multiple, sugar binding sites that provide robust attachment points for complex carbohydrate molecules present on viral envelopes. such broad antiviral activity of this lectin makes it a promising candidate for the development of a novel antiviral agent (ziolkowska & wlodawer, 2006) . lectin cv-n showed an inclusive variety of antiviral activity for influenza viruses (a and b), ebola virus, human herpes virus 6, hcv and measles virus (barrientos et al., 2003; dey et al., 2000; helle et al., 2006; o'keefe et al., 2003) . algal proteins with antiviral activities have now ''emerged'' in the anti-hiv battlefield displaying immense dormant applications as topical agents (feizi et al., 2011) . most of the research on anti-hiv activity of marine algae has converged upon red and brown macroalgae (schaeffer & krylov, 2000) . high-mannose binding nature of algal lectins makes them expedient candidates for inhibiting hiv (botos & woldawer, 2005) . they interact with glycans and cells of the host, thus disturbing proteins of viral envelope and cells of the host. a number of lectins isolated from red algae exhibit inhibitory activity against hiv. griffithsin/grft isolated from griffithsia sp. is a completely novel protein with no homology to any of the proteins listed in blast database. grft displays potent antiviral activity against both laboratory-adapted strains and primary isolates of hiv-1 (alexandre et al., 2010; charan et al., 2000; giomarelli et al., 2006; ziolkowska & wlodawer, 2006) at subnanomolar concentrations (ic 50 â¼ 0.4 nm and ec 50 â¼ 0.043-0.63) which inhibits cell fusion and cell-to-cell hiv transmission (emau et al., 2007) in contrast to several other monosaccharide-specific lectins from same structural family. grft is not only the strongest hiv inhibitor manifesting broad spectrum activity against various clades of hiv, but also acts as an initiation point for the design of smaller peptide-based antiviral minilectins which can be directed against high-mannose sugars (micewicz et al., 2010) . cv-n lectin purified from n. ellipsosporum shares no similarity with other protein sequences which are deposited so far in public protein databases. cv-n is a potential inhibitor of both laboratory adapted and clinically isolated strains of hiv-1, hiv-2 and simian immunodeficiency virus (bewley et al., 1998; dey et al., 2000) . furthermore, cv-n prevents in vitro fusion and transmission of hiv-1 between infected and non-infected cells (boyd et al., 1997) . cv-n is highly resistant to physicochemical denaturations which are caused by various denaturants, detergents, organic solvents, multiple freeze thaw cycles and heat up to 100 c with no loss of antiviral activity. these characteristics further boost its potential as an anti-hiv microbicide (bewley et al., 1998; boyd et al., 1997) . grft, cv-n and svn are mannose specific lectins found interacting with mannose-rich glycans present on the viral envelope and blocking hiv-1 entry in vitro. this supports their potential as microbicides or topical virucide to prevent sexual transmission of hiv and aids (alexandre et al., 2010; mori et al., 2005) . the envelope glycoprotein of hiv (gp120) is among the most heavily glycosylated proteins known so far. up to 50% of this 120-kda glycoprotein is contributed by n-linked carbohydrates. in particular, hiv gp120 contains 20-29 nglycosylation sites depending on the nature of the viral isolate and the type of virus clade. highly dense carbohydrate shield on gp120 has been found to be responsible for its low antigenicity and low immunogenicity. it also protects the virus against the immune system (balzarini et al., 2005) . envelope glycoprotein gp120 and gp 41 of hiv-1 forms a trimer complex that mediates virus entry into target cells through receptor binding events. as demonstrated in studies, gp120 is composed of variable region (v 1 -v 5 ) and constant regions (c 1 -c 5 ). v 3 loop acts as the major determinant of viral entry. carbohydrate moieties are affirmed to act as shields for gp120 which is highly glycosylated. thus, carbohydrate-binding agents including cv-n and griffithsin inhibit hiv-1 infection (hu et al., 2007) . algae are promising organisms to furnish novel biochemically active compounds which are of potential importance to pharmaceutical sector and general public health. lectin from a. multifida (amansin) possesses the ability to induce interferon (inf-g) production, neutrophil migration and is also a powerful stimulant of quiescent peripheral blood lymphocytes which can induce blast transformation heading for mitosis in cells in vitro. low molecular weights of algal lectins play a vital role in the study of neutrophil migration as this prevents steric problems (neves et al., 2001) . lectin bryohealin from b. plumosa has the potential of woundhealing . similarly, lectin diabolin isolated from laminaria diabolica initiates the development of a fertilization envelope around unfertilized eggs of sea urchin hemicentrotus pulcherrimus which prevents its cleavage (smit, 2004) . tcl stimulates the synthesis of pro-inflammatory cytokines tnf-a, inf-g and interleukin-6 by human whole blood cells (molchanova et al., 2010) . tcl has also been reported to be a potent mitogen for human lymphocytes. the bacteriostatic and stimulatory effects on the growth of several gram negative bacteria (serratia marcescens, salmonella typhi, klebsiella pneumonia, pseudomonas aeruginosa, enterobacter aerogenes, proteus sp) and gram positive bacteria (bacillus cereus) have been reported from solieria filiformis lectin (holanda et al., 2005) . h. japonica lectin (hypnin a) inhibits human platelet aggregation induced by adp or collagen in a dose-dependent manner (matsubara et al., 1996) . this lectin exhibits potent mitogenic activity against both lymphocytes from mouse and human. it also induces toxicity to tumor cells by inhibition of embryonic development of marine invertebrates matsubara et al., 1996) . gracilaria cornea lectin through multimechanistic approach showed acaricidal and antifeedant activity against anagasta kuehniella (flour moth), which may be important for controlling pests from a new natural source (lima et al., 2005) . interestingly, algal lectins are also used in antiadhesion trials. lectins btl and bsl have been shown to block adhesion of streptococci to their mucin receptors in acquired pellicle via competition mechanism (teixeira et al., 2007) . these lectins interfere both with bacterial adhesion and aggregation. thus, antiadhesion lectin therapy is a promising solution to the problems of caries. lectins are widely used in lectinosorbent assays which characterize cell-binding patterns (smit, 2004) . algae studied for lectins comprise only a negligible expanse of the total number of algal species and, therefore, a comprehensive province remains to be scrutinized. lectins isolated from marine resources are highly diversified not only in terms of structure, but also functional aspects including specific and unique carbohydrate specificities. the research upshot concerns the evolution of powerful tools for the study of cancer, hiv and other diseases. the ultimate goal is to develop emphatic microbicides that not only stymie the transmission of cell-free viruses but also the transmission of donar-hiv infected t-cells and guards against other stds (huskens & schols, 2012) . the sugar binding specificity of lectins towards glycoconjugates has made them captivating proteins. this property enables them to fabricate useful tools for various therapeutic applications including cancer diagnosis and prognosis, pathological markers of diseases, glycan profiling, cell-communication, bioadhesion and for controlling a variety of infections. significant research on algal lectins during past few decades has accelerated the understanding of molecularmechanism entangling adherence and recognition. the specific coupling and greater ph stability of algal lectins showed reversible linkage of algal lectins to drug enhancing penetration of drugs which can be used for targeting drugs to tumor tissue and for oral drug delivery. a number of lacunae still persist which need to be filled. even though an invigorative role of many lectins has been evident, further pharmacokinetic studies need to be endeavored before their introduction as clinical tools. distinct sources should be traversed to confine avant-grade lectins with dormant dupable properties. sanguinely, further groundwork is required to endow in vivo succor of these algal lectins equivalent to their in vitro effects and can be carried forward for the development of oral drug delivery systems, mucoadhesive formulations, lectinosorbent assays and development of efficient, safe and affordable microbicides. in case of anti-hiv drugs what is now needed is to determine precisely the distinctive features among numerous lectins that confer antiviral activity. thus, it would be possible to engineer proteins with multiple binding sites recognizing different motifs for use as anti-hiv drugs with enhanced potencies (feizi et al., 2011) . the author realizes that the need of the hour is to characterize and overcome the shortcomings in purification of algal lectins for exploring immense empire of algal lectins for biomedical applications. antinociceptive and anti-inflammatory effects of sambucus ebulus rhizome extracts in rats agglutination of enzyme treated erythrocytes by brazilian marine algae comparative study on hemagglutinins from the red algae bryothamnion seaforthii and bryothamnion triquetrum screening of brazilian marine algae for hemagglutinins mannose-rich glycosylation patterns on hiv-1 subtype c gp120 sensitivity to the lectins, griffithsin, cynovirin-n and scytovirin targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp120 cyanovirin-n binds to the several glycoprotein gp (1, 2) and inhibits infectivity of ebola virus atividade hemaglutinante na alga vermelha solieria filiformis purification and partial characterization of the lectin from the marine green alga caulerpa cupressoides (vahl) c. agardh new carbohydrate specificity and hiv-1 fusion blocking activity of the cyanobacterial protein mvl solution structure of cyanovirin-n, a potent hiv-inactivating protein agglutinins from marine macroalgae of the south eastern united states antinociceptive and anti-inflammatory effects of a mucin-binding agglutinin isolated from the red marine alga hypnea cervicornis proteins that binds high-mannose sugars of the envelope discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development agglutinins in marine algae for human erythrocytes the amino acid sequence of the agglutinin isolated from the red marine algae bryothamnion triquetrum defines a novel lectin structure isolation and characterization of myrianthus holstii lectin, a potent hiv-1 inhibitory protein from the plant myrianthus holstii algae of peter the great bay of the sea of japan as a source of lectins a comparative study of animal erythrocyte agglutinins from marine algae gracilaria tikvahiae agglutinin. partial purification and preliminary characterization of its carbohydrate specificity analysis of the agglutinating activity from unicellular algae purification and characterization of a lectin from the red marine alga amansia multifida multiple antiviral activities of cyanovirin-n: blocking of human immunodeficiency virus type 1 gp120 interaction with cd4 and coreceptor and inhibition of diverse enveloped viruses griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide bacterial, fungal and algal lectins: combants in tug of war against hiv agglutinin isolated from the red marine alga hypnea cervicornis j. agardh reduces inflammatory hypernociception: involvement of nitric oxide ligand-targeted liposomes a new survey of brazilian marine algae for agglutinins recombinant production of anti-hiv protein, griffithsin, by auto-induction in a fermentor culture antiinflammatory, analgesic and free radical scavenging activities of the marine microalgae chlorella stigmatophora and phaeodactylum tricornutum purification and characterization of a d-mannose specific lectin from the green marine alga, bryopsis plumosa bioactive proteins, peptides and amino acids from macroalgae cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans differential activity of a lectin from solieria filiformis against human pathogenic bacteria some common properties of lectins from marine algae hypnins, low molecular weight peptidic agglutinins from a marine red alga, hypnea japonica primary structures of two hemagglutinins from the marine red alga, hypnea japonica evidence for wide distribution of agglutinins in marine algae high-mannose-specific deglycosylation of hiv-1 gp120 induced by resistance to cyanovirin-n and the impact on antibody neutralization seasonal changes in growth rate, carrageenan yield and lectin content in red alga kappaphycus alvarezii cultivated in camrah bay high-mannose n-glycan-specific lectin from the red alga kappaphycus striatum (carrageenophyte) algal lectins as potential hiv microbicide candidates characterization of carbohydrate combining sites of bryohealin, an algal lectin from bryopsis plumosa isolation and characterisation of a fourth hemagglutinin from the red alga, gracilaria verrucosa, from japan a garlic lectin exerted an antitumor activity and induced apoptosis in human tumor cells the marine alga eucheuma serra j. agardh, a high yielding source of two isolectins occurrence of highly yielded lectins homologous within genus eucheuma lectins as markers for blood grouping acetic-acid conditioning stimulus induces long-lasting antinociceptive of somatic inflammatory pain purification of a lectin from the marine red alga gracilaria ornate and its effect on the development of the cowpea weevil callosobruchus maculates (coleoptera: bruchidae) algal lectins for potential prevention of hiv transmission red marine alga bryothamnion triquetrum lectin induces endothelium dependent relaxation of the rat aorta via release of nitric oxide induction and inhibition of human lymphocyte transformation by the lectin from red marine alga amansia multifida purification of a lectin from the marine red alga gracilaria cornea and its effects on the cattle tick boophilus microplus (acari: ixodidae) platelet aggregation is inhibited by phycolectins grifonin-1: a small hiv-1 entry inhibitor derived from the algal lectin purification and partial characterization of the lectin from the marine alga tichocarpus crinitus (gmelin) rupr. (rhodophyta) isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia spp hca and hml isolated from red marine algae hypnea cervicornis and hypnea musciformis define a novel lectin family an overview of lectins purification stratergies isolation and characterization of a new agglutinin from the red marine alga hypnea cervicornis neutrophil migration induced in vivo and in vitro by marine algal lectins antinociceptive properties in mice of a lectin isolated from the marine alga amansia multifida lamouroux characterization of a new lectin involved in the protoplast regeneration of bryopsis hypnoides potent anti-influenza activity of cyanovirin-n and interactions with viral hemagglutinin isolation and characterization of a new hemagglutinin from red alga gracilaria bursa-pastoris strict binding specificity of small-sized lectins from the red marine alga hypnea japonica for core (a-1, 6) fucosylated n-glycans purification and characterisation of a lectin from the red marine alga pterocladiella capillacea in vitro and in vivo anti-tumor effects of novel span 80 vescicles containing immobilized eucheuma serra agglutinin nanocarriers as an emerging platform for cancer therapy lectins from the red marine algal species bryothamnion seaforthii and bryothamnion triquetrum as tools to differentiate human colon carcinoma cells ptilota plumosa, a new source of blood group b specific lectin marine algal lectins: new developments a new isolation procedure and further characterisation of the lectin from the red marine alga ptilota serrata new affinity procedure for the isolation and further characterization of the blood group b specific lectin from the red marine alga ptilota plumosa a galactose-specific lectin from the red alga ptilota filicina cloning, expression and characterization of a novel anti-hiv lectin from the cultured cyanobacterium, oscillatoria agardhii high mannose-binding lectin with preference for the cluster of a 1-2 mannose from green alga boodlea coacta is a potent entry inhibitor of hiv-1 and influenza viruses high mannosespecific lectin (kaa-2) from the red alga kappaphycus alvarezii potently inhibits influenza virus infection in a strain-independent manner purification and characterization of novel lectin from a fresh water cyanobacterium, oscillatoria agardhii primary structure and carbohydrate binding specificity of a potent anti-hiv lectin isolated from the filamentous cyanobacterium oscillatoria agardhii anti-hiv activity of exracts and compounds from algae and cyanobacteria lectin: cell agglutinating and sugar-specific proteins purification and physiochemical properties of a hemagglutinin (gva-1) in the red alga gracilaria verrucosa biochemical properties of hemagglutinins in the red alga serraticardia maxima antibacterial activity and fatty acid composition of the extract from hypnea musciformis (gigartinales, rhodophyta) mushroom lectins: current status and future perspectives current trends of lectins from microfungi characteristics of yeast lectins and their role in cell-cell interaction lectins: sources, activities and applications medicinal and pharmaceutical uses of seaweed natural products: a review uber rizin, ein giftiges ferment aus dem samen von ricinus communis l.und einigen anderen euphorbiaceen the cytotoxic effect of eucheuma serra agglutinin (esa) on cancer cells and its applications to molecular probe for drug delivery system using lipid vesicles marine algal sources for treating bacterial diseases biological applications of plants and algae lectins: an overview in vitro inhibition of oral streptococci binding to the acquired pellicle by algal lectins immunotherapy of c3h/hej mammary adenocarcinoma with interleukin-2 mistletoe lectin or their combination: effects on tumor growth capillary leakage and nitric oxide (no) production natural products with anti-hiv activity from marine organism recent advances with liposomes as pharmaceutical carriers antinociceptive and anti-inflamatory activities of lectin from the marine green alga caulerpa cupressoides antinociceptive activity of sulfated carbohydrates from the algae bryothamnion seafortii (turner) kutz. and b. triquetrum (s.g. gmel) m. howe the alga bryothamnion seafortii contains carbohydrate with antinociceptive activity a new lectin with highly potent antihepatoma and antisarcoma activities from oyster mushrooms pleurotus ostreatus molecular characterization of a new lectin from the marine alga ulva pertusa hemagglutination activity of microcystis aeruginusa (cyanobacterium) purification and characterization of microcystis aeruginosa (freshwater cyanobacterium) lectin isolation and characterization of a mannan-binding lectin from fresh water cyanobacteria (blue-green algae) microcystis viridis domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding structural studies of algal lectins with anti-hiv activity the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. key: cord-302854-buzyani0 authors: prabakaran, ponraj; zhu, zhongyu; chen, weizao; gong, rui; feng, yang; streaker, emily; dimitrov, dimiter s. title: origin, diversity, and maturation of human antiviral antibodies analyzed by high-throughput sequencing date: 2012-08-02 journal: front microbiol doi: 10.3389/fmicb.2012.00277 sha: doc_id: 302854 cord_uid: buzyani0 our understanding of how antibodies are generated and function could help develop effective vaccines and antibody-based therapeutics against viruses such as hiv-1, sars coronavirus (sars cov), and hendra and nipah viruses (henipaviruses). although broadly neutralizing antibodies (bnabs) against the hiv-1 were observed in patients, elicitation of such bnabs remains a major challenge when compared to other viral targets. we previously hypothesized that hiv-1 could have evolved a strategy to evade the immune system due to absent or very weak binding of germline antibodies to the conserved epitopes that may not be sufficient to initiate and/or maintain an effective immune response. to further explore our hypothesis, we used the 454 sequence analysis of a large naïve library of human igm antibodies which had been used for selecting antibodies against sars cov receptor-binding domain (rbd), and soluble g proteins (sg) of henipaviruses. we found that the human igm repertoires from the 454 sequencing have diverse germline usages, recombination patterns, junction diversity, and a lower extent of somatic mutation. in this study, we identified antibody maturation intermediates that are related to bnabs against the hiv-1 and other viruses as observed in normal individuals, and compared their genetic diversity and somatic mutation level along with available structural and functional data. further computational analysis will provide framework for understanding the underlying genetic and molecular determinants related to maturation pathways of antiviral bnabs that could be useful for applying novel approaches to the design of effective vaccine immunogens and antibody-based therapeutics. broadly neutralizing antibodies (bnabs) against the hiv-1 are relatively rarely observed in patients; however, discovering hiv-1 vaccine candidates to elicit such bnabs remains a challenge due to the extensive genetic sequence variability and complex immune evasion strategies of the hiv-1 (burton, 2002; johnson and desrosiers, 2002; haynes and montefiori, 2006; prabakaran et al., 2007) . among the different factors thwarting the induction of bnabs, we previously found that all known hiv-1 bnabs are highly divergent from germline antibodies; germline antibodies of bnabs could not bind to the epitopes of respective mature antibodies, which led to a hypothesis that hiv-1 may have evolved to use the "holes" (absence of or weak binding to germline-lineaged bnabs) in the human germline b cell receptor repertoire (xiao et al., 2009) . consistent with our earlier hypothesis, we did not find any specific binders against the hiv-1 envelope glycoproteins (envs) but only identified binders against the sars cov receptorbinding domain (rbd), and soluble hendra virus g protein (sg) when combinatorial phage display libraries mimicking human antibody repertoire constructing from human igm libraries had been used for panning experiments . these findings had indicated that the major problem could be related to a high level of somatic mutations required for bnabs to accurately target the conserved structures on the hiv-1 envs. in this article, we have used high-throughput 454 sequencing of a large naïve library of human igm antibodies to explore antibody repertoire landscape for finding germline usages, somatic mutations, intermediates, and phylogenetic relationships between the intermediates and corresponding antiviral-related bnabs including the hiv-1, sars cov, and henipaviruses. this study helped to identify germline predecessors of bnabs observed in normal individuals, and find maturation pathways of antiviral bnabs. indeed, most of the known hiv-1 bnabs are highly divergent from their closest respective germlines as well as their intermediates as they undergo somatic mutations required for their neutralization function. the results corroborate that the hiv-1 may use a strategy to eliminate strong binding of germline antibodies due to the absence of closer anti-hiv antibody intermediates as an escape mechanism from adaptive immune responses, and finding of closer intermediates of bnabs from rare individuals might help designing the effective vaccines against the hiv-1 and other viral diseases. to amplify igm antibody sequences, cdna was prepared from peripheral blood b cells of 10 healthy donors as received under the research donor program of frederick national laboratory for cancer research, usa, which we previously used to construct a naïve human fab phage display library for selecting antibodies against sars cov and henipaviruses. the complete set of primers used in the pcr amplification of igm-derived heavy and light chains were described in detail elsewhere ). for 454 sequencing, primer combinations used to amplify cdna in separate reactions included the roche a and b adaptor sequences along with target amplification sequence for heavy and light chain variable domains. the gene fragments were amplified in 20 cycles of pcr using the high fidelity pcr master from roche. more detailed description of 454 sequencing can be found in our recent articles (prabakaran et al., 2011 . the standard roche 454 gs titanium shotgun library protocol was adapted as found in the roche sequencing technical bulletin. for quality control of antibody sequences, we trimmed the 454 sequence data and retained only sequences of length more than 300 nucleotides (nt), covering the entire antibody variable domains consisting of the three complementarity determining regions (cdr) along with framework regions (fr). we used imgt/highv-quest (alamyar et al., 2012) , a high-throughput version for deep sequencing ngs data analysis resource for the immunogenetic analysis. the output results from the imgt/highv-quest analysis in csv files were stored at postgresql database, and structured query language (sql) was used to retrieve the data for the further analysis. heatmap generation and statistical calculations involving distributions of antibody hcdr3 lengths and mutations were carried out using sas jmp10® statistical software (sas institute, cary, nc). translated heavy and light chain variable sequences from the 454 sequencing that shared the ighv genes of selected antiviral antibodies and associated immunogenetics data including the details of germlines, hcdr3 lengths, and mutations were retrieved from the database by using sql. sequence identities between the 454 sequence data and germlines were calculated based on the pairwise alignment using local blast as implemented in bioedit v7.0.9 (hall, 1999) . phylogenetic analysis was carried out using the archaeopteryx software (han and zmasek, 2009 ). to analyze germline origin of antiviral antibodies against the hiv-1, sars cov, and henipaviruses as expressed in the human igm repertoire, we performed 454 sequencing of a non-immune library which was previously constructed from peripheral blood b cells of 10 healthy donors and used to select antibodies against sars cov and henipaviruses (prabakaran et al., 2006; zhu et al., 2006) . a total of 113,139 sequences were obtained from which 91,528 sequences were found as unique with each had >300 nt in length. the total number of unique amino acid (aa) sequences for each v-gene subgroup in heavy and light chains that were found functionally productive as determined by imgt/highv-quest (alamyar et al., 2012) are shown in figures 1a,b , respectively. the read coverage or gene frequencies observed in the study suggested for biased germline usages and were comparable to the previous studies (glanville et al., 2009; prabakaran et al., 2012) but way far less than the theoretical diversity attainable by antibodies attributing to several factors such as library sampling, primer efficiency, and sequencing errors and limitations. nevertheless, we selected known bnabs against the viral targets including the hiv-1, sars cov, and henipaviruses (table 1) , and created sequence data sets related to those bnabs from the 454 analysis as depicted in figures 1c,d showing the germline usage frequencies of ighv genes in the v h domains, igkv, and iglv genes in the v κ and v λ domains, respectively. we found that while all antiviral-related germlines were expressed in human igm repertoire, some preferential germline usages were noted, for example, hv1-69 gene in ighv subgroups and kv3-20/lv2-14 genes in igkv/iglv subgroups were overrepresented (figures 1c,d) . the role of heavy chains of antiviral antibodies in antigen recognition is found to be associated with longer hcdr3s and extensive v h mutations ( table 1) . most of the bnabs have longer hcdr3s with aa lengths ranging from 20 to 30, except for 2g12, vrc01 and m396. all of the v h genes of anti-hiv-1 antibodies have a high degree of somatic mutations when compared to non-hiv-1 antiviral bnabs. we analyzed hcdr3 length distributions and v h mutations preexisting in germline-lineaged precursor antiviral antibodies from the ighv genes of igm repertoires from which bnabs were generated. the box plots display the distributions of hcdr3 lengths and v h mutations, figures 2a,b , respectively, which indicates a high level hcdr3 length diversity and lesser extent of somatic mutations compared to bnabs ( table 1) . to assess the vdj repertoire usage among different antiviral related ighv genes, we computed the frequencies of vdj recombination patterns as observed in the v h genes expressed in human igm repertoire involving those ighv genes of antiviral antibodies. the heatmap is shown in the figure 3 depicting the figure 3 | frequencies of vdj recombination types as observed in the human igm repertoire involving ighv genes related to the antiviral bnabs. the heatmap is colored according to the total number of unique vdj patterns existing in the corresponding ighv genes used in association with different ighd and ighj genes, and is shown on a blue-to-gray-to-red scale. the white-colored space represents the missed or absent vdj recombination types in the repertoire. august 2012 | volume 3 | article 277 | 4 most (red) and least (blue) abundant vdj types existing in the germline-lineaged repertoire for the corresponding ighv genes used in association with different ighd and ighj genes. the ighv genes v1-69 and v1-2 were frequently found to recombine with ighj genes j4 and j6, and ighd genes d3 and d6. the intermediate antibodies corresponding to bnabs against the hiv-1, sars cov, and henipaviruses were found by analyzing the human igm repertoire, and such intermediates with the closest similarities to the matured antiviral bnabs were selected for germline-linage analysis by using phylogenetic method. ighv germline gene alleles of bnabs were obtained from the imgt database. the mid-point phylogenetic neighbor-joining tree showing the evolutionary relationships of different antiviral antibodies with their corresponding germlines and intermediates is given in figure 4 . we observed that some of the anti-hiv-1 antibodies (2g12, ch01, and vrc01) were found at distal nodes in the phylogenetic tree indicating high divergence from their corresponding germline and intermediate counterparts. in contrast, bnabs against sars cov, and henipaviruses, m396 and m102, were found closer to their intermediates. we found 169 unique ighv sequences from the v1-3 gene family as intermediates of bnab b12 by using the 454 sequence analysis of a human igm library. phylogenetic analysis of those intermediates revealed two major groups, one group consisting of germline related antibodies and the other having potential intermediates closer to the bnab b12. we then constructed a phylogenetic sub-tree selecting only the potential intermediates and the v1-3 * 01 germline along with bnab 12. the tree was rooted at the known germline v1-3 * 01 of bnab b12, and phylogram showed evolutionary relationship among the different intermediates ( figure 5a) . one of the intermediates, g3jy1, had the maximum of 72% sequence identity (82% sequence similarity) at aa level to the bnab b12 ( figure 5b) . however, the hcdr3 length of that intermediate was found to be 17 aa long, which is 3 aa shorter than that of b12 antibody. to find the closest hcdr3 to that of b12, we scanned 28,925 unique hcdr3 sequences from the entire igm 454 sequence data. we identified a hcdr3 with the same length (20 aa) and 50% sequence identity to that of b12 (figure 5c) , which was found to be the most similar to the hcdr3 of b12 but the ighv gene associated with that hcdr3 was found to be v4-b. we used the hiv-1 gp120-b12 complex structure and mapped the v h somatic mutations, which showed the overlapping of three mutated residues of b12 (n36 from hcdr1, y59 from hcdr2, and w111.1 from hcdr3) that contribute to the most of binding interactions with the gp120 as previously observed (zhou et al., 2007) (figure 5d ). in this study, we have described the 454 sequence analysis of a large naïve library of human igm antibodies, and carried figure 4 | the mid-point phylogenetic neighbor-joining tree shows the evolutionary relationships between different ighvs of (bnabs) with their corresponding germlines and intermediates. the ighv germline gene alleles follow the imgt nomenclature and the closest intermediates of bnabs as found from the human igm repertoire were designated with asterisks along with names of bnabs. some of the anti-hiv-1 antibodies (2g12, ch01, and vrc01) were found at distal nodes in the phylogenetic tree indicating high divergence from their corresponding germline and intermediate counterparts. out immunogenetic analysis to study the origin, diversity, and maturation of selected known bnabs against the hiv-1, sars cov rbd, and henipaviruses sg proteins. we have found intermediates of antiviral related bnabs, of which most of those against the hiv-1 were highly diverged from their mature forms of bnabs as compared to other viral targets, sars cov, and henipaviruses. although antibodies are generated through various mechanisms involving vdj recombination, junctional modification, and hypermutations, the v-genes sculpt the most of the antigencombining sites, cdr1 and cdr2, and support frameworks for the cdr3. we found that antiviral antibodies targeting different env binding regions of the hiv-1 and other viruses utilized different germline v-genes as the origins (table 1) . we noted that, among antiviral-related bnabs, the v1-69 gene usage was dominated in the heavy chains while v3-20 and v2-14 genes of kappa and lambda were used with the highest frequencies in the light chains of human igm repertoire (figure 1) . accordingly, four of the v h genes of bnabs (4e10, x5, m102, and m396) originated the most similar hcdr3 sequence to that of bnab b12 from the igm repertoire was found to originate from v4-b * 01 gene and is shown in the pairwise alignment. (d) mapping of three of the somatically mutated residues n36, y59, and w111.1, as per imgt numbering scheme, from each of the hcdrs are shown as sticks using the complex crystal structure of hiv-1 gp120-b12 (pdb code 2ny7). from the v1-69, and three of them paired with the kappa v3-20 gene. one possible reason for dominance in the usage of those germline genes could be reflecting from the relatively higher frequencies of distributions observed in the expressed igm repertoire (figures 1a,b) . the hv3 gene was used in the three of the hiv-1 bnabs, 2g12, pg9, and ch01. the structural data for most of the bnabs selected in this analysis were known and the heavy chains of these bnabs were dominantly used. the increased number of v h mutations and longer hcdr3s are characteristics for the hvi-1 bnabs when compared to other antiviral bnabs (breden et al., 2011) . we analyzed the distribution of hcdr3 lengths and extent of somatic v h mutations in the human igm repertoire to compare with that of antiviralrelated bnabs (figure 2) . the results showed that the longer hcdr3s and low level of somatic v h mutations as compared to the hiv-1 bnabs existed in the intermediates as found from the 454 sequencing. the somatic diversity through vdj recombination involving antiviral-related v-genes in the igm repertoire was found high; the most abundant vdj combination consisted of the hv1-69 gene with certain d and j genes as depicted in gray and red (figure 3) , which might be the reason for the preferential usage of that hv1-69 in many other viral diseases (sui et al., 2009 ). further, bnabs against the sars cov and henipaviruses shared the heavy chain v-gene germline, hv1-69, with two of the hiv-1 bnabs, 4e10, and x5. all of these four bnabs were less divergent from their v-germlines and intermediates, when compared to other hiv-1 bnabs, and formed a single cluster at a mid-point rooted phylogenetic tree (figure 4) . the gp41 membrane-proximal epitope region (mper) binding site bnabs, 2f5, and m66, were moderately divergent from their vgermlines and intermediates and formed distinct clusters. the v-gene of vrc01 bnab was the most divergent from its respective germline as well as the closest intermediate, and was placed at a distal branch of hv1 subgroup of bnabs. for the mid-point rooted phylogenetic analysis, we included the closest intermediates only; however, favored maturation pathways could involve other intermediates too. we created the germline-rooted phylogenetic tree as a use-case for the bnab b12 ( figure 5a ) and analyzed the maturation pathway along different v-gene intermediates from hv1-3 gene family. the closest b12 intermediate, designated as g3jy1, had three mutations each at hcdr1 and hcdr2 compared to the germline, and were found similar though not identical to that of mature b12 ( figure 5b) . interestingly, we also identified a hcdr3 with the same length (20 aa) and 50% sequence identity to that of b12 (figure 5c) , which was found to be the most similar to the hcdr3 of b12 but the ighv gene associated with that hcdr3 was found to be v4-b. this might suggest for the possible maturation mechanism of bnabs which could be involving the vh replacement (chen et al., 1995) . these two mutated residues (n36 from hcdr1 and y59 from hcdr2) from the v-gene and a trp residue from the d-gene (w111.1 from hcdr3) contributed to the most of binding interactions with the gp120 (figure 5d ) (zhou et al., 2007) . in summary, the 454 sequence analysis of a large naïve human antibody repertoire corresponding to the selected antiviralrelated bnabs revealed the germline v-gene usage, vdj rearrangement, hcdr3 length diversity, and somatic mutations of potential intermediate antibodies of hiv-1 and other viruses such as sars cov and henipaviruses. thus, b cell germlinelineage analysis using the 454 sequence data from different sources could help finding appropriate antibody intermediates, pathways, and mechanisms useful in the development of bnabs and vaccines against the hiv-1 and other viral diseases. quest: the imgt® web portal for immunoglobulin (ig) or antibody and t cell receptor (tr) analysis from ngs high throughput and deep sequencing comparison of antibody repertoires produced by hiv-1 infection, other chronic and acute infections, and systemic autoimmune disease antibodies, viruses and vaccines immunoglobulin heavy chain gene replacement: a mechanism of receptor editing and selection of monoclonal antibodies to viral envelope glycoproteins: implications for mechanisms of immune evasion and design of vaccine immunogens precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire bioedit: a userfriendly biological sequence alignment editor and analysis program for windows 95/98/nt phyloxml: xml for evolutionary biology and comparative genomics aiming to induce broadly reactive neutralizing antibody responses with hiv-1 vaccine candidates viral persistance: hiv's strategies of immune system evasion expressed antibody repertoires in human cord blood cells: 454 sequencing and imgt/highv-quest analysis of germline gene usage, junctional diversity, and somatic mutations structure and function of the hiv envelope glycoprotein as entry mediator, vaccine immunogen, and target for inhibitors structure of severe acute respiratory syndrome coronavirus receptorbinding domain complexed with neutralizing antibody 454 antibody sequencing -error characterization and correction structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses germline-like predecessors of broadly neutralizing antibodies lack measurable binding to hiv-1 envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens structural definition of a conserved neutralization epitope on hiv-1 gp120 potent neutralization of hendra and nipah viruses by human monoclonal antibodies construction of a large naive human phage-displayed fab library through one-step cloning we thank the laboratory of molecular technology of saic-frederick inc. for providing roche 454 sequencing service. we are grateful to eltaf alamyar and to the imgt® team for providing access to imgt/highv-quest. we thank ms. maria g. singarayan for constructing the postgresql database and java applications and helping with sql. this research was supported by the intramural research program of the nih, national cancer institute, center for cancer research, and by federal funds from the nih, national cancer institute, under contract no. no1-co-12400. the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the us government. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-327135-4c2flue4 authors: chinnaswamy, s title: gene–disease association with human ifnl locus polymorphisms extends beyond hepatitis c virus infections date: 2016-06-09 journal: genes immun doi: 10.1038/gene.2016.24 sha: doc_id: 327135 cord_uid: 4c2flue4 interferon (ifn) lambda (ifn-λ or type iii ifn) gene polymorphisms were discovered in the year 2009 to have a strong association with spontaneous and treatment-induced clearance of hepatitis c virus (hcv) infection in human hosts. this landmark discovery also brought renewed interest in type iii ifn biology. after more than half a decade since this discovery, we now have reports that show that genetic association of ifnl gene polymorphisms in humans is not limited only to hcv infections but extends beyond, to include varied diseases such as non-alcoholic fatty liver disease, allergy and several other viral diseases including that caused by the human immunodeficiency virus. notably, all these conditions have strong involvement of host innate immune responses. after the discovery of a deletion polymorphism that leads to the expression of a functional ifn-λ4 as the prime ‘functional’ variant, the relevance of other polymorphisms regulating the expression of ifn-λ3 is in doubt. herein, i seek to critically address these issues and review the current literature to provide a framework to help further understanding of ifn-λ biology. supplementary information: the online version of this article (doi:10.1038/gene.2016.24) contains supplementary material, which is available to authorized users. in the year 2003, two different groups reported on the presence of three novel genes closely placed to each other, on human chromosome 19 that coded for interferons (ifns) with potent antiviral properties. 1, 2 these genes due to their relatedness to the interleukin 10 (il-10) family were initially christened il-28a, il-28b and il-29, and subsequently changed to ifnl2, ifnl3 and ifnl1. 3 the genes encode ifnλ-2, ifnl-λ3 and ifnl-λ1, respectively; together with the newly discovered ifnl-λ4 (or ifnl4) they constitute the type iii ifns or the lambda ifns (ifnl-λs). ifnl-λ1, 2 and 3 activate antiviral responses through the jak-stat (janus kinase-signal transducer and activator of transcription) pathway by utilizing a distinct receptor complex made of a heterodimer formed between ifn-λr1 and il-10r2. 2, 3 subsequent studies showed that unlike the receptors that bind to type i ifns, the ifn-λ receptors were expressed on selective cell types mainly of epithelial origin, hepatocytes and some immune cells. [3] [4] [5] [6] the discovery of ifn-λs was seminal in the sense that it showed the presence of an alternate system to the well-known type i ifns (ifn-α and ifn-β) that the different nucleated cells in the body, especially the ones on epithelial surfaces, can utilize to combat viral infections. later studies in mice have shown that type iii ifns form a strong barrier at the host-environment interface, which encompasses large regions of epithelial lining to the respiratory, gastrointestinal and urogenital tracts of mammals. [7] [8] [9] [10] a major boost in the area of ifn-λ research came after another discovery in the year 2009. three independent groups conducted genome-wide association studies (gwass) involving treatment response to chronic hepatitis c virus (hcv) infections, in three different geographical regions of the world, and reported that single-nucleotide polymorphisms (snps) in the ifnl locus (figure 1 ), had strong association with treatment-induced hcv clearance irrespective of ethnicity and geographical location of the hosts. [11] [12] [13] the search for a 'causative' or a 'functional' snp at the ifnl locus that could give a biological explanation for the hcv-gwas results was taken up rigorously by several groups, but none seem to have given a better explanation to the hcv-ifn-λ 'puzzle' than the group from the national institutes of health, usa, that discovered the presence of another ifnl upstream to ifnl3, named as ifnl4 (refs 14,15; figure 1 ; in figure 1b , the alleles for the respective snps are shown as beneficial (b) or non-beneficial (nb) with respect to the studies on hcv (reviewed in ref. 15 ; the major allele for each of the snps depicted in figure 1b has been shown to be the beneficial one in hcv infections). functional ifn-λ4 is expressed only in a subset of individuals, due to a frameshiftcausing deletion polymorphism (ifnl4-δg; rs368234815) in the first exon of ifnl4 (figure 1 ). 14 the presence of the alternate allele (ifnl4-tt) renders ifnl4 a pseudogene and this allele is seen iñ 50% of the european population and in most of the east asian population, but ifnl4 is a functional gene (ifnl4-δg allele) in majority (~95%) of the african population, 16 suggesting that human evolution has played an active role in elimination of a functional ifn-λ4 in the human species. 17 in fact, the pseudogene shows strong positive selection in human evolution, whereas the functional gene is conserved in other mammalian species except in mice and rats where the gene is completely absent. 17 in this article, i review the literature on genetic association studies that have shown the involvement of the hcv-gwas snps in non-hcv disorders that involve both viral diseases and some non-infectious conditions. i also update the progress on transcriptional studies of the ifnl4 gene and examine whether the functional ifn-λ4-generating snp is sufficient to explain the molecular mechanism of causality in the diseases it is associated with, and whether the other ifnl locus snps (mainly the ones regulating ifn-λ3 expression) may have any functional roles to play in the observed phenotypes. ifnl-λs and innate immunity against viruses innate and adaptive immunity are the two indispensable arms of the mammalian immune system. although we had a clearer understanding of the principles of functioning of the adaptive immunity arm, a lack of advanced molecular techniques and incomplete understanding of molecular mechanisms made us remain unaware of the intricacies of functioning of the innate immunity arm, for a long time. 18 with the advent of superior molecular biology techniques and the discovery of the pathogenassociated molecular pattern (pamp) or pattern recognition receptors, 19 we now have better understanding of how nucleated cells can differentially recognize different classes of pathogens and propagate signals to their surroundings, in the process raising the immediate alarm in the host. 19 large strides were made in the area of molecular recognition of viral pamps and signal transduction that leads to raising of antiviral states within virusinfected cells. 10, 20 the epithelial cells, being at the interface between the host and the environment in the respiratory, gastrointestinal and the urogenital tract, are not only prone to a variety of viral infections but are strategically located to respond and propagate alarm signals to the underlying immune cells ( figure 2) . however, the primary function of the epithelium is to provide a physical barrier between the underlying lamina propria and the lumen of the cavity or the exterior. even though they can sense and respond to pamps and damage-associated molecular patterns, 21 the epithelial cells are not professional immune cells and due to their high level of differentiation, may lack the plasticity required to send out amplified and prolonged signals to the lamina propria. therefore, a crucial link still remained missing about how an adaptive immune response is shaped within distantly located lymph nodes that have obligatory 'immune-rich environments', by taking cues from signals generated by viral infections at the epithelium ( figure 2 ). the discovery of the new class of effector immune cells called the innate lymphoid cells (ilcs) may seem to have provided the answer to this puzzle ( figure 2 ). ilcs are derivatives of common lymphoid precursors along with t and b cells. 22 these cells are stationed near the epithelial surfaces in larger numbers and respond to signals from the surrounding cells by secreting cytokines and chemokines in large quantities, thereby acting as signal amplifiers in both health and disease. 23 the ilcs are considered the innate immunity counterparts to the various th (t helper) cell (cd4+ (cluster of differentiation)) subsets of the adaptive immune system (figure 2 ; ref. 24) . for example, ilc2s respond to il-33 generated from influenza virus-infected epithelial cells by secreting il-5 and il-13, both known inducers of th2 immunity. 25 the natural killer cells, now classified as ilc1 cells, are considered the tc (t cytotoxic) cell (cd8+) counterparts. 24 even though most studies so far on ilcs have been on mice, ilcs have also been characterized in a variety of human tissues 26 and are deregulated in many human diseases. 27 with emerging roles of type iii ifns at the epithelium, 8, 10, 28 ilcs and ifn-λs now seem to be the major players in innate immunity at barrier surfaces. 10 15 ). the minor allele (mi) is the non-beneficial allele (that leads to expression of a functional ifn-λ4 and also lowers expression of ifn-λ3) and the major allele (ma) is the beneficial allele (that does not produce a functional ifn-λ4 and is associated with higher levels of expression of ifn-λ3) for all snps, again with respect to hcv studies. 15 the information on ld values has been obtained from 1000 genomes project reference panel (http://www.1000genomes.org), october 2014 release; some ld (r 2 ) values for yri (marked with *) were obtained from ref. 14. the maf values were obtained from dbsnp (national center for biotechnology information). in asian population, mafs of rs12979860, rs8099917 and rs4803217 are from jpt population; and mafs of rs368234815, rs8103142 and rs28416813 are from chb-jpt populations. yri, yoruba in ibadan, nigeria; ceu, residents with ancestry from northern and western europe; chb/jpt, han chinese in beijing, china/japanese in tokyo, japan; -, maf information not available for the ta repeat polymorphism rs59702201. how ilcs respond and interact with the epithelial cells during viral infections and how they cross-talk with other immune cells at the barrier surfaces in shaping and maintaining the optimal th responses, will form the next exciting wave in innate immunity research. similarly, research on the production, regulation and functions of ifn-λs in viral infections has also been exciting in the last decade. human ifn-λs are secreted (except ifn-λ4 that is poorly secreted 30 ) in response to the detection of viral rna intermediates from the cytoplasm of epithelial cells via the toll-like receptor and retinoic acid inducible gene i-like receptor) pathways. 4, 7, 31 ifn-λs are also known to be secreted by macrophages, plasmacytoid dendritic cells, monocyte-derived dendritic cells and hepatocytes. 4, 6, [32] [33] [34] the ifn-λr1 receptor is expressed on limited cell types including epithelial cells, hepatocytes, b cells and monocytes. 5, 35 there is currently no information on whether the newly discovered ilcs secrete any of the ifn-λs and whether they express ifn-λr1. ifn-λs act in a paracrine and/or autocrine manner to raise an antiviral state in the infected and to-be-infected cells by reprogramming the target cell gene expression patterns. 28, 36 the ifnl snps would have functional roles if they can affect the diversification of innate and adaptive immune cell subsets. diversification of ilc subsets will lead to the polarization of dendritic cells and macrophages and will eventually influence the th1/th2 balance by favoring either a th1 or a th2 response ( figure 3 ). 37 although there is no evidence for this belief, it is known that the ifnl snps do affect the expression of ifn-λ3 (ref. 15 ) and that they give rise to a new ifn (ifn-λ4). 14 ifn-λ1, 2 and 3 are known to deflect the th1/th2 balance to a th1 predominant mode in vitro and also in vivo in humans and mice 38, 39 (reviewed in ref. 37) . no such studies are reported for ifn-λ4. one hypothesis is that different levels of ifn-λ3 expression dictated by the underlying genetic polymorphisms are responsible for eliciting a th1 or a th2 response. 37 the role played by ifn-λ4 in this process is only speculative at this stage ( figure 3 ). how ifn-λs interact with the newly discovered ilcs is also unknown. a recent report showed that rotavirus infection in mice is controlled by il-22 produced by ilc3s for which the presence of an intact ifn-λ signaling pathway is required. 40 an even more important question in humans will be on what role does the ifnl snps, and therefore ifn-λ4, play in the diversification and functioning of ilcs. ifn-λ4 apart from being antiviral to hcv 14 is also known to inhibit other flaviviruses such as dengue virus, yellow fever virus 41 and human corona viruses. 30 some of the other ifn-λs are known to be induced by several human pathogens including m. tuberculosis, 42 human papilloma virus, 43 influenza virus 44 and human metapneumovirus (also induces ifn-λ4), 45 and have clear antiviral activities against some but not other viruses in mice (reviewed in ref. 46) . in a recent finding, murine ifn-λ3 but not ifn-α/ifn-β was responsible for protecting mice against norovirus persistence in mice colon, strikingly, even in the absence of t and b lymphocytes. 9 similar results were seen in reovirus infections of mice colon. 8 in conclusion, ifn-λs are potent antiviral molecules and their cross-talk with innate immune cells can potentially figure 2 . immune responses to viral infections of the epithelia. resident macrophages and dendritic cells (dcs) form the first line of cellular resistance to invading viruses. the newly discovered innate lymphoid cells (ilcs) 22 may have important roles due to their ability to respond to signaling molecules/alarmins secreted by the epithelial cells. ilcs diversify and respond by secreting large amounts of effector cytokines and chemokines locally. these effector molecules can potentially lead to the polarization of dcs and macrophages, and therefore may be critical in shaping the adaptive immunity mediated by t-helper (th) and t-cytotoxic (tc) cells that develop in the nearby lymphatic tissue (green lobed structure). mhc, major histocompatability complex. orchestrate innate immunity against several viruses and they may be particularly important at the barrier surfaces. ifn-λs also modulate adaptive immunity by affecting th1/th2 balance, tilting it to a th1-favoring response that is required for clearing viral infections. the function of the newly discovered ifn-λ4 in these immune processes remains to be determined. in cognizance of the potential that ifn-λs may hold in innate immunity, researchers across the world have got intrigued with the ifnl snps and have started to test them in candidate gene case-control studies in both infectious and non-infectious diseases. so far, association has been reported with nonalcoholic fatty liver disease (nafld), allergy and infections with several viruses. causing chronic infections and is especially a problem in immunosuppresed individuals. egli et al. 47 tested the association of the ifnl snp rs8099917 with cmv replication in a small number of solid organ transplant patients (n = 38) who were seronegative for cmv (but received an organ transplant from a cmvseropositive donor) and who had stopped receiving antiviral prophylaxis. they found that the minor allele at rs8099917 (g) was associated with decreased cmv replication (p = 0.036) in a dominant model of inheritance. further, their in vitro studies provided evidence for a beneficial effect of the minor allele against cmv replication. 47 a study in cmv-seropositive kidney transplant patients 48 also found that the minor allele (t) at rs12979860 had a dominant beneficial effect against cmv replication. although in another study in allogenic stem cell transplant recipients, 49 the minor allele t at rs12979860 protected recipients against cmv infection in a recessive model of inheritance. two more studies have also reported on the association of ifnl snps with cmv. 50, 51 the first report by bibert et al. 50 tested for the occurrence of cmv retinitis among those human immunodeficiency virus (hiv)-infected patients who were at risk of developing cmv retinitis due to low cd4 counts and cmv seropositivity. they found that carriers of two copies of the minor allele (δg/δg), increased the risk of getting cmv retinitis (p = 0.007) in multivariate regression models. in the second report by manuel et al., 51 solid organ transplant patients were tested for the association of rs368234815 with cumulative incidence of cmv replication. the results showed that the minor allele homozygotes (δg/δg), only in the pre-emptive antiviral therapy group but not among those receiving antiviral prophylaxis, had higher incidence of cmv replication. these results are indeed very interesting and sound, but the paradoxical findings of the five groups in terms of the model of inheritance of ifnl snps raise some questions. although two groups showed that their results fit best with a recessive model of inheritance of ifnl snps 50,51 in affecting cmv replication/retinitis wherein minor homozygosity was non-beneficial, the other three studies show an opposite trend where the minor allele had a beneficial effect against cmv replication in both patients and cell culture experiments, involving either dominant 47, 48 or recessive models of inheritance. 49 in stark contrast, a dominant model of inheritance (of the non-beneficial ifnl snp minor allele) has consistently given the best explanation on the observed phenotypes in association studies with both spontaneous clearance and ifn-based treatment response in chronic hcv infections. [11] [12] [13] 15 this discrepancy within the cmv studies may be partly due to statistical fluctuations owing to low sample sizes, 47, 50 and high heterogeneity among patient groups, end points and antiviral regimens that are followed in the different studies. the discrepancy needs be resolved by well-designed replication studies to gain a proper understanding of the role of ifnl snps in cmv replication and disease. human t-lymphotrophic virus. human t-lymphotrophic virus (htlv)-1 is an ancient retrovirus causing chronic infections in humans and is associated with adult t-cell leukemia/lymphoma. it is also associated with inflammatory disorders such as htlv-1associated myelopathy/tropical spastic papaparesis (ham/tsp), htlv-associated arthropathy and several other related disorders including rheumatoid arthritis. a spanish study reported for the first time that an ifnl snp (rs12979860) was associated with ham/ tsp in a small number of patients (n = 41) who had htlv-1 proviral dna in their blood cells. 52 they showed that the presence of the minor allele made 12/41 patients to have a sixfold higher risk (p = 0.03) of having symptoms of ham/tsp compared with asymptomatic carriers (n = 29) in a dominant model of inheritance. however, proviral dna load was a confounder in this association; covariate analysis suggested that both the snp and proviral dna load were linked in their association with ham/tsp. the minor allele-carrying genotypes (ct and tt) were indeed having higher proviral dna in their blood cells compared with the major homozygotes (p = 0.01). the major drawback of this study seems to be the low sample number; although the strength of the study was that there was significant effect of the minor allele on proviral dna copy number, a functional association that could be directly linked with the antiviral role of ifn-λ3. it is known that the ifnl snp minor alleles are associated with decreased expression of ifn-λ3 (reviewed in ref. 15 , although an opposite effect is evident in chronic hcv infections, where the minor allele carriers have low baseline virus levels 11 ). in a later study from brazil, 53 both rs8099917 and rs12979860 were tested in a cohort consisting of 229 htlv-positive subjects (93 ham/tsp patient and 136 asymptomatic carriers). the minor allele g of rs8099917 was significantly associated with ham/tsp in both univariate and multivariate analysis with a recessive model of inheritance (p o 0.001). in this study, the proviral dna load as a covariate did not seem to interfere with the association of rs8099917 with ham/tsp unlike the previous spanish study. 52 with respect to rs12979860, the minor allele t was associated with ham/tsp only as a heterozygote in univariate analysis (p = 0.01) and weakly in multivariate analysis (p = 0.06). however, a series of studies have also reported conflicting results to the above two reports on the association of ifnl snps with htlv-1-associated diseases. first, sanabani et al. 54 show clearly a lack of association of ifnl snp rs12979860 with ham/tsp and/or adult t-cell leukemia/lymphoma. this brazilian study had more number of samples (n = 112) than the spanish study of trevino et al. (n = 41). 52 they also did not find any correlation of proviral dna load with rs12979860 genotypes. another report from brazil with a sample size of 79 also reflected similar findings, where they found no association of rs12979860 with ham/tsp and proviral dna load. 55 this study also compared 300 healthy controls with 79 htlv-1-positive subjects and found no association with rs12979860 and htlv-1 infection. yet another recent study from brazil analyzed the genotypes at rs8099917, rs12979860 and rs8103142 in 300 healthy controls and 96 htlv-1-infected individuals, and found no association with htlv-associated arthropathy and any of the three snps when tested individually. 56 when they carried out haplotype analysis, they found some association (p = 0.01) with htlv-1 infection and one of the seven haplotypes (cct) involving the three snps (rs8099917, rs12979860 and rs8103142); with htlv-associated arthropathy and another haplotype (ttg; p = 0.05). they also found an association of the three snps individually with levels of some cytokines (such as ifn-γ) and proviral dna load (p o 0.05). 56 however, no multiple testing corrections seem to have been carried out in their analysis, thus severely undermining the results. 56 further, a japanese study also failed to see any association with adult t-cell leukemia/lymphoma and rs8099917 and also with htlv-1 and hcv mono-or co-infections. 57 last, a study from france on 95 htlv-1-positive subjects of afro-carribean lineage compared the distribution of genotypes of rs12979860 and the ifn-λ4-generating snp rs368234815, and found no association with ham/tsp. 58 in summary, the results so far are not entirely convincing on a true association of the ifnl snps with htlv infection-related diseases. further, in the two reports 52,53 that did see an association, the models of inheritance used to fit the phenotype data do not agree with each other, raising doubts on the underlying functionality of the observed genetic associations. therefore, more functional characterization of the observed association may be needed to rule out false positivity. if at all there is another human disease where ifnl snps were expected to have associations as strong as that of hcv infections, it was the case of hepatitis b virus (hbv). this expectation is because both viruses are hepatotropic and cause chronic infections; both diseases can be effectively treated using ifn-α even though they differ in their pamp ligands recognized by innate immune receptors. 59, 60 mixed results have been obtained about the role of ifnl snps in both spontaneous clearance and ifn-α-induced clearance of hbv and the literature until the year 2013-2014 has been reviewed elsewhere. [61] [62] [63] more studies have also been conducted since then (results from 10 of them are summarized in supplementary table 1 ), but have largely failed to resolve the conflict. two studies have also reported on a lack of association of ifnl snps on spontaneous as well as ifn-induced clearance of hepatitis d virus, a co-infecting satellite virus that requires hbv for replication and assembly. 64, 65 although consistent results were obtained across numerous studies with hcv-ifnl snp association mainly because they had only virological end point phenotypes 15 that correlated well with serological and biochemical parameters of the infection, several drawbacks in case of hbv studies may be responsible for the inconsistent results. some of them are: presence of different hbv genotypes as mixed infections, with some genotypes (genotype d 62 ) showing better association than others; variation in treatment regimens (ifn alone or with nucleotide analogs); end point phenotypes varying from serology to viral load, to biochemical parameters without a common quantitative parameter to assess the phenotype; prolonged clinical course of the disease with fluctuating virological, serological and biochemical markers; and prevalence rates of disease differing in different ethnicities/populations, to name a few. the reports that have shown an association of ifnl snps with hbv spontaneous or treatment-induced clearance are largely in agreement with the results from hcv studies in terms of the model of inheritance (supplementary table 1 ). although the conflicting data so far on hbv-ifnl snp association do lead to doubts about its true positive nature, the data are also not fully supportive of the notion that the association may be false positive. in fact it has been argued that if properly assessed, the association between hbv disease progression and ifnl snps could have clinical value. 62 but it appears that the effect of the ifnl snps on hbv persistence and/or progression within the human host is highly variable; it may involve more complex interactions with other variables and genes than was observed in chronic hcv infections. human immunodeficiency virus. another important viral disease that has been tested for the influence of ifnl snps is acquired immunodeficiency syndrome (aids) caused by hiv. particularly, it was of interest to see how the ifnl snp beneficial alleles are distributed in a unique group of hiv-infected individuals that can suppress the virus from replicating to high levels (defined as elite controllers/suppressors or natural viral suppressors) and defer the progression to aids (called as long-term non-progressors) without any antiretroviral therapy; and another unique group that remains hiv-seronegative despite being at high risk for infection due to intravenous drug use (highly exposed seronegative or exposed seronegative)). more interestingly, the natural viral suppressor patients have also been known to efficiently clear hcv in hcv-hiv co-infections, compared with controls in both african americans 66 and caucasians. 67 a few reports have come up in this area, some showing no correlation of hiv infection/disease progression to the ifnl snps, whereas others see clear association. [68] [69] [70] [71] [72] [73] [74] one of the earlier reports tested the association of rs12979860 in 291 high-risk seronegative and 1221 hiv-positive subjects comprising both white and black subjects in the usa. 68 no association was evident for either hiv positivity or for disease progression within the infected subjects. in another study reported by rallon et al., 69 the association of rs12979860 was tested in two different cohorts. the first comprised of 30 long-term non-progressors and 38 typical progressors to aids; the second included 29 exposed seronegative and 29 hiv-positive partners. thus, the study tested both hiv progression and protection, although in a small number of patients. no significant difference in distribution of genotypes between cases and controls was evident in both cohorts, although the beneficial cc genotype was more frequent in the exposed seronegative patients compared with hiv-positive patients (62% vs 45%), suggesting that there may be a protective role for the cc genotype against hiv that was not detected in the study, likely due to inadequate power. another study from the usa tested whether the beneficial cc genotype at rs12979860 was overrepresented in 25 african-american elite controllers/suppressors compared with hiv-infected patients with high viral loads, and found no statistically significant difference. 70 confirming this report, sajadi et al. 71 also found that cc genotype (for rs12979860) was not significantly over-represented in 48 natural viral suppressors of african-american origin compared with hivpositive (n = 124) and -negative controls (n = 173). three reports published subsequently have seen association with rs12979860 and rs368234815, and spontaneous control of hiv and/or aids progression. [72] [73] [74] interestingly, all three studies were carried out on whites/caucasians, whereas the reports that failed to see an association described above were mostly carried out on african americans (except that by rallon et al 69 that used white subjects) or mixed group of blacks and whites. 68 first, machmach et al. 72 found an association of rs12979860 with spontaneous hiv control when tested on 53 white natural viral suppressors and 389 matched non-controllers at p = 0.02 after correcting for occurrence of hla-b57 (human leukocyte antigen) protective alleles (which also have independent association with hiv and hcv spontaneous clearance) and gender, in multivariate analysis. second, machmach et al. 73 confirmed and extended their results in another report, where they found the association of rs368234815 with aids progression. last, a recent study carried out on a well-characterized spanish white cohort of hcvseropositive men exposed to hiv infection through shared needles shows a clear association of ifn-λ4-generating rs368234815 with hiv positivity. 74 the study had 213 men who were hiv seropositive and 188 were highly exposed seronegative. they found that the protective tt/tt genotype was overrepresented in the highly exposed seronegative group (0.49 vs 0.41; p = 0.006). further, this association had no interaction with the hiv-protective ccr5 (c-c chemokine receptor type 5) deletion (p = 0.7). interestingly, all three studies that found an association show data that the non-beneficial minor alleles are following a recessive model of inheritance, 72-74 suggesting a common underlying functionality in the observed genetic association between the three studies. this is however different from the dominant model of inheritance seen in hcv studies, [11] [12] [13] 15 suggesting that the two viruses may have different interactions with ifn-λ-driven immune responses. however, unlike in the case of cmv studies discussed above, [47] [48] [49] [50] [51] all three hiv studies [72] [73] [74] show that the minor alleles are non-beneficial, similar to the observations in chronic hcv infections. 15 in summary, it is evident that ifnl snps do associate with hiv replication and disease progression, even though in an ethnicity-specific manner. it appears that the beneficial alleles of ifnl snps have protective roles in whites/caucasians. in african americans, more studies with the functional ifn-λ4-generating snp rs368234815 rather than rs12979860 will reveal any true association. this is especially true as the two snps are not in strong linkage disequilibrium (ld) in this population (figure 1b) . herpes simplex virus. herpes simplex virus resides inside the human host latently in sensory neurons, but gets reactivated due to the altered host immunity and replicates efficiently in epithelial cells causing genital or oral lesions (the latter is referred to as 'cold sores'). an association of rs12979860 with the recurrence and severity of herpes simplex virus-1-induced 'cold sores' was reported from a study on a small number of individuals (n = 57). 75 a recent report carried out on a large number of individuals (n = 2192 for genital herpes; n = 1511 for oral herpes) clearly found no association of rs368234815 with recurrence of genital or oral herpes episodes. 76 among other phenotypes, the latter report ruled out an effect of the snp on 'frequency of recurrence' of oral herpes, it however did not rule out an effect of the snp on severity of infection. non-infectious diseases and miscellaneous conditions. ifnl snps have also been tried as candidate gene snps in some noninfectious inflammatory diseases. the association of ifnl snps with nafld was earlier reported by petta et al. 77 this study with 160 subjects identified an independent association with the cc genotype at rs12979860 and the severity of lobular inflammation (cc genotype positively correlated with severity of inflammation) in a cohort of patients with nafld (p = o 0.001). the study was conducted taking lead from previous reports of increased hepatic necroinflammation in hcv patients with the cc genotype at rs12979860. 78 another report showed no such association in 195 caucasian biopsy-confirmed nafld patients. 79 however, all the patients in this cohort were obese (with body mass index 430), whereas in the previous study only 40% were obese. after the latter report, petta et al. 80 revisited their data and indeed found an association with the ifnl snp only with their non-obese patients (n = 94; p = 0o0.001) but not with the obese patients (n = 66; p = 0.13). these initial doubts seem to have been resolved with a recent report by eslam et al. 81 who worked on a relatively large number of nafld patients (n = 488) and confirmed the association of the cc genotype at rs12979860 with increased severity of liver inflammation and fibrosis (p = o 0.0001). they also found a similar strong association with hepatic inflammation and fibrosis in separate cohorts of viral hepatitis c (n = 3129) and b (n = 555), strongly suggesting that even though the major allele genotype cc is beneficial against hcv and hbv, it is also responsible for excess inflammation of the liver in viral and non-viral hepatitis. all four studies 77-81 report on dominant models of inheritance for the minor alleles; but unlike in the context of chronic hcv infections, [11] [12] [13] the minor alleles in nafld are beneficial rather than being non-beneficial, as they are responsible for less severe hepatic inflammation. extending the potential role for ifnl snps in inflammatory diseases, there is also a recent report that showed association of rs12979860 and allergy in children aged o5 years. 82 although the study involved a small sample size (non-allergic, n = 35; allergic cohort 1, n = 35; food allergy cohort 2, n = 30), a large effect size (odds ratio = 4.56, p = 0.004, for cohort 1) was seen wherein the non-beneficial t allele at rs12979860 was over-represented in the allergy group in a dominant model of inheritance. another interesting finding in this study was that the effect of the ifnl snp was more pronounced in females than males. this report suggests that the non-beneficial ifnl snp minor allele-carrying genotypes may predispose children to an allergic phenotype by skewing the th1/th2 balance to a th2 predominant one. this extrapolation is also based on the findings from a recent study on mice model of allergic asthma, which showed that ifn-λ2 was able to rescue mice from allergy by suppressing th2 cytokines. 39 these conclusions would also suggest a role for ifnl snps in immune response to respiratory viral infections, which account for disease exacerbations in conditions such as asthma. 83 a study carried out on infants with respiratory syncytial virus-induced bronchiolitis shows no association of r12979860 and rs8099917 with viral load or other clinical features. 84 interestingly, the non-beneficial tt genotype of rs12979860 was associated with early age of hospitalization in the infants (p = 0.005). more studies are awaited on the association of ifnl snps with allergy and allergy-related diseases. in the autoimmune disorder multiple sclerosis, rs8099917 and rs12979860 did not show any association with ifn-β treatment. 85 as evidence for a potential role of ifn-λs and ifnl snps in adaptive immune responses, a study has linked ifnl snps with development of effective vaccine response against human influenza virus in immunosuppressed individuals. 86 the study shows that minor allele (g)-carrying individuals at rs8099917 show better vaccine responses (odds ratio = 1.99; p = 0.038) in a dominant model of inheritance, and that t cells from minor allele-carrying individuals produce more il-4 (a th2 cytokine). this study also showed that recombinant ifn-λ3 increased th1 responses from human peripheral blood mononuclear cells while inhibiting th2 responses (th2 cytokines are needed for effective seroconversion). 86 studies have accumulated evidence on the different transcription factors (tfs) that bind and drive transcription from all four ifnl genes. roles for several virus-inducible tfs such as nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells), ifn regulatory factor-3 and ifn regulatory factor-7 have been defined for ifnl1, 2 and 3 genes, and have been reviewed elsewhere. 28, 87, 88 we recently showed that apart from these three tfs, specificity protein 1 ( a gc-rich dna-binding tf) also has a role in driving transcription from the ifnl4 promoter in a549 cells 89 (figure 4a) . a cpg island of~1.5 kb is located upstream of the ifnl3 gene and overlapping the ifnl4 gene, and also includes two of the most important ifnl snps rs12979860 and rs368234815. epigenetic regulation of the transcriptional activity at the ifnl locus is likely to involve this cpg island and needs further exploration. 90, 91 even though ifnl4 messenger rna (mrna) was shown to be highly expressed from stimulated primary hepatocytes in the original report that described the discovery of ifnl4, 14 subsequent studies have not seen robust expression levels of ifnl4 gene in human samples. for example, amanzada and others found ifnl4 transcripts expressed at 4.3-5.6-fold lower levels than ifnl2/3 mrna in liver biopsies of hcv-infected patients. 92 in another report, ifnl4 mrna was detectable in only in 7/23 and 8/23 patient-derived peripheral blood mononuclear cells that were stimulated or not with ifn-poly(i:c), respectively. 93 all 23 patients carried at least one copy of the functional ifn-λ4-generating allele, δg. in the report by liang and colleagues also, ifnl4 transcripts were seen in only 33/70 liver biopsies from hcv-infected patients. 94 furthermore, lu et al. 41 found that only 2% of the transcripts generated from poly(i:c)-stimulated primary hepatocytes derived from two heterozygous (tt/δg at rs368234815) individuals, represented functional ifn-λ4 transcripts that originated from the δg allele. similarly, they also found no expression of the full-length functional ifn-λ4 mrna transcripts from poly(i: c)-stimulated a549 cells. it is possible that the qpcr assays utilized in these studies may be suffering from technical difficulties in dealing with several different mrna isoforms of ifnl4 gene. 14 all the four studies described above have used primer sets where at least one of the primers binds to the exon/intron/exon-intron junctions (figure 4b) . using a primer set that uniquely binds to the 3′-untranslated region of ifnl4 mrna, ifn-λ4 induction was seen at levels similar to that of ifn-λ2/3, upon human metapneumovirus infection of a549 cells by banos-lara et al. 45 we used the same primer set and found similar high expression levels upon poly(i:c)/hcv rna transfection in a549 cells. 89 two other studies have reported no such problems in amplifying ifnl4 transcripts from liver biopsies. 95, 96 although honda et al. 96 used the allelespecific taqman assay described earlier, 14 konishi et al. do not provide the primer sequence information that they used in their taqman assays. 95 the inconsistency in the above reports in detection of ifnl4 transcripts (either the full-length functional ifn-λ4 isoform or the other isoforms) suggests that rna-sequencing may be the optimal approach to measure ifnl4 transcripts. furthermore, the pre-mrna splicing mechanism that leads to the expression of different ifn-λ4 isoforms 14 under different stimulation and cell-type conditions needs to be examined. the relevance of other 'ifn-λ3 functional snps' after the discovery of functional ifn-λ4-generating snp till date, three functional snps have been identified that regulate ifnl3 gene transcription/translation (referred to as 'ifn-λ3 functional snps' in this section). although rs28416813 is a snp present at~37 nucleotides upstream of the start codon of ifnl3 and is known to affect downstream gene expression by differentially binding to nf-κb, 97 rs4803217 was identified in the 3′-untranslated region region of ifnl3 (figure 1a ) that affects stability of the mrna by interfering with au-rich element decay (amd). 98 both these snps are in high ld with each other and with rs12979860 and were predicted to be two of the four potential causal snps from among the hcv-gwas hits. 15, 99 a recent study defines another mechanism by which rs4803217 affects the stability of rna by remodeling its secondary structure. 100 furthermore, a third functional snp is the ta repeat polymorphism rs59702201, originally reported by the mizokami group. 101 this snp located within the proximal promoter affects transcription of ifnl3, depending on the number of repeats present. 101 recent reports have confirmed the significance of this snp in association studies involving chronic hcv infections. [102] [103] [104] apart from several reports that showed genotype-dependent differences in expression levels of ifn-λ3 (reviewed in ref. 15 ) recent reports also have confirmed this finding in ex vivo and in vivo conditions. 94, 105 these results suggest that ifn-λ3 expression levels dictated by the alleles present at the three functional snps may have a role in the observed phenotype in health and disease. however, the discovery of the ifn-λ4-generating snp (rs368234815, referred to as 'ifn-λ4 functional snp' in this section) has overshadowed the importance of the 'ifn-λ3 functional snps', as most of the new reports have chosen to test for the 'ifn-λ4 functional snp' rather than rs12979860, which is the tag-snp for the 'ifn-λ3 functional snps'. 15 another snp within the coding region of ifnl4 that leads to a non-synonymous mutation in the functional ifn-λ4 protein is thought to be a better marker of association along with rs368234815, in chronic hcv infections. 106 so the question arises: is the ifn-λ4-generating snp the sole functional snp or the other snps regulating the expression of ifn-λ3 also have independent roles? the answer to this question will be difficult to obtain under in vivo conditions due to high ld between these snps in most populations that precludes any attempts to assess their independent effects (figure 1b) . a recent report compared the strength of association of rs4803217 and rs368234815 in ifn treatment response in hcv-infected african americans, who have the lowest reported ld values among all ethnicities between rs368234815 and rs12979860 snps (figure 1b) . 107 they found that rs368234815 was more strongly associated with the outcome than rs4803217. 107 in another independent report, lu et al. applied multivariate regression to test the independence of rs368234815 from rs4803217 in predicting response to ifn treatment in hcv patients of african-american decent. 100 they found that correcting for the effect of s368234815 on rs4803217 abolished the latter snp's association with treatment response, whereas correcting for the effect of the latter snp reduced the strength of association of the former snp (from p = 0.004 to 0.065). 100 this could have resulted due to the low sample size (n = 169); nevertheless, more results are awaited to conclude that ifn-λ4-generating snp is the sole functional snp and that the observations giving credence to the functionality of the other 'ifn-λ3 functional snps may only be artifacts. studies so far point to the functional ifn-λ4-generating snp rs368234815 as the prime causal variant at the human ifnl locus. therefore, a detailed investigation is needed on the function of ifn-λ4 as both an antiviral cytokine in different viral diseases and as a potential player in diversification and maintenance of innate and adaptive immune cell subsets. in case of ifn treatment response in chronic hcv infections, even though the ifn-λ4 -generating snp can explain a large amount of variance in the observed phenotypes, 107 questions still remain on its role in spontaneous clearance of hcv. it is known that expression of functional ifn-λ4 is associated with high levels of ifn-stimulated gene (isg) expression in non-responders who further fail to upregulate their isg expression upon ifn-α treatment. 15 however, such an elegant explanation is lacking in the case of spontaneous clearance of hcv ( figure 5 ). although the majority of studies on spontaneous hcv clearance are with rs12979860 and not rs368234815, the fact that these two snps are in high ld in most populations 14 allows us to extrapolate the results. so, how does the expression of a functional ifn-λ4 in patients who get an acute hcv infection will lead them to become chronically infected? one explanation could be that similar to the ifn treatment nonresponders group, the subjects expressing functional ifn-λ4 also have higher baseline levels of isgs, which do not get further upregulated upon acute hcv infection. having a higher basal innate immune response may have helped those populations with higher frequency of the functional ifn-λ4-generating allele in dealing with prevalent viral infections. higher baseline levels of isgs in people who can express a functional ifn-λ4 may be offering protection against excess inflammation of the liver in hepatitis of non-viral origin such as nafld. [78] [79] [80] [81] however, as is evident from studies on spontaneous and treatment-induced clearance of hcv, such a pre-emptive state may become nonbeneficial in dealing with hepatitis of viral origin. further, there may be other conditions that we do not yet know in which expression of a functional ifn-λ4 may be beneficial. therefore, an epidemiological investigation to assess basal isg expression levels, in humans both in health and disease, and their correlation with expression of ifn-λ4, is needed. further, studies are also needed to examine why hepatic ifn-λ3 levels are lower in the beneficial allele/genotype-carrying individuals during chronic hcv infection and how this phenomenon is reversed once treatment is initiated (figure 5c ; ref. 94) . although ifn-λ1, 2 and 3 are known to associate with a th1 response, 38, 39, 86 no information is available in this regard for ifn-λ4. if the hypothesis that higher levels of ifn-λ3 expression (due to presence of major alleles) will lead to a th1 predominant response is to be believed, then by extrapolation, ifn-λ4 should promote th2 responses (figure 3 ; as 'ifn-λ3 functional snps' and 'ifn-λ4 functional snp' are in strong ld and the minor allele will give rise to ifn-λ4). this explanation is difficult to reconcile with the observations that ifn-λ4, except for being a poorly secreted ifn, requires the same dimeric receptor for signaling and can stimulate not only similar set of isgs but also at similar levels when compared with ifn-λ3. 14, 30, 36 besides, both ifn-λ4 and ifn-λ3 have similar antiviral potencies in vitro. 14,41 therefore, expression of ifn-λ4 and its associated isgs in a subset of individuals carrying minor alleles is less likely to favor a completely opposite phenotype to that seen in those carrying major alleles that induce higher levels of ifn-λ3 expression. human ifnl snps came to the limelight after genetic studies showed their relevance in chronic hcv infections in the year 2009. since then, case-control studies in humans have been reported involving ifnl snps and several other viral diseases, nafld, allergy and even in vaccine responses. although it appears that the associations are well replicated (such as in nafld and aids) and strong in some diseases (such as in pediatric allergy), conflicting reports weaken the association in some (such as those involving htlv and hbv), whereas further studies are needed in others (such as those involving cmv and herpes simplex virus) to clear discrepancies. the ifnl-λs may have critical roles in shaping innate and adaptive immunity in general and in viral infections in particular. however, unlike their effect in hcv infections, the ifnl snps may involve interactions with variables other than just standard end point phenotypes in many of the reported diseases/ conditions. therefore, future studies should aim at dissecting these interactions to arrive at meaningful results. well-designed replication studies and functional studies to strengthen the genetic association results are required to arrive at firm conclusions. importantly, ifn-λ4 has emerged as the key causal link to the genetic associations, but many questions remain on its functional role in the diversification and shaping of innate and adaptive immune responses in viral infections and other inflammatory conditions. 14) and therefore are expected to express ifn-λ4. 94 a recent study has documented that type iii ifns including ifn-λ2/3 and ifn-λ4 expression levels correlate with isg expression in the liver of patients undergoing anti-hcv therapy. 94 the ifn-λ2/3 and ifn-λ4 levels and their associated isg levels are higher in the liver of patients who will eventually not respond to the therapy 94 compared with those who will respond, suggesting that an unknown effect inhibits the expression of ifn-λ3 in the latter group of patients (shown as an ellipse in c). it seems that ifn-α-ribavirin treatment induces the expression of ifn-λ3 once treatment begins (depicted as a circle in c) preferably in those patients who will eventually respond to the treatment 94 (these patients have lower frequency of the functional ifn-λ4-generating alleles 14 and hence are shown without ifn-λ4 expression (c)). this induction is further dependent on whether the patients carry beneficial alleles at rs12979860. the patients who have the beneficial alleles show higher increase in their hepatic ifn-λ3 levels than those who do not, once treatment is initiated. 94 a previous report also had seen similar changes in serum ifn-λ3 levels. 109 the isg levels are shown in correlation with ifn-λ3 expression. ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il-28, il-29 and their class ii cytokine receptor il-28 r il-28a, il-28b, and il-29: promising cytokines with type i interferon-like properties viral infection and toll like receptor agonists induce a differential expression of type i andλ interferons in human plasmacytoid and monocyte-derived dendritic cells ifn-lambda (ifn-λ) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo interferon type i gene expression in chronic hepatitis c an important role for type iii interferon (ifn-lambda/il-28) in tlr-induced antiviral activity leukocytederived ifn-α/β and epithelial ifn-λ constitute a compartmentalized mucosal defense system that restricts enteric virus infections interferon-λ cures persistent murine norovirus infection in the absence of adaptive immunity guarding the frontiers: the biology of type iii interferons genetic variation in il28b predicts hepatitis c treatment-induced viral clearance il28b is associated with response to chronic hepatitis c interferon-alpha and ribavirin therapy genome-wide association of il28b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c avariant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus genetic variants at the ifnl3 locus and their association with hepatitis c virus infections reveal novel insights into host-virus interactions ifn-λ4: the paradoxical new member of the interferon lambda family selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda 4 (ifnl4) innate immunity: an overview pathogen recognition and inflammatory signaling in innate immune defenses innate immunity to virus infection intestinal epithelial cells: regulators of barrier function and immune homeostasis innate lymphoid cells in the initiation, regulation and resolution of inflammation the biology of innate lymphoid cells the 3 major types of innate and adaptive cell-mediated effector immunity il-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo human innate lymphoid cells regulation of the adaptive immune system by innate lymphoid cells interferon-λ: immune functions at barrier surfaces and beyond innate lymphoid cells: balancing immunity, inflammation and tissue repair in the intestine interferon lambda 4 signals via the ifnλ receptor to regulate antiviral activity against hcv and coronaviruses type i and type iii interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia ifn-alpha regulates tlr-dependent gene expression of ifn-alpha, ifn-beta, il-28 and il-29 type iii ifns are produced by and stimulate human plasmacytoid dendritic cells expression profiles of human interferon-alpha and interferon-lambda subtypes are ligandand cell-dependent despite ifn-lambda receptor expression, blood immune cells, but not keratinocytes or melanocytes, have an impaired response to type iii interferons: implications for therapeutic applications of these cytokines transcriptome analysis reveals a classical interferon signature induced by ifnλ4 in human primary cells the impact of the interferon-lambda family on the innate and adaptive immune response to viral infections human interferon lambda-1 (ifn-lambda1/il-29) modulates the th1/th2 response il-28 a (ifn-λ2) modulates lung dc function to promote th1 immune skewing and suppress allergic airway disease interferon-l and interleukin 22act synergistically for the induction of interferonstimulated genes and control of rotavirus infection interferon-λ4 is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden interferon lambda-2 levels in sputum of patients with pulmonary mycobacterium tuberculosis infection interferon lambda 1 expression in cervical cells differs between low-risk and high-risk human papillomavirus-positive women influenza virus activation of the interferon system impact and regulation of lambda interferon response in human metapneumovirus infection interferon-λ in the context of viral infections: production, response and therapeutic implications immunomodulatory function of interleukin 28b during primary infection with cytomegalovirus association between individual and combined snps in genes related to innate immunity and incidence of cmv infection in seropositive kidney transplant patients effect of the il28b rs12979860 c/t polymorphism on the incidence and features of active cytomegalovirus infection in allogeneic stem cell transplant patients the ifnl3/4 δg variant increases susceptibility to cytomegalovirus retinitis among hiv-infected patients influence of ifnl3/4 polymorphisms on the incidence of cytomegalovirus infection after solid-organ transplantation development of tropical spastic paraparesis in human t-lymphotropic virus type 1 carriers is influenced by interleukin 28b gene polymorphisms il28b gene polymorphism snp rs8099917 genotype gg is associated with htlv-1 carriers lack of evidence to support the association of a single il28b genotype snp rs12979860 with the htlv-1 clinical outcomes and proviral load htlv-1-associated myelopathy/ tropical spastic paraparesis is not associated with snp rs12979860 of the il-28b gene il28b gene polymorphisms and th1/th2 cytokine levels might be associated with htlv-associated arthropathy paradoxical expression of il-28b mrna in peripheral blood in human t-cell leukemia virus type-1 mono-infection and co-infection with hepatitis c virus absence of association of ifnl3/il28b rs 12979860 and ifnl4 ss 469415590 polymorphisms with the neurological status of htlv-1 afro-caribbean subjects in martinique nucleic acid sensors involved in the recognition of hbv in the liver-specific in vivo transfection mouse models-pattern recognition receptors and sensors for hbv regulation of hepatic innate immunity by hepatitis c virus systematic review with meta-analysis: do interferon lambda 3 polymorphisms predict the outcome of interferon-therapy in hepatitis b infection? one more piece in the interleukin 28b gene puzzle? the case of hepatitis b interleukin 28b genetic polymorphism and hepatitis b virus infection no impact of interleukin-28b polymorphisms on spontaneous or drug induced hepatitis delta virus clearance effects of polymorphisms in interferon λ3 (interleukin 28b) on sustained virologic response to therapy in patients with chronic hepatitis d virus infection hepatitis c infection in hiv-1 viral suppressors hepatitis c virus replication in caucasian hiv controllers il28b polymorphism does not determine outcomes of hepatitis b virus or hiv infection interleukin-28b gene polymorphisms do not influence the susceptibility to hiv-infection or cd4 cell decline protective interleukin-28b genotype affects hepatitis c virus clearance, but does not contribute to hiv-1 control in a cohort of african-american elite controllers/ suppressors il28b genotype does not correlate with hiv control in african americans il28b single-nucleotide polymorphism rs12979860 is associated with spontaneous hiv control in white subjects ifnl4 ss469415590 polymorphism is associated with unfavourable clinical and immunological status inhiv-infected individuals ifnl4 rs368234815 polymorphism is associated with innate resistance to hiv-1 infection a systematic analysis of host factors reveals a med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication interferon lambda 4 genotype is not associated with recurrence of oral or genital herpes il28b and pnpla3 polymorphisms affect histological liver damage in patients with nonalcoholic fatty liver disease genome wideassociation study identifies il28b polymorphism to be associated with baseline alt and hepatic necro-inflammatory activity in chronic hepatitis c patients enrolled in the ideal study il28b rs12979860 is not associated with histologic features of nafld in a cohort of caucasian north american patients reply to: "il28b rs12979860 is not associated with histologic features of nafld in a cohort of caucasian north american patients interferon-λ rs12979860 genotype and liver fibrosis in viral and non-viral chronic liver disease genetic variations in il28b and allergic disease in children anti-viral agents: potential utility in exacerbations of asthma evaluation of interleukin 28b single nucleotide polymorphisms in infants suffering from bronchiolitis il28b polymorphisms are not associated with the response to interferon-β in multiple sclerosis il-28b is a regulator of b-and t-cell vaccine responses against influenza mechanisms of type iii interferon expression interferon induction and function at the mucosal surface roles for transcription factors sp1, nf-ĸb, irf3, and irf7 in expression of the human ifnl4 gene il28b expression depends on a novel tt/-g polymorphism which improves hcv clearance prediction identification of improved il28b snps and haplotypes for prediction of drug response inn treatment of hepatitis c using massively parallel sequencing in a crosssectional european cohort interferon-λ4 (ifnl4) transcript expression in human liver tissue samples impaired induction of interleukin 28b and expression of interferon λ4 associated with nonresponse to interferon-based therapy in chronic hepatitis c hepatic expression levels of interferons and interferon-stimulated genes in patients with chronic hepatitis c: a phenotype-genotype correlation study interferon-lambda4 genetic polymorphism is associated with the therapy response for hepatitis c virus recurrence after a living donor liver transplant hepatic interferon-stimulated genes are differentially regulated in the liver of chronic hepatitis c patients with different interleukin-28b genotypes a single nucleotide polymorphism associated with hepatitis c virus infections located in the distal region of the il28b promoter influences nf-ĸb mediated gene transcription the favourable ifnl3 genotype escapes mrna decay mediated by au-rich elements and hepatitis c virus-induced micrornas estimating the net contribution of interleukin-28b variation to spontaneous hepatitis c virus clearance ifnl3 mrna structure is remodelled by a functional non-coding polymorphism associated with hepatitis c virus clearance genetic variation of the il-28b promoter affecting gene expression prevalence of thymine-adenine dinucleotide repeat, il28b and ifnl4 in thai population and correlation with spontaneous clearance and treatment outcome of hepatitis c infection pretreatment prediction of the outcome of response-guided peginterferon-α and ribavirin therapy for chronic hepatitis c a thymineadenine dinucleotide repeat polymorphism near il28b is associated with spontaneous clearance of hepatitis c virus ifnl3 genotype is associated with differential induction of ifnl3 in primary human hepatocytes reduced ifnλ4 activity is associated with improved hcv clearance and reduced expression of interferon-stimulated genes comparison of functional variants in ifnl4 and ifnl3 for association with hcv clearance il-28b genetic variation is associated with spontaneous clearance of hepatitis c virus, treatment response, serum il-28b levels in chinese population impact of il28b gene polymorphisms on interferon-λ3 plasma levels during pegylated interferon-α/ribavirin therapy for chronic hepatitis c in patients coinfected with hiv the author declare no conflict of interest. key: cord-339879-92esdjy9 authors: delhalle, sylvie; schmit, jean-claude; chevigné, andy title: phages and hiv-1: from display to interplay date: 2012-04-13 journal: int j mol sci doi: 10.3390/ijms13044727 sha: doc_id: 339879 cord_uid: 92esdjy9 the complex hide-and-seek game between hiv-1 and the host immune system has impaired the development of an efficient vaccine. in addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. for more than 20 years, phage display technology has been intensively used in the field of hiv-1 to explore the epitope landscape recognized by monoclonal and polyclonal hiv-1-specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. in parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify hiv-1 inhibitors. besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the hiv-1 protease. phage particles also represent valuable alternative carriers displaying various hiv-1 antigens to the immune system and eliciting antiviral responses. this review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures. in 1983, the human immunodeficiency virus (hiv-1) was identified as the causative agent of the acquired immunodeficiency syndrome (aids) [1, 2] . in 30 years of pandemic, hiv-1 has infected more than 60 million individuals and killed 25 million. thirty-three million individuals are currently living with hiv-1 making this disease a major worldwide public health problem (unaids 2010). natural sterilizing immune response against hiv-1 has never been described and despite decades of intensive research, a vaccine against hiv-1 is still lacking, mainly due to the high ability of the virus to escape from the immune response. in the absence of a vaccine, combinations of small antiviral molecules are intensively used to control hiv-1 infection. the majority of these drugs are reverse transcriptase and protease inhibitors [3] . more recently, new molecules targeting the fusion step, ccr5 or integrase were licensed for clinical use [4] [5] [6] . despite the increased life expectancy observed with the advent of these therapies, severe side effects, lack of adherence and emergence of drug-resistant virus strains still limit the long-term control of the infection [7] . hiv-1 is an enveloped virus whose genetic material consists of two identical rna strands coding for the structural genes gag, pol and env as well as the accessory genes tat, rev, nef, vif, vpr and vpu. the gag gene codes for structural proteins p17 and p24, while pol codes for viral enzymes (reverse transcriptase, integrase and protease) and env for the gp160 envelope protein precursor that is subsequently cleaved into gp120 and gp41. gp120 and gp41 proteins assemble at the surface of hiv-1 into trimeric spikes composed of three monomers of membrane-embedded gp41 complexed to free gp120. these two proteins are involved in virus entry and represent the principal targets for the humoral response. upon cd4 receptor binding, glycoprotein gp120 undergoes conformational changes exposing the v3 loop, a region that further interacts with the chemokine receptors ccr5 or cxcr4 thereby promoting viral entry [8] (figure 1 ). coreceptor binding leads to the insertion of the gp41 fusion peptide into the cell membrane, the creation of a hairpin loop intermediate and finally the fusion of both viral and cell membranes. the viral capsid then enters the cell and the genetic material is released in the cytoplasm. most viral strains use only one coreceptor to enter host cells and are classified accordingly as ccr5-(r5 strains) or cxcr4-tropic (x4 strains), although viruses with broadened coreceptor usage (dual-tropic) have also been described. r5 viruses infect macrophages and ccr5-expressing t lymphocytes, and are mainly associated with transmission. in contrast, x4 viruses infect cxcr4-expressing t-cells and t-cell lines, and often appear at the later stages of infection. the envelope glycoprotein gp120 is composed of variable and more constant regions. several studies demonstrated that the elicitation or binding of effective neutralizing antibodies are impaired by the gp120 glycan shield or steric hindrance of its constant regions [9] . moreover, variable immunodominant domains were shown to be recognized by non-neutralizing antibodies. nonetheless, it is estimated that 10% to 30% of hiv-1-positive subjects develop neutralizing antibodies (ntabs) appearing at least 1 year after infection. only 1% of infected patients develop a broad neutralizing response against heterologous virus strains [10] . among hiv-1-infected patients, such antibodies arise only rarely and tardily, thus inefficiently controlling viral replication. however, the recent identification of broadly neutralizing antibodies (bntabs) and mapping of their epitopes fueled interest in the humoral immune response against hiv-1 (reviewed by overbaugh [11] ). to better understand the reasons underlying the persistance of viral infection despite the strong and sustained immune response on the one hand, and to identify new protective immunogens, numerous studies were conducted to map the epitope landscape of both hiv-1-neutralizing and non-neutralizing antibodies isolated from infected patients. in parallel, the development of new molecules or antibody fragments capable of blocking either viral proteins or host receptors has been widely investigated. to serve this purpose, the phage display technology has been extensively exploited in the field of hiv-1 as it represents one of the most powerful technologies for epitope mapping as well as for the identification of ligand binding to many types of targets. bacteriophages (phages) are bacteria-infecting viruses whose dna or rna genome is packed in a capsid composed exclusively of surface proteins. the principle of phage display relies on cloning of exogenous dna in fusion with the phage genetic material allowing the display of foreign peptides in an immunologically and biologically competent form at the surface of phage capsid proteins [12] . the significance of phage display was first demonstrated for filamentous phages such as m13, fd or related phagemids and later extended to lytic bacteriophages λ, t4 and t7 (reviewed by beghetto [13] ). the phage biopanning process consists of iterative cycles of binding, washing and elution steps leading to the progressive selection of phages displaying peptides/proteins binding to the target of interest [14] . the target is usually immobilized on a solid support which can be plastic, beads or even cells. a significant advantage of this technology is that phages may be used to display a collection of sequences (phage library), reaching up to billions of distinct sequences. phage libraries can be constructed to express combinatorial peptides or proteins/immunoglobulin fragments/variants that may be further screened for many different purposes including drug discovery, epitope mapping, diagnosis as well as identification of therapeutic antibodies. the phage display technology is versatile as it can be applied to different domains of research; it allows the easy handling and high-throughput screening of billions of sequences. it is affordable and enables the identification of linear as well as conformational epitopes when applied to an antibody target. numerous studies have described the use of the phage display technology in the field of hiv-1 were reported. they can be classified in four main applications ( figure 2 ): i. epitope mapping, which relies on the screening of random peptide libraries on immobilized monoclonal or polyclonal antibodies to determine the linear and/or conformational epitopes recognized by these antibodies (linear epitope: sequence of continuous amino acids recognized by the paratope of a given antibody; conformational/discontinuous epitope: group of amino acids scattered along a protein sequence which come together in the folded protein and are recognized by the paratope of a given antibody). such screening usually results in the identification of sequences mimicking the natural epitope (mimotopes) and provides precise information about the location of residues forming the natural epitope. these mimotopes may in turn be used as valuable immunogens to elicit antibodies targeting the original epitope, an approach referred to as -reverse vaccinology‖. ii. inhibitor discovery, based on screening of phage libraries displaying random peptides or antibody fragments against viral or host proteins critical for viral replication. iii. -phage substrate‖ approach in which potential substrate sequences are displayed at the phage surface to enzymes such as proteases. this approach not only allows for proteolysis specificity profiling, but provides information and data to develop specific inhibitors. iv. carrier phage, in which the phage functions as a -carrier‖, -vehicle‖ or -virus-like particle‖ to display exogenous peptides such as mimotopes or even full size antigens to the immune system, to elicit specific humoral and/or cytotoxic t-cells responses. this review is intended as an overview of the different studies conducted using phages in the field of hiv-1, laying special emphasis on the nature of the phage libraries used, the target display mode, the biopanning procedure as well as the results obtained. these studies are classified according to the 4 applications described above and the main results are presented in tables. monoclonal antibodies (mabs) or polyclonal antibodies (pabs) epitopes can be identified through screening either combinatorial or antigen-fragment libraries displayed at the surface of phages. antibodies may be derived from infected/immunized animals or from hiv seropositive patients with peculiar immunological profiles, such as the long term non progressors (ltnp) [15] , leading to the identification and characterization of hiv mimotopes. the seminal paper characterizing the epitope recognized by a mab directed against hiv-1 using the phage display technology was published in 1993. keller et al. screened a 15-mer random peptide library (rpl) against the bntab 447-52d which targets the v3 loop of gp120 (krkrihigpgrafy) [16] (figure 3 ) and identified 70 clones presenting a gpxr consensus sequence [17] (table 1 ). these mimotopes were further used in rabbit immunization experiments and elicited neutralizing responses. boots et al. later investigated the linear epitope recognized by the mab 447-52d by combining gp120 competition and panning of v3-region biased/constrained libraries. such a set-up favors the selection of mimotopes in which residues surrounding the gpgr crown motif are similar to those present in the gp120 used for competition, suggesting that the use of strain-specific competitors with a mab of broad specificity can select for strain-specific mimotopes [18] . [36, 37] . in their study, a 20-mer rpl was constructed and panned against mab 58.2 according to two different protocols, either streptavidin capture of phages mixed with biotin-labeled mab 58.2 (sa-bio) or panning against mab 58.2 immobilized on microwells (micropan) [19] . phages selected with the sa-bio protocol shared a consensus sequence (y/l)(v/l/i)gpgrxf homologous to the v3 loop. the micropan protocol allowed for the identification of sequences sharing the same motif, of which two were also identified in the sa-bio panning. biopanning results were further validated in peptide array hybridization assays. hybridization of mab 58.2 to 14-mer peptides containing all possible point substitutions within the v3 loop sequence demonstrated that both phage display and peptide array experiments identified the same critical amino acids, thereby confirming the quality of the 20-mer rpl and the validity of the screenings performed. epitope mapping was also performed on a monoclonal antibody (mab 19b) isolated from an asymptomatic hiv-1-infected patient and recognizing the xxix 3 pgrafytt motif within the v3 loop sequence (krihigpgrafytt) [38] . binding of mab 19b to viral isolates presenting mutations in this sequence revealed that not all residues within this recognition motif were crucial for reactivity [20] . biopanning with a 15-mer rpl resulted in the selection of sequences compatible with the minimal binding site (-i----g--fy-t) inferred from gp120 sequence alignment from clades a to f which bound mab 19b. taken together, data from binding assays as well as phage biopanning experiments demonstrated that the mab 19b epitope spans both sides of the v3 loop. substitutions within the residues located at the crown of the loop are however tolerated, provided that the formation of a β-turn induced by the gpgr crown motif is allowed. however, one exception was reported by boots et al. who reported that the phe to trp substitution may be tolerated in the absence of a β-turn [18] . in parallel, grihalde et al. constructed and panned a 30-mer rpl against mab 1001, which recognizes a constrained linear epitope on the v3 loop [21] . several clones were obtained and presented the common motif (r/k/h)xgr mimicking the crown of the v3 loop sequence, thereby confirming the epitope sequence of mab 1001. to assess the reactivity of peptides deprived of the phage scaffold, the mimotope with the highest affinity for the mab 1001 was expressed in fusion with the e. coli alkaline phosphatase. binding of the phage and fusion protein to the mab 1001 was assessed by elisa, western blot and spr assays and highlighted that binding was independent from the scaffold, although interactions were weaker when the peptide was displayed in the fusion protein format than in the phage scaffold. in another study, laisney et al. investigated the minimal epitopes recognized by two mabs interacting with the v3 loop, 110-a and 19.26.4, whose specificity is strictly restricted to the x4-tropic lai isolate [22] . the screening of a 6-mer rpl on the mab 110-a allows the selection of numerous sequences with a consensus motif. binding assays with synthetic peptides further showed that both mabs reacted with residues 316-320 of the lai gp120. in this narrow region, the minimal epitope deduced for the mab 110-a was hyxrgp, whereas the mab 19.26.4 recognized the xq(r/k)gp motif (hy: non-aromatic aa, underlined: aa tolerating substitutions). interestingly, the essential qr residues located at positions 317-318 correspond to a qr insertion located upstream of the v3 loop gpgr crown motif that is characteristic of the lai isolate and may thus explain the restricted specificity of the two mabs. the same authors screened a 6-mer rpl on the mab 268, specific to the v3 loop of the mn isolate, and identified two groups of sequences [23] . a representative sequence from the first group (268.1, hlgpgr), corresponded to the crown of the v3 loop, a linear epitope, while two sequences of the second group (268.2, kaihri and 268.3, kslhrh), showed no homology to linear hiv-1 epitopes. both peptides 268.1 and 268.2 nevertheless inhibited the interaction of mab 268 with gp120, and were even able to compete with each other for binding to the antibody, indicating that peptide 268.2 was also a mimotope of peptide 268.1. when conjugated to klh and injected separately into rabbits, both peptides 268-1 and 268-2 were able to elicit gp120-reacting antibodies that partially competed with the homologous peptide, confirming that 268.1 and 268.2 peptides are both antigenic and immunogenic mimics of the gp120 mn v3 loop. the isolation of the bntab 2f5, which interacts with an epitope (eldkwa) located on the gp41 mper was reported in 1993 [39] . conley et al. further characterized this epitope by biopanning a 15-mer rpl on immobilized 2f5 ab. different sequences were obtained and classified in four groups, whose consensus motifs, dkw, ldxw, ed(k/r)w and eldkw, revealed information on the residues involved in 2f5 ab recognition [24] . immunization attempts with eldkwa peptides failed to elicit 2f5-like ntabs, suggesting that the epitope necessitates additional residues in order to be immunogenic. therefore, menendez et al. screened a panel of 17 libraries of linear and constrained peptides against the mab 2f5 and deduced that residues flanking the dkw core at the c-terminal side region were important for high-affinity binding to the mab [25] . they subsequently constructed and screened two phage sublibraries displaying 12 random residues either upstream or downstream of the dkw core (x 12 -aadkw and aadkw-x 12 ) and isolated three peptides displaying high affinity for 2f5 from the aadkw-x 12 library. ala substitution and deletion studies revealed that each clone bound 2f5 according to a different mechanism. this data led the authors to postulate that the 2f5 paratope was composed of two binding domains either recognizing the dkw core with strong specificity or multispecifically binding to the residues located at its c-terminus. based on this study, additional investigations were recently conducted on the bntab 2f5 epitope to assess the importance of structural constraints for mab 2f5 recognition [26] . a linear 12-mer rpl and a constrained 7-mer rpl were screened against this antibody and all the sequences selected from the 12-mer rpl contained the d(k/r)w core motif, with flanking residues l, a and s present at different frequencies. analysis of the sequence representation compared to their estimated probability of occurrence indicated a trend towards enrichment for sequences such as dkwa or ldkwa throughout panning of the 12-mer library, while all sequences selected from the constrained library contained dkwa or ldkwa. these results demonstrated that the strong epitope specificity postulated by menendez [25] is only displayed when the epitope sequence is presented in a certain structural context provided in the constrained 7-mer peptides. immunization studies performed with both linear and constrained forms of the peptide in mice and rabbits resulted in the inhibition of cell fusion only with sera of rabbits immunized with the linear peptide. rpl screening may also contribute to the elucidation of the antigen structure. to that purpose, stern et al. used a 20-mer rpl to analyze two different mouse mabs (gv1a8 and gv4d3) recognizing non-overlapping sequences between residues 1 and 142 of gp120 [27, 40] . biopanning performed on gv1a8 allowed for identification of mimotopes sharing a (l/i)w motif identical to residues 111-112 of the gp120 c1 domain and highlighted a hxxixxlw motif compatible with two turns of an α-helix. computer modeling confirmed that such a structure placed the residues recognized by gv1a8 contiguously on one face of the helix while other secondary structures did not. similarly, biopanning on mab gv4d3 yielded sequences with a trend towards an nx 3 wxxd motif. the epitope maps to the fnmwknd sequence satisfying the helical motif fxxwxxd. in this study, the use of phage display not only predicted the α-helix structure of the c1 domain of gp120, but also pinpointed the contact residues defining the surface of the helix. phage display was also applied to epitope mapping of antibody-dependent cellular cytotoxicity (adcc)-inducing mabs since hiv-1 infected cells may be targets for fc receptor-bearing effector cells interacting with hiv-1-specific abs. screening of 7-mer, 7-mer-c and 12-mer rpls against the adcc-inducing mab id6 resulted in the identification of phages with txxfxxwxxd (12-mer rpl) and fxdwxf (7-mer and 7-mer-c rpls) motifs homologous to the c1 domain of gp120 [28] . competition assays showed that binding of mab id6 to gp120 or gp160 was abrogated in the presence of 12-mer mimotopes. in contrast, heptapeptide mimics only slightly impaired this binding, supporting the hypothesis that the mab-id6 epitope probably encompasses residues additional to the fxdwxf motif. as this epitope is highly conserved among circulating hiv-1 subtypes, it might be useful to induce mab id6-like antibodies. the bntab igg1 b12 was the first neutralizing mab selected from a phage-displayed fab (antibody fragment composed of one constant and one variable domain of the heavy (ch1 and vh) and the light (cl and vl) chains linked together) library derived from an hiv-1-infected donor (see section 3.1.1.1.1.) [41] . this antibody recognizes a conformational epitope overlapping the cd4-binding site of gp120 [42] . attempts to precisely map the residues interacting with the igg1 b12 mab with 15-mer and 21-mer rpls provided no consensus sequence [18] . as previous screening of 11 cysteine-enriched peptide libraries resulted in the identification of two sequences bearing an sdl motif flanked by one or two cysteine residues (rekrwifsdlthtci and tclwsdlraqci) [30] , zwick et al. constructed two sublibraries (x 7 sdlx 3 ci and xcxxsdlx 3 ci) sharing the sdl motif and reflecting the cysteine content of the two clones [29] . a b2.1 peptide (hersymfsdlenrci) containing a unique cysteine bound b12 in fab as well as igg1 formats with a much higher affinity than the other clones. moreover, the phage-borne b2.1 peptide was used to screen the fab library from which b12 was identified. this -reverse panning‖ experiment showed that b2.1 was able to select only the fab sequence corresponding to b12, confirming the specificity of the b2.1 mimotope towards the b12 ab. b2.1 peptide was immunogenic in mice and rabbits but did not elicit significant anti-gp120 cross-reactive abs titers. dorgham et al. attempted to map the b12 epitope with a rpl of two random 10-mers joined through an allry spacer (x 10 allryx 10 ) [31] . selection resulted in the identification of clones sharing a m/varsd consensus motif (ar standing for any aromatic residue) as previously observed [29] . a second-and a third-generation of semi-rpl containing fixed consensus motifs identified in the previous panning surrounded by randomized residues were constructed (x 3 (m/v)wsdx 3 and xlxvwxdexx). phagotopes (phage particle displaying a particular peptide sequence selected on a given target) obtained from the first, second and third generation libraries showed increasing binding affinity for b12, respectively. phagotopes were able to compete with gp160 for b12 binding and triggered the production of abs capable of recognizing at least five distinct, unrelated hiv-1 strains. in contrast, the corresponding peptides were not able to compete for b12 binding and did not elicit anti-gp160 mabs. such discrepancies between phagotopes and peptides might be explained by constraints imposed by the phage scaffold. detailed characterization of the bntab b12 was conducted with the mapitope algorithm developed by enshell-seijffers et al. to facilitate the identification of discontinuous epitopes. this approach is based on the assumption that the collection of mimotopes recognized by a given antibody must in some manners reflect the antibody's paratope [32] . a constrained 12-mer rpl was screened against b12 and selected sequences were compared to those obtained from previous panning experiments performed against b12 [18, 29, 30, 43] . although no similarity was observed with the mimotopes selected by boots et al. [18] , a consensus wsdl motif was observed in the newly identified mimotopes and the sequences isolated by bonnycastle et al. [29, 30] . mapitope analysis conducted on these sequences as well as on the peptide sets isolated by boots and bonnycastle resulted for each of the three panels in the prediction of two clusters located at the periphery of the cd4 binding site. at the same time, both a linear 9-mer rpl and a constrained 10-mer rpl were used in panning experiments against another gp120 cd4 binding site mab (5145a) [33] . screening of the 9-mer rpl resulted in selection of a single sequence (wkpvvidfe), while screening of the 10-mer-c rpl on 5145a allowed for identification of a gpxepxgxwxc consensus motif. peptides were synthesized as peptide-piii fusion proteins and their affinity for 5145a was assessed in phage/mab and gp120/mab binding inhibition assays. the two most affine peptides (aecgpaeprgawvc and aecgpyeprgdwtcc) were used to immunize rabbits and elicited antibodies binding to recombinant monomeric gp120. nevertheless, generated abs seemed to target a different epitope since they were unable to compete with the 5145a cd4-binding site specific mab. the extreme c-terminus of gp120 forms a pocket which may interact with gp41 and was suggested to undergo conformational changes weakening the interaction between gp120 and gp41 upon cd4 binding. in the absence of available crystallographic information, ferrer et al. utilized the mouse mab 803-15.6 to analyze an epitope overlapping with this pocket region [34] . epitope mapping of mab 803-15.6 achieved by cross-blocking experiments on gp120 suggested that the ab recognized residues 502-516 while the screening of an heptapeptide rpl against mab 803-15.6 preincubated with gp120 allowed for the recovery of phages presenting an axxkxrh motif homologous to residues 502-508. affinity studies confirmed that ala was the n-terminal residue of the mab 803-15.6 epitope and showed that affinity increased when c-terminal residues were added. the mapitope algorithm designed by enshell-seijffers et al. was initially developed to elucidate the cd4-induced epitope recognized by the mab 17b [32] . screening of a 12-mer-c rpl yielded sequences with no homology to gp120. comparison of the mimotopes to the gp120 structure in complex with mab 17b and scd4 predicted candidate epitopes that were in agreement with the actual 17b contact residues. for further validation of the algorithm, rpl libraries were screened against the p24-specific mab 13b5 and analysis of the selected sequences predicted four clusters, the largest of which corresponded to the genuine epitope. the algorithm was finally applied to the mab cg10, an ab with an unknown epitope competing with the mab 17b for the binding to the cd4/gp120 complex. mimotopes sequences were analyzed and produced seven clusters, one of them being in accordance with previous mutation analysis impeding mab cg10 binding [44] . noteworthingly, when reconstituted in a phage scaffold, the epitope was capable of binding mab cg10. after having successfully identified linear or nearly linear epitopes [17, 20, 24] , boots et al. extended the use of the phage display technology to the identification of epitopes recognized by mabs binding to discontinuous sequences [18] . one of these abs (mab a32) binds to a cd4-induced discontinuous epitope involving residues within the c1, c2 and c4 regions of isolates from clades b, c, d, e and f [45, 46] . panning of a 15-mer rpl yielded several phages which only shared a trp residue. in the same study, panning of a 15-mer rpl against mab 50-69, which reacts with the id gklic region of gp41, resulted in the identification of sequences sharing a common trp within motifs wgcx(k/r)xlxc and fgxwfxmp. the selected consensus sequences were however not further characterized. the bntab 2g12 presents the typical feature of recognizing a cluster of high-mannose oligosaccharides of gp120 [47] [48] [49] . in an attempt to identify peptidic immunogens capable of eliciting 2g12-like abs, menendez et al. screened a set of previously described rpls [30] against 2g12 and identified one phagotope specifically binding to 2g12 (2g12.1) [35] . the crystal structure of mab 2g12 complexed to the synthetic 2g12.1 peptide was compared to structures of 2g12-oligomannose epitopes and revealed that interactions with the abs were different for the two ligands. these results showed that the peptide selected from rpl panning experiments is not a structural mimic of the 2g12 oligomannose epitope. the phagotope 2g12.1 was used in rabbit immunization experiments and elicited high titers of peptide-specific antibodies, but no cross-reactivity with gp120 was obtained, further supporting that peptide 2g12.1 is not an immunogenic mimic of the mab 2g12 epitope. the first attempt at identifying epitopes recognized by hiv-specific pabs was performed in 1999 on plasma igg from two ltnp patients (table 2 ). using linear and constrained 9-mer rpls, scala et al. identified mimotopes of the linear immunodominant (id) gklic region of gp41 or the v1 and c2 domains of gp120 [50] . these mimotopes were immunogenic when injected to mice and elicited an ntab response against hiv-1. moreover, the same mimotopes reduced viraemia to undetectable levels in immunized monkeys as shown in a subsequent study [51] . the same year, a similar study conducted on one ltnp with an rpl library of cysteine-constrained 12-mers selected for peptides defining the gp41 id epitope csgklic. the levels of reactivity of these phagotopes were further assessed against a panel of hiv positive plasma to evaluate the plasticity and polyclonality of the immune response mounted by 30 infected individuals [52] . later, palacios-rodriguez et al. evaluated the impact of factors such as highly active antiretroviral treatment (haart) or ab titers on a selection of peptides mimicking the id epitope csgklic [53] . in their study, a mix of linear 12-mer as well as linear and constrained 7-mer rpl was screened against the individual plasma samples of four hiv-1 infected patients initiating haart and presenting different titers of anti-gklic antibodies. a consensus motif cxxkxxc was obtained from the 12-mer linear rpl, and the percentage of occurrence of the motif in the selected sequences was proportional to the anti-gklic ab titers of each sample, indicating that these abs are involved in selection of the consensus motif. mice immunization experiments with the two mimotopes resembling most to the gp41 id parental epitope as well as with pools of phages eluted from the panning experiments showed that all phages elicited reactivity, and that immunization with the phage eluates induced the strongest recognition. these findings indicate that the immunogenic properties of mimotopes are different and additive, opening the possibility of immunizing animals with different mimotope combinations (see section 4). in 2007, humbert et al. investigated the immune response of eight ltnp patients presenting bntabs. by using linear and constrained rpls they identified epitopes recognized by plasma iggs captured on tosylactivated beads [54] . each panning round consisted of a positive selection performed on ltnp iggs followed by a negative selection on the iggs of healthy donors. homologies of some selected sequences to immunodominant regions such as the gp120 v3 loop or the gp41 gklic region were observed, as reported in previous studies [50, 52, 53] . further homologies to linear motifs located near the v3 loop (nnnt), downstream of the id gklic region (avpw motif) and overlapping with the 2f5 bntab epitope (ppwx 3 w motif) were also identified. additionally, the authors applied the 3dex software to compare the phage insert sequences to hiv-1 protein structure files from the rcsb protein data bank (www.pdb.org) [59, 60] . phage pools corresponding to the linear v3 loop, gklic domain and wxxxw motif, as well as pools representing potential conformational epitopes, were selected for mice immunization assays, and elicited plasma-associated neutralizing activity against primary hiv-1 strains. the highest neutralizing ability was obtained with mice immunized with the v3 mimotopes, although immunization with potential conformational epitopes also provided a modest neutralizing response. a similar approach was used by the same authors on a rhesus macaque infected with an shiv chimera encoding the env of a clade c hiv-1 strain (shiv1157ip) and presenting a broad neutralizing response against homologous shiv-c as well as heterologous hiv-1 strains of different subtypes [61] . biopanning yielded clones similar to gp120 (v2 and v3 loops or c-terminal domain) or to regions of gp41 (id gklic region, other id regions and mper domain) [55] . remaining clones showed no significant homology to linear hiv-1 regions and were analyzed with the 3dex software, which allowed the identification of a discontinuous mimotope located near the v3 loop crown. the antibodies binding to this phagotope were affinity-purified and subsequent assays demonstrated that recognition was conformation-dependent. an immunofocused immunization of mice primed with a dna vector coding for the gp160 shiv1157ip and boosted with pools of phage particles corresponding to the v3 loop, the gp120 c-terminus, the gp41 id region, the gklic region and the mper domain was set up. almost all mice developed anti-env abs and 59% of them presented a neutralizing activity. in 2009, dieltjens et al. applied the phage display technology to identify the epitopes potentially involved in the bntabs response of an hiv-1 crf02ag-infected individual (itm4) and to monitor the evolution of humoral response and viral escape through the course of infection [56] . biopanning of a 12-mer rpl against plasma samples from itm4 resulted in the identification of different peptide sequences. half of these sequences were homologous to linear epitopes on gp41, i.e., the 4e10 epitope region in the mper domain (nwfnltqtlmpr) or the lentivirus lytic peptide 2 (llp2) (slxxlrl) while the other peptides shared homologies with the c1 domain (kxwwxa) and the crown of the v3 loop (kx 3 igphxxy) of gp120. further analysis of the levels of reactivity of the phage groups against itm4 six-year follow-up samples revealed different temporal patterns of recognition, confirming the dynamic nature of the immune response. interestingly, the mper region was the only epitope retaining immunogenic properties during this period. in a more recent study, the same group investigated the antigenic landscape of an hiv-1 subtype a-infected individual with bntabs by screening an rpl library against a pool of sequential samples drawn from 1994 to 2005 [57] . the biopanning procedure yielded sequences predicted to represent autologous v2 sequence (kx 3 hx 3 y), v3 loop (kxxhxgpx 3 f) and gp41 id domain (cxgxlxctxnxp). again, follow-up sample recognition of the four phage groups showed different patterns. antibody reactivity towards gp41 id region fluctuated slightly in all plasma samples. reactivity against the v3 loop-like phages decreased over time. in contrast, the v2 loop mimotopes were not recognized before 2001, but once emerged, reactivity persisted until 2005. env sequence analysis of the follow-up samples showed that a tyr to his mutation in the v2 loop sequence coincided with the emerging antibody response against this sequence. additionally, the authors highlighted that the neutralizing activity observed in the samples was partially due to antibodies recognizing the v3 mimotopes. besides the multiple reports on the use of rpl to characterize the humoral response against hiv-1 env proteins, gupta et al. evaluated the reliability of using targeted antigen gene fragment libraries for the identification of epitopes recognized by antibodies elicited in rabbits immunized with p24. to this end, they constructed a phage library composed of dnase-digested fragments of gag dna [58] . phagotopes obtained after the first panning round displayed mainly 30-40-mer peptides, 70% of which mapped to of the n-terminus of p24 (150-240 of gag) and 30% corresponded to the c-terminal region of p24 (310-360 of gag). only one phagotope mapped to the central region of gag (269-310). at the end of the second round, selected phages displaying longer inserts of 40 to 50 aa corresponding to the n-and c-terminal regions of gag were identified, revealing the presence of two distinct antigenic regions in gag. this study demonstrated that gene-fragment phage display could be used to identify epitopes targeted by polyclonal abs. although they occur at a very low frequency in humans, antibodies targeting host proteins involved in hiv-1 infection have been reported in immunized animals. given their potential value for viral entry inhibition and the general understanding of this mechanism, rpls were screened on these mabs to gain better knowledge of their epitopes (table 3) . na: data not available. the murine mabs 3a9 and 5c7 were raised against cells transfected with the seven transmembrane-spanning domains chemokine receptor ccr5, one of the main coreceptors for hiv-1. they recognize a common epitope located near the ccr5 n-terminus [67, 68] . both mabs were used to screen a constrained 9-mer rpl [63] . phagotopes selected on 3a9 displayed the sequence chasiydfgsc while cphwlrdlrvc was the most prevalent sequence isolated on 5c7. these sequences showed homologies to residues located at the n-terminus but also within the first or third extracellular loop (ecl) of ccr5. both reacted against the targeted mab either in phage, cyclic peptide or linear peptide formats. moreover, they were able to bind to gp120 and the peptide selected on 3a9 inhibited binding of the mab to a cell line expressing ccr5. to further characterize the conformational epitope recognized by 3a9, additional screening rounds of 12-mer, 7-mer and 7-mer-c rpls were performed [62] . sequences with an hw motif homologous to the cphwlrdlrvc motif selected on the mab 5c7 were identified, and ala-scanning confirmed the importance of the hw motif and siyd motifs previously identified for 3a9 binding [63] . another murine antibody (mab 2d7) recognizing a conformational epitope on the second ecl of ccr5 [67] was explored by screening a linear 15-mer rpl [64] . three phagotopes (m14, m23 and m71) were isolated and one of them (m23) was able to inhibit cell infection by the hiv-1 sf162 isolate. the corresponding peptide (fcaldgdfgwlapac) fused to the piii phage coat protein neutralized infection mediated by the jr-fl but not the iiib strain. the fusion protein specifically bound 2d7 and was recognized in a dose-dependent manner by three ccr5 chemokine ligands, i.e., ccl5 (rantes), ccl3 (mip1α) and ccl4 (mip1ß), confirming its ccr5 mimicry. six years later, another screening campaign was conducted with a linear 12-mer rpl on 2d7 and the ewqkeglvtlwl sequence of a high-affinity binding peptide was obtained [65] , revealing that this peptide presented homologies to the n-terminal (170-qkegl-174) and c-terminal regions of the ccr5 ecl2. ala substitutions of the tl residues confirmed their crucial role in 2d7 binding. the selected peptide was used in rabbit immunization studies and elicited abs with 2d7-like biological functions, i.e., which inhibited hiv-1-mediated cell fusion and pbmc infection. the cd18 cell surface molecule, a part of the lfa-1 molecule, is involved in the syncytia formation of hiv-1-infected lymphocytes [69] . as mab mhm23, a cd18 binder, inhibits hiv-1-mediated cell fusion, poloni et al. applied the phage display technology to map the mhm23 epitope and thereby identify the cd18 domains which account for syncytia formation [66] . linear and constrained 9-mer rpl were panned on the mhm23 mab, to allow for the selection of linear and constrained sequences. a ppfxyrk consensus motif was inferred by sequence comparison, assigning the epitope recognized by mhm23 to residues 200-206 of cd18. two phagotopes inhibited in vitro hiv-1-induced syncytia formation and one of them retained this ability in the peptide format, confirming its role in syncytia formation and highlighting that mimics of this epitope could prevent cell-mediated viral propagation. as summarized in the first section of this review, phage-displayed rpls are powerful tools to determine or to characterize mabs as well as pabs epitopes. besides epitope mapping the phage display technology was also widely applied to the identification of hiv-1 inhibitors. screening of phage-displayed rpls, antibody-fragment or ligand libraries on viral or host targets contributed to the discovery of molecules interacting with the key players of hiv-1 infection. antibody libraries were particularly investigated, and repertoires of fab, scfv (antibody fragment corresponding to variable regions of the heavy (vh) and light (vl) chains of antibody connected by a short peptide linker), v hh /nanobodies (single domain antibody fragment (sdab) corresponding to the variable heavy-chain domain of a camelid heavy-chain only antibody (hcabs)) or cdr3 fragments from naï ve or hiv-1 infected subjects as well as from immunized animals were displayed at the surface of phages ( figure 4 ). most of the hiv-1 inhibitors selected with the help of phage display were identified by targeting viral proteins (table 4 ). (2) 5-helix, izn36 (3) 5-helix (1) pie12-trimer (1) tat (2) cyclin t1 [107] [108] (1) ncp7 (2) psi rna the cd4 binding site represents one of the main achille's heels of the virus since it is involved in the earliest step of hiv-1 entry and is conserved in almost all hiv-1 strains [126, 127] . numerous phage display biopannings were performed on gp120 and are classified here according to the type of antibody libraries used. burton et al. were the first to report the construction of a phage-displayed fab library from the bone marrow of an asymptomatic hiv-1-infected patient with high titers of gp120-specific abs [41] . this library was screened against recombinant gp120 from the iiib isolate and clones displaying high affinities (<10 nm) for gp120 were selected [42] . one fab (b12) (see section 2.1.1.4.) was able to neutralize the mn and iiib strains in different set-ups. this ab is the most potent neutralizing ab isolated to date, featuring neutralizing activity against 75% of 36 primary isolates of hiv-1 tested at concentrations that could be achieved by passive immunization [73] . to improve its affinity, the b12 fab was submitted to cdr walking, a procedure involving randomization of its cdr and expression of the derived libraries expressed on phages, followed by screening against on gp120 [73] . sequential cdr walking of the hcdr1 and hcdr3 domains was performed and four clones were chosen for detailed analysis of their binding affinity and neutralization potency against the iiib and mn isolates. a pro96glu mutation of the hcdr3 was identified in the clone with highest affinity, 3b3, which bound iiib gp120 with an 8-fold improved affinity (0.77 nm) compared to the parental b12 fab. similarly, fab 3b3 was able to neutralize four isolates that were insensitive to the parental b12 fab. the cdr walking mutagenesis strategy was pursued in a subsequent study and a further 420-fold improvement of the binding affinity of 3b3 for gp120 was achieved, reaching 15 pm [72] . these studies were the first to demonstrate that recombinant fabs (devoid of the typical igg contamination residual of calpain cleavage) featured neutralizing activities similar to those of whole igg. as the fabs fragments were easier to produce and their smaller size allowed them to target binding sites that were not accessible to full-length igs, this led to the construction of many fab libraries to elucidate the immune response to hiv-1 and to identify therapeutic antibodies. the extremely high binding affinity of 3b3 was also applied to develop an immunotoxin which could specifically kill hiv-1-infected lymphocytes [71] . the authors engineered 3b3 scfv fused to a truncated form of pseudomonas exotoxin a. the 3b3(fv)-pe38 fusion immunotoxin bound to the mn strain of gp120 with the same affinity as the parental fab antibody and specifically killed a gp120-expressing cell line and a chronically hiv-infected lymphocytic cell line. this study provided the proof-of-concept that high affinity anti-hiv-1 antibodies have a dual application since they may be used for their neutralizing potency but also as carriers for antiviral compounds. most antibodies obtained through screening fab libraries against monomeric gp120 targeted epitopes related to the cd4-binding domain of gp120, pointing to it as an immunodominant epitope [70] . to expedite the identification of ntabs directed against weakly immunogenic epitopes, a strategy called epitope-masking was applied in several studies. this biopanning approach is designed to mask a particular epitope with antibodies or ligands directed against the region of interest prior to addition of the phage library. ditzel et al. panned a fab library from an asymptomatic hiv-1-infected patient on immobilized recombinant gp120 and identified two dominant clones targeting the cd4-binding site [74] . these two fabs were then incubated with gp120 to mask their respective epitopes and the panning of the library was repeated, highlighting (based on sequence similarities) four groups of fabs recognizing gp120 with affinities in the range of 50 to 100 nm. epitope mapping of one representative fab for each group showed that gp120 binding of three clones was influenced by the v2 loop and the cd4-binding site and was not affected by the glycosylation status of gp120. furthermore, one of these fabs (l78) featured a broad neutralization spectrum against various hiv-1 strains. the authors performed further epitope masking by using different selection strategies with the same fab phage library [81] . the first strategy involved masking the cd4-binding site (cd4bs) epitopes either with soluble cd4 or with a cd4bs ab. all fabs selected on scd4-bound gp120 recognized the c1 region, while the fabs isolated from the cd4-bs ab-captured gp120 were classified in four different groups: (i) fabs targeting the c1 region, similarly to fabs isolated on scd4-bound gp120; (ii) fabs directed against the c1-c5-region; (iii) fabs recognizing the v2 loop; and (iv) fabs directed against a cd4bs/v2 loop region, similar to the neutralizing fab isolated by ditzel et al. [74] . multiple epitope masking was then conducted by masking the cd4bs-mab-captured gp120 with one of the c1-specific fabs selected on the scd4-bound gp120 prior to phage addition, leading to the identification of two c1/c2-dependent fabs. all isolated fabs bound their targets with affinities ranging from 4 to 300 nm. however, these fabs targeting weakly immunogenic regions were not or poorly neutralizing. more recently, koefoed et al. investigated the anti-gp120 ab repertoire of the circulating gp120-binding igg-bearing b cells of 22 hiv-1-infected patients by constructing phage displayed fabs libraries from unselected cells or from cells preselected with immobilized gp120 [128] . panning against gp120 selected for a higher number of phagotopes from the preselected library. clones from the unselected library recognized the v3 loop, while clones from the preselected library targeted the cd4 bs or a cd4-induced epitope encompassing the c1 region. these fabs displayed no significant differences with respect to epitope specificity, affinity and neutralization ability compared to fabs obtained from bone marrow libraries, and most of them were unable to neutralize hiv-1. these results were in accordance with previous findings by parren et al. (1997) concluding that the majority of the circulating hiv-1 specific antibodies were elicited by viral debris and were therefore devoid of neutralizing activity [129] . antibodies recognizing the amino acids 421-436 of the gp120 cd4bs were isolated from patients suffering from the systemic lupus erythematosus autoimmune disease [130, 131] . however, whether these antibodies neutralized hiv-1 was not known, which prompted karle et al. to quantify gp120-recognizing abs in an existing scfv phage-displayed library from the pbmcs of lupus-suffering patients [132] . biopanning selected for clones binding both gp120 and the 421-436 region of the gp120-cd4-binding site. one of these clones (jl413) neutralized r5 and x4-tropic hiv-1 primary isolates from clades b, c and d with ic 50 ranging from 0.1 to 25.6 µg/ml. a subset of gp120-binding antibodies was shown to hydrolyze gp120 by a mechanism analogous to serine protease [133] , as the nucleophilic region responsible for this activity was localized in the light chain [134, 135] , a library of light chains prepared from three lupus patients [132] was screened with an electrophilic analogue of gp120 residues 421-433 to isolate antibodies capable of binding and hydrolyzing gp120 [75] . one of the light chain clones selected (skl6) cleaved a gp120 421-433-reporter substrate as well as full-length gp120. engineering of abs composed of such a light chain coupled to a gp120-binding heavy chain might provide abs with anti-viral proteolytic activities. the advantages of reduced antibody formats were also explored with naturally occurring smaller antibodies. in addition to conventional antibodies, camelids also produce antibodies devoid of light chains ( figure 3d ). these heavy-chain only antibodies (hcabs) lack the c h1 domain and their binding specificity is provided by the variable heavy-chain domains of hcabs (v hh or nanobodies) [136] . the cdr regions of nanobodies are on average longer cdr than those of conventional abs and display a protruding conformation, thereby more easily binding clefts and active sites. nanobodies were successfully used for panning against various pathogens (reviewed by vanlandschoot [137] ). forsman et al. immunized llamas with a recombinant gp120 of subtype b/c (cn54) [78] . three different panning strategies against various gp120 resulted in the selection of three nanobodies (a12, d7 and c8) with neutralizing activity against a limited panel of clade b and c strains (ic 50 values ranging from 0.003 to 38 µg/ml). these nanobodies bound gp120 with affinities from 0.1 to 1 nm and inhibited its binding to cd4 as well as to mabs known to recognize the gp120 cd4 binding site and, last but not least, competed with each other for gp120 binding. in a follow-up study, koh et al. described a family-based approach to produce nanobodies similar to a12 and d7 [77] . they used a degenerated oligonucleotide annealing to the last six codons of cdr3 and framework (fr) 4 and an fr1-specific primer to amplify a sublibrary of related v hh clones with properties similar to the parental a12 or d7 v hh . more than 30 phagotopes were tested for their ability to neutralize 3 clade b and 3 clade c strains. all tested v hh displayed similar neutralizing activity against the clade b viruses. three different neutralization profiles (broad a12-like, intermediate and narrow d7-like potency) were observed against the clade c type strains and the breadth of neutralization potency appeared to be correlated to the presence of an yyd motif in the c terminus of a12 cdr3. when the cdr3 ydd motif from a12 was introduced within d7, gp120 binding affinity increased by 10-fold and neutralization of clade c strains increased by 5-fold. these studies were the first to demonstrate bntabs elicited in immunized animals. as v hh are stable and can be produced at a relatively low cost, they are promising hiv-1 inhibitors. the cd4 receptor recognizes gp120 through residues located within its v1 region, and engineering of this cell receptor was applied to identify cd4 variants with a better affinity for gp120, thereby displaying hiv-1 inhibitory properties. in 1997, krykbaev et al. constructed a phage-displayed library of cd4 v1 and v1-v2 variants generated by error-prone pcr and screened it against gp120 [79] . five clones with increased affinity for gp120 and presenting mutations within the cd4 v1 domain were identified. all of these clones inhibited hiv-1 entry with ic 50 ranging from 0.2 to 1 µg/ml. the phage display technology was used to improve the affinity of the mab 447-52d for its v3 loop epitope, which had also been identified through phage display [16, 17] . in 1996, thompson et al. expressed mab 447-52d as scfv on phages and combined its v h with λ and κ chains from a non-immunized pbl repertoire prior to panning on a peptide containing the v3 loop sequence [80] . additional shuffling of the hcdr1 and hcdr2 regions was combined to hcdr3 -spiking‖, i.e., the introduction of random mutations, resulting in the identification of four key residues that could be mutated to improve ab affinity. a sublibrary in which all four codons were simultaneously mutated was constructed and biopanning allowed to select for one scfv (402p5h7) with improved k d against the mab 447-52d epitope. two neutralizing fabs (fab loop 2 and fab do142-10) were obtained by screening a fab library against recombinant gp120 reaching ic 50 ranging from 0.2 to 8 µg/ml [70, 81] . in 1999, ferrer and harrison screened 7-mer, 12-mer and constrained 9-mer rpl against gp120 and identified two peptides from the 12-mer rpl [82] . the first sequence rinnipwseamm (12p1) inhibited cd4 as well as ntmab17b binding while the second peptide, tspyedwqtylm (12p2) did not affect the cd4 interaction and rather enhanced 17b binding. the 12p1 peptide was further investigated and shown to inhibit binding of monomeric yu2 gp120 to both scd4 and 17b with ic 50 values of 1.1 and 1.6 µm respectively. the 12p1 peptide also inhibited binding of these ligands to trimeric envelope glycoproteins, blocked binding of gp120 to the native coreceptor ccr5, and specifically inhibited hiv-1 infection of target cells in vitro [138] . hiv-1 entry is a multi-step process requiring the successive binding of gp120 to cd4 and to a coreceptor, ccr5 or cxcr4, and triggers successive conformational changes that expose transient epitopes. targeting of these epitopes with ntabs could therefore prevent hiv-1 infection, as has been proven with the clinically approved fusion inhibitor enfuvirtide. to identify such receptor-induced epitopes, moulard et al. screened a fab library constructed from an hiv-1 infected patient (fda-2) with high ntab titers against gp120-cd4-ccr5 complexes [83] . one fab clone, x5, bound gp120 from several strains with a low nanomolar affinity. furthermore, binding affinity was significantly increased in the presence of cd4 and slightly enhanced by ccr5. competition assays with a panel of antibodies targeting different hiv-1 epitopes revealed that x5 recognized an epitope located in close vicinity to the cd4 and coreceptor binding sites. neutralization assays with isolates from clades a, b, c, d, f and g demonstrated that x5 neutralized all isolates with potency comparable to that of b12. x5 is the first bntab recognizing a receptor-induced epitope identified to date. to select for ligands for cd4-induced epitopes, murine leukemia virus particles carrying the env protein of the dual-tropic 89.6 strain pre-incubated with scd4 were recently used to screen 7-mer, 7-mer-c and 12-mer rpls [84] . one of the selected phagotopes (xd3: hkqpwydywllr) displaying sequence similarities (in bold) with the n-terminal extracellular part of the ccr5 and cxcr4 coreceptors was identified, suggesting novel potential leads for tyrosine sulfation. both the xd3 phagotope and xd3 peptides strongly and specifically bound to 89.6 gp120 regardless of cd4 and xd3 competed with mab 17b for binding to a cd4-induced epitope. the sulfated form of the xd3 peptide recognized x5, r4 and dual-tropic strains and inhibited hiv-1 entry in the high micromolar range. the salp15 salivary protein of ixodes scapularis inhibits cd4+ t cells activation by binding to the cd4 molecule in a region that may overlap with the gp120 binding site. this inhibition is mediated by the c-terminal 95-114 gpngqtcaeknkcvghipgc sequence [139] . juncadella et al. thus analyzed salp15 as a potential hiv-1 inhibitor and demonstrated that salp15 inhibited gp120-cd4 interaction and subsequent cell fusion and that the gpngqtcaeknkcvghipgc peptide also interacted with gp120 [140] . to identify which gp120 amino acids interacted with peptide 95-114 of salp15, the authors screened a 7-mer rpl against salp15 and isolated a hvitplw sequence homologous to an (i/l)tpl motif of the gp120 c1/v1 domain which is highly conserved across hiv-1 isolates. finally, they mapped the interaction site of full-length salp15 protein or of its 95-114 c-terminal domain were able to bind to the pcvkltplcvtlnct peptide within the gp120 c1/v1 region. in addition to epitope mapping and inhibitor identification, phage display was also widely applied to elucidating the determinants of the initial response to hiv-1 antigens and more particularly the importance and different roles of igm and igg during the establishment of infection. indeed, all known bntabs are iggs that are somatically hypermutated and are thus more difficult to elicit. in contrast, igms are closer to germline antibodies and the identification of hiv-1 specific igm could be relevant for the development of vaccine immunogens. the studies listed in this section explored and emphasized the importance of the initial igm response against hiv-1 and viral strategies to skew it towards non-neutralizing or infection-enhancing antibodies. screening against gp120 with igm and igg fab repertoires constructed from a healthy donor demonstrated that only fabs isolated from igm were able to recognize gp120, although they were polyreactive, displayed low affinities and no neutralizing properties [141] . sequence analysis evidenced that selected gp120-binding fabs originated from different v h germline genes. several studies reported that gp120 displays superantigenic properties giving it the ability to bind and stimulate non-immune b cells to secrete v h 3 ig in vitro [142] . interestingly, the v h 3 antibody family is the most represented immunoglobulin gene family in healthy adults (54% of peripheral repertoire) and hiv-1 infection leads to altered v h 3 production through selective depletion of the anti-hiv-1 v h 3 antibodies [143] . toran et al. further applied the phage display technology to examine and compare the human v h 3 genes involved in igm and igg responses to gp120 to identify the correlates of long-term non progression. two igm and igg phage-displayed fab libraries from an hiv-1-infected ltnp with high gp120-specific igm and igg1 titers were constructed and screened [144] . several clones were selected from the igm library (m02, m025, 4m26 and 4m40) and three clones from the igg library (s20, s19 and s8). all igm fabs were polyreactive and had a binding affinity for gp120 in the micromolar range while the igg fabs were specific and bound gp120 with affinities in the nanomolar range, as expected. sequence analysis showed that igg fabs originated from the same germline ab. the igm fab m025 displayed the same v h region nucleotide substitutions as those of igg fab s8 and used similar d h and j h segments, suggesting that s8 arose from m025 by isotype switching. in addition, a four aminoacid difference in the hcdr3 sequence of m025 (tgqwe) and s8 (rggsi) was proposed to be associated with the 100-fold affinity increase for gp120 and to the higher neutralizing activity of igg fab s8 (id 50 = 23 ng/ml) than of igm m025 (id 50 = 3 µg/ml). in a follow-up study conducted two years later, the three igg fabs were submitted to reverse mutations to reconstitute the germline amino acid residues [145] . the higher affinity and neutralizing ability of s20 were due to the ala30arg and ala31asp somatic mutations in the hcdr1 region of the germline gene sequence, providing clues for rational modifications of cdr in human antibodies to improve affinity and hiv-1 neutralization capacity. the igm to igg isotypic switch generating high affinity neutralizing or non-neutralizing antibodies is triggered by the activation of igm-producing b cells. to characterize the epitopes recognized by hiv-1-specific igm and to assess the effects of these abs on hiv-1 infection, chen et al. constructed a fab library from blood, lymph nodes and spleen from 59 healthy donors [146] . the library was panned against gp140 (env ectodomain containing both gp120 and a truncated gp41 lacking transmembrane domain and cytoplasmic tail) of a clade b isolate and allowed for the selection of one fab clone (r3h1m) with a relatively high binding affinity for gp140 from different strains. a sublibrary derived from this clone was panned against gp140 from different isolates, resulting in the selection of clones (m19, m19a, m19b, m19c and m19d) binding with high affinity to clade b and f gp140 (ec 50 ranging from 2 nm to 80 nm). while these antibodies had weak neutralizing properties against x4-tropic isolates, they did not inhibit and in some cases even enhanced infection with r5-tropic isolates. the m19 ab, whose sequence is relatively similar to the germline ab, targeted highly conserved epitopes located near the cd4 binding site or the coreceptor binding site. the high immunogenic capacity of the conserved non-neutralizing epitopes of such antibodies could divert the immune system from actually neutralizing epitopes. the authors suggested that these newly identified mabs could be used as probes to further characterize conserved non-neutralizing or enhancing epitopes and to modify or remove them from candidate vaccine immunogens. the epitope masking strategy (section 3.1.1.1.) might be applied to these epitopes to redirect the immune system to elicitation of antibodies targeting neutralizing epitopes. to select for antibodies closely resembling the germline antibodies as a candidate source of bntabs, the same authors constructed a cord-blood-derived igm library which they submitted to parallel screening against hiv-1 env, sars coronavirus protein binding domain (rbd) and soluble hendra virus g protein (sg) [147] . although rbd and sg antigens provided enriched igm, the library could not be enriched in hiv env-binding phagotopes. these results are in accordance with the hypothesis that hiv-1 could have evolved strategies based on weak or absent binding to antibodies of the germline repertoire [146] . presenting epitopes unsuitable for binding of somatically hypermutated antibodies would enable the virus to escape from strong immune responses. . this library was screened on the mn2031 peptide encompassing the 2f5 epitope eldkwa, and led to the identification of the z13 mab [85] . epitope mapping experiments based on synthetic peptides and recombinant proteins showed that the epitope targeted by mab z13 was located downstream of the 2f5 epitope and centered on the nfwdit sequence. mab z13 neutralized primary isolates from hiv-1 b, c and e subtypes. to enhance binding potency, fabs sublibraries of z13 variants were engineered and screened against an mper peptide and gp41 [86] . the selected z13e1 variant displayed an over 100-fold increase in neutralization breadth and potency compared to the parental z13 mab. binding experiments coupled to competition assays revealed that the z13e1 mab bound to a waslwnwfditn minimal epitope overlapping the 2f5 epitope and that the asn and asp residues were essential for peptide recognition as well as hiv-1 neutralization. very recently, phage-and yeast-displayed abs libraries constructed from an hiv-1-infected patient with 2f5-like bntabs were panned against peptides containing the 2f5 epitope and against the hiv-1 jr-fl gp140 [148] . two mabs (m66 and m66.6) were identified and the most mutated variant (m66.6) neutralized hiv-1 with a higher potency than m66. ala substitutions indicated that both abs recognized the dkw core of the 2f5 epitope and two additional leucine residues located upstream (l(660,663)). fusion of viral and host membranes, the last step of hiv-1 entry, requires the initiated by the insertion of the gp41-encoded fusion peptide into the host cell membrane and the formation of an extended prehairpin intermediate (phi). the gp41 n-and c-terminal heptad repeats (nhr, chr) then collapse to form a six-helix bundle (6-hb) in which the nhr form a trimeric coiled-coil, creating grooves where the chr bind. the viral and cellular membranes are thereby brought into close proximity, enabling fusion ( figure 1 ). during phi formation, the nhr and chr do not interact and may thus be transiently targeted by compounds which prevent the formation of the six-helix bundle in a dominant-negative manner. one such compound is the synthetic chr mimic enfuvirtide (t20) [4, 149] . a hydrophobic pocket located on the n-terminal peptide trimer groove of the 6-hb is highly conserved among hiv-1 sequences and plays a critical role in membrane fusion, and therefore represents a select target for inhibitors. eckert et al. used the particular approach called -mirror-image phage display‖ to identify d-peptides targeting the gp41 hydrophobic pocket [89] . in this approach, a phage library of natural l peptides is screened against the mirror image of a target synthesized as d-peptide [150] . by symmetry, the selected d-peptide phagotope sequences will bind the natural l-form of the target. the main advantage of d-peptides over l-peptide inhibitors is their resistance to natural proteases which enhances their oral bioavailability and serum half-life. in a first study, the authors screened a constrained 10-mer rpl against the d-peptide sequence of the gp41 hydrophobic pocket fused to a soluble trimeric coiled-coil (izn17). pocket-specific binders with a consensus motif cx 5 ewxwlc were identified and inhibited cell fusion or hiv-1 entry into cells with an ic 50 in the micromolar range when synthesized using d-amino acids. in a second study, the same authors constructed a sublibrary based on the consensus sequence identified in their first study [89] which allowed the selection of sequences with a fourfold potency increase [87] . surprisingly, the most potent peptide was a 8-mer with a cx 3 ewxwlc motif which was probably selected from the 10-mer sublibrary because its smaller size favored a more compact hydrophobic core upon binding to the gp41 hydrophobic pocket. screening of second generation d-peptides from a 8-mer cx 4 wxwlc library led to the selection of pocket-specific inhibitor of entry (pie) 7 with an ic 50 of 620 nm. dimeric and trimeric forms of pie7 had respective ic 50 values of 1.9 nm and 250 pm. in a third study, the same authors constructed a phage library based on the pie7 core sequence flanked by two randomized amino acids (xxcdypewqwlcxx) and obtained phages with the h(a/p)-[pie7 core]-(r/k/e)l consensus sequence [88] . a peptide (pie12, hpcdypewqwlcel) exhibited a broad neutralizing spectrum and was even more efficient than t20, reaching an ic 50 of 0.5 nm, when trimerized. moreover, this third generation pie12-trimer displays broadened inhibitory potency and resistance to viral variants, as escape mutants required over 65 weeks of selection in vitro to emerge. the pie12 trimer is thus a promising entry inhibitor and may be used as a topical microbicide in its d conformation. in 2005, there was no evidence of abs capable of binding the highly conserved nhr region targeted by the t20 inhibitor. to determine whether antibody fragments could target this determinant on the gp41 protein, miller et al. constructed a phage-displayed naï ve scfv library and screened it against a synthetic protein mimicking the 6-hb [90] . this construct, named 5-helix, lacks one of the three chrs and the nhr trimer is partially exposed, presenting a single binding site for a chr mimic [151] . the scfv library was also panned against the izn36 compound, a homotrimerized form of 36 nhr amino acids fused to a coiled-coil peptide, therefore representing a 6-hb mimic devoid of the chr trimer [91] . the authors identified a scfv (d5), which blocks hiv-1 entry and inhibits infection in a single-cycle infectivity assay. this scfv retained its properties when produced as a whole igg1. the antibody was found to bind the hydrophobic pocket of the nhr trimer and ala scan experiments revealed the crucial role of residues l568, w571, and k574 located in the hydrophobic pocket for this interaction. igg1 d5 was able to neutralize at least five hiv-1 isolates with ic 50 ranging from 93 to 1750 nm, thereby demonstrating that the hydrophobic pocket of the nhr trimer is accessible for binding of hiv-1 inhibitors as large as iggs. the same year, another study demonstrated that antibodies binding with high affinity and specificity to heptad repeats can be isolated from synthetic fab minimalist libraries presenting tyr/ser randomization of their cdr [152] . very recently, liu et al. took advantage of these results and constructed a minimalist fab library where the lcdr3 and hcdr1-3 domains were randomized with tyr and ser [92] . screening of this library on the 5-helix mimic selected for fabs with affinity and specificity values comparable to those obtained with the scfv of the previously described d5 ab [90, 153] . huang et al. used the n34(l6)c28 polypeptide mimicking the 6-hb [154] to screen 7-mer and 12-mer rpls and selected sequences bearing a hxx(n/d)pf motif [93] . this consensus motif synthesized as peptide (jch-4) inhibited hiv-1-mediated syncytia formation [94] . fusion inhibitors were also identified by screening non-immune human fab libraries. louis et al. screened such a library [155] against antigens comprising the trimeric coiled-coil nhr fused or not to the gp41 six-helix bundle (n35ccg-n13 and nccg-gp41, respectively) [156, 157] . they identified fabs targeting (i) the 6-hb; (ii) the nhr trimeric coiled-coil or (iii) both 6-hb and trimeric coiled-coil [95] . these antibodies were tested in a cell fusion inhibition assay and the two more potent mabs, belonging to the third group, featured an ic 50 of 6-7 µg/ml. two years later, the same library was screened against nccg-gp41 and 6-hb antigens [96] . two clones, fabs 3663 and 3670, inhibited cell fusion while one fab 3674 clone selected against nccg-gp41 was also effective in infection neutralization assays. fab 3674 bound the 6-hb as well as stable nhr trimers, and recognized an epitope that partially overlapped the hydrophobic pocket targeted by the d5 ab. the same authors subsequently demonstrated that the n36 mut(e,g) peptide presenting mutations within the 5 th and 7 th aa residues of the heptad repeat [158] increased the temporal window of viral sensitivity to fab 3674 and thereby synergistically enhanced the neutralizing activity of fab 3674 as well as of the bntabs 2f5 and 4e10 [98] . a fab sublibrary was created by affinity maturation of the fab 3674 hcdr2 loop and screened against nccg-gp41, selecting for three fabs (fabs 8060, 8066 and 8068) with enhanced potency (average 5-fold decrease in ic 50 ) and neutralization breadth [97] . to follow-up a study demonstrating that affinity-purified iggs from rabbits immunized with n35 ccg n13 inhibited hiv-1-mediated fusion [156] , nelson et al. rescued a scfv antibody library from these animals [99] . three n35 ccg n13 binders were selected, and one of them, 8k8, displayed neutralizing activity against hxb2. in parallel a more complex fab library was constructed from the fda-2 hiv-1positive patient from whom z13 ab had previously been isolated [85] . screening this library against n35 ccg n13 allowed for the isolation of fab dn9 [99] . both scfv 8k8 and fab dn9 neutralized hiv-1 infection with a panel of viral strains with ic 50 ranging from 50 to 500 nm and targeted the nhr trimeric coiled-coil, presumably close to the hydrophobic pocket. three additional gp41-specific abs (m44, m46 and m48) were obtained by screening antibody phage libraries from asymptomatic seropositive patients [159] against gp140 [100] [101] [102] . a recombinant gp140 (gp140 r2) isolated from an asymptomatic seropositive patient with bntabs was reported to elicit bntabs in monkeys, further demonstrating that immunogenic epitopes were exposed on this recombinant antigen [101] . competitive antigen panning (cap) (biopanning approach designed to outcompete phagotopes binding to an immunodominant region of a multi-domain target through concomitant addition of an excess of soluble forms of this immunodominant domain) using a mixture of gp140r2 as antigen and gp120r2 as competitor resulted in the selection of a gp41-specific m46 ab [101] . m46 displayed broad neutralization properties and recognized a conformational epitope and bound weakly to 5-helix antigen but not to the trimeric nhr nor to 6-hb. in two other studies, the same libraries were panned against gp140/120 from three different isolates (89.6, cm243 and r2), which led to the identification of the m48 ab recognizing a conformational epitope of gp140 [102] and the m44 ab, which binds gp140, 5-helix and 6-hb but not to the nhr trimeric coiled-coil. m44 recognized a conserved conformational epitope and neutralized isolates from different clades with a significantly higher potency than 4e10 or z13 [100] . the competitive antigen panning approach against gp140/120 thus allows the selection of abs recognizing conformational epitopes on gp41, which are not properly folded when gp41 is used as a target. most of the studies found in the literature that apply the phage display technology to the discovery of hiv-1 inhibitors target the env protein. however, reports about the identification of peptides directed against other hiv-1 proteins involved in viral replication as well as interfering with rna sequences have been published and are summarized in this section. vpr is involved in the nuclear import of the viral preintegration complex (pic) as well as in the induction of apoptosis after cell cycle arrest and can be packaged into virions in quantities similar to the structural proteins [160] [161] [162] [163] . vpr was also reported to be associated with numerous cellular proteins such as glucocorticoid receptors, transcription factors or the uracyl dna glycosylase (udg) [160, [164] [165] [166] . to investigate whether vpr may be used as a docking protein to deliver anti-viral compounds into virions, bouhamdan et al. used a 7-mer rpl to determine a common motif involved in the interaction between vpr and its various ligands [103] . screenings rounds against vpr fusion proteins pinpointed sequences sharing a wxxf motif. since udg contains a wxxf sequence, mutants were constructed and confirmed the importance of this motif for vpr binding. cotransfection experiments indicated that the wxxf motif might be used to deliver a fusion protein into the hiv-1 virion through a new docking strategy. in 2003, krichevsky et al. conducted a study to elucidate the exact role of vpr and its contribution to the nuclear import process of the hiv-1 pic [104] . to that aim, a semi-synthetic scfv library [167] was screened against the n-terminal (aa 17-34) part of vpr (vprn) conjugated to bsa (vprn-bsa). purified scfvs fragments featuring their strong and specific binding to the vprn sequence recognized full-length vpr and inhibited vpr-mediated nuclear import, indicating that targeting vpr may lead to the development of new peptides to fight viral infection. the phage display technology has also been applied to the identification of the hiv-1 integrase inhibitors. in 2004, desjobert et al. screened a 7-mer rpl against recombinant hiv-1 integrase and identified a high affinity phagotope displaying the fhnhgkq sequence [105] . in peptide format, this sequence inhibited the strand transfer activity of in by competing with the target dna, providing the proof-of-concept that in is also a valuable target for phage display. interaction of the viral transcription activator tat with the human cyclint1 subunit of the positive transcription elongation factor (p-tefb) complex and the cooperative binding of this complex to the transactivation response element (tar) rna are prerequisites of hiv-1 transcription [168] . screening rpls or fab libraries against tat, cyclint1 or tar elements using the phage display technology identified peptides impairing tat-mediated hiv-1 replication. the first study was conducted in 1996, when pilkington et al. screened a fab library constructed from the ab repertoire of an hiv-1-infected asymptomatic patient and selected fabs recognizing a region comprised between amino acids 22 to 33 of the tat protein in a conformation-dependent manner [106] . many years later, a non-immune human scfv phage-displayed library was explored to identify peptides binding to cyclint1 [107] . clones recognizing the cyclin box domain of cyclint1 or interacting with the tat/tar recognition motif (trm) were isolated after panning against the 272 n-terminal amino acids of cyclint1. when expressed as intrabodies (antibody or antibody fragment expressed intracellularly), one of these scfvs inhibited tat-mediated transactivation without impairing cellular basal transcription or inducing apoptosis and partially inhibited hiv-1 replication in cultured cells. the tar rna sequence adopts a specific structure recognized by the basic arginine rich motif (arm) of tat and thus represents a potential target for phage display screening. in 2005, kolb and boiziau screened a 12-mer rpl against tar rna molecules and selected 12-mer and unexpectedly 57-mer sequences from the library [108] . the latter were proposed to arise from incomplete enzyme restriction during the construction of the initial library. clones were further characterized in peptide format and displayed tar-specific binding. the authors suggested that the surprisingly long peptides might have been selectively retrieved from the library because they presented a conformation that shorter 12-mer peptides were unable to adopt. the hiv-1 nucleocapsid protein p7 (ncp7) is processed from the gag precursor and is involved in the protection and encapsidation of viral rna leading to viral assembly through interaction with a specific secondary structure of the 125-base long psi rna [169] [170] [171] [172] . lener et al. screened a constrained 9-mer rpl against ncp7 and selected phagotopes sharing a ppx(d/e)r consensus motif [109] . further binding experiments suggested that the ncp7-phage interactions involved amino acids 30 to 52 of ncp7, encompassing a zinc finger domain. studies to identify inhibitors of viral packaging were also conducted. rpls were screened on the psi rna immobilized onto a streptavidin-coated surface by annealing its 5'-end to a biotinylated oligomer, leading to the selection of peptides characterized by an hwwpww motif [110] . peptide variants presenting this motif were subsequently synthesized and the most efficient binder was shown to strongly reduce virus release by infected cells, suggesting that it could serve as a lead compound to develop new anti-hiv-1 drugs [111] . a similar screening campaign was conducted, where the 5'-end of the psi rna was covalently immobilized, leaving the secondary structure intact and fully accessible [112] . screening of a 12-mer rpl selected for four clones with either whxt or hssxy motifs which were assessed for specific and dose-dependent binding to psi rna. the most prevalent sequence (syqwwwhspqtl) was expressed in fusion with the maltose-binding protein and was able to compete with ncp7 for binding to psi rna, confirming the value of the peptide as a potential hiv-1 inhibiting compound. hiv-1 accessory proteins nef and vif have an important role in hiv-1 viral replication and infectivity and, as such, represent as such interesting targets for inhibitors. in 2001, yang et al. demonstrated that vif was able to multimerize and that its 151-aalikpkqikpplp-164 domain was critical for multimerization pointing to it as an interesting target to impair vif-mediated viral replication [116] . the authors therefore, screened a 12-mer rpl library against vif and selected phages sharing a common pxp motif [117] . four of these sequences synthesized as peptides bound the c-terminus of vif with high affinity and were able to inhibit vif-vif as well as vif-hck tyrosine kinase interactions. moreover, these peptides inhibited hiv-1 replication in cultured cells. besides the identification of inhibitors from rpl, the discovery of abs targeting nef and vif applicable to intrabody-based therapy may represent an alternative way to impede viral replication [72] . in a very recent study, yoshikawa et al. evaluated the effect of two different schedules of nef and vif administration for mice immunization prior to the construction of scfvs phage-displayed libraries [113] . results demonstrated that the immunization protocol influenced the complexity of the elicited ab repertoire and thus the successful identification of abs specifically recognizing the target. a nanobody (sdab19) recognizing a conformational epitope and reacting with a high affinity (k d : 2 nm) with nef proteins from a panel of hiv-1 m, n, o and p groups was isolated through phage displaying the v hh repertoire of a llama immunized with a purified recombinant nef protein (fragment 57-205) [114] . when expressed as an intrabody, this anti-nef sdab inhibited important biologic functions of nef both in vitro and in vivo in cd4c/hiv-1nef transgenic mice. the first step in cytotoxic t lymphocytes (ctl) activation is the recognition by a t cell receptor (tcr) of the antigen-derived peptide/mhc class i complex (pmhc). to date, few studies were undertaken to examine antigen presentation at a cellular and molecular level. nunoya et al. screened pooled scfv libraries [173] on an immunodominant hla-a*2402(a24) restricted ctl epitope within the nef protein (nef138-10; rypltfgwcf) [115] . the panning procedure yielded clones binding specifically to nef 138-10/a24. clones scfv3 and scfv27 were able to bind to nef 138-10/a24 expressed at the cell surface and retained this specificity when expressed as reconstituted whole iggs. this recent study was the first to address the identification of monoclonal antibodies binding specifically to an immunodominant hiv-1 ctl epitope loaded on an hla class i molecule. reverse transcriptase is a valuable target for anti-hiv-1 compounds, as illustrated by the success of the multiple small compounds used in haart. in 1996, gargano et al. panned a phage-displayed library of synthetic combinatorial human fab fragments against recombinant hiv-1 rt [174] . two ab fragments that specifically inhibited the rna-dependent dna polymerase (rddp) activity of rt were identified. both fragments also inhibited the activities of avian and murine retroviral rts as well as the human dna polymerase α and prokaryotic dna polymerases. because of their lack of specificity, these abs fragments were not exploited further as anti-hiv-1 molecules. to develop a panel of recombinant mabs reacting with different epitopes of the rt, ohba et al. immunized mice with recombinant rt expressed in a vaccinia virus vector and constructed a phage-displayed mice fab fragments library [118] . biopanning against recombinant rt led to the identification of two fab fragments (5f and 5g) able to strongly inhibit the rddp activity of hiv-1 rt. epitope mapping and competitive elisa showed that 5f and 5g recognized an epitope similar or closely related to the epitope targeted by the mouse mab (7c4) previously described by the same authors [119] . two years later, a semisynthetic phage display library of human scfvs with randomized heavy and light chain cdr3 was screened against recombinant rt [120] . five different scfv abs directed against rt were isolated, of which three (f-6, 6e9, 5b11) inhibited the rddp activity of rt; of note, (f-6) also inhibited rt dna-dependent dna polymerase (dddp) activity. synthesis of the peptides corresponding to the cdr3 regions of the heavy and light chains showed that the heavy chain cdr3 inhibited rddp activity while the light chain peptide had no effect. these hcdr3 peptides represent the smallest antibody fragments inhibiting the rt identified to date and demonstrated that hcdr3 repertoire is a potential source of bioactive molecules (see section 3.2.1.2.). rev is a key regulatory protein. oligomerized rev binds to unspliced or singly spliced viral mrna and ensures its transport to the cytoplasm, thereby allowing the translation of viral gene products. despite considerable efforts, the structure of rev is poorly characterized since rev is refractory to crystallization, mainly because of its tendency to form insoluble aggregates [175] . in the absence of structural information, the phage display technology was used by different authors to map the domains involved in the interaction of rev with its network of partners. pilkington et al. identified two rev-specific fabs from a fab library derived from the ab repertoire of an hiv-1-infected asymptomatic patient [106] . these fabs were directed against sites adjacent to the rev basic nuclear localization signal (nls) (residues 52-64) and to the activation domain (residues 75-88). two years later, jensen et al. screened a 15-mer rpl to identify potential rev peptidic antagonists [121] . three groups of sequences sharing a srlxg(x) 2-3 r motif (group i), sharing a rvv(x) 2-4 rg/a motif (group ii) or featuring no sequence similarity (group iii), were obtained. three clones were selected based on their high frequency of occurrence (p1 and p3, group i) or on their strong binding affinity for rev (p19, group iii). they were synthesized as peptides and were shown to retain rev binding specificity. more recently, llama nanobody libraries from animals immunized with recombinant rev allowed the identification of 12 rev-binding nanobodies [123] . one of them (nb190) prevented or disrupted rev multimerization by interacting with lys20 and tyr23 of the rev n-terminal α-helix [122] . besides inhibitor discovery, fabs were recently proposed as -crystal chaperones‖ to support crystallization of their partners by locking them in specific conformations and blocking aggregation [124] . stahl et al. described the preparation, characterization, and crystallization of an equimolar complex formed between rev and a chimeric rabbit/human fab (sjs-r1) selected through phage display [124] . the rev/sjs-r1 fab complex was successfully crystallized and the fab sjs-r1 was shown to recognize a conformational epitope in the n-terminal half of rev. structural characterization of the crystallized fab/rev complex is ongoing and a corresponding scfv has been engineered and may have anti-hiv-1 properties. to identify peptides interfering with hiv-1 capsid assembly, sticht et al. screened a 12-mer rpl against the capsid (ca) protein generated by the proteolysis of the gag precursor and identified phagotopes whose sequences could be classified in four groups [125] . one of these sequences (cai, capsid assembly inhibitor) competed with phagotopes for binding to ca and inhibiting capsid assembly in vitro. interaction with cai was mapped to ca amino acids , with additional contacts in helix 4. cai did not inhibit capsid assembly in vivo, but may nevertheless serve as tool for drug screening and as a starting point for drug design based on its ca-binding properties. peptides and antibody fragments selected by means of phage display may also be used for diagnostic purposes or to assess the diversity of the immune response against hiv-1-specific antigens. de haard et al. constructed a scfv library from pbls of an hiv-1 positive patient presenting antibodies against gp120, gp41 and p24 and screened the library against gp160 and p24 [176] . one phagotope recognizing an epitope within the 2f5 and 4e10 bntabs epitopes on gp41 (ab#31) with affinities in the nanomolar range was isolated. importantly, it was shown to compete with 41 out of 42 gp160-reactive plasma samples from north-american and african hiv-1 positive patients, indicating that this antibody recognizes an epitope conserved in a large panel of isolates and might be suitable for diagnostic applications. since all vaccine candidates elicited antibodies reacting positively in hiv tests, khurana et al. applied the phage display technology to identify hiv-1 epitopes susceptible to help discriminate between successfully immunized vaccinees and seroconverters [177] . they constructed a phage library displaying the full hiv-1 genome and screened it against the sera of newly seroconverted hiv-1 positive individuals. they identified conserved epitopes present in gp41 and in gag p6 that were not part of the vaccine used at that moment and established a new detection test, named selectest, that demonstrated over 99% selectivity and sensitivity for the early detection of seroconversion. hiv selectest was able to detect antibodies against these epitopes in newly infected patients as early as 2 to 4 weeks after infection. in parallel to the targeting of viral proteins, many efforts were undertaken to identify peptides, antibody fragments or modified ligands binding to the hiv-1 host proteins and impairing their interactions with the viral proteins (table 5 ). the hiv-1 host receptors cd4, ccr5 and cxcr4 are involved in the early steps of hiv-1 infection and thus represent valuable targets for the identification of antiviral peptides or neutralizing abs. moreover, these receptors display very low variability compared to the viral env proteins facilitating the identification of neutralizing antibodies. although the cd4 receptor plays a crucial role in the entry process, the only phage display biopanning assays reported to date targeted the chemokine receptors ccr5 and cxcr4. however, to circumvent the difficulties of purifying and immobilizing such complex receptors on a solid support without losing their native structure, biopanning procedures had to be adapted. in this regard, screening strategies using biopanning on living cells [178, 179] , proteoliposomes [180] or peptides derived from the receptors extracellular parts [181, 182] were particularly successful. the cc chemokine receptor 5 (ccr5) is one of the two major hiv-1 coreceptors and binds three different endogenous chemokines ccl5 (rantes), ccl4 (mip-1β) and ccl3 (mip-1α) which were reported to prevent r5-tropic hiv-1 entry. interestingly, inhibition of ccr5 binding to hiv-1 provides an almost complete protection against r5-tropic viruses with only minor effects on the normal physiological functions of the cells [183] . the first biopanning experiment targeting ccr5 was performed using receptor embedded in paramagnetic proteoliposomes [180] . to create such proteoliposomes, magnetic beads were added to a mixture of synthetic lipids, a detergent-solubilized c9-tagged ccr5 receptor and a capture antibody, reconstituting membrane bilayers containing pure, native and properly oriented ccr5 receptor. these proteoliposomes were used in biopanning experiments with a human scfv antibody library and several antibody fragments specifically binding to ccr5-expressing cells were identified. the same year, steinberger et al. used the phage display technology to select and to humanize rabbit anti-ccr5 antibodies preventing the export of ccr5 to the cell surface [184] . following rabbit immunization with a gst-nterm ccr5 fusion protein, the authors constructed a phage displayed fab library that was screened against the antigen initially used for the immunization. a phagotope (st6) binding strongly and specifically to the immobilized antigen as well as to ccr5-positive cells was identified, expressed as a scfv and humanized by successive replacements of the rabbit light and heavy chains by their human counterparts. one humanized antibody fragment, st6/34, that retained the strong ccr5-binding capacity of the parental st6 antibody was isolated from the screening of the intermediate libraries. when expressed as intrabody the st6/34 scfv efficiently blocked the ccr5 expression at the cell surface. [185] (reviewed in chevigné et al. [191] . in this strategy, a phage library displaying randomly mutated and n-terminally extended ccl5 chemokine variants (xs#xssx###-ccl5, where # represents either a, p, s or t) was constructed and screened on ccr5-expressing cells. only intracellular phagotopes that had induced ccr5 receptor internalization were recovered. two ccl5 variants (p1 = lspvssqssa-ccl5 and p2 = fsplssqssa-ccl5) were identified. these variants displayed a higher selectivity for ccr5 and had more potent hiv-1 inhibitory abilities than the wild-type ccl5 chemokine. further characterization demonstrated that p2 acted as a ccr5 superagonist and potently induced intracellular ccr5 sequestration. p1 was less potent but significantly reduced ccr5-dependent intracellular calcium signaling. in a subsequent study, p1 and p2 variants were optimized by phage display biopanning to select for variants that retained the high anti hiv-1 potency of p1 and p2 but reduced ccr5-agonist activity [186] . three successive generations of libraries (xxpx 3 q#tp-ccl5, qgpplmx 4 -ccl5 and qgpψ$x 5 -ccl5 where ψ represents g, l or p and $ represents g, l or m) were constructed and screened as previously described [185] . the three most interesting candidates (5p12-ccl5, 5p14-ccl5 and 6p4-ccl5) produced as soluble proteins displayed highly potent antiviral activities. analogue 6p4-ccl5 acted as an agonist and sequestered ccr5, 5p12-ccl5 induced no signaling or receptor sequestration while 5p14-ccl5 induced ccr5 internalization without triggering g-protein signaling. altogether these data demonstrated that antiviral activities of similar molecules identified through phage display screening can rely on various mechanisms of action. shortly after the identification of the p1 and p2 variants, zhang et al. reported an alternative biopanning methodology relying on the use of small cyclic biotinylated peptides mimicking ccr5 extracellular loops (ecl1, ecl2 and ecl3) for the identification of ccr5-binding scfvs [181] . a mouse phage-displayed scfv library was incubated with biotinylated-ecl peptides and ecl-binding phages were specifically recovered using streptavidin-coated beads. among the ccr5-binding scfv identified, three clones (a1, b7 and l9) selected on cyclic ecl1 or ecl2 peptides inhibited r5-tropic hiv-1. screening of rpl was also applied to the discovery of small ccr5-blocking peptides through the targeting of receptor-expressing cells [178, 179] . vyroubalova et al. screened a partially randomized 10-mer phage library (cdx 3 kpcallryx 10 -piii) using competitive elution with a ccl5 analogue (nny-ccl5, 100 nm) and selected a unique peptide (allrynpfyylsfsp). this peptide was further optimized through n-terminal extension, exon shuffling and biopanning. by applying successive treatments consisting of a preselection using a low amount of nny-ccl5 (100 pm) to discard low-affinity binding phages followed by classical alkaline elution using tea and a competitive elution containing nny-ccl5 (1 µm) they identified an extended peptide (lldstfftadallrynpfyyls-fsp) inhibiting r5-tropic hiv-1 cell fusion with an ic 50 of 5 μm. in parallel, wang et al. screened a fully randomized 12-mer rpl using acidic elution and identified phagotopes binding specifically to ccr5-expressing cells and sharing the afdwtfvpslil sequence [178] . in peptide and phage formats this sequence blocked the binding of the anti-ccr5 neutralizing 2d7 mab and completely inhibited binding of the chemokine ccl5 to the receptor [179] . the cxc chemokine receptor 4 (cxcr4) is the second major hiv-1 coreceptor. cxcr4 binds only to one endogenous chemokine ligand (cxcl12) and is also expressed at the surface of numerous cancer cell types underlining its high value as therapeutic target [192, 193] . despite this importance and the relative success of phage biopanning on ccr5, only two recent studies reported the use of phage display to search cxcr4 inhibitors [182, 187] . in 2010, jahnichen et al. isolated llama-derived v hh binding specifically to cxcr4 and inhibiting the entry of x4-tropic virus [187] . to select v hh binding exclusively to functional and properly folded receptor, llamas were immunized with cxcr4-expressing hek293t cells. a phage library was subsequently constructed from the pbmcs of immunized camelids and several phage clones inhibiting the binding of labeled cxcl12 chemokine to the receptor were identified. in particular, two v hhs (238d2 and 238d4) showed low nanomolar affinity for the receptor and inhibited entry of x4 and x4/r5-viruses into different cxcr4 + cell types with ic 50 values ranging from 10 to 100 nm. dimerization of 238d2 and 238d4 to form biparatopic proteins increased their antiviral properties to ic 50 values in the picomolar range. epitope mapping revealed that the two v hh s inhibited cxcr4 mainly through binding to the second extracellular loop. very recently, we used a peptide corresponding to this particular extracellular loop (ecl2) as target to identify short cxcr4 antagonists [182] . by screening a non-immune phage library displaying the human hcdr3 peptide repertoire [194] , several small peptides binding to the ecl2 peptide that specifically recognized cxcr4-expressing cells were identified. notably, one of these hcdr3 peptides (typgry) acted as a cxcr4 antagonist with potency in the micromolar range. in addition to targeting host receptors, a few studies reported the identification of peptides or antibody fragments directed against other host proteins such as cell surface determinants (cd) or intracellular enzymes [188, 190, 195, 196] . the cd40 receptor, a member of the tumor necrosis factor receptor superfamily, and its cd40l ligand are involved in several biological processes including cell proliferation, activation and production of cytokines and chemokines. during hiv-1 infection, viruses were proposed to selectively downregulate or even deplete the pool of cd40l-expressing cd4+ t cells. in this context, antibodies binding to cd40 and restoring the hiv-1-induced cd40l downregulation might be of interest. in 2002, ellmark et al. identified a set of anti-cd40 antibody fragments through biopanning of a human scfv phage library against a biotinylated cd40 antigen [189] . when expressed as full-length igg1 one antibody (clone b44) suppressed hiv-1 infection by a r5-tropic virus, most probably through induction of cc chemokine production [188] . more recently, the ddx3 protein, a cellular rna helicase involved in rna unwinding was shown to play important roles in hiv-1 replication. this protein presents a unique region (alramkeng) responsible for high affinity binding to the hiv-1 rna. garbelli et al. carried out a biopanning experiment with a mix of linear 12-mer and linear and constrained 7-mer rpl against a peptide mimicking the ddx3 region interacting with hiv-1. they identified a 7-mer peptide (sdvptqv) blocking the replication of an x4-tropic virus with an ic 50 of 20 μm [190] . besides therapeutic purposes, the phage display technology has also been applied for fundamental studies of host proteins. in particular, a study focusing on the roles and the diversity of the anti-cd34 autoimmune repertoire in the myelosuppression appearing in hiv-1 infected individuals was reported by rubinstein et al. [195] . using a substractive biopanning procedure from the immune repertoire of a hiv-1 seropositive patient, the authors selected fab fragments binding specifically to the cd34 receptor. sequencing and binding analyses of these antibody fragments demonstrated the heterogeneous origin of the anti-cd34 autoimmune repertoire and suggested that these autoantibodies might be generated through antigen-specific driven processes. the use of phages for protease cleavage specificity profiling was first described by matthews and wells in 1993 [197] . this -phage substrate‖ approach relies on the use of phage particles to screen for enzyme substrates instead of classical binder selection. protease cleavage profiling using phage takes advantage of the natural resistance of phage particles to proteolysis. phage particles displaying random peptides are immobilized on a solid support and submitted to proteolytic elution to specifically liberate phages presenting peptides corresponding to the protease cleavage site. the phage substrate approach allows thus to rapidly determine the cleavage profile of a given protease and provides optimized substrate candidates which can be further used as leads for the development of specific inhibitors. over the last two decades, phage substrate has been applied to a large variety of proteases including the hiv-1 protease (pr) [198, 199] . the hiv-1 pr is a homodimeric aspartic protease responsible for nine critical cleavage steps within both the structural (gag) and the non-structural (gag/pol) polyproteins. the hiv-1 protease recognizes substrate residues encompassing p4 to p3' positions (schechter and berger's nomenclature), with the primary determinants from positions p2 to p2' positions [200, 201] . interestingly, alignment of the nine natural substrate cleavage sites of the hiv-1 pr shows a high sequence diversity suggesting a broad proteolytic specificity. in 2000, beck et al. reported the use of hexapeptide phage library to unravel the hiv-1 pr specificity and develop new protease inhibitors (table 6 ) [199] . this library was constructed by fusing the mab 3-e7 epitope upstream of the randomized sequences. phages were first incubated with the hiv-1 pr and uncleaved phages were removed by addition of pansorbin cells. biopanning selected for highly diverse sequences consistent with the suggested broad substrate specificity of the hiv-1 protease. however, none of the selected peptides corresponded to the hiv-1 polyproteins cleavage sites. nonetheless, several phage-displayed peptides were very efficiently cleaved by the hiv-1 protease. in peptide format, most of them displayed a k m value lower than the one determined for a peptide mimicking the natural substrate at the matrix/capsid junction (irkil↓fldg) ( table 6 , italic). the most potent selected substrate (gsgif↓letsl) was cleaved 60 times more efficiently and had a k m value of 5 μm i.e., 260 times lower than the natural substrate (k m = 1300 μm). interestingly, the gsgif↓letsl substrate displayed only two residues (in bold) of the optimal cleavage site model designed based on the most frequently selected residues for each position (sgvy↓fvts) ( table 7) . table 6 . phage substrate analysis of the hiv-1 protease cleavage specificity. arrow denotes cleavage sites in the natural or the selected substrates sequences. underlined substrates correspond to sequences used to derived inhibitors. italic sequences correspond to natural substrate (matrix/capsid junction) used as reference for the assessment of the cleavage efficiently. ψ: amid-reduced bound. table 7 . hiv-1 protease specificity model. the model was build based on the 11 most efficiently cleaved phage substrates. underscript numbers represent the frequency of appearance of the amino acid at a given position. sequences corresponding to the four most potent substrates were synthesized as peptidic transition state analogues and presented inhibitory activity in the nanomolar range (table 6 , underlined sequences). in 2001, the same authors applied the phage substrate approach to compare the relative specificities of the human (hiv-1) and feline (fiv) immunodeficiency virus prs [198] . hexapeptides specifically processed by each of the two proteases as well as peptides cleaved by both enzymes were identified. further mutational analysis of synthetic peptides derived from a sequence processed with the same efficiency by the two proteases (ksgvf↓vvng) was performed to assess the influence of amino acid substitutions on the catalytic process of each pr (table 6 , underlined sequence). they showed that substitutions for a val at position p2 or p2' increased the cleavage by the hiv-1 protease whereas the introduction of a val at position p1 was more favorable to fiv protease activity. in particular, the gsgvfψ(ch 2 nh)vvngl inhibitor identified in the first study was active against both prs with different potencies and replacement of its amide group by a hydroxyethylene group resulted in a peptide with equivalent inhibitory activity towards both the hiv-1 and the fiv proteases. in parallel to epitope mapping, inhibitor discovery and enzyme profiling applications, bacteriophage particles were exploited as carriers for the development of anti-hiv-1 vaccines. for this particular purpose, phages are not used as affinity selection tools but rather to display antigens to the immune system, aiming to elicit specific neutralizing antibodies or/and efficient cytotoxic t-cell responses. indeed, phages can also be considered as biologically inert particles characterized by a dense and repetitive organization capable of displaying a wide range of exogenous proteins at a precise valency and in a controlled manner. moreover, phages were shown to be naturally immuno-stimulatory [202, 203] and are particularly affordable, easy and rapid to produce and to administer in many animal models [204] . altogether these characteristics make bacteriophages a suitable and valuable carrier for hiv-1 vaccine development. anti-hiv-1 vaccination trials to elicit humoral and cellular responses against various hiv-1 proteins such as the envelope proteins gp120 and gp41 as well as the rt and the p17 proteins [205] [206] [207] [208] [209] were conducted with a large diversity of bacteriophages including m13, t4, ms2 and lambda phages [205, [210] [211] [212] [213] ( table 8) . numerous phage-vaccination trials using mimotopes identified on anti-hiv-1 antibodies were reported (see tables 1-3 ). all these attempts aimed mainly at eliciting antibodies directed against the genuine epitope of an existing antibody. besides these descriptions, a few studies were exclusively dedicated to the use of phage particles as vehicles to display fragments or complete hiv-1 proteins to the immune system to prime new immune responses. the pioneer attempt was reported by minenkova et al. in 1993 [205] . immunization of rabbits with filamentous m13 phage particles displaying a small peptide (gedrw) derived from the p17 gag protein in fusion with piii minor coat protein elicited the production of specific iggs reacting with the natural p17 antigen as well as with the gag precursor protein. shortly afterwards, two studies reported that mice immunization with fd phages displaying the gp120 v3 loop fused to the piii protein induced high titers of antibodies cross-reacting with v3 loops of different strains and featuring neutralizing activity [207, 210] . the major coat protein pviii of the fd phage (2700 copies per particles) was also investigated as scaffold protein for vaccination. immunization with phages displaying a pviii-fused peptide corresponding to residues 309-317 of the hiv-1 rt resulted in the induction of a specific cytotoxic t-cell response (ctl) against the displayed peptide. priming nevertheless required a co-immunization with a t-helper epitope from the same protein (kdswtvndiqklvgk) provided by either the same or a separate phage particle [206] . more recently attempts to prime ctl response were conducted with m13 phages displaying a mixture of thousands of variants of ctl epitopes (rgpgxax 4 or xgxgxaxvxi) derived from the gp120 v3 loop (residues 311-320; rgpgrafvti) presented in an immunoglobulin v h domain scaffold [215] . mice immunization provided potent and broad epitope-specific long lasting response (12 months) and effector memory t cells were induced. moreover, recent studies demonstrated that mice immunized with these variable epitope libraries are capable of neutralizing half of the subtype b viral isolates used for challenge, including hiv-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies [218] . although they are versatile and easy to manipulate, filamentous phages present limitations for vaccination, and namely low display level and the limited size of the foreign peptides that can be incorporated without interfering with the natural functions of the phage coat proteins. therefore, alternative bacteriophage models were explored. t4 phage and its two accessory capsid-decorating proteins soc and hoc were demonstrated to be particularly efficient for high copy display of various large hiv-1 antigens [211, 214, 216] . in contrast to piii and pviii proteins, soc and hoc present the major advantage of being dispensable for phage assembly yet of being able to strongly bind to the outer surface of the capsid, if added subsequently [219] . soc and hoc self-associate to the capsid at a display level of 960 and 150 copies per capsid, a far higher density than that of filamentous minor coat protein (3 to 5 copies). hoc protein provides high-density display of single as well as multiple antigens and immunization of mice with the p24-hoc-t4 elicits strong humoral and cellular responses. moreover, fusion of different antigens to soc and hoc offer the possibility to develop multicomponent hiv-1 vaccine particles [216] t4 phage displaying v3 loop of the gp120 protein fused to soc protein was reported to elicit antibodies capable of recognizing the native antigen [214] . further study demonstrated that hoc protein can also be used to display various hiv-1 antigens including the p24, nef or a trimeric peptide derived from the c-terminus of gp41 [216] . more recently, lambda phage and its decorating capsid protein were used for dense display of glycosylated mammalian cell-derived trimers of gp140 protein [213] . lambda phage provided a display level of 30 copies of gp140 trimer per particle, a 20-fold higher display than observed on native hiv-1 virions (14 ± 7 spikes per virion) [220] . rabbit immunization trials were rather disappointing and higher antibody titers were elicited in animals receiving soluble oligomeric gp140. these results were proposed to be due to the sequential immunization process and to the strong and immunodominant response against phage capsid proteins, which is most probably boosted upon sequential immunization, and could lead to a decrease of the humoral immune response to the displayed antigen. virus like particles (vlp) derived from the ms2 bacteriophage coat protein displaying the v3 loop of gp120 and devoid of phage genome were also reported [212] . although characterized by a low display level (90 copies per vlp) these particles were nevertheless able to elicit high titers of specific antibodies when injected in mice and provided neutralizing activity at 1/10 sera dilution. more recently, pp7 phage-derived vlps displaying the v3 loop of gp120 were used to immunize mice providing high-titer antibody response [217] . aids was first described in 1981 [1] , and two years later hiv-1, its causative agent, was isolated [2] . the phage display technology was first published almost concomitantly in 1985 [12] . both protagonists lived parallel lives until 1991, when burton et al. identified a human fab recognizing the cd4-binding site of hiv-1 gp120 through phage display [41] . this first antibody, b12, turned out to be one of the rare bntabs characterized to date, and many studies are still ongoing to set up scaffolds presenting its epitope in a conformation capable of eliciting abs with b12-like hiv-1 inhibiting properties. this article was the starting point of a dense literature exploiting the different applications of the phage display technology to gain as much knowledge as possible on the hiv-1 infection process. the complex hide-and-seek game between hiv and the host immune system and the absence of an efficient vaccine candidate emphasizes the need to better comprehend the hiv-1-related immune response and to identify antiviral molecules. hiv-1 is to the best of our knowledge the only pathogen to which all four applications of the phage display technology described in this review-i.e., epitope mapping, inhibitor discovery, substrate presentation and carrier phage-have been applied. parallel improvements in the technology on one hand, and crucial discoveries in the hiv-1 field on the other hand, most likely account for such a tight interplay. the phage display technology applied to hiv-1 has provided precious knowledge concerning the regions recognized by the ab repertoire of immune patients, and allowed to identify immunodominant regions. it has also allowed to map the epitopes of numerous monoclonal antibodies directed against viral and host proteins, and, most importantly, discontinuous epitopes. alternative phage display biopanning substractive procedures such as epitope masking or competitive antigen panning also precisely highlighted non-immunodominant regions of the hiv-1 antigen, which had remained occult using classical biopanning approaches. although the antibodies identified through epitope-masking are mostly devoid of neutralizing activity, this strategy led to the identification of fabs binding with high affinity to specific targets. taking advantage of this affinity, approaches aiming at engineering bispecific antibodies, coupling these inhibitors to toxins, or multimerizing them to increase their binding avidity could lead to the development of antiviral compounds or more efficient vaccine candidates. although highly informative, immunization trials performed with mimotopes/phagotopes selected through phage display remained altogether rather disappointing as no long-lasting neutralizing response was reported, and provided yet further proof that antigenicity does not necessarily imply immunogenicity [221] . among the bottlenecks in the field of hiv-1 vaccine research and development are the weak immunogenic properties of the identified mimotopes or native antigens. whether the difficulty of eliciting and/or isolating strong or broad neutralizing antibodies from hiv-1-infected patients, be they normal progressors or ltnps, is a caveat of phage display or a real reflect of the restricted humoral response in fighting hiv, or to both, remains to be established. nevertheless, these discrepancies are not exclusive of hiv-1 mimotopes and were observed with other antigens. from a fundamental prospect, the use of phage-displayed igm libraries to model abs close to the germline abs allowed to elegantly expore the poorly characterized determinants of the initial antiviral immune response. phage display also contributed to a better knowledge on the structure of the diverse hiv-1 proteins as well as a gain of insight on non-structural proteins involved in replication mechanisms. a highlight example would be the phage substrate approach which allowed the precise characterization of the hiv-1 protease cleavage specificity and thereby provided valuable inhibitor candidates. unraveling viral replication steps or protein interactions led to exploit phage display to engineer various types of inhibitors targeting either viral or host proteins. in parallel, phage display allowed the identification of various types of inhibitors targeting either viral or host proteins. nevertheless, only a few strong antiviral candidates with broad neutralizing activities and/or potencies in the nanomolar or even picomolar range were identified with the phage display technology while most of them presented inhibiting activities that ranged at the best in the micromolar range and could not be further exploited. some of the most potent inhibitors originated from fab libraries derived from asymptomatic hiv-1-infected patients whose reactivity against hiv-1 was previously assessed (b12, x5, z13) or from immunized camelids (v hh anti cxcr4). other inhibitors were identified from semi-synthetic (ligand analogues of ccl5) or randomized peptide libraries (d-peptides: pie12 3 ) and their affinity was improved through secondary libraries. these inhibitors were selected against different targets (env proteins, coreceptors) by biopanning carried out using different types of support (immobilized proteins, cells, peptides), illustrating the power and versatility of the phage display technology [41, 83, 85] (figure 5 ). inhibitors blocking key steps in the entry process were identified using the phage display technology. these inhibitors target: the cd4 binding site (fab b12 and z13), the coreceptors ccr5 (ccl5 variants) or cxcr4 (v hh 238d2 and 238d4), the cd4-induced epitope of gp120 (fab x5) or the heptad repeat region of gp41 (peptide pie12 3 ). despite the variable success of the phage display technology per se in developing a direct antiviral agent or immune response, this technology continues to contribute precious and otherwise inaccessible developments in the field of hiv-1 research, mainly owing to the ability of phages to display a high density of either single antigens or libraries of antigens/mimotopes. to this regard, phages are being extensively explored to increase immune responsiveness. the latest developments include exploiting the phage as dna vaccine carrier or hybrid phage and pursuing the symbiotic interplay between phage display and hiv-1. pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) running a tightrope: regulatory challenges in the development of antiretrovirals potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry maraviroc (uk-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor ccr5 with broad-spectrum anti-human immunodeficiency virus type 1 activity 5-dihydroxypyrimidine carboxamides and n-alkyl-5-hydroxypyrimidinone carboxamides are potent, selective hiv integrase inhibitors with good pharmacokinetic profiles in preclinical species plasma hiv rna decline and emergence of drug resistance mutations among patients with multiple virologic failures receiving resistance testing-guided haart an overview of the determinants of ccr5 and cxcr4 co-receptor function the human immunodeficiency virus type 1 envelope spike of primary viruses can suppress antibody access to variable regions continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers the antibody response against hiv-1. cold spring harb. perspect filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface lambda-display: a powerful tool for antigen discovery antibody-selectable filamentous fd phage vectors: affinity purification of target genes human immunodeficiency virus type 1 long-term non-progressors: the viral, genetic and immunological basis for disease non-progression production of site-selected neutralizing human monoclonal antibodies against the third variable domain of the human immunodeficiency virus type 1 envelope glycoprotein identification of hiv vaccine candidate peptides by screening random phage epitope libraries anti-human immunodeficiency virus type 1 human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries defining critical residues in the epitope for a hiv-neutralizing monoclonal antibody using phage display and peptide array technologies a human monoclonal antibody to a complex epitope in the v3 region of gp120 of human immunodeficiency virus type 1 has broad reactivity within and outside clade b epitope mapping of anti-hiv and anti-hcv monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library permissive residues within the minimal epitopes of neutralizing monoclonal antibodies to the v3 loop of hiv-1 neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by iam-41-2f5, an anti-gp41 human monoclonal antibody human immunodeficiency virus type 1-neutralizing monoclonal antibody 2f5 is multispecific for sequences flanking the dkw core epitope constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-hiv-1 mab 2f5 helical epitopes determined by low-stringency antibody screening of a combinatorial peptide library phage-displayed mimotopes recognizing a biologically active anti-hiv-1 gp120 murine monoclonal antibody identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12 probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage immunogenicity of hiv type 1 gp120 cd4 binding site phage mimotopes the mapping and reconstitution of a conformational discontinuous b-cell epitope of hiv-1 peptides selected from a phage display library with an hiv-neutralizing antibody elicit antibodies to hiv gp120 in rabbits, but not to the same epitope structural and functional characterization of an epitope in the conserved c-terminal region of hiv-1 gp120 a peptide inhibitor of hiv-1 neutralizing antibody 2g12 is not a structural mimic of the natural carbohydrate epitope on gp120 antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gp120 principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein human monoclonal antibody that recognizes the v3 region of human immunodeficiency virus gp120 and neutralizes the human t-lymphotropic virus type iiimn strain a conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1 humoral immune response to immunocomplexed hiv envelope glycoprotein 120 a large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals recombinant human fab fragments neutralize human type 1 immunodeficiency virus in vitro computational prediction of the cross-reactive neutralizing epitope corresponding to the [corrected] monclonal [corrected] antibody b12 specific for hiv-1 gp120 a conserved hiv gp120 glycoprotein structure involved in chemokine receptor binding exploration of antigenic variation in gp120 from clades a through f of human immunodeficiency virus type 1 by using monoclonal antibodies characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-cd4 binding the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2g12 recognizes a cluster of α1→2 mannose residues on the outer face of gp120 human monoclonal antibody 2g12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1 the mannose-dependent epitope for neutralizing antibody 2g12 on human immunodeficiency virus type 1 glycoprotein gp120 selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv-1-positive sera protection of rhesus macaques against disease progression from pathogenic shiv-89.6pd by vaccination with phage-displayed hiv-1 epitopes dissection of the humoral immune response toward an immunodominant epitope of hiv: a model for the analysis of antibody diversity in hiv+ individuals collection of phage-peptide probes for hiv-1 immunodominant loop-epitope mimotopes selected with antibodies from hiv-1-neutralizing long-term non-progressor plasma inducing cross-clade neutralizing antibodies against hiv-1 by immunofocusing evolution of antibody landscape and viral envelope escape in an hiv-1 crf02_ag infected patient with 4e10-like antibodies unravelling the antigenic landscape of the hiv-1 subtype a envelope of an individual with broad cross-neutralizing antibodies using phage display peptide libraries mapping of hiv-1 gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system the protein data bank u. 3d-epitope-explorer (3dex): localization of conformational epitopes within three-dimensional structures of proteins molecularly cloned shiv-1157ipd3n4: a highly replication-competent, mucosally transmissible r5 simian-human immunodeficiency virus encoding hiv clade c env requirement of multiple phage displayed peptide libraries for optimal mapping of a conformational antibody epitope on ccr5 monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor ccr5: implications for the tertiary structure of the receptor and for an n-terminal binding site for hiv-1 gp120 inhibition of m-tropic hiv-1 infection by the fd phage-gene 3 protein with mip-1α-binding activity identification of a linear peptide recognized by monoclonal antibody 2d7 capable of generating ccr5-specific antibodies with human immunodeficiency virus-neutralizing activity identification of a lfa-1 region involved in the hiv-1-induced syncytia formation through phage-display technology interaction of chemokine receptor ccr5 with its ligands: multiple domains for hiv-1 gp120 binding and a single domain for chemokine binding ccr5 levels and expression pattern correlate with infectability by macrophage-tropic hiv-1, in vitro human immunodeficiency virus (hiv) infection in cd4+ t lymphocytes genetically deficient in lfa-1: lfa-1 is required for hiv-mediated cell fusion but not for viral transmission molecular profile of an antibody response to hiv-1 as probed by combinatorial libraries specific killing of hiv-infected lymphocytes by a recombinant immunotoxin directed against the hiv-1 envelope glycoprotein cdr walking mutagenesis for the affinity maturation of a potent human anti-hiv-1 antibody into the picomolar range in vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity neutralizing recombinant human antibodies to a conformational v2-and cd4-binding site-sensitive epitope of hiv-1 gp120 isolated by using an epitope-masking procedure antibodies to the superantigenic site of hiv-1 gp120: hydrolytic and binding activities of the light chain subunit cross-clade hiv-1 neutralization by an antibody fragment from a lupus phage display library generation of a family-specific phage library of llama single chain antibody fragments that neutralize hiv-1 llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (hiv-1)-neutralizing properties and high affinity for hiv-1 gp120 jones, i. mutant cd4 molecules with improved binding to hiv envelope protein gp120 selected by phage display affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity mapping the protein surface of human immunodeficiency virus type 1 gp120 using human monoclonal antibodies from phage display libraries peptide ligands to human immunodeficiency virus type 1 gp120 identified from phage display libraries broadly cross-reactive hiv-1-neutralizing human monoclonal fab selected for binding to gp120-cd4-ccr5 complexes peptide ligands selected with cd4-induced epitopes on native dualtropic hiv-1 envelope proteins mimic extracellular coreceptor domains and bind to hiv-1 gp120 independently of coreceptor usage broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41 an affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2f5 and 4e10 potent d-peptide inhibitors of hiv-1 entry design of a potent d-peptide hiv-1 entry inhibitor with a strong barrier to resistance inhibiting hiv-1 entry: discovery of d-peptide inhibitors that target the gp41 coiled-coil pocket a human monoclonal antibody neutralizes diverse hiv-1 isolates by binding a critical gp41 epitope design of potent inhibitors of hiv-1 entry from the gp41 n-peptide region synthetic fab fragments that bind the hiv-1 gp41 heptad repeat regions identification of the hiv-1 gp41 core-binding motif-hxxnpf the mechanism by which molecules containing the hiv gp41 core-binding motif hxxnpf inhibit hiv-1 envelope glycoprotein-mediated syncytium formation characterization and hiv-1 fusion inhibitory properties of monoclonal fabs obtained from a human non-immune phage library selected against diverse epitopes of the ectodomain of hiv-1 gp41 a monoclonal fab derived from a human nonimmune phage library reveals a new epitope on gp41 and neutralizes diverse human immunodeficiency virus type 1 strains affinity maturation by targeted diversification of the cdr-h2 loop of a monoclonal fab derived from a synthetic naive human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of fabs with improved hiv-1 neutralization potency and breadth sequestering of the prehairpin intermediate of gp41 by peptide n36mut(e,g) potentiates the human immunodeficiency virus type 1 neutralizing activity of monoclonal antibodies directed against the n-terminal helical repeat of gp41 antibody elicited against the gp41 n-heptad repeat (nhr) coiled-coil can neutralize hiv-1 with modest potency but non-neutralizing antibodies also bind to nhr mimetics cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens cross-reactive hiv-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (r2) with broadly hiv-1 neutralizing antibodies selection of a novel gp41-specific hiv-1 neutralizing human antibody by competitive antigen panning diversity of hiv-1 vpr interactions involves usage of the wxxf motif of host cell proteins antibody fragments selected by phage display against the nuclear localization signal of the hiv-1 vpr protein inhibit nuclear import in permeabilized and intact cultured cells identification by phage display selection of a short peptide able to inhibit only the strand transfer reaction catalyzed by human immunodeficiency virus type 1 integrase recombinant human fab antibody fragments to hiv-1 rev and tat regulatory proteins: direct selection from a combinatorial phage display library inhibition of tat-mediated transactivation and hiv-1 replication by human anti-hcyclint1 intrabodies selection by phage display of peptides targeting the hiv-1 tar element use of a constrain phage displayed-peptide library for the isolation of peptides binding to hiv-1 nucleocapsid protein (ncp7) identification of peptide ligands for target rna structures derived from the hiv-1 packaging signal ψ by screening phage-displayed peptide libraries inhibition of hiv-1 by a peptide ligand of the genomic rna packaging signal ψ selection and characterization of peptides specifically binding to hiv-1 ψ (ψ) rna modifying the antigen-immunization schedule improves the variety of monoclonal antibodies obtained from immune-phage antibody libraries against hiv-1 nef and vif inhibition of the nef regulatory protein of hiv-1 by a single-domain antibody short communication: generation of recombinant monoclonal antibodies against an immunodominant hla-a*2402-restricted hiv type 1 ctl epitope the multimerization of human immunodeficiency virus type i vif protein: a requirement for vif function in the viral life cycle potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (hiv-1) vif proteins an immunodominant neutralization epitope on the -thumb‖ subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by phage display antibodies a novel neutralization epitope on the -thumb‖ subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by a monoclonal antibody recombinant human antibodies against the reverse transcriptase of human immunodeficiency virus type-1 hiv-1 rev nuclear export signal binding peptides isolated by phage display measuring cooperative rev protein-protein interactions on rev responsive rna by fluorescence resonance energy transfer an intrabody based on a llama single-domain antibody targeting the n-terminal α-helical multimerization domain of hiv-1 rev prevents viral production generation and characterization of a chimeric rabbit/human fab for co-crystallization of hiv-1 rev a peptide inhibitor of hiv-1 assembly in vitro potential drug targets on the hiv-1 envelope glycoproteins, gp120 and gp41 small-molecule hiv-1 gp120 inhibitors to prevent hiv-1 entry: an emerging opportunity for drug development molecular characterization of the circulating anti-hiv-1 gp120-specific b cell repertoire using antibody phage display libraries generated from pre-selected hiv-1 gp120 binding pbls relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design binding of glycoprotein 120 and peptides from the hiv-1 envelope by autoantibodies in mice with experimentally induced systemic lupus erythematosus and in patients with the disease prospects for immunotherapeutic proteolytic antibodies phosphonate ester probes for proteolytic antibodies naturally occurring proteolytic antibodies: selective immunoglobulin m-catalyzed hydrolysis of hiv gp120 theory of proteolytic antibody occurrence site-directed mutagenesis of proteolytic antibody light chain naturally occurring antibodies devoid of light chains nanobodies(r): new ammunition to battle viruses mode of action for linear peptide inhibitors of hiv-1 gp120 interactions cutting edge: cd4 is the receptor for the tick saliva immunosuppressor, salp15 the ixodes scapularis salivary protein, salp15, prevents the association of hiv-1 gp120 and cd4 natural human antibodies retrieved by phage display libraries from healthy donors: polyreactivity and recognition of human immunodeficiency virus type 1gp120 epitopes a vh clonal deficit in human immunodeficiency virus-positive individuals reflects a b-cell maturational arrest selective variations in vivo of vh3 and vh1 gene family expression in peripheral b cell igm, igd and igg during hiv infection molecular analysis of hiv-1 gp120 antibody response using isotype igm and igg phage display libraries from a long-term non-progressor hiv-1-infected individual improvement in affinity and hiv-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by hiv-1 infection cross-reactive human igm-derived monoclonal antibodies that bind to hiv-1 envelope glycoproteins characterization of germline antibody libraries from human umbilical cord blood and selection of monoclonal antibodies to viral envelope glycoproteins: implications for mechanisms of immune evasion and design of vaccine immunogens cross-reactive hiv-1-neutralizing human monoclonal antibodies identified from a patient with 2f5-like antibodies enfuvirtide: the first therapy to inhibit the entry of hiv-1 into host cd4 lymphocytes identification of d-peptide ligands through mirror-image phage display protein design of an hiv-1 entry inhibitor molecular recognition by a binary code structural basis for hiv-1 neutralization by a gp41 fusion intermediate-directed antibody subdomain folding and biological activity of the core structure from human immunodeficiency virus type 1 gp41: implications for viral membrane fusion fully synthetic human combinatorial antibody libraries (hucal) based on modular consensus frameworks and cdrs randomized with trinucleotides covalent trimers of the internal n-terminal trimeric coiled-coil of gp41 and antibodies directed against them are potent inhibitors of hiv envelope-mediated cell fusion design and properties of n(ccg)-gp41, a chimeric gp41 molecule with nanomolar hiv fusion inhibitory activity design of a novel peptide inhibitor of hiv fusion that disrupts the internal trimeric coiled-coil of gp41 neutralizing and infection-enhancing antibody responses to human immunodeficiency virus type 1 in long-term nonprogressors the human immunodeficiency virus type 1 vpr transactivator: cooperation with promoter-bound activator domains and binding to tfiib human immunodeficiency virus type 1 vpr induces apoptosis following cell cycle arrest human immunodeficiency virus vpr product is a virion-associated regulatory protein identification of hiv-1 vpr product and function human immunodeficiency virus type 1 vpr protein binds to the uracil dna glycosylase dna repair enzyme the glucocorticoid receptor type ii complex is a target of the hiv-1 vpr gene product interaction of virion protein vpr of human immunodeficiency virus type 1 with cellular transcription factor sp1 and trans-activation of viral long terminal repeat antibody fragments from a -single pot‖ phage display library as immunochemical reagents hiv-1 tat interacts with cyclin t1 to direct the p-tefb ctd kinase complex to tar rna a mimic of hiv-1 nucleocapsid protein impairs reverse transcription and displays antiviral activity evidence of interactions between the nucleocapsid protein ncp7 and the reverse transcriptase of hiv-1 hiv-1: fifteen proteins and an rna basic residues in human immunodeficiency virus type 1 nucleocapsid promote virion assembly via interaction with rna human fc epsilon rialpha-specific human single-chain fv (scfv) antibody with antagonistic activity toward ige/fc epsilon rialpha-binding human recombinant antibody fragments neutralizing human immunodeficiency virus type 1 reverse transcriptase provide an experimental basis for the structural classification of the dna polymerase family human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich -bubble‖ located within the rev-responsive region of viral mrnas selection of human anti-human immunodeficiency virus type 1 envelope single-chain antibodies from a peripheral blood cell-based phage repertoire human immunodeficiency virus (hiv) vaccine trials: a novel assay for differential diagnosis of hiv infections in the face of vaccine-generated antibodies identification of peptide ligands to the chemokine receptor ccr5 and their maturation by gene shuffling selection of cc chemokine receptor 5-binding peptide from a phage display peptide library paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, ccr5 selection of active scfv to g-protein-coupled receptor ccr5 using surface antigen-mimicking peptides selection of a cxcr4 antagonist from a human heavy chain cdr3-derived phage library the role of a mutant ccr5 allele in hiv-1 transmission and disease progression generation and characterization of a recombinant human ccr5-specific antibody. a phage display approach for rabbit antibody humanization human immunodeficiency virus type 1 entry inhibitors selected on living cells from a library of phage chemokines highly potent, fully recombinant anti-hiv chemokines: reengineering a low-cost microbicide cxcr4 nanobodies (vhh-based single variable domains) potently inhibit chemotaxis and hiv-1 replication and mobilize stem cells identification of a strongly activating human anti-cd40 antibody that suppresses hiv type 1 infection modulation of the cd40-cd40 ligand interaction using human anti-cd40 single-chain antibody fragments obtained from the n-coder phage display library a motif unique to the human dead-box protein ddx3 is important for nucleic acid binding, atp hydrolysis, rna/dna unwinding and hiv-1 replication engineering and screening the n-terminus of chemokines for drug discovery the therapeutic potential of cxcr4 antagonists in the treatment of hiv infection, cancer metastasis and rheumatoid arthritis targeting cxcr4 in hiv cell-entry inhibition non-immunized natural human heavy chain cdr3 repertoires allow the isolation of high affinity peptides mimicking a human influenza hemagglutinin epitope anti-cd34+ fabs generated against hematopoietic stem cells in hiv-derived combinatorial immunoglobulin library suggest antigen-selected autoantibodies sensitivity of hiv type 1 primary isolates to human anti-cd40 antibody-mediated suppression is related to coreceptor use substrate phage: selection of protease substrates by monovalent phage display molecular basis for the relative substrate specificity of human immunodeficiency virus type 1 and feline immunodeficiency virus proteases identification of efficiently cleaved substrates for hiv-1 protease using a phage display library and use in inhibitor development on the size of the active site in proteases. i. papain kinetic and modeling studies of s3-s3' subsites of hiv proteinases interferon production by t4 coliphage bacterial viruses as human vaccines? bacteriophage lambda is a highly stable dna vaccine delivery vehicle design of specific immunogens using filamentous phage as the carrier phage display of peptide epitopes from hiv-1 elicits strong cytolytic responses structural mimicry and enhanced immunogenicity of peptide epitopes displayed on filamentous bacteriophage. the v3 loop of hiv-1 gp120 antigenic properties of phage displayed peptides comprising disulfide-bonded loop of the immunodominant region of hiv-1 gp41 phage display of functional, full-length human and viral membrane proteins engineering a peptide epitope display system on filamentous bacteriophage phage t4 soc and hoc display of biologically active, full-length proteins on the viral capsid immunogenic display of diverse peptides on virus-like particles of rna phage ms2 dense display of hiv-1 envelope spikes on the lambda phage scaffold does not result in the generation of improved antibody responses to hiv-1 env phage display of intact domains at high copy number: a system based on soc, the small outer capsid protein of bacteriophage t4 variable epitope library-based vaccines: shooting moving targets assembly of human immunodeficiency virus (hiv) antigens on bacteriophage t4: a novel in vitro approach to construct multicomponent hiv vaccines immunogenic display of diverse peptides, including a broadly cross-type neutralizing human papillomavirus l2 epitope, on virus-like particles of the rna bacteriophage pp7 variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response the two dispensable structural proteins (soc and hoc) of the t4 phage capsid; their purification and properties, isolation and characterization of the defective mutants, and their binding with the defective heads in vitro distribution and three-dimensional structure of aids virus envelope spikes antigenic and immunogenic phage displayed mimotopes as substitute antigens: applications and limitations this manuscript is supported by the -centre de recherche public-santé‖, luxembourg (grants 20100708 gpcr47 and 20101002 eumimo). the authors are grateful to danielle perez-bercoff for thorough reading and helpful discussions. the authors also want to thank carole devaux, virginie fievez, marie-eve dumez and martyna szpakowska for critically reading the manuscript. key: cord-326558-6tss9ydx authors: chen, jiao; shang, jiayu; wang, jianrong; sun, yanni title: a binning tool to reconstruct viral haplotypes from assembled contigs date: 2019-11-04 journal: bmc bioinformatics doi: 10.1186/s12859-019-3138-1 sha: doc_id: 326558 cord_uid: 6tss9ydx background: infections by rna viruses such as influenza, hiv still pose a serious threat to human health despite extensive research on viral diseases. one challenge for producing effective prevention and treatment strategies is high intra-species genetic diversity. as different strains may have different biological properties, characterizing the genetic diversity is thus important to vaccine and drug design. next-generation sequencing technology enables comprehensive characterization of both known and novel strains and has been widely adopted for sequencing viral populations. however, genome-scale reconstruction of haplotypes is still a challenging problem. in particular, haplotype assembly programs often produce contigs rather than full genomes. as a mutation in one gene can mask the phenotypic effects of a mutation at another locus, clustering these contigs into genome-scale haplotypes is still needed. results: we developed a contig binning tool, virbin, which clusters contigs into different groups so that each group represents a haplotype. commonly used features based on sequence composition and contig coverage cannot effectively distinguish viral haplotypes because of their high sequence similarity and heterogeneous sequencing coverage for rna viruses. virbin applied prototype-based clustering to cluster regions that are more likely to contain mutations specific to a haplotype. the tool was tested on multiple simulated sequencing data with different haplotype abundance distributions and contig sizes, and also on mock quasispecies sequencing data. the benchmark results with other contig binning tools demonstrated the superior sensitivity and precision of virbin in contig binning for viral haplotype reconstruction. conclusions: in this work, we presented virbin, a new contig binning tool for distinguishing contigs from different viral haplotypes with high sequence similarity. it competes favorably with other tools on viral contig binning. the source codes are available at: https://github.com/chjiao/virbin. high genetic diversity within viral populations has been observed in patients with chronic infection with rna viruses such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), etc [1] [2] [3] [4] [5] . the genetic diversity could be caused by multiple infections of different strains or by mutations during the virus replication inside the host. in the latter case, the high replication rate, coupled with the low fidelity of the viral polymerase in most rna viruses, results in a group of different but related strains infecting the same host, which is often termed as *correspondence: yannisun@cityu.edu.hk 2 electrical engineering, city university of hong kong, hong kong, china full list of author information is available at the end of the article "quasispecies" [6] . previous studies have revealed that patients with chronic virus infections, such as acquired immune deficiency syndrome (aids), are often the reservoir of new viral variants, which are likely produced during the replication process [7] . because different strains could have very different biological properties such as virulence, transmissibility, antiviral drug resistance etc, characterizing the genetic diversity within viral populations is very important for developing effective prevention and treatment strategies. for example, if some strains have developed antiviral drug resistance, they may become the dominant strains and lead to treatment failure. thus, characterization of the strain-level diversity of viral populations is indispensable for understanding the viruses and is of great clinical importance. sequencing viral quasispecies for genetic diversity analysis was one of the first applications of nextgeneration sequencing (ngs) technologies [3] . applying whole genome shot-gun sequencing to viral quasispecies does not require cultivation and can sequence divergent strains from known virus families. it thus has become a favored choice for characterizing the diversity of quasispecies. given the sequenced viral quasispecies, different types of analysis can be conducted to probe the genetic diversity. a relatively straightforward analysis is to understand the local diversity of known viruses by performing read mapping against reference genomes. while this type of analysis can produce a collection of local changes (mutations, insertions, or deletions) of the strains inside the quasispecies, it is not sufficient to infer the biological properties of the strains, which are more likely to be determined by multiple genes. in particular, epistatic interactions are abundant in rna viruses, where the mutation of one gene masks the phenotypic effects of a mutation at another locus. thus genome-scale reconstruction of the strains is essential for phenotype prediction of viruses [8] . reconstructing the genome-scale strain sequences inside quasispecies is often referred to as genome-scale haplotype reconstruction, where the genomes of strains are called haplotypes. the goal is to assemble short reads from sequenced viral populations into correct haplotype sequences. when the reference genome is available, read mapping can be conducted first to identify local mutations and then cluster the local mutations (or short contigs) into genome-scale haplotypes. when quality reference genomes are not available, which is often the case for emerging viruses such as severe acute respiratory syndrome (sars) coronavirus, read mapping is not a very effective strategy to identify all mutations. thus, de novo assembly is needed to stitch the reads into haplotypes. with or without reference genomes, genome-scale haplotype reconstruction in quasispecies remains a computationally challenging problem. high similarity between haplotypes in the same quasispecies and the heterogeneous sequencing depth along the viral genomes present barriers to adoption of existing assembly programs. a recently published comparison showed that none of the tested haplotype reconstruction tools were able to successfully reconstruct the five known strains for a mock hiv quasispecies [9] . we had the same observations when comparing several popular metagenomic assembly tools and haplotype assembly tools such as idba-ud [10] , iva [11] , savage [12] , mlehaplo [13] on the same data set [14] . many methods output a set of contigs with various sizes that are much shorter than the genomes. with these outputted contigs from assembly programs, it still remains to infer the number of haplotypes and to match the contigs to their originating haplotypes. thus, there is a need to cluster the contigs into different groups so that each group represents a haplotype. this step is called contig scaffolding or binning and has been applied for bacterial strain characterization. contig binning for viral quasispecies has its unique challenges. first, the goal of binning is to distinguish contigs from different viral strains rather than species. thus, composition-based features such as tetranucleotide frequencies or gc contents are not informative enough to separate contigs from different haplotypes, which usually share high sequence similarity (over 90%). tools that heavily rely on sequence composition-based features will not be able to estimate the number of haplotypes correctly. second, rna virus sequencing tends to be compounded by gene expression and fast degradation and thus the observed sequencing coverage along each haplotype, or even a contig, can be more heterogeneous than expected. in addition, if a contig contains a region that is common to multiple haplotypes, that region tends to have higher coverage than a haplotype-specific segment. all these challenges require carefully designed methods to use the coverage information for contig binning. although a number of contig binning algorithms have been developed [15] [16] [17] [18] [19] [20] [21] , they all possess limitations in distinguishing contigs from different viral strains of the same species. most of the existing contig binning tools for microbiome sequencing data are designed for bacteria. these methods usually estimate the bin number by aligning metagenomic data to a pre-established marker gene database, and then assign assembled contigs to different bins using sequence composition information and read coverage levels. for example, maxbin [15] uses both tetranucleotide frequencies and contig coverage levels to assign assembled contigs into different bins. some binning tools [18] leverage co-abundance of genes across multiple metagenomic samples. the rationale is that if two contigs are from the same bin, their coverage profiles across multiple samples should be highly correlated. recently, there are a couple of newly developed tools for strain level analysis from metagenomic data, such as constrain [20] and strainphlan [21] . both rely on species identification using clade-specific genes, then zoom in to identify the strains. however, both tools were mainly tested on bacteria. our method is designed to cluster contigs produced by existing assembly tools. there are another group of methods conducting haplotype reconstruction via read clustering [22, 23] , which groups variant sites obtained by read mapping against reference genomes. these tools don't usually output contigs and thus do not use contig binning. here we present virbin, a method designed specifically for binning contigs derived from viral quasispecies data. the input to virbin is a set of contigs derived from assembly tools. the output includes the estimated number of haplotypes, the grouped contigs for each haplotype, and the corresponding relative abundances. unlike many bacterial contig binning tools that require multiple samples, our method works on a single sample. we evaluate the haplotype number estimation and clustering performance of virbin on both simulated and mock hiv quasispecies sequencing data. the simulated data provide us with known ground-truth for accurate evaluation of the clustering performance. to evaluate how the number of haplotypes affects the haplotype inference performance, we produced simulated quasispecies sequencing data consisting of 5 haplotypes and 10 haplotypes, respectively. for each experiment, we evaluate the performance of virbin from three aspects: haplotype number estimation, clustering performance, and the computed haplotype abundance. when the originating haplotypes of the input contigs are known, we can evaluate both the recall and precision for the clustering step. first, we map the clusters to haplotypes based on the consensus haplotype label of the component contigs. if there is no consensus haplotype membership (e.g. a tie), we map the clusters to haplotypes based on the ranking of the abundance. let a cluster be b and its paired haplotype be h. as the input to our program is a set of contigs, let the contig set originating from h be c h . define b ∩ c h as the common regions between the two contig sets. following other contig binning tools [15, 18] , the base-level recall for h is thus |b∩c h | |c h | , which quantified how many of the bases in c h are correctly clustered in b. the base-level precision is defined as |b∩c h | |b| , which quantifies how many of the bases in cluster b are from contig set c h . similar metrics can be defined for contig-level, which can be found in the additional file 1. first, we constructed 5 haplotypes with average sequence similarity around 93%. second, in order to simulate haplotypes of different relative abundances, we generated 3 sets of reads following different abundance distributions. third, we generated contigs using two different methods. in method 1, we simulated 5 sets of error-free contigs of different sizes directly from the reference genomes. in method 2, we applied available assembly tools to generate contigs from the reads. the simulated contigs are not dependent on any assembly tool and thus are ideal for evaluating the binning method. for simulated contigs, we have 3 (sets of reads) ×5 (sets of simulated contigs), i.e., 15 sets of input to our program. for assembled contigs, we have 3 (sets of reads) ×2 (sets of assembled contigs) as input to virbin because we applied two assembly tools. additional file 1: figure s1 sketches the process of input data generation. the data simulation details can be found below. hiv haplotype construction: there are many sequenced hiv strains in the hiv sequence database [24] . however, many of the strains do not possess sufficiently high similarity to be included in simulated quasispecies. thus, we use both real and simulated strain sequences to simulate haplotypes of high similarity. simulated strains were produced by mutating, deleting, or inserting bases at random positions from a real strain in the hiv database. as a result, the five-haplotype dataset contains a hiv-1 strain fj061 from hiv sequence database, 3 simulated haplotypes from fj061, and another hiv-1 strain fj066. the sequence similarity between the simulated haplotypes and its originating sequence is 97%. the average sequence similarity between all the five haplotypes is around 93%, which is comparable to the sequence similarity between haplotypes in a mock hiv quasispecies dataset [9] . reads simulation using different haplotype abundance distributions: with available hiv haplotypes, simulated reads were generated from them by art-illumina [25] as error-containing miseq paired-end reads, with read length of 250 bp, average insert size of 600 bp, and standard deviation of 150 bp. with the total coverage of 1000-x, three sets of reads are produced using different abundance distributions. the first one is based on the power law equation [26] . the second and the third sets of reads represent challenging cases where different haplotypes have similar abundances, which create difficulties for abundance-based binning algorithms. the abundance differences in the second and third data set are 0.06 and 0.03, respectively. in total, there are 38,914 simulated reads for 5 hiv haplotypes. the relative abundance for five haplotypes in each read set can be found in table 1 . "power" is the read sets generated based on the power law equation. "equal (6%)" is the abundance distribution with equal difference of 0.06. "equal (3%)" is the abundance distribution with equal difference of 0.03 as the total coverage is 1000-x, the sequencing coverage of each haplotype is the product of the total coverage and the relative abundance. contig simulation: for each reference genome (denote its length as l), we randomly generated a list of location pairs (p 1 , p 2 ), where 1 ≤ p 1 < p 2 ≤ l. each location pair represents a candidate contig's starting and ending position. then, in the simulated contigs, we only keep the ones above 500 bp (i.e. p 2 − p 1 + 1 ≥ 500). in addition, we would like to simulate the hard case where the contigs cannot be extended any more using large overlaps. thus, we sort all the remaining contigs by p 1 and remove the ones that have overlaps of size above 100 bp with previous contigs in the sorted list. the five sets of simulated contigs have different n50 values and are referred to as "1000" to "5000", indicating the upper bound of the contig length in each set. additional file 1: table s1 shows the detailed properties of the five sets of contigs. all the simulated data sets can be downloaded from virbin's github repository. according to our methods, the haplotype number estimation only depends on the alignment results of contigs. for all five sets of simulated contigs with different average lengths, the consensus window depth of the 50 longest windows is 5 for all. the histogram of window depth for 5 simulated contig sets is shown in fig. 1 . it is clear that window depth 5 dominates longest windows. thus, the estimated number of haplotypes is 5, reflecting the truth for our data sets. in general, the percentage of windows with depth 5 increases with increasing contig lengths. we applied virbin to cluster contigs into 5 groups. since the ground truth about the haplotype membership of each contig is known, we were able to evaluate the clustering results by calculating the precision and recall at the base level. the evaluation results are shown in fig. 2 . the performance of clustering is worst for shortest contig set (denoted as 1000 along the y-axis). with increasing contig lengths, the clustering performance becomes better for all three different abundance distributions. when the contigs are long, the clustering performance for haplotypes with different abundance distributions is comparable. the results were compared with maxbin, which is a binning tool for metagenomic contigs based on tetranucleotide frequencies and reads coverage levels. maxbin requires marker genes to identify seed contigs for binning. we were able to run the core clustering program of maxbin by inputting both the number of haplotypes (i.e. 5) and the seed contigs manually. we randomly chose one contig from each haplotype as the seed contig and calculated the contigs' abundances by mapping reads to them using bowtie2. although the haplotype number was explicitly provided to maxbin, empty clusters can be produced by maxbin. the results from maxbin are shown in fig. 3 . for the shortest contig set, maxbin only reported two clusters with one contig for each cluster, leaving 59 (96.7%) contigs unclassified. for contigs sets from 2000 -5000, maxbin was able to generate five clusters, but with~30% contigs unclassified. the results of maxbin usually have lower precision or recall values than virbini̇n addition, fig. 1 the histogram of window depth for the 50 longest windows. x-axis: the observed window depth, from 3 to 7. y-axis: the number of windows with the corresponding window depth on x-axis. "1000" to "5000" represent the five sets of contigs for contig sets from 1000 -4000, there are haplotypes without correctly assigned contigs. the lower sensitivity of maxbin could be caused by its dependency on both sequence composition and contig coverage for clustering. due to high sequence similarities between viral haplotypes, sequence composition is not informative enough in differentiating contigs from different viral strains. instead, maxbin could mistakenly cluster contigs from the homogeneous regions of the viral genome, leading to more chimeric clusters. strainphlan [21] is also able to to characterize the genetic structure of viral strains in metagenomes. it takes the raw sequencing reads and metaphlan2 [27] database of species-specific reference sequences as input and aims to output the most abundant strain for each sample. however, it failed to detect any viral species at the first step running metaphlan2. constrains [20] is another tool designed to identify strain structures from metagenomic data. it uses bowtie2 to map reads to a set of universal genes and infers the within-species strains abundances by fig. 3 the recall and precision of contig binning results by maxbin. x-axis represents each haplotype, in decreasing order of relative abundance. y-axis is the index of the 15 data sets utilizing single-nucleotide polymorphism (snp) patterns. this tool again did not get enough mapped reads to report any strain abundance. and it takes considerable efforts for us to modify their codes for our inputs. thus, we cannot report the results from strainphlan or constrains. relative abundance computation: once the iterative clustering algorithm converges, the abundance of each cluster can be computed as the weighted average abundances for all contigs from this cluster. fig. 4 compares the known haplotype abundance profiles with our computed ones. there are three read sets with different abundance distributions (table 1) . for each distribution, there are five sets of contigs (additional file 1: table s1 ). thus, three plots of five curves are presented to compare the ground truth and the computed abundance. in addition, we applied χ 2 -test to compare the ground-truth distribution and each computed abundance distribution. the p-values from all the tests are larger than 0.99, indicating that the distributions are not statistically different. as maxbin only correctly clustered several contigs, we did not include the abundance comparison. fig. 4 compare the known haplotype abundance distributions with computed ones by virbin. x-axis represents each haplotype. y-axis is the ground truth or predicted abundance for each haplotype in addition to simulated contigs, we also tested virbin on assembled contigs by two de novo assembly tools: a generic assembly tool sga [28] and a viral haplotype reconstruction tool pehaplo. the assembled contigs were evaluated by metaquast [29] and the results are listed in additional file 1: table s2 . both pehaplo and sga produced enough contigs to cover almost all the five haplotypes. contigs produced by pehaplo have larger n50 values than contigs by sga. while both tools apply overlap graph for assembly, pehaplo utilizes a pairedend information guided method for path finding, which can potentially connect some of the nodes together. therefore, it produced longer contigs than sga. the contigs are paired with haplotypes based on the highest sequence similarity. all of the contigs and their originating haplotypes have similarity of at least 98%. for all three sets of contigs assembled by pehaplo and sga on three sets of reads, the consensus window depth of the 50 longest windows is 5, revealing the actual haplotype number. figure 5a presents the clustering results on contigs generated by pehaplo and sga. it shows that virbin achieved good clustering results on contigs assembled by both assembly tools. the clustering results on sga's contigs are similar to pehaplo's contigs, with both high precision and recall. this observation is consistent with the results on simulated contigs that when the contigs are long enough, virbin can produce good results. again we compared our results with maxbin. the clustering results of maxbin on assembled contigs are shown in fig. 5b . for contigs assembled by pehaplo, maxbin correctly clustered all corresponding contigs to the strain fj061-h2 as the recall is 1.0. however, this cluster also the comparison between the predicted abundance by virbin and the ground-truth on two sets of assembled contigs is presented in additional file 1: figure s2 . we also simulated reads from 10 haplotypes and tested virbin on this data set. the data generation and also the detailed results, clustering results is presented in additional file 1: table s3 and the relative abundances for each haplotype are shown in additional file 1: figure s3 , can be found in the section 3 of additional file 1. in this experiment, we applied virbin to a mock hiv quasispecies data set (srr961514), sequenced from the mix of five hiv-1 strains (89.6, hxb2, jrcsf, nl43, yu2) with illumina miseq sequencing technology [30] . this data set contains 1,429,988 (250 bp) of reads that cover the five strains to 20,000x. the raw data set was preprocessed with faqcs/1.3 [31] and trimmomatic [32] to trim and filter low-quality reads or adapters. the remaining reads were then error-corrected with karect [33] . after pre-processing, 774,044 reads were left. by mapping pre-processed reads to the available 5 reference genomes by bowtie2, we were able to estimate the abundance for each haplotype as shown in fig. 6 . we use the contigs assembled by pehaplo as input for virbin. pehaplo produced 24 contigs from the real miseq hiv data set that can cover about 92% of the five hiv-1 strains. these contigs have a n50 value of 2223 bp and the longest contig is 9133 bp. virbin was applied to the aligned contigs for haplotype number estimation. all the windows were sorted in descending order of window length. out of the top 50 windows, 27 contain 5 contigs, 16 contain 6 contigs, and 2 contain 4 contigs. out of the top 25 windows, 17 contain 5 contigs, 5 contain 6 contigs, and 1 contains 4 contigs. similar to the simulated data, using the consensus window depth (i.e. 5) correctly predicted the haplotype number. clustering results: the clustering algorithm was applied to cluster the contigs into 5 groups. for each contig, its originating haplotype is determined by comparing the contig with all reference genomes. the haplotype with the highest similarity and above 98% is assigned. the outputs of virbin and maxbin are shown in table 2 . strainphlan and constrains were applied on this real hiv data set. strainphlan was able to identify the hiv species, but could not report any strain information. constrains could not align enough reads to marker genes for further strain-level analysis. compared to the simulated contigs or assembled contigs using simulated reads, the results of virbin on the real sequencing data have generally lower sensitivity and precision. there are two major reasons. first, the assembled contigs for real reads are more likely to contain errors. second, this data set has several haplotypes with very similar average abundances. referring to fig. 6 , the abundance difference between the 2 least abundant haplotypes is < 2%. thus, the clustering algorithm could mix contigs from these haplotypes. for the mock data experiment, we also present the recall and precision at contig level in additional file 1: table s4 . we again compared the predicted abundance profile with the known one in fig. 6 . the χ 2 -test output p-value 0.9999 and 0.9995 for virbin and maxbin, respectively, indicating that the predicted abundance distributions by virbin and maxbin are not statistically different from the ground truth. overall, virbin can cluster more contigs into their originating haplotypes than maxbin. while virbin focuses on sub-contigs that are more likely unique to one haplotype, maxbin clusters whole contigs, which could contain regions common to multiple haplotypes and makes read coverage more heterogeneous. in addition, sequence composition-based features such as tetranucleotide frequencies are not effective in distinguishing highly similar viral strains. our experimental results show that virbin works better for longer contigs that can cover bigger regions of the underlying genomes. when the genome coverage by the contigs is below 70%, the performance of virbin deteriorates because it becomes harder to estimate the correct number of haplotypes. in addition, the empirical experience shows that it is difficult to classify two viral strains when the abundance difference between them is below 3%. thus, although we have demonstrated much better contig binning performance for distinguishing viral in this work, we presented virbin, a new contig binning tool for distinguishing contigs from different viral haplotypes with high sequence similarity. by conducting experiments on multiple simulated data sets of different haplotype abundance distribution, different number of haplotypes, and different sets of simulated or assembled contigs, we demonstrated that our tool can produce more accurate clustering results than popular contig binning tools for viral haplotype reconstruction. the overall pipeline of our method is shown in fig. 7 . there are mainly two steps: (1) estimate the number of haplotypes by aligning contigs and identifying windows; (2) calculate relative abundances in each window and apply a clustering algorithm to group clusters of the same haplotype. the underlying algorithm of grouping contigs into haplotypes is prototype-based clustering [34] . features such as the overlaps and paired-end connections have limited usage in grouping distant contigs from the same haplotype. the clustering will mainly use the features based on the abundance distributions. although abundance-based clustering has been used for contig binning from multiple samples [15, 19] , existing tools are not designed fig. 7 the pipeline of virbin to tackle key challenges of distinguishing contigs of different haplotypes. first, the observed coverage of each contig not only depends on the abundance of the underlying haplotype, but also depends on whether it is a unique or shared region by two or more haplotypes. second, heterogeneous coverage of each haplotype in an rna viral quasispecies is common, which is caused by sequencing-related biases and compounded by gene expression. thus, directly applying existing prototypebased clustering models such as gaussian-mixture model to contigs is not expected to produce accurate clustering. our solution to this problem is to cut the contigs into "windows" and to apply the clustering on sub-contigs that are more likely to represent one haplotype. in addition, instead of assuming any parametric distribution, which is usually not the case for haplotype contigs, we will use a non-parametric distribution. although the high similarity between haplotypes presents a barrier to adoption of kmer-based features for distinguishing contigs from different haplotypes, it brings opportunities for haplotype number estimation. with stringent alignment threshold, contigs that can be aligned with each other usually come from the same region of the virus and thus the number of aligned contigs can be carefully used for haplotype number estimation. we progressively build multiple sequence alignments using contigs' pairwise alignments. in this step, base-level accuracy of the alignment is not a major concern and thus progressive construction of the alignment between contigs can serve the purpose well. we first sort the contigs by their lengths in descending order. the longest contig will be used as the first reference. all the other contigs will be aligned to the reference using blast+ [35] to generate an alignment profile similar to multiple sequence alignment. two types of alignments are kept from the output of blast+. one is the alignment that is local to the reference but global to the shorter contigs. the other is overlap alignment, which is the alignment between the suffix/prefix strings of the contigs. if not all the shorter contigs can be aligned to the reference contig, this process will continue by using the second longest contig as the reference until all the contigs are used. figure 8c shows the alignment between contigs from three haplotypes in fig. 8a using the longest contig as the reference, which is usually produced for the most abundant haplotype. each multiple alignment can be divided into many windows, which are formed whenever there is a change of the sequences in the alignment. we define the number of contigs inside each window as the window depth, d. based on these definitions, we have the following observations. when each position of the underlying haplotypes can be covered by at least one contig, d is equal to or larger than the number of haplotypes. note that the common regions between different haplotypes are regarded as different positions and thus should be covered by different contigs. this conclusion can be proved by contradiction easily. fig. 8b shows the contigs satisfying the conditions in the ideal case. there are three haplotypes with different abundances. they only contain mutations at three positions that are far away from each other. because of the long common regions among them, assembly programs usually won't be able to recover all the three genomes. instead, they can generate short but correct contigs. in fig. 8b , each position in the three haplotypes is covered by at least one contig. in this case, all the windows have depth of at least 3. if every position of a haplotype is only covered by one contig, the windows will have depth n, which is the number of haplotypes. as some positions can be covered by multiple contigs, the overlaps between contigs contribute to window depth larger than n. for example, in fig. 8b , the middle window contains the overlaps between fig. 8 window construction from aligned contigs. a three haplotypes with mutations at three locations. line weights represent the haplotype abundance. s 1 and s 2 are two mutation-free regions common to three haplotypes. s 1 and s 2 are at least 1k bp. b the alignment of contigs that satisfy the ideal condition. the grey-scale intensity represents the coverage of a contig. three windows are produced. c the contigs that cannot cover all the three haplotypes. there are six windows. their depth values are denoted below each window. for contig marked with "a s 2 ", its sequencing coverage is plotted above the contig two contigs from each haplotype and thus has depth 6. in this ideal case, we can choose the smallest window depth as the number of haplotypes in a sample. in practice though, the assumptions about the contigs' properties are not always true. thus, in our implementation, we will use the consensus window depth as the number of haplotypes, by assuming that most windows cover all haplotypes and contain haplotype-specific mutations. for the contigs shown in fig. 8c, window depth 3 is the most frequent one. in the implementation, we first sort all the windows in descending order of window length. then we choose the most frequent window depth of the top x windows as the number of haplotypes. the default value of x is 50 in our implementation. we will present the results of haplotype number estimation using consensus window depth for both simulated and real quasispecies data. step 2: contig clustering based on relative abundance distribution let the number of haplotypes estimated by step 1 be n. the final goal of the pipeline is to divide the contigs into n groups so that each group contains contigs originating from the same haplotype. in this step, we conduct clustering on subcontigs from windows with depth n. unlike many existing contig clustering tools, our clustering is not applied to a complete contig. because each contig can contain both haplotype-specific region and shared regions among different haplotypes, using the read coverage profile of the whole contig will confuse the clustering algorithm and makes the convergence slow or leads to wrong assignment of the objects. for example, in fig. 8c , the contig "a s 2 " contains mutation a from one haplotype and also a shared region s 2 . thus, significantly more reads will be mapped to the shared region and make the coverage for this contig highly heterogeneous. thus, the objects as input to the clustering algorithm are "subcontigs" in windows of depth n, where the sub-contigs are substrings of the contigs in these windows. they are more likely to represent the relative abundance of one haplotype. after clustering on sub-contigs, a post-processing step will be applied to get the cluster membership for each contig based on its sub-contigs' memberships. the clustering algorithm we adopt is prototype-based clustering and is essentially an augmented k-means algorithm. in a standard k-means algorithm, the centroid of the objects in a cluster is the prototype of the cluster. in our algorithm, the prototype is a distribution that is derived from the sub-contigs and empirically describes the relative abundance distribution. the clustering algorithm will assign each sub-contig to one cluster based on the posterior probability of the abundance distribution. although different clustering methods such as gaussian mixture model can be applied to cluster the sub-contigs, the augmented k-means as shown below has the fastest convergence with better clustering accuracy according to our tests. before we describe the main components, we first introduce the notations. the average relative abundance (denote asc) for a sub-contig c i in a window of depth n is calculated as: where s(c i ) is the total number of reads covering subcontig c i . similarly, we can calculate the position-specific relative abundance vector c for a sub-contig c i as where c i [k] represents the reads coverage at position k of sub-contig c i . |c i | is the number of bases in the sub-contig. n is the number of bins or haplotypes estimated by step 1. virbin utilizes the position-specific relative abundances of sub-contigs in windows with depth n to estimate the probability that a sub-contig belongs to a bin. let the n bins be h 1 , h 2 , . . . , h n . letẽ i (x) be the probability density function of relative abundance for sub-contigs from bin i, where x is an observed abundance. the iterative clustering algorithm contains four steps as shown below: initialization: initialize n groups by randomly assign sub-contigs to them. updating the empirical abundance distributionẽ i : for each bin i, the component sub-contigs' relative abundance profiles cs are aggregated to calculate the empirical probability density functionẽ i . the aggregation is performed by calculating the normalized histograms for these relative abundance profiles, so that the summation of histogram values will be 1. the number of bars of the histogram is a parameter that can be set by users. the default number is 1000. given an abundance value x, it is normalized to its closest bar value to get the corresponding probabilityẽ i (x). re-assignment of the sub-contigs: onceẽ i is derived, the relative likelihood of c j being produced from the ith prototype distribution can be calculated asẽ i (c j ). the prior probability of each bin (or haplotype) is a weighted sum of the likelihoods of all the component sub-contigs. the weights are determined by the total bases in the sub-contigs. this weighted sum enables us to incorporate both the total sub-contig bases and also the associated abundance generation likelihood for estimating the prior probability of a particular haplotype. the prior probability pr(h i ) for bin i is: with both likelihood and prior from iteration t, the expected probability that c j belongs to haplotype h i at iteration t +1 can be calculated as likelihood * prior, that is with the posterior probabilities calculated for each group distribution, we can reassign the sub-contig c i to the haplotype with the maximum posterior probability. the same reassigning procedures are applied for all the subcontigs. with the assignment results, the distribution e i and prior probability p(h i ) can be updated. iteration: iterate step 2 and 3 until the clustering results do not change or the maximum number of runs have been achieved. the default maximum number of runs is 100. step 3: post-processing the output of the augmented k-means is the clustered sub-contigs. for each cluster, its average abundance is calculated as the weighted average of the abundances of all sub-contigs in the cluster and the weight is determined by the length of a sub-contig. the haplotypes' abundances are the average abundances of the clusters. as each contig can contain multiple sub-contigs, which could have different membership, the contig's membership is determined by the dominant membership of its sub-contigs. for example, if a sub-contig is not in the window of depth n, it is not an input to the clustering step and will not be clustered. this could happen when a region of a contig is common to multiple haplotypes. it is also possible that the sub-contigs of a contig are assigned to different clusters, which could be caused by assembly errors. hepatitis c virus dynamics during natural infection are associated with long-term histological outcome of chronic hepatitis c disease hiv treatment failure: testing for hiv resistance in clinical practice deep sequencing of evolving pathogen populations: applications, errors, and bioinformatic solutions contribution of intra-and interhost dynamics to norovirus evolution deep sequencing reveals mixed infection with 2009 pandemic influenza a (h1n1) virus strains and the emergence of oseltamivir resistance evolutionary dynamics: exploring the equations of life chapter 3 -pathogenesis of viral infections and diseases viral quasispecies assembly via maximal clique enumeration viquas: an improved reconstruction pipeline for viral quasispecies spectra generated by next-generation sequencing idba-ud: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth iva: accurate de novo assembly of rna virus genomes de novo assembly of viral quasispecies using overlap graphs maximum likelihood de novo reconstruction of viral populations using paired end sequencing data de novo haplotype reconstruction in viral quasispecies using paired-end read guided path finding maxbin: an automated binning method to recover individual genomes from metagenomes using an expectation-maximization algorithm binning metagenomic contigs by coverage and composition metabat, an efficient tool for accurately reconstructing single genomes from complex microbial communities cocacola: binning metagenomic contigs using sequence composition, read coverage, co-alignment and paired-end read linkage desman: a new tool for de novo extraction of strains from metagenomes constrains identifies microbial strains in metagenomic datasets microbial strain-level population structure and genetic diversity from metagenomes viral quasispecies reconstruction via tensor factorization with successive read removal qsdpr: viral quasispecies reconstruction via correlation clustering art: a next-generation sequencing read simulator quasispecies dynamics with network constraints metaphlan2 for enhanced metagenomic taxonomic profiling efficient de novo assembly of large genomes using compressed data structures metaquast: evaluation of metagenome assemblies full-length haplotype reconstruction to infer the structure of heterogeneous virus populations rapid evaluation and quality control of next generation sequencing data with faqcs trimmomatic: a flexible trimmer for illumina sequence data karect: accurate correction of substitution, insertion and deletion errors for next-generation sequencing data introduction to data mining blast+: architecture and applications publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. supplementary information accompanies this paper at https://doi.org/10.1186/s12859-019-3138-1. this work was supported by michigan state university (east lansing, usa) and city university of hong kong (hong kong, china sar) project 7200620. the funding did not play any role in design/conclusion. virbin can be download from https://github.com/chjiao/virbin. data are available upon request.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord-304427-r7jt95ko authors: pasquato, a.; dettin, m.; basak, a.; gambaretto, r.; tonin, l.; seidah, n.g.; di bello, c. title: heparin enhances the furin cleavage of hiv-1 gp160 peptides date: 2007-12-22 journal: febs lett doi: 10.1016/j.febslet.2007.11.050 sha: doc_id: 304427 cord_uid: r7jt95ko infectious hiv-1 requires gp160 cleavage by furin at the rekr(511)↓ motif (site1) into the gp120/gp41 complex, whereas the kakr(503) (site2) sequence remains uncleaved. we synthesized 41mer and 51mer peptides, comprising site1 and site2, to study their conformation and in vitro furin processing. we found that, while the previously reported 19mer and 13mer analogues represent excellent in vitro furin substrates, the present extended sequences require heparin for optimal processing. our data support the hypothesis of a direct binding of heparin with site1 and site2, allowing selective exposure/accessibility of the rekr sequence, which is only then optimally cleaved by furin. human immunodeficiency virus type-1 (hiv-1) is the etiological agent for the acquired immunodeficiency syndrome (aids). the envelope glycoprotein gp160 is processed by host proteases into the gp120/gp41 heterodimer. this allows the virus to infect target cells following the cell surface binding of the trimeric complex gp120/gp41 to the human cd4 receptor [1] , and subsequently to the cxcr4/ccr5 co-receptors [2, 3] . this interaction induces conformational change, which leads to the dissociation of gp120 from gp41, allowing the n-terminal gp41 fusion peptide to be inserted into the host cell membrane [4] . though the structures of a monomeric gp120 core in complex with the cd4 receptor/fab 17b [5] and that of a post-fusion form of gp41 were solved [6] , little is known about the entire env conformation. in fact, individually gp120 does not mimic its precursor conformation, as two monoclonal antibodies directed against the v3 loop recognized gp160, but not gp120 [7] . the env precursor is cleaved by the proprotein convertase (pc) furin or furin like-proteases at the rekr 511 fl site1 [8] , resulting in the fusogenic gp120/gp41 complex. since the r511t mutation results in uncleaved gp160 and non-infectious virus, cleavage is essential for viral entry [9] . in vitro investigations on peptides encompassing the gp120/gp41 junction confirmed that cleavage occurs at arg 511 fl [10] . interestingly, upstream of the physiological processing site, a second site2 potential furin motif (kakr 503 ) is inefficiently cleaved. the exact role of site2 is unknown, though mutations of gp160 kakr 503 sequence result in an unprocessed precursor [11] . conformational differences between site1 and site2 may explain the preference of furin for site1 [12] . even though short gp160 peptides are efficiently cleaved in vitro by furin at arg 511 , the full length gp160 was shown not to be efficiently cleaved ex vivo. this suggests that gp160 structure [11, 13] , post-translational modifications [14] , as well as cellular and/or extracellular factors, may also influence the efficacy and selectivity of the furin mediated cleavage [15] . viruses, such as sindbis [16] and coronavirus [17] , bind to target cells via cell-surface glycosaminoglycans (gags). proteoglycans were also shown to facilitate hiv-1 binding to and/ or entry into cells lacking the cd4 receptor [18] . moreover, enzymatic removal of cell surface heparan sulfate chains drastically impairs hiv-1 infection of cd4+ cells [19] . this effect likely implicates gp160 and/or gp120 interaction with gags [20] [21] [22] . many authors identified the binding site for heparin or its derivatives within the gp120 v3 loop [21] . however, an increasing body of evidence points to a possible gp160-gags interaction [22] . to investigate the influence of the sequence surrounding the rekr 511 motif on gp160 processing efficiency, we synthesized various peptides derived from the gp120/gp41 junction (table 1) . moreover, as a measure of the modulating role of gags on the furin processing, we evaluated the effect of heparin by both circular dichroism (cd) and hplc. we found that the gp120/ gp41 junction itself binds heparin, thus enhancing its furin processing. the synthesis of the 14mer and 19mer was reported [10] . the 18mer, 41mer, and 51mer were synthesized on a semi-automatic synthesizer (applied biosystems, mod. 431a) using a rink amide mbha resin abbreviations: hiv-1, human immunodeficiency virus type 1; gags, glycosaminoglycans; cd, circular dichroism; aids, acquired immunodeficiency syndrome; rp-hplc, reverse phase high performance liquid chromatography; pc, proprotein convertase; btmd, before trans membrane domain; ms, mass spectrometry; amc, 7-amino-4methyl-coumarin; mca, 7-amido-4-methylcoumarin; tfe, trifluoroethanol; sds, sodium dodecyl sulfate; tris-hcl, tris-(hydroxymethyl) aminomethane-hcl; dmso, dimethyl sulfoxide; hf, hydrofluoric acid; cmk, chloromethylketone; pbs, phosphate buffer saline (novabiochem, la jolla, 0.48 mmol/g, 0.25 mmol), boc chemistry and hbtu/hobt activation. detachment from the solid support and removal of the side chain protecting groups were achieved treating with hf:anisole:dmso:2-mercaptopyridine/10:1:1:1 (1 h, 0°c). crude products were purified by reverse phase high performance liquid chromatography (rp-hplc) on a delta pak hr c 18 column (waters, 6 lm, 60å , 7.8 · 300 mm). homogeneity grade was evaluated by rp-hplc on a vydac c 18 column (waters, 5 lm, 300 å , 4.6 · 250 mm). molecular mass was checked by electrospray-time of flight (tof) mass spectrometry (ms; mariner 5120 api-tof). cd spectra were recorded on a jasco cd spectropolarimeter model j-710 with a cylindrical fused quartz cell (path length 0.1 cm). the spectra are reported in units of mean ellipticity (peptide molecular weight/number of amide bonds), [h] r (deg cm 2 dmol à1 ) or ellipticity, [h] (deg cm 2 ). the measurements were performed in water, 10 mm phosphate buffer ph 7, 14 mm sodium dodecyl sulfate (sds) in 10 mm phosphate buffer ph 7, trifluoroethanol (tfe) 98%, 20 lm heparin (sigma, low molecular weight) in 0.15 m nacl + 25 mm tris-(hydroxymethyl) aminomethane-hcl (tris-hcl) buffer ph 7, and in 0.15 m nacl + 25 mm tris-hcl buffer ph 7. peptide concentrations, determined by amino acid analysis or uv absorption, varied from 18 to 43 lm. the spectra were corrected for the solvents, salts and heparin minor contributions. the media of bsc40 cells infected with either wild type vaccinia virus (vv:wt, control) or a soluble form of hfurin (vv:hfurin-btmd) [8] were collected 18 h post-infection and concentrated (centriprep ym-30). activity was measured with the fluorogenic substrate pyr-rtkr-mca. assays were performed in 100 ll at 37°c on 100 lm peptide in 2 mm cacl 2 , 25 mm tris-hcl buffer ph 7.0, 1 mm b-mercaptoethanol, 2 ll furin ($2 relative fluorescence units (rfu); where 1 rfu is defined as 1 pmol 7-amino-4-methyl-coumarin (amc) released/min/ll enzyme acting on 100 lm of the fluorogenic substrate pyr-rtkr-mca) or as control 2 ll of media from vv:wt culture supernatant. when specified, incubation media also contained 2.5 lm, 20 lm or 25 lm heparin. heparin alone shows no enzymatic activity (not shown). at various time points, 20 ll samples were analyzed by rp-hplc on a varian c 18 column (5 lm, 100 å , 4.5 · 250 mm) and digestion products identified by mass spectrometry (ms). percent cleavage was calculated from precursor areas. reactions, performed in 100 ll (25 mm tris-hcl, 1 mm b-mercaptoethanol and 2 mm cacl 2 , ph 7.0) at 37°c, contained 50 lm pyr-rtkr-mca or 100 lm 19mer as substrate, 2 ll of furin and differ-ent concentrations (1-100 lm) of the 18mer as inhibitor or 5 lm dec-rvkr-cmk [23] . enzymatic activity with mca-conjugated peptidyl substrate was monitored (360 nm excitation, 460 nm emission) with a spectra max gemini em microplate spectrofluorometer (molecular devices), in the presence or absence of either 20 lm or 100 lm heparin. inhibition assays with the 19mer were monitored by rp-hplc. the ic 50 were calculated using grafit version 4.09 software. four peptides, 51mer, 41mer, 19mer and 13mer, spanning the gp160 cleavage sequence, were synthesized ( table 1 ). the 13mer containing site1, and the 19mer, which includes site1 and site2, were chosen as ref. [10] . the extended 41mer and 51mer were synthesized to investigate the influence of the regions surrounding the physiological cleavage site on furin processing. it was reported that a cell-permeable 22mer sequence kieplgvaptkakrrvvqrekr 511 , which does not contain p 0 residues, interferes with gp160 processing [24] . thus, to test for a possible in vitro inhibitory function we also synthesized a 18mer peptide (lgvaptkakrrvvq-rekr 511 ), mimicking the furin-processing product of the 41mer (table 1) . the spectra in phosphate buffer ph 7.0 and water showed a diagnostic band with a minimum at 198 nm, suggesting that the 51mer, 41mer and 19mer are unstructured ( fig. 1a-c) . in the presence of sds, a red shift of the negative band was observed with a minimum at 201 nm for the 19mer (fig. 1a) . interestingly, based on the 220 nm band intensity, the same micellar solution induced the 41mer and 51mer to assume a 20% and 21% a-helix conformation respectively (fig. 1b,c) . this indicates a likely sds interaction that could be due to the insertion of the hydrophobic c-terminus into micelles and/or could result from the electrostatic binding of the positively charged site1 and/or site2 to the negatively charged micellar surface. tfe induced order in the structure of the 19mer, 41mer, and 51mer (positive band at 190 nm, two negatives at 206 and 220 nm). the a-helix content was 30% in 98% tfe/h 2 o for the 19mer, 59% for 41mer and 62% for 51mer (fig. 1a-c) . since cd profiles in negatively charged sds micellar solutions showed a transition of conformers towards a more structured population, and gp160 cleavage site is positively charged, further conformational investigations were performed. the cd profile of 20 lm heparin is similar to that of water (fig. 1d) and that of the 19mer does change in the presence of heparin (fig. 2a) . in contrast, the cd spectra of the 41mer and the 51mer were significantly modified in the pres-ence versus absence of heparin (fig. 2b,c) . similar results were obtained with higher heparin concentrations (100 lm). the 13mer and 19mer peptides were digested equally well by furin at site1 (tables 2 and 3) , showing complete processing at 5 h (fig. 3a,b) . in contrast, the 41mer and 51mer peptides were either barely or unprocessed, respectively, even after 24 h digestion at ph 7 (fig. 4a,b; tables 1-3 ). since in vitro gp160 cleavage was reported to be optimal at phs 6-7 [25] , further assays were performed on the 41mer and 51mer at acidic conditions (ph 6.3, 6.7), again revealing no differences with respect to the results obtained at ph 7. furthermore, similar data were observed in presence of low levels of denaturants (0.05% tx-100 or sds) (not shown). we first hypothesized that product inhibition could explain these results. we thus tested the in vitro ability of the 18mer peptide, representing the furin-derived product of the 41mer (table 1) , to inhibit the processing of either the fluorogenic pyr-rtkr-mca or the 19mer peptides. while the 18mer peptide effectively reduced the release of free amc with an estimated ic 50 of 1.6 lm (fig. 5a) , it could only partially inhibit the 19mer processing with an ic 50 > 100 lm (fig. 5b) . we conclude that product inhibition cannot explain the inability of furin to process the 41mer and 51mer peptides. because cd investigations showed a likely binding between heparin and the 41mer or 51mer (fig. 2b,c) , all four gp160-derived analogues were digested overnight in the absence or presence of 2.5 lm heparin. under these conditions, the 13mer and 19mer were digested at site1 with similar rates independent of the presence of heparin. in contrast, while no significant processing occurred in the absence of heparin, $40% and $60% processing at the rekrfl site of the 41mer (into an 18mer product with identical retention time on rp-hplc to the synthetic version) and 51mer peptides, respectively, were observed in the presence of 2.5 lm heparin (fig. 6) . as control, we confirmed that the 41mer peptide is not cleaved by the recombi-nant vv:wt-infected culture supernatant (fig. 6 , upper center panel). furthermore, cleavage was inhibited by adding a well known pc-inhibitor, dec-rvkr-cmk (fig. 6 , upper right panel) [23] . at 25 lm heparin we obtained a more extensive processing, but also noticed precipitation of the peptides (not shown). finally, in a separate 6 h furin incubation experiment, the processing of the 41mer and 51mer peptides also showed a similar enhancement effect of heparin (not shown). in conclusion, these data indicate a likely heparin-peptide interaction that may better expose site1, and hence allow more effective furin cleavage. five peptides (table 1) spanning the gp120/gp41 junction were investigated to better define the gp160 glycoprotein cleavage. the 19mer and its shorter analogue 13mer were processed by furin at site1 (rekr 511 ), while site2 (kakr 503 ), which is included only in the 19mer, was uncleaved (fig. 3) . the lack of processing at site2 may be rationalized on the basis of structural motifs. in fact, the 19mer nmr molecular model in tfe revealed that site2 is embedded in a helical segment, whereas site1 is in a exposed loop at the c-terminus of the peptide [12] . in contrast, the 41mer and 51mer, spanning extensive sequence of the gp160 cleavage region, were shown to represent very poor furin substrates. this suggests that the generated fragments could either act as inhibitors or that the more extended regions surrounding the physiological cleavage site prevents effective processing. since the possibility of product inhibition by the 18mer was excluded, we turned our attention towards structural restrictions and/or the need of other factors to rationalize the noncleavability of the 41mer and 51mer peptides. cd analysis on the 19mer, 41mer and 51mer in aqueous solution revealed that the three analogues are unstructured, and yet only the 19mer is digested by furin. thus, some structural constraints must exist in the 41mer and 51mer, at least around site1. the same argument may explain the inability of furin to cleave at site2 in any substrates used. in an attempt to increase the 41mer and 51mer processing, we added some detergents to enhance the peptides backbone flexibility without affecting enzyme activity. however, neither tx-100 nor sds had any effect. therefore, we suspected that cellular/extracellular factors may influence the cleavability of gp160 by furin. indeed, surface proteins containing heparin-binding motifs processed by furin were reported [16, 26] . in particular, sindbis virus attachment to target cells was enhanced in the presence of heparan sulfate (hs) via the furin recognition motif of the unprocessed envelope glycoprotein pe2 [16] . similarly, peptides derived from the cleavage site of the human respiratory syncytial virus (rsv) fusion glycoprotein bind heparin and cellular gags [26] . since the gp120/gp41 does not form stable trimers, while unprocessed gp160 does, it was hypothesized that gp160 oligomer attachment to the plasma membrane heparin sulfate occurs via its furin cleavage site [27] . indeed, the kakr 503 rvvqrekr 511 sequence exhibits a basic region, which contains two potential inverted consensus hs-binding domains. thus, it was shown that the affinity of gp160 for heparin is about 3-times higher than that observed for gp120, implying that gp41 and/or hidden motifs in the mature gp120 may be involved in heparin binding [22] . cd spectra of the 41mer and 51mer suggest these peptides could interact with heparin (fig. 2) and undergo structural reorganization. in fact, in presence of heparin, the profiles change with respect to those of the peptides alone. the nega-tive band at 198 nm, diagnostic for aperiodic structures, is replaced by a positive one. in contrast, it is noteworthy that the shorter 19mer does not change its cd profile in the presence of heparin. since the difference between the 41mer and the 19mer lies in 22 hydrophobic residues and the interaction between heparin and polypeptides is supposed to be electrostatic, these additional residues may support a favorable peptide conformation that optimally orients the positively charged side chains towards the negatively charged sulfate moieties. our results agree with a probable glycosaminoglycans-gp160 interaction, as proposed [22, 27] , and suggest that the residues spanning the gp120/gp41 junction may contribute in gp160-gags binding. moreover, given that heparin induces a change in the 41mer and 51mer conformation, which could play a key role in the enzyme-substrate recognition, we analyzed how it may influence their furin processing. surprisingly, while up to 100 lm heparin did not influence furin activity on pyr-rtkr-mca processing (fig. 6, left upper panel) , the 41mer and 51mer peptides were digested at site1 (fig. 6) . therefore, we hypothesize that heparin induces conformational change, optimally exposing the furin-cleavage rekr 511 site. this is the first time that heparin is shown to enhance the in vitro cleavage of precursors by furin. in conclusion, this study has shown that, in the absence of heparin, the 41mer and 51mer gp160 derived peptides represent very poor furin substrates in vitro, in contrast to the shorter analogues (13mer and 19mer) that are efficiently processed. heparin was shown to strongly interact with the 41mer and 51mer peptides, inducing conformational changes, thereby exposing site1 for cleavage. since the 41mer and 51mer peptides may not faithfully mimic the conformation around the cleavage site within the complete gp160 precursor, more analyses are required to assess if heparin is essential in vivo during gp160 maturation and how gags modulate hiv-1 activity. induction of cd4-dependent cell fusion by the htlv-iii/lav envelope glycoprotein hiv-1 entry cofactor: functional cdna cloning of a seventransmembrane, g protein-coupled receptor fusin-a place for hiv-1 and t4 cells to meet mechanisms of viral membrane fusion and its inhibition structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody core structure of gp41 from the hiv envelope glycoprotein conformational changes affecting the v3 and cd4-binding domains of human immunodeficiency virus type 1 gp120 associated with env processing and with binding of ligands to these sites comparative cellular processing of the human immunodeficiency virus (hiv-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus structural investigation and kinetic characterization of potential cleavage sites of hiv gp160 by human furin and pc1 mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site structural investigation of the hiv-1 envelope glycoprotein gp160 cleavage site functional roles of the v3 hypervariable region of hiv-1 gp160 in the processing of gp160 and in the formation of syncytia in cd4+ cells hiv-1 acute infection env glycomutants designed from 3d model: effects on processing, antigenicity, and neutralization sensitivity comparative functional role of pc7 and furin in the processing of the hiv envelope glycoprotein gp160 attenuation of sindbis virus variants incorporating uncleaved pe2 glycoprotein is correlated with attachment to cell-surface heparan sulfate murine coronavirus with an extended host range uses heparan sulfate as an entry receptor human immunodeficiency virus type 1 enters primary human brain microvascular endothelial cells by a mechanism involving cell surface proteoglycans independent of lipid rafts cellsurface heparan sulfate proteoglycan mediates hiv-1 infection of t-cell lines heparin and its derivatives bind to hiv-1 recombinant envelope glycoproteins, rather than to recombinant hiv-1 receptor the v3 region of the envelope glycoprotein of human immunodeficiency virus type 1 binds sulfated polysaccharides and cd4-derived synthetic peptides the interaction of a glycosaminoglycan, heparin, with hiv-1 major envelope glycoprotein processing of viral glycoproteins by the subtilisin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones an anti-hiv peptide construct derived from the cleavage region of the env precursor acts on env fusogenicity through the presence of a functional cleavage sequence biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160 identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity processing, stability, and receptor binding properties of oligomeric envelope glycoprotein from a primary hiv-1 isolate acknowledgement: we would like to thank brigitte mary for her secretarial help. this work was supported by a cihr grant (#mgp-44363), a canada chair (#201652) and by a generous gift from the strauss foundation. key: cord-337659-x4oywbrj authors: wilson, brenda a. title: global biosecurity in a complex, dynamic world date: 2008-07-31 journal: complexity doi: 10.1002/cplx.20246 sha: doc_id: 337659 cord_uid: x4oywbrj biosecurity is emerging as a major global health priority for which innovative and unprecedented solutions are needed. biosecurity is a challenging biocomplexity problem involving multifaceted processes such as interactions between humans and nonhuman biota, anthropogenic environmental and ecological factors, and socioeconomic and political pressures. key to an effective biosecurity strategy will be fundamental understanding of evolutionary, anthropogenic and environmental driving forces at play in transmission and perpetuation of infectious diseases. biosecurity solutions will depend on increased support of basic biomedical research and public education, enhanced healthcare preparedness, alternative strategies for ensuringsafety, and improved interagency cooperation regarding global health policy. © 2008 wiley periodicals, inc. complexity, 2008. i t is widely accepted by historians that there are certain dates upon which history seems to pivot, turning points that forever change the course of future events. the momentous months of september and october 2001 mark such a critical period in our recent history. our world as we knew it shifted, not just for the united states, but all nations. as a consequence, defense against bioterror agents came to the forefront as a major health priority in the u.s. and elsewhere. indeed, we were still reeling from the impact when sars swept across the globe in 2002-2003, followed closely behind by the still ongoing worldwide spread of avian flu and the concomitant fear of it transforming into a human flu pandemic on the scale of that experienced in 1918. but, these events are only at the pinnacle of a mounting number of impinging natural and imposed biohazards (table 1) [1] [2] [3] [4] [5] . importantly, these manmade and natural events have revealed a number of glaring gaps in our knowledge about infectious diseases, their transmission and perpetuation, and how to effectively combat them. existing and looming biological threats have now made biosecurity, which includes biodefense, the most pressing global health priority. in a rapidly changing world, biosecurity is at the intersection of every sphere of medical, biological, ecological, socioeconomic, and political system. although one might argue that the principal difference in the infectious disease threat today versus say 10, 25, or 50 years ago is bioterrorism, the resources spend on preparing for a bioterror attack is viewed by most scientists as grossly exorbitant [6] , particularly considering the small numbers of individuals who have been or could be affected by this type of attack and considering the relatively low medical relevance or prevalence of the diseases caused by the limited number of highpriority bioterror bioagents, the socalled ''category a select agents.'' and, while admittedly the preparedness and surveillance measures put in place for one has certainly helped to protect against the other (the improved global response to and curtailment of sars coming after the anthrax bioterrorist attacks is a prime example of this), most scientists feel that the limited resources available from an already overburdened system should instead be used for studying and preparing against the looming and potentially more devastating infectious disease threats from natural or accidental exposure [7] , which could affect millions of people and animals and could have huge health and economic consequences. and thus, while the threat of bioterrorism must be considered, many scientists propound that the focus should be on the more urgent and dire problem of biosecurity, rather than just bioterrorism. it has been argued by many that there is no better creator of new highly potent biological threats than nature itself. however, there is also little doubt that anthropogenic environmental, socioeconomic, and ecological influences can have devastating impact on the extent and severity of the outcome of these natural biological hazards. risks to public health come from diverse scenarios ranging from epidemics to outbreaks during natural disasters to accidental exposures through poor food processing to deliberate releases or fear thereof ( table 2) . finding solutions to these challenging biocomplexity problems will require integrated, multilevel, flexible, and interdisciplinary approaches that stretch traditional concepts. key to an effective global biosecurity strategy will be improved detection, prevention, treatment, and management of infectious diseases, but also better understanding of the intrinsic and extrinsic factors that contribute to their virulence and influence their transmission, prevalence, and perpetuation. thus to achieve this, we first need a better understanding of the critical evolutionary, anthropogenic, and environmental driving forces that contribute to natural and man-made biological threats. to gain a sense of the potential impact of biological threats on biosecurity, it is best to begin by considering the source and nature of the biological agents that pose biosecurity risks (table 3) . manmade biological threats come in two flavors, deliberate and accidental. the concept of intentionally using biological agents as weapons is nothing new to warfare, and we are all aware of the american, russian, and other state-sponsored programs to develop biological agents as ''weapons of 22 cases/5 deaths total in 2001 from anthrax (perpetrator still at large) >2,000 hospitalized/18 deaths total in 1994-5 from sarin gas (aum shinrikyo) 751 cases/0 deaths total in 1984 from salmonella (rajneeshee) 1 death in 1978 from ricin (assassination of georgi markov) natural casualities: 300-500m cases/2 million deaths per year from malaria world-wide 76m cases/325,000 hospitalizations/5,000 deaths per year from foodborne illnesses in usa 8-10m cases/2 million deaths per year from tuberculosis world-wide >60m cases/>20m deaths total from hiv/aids world-wide, >0.5m deaths in usa 170m cases/10,000 deaths total from hepatitis c in usa mass destruction'' (wmd). these state-sponsored wmd programs have been largely dismantled [8] , and instead the concept of using biological agents as bioweapons has now been usurped by individuals or small groups acting as terrorists engaged in a different form of warfare, where these agents are perhaps more accurately described as ''weapons of mass disruption'' (still wmd). we experienced a vivid and horrible display of this new brand of wmd with the anthrax attacks of 2001, in which the u.s. postal system was utilized to dispense deadly disease, but even more notably, fear and turmoil. the cost of mounting a response to this new wmd has been enormous, not only in terms of billions of u.s. taxpayers' dollars, particularly directed toward biodefense (table 4 ), but also in countless man-hours expended in ramping up other areas of security and healthcare preparedness and in the astounding disruption of lifestyle (e.g. inconveniences caused by intensified travel-related security measures). a growing number of scientists feel that the bioterror threat is exaggerated [7] and that it is highly unlikely that any terrorist organization, foreign or domestic, could on their own develop from scratch a bioweapon capable of causing mass casualties. instead, it is more likely that the potential terrorists would steal or procure existing material and deploy it on a much smaller scale. in contrast, manmade threats resulting from inadvertent release, accidental contamination, or even from non-malicious intentional introduction of biological agents represent much more measurable concerns with known likelihood of risk. there have been a number of recent high-profile incidences that illustrate the havoc, alarm, and economic consequences that can result from widespread distribution of contaminated food because of accidental introduction of harmful microbes during food processing. what previously was seen only sporadically, such as at church socials, family gatherings or community picnics, moved abruptly into the public's eye with the largescale problem of undercooked fastfood hamburger meat contaminated with e. coli o157:h7, a toxin-producing bacterium that causes dysentery-like diarrhea and can cause kidney failure and death, especially in children and the elderly. this dangerous microbe has since been associated with over 400 multistate or multination outbreaks of contaminated food, including meat, radish sprouts, apple juice, let-tuce, and most recently spinach. late last summer, e. coli o157:h7 contamination of prepackaged fresh spinach led to 204 cases of illness across 26 states, with 104 hospitalizations, 31 kidney failures, and 3 deaths [9]. the outbreak, which was traced back to spinach obtained from a few fields in california [10] , shook consumer confidence and cost the industry an estimated $150m in economic losses [11] . there are many other examples of how the spread of natural threats can be greatly facilitated by our modern technologies, practices, and behaviors. viruses such as the marburg and ebola viruses, first discovered in the 1960s and 1970s, are examples of biological agents responsible for recently emerged diseases [12] . ebola and marburg viruses are considered to be zoonotic diseases that are transmissible by close contact with animal species, but their spread has been facilitated by conditions in the country of outbreak, including political upheavals, reuse of needles, and cultural burial practices. zoonotic diseases are often perceived as only a problem of developing countries, where there is much closer contact with animals, both domestic and wild. however, living with animals is not limited to the third world. consider how many americans alone live with pets, sleeping with them, and even kissing them. it is interesting to consider that measles virus is closely related to canine distemper virus [13, 14] , suggesting that at some point a dog-human transmission occurred or a common ancestral virus may have infected both. of particular and growing concern are high impact, foreign animal diseases, such as mad cow and foot-and-mouth diseases (table 5 ). bovine spongiform encephalopathy (bse), commonly known as mad cow disease, is a fatal, progressive neurodegenerative disease of cattle caused by an infectious form of misfolded protein called a prion [15] . transmission of bse occurs when healthy animals come in close contact, usually through ingestion, with prion-containing tissues from animals that have the disease. the first probable occurrence in cattle was in the early 1980s, possibly as a result of feeding cattle meat and bone meal that contained scrapie-infected sheep products. scrapie is a prion disease of sheep and goats. industrial cattle-farming practices in europe prior to 1987 used rendered meat and bone meal, instead of the more common soybean meal used elsewhere, as a protein supplement in cattle feed. in the early 1980s, a change in the rendering process in the u.k., in which a lower sterilization temperature was used for the steam boiling step in the process, is thought to be the major contributing factor to an increase in prions in the cattle feed that resulted in the bse epizoonotic outbreak. the uk epidemic peaked in 1993 with nearly 1,000 new cases per week, and by the end of 2005 there were over 184,000 cases of bse confirmed in the u.k. [16, 17] . bse attracted particular attention because it now appears that it can also be transmitted to humans that consume tainted meat. since the first reported case in 1996, at least 188 people, 160 in the u.k. and 28 elsewhere, have died of a disease with similar neurological symptoms to bse, called variant creutzfeldt-jakob disease (vcjd) [18] . for many of the vcjd cases, there is direct evidence that they had consumed tainted beef years before. the connection between bse and vcjd has a wider impact than just food safety--blood, tissues and organ donation programs are also affected and anyone having exposure to bse is a potential carrier [19] . because of the long incubation period for prion diseases (years to decades), the full extent of the human vcjd outbreak is still not fully known, although the number of new cases appears to be declining. the long incubation period also makes testing for the disease in animals difficult because most livestock are slaughtered long before noticeable symptoms occur or even before plaques in the brain can be readily detected during inspection by necropsy. although many foreign animal diseases are not of serious concern to human health (i.e. humans may be affected only very rarely through direct contact with infected animals), they do pose considerable threat to our agricultural and food industries and could cost millions or even billions of dollars in economic and trade losses (hence their status as ''high impact'' diseases). foot-and-mouth disease (fmd) is a highly contagious, sometimes fatal viral disease primarily of cattle and pigs, but it has a wide host range. fmd occurs worldwide, but a number of areas, including north america, australia, new zealand, japan, most of europe, and parts of south america have been fmd-free for some time, mainly due to eradication through rigorous vaccination and culling programs. however, in 2001, a major outbreak of fmd in the u.k. resulted in the slaughter of millions of animals, huge economic and trade losses estimated in the range of £20m, the temporary cancellation of sporting events and other outdoor events attended by farmers or those living in the country, and the implementation of strict policies on the sale and trade of livestock, as well as disinfection of all persons entering or leaving farming areas [20, 21] . countries are recognized to be in one of three fmd categories: fmd present with or without vaccination, fmd free with vaccination, and fmd free without vaccination [22, 23] . understandably, countries designated as fmd free without vaccination have the greatest export markets, so most developed countries have greatly enhanced their agricultural surveillance and trade policies to maintain their fmd-free status. innocuous introduction of foreign plants, animals, or insects can also have considerable ecological and economic impact on horticultural and agricultural industries. kudzu, a member of the pea family that is native to southeast asia, is an example of an invasive alien plant species that has caused considerable damage since its introduction to the southeastern regions of the u.s., where it is sometimes referred to as ''the plant that ate the south'' [24] . kudzu was first intro-duced from japan into the u.s. in 1876 at the philadelphia centennial exposition, after which it gained some popularity as an ornamental shade vine. but, from 1935 to 1953, the soil conservation service promoted its use as a means for controlling soil erosion. once established with a root system that can reach depths of up to 12 feet, kudzu vines grow as much as a foot per day during a season with lengths up to 100 feet. kudzu now covers over 1m hectares and poses a considerable threat to the otherwise high biodiversity of flora found in the south [25] . a tremendous amount of money and effort is spent each growing season to prevent the highly prolific kudzu from overtaking roads, bridges, powerlines, local vegetation, and even homes, barns and other buildings. it costs an estimated $500m annually in lost cropland and management resources [26] . for successful long-term control, the entire root system must be destroyed or the plant will grow back, and considerable effort has been made to find pesticides that can control this plant pest. key to an effective strategy to combat complex biological problems to ensure biosecurity will be a greater understanding of the driving forces that are important for transmission and perpetration of infectious disease and then the management and implementation of effective preventive or containment measures. the potential power of natural selection is obvious. time and again we have seen how rapidly microbes can evolve in response to selective pressure, such that certain behaviors or genetic traits tend to be eliminated from the gene pool, while others are maintained or changed. as a consequence, new biological threats are bound to emerge as we impose our influence on the environment. the goal of most pathogenesis research, of course, is to use our understanding of the ecology of host-microbe interactions and their role in pathogenesis to develop predictive models for disease progression and transmission. unfortunately, our current understanding is rudimentary at best, and we are just now beginning to tease out the intricacies of the co-evolution of pathogens with their hosts and environment and the role that these host-microbe interactions play in emergence of disease. human-induced evolution can be extraordinarily rapid and pervasive. the natural history of myxoma virus in american rabbits and its introduction into european rabbits as a means for controlling the rabbit populations in australia provides an interesting example of the co-evolution of a virus and its animal host [27] . it also provides a glimpse into understanding the emergence of infectious disease. shortly after european rabbits were first brought to the americas in 1895, they were found to succumb to a deadly and extremely infectious disease, which nearly half a century later was found to be caused by a vector-borne myxoma virus acquired through contact with the native, more resistant host, the common wild rabbit of south america. european rabbits were first introduced into australia in 1859 for hunting, but by the 1880s they had become a major pest. to help in controlling the rabbit population, rabbits infected with this deadly virus were introduced in 1950 and the favorable weather conditions for mosquitoes that year helped to rapidly spread the disease, killing millions of rabbits. but, evolution and the power of natural selection then came into play. the myxoma virus that was first introduced killed over 99.9% of infected rabbits, yet a few rabbits survived the exposure. within a year, new strains of the virus appeared that killed only 90% of infected rabbits, and in subsequent years even more attenuated viral strains appeared. under such strong selective pressure, the rabbits, too, evolved to gain increased resistance such that the original, highly lethal virus would no longer kill more than 30% of the rabbit offspring. clearly, genetic changes in both the viral and rabbit populations quickly altered the outcome of the disease in terms of severity and persistence. today, only 50% of infected rabbits succumb during a myxomatosis epidemic. one of the most devastating recently emerged diseases, whose initial and continuing spread can be attributed to human behavior, is that of hiv/aids. hiv is thought to have originated in nonhuman primates [28] , but has become established in humans and is now transmitted human-tohuman through unprotected sexual practices, reuse of needles, and at first (although no longer) through contaminated blood supplies [29, 30] . aids was first noticed in the early 1980s as unusual occurrences of a rare cancer, kaposi's sarcoma, in young homosexual men. the social stigma associated with the disease gradually shifted with the realization that other populations were also at risk, including heterosexual and bisexual women, drug addicts, hemophiliacs, blood transfusion recipients, and babies born to hiv-positive mothers. the annual death toll rose linearly from 1987 with over 13,000 deaths to a height of nearly 42,000 in 1995, before a noticeable decline was observed with the introduction in 1996 of a cocktail of three anti-hiv drugs. by 1997 the annual death toll was down to 15,500 and has since declined to around the 12,000 mark [31] . however, although the death rate has declined, the number of cases reported annually in the u.s. still hovers around 40,000 [4, 32] . host-microbe co-evolution over time appears to be in effect for hiv/ aids. there are now a considerable number of hiv-positive individuals who have survived for many years without acquiring aids. a large part of this is due to advances in anti-hiv medications and improved healthcare. however, even before the increased availability of hiv medications, there were a significant number of individuals engaging in high-risk behavior that appeared to be resistant to acquiring hiv. by examining these ''survivors,'' scientists found a genetic mutation (allele) in a surface receptor, called ccr-5, which prevents the hiv virus from entering host cells [33] . these individuals having the mutant receptor allele are mostly of european decent. in some parts of europe, up to 20% of the population carry at least one copy of the mutant receptor allele, while populations in the rest of the world do not carry the same allele [34] . it is believed that this allele might have arisen through selective pressure from previous exposure of the population to another plague (perhaps bubonic plague, although this is not certain). but, importantly, the strong selective pressure that the new antiviral medications have placed on hiv has led to an accelerated deadly arms race. current antivirals, at an annual cost of over $10,000 per person, are targeted mainly against the viral outer coat proteins gp120 and gp41, the processing protease, and the reverse transcriptase. although these viral protein targets are less variable than others in the viral genome, hiv has a high mutation rate and the strong selective pressure has caused the virus to rapidly evolve in response [29] . the ever-evolving hiv makes developing new drugs a constant, and costly, challenge [35] . other pathogens have been around for quite some time, but have recently acquired new properties making them increasingly more deadly. during the 1990s, staphylococcus aureus emerged as one of the most common causes of hospital-acquired infections in the us. drug-resistant infections increase the risk of death, as well as the cost and duration of hospital stays. over a relatively short period of time s. aureus acquired genes that increased the bacterium's resistance to antibiotics [36] . a timeline of the emergence of these new strains of s. aureus clearly demonstrates the evolution of a pathogen that is under strong selective pressure to survive (table 6 ). once acquired, antibiotic genes can be spread from one microorganism to another through a process known as horizontal gene transfer, which involves uptake or transfer of dna encoding those resistance genes within or between different bacterial species. importantly, the antimicrobial resistance is maintained even after the selective pressure is removed. this exchange of genetic information is believed to contribute to the alarming rise in multidrug resistant bacteria [37] . rapid development of antimicrobial resistance is forcing clinical and pharmaceutical researchers to devise alternative, innovative approaches to respond to this threat [38] . hospitals are thought to be a major source of multidrug resistant bacteria, but agricultural practices involving usage of antibiotics as prophylactics and growth promoters in feed and crops have also played a role in its emergence [39] . eye-opening evidence for just how prevalent genetic exchange occurs in natural environments is provided by the example of the substantial increase in antibiotic resistance among both community and clinical isolates of bacteria in the gut [40] . tuberculosis (tb) is the leading cause of death in the world, and after a century of decline in the u.s., tb is once again on the rise, and alarmingly multiple drug-resistant strains have emerged. this increase in cases worldwide is attributable to a number of complex factors, including changes in the social structure and socioeconomic upheaval, the hiv epidemic, and a failure in some countries to improve public treatment programs. multidrug resistance in tb is a growing international health concern, because it has dramatically increased the difficulty in controlling the spread of tb and because of the high mortality rate associated with co-infection with hiv [41] . co-infection of multidrug-resistant tb with hiv fuels the transmission of tb by accelerating the progression of latent tb into active disease because of the damage that hiv causes to the host immune system, which normally controls tb infection. individuals that test positive for both tb and hiv often die with 1-6 months. a major epidemic of tb-hiv infections has spread across the former soviet union due to socio-economic changes in the country that have led to overcrowded housing, and in particular overcrowded prisons [42] . unfortunately, the pharmaceutical industry has largely abandoned tb drug development due to perceived nonprofitable consumer market--at risk populations are also the poorest [43] . the spread of any disease that is transmitted from human to human is greatly facilitated under crowded conditions, which allow for efficient exposure to a higher initial inoculum of the infectious agent. sporting events, concerts, and other gatherings of large numbers of people in a confined area can promote human-to-human transmission, as well as exposure to new populations. legionnaire's disease, caused by the bacterium legionella pneumophila, was first recognized in 1976 when it struck a group of american legion conference attendees in philadelphia [44] . this bacterial pathogen normally lives in fresh water as a parasite of amoeba, but unfortunately for us it can also live and thrive inside one type of our immune cells called a macrophage. the disease is acquired through aerosol exposure from contaminated water in ventilation systems, such as the air conditioning units in a hotel, or through aspiration during nasogastric tube feedings diluted with contaminated potable water in hospital or nursing-home settings [45] . since the first widely publicized incident on the holland-america cruise line in 2002, there have been numerous reports of cruise ship passengers succumbing to acute gastroenteritis caused by the norwalk virus [46] . a cruise, where hundreds of passengers and crew mingle in close contact, can provide optimum conditions for a virus to spread through food, water, and direct contact. cruise ships are now required to report all gastrointestinal illnesses to the cdc before entering a u.s. port, especially if 2% or more of the passengers or crew are ill. what has been most economically troubling for the cruise line industry is the recalcitrant nature of the virus to decontamination efforts [47] . overcrowding often leads to poor sanitation, resulting in accumulation of refuse, sewage, and vermin that thrive under such unsanitary conditions. natural disasters, civil disturbances, and war have caused large population displacements, with millions of people (and their animals) worldwide today living in refugee camps, which are overcrowded with poor sanitation and often without adequate food or clean water. these crowded camps provide ideal conditions for brewing and transmitting new infectious agents in malnourished or immunocompromised populations of humans and animals. the norwalk virus reared its ugly head once again during the katrina crisis in 2005 [48] . of the estimated 24,000 evacuees sheltered temporarily at facilities in reliant park, a sports and convention complex in houston, texas, 1,169 persons reported symptoms of acute gastroenteritis during a 2-week period at the beginning of september. medical personnel, police, and volunteers having direct contact with patients also reported symptoms, suggesting secondary transmission. although the local public health officials and the cdc implemented extensive infection-control measures, including publicizing the need for enhanced hygiene techniques, the outbreak continued for an additional week before declining. habitat destruction (e.g. deforestation, slash-burn practices) and urban expansion can uncover natural reservoirs and expose humans and domestic animals to new disease-causing microbes. each year 100-200 zoonotic cases of the pneumonic disease tularemia, caused by the bacterium francisella tularenesis, are reported in the us, primarily in arkansas, missouri, and oklahoma [49] . transmission usually occurs through arthropod bites, especially ticks or deerflies, but it can also occur through inhalation of contaiminated aerosols. in the late 1930s, rabbits from arkansas and missouri were introduced to cape cod and martha's vineyard, and cases of tularemia in massachusetts were reported shortly thereafter. martha's vineyard experienced two larger outbreaks in 1978 and 2000, which were linked to outdoor activities of mowing lawns and cutting brush [50] . the humans were presumably infected by inhalation of microbe-contaminated animal remains mechanically aerosolized by the cutting action of the mowers or brush cutters. pollution and exposure to waste water or sewage can also lead to the emergence of new diseases. coral black-band disease is a globally distributed disease that has been causing the degradation of coral reef ecosystems. first reported in the 1970s, the disease is observed as a pathogenic microbial consortium (mat) that migrates from the top to bottom of healthy coral, leaving behind dead exposed skeleton that disrupts the ecological and geological structures of coral reefs (see figure 1 ) [51] . a factor that appears to be contributing to the development and spread of coral black band disease is the pollution of seawater from industrial, municipal, and other terrestrial waste sites near the coral reefs [52] . modern technologies have led to greater efficiency in production, marketing, and commerce of goods around the world. rapid transport of imported material and tourism related travel facilitate the spread of infectious diseases around the globe and are clearly contributing to the increased prevalence and severity of the diseases. exotic souvenirs, including wild animals and their associated microbes, have been imported illegally into the u.s. from various parts of the world. an outbreak of monkeypox in 2003 among residents of wisconsin, northern illinois, and northwestern indiana was the result of infection from prairie dogs bought at a pet shop in texas that became infected after contact with various exotic african rodents shipped from ghana and then distributed by other pet shop outlets in the midwest [53] [54] [55] [56] . rare zoonotic cases of monkeypox in humans had been reported previously only in remote villages of central and western africa near tropical rainforests where there is close contact with infected animals [57, 58] . recent studies suggest that exposure to monkeypox in these areas has increased due to encroachment of humans into animal habitats. the cdc and fda subsequently embargoed all african rodents into the u.s. and banned the distribution or sale of african rodents and prairie dogs in the u.s. [59] . trade routes and human practices have contributed to the spread of numerous diseases throughout history, but the speed with which they are spreading today have demanded the need for ever more rapid response and containment measures to be in place. an interesting example is that of cholera, caused by the cholera toxin-producing bacterium vibrio cholerae. in asia, cholera has been endemic for hundreds, maybe thousands of years, and cholera-like disease has been described in a number of ancient texts. the first well-documented epidemic in europe occurred in 1871. since 1871, seven major cholera pandemics have occurred [60] . the first six were caused by the classical o1 biotype, whereas the seventh, which began in 1961 and persists today, is caused by the el tor o1 biotype. in 1991, el tor reemerged in peru after a hiatus of over 100 years, and rapidly spread throughout central and south america over the following couple of years, with more than 1.5 million cases and over 10,000 deaths [61] . the spread of el tor in these countries could be traced along the major north-south coastal trucking route and is attributed to poor sanitation in these areas. the most recent cholera outbreaks have occurred in developing countries, such as angola, where civil strife has hindered water treatment and sanitation efforts [62, 63] . the extent of the global cholera burden has been grossly underreported [62] , in part due to limited resources, but also due to the detrimental effects such news can have on trade and travel to those regions. in some endemic areas, such as bangladesh, improved management strategies by the government and who, including aggressive rehydration therapy and antibiotics, have shortened the duration of illness and have reduced the fatality rates from natural cholera epidemics, which are largely seasonal in nature. in 1992, a new strain of vibrio cholerae, designated o139 or ''bengal,'' caused a massive cholera epidemic in south asia [64] . what was most disturbing about this new strain was its high prevalence in adults, suggesting that prior immunity gained during childhood through exposure to the classical or el tor o1 strains offered little or no protection against this new o139 strain. its subsequent spread to other asian countries lead some to worry that it may cause an eighth cholera pandemic, but luckily so far this has not materialized due to timely mobilization of effective control measures by researchers and healthcare officials. however, an emerging concern is the increased incidence of antibiotic resistant strains of vibrio cholerae in bangladesh. nearly all isolates are now resistant to the less expensive antibiotics, tetracycline, trimethoprinsulfamethoxazole, and erythromycin. although most are still sensitive to ciprofloxacin, the effective doses needed for treatment are increasing. seasonal changes in rainfall and sunlight can trigger periodic or transient emergence of some human pathogens such as cholera. an intriguing observation comes from the study of the annual epidemic profile of endemic cholera in the bengal region of bangladesh and india, where nearly all cases occur in a synchronized, explosive outbreak during major transitions of climate in the post-monsoon months of october and november [65] . as the rains decline and sunlight increases there is a burst of algal and zooplankton bloom. it has been proposed that the increased concentrations of these particles (surfaces to which the bacteria adhere) in drinking water sources consequently increase the rates of ingestion [66, 67] . during other times in the year, cholera cases occur only sporadically because the zooplankton sediment and there is less ingestion of bacteria-coated particles. recently, an additional factor has been credited toward the seasonal cholera epidemics, namely predation of the v. cholerae bacteria by bacteriophage (viruses that infect bacteria) due to amplification of the phage in the intestines of humans, followed by release into the environment [68] . support for this model comes from the inverse correlation of the phage count with the abundance of toxigenic v. cholerae in water samples and with the incidence rates of cholera [65, 69] . climate change can also dramatically alter the spread of arthropodborne diseases, which are most prevalent in a limited range of temperatures or environments preferred by these vectors. shifts in warming or cooling trends may extend or narrow the range of such vectors and the diseases they transmit. drought or flooding can also lead to spread of disease into new populations of animals or humans. the west nile virus is an example of a recently emerged vector-borne disease that has been introduced to a new geographic area. the virus was first isolated in uganda in 1937 and has since been known to cause disease in africa, west asia, europe, and the middle east [70] . until 1999 when it caused a deadly outbreak in the new york metropolitan area, it had never been observed in the u.s., but now it has spread to every state, except alaska and hawaii, as well as canada and mexico. as of march 2007, the cumulative number of human disease cases in the u.s. is 4,256 [71] . the west nile virus is usually transmitted between birds by mosquitoes, but can be transmitted to humans and other hosts, particularly during favorable seasonal conditions with a hot dry summer followed by a wet fall, as what occurred in the new york area in 1999. its introduction into the u.s. is thought to have occurred recently since the genetic profiles of the new york virus isolates suggest they came from a single source, which is related to a virus isolated in 1998 in israel [72] . although not known for certain, it is possible that an infected bird could have been imported or an infected mosquito or tick may have hitched a ride on an international flight or on a ship carrying old imported tires infested with mosquito larvae. large holding and storage facilities for meat, grains, dairy and produce provide new habitats and breeding grounds for insects and vermin such as mice and rats. humans can be infected with the deadly hantavirus through inhalation of aerosolized virus present in dried rodent urine in grains or feedstuffs. in 1993 the southwestern u.s. experienced a mysterious outbreak of a new deadly respiratory illness in healthy people, which within a couple of months was identified by the cdc as a previously unknown type of hantavirus [73, 74] . because the researchers knew that other hantaviruses were transmitted by rodents, they began trapping mice and rats in the area around the victims' homes and discovered that the deer mouse was the primary natural reservoir. further investigation revealed that there had been earlier unexplained deaths due to this hantavirus, but these cases were sporadic. the reason for the clustered outbreak in the 1993 could be connected to the unusually high numbers of mice in the area during that season [75, 76] . for several years, the region had experienced drought, but in early 1993, heavy snows melting and rainfall helped revive the flora and fauna in the region, such that the deer mice had plenty to eat. the mice increased dramatically in numbers, and consequently increased the likelihood of transmission to humans. intense cross and intraspecies interactions are conducive to transmission of a pathogen from one host to another. the rapidity with which microbes and viruses are able to evolve increases the likelihood of such close host-host contacts to cause the pathogen to ''jump'' across species barriers. like bse, chronic wasting disease (cwd) is a prion-mediated transmissible spongiform encephalopathy of cervids, such as mule deer, white-tailed deer, and rocky mountain elk [77] . the potential for cwd to similarly cross the species barrier from cervids to humans is considered unlikely. but, because bse has been transmitted from cattle to humans (as vcjd), it is feared by some that cwd might also ''jump'' the species barrier. although cwd can be transmitted to cattle, sheep, and goats by direct inoculation into the brain [78] , studies have not yet demonstrated that domestic livestock are susceptible via oral exposure, the presumed natural route of exposure to bse [79] . it is feared that homology within critical amino acid sequences of the human and cervid proteins might facilitate cross-species transmission of cwd to humans, as what appears to have occurred for bse. thus, understanding how prions overcome resistance to transmission between species is crucial if we are to prevent future epidemics. although surveillance efforts for cwd in captive and free-ranging cervids are continu-ing, eradication of cwd from wild populations of cervids is unlikely with currently available management techniques. the potential emergence of a new disease-causing zoonotic agent that is transmissible between humans is a major concern. constant exposure and certain behaviors increase the likelihood that a virus will ''jump'' species. we experienced a frightening example of this with the rapid worldwide spread of the sars virus. alarmingly, we are currently at the brink of experiencing another such emergence, which could have devastating consequences on the human population. already the current spread of avian influenza a virus has resulted in the death from disease or culling of over 300m domestic poultry in asia, with an estimated $10b in economic losses to the asian poultry sector (table 7 ) [5] . a question on many people's mind today is whether another pandemic flu like the one in 1918 is inevitable [80] . we already know that the circulating influenza h5n1 virus can ''jump'' from birds to humans [81] , but luckily we have not yet observed significant human-tohuman transmission other than a few cases through intimate unprotected contact with a critically ill index patient [82] . will this fine dividing line be crossed soon? or, will this threat diminish before it evolves into a more human-specific virus? the truth is that we know very little about the specific factors that trigger a ''jump'' between species or a transition into a rapidly transmissible virus [83] . we know even less about how to prevent these events from occurring or how to predict when they will occur. the current h5n1 strain, first limited to poultry, quickly spread to migrating birds, but has now emerged in mammals and humans mostly through zoonotic contact. previously, it was widely accepted that avian viral strains could only readily infect humans after first having undergone genetic shuffling within swine, but now it appears that direct transmission from bird to human can occur [84] . although its transmission from human-to-human is (luckily) still inefficient, the who, the cdc, and other organizations have already mobilized for just such an event [85] . distinguishing a deliberately introduced infectious disease from a naturally occurring or emerging infectious disease is inherently more difficult due to their ''dual-use'' nature. the good news, though, is that effective medical treatment and prevention strategies for combating a naturally occurring infectious disease will most likely work just as well for one that is deliberately introduced. the exponential advances that have been made in the life sciences, medicine, and biotechnology have not only dramatically enabled our ability to respond to biological threats, but, sadly they have also increased the potential risks of malevolent exploitation and inadvertent misuse. indeed, a report from the u.s. national research council and the institute of medicine concluded that the breadth of potential biological threats is far wider than is commonly appreciated and will continue to expand in the future [86] . the nih invests over $28b annually in medical research. since 2001, nih has directed over $10b toward countering bioterrorism alone and currently spends over $3b of its annual budget on infectious diseases with over $1.8b going toward emerging infectious diseases (table 4) [87] . considerable attention has been recently focused on developing better preparedness and surveillance (early warning) as strategies for more effective response to biological threats. this depends on having reliable, sensitive and rapid means for recognition of unusual events or unexpectedly high levels of common events. a number of animal and human health laboratory response networks have been established with the goal of maintaining an integrated national and international system for facilitating standardization and movement of information, for expansion of detection and diagnostics measures, for coordinating responses among federal, state, university and commercial clinical laboratories, and for identification of common source outbreaks. on the international front, the who and cdc have increased activities to build capacity for global disease detection and response, with immediate focus on and strengthening of influenza surveillance. the global alert and response network (goarn) was established in 2000 by who as a partnership of >140 institutions and networks to mobilize human and technical resources for the rapid identification and control of disease outbreaks that are of international importance [88] . the global livestock early warning system (glews) has been formed by the food and agricultural organization (fao) of the united nations and the world health organization for animal health (oie) to strengthen epidemiological analysis and prediction of major animal diseases and zoonoses and to improve reporting from a variety of data sources that might impact disease transmission from animals to humans [89] . the recent pandemics have also mobilized strengthening of the international health regulations, which were first established by who in 1969 to ensure maximum security against international spread of certain diseases (cholera, plague, yellow fever, and smallpox--although smallpox was removed from the list in 1981) with minimum interference of world commerce. in 2005 a revised set of regulations was adopted unanimously at the world health assembly to increase the roles and responsibilities of who and member states, including financing, developing, strengthening, maintaining and implementing core surveillance, and response capacities [90] . biosecurity requires multipronged, flexible, and interdisciplinary approaches to combat the perpetuation and spread of infectious diseases. in all, simultaneous use of multiple strategies will be necessary for effective infection control and disease management. one such strategy is to use evolutionary engineering (combining predictive evolution and genetic engineering) to design vaccines and drugs based on predictive targets. for example, an innovative approach for control of e. coli o157:h7 contamination of food and water was recently employed to eliminate the source for the organism. by vaccinating the animal reservoir--cattle--the researchers were able to prevent colonization of the cattle with the microbe, reducing the levels of bacteria shed in feces and thereby reducing the risk of human disease [91] . this strategy was shown to significantly decrease the prevalence of e. coli o157:h7 in a clinical trial conducted in a feedlot setting. improved sanitation and use of chlorinated drinking water has dramatically reduced the incidence of waterborne disease. evidence suggests that the cholera epidemic in south and central america was caused by a complex set of circumstances, including poor sanitation conditions, poor separation of drinking water and waste streams, and inadequate water treatment and distribution systems [61] . indeed, outside of peru's capital lima, chlorination of drinking water supplies at the time of the epidemic was limited at best. improved water quality and sanitation have since reduced the incidence of cholera in south american countries. another simple, yet surprisingly effective strategy recently implemented has been the use of filtering water through multilayered cloth filters to remove the plankton and other particles to which the vibrio bacteria adhere [92] . over the past 25 years an unprecedented mobilization of resources have been directed at stopping the hiv pandemic, ranging from preventive strategies for persons at high risk for contracting hiv, such as educational counseling, testing, and referral services, to treatment with multiple drug regimens, to management measures aimed at improving the healthcare of persons living with hiv and preventing further transmission [93] . although enormous success in prevention of hiv/aids in the u.s. has been achieved and we have learned a lot about how to approach rapidly evolving diseases from this experience, a number of prevention and treatment challenges remain. hiv prevalence remains high among homosexual men [94] and racial/ethnic disparities have increased, especially among african-american men and women [95] with prevalence among african-american men reported as high as 46% [96] . new programs are needed to more effectively reach these populations [93] . one approach toward improved treatment and reduction of drug resistance is the use of multidrug overkill (triple drug therapy). administration of just a single drug often leads to the development of resistance to that drug, but strong multidrug doses decrease the likelihood of multidrug resistance. for example, recent studies have shown that triple drug combination antiviral therapy in treating hivinfected persons offer superior viral suppression over other drug regimens [97] . when two or more drugs are used simultaneously, each helps prevent the emergence of resistance to the other drug. effective regimens for the treatment of tb must contain four different drugs to which the organisms are susceptible. to illustrate how this might be so effective, consider that mutation rates in the tb-causing bacterium lead to a frequency of resistance to isonazid of 1 in 10 8 , to streptomycin of 1 to 10 8 , to ethambutol of 1 to 10 7 , and to rifampicin of 1 to 10 9 . bacterial mutants resistant to any single drug are naturally present in any large bacterial population. an inactive tb granuloma contains 10 2 -10 4 bacteria, whereas an active tb lesion contains 10 7 -10 9 bacteria. this means that the chance of gaining resistance to any one of the drugs is relatively high in an active lesion, but the chance of gaining resistance to multiple drugs is considerably less [98] . the course of the four-drug treatment for tb usually lasts from 6 to 9 months. when adherence with the regimen is assured, the four-drug regimen is highly effective; however, a problem with tb treatment is that the drugs used are often counter-indicated, cause unpleasant side effects, and must be administered in series over a long period of time rather than simultaneously, which leads to problems with patient compliance [43] . nearly half of individuals with tb do not complete their treatments. reduction of noncompliance can be achieved by direct observation of the patient to ensure full dosage. in developed countries, such as the u.s., this is relatively easy to achieve with the use of healthcare workers and family members. however, in developing countries, there are many obstacles to adhering to treatment regimens, and alternative strategies to improve compliance are needed. in addition to direct observation of treatment, other tactics include sending reminder cards or phone calls, monetary incentives, health education and counseling, and making access to clinic facilities easier [99] . another strategy for reducing the prevalence of antimicrobial resistance is to remove the overall selection pressure by minimizing exposure to the drug, and especially withholding the most effective drugs, i.e. the ''drugs of last resort,'' until absolutely needed. this is becoming more and more difficult to accomplish with the accelerated rate of spread of antibiotic resistance through the overuse and over-prescription of antibiotics [37, 39, 100] . indeed, many researchers were dismayed at the large distribution of ciprofloxacin (cipro tm ) to treat over 60,000 people after the anthrax attack in 2001 and warned that this widespread use could lead to resistance in other bacteria [101] . the strain of bacillus anthracis strain used in the attack was equally sensitive to other less expensive and more commonly used drugs. ciprofloxacin is considered a ''drug of last resort'' because of its broad-spectrum and efficacy against many pathogenic bacteria, particularly those (such as staphylococcus) that are already resistant to other drugs. prescreening for sensitivity is another approach that allows for use of narrow-range rather than broad-spectrum antimicrobials, which further reduces the likelihood of resistance developing and spreading to other pathogens. management and implementation of effective preventive or containment measures in the event of natural or man-made biological threats will require increased infrastructure and diagnostic and surveillance capabilities. for example, the tremendous scale-up in food production, from the vast herds of cattle to the huge confined feedlots to the slaughterhouses to the many hundreds of distributors and supermarkets, has undoubtedly contributed to the emergence and prevalence of food-borne diseases such as that caused by e. coli o157:h7. the complexity of the modern food preparation and distribution process makes epidemiological tracking of the sources of contamination difficult, although there have been noticeable advances. in response to the need for improved agriculture and food biosecurity, congress passed the national agriculture and food defense act of 2007, which will require enormous effort and financial commitment on the part of the government and agricultural and food industries. the logistics involved are daunting, particularly in coordinating efforts among many different agencies. one example is the food-borne diseases active surveillance network (foodnet), which is a collaborative project of the cdc, the usda, and the fda to monitor and q 2008 wiley periodicals, inc. study the epidemiology of food-borne diseases [102] . in response to the growing need for better food surveillance, the fda implemented the hazard analysis and critical control point (haccp) program for prevention of food-borne diseases, which involves monitoring food distribution at critical control points where contamination is most likely to occur [103, 104] . in 2005 the food safety and inspection service (fsis) of the usda established the food emergency response network (fern), which will work with the fda and its haccp program to integrate a laboratory network across the u.s. that can quickly respond to food-related emergencies [105] . the fsis, as part of its task of protecting public health through food safety and defense, recently implemented a new assessment method, called carver1shock [106] , for identifying the most vulnerable target sites within a food processing system (table 8 ). in considering the gaps in the u.s. agriculture and food defense capabilities, the main question that must be addressed is: exactly what level of security do the people of the usa want the agricultural industry to achieve, and our government to enforce, in terms of food safety and biosecurity? it is clear that we (as a nation and international community) are not happy with the current security status of our food and agricultural supplies. the outbreaks and ensuing deaths, economic losses and drop in consumer confidence resulting from contaminated spinach by e. coli o157:h7 [11] , mentioned above, amply demonstrate this point. but, if we take this episode as an example, one must question exactly what could have been done to prevent or further mitigate this outbreak than was already done. by all accounts, the surveillance, detection, evaluation, containment, and recovery measures were remarkably fast, accurate, and as good as one could possibly hope, considering the circumstances-far better than in previous incidents of a similar nature, thanks to improved measures newly in place. this was considered an accidental outbreak, but what if it had been a deliberate attack? undoubtedly, the response would have been little different. yet, what happened is still deemed by most as unacceptable. so, how could this be improved? most current measures are aimed at further improvements to detect or con-tain a future incident by increasing surveillance at the front-end, increasing diagnostics or enhancing epidemiological monitoring capabilities once an incident has occurred. but, just how much of an improvement would that be? clearly, what the public wants is a near certainty that such an event as occurred with the spinach will not happen again. part of the difficulty in adequately addressing this need is that current efforts are focused on too broad and vast a target (there are just too many steps in the food processing, where things can go wrong and something could slip through the cracks). instead, the focus for ensuring near complete biosecurity should be at the very end of the food processing chain, namely at the packaging and delivery stage. while all the proposed surveillance, detection, evaluation, containment and recovery measure will significantly reduce the possibility of future contamination, and thus should be done to the extent possible, they will not ensure the desired near-complete protection from an incident (which is what the consumer is demanding). so, how can this be achieved? many scientists see food irradiation as one viable solution. food irradiation of already processed and packaged food through promising new technology can eliminate disease-causing microbes from foods. the effects of ionizing radiation on food and on animals and people eating the irradiated food have been extensively studied and deemed safe [107] [108] [109] , and indeed this technology has been implemented already for certain foodstuffs. yet, bringing this technology into use for most foods has been challenging, primarily due to misconceptions and fear about its safety on the part of the public and policymakers. closing this gap will require a ramping up of public engagement by the scientific community. somehow scientists must better communicate with the public and policymakers, convincing them of the benefit criticality-measure of public health or economic impacts of an attack to achieve terror goals accessibility-ability to physically access and egress from the target recuperability-ability of system to recover from an attack vulnerability-ease in accomplishing the attack effect-amount of actual direct economic loss from an attack as measured by loss in production recognizability-ease of identifying the target shock-the combined physical, health, economic and psychological effects of an attack the carver1shock method rates each of seven attributes on a scale of 1-10 for identification of vulnerable sites in food processing and distribution system that might be targets for attack or points of contamination. of this technology and at the same time alleviating their fears of its use. to achieve global health biosecurity it is critical that the communication and educational barriers between nonscientists and scientists are overcome and that efforts by public healthcare, scientific and security policy communities are better integrated. enhanced coordination between multiple agencies is essential for implementing and maintaining global disease surveillance systems, public healthcare, and diagnostic and basic research laboratories. thus far, many redundancies in effort remain and interagency cooperation is still fragmentary. international cooperation has improved with regard to regulatory and containment (import/ export) policies, particularly in the area of travel restrictions during pandemics, but cooperation is still a bit shaky in other areas where commerce and political issues are concerned. and, much work remains in the areas of regulation of agricultural, societal and medical practices, pollution control, and prevention of habitat destruction. strengthening national and international capacities to prevent and control disease epidemics will require continued promotion of international cooperation and technical partnerships with institutions and networks around the globe to mobilize resources for control of disease outbreaks. regulation and oversight of research and use of potentially dangerous bioagents (''select agents'' or highrisk or high-impact bioagents) is well underway, but there remain disparities between that conducted in the u.s. and that done elsewhere in the world. biosafety and biosecurity of pathogens in laboratories and healthcare settings will require enhanced education for better preparedness and increased confidence of the public and policy makers in science. many scientists feel that policymakers have not fully understood the difference between biosafety and biosecurity and have consequently imposed a number of mandates and regulations that equate enhanced need for security with enhanced danger, i.e. greater biosafety risk. balancing biomedical and biotechnological advancement with biosecurity will always be at odds due to the ambiguous definition of what constitutes a potential biosecurity threat, but improved education will help with making those decisions. although warning and prevention are preferable to coping with the consequences of an attack, an emphasis must also be placed on improving public healthcare and basic research and education. it is critical that we develop a homeland and global biosecurity strategy that is applicable to both intentional and unintentional disease outbreaks. the best defense against any microbial threat is a robust public healthcare system in regard to its science understanding, capacity, and practice. there has been considerable advancement in the area of prevention, with improved surveillance and detection, and with new drugs becoming available at a remarkably rapid pace, but we are still only at the tip of the iceberg in our understanding of pathogen evolution, post-exposure treatment and control, prediction of epidemic versus pandemic spread, rates of transmission, impact of climate change, and preparation for handling a biothreat agent of unknown origin, whether it be from natural, accidental, or deliberate exposure. h2n2) epidemic 1963 (h7n3) england, turkey 1902 (h3n2) 1966 (h5n9) canada, turkey 1918 (h1n1) ''spanish'' pandemic 1976 (h7n7) australia, chicken 1933 (h1n1) 1979 (h7n7) germany, chicken 1947 (h1n1) 1979 (h7n7) england, chicken 1957 (h2n2) ''asian'' 1983-1985 (h7n7) pennsylvania (17m killed) 1968 (h3n2) swine'' non-epidemic 2002 (h7n7) hong kong (>1m killed) 2003 (h7n7) netherlands (30m killed) 2004 (h7n3) canada (17m killed) 2004-present (h5n1) asia, pandemic report of the committee on emerging microbial threats to health in the 21st century on ''microbial threats to health: emergence, detection, and response global and regional burden of disease and risk factors measuring the global burden of disease and epidemiological transitions: 2002-2030 deaths: preliminary data for avian influenza: an omnipresent pandemic threat an open letter to elias zerhouni bioterror threat exaggerated fda's role in tracking and resolving the recent e. coli spinach outbreak food safety system: contaminated spinach prompts calls for major overhaul. c & en news on the trail of ebola and marburg viruses molecular evolution of viral fusion and matrix protein genes and phylogenetic relationships among the paramyxoviridae morbillivirus infections, with special emphasis on morbilliviruses of carnivores the transmissible spongiform encephalopathies: emerging and declining epidemics variant cjd (vcjd) and bovine spongiform encephalopathy (bse): 10 and 20 years on: part 1 variant cjd (vcjd) and bovine spongiform encephalopathy (bse): 10 and 20 years on: part 2 public health and the bse epidemic foot and mouth disease: the human consequences. the health consequences are slight, the economic ones huge carnage by computer: the blackboard economics of the 2001 foot and mouth epidemic international union for conservation of nature and natural resources the vine to love or hate controlling exotic plants in your forest biological control of vertebrate pests: the history of myxomatosis, an experiment in evolution. cambridge aids as a zoonosis: scientific and public health implications the causes and consequences of hiv evolution the biology and evolution of hiv aids deaths fall by nearly one half deaths: leading causes for ccr-5 genotype and sexual transmission of hiv-1 resistance to hiv-1 infection in caucasian individuals bearing mutant alleles of the ccr-5 chemokine receptor gene the rationale and development of new drugs to treat hiv infection emergence and resurgence of meticillin-resistant staphylococcus aureus as a public-health threat changing paradigms in combating antibiotic-resistant bacteria ecology and physiology of infectious bacteria-implications for biotechnology veterinary use and antibiotic resistance evidence for extensive resistance gene transfer among bacteroides spp. and among bacteroides and other genera in the human colon current status of some antituberculosis drugs and the development of new antituberculous agents with special reference to their in vitro and in vivo antimicrobial activities epidemic of tuberculosis in the former soviet union: social and biological reasons new tuberculosis drugs in development legionella pneumophila pathogesesis: a fateful journey from amoebae to macrophages legionnaires' disease in long-term care facilities: overview and proposed solutions outbreaks of gastroenteritis associated with noroviruses on cruise ships -united states norwalk-like virus: public health consequences and outbreak management norovirus outbreak among evacuees from hurricane katrina epidemiologic and molecular analysis of human tularemia an outbreak of primary pneumonic tularemia on martha's vineyard cyanobacteria associated with coral black band disease in caribbean and indo-pacific reefs partitioning of bacterial communities between seawater and healthy, black band diseased, and dead coral surfaces update: multistate outbreak of monkeypox -illinois the detection of monkeypox in humans in the western hemisphere monkeypox transmission and pathogenesis in prairie dogs re-emergence of monkeypox in africa: a review of the past six years outbreak of human monkeypox, democratic republic of congo cdc. monkeypox factsheet -embargoed african rodents and monkeypox virus the 1991 cholera epidemic in peru: not a case of precaution gone awry review of reported cholera outbreaks worldwide getting serious about cholera emergence and evolution of vibrio cholerae o139 seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages critical factors influencing the occurrence of vibrio cholerae in the environment of bangladesh cholera and climate: revisiting the quantitative evidence self-limiting nature of seasonal cholera epidemics: role of host-mediated amplification of phage hostinduced epidemic spread of the cholera bacterium west nile encephalitis epidemic in southeastern romania origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states outbreak of acute illness -southwestern united states update: hantavirus disease -united states etiology and epidemiology of the four corners hantavirus outbreak hantavirus infections and outbreaks in 1993 chronic wasting disease experimental transmission of chronic wasting disease agent from mule deer to cattle by the intracerebral route species barriers in prion diseases-brief review the origins of pandemic influenza-lessons from the 1918 virus avian influenza a (h5n1) infection in humans probable person-to-person transmission of avian influenza a (h5n1) transmission of influenza a in human beings review: molecular evolution and the feasibility of an avian influenza virus becoming a pandemic strain -a conceptual shift pandemic influenza: are we ready? disaster manag response report of the committee on advances in technology and the prevention of their application to next generation biowarfare threats on ''globalization, biosecurity, and the future of the life sciences global outbreak alert and response network (goarn) world health organization, global early warning system for major animal diseases world health organization decreased shedding of escherichia coli o157:h7 by cattle following vaccination with type iii secreted proteins reduction of cholera in bangladeshi villages by simple filtration evolution of hiv/aids prevention programs -united states hiv/aids diagnoses-33 states racial/ethnic disparities in diagnoses of hiv/aids-33 states unrecognized infectin, and hiv testing among men who have sex with men -five us cities an updated systematic overview of triple combination therapy in antiretroviral-naive hiv-infected adults the resumption of consumption -a review on tuberculosis interventions for promoting adherence to tuberculosis management playing chicken with our antibiotics. overtreatment is creating dangerously resistent germs researchers question obsession with cipro foodborne diseases active surveillance network the integration of process analytical technologies, concurrent validation, and parametric release programs in aseptic processing use of microbial data for hazard analysis and critical control point verification-food and drug administration perspective food emergency response network -fern cooperative agreement food and drug administration center for food safety and applied nutrition, carver 1 shock software tool for food defense and terrorism the role of irradiation in food safety irradiation of food-helping to ensure food safety food irradiation: a safe and useful technology key: cord-332588-k4tghibp authors: d’alessandro, sarah; scaccabarozzi, diletta; signorini, lucia; perego, federica; ilboudo, denise p.; ferrante, pasquale; delbue, serena title: the use of antimalarial drugs against viral infection date: 2020-01-08 journal: microorganisms doi: 10.3390/microorganisms8010085 sha: doc_id: 332588 cord_uid: k4tghibp in recent decades, drugs used to treat malaria infection have been shown to be beneficial for many other diseases, including viral infections. in particular, they have received special attention due to the lack of effective antiviral drugs against new emerging viruses (i.e., hiv, dengue virus, chikungunya virus, ebola virus, etc.) or against classic infections due to drug-resistant viral strains (i.e., human cytomegalovirus). here, we reviewed the in vitro/in vivo and clinical studies conducted to evaluate the antiviral activities of four classes of antimalarial drugs: artemisinin derivatives, aryl-aminoalcohols, aminoquinolines, and antimicrobial drugs. antimalarial drugs used for the treatment and prevention of malaria are classified in a heterogenic group [1] . they are usually divided based on the chemical structure or the source of the drugs. most of them derive from traditional medicine and plants. after identification of the active principles, chemical modifications are introduced to increase the activity and ameliorate the selectivity index. they present different modes and various mechanisms of action, which are often still not elucidated, against malaria parasites. furthermore, due to the complexity of these molecules, additional side activities have been reported. for these reasons, antimalarial drugs have been studied, proposed, and sometimes used for the treatment of other pathologies, such as cancer, autoimmune diseases, and nonmalaria infectious diseases [2] [3] [4] . moreover, the geographical overlaps between malaria and viral-related diseases [5] [6] [7] have led to the consideration of possible use of antimalarial drugs as new antiviral drugs. finally, the lack of new effective antiviral drugs and vaccines against many viral infections has strengthened interest in the potential antiviral activity of antimalarial drugs. in the present review, the authors present the use and the efficacy against human viruses of the principal antimalarial drugs, divided into four main groups: artemisinin derivatives, aryl-aminoalcohols, aminoquinolines, and antimicrobial drugs. the chemical structures of the cited compounds are summarized in figure 1 . works on newly synthesized derivatives, which are not licensed, were not taken into consideration. when possible, original manuscripts were cited. however, previous works of other scientists were acknowledged also by citing review articles, aiming to provide a comprehensive list of published papers on the proposed field. artemisia annua (qinghao) is a plant of the asteraceae family, which has been used for ages in traditional chinese medicine [8] . the sesquiterpene lactone artemisinin (art), the active principle, was discovered in the 1970s. since then, chemical structural modification studies have been performed to obtain new compounds with enhanced antimalarial activity and improved pharmacological properties. art derivatives are safe and well-tolerated drugs. this safety is one of the reasons why they have been studied for their efficacy in other diseases beyond malaria. art derivatives are active against other parasites, cancer cells and viruses, although with lower potency, with effective concentration50s (ec50s) in the micromolar range, compared to the nanomolar range as antimalarials [9] . the majority of the literature describes the antiviral effect of art derivatives in vitro toward human cytomegalovirus (hcmv). a large contribution to the field was given by thomas efferth and his collaborators, with both original research articles [10] [11] [12] [13] and reviews [14, 15] . art, the active principle extracted from artemisia annua, is poorly soluble in water and oil, thus, the development of semisynthetic derivatives was necessary for appropriate formulation. poor physicochemical properties may account for the scarce literature about the use of art as an antiviral. art, dihydroartemisinin (dha) and artesunate (as) were compared for their anti-hcmv effect in a fibroblast cell model by measuring viral dna synthesis in cellular lysates. art showed the lowest artemisia annua (qinghao) is a plant of the asteraceae family, which has been used for ages in traditional chinese medicine [8] . the sesquiterpene lactone artemisinin (art), the active principle, was discovered in the 1970s. since then, chemical structural modification studies have been performed to obtain new compounds with enhanced antimalarial activity and improved pharmacological properties. art derivatives are safe and well-tolerated drugs. this safety is one of the reasons why they have been studied for their efficacy in other diseases beyond malaria. art derivatives are active against other parasites, cancer cells and viruses, although with lower potency, with effective concentration 50s (ec 50 s) in the micromolar range, compared to the nanomolar range as antimalarials [9] . the majority of the literature describes the antiviral effect of art derivatives in vitro toward human cytomegalovirus (hcmv). a large contribution to the field was given by thomas efferth and his collaborators, with both original research articles [10] [11] [12] [13] and reviews [14, 15] . art, the active principle extracted from artemisia annua, is poorly soluble in water and oil, thus, the development of semisynthetic derivatives was necessary for appropriate formulation. poor physicochemical properties may account for the scarce literature about the use of art as an antiviral. art, dihydroartemisinin (dha) and artesunate (as) were compared for their anti-hcmv effect in a fibroblast cell model by measuring viral dna synthesis in cellular lysates. art showed the lowest activity, even when fractional doses and daily repeated administration were used to counteract the problem of instability of the compounds in a culture medium [16] . compared to other compounds from traditional chinese medicine, art and as were also the most active, with low toxicity, for the inhibition of the hepatitis b virus (hbv), measured by hepatitis b surface antigen (hbsag) and dna release in a culture medium. moreover, synergism with the antiviral lamivudine was demonstrated [17] . art downregulated the oncogenic human papillomavirus (hpv) 39 proteins e6 and e7 in an in vitro model of cervical carcinoma [18] . these results partially confirmed the report by disbrow and colleagues, who observed an antiproliferative effect of dha on canine oral papillomavirus [19] . art also inhibits hepatitis c virus (hcv) replicon replication [20] , and the effect is synergistic with hemin, an iron donor [21, 22] . finally, art showed inhibition of human immunodeficiency virus (hiv) replication, but the effect was not reproducible in different cell models [23] . the activity of as on the replication of hcmv was demonstrated in different in vitro cell models, such as fibroblasts [11, 16, 24] and tumor cells [25, 26] . compared to other art derivatives, as had the highest activity [16, 27] and an activity comparable to or even higher than that of classical antiviral drugs, such as ganciclovir [16, 25, 28] . the antiviral effect was confirmed against different, multidrug-resistant strains [10, 25, 27, 29] and with different methods, such as the plaque assay and luminescent/fluorescent methods based on the use of transgenic viral strains [24, 30, 31] . in some cases, the effect of as was synergistic with antiviral drugs such as maribavir [27, 32, 33] , lamivudine [17] , ganciclovir [34] , foscarnet, cidofovir, letermovir [33] . however, in vitro data regarding synergism are not always confirmed in other experimental conditions, as for the case reported by morère, where synergism between maribavir and as was not confirmed in an ex vivo placenta model [32] . moreover, few data obtained with animal models are available. in the rat cmv/rat model, as demonstrated antiviral activity, measured as the dissemination of virus to the salivary glands, the number of viral genome copies, and virus titers in salivary glands. this activity was exerted only when as was coadministered with iron in the ferrosanol™ formulation [11] . as in association with valacyclovir was tested in a murine model of herpes simplex virus encephalitis (hse) to evaluate not only the antiviral but also the immunomodulating activity, which would be beneficial in this disease model. the survival rates of mice treated with both drugs were higher than those of mice treated with valacyclovir alone, but no significant difference was observed in the brain viral loads. levels of cytokines such as interleukine (il)-1β, il-2, il-6, interferon (ifn)-γ, and ccl2 were reduced in mice treated with valacyclovir combined with art versus those in mice treated with valacyclovir alone [35] . clinical data supporting the use of as against hcmv are contrasting. in a case report, a rapid decrease in viral load was observed in a stem cell transplant recipient infected with a newly identified foscarnet-resistant and ganciclovir-resistant hcmv strain and treated with as [12] . in this regard, a clinical trial was registered for the use of as in stem cell transplant recipients, but although the recruitment of 20 patients in israel has been completed, no results are available yet (table 1) . no difference between cqand placebo-treated groups [37] orthomyxoviridae iav chloroquine nct01078779 no prevention of iav infection [38] retroviridae hiv hydroxychloroquine [39, 40] reduction in hiv-1 rna load in plasma hydroxychloroquine isrctn30019040 increased hiv-1 replication and decreased cd4 numbers [41] chloroquine nct00819390 modest reduction in immune activation [42] chloroquine nct02004314 patients did not experience any improvement after cq treatment [43] chloroquine nct01650558 terminated, awaiting results [44, 45] on the other hand, a renal transplant recipient patient with documented valganciclovir resistance mutations in hcmv was treated with as, with no positive effects [46] . a third case report of a patient with multidrug-resistant hcmv infection is difficult to interpret due to the complicated series of treatments after two hematopoietic stem cell transplantations and consequent acute graft-versus-host disease episodes. in this patient, as given in association with maribavir was withdrawn two weeks after initiation because of orthostatic hypotension [47] . wolf and colleagues described six cases of stem cell transplant recipients who received pre-emptive as treatment for hcmv infection. two of these showed a decrease in the viral load [13] . in another study, five transplanted patients infected with hcmv strains resistant to different antiviral drugs were treated with as after many unsuccessful cycles of antiviral treatments. three out of five had a favorable outcome [48] . at present, art derivatives are recommended as antimalarial treatments in combination with another molecule with a different mechanism of action and longer half-life (art combination therapy, act) to avoid the onset of resistance and to prevent recrudescence. one of these combinations, as plus amodiaquine (aq), was tested against hcmv in a clinical study conducted on 494 ugandan children treated for acute malaria either with the act or with sulfadoxine-pyrimethamine plus aq. no measurable difference was observed in either the hcmv detection frequency or load in the blood of children in the two groups [49] . as was effective at a low micromolar range against epstein barr virus (ebv) in both epithelial cells and lymphocytes [50] . antiviral activity against human herpes virus-6 (hhv-6) was demonstrated not only in vitro [51] but also in a child affected by hhv-6b-associated myocarditis. as treatment was associated with a decrease in the levels of hhv-6b dna in the myocardium [52] . however, data on the effect of as against hhv-6 are contrasting in the literature [53] . as affects human bk polyomavirus (bkpyv) and jc polyomavirus (jcpyv) replication in vitro [54, 55] . both viruses latently and asymptomatically infect the human host and are able to reactivate in immunosuppressed hosts, such as hiv-positive patients or transplant recipients. jcpyv causes a rare and fatal disease known as progressive multifocal leukoencephalopathy (pml), while bkpyv is associated with nephropathy [56] . as mentioned above, art and as were more active against hbv and less toxic than other compounds from traditional chinese medicine [17] . the reduction in hcv replicons caused by as was dose and time-dependent in vitro and increased when as was given in association with ifn [57] . interestingly, in the usa, an open-label study is currently investigating a novel nonsurgical approach to the treatment of hpv-associated anal intraepithelial high-grade neoplasia using as suppositories. the outcomes that will be verified will be the regression of the lesions and the clearance of hpv. similarly, a phase ii double-blind, placebo-controlled, randomized study of as vaginal inserts has been designed for the treatment of women who have cervical high-grade intraepithelial neoplasia, but recruitment has not yet started (table 1) . finally, the combination as-aq was also used in patients infected with the ebola virus (ebov), reducing the mortality risk more than the other act used (artemether-lumefantrine) [58] . however, moderate to serious risk of bias and small sample sizes preclude conclusions [59] . during the ebov disease epidemic in west africa in 2014-2016, two mass drug administrations of as-aq were implemented to decrease the burden of malaria. garbern and colleagues performed a retrospective study to assess the potential effect of this treatment on the mortality of patients with ebov. although the risk of mortality for treated patients compared to that of ebov infected patients not exposed to as-aq was decreased, the effect was not significant. prospective trials are needed [60] . the activity of different art derivatives was evaluated against hcmv in a fibroblast model using a luminescent assay based on transgenic viral strain. compared to as and/or art, artemether was the most active. however, the activity was only seen at the micromolar range of concentrations, and only the synthesis of dimers allowed the activity to be effective at nanomolar concentrations [61, 62] . dha is the active metabolite of most art derivatives and an antimalarial drug itself. dha and art were tested against bovine viral diarrhea virus (bvdv), a surrogate in vitro model of hcv, showing moderate activity in the micromolar concentration range [63] . artemether is often used in combination with lumefantrine. one of the commercial versions of this combination, coartem ® , was used in a prospective observational study in mali in children (6 months-10 years) coinfected with hcmv and malaria. viral load in the urine decreased but only in high virus shedders [64] . the artemether-lumefantrine combination was also used in patients infected with ebov, showing reduced efficacy in reducing the risk of death compared to that of as-aq, as already described in the "artesunate" paragraph [58] . the antiviral effect of the aryl-aminoalcohol compounds quinine sulfate, mefloquine, halofantrine, and lumefantrine on both classical and emerging viruses has been studied. quinine, an alkaloid extract from chinchona (quina-quina) tree bark, was discovered in the 17th century and has been used to treat malaria since the early 1600s, currently still playing a pivotal role, especially in the treatment of chloroquine (cq)-resistant plasmodium falciparum [65, 66] . due to the benefit derived from antimalarial drugs in other pathologies, possible antiviral effects of quinine were investigated. the first manuscript regarding the effect of quinine on influenza virus infections in mice was published in 1946 [67] . subsequently, in vitro evaluation of quinine sulfate has been conducted with other viruses, such as herpes simplex virus-1 (hsv-1) and influenza a virus (iav). quinine sulfate at micromolar but not toxic doses reduced the number of plaques formed by hsv-1 in vitro in vero and hacat cell models, although no viricidal activity was observed [68, 69] . quinine sulfate in vitro activity was also tested against iav by means of viral plaque inhibition assay, evaluating its prophylactic activity and showing different effects with an ec 50 within the micromolar range, depending on the viral strains [70] . recently, quinine sulfate was tested in vitro against emerging dengue virus (denv) strains in different cell lines, showing a reduction in denv-2 virion production up to 80% compared to that of the untreated control and a concentration-dependent reduction in denv rna and viral proteins. the inhibition of replication was then confirmed for all four different serotypes of denv [66] . mefloquine (mq), a synthetic analog of quinine with a long history of use and good safety in humans, has been widely tested as an antiviral. one of the first reports refers to jcpyv. in 2009, brickelmaier et al. chose mq because of its high blood-brain barrier penetration capability since it accumulates in brain tissue at a six-fold higher concentration than its ec 50 . mq was active against different strains of jcpyv in three different cell models, with ec 50 s within the low micromolar range [71] . because of the absence of a suitable animal model, this first paper represented the starting point for a series of trials in human populations, which obtained contrasting results and are summarized in table 2 . seventeen case reports described the use of mq at different doses [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] . although in 12 cases there was no progression of the disease at follow-up [72] [73] [74] [77] [78] [79] [81] [82] [83] [84] 86, 87] , it is difficult to draw conclusions due to the challenging treatment protocols and compromised health of the patients. in many cases, mq was combined with mirtazapine, an antidepressant that, acting on the 5-ht2a serotonin receptor, is able to inhibit jcpyv entry into glial cells, preventing the diffusion of the infection in oligodendrocytes. the outcomes of this treatment are controversial, leading to the resolution of the infection, with a claimed effect of mq and mirtazapine treatment [86, 87, [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] , and to the resolution of the infection probably due to other factors [96, [106] [107] [108] [109] or to the death of the patient, which was not always directly related to the unsuccessful therapy [87, 96, [110] [111] [112] [113] [114] [115] [116] . in one case, the suspension of the therapy was necessary due to the side effects [117] . in a few cases, a third partner drug was added to mq and mirtazapine. again, the outcome was variable, and the contribution of the single drugs was difficult to determine [118, 119] . mefloquine + risperidone fatal outcome [75] mefloquine + risperidone + cytarabine pml resolution [120] mefloquine + cidofovir pml resolution due to other factors [121] hiv positive mefloquine no pml progression [122] [123] [124] [125] fatal outcome [126] mefloquine + mirtazapine premature fatal outcome [127, 128] clinical trial standard of care (soc) versus soc + mefloquine lack of differences: study ended prematurely [129] mq has also been combined with risperidone [75] or risperidone and cytarabine [120] , and the final outcomes were opposing, either death or recovery of the patient. in one case, mq was administered with the antiviral cidofovir, resulting in final remission of the pathology, most likely due to synergy with other factors [121] . different case reports have described the use of mq by itself in hiv-positive pml patients, sometimes with repression of jcpyv replication [122] [123] [124] [125] and rarely with the death of the patient [126] . the combined treatment mq and mirtazapine was administered to hiv-positive patients, leading to a failure because of premature death and not always due to the pml itself [127, 128] . one randomized study was conducted with hiv-positive and hiv-negative patients, comparing the efficacy of the standard of care normally used to treat pml to the standard of care supplemented with mq. this study ended prematurely because of a lack of significant differences between the two groups [129] . a study on the antiviral effect of mq on iav was performed by marois et al. mq showed different grades of efficacy, depending on the viral strain and ranging from partial inhibition of replication to total ineffectiveness [70] . more recently, the effect of mq on some emerging viruses was studied. mq was tested for the first time against the zika virus (zikv) in 2016, showing different reductions in infection rate, depending on the cell model used, and different cytotoxicities, thus making it difficult to draw a conclusion [130] . balasubramanian et al. confirmed the in vitro effect of mq on zikv infection and evaluated it on denv, performing several in vitro assays [131] . sun et al. performed an in vitro screening of 795 fixed-dose drug combinations of three molecules, choosing those able to block more than 90% ebov-like particle entry into hela cells. one of the three best combinations was composed of mq with toremifene (an antagonist of estrogen receptors) and the antifungal posaconazole, whose activity was confirmed by dose-response experiments in vero cells infected with ebov [132] . although some benefits could be seen in the use of mq as antiviral drug, its neurotoxicity should be taken into account: when it is used for malaria prophylaxis, it is known to cause serious neuropsychiatric adverse reactions, and to date, international mq labels warn patients to discontinue it at the onset of prodromal psychiatric and neurologic symptoms [133] . however, to date, to the best of our knowledge, there are no systematic studies concerning neurotoxic manifestations of mq, when used ad antiviral drug, and not for the antimalarial prophylaxis. it could be speculated that in some conditions the benefit:risk ratio would look more favorable than for mq used for malaria chemoprophylaxis. while good results were obtained using quinine sulfate and mq as antivirals, the same cannot be stated for halofantrine and lumefantrine, which failed in the inhibition of viral replication, based on the few studies conducted to date. mazzon and colleagues, performing an in vitro screen of 2500 compounds, were able to describe inhibition activity of halofantrine on semliki forest virus (sfv) and denv-2, but, due to the low selectivity index of the drug, further investigations were not conducted [134] . lumefantrine has been tested only in a commercial combination with artemether, known as coartem ® , as previously described in the paragraph about art derivatives. chloroquine (cq) is an aminoquinoline known since 1934. it was synthesized to be used as an antimalarial drug, but its properties and mechanism of action encouraged its use for the treatment of different diseases. currently, cq and its hydroxy-analog hydroxychloroquine (hydroxycq) cannot be used as antimalarial drugs in wide areas where the resistance of malaria parasites emerged. they are commonly used for connective tissue disorders, such as rheumatoid arthritis. due to low toxicity and cost, high tolerability and immunomodulatory properties, cq and hydroxycq have also been proposed for use against viral infections. even if their specific mechanisms in individual diseases are not clear, it is well assessed that the antiviral activities of the aminoquinoline take advantage of their strong anti-inflammatory activity. the major proposed mechanisms of actions of cq analogs which are suggested to influence the anti-viral activity are, among the others: the inhibition of cytokine production and release by t cells: il-1, 2, 6, or 18, tumor necrosis factor tnf-α and ifn-γ, reduced levels of chemokines ccl2 and cxcl10, inhibition of micro-rna expression, decreased th17-related cytokines, decreased dna, rna and protein synthesis in thymocytes (reviewed in [135] ). the in vitro antiviral effect of cq was first reported approximately 40 years ago [136, 137] , and since that time, its use as an antiviral drug has been extensively discussed. in particular, cq/hydroxycq have been used for the treatment of emerging chikungunya virus (chikv) infection, recently causing numerous outbreaks in the world. khan et al. showed that the treatment of infected vero cells with different micromolar concentrations of cq reduced virus yield and viral rna copy number [138] . de lamballerie and colleagues confirmed the inhibition of chikv replication in vero-e6 cells using cq. the efficacy of cq was inversely related to the concentration of the viral inoculum used, an unfavorable observation, considering the high viremia measured at the acute stage of chikv infection (up to 10 10 virus copies/ml serum) [37] . sourisseau and colleagues treated hela cells with cq, obtaining a potent inhibition of chikv replication and its relative cytopathic effects [139] . a double-blind placebo-controlled trial was designed to evaluate the efficacy and safety of cq for the treatment of chikv infection in 2006 in french reunion island (indian ocean). no significant difference was observed between the cq and placebo groups, either in the mean duration of febrile arthralgia or in the rate of viremia decrease [37] . however, the number of patients included in the study was too small to draw definitive conclusions regarding the efficacy of cq treatment (table 1 ) [37] . aminoquinolines were proposed for the treatment of other viral infections, such as zikv. in 2017, it was demonstrated that cq and aq exerted anti-zikv activity in vero cells, with low micromolar ic 50 s [140] . these results were in agreement with the decreasing number of zikv-infected cells after cq treatment. additionally, cq protected the cells from further zikv infection, as measured by cell viability at noncytotoxic concentrations [141] . the activity of cq has also been indirectly demonstrated against denv infection. the results obtained by kleber and colleagues showed that cq suppressed tnf-α and ifn-γ production, and it was hypothesized that cq might be used to treat patients suspected of having dengue disease, avoiding the more severe form of dengue hemorrhagic fever and/or shock. a clinical trial was also established to verify the effect of cq versus placebo in denv-infected patients in brazil. cq promoted a reduction in the intensity of pain and an improvement in the well-being of patients with denv infection but did not alter the duration of the disease or the intensity and days of fever (table 1 ) [36] . to study the effects of cq against ebov, a group led by dowall conducted an in vitro investigation using the human cell line mrc-5 and in vivo studies with the well-characterized guinea pig model [142] . they were able to demonstrate that cq reduced ebov replication in mrc-5 cells. in contrast, the administration of cq to 12 guinea pigs did not protect the infected animals against the ebola disease [142] . madrid and colleagues suggested that cq could interfere with the late stages of ebov replication and assembly [143] . despite these positive in vitro results, the clinical trials were sometimes conflicting. for this reason, later, the literature was reviewed to clarify the efficacy of cq in the treatment of filovirus infection [144] . it was concluded that the efficacy of cq against the viruses belonging to this family was dependent on the cq plasma concentrations, which must be sustained in patients until the clearance of the viremia [144] . cq was shown to inhibit the replication and spread of coronavirus (cov) in vitro and to prevent infection with cov in newborn mice. since the suppressive effect of cq was also present when the cells were treated before the infection, a prophylactic advantage of cq use was suggested [145] [146] [147] . cq and its analogs have effects against hcv. in particular, the treatment of jfh-1 or huh-7 cells with cq reduced hcv entry, replication, and infection in a dose-dependent manner [148] [149] [150] . furthermore, cq, in combination with ifn-α, prevented the replication of hcv and enhanced the antiviral effect of ifn-α [149] . in this regard, two phase i/ii clinical trials were initiated to verify the efficacy of the combination treatment of hydroxycq and ribavirin, but no results were posted due to limited recruitment. the anti-hiv-1 and anti-hiv-2 activities of cq and its analogs were tested in vitro and in vivo. the first report about the in vitro use of cq as an anti-hiv-1 agent was published in 1990 by tsai et al., which showed the suppressive effects of cq on the replication of hiv-1 in a t cell line [151] . a few years later, sperber and colleagues confirmed these results, showing the ability of cq and hydroxycq to inhibit hiv-1 replication not only in t cells but also in monocytes [152, 153] . subsequently, the same group demonstrated the cq and hydroxycq anti-hiv-1 and anti-hiv-2 in vitro effects at concentrations that are clinically achievable [154] . cq had an additive effect against hiv-1 when used in combination with other antiretroviral agents [155, 156] . naarding et al. demonstrated that cq reduced hiv-1 transmission to and replication in cd4 + t-lymphocytes [157] . similarly, martinson et al. observed that cq had a preventive role in hiv infection, reducing cd8 + t cell activation upon hiv replication [158] . the antiviral activity of hydroxycq was demonstrated in vivo by several clinical trials. the somministration of hydroxycq was able to reduce the amounts of plasma hiv-1 rna and il-6 in patients treated for eight weeks compared to those of the placebo group [39] . during a second clinical trial, hydroxycq was shown to reduce the hiv-1 rna plasma level, although at a lower level than the antiviral zidovudine [40] . in contrast, the results published in 2012 by paton and colleagues showed negative results, with an increase in viral load and a decrease in cd4 number [41] . another double-blind, randomized placebo-controlled trial testing the effects of cq in 13 chronically hiv-infected persons was conducted in minnesota. the results showed that the administration of cq during chronic hiv infection resulted in decreased immune activation, but no data regarding hiv status were reported [43] . very recently, the aids clinical trials group a5258 was completed. it was a randomized, double-blind, placebo-controlled study in 33 hiv-1-infected participants off antiretroviral therapy and 37 participants on antiretroviral therapy. cq modestly reduced immune activation in antiretroviral therapy-treated hiv-infected participants [42] . finally, the recruitment of 1499 patients was concluded a few months ago in a randomized, controlled, open-label, phase iii trial of the standard of care with cq prophylaxis compared to no prophylaxis in hiv-positive patients in malawi (table 1) [44, 45] . chloroquine and hydroxychloroquine and other rna viruses cq was shown to inhibit the in vitro replication of h1n1 and h3n2 iav strains [159] . a phase ii clinical trial aiming to verify the effect of cq compared to that of placebo on iav was started in singapore in 2005, and 1516 patients were recruited. however, cq was not shown to prevent infection with iav (table 1 ) [38] . cq had inhibitory effects on the entry and replication of enterovirus (ev)-a71 in cell-based systems [160] . yong and colleagues studied the efficacy of cq against several ev serotypes and evaluated its therapeutic capacity in vitro in rd cells and in vivo in a murine model [161] . they demonstrated the potential of cq as an antiviral in the treatment of hand, foot, and mouth disease caused by ev infection. the positive results obtained in the murine model of infection were indicative of the fact that cq may mitigate the disease severity in mammals [161] . amodiaquine (aq) was originally developed and has been widely used for the treatment of malaria. however, subsequent studies revealed that it was active against a wide range of human pathogens, including several viruses. in a study published in 2014, it was investigated whether quinolone derivatives could inhibit the replication of denv [162] . the time-course analysis suggested that aq was stable and that it reproducibly inhibited denv infectivity. the data also showed that viral entry and internalization were partially inhibited by the drug, but the major effect occurred at a later stage of the viral life cycle [162] . it is known that aq inhibits ebov replication in vivo [58] . a recent study demonstrated that aq was active against severe fever with thrombocytopenia syndrome (sfts) caused by sfts virus (sftsv) [163] . primaquine was tested as an antiviral on primary chicken embryo cells (cecs) infected by newcastle disease virus [164] . it was demonstrated that primaquine had an effect on the accumulation of viral hemagglutinin on the cell surface. in addition, primaquine inhibited protein synthesis in virus-infected cells [164] . atovaquone, a naphthoquinone antimalarial drug often used in pregnant women, is a ubiquinone (coenzyme q) analog that inhibits mitochondrial cytochrome complex iii (bc1 complex). it is also able to deplete the intracellular nucleotide pools by inhibiting dihydroorotate dehydrogenase, an enzyme for de novo pyrimidine synthesis [165] . in 2019, one published work reported the in vitro antiviral action of atovaquone against chikv. low micromolar doses inhibited the number of infected cells, as evaluated by high-content fluorescence microscopy. the result was then confirmed by the reduction in chikv virions in different cells by plaque assay [164] . in the same paper, kottkamp and colleagues evaluated the ability of atovaquone to reduce the infectivity of both a brazilian and a ugandan strain of zikv by immunostaining the envelope after treatment and infection and subsequent plaque assay in different cells. these data were further confirmed ex vivo in a human placenta tissue model, with a dose-dependent reduction in infection and virion production by the ugandan strain [166] . antibiotic drugs, such as doxycycline and sulfonamides, are widely used in the chemoprophylaxis of malaria in all malaria areas [167] . it has been reported that these therapies could have activities beyond antimalarial activity, also against viral infectious agents. surprisingly, in many cases, the administration of antibiotics alone or in combination with other antiviral agents showed significant antiviral activities against different types of viral infections [168] [169] [170] [171] . despite this evidence, the antiviral mechanisms of action have not been completely investigated. doxycycline (dox) is a semisynthetic tetracycline antibiotic that prevents bacterial protein synthesis by acting on ribosomes (30s subunit) [167, [172] [173] [174] [175] . dox, widely used alone or in combination with quinine for chemoprophylaxis in cq-resistant p. falciparum cases of malaria [176] , has been shown to exert inhibitory effects against different microbial infections. dox significantly inhibited the proliferation of a panel of hpv-positive cervical cancer cell lines, also inducing apoptosis of cervical cancer cells in a time-and dose-dependent manner [177] . concerning emerging infections, in a recent work, researchers tried to identify candidate antibiotics that can block the function of denv viral envelope proteins to prevent viral entry. using an innovative visual screening approach coupling computational studies and biologic assays on ten nontoxic candidates, they suggested that dox significantly inhibited plaque formation, demonstrating an inhibitory effect on denv propagation [178] . this result was confirmed by rothan and colleagues, who evaluated the replication rate of denv in an in vitro model of infected cells. they reported that dox inhibited denv replication in vitro by reducing viral protease activity and entry into host cells [168] . the same research group determined the inhibitory effects of dox against chikv as well as its possible effect on the virus life cycle in vero cells when the virus and the drug were administered concurrently to the cells. computational studies indicated that dox might be a noncompetitive inhibitor of chikv protease. this hypothesis was confirmed by in vitro experiments demonstrating that the effect of dox was directed more toward viral entry than toward viral replication [169] . furthermore, the inhibitory effect of dox on the replication of vesicular stomatitis virus (vsv) in different stably infected cell lines was reported [170] . the results showed significant inhibition of the replication of vsv in a dose-dependent but not cell-type dependent manner, suggesting that dox exerted its antiviral activity at the early-mid stage of vsv infection [170] . these results indicated a different mode of action of dox against vsv than that against denv and chikv, suggesting that the antiviral activity of dox could be dependent on the virus species. finally, only an in vivo study was published on iav. treatment with dox attenuated acute lung injury in mice infected with a virulent iav h3n2 strain, with no effects on virus titers, suggesting that the antibiotic treatment was able to alleviate severe influenza pneumonia symptoms [171] . it is, therefore, possible to conclude that despite the role of dox as a potential antiviral agent in vitro, the mechanisms of viral replication inhibition and the targeted virus species have yet to be clarified. clinical trials confirming in vitro observations are needed. sulfonamides constitute an important class of drugs containing many types of pharmacological agents with broad-spectrum bacteriostatic activity. sulfonamides interrupt the synthesis of folic acid, interfering with the dihydropteroate synthase and dihydrofolate reductase enzymes of bacteria (and protozoa) and inhibiting bacterial growth [179, 180] . the sulfadoxine-pyrimethamine combination is used in some settings for the treatment of uncomplicated malaria in pregnant women, and it is the only drug currently recommended for intermittent preventive therapy during pregnancy [181] . a study investigated the effect of this combination on hiv replication. experiments were conducted in vitro using peripheral blood mononuclear cells, mt-2 cells, mt-4 cells, and a latently infected cell line named u1. the results showed that the sulfadoxine-pyrimethamine combination combined with antiretroviral therapy significantly enhanced hiv replication in mt-2 cells, while it inhibited hiv replication in peripheral blood mononuclear cells [23] . moreover, recent studies have demonstrated that sulfonamides can act on latent herpesviruses, such as ebv and kaposi sarcoma herpesvirus (kshv) [182, 183] . furthermore, a work conducted by angius and colleagues reported that sulfonamide antibiotics suppressed the kshv latent state in permanently infected lymphoma cells [183] . this result suggested that sulfonamides might play a potential role in clearing kshv-infected lymphoma cells. since conventional antiherpes drugs are able to slightly suppress viral replication in the lytic phase but do not clear the latent state, the finding of potential new small molecules, such as sulfonamides drugs, could initiate a promising program of studies on both oncogenic and degenerative diseases, in which herpesvirus latency is suspected to be involved [184] [185] [186] . antimalarial drugs have been widely tested against a large number of viruses, especially in vitro, with variable outcomes (table 3) . among the art derivates, some showed strong activities against viruses, such as as against hcmv, art, and as against hbv and hcv, and as against hpv and hpyv, whereas data regarding the activity against hiv are uncertain. sulfonamides in vitro [182] hcmv artemisinin in vitro [16] artesunate in vitro [10, 11, 16, 17, [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] arthemeter in vitro [61, 62] hhv-6 artesunate in vitro [51, 53] hhv-6b artesunate in vitro [52] kshv doxycycline in vitro [183] hse artemisinin in vivo (mouse) [35] dihydroartemisinin in vitro [16] kshv sulfonamides in vitro [183] rcmv artesunate in vivo (rat) [11] polyomaviridae bkpyv artesunate in vitro [55] jcpyv artesunate in vitro [54] mefloquine in vitro [71] hepadnaviridae hbv artemisinin, artesunate in vitro [17] papillomaviridae hpv artemisinin in vitro [18] dihydroartemisinin in vivo (dog) [19] doxycycline in vitro [177] [187, 188] dihydroartemisinin in vitro [63] hcv artemisinin in vitro [20] [21] [22] artesunate in vitro [57] chloroquine in vitro [148] [149] [150] zikv mefloquine in vitro [130, 131] chloroquine in vitro [140, 141] amodiaquine in vitro [140] atovaquone in vitro [166] denv quinine sulfate in vitro [66] mefloquine in vitro [131] halofantrine in vitro [134] doxycycline in vitro [168, 178] amodiaquine in vitro [162] chikv doxycycline in vitro [169] chloroquine in vitro [37, 138, 139] atovaquone in vitro [166] togaviridae sfv halofantrine in vitro [134] rhabdoviridae vsv doxycycline in vitro [170] orthomyxoviridae iav quinine sulfate in vitro [70] in vivo (mouse) [67] mefloquine in vitro [70] doxycycline in vivo (mouse) [171] chloroquine in vitro [159] [145] [146] [147] picornaviridae enteroviruses chloroquine in vitro [160] chloroquine in vitro/in vivo (mouse) [161] filoviridae ebov chloroquine in vitro [142, 143] in vivo [142] artesunate, amodiaquine in vivo [58] mefloquine in vitro [132] retroviridae hiv artemisinin in vitro [23] doxycycline in vitro [23] mefloquine, toremifene, posaconazole in vitro [132] chloroquine in vitro [151, 157, 158] in vivo [155] chloroquine, hydroxychloroquine in vitro [152] [153] [154] 156] phenuiviridae sftsv amodiaquine in vitro [163] among the aryl-aminoalcohols, the use of mq in the treatment of jcpyv infection has been extensive, although with contradictory outcomes. among the aminoquinolines, both cq and hydroxycq showed promising results in reducing the replication of some emerging viruses, such as denv and zikv. emerging infections have also been targeted by antibacterial drugs, such as dox. however, the successful use of antimalarial drugs in vitro did not always lead to a satisfactory outcome in their clinical application (table 4 ). table 4 . clinical studies for the assessment of antimalarial drug activity against viral infection. herpesviridae hcmv artesunate [12, 13, [46] [47] [48] artesunate-amodiaquine [49] arthemeter-lumefantrine [64] hhv6 artesunate [52] polyomaviridae jcpyv artesunate [54, 55] mefloquine [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [122] [123] [124] [125] [126] mefloquine and mirtazapine [86, 87, 127, 128] mefloquine, mirtazapine and a third drug [118, 119] mefloquine and risperidone [75] mefloquine, risperidone and cytarabine [120] mefloquine and cidofovir [121] mefloquine and pml standard of care [129] filoviridae ebov artesunate-amodiaquine [58, 60] nevertheless, some drugs have already been used in several clinical trials, as summarized in table 1 . most of them regard the use of antimalarial drugs against hiv infection, but some of them failed, and for others, the final results are not available. although these outcomes can seem discouraging, at least four clinical trials deserve attention: the one on the use of as against hcmv, with particular regard to the drug-resistant strains, the one targeting chikv with cq, and the other two very innovative and ongoing trials on the use of as against hpv for the treatment of anal and cervical intraepithelial high-grade neoplasia. based on these observations, we can state that the use of antimalarial drugs might be useful, especially in cases of antiviral resistance and in light of the emergence of many viruses against which effective drugs are not available. world health organization. guidelines for the treatment of malaria; world health organization antimalarials-are they effective and safe in rheumatic diseases? reumatologia anticancer effect of antimalarial artemisinin compounds quinine sulfate inhibits invasion of some bacterial skin pathogens spatial overlaps in the distribution of hiv/aids and malaria in zimbabwe concurrent dengue and malaria in the amazon region global prevalence and distribution of coinfection of malaria, dengue and chikungunya: a systematic review artemisinin-a gift from traditional chinese medicine to the world (nobel lecture) antiplasmodial and antitumor activity of artemisinin-from bench to bedside antiviral activity of artesunate towards wild-type, recombinant, and ganciclovir-resistant human cytomegaloviruses the anti-malaria drug artesunate inhibits replication of cytomegalovirus in vitro and in vivo artesunate as a potent antiviral agent in a patient with late drug-resistant cytomegalovirus infection after hematopoietic stem cell transplantation human cytomegalovirus kinetics following institution of artesunate after hematopoietic stem cell transplantation the antiviral activities of artemisinin and artesunate beyond malaria: the inhibition of viruses by artemisinin-type compounds stability and antiviral activity against human cytomegalovirus of artemisinin derivatives effect of artemisinin/artesunate as inhibitors of hepatitis b virus production in an "in vitro" replicative system artemisinin represses telomerase subunits and induces apoptosis in hpv-39 infected human cervical cancer cells dihydroartemisinin is cytotoxic to papillomavirus-expressing epithelial cells in vitro and in vivo artemisinin analogues as potent inhibitors of in vitro hepatitis c virus replication hemin potentiates the anti-hepatitis c virus activity of the antimalarial drug artemisinin iron inactivates the rna polymerase ns5b and suppresses subgenomic replication of hepatitis c virus evaluation of the effect of pyrimethamine, an anti-malarial drug, on hiv-1 replication human embryonic lung fibroblasts treated with artesunate exhibit reduced rates of proliferation and human cytomegalovirus infection in vitro antiviral activity of ganciclovir and artesunate towards human cytomegalovirus in astrocytoma cells unique and highly selective anticytomegalovirus activities of artemisinin-derived dimer diphenyl phosphate stem from combination of dimer unit and a diphenyl phosphate moiety the unique antiviral activity of artesunate is broadly effective against human cytomegaloviruses including therapy-resistant mutants new efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids the broad-spectrum antiinfective drug artesunate interferes with the canonical nuclear factor kappa b (nf-κb) pathway by targeting rela/p65 an artemisinin-derived dimer has highly potent anti-cytomegalovirus (cmv) and anti-cancer activities highly potent artemisinin-derived dimers and trimers: synthesis and evaluation of their antimalarial, antileukemia and antiviral activities ex vivo model of congenital cytomegalovirus infection and new combination therapies artesunate demonstrates in vitro synergism with several antiviral agents against human cytomegalovirus in vitro combination of anti-cytomegalovirus compounds acting through different targets: role of the slope parameter and insights into mechanisms of action valacyclovir combined with artesunate or rapamycin improves the outcome of herpes simplex virus encephalitis in mice compared to antiviral therapy alone chloroquine use improves dengue-related symptoms on chikungunya acute infection and chloroquine treatment. vector borne zoonotic dis chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial hydroxychloroquine treatment of patients with human immunodeficiency virus type 1 comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type 1 effects of hydroxychloroquine on immune activation and disease progression among hiv-infected patients not receiving antiretroviral therapy: a randomized controlled trial the effect of chloroquine on immune activation and interferon signatures associated with hiv-1 reduction of immune activation with chloroquine therapy during chronic hiv infection assessment of chloroquine as a modulator of immune activation to improve cd4 recovery in immune nonresponding hiv-infected patients receiving antiretroviral therapy tscq study: a randomized, controlled, open-label trial of daily trimethoprim-sulfamethoxazole or weekly chloroquine among adults on antiretroviral therapy in malawi: study protocol for a randomized controlled trial artesunate is ineffective in controlling valganciclovir-resistant cytomegalovirus infection combination therapy for multidrug-resistant cytomegalovirus disease success and failure of artesunate treatment in five transplant recipients with disease caused by drug-resistant cytomegalovirus an artesunate-containing antimalarial treatment regimen did not suppress cytomegalovirus viremia a reporter system for epstein-barr virus (ebv) lytic replication: anti-ebv activity of the broad anti-herpesviral drug artesunate sensitivity of human herpesvirus 6 and other human herpesviruses to the broad-spectrum antiinfective drug artesunate first therapeutic use of artesunate in treatment of human herpesvirus 6b myocarditis in a child antiviral activity of diverse classes of broad-acting agents and natural compounds in hhv-6-infected lymphoblasts antiviral effects of artesunate on jc polyomavirus replication in cos-7 cells antiviral effects of artesunate on polyomavirus bk replication in primary human kidney cells artesunate, an anti-malarial drug, has a potential to inhibit hcv replication effect of artesunate-amodiaquine on mortality related to ebola virus disease anti-ebola therapy for patients with ebola virus disease: a systematic review effect of mass artesunate-amodiaquine distribution on mortality of patients with ebola virus disease during west african outbreak. open forum infect artemisinin-derived dimers have greatly improved anti-cytomegalovirus activity compared to artemisinin monomers artemisinin-derived dimer phosphate esters as potent anti-cytomegalovirus (anti-cmv) and anti-cancer agents: a structure-activity study novel artemisinin derivatives with potential usefulness against liver/colon cancer and viral hepatitis effect of artemether-lumefantrine (coartem) on cytomegalovirus urine viral load during and following treatment for malaria in children quinine, an old anti-malarial drug in a modern world: role in the treatment of malaria drug repurposing of quinine as antiviral against dengue virus infection effect of quinine on influenza virus infections in mice quinine sulfate and hsv replication antiviral effects of quinine sulfate on hsv-1 hacat cells infected: analysis of the molecular mechanisms involved inhibition of influenza virus replication by targeting broad host cell pathways identification and characterization of mefloquine efficacy against jc virus in vitro mefloquine treatment in a patient suffering from progressive multifocal leukoencephalopathy after umbilical cord blood transplant mefloquine in the treatment of progressive multifocal leukoencephalopathy mefloquine improved progressive multifocal leukoencephalopathy in a patient with systemic lupus erythematosus failure of mefloquine therapy in progressive multifocal leukoencephalopathy: report of two japanese patients without human immunodeficiency virus infection a chronic lymphocytic leukemia patient with progressive multifocal leukoencephalopathy caused by john cunningham virus fingolimod-associated pml with mild iris in ms: a clinicopathologic study a punctate magnetic resonance imaging pattern in a patient with systemic lupus erythematosus is an early sign of progressive multifocal leukoencephalopathy: a clinicopathological study use of interleukin-2 for management of natalizumab-associated progressive multifocal leukoencephalopathy: case report and review of literature. ther rituximab-associated progressive multifocal leukoencephalopathy derived from non-hodgkin lymphoma: neuropathological findings and results of mefloquine treatment long-term survival in a patient with progressive multifocal leukoencephalopathy after therapy with rituximab, fludarabine and cyclophosphamide for chronic lymphocytic leukemia progressive multifocal leukoencephalopathy developing after liver transplantation showing marked neurological symptom improvement and arrest of further deterioration of imaging findings: a case report efficacy of mefloquine to progressive multifocal leukoencephalopathy initially presented with parkinsonism mefloquine improved progressive multifocal leukoencephalopathy in a patient with immunoglobulin a nephropathy progressive multifocal leukoencephalopathy in a 62-year-old immunocompetent woman usefulness of 11c-methionine-positron emission tomography for the diagnosis of progressive multifocal leukoencephalopathy natalizumab-related progressive multifocal leukoencephalopathy in greece late relapse of progressive multifocal leucoencephalopathy postallogenic transplant in a young patient with cll successful management of natalizumab-associated progressive multifocal leukoencephalopathy and immune reconstitution syndrome in a patient with multiple sclerosis progressive multifocal leukoencephalopathy associated with isolated cd8+ t-lymphocyte deficiency mimicking tumefactive ms a case of progressive multifocal leukoencephalopathy in a patient with sarcoidosis progressive multifocal leukoencephalopathy in an immunocompetent patient presymptomatic diagnosis with mri and adequate treatment ameliorate the outcome after natalizumab-associated progressive multifocal leukoencephalopathy mirtazapine and mefloquine therapy for non-aids-related progressive multifocal leukoencephalopathy progressive multifocal leukoencephalopathy in common variable immunodeficiency: mitigated course under mirtazapine and mefloquine progressive multifocal leukoencephalopathy associated with fumaric acid esters treatment in psoriasis patients significant improvement following combination treatment with mefloquine and mirtazapine in a patient with progressive multifocal leukoencephalopathy after allogeneic peripheral blood stem cell transplantation treatment of progressive multifocal leukoencephalopathy-immune reconstitution inflammatory syndrome with intravenous immunoglobulin in a patient with multiple sclerosis treated with fingolimod after discontinuation of natalizumab progressive multifocal leukoencephalopathy with negative jc virus pcr following treatment of follicular lymphoma: implications for biologics in the era of targeted cancer therapy asymptomatic progressive multifocal leukoencephalopathy during natalizumab therapy with treatment brain biopsy is more reliable than the dna test for jc virus in cerebrospinal fluid for the diagnosis of progressive multifocal leukoencephalopathy progressive multifocal leukoencephalopathy in a 44-year old male with idiopathic cd4+ t-lymphocytopenia treated with mirtazapine and mefloquine a case of developing progressive multifocal leukoencephalopathy while using rituximab and mycophenolate mofetil in refractory systemic lupus erythematosus inflammatory cerebellar pml with a cd4/cd8 ratio of 2.9 showed a favorable prognosis in a patient with rheumatoid arthritis: a case report progressive multifocal leukoencephalopathy in the absence of typical radiological changes: can we make a diagnosis? successful treatment of progressive multifocal leukoencephalopathy with recombinant interleukin-7 and maraviroc in a patient with idiopathic cd4 lymphocytopenia asymptomatic progressive multifocal leukoencephalopathy: a case report and review of the literature jc virus granule cell neuronopathy in the setting of chronic lymphopenia treated with recombinant interleukin-7 progressive multifocal leukoencephalopathy complicating untreated chronic lymphatic leukemia: case report and review of the literature progressive multifocal leukoencephalopathy associated with thymoma with immunodeficiency: a case report and literature review progressive multifocal leukoencephalopathy after t-cell replete hla-haploidentical transplantation with post-transplantation cyclophosphamide graft-versus-host disease prophylaxis progressive multifocal leukoencephalopathy after ibrutinib therapy for chronic lymphocytic leukemia natalizumab-associated progressive multifocal leukoencephalopathy in a patient with multiple sclerosis: a postmortem study progressive multifocal leukoencephalopathy in patients with a hematological malignancy: review of therapeutic options progressive multifocal leukoencephalopathy in a patient with pre-clinical primary biliary cirrhosis detection of jc virus archetype in cerebrospinal fluid in a ms patient with dimethylfumarate treatment without lymphopenia or signs of pml progressive multifocal leukoencephalopathy in the absence of immunosuppression progressive multifocal leukoencephalopathy with balanced cd4/cd8 t-cell infiltration and good response to mefloquine treatment progressive multifocal leukoencephalopathy associated to natalizumab extended dosing regimen does anti-jcv therapy improve the prognosis of aids-related pml? progressive multifocal leukoencephalopathy with immune reconstitution inflammatory syndrome (pml-iris): two case reports of successful treatment with mefloquine and a review of the literature favourable outcome of progressive multifocal leukoencephalopathy with mefloquine treatment in combination with antiretroviral therapy in an hiv-infected patient akinetic mutism caused by hiv-associated progressive multifocal leukoencephalopathy was successfully treated with mefloquine: a serial multimodal mri study development of primary central nervous system lymphoma associated with human immunodeficiency virus and jc virus infection mirtazapine and mefloquine therapy for progressive multifocal leukoencephalopathy in a patient infected with human immunodeficiency virus hiv-associated progressive multifocal leukoencephalopathy: longitudinal study of jc virus non-coding control region rearrangements and host immunity a study of mefloquine treatment for progressive multifocal leukoencephalopathy: results and exploration of predictors of pml outcomes a screen of fda-approved drugs for inhibitors of zika virus infection antiviral activities of selected antimalarials against dengue virus type 2 and zika virus synergistic drug combination effectively blocks ebola virus infection a serious nightmare: psychiatric and neurologic adverse reactions to mefloquine are serious adverse reactions identification of broad-spectrum antiviral compounds by targeting viral entry chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases effect of chloroquine on the growth of animal viruses comparison of the antiviral activity in vitro of some non-steroidal anti-inflammatory drugs assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in vero cells characterization of reemerging chikungunya virus the antimalarial drug amodiaquine possesses anti-zika virus activities an endocytosis blocking agent, inhibits zika virus infection in different cell models. viruses chloroquine inhibited ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model a systematic screen of fda-approved drugs for inhibitors of biological threat agents chloroquine could be used for the treatment of filoviral infections and other viral infections that emerge or emerged from viruses requiring an acidic ph for infectivity chloroquine is a potent inhibitor of sars coronavirus infection and spread antiviral activity of chloroquine against human coronavirus oc43 infection in newborn mice in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine hepatitis c virus entry depends on clathrin-mediated endocytosis inhibition of hepatitis c virus replication by chloroquine targeting virus-associated autophagy targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases inhibition of human immunodeficiency virus infectivity by chloroquine inhibition of human immunodeficiency virus type 1 replication by hydroxychloroquine in t cells and monocytes inhibition of hiv-1 replication by hydroxychloroquine: mechanism of action and comparison with zidovudine the anti-hiv-1 activity of chloroquine the potential place of chloroquine in the treatment of hiv-1-infected patients anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors effect of chloroquine on reducing hiv-1 replication in vitro and the dc-sign mediated transfer of virus to cd4+ t-lymphocytes dendritic cells from hiv-1 infected individuals are less responsive to toll-like receptor (tlr) ligands in vitro inhibition of human influenza a virus replication by chloroquine caveolar endocytosis is required for human psgl-1-mediated enterovirus 71 infection an evaluation of chloroquine as a broad-acting antiviral against hand, foot and mouth disease amodiaquine, an antimalarial drug, inhibits dengue virus type 2 replication and infectivity establishment of an antiviral assay system and identification of severe fever with thrombocytopenia syndrome virus inhibitors primaquine diphosphate: inhibition of newcastle disease virus replication antimalarial pharmacology and therapeutics of atovaquone atovaquone inhibits arbovirus replication through the depletion of intracellular nucleotides inhibitory effect of doxycycline against dengue virus replication in vitro a combination of doxycycline and ribavirin alleviated chikungunya infection antiviral activity of doxycycline against vesicular stomatitis virus in vitro doxycycline treatment attenuates acute lung injury in mice infected with virulent influenza h3n2 virus: involvement of matrix metalloproteinases a highly sensitive and selective assay of doxycycline by dualwavelength overlapping resonance rayleigh scattering brucella infection with pancytopenia after pediatric liver transplantation bioactive implant surface with electrochemically bound doxycycline promotes bone formation markers in vitro and in vivo susceptibility of plasmodium falciparum isolates to doxycycline is associated with pftetq sequence polymorphisms and pftetq and pfmdt copy numbers doxycycline inhibits proliferation and induces apoptosis of both human papillomavirus positive and negative cervical cancer cell lines. can combinatorial computational approaches to identify tetracycline derivatives as flavivirus inhibitors antibiotics that inhibit nucleotide synthesis special issue: sulfonamides. molecules pharmacokinetics of sulfadoxine-pyrimethamine in hiv-infected and uninfected pregnant women in western kenya discovery of selective inhibitors against ebna1 via high throughput in silico virtual screening antimicrobial sulfonamides clear latent kaposi sarcoma herpesvirus infection and impair mdm2-p53 complex formation retinoic acid analogues inhibit human herpesvirus 8 replication inhibition of infection and replication of human herpesvirus 8 in microvascular endothelial cells by alpha interferon and phosphonoformic acid evidence for viral etiology of multiple sclerosis bovine viral diarrhea virus as a surrogate model of hepatitis c virus for the evaluation of antiviral agents antiviral effect of artemisinin from artemisia annua against a model member of the flaviviridae family, the bovine viral diarrhoea virus (bvdv) key: cord-320116-63yvpuqx authors: bancroft, tara; debuysscher, blair l.; weidle, connor; schwartz, allison; wall, abigail; gray, matthew d.; feng, junli; steach, holly r.; fitzpatrick, kristin s.; gewe, mesfin m.; skog, patrick d.; doyle-cooper, colleen; ota, takayuki; strong, roland k.; nemazee, david; pancera, marie; stamatatos, leonidas; mcguire, andrew t.; taylor, justin j. title: detection and activation of hiv broadly neutralizing antibody precursor b cells using anti-idiotypes date: 2019-10-07 journal: j exp med doi: 10.1084/jem.20190164 sha: doc_id: 320116 cord_uid: 63yvpuqx many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest. in light of this, there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes. here, we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus 1 (hiv-1) broadly neutralizing antibody b12 (iglb12). we determined the crystal structure of two anti-idiotypes in complex with iglb12 and used these anti-idiotypes to identify rare naive human b cells expressing b cell receptors with similarity to iglb12. immunization with a multimerized version of this anti-idiotype induced the proliferation of transgenic murine b cells expressing the iglb12 heavy chain in vivo, despite the presence of deletion and anergy within this population. together, our data indicate that anti-idiotypes are a valuable tool for the study and induction of potentially protective antibodies. there are many pathogens, such as hiv-1, respiratory syncytial virus, and influenza virus, for which the development of a protective vaccine has been elusive despite decades of effort. in many cases, the failure does not appear to be an absolute inability of the immune system to produce protective antibodies, since they have been characterized in some infected individuals (beeler and van wyke coelingh, 1989; burton et al., 1991; okuno et al., 1993; johnson et al., 1997; karron et al., 1997; scanlan et al., 2002; kashyap et al., 2008; throsby et al., 2008; ekiert et al., 2009 ekiert et al., , 2011 scheid et al., 2009 scheid et al., , 2011 sui et al., 2009; walker et al., 2009 walker et al., , 2011 wu et al., 2010; corti et al., 2011; dreyfus et al., 2012 dreyfus et al., , 2013 huang et al., 2012 huang et al., , 2014 magro et al., 2012; mouquet et al., 2012; liao et al., 2013; ngwuta et al., 2015) . it is not entirely clear why traditional vaccine development approaches have failed to induce similar protective antibodies, but mounting evidence suggests that protective vaccines for these pathogens will likely need to stimulate specific lineages of antibodies targeting defined epitopes (pantaleo and koup, 2004; rappuoli et al., 2016; lang et al., 2017; robbiani et al., 2017; goodwin et al., 2018; kwong and mascola, 2018) . in contrast, traditional vaccines generally stimulate polyclonal antibody responses against whole pathogens, or large subunits from pathogens, rather than focus the immune response toward known protective epitopes. the observation that a subset of hiv-1-infected individuals develop broadly neutralizing antibodies (bnabs) targeting the hiv-1 envelope protein (env) that are capable of neutralizing diverse hiv-1 viral isolates over the course of infection demonstrates that the human immune system is capable of generating bnab responses provided it receives the appropriate antigenic stimulation. indeed, numerous bnabs have been isolated from infected individuals that define the sites of vulnerability and mechanisms of neutralization of hiv-1 (west et al., 2014; burton and hangartner, 2016; kwong and mascola, 2018) . importantly, bnabs have been shown to protect from experimental infection in animal models, suggesting that they will be an important component of an effective vaccine against hiv-1 (mascola et al., 1999; shibata et al., 1999; parren et al., 2001; hessell et al., 2009; moldt et al., 2012; pietzsch et al., 2012; gruell et al., 2013; balazs et al., 2014; shingai et al., 2014; gautam et al., 2016; liu et al., 2016) . unfortunately, bnabs have not been elicited by vaccination in humans, despite the use of diverse recombinant env-derived immunogens and immunization schemes (burton et al., 2004; cohen and dolin, 2013; schiffner et al., 2013) . recent advances in the amplification of antibody genes from individual b cells have revealed that bnabs often require extensive somatic mutation to achieve broad and potent neutralizing activity (scheid et al., 2009; dimitrov, 2010; klein et al., 2013a; kwong et al., 2013; mascola and haynes, 2013; west et al., 2014) . although mutated bnabs are capable of broad env recognition and virus neutralization, most inferred germline bnab variants do not display reactivity with any env variant tested, which led us and others to propose that one reason for the lack of bnab elicitation through immunization is the lack of stimulation of b cells expressing bnab progenitor b cell receptors (bcrs) by env-based immunogens (xiao et al., 2009; hoot et al., 2013; jardine et al., 2013; mcguire et al., 2013; andrabi et al., 2015; zhou et al., 2015; gorman et al., 2016) . env-based immunogens capable of binding to certain germline bnabs have been recently developed that effectively activate b cells in knock-in mice expressing the desired bcrs in vivo (dosenovic et al., 2015; escolano et al., 2016; jardine et al., 2016; mcguire et al., 2016; steichen et al., 2016; abbott et al., 2018) . for many bnabs, however, antigens capable of being bound by the corresponding germline forms have not yet been identified or designed. here, we demonstrate the use of anti-idiotypes as an alternative approach to structure-based immunogen design to target the inferred germline version of the hiv-1 bnab b12. we focused upon b12 because despite being one of the first hiv-1 bnabs isolated, env-based immunogens have not been identified or developed to recognize its ancestral germline form (xiao et al., 2009; hoot et al., 2013) . the mature form of b12 was isolated through phage display (barbas et al., 1992) , and it used a heavy chain derived from v h 1-3*02/d h 2-21/j h 6*03 and light chain derived from v k 3-20*01/j k 2 (xiao et al., 2009; hoot et al., 2013) . using anti-idiotypes specific for the inferred germline version of b12 (iglb12) as baits for single b cell sorting, we identified a subset of human germline bcrs using v h 1-3 with some heavy chain cdrh3 similarity to iglb12. while crystal structures indicated that one of these anti-iglb12 idiotypes made extensive contacts with the iglb12 cdrh3 region encoded by d h 2-21, this gene segment was not enriched in sorted b cells. moreover, we could not identify any cdrh3 regions with >50% amino acid sequence identity to iglb12 within a dataset including hundreds of bcrs. in light of this, we hypothesized that b cells expressing bcrs containing cdrh3s with high similarity to iglb12 may be deleted from the repertoire as a result of autoreactivity with self-antigens. in line with this notion, we demonstrate that the iglb12 antibody stains hep-2 cells, a feature of other autoreactive hiv-1-specific antibodies (haynes et al., 2005; verkoczy et al., 2010; ota et al., 2013) . to evaluate the consequence of this autoreactivity in vivo, we generated mice with the iglb12 heavy chain knocked into the endogenous murine heavy chain locus. the iglb12 heavy chain knock-in mice exhibited b cell deletion during development, with the surviving b cells exhibiting bcr down-regulation and anergy. despite the presence of anergy, immunization with a multimerized version of an anti-iglb12 idiotype stimulated the proliferation and differentiation of transgenic b cells expressing the iglb12 heavy chain. in summary, our results establish a proof of concept that anti-idiotypes can be used as immunogens to identify and stimulate the ancestral germline version of protective antibodies. to generate anti-idiotype antibodies to iglb12 , we immunized and boosted mice intraperitoneally with iglb12 in adjuvant. 3 d after the final immunization, splenocytes were isolated and fused to myeloma cells, resulting in the generation of hundreds of hybridomas producing antibodies able to bind iglb12. nearly all of the clones were excluded because they bound to a panel of control germline antibodies as well as iglb12, suggesting specificity for the heavy or light chain constant regions of human ig (data not shown). importantly, four clones (ib1, ib2, ib3, and ib5) exhibited binding to iglb12 with similar apparent affinities (dissociation constants [k d ] of ∼10 −9 ) but did not bind a panel of control antibodies derived from germline bcr sequences of anti-hiv-1 antibodies (fig. 1 , a-c; and table s1 ). the anti-iglb12 idiotypes also failed to bind the fully mature version of b12, indicating a preference for the unmutated sequence ( fig. 1 c) . one clone, ib2, bound a chimera expressing the iglb12 heavy chain and a noncognate control light chain, while the other three clones, ib1, ib3, and ib5, did not bind this chimera ( fig. 1 c) , suggesting different modes of recognition between ib2 and ib1, ib3, and ib5. we next assessed whether binding of these clones was specific for the iglb12 heavy chain cdrh3 region as opposed to a dependence on v h 1-3, the variable gene segment used by iglb12. consistent with our clones specifically recognizing features unique to iglb12, all four clones failed to bind naturally paired control antibodies using v h 1-3 (dekosky et al., 2016) whether or not they were paired with a v k 3-20 light chain, from which the b12 light chain is derived ( fig. 1 c) . together, these data suggest that these four clones preferentially recognized the iglb12 idiotype. among the four anti-iglb12 idiotypes identified by our screen, we focused upon ib2 and ib3 because we solved their crystal structures in complex with iglb12 at a resolution of 2.6å and 3.0å, respectively. the crystal structures revealed that ib2 and ib3 displayed distinct modes of interaction with iglb12 ( fig. 2 ). ib3 interacted with both the heavy and light chains of iglb12 (fig. 2 , a-c), consistent with biolayer interferometry (bli) data ( fig. 1 c) . most interactions occurred within the heavy chain of iglb12 (fig. 2 , b and c), which contributed 76% of the buried surface area (bsa) and six of the eight hydrogen bonds. one hydrogen bond occurred in each of the cdrh3 and cdrl3 regions of iglb12, with the remaining six hydrogen bonds and bsa occurring in the cdr1, cdr2, and framework regions of iglb12, encoded by v h 1-3 or v k 3-20 (fig. 2 , d and e). in contrast, ib2 approached iglb12 at a 45°angle relative to ib3 and bound exclusively to the iglb12 heavy chain (fig. 2, a and f) . the cdrh3 region of iglb12 contributed nearly half of the bsa and two of the six hydrogen bonds, with the remaining bsa and hydrogen bonds largely focused upon cdrh1 and cdrh2, which are encoded by v h 1-3 (fig. 2 , g-j). the extensive interactions with the iglb12 heavy chain cdrh3 likely explain why ib2 and ib3 do not bind other antibodies using v h 1-3 ( fig. 1 c) . identification of iglb12-like bcrs using anti-iglb12 idiotypes we next assessed whether the anti-iglb12 idiotypes could be used to identify b cells expressing iglb12-like bcrs from within the polyclonal human b cell repertoire of hiv-uninfected individuals using single b cell sorting followed by rt-pcr amplification and sequencing of heavy and light chain genes. in an initial set of experiments, ib1, ib2, and ib3 were biotinylated and tetramerized using streptavidin conjugated to allophycocyanin (apc) and incubated together with 100 million human peripheral blood mononuclear cells (pbmcs) before enrichment using anti-apc microbeads. using this approach ∼2% of b cells in the apc-enriched fraction bound to ib1/2/3-apc, but not apc-dylight755 (apc755)-conjugated isotype control tetramers (fig. 3 a) . highlighting the efficiency of this enrichment approach, few ib1/2/3-apc + b cells could be detected in the apcdepleted fraction (fig. 3 a) . using this approach, single b cells binding ib1/2/3 tetramers were sorted into individual wells of a 96-well plate, and the heavy and light chains expressed by these cells were sequenced using nested rt-pcr (tiller et al., 2008) . in total, we facs-purified 91 single ib1/2/3-binding b cells from an individual. this yielded 66 heavy chain and 38 light chain sequences, with 31 heavy and light chain pairs (table s2) . to confirm the specificity of our flow cytometry approach, antibodies were produced from paired heavy and light chains from 10 cells. 80% of these antibodies bound ib2, but not ib1, ib3, or ib5, when assessed by bli (fig. 3 b) . interestingly, the eight antibodies that bound ib2 all used the same heavy chain variable region as igbl12, v h 1-3, while the two antibodies that failed to bind any of the anti-idiotypes used other v h alleles (table s2 ). in two of the cloned antibodies, the v h 1-3 + heavy chains were paired with v k 3-20 + light chains, suggesting that the lack of binding to ib1, ib3, and ib5 was not due to the absence of this segment ( fig. 1 c) . these results highlight the high specificity of our flow cytometry approach and suggest that ib2 is bound by a higher number of cells compared with other anti-idiotypes. to gain a more in-depth understanding of the bcrs recognized by individual anti-idiotypic antibodies, we next conducted sorts with ib2 and ib3 labeled with different fluorophores. we focused our analysis on these anti-idiotypes because the highresolution crystal structures of ib2 and ib3 complexed with iglb12 allowed us to make predictions about the bcrs they may preferentially recognize. from the crystal structures, we predicted that ib2 would be highly selective for b cells expressing bcrs using v h 1-3 with cdrh3 regions similar to iglb12. in contrast, we predicted that ib3 would be selective for b cells expressing bcrs using v h 1-3 with more diverse cdrh3s paired with v k 3-20 light chains with some cdrl3 similarity to iglb12. due to the random nature of v(d)j recombination that gives rise to cdr3 regions, we did not expect to find b cells expressing cdrh3s or cdrl3s identical to those of iglb12. to test these predictions, ib2 and ib3 were biotinylated and tetramerized using streptavidin conjugated to apc or r-phycoerythrin (pe). staining pbmcs with anti-idiotype tetramers revealed that <0.05% and 0.008% of the live b cell population from hiv-uninfected individuals bound ib2-apc or ib3-pe, respectively, but not apc755-or pe-dylight594 (pe594)-conjugated isotype control tetramers (fig. 4 a) . to the affinity (k d ) of the anti-idiotype for iglb12 was calculated based upon the on rate (k on ) and off rate (k off ) determined by bli. (c) the heat map displays the maximum shift (nm) measured by bli when ib1, ib2, ib3, or ib5 were incubated with the listed antibodies and antibody groups. detailed information on sequences of the non-v h 1-3 and v h 1-3 + control antibodies can be found in table s1 . data are representative of two to three similar experiments. more easily identify rare b cells binding ib2 and/or ib3, tetramer-binding cells were enriched using apc-specific and pe-specific magnetic microbeads from samples of 200 million pbmcs before flow cytometry (fig. 4 a) . using this approach, single b cells binding ib2 and/or ib3 tetramers were sorted into individual wells of a 96-well plate, and the heavy and kappa light chains expressed by these cells were sequenced using nested rt-pcr (tiller et al., 2008) . in total, we facs-purified 576 single ib2-binding b cells and 150 single ib3-binding b cells from three individuals. from ib2-binding b cells, we obtained man pbmcs that bound a cocktail containing ib1-apc, ib2-apc, and ib3-apc tetramers in fractions enriched or depleted of apc + cells using anti-apc microbeads before flow cytometry. apc755 tetramers containing isotype control antibodies were included in these experiments to exclude b cells specific for the apc, streptavidin, and conserved portions of the anti-idiotypes. the displayed plots were derived from ∼100,000 apc-depleted or ∼400,000 apc-enriched cells derived from 100 million pbmcs and representative of three similar experiments. the mean percentage ± sd of ib1/2/3-apc + apc755 − b cells in the enriched and depleted fractions from three individuals is shown on the plots. (b) the frequency of ten antibodies cloned from ib1/ib2/ib3-apc + apc755 − b cells from an individual were assayed for binding to ib1, ib2, ib3, or ib5 by bli. each antibody was assessed for binding in two to three independent experiments. 302 heavy chain and 174 kappa light chain sequences, with 136 heavy and light chain pairs (table s3 ). ib3-binding b cells yielded 84 heavy chain and 44 kappa light chain sequences, with 27 heavy and light chain pairs (table s4) . we were unable to recover heavy or light chain sequences from the small number of sorted cells binding both ib2 and ib3 tetramers. the bcr sequences from b cells binding to ib2 or ib3 were next compared with iglb12. on average, over 71% of the ib2binding b cells from all three individuals used the same v h gene as iglb12, v h 1-3 (fig. 4 b) . in contrast, only 4.6% of individually sorted b cells expressed v h 1-3 when neither antiidiotype was used to select cells for sorting ( fig. 4 b) , consistent with previous studies . in terms of the entire b cell repertoire, an average of 0.04% of the b cells bound ib2 and used v h 1-3 ( fig. 4 c) . in contrast, only 11% of the ib3binding b cells used v h 1-3, which amounted to ∼0.001% of the b cell repertoire (fig. 4 , b and c). consistent with the crystal structure revealing that ib3 interacts with both the heavy and light chains of iglb12 (fig. 2 , b and c), four out of the five ib3 + v h 1-3 + bcrs in which light chain sequences were recovered used the same light chain as igbl12, v k 3-20, or the closely related v k 3d-20 ( fig. 4 d) . surprisingly, ib2 + v h 1-3 + bcrs exhibited a slight preference for v k 3-20 ( fig. 4 d) , which was not predicted by the heavy chain-only mode of binding indicated by the crystal structure (fig. 2 , f-j). we next examined the unique heavy chain cdrh3 regions used by ib2 + v h 1-3 + bcrs in search of further similarity to iglb12. since we were only able to detect 10 ib3 + v h 1-3 + bcrs in this dataset, these bcrs were not included in this analysis. the crystal structure of ib2 bound to iglb12 revealed that 44% of the bsa was contributed by the cdrh3 of iglb12 (fig. 2 j) . nearly all of the cdrh3 bsa, 82% (i.e., 36% of the total bsa), is focused on the portion encoded by d h 2-21 ( fig. 4 e) . however, only 3.6% of ib2 + v h 1-3 + bcrs used d h 2-21, which was not different from control v h 1-3 + bcrs (fig. 4 f) . of the cdrh3 regions in ib2 + v h 1-3 + bcrs, the two most similar to iglb12 cdrh3 only displayed 50% amino acid identity ( fig. 4 g) . the moderate similarity of ib2 + bcr sequences to iglb12 led us to consider whether there were biological mechanisms selecting against cdrh3s that more closely matched iglb12. specifically, we hypothesized that the iglb12 heavy chain was autoreactive, resulting in deletion of similar sequences from the b cell repertoire. autoreactivity of iglb12 has not been reported, and conflicting reports of autoreactivity have been reported for the mature form of b12. one study reported that mature b12 exhibited autoreactivity using an in vitro assay (haynes et al., 2005; ota et al., 2013) , however b cell development in transgenic mice expressing this antibody was normal (ota et al., figure 4 . identification and analysis of human b cells able to bind anti-iglb12 idiotypes. (a) detection of live cd19 + cd3 − cd14 − cd16 − b cells from human pbmcs that bound ib2-pe and ib3-apc tetramers with or without enrichment using anti-pe and anti-apc microbeads before flow cytometry. pe594 and apc755 tetramers containing isotype control antibodies were included in these experiments to exclude b cells specific for the pe, apc, streptavidin, and conserved portions of the anti-idiotypes. the displayed plots were derived from ∼400,000 unfractionated pbmcs or ∼400,000 peand apc-enriched cells derived from 200 million pbmcs and representative of three individuals in three independent experiments. the percentages of single and double positive b cells in the enriched and unenriched fractions from one individual are shown on the plots. (b) the frequency of heavy chains using v h 1-3 within the population of ib2 + b cells, ib3 + b cells, and a population of control b cells that was not selected based upon antigen binding are displayed for three individuals. (c) the frequency of total v h 1-3 + , ib2 + v h 1-3 + , and ib3 + v h 1-3 + within the entire b cell repertoire is displayed for three individuals. (d) the frequency of v k 3-20/v k 3d-20 usage among 120 ib2 + v h 1-3 + bcrs and 10 ib3 + v h 1-3 + bcrs using kappa light chains pooled from three individuals are compared with 259 v h 1-3 + bcrs using kappa light chains from a control dataset derived from naive b cells . (e) quantitation of the percent bsa between ib2 and the segments derived from v h 1-3, d h 2-21, j h 6, and n nucleotide additions within iglb12 heavy chain cdrh3 from the crystal structures displayed in fig. 2 . (f) d h 2-21 usage among 228 ib2 + v h 1-3 + bcrs pooled from three individuals are compared with 467 v h 1-3 + bcrs from a control dataset derived from naive b cells . (g) comparison of cdrh3 similarity from 228 ib2 + v h 1-3 + bcrs compared with the cdrh3 of iglb12 using pairwise alignment. the p values (*, p < 0.05; **, p < 0.01; ***, p < 0.001) in b and c were determined using an unpaired two-tailed student's t test, and the p values in d were determined by fisher's exact test. 2013). as a preliminary assessment of potential autoreactivity, we measured iglb12 binding to human hep-2 cells using immunofluorescence. this approach revealed that iglb12 bound moderately to hep-2 cells compared with 4e10 (fig. 5 , a and b), an hiv-specific antibody previously shown to exhibit binding in this assay (haynes et al., 2005; verkoczy et al., 2010) . the iglb12 staining was similar to the mature form of b12, both of which were brighter than that of 10e8 (fig. 5 , a and b), an antibody that was previously shown to be nonreactive in this assay (haynes et al., 2005; verkoczy et al., 2010) . together, these data suggest that iglb12 is autoreactive. deletion and anergy in transgenic mice expressing iglb12 heavy chain to assess whether the autoreactivity detected in the hep-2 assay resulted in functional consequences in vivo, we generated a transgenic mouse in which all b cells expressed the iglb12 heavy chain. previous work has demonstrated that b cells from bcr knock-in mice expressing 4e10, but not mature b12, are deleted from the repertoire as a consequence of autoreactivity ota et al., 2013) . we found that iglb12 heavy chain transgenic mice displayed a three-to fourfold reduction in the number of cd19 + b220 + cd93 − cd3 − f4/80 − gr-1 − mature b cells in the spleen compared with wt mice or mice expressing a control inferred germline heavy chain generated using the same knock-in strategy fig. 6, a and b) . deletion could be traced back to the immature stage in the bone marrow, where iglb12 heavy chain transgenic mice contained approximately twofold fewer cd19 + b220 + cd93 + igm + cd3 − f4/ 80 − gr-1 − mature b cells compared with both wt and control transgenic animals (fig. 6, c and d) . together, the data indicate that a significant portion of murine b cells expressing an iglb12 heavy chain are deleted in the bone marrow. previous studies have shown that autoreactive b cells that escape deletion are often rendered functionally unresponsive to antigenic stimulation, a state referred to as anergy (goodnow et al., 1988 cyster et al., 1994; fulcher and basten, 1994; klinman, 1996; nemazee, 2006 nemazee, , 2017 . given this, we next considered whether the b cells remaining in iglb12 heavy chain transgenic mice were anergic. previously, several phenotypes of anergic b cells have been described in mice, including cd93 + cd23 + igm low "t3" transitional phenotype, as well as mature b cells expressing low levels of igm (allman et al., 2001; merrell et al., 2006; taylor et al., 2012; agrawal et al., 2013; sabouri et al., 2016) . in agreement with the hypothesis that circulating iglb12 heavy chain transgenic b cells were anergic, the number of t3 cells was increased 29-fold in these animals compared with control transgenic animals (fig. 6 , c and e). in addition, igm expression was 6.4-fold lower on mature b cells from iglb12 heavy chain transgenic mice compared with control heavy chain transgenic animals (fig. 7 a) . igd expression was also reduced 1.9-fold on mature b cells from iglb12 heavy chain transgenic mice compared with control transgenic animals ( fig. 7 b) , which suggested a global down-regulation of surface bcr on these cells. down-regulated surface bcr expression by iglb12 heavy chain transgenic mature b cells was confirmed through the assessment of cd79β (igβ; fig. 7 c) , a component of the bcr signaling complex required for surface bcr expression. the increased frequency of iglb12 heavy chain transgenic b cells exhibiting an anergic phenotype in combination with decreased bcr expression suggested that these cells would respond poorly to bcr stimulation. to test this, we labeled purified mature cd93 − b cells from wt and iglb12 heavy chain transgenic mice with celltrace violet (ctv) and incubated them with varying concentrations of anti-ig for 72 h in vitro before analysis. at all concentrations of anti-ig, fewer iglb12 heavy chain transgenic b cells had diluted ctv compared with b cells from wt mice (fig. 8, a and b) . these data suggest that the iglb12 heavy chain transgenic b cells that escape deletion are functionally anergic. combined with the hep-2 staining data, our murine data suggest that the iglb12 heavy chain is autoreactive, supporting the hypothesis that deletion and anergy of iglb12-like bcrs occurs in humans. the presence of anergy within iglb12 heavy chain transgenic b cells suggests that immunogens targeting this population would need to overcome these functional defects. before performing immunization experiments, we modified ib2 to ensure optimal murine responses. since ib2 was generated in a mouse, it would likely generate a poor response if injected unmodified, since tolerance mechanisms would be expected to eliminate most sources of t cell help targeting it. to increase possible cd4 + t cell help, the heavy and light chain constant regions used by this anti-idiotype were humanized (tiller et al., 2009 ). since multimerization can overcome anergic responses (cooke et al., 1994; gautam et al., 2016; mcguire et al., 2016) , humanized ib2 antigen-binding fragment (fab) was fused to a modified multimerization domain of the chicken c4b-binding protein (huib2-c4b), which self-assembles into a multimer containing up to seven humanized ib2 fab domains (ogun et al., 2008; hofmeyer et al., 2013) . to further garner t cell help, the 2w epitope (rees et al., 1999) was added to the c-terminus of c4b separated by a cathepsin consensus sequence in order to promote endosomal processing and mhc presentation (schneider . for immunization experiments, 4 × 10 5 b cells from cd45.2 + iglb12 heavy chain transgenic mice were labeled with ctv and transferred into cd45.1 + wt recipient mice. 1 d after transfer, the mice were immunized with huib2-c4b or an irrelevant humanized isotype control-c4b fusion (control-c4b). 5 or 14 d following the injection of the control-c4b, cd45.2 + donor b cells accounted for an average of ∼0.0006% of total b cells in control recipient animals, which we detected by enriching for cd45.2 + cells before analysis (fig. 9, a and b) . as expected of resting cells that had not encountered antigen and are therefore not proliferating, these cells largely retained high levels of ctv (fig. 9 , c and d). in contrast, 5 d following injection of huib2-c4b, donor b cells increased twofold to an average of ∼0.001% of total b cells (fig. 9, b-d) . approximately half of the donor b cells had low levels of ctv, indicating that proliferation accounted for the increased frequency of donor cells (fig. 9, b-d) . in addition to expansion, ∼33% of transgenic b cells in huib2-c4b-immunized mice bound gl7 and down-regulated cd38 (fig. 9 , e and f), a phenotype of germinal center b cells (allen et al., 2007; kurosaki et al., 2010; victora et al., 2010; vinuesa et al., 2010; kitano et al., 2011) . in these experiments, ib2binding germinal center b cells derived from both the transgenic donor and recipient animals could be detected using ib2 tetramers (fig. 10 , a-c). this analysis revealed that transgenic cd45.2 + iglb12 b cells accounted for 5.8% of the ib2binding germinal center b cells (fig. 10 d) . this frequency represented an increase from the 1-2% of transgenic cells found in cells outside the germinal center in both huib2-c4b and control-c4b injected animals (fig. 10 d) . together, these results indicate that anti-iglb12 idiotype antibodies can be used as immunogens that can overcome anergy and induce b cell activation and entry into germinal centers. our results demonstrate that anti-idiotype antibodies can identify and activate bnab precursor b cells in vivo and represent a potential alternative or complement to structure-based immunogen design. anti-idiotypes present several advantages. first, antigens derived from pathogens are generally difficult to produce and are often highly unstable or difficult to maintain in the desired conformation, especially after injection. from a manufacturing standpoint, anti-idiotypes are easily produced once identified and would be expected to be highly stable the bar indicates the mean, and the p values (*, p < 0.04; **, p < 0.01; ***, p < 0.001) were determined using an unpaired two-tailed student's t test. bancroft et al. in vivo, as evidenced by the licensure of several therapeutic antibodies. another advantage to this approach is that one could simultaneously use numerous anti-idiotypes to stimulate multiple potentially protective lineages. however, it must be recognized that it is unlikely that the injection of a single antiidiotype containing immunogen would induce the production of a protective antibody in situations where high levels of somatic mutation are required (scheid et al., 2009; dimitrov, 2010; pancera et al., 2010; zhou et al., 2010; haynes et al., 2012; mouquet et al., 2012; jardine et al., 2013; klein et al., 2013a,b; mcguire et al., 2013; escolano et al., 2016) . in these situations, successive injections of immunogens containing anti-idiotypes of increasingly mature antibodies may guide antibody maturation toward highly protective antibodies. alternatively, anti-idiotypes may be better suited for activation of protective antibodies that require less somatic mutation, such as those that can neutralize middle east respiratory syndrome (agrawal et al., 2016) and respiratory syncytial virus (goodwin et al., 2018) , or as the first step in a maturation pathway that includes pathogen-derived antigen in subsequent boosts. to identify four clones that produced anti-iglb12 idiotypes, we used multiplexed screening approaches to evaluate thousands of hybridomas. in future studies the screening burden can be further reduced by enrichment or sorting of b cells expressing bcrs with desirable binding properties before and/or after hybridoma fusion (taylor et al., 2012; spanier et al., 2016) . to our knowledge, this study represents the first time antiidiotypes have been used to identify and analyze naive b cells expressing bcrs with similarities to the inferred germline sequence of a broadly neutralizing hiv-1-specific antibody. an important result within these analyses is the finding that different anti-idiotypic antibodies can have different modes of interaction with bcrs similar to the one of interest. the binding profile analysis (fig. 1 c) and crystal structure data (fig. 2) suggested that ib2 would select only for bcrs with iglb12-like v h 1-3 + heavy chains, while ib3 would select for bcrs with iglb12-like v h 1-3 + heavy and v k 3-20 + light chains, perhaps as a subset of the ib2-binding population. sorting and sequencing bcrs from human b cells was therefore important and revealed that ib2 and ib3 selected for completely different populations of cells, with ib2 selecting far more stringently for v h 1-3 + bcrs compared with ib3. given these findings, we are exploring ways to better predict which anti-idiotypes will be better at identifying bcrs of interest. nevertheless, our results indicate that data are pooled from four independent experiments, which were normalized to account for experiment-to-experiment variability by displaying the data as a geometric mean fluorescence intensity (gmfi) fold increase over background fluorescence in cd19 − b220 − non-b cells. the bar indicates the mean, and p values (**, p < 0.001; ***, p < 0.001) were determined using an unpaired two-tailed student's t test (n = 3-11). the p values (*, p < 0.05; **, p < 0.01; ***, p < 0.001) were determined using an unpaired two-tailed student's t test. probing the preimmune repertoire with anti-idiotypes or other potential immunogens is an informative step in vaccine design. in the course of our sequencing experiments, we screened through the equivalent of 30 million b cells and only identified a few cdrh3 sequences recognized by the anti-idiotypes that showed >50% amino acid identity to iglb12. it is conceivable that the antiidiotypes were able to recognize rare cdrh3 sequences with higher identity to iglb12 but that we were unable to identify them due to limitations in the efficiency of cell sorting and bcr sequencing. the ability to screen a larger number of cells currently exceeds our capacity but is obtainable using enrichment and cell sorting in combination with droplet-based bcr sequencing approaches. nevertheless, the absence of cdrh3 sequences with high similarity to iglb12 led us to hypothesize that these sequences may be purged from the repertoire due to autoreactivity. support for this hypothesis came from iglb12 staining of hep-2 cells and experiments in which b cell deletion and anergy were observed in transgenic mice expressing the iglb12 heavy chain. these tolerance effects may explain why b12-like bnabs have not been commonly identified from hiv-infected individuals in several studies. in contrast, bnabs with other specificities are present and are identified in a large fraction of hiv-1 subjects who develop bnab responses, such as those targeting the n332 supersite landais et al., 2016; rusert et al., 2016; ditse et al., 2018) . the existence of deletion and anergy in mice suggested that immunization strategies able to overcome tolerance may be required to stimulate the production of b12-like bnabs in humans. the need to overcome tolerance is not unique to iglb12 knock-in mice, since deletion and anergy have also been detected in mice expressing the inferred germline forms of the hiv-1 bnabs 4e10 and 3bnc60 (mcguire et al., 2016; zhang et al., 2016) . autoreactivity is not a unique feature of some hiv-specific lineages and has been detected in assessments of influenza bnabs (andrews et al., 2015; bajic et al., 2019) . notably, it remains possible that the tolerance exhibited in transgenic mice expressing the iglb12 heavy chain is an artifact of the inability to accurately infer the true unmutated heavy chain cdrh3 from the mature b12 antibody. for example, the cdrh3 in this sequence contains two n regions that cannot be inferred and are instead carried over from the mature sequence. it is possible that the true germline heavy chain cdrh3 had different sequences in these locations that would not have resulted in deletion and anergy. importantly, activation and germinal center entry of iglb12expressing b cells is just the first step of the maturation process. this lineage would need to be guided toward broad env binding and neutralization, which will likely require many rounds of mutation and selection. future work will explore whether prime boost experiments with ib2/ib3 and env can be used to guide the maturation of bnab responses. overall, our study demonstrates the utility of using antiidiotypes to identify and stimulate b cells expressing desirable bcrs. future work is aimed at including anti-idiotypes in the vaccine design process, both as tools to identify cells of interest and as immunogens themselves. human pbmc collection and storage blood was obtained by venipuncture from healthy, hivseronegative adult volunteers enrolled in the general quality control study in seattle, wa, which was approved by the fred hutchinson cancer research center institutional review board. informed consent was obtained from all study participants before enrollment into the parent protocols. pbmcs were isolated from whole blood using accuspin system histopaque-1077 (sigma-aldrich) resuspended in 10% dimethylsulfoxide in heat-inactivated fetal bovine serum and cryopreserved in liquid nitrogen before use. . briefly, the d q52 -j h cluster was replaced in a c57bl/6derived c2 embryonic stem cell line with the iglb12 heavy chain vdj exon or inferred germline vrc01 heavy chain vdj exon. mice derived from these modified embryonic cells carrying the transgenic heavy chain were interbred and homozygous transgenic heavy chain +/+ male or female mice were used for experiments. control transgenic animals expressing the iglvrc01 heavy chain were generated in an identical manner . the sequence of iglb12 has been previously reported , and the cdrh3 and cdrl3 sequences are listed in table s2 . anti-idiotypic antibodies were generated by the fred hutchinson cancer research center antibody technology core. briefly, 8-12-wk-old female balb/c mice (jackson laboratory) were immunized and boosted intraperitoneally with iglb12 in adjuvant five times over a 5-mo period. 3 d following the final boost, splenocytes were electrofused to myeloma cells, and thousands of hybridoma colonies were screened for binding to iglb12 and a panel of control germline antibodies. hybridomas producing antibodies specific for iglb12 were subcloned from single cells and expanded before anti-idiotype purification. supernatant from these expanded hybridomas was harvested and purified over protein g resin (pierce) following the manufacturer's recommendations. to produce recombinant antiidiotypes, rna was extracted from 1 × 10 6 cells using the rneasy kit (qiagen) and the heavy and light chain sequences of the murine hybridomas were by obtained using the mouse ig-primer set (69831; emd millipore) using the protocol developed by seigel et al. (siegel, 2009) . sequences were codon optimized and cloned into ptt3-based igg expression vectors with human constant regions (snijder et al., 2018) using in-fusion cloning (clontech). purified anti-idiotype antibodies were biotinylated using an ezlink sulfo-nhs-lc-biotinylation kit (thermo fisher scientific) using a 1:1 molar ratio of biotin to anti-idiotype. unconjugated biotin was removed by centrifugation using a 50-kd amicon ultra size exclusion column (emd millipore). to determine the average number of biotin molecules bound to each anti-idiotype molecule, streptavidin-pe (prozyme) was titrated into a fixed amount of biotinylated anti-idiotype in increasing concentrations and incubated at room temperature for 30 min. samples were run on an sds-page gel (bio-rad laboratories) and transferred to nitrocellulose and incubated with streptavidin-alexa fluor 680 (1:10,000; thermo fisher scientific) to determine the point at which there was excess biotin available for the streptavidin-alexa fluor 680 reagent to bind. biotinylated antiidiotypes were mixed with streptavidin-pe or streptavidin-apc at the ratio determined above to fully saturate streptavidin and incubated for 30 min at room temperature. unconjugated antiidiotypes were removed by several rounds of dilution and concentration using a 300k nanosep centrifugal device (pall corporation). control pe594 and apc755 tetramers were created by mixing isotype control antibodies with streptavidin-pe preconjugated with dylight 594 (pe594) or streptavidin-apc preconjugated with dylight 755 (apc755; both from thermo fisher scientific) following the manufacturer's instructions. on average, pe594 and apc755 contained 4-8 dylight molecules per pe/ apc. the concentration of each anti-idiotype tetramer was calculated by measuring the absorbance of pe (565 nm, extinction coefficient = 1.96 µm −1 cm −1 ) or apc (650 nm, extinction coefficient = 0.7 µm −1 cm −1 ) and the lot-specific ratio of streptavidin-pe or streptavidin-apc. figure 10 . transgenic b cells expressing the iglb12 heavy chain enter germinal centers despite competition from wt cells. data from two experiments in which 4 × 10 5 purified b cells from cd45.2 + iglb12 heavy chain transgenic mice were labeled with ctv and adoptively transferred retro-orbitally into wt cd45.1 + recipients 1 d before intraperitoneal immunization with 20 µg huib2-c4b or control-c4b and 25 µg sigma adjuvant system. 14 d later, spleen and lymph nodes from individual mice were pooled and enriched for ib2-pe + and cd45.2-biotin + iglb12 heavy chain transgenic donor cells using anti-pe and anti-biotin microbeads before analysis by flow cytometry. (a) representative flow cytometric analysis of live cd19 + cd3 − gr-1 − f4/80 − b cells binding ib2-pe tetramers, but not isotype control pe594 tetramers. (b) representative gating of gl7 + cd38 − germinal center b cells in the ib2 + population described in a. (c and d) representative gating (c) and quantitation(d) of the frequency of cd45.2 + cd45.1 − donor b cells within the total ib2 + and ib2 + gl7 + cd38 − germinal center populations described in a and b. the percentages of cells within gates in a-c are from representative animals. the bars in d represent the mean from individual recipient mice (n = 6-7), and p values (*, p < 0.05) were determined using an unpaired two-tailed student's t test. bancroft et al. tetramer enrichment for human pbmc analysis, 200 × 10 6 frozen cells were thawed into rpmi with 10% fetal bovine serum (thermo fisher scientific), 100 u/ml penicillin (thermo fisher scientific), 100 µg/ml streptomycin (thermo fisher scientific), and 0.0275 mm 2-mercaptoethanol (sigma-aldrich). cells were centrifuged and resuspended to 0.2 ml in ice-cold facs buffer composed of 1× dulbecco's pbs (dpbs) with 1% newborn calf serum (thermo fisher scientific) containing 2% rat and mouse serums. pe594-and apc755-conjugated control antiidiotype tetramers were added at a final concentration of 5-15 nm and incubated on ice for 5 min to allow binding to b cells of unwanted specificities. ib2-pe and ib3-apc or ib1-apc, ib2-apc, and ib3-apc tetramers were added at a final concentration of 5 nm and incubated on ice for 25 min, followed by a 10-ml wash with ice-cold facs buffer. 25 μl of both anti-peand/or anti-apc-conjugated microbeads (miltenyi biotec) were added and incubated on ice for 30 min. 3 ml of facs buffer was then added to the cell mixture and passed over a magnetized ls column (miltenyi biotec). the column was washed once with 5 ml ice-cold facs buffer and then removed from the magnetic field. 5 ml ice-cold facs buffer was pushed through the unmagnetized column twice using a plunger to elute the bound cell fraction. purified naive mature b cells were prepared from spleens of iglb12 heavy chain transgenic mice using a b cell negative selection kit (miltenyi biotec) supplemented with 1.25 µg/ml biotinylated anti-cd93 (aa4.1; ebioscience) to remove transitional b cells. purified b cells were washed in 1× dpbs, adjusted to a concentration of 5 × 10 7 cells/ml in warm 1× dpbs and incubated with 5 µg ctv (thermo fisher scientific) for 12 min at 37°c before being washed with media containing 10% fetal bovine serum (thermo fisher scientific). cells were resuspended in warm 1× dpbs and 4 × 10 5 cells per mouse were injected retroorbitally into cd45.1 + recipients. 1 d following the transfer, recipient mice were injected intraperitoneally with 0.2 ml of the following solution: 0.1 ml (25 µg) diluted sigma adjuvant system (sigma-aldrich) and 20 µg of a humanized version of ib2 containing fabs fused to the 2w epitope (eawgalanwavdsa) heptamerized on the c4b nanoparticle as described previously (hofmeyer et al., 2013) in 0.1 ml 1× dpbs. a humanized isotype control fab-c4b in diluted sigma adjuvant system was used as a control. 5 d following injection, the spleen, inguinal, brachial, cervical, and mesenteric lymph nodes were harvested into 1× dpbs, minced, and then digested as described previously in dispase (0.8 mg/ml; thermo fisher scientific), collagenase (0.2 mg/ml; roche), and dnase (0.1 mg/ml; roche) for 20 min at 37°c. enzymes were inactivated with 5 mm edta, and single-cell suspensions were obtained. samples were centrifuged and cell pellets resuspended to 0.2 ml in 1× dpbs containing 1% newborn calf serum, 5 µg/ml of anti-cd16/cd3 (2.4g2, bioxcell), and 1.25 µg/ml anti-cd45.2 biotin (104; ebioscience) and enriched using 25 µl anti-biotin microbeads (miltenyi biotec) as described for tetramer enrichment. after centrifugation, supernatant was discarded and cell pellets were resuspended in facs buffer. the entire enriched fraction and 1/40th of the flow-through or unenriched fractions and were incubated with antibodies against surface markers in a total volume of 50 µl for 25 min on ice and washed with facs buffer. human pbmcs were labeled with 6.24 µg/ml anti-igm fitc (g20-127; bd biosciences), 4 µg/ml anti-igd percp-cy5.5 (ia6-2; bd biosciences), 2 µg/ml anti-cd20 efluor 450 (2h7; ebioscience), 1 µl anti-cd27 bv650 or bv480 (l128; bd biosciences), 1 µl anti-cd3 bv711 (uct1; bd biosciences), 1 µl anti-cd14 bv711 (mϕp9; bd biosciences), 2.8 µg/ml anti-cd16 bv711 (3g8; biolegend), and 1 µl anti-cd19 pe-cy7 (hib19; bd biosciences). in adoptive transfer experiments, mouse spleen and lymph node cells were labeled with 2.5 µg/ml gl7 fitc (bd biosciences), 2 µg/ml anti-cd45.1 percp-cy5.5 (a20; biolegend), 2 µg/ml anti-cd38 alexa fluor 700 (90; ebioscience), 2 µg/ml anti-cd45.2 pe-cy7 (104; biolegend), 2 µg/ml anti-cd3 bv510 (145-2c11; bd biosciences), 2 µg/ml anti-f4/80 bv510 (bm8; biolegend), 2 µg/ml anti-gr-1 bv510 (rb6-8c5; bd biosciences), 2 µg/ml anti-b220 bv786 (ra3-6b2; bd biosciences), and 2 µg/ml anti-cd19 buv395 (1d3; bd biosciences). 1 µg/ml streptavidin-bv650 (bd biosciences) was also included to detect donor cells that were enriched using anti-cd45.2 biotin. for b cell subset analysis, cells were labeled with 2.5 µg/ml anti-cd79β fitc (hm79-12; bd biosciences), 2 µg/ml anti-igd percp-cy5.5 (11-26c.2a; bd biosciences), 2 µg/ml anti-cd21 pe-cy7 (ebio8d9; ebioscience), 2 µg/ml anti-cd93 bv421 (aa4.1; bd biosciences), 2 µg/ml anti-cd3 bv510 (145-2c11; bd biosciences), 2 µg/ml anti-f4/80 bv510 (bm8; biolegend), 2 µg/ml anti-gr-1 bv510 (rb6-8c5; bd biosciences), 2 µg/ml anti-igm b bv650 (af6-78; bd biosciences), 2 µg/ml anti-cd23 bv786 (b3b4; bd biosciences), 2 µg/ml anti-b220 buv395 (ra3-6b2; bd biosciences), and 2 µg/ml anti-cd19 buv737 (1d3; bd biosciences). for in vitro proliferation experiments, cells were labeled with 2 µg/ml anti-cd3 bv510 (145-2c11; bd biosciences), 2 µg/ ml anti-f4/80 bv510 (bm8; biolegend), 2 µg/ml anti-gr-1 bv510 (rb6-8c5; bd biosciences), and 2 µg/ml anti-cd19 buv395 (1d3; bd biosciences). for all experiments, 0.25 µl fixable viability dye efluor 506 or efluor 780 (ebioscience) or fixable viability stain 700 (bd biosciences) was included in the surface antibody cocktail to distinguish live from dead cells. flow cytometry was performed on a five-laser (355 nm, 405 nm, 488 nm, 561 nm, and 640 nm) lsr ii, lsrfortessa, facsaria ii, or facsymphony device (bd biosciences) and analyzed with flowjo 10 software (tree star). 2 × 10 4 fluorescent accucheck counting beads (thermo fisher scientific) were added to the samples before flow cytometry and used to calculate total numbers of cell subtypes in the columnbound and flow-through suspensions, as previously described . in vitro proliferation assays naive mature b cells were purified and labeled with ctv as described for adoptive transfer experiments. cells were adjusted to a concentration of 2 × 10 6 cells/ml in rpmi (thermo fisher scientific) containing 10% fetal bovine serum (thermo fisher scientific), 100 u/ml penicillin (thermo fisher scientific), 0.1 mg/ml streptomycin, 2 mm l-glutamine (thermo fisher scientific), 0.02 mg/ml gentamicin (thermo fisher scientific), and 0.027.5 mm 2-mercaptoethanol (sigma-aldrich). 2 × 10 5 cells from each sample were added per well of a 96-well flat bottomed plate, and cells were cultured in the presence or absence of 2, 5, 10, or 25 µg/ml f(ab9) 2 goat anti-mouse ig (jackson immunoresearch) in a final volume of 0.2 ml and incubated for 72 h at 37°c before analysis by flow cytometry. single-cell sorting single human b cells were isolated using a facsaria ii cell sorter with diva configuration (bd biosciences) following tetramer enrichment and cell surface marker staining using human pbmcs as described above. single b cells from defined subpopulations were sorted according to surface marker expression patterns. specifically, ib2-or ib3-specific b cells were sorted using side scatter-based doublet discrimination, followed by gating on fvd − cd19 + cd20 + cd3 − cd14 − cd16 − b cells that bound ib2-pe or ib3-apc tetramers but not pe650 or apc755 tetramers containing isotype control antibodies. we also sorted 576 control fvd − cd19 + cd20 + cd3 − cd14 − cd16 − cd27 − igm/d + b cells from two individuals that were not stained with an antiidiotype. single cells were sorted into individual wells of 96-well pcr plates (eppendorf) containing 10 µl/well ice-cold lysis buffer containing 0.25 µl (12.5 u) rnaseout (thermo fisher scientific), 2.5 µl 5× superscript iv first strand buffer (thermo fisher scientific), 0.625 µl 0.1 m dithiothreitol (thermo fisher scientific), 0.3125 µl 10% igepal detergent (sigma-aldrich), and 6.625 µl diethyl pyrocarbonate-treated water. plates were sealed with adhesive pcr plate seals (thermo fisher scientific), centrifuged briefly, and immediately frozen on dry ice before storage at −80°c. nested rt-pcr bcr sequencing and analysis rt was performed using superscript iv (thermo fisher scientific) as previously described (tiller et al., 2009; wu et al., 2010) . briefly, 3 µl rt reaction mix consisting of 1.5 µl 50 µm random hexamers (thermo fisher scientific), 0.4 µl 25 mm deoxyribonucleotide triphosphates (dntps; thermo fisher scientific), 0.5 µl (10 u) superscript iv rt, and 0.6 µl water was added to each well containing a single sorted b cell in 10 µl lysis buffer and incubated at 50°c for 1 h. following rt, 2 µl cdna was added to 19 µl pcr reaction mix so that the final reaction contained 0.2 µl (0.5 u) hotstartaq polymerase (qiagen), 0.075 µl 50 µm 39 reverse primers, 0.115 µl 50 µm 59 forward primers, 0.24 µl 25 mm dntps, 1.9 µl 10× buffer (qiagen), and 16.5 µl water. the pcr program was 94°c 30 s, 57°c 30 s, 72°c 55 s, 50 cycles, 72°c 10 min for igm/igg/kappa light chains and 94°c 30 s, 60°c 30 s, 72°c 55 s, 50 cycles, 72°c 10 min for lambda light chains. after the first round of pcr, 2 µl of the pcr product was added to 19 µl of the second-round pcr reaction so that the final reaction contained 0.2 µl (0.5 u) hotstartaq polymerase, 0.075 µl 50 µm 39 reverse primers, 0.075 µl 50 µm 59 forward primers, 0.24 µl 25 mm dntps, 1.9 µl 10× buffer, and 16.5 µl water. pcr programs were the same as the first round of pcr. 4 μl of the pcr product was run on an agarose gel to confirm the presence of a ∼500-bp heavy chain band or 450-bp light chain band. 5 μl from pcr reactions showing the presence of heavy or light chain amplicons was mixed with 2 µl of exosap-it (thermo fisher scientific) and incubated at 37°c 15 min, followed by 80°c for 15 min to hydrolyze excess primers and nucleotides. hydrolyzed second-round pcr products were sequenced by genewiz with the respective reverse primer, and sequences were analyzed using imgt/v-quest to identify v, d, and j gene segments. non-v h 1-3-derived control antibodies were previous described liao et al., 2013; mcguire et al., 2014a,b) . paired heavy chain vdj and light chain vj sequences from bcr sequencing experiments or naive v h 1-3 + b cells identified previously were codon optimized and cloned into ptt3-derived expression vectors containing the human igg1, igk, or igl constant regions using in-fusion cloning (clontech). bli assays were performed on the octet.red instrument (for-tebio). for anti-idiotype binding screens, anti-mouse igg capture sensors (fortebio) were immersed in kinetics buffer (1× pbs, 0.01% bsa, 0.02% tween 20, and 0.005% nan 3 , ph 7.4) containing 5 µg/ml purified anti-idiotypic antibody for 100 s. after loading, the baseline signal was then recorded for 60 s in kinetics buffer. the sensors were then immersed in kinetics buffer containing 20 µg/ml purified human antibody for a 100 s association step. the maximum response was determined by averaging the nanometer shift over the last 5 s of the association step after subtracting the background signal from each analytecontaining well using empty anti-mouse igg fc capture sensors at each time point. kinetic analyses were performed at room temperature with shaking at 500 rpm. anti-mouse ig capture sensors were immersed in kinetics buffer containing 20 µg/ml purified antiidiotypic antibody for 240 s. after loading, the baseline signal was then recorded for 60 s in kinetics buffer. the sensors were next immersed into wells containing serial dilutions of purified iglb12 fab in kinetics buffer for a 300-s association phase, followed by immersion in kinetics buffer for an additional 600-s dissociation phase. the background was signal measured from each analyte-containing well using sensors loaded with a negative control antibody and subtracted from the signal obtained with each corresponding ligand-coupled sensor at each time point. kinetic analyses were performed at least twice with an independently prepared analyte dilution series. curve fitting was performed using a 1:1 binding model and fortebio data analysis software. the mean on rate and off rate values were determined by averaging all binding curves in each dilution series that matched the theoretical fit with an r 2 value of ≥0.95. the zeus ifa ana hep-2 test system was used as recommended by the manufacturer (zeus scientific) with minor changes. briefly, hep-2-coated slides were incubated with 25 µl antibody at 0.1 mg/ml for 30 min at room temperature. slides were then washed three times in 1× dpbs followed by the incubation of 25 µl of 0.01 mg/ml goat anti-human igg alexa fluor 594 (thermo fisher scientific) in 1× dpbs for 30 min at room temperature in the dark. after washing three times with 1× dpbs, slides incubated with zeus ifa ana hep-2 test system fixative, and coverslips were applied. images were acquired using the evos cell imaging system (thermo fisher scientific) and analyzed using imagej to determine the average alexa fluor 594 fluorescence per hep-2 cell for each image. fab and cleavable single-chain variable fragment (c/scfv) expression, purification, and complex preparation ib2 and ib3 igg purified from hybridomas were digested over activated ficin on agarose resin (thermo fisher scientific) for 48 h to obtain fabs. fabs for iglb12 were generated by digesting with 1 µg lys c (roche) for 10 mg of igg overnight at 37°c. the fabs were purified from fc regions by collecting the fraction that did not bind to a protein a column (thermo fisher scientific) and by size exclusion chromatography using superdex 200 (ge healthcare life sciences) in 5 mm hepes and 150 mm nacl. a c/scfv iglb12 was cloned and expressed using lentiviralbased stable transduction of human embryonic kidney suspension adapted freestyle 293f cells (thermo fisher scientific) as described previously (bandaranayake et al., 2011) . briefly, plasmids containing appropriate constructs were incorporated into replication-incompetent lentiviral particles and transduced into human embryonic kidney suspension adapted freestyle 293f cells plated in fresh media at 10 6 /ml in 10 ml and incubated at 37°c with 8% co 2 and 80% humidity. cells were supplemented with 25 ml fresh freestyle media 8-16 h after transduction. 3 d following transduction, cells were replated at a cell density of 0.5 × 10 6 /ml in cultures as large as 2 liters. culture supernatants were harvested by centrifugation at 8,000 rpm to remove cells and clarified by passing through 0.22-μm filter (thermo fisher scientific). clarified supernatants were supplemented with 150 mm nacl and concentrated using a 10,000 molecular weight cutoff amicon stirred cell concentrator (emd millipore). recombinant proteins were further purified by size exclusion chromatography using a superdex 75 and/or superdex 200 column (ge healthcare) running in 25 mm piperazine-n,n9-bis(2-ethanesylfonic acid) ph 7.0, 150 mm nacl, 1 mm edta, and 0.02% nan 3 . eluted fractions were analyzed using sds-page to assess purity. protein concentrations were determined using molar extinction coefficients and absorbance at 280 nm. complexes of ib2 fab + iglb12 c/scfv and ib3 fab + iglb12 fab were formed by mixing both components at a 1:1 molar ratio and incubating overnight at 4°c. complexes were purified by size exclusion chromatography using superdex 200 in 5 mm hepes and 150 mm nacl at ph 7.4. crystals of ib2 fab + iglb12 c/scfv and ib3 fab + iglb12 fab were obtained using a mosquito and an nt8 dispensing robots, and screening was done using commercially available screens (rigaku wizard precipitant synergy block #2, molecular dimensions proplex screen ht-96, hampton research crystal screen ht) by mixing 0.1 µl/0.1 µl (protein/reservoir) by the vapor diffusion method. crystals used for diffraction data were grown in the following conditions: ib3 fab + iglb12 fab crystals were grown in solution containing 0.2 m ammonium phosphate monobasic, 0.1 m tris, ph 8.5, and 50%(+/−) 2-methyl-2,4-pentanediol. ib2 fab + iglb12 c/scfv crystals were grown in solution containing 13.4% polyethylene glycol 8000, 13.4 peg 400, 0.1 m mgcl 2 , and 0.1 m tris, ph 8.5, using the vapor diffusion method. crystals were cryoprotected in solutions containing 30% molar excess of their original reagents and 20% glycerol for ib3 fab + iglb12 fab and 20% 2r-3r butanediol for ib2 fab + iglb12 c/scfv. crystal diffracted to 3å for ib3 fab + iglb12 fab and 2.6å for ib2 fab + iglb12 c/scfv. data were collected with advanced light source 5.1 and 5.2 and processed using hkl2000 (otwinowski and minor, 1997) . the structures were solved by molecular replacement using phaser in ccp4 (collaborative computational project, number 4, 1994) and pdb accession number 2ny7 (zhou et al., 2007) for the mature b12 portion as a search model. iterating rounds of structure building and refinement was performed in coot (emsley and cowtan, 2004) and phenix (adams et al., 2010) . the refinement statistics are summarized in table s5 , and the structural figures were made with pymol (delano, 2002) . atomic coordinates and structure factors of the ib2/iglb12 (6ol6) and ib3/iglb12 (6ol5) complex x-ray crystal structures have been deposited in the protein data bank. the heavy and light chain sequences of ib1 (mk775682 and mk775686), ib2 (mk775683 and mk775687), ib3 (mk775684 and mk775688), and ib5 (mk775685 and mk775689) have been deposited in genbank. table s1 contains sequence information for the control antibodies used in fig. 1 c. table s2 contains sequence information for the heavy and light chain sequences derived from b cells binding the ib1/2/3 tetramer cocktail. table s3 contains sequence information for the heavy and light chain sequences derived from human b cells binding the ib2 tetramers. table s4 contains sequence information for the heavy and light chain sequences derived from human b cells binding the ib3 tetramers. table s5 contains data collection and refinement statistics for the crystal structures shown in precursor frequency and affinity determine b cell competitive fitness in germinal centers, tested with germline-targeting hiv vaccine immunogens phenix: a comprehensive python-based system for macromolecular structure solution passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection transitional b cell subsets in human bone marrow germinal-center organization and cellular dynamics. immunity resolution of three nonproliferative immature splenic b cell subsets reveals multiple selection points during peripheral b cell maturation identification of common features in prototype broadly neutralizing antibodies to hiv envelope v2 apex to facilitate vaccine design immune history profoundly affects broadly protective b cell responses to influenza autoreactivity profiles of influenza hemagglutinin broadly neutralizing antibodies vectored immunoprophylaxis protects humanized mice from mucosal hiv transmission daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors recombinant human fab fragments neutralize human type 1 immunodeficiency virus in vitro neutralization epitopes of the f glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function broadly neutralizing antibodies to hiv and their role in vaccine design a large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals hiv vaccine design and the neutralizing antibody problem novel hiv vaccine strategies: overview and perspective the ccp4 suite: programs for protein crystallography immunoglobulin signal transduction guides the specificity of b cell-t cell interactions and is blocked in tolerant self-reactive b cells a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins competition for follicular niches excludes self-reactive cells from the recirculating b-cell repertoire large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires unraveling hot spots in binding interfaces: progress and challenges therapeutic antibodies, vaccines and antibodyomes hiv-1 subtype c-infected children with exceptional neutralization breadth exhibit polyclonal responses targeting known epitopes immunization for hiv-1 broadly neutralizing antibodies in human ig knockin mice immune tolerance negatively regulates b cells in knock-in mice expressing broadly neutralizing hiv antibody 4e10 highly conserved protective epitopes on influenza b viruses structure of a classical broadly neutralizing stem antibody in complex with a pandemic h2 influenza virus hemagglutinin antibody recognition of a highly conserved influenza virus epitope a highly conserved neutralizing epitope on group 2 influenza a viruses coot: model-building tools for molecular graphics sequential immunization elicits broadly neutralizing anti-hiv-1 antibodies in ig knockin mice reduced life span of anergic self-reactive b cells in a double-transgenic model a single injection of anti-hiv-1 antibodies protects against repeated shiv challenges altered immunoglobulin expression and functional silencing of self-reactive b lymphocytes in transgenic mice control systems and decision making for antibody production infants infected with respiratory syncytial virus generate potent neutralizing antibodies that lack somatic hypermutation structures of hiv-1 env v1v2 with broadly neutralizing antibodies reveal commonalities that enable vaccine design antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal hiv-1 infection in a transgenic mouse model cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv-1 antibodies b-cell-lineage immunogen design in vaccine development with hiv-1 as a case study broadly neutralizing human anti-hiv antibody 2g12 is effective in protection against mucosal shiv challenge even at low serum neutralizing titers arranged sevenfold: structural insights into the c-terminal oligomerization domain of human c4b-binding protein recombinant hiv envelope proteins fail to engage germline versions of anti-cd4bs bnabs broad and potent neutralization of hiv-1 by a gp41-specific human antibody broad and potent hiv-1 neutralization by a human antibody that binds the gp41-gp120 interface rational hiv immunogen design to target specific germline b cell receptors hiv-1 vaccines. priming a broadly neutralizing antibody response to hiv-1 using a germline-targeting immunogen hiv-1 broadly neutralizing antibody precursor b cells revealed by germlinetargeting immunogen development of a humanized monoclonal antibody (medi-493) with potent in vitro and in vivo activity against respiratory syncytial virus respiratory syncytial virus (rsv) sh and g proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated rsv subgroup b mutant combinatorial antibody libraries from survivors of the turkish h5n1 avian influenza outbreak reveal virus neutralization strategies bcl6 protein expression shapes pre-germinal center b cell dynamics and follicular helper t cell heterogeneity somatic mutations of the immunoglobulin framework are generally required for broad and potent hiv-1 neutralization antibodies in hiv-1 vaccine development and therapy the "clonal selection hypothesis" and current concepts of b cell tolerance unique properties of memory b cells of different isotypes hiv-1 vaccines based on antibody identification, b cell ontogeny, and epitope structure broadly neutralizing antibodies and the search for an hiv-1 vaccine: the end of the beginning broadly neutralizing antibody responses in a large longitudinal sub-saharan hiv primary infection cohort antibody 27f3 broadly targets influenza a group 1 and 2 hemagglutinins through a further variation in v h 1-69 antibody orientation on the ha stem co-evolution of a broadly neutralizing hiv-1 antibody and founder virus antibody-mediated protection against shiv challenge includes systemic clearance of distal virus neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention hiv-1 neutralizing antibodies: understanding nature's pathways protection of macaques against pathogenic simian/human immunodeficiency virus 89.6pd by passive transfer of neutralizing antibodies engineering hiv envelope protein to activate germline b cell receptors of broadly neutralizing anti-cd4 binding site antibodies hiv antibodies. antigen modification regulates competition of broad and narrow neutralizing hiv antibodies diverse recombinant hiv-1 envs fail to activate b cells expressing the germline b cell receptors of the broadly neutralizing anti-hiv-1 antibodies pg9 and 447-52d specifically modified env immunogens activate b-cell precursors of broadly neutralizing hiv-1 antibodies in transgenic mice identification of anergic b cells within a wild-type repertoire highly potent hiv-specific antibody neutralization in vitro translates into effective protection against mucosal shiv challenge in vivo complex-type n-glycan recognition by potent broadly neutralizing hiv antibodies receptor editing in lymphocyte development and central tolerance mechanisms of central tolerance for b cells prefusion f-specific antibodies determine the magnitude of rsv neutralizing activity in human sera the oligomerization domain of c4-binding protein (c4bp) acts as an adjuvant, and the fusion protein comprised of the 19-kilodalton merozoite surface protein 1 fused with the murine c4bp domain protects mice against malaria a common neutralizing epitope conserved between the hemagglutinins of influenza a virus h1 and h2 strains b cells from knock-in mice expressing broadly neutralizing hiv antibody b12 carry an innocuous b cell receptor responsive to hiv vaccine candidates processing of x-ray diffraction data collected in oscillation mode crystal structure of pg16 and chimeric dissection with somatically related pg9: structure-function analysis of two quaternary-specific antibodies that effectively neutralize hiv-1 structure and immune recognition of trimeric pre-fusion hiv-1 env correlates of immune protection in hiv-1 infection: what we know, what we don't know, what we should know antibody protects macaques against vaginal challenge with a pathogenic r5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro a mouse model for hiv-1 entry reverse vaccinology 2.0: human immunology instructs vaccine antigen design an inverse relationship between t cell receptor affinity and antigen dose during cd4(+) t cell responses in vivo and in vitro recurrent potent human neutralizing antibodies to zika virus in brazil and mexico determinants of hiv-1 broadly neutralizing antibody induction igd attenuates the igm-induced anergy response in transitional and mature b cells the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2g12 recognizes a cluster of alpha1-->2 mannose residues on the outer face of gp120 broad diversity of neutralizing antibodies isolated from memory b cells in hivinfected individuals sequence and structural convergence of broad and potent hiv antibodies that mimic cd4 binding development of prophylactic vaccines against hiv-1 cutting edge: introduction of an endopeptidase cleavage motif into a determinant flanking region of hen egg lysozyme results in enhanced t cell determinant display neutralizing antibody directed against the hiv-1 envelope glycoprotein can completely block hiv-1/siv chimeric virus infections of macaque monkeys passive transfer of modest titers of potent and broadly neutralizing anti-hiv monoclonal antibodies block shiv infection in macaques antibody affinity optimization using yeast cell surface display an antibody targeting the fusion machinery neutralizes dual-tropic infection and defines a site of vulnerability on epstein-barr virus efficient generation of monoclonal antibodies against peptide in the context of mhcii using magnetic enrichment hiv vaccine design to target germline precursors of glycan-dependent broadly neutralizing antibodies structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses deletion and anergy of polyclonal b cells specific for ubiquitous membrane-bound self-antigen humoral immunity. apoptosis and antigen affinity limit effector cell differentiation of a single naïve b cell heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm+ memory b cells efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning cloning and expression of murine ig genes from single b cells autoreactivity in an hiv-1 broadly reactive neutralizing antibody variable region heavy chain induces immunologic tolerance germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter t cells and follicular dendritic cells in germinal center b-cell formation and selection protocol g principal investigators. 2009. broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target broad neutralization coverage of hiv by multiple highly potent antibodies structural insights on the role of antibodies in hiv-1 vaccine and therapy rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1 germline-like predecessors of broadly neutralizing antibodies lack measurable binding to hiv-1 envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens initiation of immune tolerance-controlled hiv gp41 neutralizing b cell lineages structural definition of a conserved neutralization epitope on hiv-1 gp120 structural basis for broad and potent neutralization of hiv-1 by antibody vrc01 structural repertoire of hiv-1-neutralizing antibodies targeting the cd4 supersite in 14 donors we thank a. macy, m. baker, d. alwan, and the fred hutchinson cancer research center flow cytometry shared resource for technical assistance and s. voght for careful reading of the manuscript. we thank the james b. pendleton charitable trust for generous support of formulatrix robotic instruments. we also thank j. mcelrath for pbmc (fred hutchinson cancer research center, seattle, washington). key: cord-350221-8u6q3wfa authors: yang, sung-tae; kreutzberger, alex j. b.; kiessling, volker; ganser-pornillos, barbie k.; white, judith m.; tamm, lukas k. title: hiv virions sense plasma membrane heterogeneity for cell entry date: 2017-06-28 journal: sci adv doi: 10.1126/sciadv.1700338 sha: doc_id: 350221 cord_uid: 8u6q3wfa it has been proposed that cholesterol in host cell membranes plays a pivotal role for cell entry of hiv. however, it remains largely unknown why virions prefer cholesterol-rich heterogeneous membranes to uniformly fluid membranes for membrane fusion. using giant plasma membrane vesicles containing cholesterol-rich ordered and cholesterol-poor fluid lipid domains, we demonstrate that the hiv receptor cd4 is substantially sequestered into ordered domains, whereas the co-receptor ccr5 localizes preferentially at ordered/disordered domain boundaries. we also show that hiv does not fuse from within ordered regions of the plasma membrane but rather at their boundaries. ordered/disordered lipid domain coexistence is not required for hiv attachment but is a prerequisite for successful fusion. we propose that hiv virions sense and exploit membrane discontinuities to gain entry into cells. this study provides surprising answers to the long-standing question about the roles of cholesterol and ordered lipid domains in cell entry of hiv and perhaps other enveloped viruses. cell membranes consist of numerous proteins and lipids exhibiting complex behavior that includes organization into dynamic nanodomains enriched in sphingolipids and cholesterol that are sometimes referred to as "lipid rafts" (1, 2) . accumulating evidence indicates that membrane domain organization plays a vital role in cell signaling and adaptive immune responses to combat infections by many pathogens (3) (4) (5) . for example, hiv has evolved to exploit organized membrane domains to gain entry into host cells (6) (7) (8) (9) . it is well established that recognition and attachment of hiv to host cells are mediated by the binding of the viral envelope glycoprotein gp120 to the cell-surface receptor cd4 and a co-receptor ccr5 or cxcr4 (10, 11) . after attachment, membrane fusion mediated by subunit gp41 of the envelope glycoprotein leads to cell entry. because cd4 has been found to associate with detergent-resistant fractions of the cell membrane, it has been assumed that cholesterol-and sphingomyelin-rich rafts are platforms for hiv entry (12, 13) . however, because lipids in cholesterolrich regions are more tightly packed and more ordered than those in the surrounding membrane, these sites may seem energetically unfavorable for membrane fusion, thereby raising doubts about the involvement of ordered lipid regions in the fusion step of hiv entry. because ordered lipid domains are thought to be dynamically fluctuating nanoscopic assemblies of lipids and resident proteins in living cells, visualization of the potential involvement of these membrane regions upon entry of viral particles into intact cells remains technically challenging (14, 15) . in a first step toward solving this problem, we showed recently that model membranes with coexisting microscopic ordered and fluid lipid domains were useful in proving that the fusion peptide of gp41 interacts preferentially with ordered/disordered lipid domain boundaries and that these boundaries are also the preferred sites for fusion peptide-mediated membrane fusion (16) . however, these discoveries raised the important question of whether the results obtained in this highly artificial model system could be translated to biological membranes that contain the hiv receptor and co-receptor and to virus particles bearing the full, trimeric, gp120/gp41 complex, that is, whether membrane domain boundaries would still be the preferred sites for virus attachment and/or membrane fusion at the plasma membranes of appropriate target cells. therefore, we developed a new approach to measuring binding and fusion of hiv particles with plasma membranes to assess the role of membrane heterogeneity in these processes. we used giant plasma membrane vesicles (gpmvs) derived from hela cells that stably express the cd4 receptor and ccr5 co-receptor (cd4 + /ccr5 + ), investigated the lateral partitioning of both receptors in these membranes, and imaged the preferred regions of hiv binding and fusion at the single-particle level (fig. 1a) . gpmvs show temperaturedependent, micrometer-scale liquid-ordered/liquid-disordered (lo/ld) phase separation and have been extremely useful to study the dynamics and lateral distribution of resident membrane proteins (17, 18) . using this approach, we found that lipid order discontinuities in heterogeneous receptor-and co-receptor-containing plasma membranes are important for gp120-mediated receptor/co-receptor targeting and gp41-mediated membrane fusion. a number of studies have been conducted to localize cd4 and ccr5 in lymphocyte cell membranes. although the association of cd4 with lipid rafts is generally accepted, divergent conclusions have been reached regarding the raft localization of ccr5 (6, 12) . confirming previous results, we observed by immunofluorescence that cd4 colocalized with raft-resident ganglioside gm1 in cd4 + /ccr5 + hela cells, whereas ccr5 colocalized only partially with gm1 ( fig. s1 ). when we prepared gpmvs from cd4 + /ccr5 + hela cells by gentle formaldehyde and dithiothreitol (dtt) treatment (movie s1 and fig. s2, a and b), which forms membrane blebs by releasing otherwise intact cell membranes from the cytoskeleton (17, 18) , we found that most gpmvs showed microscopic lo/ld phase separation below 25°c (movie s2 and fig. s2 , c to e). as expected from immunostaining of intact cells, cd4 and gm1 were substantially excluded from the ld region in gpmvs, indicating the partitioning of cd4 into the lo region ( fig. 1 , b and c). preferential localization of ccr5 at the boundaries between lo and ld phases occurred and was evident in cell-attached blebs and cell-detached gpmvs (fig. 1, b and c) . this result might explain the debate about the controversial association of ccr5 with lipid rafts: ccr5 is not associated with lo or with ld lipid phases but accumulates at the boundaries between them. very similar results were obtained with an alternative procedure to induce membrane blebs by the application of small concentrations of n-ethylmaleimide (nem) ( fig. s3 ), suggesting that the results are independent of the blebbing agent and that the resulting gpmvs do not have serious membrane defects. recognition of lo/ld membrane boundaries by hiv next, we examined whether and how hiv envelope (env) particles interact with gpmvs. murine leukemia viruses (mlvs) pseudotyped with hiv gp120/gp41 and fluorescently labeled with mko-gag were incubated with cd4 + /ccr5 + gpmvs. we visualized bound particles by epifluorescence microscopy and found that they migrate along the preparation of large-scale phase-separated gpmvs facilitates the study of the lateral distribution of cd4 and ccr5 and the role of ordered lipid domains in hiv entry. lo and ld phases on gpmvs are indicated with red and blue lines, respectively. (b and c) partitioning of cd4 and ccr5 in cell-attached (b) or cell-detached (c) gpmvs. gpmvs were first stained with 1,1′-didodecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (dii) at 4°c for 60 min and then incubated with fluorescent-labeled ctxb (alexa fluor 555), anti-cd4 (alexa fluor 488), or anti-ccr5 (alexa fluor 647) antibodies at 4°c for 60 min. epifluorescence images of gpmvs performed at room temperature (~22°c) show large-scale lo/ld phase coexistence in which dii and ctxb are used as markers of ld and lo phases, respectively. the selected rectangles are enlarged and shown below. (d) hiv binding to cell-attached gpmvs. cell-attached gpmvs (left) and viruses labeled with mko-gag (center) are visualized by bright-field and epifluorescence microscopy, respectively. image overlay (right) shows that some bright dots (hiv env particles) indicated by arrows are sitting on the gpmv surface (top). time series of images from the selected box region shows that the particles bound to gpmv are free to move laterally along the gpmv surface (bottom). a movie version of this figure is available (movie s3). (e and f) hiv binding to the boundaries between lo and ld phases in cell-attached (e) or cell-detached (f) gpmvs. gpmvs (left) and hiv env particles (center) were labeled with dio and mko-gag, respectively. (g) quantification of virions bound to three different regions (lo, ld, and lo/ld boundary) of the gpmvs. data are means ± sd of triplicates. a total of 120 virions were analyzed for their distribution between the three compartments. the largest fraction of virions was found in lo/ld boundary regions. scale bars, 10 mm. the gpmv surfaces ( fig. 1d and movie s3) . staining the gpmvs with the ld marker 3,3′-dilinoleyloxacarbocyanine perchlorate (dio) showed that most bound particles localize to lo/ld boundary regions in cellattached (fig. 1e, movie s4, and fig. s4 ) and cell-detached gpmvs ( fig. 1f and movie s5). we quantified the virions bound to three different regions (lo, ld, and lo/ld boundaries), as shown in fig. 1g . to assess whether the targeting of the virions to the lo/ld interface is specific to hiv env, we also performed similar experiments with mlvs pseudotyped with the env g protein (glycoprotein) from vesicular stomatitis virus (vsv-g). vsv-g virions preferentially bound to the ld phase in gpmvs ( fig. s5 ), indicating that the lo/ld boundary interaction is not a general feature of all viral particles and requires hiv env. given that cd4 is initially localized in lo phase regions of the membrane, it seems reasonable to propose that hiv virions bind to cd4-rich lo phase membrane regions and then move to domain boundaries where they also engage with ccr5. ccr5 binding and the preferential targeting of the gp41 fusion peptide to lipid phase boundaries (16) likely combine to direct the particles for fusion at these boundaries. to determine whether hiv env particles indeed fuse with gpmvs at lipid phase boundaries, we first examined whether they fuse with gpmvs in a bulk lipid mixing assay. virions were labeled with selfquenching concentrations of the fluorescent lipid probe r18 and mixed with unlabeled gpmvs. lipid mixing was only observed when the gpmvs contained both cd4 and ccr5 but was negligible with gpmvs that were derived from hela cells lacking receptors and coreceptors ( fig. 2a) . demonstrating the specificity of the hiv/gpmv fusion system, we found that lipid mixing is strongly suppressed by the well-known hiv entry inhibitors maraviroc and enfuvirtide (fig. 2b ). maraviroc inhibits binding to the co-receptor ccr5 (19) , and enfuvirtide inhibits fusion by blocking the required conformational change of gp41 (20) . maraviroc reduced the targeting of hiv virions to phase boundaries (fig. 2c) , further supporting the notion that ccr5 recognition occurs at these boundaries. by contrast, enfuvirtide had little effect on the boundary preference of hiv binding while still blocking fusion (fig. 2d ), suggesting that boundary binding alone is not sufficient for fusion; it requires the energy from refolding gp41 into the six-helix bundle structure as well. we also observed fusion with gpmvs at the single-particle level. the fluorescence of many hiv env particles that were bound at lo/ld phase boundaries spread over time, indicating that the particles fused with the gpmvs (fig. 2e ). in addition, we carried out electron cryo-microscopy (cryoem) in an attempt to directly visualize the process of fusion of viral particles with gpmvs. as previously observed for bare mlvs containing only gag and gag-pol (21), the spherical or tennis racket-shaped particles showed a characteristic morphology with a capsid surrounded by the viral envelope (fig. 2f ). representative electron micrographs obtained from four different incubations with two separate viral and two separate gpmv preparations show hiv env particles in intimate contact with the gpmv surfaces ( fig. 2g and fig. s6 ). we also observed membrane perturbations in very confined areas of the gpmv lipid bilayer that may represent fusion intermediates (fig. 2g ). to ensure that the observed contacts were specific, controls with gpmvs lacking cd4 and ccr5 were mixed with hiv env particles. fifteen to 30 randomly selected gpmvs per grid were imaged for three different samples, and no virus-gpmv fusion events were observed. however, the present resolution of the cryoem images does not permit a distinction between lo and ld phases and is thus not sufficient to determine whether fusion occurs at the lo/ld phase boundaries. next, we examined whether the lo/ld phase coexistence on gpmvs is required for hiv env particle binding and/or fusion. to disrupt lo phase domains, we treated gpmvs with methyl-b-cyclodextrin (mbcd), which depletes cholesterol from the membranes. mbcd treatment of gpmvs led to a marked change from approximately circular lo domains to corrugated shapes that are characteristic for bilayers in the gel phase ( fig. 3a and movie s6). however, cholesterol extraction did not significantly alter the overall surface expression levels of cd4 or ccr5 on gpmvs (fig. 3b ). virion binding was quantified by directly counting the number of particles attached to gpmvs (fig. 3c ) or by a centrifugation-based assay ( fig. s7 ). regardless of the method, particle binding was high and not diminished by cholesterol depletion of the gpmvs, indicating that the presence of lo domains is not required for the binding of virions. however, hiv env particles did not bind to gpmvs lacking cd4 and ccr5, further confirming that their attachment is mediated by these receptors rather than lipid phase separation. contrary to virion attachment, disruption of the lo phase domains in gpmvs by mbcd significantly decreased the efficiency of fusion of hiv env virions with gpmvs ( fig. 3d ). this result agrees with a previous report demonstrating a decrease of hiv cell entry after depletion of cholesterol from the host cell membrane (22) . the inhibitory effect on fusion by the disruption of lo phases and lo/ld phase boundaries in gpmvs suggests that the unique properties of these boundaries are responsible for attracting receptors and co-receptors and the high fusogenicity of hiv env virions at these sites. thus, the same physical principles that promote fusion peptide-mediated fusion in lipid model membranes, namely, line tension and lipid packing defects at lo/ld phase boundaries (23), also provide a significant driving force for membrane fusion of hiv env viral particles with biological membranes. lysolipids, which adopt an inverted-cone molecular shape, have been reported to inhibit fusion in a wide variety of systems owing to their propensity to induce positive intrinsic membrane curvature (24, 25) . here, we tested whether lysolipids affect the lipid phase behavior of gpmvs and the ability of hiv to fuse with them. lysosphingomyelin (lysosm) partially dissolved the lo domains of gpmvs into much smaller domains, whereas lysophosphatidylcholine (lysopc) had little effect on lo domains ( fig. 4a and movie s7). although both lysosm and lysopc share a choline headgroup and a similar molecular shape, they may differently affect line tension at phase boundaries. lysosm is a linactant that appears to reduce line tension, whereas lysopc probably partitions more uniformly into the ld phase with a lesser effect on line tension. hiv env virion binding was not affected by lysosm or lysopc (fig. 4b ). however, lysosm significantly reduced the fusion efficiency of hiv particles, whereas lysopc had only a moderate effect (fig. 4b ). together, these results again support the notion that the site of fusion is the domain boundary region where inhibitory lysosm is enriched, but lysopc is not enriched. the same effect could be triggered enzymatically. treatment of gpmvs with phospholipase a2 (pla2), which hydrolyzes glycerophospholipids, including pc to lysopc, had little effect on the lipid phase behavior of gpmvs and hiv fusion, whereas sphingolipid ceramide n-deacylase (scdase), which converts sm into lysosm, dispersed the lo domains, which eventually spread over the entire gpmv surface and significantly inhibited fusion (fig. 4 , c and d). note that the fluorescent lipid probe dii always marks the ld phases in these images. the apparent reversal of contrast in some images is due to different levels of cholesterol that can change the connectivity of lo phases, as previously demonstrated in model systems (26) . to further probe the influence of lysolipids on lipid phases and hiv fusion in well-defined model membranes, we used giant unilamellar vesicles (guvs) and large unilamellar vesicles (luvs) composed of bsm/bpc/bps/cholesterol (2:1:1:1) as models (16) . only lysosm significantly changed the lo domains in guvs (fig. 4e ) and inhibited the fusion of luvs with hiv fusion peptide-decorated virosomes, whereas lysopc had only a small effect at the highest concentration (fig. 4f ). the domain edges first undulated before larger invaginations formed, eventually dispersing the domains into much smaller ones (movie s8). regulating the size of lipid rafts by using lysosphingolipids has significant consequences not only on hiv entry but also on cell signaling. for example, it is well known that sphingosine-1-phosphate is a potent mediator of numerous signaling pathways (27) and the finding that these signaling mechanisms could be potentiated by raft regulation may be an interesting topic for future investigations. as previously reported, the recruitment of some proteins such as ras and rac-1 to domain boundaries may be more general and may contribute to the regulation of signaling pathways by regulating the nature and size of lipid rafts in these systems (28, 29) . supported lipid bilayers generated from synthetic and natural lipids have been widely applied since their original introduction (30) , including more recently to study fusion of single particles (16, 31) . the planar geometry of supported membranes has the advantage over guvs that a large extended membrane can be observed by total internal reflection fluorescence (tirf) microscopy and, therefore, that large numbers of single events can be simultaneously recorded with high signal-to-noise ratio in each experiment, leading to greatly improved statistics (32) . to exploit this advantage for biological membranes as well, we have developed supported planar plasma membranes (sppms) for single-particle fusion studies (fig. 5a ). our strategy for preparing sppms is to fuse gpmvs onto polymer-supported lipid monolayers that have been transferred from a langmuir trough (33) , which is different from previous methods (34) and preferentially orients membrane receptors with their binding sites accessible from solution. similar to plain supported lipid bilayers (26, 35) , sppms prepared in this fashion display micrometersized domains, in which raft and nonraft lipid components are laterally mobile ( fig. s8 ). in agreement with the observations with gpmvs, the overlay of cd4 and ccr5 images in sppms shows that cd4 was substantially associated with lo domains, whereas ccr5 was preferentially located at the edges of the domains (fig. 5b and fig. s9 ). we observed by tirf microscopy that single hiv env particles bound predominantly to domain boundaries, whereas most vsv-g particles bound to ld phases on sppms (fig. 5 , c and d). in addition, these experiments recapitulate the observation made with gpmvs that the location of hiv binding to sppms is determined primarily by the location of the receptor and co-receptor in these membranes and not by the lipid phases (fig. 5e ). we observed large numbers of single-membrane fusion events of hiv env particles with sppms ( fig. 5f and movie s9) and classified them as docking, hemifusion, or full fusion (fig. 5g) in lo, ld, and lo/ld compartments of the sppm. we observed no direct full fusion and very little hemifusion in ld phases and only 11 ± 3% direct full fusion and 13 ± 4% hemifusion of hiv virions in the lo phase domains (fig. 5h ). in contrast, hiv virions fused very efficiently (56 ± 3% of all particles) at the lo/ld domain boundaries and an additional 9 ± 4% hemifused in these locations (fig. 5h ). these results are qualitatively similar to but quantitatively more robust than those obtained in gpmvs and therefore validate sppms as a useful additional system to quantitatively analyze fusion in heterogeneous biological membranes at the single-particle level. membrane fusion in hiv entry is a highly cooperative process that must overcome several energy barriers associated with different fusion intermediates. this energy must be provided and released at the right time and at the right place to drive the merger between the viral and target membranes. although cholesterol-rich lipid domains have been implicated in cell entry, their proposed involvement was puzzling because ordered lipids are thought to be detrimental to membrane bending and fusion. however, we recently discovered that lo/ld phase boundaries facilitate fusion in model systems and demonstrated that line tension at these boundaries might provide an additional previously unrecognized driving force for fusion (23) . this previous work was limited because no receptors or co-receptors were present and because it could be argued that pure lipid model membranes oversimplify biological membranes. to overcome these limitations, we demonstrated in this work that a very similar mechanism governs hiv fusion with biological host membranes containing its natural receptors. we directly observed that lipid domain boundaries are the portals for hiv binding and membrane fusion, presumably because they present the weakest points in the host membrane. on the basis of these results, we propose the following model: hiv gp120 binds to cd4 receptors located in cholesterol-rich lipid domains, which exist transiently in the plasma membranes of living cells. if located near ccr5 co-receptors at domain boundaries, this initial binding leads to structural changes of gp120 and binding to the ccr5 co-receptor, enriching bound virions at the boundaries (fig. 6a) . after cd4 and ccr5 binding, gp41 changes its conformation to expose the fusion peptide to lipids in the boundary region (16) . the predisposed preference of ccr5 for membrane domain boundaries greatly facilitates the targeting of the fusion peptide to these same fusion-promoting areas of the host membrane. although we did not examine the other hiv co-receptor, cxcr4, in this study, it might function equivalently to ccr5 in recruiting virions to domain boundary regions for more efficient fusion. because receptors cooperate with membrane domain boundaries to form hiv entry sites, it would be interesting to examine in future studies the binding and fusion efficiencies of hiv particles with gpmvs or sppms, where cd4 and ccr5 are distributed differently or randomly. in contrast to many other enveloped viruses, hiv displays only a few (~14) trimeric glycoprotein spikes on its envelope (36) . this small number is certainly inefficient for hiv entry and fusion. however, the cooperativity of hiv recognition and fusion at membrane domain boundaries may be a unique way to overcome this deficiency and may provide enough energy to drive fusion through its transition states with low env copy numbers. just as in the model systems (23) , line tension at the boundary likely serves as an additional driving force for fusion. it is well known that cd4 + t cells play a central role in the immune response following t cell antigen receptor engagement, which triggers t cell activation and proliferation against invading pathogens (37, 38) . t cell activation elicits the recruitment of a number of proteins to ordered lipid regions (39) and results in the formation of larger membrane domains, which can be exploited by hiv to facilitate entry (fig. 6, b and c) . hiv exhibits infection at a much lower frequency in naïve cd4 + t cells than in activated cd4 + t cells (40, 41) , suggesting that hiv infection may be associated with the coalescence of cholesterol-rich lipid domains. just as in model systems, we have shown here that line tension may also be a dominant force that promotes membrane fusion in biological membranes. the boundaries of the larger lipid domains in activated t cells could contribute to the elevated fusogenic capacity of activated t cells versus resting t cells. it is possible that activation of the immune system against other invading pathogens makes the cells more prone to hiv entry through the described mechanism. therefore, enhanced domain activation could lead to an elevated production of hiv in people infected by other pathogens and thus an acceleration of the progression of aids (fig. 6d) . finally, this work also showed that gpmvs and a novel procedure to produce supported planar gpmv-derived membranes that we call sppms are very useful to study the selected targeting and membrane fusion of hiv virions. we anticipate that these methods will also be useful to study the binding, fusion, and properties of many other particles on cell-derived plasma membranes. the new approaches are versatile and should lead to many new discoveries regarding the role of lipid and protein heterogeneity of cell membranes. they may also serve as novel platforms for screening of viral entry inhibitors. most lipids and fluorescent probes were from avanti polar lipids, and dio, dii, did, and octadecyl rhodamine b chloride (r18) were from invitrogen. mbcd, nem, and fluorescein isothiocyanate-, alexa 647-, or alexa 555-labeled ctxb were purchased from sigma-aldrich. formaldehyde and dtt were purchased from avantor and rpi, respectively. alexa 488-labeled cd4 antibody and alexa 647labeled ccr5 antibody were purchased from novus biologicals. hiv entry inhibitors (maraviroc and enfuvirtide), scdase, and pla2 were purchased from sigma-aldrich. hiv fusion peptide was custom-synthesized by the yale w.m. keck biotechnology resource laboratory. hiv pseudovirus production hiv env particles pseudotyped with envelope gp160 protein and containing mlv core proteins were prepared by cotransfection of 293t cells with 3 mg of pfb-luc (mlv vector plasmid), 2 mg of phit60 (packaging construct), 1 mg of mlv mko-gag, and 3 mg of hiv jrfl env plasmids using the polyfect reagent (qiagen) (16) . the viruscontaining medium was collected at 48 hours after transfection and centrifuged at 2500 rpm for 10 min at 4°c to clear debris. to prepare did-labeled hiv particles, 293t medium was replaced with opti-mem i (gibco life technologies) containing 10 mm did at 16 to 20 hours after transfection. after 4 hours of incubation at 37°c, unincorporated dye was removed by washing, and cells were returned to regular growth medium. after 24 hours of incubation, the extracellular medium was collected, centrifuged at low speed to remove cell debris, filtered through a 0.45-mm syringe filter, and stored at −80°c until use. for control experiments, mlvs pseudotyped with vsv-g and mlv core proteins were also prepared. for bulk binding and lipid mixing assays, membranes of viral particles were labeled with 20 ml of octadecyl rhodamine b chloride (1 mg/ml; r18) in 50 ml of virus for 60 min at room temperature and then free r18 was removed on a sephadex pd-10 desalting column (ge healthcare). the infectious titer was determined by a luciferase assay, as previously described (42) , in cd4 + /ccr5 + hela cells for hiv env pseudotyped viruses. preparation of gpmvs derived from cd4 + /ccr5 + hela cells cd4 + /ccr5 + hela cells were provided by d. m. rekosh (university of virginia). gpmvs were produced and isolated from the cells, as previously described (17, 18) , with slight modifications. briefly, cd4 + /ccr5 + hela cells were cultured in iscove's modified dulbecco's medium with 10% fetal bovine serum (fbs), g418 (0.2 mg/ml), 1% penicillinstreptomycin (penstrep), and puromycin (1 mg/ml) in an incubator at 37°c with 5% co 2 . plain (cd4 − /ccr5 − ) hela cells, which lack cd4 and ccr5, were grown in dulbecco's modified eagle's medium, 10% fbs, and 1% penstrep in the same incubator. after growing the cells to a confluence of 70 to 90% in culture dishes, cells were washed three times with blebbing buffer [10 mm hepes, 150 mm nacl, and 2 mm cacl 2 (ph 7.4)]. membrane vesiculation was then induced by adding 25 mm formaldehyde and 2 mm dtt or 2 mm nem in ordered membrane domains in resting t cell plasma membranes are nanoscopic and short-lived, but small dynamic domains can coalesce to create larger ones to function as signaling platforms upon pathogen invasion. helper cd4 + t cells recognize the pathogen-derived antigens on the surface of antigen-presenting cells (apc) and become activated by coalescence of membrane domains. the activation of cd4 + t cells stimulates the ability of b cells and cd8 + t cells to defend against the invading pathogens. in this model, we propose that cd4 + t cells that are challenged by pathogens are more prone to hiv infection than resting t cells, as indicated by the red arrows. the hiv infection leads to apoptotic t cell death and ultimately results in the progression to aids. blebbing buffer for 1 hour at 37°c. following shaking, solutions containing cell-detached gpmvs were gently decanted into a 50-ml tube. the gpmvs were placed on ice for 30 min to settle relatively large gpmvs (~10 mm in diameter), which were then transferred from the bottom of the tube to coverslips for observation on an epifluorescence microscope. lateral distribution and relative amount of cd4 and ccr5 on gpmvs we observed the lateral distributions of cd4 and ccr5 on cell-attached and isolated gpmvs. for the preparation of cell-attached gpmvs, cd4 + /ccr5 + hela cells were seeded on quartz slides or 18-mm glass coverslips and grown to about 40% confluence. after inducing gpmvs from the cells, as described above, they were rinsed extensively with phosphate-buffered saline (pbs) to remove gpmvinducing chemicals. cell-attached gpmvs or isolated gpmvs were blocked with 0.1% bovine serum albumin in pbs and then the vesicles were stained with the ld marker dii (0.2 mm) or dio (0.5 mm) and/or the lo marker alexa 555-conjugated ctxb (1 mg/ml) for 60 min on ice. after labeling, the gpmvs were incubated with an anti-cd4 antibody and/or an anti-ccr5 antibody conjugated with alexa 488 and/or alexa 647, respectively, at a concentration of 5 mg/ml at 4°c for 60 min. the lateral distribution of fluorescent lipids and proteins on cells or gpmvs was visualized using an epifluorescence microscope. the immunofluorescence staining was also used to evaluate the amount of cd4 and ccr5 on the surface of gpmvs. for quantification, isolated gpmvs were incubated with antibodies at 4°c for 60 min and then unbound antibodies were removed by centrifugation (16,000g) for 30 min at 4°c. gpmv pellets were resuspended in 400 ml of hepes buffer. relative amounts of cd4 and ccr5 were measured at room temperature by fluorescence emission intensities of alexa 488 at 520 nm and alexa 647 at 667 nm in a jobin-yvon fluorolog-3 spectrofluorometer (jobin-yvon) with excitation wavelength at 495 and 650 nm, respectively. gpmvs from cd4 − /ccr5 − hela cells were used as negative controls and displayed only low or negligible fluorescence intensity. single and bulk binding assay of hiv env particles to gpmvs for the binding of single hiv env particles to gpmvs, gpmvs were induced from cd4 + /ccr5 + hela cells on quartz or glass slides. after staining with dio or 7-nitro-2-1,3-benzoxadiazol-4-yl (nbd)-dope, gpmvs were rinsed with buffer in a fabricated flow-through chamber. hiv particles labeled with mko (mko-gag) were injected into the chamber containing the cell-attached or isolated gpmvs and incubated at 4°c for 60 min. binding of the particles to gpmvs was visualized by epifluorescence microscopy for binding sites of virions, and the number of virions bound to gpmvs was counted for binding efficiency. binding sites of vsv-g env particles on gpmvs were also performed for control experiments. to quantify bulk binding efficiency of hiv, r18labeled viral particles were added to gpmvs labeled in a tube with nbd-dope and incubated at 4°c for 60 min. unbound particles were removed by centrifugation (16,000g) for 30 min at 4°c. the pellets containing virus-bound gpmvs were resuspended in 400 ml of 0.5% (v/v) triton x-100. fluorescence emission intensities of r18 at 590 nm and nbd at 535 nm were recorded at room temperature in a jobin-yvon fluorolog-3 spectrofluorometer (jobin-yvon) with excitation wavelength at 560 and 460 nm, respectively. the ratio of fluorescence intensity at 590 nm/535 nm was used to measure relative binding efficiencies of hiv env particles to gpmvs. fusion between r18-labeled virus particles and unlabeled gpmvs was measured by the dequenching of r18 fluorescence. unlabeled gpmvs were added to the r18-labeled virions (1 × 10 8 particles) in hepes buffer at room temperature. concentration of gpmvs was estimated by total protein concentration in gpmvs using a bicinchoninic acid assay. protein concentrations of gpmvs derived from cd4 + /ccr5 + , mbcd-treated cd4 + /ccr5 + , and plain cd4 − /ccr5 − hela cells were 101, 87, and 62 mg/ml, respectively. fluorescence intensities were measured under constant stirring using a fluorolog-3 spectrofluorometer (jobin-yvon) with excitation and emission wavelengths of 555 and 590 nm, respectively. dequenching of r18 fluorescence was normalized to the initial fluorescence (f/f 0 ). to prepare the samples for electron cryo-microscopy (cryoem), hiv env particles were incubated with gpmvs for 60 s at room temperature to capture early stages of membrane fusion between hiv particles and gpmvs. samples (~3.5 ml) were applied to either c-flat or quantifoil holey carbon grids, manually blotted to near dryness with filter paper, and rapidly plunged into a slurry of liquid ethane. the grids were then transferred under liquid nitrogen to a tecnai f20 cyroem (philips/fei) operating at 120 kv. images were recorded at magnifications of ×11,000 or ×30,000 under low-electron dose conditions (~20 e − /å 2 ) using a 4k × 4k chargecoupled device (ccd) camera (gatan). for cholesterol depletion, isolated gpmvs from cd4 + /ccr5 + hela cells were incubated with 5 mm mbcd in hepes buffer for 30 min at room temperature. other gpmvs were incubated with 5 mm lysosm or 20 mm lysopc in hepes buffer for 30 min at room temperature to examine the effect of lysolipids on lipid phases. yet, other gpmvs were added to 10 u of pla2 in 10 mm tris-hcl buffer (ph 8.5) or 10 mu of scdase in 50 mm sodium acetate buffer (ph 6.0), which was adjusted to an osmolality of 300 mmol/kg by the addition of nacl, and incubated at room temperature for 30 min. after incubation, gpmvs were isolated by centrifugation (16,000g) for 30 min at 4°c and were used for measurements of hiv binding and fusion. fluorescence images were recorded on a zeiss axiovert 200 fluorescence microscope (carl zeiss) with a mercury lamp as a light source, 40× or 63× water immersion objectives (carl zeiss; numerical aperture, 0.95), and an electron-multiplying ccd cooled to −70°c (ixon dv887esc-bv, andor) as a detector. images were acquired using homemade software written in labview (national instruments). membranes stained with dio or nbd were epi-illuminated through a 480-nm band-pass filter (d480/30, chroma) and via a dichroic mirror (505dclp, chroma). fluorescence was observed through a 535-nm band-pass filter (d535/40, chroma). dii-or rhodamine-stained membranes and mko-labeled viral particles were epi-illuminated through a 540-nm band-pass filter (d540/25, chroma) and via a dichroic mirror (565dclp, chroma). fluorescence was observed through a 605-nm band-pass filter (d605/55, chroma). viral particles or luvs stained with did were illuminated through a 620-nm band-pass filter (et620/60, chroma) and observed through a 665-nm band-pass filter (hq665/60, chroma). all fluorescence imaging was performed at room temperature (~22°c). images were processed and assessed for colocalization using imagej. guvs were prepared via the electroformation technique. briefly, 25 ml of a 10 mm lipid mixture composed of bsm/bpc/bps/cholesterol (2:1:1:1) containing the fluorescent lipid probe rh-pe [0.1 mole percent (mol %)] was deposited on the conducting surfaces of two indium tin oxide-coated glass slides. after the slides were placed in a vacuum desiccator for 90 min to remove residual solvent, a fabricated chamber composed of two conducting slides facing each other separated by a 0.5-mm spacer was filled with 300 mm sucrose in h 2 o. guvs were generated by applying alternating electric current (3 v, 10 hz) through a function generator for 120 min at 60°c and transferred into a 300 mm glucose solution to settle by gravity on the microscope slide. lipid mixing of luvs mediated by hiv fusion peptide luvs were prepared by extrusion. phospholipids composed of bsm/ bpc/bps/cholesterol (2:1:1:1) were dissolved in a mixture of chloroform and methanol, and the solvent was evaporated under a stream of nitrogen gas in a glass test tube. the thin lipid film was further dried overnight under vacuum and hydrated with 0.5 ml of hepes buffer [10 mm hepes and 120 mm nacl (ph 7.2)]. the suspension was vigorously vortexed for 5 min, was subjected to 10 freeze-thaw cycles, and was then extruded 21 times through two stacked polycarbonate filters with a pore size of 100 nm (avestin). the lipid mixing assay was based on fluorescence resonance energy transfer between nbd-pe and rhodamine-pe. the hiv fusion peptide was added to 50 mm luvs with a ratio of 1:9 of labeled (1 mol % of nbd-pe and rhodamine-pe each) to unlabeled luvs in hepes buffer at room temperature. the fluorescence was recorded under constant stirring in a fluorolog-3 spectrofluorometer (jobin-yvon) with the excitation and emission wavelengths set at 460 and 535 nm, respectively. the value for 0% lipid mixing was the fluorescence intensity of the luv suspension before the fusion peptide was added, whereas the value for 100% lipid mixing was obtained by adding triton x-100 [final concentration, 0.5% (v/v)] to the suspension at the end of each experiment. quartz slides were cleaned by boiling in contrad detergent for 15 min and by sonication in a hot bath for 30 min. after rinsing with deionized water and ethanol, the slides were immersed in piranha solution (3:1 mixture of 95% sulfuric acid and 30% hydrogen peroxide) to remove remaining organic residues, followed by extensive rinsing in pure water. the first leaflet of the sppm was prepared by langmuir-blodgett (lb) transfer directly onto the quartz slide. lipid mixtures composed of bsm/ bpc/cholesterol (2:2:1) with 3 mol % of dmpe (1,2-dimyristoyl-snglycero-3-phosphoethanolamine)-polyethyleneglycol-triethoxysilane (43) were spread onto a pure water surface in a nima 611 lb trough (ksv nima). after allowing the solvent to evaporate for 10 min, the monolayer was compressed at a rate of 10 cm 2 /min to reach a surface pressure of 32 mn/m. immediately before use, quartz slides were further cleaned for 10 min in an argon plasma sterilizer (harrick scientific) and then dipped into the trough at a speed of 200 mm/min and withdrawn at 5 mm/min while keeping the monolayer surface pressure constant at 32 mn/m. the transferred lipid monolayer was dried in a vacuum desiccator at room temperature overnight and cured in a 70°c oven for 40 min to covalently link the polymer to the sio 2 slide surface. after equilibration in the desiccator to room temperature, the slide with the tethered polymer-supported lb monolayer was placed in a custombuilt flow-through chamber. a suspension of gpmvs in hepes buffer was injected into the chamber and incubated for at least 2 hours at room temperature to form the distal leaflet of the sppm bilayer. excess unfused gpmvs were then washed out by extensive rinsing with hepes buffer. the integrity of the sppms and the diffusion of the lipids within them were examined by fluorescence recovery after photobleaching (frap). bilayers were bleached in a pattern of parallel stripes, and the data were fit to the model f(t) = f ∞ + (f 0 − f ∞ )exp(−da 2 t), where f 0 and f ∞ are the initial and final fluorescence intensities after bleaching, respectively, a = 2p/p, p is the stripe period (12.7 or 3.2 mm), and d is the lateral diffusion coefficient. the mobile fraction (mf), which reflects the percentage of observed fluorescence recovery within the time frame of a frap experiment (<1 min), is given by mf ¼ f∞àf0 f pre àf 0 â 200, where f pre is the fluorescence intensity before photobleaching. ten regions on three independently prepared bilayers were sampled to determine the reported average values. binding and fusion of single hiv env particles on sppms by tirf microscopy to measure binding to sppms, mko-labeled (mko-gag) hiv env particles were injected into a chamber with unlabeled sppms, and the fluorescence increase by hiv binding to sppms was monitored over time by prism-based tirf microscopy. the prism-quartz interface was lubricated with glycerol to allow easy translocation of the sample cell on the microscope stage. the light source was an argon ion laser (innova 90c, coherent) emitting light at 514 nm, and fluorescence was observed through a 610-nm band-pass filter (d610/60, chroma). the beam was totally internally reflected at an angle of 72°from the normal to produce an evanescent wave. the intensity of the laser beam was computer-controlled through an acousto-optic modulator (isomet) or could be blocked entirely by a computer-controlled shutter. the laser intensity, shutter, and camera were controlled by a homemade program written in labview (national instruments). to investigate the targeting of hiv particles to different regions of ld/lo phase-separated sppms, mko-labeled particles were incubated with sppms that were stained with dio for 60 min at room temperature. after unbound particles were washed away with hepes buffer, phase-separated sppms were visualized by epifluorescence microscopy, and bound particles were visualized by tirf microscopy. to analyze and quantify the distribution of sppm-bound particles, we distinguished three regions of the sppm: lo domains, ld phase areas, and lo/ld boundary regions with a 0.75-mm width centered on the perimeter of each lo domain. custom-built particle-tracking software (35) was used to automatically detect the position (x and y coordinates) of each particle. to monitor the fusion of single hiv particles with the sppm, did-labeled hiv env particles were injected into a chamber, one large window wall of which was formed by the sppm. the light source for tirf illumination was a diode laser (cube640, coherent) to excite the lipid dye did through a 620-nm filter (et620/60, chroma) and via a dichroic mirror (660dclp, chroma). did fluorescence was observed through a 665-nm band-pass filter (hq665/60, chroma). data acquisition and image analysis were accomplished through custom-built software written in labview (national instruments) (35) . single events that included docking, hemifusion, and full fusion were measured by analyzing the peak fluorescence intensities from each bound particle as a function of time: events with no fluorescence change over time were classified as docking, those with decays to around one-half of the original peak intensity were classified as hemifusion, and those with complete decays were classified as direct full fusion events (44) . supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/3/6/e1700338/dc1 fig. s1 . association of cd4 and ccr5 with lipid rafts in cd4 + /ccr5 + hela cells with stably expressed cd4 and ccr5. fig. s2 . formation of gpmvs by treatment of cd4 + /ccr5 + hela cells with small amounts of formaldehyde and dtt. fig. s3 . partitioning of cd4 and ccr5 in gpmvs induced by nem instead of formaldehyde and dtt from cd4 + /ccr5 + cells. fig. s4 . hiv env pseudovirus particles bind preferentially at boundaries between coexisting lo and ld domains in gpmvs. fig. s5 . vsv-g pseudovirus particles bind to ld membrane regions in gpmvs. fig. s6 . electron cryo-micrographs of hiv env particles bound/fused to gpmvs. fig. s7 . modulation of lipid phases does not affect binding of hiv to gpmvs. fig. s8 . preparation of sppms with gpmvs. fig. s9 . lateral distribution of cd4/ccr5 in sppms. movie s1. growing gpmvs on cell surfaces. functional rafts in cell membranes membrane lipids: where they are and how they behave lipid rafts as functional heterogeneity in cell membranes functions of lipid rafts in biological membranes a role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains pathogens: raft hijackers hiv-1 assembly, release and maturation hiv-1, lipid rafts, and antibodies to liposomes: implications for anti-viral-neutralizing antibodies role of lipid rafts in virus replication cd4-induced interaction of primary hiv-1 gp120 glycoproteins with the chemokine receptor ccr-5 hiv: cell binding and entry. cold spring harb lipid rafts and hiv-1: from viral entry to assembly of progeny virions lipid rafts: at a crossroad between cell biology and physics separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol imaging coexisting fluid domains in biomembrane models coupling curvature and line tension hiv gp41-mediated membrane fusion occurs at edges of cholesterol-rich lipid domains large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles elucidating membrane structure and protein behavior using giant plasma membrane vesicles structure of the ccr5 chemokine receptor-hiv entry inhibitor maraviroc complex hiv entry and its inhibition noninfectious virus-like particles produced by moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents lipid rafts and hiv pathogenesis: host membrane cholesterol is required for infection by hiv type 1 line tension at lipid phase boundaries as driving force for hiv fusion peptide-mediated fusion mechanics of membrane fusion lipid polymorphisms and membrane shape role of cholesterol in the formation and nature of lipid rafts in planar and spherical model membranes sphingosine-1-phosphate: an enigmatic signalling lipid membrane-mediated induction and sorting of k-ras microdomain signaling platforms regulation of rac1 translocation and activation by membrane domains and their boundaries supported phospholipid bilayers single vesicle millisecond fusion kinetics reveals number of snare complexes optimal for fast snare-mediated membrane fusion reconstituting snare-mediated membrane fusion at the single liposome level formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking distribution and three-dimensional structure of aids virus envelope spikes the immunological synapse: a molecular machine controlling t cell activation three-dimensional segregation of supramolecular activation clusters in t cells lipid rafts and signal transduction concentration of mhc class ii molecules in lipid rafts facilitates antigen presentation aggregation of lipid rafts accompanies signaling via the t cell antigen receptor clomiphene and its isomers block ebola virus particle entry and infection with similar potency: potential therapeutic implications tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker high cholesterol obviates a prolonged hemifusion intermediate in fast snare-mediated membrane fusion acknowledgments funding: this work was supported by nih grants r01 ai30557 wrote the manuscript. competing interests: the authors declare that they have no competing interests. data and materials availability: all data needed to evaluate the conclusions of this paper are present in the paper and/or supplementary materials hiv virions sense plasma membrane heterogeneity for cell entry key: cord-302082-aaokc182 authors: stanberry, lawrence r.; strugnell, richard title: vaccines of the future date: 2011-08-31 journal: perspectives in vaccinology doi: 10.1016/j.pervac.2011.05.006 sha: doc_id: 302082 cord_uid: aaokc182 nan the advances made in vaccine technology since edward jenner vaccinated the young james phipps against smallpox have had a spectacular impact on human health over the last two centuries (see chapter 1 e vaccine evolution). vaccines have been fundamental in the control and elimination of many debilitating and lethal diseases, and more diseases are currently targeted for eradication by vaccination. recent major breakthroughs in immunology, molecular biology, genomics, proteomics, biochemistry and computing sciences have driven vaccine technology forward, and will continue to do so. many challenges remain, however, including persistent or latent infections, pathogens with complex life cycles, antigenic drift and shift in pathogens subject to selective pressures, challenging populations and emerging infections. to address these challenges researchers are exploring many avenues: novel adjuvants are being developed that enhance the immune response elicited by a vaccine while maintaining high levels of tolerability; methods of protective antigen identification are iterated with every success; vaccine storage and transport systems are improving (including optimising the cold chain and developing temperature-stable vaccines); and new and potentially more convenient methods of vaccine administration are being pursued. high priority targets include life-threatening diseases, such as malaria, tuberculosis (tb) and human immunodeficiency virus (hiv), as well as problematic infections caused by ubiquitous agents, such as respiratory syncytial virus (rsv), cytomegalovirus (cmv) and staphylococcus aureus. non-traditional vaccines are also likely to become available for the management of addiction, and the prevention, treatment and cure of malignancies. this chapter is not meant as a compendium of all new-generation vaccines, but rather as an outline of the modern principles that will likely facilitate the development of future vaccines. as shown in figure 6 .1, there are several key elements that are likely to be 152 understanding modern vaccines the foundation for the development of future vaccines. this chapter will illustrate these elements and provide examples that show promise. since the first use of an adjuvant in a human vaccine over 80 years ago, adjuvant technology has improved significantly with respect to improving vaccine immunogenicity and efficacy. over the advances in adjuvant design have been driven by parallel advances in vaccine technology as many modern vaccines consist of highly purified antigens e with low non-specific reactogenicity which require combination with adjuvants to enhance the immune response. future developments in adjuvant technology are expected to provide stronger immune priming, enhance immune responses in specific populations, and lead to antigen sparing. adjuvants to date have demonstrated an ability new adjuvants must aim to drive the immune response that is associated with lifelong protection. new adjuvants and adjuvant combinations will play many roles in future vaccines as illustrated in figure 6 .2. adjuvants will need to be individually selected for specific vaccine targets in order to achieve the desired goal (ie enhanced immunogenicity, induction of specific immune profile etc). to deliver this aim, some adjuvants will be mixed with free antigens, while others will need to be covalently linked to the antigenic moiety as part of a complex molecule. some examples of new adjuvants that have been evaluated in humans or that are in clinical trials are listed in table 6 .1 (also see chapter 4 e vaccine adjuvants). modern approaches to antigen design tend to eschew classical trial and error techniques in favour of identifying the type of pathogenic structures (ie antigens) that are most likely to be important immunogens based on their structural signature or physical location within the pathogen (table 6 .2) (see chapter 3 e vaccine antigens). the t or b cell immune responses to an antigen are targeted to precise regions of the antigen (ie epitopes e either linear or three-dimensional conformational structures; in the case of protein antigens these are specific peptide epitopes). historically, simple, linear, synthetic peptide epitope vaccines have been poorly immunogenic because they lack a specific conformation and are easily degraded by a variety of extracellular and cell-surface proteases that serve to limit epitope presentation to t cells and/or result in destruction of the b-cell epitope. peptide vaccines need to union. every effort has been made to verify the information in the above table. the information included is not meant to be exhaustive but is intended to provide an overview of the subject matter. subunit and individual epitope vaccines need to be optimised to ensure adequate immunogenicity. novel strategies are being developed and exploited in order to identify antigens recognised by t and b cells, thus facilitating a more knowledge-based vaccine design. one of the most common ways to identify these antigens is to measure cellular proliferation (t or b cells) upon in vitro stimulation with antigen. high-throughput screening assays of candidate synthetic peptides that drive cellular proliferation help speed the rate of antigen discovery. reverse vaccinology combines knowledge of the pathogen's genome sequence with known protein sequences via computer analysis, to predict protein expression and post-translational modifications and identify likely vaccine candidates (see chapter 3 e vaccine antigens; figure 3 .5). the development of epitope-based vaccines is one example of reverse vaccinology where computer software combines prediction algorithms to suggest sequences similar to those for pathogenic components. epitope mapping, combined with the creation of more stable poly-epitope vaccines, may lead to the successful translation of this technology into products. mhc molecules exhibit widely varying binding specificities; a vaccine expressing a single peptide antigen would therefore only target a few mhc molecules and thus only be recognised by the t cells of individuals carrying a specific mhc phenotype. poly-epitope technology could be used to generate a synthetic protein carrying antigenic epitopes from multiple strains or pathogens. this would overcome the mhc restriction and afford protection in individuals carrying different mhc types. the screening of pathogen peptide libraries is another example of new approaches to antigen discovery. screening methods are used to identify antigens that can stimulate cd4 þ or cd8 þ t cells, or which bind to antibodies from humans known to have been infected with the relevant pathogen. where peptide screening uses antibodies, an additional consideration is the synthesis of antigens that contain the tertiary (folding/three-dimensional) structure of the native immunogen, since vaccine efficacy can be impacted by infidelities in the structure of the final product. incorrect protein folding may result in a less immunogenic antigen or an antigen that induces an immune response that differs from that of the native immunogen. the mimicking of the three-dimensional structure of the native immunogen is important during the synthesis of antigens that are being used to target b-cell responses. conversely, the requirement for folding is reduced for t cells since t cells bind only processed peptides, from degraded proteins. likewise, dna expression libraries using the pathogen genomic dna have been screened using animal model systems to identify genes encoding proteins that afford protection against infection or disease caused by the pathogen. one example is genocea's vaccine development programmes that are built around a broad platform for the rapid discovery of t-cell antigens. the process is explained in figure 6 .3. t-cell antigens, specifically antigens that stimulate cd4 þ and cd8 þ t cells, are critical to generating disease-specific cellular immune responses and long-term t-cell memory. stability of the final product is another important consideration. adverse environmental conditions can result in degradation of the vaccine, rendering it non-immunogenic. in order to maintain product integrity many vaccines (particularly live vaccines) must be stored at cold temperatures ( 4 c). the maintenance of the vaccine at this temperature from production site to distribution site, and medical office or clinic, is referred to as the 'cold chain'. maintaining the cold chain is much less of a challenge in resource-rich countries, but can be a major barrier to vaccine implementation in resource-limited areas. ongoing research designed to increase our understanding of vaccine degradation may address the problems associated with cold chain management and lead to the development of thermostable vaccines. the level of antigen presentation which occurs with some current vaccines may sometimes be insufficient to drive long-lasting immune responses of high quality (see chapter 3 e vaccine antigens). this may be due to inadequate exposure of the antigen to immature antigen-presenting cells (apcs) rapid or subimmunogenic degradation or sequestration of antigens, or lack of immunogenicity due to the physical presentation of the antigen. the discovery and modifying vaccine formulations to increase tolerance to temperature fluctuations is likely to increase the shelf-life of the product and reduce transport and wastage issues. understanding modern vaccines refinement of new and varied options for antigen presentation is expected to allow the design of vaccines to produce specific immune profiles. some of these technologies have been shown to facilitate oral delivery to target mucosal immune responses and also trigger both innate and adaptive immune systems, including t-and b-cell effector and memory responses. candidate viral vector vaccines utilise a non-pathogenic virus to carry and subsequently induce expression of genes that produce immunogenic foreign proteins at high levels in the host. these are taken up by immature apcs, and have been shown to lead to a robust, long-lasting immune response to the target antigen ( figure 6 .4). viral vector vaccines, eg recombinant poxvirus vaccines, can be administered mucosally to stimulate mucosal immune responses. the attenuated modified vaccinia virus ankara (rmva) vectors are showing promise as mucosal delivery vectors. pre-existing immunity to the viral vaccine vector is an impediment to successful use of this approach. as ways to avoid anti-vector immunity, viruses can be attenuated or inactivated, by deleting or replacing pathogenic genes. figure 6 .4 demonstrates how viral vaccine vectors are made. dna expressing an immunogenic transgene (the vaccine antigen) is inserted into the viral vector genome for expression following administration into the recipient; expression of the vaccine antigen can be boosted by using a variety of dna promoters. if the viral vector is no longer able to grow and replicate, the virus is grown using a cell line (a so-called complementing cell line) that has been engineered to produce the missing viral product. often, viral genes are removed in an effort to reduce or eliminate the pathogenicity of the vector and in some cases viral genes are removed to make the vector itself less immunogenic; an anti-vector immune response would greatly reduce the ability of the vector to induce an antigen-specific response. examples of viral vector candidate vaccines in clinical development are listed in table 6 .3. non-pathogenic bacterial vectors have many features that make them an attractive vaccine platform. bacterial vectors can be engineered for maximum safety (eg deletion of two or more genes from the same metabolic pathway), and to express large numbers of foreign antigens ( figure 6 .5). two key issues affecting bacterial vaccine vectors are: a) to decide whether the optimal platform should be a bacterial vaccine in its figure 6 .4 viral vectors for vaccines. viral vector vaccines exploit the natural ability of viruses to infect or otherwise enter (in the case of disabled viral vectors) host cells, and then deliver pathogen-specific antigens. antigen-encoding genes are isolated from the pathogen and inserted into the viral vector genome. the viral vector can then be used as a factory for production of large quantities of pathogen antigen in vivo, following introduction of the vector into the vaccine recipient, with the pathogen antigen then expressed on the surface of the infected/transduced host cells or exported out of the producer cell. mhc, major histocompatibility complex. own right or a bacterial vector system to deliver exogenous antigens; and b) to determine whether re-administration of the vector, either with the same or different target antigens, will fail because of the immune response to the bacterial vector vaccine at the time of its initial administration. initial assessments of the feasibility of using attenuated bacterial vectors for the delivery of foreign antigens have focused on salmonella species. bacterial vaccine vectors for humans, however, have been disappointing so far. it may be necessary to develop unique bacterial vaccine vectors for delivering exogenous antigens, in which case the vectors can be modified to allow for re-use. for example, if immunity against the vector, which is a major impediment to vaccine re-use, is determined by antibodies against the surface structures of the bacterium (such as lipopolysaccharide [lps]), the dedicated vaccine vector could be developed to lack expression of lps or to express truncated/ different forms of lps to the target, thereby avoiding priming of the immune response and allowing for re-use of the vector and/or vaccine. some potential options for live, attenuated bacterial vectors are shown in table 6 .4. dna vaccines are the result of the discovery in the early 1990s that the gene, rather than the encoded protein, if delivered in an 'expressible' form, could induce an immune response (see chapter 1 e vaccine evolution). the principle behind dna vaccines is that the antigenic molecule is produced within the host from the dna or rna that is injected, in contrast to more traditional vaccination where the antigen is supplied in the vaccine formulation. the gene(s) for target antigen(s) is/are usually encoded in a circular plasmid expression vector under the control of promoter sequences that direct gene expression in mammalian cells, which is achieved after injection into mammals. the dna vaccine process can circumvent some of the major issues resulting from recombinant protein administration. the construction and production of the plasmids carrying the gene of interest together with the promoter sequences is relatively simple; antigens expressed from plasmids retain their native conformation, the gene can be readily modified to produce tailored antigens, and bacterial plasmid dna is intrinsically immunogenic (subsequently shown to result from the pathogen-associated molecular patterns [pamps] it carries). additional desirable features include the ability to engineer and deliver genetic adjuvants in tandem or parallel with the antigen, the potential to deliver multiple antigen genes in one construct or within other constructs that encode adjuvanting protein(s), and the ability to induce both cellular and humoral immune responses. despite promising data in pre-clinical testing, dna vaccine candidates have shown only limited success in clinical settings so far. one of the current drawbacks of dna vaccines is the inefficiency of conventional delivery methods for the plasmid dna; however, understanding modern vaccines emerging proprietary particle-mediated delivery technology or electroporation technology seeks to improve this situation. with the electroporation method, brief electrical pulses are applied at the site of immunisation which causes a transient disruption of cell membranes. this results in an enhancement in uptake of the dna vaccine between 10e100-fold. examples of dna candidate vaccines in clinical development are presented in table 6 .5. dendritic cell (dc) vaccines typically use monocytes harvested from the blood (in most cases from the individual who will receive the vaccine) to produce immature dcs in vitro. the monocytes are antigen-loaded and treated to induce their maturation into apcs and infused back into the patient. the first food and drug administration (fda)-approved dc vaccine, designed for the treatment of prostate cancer, was licensed in 2010 (sipuleucel-t); examples of other targets for dc vaccine therapy are presented in table 6 .6. dc vaccines offer an individualised approach to therapeutic vaccine development, but represent a specialised method of vaccination that is currently limited to aggressive cancers, and the treatment of serious, intractable infections. a comparison between the strengths and weaknesses of selected new vaccine platforms is presented in table 6 .7. developing administration techniques that place the vaccine directly at the site(s) where pathogens are most likely to initiate an infection (eg mucosal or respiratory sites) is likely to improve vaccine efficacy and safety. traditional methods of vaccine administration can potentially pose a number of limitations with respect to reactogenicity, immunogenicity, convenience, efficacy, safety and cost-effectiveness. the information included is not meant to be exhaustive but is intended to provide an overview of the subject matter. ongoing research on alternative experimental administration strategies includes ballistic delivery to skin (the gene gun), the transdermal patch and other intradermal methods, plus sublingual, aerosol, rectal and vaginal mucosal vaccines. the main advantages of alternative delivery strategies are the potential to induce immune responses at the common portals of pathogen entry (eg oral polio vaccine replicating in the gut), potential convenience (eg ease of use of the transdermal patch), potential combination of vaccines to reduce or simplify the vaccination schedule, and reduction or elimination of administration via standard hypodermic needle injection. despite the intuitive value of these approaches, few vaccines today are administered via non-im routes. this is for several reasons including feasibility, lack of proven efficacy and limited safety data. some problems have been observed with new routes of delivery, for example, after the 2000 launch of an inactivated intranasal influenza vaccine (a virosome formulation adjuvanted by heat labile enterotoxoid of escherichia coli), post-licensure data indicated a significantly increased risk of bell's palsy in vaccinees and forced its withdrawal from the market. this experience led to a higher level of caution in the development of intranasal vaccines. today, the only example of a licensed vaccine against a latent infection is the zoster vaccine; the vaccine formulation is the high potency (about 15-fold) version of the live, attenuated varicella zoster virus (vzv) vaccine. this vaccine has been used to boost the anti-vzv cell-mediated immune response in older subjects and has been shown to reduce the overall incidence of zoster by 50% in subjects aged 60 years or older (oxman et al., 2005) . future vaccines may control persistent infections either by preventing the initial infection or disease (ie prophylactic vaccines) aerosol delivery: 'mass immunization of almost all susceptible children in a short period of time, has the potential of rapidly eliminating measles as a public health problem. immunization by inhalation of aerosolised measles vaccine provides a procedure that could make such a mass programme possible, especially in parts of the world where measles continues to be a serious problem.' (sabin et al., 1983) . administering the measles vaccine as an aerosol, either as nebulised vaccine or as an increased understanding of human immunology and of hostepathogen interactions should enable the identification of the type(s) of immunity required to effectively prevent or control persistent infections (see chapter 2 e vaccine immunology). some examples of persistent infections are shown in table 6 .8. mycobacterium tuberculosis can persist in a latent state within the human host for years without causing disease (latent tb). protection against miliary (disseminated) tb in children is provided by the bacille calmetteeguérin (bcg) vaccine, developed through culture attenuation of mycobacterium bovis early in the 20th century, which is routinely given in many countries. the vaccine, however, provides only modest and often temporary protection against pulmonary tb, and provides lower efficacy in resource-limited regions closer to the equator. in addition, vaccination with live, attenuated mycobacterium bovis is a particular concern in hiv-positive individuals, especially those with advanced immune suppression; this population would particularly benefit from tb vaccination as tb is a leading cause of death worldwide for people with hiv/acquired immunodeficiency syndrome (aids). however, a recent phase iii trial demonstrated that protection against tb can be provided to individuals with hiv by using an inactivated whole-cell mycobacterial vaccine (von reyn et al., 2010) . the current state of tb vaccine development has been summarised in reviews by walker et al. (2010) and lambert et al. (2009) and examples of vaccines in development are shown in table 6 .9. cytomegalovirus, a herpes virus, establishes latent infection in cells in the bone marrow and peripheral blood. primary infection during pregnancy is associated with congenital infection that frequently causes a well-characterised spectrum of abnormalities and disabilities, which may be severe or fatal. reactivation in pregnancy is common, but is unlikely to cause severe congenital infection, although some manifestations, especially hearing loss, remain common. reactivation of cmv is of special concern in immunocompromised individuals, where severe and fatal pulmonary, hepatic and central nervous system infections are common. gastrointestinal disease and retinitis are common in association with hiv. a successful cmv vaccine has proved elusive for more than 30 years. based upon the observation that antibodies to the cmv envelope glycoprotein b (gb) could pass et al., 1999) . a recent phase ii clinical trial in cmv-seronegative women 1 year post-partum has shown the potential of gb/mf59 in decreasing incident cases of maternal and congenital cmv infection (pass et al., 2009) . this is the first evidence that a cmv vaccine can protect against infection. an alternative approach to the development of a cmv vaccine has been to utilise dna vaccination to induce host responses to cmv gb and phosphoprotein 65 (pp65 is another viral target). recent studies have shown that injection of combinations of plasmids, formulated with an adjuvant, can induce vaccine-specific immune responses, and can prime for effective memory responses. the hallmark of herpes simplex virus types 1 and 2 (hsv-1 and hsv-2) is their ability to establish and maintain latent infection in to be exhaustive but is intended to provide an overview of the subject matter. sensory ganglion neurons. periodic reactivation of the latent infection results in recurrent infections. both hsv-1 and hsv-2 can cause myriad diseases but the greatest public health problem is genital herpes. genital hsv-2 infection increases the risk of hiv acquisition and transmission, and control of genital herpes has been predicted to significantly impact the hiv epidemic. given the complex natural history of hsv infections, vaccines could have a variety of possible risks and benefits (table 6 .10). an effective hsv vaccine has been sought for more than 80 years. recently, an hsv-2 glycoprotein d (gd2) candidate vaccine containing the as04 adjuvant (see chapter 4 e vaccine adjuvants), was tested in three large, double-blind, phase iii controlled trials. the first two studies recruited volunteers with a partner with genital herpes disease and found the candidate vaccine was 73% effective against genital herpes disease in women seronegative for both hsv-1 and hsv-2 (stanberry et al., 2002) . trends towards protection against infection were also observed, but were not statistically significant. the candidate vaccine was not effective in hsv-1 seropositive women; or in understanding modern vaccines men, regardless of their hsv seropositivity status. these were the first studies to report a significant difference in vaccine efficacy between men and women. this finding could have important implications for other vaccines targeting sexually transmitted diseases. the basis for this difference could relate to differences in how men and women respond to novel adjuvants or may reflect differences in the acquisition and natural history of genital herpes in men and women. a third phase iii efficacy trial of the gd2 candidate vaccine in hsv-1 and hsv-2 negative women who thought themselves possibly at risk of acquiring genital herpes (a different risk population than in the original two trials) has been completed and is being analysed. an initial assessment of the results of the third trial showed that the vaccine had an acceptable safety profile but the primary trial endpoint, prevention of genital herpes disease, was not met (niaid, 2010) . although the development of the vaccine has been stopped, further analyses and comparison of the trials may guide researchers as they continue seeking vaccines to control hsv infections. as discussed in chapter 2 e vaccine immunology, some pathogens have complex life cycles. one specific example is parasites, sometimes using more than a single host, where each development phase is marked by differential expression of major proteins, meaning that possible antigen targets are host-and development-phase specific. taenid worms aside, vaccines against parasites have been extremely difficult to develop and only a limited number have performed well in later-stage clinical trials. the protozoan parasite plasmodium falciparum, the most common cause of malaria, has a complex life cycle, as shown in figure 6 .7. the plasmodium parasite has a genome encoding more than 5000 proteins, and presents different allelic and immunogenic table 7) . one of the furthest advanced of these new candidate vaccines is rts,s/as01. the vaccine targets the pre-erythrocytic stage of the parasite (figure 6.7) . to be protective, a vaccine targeted at this phase needs to induce humoral immunity, to prevent parasites from invading the liver, and cell-mediated immunity to destroy hepatocytes that become infected in the face of the humoral immune response. the rts,s antigen, produced in saccharomyces cerevisiae, contains sequences of the p. falciparum circumsporozoite protein, linked to the hepatitis b surface antigen (hbsag). this chimeric protein spontaneously assembles into mixed polymeric particulate structures. in phase ii studies, the rts,s/as01 candidate vaccine induced a strong neutralising antibody response and cell-mediated immunity, and afforded protection against malaria (bejon et al., 2008; abdulla et al., 2008) . rts,s/as01 has been selected to proceed to phase iii clinical testing due to its higher efficacy compared with alternative formulations. if successful, the rts,s/as01 candidate vaccine could be the first licensed human vaccine against a parasite. other malaria candidate vaccines in development are shown in appendices, supplementary table 7 . pathogens may mutate or recombine to change their antigenic profile. antigenic drift refers to a gradual process whereby point mutations in genes encoding antigenic proteins change the antigen sufficiently so that over time previously effective antibodies and vaccines no longer effectively control the pathogen and hence new vaccines need to be created. antigenic shift is a more dramatic event where there is a recombination of genes between different pathogen strains that gives rise to a new strain with a unique antigenic profile. in theory, pathogens are susceptible to selective pressure and an immunological environment that provides strong selective pressures should provide the 'bottleneck' that drives selection. this occurs with influenza viruses, where the high mutation frequency allows for the selection of mutants that are not neutralised. the risk of vaccine-mediated immune selection of pathogens, though certainly present, is difficult to demonstrate. moreover, peptide vaccines only use the antigenic epitope so the risk of pathogen evolution is theoretically increased. however, this phenomenon has not been regularly observed in experimental studies and may reflect the complex nature of most vaccine antigens and the presence of immune responses against multiple antigens and multiple epitopes within antigens. serotype replacement, where the distribution of specific microbial serotypes within communities changes after the introduction of vaccines, has occurred for some bacterial pathogens and may be a consequence of the use of capsular vaccines that address only a limited number of serotypes. similarly, since their introduction in the 1940s, the use of antibiotics has exerted a selective pressure on bacterial strains leading to selection for common resistance alleles (eg the extended-spectrum beta-lactamase [esbl] resistance of enteric bacteria and beta-lactamase resistance in gonococci). to date, there has been no requirement to remodel a vaccine because of vaccine-mediated immune escape; however, new vaccines against the pneumococcus have been licensed, including additional capsular types, to expand the geographical coverage of most frequent types and, in part, to counter the observed phenomenon of serotype replacement. annual seasonal influenza infections are subject to natural antigenic drift which requires the reformulation of the vaccine when drifts occur, but there is no evidence that the deployment of the vaccine accelerates this drift. antigenic shift, while not the result of selective pressure, gives rise to viral strains containing a mixture of the surface antigens from the parent strains. pathogens that can undergo antigenic shift, including influenza viruses (figure 6 .8), present major challenges for vaccine developers. chapter 4 e vaccine adjuvants, there has been progress in the another approach to the problem of influenza genome shifts has been to target weakly immunogenic conserved antigens such as the influenza m2e protein. one approach to addressing the weak immunogenicity of the antigen has been to link it to a potent toll-like receptor adjuvant such as flagellin, an approach developed by vaxinnate inc. during primary infection of a single individual with hiv, mutations in surface proteins of the virus lead to selection of a 'cloud' of antigenic variants that can evade the cell-mediated immune responses complicating the development of broadly effective vaccines. this propensity for mutation has given rise to many strains of hiv (figure 6 .9). two types of hiv, hiv-1 and hiv-2, have been identified, with hiv-1 being the most common. on a global scale, hiv-1 strains are differentiated according to their respective group and subtypes (or 'clades') within groups. the amino acid sequence of the viral envelope glycoprotein gp120 shows 25e35% divergence between clades and up to 20% divergence within any given clade, which constitutes a formidable hurdle to vaccine development. this is made worse by recombination between clades of hiv-1, which has produced circulating recombinant forms (crfs) which differ in antigenicity depending on the geographical region. since the initiation of hiv vaccine programmes, more than 30 candidate vaccines have been tested in over 80 phase i/ii clinical trials involving more than 10,000 healthy human volunteers. regrettably, all attempts to date have failed to yield a licensed hiv vaccine. questions remain concerning the immune mechanisms behind vaccines that achieve partial protection. regardless of the unknowns, the ability to prevent infection in at least some individuals still offers real hope that a globally effective hiv vaccine might be possible. current research is comparing the immune responses of subjects who are naturally protected against hiv with those who were infected, seeking to find the elusive immunological mechanisms of protection to help guide the design of future t-cell vaccines against the virus. infections of group a streptococcal serotypes (ie streptococcus pyogenes) account for approximately 85% of cases of uncomplicated bacterial pharyngitis and streptococcal invasive infections in north america. the m protein of group a streptococci is a major virulence determinant of these organisms and also functions as a major target for protective antibodies. one of several strategies for vaccine prevention of these infections is based on type-specific m protein epitopes. however, group a streptococcal vaccine development faces many obstacles: i) the widespread diversity of circulating m protein types; ii) immunological cross-reactivity between epitopes in the m protein and several human tissues introducing an autoimmune risk; and iii) animal models are of limited value because humans are the only hosts for group a streptococci. in an attempt to partially overcome some of these obstacles, a design strategy akin to that of the pneumococcal polysaccharide vaccines has been employed to generate a group a streptococci multivalent m protein-based vaccine containing type-specific determinants from 26 different m serotypes. this multivalent vaccine is currently in clinical development. the 'prime-boost' approach the term 'prime boost' (or heterologous boosting) describes an approach to vaccination where one type of vaccine, such as a live-vector vaccine, is administered followed by a second type of vaccine, such as a recombinant subunit vaccine. this is in contrast with the traditional method of homologous boosting in which two or more doses of the same vaccine are given successively. the intent of prime-boost vaccination is to induce different types of immune responses and enhance the overall immune response, a result that may not occur if only one type of vaccine were to be given for all doses. this approach has been employed in trials with, for example, tb, cmv, malaria and hiv candidate vaccines. for example, in studies on new tb vaccines, subjects already primed with the live, attenuated bcg vaccine have been boosted with a subunit adjuvanted vaccine (see tuberculosis). respiratory syncytial virus is a common cause of bronchiolitis and pneumonia in infants, and exacerbations of chronic obstructive pulmonary disease in the elderly. the development of an effective vaccine has been challenging; natural immunity to rsv infection is incomplete and re-infections occur in all age groups. moreover, the primary target population for vaccination is newborns and young infants, and they are a challenging population as they have relatively immature immune systems and the presence of maternal antibodies may interfere with vaccination of the young infant (see chapter 2 e vaccine immunology). the initial efforts to develop a formalin-inactivated cell culture-derived rsv vaccine resulted in an unanticipated enhancement of natural rsv disease in some of the rsv-naïve infants who received the vaccine in a clinical trial and subsequently were exposed to rsv. the exacerbated disease is thought to be due to an exaggerated t helper type 2 cell immune response (see chapter 2 e vaccine immunology). safety concerns regarding the potential of vaccines to trigger or prime for immunopathological responses has resulted in a cautious approach to the development of rsv vaccines. the vaccine candidates most advanced in clinical development use two different approaches e one uses a live, attenuated virus with a gene deletion deliberately targeted to minimise immunopathological responses. the other approach uses a live viral vector to deliver only a key rsv surface antigen, thereby avoiding the risk of an immunopathological response arising from exposure to the rsv virus itself. infectious illnesses exert a major burden of disease in developing countries. the greatest burden is caused by diseases for which we currently have no vaccines, eg taeniid cestode parasites are associated with high human morbidity and losses in livestock. global efforts to reduce these infections in humans are ongoing through the use of antihelminthics and the implementation of lifestyle changes, but this is having little effect. however, substantial progress has been made towards developing veterinary vaccines which encourages investigation of the potential use of similar vaccines in humans to prevent, for example, hydatid disease (arising from infection with echinococcus granulosus) and cysticercosis (from infection with taenia solium). relative to their burden on society, such diseases have a low priority for funding. unless comprehensive measures are taken to address the gaps in funding, research and global immunisation coverage, developing countries will continue to be overwhelmed by some of the most devastating diseases. in order to improve the situation, collaborative schemes are underway that bring together academic institutions, industry and public/charitable financing organisations. microbiome project is a national institutes of health initiative that seeks to determine the relationship between human health and changes in the human microbiome. by using revolutionary sequencing technologies to characterise the microbiology of five body sites e oral cavity, skin, vagina, gut and nasal tract/lung e an association may be made between the microbiomes associated with either the healthy body state or disease. characterising microbes associated with disease-related pathogens may allow for the development of new vaccines that preserve or protect the healthy microbiome and hence could protect human health. some of the areas of current research are outlined in the box, right. some conditions traditionally thought of as non-infectious may in fact have infectious origins (table 6 .12); therefore, vaccination could be a strategy to prevent these diseases. other diseases may result from an interaction between the host's genetic background and a particular microbe (a so-called gene-environment interaction). some diseases have an established link with an identified infectious agent. for example, primary cmv infection is a known cause of congenital mental retardation; similarly the link between bacterial vaginosis and foetal prematurity is widely accepted. while some links have been established, others remain speculative (table 6 .12). candidate vaccines are in development for the prevention and treatment of various types of addiction. the basic concept is to induce the production of antibodies which will bind the drug and impede its crossing the bloodebrain barrier to exert its psychoactive effects. several nicotine candidate vaccines have now entered clinical trials. a cocaine candidate vaccine has also shown some benefit in a phase iib clinical trial. the key issue to date for both nicotine and cocaine candidate vaccines has been to induce continued on next page high immunoglobulin (ig)g anti-drug antibody levels, which appear to be critical in achieving some degree of efficacy. candidate vaccines against methamphetamine addiction are also in early development. to date, the approach to developing prophylactic cancer vaccines has been to target infectious diseases that cause or contribute to the development of cancer such as hpv (cervical cancer) and hbv (hepatocellular carcinoma). examples of infectious diseases associated with cancer are shown in table 6 .13. the successful development of a nicotine vaccine would be expected to reduce cigarette smoking-related lung cancer. some cancers express tissue-specific antigens that can be targeted by the immune system. therapeutic cancer vaccines aim to target tumour-associated antigens (taa) with t-cell mediated immune responses. taa can be related to the genetic changes that drive the cancer (eg ras oncogene), or inappropriate up-regulation/ expression of genes (eg carcinoembryonic antigen). with such taa targets, vaccines aim to maximally stimulate a cytotoxic t-cell response and their design often includes adjuvants to enhance antigen presentation. tumours develop in a multistep process in the face of the host immune response and frequently evolve to escape immune control. mechanisms of evasion include genetic changes (loss of human leukocyte antigen/taa expression) and induction of immune regulatory systems (t-cell anergy due to the activity of t reg cells) which limit anti-tumour immunity. the key approach for therapeutic cancer vaccines is resetting the immune response to deliver anti-tumour immunity that alters or destroys cancer cells and hence eliminates or reduces the tumour. one strategy uses the patient's own tumour as the immunogen, thereby providing all the potential idiotypic changes that might act as taa, in conjunction with antigen-presenting dcs harvested from the same patient and activated in vitro (see dendritic cell vaccines). there are different types of therapeutic candidate vaccines currently undergoing clinical trials for numerous types of cancer (table 6 .14). the most advanced candidates currently in phase iii are described in chapter 4 e vaccine adjuvants. there has been some success in the development of therapeutic cancer vaccines, with the fda approval of the first dc vaccine oxford biomedica muc1, mucin 1 cell surface; hil2, human cytokine interleukin-2; asci, antigen-specific cancer immunotherapeutics; mage, melanomaassociated antigen; ctl, cytotoxic t lymphocyte. every effort has been made to verify the information in this table. the information included is not meant to be exhaustive but is intended to provide an overview of the subject matter. vaccine approaches. however, this presents opportunities for the application of novel technologies and adjuvants. some of the considerations for vaccines designed for use in special populations include: immunosenescence in the elderly; the poor immunological response to traditional vaccines seen in immunocompromised individuals (patients with hiv, transplant recipients); the crossing of vaccine components into the foetal bloodstream when vaccines are administered to pregnant women; and the safety and immunogenicity concerns surrounding vaccines for neonates due to their naïve and immature immune system. cell-mediated immunity is depressed in pregnant women, leaving them at high risk of infection from pathogens, including those harmful to the foetus. most live, attenuated vaccines are contraindicated during pregnancy because of the theoretical risk of foetal infection from the vaccine. however, inactivated viral or bacterial vaccines can be administered. pregnant women can, therefore, be vaccinated against some infections, including several that pass from mother to foetus (such as hepatitis a and b), and against infections acquired by the infant in the first few months of life (often from close contact with the mother). in the latter case, the infant can be protected by transfer of maternal antibodies during late gestation. examples of diseases that can be prevented in pregnant women include influenza, tetanus, diphtheria and probably pertussis. other diseases, such as those caused by the so-called torch pathogens (toxoplasma, others including syphilis, cmv and hsv), are not yet preventable through vaccination though encouraging phase ii results have been presented for a vaccine to prevent group b streptococcus carriage in pregnant women (hillier et al., 2009; smith, 2009 ). the 2009 h1n1 pandemic influenza outbreak posed an increased risk to pregnant women and vaccination was specifically recommended in pregnant women as one of the high-risk groups. the pandemic example has emphasised once more the importance of protecting pregnant women against influenza. seasonal influenza vaccination in pregnancy is well tolerated and the benefiterisk profile when administered to pregnant women supports its use during pregnancy. many public health authorities worldwide recommend seasonal influenza vaccination in pregnant women and this recommendation is motivated not only by the potentially severe course of influenza during pregnancy, but also by the need to protect vulnerable infants against influenza during their first months of life. boosting rsv immunity in pregnant women through vaccination may be another approach to protecting the newborn against rsv infection during the most vulnerable early months after birth. neonatal immunisation is a strategy to protect infants against infections during a particularly vulnerable period. a recent study showed that immunisation with an acellular pertussis vaccine at birth and 1 month of age induces high igg anti-pertussis antibody titres by 2 months of age (wood et al., 2010) . it is hoped that this approach may reduce death and morbidity from bordetella pertussis infection in the first 3 months of life. the elderly respond poorly to vaccination as the immune system becomes more senescent with increasing age and, therefore, new vaccine technologies are needed to improve the response to vaccination in this population. in the late 1990s, an influenza vaccine adjuvanted with the oil-in-water emulsion, mf59ô, was shown to be more effective at inducing high immune responses in the elderly (minutello et al., 1999) . alternative vaccine administration techniques have also been studied in the elderly. research showed that in subjects 60 years of age or older, an influenza vaccine administered with an intradermal microinjection system induced significantly higher antibody titres compared with im vaccination (arnou et al., 2009 ). subsequently, a microinjection system influenza vaccine was licensed for use in europe, and a high antigen dose formulation has been licensed for the elderly in the usa. individuals with cancer, hiv infection or who are asplenic can be immunocompromised as a result of their condition. patients can also be immunocompromised as a result of therapy, eg when receiving an organ transplant, radiation therapy or immunosuppressive medication. such patients are therefore at an elevated risk of infection from pathogens such as herpesviruses (particularly cmv and epsteinebarr virus), hbv, hcv, pneumocystis and coinfections and represent a special population regarding immunisation. despite a likely reduction in the efficacy of vaccinations in immunocompromised individuals, immunisation remains a frequent recommendation in the hope that at least partial immunity will be achieved. eliciting a response from vaccination in immunocompromised patients may require an increase in the dose and/or number of doses; altering the dosing interval; selecting a different vaccine formulation; or administration via an alternative route. evidence in this patient population is lacking and guidelines are often based on theoretical assumptions. live vaccines are generally contraindicated in immunocompromised or immunosuppressed individuals due to the risk of an active and symptomatic infection resulting from the vaccine itself (non-controlled replication process). encouragingly, vaccine formulations with highly purified antigens and novel adjuvants or alternative deliveries have been shown to induce more effective immune responses than the classical inactivated vaccines in immunocompromised hosts, including patients with end-stage renal diseases in pre-haemodialysis and haemodialysis (see chapter 4 e vaccine adjuvants), patients with hiv and those who have received haematological stem cell transplants. the future of vaccine development can build on the knowledge and experience gained over the last 200 years, and at the same time can take advantage of the most cutting-edge technologies and 194 understanding modern vaccines research. new approaches to antigen selection and production, antigen delivery, adjuvantation and vaccine administration will allow us to target established and emerging diseases, and populations with complex needs. vaccination has been one of the most successful and cost-effective health interventions ever conceived and is now expanding further into cancer and chronic diseases. this expansion of scope and the subsequent impact on human disease is likely to continue into the future in currently unforeseen ways, further increasing the importance of vaccine science and engineering in improving human health. safety and immunogenicity of rts,s/as02d malaria vaccine in infants intradermal influenza vaccine for older adults: a randomized controlled multicenter phase iii study efficacy of rts,s/as01e vaccine against malaria in children 5 to 17 months of age women receiving group b streptococcus serotype iii tetanus toxoid (gbs iii-tt) vaccine have reduced vaginal and rectal acquisition of gbs type iii new vaccines against tuberculosis safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with mf59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults a subunit cytomegalovirus vaccine based on recombinant envelope glycoprotein b and a new adjuvant vaccine prevention of maternal cytomegalovirus infection vaccination with alvac and aidsvax to prevent hiv-1 infection in thailand successful immunization of children with and without maternal antibody by aerosolized measles vaccine. i. different results with undiluted human diploid cell and chick embryo fibroblast vaccines associate clinical professor of medicine glaxosmithkline herpes vaccine efficacy study group. glycoprotein-d-adjuvant vaccine to prevent genital herpes prevention of tuberculosis in bacille calmette-guérin-primed, hiv-infected adults boosted with an inactivated whole-cell mycobacterial vaccine the second geneva consensus: recommendations for novel live tb vaccines acellular pertussis vaccine at birth and one month induces antibody responses by two months of age genocea technology developing cell culture-derived pandemic vaccines seeking new pathways for hiv vaccine discovery infectious diseases and global cancer control intrauterine infection and preterm delivery the nih human microbiome project alliance for case studies for global health. case studies for global health: building relationships alliance for case studies international aids vaccine initiative vaccines of the future national institute of allergy and infectious diseases (niaid). statement: study finds genital herpes vaccine ineffective in women world health organization's special programme for research and training in tropical diseases aidsvaxô is a trademark of global solutions for infectious diseases corporation; alvacô is a trademark of connaught technology corporation mf59ô is a trademark of novartis; montanideô is a trademark of seppic; nanostatô is a trademark of nanobio corporation. for details of trademarks not listed above please see the manufacturer's website (see section internet resources) vaccines of the future key: cord-306315-vt2e0crh authors: elabbadi, alexandre; pichon, jérémie; visseaux, benoit; schnuriger, aurélie; bouadma, lila; philippot, quentin; patrier, juliette; labbé, vincent; ruckly, stéphane; fartoukh, muriel; timsit, jean-françois; voiriot, guillaume title: respiratory virus-associated infections in hiv-infected adults admitted to the intensive care unit for acute respiratory failure: a 6-year bicenter retrospective study (hiv-vir study) date: 2020-09-14 journal: ann intensive care doi: 10.1186/s13613-020-00738-9 sha: doc_id: 306315 cord_uid: vt2e0crh introduction: acute respiratory failure is the main reason for admission to the intensive care unit (icu) in hiv-infected adults. there is little data about the epidemiology of respiratory viruses in this population. methods: hiv-infected adults admitted to two intensive care units over a 6-year period for an acute respiratory failure and explored for respiratory viruses with multiplex polymerase chain reaction (mpcr) were retrospectively selected. objectives were to describe the prevalence of respiratory viruses, coinfections with non-viral pathogens, and hospital outcome. results: a total of 123 episodes were included. an hiv infection was newly diagnosed in 9% of cases and 72% of the population were on antiretroviral therapy. real-time mpcr tests identified at least one respiratory virus in the respiratory tract of 33 (27%) patients, but with a non-viral copathogen in two-thirds of cases. rhinovirus was predominant, documented in 15 patients, followed by influenza and respiratory syncytial viruses (both n = 6). the prevalence of respiratory virus-associated infection did not vary along with the level of the cd4 t-cell deficiency, except for rhinovirus which was more prevalent in patients with a cd4 lymphocyte count below 200 cells/µl (n = 13 (20%) vs. n = 2 (4%), p < 0.01). in multivariate analysis, respiratory virus-associated infection was not associated with a worse prognosis. conclusions: viruses are frequently identified in the respiratory tract of hiv-infected patients with acute respiratory failure that requires icu admission, but with a non-viral copathogen in two-thirds of cases. rhinovirus is the predominant viral specie; its prevalence is highest in patients with a cd4 lymphocyte count below 200 cells/µl. acute respiratory failure (arf) is the leading cause of admission to the intensive care unit (icu) in hivinfected patients [1] [2] [3] . infectious causes are predominant, although the proportion of opportunistic infections has decreased significantly in the era of combination antiretroviral therapy (art) [2, 4, 5] . in contrast, the open access *correspondence: guillaume.voiriot@aphp.fr 1 assistance publique -hôpitaux de paris, service de médecine intensive réanimation, hôpital tenon, sorbonne université, paris, france full list of author information is available at the end of the article burden of non-hiv-related pulmonary events, such as bacterial pneumonia, acute bronchitis and acute exacerbation of chronic obstructive pulmonary disease (copd) has been shown increasing [2, 3, 6] . these important changes in the etiologic panel of arf in hiv-infected patients question the role of respiratory viruses. indeed, using nucleic acid amplification test such as multiplex polymerase chain reaction (mpcr), these pathogens have been shown highly prevalent (20-56%) in large cohorts of adult patients admitted to the icu for all-cause arf [7, 8] , community-acquired pneumonia [9, 10] , hospitalacquired pneumonia [11] , acute exacerbation of copd [12, 13] , and asthma [14] , compared to asymptomatic adults [15, 16] . high prevalence has also been described in specific immunocompromised populations, such as cancer and hematology patients [17, 18] . in contrast, little is known about the epidemiology of respiratory viruses in hiv-infected patients [19, 20] , especially those admitted to the icu, and the prevalence of respiratory viruses according to the cd4 t-cell deficiency. moreover, coinfections with virus and opportunistic pathogens may occur. overall, respiratory virus-associated infections may affect prognosis. therefore, we conducted a comprehensive observational study among adult hiv-infected icu patients with arf explored with respiratory mpcr. our goals were to describe the prevalence of respiratory viruses, coinfections with non-viral pathogens, and hospital outcome. we conducted a retrospective bicenter observational study in two icu of the paris area (the 26-bed icu of the bichat university hospital and the 20-bed icu of the tenon university hospital). from april 2011 to april 2017, all consecutive hiv-infected patients admitted to icu having undergone an mpcr in the respiratory tract within 72 h following their icu admission were screened. medical records were independently reviewed by two physicians (ae and gv). all patients with arf at icu admission were included. arf was defined by the presence of at least two of the following criteria: cough, expectoration, dyspnea, rales, signs of respiratory distress (tachypnea exceeding 30/min, paradoxical abdominal breathing), chest pain, hypoxemia requiring oxygen therapy, noninvasive ventilation or intubation. in case of multiple admissions over the 6-year study period, only the first admission was analyzed. at icu admission and during icu stay, data regarding demographics, comorbidity, hiv-related characteristics, clinical examinations, laboratory and radiological findings, microbiologic investigations, and therapeutic management were collected (for details, see additional file 1). mortality was defined as death from any cause within 28 days following the icu admission. respiratory mpcrs were performed either in nasopharyngeal (np) swabs or in lower respiratory tract (lrt) specimen, usually bronchoalveolar lavage (bal) fluid otherwise endotracheal aspirate. during the study period, different respiratory mpcr kits (additional file 1: table s1 ) were used (for more details about microbiological evaluation, see additional file 1). medical charts were independently reviewed by two clinicians (ae and gv). they determined the causative diagnosis of arf for each patient, using a 5-class classification. in case of an inter-reviewer discordance, a shared review of the medical charts was performed, and an agreement was found. the five mutually exclusive classes of causative diagnosis for arf were: (i) pneumocystis jirovecii pneumonia (pcp); (ii) other opportunistic lung infections; (iii) non-opportunistic acute lung infection; (iv) non-infectious lung disease, and (v) extra-pulmonary cause (for details, see additional file 1). the primary endpoint was to determine the prevalence of respiratory viruses according to the cd4 lymphocyte count. a respiratory virus documented with mpcr was always considered as a pathogen of the respiratory tract, regardless of the type of specimen (np or lrt). the cd4 lymphocyte count measured during the icu stay was used to group patients, with a cut-off of 200 cells/µl (≤ 200 cells/µl for the low-cd4 group; > 200 cells/µl for the high-cd4 group) [19, 21] . secondary endpoints were to describe the epidemiology of respiratory viruses and coinfections with non-viral pathogens, to identify risk factors for respiratory virusassociated infection, and to study outcome. a composite criterion named "complicated course" included death from any cause within 28 days following the icu admission or mechanical ventilation for more than 7 days. continuous data were expressed as median [first through third quartiles] and were compared using the pairwise mann and whitney test. categorical data were expressed as number (percentage) and were evaluated using the chi-square test or fisher exact test. p values less than 0.05 were considered significant. a univariate logistic regression with clinically relevant variables was used to identify variables associated with a respiratory virusassociated infection. a multivariate conditional logistic regression, including variables with p value less than 0.10 in the previous step, was used to identify variables independently associated with a respiratory virus-associated infection. similar statistical analyses were performed to identify variables independently associated with death from any cause within 28 days following the icu admission and mechanical ventilation for more than 7 days in survivors at day-28. quantitative variables that did not validate the log-linearity assumption were transformed into categorical variables according to their median value. missing data were imputed to the median or the more frequent value. the accuracy of the final model was tested using area under the receiver operating characteristic curve analysis and the hosmer-lemeshow chi-square test. analyses were performed using the sas software package (sas institute, cary, nc, usa). during the 6-year study period, 135 hiv-infected adult patients were admitted at least once to icu and underwent a respiratory mpcr in the first 72 h of the icu stay. among them, 12 did not fulfill criteria of arf. the final study group consisted of 123 patients. their main characteristics, stratified by the cd4 lymphocyte count at icu admission, are presented in table 1 . of these 123 patients, 2 were admitted twice during the study period and one was admitted thrice. eleven patients (9%) were newly diagnosed as having hiv infection on icu admission; the remaining 112 had been previously diagnosed, and 88 were on art but with poor adherence to the treatment in 21 patients, as mentioned by the infectiologist in the medical charts. latest available median cd4 lymphocyte count and hiv viral load were 351 cells/µl [140-600] and 0 log copies/ml [0-3.4], respectively. at least one additional factor of immunosuppression was identified in 10 (8%) patients. at icu admission, median cd4 lymphocyte count was 170 cells/µl , with 66 patients (54%) equal or below 200 cells/µl (low-cd4 group) and 57 (46%) above 200 cells/µl (high-cd4 group). both these groups did not differ regarding demographics, performance status, factors of immunosuppression other than hiv and comorbidity, except for copd which was more prevalent in the high-cd4 group (n = 12 (21%) vs. n = 4 (6%), p = 0.01). the microbiological investigations are displayed in additional file 1: table s2 . mpcr was performed in np swabs exclusively (n = 46, 37%) or in lrt specimen exclusively (n = 50, 41%), or both (n = 27, 22%). respiratory tract specimens for bacterial culture have been obtained in 110 (91%) patients. bal fluid has been obtained in 77 (63%) patients. causative diagnoses of arf are displayed in table 2 . an opportunistic lung infection was diagnosed in 38 (31%) patients. seven of the 11 patients with newly diagnosed hiv infection and 8 patients receiving art, but with a poor adherence to the treatment had pcp. non-opportunistic acute lung infections were identified as causative diagnosis of arf in 59 (48%) patients. all the bacteria-infected patients received an appropriate antimicrobial regimen within the first 24 h of icu stay. eight patients had a clinical presentation suggestive of lung infection, but without microbiological documentation. the arf was attributed to a non-infectious lung disease in 19 (15%) patients, mainly related to a decompensated chronic condition, i.e., acute exacerbation of copd and pulmonary edema. overall, 36 respiratory viruses were identified in 33 (27%) patients (table 3) . rhinovirus was predominant (n = 15), followed by influenza (n = 6), respiratory syncytial virus (n = 6) and parainfluenza virus (n = 5). only one pure virus-virus coinfection was found. the prevalence of respiratory virus-associated infection did not differ among low-and high-cd4 groups (table 1) ; therefore, the median cd4 lymphocyte count in respiratory virus-infected patients was 109 cells/µl, in comparison with 192 cells/µl in non-infected patients (fig. 1) . however, the prevalence of rhinovirus-associated infection was higher in the low-cd4 group, and three-quarters of rhinovirus-infected patients exhibited a cd4 lymphocyte count below 200 cells/µl (fig. 2) . in 22 patients, the viral documentation was accompanied by a non-viral documentation (additional file 1: figure s1), with bacteria-virus coinfection in 11 patients, bacteria-virus-virus in 2 patients, p. jirovecii-virus in 7 patients and p. jirovecii-virus-virus in one patient. the rate of viral documentation among patients explored with np swab exclusively, lrt specimen exclusively, or both, did not differ significantly (30%, 26% and 22%, respectively; p = 0.73). documentation of respiratory viruses was more frequent during the winter period (october to march) (additional file 1: figure s2 ). conversely, rhinovirus documentation did not follow a seasonal distribution, since only 7/15 were observed during the period from october to march. characteristics of the population, stratified by respiratory virus-associated infection are presented in additional file 1: table s3 . at icu admission, respiratory virus-infected patients displayed higher respiratory rate and fever. in multivariate analysis, female gender, smoking and steroid therapy were shown as independently associated with respiratory virus-associated infection ( table 4) . mortality at day-28 was 12%, and 26% of patients displayed a complicated course, without difference between high-cd4 and low-cd4 groups (table 1) . we investigated whether a respiratory virus-associated infection table 2 causative diagnosis of acute respiratory failure in 123 hiv-infected patients admitted to the icu data are presented as number (%). cd4 refers to cd4 lymphocyte count (cells/µl) a other opportunistic lung infections included cmv-associated pneumonia (n = 5) and pulmonary tuberculosis (n = 4) b non-infectious lung diseases included acute exacerbation of copd of non-infectious etiology (n = 6), cardiogenic lung edema without underlying lung agent (n = 5), cryptogenic hemoptysis (n = 1), intra-alveolar hemorrhage (n = 1); acute interstitial pneumonia (n = 2), mendelson syndrome (n = 1), sickle cell disease (acute chest syndrome) (n = 1); neoplastic pleural effusion (n = 1) and castleman disease (n = 1) c extra-pulmonary causes included histoplasmosis (n = 1), cryptococcus neoformans meningitis (n = 1), bacterial meningitis (n = 2), pyelonephritis (n = 2) and bacteremia of unknown origin (n = 1) all extra-pulmonary cause c 7 (5.7) 6 (9.1) 1 (1.8) affected prognosis. in the analysis stratified by respiratory virus-associated infection, outcome was similar between infected and non-infected patients (additional file 1: table s3 ). in multivariate analysis, a respiratory virus-associated infection was not identified as an independent factor of either a complicated course (table 5 ) or death at day-28 (additional file 1: table s4 ). this retrospective study investigated the epidemiology of respiratory viruses in hiv-infected adults admitted to the icu for arf. real-time mpcr tests identified at least one virus in the respiratory tract of 27% of patients, but with a non-viral copathogen in two-thirds of cases. the prevalence of respiratory virus-associated infection did not vary along with the level of the cd4 t-cell deficiency, except for rhinovirus which was more prevalent in patients with a cd4 lymphocyte count below 200 cells/ µl. in multivariate analysis, respiratory virus-associated infection was not associated with a worse prognosis. in this study, more than one patient out of four (27%) were infected with at least one respiratory virus. this finding illustrated the high yield of an aggressive diagnostic strategy with a broad panel mpcr on respiratory tract specimens. our results are original since prior works having described the etiological panel of arf in hivinfected icu patients were conducted before the era of real-time mpcr [2, 4, 22] . interestingly, the rate of viral documentation that we observed was similar to what had been described (27 to 29%) previously in non-hiv adults admitted to the icu for an acute respiratory disorder requiring intubation [7, 8] . we identified at least one non-viral copathogen in more than two-thirds of the patients with a viral documentation, in line with a recent report in a population with a high prevalence of tuberculosis [19, 23] . nonopportunistic acute lung infections, including pneumonia, acute bronchitis and exacerbation of copd, were the first cause of arf, consistent with previous reports in western countries [2, 4] . this finding highlights the burden of chronic respiratory conditions in aging hivinfected populations [6] . here, more than 40% of patients were smokers. synergistic effects of tobacco and hiv [24] in promoting chronic bronchitis and pro-copd changes in the lung [25] have been demonstrated. moreover, high rates of viral documentation within airways of copd patients both at stable state and during exacerbation have been reported [26] . these data may explain the high rate of viral documentation that we observed. in multivariate analysis, smoking was independently associated with respiratory virus-associated infection. this finding is in line with previous works demonstrating that tobacco exposure alters immune responses to rhinovirus [27] , influenza virus [28] and respiratory syncytial virus [29] . interestingly, female gender was associated with an increased risk of respiratory virus-associated infection on multivariate analysis. prior cohort studies in primary care described an increased risk for development of influenza-like illnesses in women compared to men [30, 31] . however, to our knowledge, no prior study has specifically explored this point in hiv-infected populations admitted for arf. in this study, we also aimed to investigate a putative role of the hiv-related cd4 t-cell deficiency in promoting respiratory virus-associated infection. previous studies explored this point in children, but with conflicting results. annamalay et al. described similar rates of viral documentation in hiv-infected and non-infected children admitted for lower respiratory tract infections [32] , whereas o'callaghan-gordo et al. observed that respiratory virus-associated infections were 6 to 16 times more prevalent among hiv-infected children admitted for pneumonia [33] . as we did not include a comparative non-hiv population, we rather examined whether or not the rate of viral documentation varied along with the level of cd4 t-cell deficiency. finally, we found no association between the cd4 lymphocyte count and the risk of respiratory virus-associated infection, in line with a previous report focusing on influenza viruses [34] . rhinovirus was the predominant virus, accounting for more than 40% of viral documentations. this high prevalence was consistent with that of previous reports in icu patients with arf [7] , community-acquired pneumonia [35] or acute exacerbation of copd [13] . surprisingly, rhinovirus was much more prevalent in low-cd4 patients. this finding is original, since no prior work has specifically explored this point in adults. in hiv-infected children, rhinovirus has been shown highly prevalent, during both pneumonia and bronchiolitis, but without data regarding a putative association with the level of cd4 t-cell deficiency [32, 36] . other data in hematology and cancer adults demonstrated high rates of rhinovirus documentation within airways during respiratory tract infections [37, 38] . to explain this high prevalence in immunocompromised patients, a mechanism of prolonged viral shedding has been proposed, rather than iterative reinfections as observed in copd patients [39] . the prolonged rhinovirus shedding may be attributable to an inefficient immunological control of a single infectious episode [40, 41] . therefore, in pediatric hematopoietic stem cell transplant recipients with a persistent rhinovirus shedding (≥ 30 days), piralla et al. demonstrated significant lower cd4, cd8 and nk lymphocyte counts at the onset of infection, as compared to children with a brief rhinovirus shedding. moreover, a decrease in rhinovirus load was associated with significant increases of the same lymphocyte counts [42] . these data would suggest an important role for the t-cell immunity in the control of rhinovirus infection, and subsequently, may explain a delayed rhinovirus clearance in low-cd4 hivinfected patients, resulting in persistent shedding and increased prevalence. we observed a high rate of dual infection, either virusbacteria or virus-opportunistic pathogen. these findings made us consider the prognostic impact of such coinfections. studies in icu adult patients with pneumonia suggested that virus-bacteria coinfection was associated with a worse prognosis [43] . in mice, the coinfection of influenza with streptococcus pneumoniae [44] , legionella pneumophila [45] or staphylococcus aureus [46] impaired the anti-influenza immune response and increased mortality. whether similar synergistic effects exist in virusopportunistic pathogen coinfection remain unknown. only one animal study has explored the couple pneumocystis jirovecii-influenza, but in a successive rather than concomitant model [47] . unfortunately, in our study, the low number of observations prevented us from analyzing the prognosis according to the presence of coinfections. our study has several limitations. first, this study included adult patients with arf that required icu admission, preventing any conclusion on other populations such as hiv-infected children or hiv-infected adults with arf that did not require icu admission. second, the study was retrospective, so we did not control the microbiological investigations. the preferred sample for mpcr test in non-intubated patients was not the sputum, but the nasopharyngeal swab [48] . several factors may have discouraged clinicians to use sputum, including the high number of patients unable to produce sputum [49] and the highly viscous nature of this sample that can make nucleic acid extraction difficult [50] . by definition, an mpcr was performed in the respiratory tract of every included patient because it was a criterion for patient screening. but some other microbiological tests were only occasionally performed, i.e., cytomegalovirus pcr. furthermore, the retrospective design prevented us from obtaining a number of data, which were rarely reported in medical records by physicians, including vaccine history, pneumocystis jirovecii prophylaxis, symptoms before hospital referral, and duration of symptoms before icu admission. third, only patients having undergone an mpcr in the respiratory tract within the 72 h following their icu admission were screened; this might suggest a confounding of indication. fourth, the choice to classify patients according to their cd4 lymphocyte count on the icu admission, instead of the latest known value, might be criticized. however, this choice was guided by the high number of missing values in the latest cd4 lymphocyte count as well as the number of newly diagnosed patients without any prior cd4 lymphocyte count. fifth, we assumed that a virus identified with pcr in nasopharyngeal or lower respiratory tract samples was always a pathogen of the respiratory tract, whatever the clinical picture and radiological features. this might be criticized since respiratory viruses might be present in asymptomatic adult subjects [15] , even if it seems rare, about 2% of asymptomatic adults seen at the emergency department [16] . sixth, to avoid overinterpreting the data, we decided to consider respiratory viruses as a homogeneous group of pathogens in the analysis stratified by respiratory virus-associated infection. this might be criticized since the pathogenicity differs from one viral species to another. viruses are frequently identified in the respiratory tract of hiv-infected patients with arf that required icu admission, but with a non-viral copathogen in two-thirds of cases. rhinovirus is the predominant viral specie; its prevalence is highest in patients with a cd4 lymphocyte count below 200 cells/µl. supplementary information accompanies this paper at https ://doi. org/10.1186/s1361 3-020-00738 -9. additional file 1. additional information on material and methods, table s1 (panels of mpcr kits used in the two participating icus over the 6-year study period), table s2 (microbiological investigations performed in 123 hiv-infected patients admitted to the icu for acute respiratory failure, according to the diagnosis of respiratory virus-associated infection), table s3 (baseline characteristics, behavior during icu stay, and outcome of 123 hiv-infected patients admitted to the icu for acute respiratory failure, according to the diagnosis of respiratory virus-associated infection), table s4 (multivariate analysis of the risk factors for death at day-28 in 123 hiv-infected patients admitted to the icu for acute respiratory failure), figure s1 (distribution of the microbiological documentations in 123 hivinfected patients admitted to the icu for acute respiratory failure), figure s2 admissions to intensive care unit of hiv-infected patients in the era of highly active antiretroviral therapy: etiology and prognostic factors etiologies and outcome of acute respiratory failure in hivinfected patients temporal trends in critical events complicating hiv infection: 1999-2010 multicentre cohort study in france survival for patients with hiv admitted to the icu continues to improve in the current era of combination antiretroviral therapy pulmonary infections in hiv-infected patients: an update in the 21st century hiv infection and risk for incident pulmonary diseases in the combination antiretroviral therapy era epidemiology and clinical outcome of virus-positive respiratory samples in ventilated patients: a prospective cohort study respiratory viruses in invasively ventilated critically ill patients-a prospective multicenter observational study viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia impact of respiratory viruses in hospital-acquired pneumonia in the intensive care unit: a single-center retrospective study virus infection in exacerbations of chronic obstructive pulmonary disease requiring ventilation procalcitonin algorithm to guide initial antibiotic therapy in acute exacerbations of copd admitted to the icu: a randomized multicenter study epidemiology of respiratory viruses in patients hospitalized with near-fatal asthma, acute exacerbations of asthma, or chronic obstructive pulmonary disease community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study community-acquired pneumonia requiring hospitalization among u.s. adults clinical significance of upper airway virus detection in critically ill hematology patients management of respiratory viral infections in hematopoietic cell transplant recipients and patients with hematologic malignancies prospective etiological investigation of community-acquired pulmonary infections in hospitalized people living with hiv. medicine (baltimore) respiratory viruses in hiv-infected patients with suspected respiratory opportunistic infection outcome of critically ill human immunodeficiency virus-infected patients in the era of highly active antiretroviral therapy survival of hiv-infected patients in the intensive care unit in the era of highly active antiretroviral therapy etiology of pulmonary infections in human immunodeficiency virus-infected inpatients using sputum multiplex real-time polymerase chain reaction impact of hiv infection and smoking on lung immunity and related disorders cigarette smoke and hiv synergistically affect lung pathology in cynomolgus macaques a prospective, observational cohort study of the seasonal dynamics of airway pathogens in the aetiology of exacerbations in copd cigarette smoke decreases innate responses of epithelial cells to rhinovirus infection cigarette smoke attenuates the rig-i-initiated innate antiviral response to influenza infection in two murine models cigarette smoke suppresses tlr-7 stimulation in response to virus infection in plasmacytoid dendritic cells recording of influenza-like illness in uk primary care 1995-2013: cohort study incidence and risk factors for influenza-like-illness in the uk: online surveillance using flusurvey respiratory viruses in young south african children with acute lower respiratory infections and interactions with hiv etiology and epidemiology of viral pneumonia among hospitalized children in rural mozambique: a malaria endemic area with high prevalence of human immunodeficiency virus pandemic (h1n1) 2009 influenza virus seroconversion rates in hiv-infected individuals incidence of respiratory viruses in patients with community-acquired pneumonia admitted to the intensive care unit: results from the severe influenza pneumonia surveillance (sips) project clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in hiv-infected and hiv-uninfected south african children respiratory virus detection in immunocompromised patients with filmarray respiratory panel compared to conventional methods virological diagnosis in community-acquired pneumonia in immunocompromised patients prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness chronic rhinoviral infection in lung transplant recipients persistent human rhinovirus type c infection of the lower respiratory tract in a pediatric cord blood transplant recipient persistent rhinovirus infection in pediatric hematopoietic stem cell transplant recipients with impaired cellular immunity viral infection in community-acquired pneumonia: a systematic review and meta-analysis coinfection with streptococcus pneumoniae negatively modulates the size and composition of the ongoing influenza-specific cd8 + t cell response role of tissue protection in lethal respiratory viral-bacterial coinfection influenza virus primes mice for pneumonia from staphylococcus aureus pneumocystis infection enhances antibodymediated resistance to a subsequent influenza infection diagnosis and management of respiratory viruses in critically ill adult patients: an international survey of knowledge and practice among intensivists diagnostic value of microscopic examination of gram-stained sputum and sputum cultures in patients with bacteremic pneumococcal pneumonia comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations none. gv had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis, including and especially any adverse effects. ae participated in the design of the study, participated in the data acquisition, analysis and interpretation, and the statistical analysis, and drafted the manuscript. jp, bv, as, lb, qp, jp and vl participated in the data acquisition, analysis and interpretation, and helped to revise the manuscript. rs participated in the data analysis and interpretation, and the statistical analysis. mf and jft participated in the design of the study, participated in the data analysis and interpretation, and helped to revise the manuscript. gv designed the study, participated in the data analysis and interpretation, and the statistical analysis, and revised the manuscript. all authors read and approved the final manuscript. none. data and materials supporting the findings of this study can be entirely shared if asked. not applicable. the authors have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. key: cord-329223-f84gjxm1 authors: kouokam, joseph calvin; huskens, dana; schols, dominique; johannemann, andrew; riedell, shonna k.; walter, wendye; walker, janice m.; matoba, nobuyuki; o'keefe, barry r.; palmer, kenneth e. title: investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate date: 2011-08-02 journal: plos one doi: 10.1371/journal.pone.0022635 sha: doc_id: 329223 cord_uid: f84gjxm1 many natural product-derived lectins such as the red algal lectin griffithsin (grft) have potent in vitro activity against viruses that display dense clusters of oligomannose n-linked glycans (nlg) on their surface envelope glycoproteins. however, since oligomannose nlg are also found on some host proteins it is possible that treatment with antiviral lectins may trigger undesirable side effects. for other antiviral lectins such as concanavalin a, banana lectin and cyanovirin-n (cv-n), interactions between the lectin and as yet undescribed cellular moieties have been reported to induce undesirable side effects including secretion of inflammatory cytokines and activation of host t-cells. we show that grft, unlike cv-n, binds the surface of human epithelial and peripheral blood mononuclear cells (pbmc) through an exclusively oligosaccharide-dependent interaction. in contrast to several other antiviral lectins however, grft treatment induces only minimal changes in secretion of inflammatory cytokines and chemokines by epithelial cells or human pbmc, has no measureable effect on cell viability and does not significantly upregulate markers of t-cell activation. in addition, grft appears to retain antiviral activity once bound to the surface of pbmc. finally, rna microarray studies show that, while cv-n and cona regulate expression of a multitude of cellular genes, grft treatment effects only minimal alterations in the gene expression profile of a human ectocervical cell line. these studies indicate that grft has an outstanding safety profile with little evidence of induced toxicity, t-cell activation or deleterious immunological consequence, unique attributes for a natural product-derived lectin. hiv-1 is the prototype example of a virus that utilizes an oligomannose-rich ''glycan shield'' to occlude functionally important domains of the envelope glycoproteins from antibodies, and evade the immune response [1, 2] . recently doores et al. [3] showed that previous measurement of the proportion of oligomannose nlg relative to complex nlg on recombinant hiv envelope glycoproteins underestimated the representation of oligomannose nlg on the native envelope spikes of hiv-1, which appear to display nlg that are almost exclusively mannoseterminal man 5-9 -glcnac 2 structures. it is likely that limited access to the high density of nlg presented on the hiv-1 trimeric glycoprotein spike by golgi and endoplasmic reticulum (er) a1r2 mannosidases results in an atypical preponderance of oligomannose glycans rather than complex nlg on hiv-1 surface glycoproteins [3, 4, 5] . given that man 5-9 -glcnac 2 structures are present on less than 4% of the normal human n-glycome [6, 7] , dense clusters of oligomannose nlg appear to be a feature specific to viral envelope glycoproteins, particularly those of hiv-1 and other immunodeficiency lentiviruses [3] . consequently, clusters of oligomannose nlg may be attractive molecular targets for antiviral drugs and vaccines that act to interrupt hiv-1 infection of target cells by: (i) binding on the virus envelope and thereby interfering with the structural transitions involved in receptor and co-receptor docking and virus entry into t-cells and (ii) blocking access to viral envelope oligomannose nlg targeted by c-type lectin receptors dc-sign and mmr on dendritic cells and macrophages [5, 8, 9] . it has long been known that a variety of oligomannose-specific lectins have potent in vitro hiv-1 inhibitory activities, and therefore have been proposed as microbicide candidates for topical prophylaxis of hiv-1 infection, and as potential therapeutics [8, 9] . however many lectins possess lymphocyte mitogenic activities incompatible with their use as pharmaceuticals, and some are known human and animal toxins, although the pharmacological basis for their toxicity is poorly characterized [10, 11] . the antiviral potency of lectins has been correlated to their capacity to bind multiple glycans simultaneously, often facilitated by their ability to form dimers and higher order multimers [12, 13, 14] . the most extensively characterized antiviral lectin is cv-n, a small protein that exists in both monomeric and homodimeric configurations and has exceptionally potent anti-hiv activity, in the low nanomolar range [15] . cv-n targets the mana1r2man terminating glycans displayed in the man 6-9 glcnac 2 structures on the surface of many viral envelope glycoproteins. each monomer of cv-n has the capacity to bind two oligomannose structures [16] . when formulated into a carboxyethylcellulose gel matrix, cv-n provided almost complete protection against a single high dose intrarectal or intravaginal challenge with a pathogenic simian-human immunodeficiency virus (shiv) [17, 18] . however, subsequent in vitro toxicity studies have raised concerns about the safety of cv-n based microbicides, finding that in vitro, cv-n has the capacity to promote secretion of pro-inflammatory cytokines and chemokines from human peripheral blood mononuclear cells (pbmc), activate quiescent cd4 + t-cells, and promote t-cell proliferation [19, 20, 21, 22] . similar results were also reported for other lectins such as microvirin (mvn) (17) and concanavalin a (con a) (18) . it should be noted that the toxicities of cv-n were much milder in treated cervical explants in comparison with pbmc [20, 21] . the possible pathogenic consequences associated with these off-target activities have raised concerns about all other members of the natural product-based class of antiviral lectins [19, 20, 21] . grft has the most potent and broad spectrum hiv-1 inhibitory activity yet described for any antiviral lectin [15, 23, 24, 25] . it is a 25 kda domain-swapped homodimer, with the first 16 amino acids of each 12.7 kda monomer completing the b-prism fold of the other [26] . the homodimer has six carbohydrate binding pockets, 3 located at each of the opposite ends of the double-prism homodimer. atomic resolution crystal structures of an engineered monomeric grft showed that each monomer can bind to two different nonamannoside molecules through all three carbohydrate binding sites [13, 26] . the antiviral activity of monomeric grft is substantially lower than that of the homodimeric form, confirming that the grft potency is dependent on its ability to bind multiple oligomannose structures simultaneously, with strong avidity [13] . we showed recently that grft causes no mitogenic stimulation of pbmc exposed to the drug [23] . grft is fully active in the presence of macaque vaginal secretions [25] , and was shown to have a good safety profile in the rabbit vaginal irritation model, the gold standard preclinical safety test for vaginal products [23] . moreover, treatment of human cervical explants with grft induced minimal alterations in the expression profile of a panel of proinflammatory chemokines and cytokines. grft also strongly inhibited hiv-1 infection of the cervical explants, and dissemination of hiv-1 infection from cells resident in the explants to donor t-cells [23] . in the present study, we performed a comprehensive set of experiments to interrogate the molecular response of cultured human cervicovaginal cells and pbmc to grft exposure. our investigations employed comparisons between the biological activities of grft, which binds mannose-terminal man 5-9 -glcnac 2 , with other lectins of well-defined carbohydrate binding specificity: (1) cv-n, which binds mana1r2man terminating glycans on man 6-9 -glcnac 2 structures; (2) phytohemagglutinin a (pha), targeting d-galactose and n-acetyl-d-galactosamine on gycan structures; (3) cona, specific for terminal (tri) mannose on high mannose glycans and (4) pokeweed agglutinin (pkm), which binds n-acetylglucosamine. our studies reveal clear distinctions in biological and toxicological properties of these lectins, and confirm grft's superior safety profile for use as a topical microbicide. grft and cv-n binding to human cervical epithelium, cultured human cervicovaginal cells, and pbmc we used paraffin-embedded cervical epithelial sections from a 21year old donor to evaluate the binding pattern of fluorescently labeled grft and cv-n to human mucosal epithelia. grft lec-, a mutant form of grft where we eliminated the lectin activity through mutation of all six mannose binding sites, was used as a control to help distinguish binding mediated by the grft carbohydrate binding pockets versus binding associated with other grft structures. the light micrograph in figure 1a shows an h&e stained cervical tissue section to orient the observer to the microanatomy of the human cervical epithelium. different layers of the squamous epithelium starting at the basement membrane (basal, parabasal, intermediate, superficial) are evident; cervical connective tissue or stroma is beneath the basement membrane. tissues incubated with labeled grft, grft lecand cv-n are shown in fig. 1b , c and d, respectively. minimal fluorescence seen in tissues exposed to grft lec(fig. 1c ) compared with grft-stained tissues (fig. 1b) confirmed that the binding of grft to the outermost layer of the squamous epithelium was via its carbohydrate binding activity. there were distinct differences evident in the binding pattern of grft ( fig. 1c ) relative to cv-n (fig. 1d) , which bound far more extensively than grft throughout all layers of the squamous epithelium, basement membrane and underlying stromal tissue. additional fluorescence micrographs are provided in fig. s1 . in cultured cervicovaginal epithelial cells we also observed binding of both grft and cv-n, but not grft lecto ect1/e6e7 cells (compare fig. 1e through h), as well as end1/e6e7 and vk2/e6e7 cells (data not shown). we used flow cytometry to evaluate binding of fluorescently labeled grft, grft lecand cv-n to human pbmc. clear shifts in fluorescence intensity show that grft (fig. 1 i) and cv-n ( fig. 1k ) efficiently bind the surface of human pbmc relative to grft lec(fig. 1j ), for which we observed only minimal binding. binding of grft to the surface of pbmc was significantly reduced when occluding the glycan binding pockets by pre-incubation with yeast mannan (fig. 1i) . interestingly, the binding of cv-n to pbmc was reduced, but not eliminated, by mannan binding, which implies a second mode of binding between cv-n and the cell surface (fig. 1k ). we assume that distinct populations of differentially labeled cells seen in the flow histograms reflect differences in the amount of labeled protein that binds different subpopulations of leukocytes in the unfractionated pbmc samples. when freshly-isolated pbmc were pre-incubated for 24 hrs with grft at various concentrations, washed and then infected with hiv-1 r5 strain bal (without adding new compound), grft inhibited viral replication for 9 days of cell culture (fig. 2) . as a control maraviroc (mvr) at 2 mm was included and this also showed anti-hiv activity after 9 days in culture, as this compound is known to bind specifically to the ccr5 receptor. however, lower concentrations of maraviroc (at 0.4 mm) did not retain its antiviral activity in this assay protocol (data not shown). an mtt assay was used to assess the effects of grft on end1/ e6e7, ec1/e6e7 and vk2/e6e7 cell viability by measuring the metabolic activity of treated cells. in these experiments (fig. 3) we observed no loss in cell viability after a 3 day exposure of the endocervical and ectocervical cell lines to concentrations of up to 1 mg/ml (84 mm) grft, at least 10-times more concentrated than a likely microbicide formulation [23] . high doses of grft did, however, slightly reduce viability of the vaginal keratinocyte (vk2) cells. in marked contrast to grft, the mannose-specific mitogenic lectin cona showed clear concentration-dependent cytotoxicity towards all three cell lines. as shown in fig. 3 , treatment with 1 mm cona killed approximately 94% of the ectocervical cells and vaginal keratinocytes, and 80% of the endocervical cells. pha, another lectin known to be mitogenic in vivo and in vitro also caused doseresponsive cell death, but to a lesser extent than cona. we previously showed that grft exhibits no mitogenic stimulatory effect in human pbmc, using incorporation of tritiated thymidine as a marker for cell proliferation [23] . in the present study we investigated the mitogenic activity of grft on pbmc by flow cytometry, evaluating changes in size and morphology of cells treated with grft in comparison with cells treated with the vehicle (pbs). cells treated with 1 or 4 mm grft had flow cytometry profiles similar to the control cells (fig. 4a , b and c). in contrast treatment with lectins cona and pha at doses (0.37 mm cona and 10 mg/ml pha) that do not negatively affect cell viability, resulted in completely different flow cytometric plots, with emergence of a subpopulation composed of larger cells (higher forward scatter fsc) with perceptibly higher side scatter (ssc) values gated in region r2, as shown in fig. 4d and e. in addition, a clear decrease in cell number was observed in region r1 after treatment with cona and pha, as quantified in fig. 4f . in fig. 5 , we show that grft also does not induce cell proliferation in any of the three cultured human cervical and vaginal epithelial cells. cell division was assessed by monitoring brdu incorporation in newly synthesized dna of actively dividing cervicovaginal cell lines. treatment with 1 or 8 mm grft did not induce proliferation of any of the cell lines since brdu counts were not elevated in comparison with control cells treated with vehicle alone (pbs). in contrast pokeweed agglutinin (pkm), a well characterized mitogen, caused concentration-dependent increase in cell division, especially in the cervical cell lines end1/e6e7 and ect1/e6e7. to evaluate the effect of grft on cell surface markers of immune activation, we measured expression of the following membrane proteins: (i) cd69, a marker of activated tlymphocytes, considered an ''early'' marker of t-cell activation; (ii) cd25, the alpha chain of the il-2 receptor, upregulated on activated t-cells are highly susceptible to hiv-1 infection, and hence induction of these markers indicates an undesirable side effect of lectin treatment of pbmc. pbmc were incubated in the presence of the test compounds for 72 hours. the vehicle control was pbs, and positive controls were 10 mg/ml pha [20] , as well as 0.37 mm cona, chosen as this concentration was not cytotoxic. in pbs treated cells, 1.2% pbmc in average were double positive (cd4 + /cd25 + ) and a non significant increase of this population was observed after incubation in presence of 1 or 4 mm grft (fig. 6 , left panel and fig. s2a ). treatment with pha and cona resulted in an impressive increase in the number of cd4 + /cd25 + cells (fig. 6 , left panel and fig. s2a ). in addition, the numbers of cd4 2 /cd25 + cells were elevated when pbmc were cultured in presence of pha or cona compared to their pbs and grft counterpart (fig. 6 , left panel and data not shown). similarly, grft treatment did not affect significantly the proportion of cd4 + /cd69 + pbmc compared with pbs treatment, whereas cona and pha treatment resulted in an increase of more than 10 times in this sub-population (fig. 6 , middle panel and fig. s2b ). the numbers of cd4 2 /cd69 + were also elevated after treatment with cona and pha (fig. 6 , middle panel, and data not shown). in unstimulated cells, about 15% of cd4 positive pbmc expressed the late activation marker hla-dr and treatment with grft and cona did not affect this number in a significant fashion (fig. 6 , right panel and fig. s2c ). pha treatment of cells yielded more than twice the number of cd4 + /hla-dr + cells (33.4%) compared with unstimulated pbmc. activation of pbmc may also be reflected by the production of cytokines and chemokines. to investigate potential activating properties of grft more thoroughly, pbmc were cultured in the presence of 10 mg/ml grft (788 nm) for 72 h. in the culture supernatant, the concentrations of il-1a, il-1ra, il-2, il-4, il-5, il-6, il-7, il-8, il-9, il-10, il-12, il-13, il-15, il-17, eotaxin, fgf, g-csf, gm-csf, ifn-c, ip-10, mcp-1, mip-1a, mip-1b, pdgf-bb, rantes, tnf-a, and vegf were determined. a detailed overview of the cytokine profiles of grft-treated pbmc from multiple blood donors is given in fig. 7 . for reference, we provide previously published data where pbmc with the same donor origins were treated with cv-n at 2 mg/ml (182 nm), lower than the grft concentration tested, since higher concentrations of cv-n proved too toxic to pbmc [19] . the concentration of the separate cytokines was compared with that of the untreated pbmc and calculated as a fold increase value. in the previous studies on pbmc treatment with cv-n and mvn, considerable variability in the lectin-induced cytokine profile was observed between the different pbmc donors. therefore, the fold increase values obtained from the different donors were divided over different ranking groups (i.e. 1-3-, 3-10-, 10-100-, 100-500-, and .500-fold increase), and the number in each rank is given as a percentage of the total and indicated by a specific color (fig. 7) . confirming our data in fig. 6 that grft has very little effect on lymphocyte activation markers, we also see minimal alterations in the cytokine and chemokine release for the majority of markers, in most donor pbmc. this profile indicates that grft induces significantly less response from pbmc than has been previously reported for cv-n, mvn and cona [19] . the only chemokine induced weakly by grft in the majority of donors (75%) was mcp-1 [19] . although these studies were performed at a later date than the previously published cv-n and mvn studies, we used exactly the same donor panel, and the assays were performed in the same laboratory (d. schols), justifying comparison between the experiments. in our previously published studies, we showed that treatment of human cervical explants with a range of grft concentrations had no significant effects on expression of a panel of chemokines and cytokines [23] . in the present studies, we used cultured human endocervical, ectocervical and vaginal cell lines for more detailed analysis of potential ''off-target'' effects of grft treatment, since the cell lines are easier to procure than fresh human cervical tissues, and experiments with the cell lines are more easily reproduced without question of variability in genetic background and physiological conditions of the donor. to validate the utility of these cell lines (which are immortalized by induction of papillomavirus e6 and e7 oncogenes) for analysis of any ''off-target'' effects of grft treatment, we wished to confirm that the cell lines behaved similarly to cervical explants after treatment with grft, and cona, which induced inflammatory responses in our pbmc studies. we assessed the secretion of many key mediators of inflammatory responses, including il-1b, il-2, il-6 and il-8 by all three cervico-vaginal cell lines. elisa kits specific for these cytokines were used for their detection in end1/e6e7, ect1/ e6e7 and vk2e6e7 cell culture supernatants after 24 h of incubation in presence of the test compounds. after treatment with 8 mm grft, the supernatants showed barely detectable levels of il-1b and il-2, similar to the cells treated with pbs ( fig. 8a and b) . in contrast, all three cell lines produced impressive amounts of these cytokines upon incubation in presence of cona ( fig. 8a and b) . treatment of cervical epithelial cell lines end1/e6e7and ect1/ e6e7 with grft (8 mm), cona (2 mm) or pbs resulted in similar levels of il-6 secretion (fig. 8c ), but in vk2/e6e7, il-6 release was approximately three times higher after cona treatment compared to pbs, whereas grft did not induce the secretion of this cytokine (fig. 8c ). in the case of il-8, each of the three cell lines showed a distinct pattern of secretion of this cytokine, although no induction was observed after treatment with 8 mm grft: vk2/ e6e7 showed barely detectable levels of il-8 regardless of the treatment; il-8 levels in ect1/e6e7 were relatively high after pbs and grft treatment and these amounts were about ten times reduced after treatment with cona; grft treatment resulted in a slight decrease of il-8 in end1/e6e7 cell culture supernatants whereas the concentrations of this cytokine were significantly increased following treatment with cona (fig. 8d ). in addition, grft treatment did not alter the production of il-10, ip-10, mip-1b and tgf-a in the cervico-vaginal cells studied (data not shown). these data indicated that all 3 cell lines behaved as predicted in response to grft and cona treatment. since the ectocervical epithelium is an important point of mucosal transmission of hiv-1, we focused on the ect1/e6e7 cell line to derive a more comprehensive assessment of the effect of grft treatment on cytokine production by the cultured ectocervical cells. a multiplexed luminex assay of the ect1/e6e7 cell line treated with grft confirmed that the ect1 cells behaved very similarly to human cervical explants (data not shown), and validated our use of these cells in our microarray studies. gene expression in ect1/e6e7 cells in response to treatment with grft was compared with vehicle (pbs), grft lec-, cv-n and cona treatments (all qualified with endotoxin levels less than 0.05 eu per milligram). this was studied using a microarray experiment in which the whole human genome was represented by the 41,000 known genes and transcripts [27] . the microarray data were deposited in the gene expression omnibus database, under accession number gse28584. the heat map shown in fig. 9a indicates that cells exposed for 24 hours to grft lec-(1 and 8 mm), and low concentrations of grft (0.1 mm ) and cv-n (0.05 mm) showed comparable gene expression profiles to those that were incubated in presence of pbs alone. treatment of ect1/ e6e7 with 1 mm grft resulted in minor changes in the expression profile while 4 mm grft appeared to alter the expression of many genes but to a much lesser extent compared to cv-n (0.5 and 4 mm) and cona at 1 mm (fig. 9) . thus, the microarray studies confirm the data showing that grft has substantially lower ''off-target'' effect in comparison with cv-n and cona, although there are clearly several genes that are regulated by the carbohydrate binding activity of grft (compare with grft lec-). as predicted from the heat map (fig. 9a) , no gene was identified as regulated by 0.1 mm grft, even when the cutoff was brought to 1.0. however, a first analysis using benjamini-hochberg low stringency correction with a cutoff of 2.0 yielded 107 and 35 entries as differentially expressed in samples treated with 1 and 4 mm grft, respectively (fig. 9b) . treatment with cv-n (0.5 and 4 mm) or cona (1 mm) resulted in regulation of impressive numbers of human genes (fig. 9b) . we then employed stricter criteria by keeping only the positive entries that showed grft concentration dependent gene expression. this yielded 2 and 32 genes for 1 and 4 mm grft, respectively. the entries which showed an increased gene expression after treatment with 1 mm grft included a gene annotated as immunoglobulin-like and fibronectin type iii domain containing 1 (igfn1). the entries that were found to be affected by 4 mm grft are summarized in table s2 . among the 26 genes mapped by the ingenuity database, there was overrepresentation of genes in the following canonical pathways: nrf2-mediated oxidative stress response (maf, hmox1, sod2), phospholipid degradation, glycerophospholipid metabolism and endothelin-1 signaling (hmox1, wisp2), camp mediated signaling (calml5, pkib) and acute phase response signaling (hmox1, sod2). furthermore using the ingenuity software we identified five toxicological functions with an overrepresentation of genes including liver hyperbilirubinemia and steatohepatitis, cardiac arteriopathy, renal and liver necrosis (data not shown). none of these is relevant to mucosal treatment with grft. we used quantitative rt-pcr (q-rt-pcr) to validate the microarray results. expression of 14 genes was studied using 18s rna and b-actin mrna as controls. with the exception of mycn, all genes studied showed comparable expression levels in both experimental systems including microarrays and q-pcr (table s3 ). natural product lectins have received considerable attention as potential antiviral drugs, particularly in the context of prevention of hiv-1 transmission via mucosal surfaces (reviewed recently in [8] ). although the volume of literature supporting their use as antivirals in vivo is dwarfed by a comprehensive set of data showing potent in vitro antiviral activity, there are reports of impressive in vivo efficacy of cv-n in animal models of hiv-1 prevention [17, 18] , influenza prevention and treatment [28] , and ebola virus prophylaxis and treatment [29] , and of grft in prevention of sars-cov infection [30] . despite the myriad of potential prophylactic and therapeutic applications of antiviral lectins, enthusiasm for their development as pharmaceuticals is tempered by a long history of research into natural product lectins, which characterizes many members of this broad class as erythrocyte agglutinins, lymphocyte mitogens, and potentially lethal toxins [10, 11] . the pharmacological basis of natural product lectin toxicity is generally poorly understood, but at a basic mechanistic level is thought to reside in the lectins' capacity for multivalency of binding to cell surface glycans, resulting in cell agglutination and/ or cross-linking of cell surface receptor molecules with consequent activation of signaling pathways. why different lectins that bind identical glycan moieties have quite distinct biological effects in vitro remains a paradox that is well illustrated by our data which, show very different in vitro activity profiles of four different oligomannose-binding lectins: grft, cv-n, mvn [19] and cona. characterization of grft is the primary focus of our studies, but understanding the molecular pharmacology and toxicology of this potent antiviral lectin is informed by comparison to cv-n, for which a rich set of in vitro and in vivo data is available. both molecules have comparable hiv-1 neutralization activities, with mean ic 80 values against clade c viruses of 42.764.4 nm for grft and 77.0618.2 for cv-n [15] . both proteins bind oligomannose glycans, with grft targeting terminal mannose residues found on man 5-9 -glcnac 2 [13] and cv-n specific for the mana1r2man linkages found on man 6-9 -glcnac 2 [16, 31, 32] . they thus share overlapping binding specificities, and should target identical cell surface and viral glycans. if anything, grft would be predicted to bind to a larger number of glycan targets than cv-n since it can bind pentamannose structures that lack the a1r2 mannose linkages that cv-n targets [13] . in this context, it is surprising that cv-n appears to bind more promiscuously than grft throughout the cervical epithelium and sub-epithelial stroma (fig. 1a-d, and fig. s1 ). given that grft lec-(the carbohydrate-binding deficient form of grft) hardly bound the epithelial sections or cultured cervical and vaginal epithelial cells (fig. 1) , and grft binding to cultured epithelial cells was blocked by mannan, we conclude that grft's binding activity to the cell surface is exclusively via its carbohydrate binding activity. it is very interesting to note that cv-n binding to the surface of pbmc is not entirely eliminated by blocking its carbohydrate binding sites with mannan (fig. 1k ). this implies that its capacity to bind and induce signaling in cells may not reside entirely in its lectin activities, and supports prior studies with cv-n [21] . another important result showed that grft seems to bind somewhat selectively to terminally differentiated keratinocytes on the epithelial surface, presumably reflecting presence of optimal glycoprotein binding targets on the surface of those cells only in the intact epithelium ( fig. 1c and d; and fig. s1 ). the data presented in fig. 2 strongly support grft's candidacy as a microbicide and antiviral, since they show that grft retains antiviral activity even when complexed to the surface of pbmc. this contrasts with many other lectins or compounds we have tested in this assay. the most plausible mechanism that might explain grft's retention of hiv-1 entry inhibition activity is that fewer than all six carbohydrate binding sites are occupied when the lectin is docked on cell surface glycoprotein/s, leaving sites available for binding to the viral envelope glycoprotein. the crystal structure of grft suggests that the three glycan-binding pockets of each grft monomer are on opposite ends of the double prism homodimer [13] , suggesting that only one monomer of the homodimer is engaged in binding the cell surface, leaving the ''free'' monomer competent to bind and cross-link two oligomannose structures on the surface of hiv-1. since the pbmc were washed extensively 24 hours after treatment and prior to addition of the hiv-1 inoculum, this suggests that cell surface-bound grft irreversibly inactivated the inoculum, with no evidence of breakthrough infection at 9 days post infection. this duration of antiviral activity is unprecedented. conventionally, antiviral potencies of virus-targeted entry inhibitor drugs are measured without washing the cells prior to addition of viruses. in traditional antiviral assays the ic 50 of grft against hiv-1 bal is 0.2 nm in pbmc, and 0.1 nm against hiv-1 bal in monocytes/macrophages (data not shown). remarkably, the ic 50 for grft in the washed pbmc assay (fig. 2) , when the test agent is applied 24 hours prior to cell washing and infection, is 0.78 nm, showing quite exceptional antiviral activity of grft. in the same assay we show that activity of ccr5 antagonist maraviroc persists for 24 hrs, although a high concentrations, but grft's activity persists substantially longer, a property that may facilitate non-coitally linked administration of microbicides containing grft as an active ingredient. the cardinal rule in development of an anti-hiv-1 microbicide must be ''first, do no harm''. randomized controlled preclinical and clinical studies with detergent-based microbicides such as nonoxynol-9 showed a trend towards evidence of harm, with increased incidence of not only hiv-1 infection, but also hsv-2 and hpv seen in the experimental arms [33, 34, 35, 36, 37] . the microbicides field therefore requires stringent and extensive in vitro and in vivo safety studies before human trials initiate. initial studies showed that grft was not cytotoxic; had no mitogenic activity; did not induce secretion of chemokine and cytokine-mediators of inflammation in treated cervical explants; and showed a good safety profile in the rabbit vaginal irritation test [23, 24, 25] . the first tissues that grft would be exposed to in the human vagina are keratinocytes in the outer surface of the vaginal and cervical epithelia. grft would also encounter hiv-1 target cells such as dendritic cells and macrophages resident in the epithelium and submucosal stroma as well as the primary hiv-1 target cells, mucosal cd4+ t-cells. in this work we expanded the safety studies to include analyses of off-target effects derived from grft binding cell surface oligomannose glycans on human pbmc as well as characterized ectocervical, endocervical and vaginal keratinocyte cell lines end1/e6e7, ect1/e6e7 and vk2e6e7, which were originally established from normal human endocervical, ectocervical, and vaginal epithelia, respectively, and immortalized by expression of human papillomavirus 16/e6e7 [38] . the immortalized cell lines were shown to have close resemblance to those of their respective tissues of origin and primary cultures in their morphological and immunocytochemical characteristics and therefore proposed for studies dealing with testing pharmacological agents for intravaginal application [38] . first we evaluated the effects of grft on cell viability and we showed that up to 84 mm grft did not decrease cell viability in the cervical cell lines end1/e6e7and ect1/e6e7 whereas vk2/ e6e7 was somewhat sensitive to grft at high concentrations of 8 mm and above. these results are consistent with the pioneering work of mori and coworkers in which no significant cellular toxicity was found when a variety of human cell types were treated with grft at concentrations of up to 0.783 mm [24] . since many lectins have been reported to be mitogenic [39, 40] , we examined if grft would have an effect on the cervico-vaginal cell proliferation. using a colorimetric assay for detection of brdu in newly synthesized dna, we showed that high concentrations of grft did not increase the proliferation of either end1/e6e7, ect1/e6e7 or vk2/e6e7. we then evaluated the concentrations of a panel of cytokines and chemokines in cervico-vaginal cell culture supernatants using elisa. we found that high concentrations of grft had little to no effect on the secretion of cytokines/chemokines studied including il-1b, il-2, il-6, il-8, il-10, ip-10, mip-1b and tgfa in all the three cervico-vaginal cell lines including end1/e6e7, ect1/e6e7 and vk2/e6e7. therefore, our results, taken together suggest that grft at these concentrations does not alter the secretion of the immune system mediators by cervico-vaginal cells in a significant fashion. using the ect1/e6e7 cell model, we tested grft in order to evaluate its effects on gene expression after over night incubation. to our knowledge, this is the first study evaluating the effect of an anti-hiv lectin on gene expression using human genome microarrays. our data revealed that exposure to grft at 0.1 mm did not affect gene expression whereas treatment of ect1/ e6e7 with 1 mm grft resulted in minor alteration of the gene expression profile, showing only 2 genes that were considered as significantly upregulated (cutoff of 2.0). of note our working concentrations were very high. for instance, 1 mm corresponds to about 1,600 times and more than 23,000 times the ec 50 found for the most resistant and the most sensitive hiv-1 strain, respectively. at higher concentrations of 4 mm grft, 32 genes were considered as differentially expressed (cutoff = 2.0). the fold changes found were less or equal to 3.2. q-rt-pcr evaluation of the expression of selected genes validated the microarray data presented here. it is unclear what is the biological relevance and significance of this level of regulation. using the same cell line (ect1/e6e7) sharkey et al. classified a gene as differentially expressed when the fold change was more than 2.0 and found a total of 444 probe sets that fell in this category after treating the cells 12 h with 10% human seminal plasma [41] . this is in sharp contrast to treatment with 4 mm grft, a level at least 1,000 fold greater than the average antiviral ec 50 , which regulated expression of only 32 genes. in summary, our data provide further evidence that grft, an exceptionally potent antiviral lectin, has very minor effects on the molecular physiology of human cells. at this point, the molecular basis for the distinct biological activities of different antiviral lectins is uncharacterized, we propose that the specific spatial arrangement of the carbohydrate binding sites may determine the nature and extent of cross-linking of cell surface glycoproteins. grft clearly has superior binding and cross-linking activity with the hiv-1 envelope glycoprotein, which displays dense clusters of oligomannose nlg, but does not induce off-target cellular signaling to the extent that other lectins do. we believe this provides further data in strong support of focused clinical development of hiv-1 microbicides containing grft as an active ingredient. recombinant grft was produced in nicotiana benthamiana plants. recombinant cv-n was produced in escherichia coli. methods for expression and purification of both products have been described previously [23, 42] . a synthetic cdna encoding a lectin activity-deficient mutant of grft, termed grft lec-, was designed with a conservative amino acid substitution of aspartic acid to asparagine in each of the 3 carbohydrate binding pockets identified in the primary amino acid sequence and crystal structures of grft [24, 26] . grft lecwas expressed in n. benthamiana and purified exactly as described for grft [23] . proteins were purified to .99% purity, and formulated in phosphate buffered saline (pbs), ph 7.4 at 10 mg/ml protein concentration. endotoxin was removed from grft, grft lecand cv-n protein samples using detoxi-gel endotoxin-removing gel gravity flow columns (thermo scientific). endotoxin levels were measured using the toxinsensor tm chromogenic lal endotoxin assay kit from genscript (piscataway, nj). only products with final endotoxin readings less than 0.05 endotoxin units (eu) per milligram were used in the in vitro studies, and all dilutions were performed in endotoxin-free buffers. grft, grft lecand cv-n were fluorescently-labeled with amine-reactive alexa fluor 488 carboxylic acid, succinimidyl ester using a kit from molecular probes/invitrogen, according to the manufacturer's specifications. control lectins concanavalin a (cona), phytohemagglutinin a (pha) and pokeweed agglutinin (pkm) were purchased from sigma. lectin activity measurements using hiv-1 gp120-binding elisa immobilized hiv-1 gp120 (protein sciences corporation) was used to measure the lectin activity of purified grft, cv-n and fluorescently labeled conjugates thereof, and to confirm that grft leclacked gp120 binding activity. nunc maxisorp elisa plates were coated overnight with 1 mg/ml gp120 (strain iiib, protein sciences) diluted in pbs. the wells were blocked with 5% (w/v) non-fat dry milk in pbs+0.05% tween (pbs-t; immunowash, bio-rad) and washed before addition of serial dilutions of lectin analyte (grft, grft lec-, cv-n or alexa-fluor 488-labelled conjugates thereof) diluted in 16 pbs for 1 h. after three washes with pbs-t, a primary polyclonal antiserum (rabbit anti-grft or cv-n or alexa-fluor 488 (invitrogen), as appropriate) diluted 1:10,000 in pbs was added for 1 h at room temperature. the wells were again washed before goat anti-rabbit igg-hrp (southern biotech) was added at a 1:10,000 dilution. colorimetric values reflecting hrp activity were derived using kpl sureblue tmb microwell peroxidase substrate, with the reaction stopped by addition of 1 n h 2 so 4 . the plates were read at 450 nm on a biotek synergy ht reader with data collected using gemini software. we confirmed that the labeled products retained lectin activities comparable to the unlabeled product by gp120-binding elisa. we used elisa with anti-alexafluor 488 detection to measure the total amount of label conjugated to grft, grft lecand cv-n, which displayed quantitatively similar labeling efficiency. end1/e6e7, ect1/e6e7 and vk2/e6e7 are well characterized immortalized cell lines derived from normal human endocervical, ectocervical and vaginal epithelia, respectively [38] . all 3 cell lines were purchased from the american type culture collection (atcc, rockville, md). the cervico-vaginal cell lines were grown as previously described [38] in keratinocyte serum-free medium (ksfm) supplemented with recombinant human epidermal growth factor (0.1 ng/ml), bovine pituitary extract (50 mg/ml), calcium chloride (0.4 mm) and an antibiotic cocktail composed of penicillin and streptomycin at final concentrations of 100 u/ml and 100 mg/ml, respectively. reagents were obtained from invitrogen (sandiego ca, or from sigma chemical company). cryopreserved human pbmc used in assays of inflammatory cytokines and chemokines were purchased from seracare life sciences inc. (milford, ma) and were immediately cultured for the experiments in rpmi 1640 supplemented with 10% fetal bovine serum (fbs) and the penicillin-streptomycin antibiotic cocktail (to 100 u/ml and 100 mg/ml final concentrations, respectively). slides with paraffin embedded human cervical tissue sections from a healthy 21 year old female (us biomax, inc.) were deparaffinized and rehydrated. alexa-fluor 488-labelled proteins of interest were added to the slides and incubated overnight at 4uc in a humidity chamber. the slides were rinsed with pbs twice for 10 minutes and coverslipped using vectashield mounting media with dapi (vector laboratories, burlingame, ca). for light microscopy, tissue sections were stained with hematoxylin and eosin by standard methods. ect1/e6e7, end1/e6e7 and vk2/ e6e7 cultured cells were seeded onto eight-well lab-tek chamber slides (nalgene nunc) in duplicate at 10,000 cells per well and allowed to incubate at 37uc with 5% co 2 . after eight hours the fluorescently labeled proteins of interest were added to the wells and incubated overnight at 37uc with 5% co 2 . the slides were washed twice for 10 minutes with pbs and coverslipped using vectashield mounting media with dapi (vector laboratories, burlingame, ca). slides were visualized using the axio observer z1 microscope with apotome assembly (carl zeiss, thornwood, ny). human pbmc (seracare life sciences, md) were thawed and seeded onto 48-well culture plates (celltreat, ma) at 2.5610 5 cells per well. three dilutions of each alexa-fluor 488-labeled protein were added to the cells for overnight incubation at 37uc with 5% co 2 . samples were also prepared with 5 and 10 mg of mannan (sigma, st. louis mo) at each protein dilution. all samples were analyzed in duplicate. following incubation, cells were briefly trypsinized (tryple express, gibco) and placed in 5 ml polystyrene tubes (bd falcon, ma). cells were washed twice with pbs and analyzed using the bd facsaria (bd biosciences, nj) flow cytometer. freshly isolated pbmc were cultured in the presence of grft, cv-n and maraviroc for 24 hrs. then the cells were collected, washed in culture medium, suspended in rpmi medium with 2 ng/ml il-2 and seeded in a 48-well flat bottom plate (5610 5 cells in 450 ml) and 50 ml of the ccr5-tropic clade b hiv-1 bal stock was added at 100 tcid 50 . the supernatant of each sample was collected after 9 days and viral replication measured by a specific p24 ag elisa (perkin elmer, zaventem, belgium). viability of cervico-vaginal cell lines was measured using a colorimetric mtt [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay kit from biotium inc. (hayward, ca) following the manufacturer's instructions. briefly, 10 4 cells per well were seeded (96 well plates), the test reagents added and the cultures incubated for three days in a humid environment with 5% co 2 at 37uc. afterwards, 10 ml of mtt solution were added to each well followed by 4 hours incubation at 37uc. then the medium was gently removed and the insoluble purple formazan product dissolved in dmso to yield a colored solution which absorbance was read at 570 nm with a background at 630 nm. proliferation of cervicovaginal cell lines was evaluated by bromodeoxyuridine (brdu, a thymidine analog) incorporation in newly synthesized dna using a cell proliferation elisa kit from roche, according to the manufacturer's instructions. for human pbmc, cells were treated with grft for three days and analyzed by flow cytometry for any changes in size and/or morphology using forward scatter (fsc) and side scatter (ssc) with a facscalibur (bd, san jose, ca) counting 10,000 events per sample. data were acquired and analyzed using cellquest pro from bd. cona (0.37 mm), pha (10 mg/ml) and pbs were used as controls. three day old pbmc were analyzed flow-cytometrically after dual fluorescent staining with anti-mouse antibodies purchased from bd pharmingen (san diego, ca). briefly, cell cultures were transferred from plates to a 5 ml round bottom tubes and washed with pbs containing 5% inactivated fbs (washing solution). after 10 min blocking with purified rat anti-mouse cd16/cd32 (mouse bd fc block), cells were incubated in dark with fitcconjugated anti-cd4 mab in combination with pe-conjugated anti-cd25, anti-cd69 or anti hla-dr mab for 30 min on ice. finally pbmc were washed and analyzed with a facscalibur (bd, san jose, ca), counting 10 000 events per sample. data were acquired and analyzed using cellquest pro from bd. cona (0.37 mm) and pha (10 mg/ml), and pbs were used as positive and negative controls, respectively. multiplex immunoassays were carried out on a bio-plex instrument (biorad) using a milliplex human cytokine/chemo-kine 42-plex luminex bead-based assay (millipore), following the manufacturer's instructions. each luminex immunoassay was repeated three times. the kit allows simultaneous quantification of human-epidermal growth factor (egf), eotaxin, fibroblast growth factor-2 (fgf-2), fms-like tyrosine-kinase 3 ligand (flt-3 ligand), fractalkine, granulocyte colony-stimulating factor (g-csf), granulocyte-macrophage-csf (gm-csf), gro, interferon-a2 (ifn-a2) , ifn-c, interleukin-10 (il-10), il-12 (p40), il-12 (p70), il-13, il-15, il-17, il-1a, il-1b, il-2, il-3, il-4, il-5, il-6, il-7, il-8, il-9, il-1 receptor antagonist (il-1ra), interferoninducible protein-10 (ip-10), monocyte chemoattractant protein-1 (mcp-1), mcp-3, macrophage-derived chemokine (mdc), macrophage inflammatory protein-1a (mip-a), mip-1b, plateletderived growth factor-aa (pdgf-aa), pdgf-ab/bb, regulated upon activation normal t-cell expressed and secreted (rantes), soluble cd40 ligand (scd40l), soluble il-2ra (sil-2ra), transforming growth factor a (tgf-a), tumor necrosis factor-a (tnf-a), tnf-b and vascular endothelial growth factor (vegf). supernatants collected from ect1/e6e7 after 24, 48 and 72 hours of culture in presence of test compounds were studied in the multiplex-immuno assays and data were analyzed using the luminex 6ponent r software. individual elisa experiments were performed on end1/e6e7, ect1/e6e7 and vk2e6e7 cell culture supernatants collected after 24 hours of incubation in presence of grft or controls. elisa ready-set-go! kits designed for accurate and precise measurement of human il-1b, il-2, il-6 and il-10 were purchased from ebioscience, inc. quantikineh elisa kits from r&d systems, inc. were used for the detection of il-8, ip-10 and tgf-a. mip-1b was quantified using an elisa kit from cellsciences. all elisas were performed according to the manufacturer's specifications. bio-plex cytokine assay in human pbmc supernatants pbmc from multiple blood donors [19] were cultured for 72 h in presence of 10 mg/ml grft (788 nm). in the culture supernatants, the concentrations of il-1a, il-1ra, il-2, il-4, ilgene expression analysis in ect1/e6e7 cells cultured ect1/e6e7 cells were incubated with dilutions of grft, grft lec-, cv-n, cona, or vehicle only (pbs, ph 7.4) for 16 hours. the cells were lysed using a qiagen qiashredder, and total rna was extracted using a qiagen rneasy mini kit. total rna was quantified spectrophotometrically and the sample quality was checked on an agilent 2100 bioanalyzer (agilent technologies, wilmington, de). 200 ng rna were used to generate cyanine 3 (cy3) crna with the aid of low rna input linear amplification kit, one-color (agilent technologies, wilmington, de) according to the manufacturer's instructions. 1.65 ug of each labeled crna sample were fragmented at 60uc for 30 min using an agilent gene expression hybridization kit (agilent technologies, wilmington, de) followed by hybridization to a whole human genome agilent oligonucleotide slide containing four high-definition 44 k microarray (agilent technologies, wilmington, de) at 65uc for 17 hours. after hybridization, the slides were washed with agilent gene expression wash buffers (agilent technologies, wilmington, de) and scanned using an agilent g2565ba microarray scanner system (agilent technolo-gies, wilmington, de). then, one-color microarray images were extracted with the feature extraction software v 9.5.1 (agilent technologies, wilmington, de) and the raw data imported into genespring gx 10 software (agilent technologies, wilmington, de) for normalization and further analysis to yield a list of differentially expressed genes. benjammini and hocheberg correction controlling the false discovery rate fdr [43, 44, 45] was used and we considered only a fold change of at least 2.0 compared to the pbs treated samples to represent a meaningfully altered gene expression [41, 46, 47] . the raw microarray data were uploaded into the gene expression ominbus (geo) database, a miame-compliant database as detailed on the mged society website http://www.mged.org/workgroups/miame.html. the geo accession number is gse28584. further analysis of the regulated genes was carried out using the ingenuity knowledge database (ingenuity systems inc., redwood city, ca) yielding putative toxicological functions, and canonical pathways. quantitative rt-pcr was carried out in order to validate the microarray results. first strand cdna was reverse transcribed from 250 ng rna employing a high capacity rna-to-cdna kit (applied biosystems) following the manufacturer's instructions. optimal amounts of template cdna were added to a reaction mixture containing 10 ml of 26taqmanh gene expression master mix (applied biosystems) and water to 20 ml and this mixture was used to set the pcr reactions in taqmanh array standard 96 well plates (applied biosystems). these plates contain pre-spotted individual taqmanh gene expression assays for detection of human cysteine-aspartic acid protease (caspase14, casp14), beta defensin 103a (defb103a), immunoglobulin-like and fibronectin type iii domain containing 1 (igfn1), interleukin-1b (il-1b), il-2, il-6, il-8, il-10, il-33, ip-10, v-myc myelocytomatosis viral related oncogene (mycn), thyroglobulin (tg ), tgfa, tripartite motif-containing 63 (trim63) as well as the house keeping genes 18 s and beta actin (actb). individual probes used in these experiments are described in table s1 . pcr amplification was performed in an 7900ht fast real-time pcr system (appliedbiosystems) as follows: initial activation step (95uc, 20 min), 40 cycles (95uc, 1 min) and 20 min at 60uc. sds 2.3 or rq manager 1.2 software from applied biosystems was used to evaluate the cycle threshold (ct) for each reaction, which is the cycle number at which 50% maximal amplicon synthesis is achieved. ratios were derived as proposed elsewhere [48] . group means and standard deviations were derived from the values obtained in three individual replicates. statistical signifi-cance was analyzed by a one-way analysis of variance (anova) and student's t-test unless otherwise stated, using graphpad software (san diego, ca). differences were considered statistically significant if p,0.05. for microarray data, the statistical analysis was carried out as described in the gene expression analysis methods above. figure s1 analysis of binding specificity of grft in comparison with grft lecand cv-n. in the first column we depict the identical fluorescence micrographs to fig. 1 antibody neutralization and escape by hiv-1 hiv-1 gp120 mannoses induce immunosuppressive responses from dendritic cells envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens glycosylation: heterogeneity and the 3d structure of proteins exploiting the defensive sugars of hiv-1 for drug and vaccine design ) variability, heritability and environmental determinants of human plasma nglycome profile of native n-linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time-of-flight mass spectrometry potential of carbohydrate-binding agents as therapeutics against enveloped viruses carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp120: a new therapeutic concept to hit the achilles heel of hiv history of lectins: from hemagglutinins to biological recognition molecules anti-hiv activity of defective cyanovirin-n mutants is restored by dimerization monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-hiv activity actinohivin: specific amino acid residues essential for anti-hiv activity mannose-rich glycosylation patterns on hiv-1 subtype c gp120 and sensitivity to the lectins, griffithsin, cyanovirin-n and scytovirin multivalent interactions with gp120 are required for the anti-hiv activity of cyanovirin cyanovirin-n inhibits aids virus infections in vaginal transmission models cyanovirin-n gel as a topical microbicide prevents rectal transmission of shiv89.6p in macaques microvirin, a novel alpha(1,2)-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv-1 activity comparable with that of cyanovirin-n but a much higher safety profile safety concerns for the potential use of cyanovirin-n as a microbicidal anti-hiv agent mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type 1 strains with n-glycan deletions in their gp120 envelopes cyanovirin-n potently inhibits human immunodeficiency virus type 1 infection in cellular and cervical explant models scaleable manufacture of hiv-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding igfbp-3 hypermethylation-derived deficiency mediates cisplatin resistance in non-small-cell lung cancer treatment of influenza a (h1n1) virus infections in mice and ferrets with cyanovirin-n cyanovirin-n binds to the viral surface glycoprotein, gp1,2 and inhibits infectivity of ebola virus broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae dissecting carbohydrate-cyanovirin-n binding by structure-guided mutagenesis: functional implications for viral entry inhibition crystal structure of cyanovirin-n, a potent hiv-inactivating protein, shows unexpected domain swapping genital transmission of hpv in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan in vitro preclinical testing of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide: a retrospective analysis of results from five laboratories safety study of nonoxynol-9 as a vaginal microbicide: evidence of adverse effects a comprehensive murine model to evaluate topical vaginal microbicides: mucosal inflammation and susceptibility to genital herpes as surrogate markers of safety the impact of the use of col-1492, a nonoxynol-9 vaginal gel, on the presence of cervical human papillomavirus in female sex workers generation of papillomavirus-immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue-specific differentiation proteins inhibition of hiv entry by carbohydrate-binding proteins molecular cloning of the mitogenic mannose/maltose-specific rhizome lectin from calystegia sepium seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells recombinant production of cyanovirin-n, a potent human immunodeficiency virus-inactivating protein derived from a cultured cyanobacterium controlling the false discovery rate in behavior genetics research identifying differentially expressed genes using false discovery rate controlling procedures biological microarray interpretation: the rules of engagement a new strategy to identify and annotate human rpe-specific gene expression gene expression profiling in autoimmune noninfectious uveitis disease analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method key: cord-292546-un0blb3w authors: dandachi, dima; geiger, grant; montgomery, mary w; karmen-tuohy, savannah; golzy, mojgan; antar, annukka a r; llibre, josep m; camazine, maraya; díaz-de santiago, alberto; carlucci, philip m; zacharioudakis, ioannis m; rahimian, joseph; wanjalla, celestine n; slim, jihad; arinze, folasade; kratz, ann marie porreca; jones, joyce l; patel, shital m; kitchell, ellen; francis, adero; ray, manoj; koren, david e; baddley, john w; hill, brannon; sax, paul e; chow, jeremy title: characteristics, comorbidities, and outcomes in a multicenter registry of patients with hiv and coronavirus disease-19 date: 2020-09-09 journal: clin infect dis doi: 10.1093/cid/ciaa1339 sha: doc_id: 292546 cord_uid: un0blb3w background: people with hiv (pwh) may have numerous risk factors for acquiring coronavirus disease-19 (covid-19) and developing severe outcomes, but current data are conflicting. methods: healthcare providers enrolled consecutively by non-random sampling pwh with lab-confirmed covid-19, diagnosed at their facilities between april 1st and july 1st, 2020. de-identified data were entered into an electronic research electronic data capture (redcap). the primary endpoint was severe outcome, defined as a composite endpoint of intensive care unit (icu) admission, mechanical ventilation, or death. the secondary outcome was the need for hospitalization. results: 286 patients were included; the mean age was 51.4 years (sd, 14.4), 25.9% were female, and 75.4% were african-american or hispanic. most patients (94.3%) were on antiretroviral therapy (art), 88.7% had hiv virologic suppression, and 80.8% had comorbidities. within 30 days of positive sars-cov-2 testing, 164 (57.3%) patients were hospitalized, and 47 (16.5%) required icu admission. mortality rates were 9.4% (27/286) overall, 16.5% (27/164) among those hospitalized, and 51.5% (24/47) among those admitted to an icu. the primary composite endpoint occurred in 17.5% (50/286) of all patients and 30.5% (50/164) of hospitalized patients. older age, chronic lung disease, and hypertension were associated with severe outcomes. a lower cd4 count (<200 cells/mm³) was associated with the primary and secondary endpoints. there was no association between the antiretroviral regimen or lack of viral suppression and predefined outcomes. conclusion: severe clinical outcomes occurred commonly in pwh and covid-19. the risk for poor outcomes was higher in those with comorbidities and lower cd4 cell counts, despite hiv viral suppression. patients with severe coronavirus disease-19 requiring hospital admission are more likely to be older, male, and have underlying comorbidities. [1] [2] [3] additionally, immunocompromising conditions such as malignancy and solid organ transplantation may increase patients' risk for severe covid-19 and death. [4] [5] [6] [7] [8] data are conflicting whether people with hiv (pwh) are also at increased risk. pwh may have numerous factors that could increase their risk of exposure to and acquisition of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). first, pwh are aging and have high rates of smoking, chronic cardiovascular and lung disease, and obesity. 9 in addition, racial and ethnic minorities are disproportionately affected by hiv disease and covid-19. ongoing research aims to determine the impact of structural racism in covid-19 outcomes. while the causes of health disparities are multifaceted, lack of access to healthcare, differences in socioeconomic, and prevalence of chronic diseases are potential contributors. 10, 11 several case series have described clinical characteristics of pwh and covid19 . many have been either limited to single-center studies, hospitalized patients, or included patients with suspected but not confirmed covid-19. most were also limited by small numbers of patients or lacked important hiv-specific variables such as cd4 cell counts, antiretroviral therapy (art), and plasma hiv viral load. [12] [13] [14] [15] some of these studies reported that pwh with covid-19 had similar clinical characteristics and comparable risk of severe disease to the general population. [16] [17] [18] [19] [20] [21] however, other studies found higher rates of sars-cov-2 infection and worse outcomes among pwh. [22] [23] [24] because current data are conflicting and limited, further investigation in hiv and covid-19 is warranted. we aim to describe the clinical characteristics and outcomes of covid-19 in pwh and to characterize pwh at the highest risk for severe covid-19-associated outcomes through an extensive multicenter registry. a c c e p t e d m a n u s c r i p t the covid-19 in pwh registry was sponsored by the university of missouri, columbia. the study was reviewed by the university of missouri institutional review board and considered to be exempt. anonymized patient data were collected without the need for informed consent. this multicenter registry was for pwh and covid-19, who received care between april 1 st and july in both inpatient and outpatient settings were eligible for study inclusion. healthcare providers collected study data by chart review of pwh and covid-19 diagnosed at their facilities and entered anonymous information into a secure electronic research electronic data capture (redcap). 25, 26 patients were enrolled consecutively by non-random sampling. study variables included patient demographics, hiv-associated variables, underlying medical problems, covid-19 clinical presentation as reported by patients, laboratory values, treatment, and clinical outcomes. providers certified that the information submitted was accurate to the best of their knowledge. two reviewers cross-validated the data for duplicity by age, gender, race, location, and hiv-1 rna (viral load). a c c e p t e d m a n u s c r i p t laboratory-confirmed covid-19 was defined as positive reverse-transcriptase-polymerase-chainreaction (rt-pcr) in respiratory samples or serum sars-cov-2 specific igg/igm. the us geographical region of residence was based on the cdc's national hiv surveillance system region distribution. 27 chronic lung disease included asthma and copd. cardiovascular disease included coronary artery disease and congestive heart failure. chronic liver disease included cirrhosis, chronic hepatitis b, and chronic untreated hepatitis c. active malignancy excluded non-melanoma skin cancer. we defined obesity as body mass index (bmi) of ≥ 30. 28 we defined virologic suppression as hiv-1 rna (viral load) < 200 copies/ml. 29 we collected the most recent hiv viral load measured before or at the time of covid-19 presentation. art categorizations were mutually exclusive. the primary endpoint was severe clinical outcome, defined as a composite endpoint of icu admission, use of mechanical ventilation, or death. 1 outcomes for survival analyses were time from positive sars-cov-2 test to icu admission and death. the secondary outcome of interest was hospitalization. we used descriptive statistics to summarize patient data. for categorical variables, we used frequency and calculated proportions using the number of patients with data available as the denominator. for continuous variables, we used the mean with standard deviation (sd) or the median with interquartile range (iqr). we analyzed the association between baseline variables with the defined outcomes by univariate analysis using the chi-square test, fisher's exact test, or t-tests as indicated. a multivariable logistic regression model was used to assess the association between each outcome of interest, severe outcome a c c e p t e d m a n u s c r i p t and hospitalization, and independent variables. to obtain the parsimonious model for each multivariable analysis, we identified the significant independent variables using the backward selection method. variables with a p-value ≤ 0.2 were included in the model in addition to clinically relevant variables. to adjust for within-region differences, we fitted a generalized estimating equation (gee) logistic regression model, assuming an exchangeable correlation structure and regions as clusters, for each outcome. to test whether there was a differential effect of cd4 count on icu-free survival and overall survival, we used the kaplan-meier method to analyze survival outcomes and log-rank statistics to compare the survival distribution of the cd4 groups (< 200, 200 -500, > 500 cells/mm³) with respect to time from positive sars-cov-2 test to icu admission and death. in a post-hoc sub-analysis, we included patients from the us to examine the clinical presentation and study outcomes, excluding international locations. user-defined missing values were treated as missing and not imputed. all tests were two-sided, with a level of significance defined as ≤ 0.05. sas ® software was used for statistical analysis of data. between april 1 st and july 1 st , 2020, we identified 286 unique pwh and laboratory-confirmed covid-19. thirty-six institutions from 21 states and three international locations contributed to the data; five duplicate cases were removed. a c c e p t e d m a n u s c r i p t art regimen was an integrase inhibitor with two nucleoside reverse transcriptase inhibitors (61.3%; 171/279). hypertension (46.5%), obesity (32.3%), and diabetes (21.3%) were the most common underlying medical problems. when stratified by hospitalization status, older age, lower cd4 counts, number of years living with hiv, not being on art or virally suppressed, and high comorbidity burden were associated with higher hospitalization rates (table 1) . a sars-cov-2 rt-pcr test was used to diagnose all but one patient, who was diagnosed based on serologic testing. the most frequently reported symptoms within 72 hours of diagnostic testing were cough (76.2%; 205/269), fever (70.7%; 198/280), and fatigue (66.0%; 140/212). pwh hospitalized with covid-19 were significantly more likely to have fever, fatigue, dyspnea, gastrointestinal symptoms, and altered mental status. whereas, pwh who were not hospitalized were more likely to have upper respiratory symptoms such as sore throat and nasal congestion, and headache. at presentation, all patients with a peripheral oxygen saturation of < 94% on room air (30.6%; 72/235) and a quick sequential organ failure assessment (q-sofa) score of ≥ 2 (8.3%;17/206) were hospitalized. a chest x-ray was obtained in 194 (67.8%) patients, of whom 77.8% had abnormal findings, and a computerized tomography (ct) scan was performed for 44 (15.4%) patients of whom 86.4% had abnormal findings. patients who were hospitalized were more likely to have imaging done and to have abnormal findings (table 2) . within 30 days of positive sars-cov-2 testing, 164 (57.3%) patients were hospitalized. among hospitalized pwh, potential sars-cov-2 targeted treatments, excluding placebo-controlled clinical trials, were given to 99 (60.4%) patients, the most common medication being hydroxychloroquine (40.9%) ( table 3) . c c e p t e d m a n u s c r i p t testing. when stratified by age groups (<40, 40 -60, >60 yrs.), the relative risk for all poor outcomes was more than one for older age groups compared to less than 40. patients who are older than 60 were significantly more likely to require respiratory support, develop acute kidney injury, and die, compared to patients who were less than 40 years old (table 3) . multivariable analysis identified higher age, lower cd4 counts, chronic kidney disease, and chronic lung disease as independent predictors of hospitalization. phw with three or more comorbidities, compared to having only hiv disease, were also more likely to be hospitalized. (table 4) . chronic lung disease (32.0 vs. 14.0%; p<0.01), and ckd (30.0 vs. 14.0%; p<0.01). in a multivariable analysis, older age, lower cd4 count, chronic lung disease, hypertension, and high comorbidity burden were significantly associated with severe outcomes (table 4) . based on 47 patients admitted to an icu and 27 deceased patients, cd4 cell count had a significant effect on survival. the pairwise comparison showed a significant difference between the cd4 count < 200 and cd4 count > 500 cells/mm3 for both icu-free survival (p=0.04) and overall survival (p=0.05) (figure 1 ). in post-hoc analysis, we included pwh diagnosed with covid-19 from the us (n=265) and excluded those from international locations (n=21). there were no significant differences in presentation and predictors of outcomes, except for hypertension. hypertension was not significantly associated with severe outcome, after adjusting for other variables. analysis presented in supplementary material (tables 1-4 a c c e p t e d m a n u s c r i p t in this multicenter analysis, severe clinical outcomes occurred commonly in pwh and covid-19. as reported in multiple other studies in people without hiv, we found that age, chronic lung disease, and comorbidity burden were associated with increased rates of severe outcomes. in addition, among hiv-specific factors, we found that a lower cd4 count (< 200 cells/mm3) was associated with poor outcomes, including higher hospitalization rates, lower icu-free survival, and overall survival. our study is the first to characterize outcomes in a large number of geographically diverse pwh with laboratory-confirmed covid-19. the clinical and radiologic presentations of the pwh in our study were similar to those reported in other studies of patients with covid-19, with or without hiv co-infection. 1, 17, 22, 30 our study confirms the unequal racial and gender distribution of pwh and covid-19. it mirrors the demographics of pwh in the us with a higher proportion of men, african american, and hispanic patients. 31 however, we did not find that race and gender were associated with worse outcomes in this cohort. our findings demonstrate a high prevalence of comorbidities among pwh and covid-19. consistent with other studies, 22, 32 we found that underlying comorbidities constitute a significant risk factor for hospitalization and poor outcomes in pwh. available data indicate considerable variability in mortality rates, icu admission rates and need for invasive mechanical ventilation among pwh diagnosed with covid-19. 16, [18] [19] [20] 22 based on our analyses, rates of icu admission, mechanical ventilation use, and death among pwh and covid-19 were consistent with the general us data. 30 we did not identify hiv viremia (a proxy for not taking art) as a risk factor for severe covid-19, but the proportion of study participants with hiv viremia was small and more than 90% of our study enrollees were receiving art. hence our ability to compare the outcomes between those with and without hiv control was limited. a c c e p t e d m a n u s c r i p t it has been postulated that patients with advanced hiv, low cd4 counts, and severe immunosuppression cannot mount the robust inflammatory response responsible for covid-19associated complications. 15, 16, 18, 21 our study does not support this hypothesis for those who are virologically suppressed but have low cd4 cell counts. although we did not collect information about nadir cd4 cell count or duration of art, this population (low cd4 but virally suppressed) usually has a history of severe immunosuppression, recent art initiation, or both. our findings show a significant association between low cd4 counts and poor outcomes contrasting recently published cases series. the study by karmen-tuohy et al. showed that 6 patients out of 19 had cd4 < 200 cells/mm3 and the cd4 count was not associated with mortality in hiv-positive patients. 19 a recent study from spain suggested that pwh receiving tenofovir disoproxil fumarate (tdf) in combination with emtricitabine as part of their art regimen have a lower risk for sars-cov-2 acquisition and related hospitalizations than those on other art regimens. 34 although the data we gathered were not specific to address the differences between those on tdf and other nucleoside reverse transcriptase inhibitors, in our study, we did not find an association between the class of art or the use of darunavir-containing regimens and predefined outcomes. this study has several limitations. first, this study cannot comment on the prevalence of sars-cov-2 infection among pwh. second, covid-19 testing, treatment, and hospitalization were all done at the discretion of individual healthcare providers and may have varied widely between sites as well as between different time points during the pandemic, reflective of local test availability and policies. there may also be selection bias, as contributors entered cases voluntarily and may not have entered every case from their institutions or clinics. however, 17 out of 36 institutions (accounting for 246 patients out of 286), have included systematically all hiv patients with covid identified through the search performed in their corresponding centers during the study period. we could not control for a c c e p t e d m a n u s c r i p t covid-19 therapies because we did not collect data on steroid use and clinical trial participation, as well as the small number of patients in each treatment group. we also did not collect data on social determinants of health, which may have impacted the clinical course of covid-19. future studies should assess whether specific socioeconomic factors impart a greater risk related to covid-19 for pwh. finally, death is counted as all-cause mortality; we did not verify the exact cause of death. despite these limitations, evaluating outcomes for pwh with covid-19 who were hospitalized or non-hospitalized from multiple sites and settings (community hospitals, clinics, and large academic centers) make these results more generalizable. in conclusion, our study adds to existing reports of the clinical characteristics of covid-19 among as principal investigator, dr. dandachi had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. analysis and interpretation of data: all authors. critical revision of the manuscript for important intellectual content: all authors. m a n u s c r i p t m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t the model for hospitalization outcome is adjusted for age, sex, race/ethnicity, years with hiv, cd4 count, hiv viral load suppression, antiretroviral regimen, hypertension, diabetes, chronic lung disease, chronic kidney disease, cardiovascular disease, active malignancy, and chronic liver disease. the model for severe outcome is adjusted for age, sex, race/ethnicity, cd4 count, hiv viral load suppression, hypertension, diabetes, chronic lung disease, chronic kidney disease, and chronic liver disease. the model for the association between hospitalization, severe outcome, and comorbidity burden is adjusted for age, sex, and race/ethnicity. † severe outcome, defined as a composite outcome of intensive care admission, invasive mechanical ventilation, or death. m a n u s c r i p t a c c e p t e d m a n u s c r i p t figure 1 comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region obesity in patients younger than 60 years is a risk factor for covid-19 hospital admission covid-19 in immunocompromised hosts: what we know so far patients with cancer appear more vulnerable to sars-cov-2: a multicenter study during the covid-19 outbreak covid-19 in persons with haematological cancers covid-19 and kidney transplantation cdc. hiv public health partners. policy, planning, and strategic communication. 2020. 10. price-haywood eg covid-19 and african americans co-infection of sars-cov-2 and hiv in a patient in wuhan city covid-19 in patients with hiv: clinical case series presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area a survey for covid-19 among hiv/aids patients in two districts of wuhan, china (3/4/2020) covid-19 in people living with human immunodeficiency virus: a case series of 33 patients. infection clinical features and outcomes of hiv patients with coronavirus disease 2019 outcomes among hiv-positive patients hospitalized with covid-19 covid-19 and people with hiv infection: outcomes for hospitalized patients clinical characteristics, comorbidities and outcomes among persons with hiv hospitalized with coronavirus disease description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort. lancet hiv risk of covid-19 death: a population cohort study from the western cape province characterization and clinical course of 1000 patients with coronavirus disease 2019 in new york: retrospective case series research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support the redcap consortium: building an international community of software platform partners defining treatment failure in resource-rich settings interim analysis of risk factors for severe outcomes among a cohort of hospitalized adults identified through the u.s. coronavirus disease 2019 (covid-19)-associated hospitalization surveillance network (covid-net) disproportionate burden of covid-19 among racial minorities and those in congregate settings among a large cohort of people with hiv updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan incidence and severity of covid-19 in hiv-positive persons receiving antiretroviral therapy: a cohort study a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-330800-s91zfzfi authors: reta, daniel hussien; tessema, tesfaye sisay; ashenef, addis simachew; desta, adey feleke; labisso, wajana lako; gizaw, solomon tebeje; abay, solomon mequanente; melka, daniel seifu; reta, fisseha alemu title: molecular and immunological diagnostic techniques of medical viruses date: 2020-09-04 journal: int j microbiol doi: 10.1155/2020/8832728 sha: doc_id: 330800 cord_uid: s91zfzfi viral infections are causing serious problems in human population worldwide. the recent outbreak of coronavirus disease 2019 caused by sars-cov-2 is a perfect example how viral infection could pose a great threat to global public health and economic sectors. therefore, the first step in combating viral pathogens is to get a timely and accurate diagnosis. early and accurate detection of the viral presence in patient sample is crucial for appropriate treatment, control, and prevention of epidemics. here, we summarize some of the molecular and immunological diagnostic approaches available for the detection of viral infections of humans. molecular diagnostic techniques provide rapid viral detection in patient sample. they are also relatively inexpensive and highly sensitive and specific diagnostic methods. immunological-based techniques have been extensively utilized for the detection and epidemiological studies of human viral infections. they can detect antiviral antibodies or viral antigens in clinical samples. there are several commercially available molecular and immunological diagnostic kits that facilitate the use of these methods in the majority of clinical laboratories worldwide. in developing countries including ethiopia where most of viral infections are endemic, exposure to improved or new methods is highly limited as these methods are very costly to use and also require technical skills. since researchers and clinicians in all corners of the globe are working hard, it is hoped that in the near future, they will develop good quality tests that can be accessible in low-income countries. viruses are small segments of nucleic acid, deoxyribonucleic acid (dna), or ribonucleic acid (rna) within a protein coat or lipoprotein coat (envelope). viruses require host resources for their replication because they are obligate intracellular parasites. once viruses enter the host cells, they take over or hijack the cells' biosynthetic machineries for the replication of their genomes and other components [1, 2] . recently, among others, have caused several outbreaks in different countries [3] [4] [5] [6] [7] [8] [9] . e morbidity and mortality rates of human viral infections are significantly high [10] . for example, the pandemic swine influenza a (h1n1) infection in 2009 occurred in 214 countries with more than 18,036 deaths [5] . in 2010 alone, the number of human deaths due to rabies globally was estimated to be 61,000, with 84% of the deaths occurred in rural areas [11] . in 2013, approximately, 35,000,000 people were infected with hiv worldwide [10] . e world health organization (who) reported 1.34 million deaths of viral hepatitis in 2015 [12] . as on 6 th january 2015, h5n1 viruses have killed 402 out of 694 laboratory-confirmed human infections in 16 countries [13] , with a mortality rate of around 58%. recently, the world is challenged by the novel coronavirus disease 2019 . e disease is caused by the novel coronavirus (sars-cov-2). e pathogen first emerged in wuhan city, hubei province, china, which has now quickly gained worldwide spread [9, 14] . on 11 th march 2020, the who declared the covid-19 outbreak a global pandemic. according to the who, 9, 129, 146 confirmed cases of covid-19 have been reported worldwide, including 473, 797 deaths since 31 st december 2019 and as of 24 th june 2020 [15] . erefore, good diagnostic techniques are required to detect these viral infections early and accurately. early and accurate detection of viral diseases plays a significant role in selecting appropriate therapy timely, minimizing therapy costs, minimizing unnecessary loss of human lives, and controlling the disease. it also helps to develop appropriate disease prevention and treatment strategies, like development of antiviral vaccines and new therapeutic agents [14, 16, 17] . traditionally, laboratory diagnoses of medical viruses are carried out by isolating viruses in embryonated chicken eggs, in tissue culture, or in laboratory animals and visual examination of viral particles in sample using electron microscopy among others [16] . many conventional diagnostic tools tend to be cumbersome, time-consuming, expensive, and poorly reproducible [18, 19] . in contrast, molecular techniques have revolutionized diagnostic virology by detecting the presence or absence of viral nucleic acids in a patient's sample [18] . immuno-based techniques still play a great role for the detection and serosurveillance of human viral infections despite the fact that many of the traditional methods are replaced by nucleic acid-based techniques [20] . immunological methods detect viral infections by identifying antiviral antibodies or viral antigens in clinical samples [21, 22] . here, we describe some of the molecular and immunological diagnostic approaches for the detection of medical viruses. nucleic acid-based molecular detection techniques have revolutionized diagnostic virology with their faster, highly sensitive, and highly specific diagnosis [14, 23, 24] . since these methods detect specific nucleic acid sequences, nucleic acid-based diagnostic tests can be applied for the detection of virtually any virus that affects humans [1] . techniques. molecular techniques that involve the amplification of viral genomic material are extremely sensitive and specific, provide rapid diagnosis, and allow the detection of several viruses at same time [16] . nucleic acid amplification techniques are very useful for the detection of viruses that are uncultivable or difficult and harmful to culture, slow growing viruses in culture, and viruses that display antigenic variations [1, 25] . e nucleic acid amplification tests are very popular in the diagnosis of viral infections caused by several viruses, including hepatitis c virus (hcv), human immunodeficiency virus (hiv), dengue virus, epstein-barr virus (ebv), influenza viruses, zika virus (zikv), ebola virus, and coronavirus [26] [27] [28] [29] [30] [31] [32] . several nucleic acid amplification methods are currently available for the laboratory diagnosis of viral infections worldwide, and their advantages and limitations will be summarized in table 1 . . pcr is a typical example of nucleic acid amplification assay. it has revolutionized the field of molecular diagnosis since developed by mullis and faloona [50] . pcr is based on extraction and purification of dna molecule and exponential amplification of the target sequence, using a thermostable dna polymerase and two specific oligonucleotide primers. after the pcr reaction, the amplified product can be detected by several techniques, including gel electrophoresis, colorimetric methods, and sequencing [10, 51, 52] . since its inception, pcr has been used for the detection of human viral infections with overall clinical sensitivity ranging from 77.8% to 100% and clinical specificity ranging from 89% to 100% [28, [53] [54] [55] . ese reports suggest that pcr can be employed for the detection of medical viruses in a variety of specimen types. conventional pcr is still in use by some clinical laboratories worldwide, but it is rapidly replaced by more advanced variants of the technique. pcr is a highly versatile technique. a number of variants of the conventional pcr have been developed, but the most important variants are reverse transcription-pcr and realtime pcr [1, 10] . e first method was devised to amplify ribonucleic acid (rna) targets [1] ; the second technique was introduced to quantify deoxyribonucleic acid (dna) in real time throughout the pcr reactions [56] . rt-pcr was designed to amplify rna targets. in this technique, reverse transcriptase (rt) is used to convert viral rna targets into complementary dna (cdna), and then the resulting cdna is amplified by conventional pcr. since its development, rt-pcr has been used for the diagnosis of human infection by rna viruses. conventional rt-pcr demonstrated overall sensitivity ranging from 73% to 100% and specificity ranging from 99% to 100% in the detection of viral infection [29, 57, 58] . ese data indicate that rt-pcr is an excellent technique for the diagnosis of human infection by rna viruses. nowadays, however, the method is not used commonly in clinical specimens owing to its high cost and timeconsuming process [14] . in real-time pcr system, viral nucleic acid amplification and detection steps are carried out at the same time. e detection of the amplification product is relied on the amount of fluorescence emission from the specimen. e fluorescence emission from the specimen is monitored by special thermal cycler. e computer, with appropriate software connected to the thermal cycler, records the data and produces an amplification plot at every reaction cycle [51, 59] . e detection and quantification of amplification products can be done by using sybr green, the taqman, and molecular beacon chemistries. e sybr green dye binds to the minor groove of double-stranded dna (dsdna) product and upon excitation by appropriate light, it exhibits improved fluorescence, which is directly proportional to the accumulated dsdna product. e taqman probe is a dna oligonucleotide with a fluorescent dye termed reporter attached to one end (5′ base) and quencher on the other (3′ base) ( figure 1 ). taqman probes are designed to hybridize to an internal region of a pcr product. during the annealing stage of the pcr, both the primer and the taqman probe bind to the template strand. when the taq dna polymerase extends the primer, the polymerase cleaves the probe by its the 5′-3′ exonuclease activity. cleavage of the probe leads to the release of the fluorescent dye (figure 1 ), resulting in fluorescence emission. e amount of fluorescence is directly proportional to the pcr product. molecular beacon is a small dna molecule with a fluorescent dye at the 5′ end and a quencher at the 3′ end. e sequences at the very 3′ and 5′ ends are complementary to each other. e internal part of the molecule is designed to be complementary to the target sequence of a pcr product. when molecular beacon is free in solution, it will adopt a hairpin structure. is brings the fluorophore and quencher in close proximity, leading to absorption of emitted light of the florescent dye by the quencher and hence fluorescence is not detected (figure 2 a). however, when a molecular beacon hybridizes to the target sequence, the fluorophore and quencher are separated, leading to the emission of fluorescence (figure 2 b ). e amount of fluorescence is directly proportional to the pcr product [16, 42, 51, 60] . owing to high sensitivity and specificity, short turnaround time for results, and ease of performance [33, 61] , most laboratories across the globe employ real-time pcr for the detection and quantification of medical dna and rna viruses in clinical specimens. for example, boppana et al. [39] used real-time pcr for the detection of cytomegalovirus (cmv) in liquid saliva with overall sensitivity of 100% and specificity of 99.9%, compared with standard rapid culture. e method was also employed for the diagnosis of primary of ebv infection with overall sensitivity of 95.7% and specificity of 100%, compared to serologic assays [62] . realtime pcr was also served to determine viral load in herpes simplex encephalitis patients [40] . e determination of viral loads in patient specimens is crucial as it provides prognostic and predictive information. in this study, patients with higher viral loads in their cerebrospinal fluid (csf) found to require acyclovir therapy for a longer duration and had a poorer clinical outcome than the patients with lower viral loads in their csf [40] . e assay can also be used for the multiplex identification of different viruses. both taqman probe and molecular beacon play crucial roles for multiplex identification of different viruses in a single pcr reaction. in multiplexing assays, different probes/ beacons are labeled with different fluorescent dyes [41] . in multiplex assay, sensitivity of 100% and specificity of 99.6% were reported, compared to immunofluorescence assay, for real-time pcr in the detection of human adenovirus b, c, and e in the throat swab samples [63] . ramamurthy and his colleagues [33] compared multiplex real-time pcr with multiplex conventional pcr for the detection of neurotropic viruses (cmv, ebv, herpes simplex virus types 1 and 2 (hsv-1 and hsv-2) japanese encephalitis virus (jev), and varicella-zoster virus (vzv)) in csf. out of 147 csf samples collected from patients with neurological disorders, real-time pcr detected viral pathogens in 88 samples while conventional pcr could only detect the viruses in six samples, suggesting that real-time pcr has higher sensitivity than conventional pcr. qiu et al. [64] developed a triplex quantitative real-time pcr assay for rapid and differential detection of human adenovirus (hadv) serotypes 2, 3, and 7 for potential clinical use. e analytical sensitivity (limit of detection; lod) of this assay was 10 2 dna copies/reaction for each of serotypes and no cross-reactions with other respiratory pathogens. e authors concluded that the assay is sensitive and specific and has the potential for clinical use in the rapid and differential detection and quantitation of hadv serotypes 2, 3, and 7 in human specimens. by the incorporation of reverse transcription step, realtime pcr can be combined with the conventional reverse transcription pcr (rt-pcr) to form reverse transcription quantitative real-time pcr (rt-qpcr). rt-qpcr has a number of advantages over the conventional rt-pcr technique, including reduction of contamination, possibility of quantifying the amplicons, and quick assay time since there are no post-pcr processing activities [14, 51] . as a result, rt-qpcr is widely deployed for the detection and quantification of several rna viruses in clinical specimens, including zikv, ebola virus, coronavirus, hcv, respiratory syncytial virus (rsv), dengue virus, hiv-1, and influenza a virus [30] [31] [32] [65] [66] [67] [68] [69] . recently, corman et al. [32] developed rt-qpcr for the detection of sars-cov-2. e assay targeted envelope protein (e) gene and rna-dependent rna polymerase (rdrp) gene of sars-cov-2. high lods of 5.2 copies/reaction for e-gene and 3.8 copies/reaction for rdrp gene were demonstrated and no cross-reaction with other coronaviruses, suggesting the usefulness of the method for sensitive and specific diagnosis of covid-19. e rt-qpcr assay (quanty zebov fast assay) was evaluated for the detection of ebola virus in clinical samples. clonit srl (italy) developed the assay during outbreak of ebola in sierra leone, and it had overall sensitivity of 100% and specificity of 98.63%, compared to realstar ® filovirus screen rt-qpcr kit 1.0 (altona diagnostics) [31] . gueudin et al. [68] used rt-qpcr for diagnosis and monitoring of hiv-1 group o infection with lod of 40 copies/ml and specificity of 100%. e method was applied for zikv detection in human serum and urine, and it had lods of 2.5 pfu/ml and 250 pfu/ml in urine and serum, respectively [30] . júnior et al. [70] used rt-qpcr for the detection of respiratory viruses in outpatients with acute respiratory infection. ey also compared the performance of rt-qpcr with indirect immunofluorescence assay (ifa). accordingly, rt-qpcr managed to detect viral pathogens in 88 (88/162) nasopharyngeal aspirates, but ifa detected viral pathogens in only 33 (33/162) specimens. e data indicated that the use of rt-qpcr increased the viral detection by 33.9%. today, several real-time rt-pcr kits are available commercially. for example, real-time-qpcr test developed by cepheid ab (sunnyvale, ca, usa) is commercially available for the qualitative detection of ebola virus in edta venous whole blood or buccal swabs. e assay targets viral nucleoprotein and glycoprotein genes of ebola zaire virus. e assay has a lod of 82 rna copies/reaction with turnaround time of 98 minutes. simplexa ™ dengue rt-pcr assay developed by focus diagnostics (cypress, ca, usa) is a commercial kit for detection and typing of dengue virus serotypes 1, 2, 3, and 4 in human serum. e assay targets four serotype specific regions, namely, dengue 1 (nonstructural (ns)5 gene ), dengue 2 (ns3 gene), dengue 3 (ns5 gene), and dengue 4 (capsid gene). e lods of the assay are 0.16 pfu/ ml, 2.0 pfu/ml, 0.2 pfu/ml, and 0.2 pfu/ml for dengue 1, dengue 2, dengue 3, and dengue 4, respectively. real-star zika virus rt-pcr kit 1.0 is available, developed by altona diagnostics (hamburg, germany), for qualitative detection of zikv specific rna in human serum or urine. e lod of the assay is 0.61 rna copies/μl. abbott realtime hcv quantitative assay developed by abbott laboratories (rungis, france) is commercially available for hcv rna quantitation in human serum and plasma. e target sequence for the assay is in the highly conserved 5′untranslated region (utr) of the hcv genome. e lod of the assay is 12 iu/ml when testing human plasma or serum. cobas taqman hiv-1 test developed by roche diagnostics (branchburg, usa) is commercially available for the quantitation of hiv-1 in human plasma. e realtime rt-pcr targets two highly conserved regions of the hiv-1 genome, namely, gag and long terminal repeat (ltr). e assay has lod of 20 hiv-1 rna copies/ml. recently, several developers of diagnostic tests have developed realtime rt-pcr kits for covid-19, and they are now seeking marking and emergency use authorization (eua) from regulatory agencies. for example, co-diagnostics (salt lake city, usa) has developed real-time rt-pcr kit (logix smart covid-19 test) for qualitative detection of nucleic acid from the sars-cov-2 in lower respiratory samples (e.g., bronchoalveolar lavage, sputum, and tracheal aspirate) and upper respiratory specimens (e.g., oropharyngeal swabs, nasal swabs, and nasopharyngeal swabs). e kit has received eua from united states food and drug administration (us fda) and ce-ivd marking approval. e assay targets rdrp gene of sars-cov-2. e lod of the assay is 9. methods. transcription-based amplification method includes nucleic acid sequence-based amplification (nasba) and transcription-mediated amplification (tma). nasba and tma are similar to each other. ey are isothermal amplification methods. e entire amplification process is carried out at the temperature of 41°c. in both cases, the viral rna target is first converted into cdna with rt and then rna polymerase synthesizes multiple copies of viral rna product. e only difference between tma and nasba in the amplification process is two enzymes (rt and rna polymerase) are utilized in case of tma while nasba utilizes three enzymes (avian myeloblastosis virus reverse transcriptase (amv-rt), rnase h, and t7 rna polymerase) [42, 51] . as depicted in figure 3 , in the nasba process, three enzymes and two primers work together to exponentially amplify a target viral rna. primer 1 (p1) carries at its 5′ end t7 rna polymerase promotor region and at its 3′ end, p1 carries sequence that is complementary to a target viral rna sequence. primer 2 (p2) carries a sequence complementary to cdna strand. e amplification reaction begins with the production of cdna copy of the viral rna by rt using p1. rnase h degrades the viral rna from rna-dna hybrid molecules. en, rt synthesizes dsdna molecules using p2 and the released dna strand. finally, t7 rna polymerase uses dsdna molecules as templates to transcribe many viral rna copies. e above cycle is repeated several times, resulting in the accumulation of many viral rna copies and ds dna molecules. e amplified product can either be detected by gel electrophoresis at the end of the assay or in real time using molecular beacon [16, 42, 43, 71] . transcription-based amplification methods have several advantages, for example, they do not require a thermal cycler, so developing countries and budget-restricted laboratories can afford to perform the assays, they have rapid kinetics (requires fewer cycles), and they produce a single-stranded rna product that is suitable for detection by various techniques [42, 51, 71, 72] . transcription-based amplification methods are suitable for the diagnosis of human viral infections caused by rna viruses. ey can amplify viral genomic rna, messenger rna, or ribosomal rna [51, 71, 73] . ayele et al. [44] developed nasba assay that uses gag-based molecular beacons to distinguish between hiv-1 subtype c (c and c′) circulating in ethiopia. e assay demonstrated high levels of sensitivity and specificity for both beacons (90.5% sensitivity and 100% specificity for the c beacon and 100% sensitivity and 95.2% specificity for the c′ beacon) by considering sequencing as gold standard for genotyping. moore et al. [74] also used nsaba for the detection of influenza a h5n1 virus in clinical specimens with a lod of 10 rna copies/μl along with the same sensitivity as rt-pcr and average turnaround time of 4 hours. e nasba assay was also used for the detection of dengue viral rna with lod of 1 pfu/ml for all of 4 dengue virus serotypes, no cross-reaction with jev, and turnaround time of 3 hours [27] . ender et al. [26] used tma for screening of blood donations for hiv-1 and hcv rna. e tma assay had lods of 16.2 iu/ml for hiv-1 and 3.5 iu/ml for hcv. a multiplex nasba assay was used for simultaneous detection of hiv-1 and hcv in plasma samples. e lod of the assay for both hiv-1 and hcv was determined to be 1000 copies/ml and no crossreactions with other selected viruses [45] . swenson and his colleagues [75] used real-time tma for the detection of hsv-1 and hsv-2 in lesion swab specimens with overall sensitivities of 98.2% and 99.4%, respectively, and specificity of 97.8% and 94.5%, respectively, compared to culture. in one study, real-time nasba assay was more sensitive than the conventional rt-pcr in the detection of norovirus. in this study, rt-pcr detected 10 pg of standard viral rna, while the real-time nasba assay could detect 100 fg of standard viral rna [76] . ese data indicate that the assays are sensitive, specific, and costeffective for the detection of human infection by rna viruses. tma-based assays for the detection of hcv and hiv-1 are commercially available, developed by hologic (san diego, ca, usa). e aptima hcv rna qualitative assay is used for the detection of hcv rna in human plasma or serum. e assay utilizes tma to amplify conserved regions within the 5′-utr of the hcv genome. e assay has lod of 7.5 iu/ml with a specificity of 99.6%. nasbabased kits for detection of hiv-1, cmv, enterovirus, and rsv are also commercially available, developed by bio-mérieux clinical diagnostics. e nuclisens easy q rsv a and b assay is developed by biomérieux (marcy l'etoile, france), and it is used for qualitative detection of rsv in respiratory samples of different types. e assay is based on real-time nasba, and it targets f gene of rsv. moore et al. [77] evaluated the performance of the commercial test kit using 508 respiratory specimens that were tested by direct immunofluorescence and culture. e assay was found to be more sensitive than culture and immunofluorescence assay. e sensitivity and specificity of the assay were determined to be 99% and 87%, respectively, compared to immunofluorescence assay with turnaround time of <4 hours. lamp is another isothermal nucleic acid amplification method that is extensively utilized for sensitive, specific, rapid, and cost-effective detection of both dna and rna viruses in human specimens. e method was first developed by notomi et al. [78] and rapidly gained popularity in diagnostic virology. e method employs four to six unique primers and dna polymerase with stranddisplacement activity to amplify target dna [78, 79] . e addition of rt in lamp reaction (rt-lamp) permits the amplification of rna target [80] . primer sets for lamp initially reported by notomi et al. [78] include the forward inner primer (fip), backward inner primer (bip), forward outer primer (f3), and backward outer primer (b3). e primers are specifically designed to recognize six precise regions from a targeted nucleic acid sequence. nagamine et al. [79] later added two loop primers, namely, forward loop primer (lf) and backward loop primer (lb), to accelerate lamp assay. owing to the use of four to six specific primers, lamp assay has outstanding sensitivity and specificity in the detection of target nucleic acids [79, 81] . a detailed description of the lamp reaction mechanism is available in reviews by becherer et al. [81] , tomita et al. [82] , and silva et al. [83] , which use illustrations to explain the mechanism. e lamp reaction is performed in constant temperature between 60-65°c, alleviating the need for expensive specialized equipment. e method requires only inexpensive heating block or water bath, making it very useful under poor laboratory settings [84] . e lamp reaction takes turnaround time of <1 hour and the amplified product can be detected by several methods, including the real-time measurement of the turbidity caused by precipitated magnesium pyrophosphate using a turbidimeter, visual detection of magnesium pyrophosphate precipitation following completion of the reaction, detection of fluorescence under ultraviolet light or natural light by adding an intercalating fluorescent dye to the final reaction mixture, and visualization of the bands with various sizes using agarose gel electrophoresis [84] [85] [86] [87] . lamp assay has been successfully utilized to the rapid detection of a number of dna viruses in human specimens, such as hsv-1 with lod of 10 copies of hsv-1 dna/μl and no cross-reactions with other selected viruses [85] , hadv40 and hadv41 with lod of between 50 and 100 copies of dna/reaction, no cross-reactions with other selected viruses, and turnaround time of 60 minutes [88] , ebv with sensitivity of 86.4%, specificity of 100%, compared to serological assay, and only 45 minutes of amplification of the target sequences [89] , and cmv with lod of 10 dna copies/μl, no cross-reactivity with other viruses, and turnaround time of 1 hour after rna extraction [90] . e utility of lamp is expanded by merging it with reverse transcription (rt) into rt-lamp to allow the rapid detection of rna viruses in clinical specimens. recently, for instance, huang et al. [91] developed a rapid rt-lamp assay for diagnosis of sars-cov-2 with lod of 80 copies of viral rna/ml in a sample within a 30 minutes reaction. is assay was validated by using 16 clinical samples (8 positives and 8 negatives) that were also tested by rt-qpcr. e testing results of the assay were consistent with rt-qpcr method, suggesting rt-lamp assay can be used for rapid, simple, cost-effective, and sensitive detection of sars-cov-2 in respiratory samples. similarly, lu et al. [92] developed the rt-lamp method for rapid detection of sars-cov-2 with lod of 30 rna copies/reaction and turnaround time of 40 minutes . further, baek et al. [93] developed a rapid rt-lamp assay for early detection of sars-cov-2. e assay has lod of 100 rna copies/reaction, which is close to that of rt-qpcr with a rapid detection span of 30 minutes . rt-lamp assay has also been developed to detect middle east respiratory syndrome coronavirus (mers-cov) with lod of 3.4 copies of mers-cov rna/ reaction along with the same sensitivity as mers-cov rt-qpcr, no cross-reaction to other respiratory viruses, and results available in <1 hour [87] . kurosaki et al. [94] detected acute ebola virus infection by rt-lamp coupled with a portable device. e sensitivity and specificity of the assay was 100% each, compared to rt-qpcr and turnaround time of 35 minutes . in one study, rt-lamp was more sensitive than conventional rt-pcr and nasba [95] . e assay has been also developed for rapid detection of dengue virus [84] , influenza a (h1n1) pdm09 virus [96] , h5n1 avian influenza virus [97] , hcv [98] , hiv-1 [99] , rsv [46] , and zikv [100] in clinical samples. rt-lamp-based commercial test kits are available for the detection of sars-cov-2 in respiratory specimens. e assay is developed by color genomics (usa), and it uses three sars-cov-2 specific primer sets targeting n gene , e gene , and orf1a region, respectively, and a fourth control primer set targeting human ribonuclease p [101] . in dna microarray diagnosis, fluorescently labeled viral nucleic acids in a test sample are used to screen an array of oligonucleotide probes immobilized on a solid surface (e.g., glass slide). e oligonucleotide probes used here are specific for the genome of the target virus. e results of hybridization between immobilized probes and target sequences labeled with fluorescent dyes are detected and quantified by fluorescence-based detection [16, 51, 72] . extensive literature exists demonstrating the utility of dna microarray for the detection of medical viruses in human specimens. chiu et al. [102] used dna microarray for high-throughput multiplex detection of viruses in nasopharyngeal aspirate samples originated from children infected with respiratory viruses. e assay demonstrated overall sensitivity of 87% to 90% and specificity of ≥99% in the detection of rsv, influenza a virus, and rhinovirus/ enterovirus compared to rt-pcr. in one study, dna microarray was utilized for simultaneous detection of hsv-1/2, vzv, ebv, cmv, human herpesvirus-6 types a and b (hhv-6 a/b), and adenovirus in clinical samples with lod of 10 ge/reaction for each virus without crossreactivity [103] . dna microarray was also used to identify viral causes of meningitis and encephalitis with overall sensitivity of 93% and specificity of 100%, compared to single-virus pcr [104] . dna microarray was also utilized for high-throughput multiplex detection of gastrointestinal viruses [105] , viruses transmitted by small mammals and arthropods [106] , herpesviruses, enteroviruses and flaviviruses [107] , hiv-1, hiv-2, and hepatitis viruses [108] and dual infection with two dengue virus serotypes [109] in human specimens. dna microarray was used to identify and genotype drug-resistant mutations of hiv [110, 111] to detect and genotype drug-resistant hepatitis b virus (hbv) mutations [112] , to detect and genotype sars coronavirus [113] , and to detect and determine lineage of influenza b viruses [114] . during an outbreak of sars in china in 2002, dna microarray also served for the discovery of a new member of the coronavirus family [115] . dna microarray technology is a high-throughput tool as it allows multiplex detection of a large number of potential viral pathogens in clinical specimens [102] [103] [104] [105] . e technique does have a number of limitations nevertheless, including being too expensive to be used for routine clinical diagnosis, labor-intensive, and time-consuming (the hybridization process may take hours to days to complete). nonspecific hybridization between test materials and immobilized probes can affect the sensitivity of the assay. in addition, designing of specific probes requires almost complete information of the genetic makeup the virus of interest. e assay detects only those viral pathogens that have target probes on the array [72, 116, 117 ]. (ngs) . ngs finds itself very useful in diagnostic virology as it can directly analyze viral nucleic acid fragments extracted from clinical specimens [118, 119] . generally, ngs involves preparation of test sample, sequencing of the target nucleic acid fragments using one of the available ngs platforms, and analysis of the sequence data using suitable bioinformatic tools [10, 120] . several companies produce different ngs machines that use different methods of sequencing, reagents, and data analysis tools [119] . for example, pyrosequencing (roche 454) detects release of pyrophosphate following incorporation of nucleotides in a dna polymerization process. illumina's ngs platforms detect release of fluorescent labels from incorporated nucleotides in a dna polymerization process. e emerging technologies like oxford nanopore (minion) platform sequences the target nucleic acid by sensing the ionic current of dna/rna molecules that pass through the nanopores [10, 119, 120] . despite its high sequence error rate (up to 38.2%) [121] , minion nanopore sequencer has merits over other ngs platforms. firstly, it can generate longer read lengths (up to 882 kb) in real time [122] , making it suitable for whole genome sequencing of viral pathogens with short turnaround time [123, 124] . secondly, it is portable and no internet is required for analysis, making it deployable in the field during outbreaks of viral infections [125] . irdly, it has low capital cost, making it affordable in low-income countries and budget-restricted laboratories across the globe [121] . ngs has been used in diagnostic virology for several applications. recently, kustin et al. [126] used ngs for rapid and robust identification of respiratory viruses in clinical samples. it was applied to track influenza a (h1n1) pdm09 virus [127] . dessilly et al. [128] used ngs for the detection of hiv-1 drug resistance mutations. ngs was also conducted to discover a new ebola virus [129] . unlike pcr and dna microarray methods, ngs does not require prior knowledge of genomic sequences of the viral pathogens. it does not also require target specific pcr primers and oligonucleotide probes [126, 130] . however, the use of ngs in clinical laboratories is limited because of the following reasons: the turnaround time, the number of samples per run, cost of sequencers, and requirement of skills in bioinformatics [10, 128, 131] . medical viruses e humoral branch of the immune system makes antibodies in response to viral infections. is natural response of the human body against viral infection is utilized for the development of immunological diagnostic methods. several immunological diagnostic techniques are available for the detection human viral infections in clinical samples, including enzyme-linked immunosorbent assay, western blotting, immunofluorescence assay, and hemagglutination inhibition assay. e principles of these assays rely on the formation of antigen-antibody complex and consist of clinical specimens, whole virus or viral antigen, and an indicator [10, 19, 20] . 8 international journal of microbiology in elisa, enzyme conjugated antibody is utilized to detect the presence of specific antiviral antibody or viral antigen in human specimens. in positive sample, the reaction between an enzyme conjugated with an antibody and colorless chromogenic substrate leads to the formation of a colorful product. in the absence of antigen/antibody in the clinical specimen, no color is produced. e intensity of the color is directly proportional to the amount of antigen-antibody complex formed. e color change can be observed by the naked eye or read by a spectrophotometer, which can measure the absorbance. several enzymes, including alkaline phosphatase, horseradish peroxidase (hrp), and β-galactosidase, have been used for elisa. ere are several variants of elisa, but the two main types are antigen-capture elisa (also called sandwich elisa) and antibody-capture elisa (also called indirect elisa) [19, 132] . as illustrated in figure 4 , the first method detects viral antigen by immobilizing antibody specific for the viral protein of interest on a microtiter well [22] ; the second technique detects antiviral antibody in a patient sample by coating whole virus or viral protein on a microtiter well [133] . elisa is sensitive and specific, easy to perform, and has a short turnaround time for results. consequently, the assay has been developed and extensively utilized for the detection and serosurveillance of human viral pathogens. recently, adams et al. [134] developed antibody-capture elisa for the detection of sars-cov-2 igm or igg in human plasma samples. e assay was tested by using 40 plasma samples from rt-pcr-confirmed sars-cov-2 infected patients and 50 plasma samples from healthy control. it demonstrated overall sensitivity of 85%, compared to rt-pcr and specificity of 100% in the detection of anti-sars-cov-2 igg or igm in plasma samples. is assay detected igg levels in all of rt-pcr positive individuals from ≥10 days following symptoms onset with a sensitivity of 100%. similarly, colavita et al. [135] developed antibodycapture elisa for the detection of anti-sars-cov-2 igg, igm, and iga in serum samples. e assay was validated using 553 serum samples collected from suspected and confirmed sars-cov-2 infection cases, healthy donors, and patients positive for other infections or autoimmune conditions. e assay showed overall sensitivity of 91.7% and 97.9% for the detection of igg and iga in serum sample, respectively, and specificity of >96% for all antibody types, compared to ifa reference test. chen et al. [22] also developed antigen-capture elisa for the detection of mers-cov in clinical specimens. e assay demonstrated a lod of <1 ng of mers-cov-recombinant nucleocapsid protein/ml and specificity of 100%. antigen-capture elisa was developed for rapid detection of dengue virus ns1 and differentiation of denv serotypes in human specimens. e overall sensitivity and specificity of the test were 84.85% and 100%, respectively, compared to rt-qpcr, and the sensitivity rates for serotyping were 88.2%, 94.7%, 75%, and 66.6% for denv serotype 1 (denv1), denv2, denv3, and denv4, respectively, with no cross-reactivity among serotypes [136] . elisa was also employed for the detection of several other medical viruses, including ebola virus [133] , hsv-2 [137] , sars-cov [138] , hepatitis viruses [139] , h5n1 influenza virus [140] , and zikv [141] . commercial antibody-capture elisa-based test kit (anti-zikv iga, igg or igm elisa) is available, developed by euroimmun ag (germany), for serodiagnosis of acute and past zikv infections. e assay uses zikv-specific ns1 recombinant antigen. e overall sensitivity and specificity of the assay are 100% and 94%, respectively, compared to rt-pcr. creative diagnostics (usa) also developed sandwich elisa-based commercial kit (hiv 1 and 2 ag/ab elisa kit) for qualitative determination of antigens or antibodies to hiv-1 and hiv-2 in human serum or plasma samples. e assay uses recombinant hiv antigens (hiv-1 glycoprotein (gp)41, gp120, and hiv-2 gp36) and anti-hiv viral gag protein p24 antibodies. e lod of the assay for the detection of hiv p24 antigen is about 1pg/ml. moreover, bio-rad (france) developed ns1 ag capture elisa-based commercial kit (platelia dengue ns1 ag) for the qualitative or semiquantitative detection of dengue virus ns1 antigen in human serum or plasma samples. e assay employs anti-ns1 monoclonal antibody (mab) as capture antibody and anti-ns1 mab-hrp conjugate as detection antibody. e sensitivity rates of the assay related to virus serotype are 88.9%, 87.1%, 100%, and 93.3% for denv1, denv2, denv3, and denv4, respectively, compared to rt-pcr, and specificity of the assay is 100% for all serotypes. recently, euroimmun ag (germany) has developed antibodycapture elisa-based kit (anti-sars-cov-2 elisa igg) for qualitative detection of igg to sars-cov-2 in human serum or plasma samples. e assay uses recombinant s1 protein of sars-cov-2 as capture antigen. e assay has received eua from us fda for use in authorized laboratories. e estimated sensitivity and specificity of the assay are 90% and 100%, respectively, compared to nucleic acid amplification test. epitope diagnostics, inc. (usa) has also developed two types of elisa kits (covid-19 igg elisa and covid-19 igm elisa kits) for the detection of anti-sars-cov-2 igg and igm in human serum samples, respectively. covid-19 igg elisa kit uses sars-cov-2 recombinant antigen and hrp labeled anti-human igg antibody. covid-19 igm elisa employs anti-human igm antibody and hrp labeled sars-cov-2 recombinant antigen. e assays have a lod of 5iu/ml. e kits are approved by fda for clinical and research use. western blotting (also known as immunoblotting) assay detects viral proteins or antiviral antibodies. for detection of viral proteins, denatured whole viral proteins are first separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). viral proteins are then electrotransferred onto nitrocellulose membrane. e membrane is then incubated with enzyme conjugated antibodies specific for the viral proteins. if the viral proteins are bound by enzyme labeled antibody, addition of a chromogenic substrate leads to the formation of colored bands at the sites of the viral antigens ( figure 5 ) [19, 132] . for detection of antiviral antibodies, viral specific denatured proteins are electrophoretically blotted onto nitrocellulose membrane after subjected to sds-page. e membrane is then incubated with patient serum. if the patient serum contains antibodies against the viral proteins, they will bind to their specific viral proteins. e addition of enzyme conjugated secondary anti-human antibody and a chromogenic substrate results in the production of colored bands at the locations of the viral proteins [142] . immunoblotting has been used in clinical diagnosis for serosurveillance and as confirmatory tests for human viral infection. he et al. [143] developed western blot assay for detection of antibodies against sars-cov in human serum samples. e assay demonstrated a sensitivity of 98.3% and specificity of 90.9%, compared to ifa. western blotting assay was also used for the detection of anti-chikungunya virus antibody in human serum. sensitivity of 83.3% and specificity of 96.7% were demonstrated by the assay using 30 sera from confirmed chikungunya virus infected patient and 30 normal sera [144] . in one study, western blotting was a promising method for surveillance of hiv-1 infection in resource-limited regions [145] . e assay was also used for the detection and confirmation of hcv and hiv infections [146] [147] [148] . western blotting assay is also commercially available. for example, j. mitra and co. pvt. ltd (mumbai, india) developed commercial kit (hiv 1 and hiv 2 western blot) for the detection of antibodies to hiv-1 and hiv-2 in human serum or plasma samples. e assay uses preblotted nitrocellulose membrane strips with resolved hiv-1 viral lysate and hiv-2 antigen (gp36). e assay has 100% sensitivity and 100% specificity when compared with licensed western blot test. gs hiv-1 western blot kit for the detection of antibodies to hiv-1 in human serum, plasma, or dried blood spots is also available developed by bio-rad laboratories (redmond, usa). e assay uses preblotted nitrocellulose membrane strips with resolved hiv-1 viral proteins. e assay has 100% sensitivity and 87.2% specificity, compared to licensed hiv-1 western blot test. immunofluorescence assay is commonly conducted for the detection of viral antigens or antiviral antibodies in clinical samples. e assay is conducted in two formats: direct immunofluorescence assay (dfa) that detects viral antigens in patient sample [149] and indirect immunofluorescence assay (ifa) that detects antiviral antibody [150] or viral antigen [151] in clinical specimen. in the dfa, antibody that recognizes viral antigen is directly conjugated to fluorescent dye. in the ifa, viral antigen specific antibody is unlabeled and is detected with a second fluorescently labeled anti-human antibody (figure 6 ). ifa is more sensitive than dfa because several fluorescently labeled anti-immunoglobulin antibodies bind to each antiviral antibody, increasing the intensity of fluorescence at the site of each antiviral antibody. e most widely used fluorescent dye in diagnostic virology is fluorescein isothiocyanate (fitc), which emits an intense yellow-green fluorescence, but rhodamine, which emits a deep red fluorescence, is also available. after staining, the specimen is examined under fluorescence microscope with a source of incident uv light [1, 16, 132] . ifa was used for the diagnosis of sars. e assay showed 100% sensitivity and 100% specificity in the detection of anti-sars-cov igg in human serum samples when compared to rt-pcr [150] . madhusudana et al. [152] developed ifa for the detection of anti-rabies virus antibodies in human serum and csf. when compared to the mouse neutralization test, the assay demonstrated a sensitivity of 97.2% and a specificity of 97.9%. ifa was also used for direct detection of hsv antigen in clinical specimens with sensitivity of 84.6% and specificity of 95.7%, compared to the tissue culture method [151] . moreover, ifa was applied for subtyping of influenza a virus with 100% agreement to rt-pcr [153] . ifa was also used for the detection of ebv [21] and as a confirmatory test for hiv-1 [154] . concerning dfa, in one study, it showed 60% sensitivity and 96% specificity in the detection of pandemic influenza a (h1n1) pdm09 in children when compared to rt-qpcr [149] . in another study, dfa showed high specificity (99-100%) in comparison to rt-qpcr for the detection of rsv in children [155] . ifa-based commercial test kit (anti-zikv iift) is available, developed by euroimmun ag (germany), for the detection of zikv infection. e assay uses the complete zikv particles as antigen. consequently, cross-reactivities with antibodies against viruses of the flavivirus family can occur. de ory et al. [156] (figure 7 ). e dilution rate where complete inhibition of agglutination of rbcs occurred is recorded. e hi titer, therefore, is the reciprocal of the last serum dilution which completely inhibits ha [10, 132, 157] . hi was utilized for a number of applications in diagnostic virology. e assay was used for serosurveillance of influenza a (h1n1) pdm09 virus [158] and measles virus [159] . in one study, hi assay was applied to assess the efficacy of pandemic influenza vaccine [160] . in a validation study using sera from 79 rt-qpcr-confirmed cases and 176 sera from a nonexposed population, hi assay showed high sensitivity (92%) and specificity (91%) for the detection of human infection with 2009 pandemic h1n1 virus [161] . immunological diagnostic methods are widely employed in routine clinical diagnosis of human viral infections worldwide. e methods have several advantages, such as high sensitivity and specificity, relatively simple to conduct, rapidity, and possibility of testing several specimens simultaneously [10, 138] . however, immunological-based assays do have several limitations. e assays are subject to interferences. interferences in immunoassays may result from the presence of (a) cross-reactive agents in the sample that carry similar or the same epitopes as the viral antigen of interest, leading to false-positive result [10, 162] ; (b) endogenous antibodies, like autoantibodies, heterophilic antibodies, or human anti-animal antibodies in the specimen. despite the fact that viral antigen is not present in the sample, endogenous antibodies may interact with antiviral antibodies or detection antibodies, leading to false-positive result [162, 163] . e specificity of immunoassay may be affected when they are used in malaria-endemic areas. as it is known, plasmodium induces nonspecific polyclonal b-cell activation that leads to generation of nonspecific antibodies [164] . ese broad specific antibodies may react with a variety of antigens, leading to false-positive test. in one study, of 34 samples from pcr confirmed malaria patients, 14 samples were positive or borderline for anti-zikv antibodies in commercially available zikv elisa test kit. when these 14 samples were tested using virus neutralization assay, zikv infection was not demonstrated in the 11 samples [165] . hi assay is laborious and time-consuming. e interpretation of the assay results between laboratories may be different as no standard reagents are available for the assay [153, 157, 166] . in case of if assay, prolonged exposure where specific antibody is absent in the clinical specimen, the viral hemagglutinin is free to agglutinate the rbcs. e hi titer of s1-s5 is 2 10 . abbreviations: r, rbcs only; v + r, virus and rbcs; pc, positive control; s, sample (unpublished data). of specimen to uv light leads to fading of fluorescence that could result in false-negative test [167] . reagents and equipment that are used in some of the immunoassays are expensive [10, 11] . most of viral diseases are endemic to ethiopia [168] . serosurveys have demonstrated the high prevalence rate of hbv [169, 170] , hcv [170] , hiv [171] , and hsv-2 [137] . e population is vulnerable to rabies [172] and influenza [173] . rotaviral diarrhea is the leading cause of morbidity and mortality in children [174] . recently, like other nations in the globe, public health and economic sectors of ethiopia are heavily challenged by covid-19 pandemic. immunological methods, mostly commercial elisa test kits [137, 169] and immunochromatographic test kits [170, 175] , are used for the detection of viral infections in most clinical laboratories in the country. ifa technology is available only in ethiopian public health institute for the detection of rabies virus infection in suspected dogs that bit humans [176] . conventional rt-pcr is used for the detection of influenza virus in human specimens in national influenza laboratory [173] . recently, the rt-qpcr technique is widely used in several research institutes, universities, and clinical laboratories for the detection of sars-cov-2 in clinical samples. in general, few molecular techniques such as conventional pcr and rt-pcr are utilized in research institutes and universities for research purposes. since most laboratories are budget-restricted and do not have trained laboratory personnel, molecular methods are not used for routine clinical diagnosis of human viral infections in the country. nationwide use of rt-qpcr technologies for the diagnosis of covid-19 and the experiences obtained will open the door to introduce molecular techniques for routine laboratory testing of other human viral infections. e introduction of nucleic acid-based diagnostic tests into diagnostic virology has made tremendous improvement in the detection of human viral infections. since nucleic acidbased diagnostic tests are highly sensitive and specific, they play a crucial role in the diagnosis and control of medical viruses. molecular diagnostic methods diagnose viral infections by detecting viral rna or dna. erefore, these techniques can pick infected individuals before antibody response is mounted against the virus in question. is is especially important in young, elderly, and immunosuppressed patients. however, they are beyond the reach of resource-limited nations due to their high cost, instrumentation complexity, and requirement for technical expertise. immunoassays also play a significant role in the diagnosis and serosurveillance of viral infections worldwide. although immunotechniques are easy to perform and inexpensive compared to molecular methods, they are not widely available in low-income countries. consequently, scientists are working hard to develop inexpensive good quality tests in low-income nations. moreover, most countries in the developing world are training their citizens abroad and inland at postgraduate level by opening relevant departments and institutes. e authors declare that there are no conflicts of interest. diagnostic approaches for viruses and prions in stem cell banks comparisons of highly virulent h5n1 influenza a viruses isolated from humans and chickens from hong kong transmission dynamics and control of severe acute respiratory syndrome pandemic (h1n1) 2009-update 100. weekly update, who zika virus infection in pregnant women in rio de janeiro diagnosis of ebola virus disease: past, present, and future stability of zika virus in urine: specimen processing considerations and implications for the detection of rna targets in urine early epidemiological analysis of the coronavirus disease 2019 outbreak based on crowdsourced data: a population-level observational study recent advances in diagnostic testing for viral infections laboratory diagnosis of human rabies: recent advances global hepatitis report cumulative number of confirmed human cases of avian influenza a (h5n1) reported to who recent advances and perspectives of nucleic acid detection for coronavirus coronavirus disease 2019 (covid-19) situation report-156 detection of rna viruses: current technologies and future perspectives immunologic and molecular methods for viral diagnosis biotechnology to improve health in developing countries: a review methods for rapid virus identification and quantification immunological detection and characterization detection of epstein-barr virus infection subtype in patients with multiple sclerosis by indirect immunofluorescence assay a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus molecular detection of bacterial and viral pathogens-where do we go from here? molecular diagnosis of infectious diseases using cytological specimens point-counterpoint: is the era of viral culture over in the clinical microbiology laboratory? screening of blood donations for hiv-1 and hcv rna by transcription-mediated amplification assay: one year experience rapid detection of dengue viral rna by nucleic acid sequence-based amplification (nasba) epstein-barr virus (ebv) detection and typing by pcr: a contribution to diagnostic screening of ebv-positive burkitt's lymphoma diagnostic accuracy of a rapid rt-pcr assay for point-of-care detection of influenza a/b virus at emergency department admission: a prospective evaluation during the 2017/2018 influenza season optimization of qrt-pcr assay for zika virus detection in human serum and urine evaluation of a rapid and sensitive rt-qpcr assay for the detection of ebola virus detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr comparison of a conventional polymerase chain reaction with real-time polymerase chain reaction for the detection of neurotropic viruses in cerebrospinal fluid samples real-time pcr in the microbiology laboratory loop mediated isothermal amplification (lamp) assays as a rapid diagnostic for covid-19 potentials and limitations of molecular diagnostic methods in food safety e use of next generation sequencing in the diagnosis and typing of respiratory infections development of multiplex reverse transcription-polymerase chain reaction for simultaneous detection of influenza a, b and adenoviruses saliva polymerase-chain-reaction assay for cytomegalovirus screening in newborns determination of viral load by quantitative real-time pcr in herpes simplex encephalitis patients nucleic acid sequence based amplification (nasba)-prospects and applications nucleic acid sequence-based amplification development of a nucleic acid sequence-based amplification assay that uses gag-based molecular beacons to distinguish between human immunodeficiency virus type 1 subtype c and c' infections in ethiopia a simple and rapid method for the detection of hiv-1/hcv in co-infected patients reverse-transcription loop-14 international journal of microbiology mediated isothermal amplification for rapid detection of respiratory syncytial virus directly from nasopharyngeal swabs development of a loop-mediated isothermal amplification assay for rapid detection of bk virus new closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination loop-mediated isothermal amplification (lamp): principle, features, and future prospects specific synthesis of dna in vitro via a polymerase-catalyzed chain reaction application of molecular diagnostic techniques for viral testing detection of hemagglutinin variants of the pandemic influenza a (h1n1) 2009 virus by pyrosequencing detection of cytomegalovirus in urine from newborns by using polymerase chain reaction dna amplification diagnosis of cytomegalovirus pneumonia by the polymerase chain reaction with archived frozen lung tissue and bronchoalveolar lavage fluid investigational approach to adenoviral conjunctivitis: comparison of three diagnostic tests using a bayesian latent class model quantitation of viral dna by real-time pcr applying duplex amplification, internal standardization, and twocolor fluorescence detection diagnosis of respiratory syncytial virus infection: comparison of reverse transcription-pcr to viral culture and serology in adults with respiratory illness detection of ebola virus in oral fluid specimens during outbreaks of ebola virus hemorrhagic fever in the republic of congo kinetic pcr analysis: real-time monitoring of dna amplification reactions detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of ermus aquaticus dna polymerase real-time pcr in clinical microbiology: applications for routine laboratory testing utility of real-time pcr in the diagnosis of primary epstein-barr virus infection rapid diagnosis of human adenovirus b, c and e in the respiratory tract using multiplex quantitative polymerase chain reaction a triplex quantitative real-time pcr assay for differential detection of human adenovirus serotypes 2, 3 and 7 real-time rt-pcr for quantitation of hepatitis c virus rna real-time quantitative pcr assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients real-time pcr for detecting circulating dengue virus in the guangdong province of china in 2006 a new real-time quantitative pcr for diagnosis and monitoring of hiv-1 group o infection a diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type a detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection molecular beacon probes-base multiplex nasba real-time for detection of hiv-1 and hcv nucleic acid-based diagnostics for infectious diseases in public health affairs higher specificity of nucleic acid sequence-based amplification isothermal technology than of real-time pcr for quantification of hiv-1 rna on dried blood spots development and validation of a commercial real-time nasba assay for the rapid confirmation of influenza a h5n1 virus in clinical samples evaluation of a transcription mediated amplification assay for detection of herpes simplex virus types 1 and 2 mrna in clinical specimens real-time nucleic acid sequence based amplification (real-time nasba) for detection of norovirus enhanced clinical utility of the nuclisens easyq rsv a+b assay for rapid detection of respiratory syncytial virus in clinical samples loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers detection of japanese yam mosaic virus by rt-lamp loop-mediated isothermal amplification (lamp) -review and classification of methods for sequence specific detection loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products loop-mediated isothermal amplification (lamp) for the diagnosis of zika virus: a review detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification loop-mediated isothermal amplification assay for the diagnosis of retinitis caused by herpes simplex virus-1 detection of hepatitis c virus by an improved loop-mediated isothermal amplification assay detection of middle east respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (rt-lamp) development and evaluation of a loop-mediated isothermal amplification assay for the detection of adenovirus 40 and 41 rapid detection of epstein-barr virus dna by loop-mediated isothermal amplification method development and evaluation of loop-mediated isothermal amplification assay for rapid and inexpensive detection of cytomegalovirus dna in vitreous specimens from suspected cases of viral retinitis rt-lamp for rapid diagnosis of coronavirus sars-cov-2 development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov-2 development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel sars-cov-2 development and evaluation of reverse transcription-loopmediated isothermal amplification (rt-lamp) assay coupled with a portable device for rapid diagnosis of ebola virus disease in guinea comparison between rt-pcr, nasba and rt-lamp methods for detection of coxsackievirus b3 visual detection of pandemic influenza a h1n1 virus 2009 by reverse-transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye an updated loop-mediated isothermal amplification method for rapid diagnosis of h5n1 avian influenza viruses loop-mediated isothermal amplification assay for rapid detection of hepatitis c virus detection of acute hiv-1 infection by rt-lamp detection of zika virus using reverse-transcription lamp coupled with reverse dot blot analysis in saliva infectious diseases detection by microarray: an overview of clinical relevant infections utility of dna microarrays for detection of viruses in acute respiratory tract infections in children detection of herpesvirus and adenovirus co-infections with diagnostic dna-microarrays dna microarrays for virus detection in cases of central nervous system infection dna microarray for detection of gastrointestinal viruses dna microarray platform for detection and surveillance of viruses transmitted by small mammals and arthropods dna probe array for the simultaneous identification of herpesviruses, enteroviruses, and flaviviruses characterization of real-time microarrays for simultaneous detection of hiv-1, hiv-2, and hepatitis viruses a dna microarray-based assay to detect dual infection with two dengue virus serotypes an efficient microarray-based genotyping platform for the identification of drug-resistance mutations in majority and minority subpopulations of hiv-1 quasispecies development of a simple microarray for genotyping hiv-1 drug resistance mutations in the reverse transcriptase gene in rural tanzania microarray-based genotyping and detection of drug-resistant hbv mutations from 620 chinese patients with chronic hbv infection development of a single nucleotide polymorphism dna microarray for the detection and genotyping of the sars coronavirus diagnostic microarray for influenza b viruses viral discovery and sequence recovery using dna microarrays metagenomics and the molecular identification of novel viruses dna microarrays: types, applications and their future next-generation sequencing for infectious disease diagnosis and management current approaches for diagnosis of influenza virus infections in humans application of next generation sequencing in clinical microbiology and infection prevention assessing the performance of the oxford nanopore technologies minion nanopore sequencing and assembly of a human genome with ultra-long reads rapid and accurate sequencing of enterovirus genomes using minion nanopore sequencer whole genome sequencing of influenza a and b viruses with the minion sequencer in the clinical setting: a pilot study multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples a method to identify respiratory virus infections in clinical samples using nextgeneration sequencing evolutionary dynamics of local pandemic h1n1/2009 influenza virus lineages revealed by whole-genome analysis first evaluation of the next-generation sequencing platform for the detection of hiv-1 drug resistance mutations in belgium newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda next-generation sequencing and bioinformatic approaches to detect and analyze influenza virus in ferrets metagenomic nextgeneration sequencing aids the diagnosis of viral infections in febrile returning travellers nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect elisa) for detecting antibodies specific to ebola virus and marbug virus evaluation of antibody testing for sars-cov-2 using elisa and lateral flow immunoassays evaluation of elisa tests for the qualitative determination of igg, igm and iga to sars-cov-2 development of an enzyme-linked immunosorbent assay for rapid detection of dengue virus (denv) ns1 and differentiation of denv serotypes during early infection transmission of herpes simplex virus type 2 among factory workers in ethiopia detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay detection of hepatitis a, b, and c virus-specific antibodies using oral fluid for epidemiological studies newly established monoclonal antibodies for immunological detection of h5n1 influenza virus high specificity of a novel zika virus elisa in european patients after exposure to different flaviviruses laboratory diagnostics for hiv infection development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome western blot detection of human anti-chikungunya virus antibody with recombinant envelope 2 protein western blot-based logistic regression model for the identification of recent hiv-1 infection: a promising hiv-1 surveillance approach for resource-limited regions early detection of anti-hcc antibody in acute hepatitis c virus (hcv) by western blot (immunoblot) using a recombinant hcv core protein fragment detection of human immunodeficiency virus type 1 (hiv-1) antibody by western blotting and hiv-1 dna by pcr in patients with aids western blot profile in hiv infection accuracy of rapid influenza diagnostic test and immunofluorescence assay compared to real time rt-pcr in children with influenza a(h1n1)pdm09 infection immunofluorescence assay for serologic diagnosis of sars detection of herpes simplex virus in direct specimens by immunofluorescence assay using a monoclonal antibody comparative evaluation of a simple indirect immunofluorescence test and mouse neutralization test for assaying rabies antibodies subtyping influenza a virus with monoclonal antibodies and an indirect immunofluorescence assay new indirect immunofluorescence assay as a confirmatory test for human immunodeficiency virus type 1 comparison of direct fluorescence assay and real-time rt-pcr as diagnostics for respiratory syncytial virus in young children comparative evaluation of indirect immunofluorescence and ns-1-based elisa to determine zika virus-specific igm current progress with serological assays for exotic emerging/re-emerging viruses hemagglutination inhibition antibody response following influenza a(h1n1)pdm09 virus natural infection: a crosssectional study from thirthahalli study on assays for the detection of serum antibodies to measles from children and its standardization qualification of the hemagglutination inhibition assay in support of pandemic influenza vaccine licensure sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic h1n1 virus in u.s. populations interferences in immunoassays endogenous antibody interferences in immunoassays how malaria modulates memory: activation and dysregulation of b cells in plasmodium infection letter to the editor: specificity of zika 18 international journal of microbiology virus elisa: interference with malaria detection of np, n3 and n7 antibodies to avian influenza virus by indirect elisa using yeast-expressed antigens microscopy in virology: application and sample preparation viral diseases in ethiopia: a review seroepidemiology of hepatitis b virus in addis ababa, ethiopia: transmission patterns and vaccine control prevalence and risk factors of hepatitis b and hepatitis c virus infections among patients with chronic liver diseases in public hospitals in addis ababa, ethiopia hiv prevalence correlates with high-risk sexual behavior in ethiopia's regions rabies: a major fatal viral disease of humans and animals in ethiopia challenges of establishing routine influenza sentinel surveillance in ethiopia epidemiology of rotavirus diarrhea among children aged less than 5 years in rural southern ethiopia evaluation of diagnostic performance of non-invasive hiv self-testing kit using oral fluid in addis ababa, ethiopia: a facility-based cross-sectional study overview of rabies in and around addis ababa key: cord-270399-yfko8mpc authors: foster, allison; khan, zohaib; siddiqui, aisha; singh, sukhdev; atere, muhammed; nfonoyim, jay m title: it’s complicated: a case report on a covid-19-positive hiv patient presenting with rhabdomyolysis and acute kidney injury date: 2020-10-15 journal: sage open med case rep doi: 10.1177/2050313x20965423 sha: doc_id: 270399 cord_uid: yfko8mpc the sars-cov-2/covid-19 pandemic in early 2020 has had a devastating impact on health systems around the world. while viral pneumonia remains the most common complication, reports are surfacing of cases with neurological, cardiac, and renal involvement. even less is known about the implications in special high-risk populations. in this report, we discuss a unique case of an hiv-positive patient in new york city who presented with a 2-week history of worsening fatigue, cough, dyspnea, and myalgias and was found to have covid-19 pneumonia and acute kidney injury. he was managed for severe uremic metabolic acidosis and electrolyte abnormalities with emergent hemodialysis and supportive therapy with subsequent improvement. direct involvement of sars-cov-2 and pneumonia-induced rhabdomyolysis were identified as the precipitating factors of his acute kidney injury. the pathophysiologic mechanisms of acute kidney injury, sars-cov-2 renal tropism, and the impact of highly active antiretroviral therapy on covid-19 pneumonia are discussed. we highlight the importance of clinician awareness of this potentially fatal complication of covid-19 pneumonia, particularly in the hiv-positive population as early recognition and management can have favorable outcomes. in december 2019, sars-cov-2 a coronavirus caused an outbreak of viral pneumonia in wuhan, china. the pandemic illness termed covid-19 has now infected over 2 million people and has caused over 140,000 deaths worldwide. 1 recent studies have demonstrated that approximately 20%-40% of covid-19 patients admitted to the intensive care unit (icu) develop acute kidney injury (aki). 2,3 covid-19-induced aki presents with a more severe clinical course, particularly in the icu setting, and is a negative prognostic factor with respect to survival. the mechanisms of kidney injury in covid-19 infection have been hypothesized to be due to: (1) hypoperfusion-related injury of the renal tubule, (2) viral tropism for the kidney due to highly expressed angiotensin-converting enzyme (ace)-2 receptors on podocytes and epithelial cells that are essential for viral uptake, and (3) cytokine-induced nephropathy. 4 reports are limited regarding patients on antiviral therapy who present with covid-19 and subsequently develop kidney injury. furthermore, a common complication of infection in hiv-positive patients is rhabdomyolysis which tends to precede aki. hiv, overlapping infections, and antiviral medications used to treat hiv are known to be nephrotoxic. considering the potential of nephrotoxicity of sars-cov2, we must attempt to distinguish the causes and manifestations of kidney injury as well as the possibility of combined effects. early recognition of covid19 and aki in vulnerable populations is crucial to intervening early and lowering mortality. a man in his 40s with a history of hiv on highly active antiretroviral therapy (haart) and hypertension with a body mass index (bmi) of 34.9 presented to the emergency department (ed) after being seen at an urgent care clinic with hypotension and dyspnea. he visited his primary care provider 2 weeks prior for fever, chills, and cough productive of grayish sputum; however, his symptoms persisted despite completing a course of azithromycin. during the interview, he denied recent travel or covid-19 exposure. he endorsed fatigue, leg cramping, and poor appetite. cd4+ count and viral load were unknown to the patient. his vitals were unstable in ed with tachypnea (58 breaths/min) and hypotension (70/50 mmhg), heart rate of 91 beats/min, temperature of 37.1°c, and an oxygen saturation of 92% while on a non-rebreather mask at a flow rate of 15 l. on examination, he was in respiratory distress with decreased breath sounds in bilateral lung fields. the patients' baseline creatinine was unknown at the time of presentation. the remainder of the examination was noncontributory; patient had good skin turgor and appropriate capillary refill. vital signs for the duration of the visit are provided in table 1 . fluid resuscitation with iv normal saline was initiated. on re-examination, patient was hemodynamically stable with persistent tachypnea (28 breaths/min). he had multiple electrolyte abnormalities which are presented in table 2 . inflammatory markers, troponin, and coagulopathy studies were not performed. urinalysis was significant for a white blood cell (wbc) > 100/high-power field (hpf), red blood cell (rbc) 3/hpf, and ph 5.0. bedside electrocardiogram (ekg) showed tachycardia (105 beats/min) and a prolonged qtc (518 ms) ( figure 1) . a rapid nasopharyngeal swab collected was positive for sars-cov-2, prompting contact and droplet precautions. patient was tested for hepatitis b and c which were both negative; however, the patient was not tested for concomitant influenza. chest x-ray showed patchy infiltrates laterally in the right midlung and the right base with lesser peripheral patchy infiltrates laterally from left mid to lower lung. in the ed, iv magnesium and iv terbutaline were administered. patient was admitted to the icu, for management of acute renal failure with electrolyte imbalance, rhabdomyolysis, anion gap metabolic acidosis secondary to uremia, and covid-19 pneumonia. patient denied prior history of renal disease. in the icu, 3 amps of iv sodium bicarbonate was administered followed by iv bicarbonate drip. a straight catheter drained 25 ml of tea-colored urine prompting the insertion of a foley catheter. all nephrotoxic agents including haart were held. prolongation of the qtc on ekg contraindicated the use of hydroxychloroquine and azithromycin. on hospital day 1, despite continued management, he showed no significant improvement with an elevated uric acid of 13.8 mg/dl. his renal function remained severely compromised with persistent severe metabolic acidosis; creatine kinase (ck) remained elevated at 2185 u/l with continued electrolyte abnormalities (hypokalemia, hypocalcemia, hyperphosphatemia, hypermagnesemia). chest x-ray showed persistent patchy peripheral infiltrates in the right lung with lesser patchy peripheral infiltrates just above the left lung base ( figure 2 ). given his intractable acidosis, a hemodialysis catheter was placed for emergent dialysis. lopinavir and ritonavir were held due to nephrotoxicity. viral hiv load was low at 47 hiv-1 rna copies/ ml. patient was closely monitored in supplementary icu area until discharged on hospital day 7. during the hospital stay, following initial fluid resuscitation with lactated ringers at a rate of 100 ml/h for 10 h, normal saline was administered in three 1000 ml boluses initially and then at a rate of 150 ml/h for 6 h. for the remainder of the hospital stay, 6000 ml of lactated ringer's was administered at a rate of 150 ml/h for 40 h and 500 ml of normal saline on the final day of admission at a rate of 100 ml/h. patient was dialyzed via hemodialysis and electrolyte imbalances were corrected resulting in normalization of the patient's vital signs except for persistent tachycardia (113 beats/min). patients' blood pressure was 123/70 postdialysis. electrolyte imbalances, glomerular filtration rate (gfr), uric acid level, and ck level improved; hypokalemia and patient's anion gap metabolic acidosis resolved. improvement in renal function prompted discontinuation of hemodialysis. patient's foley catheter was removed for a voiding trial, and he was in no acute distress on 4-l nasal cannula saturating at 96%. a renal biopsy was unable to be obtained from the patient. covid-19 has challenged health care systems globally with a myriad of presentations. with an estimated 14-day incubation period, most individuals show symptoms 4-5 days postexposure. symptomatic covid-19 patients generally present with fever, cough, malaise, and shortness of breath, usually managed at home with supportive care. other commonly reported symptoms are headache, sore throat, rhinorrhea, and diarrhea. 5 well-recognized risk factors for severe presentations include age, comorbidities such as lung disease, coronary artery disease, hypertension, diabetes, chronic kidney disease, and cancer. 6 in hospitalized patients, lymphocytopenia, elevation of liver enzymes, and ck are associated with icu admission and higher mortality. 7 this patient had an elevated white blood cell count and his ck was significantly elevated at infiltrates with peripheral, lower zone distribution and bilateral involvement, which is commonly seen in covid-19 pneumonia. 8 the patient presented with significant elevations in ck; his imaging revealed severe covid-19 pneumonia, he was hiv positive, and he had a history of hypertension and was obese. despite many risk factors and findings that would lend to poor prognosis, the patient was hospitalized for 7 days and required only a single round of hemodialysis. this lends the question of why his clinical course was brief compared with the average of 10 days for his age group and risk group. 9 recently, hemodialysis has been proposed as a prospective treatment for sars-cov-2 due to the affinity of the virus for renal parenchyma. our patient did have a remarkable outcome following a sole round of hemodialysis via shiley catheter. future large-scale studies will be needed to provide insight to the efficacy of hemodialysis as a treatment for covid-19. in a recent histopathologic examination of covid-19 patients, 9 sars-cov-2-associated pulmonary lesions manifest as inflammation of the parenchyma and interstitium, with hyperplasia of the alveolar epithelium and formation of hyaline membranes. damage was evidenced in areas outside of the lungs with lesions apparent in the cells of the immune, cardiac, hepatic, and renal systems. 8 this tissue tropism is due to high expression of ace-2 receptors on the alveoli. sars-cov-2 potentially gains entry into cells through binding of a spike protein on its envelope to ace-2 receptors; uptake is via a clathrin-dependent endocytosis mechanism. 10 reports show that there are numerous ace-2 receptors in podocytes and glomerular mesangial cells of the kidney. 11 given these findings, renal involvement of covid-19 is not an unexpected phenomenon. in a retrospective study of patients from wuhan from 17 january 2020 to 3 march 2020 with lab-confirmed covid-19, 27% of patients exhibited acute renal failure. 12 post-mortem analysis showed severe acute tubular necrosis (atn) and lymphocyte infiltration in the kidneys with sars-cov-2 np antigen identified via immunohistochemistry and accumulated viral particles on electron microscopy. 11 reports have also indicated severe hyponatremia and hypokalemia in patients infected with sars-cov-2, possibly due to an inflammatory response pathologically causing electrolyte impairment secondary to non-osmotic release of vasopressin. 13 a transtubular potassium gradient for this patient was scored as 8 indicating hypokalemia as a result of renal potassium wasting supporting the idea that sars-cov-2 renal damage was responsible for this patient's electrolyte abnormalities. these findings lend support to the hypothesis that sars-cov-2 may directly induce renal damage. aki is an abrupt decrease in the gfr resulting in electrolyte disturbances, retained metabolic waste, and is a well-recognized complication of hiv. 14 the symptoms of aki are generally nonspecific apart from oliguria, making blood urea nitrogen (bun) and creatinine essential for diagnosis. 14 diagnostic criteria for aki are defined as either a 50% increase in serum creatinine from the patient's baseline or a reduction in creatinine clearance by 50%. 14 a strong correlation exists between the onset of aki symptoms and the patient requiring intubation with mechanical ventilation. 15 the rate of aki in patients receiving mechanical ventilation was noted to be as high as 89.7% in some studies compared to that of patients not receiving mechanical ventilation which was 21.7%. 15 despite previous studies from italy and china which determined the rate of aki to be from 0.5% to 29%, with many studies demonstrating percentages on the lower end of the spectrum, the united states had an astronomical number of aki cases. 15 since china and italy had experienced surges in cases of sars-cov-2 prior to the united states, it is perplexing as to why the covid-19 patients in the united states were more likely to suffer from renal injury. 15 various risk factors have been studied such as hypertension, diabetes mellitus type ii, age as well as obesity; however, mechanical ventilation and the use of vasopressors demonstrate the strongest correlations in terms of risk of developing aki. 15 our patient was maintained on nasal cannula for the duration of his hospitalization and his hypotension was treated with fluid resuscitation instead of vasopressors. although our patients' baseline creatinine was unknown, his elevations in ck, electrolyte derangements, bun, and acute clinical presentation were suggestive of aki. aki in hiv patients has a wide range of causes including prerenal azotemia, rhabdomyolysis, microangiopathy, glomerulonephritis, hepatitis b or c, atn, acute interstitial nephritis, crystal-induced nephropathy, nephrotoxicity from haart, and hiv-associated nephropathy. 13, 14, 16 atn is an important cause to investigate in this patient particularly due to its association with aki, covid-19, and hiv. in our patient, his urinalysis only contained 3 rbc/hpf and no casts which made atn, glomerulonephritis, microangiopathy, and crystal-induced nephropathy less likely. thrombotic microangiopathy (tma) has been noted in patients with covid-19-induced aki. 17 underlying complement disorders and gemcitabine use have been demonstrated as the potential causes of tma in such patients. 17 new data have shown that sars-cov-2 can activate the alternate and lectin pathways of the complement system resulting in an inflammatory response. 17 the inflammation leads to the development of coagulopathy and subsequently tma in some cases. 17 it is a point of contention as to whether infection with covid-19 is a secondary insult to patients with underlying predispositions to the development of tma such as complement disorders or prior use of medications that have been associated with the development of tma. 17 while aki in this patient can be attributed to his initial hypotension and hemodynamic instability resulting in decreased renal artery perfusion, it is crucial to consider that sars-cov-2 infection of the kidneys with subsequent replication in the renal tissue may have played a substantial role in the patients' abrupt decline in renal function. 3 rhabdomyolysis is a common cause of aki among hivpositive patients with an incidence reported to be of 943 per 100,000 person-years. 18 infection is most commonly identified as the precipitating etiology, particularly pneumonia which constitutes 38% of infectious causes. 16 in the hiv population, rhabdomyolysis is associated with increased mortality compared to the rest of the population. 18 rhabdomyolysis can be evoked by various alternative figure 2 . initial chest x-ray. posterior anterior chest x-ray demonstrating patchy peripheral infiltrates in the right lung with lesser patchy peripheral infiltrates just above the left lung base. mechanisms including trauma, crush injuries, use of statins, cocaine, amphetamines, heroin, phencyclidine, musculoskeletal diseases, hyperthermia, hypothermia, burns, muscular ischemia, hypophosphatemia, seizures, prolonged operations, and severe dehydration. 19 the patient stated he had not experienced any trauma, seizures, temperature extremes, prolonged operations, burns, or crush injuries. the patient was screened for illicit drug use upon presentation which was negative and phosphate levels were elevated on presentation. severe dehydration was the only potential cause apart from infection which was significant in this patient as a cause for rhabdomyolysis. studies have demonstrated that covid-19 has been known to cause hypotension in greater than 50% of cases. 20 whether directly through involvement of sars-cov-2 interaction with ace-2 receptor or indirectly from causing hypotension from an undetermined mechanism, this patient's decline in renal function can be attributed to his acute infection with covid-19. in a patient with hiv on haart, it is important to consider the potential impact of therapy on both renal function and the patients' ability to counter infections. although haart increases cd4+ counts and subsequently decreases the risk of certain infections, it is associated with an increased incidence of aki from 2.9% to 6.0% in hiv patients. 16 despite the concern of aki from haart, 52% of all renal injury in hiv patients are primarily caused by infection. 21 delaying administration of haart in hiv patients with aki prevents further renal injury from the medications' nephrotoxic nature. 22 prior to discontinuation upon hospitalization, our patient's haart regimen consisted of lopinavir and ritonavir. the patient endorsed compliance with his haart regimen prior to hospitalization. lopinavir acts via inhibition of viral 3 chromotrypsin-like (3cl) proteases which has been hypothesized as the mechanism for efficacy against sars-cov-2 since 3cl proteases play a fundamental role in the processing of viral rna which interferes with the viral replication process. [23] [24] [25] although the lopinavir/ritonavir combination drug has been investigated as a potential treatment regimen for covid-19, there has been conflicting evidence in terms of efficacy. [21] [22] [23] [24] [25] [26] some studies have reported a reduction of symptoms and sars-cov-2 viral load when initiated early in the treatment course. 2 despite the recommendation by early studies that lopinavir/ritonavir should be used in vulnerable populations with covid-19 pneumonia, more evidence is needed to enforce this practice. [24] [25] [26] our patient's exceptional clinical course despite having hiv lends to the idea that his haart regimen as well as his azithromycin use prior to presentation may have decreased the total amount of sars-cov-2 viral replication in both the renal parenchyma and pulmonary tissue resulting in a rapid recovery and subsequent hospital discharge. the potential of covid-19 to antagonize renal function is crucial to note given most cases of both rhabdomyolysis and aki in hiv patients are precipitated by infections. in the case presented, our patient had significantly elevated ck of 2185 u/l, with drastic elevations in creatinine and significant uremia. it is likely that the aki in our patient was multifactorial involving direct tissue damage from sars-cov-2, covid-19-induced hypotension, and indirect injury through another mechanism compounded by rhabdomyolysis. in this report, we present a case of aki in an hiv-positive patient with covid-19 with a successful outcome. clinicians should be cautious of this complication in hiv patients; those presenting with symptoms consistent with sars-cov-2 infection may be at an increased risk of aki given they are immunocompromised. in our case, the damage to the kidney was likely a combination of pneumonia-induced rhabdomyolysis and direct effects of sars-cov-2 on renal parenchyma. the use of haart therapy and azithromycin prior to admission may have been a contributing factor in the remarkably quick recovery of our patient. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. our institution does not require ethical approval for reporting individual cases or case series. the author(s) received no financial support for the research, authorship, and/or publication of this article. written informed consent was obtained from the patient(s) for their anonymized information to be published in this article. who. novel coronavirus management of acute kidney injury in patients with covid-19 acute kidney injury in covid-19: emerging evidence of a distinct pathophysiology covid-19 and kidney failure in the acute care setting: our experience from seattle world health organization: who. coronavirus who int people who are at higher risk for severe illness clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study frequency and distribution of chest radiographic findings in covid-19 positive patients treatment efficacy analysis of traditional chinese medicine for novel coronavirus pneumonia (covid-19): an empirical study from wuhan histopathology study of multiple coronavirus puncture in three patients with novel coronavirus pneumonia (covid-19 should covid-19 concern nephrologists? why and to what extent? the emerging impasse of angiotensin blockade glomerular localization and expression of angiotensin-converting enzyme 2 and angiotensin-converting enzyme: implications for albuminuria in diabetes il-6, and sars-cov-2 (covid-19) infection: may all fit together? human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. medrxiv. epub ahead of print 6 acute kidney injury in patients hospitalized with covid-19 renal and urologic emergencies in the hiv-infected patient covid-19-associated kidney injury: a case series of kidney biopsy findings acute renal failure in hospitalized patients with hiv: risk factors and impact on in-hospital mortality cardiac and arrhythmic complications in patients with covid-19 rhabdomyolysis in an hiv cohort: epidemiology, causes and outcomes severe recurrent rhabdomyolysis-induced acute kidney injury in a hivinfected patient on antiretroviral therapy acute kidney injury in hiv-infected patients covid-19-the search for effective therapy lopinavir ritonavir: a rapid review of effectiveness in covid-19-cebm. cebm case of the index patient who caused tertiary transmission of coronavirus disease 2019 in korea: the application of lopinavir/ritonavir for the treatment of covid-19 pneumonia monitored by quantitative rt-pcr key: cord-330852-n7j0c4ne authors: fischer, wolfgang b.; wang, yi-ting; schindler, christina; chen, chin-pei title: mechanism of function of viral channel proteins and implications for drug development date: 2012-02-23 journal: int rev cell mol biol doi: 10.1016/b978-0-12-394305-7.00006-9 sha: doc_id: 330852 cord_uid: n7j0c4ne viral channel-forming proteins comprise a class of viral proteins which, similar to their host companions, are made to alter electrochemical or substrate gradients across lipid membranes. these proteins are active during all stages of the cellular life cycle of viruses. an increasing number of proteins are identified as channel proteins, but the precise role in the viral life cycle is yet unknown for the majority of them. this review presents an overview about these proteins with an emphasis on those with available structural information. a concept is introduced which aligns the transmembrane domains of viral channel proteins with those of host channels and toxins to give insights into the mechanism of function of the viral proteins from potential sequence identities. a summary of to date investigations on drugs targeting these proteins is given and discussed in respect of their mode of action in vivo. all rights reserved. the cellular life cycle of viruses is tightly connected with lipid membranes. during viral entry, membranes have to be crossed, and within the cell, the viral life depends on subcellular membranes, since viruses build their proteins along lipid membranes. they also use the principle of compartmentalizing for optimized replication and therefore deform subcellular membranes. another strategy is to capitalize on electrochemical and substrate gradients formed across membranes. for the use of these gradients in the most sensible way, channel-forming proteins are encoded in the genome of the virus. channel-forming proteins are active at almost every stage of the viral replication. they are involved during the entry phase of the virus into the host and also within the infected cell. while the roles of some channels are well identified, for others this role still has to be elucidated. the assumption that similar tasks on a molecular level can only be accomplished by the same type of molecule in almost the same conformational and spatial arrangements inspires this review on the mechanism of function of viral channels in comparison with host channels. what makes knowledge of the mechanism of function so important? there is the obvious reason that we are simply curious about it. one simply cannot stop wondering how mechanics can be achieved in an environment in which we have to leave the concepts of movements in viscous media and can only rely on electrostatic and quantum mechanical principles to get a glimpse of what is happening. learning more about the time domain of the mechanics is highly desirable as well. maybe we can understand the biological "nanomachines" and design new ones, which are "longer lasting," capable of handling bigger loads, and show other improved characteristics. since the viral channel-forming proteins are smaller than their host companions, their architecture depicts miniature design in an already miniaturized world, or going down-scale in an already down-scaled world. on the other hand, there is crucial wish to secure and cure our health as well as to ease our life. how does knowledge about the mechanism of function fit into this wish? if we know the function of an enzyme, which cleaves a specific substrate, we can design a substrate which cannot be cleaved but rather jammed into the active site of the enzyme. if we know the conformation transition the molecule undergoes, we are able to generate more potent drugs. dealing with other proteins than enzymes, such as the channel proteins mentioned here, the site of drug action is not so easy to elucidate. in principle, we "see" the protein but do not know where to go with our drug molecule. consequently, an active site may be one of the many conformations the protein adopts during its "working cycle." from this particular perspective, one has to explore the entire conformational space of the protein as a prerequisite for improved drug development. crystal structures serve as an important starting point for computational drug development representing "frozen" conformations. computational modeling is able to screen and visualize more conformational space and in this respect possibly "link" individual crystal structures. the idea is to analyze the viral channels in respect to larger host channels. therefore, the architecture of those channels, which are anticipated to have a relation to the viral channels, is introduced. the relation is driven by sequence alignment of viral channel forming proteins (vcps) with those of host proteins . identifying related host proteins, specifically gating mechanics are elucidated and cross-related to the viral channels. in general, there are several mechanical movements especially within the lipid membrane known for ion channels, such as moderate conformational changes (cymes and grosman, 2008; miyazawa et al., 2003 ; and references therein) and sliding of helices (chang et al., 1998; kuo et al., 2003) . openings of pores designed by toxins seem to be made irreversibly by assembly (mueller et al., 2009) . finally, the idea of "drug hunting" is outlined in respect to small molecule drugs and peptide drugs. the proteins are introduced with respect to the mechanism of function. all citations are driven by the idea to deliver structural information. for extended references about molecular biological information, it is referred to respective reviews. the work on viral channel-forming proteins has been reviewed in great detail in the literature gonzales and carrasco, 2003 ; and referenced literature below). generally, the name "viroporin" or "vcp" is established in the literature for this class of proteins. so far, some of the channels are very selective for protons, others are permeable for small molecules, while the majority though are weakly selective for ions. as to date, the biophysical role of the vcps can be summarized to alter electrostatic repulsion of proteins so that lipid membranes can approach each other during fusion and budding, as well as alteration of cell homeostasis and membrane depolarization induced cell apoptosis (franco and bortner, 2006) . vcps comprise a series of proteins with different transmembrane (tm) topology. with the discovery of m2 from influenza a and vpu from human immunodeficiency virus type 1 (hiv-1), proteins with a single tm topology have been found. later, proteins such as 2b from polio virus and p7 from hepatitis c virus (hcv) have been proposed to harbor two transmembrane domains (tmds). the recently suggested viral ion channel from severe acute respiratory syndrome coronavirus (sars-cov), 3a, is now the longest channel with three tmds. other proteins found to form channels are belonging to either one of these classes of channels. how can a protein be defined as a channel? channels are made of subunits of proteins forming a circular tertiary structure around a symmetry axis. the channels per se could be either homo-or hetero-oligomeric. they are membrane embedded and conduct more or less selectively either one of the physiological relevant ions. the degree of selectivity comes with the diameter and side chain composition of the pore formed by the assembled proteins, the lumen of the pore. the wider the pore is, the less selective it should be. less selective channel proteins are likely to be called pores (gonzales and carrasco, 2003) , such as, 2b from polio virus, which is able to conduct besides ions also small molecules. in the following, the focus is on those vcps for which detailed structural information is available either from experiments or from computational studies. 2.1. m2, vpu, and p7 2.1.1. m2 from influenza a the genome of influence is spread over eight single-stranded rna segments. on the seventh segment, which encodes the matrix protein m1, a second overlapping reading frame has been identified coding a protein of about 11 kda called m2 (allen et al., 1980; lamb and choppin, 1981; lamb and lai, 1981; winter and fields, 1980) (fig. 6.1a ). its topology is found to be monotopic comprising an n terminal side on the extracellular side marking it a type iii integral membrane protein present at the plasmamembrane of infected cells (lamb et al., 1985) . the amino acid distribution indicates about 18-23 amino acids on the extramembrane side, 19 amino acids membrane spanning, and 54 amino acids toward the c terminus (zebedee and lamb, figure 6 .1 available structures of m2 and bm2 from experimental sources. the structures are shown in "gaussian contact mode" (moe software) indicating hydrophilic residues in blue and hydrophobic residues in green. in addition, helices are shown as yellow bands and specific amino acids are represented in a stick modus (histidines in red and tryptophans in violet). (a) m2 channel: 2l0j from solid state nmr (sharma et al., 2010) ; 2rlf from solution nmr (schnell and chou, 2008) , the drug rimantadine is shown in stick modus (blue); 3bkd from x-ray and expressed protein (stouffer et al., 2008) ; 3c9j from the same source as 3bkd with the drug amantadine shown in stick modus (green); 3lbw from spps (acharya et al., 2010, from spps) ; (b) tmd (2kix) and cytoplasmic domain (2kj1) of bm2 (wang et al., 2009b) . (c) structure of a synthetic peptide corresponding to pb1-f2 solved by nmr spectroscopy (bruns et al., 2007). 1988). it is also present in the virion with about 14-68 molecules (zebedee and lamb, 1988) . m2 assembles into cystein-linked homodimers which noncovalently associate into homotetramers (holsinger and lamb, 1991; sugrue and hay, 1991) . some of the m2 proteins of various subtypes have been identified to be palmitylated at cys-50 (sugrue et al., 1990) . amantadine resistance mutant studies of influenza viruses correlated resistance to mutations in the seventh segment, especially to the tmd of m2 (hay et al., 1985) . mapping the mutations on a helical wheel reveals that the mutations are along one side of the helical structure. the mapping supports the idea that the protein assembles around a kind of central axis which is relevant for its functioning as a proton channel (sugrue and hay, 1991) . experiments with a synthetic peptide construct representing the tmd of m2 reconstituted into artificial bilayers have verified its proton activity (duff and ashley, 1992) . also, whole-cell recordings of m2 and mutant m2 expressed in xenopus laevis show that m2 activity is enabled by low ph and that amantadine is interacting with the channel (wang et al., 1993) . channel activity has also been demonstrated in mammalian cells (wang et al., 1994) and vesicle-based fluorescence essays (schroeder et al., 1994) , with the latter technique confirming proton conductance (lin and schroeder, 2001) . reconstitution of the protein into artificial bilayers also reveals channel activity of m2 (tosteson et al., 1994) . electrophysiological experiments with m2 in mouse erythroleukemia cells have been conducted on ion selectivity confirming that m2 is 10 7 times more selective for protons than for monovalent cations (chizhmakov et al., 1996) . whole-cell recordings with m2 expressed in xenopus oocytes reveal a very low conductance of about 1-10 fa, which allows about 104 protons per second to pass (mould et al., 2000) . these measurements are flanked by results from studies with x. laevis (shimbo et al., 1996) . using the whole-cell patch clamp technique on x. leavis, first evidence is given that the protonation state of the one histidine within the tmd is important for conductance (wang et al., 1995) . proton conductance of full-length m2 expressed in bl21 cells and reconstituted into liposomes has also been shown to be independent on the presence of electrolytes (vijayvergiya et al., 2004) . using a fluorescence essay based study, it is reported that proton conductance is independent of the content of cholesterol in the liposomes (lin and schroeder, 2001) . with this finding, it is proposed that m2 is active in lipid raft free environment and would just be associated with a lipid raft due to its cytoplasmic part. the authors suggest that raft association may control the number of m2 proteins to be incorporated into the virion. experimental evidence that the tmd of m2 is helical has been achieved with m2 peptide reconstituted into dopc liposomes using cd spectroscopy . solid nmr spectroscopic measurements with m2-tmd peptides confirm the helical motif, deliver tilt angles of the peptide within dmpc bicelles of about 33 , and suggest a left-handedness of the putative bundle (kovacs and cross, 1997) . by adding data from functional studies like cys scanning and electrophysiological measurements as mentioned as well as computational modeling data (sansom and kerr, 1993; sansom et al., 1997; zhong et al., 1998) , an approximate structural model of the tetrameric assembly of the tmds of m2 with the histidines and tryptophans as important pore lining residues has been generated. in a combined study using ftir spectroscopy with sitespecific labeled amino acids and a dynamic molecular global search protocol, further strong support for the tetrameric model has been achieved . extensive solid state nmr spectroscopic investigations on chemically synthesized tmd peptides of m2 delivered "high-resolution" distance and orientation data sets of the residues at ph 7.0 (nishimura et al., 2002) . a precise proposal has been given for the orientations of the tryptophans and histidines in a tetrameric assemble. in addition, helix stability has been confirmed. according to models of assembled helices, a water filled cavity toward the n terminal side has been suggested. solid state nmr measurements of full-length m2 expressed in escherichia coli and reconstituted into dmpc bicelles confirm the findings on the studies with peptides (tian et al., 2002 (tian et al., , 2003 . these studies indicate a mode of assembly of the tmds which is independent of the rest of the protein . studies based on tryptophan fluorescence on fully expressed m2 and mutants reveal that trp-41 and his-37 work in concert upon low ph activation (czabotar et al., 2004) . together with other experimental wang et al., 1995) and computational studies schweighofer and pohorille, 2000) , it is proposed that several mechanisms of function shuttle the proton across a selective and narrow part around the histidines of the channel. in one mechanism, called "shuttle" mechanism, the incoming proton is taken up by his-37 on its d-nitrogen which induces a release of the hydrogen on the e-nitrogen. via a tautomerization, the sidechain of his-37 is reloaded. in another mechanism, the "water-wire" mechanism, the protonated histidines will repel each other allowing for an opening and transport via a hydrogen wire. computational modeling combining classical molecular dynamics simulations with multistate empirical valence bond (ms-evb) approach (schmitt and voth, 1998) has shown that a hydronium ion will not move through the histidine ring. it is rather anticipated that the proton could shuttle even if the histidines are not fully removed to generate a kind of "open" channel (smondyrev and voth, 2002) . the consequence of this study is that not all of the histidines need to be protonated and consequently charged to generate an "open" channel. solid state nmr spectroscopic investigations have shown that at high ph, the channel can exist in a closed state when a neighboring pair of the histidines share a proton, and consequently, a third one releases this pairing and opens for a putative water-wire to conduct the proton (hu et al., 2006) . with the detection of conformational distinct tryptophans upon low and high ph conditions by 19f nmr spectroscopy, a contribution of trp-41 during channel gating has been verified (winter et al., 2008) . finally, to date a cooperative gating model is proposed in which upon low ph on the n terminal side one histidine gets trapped via a cation-p interaction with the tryptophan while the other histidine is able to offer one of its protons to the water on the c terminal side (sharma et al., 2010) . structural models at atomic resolution are available based on nmr spectroscopy (pielak and chou, 2010; schnell and chou, 2008 ) and x-ray crystallography (acharya et al., 2010; stouffer et al., 2008; wang et al., 2011) (fig. 6.1a) . all structures reveal a left-handed bundle. for the nmr investigations, a tmd m2 construct with an amphiphatic helix at the c terminal side has been used to stabilize the helix bundle which otherwise could not have been investigated (schnell and chou, 2008) . the expressed m2 construct is reconstituted into dhpc detergent micelles and due to solution nmr spectroscopic investigations at high ph (ph 7.5). the construct is recorded in the presence of the antiviral drug rimantadine which interacts with the helix bundle at the lipid protein interface. the bundle forms a narrow pore which has a wider pocket of about 0.6 nm around gly-34 and is highly restricted around his-37 and trp-41. the structure is referred to as a potential closed form of the channel (pielak and chou, 2010) . solely, the tmd of m2 has been crystallized and its structure resolved to 0.2 nm in octyl-b-d-glucopyranoside (og) detergents at high ph (ph 7.3) (stouffer et al., 2008) . the pore is opened into a tepee-like shape with its restriction on the n terminal side and the trp-41 apart from each other. a mutant m2 (gly-34 is replaced by alanine) cocrystallized with amantadine at lower ph (ph 5.3) identifies a structure which is similar in shape as the one crystallized at ph 7.3. the binding site of amantadine is marked to be within the lumen of the pore. the shape of the bundle is considered to represent an open form of the channel. solution nmr studies reveal that at low ph the tmds are dynamic (hu et al., 2011; li et al., 2007; pielak et al., 2009) . it is a common theme of all models that, besides the his-x-x-x-trp motif, asp-44 and arg-45 play an additional role in the gating mechanism. similar to influenza a, segment 7 of the influenza b genome also harbors two proteins m1 and m2 with the latter to be a proton channel called bm2 (briedis et al., 1982) of about 15 kda (109 amino acids) (horvath et al., 1990) . the sequence of bm2 is highly conserved which indicates its importance for the virus (hiebert et al., 1986) . the protein is phosphorylated and localized in the cytoplasm but also transported to the plasma membrane where it is incorporated into virions (odagiri et al., 1999) . like m2, bm2 is a type iii integral membrane protein with an h-x-x-x-w motif but with no further sequence homology with m2 including cysteins in the ectodomain . its role is suggested to equilibrate the ph gradient between the golgi and the cytoplasm by forming proton channels (mould et al., 2003) . proton conductance of bm2 is found to be higher than for m2. mutation studies identify polar serine residues (serines 9, 12, and 16) facing the lumen of the pore which is discussed as an explanation for the resistance of bm2 against amantadine (ma et al., 2008; paterson et al., 2003) . the oligomerization state is experimentally confirmed to be a tetramer together with functional studies using the two electrode voltage clamp method (balannik et al., 2008) . despite functional similarity with m2 protein, bm2 cannot form oligomers with m2. not forming a tetramer via the "dimer of a dimer" concept, the route of assembly seems to follow the same as for other vcps such as vpu, p7, or 2b. solution nmr spectroscopy identifies a large left-handed coiled-coil tetramer consisting of two segments, separated by a 10-amino-acid region (residues 34-43) (wang et al., 2009b) (fig. 6 .1b). the "full-length" structural model is derived from experimental results of two overlapping constructs: a tmd containing construct bm2 1-33 (2kix) and a cytoplasmic domain containing construct bm2 26-109 (2kj1). with its large cytoplasmic domain which generates a large dipole moment, it is suggested that bm2 is also involved in the recruitment of matrix proteins at the cell surface and in combination with that involved in the viral assembly and budding process. the channel domain consists of two heptad repeats leu-8 to ile-14 and leu-15 to ile-21, and the data confirm serines 9, 12, and 16 as pore lining. ser-12 completely faces the pore while the other two are also involved in interhelix packing. the pore is occluded by a ring of four phe-5 and trp-23. interesting to note is that mutations of ser-12 and ser-16 into alanines affect proton conductance much more than mutations of his-19 and his-27 into alanine (wang et al., 2009b) . computational modes of the tmds of bm2 have been modeled using coarse-grained simulation techniques in combination with united atom simulations (rouse et al., 2009) . the suggestion in this study is that, also in this channel, a minimum of three histidines need to be protonated to induce conformational changes which lead to an open conformation. antiviral amantadine does not inhibit the activity of bm2 . the reason is found to be due to the polar serines facing the lumen of the pore. more polar drugs seem to be necessary to block the channel (ma et al., 2008) . recently, a monoclonal antibody has been identified to target the ectodomain of bm2 comprising antiviral activity successfully (wang et al., 2010b) . with pb1-f2 encoded by influenza a, a second channel protein has recently been identified (chanturiya et al., 2004; henkel et al., 2010) of which a structural model for its tmd is reported (bruns et al., 2007) ( fig. 6.1c ). this protein has been found with a strong tendency to oligomerize. this would be the second virus, next to sars-cov which encodes more than one channel protein. the studies have been done with synthetic peptides reconstituted into planar lipid bilayers and microsomes. the structural integrity has been demonstrated with a united atom md simulation in a fully hydrated lipid bilayer. to date, investigations on m2 are very advanced in respect to structural and functional studies. albeit, structural information about the cytoplasmic domain is still lacking, the tmd is very well characterized by nmr and x-ray data. the protein has been for a long time a potent drug target (davies et al., 1964) . in the late 1980, a novel open reading frame hosting a 16-kda protein has been detected independently by two groups (cohen et al., 1988; strebel et al., 1988) (fig. 6.2 figure 6 .2 available structures of vpu from experimental sources. the structures are shown in "gaussian contact mode" (moe software) indicating hydrophilic residues in blue and hydrophobic residues in green. in addition, helices are shown as yellow bands and specific amino acids are represented in a stick modus (histidines in red, tryptophans in violet, and serines in orange). 1pi7 tmd from solid state nmr (park et al., 2003) , 1vpu (willbold et al., 1997) and 2k7y (wittlich et al., 2009) of a frame shift mutation into the vpu gene, an up to 10-fold reduction of viral load has been observed (strebel et al., 1988) . the conclusion drawn from the experiments has been that vpu is involved in virus assembly and particle release. the new protein has not been detected in hiv-2 and siv (cohen et al., 1988) . vpu's involvement in inducing particle release (strebel et al., 1988 (strebel et al., , 1989 terwilliger et al., 1989) has been attributed to a downregulation of the receptor protein cd4 via the proteasome pathway (ruiz et al., 2010a; willey et al., 1992a,b) and the formation of ion channels (ewart et al., 1996; schubert et al., 1996b) . the two routes of action are attributed to two distinct domains of the protein (schubert et al., 1996a) : the cytoplasmic domain for the former (bour et al., 1995) and the tmd for the latter (schubert et al., 1996b) . the mechanism of how the protein enhances virus particle release is anticipated by the formation of a homooligomer at the site of the plasma membrane which renders the membrane permeable for ions (schubert et al., 1996b) . cd4 degradation is found to be due to the interaction of a short sequence, krllsekkt, in the cytoplasmic tail of cd4 with a helical domain of the cytoplasmic part of vpu (tiganos et al., 1997) . also, residues at the linker region between the tmd and cytoplasmic domain of vpu contribute to cd4 degradation . for cd4 downregulation, the two phosphorylation sites of vpu, ser-52 and ser-56, are identified to be essential (paul and jabbar, 1997; schubert et al., 1994) . downregulation via interaction with vpu has also been reported for btrcp (margottin et al., 1996) to hand over cd4 to the proteosomal degradation pathway. also, other host proteins such as vpu binding protein (ubp) (callahan et al., 1998) , cd74 (hussain et al., 2008) , and cd317 (bolduan et al., 2011; neil et al., 2008; van damme et al., 2008) are "marked" by vpu for downregulation. interaction of vpu with host factors at the site of the plasmamembrane are reported for task channels (hsu et al., 2004) and bst-2/tetherin (also called cd317) (neil et al., 2008; van damme et al., 2008) . in both host proteins, the tmd of vpu is responsible for the interaction (hsu et al., 2004; skasko et al., 2011) . for bst-2, it has been shown that vpu slows down bst-2 transport to the plasma membrane (dubã© et al., 2010) and that the vpu-bst-2 complex is retained in the er (skasko et al., 2011) . the interaction is reported to be due to the tmds of both proteins. bst-2 itself is known to form a dimer and crystallographic data identify for the extramembrane part an elongated helical motif which forms a coiled-coil toward the c terminal side and a flexible region able to assemble into a tetramer on the n terminal side (hinz et al., 2010; schubert et al., 2010) . the loop sequence, eyrkll, connecting the tmd of vpu with its cytoplasmic domain has been shown to be responsible of transporting vpu to the plasma membrane (ruiz et al., 2008) . at the site of the plasma membrane, it is anticipated that vpu forms channels via oligomerization. expression of vpu in amphibian oocytes induces conductance of cations measured under voltage clamp conditions (coady et al., 1998; schubert et al., 1996b) which shows marginal effect on divalent ions such as ba 2ã¾ and ca 2ã¾ . together with peptides representing the tmd and a scrambled sequence, it has been shown that solely the tmd exhibits cation specific channel activity. a series of experiments has been performed which show that expressed vpu in e. coli purified and reconstituted into artificial bilayers exhibit channel activity (ewart et al., 1996; mehnert et al., 2007) . recordings solely with the tmd of vpu reveal similar conductivity compared to full-length protein with minimal changes in the kinetics (ma et al., 2002; mehnert et al., 2007) . replacing trp-23 by leu in a synthetic peptide construct, vpu 132 , reconstituted into artificial lipid bilayers, alters the open time duration and shut kinetics of the peptide, while a change of ser-24 to leu abolishes channel activity completely (mehnert et al., 2008) . no affect has been reported when arg-31 is exchanged into val. channel recordings of the tmd of vpu in solutions of different ions reveal that the vpu channels which are only slightly selective indicate almost pore-like characteristics. it has been suggested that vpu shows a channel-pore dualism (mehnert et al., 2008) which means that vpu can either act as a more or less selective channel or nonselective pore depending on specific in vivo conditions. these conditions could possibly be due to changes in lipid environment. in a recent electrophysiological study, using whole-cell clamp conditions with 293t cells expressing full-length vpu mutant s24a does also not exhibit channel activity (bolduan et al., 2011) . in another study, in which vpu has been expressed in e. coli, the protein renders the membrane of the cells susceptible to a series of molecules as well (gonzales and carrasco, 1998) . 2-nitrophenyl-d-galactopyranoside, uridine, translation inhibitor hygromycin b, and lysozyme are reported to pass the membrane as well as hygromycin b and neurobiotin when expressed in eukaryotic cos cells. channel activity allows the change of electrochemical gradients, depolarization, across the lipid membrane. the effect of depolarization of the membrane is reported to affect the fission of the budding hiv-1 virion from the infected hela cells (hsu et al., 2010 ) (e.g., lowering electrostatic repulsion to enable fission): blocking two-pore k ã¾ (k 2p ) channel task virion release with vpu inactive hiv leads to an enhancement of virion release. since task channel activity in hela cells measured under wholecell voltage clamp conditions is not affected in the presence of vpu, it is concluded that vpu reduces the number of task channels at the plasmamembrane by interacting with the k ã¾ channel making it susceptible for degradation. sequence similarity of the tmd with the first tmd of task implies physical interaction of the two proteins (hsu et al., 2004) . it can be concluded at this state that vpu interacts with host proteins, which leads to downregulation via the proteasomal pathway or redirecting them. consequently, channel activity of vpu per se does not seem to be necessary to fulfill its auxiliary role in enhancing viral release. yet, vpu especially when inserted into lipid membranes shows channel activity which can be modulated by mutations. escape mutant studies as done for m2 from influenza a (hay et al., 1985) are lacking and a selective "channel blocker" is not yet identified (lamb and pinto, 1997) . the recent anti-channel drug bit225, n-[5-(1-methyl-1h-pyrazol-4-yl)naphtalene-2-carbonyl]-guanidine, seems to fulfill the role as a vpu channel blocker when administered in a concentration of 40 mm to vpu reconstituted into artificial lipid membranes of a channel recording device (khoury et al., 2010) . structural information of vpu has emerged from solution and solid state nmr spectroscopy ( fig. 6 .2). in the late 1990s, two structural models of the cytoplasmic domain of vpu have been published using solution nmr spectroscopy ( fig. 6 .2). the structural features discovered have been a helix-loop-helix motif followed either by another short helix (willbold et al., 1997) or a reverse turn (federau et al., 1996; wray et al., 1995) . while the former data derived from recordings of vpu expressed in e. coli purified and dissolved at high salt solution, the latter spectra have been recorded from a synthetic peptide dissolved in aqueous tfe solution. it is debated that the high salt solution induces the tertiary fold and thus the formation of the third helix. tfe is known to support helix formation but weakens the formation of a fold. more recent data in low salt solution confirm two helical motifs in the cytoplasmic domain (wittlich et al., 2009 ). measurements in the presence of micelles formed by dodecylphosphatidycholine (dpc) induce secondary elements (two helices) and a tertiary fold. secondary structures are supported by cd spectroscopy (wittlich et al., 2009; wray et al., 1995) . all structural investigations have in common that the two series 52 and 56 have not been phosphorylated. investigations on the structural implications of phosphorylation of the two serines to the structure have been done either on a synthetic peptide in aqueous tfe solution (coadou et al., 2001 (coadou et al., , 2002 or on a peptide expressed in e. coli, purified and measured in the presence of dpc micelles (wittlich et al., 2008) . without dpc upon posphorylation, parts of the cytoplasmic helices unwind into a b-strand (coadou et al., 2002) , while in the presence of dpc micelles, structural changes are limited to some loss of helicity of helix1 toward the c terminus and extension of helicity toward the n terminal side of helix2 (wittlich et al., 2008) . the helix motif for the tmd of vpu has first been suggested by solid state nmr spectroscopy (marassi et al., 1999; wray et al., 1999) . applying the same technique to extended constructs of the tmd of vpu with residues of the cytoplasmic domain indicates that the second helix is aligned parallel to the membrane surface (marassi et al., 1999; park et al., 2003) . x-ray reflectivity measurements done with full-length vpu on a lipid monolayer reveal that the second helix in the cytoplasmic domain is bound loosely (zheng et al., 2001) . the helical motif of the tmd of vpu has been supported by site-specific ftir spectroscopy . ssnmr data show that the tmd vpu construct shows rotational dynamics within the lipid membrane (park et al., 2006) . the extent of the tmd helix is still controversial, when comparing all the ssnmr data. in a recent study, the length of the helical motif toward the c terminal side of the tmd is reported to extend beyond the hydrophobic slab of the bilayer (sharpe et al., 2006) . the tmd of vpu is found to exhibit a weak kink around ile-17, a result obtained using a vpu 2-30ã¾ construct measured by solid state nmr spectroscopy (park et al., 2003) . md simulations using a vpu 1-52 construct support the findings reporting kink angles to vary between 7 and 15 around the same amino acids found experimentally (sramala et al., 2003) . computer simulations in various lipids identify a kink around ser-24 due to the compensation of hydrogen bonding of the side chain with adjacent backbone residues toward the n terminal side (krã¼ger and fischer, 2008) . further bending of the helix is found from ser-24 to ile-20. kinking is seen as a mechanism to compensate for varying lipid thicknesses. to summarize the findings, there could be hinge regions around ile-17 and ser-24 proposing modular segments of the protein within the lipid bilayer. according to the hypotheses of vpu being released after manufacturing into the er membrane, the protein could oligomerize prior to any interaction with other host proteins. oligomerization is also seen as a prerequisite of channel formation. earliest studies point toward an oligomeric assembly using sds-page (maldarelli et al., 1993) . on the bases of synthetic peptides linked together as either tetramers or pentamers and their channel activity, the latter state is reported to be the favored one (becker et al., 2004) . full-length vpu and its tmd alone, both expressed in vitro and analyzed using gel permeation chromatography, are found to be in a pentameric state (hussain et al., 2007) . computational modeling of the tmd based on ssnmr data on expressed tmd of vpu supports the pentameric state (park et al., 2003) but a tetrameric state is also proposed. a combined experimental study using bilayer recordings on synthetic peptide based on the tmd and computational modeling using md simulations is in favor of the pentameric assembly (cordes et al., 2002) . pentameric assemblies have been used for the computational modeling of the vpu ion channel embedded in hydrated lipid bilayers (cordes et al., 2001; grice et al., 1997; moore et al., 1998) . simulations of hexameric assemblies in a hydrated slab of octan, mimicking a lipid bilayer, have been reported to collapse and are disregarded as a potential assembly (lopez et al., 2002) . in another simulation study in a hydrated popc bilayer system, the tetrameric and hexameric assemblies have been ruled out based on the derived pore radii and the estimated conductance these pores would show (cordes et al., 2002) . simulations on extended models including the first cytoplasmic helix (sramala et al., 2003) and the entire cytoplasmic part have been reported with the proteins embedded either in a lipid monolayer at the air water interface (sun, 2003) or in hydrated popc sramala et al., 2003) . in one simulation of full-length vpu, the cytoplasmic domain has been generated artificially based on the available nmr data (sun, 2003) while for the other simulations experimentally derived cytoplasmic domain data have been artificially merged with simulated extended vpu ; tmd and first cytoplasmic helix taken from sramala et al., 2003) . vpu is predominantly acting via interactions with other host proteins. structural information of the tmd is in lack of x-ray crystallographic data, but in contrast to m2, the structural details of the cytoplasmic domain are well resolved. ion channel functionality is still being debated and it seems that vpu may emerge as a potential drug target. the genome of hcv is expressed as a large polyprotein and cleaved by cellular and viral proteases into 10 cleavage products among which is p7, a 63 residue 6-7 kda bitopic membrane protein (elbers et al., 1996; lin et al., 1994) (fig. 6 .3). the protein is preceded by the structural protein e2 from which it is incompletely cleaved and succeeded by the nonstructural protein ns2. it is not clear at this moment to which side structural or nonstructural p7 belongs to. abolishing the cleavage between e2 and p7 results in noninfections virions (harada et al., 2000) . expressing e2 and p7 independently recovers the production of infectious virions making p7 an essential part of the life cycle of hcv. also, functional relevant interaction of p7 with ns2 has been reported (tedbury et al., 2011) . ns2 is located to sites where viral replication and particle assembly take place. in the absence of p7, the location of ns2 to these sites is lost. this function of p7 is reported to be independent of its function as a channel protein. it has been suggested that p7 plays an accessory role in altering the topology of ns2 which consequently affects ns2 function (ma et al., 2011) . as a channel-forming protein, p7 is proposed to have the same role in the life cycle of the virus as m2 supporting cell entry (griffin, 2009) . it has been discovered that p7 is necessary during the early stage of particle assembly ( jones et al., 2007; steinmann et al., 2007a) . using a so-called j6/jfh chimeric genome which can be used to study the hcv life cycle in human hepatoma cells (huh-7.5), it has been shown that p7, but not its precursor forms e2-p7 and p7-ns2, is required for infectious virus production ( jones et al., 2007) . mutations in the basic loop between the two tmds (kr33/35qq or kr33/35aa) lead to a decrease of the amount of released infectious virions (steinmann et al., 2007a) . it is proposed that these mutations affect channel activity as they are at the mouth of the putative pore. other mutations within the tmds of p7 (tmd1 and tmd2), such as the highly conserved trp-30 and tyr-42 each of them being separately mutated into phenylalanine, impair the infectivity of the virions. mutations of his-31 also do not have an effect on rna replication (steinmann et al., 2007a) , similar to the wild-type p7 (lohmann et al., 1999) . concluding from these data, p7 is not essential for viral entry, supporting the essence of p7 within the later stage of infectivity which is linked to virus assembly. nevertheless, p7 is essential for the virus but the mechanism of ion channel activity is not yet linked to either of these events. the topology of p7 has been identified to contain two tmds connected via a short hydrophilic segment. both ends of the tmds are found to point into the er lumen (carrã¨re-kremer et al., 2002) . the protein is detected within the plasma membrane with both termini pointing toward the extracellular environment. experiments with p7 expressed in e. coli (clarke et al., 2006; griffin et al., 2003) , as well as p7 protein derived from solid phase peptide synthesis (spps) (pavlovic et al., 2003; premkumar et al., 2004) , both individually reconstituted into artificial lipid bilayers disturb the 2k8j tmd2 tmd1 his-17 tmd1 tmd2 tmd2 figure 6 .3 available structure of p7 from experimental and computational sources. the structures are shown in "gaussian contact mode" (moe software) indicating hydrophilic residues in blue and hydrophobic residues in green. in addition helices are shown as yellow bands with histidine residue represented in a stick modus (red). 2k8j: tmd2 from solution nmr (montserret et al., 2010) , boxed; top row to the right is a computationally assembled monomer, lower row a potential hexameric assembly of the monomers forming a channel. bilayer, making it susceptible to ion conductance. most recent data reveal that channel activity is dependent on lipid composition and they are discussed together with data from cd in terms of a change in topology of the protein (whitfield et al., 2011) . it is proposed that a lipid dependent equilibrium between an antiparallel alignment and a l-shape alignment (one helix parallel the other perpendicular to the membrane normal) of the two tmds exists. in another study of p7 purified from the same expression system p7, it has been reported using transmission electron microscopy (tem) that the protein assembles into a hexamer (griffin et al., 2003) . with a flag-p7 construct at the n terminal side also expressed in e. coli, purified and measured with tem, a heptameric assembly is proposed (clarke et al., 2006) . the heptameric assembly has been supported by sds-page and mass spectrometry. most recently, electron micrographs of p7 synthesized by spps are in support of a hexameric assembly of the protein (luik et al., 2009) . the first computational model of p7 has been generated using a coarse grained conformational search protocol (patargias et al., 2006 ) (see also fig. 6 .3). the two tmds and the short link between them have been identified as a consensus from the results of multiple secondary structure prediction programs (cuthbertson et al., 2005) . the antiparallel aligned p7 protein is hither forth called the monomer. the alignment as a monomer positions the tmds so that a bundle could be formed in which the histidines of tmd1 of all the monomers point into the putative lumen. consequently, a pivotal role for this particular histidine in gating has been suggested (patargias et al., 2006) . other groups have also challenged computational modeling to fill the data from electron microscopy (em) with structural information (griffin et al., 2003) . most recent md simulation studies with an extended p7 protein randomly positioned in a simulation box together with lipid molecules using coarse grained techniques support the antiparallel alignment of the tmds in the p7 monomer (luik et al., 2009) . the role of histidine in the conductance of ions across the protein bundle has been supported by mutant studies of chemical synthesized p7 reconstituted into lipid bilayers . studies with cu 2ã¾ containing solutions in combination with wild-type and h17a mutants have allowed the conclusion that histidine is pore lining, as predicted earlier (patargias et al., 2006) . solution nmr spectroscopic experiments with p7 expressed in e. coli and reconstituted into dhpc micelles deliver data which support its bitopic topology opella, 2010, 2011) . both of the two tmds are split into two segments with specific dynamics comprising four helical segments. the two unstructured n and c termini as well as the short and mobile loop segment between the tmds comprising residues 28-36 are outside of the membrane. residues 5-15 constitute a helical segment embedded into the micelles which is similar to the third segment in its dynamics. while the first helix has no membrane anchoring amino acids, the third helical segments including residues 41-48 seem to be affected by the dynamics of the loop segment. the second helix (residues 17-27) is found to be less mobile and it is suggested that this is due to the membrane anchoring aromatic residues trp-30 and tyr-31. similarly, low mobility is also found for the fourth helix which is flanked by two prolines, pro-49 and pro-58, containing a stretch of five leucines (leu-50-leu-54). in another solution nmr spectroscopic study, data of full-length p7 derived from peptide synthesis and recorded in a mixture of trifluoroethanol and water also mainly identify helical motifs (montserret et al., 2010) ( fig. 6.3, boxed) . flexible regions around residues gly-15 and gly-18 of tmd1 and at the c terminal end of tmd2 are observed. cd measurements in various membrane mimetic environments support the overall helicity of the protein in its monomeric state. distance restraints from the nmr measurements have been used as input for computational modeling using a docking approach in combination with extended molecular dynamics simulations to model the assembly of the two tmds. the model reveals that the aromatic residues phe-25, trp-30, tyr-42, tyr-45, and trp-48 form a staged p-system. channel characteristics of p7 reconstituted into asolecitin bilayers identify a voltage-dependent current behavior which is rectifying and showing an 11 times higher selectivity for cations (montserret et al., 2010) . main conductance levels are reported to be 22 and 41 ps at ã¾60 and ã¾140 mv, respectively. patch recordings with liposome identify slightly higher conductance levels at around 35, 57 120, and 184 ps when a pipette holding potential of ã¾140 mv is applied. channel recordings are reported to be blocked by hexamethylene amiloride (hma) but not by amantadine. the pore lining tmds is identified to be tmd1, since a major conductance level of about 60 ps has been found at a holding potential of ã¾100 mv when reconstituted into lipid bilayers. in contrast, there could be no channel recordings observed for tmd2. a synthetic full-length p7 construct derived from spps shows a reversal potential of 44.3 mv in a 10:1 (cis: trans) gradient buffer, suggesting a moderate cation selectivity (premkumar et al., 2004) . the experiments indicate that the protein bundle shows lower permeability for calcium ions and considerable chloride conductance. the oligomerization state of p7 is dependent on the protein:detergent ratio (c 12 e 8 ) when using sedimentation experiments (montserret et al., 2010) . at lower ratio, a hexameric assembly is proposed which can shift to slightly higher numbers at higher ratios. so far, crystallization trials failed to deliver any conclusive structural information ( jones et al., 2007) . his-17 has been identified to point into the lumen of the pore (patargias et al., 2006) . this residue has also been found in m2, which is identified as proton sensor involved in gating of the channel. conductance studies of p7 when reconstituted into artificial lipid bilayers reveal that the presence of cu 2ã¾ in the bath solution blocks the activity of p7 ). since copper ions can be complexed by histidines, this study confirms the orientation of these residues toward the pore. with histidines in the pore, it has also been suggested that this allows ph sensitivity in any kind of form (patargias et al., 2006) : is the p7 bundle just sensitive to protons but does not conduct them? or is p7 also a proton conductor similar to m2? it has been now suggested that protonation of the histidines will lead to an opening of the pore (foster et al., 2011) . with further analogy to m2, the question arises, can the protein also be built by less than the six units as it is proposed to date? of course, experimental evidence is given for larger units, such as hexamers and possibly heptamers. other channel-forming proteins 2.2.1. 6k from alphavirus a series of proteins in other viruses have also been reported as vcps (carrasco, 1995; fischer, 2005; fischer and sansom, 2002; gonzales and carrasco, 2003) . one of these proteins is 6k, which is found in several viruses of alphavirus genus belonging to the family togaviridae (carrasco, 1995; garoff et al., 1982; madan et al., 2005; schlesinger and schlesinger, 1986; strauss and strauss, 1994; wang et al., 2010a) . alphaviruses express their structural proteins in a polyprotein consisting of e3-e2-6k-e1-c which is proteolytically processed into its constituents (liljestrom and garoff, 1991) . the 6k protein is a 6 kda hydrophobic protein of 60 amino acids which is palmitylated (gaedigk-nitschko et al., 1990; lusa et al., 1991) . it is found to be transported to the site of the plasma membrane via a p62/e1 complex and only to a lower extend incorporated into the budding virion (lusa et al., 1991) . albeit sitespecific mutation in the 6k region identifies viruses with reduced budding capabilities, a full deletion of the protein does not result in blocking viral replication. the lack of 6k leads to an altered spike structure of the virion and it is concluded that the protein is important for the correct assembly of the virion (mcinerney et al., 2004) . these findings make 6k another accessory protein which amplifies the release of virions. two hydrophobic stretches in the sequence of 6k suggest two tmds one of which would be long enough to span the bilayer (liljestrom and garoff, 1991) . a bioinformatics approach using a hidden markov model reduces the number to only one tmd (sonnhammer et al., 1998) . a single tmd is also the conclusion drawn from mutation studies of semliki forest virus 6k protein in its interfacial region (sanz et al., 2003) . ross river and barmah forest virus 6k proteins expressed in e. coli and reconstituted into artificial lipid bilayers form channels with conductance ranging from 40 to 800 ps (melton et al., 2002) . the large conductance is attributed to the formation of larger oligomeric units of the protein. no other roles than permeabilizing the lipid membrane and being involved in the budding process are known to date. it has been discussed that topology and mechanism of function of 6k is dependent on lipid thickness and constitution which the protein experiences during trafficking (sanz et al., 2003) , a mode of action which has also been suggested for vpu from hiv-1 (mehnert et al., 2008) . 2.2.2. e, 3a, and 8a from severe acute respiratory syndrome-corona virus coronaviruses belong to the family of coronaviridae and replicate in various animal species (marra et al., 2003; stadler et al., 2003) . human coronaviruses have been known since the 1960s (myint, 1995) . in 2002, a member of the virus has been found to cross the species barrier and infected humans with a mortality rate of about 5%, and even higher for people above their sixties (zhong et al., 2003) . the symptoms are described by a sars which are almost flu like and come with severe fever (stadler et al., 2003) . the name of the family derives from a corona like shape of the virion when observed in electron microscopy (beniac et al., 2006) . coronaviruses belong to the enveloped viruses and harbor the largest positive-sense single-stranded rna genome of 30-32 kb amongst the rna viruses (narayanan et al., 2008) . the genome is transcribed into a large polyprotein which is cleaved by virus-encoded proteases. the structural proteins are spike (s), matrix (m), and nucleocapside proteins (n) as well as the envelope protein (e). a large series of accessory proteins is distributed toward the 3 0 end of the genome (narayanan et al., 2008) among several proteins exhibiting channel activity, as listed below. protein e has a length of 76 amino acids with a short n terminus of 7-9 amino acids, a long tmd of 21-29 amino acids and a c terminus (shen et al., 2003) . when e protein is synthesized and reconstituted into artificial lipid bilayers, channel activity with conductance in the range of 50 ps is observed . sds-page experiments identify e protein to form pentamers (parthasarathy et al., 2008) . electrophysiological measurements using whole-cell clamp conditions of e protein gene infected hek-293 cells reveal a current which is sensitive to the administration of hma (pervushin et al., 2009) . ftir spectroscopic investigations suggest a helical hairpin-like structure for the long tmd of e protein (arbely et al., 2004) . it is argued that, the peptide is highly helical with hardly any tilt and has a phenylalanine (phe-23) located toward the lipid head group region. in addition to that, an asparagine (asn-15) would support the n and c terminal ends to assemble. nmr spectroscopic investigations support a helical tmd without any turns (pervushin et al., 2009 ). merely, a leucine residue (leu-18) does not fit into the residual dipole coupling calculations proposing a non-helical section or a conformational flexible region. computational modeling studies of the e protein in an implicit bilayer model using monte carlo simulations support the experimental results of the secondary structure of the membrane traversing helical domain to be helical (chen et al., 2010) . the calculation starts from a random conformation of the tmd fully embedded in the hydrophobic slab of the bilayer. the presence of hma differences in the 1hn chemical shifts of two residues on either side of the tmd is found when compared to those without any drug (pervushin et al., 2009) . this is in contrast to measurements in the presence of amiloride. consequently, hma is suggested to be located within the pore most likely at the c terminal mouth. orf 3a of sars-cov is another protein identified to exhibit channel activity when expressed in xenopus oocytes (lu et al., 2006) . it is located between s and e proteins and built by 274 amino acids (zeng et al., 2004) . the protein is found at the site of the plasma membrane and in the cell (tan et al., 2004; yuan et al., 2005) as well as in intracellular virus particles (ito et al., 2005; yu et al., 2004; yuan et al., 2005) . the protein is also released in membranous structures from infected and 3a expressing cells (huang et al., 2006) . similar to other channel-forming proteins of other viruses, 3a also interacts with a series of other host and viral proteins (narayanan et al., 2008) . three tmds are proposed at the n terminal side preceding a longer c terminal cytoplasmic domain of about 148 amino acids. residues 127-133 harbor a cystein rich region which is involved in connecting to monomeric 3a proteins into a covalently linked dimer (lu et al., 2006) . two dimers assemble into another dimer via noncovalent bonds forming a tetrameric unit. experimental data of the structure of 3a are not yet available. up to now, only computational models are generated krã¼ger and fischer, 2009) . a variety of assembly protocols have been followed which can be categorized into protocols which screen the conformational space of the monomeric unit by a concerted movement of all tmds and in those which build up the monomeric unit in a sequential manner. the latter means, two tmds are assembled first following by the third tmd. finally, in both protocols, the tetrameric assembly is done in a concerted screening protocol. while a concerted protocol proposes the third tmd to be pore lining ), one of the sequential protocols (assembling tmd1 and tmd2 first followed by adding tmd3) proposes the second tmd to be pore lining in the tetramer . the rational for using the sequential assembly for the monomers is that it most likely resembles a biological relevant translocon based pathway of assembly. along these investigations, the finding of unusual residues lining the lumen of the pore such as tyrosines and histidines is a common feature. in addition for tmd2 lining the pore, a ring of histidines is formed in the tetramer . with histidines pore lining, the channel is getting in line with m2 of influenza a and computational models of p7 from hcv (patargias et al., 2006) . it needs to be evaluated whether 3a is sensitive to protons. the role of 3a in the life cycle of sars-cov is not elucidated yet. more recently, another open reading frame of sars-cov has been identified to exhibit channel activity. orf 8 is separated into two orfs 8a and 8b in human isolates with a 29-nt deletion (guan et al., 2003) . protein 8a has been identified to amplify viral release and to induce the hyperpolarization of the mitochondrial membrane (chen et al., 2007a) . consequently, it is concluded that 8a is involved in cellular apoptosis. patients with antibodies against 8a recovered from sars-cov infection. 8a is identified as a 39 amino acid transmembrane protein (guan et al., 2003) . secondary structure prediction programs suggest the n terminus to be transmembrane followed by an extramembrane domain containing approximately 15-20 . a peptide corresponding to full-length 8a derived from spps and reconstituted into artificial lipid membranes exhibits weak cation specific channel activity with conductance of around 9 ps at elevated temperatures (38.5 c). since the sequence of 8a is rich of cysteins, experiments have also been done under reducing conditions. computational modeling of the tmd reveals a sequence of cysteins on one side parallel to the helix axis and a hydrophobic stretch of serines and threonines on the opposing side. assembling the single tmd around a central axis into tetra-, penta-, and hexamers delivers potential structures for the putative pore. consequent molecular dynamics simulations show a pentameric model maintaining a continuous water column. this model shows ser-11 and thr-8 as well as cys-15 to be pore lining. in addition, the c terminal mouth is surrounded by summarizing the findings about sars-cov, with its large genome it seems that the virus hosts three potential channel-forming proteins, e, 3a, and 8a. all three of them are identified as auxiliary proteins, also interacting with a series of other viral and host proteins (narayanan et al., 2008) . all three proteins have in common, that their roles within the viral life cycle have yet to be identified. channel activity is found either with expression of the protein in other cells or by reconstitution of the proteins into artificial lipid membranes. the question emerges why the virus needs three channels while all other channel expressing viruses known to date proteins only need one type of channel protein. entero viruses among which poliovirus, coxsackie virus, and echo virus are listed encode a nonstructural 97-99 amino acid protein called 2b (aldabe et al., 1996; doedens and kirkegaard, 1995; van kuppeveld et al., 1995) . it has been identified to render the membrane of the endoplasmic reticulum and the plasma membrane permeable for small molecules and especially calcium ions (aldabe et al., 1996; doedens and kirkegaard, 1995; van kuppeveld et al., 1997a) . it has also been reported that 2b is retained in the golgi apparatus where it increases the release of ca ions, a role which is also found for the endoplasmic reticulum in the presence of 2b (de jong et al., 2006) . more recently, it has been reported that 2b is also able to conduct monovalent ions and its conductance can be blocked by a known chloride channel blockers such as 4,4 0 -diisothiocyanatostilben-2,2 0 -disulfonic acid (xie et al., 2011) . the measurements have been done using two electrode voltage clamp conditions and with 2b expressed in xenopus oocytes. 2b is composed of two hydrophobic domains (van kuppeveld et al., 1995 (van kuppeveld et al., , 1997b and is found to homooligomerize into tetramers (cuconati et al., 1998; de jong et al., 2002; van kuppeveld et al., 2002) . in a computational approach, a dimeric and tetrameric structure of 2b from polio virus have been proposed (patargias et al., 2009) . the individual tmds have been modeled into an ideal helix and inserted into a fully hydrated lipid bilayer to relax the structure during a short molecular dynamics simulation. the individual tmds have then been taken to form the monomer. after another md simulation, the tetramers have been produced and also relaxed for many nanoseconds of md simulations. from those models, it is suggested that both tmds contribute to form the lumen of the pore. it is further suggested that lysines contribute to the inner face of the pore. a short peptide based on 20 amino acids of the second tmd has been able to permeabilize the plasmamembrane of cells (madan et al., 2007) . the experiments show that tmd2 alone could form a pore and possibly is at least part of the pore lining motif of the bundle made of the full-length protein. for extended reviews, please consult fischer and krã¼ger (2009) and gonzales and carrasco (2003) . plant viruses such as green algae infecting p. bursaria chlorella virus (pbcv-1; yamada et al., 2006 ) express a 94 amino acid potassium selective channel, called kcv (gazzarrini et al., 2003; plugge et al., 2000) . sequence homology with the potassium kcsa (doyle et al., 1998) has been proposed and consequently a structural model is suggested to be similar to kcsa. in xenopus oocytes, the protein induces channel activity and it is suggested that the channel is important for viral entry (mehmel et al., 2003; neupã¤rtl et al., 2008) . it can be blocked by amantadine and barium ions (plugge et al., 2000) . a series of mutants have been analyzed in respect to the consequences on the physiology of the channel and its mechanism of function (gazzarrini et al., 2004) . extended physiological measurements reveal two types of kinetics of the channel (abenavoli et al., 2009 ). this identifies that despite of the small size and without any extramembrane domains, the protein undergoes diverse gating behavior similar to other k ã¾ channels. computational models of kcv built on homology modeling and implementation of nmr spectroscopic data have been generated (tayefeh et al., 2007 (tayefeh et al., , 2009 ). the simulations show functional relevant amino acids and facilitated ion movement through the selectivity filter. another k ã¾ channel has also been found in the genome of brown algae infecting ectocarpus siliculosus and called kesv . with its 124 amino acids, it is slightly longer than kcv. three potential hydrophobic regions have been identified by sequence analysis. the channel is blocked by barium ions and amantadine. structural studies are most advanced for m2 and vpu. also topological information is available for p7. these studies on the structure of the channel deliver a snapshot of the overall dynamics the protein exhibits. usually, special biochemical conditions are applied to grab the protein for structural studies or to trap the protein into a specific conformational state. the combination of the pictures allows assessment to the mechanics of the protein. inevitable to these investigations is the use of computational tools to catch a glimpse about the dynamics on an atomistic scale. simulations on gating are widely applied to m2 to identify the roles of the histidines chen et al., 2007b; khurana et al., 2009) . in case of other vcps, the studies are focusing on the generation of structural models . there are many questions linked with investigations on vcps. some of the vcps form covalently linked dimmers which assemble into proton conducting tetramers for which gating is achieved by a ring of histidines. other vcps assemble into oligomers and it is anticipated that any hydrophilic residue within the tmd will point toward a putative pore (vpu, e, 8a). these channels neither are covalently linked nor conduct protons. the question about what triggers a defined opening is unknown. to summarize the mechanics of the proteins are fully in the dark. for p7, it can be speculated that, with the suggestion of histidines facing a pore or being aligned at a helix-helix interface within the membrane, the protein is sensitive to the surrounding ph as well. in addition to that, histidines can interact with metal to stabilize the assembly. with the tritopic 3a forming covalently linked dimers which assemble into tetramers, speculations about more sophisticated gating mechanisms are allowed. reviewing the mechanism of function at this stage of research on viral channel-forming proteins is still very speculative due to a lack of structural details for most of the proteins. the accumulation of structural information for proton channel m2 and with it clues about its mechanics is definitively sparked by the fact that m2 has been very quickly identified as a target for antiviral therapy due to its importance in the viral life cycle (hay et al., 1985) . it is also this circumstance which has ignited the hunt for channel proteins in other viruses and their potency to serve as antiviral target. evaluating potential mechanics of these proteins (fig. 6 .5), comparative investigations are the route to go at this stage. in doing so, the sequence of the tmds of individual viral channels are compared with those of the host channels and toxins for which structural and more functional information are available schindler and fischer, 2011) . based on sequence alignment of the tmds of these proteins, speculation and links of how the viral proteins may work are given. in the following, host channel proteins and toxins are chosen to be reviewed which have been identified to show sequence identity with the tmds of selected viral channels such as vpu, p7, 2b, and 3a schindler and fischer, 2011) . the introduction of the host channels and toxins follows a review of ideas about referring to the mechanics of the respective viral channels. pore forming toxins (pfts) are a class of proteins which are expressed by bacteria as well as higher organisms (iacovache et al., 2008) and even human cells (agerberth et al., 1995) to protect them against or support an attack. their role is to punch holes into the membrane and enable the draining of the cytoplasmic interior and thereby leading to cell death. pores formed by this class of proteins tend to be highly unselective. they are considered in this review since in a recent study about sequence alignment of vpu from hiv-1 with clya identified an alignment of the tmd of vpu with the membrane spanning domain of clya, aa . there are two classes of pfts depending on the membrane spanning motif they use: those which adopt a helical motif, a-pfts, such as clya (mueller et al., 2009) , and those which adopt a b-barrel motif, b-pfts, such as a-hemolysin of staphylococcus aureus (song et al., 1996) . (for an overview, refer to http:// blanco.biomol.uci.edu/membrane_proteins_xtal.html.) the mode of action of pfts goes through various steps. the proteins are released, they will "see" an aqueous environment, have to attach to the membrane, and assemble to finally convert into a membrane protein which intrudes into the membrane. many of these modes are not comparable to the mode of action of vpu except when the toxins are embedded in the membrane to form unselective pores. clya belongs to the class of a-pfts. in its functional form, it forms a 400 kda pore of 12 monomers with a height of 13 nm and an inner pore diameter of 7 nm (mueller et al., 2009 ). the single protein consists of four helices, aa, ab, ac, and af which are oriented parallel to the membrane surface. upon membrane attachment of the monomer, its b-tongue, bt, named like that because of the short b-fold found in this region, initiates membrane anchoring and a rise of the monomer aligning the helices perpendicular to the membrane surface. at this stage, assembly is also initiated and the monomers align head-to-head into a dodecamer. assembly facilitates the interlocking of the 12 aas into an iris-like arrangement which intrudes into the membrane stabilizing the pore. due to the enormous size, this pore can then be considered to be "open" leading to the vanishing of the hydrophobic permeation barrier of the lipid membrane and with it of any electrochemical or substrate gradients across the plasma membrane. smaller pfts such as alamethicin, magainin, or melittin follow the same strategy with the only difference that they form either a pore fully mantled by protein (barrel-stave model) or a pore with the phospholipid head groups part of the mantling wall (toroidal model) (huang, 2000; shai and oren, 2001) . at this stage, there is no available information on a gating mechanism of the smaller pfts unless due to thermal fluctuations within the membrane, consequent membrane curvature, and stress alterations the pore may collapse. this may not be the case for the larger pfts such as clya or ahemolysin. due to the role of these proteins, any "sophistication" of the mechanics of gating would not be necessary anyway. due to the similarity of vpu with aa of clya, the following speculations are allowed: vpu assembles into homooligomers. numbers range from 4 to 5 as minimal assembly units. with the tetramer, most likely no channel activity should be possible adopting a tight helix packing motif. with a pentameric assembly, ions would be able to pass through and with hexameric or larger bundles ion selectivity should be lost and substrates may also be able to pass through. with 5 and more monomers, the pore could be mantled by the tmds. tilts of the tmd of vpu have been measured and range from about 6 (in dmpc; to 13 (park et al., 2003) , 18 (park and opella, 2005) , and lower than 20 (sharpe et al., 2006) with the latter three values obtained with peptide reconstituted into dopc. upon thinning of the membrane, the tilt of vpu will increase because of compensating for the mismatch with the lipid bilayer (park and opella, 2005) . the angles obtained for vpu are lower than the observed tilt in the crystal structure of clya 2wcd (mueller et al., 2009) which adopts values around 45 in detergents. it is possible that in a lipid bilayer this value may be lower as well. it is therefore speculated for vpu that possibly lipid composition and lipid dynamics may act as trigger to gate the protein bundle (mehnert et al., 2007 (mehnert et al., , 2008 (fig. 6.4) . conductance values for vpu are within a range of 30-60 ps (schubert et al., 1996b) and exhibit gating into subconductance states (ma et al., 2002; mehnert et al., 2008) . nevertheless, these values rather indicate ion channel activity than "brute" conductance through a hole in the bilayer. concluding from the above, gating may be driven by stochastic events triggered by the thermodynamics of the environment (fig. 6.5a and b) . assuming a "sliding mechanism" to rise the tilted tmds , possibly in combination with minimal rotational movements, since the peptide is dynamic in the lipid bilayer (park et al., 2006) , it can easily be triggered by membrane thickening or thinning. a concerted rotation around each of the helix axis (bour and strebel, 2003; montal, 2003) needs to cross large energy barriers but would on the other hand be mostly independent of lipid thickness. the overall number of monomers forming the pore is still a matter of debate since conflicting pore geometries and oligomeric states are proposed. the zipper motif (kim et al., 2005) composed by a line of alanines, formed when adopting a helical conformation (fischer, 2003) , is an important tool for membrane protein packing. nmr spectroscopic data reveal a splitting of nmr signals especially for ala-18 which is interpreted as that the 2wcd figure 6 .4 crystal structure of toxin clya (2wcd) (mueller et al., 2009) . the structure is shown in "gaussian contact mode" (moe software) indicating hydrophilic residues in blue and hydrophobic residues in green. the membrane inserted segment aa, which shows sequence alignment with the tmd of vpu, is either highlighted in orange or in red . left: side view and below view from the extracellular side; middle: view inside the pore, omitting several units and below view from the cytoplasmic side; right: backbone representation of the protein in side view and cytoplasmic view below. splitting is caused by interhelix packing (sharpe et al., 2006) . this information has to be taken into account when assembling vpu. with the emergence of the crystal structure (0.32 nm) of the bacterial (streptomyces lividans) potassium channel kcsa, a keystone for ion channel structure has been delivered (doyle et al., 1998; mackinnon, 2003) . its topology has been identified to be bitopic with an inner and outer helix. the two helices are linked by the "pore helix" and the selectivity filter. four subunits form the channel by lining a pathway for ions to cross the bilayer. the passage of an ion can be described as passing a gate on the cytoplasmic side and moving into a vestibule which stabilizes the ion via the dipole moment of a so-called inner helix. the ions need to pass the selectivity filer in a concerted motion with already potassium ions located within the filter already. with its high selectivity, kcsa shows similarity with voltage-gated k channels (kv) in terms of ion permeation and similarity in topology with inward rectifying k channels (kir). important to note is that kcsa channels are triggered by ph changes. deciphering the cause of the enormous selectivity of k channels for potassium over sodium is a challenge up to date (noskov and roux, 2006) . it is anticipated that the structure of kcsa represents a closed stage. with the structure (0.33 nm) of a prokaryotic (methanobacterium thermoautotrophicum) calcium-gated k channel, a model in the open stage is described ( jiang et al., 2002a,b) . with this discovery and in comparison with the ksca structure, a gating mechanism can be proposed . during activation, the inner helices push radial outward and open the constriction at the cytoplasmic side. this opening is accompanied by a hinge around a glycine residue at position 99 (g99). this is the most pronounced conformational change so far described for ion channels. the selectivity filter remains unchanged in its structural position and the outer helix only undergoes marginal conformational changes ( jiang et al., 2002b) . also, hydrogen bonding seems to be essential for the gating mechanism (rapedius et al., 2007) . as a conclusion for the context of this review and in respect to the findings for vpu in relation to kcsa , the four outer helices seem to fulfill the role as a sheltering corona toward the lipid environment. generation of computational models of a conducting pore is biased toward the idea, and findings in other channels, that hydrophilic residues have to face the lumen of a pore. sequence identity of the tmd of vpu is found with the outer helix of the k channel task (hsu et al., 2004) . it has been speculated that vpu results from molecular piracy. due to similarity with the outer helix of task, it could rather be interpreted that vpu also mainly acts on the lipid membrane. mechanosensitive channels (msc) belong to a class of ion channels which respond on the mechanical stress within a lipid bilayer (anishkin and sukharev, 2009; kloda et al., 2008; perozo and rees, 2003) . they are designed by nature to sense alterations in osmotic and mechanic pressures imposed onto the lipid membrane. by sensing the change, they enable the physiological relevant ions to diffuse passively through their interior to alter electrochemical and substrate gradients of relevant cells (zhou et al., 1991) . consequently, these membrane proteins convert the mechanical stress into readable signals for our nervous system. two types of msc can be distinguished according to the levels of conductance they generate: mechanosensitive channels of small (mscs) and large conductance (mscl). for both types of channels, crystallographic data from bacterial channels are available (bass et al., 2002; chang et al., 1998; liu et al., 2009; steinbacher et al., 2007; wang et al., 2008) . focusing on mscl and the crystal structure of a homologous protein from mycobacterium tuberculosis (chang et al., 1998) , the protein has been identified to consist of a pentameric homoassembly with two tmds. its water filled pore is aligned with hydrophilic residues and has a diameter of about 1.8 nm. it is assumed that this structure catches the closed state of the channel. other structures are reported to be due to intermediate states of the opening (liu et al., 2009 ). this structure (liu et al., 2009 ) includes a truncation in the cytoplasmic domain which allows the tmds to adopt an increased tilt compared to the "closed" and stable state of the protein. this protein has shown increased current and therefore supports the idea of now having a structure in a more open state, albeit the still very narrow pore diameter. however, the protein is reported to be a tetramer in contrast to the earlier studies of the mscl in the closed state (chang et al., 1998) . nevertheless, the larger tilt of the tmds in a partially open state compared to the tilt in the closed structure indicates a sliding mechanism for opening ( fig. 6.5b ). interesting to note is that similar to p7, over time, and with different experiments, several oligomeric states have been proposed. biochemical data (sukharev et al., 1999) and two-dimensional crystallography (saint et al., 1998) support the formation of hexameric assemblies of the protein. the crystallographic data identify pentameric (chang et al., 1998) and tetrameric structures (liu et al., 2009) . it is up to further investigations to decide about the implications of the findings on the mechanism of function of this channel protein. proteins p7 and 2b align particularly good with the tmds of the mechanosensitive channel mscl (schindler and fischer, 2011) . this ignites a discussion of whether the two proteins would also respond to mechanical stress profiles of the lipid bilayer and gate in a similar way (fig. 6.5b ). the nachr is part of the cystein-loop receptor super family of pentameric neurotransmitter-gated ion channels (le novã¨re and changeux, 1995) triggered by acetylcholine and susceptible to nicotine. the channel protein assembles symmetrically around a fivefold central axis. within the family, the channel can exist in homo-and heteromeric assemblies of about 290 kda (unwin, 2005) . upon acetylcholine binding, the channel converts into an open state enabling a flux of mostly monovalent cations across the lipid membrane. with its location at nerve-muscle synapses, its action induces fast chemical transmission of nerve signals. information about the morphology of the protein has mostly been derived from electron microscopy (unwin, 2003 (unwin, , 2005 unwin et al., 1988) . the channel can be divided into three parts, a large extracellular domain hosting the n termini of the subunits, a membrane spanning part which contains the gate, and a smaller cytoplasmic domain. the overall length of the protein is 16 nm, with a pore of about 2 nm in diameter. the two binding sites of the neurotransmitter are about 4 nm above the membrane surface and opposing each other. cryo-electron microscopy has been used to derive structural features of the receptor when briefly exposed to acetylcholine and immediately frozen (unwin, 1995 (unwin, , 1998 . the data yield a resolution of 0.9 nm. in comparison with earlier structures, which are related to the closed state, the major changes in the open state are due to an alteration of the ligand binding domain which initiates a small axial rotation in each of the two tmds of the respective subunits almost 5 nm away. imposing structural data into the models suggest that the closed state is due to a constriction of the pore in the middle of the membrane caused by leu-251 from each subunit, so to speak a ring of leucines. the residues are part of the pore lining m2 helix of each subunit. the constriction does not result from an almost overlap of the five residues; it rather follows from an approach of these residues. the consequence of this approach is a hydrophobic barrier which interrupts the water column which would otherwise fit through the pore. upon activation, the ring widens through a rotational motion, twist-to-open (miyazawa et al., 2003; unwin, 2003) , giving enough space so water can pass this barrier, which represents the gate of the channel. passing of water in the open stage has been shown using molecular dynamics simulations on the tmds of the receptor embedded in a hydrated lipid bilayer (beckstein and sansom, 2006) . it has been suggested that this mechanism is a blue print for other members of the ligand-gated ion channels (unwin, 2003) . the bacterium erwinia chrysanthemi also expresses a ligand-gated ion channel which belongs to the family of pentameric ligand-gated ion channels (elic). crystal structures of this channel in its potential closed state have been obtained (hilf and dutzler, 2008) . the elic channels show 16% sequence homology with nachra and comprise the closest to a structure representing this class of ligand-gated ion channels. in terms of the extramembrane and transmembrane part, this channel approximately adopts the same dimensions (9.5ã�11 nm) as the nachr. in contrast to the nachr, the channel has no cytoplasmic domain. the 10 b-strands of each of the subuntis mantle a cylindrical vestibule of approximately 1.6 nm diameter. within the membrane, the pore narrows down to a diameter of 0.7 nm lined by one of four helical tmds per subunit. toward the extramembranes segment, the transmembrane passage is confined by a ring of phenylalanines (f246) and leucines (l239). it is anticipated that these two constriction sites form a gate similar to the ring of leucines in the nachr. similar to the nachr, the respective tmds a1 and a3 are involved in inter-subunit contact, whereas a2 lines the pore. the tmd a4 is positioned at the periphery of the transmembrane subunit. since the ligand triggering of the channel is not known, a study about the potential open state cannot be conducted. cyanobacterium gloebacter violaceus encodes a pentameric ion channel (glic) (hilf and dutzler, 2009) homologous to elic which is activated by low ph and does not desensitize after activation. crystal structure of the channel at ph 4 with a resolution of 0.31 nm is therefore considered to show the open state of this channel. the structure of glic is "very similar" to the structure of elic and therefore the data are taken to identify a novel gating mechanism for the pentameric ligand-gated ion channels. the lumen of the pore in the transmembrane region confines gradually toward the cytoplasmic side. this is due to a2 and a3 helices which are rotated as a rigid unit around val-267 by about 9 . the extramembrane part of the pore is flanked by hydrophobic residues while the cytoplasmic part is flanked by polar side chains. both ends of the channel are guarded by rings of acidic residues. the narrow part at the cytoplasmic end is due to a ring of glutamate residues (e221). crystallization of a mutant channel in which the glutamate residues are replaced by alanine under the same experimental conditions (low ph) reveals a similar overall conformation of the protein backbone but with the restriction derived from the glutamic acids being removed. this indicates that the local change of the amino acid residues does not alter their conformation and that only minor changes of the side chains allow an increased pore radius. the conclusion is that the glutamates interact directly with the permeant cations. the studies on both elic and glic are considered as studies on a closed (elic) and open (glic) ligand-gated ion channel. the opening is shown to result from a change in the tilt of two of the tmds, a2 and a3, of each of the subunits around a pivot point two-third across the transmembrane pore. this is proposed for nachr in contrast to the rotational motion of m2, which is similar to a2. protein 3a identifies identity with ligand-gated channels such as the nachr and pglic/elic (schindler and fischer, 2011) . this finding proposes that this large viral channel could be triggered by a ligand and also allows more complex gating behavior (fig. 6 .5a and c) for viral monotopic proteins such as vpu, e protein, or 8a. there is no identity with the pore lining part of the two types of toxins, such as the helical toxin clya and the b sheet structured ahemolysin. with m2 and bm2 proteins of influenza a and b, respectively, known to conduct protons, it is tempting to compare the mechanism of function of these proteins with other known proton conducting or transporting membrane proteins. one of the proteins, which allow protons to move across the membrane, is the light driven proton pump bacteriorhodopsin (br) from halobacterium halobium (oesterhelt and stoeckenius, 1971) . bacteriohodopsin is part of a family of light activated membrane proteins such as halorhodopsin and rhodospin. albeit the fact that this protein needs light to activate a vectorial proton transport to build up a proton motif force, the movement of the protons along certain amino acids within the protein can give an idea of what is necessary for proton translocation. bacteriorhodopsin has been analyzed in great details using techniques like cryo-electron microscopy (henderson et al., 1990) , ftir spectroscopy (rã¶dig et al., 1999; rothschild, 1992) , nmr spectroscopy (herzfeld and lansing, 2002) , x-ray crystallography (edman et al., 1999) , kinetic analysis (kã¶tting and gerwert, 2005) , and other techniques. describing its mechanism in short, the proton translocation mechanism is triggered by light leading to an isomerization of a retinal linked via a schiff base to lys-216 from its all trans into 13-cis conformation. this very fast event is followed by structural rearrangements of the protein which are characterized by specific spectral changes known as the photocycle of br (neutze et al., 2002) . structural rearrangements include kinks in two of the helices (c and f). further, the action of localized water molecules (fischer et al., 1994) in combination with a series of well-aligned titratable amino acids are necessary to achieve vectorial grotthuss-type proton transport. another highly selective human voltage-gated proton channel is hv1 (decoursey, 2008). hv1 is polytopic membrane protein with four tmds (ramsey et al., 2010) . pore lining residues are also identified to be titratable ones. however, if these titratable residues are mutated, proton conductivity is not fully abrogated. computational modeling studies using homologous models of voltage dependent potassium channels reveal that the structure allows water molecules within the pore of the channel. combining the experimental and computational data, it is suggested that a water-wire exists to transfer the proton as selectively as this channel does, but that proton translocation does not necessarily require titratable residues. what is the mechanism of function of m2, a proton channel of minimal design (fig. 6.5d) ? in comparison to other proton translocating and channeling proteins, it is missing a sequence of titratable residues within the pore. localized and structurally defined water molecules are not yet reported. this may be due to the fact that a structure of full-length m2 protein is not yet at hand. the gating mechanism may find some relation to br in as much the histidines of m2 rotate or "flip," similar to cis/trans isomerization in br, and "shuffle" the proton to the cytoplasmic side to be picked up by trp-41 and asp-44. structural changes for m2 seem to involve a change of the overall tilt of the protein assembly. similarities are thus far speculative and it remains the question in how far the design of m2 resembles a novel structural concept of proton translocation. most of the vcps are smaller in size than their host channels. with about 100 amino acids in length, they harbor a maximum of two tmds. only 3a from sars-cov harbors three domains but is with 274 amino acids almost three times as long. with one or two tmds per protein which itself needs to self-assemble to form a channel, it is anticipated that the channel should be sensitive to the lipid environment, which is mentioned for vpu (mehnert et al., 2008) . additional tmds per protein, as found in many other host channels, could act as a "mechanical buffer" toward the lipid membrane. functional information is mainly done either with the proteins reconstituted into artificial lipid compositions or with the protein expressed in xenopus oocytes (table 6 .1). structural data from nmr or x-ray sources are also recorded in artificial environments. there is emerging evidence that lipid rafts are playing a highly important role in the cellular life cycle of enveloped viruses (brã¼gger et al., 2006; campbell et al., 2001) . lipid rafts are cholesterol and glycosphingolipid rich detergent-resistant membrane patches (simons and ikonen, 1997; stier and sackmann, 1973) . purification of m2 from infected cells or eukaryotic expression systems has identified the presence of cholesterol (schroeder, 2010; . therefore, it has been suggested that m2 from influenza a is attached to lipid rafts in vivo (lin and schroeder, 2001; . since all of the m2 channel recording data identify the function of m2, even when taking just its tmd and not even covalently linked together, it seems that rafts just only serve as a scaffold for in vivo function. to combine these findings, it is suggested that in the case of a raft attachment of a monomeric protein, or dimeric protein in case of m2, several of the raft patches can form a trapped cholesterol free "patch" in which the protein can function as a proton channel (lin and schroeder, 2001) . raft association has also been reported for vpu (ruiz et al., 2010b) . expression of vpu in human 293 cell lines reveals that it partitioned into detergent-resistant membrane microdomains. its partitioning could be abolished when residues in the tmd are substituted by alanines. mutations of trp22 and a sequence on the middle of the domain, ivv19, are key residues responsible for microdomain integration. taken together with computational modeling data in which it is suggested that vpu is flexible and adopts to its environment via residues in the region from ile-17 to ser-24 (krã¼ger and fischer, 2008; park et al., 2003) , replacement of these the code for structural information refers to the four letter code used by the rcsb protein data bank (www.pdb.org). c, cytoplasmic domain. for the x-ray structures the resolution is given in ã� . residues hampers essential flexibility necessary for domain integration. important to note is that raft association is not required for cd4 downregulation and that the above mutations can be correlated with a reduced particle release (ruiz et al., 2010b) . the exact placement within the microdomain is unclear and it could still be suggested, similar to m2, that channel activity of vpu, which is independent of the cholesterol or sphingolipid content, could be achieved with the protein "released" from the raft into the confined space of an entrapped cholesterol free patch surrounded by rafts. 3a has been detected in membranous structures from expressing and infected cells (huang et al., 2006) . membrane flotation essay identified 3a at relevant gradients indicating that the protein is associated with lipid membranes. when the membranous structures are treated with "raft" detergents, membrane floatation essay unravels that still some fraction of 3a is bound to membranes. it is therefore concluded that some of the 3a proteins are located within or at raft-like domains. additionally, e protein from sars-co has been identified to be located in lipid rafts expressed in hela cells as well (liao et al., 2006) . the role of lipid composition on the mechanism of function of these channels is still little explored. with the channel proteins being raft associated, novel routes for antiviral therapy can be envisioned as discussed below (ruiz et al., 2010b) . when blocking enzymes, the site of interference is well defined. the prime target site is the active region of the enzyme and eventually the outer side of the protein for, noncompetitive modulators. membrane proteins contribute in drug therapy as drug targets to a large extent. for these proteins, the sites of interference are similar to enzymes. they are, for example, the docking sites of the biological ligand relevant for the receptor or ion channel. in cases of the latter, physical blocking of the channel, like a plough, is another option. for the extramembrane parts of the membrane proteins, other allosteric sites are equally important to be targeted by modulators. anticipating vcps as targets, one can imagine that with their small size the lipid membrane plays an important role in the mechanism of function of these channels. the interaction of proteins with each other is also highly dependent on the dynamics of the lipid membrane (langosch and arkin, 2009 ). in other words, a potential drug does not necessarily need to interact with the protein but can also alter lipid dynamics which in turn modulates protein function. consequently, targeting, for example, lipid rafts could also have potential antiviral effects. for some of the channel proteins, it emerges that they are interacting with a series of host factors via protein-protein interactions. the interaction is not confined to the extramembrane parts of the viral protein but also includes the tmds. this is especially the case for vpu and its interaction with the host factor bst-2. hampering with protein-protein interaction is consequently a promising path for drug development. small molecule design is the way how man is trying to steer proteins for the cure of diseases. viruses do not use small molecules, they rather create their own proteins to interfere with the mechanism of function of the host proteins. this concept inspires the development of drugs based on specific peptide-peptide interaction, such as peptide drugs. the idea of a peptide drugs is to mimic the fold of the viral "protein ligand" which targets the host protein. the consequent blocking of the "protein ligand" interrupts the pathway of the viral protein within the cellular life cycle. the "coating" of the protein with peptides could either involve coating of the host or the viral protein. potential peptide candidates as "coats" can be designed specifically by analyzing protein mechanics of both host and viral protein. another option would be to search related proteins from data banks of phylomers , proteins/peptides known to interrupt protein-protein interactions and used for protein silencing to validate targets (watt et al., 2006 ). an example of such an approach which has been done for hiv-protease (park, 2000) and hiv-integrase (maroun et al., 2001; zhao et al., 2003) indicates that this approach is a valid one for globular proteins. up to now, peptide drugs are used in a limited number of cases (rishabh et al., 2009) . however, their potential use is hypothesized to increase in the near future. most striking is their highly specific mode of action which is expected to require a low dose to be administered. in addition, peptide drugs seem to be toxicologically safe and are found to exhibit lower side effects than their small molecule counterparts (agyei and danquah, 2011) . peptide drugs, despite the benefit, harbor some draw backs. even though they are now more comfortably manufactured, administering of the drugs is still a major hurdle for their use on a broader scale (shaji and patole, 2008) . injection still is the major route for entering the drug into the body and up to now mostly used for treating diabetes. among the routes of administering, oral or nasal routes are still the most favorable ones. once administered, they also face difficulties such as fast enzymatic degradation, membrane impermeability due to their size, and metabolic stability. thus, chemical modifications are also necessary not only to generate a deliverable form of the drug but also to increase the retention time of the drugs in the body to improve their therapeutic value. one of the routes to improve the therapeutic value is to use peptides and develop peptidomimetics. the latter step is used to develop antimicrobial peptides (godballe et al., 2011; scott et al., 2008) . one of the first peptide like drugs proposed for antiviral therapy against membrane proteins, called 5-helix, has targeted gp41 from hiv-1, (root et al., 2001) . the idea of this peptide is to mimic the trimer-of-hairpins state of gp41 which consists of six helical elements of gp41 tightly packed. with only five helices, it is anticipated that the steric vacancy for the 6th helix will be taken up by the c-peptide region of the viral gp41 protein under native conditions. this interaction hampers the fold of gp41 into the trimer-ofhairpin state. shorter peptides have been suggested to target the c terminal region further (eckert and kim, 2001) . also, conserved hydrophobic pockets in the n terminal region of gp41 are targeted by peptides including small molecules (debnath, 2006) . currently, computational methods such as md simulations have been applied to assess the binding and search for novel peptides (strockbine and rizzo, 2007) . these developments have led to the first antifusion drug enfuvirtide (poveda et al., 2005; steffen and pã¶hlmann, 2010) . d-peptides, which show better affinity and bioavailability, are also reported to interfere with gp41 (welch et al., 2007) . most recently, a peptide based on a 1 -antitrypsin has shown promising results in patients (forssmann et al., 2010) . this 20-amino-acid peptide targets the fusion peptide of gp41. another strategy is to use peptides as a "molecular shield" of the host protein to protect it against interaction with viral proteins (strangler et al., 2007) . this concept is introduced with studies on preventing membrane associated nef protein from hiv-1 to interact with the cellular transduction protein hemato-poietic-cell kinase (hck). an artificial 12 amino acid proline-rich peptide abolishes the interaction between these two proteins. amantadine (1-aminoadamantan) (davies et al., 1964) and its derivatives have been used as one of the first antiviral drugs targeting m2, from influenza (hay et al., 1985) (fig. 6.6 ). activity occurs in two stages. in a nonspecific way at higher concentrations (>0.1 mm), it affects the fusion event hampering the conformation of hemagglutinin to the fusion relevant conformational state. at a later stage of the viral life cycle and at much lower concentrations (0.1-5 mm), the drug acts strain specific against virus assembly. at this stage, it prevents the transformation of the fusion protein to forming a so-called high ph conformational stage. the nonspecific action can also be achieved by a series of amines (hay et al., 1985) . mutant escape studies have shown that a series of hydrophobic residues at positions 27, 30, 31, and 34 of m2 (hay et al., 1985) as well as 26 (wang et al., 1993) are mutated in respective strains. mapping the residues onto a helical wheel indicates that they are clustered on one side and assumed to face the inside of the bundle rather than the lipid environment (sugrue and hay, 1991) . thus, amantadine is assumed to intrude into the pore and block the protein by occlusion of the pore. computational modeling of the drug in the center of an in silico model of the pore have been undertaken along this line (sansom and kerr, 1993 ). the m2 model has been built by copy-rotation of the monomer so that the hydrophilic residues face the lumen of the pore resulting in a slightly wider pore at the n terminal side than on the c terminal side. by positioning amantadine along the center of the pore and calculating for each position the interaction energy, a profile has been generated, which indicates a favorable binding site at the level of ser-31 with the amantadine cube facing the n terminal mouth and the amino group the c terminal side. the same experiments with a cyclic-pentylamine have not revealed a favorable binding site at the same position. neutron diffraction experiments confirmed a position in the area between val-27 and ser-31 (duff et al., 1994) . in a similar approach, using molecular dynamics simulations, amantadine has been positioned in the same way as reported (sansom and kerr, 1993) within the pore of m2 (yi et al., 2008) . the computational model of m2 has been leaned on an assembly based on nmr spectroscopic investigations (hu et al., 2007) . during the molecular dynamics simulation of 15 ns, the drug remains at its position around ser-31 supporting the site of interaction of amantadine with m2. in another proposal, amantadine is modeled to be within the central cavity with the amino group closer to the ring of histidines (gandhi et al., 1999) . the proposal that the amino group is pointing toward the histidines from various sites within the n terminal side of the pore is verified by crystallographic studies (stouffer et al., 2008) . in this crystallographic study with a peptide corresponding to the tmd of m2 with only one mutation at position 34, where glycine is replaced by alanine, the location of amantadine is in the lumen of the pore on the n terminal side (3c9j; stouffer et al. 2008) . the crystal structure is obtained at low ph (ph 5.3) representing an open-like or conducting channel. residues such as val-27, ala-30, ser-31, and gly-34 "trap" the drug so that its amino group points toward the c terminal side. it has been shown that mutation of these residues makes the channel insensitive to amantadine (deyde et al., 2007) . since amantadine shows no cooperativity (wang et al., 1993) , this binding site is seen as the single site and the mode of action as an "occluding the pore." based on solid state nmr spectroscopic studies of a peptide corresponding to the tmd of m2 derived from spps and reconstituted into dlpc (1,2-dilauroyl-snglycero-3-phosphatidylcholine), a binding site near ser-31 has been suggested (cady et al., 2009) , which has been confirmed in a later experiment (cady et al., 2010) . also, at higher amatadine concentrations, solid state nmr experiments suggest a second site at the c terminal side which should be considered as a weak binding site with low affinity (cady et al., 2010) . molecular dynamics simulations over 15 ns with the amantadine bound m2 model from crystallography show that the drug remains at the position disrupting the continuous water column similar to the ring of valines (at position 27). therefore, it is concluded that the mode of amantadine blocking is by interfering with the water molecules at the mouth of the pore. in a solution, nmr spectroscopic investigation of a m2 peptide fusion construct corresponding to the tmd of the protein, which is reconstituted into dhpc (1,2-diheptanoyl-sn-glycero-3-phosphocholine), the amantadine derivative rimantadine has been detected to bind at the outside of the bundle at the c terminal side (schnell and chou, 2008) . the experiments have been performed at ph of 7.5 which implies that the structural model represents a "closed" or non-active state of the protein. rimantadine has a larger portioning coefficient between octanol and water than amantadine (belshe et al., 1989) and possibly diffuses from the site of the membrane toward the protein. however, in this experiment four rimantadines per bundle are reported. visualization of the binding site reveals that rimantadine sits within a pocket of hydrophobic residues ile-42, leu-40, and leu-43 at the helix-helix interface. a blocking mechanism is suggested in which the drug is hampering the transition of the bundle into the open or h ã¾ gating state. based on these studies and earlier suggestions , it is reasonable to conclude that there are allosteric effects (schnell and chou, 2008) involved in the binding of amantadine derivatives to m2. the mode of action for all drugs possibly involves an alteration of protein dynamics and based on the type of drug allosteric interactions rather than single-site blocking. amantadine is not reported to be active against bm2 . amantadine has been reported to block p7 when the protein is expressed in e. coli, purified and reconstituted into lipid bilayers (griffin et al., 2003) . the protein, a his-p7 construct, shows burst activities at a holding potential of ã�120 mv. adding amantadine to a final concentration of 1 mm into both chambers of the setup leads to a complete abrogation of the bursts after about 10 s. application of amantadine to hcv replication cell cultures (lohmann et al., 1999) revealed no effect of the drug on rna replication, virus release, and infectivity of the virions (steinmann et al., 2007b) . using a peptide derived from spps, corresponding to specific strain of hcv (gt 1a, isolate h77) which also is reconstituted into artificial lipid bilayer, addition of more than 10.3 mg/ml of amantadine has been necessary to affect channel activity of the p7 peptide. a full blocking, showing a zero channel activity, could not be achieved. a docking approach using auto-doc 3.0 has been used to dock amantadine onto a computationally derived hexameric model of p7 (patargias et al., 2006) . the study proposes a binding site of the drug within the lumen of the pore between residues h17 and ser21, with the amino group facing ser21. the binding constant has been estimated to be around k i â¼ 68 mm. since amantadine is located toward two monomers, it allows plenty of space for ions to pass the pore. so far, amantadine has shown little effect in clinical trials (deltenre et al., 2004; maynard et al., 2006) . currently, a series of derivatives of amantadine have been tested (foster et al., 2011) . channel activity of a peptide corresponding the n terminus of vpu including the tmd, vpu 1-32 , derived from spps, reconstituted into artificial lipid bilayers is not affected by amantadine . amantadine has also been reported to block kcv (plugge et al., 2000) . due to the emerging resistance of influenza strains against amantadines and derivatives and also due to the side effects caused in the central nervous system of the latter compounds, a novel class of drugs, spiropiperidines, is currently investigated (wang et al., 2009a ) (bl-1743, fig. 6 .6). solid state nmr studies reveal that the derivative 3-azaspiro[5.5]undecane hydrochloride alters the dynamics of the protein and affects a larger series of amino residues within the pore. using a docking approach (autodock) with the drug bound crystal structure of m2 (3c9j (stouffer et al., 2008) , a binding site similar to amantadine is proposed. iminosugars are more effective than amantadine against p7, as indicated by channel recordings measurements which is also confirmed by cell based essays (steinmann et al., 2007b) . especially, long alkyl chain iminosugars block p7 much stronger than iminosugars with short alkyl chains. it has been reported that vpu is sensitive to derivatives of amiloride . experiments have been done with vpu expressed in hela cells together with the gag protein of hiv-1. budding virus like particles have been observed by electron microscopy. experiments in the presence of hma in the culture medium after transfection of the cells with the expression plasmid lead to an almost complete inhibition of the budding process. channel recording of a recombinant vpu protein, expressed in e. coli, could be blocked with the addition of hma in a range of 25-125 mm. an allosteric blocking could be anticipated since it is reported that at lower hma concentrations blockage is not complete. blocking has also been dependent of the applied potential. therefore, it seems that blocking also depends on the side of which the drug approaches the channel. in the same type of experiments, a derivative dimethyl amiloride (dma) also affects channel recordings in a similar way. since blocking by dma has been less complete than by hma at higher concentrations, a lower potency for dma has been concluded. amiloride itself has shown no effect in this study. the data have been confirmed in a study using a vpu peptide (vpu 1-32 ) reconstituted into artificial "micro" bilayers which are spanning a porous silicon device (rã¶mer et al., 2004) . adding 100 mm of both hma and dma to a measurement setup results in a full blocking of channel activity, while amiloride does not show any effect on the activity. in a computational study with a helical model of the monomeric tmd of vpu and in an assembly of five and six tmds forming bundles, putative binding sites of hma and amiloride have been evaluated (kim et al., 2006) . most striking are calculations of an estimated free energy using the docking program autodock 3.0. the calculations reveal lowest binding constant for hma interacting with the pentameric bundle. binding sites of hma within the bundle identify an interaction of the guanidinium group of hma at the site of ser-24. interaction of hma with a monomeric tmd identifies trp-23 as a potential site for interactions, suggesting p-p interactions. the computational data support the currently emerging idea of multiple binding sites of antiviral channel drugs and with it the suggestion of allosteric binding modes (pielak et al., 2009; schnell and chou, 2008) . from investigations on vpu interacting with hma-two different inhibition levels at various drug concentrations in bilayer recording studies and two putative binding sites identified in a docking approach-it seems very likely that vpu also follows this path. hma is reported to block channel activity of full-length p7 when derived from spps, purified and reconstituted into artificial lipid bilayers (premkumar et al., 2004) . in repeatedly, recorded experiments hma has been added to the cis side of a bilayer recording setup which contained 500 mm kcl while the trans side contains 10 times less kcl. to date, n-[5-(1-methyl-1h-pyrazol-4-yl)-naphtalene-2-carbonyl]-guanidine (bit225, fig. 6 .6) is the second most advanced class of antiviral drugs targeting a vcp. synthetic p7 derived from spps and reconstituted into lipid bilayers can be blocked by bit225 at a concentration of 100 mm bit225 on both sides of the measurement chamber and both filled with 50 mm kcl ). an ic 50 in the submicromolar range has been reported with a cell-based essay system using the model virus bvdv (bovine viral diarrhea virus). in combination with interferon alpha-2b, (rifna-2b) even a synergistic affect has been observed. bit225 has now successfully completed phase ib/iia trials. the drug is also evaluated for its affection of vpu (khoury et al., 2010) . a synthetic peptide construct representing the first 32 amino acids of vpu including the tmd (vpu 1-32 ), reconstituted into lipid bilayers shows channel activity which is knocked out by a 40 mm solution of bit225. in monocyte-derived macrophages chronically infected with hiv-2, which does not encode vpu, no affect has been observed, which supports that vpu is the target. a wide range of hiv-1 isolates are susceptible to bit225 as well. based on a computational evaluation of several guanidines on a pentameric vpu 1-32 , bit225 has come out as a highly potential candidate (patargias et al., 2010) . the study uses models of vpu tmds in which the serines (ser-24) are facing the potential lumen of the pore. for this investigation, the docking software flexx (biosolvit) has been used. the small molecule 4,4 0 -diisothiocyanatostilben-2,2 0 -disulfonic acid has been reported to block 2b (xie et al., 2011) . amphotericin b methyl ester (ame) is a water soluble derivative of amphotericin b (waheed et al., 2008) (fig. 6.6 ). it is water soluble, shows low toxicity, and is able to bind to cholesterol within the membrane. viral membranes, especially those in hiv-1 virions, are found to exhibit some similarity in the composition to lipid rafts (aloia et al., 1993; brã¼gger et al., 2006) . with the rafts containing cholesterol, ame alters the properties of the viral membrane. when jurkat cells are infected with a plasmid containing an infectious full-length hiv-1 and continuously exposed to 10 mm ame escape mutants indicate mutations in an endocytoses motif in the cytoplasmic part of gp41, the viral fusion protein (waheed et al., 2006) . in another study, it has been shown that ame lowers virus production (waheed et al., 2008) . ame seems to be noneffective against the gagmembrane protein assembly and gag-association with detergent-resistant membranes. it rather lowers the amount of virion release by approximately fivefold. vpu-deficient hiv mutants are insensitive to ame treatment also in the presence of an overexpression of cd317/bst-2/tetherin. reinjection of the cells with vpu plasmid makes the cell sensitive to ame again. this indicates that ame most likely disrupts the vpu-cd317/dst-2/ tetherin interaction. it is speculated that ame blocks ion channel activity of vpu. further, it may be speculated that vpu may need raft association to be fully functional. this study (waheed et al., 2006 (waheed et al., , 2008 shows that there could also be a conceptual different pathway to block a membrane protein. it could be anticipated that ame locates itself at those sites of lipid rafts which "show" the cholesterol, and with this, the interaction of vpu or other viral proteins with these sites is not possible anymore. if ame does not interact with vpu, it cannot act as a "replacement" of cholesterol and vpu function is abrogated. lipid membrane composition is an integral part for the mechanism of function of some of the vcps such as m2 and vpu. especially, the latter is found in lipid rafts. since rafts are lipid patches with a high content of cholesterol, cholesterol depleting drugs are suggested to combat the virus (ruiz et al., 2010b) . experiments have shown that cells which express vpu but are treated with a combination of lovastatin and m-b-cd show reduced levels of vpu in detergent-resistant membrane microdomains. it has been shown that lipid composition and with it the existence of rafts is essential for viral entry and budding (brã¼gger et al., 2006) . anti-raft drugs have been proposed to target rafts and with it especially to combat the replication of hiv-1 (verma, 2009 ). plant-derived drugs have been found exhibiting potential preventive effects of hiv-1 progression. compounds such as o-3-fatty acids and plant-derived triterpenes are investigated. since involvement of rafts in the mechanism of function of the viral channels is gradually emerging, these drug candidates could potentially be affecting the vcps. other plant-derived drugs such as polyphenols are proposed to target extramembrane parts of channel proteins while lipophilic terpenoids act within the membrane either directly or indirectly (wink, 2008) . saponins target cholesterol and could also act in a dual mode as mentioned: directly by protein-drug interaction or by an indirect mode of action, such as distorting raft composition. it is up to further studies to modify these candidates to address them more specifically to viral channels. a promising plant-derived drug candidate is emodin (6-methyl-1,3,8trihydroxyanthraquinone, fig. 6 .6), targeting 3a not only of sars-cov but also hcov-oc43 (schwarz et al., 2011) . the binding constant has been calculated to be 20 mm using voltage clamp conditions on xenopus oocytes expressing 3a. to date, m2 is the only target channel protein used in antiviral therapy. protein p7 and eventually vpu are in closer reach. for other proteins, investigations are still very much on a laboratory level reporting interactions of known drugs with the channel proteins. the current findings on the viral channel-forming proteins are reviewed in respect of the mechanism of function and their role of affecting electrochemical or substrate gradients. while for m2, a detailed picture of the mechanics of proton translocation is emerging due to structural work, the reason for weak cation selectivity of most of the channels is still in the dark. based on sequence alignment studies, a relation to host channels and toxins is drawn for some of the channel proteins. with vpu relating to toxin clya, and ditopic channels like p7 and 2b relating to mcsl and tritopic 3a likely to ligand-gated channels, the picture emerges that this type of protein covers the range from pores to channels. m2 as a proton channel may "borrow" mechanisms from other proton conducting and translocating membrane proteins. the evidence is growing that some of these proteins are raft associated. also, the evidence emerges that channel functionality is not the only role these proteins inherit. many of them interact with host factors to steer the cell toward the benefit of the virus (stage of steering). questions arise whether this "steering of the host" is done as monomeric or oligomeric units. in addition to it, there is some kind of equilibrium between the "stage of steering" and the proper channel state as an oligomeric unit. or is the interaction with the host to be seen as a kind of "host based ligand" closing the channel while without the host protein the channel is open; or do other ligands trigger activity? more surprising, with influenza a and sars-cov two viruses emerge which harbor more than one channel protein. on the other end of this scale, for dengue virus there is no report of any channel protein up to now despite its membership to the same family, flaviviridae, as hcv. drug development is yet still to be fully explored. with m2 as the only target so far, two more channel proteins are on the horizon to be potential candidates. since structural information is sparse for most of the channels, development of pore occluding drugs is difficult. drug-protein interaction seems to obey, in the same sophisticated way as for any other host or protein of pathogens, allosteric binding modes. on the other hand, drug development is challenged, due to the fact that protein-protein interactions between viral and host proteins are also happening within the lipid membrane. thus, novel concepts for finding drugs are essential. this may put peptide drugs, peptidomimetics, and plant-derived drugs in an elevated position for lead discovery. fast and slow gating are inherent properties of the pore module of the kã¾ channel kcv structure and mechanism of proton transport through the transmembrane tetrameric m2 protein bundle of the influenza a virus fall-39, a putative human peptide antibiotic, is cystein-free and expressed in bone marrow and testis industrial-scale manufacturing of pharmaceutical-grade bioactive peptides membrane permeabilization by poliovirus proteins 2b and 2bc influenza virus rna segment 7 has the coding capacity for two polypeptides lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membrane state-stabilizing interactions in bacterial mechanosensitive channel gating and adaptation a highly unusual palindromic transmembrane helical hairpin formed by sars coronavirus e protein the oligomeric state of the active bm2 ion channel protein of influenza b virus crystal structure of escherichia coli mscs, a voltage-modulated and mechanosensitive channel chemical synthesis and single channel properties of tetrameric and pentameric tasps (templateassembled synthetic proteins) derived from the transmembrane domain of hiv virus protein u (vpu) a hydrophobic gate in an ion channel: the closed state of the nicotinic acetylcholine receptor resistance of influenza a virus to amantadine and rimantadine: results of one decade of surveillance architecture of the sars coronavirus prefusion spike ion channel activity of hiv-1 vpu is dispensable for counteraction of cd317 the hiv-1 vpu protein: a multifunctional enhancer of viral particle release the human immunodeficiency virus type 1 vpu protein specifically binds to the cytoplasmic domain of cd4: implications for the mechanism of degradation sequence of rna segment 7 of the influenza b virus genome: partial amino acid identity between the membrane protein (m1 of influenza a and b viruses and conservation of a second open reading frame the hiv lipidome: a raft with an unusual composition structural characterization and oligomerization of pb1-f2, a pro-apoptotic influenza a virus protein structure of amantadine-bound m2 transmembrane peptide of influenza a in lipid bilayers from magic-angle-spinning solid-state nmr: the role of ser31 in amantadine binding structure of the amantadine binding site of influenza m2 proton channels in lipid bilayers functional interaction of human immunodeficiency virus type 1 vpu and gag with a novel membrane of the tetratricopeptide repeat protein family lipid rafts and hiv-1: from viral entry to assembly of progeny virions multiple proton confinement in the m2 channel from the influenza a virus modification of membrane permeability by animal viruses subcellular localization and topology of the p7 polypeptide of hepatitis c virus structure of the mscl homolog from mycobacterium tuberculosis: a gated mechanosensitive ion channel pb1-f2, an influenza a virus-encoded proapoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes a novel potassium channel encoded by ectocarpus siliculosus virus open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis proton transport behaviour through the influenza a m2 channel: insights from molecular simulations construction of an implicit membrane environment for the lattice monte carlo simulation of transmembrane protein orf 8a of severe acute respiratory syndrome coronavirus forms an ion channel: experiments and molecular dynamics simulations determination of pore-lining residues in the hepatitis c virus p7 protein selctive proton permeability and ph regulation of the influenza virus m2 channel expressed in mouse erythroleukaemia cells evidence for the formation of a heptameric ion channel complex by the hepatitis c virus p7 protein in vitro conformational analysis by nmr and molecular modelling of the 41-62 hydrophilic region of hiv-1 encoded virus protein u (vpu) hiv-1 encoded virus protein u (vpu) solution structure of the 41-62 hydrophilic region containing the phosphorylated sites ser 52 and ser 56 effects of vpu expression on xenopus oocyte membrane conductance identification of a protein encoded by the vpu gene of hiv-1 nmr studies of the p7 protein from hepatitis c virus secondary structure, dynamics, and architecture of the p7 membrane protein from hepatitis c virus by nmr spectroscopy the structure of the hiv-1 vpu ion channel: modelling and simulation studies bundles consisting of extended transmembrane segments of vpu from hiv-1: computer simulations and conductance measurements a protein linkage map of the p2 nonstructural proteins of poliovirus transmembrane helix prediction: a comparative evaluation and analysis pore-opening mechanism of the nicotinic acetylcholine receptor evinced by proton transfer studies of structural changes in the m2 proton channel of influenza a virus by tryptophan fluorescence antiviral activity of 1-adamantanamine (amantadine) multimerization reactions of coxsackievirus proteins 2b, 2c and 2bc: a mammalian two-hybrid analysis the coxsackievirus 2b protein increases efflux of ions from the endoplasmic reticulum and golgi, thereby inhibiting protein trafficking through the golgi progress in identifying peptides and small-molecule inhibitors targeted to gp41 of hiv-1 voltage-gated proton channels evaluation of amantadine in chronic hepatitis c: a meta-analysis surveillance of resistance to adamantanes among influenza a (h3n2) and a (h1n1) virus isolated worldwide inhibition of cellular protein secretion by poliovirus proteins 2b and 3a the structure of the potassium channel: molecular basis of k ã¾ conduction and selectivity antagonism of tetherin restriction of hiv-1 release by vpu invovles binding and sequestration of the restriction factor in a perinuclear compartment the transmembrane domain of influenza a m2 protein forms amantadine-sensitive proton channels in planar lipid bilayers the secondary structure of influenza a m2 transmembrane domain. a circular dichroism study neutron diffraction reveals the site of amantadine blockade in the influenza a m2 ion channel mechanisms of viral membrane fusion and its inhibition high-resolution x-ray structure of an early intermediate in the bacteriorhodopsin photocycle processing in the pestivirus e2-ns2 region: identification of proteins p7 and e2p7 the vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by hiv-1 protein vpu solution structure of the cytoplasmic domain of the human immunodeficiency virus type 1 encoded virus protein u (vpu) vpu from hiv-1 on an atomic scale: experiments and computer simulations viral membrane proteins: structure, function and drug design viral channel forming proteins-modelling the target viral channel forming proteins viral ion channels: structure and function detection of a water molecule in the active-site of bacteriorhodopsin: hydrogen bonding changes during the primary photoreaction short-term monotherapy in hiv-infected patients with a virus entry inhibitor against the gp41 fusion peptide resistance mutations define specific antiviral effects for inhibitors of the hepatitis c virus p7 ion channel potential roles of electrogenic ion transport and plasma membrane depolarization in apoptosis site-directed mutations in the sinbis virus 6k protein reveal sites for fatty acylation and the underactlated protein affects virus release and virion structure cu(ii) inhibition of the proton translocation machinery of the influenza a virus m2 protein structure and assembly of alphaviruses the viral potassium channel kcv: structural and functional features long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins antimicrobial b-peptides and a-peptoids the human immunodeficiency virus type 1 vpu protein enhances membrane permeability ion channels formed by hiv-1 vpu: a modelling and simulation study plugging the holes in hepatitis c virus antiviral therapy the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug, amantadine isolation and characterization of viruses related to the sars coronavirus from animals in southern china e2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies the molecular basis of the specific anti-influenza action of amantadine model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy the proapoptotic influenza a virus protein pb1-f2 forms a nonselective ion channel magnetic resonance studies of the bacteriorhodopsin pump cycle nucleotide sequence of rna segment 7 of influenza b/singapore/22/79: maintenance of a second large open reading frame x-ray structure of a prokaryotic pentameric ligand-gated ion channel structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel structural basis of hiv-1 tethering to membranes by the bst-2/tetherin ectodomain influenza virus m2 integral membrane protein is a homotetramer stabilized by formation of disulphide bonds eukaryotic coupled translation of tandem cistrons: identification of the influenza b virus bm 2 polypeptide in silico investigations of possible routes of assembly of orf 3a from sars-cov mutual functional destruction of hiv-1 vpu and host task-1 channel membrane potential depolarization as a triggering mechanism for vpu-mediated hiv-1 release histidines, heart of the hydrogen ion channel from influenza a virus: toward an understanding of conductance and proton selectivity backbone structure of the amantadine-blocked trans-membrane domain m2 protein channel from influenza a virus conformational plasticity of the influenza a m2 transmembrane helix in lipid bilayers under varying ph, drug binding, and membrane thickness action of antimicrobial peptides: two-state model sever acute respiratory syndrome coronavirus 3a protein is released in membranous structures from 3a protein-expressing cells and infected cells oligomerization of the human immunodeficiency virus type i (hiv-1) vpu protein-a genetic, biochemical and biophysical analysis human immunodeficiency virus type 1 vpu protein interacts with cd74 and modulates major histocompatibility complex class ii presentation pore formation: an ancient yet complex form of attack severe acute respiratory syndrome coronavirus 3a protein is a viral structural protein crystal structure and mechanism of a calcium-gated potassium channel the open pore conformation of potassium channels hepatitis c virus p7 and ns2 proteins are essential for production of infectious virus antiviral efficacy of the novel compound bit225 against hiv-1 release from human macrophages molecular dynamics calculations suggest a conductance mechanism for the m2 proton channel from influenza a virus transmembrane glycine zippers: physiological and pathological roles in membrane proteins drug-protein interaction with vpu from hiv-1: proposing binding sites for amiloride and one of its derivatives mechanosensitive channel of large conductance proteins in action monitored by time-resolved ftir spectroscopy transmembrane four-helix bundle of influenza a m2 protein channel: structural implications from helix tilt and orientation exploring the conformational space of vpu from hiv-1: a versatile and adaptable protein assembly of viral membrane proteins vpu transmembrane peptide structure obtained by site-specific fourier transform infrared dichroism and global molecular dynamics searching experimentally based orientational refinement of membrane protein models: a structure for the influenza a m2 h ã¾ channel crystal structure of the potassium channel kirbac1.1 in the closed state identification of a second protein (m 2 ) encoded by rna segment 7 of influenza virus conservation of the influenza virus membrane proteins (m 1 ) amino acid sequence and an open readig frame of rna segment 7 encoding a second protein (m 2 ) in h1n1 and h3n2 strains do vpu and vpr of human immunodeficiency virus type 1 and nb of influenza b virus have ion channel activities in the viral life cycles influenza virus m2 protein is an integral membrane protein expressed on the infected-cell surface interaction and conformational dynamics of membranespanning protein helices molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells full length vpu from hiv-1: combining molecular dynamics simulations with nmr spectroscopy solid-state nmr characterization of conformational plasticity within the transmembrane domain of the influenza a m2 proton channel biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein internally located cleavable signal sequences direct the formation of semliki forest virus membrane proteins from a polyprotein precursor definitive assignment of proton selectivity and attoampere unitary current to the m2 ion channel protein of influenza a virus processing in the hepatitis c virus e2-ns2 region: identification of p7 and two distinct e2-specific products with different c termini structure of a tetrameric mscl in an expanded intermediate state replication of subgenomic hepatitis c virus rnas in a hepatoma cell line molecular dynamics investigation of membrane-bound bundles of the channel-forming transmembrane domain of viral protein u from the human immunodeficiency virus hiv-1 severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release the 3-dimensional structure of the hepatitis c virus p7 ion channel by electron microscopy fate of the 6k membrane protein of smliki forest virus during virus assembly a novel hepatitis c virus p7 ion channel inhibitor, bit225, inhibits bovine viral diarrhea virus in vitro and shows synergism with recombinant interferon-a-2b and nucleaoside analogues expression, purification, and activities of full-length and truncated versions of the integral membrane protein vpu from hiv-1 identification of the pore-lining residues of the bm2 ion channel protien of influenza b virus hepatitis c virus ns2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins potassium channels requirement of the vesicular system for membrane permeabilization by sindbis virus plasma membrane-porating domain in polio virus 2b protein. a short peptide mimics viroporin activity human-immunodeficiencyvirus type-1 vpu protein is an oligomeric type-i integral membrane protein correlation of the structural and functional domains in the membrane protein vpu from hiv-1 interaction between the cytoplasmic domains of hiv-1 vpu and cd4: role of vpu residues involved in cd4 interaction and in vitro cd4 degradation peptide inhibitors of hiv-1 integrase dissociate the enzyme oligomers the genome sequence of the sars-associated coronavirus amantadine triple therapy for non-responder hepatitis c patients. clues for controversies (anrs hc 03 bitri) semliki forest virus produced in the absence of the 6k protein has an altered spiek structure as revealed by decreased membrane fusion capability possible function for virus encoded k ã¾ channel kcv in the replication of chlorella virus pbcv-1 towards a mechanism of function of the viral ion channel vpu from hiv-1 biophysical characterisation of vpu from hiv-1 suggests a channel-pore dualism alphavirus 6k proteins form ion channels structure and gating mechanism of the acetylcholine receptor pore structure-function correlates of vpu, a membrane protein of hiv-1 nmr structure and ion channel activity of the p7 protein from hepatitis c virus simulation of the hiv-1 vpu transmembrane domain as a pentameric bundle mechanism for proton conduction of the m 2 ion channel of influenza a virus influenza b virus bm2 protein has ion channel activity that conducts protons across membranes the structrure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism human coronavirus infection sars coronavirus accessory proteins tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu chlorella viruses evoke a rapid release of kã¾ from host cells during the early phase of infection bacteriorhodopsin: a high-resolution structural view of vectorial proton transport the closed state of a h ã¾ channel helical bundle combining precise orientational and distance restraints from solid state nmr ion selectivity in potassium channels the bm2 protein of influenza b virus is synthesized in the late phase of infection and incorporated into virions as a subviral component rhodopsin-like protein from purple membrane of halobacterium halobium genetic selection for dissociative inhibitors of designated protein/protein interactions tilt angle of a trans-membrane helix is determined by hydrophobic mismatch three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (vpu) from hiv-1 rotational diffusion of membrane proteins in aligned phospholipid bilayers by solid-state nmr spectroscopy structural flexibility of the pentameric sars coronavirus envelope protein ion channel protein-protein interactions: modeling the hepatitis c virus ion channel p7 model generation of viral channel forming 2b protein bundles from polio and coxsackie viruses ligand-protein docking studies of potential hiv-1 drug compounds using the algorithm flexx influenza b virus bm2 protein is an oligomeric integral membrane protein expressed at the cell surface phosphorylation of both phosphoacceptor sites in the hiv-1 vpu cytoplasmic domain is essential for vpu-mediated er degradation of cd4 the hepatitis c virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives structure and mechanism in prokaryotic mechanosensitive channels structure and inhibition of the sars coronavirus envelope protein ion channel flu channel drug resistance: a tale of two sides mechanism of drug inhibition and drug resistance of influenza a m2 channel understanding the mechanism of action of the anti influenza virus drug amantadine a functionally defined model for the m2 proton channel of influenza a virus suggests a mechanism for its ion selectivity a potassium channel protein encoded by chlorella virus pbcv-1 enfuvirtide, the first fusion inhibitor to treat hiv infection cation-selective ion channels formed by p7 of hepatitis c virus are blocked by hexamethylene amiloride an aqueous h ã¾ permeation pathway in the voltage-gated proton channel hv1 h bonding at the helix-bundle crossing controls gating in kir potassium channels protein and peptide drugs: a brief review time-resolved step-scan fourier transform infrared spectroscopy reveals differences between early and late m intermediates of bacteriorhodopsin channel activity of a viral transmembrane peptide in micro-blms: vpu 1-32 from hiv-1 protein design of an hiv-1 entry inhibitor ftir difference spectroscopy of bacteriorhodopsin: towards a molecular model simulations of the bm2 proton channel transmembrane domain from influenza virus b requirements of the membrane proximal tyrosine and dileucine-based sorting signals for effective transport of the subtype c vpu protein to the plasma membrane and virus release the vpu protein: new concepts in virus release and cd4 down-modulation membrane raft association of the vpu protein of human immunodeficiency virus type 1 correlates with enhanced virus release a hexameric transmembrane pore revealed by two-dimensional crystallization of the large mechanosensitive ion channel (mscl) of escherichia coli influenza virus m 2 protein: a molecular modelling study on the ion channel the influenza a virus m2 channel: a molecular modelling and simulation study potassium channels: structures, models, simulations interfacial domains in sindbis virus 6k protein sequence alignment of viral channel proteins with cellular ion channels and toxins formation and assembly of alphavirus glycoproteins multistate empirical valence bond model for proton transport in water structure and mechanism of the m2 proton channel of influenza a virus cholesterol-binding viral proteins in virus entry and morphogenesis influenza a virus m2 protein: proton selectivity of the ion channel, cytotoxicity, and a hypothesis on peripheral raft association and virus budding functional reconstitution in lipid vesicles of influenza virus m2 protein expressed by baculovirus: evidence for proton transfer activity the influenza virus ion channel and maturation cofactor m2 is a cholesterol-binding protein the human immunodeficiency virus type 1 encoded vpu protein is phosphorylated by casein kinase-2 (ck-2) at positions ser52 and ser54 within a predicted alpha-helix-turnalpha-helix-motif the two biological activities of human immunodeficiency virus type 1 vpu protein involve two separable structural domains identification of an ion channel activity of the vpu transmembrane domain and its involvement in the regulation of virus release from hiv-1-infected cells structural and functional studies on the extracellular domain of bst2/tetherin in reduced and oxidized conformations emodin inhibits current through sars-associated coronavirus 3a protein computer simulation of ion channel gating: the m 2 channel of influenza a virus in a lipid bilayer de novo designed synthetic mimics of antimicrobial peptides from 'carpet' mechanism to de-novo designed diastereomeric cell-selective antimicrobial peptides protein and peptide drug delivery: oral approaches insight into the mechanism of the influenza a proton channel from a structure in a lipid bilayer structure and dynamics of the hiv-1 vpu transmembrane domain revealed by solid-state nmr with magic-angle spinning small envelope protein e of sars: cloning, expression, purification, cd determination, and bioinformatics analysis ion selectivity and activation of the m 2 ion channel of influenza virus functional rafts in cell membranes bst-2 is rapidly down-regulated from the cell surface by the hiv-1 protein vpu: evidence for a post-er mechanism of vpu-action molecular dynamics simulation of proton transport through the influenza a virus m2 channel structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore a hidden markov model for predicting transmembrane helices in protein sequences molecular dynamics simulations on the first two helices of vpu from hiv-1 sars-beginning to understand a new virus peptide-based inhibitors of the hiv envelope protein and other class i viral fusion proteins structures of the prokaryotic mechanosensitive channels mscl and mscs hepatitis c virus p7 protein is crucial for assembly and release of infectious virions antiviral effects of amantadine and iminosugar derivatives against hepatitis c virus spin labels as enzyme substrates. heterogeneous lipid distribution in liver microsomal membranes structural basis for the function and inhibition of an influenza virus proton channel competitive displacement of full-length hiv-1 nef from the hck sh3 domain by a high-affinity artificial peptide the alphaviruses: gene expression, replication, and evolution novel gene of hiv-1, vpu, and its 16-kilodalton product molecular and biochemical analysis of human deficiency virus type 1 vpu protein binding of antifusion peptides with hivgp41 from molecular dynamics simulations: quantitative correlation with experiments structural characteristics of the m2 protein of influenza a viruses: evidence that it forms a tetrameric channel palmitoylation of the influenza a virus m 2 protein stoichiometry of the large conductance bacterial mechanosensitive channel of e. coli. a biochemical study molecular dynamics simulation of human immunodificiencey virus protein u (vpu) in lipid/water lamgmuir monolayer a novel severe acute respiratory syndrome coronavirus protein, u274, is transported to the cell surface and undergoes endocytosis molecular dynamics simulation study of the cytosolic mouth in kcv-type potassium channels model development for the viral kcv potassium channel the sub-cellular localisation of the hepatitis c virus non-structural protein ns2 is regulated by an ion channel-independent function of the p7 protein functional role of human immunodeficiency virus type 1 vpu expression and initial structural insights from solid-state nmr of the m2 proton channel from influenza a virus initial structural and dynamic characterization of the m2 protein transmembrane and amphipathic helices in lipid bilayers putative a-helical structures in the human immunodeficiency virus type 1 vpu protein and cd4 are involved in binding and degradation of the cd4 molecule structural and functional analyis of the membrane-spanning domain of the human immunodeficiency virus type 1 vpu protein reconstitution of the influenza virus m 2 ion channel in lipid bilayers acetylcholine receptor channel imaged in the open state the nicotinic acetylcholine receptor of torpedo electric ray structure and action of the nicotinic acetylcholine receptor explored by electron microscopy refined structure of the nicotinic acetylcholine receptor at 4 ã� resolution arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized torpedo postsynaptic membranes the interferon-induced protein bst-2 restricts hiv-1 release and is downregulated from the cell surface by the viral vpu protein genetic analysis of a hydrophobic domain of coxsackie b3 virus protein 2b: a moderate degree of hydrophobicity is required for a cis-acting function in viral rna synthesis coxsackievirus protein 2b modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release structurefunction analysis of coxsackie b3 virus protein 2b homomultimerization of the coxsackievirus 2b protein in living cells visualized by fluorescence resonance energy transfer microscopy hiv: a raft-targeting approach for prevention and therapy using plantderived compounds proton conductance of influenza virus m2 protein in planar lipid bilayers inhibition of hiv-1 replication by amphotericin b methyl ester: selection for resistant variants inhibition of human immunodeficiency virus type 1 assembly and release by cholesterol-binding compound amphotericin b methyl ester: evidence for vpu dependence ion channel activity of influenza a virus m 2 protein: characterization of the amantadine block direct measurement of the influenza a virus m 2 protein ion channel activity in mammalian cells activation of the m 2 ion channel of influenza virus: a role for the transmembrane domain histidine residue the structure of an open form of an e. coli mechanosensitive channel at 3.45 ã� resolution discovery of spiro-piperidine inhibitors and their modulation of the dynamics of the m2 proton channel from influenza a virus solution structure and functional analysis of the influenza b proton channel viral proteins function as ion channels monoclonal antibody, but not synthetic peptide, targeting the ectodomain of influenza b virus m2 proton channel has antiviral activity structural and dynamic mechanisms for the function and inhibition of the m2 proton channel from influenza a virus protein silencing with phylomers: a new tool for target validation and generating lead biologicals targeting protein interactions potent d-peptide inhibitors of hiv-1 entry the influence of different lipid environment on the structure and function of the hepatitis c virus p7 ion channel protein secondary structure and tertiary fold of the human immunodeficiency virus protein u (vpu) cytoplasmatic domain in solution human immunodeficiency virus type 1 vpu protein induces rapid degradation of cd4 human immunodeficiency virus type 1 vpu protein regulates the formation of intracellular gp160-cd4 complexes sars coronavirus e protein forms cation-selective ion channels evolutionary advantage and molecular modes of action of multi-component mixtures used in phytomedicine cloning of influenza cdna into m13: the sequence of the rna segment encoding the a/pr/8/34 matrix protein solid-state 19f nmr spectroscopy reveals that trp41 participates in the gating mechanism of the m2 proton channel of influenza a virus structural consequences of phosphorylation of two serine residues in the cytiplasmic domain of hiv-1 vpu nmr structural characterization of hiv-1 virus protein u cytoplasmic domain in the presence of dodecylphosphatidylcholine micelles solution structure of the hydrophilic region of hiv-1 encoded virus protein u (vpu) by cd and 1 h nmr-spectroscopy solution structure and orientation of the transmembrane anchor domain of the hiv-1 -encoded virus protein u by high resolution and solid-state nmr spectroscopy 2b protein of enterovirus 71 induces chloride-dependent current and can be blocked by dids chlorella viruses a secondary gate as a mechanism for inhibition of the m2 proton channel by amantadine identification of a novel protein 3a from severe acute respiratory syndrome coronavirus subcellular localization and membrane association of sars-cov 3a protein influenza a virus m 2 protein: monoclonal antibody restriction of virus growth and detection of m 2 in virions characterization of the 3a protein of sars-associated coronavirus in infected vero e6 cells and sars patients interfacial peptide inhibitors of hiv-1 integrase activity and dimerization structural studies of the hiv-1 accessory protein vpu in langmuir monolayers: synchroton x-ray reflectivity the m2 channel of influenza a virus: a molecular dynamics study epidemiology and cause of severe acute respiratory syndrom (sars) in guangdong, people's republic of china a mechanosensitive channel in whole cells and in membrane patches of the fungus uromyces key: cord-304748-ddwawfv2 authors: mendelsohn, andrea s.; ritchwood, tiarney title: covid-19 and antiretroviral therapies: south africa’s charge towards 90–90–90 in the midst of a second pandemic date: 2020-04-30 journal: aids behav doi: 10.1007/s10461-020-02898-y sha: doc_id: 304748 cord_uid: ddwawfv2 nan the covid-19 pandemic has spurred panic in south africa, a country with the highest number of hiv patients in the world and a persistent tb epidemic. south africa had its first covid-19 case on march 5, 2020 . fearful that a significant segment of the population was particularly vulnerable-7.7 million south africans have hiv and even more live in crowded conditions-the president declared a national state of disaster on march 15th when the caseload rose to 51 [1] . the country initially implemented a 3-week lockdown to contain covid-19's spread, which was later extended to 5 weeks. in preparation for a future swell of covid-19 patients, the western cape department of health (wc doh) implemented a plan to "de-escalate" healthcare services to reduce the spread of infection and increase capacity to accommodate covid-19 patients [2] . all non-urgent elective surgeries and outpatient appointments were postponed. stable chronic patients were given 2-months supplies of medication with up to 6 months of refills. only emergency life-saving care, contraception, antenatal care, and immunizations have continued as normal. telemedicine is not a viable alternative in the south african public health system. consequently, all patients are screened at the hospital gate for acute respiratory symptoms before entering. those with possible covid-19 symptoms are given surgical masks and isolated for clinical assessment, treatment and testing. inside the hospital, social distancing is adhered to as strictly as possible. in one clinic, rather than sitting in a crowded waiting area, patients are asked to wait 1.5 m apart from each other outside and brought into the building for their visit or pharmacy collection 5 patients at a time. as much as possible, chronic medications are packaged and delivered by community health workers to community-based locations. the same de-escalation plan makes provision for the distribution of anti-retroviral medication (arvs) to the 5 million south africans currently on arvs [1] . while there is no evidence that people living with hiv (plhiv) who are virally suppressed on arvs are at increased risk of severe covid-19 disease, there is genuine concern that plhiv with advanced immunocompromise or unsuppressed hiv may suffer worse covid-19 outcomes [3] . therefore, there is extra pressure in south africa to decrease the exposure of plhiv to covid-19 infection and rapidly increase viral suppression rates. further complicating the pandemic is south africa's rollout of a new first-line arv, a fixed-dose combination pill containing tenofovir disoproxil fumarate, lamivudine, and dolutegravir (tld). south africa has changed to a first line dolutegravir-based regimen because of its higher barrier to resistance and reduction in associated side effects compared to efavirenz. in 2019 the change to tld was delayed over concerns of increased neural tube defects when women conceive on tld. however, reassured by improved botswana data and persuaded that arv programs must respect a woman's right to make her own healthcare decisions, the south african national department of health launched tld on december 1, 2019 [4] [5] [6] . providers began prescribing tld nationwide in january 2020. prior to the covid-19 outbreak, the plan in south africa was to phase out the old first-line fixed dose combination pill of tenofovir disoproxil fumarate, emtricitabine, and efavirenz (tee) slowly over 1-2 years. since tee stocks in the western cape are limited and tld is plentiful, it is essential to roll-out tld as planned [7] . what's more, tld is a more effective arv [8, 9] . during a highly infectious respiratory pandemic, plhiv should be on the best arv regimen available to minimize their risk of virologic failure, immunocompromise, and severe covid-19 infection. to facilitate the rollout of tld with de-escalated hiv services, the wc doh and the southern african hiv clinicians society (sahcs) issued specific recommendations [7, 10] . the sahcs strongly advocated that 6-month supplies of arvs be issued to stable patients, reducing the risk of covid-19 exposure inherent in seeking in-person treatment at healthcare facilities as well as to minimize patient flow in a clinic [10] . ideally, to change from tee to tld a patient should have a vl < 50 copies/ml within 6 months, as stipulated by national guidelines. however, the sahcs and wc doh advocated switching a patient to tld, even without a recent vl, if the patient has been on arvs for > 1 year, the past 2 vls were < 50 copies/ml, and they have regularly collected arvs over the past year [7, 10] . patients fulfilling these criteria are very likely to be suppressed and can be switched to tld without delay [10] . additionally, the wc doh urged same day initiation of arvs when not medically contraindicated, arv initiation for all co-infected tb patients 2 weeks after starting tb treatment, and urgently changing all patients failing 1 st line tee to a 2 nd line arv regimen, preferably with dolutegravir [7] . patients unsuppressed on arvs will need additional clinical contact and adherence support. the sahcs and wc doh recommendations seek to fast-track plhiv onto a robust arv regimen during the pandemic to maximize their possibility of viral suppression and minimize their exposure to the healthcare system and, potentially, covid-19. the uncertainty is what impact this de-escalated hiv program will have on hiv outcomes in south africa. unsuppressed patients in need of additional adherence support are likely to suffer the most without the help of auxiliary services such as social work, treatment support groups, addiction rehabilitation, and psychotherapy. likewise, many of south africa's lay hiv-counselors have been re-deployed to community covid-19 screening. although hiv testing is ongoing, anecdotally testing numbers and new diagnoses of hiv are lower since the wc doh began discouraging nonessential healthcare visits. hiv self-testing at home or hiv testing in coordination with community covid-19 screens could potentially fill that gap. it is well documented that differentiated models of care, such as south africa's adherence clubs, for stable arv patients have improved long-term virologic suppression and retention in care in comparison to usual care [11] [12] [13] . plhiv are more likely to continue treatment if it is convenient and they can keep working without sitting all day in un-friendly, stigmatizing clinics to collect medication. in fact, the who recommends that clinically stable patients receive arv refills every 3-6 months to decrease the burden of care [14] . forced by the pandemic, the wc doh has advised clinicians to give stable patients up to 6 months of refills of a highly efficacious arv-tld-with minimal clinic contact. we have effectively turned most of the hiv service into one giant alternative model of care. it is possible that without the burden of frequent visits, there might be improved retention in care during the pandemic because patients are given the medication and empowered to manage their own health. the roll-out of tld will confound outcomes, because it will be impossible to tease out if viral suppression rates are due to the better arv, less burdensome healthcare system, or both. crisis is often the catalyst for ingenuity. depending on outcomes, we suspect that south africa will ultimately return to a combination of the old and the new model of care. stable arv patients will be empowered to live their lives with 3-6 month supplies of medication and minimal clinic interaction. those struggling with arv adherence should be reabsorbed into multi-disciplinary teams to meet their medical, psychological, and social needs. universal hiv testing should be implemented and tld should be offered to all eligible plhiv given its limited side effects and greater accessibility. south africa country data health services response to covid-19. 2020. western cape department of health staff correspondence q&a on covid-19, hiv and antiretrovirals neural-tube defects and antiretroviral treatment regimens in botswana consolidated guidelines on sexual and reproductive health and rights of women living with hiv press release: south africa to introduce state-of-theart hiv treatment circular h37/2020. management of patients with hiv, tb and non-communicable chronic diseases during outbreak of covid-19; multi-month dispensing of chronic medicines, including art. western cape department of health dolutegravir plus abacavir-lamivudine for the treatment of hiv-1 infection dolutegravir plus two different prodrugs of tenofovir to treat hiv provision of 6 months of antiretroviral treatment-position statement from the southern african hiv clinician society effectiveness of patient adherence groups as a model of care for sta high rates of retention and viral suppression in the scale-up of antiretroviral therapy adherence clubs in cape town, south africa art adherence clubs: a long-term retention strategy for clinically stable patients on antiretroviral therapy world health organization publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-310931-5165078t authors: oppong, joseph r. title: globalization of communicable diseases date: 2019-12-04 journal: international encyclopedia of human geography doi: 10.1016/b978-0-08-102295-5.10438-x sha: doc_id: 310931 cord_uid: 5165078t fueled by globalization and human behavior, communicable diseases pose a serious threat to humankind despite unparalleled technological advances. new viruses and devastating communicable diseases such as ebola and zika are emerging; diseases previously considered eradicated such as measles are reemerging, while antibiotic resistance is increasing to dangerously high levels worldwide. increased human population and accelerated global travel make local outbreaks instant global threats. researchers are concerned that an avian influenza outbreak could kill many more people when it emerges because of the absence of immunity and human travel interaction patterns. yet the threat of communicable disease varies by geographic location—where you live matters. this entry examines the spatial patterns of familiar communicable diseases, including the syndemic of human immunodeficiency virus (hiv) and tuberculosis, as well as new diseases such as ebola, zika, and dengue. it highlights the huge potential of mapping communicable disease genotypes while raising the alarm on the urgent need for effective global disease surveillance systems and new tools for fighting communicable diseases. because communicable diseases do not respect political boundaries, global cooperation is vital to prevent this threat to humankind. rates are much lower, increasing numbers of people are living with hiv/aids, and prevalence is particularly high among subpopulations such as blacks, men who have sex with men, and intravenous drug users. at the end of 2016, about 36.7 million people were living with hiv globally, with 25.6 million cases in sub-saharan africa, a region with 11% of the world's population, and 91% of the world's children living with the disease. hiv prevalence rates vary widely between african countries, ranging from a low of less than 1% of the adult population in senegal and somalia to more than 15% in south africa and zambia. but the world's most affected region remains southern and eastern africa where an estimated 19.4 million people were living with hiv in 2016, 59% of them being female. the region also had 43% of the global total of new hiv infections, but only 60% of the people living with hiv in the region were accessing antiretroviral therapy in 2016. women are more vulnerable and thus more severely affected than are men, and at much younger ages. the major driving forces of hiv/aids include poverty and economic inequality which necessitate transactional sex, and gender inequality enshrined in cultural practices such as widow inheritance. global efforts to control the disease through improved testing and supply of inexpensive medications and other programs are beginning to yield positive results including plummeting death rates, and unaids has set a goal of ending aids by 2030. tuberculosis (tb) is an airborne respiratory disease caused by mycobacterium tuberculosis. tb bacilli are propelled into the air when people who are sick with pulmonary tb cough, sneeze, talk, or during normal breathing. inhaling even a small number of these airborne bacilli is enough to cause infection. most exposed persons never become ill but develop latent tb. an important minority (5%-10%) progress from latent to active tb due, for example, to immunosuppression from coinfection with the hiv. despite treatment advances, tb remains an uncontrolled communicable disease worldwide and about 9 million persons are diagnosed and 2 million die yearly. currently, more than 90% of the global tb burden occurs within developing countries due to widespread hiv/aids infection, crowding, medication shortages, and poor healthcare programs. in developed countries, newly diagnosed tb occurs most frequently among foreign-born immigrants, and native-born homeless who typically live in extremely crowded and unsanitary conditions. for example, in germany and france immigrants are three times and six times, respectively, more likely to be diagnosed with tb than are native-born persons. in 2016, 10 countries in eastern and southern africa (angola, ethiopia, kenya, lesotho, mozambique, namibia, south africa, tanzania, zambia, and zimbabwe) were listed among the countries with the highest tb burden. the two epidemicsdhiv/aids and tbdare intertwined in such a catastrophic way that they have been termed syndemic. hiv is now associated with epidemic outbreaks of tb, multidrug-resistant tb (mdrtb), and extensively drug-resistant tb (xdr-tb). similarly, tb is now the leading cause of death in hiv-positive adults, and in 2017, people living with hiv were 20 times more likely to have tb worldwide. polio is a viral disease that is usually spread through fecal contaminated water and food. early symptoms of infection include fatigue, fever, vomiting, headache and pain in the neck, and extremities. the virus invades the nervous system and can cause paralysis in hours. incubation takes between 3 and 30 days, but a person begins to shed the virus in their stool 7-10 days before, and 3-6 weeks after the onset of symptoms. a small minority of cases (0.5%-1%) result in paralysis, and at least 50% of cases occur in children 3-5 years old. because most infected people have no symptoms at all while spreading the highly contagious virus for weeks, once introduced, polio usually infects many people before an outbreak is detected. polio provides an illustrative example of the challenges in global disease control. once considered almost eradicated worldwide, polio resurged not only in the six countries with endemic polio (nigeria, india, pakistan, niger, afghanistan, and egypt) but transmission was reestablished in an additional six countries (burkina faso, central african republic, chad, cote d'ivoire, mali, and sudan). despite massive efforts, total eradication continues to be an elusive goal. the global campaign to eradicate polio by 2005 suffered a severe setback when parts of northern nigeria rejected the polio vaccine because muslim clerics claimed that it causes infertility and spreads hiv/aids and cancer. from those few states, polio spread through large parts of the country, neighboring countries, and eventually to saudi arabia through nigerian muslims on pilgrimage to mecca. indonesian muslims, who were exposed during their pilgrimage in mecca, carried the disease with them to indonesia where it became reestablished. polio reminds us that global cooperation is critical for controlling communicable diseases. sexual behavior of travelers is contributing to spatial mixing of different strains of sexually transmitted pathogens worldwide. consequently, sexually active travelers risk being infected with new strains of known sexually transmitted infections. for example, gonorrhea, which causes an estimated 78 million cases worldwide each yeardabout 820,000 in the united statesdis becoming more and more resistant to antibiotics. early in 2018, a superresistant strain was reported in the united kingdom, diagnosed in a man, who developed symptoms after sexual contact with a woman in southeast asia. the bacterial infection was resistant to the two major antibiotics for treating the diseasedazithromycin and ceftriaxone. this case confirms one major concern for sexually active humanity: drug-resistant gonorrhea is spreading around the globe. long known to be endemic in africa, southeast asia, and the pacific islands, zika virus (zikav) was first identified in brazil in 2015 (where it threatened cancellation of the rio olympics) and subsequently spread throughout the region of the americas. zikav is spread primarily by the mosquito vector, aedes aegypti, and also by sexual contact, blood transfusion, and congenitallydfrom a pregnant woman to her unborn baby during pregnancy or around birth. symptoms include mild rash, fever, red eyes, body pains, and headache. infection during pregnancy can cause miscarriage; congenital microcephalydbabies with abnormally small heads; or other serious brain anomalies. in 2016, a total of 5,168 noncongenital zikav disease cases were reported in the united states, mostly in travelers returning from zikav-affected areas, but local mosquito-borne transmission increased. no local mosquito-borne transmission has been reported in the continental united states in 2018. despite the presence of aedes aegypti in multiple states, other environmental conditions (e.g., use of air conditioning and window screens and temperate climate) likely limit the transmission risk in us states. as of march 2017, a total of 84 countries globally had reported local mosquito-borne zikav transmission. determining accurately the complete global burden of zikav disease is extremely difficult due to significant underreporting. because disease is often clinically mild, patients might not seek medical care. among those who do, the disease may be easily mistaken for another illness with similar presentation (e.g., dengue or chikungunya virus disease), testing may not be available or requested, and diagnosed cases may not be reported. severe acute respiratory syndrome (sars) illustrates how migration facilitates the spatial spread of disease. from its source, guangdong province in south china, sars spread initially to hong kong, from where it diffused worldwide. when it was over, a total of 8439 probable cases and 812 deaths had been reported in 30 countries. the devastation of sars followed a path of spatially linked places and people facilitated by international travel. canadians returning from asia imported sars into canada. the first cases involved a canadian family of hong kong descent who lived in toronto. a 78-year-old woman and her husband traveled to hong kong and stayed at a hotel where a cluster of 13 persons with suspected or probable sars are known to have stayed. two days after returning home, the woman developed what is now known as sars and died. several family members who had close contact with the index case then developed sars symptoms, and one was later admitted to a hospital that subsequently became the epicenter for the toronto outbreak. sars killed 44 canadians, caused illness in hundreds more, paralyzed a major segment of ontario's healthcare system for weeks, and put more than 25,000 residents of the greater toronto area in quarantine. dengue is transmitted by the mosquitoes aedes aegypti and aedes albopictus, which are found throughout the world. in many parts of the tropics and subtropics, dengue is endemic, that is, it occurs every year, usually during a season when aedes mosquito populations are high, often when rainfall is optimal for breeding. these areas are, however, additionally at periodic risk for epidemic dengue, when large numbers of people become infected during a short period. dengue epidemics require a coincidence of large numbers of vector mosquitoes, large numbers of people with no immunity to one of the four virus types (denv 1, denv 2, denv 3, denv 4), and the opportunity for contact between the two. although aedes are common in the southern united states, dengue is endemic in northern mexico, and the us population has no immunity, dengue transmission in the continental united states is low primarily because contact between people and the vector is too infrequent to sustain transmission. today about 2.5 billion people, or 40% of the world's population, live in areas with a risk of dengue transmission. dengue is endemic in at least 100 countries in asia, the pacific, the americas, africa, and the caribbean. the world health organization (who) estimates that 50 to 100 million infections occur yearly, including 500,000 dhf cases and 22,000 deaths, mostly among children. chikungunya virus is most often spread to people by aedes aegypti and aedes albopictus mosquitoes, the same mosquitoes that transmit dengue virus. chikungunya was first described during an outbreak in southern tanzania in 1952. the name "chikungunya" derives from a word in the kimakonde language, meaning "to become contorted," and describes the stooped appearance of sufferers with very debilitating joint pain that usually lasts for a few days or may be prolonged to weeks. occasional cases of eye, neurological, and heart complications have been reported, as well as gastrointestinal complaints. outbreaks have occurred across africa, asia, europe, and the indian and pacific oceans. in the late 2013, chikungunya virus was found for the first time in the americas on islands in the caribbean. there is no vaccine to prevent or medicine to treat chikungunya virus infection. the symptoms of chikungunya are similar to those of dengue and zika. some communicable diseases originate in animals and occasionally mutate to infect humans. an excellent example is avian influenza. it occurs naturally among wild birds; they shed it in their saliva, nasal secretions, and feces, but usually do not get sick from it. domesticated birds, including chickens, ducks, and turkeys, become infected through contact with contaminated surfaces, birds, or their secretions. human contact with infected birds or contaminated surfaces produces infection, but human-to-human spread of avian influenza viruses is rare, typically limited, and unsustained. symptoms of avian influenza in humans include fever, cough, sore throat, muscle aches, eye infections, severe respiratory diseases, and other life-threatening complications. human influenza virus refers to those subtypes (currently h1n1, h1n2, and h3n2) that spread widely among humans. in the united states, human flu kills 36,000 people and hospitalizes more than 200,000 people every year. research continues into new vaccines to counter a possible flu pandemic, but unfortunately, current vaccine production methods require an outbreak before an effective vaccine can be developed and even then it takes between 5 and 7 months. to avoid a global pandemic of flu virus, new vaccine technologies are required, to respond quickly and effectively to new challenges of communicable disease outbreaks. ever since the seminal work of dr. john snow, mapping has been a key tool in fighting communicable diseases. thanks to recent technological advances, particularly in geographic information system (gis), the role of mapping in disease tracking, surveillance, and control has burgeoned. recent examples include west nile virus and tb control. cromley and mclafferty provide an excellent overview of gis applications in public health generally. one particularly fascinating recent development is mapping genotypes of communicable disease. each disease-causing organism has a specific phenotype, and the species members have a more unique genotype. tools exist for separating and categorizing disease organisms with these more unique genotypes into groups based on their genetic structure (isolates). two people with identical or matching isolates indicate recent transmission from a common source; unique isolates indicate remote transmission from a different source. analyzing and mapping these genotypes allows researchers to distinguish between not just disease species (phenotype) but also locate areas with high incidence and different strains of the disease, including recent versus remote transmission of disease. areas with high rates of clustered genotypes indicate ongoing transmission (i.e., an outbreak), whereas multiple unique isolates indicate infections that were acquired elsewhere (are imported). moonan et al. based a recently successful tb intervention on geographically targeted screening of clustered tb isolates. because it assumed that persons with clustered strains of disease from geographically related areas represent ongoing community transmission that can be identified and interrupted through treatment, the study focused resources on zip codes with high incidence and clustered isolates. during the 28-month intervention, the number of cases of tb decreased from 28.5 cases per 1000 screenings to 2.4 cases per 1000. the rate of developing latent tb infection fell from 14.3 to 2.2 per 100 person-years of exposure. the intervention was successful because it distinguished between high incidence areas with high and low levels of clustered strains and deployed resources not just on the basis of incidence but on the basis of genotype. since unique isolates represent remote transmission, location-based screening in areas with high rates of unique isolates would be less likely to identify either persons with active tb or recent acquired latent tb infection and thus not as cost-effective. despite their incredible potential, these approaches are rarely employed in developing countries where they are most needed. before the measles vaccine existed, 9 out of every 10 children got the disease before age 15 and 2 million people died from it every year. similarly, small pox killed 3 out of every 10 people and left survivors with unsightly scars. sadly, as vaccinations have successfully eliminated such previously deadly or terribly debilitating diseases, people have become indifferent or even hostile to them. the number of parents refusing to vaccinate their children, and the surge in the opposition to vaccines, in general, what has been termed the anti-vaccination movement, particularly in developed countries, should be a major concern. recent measles outbreaks in the united states, the united kingdom, and france should be a shocking reminder that surging opposition to vaccines surely paves the way for the return of deadly diseases. communicable diseases remain a major threat to humankind. effective surveillance and response remain our best protection against these deadly threats. unfortunately, surveillance remains poor and rudimentary in areas where outbreaks are most likely. strengthening the health and surveillance systems of poor developing countries is critically important because communicable diseases do not respect political boundaries. sadly, as vaccinations have successfully eliminated previously deadly or terribly debilitating diseases, people have become indifferent or even hostile to vaccinations, making it more likely the deadly diseases return. global cooperation is vital to prevent this threat to humankind. see also: diffusion; health geography; medical geography. clinical features and short-term outcomes of 144 patients with sars in the greater toronto area mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (spoldb4) for classification, population genetics and epidemiology controlling tuberculosis in the united states recommendations from the american thoracic society, cdc, and the infectious diseases society of america severe acute respiratory syndrome the pristine myth: the landscape of the americas in 1492 international travel and sexually transmitted disease the burden of dengue and chikungunya worldwide: implications for the southern united states and california update: noncongenital zika virus disease cases d 50 u.s. states and the district of columbia tuberculosis in canada annual report on tuberculosis cases zika virus disease in travelers returning to the united states continued transmission of zika virus in humans in west africa, 1992-2016 a brief history of evolving diagnostics and therapy for gonorrhea: lessons learned the anti-vaccination movement: a regression in modern medicine zika virus transmissiondregion of the americas place and health: towards a reformed medical geography putting health and health care into place: an invitation accepted and declined to reform is not discard: a reply to paul hiv and tuberculosis: a deadly human syndemic tuberculosis: epidemiology of the disease in canada a reformed medical geography reconsidered using gis technology to identify areas of tuberculosis transmission and incidence what is the outcome of location-based targeted tuberculosis screening based on universal genotyping? foreign-born status and geographic patterns of tuberculosis in tarrant county the global health threat of african urban slums: the example of urban tuberculosis commentary on kearns's 'place and health: toward a reformed medical geography epidemiology of tuberculosis in the united states on the mode of communication of cholera the risk of cancer associated with specific mutations of brca1 and brca2 among ashkenazi jews travel and the introduction of human immunodeficiency virus type 1 non-b subtype genetic forms into western countries molecular detection and genotyping of pathogens: more accurate and rapid answers dead crow reports and location of human west nile virus cases who guidelines for the global surveillance of severe acute respiratory syndrome (sars) global polio eradication initiative. health and medical geography specialty group of association of american geographers the impact of globalization on infectious disease emergence and control: exploring the consequences and opportunities, workshop summary -forum on microbial threats key: cord-304214-66nxk4e8 authors: sanders, john w.; ponzio, todd a. title: vectored immunoprophylaxis: an emerging adjunct to traditional vaccination date: 2017-02-10 journal: trop dis travel med vaccines doi: 10.1186/s40794-017-0046-0 sha: doc_id: 304214 cord_uid: 66nxk4e8 the successful development of effective vaccines has been elusive for many of the world’s most important infectious diseases. additionally, much of the population, such as the aged or immunocompromised, are unable to mount an effective immunologic response for existing vaccines. vectored immunoprophylaxis (vip) is a novel approach designed to address these challenges. rather than utilizing an antigen to trigger a response from the host’s immune system as is normally done with traditional vaccines, vip genetically engineers the production of tailored antibodies from non-hematopoietic cells, bypassing the humoral immune system. direct administration of genes encoding for neutralizing antibodies has proven to be effective in both preventing and treating several infectious diseases in animal models. while, a significant amount of work has focused on hiv, including an ongoing clinical trial, the approach has also been shown to be effective for malaria, dengue, hepatitis c, influenza, and more. in addition to presenting itself as a potentially efficient approach to solving long-standing vaccine challenges, the approach may be the best, if not only, method to vaccinate immunocompromised individuals. many issues still need to be addressed, including which tissue(s) makes the most suitable platform, which vector(s) are most efficient at transducing the platform tissue used to secrete the antibodies, and what are the long-term effects of such a treatment. here we provide a brief overview of this approach, and its potential application in treating some of the world’s most intractable infectious diseases. from the early practice of scarification to prevent smallpox through the creation of targeted, recombinant vaccines, the development of effective vaccines has been one of the great achievements in public health and medicine, resulting in millions of lives saved. modern vaccines typically protect by eliciting immunity following exposure to an inactivated or attenuated whole pathogen or recombinant components of a pathogen [1] . this approach works well for diseases in which natural infection leads to immunity and protection against re-infection and has resulted in the eradication of smallpox and dramatic declines in such diseases as diphtheria, measles, and polio [2] . however, it has been more challenging to develop effective vaccines against diseases for which prior infection does not offer full future protection, such as hiv, malaria, hepatitis c virus, and influenza a [1] . although cellular immunity is certainly important, humoral immunity appears to play the most significant role in the protection associated with most vaccines [3] . passive immunization achieved through the infusion of serum has played a significant historical role in the treatment and prevention of infection [4, 5] . the recent development of hybridoma technology and humanized monoclonal antibodies have resulted in a new class of antibody-based drugs with demonstrated and potential efficacy in cancer, inflammatory diseases, addiction, and infectious diseases [6] . within this context, there has been an increased interest in passive immunization utilizing monoclonal antibodies produced in plants or transgenic animals for infections such as ebola virus and mers-cov [7, 8] . however, logistical requirements including the need for high antibody concentrations requiring repeated injections due to the short half-life of antibodies, a cold-chain for delivery, and trained medical personnel for delivery create potential limitations to the use of this therapy, especially in low resource areas [1, 9] . the development of passive immunization by gene therapy could be a solution to some of those logistical issues and holds potential promise as either an adjunct to standard vaccination in populations who do not generate a sufficient immune response or for pathogens able to evade current vaccination strategies due to antigenic variability. originally proposed as a concept in 2002 [10] , passive immunization by vector-mediated delivery of genes encoding broadly neutralizing antibodies for in vivo expression has been referred to as immunoprophylaxis by gene transfer (igt) [11] , vector-mediated antibody gene transfer [11] , or vectored immunoprophylaxis (vip) [6, 12] ; and for sake of consistency, 'vip' is used here. rather than passively transfering pre-formed antibodies, vip is a process in which genes encoding previously characterized neutralizing antibodies are vectored into non-hematopoietic cells which then secrete the monoclonal antibodes encoded by those genes [1] (see fig. 1 .) this vectored delivery and production of specified antibodies allows for protection without generating a standard immune response and results in endogenous antibody production that has the potential to be sustained [9] . the approach has several benefits, including: 1) it does not require the host have the ability to respond immunologically, 2) the antibody can naturally be selected for a specific pathogen targets, as well as specific epitopes, 3) the antibody can be genetically modified to further enhance its activity, and, 4) vectors can be selected or engineered to have tropic characteristics targeting specific tissues and cells, potentially allowing either systemic or enhanced localized antibody production [9] . vip has been demonstrated to be effective in a host of animal models for the prevention of infection with several pathogens, especially those commonly afflicting travelers (see table 1 ), including influenza a virus [13, 14] , malaria (plasmodium falciparum) [15] , hepatitis c virus [16] , respiratory syncitial virus [17] , bacillus anthracis [18] , dengue virus [19] , and chickungunya virus [20] . in addition to the protection conferred by systemic neutralizing antibodies, protection against infection with influenza a virus has also been demonstrated following intranasal administration of vectored local antibody production [21] . by far, the most extensive and promising exploration of vip for an infectious disease has been against hiv. in the initial study demonstrating the potential of vip, a recombinant adeno-associated virus (raav) vector using a dual-promoter system generated both light and heavy chains of igg1b12, one of the early broadly neutralizing antibodies described for hiv. the raav was injected into the quadricep muscles of immunodeficient mice and biologically active antibody was found in sera for over 6 months [10] . this study provided the first evidence that raav vectors could transfer antibody genes to muscle, and muscle tissue was a suitable platform to produce and distribute the antibodies throughout the circulation [11] . follow-on studies used a native macaque siv gp120-specific fab molecule as an immunoadhesin, a chimeric, antibody-like molecules that combine the functional domain of a binding protein with immunoglobulin constant domains, which were considered to be superior to single chain (scfv) or whole antibody (igg) molecules with respect to achievable steady-state serum concentrations [22] . six of nine rhesus macaques were completely protected against intravenous challenge with virulent siv and still had stable immunoadhesin levels 6 years after injection [11] . the three subjects not protected were found to have developed an immune response to the immunoadhesin by 3 weeks after injection [11] . another group used an raav vector injected into the quadriceps muscle of a humanized mouse to express an array of broadly neutralizing antibodies: 2g12, igg1b12, 2f5, 4e10 and vrc01. though vrc01 serum levels as low as 8.3 μg/ml provided the genetic sequences of the antibody variable regions are determined. c the genetic sequence for the bna can then be placed downstream from an appropriate promoter (prom) within a suitable vector. d the vector can then be administered to the subject in an appropriate tissue platform, such as muscle. the bna produced by the vector and secreted by the tissue confers the host subject with broad and lasting protection from the targeted pathogen protection from an intravenous challenge with hiv, they achieved concentrations as high as 100 μg/ml for at least 12 months [12] . they followed-up that study by optimizing the broadly neutralizing antibody, and although muscle was chosen as a platform for expression and secretion of the igg1 isotype, antibodies were found to effectively reach the vaginal mucosa. animals receiving vip that expressed a modified vrc07 antibody (concentration of nearly 100 μg/ml in the serum and 1 μg/ml in vaginal wash fluid) were completely resistant to repetitive intravaginal challenge by a heterosexually transmitted founder hiv strain [23] . saunders, et al., used an raav serotype 8 vector to produce a full length igg of a simianized form of the broadly neutralizing antibody vrc07 in macaques which was protective against simian-human immunodeficiency virus (shiv) infection 5.5 weeks after treatment [24] . shivs are chimeric viruses constructed to express the hiv envelope glycoprotein to be used in vaccine experiments to evaluate neutralizing antibodies. the antibody reached levels up to 66 μg/ml for 16 weeks, but immune suppression with cyclosporine was needed to sustain expression due to the development of anti-idiotypic antibodies [24] . the approach to preventing hiv was enhanced further by fusing the immunoadhesin form of cd4-ig with a small ccr5-mimetic sulfopeptide at the carboxy-terminus (ecd4-ig). ecd4-ig is more potent than the best broadly neutralizing antiody and binds avidly to the hiv-1 envelope glycoprotein. rhesus macaques expressed 17-77 μg/ml of fully functional rhesus ecd4-ig for more than 40 weeks after injection with a self complimentary serotype 1 aav (scaav1) vector and were completely protected from multiple challenges with a simian/human immunodeficiency virus, shiv-ad8 [25] . of note, the rhesus ecd4-ig was also markedly less immunogenic than rhesus forms of four well-characterized broadly neutralizing antibodies [25] . in addition to disease prevention as noted above, studies have also demonstrated an application for vip in the effective treatment of previously-infected animals. using hiv-1-infected humanized mice, horwitz, et al., demonstrated that following initial treatment with anti-retroviral therapy (art), a single injection of adeno-associated virus directing expression of broadly neutralizing antibody 10-1074, produced durable viremic control after the art was stopped [26] . the first human trial using the vip approach started in january 2014 and is a phase 1, randomized, blinded, doseescalation study of an raav1 vector coding for pg9, a potent broadly neutralizing antibody, in high risk, healthy adult males (clinicaltrials.gov number, nct01937455). another study evaluating using vip in hiv-positive subjects is scheduled to get underway soon [6] . many options exist for vectoring the transgene into the host tissue, each with distinct advantages and limitations. naked plasmid dna is relatively easy to use, does not elicit significant immunogenicity, and has the potential for inexpensive large-scale production [20, 27] . recent advances in both the mechanism of delivery [28] and optimization of plasmid and electroporation conditions [29] have improved the concentration and duration of antibody production, but it has yet to prove as potent as viral vectoring. viral vectors offer the advantage of efficient, rapid delivery of the transgene into host cells and the potential for integration into the host genome, allowing for sustained expression [1] . the life cycle of a virus consists of attachment, penetration, uncoating, replication, gene expression, assembly and budding. replication and gene expression typically take place in the nucleus where viral genomes persist episomally or integrate into the host genome (i.e., a provirus). vectors that persist episomally can provide sustained transgene expression in post-mitotic tissue, but since they do not alter the host genome, they may be lost if and when the cells divide. vectors that integrate into to the host genome may provide life-long transgene expression in dividing cells but could also lead to insertional mutagenesis resulting in apoptosis or malignant transformation [30] . adenoviral vectors produce rapid, but transient, gene expression that could be ideal for responding to a disease outbreak, but would have limitations for long term protection [1] . adenovirus serotype 5 (ad5) has successfully transduced protective antibodies for respiratory syncytial virus (rsv), influenza a virus (iav), and bacillus anthracis [14, 17, 18] . the ad5 genome is easy to engineer and remains episomal, but there is significant pre-existing immunity to ad5, estimated at 50% of the adult population worldwide and even higher in sub-saharan arica, which decreases the ability to transfer the transgene. additionally, it can result in systemic cytokine release creating a sepsis presentation and there is significant tissue tropism for the liver when delivered intravenously. alternative adenoviral vectors are being researched [31] . lentivral vectors are better suited for long term expression since they typically integrate into the genome and can transduce dividing and non-dividing cells. they have successfully been utilized to transduce hematopoietic stem cells to produce broadly neutralizing antibodies against hiv in mouse models [32, 33] . however, because they can integrate into the host genome, there is concern for mutagenesis. newer generation lentiviral vectors contain deletions in their long-terminal repeat (ltr) and a self-inactivating (sin) ltr, leaving them replication incompetent, which should make them much safer, but this question is not fully answered [30] . although other viral vectors are being explored, raav vectors are currently the favored vehicle for delivering the antiody genes into the host tissue due to their efficiency in gene transfer [34] . in contrast to other viral vectors, such as adenovirus, raav's have not been associated with any human diseases and do not stimulate signficant immunologic reaction, and are therefore able to induce long-term expression of non-self-proteins [34] . they are engineered to consist of the antibody gene expression cassette flanked by the aav itrs (inverted terminal repeats), which are the only part of the aav genome present in the raav vector and are required for raav vector genome replication and packaging. despite a relativley small packaging capacity of 5 kb, both heavy-and light-chain antibody genes can be incorporated into a single vector, either using a promoter for each gene cassette or a single promoter for expression with the heavy and light chain separated by a foot-and-mouth disease virus 2a peptide [9] . all studies to date have targeted skeletal muscle as the platform for transfection and antibody production. muscle offers some significant advantages. it is easily accessible for localized vector administration, and some muscle groups can be removed in the event of mutagenesis or auto-immunity without functional consequence. however, muscle has certain disadvantages as well. it is a tissue that does not normally produce circulating proteins and therefore may not do it efficiently. it also contains antigenpresenting dendritic cells that could induce immune responses which might eliminate transduced cells or induce auto-immunity. additionally, the removal of muscle tissue would likely have a significant effect on a subject's lifestyle in the event of a potential unexpected vip-induced pathology. other platforms have been considered. for example, some authors have suggested the liver as an alternative site [35] . unlike muscle, it is designed to secrete circulating proteins. it is also thought to be less immunogenic. however, transduction would require systemic administration of the vector, and there would be no simple means of eliminating expression in the event of a complication. another potential site could be the salivary glands. while it is well-know that the salivary glands secrete proteins into the oral cavity, it may be less well appreciated that they have also been used as a platform to deliver therapeutic proteins, including the igg fc fragment and a host of other proteins, into the systemic circulation [36, 37] . transgenes delivered to the salivary gland tend to favor being sorted either into the saliva or the blood, though it is currently a challenge to predict which direction a particular protein will sort [7] . the major paired salivary glands are also easily accessible, and the parotid glands are encapsulated, which minimizes vector spillage into the general circulation. futhermore, in the event of complications, the transfected glands could be removed without creating major disability. safety concerns associated with vip include genotoxic events typically associated with any viral vector mediated gene therapy, such as inflammation, a random insertion disrupting normal genes, activation of proto-oncogenes, and insertional mutagenesis [38] . there are many factors which affect the likelihood of developing a genotoxic event including the vector, the targeted insertion site, the transgene, the targeted cell type, and host factors including age and underlying disease [39] . the risk of genotoxicity or carcinogenicity can potentially be decreased by selection of the promoter and the integration site, using novel techniques such as clustered regularly interspaced short palindromic repeat (crispr)-cas9 (an rna-guided gene-editing platform that allows for cutting of dna in a specified gene), but much more work needs to be done to better characterize safety and efficacy of these methods [39] . as the purpose of vip is to produce a monoclonal antibody, the possibility of producing a paraproteinemia similar to that caused by multiple myeloma, other hematologic malignancies, primary amyloidosis, or a monoclonal gammopathy of undertermined significance (mgus) is a concern. the most benign of these is mgus, but it has been increasingly recognized to have pathologic associations including is nephropathy secondary to monoclonal gammopathy of renal significance (mgrs), neuropathy, oculopathy, and dermopathy as well as possible associations with autoimmunity and coagulopathy and an epidemiologic association with early mortality from a variety of apparently unrelated causes [40] [41] [42] . any of these conditions could result from a monoclonal gammopathy produced by vip. however, it should be noted that mgus is very common, occurring in 3% of the population older than 50 years old, and most of these associations remain either unclear or uncommon [40] . however, the potential for autoimmunity should be of particular concern. it is possible that the monoclonal antibody could interact with self-antigen and either stimulate an autoimmune antibody that interacts with self-antigen [40] or neutralizes the intended effect of the monoclonal antibody. vectored immunoprophylaxis has demonstrated great promise in a variety of pre-clinical studies as a potential adjunct to vaccination in patients not able to respond effectively to immunization or as an alternative to vaccination for infectious diseases not effectively covered by current vaccines. the rapid identification of specific neutralizing antibodies is likely to increase the potential for this method. one could imagine uses for vip such as an adjunct to vaccination for influenza in the elderly and immunocompromised, for hiv protection in high risk populations, or as part of a ring vaccination strategy in an outbreak of a disease such as ebola. many important questions remain, including the ability to produce equally effective clinical results in human trials, the duration of response, and the potential for side-effects. mutagenesis at the site of transfection is a common concern, but the development of an immune response to the transgene product or the off-target binding of the antibodies are more likely scenarios, either of which could result in decreased efficacy of the procedure or a significant auto-immune reaction. questions also remain concerning the best vector and the optimal tissue site for transfection. despite these questions and concerns, the advantages offered in settings ranging from chronic protection of the aged or immunocompromised to rapid protection for early responders in the event of a bioterror or emerging infection event are significant and intriguing. further preclinical and clinical studies are certainly warranted. engineering humoral immunity as prophylaxis or therapy vaccine-preventable disease table working g. historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states contributions of humoral and cellular immunity to vaccine-induced protection in humans hark back: passive immunotherapy for influenza and other serious infections meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? passive immunization against hiv/aids by antibody gene transfer human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo plant-based vaccines against viruses emerging vaccine technologies. vaccines (basel) generation of neutralizing activity against human immunodeficiency virus type 1 in serum by antibody gene transfer vector-mediated in vivo antibody expression antibody-based protection against hiv infection by vectored immunoprophylaxis broad protection against influenza infection by vectored immunoprophylaxis in mice passive immunization with a recombinant adenovirus expressing an ha (h5)-specific single-domain antibody protects mice from lethal influenza infection vectored antibody gene delivery protects against plasmodium falciparum sporozoite challenge in mice broadly neutralizing antibodies abrogate established hepatitis c virus infection genetic delivery of an anti-rsv antibody to protect against pulmonary infection with rsv rapid/sustained anti-anthrax passive immunity mediated by co-administration of ad/aav protection against dengue disease by synthetic nucleic acid antibody prophylaxis rapid and long-term immunity elicited by dna-encoded antibody prophylaxis and dna vaccination against chikungunya virus intranasal antibody gene transfer in mice and ferrets elicits broad protection against pandemic influenza vector-mediated gene transfer engenders long-lived neutralizing activity and protection against siv infection in monkeys vectored immunoprophylaxis protects humanized mice from mucosal hiv transmission broadly neutralizing human immunodeficiency virus type 1 antibody gene transfer protects nonhuman primates from mucosal simian-human immunodeficiency virus infection aavexpressed ecd4-ig provides durable protection from multiple shiv challenges hiv-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice monoclonal antibodies produced by muscle after plasmid injection and electroporation a novel electroporation device for gene delivery in large animals and humans optimized and enhanced dna plasmid vector based in vivo construction of a neutralizing anti-hiv-1 envelope glycoprotein fab interaction of vectors and parental viruses with the host genome development of novel adenoviral vectors to overcome challenges observed with hadv-5-based constructs inhibition of in vivo hiv infection in humanized mice by gene therapy of human hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti-hiv antibody engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-hiv antibody after in vitro maturation to human b lymphocytes aav vectors vaccines against infectious diseases viral vectors take on hiv infection advances in salivary gland gene therapy -oral and systemic implications in vivo secretion of the mouse immunoglobulin g fc fragment from rat submandibular glands general considerations on the biosafety of virus-derived vectors used in gene therapy and vaccination viral vectors: the road to reducing genotoxicity monoclonal gammopathy: the good, the bad and the ugly criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the international myeloma working group monoclonal gammopathy of renal significance: when mgus is no longer undetermined or insignificant none. none. availability of data and materials not applicable.authors' contributions jws and tap contributed equally to the development of this manuscript. both authors read and approved the final manuscript. the authors declare that they have no competing interests. ethics approval and consent to participate not applicable.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-304157-u0mlee6u authors: nyasulu, juliet; pandya, himani title: the effects of coronavirus disease 2019 pandemic on the south african health system: a call to maintain essential health services date: 2020-07-22 journal: afr j prim health care fam med doi: 10.4102/phcfm.v12i1.2480 sha: doc_id: 304157 cord_uid: u0mlee6u south africa had its first coronavirus disease 2019 (covid-19) case on 06 march 2020 in an individual who travelled overseas. since then, cases have constantly increased and the pandemic has taken a toll on the health system. this requires extra mobilisation of resources to curb the disease and overcome financial loses whilst providing social protection to the poor. assessing the effects of covid-19 on south african health system is critical to identify challenges and act timely to strike a balance between managing the emergency and maintaining essential health services. we applied the world health organization (who) health systems framework to assess the effects of covid-19 on south african health system, and proposed solutions to address the gaps, with a focus on human immunodeficiency virus (hiv) and expanded programme on immunisation (epi) programmes. the emergence of covid-19 pandemic has direct impact on the health system, negatively affecting its functionality, as depletion of resources to curb the emergency is eminent. diversion of health workforce, suspension of services, reduced health-seeking behaviour, unavailability of supplies, deterioration in data monitoring and funding crunches are some of the noted challenges. in such emergencies, the ability to deliver essential services is dependent on baseline capacity of health system. our approach advocates for close collaboration between essential services and covid-19 teams to identify priorities, restructure essential services to accommodate physical distancing, promote task shifting at primary level, optimise the use of mobile/web-based technologies for service delivery/training/monitoring and involve private sector and non-health departments to increase management capacity. strategic responses thus planned can assist in mitigating the adverse effects of the pandemic whilst preventing morbidity and mortality from preventable diseases in the population. in south africa, since march 06, when the first coronavirus disease 2019 (covid-19) case was reported, cases have increased to over 188 000 at the time of writing this article. 1 this pandemic has called for extra mobilisation of resources to curb the disease and overcome financial loses whilst providing social protection to the poor. 2 for decades, the south african health system has shouldered a quadruple burden of diseases (much of which is preventable), with mother and child health indicators far from accepatable targets. 3, 4 the south african primary healthcare provides both curative and preventive health services. these include under-five child health services, such as growth monitoring and expanded programme on immunisation (epi); reproductive health services such as family planning, cervical and breast cancer screening, antenatal, labour and postnatal care services; chronic disease care for both communicable and non-communicable diseases including human immunodeficiency virus (hiv) services; and many other health promotion, preventative and curative services. 5 it is concerning that out of 7.7 million hiv-positive people living in south africa, about 4.9 million people are on antiretroviral treatment (art), with one-third (1.6 million) not virally suppressed. 6 the current evidence indicates that those virally suppressed are not at higher risk. however, in addition to 1.6 million, we do not know about the other 2 million not on art, who probably will be at higher risk of severe covid-19 infection. 6, 7 south africa has existing antiretroviral (arv) and vaccine stock-out challenges because of supply chain constraints. 8, 9, 10, 11 in addition, gaps have been identified around low routine immunisation coverage resulting in outbreaks of vaccine south africa had its first coronavirus disease 2019 (covid-19) case on 06 march 2020 in an individual who travelled overseas. since then, cases have constantly increased and the pandemic has taken a toll on the health system. this requires extra mobilisation of resources to curb the disease and overcome financial loses whilst providing social protection to the poor. assessing the effects of covid-19 on south african health system is critical to identify challenges and act timely to strike a balance between managing the emergency and maintaining essential health services. we applied the world health organization (who) health systems framework to assess the effects of covid-19 on south african health system, and proposed solutions to address the gaps, with a focus on human immunodeficiency virus (hiv) and expanded programme on immunisation (epi) programmes. the emergence of covid-19 pandemic has direct impact on the health system, negatively affecting its functionality, as depletion of resources to curb the emergency is eminent. diversion of health workforce, suspension of services, reduced health-seeking behaviour, unavailability of supplies, deterioration in data monitoring and funding crunches are some of the noted challenges. in such emergencies, the ability to deliver essential services is dependent on baseline capacity of health system. our approach advocates for close collaboration between essential services and covid-19 teams to identify priorities, restructure essential services to accommodate physical distancing, promote task shifting at primary level, optimise the use of mobile/web-based technologies for service delivery/training/monitoring and involve private sector and nonhealth departments to increase management capacity. strategic responses thus planned can assist in mitigating the adverse effects of the pandemic whilst preventing morbidity and mortality from preventable diseases in the population. preventable diseases, such as measles, in south africa. 12 anecdotal reports show that there is a decline in access to art from march 2020 by those already initiated on art. for example, some districts supported by right to care (rtc), a president's emergency plan for aids relief (pepfar) partner for gauteng province, show increasing numbers of missed appointments to collect art. 13 similarly, epi and other essential services are also likely to be affected. fear of contracting covid-19, the physical distancing policy and a shift in focus of service providers from basic essential services to covid-19 pandemic demands may be the reasons for the decline in access to essential services. 14 emergence of the covid-19 pandemic risks the worsening of existing gaps and increasing deaths as shown in the previous ebola outbreaks. 15, 16 the resilience of health system to timely adapt and strike a balance between maintaining routine services and coping with the pandemic is crucial to mitigate the damage. 17 currently, there is a rapid and overwhelming increase in strain on the health system because of covid-19, overstretching the capacity of healthcare workers to operate effectively. 18, 19 this article looks at the possible effects of covid-19 pandemic on the south african health system and proposes possible solutions to maintain the delivery of essential health services whilst fighting the pandemic, with a specific focus on hiv and epi. we believe that maternity care, child care (e.g. immunisation) and hiv services stand out as the most critical markers and a proxy for the strength of a health system, particularly in the context of south africa. 6, 8, 12 resilience of a health system is indicated by its ability to offer basic healthcare services to pregnant women, children and people with hiv. these groups are comparatively more vulnerable and contribute to a high burden of morbidity and mortality at a population level. lessons from previous epidemics such as ebola have shown that when there is a threat to a health system, these groups are affected first and to a higher extent. 16 moreover, because of our experience and expertise in working with hiv and immunisation, we selected them as priority services and focus of this article. we applied the world health organisation (who) health systems framework and its six building blocks to assess how covid-19 pandemic has affected the south african health system ( figure 1 ). 20 using the documented existing service delivery gaps, we analysed epi and hiv programmes as examples of priority essential health services to be maintained by south africa during this emergency period. 6, 8, 18 in addition, solutions to strike a balance between responding to covid-19 pandemic and maintenance of these essential services are proposed. this article followed all ethical standards for a research without direct contact with human or animal subjects. pandemic and essenɵal health service delivery how can emergency and essenɵal health services teams collaborate? which essenɵal services need to be prioriɵzed, postponed and suspended? how do we tap from resources outside the health systems and ensure mulɵsectoral collaboraɵon? how do we maximize the potenɵal of web and mobile based technologies for service delivery? how do we maintain quality monitoring of exisɵng essenɵal services during emergency? how do we ɵmely integrate emergency responses with priority essenɵal services? what are the exisɵng gaps and strengths in essenɵal services (within each who building block)? what is the current capacity of the health system to mobilize resources? currently, which are the worst performing provinces, districts and faciliɵes needing more aʃenɵon? which populaɵon group will be most affected? (e.g. infants, migrants) what emergency responses will be needed for covid-19? how will covid-19 responses affect health system's delivery of essenɵal services? a. essential services not prioritised because of competing interests, 18 e.g., immunisation campaigns paused world health organization proposes outreach mechanisms to ensure delivery of essential services, 18 including immunisations and hiv services. for example, auxiliary nurses can do field-based immunisations (at rural health posts closer to community) rather than children crowding at clinics. human immunodeficiency virus experts advise that male circumcision can be paused whilst harm reduction and condom distribution and hiv treatment services need to be maintained with modifications that will reduce contact with service providers. 21 currently, south africa is implementing central chronic medicines dispensing and distribution (ccmdd) programme whereby stable patients collect their chronic medications at different pick-up points near them outside the health facility. we recommend that ccmdd must be maximised to reduce physical contacts with service providers. 22 immunisation campaigns can be modified to reduce huge numbers at once. consider integrated community-based outreach platforms offering immunisation services. 23 b. covid-19 physical distancing policy compels population to defer healthcare seeking for essential routine services like hiv and epi 14 integrate essential services with covid-19 services at facility and community levels. for example, involve nurses delivering epi and hiv services in screening for covid-19 and reporting cases. 23 identify and prioritise vulnerable communities including infants, poor and the elderly for essential services. for example, maximise the use of social protection grants available during the emergency to promote access by the vulnerable groups. generate a country-specific list of essential services for sa (based on context and supported by who guidance and tools). prioritise current worse-performing provinces, districts and facilities, which need more attention and resources for delivery of essential services. shift focus from conducting face-to-face, manual and paper-based routine operations and monitoring to utilising information technology and web-based platforms for maintaining services, for example, health promotion and prevention messages through mobile technology. ensure positive health-seeking behaviour and adherence to care by maintaining population's trust in the capacity of the health system, to safely meet essential needs and to control infection risk in health facilities. the communities should be sensitised and reassured through media, text messages and platforms like religious and other existing community structures. (who operational guidelines) 18 intensive covid-19 screening for health service providers. prioritise and ensure adequate supply of personal protective equipment (ppe) for health workers. explore ways to support those needing self-isolation and quarantine whilst protecting their family/household. consider short, web-based training for health workers in covid-19 screening, first-line treatment, referral guidelines, quarantine/ isolation policies and personal protection through smart phones (based on videos/apps). they also need to be trained on how to assure/motivate/counsel the clients because they are the frontline contacts. c. shortage of staff from essential services because of redeployment towards covid-19 response consider task shifting and scope expansion where possible to improve access to care (24) -for example, enrolled nurses and enrolled assistant nurses could take up health prevention/promotion as well as curative tasks from professional nurses, for example, immunisation. use of qualified health workforce resident in south africa but not working, to be recruited. part time health workforce to be asked to work full time. utilise the senior health workforce students from training institutions to alleviate staff shortage pressures. maximise health workforce from the non-governmental partners like provincial and district pepfar collaborations, defence, red cross, etc. clinical associates, senior students from nursing colleges and interns can be deployed on a short-term basis and, if possible, accelerate early certification without compromising quality. redistribute and redeploy staff from non-affected areas, or high-performing districts to low-performing districts. reassurance from department of health, small incentives for those health workers who contribute to both phc and covid-19 response. explore ways of acknowledging and appreciating the health workforce. a worsening of the quality of existing data in public health system 25 minimise paper-based reporting and data collection considering physical distancing strengthen online, web-based information systems for monitoring and progress of hiv and epi programmes, which can be directly used by health workers and data can be submitted through smart phones to a centralised server, which is accessible to all project managers and decision-makers. b. competing interests leading to a shift in focus to monitor the covid-19 data currently in greatest demand during the emergency, ensure monitoring of ongoing delivery of essential health services to identify gaps and provide timely response. prioritise, in this case, epi, hiv and other critical indicators in the dhis that need to be essentially monitored and leave out those indicators the monitoring of which can be delayed, such as male circumcision. c. lack of time for quarterly reviews to monitor progress on essential services to identify and address gaps, for example, health facility assessments, imci health worker supervision, etc. decentralise quarterly reviews at facility level -promote internal reviews of routine essential services (designate a team of nurses led by facility managers) if supervisors cannot visit the clinics and provide online feedback to managers. web-based data reviews through zoom/ms team/skype/google meet, etc., and other platforms will save time without disturbing physical distancing. d. surveillance and reporting of afp and vaccine preventable diseases might not be ensured maximise online tools for monitoring and reporting of cases of acute flaccid paralysis (afp) for polio, measles, etc. (e.g. apps, web-based software) involve private clinics and gps in reporting and surveillance. a. south africa has existing arv and vaccine stock-out challenges because of supply chain constraints. 8, 9, 10, 11 prioritise the worst-performing provinces on arvs, vaccines and other essential medicines stock-outs. collaborate with private health sector, pharmaceutical companies to maximise contribution and utilise their platforms. use of advances in technology to improve supply chain management could be linked with current initiatives such as momconnect. stock-outs for medicines and vaccines can be reported by facilities or districts online through web-based platforms which are monitored by the district supply chain managers and supplies could be procured accordingly. for example, blood information and management application (bima) in bangladesh takes online demand for blood and manages procurement. 26 b. shortage of covid-19 essential protective wear for healthcare workers has already been reported 27 as manufacturers fail to meet demands enhance and promote local manufacturing of ppes. capitalise on buffer system. economy shrinking coupled with high financial constraints to cope with the pandemic may lead to fiscal constraints on essential health services spending for hiv and epi 28 presidency and department of finance need to coordinate with department of health and decide on diverting any funds available in contingency or from other non-essential departments, for example, tourism, and create extra budget heads for maintaining essential health services such as procuring arvs or vaccines. divert surplus funds under hiv and epi heads towards poor performing districts and provinces for extra support (e.g. run a mobile unit for vaccination or conduct a community-based catch-up campaign). strengthen private and public health sector partnership to ensure that the public health system taps from the available resources in the private sector. initiate and promote covid-19 fundraising activities at local, regional and national levels. for example, sa has introduced solidarity fund where individuals and firms are donating resources to meet the needs of the poor, and at the time of writing this article, r2.5 billion had been raised with a target of r4bn. 29 table 1 continues on the next page → we applied the who health systems framework to highlight strengths and gaps in the epi and hiv service delivery system and explain how these are affected with the emergence of the covid-19 pandemic. we have also proposed possible solutions on how to deal with these challenges. the questions raised in the above conceptual framework do not specifically direct our analysis; rather, they serve as a generic guideline for consideration by health managers and stakeholders in order to maintain primary health services during a pandemic. table 1 summarises these gaps and possible solutions to maintain essential service delivery, with a focus on hiv and epi. the emergence of the covid-19 pandemic has put great burden on the health system, negatively affecting its functionality. we propose the who health systems framework as an approach for assessing and prioritising services by health systems to strike a balance between the responses to covid-19 pandemic and delivery of quality essential healthcare services, with a focus on epi and hiv programmes. firstly, representation and close collaboration between the covid-19 and the essential services teams at all levels are recommended. these teams together will need to identify priority essential services within the two programmes and decide which services are to be continued, postponed or suspended. 18 at this time, identification of how the emergency is affecting the health system and which geographic areas and vulnerable groups should be prioritised are critical. 18, 30 application of the who health systems building blocks will provide a systematic and comprehensive approach to the identification of these gaps. 20 the next step is to identify and implement solutions to address the gaps worsened or caused by the covid-19 emergency response. for instance, the redeployment of health workforce, coupled with others being infected with covid-19, depleted the already existing shortage. 19 therefore, considering task shifting, integration of services, utilisation of senior students, tapping from ngo partners and government workforce outside the department of health would alleviate the health workforce shortage. 18, 19, 20, 21, 22, 23, 24 the most important task is to provide support to the available health workforce in different aspects needed. 31 admittedly, the covid-19 physical distancing currently advocated for puts hiv-positive individuals and parents in a dilemma to defer routine appointments, worsening the current gaps in hiv/epi programme. 21 therefore, in order to ensure positive health-seeking behaviour and adherence to care, there is a need to maintain population's trust in the capacity of the health system to safely meet essential needs and to control infection risk in health facilities. 18 this requires training of health service providers in providing essential services with extra safety to control the spread of covid-19, 18 accompanied by communication with the users and reassurance of access and safety of health services. in times of such pandemics, it is important to ensure that the vulnerable communities including the poor and the elderly have the ability to access these essential services. 18, 30 furthermore, existing gaps in immunisation and hiv services including stock-outs have been established, which can worsen when dealing with the covid-19 pandemic. 8, 9 we therefore propose service delivery approaches that do not attract crowds like ccmdd and outreach services. 18, 22, 23 we also suggest the need to minimise relying on manual operations and paper-based service delivery and monitoring, and utilise information technology and web-based platforms to monitor and conduct its routine operations. 26, 32 end-user monitoring of the supply chain by patients and civil society has the potential to increase transparency and complement public sector monitoring systems. 8 in conclusion, the resilience of the health system is a critical determinant of how a country responds to a pandemic. 33 in emergencies like covid-19, the ability of a health system to organization health systems framework). proposed solutions to maintain essential health services whilst responding to the pandemic a. depleted leadership capacity for essential services as programme managers had been redeployed to covid-19. inter-sectoral collaboration -human resources from other non-health departments need to be involved to provide the required leadership and coordinate with health department. these could include department of finance, department of agriculture, department of education, ngo and multi-national partner institutions, for example, unicef and who. the use of other ministerial departments to complement the containment of the pandemic. for example, the ministry of water and sanitation to ensure that population including the hardest to reach ones have access to clean water and soap for handwashing. this can be accomplished in collaboration with the ministry of defence that can help in distribution. in addition, the ministry of information and education can support free online education, the ministry of telecommunications can generate awareness by media campaigns and telkom companies can be involved to provide mobile data free of cost to support information exchange and online management of health information, etc. department of health can also utilize senior students from medical, nursing and public health universities, clinical associates, interns and paediatrics registrars to assist with programme management and operations. they are better trained and equipped and can work in coordination with existing programme managers and leaders on a short-term voluntary basis to get a hands-on experience in public health and emergency response. b. decisions to navigate and strike a balance between the emergency covid-19 and essential services close collaboration between the covid-19 and essential services teams at all levels of management (national, provincial, district, sub-district and below) to identify and agree on the priority essential services that must maintain continuity during emergency period. national coordinators for hiv and epi need to adapt and implement who essential services guidelines to south african context and communicate with provincial and district-level programme managers on how to operationalise the modified guidelines in their respective areas. deliver essential services is dependent on the existing burden and baseline capacity of the health system. the existing high disease burden would put the south african health system in a fragile state to cope with the pandemic if timely adaptation actions are not taken. the approach proposed in this article, about using who building blocks to identify existing gaps, challenges and possible solutions, can be adopted by other low-and middle-income settings to identify priority actions in order to strike a balance between attending to a pandemic and simultaneously maintaining essential services. the authors envisage that applying these principles during such pandemics will lead to informed health systems decisions in striking a balance between emergency response and essential health service delivery, and maintaining of curative and preventive essential health services, which in turn will reduce morbidity and mortality from preventable and treatable diseases. national institute of communicable diseases (nicd); c2019 escalation of measures to combat coronavirus covid-19 pandemic [press release]. government of south africa mortality trends and differentials in south africa from 1997 to 2012: second national burden of disease study initial burden of disease estimates for south africa evaluating the performance of south african primary care: a cross-sectional descriptive survey setting the scene: some data on the hiv epidemic. 9th sa aids conference systematic review of the efficacy and safety of antiretroviral drugs against sars, mers, or covid-19: initial assessment stock-outs of antiretroviral and tuberculosis medicines in south africa: a national cross-sectional survey nationwide shortage of vital vaccines causes concern the status of vaccine availability and associated factors in tshwane government clinics impact of vaccine stock-outs on infant vaccination coverage: a hospital-based survey from south africa annual measles and rubella surveillance review, south africa. natl inst commun dis public health surveillance bull right to care: coronavirus in sa: hiv-positives are skipping treatment and drastic drop in testing. africa's medical media digest 2020 life-saving vaccinations must not 'fall victim' to covid-19 pandemic -unicef chief no increased coronavirus risk for people with well-controlled hiv says who, but how will health systems cope? the health impact of the 2014-15 ebola outbreak public health emergency preparedness: a framework to promote resilience covid-19: operational guidance for maintaining essential health services during an outbreak: interim guidance 25 geneva: who; c2020 effects of brain drain on the south african health sector: analysis of the dynamics of its push factors everybody's business, strengthening health systems to improve health outcomes. who's framework for action latest who updates and guidance on covid-19 and hiv ongoing initiatives to improve the quality and efficiency of medicine use within the public healthcare system in south africa: a preliminary study integrated delivery of health services during outreach visits: a literature review of program experience through a routine immunization lens nurses art initiations (nimart) a tool to promoting art access and reducing the art service burden at referral hospitals improving health information systems for decision making across five sub-saharan african countries: implementation strategies from the african health initiative can mhealth improve access to safe blood for transfusion during obstetric emergency? coronavirus personal protective equipment shortage. maverick: styli charalambous health spending at a time of low economic growth and fiscal constraint. south afr health rev solidarity fund: unity in action ensuring access to quality health care in vulnerable communities supporting the health care workforce during the covid-19 global epidemic mobile devices and apps for health care professionals: uses and benefits what makes health systems resilient against infectious disease outbreaks and natural hazards? results from a scoping review the authors would like to thank prof. susan goldstein for her input in conceptualising the health systems gaps. this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data sharing is not applicable to this article as no new data were created or analysed in this study. the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. the authors have declared that no competing interests exist. j.n. conceptualised the study and wrote the first draft. j.n. and h.p. were both involved in the writing of the manuscript and approval of the final version. key: cord-316904-g7dli0a8 authors: chang, hernan r.; dulloo, abdul g.; bistrian, bruce r. title: role of cytokines in aids wasting date: 1998-12-31 journal: nutrition doi: 10.1016/s0899-9007(98)00108-7 sha: doc_id: 316904 cord_uid: g7dli0a8 abstract there is now a large literature implicating cytokines in the development of wasting and cachexia commonly observed in a variety of pathophysiologic conditions. in the acquired immunodeficiency syndrome (aids), cytokines elicited by primary and secondary infections seem to exert subtle and sustained effects on behavioral, hormonal, and metabolic axes, and their combined effects on appetite and metabolism have been postulated to drive wasting. however, correlations of increased blood levels of a particular cytokine with wasting in aids have not been consistent observations, perhaps because cytokines act principally as paracrine and autocrine hormones, as well as indirectly by activating other systems. a better understanding of the mechanisms underlying the catabolic effects of cytokines is clearly needed if more efficacious strategies are to be developed for the prevention and treatment of wasting in aids. in this review we first examine the interacting factors contributing to the aids wasting syndrome. we then analyze the complex and overlapping role of cytokines in the pathophysiology of this condition, and put forward a number of hypotheses to explain some of the most important features of this syndrome. there are close interrelationships between malnutrition and infection. malnutrition enhances susceptibility to infections, exacerbates their harmful effects, and influences their outcome. infections also can produce malnutrition: the wasting syndrome and cachexia are common complications of infections, and they play an important role in the morbidity and mortality of the aids. indeed, although wasting is not universally observed in aids patients, the wasting syndrome in a human immunodeficiency virus (hiv)-seropositive individual is generally utilized to establish the diagnosis of aids 1 and is defined by a decrease in body mass greater than 10% in the absence of concomitant opportunistic infections, malignancies, and other identifiable causes of weight loss. 1 independent of the causes of wasting, aids patients who experience weight loss beyond a certain percentage of ideal body weight are at greater risk of death, thereby establishing a link between survival and the extent of body cell mass depletion. [2] [3] [4] [5] consequently, reversing wasting should improve the life expectancy and quality of life. 6 however, this has proved to be difficult and the outcome of nutritional supplementation is poor, with the tendency for weight gain to be fat and water and not lean tissue. the causes of malnutrition leading to wasting in aids are believed to be a combination of several factors, which, as shown in table i , can be classified under three main categories: 1) reduced nutrient intake, 2) malabsorption, and 3) metabolic disturbances. of the factors that may underlie the reduction in nutrient intake in hiv patients, anorexia is usually the most prominent. it is primarily the result of the cytokine responses to the infection per se, but it can also be caused or exacerbated (as an unwanted side effect) by certain medications employed in hiv patients receiving multidrug therapy for concomitant medical problems. a diminished ability to ingest nutrients can also be observed in hiv patients suffering from some central nervous system (cns) processes such as dementia or tumors. furthermore, the almost universal presence of inflammatory processes, infections, or both, involving the digestive tract of aids patients producing gingivitis, stomatitis, esophagitis, or enteritis, often impair the ability to ingest, in large part due to pain preventing intake or aggravated by food intake, as well as absorb nutrients. the net effect is that all these factors contribute to a deficit of energy and nutrient supply to meet the body's needs. in addition to reduced nutrient intake, intestinal malabsorption (due to intestinal dysfunction and inflammation) can also be an important contributory factor to malnutrition in aids cases. [7] [8] [9] [10] [11] however, a causal link between abnormal malabsorption tests and wasting is not clear. in hiv-infected children, although postnatal gain in weight and lean body mass were found to be lower than in an hiv-negative comparison group, 12 an earlier study by the same group 13 found that lactose malabsorption was not associated with higher rates of diarrhea or growth failure. this underlies the fact that carbohydrate malabsorption in hiv-infected children is not the only factor responsible for growth failure. moreover, fat malabsorption is generally more clearly related to development of malnutrition. in fact, malabsorption, particularly of fat but also certain minerals and vitamins, is common in patients with protracted diarrhea, and patients with hiv infection are prone to develop a variety of enteric infectious processes that cause diarrhea. these include those due to protozoan, viral, bacterial, and fungal pathogens (see table ii ), as well as neoplasm (lymphoma, kaposi sarcoma) and disseminated infectious processes. the role of several additional enteric pathogens (including enteroaggregative escherichia coli) in the pathogenesis of diarrhea and malabsorption has not been adequately studied and certainly deserves more attention. 14, 15 furthermore, the paucity of food in the intestine over long periods of time, in conjunction with protein-energy malnutrition, may provoke structural changes and functional intestinal atrophy. the resulting decline in digestive functions could further impair absorption of both macronutrients (fat, protein, and carbohydrates) and micronutrients (vitamins and minerals). thus, absorptive dysfunction compounds the problem of reduced nutrient intake and often aggravates anorexia through abdominal symptoms. in fact, in many if not most malabsorption syndromes, it is generally the reduction in oral intake induced by symptoms related to malabsorption (cramps, bloating, pain, and diarrhea) that is responsible for most of the energy gap between intake and total expenditure. like many pathophysiologic conditions such as sepsis and cancer, a hypermetabolic state, characterized by an increase in resting energy expenditure (ree) and disturbances in the metabolism of protein (muscle proteolysis) and fat (hypertriacyglycerolemia), has also been implicated in the aids wasting syndrome. in fact, an earlier notion that hypermetabolism is the major driving force behind wasting in aids has been particularly attractive because it provided an explanation for the excessive loss of muscle, and the difficulty of reversing the wasting syndrome (with nutritional therapy most often resulting in gain in body fat and water, with marginal or no gain in lean tissue). these metabolic abnormalities have been associated with futile cycling, such as that occurring when fatty acids are being mobilized at an accelerated rate from adipose tissue, and then reesterified into triacylglycerol for storage again in fat, 16 as well as the wasteful use of substrates underlying the conversion of glucose into fatty acids, before being stored as fat (i.e., de novo lipogenesis). 17 the contribution of these metabolic pathways to the hypermetabolic state is, however, unclear and hypertriacylglycerolemia in the hiv patients does not correlate with wasting. 18 -21 moreover, although an increase in ree has been reported in all stages of hiv infection, 22-27 the notion that hypermetabolism is the primary factor underlying wasting in aids has been challenged by more recent studies in patients with hiv indicating that the increase in ree per se is not sufficient to cause wasting. 25 first, ree is increased even in asymptomatic hiv-infected patients with normal cd4 cell counts, but such individuals can usually sustain their weight and lean body mass for prolonged periods. second, in patients who were actively losing weight or had stable weight, ree was about 10% higher than normal, but their total energy expenditure (tee) was found to be no greater than that predicted for healthy individuals. these apparent discrepancies between increased ree coexisting with normal or low tee have been reconciled with data indicating that the increased ree is largely compensated by energy saved as a result of reduced physical activity, and it is loss of appetite leading to decreased intake, coupled with malabsorption, that primarily drives wasting in aids. to what extent such reduction in physical activity is the result of lethargy and fatigue from the illness per se or that of a normal adaptive physiologic response to save energy is unknown. however, it is clear that the decrease in physical activity will alter the quality of life, which may further deteriorate because a drastic reduction in locomotor activities might lead not only to muscle atrophy and consequential functional impairments, but can also contribute to the failure to rebuild lean body mass during realimentation. furthermore, an increase in ree during active weight loss is by no means trivial, because this is counterproductive to the adaptive reduction in ree that is the normal response in order to buffer the energy deficit and hence contributes to exacerbate the negative energy balance even further. the most likely scenario in the development of the wasting in hiv-infected patients can be summarized as follows: • despite increased ree in response to hiv-infection, the patients can maintain their weight often for long periods of time, most probably through reductions in the amount of energy they expend on physical activity. this "compensation," however, may still be deleterious because it interferes with lifestyle and increases the susceptibility to muscle atrophy, and consequential functional impairment. • subsequent weight loss is primarily caused by poor appetite, malabsorption, or both, generally triggered by secondary infections. 22,25 furthermore, the persisting hypermetabolic state is counterproductive to the adaptive down-regulation of resting metabolism that normally occurs in uncomplicated starvation, thereby exacerbating the wasting process. • the rate of weight loss and the severity of wasting is likely to be dependent upon the type, severity, and outcome of the secondary infection. aids patients with malnutrition due principally to malabsorptive symptoms seem to restore body weight and lean body mass when recovering from secondary opportunistic infection or when receiving nutritional support, 28,29 similar to what has been observed in non-hiv individuals recuperating from starvation, anorexia nervosa, and other clinical conditions, although both body fat and lean tissue are being recovered, fat is being restored at a disproportionately faster rate relative to lean tissue repletion. 30 -34 however, when protein energy malnutrition is largely a reflection of systemic illness and inflammation, the recovery of lean tissue seems to be even poorer or delayed, such that body weight repletion is more as fat than body protein, similar to patients with sepsis, presumably related to inefficient protein anabolism. 33 it is against this background presentation of the interacting factors contributing to malnutrition and functional impairment in hivinfected patients-namely anorexia, malabsorption, hypermetabolism, lethargy, and impaired fat and protein metabolism-that the role of cytokines in the aids wasting syndrome is discussed in the following sections. in the 1980s, beutler and cerami 35 isolated a 17-kda protein while searching for a mediator to account for the metabolic changes (particularly hypertriacylglycerolemia) observed in animals infected with the protozoan parasite trypanosoma cruzi (chagas' disease agent). this protein was termed cachectin, as it was supposed to mediate the wasting and hypertriacylglycerolemia found in those experimental animals. 35 cachectin was subsequently found to reduce the activity of lipoprotein lipase (lpl) (potentially accounting for lower triacylglycerol turnover in vivo) and to promote lipolysis in vitro. 36 in addition, purified cachectin was able to produce anorexia, weight loss, and fever when injected into experimental animals. subsequently, when the cachectin dna sequence was isolated, it become apparent that its dna sequence was identical to that coding for tumor necrosis factor (tnf). 36 tnf had been previously isolated from the serum of animals injected with bacterial endotoxins and was able to produce necrosis of transplanted tumors in animals. 37 furthermore, administration of purified or recombinant tnf to experimental animals provokes very similar metabolic and hemodynamic changes to those observed during bacterial sepsis that could be blocked by the use of specific antisera or monoclonal antibodies. 38,39 administration of recombinant tnf to humans suggested that this cytokine could play an important role in the early activation of the hemostatic mechanism in septicemia. 40 recent experimental studies with knockout mice (for the 55-kda tnf receptor) have confirmed the importance of tnf in the pathophysiology of endotoxic shock. 41, 42 it is now well established that tnf and other cytokines (e.g., interleukins, interferons) are a group of hormonelike polypeptide mediators released by various cell types having pleiotropic actions on many cell types, and they play a regulatory role in normal and abnormal homeostasis and in host defense mechanisms (inflammatory and immune responses). cytokines share some general characteristics in their mode of action: different cytokines can have a similar effect on several target cells (redundancy); they can activate the secretion of other cytokines (producing a "cascade"); and they can also initiate their own secretion (autocrine), act in a paracrine fashion on neighboring cells, or act on distant cells as hormones. in addition to their pleiotropic actions on many body systems, they could potentially contribute to the wasting and cachexia of aids by their ability to induce anorexia, alter energy expenditure, increase muscle proteolysis and net protein breakdown, and initiate various abnormalities of lipid metabolism. a role for tnf in aids wasting syndrome was postulated in earlier studies indicating that tnf serum levels were elevated in patients with aids. 43 however, subsequent studies failed to show high-serum tnf levels in most aids patients, and no correlation appeared to exist between serum tnf levels and the magnitude of weight loss in aids patients. 44 -48 at least in some studies the choice of the methods to determine tnf activity (immunoassay versus bioassay) might account for the differences observed. pentoxifylline, a xanthine that inhibits the production of tnf by decreasing activity of the transcription factor nf-kb, has been used to highlight indirectly the possible importance of tnf in the hypertriacylglycerolemia observed in aids patients. administration of pentoxifylline was shown to reduce the triacylglycerol levels in aids patients, although this reduction was only marginally significant (p ϭ 0.06) and the study was open label (nonblinded). 49 more importantly, pentoxifylline does not alter other aspects of aids wasting, emphasizing the fact that aids wasting is not entirely tnf dependent. interleukin-1 (il-1) shares many of the characteristics of tnf and can also produce anorexia, hypertriacylglycerolemia, and stimulate hepatic fatty acid synthesis. 50,51 in addition, il-1 reduces lpl activity and produces lipolysis. 19,50 moreover, both tnf and il-1 can promote hiv-1 replication in in vitro cellular systems, which has led to the suggestion that cytokines may be important for the progression of hiv infection to aids. thus, tnf production is linked to hiv infection and the potential role of tnf in this setting is a source of this enhanced production. 52,53 naturally occurring cytokine antagonists such as the soluble form of the p55 (type i) tnf receptor (tnfsrp55) and the il-1␤ receptor antagonist (il-1ra) are produced in the body to counteract the potentially harmful effects of excessive tnf and il-1 production, respectively. 54,55 enhanced plasma levels of soluble tnf receptors have been reported to be correlated with rapid progression toward aids in hiv-1 infected patients. 56 moreover, a study suggested that enhanced tnfsrp55 and tnfsrp75 (type ii) were predictive of worsening nutritional status in hiv patients. 57 a more recent study showed that high serum levels of il-1␤, tnf, and il-8 together with an excess of the natural inhibitors il-1ra and tnfsrp55 were seen in asymptomatic hiv-1-positive african women but not in african women with aids or in hiv-negative controls. 46 this study suggests that cytokine antagonists may play a role in modulating cytokineassociated symptoms in the early phases of hiv infection. 46 alternatively, because most of the aids patients in that study were at the endstage of their disease and therefore likely to have significant protein-energy malnutrition, it might only reflect the inability or reduced ability to synthesize new proteins, including cytokines. on the other hand, another study has found no correlation between elevated soluble tnf receptor types i and ii levels and metabolic disturbances in hiv infections. 58 other studies have shown increased serum/plasma levels of il-1, tnf, il-6, and interferon-␥ in some populations of hivinfected patients. 47,48,58,59 il-6, an important mediator of the acute-phase response, reduces lpl activity in vitro and in vivo and promotes fatty acid synthesis. 60,61 in contrast to tnf and il-1, il-6 serum levels are consistently raised in aids and il-6 has been implicated in the development of cachexia in inflammatory and neoplastic processes. 47,48,59,62-64 serum levels of il-6 in hiv-infected patients are high when compared with non-infected normal subjects. 65 the levels of il-6 appear to increase according to the stage of hiv disease and appear to be higher in terminal stages of the disease. 65, 66 however, no data has been provided yet to link il-6 blood levels directly with the development of wasting and cachexia in aids patients. a major problem with studies regarding cytokines and circulating soluble receptors in the bloodstream of patients with hiv is that cytokines principally act in an autocrine and paracrine manner, thus making blood levels not necessarily relevant for a proper interpretation of their effects on tissues, organs, or body systems. moreover, cytokines are rapidly internalized by cells and they can activate the release of other substances. with respect to cytokine actions, it is therefore more adequate to think in terms of effects on tissues, organs, or systems rather than trying to simply correlate a complex clinical syndrome such as wasting with elevated circulating cytokine levels. for instance, il-6 has been more consistently found in the blood of hiv patients, and this is probably due to its longer half-life in serum as well as related to its major role in the acute-phase response as compared with il-1 and tnf, which are rapidly cleared from the bloodstream. the role of some cytokines such as tnf, il-1, il-2, il-6, and interferon-␥ in controlling food intake, energy expenditure, or both, have been underscored by many experimental studies. [67] [68] [69] [70] [71] [72] [73] these studies have demonstrated that exogenous administration of those cytokines may mimic the hypermetabolism and anorexia associated with infection. in addition, pretreatment with specific anticytokine antibodies blocked the anorectic and thermogenic effects to the exogenous administration of cytokines as well as those of cytokine-secreting tumors. furthermore, other studies utilizing techniques of intracerebroventricular microinjection have demonstrated the anorexigenic effects of several substances that can be induced by cytokines. those substances include plateletactivating factor, several chemokines/intercrines such as il-8, platelet factor-4, interferon-inducible protein-10, monocyte chemotactic protein-1/monocyte chemotactic and activating factor (mcp-1/mcaf), regulated-upon-activation of normal t cell ex-pressed and presumably secreted (rantes), as well as ␤2microglobulin, a marker for immune activation. 74 -76 these findings suggest an interaction (and perhaps redundancy or synergistic action) of several immunomodulators that are released during inflammatory and immune processes to induce anorexia. what are mechanisms (and mediators) by which anorexia and hypermetabolism are produced? several neurotransmitters, amino acids, peptides, and cytokines can potentially influence food intake during starvation and infectious processes. in addition, some neurotransmitters function during normal regulation of food behavior. prominent among them are corticotropin-releasing hormone (crh), neuropeptide y (npy), cholecystokinin (cck), norepinephrine, acetylcholine, serotonin, dopamine, glutamate, ␥-aminobutyric acid, and others. [77] [78] [79] the paraventricular nuclei (pvn) and the hypothalamus appear to be important areas for the control of compensatory feeding behavior in response to changes in energy homeostasis. 80 crh and npy appear to play a prominent role in the responses to starvation. increases of npy in the pvn area produce hunger together with activation of the hypothalamic-pituitary-adrenal (hpa) axis. 81 increases of crh produce a reduction in messenger rna (mrna) for npy together with anorexia and activation of the hpa axis, as well as increased thermogenesis, lipolysis, hyperglycemia, and inhibition of expected insulin secretion. 79, 80, 82 the paradoxical activation of the hpa axis (together with the secretion of glucocorticoids) during the secretion of both substances, crh and npy, which have opposite effects on feeding behavior, might be explained by the fact that when the hpa axis is activated, it is done so in the setting of different orchestrated responses. 83, 84 several cytokines, such as tnf, il-1, and il-6, as well as other mediators of inflammation, which were initially and collectively called "tissue corticotropin-releasing factor," can activate the hpa axis during inflammatory states. 85, 86 activation of the hpa axis by cytokines leads to the secretion of glucocorticoids, an action believed to participate in the negative feedback control of the immune response. 85, 86 during inflammatory states tnf is secreted first and promotes the cellular secretion of il-1; and the release of both cytokines leads to the secretion of il-6, which in turn acts in conjunction with glucocorticoids to elicit the production of many mediators of the acute-phase response by the liver. 62,85,87 tnf, il-1, and il-6 act in a synergistic manner, whereas glucocorticoids down-regulate the secretion of those cytokines 63,87-89 and other inflammatory mediators including nitric oxide, platelet activating factor, and prostanoids. 90 -92 tnf, il-1, and il-6 also participate in the stimulation of the hpa axis during endotoxin administration. 93 noteworthy, anti-il-6 antibodies can almost completely block the stimulation of the hpa axis by endotoxin. 93 as these cytokines do not appear to cross the blood-brain barrier in significant amounts, how can these cytokines be acting at the cns level? one explanation is that tnf, il-1, and il-6, produced peripherally, can act on crh neurons through the activation of other cells such as astrocytes or microglial cells in the brain or cells in the area postrema, which are not protected by the blood-brain barrier, to secrete cytokines or other substances. alternatively, activated endothelial cells, phagocytic cells, or activated t cells migrating through the blood-brain barrier can initiate further cellular production of cytokines to act on crh-producing neurons. to gain an insight into these mechanisms we performed a study in which transgenic mice expressing high levels of soluble tnf-r1 fusion protein (and therefore having blunted circulating tnf levels) showed reduced thermogenesis and blunted mrna expression for tnf, il-1, il-6, and crh in the brain in response to a parenteral challenge (d. arsenijevic, i. garcia, h. r. chang, and a. g. dulloo, unpublished observations). these results support the hypothesis that local production of tnf, il-1, and il-6 in the brain may mediate the increased brain production of crh to produce anorexia and hypermetabolism during endotoxin administration. furthermore, that study demonstrates that tnf is necessary for eliciting the increased production of crh and that tnf is indeed needed as an amplifier of the inflammation cascade through up-regulation of il-1 and il-6 production. this finding is in agreement with other data showing that cns administration of antibodies to neutralize il-1␤, il-6, or tnf inhibits the thermogenic and anorectic responses to peripherally injected endotoxin in the rat. 94 further systems such as the noradrenergic system may be activated during inflammatory stress. 95 the overall effect should be the enhancement of crh production, which carries the previously mentioned effects including anorexia, lipolysis, and increased thermogenesis and therefore weight loss. systemic administration of il-1 also stimulates the expression of crh mrna in the pvn together with dose-dependent activation of the hpa axis and sustained suppression of food intake. 96, 97 this effect of il-1 is partially reversed by crh antisera administration. 98 il-1 receptors have been demonstrated in hypothalamic structures. 99, 100 almost identical effects on crh release and food intake have been reported for tnf, il-6, il-2, and interferon-␥, 100 suggesting that sustained and moderate increases in the levels of those cytokines (which act synergistically) can potentially increase crh, thereby blocking the normal compensatory hypothalamic response to weight loss (i.e., increased appetite and reduced thermogenesis). and indeed, elevated levels of several cytokines, including il-1 and il-6, have been reported in the cerebrospinal fluid of aids patients. 101 thus, local production of cytokines within the cns can contribute to the wasting syndrome and cachexia observed in hiv infection and aids through chronic release of crh. another potential mechanism by which sustained production of cytokines could enhance the secretion of crh is through reduction of the sensitivity of target tissues to the effects of glucocorticoids. for instance, a decreased affinity of glucocorticoid receptors for cortisol has been described in phagocytic cells from some aids patients. 102 in those aids patients, there were elevated levels of cortisol and corticotropin associated with signs of glucocorticoid deficiency including hyponatremia and postural hypotension pointing to a glucocorticoid-resistant condition. 102 by this mechanism, hpa axis activation would be resistant to the negative feedback mechanism provided by the secretion of glucocorticoids. interestingly, il-2 in combination with il-4 has been described to produce resistance of t cells to the action of glucocorticoids by reducing the affinity of the glucocorticoid receptor for its ligand. 103 il-4 is involved in antiinflammatory mechanisms together with other cytokines, such as il-10 and il-13, and they are able to augment the expression of il-1ra. 104 -108 both insulin and glucocorticoids play an important role in the peripheral response to fasting and starvation. they also appear to play a role in the cns regulation of energy balance by regulating npy synthesis and release. 83 thus, insulin, as opposed to its peripheral anabolic effects, promotes a state of negative energy balance (through anorexia and increased energy expenditure) by reducing npy gene expression in the hypothalamus. 83 fasting (which lowers insulin levels) increases hypothalamic npy gene expression. 109 glucocorticoids have opposing effects to insulin, thus promoting a state of positive energy balance with an increase of caloric intake. 83, 110 peripherally, glucocorticoids, as well as glucagon, catecholamines, and growth hormone, may induce insulin resistance when these hormones are present in enhanced levels and in the presence of stress or infections such as in sepsis. 111 current data, however, suggest that glucagon and catecholamines are not highly elevated in aids, whereas elevated basal cortisol levels have been found frequently. 112, 113 administration of tnf to humans has been found to produce a hyperglycemic state without alterations in insulin levels, suggesting an insulin resistance-like state. 114 moreover, infusion of tnf into experimental animals produces marked insulin resistance. 115 these findings may help to partly explain the hyperglycemia and the relative insulin resistance observed in several infectious processes. however, hiv infection is characterized by high rates of insulin clearance as well as an increased sensitivity of peripheral tissues to insulin. 112 is there a role for leptin? the newly described molecule leptin and its putative receptor also appear to play a role in maintaining normal weight. leptin is the protein product of the ob gene and its circulating levels reflect energy stores in adipocytes, suggesting its role as an "adipostat." 116 -118 the leptin receptor gene is highly expressed at the hypothalamic level. 119, 120 increased leptin levels produce anorexia and increased energy expenditure, possibly by reducing the release of npy 119,121 at the hypothalamic level. thus, reduced leptin levels induce hunger and reduced thermogenesis, pointing to a role for reduced levels of leptin in the adaptive changes to fasting/starvation. on the other hand increased leptin levels may be related to resistance to obesity (anorexia and enhanced thermogenesis), and may be acting on melanocyte-stimulating hormone and the melanocortin-4 receptor, in other regions of the brain. [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] glucocorticoids and insulin have been demonstrated to up-regulate the production of leptin. [125] [126] [127] cytokines, such as tnf and il-1, and endotoxin, are able to stimulate leptin production 128 and anorexia is directly proportional to the increase of leptin pointing to a potential role of this molecule in the anorexia associated with infection. 128 however, a first study measuring leptin levels in patients with aids found that leptin levels were not increased relative to body fat in patients who were anorexic, were losing weight, or had a history of weight loss. also, leptin levels were not elevated during secondary infection, suggesting a lack of a link between increased leptin levels and anorexia in aids. 129 another study comparing the levels of leptin in hiv-infected men to age-and body-fatmatched uninfected individuals showed no differences in serum leptin levels and there was no correlation with lean body mass. 130 there was, however, correlation between leptin concentrations and percent body fat and body fat content, extending the notion that circulating leptin levels directly reflect adipose tissue mass, even in hiv-infected men with low body-fat content. 130 a significant loss of lean body mass mainly due to muscle proteolysis has long been appreciated to be characteristic of wasting and cachexia in trauma and sepsis (as well as in aids, cancer, fasting, and acidosis). well before the wealth of information that currently exists on cytokine pathophysiology, several investigators were searching for mediators, such as the so-called "muscle proteolysis factor," that could be responsible for the metabolic disturbances and wasting observed. 131 theoretically the loss of lean body mass could be due to an increase in tissue protein degradation or a decrease in tissue protein synthesis or a combination of both. accelerated protein breakdown in muscle is necessary to meet the needs of the anabolic response in liver, hemotopoietic, and wound tissue during stress and infection. on the other hand, in starvation (and in the absence of infection) there is increased appetite on refeeding, reduced energy expenditure, and relative preservation of muscle mass, but also no requirement for enhanced anabolism in those select tissues. in the wasting of aids and in the presence of cytokines there might be maintenance or declines in basal metabolic rate together with anorexia and muscle catabolism. what, therefore, would be the cause for muscle wast-ing in aids? tnf and il-1 have been shown to produce skeletal muscle catabolism in addition to their anorectic and net nitrogen loss effects, 132,133 but by different mechanisms. moreover, administration of tnf and il-1 to experimental animals have been found to produce weight loss, net nitrogen loss, skeletal muscle catabolism, and increased liver weight. 134, 135 the effects were observed independent from and additive to those resulting from semistarvation. 134, 135 recent evidence strongly suggests that activation of the ubiquitin-proteasome pathway is responsible for muscle wasting in several catabolic states. 136 the activation or the suppression of the pathway is related to the rate of ubiquitin conjugation to proteins. higher rates of ubiquitin conjugation result in enhanced muscle proteolysis. glucocorticoids promote muscle proteolysis, an action that opposes the anabolic effects of insulin, by increasing mrnas encoding ubiquitin and proteasomes subunits and therefore increasing ubiquitin-protein conjugates. 136 in the fasting state, muscle proteolysis may take place in the presence of glucocorticoids and when insulin levels are low. glucocorticoids in combination with several cytokines such as tnf, il-1, and il-6 participate in stimulation of the ubiquitinproteasome pathway producing muscle proteolysis. [137] [138] [139] in aids, all the requisite conditions for muscle proteolysis are present: increased basal levels of cortisol, 113 low circulating insulin levels, 113 and subtle release of several cytokines that may be acting synergistically. interference with tnf production by anti-tnf antibodies or by administration of pentoxifylline produces blockade of muscle proteolysis in vivo. 140, 141 pentoxifylline may be acting to block muscle proteolysis through inhibition of the proteasome-dependent activation of the transcription factor nf-kb, which is also needed for tnf production. 142 blockade of il-1 action by administering soluble il-1ra to experimental animals prevents muscle proteolysis in response to endotoxin. 143 recently, a novel glycoprotein, able to produce muscle proteolysis in vitro, as opposed to tnf and il-6, which are unable to do so and are only effective in vivo, has been described in rodents and in the urine of cachectic patients with certain cancers. 144 whether this glycoprotein also is produced in aids patients with cachexia is unknown. because cytokines such as tnf and il-1 are not able to produce muscle proteolysis in vitro, their effects on muscle proteolysis may be indirect. 143, 145, 146 several subtle endocrine alterations have been demonstrated in hiv patients that potentially may be related to cytokine production. 113 thyroid hormone, adrenal, and gonadal homeostasis could be altered during hiv infection by cytokines. the euthyroid sick syndrome can be observed with severe caloric depletion and severe illnesses, and is characterized by impaired peripheral conversion of thyroxine to t3, resulting in high normal or normal circulating levels of thyroxine and lower levels of t3. 147 in addition, enhanced rt3 levels are present due to reduced clearance, 147 whereas thyrotropin (tsh) levels appear to be within normal limits. in aids patients with anorexia and weight loss, conversion of thyroxine to t3 is decreased (euthyroid sick syndrome) as well as the levels of insulin-like growth factor-i (igf-i), whereas in stable hiv patients t3 levels are normal. 148, 149 the reduction in t3 in those patients might be the consequence of an adaptive response to caloric deprivation, as is also observed during fasting and malnourished states. maintenance of such low levels of t3 during nutritional rehabilitation may hamper the buildup of lean body mass. infusion of il-6 to patients with cancer and normal thyroid function has been shown to acutely decrease tsh and t3 and to enhance levels of rt3, but after several weeks of il-6 administration only tsh was found to be elevated. 150 similarly, infusion of tnf to normal persons produced acute de-creases of tsh and t3 and increased rt3. 151 the clinical significance of such changes in thyroid function upon cytokine administration are not completely understood but suggest that the changes observed in aids patients may be due to the effects of cytokines. in aids, increased basal levels of cortisol have been demonstrated, 113 and this might reflect the activation of the stress response. as a result of such cortisol levels, the cortisol response to provocative testing may be abnormal. cytokines can directly stimulate the release of corticotropin (acth), crh, and cortisol, 96 -100,152 which might suggest their potential role for the mild cortisol elevations in hiv patients. the demonstration of glucocorticoid resistance in some hiv patients 102 may provide an additional explanation for cortisol elevations in the serum of some aids patients. data on gonadal function in aids has been gathered mostly from male patients. there are substantial data to indicate that hiv infection is accompanied by hypogonadism. 113 hypogonadism in aids can be primary (testicular) or central in etiology, and may be due to the release of cytokines. for instance, administration of tnf to healthy men produces a rise in luteinizing hormone (lh) followed by a decrease in testosterone levels. 153 moreover, il-1 has been shown at high levels to block steroidogenesis by inhibiting the binding of lh to leydig cells. 154 a particularly noteworthy relationship is that crh, which is induced by cytokines, is also produced by leydig cells of the testes and produces autocrine effects by inhibiting testosterone biosynthesis. decreases in testosterone levels may make difficult any attempt to increase muscular mass. indeed, administration to aids patients of megestrol acetate, which stimulates appetite, also lowers testosterone levels, and results in weight gain but mainly of fat mass. 155 recent experimental data suggest that the appetite-stimulant effect of megestrol acetate involve stimulation of synthesis, transport, and release of neuropeptide y in the hypothalamus. 156 the nutritional status of an individual can be one of the major determining factors in resistance to infection. hiv infection, on the other hand, often produces malnutrition leading to wasting and cachexia. wasting syndrome in aids is multifactorial: hiv infection of gastrointestinal tissue and lymphoid tissue particularly, anorexia, inadequate nutrient intake and malabsorption, and catabolic effects on intermediary metabolism play important roles along with similar effects of repeated secondary infections. currently available preventive/therapeutic approaches for wasting in aids include baseline nutritional assessment 157 (table iii) , early diagnosis of malnutrition and maintenance of adequate nutritional intake, early diagnosis/prevention of opportunistic infections, and appetite stimulants as well as anabolic hormonal therapy. 158 -160 adequate antiretroviral therapy might be considered during early stages of hiv infection as part of the wasting preventive measures, because successful treatment of the primary infection with hiv or secondary infections is the most potent of anabolic therapies. cytokines play a complex and overlapping role in the development of wasting and cachexia in aids. they can exert behavioral, hormonal, and endocrine effects that can persist for long periods of time to produce wasting and cachexia. multipronged therapeutic approaches are therefore required to counteract their effects on different body systems. attempts have been made to modulate the release of certain cytokines such as tnf by pharmacologic means in aids patients, with the aim of arresting or reversing the wasting process. for instance, pentoxifylline has been found in pilot studies to decrease tnf serum levels as well as serum triacylglycerols in aids patients. 49, 161 however, viral load was not altered and there was no benefit in terms of weight gain. 49, 161 other pilot studies have failed to show any benefits of pentoxifylline administration to aids patients in terms of weight gain or effect on serum tnf levels. 162 similar observations have been made in cancer patients with cachexia, where pentoxifylline failed to improve anorexia or cachexia. 163 thalidomide, a drug with sedative effects that has been used for many years for the therapy of some reactive forms of leprosy (erythema nodosum leprosum), has been found to down-regulate tnf production in vitro. 164 thalidomide has shown in pilot studies to promote weight gain in hiv patients with wasting. 165, 166 thalidomide administration produced a reduction in serum tnf levels in a pilot study of hiv patients with wasting and tuberculosis. 166 however, in a recent trial in which thalidomide was effective for the therapy of aphthous ulceration of the mouth, there were increases in tnf and soluble tnf receptor type ii as well as viral load. 167 thalidomide has potent antiinflammatory properties and has been successfully used for therapy of certain types of aphthous ulcerations in aids patients as well as for the treatment of graft-versus-host disease in allogeneic bone marrow transplantations. 168, 169 an additional mechanism by which thalidomide might promote weight gain is by improving the absorption of nutrients through the gastrointestinal tract. to date, no firm evidence has been provided to indicate that pharmacologic blockade or manipulation of cytokine production is useful in the prevention or therapy of wasting and cachexia in aids. in fact, sustained blockade of cytokine production may produce harmful effects as they are needed for a proper functioning and tuning of the immune system and blockade of a particular cytokine may in turn produce blockade of other cytokines, which may have unpredictable results. for instance, administration of pentoxifylline produced an increase in mycobacterial load in macrophages from aids patients with disseminated mycobacteriumavium-intracellulare complex infection. 170 an alternative way of counteracting the effects of cytokines may include use of a cytokine with inhibitory actions on other cytokines or a drug or drugs that enhance the release of inhibitors, although these agents would be subject to the same concerns. for instance, epinephrine has been shown to increase the release of the antiinflammatory cytokine il-10, which has been previously found to inhibit tnf. 171, 172 however, it is difficult to see how this could be clinically applied, although oral or b-agonist therapy would be theoretically possible. finally, the use of antiinflammatory cytokines might be possible. potentially new approaches for prevention/therapy of wasting and cachexia of aids may include drugs that selectively can modulate the release of mediators acting at the target tissues, organs, or systems. for example, selective inhibitors or antagonists of crh that can go across the blood-brain barrier or agents that can down-regulate hypothalamic release of crh may effectively combat anorexia. also, specific inhibitors of the ubiquitinproteasome pathway might be useful to arrest muscle catabolism. however, the most effective therapy for wasting induced by infectious agents is always the successful treatment of the infection rather than the body's cytokine response to infection. ultimately the ideal nutritional solution to aids wasting will be successful antiretroviral therapy. further work in this area is greatly needed and hopefully the results of those studies will enhance our knowledge and help us to explore new therapeutic avenues for wasting and cachexia of aids. revised classification system for hiv infection and expanded surveillance case definition for aids among adolescents and adults nutritional status, gastrointestinal dysfunction, and survival in patients with aids magnitude of body-cell-mass depletion and the timing of death from wasting in aids body weight as an essential data in the management of patients with human immunodeficiency virus infection and the acquired immunodeficiency syndrome body composition studies in patients with the acquired immunodeficiency syndrome quality of life in persons with human immunodeficiency virus infection. measurement by the medical outcomes study instrument d-xylose malabsorption: characteristic finding in patients with the aids wasting syndrome and chronic diarrhea enteropathy associated with the acquired immunodeficiency syndrome malabsorption and mucosal abnormalities of the small intestine in the acquired immunodeficiency syndrome small intestinal structure and function in patients infected with human immunodeficiency virus (hiv): evidence for hiv-induced enteropathy steatorrhea: a common manifestation in patients with hiv/aids growth and body composition in children infected with human immunodeficiency virus-1 malnutrition and carbohydrate malabsorption in children with vertically transmitted human immunodeficiency virus-1 infection gastrointestinal manifestations of hiv infection summary of the 31st united states-japan interleukin-6: an overview cachexia and the acute-phase protein response in inflammation are regulated by interleukin-6 quantitative analysis of serum il-6 and its correlation with increased levels of serum il-2r in hiv-induced diseases increased interleukin-6 production is associated with disease progression in hiv infection interferon-␥, more a cachectin than tumor necrosis factor cytokines, muscle proteolysis and the catabolic response to infection and inflammation the role of interleukin-6 in lipopolysaccharide-induced weight loss, hypoglycemia and fibrinogen production in vivo inhibition of central actions of cytokines on fever and thermogenesis by lipocortin-1 involves crf metabolic effects of cachectin/tumor necrosis factor are modified by site of production tumor necrosis factor and regulation of metabolism in infection role of systemic versus tissue levels tumor necrosis factor in the malnutrition (cachexia) of infection and cancer immunomodulators and feeding regulation: a humoral link between the immune and nervous systems chemokines/intercrines and central regulation of feeding modulation of feeding by ␤ 2 -microglobulin, a marker of immune activation the lateral hypothalamus: a primary site mediating excitatory amino acid-elicited eating neuropeptide regulation of appetite and weight central effects of crf on metabolism and energy balance the hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism food deprivation and ingestion induce reciprocal changes in neuropeptide y concentrations in the paraventricular nucleus hypothalamic neuropeptide y messenger ribonucleic acid levels in pre-obese and genetically obese (fa/fa) rats: potential regulation thereof by corticotropin-releasing factor feast and famine: critical role of glucocorticoids with insulin in daily energy flow hypothalamic response to starvation: implications for the study of wasting disorders the hypothalamic-pituitary-adrenal axis and immunemediated inflammation neuroendocrine-immune system interactions. the immune-hypothalamo-pituitary-adrenal axis glucocorticoid therapy for immune-mediated diseases: basic and clinical correlates glucocorticoids selectively inhibit the transcription of interleukin-1␤ gene and decrease the stability of interleukin-1␤ mrna glucocorticoid inhibition of interleukin-1 induced interleukin-6 production by human lung fibroblasts: evidence for transcriptional and post-transcriptional regulatory mechanisms the arginine-nitric oxide pathway glucocorticoids suppress group ii phospholipase a 2 production by blocking mrna synthesis and post-transcriptional expression cdna cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase synergistic roles of interleukin-6, interleukin-1, and tumor necrosis factor in adrenocorticotropin response to bacterial lipopolysaccharide in vivo cytokines and thermogenesis the participation of the nervous system in the inflammatory reaction interleukin-1 stimulates corticotropin-releasing factor gene expression in rat hypothalamus interleukin-1 stimulates the secretion of of hypothalamic corticotropinreleasing factor anorexia induced by interleukin 1: involvement of corticotropin-releasing factor interleukin-1 immunoreactive inervation of the human hypothalamus immunoregulators in the nervous system human immunodeficiency virus type 1 (hiv-1) infection of the central nervous system: an evaluation of cytokines in cerebrospinal fluid cortisol resistance in acquired immunodeficiency syndrome combination of il-1 with il-4 reduces glucocorticoid receptor-binding affinity and t cell response to glucocorticoids coordinated anti-inflammatory effects of interleukin-4. interleukin-4 suppresses interleukin-1 production but up-regulates gene expression and synthesis of interleukin-1 receptor antagonist il-10 inhibits cytokine production by activated macrophages interleukin-13 is a new human lymphokine regulating inflammatory and immune responses biologic control of the tumor necrosis factor and interleukin-1 signaling cascade update on cytokines altered expression of hypothalamic neuropeptide mrnas in food-restricted and fooddeprived rats neuroendocrine control of the development of obesity: understanding gained from studies of experimental animal models insulin resistance-mechanisms, syndromes and implications evidence of endocrine involvement early in the course of human deficiency virus infection endocrine and metabolic disturbances in human immunodeficiency virus infection and the acquired immune deficiency syndrome tumor necrosis factor mimics the metabolic response to acute infection in healthy humans tumor necrosis factor impairs insulin action on peripheral glucose disposal and hepatic glucose output positional cloning of the mouse obese gene and its human homologue leptin levels in human and rodent: measurement of plasma leptin and ob rna in obese and weight-reduced subjects recombinant mouse ob protein: evidence for a peripheral signal linking adiposity and central neural networks the hypothalamic leptin receptor in humans. identification of incidental sequence polymorphisms and absence of the ob/ob mouse and fa/fa rat mutations localization of leptin receptor mrna and the long form splice variant (ob-rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization activation of ␤ 3 adrenergic receptors suppresses leptin expression and mediates a leptinindependent inhibition of food intake in mice the role of neuropeptide y in the antiobesity action of the obese gene product role of the melanocortinergic neurons in feeding and the agouti obesity syndrome the alphabet of weight control transient increase in obese gene expression after food intake or insulin administration induction of ob gene expression by corticosteroids is accompanied by body weight loss and reduced food intake acute and chronic effect of insulin on leptin production in humans: studies in vivo and in vitro endotoxin and cytokines induces expression of leptin, the ob gene product, in hamsters: a role for leptin in the anorexia of infection serum leptin levels in the acquired immunodeficiency syndrome serum leptin concentrations in human immunodeficiency virus-infected men with low adiposity muscle proteolysis induced by a circulating peptide in patients with sepsis or trauma infusion of tumor necrosis factor/cachectin promotes muscle catabolism in the rat. a synergistic effect with interleukin-1 cachectic effects of recombinant human tumor necrosis factor in rats metabolic changes in rats during a continuous infusion of recombinant interleukin-1 mechanisms of host wasting induced by administration of cytokines in rats mechanisms of muscle wasting. the role of the ubiquitin-proteasome pathway evidence that tumor necrosis factor participates in the regulation of muscle proteolysis during sepsis effects of tumor necrosis factor or interleukin-1 on muscle amino acid uptake and the role of glucocorticoids interleukin-6 induces skeletal muscle protein breakdown in rats tumor necrosis factor-␣ mediates changes in tissue protein turnover in a rat cancer cachexia model pentoxifylline decreases body weight loss and muscle protein wasting characteristics of sepsis the ubiquitinproteasome pathway is required for processing of the nf-b1 precursor protein and the activation of nf-b reduced muscle protein breakdown in septic rats following treatment with interleukin-1 receptor antagonist characterization of a cancer cachectin factor the toxic effects of tumor necrosis factor in vivo and their prevention by cyclooxygenase inhibitors tumor necrosis factor can induce fever in rats without activating protein breakdown in muscle or lipolysis in adipose tissue alterations of thyroid function in patients with systemic illnesses: the euthyroid sick syndrome thyroid hormone levels in the acquired immunodeficiency syndrome (aids) or aids-related complex elevation of serum thyroxine-binding globulin (but not of cortisol-binding globulin and sex hormone-binding globulin) associated with the progression of human immunodeficiency virus infection effects of acute and chronic interleukin-6 administration on thyroid metabolism in humans tumor necrosis factor: a putative mediator of the sick euthyroid syndrome in man role of endotoxin and interleukin-1 in modulating acth, lh, and sex steroid secretion effects of tumor necrosis factor on the hypothalamic-pituitary-testicular axis in healthy men interleukin-1 inhibits leydig cell steroidogenesis in primary culture effects of megestrol acetate therapy on body composition and circulating testosterone concentrations in patients with aids megestrol acetate stimulates food and water in the rat: effects on regional hypothalamic neuropeptide y concentrations nutrition support and the human immunodeficiency virus (hiv) nutrition and hiv infection nutritional aspects of hiv infection wasting syndrome in aids: pathophysiologic mechanisms and therapeutic approaches use of pentoxifylline therapy for patients with aids-related wasting: pilot studies pentoxifylline therapy in hiv seropositive subjects with elevated tnf pentoxifylline for treatment of cancer anorexia and cachexia? a randomized, doubleblind, placebo-controlled trail thalidomide exerts its inhibitory action on tumor necrosis factor-␣ by enhancing mrna degradation effects of thalidomide on wasting syndrome in patients with aids: a randomized, double blind, placebo controlled clinical trial (abstract 536b) the effect of thalidomide on the pathogenesis of human immunodeficiency virus type 1 and m. tuberculosis infection thalidomide for the treatment of oral aphthous ulcers in patients with human immunodeficiency virus infection treatment of resistant aphthous ulceration with thalidomide in patients positive for hiv antibody thalidomide: rationale for renewed use in immunological disorders pentoxifylline aggravates impairment in tumor necrosis factor-␣ secretion and increases mycobacterial load in macrophages from aids patients with disseminated mycobacterium-avium intracellulare complex infection epinephrine inhibits tumor necrosis factor-␣ and potentiates interleukin-10 production during human endotoxemia controlling the production of interleukin-1 and tumor necrosis factor in disease for an additional perspective key: cord-354790-xx6imhzb authors: lambour, jennifer; naranjo-gomez, mar; piechaczyk, marc; pelegrin, mireia title: converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play date: 2016-08-17 journal: emerg microbes infect doi: 10.1038/emi.2016.97 sha: doc_id: 354790 cord_uid: xx6imhzb monoclonal antibodies (mabs), which currently constitute the main class of biotherapeutics, are now recognized as major medical tools that are increasingly being considered to fight severe viral infections. indeed, the number of antiviral mabs developed in recent years has grown exponentially. although their direct effects on viral blunting have been studied in detail, their potential immunomodulatory actions have been overlooked until recently. the ability of antiviral mabs to modulate antiviral immune responses in infected organisms has recently been revealed. more specifically, upon recognition of their cognate antigens, mabs form immune complexes (ics) that can be recognized by the fc receptors expressed on different immune cells of infected individuals. this binding may be followed by the modulation of the host immune responses. harnessing this immunomodulatory property may facilitate improvements in the therapeutic potential of antiviral mabs. this review focuses on the role of ics formed with different viral determinants and mabs in the induction of antiviral immune responses in the context of both passive immunotherapies and vaccination strategies. potential deleterious effects of ics on the host immune response are also discussed. therapeutic potential of antiviral monoclonal antibodies monoclonal antibodies (mabs) have gained an important place in the therapeutic arsenal against severe human diseases. more than 50 mabs have been approved or are under review for human use, and several hundred are currently being tested in the clinic, 1,2 most of them to treat patients suffering from a variety of cancers or inflammatory diseases. concerning antiviral mabs, only one, directed against respiratory syncytial virus (rsv), has been approved for the prophylactic treatment of pediatric infections. however, employing mabs as antiviral drugs is under consideration for the treatment of several chronic and acute severe viral infections, especially to address the public health emergencies such as the recent ebola virus and middle east respiratory syndrome coronavirus outbreaks. [3] [4] [5] [6] [7] illustrating this trend, the number of antiviral mabs developed and tested in preclinical and clinical trials has grown exponentially in the past 10 years and includes mabs directed against life-threatening agents, such as human immunodeficiency virus (hiv), hepatitis b virus (hbv), hepatitis c virus (hcv), influenza virus, dengue virus, ebola virus and severe acute respiratory syndrome virus coronavirus, among others. [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] importantly, recent clinical data have also demonstrated the efficacy of anti-hiv mabs in controlling viremia, when administered to hiv-infected patients, lending strong support to the idea that mabs could broaden the therapeutic arsenal against severe viral infections. 23, 24 their use as antiviral agents is all the more likely to be considered given that multiple biological activities may account for their therapeutic effects. although a few mabs have been developed to inhibit the recognition of viral receptors or co-receptors on the surface of target cells, most antiviral mabs have been selected for their ability to neutralize virions through the binding of their antigen-binding (fab) fragment to viral surface antigens essential for entry into host cells. however, the biological activity of antibodies is also mediated by the fragment crystallizable region (fc) moiety. thus, it is interesting to note that most antiviral mabs in use are immunoglobulin (ig)-gs displaying a variety of effector functions, including binding to both complement and fcγ receptors (fcγrs). different types of fcγrs are expressed in a regulated manner by many cells of the immune system, including b cells, natural killer cells, dendritic cells (dcs), monocytes/macrophages, granulocytes and mast cells, and their engagement by the fc antibody moiety is essential for regulating the antibody effector functions. 25, 26 upon recognition of their target antigens, antiviral mabs can facilitate virus elimination via two types of complementmediated mechanisms: (i) inactivation of viral particles and/or phagocytosis of opsonized virus mediated by cells of the innate immune system ( figure 1 ) and (ii) opsonization and subsequent lysis of infected cells, when viral antigens are also expressed on the cell surface (for example, envelope (env) glycoprotein of lentiviruses such as hiv) via complement-dependent cytotoxicity. in addition to complementmediated actions, recognition of fcγrs can entail antibody-dependent cellular phagocytosis and antibody-dependent cell-mediated cytotoxicity ( figure 1 ). 20, [27] [28] [29] [30] finally, antiviral mabs also have a role in viral blunting by inhibiting cell-to-cell viral transmission. 31 in addition to controlling the viral propagation by these mechanisms, the opsonization of viral particles and/or infected cells by therapeutic antiviral mabs of the igg type leads to the formation of immune complexes (ics) recognizable by the fcγrs expressed on antigen-presenting cells (apcs) such as dcs. this can potentially affect the endogenous antiviral adaptive immune response of passive immunotherapy-treated individuals. despite the fact that the immunoregulatory functions of antibodies (as well as ics) have been known for a long time, and have been reported in different experimental settings and physiopathological situations, 25,32-38 the immunomodulatory role of mabs with clinical potential as antiviral drugs has only recently been considered. this review mainly focuses on the induction of antiviral immune responses by ics in both passive immunotherapies and vaccination strategies. the potential deleterious effects of antiviral antibodies on the host immune dysfunction and/or viral propagation are also discussed. only recently have studies addressed whether and how passive immunotherapies utilizing antiviral mabs are able to enhance the antiviral immunity in infected individuals. this is largely due to the limited availability of suitable immunocompetent animal models of viral infection that allow in-depth investigations of the endogenous immune response. the concept that passive immunotherapies utilizing antiviral mabs can induce long-term protective immunity has recently been established using an immunocompetent mouse model, consisting of short immunotherapies given to young animals infected with the frcase murine leukemia virus. the induction of such 'vaccine-like' effects by antiviral mabs, as well as some of the mechanisms involved, are reviewed in detail elsewhere. 39 in brief, the inoculation of mouse pups with frcase is fatal because the antiviral immune response is too weak to control the viral propagation. in contrast, treatment with a neutralizing mab for several days shortly after infection blunts viral propagation and induces a lifelong protective antiviral immunity composed of both a highly neutralizing humoral response and a cytotoxic cd8 + t-cell response. [40] [41] [42] [43] [44] [45] this induction of protective immunity strictly depends on the fc fragment of the neutralizing mabs. 43, 44 moreover, the formation of ics composed of the administered mabs and infected cells rather than virions is crucial for the enhanced antiviral immune response. 43 such ics are recognized by the fcγrs expressed by dcs, which facilitate ics internalization and lead to stronger activation and more efficient antigen presentation by dcs, eventually leading to stronger cytotoxic t-lymphocyte (ctl) responses. an fc-mediated effect that occurs concurrently is the inhibition of regulatory t-cell (treg) expansion. this depends on the mab effector functions 45 and occurs rapidly. moreover, it is necessary for the development of the protective humoral and cellular responses, as treg-mediated immunosuppression is observed in all cases of chronic viral infections, where it dampens antiviral immune responses, thereby permitting the establishment of chronicity. finally, breastfeeding and placental transfer of maternal anti-frcase igs induced by mab immunotherapy not only led to the viral propagation blunting in infected pups, but also to the induction of long-lasting protective humoral immunity in these animals. 42 this is a particularly interesting observation when one considers that the frcase model is reminiscent of perinatal infection by hiv, including breastfeeding-mediated mother-to-child virus transmission. other evidence for the induction of 'vaccine-like' effects by antiviral mabs comes from studies in several preclinical models of human viral infections and from hiv-infected patients. in a mouse model of rsv infection, the administration of a neutralizing mab directed against the virus attachment protein g induced a shift in the adaptive immune response from th2-to th1-type, leading to sustained and enhanced humoral and cd8 + t-cell responses. 46 however, this effect was not fc-dependent, but rather due to the ability of the therapeutic mab to counteract the intrinsic immunosuppressive activity of the rsv g protein. mab-driven enhancement of the humoral response has also been reported in two preclinical models of henipavirus infection in african green monkeys. 47, 48 recovery from both hendra and nipah virusinduced disease correlated with the development of host antibody responses consequent to the administration of the highly neutralizing 102.4 mab. this hendra and nipah virus cross-reactive mab is currently being considered for human use. finally, anti-hiv antibodies can modulate immune responses in infected organisms. such effects were initially reported in several nonhuman primate models of hiv infection and then observed in infected humans. macaques were infected with different strains of simian immunodeficiency virus (siv) or simian hiv (shiv, a chimeric virus in which hiv env substitutes for that of siv and allows for the assessment of the antiviral effects of anti-hiv antibodies) following different protocols. these experiments showed that the administration of highly neutralizing antibodies (either mabs or polyclonal igs) enhanced both the humoral and cellular antiviral immune responses of treated animals. 8, [49] [50] [51] interestingly, recent clinical data describe the elicitation of host humoral responses in viremic subjects upon single injection of the potent 3bnc117 anti-hiv mab. 52 however, the mechanisms leading to the stimulation of antiviral immune responses in these preclinical models of hiv infection or in infected patients remain uncharacterized. moreover, it is unknown whether these antiviral responses have genuine protective vaccine-like effects. in any case, these important observations open new avenues for the improvement of mab-based antiviral hiv therapies. moreover, as the in vivo activity of anti-hiv-1 bnabs, including viral load control, was recently shown to crucially depend on fc effector functions, 53,54 an important issue is identifying that fc-fcγrs interactions are involved in the induction of vaccinelike effects by antiviral mabs. to understand the mechanisms underlying the enhancement of antiviral responses by ics, several in vitro studies have addressed whether antibody-mediated viral uptake by dcs could lead to stronger activation of these cells and the development of stronger virus-specific cd4 + and cd8 + t-cell responses in an fc-dependent manner. such an increase in the cellular immune response has been reported in different infectious settings using ics made with different types of antigens, including recombinant viral proteins and whole virions, as well as infected cells (table 1) . concerning ics made with viral proteins, several reports have shown that ics made up of anti-hbv mabs and the hepatitis b surface antigen (hbsag) can affect dc function and enhance t-cell responses. hbsag/anti-hbv ics significantly increased the uptake of the immunocomplexed hbsag antigen, and augmented the in vitro proliferation of virus-specific t cells and their production of interferon (ifn)-γ. 55 moreover, dcs from hbv-infected patients incubated with hbsag/anti-hbv ics showed higher expression of major histocompatibility complex (mhc)-ii molecules and higher production of interleukin (il)-12. ic-loaded dcs also enhanced production of il-2 and ifn-γ by co-cultured t cells. 56 interestingly, the therapeutic efficacy of hbsag/anti-hbv ics has been tested in clinical trials (see below) in hbv-infected patients with encouraging results. [60] [61] [62] more recently, in experiments aimed at visualizing immunopotentialization by hbsag/anti-hbv ics (see below), live-cell imaging revealed that ics were internalized via the fcγrs of apcs and were subsequently transported through early and late endosomes into lysosomes, where they co-localized with mhc-i and mhc-ii molecules. 63 consistent with the latter observation, the administration of dcs loaded with hbsag/anti-hbv ics to mice increased the number of ifn-γ-and tumor necrosis factor-α-producing cd8 + and cd4 + t cells. similarly, in an siv infection setting, the incubation of apcs with ics made with a recombinant full-length gag p55 protein and an anti-p55 igg increased siv capsid cross-presentation. capsid cross-presentation was dependent on fcγr-mediated uptake of the immunocomplexed siv capsid protein, and required its proteasomal and endosomal degradation to generate stronger gag-specific cd8 + t-cell responses. 57 from a mechanistic standpoint, these studies indicate that antiviral antibodies might enhance the priming and expansion of virus-specific cd4 + and cd8 + t cells by both promoting the secretion of key cytokines and facilitating the uptake and crosspresentation of viral ags by fcγr-expressing dcs. immune-complexed whole virions have also been shown to affect the functional activation of dcs. the stimulation of dcs with ics composed of siv virions and highly neutralizing siv-hyperimmune sera (svig) led to the increased virus-specific cd4 + t-cell responses in an fc-dependent manner. 58 in contrast, dcs stimulated with ics made of hiv-1 and a polyclonal igg pool from hiv-infected subjects showed only weak hiv-specific ctl-stimulating activity. this suggested that opsonization of hiv-1 by iggs might be associated with decreased ctl-stimulatory dc activity. 59 however, not all igg isotypes display equivalent effector functions. therefore, the undefined nature of the antibodies (both in terms of predominant isotypes and neutralization potential) used to form the hiv-ics in these experiments might explain these observations. whether hiv-ics made with highly neutralizing anti-hiv mabs of a specific igg isotype might induce stronger cd8 + t-cell responses is an important issue deserving further investigation (figure 2) . moreover, the nature of the viral determinant present in ics might also be crucial in the stimulation of antiviral responses. interestingly, as mentioned above, in the mouse frcase infection model, ics made up of a neutralizing mab and infected cells, but not those made with virions, efficiently induce strong gag-specific cd8 + t-cell responses with high cytotoxic activity. 43 this observation shows that the viral and cellular ics can trigger different immunologic outcomes. in the case of frcase, this is explained by the fact the frcase-gagl ctl immunodominant epitope is, at best, poorly incorporated into virions. taken together, these data indicate that the uptake of cellular ics might allow the presentation of a broader viral antigenic repertoire, leading to stronger effects on immunity ( figure 2 ). ics formed with endogenous antibodies generated in virally infected mice have been shown to influence antiviral cellular immune responses in several models (table 2) . notably, the highly neutralizing humoral response generated against the frcase retrovirus in mab-treated-infected mice was demonstrated to limit the viral propagation and to enhance memory cellular responses long after the disappearance of the therapeutic mab (which occurs within two weeks post administration, reflecting the natural igg lifespan in vivo). ic-mediated activation of dcs upon binding to fcγr was key for this effect. 43 similarly, in an influenza virus infection model, ics formed with endogenous antiviral antibodies promoted more sustained antigen presentation by dcs, resulting in stronger cd8 + t-cell proliferation. 64 interestingly, such prolonged antigen presentation by dcs was dependent on virus-specific, isotype-switched antibodies that facilitated the capture and cross-presentation of viral antigens by figure 2 parameters to consider for achieving optimal ic-mediated modulation of antiviral immune responses. the optimization of vaccine-likeeffect-inducing protocols will require the consideration of several parameters such as the nature of the antigen (that is, purified viral proteins, whole virions and infected cells) and the antibody (that is, monoclonal vs polyclonal, nature of the isotype, engineered fc domain with improved effector functions and so on) used to form the immunogenic ics, as differences in these parameters might impact immunological outcomes. in addition, whether the optimized ics are used alone or in combination with immunostimulatory molecules might also be of paramount importance. several other parameters, including the ic dosage, the route of administration, the choice of adjuvant and the immunological status of patients, will also have to be considered. fcγr-expressing dcs. in addition, serum antibodies can affect the virus-specific cd4 + /cd8 + t-cell balance in an fc-dependent manner during rsv infection. 65 an enhanced ratio of rsv-neutralizing to -non-neutralizing antibodies profoundly enhanced the cd4 + t-cell response. in a murine lymphocytic choriomeningitis virus (lcmv) infection model, endogenous virus-specific antibodies could stimulate innate immunity and thereby positively affect both the induction and the maintenance of the virus-specific cd8 + t-cell response. notably, anti-lcmv antibodies limited viral replication in peripheral organs, but allowed replication of the virus in the marginal zone of the spleen, promoting cd8 + t-cell priming. 66 interestingly, anti-lcmv antibodies were also reported to be essential for long-term maintenance of the memory ctl response in infected mice. 67, 68 these observations, together with the in vitro studies described above, demonstrate that virus-specific antibodies can promote the acquisition, processing and presentation of antigens that are subsequently instrumental for priming t-cell responses and programming functional cd8 + memory in an fc-dependent manner. they strengthen the concept that antiviral antibodies can regulate the quality and function of antiviral t-cell responses through the formation of ics. moreover, they also provide a rationale for developing novel ic-based therapeutic vaccination strategies. in 1961, terres and wolins 69 demonstrated the ability of ics to induce higher antibody titers than antigens alone. since then, the immunogenic potential of ics, alone or in combination with different types of adjuvants, has been tested in several viral infection systems, including animal models of human infections, for example, those involving hbv, hiv, rsv or flaviviruses. the immunostimulatory effects of ics are principally attributed to the ability of fc antibody fragments to recruit the host immune system. however, evidence also implicates fab fragments in modulation of the antiviral immune response, although the outcomes are less documented and were proposed to occur via alterations in antigen conformation and/or in the exposure of specific epitopes. we describe the enhancement in antiviral immune responses observed in ic-based vaccination experiments below (table 3) . ics have been tested as vaccines to augment protective immune responses in different animal models of hbv infection. in ducks, ics made with duck hbsag and rabbit anti-duck hbsag (dhbsag/ anti-dhb) were used as immunogens in the form of solid matrixantibody-antigen complexes (smaa). such smaas contained killed staphylococcus aureus as a solid matrix and mab-opsonized viruses. 79 they were initially shown to induce both humoral and ctl responses against the paramyxovirus simian virus 5 in immunized mice. 80 immunization of hbv-infected ducks with smaa-based dhbsag/anti-dhb ics led to the viral clearance in 60% of infected ducks. notably, the administration of dhbsag/anti-dhb ics lacking staphylococcus aureus showed decreased immunization efficiency, suggesting that the bacteria-based solid matrix functions as an adjuvant. 70 ics have also been tested as a therapeutic vaccine against hbv infection in mice 63, 71, 72 and woodchucks. 73 balb/c mice immunized with hbsag/anti-hbv ics produced the increased levels of virusspecific antibodies. 71 moreover, administering hbsag/anti-hbv ics to balb/c mice via intranasal inhalation induced both mucosal and systemic th1-polarized immune responses, when administered with adjuvants such as cholera toxin or oligodeoxynucleotides containing modulation of antiviral immune responses by immune complexes j lambour et al immunostimulatory cpg motifs (cpg). this was not observed using hbsag alone. 74 in addition, the administration of hbsag/anti-hbv ics to hbsag-positive transgenic mice decreased the serum hbsag levels and induced stronger ctl responses than hbsag alone. 72 notably, the co-administration of ics and a plasmid coding for hbsag increased the antiviral immune response induced by ics, indicating the adjuvant effect of dna in this setting. a similar effect was also reported in a woodchuck model of hbv infection: immunization of woodchuck hepatitis virus (whv)-infected animals with whv surface antigen/anti-whv antibody ics combined with a dna vaccine resulted in a higher reduction of both viral load and antigenemia relative to ics alone. 73 interestingly, the whv-infected animals were pretreated with lamivudine (a potent hbv antiviral drug able to enhance t-cell responses in chronically hbv-infected patients) before ic/dna immunization, suggesting that combination strategies should be considered in treating chronic hbv infections (figure 2 ). the enhancement of antiviral immune responses by ics in vitro and in preclinical models of hbv infection paved the way for the development of ic-based therapeutic vaccination strategies against chronic viral hepatitis b infection. a therapeutic vaccine composed of yeast-derived recombinant hbsag/anti-hbv immunogenic complexes (yics) has been tested in a series of clinical trials. this vaccine approach was initially shown to be safe and to induce higher titers of hbsag antibodies, as well as to increase serum ifn-γ and il-2 levels in a phase i trial. 60 importantly, a subpopulation of chronic viral hepatitis b patients showed a decrease in serum hbv viral load and hbsag levels together with an increase in anti-hbsag antibody titers in subsequent phase ii trials. 61, 62 from a mechanistic standpoint, recent data showing that the administration of yic-loaded dcs to mice increased both cd8 + and cd4 + t-cell responses 63 suggest that the improved immune responses induced by yics might account for the antiviral effect observed in a fraction of patients. in an attempt to enhance the immunogenic potential of yic-based vaccines, a phase iii trial tested the therapeutic effect of a higher number of ic doses. unfortunately, overstimulation with yic decreased the vaccine efficiency due to host immune fatigue. 81 this suggests that vaccination protocols must be optimized and must take into account both the nature and the dose of ics, as well as other parameters such as the route of administration, the type of adjuvant and the immunological status of patients to achieve efficient protective immunization ( figure 2 ). in 1988, a study immunized healthy volunteers with hiv peptides. 82 the authors found that compared with free antigen, recall immunization with ics induced stronger t-cell responses through uncharacterized mechanisms. other reports also describe alteration of the anti-hiv response by ics. [75] [76] [77] 83 in particular, the immunization of immunocompetent mice with ics containing a recombinant hiv-1 gp120 env glycoprotein and a mab (654-d mab) directed to the cd4-binding site induced a higher virus-neutralizing antibody response relative to free antigen. as described above, humoral responses were further increased upon the co-administration of ics and monophosphoryl lipid-a/dimethyldioctadecylammonium adjuvants. notably, the interaction of the anti-cd4-binding site mab with hiv-1 gp120 induced conformational changes in the latter, leading to the enhanced antigenicity and immunogenicity of neutralizing epitopes localized in the hiv-1 v3 loop. 75 these observations highlight the ability of anti-hiv-1 antibodies to induce antigenic alterations in specific hiv-1 gp120 epitopes upon ic formation. interestingly, further improvement in the immunogenicity of the v3 loop was obtained in ics generated with gp120 mutants lacking site-specific n-linked glycans. 76, 77, 83 taken together, these observations suggest that the ability of ics to stimulate the induction of neutralizing antibodies is dictated by the nature of the antigen, as well as the specificity and affinity of the mabs utilized. these results also indicate the potential contribution of fab-mediated activities in the enhancement of antiviral humoral responses by ics. tsouchnikas et al. 84 investigated the influence of immunization with ics on the specificity of antibody responses using the e protein of the tick-borne encephalitis virus as an immunogen. mice were immunized with a dimeric soluble form of e (se) alone or in complex with mabs specific for each of the three domains of e. the antibody response induced by these ics was compared with that observed after immunization with se alone. unexpectedly, immunization with ics did not change the extent of the overall antibody response in immunized mice. however, substantially different antibody responses were observed between the different ics. these differences most likely reflected an epitope-shielding phenomenon and antibody-mediated structural changes that led to the dissociation of the se dimer. thus, such phenomena can profoundly influence the fine specificity of antibody responses to the same immunogen and must be considered in ic-based vaccination strategies. as mentioned above, serum anti-rsv antibodies can affect virusspecific t-cell responses. 65 on the basis of this, kruijsen et al. 78 tested whether ics made with the commercial rsv-neutralizing mab palivizumab could influence adaptive immune response priming after intranasal administration. substantial anti-rsv t-cell priming and b-cell responses were observed in mice receiving rsv-ics, resulting in predominant th1-type cd4 + t-cell response and igg2c antibody responses. importantly, the ics also primed anti-rsv cd8 + t cells. these data have important implications for the prophylaxis and treatment of pediatric rsv infections. nevertheless, interactions between ics and neonatal versus adult innate and adaptive immune systems still need to be investigated because mouse studies have revealed potential antibody-induced neonatal autoimmunity in certain settings. 85, 86 ics and immune dysfunction in the course of viral infections, the formation of ics composed of viral determinants and the resulting host humoral responses can potentially produce deleterious effects. persistent ics are formed in a variety of chronic viral infections and may lead to unregulated and protracted fcγr signaling. this may lead to immune dysfunction instead of stimulating antiviral immune responses. in this regard, the high levels of ics formed during lcmv infection interfere with fcγr-mediated activities. 87, 88 these endogenously formed ics were shown to outcompete the effector functions of exogenously administered therapeutic mabs, in particular binding to fcγrs expressed by immune cells. persistent endogenous ics are also linked to dysfunctional b-cell responses in hiv infection, including the suppression of antiviral iga responses and impaired production of neutralizing antibodies (reviewed in moir et al. 89 ). the composition of ics might also negatively affect the efficiency of the antiviral immune response. for instance, the composition of ics has been shown to be dynamic throughout the course of hiv infections due to changes in both antibody specificities and virion levels. notably, circulating ics are initially comprised of antibodies that opsonize both infectious and non-infectious virions. this results in a decrease in the availability of antibodies able to blunt viral propagation. this phenomenon probably contributes to the reduced efficiency of the antibodies generated during acute infection. 90 changes in circulating ics have also been reported in hcv infections. the level of circulating ics is low in acutely infected patients, whereas chronically infected individuals show a high proportion of immunocomplexed hcv, raising the possibility that ics may have a role in the pathogenesis of hcv, namely liver damage. 91 moreover, the formation of ics with non-neutralizing antibodies may also lead to the antibody-dependent enhancement of viral infection of fcγr-expressing cells. this happens in several viral infections, including those by the dengue virus. [92] [93] [94] [95] along this line, the binding of ics to fcγrs on monocytes/macrophages can paradoxically suppress innate immunity, induce il-10 production and bias responses from th1 toward th2. this in turn leads to the increased infectious outputs by infected cells via intrinsic antibodydependent enhancement. 94, 96 finally, ics have also been reported to have a role in increasing viral loads in the context of gene transfer-based vaccination strategies. in the step hiv-1 vaccine trial, which evaluated a replication-defective adenovirus type 5 vector vaccine, the ics formed with pre-existing anti-adv5 antibodies improved the environment for hiv-1 replication in t cells. this may have been due to the ic-driven activation of a dc-t-cell axis that induces the activation of cd4 + t cells and leads to a permissive environment for hiv-1 infection. this environment probably explains the increased propagation of hiv-1 infection among adenovirus type 5-seropositive vaccine recipients. 97 several approaches can be considered to enhance the immunomodulatory potential of antiviral mabs, both alone and in the form of ics, in particular through combining neutralizing mabs and ic-based vaccination strategies with other therapies. a first possibility would consist of inhibiting immunosuppressive mechanisms in infected individuals by either depleting the treg response, as suggested by nasser et al. 45 or targeting immune checkpoints, the latter strategy having already led to improved immune responses against both viral infections and cancer. [98] [99] [100] [101] in addition, the combination of antiviral mabs with different immunostimulatory agents can also be envisaged. because the primary structure and glycosylation pattern of the fc fragment are both essential for antibody effector functions due to their impact on the engagement of type i and type ii fcr family members, 26, 27 fc engineering might also represent another approach, not only to improve direct antiviral effects, but also to induce stronger vaccine-like effects. in this regard, identification of the main fcrs and fcr-mediated mechanisms involved in enhancing the antiviral immune response will be of utmost importance. taking into account that the various igg isotypes display different effector functions and interact differently with fcγrs, the careful selection of antiviral mab subclasses is also crucial for enhancing antiviral immune responses. finally, as fcγr polymorphisms have already been associated with differences in viral disease progression and the therapeutic efficiency of anticancer mabs, it will be important to evaluate the extent to which such polymorphisms can affect the vaccine-like effects induced by mab-based antiviral immunotherapies. the therapeutic potential of antiviral mabs is now widely accepted, and their use as antiviral drugs is increasingly under consideration. the diverse biological activities of these mabs lead to the direct control of viral propagation and the modulation of antiviral immunity. this provides a novel rationale for their use in diverse prophylactic and therapeutic approaches. the improvement in both humoral and cellular responses achieved through the administration of mabs, either free or in the form of immunogenic ics, offers new therapeutic options. the challenge now is to improve our understanding of how ics convert mab-based immunotherapies from 'passive' to 'active' and to exploit the underlying mechanisms. this conversion will be crucial in reaching the goal of using antiviral mabs to induce longlasting protective immunity against life-threatening viral infections. molecular properties of human igg subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases trial watch: monoclonal antibodies in cancer therapy isolation of potent neutralizing antibodies from a survivor of the 2014 ebola virus outbreak protective monotherapy against lethal ebola virus infection by a potently neutralizing antibody prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 reversion of advanced ebola virus disease in nonhuman primates with zmapp therapeutic efficacy of potent neutralizing hiv-1-specific monoclonal antibodies in shiv-infected rhesus monkeys antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology monoclonal antibodies for prophylactic and therapeutic use against viral infections broadly neutralizing antiviral antibodies broadly neutralizing antibodies abrogate established hepatitis c virus infection a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus clinical development of monoclonal antibody-based drugs in hiv and hcv diseases mechanism of human antibody-mediated neutralization of marburg virus cross-reactive and potent neutralizing antibody responses in human survivors of natural ebolavirus infection a single injection of anti-hiv-1 antibodies protects against repeated shiv challenges early short-term treatment with neutralizing human monoclonal antibodies halts shiv infection in infant macaques overcoming drug-resistant herpes simplex virus (hsv) infection by a humanized antibody the growth and potential of human antiviral monoclonal antibody therapeutics a novel humanized antibody neutralizes h5n1 influenza virus via two different mechanisms therapeutic efficacy of antibodies lacking fcgamma receptor binding against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies viraemia suppressed in hiv-1-infected humans by broadly neutralizing antibody 3bnc117 virologic effects of broadly neutralizing antibody vrc01 administration during chronic hiv-1 infection mouse and human fcr effector functions type i and type ii fc receptors regulate innate and adaptive immunity exploring the potential of monoclonal antibody therapeutics for hiv-1 eradication neutralizing antibodies and control of hiv: moves and countermoves which antibody functions are important for an hiv vaccine? fcgammar dependent mechanisms of cytotoxic, agonistic, and neutralizing antibody activities broadly neutralizing antibodies that inhibit hiv-1 cell to cell transmission properties of mouse and human igg receptors and their contribution to disease models how antibodies act as natural adjuvants antibodies as natural adjuvants antibody-mediated regulation of the immune response fcgamma receptors as regulators of immune responses intravenous immunoglobulin therapy: how does igg modulate the immune system? antibody-mediated immunomodulation: a strategy to improve host responses against microbial antigens antiviral monoclonal antibodies: can they be more than simple neutralizing agents? induction of long-term protective antiviral endogenous immune response by short neutralizing monoclonal antibody treatment endogenous cytotoxic t-cell response contributes to the long-term antiretroviral protection induced by a short period of antibody-based immunotherapy of neonatally infected mice efficient mother-to-child transfer of antiretroviral immunity in the context of preclinical monoclonal antibody-based immunotherapy a crucial role for infected-cell/antibody immune complexes in the enhancement of endogenous antiviral immunity by short passive immunotherapy long-lasting protective antiviral immunity induced by passive immunotherapies requires both neutralizing and effector functions of the administered monoclonal antibody control of regulatory t cells is necessary for vaccine-like effects of antiviral immunotherapy by monoclonal antibodies prophylaxis with a respiratory syncytial virus (rsv) anti-g protein monoclonal antibody shifts the adaptive immune response to rsv ra2-line19f infection from th2 to th1 in balb/c mice a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody neutralizing polyclonal igg present during acute infection prevents rapid disease onset in simian-human immunodeficiency virus shivsf162p3-infected infant rhesus macaques passive neutralizing antibody controls shiv viremia and enhances b cell responses in infant macaques an anti-hiv-1 v3 loop antibody fully protects cross-clade and elicits t-cell immunity in macaques mucosally challenged with an r5 clade c shiv hiv-1 therapy with monoclonal antibody 3bnc117 elicits hoost immune responses against hiv-1 broadly neutralizing anti-hiv-1 antibodies require fc effector functions for in vivo activity broadly neutralizing antibodies and viral inducers decrease rebound from hiv-1 latent reservoirs in humanized mice hbsag-serum protein complexes stimulate immune t lymphocytes more efficiently than do pure hbsag selective functional deficit in dendritic cell-t cell interaction is a crucial mechanism in chronic hepatitis b virus infection evidence for antibody-mediated enhancement of simian immunodeficiency virus (siv) gag antigen processing and cross presentation in siv-infected rhesus macaques polyfunctional cd4+ t-cell induction in neutralizing antibody-triggered control of simian immunodeficiency virus infection antibodies attenuate the capacity of dendritic cells to stimulate hiv-specific cytotoxic t lymphocytes vaccination with recombinant hbsag-hbig complex in healthy adults therapeutic effect of hepatitis b surface antigenantibody complex is associated with cytolytic and non-cytolytic immune responses in hepatitis b patients a randomized controlled phase iib trial of antigenantibody immunogenic complex therapeutic vaccine in chronic hepatitis b patients immuno-potentiating pathway of hbsag-hbig immunogenic complex visualized prolonged antigen presentation by immune complex-binding dendritic cells programs the proliferative capacity of memory cd8 t cells serum antibodies critically affect virus-specific cd4+/cd8+ t cell balance during respiratory syncytial virus infections virus-specific antibodies allow viral replication in the marginal zone, thereby promoting cd8(+) t-cell priming and viral control maintenance of memory ctl responses by t helper cells and cd40-cd40 ligand: antibodies provide the key impaired antibody response causes persistence of prototypic t cell-contained virus enhanced immunological sensitization of mice by the simultaneous injection of antigen and specific antiserum. i. effect of varying the amount of antigen used relative to the antiserum antigen-antibody complex as therapeutic vaccine for viral hepatitis b enhancement of the immune response to hepatitis b virus vaccine by antigen specific igm therapeutic efficacy of hepatitis b surface antigenantibodies-recombinant dna composite in hbsag transgenic mice combination of an antiviral drug and immunomodulation against hepadnaviral infection in the woodchuck model immunization against hepatitis b virus by mucosal administration of antigen-antibody complexes the use of immune complex vaccines to enhance antibody responses against neutralizing epitopes on hiv-1 envelope gp120 improving immunogenicity of hiv-1 envelope gp120 by glycan removal and immune complex formation elicitation of broadly reactive antibodies against glycanmodulated neutralizing v3 epitopes of hiv-1 by immune complex vaccines intranasal administration of antibodybound respiratory syncytial virus particles efficiently primes virus-specific immune responses in mice immunization with solid matrix-antibody-antigen complexes containing surface or internal virus structural proteins protects mice from infection with the paramyxovirus, simian virus 5 solid matrix-antibody-antigen complexes induce antigenspecific cd8+ cells that clear a persistent paramyxovirus infection results of a phase iii clinical trial with an hbsag-hbig immunogenic complex therapeutic vaccine for chronic hepatitis b patients: experiences and findings antigenic peptides recognized by t lymphocytes from aids viral envelope-immune humans targeting a neutralizing epitope of hiv envelope gp120 by immune complex vaccine immunization with immune complexes modulates the fine specificity of antibody responses to a flavivirus antigen cutting edge: ly49c/i(-) neonatal nk cells predispose newborns to autoimmune ovarian disease induced by maternal autoantibody the unique neonatal nk cells: a critical component required for neonatal autoimmune disease induction by maternal autoantibody antibody effector functions mediated by fcgamma-receptors are compromised during persistent viral infection suppression of fcgamma-receptor-mediated antibody effector function during persistent viral infection b cells in hiv infection and disease dynamic antibody specificities and virion concentrations in circulating immune complexes in acute to chronic hiv-1 infection immune complexed (ic) hepatitis c virus (hcv) in chronically and acutely hcv-infected patients paradoxical role of antibodies in dengue virus infections: considerations for prophylactic vaccine development dengue antibody-dependent enhancement: knowns and unknowns intrinsic antibodydependent enhancement of microbial infection in macrophages: disease regulation by immune complexes how innate immune mechanisms contribute to antibodyenhanced viral infections antibody-dependent enhancement infection facilitates dengue virus-regulated signaling of il-10 production in monocytes activation of a dendritic cell-t cell axis by ad5 immune complexes creates an improved environment for replication of hiv in t cells restoration of hbv-specific cd8+ t cell function by pd-1 blockade in inactive carrier patients is linked to t cell differentiation a randomized, double-blind, placebo-controlled assessment of bms-936558, a fully human monoclonal antibody to programmed death-1 (pd-1), in patients with chronic hepatitis c virus infection immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential augmentation of hepatitis b virusspecific cellular immunity with programmed death receptor-1/programmed death receptor-l1 blockade in hepatitis b virus and hiv/hepatitis b virus coinfected patients treated with adefovir key: cord-315687-stgj6olw authors: demma, lj; vanderford, th; logsdon, jm; feinberg, mb; staprans, si title: evolution of the uniquely adaptable lentiviral envelope in a natural reservoir host date: 2006-03-20 journal: retrovirology doi: 10.1186/1742-4690-3-19 sha: doc_id: 315687 cord_uid: stgj6olw background: the ability of emerging pathogens to infect new species is likely related to the diversity of pathogen variants present in existing reservoirs and their degree of genomic plasticity, which determines their ability to adapt to new environments. certain simian immunodeficiency viruses (sivcpz, sivsm) have demonstrated tremendous success in infecting new species, including humans, resulting in the hiv-1 and hiv-2 epidemics. although siv diversification has been studied on a population level, the essential substrates for cross-species transmission, namely siv sequence diversity and the types and extent of viral diversification present in individual reservoir animals have not been elucidated. to characterize this intra-host siv diversity, we performed sequence analyses of clonal viral envelope (env) v1v2 and gag p27 variants present in individual sivsm-infected sooty mangabeys over time. results: sivsm demonstrated extensive intra-animal v1v2 length variation and amino acid diversity (le38%), and continual variation in v1v2 n-linked glycosylation consensus sequence frequency and location. positive selection was the predominant evolutionary force. temporal sequence shifts suggested continual selection, likely due to evolving antibody responses. in contrast, gag p27 was predominantly under purifying selection. sivsm v1v2 sequence diversification is at least as great as that in hiv-1 infected humans, indicating that extensive viral diversification in and of itself does not inevitably lead to aids. conclusion: positive diversifying selection in this natural reservoir host is the engine that has driven the evolution of the uniquely adaptable siv/hiv envelope protein. these studies emphasize the importance of retroviral diversification within individual host reservoir animals as a critical substrate in facilitating cross-species transmission. most newly emerging human pathogens are zoonotic [1] , yet little is known about the natural reservoirs from which these zoonoses emerge. rna viruses, due to their extraordinary genomic variability, have been particularly capable of establishing infection in new host species [1] [2] [3] [4] [5] . as examples, the transfer of avian influenza a [6] [7] [8] and rodent hantavirus [9] [10] [11] [12] from their natural reservoirs to create novel human outbreaks has been documented on several occasions [13, 14] . nonetheless, successful breaching of the host range barrier is relatively rare, with self-sustaining outbreaks in a new host species presumably requiring multiple mutational events. two different simian immunodeficiency viruses (sivs) from central african chimpanzees and west african sooty mangabeys (sm) are inferred to have been transferred to humans by several independent zoonotic events, resulting in the introduction to humans of hiv-1 and hiv-2, respectively [15] [16] [17] [18] . although phylogenetic analyses of siv sequences reveal considerable viral genetic diversity between different infected individuals [19] , the magnitude of intra-animal viral diversity, the substrate for selection in cross-species transmission events, has not been studied. furthermore, the mechanisms and tempo of the generation of viral variation in natural reservoir hosts are poorly understood. over 40 different species of african non-human primates harbor the cd4+ t cell tropic lentiviruses [20] . in these natural reservoir hosts, the sivs do not cause aids, despite high viremia. disease only develops upon transmission of siv to new non-natural hosts such as humans or asian macaques [21] . we have been studying the virologic and immunologic aspects of natural siv infection in a colony of siv-infected sms at the yerkes national primate research center [22] [23] [24] . although siv-infected sms are highly viremic, they manifest far lower levels of aberrant immune activation and apoptosis than are seen in pathogenic siv and hiv infections and maintain preserved t lymphocyte populations and regenerative capacity [22, 23] . studies of the sivsm viral variants obtained from different sms demonstrate magnitudes of inter-animal viral diversity similar to that observed with different hiv-1 group m subtypes [19] . variation in the viral surface proteins of zoonotic viruses is likely key to the ability of these agents to engage new host cell receptors and gain a foothold in new species. for influenza virus, amino acid changes and changes in glycosylation patterns in the viral hemagglutinin affect receptor binding specificity and host range [25, 26] . for the sars coronavirus (sars-cov) discreet variations in the spike protein are proposed to be important for viral tropism and animal-to-human transmission [27] . the hiv and siv envelope (env) proteins are extraordinarily genetically variable and highly glycosylated. hiv env has evolved to tolerate considerable aa sequence flexibility, including variation in n-glyc sites, and to conformationally shield key receptor-binding domains [28] . this genetic and functional flexibility enables env to escape from antibody responses and to utilize different co-receptors to gain efficient entry into target cells [29] [30] [31] [32] [33] [34] [35] . in our studies of the adaptation of sivsm from a naturally infected sm to a new simian host (rhesus macaques) we observed that one of three phylogenetically distinct env variants could replicate to high levels in the newly infected macaques. these variants encoded a shorter variable region 1 loop and lacked two specific n-linked glycosylation sites (n-glyc sites) [24] . the pre-existence of viral env variants in naturally infected sms that are capable of replicating to high levels in a new host species pointed to the importance of sivsm diversity in the reservoir host in enabling cross-species transmission. studies of zoonotic rna virus diversity have not focused on the variation that already exists in the source reservoir hosts; rather, the focus has largely been on the genetic variation and specific adaptive mutations that are observed in the newly emerged human pathogen [36, 37] . while adaptive mutations are critical for efficient host-to-host propagation in the newly-infected species, viral diversity that is already extant in reservoir hosts is another important source of the genetic variation necessary for successful cross-species transmission. here we describe extraordinarily high intra-host sivsm env v1v2 diversity in naturally infected sms, maintained by its high replication rate and positive selection most likely mediated by antibody responses. ongoing evolution of an extremely mutable siv env in the natural host explains the ease with which five naturally siv-infected sms (table 1) were sampled three times over a 2-year period. viral rna in plasma obtained in 3/99, 5/99, and 5/01 was measured by a realtime rt-pcr assay designed to quantitatively detect the diverse sivsm variants [23] . time points were chosen so that evolution could be assessed over both shorter and longer time intervals. viral load averaged 1.5 × 10 6 siv rna copies/ml plasma, and fluctuated modestly over the 2-year period ( figure 1 ). no clinical signs of aids were observed in any of the infected sms over the study period. multiple v1v2 env clones (range [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] and p27 gag clones (range [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] were sampled from each animal at each time point (genbank accession numbers ay733102-ay733566). env and gag were chosen for analysis since they were thought to represent the extremes of diversity in siv populations. these genes also differ in how the immune system detects them, with env v1v2 being exposed primarily to neutralizing antibodies [38] and gag p27 being recognized mostly through cellular immune responses [39] . the number of individual viral sequences analyzed (table 1 ) combined with the sampling of variants over a short time interval (2 months) and a longer time interval (2 years) exceeds that reported in previous studies of siv diversity in natural hosts [40] [41] [42] [43] . to characterize the overall evolutionary dynamics of natural siv variation, we built maximum likelihood trees of both env v1v2 ( figure 2a ) and gag p27 ( figure 2b ) sequences. the sivsm variants from each sm formed distinct clades in both genes, and the env and gag trees showed the same relationship between virus populations of the 5 animals. these results demonstrate that each host harbors a phylogenetically distinct population of sivs, presumably as the result of infection with distinct viral populations and subsequent host-specific viral evolution. viral load quantification for five naturally infected sooty mangabeys the translated env aa sequences (fjo, figure 3 ; data from all animals can be obtained from thv) demonstrate significant v1v2 heterogeneity, including heterogeneity in numerous predicted n-glyc sites (nxs/t, where x can be any aa but proline). considerable v1 length variations were observed (table 2 and for example, figure 3 ), such that alignment of this region required manual adjustment, and may not represent precise homology. there were no trends in v1v2 sequence length variation over time (data not shown). gag aa alignments (available from thv) showed significantly less aa variation reflecting its highly conserved nature. pairwise nt and aa diversity was calculated after removing regions of uncertain homology (gap-stripping) in v1, such that the values obtained for intra-host diversity represent minimum values. average pairwise aa diversity was high in env v1v2 (average: 5.6%, range: 0 and 37.7%; table 1 ) and low in gag p27 (average 1%; range: 0 and 7.1%, data not shown). the minimal diversity detected in gag, which was amplified under identical conditions, confirms that the observed v1v2 diversity is not the result of pcr-introduced mutation. in individual animals, the magnitude of nt and aa diversity did not change significantly over the 2-year observation period ( table 2) . however, there appeared to be animal-to-animal variation in the extent of v1v2 diversity, with animals ffj and fdo exhibiting lower v1v2 nt and aa diversity than fjo and fbo (anova p < 0.01, with bonferroni adjustment). nt and aa diversity were not correlated with viremia, suggesting that mechanisms other than or in addition to the magnitude of virus replication determine the extent of viral diversity. we cannot rule out that reduced diversity in ffj although the magnitude of sequence diversity did not change over time, it was likely that env sequences at later time points had diverged from those sampled earlier. to investigate the temporal pattern of sequence evolution within each animal, all available samples from all three time-points for each animal were pooled and analyzed by maximum likelihood (fig. 4; fqi) . sixteen of the nineteen (85%) bootstrap-supported clades from fqi contain variants from a single time point only. this pattern was repeatable amongst variants from all other animals; 100%, 80%, 69%, and 63% of bootstrap supported clades consisted of a single time point in animals fdo, ffj, fjo, and fbo, respectively. in an analysis of random trees, the number of matching time-point sequences that comprise a monophyletic group showed a poisson distribution; 86% of variants did not form monophyletic clades with any other matching time-point variant (i.e., these sequences stood alone). thus, the observed temporal clustering of sivsm viral populations does not occur by chance alone (kolmogorov-smirnov test, p < 0.01). temporal phylogenetic structure in v1v2 suggested that continual v1v2 diversification was occurring. to look for evidence of positive selection, dn and ds were calculated at each site and averaged over a 3-codon sliding window for viv2 (fig. 5a ) or 30-codon sliding window for p27 (fig. 5b) . these results confirmed that dn-ds>0 (p = 0.003, t-test) in v1 (aa's 25-55) in all animals, indicating positive selection. for p27, the same test showed that ds>dn along this gene (t-test, p < 0.001), indicating that purifying selection limits its diversity. v1 was consistently found to be under significant positive selection in all animals, except ffj (data not shown). by contrast, the few aa changes in p27 sequences in the different animals over time appeared random in nature except for a single partially fixed mutation in fdo. up to 10 n-glyc sites are contained within the sivsm v1v2 regions sequenced in this study. in multiple locations overlapping consensus motifs (aa's 42-44, 52-54, and 95-107) are present, such that the exact site of glycosylation varies (fig. 3) . these overlapping consensus motifs are in particularly diverse regions of v1v2 and in regions of strong positive selection. v1v2 clones from the five sms contained variable numbers of n-glyc sites, ranging from 3 to 10. the average number of n-glyc sites among all animals was 7.2. there was no clear pattern of increased or decreased v1v2 env glycosylation with time. however, the mean number of n-glyc sites for ffj and fdo (7.8 and 8.2, respectively) was significantly higher than the other animals (average between 6.5 and 6.9; anova, tukey b, p < 0.001). an additional n-glyc site is found in v1 in the majority of sequences in ffj and fdo at position 45, but not in the other animals. there was also a smaller range of n-glyc sites per set of sequences in ffj and fdo (6-9) compared to other animals (3-10). as described, the fdo and ffj sivsm populations were less diverse and had lower average dn compared to the virus populations found in the other 3 animals (table 2) . a significant inverse correlation between the mean number of n-glyc sites and both pairwise nt diversity and nonsynonymous substitutions was observed when combining data from all five sms (p < 0.001, fig. 6 ). env amino acid diversity of fjo sivsmm sequences may 1999 diversification of the hiv genome in humans underlies its success in evading pharmacologic and immunologic selection pressures, and likely facilitates human-tohuman transmission events. it has also been suggested that extensive virus diversification actually drives disease progression and the destruction of the immune system [44, 45] . to compare the sivsm genome diversity observed in natural hosts with that of hiv-1 in humans, longitudinally sampled env aa sequences from proviral dna representing 9 untreated, chronically hiv-infected humans [46] were compared to our plasma rna-derived sivsm env data. two time points were chosen from both the sm and the human dataset so that the interval between observations was approximately 2.5 years. for the comparison of nucleotide sequence diversity, homologous regions surrounding v1v2 were aligned and gap-stripped. average pairwise nucleotide diversity was calculated separately in each host at both time points (figure 7a ). measures of sivsm and hiv-1 nt diversity were not significantly different from each other within each time point ( figure 7b ; p > 0.05, mann-whitney u test). thus sivsm v1v2 sequence diversity in the natural sm host is at least as great as, if not greater than that observed in hiv-1-infected humans, especially given that the archival nature of proviral sequences may overestimate the diversity of the actively replicating viral rna population [47] [48] [49] . env adapts not only through raw nt sequence variability, but also through variation in both sequence length and nglyc site density and position. substantial changes in these phenotypic parameters will affect the ability of env to utilize different co-receptors [50, 51] , evade neutralizing antibodies [52, 53] and establish new infections in naïve hosts [54, 55] . to elucidate differences in sivsm and hiv-1 v1v2 sequence length and n-glyc site density variation, a pooled estimate of variance within each species was compared. neither the variances of sequence length nor glycosylation density differed significantly between species at time point 1 although although humans had a greater variance in both parameters at time point 2 (f max test, p < 0.01). the variance of sequence length of sivsm v1 decreased between the two time points (f max test, p < 0.005) suggesting that the magnitude of selection in sms shifts over time, while in humans the variance remained stable ( figure 7c ). the variation in glycosylation density ( figure 7d -e) remained relatively stable over time within both species except for a slight but non-significant expansion of variance in humans at time point 2. to identify viral characteristics that may explain how the sivs have successfully infected other primate species, we analyzed the types and extent of sivsm diversification in naturally infected sms. our findings of high intra-host extremes of sivsm v1v2 nt diversity extend previous studies of naturally siv-infected sms and african green monkeys (agms) [56] [57] [58] [59] [60] [61] [62] [63] by demonstrating that viruses found within a single animal can vary by greater than 35% at the aa level. the ranges of aa diversity in some intra-host pairwise sivsm v1v2 sequence comparisons in this study rival that of inter-animal comparisons [40] . as our diversity calculations exclude v1v2 length variation, they represent an underestimate of the true magnitude of viral diversity. v1v2 length polymorphisms would be predicted to have dramatic effects on sivsm env conformation and phenotypic diversity [64, 65] . positive selection in v1v2 appears to explain the observed env diversification. specific sites in v1 were consistently selected for in four of the five animals. our results agree with other studies of siv and hiv selection, in which dn-ds was consistently greater than 0 [66] [67] [68] . however, the majority of previous studies of nonpathogenic siv infection [56, 69, 70] detected temporal shifts in sivsm populations, some of which involved the gain or loss of n-glyc sites. beyond aa sequence variation, the extensive glycosylation of the hiv and siv envelope glycoprotein is thought to reduce protein epitope exposure and to facilitate viral evasion of antibody neutralization [28, 52, 53, 55] . ten potential n-glyc sites were recognized in the sivsm v1v2 region, with the average virus encoding 7.2 n-glyc sites. the neutralization resistant sivmac239 strain contains 8 predicted glycosylation sequences in the same region, while some other macaque-adapted sivs appear to have fewer n-glyc sites, especially in the v1 region [28] . thus, like sivcpz in a naturally infected chimpanzee [71] , sivsm appears to be highly glycosylated in naturally infected sms. presumably, continually evolving antibody responses in these natural hosts maintain a highly glycosylated surface protein, albeit without effectively suppressing virus replication. our observation of an inverse relationship between n-glyc site density and sivsm v1v2 sequence diversity might result from the more highly gly-cosylated viral variants being better shielded from the diversifying selection pressures of anti-siv antibodies than less glycosylated variants, as recently suggested for hiv [55] . thus, antibody-mediated pressures on the sivsm envelope glycoprotein appear to exist in this natural host reservoir species, and serve to continually select for adaptations in envelope sequence and structure. in contrast to env, sivsm gag p27 was under strong purifying selection in infected sms. temporal analyses of gag p27 demonstrated no evidence of the fixation of specific aa substitutions, suggesting that gag p27 is not the target of strong selective pressures such as those that might be expected if anti-gag cellular immune responses were present. these observations corroborate our findings that natural sm hosts mount limited cellular immune responses to siv infection [22, 23, 72] . comparison of our sivsm plasma rna-derived v1v2 sequences and a set of hiv-1 envelope sequences obtained from proviral dna [46] , while not the ideal glycosylation of sivsmm v1v2 is inversely correlated with pairwise nucleotide diversity figure 6 glycosylation of sivsmm v1v2 is inversely correlated with pairwise nucleotide diversity. comparison, demonstrates that natural sivsm v1v2 diversity is as great, if not greater than that observed in hiv-1infected humans. since average pairwise diversity is an indirect measure of viral effective population size [73] , these results suggest that an equivalent number of target cells are infected in both sm and human immunodeficiency virus infections. the similar levels of viral variation may also indicate that selective forces acting on env v1v2 are comparable in both sivsm-infected natural mangabey reservoir hosts and in hiv-infected humans. a caveat of these siv and hiv sequence comparisons is that this protein is quite divergent between the two viruses, and it is possible that this region of env could be under different functional and immune selection pressures in the two hosts. as v1v2 is primarily a target of the antibody response, it will be important to more thoroughly characterize in natural hosts sivsm variation in viral genome regions known to encode multiple cytotoxic t lymphocyte (ctl) epitopes in non-natural hosts (such as humans and macaques). such studies could help to elucidate the selective pressures exerted by the natural host on other genome regions and inform us as to the potential for genetic plasticity in viral genes that are targeted by current ctl-focused hiv vaccine strategies. the observation that high-level virus replication and extensive sequence diversification do not harm sms is consistent with the notion that the direct effects of siv replication are not sufficient to explain aids [44, 45, 74] . instead, our studies of natural host responses to infection indicate that indirect mechanisms, such as host inflammatory immune responses elicited by virus infection, likely play a role in the development of aids in new nonnatural hosts [22, 23] . because the humoral immune responses in naturally infected sms do not significantly suppress virus replication, they may actually serve to pro sooty mangabey human mote the continuous selection of env sequences and structures [75] . this helps to explain how the unique siv/hiv env structure has evolved in lower primates, resulting in a virus that is extremely difficult to neutralize [75, 76] . this continuous diversifying selection pressure likely also serves to generate variants with expanded cell tropisms that are well suited to adapt to new host cellular environments [24] . for instance, a spectrum of variant siv env conformations with differing requirements for the levels of cd4 on target cells might help to breach species differences in cd4 molecules, which are generally not as well conserved as the viral co-receptors such as ccr5 [77, 78] . thus, high viral variability and recombination within a natural reservoir host or host population will increase the likelihood that variants with the ability to replicate in new host species exist. the ongoing intra-host diversification of human-adapted rna viruses, such as hiv and hepatitis c virus, enables these viruses to continually respond to changing pressures, such as those imposed by immune responses and antiviral therapies, making treatment of these human diseases a formidable challenge [52, 79, 80] . the extent of intra-host sivsm env diversification in its natural reservoir likely underlies the ease with which certain sivs infect new host species [20, 24] . as new human pathogens emerge, much focus is placed on viral evolution in the newly infected hosts, such as adaptive mutations that facilitate robust replication and pathogenesis. however, our studies of sivsm demonstrate that an important source of viral variation and thus adaptive potential can be found within the viral populations of individual reservoir host animals. this extensive intra-animal viral variation, which is likely key to facilitating crossspecies transmission events, may be a common zoonotic signature among diverse emergent pathogens. five age-matched, naturally siv-infected sms from the colony at the yerkes national primate research center, atlanta, ga were chosen for study. individual animals were between 8 and 12 years of age and were estimated to have been infected for approximately 3 to 9 years, based on available hiv-2 seroconversion data. thus, all animals were born in, and acquired their sivsmm infection in, captivity. group housing of the animals confounds identification of potential donor-recipient pairs. plasma from animals fqi, fjo, ffj, fdo, and fbo was obtained on 3-13-99, 5-12-99, and 5-10-01 and viral rna was extracted and quantified using a real-time rt-pcr assay designed to quantitatively detect the diverse sivsmm variants [23] . viral rna was diluted such that approximately 2500 copies of viral rna were used in a superscript™ first-strand synthesis system for rt-pcr (invitrogen corporation, carlsbad, ca.), following the protocol provided, primed by random hexamers. 2 µl of cdna from the rt-pcr was used for pcr amplification of both env v1v2 and gag p27 with qiagen hotstar taq (qiagen inc., chatsworth, ca.). the env v1v2 region was amplified with the forward primer v1v2df (5'-tttgatgcntggaayaayac-3') corresponding to bp 6774-6792 of the sivsmmh4 genome (genbank accession no. x14307), and the reverse primer v1v2dr (5'-catagcatcccartartgctt-3') corresponding to bp 7217-7238 of the sivsmmh4 genome. the primer pair amplified a 421 bp fragment spanning the v1-v2 hypervariable region of envelope. the gag region was amplified using shortgagf1 (5'ttaagtccaagaa-cattaaatgc-3') and shortgagr (5'gtagaacctgtcta-catagct-3') which correspond to bp 1493-1515 and 19371957 of sivsmmh4, respectively, yielding a 421 bp product of the 5' end of the p27 capsid protein. primers were designed by choosing highly conserved regions from an alignment of all siv and hiv2 env and gag sequences from the hiv sequence database [81] . conditions for each reaction were 30 min. at 50°c, 15 min. at 95°c, followed by 40 cycles of 94°c for 1 min., 52°c for 30 s, and 72°c for 1 min. a final extension time was carried out for 5 min. at 72°c. no-template controls and negative controls from the rna extraction were used in each set of reactions, both rt and pcr, to ensure that no cross contamination occurred at either step. rt-pcr sensitivity was determined to be = 500 copies per reaction. cloning and dna sequencing pcr products from each sample were run on a 1.5% lowmelt agarose gel. the resulting 425 bp v1v2 or 421 bp gag product was extracted and cloned into the pcr4-topo vector (topo ta cloning kit, invitrogen). from rodrigo et al. [82] it was determined that if 2500 copies of viral rna are used in the rt-pcr reaction, 20 clones picked from the pcr product will be unique. therefore, approximately 20 clones from v1v2 and 10 from gag (due to lower expected diversity in this conserved gene) at each time point and each animal were randomly selected and sequenced using the m13f and m13r primers using the dye terminator cycle sequencing method with an mj research automated sequencer. sequences were aligned using the program clustal x [83] , followed by manual adjustment using macclade 4.0 [84] and bioedit sequence alignment editor [85] . nonaligned regions of length variation in v1 and v2 were removed (corresponding to nucleotides 6932-6974), and sequences containing internal stop codons or frame shifts were also excluded from analysis as these are thought to be pcr artifacts [86] . for tree construction, the modeltest program [87] was used to construct and evaluate the dna substitution models used. based on the modeltest results phylogenetic analysis on sequences obtained from successive time points during the acute infection was performed by maximum likelihood (ml) using the program treefinder [88] . the general-time-reversible model, which allows for rate variation between sites [89] [90] [91] , was used, and the shape parameter (α) of the gamma distribution used in this model was estimated, as were base frequencies and substitution rate parameters. bootstrap support was determined with 1,000 resamplings of the ml tree using distance methods in paup4.0b10*, incorporating the estimated rate parameters. phylogenetic trees were constructed from all clones obtained from v1v2 and gag and also separately on v1v2 and gag sequences obtained from each animal at each time point by maximum likelihood (ml) using the program treefinder. the cumulative number of nonsynonymous (dn) and synonymous (ds) nucleotide substitutions was estimated using snap, synonymous/non-synonymous analysis [81] which calculates rates of nucleotide substitution based on the method of nei and gojobori [92] , and incorporating a statistic developed in ota and nei [93] . viral diversity at each time point was determined by calculating the pairwise nucleotide distances for each of the clones using the method of tamura and nei [94] , and pairwise amino acid distances using the gamma distance method in the program mega 2.1 [95] . average dn and ds were calculated using the modified nei-gojobori method in mega 2.1. phylogenetic trees constructed with synonymous or nonsynonymous sites only were constructed in mega 2.1 using distance methods, incorporating the tamura-nei model of nucleotide substitution with gamma-distributed rates. all statistics were computed using systat 10. in order to show that viral populations do not vary randomly through time, random trees of all variants from each animal were generated and the number of matching time-point sequences that formed a monophyletic clade was counted for each random tree. for the random trees, the number of matching time-point sequences that comprise a monophyletic group are poisson distributed. the kolmogorov-smirnov test was used to compare our observed trees with those built from randomly sampled sequences. env nt sequences from 9 patients of a study of 10 hivinfected patients [46] were compared to our sivsm env data with respect to nt diversity, sequence length varia-tion, and predicted n-linked glycosylation site diversity. for v1v2 nt diversity comparisons, sequences from both sms and patients were aligned and stripped of gaps. pairwise estimates of intra-host nt diversity were calculated using mega 2.1 [96] . for sequence length variation, alignments (including gaps) of both sivsm and hiv-1 were pared down to the v1v2 region as defined by the flanking regions of extreme conservation. for this test, homology of each amino acid site was not as important as the overall homology of the region. mean-squared error variance was determined by anova in r [97] for both glycosylation density and sequence length in each species at each time point. variances were compared manually using an f max test. genbank accession nos: ay733102-ay733566 population biology of emerging and reemerging pathogens human viral infections: an expanding frontier. antivir res viruses at the edge of adaptation mutation rates and rapid evolution of rna viruses viral escape mechanisms-escapology taught by viruses h9n2 influenza a viruses from poultry in asia have human virus-like receptor specificity emergence of influenza a viruses receptor binding and membrane fusion in virus entry: the influenza hemagglutinin genetic analysis of the diversity and origin of hantaviruses in persomyscus luecopus mice in north america evolutionary diversification of proteincoding genes of hantaviruses temporal and spatial analysis of sin nombre virus quasispecies in naturally infected rodents hantavirus pulmonary syndrome: an emerging infectious disease serologic and genetic identification of peromyscus maniculatus as the primary rodent reservoir for a new hantavirus in the southwestern united states the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses epidemiology and the emergence of human immunodeficiency virus and acquired immune deficiency syndrome origins and evolution of aids viruses: estimating the time-scale origins and evolution of aids viruses tracing the origin and history of the hiv-2 epidemic molecular epidemiology of simian immunodeficiency virus sivsm in u.s. primate centers unravels the origin of sivmac and sivstm the history of sivs and aids: epidemiology, phylogeny and biology of isolates from naturally siv infected non-human primates (nhp) in africa aids as a zoonosis: scientific and public health implications staprans si: divergent host responses during primary sivsmm infection of natural mangabey and non-natural rhesus macaque hosts non-pathogenic simian immunodeficiency virus infection of sooty mangabey mokeys is characterized by limited bystander immunopathology despite chronic high-level viremia siv quasispecies adaptation to a simian new host monoclonal antibodies differentially affect the interaction between the hemagglutinin of h9 influenza virus escape mutants and sialic receptors sialobioloogy of influenza: molecular mechanism of host range variation of influenza viruses identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy korber b: tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin specific n-linked and o-linked glycosylation modifications in the envelope v1 domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies a role for carbohydrates in immune evasion in aids role of n-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response location-specific, unequal contribution of the n glycans in simian immunodeficiency virus gp120 to viral infectivity and removal of multiple glycans without disturbing infectivity antigenic variation of siv: mutations in v4 alter the neutralization profile hiv-1 evades antibodymediated neutralization through conformational masking of receptor-binding sites selection for neutralization resistance of the simian/human immunodeficiency virus shivsf33a variant in vivo by virtue of sequence changes in the extracellular envelope glycoprotein that modify n-linked glycosylation adaptation of sars coronavirus to humans the role of evolution in the emergence of infectious diseases neutralizing antibody responses drive the evolution of human immunodeficiency virus type 1 envelope during recent hiv infection clustering patterns of cytotoxic t-lymphocyte epitopes in human immunodeficiency virus type 1 (hiv-1) reveal imprints of immune evasion on hiv-1 global variation patterns of genomic sequence diversity among their simian immunodeficiency viruses suggest that l'hoest monkeys (cercopithecus lhoesti) are a natural lentivirus reservoir simian immunodeficiency virus (siv) from sun-tailed monkeys (ceropithecus solatus): evidence for host-dependent evolution of siv within the c. l'hoesti superspecies isolation of simian immunodeficiency viruses from two sooty mangabeys in cote d'ivoire: virological and genetic characterization and relationship to other hiv type 2 and sivsm/mac strains isolation and characterization of simian immunodeficiency viruses from two subspecies of african green monkeys antigenic diversity thresholds and the development of aids mathematical biology of hiv infections: antigenic variation and diversity threshold evolution of human immunodeficiency virus type 1 env sequence variation in patients with diverse rates of disease progression and t-cell function consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection the lymphocyte hiv reservoir in patients on long-term haart is a memory of virus evolution the reservoir for hiv-1 in human peripheral blood is a t cell that maintains expression of cd4 determinants within gp120 and gp41 contribute to cd4 independence of siv envs evolution of coreceptor use and cd4-independence in envelope clones derived from sivsm-infected macaques rapid evolution of the neutralizing antibody response to hiv type 1 infection antibody neutralization and escape by hiv-1 envelope-constrained neutralizationsensitive hiv-1 after heterosexual transmission the sequential introduction of hiv-1 subtype b and crf01_ae in singapore by sexual transmission: accelerated v3 region evolution in a subpopulation of asian crf01 viruses the evolutionary rate of nonpathogenic simian immunodeficiency virus (sivagm) is in agreement with a rapid and continuous replication in vivo simian immunodeficiency virus replicates to high levels in sooty mangabeys without inducing disease development in vivo of genetic variability of simian immunodeficiency virus simian immunodeficiency viruses from african green monkeys display unusual genetic diversity sivagm infection of its natural african geen monkey host simian immunodeficiency viruses of african green monkeys wide range of viral load in healthy african green monkeys naturally infected with simian immunodeficiency virus simian immunodeficiency virus replicates to high levels in naturally infected african green monkeys without inducing immunologic or neurologic disease temporal relationship between v1v2 variation, macrophage replication, and coreceptor adaptation during hiv-1 disease progression functional characterization of the v1v2 region of human immunodeficiency virus type 1 shortening of the symptom-free period in rhesus macaques is associated with decreasing nonsynonymous variation in the env variable regions of simian immunodeficiency viurs sivsm during passage evolution of envelope sequences from the genital tract peripheral blood of women infected with clade a human immunodeficiency virus type 1 evolution of human immunodeficiency virus type 1 sequences in infected individuals with differing disease progression profiles genetic differences accounting for evolution and pathogenicity of simian immunodeficiency virus from a sooty mangabey monkey after cross-species transmission to a pigtailed macaque genetic variation of the sivagm transmembrane glycoprotein in naturally and experimentally infected primates genetic variability of the v1 and v2 env domains of sivcpz-ant and neutralization pattern of plasma viruses in a chimpanzee infected naturally turnover of lymphocytes and conceptual paradigms in hiv infection coalescent approaches to hiv population genetics naturally siv-infected sooty mangabeys: are we closer to understanding why they do not develop aids? evolution of resistance to drugs in hiv-1-infected patients failing antiretroviral therapy the antigenic structure of the hivgp120 envelope glycoprotein structure and function of cc-chemokine receptor 5 homologues derived from representative primate species and subspecies of the taxonomic suborders prosimii and anthropoidea cloning and sequences of primate cd4 molecules: diversity of the cellular receptor for simian immunodeficiency virus/human immunodeficiency virus early changes in hepatitis c viral quasispecies during interferon therapy predict the therapeutic outcome the origin of quasispecies: cause or consequence of chronic hepatitis c viral infection? sampling and processing hiv molecular sequences: a computational evolutionary biologist's perspective. in computational and evolutionary analysis of hiv molecular sequences edited by: rodrigo ag, learn gh clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice interactive analysis of phylogeny and character evolution using the computer program mac-clade bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt long-term evolution of the hypervariable region of hepatitis c in a common-source-infected cohort modeltest: testing the model of dna substitution a general additive distance with time-reversibility and rate variation among nucleotide sites estimation of evolutionary distances under stationary and nonstationary models of nucleotide substitution estimating the pattern of nucleotide substitution simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions variance and covariances of the numbers of synonymous and nonsynonymous substitutions per site estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees molecular evolutionary genetic analysis. 2.0th edition mega2: molecular evolutionary genetics analysis software r: a language for data analysis and graphics we dedicate this paper to the memory of dr. h. mcclure, for his selfless devotion to advancing aids research in the nonhuman primate models, and for his genuine and warm collegiality. we also thank drs. f. novembre and s. garg for assistance with serologic and virologic measures, and dr. b. korber for helpful discussions. this work was supported by grant ai4915502 to m.b.f, and rr00165 to the yerkes primate center and niaid statistical training on aids grant t32-ai07442. siv, simian immunodeficiency virus; sm, sooty mangabey; rm, rhesus macaque; nt, nucleotide; aa, amino acid; ab, antibody; nab, neutralizing antibody. the author(s) declare that they have no competing interests. ljd, mbf, and sis conceived and designed the experiments. ljd carried out the reverse transcription, pcr, and cloning. jml contributed reagents and manpower for sequencing. ljd, thv, and jml conceived and performed statistical and phylogenetic analyses of the sequence data. ljd, thv, and sis wrote the manuscript. all authors read and approved the final manuscript. key: cord-320156-xs936r6u authors: nunes, marta c.; kuschner, zachary; rabede, zelda; cutland, clare l.; madimabe, richard; kuwanda, locadiah; klugman, keith p.; adrian, peter v.; madhi, shabir a. title: polyomaviruses-associated respiratory infections in hiv-infected and hiv-uninfected children date: 2014-10-28 journal: j clin virol doi: 10.1016/j.jcv.2014.10.013 sha: doc_id: 320156 cord_uid: xs936r6u background: two recently discovered polyomaviruses (pyv), wu and ki, have been identified in respiratory-tract specimens from children with acute respiratory infections, although there are limited data in hiv-infected children. objectives: to determine the prevalence and clinical manifestations of wupyv and kipyv-associated lower respiratory tract infections (lrtis) hospitalization in hiv-infected and -uninfected children; and probe the role of pneumococcal co-infection. study design: nasopharyngeal aspirates were collected from a cohort of 39,836 children randomized to receive 9-valent pneumococcal conjugate vaccine (pcv9) or placebo when hospitalized for lrtis, and were screened by pcr for wupyv, kipyv and other respiratory viruses. results: in placebo-recipients the prevalence of wupyv was 6.3% (18/285) in hiv-infected and 13.9% (66/476) in hiv-uninfected children (p = 0.002). in wupyv-positive lrtis hiv-infected children had lower oxygen saturation at admission and a higher case fatality rate (11.1% vs. 0%; p = 0.04). kipyv was identified in 10.2% (29/285) of hiv-infected and in 7.4% (35/476) of hiv-uninfected placebo-recipients with lrtis (p = 0.13). hiv-infected compared to hiv-uninfected children with kipyv-positive lrtis had lower oxygen saturation, higher respiratory rate and longer duration of hospitalization. co-infections with other respiratory-viruses were detected in 65.5% of wupyv-positive lrtis and in 75.0% of kipyv-positive lrtis. among hiv-uninfected children, there was a lower incidence of hospitalization for clinical pneumonia episodes in which kipyv (80%; 95% ci: 41, 93) and wupyv (49%; 95% ci: 9, 71) were identified among pcv9-recipients compared to placebo-recipients. conclusions: polyomaviruses were commonly identified in hiv-infected and -uninfected children hospitalized for lrtis, frequently in association with other viruses and may contribute to the pathogenesis of pneumococcal pneumonia. lower respiratory tract-infections (lrtis) are a major cause of hospitalizations during childhood [1] . determining pathogen specific causality of lrtis is hampered by lack of sensitive methods for diagnosing bacterial pneumonia, as well as the concurrent identification of multiple respiratory-viral pathogens, particularly when using molecular assays [2] . nevertheless, worldwide studies attribute a large proportion of lrtis to viral infections [3, 4] . largescale molecular screening based technologies have contributed to the discovery of new infectious pathogens, including in 2007 two polyomaviruses (pyv), wu-and ki-polyomavirus [5, 6] . these pyv belong to the polyomaviridae family and have been associated with respiratory disease in humans, although direct evidence of causality is lacking [7] . two other polyomaviruses (bkpyv and jcpyv) have been implicated in disease in immunocompromised patients [8] [9] [10] . there are, however, conflicting data with regard to the role of wupyv and kipyv in immunocompromised individuals [11] [12] [13] . previous studies have used pneumococcal-conjugate-vaccine (pcv) randomized placebo-controlled trials (rct) as a probe to establish the probability of co-infections by streptococcus pneumoniae (pneumococcus) vaccine-serotypes and respiratory-virus in children hospitalized for pneumonia [14, 15] . the rational of this approach is that any biological-plausible difference between pcv-and placebo-recipients in the incidence of any disease which could be associated with vaccine-serotype pneumococcal infection would indicate a role of vaccine-serotypes in the outcome of interest (for review [16] ). for example, we have previously reported that pcv-recipients, had a 45% lower incidence of pneumonia hospitalizations in which influenza-virus was identified, and as such concluded that at least a similar proportion of the influenza-associated pneumonias among placebo-recipients was precipitated by co-infection with pcv-serotypes [14] . the aim of this study was to determine the burden and clinical features of wupyv and kipyv infections in hiv-infected and hiv-uninfected children hospitalized for lrtis. furthermore, as an exploratory analysis we used the design of a rct of a 9-valent pcv (pcv9) to probe whether pneumococcal co-infection may contribute to hospitalization for pyv-associated pneumonia. we analyzed respiratory specimens collected from children who participated in rct in south africa as previously described [14, 17] . briefly, 39,836 children were randomized (1:1) from 1998 to 2000 to receive 3 doses of pcv9 or placebo [17] . hospitalbased surveillance for all-cause hospitalization was undertaken, all hospitalized children underwent hiv testing [17] . nasopharyngeal aspirates (npas) were obtained from children hospitalized with lrtis for identification of selected respiratory-viruses [14] and archived from february 2000 onward. in this study only npas collected from february 2000 to january 2002 from children <2 years old were analyzed. if a child had recurrent lrti hospitalizations, only npas collected >28 days apart were included in the analysis. these samples had been previously investigated for respiratory syncytial virus (rsv), influenza a/b, parainfluenza viruses (piv) and adenovirus using immunofluorescence and for humanmetapneumovirus (hmpv) by nested-pcr, as described [14, 15] . archived npas were tested by real-time reverse transcriptase-pcr (rrt-pcr) using the primers and probes as described [18] . we tested for wu-and ki-polyomavirus, as well as for human-bocavirus (hbov), human-rhinovirus (hrv), and four human-coronaviruses (cov). a comprehensive overview of the identified individual viruses has been reported separately [18] . the current analysis details the epidemiology of wupyv and kipyv-positive lrtis in the study cohort. the clinical definitions used in this study have been described [14] . the analyses on the epidemiology of wupyv and kipyv in children hospitalized for lrtis were restricted to placebo-recipients in the context of the initial trial [17] . proportions were compared by chi-square or fisher's exact tests and continuous variables by student's t-test or mann-whitney test. regression analyses were performed to compare clinical features between hiv-infected and -uninfected children. multiple regressions were controlled for age at hospitalization, detection of a virus previously-tested and year of collection. in an exploratory analysis using the concept of vaccine-probe studies [14, 19] , we explored whether there was any association between pcv9 and the risk of hospitalization for lrtis in which wupyv or kipyv were detected by estimating vaccine efficacy (ve) based on the formula: incidence rate in the unvaccinated − incidence rate in the vaccinated incidence rate in the unvaccinated × 100 children were included in the per-protocol analysis if they received all the study-vaccines as per planned schedule and the lrti event occurred >14 days after the third dose of study-vaccine. only the first episode of viral detection was included in the ve calculation for an individual participant. p-values <0.05 were considered significant. analyses were performed using stata version 12.1 (college station, tx, usa). a total of 1460 npas were analyzed by rrt-pcr, including 699 from pcv9-recipients (48.8%) and 761 (52.1%) from placeborecipients [18] . wupyv was detected less frequently in hiv-infected (6.3%) compared to hiv-uninfected children (13.9%, p = 0.002) ( table 1) . one hiv-infected and three hiv-uninfected children had at least two episodes of wupyv-positive lrtis more than 28 days apart. among hiv-uninfected children, wupyv was generally detected concurrently with other respiratory-viruses (72.7%), which was at a higher frequency compared to hiv-infected children (38.9%, p = 0.005) ( table 1 ). the most common co-detected viruses were hrv (31.8%, n = 21), hbov (16.7%, n = 11), rsv (12.1%, n = 8) and hmpv (10.6%, n = 7) in hiv-uninfected children; and hrv (27.8%, n = 5) and kipyv (16.7%, n = 3) in hiv-infected children. by multivariate analysis of wupyv-associated lrtis, hivinfected compared to hiv-uninfected children were more likely to have oxygen saturation <90%, present clinicallyas pneumonia f other laboratory investigations assessed that were not significantly different between hiv-infected and hiv-uninfected children included: percentage of children presenting with c-reactive protein levels ≥40 mg/l or procalcitonin levels ≥2 ng/ml and mean white cell count. g streptococcus pneumoniae was isolated from one hiv-infected child and in two hiv-uninfected children in whom wupyv was detected. h bacteria isolated from hiv-infected children in whom kipyv was detected included: streptococcus pneumoniae (n = 3), escherichia coli (n = 2) and haemophilus parainfluenzae (n = 1). rather than bronchiolitis. pneumococci were isolated by blood culture in one hiv-infected child and two hiv-uninfected children with wupyv-associated lrti (table 1) . hiv-infected (11.1%) compared to hiv-uninfected children (0%, p = 0.04) had a higher case fatality rate (cfr). the two hiv-infected children who died were 13 and 15 months old, both presented with pneumonia and wupyv was the only respiratory-virus identified, neither had evidence of bacteraemia nor pulmonary tuberculosis and the one child tested for pneumocystis jiroveci (pcp) infection was negative (table 2) . kipyv was detected in 10.2% of the specimens available from hiv-infected and in 7.4% from hiv-uninfected children (p = 0.13) ( table 1) . recurrent kipyv-associated lrti episodes occurred in five hiv-infected children, including three children with two episodes each, one child with 3 episodes and one child with 5 episodes. there was a single hiv-uninfected child with two kipyv-positive lrtis. identification of kipyv was perennial, albeit uncommon during november and december 2001 (fig. 1) . there was a lower prevalence of kipyv detection in 2000 compared to 2001 among hiv-infected (6.6% [13/198] kipyv was frequently detected in combination with other respiratory-viruses in both hiv-infected (75.9%) and hivuninfected children (74.3%) ( table 1 ). in hiv-infected the most common co-detected viruses included hrv (51.7%, n = 15), wupyv and hmpv (10.3%, n = 3 each). in hiv-uninfected the most common viruses associated with kipyv were hrv (31.4%, n = 11), hmpv (14.3%, n = 5) and wupyv (11.4%, n = 4). hiv-infected compared to hiv-uninfected children with kipyvassociated lrtis were more likely to have oxygen saturation <90%, higher respiratory rate, longer duration of hospitalization (median [range]: 5.0 days vs. 1.0 day [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] ; p = 0.008) and were more likely to have alveolar consolidation on chest x-ray (cxr-ac) and concurrent bacteraemia (table 1) . pneumococci were isolated from three hiv-infected children. four hiv-infected (13.8%) and two hiv-uninfected (5.7%) children in whom kipyv was detected died during hospitalization. hiv-uninfected children with kipyv-associated lrti who died compared with those who survived were younger (3 vs. 12 months; p = 0.03). both hiv-uninfected children, aged 3 months, presented with pneumonia, had co-infections with other viruses and one child (who was malnourished) also had pcp infection (table 2) . of the four hiv-infected children who died, kipyv was the only respiratory-virus identified in two of them although at least one was co-infected with pcp. overall, five of the six children who died had indirect markers of bacterial infection, including three hiv-infected with raised c-reactive protein and procalcitonin levels (two of whom also had escherichia coli bacteraemia) and the other two (one hiv-infected and one hiv-uninfected) with cxr-ac ( table 2) . as an exploratory analysis the effect of pcv9-vaccination on the incidence of polyomavirus-associated pneumonia hospitalizations was evaluated. in fully vaccinated hiv-uninfected children, the incidence of wupyv-positive clinical pneumonia hospitalizations was 48.5% (95% ci: 9.1, 70.8) lower in children who received pcv9 compared to placebo-recipients (table 3) . a similar ve pointestimate was observed in cases restricted to episodes in which wupyv was the sole detected virus (45.4%, 95% ci: −47.6, 79.8). there were also reductions in the incidence of hospitalization for kipyv-associated clinical pneumonia in pcv9-recipients compared to placebo-recipients overall (51.3%, 95% ci: 14.5, 72.3) and specific in hiv-uninfected children (80.0%, 95% ci: 41.4, 93.2). similar reductions were observed when kipyv was the only identified virus (table 3) . no differences in incidence were observed between pcv9-and placebo-recipients in hospitalizations for wupyv-or ki-associated clinical pneumonia in hiv-infected children and in hospitalizations for wupyv-or kipyv-associated bronchiolitis. to our knowledge, this is the most detailed report on the prevalence and clinical features of lrtis where polyomaviruses were detected in hiv-infected and -uninfected children. among hospitalized hiv-infected children, wupyv was detected in 6% of the cases and kipyv in 10%. in hiv-uninfected children the prevalence of wupyv was 14% and of kipyv was 7%. in hiv-infected children 11-14% of the cases positive for at least one polyomavirus were fatal and in 67% (4 cases in total) of these wupyv or kipyv were identified as single-detections. our study also established a possible interaction between polyomaviruses and pneumococcus in hiv-uninfected children, although this may have been masked in hiv-infected children in whom pcv9-vaccination was demonstrated to be less efficacious against pneumonia [17] . wupyv was detected in 9% of respiratory samples from children presenting with upper or lrti in germany, 7% in south korea and south africa and 6% in thailand; kipyv has been identified in 1-6% of children presenting with respiratory-tract infections [12, 13, [20] [21] [22] . another study from south africa reported that 57% of 21 children with lrtis with wupyv and 33% of 3 with kipyv were hiv-infected, however, the hiv-status was unknown in 50% of the cases [12] . in that same study, both polyomaviruses were table 3 differences in incidence of wupyv and kipyv-associated lower respiratory tract infections between fully-immunized children who received 9-valent pneumococcal conjugated vaccine and placebo recipients; per-protocol analysis. absent among 50 healthy immunocompetent controls [12] . the role of wu/kipyv as more serious pathogens in immunocompromised individuals is uncertain. higher viral loads of pyv were documented in lymphoid tissues and in the brains of hiv-infected adults compared to those from immunocompetent individuals [23, 24] . similarly higher kipyv viral loads were found in respiratory-tract specimens from haematology/oncology paediatric patients with respiratory infections, however, whether the viral loads correlated with disease severity was not established [13] . these observations suggest that there may be more viral replication and/or that polyomaviruses might reactivate in immunosuppressed individuals, implying that t-cell impairment might be a factor in facilitating polyomavirus replication. norja et al. found that wu/kipyv detection on respiratory samples occurred predominantly in two groups of subjects: immunocompetent <2 years of age with lrtis (7%) in whom there was a high frequency of co-infection (75%), and among older, generally immunocompromised individuals without respiratory illness (11%) or with mild upper respiratory-tract infections (7%) [7] . although our study was not designed to establish whether polyomavirus infections caused more severe disease in hiv-infected children, among hospitalized children in whom polyomaviruses were detected hiv-infected were more likely to present with pneumonia rather than bronchiolitis, had a longer duration of hospitalization and higher cfr compared to hiv-uninfected children. similar observations have been detected between hiv-infected and hiv-uninfected children for other viruses [18] , and consequently may not necessarily infer causality for polyomaviruses precipitate more severe illness in hiv-infected children. the increased morbidity and mortality in hiv-infected children could have been related to other co-morbidities such as bacterial or other non-viral co-infections. in the absence of sensitive tools for diagnosing bacterial pneumonia, as well as lack of investigating for other non-viral causes of pneumonia, our study is unable to conclude the actual role of polyomaviruses to more severe disease in hiv-infected children. nonetheless, the higher cfr, as well as that four fatal cases with polyomavirus as the sole respiratory-virus detected were all hiv-infected children suggest a possible association of polyomavirus causing severe disease in hiv-infected. furthermore, five hiv-infected children had recurrent kipyv-positive lrtis compared to only one hiv-uninfected child. this could have been due to extended viral shedding, viral reactivation or reinfection. a study from germany detected kipyv dna in the respiratory-tract of an immunocompromised child for 7 months and hypothesized that a kipyv infection during childhood could result in latency of the virus in normal individuals, however, an immune impairment could result in viral reactivation [25] . in 2001 the detection rate of kipyv in hiv-infected children was higher than in 2000 this may be because of the older cohort in 2001, hence possibly different risk of infection, or differences in year-to-year epidemics. the concept of a vaccine-probe analysis was initially used in attributing the contribution of haemophilus influenza-type b in the aetiology of radiological-confirmed pneumonia, as well as subsequently in pcv trials [14, 16, 19] . we have used the same approach in probing the likelihood of co-infection by pcv9-serotypes in children hospitalized for pneumonia associated with influenza virus, piv and hmpv [14, 15] ; and we demonstrated that 44-58% of children hospitalized for pneumonia-associated with these viruses were likely to have pneumococcus co-infection [14, 15] . using this type of analysis, our study suggests that co-infections with pcv9serotypes contribute to hospitalizations for clinical pneumonia in which wu/kipyv were identified. the imputed rate of coinfection of pneumococci in children with polyomaviruses-positive pneumonia from our study provides a conservative estimate of this possible interaction as only 9 serotypes were included in the vaccine and ve even against vaccine-serotypes pneumococcal pneumonia was not 100%. the high ve estimate for kipyv-positive disease needs to be contextualized within the wide uncertainty bounds of this estimate. the suggested interaction between polyomaviruses and pneumococcus warrants further study. high co-infection rates with other respiratory-viruses were found for wupyv and kipyv which are similar to previous reports [6, 13, 20] . in the absence of a control group of children without lrtis a definitive causal relationship between polyomavirus detection and disease could not be inferred and this constitutes a limitation of our study. another limitation of our study is that we relied on npas, where identification of an organism does not necessarily imply infection. also in our study the hiv-infected children were not treated with anti-retroviral treatment (art), which may have contributed to their clinical course and may differ to hiv-infected children treated with art. determining the aetiology of pneumonia remains a challenge, especially in children and the pathogenic potential of some of the newly-described respiratory-viruses is difficult to address in the context of multiple infections and without specific symptoms. although polyomaviruses were frequently detected in children hospitalized for lrtis in our study, further studies which include autopsy samples from fatal lrti cases, lung aspirate ant-mortem and also enrolment of healthy controls [26] , are required to clarify the role of polyomaviruses in the pathogenesis of pneumonia. global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis etiology of community-acquired pneumonia in 254 hospitalized children viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis viral pneumonia identification of a third human polyomavirus identification of a novel polyomavirus from patients with acute respiratory tract infections no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease human polyomavirus (bk) infection and ureteric stenosis in renal allograft recipients renal failure due to bk virus infection in an immunodeficient child cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy polyomaviruses ki and wu in immunocompromised patients with respiratory disease human polyomaviruses: wu and ki in hiv exposed children with acute lower respiratory tract infections in hospitals in south africa wu and ki polyomavirus infections in pediatric hematology/oncology patients with acute respiratory tract illness a role for streptococcus pneumoniae in virus-associated pneumonia pneumococcal coinfection with human metapneumovirus use of vaccines as probes to define disease burden a trial of a 9-valent pneumococcal conjugate vaccine in children with and those without hiv infection clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in hiv-infected and hiv-uninfected south african children randomised trial of haemophilus influenzae type-b tetanus protein conjugate vaccine [corrected] for prevention of pneumonia and meningitis in gambian infants presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection wu polyomavirus in children with acute lower respiratory tract infections prevalence and molecular characterization of wu/ki polyomaviruses isolated from pediatric patients with respiratory disease in thailand reactivation and mutation of newly discovered wu: ki, and merkel cell carcinoma polyomaviruses in immunosuppressed individuals wu and ki polyomaviruses in the brains of hiv-positive patients with and without progressive multifocal leukoencephalopathy prolonged ki polyomavirus infection in immunodeficient child pneumonia etiology research for child health. introduction this work is based upon research supported in-part by the south african research chairs initiative of the department of science and technology (dst) and national research foundation (nrf) in vaccine preventable diseases. additional funding support was received from the national health laboratory service research fund and medical research council (respiratory and meningeal pathogens research unit). any opinion, findings and conclusions or recommendations expressed in this material are those of the author(s) and therefore the nrf and dst do not accept any liability with regard thereto. mcn had financial support from the university of the witwatersrand. the authors thank the essential contribution of the members of the vaccine trialist group [17] for their involvement in the original study, all the trial participants and all rmpru staff involved in the study. the authors also thank john w. rossen for technical assistance and biomérieux south africa for providing reagents. the authors have no conflicts of interest to disclose. the main efficacy trial and subsequent retrospective analyses were approved by the human research ethics committee (hrec) of the university of the witwatersrand. signed written informed consent was obtained from the parent/legal guardians as part of the trial. hrec did not require additional consent for this analysis. the main study was not registered under any clinical trial registry as it was undertaken prior to registration being mandatory. key: cord-305039-grsv06j7 authors: flego, michela; ascione, alessandro; cianfriglia, maurizio; vella, stefano title: clinical development of monoclonal antibody-based drugs in hiv and hcv diseases date: 2013-01-04 journal: bmc med doi: 10.1186/1741-7015-11-4 sha: doc_id: 305039 cord_uid: grsv06j7 today there are many licensed antiviral drugs, but the emergence of drug resistant strains sometimes invalidates the effects of the current therapies used in the treatment of infectious diseases. compared to conventional antiviral drugs, monoclonal antibodies (mabs) used as pharmacological molecules have particular physical characteristics and modes of action, and, therefore, they should be considered as a distinct therapeutic class. despite being historically validated, antibodies may represent a novel tool for combatting infectious diseases. the current high cost of mabs' production, storage and administration (by injection only) and the consequent obstacles to development are outweighed by mabs' clinical advantages. these are related to a low toxicity combined with high specificity and versatility, which allows a specific antibody to mediate various biological effects, ranging from the virus neutralization mechanisms to the modulation of immune responses. this review briefly summarizes the recent technological advances in the field of immunoglobulin research, and the current status of mab-based drugs in clinical trials for hiv and hcv diseases. for each clinical trial the available data are reported and the emerging conceptual problems of the employed mabs are highlighted. this overview helps to give a clear picture of the efficacy and challenges of the mabs in the field of these two infectious diseases which have such a global impact. the innate immune response is the first-line defense in determining the outcome of an infection. infectious agents contain conserved motifs on their surface that react with conserved pattern recognition toll-like receptors of the host. this interaction initiates a powerful innate immune response. moreover, the infectious agent's surface proteins and carbohydrates come into contact with b-cell receptors, membrane-bound immunoglobulin of isotype m (igm) or d (igd), and often induce potent antibody responses, which take some weeks to fully develop [1] . when a vertebrate organism encounters a pathogen, such as a virus or bacteria, it generates a polyclonal antibody response against numerous epitopes on different antigens during infection; therefore, polyclonal serum contains a large and diverse population of antibodies, which also include neutralizing antibodies (nabs). thus, polyclonal serum-derived biotherapeutic products can contain various nabs against multiple and distinct epitopes; these nabs provide strong protective activity due to additive or even synergistic effects on neutralization. however, in this type of product the vast majority of their constituent specific antibodies are non-neutralizing, since they are directed against misfolded protein or against epitopes on native surface proteins for which antibody binding is not protective [2, 3] . furthermore, for some viral and bacterial infections, no correlates of protection have been established; therefore, the significance of antibody titers, apart from indicating past exposure, is not clear. mechanisms of immunological escape can explain why total antibody titers are not always protective. many infectious organisms, including viruses, can constantly mutate surface proteins and exploit glycans to shield important epitopes, diverting the antibody response away from functionally important epitopes in favor of immunogenic irrelevant epitopes [4] . thanks to their protective properties, the administration of hyperimmune sera from immunized animals or immune human donors, named 'serum therapy', was the first effective treatment of infectious diseases. later, the advent of antibiotic therapy with the advances in vaccine design has meant that serum therapy was almost abandoned for many infectious diseases. nevertheless, hyperimmune human sera immunoglobulin preparations are still used to treat different bacterial toxins and virus related diseases, including those caused by cytomegalovirus (cmv), respiratory syncytial virus (rsv), hepatitis a virus (hav), hepatitis b virus (hbv), rabies, vaccinia, vesicular stomatitis virus (vsv) and measles, underscoring the fact that antibody therapy remains an effective means of treatment [5, 6] . today, the ability to rapidly generate and manipulate antibodies with a defined epitope recognition, named "monoclonal antibodies" (mabs) (figure 1 ), has opened a new window of opportunity for a rematch of antibodies in clinical practice. this achievement has been possible thanks to advances in cellular biology and biotechnology (figure 2) , and also to improved purification techniques which have made these therapeutics safer, less immunogenic and more effective. mab preparations have many advantages over immune sera-derived preparations which can vary due to both time and the source of origin, since different hosts mount different antibody responses. one advantage is that mabs, by virtue of the fact that they are chemically defined reagents, exhibit relatively low lot-tolot variability and low risk of pathogen transmission. another advantage for mab preparations is the much figure 1 schematic structure of a mab. all immunoglobulins are composed of two identical light (l) chains and two identical heavy (h) chains, linked by disulphide bonds (black dashed bars). the heavy chains contain one variable domain (vh) and three or four constant domains (ch1, ch2, ch3 and ch4) depending on antibody isotype. by contrast, the light chains contain only one variable domain (vl) and a single constant domain (cl). within the fab region, at the end of the two arms of the y-shaped molecule, the variable domain of a heavy chain pairs with the light chain variable domain to form the antigen-binding site. in more detail, within the matched v regions, three short polypeptide segments on the heavy chain and three on the light chain form the complementarity-determining regions (cdrs), which dictate the precise antigen-binding characteristics of the antibody. on the other end, the fc domain, which includes the sites for interaction with the complement system and fc receptors, mediates effector functions determining the fate of the bound antigen. greater activity per mass of protein since all the ig molecules are specific for the desired target. this phenomenon is illustrated by the report that two 0.7 mg doses of two mabs provided the same protection against tetanus toxin as 100 to 170 mg of tetanus immunoglobulins [7] . neither does mab therapy have the immunological complications associated with the use of heterologous sera in humans, such as serum sickness and immediate hypersensitivity, which significantly limited the latter's usefulness [8] . in recent years, mabs have emerged as a new class of biological drugs in oncology as well as in immune and inflammatory diseases, albeit their development in infectious diseases has been slower. to date, the only mab approved in this field is palivizumab, an anti-rsv mab licensed for prevention of severe respiratory disease in high-risk infants and immunocompromised adults. now the scenario is gradually changing and there are many antibodies against viruses and bacteria in various stages of clinical development. this trend has also been influenced by the development of different scientific disciplines, which makes it possible to study and dissect the function of individual microbial structures supporting figure 2 evolution of mabs linked to the need to decrease their immunogenicity. different methods to obtain mabs are depicted. mouse mabs, the 'hybridoma' cells derived from the stable fusion of immortalized mouse myeloma cells with lymphocytes from immunized mice, are screened to identify individual clones producing identical antibody to a single antigenic determinant [118] . chimeric mabs, the murine constant regions of both heavy and light antibody chains (mch and mcl), are replaced with human counterparts (hch1, hch2, hch3 and hcl1), leaving intact the murine variable portions (mvh and mvl) [119] . humanized mabs, only the cdrs of the murine mabs (mcdrs) from both the mvh and the mvl, are 'grafted' into a human backbone antibody [120, 121] . human mabs, 1)human memory b-cells isolated from patients are immortalized by epstein barr virus (ebv) and cpg oligodeoxynucleotide, and then screened for specific antibody production [122] . 2) transgenic mice, obtained by a genetic replacement of the mouse immunoglobulin genes with human counterparts, are used to obtain fully human mabs by traditional hybridoma technology [123] . 3) antibody libraries, constructed by in vitro combinatorial assembly of human immunoglobulin variable-region gene (v genes) and cloned to provide the display onto phage surfaces, are subjected to a panning against an antigen in order to select specific clones [124] . the first mabs of each category approved for clinical use are shown. palivizumab is the first and, so far, only mab approved for infectious diseases. the endings used to name the different types of mabs are also indicated. the development of more targeted drugs. there are excellent reviews about this topic [6, 9] . in this review we focus on the mab-therapies now underway in clinical trials (table 1 ) designed for human immunodeficiency virus (hiv) and hepatitis c virus (hcv) infectious diseases. both these worldwide epidemics require new strategies due to the lack of a definitive cure and effective vaccines, to the continuous emergence of drug resistant variants, to the toxicities of licensed drugs and to the need to ensure a treatment for all patients. in this context, antibodies represent an intriguing alternative as therapeutics; in their favor are their different resistance mechanisms and a more favorable toxicity profile when compared to other available drug classes, fitting them for use in conjunction with the current chemotherapy by slowing the onset of resistance and possibly enhancing therapeutic efficacy. the antibody structure comprises a pair of identical heavy and light chains linked by disulphide bonds held in a yshaped arrangement (figure 1 ). the fragment antigen-binding (fab) portion, the region that binds the antigen, is composed of one variable and one constant domain of both the heavy and the light chain. the remaining constant sections of the longer heavy chains form the tail of the y, termed the crystallizable fragment (fc) region, which provides the signal for effector functions. antibodies can provide protective effects through various mechanisms [10] . viral neutralization is generally meant as the ability of an antibody to provide sufficient steric interference to disrupt the interaction between a microbic antigen and its ligand in experimental conditions in vitro. this activity is clearly associated with protection, thanks to their fab domain alone, both in natural infection and after immunization. virus infection includes sequential steps beginning with attachment to cell-surface receptors and ending with delivery of the viral genetic material into the cytoplasm [11] . fusion of viral and cellular membranes is a basic entry mode for enveloped viruses, such as hiv and hcv [12] , which still differ in specific aspects of viral entry and assembly, thus offering unique therapeutic opportunities. the cell surface is certainly more directly accessible for the action of the antibodies; therefore, the phase of virus entry is one of the most important targets in preventing viral infection at the origin, and many known nabs act at this step. for the same reason, inhibition of the release of progeny virus is another possible mechanism of neutralization, as demonstrated by antibodies directed against influenza a virion surface neuraminidase [13] . in hiv and hcv fields, no virus release inhibiting antibodies have been identified to date. the interaction of hiv envelope surface protein gp120 with its host receptor, cd4, on human t cells triggers conformational changes in the envelope, resulting in exposure of a transient binding site for co-receptor ccr5 or cxcr4. this in turn promotes additional conformational changes in virus gp41 protein which allow it to insert its fusion peptide into the target cell membrane to initiate membrane fusion and viral entry into host cells. nabs can inhibit viral infection by several different mechanisms in parallel with the steps that allow the viruses to enter into cells ( figure 3 ). they can directly block virus attachment to target cells by interfering with virus-receptor interactions, as in the case of nabs against the cd4-binding site on hiv gp120 [14] . this same goal can also be achieved by directing the antibodies to the virus receptor and/or co-receptor on host cells. mabs can also block fusion at the cell membrane at the post-binding/pre-fusion stage, as exemplified by anti-cd4 [15] and/or anti-ccr5/cxcr4 (ccmotif receptor 5/cxc-motif receptor 4) mabs, under development [16] . again, mabs directed to the external proximal membrane region of hiv gp41 can interfere with conformational changes needed for membrane fusion [17] . unlike hiv, hcv entry into target cells occurs via clathrin-mediated endocytosis of the viral particle [18] . subsequent release of the viral genome into the cytosol requires the ph-dependent fusion of viral and cellular membranes. current models suggest that hcv circulate as lipoviral-particles (lvps) in the vascular system, these consisting of lipoproteins in complex with virus particles. following localization to the surface of hepatocytes through interactions of lvps with glycosaminoglycans and the low density lipoprotein receptor, specific binding of the e1 and e2 virus surface glycoproteins with the host sr-b1 scavenger receptor and cd81 occur [19] . subsequently, viral particles are translocated to regions of the membrane possessing tight junction proteins occludin and claudins; the binding to these receptors results in clathrin-mediated endocytosis. as for hiv, mabs directed against spike viral proteins, as well as against host receptors, may act at an early stage of infection by preventing the binding of the virus on the cell surface. for example, antibodies recognizing the cd81-binding site within the envelope glycoprotein e2 have been shown to block viral entry, as have a number of anti-receptor antibodies targeting cd81 [20, 21] and sr-bi [22] . some antibodies may act by blocking conformational changes and/or the requisite interactions between the viral and endosomal membranes required for fusion; although as yet no fusion determinant within the envelope glycoproteins has been defined. host protection in vivo is more complex and involves the interaction of antibodies with cells and molecules of the innate immune system. the antibody can exert protective actions through an fc region-mediated recruitment of other components of the immune system, including antibody-dependent cell-mediated cytotoxicity (adcc), complement-dependent cytotoxicity (cdc) and antibody-dependent cellular phagocytosis. receptors for the fc segment of igg (fcy receptors; fcγrs) are expressed on the surface of different types of cells, including natural killer cells (nk), monocytes, macrophages, dendritic cells and neutrophils. with the exception of γδt cells, fcγrs are not normally found on t lymphocytes. similarly, the receptor for fc segment of iga, the fcαr, involved in phagocytosis and induction of microbe killing, is expressed on monocytes, macrophages and neutrophils [23] . the adcc process is triggered by the interaction between the fc region of an antibody bound to a nonself antigen exposed on host cells, and the fc receptors on immune effector cells. the subsequent release of cytokines and cytotoxic granules containing perforins and granzymes promotes the death of the target cell. cdc is initiated by complement component c1q binding to the fc region of igg, which is in turn bound to the foreign antigen on the cell surface. this triggers a proteolytic cascade to activate the complement, so leading to the formation of a membrane attack complex that kills the target cell by disrupting its cell membrane. the fc region can also mediate complement binding to and deposition on free virions, which can cause a direct virotoxic effect or inhibit virus binding to cells. moreover, the so-called opsonization process, consisting of the binding of antibody fab portion to the antigen following by the interaction of fc domain to an fc receptor on phagocytes, is a powerful mechanism to enhance the phagocytosis [9] . with respect to hiv, a potential role for adcc in modulating the course of hiv infection was first proposed on the basis of studies showing an inverse association between adcc antibody levels and the clinical stage of disease. the strongest evidence for a role for adcc antibody in disease progression comes from a study by baum et al. of the multicenter aids cohort study [24] . in that study, rapid progressors had significantly lower adcc antibody titers against cem. nkr cells coated with gp120 than did non-rapid progressors at corresponding visits or non-progressors at any visit. morever, hiv-infected individuals with spontaneously undetectable viremia were shown to have higher adcc antibody levels than viremic subjects [25] . in the context of hcv infection, fc-mediated effector functions, although less well understood, can still have an important role. sera from both the acute and chronic phase of infection can mediate adcc via binding to viral protein e2 expressed at the cell surface [26] , while several e2-specific mabs are able to induce cdc of e2-expressing cells [27] . optimizing non-nab effector functions, such as adcc, cdc and fagogocytosis, may prove critical in the design of new effective anti-hcv therapeutic antibodies [28] . (1), as well as by binding to the viral receptor or co-receptor on host cell surface (2) . some antibodies, can neutralize viral infection through interfering with conformational changes required for membrane fusion and subsequent release of the viral core into the target-cell cytoplasm; this postbinding neutralization may occur at the cell surface (3), or inside the endosomes for the viruses (for example, hcv) whose entry into the cell requires an endocytosis step (4). antibodies recognizing viral or host proteins expressed on infected cell surface can exert protective actions through the fc-mediated effector functions (for example, cdc, adcc) (5). again, mabs may prevent the release of progeny virions (6) . at the bottom the antibody neutralizing effects on the viruses before cell binding, including the direct virolysis by cdc and the mab-mediated enhanced phagocitosis, are shown (7) . in panel b, the possible mab-mediated immunomodulary therapies are depicted. in some chronic viral infections, virus-specific immune cells may persist in a 'non-functional' state, because of an imbalance of immunoregulatory signals involving multiple inhibitory and activating receptors, triggered by soluble factors and/or cell surface ligands. therapeutic approaches using specific mabs to block host immunosuppressive molecules (antagonism) or to trigger activating receptors (agonism) may be a valid strategy to restore immune cell function and treat various chronic viral infections. system is the exhaustion of virus-specific t cells. exhaustion consists of a progressive dysfunction characterized by the inability to proliferate and to produce key antiviral and immune stimulating cytokines (for example, interleukin (il)-2, tumor necrosis factor (tnf)-a, interferon (ifn)-γ), or to lyse infected cells [29] . a feature of functional exhaustion is that it affects many antiviral properties of both mouse and human cd8+ t cells. loss of effector functions proceeds in a hierarchical manner starting with defects in il-2 production and proliferation, followed by the decrease of tnf production. cytotoxic activity is also lacking in exhausted human cd8 + t cells. at a severe stage of exhaustion, ifn-γ production is eventually compromised, with exhausted t cells ending up deleted if the high antigenic load persists [30] . exhaustion can also occur in cd4+ t cells in both mice [31] and humans [32] . probably the best explanation for this progressive dysfunction and loss of effector t cells is the continuous triggering of virus-specific t cell receptors owing to a high antigenic load in persistently infected hosts without a critical rest period. the current consensus is that functional exhaustion is a way of limiting the magnitude of effector t cell responses. although this may safeguard against autoimmune responses, it may also compromise effective immunity against persistent infectious agents and tumors [29] . exhausted t cells are subject to complex layers of negative regulation. this involves signaling through multiple inhibitory receptors that inhibit functional and proliferative responses. the cd28 family member programmed cell death 1 (pd-1) has been shown to be the most highly expressed inhibitory receptor on cd8+ t cells during chronic infection, and to have a major role in regulating t cell exhaustion during infection [33, 34] . increased expression of pd-1 by t cells also occurs during hbv and hcv infections [35] [36] [37] . several other inhibitory receptors have also been shown to induce t cell unresponsiveness during chronic infections. these receptors include cytotoxic t lymphocyte antigen 4 (ctla-4) [31, 38, 39] , t cell immunoglobulin domain and mucin domain protein 3 (tim3) [40, 41] , and lymphocyte activation gene 3 (lag-3) [38] . in addition, certain cytokines, such as il-10 and transforming growth factor-β (tgfβ) as well as regulatory t cells, may also contribute to the lack of t cell functionality during situations of high antigenic burden [42] . there is intriguing evidence that blockade of the inhibitory receptor could restore antigen t cell responses. for example, blockade of the pd-1 signaling pathway improves antigen-specific t cell proliferation and cytokine secretion in lymphocytic choriomeningitis virus (lcmv)-infected mice [31, 34] and in humans with chronic hiv [32, 43, 44] , hbv [45] and hcv [36] infections. this effect was synergistically improved in lcmv infected mouse following the simultaneous blockade of the t cell inhibitory receptors pd-1 and lag-3, thanks to which a diminished viral load in vivo was observed, although blocking lag-3 pathway alone had little effect on the severity of exhaustion [34] . moreover, mabmediated blocking of ctla-4 pathway in vitro augments hiv-specific cd4+ t-cell function suggesting that the immune modulation of this target may also provide a clinical benefit in infected individuals [39] . another example is the manipulation of signals mediated by glucocorticoid-induced tnf receptor (gitr), a recently identified member of the tnf receptor superfamily, preferentially expressed on subset cd4 +cd25+ regulatory t cells. gitr signals break the suppressive activity of this subset. in fact, an agonistic anti-gitr mab immediately injected after viral infection significantly increased the number of activated cd4+ and cd8+ t cells secreting ifn-γ [46] . one must remember that the manipulation of immunological responses could have detrimental effects on the host, as highlighted by the recent tragic human trial of tgn1412. this is a mab against human t cell costimulatory molecule cd28 developed by tegenero to treat b-cell chronic lymphocytic leukaemia, autoimmune and inflammatory diseases, on the basis of its capability of inducing preferential activation and expansion of immunosuppressive regulatory t (treg) cells, as observed in rodent models. tgn1412 has been termed a 'superagonist' because it binds to cd28 and activates t cells without the need for prior t cell antigen receptor (tcr) signaling. in a phase i clinical trial (in march 2006), following administration of tgn1412, six healthy young men suffered a life-threatening cytokine-release syndrome (crs) involving multi-organ failure, something unpredicted by the preclinical studies. it is now clear that in the presence of tgn1412, activated cd4+ effector memory t (t em ) cells were the source of the cytokines that mediated the crs observed in the volunteers. treg cells were not able to prevent systemic inflammation, probably because the balance between activated treg cell and t em cell numbers is disadvantageous for humans compared with laboratory rodents. furthermore, in macaques, but not in humans, cd4+ t cells lose cd28 expression during their differentiation into t em cells; this detail, however, had gone unnoticed despite many years of primate testing. in conclusion, this model failed to prevent the disastrous case above [47] . in view of these events, such a risk needs to be carefully assessed if the modulation of immune inhibitory or activating receptors is used for increasing the functional activity of virus-specific t cells in order to avoid nonspecific inflammation. these therapeutic approaches are being carefully evaluated for cancer as well as for hiv and hcv chronic viral disease. given the potential antiviral effect of the antibodies, viruses have evolved multiple mechanisms to protect themselves from antibody binding. one of these, the viral receptor glycosylation, is widely shared among different viruses. carbohydrates are poorly immunogenic and, therefore, do not stimulate the response of type b lymphocytes and simultaneously hide the underlying protein structures. hcv e2 protein contains up to 11 potential nlinked glycosylation sites. specific glycans mask the cd81binding site and, therefore, nab epitopes [48] . lipid shielding may represent an additional strategy used by hcv to evade the antibody response. current data suggest that key neutralizing epitopes are less accessible on lvps. more recently, hcv has been found capable of direct cellto-cell transmission, which is largely resistant to antibody neutralization [49, 50] . hiv envelope protein is also glycosilated and changes occur in the frequency and position of glycans hiv gp120; these 'evolving glycan shields' have been shown to decrease sensitivity to antibody neutralization [51] . other factors of antibody escape for hiv are: trimerization of the gp 120 and gp 41 that can shield vulnerable epitopes better exposed on the individual monomeric subunits; kinetic and spatial constraints that impede antibodies from accessing potentially vulnerable sites during receptor binding and membrane fusion process; the variable loops of gp120 that are a prime target for nabs, which usually have a very narrow breadth of reactivity [52] . finally, the high mutation rate of many viruses, including hiv and hcv, which undergo rapid antigenic variation, allows them to escape neutralization, constituting a significant hurdle for nabs development. all these problems may be counter-balanced by selecting nabs which target conserved and more accessible areas of viral particles, and/or by using mixtures of nabs which target various key epitopes. in fact, it has been demonstrated that combination therapy with mab cocktails prevents escape variants for many viruses, including influenza [53] , coronavirus [54] and lcmv [55] , and that broad neutralization in the sera of most of some individual hiv infected donors can be associated with single or four to five principal specificities [56] . recent studies have indicated that nabs play a critical role in hcv disease outcome. viral clearance was associated with a rapid induction of neutralizing antibodies in the early phase of infection with some evidence that these antibodies are broadly reactive [57, 58] . in contrast, chronic hcv infection was characterized by absent or low-titer neutralizing antibodies in the early phase of infection and the persistence of infection, despite the induction of crossneutralizing antibodies in the later phase of infection. current understanding of the nab response raised against hcv suggests that e2 is the major target, and that multiple epitopes within e2 may be targeted by both linear-and conformation-dependent antibodies. predominantly, these neutralization epitopes overlap with cd81binding sites and clearly demonstrate a role in inhibition of entry. currently, one of these mabs, mbl-hcv1, is being investigated in clinical trials in the prevention of liver re-infection after transplantation, for which novel antiviral preventive and therapeutic strategies are urgently needed. in fact, re-infection of the graft is universal, being characterized by accelerated progression of liver disease; ifn-based therapies exhibit enhanced adverse effects and limited efficacy in these patients [59, 60] . mbl-hcv1 is a fully human monoclonal antibody isolated from transgenic mice and directed to a highly conserved linear epitope of hcv e2 glycoprotein. it is able to neutralize pseudoviruses from multiple hcv genotypes and has demonstrated efficacy in preventing hcv genotype-1 infection in hcv naïve chimpanzees. a phase i open-labeled, dose escalation study was performed in healthy adult volunteers starting with 1 mg/kg and escalating to 3, 10, 30 and 50 mg/kg after a 10-day post-infusion safety review. mbl-hcv1 was well-tolerated without any seriously adverse effect event. based on the favorable safety, tolerability and pharmacokinetics data, a phase ii study of mbl-hcv1 in chronically infected hcv patients undergoing liver transplantation has been planned [61] . in the context of hiv disease, despite intensive study over two decades, only a small number of broadly neutralizing mabs have been identified from infected patients and little is known about their activity in vivo. these antibodies are able to inhibit viral entry of most primary hiv isolates in vitro [17, [62] [63] [64] and the exceptionally high level of mutation found in their genes may reflect chronic immune responses to hiv and persistent hypermutation and selection [65] . a number of trials evaluating different formulations of anti-hiv monoclonal antibodies are now in progress. the first trial assessed a chimeric monoclonal antibody cgp 47,439 to the v3 loop of the hiv-1 envelope gp120 over 21 weeks [66, 67] . subsequent studies evaluated the kinetics of monoclonal antibody f105 directed to the cd4-binding site of gp120 [68, 69] , a humanized antibody binding to the v3 epitope gpgraf [70] . finally, a humanized mab, kd-247 is under evaluation in clinical trials. its epitope was mapped to 6 aa, igpgra, at the tip of the v3 loop of envelope protein and demonstrates crossneutralizing activity against hiv-1 isolates in clade b [71] . a drug based on the mab cocktail mode is also currently in clinical development. in this regard it has already been observed that in hiv neutralization assays the effectiveness of a mix of broadly neutralizing antibodies increased synergistically compared to the effect of the individual antibody. the synergy effect was relatively weak, with a maximum of two-to four-fold enhancement, between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations [72] . however, the use of antibodies in the cocktail mode, as an approach to improve their effectiveness, is already recognized for other pathogens or toxins. in the case of tetanus toxin, it has been reported that combining the action of three out of four antibodies increased the neutralizing activity up to 200 times [73] . in the case of botulinum toxin, neutralizing activity has been reported up to 20,000 times higher when using a mixture of three monoclonal antibodies [74] . instead, other studies have demonstrated that the combination of two potent neutralizing mabs against hiv, vrc01 and pg9, although not synergistic, can mediate additive neutralization viral activity and provides an improved neutralization coverage of 90% to 97% of viral strains by combining independent epitope targeting [75] . in a proof-of-concept passive immunization trial with humans, it has been demonstrated that a cocktail of the three broadly neutralizing mabs -2g12, 4e10 and 2f5was able to delay viral rebound in patients whose infections were fully suppressed by antiretroviral treatment before administration of the antibodies [76] . interestingly, the main antiviral effect observed was primarily attributable to the 2g12 antibody, a mab that binds to a noncontinuous epitope composed of glycosylation residues distributed over the envelope protein gp120 [64] , whereas the other two mabs, 4e10 [77] and 2f5 [78] , recognize two adjacent highly conserved epitopes on the membrane-proximal ectodomain of the hiv-1 envelope protein gp41. in earlier phase i clinical trials, safety and tolerability were demonstrated [79, 80] . during a longterm multiple dose phase ii clinical trial, high doses of the three neutralizing antibodies were given in combination to 14 hiv-1-infected individuals at weekly intervals over three months. pharmacokinetic analysis revealed that repeated infusions at high dose levels were well tolerated by the patients and did not elicit an endogenous immune response against the monoclonal antibodies. the antibodies showed distribution and elimination kinetics similar to those seen for other human-like antibodies, though monoclonal antibody 2g12 had a significantly longer elimination half-life (21.8 +/-7.2 days) than monoclonal antibodies 4e10 (5.5 +/-2.2 days) and 2f5 (4.3 +/-1.1 days) [81] . furthermore, analyses of the emergence of mutations conferring resistance to these three mabs were performed. sequence analysis of the 2g12 epitope relevant n-glycosylation sites of viruses derived from 13 patients demonstrated that mutations in these sites are associated with resistance. in vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape 2g12 pressure. importantly, in vitro selection with 2f5 and 4e10 demonstrated that resistance to these nabs can be difficult to achieve and can lead to selection of variants with impaired infectivity [82] . moreover, generation of viruses resistant to the triple-combination was a slower process characterized by recurrent loss of virus replication; some generated triple-resistant viruses seemed to be impaired in their replicative fitness, and none of the patients developed detectable viruses that escaped neutralization by all three mabs within the 77day observation period [83] . as is true with all mabs designed for infectious disease, the development of a successful vaccine would reduce the need for them. however, given the scarcity of drugs in the field of virology and given the slow progress on the hiv vaccine front, the development and use of microbicides, compounds that could be applied topically to prevent hiv transmission, is one of the possible strategies to counter the spread of hiv. in this regard, mabs could be proposed as suitable components of microbicides to fight hiv entry at mucosal surface. a safety study of p2g12 mab administered vaginally in healthy women has been completed. p2g12 is the broadly neutralizing 2g12 mab manufactured from tobacco plants [84] . most mabs in clinical trials have been produced using a system called chinese hamster ovary cell (cho-cell) fermentation [85] , including 2g12 used along with 2f5 and 4e10 antibodies as a cocktail. the cho-cell fermentation production method is very expensive and cannot produce enough mabs on a scale required for the global market; therefore, plant manufacture of such mabs may hopefully offer some solutions to lower production costs and improve output. the process yields five grams of purified antibody from 250 kg of tobacco and production costs could be 10 to 100 times lower than when using conventional bioreactors. this study has been designed to confirm the safety of a vaginally delivered mab p2g12 derived from plants and manufactured to good manufacturing practice (a quality standard used for the manufacture of medicinal products). the medicine is the first plant-produced antibody to be greenlit for clinical testing by britain's medicines and healthcare products agency (mhra). it took about a year to get that agency's stamp of approval because it required assurances that the drugs did not contain allergenic plant sugars or pesticides. no matter how it is produced, p2g12 antibody has not been shown to actually prevent hiv-1 infection in clinical trials; thus a version made from tobacco plants would not see approval any time soon. p2g12 would also likely be just one ingredient in a cocktail of plant-produced antibodies [84] . to eliminate or reduce the development of escape variants it has been proposed that targeting the conserved cellular receptors of the virus may open new avenues for a viable antibody therapy for hiv infection. hiv entry into cd4+ t cells requires the presence of a co-receptor, either ccr5 or cxcr4, on the target cell. thus, based on this hypothesis, mabs directed against cd4 and against the co-receptor ccr5, have been developed and are being analyzed in clinical trials. cd4 functions as a co-receptor, physically associating with the tcr during ag recognition by binding to a nonpolymorphic component of the major histocompatibility complex (mhc) class ii molecules on the surface of the antigen-presenting cell. ibalizumab, a humanized mab, binds cd4 on t cell surface away from the binding site for mhc class ii molecules. it does not inhibit gp120 binding to cd4 but appears to exert its antiviral property by post-binding conformational effects that prevent cd4bound gp120 from interacting with ccr5 or cxcr4 [19, 86] . by contrast, other monoclonal antibodies, that competitively inhibit gp120 binding, interfere with mhc class ii immune function [87, 88] . the reported human experience with ibalizumab consists of three clinical trials. during phase i study, it was observed that peak mean reductions in viral load occurred later in the higher dose cohorts, whereas the extent and duration of viral suppression correlated with the degree of cd4+ cell coating by ibalizumab, which was maintained longer in the higher dose cohorts, with a duration of 15 to 34 days. peak increases in cd4 counts at one day after infusion, well before the peak declines in viral load; this suggests that the increase may have been due to redistribution of cd4+ cells from lymphoid tissue rather than regeneration of cd4+ cells in the setting of viral suppression. a multidose study demonstrated continued safety over an extended treatment period and provided data on the development of ibalizumab resistance. resistance testing showed reduced susceptibility relative to baseline. resistant isolates remained dependent on cd4 for viral entry, suggesting that resistance did not develop through the use of alternative receptors. genotypic analysis was unable to identify mutations, diagnostic of ibalizumab resistance. consistent with the allosteric mechanism of ibalizumab's anti-hiv-1 effect, the development of resistance is associated with a reduction in the maximum percentage inhibition rather than the shift in the ic50 characteristic of competitive inhibitors [89, 90] . the half-life of iggs under normal physiological circumstances is two to three weeks [91] . in contrast, the average half-life of ibalizumab is 3 to 3.5 days [89] . this is consistent with observations of other anti-cd4 antibodies, in which internalization or shedding of the receptor results in more rapid antibody degradation. a randomized, double-blind, placebo controlled, phase iia study has evaluated the ibalizumab efficacy, the results showing a considerable viral load reduction with respect to the placebo arm [92] . ccr5 is a chemokine receptor that mediates activation and migration of t cells and other leukocytes. ccr5using (r5) viruses typically mediate transmission and then predominate through the progression to symptomatic disease. viruses can use an alternative chemokine receptor, cxcr4, either exclusively or in addition to ccr5. the cxcr4-using virus can be present initially, but tends to result in an increasing proportion of subjects in the later stages of the disease [93] . ccr5 co-receptor antagonists represent an emerging antiretroviral treatment class and the first to target a host molecule. currently, two anti-ccr5 mabs are being investigated. one of these is ccr5mab004, a fully human igg4 monoclonal antibody with robust activity against a diverse panel of hiv-1 isolates; it synergizes in vitro with other arv classes and appears safe and effective in reducing hiv viral load. high levels of receptor occupancy were observed for 14 to 28 days with the highest dose cohorts, suggesting the potential for weekly, fortnightly or even monthly dosing [94] . the other anti-ccr5 mab is pro 140, a humanized mab that also synergizes with small-molecule ccr5 antagonists in laboratory studies [95] . pro140 is being investigated in two modes of administration: the classical intravenous (iv) form, and subcutaneous (sc) form. the trial involving sc administration is the first to bear the proof of concept for a mab administered subcutaneously in hiv-1 infected subjects as a potent and longacting antiretroviral agent. an iv form of pro 140 tested as monotherapy in hiv-1 subjects with only r5 virus detectable [96] demonstrated potent and prolonged antiviral activity, with a 1.83 log10 mean reduction in hiv-1 rna and safety relative to placebo. the successive randomized, double-blind, placebo-controlled iia trial examined the antiviral activity, tolerability and pharmacokinetics of single intravenous infusions of up to 10-mg/kg of mabs. all pro 140-treated subjects treated with 10 mg/kg experienced a 1-log10-unit reduction in hiv-1 rna level, there being just one exception; a post-study analysis using the enhanced-sensitivity trofile assay determined that this subject had dual/mixed virus at screening. there was no change in co-receptor tropism or emergence of pro 140-resistant virus during the course of this study, supporting the view that pro 140 broadly inhibits r5 hiv-1 with a high barrier to resistance. the maximum tolerated dose of iv pro 140 has not been determined, suggesting a sizeable margin of safety for pro 140 sc administration study [97] . the study involving pro 140 sc administration showed virologic suppression between successive doses and no changes in r5 viral susceptibility to pro 140 following three weeks of monotherapy, indicating no adaptation of virus to use ccr5 in the presence of drug. pharmacokinetic data suggest the possibility of a drug regimen administered fortnightly for hiv infected individuals. proteins and other macromolecules drain from sc sites into both blood capillaries and the lymphatic system. in animals, proteins with molecular weights of greater than 16,000 daltons have been observed to drain primarily into the lymphatic system following sc administration [98] . such proteins transit through lymph fluid and typically are not absorbed significantly into the blood until they reach the thoracic duct. since the molecular weight of pro 140 is approximately 150,000 daltons, a substantial amount of sc pro 140 can be expected to drain into the lymphatic system and potentially encounter ccr5+ cells in lymphoid tissues prior to reaching the bloodstream. for these reasons, serum concentrations may not provide a full picture of the overall exposure following sc dosing of pro 140. sc infusion is currently used by individuals with primary immunodeficiency to self-administer at home significantly larger amounts (approximately 11 grams) and volumes (approximately 70 ml total, up to 15 ml/site) of the weekly sc-administered immunoglobulin [99] . self-administration of 324 mg sc pro 140 would be much simpler in comparison. therefore, sc pro 140 offers the potential for significant dose-dependent hiv-1 rna suppression and may offer greater convenience for many patients in terms of patient selfadministration [100] . the sc injection mode was chosen in order to evaluate pro140 safety and efficacy as an adjunct to an oral antiretroviral regimen in hiv-infected injection drug users with viral rebound and documented poor adherence to the previous antiretroviral regimen. therefore, a phase iib, national, multicenter, randomized, doubleblind, placebo-controlled study was initiated and is currently recruiting participants. given the complications that arise from the occurrence of drug resistances, the use of antibodies together with combined therapy increases the drug number and, therefore, the therapeutic opportunities. in particular, in the case of ccr5 inhibitors, one report has demonstrated that resistance to ccr5 inhibitors may increase the sensitivity of the resistant virus to certain neutralizing antibodies [101] . compared to ccr5, cxcr4-based blocking agents as therapy against hiv are less attractive due to the crucial role of cxcr4 in many biological processes, and the absence to date of known naturally occurring mutations leading to the inactivation of cxcr4 gene in humans. moreover, one major problem is linked to the fact that, whereas r5 viruses are found on their own in 50% or more of patients, viruses that using cxcr4 co-receptor (x4) usually are present mixed together with r5 viruses; therefore, the use of cxcr4 specific mabs could result in only little or transient effect on the overall viremia, also complicating the evaluation of pharmacological activity. however, antibodies against cxcr4 might still provide some benefits for some hiv positive patients when co-administrated with ccr5 antagonists, if the safety of such combinations is established [93] . there is a pressing need for antiviral agents that are effective against multiple classes of viruses. broad specificity might be achieved by targeting phospholipids that are widely expressed on infected host cells or on viral envelopes. phosphatidylserine (ps), the most abundant anionic phospholipid of the plasma membrane, is segregated at the inner leaflet of the plasma membrane of resting mammalian cells. loss of ps asymmetry occurs during apoptosis, cell injury, cell activation and malignant transformation, and results from inhibition of the translocases or activation of ps exporters, or lipid scrambling enzymes, such as scramblases. after enveloped viruses replicate within the host cell, they create their 'envelope' by carrying along part of the host cell's membrane upon exiting. as a result, the target phospholipid becomes exposed on the surface of the virus as well as on the infected host cell [102] . bavituximab is the first in a new class of patented antibody therapeutics that target and, preferentially bind, to these exposed phospholipids. it has demonstrated broad therapeutic potential across multiple oncology indications and represents a new approach to treating viral disease, too. bavituximab is currently being evaluated in randomized phase ii clinical trials for non-small cell lung cancer and pancreatic cancer, for therapy of chronic hcv infection and for hiv/hcv co-infection. the therapeutic effect of bavituximab appears to be due to adcc of tumor and virus-infected cells. since ps exposure is an early event during virus infection, adcc may limit virus spread. furthermore, in the infectious disease setting, bavituximab causes opsonization and clearance of infectious virus from the bloodstream, leaving less virus to infect other tissues. three completed phase i hcv clinical trials have shown that bavituximab is generally safe and well-tolerated. reductions in serum hcv rna levels were also observed. a randomized phase ii clinical trial with previously untreated hcv genotype-1 infected patients was designed to determine the early virologic response (evr) rate after 12 weeks of therapy with bavituximab in combination with the antiviral drug ribavirin and safety profile versus pegylated ifn-α-2a and ribavirin. the results show that the combination of bavituximab with ribavirin has a better safety profile than an ifn-containing regimen. however, the evr development in the bavituximab-containing arm was later than the ifn-containing group; therefore, a longer-term evaluation is needed to adequately compare their effectiveness. in addition, the lower dose level appears to be more active in hcv patients than the high dose does. such results suggest that future studies evaluating longer bavituximab treatment durations at or around the lower dose level in combination with ribavirin and potentially direct acting antivirals in certain patient populations may hold promise as ifn-free hcv therapeutic regimens [103] . targeting ps on cells infected with multiple different viruses and on virions themselves is a promising antiviral strategy. although resistance has developed in monotherapy trials with ibalizumab (an anti-cd4 antibody), host-derived antigen, such as anionic phospholipids, on virus-infected cells are independent of the viral genome and as a consequence the acquisition of drug resistance should be theoretically less problematic than with agents that target virus-encoded components. since the discovery of pd-1 as an inhibitory receptor associated with t-cell dysfunction, the roles of various inhibitory receptors on virus-specific cd8+ t cells have been extensively studied in human chronic viral infections, such as hcv, hbv and hiv infections. as blocking the inhibitory receptors in vitro restores the functions of virus-specific t cells, novel hiv and hcv treatments based on blockade of several immune checkpoint molecules are being investigated. in particular, mabs interfering with two major inhibitory networks of the b7:cd28 family, namely the pd-1 and ctla-4 pathways [104] , are currently being studied in clinical trials, to evaluate their safety and efficacy. these mabs recognize the pd-1 or ctla-4 receptor and neutralize the binding with their respective ligands. the pd-1:pd-l1 pathway delivers inhibitory signals which regulate t cell activation. as a result it performs a key role in various processes, namely in multiple tolerance checkpoints that prevent autoimmunity, in the suppressive tumor microenvironment, in the immune-mediated tissue damage, in host defenses aimed at eradicating microbial pathogens and tumors and finally, in t cell exhaustion that contributes to both lack of viral control during chronic infections and to t cell unresponsiveness [105] . in cancers, a strong correlation between increased pd-l1 expression on tumors and a negative survival prognosis in patients has already been observed. various studies indicate that mabs targeting the pd-1 signaling pathway reinvigorate antigen-specific t-cell responses and promote an immune response to fight tumors [106] . in hcv infection the relationship between the pd-1 expression and the outcome of the acute hcv infection was questioned; subsequently, recent studies have shown that the progression of acute hcv infection to the chronic stage is associated with a high level of pd-1 on hcv-specific cd8+ t cells, whereas the clearance of hcv infection is associated with lower levels of pd-1 expression [36] . given these premises, mdx-1106, a fully human antibody also known as ono-4538, and ct-011, a humanized antibody, both interacting with pd-1 receptor, are being developed as a treatment for cancer disease and for therapy of chronic hcv infection [107] . to date, most clinical experience with pd-1 blockade has been gained with mdx-1106 in the tumor setting. drug-related grade 3 or 4 toxic effects occurred in 14% of patients, in whom there were drug-related adverse events of special interest, those with potential immune-related causes; they included pneumonitis, vitiligo, colitis, hepatitis, hypophysitis and thyroiditis. pneumonitis (3%) ranged from isolated radiographic abnormalities to progressive, diffuse infiltrates associated with clinical symptoms in a small number of patients. although three deaths occurred, mild-to-moderate pneumonitis was managed successfully with either observation or glucocorticoids. however, objective responses were observed in approximately one in four to one in five patients with non-small-cell lung cancer, melanoma, or renal-cell cancer; overall, an adverse-event profile does not appear to preclude its use [108] . besides these studies, an ongoing phase i safety trial with active hepatitis c genotype 1 infected patients has been designed to assess the safety and tolerability profile of mdx-1106 [109] . clinical studies to evaluate the use of ct-011 in hcv disease have also been initiated [110] . ctla-4 is up-regulated on activated t cells and inhibits t cell activation by reducing the production of il-2 and arresting cell cycle progression. ctla-4 has also been shown to have an impact on t cell responses in animal tumor models and humans [111, 112] . human trials that used a blocking anti-ctla-4 mab demonstrated a reduction in tumor mass and clinical benefit in a substantial minority of treated subjects. studies of the role for ctla-4 in chronic infections have produced mixed results. in chronic hiv infection, many studies indicate that impaired cd4+ t cell function is associated with viral persistence [113] , although the function of ctla-4 in causing hiv persistence by suppressing t cell function remains unclear [114] . on the other hand, ctla-4's role in chronic hcv infection seems to be more defined. the hcv-specific cd8+ t cells found in the livers of chronic hcv patients overexpressed not only pd-1, but also ctla-4. co-expression of pd-1 and ctla-4 was observed in liverinfiltrating lymphocytes, but not in peripheral blood lymphocytes [36] , suggesting the phenotypic differences of virus-specific cd8+ t cells in different in vivo compartments. pd-1 and ctla-4 expressing hcv-specific t cells were profoundly dysfunctional [115] . tremelimumab is a fully human igg2 mab directed against ctla-4. while a phase ii study for hiv disease with this drug has been withdrawn prior to enrollment, clinical trials for hcv disease are still underway. tremelimumab binds to activated t lymphocytes and results in inhibition of b7-ctla-4-mediated down-regulation of tcell activation. it also acts as an il-2 stimulant. it was generated, using xenomouse technology (figure 2) , as an anticancer agent and is currently in worldwide phase iii development for malignant melanoma, phase ii development for colorectal cancer, gastrointestinal cancer, gynecological cancer and non-small cell lung cancer in the us and other countries. it is also being investigated for prostate, breast and pancreatic cancer in various countries. as for anti-pd-1 antibodies, immune-related adverse effects of tremelimumab are of special interest because of its presumed mechanism of action. most of the experience in identifying and managing ctla-4 treatment-related side effects has derived from studies in cancer, particularly in melanoma. these effects mainly include colitis/diarrhea, dermatitis, hepatitis and endocrinopathies; uveitis, nephritis and inflammatory myopathy also have been occasionally reported. these unique side effects are likely a direct result of breaking immune tolerance upon ctla-4 blockade; they are generally mild, reversible and manageable, following specific treatment guidelines that include symptomatic therapies or systemic corticosteroids [116] . in december 2008, pfizer initiated a phase ii trial in patients with latestage unresectable liver cancer who also have hepatitis c infections. the primary endpoint of this single-armed study is the ability of tremelimumab to produce tumor responses among hcv-infected patients with hepatocellular carcinoma and to produce changes in hepatitis c viral load. the first results indicate that tremelimumab demonstrated an excellent safety profile, with a promising antitumor efficacy against hcc in 17 patients, as well as an intense antiviral activity. in fact, a significant and progressive decline in serum hcv viral load was observed, this being associated with an increase in anti-hcv immune response in 76% of patients [117] . since there are multiple levels of immunoregulation, a synergistic use of antibodies against different checkpoint molecules might represent the next stage in immunotherapy for chronic infectious diseases, as evidenced from ex vivo studies about the combined pd-1/ctla-4 blockade in hcv disease [36] . furthermore, because the host mechanisms that inhibit t cell activity are common and conserved aside from specific virus-encoded immune evasion strategies, the antibodies targeting inhibitory receptors may prove extremely versatile drugs potentially effective against multiple classes of viruses. the need to treat hiv and hcv infectious diseases, two epidemics of global impact, has reawakened interest in mab-based therapy, supporting a variety of clinical studies. the results that are emerging, will help to create models for the further development of such drugs and extend their use against other viruses as well. although the mab production costs are high, increasing advances of biotechnology and production systems will make them more competitive on the market, and new approaches, such as using mab cocktails or combining mabs with available drugs, will improve effectiveness. treatment with mabs as part of a drug regimen is the most likely future for mabs that block hcv and hiv infection in order to avoid viral escape, while chronic treatment could attract further investments from pharmaceutical companies. furthermore, broad spectrum mabs, such as bavituximab and immunomodulatory mabs, could be useful against a whole range of diseases, thus extending marketability and profit margins. this review has focused on the use of intact mabs as a novel emerging and versatile class of pharmaceuticals. it is important to note, however, that biotechnology also provides the opportunity to build various antibody formats whose improved pharmacokinetics and pharmacodynamic properties could be co-opted in the fight against infectious diseases. resistance to antiretroviral drugs; the mechanisms of immune reconstitution; and, finally, operational and implementation research in resource limited settings. he chaired or participated in many international clinical studies on antiretroviral therapy, and he is currently the coordinator of the -european commission-funded hiv clinical trials network (neat). cellular and molecular immunology hemming vg: use of intravenous immunoglobulins for prophylaxis or treatment of infectious diseases passive antibody therapy for infectious diseases carbohydrate dramatically influences immune reactivity of antisera to viral glycoprotein antigens passive immunity in prevention and treatment of infectious diseases monoclonal antibody-based therapies for microbial diseases immunotherapy with human monoclonal antibodies. fragment a specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin polyclonal immunoglobulins and hyperimmune globulins in prevention and management of infectious diseases the growth and potential of human antiviral monoclonal antibody therapeutics potent antibody therapeutics by design virus entry: molecular mechanisms and biomedical applications mechanism of membrane fusion by viral envelope proteins preparation and properties of antibody directed specifically against the neuraminidase of influenza virus the antiviral activity of antibodies in vitro and in vivo a monoclonal antibody to cd4 domain 2 blocks soluble cd4-induced conformational changes in the envelope glycoproteins of human immunodeficiency virus type 1 (hiv-1) and hiv-1 infection of cd4+ cells potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the ccr5 monoclonal antibody pro 140 anti-human immunodeficiency virus type 1 (hiv-1) antibodies 2f5 and 4e10 require surprisingly few crucial residues in the membraneproximal external region of glycoprotein gp41 to neutralize hiv-1 hepatitis c virus entry depends on clathrin-mediated endocytosis hepatitis c virus cell entry: role of lipoproteins and cellular receptors anti-cd81 antibodies can prevent a hepatitis c virus infection in vivo serum-derived hepatitis c virus infection of primary human hepatocytes is tetraspanin cd81 dependent role of scavenger receptor class b type i in hepatitis c virus entry: kinetics and molecular determinants fc receptors and their role in immune regulation and autoimmunity hiv-1 gp120-specific antibody-dependent cell-mediated cytotoxicity correlates with rate of disease progression heterogeneous neutralizing antibody and antibody-dependent cell cytotoxicity responses in hiv-1 elite controllers serum antibodies against the hepatitis c virus e2 protein mediate antibody-dependent cellular cytotoxicity (adcc) hepatitis c virus (hcv)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against hcv envelope protein advances in the assessment and control of the effector functions of therapeutic antibodies the function of programmed cell death 1 and its ligands in regulating autoimmunity and infection viral persistence alters cd8 t -cell immunodominance and tissue distribution and results in distinct stages of functional impairment t cells and viral persistence: lessons from diverse infections pd-1 expression on hivspecific t cells is associated with t -cell exhaustion and disease progression restoring function in exhausted cd8 t cells during chronic viral infection coregulation of cd8+ t cell exhaustion by multiple inhibitory receptors during chronic viral infection pd-1 expression in acute hepatitis c virus (hcv) infection is associated with hcv-specific cd8 exhaustion synergistic reversal of intrahepatic hcv-specific cd8 t cell exhaustion by combined pd-1/ ctla-4 blockade characterization of hepatitis b virus (hbv)-specific t -cell dysfunction in chronic hbv infection molecular signature of cd8+ t cell exhaustion during chronic viral infection upregulation of ctla-4 by hiv-specific cd4+ t cells correlates with disease progression and defines a reversible immune dysfunction tim-3 expression defines a novel population of dysfunctional t cells with highly elevated frequencies in progressive hiv-1 infection upregulation of tim-3 and pd-1 expression is associated with tumor antigen-specific cd8+ t cell dysfunction in melanoma patients t -cell exhaustion: characteristics, causes and conversion pd-1 is a regulator of virus-specific cd8+ t cell survival in hiv infection upregulation of pd-1 expression on hiv-specific cd8+ t cells leads to reversible immune dysfunction expression of the interleukin-7 receptor α chain (cd127) on virus-specific cd8+ t cells identifies functionally and phenotypically defined memory t cells during acute resolving hepatitis b virus infection in vivo ligation of glucocorticoid-induced tnf receptor enhances the t-cell immunity to herpes simplex virus type 1 the storm has cleared: lessons from the cd28 superagonist tgn1412 trial hepatitis c virus envelope glycoprotein e2 glycans modulate entry, cd81 binding, and neutralization hepatitis c virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies cd81 is dispensable for hepatitis c virus cell-to-cell transmission in hepatoma cells antibody neutralization and escape by hiv-1 identifying epitopes of hiv-1 that induce protective antibodies combination therapy using chimeric monoclonal antibodies protects mice from lethal h5n1 infection and prevents formation of escape mutants human monoclonalantibody combination against sars coronavirus: synergy and coverage of escape mutants in vivo selection of neutralization-resistant virus variants but no evidence of b cell tolerance in lymphocyticchoriomeningitis virus carrier mice expressing a transgenic virus-neutralizing antibody a limited number of antibody specificities mediate broad and potent serum neutralization in selected hiv-1 infected individuals human serum facilitates hepatitis c virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis c virus infection rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis c treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus recurrence of hepatitis c virus after loss of virus-specific cd4(+) t-cell response in acute hepatitis c safety and pharmacokinetics of a novel human monoclonal antibody directed against e2 glycoprotein of hepatitis c virus (mbl-hcv1) in healthy volunteers. easl the international liver congress™ crystal structure of a neutralizing human igg against hiv-1: a template for vaccine design a conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1 human monoclonal antibody 2g12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1 broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals a phase i/iia clinical study with a chimeric mouse-human monoclonal antibody to the v3 loop of human immunodeficiency virus type 1 gp120 pharmacokinetics of an hiv-1 gp120-specific chimeric antibody in patients with hiv-1 disease phase i study of a human monoclonal antibody directed against the cd4-binding site of hiv type 1 glycoprotein 120 pharmacokinetics of f105, a human monoclonal antibody, in persons infected with human immunodeficiency virus type 1 monoclonal antibody hnm01 in hivinfected patients: a phase i study sequential immunization with v3 peptides from primary human immunodeficiency virus type 1 produces cross-neutralizing antibodies against primary flego et al isolates with a matching narrow-neutralization sequence motif neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies neutralization of tetanus toxin by distinct monoclonal antibodies binding to multiple epitopes on the toxin molecule potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody mascola neutralization coverage is improved by combining monoclonal antibodies that target independent epitopes hiv-1 delay of hiv-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1 a broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1 passive immunization with the anti-hiv-1 human monoclonal antibody (hmab) 4e10 and the hmab combination 4e10/2f5/2g12 a phase i trial with two human monoclo-nal antibodies (hmab 2f5, 2g12) against hiv-1 long-term multiple-dose pharmacokinetics of human monoclonal antibodies (mabs) against human immunodeficiency virus type 1 envelope gp120 (mab 2g12) and gp41 (mabs 4e10 and 2f5) in vivo and in vitro escape from neutralizing antibodies 2g12, 2f5, and 4e10 hiv-1 mutants escaping neutralization by the human antibodies 2f5, 2g12, and 4e10: in vitro experiments versus clinical studies antibody production clinical trial of farmed hiv drug finally gets underway epitope mapping of ibalizumab, a humanized anti-cd4 monoclonal antibody with anti-hiv-1 activity in infected patients immunosuppression of cynomolgus renal allograft recipients with humanized okt4a monoclonal antibodies functional epitope analysis of the human cd4 molecule. the mhc class ii-dependent activation of resting t cells is inhibited by monoclonal antibodies to cd4 regardless whether or not they recognize epitopes involved in the binding of mhc class ii or hiv gp120 safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly tnx-355), an anti-cd4 monoclonal antibody, in human immuno-deficiency virus type 1-infected adults antiretroviral activity of the anti-cd4 monoclonal antibody tnx-355 in patients infected with hiv type 1 mouse/human chimeric monoclonal antibody in man: kinetics and immune response phase 2 efficacy and safety of the novel entry inhibitor, tnx-355 the biology of ccr5 and cxcr4 a phase 1, doseescalation, placebo-controlled study of a fully human monoclonal antibody (ccr5mab004) against ccr5 in patients with ccr5-tropic hiv-1 infection potent antiviral synergy between monoclonal antibody and small-molecule ccr5 inhibitors of human immunodeficiency virus type1 a phase i study to explore the activity and safety of sch532706, a small molecule chemokine receptor-5 antagonist in hiv type-1-infected patients phase 2a study of the ccr5 monoclonal antibody pro 140 administered intravenously to hiv-infected adults transport and absorption of drugs via the lymphatic system safety and efficacy of self-administered subcutaneous immunoglobulin in patients with primary immunodeficiency diseases anti-hiv-1 activity of weekly or biweekly treatment with subcutaneous pro 140, a ccr5 monoclonal antibody neutralizing antibody and antiretroviral drug sensitivities of hiv-1 isolates resistant to small molecule ccr5 inhibitors targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases advancing clinical trials in cancer and viral infection: bavituximab antiviral the pd-1 pathway in tolerance and autoimmunity tumor b7-h1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up targeting the pd-1/b7-h1(pd-l1) pathway to activate anti tumor immunity sznol m: safety, activity, and immune correlates of anti-pd-1 antibody in cancer clinical trial.gov a service of the u.s. national institutes of health: a study of mdx-1106 to treat patients with hepatitis c infection national institutes of health: safety and tolerability study of the monoclonal antibody ct-011 in patients with chronic hepatitis c genotype 1 infection biologic activity of cytotoxic t lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients cancer regression and autoimmunity induced by cytotoxic t lymphocyteassociated antigen 4 blockade in patients with metastatic melanoma progress in defining cd4 helper cell responses in chronic viral infections pd-1 and ctla-4 inhibitory cosignaling pathways in hiv infection and the potential for therapeutic intervention functional restoration of hcv-specific cd8 t cells by pd-1 blockade is defined by pd-1 expression and compartmentalization the emerging toxicity profiles of anti-ctla-4 antibodies across clinical indications antiviral and antitumoral effects of the anti-ctla4 agent tremelimumab in patients with hepatocellular carcinoma (hcc) and chronic hepatitis c virus (hcv) infection: results from a phase ii clinical trial aacr annual meeting continuous cultures of fused cells secreting antibody of predefined specificity chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains replacing the complementarity-determining regions in a human antibody with those from a mouse humanized antibodies as potential therapeutic drugs an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus antibody engineering via genetic engineering of the mouse: xenomouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies selecting and screening recombinant antibody libraries pre-publication history the pre-publication history for this paper can be accessed here clinical development of monoclonal antibody-based drugs in hiv and hcv diseases authors' contributions mf and aa contributed equally to writing and editing the manuscript. mc and sv critically reviewed the manuscript and made final changes. all authors have read and approved the final version of the manuscript.authors' information mf, after her doctorate (immunology, 2006, university of rome, tor vergata, italy), was enrolled as a researcher at the department of therapeutic research and medicine evaluation at the istituto superiore di sanità, rome, italy (iss). she has acquired extensive expertise in biotechnologies, such as the isolation, characterization and genetic manipulation of human mabs in single chain format for the development of biological constructs designed for clinical use. the authors declare that they have no competing interests. mc is the author of a patent about a mab against p-glycoprotein (patent number: 6063621), but this mab is not suitable for clinical applications in hiv and/or hcv diseases submit your next manuscript to biomed central and take full advantage of: key: cord-354050-kcn67stj authors: shi, guoli; schwartz, olivier; compton, alex a. title: more than meets the i: the diverse antiviral and cellular functions of interferon-induced transmembrane proteins date: 2017-11-21 journal: retrovirology doi: 10.1186/s12977-017-0377-y sha: doc_id: 354050 cord_uid: kcn67stj the first responders of human antiviral immunity are components of the intrinsic immune response that reside within each and every one of our cells. this cell-autonomous arsenal consists of nucleic acid sensors and antiviral effectors strategically placed by evolution to detect and restrict invading viruses. while some factors are present at baseline to allow for constant surveillance of the cell interior, others are upregulated by cytokines (such as interferons) that signal a viral infection underway in neighboring cells. in this review, we highlight the multiple roles played by the interferon-induced transmembrane (ifitm) proteins during viral infection, with focuses on ifitm3 and hiv-1. moreover, we discuss the cellular pathways in which ifitm proteins are intertwined and the various functions they have been ascribed outside the context of infection. while appreciated as broadly-acting, potent restriction factors that prevent virus infection and pathogenesis in cell culture and in vivo, questions remain regarding their precise mode of action and importance in certain viral contexts. continued efforts to study ifitm protein function will further cement their status as critical host determinants of virus susceptibility and prioritize them in the development of new antiviral therapies. the ifitm protein family is encoded by five genes in humans, including the immune-related ifitm1, ifitm2, and ifitm3, as well as ifitm5 and ifitm10 which have no characterized roles in immunity [1] . today, ifitm genes are present in many vertebrate animal species yet they likely emerged in early unicellular eukaryotes via horizontal gene transmission from a bacterium [2] . since then, species-specific gene expansions have given rise to unique ifitm gene repertoires that vary at the level of sequence and copy number [3] [4] [5] . in addition to the canonical ifitm gene locus on chromosome 11 in humans, there are a number of ifitm-like genes dispersed throughout our genome for which a functional understanding is lacking [2] . despite what their name implies, only the immune-related ifitm genes are interferon-inducible, and furthermore, moderate to high levels of expression may be seen in several tissue types even in the absence of interferon. the immunological and clinical importance of ifitm proteins to innate immunity is tied to their unique ability to inhibit the earliest step of the virus life cycle: entry into cells. as a result, they prevent not only viral replication but also the sequelae of virus-associated disease, such as cytopathicity (cell death) and inflammation. initially revealed to be endogenous inhibitors of influenza a virus (iav), west nile virus (wnv), and dengue virus, today it is recognized that a growing list of viruses are sensitive to ifitm-mediated restriction [6, 7] . the use of retroviral "pseudotypes, " in which different viral envelope glycoproteins are swapped into the same retrovirus capsid core, demonstrated that the route of cellular entry is a major determinant of restriction and identified strains exhibiting resistance. in general, those that require a ph-dependent triggering of viral fusion machinery in endosomes are the most affected when ifitm proteins, in particular ifitm3, are overexpressed or silenced in cells [8] [9] [10] . these findings suggested that ifitm-mediated antiviral activity manifests at the entry stage. the subcellular localization of ifitm proteins is important to our understanding of how virus entry is inhibited. they are regularly detected in endosomes, lysosomes, autophagosomes, and the plasma membrane, and the extent of which can vary by cell type and by ifitm family member. their presence at various cellular compartments is the result of dynamic protein trafficking that begins with de novo synthesis in the endoplasmic reticulum and ends with degradation in lysosomes [11] . multiple pieces of evidence suggest that ifitm3 takes on a type ii transmembrane protein topology, with a cytosolic aminoterminus, a luminal/extracellular carboxy-terminus, and two hydrophobic domains: one intramembrane domain (hd1) and one transmembrane domain (hd2) [12] [13] [14] . interestingly, there is also support for alternative membrane topologies in which the termini orientations are reversed [13] . the hydrophobic domains of ifitm members ensure movement between membrane-enclosed vesicles in the biosynthetic-secretory pathway, and posttranslational lipidification with an s-palmitoyl group promotes durable membrane associations [15, 16] . after passage through the endoplasmic reticulum, golgi complex, and plasma membrane, endocytic sorting motifs in the amino-terminus of ifitm2 and ifitm3 allow internalization into endosomes, and this positioning is crucial for antiviral activity [5, 17, 18] . late endosomes, multivesicular bodies, and lysosomes form hybrid organelles in cells and are nearly indistinguishable by conventional methods, and hence the subcellular localization of ifitm3 is often described as endolysosomal. however, some of the overlap with lysosomes must be attributed to the degradative pathway controlling ifitm3 protein turnover, which is mediated by the e3 ubiquitin ligase nedd4 [19] . furthermore, recent reports highlighting roles for ifitm3 in autophagy, a catabolic process involving the degradation of cellular cargo in lysosomes, may explain the apparent association with autophagosomes [20] . in summary, while endolysosomes appear to be the key compartment whereby ifitm3 restricts virus entry, its detection in other organelles may be indicative of uncharacterized cellular functions that are only now being investigated. as residents of cellular membranes, an attractive explanation for the effects of ifitm proteins on virus entry involves direct modification of membrane rigidity and curvature, but other indirect mechanisms have also been proposed (fig. 1 membrane proteins can alter the shape and fluidity of lipid bilayers, and furthermore, that virus-cell fusion is affected by these factors [21] . most of what we know about the antiviral activities of ifitm proteins results from work using iav and vesicular stomatitis virus (vsv), which perform ph-dependent fusion reactions in endosomes to gain access to the cell interior [22] . early findings using fluorescent microscopy and flow cytometry of single cells implicated ifitm3 as an obstacle to virus-cell fusion. when cells overexpressing ifitm3 are challenged with iav, productive infection is inhibited [8] . upon close examination, virions undergo attachment and internalization into cells before becoming cleared from the cell interior [9] . parallel experiments further showed that ifitm3 may expand the size and acidity of the endolysomal compartment itself. in effect, ifitm3 appears to trap endocytosed virions inside vesicles slated for destruction in a degradative pathway [6] . it remains to be determined whether ifitm3-containing structures represent a distinct, hostile subset of endolysosomes that are not conducive to virus-cell fusion. indeed, an article reported that iav sensitivity to ifitm3 is determined by the acidic threshold at which viral hemagglutinin (ha) triggers virus-cell fusion in endosomes. that is, ha variants which drive fusion at higher ph (less acidic) are more resistant to the block by ifitm3, suggesting that evasion of more acidic, late endosomes (where ifitm3 resides in most cell types) enables infection [23] . this assertion is supported further by the observation that vsv infection, which requires virus-cell fusion in early endosomes, is less affected by the presence of ifitm3 [8, 22, 24] . the effects of ifitm proteins on the makeup of the cell interior may present important clues about its principal mechanism of action [9] . simple experiments examining cell-cell fusion, in which ifitm proteins and viral envelope proteins are co-expressed, have been instrumental in testing different mechanistic possibilities. initial reports showed that ifitm members inhibit cell-cell fusion mediated by three classes of viral fusion proteins (some more than others) in a way that did not affect envelope expression itself [25] . this finding suggests that ifitm proteins may affect the physical aspects of membranes in a way that prevents fusion by diverse viral fusion proteins. while informative, an important caveat is that cell-cell fusion experiments in tissue culture do not accurately reproduce the true nature of virus-cell encounters, since ifitm and viral fusion proteins may not be expressed at physiologically relevant levels or at correct subcellular sites. attempts at identifying the precise step of the fusion sequence inhibited by ifitm suggested a block prior to hemifusion, the point at which lipid mixing begins between leaflets of two juxtaposed membranes [25] . however, other techniques including virus particle tracking detected lipid mixing between cell and virus in the presence of ifitm but no viral escape into the cytoplasm, suggesting that dilation of the fusion pore is restricted [26] . the basis for these observations may lie with increased lipid density or increased positive curvature of ifitm-containing membranes [25] . another piece of evidence in support of ifitm proteins as membrane remodelers is the finding that amphotericin b, an antifungal compound known to enhance membrane fluidity, counteracts the activity of ifitm3 to render cells permissive to infection [27] . furthermore, the discovery of an amphipathic helix within the first hydrophobic domain of ifitm3 was found to be crucial to the inhibition of virus entry [14] . this previously unappreciated structure is positioned adjacent to palmitoylated cysteine residues involved in membrane targeting and is also important for the inhibition of cell-cell fusion. based on previously recognized functions of other proteins endowed with an amphipathic helix, ifitm proteins may sense membrane changes occurring during virus-driven hemifusion and prevent fusion pore dilation. these latest findings implicate the first hydrophobic domain (hd1) of ifitm3 as the functional "arm" responsible for its antiviral activity. however, this contrasts with a previous finding that proposed a role for hd2 in cholesterol augmentation in endosomes [28] . residues within hd2 of ifitm3 were reported to interact with the vesicle-associated membrane protein-associated a (vapa), previously linked to cholesterol trafficking. while the effects of cholesterol on membrane fluidity and virus-cell fusion are well-characterized, a number of studies have failed to draw a mechanistic link between ifitm3 activity and cholesterol levels [26, 27, 29, 30] . therefore, effects of ifitm3 on lipid trafficking may not underlie virus inhibition, yet may be indicative of presently uncharacterized activities in the cell. interestingly, vapa and the related protein vapb can enhance the replication of some viruses, most likely through the manipulation of the lipid composition of cell membranes used for virus replication [31, 32] . overall, the study of pathogenic rna viruses demonstrating a high degree of sensitivity to ifitm-mediated restriction has provided some mechanistic insight behind the block they impose to virus entry. nevertheless, the demonstration that certain viruses are resistant to, or even benefit from, the ifitm proteins indicates that antiviral activity may be achieved through the coordination of other cellular proteins. ifitm proteins, especially ifitm1, are known to reside in the plasma membrane and to interact with transmembrane proteins, such as tetraspanins. hepatitis c virus (hcv) utilizes cd81 and occludin as co-receptors, which are components of cellular tight junctions and also happen to interact with ifitm1 [33] . overexpression of ifitm1 leads to inhibition of hcv entry into cells as well as disruption of tight junction complexes, suggesting that these two effects are functionally linked [34] . since cd81 and other tetraspanin proteins can modulate fusion events in multiple virus infections [35] [36] [37] [38] , the ability for ifitm proteins to alter the location or clustering of membrane protein complexes may be central to their antiviral activity. an 'indirect' mode of action is in agreement with the observation that cellular entry of the coronavirus hcov-oc43 is promoted by ifitm proteins [39] . this exceptional case may be explained by the fact that different viruses take advantage of distinct cellular receptors and thus distinct entry routes that are differentially impacted by the presence of ifitm proteins. a transmembrane metalloprotease known as zmp-ste24 (also known as face1) has recently emerged as a downstream effector of ifitm3 [40] . the authors posit that ifitm3 traffics zmpste24 to the sites of virus fusion at endosomes via a direct protein-protein interaction. importantly, in zmpste24 knockout cells, ifitm3 overexpression no longer provides protection from virus challenge. no mechanistic information is available for the antiviral activities of zmpste24 and thus future studies must address the specific determinants of how it binds to ifitm3. it is possible that domains of ifitm3 previously shown to be essential for antiviral function play roles in modulating the location or function of this and other cellular factors, resulting in an organized block to virus entry. the path to identifying the roles played by ifitm proteins during hiv-1 infection was less straightforward for multiple reasons. first, while the impact of ifitm proteins on viruses that require endocytosis and ph-dependent fusion is clearly appreciated, the role for endocytosis in hiv-1 cellular entry has been widely debated [41] . second, hiv-1 exhibits the capacity to spread between cells in a process known as cell-to-cell transmission, which likely allows escape from immune barriers [42] . despite these challenges, the study of ifitm and hiv-1 was mutually instructive: a new antiviral function was described for the former and the ports of cellular entry were defined for the latter. in the first formal description of the antiviral properties of ifitm family members, ifitm3 silencing had little to no effect on hiv-1 infection in hela-cd4 cells, thus grouping it with another retrovirus (amphotropic murine leukemia virus) deemed to be resistant [8] . also, in the paper that identified tetherin/bst-2 as the target of viral accessory protein vpu, which promotes hiv-1 release from cells, it was shown that ifitm proteins exhibited no impact on hiv-1 egress [43] . however, two highthroughput screens studying the activities of interferon stimulated genes (isgs) provided evidence that ifitm proteins impact hiv-1 replication when silenced or overexpressed [44, 45] . lu et al. provided the first in-depth functional demonstration of ifitm-mediated hiv-1 restriction at the level of entry in t cells, and this work was later extended to other lentiviruses of non-human primates [46] . in addition, experiments allowing ongoing replication in tissue culture revealed that the antiviral properties of ifitm proteins may not be limited to the inhibition of virus entry (see next section). as the primary determinant for virus-cell attachment and the subsequent fusion reaction, the viral envelope glycoprotein (env) was suspected to play an important role in whether or not hiv-1 and related lentiviruses are subject to inhibition by ifitm proteins. a report taking advantage of retroviral env pseudotyping provided key insight into viral factors that govern susceptibility and resistance. the authors showed that one can decrease sensitivity to ifitm proteins by increasing amounts of lentiviral env incorporated into virions [47] . however, they pointed out that the capsid core (vector) is also a determinant, indicating that the structure of the viruslike particle and/or its coordination with env also affects the degree of restriction. another study corroborated the importance of env with regards to inhibition by ifitm proteins, this time via examination of patient-derived hiv-1 clones known as transmitted/founder (tf) strains [48] . these viral variants represent a close approximation of the viral sequence that seeds infection in a newlyinfected individual, and they tend to utilize a specific co-receptor on the cell surface known as ccr5. here, it was revealed that the sensitivity of hiv-1 entry to ifitmmediated restriction depends on coreceptor usage and the subcellular localization of ifitm in the host cell. cxcr4-tropic hiv-1 strains were shown to exhibit sensitivity to ifitm2 and ifitm3, which are mostly localized to endolysosomes, while ccr5-tropic strains were sensitive to ifitm1 at the plasma membrane. this differential outcome suggests that hiv-1 fusion may occur in endosomes or at the plasma membrane depending on which virus coreceptor is engaged at the cell surface. tf strains are relatively resistant to ifitm-mediated restriction, yet matched viral clones derived 6 months following initial infection exhibited a gain of sensitivity to ifitm2 and ifitm3 [49] . this finding suggests that founder viruses enter cells at the plasma membrane, while viruses isolated at later stages of infection might increasingly rely on endosomal entry. however, ifitm2 and ifitm3 also transit to the plasma membrane before endocytosis, and thus the varying sensitivity of hiv-1 strains may result from fusion at different plasma membrane microdomains. furthermore, the authors show that the site of hiv-1 entry, as inferred by sensitivity to ifitm proteins, may also depend on cell surface levels of cd4. another report reinforced the link between ifitm and hiv-1 entry by demonstrating that cxcr4-tropic, but not ccr5-tropic hiv-1, is hypersensitive to a splice variant of ifitm2 lacking the amino terminus. notably, this ifitm variant is especially abundant in primary human cells (cd4+ t cells and monocytes) that serve as targets for hiv-1 infection in vivo [50] . together, these recent data suggest that ifitm2 and ifitm3 may be major selective pressures responsible for the use of ccr5 during primary hiv-1 infection. in addition to restricting virus entry, recent findings indicate that ifitm proteins perform antiviral functions impacting late stages of the hiv-1 life cycle. in contrast to previous strategies in which infections were launched using only cell-free virus preparations, the use of cell coculture experiments using infected cells (donors) and uninfected cells (targets) revealed new antiviral functions [51] . we found that ifitm overexpression in targets had little to no consequence for hiv-1 transmission and spread in this context, while, surprisingly, overexpression in donor cells led to potent decreases [51] . further experiments showed that the block to virus spread is attributed to an inhibition of virion infectivity, with ifitm3 exhibiting the most potent restriction. virus-cell fusions assays showed that hiv-1 virions produced in the presence of ifitm3 are less fusogenic when incubated with fresh target cells, and assessment of virion content indicated that ifitm3 incorporates into the viral lipid bilayer. this antiviral activity is enhanced upon expression of an ifitm3 mutant that is defective for endocytosis, indicating that restriction of hiv-1 virion infectivity is performed at the plasma membrane [5] . two other teams reported similar findings on this phenomenon in hiv-1 producing cells, with one adding a layer of mechanistic insight involving hiv-1 env glycoprotein [52] [53] [54] . here, the production of hiv-1 particles in the presence of ifitm3 via co-transfection of 293t cells resulted in defects in env maturation and decreases in virion-associated gp120, the infectious form of env [53] . the authors posited that ifitm3 interferes with env via a proteinprotein interaction in virus-producing cells. of note, this relationship between antiviral protein and env appears to hold true upon examination of non-human primate ifitm3 and their lentiviral counterparts [54] . nonetheless, there exist certain discrepancies that cloud the potential importance of this observation. first, ifitm members inhibit hiv-1 infectivity to varying degrees (ifitm3 > ifitm2 > ifitm1) [51] yet all three proteins inhibit certain env proteins to a similar extent [55] . second, ifitm3 overexpression in t cells reduces virus infectivity but has no detectable impact on hiv-1 env levels in infected cells or purified virions [51] , and a lack of effect has been reported by others using diverse experimental systems [5, 48, 56] . third, we now know that the negative "imprinting" of virions by ifitm3 occurs with various dna and rna viruses, apparently in the absence of envelope glycoprotein perturbation [56] . therefore, follow-up experiments will require the study of endogenous ifitm3 in various cell types as well as the effects of type-i interferon on hiv-1 env maturation and virion incorporation. overall, it is unclear whether modification of env is directly responsible for the inhibition of hiv-1 virion infectivity, nor is it known whether the virion incorporation of ifitm3 is critical for this effect. it is possible that both play mechanistic roles which are not mutually exclusive. of note, enriched plasma membrane localization of ifitm3 enhances both anti-hiv-1 activity as well as ifitm3 incorporation into virions, suggesting a functional link [5] . nonetheless, the recent identification of env variants that are resistant to the ifitm3-mediated restriction of virion infectivity confirms this viral protein as an important determinant. it was shown that a particular ccr5-tropic strain (ad8) of env is unaffected by ifitm3 overexpression in virus-producing cells, exhibiting no defects in virion infectivity. this resistance phenotype maps to the v3 loop of gp120 and is transferrable to other env isolates [57] . another feature of hiv-1 strains that are most sensitive to the ifitm3-imposed block to virus fusogenicity is the use of env proteins with high sensitivity to both soluble cd4 and the neutralizing antibody 17b, which recognizes a cd4-induced epitope. therefore, virus susceptibility to the infectivity defect caused by ifitm3 is linked to conformational changes in the cd4-binding site of env, although the stability or clustering of virion-incorporated env trimers may also be involved. based on what we know about the changes to cellular membrane fluidity caused by ifitm proteins, it is possible that similar disturbances in the viral lipid bilayer containing env are involved in the restriction of virion infectivity. another important observation made in these studies is that resistance to one antiviral function of ifitm3 is associated with resistance to another, since the ad8 strain exhibits relatively less sensitivity to the effects of ifitm3 in both target cells and producer cells, and tf strains are completely resistant to both modes of restriction [48, 56] . thus, ifitm3 performs at least two antiviral activities against hiv-1 for which there may be mechanistic overlap. for reasons that are not yet clear, cd4 engagement by env is an important determinant for whether virus is restricted at the level of target cells and producer cells [48, 55] . it is possible that cd4 binding and the conformational changes in env that follow (which dictate interactions with co-receptors) affect the route and kinetics of hiv-1 entry into cells [58] , and in turn, affect sensitivity to ifitm-mediated antiviral activities. globally, the multiple constraints that ifitm proteins place on hiv-1 env suggest that these cellular factors contribute to the genetic bottleneck that selects for interferon-resistant virus at the earliest stages of hiv-1 infection in vivo [59] . the study of diverse viruses has been central to discoveries involving the ifitm protein family. the physiological importance of ifitm activity has been clearly demonstrated for those viruses that exhibit the greatest sensitivity in cell culture experiments, such as iav. however, additional functions were found using viruses that are actually resistant to the block at cellular entry. in both cases, the use of transgenic mice deficient for the murine ifitm locus was central to establishing the in vivo significance of ifitm proteins in diseased and healthy states. downregulation of ifitm3 in target cells can lead to increased cytopathicity upon virus challenge in vitro, which has been attributed to its role as a protective barrier preventing virus entry and subsequent replication [60, 61] . nonetheless, in vivo experiments using mouseadapted virus strains have been critically important to understanding the full spectrum of downstream consequences resulting from ifitm deletion, ranging from cell death to the manifestation of virus-associated disease propagated throughout the host organism. for example, accelerated disease progression and mortality are observed in ifitm3-deficient mice challenged with iav, suggesting that ifitm3 confers a survival advantage to cells exposed to virus [62, 63] . indeed, the expression of ifitm3 may preserve the integrity and function of cells that would otherwise be destroyed by uninterrupted viral replication [64] . it was reported that elevated levels of ifitm3 protein in memory cd8+ t lymphocytes, which kill virus-infected cells and serve as important targets themselves for iav infection in the lung, promotes their survival during infection and enables long-term defense against future viral exposures [65] . in a murine model of chikungunya virus (chikv), mice lacking ifitm3 sustained greater joint swelling which was correlated to increased viral burden and pro-inflammatory cytokine production [66] . ifitm3-deficient mice were also found to be more vulnerable to lethality in the context of wnv. while wnv disease is associated with neurotropism, ifitm3 limited pathogenesis by suppressing viremia first in peripheral organs [67] . thus, by acting as the first line of defense in cells exposed to invading viruses, ifitm proteins prevent a plethora of adverse events that can lead to disease and death. yet unexpected antiviral activities associated with ifitm3 were revealed during murine cytomegalovirus (cmv) infection. in this case, ifitm3 does not limit virus entry into cells (the protein was previously shown to facilitate cmv virion morphogenesis [68] ). rather, cmv infection in ifitm3-deficient mice led to much higher production of the pro-inflammatory cytokine interleukin-6 (il-6) [69] . the result was dysregulation of cellular immunity and impaired control of virus replication, suggesting that murine ifitm3 plays a part in regulating cytokine production important for resolution of virus infection. another example in which ifitm3 could be found performing a non-canonical activity was in the setting of sendai virus (sev) infection. this murine paramyxovirus has been shown to be insensitive to the ifitm3-mediated block to virus entry [70] . notwithstanding, it was shown that ifitm3 regulates interferonbeta production triggered by sev infection in human cell lines [71] . that is, overexpression of ifitm3 inhibited production of the cytokine while knockdown had the opposite effect. the authors propose that ifitm3 associates with the transcription factor driving interferon-beta gene expression, irf3, and accelerates its turnover in autophagosomes. since interferon-beta itself induces the expression of ifitm3, this implies a role in negative feedback of the interferon pathway [71] . this finding warrants testing in an in vivo murine model, but it seems likely that the effects of ifitm proteins on interferon signaling will yield much interest from researchers and clinicians in the field of microbial pathogenesis. naturally occurring variation in ifitm genes has provided an additional genetic platform for the study of pathogenic virus infections important to human public health. population-level associations have been drawn between single-nucleotide polymorphisms (snp) in ifitm3 and severe outcomes following iav infection, corroborating an important in vivo function but lacking a mechanistic explanation. the most cited example is rs12252-c, a nonsynonymous mutation in the first coding exon of ifitm3, which was predicted to affect mrna splicing and to produce protein with an amino-terminal truncation [63] . this minor variant was found to be enriched in a group of individuals hospitalized following iav infection, and it is found at relatively high frequency among asian ethnic groups. however, the reasons for the disease association has remained elusive, as the truncated isoform of ifitm3 predicted to result from the snp had not been detected in cells or individuals. it has now been reported that cell lines derived from individuals who are homozygous for rs12252-c express a mrna transcript encoding full-length ifitm3, whereas the shorter isoform was not found [50] . over the course of several years, a slew of publications has either confirmed or refuted the genetic association between rs12252-c and iav infection outcome. a recent and comprehensive study now links an additional variant in the ifitm3 locus to severe influenzaassociated illness in three independent cohorts, albeit the snp identified is distinct from rs12252-c. known as rs34481144-a, it is found in the 5' untranslated region and is linked to lower ifitm3 protein levels in cells [72] . experimental evidence showed that the snp controls gene promoter activity via decreased binding of transcription factor irf3 and increased binding of ctcf, which promote and repress ifitm3 transcription, respectively. in agreement with the previous finding that ifitm3 preserves antiviral cd8 + t cells [65] , individuals harboring rs34481144-a contained reduced numbers of these cells in lung airways during infection [72] . the evolutionary pressures responsible for the maintenance of this 'defective' allele in humans are unclear, but its existence may allude to the involvement of ifitm3 in cellular processes requiring fine-tuned protein expression. before it was realized that ifitm proteins perform broad-spectrum antiviral activities, they were implicated in pathways important to embryonic development and cancer (exhaustively reviewed in [73] ). a crucial contribution to developmental processes seems unlikely, since transgenic mice in which the murine ifitm locus is knocked out exhibit no obvious abnormalities apart from being fat, suggesting a metabolic irregularity [74] . there is ample evidence, on the other hand, for both positive and negative regulation of ifitm expression during tumorigenesis. while detailed descriptions were previously lacking, recent developments have provided a mechanistic grounding to many observations linking ifitm proteins to cell proliferation, adhesion, and migration. in addition to the interferon signaling pathways, ifitm gene expression is controlled by a number of cascades involved in cellular homeostasis (fig. 2) . for example, growth factor receptors lead to downstream upregulation of ifitm2 upon triggering by insulin-like growth factor-1 (igf1), which relies on signaling via phosphatidylinositol-3-kinase (pi3k) and akt kinase. in gastric cancer cohorts, the upregulation of ifitm2 is associated with accelerated disease progression and shorter survival time [75] . silencing of ifitm2 in gastric cancer cells decreased cell proliferation, migration, and metastasis, while ifitm2 depletion in a mouse model resulted in dramatic decreases in tumor size [75] . ifitm3 has also been functionally implicated in this cancer type, as its knockdown suppressed tumor cell migration, invasion and proliferation capacity [76] . there is also evidence that ifitm3 acts indirectly to affect these cellular properties through regulation of other cellular proteins, such as osteopontin [77] . recently, the involvement of ifitm3 in cancer was further extended to include spatial regulation of the src oncoprotein. the trafficking of src between focal adhesions and the cell interior, which is regulated by activating molecule in beclin1-regulated autophagy (ambra1) and focal adhesion kinase (fak), is important for cell migration and metastasis. ambra1 redirects src towards autophagosomes to disfavor substrate attachment and favor cell movement in an ifitm3-dependent manner [78] (fig. 2) . collectively, the twin antiviral proteins ifitm2 and ifitm3 may serve as biomarkers for tumorigenic phenotypes as well as targets for anticancer interventions. it remains to be determined which domains of ifitm proteins are involved in the control of cellular properties and if they overlap with those known to be involved in antiviral immunity. for example, just as the subcellular localization of ifitm proteins is important to their antiviral activities [11] , it may also affect their involvement in cellular housekeeping functions. as a proof of principle, ifitm2 can act as a cell surface receptor for a secreted form of bag3, which promotes pro-survival signaling through the pi3k and p38 mapk pathways [79] . several reports highlighting ifitm-mediated impacts on basic cellular processes provide further opportunities to test how these proteins inhibit virus infections. a focus on functional roles of endogenous, rather than overexpressed, ifitm proteins has been crucial to this end. a prime example is the observation that deletion of the ifitm locus in murine cells led to interruption of clathrin-mediated endocytosis and loss of acidity within endosomes, suggesting that endogenous ifitm proteins might positively regulate these processes in vivo [80] . this possibility is further supported by work in astrocytes showing that knockdown of ifitm3 inhibits clathrindependent endocytosis [81] . as previously mentioned, the ifitm-deficient mice exhibit an age-related obesity associated with defects in leptin signaling, which could be explained by disturbances in ligand-receptor internalization [74] . interestingly, the ifitm proteins are also involved with the endocytosis-associated protein caveolin-1 (cav-1), with consequences for cell signaling events [82, 83] . together, these data hint that impacts on endocytic trafficking may also contribute to the mechanisms by which ifitm proteins inhibit virus entry. while the antiviral effects of ifitm proteins are generally assumed to manifest at the stage of virus-cell fusion, these data suggest that an effect on virus internalization must be carefully considered on a case-by-case basis. for example, since knockdown or knockout of ifitm3 leads to decreases in clathrin-mediated endocytosis and increases in virus entry, it is possible that ifitm3 promotes the trafficking of incoming virions into endocytic pathways that are acidified, degradatory and/or otherwise non-productive. since ifitm proteins share a high degree of sequence homology (especially ifitm2 and ifitm3, which differ by only 13 amino acids), future efforts must assess the specific or redundant role for each ifitm family member when describing new activities. only then can the functional utility of each be properly understood, be it during virus infection or basal cellular homeostasis. furthermore, it is important to test whether interferon signaling serves as a 'switch' to promote antiviral functions over housekeeping ones. we expect that boosting efforts towards understanding the host factors interacting with ifitm proteins will expose novel purposes in cells, which may, in turn, reveal additional ways by which these proteins interfere with virus infections. cell-based experiments have identified many binding partners of ifitm proteins, but in most cases, it is unclear whether cytokine regulation; ifitm3 is a negative regulator of the interferon response because it accelerates the turnover of irf3 in autophagosomes. it also suppresses the production of il-6, a pro-inflammatory cytokine which, itself, can also induce the expression of ifitm genes. ifitm2, in contrast, promotes the upregulation of il-6 by acting as a cell surface receptor for secreted bag3. as bag3 is also a well-characterized chaperone for selective autophagy, it will be of interest to determine if ifitm2 also participates with bag3 in autophagy-related processes. cell migration and invasion; ifitm3 is a central component of a multi-protein interaction involving src, fak, and ambra1, which is important for regulating cell adhesion and movement. ifitm3 assists in the subcellular trafficking of src between focal adhesion points and autophagosomes. cell growth, proliferation, and cell cycle regulation; interferon signaling is known to negatively regulate cell division and growth via stat signaling, with ifitm proteins serving as downstream effectors. ifitm1 interacts with caveolin-1 (cav-1) to inhibit erk/mapk signaling, a pathway which stimulates cell proliferation when active. ifitm1 also stabilizes p53, a tumor suppressor with anti-proliferative functions these interactions are direct or specific. interrogation of potential interactors in transgenic mice will help clarify their precise role and enable prioritization of different hypotheses. in general, increased experimental crosstalk between virology and cell biology will provide much needed opportunities for discovery and will facilitate the development of host-directed therapies targeting ifitm proteins in the setting of infection and cancer. the broad-spectrum antiviral functions of ifit and ifitm proteins the dispanins: a novel gene family of ancient origin that contains 14 human members evolution of vertebrate interferon inducible transmembrane proteins evolutionary dynamics of the interferon-induced transmembrane gene family in vertebrates natural mutations in ifitm3 modulate post-translational regulation and toggle antiviral specificity ifitms restrict the replication of multiple pathogenic viruses ifitm proteins-cellular inhibitors of viral entry the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitm3 inhibits influenza a virus infection by preventing cytosolic entry distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus regulation of the trafficking and antiviral activity of ifitm3 by post-translational modifications combined approaches of epr and nmr illustrate only one transmembrane helix in the human ifitm3 interferon-induced transmembrane protein 3 is a type ii transmembrane protein ifitm3 requires an amphipathic helix for antiviral activity palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 the palmitoyltransferase zdhhc20 enhances interferoninduced transmembrane protein 3 (ifitm3) palmitoylation and antiviral activity phosphorylation of the antiviral protein interferon-inducible transmembrane protein 3 (ifitm3) dually regulates its endocytosis and ubiquitination identification of an endocytic signal essential for the antiviral action of ifitm3 e3 ubiquitin ligase nedd4 promotes influenza virus infection by decreasing levels of the antiviral protein ifitm3 a balancing act between ifitm3 and irf3 membrane curvature and its generation by bar proteins acid-dependent viral entry ph optimum of hemagglutinin-mediated membrane fusion determines sensitivity of influenza a viruses to the interferon-induced antiviral state and ifitms host cell factors and functions involved in vesicular stomatitis virus entry ifitm proteins restrict viral membrane hemifusion ifitm3 restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion amphotericin b increases influenza a virus infection by preventing ifitm3-mediated restriction the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms the interferon-induced transmembrane proteins, ifitm1, ifitm2, and ifitm3 inhibit hepatitis c virus entry rhinovirus uses a phosphatidylinositol 4-phosphate/cholesterol counter-current for the formation of replication compartments at the er-golgi interface noroviruses co-opt the function of host proteins vapa and vapb for replication via a phenylalaninephenylalanine-acidic-tract-motif mimic in nonstructural viral protein ns1/2 cd81 (tapa-1): a molecule involved in signal transduction and cell adhesion in the immune system ifitm1 is a tight junction protein that inhibits hepatitis c virus entry the role of tetraspanins in fusion evidence showing that tetraspanins inhibit hiv-1-induced cell-cell fusion at a post-hemifusion stage tetraspanins cd9 and cd81 function to prevent the fusion of mononuclear phagocytes formation of syncytia is repressed by tetraspanins in human immunodeficiency virus type 1-producing cells interferon induction of ifitm proteins promotes infection by human coronavirus oc43 zmpste24 defends against influenza and other pathogenic viruses on the whereabouts of hiv-1 cellular entry and its fusion ports unique features of hiv-1 spread through t cell virological synapses tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu the ifitm proteins inhibit hiv-1 infection a diverse range of gene products are effectors of the type i interferon antiviral response primate lentiviruses are differentially inhibited by interferon-induced transmembrane proteins virion background and efficiency of virion incorporation determine susceptibility of simian immunodeficiency virus env-driven viral entry to inhibition by ifitm proteins resistance of transmitted founder hiv-1 to ifitm-mediated restriction coupling of replication and assembly in flaviviruses î�20 ifitm2 differentially restricts x4 and r5 hiv-1 ifitm proteins incorporated into hiv-1 virions impair viral fusion and spread ifitm proteins are incorporated onto hiv-1 virion particles and negatively imprint their infectivity ifitm proteins restrict hiv-1 infection by antagonizing the envelope glycoprotein nonhuman primate ifitm proteins are potent inhibitors of hiv and siv the v3 loop of hiv-1 env determines viral susceptibility to ifitm3 impairment of viral infectivity interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of ifitms the v3-loop of hiv-1 env determines viral susceptibility to ifitm3 impairment of viral infectivity delineating cd4 dependency of hiv-1: adaptation to infect low level cd4 expressing target cells widens cellular tropism but severely impacts on envelope functionality resistance to type 1 interferons is a major determinant of hiv-1 transmission fitness the ifitms inhibit zika virus replication zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells ifitm3 limits the severity of acute influenza in mice ifitm3 restricts the morbidity and mortality associated with influenza respiratory dc use ifitm3 to avoid direct viral infection and safeguard virus-specific cd8 + t cell priming enhanced survival of lung tissue-resident memory cd8 + t cells during infection with influenza virus due to selective expression of ifitm3 the interferonstimulated gene ifitm3 restricts infection and pathogenesis of arthritogenic and encephalitic alphaviruses the interferon-stimulated gene ifitm3 restricts west nile virus infection and pathogenesis human cytomegalovirus exploits interferon-induced transmembrane proteins to facilitate morphogenesis of the virion assembly compartment the antiviral restriction factor ifn-induced transmembrane protein 3 prevents cytokine-driven cmv pathogenesis palmitoylation on conserved and nonconserved cysteines of murine ifitm1 regulates its stability and anti-influenza a virus activity ifitm3 inhibits virus-triggered induction of type i interferon by mediating submit your next manuscript to biomed central and we will help you at every step: autophagosome-dependent degradation of irf3 snp-mediated disruption of ctcf binding at the ifitm3 promoter is associated with risk of severe influenza in humans the small interferon-induced transmembrane genes and proteins age-related onset of obesity corresponds with metabolic dysregulation and altered microglia morphology in mice deficient for ifitm proteins igf1/igf1r/stat3 signaling-inducible ifitm2 promotes gastric cancer growth and metastasis mechanism and biological significance of the overexpression of ifitm3 in gastric cancer interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo ifitm3 reduced opn expression ambra1 spatially regulates src activity and src/fak-mediated cancer cell invasion via trafficking networks bag3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages interferon-inducible transmembrane proteins of the innate immune response act as membrane organizers by influencing clathrin and v-atpase localization and function astroglial ifitm3 mediates neuronal impairments following neonatal immune challenge in mice binding of ifitm1 enhances the inhibiting effect of caveolin-1 on erk activation ifitm1 promotes the metastasis of human colorectal cancer via cav-1 we thank the late mark wainberg for his support and for his efforts at the retrovirology journal. os and aac conceived the manuscript. gs os and aac wrote and edited the manuscript. all authors read and approved the final manuscript. the authors hereby state that no competing interests exist. not applicable. all authors consent to the publication of this manuscript. not applicable. work in the lab of aac is supported by the intramural research program of the nih, national cancer institute, center for cancer research. work in the lab of os is supported by grants from the anrs, the vaccine research institute, the "chikv-viro-immuno" anr-14-ce14-0015-01 and "timtamden" anr-14-ce14-0029 projects, the labex ibeid program anr-10-ihub-0002, a gilead hiv cure grant and institut pasteur. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-309900-4nln90jn authors: doornekamp, laura; stegers-jager, karen m.; vlek, odette m.; klop, tanja; goeijenbier, marco; van gorp, eric c. m. title: experience with a multinational, secondary school education module with a focus on prevention of virus infections date: 2017-07-12 journal: am j trop med hyg doi: 10.4269/ajtmh.16-0661 sha: doc_id: 309900 cord_uid: 4nln90jn worldwide, virus infections are responsible for many diseases in terms of morbidity and mortality. vaccinations and therapies are only available for relatively few virus infections and not always where they are needed. however, knowledge of transmission routes can prevent virus infection. in the context of this study, we measured the effects of a secondary school education module, named viruskenner, on knowledge, attitude, and risk behavior as these relate to virus infections. a nonrandomized intervention study was conducted between april and august 2015 to assess the effect of this 2-month education module on knowledge, attitude, and behavior of 684 secondary school students in the netherlands, suriname, and indonesia. for the netherlands, a control group of a further 184 students was added. factor analysis was performed on questions pertaining to attitude and behavior. comparative analyses between preand posttest per country were done using multiple linear regression, independent sample t-tests, and one-way analysis of variance. these showed a significant increase in knowledge about virus infections and the prevention of infectious diseases among the dutch and surinamese groups, whereas a trend of increased knowledge was evident among the indonesian participants. the dutch control group showed an overall decrease in knowledge. regression analyses showed that there was a significant interaction effect between participation and time on knowledge, attitude, and awareness and behavior and risk infection. attitudes improved significantly in the intervention group. pearson correlation coefficients between knowledge, attitude, and behavior were found to be positive. viruses are responsible for almost half of all emerging infections worldwide and are among the most emerging pathogens. [1] [2] [3] most virus infections are not treatable with antivirals and neither are they preventable with vaccines. therefore, education plays a key role in raising awareness for infectious diseases and preventing the spread of virus infections. 4 a population that is aware of the different ways a virus can be transmitted and does know how to embed effective preventive methods in daily life can protect themselves against virus infections. this principle is based on the knowledge, attitude, behavior (kab) model, showing that increased knowledge can change people's attitudes and lead to behavioral change. [5] [6] [7] a foundation for health-related attitudes and behavior is laid in early stages of life. following many theories, the likelihood of changing attitude is high in adolescence. 8 also, the world health organization (who) states with their health promoting school framework that schools are a good environment to start promoting health. 9 five years ago, a consortium of scientists and teachers developed a school-based education module named viruskenner, regarding virus infections. this module aimed to teach students how to prevent virus infections. the module started with one secondary school in the netherlands and evolved to a project with eight different secondary schools in the netherlands. in 2014, the first surinamese school joined and in 2015 the first indonesian school. these two countries were already involved with the organizing institute by an international collaboration on emerging infectious disease population studies, facilitating easy communication and logistics. the viruskenner module was extensively evaluated by independent researchers in the early years of the project. the conclusions of these evaluations led to improvement of the module and the questionnaires used. for example, in 2012, the concept of students being coached by an infectious disease expert was introduced. when becoming an international education module, it was interesting to see the impact of viruskenner in different countries on knowledge, attitudes, and behavior as they relate to virus infections, and find out which educational factors play a role in these changes. educational programs that address infectious diseases are quite common, although most education is focused on a specific infection or a group of infections, particularly human immunodeficiency virus (hiv) and other sexually transmitted infections. a recent systematic review and meta-analysis evaluated 64 school-based sex education programs in middleand low-income countries. most of these programs (55 of 64) focused on comprehensive sex education, with the remaining nine focusing on sexual abstinence. about half of the studies (33) were included in the meta-analysis and showed an overall positive effect on hiv-related knowledge, condom use, the initiation of sexual intercourse, the number of sexual partners, and self-efficacy. 10 although hiv is among the virus infections that place the highest burden on society, it is not the only virus that significantly impacts global health. besides hiv, lower respiratory tract infections (e.g., influenza), and diarrheal diseases (like norovirus) also belong to the 10 leading contributors to the global burden of disease. 11 furthermore, arthropodborne diseases (like dengue) have a very high incidence. 12 remarkably, virus infections other than hiv are less frequently addressed in education modules. for example, only a few trials were carried out to measure the impact of an educational intervention for viral hepatitis, human papillomavirus (hpv), dengue, and influenza. [13] [14] [15] [16] [17] most of the educational interventions that were analyzed showed positive results in improving knowledge and attitude pertaining to the subject of the intervention. given the success of education programs about hiv, education modules about other virus infections that have global impact might also work. we developed an education module that focuses on multiple viruses with different transmission routes and all with global impact: hiv, dengue, hantavirus, chikungunya, middle east respiratory syndrome (mers) coronavirus, hpv, norovirus, viral hepatitis, measles, and influenza. we studied the efficacy and success of the education module in three countries, netherlands, suriname, and indonesia, each differing in culture, circulating viruses, and infection pressure. the education module aims to effectively increase knowledge, attitudes, and behavior regarding several virus infections in each of these different circumstances. participants and setting. schools in three countries participated in this nonrandomized intervention study: four in the netherlands, a high-income country in europe; one in suriname, an upper middle-income country in latin-america; and one in indonesia, a lower middle income country in southeast asia. 18 the effect of the education module was measured per country, by comparing the results of a pre-and posttest. the situations per country, for example, culture and school system, were too different to make a fair comparison between countries. however, the target group for the education module is the same in each country and the concept of the module and measurements were as comparable as possible. secondary schools in the netherlands, suriname, and indonesia had been invited to apply to participate in the education module with their 10th grade students (generally 14 or 15 years of age). all schools were well-known public schools for students with an above-average socioeconomic status. the school in suriname that participated had about 840 students and was located in paramaribo, the capital and largest city of the country in inhabitants. the indonesian school that participated was located in surabaya, the second largest city in the country. this was a senior high school (grade [10] [11] [12] and had about 1,200 students. the four schools in the netherlands ranged in number of students from 1,600 to 2,400 and were from different regions but all in the dutch urban agglomeration, including one school from amsterdam, the capital city of the netherlands. the 10th grade is the final stage of the junior high school in the netherlands, which means that all students have, until then, followed the same subjects and have expressed their interest in the choice for a special curriculum. for example, a beta scientific curriculum, which includes the following subjects: biology, physics, chemistry, and mathematics. in the netherlands, the schools that were invited to participate were all schools that offer students an option for technasium, which is an elective course for students interested in beta scientific subjects. 19 the participating students had all chosen this special curriculum with additional technical courses. information about the module was disseminated via the project website and the technasium network coordinator. a control group for the dutch intervention group was selected at one of the participating dutch schools. thus, although they had not opted for the technasium curriculum, they do have a similar background and social environment. school curricula are defined differently in each country. in the netherlands, students choose a profile and we defined "nature and science" and "nature and health" as scientific profiles. in suriname, students can choose biology, and we defined this as a scientific profile. indonesian students can choose between a social profile (ilmu pengetahuan sosial [ips]) and a science profile (ilmu pengetahuan alam [ipa]). we defined ipa as a scientific profile. in both suriname and indonesia, schools that matched most closely, in terms of grade and education level, with the dutch intervention group were invited to participate in the study. of the dutch schools, two were preuniversity education level (known in the netherlands as "vwo") and two were mixed preuniversity education level and advanced general secondary education (known in the netherlands as "havo"). the surinamese participants were from one vwo school, which is comparable with the dutch vwo education level. these surinamese participants can therefore be seen as preuniversity education level. 20 the indonesian participants were from one sekolah menengah atas (sma) (high school), which is comparable with havo in the netherlands and internationally known as advanced general secondary education. 21 the dutch control group consisted of students with preuniversity education level and advanced general secondary education level. design of the intervention. the viruskenner education module is based on the "learning-by-doing" principle. students are challenged to create a prevention tool for a specific virus infection. by involving students in real-life science-based problems and stimulating active learning (searching for information, test possible solutions and present their idea) a high impact can be achieved. [22] [23] [24] in each country, the 2-month module started with a national opening day, during which all participants of that country were introduced to the field of infectious diseases and viruses by means of four short lectures from experts in the field of virology, public health, and infectious diseases. an optimal learning effect can be reached by bringing students in contact with experts. 25 so, in all countries one or two dutch experts from the department of viroscience in the erasmus medical center in rotterdam were assigned to a class to coach them during the project. the students were supposed to work in groups of four to six students in competition with the other groups. 22, 26 each group worked on one of the viruses of the subject list including hiv, dengue, hantavirus, chikungunya, mers coronavirus, hpv, norovirus, viral hepatitis, measles, and influenza. students developed a prevention tool to disseminate this knowledge among their peers and, in doing so, help prevent virus infections that impact local or global health. during the three national final days (one in each country), the best groups per class, selected by the teachers and coach during a school final, presented their results and final product to their peer students and a jury. this independent jury was selected per country and based on proven expertise in virology, communication strategy, and/or overall creativity. in each country, the jury chose two winners: the most informative presented prevention tool and the most creative prevention tool. the study was conducted between april and august 2015. a pretest was performed 1 or 2 days before the start of the module to assess their basic knowledge, attitude, and behavior; a posttest 5-7 days after its completion to let the information settle in their memory and give the students some time to evaluate their attitude and behavior a few days after the final day. other measurement instruments were used to get additional information ( figure 1 ). during the intervention, students could use the modules' website (www.viruskenner.nl) and other supportive resources, like a youtube channel and a facebook page (all in dutch and english and available for all participating countries), to find more information on the project and on virus infections and to disseminate information about their prevention tools. [27] [28] [29] instruments. the effect of the education module was measured by the kab model. given that there was no validated instrument to assess knowledge, attitude, and behavior regarding several viral infections, we used a self-designed questionnaire (supplemental table 1 ). the questionnaire was based on 5 years' experience with the education module. a team comprising two senior virologists, a communication scientist, and an education expert developed the questionnaire, which was refined after a pilot with a group of 60 students from a school similar to the participating schools. the questionnaires used for the dutch and surinamese schools were in dutch. the questionnaires for the indonesian students were first translated into english by a dutch researcher and then into indonesian by a native speaker of the language. the pre-and posttest questionnaires addressed five areas: 1) sociodemographic factors, 2) stigma and fear, 3) attitude and behavior, 4) knowledge on viruses and infectious diseases in general, and 5) the opportunity to write down questions or comments about the questionnaire or module. the posttest had an additional category-6) perceptions of the project. a principal component analysis (pca) with varimax rotation was performed for the attitude and behavioral questions on the results of the pretest questionnaires, as suggested in literature. 30 varimax was the preferred rotation because this results in a small number of factors per variable and a small number of variables per factor. this is the most popular type of rotation because it makes the interpretation of the data more reliable and easier. 31 one of the behavior items ("i do not use a condom when i have sexual intercourse") was excluded because of more than 10% missing values. the remaining missing values were randomly spread over the sample population. the sample size was big enough to delete these cases list wise. the kaiser-meyer-olkin (kmo) value is a statistic that measures how much two random variables correlate. a kmo value greater than 0.8 represents a small partial correlation which makes a factor analysis more useful. in this study, the kmo value was 0.849, which means there were relatively compact patterns of correlations and the factor analysis would provide reliable components. 32 the number of extracted factors was based on the objective and interpretability criteria mentioned in schönrock-adema and others: 1) the screen test, 2) eigenvalues > 1.5, 3) > 5% of the variance explained by all factors, and 4) interpretability. however, the criterion of eigenvalue > 1.5 led to only two components, which was not interpretable. therefore, we set the norm of an eigenvalue back to greater than one (kaiser's criterion). 33, 34 the pca with varimax rotation finally resulted in four components. the reliability per component was calculated by cronbach's alpha (table 1 ). internal consistency for the components "attitude and awareness" and "behavior and life science" was above 0.7 and therefore acceptable. the components "attitude and risk infections" and "behavior and risk infection" should be interpreted with caution, because of the diversity of the constructs. 35 an additional instrument to measure knowledge was a live multiple-choice quiz, which was implemented at the end of the final day. in the netherlands and suriname, portable electronic devices (keypad and software from interactive voting system ® ) were used by the students to answer 40 knowledge questions. in indonesia, these portable electronic devices were not available, so the knowledge quiz was done by voting with colored papers; therefore, recording these results was not possible. to obtain more information about factors that influenced the impact of the education module, teachers of all four participating schools in the netherlands were interviewed when they had completed the education module. the aim of this additional qualitative component was to determine possible confounders which might have influenced the difference in outcomes between the pre-and posttests and to find out whether the teachers noticed increased knowledge or improved attitude and/or behavior among their students. although the teacher interviews were carried out in the netherlands only, the module was evaluated in each country. in suriname and indonesia, the project was evaluated with the local organizing teams but not per individual teacher. in the indonesian and surinamese culture, hierarchy is strong and extensive evaluation uncommon. therefore, the teachers preferred a general evaluation with the head of the school. however, we do feel these interviews were less helpful because the heads of the schools were not closely involved in the project. the dutch teacher interviews were semistructured and took about 30 minutes each. questions that were asked included "how was the contact with the coaches?" and "what have the students learned during the project?" teachers were interviewed in their classrooms after the classes had filled out the posttest questionnaire. finally, user data from the website and social media were analyzed after the completion of the module to find out which supportive resources were most popular during the education module (supplemental figure 1) . outcomes. the primary outcomes in this study were knowledge, attitude, and behavior and stigma and fear. stigma and fear, attitude, behavior were measured on a 5point likert scale. all these outcomes ranged from 1 (strongly disagree) to 5 (strongly agree). stigma and fear were measured with two questions and mean values were calculated. the outcome "stigma" was used in this study to describe a negative thought regarding people with a hiv infection. the stigma was expected to be high before the module started. by gaining knowledge, the stigma could be decreased. the outcome "fear" in this study aims to measure how afraid people are to get infected in case of a large outbreak; at the time of this study, ebola was the best example. the attitude and behavior questions were subdivided into four components by the factor analysis and the mean score per component was calculated. (table 1) . unstandardized coefficients (b) as outcomes of the regression analysis showed which factors contribute significantly to these four attitude and behavior components and to knowledge. the outcome knowledge represented the student's knowledge regarding infectious diseases in general and the viruses in specific that were included in the education module. knowledge was measured in the questionnaire by means of the responses to 32 questions, which had to be answered with "true" or "false." each correct answer resulted in one point, an incorrect answer in zero points. the mean percentage of all knowledge questions that were answered correctly was calculated per group and ranged from 0% to 100%. knowledge outcomes from the quiz were calculated in percentages. as a secondary endpoint, the perceptions of the students about their participation in the project were evaluated. ten statements measured if students enjoyed working on the project and if they thought the project was informative. this was scored on a 5-point likert scale and ranged from 1 (strongly disagree) to 5 (strongly agree). data management and analysis. the questionnaires were read by the open source optical mark recognition program sdaps (benjamin berg, karlsruhe). correct reading was checked manually by two different persons. all data were imported into one database and analyzed with ibm spss version 21. all questionnaires in which less than 90% of the knowledge questions had been answered were excluded. cases that showed a variance equal to zero in the likert scale questions were excluded for analysis on these outcomes. items with more than 10% missing values were deleted. in all analyses, p < 0.05 was considered significant. descriptive analyses were performed to calculate the frequencies of students' characteristics and pearson's χ 2 test was used to identify significant differences between the characteristics of the groups in the pre-and posttest. correlations between knowledge, attitude, and behavior were calculated for all students in the intervention group, with a pearson's coefficient. the average knowledge per country in the pre-and posttest situations was compared by an independent sample t-test. effect sizes (es) were calculated with cohen's d. effect sizes greater than or equal to 0.30 were considered medium, and those greater than or equal to 0.50 as large. 36 multiple linear regression analysis was used to find factors that influenced the knowledge, attitude, and behavior outcomes. time point (pre-and posttest) and participation (intervention and control group) and the interaction between these two variables were added as independent variables, as well as gender, age, education level, school, and country. tolerance values were computed to assess multicollinearity. values below 0.2 were viewed as potentially problematic. 30 stigma and fear were compared between pre-and posttest with a one-way analysis of variance (anova). the sample size allowed us to calculate differences between the components of the factor analysis in pre-and posttest per country with a one-way anova test. perceptions of the project were measured only after the module had finished. means and standard deviation were summarized per country. the data set is provided in the supplementary materials. ethics. the study was carried out in accordance with the declaration of helsinki. according to dutch law, this study was exempt from medical ethical approval requirements. the technasium network in the netherlands approved this study to be performed at the dutch technasium schools and informed the students and parents. in suriname and indonesia, the headmasters of the schools approved conducting the viruskenner module and evaluations at their schools and informed the students and their parents. participation was voluntary and anonymity was guaranteed. participants and setting. in 2015, a total of 684 (of 738) secondary school students participated in the viruskenner education module. two of the participating schools in the netherlands, dropped out (54 of 260 dutch students, representing 20.7% of them) because of noncompletion of the module and evaluation program. one school dropped out because the teacher got sick after the kick-off and the other school could not attend the final day because it clashed with another school activity that day (figure 2 ). in suriname, 158 students participated and there was no dropout. this was also the case in indonesia, where all 320 students completed the education module. response rates for the netherlands were 95.6% of participants for the pretest and 70.9% for the posttest. in suriname, these percentages were 90.5 and 96.8%, respectively, and for indonesia 97.2 and 99.1%, respectively. the control group had a response rate of 100% for the pretest and 73.4% for the posttest. table 2 presents the pre-and posttest characteristics of the module participants from all three countries and the control group. in all groups, except the surinamese group, the age category in the posttest was significantly higher than in the pretest. however, for gender and education, the characteristics did not show any significant differences between the pre-and posttest per country. in indonesia, the preference for science was significantly higher in the posttest than in the pretest. on average, in the netherlands, most of the participating students were male, whereas in indonesia, they were mostly female. generally speaking, in the netherlands, the students from both the control group and the intervention group were significantly younger than average. the pretest showed that on average more students attended preuniversity education in the netherlands, suriname, and in the control group. in indonesia, all students attended advanced general secondary education. in the posttest, the percentage of preuniversity education students in the dutch intervention group rose to 65.3%. in the control group, this percentage decreased to 64.1%. in both the pre-and posttest, the amount of participants from the intervention group in the netherlands and suriname that chose scientific profiles was not significantly different from the average. the control group consisted of less students with scientific profiles than average and indonesia had more students with science-related profiles. correlations between knowledge score and attitude and behavior. pearson's coefficients showed a positive and significant correlation between the knowledge scores and all four components regarding attitude and behavior (table 3) . knowledge was most strongly correlated with attitude and awareness (r = 0.20). students who scored higher on attitude and awareness also scored higher on behavior regarding risk of infection (r = 0.47) and behavior regarding life sciences (r = 0.51). knowledge. during the project, the answer to one of the 32 knowledge questions changed, due to the mers epidemic in south korea. because of the confusion surrounding this question, we decided to exclude it from the analysis. analyses per country showed differences in achieved knowledge ( figure 3) , with mean knowledge increasing in all three participating countries. for suriname and the netherlands, this increase was significant (p < 0.001). the overall effect size (cohen's d) for all intervention groups was 0.43, which represents a medium effect. at 0.77, the effect size for suriname was the highest. the effect size for the netherlands was 0.52, which also represents a large the given percentages have been calculated from the number of students for which data is available for that variable. the percentages have been rounded off to one decimal place. pearson chisquare was used to calculate differences per country and the control group between pre-and post-test. t = 0 represents the pre-test and t = 1 represents the post-test after 10 weeks. education represents the level of education, in which havo stands for advanced general secondary education and vwo stands for pre-university education level. science represents the interest of the students, measured by their (preferred) choice of curriculum. figure 3. the impact of the viruskenner on students' knowledge. the knowledge of the participating and nonparticipating students per country before and after the intervention is represented by the mean percentage of the true/false questions in the questionnaire that were answered correctly. the blue line represents the netherlands, without the control group. the orange line represents all intervention groups, so from the netherlands, suriname, and indonesia. * p < 0.05; ** p < 0.01; *** p < 0.001. this figure appears in color at www.ajtmh.org. the correlation coefficients shown have been calculated from all values in the intervention groups at both the pre-and posttest. * p < 0.05 ** p < 0.01 *** p < 0.001. effect. 36 for example, in the netherlands, the percentage of correct answers on the statement "dengue is a virus infection that is transmitted by a tiger mosquito" raised from 71% correct in the pretest to 90% correct in the posttest (the correct answer is true). the score for "if someone is infected with hiv this person has aids" raised from 76% to 83% (the correct answer is false because acquired immunodeficiency syndrome is a syndrome in which the immune system is suppressed and opportunistic infections can cause illness, which can be prevented in hiv infected individuals by taking antiretrovirals).although in some other questions the percentage of correct answers differs only one percentage point between the pre-and posttest. the mean percentage of correct answers on a few questions declined. the mean total knowledge in the control group decreased significantly (p = 0.032). in the multiple regression analyses, the variable participation (control group or intervention group) contributed significantly (p < 0.001; b = 0.078) to the knowledge outcome. the variable time point (pre-or posttest), however, did not. most information about the impact of the module on knowledge is given by the interaction between participation and time point, which was significant (p < 0.001; b = 0.053). other variables that contributed significantly to knowledge were gender, age > 16 years, and the school ( table 4 ). the mean tolerance of all variables in the regression analyses is 0.3. although this suggests that there is some multicollinearity between predictors, this value is no reason for concern. 30 the data from the knowledge quiz showed that the netherlands had a mean score of 70.7%, with suriname scoring 59.6%. stigma and fear. the first question regarding stigma and fear was "i don't want to mix with people who have hiv" and the second one was "i am afraid that i will get infected by ebola." generally speaking, the module participants' answers did not change significantly. however, the results per country showed a significant decrease in suriname on both questions (p = 0.009; effect size [es] = 0.31 and p = 0.001; es = 0.42, respectively); other countries showed no significant differences in separate analyses, neither did the control group. figure 4 shows the changes in the four components regarding attitude and behavior. in the intervention group (all countries combined), attitude and awareness increased significantly (p = 0.028; es = 0.12) and so did attitude and risk infection (p < 0.001; es = 0.30). behavior and risk infection increased, but with the chosen p value of 0.05, the increase was on the borderline of significance (p = 0.062; es = 0.11). behavior and life sciences also increased with borderline significance (p = 0.060; es = 0.10). in the control group, attitude and awareness and behavior and risk infection decreased significantly (p < 0.001; es = 0.67 and p < 0.001; es = 0.51, respectively). attitude and risk infection and behavior and life sciences both showed a slight, but nonsignificant increase. although attitude, and even behavior, in the intervention group seemed to increase, in the subanalysis per country, we only found a significant increase in attitude and risk infection for the netherlands and suriname (figure 4) . the multiple regression analysis showed that as main effects, participation and time point both contributed significantly to attitude and awareness. we also found significant interaction between participation and time point for this outcome (p < 0.001; b = 0.430). the independent variables gender, school 2, education level, and countries also contributed significantly to attitude and awareness. for the attitude and risk infection outcome, only surinamese students had higher scores (p < 0.05; b = 0.213). for behavior and risk infection, the main variable participation was not significant. but time point was, and it had a negative effect (p < 0.001; b = _ 0.288). the interaction between these two resulted in a significantly positive effect (p < 0.001; b = 0.310). being older, the school and the country also contributed significantly, to behavior and risk infection. behavior and life sciences were influenced by participation in the module; however, time point had no significant effect and the interaction was not significant either. countries and education level, however, did contribute significantly to behavior and life sciences ( table 4) . appreciation of the project. generally speaking, the students enjoyed participating in the education module and said that it taught them a lot about infectious diseases. the score on the statement "i enjoyed working on the project viruskenner" was measured on a scale from 1 (totally disagree) to 5 (totally agree). the mean score in the netherlands was 3.2, in suriname 4.37, and in indonesia 3.93. in total, 90% of all students that participated gave a score of 3 or higher. supplemental table 1 in the supplementary data reports how they answered the other evaluation questions. teacher interviews. the first and second school participated in the project for 5 and 4 years, respectively, but the head teacher of the first school was involved for the first time. the project was completely new for the third and fourth schools. the first school allowed the most time for students to work on the project, 6 hours a week for 8 weeks. the fourth school allowed 5 hours a week for 8 weeks. the second and third schools allowed 5 and 4 hours a week, respectively, for 6 weeks in each case. in the first three schools, the students had no other lessons about viruses during the project period; only in school four did the biology teacher pay some extra attention to them. none of the teachers let the students prepare for the kick-off, but the teacher in the fourth school told them to read the manual. in the first and fourth schools the students themselves decided on the composition of the collaboration groups and chose their subject of preference. students in the second school also decided their group composition, but straws were drawn to allocate the subjects. the teacher in the third school divided the students and subjects over the groups randomly. all teachers reported that contact with their coaches during the project was good, although it has to be said that there were some communication problems with the teachers in the second school. and while school number three's teacher said that the contact with the coach was very helpful and amicable, he added that the students got to learn more about the world of scientific research, and that this aspect might have been emphasized even more. another remark was made by a teacher in the first school, who said that the website should be promoted for learning purposes more frequently and contact with the coaches could be more intensive. all teachers responded positively to the question: "what do you think the students learned from the module?" the teacher of the first school said he thought students are now more aware of the worldwide impact of infectious diseases. he even remarked that during the break on the final day he noticed that more students washed their hands after going to the bathroom. the teacher in the second school insisted that students are now more focused on viruses in the news, such as ebola or mers, and that there is a gap between knowing and doing. finally, a teacher in the fourth school concluded that during the completion of the posttest questionnaires he got the distinct impression that the students learned a lot. user data online resources. although the education was mainly face to face, online supportive resources were available to increase the educational impact. the graph in s1 shows the use of the website and social media in time. 27 after adjusting for age, sex, education level, school, and country, viruskenner proved to be an effective education module for increasing the knowledge of young people in the netherlands and suriname of virus infections, according to this nonrandomized intervention study. with all limitations of this study design taken in mind, we describe a positive correlation in knowledge, attitude, and behavior in the participating secondary school students. participation had a positive effect on attitude and awareness. this effect was higher among females and students who had attained a higher level of education. knowledge, behavior, and risk infection were higher in female and older students (16+). and while the attitude components increased in the figure 4 . the impact of the education module on students' attitude and behavior. the graphs illustrate the changes per country in the four different components of the attitude and behavior questions that were answered on a 5-point likert scale. a higher score represents a more positive attitude or healthier behavior. panel a shows the score for the attitude and aswareness component, panel b for attitude and risk infection, panel c for behavior and risk infection and panel d for behavior and life sciences. the blue line represents the netherlands, without the control group. the orange line represents all intervention groups, so from the netherlands, suriname, and indonesia. * p < 0.05; ** p < 0.01; *** p < 0.001. this figure appears in color at www.ajtmh.org. intervention group, the behavior components only showed an increasing trend. there was no significant effect of participation on attitude and risk infection, but there was on behavior and risk infection. this might be explained by the positive effect in the control group for attitude and risk infection but negative for behavior and risk infection. it might be due to there being less motivation in the control group to fill in the questionnaires. the education module had less impact on students' knowledge in indonesia. the somewhat limited impact on indonesian participants could be explained by their lower level of involvement. 37 all students in the netherlands and suriname developed a prevention tool and prepared a presentation. however, the evaluations found that in indonesia only a selection of the students did. additionally, in suriname (and partly in the netherlands), family members were invited to attend the final day. in indonesia, this was not possible due to the limited space. the engagement of families could well have had a positive effect. another striking fact in indonesia was the relatively high scores for attitude and awareness and behavior, in both pre-and posttests. the same was true for suriname, which might point to cultural differences with the netherlands. collectivistic countries, like suriname and indonesia, tend to give more socially desirable answers to questionnaires than individualistic countries like the netherlands. 38 overall, most students of all countries enjoyed working on the project. although most outcomes in the intervention group showed a positive trend or change, in the control group knowledge, attitude, and awareness and behavior and risk infection decreased. these students did not differ significantly in gender, education level, or profile between pre-and posttest. the decreased outcomes might be explained by reduced motivation in doing the same test twice. to our knowledge, this is the first study to evaluate an education module on several viruses in several continents. the heterogeneity of the study population increases the external validity of the study. comparing the results of the same education module in different countries gave insights in the importance of educational factors on the impact. in each country, the pre-and posttests were compared. however, a limitation of the study is that only the netherlands had a control group. the control group consisted of more students that had chosen a nonscience curriculum than the intervention group. however, there was no significant difference in knowledge score between the nonscience and science students in the control group. although science students scored higher on attitude and awareness and behavior and life science questions. although the time that schools spend on the project differed, no direct relationship was found between the hours spent and the results achieved. making the results translatable to schools that could participate to the module and would spend at least 4 hours per week during 6 weeks. due to logistics, randomization of schools was not possible. for a maximum effect, it is important to embed the project in the curriculum, so schools were chosen by a curriculum in which it would fit, as it is in the netherlands with technasium. multiple participating schools per country would have added value as it would have enabled us to perform a proper multilevel analysis, instead of a multiple linear regression analysis. furthermore, it might be good to measure any balancing measure, for example, the mean grades, to determine whether there are any unanticipated harms to scores on other subjects in school, due to the time the students spent on the project. although the project is embedded in the curriculum, the harms to other subject would be minimized. the questionnaires were composed with accuracy in dutch (the national language in the netherlands as well as in suriname). the ones that were used in indonesia were translated to bahasa indonesia without back translation. self-reported questionnaires are useful to measure knowledge changes. however, self-reported attitude and behavior have to be interpreted with caution. the effect was little and could even be due to overestimation. the effect of participation in the module on knowledge, however, was large in two of three countries. measuring a long-term effect in these countries as well would be of additional value. a clear effect on knowledge, but a negligible or nonexistent effect on attitude and behavior is common in educational research. several studies pertaining to hiv or sex education show that knowledge increased after participation in an education module. 10, 39, 40 we only found a few studies in which peer education did not increase knowledge. 41, 42 the literature about stigma, awareness, and attitude is inconclusive. some studies conclude that awareness can be increased or that attitudes can be changed, whereas others conclude that the effects on these is limited. [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] according to the available literature, behavior is the most difficult part to measure and improve. most studies about virus education evaluate hiv prevention programs. condom use or the intention to use condoms are measured most frequently. in self-reported questionnaires these outcomes improved significantly in some studies, which is promising. however, other studies did not find a significant improvement. 10, 41, 45, 47, 52 we found two studies, both conducted in africa, that tested behavioral change based on the prevalence of virus infections. in them, participants' blood samples were tested for hiv and herpes simplex virus antibodies, before and 3-8 years after an education module or compared with a control group. however, no significant differences were evident in infection rates. 53, 54 so what factors play a role in making an educational intervention effective in changing behavior? we found some studies that based their intervention on the health promoting school framework of the who proved to be successful in changing health-related behavior. 55 important elements of this framework which were applied in these studies were implementation of the intervention in the school curriculum, involvement of the school environment in the project, and involvement of family and society in the intervention. 55, 56 the viruskenner education module was implemented in the technasium curriculum in the netherlands, but was not part of the curriculum in suriname and indonesia. the family and society were involved in the project, particularly in the netherlands and suriname. however, stronger involvement of the school environment and ethos in prevention of infectious diseases might increase the impact of the intervention on attitude and behavior in all countries. this might be reached by additional interventions like handwashing posters in the sanitary facilities or selling machines for mosquito nets in the schoolyard, for example. knowledge that is not translated into behavior change would not make a difference in numbers of virus infections. so adjustments to the viruskenner module are needed to have a greater impact on attitude and behavior. active learning has the best chance of being successful if every individual student participates. students' families have to be closer involved and a sharper focus on infection prevention in school environments is needed. increasing knowledge is a great first step, because it correlates with attitude and behavior. however, significant improvements in attitude and behavior must be reached to have a possible impact on infection rates. therefore, further exploration of contributing elements of education modules that reached behavioral changes would be very useful. global trends in emerging infectious diseases diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence population biology of emerging and reemerging pathogens the spread of awareness and its impact on epidemic outbreaks health impacts of education: a review matching the message to the process: the relative ordering of knowledge, attitudes, and practices in behavior change research the knowledge, attitudes, and behaviors approach how to evaluate performance and learning in complex environments development of attitude strength over the life cycle: surge and decline the world health organization's health promoting schools framework: a cochrane systematic review and meta-analysis school based sex education and hiv prevention in lowand middle-income countries: a systematic review and metaanalysis dengue and severe dengue evaluation of the hepatitis b prevention education programme in poland an intervention study for viral hepatitis. peer-led health education among high school students the effect of school-based cervical cancer education on perceptions towards human papillomavirus vaccination among hong kong chinese adolescent girls an educational programme on dengue fever prevention and control for females in jeddah high schools effects of an influenza prevention program using non-pharmaceutical prevention measures to improve the knowledge, attitudes and practices of elementary school students in nakhon phanom province countries and economies stichting technasium the surinamese education system described and compared with the dutch system the indonesian education system described and compared with the dutch system learning effects of an international group competition project where's the evidence that active learning works? a conceptual framework for organizing active learning experiences in biology instruction doing science at the elbows of experts: issues related to the science apprenticeship camp is peer interaction necessary for optimal active learning? viruskenner: knowledge is the antidote viruskenner facebook page discovering statistics with spss factor rotations in factor analysis. encyclopedia of social sciences research methods an index of factorial simplicity necessary steps in factor analysis: enhancing validation studies of educational instruments. the pheem applied to clerks as an example the application of electronic computers to factor analysis the handbook of psychological testing a visitor's guide to effect sizes: statistical significance versus practical (clinical) importance of research findings does active learning work? a review of the research social desirability in crosscultural research evaluation of a school-based hiv prevention intervention among yemeni adolescents effects of a rapid peer-based hiv/aids educational intervention on knowledge and attitudes of high school students in a high-income arab country effectiveness of a peer-led hiv prevention intervention in secondary schools in rwanda: results from a non-randomized controlled trial an evaluation of a peerbased hiv/aids education program as implemented in a suburban high school setting effectiveness of school-based education on hiv/aids knowledge, attitude, and behavior among secondary school students in wuhan characteristics of effective interventions in improving young people's sexual health: a review of reviews effectiveness of a school hiv/aids prevention program for spanish adolescents evaluation of hiv/aids secondary school peer education in rural nigeria longitudinal study of a school based hiv/aids early prevention program for mexican adolescents effectiveness of an hiv prevention program for secondary school students in mongolia the effects of a social-cognitive method based education on knowledge and attitudes intentions with respect to hiv transmition among school learners in maragheh acquired immunodeficiency syndrome educational program: effects on adolescents' knowledge and attitudes a systematic review of school-based sexual health interventions to prevent sti/hiv in sub-saharan africa effects of the culturallysensitive comprehensive sex education programme among thai secondary school students long-term biological and behavioural impact of an adolescent sexual health intervention in tanzania: follow-up survey of the community-based mema kwa vijana trial the regai dzive shiri project: results of a randomized trial of an hiv prevention intervention for youth the who health promoting school framework for improving the health and well-being of students and their academic achievement reducing obesity via a school-based interdisciplinary intervention among youth: planet health acknowledgments: we would like to thank the european union for the assignment of the erasmus+ grant (ka2-cooperation for innovation and the exchange of good practices-capacity building in the field of youth), the viroscience department, and cirion foundation for sponsoring the project. we would also like to thank erik sickmann, georgina arron, and wilco zwennis for their contributions in the planning and management of the module. furthermore, we would like to thank benjamin berg, who developed the sdaps software to process the questionnaires and henri starmans for processing the scans of the questionnaires. finally, we would, of course, like to thank all participating schools and students for their invaluable input and time. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord-334855-s0ci3r8w authors: andersen, petter i.; krpina, klara; ianevski, aleksandr; shtaida, nastassia; jo, eunji; yang, jaewon; koit, sandra; tenson, tanel; hukkanen, veijo; anthonsen, marit w.; bjoras, magnar; evander, magnus; windisch, marc p.; zusinaite, eva; kainov, denis e. title: novel antiviral activities of obatoclax, emetine, niclosamide, brequinar, and homoharringtonine date: 2019-10-18 journal: viruses doi: 10.3390/v11100964 sha: doc_id: 334855 cord_uid: s0ci3r8w viruses are the major causes of acute and chronic infectious diseases in the world. according to the world health organization, there is an urgent need for better control of viral diseases. repurposing existing antiviral agents from one viral disease to another could play a pivotal role in this process. here, we identified novel activities of obatoclax and emetine against herpes simplex virus type 2 (hsv-2), echovirus 1 (ev1), human metapneumovirus (hmpv) and rift valley fever virus (rvfv) in cell cultures. moreover, we demonstrated novel activities of emetine against influenza a virus (fluav), niclosamide against hsv-2, brequinar against human immunodeficiency virus 1 (hiv-1), and homoharringtonine against ev1. our findings may expand the spectrum of indications of these safe-in-man agents and reinforce the arsenal of available antiviral therapeutics pending the results of further in vitro and in vivo tests. every year, emerging and re-emerging viruses, such as ebola virus (ebov), marburg virus (marv), and rift valley fever virus (rvfv), surface from natural reservoirs and kill people [1, 2] . in addition, influenza a virus (fluav), human immunodeficiency (hiv-1), herpes simplex (hsv), and other viruses regularly infect human population and represent substantial public health and economic burden [3, 4] . the world health organization (who) and the united nations (un) have called for better control of viral diseases (https://www.who.int/blueprint/priority-diseases/en/; https://sustainabledevelopment.un.org/). developing novel virus-specific vaccines and antiviral drugs can be time-consuming and costly [5, 6] . in order to overcome these time and cost issues, academic institutions and pharmaceutical companies have focused on the repositioning of existing antivirals from one viral disease to another, considering that many viruses utilize the same host factors and pathways to replicate inside a cell [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . broad-spectrum antiviral agents (bsaas) are small-molecules that inhibit a wide range of human viruses. we have recently reviewed approved, investigational and experimental antiviral compounds and identified 108 bsaas, whose pharmacokinetics (pk) and toxicity had been studied in clinical trials [16] . we tested 40 of these bsaas against human metapneumovirus (hmpv), hepatitis c virus (hcv), cytomegalovirus (cmv), and hepatitis b virus (hbv). we demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against hmpv, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against hcv [17] . we also tested 55 bsaas, including these 40, against fluav, rvfv, echovirus 1 (ev1), zikv, chikv, rrv, hiv-1 and hsv-1. we identified novel activities for dalbavancin against ev1, ezetimibe against hiv-1 and zikv, and azacytidine, cyclosporine, minocycline, oritavancin and ritonavir against rvfv [18] . here, we evaluated the efficacy of 43 bsaas, which do not overlap with 55 agents we tested before. we identified novel in vitro activities of obatoclax and emetine against hsv-2, ev1, hmpv and rvfv. moreover, we demonstrated novel antiviral effects of emetine against fluav, niclosamide against hsv-2, brequinar against hiv-1, and homoharringtonine against ev1 in vitro. to identify potential bsaas, we have reviewed approved and investigational safe-in-man antiviral agents using drug bank and clinical trials websites, respectively. in addition, we reviewed investigational and approved safe-in-man antibacterial, antifungal, antiprotozoal, antiemetic, etc. agents, for which antiviral activities have been reported in pubmed. by excluding vaccines and interferons, we identified 108 molecules that inhibit the replication of several viruses in man [16] . most recently, novel antiviral activities have been reported for some of these agents [17] . forty-three compounds were used in this study, and their suppliers and catalogue numbers are summarized in table s1 . to obtain 10 mm stock solutions compounds were dissolved in dimethyl sulfoxide (dmso, sigma-aldrich, steinheim, germany) or milli-q water. the solutions were stored at −80 • c until use. madin-darby canine kidney (mdck, american type culture collection (atcc)) and african green monkey kidney epithelial (vero-e6, atcc) cells were grown in dulbecco's modified eagle's medium (dmem; gibco, paisley, scotland) supplemented with 100 u/ml penicillin and 100 µg/ml streptomycin mixture (pen/strep; lonza, cologne, germany), 2 mm l-glutamine, and 10% heat-inactivated fetal bovine serum (fbs; lonza, cologne, germany). human telomerase reverse transcriptase-immortalized retinal pigment epithelial (rpe, atcc) cells were grown in dmem-f12 medium supplemented with pen/strep, 2 mm l-glutamine, 10% fbs, and 0.25% sodium bicarbonate (sigma-aldrich, st. louis, usa). ach-2 cells, which possess a single integrated copy of the provirus hiv-1 strain lai (nih aids reagent program), were grown in rpmi-1640 medium supplemented with 10% fbs and pen/strep. tzm-bl cells were grown in dmem supplemented with 10% fbs and pen/strep. human lung adenocarcinoma epithelial a549 cells were cultured in dmem medium containing 10% fbs and pen/strep. a549-npro cells (kindly provided by prof. steve goodbourn, university of london), which stably express bvdv npro protein, which inhibits ifn production, were cultured in dmem containing 10% fbs, pen/strep, and 10 µg/ml puromycin. all cell lines were grown in a humidified incubator at 37 • c in the presence of 5% co2. hsv-2 strain g was from the atcc. ev1 (farouk strain; atcc) was from prof. marjomäki (university of of jyväskylä) [19] . rvfv encoding the far-red fluorescent protein instead of non-structural (ns) protein (rvfv-rfp) was from profs. hartmut hengel and friedemann weber (university medical center freiburg) [20] . hmpv nl/1/00 strain, encoding green fluorescent protein (hmpv-gfp), was from vironovative and erasmus mc [21] . the gfp-expressing influenza a/pr/8-ns116-gfp strain (fluav-gfp) was generated by drs. andrej egorov (vienna) [22] . all the experiments with viruses were performed in compliance with the guidelines of the national authorities using appropriate biosafety laboratories under appropriate ethical and safety approvals. fluav-gfp was amplified in a monolayer of mdck cells in dmem containing pen/strep, 0.2% bovine serum albumin, 2 mm l-glutamine, and 1 µg/ml l-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (tpck)-trypsin (sigma-aldrich, st. louis, usa). hmpv-gfp, rvfv-rfp and the wild-type hsv-2 strain were amplified in a monolayer of vero-e6 cells in the dmem medium containing pen/strep, 0.2% bovine serum albumin, 2 mm l-glutamine, and 1 µg/ml tpck-trypsin. ev1 was amplified in a monolayer of a549 cells in the dmem media containing pen/strep, 0.2% bovine serum albumin, and 2 mm l-glutamine. for the production of hiv-1, 6 × 10 6 ach-2 cells were seeded in 10 ml medium. virus production was induced by the addition of 100 nm phorbol-12-myristate-13-acetate. the cells were incubated for 48 h. the hiv-1-containing medium was collected. the hiv-1 concentration was estimated by measuring the concentration of hiv-1 p24 in the medium using anti-p24-elisa, which was developed in-house. recombinant purified p24 protein was used as reference. the virus stocks were stored at −80 • c. approximately 4 × 10 4 rpe cells were seeded per well in 96-well plates. the cells were grown for 24 h in dmem-f12 medium supplemented with 10% fbs, and pen/strep. the medium was replaced with dmem-f12 medium containing 0.2% bovine serum albumin, 2 mm l-glutamine, and 1 µg/ml tpsk-trypsin. the compounds were added to the cells in 3-fold dilutions at seven different concentrations starting from 10 or 30 µm. saliphenylhalamide, abt-263 and dmso were added to the control wells. saliphenylhalamide is an inhibitor of cellular vacuolar atpase, which protects cells from virus-mediated death, whereas abt-263 is an inhibitor of anti-apoptotic bcl-2 proteins, which facilitates death of cells with viral nucleic acids [23] [24] [25] [26] [27] [28] . rpe cells were infected with hsv-2, fluav-gfp, hmpv-gfp or rvfv-rfp viruses at multiplicity of infections (moi) of 0.1, 0.5, 0.1 and 1, respectively. hsv-2-infected rpe cells were imaged after 72 h in the phase-contrast mode. rvfv-mediated rfp expression and fluav-mediated gfp expression were visualized after 24 h, whereas hmpv-mediated gfp expression was recorded after 96 h using fluorescent microscopy (zeiss observer z1, zaventem, belgium). image j software (v.ij 1.46r, nih) was used to determine fluorescent intensities. rpe cells were treated with bsaas or control compounds as described above and infected with hsv-2, ev1, fluav-gfp, hmpv-gfp or rvfv-rfp viruses at multiplicity of infections (moi) of 0.1, 0.1, 0.5, 0.1 and 1, respectively. after 48 h of infection, the medium was removed from the cells. the viability of mock-and virus-infected cells were measured using cell titer glow assay (ctg; promega, madison, usa). the luminescence/fluorescence were read with a pherastar fs plate reader (bmg labtech, ortenberg, germany). for testing compound toxicity and efficacy against hiv-1, approximately 4 × 10 4 tzm-bl cells were seeded in each well of a 96-well plate. tzm-bl cells express firefly luciferase under control of hiv-1 long terminal repeat (ltr) promoter allowing quantitation of the viral infection (tat-protein expression by integrated hiv-1 provirus) using firefly luciferase assay. the cells were grown for 24 h in cell growth medium. compounds were added to the cells in three-fold dilutions at seven different concentrations starting from 30 µm. no compounds were added to the control wells. the cells were infected with hiv-1 (corresponding to 300 ng/ml of hiv-1 p24) or mock. at 48 hpi, the media was removed from the cells, the cells were lysed, and firefly luciferase activity was measured using the luciferase assay system (promega, madison, wi, usa) and pherastar fs plate reader. in a parallel experiment, cell tox green reagent (ctxg; promega, madison, wi, usa) was added to the cells and fluorescence was measured with a plate reader. the half-maximal cytotoxic concentration (cc 50 ) for each compound was calculated based on viability/death curves obtained on mock-infected cells after non-linear regression analysis with a variable slope using graphpad prism software version 7.0a. the half-maximal effective concentrations (ec 50 ) were calculated based on the analysis of reporter protein expression or the viability/death of infected cells by fitting drug dose-response curves using four-parameter (4pl) logistic function f (x): where f (x) is a response value at dose x, a min and a max are the upper and lower asymptotes (minimal and maximal drug effects), m is the dose that produces the half-maximal effect (ec 50 or cc 50 ), and λ is the steepness (slope) of the curve. a relative effectiveness of the drug was defined as selectivity index (si = cc 50 /ec 50 ). the threshold of si used to differentiate between active and inactive compounds was 3. rpe cells were treated with combinations of increasing concentrations of obatoclax and emetine. the cells were infected with fluav-gfp at moi 0.5. after 24 h, gfp fluorescence was recorded using fluorescent microscopy. in a parallel experiment, the viability of infected cells was measured using the ctg assay. to test whether the drug combinations act synergistically, the observed responses were compared with expected combination responses. the expected response of the emetine-obatoclax drug combination on the viability of fluav-and mock-infected rpe cells was calculated using bliss reference model [29] . for testing the production of hsv-2 and ev1 viruses in compound-treated and non-treated rpe cells, the media from the cells were serially (10-fold) diluted, starting from 10 −3 to 10 −8 in serum-free growth media containing 0.2% bovine serum albumin, and applied to a monolayer of a549-npro cells in 12-well plates. after one hour, cells were overlaid with growth medium containing 1% carboxymethyl cellulose and 1% fbs and incubated for 72 h. the cells were fixed and stained with crystal violet dye. the plaques were calculated in each well. the titers were expressed as plaque-forming units per ml (pfu/ml). forty-three safe-in-man bsaas used in this study reached different stages of drug development process (figure 1 , tables s2 and s3). altogether, these bsaas inhibit the replication of 52 viruses belonging to (−) single-stranded (ss)rna, (+)ssrna, ssrna-reverse transcriptase (rt), ssdna, double-stranded (ds)dna, or dsdna-rt virus groups. we tested 43 bsaas against wild-type hsv-2 in rpe cells. seven different concentrations of the compounds were added to virus-or mock-infected cells. cell viability was monitored by microscopy and the ctg assay. after the initial screening, we identified four compounds (obatoclax, emetine, niclosamide and ganciclovir) that at none-cytotoxic concentrations rescued cells from virus-mediated death (figure 2a) . to determine the efficiency and toxicity of these hsv-2 inhibitors, we measured the viability of mock-and virus-infected cells after 72 h using the ctg assay ( figure 2b ). the sis for obatoclax, emetine, niclosamide and ganciclovir were 12, 37, 3 and >750, respectively (table s4) . we titrated hsv-2 produced from drug-treated and non-treated cells in a540-npro cells ( figure 2c ). the experiment revealed that 0.12 µm obatoclax, 0.04 µm emetine, 0.37 µm niclosamide and 0.37 µm ganciclovir lowered the production of hsv-2 in rpe cells. thus, we identified novel anti-hsv-2 activities of obatoclax, emetine and niclosamide and confirmed the known anti-hsv-2 activity of ganciclovir. similarly, we examined 43 bsaas against wild-type ev1 in rpe cells. we monitored the viability of mock-and virus-infected cells by microscopy and the ctg assay. after the initial screening, we identified three compounds (obatoclax, emetine, and homoharringtonine), which rescued cells from virus-mediated death at none-cytotoxic concentrations. to determine the efficiency and toxicity of novel ev1 inhibitors, we measured the viability of mock-and virus-infected cells after 48 h using the ctg assay ( figure 3a ). the sis for obatoclax, emetine, and homoharringtonine were 25, >300, and >300, respectively (table s4) . we titrated ev1 produced from drug-treated and non-treated cells in a549-npro cells ( figure 3b ). the experiment revealed that all three compounds lowered the production of ev1, confirming novel antiviral activities of obatoclax, emetine, and homoharringtonine. we also examined the toxicity and antiviral activity of 43 bsaas against hiv-1-mediated firefly luciferase expression. the firefly luciferase open reading frame is integrated into the genome of tzm-bl cells under the hiv-1 ltr promoter. our primary screen identified two compounds (brequinar and suramin) that suppressed hiv-1-mediated firefly luciferase expression without detectable cytotoxicity. we performed a validation experiment with anti-hiv-1 compounds and calculated selectivity. the si for brequinar was >750, whereas the si for suramin was >375 (figure 4 ; table s4 ). thus, we identified novel activity of brequinar and confirmed known activity of suramin. in addition, we examined the toxicity and antiviral activity of 43 bsaas against rfp-expressing rvfv in rpe cells. both fluorescent microscopy and the ctg assay showed that obatoclax and emetine inhibited rvfv-mediated rfp expression at non-cytotoxic concentrations ( figure 5a-c) . the si for obatoclax was >100, and the si for emetine was >75 (table s4 ). next, we tested 43 bsaas against gfp-expressing hmpv. hmpv-mediated gfp expression and cell viability were measured after 72 h. after the initial screening, we identified two compounds, obatoclax and emetine, which lowered gfp expression without detectable cytotoxicity. replicate experiments confirmed these hits ( figure 6a-c) . the si for obatoclax was six and for emetine was 10 (table s4 ). next, we tested 43 bsaas against gfp-expressing fluav in rpe cells. after the initial screening, we identified two compounds, obatoclax and emetine, which lowered gfp expression without detectable cytotoxicity. we repeated the experiment with these compounds and confirmed initial hits ( figure 7a -c). the si for obatoclax was 31 and for emetine was >300 (table s4) . thus, we identified novel anti-fluav activity of emetine and confirmed known activity of obatoclax. to test whether the obatoclax-emetine combinations act synergistically against fluav-mediated gfp expression and rescue infected cells from death, the observed responses were compared with expected combination responses. the deviations in observed and expected responses showed no synergistic effect ( figure 7d ,e; figure s1 ). this result indicates that obatoclax and emetine target distinct cellular pathways essential for virus infection. here, we tested 43 safe-in-man bsaas against (−)ssrna, (+)ssrna, rt-ssrna and dsdna viruses and identified novel activities for five agents (table 1, figure s2 ). we identified novel activities of niclosamide against hsv-2, brequinar against hiv-1, homoharringtonine against ev1, obatoclax against hsv-2, ev1, hmpv and rvfv, and emetine against hsv-2, ev1, hmpv, rvf and fluav. we also confirmed antiviral activities of ganciclovir against hsv-2, suramin against hiv-1, and obatoclax against fluav [24, 30, 31] . our results pointed out that an evasion mechanism observed in one virus could be relevant for other viruses and that existing bsaas could be re-positioned to other viral infections. obatoclax was originally developed as an anticancer agent. several phase ii clinical trials were completed that investigated the use of obatoclax in the treatment of leukemia, lymphoma, myelofibrosis, and mastocytosis. in addition, its antiviral activity was reported against fluav, zikv, wnv, yfv, sinv, junv, lasv, and lcmv in vitro [24, 26, 32, 33] . it was shown that obatoclax inhibited viral endocytic uptake by targeting cellular induced myeloid leukemia cell differentiation protein mcl-1 [24] . given that obatoclax also inhibits rvfv, ev1, hmpv and hsv-2, it could be pursued as a potential bsaa candidate. emetine is an anti-protozoal drug. it is also used to induce vomiting. in addition, it possesses antiviral effects against zikv, ebov, rabv, cmv, hcov-oc43 and hiv-1 [34] [35] [36] [37] [38] . it was proposed that emetine can directly inhibit viral polymerases, though it may have some other targets as well [39] . given that emetine also inhibits fluav, rvfv, ev1, hmpv and hsv-2, it may represent a promising bsaa candidate. niclosamide is an orally bioavailable anthelmintic drug and potential antineoplastic agent. in addition, it inhibits the broadest range of viruses, including hsv-2, in vitro and, in some cases, in vivo [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . it was shown that niclosamide induces endosomal neutralization and prevents virus entry into host cells. this supports the further development of niclosamide as a bsaa. homoharringtonine is an anticancer drug which is indicated for treatment of chronic myeloid leukemia. it also possesses antiviral activities against hbv, mers-cov, hsv-1 and vzv [50] [51] [52] [53] . homoharringtonine binds to the 80s ribosome and inhibits viral protein synthesis by interfering with chain elongation [51] . given that homoharringtonine also inhibits ev1, it may represent a promising bsaa candidate. brequinar is an investigational anticancer agent (phase i/ii). brequinar attenuates the replication of denv, wnv, yfv, lasv, junv, lcmv, vsv, hiv-1, and powv (nct03760666) [32, 54] . it inhibits dihydroorotate dehydrogenase, thereby blocking de novo pyrimidine biosynthesis, which is essential for the transcription and replication of viral rna. given that brequinar also inhibits hiv-1, it may represent a promising bsaa candidate. the human non-malignant rpe cell line represents an excellent model system for studying the infection of different viruses [17, 18, 24, 26] . in addition, different viral strains expressing reporter proteins, such as rvfv-rfp, hmpv-gfp and fluav-gfp, are excellent tools for drug screening [17, 18, 24, 27] . however, the number of novel and confirmed antiviral activities of bsas could be higher if we had used other cell lines and viral strains, different virus loads, different measurement endpoints, a different time of compound addition, as well as a higher purity and concentration range of 43 bsaas. moreover, antiviral properties of bsaas detected in cell-line-based assays might not be reproduced in vivo because systemic mechanisms may compensate the blocked target effect. thus, follow-up studies are needed to validate our initial hits. altogether, we expanded the spectrum of antiviral actions of niclosamide, brequinar, homoharringtonine, obatoclax and emetine in vitro. importantly, pk and safety studies have been performed on these compounds in laboratory animals and humans. this information could be used to initiate efficacy studies in vivo, saving time and resources. the most effective and tolerable bsaas or their combinations will have a global impact, improving the preparedness and protection of the general population from emerging and re-emerging viral threats and the rapid management of drug-resistant strains, as well as being used for first-line treatment or for prophylaxis of viral co-infections. bsaas could have a pivotal role in the battle against emerging and re-emerging viral diseases. the development of novel bsaas could save time and resources which are required for the development of their alternatives-virus-specific drugs and vaccines. in future, bsaas could have a global impact by decreasing morbidity and mortality from viral and other diseases, maximizing the number of healthy life years, improving the quality of life of infected patients, and decreasing the costs of patient care. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/11/10/964/s1. table s1 : compounds, their suppliers and catalogue numbers; table s2 : developmental status of broad-spectrum antivirals used in the study; table s3 : human viruses and associated diseases; figure s1 : the expected response of the emetine-obatoclax drug combination on the viability of fluav-and mock-infected rpe cells, as measured with the ctg assay using the bliss reference model; figure s2 available online: wwwwhoint/medicines/ebola-treatment/who-list-of-top-emerging-diseases/en emerging virus diseases: can we ever expect the unexpected? emerg. microbes infect global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study global, regional, and national disability-adjusted life-years (dalys) for 359 diseases and injuries and healthy life expectancy (hale) for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study approved antiviral drugs over the past 50 years infectious disease. combating emerging viral threats srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target experimental drugs poised for use in ebola outbreak an off-target effect of bx795 blocks herpes simplex virus type 1 infection of the eye repurposing of kinase inhibitors as broad-spectrum antiviral drugs the future of antivirals: broad-spectrum inhibitors approaches for identification of hiv-1 entry inhibitors targeting gp41 pocket drug repurposing: progress, challenges and recommendations editorial: drug repositioning: current advances and future perspectives review of drug repositioning approaches and resources expanding the activity spectrum of antiviral agents common nodes of virus-host interaction revealed through an integrated network analysis. front novel activities of safe-in-human broad-spectrum antiviral agents internalization of echovirus 1 in caveolae t7 rna polymerase-dependent and -independent systems for cdna-based rescue of rift valley fever virus an improved plaque reduction virus neutralization assay for human metapneumovirus rescue of influenza virus expressing gfp from the ns1 reading frame antiviral properties of chemical inhibitors of cellular anti-apoptotic bcl-2 proteins saliphenylhalamide, and gemcitabine inhibit influenza a virus infection anticancer compound abt-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism the proton translocation domain of cellular vacuolar atpase provides a target for the treatment of influenza a virus infections inhibition by cellular vacuolar atpase impairs human papillomavirus uncoating and infection synergyfinder: a web application for analyzing drug combination dose-response matrix data the anti-parasitic drug suramin potently inhibits formation of seminal amyloid fibrils and their interaction with hiv-1 ]methyl)guanine: a selective inhibitor of herpes group virus replication the reframe library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry efficacy and mechanism of action of low dose emetine against human cytomegalovirus natural plant alkaloid (emetine) inhibits hiv-1 replication by interfering with reverse transcriptase activity emetine inhibits replication of rna and dna viruses without generating drug-resistant virus variants identification of broad-spectrum antiviral compounds by targeting viral entry arbidol and other low-molecular-weight drugs that inhibit lassa and ebola viruses the antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mtor niclosamide rescues microcephaly in a humanized in vivo model of zika infection using human induced neural stem cells niclosamide inhibits lytic replication of epstein-barr virus by disrupting mtor activation antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission identification of three antiviral inhibitors against japanese encephalitis virus from library of pharmacologically active compounds 1280 niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects thiazolides as novel antiviral agents. 2. inhibition of hepatitis c virus replication inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide anti-varicella-zoster virus activity of cephalotaxine esters in vitro the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs effect of cantharidin, cephalotaxine and homoharringtonine on "in vitro" models of hepatitis b virus (hbv) and bovine viral diarrhoea virus (bvdv) replication characterization of dengue virus resistance to brequinar in cell culture we thank jelena kiprovskaja and laivi karu for ordering consumables and handling mtas. we thank andres merits for creating excellent infrastructure to work with viruses at tuit. the authors declare no conflict of interest. key: cord-318570-wj7r6953 authors: xiao, yinzong; thompson, alexander j.; howell, jessica title: point-of-care tests for hepatitis b: an overview date: 2020-10-02 journal: cells doi: 10.3390/cells9102233 sha: doc_id: 318570 cord_uid: wj7r6953 despite the heavy disease burden posed by hepatitis b, around 90% of people living with hepatitis b are not diagnosed globally. many of the affected populations still have limited or no access to essential blood tests for hepatitis b. compared to conventional blood tests which heavily rely on centralised laboratory facilities, point-of-care testing for hepatitis b has the potential to broaden testing access in low-resource settings and to engage hard-to-reach populations. few hepatitis b point-of-care tests have been ratified for clinical use by international and regional regulatory bodies, and countries have been slow to adopt point-of-care testing into hepatitis b programs. this review presents currently available point-of-care tests for hepatitis b and their roles in the care cascade, reviewing evidence for testing performance, utility, acceptability, costs and cost-effectiveness when integrated into hepatitis b diagnosis and monitoring programs. we further discuss challenges and future directions in aspects of technology, implementation, and regulation when adopting point-of-care testing in hepatitis b programs. more than 257 million people, or 3.2% of the world's population, are estimated to be living with chronic hepatitis b virus infection, with the greatest disease burden in low-resource countries in the asia-pacific and sub-saharan africa [1] . without treatment, one in every four persons infected with chronic hepatitis b will develop liver cirrhosis over 20-30 years, and 2-5% of people with cirrhosis will develop liver cancer annually [2] . globally, over 800,000 deaths annually are attributable to hepatitis b infection [1, 3] . most of this disease burden is preventable by appropriate guideline-based treatment [4] [5] [6] [7] [8] . given the magnitude of the global public health burden from hepatitis b, the world health organization (who) has outlined ambitious hepatitis b elimination targets of a 65% reduction in mortality and a 90% reduction in incidence from baseline (2015) by 2030 [9] . however, current estimates suggest that we are a long way from achieving these goals unless investment and care cascade are scaled up [1, 10] . the hepatitis b vaccination has greatly contributed to preventing transmission and reducing hepatitis b incidence globally; however, vaccination coverage is still suboptimal in resource-limited regions [11] , and most countries in africa have been unable to implement the hepatitis b birth-dose vaccine due to multiple logistical and cost barriers [12, 13] . meanwhile, for people who are already living with hepatitis b, receiving early diagnosis and clinical care is the key to reducing morbidity and mortality. however, in 2016, the who estimated that only 11% of people living with hepatitis b were diagnosed, among whom only 17% of those eligible were on treatment [1]. the hepatitis b cascade of care involves multiple steps: screening, diagnosis, linkage to care, assessment of liver disease stage and treatment eligibility, then treatment and/or monitoring, including surveillance for hepatocellular carcinoma (hcc) (figure 1) . laboratory blood tests are required at every step of the care cascade, including blood tests for hepatitis b serology, quantitative hepatitis b virus (hbv) dna level by polymerase chain reaction (pcr) and liver function tests (figure 1 ). these tests require laboratory resourcing, technology and expertise beyond existing peripheral laboratory capabilities in many low-resource and geographically isolated regions [14] [15] [16] . in many countries, laboratory services are centralised due to high costs and limited skilled technician capacity; however, transport of blood samples from regional to centralised laboratories presents its own challenges in geographically isolated or insecure regions, particularly if cold chain supply must be preserved [14] . cost is another major limitation: price reductions for diagnostics have fallen slowly over time compared with medication costs, and hepatitis b diagnostic tests cost more than therapy in many low-income countries [16, 17] . moreover, the requirement for lifelong monitoring for most people living with hepatitis b that involves regular blood tests [4, 5] , combined with barriers to timely healthcare access such as hepatitis b-related stigma [18, 19] , healthcare costs for users and providers [20] and the logistics of accessing consistent, high-quality, affordable healthcare services in a timely manner are major barriers for people to receive guideline-based care [16] . these barriers lead to significant attrition from every step of the hepatitis b care cascade over time, and those lost from care represent missed opportunities for treatment and liver cancer prevention [16, 21] . laboratory-based blood tests are required at every stage of the hepatitis b cascade of care for diagnosis, assessment of liver disease stage, treatment eligibility and long-term monitoring of disease progression. diagnostic testing for hepatitis b involves detection of hepatitis b surface antigen (hbsag) in blood, which indicates active infection with the virus. standard laboratory electro-chemiluminescence immunoassay-based hbsag testing is performed on serum or plasma samples derived from whole blood. if active infection is confirmed, subsequent blood tests are performed to determine the stage of disease and need for treatment, including a hepatitis b virus (hbv) polymerase chain reaction (pcr)-based quantitative dna level or viral load, a hepatitis b eag and eab assay and liver function tests to determine whether an elevated aminotransferase (alt) indicative of liver inflammation or other signs of impaired liver function are present. further assessment for the presence of liver fibrosis and cirrhosis is also required, most commonly by transient elastography and/or liver biopsy. all patients irrespective of treatment require ongoing disease monitoring, including at minimum an hbv dna level, hbeag and hbeab (if not already seroconverted from hbeag positive to hbeab positive) and alt levels every 3-6 months. point-of-care tests (pocs; also known as rapid diagnostic tests, rdts) are simplified versions of laboratory-based tests that have the potential to circumvent major barriers people face to accessing hepatitis b blood-based testing in various settings. pocs usually require small amounts of body fluids (for example, a finger-prick blood sample or oral swab), short turn-around time, and are generally easy to use with minimal required training and therefore can be provided to people in a variety of community and outreach settings by a broad range of trained workers [22] and are scalable to rapidly reach large populations as has been seen with the highly successful egyptian national hepatitis c screening program [23] . the simple collection process (finger-prick or mouth swab) is also highly acceptable, feasible and attractive to people undergoing testing [22, 24, 25] . a key benefit of pocs in the field of hepatitis b is to engage hard-to-reach communities for testing, such as using hbsag poc tests for hepatitis b screening in remote areas, or harm reduction programs [24] [25] [26] [27] . pocs also have great potential for retaining patients in care when used in the community for chronic hepatitis b stage evaluation and disease monitoring [26, 27] . figure 2 outlines the key phases of disease in chronic hepatitis b infection and the indicators for blood testing in each stage. the who recommends that an ideal poc test needs to meet the assured criteria of being "affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users" [28] . since 1998, the who has implemented an evaluation and performance assessment program for all pocs in viral hepatitis to report on accepted quality parameters for widespread clinical use [29] . many pocs have been developed in the field of hepatitis b, particularly for screening and diagnosis; however, only three pocs for detecting hbsag have been prequalified by the who [29] . there is currently a lack of pocs for hepatitis b stage assessment or monitoring that have been endorsed to use by the who; however, several novel tests are now in clinical trials that may fill this important care delivery gap. typically, poc tests have lower accuracy than traditional laboratory-based tests, but they facilitate the triage of people who require more complex and expensive laboratory assays to confirm a positive poc test result and thereby reduce costs. regulatory and economic constraints are additional barriers to transferring pocs to field use. in different settings, they therefore require a comprehensive appraisal of factors including testing performance, feasibility (such as storage requirements, power supply), acceptability and cost-effectiveness when using pocs to scale up access to hepatitis b diagnosis and management under real-life conditions. in this review, we outline the accuracy of available poc tests for hepatitis b and explore the evidence for utility and cost-effectiveness when integrated into hepatitis b diagnosis and monitoring programs. we also describe future technologies and explore how poc tests might best be used to achieve who 2030 hepatitis b elimination goals. practically, the three key clinical requirements for poc hepatitis b assays in the field are for diagnosis of current infection, determining treatment eligibility and also monitoring, as well as diagnosis of hepatitis b immunity and the need for hepatitis b vaccination in the uninfected ( figure 2 ). detection of hepatitis b surface antigen (hbsag) is the primary step to diagnose current hepatitis b infection, and multiple hbsag pocs are commercially available. most are qualitative lateral-flow chromatographic immunoassays which are one-step, easy to use, can be used with a variety of different specimens (whole blood, serum and plasma) and provide rapid semiquantitative visible results (usually within 15-30 min). to date, three hbsag rapid tests (determine hbsag 2, alere medical co. ltd, chiba-ken, japan; vikia hbsag, biomérieux sa, marcy-l'étoile, france; and sd bioline wb, abbott diagnostics korea inc. giheung-gu, republic of korea) have met who prequalification criteria [29] , with multiple studies showing their high accuracy for determining hbsag positivity in various populations, particularly in moderate-high-prevalence populations (table s1) . determine hbsag poc test is the one of the most widely-used hbsag poc tests [30] with the most published data on clinical performance. a 2017 meta-analysis [31] including 9 studies with 7730 samples showed a pooled sensitivity of 90.8% and specificity of 99.1% using determine. though most studies [32] [33] [34] [35] [36] [37] [38] showed high clinical sensitivity of 89-100% in the general population, the reported sensitivity varied widely in hiv-infected populations (56-100%) [39] [40] [41] [42] [43] [44] . the cause of the reported lower sensitivity in hiv-coinfected populations [39, 40, 43, 44] is unclear, but potential reasons may include the cross reaction of hiv-reverse transcriptase inhibitors and hepatitis b virus, a higher rate of occult hepatitis b infection in early hiv cohorts, a higher reported rate of hbsag loss in both untreated and treated hiv-infected populations and the use of tenofovir-based hiv regimens that effectively suppress hepatitis b virus dna levels and a large decline in hbsag titres [31, 45, 46] . sd bioline hbsag [38, [47] [48] [49] and vikia hbsag poc test [32, 33, 38, 50, 51] have also been shown to have good sensitivity (above 90%) and excellent specificity (above 99%) in general populations; however, lower sensitivity was also reported in hiv-infected populations [40] . a common application of these hbsag pocs is to measure seroprevalence in general or specific subpopulations in low-resource settings [52] [53] [54] [55] [56] . they have also been used in mass screening programs for hepatitis b in both community outreach [24, 57] and health-facility-based screening [52, 53, [58] [59] [60] [61] [62] in low-resource settings and shown great public health benefits. for example, in a community-based outreach screening program conducted in 75 camps in southern india [63] , the "screen and vaccinate/linkage to care" strategy led to over 7700 vaccinations in the camps and 162 people with high viral load getting treatment. the program increased the accessibility of hepatitis b diagnostic testing in a low-resource setting, and the timely results of pocs contributed to people's engagement in post screening interventions [63] . the hbsag pocs were also used in programs to engage hard-to-reach populations such as people who inject drugs, sex workers [64] , disadvantaged groups or some ethnic groups [65, 66] by providing self-testing, community or health-facility-based testing services. in a randomised control study conducted in a clinic engaging mostly african immigrants in france [65] , people without health cover attending a clinic were provided free testing for hepatitis b, hepatitis c and hiv using either pocs or prescriptions of testing at a pathologist; a higher rate of testing and linkage to care was observed among people allocated to receive pocs. however, another multicentre randomised control study in france [66] found no difference in effectiveness of linkage to care using the approach of an hbsag rapid test plus a standard lab-based confirmatory serology test versus lab-based standard serology in five clinics. in this study, it was described that participants received testing results via mail or phone call, but it was unclear whether participants received testing results at the same visit if they were in the poc testing group [66] . other than the who-prequalified hbsag pocs, emerging new brands of hbsag have been reported in field studies including the drw-hbsag assay, diagnostics for the real world ltd., (ce-marked) [35, 67] , first response hbsag card test, premier medical corporation [68] (ce-marked), naosign(r) hbs poc strips, bioland [69] , and one step hbsag test, general biologicals corporation [70, 71] (non-exhaustive list). a study in mongolia [72] which tested 19 commercially available hbsag pocs using a serum sample showed the average sensitivity and specificity being 100% and 99%, respectively. whilst most hbsag pocs of various brands have shown promising clinical performance [35, 38, 67, 70, 73] , available validation data are limited and further studies with large sample size and in diverse populations including different ethnicity and hepatitis b prevalence populations and people living with hiv are needed. multiplex diagnostic pocs can be highly attractive for low-middle-resource settings with the capacity to detect multiple pathogens using a single testing strip. some multiplex pocs which detect hbsag are commercially available and some are ce-marked (hbsag/hcv/hiv/syphilis combo test, euro genomas; hbsag and hcv combo test, euro genomas; artron detect 3 hiv/hcv/hbv combo, artron laboratories; hiv, hbsag and hcv rapid test, maternova inc., providence, ri, usa), but none have been listed by who prequalification [16, 29, 74] . accuracy of hbsag detection using multiplex has been shown to be high [75] , but limited clinical validation data are available. innovations in sampling technique have provided more convenient specimen collection methods, such as using oral fluid as specimen collected by an oral swab [25, 48, 51] . the simplified process was highly acceptable to individuals [25, 48] , but testing accuracy is a challenge to overcome [48] , and it may additionally require trained technicians or lab-based enzyme immunoassays or equipment for sample preparation such as requiring a centrifuge for target analyte separation [51] . future development needs to consider combining sample preparation steps together with detection and readout into one single device, without sacrificing testing accuracy. although many studies of different hbsag pocs showed very good sensitivity [31, 38, 73] , false negativity is still among the biggest concerns: around one in ten negative test results on average could be hbsag positive [31] . most cases with false negative results were reported to have low titres of hbsag, such as in studies using determine/vikia hbsag pocs, where most false negative cases had hbsag titres lower than 30 iu/ml [32, 33] . as hbsag level does not correlate with severity of liver damage, there is a chance that people with advanced liver disease may be missed. other potential factors affecting the accuracy of hbsag pocs may include hbv dna level, different genotypes, co-infection with hepatitis c or hiv and hepatitis b variants with s gene mutations that are not detected by the poc hbsag test [31, 33, 76, 77] . as only a few studies have obtained comprehensive serological and genetic profiles of false negative cases, more data are needed to explore these associations and determine the implications for clinical practice. specimen type is unlikely to affect the efficacy of hbsag poc tests: a meta-analysis showed similar pooled sensitivity of studies using whole blood sample compared to plasma or serum [31] , and studies evaluating hbsag pocs using capillary whole blood collected by finger-prick all showed reasonably high sensitivity (88-90%) [32, 44, 78] . in practice, there is no absolute cut-off for testing performance when choosing pocs for hepatitis b programs, and the increased access to testing might mitigate the harm caused by reduced accuracy; however, sensitive pocs that have been validated in similar contexts to their planned use should be prioritised [79] . hepatitis b surface antibody (anti-hbs) is the key marker to determine an individual's immunity status to hepatitis b virus and triage the need for vaccination. a few anti-hbs pocs are commercially available, but most have poor reported sensitivity ranging from 20% to 70% [33, [80] [81] [82] . one study reported a sensitivity of 91.8% using an anti-hbs rapid test card among 1272 samples [70] ; however, these findings require further validation. a study [66] showed using hbsag/ anti-hbs pocs was not effective in increasing vaccination rate due to poor sensitivity of anti-hbs poc and high reliance on confirmatory enzyme immunoassay. though a poc test for anti-hbs can help with triage vaccination need, given hepatitis b vaccination is relatively cheap, context-specific cost-effectiveness analyses would be needed to determine settings where the use of pocs of anti-hbs would be cost-effective. treatment decisions in hepatitis b are guided by patient age, hepatitis b dna viral load and the degree of liver inflammation and fibrosis, as measured by alanine aminotransferase (alt) levels and either transient elastography or liver biopsy, respectively [4, 5, 83] . however, there are few poc tests currently available for these parameters, and none have been widely validated and who prequalification approved. hepatitis b virus (hbv) dna level is the critical indicator when deciding an individual's management plan as per clinical guidelines. polymerase chain reaction (pcr) platforms for nucleic acid detection are still the main technique of quantitative assessment hbv dna levels; conventional pcr platforms are usually built in laboratories and require high manual input and pose barriers for accessibility in remote areas and other resource-limited areas areas [12] . a rapid molecular test, xpert ® hbv viral load (cepheid inc., sunnyvale, ca, usa, ce-marked, approved by american fda and tga in australia), is commercially available for hbv dna quantification that provides test results in less than one hour [84, 85] . the test is a cartridge-based, real-time pcr assay which is run on the genexpert instrument, a molecular diagnostic platform. the processing unit of the system is around the size of a coffee machine, and it also runs a range of other rapid molecular tests such as who prequalified xpert hcv viral load, hiv-1 qual and hiv-1 viral load tests [29] , which poses an opportunity for hepatitis b viral load test to be adopted in areas with existing platforms at a low additional cost. so far, limited data are available on the analytical performance of the assay [84, 85] . two recent studies [84] using serum samples showed a good correlation between hbv dna quantification by using xpert hbv viral load assay with the results of the laboratory reference assay; they also have a low limit of detection (lod) of 7.5 iu/ml, which is similar to most commonly used hbv dna platforms (usually with lod of 10iu/ml) [84] . in practice, xpert testing for hbv dna led to a faster workflow with a mean time to result being 6-8 h, which provided a near-poc solution [84, 86] . however, as a new unit, genexpert facilities are still expensive; the operation requires uninterrupted power supply, as well as technician training and skills for system running, services and reagent maintenance. hepatitis e antigen (hbeag) is a key indicator to determine phase of chronic hepatitis b infection (figure 2) , treatment initiation and is used as a surrogate of hbv dna measurement for evaluating risks of maternal-to-child-transmission [2, 4, 83, 87] . several hbeag pocs are commercially available; however, published data show the accuracy of hbeag pocs has a wide range, with sensitivity of 30-82% and specificity of 67-100% [33, 80, 88] . similarly, anti-hbe pocs are reported to have poor sensitivity but excellent specificity in studies [33, 81] . given the high costs and challenges in accessing hbv dna testing in low-resource settings, the who recommends hbeag to triage treatment [83, 89, 90] ; therefore, the low testing accuracy of hbeag pocs is an urgent issue to be addressed. novel serum biomarkers such as hbv core antigen (hbcrag) have been shown to correlate with serum hbv dna levels and intrahepatic cccdna levels, a marker of hepatitis b-related hcc risk, and have therefore been explored as a potential indicator for treatment determination, off-therapy virologic suppression and hcc risk evaluation [91, 92] . however, there is no rapid lateral flow assay for hbcrag so far. another novel biomarker, serum hbv rna [93] , has been shown to be positively correlated with hbv dna level, and levels were higher than hbv dna in patients on nucleos(t)ide analogues and can thus be a potential marker for off-therapy hbv suppression. however, its clinical predictive utility is not yet well defined, and the measurement of serum hbv rna presents its own challenges even in routine lab-based testing; thus, further studies are needed to guide defining the clinical role and development of hbv rna pocs. alt is part of the liver function test assay panel and is a key marker of liver inflammation, used to determine hepatitis b treatment eligibility [4, 5, 83] (figure 2 ). alt has been proposed as an indicator for treatment in people with positive hbsag in low-resource settings where hbv dna testing is unavailable. a semiquantitative poc using alt 40u/l as the cut-off (biopoint ® alt-1) has been developed and manufacturer data suggest a high sensitivity of 94% and specificity of 85% [94] . several serological biomarkers have been combined in algorithms to offer indirect non-invasive assessment of liver fibrosis and have been validated to varying degrees in hepatitis b populations, such as the ast to platelet ratio index (apri) and fibrosis 4 index (fib-4) [95, 96] . these indices require quantitative testing results of ast, platelets with or without alt, unfortunately none of which are currently available in a poc test format. dried blood spot (dbs), while not a poc test, is a sampling method which offers viable solutions for mass screening or testing in low-resource settings where testing capacity or access are limited. in practice, a single finger-prick blood sample is applied to a chemically modified paper card which collects and store serological markers and nucleic acid; specimens obtained in the field can be transported to a laboratory at ambient temperatures, where the blood sample is processed following a dbs protocol and tested using immunoassays or molecular techniques [83, 97] . dbs samples have a relatively long shelf-life at ambient temperature without sample degradation [98] , which is attractive for regions that are geographically isolated or have varying security situations precluding rapid transport to a central laboratory. this method is now recommended by the 2017 who hepatitis b testing guidelines [83] in settings where no access to venous blood sampling or quality-assured testing assays is available. dbs testing has been used to detect hbsag, hbeag, anti-hbc, hbv dna and even for viral genotyping [99, 100] . a meta-analysis [101] evaluating dbs for hbv dna quantification showed pooled sensitivity of 95% (83-99%) and specificity of 99% (53-100%); however, most of the included studies used cold chain to store samples, which might limit the generalisation of the accuracy estimates in field conditions. although dbs testing increases testing access in low-resource and geographically isolated settings, it still requires high technical expertise and standard laboratory assays that may not be routinely available. cost-effectiveness and affordability are key considerations when adopting poc tests in hepatitis b programs. quoted costs of lateral flow-based hbsag pocs are generally lower than laboratory-based immunoassays, with the estimated procurement costs being us$0.2-0.95 and us$0.4 to 2.8 per test, respectively [72, 83] . conventional lab-based testing usually requires additional costs such as a reading machine, professional laboratory staff and technical training; therefore, the total costs for testing are often much higher than using pocs in low-resource settings. multiplex poc testing is expected to be cheaper than multiple poc tests. for example, the manufacturing costs of a hiv/hcv/hbsag poc is around us$1 [102] ; thus, using multiplex in high-risk populations who require broad spectrum pathogen screening is expected to be resource-saving. costs for conventional hepatitis nucleic acid testing are estimated to range from us$30 to 120 [15] , and the cost can be up to us$400 per assay in resource-limited countries and regions [12] . in 2018, a viral load testing program was introduced in sub-saharan african countries to access an integrated molecular diagnostics instrument (hologic panther system), at an all-inclusive ceiling price of us$12 per patient sample [103] . the foundation for innovative new diagnostics (find) has negotiated the price of xpert hbv viral load assay for 145 developing countries, and it costs us$14.9 per cartridge excluding shipment; however, the testing instrument costs between us$11,530 to us$64,350 depending on the throughput capacity of the processing unit [104, 105] . however, these costs may still be higher than what programs could afford in some settings. in addition, due to reduced accuracy compared with standard assays, diagnostic poc testing is often used as a screening tool to triage those requiring more expensive laboratory-based testing confirmation [15] , which means many of the costs for centralised laboratory services are only partially offset by poc test use. while novel poc testing may have increased testing performance, costs usually fall slowly due to patent protection laws [16] . even for countries that could afford these pocs, it may cost more than lab-based testing where well-established laboratory services are available; therefore, the main demand for pocs is limited to self-testing or outreach programs to improve testing uptake. the cost-effectiveness of using pocs for hepatitis b can therefore be different in different settings. using hbsag pocs as a screening tool was found to be cost-effective in community-based approach in hbv-endemic but low-resource settings. nayagam et al. [106] assessed a community-based hbv screening and treating program in the gambia where hbsag pocs were provided to adult participants door-to-door at a total screening cost of us$7.4 per person. the program was found to be highly cost-effective, with an icer of us$540 per daly averted compared to status quo where no publicly provided hbv screening or treatment was available. integrating low-cost hbv pocs into existed healthcare services such as antenatal screening [52, 58, 107] , blood donor screening [62] and hiv clinics [59] [60] [61] can be another solution to achieve scale-up of hbv testing [108, 109] . zhang et al. [109] showed the integration of hbv screening within the existing antenatal care in cambodia was highly cost-effective. in their model, the unit cost of hbsag and dna test (estimated to be us$1 and us$30) was one of the key parameters driving cost-effectiveness; in such cases, cheap pocs could potentially improve the cost-effectiveness of such an integration program even further. studies in low hbv endemicity countries showed programs offering hepatitis b screening followed by vaccination or linkage to clinical care among people with increased risks are likely to be cost-effective [110] ; however, there is a lack of programs adopting pocs in hepatitis b screening strategy, and thus, a lack of evidence suggesting economic impacts using pocs for hepatitis b in populations who have regular access to healthcare services. however, a few studies have shown rapid hepatitis c or hiv testing nested in harm reduction programs or among priority populations can be cost-effective [111] [112] [113] . more evidence on using pocs in hbv screening or monitoring programs in the field is needed, especially covering the implementation costs and the effects of broader testing access compared to standard testing services or no testing services (where there being no access to testing is the current practice). whilst poc testing theoretically circumvents many test access barriers, acceptability from targeted population remains a key determinant of successful implementation of hepatitis b programs. however, limited data are available on the satisfaction appraisal from users and stakeholders. in general, pocs are highly acceptable to customers due to their easy-to-use nature, short turnaround time, minimal bio sample requirement and provision of testing capacity to familiar staff in contexts where people want to be tested [114] . in a survey conducted among implementers and users of hep b and c testing services from 43 countries, almost half of respondents from low-and middle-income countries preferred a poc test method using capillary whole blood [83] . while there is no agreement on what accuracy would be considered acceptable, half of the respondents would accept an assay with a minimal sensitivity of 95% [83] . acceptability of rapid testing for hepatitis b or other blood-borne viruses and sexual transmitted diseases can be varied in different populations. a survey done in a prison setting showed hcv pocs were highly accepted [115] . another study showed that people may find it stressful when testing hiv, hcv and syphilis using a poc test [116] . when using pocs for blood-borne virus screening in public events or community outreach programs, the acceptance rate varied widely in customers with different socioeconomic status, ethnic or geographic backgrounds [63, 117, 118] . in health facility settings, healthcare providers find pocs generally speed up decision making and improve patients' compliance with chronic management plans requiring repeat testing over time [30] ; however, there are also general concerns such as suboptimal testing accuracy and increased workload for healthcare workers [119] . other than getting tested from healthcare providers or trained personnel, pocs also have the potential to be a self-testing tool with universal access. in some countries, rapid tests for hepatitis b and/or hiv and hepatitis c can be purchased online or over the counter. while self-testing offers a confidential testing solution for customers, a standard approach will be needed to ensure that people having accessible pre-and post-counselling, as well as pathways of linkage to care. a general limitation of pocs for hepatitis b is reduced accuracy compared to standard lab-based testing. there are also specific limitations for individual poc tests which were highlighted above (section 2), such as hbsag poc tests which were shown to have varied sensitivity in hiv-infected populations. in addition, there is still a lack of poc tests for liver cirrhosis and hbv dna levels required to determine treatment eligibility for patients with hepatitis b. there are also limitations in the aspects of regulatory process, procurement and storage management for poc tests, as well as costs when implementing pocs for hepatitis b in different settings. the who prequalification process for in vitro diagnostic tests for diseases with a high individual or public health risk, including hepatitis b, assesses both the test's performance and manufacturing quality. for countries without regulatory procedures in place, this provides a thorough review of potential diagnostic tests they could select based on specific needs; however, the process of getting prequalified approval by the who can be slow. for low-resource settings, stock-out and supply issues can be a barrier for use of poc tests; lack of scale may also mean they can be more expensive than high throughput assays in some settings; testing accuracy as well as instrument maintenance can be impacted by extreme weather conditions (heat, humidity) in the field; novel testing platforms such as genexpert can still be expensive, and the use of the instruments can be subject to field conditions such as power supply. on the other side, for high-income countries, a main challenge for the introduction and implementation of poc tests is the regulatory and reimbursement approval process for new diagnostics, which require demonstration of analytic and clinical validity, as well as clinical usefulness and cost-effectiveness data. as an example, the fda regulatory process can be long and expensive [120] , and the return for investment in high-income countries where poc tests will compete for market with standard diagnostic pathways can be challenging. increasing testing accuracy is the major challenge for poc tests that are already compact and easy to use. when developing poc diagnostics, features targeting resource-limited settings without basic infrastructures or cold chain need to be included; tests with high quality need to be validated across populations and specimen type. rapid affordable serology tests of high accuracy for novel biomarkers which could be alternatives for molecular testing are a major need. technology is needed to integrate convenient sampling and specimen preparation into a one-step testing assay. inter-user variability is another challenge to address if poc tests require technique training or multiple steps; a standard protocol or mobile apps can be used to overcome this problem where suitable. miniaturisation of testing instruments is the trend, especially for instruments that could perform molecular analysis such as portable hand-held devices, without sacrificing testing accuracy. dry blood spot kits for hiv and hepatitis c can already be ordered online to be sent to a home address as a private way to test for infection [121] . faecal occult blood tests are mailed out to all older adults in some regions as a public health initiative to screen for bowel cancer [122] . a similar approach could be evaluated to screen for hepatitis b among populations that are disengaged from traditional health services. in resource-limited settings, adopting hepatitis b testing in existing platforms or programs can be more cost-effective than starting a new initiative [109] . mobile phone technology has the potential to be used for screening and monitoring health conditions [123] . mobile phones are now being used around the world for contact tracing for sars-cov-2, an approach that is immediately applicable to hepatitis b. recently, google searches for anosmia have been linked to the epidemiology of sars-cov-2 [124] . there is a need for the streamlining of regulatory and reimbursement approval processes in high-income countries where the traditional approval process is expensive and slow, particularly for poc diagnostics suitable for use as public health tools to promote the engagement of marginalised individuals, including people who inject drugs, migrants and culturally and linguistically diverse communities affected by hepatitis b. in low-and middle-income countries where regulatory processes can be less demanding, the key is to ensure the quality and performance of tests as they come to market. more than 60 products have been prequalified since the who prequalification process started in 2010 [29] . it has been proposed that a model list of essential diagnostics be developed, comparable with the model list of essential medicines maintained by the who. such a list would help in the selection of diagnostic methods and would facilitate improvements in the regulation and affordability of in vitro diagnostic tests and in training in their use. the who has set ambitious goals for the elimination of hepatitis b as a public health threat by 2030. birth dose vaccination is the most important public health intervention to reduce incidence and will also reduce mortality level long term. for the individual already infected with hepatitis b, the key to preventing liver-related harm is the maintenance of sustained viral suppression. this requires diagnosis and linkage to care; in some people, antiviral therapy will be necessary. hepatitis b is typically asymptomatic until advanced disease has developed. therefore, screening is required. the risk factors and epidemiology of hepatitis b are well described, but screening rates are suboptimal and often occur in the context of opportunistic doctor-patient consultations following presentation with an unrelated problem. testing typically involves venesection followed by centralised testing in a laboratory with batch processing and automation to improve efficiency. this system works well for the engaged individual being cared for by a motivated health care practitioner. however, even in high-income countries, up to 80% of infected patients remain unaware of their infection [125] . thus, there is a need to scale up screening for hepatitis b in high-risk populations, and a need to reconsider current models of care for screening. now that hepatitis b treatment is cheap, safe and highly effective and durable, there is an urgent need to reconsider a public health approach to the management of hepatitis b. point-of-care tests provide a tool for mass screening in community settings. they also provide the opportunity to reduce the care cascade to one of same-day "test and treat". the effective employment of such strategies will be necessary for achievement of who elimination goals. natural history of chronic hepatitis b: special emphasis on disease progression and prognostic factors global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the global burden of disease study clinical practice guidelines on the management of hepatitis b virus infection update on prevention, diagnosis, and treatment of chronic hepatitis b: aasld 2018 hepatitis b guidance long-term suppression of hepatitis b e antigen-negative chronic hepatitis b by 24-month interferon therapy the long-term benefits of nucleos(t)ide analogs in compensated hbv cirrhotic patients with no or small esophageal varices: a 12-year prospective cohort study the risk of hepatocellular carcinoma decreases after the first 5 years of entecavir or tenofovir in caucasians with chronic hepatitis b requirements for global elimination of hepatitis b: a modelling study third dose of hepatitis b vaccine. reported estimates of hepb3 coverage. secondary third dose of hepatitis b vaccine. reported estimates of hepb3 coverage hepatitis b in sub-saharan africa: strategies to achieve the 2030 elimination targets reported estimates of hepb_bd coverage. secondary hepb birth dose. reported estimates of hepb_bd coverage aiming for the elimination of viral hepatitis in australia, new zealand, and the pacific islands and territories: where are we now and barriers to meeting world health organization targets by 2030 the future of viral hepatitis testing: innovations in testing technologies and approaches accelerating the elimination of viral hepatitis: a lancet gastroenterology & hepatology commission diagnosis of viral hepatitis more than a virus: a qualitative study of the social implications of hepatitis b infection in china managing chronic hepatitis b: a qualitative study exploring the perspectives of people living with chronic hepatitis b in australia innovative strategies for the elimination of viral hepatitis at a national level: a country case series pdf;jsessionid=9deca1ff83bc4a8c41b74e3be2649662?sequence=12017 the rapid-ec study-a feasibility study of point-of-care testing in community clinics targeted to people who inject drugs in screening and treatment program to eliminate hepatitis c in egypt acceptability and feasibility of a screen-and-treat programme for hepatitis b virus infection in the gambia: the prevention of liver fibrosis and cancer in africa (prolifica) study implementation of rapid hiv and hcv testing within harm reduction programmes for people who inject drugs: a pilot study hepatitis b screening in an argentine emergency department: a pilot study to increase vaccination in a resource-limited setting point-of-care hepatitis c testing from needle and syringe programs: an australian feasibility study rapid tests for sexually transmitted infections (stis): the way forward helath organisation list of prequalified in vitro diagnostic products. secondary world helath organisation list of prequalified in vitro diagnostic products 2020 a practical guide to global point-of-care testing diagnostic accuracy of tests to detect hepatitis b surface antigen: a systematic review of the literature and meta-analysis validation of rapid point-of-care (poc) tests for detection of hepatitis b surface antigen in field and laboratory settings in the gambia, western africa performance of rapid tests for detection of hbsag and anti-hbsab in a large cohort evaluation of rapid diagnostic tests for the detection of human immunodeficiency virus types 1 and 2, hepatitis b surface antigen, and syphilis in ho chi minh city, vietnam. am evaluation of a new hepatitis b virus surface antigen rapid test with improved sensitivity evaluation of the performance of four rapid tests for detection of hepatitis b surface antigen in antananarivo, madagascar hepatitis b surface antigen seroprevalence among prevaccine and vaccine era children in bangladesh evaluation of four rapid tests for detection of hepatitis b surface antigen in ivory coast prevalence of infection with hepatitis b and c virus and coinfection with hiv in medical inpatients in malawi detection of highly prevalent hepatitis b virus coinfection among hiv-seropositive persons in ghana reliability of rapid testing for hepatitis b in a region of high hiv endemicity viral hepatitis and rapid diagnostic test based screening for hbsag in hiv-infected patients in rural tanzania prevalence and associations with hepatitis b and hepatitis c infection among hiv-infected adults in south africa field performance of the determine hbsag point-of-care test for diagnosis of hepatitis b virus co-infection among hiv patients in zambia occult hepatitis b and hiv infection hiv-hepatitis b virus coinfection prevalence of chronic hepatitis b virus infection before and after implementation of a hepatitis b vaccination program among children in nepal point of care and oral fluid hepatitis b testing in remote indigenous communities of northern australia evaluation of the analytical performance of six rapid diagnostic tests for the detection of viral hepatitis b and c in lubumbashi, democratic republic of congo performance of point of care assays for hepatitis b and c viruses in chronic kidney disease patients evaluating hbsag rapid test performance for different biological samples from low and high infection rate settings & populations hepatitis b virus sero-prevalence amongst pregnant women in the gambia seroprevalence of hbv among people living with hiv in anyigba assessment of hepatitis b immunization programme among school students in qatar seroprevalence of hepatitis b virus infection and associated factors among healthcare workers in northern tanzania seroprevalence of hepatitis b and c virus infections among diabetic patients in kisangani (north-eastern democratic republic of congo) eligibility for hepatitis b antiviral therapy among adults in the general population in zambia maternal hepatitis b infection burden, comorbidity and pregnancy outcome in a low-income population on the myanmar-thailand border: a retrospective cohort study the prevalence of hepatitis b virus among hiv-positive patients at kilimanjaro christian medical centre referral hospital sero-prevalence of hbv and associated risk factors among hiv positive individuals attending art clinic at mekelle hospital hepatitis b infection, viral load and resistance in hiv-infected patients in mozambique and zambia palliduminfections among blood donors and transfusion-related complications among recipients at the laquintinie hospital in douala prevalence of hepatitis b and hepatitis c infection from a population-based study in southern india prevalence estimates of hiv, syphilis, hepatitis b and c among female sex workers (fsw) in brazil simultaneous human immunodeficiency virus-hepatitis b-hepatitis c point-of-care tests improve outcomes in linkage-to-care: results of a randomized control trial in persons without healthcare coverage. open forum infect effectiveness of hepatitis b rapid tests toward linkage-to-care performance of a new rapid test for the detection of hepatitis b surface antigen in various patient populations hepatitis b vaccination in burkina faso: prevalence of hbsag carriage and immune response in children in the western region a simple and inexpensive point-of-care test for hepatitis b surface antigen detection: serological and molecular evaluation a simple and rapid test-card method to detect hepatitis b surface antigen and antibody: potential application in young children and infants evaluation of the performance of two rapid immunochromatographic tests for detection of hepatitis b surface antigen and anti hcv antibodies using elisa tested samples sensitivity and specificity of commercially available rapid diagnostic tests for viral hepatitis b and c screening in serum samples evaluation of performance testing of different rapid diagnostic kits in comparison with eias to validate detection of hepatitis b virus among high risk group in nigeria multi-disease diagnostics landscape for integrated management of hiv, hcv, tb and other coinfections analytical performances of simultaneous detection of hiv-1, hiv-2 and hepatitis cspecific antibodies and hepatitis b surface antigen (hbsag) by multiplex immunochromatographic rapid test with serum samples: a cross-sectional study performance evaluation of 70 hepatitis b virus (hbv) surface antigen (hbsag) assays from around the world by a geographically diverse panel with an array of hbv genotypes and hbsag subtypes comparative performance of three rapid hbsag assays for detection of hbs diagnostic escape mutants in clinical samples: table 1 point-of-care screening for hepatitis b virus infection in pregnant women at an antenatal clinic: a south african experience a guide to aid the selection of diagnostic tests evaluation of rapid diagnostic tests for assessment of hepatitis b in resource-limited settings poor sensitivity of rapid tests for the detection of antibodies to the hepatitis b virus: implications for field studies performance of rapid diagnostic tests for the detection of anti-hbs in various patient populations performance of the xpert hbv viral load assay versus the aptima quant assay for quantifying hepatitis b virus dna evaluation of the xpert hbv viral load for hepatitis b virus molecular testing rapid, random-access, quantification of hepatitis b virus using the cepheid xpert®hbv viral load assay prevention of materno-foetal transmission of hepatitis b in sub-saharan africa: the evidence, current practice and future challenges poor sensitivity of commercial rapid diagnostic tests for hepatitis b e antigen in senegal, west africa validation of the treat-b score for hepatitis b treatment eligibility in a large asian cohort: treat-b improves with age development of a simple score based on hbeag and alt for selecting patients for hbv treatment in africa hepatitis b core-related antigen (hbcrag): an alternative to hbv dna to assess treatment eligibility in africa serum hepatitis b virus rna: a new potential biomarker for chronic hepatitis b virus infection validation of a novel rapid point-of-care alt test in patients with viral hepatitis non-invasive assessment of liver fibrosis and prognosis: an update on serum and elastography markers comparison of diagnostic accuracy of aspartate aminotransferase to platelet ratio index and fibrosis-4 index for detecting liver fibrosis in adult patients with chronic hepatitis b virus infection: a systemic review and meta-analysis dried blood spots-preparing and processing for use in immunoassays and in molecular techniques assessment of dried blood spot samples as a simple method for detection of hepatitis b virus markers diagnostic accuracy of serological diagnosis of hepatitis c and b using dried blood spot samples (dbs): two systematic reviews and meta-analyses evaluation of the efficiency of dried blood spot-based measurement of hepatitis b and hepatitis c virus seromarkers diagnostic accuracy of detection and quantification of hbv-dna and hcv-rna using dried blood spot (dbs) samples-a systematic review and meta-analysis usefulness of simultaneous screening for hiv-and hepatitis c-specific antibodies and hepatitis b surface antigen by capillary-based multiplex immunochromatographic rapid test to strengthen prevention strategies and linkage to care in childbearing-aged women living in resource-limited settings breakthrough agreement will reduce costs and increase access to diagnostic technology for millions in low-and middle-income countries technologies to support diagnosis and linkage to care: knowing your status and getting care 2.0 secondary genexpert®negotiated prices cost-effectiveness of community-based screening and treatment for chronic hepatitis b in the gambia: an economic modelling analysis seroprevalence of hepatitis b virus surface antigen and factors associated among pregnant women in dawuro zone, snnpr, southwest ethiopia: a cross sectional study aile, i. the cost-effectiveness of predonation screening for transfusion transmissible infections using rapid test kits in a hospital-based blood transfusion centre integrated approach for triple elimination of mother-to-child transmission of hiv, hepatitis b and syphilis is highly effective and cost-effective: an economic evaluation the effectiveness and cost-effectiveness of screening for and vaccination against hepatitis b virus among migrants in the eu/eea: a systematic review cost-effectiveness of rapid hepatitis c virus (hcv) testing and simultaneous rapid hcv and hiv testing in substance abuse treatment programs cost-effectiveness of one-time hepatitis c screening strategies among adolescents and young adults in primary care settings cost-effectiveness of strategies for testing current hepatitis c virus infection community-based, point-of-care hepatitis c testing: perspectives and preferences of people who inject drugs time matters: point of care screening and streamlined linkage to care dramatically improves hepatitis c treatment uptake in prisoners in england stressful point-of-care rapid testing for human immunodeficiency virus, hepatitis c virus, and syphilis implementing rapid hiv testing in outreach and community settings: results from an advancing hiv prevention demonstration project conducted in seven u acceptability of hiv self-testing: a systematic literature review exploring the barriers and facilitators to use of point of care tests in family medicine clinics in the united states innovation under regulatory uncertainty: evidence from medical technology do you need a dbs test? secondary do you need a dbs test national bowel cancer screening program secondary national bowel cancer screening program will an innovative connected aidesmart! app-based multiplex, point-of-care screening strategy for hiv and related coinfections affect timely quality antenatal screening of rural indian women? results from a cross-sectional study in india use of google trends to investigate loss-of-smell-related searches during the covid-19 outbreak new virological tools for screening, diagnosis and monitoring of hepatitis b and c in resource-limited settings this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-304816-7gg6pxnt authors: li, wei; ma, qiang; wang, xiao; tang, min; lin, jie; xiao, bin title: letter to the editor: the characteristics of two patients co‐infected with sars‐cov‐2 and hiv in wuhan, china date: 2020-06-10 journal: j med virol doi: 10.1002/jmv.26155 sha: doc_id: 304816 cord_uid: 7gg6pxnt we reported two cases of covid‐19 patients co‐infected with hiv. the sars‐cov‐2 nucleic acid test of patients turned negative while the clinical symptoms persisted, and interstitial pneumonia gradually deteriorated. the cases provided evidences to clinicians for the diagnosis and treatment of covid‐19 patients co‐infected with hiv. this article is protected by copyright. all rights reserved. the outbreak of coronavirus disease 2019 in early december 2019 has rapidly spread to all over the world. as announced by world health organization (https://www.who.int), there were approximately 4,628,900 people infected with severe acute respiratory syndrome corona virus 2 (sars-cov-2), which has killed more than 312,000 people worldwide until may 18, 2020. however, clinical manifestations vary widely from patient to patient [1] . the adaptive immune response plays a key role in all viral infections, during which t cells mediated cell immunity and b lymphocytes mediated humoral immunity. as is known to all, the human immunodeficiency virus (hiv) targets the immune system and mainly destroys cd4 + t lymphocytes. recently, covid-19 patients co-infected with hiv have attracted the interests of some researchers. a 61-year-old male patient co-infected with covid-19 and hiv was in stable condition and discharged after 20 days of treatment [2] . another study concluded that sars-cov-2/hiv co-infected patients having a more favorable prognosis when already under antiretroviral therapy [3] . in contrast, a study from new york revealed that the mortality of covid-19 patients with hiv infection reached up to 78% [4] . given that, the outcome of sars-cov-2/hiv co-infected patients seems controversial, and how to uncover the underlying hiv infected in covid-19 patients remains to be elucidated. in the present study, we reported two young male covid-19 patients co-infected with hiv and analyzed the clinical and laboratory features of them. it will provide clues for the diagnosis and treatment of covid-19 patients co-infected with hiv. (supplemental figure 1a) . the laboratory test results showed that the protein synthesis by hepatocytes like tp or alb were decreased and the hepatocellular enzymes alt or ast were significantly increased, the absolute count of lymphocytes was lower than the normal reference value. however, the infection had a limited effect on heart function ( figure 1a ) or renal function (supplemental figure 1b) . the detailed characteristics of case 1 were shown in supplemental table 1 . moreover, ct scan images of the lung showed that the high-density area was gradually increased from february 2 to february 20 ( figure 1b) . the patient had an intermittent fever and chest pain, and the highest body temperature was 39.4℃. most importantly, the patient presented fluctuating dyspnea symptoms for a long time. the number of lymphocytes also fluctuated from normal to far below the reference value, and the lowest lymphocyte count was 0.56×10 9 /l. the clinicians evaluated the symptoms and examinations comprehensively and speculated that the patients might suffer from immunodeficiency diseases. then hiv detection results showed that the patient was hiv positive. at last, the patient was transferred to a special hospital for infectious diseases and received further therapy. that the myocardial enzymes such as ldh, hbdh, ck, and ck-mb were gradually increased, the liver function index tp or alb was decreased and alt or ast was increased, the absolute count of lymphocytes was lower than the normal reference value ( figure 1c ). the level of il-6 was significantly increased (supplemental figure 1d) , while the effect of infection on renal function was limited (supplemental figure 1e) . the detailed characteristics of case 2 were this article is protected by copyright. all rights reserved. shown in supplemental table 1 . moreover, the ct scan of the lung showed that the high density area was gradually increased since from march 2 to march 15 ( figure 1d) . the patient had an intermittent fever and cough, and the highest body temperature was 40.2℃. most importantly, the symptom of dyspnea had gradually worsened. to validate whether the patient simultaneously suffered from immune deficiency diseases, hiv detection tests were performed. the results showed that the patient was co-infected with hiv. at last, the patient was transferred to a special hospital for infectious diseases and received further therapy. we reported two cases of covid-19 patients co-infected with hiv. although there is a lack of epidemiological investigation on whether hiv patients are susceptible to covid-19, the above cases presented the following distinctive clinical course and manifestations. firstly, the symptom of dyspnea persisted, and the exudative inflammation of lung was gradually increased, though the test of sars-cov-2 nucleic acid and specific igm or igg rapidly turned negative after receiving standard combination therapy. secondly, previous studies showed that the median hospital time of covid-19 patients was 12.32 or 14.8 days [5, 6] , however, these two young patients had a longer course of the disease and more severe symptoms. thirdly, the laboratory test showed that the liver function of case 1 and heart function of case 2 was obviously damaged. it has been reported that sars-cov-2 not only caused lung dysfunction, but also liver, heart, and this article is protected by copyright. all rights reserved. clinical features of patients infected with 2019 novel coronavirus in wuhan, china co-infection of sars-cov-2 and hiv in a patient in wuhan city covid-19 in a patient with hiv infection clinical features and outcome of hiv/sars-cov-2 co-infected patients in the bronx epidemiological characteristics and clinical features of 32 critical and 67 noncritical cases of covid-19 in chengdu epidemiological and clinical features of 125 hospitalized patients with covid-19 in this article is protected by copyright. all rights reserved we thank for the work and contribution of all the health providers from huoshenshan hospital. we declare no competing interests. accepted article accepted article key: cord-337458-dc90ecfe authors: markwalter, christine f.; kantor, andrew g.; moore, carson p.; richardson, kelly a.; wright, david w. title: inorganic complexes and metal-based nanomaterials for infectious disease diagnostics date: 2018-12-04 journal: chem rev doi: 10.1021/acs.chemrev.8b00136 sha: doc_id: 337458 cord_uid: dc90ecfe [image: see text] infectious diseases claim millions of lives each year. robust and accurate diagnostics are essential tools for identifying those who are at risk and in need of treatment in low-resource settings. inorganic complexes and metal-based nanomaterials continue to drive the development of diagnostic platforms and strategies that enable infectious disease detection in low-resource settings. in this review, we highlight works from the past 20 years in which inorganic chemistry and nanotechnology were implemented in each of the core components that make up a diagnostic test. first, we present how inorganic biomarkers and their properties are leveraged for infectious disease detection. in the following section, we detail metal-based technologies that have been employed for sample preparation and biomarker isolation from sample matrices. we then describe how inorganicand nanomaterial-based probes have been utilized in point-of-care diagnostics for signal generation. the following section discusses instrumentation for signal readout in resource-limited settings. next, we highlight the detection of nucleic acids at the point of care as an emerging application of inorganic chemistry. lastly, we consider the challenges that remain for translation of the aforementioned diagnostic platforms to low-resource settings. in 1906, william osler, one of the founders of modern american medicine, stated, "diagnosis, not drugging, is our chief weapon of offence." 1 despite the medical advances of the 20th and 21st centuries, infectious diseases remain major global health issues and continue to claim millions of lives each year in low-and middle-income countries (lmics). 2−4 as the global community moves toward control and elimination of infectious disease, it has become evident that there is a pressing need for diagnostic strategies that can be applied in primary health care settings. 5−7 this review shines a spotlight on how the applied uses of inorganic chemistry advance the concepts of metals-in-medicine beyond therapeutics and vaccines and into the realm of diagnostics, enabling new tools to meet these global challenges. the world health organization depicts lmic health care accessibility and infrastructure as a tiered pyramid structure, in which the best-equipped facilities are the least accessible and facilities with the least amount of resources are most common ( figure 1 ). 8 in this system, a level 1 facility is a primary care setting with little laboratory infrastructure, trained personnel, or advanced diagnostic technology. in lmics, both urban and rural level 1 facilities frequently lack essential resources such as consistent electricity and clean running water. district hospitals (level 2), regional laboratories (level 3), and national reference laboratories (level 4) have more advanced diagnostic technology available with increasing infrastructure; however, these facilities are often inaccessible to most patients in need due to geography, cost, and lack of transportation. 8 consider the case of early human immunodeficiency virus (hiv) diagnosis in exposed infants. coulibaly et al. highlight the challenges faced by this population in their 2011 burkina faso study. 9 hiv-positive mothers were advised to attend a 6week postnatal appointment at their nearest primary healthcare facility for collection of dried blood spot samples from their exposed infants. qualified sample collection technicians were often only available once each month, so mothers would have to return to the clinic on that day for testing. once collected, the dried blood spot samples were sent to district-level hospitals (level 2), which then sent the samples to a reference laboratory (level 3). the tertiary laboratory then determined viral load by performing polymerase chain reaction (pcr) to detect hiv genetic material. test results followed the same circuitous path back to the patients, a process that could take as long as four months. additionally, in the case of a positive dried blood spot result, the process had to be repeated in order to validate the original results. 9 in contrast, the same high-risk newborns in the united states are screened for hiv at birth with additional tests at 2−3 weeks, 1−2 months, and 4−6 months. 10 for these patients, results are usually available within 1 or 2 days of sample collection, allowing immediate antiretroviral treatment for hiv-positive children. 11 the consequences of diagnostic efficiency versus inefficiency could not be more striking; in the burkina faso study, 10% of the hiv-positive infants died before antiretroviral therapy could even be started, while the early treatment initiated in the u.s. improved infant survival by a remarkable 76%. 9, 12 the stark disparity in response time between these two settings shows just how devastating outcomes can be for patients relying on level 1 facilities for their healthcare needs. disease diagnosis in a level 1 setting represents a challenge that often requires simple tools that can be used without skilled personnel or significant laboratory or physical infrastructure. the world health organization has developed a set of criteria ("assured") that defines the ideal characteristics for pointof-care (poc) tests in low-resource settings. 13 in accordance with these criteria, an ideal test should be affordable to those who are at risk of infection, result in few false-negatives (sensitive) and false-positives (specific), and be user-friendly, rapid and robust, equipment-free, and deliverable to the populations in need of the test. additionally, the required performance parameters of a test depend on its intended usecase scenario. for individual patient case management, sensitive multiplexed tests are advantageous for determining the source(s) of nonspecific symptoms and selection of appropriate treatment. from an epidemiological standpoint, if the goal is simply to reduce the prevalence of high-intensity infections within a geographic region, the need to quantify disease burden outweighs the need for high analytical sensitivity. if the goal is disease elimination, high analytical sensitivity is one of the most important parameters, since the interruption of local transmission requires the detection of every infection, including extremely low-intensity infections. the most sensible approach to designing and developing a poc diagnostic is to examine and optimize each individual component before integrating into a single diagnostic device. in this review, we define the components of a diagnostic to include: (1) the target biomarker, an endogenous indicator of a disease state, which is most often a pathogen or host protein, carbohydrate, or nucleic acid sequence, (2) sample preparation, which allows for biomarker isolation, purification, and/or concentration from complex biological matrices, (3) molecular recognition elements, which specifically capture and detect the target biomarker, (4) signal generation and amplification, and (5) instrumentation for signal read-out. simple components requiring only a single user step are generally preferable in lowresource level 1 settings. however, there are few diagnostic technologies that currently can be deployed in these settings, and the lack of appropriate diagnostics in resource-limited settings can lead to tragic outcomes. fortunately, emerging technologies based on inorganic chemistry and nanomaterials can be exploited as potential solutions. in this review, we focus on how inorganic chemistry and nanomaterials have been utilized in each component of a poc diagnostic for infectious disease detection. section 2 describes inorganic biomarkers that are associated with infectious diseases and how their properties have been exploited in diagnostic applications. in section 3, we discuss how coordination chemistry has been harnessed for sample preparation and protein biomarker enrichment for poc tests. section 4 details the critical advancements in inorganic chemistry and nanomaterials for signal generation probes that have allowed the field of poc diagnostics to rapidly progress. in section 5, we provide an overview of field-deployable instrumentation that incorporates the fundamental inorganic chemistries discussed in sections 2−4 for poc diagnosis of infectious disease. finally, section 6 discusses the emerging trend of using inorganic chemistry and nanomaterials for nucleic acid detection at the point of care. biomarkers are quantifiable characteristics of a disease state that can be measured accurately and reproducibly. the vast majority of biomarker targets for infectious diseases fall into three main categories: (1) pathogen genetic material, (2) protein and carbohydrate antigens produced by a pathogen, or (3) human host responses to the presence of infection, such as pathogen-specific antibodies. while inorganic markers of infectious disease are far less common than their bioorganic counterparts, metal-based biomarkers have unique properties that can be leveraged for innovative diagnostic strategies, as illustrated in the following two examples. hemozoin, often called malaria pigment, is a biomineral produced by some parasites that rely on hematophagy as their primary source of nutrients. 14 hemozoin was first described for the malaria parasite plasmodium falciparum over a century ago and is produced by all plasmodium species. 15−17 in the erythrocytic life stages, malaria parasites digest host hemoglobin in the acidic digestive food vacuole as a source of amino acids. the breakdown of one hemoglobin molecule releases four molecules of heme (ferriprotoporphyrin ix [fe(iii)-ppix]), which are highly toxic to the parasite. 17 as plasmodium spp. lack functional heme oxygenase activity, the parasites remove the free heme by crystallizing it into insoluble, inert hemozoin crystals. 17, 18 these crystals accumulate in the parasite over the course of its erythrocytic life cycle. after the mature schizont ruptures, hemozoin is released into circulation and rapidly taken up by phagocytes. 19 the heme detoxification pathway that produces hemozoin is crucial for malaria parasite survival; therefore, hemozoin indicates the presence of plasmodia parasites, making it an attractive biomarker for malaria detection. 19 structurally, hemozoin crystals consist of reciprocating fe(iii)ppix dimers linked through coordination between the central ferric ions and a carboxylate group of one of the propionate moieties on protoporphyrin iv. 17, 20, 21 dimers assemble into chains through hydrogen bonding of the second propionate porphyrin side chain, and these networks of crosslinked dimers are held together via π−π interactions ( figure 2 ), resulting in an anisotropic rectangular crystal with unique magnetic, optical, and photoacoustic properties. 22 one such property of hemozoin is its paramagnetism, which is derived from the unpaired electrons on the central fe (iii) ions. 23 like all paramagnetic substances, hemozoin has a magnetic moment and is thus attracted up a magnetic gradient in the presence of an externally applied magnetic field. the paramagnetism of hemozoin has been leveraged for malaria diagnosis in several ways. first, the magnetic properties have been exploited for separation, purification, and concentration of infected red blood cells (irbcs) from blood samples. with the use of a high-gradient external magnetic field, irbc separation was first performed by heidelberger et al. 24 in 1946 for the purpose of vaccine development, though at the time the method was described as "applicable only to small blood volumes, [and requiring] apparatus difficult of access." 24 since then, the availability of high-field permanent magnets and electromagnets has substantially improved, and the time required for magnetic separation of irbcs has been reduced. both commercially available systems as well as custom-made microfluidic devices have been used to enrich irbcs from whole blood samples for analysis by microscopy. 25−32 three groups have explored detection of hemozoin based on the fact that paramagnetic materials decrease nuclear magnetic resonance (nmr) transverse relaxation time (t 2 ) of water, the same phenomenon leveraged for magnetic resonance imaging (mri) contrast agents. karl et al. 33 first demonstrated that hemozoin could be detected based on decreased t 2 , though only at parasitemias greater than 10000 parasites/microliter, which is 2 orders of magnitude greater than the detection limits of commercial rapid diagnostic tests. several years later, peng et al. 34 and kong et al. 35 developed a low-cost (<$2000), homemade micromagnetic resonance device for hemozoinbased malaria detection. they reported detection limits less than 10 parasites/microliter using their method and attributed this drastic improvement to the use of ultrashort echo times and a premeasurement centrifugation step that pelleted red blood cells from whole blood. 34 these discordant conclusions led to a reaction by karl et al. 36 and a response by peng et al. 37 recently, gossuin et al. 38 confirmed the results of karl et al., indicating that conventional relaxometry alone is insufficient for sensitive malaria detection. these findings led to two hypotheses related to the superior sensitivity measured by peng et al.: (1) the premeasurement centrifugation of higherdensity irbcs (compared to uninfected rbcs) significantly enriched hemozoin in the portion of the pellet measured, and (2) ultrashort echo times may have allowed for probing of other protons, such as macromolecular protons, which may be more susceptible to the presence of hemozoin. 38 a simple, lowresource relaxometry-based detection device remains to be developed, and such a device would ultimately require resources typically not available in primary healthcare settings in lmics (i.e., consistent availability of electricity and clean water). however, these studies highlight the importance and advantages of sample preparation and enrichment steps in the development of sensitive infectious disease diagnostics. in addition to its paramagnetic properties, the unique optical properties of hemozoin enable the development of new diagnostic tools for malaria detection. in particular, hemozoin strongly scatters and absorbs light. 39 the latter characteristic led to the remarkable development of the microvascular microscope (mvm) for needle-free, in vivo detection of hemozoin. 40, 41 the mvm employed cross-polarized epiillumination to optically access vessels below the surface of the skin, with green light (λ = 532 nm) illumination for locating vessels and red light (λ = 655 nm) illumination for detecting hemozoin. this method was able to detect parasitemias as low as 0.03% in mouse models, corresponding to approximately 1000 parasites/microliter in humans. unfortunately, lower parasite density infections were not explored. 40 the mvm was able to differentiate between irbcs and hemozoin-containing white blood cells, which can persist in circulation for weeks after an infection is cleared, based on the diameter and relative intensity of the detected hemozoin. 41 additionally, hemozoin particle velocity could be measured in vivo (figure 3 ), potentially allowing for identification of cytoadhesion and sequestration, two phenomena related to disease severity. 41, 42 though further sensitivity analysis and field testing is required, the mvm represents a promising, noninvasive tool for malaria detection, particularly if a portable and battery-powered device is developed. photoacoustic spectroscopy has also demonstrated potential for transdermal malaria detection. 43−48 photoacoustic (pa) hemozoin detection requires excitation with intense light and measurement of the resulting acoustic signals generated by local thermal expansion of the surrounding medium. the utility of in vitro pa detection of hemozoin was first demonstrated by balasubramanian et al. in 1984 . 43 since then, it has been shown that short, intense laser excitation of hemozoin in the parasite can localize heat around the crystal, evaporating the surrounding medium and creating transient, nanosized vapor bubbles. the expansion and collapse of these nanobubbles enhance acoustic detection. 44 this technique has allowed for transdermal malaria detection with exceptional sensitivity in mice. this method was also applied to one human patient with high parasite density (8900−65000 parasites/microliter). 45−48 further studies with increased patient sample size will be necessary to determine the true limits of the technology. magneto-optical detection exploits both the paramagnetic and optical properties of malarial hemozoin crystals, which have a rectangular shape that affords a high level of both magnetic anisotropy and optical dichroism. in this technique, whole blood (or lysed whole blood) is inserted into a magnetic field, orienting the paramagnetic crystals along the applied field direction. this phenomenon, known as the cotton-mouton effect, induces an optical dichroism across the dispersion of crystals, resulting in a detectable change in transmittance of modulated polarized light (λ = 660 nm) ( figure 4 ). 49, 50 when applied to patient whole blood samples, the diagnostic sensitivity and specificity of magneto-optical detection were not competitive with rapid diagnostic tests and pcr, though the ease of use is encouraging for poc applications. 49, 50 in particular, model simulations have suggested that magnetooptic detection of hemozoin could be exploited for the development of a noninvasive probe capable of measuring transmittance of polarized light through a fingertip. 51 a similar magneto-optic strategy that employs a fluctuating magnetic field and measures the change in amplitude of transmittance has shown potential in mouse models, but further evaluation of the diagnostic sensitivity and specificity in human samples is needed. 52−55 recently, a commercial magneto-optic device for malaria detection based on hemozoin has demonstrated promise in field evaluations, detecting clinical parasite densities as low as 5 parasites/microliter (hemex health). 56 additional techniques used to detect malarial hemozoin include flow cytometry, 57−60 laser-desorption mass spectrometry, 61−64 and raman spectroscopy. 65−69 while these strategies are highly sensitive, they require expensive and resourceintensive instrumentation that typically prevent their use in a district hospital setting, local health outpost, or field setting. however, as instruments become more compact, robust, and affordable, similar to some of the technologies detailed in this section, their implementation in field settings could become more feasible. importantly, hemozoin is synthesized by other hematophagous parasites that infect humans, including the schistosoma, echinostoma, and opisthorchis trematodes, as well as blood feeding insects, such as rhodnius and boophilus. 17, 70, 71 despite being produced by these additional parasites, hemozoin has not yet been used as a diagnostic biomarker for nonmalarial pathogens. further, it is important to note that in all of the aforementioned hemozoin-based malarial detection strategies, hemozoin originating from trematodes would produce a positive signal. this is particularly important to consider regarding false positive tests since the geographic distribution of malaria overlaps with schistosoma. 72, 73 in addition to confounding signal originating from other hematophagous parasites, hemozoin as a biomarker presents some unique challenges to malaria diagnosis. first, the biocrystal is present at much lower concentrations in the ring stage when compared to the later trophozoite and schizont stages of the erythrocytic cycle. 32 thus, magnetic enrichment or malaria diagnosis strategies based on hemozoin could miss early stages of infection, potentially resulting in false-negatives. additionally, phagocytic cells ingest irbcs and hemozoin released into circulation after schizont rupture, and hemozoincontaining white blood cells have been shown to persist in circulation up to 17 days after parasite clearance. 74 this persistence can lead to false-positive results in hemozoin-based malarial diagnostics unless an algorithm for distinguishing pigment-containing white blood cells and irbcs is devised, such as the one developed in the aforementioned work by burnett et al. 41 despite these challenges, researchers continue to develop innovative techniques for malaria detection using hemozoin as a biomarker. implementation of these methods will require careful design with cost and equipment reduction in mind in addition to rigorous field evaluation. schistosome eggs are the most common biomarker for schistosomiasis detection. they are produced by adult schistosoma worm pairs that reside in the intestine and bladder and deposited into the vessels of the gut wall. 75 many eggs become permanently lodged in the intestine, liver, bladder, or urogenital system, leading to chronic inflammation and systemic tissue damage; 76 however, some mature eggs are excreted in urine or feces to allow the parasites to continue the transmission cycle. 75 eggs from distinct species are distinguishable by their morphologies. differences include the overall egg shape (round to oval), spine position, and extent of filamentous coating. 77 all of these features are observable by microscopy, which is the gold standard for schistosomiasis diagnosis. voided eggs from all schistosoma species contain mature miracidia within a relatively impermeable eggshell. 75 while the dense biopolymer composition of schistosoma eggshells has been studied since the 1960s, 78 the inorganic components of the eggshells were not investigated until 2007, when jones et al. 79 demonstrated the presence of iron in schistosoma japonicum eggshells ( figure 5a ). the group used inductively coupled plasma mass spectrometry (icp-ms) and energy dispersive spectroscopy (eds) to provide evidence that iron localized to the eggshell. although these elemental characterization techniques did not provide specific information about the oxidation state or organization of iron present, the group found that the eggshells contained 121 mg/kg (dry weight) fe, making up nearly 89% of the iron found in intact eggs. 79 in the same year as the jones study, teixeira et al. 80 set out to increase the sensitivity of microscopy for schistosoma detection using magnetic microparticles to enrich eggs from fecal samples. to accomplish this, stool samples were suspended in water and repeatedly sieved and washed before the sediment was incubated with magnetic particles that bound to the schistosoma eggs. this technique for egg concentration was dubbed the helmintex method 80 and was demonstrated to be more sensitive than kato-katz, the current who recommended sample preparation method for schistosome diagnosis in a field setting. 81 the initial hypotheses of the helmintex method were that biotinylated lectins would bind to carbohydrates on the surface of schistosoma mansoni eggs and that streptavidin-coated magnetic beads could bind the biotinlectin-egg complexes. using an external magnet, the eggs could then be isolated and concentrated before detection by microscopy. however, control experiments demonstrated the following: (1) lectins were not necessary for egg binding to magnetic particles, (2) nonmagnetic latex particles did not bind to s. mansoni eggs, and (3) the eggs alone did not migrate to an external magnet ( figure 5b ). 80 these observations, coupled with the discovery of iron localized to eggshells, led some to hypothesize that, in the presence of an external magnetic field, the paramagnetic beads act as weak bar magnets, enabling attraction of the iron in the shells of the eggs. 82 in 2013, karl et al. 83 further probed the iron distribution and magnetic properties of schistosome eggshells to elucidate the mechanism of the helmintex method. the study found that iron was localized to pores in eggshells, and schistosome eggs demonstrated moderate paramagnetic behavior. however, the eggs did not move in a magnetic field, and the paramagnetic particles bound to parasite eggs in the absence of a magnetic field, suggesting that the mechanism of binding was not magnetic in origin. 83 the authors also found that s. japonicum eggs bound more microparticles than s. mansoni, leading to the hypothesis that the filamentous outer structure of the eggs, more prominent in s. japonicum compared to s. mansoni eggs, provided strong, nonmagnetic adhesion based on many relatively weak van der waals interactions. 83 in a separate study, candido et al. 84 also observed that s. japonicum eggs bound more microparticles than s. mansoni. the group used poisson analysis and found two distinct populations within each species: a high "affinity" group and a low "affinity" group, where affinity was determined by the number of microparticles bound to the visible edge of a single egg. interestingly, the magnitudes of the high affinity values were similar between the two species, but the fraction of eggs in the high affinity group for s. japonicum was greater than for s. mansoni. 84 collectively, these results suggest that differences in filamentous microspines between the two species do not fully explain the mechanism of egg-microparticle binding. additionally, the work by candido et al. opens up many questions, the answers to which could greatly inform further optimization of the helmintex method. for instance, what are the physiochemical differences between high and low affinity egg groups? is there a biological difference between the two groups, such as egg maturity or surface antigen expression? both the karl et al. and candido et al. studies were performed on eggs isolated from livers of infected mice, but do these two egg populations appear in the urine or feces of infected humans? if so, should two types of magnetic microspheres be used to ensure the helmintex method captures all egg types present in a sample? these questions should be answered carefully and systematically. given the greater number of microspheres bound to eggs in the high affinity group, the populations should be separable using external magnetic fields of differing strengths. egg-microparticle binding mechanisms for each population could be interrogated by determining solution properties (e.g., ph, salt, surfactant, chelators, blocking proteins, etc.) required to interrupt the interactions. additionally, functionalizing microspheres in a way that systematically varies their zeta potential and hydrophobicity could determine the role these physiochemical properties play in binding. these studies will be crucial if the helmintex method aims to determine infection intensity using quantitative egg enrichment. this is particularly important for schistosomiasis surveillance and morbidity control campaigns, which aim to quantify and reduce the prevalence of high-intensity infections. the helmintex method originated with the idea that iron in the eggshells of schistosomes could serve as a handle for magnetically enriching schistosoma eggs for more sensitive detection in the field setting. while the helmintex method has already been shown to improve the sensitivity of kato-katz, elucidation and optimization of the true binding mechanisms could lead to an even more impactful method for diagnosing low-intensity schistosomiasis infections. metal-based sample preparation techniques that enrich biomarker concentration improve the sensitivity of diagnostics by delivering more biomarker to the test. these strategies can be applied to existing assured diagnostic tests, such as lateral flow assays (lfas). while the easiest way to deliver more biomarker to the tests would be to add larger sample volumes (100−500 μl), many lfas cannot accommodate large sample volumes for several reasons: 85 first, limited bed volumes in porous paper substrates physically limit the amount of liquid a test can hold. second, the increase in the number of interfering molecules that results from an increase in sample volume could cause nonspecific cross reaction with the test's molecular recognition elements (e.g., antibodies). third, colored biological matrices, such as whole blood, can increase the background signal and decrease the user's ability to distinguish between positive and negative results. metal-based sample preparation techniques mitigate these shortcomings, allowing for the concentration of a target from a large volume sample into a smaller, test-compatible volume. moreover, metal-based sample preparation also removes the target from its original complex biological matrix, decreasing the potential for nonspecific cross-reactivity. 85 these low-cost, simple, and robust methods represent a powerful tool for use-case scenarios in which improved sensitivity is needed, such as elimination campaigns. this section will discuss how metal coordination chemistry has been leveraged for simple sample preparation methods applicable to poc diagnostics. coordination chemistry, which is defined as the chemistry of metal atoms or ions that form complexes with one or more ligands, 86 can be utilized to coordinate biomarkers with a particular affinity for metal ions. the most significant illustration of this capacity is the isolation of proteins via immobilized metal affinity chromatography (imac). 87,88 this technique involves the conjugation of various ligands to a solid support matrix such as agarose, cellulose, or silica. these ligands act as lewis bases, chelating metal ions with a high affinity. the fixed metal ions serve as lewis acids, coordinating to both the immobilized ligands and specific amino acid residues in a protein biomarker. 89 while amino acid affinities toward metal ions vary, histidine demonstrates a particularly high affinity toward metal ions in this format. these interactions can be leveraged to isolate a biomarker from a complex biological matrix. once separated, the target can be competitively eluted and concentrated into a smaller volume suitable for a poc diagnostic test. 87, 88 the choice of chelating ligand is critical to imac protein separation efficiency. ligands both fix the metal ion to the solid support and modulate metal-protein binding based on the number and conformation of chelation sites. 88 ,90−92 a multitude of ligands with varying coordination numbers have been employed. most imac platforms contain tridentate or tetradentate chelators, although some bidentate and pentadentate options exist ( figure 6 ). tetradentate nitrilotriacetic acid (nta) 93 and tridentate iminodiacetic acid (ida) 94 are two of the most common commercial ligands, but many companies have also developed their own proprietary structures such as talon 95 or ni-penta. 96 the variety of chelating ligands, many of which possess different coordination numbers, make imac tunable, which is beneficial when tailoring the chemistry to different diagnostic applications. 87,88,97−100 guided by pearson's hard−soft acid−base theory, numerous metal ion and ligand combinations have been exploited in imac. borderline acids such as co 2+ , ni 2+ , cu 2+ , and zn 2+ have predominated, 86−88 displaying varying levels of affinity and specificity. the determination of which ion will be optimal in a given isolation often depends on the protein in question. ni(ii)-nta has been one of the most widely utilized metal− ligand combinations in imac applications. until recently, a thorough investigation that compared divalent metals and their molecular recognition capability was lacking. bauer et al. 99 filled this void by conducting a systematic investigation of the affinity of malarial biomarker histidine-rich protein 2 (hrp2) to m 2+ -functionalized biosensors and solid phase resins (m 2+ = co 2+ , ni 2+ , cu 2+ , or zn 2+ ). hrp2 is the most frequently detected target in malarial rapid diagnostic tests and possesses a unique primary structure containing 35% histidine, making it an ideal histidine-rich target. 101 the authors used biolayer interferometry (bli), an optical biosensor technique, to determine kinetic constants (e.g., k on , k d ) for the binding of the various metals to hrp2. the authors found that co 2+ and zn 2+ exhibited the fastest binding kinetics (highest k on ) and the highest hrp2 binding affinity (lowest k d ) on bli biosensors. 99 when the same imac complexes were incorporated into a loose solid phase resin, zn(ii)-nta resulted in the fastest complete binding of hrp2 per mole of divalent metal while also allowing for the most efficient elution of hrp2 upon the addition of imidazole. once a metal had been selected, hrp2's binding and elution behavior was evaluated against a variety of ligands and solid supports. the selected zn(ii)-ida resin was then integrated into a pipet tip format, enabling direct hrp2 capture from spiked lysed blood samples. this field-deployable sample isolation and concentration tool displayed up to a 4-fold signal enhancement in commercial rapid diagnostic tests (figure 7 ). this study 99 demonstrated that optimizing the imac metalligand combination for a particular application is critical for efficient biomarker capture and elution; the initial binding cannot be too tight so as to prevent biomarker elution with a competing ligand (e.g., imidazole) nor too weak so as to attain suboptimal biomarker enrichment. 88, 99 imac has been adapted to a variety of solid support platforms, from agarose gels to rigid silica particles. 102, 103 although many of these platforms have become wellestablished research tools, the emergence of novel materifigure 6 . coordination of imac ligands (ida, nta, and ted) to a divalent metal for binding histidine residues in proteins. co 2+ , ni 2+ , cu 2+ , and zn 2+ have predominantly been utilized in this application. ida (iminodiacetic acid) is a tridentate ligand (coordination number = 3), with the remaining three coordination sites in the metal available to bind two histidine residues and a water molecule. nta (nitrilotriacetic acid) is a tetradentate ligand (coordination number = 4) with two coordination sites for histidine residue binding. ted (tris(carboxymethyl)ethylene diamine) is a pentadentate ligand (coordination number = 5) with one remaining coordination site for protein binding. als 104−108 has enabled imac chemistry in new applications. for example, wang et al. 109 polymerized polyhedral oligomeric silsesquioxanes (posss) into micron-sized particles with nanosized pores, which were subsequently functionalized with ida and charged with cu 2+ . this material was then used for specific capture of hemoglobin, which is rich in surface-exposed histidine residues. incubation of the test solution with the cu(ii)-ida functionalized material enabled selective capture of hemoglobin versus other proteins without these accessible histidine residues. 109 the development of this novel material using the principles of imac demonstrates that nanomaterials can be modified for molecular recognition of histidine-rich proteins. these modified nanomaterials have the potential to be incorporated into poc diagnostic frameworks for biomarker capture. imac-functionalized cellulose membranes could provide another excellent option for sample preparation in lowresource settings. given that these membranes already hold an established place as a laboratory research tool, they could readily be incorporated into field-friendly paper fluidic devices. opitz et al. 110 utilized commercial ida-functionalized cellulose charged with zn 2+ as a platform for separating influenza virus particles from culture lysate. after an initial metal screening, they saw that zn 2+ -charged membranes separated particles with significantly higher affinity the other metals tested. the authors postulated that the influenza hemagglutinin glycoprotein on the particular strain studied (influenza virus a/ puerto rico/8/34) contained histidine-rich zn 2+ -coordinating sequences on each subunit and that the repeated configuration of these subunits as part of the capsid structure contributed to a higher overall binding avidity. 110 although this imac-based method for separating virus particles from cell culture was developed to improve the efficiency of laboratory-based research, it is not difficult to imagine extending this concept to a simple sample preparation method for increasing the sensitivity of an influenza rapid test. applying the developed protocol to a diagnostic test would require feasibility testing on clinical patient samples, ideally, resulting in the design of an integrated device for both sample preparation and virus detection. imac-functionalized cellulose membranes present a unique advantage in relation to other materials because of the ease in which they can be incorporated into paper-based diagnostic formats that are usable in low-resource poc settings. magnetic particles hold a number of advantages over other solid imac supports. the increased surface area of the particles provides increased availability for chemical reactivity and functionalization. 111, 112 the independent response of paramagnetic particles to an externally applied magnetic field enables active mixing, which has proven especially useful in microfluidic applications. this magnetic susceptibility also allows for easy manipulation of the particles once a targeted biomarker is bound, permitting biomarker isolation and concentration before downstream detection. 111, 112 multistep protocols that would otherwise rely heavily on laboratory techniques such as filtration, centrifugation, and column chromatography can easily be performed using magnetic beads and a hand-held external magnet. aside from chromatographic capabilities, magnetic particles have been employed as signal generation labels that are detected by inductive or magnetoresistive sensors. this application is discussed in detail in sections 4.2.6 and 5.4. magnetic particles thus represent a promising vehicle for imac chemistries in low-resource settings. the wright research group recently demonstrated that imac-functionalized magnetic beads have the potential to improve commercial poc diagnostic sensitivity for malaria. 113 hrp2 was captured from large-volume (100 μl whole blood samples diluted and lysed to 200 μl) p. falciparum-spiked samples via chelation to ni(ii)-nta on the surface of . flow-through device for capture of malarial biomarker hrp2 via zn(ii)-ida chelation to histidine residues. the device incorporated a silica resin solid phase functionalized with zn(ii)-ida into a pipet tip, which enabled rapid capture of hrp2 from whole blood samples using the principles of imac. subsequent addition of imidazole at a high concentration competitively eluted the hrp2 from the zn(ii)-ida resin into a smaller volume. this small volume sample was then deposited onto a commercially available rapid diagnostic test which enhanced sensitivity for hrp2 detection in a poc setting. adapted with permission from ref 99. copyright 2017 american institute of physics. commercially available magnetic particles. then, using a specially designed extraction cassette and a hand-held magnet, the hrp2-bound particles were guided through stationary liquid chambers separated by surface-tension valves to wash the sample and remove background. once purified, the magnetic beads with bound hrp2 were transferred to a final 10 μl elution chamber preloaded with imidazole to release the biomarker. this resulted in a 10-fold theoretical concentration factor for hrp2 due to the transfer from a 100 μl blood sample to a 10 μl elution chamber. after extraction using this self-contained system, the authors added the 10 μl elution volume to a variety of commercial hrp2-based malaria rapid diagnostic tests and found significant signal enhancement across all brands. 113, 114 in subsequent studies, the group developed and optimized a hand-held, easy-to-use device 85, 115 (figure 8a ) in which hrp2-bound, imac-functionalized magnetic beads were directly transferred to the sample pad of commercial malaria lateral flow assays. the biomarker was eluted with an imidazole-spiked running buffer. as shown in figure 8b , this device enabled highly sensitive hrp2 detection with few added user steps, improving the detection limits of commercial malaria tests by over an order of magnitude with minimal added cost per test. 85, 115 this enhanced sensitivity could make a substantial difference in malaria elimination campaigns, which rely on detecting and treating all infected individuals, including low-density carriers. one of the primary limitations of imac chemistry is its ability to recognize only those proteins with affinities for metal ions. for purification of recombinant proteins from cell culture, this drawback has been overcome via incorporation of short hexahistidine tags (his 6 -tags) into expressed protein sequences. 116,117 using a similar principle, bauer et al. 117 developed a platform in which target-specific antibodies modified with his 6 -tags were combined with imac magnetic beads to isolate and concentrate biomarkers from biological samples. thus, the advantages of imac for biomarker enrichment were no longer limited to histidine-rich proteins but rather could be extended to any protein biomarker target. in the context of malaria diagnosis, this expanded capability allows for the detection of the nonhistidine-rich biomarker plasmodium lactate dehydrogenase (pldh) alongside hrp2, which is beneficial for several reasons. plasmodium ldh is essential to parasite survival and is conserved across all species of malaria known to infect humans, whereas hrp2 is produced only by p. falciparum. 118 in addition, hrp2-only tests fail to account for parasitic infections containing pf hrp2 gene deletions, which are increasing in prevalence worldwide. 72 finally, hrp2 has been shown to persist in host circulation up to 52 days after successful treatment, but pldh clears within 24 h after parasite clearance, so a dual assay can distinguish between resolved and active p. falciparum infections. 119−122 for these reasons, bauer et al. 117 employed their imacbased his 6 -tagged antibody approach to concentrate both pldh and hrp2 from large volume samples (200 μl parasitespiked whole blood) utilizing commercially available imac magnetic beads. 117 in this dual capture system, hrp2 coordinated directly to the co(ii)-nta-functionalized surface of the magnetic bead, while pldh was bound using his 6tagged anti-pldh antibodies ( figure 9 ). following isolation of both biomarkers, hrp2 and the his 6 -tagged-antibody/pldh complex were simultaneously eluted with imidazole. when applied to whole blood samples spiked with the p. falciparum parasite, this strategy resulted in a nearly 20-fold improvement in the limit of detection of commercial lfas. 117 this unique platform demonstrates that imac-based biomarker enrichment is not limited to histidine-rich proteins and could be expanded to virtually any biomarker for which there is a commercially available antibody. one of the challenges associated with employing a his 6tagged antibody/imac concentration strategy is that the biomarker remains bound to the antibody during the detection step. the antibody used for sample preparation has the potential to block target binding (via overlapping epitopes or steric effects) to the molecular recognition elements employed in the downstream assay, leading to false-negative results. this challenge can be addressed by designing the diagnostic such that the his 6 -tagged antibody and downstream detection elements bind to orthogonal epitopes of the target. furthermore, other his 6 -tagged molecular recognition elements such as aptamers could be used in place of his 6 -tagged antibodies. aptamers are short, single-stranded nucleic acid sequences with secondary structures that afford high (∼nanomolar−picomolar) binding affinities. compared to antibodies, aptamers are smaller and may target unique epitopes, potentially allowing for this innovative sample preparation strategy to be more universally applied. 123−126 coordination chemistry-based sample preparation technologies have enabled significant improvements in the sensitivities of point-of-care diagnostics. the enhanced sensitivities, which in several examples were improved by a whole order of magnitude, make a strong case for the use of these technologies in elimination campaigns. 85, 115, 117 previously, biomarker enrichment strategies were not incorporated into low-resource diagnostics because of the restrictive format of the tests. the imac-based chemistries described in this section represent innovative yet simple solutions that open the door for the development of poc diagnostics that incorporate sample preparation. while some of the sample preparation methods presented in this section increased the total number of assay steps, these methods were not designed in tandem with the diagnostic devices that were utilized for detection. moving forward, human-centered device design that integrates sample preparation with detection elements will permit the next generation of poc diagnostics to be developed with minimized user steps and improved sensitivities. inorganic metal complexes and nanoparticles have been instrumental in the development of targeted reagents for disease-associated dna, proteins, or cells. their biomedical applications include the development of metallodrugs, 127, 128 enzyme inhibitors, 129−131 photothermal 132,133 and photodynamic 133 therapy agents, and mri contrast, 127, 132 in vivo bioimaging, 133, 134 and protein/cell imaging agents. 135, 136 their unique optical, electrochemical, magnetic, and catalytic properties have also enabled their use as signaling modalities in diagnostics. this section focuses on the application of metalbased signal generation probes for the detection of protein biomarkers at the point of care (summarized in table 1 by biomarker and in table 2 by signal generation method). (the detection of pathogenic nucleic acids via metal-based signal generation probes is discussed in detail in section 6). first, we describe how inorganic complexes have been utilized in photoluminescent, electrochemical, and electrochemiluminescent applications, respectively. subsequently, we detail the essential bioconjugation chemistries that permit nanoparticles to be functionalized with molecular recognition elements to generate useful diagnostic reagents. we then discuss how various classes of these functionalized nanoparticles have been used in poc diagnostic assays. lastly, we consider the catalytic properties of metalloenzymes and metal nanoparticles that have been utilized for signal amplification in poc diagnostics for infectious diseases. the broad array of properties that result when a ligand coordinates to a metal has been leveraged for signal production in a variety of diagnostic formats. inorganic coordination compounds are valuable as signal generation probes due to their low molecular weight, tunability, and unique catalytic and photophysical properties. 130 these compounds have been used in photoluminescent (i.e., fluorescent and phosphorescent), 137−142 electrochemical, 143−148 and electrochemiluminescent (ecl) 149−157 sensing platforms. each signal generation method has trade-offs with regard to ease-of-use and interpretation, as well as complexity of instrumentation required to measure signal output. when designing a lowresource diagnostic, these trade-offs must be evaluated based on the use-case scenario and testing environment. collectively, these considerations better inform the developer as to which inorganic complexes are suitable for a particular signal generation method. 4.1.1. luminescence. photoluminescent inorganic complexes possess unique properties and offer several advantages over organic fluorophores as signaling molecules for diagnostics. these advantages include long-lived phosphorescence, significant stokes shifts, and emission tuneability via ligand composition. 137, 158, 159 long-lived phosphorescence is beneficial because it is spectrally distinct from the autofluorfigure 9 . a his 6 -tag was coupled to an anti-pldh antibody via sulfo-smcc to allow for chelation of the his 6 -tagged antibody to co(ii)nta-functionalized magnetic bead. remaining sites on the bead surface (unoccupied by antibody) permitted coordination of hrp2 directly to the bead surface. subsequent to biomarker capture, imidazole was added to elute hrp2 and the his-tagged antibody/ pldh complex. adapted from ref 117 158 the large stokes shifts of metal complexes also result in improved sensitivity because self-quenching of signaling molecules is minimized. 158 additionally, the tunable emissions of metal complexes enable their use as signal transducers, whereby ligand exchange reactions that induce changes in emission spectra can be correlated to biomarker concentration. 159 to date, cyclometalated d 6 complexes of ir (iii) are the most commonly utilized photoluminescent signal generation probes. [137] [138] [139] 141, 142, 160 ligands in these cyclometalated ir (iii) complexes (e.g., deprotonated 2-phenylpyridine) demonstrate strong σ-donor effects, which significantly increase the energy of the 1 mc (metal-centered) excited state (i.e., the ligand field stabilization energy) that gives rise to nonradiative decay pathways. as a result, excited electrons in these complexes preferentially populate the 1 mlct (metal-toligand charge transfer) excited state (as opposed to 1 mc). the excited electrons then undergo intersystem crossing and subsequently phosphoresce from a mixture of the 3 lc (ligand-centered) and 3 mlct excited states ( figure 10 ). the high phosphorescent quantum yields with large stokes shifts make these complexes ideal for signal generation. 160, 161 davis et al. 138 designed a "switch-on" ir(iii) detection probe that selectively produced phosphorescent signal in the presence of malarial biomarker hrp2. the cyclometalated ir(iii) complex, [ir(ppy) 2 (h 2 o) 2 ] + , was poorly luminescent in the absence of hrp2; however, when aqua ligands were displaced by hrp2's histidine residues, a biomarker-dependent 3 mlct/ 3 lc phosphorescent signal was produced ( figure 11 ). similar to the principles of imac chemistry discussed in section 3, this approach capitalized on the chelation of hrp2's histidine repeats to the inorganic coordination compound. the assay design aptly showed how the tunable luminescence of ir(iii) complexes could be harnessed for signal transduction, demonstrating that a ligand exchange reaction's phosphorescent signal could be correlated to biomarker concentration. in addition, the ir(iii) complex displayed a significant stokes shift (δλ = 145 nm), 138 making it less prone to self-quenching effects and decreases in sensitivity associated with its organic fluorophore counterparts. 162 this study 138 also incorporated the ir(iii) probe into an imac magnetic bead-based biomarker isolation and detection assay for recombinant hrp2 which had a detection limit of 14.5 nm. although less sensitive than an elisa (enzymelinked immunosorbent assay), this hrp2 concentration was within the relevant range for clinical diagnosis of malaria. moreover, the bead-based assay could be completed in less than 2 h, whereas traditional elisas require 6−8 h. while the current assay format would only permit its application in better-equipped laboratories, the developed cyclometalated ir(iii) probe could be an exciting detection element in an lfa with hrp2-specific antibodies at the test line. however, this photoluminescence detection mechanism is dependent on existence of a histidine-rich biomarker, so further applications of this particular strategy are limited. a more generalizable platform using photoluminescent ir (iii) probes was developed by lin et al. 139 for the detection of interferon-gamma (ifn-γ), an inflammatory cytokine biomarker for immunological diseases such as hiv. for molecular recognition and detection of ifn-γ, the assay used two aptamers: one was highly specific for ifn-γ, and the second was a guanine-rich nucleic acid sequence. the two aptamers were designed to be partially complementary and were prehybridized prior to the introduction of a sample containing ifn-γ. in the presence of ifn-γ, the ifn-γ-specific aptamer bound to its target and liberated the guanine-rich sequence. introduction of k + ions induced the formation of guanine quadruplexes, which subsequently bound an ir (iii) probe for "switch-on" luminescence that correlated biomarker concentration with phosphorescent signal (figure 12 ). this ir(iii) probe possessed a long phosphorescence lifetime (>2.9 μs) and significant stokes shift (δλ = 215 nm), thus exhibiting the canonical advantages of inorganic probes over organic fluorophores. the assay demonstrated adequate specificity for ifn-γ versus other proteins commonly found in biological matrices (e.g., human serum albumin and immunoglobulins) and had a limit of detection of 0.12 nm and a dynamic range of 1−300 nm. however, the assay suffered a reduction in sensitivity when conducted in a 0.5% cell extract, and no attempts were made in the complex biological matrices that are pertinent to hiv diagnosis (e.g., blood, urine, saliva, etc.). nonetheless, the assay established a proof-of-principle for using a photoluminescent cyclometalated ir(iii) complex as a detection probe for an hiv biomarker. 139 moreover, the assay format is generalizable such that it could be applied to virtually any biomarker for which there is a high-affinity aptamer. if combined with one of the sample preparation strategies discussed previously (section 3) or integrated with paper or another field-ready substrate, this ir(iii)-based detection strategy could produce a robust and sensitive assay that is applicable in low-resource diagnostic settings. 4.1.2. electrochemistry and electrochemiluminescence (ecl). by applying a potential (or range of potentials) to a sample containing an electroactive inorganic probe, current or luminescence can be measured as an output for electrochemical or ecl detection, respectively. electrochemical or ecl-based assays that combine molecular recognition elements, such as antibodies or aptamers, with electroactive inorganic probes have shown promise for infectious disease detection. 149,163−165 in particular, inorganic low-spin d 6 complexes of ir (iii) and ru(ii), as well as hemin (ferriprotoporphyrin ix), have been utilized in developing electrochemical-and ecl-based probes. 149 both electrochemistry and ecl are signal generation strategies that are well-suited to the development of poc diagnostics, largely due to (1) capability for miniaturization, (2) low cost, (3) simplicity, (4) rapid time-to-result, and (5) high sensitivity. 149, 163 this has been aptly demonstrated with the development of paper-based electrochemical diagnostic platforms, 166 often termed microfluidic paper-based electrochemical devices (μpeds) 167−169 or electrochemical paper analytical devices (epads). 170 these tests utilize paper as the assay substrate and have been combined with instrumentation amenable to low-resource settings, such as cell phones, for detection. incorporation of existing electrochemical-and eclbased assays into these formats could result in robust and highly sensitive infectious disease diagnostics. this would be impactful for disease surveillance or case management, where robust and sensitive diagnostics are needed. electrochemistry. diagnostic assays with electrochemical detection commonly consist of a three-electrode system: a working electrode (often gold or carbon modified with a molecular recognition element for biomarker capture), a counter electrode (typically platinum), and a reference electrode (commonly a saturated calomel electrode or ag/ agcl electrode). 171 in this format, incubation of a biological sample in an electrochemical cell initially results in capture of the biomarker at the working electrode. introduction of a biomarker-specific inorganic probe and a supporting electrolyte and subsequent application of an electrochemical method (e.g., cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, etc.) results in significant current production at the working electrode. this current can then be correlated to biomarker concentration. with regard to infectious disease biomarker detection, hemin, ir(iii) complexes and ru(ii) complexes have been utilized as electrochemical probes. 144−146 hemin (ferriprotoporphyrin ix) is a fe 3+ -containing porphyrin derivative that mimics the enzymatic activity of horseradish peroxidase in catalyzing the reduction of h 2 o 2 (see section 4.3) . this enzymatic activity is further enhanced when hemin is incorporated into a g-quadruplex aptamer. 143, 144, 172 zhang et al. 144 developed an electrochemical assay for the detection of hiv-associated ifn-γ that utilized hemin as a key component of the signal generation mechanism. a 3′-thiolated dna construct was conjugated to a gold working electrode, where the dna construct contained a gquadruplex sequence that was specific for hemin and an ifn-γspecific aptamer. both the g-quadruplex and the ifn-γ-specific aptamer were prehybridized by an 8-nucleotide hairpin. the authors utilized this functionalized working electrode to the assay utilized two aptamers, the first being an ifn-γ-specific aptamer (green) that was prehybridized to a g-quadruplex aptamer (blue). addition of a sample resulted in the capture of ifn-γ by the first aptamer and liberation of the g-quadruplex aptamer, which underwent a structural switch to form guanine tetrads upon introduction of k + ions. the gquadruplexes were specific for a cyclometalated ir (iii) perform cyclic voltammetry on samples containing ifn-γ to monitor the reduction of h 2 o 2 , which could be correlated to ifn-γ concentration ( figure 13 ). in samples containing ifn-γ, the ifn-γ-specific aptamer bound to ifn-γ, opening the hairpin and subsequently allowing for hemin to bind to the g-quadruplex. the potential sweep catalyzed the reduction of h 2 o 2 by the hemin/gquadruplex dnazyme, producing a cathodic current that was proportional to the concentration of ifn-γ in the sample. the assay yielded a detection limit of 0.1 nm and a dynamic range of 0.1−120 nm 144 and thus performed similarly to the luminescent assay developed by lin et al. 139 (section 4.1.1). though the authors demonstrated the specificity of the assay for ifn-γ versus nontarget proteins bsa and igg, they did not report whether the assay could be performed in more complex matrices, which can significantly compromise assay performance. more rigorous testing of the assay in biological matrices is therefore needed to demonstrate the robustness of the assay. miao and colleagues 145 synthesized and implemented an ir(iii) complex-based electrochemical probe for detection of ifn-γ ( figure 14 ). first, a gold working electrode was functionalized with a molecular beacon (mb) that contained an ifn-γ-specific aptamer as well as a sequence that was partially complementary to a second oligonucleotide strand (h 1 ). in the absence of ifn-γ, the portion of the mb complementary to h 1 was hybridized into a hairpin structure. when ifn-γ bound to the aptamer, the h 1 sequence was freed and available to hybridize with its complement, h 2 . the developed ir (iii) probe then bound to the major and minor grooves of the h 1 −h 2 double helix. oxidation and reduction of the 1,10-phenanthroline-5,6-dione ligand via differential pulse voltammetry (dpv) resulted in ifn-γ concentrationdependent current, thus correlating an electrochemical signal to the presence of target biomarker. the electrochemical assay had a detection limit of 16.3 fm and a dynamic range of 50 fm−3.0 pm and could be conducted in samples that were 5.0% human serum. importantly, this assay yielded an ∼7000-fold improvement in limit of detection compared to the two previously discussed assays for detection of ifn-γ. 145 this is largely due to the signal amplification that resulted from the groove-binding of numerous ir (iii) probes to the h 1 −h 2 double helix, which provided multiple signal-generating elements for every one ifn-γ biomarker bound. in its present form, this assay could not be applied in a primary healthcare setting because it utilizes an electrochemical workstation that requires trained personnel and significant laboratory resources. however, a portable electrochemical detection system such as the one described in section 5.5 173 could allow for this highly sensitive assay to be applied in a poc setting. electrochemiluminescence. similar to most electrochemical assays, ecl systems employ a working electrode that is functionalized with a target-specific molecular recognition element. the potential manipulation is similar to voltammetric measurements; however, in ecl, the applied potential also generates an excited state in the ecl probe, which then produces luminescence upon relaxation. 149, 165 the canonical probe for ecl-based detection is [ru(bpy) 3 ] 2+ , a complex (or derivative thereof) which has been used extensively in the development of ecl-based sensors for infectious disease detection. [150] [151] [152] [153] [154] 156 in diagnostic assays, ecl with [ru-(bpy) 3 ] 2+ occurs primarily through a coreactant mechanism, where the coreactant is most commonly n-tripropylamine (tpra) (figure 15 ). in coreactant ecl, simultaneous figure 13 . hemin dnazyme-based catalytic assay for electrochemical detection of ifn-γ. a thiolated dna construct was conjugated to a au working electrode. the dna construct consisted of a gquadruplex aptamer specific for hemin (purple) and an ifn-γ-specific aptamer (blue). both aptamers in the dna construct were prehybridized by an 8-nucleotide hairpin. in the presence of ifn-γ, the ifn-γ-specific aptamer bound to ifn-γ and opened the hairpin. hemin was then added, which bound to the g-quadruplex, forming a dnazyme that could catalyze the reduction of h 2 o 2 . this reduction produced a current that was proportional to ifn-γ concentration in the sample. 3 ]• 2+ that is analogous to the 3 mlct state that is populated through absorption of a photon. the triplet excited state then undergoes phosphorescent decay to the ground state, allowing for luminescent detection of target biomarkers. 149 of the various mechanisms for electrogeneration of a luminescent excited state, coreactant ecl has proven to be the most advantageous for the development of diagnostic assays because it permits measurements that can be obtained in aqueous solvents and matrices. one platform for detection of infectious diseases, pioneered by yoon et al., 150 used immunoliposomes to encapsulate ecl signal generation probes. liposomes equipped with a maleimide handle were synthesized by the freeze−thaw method and loaded with [ru(bpy) 3 ] 2+ . the maleimidefunctionalized liposomes were then conjugated to thiolactivated antibodies specific for the target antigen. the resulting immunoliposomes then served as a target-specific reagent containing an ecl probe that could be released with the application of detergents such as sds or triton. the authors 150 incorporated the [ru(bpy) 3 ] 2+ -loaded immunoliposome into a membrane strip test for detection of the legionella antigen. the strip test contained the following components: (1) a nitrocellulose membrane impregnated with legionella antigen, (2) a glass fiber pad presoaked in sds, and (3) screen-printed electrodes that could interface with an ecl analyzer. the immunoliposomes were incubated with a buffer solution containing the legionella antigen, and then the sample solution was allowed to wick up the nitrocellulose strip. both the antigen in the sample and the antigen embedded in the nitrocellulose competed for binding with the immunoliposome. in positive samples, the immunoliposome was bound by the legionella antigen in solution and migrated past the nitrocellulose strip to the glass fiber pad. the sds in the glass fiber pad lysed the immunoliposome, releasing [ru(bpy) 3 ] 2+ , which was detected by coreactant ecl and correlated to the antigen concentration ( figure 16 ). the assay could be completed in 20 min and had a limit of detection of 2 ng/ml. 150 compared to a traditional lateral flow assay, the test required additional user manipulation steps (preincubation of the sample and immunoliposome, addition of coreactant, etc.). the assay was also dependent on a benchtop ecl analyzer and, therefore, ill-suited to a level 1 healthcare setting in its current format. however, this novel platform demonstrated the capabilities of ecl for detecting a target antigen and showed how a [ru(bpy) 3 ] 2+ -loaded immunoliposome could be utilized to amplify signal. variations of this loaded immunoliposome platform have since been applied to the detection of the influenza virus biomarker hemagglutinin. 151, 152 recently, zhou et al. 153 developed an ecl-based immunosensor for p24, a hiv biomarker associated with early stage infection and detection of which promotes earlier intervention in hiv cases. the detection probe in this assay was a nanocomposite consisting of gold nanoparticle-decorated graphene (p-rgo@au) and [ru(bpy) 3 ] 2+ -doped silica nanoparticles (ru-sio 2 ) ( figure 17 ). a gold nanoparticle-modified glassy carbon electrode (gce) was functionalized with an anti-p24 antibody (ab1) for molecular recognition of p24. a second anti-p24 antibody (ab2) conjugated to the p-rgo@ au@ru-sio 2 nanocomposite was used as the detection element. once the ab1-p24-ab2 sandwich was formed on the electrode, coreactant n-tripropylamine (tpa) was added to produce the ecl signal through the mechanism previously discussed. 149 the p-rgo@au@ru-sio 2 nanocomposite offered several benefits to the system, namely an increased surface area for the ecl reaction and an increased rate of electron transfer. the sio 2 nanoparticles permitted greater [ru(bpy) 3 ] 2+ loading, and the gold nanoparticle-graphene composite increased loading of the [ru(bpy) 3 ] 2+ -doped sio 2 nanoparticles. the authors contended that these aspects led to an increased concentration of ecl-probe at the working electrode surface, which ultimately improved the assay's review detection limit (lod = 1.0 pg/ml) and linear range. additionally, the authors demonstrated that the p24 antigen could be detected in diluted human serum. 153 a remaining challenge for developing luminescent, electrochemical, and ecl assays that incorporate inorganic complexes is thorough validation in complex biological matrices and clinical samples. the aforementioned luminescent assays 138, 139 were not rigorously evaluated in biological samples. this is an important consideration in developing luminescent assays because of the high background that can result from the presence of interferents in biological samples. biofouling of electrodes is a known challenge in developing electrochemical sensors as well. 174, 175 although several of the previously mentioned electrochemical and ecl assays were tested in dilute biological matrices, 145,153 a more thorough evaluation of how the biological matrix affects the limit of detection is still needed. however, the issue of interference and biofouling could potentially be addressed by integrating these detection platforms with the metal-based sample preparation strategies discussed in section 3, resulting in more robust and sensitive assays. inorganic complexes have demonstrated promise for detection of infectious disease biomarkers. the luminescent, electrochemical, and ecl detection platforms discussed in this section all capitalized on the unique properties that result from the interplay between ligands and metal centers. however, inorganic complexes are rarely used as stand-alone probes for biomarker detection. instead, inorganic nanoparticles have emerged as the predominant probes in infectious disease diagnostics. 176, 177 integration of inorganic complexes with nanomaterials represents a potential avenue to developing the next generation of detection probes. one interesting nanomaterial that incorporates inorganic complexes is the metal− organic framework (mof), which is a nanosized network of organic ligands and metal ion/complex connecting points. 178, 179 the ligands provide functional handles for the conjugation of molecular recognition elements, while signalgenerating metal complexes are installed at the connecting points or encapsulated within the network. recently, electrochemical aptasensors have been developed that contain aptamer-conjugated mofs for specific recognition and detection of various protein or small molecule targets. 180−182 moreover, two of the complexes discussed in this section, ir(ppy) 3 and [ru(bpy) 3 ] 2+ , have already been incorporated into mofs for sensing and imaging applications. 183, 184 by integrating molecular recognition elements into the mof architecture, the applications of these ir(iii)-and ru(ii)-based mofs could be further expanded to include luminescent, electrochemical, or ecl detection of infectious disease biomarkers. the recent expansion of nanochemistry has drastically influenced many areas of science. in diagnostics, inorganic nanoparticles have become ubiquitous as detection elements in numerous platforms (e.g., colorimetric, luminescent, electrochemical, thermal, etc.), and each distinct class of nanoparticles possesses unique advantages and caveats. 176,177,185−187 the diagnostic setting and use-case scenario determine the applicable detection methods which, in turn, direct which nanoparticles to use. in order to apply inorganic nanoparticles as diagnostic reagents, target-specific molecular recognition elements (i.e., antibodies, aptamers, and oligonucleotide probes) must be functionalized to the nanoparticle surface. this section will first review bioconjugation reactions and nanoparticle characterization methods (section 4.2.1). the list of reactions is not meant to be exhaustive but, rather, to highlight the most essential covalent and affinity-based coupling strategies for nanoparticle bioconjugation. then, we survey the various types of nanoparticles that have been applied to the detection of infectious diseases with potential applications at the point of care (sections 4.2.2−4.2.6). 4.2.1. functionalization and characterization of nanoparticles. developing a nanoparticle detection reagent that can be targeted toward a biomarker requires conjugation of molecular recognition elements to its surface. these types of conjugations often require linkers that facilitate attachment to the nanoparticle surface. the chemistries employed for coupling molecular recognition elements to nanoparticle surfaces for diagnostic applications generally rely on a few key bioconjugation reactions and are similar to those used for planar solid supports. however, the smaller size regime of nanoparticles significantly influences the reaction kinetics on nanoparticle surfaces; therefore, bioconjugation reactions must be optimized for a given class and size of nanoparticle. 188, 189 covalent coupling. given the prevalence of carboxylates and primary amines on nanoparticle surfaces and molecular recognition elements, one of the most common bioconjugation strategies for generating functionalized nanoparticle detection probes is the formation of amide bonds. the conventional reagent for forming amide bonds on nanoparticle surfaces is 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (edc), which produces an activated ester that can subsequently react with a primary amine. however, alone, the edcactivated ester reacts slowly with amines, often leading to hydrolysis products that inhibit amide coupling. for this reason, edc is usually paired with sulfo-nhs (n-hydroxysuccinimide) esters to improve the stability of the activated ester intermediate. the sulfo-nhs ester-activated intermediate readily reacts with primary amine nucleophiles to form stable amide linkages ( figure 18a ). 190, 191 edc/sulfo-nhs coupling is useful in generating both antibody-and nucleic acid-nanoparticle conjugates as diagnostic detection probes. due to the intrinsic abundance of primary amines in antibodies, they are readily conjugated to carboxylate nanoparticles without the need for initial derivatization. 188, 192 edc coupling with imidazole enables conjugation of ethylene diamine to the 5′ phosphate group of nucleic acids, yielding 5′-amine-functionalized oligonucleotides that can be coupled to carboxylated particles. 193 quantum dots, magnetic nanoparticles, and polymeric (latex) particles have found applications in poc diagnostics and are commonly equipped with carboxylates or amines on the surface to enable conjugation via edc coupling. quantum dots are conventionally stabilized by dihydrolipoic acid (dhla) derivatives or various copolymers that install a carboxylate on the surface, and magnetic nanoparticles are coated with carboxylatecontaining polymers to enable further functionalization with primary amine-containing biomolecules. 188,194−196 alternatively, both quantum dots and magnetic nanoparticles can be coated with a silica shell derivatized with carboxylate or amine functional groups. silica nanoshells are formed via the condensation of silica oxide monomers, such as tetraethyl orthosilicate (teos) or 3-aminopropyl-triethoxysilane (aptes), yielding surface aminopropyl-silanols ( figure 18b ). the surface of the silica can subsequently be coupled to molecular recognition elements via edc. 188 in addition to supplying functional groups for further bioconjugation, the silica encapsulation of other nanoparticles provides biocompatibility and protects the core material from degradation, making it a suitable strategy for the preparation of diagnostic nanoparticle detection probes. 197 the other significant reaction class for generating nanoparticle detection probes is the bioconjugation of thiols present either in molecular recognition elements or on nanoparticle surfaces. thiolated nucleic acids are prepared by the same method as amine-functionalized nucleic acids, except cystamine is used in place of ethylene diamine for the addition of a disulfide that can be reduced with dithiothreitol (dtt) to give a 5′-thiolated oligonucleotide probe. 193, 198 on the other hand, antibodies generally do not contain free thiols and, therefore, either have to be thiolated (often with traut's reagent) or cleaved at the interchain disulfides (via dtt or papain) to generate thiols for subsequent bioconjugation to a nanoparticle surface. 192, 198 coupling of thiolated biomolecules to noble metal nanoparticles (referring to gold and silver nanoparticles) is conventionally done via coordinate covalent (dative) bonds, where the lone pairs of the sulfur atoms form stable linkages directly to the nanoparticle surface ( figure 18c ). 188, 191 alternatively, thiol-containing affinity reagents or nanoparticles can be coupled via a maleimide-derived heterobifunctional cross-linking agent, commonly sulfo-smcc (succinimidyl-4-(n-maleimidomethyl)cyclohexane-1-carboxylate). thiols react chemical reviews with the maleimide functionality of sulfo-smcc to form stable thioethers, whereas the nhs-ester component of sulfo-smcc reacts with primary amines in molecular recognition elements or on nanoparticle surfaces ( figure 18d ). 191, 194, 195, 197 photochemical cross-linking and "click chemistry" have also been utilized to produce nanoparticle-based detection probes. photosensitive functional groups, including phenyl azides and diazirines, generate reactive nitrenes and carbenes, respectively, upon exposure to uv light and allow for insertion into carbon−hydrogen and nitrogen−hydrogen bonds ( figure 18e ). photochemical agents can be integrated into heterobifunctional cross-linkers, such as sulfo-nhs-lc-diazirine, for conjugation of nanoparticles to molecular recognition elements. 191, 199, 200 "click" chemistry generically refers to bioorthogonal reactions with high yields, minimal side products, and mild conditions. while the most common "click" reaction is the conjugation of an azide to an alkyne in the presence of a cu(i) catalyst ( figure 18f ), there are many classes of "click" reactions that couple nanoparticles and molecular recognition elements. 191,201−205 biotin−streptavidin. the predominant affinity-based conjugation strategy for nanoparticle functionalization is the biotin−streptavidin system. biotin is a small molecule that binds with an extraordinarily high affinity (k d = 1.3 × 10 −15 m) to the bacterial protein streptavidin. 206 streptavidin is an approximately 60 kda tetrameric protein, permitting a 4:1 stoichiometry of biotin to streptavidin. 206 in general, streptavidin is added to the nanoparticle surface via passive adsorption or a heterobifunctional cross-linker such as sulfo-smcc. the most common method for biotinylating a molecular recognition element is the coupling of sulfo-nhsbiotin or nhs-(peg) n -biotin to a primary amine. although streptavidin−biotin bioconjugation is generally easy, a (peg) n spacer may be included to optimize for the depth of the streptavidin binding pocket (9 å) as well as the low relative solubility of biotin. 206 nanoparticle characterization. characterization of nanoparticle conjugates is essential for determining the success and extent of particle functionalization, which can drastically affect particle performance in a diagnostic format. there are several characterization techniques applicable to the development of detection probes for diagnostics. uv−visible spectroscopy (uv−vis) is one of the most commonly used methods for biomolecule-functionalized nanoparticles given its simplicity and applicability to the detection of nucleic acids and proteins. 207, 208 particle size and distribution can be assessed using dynamic light scattering (dls) and transmission electron microscopy (tem), 209−211 while functional group characterization on the nanoparticle surface can be evaluated with nmr and/or infrared (ir) spectroscopy. 212−215 all of these analytical methods have trade-offs with respect to cost, sensitivity, time of analysis, and complexity of sample preparation. however, because many of the techniques offer complementary information, they can be used in combination to ensure an extensive characterization of inorganic nanoparticle detection probes. the principal concern associated with antibody modification is decreased antigen-binding activity as a result of overfunctionalization of the variable region of the antibody. optimization of the bioconjugation reaction minimizes this effect by achieving a balance between sufficient nanoparticle coupling efficiency and retention of antigen binding. the extent of antibody modification can be monitored with functional group-specific chromophores that are detectable by uv−vis, such as ellman's reagent for free-thiol detection or haba (4′-hydroxyazobenzene-2-carboxylic acid) for biotin detection. 192, 198 also, biosensor techniques, such as surface plasmon resonance (spr), quartz crystal microbalance (qcm), and bli, can be employed to assess the impact of modification on the binding affinity for the antigen. 216−218 decreased target affinity can also originate from suboptimal loading of antibody or nucleic acid on the nanoparticle surface. insufficient coverage may lead to the nonspecific binding of interferents, whereas overloading the nanoparticle with molecular recognition elements can result in steric effects, decreasing target binding. simple absorbance-based supernatant assays that measure antibody or nucleic acid concentration both before and after nanoparticle functionalization reactions give an estimation of the element's surface density and aid in determining the nanoparticle surface saturation. 207, 208 the straightforward nature of these procedures make them popular strategies for characterizing the extent of functionalization of nanoparticle conjugates for use in infectious disease diagnostics. the bioconjugation chemistry for attaching a molecular recognition element to a nanoparticle surface is the key component for transforming a nanoparticle with interesting signaling properties into a functional reagent that can be employed in diagnostic assays. the characterization of the nanoparticle postfunctionalization is critical to ensuring optimal signal in downstream diagnostic application. several classes of functionalized nanoparticles can be utilized in developing poc diagnostics for infectious disease, the choice of which depends on the use-case scenario and desired signal output. sections 4.2.2−4.2.6 describe the fundamental principles and poc applications for these classes of nanoparticles. 4.2.2. noble metal nanoparticles. both gold nanoparticles (aunps) and silver nanoparticles (agnps), collectively referred to as noble metal nanoparticles, possess many unique properties that make them advantageous probes for signal generation in infectious disease diagnostics. noble metal nanoparticles display a vivid color that is observable with the naked eye due to surface plasmon resonance (spr). the phenomenon of spr, illustrated in figure 19a , occurs when incoming photons strike the nanoparticle surface and generate a dipole that causes electron oscillations (i.e., surface plasmons) with a frequency that resonates with the frequency of the incoming light. visible light fulfills this resonance condition for noble metal nanoparticles, giving rise to large molar extinction coefficients (on the order of ∼10 9 m −1 cm −1 ) at visible wavelengths. 219 moreover, a nanoparticle's color and λ max can be manipulated by controlling its size ( figure 19b ). 220, 221 noble metal nanoparticles are also easily functionalized with antibodies, aptamers, or oligonucleotide probes to afford specificity to a target biomolecule. 177, 188 altogether, these qualities have made noble metal nanoparticles a frequent choice for colorimetric labels in diagnostic assays designed to be interpreted by the naked eye. it is important to note that we have limited further discussion of aunps since their use in diagnostics and sensing applications has been extensively reviewed. 177,219,222−226 given the vast literature that exists for aunp applications, we have elected to focus on the applications of agnps as signal generation probes for infectious disease diagnosis. similar to aunps, localized spr of agnps has been capitalized upon to chemical reviews review develop colorimetric assays for infectious disease protein and nucleic acid biomarkers. 227−231 additionally, the conjugation of fluorescent probes (e.g., fam) to agnp surfaces, as well as the intrinsic luminescence of some agnps, has permitted the development of luminescence-based assays. 232, 233 the high agnp conductivity has also been exploited for electrochemical detection of nucleic acids. 234−236 lastly, surface-enhanced raman spectroscopy (sers) of agnps has been employed for the direct detection of malaria-infected red blood cells because of the significant light scattering of agnps and the high specificity of raman signatures. 69,237−240 colorimetric detection using nanoparticles in paper-based assays has been a widely implemented method for signal readout in primary healthcare settings. in fact, the success of lateral flow assays can be credited in part to the nanoparticlebased detection systems that allow for visual interpretation of results by the end user. the use of functionalized agnps as colorimetric detection probes in paper-based diagnostics for infectious disease has been reported for both protein and nucleic acid biomarkers. 227, 241 for example, yen et al. 229 employed agnps in a multiplexed lateral flow assay for detection of dengue, yellow fever, and ebola virus. the authors synthesized 30-, 41-, and 47 nm agnps, which were visually detectable as orange, red, and green, respectively, due to sizedependent spr shifts. each nanoparticle of a particular size was specifically functionalized with a distinct target-specific antibody associated with one of the viruses, allowing for detection of all three diseases in a single assay ( figure 20) . the detection limit for each biomarker was found to be 150 ng/ml, which is within clinically relevant ranges for each disease but is higher than other published methods. 242, 243 such multiplexed diagnostic assays are critical for discriminating between febrile illnesses at the point of care to allow for selection of the appropriate treatment course. colorimetric agnp aggregation assays also rely on the sizedependence of spr. when agnps aggregate, the decrease in distance between nanoparticles results in interparticle spr, producing a large red-shift in absorption and distinct color change that is visible to the unaided eye. 222, 227 these reaction mechanisms are classified as either "cross-linking" or "noncross-linking." in cross-linking assays, agnps are functionalized with both molecular recognition elements and capping ligands that stabilize the nanoparticles in solution. addition of a sample of target biomarker results in cross-linking of the functionalized nanoparticles, drastically decreasing the nano review particle−nanoparticle distance and producing in a spectral shift. 244, 245 agnp aggregation assays in this format have been implemented for detection of both infectious disease protein 228 and nucleic acid 230 biomarkers; however, to the best of our knowledge, cross-linking-based agnp aggregation has yet to be employed in paper diagnostics or other lowresource platforms. in non-cross-linking aggregation assays, nanoparticles are either aggregated or stabilized with molecular recognition elements that electrostatically adsorb to the nanoparticle surface. subsequent addition of a sample containing the diagnostic target then either disrupts or promotes nanoparticle stabilization, producing an observable color change. 222 for example, teengam et al. 231 developed a non-cross-linking aggregation assay using agnps for multiplexed visual detection of middle east respiratory syndrome coronavirus, mycobacterium tuberculosis, and human papillomavirus oligonucleotides in a microfluidic paper analytical device (μpad). the assay lacked the sensitivity required for detection of pathogenic nucleic acids; however, most sensitive nucleic acid diagnostics incorporate amplification techniques (e.g., pcr) that require extensive resources and infrastructure only available in betterequipped laboratories. 246 nonetheless, the use of a agnp aggregation-assay in a μpad enabled multiplexed visual detection of three different nucleic acid biomarkers in a format that could be applicable to a primary healthcare setting. taking advantage of the intrinsic fluorescence of some agnps, kurdekar et al. 233 developed a fluorescent immunoassay for detection of p24 that utilized a streptavidin-coated agnp as the detection probe. using a standard well-plate immunoassay format, adsorbed anti-p24 antibodies captured p24 from samples. a biotinylated anti-p24 detection antibody and the streptavidin-conjugated fluorescent agnp were subsequently added to generate a fluorescent signal that correlated with p24 concentration. the assay successfully detected p24 standard spiked into plasma as well as the p24 present in hiv-positive patient plasma samples. moreover, no cross reactivity was observed in samples that were hivnegative but hbv-or hcv-positive. the limit of detection of the assay was 10 pg/ml, which is an order of magnitude higher than a previously discussed assay for p24 (section 4.1.2). 153 this discrepancy could be explained by the lack of signal amplification in the fluorescent agnp-based immunoassay. in its current format, the fluorescent agnp immunoassay is not amenable to a resource-limited primary healthcare setting; however, fluorescent agnps could be employed in a lateral flow assay format and combined with a portable fluorescent reader, allowing for implementation at the point of care. 247 4.2.3. quantum dots (qds). quantum dots (qds) are semiconductor nanocrystals with luminescent and conductive properties that make them advantageous as detection probes in infectious diseases diagnostics. canonically, qds consist of combinations of elements from groups ii and vi or groups iii and v and range in size from 1 to 100 nm. 248, 249 at these small sizes, qds absorb photons, generating excited electrons in the conduction band and positively charged holes in the valence band of the semiconductor ( figure 21a ). the distance that separates the electron−hole pair is confined to less than the diameter of the nanocrystal, resulting in "particle in a box" behavior known as quantum confinement. 249−251 as a result of quantum confinement, qd emission wavelength increases as nanoparticle size increases; thus, qd emission can be tuned via size-controlled synthesis ( figure 21b ). 252 because qds exhibit absorption across a broad spectrum yet have emission spectra with very narrow and tunable bandwidths, they are particularly useful for multiplexed detection of disease biomarkers. 194, 253 additionally, qds demonstrate increased biocompatibility, photostability, quantum yield, and semiconducting properties compared to traditional organic fluorophores, further rendering qds advantageous as signal generation probes for diagnostics. 254 265 escherichia coli, 266 and cholera 267, 268 in fluorescent assays for laboratory and resource-limited settings. functionalized qds or qd-loaded microparticles have been utilized as fluorescent probes for both protein and nucleic acid biomarkers. 194, 256, 269 the surface of qds can be functionalized with antibodies or antigens for protein detection or oligonucleotide probes for target dna detection using the bioconjugation strategies discussed in section 4.2.1. for a thorough evaluation of the bioconjugation of qds, the reader is directed to a number of reviews. 194, 196, 270 goldman et al. 271, 272 pioneered the earliest uses of antibody-functionalized qds as fluorescent detection probes. the authors utilized affinity-based coupling chemistry (i.e., biotin−avidin) as well as electrostatic interactions between charged moieties to functionalize antibodies to the metal surfaces of cdse/zns core−shell quantum dots. the biotin−avidin system was found to be the more simple, reproducible, and robust method for qd bioconjugation of the two, and these functionalized qds were successfully utilized in a multiplexed fluorescent assay for cholera toxin and staphylococcal enterotoxin b. 272 these early works exploited the narrow emission profile and enhanced quantum yield of qds for antigen detection and laid the groundwork for the development of enhanced, fluorescencebased diagnostics for a variety of targets. building upon this previous work, klostranec et al. 253 constructed a multiplexed fluorescence-based microfluidic device for detecting hiv, hepatitis b, and hepatitis c antibodies utilizing antigen-functionalized qd barcode particles. two types of cdse/zns qds were synthesized and loaded into polystyrene beads at varying ratios, resulting in three distinct qd barcode particles. each qd barcode particle was covalently bound to a disease-specific antigen via edc coupling. the assay employed a "sandwich" format where disease-specific antibodies were captured by the antigenfunctionalized qd barcode particles, and a fluorophoreconjugated (alexafluor 488) antihuman antibody was used to detect the presence of a target antibody. the narrow peak width of qd emission enabled detection of both the antibodyconjugated fluorophore at ∼520 nm to indicate the presence of target antibody as well as both of the qds at 570 nm (yellow) or 615 nm (red). the ratio of the red:yellow signal intensity allowed for barcode identification and determination of which of the three target antibodies was present ( figure 22 ). 253 the detection limits for antibodies correlating to all three diseases were in the picomolar range, which is a 50-fold improvement compared to commercial elisas. 253 while the assay enabled multiplexed and sensitive detection of infectious disease biomarkers, the microfluidic platform employed by the authors required extensive user manipulations and complex instrumentation and analysis that would only be viable in higher level healthcare settings. given the improvements in sensitivity of the assay, adaptation of the technique to a handheld fluorimeter or cell phone-adapted fluorescence device could potentially allow for deployment in a primary healthcare low-resource setting. the benefit of this strategy is that the biomarker signal and barcode signal are both fluorescencebased, resulting in a multiplexed detection platform that is simplified compared to assays with different detection schemes for biomarkers and barcodes. the authors hypothesized that up to 10 6 barcode combinations are theoretically possible using this approach due to the narrow and distinct fluorescent peak widths generated by different qd particles, allowing for even greater multiplexing. 253 however, such hypotheses should be approached with caution, as highly multiplexed assays can be more susceptible to cross-reactivity, thereby reducing the diagnostic specificity of the individual assays. qd-loaded particles have also been functionalized with antibodies for the detection of protein biomarkers for infectious disease. recently, hu et al. 257 developed a lateral flow assay that utilized antibody-coated polymeric particles (260.9 nm) loaded with both qds (3−6 nm) and aunps (20 nm) for detection of ebola virus (ebov) glycoprotein. the reporter probes, which the authors dubbed "dual signal readout nanospheres" (rns), enabled detection of the ebov glycoprotein either visually via aunps or fluorescently via qd emission. characterization of the rns revealed that there were dozens of aunps adsorbed and hundreds of qds embedded per every one antibody-coated polymeric particle ( figure 23 , panels a and b); thus, for every one biomarker arrested at the test line, there were significantly more signaling review moieties to permit more sensitive detection. additionally, two sets of antibody-functionalized rns were fabricated and used for each lfa: (1) streptavidin-conjugated and (2) biotinconjugated rns. application of the sample and streptavidinconjugated rns to the sample pad resulted in migration of the sample and rns to the test line and formation of an antibody "sandwich" at the test line. subsequently, the biotin-conjugated rns were added and bound to the streptavidin-coated rns, enhancing the signal based on the high affinity of the biotin− streptavidin interaction. the authors 257 utilized the lfa to detect either the ebov glycoprotein or the whole virion spiked into samples. the assay demonstrated a broad dynamic range that spanned 3 orders of magnitude for detection of aunps with a limit of detection by visual inspection of 2 ng/ml. when the qd fluorescence signal was used, the detection limit was improved to 0.18 ng/ml (figure 23 , panels c and d). the fluorescent detection of this assay was 1−2 orders of magnitude more sensitive than 2 commercial brands of rapid tests for ebov glycoprotein. moreover, the fluorescent signal could be quantitated by image processing software that is readily available on a smartphone. though the authors utilized a benchtop fluorimeter for qd excitation and emission, the use of a smartphone adapted hand-held fluorimeter or dedicated fluorescent lateral flow reader could enable measurements at the point of care. 257 the sensitive dual signal readout provides versatility to this platform such that it could be employed in a number of use-case scenarios. if morbidity control were the primary concern, visual detection of aunps would suffice. if more sensitive and quantitative measurements were needed for surveillance, intervention management, or an elimination campaign, the fluorescent capabilities of the detection probes could be exploited. however, for a given use-case scenario, likely only one signal readout modality is needed. as a consequence, the additional cost of implementing a dual signal rn, when using a single signal (visual or fluorescent) rn would suffice, needs to be considered. nonetheless, the assay demonstrated that quantum dots could be incorporated into tests that are readily deployable in primary healthcare settings, though more rigorous field evaluation and validation of the assay would be necessary for clinical use. there have been significant advances in the use of qd-based detection methods for infectious disease biomarkers. while the complexity of instrumentation required for detecting quantum dots is often still a barrier to their implementation at the point of care, developments in smartphone-based devices are currently enabling the detection of fluorescent labels such as qds at the point of care. 273−275 fluorescent assays have demonstrated a capacity for multiplexing. because of the narrow emission profiles demonstrated by qds, the potential for continued expansion in multiplexing likely lies with qdbased fluorescent assays. given the importance of detecting comorbidities and coinfections or discriminating between febrile illnesses, 276,277 the qd-based technologies represent a platform that has the potential to make a significant impact at the point of care. 4.2.4. lanthanide chelate-doped nanoparticles. nanoparticles doped with lanthanide chelates are advantageous as detection labels owing to their large stokes shifts, sharp emission peaks, stable luminescence, long fluorescence lifetime, and biocompatibility. 278, 279 in a lateral flow assay format, these particles are often functionalized with targetspecific antibodies and then embedded in the conjugate pad of the test. like qds, detection of lanthanide chelate-doped nanoparticles requires additional instrumentation for particle excitation and measurement of emission. 280, 281 despite this need for additional equipment, these particles are attractive labels for poc diagnostics because they enable highly sensitive biomolecule detection. in the context of elimination or surveillance of a particular infectious disease, the added sensitivity provided by lanthanide chelate-doped nanoparticles could allow for detection of low-intensity infections that would otherwise be missed. except lanthanum, all lanthanides are f-block elements; the 4f orbital is gradually filled as the atomic number increases. because the 4f orbital is largely shielded from the chemical environment by the filled 5s and 5p shells, the lanthanides are very similar in their chemical properties. for example, the most common oxidation state for aqueous lanthanide ions is +3. 282, 283 most of these ions are luminescent, though some lanthanides have greater quantum yields than others due to the large energy gaps between the lowest emissive energy level and the highest sublevel of the ground state. as the size of the energy gap increases, lanthanide ions become less prone to lower energy nonradiative decay processes, resulting in greater quantum yields. 278 eu 3+ is the most commonly utilized lanthanide for labels in lateral flow assays because of its (1) ideal energy gap between the nondegenerate emissive state of 5 d 0 and the ground state of 7 f j and (2) convenient emission in the visible region. consequently, eu 3+ will be the focus of this section. 279 europium(iii) itself is not strongly luminescent because most excitations involve forbidden electric dipole f-f transitions, which have low absorption cross sections. 278 however, this can be overcome when the ion is coordinated to a sensitizing structure, such as an organic ligand. a simplified jablonski diagram of the luminescence process for a eu 3+ chelate is depicted in figure 24 . the ligand absorbs the excitation energy and transfers it to a triplet state (t 1 ) via intersystem crossing (isc). next, this energy is transferred to one of the 5 d j levels of eu 3+ , and, after internal conversion (iet), emission occurs from the 5 d 0 energy level to any of the 7 f j levels. 278, 279 in addition to sensitization, chelating ligands also promote luminescence by shielding eu 3+ from aqueous quenching effects and allow for coupling to biomolecules or polymeric materials. 278 chelating ligands typically absorb in the uv (330−370 nm), and the subsequent eu 3+ emission occurs at 580−690 nm. 279 ligands usually contain a chromophore portion (e.g., pyridine, salicylate, phenanthroline, coumarin, pyrazole, triphenylene, and quinolone) and a chelating structure based on multidentate polyacids or macrocycles (e.g., edta). 278 tetracycline has also been employed as a ligand for eu 3+ . 284 these chelate dyes are incorporated into nanoparticles functionalized with target-specific molecular recognition elements for use in lateral flow assays. most lateral flow assays that employ eu 3+ chelates use commercially available particles, which are often superior due to their monodispersity and quality control standards. the majority of commercially available eu 3+ chelate lateral flow labels are 100−300 nm carboxyl-or streptavidin-decorated polystyrene particles and are readily available from large vendors such as thermo-fisher 285 and expedeon ltd. 286 eu 3+ chelates are easily adsorbed into polymeric (e.g., polystyrene) nanoparticles based on hydrophobic interactions between the polymers and the organic chelating ligands of the complexes. for example, huhtinen et al. 287 found that simply incubating 50 nm polystyrene particles with eu 3+ chelated with dipicolinic acid derivative ligands resulted in 1000−2000 chelates per nanoparticle. this represents a density of approximately 0.02 chelates/nm 3 . harmäet al. 288 found a similarly high density of approximately 0.05 chelates/nm 3 (31000 chelates/particle) in 107 nm commercially available particles. thus, for each biomolecule bound in a lateral flow format, thousands of associated eu 3+ chelates produce a signal, resulting in a highly sensitive measurement. the advantages of eu 3+ chelate-loaded polystyrene nanoparticles as labels for lateral flow assays were demonstrated by yeo et al., 289 who developed a sensitive lateral flow assay for the detection of avian influenza h7 subtype virus. these viruses can be highly pathogenic; for a 2013 h7n9 outbreak in china, a mortality rate of roughly 36% was estimated for hospital patients with laboratory-confirmed infections. 290 currently, laboratory-based molecular testing is required to differentiate between h7 subtype avian influenza and other viral infections. thus, there is a need for rapid tests that can be performed at the point of care. the authors approached this problem by developing and evaluating a panel of 10 h7 subtype-specific monoclonal antibodies against hemagglutinin 1 of h7n9 influenza. after the best antibody pair was determined, h7 subtype-specific lateral flow assays were constructed with both europium-chelate and gold nanoparticle (aunp) conjugates. using a benchtop time-resolved fluorescence reader, the authors found that the detection limits for the eu 3+ chelate-based tests were 25-fold better than those of aunp-based tests. additionally, the developed eu 3+ -based tests performed better than commercially available tests detecting influenza a nucleoprotein. 289 these enhancements over gold particles are impressive, although given the high density of chelates per nanoparticle, it is surprising that larger enhancements were not achieved. one possible explanation could be the efficiency of molecular recognition element adsorption for the two types of nanoparticles. in addition to avian influenza, 289, 291 eu 3+ chelate-embedded polymeric nanoparticles have been employed for the detection of e. coli, 292 hepatitis b, 293 and procalcitonin, 281 a host marker for bacterial infections. several commercial platforms based on these particles have been developed as well, including the sofia influenza a+b tests (quidel, inc.) 294−299 and the aqcare chlamydia trf test (medisensor, inc.), 300, 301 which have been extensively evaluated in the literature. the high sensitivity of these assays is derived not only from the high number of eu 3+ chelates embedded in the polymeric nanoparticles but also from the use of time-resolved fluorescence measurements. one of the primary disadvantages of using luminescent probes as labels in lateral flow assays is the high autofluorescence of biological samples and assay components occurring at excitation and emission wavelengths similar to those of the probes. 302 this fluorescence can interfere with lateral flow readings, causing high background that leads to low-sensitivity measurements. however, lanthanide chelates overcome these effects because their fluorescence lifetimes are several orders of magnitude longer than conventional fluorescent materials (microseconds or milliseconds compared to nanoseconds). 278, 279 this allows for the use of time-resolved measurements in which samples are excited with a pulse of uv light, and emission signal is collected after a time delay on the order of 100 ns for a given time period. 284 by delaying the collection of emitted signal, the short-lived autofluorescence from biological and assay components is excluded from measurements. while the intensity of the delayed luminescence is slightly decayed from the initial signal output of the chelates, these measurements can be cycled to improve signalto-noise. 303 of course, the unique instrumentation required for time-resolved measurements makes field-deployment technically challenging, though currently available benchtop devices could allow for measurements in district clinics and laboratories. recently, paterson et al. 304 developed a promising smartphone-based platform for time-resolved luminescent measurements, which could allow for point-of-care measurements based on eu 3+ chelate particles. however, the lengthy 100 ms time-delay employed in their device is only compatible with the most persistent luminescent nanophosphors such as crystalline sral 2 o 4 :eu 2+ ,dy 3+ particles. while polymeric eu 3+ chelate nanoparticles are the primary materials employed by lanthanide-labeled lateral flow assays, these particles present some disadvantages. their large size and tendency to swell can cause aggregation in aqueous solutions. additionally, since they are not covalently bound to the polymers, eu 3+ chelates can leach from the particles. 284 silica particles avoid some of these problems. in one example, xia et al. 305 developed a lateral flow assay for hepatitis b detection using eu 3+ chelate-loaded silica nanoparticles. to synthesize these particles, the authors relied on an iterative method involving silica condensation from teos, surface-amination using aptms, covalent functionalization of chelating ligands to the particles, and loading of eu 3+ . this process was repeated five times to achieve a high density of eu 3+ chelates. the resulting 55 nm nanoparticles contained a remarkable 686000 chelates/particle, corresponding to a density of roughly 7.9 eu 3+ chelates/nm 3 , more than 100 times greater than commercially available polymeric particles. 306 these particles were then functionalized with oxidized dextran to serve as a hydrophilic linker, and free amine moieties on antihepatitis b surface antigen (hbsag)-specific antibodies were coupled to the surface via reductive amination of the aldehyde groups. after synthesis and characterization, the particles were used as labels in a traditionally formatted lateral flow assay for hbsag. to measure signal, the strips were illuminated with a uv lamp and imaged using a digital camera. signal intensity was determined using image analysis in adobe photoshop. unlike the previous example presented, this imaging technique does not take advantage of the long fluorescence lifetime of eu 3+ chelates. however, even without time-resolved measurements, the detection limit of the lfa was found to be 30 pg/ ml hbsag, 100-fold better than a similarly formatted goldbased lateral flow assay. 305 the study was then validated by analyzing 286 clinical serum samples using the developed eu 3+ chelate-based lateral flow assay as well as a quantitative elisa. the two methods were concordant for all samples analyzed and had similar sensitivities. 305 thus, the developed method represents an innovative tool for diagnosis of hepatitis b and provides sufficient sensitivity when compared to clinical methods. in order to implement this assay directly at the point of care, two modifications are necessary: (1) the assay should be optimized for field-friendly whole-blood specimens, and (2) a mobile phone-based detection strategy should be implemented. lanthanide chelate-based nanoparticle probes are a promising avenue for developing highly sensitive lateral flow assays. similar to other fluorescent probes, instrumentation remains the primary barrier to implementation of eu 3+ chelate nanoparticles at the point of care. however, as optical devices are miniaturized and become more affordable, these probes could become commonplace among rapid tests for infectious diseases. 4.2.5. up-converting phosphor nanoparticles. upconverting phosphor (ucp) nanoparticles represent a unique and exciting reporter technology for bioassays. unlike typical fluorescent labels, these ceramic nanoparticles doped with rare earth elements absorb low-energy ir radiation and emit higher-energy visible light. 307−309 ucps are often superior to conventional colorimetric and fluorescent reporters for a number of reasons. first, the high anti-stokes shift from ir to visible does not occur in nature. thus, the inherent autofluorescence typically associated with biological samples and assay components does not interfere with ucp measurements. second, ucp signal is highly stable and does not photobleach, allowing for indefinite storage and repetitive analysis of a single test. this is especially useful for confirming field results in a central laboratory. 309 third, assays are easily multiplexed; distinct particles absorb at the same ir excitation energy and emit wavelengths defined by the dopant. finally, optical detection of ucp particles offers an inherent boost in analytical sensitivity over visual detection of gold; as few as 10−100 ucp particles can be detected at the test line of a lateral flow strip, while approximately 40000 gold particles are needed for visual detection. 310 this increase in sensitivity makes ucp particles attractive labels for diagnostic tests employed in surveillance or elimination campaigns in resourcelimited settings, since they would enable the detection of lowintensity or asymptomatic infections. however, similar to qds and lanthanide-chelate particles discussed in the preceding sections, these benefits of ucp particles must be weighed against the additional cost and resource requirements needed for detection instrumentation. the primary mechanisms for up-conversion include excited state absorption, energy transfer, and cooperative sensitization. excited state absorption is the absorption of light by electrons already in an excited state. this requires two photons and equal separation between the ground state, first excited state, and second excited state of a single ion. energy transfer upconversion requires two ions, a sensitizer and an activator. in this process, the sensitizer ion is excited and sequentially transfers its energy to the ground state and first excited state of the activator ion. 307 cooperative sensitization is similar to energy transfer, though it involves excitation of two sensitizer ions which simultaneously transfer their energy to the activator ion. other up-conversion mechanisms include photon avalanche and cross relaxation. 311 energy transfer up-conversion is the most common upconversion mechanism for bioassay reporter particles, which typically involve a yb 3+ sensitizer (excitation 980 nm) and a rare earth (er 3+ , tm 3+ , pr 3+ , ho 3+ , and gd 3+ ) activator codoped in yttrium fluoride (yf 3 ), yttrium oxide (y 2 o 3 ), yttrium oxysulfide (y 2 o 2 s), or sodium yttrium fluoride (nayf 4 ) crystals. 307, 309 an illustration of the up-conversion processes for two of these materials are shown in figure 25 . ucps have been utilized as labels for both protein and nucleic acid targets in lateral flow assays for a variety of infectious diseases. recently, corstjens et al. 312−317 developed an ultrasensitive, ucp-based lateral flow assay for schistosomiasis that detects schistosoma circulating anodic antigen (caa), a proteoglycan biomarker waste product produced by the parasite. caa is present in the serum and urine of patients with schistosoma infections of all known species and has been found to correspond well with worm burden, clearing soon after successful treatment. 312, 317, 318 the developed lateral flow assay employed 400 nm y 2 o 2 s:yb 3+ , er 3+ ucps, which are excited at 980 nm and emit at 550 nm (green) and can detect caa for a single schistosoma worm pair in serum. 314, 317 this assay has been applied to patient samples from endemic areas, and due to its superior sensitivity, it has demonstrated much higher schistosomiasis prevalence than microscopy, serology, and nucleic acid-based tests. [315] [316] [317] 319 the caa lateral flow assay format is similar to conventional lateral flow assays. caa-specific antibodies are printed on the test line, and antimouse igg is printed on the control line. the workflow of the assay in its current form, however, differs from a typical field-ready test. first, a trichloroacetic acid (tca) extraction is performed on a urine or serum sample, requiring a centrifugation step. the extract supernatant is then combined with running buffer and anti-caa-functionalized ucp particles and incubated for 1 h on an orbital shaker at 37°c before the lateral flow strip is added to the solution. the test is allowed to develop and must dry completely (at least 3 h) before scanning and analyzing the strip. 312, 313 in order to simplify the preparation procedure and decrease the number of steps, a dry reagent kit was later developed in which all buffers and particles were lyophilized. 313 this kit was shipped to a tertiary lab in south africa, where nearly 2000 samples were evaluated by local technicians using both the kit and a caa elisa. the researchers found that the ucp-based lfa successfully identified more low-level infections than the caa elisa. 320 to increase the analytical sensitivity of the assay, corstjens et al. 317 added a spin-filter concentration step to the sample preparation method. this allowed for caa in urine sample volumes of 0.5−7.5 ml to be concentrated into 20 μl before addition to the lateral flow strip. the resulting detection limits improved as sample volume increased, reaching as low as 0.03 pg/ml for the 7.5 ml assay. to demonstrate clinical applicability, the concentration step was successfully performed on 2 ml patient urine samples from kenya (high-endemic, s. mansoni) and china (low-endemic, s. japonicum) 316, 317 it is important to note that, though this sample concentration step improved the sensitivity of the assay, it required laboratory equipment to carry out; all patient samples in these studies were processed in well-equipped laboratories. further, the additional concentration step increased the cost of this ultrasensitive caa assay, though sample pooling could make this test more cost-effective and allow for monitoring of worm burdens at the subpopulation level for large-scale surveillance. 320 for this ucp-based ultrasensitive assay to be utilized in a field setting, a more robust, field-ready sample preparation method is needed. lateral flow assays with ucp reporters have also been developed for detection of protein markers of other infectious diseases, including neurocysticercosis, 321 334 and hiv. 335 additionally, ucp-based multiplexed lateral flow assays have been developed for detection of protein biomarkers for multiple infectious diseases. 336, 337 these multiplexed diagnostics typically consist of one strip with multiple test lines, each capturing a distinct protein biomarker. in this format, the ucp crystals are the same for each biomarker, though they are functionalized with target-specific antibodies. one disadvantage of highly multiplexed assays on a single strip is the potential increase in cross-reactivity and nonspecific binding that could lead to false-positive results. to mitigate this risk, hong et al. 330 developed a unique, circular cassette with 10 channels for 10 different singleplex lateral flow assays using ucp particles as the reporter labels. one unexplored application of multiplexed detection on a lateral flow assay is the use of ucp particles with identical excitation but differing emission profiles. this could be particularly advantageous for large biomarkers with multiple accessible epitopes and could provide additional clinical information. for example, an assay that captures and detects whole organisms could include a second ucp probe for detecting surface proteins that confer drug resistance, providing both detection and susceptibility results in one assay. ucp nanoparticles have also been used in nonlateral flow diagnostic formats, including immunohistochemistry, 338 microarrays, 339 magnetic bead assays, 340, 341 and plate immunoassays, 309 though these platforms are not readily amenable to low-resource, point-of-care settings. even in the lateral flow format, ucp nanoparticles present interesting challenges for field deployment. clearly, sample preparation methods, including purification, concentration, and amplification, must be adapted for environments lacking controlled laboratory conditions and should rely on as little electricity as possible. while hand-powered centrifuges 342, 343 and battery-powered mixers 115, 344 have been developed, the ideal poc assay will be optimized to preclude these additional resources. another issue, often unaddressed, is that the lateral flow strips must be completely dry before optical measurements and analysis. this is because water also absorbs in the near-ir, decreasing excitation efficiency of the sensitizer. 345 typically, protocols require at least 3 h of drying time after the lateral flow assay has developed, a long time-to-result for a point-of-care test. finally, detection of ucp particles relies on optical instrumentation. although the most sensitive of these devices are benchtop instruments, portable battery-powered devices have been developed, such as the ucp-modified version of qiagen's esequant lateral flow reader ("ucp-quant"). 313, 314, 346 many of the sample preparation methods described in section 3 can be applied to ucp lateral flow formats. additionally, innovations in hand-held optical readers discussed in section 5 require only simple adaptations to detect the anti-stokes shift of ucp particles. there are clear advantages of using ucp nanoparticles as labels in lateral flow assays, including their inherently low background interference and high analytical sensitivity. innovations in sample preparation methods and the development of portable optical readers will allow for these advantages to be exploited in low-resource, poc settings. 4.2.6. magnetic nanoparticles. while most frequently used for sample preparation and biomarker enrichment, magnetic nanoparticles are emerging as promising labels in poc assays. similar to noble metal nanoparticles, magnetic nanoparticles can be easily functionalized and possess strong optical absorbance, which has led to their use as visual labels on lateral flow assays. 347 however, their magnetic properties can also be leveraged for detection, which has advantages over traditional optical detection in a lateral flow format. first, when visual or optical detection of absorbent or fluorescent particles is employed, only signal from the surface (∼10 μm) of the membrane is measurable due to the opacity of the membrane. typically, nitrocellulose membranes used in lfas are 100− 200 μm thick, so optical detection only allows for signal from the top 10% of the membrane to be measured. in contrast, magnetization measurements can be performed regardless of the opacity of the substrate, taking advantage of the entire volume of the test line on a lateral flow assay. 348 in addition, metal-oxide nanoparticles are highly stable, and drying and aggregation do not influence the intensity of magnetic measurements. 349 finally, there are very few biological or assay components that interfere with magnetic measurements. 350 the properties of magnetic particles are size-and temperature-dependent. 195, 351 iron oxide (typically magnetite or maghemite) particles are the most common magnetic nanoparticles employed in diagnostic applications. in the bulk, these materials are ferri-or ferromagnetic and thus retain magnetization after an external field has been applied. this hysteresis reaches a maximum when particle size is decreased to the point that the material becomes single-domain. as particle size is even further reduced, the hysteresis effect decreases until the particles reach a critical diameter, below which brownian forces are strong enough to overcome magnetic forces. thus, at these small sizes, the particles are superparamagnetic; the magnetic moments of the particles are aligned in the presence of an external magnetic field, but they revert back to a nonmagnetic state when the field is removed. 351 in the lateral flow format, magnetic nanoparticles are labeled with target-specific molecular recognition elements and employed as conjugates for analyte detection. 352 superparamagnetic behavior is ideal for particles applied as labels review for lateral flow assays because the lack of magnetization in the absence of an external field allows for the particles to wick along the strip without aggregating due to magnetic effects. 353 then, the magnetic properties of the particles can be leveraged after labels have bound to the test and control lines. magnetic nanoparticles are typically detected on lateral flow assays based on induction (figure 26 ) or using magnetoresistive sensors. 350 in the inductive format, which employs the principles of faraday's law, a magnetic particle-based test is placed in an oscillating magnetic field above a set of induction coils. in the absence of magnetic particles, the net current induced in the coils is zero. however, when particles are present, the direction of their magnetic moments oscillates with the external magnetic field, resulting in a measurable net voltage induced across the coils that is proportional to the total number of particles at the test or control line. 354, 355 in contrast, magnetoresistive sensors are based on the principle that the electrical resistance of certain materials, which are incorporated in the sensors, can change upon application of an external magnetic field. 350 in this format, a magnetic field is applied to para-or superparamagnetic detection elements in order to align their magnetic moments and produce a fringe field. magnetoresistive sensors are placed close enough to the particles to detect the fringe field they produce based on the change in resistance of the materials within the sensor. 356, 357 a detailed description of the instrumentation required for these detection methods is provided in section 5.4. magnetic nanoparticles have been employed as detection labels in lateral flow assays for protein and whole-cell targets. for example, handali et al. 349 developed two magnetic particle-based lateral flow assays for detection of taenia solium, a cestode that is prevalent around the world for which swine are intermediate hosts. when contaminated, undercooked pork is ingested, the parasites develop into tapeworms in the human gut (taeniasis). however, if eggs are ingested, the larval stage can infect the human nervous system, potentially forming cysts in the brain. this severe form of the disease, called neurocysticercosis, is a leading cause of epilepsy worldwide. 358 the gold standard for diagnosis of taeniasis is observation of eggs in stool by microscopy, though this method is insensitive and labor-intensive. diagnosis of neurocysticercosis currently requires ct scans of the brain, a technology that is unavailable in low-resource settings. 359 as such, there is a pressing need for t. solium rapid diagnostic tests. handali et al. 349 developed two serological magnetic beadbased lateral flow assays that detected the immune response (i.e., host antibodies) against taeniasis-specific (es33) and cysticercosis-specific (t24) antigens. for each test, one batch of the recombinant antigen was printed at the test line, and a separate batch was coupled to commercially available carboxylated superparamagnetic particles via edc/nhs chemistry. the recombinant antigen at the test line and the antigen conjugated to magnetic particles were able to simultaneously bind to host antibodies in the sample, taking advantage of the multivalent nature of anti-es33 and anti-t24 igm and igg antibodies. the lateral flow assays were evaluated using an inductionbased reader and visual reading to detect antibodies in serum samples from endemic areas and nonendemic control regions ( figure 27 ). 349 it was found that the taeniasis-specific es33 assay had a diagnostic sensitivity and specificity of 95% and 96%, respectively, when evaluated with the magnetic reader. the sensitivity and specificity of the neurocysticercosis t24 test using the magnetic reader were 94% and 99%, respectively, for patients with two or more viable cysts as determined by ct scan or mri of the brain. for patients with only one viable cyst, the t24 assay only identified 5/15 cases of neurocysticercosis, indicating that the burden of infection was not great enough to elicit an immune response. 349 despite this decrease in sensitivity for single-cyst infections, the developed lateral flow assays are promising alternatives to the current gold standards. further, because the assays were not speciesspecific, they could be used to detect porcine cysticercosis as a marker of disease control and transmission. one disadvantage of utilizing superparamagnetic labels is that many magnetic readers are benchtop devices requiring electricity and a laboratory setting. however, in over 70% of the cases, the magnetic nanoparticles could be detected visually. 349 while this sensitivity is not ideal, it is possible that initial visual results could be applied in the field with follow-up laboratory-based measurements assessed for the visually negative assays. as hand-held magnetic readers become more commonplace, these assays will become truly impactful. in addition to t. solium, magnetic particles have been employed in protein-detecting lateral flow assays for hiv 360, 361 and h1n1 influenza. 362 whole cell-detecting magnetic particle-based assays have also been developed for a number of targets, including vibrio parahemolyticus, 363,364 listeria monocytogenes, 365 and bacillus anthracis. 366, 367 the demonstrated sensitivity of these magnetic particle-based lateral flow assays makes them promising alternatives to traditional assays with colorimetric or fluorescent labels. however, current instrumentation, which will be discussed further in section 5.4, limits their utility at the point of care. one exciting avenue that has yet to be explored for infectious disease detection is the employment of the same superparamagnetic nanoparticles for dual purposes: immunomagnetic biomarker enrichment from large-volume samples and visual labeling for lateral flow assays. ideally, such a diagnostic would integrate sample preparation and assay into one device. once developed, the combined effects of target enrichment and magnetic detection could lead to a highly sensitive test capable of detecting low-density infections. one of the most widely utilized techniques for improving the sensitivity of diagnostics is signal amplification, where thousands of signaling molecules are generated for every one biomarker molecule. though there are now numerous methods for amplifying signal in diagnostic assays, the use of metalloenzymes was one of the first approaches. 368, 369 metalloenzymes are metal-containing catalytic proteins in which the presence of particular metals or metal complexes in the tertiary structure is critical to the catalytic turnover of the substrate. 370 the predominant metalloenzymes employed in diagnostics are alkaline phosphatase (alp), horseradish peroxidase (hrp), and catalase. the high catalytic efficiencies of these enzymes enable the rapid conversion of substrate to detectable products. 371 using cross-linking chemistry discussed in section 4.2.1, these metalloenzymes can be conjugated to antibodies or nucleic acids that afford specificity for a target biomarker. 192, 193 the three primary enzymes used in metalloenzyme detection conjugates rely on different metal ions for catalytic activity. alp possesses two zn(ii) ions and one mg(ii) ion per enzyme monomer and hydrolyzes phosphate monoesters. 372, 373 the conventional substrates for alp in point-of-care diagnostic applications are bcip (5-bromo-4-chloro-3-indolyl phosphate) and nbt (nitro blue tetrazolium), where dephosphorylation and oxidation of bcip allows for reduction of yellow nbt dye to a blue/purple formazan precipitate. 371 both hrp and catalase are heme-containing monooxygenases that catalyze reactions with h 2 o 2 . 374 4.3.1. elisas (enzyme-linked immunosorbent assays). metalloenzyme-antibody conjugates have been widely implemented in elisas for the detection of protein biomarkers for disease. this is due to the sensitivity afforded by the use of enzymes for signal amplification as well as the specificity of the antibody−antigen interactions used for molecular recognition. 371, 377, 378 despite the high sensitivities of elisas, their application in poc settings is limited by several factors: 378−381 signal readout for elisas typically requires a spectrophotometer unavailable in a primary healthcare setting, though there are a growing number of portable alternatives 382 (e.g., cellphones or hand-held scanners; see section 5.1.3). elisas are time-consuming (∼6−8 h), demand extensive sample handling and solution manipulation, and often require specialized training. lastly, the lack of thermal and long-term stability of metalloenzymes in elisas can lead to suboptimal assay performance, as elisas are intrinsically dependent on these metalloenzymes for signal amplification and readout. thus, elisas are usually limited to well-equipped tertiary laboratories. to mitigate these issues, investigators have begun to use paper as the elisa solid support, increasing the likelihood that these sensitive assays could be used in resource-limited settings. paper-based elisas have been developed for detection of diagnostic markers of hiv and malaria. 381, 383, 384 long-term stabilization conditions of metalloenzymes on paper have also been investigated to maximize catalytic activity in poc settings. 385−387 paper-based elisas seek to address many of the issues that inhibit the use of elisas in more resource-limited poc settings. the time to result for paper-based elisas is ∼1 h due to the comparatively smaller volumes required when contrasted with conventional benchtop elisas. these smaller reagent volumes also significantly reduce the overall cost of elisas. cheng et al. 381 first pioneered this method for the detection of serum antibodies against hiv-1 envelope antigen gp41, where an alp-antibody conjugate was utilized to turn over bcip/nbt substrates to purple precipitate products. the authors used hydrophilic paper that was patterned with hydrophobic photoresist to fabricate 96 test "wells" that were spatially separated just as in a polystyrene 96-well plate. samples containing biomarker were added to the paper elisa card, which was placed on top of a blotting pad to enable wicking of the reagents through the test wells. though the assay boasted a faster time to result and a lower cost than the analogous conventional elisa, the sensitivity of the paperbased elisa was decreased by an order of magnitude compared to the conventional elisa format. lathwal and sikes 383 conducted a systematic investigation of several signal amplification methods for paper-based colorimetric detection of malarial biomarker hrp2. to investigate multiple metalloenzyme−substrate combinations, the same capture antibody was immobilized on aldehyde-modified cellulose for all experiments, and the following detection systems were evaluated: (1) hrp with substrates dab and h 2 o 2 , (2) hrp with substrates tmb and h 2 o 2 , (3) alp with substrates bcip and nbt, (4) ag deposition onto aunps, and (5) polymerization-based amplification. because all factors (i.e., capture antibody, capture antibody loading, biomarker, detection antibody) were held constant, the differences review observed in each assay were presumed to be the direct result of the signal amplification system used. positive and negative controls were evaluated at various time points for each signal amplification method to determine an optimal window for visual readout of the signal ( figure 28 ). 383 all of the methods had an optimal time point for visual signal readout with the exception of hrp-tmb/h 2 o 2 , which produced significant false-positive signals and a color change from blue to brown that was difficult to interpret. of the 4 remaining methods, the metalloenzyme−substrate combinations (hrp-dab/h 2 o 2 and alp-bcip/nbt) had detection limits an order of magnitude better than ag deposition and polymerization-based amplification methods when quantified by rgb color intensity. 383 any portable colorimetric reader, even a cell phone, could enable the use of these highly sensitive metalloenzyme conjugates in a primary healthcare setting. the advantages of paper-based elisas are potentially compromised by the instability of metalloenzyme conjugates, since denaturation of metalloenzymes leads to poor turnover of substrate and lower sensitivities. 388 for this reason, notable research efforts have been devoted to optimizing long-term storage of metalloenzymes, particularly hrp, for use in microfluidic paper analytical devices (μpads) or two-dimensional paper networks (2dpns). 385−387 one method involves vacuum drying or freeze-drying enzymes and substrates in the presence of trehalose, a disaccharide often used as a cryoprotectant because of its capacity to stabilize the protein's interaction with solvents, forming a protective "glass" as it dries. 389, 390 ramachandran et al. used a combination of trehalose, bovine serum albumin (bsa), and feso 4 -edta for vacuum drying of hrp-conjugated antibodies against malarial biomarker hrp2 into glass fiber pads. 387 the stabilized hrp could be stored for 5 months at 45°c under these conditions without a significant loss in catalytic activity. a glass fiber pad containing all of these reagents was incorporated into a 2dpn for detection of hrp2 with a lod of 6.5 ng/ml, a value well within clinically relevant concentrations. this assay also demonstrated the utility of metalloenzymes in paper-based poc diagnostics, providing a signal amplification step that is rarely present in conventional paper diagnostics (e.g., lateral flow assays). these advances in enzyme stabilization when combined with simplified paperbased assay formats could potentially allow for elisa sensitivity to be translated for direct use at the point of care. 4.3.2. nanoparticle-assisted enzymatic signal amplification. the integration of noble metal nanoparticles has further augmented the signal amplification capabilities of metalloenzymes in infectious disease diagnostics. nanoparticles have served as surfaces for the coupling of antibody-metalloenzyme conjugates and have been implemented in electrochemical sensors for detection of infectious disease-associated protein biomarkers. 391−393 zheng et al. 392 developed an amperometric immunosensor for detection of hiv antigen p24 that employed a standard "sandwich" format. a glassy carbon electrode was modified with gold nanoparticles to allow for immobilization of anti-p24 capture antibodies. hrp-conjugated anti-p24 antibodies were used for detection, catalyzing the oxidation of substrate hydroquinone in the presence of h 2 o 2 . the reaction generated a reductive current at the electrode surface proportional to p24 concentration. the detection limit of the assay was 8 pg/ml, similar to the p24 assays 153, 233 previously discussed in sections 4.1.2 and 4.2.2. while the assay was robust to human serum samples spiked with p24, the serum samples tested contained a p24 concentration 3 orders of magnitude higher than the lod. 392 the currently required electrochemical workstation would limit the assay's application to higher level laboratories. nonetheless, this amperometric method demonstrated the value of integrating noble metal nanoparticles with metalloenzyme conjugates for signal amplification in diagnosis of infectious diseases. noble metal nanoparticles have also been utilized as colorimetric signaling probes in elisas for detection of protein biomarkers for disease. 394−398 so-called "plasmonic" elisas utilize the standard "sandwich" assay format with an immobilized capture antibody and a detection antibody that is conjugated to a metalloenzyme. however, as opposed to using conventional metalloenzyme substrates for colorimetric detection, plasmonic elisas employ the enzyme as a kinetic tool for nanoparticle nucleation. this causes drastic shifts in spr and in the absorbance spectra of the nanoparticles that are detectable with the naked eye. 399 the plasmonic elisa pioneered by de la rica and stevens 396 utilized a catalase-conjugated detection antibody to control the growth of aunps in such a manner that was proportional to the concentration of hiv biomarker p24. in the absence of biomarker, bulk au(iii) was reduced by h 2 o 2 to form stable, spherical aunps that appeared red in color. when p24 was present in a sample, the catalase-conjugated (figure 29) . the red-shift in the spr was highly sensitive toward h 2 o 2 concentration; thus, depletion of h 2 o 2 with catalase that was only present in p24-positive samples allowed for very sensitive detection of p24 with the naked eye. the absorbance was also quantified using a simple spectrophotometric readout, yielding a detection limit of 1.0 ag/ml, 396 nearly 6 orders of magnitude more sensitive than the previously discussed detection methods for p24 antigen. 153, 233, 392 the authors also demonstrated that the plasmonic elisa could detect p24 in hivpositive serum samples, even identifying p24 in samples from hiv-positive patients with viral loads less than 50 copies/ ml. 396 the remarkable sensitivity of this assay for naked-eye detection of p24 provides the necessary improvement required for elimination campaigns and active case management. it would permit earlier detection of p24, leading to improved outcomes for hiv patients. the assay could be particularly impactful for the challenges associated with early infant hiv diagnosis in low-resource settings. the plasmonic elisa presented by the authors still calls for extensive sample handling and user manipulation and is currently unsuitable to a primary healthcare setting. adaptation of the previously discussed enzyme stabilization measures and integration of the assay to a field-ready format (e.g., paper-based elisa or 2dpn) could enable its translation to an ultrasensitive fieldready diagnostic for hiv detection. metalloenzymes play a critical role for signal amplification in a number of assays, enabling the sensitive detection of infectious diseases. several research efforts have been aimed at translating the sensitivity of metalloenzyme-based assays to formats such as paper-based diagnostics that are amenable to resource-limited settings. although the issue of enzyme storage in paper has been addressed to certain extent, 385−387 the longterm stability of enzymes remains a concern for poc diagnostics, especially in climates with elevated temperatures and prevalent infectious disease. there are numerous methods that utilize nonenzymatic means for signal amplification that eliminate the issue of long-term enzyme storage altogether, including nanoparticles that act as enzyme mimics and other metal-based methods. these will be covered in the following section. while the majority of signal amplification in bioassays is enzyme-based, several interesting metal-based amplification strategies have been developed. these strategies include metal nanoparticle dissolution, nanocrystal ion exchange, enzyme mimics, and reductive nanoparticle enlargement. 394,400−403 compared to enzymes, metal-based signal-amplification has the distinct advantage of long-term storage and thermal stability. however, many of these strategies suffer from the following drawbacks: (1) amplification often requires additional steps to be integrated into point-of-care test workflow, and (2) incorporation into a paper-based format can be technically challenging. however, innovative assay design can overcome these challenges. integration of signal amplification into point-of-care tests can drastically improve analytical and diagnostic sensitivity, resulting in earlier diagnoses and detection of low-density infections. therefore, diagnostics which incorporate novel and user-friendly signal amplification steps could fill a need for elimination campaigns and surveillance programs. this section reviews metal-based signal amplification strategies that show promise for application to low-resource infectious disease diagnostics. 4.4.1. nanoparticle dissolution and cation exchange. at the heart of signal amplification is the principle that, for each biomarker target captured in an assay, many signalgenerating elements (particles, molecules, atoms, electrons, photons, etc.) are produced. in a typical nanoparticle-based assay, a single nanoparticle often indicates the presence of one biomolecule. however, a single nanoparticle is a densely packed lattice of thousands to millions of metal atoms, each of which, after nanoparticle dissolution or cation exchange, can be leveraged for signal generation (figure 30 ). 199,404−406 metal ion chelating reagents that result in chromogenic or fluorescent signal have long been used for detection of trace metals for a myriad of applications. 407, 408 recently, tong et al. 405 exploited ferrous ion-chelating ferrozine in an iron oxide nanoparticle-linked immunosorbent assay (ilisa). antibodyfunctionalized wustite (feo) particles were used as detection elements in direct, competitive, indirect, and sandwich wellplate assays. after functionalized iron-oxide particles bound to the target, the nanocrystals were treated with acid and reducing agents, resulting in stoichiometric conversion to ferrous ions. thus, thousands of fe 2+ ions were released for each biomarker present. when excess ferrozine was added, solutions changed from transparent to purple upon chelation, with intensities directly proportional to the iron concentration in solution. proof-of-concept assays detecting mouse igg had low picomolar detection limits, similar to assays that depend on enzymatic signal amplification. to extend the applicability of their amplification technology, the group also demonstrated its use in a western blot. by switching the chelating reagent to potassium ferrocyanide (prussian blue), the reagent could easily be precipitated onto a cellulose membrane when iron oxide particles were used as detection elements. 405 while wellplate assays are far from applicable to field settings, the ilisa system could be translated to an aptly designed paper diagnostic format. another avenue for signal amplification similar to nanoparticle dissolution is nanocrystal cation exchange, in which the cations within a nanocrystal are place-exchanged with different cations. 409 while bulk, solid-phase cation exchange often requires elevated temperatures and several weeks, complete nanoscale exchange can occur within seconds due to increased surface area and lower energy barriers to diffusing ions. 410 for cation exchange to be employed in a bioassay, nanocrystals (znse, zns, cdse, or cus) are first functionalized with targetspecific molecular recognition elements, such as antibodies or aptamers, which are utilized as detection probes. using rapid silver ion exchange, each bound nanocrystal releases thousands of zn 2+ , cd 2+ , or cu 2+ ions. zinc and cadmium ions are then detectable using fluorogenic chelating reagents such as fluozin-3 or rhod-5n, respectively, and cu 2+ can be detected in a chemiluminescent reaction with luminol and h 2 o 2 . these reactions produce an amplified, stoichiometric signal and have been employed in well-plate 403, 411 and magnetic beadbased 412−415 immunoassays for protein 403, 411, 415 and cell 412 detection as well as rolling circle amplification for dna/ mirna 413, 414 detection. despite this versatility, the cation exchange method has yet to be applied in paper-based formats. similar to the ilisa example discussed previously, signal amplification using nanocrystal cation exchange would require a precipitating reagent for metal ion detection as well as clever design of a paper microfluidic device. enzymatic signal amplification results in high analytical sensitivity and is easily performed in a controlled laboratory environment. however, as discussed in section 4.3, elevated temperatures often lead to decreased catalytic turnover, making it difficult to incorporate enzymatic signal amplification into low-resource infectious disease diagnostics. in recent years, some inorganic nanoparticles, including noble metal, rare earth, and magnetic nanoparticles, have been found to display surprising enzyme-like catalytic activity. 416−418 these nanomaterial-based artificial enzymes, originally dubbed "nanozymes" by manea et al., 419 are highly stable toward a range of temperatures and phs and cannot be degraded by proteases. further, catalytic activity of inorganic nanoparticles can be tuned with particle size, shape, coating, modification, and composition. for these reasons, nanozymes have been incorporated into a myriad of sensors for various applications. in 2007, gao et al. 420 made the surprising discovery that magnetite (fe 3 o 4 ) nanoparticles have intrinsic peroxidase-like activity, displaying michaelis−menten kinetics consistent with a ping-pong mechanism. fe 3 o 4 magnetic nanoparticles were found to catalytically turn over several common horseradish peroxidase substrates, including tmb, dab, and o-phenylenediamine (opd), with catalytic turnover numbers equal to or improved over horseradish peroxidase. the group demonstrated that the particles could be used in place of horseradish peroxidase in a traditional immunoassay and that the magnetic properties of fe 3 o 4 could be leveraged further to enrich biomarkers before detection. 420 as a result of this seminal work, magnetic nanoparticles have been employed as signal amplification elements in protein and dna bioassays for a variety of infectious diseases, including ebola, 242 rotavirus, 402 mycoplasma pneumonia, 421 vibrio cholerae, 422 chlamydia trachomatis, 401 l. monocytogenes, 423 enterobacter sakazakii, 424 and salmonella typhimurium. 425 beyond magnetite, several other types of nanoparticles, including gold, 426 platinum, 427 hybrid particles, 428−432 and mofs, 433 have been utilized as enzyme mimics in bioassays for targets such as respiratory syncytial virus, 432 avian influenza a, 426 salmonella, and e. coli, 431 hepatitis c and hiv, 428 and staphylococcus aureus. 433 many of the assays that utilize review nanozymes as catalytic turnover reagents for detection are formatted similarly to immunoassays on the surface of well plates. 402,421−423 several groups have translated this technology to the lateral flow format and found that the nanozyme detection element resulted in at least 100-fold improvement in limits of detection when compared to traditional gold nanoparticle-based lateral flow strips. 242, 424, 427, 431, 434 for example, loynachan et al. 435 developed an ultrasensitive lateral flow assay for the detection of hiv p24 antigen using porous platinum au@pt core−shell nanozymes that catalyzed the precipitation of cn/dab (4-chloro-1-naphthol/3,3′-diaminobenzidine tetrahydrochloride) in the presence of h 2 o 2 ( figure 31 ). incorporation of this detection scheme, as well as careful and systematic design of the affinity reagents for p24 capture and detection, resulted in a lateral flow assay with femtomolar detection limits, more sensitive than laboratory-based elisa methods and nearly 2 orders of magnitude more sensitive than commercially available rapid tests. 435 because visual signal varied linearly with concentration before and after catalytic signal amplification, the test also had an ultrabroad dynamic range. the advantages of the nanoparticle enzyme mimics were directly demonstrated in a stability test in which the activities of lyophilized porous pt core−shell nanocatalyst conjugate and lyophilized hrp conjugates were measured over time. while the enzyme conjugates lost nearly all activity after 15 days of storage at room temperature, the nanocatalysts maintained 100% of their initial activity while stored at room temperature and nearly 80% of their initial activity while stored at 44°c for up to 42 days. 435 however, it should be noted that the optimal hrp lyophilization and storage conditions 387,389 discussed in section 4.3.1 were not employed in this study. to achieve such significant improvements in sensitivity using nanozymes, a wash step, a substrate addition step, and a reaction quenching step were inserted into the lateral flow assay workflow after the initial signal development. 435 these additional steps make this assay and other similar diagnostics more difficult to perform in the field. redesigned paper devices or automated lateral flow cassettes could simplify this workflow review and allow for application of nanozymes in field settings, providing much-needed signal amplification and improved sensitivity for low-resource infectious disease diagnostics. 4.4.3. reductive nanoparticle enlargement. the use of gold nanoparticles as detection elements in commercially available lateral flow assays is ubiquitous. 436 particles used in these assays are typically 15−50 nm and can be detected either visually (without optical equipment) or using simple hand-held readers. 177, 219, 437 often, the detection limits of these assays are not low enough to diagnose low-density infections. one simple solution for improving sensitivity is chemically enlarging the particles, making them more visually intense. in this process, a lateral flow assay is run in the typical manner such that, if the antigen is present, a gold nanoparticle-containing sandwich complex is formed at the test line. next, an enhancement solution consisting of a molecular precursor and reducing agent is added to the test. the gold nanoparticles at the test and control lines serve as nucleation sites for solid metal deposition. the most common format for particle enlargement, silver enhancement, is based on 19th-century photographic techniques and involves the reduction of silver ions (e.g., silver nitrate) to silver metal on the surface of gold particles in an acidic buffered solution containing hydroquinone. 438 this process is similar to the seeded-growth nanoparticle synthesis method and can result in particles from hundreds of nanometers to several micrometers in diameter ( figure 32 ). 439 because silver enhancement found its first biological application in tissue staining in 1935, 440 there are many commercially available silver enhancement solutions designated for microscopy applications (sigma, thermo, ted pella, nano, etc.). the first application of silver enhancement to paper diagnostics was in 1991 when horton et al. 441 developed a proof-of-concept lateral flow assay for the detection of mouse igg and demonstrated that silver treatment resulted in a 100fold improvement in detection limits. since then, silver enhancement has successfully improved the detection of many infectious diseases by several orders of magnitude, including influenza, 439, 442 hiv, 443 malaria, 444−449 y. pestis, 450 and e. coli. 451 one of the drawbacks to silver enhancement on lateral flow assays is that it must be performed after the test has completed its initial development. increasing the total number of steps required reduces the likelihood that the test could be applied in the field. for example, one group 450 found that silver enhancement improved the detection limits of their y. pestis test 1000-fold. in order to take advantage of this impressive enhancement, the authors allowed the gold test to develop as usual, washed the strip three times, submerged it into enhancing reagents for 5 min, and then stopped the reaction with sodium thiosulfate. this procedure is feasible in a district hospital or even a local health outpost with minimal resources, and the enhanced assay would be a true asset in these settings should a plague pandemic occur. however, the additional steps required for signal amplification make poc application in a low-resource field setting unlikely. for this reason, some groups who employ a silver enhancement step in their paper diagnostic assays have moved away from the traditional lateral flow assay format and toward alternative formats that allow for automated delivery of silver enhancement reagents to the test line. fujifilm 439, 452 applied their expertise in photo technology to develop a benchtop workstation that automated silver enhancement for influenza rapid diagnostic tests. in this format, a user added the sample to a lateral flow cartridge that contained all reagents necessary for test washing and silver enhancement. the cartridge was inserted into the benchtop workstation, which released the solutions in a timed, automated manner and provided a quantitative readout of the strip after test development was completed. despite the fact that the instrument greatly simplified the assay protocol for the user, the requirement of a reliable source of electricity greatly inhibits its application to a field setting. recently, the yager and fu groups have designed 2dpns that rely on unique paper geometry or embedded paper-fluidic valves to enable programmed, electricity-free delivery of silver enhancement reagents to the assay test line. [444] [445] [446] [447] [448] 453, 454 these 2dpns simply require the addition of sample and buffer to pads that have been preloaded with assay reagents, mirroring the same number of steps required to run a traditional lateral flow assay. the unique physical structures of 2dpns allow for precisely timed delivery of sample, then gold conjugate, and finally silver enhancement reagents to the test line. similarly, cho et al. 455 developed a cross-flow chromatography system, in which silver enhancement reagents were embedded into paper and then rehydrated after the lateral flow assay developed. the enhancement reagents flowed perpendicular to the initial immunochromatographic test over the test and control lines. these creative approaches to redesigning the lateral flow assay make silver enhancement the most field-deployable metal-based signal amplification strategy to date, although large-scale manufacturing requirements must be considered to determine the scalability of such test designs. finally, it should be noted that while silver enhancement on gold particles is most common, other metals have been used for reductive nanoparticle enlargement for lateral flow enhancement. gold has been deposited onto gold and silver nanoparticles, 443,456−458 and silver onto silver and platinum nanoparticles. 459 while there are many other examples of solution and microfluidic assays employing reductive nanoparticle enlargement for visual and electrochemical signal amplification, these formats are less conducive to low-resource disease diagnosis. 451, 457, 460 4.4.4. bio-barcodes. the amplification methods discussed thus far increase the number of signaling elements after the initial assay has developed, requiring additional steps post hoc. another amplification strategy is to generate a greater number of detectable molecules midassay, thereby increasing the downstream signal output of the assay. in 2002, the mirkin group 461 demonstrated that protein biomarker targets could be detected indirectly in a highly sensitive manner by employing gold nanoparticles functionalized with dna tags. in this strategy, magnetic microparticles functionalized with targetspecific antibodies were added to a biological sample. after biomarker capture, the supernatant was removed and intermediate detection elements were added to the particles. these detection elements consisted of gold nanoparticles functionalized with a low density of target-specific antibodies and a very high density of double-stranded barcode dna. using an external magnet, the gold nanoparticles that bound the target were separated, and then the barcode dna was dehybridized and detected downstream ( figure 33 ). 462 because the number of barcode tags was much higher than the number of captured biomarkers, the resulting signal was amplified when compared to a direct detection method. one powerful advantage of this modular assay format is it could be easily multiplexed, with distinct barcode tags for each target of interest. 463 further, while the biobarcode method was initially applied to the detection of proteins, it has also been applied to nucleic acid detection. simply replacing the biomarker-specific antibodies with dna complementary to the nucleic acid target allows for detection of dna or rna targets with pcr-like sensitivity. 464 since its development, the biobarcode assay format has been utilized for the detection of infectious diseases such as hepatitis b, 463 hepatitis c, 465, 466 variola virus, 463 ebola, 463 hiv, 463, 467, 468 b. anthracis, 464 and salmonella, 469 as well as c-reactive protein 470 and a variety of cytokines. 471, 472 despite its promise for sensitive detection, the biobarcode assay format suffers from two major drawbacks: (1) the high number of user steps required before the barcode tags are released for detection makes it extremely difficult to adapt to a lowresource format, and (2) low-resource detection of the nucleic acid tags remains a challenge. initial detection strategies for the biobarcode assay were scanometric; the barcode tags were detected in a sandwich hybridization assay on the surface of a glass slide, with gold nanoparticles as the detection elements. signal was further enhanced using reductive silver deposition before the slides were evaluated using image analysis. [461] [462] [463] [464] 467, 473 methods such as pcr, 466,468 mass spectrometry, 474 and electrical detection 465 have been employed to detect the barcode tags as well. nam et al. 461 ,472 developed a colorimetric assay in which the released gold nanoparticle biobarcode tags were introduced to a second solution of gold nanoparticles functionalized with single-stranded dna complementary to the tags. the hybridization of the biobarcode tags with complementary dna resulted in aggregation of the two gold nanoparticle-dna conjugates, yielding a detectable color change. none of these detection methods can be employed in a field setting; however, it is not difficult to imagine developing a hybridization-based lateral flow assay for barcode tag detection or functionalizing the tags themselves with moieties such as biotin or a flag peptide that could make them readily detectable in a paper diagnostic format. as is evidenced by sections 4.3 and 4.4, significant advances have been made in developing metalloenzyme-and metal nanoparticle-based signal amplification strategies for infectious disease diagnosis. however, even with the advancements in metalloenzyme stabilization and highly stable nanoparticlebased approaches, poc assays utilizing signal amplification are still scarce. most of the assays discussed, even those that employ paper substrates or lateral flow formats, require too many user steps before the signal is generated. future work that minimizes user steps, for instance, through use of 2dpns or vertical flow assay formats, will be critical in translating these signal amplification strategies from laboratory tests to assays that can be run at a primary healthcare facility. moreover, most of the assays discussed in sections 4.3 and 4.4 were also heavily reliant on benchtop, laboratory-based instrumentation for signal readout. the next section will discuss the various types of field-deployable instrumentation that have been developed in an effort to bring quantitative and sensitive assays, such as those discussed in section 4, to low-resource settings. traditionally, point-of-care diagnostics such as lateral flow assays have delivered qualitative visual results. although this simplicity is advantageous in the field, the diagnostic sensitivity and specificity can suffer from subjective interpretation and user bias errors. quantitative measurements mitigate these factors, avoiding the data loss associated with qualitative tests and providing insight into important variables such as infection intensities and biomarker expression patterns. in an elimination setting, these parameters allow for robust epidemiological studies into transmission dynamics and intervention efficacy. novel detection strategies that offer highly sensitive and quantitative results, including many of those outlined in section 4, require instrumentation for signal readout. as a result, the development of appropriate and simple instrumentation will likely become an integral part of the application of diagnostic technologies in low-resource settings. incorporation of detection devices into point-of-care diagnostics effectively eliminates the possibility of "equipment-free" tests (e in assured criteria) and reduces the "affordable" nature of simple tests (a in assured criteria). 13 however, the potential benefits of instrumentation, including improved sensitivity and reduced bias, will outweigh these disadvantages in many use-case scenarios, and the overall cost of instrumentation per test can be reduced when a device is used to evaluate many tests over time. additionally, many instruments incorporate common consumer electronic devices, such as mobile phones and smart phones, thereby reducing the equipment burden. this section outlines some of the devices developed for point-of-care infectious disease diagnosis. table 3 is included at the beginning of section 5 to highlight selected examples of point-of-care instrumentation. a vast majority of the metal-based probes and nanoparticles highlighted in section 4 produce optical signals such as absorbance, fluorescence, or phosphorescence. as a result, a diverse array of instrumentation has been developed to facilitate assay readout for imaging and quantitative purposes. this section provides an overview of efforts to develop portable, affordable, and sensitive instruments for optical detection in low-resource settings. 5.1.1. microscopy. conventional microscopy remains the gold standard diagnostic technique for many infectious diseases, including malaria, tuberculosis, schistosomiasis, and intestinal protozoa. 475 to detect these diseases, a variety of microscopic techniques, such as bright field, dark field, and fluorescence microscopy, are used for direct inspection of stained or unstained smears (blood, sputum, urine, etc.). while microscopy can be a useful diagnostic tool, results depend strongly on infection intensities, sample preparation methods, and the training level of the microscopist. 476 these challenges lead to variability that reduces the utility of microscopy-based diagnosis in field settings, especially in low-transmission areas. additionally, microscope instrumentation can be expensive and bulky. to address these drawbacks, many groups are developing portable, affordable, and easy-to-use microscopy tools suitable for low-resource settings. one approach to bringing laboratory-based microscopy tools to low-resource settings involves building simple, portable, and/or miniature microscopes from low-cost materials. while this solution does not address some of the primary disadvantages of microscopy as a poc diagnostic (i.e., the need for skilled microscopists and sample preparation requirements), it does allow for conventional microscope platforms to be deployed in difficult settings at low cost. such devices typically replace high-energy, expensive light sources with leds, reducing the expense and power required for illumination in addition to increasing the light source lifetime. 477 structural components of these microscopes are often three-dimensionally (3d) printed, 477−480 and affordable plastic or hybrid lenses and fiber optics can replace expensive and delicate glass lenses. 477 tuberculosis. when samples were evaluated as positive or negative for m. tuberculosis, results from the global focus microscope and a laboratory-grade fluorescence microscope were in agreement for 98.4% of cases. 485 many similarly portable and lightweight microscopes have been developed, including tomographic, 482 holographic, 483 wide-field, 484 and fluorescence 486 microscopes, some impressively weighing in at less than 200 g. 479,482−484 these microscopes have been applied not only to the detection of tuberculosis 480, 485, 486 but also to malaria, 481 soil-transmitted helminths, 487 schistosomiasis, 487 and hymenolepis nana. 482 the foldscope is an example of a low-resource microscope that most embodies the assured criteria. 488 assembled using the principles of origami, the foldscope is constructed from flat paper, copper tape, a button-cell battery, an led, polymer filters, and a ball lens ( figure 34b ). the entire device costs less than $1, can achieve magnification up to 2180×, and was shown to be capable of detecting giardia lamblia, leishmania donovani, trypanosoma cruzi, e. coli, and schistosoma hematobium eggs. 488 despite these accomplishments, when the foldscope was applied to s. hematobium detection in a field setting, the diagnostic sensitivity was just 55.9% when compared to conventional light microscopy. 489 this limited sensitivity was likely a result of challenges with physical manipulation of the foldscope when integrated with a smartphone display. in other words, the foldscope failed to satisfy the "u" for user-friendly in the assured criteria for this particular use-case. this example demonstrates the importance of rigorous field evaluation of novel diagnostic devices and indicates that improvements to the foldscope must be made before implementation for case management. recently, mobile phone-based microscopy has emerged as a potential alternative to conventional microscopes for infectious disease diagnostics. phone-based bright-field, fluorescence, and dark-field microscopy devices have been developed, often employing compact, pocket-sized attachments ( figure 34c ). 490 these devices have been applied to the detection of a myriad of infectious diseases, including malaria, 491, 492 tuberculosis, 492 schistosomiasis, 493, 494 human papillomavirus (hpv), 495 soil-transmitted helminths, 496 filarial diseases, 497, 498 and intestinal protozoa. 494, 499 repurposing mobile phones is advantageous due to their widespread adoption and growing connectivity in low-resource settings. additionally, the acquisition of digital images could allow for image-processing (locally or remotely), automated diagnoses, and near real-time data transmission. these advantages, coupled with diseasespecific software applications, could potentially decrease the user variability typically associated with microscopy, improving case management and epidemiological studies. one example that leverages these advantages is the loascope, developed by the fletcher group ( figure 34d ). 500, 497, 498 the loascope is a smartphone-based device that combines video microscopy and automated software with onboard quantitative detection based on the movements of filarial parasite loa loa in whole blood. an application downloaded to the smartphone is used for stage control, video capture, image analysis, and data reporting of quantitative l. loa microfilaria densities. detection of l. loa is important because high levels of microfilariae are associated with potentially fatal adverse events after treatment with ivermectin, a drug frequently used in mass drug administration efforts against onchocerciasis and lymphatic filariasis (lf). these adverse events have led to the suspension of mass drug administration campaigns and subsequent major setbacks in onchocerciasis and lf elimination efforts. rapid, point-of-care measurement of l. loa infection intensity could allow for these elimination campaigns to resume. this device was validated in a field setting and yielded results similar to those of thick smears. 497 it was then applied in a large-scale (16,259 participants) test-and-not-treat approach for onchocerciasis in an area with high prevalence that had not participated in mass drug administration campaigns since 1999 due to the prevalence of l. loa. 498 each patient was screened for l. loa burden using the loascope, and those with high counts were not given ivermectin but were treated for l. loa with albendazole. this is a striking example of mobile phone-based microscopy in the field; all loascope measurements were performed by operators with only one hour of training, and the application-specific software significantly decreased the probability of user error by eliminating the need for subjective interpretation of results. in total, the loascope enabled ivermectin delivery to 15522 participants that otherwise would not have received treatment for onchocerciasis, demonstrating the incredible impact mobile phone microscopy can have when applied to detection of infectious diseases. 498 despite the fact that many mobile phone-based microscopes lack the analytical and diagnostic sensitivity of conventional microscopes, the loascope is an excellent demonstration that mobile phone microscopy can be highly impactful if applied in an appropriate use-case scenario. because this particular study focused on identifying patients with very high l. loa burdens, it is unclear to what extent this device could detect low-burden infections. generalization of this technology to other filarial diseases will also require further study. additionally, potential interference from other filarial parasites needs to be studied. as work continues in the field of low-resource microscopy, it will be important to develop new tools with specific use-case scenarios in mind. one potential avenue for application-based innovation involves development of low-resource sample preparation methods coupled with novel probes, such as the inorganic complexes and nanoparticles discussed in section 4. 5.1.2. lateral flow analysis. while much progress has been made in the development of rugged, field-applicable microscopy devices, lateral flow assays currently remain the most field-friendly format for infectious disease diagnosis. however, the simple, direct visual readout of lateral flow assays, often cited as one of the primary advantages of this format, is also considered its achilles heel; difficulties in interpretation resulting from visual impairment and operator bias can result in user-to-user variation. after tests are interpreted, maintaining accurate records and communicating test results to local and national health organizations remain substantial challenges. to overcome these challenges, a number of optical readers have been developed and commercialized to provide rapid, objective, and quantitative measurements of signal on lateral flow assays. the underlying instrumentation within these readers can generally be grouped into two categories: (1) optomechanical scanning and (2) imaging. for scanning readers, the primary optical components include a light source (led or laser) and photodiode. 501 the specifications of these components are tailored to the specific detection element used in a particular assay. light source power and wavelength are determined by the absorbance or excitation wavelength of the labels employed in each specific test. the light source is rastered along the lateral flow strip, and a photodiode then detects reflectance or emission, depending on whether colorimetric or fluorescent labels are used. measurements are typically recorded with millivolt (mv) units, though biomarker concentration can be obtained if a calibration curve is established. when colored particles are present at the control and test lines, they absorb incident light, resulting in a quantifiable decrease in reflectance. for fluorescent labels, the intensity of emitted light increases as the light source passes over the control and test lines. optics are configured in either a specular or confocal arrangement. 501 in the specular arrangement, which is primarily used for colorimetric labels, the photodiode is positioned such that it collects light that is reflected from the test at the same angle as the incident light from the source. confocal scanning systems can be used to measure reflectance or fluorescence. in this case, incident light is focused to a point on the lateral flow strip, and the photodiode is placed above this focal point. light returning from the strip, whether reflected or emitted, passes through an aperture that excludes rays that were not directly from the focal point, eliminating background scattering. 502 in the case of fluorescent labels, a dichroic mirror or filter is often placed before the aperture. both specular and confocal scanning configurations are sensitive to the z position of the lateral flow strip. in the specular arrangement, any small deviation along the z axis can result in the reflected light completely missing the photodiode due to misalignment. confocal systems are slightly more tolerant to small variations along the z axis, depending on the size of the pinhole aperture. however, even slight variations off the focal plane can drastically decrease the number of photons reaching the photodiode, altering the quantitative output. another challenge associated with confocal and some specular opto-mechanical scanning strip readers is that a point of incident light only covers 10−20% of the test and control lines. 503 this is especially problematic if the concentration of particles varies along the test or control line due to improper flow or manufacturing variations. despite these challenges, scanning systems are advantageous due to their low cost and relative simplicity. one of the most widely used commercial systems is the esequant line of lateral flow readers manufactured by qiagen, inc. ( figure 35a ). 346 qiagen offers customization of optics for colorimetric, fluorescent, chemiluminescent, and upconverting phosphor labels. the user should take into account that these systems often vary in sensitivity. as a general rule, smaller portable systems rarely perform as well as their benchtop counterparts. for example, van dam et al. 313 found that the portable commercial reader, the ucp-quant (qiagen) , was at least an order of magnitude less sensitive than a custom-modified benchtop reader for evaluating upconverting phosphor-based schistosomiasis lateral flow assays. consequently, selecting a scanning lateral flow strip reader for a particular application requires consideration of both portability and sensitivity. imaging-based lateral flow readers greatly reduce the optical equipment required for quantifying lateral flow assays. these systems produce a picture record of a lateral flow assay using a charge coupled device (ccd) or complementary metal-oxidesemiconductor (cmos) detector and rely on image processing software to detect and/or quantify the intensity of the test line. 503 benefits of this format include the ability to store test images alongside patient records and a lack of moving parts, making imaging-based readers more robust to field conditions. further, image analysis software can identify and correct for nonuniform signal, allowing for analysis of the full test line. imaging-based readers can be used for detection of colorimetric and fluorescent labels. in the latter case, hardware for excitation is required, and filters can be added to improve image quality. since test position and illumination can affect image analysis, many camera-based lateral flow readers include housing units for run-to-run consistency. commercial lateral flow imaging devices include the deki reader (fio), ax-2x (axxin inc.), skansmart (skannex), and the rds-2500 pro (detekt biomedical llc) ( figure 35b ). 504−506 these devices have been used for quantification of malaria, 507−510 dengue, 509 burkholderia pseudomallei, 509 and y. pestis 509 lateral flow assays, and some, such as the deki reader, have been tested extensively in the field. 507−510 image-based lateral flow quantification translates well to mobile phone technology, especially as smartphone camera quality improves. mobile phones have been used to quantify lateral flow assays for numerous diseases, including malaria, 511−514 hiv, 511 and tuberculosis. 511 similar to commercially available imaging-based readers, many of these studies required external devices to ensure consistent illumination and distance from the camera in order to maintain consistent image analysis. recently, scherr et al. developed a lateral flow imaging platform using an unmodified mobile phone as the only hardware. 512 relying only on software for image analysis, lateral flow images were captured under ambient lighting conditions. the images were sent to a secure web database (redcap) and analyzed using a matlab-based algorithm. quantitative results were sent back to redcap and delivered to the phone in real time. this software-based imaging platform was tolerant to moderate variations in test orientation, distance, and illumination, making data collection as easy as taking a cell phone picture. when compared to the scanning esequant lateral flow reader, this processing platform was found to have improved detection limits. additionally, the algorithm reliably worked for camera resolutions between 0.5 and 8 megapixels, suggesting that any modern mobile phone camera could capture images with sufficient quality for the image processing software to work. 512 as mobile phones are incorporated into low-resource diagnostic workflows, it is important to ask whether the conventional lateral flow format can be redesigned to take full advantage of potential benefits smartphones can offer. in other words, what changes can be made to the diagnostic test format that maintain the simplicity of lateral flow assays but will enable improved quantification, communication, and aggregation of test results? one option is to embed quick response (qr) codes into the lateral flow test. this idea has been approached in a number of ways, including placing stickers on lateral flow tests and adding a transparent overlay in which the test and control lines become part of the qr code. 514, 515 in these formats, the embedded barcode is either static, providing useful information about the brand, lot, and patient id, or dynamic, producing a positive/negative read-out. scherr et al. developed a novel platform in which the control line of the lateral flow assay was replaced with a control grid patterned in the shape of a qr code ( figure 35c ). 513 the qr code functioned as a flow control, appearing on both positive and negative tests, but not invalid tests. the qr codes also triggered data analysis and enabled corrections for lighting effects. when scanned, manufacturing details were quickly and easily accessed at the same time as the test line was quantified. 513 it is not difficult to imagine the impact such a test format could have when combined with the data communication and aggregation abilities of smartphones. epidemiological and surveillance studies could account for variables that are not even currently considered in most studies, such as manufacturing batch-to-batch variation. geographic data could also be coupled to specific rapid tests. as tools for automated reading of lateral flow assays become more prevalent, establishing standards for consistent reporting and data management will be important. just like with the lateral flow assays themselves, test-reading instrumentation will need to be validated to ensure proper and consistent biomarker quantification across different devices and test brands. although optical lateral flow readers may detect test lines invisible to the human eye, readers alone will not replace the need for ultrasensitive lateral flow tests. 5.1.3. other smartphone-enabled optical measurements. mobile phones have been adapted for optical measurements beyond lateral flow quantification. one example is the mobile phone-based, hand-held microplate reader developed by berg et al. 382 this device consists of a 3dprinted attachment in which an led array illuminates each well, and the transmitted light is collected via 96 individual optical fibers. results are visualized and delivered to the user in one minute within a custom mobile phone app. the device was shown to be more than 98% accurate for immunoassays detecting mumps igg, measles igg, and herpes simplex virus igg (hsv-1 and hsv-2) when compared to an fda-approved clinical spectrophotometer. 382 while microplate-based immunoassays are not feasible in a field setting, this device could make a true impact in resource-limited clinics as an excellent alternative to traditional bulky and expensive spectrophotometers. additionally, because the device is attached to a mobile phone, it offers the potential to rapidly communicate and aggregate results, further enabling large-scale epidemiological studies. mobile phone attachments for other optical detection modalities have been developed as well. in one exciting example, long et al. 516 developed the tri-analyzer, a trimodal smartphone attachment capable of measuring transmission, reflection, and emission intensities. this multifunctional device leveraged white light from the smartphone's rear-facing flash led or an integrated green laser diode for sample illumination via optical fibers. the optical fibers collected light transmitted through or emitted from the sample or light reflected from a photonic crystal biosensor. this collected light was then collimated and focused before being dispersed by the diffraction grating onto the smartphone cmos sensor. the intensity at each wavelength was determined by the integration of pixel intensity at the corresponding position along the sensor. this clever detection strategy allowed for full absorbance and emission spectra to be collected with a simple smartphone-based device. in two proof-of-concept assays, the developed device could detect analytes at lower concentrations than those detected using conventional laboratory instrumentation, indicating that the miniaturization of the technology did not result in a trade-off of sensitivity. 516 thus, this trimodal smartphone attachment enables quantitative laboratory-quality optical measurements at one's fingertips. similarly formatted mobile phone-based spectrophotometers, 517,518 surface plasmon resonance sensors, 519−521 and flow cytometers 522, 523 have also been developed. the miniaturization and decrease in cost associated with these smartphone-enabled tools will make chemical reviews laboratory techniques possible that would otherwise be inaccessible in low-resource settings. noble metal nanoparticles have extremely high molar absorptivities, making them ideal visual labels for lateral flow assays. this intense absorbance occurs when particles are excited with light that matches the resonant oscillation of their free electrons. as these electrons relax down to the ground state, they release energy in the form of heat to the surroundings. 524 the amount of heat generated is equal to the product of the number of noble metal nanoparticles, the cross-sectional area of the nanoparticles, and the intensity of the incident light. 524 recently, bischof's group took advantage of these unique thermophysical properties to develop a novel method for lateral flow quantification based on the thermal contrast of gold nanoparticles. 524−526 in this method, a highpowered green laser was used to excite the gold nanoparticles, and an infrared-detecting camera was employed to measure the resulting thermal contrast. an early benchtop prototype of the device demonstrated a 32-fold improvement in detection limits of an fda approved lateral flow assay for cryptococcal capsular polysaccharide glucuronoxylomannan antigen (crag). 525 crag is a biomarker for cryptococcal infections, which are opportunistic fungal infections that are among the leading causes of death for patients with aids and a common cause of adult meningitis in sub-saharan africa. 525 the thermal contrast-coupled crag lateral flow assay was further validated in a second study, displaying 92% accuracy in quantifying crag levels in patient samples. 527 after successful validation of their prototype device, the group developed a reader that incorporated a green laser, infrared camera, and automated control system into a benchtop box device ( figure 36 ). 526 the study demonstrated the utility and versatility of thermal contrast as a method for quantification of lateral flow assays from a variety of vendors and for multiple diseases. an 8-fold improvement in detection threshold compared to visual inspection was found for lateral flow assays for influenza (bd veritor), malaria (first response, sd bioline, and parahit), and clostridium difficile (ridaquick). 526 in a separate study, the group found that this enhancement factor was greater as nanoparticle size increased, suggesting that redesign of lateral flow assays could further improve test sensitivity using a thermal contrast reader. 528 while thermal contrast represents a unique and versatile method for lateral flow quantification, several issues must be addressed before large-scale implementation. these include cost, portability, and background signal reduction from biological interferences such as blood. additionally, nitrocellulose membranes are highly flammable; while it does not appear that full strip ignition has occurred in the currently published studies, visible burn marks along the nitrocellulose membrane 526 suggest that safety studies to determine the limits of the thermal contrast system should be undertaken before widespread implementation. nevertheless, thermal contrast represents a highly sensitive and promising tool for lateral flow quantification. surface-enhanced raman spectroscopy (sers) is a highly sensitive vibrational spectroscopy technique for structurebased analyte detection. in this technique, excited localized surface plasmons, typically from noble metal (e.g., gold and silver) substrates with nanoscale features, generate amplified electromagnetic fields, enhancing the inelastic raman scattering signals of analytes of interest up to 10 14 times. 529 instrumentation for sers detection requires a high-powered laser excitation source, which is typically tuned according to the resonant frequency of the plasmonic substrate. after excitation, filters are used to exclude elastic rayleigh scattering, allowing only raman-scattered light to reach the detector. 530 the high sensitivity of sers makes it an attractive detection platform for infectious disease diagnostics, particularly for the detection of low-density infections. many laboratory-based bioassays for protein, nucleic acid, and whole-cell biomarkers that leverage sers detection have been developed for a myriad of infectious diseases. 68,531−536 these assays are generally either performed on the surface of particles in solution or on a bulk solid substrate. detection elements typically consist of nanoparticles functionalized with raman reporter molecules and target-specific molecular recognition elements. 529 once a complex is formed, the nanoparticles provide the plasmonic surface for raman signal enhancement of the reporter molecule. nanoparticles are typically gold, silver, or au@ag particles and come in a variety of shapes that provide optimized signal enhancement. 537 small-molecule raman reporters, such as 4-mercaptobenzoic acid, malachite green, and 4-aminothiophenol, are often employed because biomolecule targets typically have complex vibrational signatures and are frequently too far away from the plasmonic surface for sensitive detection and identification. 529 the bulky nature and resource requirements of sers detection remain its primary barrier to implementation in point-of-care settings. several hand-held raman spectrometers have been developed and commercialized, including the firstdefender (thermofisher scientific, inc.), 538, 539 nano-ram (b&w tek), 540, 541 and progeny (rigaku corp.). these devices are advertised for identification of unknown substances in forensic, environmental, military, and first-response contexts and for quality control in industrial settings. while many studies that utilize sers-based detection for infectious disease biomarkers suggest that these commercially available portable devices could be utilized for analysis of a sers-based lateral flow assay, none of the studies have actually implemented a hand-held device. 542−547 accordingly, it is unknown whether such devices have sufficient sensitivity or spatial resolution to use effectively with low-resource diagnostics. these parameters, along with cost analysis, should be studied to determine whether current commercial devices could be implemented. magnetic nanoparticles possess advantages over optical lateral flow labels because their signal is rarely hindered by test opacity or biological interferents. a detailed discussion of these qualities as well as examples of specific assays that employ magnetic nanoparticles are provided in section 4.2.6. here, we discuss instrumentation required for magnetic particle detection. there are a variety of methods for detecting paramagnetic and superparamagnetic nanoparticles on lateral flow assays based on either the inductive effect or magnetoresistance. for example, magnabiosciences (usa) has developed an induction-based device that has been used extensively in the literature for detection of infectious diseases such as anthrax, 366, 367 t. solium, 349 hiv, 360,361 v. parahemolyticus, 364 and l. monocytogenes. 365 in this format, the lateral flow assay is placed inside an oscillating magnetic field on a set of induction coils, and the instrument measures the induced electromotive force (v) on the coils. assays quantified by this technology generally had a greater sensitivity compared to visual detection of iron oxide particles. 548 while the current iteration of the magnabiosciences reader is a relatively large benchtop device that is unsuitable for use in low-resource settings, a more portable version is being developed. 549, 550 other companies, such as magnasense (finland) and magnisense (france), have developed portable, hand-held devices based on inductive detection of paramagnetic particles, though these devices have not been as extensively tested in the literature. several groups have developed lateral flow assay readers using magnetoresistive sensors. these devices are based on the principle that the electrical resistance of certain materials, which are incorporated in the sensors, can change upon application of an external magnetic field. 350 there are several types of sensors based on magnetoresistance, but the most common are giant magnetoresistive (gmr) sensors, which were once used in the reading heads for hard disk drives. 551 when applied to lateral flow assays, gmr sensors are placed in close proximity to the test and control lines, and an external magnetic field is applied to the test. this external field aligns the magnetic moments of paramagnetic or superparamagnetic lateral flow detection reporters. this alignment then produces a fringe field that is detectable by a change in resistance when a constant current is applied across the gmr sensor. 551 magnetoresistive sensors have been applied extensively by researchers to the detection of magnetic particle labels on lateral flow assays; however, most have been proof-of-principle studies using imitative lateral flow assays or tests for biomarkers such as hcg, cardiac troponin 1, and ifnγ. 357,552−555 the high sensitivity of magnetic detection on lateral flow assays makes this technique attractive for infectious disease diagnosis. while magnetic detection necessitates instrumentation for result readout, iron-containing magnetic nanoparticles also have the added advantage of being highly absorbent visual labels. 347 thus, magnetic nanoparticles can potentially be applied to different use-case scenarios with differing sensitivity requirements. in a use-case scenario such as morbidity control, where it is most important to identify high-intensity infections in the field, the visual detection of magnetic particles would be sufficient. when improved sensitivity is desired to detect lowintensity infections for epidemiological or surveillance purposes, analysis using the current instrumentation for reading magnetic nanoparticle labels could be performed in a district hospital or laboratory. however, further work on miniaturization of instrumentation and validation of hand-held readers will be required for field application of magnetic detection on lateral flow assays. as discussed in previous sections, electrochemical detection offers several benefits for poc infectious disease detection, namely that it is inexpensive, sensitive, rapid, and quantitative. the electrochemical detection systems previously discussed were largely proof-of-concept diagnostics for detection of protein or nucleic acid biomarkers; the infrastructure required to carry out these assays would limit their utility in a primary healthcare setting in low-and middle-income countries. integration of validated and field-ready electrochemical detection systems with developed electrochemical assays represents a viable strategy for implementing the next generation of poc diagnostics. the blood glucose biosensor is a remarkably successful electrochemical device that is ubiquitously applied at the point of care. generally, electrochemical glucose meter kits consist of three components: a lancet for sample collection, plastic test strips for wicking of the glucose-containing sample and generation of current, and a device for digital readout of the generated current. 556 the test strips house an electrochemical cell that contains a counter-reference electrode, fill-detection electrode, enzyme-coated working electrode, and mediators ( figure 37a ). in these devices, the working electrode is coated with the enzyme glucose oxidase (gox) or glucose dehydrogenase (gdh), and the enzyme oxidizes the glucose present in a blood sample. ferrocene derivatives or other group viii metal complexes act as mediators to facilitate electron transfer from the enzyme redox center to the electrode surface, thereby producing a measurable current. 556 since the development of the first commercial blood glucose biosensor in 1987, the use of this technology has drastically expanded with the current market now offering numerous blood glucose meters that are portable, rapid, sensitive, and quantitative. 557 the blood glucose biosensor has had an enormous impact on management of types i and ii diabetes where constant monitoring and a rapid time to result are critical to preventing incidences of hyper-and hypoglycemia. successful adaptation of this technology for field detection of infectious diseases would have a profound impact in disease surveillance and elimination campaigns, where sensitive and quantitative results are desired to best inform healthcare professionals about disease distribution. 556, 557 several groups have repurposed commercial blood glucose meters for detection of a variety of targets. 167,558−562 xiang and lu 558 developed a platform that utilized a personal glucose meter for signal output to detect protein (ifn-γ), nucleic acid (adenosine), small molecule (cocaine), or toxic ion (uranium) targets. their strategy employed a magnetic bead-conjugated aptamer specific for the biomarker of interest. the aptamer was initially prehybridized to a partially complementary dna strand that was linked to invertase, an enzyme that catalyzes the hydrolysis of sucrose into its fructose and glucose subunits. in biomarker-containing samples, the aptamer bound to the biomarker, releasing the invertase-conjugated complementary dna into solution. the magnetic beads were then removed from solution using an external magnet, and the addition of sucrose to the supernatant resulted in glucose production that was readily detectable by a personal glucose meter. while this creative application of an extant platform expanded its functionality, it also increased the number of assay steps, potentially limiting its use directly at the point of care. to address this concern, the lu group 562 has since integrated this solution-based assay into a lateral flow strip with dried reagents, eliminating several user steps and further simplifying the platform such that it could be applied at the point of care. this innovative approach is an excellent example of how metalbased sample preparation using magnetic beads can be combined with an existing electrochemical platform for rapid detection of a variety of disease targets. as the blood glucose meter clearly demonstrates, portable electrochemical instrumentation has enabled minimally trained users to measure analytes at the point of care and rapidly obtain information regarding their health status. mobile health technologies (i.e., mobile phones and telemedicine) augment these advances in instrumentation, connecting individual users to an entire healthcare infrastructure. mobile health technologies are useful for tracking patient information and facilitating communication between the patient and the care provider. as these technologies mature, they are becoming increasingly important for modeling diseases and disease trends throughout various populations. 563 integration of portable electrochemical diagnostics with mobile health technologies in so-called "connected" diagnostics has been pursued by interfacing electrochemical devices with mobile phones. 173, 564 nemiroski and colleagues 173 developed a universal mobile electrochemical detector (umed) that combined a potentiostat with virtually any mobile phone using audio-based data transmission ( figure 37b ). the device could accommodate several electrochemical techniques (amperometry, coulometry, voltammetry, and potentiometry), and the voice system-based data ensured broad compatibility with a variety of mobile phones and networks. the authors employed the umed to detect blood glucose, heavy metals in water, electrolytes, and malarial biomarker hrp2. following sample collection and measurement using the potentiostat, the user placed a phone call to a skype number in a remote location to vocally report the value of the analyte measured. the remote application then extracted the value from the audio data and sent an sms message back to the user with additional diagnostic information (e.g., "low" if the reported blood glucose level was low). although the umed was applied to detect an infectious disease biomarker (hrp2), it is limited by the availability of existing compatible testing technology. for instance, hrp2 detection required an offline 96-well plate elisa to be conducted prior to analysis on the umed and transmission over a mobile network. as more progress is made in electrochemical diagnostic design, the umed and other similar devices will be able to reach their full potential as "connected" diagnostics. as battery-operated and portable devices become more robust, electrochemical detection devices are becoming increasingly viable as poc platforms. the success of the personal glucose meter is a testament to the effective and reliable diagnostic performance that is possible with electrochemical measurements. with the concurrent expansion of information technologies, "connected" electrochemical diagnostics could serve as the link between the chemistries utilized at the point of care and the remote higher level healthcare systems that map disease trends and inform treatment decisions. in high-income countries, advances in portable electronics and instrumentation have enabled live health tracking at consumers' fingertips. wearable commercial health diagnostics such as apple watch, garmin, and fitbit are simple to use and provide real-time updates of heart rate, activities completed, or calories burned. in a similar vein, there are several emerging healthcentered tools, such as press-on tattoos, 565,566 skin patches, 567 and face masks, 568 capable of detecting changes in human physiology at the point of use. in addition, the advent of technologies such as google glass have allowed for "connected" interpretation of rapid diagnostic tests. 515 overall, wearable instruments combine sample collection, sample preparation, analysis and readout, and data transmission into one device, representing significant progress in diagnosis and health management at the point of care. small electrochemical instrumentation has become a focus of innovation, and research in this field has led to a number of skin-based wearable diagnostics. jia et al. 565 566 detected glucose in the interstitial fluid of the skin ( figure 38a ). these technologies are extraordinarily promising for future diagnostic devices, particularly as new biomarkers of infectious diseases are discovered in surface biofluids such as sweat. 569 recent advances in microprojection 570,571 and microneedle 572,573 technology have allowed researchers to expand the use of skin-based wearables beneath the surface of skin. in several applications, patches consisting of many microprojections were easily applied to the skin with a gentle press and could draw hundreds of microliters of blood with little-to-no pain. 567,574−576 in 2014, lee et al. 567 outlined the design of a multiplexed patch that captured both igg and malarial biomarker hrp2 from the skin of inoculated mice. the patch consisted of microprojection arrays (mpas) that were subsequently functionalized with anti-hrp2 antibodies, fc-specific antimouse igg antibodies, and antidengue antibodies. the anti-hrp2-functionalized mpas detected the biomarker, while the anti-igg-functionalized mpas acted as a positive control for skin penetration, and the antidenguefunctionalized mpa served as a negative control. though sample collection and preparation were applicable at the point of use, the assay required an elisa for colorimetric readout of the captured antigen. further research into integration of this technology with a diagnostic such as a stacked paper-based assay would allow for real-time skin-based monitoring of infectious diseases. another forthcoming application for wearable instrumentation is in breath analysis via paper masks. guder et al. 568 developed an electrical sensor to monitor respiration. the sensor consisted of a paper surgical mask printed with graphite electrodes that were attached to a battery and signal amplifier. as a user breathed, the humidity changes from the inhalation and exhalation generated a measurable current across the electrode, which was then detected using a bluetoothconnected mobile device ( figure 38b ). as this technology evolves, it may be possible to adapt it for analysis of breathbased biomarkers, particularly for respiratory infectious diseases such as tuberculosis and influenza. in addition, incorporating the external battery/amplifier pack into the material of the mask may open avenues for broader distribution, particularly to low-resource areas. likewise, personal computer displays such as google glass have enabled users to harness the power of a technological workstation in a hands-free glasses format. this capacity could significantly improve diagnostic capabilities and effectiveness. in 2014, feng et al. 515 developed a method of lateral flow assay analysis which combined visual examination of a lateral flow test with computerized image analysis and wireless storage capability ( figure 38c ). this wearable imaging technique allowed users to collect images of tests and store them in a cloud-based system or locally on the google glass headset if internet access was unavailable. once the google glass headset was connected to a wireless network, the test images could be sent to a secure server to be analyzed, and then the test's results and analysis were sent back to the viewer's eyepiece. this method has been demonstrated using rapid diagnostic tests for both hiv antibodies and prostate specific antigen (psa), 515 though it has the potential to be adapted for any lateral flow assay with visual labels. while the use of computer-based imaging may remove some of the inaccuracies that arise from subjective human-based interpretation of results, it must also be noted that there are a number of technological hurdles that should first be addressed. the google glass is not capable of autofocusing, and the images were taken under good lighting against white and universally solid, simple backdrops. thus, this technology is limited in its field application. in addition, test recognition was dependent on preprinted qr codes that were prepared and attached to the tests by researchers before use. in order to more efficiently utilize this technology, the google glass application could be adapted to identify test features without the need for a usersupplied qr code and web-based test input. this would simplify the workflow and allow for more high-throughput analysis of diagnostic tests. overall, there is significant room for growth and development in the field of wearable instrumentation. wearable diagnostics, which may have previously seemed more at home in the world of science fiction, are becoming commonplace in health-related research efforts. the development of wearable diagnostic technologies could drastically improve the simplicity of diagnostic measurements and interpretation in regions with little or no laboratory infrastructure. as these technologies continue to be developed for applications in high-resource areas of the world, the scientific community would be remiss not to consider the impact they could have on infectious disease diagnosis in resource-limited settings. inorganic chemistry for nucleic acid detection thus far, we have highlighted innovative examples of metalbased strategies for improving point-of-care diagnosis of infectious diseases based primarily on protein and inorganic disease biomarkers. in most cases, leveraging inorganic chemistry for sample preparation methods and detection probes would require minimal modification for application at the point of care. it is clear that, despite the potential trade-offs in complexity of workflow and instrumentation, the increased sensitivity offered by these metal-based strategies for biomarker detection could significantly improve morbidity control and elimination efforts. in many instances, though, sensitive and quantitative detection of the presence or absence of a disease does not paint the full picture needed to implement successful control or elimination campaigns. in some cases, the pathogen's own genetic material must be detected and extensively studied on a population scale in order to inform strategy. this research provides valuable information on pathogen species and strain distribution and drug resistance, which can inform treatment and diagnostic strategies in a control or elimination campaign. 579 the information-rich and highly specific nature of nucleic acid detection makes these biomarkers attractive targets for point-of-care diagnostics. in fact, several groups have begun to apply some of the metal-based detection strategies for proteins and inorganic biomarkers discussed in the preceding sections t o t h e d e t e c t i o n o f p a t h o g e n i c d n a o r rna. 231,234,235,273,580−587 for instance, many metal-containing nanoparticle probes have been functionalized to detect pathogen-specific nucleic acid sequences in a variety of formats. the size-dependence of spr for noble-metal nanoparticles has been leveraged in an aggregation assay for multiplexed detection of middle east respiratory syndrome coronavirus, m. tuberculosis, and hpv in a μpad format. 231 the high conductivity of agnps has been exploited for electrochemical detection of m. tuberculosis and hiv. 234, 235 luminescent particles (i.e., quantum dots, europium chelatedoped particles, and upconverting phosphors) have been functionalized for the detection of a myriad of diseases, including hiv, 273,580−583 hbv, 273 hcv, 273 hpv, 584 p. falciparum malaria, 585 bacillus cereus, 581, 582, 586, 587 and streptococcus pyogenes. 581 moreover, these nanoparticle-based methods occur in a variety of formats, including lateral flow assays. because specific nucleic acid sequences are typically present at much lower concentrations than protein biomarkers in patient samples, a target amplification step is often required before the detection method can be applied. the most common target amplification technique is pcr, a timeconsuming enzyme-based method that requires technical expertise, expensive equipment, and significant laboratory infrastructure. these requirements are the primary barriers to sensitive detection of pathogen genetic material in the field. fortunately, alternative target amplification techniques with potential point-of-care applications have emerged. for example, the biobarcode signal amplification strategy discussed in section 4.4.4 has been adapted for amplification of nucleic acid targets. 464 however, in general, isothermal enzymatic amplification techniques have demonstrated the most sensitivity and promise for point-of-care diagnostic applications. isothermal amplification techniques do not require any thermal cycling, are simpler to operate, and require fewer resources when compared to traditional pcr. this class of enzymatic amplification techniques includes loop-mediated isothermal amplification (lamp), helicase-dependent amplification (hda), rolling circle amplification (rca), multiple displacement amplification (mda), recombinase polymerase amplification (rpa), and nucleic acid sequence-based amplification (nasba). 588 among these strategies, lamp is one of the most widely validated, having been employed for amplification of nucleic acid targets for a variety of diseases and infections. these include targets for malaria, 589−592 hiv, 593 hpv, 594 bordetella pertussis, 595 c. trachomatis, 595,596 neisserie gonorrheae, 595 h1n1 influenza, 595,597 e. coli, 598 and several filarial parasites. 599 in addition to malaria diagnosis, lamp has also been used to detect artemisinin resistance in p. falciparum malaria. 600 this technique consists of iterative elongation and recycling of stem-loop dna structures, usually requiring a constant temperature between 60 and 65°c. 588 the relatively simple workflow of lamp (when compared to other enzymatic amplification techniques) and its potential for colorimetric visual readout have led to the development of several low-resource devices for nucleic acid detection. one example is the noninstrumented nucleic acid amplification device (nina), which relies on an exothermal chemical reaction (mgfe powder, saline, and oxygen) coupled to a phase change material for prolonged and precise heating inside a thermos cup. 589, 601 despite its streamlined workflow, the nina device does not account for sample preparation. the preamplification nucleic acid extraction step still requires laboratory equipment such as a heated water bath and a centrifuge. 589 although the amplification step itself has been modified for use at the point of care, the complete protocol relegates it to the laboratory environment. in an effort to mitigate these limitations, the klapperich group 594 has developed an inventive method in which nucleic acid extraction, amplification, and detection are fully integrated into a single paper fluidic device. to accomplish this, dna from clinical samples was precipitated from solution and applied to a paper membrane, which was washed with ethanol to remove impurities. lamp reaction reagents, which included fitc-conjugated forward and biotinylated reverse loop primers, were then applied to the sample port, which was sealed for a 30 min heating step on a hot plate. the amplified solution was then wicked onto a lateral flow assay equipped with anti-fitc antibodies on the test line and streptavidincoated aunps for detection ( figure 39 ). in the presence of the target, both primers hybridized to the amplified genetic material, forming a "sandwich" on the lateral flow assay and resulting in a visible test line signal. 594 this all-in-one paper-based nucleic acid test is a perfect example of how target extraction and amplification can be innovatively combined with metal-based detection elements. this integration will allow for complex nucleic acid detection protocols to be performed at the point of care that previously were only possible in well-equipped laboratories. moving forward, several considerations must be addressed, including integration of laboratory-free heating elements, reducing the number of user steps, and large-scale manufacturing feasibility. nonetheless, combination of the paper-based devices developed by the klapperich group with simple, integrated heating elements similar to the nina device could allow for sensitive and information-rich nucleic acid detection in low resource settings. this would enable case management and elimination strategies to be precisely carried out in nearly any healthcare setting. the application of inorganic chemistry and nanomaterials to point-of-care infectious disease diagnosis has enhanced the sensitivity and specificity of diagnostic tests, leading to improved outcomes and reduced transmission. the sample preparation and detection methods presented herein are promising examples of how metal-based strategies can expand the use-case scenarios of diagnostic tests, resulting in access to care that would otherwise be unattainable. however, we have also seen that these same metal-based strategies that improve review diagnostic sensitivity and specificity can lead to more complex tests, potentially requiring more user steps, expertise, and time to complete. these trade-offs of complexity, cost, and speed must be weighed against the potential impact that improved sensitivity and specificity could have in a given use-case scenario. for example, in the case of morbidity control, where the goal is to reduce the prevalence of high-intensity infections, the demand for simple, affordable, and rapid tests likely outweighs the need for highly sensitive tests. once in the elimination phase, however, where the goal is to completely interrupt local transmission, high sensitivity tests are imperative for detecting and treating low-density infections. consequently, the need for more sensitive diagnostics likely outweighs small increases in test complexity or cost for elimination campaigns. in our evaluation of various inorganic complexes and nanomaterials, advantages and caveats became apparent for different classes of materials. the properties of these materials directly affect how they can be applied in diagnostics. noble metal nanoparticles have frequently been incorporated into paper-based assays, such as lateral flow assays, with visual signal readout because of their stability and superior molar absorptivities. 219, 221, 229, 231 despite the strong optical signal that noble metal nanoparticles afford, these colorimetric assays are often still limited by poor sensitivity. this potentially limits their impact in a use-case scenario such as an elimination campaign. if better sensitivity is needed, there are essentially three metal-based strategies that we have discussed in this review for further improvement: (1) sample preparation for biomarker enrichment, (2) luminescent detection probes, and (3) signal amplification techniques. the imac-functionalized materials used for sample preparation collectively represent one approach for improving assay sensitivity by enriching the biomarker concentration. while some of the sample preparation methods demonstrated increased diagnostic sensitivity, these methods did add to the number of user steps. 85, 99, [113] [114] [115] 117 metal-based sample preparation is perhaps the simplest of the three metal-based approaches to increasing sensitivity; however, for it to be truly impactful, it will need to be incorporated into diagnostics that are developed with the end user in mind such that the overall workflow is considered and the assay steps are minimized. alternatively, noble metal nanoparticles can be replaced with luminescent detection probes to afford greater sensitivity. quantum dots, lanthanide chelate-doped nanoparticles, and up-converting phosphor nanoparticles all potentially offer improvements in sensitivity over colorimetric labels, albeit through different mechanisms. 257, 289, 305, 309 while luminescent inorganic complexes were incorporated into proof-of-principle assays for the detection of infectious disease biomarkers, they have yet to be utilized in a field-deployable format and lack the sensitivity of the aforementioned nanomaterials. 138, 139 quantum dots and up-converting phosphor nanoparticles also have the capability for multiplexing that could be beneficial for case management. 252, 253, 310 although the various luminescent nanoparticles offer improvements in sensitivity, they also require more instrumentation for signal readout. the increased cost and complexity of signal readout for luminescent nanoparticles must therefore be considered when developing diagnostic strategies to combat infectious diseases. lastly, metal-based signal amplification techniques could be incorporated to improve sensitivity. almost all of the signal amplification techniques discussed in this review (metalloenzyme-based amplification, nanoparticle dissolution, nanoparticle enzyme mimics, reductive nanoparticle enlargement, and biobarcodes) offer colorimetric readouts with improved sensitivity. however, these improvements come at the cost of an increased number of assay steps and thus longer total assay times. while some efforts, such as the p24 lfas developed by loynachan et al., 435 demonstrated progress toward more fieldfriendly signal amplification methods, further improvements in the ease of use and time to result must be made to translate these methods to primary healthcare facilities. for these metal-based strategies that improve sensitivity, it is clear that simplifying the user interface is a significant challenge that remains. aside from the user interface, there are several other challenges to translating novel diagnostic strategies that must be considered before implementation. these include: (1) the need for clinical validation, (2) the potential for large-scale manufacturability, (3) the availability of distribution channels, figure 39 . workflow for detecting nucleic acids on a paper fluidic device. different dyes were used to illustrate the various components of the assay. (a) the dna-containing sample (blue dye) is added to the sample port, where it wicks down the test by capillary action (b). the test is then washed twice with ethanol (yellow), which also flows down the test (c−f). (g) the absorbent pad is then removed and discarded. (h) the lamp reaction mix is then added to the sample port, and the test is folded to protect the sample/lamp mixture from evaporation. (i) once the test has been heated on a hot plate for 30 min, the hydrophobic barrier is removed so that the sample port is exposed on top and bottom, allowing it to come into contact with the lfd strip for dna elution. (j) the sample, once eluted, can then wick down the length of the test strip to generate a signal. adapted with permission from ref 594. copyright 2016 royal society of chemistry. review and (4) integration into current health systems. most of the metal-based strategies for improved infectious disease diagnostics presented in this review have demonstrated some level of feasibility in a small set of patient or mock clinical samples. however, new technology must be clinically validated on a large set of patient samples from endemic regions before implementation. such studies provide insight into the diagnostic performance and tolerance of day-to-day and interpatient variation in the appropriate clinical context. this rigor can draw attention to challenges and weaknesses that would otherwise go unnoticed in a research laboratory setting. production scalability of novel diagnostic technology is another important consideration for successful translation. even in the research phase of diagnostic development, material choice, device design, and fabrication should be based, in part, on manufacturing scalability in order to streamline implementation. many of the metal-based strategies presented in this review, particularly the diverse inorganic nanoparticle detection probes used in lateral flow assays, are advantageous in this context because they are easily integrated into existing lfa manufacturing infrastructure. beyond the laboratory, it is important to consider distribution channels and how novel tools will be integrated into existing health systems. region-specific health delivery systems and their respective regulatory approvals are ultimately what define a novel diagnostic device's implementation. for example, in one region, most rapid testing for infectious diseases may be performed at local health clinics run by a national ministry of health, whereas in another area, privately run dispensaries may be the most common source of primary health care. understanding the structural differences between such health delivery systems as well as the respective availability of resources could impact device design in the research phase. although there are many considerations and barriers to translating novel technologies to the clinical setting, we are optimistic that the continued development of metal-based point-of-care diagnostic strategies will have a profound impact on global health. returning to the example presented in the introduction, if some of the ultrasensitive p24 detection methods presented in this review 396, 435 were adapted for use at primary healthcare facilities, the time to result for infant hiv diagnosis at the point of need would be significantly reduced. in areas like burkina faso, this would enable earlier initiation of very effective antiretroviral therapies and drastically reduce infant mortality stemming from opportunistic infections. finally, the continually increasing connectivity in low-and middle-income countries coupled to sensitive diagnostics will allow for real-time disease surveillance, intervention analysis, and epidemiological studies. "connected" diagnostics have the potential to accelerate communications between field workers, local health outposts, district hospitals, and national health ministries. the ability to rapidly convey diagnostic results could not only enable active case detection strategies but also would provide faster responses to epidemics such as the 2014− 2016 ebola and 2015−2016 zika outbreaks. these faster responses could potentially reduce mortality and improve clinical outcomes. metal-based strategies for sample preparation and signal generation show great promise for use in the development and improvement of sensitive diagnostic devices for low-resource settings. these devices, coupled with connected, fielddeployable instrumentation, will empower rapid response, effective control, and successful elimination of infectious diseases worldwide, ushering in an era of sustainable and improved global health. (2011) and an american association for the advancement of science fellow (2014). for the past decade, he has unraveled the mechanism of heme detoxification within the parasite plasmodium falciparum, the causative agent of malaria. additionally, he has made important contributions in understanding hemozoin's immunomodulatory activity in parasite-host interactions, the mechanism of action for antimalarial compounds, and the development of biomarkers for low-resource diagnostics. more recent efforts have focused on the challenges of innovation in low-resource diagnostics. he has focused primarily on malaria biomarkers and new approaches to formatting and improving performance of diagnostics for the detection of asymptomatic patients, drug-resistant infections, and drug-sensitive patients. his lab works in the field in macha, zambia; cape town, south africa; and san marcos, peru. this work is supported by the national institutes of health (r01 ai135937; r21 tw010635-01), nih/fogarty international center (d43 tw009348), the bill and melinda gates foundation (opp1123092), and vanderbilt university through the laboratories for innovations in global health technologies. c.f.m. and k.a.r. acknowledge support from the national science foundation graduate research fellowship program (dge-1145197). we gratefully acknowledge shellie richards for her tireless proofreading efforts that sharpened the manuscript. aequanimitas, with other addresses to medical students trends in infectious disease mortality in the united states during the 20th century convergence of non-communicable and infectious diseases in lowand middle-income countries uneven progress in preventing and treating hiv infections worldwide disease eradication, elimination and control: the need for accurate and consistent usage the principles of disease elimination and eradication lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. a literature survey no. e111240. (10) u.s. department of health and human services. diagnosis of hiv infection in infants and children early antiretroviral therapy and mortality among hiv-infected infants emergence of a peak in early infant mortality due to hiv/aids in south africa evaluating diagnostic point-of-care tests in resource-limited settings a newly discovered parasite in the blood of patients suffering from malaria. parasitic etiology of attacks of malaria the shape and size of hemozoin crystals distinguishes diverse plasmodium species hemozoin and antimalarial drug discovery direct tests of enzymatic heme degradation by the malaria parasite plasmodium falciparum malarial hemozoin: from target to tool an iron-carboxylate bond links the heme units of malaria pigment the structure of malaria pigment β-haematin structure and formation of synthetic hemozoin: insights from first-principles calculations. cryst. growth des. 2011, 11, 3332−3341 separation of malaria-infected erythrocytes from whole blood: use of a selective high-gradient magnetic separation technique one-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells diagnosis of malaria by magnetic deposition microscopy removal of malaria-infected red blood cells using magnetic cell separators: a computational study magnetic separation of malaria-infected red blood cells in various developmental stages application of magnetic cytosmear for the estimation of plasmodium falciparum gametocyte density and detection of asexual stages in asymptomatic children nuclear magnetic resonance: a tool for malaria diagnosis? micromagnetic resonance relaxometry for rapid label-free malaria diagnosis enhancing malaria diagnosis through microfluidic cell enrichment and magnetic resonance relaxometry detection reply to "considerations regarding the micromagnetic resonance relaxometry technique for rapid label-free malaria diagnosis detection of malarial byproduct hemozoin utilizing its unique scattering properties in vivo microscopy of hemozoin: towards a needle free diagnostic for malaria towards a needle-free diagnosis of malaria: in vivo identification and classification of red and white blood cells containing haemozoin hemozoin-generated vapor nanobubbles for transdermal reagentand needle-free detection of malaria malaria theranostics using hemozoin-generated vapor nanobubbles. theranostics transdermal diagnosis of malaria using vapor nanobubbles vivo photoacoustic flow cytometry for early malaria diagnosis preclinical photoacoustic models: application for ultrasensitive single cell malaria diagnosis in large vein and artery a magneto-optic route toward the in vivo diagnosis of malaria: preliminary results and preclinical trial data laboratory evaluation on the sensitivity and specificity of a novel and rapid detection method for malaria diagnosis based on magneto-optical technology (mot) the in vivo diagnosis of malaria: feasibility study into a magneto-optic fingertip probe rotatingcrystal malaria diagnosis: pre-clinical validation evaluation of a novel magneto-optical method for the detection of malaria parasites keźsmaŕki, i. efficient monitoring of the blood-stage infection in a malaria rodent model by the rotating-crystal magneto-optical method sensitivity of hemozoin detection by automated flow cytometry in non-and semi-immune malaria patients full blood count and haemozoin-containing leukocytes in children with malaria: diagnostic value and association with disease severity a novel flow cytometric hemozoin detection assay for real-time sensitivity testing of plasmodium falciparum mass spectrometry for malaria diagnosis rapid detection of malaria infection in vivo by laser desorption mass spectrometry detection of plasmodium falciparum in pregnancy by laser desorption mass spectrometry magnetic field enriched surface enhanced resonance raman spectroscopy for early malaria diagnosis raman spectroscopic analysis of malaria disease progression via blood and plasma samples optical diagnosis of malaria infection in human plasma using raman spectroscopy bio-sensing with butterfly wings: naturally occurring nano-structures for sers-based malaria parasite detection haemoproteus and schistosoma synthesize heme polymers similar to plasmodium hemozoin and β-hematin knobs" in opisthorchis felineus infected liver. parasites vectors 2015, 8, 459. (72) world health organization clearance kinetics of parasites and pigment-containing leukocytes in severe malaria the schistosome egg: development and secretions fine structure of schistosome eggs as seen through the scanning electron microscope tracking the fate of iron in early development of human blood flukes graeff-teixeira, c. detection of schistosoma mansoni eggs in feces through their interaction with paramagnetic beads in a magnetic field comparison of the kato-katz and helmintex methods for the diagnosis of schistosomiasis in a low-intensity transmission focus in bandeirantes magnetic beads for schistosomiasis diagnosis the iron distribution and magnetic properties of schistosome eggshells: implications for improved diagnostics the affinity of magnetic microspheres for schistosoma eggs direct transfer of hrpii-magnetic bead complexes to malaria rapid diagnostic tests significantly improves test sensitivity guide to protein purification immobilized metal ion affinity chromatography chapter 1: affinity chromatography guide to protein purification metal-affinity separations: a new dimension in protein processing new metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues metal chelate affinity chromatography, a new approach to protein fractionation edta-resistant, chemical-tolerant ni-penta agarose-based resins immobilized metal ion affinity chromatography (imac) chemistry and bioseparation applications immobilized metal ion affinity chromatography rapid concentration and elution of malarial antigen histidine-rich protein ii using solid phase zn(ii) resin in a simple flow-through pipette tip format purification and partial characterization of an unusual protein of plasmodium falciparum: histidine-rich protein ii perspectives of immobilized-metal affinity chromatography chitosan-coated silica beads as immobilized metal affinity support for protein adsorption a novel hierarchical nanozeolite composite as sorbent for protein separation in immobilized metal-ion affinity chromatography the design and synthesis of a hydrophilic core−shell−shell structured magnetic metal−organic chemical reviews review framework as a novel immobilized metal ion affinity platform for phosphoproteome research hydrophilic nb5+-immobilized magnetic core−shell microsphere − a novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides zirconium-cation-immobilized core/shell (fe 3 o 4 @polymer) microspheres as an imac material for the selective enrichment of phosphopeptides hydrophilic polydopamine-coated graphene for metal ion immobilization as a novel immobilized metal ion affinity chromatography platform for phosphoproteome analysis facile synthesis of copper(ii)-decorated functional mesoporous material for specific adsorption of histidine-rich proteins purification of cell culture-derived influenza virus a/puerto rico/8/34 by membrane-based immobilized metal affinity chromatography biofunctional magnetic nanoparticles for protein separation and pathogen detection low-resource method for extracting the malarial biomarker histidine-rich protein ii to enhance diagnostic test performance simple sample processing enhances malaria rapid diagnostic test performance magnetically-enabled biomarker extraction and delivery system: towards integrated assured diagnostic tools nickel nanoparticle-doped paper as a bioactive scaffold for targeted and robust immobilization of functional proteins metal affinity-enabled capture and release antibody reagents generate a multiplex biomarker enrichment system that improves detection limits of rapid diagnostic tests immunomagnetic capture and colorimetric detection of malarial biomarker plasmodium falciparum lactate dehydrogenase a review of practical techniques for the diagnosis of malaria simultaneous capture and sequential detection of two malarial biomarkers on magnetic microparticles plasmodium falciparum hrp2 elisa for analysis of dried blood spot samples in rural zambia characterization of plasmodium lactate dehydrogenase and histidine-rich protein 2 clearance patterns via rapid on-bead detection from a single dried blood spot aptamers: an emerging class of molecules that rival antibodies in diagnostics aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay recognition of biomarkers and cell-specific molecular signatures: aptamers as capture agents in vitro selection of rna molecules that bind specific ligands design of targeting ligands in medicinal inorganic chemistry metallodrugs in medicinal inorganic chemistry exploring biologically relevant chemical space with metal complexes targeting proteins with metal complexes dicarba-closo-dodecarborane-containing half-sandwich complexes of ruthenium, osmium, rhodium and iridium: biological relevance and synthetic strategies magnetic nanoparticle design for medical diagnosis and therapy chemistry, biology, and medicine of fluorescent nanomaterials and related systems: new insights into biosensing, bioimaging, genomics, diagnostics, and therapy nanomaterials for in vivo imaging metals on the nanoscale: optical and photothermal properties and some applications in imaging, sensing metal complexes for the detection of disease-related protein biomarkers on-particle detection of plasmodium falciparum histidine-rich protein ii by a "switch-on" iridium(iii) probe graphene quantum dot based "switch-on" nanosensors for intracellular cytokine monitoring luminescent turn-on detection of hg(ii) via the quenching of an iridium(iii) complex by hg(ii)-mediated silver nanoparticles. sci. rep. 2017 the application of a g-quadruplex based assay with an iridium(iii) complex to arsenic ion detection and its utilization in a microfluidic chip amplified biosensing using the horseradish peroxidase-mimicking dnazyme as an electrocatalyst label-free and amplified electrochemical detection of cytokine based on hairpin aptamer and catalytic dnazyme a cyclometalated iridium(iii) complex used as a conductor for the electrochemical sensing of ifn-γ exonuclease iii-based and gold nanoparticle-assisted dna detection with dual signal amplification sensitive detection of hiv gene by coupling exonuclease iii-assisted target recycling and guanine nanowire amplification triple-helix molecular switch electrochemical ratiometric biosensor for ultrasensitive detection of nucleic acids electrochemiluminescence (ecl) development of a membrane strip immunosensor utilizing ruthenium as an electro-chemiluminescent signal generator attomole detection of hemagglutinin molecule of influenza virus by combining an electrochemiluminescence sensor with an immunoliposome that encapsulates a ru complex detection of influenza virus by a biosensor based on the method combining electrochemiluminescence on binary sams modified au electrode with an immunoliposome encapsulating ru (ii) complex you, t. a novel electrochemiluminescence immunosensor for the analysis of hiv-1 antigen based on p-rgo@au@ru-sio 2 composite stretch−stowage−growth strategy to fabricate tunable triply-amplified electrochemiluminescence immunosensor for ultrasensitive detection of pseudorabies virus antibody design of sensitive biocompatible quantum-dots embedded in mesoporous silica microspheres for the quantitative immunoassay of human immunodeficiency virus-1 antibodies an ultrasensitive electrogenerated chemiluminescence-based immunoassay for specific detection of zika virus novel electrochemiluminescence-sensing platform for the precise analysis of multiple latent tuberculosis infection markers phosphorescent chemosensors based on heavy-metal complexes transition metal complexes with strong absorption of visible light and long-lived triplet excited states: from molecular design to applications photochemistry and photophysics of coordination compounds: iridium. in photochemistry and photophysics of coordination compounds ii metal centered ligand field excited states: their roles in the design and performance of transition metal based photochemical molecular devices bright building blocks for chemical biology electrochemical biosensors: towards point-of-care cancer diagnostics electrochemiluminescence detection for development of immunoassays and dna probe assays for clinical diagnostics electrochemiluminescence detection in paper-based and other inexpensive microfluidic devices paper-based bipolar electrode-electrochemiluminescence (bpe-ecl) device with battery energy supply and smartphone read-out: a handheld ecl system for biochemical analysis at the point-of-care level electrochemical sensing in paper-based microfluidic devices aptamer-based origami paper analytical device for electrochemical detection of adenosine electrogenerated chemiluminescence detection in paper-based microfluidic sensors electrochemistry on paper-based analytical devices: a review quantitative chemical analysis dna-enhanced peroxidase activity of a dna aptamer-hemin complex universal mobile electrochemical detector designed for use in resource-limited applications comparative study of the effect of various electrode membranes on biofouling and electrochemical measurements label-free biomarker detection from whole blood nanochemistry and nanomedicine for nanoparticle-based diagnostics and therapy gold nanoparticles for in vitro diagnostics metal−organic frameworks as sensory materials and imaging agents introduction to metal− organic frameworks an electrochemical aptasensor for multiplex antibiotics detection using y-shaped dna-based metal ions encoded probes with nmof substrate and csrp target-triggered amplification strategy two-dimensional zirconium-based metal−organic framework nanosheet composites embedded with au nanoclusters: a highly sensitive electrochemical aptasensor toward detecting cocaine nh 2 -ni-mof electrocatalysts with tunable size/ morphology for ultrasensitive c-reactive protein detection via an aptamer binding induced dna walker−antibody sandwich assay porous phosphorescent coordination polymers for oxygen sensing phosphorescent nanoscale coordination polymers as contrast agents for optical imaging a review on the classification, characterisation, synthesis of nanoparticles and their application a review of silver nanoparticles: research trends, global consumption, synthesis, properties, and future challenges nanotechnology in diagnosis: a review microparticles and nanoparticles conjugation of nanoparticles to proteins zero-length crosslinkers the reactions of bioconjugation antibody modification and conjugation nucleic acid and oligonucleotide modification and conjugation quantum dot bioconjugates for imaging, labelling and sensing magnetic nanoparticles: synthesis, protection, functionalization, and application bioconjugation of quantum dots: review & impact on future application chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy functional targets for bioconjugation sensitive method for biomolecule detection utilizing signal amplification with porphyrin nanoparticles retroreflective janus microparticle as a nonspectroscopic optical immunosensing probe applications of orthogonal "click" chemistries in the synthesis of functional soft materials bioorthogonal chemistry amplifies nanoparticle binding and enhances the sensitivity of cell detection growing applications of "click chemistry" for bioconjugation in contemporary biomedical research click chemistry in complex mixtures: bioorthogonal bioconjugation strept)avidin−biotin systems accurate quantification of nucleic acids using hypochromicity measurements in conjunction with uv spectrophotometry how antibody surface coverage on nanoparticles determines the activity and kinetics of antigen capturing for biosensing techniques for physicochemical characterization of nanomaterials zeta potential − what they are and what they are not? time evolution of the nanoparticle protein corona protein-nanoparticle interactions poly-hetero-ω-functionalized alkanethiolate-stabilized gold cluster compounds multifunctional nanoparticles as simulants for a gravimetric immunoassay ph-dependent protein conformational changes in albumin:gold nanoparticle bioconjugates: a spectroscopic study present and future of surface plasmon resonance biosensors label-free methods of reporting biomolecular interactions by optical biosensors quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactions gold nanoparticles in chemical and biological sensing size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles tailoring surface plasmons through the morphology and assembly of metal nanoparticles colorimetric biosensing using smart materials the use of gold nanoparticles in diagnostics and detection gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity gold nanoparticles in biomedical applications: recent advances and perspectives the role of nanogold in human tropical diseases: research, detection and therapy development of a histidine-targeted spectrophotometric sensor using ni(ii)nta-functionalized au and ag nanoparticles multicolored silver nanoparticles for multiplexed disease diagnostics: distinguishing dengue, yellow fever, and ebola viruses multiplexed colorimetric detection of kaposi's sarcoma associated herpesvirus and bartonella dna using gold and silver nanoparticles multiplex paper-based colorimetric dna sensor using pyrrolidinyl peptide nucleic acid-induced agnps aggregation for detecting mers-cov, mtb, and hpv oligonucleotides highly selective and sensitive nucleic acid detection based on polysaccharide-functionalized silver nanoparticles fluorescent silver nanoparticle based highly sensitive immunoassay for early detection of hiv infection one-pot synthesis of luminol functionalized silver nanoparticles with chemiluminescence activity for ultrasensitive dna sensing ultratrace voltammetric method for the detection of dna sequence related to human immunodeficiency virus type 1 development of an electrochemical surface-enhanced raman spectroscopy (ec-sers) aptasensor for direct detection of dna hybridization biosensing with plasmonic nanosensors surface-enhanced raman scattering in local optical fields of silver and gold nano-aggregatesfrom single-molecule raman spectroscopy to ultrasensitive probing in live cells direct detection of malaria infected red blood cells by surface enhanced raman spectroscopy tailored periodic si nanopillar based architectures as highly sensitive universal sers biosensing platform silver nanoparticle−oligonucleotide conjugates based on dna with triple cyclic disulfide moieties nanozyme-strip for rapid local diagnosis of ebola a sensor tip based on carbon nanotube-ink printed electrode for the dengue virus ns1 protein sensing colorimetric approaches based on gold and silver nanoparticles aggregation: chemical creativity behind the assay simple silver nanoparticle colorimetric sensing for copper by paper-based devices point-of-care nucleic acid testing for infectious diseases based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device quantum dots for live cells semiconductor clusters, nanocrystals, and quantum dots semiconductor nanocrystals: a powerful visual aid for introducing the particle in a box electron−electron and electron-hole interactions in small semiconductor crystallites: the size dependence of the lowest excited electronic state semiconductor nanocrystals: structure, properties, and band gap engineering convergence of quantum dot barcodes with microfluidics and signal processing for multiplexed high-throughput infectious disease diagnostics semiconductor nanocrystals as fluorescent biological labels quantum dot bioconjugates for ultrasensitive nonisotopic detection biocompatible quantum dots for biological applications dual-signal readout nanospheres for rapid point-of-care detection of ebola virus glycoprotein a sandwich-hybridization assay for simultaneous determination of hiv and tuberculosis dna targets based on signal amplification by quantum dots-powervision polymer coding nanotracers quantum dots nanoparticle-based lateral flow assay for rapid detection of mycobacterium species using anti-fpra antibodies automating quantum dot barcode assays using microfluidics and magnetism for the development of a point-of-care device quantum dots: a new tool for anti-malarial drug assays fabrication of metal nanoshell quantum-dot barcodes for biomolecular detection nanoparticle-based histidine-rich protein-2 assay for the detection of the malaria parasite plasmodium falciparum integrated magnetic bead−quantum dot immunoassay for malaria detection a fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip quantum dot enabled detection of escherichia coli using a cell-phone multiplexed toxin analysis using four colors of quantum dot fluororeagents mediated multicolor immunochromatographic rapid test strip for visual and instrumental simultaneous detection of vibrio cholera o1 (ogawa) and clostridium botulinum toxin a nanotechnology diagnostics for infectious diseases prevalent in developing countries recent progress in the bioconjugation of quantum dots avidin: a natural bridge for quantum dot-antibody conjugates conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunoassays integrated quantum dot barcode smartphone optical device for wireless multiplexed diagnosis of infected patients toward point-of-care diagnostics with consumer electronic devices: the expanding role of nanoparticles multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots early diagnosis of febrile illness: the need of the hour challenges in the etiology and diagnosis of acute febrile illness in children in lowand middle-income countries luminescent lanthanide reporters: new concepts for use in bioanalytical applications europium enabled luminescent nanoparticles for biomedical applications recent advances in nanoparticle-based lateral flow immunoassay as a point-of-care diagnostic tool for infectious agents and diseases rapid and sensitive lateral flow immunoassay method for procalcitonin (pct) based on time-resolved immunochromatography lanthanide chemistry: from coordination in chemical complexes shaping our technology to coordination in enzymes shaping bacterial metabolism lanthanide nanoparticles: from design toward bioimaging and therapy nanoparticle labels in immunosensing using optical detection methods thermo scientific dyed and fluorescent particles brochure sm(iii), and dy(iii) lanthanide chelate nanoparticle labels europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostate-specific antigen improvement of a rapid diagnostic application of monoclonal antibodies against avian influenza h7 subtype virus using europium nanoparticles human infection with avian influenza a h7n9 virus: an assessment of clinical severity rapid detection of avian influenza virus by fluorescent diagnostic assay using an epitope-derived peptide novel immunochromatographic assay based on eu (iii)-doped polystyrene nanoparticle-linker-monoclonal antibody for sensitive detection of escherichia coli o157:h7 chelate microparticle-based lateral flow immunoassay strips for rapid and quantitative detection of antibody to hepatitis b core antigen. sci. rep. 2017 evaluation of sofia fluorescent immunoassay analyzer for influenza a/b virus detection of influenza a and b viruses with the sofia analyzer: a novel, rapid immunofluorescence-based in vitro diagnostic device sensitivity of the quidel sofia fluorescent immunoassay compared with 2 nucleic acid assays and viral culture to detect pandemic influenza a(h1n1)pdm09 point-counterpoint: can newly developed, rapid immunochromatographic antigen detection tests be reliably used for the laboratory diagnosis of influenza virus infections? evaluation of the sofia influenza a + b fluorescent immunoassay for the rapid diagnosis of influenza a and b a prospective study to assess the diagnostic performance of the sofia® immunoassay for influenza and rsv detection highly sensitive and novel point-of-care system systematic reviews of point-of-care tests for the diagnosis of urogenital chlamydia trachomatis infections autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis time-resolved luminescent lateral flow assay technology a low-cost smartphone-based platform for highly sensitive point-of-care testing with persistent luminescent phosphors lateral flow immunoassay using europium chelate-loaded silica nanoparticles as labels multiple fluorescent labeling of silica nanoparticles with lanthanide chelates for highly sensitive time-resolved immunofluorometric assays upconversion nanomaterials: synthesis, mechanism, and applications in sensing sensing using rare-earth-doped upconversion nanoparticles infrared up-converting phosphors for bioassays detection of analytes by immunoassay using up-converting phosphor technology upconversion nanoparticles: design, nanochemistry, and applications in theranostics up-converting phosphor technology-based lateral flow assay for detection of schistosoma circulating anodic antigen in serum a robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of schistosoma circulating anodic antigen tools for diagnosis, monitoring and screening of schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels an ultra-sensitive assay targeting the circulating anodic antigen for the diagnosis of schistosoma japonicum in a low-endemic area, people's republic of china improved sensitivity of the urine caa lateral-flow assay for diagnosing active schistosoma infections by using larger sample volumes. parasites vectors new diagnostic tools in schistosomiasis evaluation of banked urine samples for the detection of circulating anodic and cathodic antigens in schistosoma mekongi and s. japonicum infections: a proof-of-concept study utilizing the ultrasensitive schistosoma up-converting phosphor lateral flow circulating anodic antigen (ucp-lf caa) assay for sample pooling-strategies feasibility of a lateral flow test for neurocysticercosis using novel up-converting nanomaterials and a lightweight strip analyzer multi-center evaluation of a user-friendly lateral flow assay to determine ip-10 and ccl4 levels in blood of tb and non-tb cases in africa use of lateral flow assays to determine ip-10 and ccl4 levels in pleural effusions and whole blood for tb diagnosis a user-friendly, highly sensitive assay to detect the ifn-γ secretion by t cells lateral flow assay for simultaneous detection of cellular-and humoral immune responses field-evaluation of a new lateral flow assay for detection of cellular and humoral immunity against mycobacterium leprae field-friendly test for monitoring multiple immune response markers during onset and treatment of exacerbated immunity in leprosy rapid quantitative detection of yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor development of an up-converting phosphor technology-based 10-channel lateral flow assay for profiling antibodies against yersinia pestis rapid and quantitative detection of brucella by up-converting phosphor technology-based lateral-flow assay upconversion fluorescent strip sensor for rapid determination of vibrio anguillarum development of up-converting phosphor technology-based lateral-flow assay for rapidly quantitative detection of hepatitis b surface antibody evaluation of uplink− rsv: prototype rapid antigen test for detection of respiratory syncytial virus infection rapid assay format for multiplex detection of humoral immune responses to infectious disease pathogens evaluation of up-converting phosphor technology-based lateral flow strips for rapid detection of bacillus anthracis spore, brucella spp., and yersinia pestis detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology up-converting phosphor reporters for nucleic acid microarrays dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of salmonella typhimurium and staphylococcus aureus simultaneous aptasensor for multiplex pathogenic bacteria detection based on multicolor upconversion nanoparticles labels a hand-powered, portable, low-cost centrifuge for diagnosing anemia in low-resource settings hand-powered ultralow-cost paper centrifuge a handheld orbital mixer for processing viscous samples in low resource settings lanthanide-doped upconversion nano-bioprobes: electronic structures, optical properties, and biodetection setting up the cut-off level of a sensitive barcode lateral flow assay with magnetic nanoparticles study of superparamagnetic nanoparticles as labels in the quantitative lateral flow immunoassay development and evaluation of a magnetic immunochromatographic test to detect taenia solium, which causes taeniasis and neurocysticercosis in humans magnetic sensing platform technologies for biomedical applications magnetic nanoparticles: preparation, physical properties, and applications in biomedicine application of biomolecular recognition via magnetic nanoparticle in nanobiotechnology modeling and simulation of magnetic nanoparticle sensor biosensing using magnetic particle detection techniques magnetoresistive performance and comparison of supermagnetic nanoparticles on giant magnetoresistive sensor-based detection system lateral flow immunoassay using magnetoresistive sensors taeniasis/cysticercosis fact sheet diagnosis of taeniasis and cysticercosis antigen using magnetic immuno-chromatography (mict) rapid detection and differentiation of antibodies to hiv-1 and hiv-2 using multivalent antigens and magnetic immunochromatography testing development and evaluation of a paramagnetic nanoparticle based immunochromatographic strip for specific detection of 2009 h1n1 influenza virus effect of physiochemical property of fe 3 o 4 particle on magnetic lateral flow immunochromatographic assay a highly sensitive and flexible magnetic nanoprobe labeled immunochromatographic assay platform for pathogen vibrio parahaemolyticus a novel method to detect listeria monocytogenes via superparamagnetic lateral flow immunoassay rapid detection of bacillus anthracis spores using a super-paramagnetic lateral-flow immunological detectionsystem detection of bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on "road closure fundamentals and history of elisa: the evolution of the immunoassays until invention of elisa coupling of enzymes to proteins with glutaraldehyde: use of the conjugates for the detection of antigens and antibodies mysteries of metals in metalloenzymes practical guide to elisa development the reaction pathways of zinc enzymes and related biological catalysts structure and mechanism of alkaline phosphatase crystal structure of horseradish peroxidase c at 2.15 å resolution understanding the structure and function of catalases: clues from molecular evolution and in vitro mutagenesis bioinorganic chemistry enzyme immunoassay techniques: an overview formats and applications of lateral flow assay: a literature review point-of-care testing for infectious diseases: past, present, and future paper-based elisa cellphone-based hand-held microplate reader for point-of-care testing of enzyme-linked immunosorbent assays assessment of colorimetric amplification methods in a paper-based immunoassay for diagnosis of malaria folding analytical devices for electrochemical elisa in hydrophobic r h paper fluidic timers for time-dependent, point-of-care assays on paper use of multiple colorimetric indicators for paper-based microfluidic devices long-term dry storage of an enzyme-based reagent system for elisa in point-of-care devices the denaturation and degradation of stable enzymes at high temperatures freeze-drying of nanoparticles: formulation, process and storage considerations why is trehalose an exceptional protein stabilizer? an analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose magneto immunoassays for plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles a sandwich hiv p24 amperometric immunosensor based on a direct gold electroplating-modified electrode electrochemical immunosensor for interferon-γ based on disposable ito detector and hrp-antibody-conjugated nano gold as signal tag enzyme-responsive nanoparticles for drug release and diagnostics plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth plasmonic elisa for the ultrasensitive detection of disease biomarkers with the naked eye alcohol dehydrogenase-catalyzed gold nanoparticle seed-mediated growth allows reliable detection of disease biomarkers with the naked eye plasmonic elisa for the detection of analytes at ultralow concentrations with the naked eye expanding horizons. rsc adv signal amplification using functional nanomaterials for biosensing label-free colorimetric detection of nucleic acids based on target-induced shielding against the peroxidase-mimicking activity of magnetic nanoparticles a novel colorimetric immunoassay utilizing the peroxidase mimicking activity of magnetic nanoparticles sensitive immunoassay for porcine pseudorabies antibody based on fluorescence signal amplification induced by cation exchange in cdse nanocrystals nanoparticle dissolution from the particle perspective: insights from particle sizing measurements tiny grains give huge gains: nanocrystal-based signal amplification for biomolecule detection nanomedicine: tiny particles and machines give huge gains the determination of trace metal pollutants in environmental matrices using ion chromatography use of a chelating agent to determine the metal availability for leaching from soils and wastes. waste manage cation exchange reactions in ionic nanocrystals cation exchange on the nanoscale: an emerging technique for new material synthesis, device fabrication, and chemical sensing cation exchange in znse nanocrystals for signal amplification in bioassays cation exchange in aptamer-conjugated cdse nanoclusters: a novel fluorescence signal amplification for cancer cell detection tagging the rolling circle products with nanocrystal clusters for cascade signal increase in the detection of mirna chemiluminescence detection of dna/microrna based on cation-exchange of cus nanoparticles and rolling circle amplification high sensitive detection method for protein by combining the magnetic separation with cation exchange based signal amplification redox enzyme-mimicking activities of ceo 2 nanostructures: intrinsic influence of exposed facets solids go bio: inorganic nanoparticles as enzyme mimics metal-based nanomaterials for nanozymes nanozymes: gold-nanoparticle-based transphosphorylation catalysts intrinsic peroxidase-like activity of ferromagnetic nanoparticles a sensitive and rapid immunoassay for mycoplasma pneumonia based on fe 3 o 4 nanoparticles detection of vibrio cholerae using the intrinsic catalytic activity of a magnetic polymeric nanoparticle rapid and visual detection of listeria monocytogenes based on nanoparticle cluster catalyzed signal amplification ultrasensitive detection of viable enterobacter sakazakii by a continual cascade nanozyme biosensor colorimetric detection system for salmonella typhimurium based on peroxidase-like activity of magnetic nanoparticles with dna aptamers amplified visual immunosensor integrated with nanozyme for ultrasensitive detection of avian influenza virus simple and sensitive point-of-care bioassay system based on hierarchically structured enzyme-mimetic nanoparticles nanozyme-based bio-barcode assay for high sensitive and logic-controlled specific detection of multiple dnas an enzyme-free signal amplification technique for ultrasensitive colorimetric assay of disease biomarkers platinum-decorated gold nanoparticles with dual functionalities for ultrasensitive colorimetric in vitro diagnostics nanozyme-mediated dual immunoassay integrated with smartphone for use in simultaneous detection of pathogens a colorimetric immunoassay for respiratory syncytial virus detection based on gold nanoparticles−graphene oxide hybrids with mercury-enhanced peroxidase-like activity copper-based metal−organic framework nanoparticles with peroxidase-like activity for sensitive colorimetric detection of staphylococcus aureus pt-decorated magnetic nanozymes for facile and sensitive point-of-care bioassay platinum nanocatalyst amplification: redefining the gold standard for lateral flow immunoassays with ultrabroad dynamic range paper-based microfluidic point-of-care diagnostic devices nanoparticles for diagnostics: advances towards points of care silver acetate autometallography: an alternative enhancement technique for immunogold-silver staining (igss) and silver amplification of gold, silver, mercury and zinc in tissues development of a highly sensitive immunochromatographic detection kit for h5 influenza virus hemagglutinin using silver amplification use of immunogold with silver enhancement rapid, single-step immunochromatographic procedure for the detection of mouse immunoglobulin clinical evaluation of highly sensitive silver amplification immunochromatography systems for rapid diagnosis of influenza a lateral flow assay for quantitative detection of amplified hiv-1 rna two-dimensional paper network format that enables simple multistep assays for use in low-resource settings in the context of malaria antigen detection dissolvable fluidic time delays for programming multi-step assays in instrument-free paper diagnostics tunable-delay shunts for paper microfluidic devices highly sensitive immunoassay based on controlled rehydration of patterned reagents in a 2-dimensional paper network a versatile valving toolkit for automating fluidic operations in paper microfluidic devices investigation of reagent delivery formats in a multivalent malaria sandwich immunoassay and implications for assay performance rapid detection of yersinia pestis recombinant fraction 1 capsular antigen colorimetric detection of escherichia coli based on the enzyme-induced metallization of gold nanorods development of highly sensitive immunochromatographic detection kit for seasonal influenza virus using silver amplification chemical signal amplification in two-dimensional paper networks enhanced sensitivity of lateral flow tests using a two-dimensional paper network format immunogold− silver staining-on-a-chip biosensor based on cross-flow chromatography dual enlargement of gold nanoparticles: from mechanism to scanometric detection of pathogenic bacteria quantitative electrical detection of immobilized protein using gold nanoparticles and gold enhancement on a biochip silver and gold enhancement methods for lateral flow immunoassays enhancement of an immunoassay using platinum nanoparticles and an optical detection an integrated approach to a portable and low-cost immunoassay for resource-poor settings bio-barcodes based on oligonucleotide-modified nanoparticles nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins bio-bar-code-based dna detection with pcr-like sensitivity ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based dna amplification an improved gold nanoparticle probe-based assay for hcv core antigen ultrasensitive detection nanoparticle-based biobarcode amplification assay (bca) for sensitive and early detection of human immunodeficiency type 1 acquired immune defic fluorescent bio-barcode dna assay for the detection of salmonella enterica serovar enteritidis sandwich np-based biobarcode assay for quantification c-reactive protein in plasma samples colorimetric bio-barcode amplification assay for cytokines detection of proteins using a colorimetric bio-barcode assay multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes barcode dna-mediated signal amplifying strategy for ultrasensitive biomolecular detection on matrix-assisted laser desorption ionization time of flight (maldi-tof) mass spectrometry professional/infectious-diseases/laboratory-diagnosis-of-infectiousdisease/introduction-to-laboratory-diagnosis-of-infectious-disease (accessed asymptomatic malaria infections: detectability, transmissibility and public health relevance all-plastic miniature fluorescence microscope for point-of-care readout of bead-based bioassays all-plastic, miniature, digital fluorescence microscope for three part white blood cell differential measurements at the point of care field-portable pixel super-resolution colour microscope battery-operated, fluorescence field microscope for the developing world quality for biomedical technologies iii optical systems for point-of-care diagnostic instrumentation: analysis of imaging performance and cost field-portable lensfree tomographic microscope holographic pixel super-resolution in portable lensless on-chip microscopy using a fiber-optic array field-portable wide-field microscopy of dense samples using multi-height pixel super-resolution based lensfree imaging bright field and fluorescence microscope mobile digital fluorescence microscopy for diagnosis of tuberculosis point-of-care mobile digital microscopy and deep learning for the detection of soil-transmitted helminths and schistosoma haematobium foldscope: origami-based paper microscope diagnosis of schistosoma haematobium infection with a mobile phone-mounted foldscope and a reversed-lens cellscope in ghana mobile phone-based microscopy, sensing, and diagnostics evaluation of malaria diagnoses using a handheld light microscope in a community-based setting in rural cote d'ivoire mobile phone based clinical microscopy for global health applications evaluation of a mobile phone-based microscope for screening of schistosoma haematobium infection in rural ghana accuracy of mobile phone and handheld light microscopy for the diagnosis of schistosomiasis and intestinal protozoa infections in cote d'ivoire mobile phone microscopy for the diagnosis of soil-transmitted helminth infections: a proof-of-concept study point-of-care quantification of blood-borne filarial parasites with a mobile phone microscope cost-effective and compact wide-field fluorescent imaging on a cell-phone low-cost mobile phone microscopy with a reversed mobile phone camera lens handheld and portable reader devices for lateral flow immunoassays lateral flow immunoassay systems: evolution from the current state of the art to the next generation of highly sensitive, quantitative rapid assays lateral flow reader systems > rds-1500 pro detekt rds-2500 brochure clinical performance of an automated reader in interpreting malaria rapid diagnostic tests in tanzania field evaluation of an automated rdt reader and data management device for plasmodium falciparum/ plasmodium vivax malaria in endemic areas of colombia point-of-need diagnostics: biosurveillance with a device2cloud capability in sierra leone evaluation of the deki reader, an automated rdt reader and data management device, in a household survey setting in low malaria endemic southwestern uganda integrated rapid-diagnostic-test reader platform on a cellphone mobile phone imaging and cloud-based analysis for standardized malaria detection and reporting an embedded barcode for "connected" malaria rapid diagnostic tests google analytics and quick response for advancement of gold nanoparticle-based dual lateral flow immunoassay for malaria − plasmodium lactate dehydrogenase (pldh) immunochromatographic diagnostic test analysis using google glass multimode smartphone biosensing: the transmission, reflection, and intensity spectral (tri)-analyzer smartphone-based portable wireless optical system for the detection of target analytes surface plasmon resonance chemical sensing on cell phones a smartphone based surface plasmon resonance imaging (spri) platform for on-site biodetection lipopolysaccharides detection on a grating-coupled surface plasmon resonance smartphone biosensor optofluidic fluorescent imaging cytometry on a cell phone 3d-printed smartphone-based point of care tool for fluorescenceand magnetophoresis-based cytometry thermophysical and biological responses of gold nanoparticle laser heating significantly improved analytical sensitivity of lateral flow immunoassays by using thermal contrast thermal contrast amplification reader yielding 8-fold analytical improvement for disease detection with lateral flow assays multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contrast the role of nanoparticle design in determining analytical performance of lateral flow immunoassays the development of surface-enhanced raman scattering as a detection modality for portable in vitro diagnostics: progress and challenges materials, applications, and the future. mater. today separation and detection of multiple pathogens in a food matrix by magnetic sers nanoprobes sers driven cross-platform based multiplex pathogen detection fabrication of magnetic gold nanorod particles for immunomagnetic separation and sers application sers-based sandwich immunoassay using antibody coated magnetic nanoparticles for escherichia coli enumeration multifunctional magnetic−plasmonic nanoparticles for fast concentration and sensitive detection of bacteria using sers detection of mycobacterium avium subsp. paratuberculosis by a sonicate immunoassay based on surface-enhanced raman scattering surface-enhanced raman scattering-based immunoassay technologies for detection of disease biomarkers thermo scientific firstdefender rmx handheld chemical identification pricing chemical identification a sers-based lateral flow assay biosensor for highly sensitive detection of hiv-1 dna application of a sers-based lateral flow immunoassay strip for the rapid and sensitive detection of surface-enhanced raman scattering based lateral flow immunochromatographic assay for sensitive influenza detection simultaneous detection of dual nucleic acids using a sers-based lateral flow assay biosensor au@ag serrs tags coupled to a lateral flow immunoassay for the sensitive detection of pneumolysin surface-enhanced raman spectroscopy-based sandwich immunoassays for multiplexed detection of zika and dengue viral biomarkers multiplexed detection with magnetic nanoparticles nanomaterials for the life sciences magnetoresistive-based biosensors and biochips contactless measurement of magnetic nanoparticles on lateral flow strips using tunneling magnetoresistance (tmr) sensors in differential configuration gmr sensors and magnetic nanoparticles for immuno-chromatographic assays a giant magnetoresistive reader platform for quantitative lateral flow immunoassays increase in the detection sensitivity of a lateral flow assay for a cardiac marker by oriented immobilization of antibody electrochemical glucose sensors and their applications in diabetes management a history of blood glucose meters and their role in self-monitoring of diabetes mellitus using personal glucose meters and functional dna sensors to quantify a variety of analytical targets using commercially available personal glucose meters for portable quantification of dna portable and quantitative detection of protein biomarkers and small molecular toxins using antibodies and ubiquitous personal glucose meters detection of protein biomarker using a blood glucose meter. in mobile health technologies integration of solution-based assays onto lateral flow device for one-step quantitative point-of-care diagnostics using personal glucose meter connecting for health: global vision, local insight; world health organization: geneva rapid electrochemical detection on a mobile phone electrochemical tattoo biosensors for real-time noninvasive lactate monitoring in human perspiration tattoo-based noninvasive glucose monitoring: a proof-of-concept study capture of the circulating plasmodium falciparum biomarker hrp2 in a multiplexed format, via a wearable skin patch paper-based electrical respiration sensor proteomic profiling of eccrine sweat reveals its potential as a diagnostic biofluid for active tuberculosis needle-free vaccinations in skin using multilayered, densely-packed dissolving microprojection arrays surface modifications of microprojection arrays for improved biomarker capture in the skin of live mice dissolving microneedle vaccine delivery systems vaccine efficacy of transcutaneous immunization with amyloid β using a dissolving microneedle array in a mouse model of alzheimer's disease towards pain-free diagnosis of skin diseases through multiplexed microneedles: biomarker extraction and detection using a highly sensitive blotting method dynamic application of microprojection arrays to skin induces circulating protein extravasation for enhanced biomarker capture and detection a three-dimensional and bevel-angled ultrahigh aspect ratio microneedle for minimally invasive and painless blood sampling in-depth interviews to understand the feasibility of using the mlab app for promotion of hiv-self testing in young men esequant lateral flow system technical flyer 1070062 towards a genomics-informed, real-time, global pathogen surveillance system single quantum dot-based nanosensor for multiple dna detection development of a microfluidic device for detection of pathogens in oral samples using upconverting phosphor technology (upt) an integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids development of a generic microfluidic device for simultaneous detection of antibodies and nucleic acids in oral fluids use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: a rapid, sensitive dna test to identify human papillomavirus type 16 infection improved assay to detect plasmodium falciparum using an uninterrupted, semi-nested pcr and quantitative lateral flow analysis a disposable microfluidic cassette for dna amplification and detection a portable analyzer for pouch-actuated, immunoassay cassettes isothermal amplification methods for the detection of nucleic acids in microfluidic devices evaluation of non-instrumented nucleic acid amplification by loop-mediated isothermal amplification (nina-lamp) for the diagnosis of malaria in northwest ethiopia high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (htlamp) assay for malaria elimination evaluation of the illumigene malaria lamp: a robust molecular diagnostic tool for malaria parasites nina-lamp compared to microscopy, rdt, and nested pcr for the detection of imported malaria electricity-free amplification device for detection of hiv-1 a fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics paper-based molecular diagnostic for chlamydia trachomatis paper-based rna extraction, in situ isothermal amplification, and lateral flow detection for low-cost, rapid diagnosis of influenza a (h1n1) from clinical specimens paper machine" for molecular diagnostics colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (nina-lamp) a novel snp-lamp assay for detection of artemisinin-resistant plasmodium falciparum malaria noninstrumented nucleic acid amplification assay. in microfluidics, biomems, and medical microsystems vi key: cord-337720-kmwft059 authors: closson, kalysha; lee, melanie; gibbs, andrew; kaida, angela title: when home is not a safe place: impacts of social distancing directives on women living with hiv date: 2020-06-02 journal: aids behav doi: 10.1007/s10461-020-02941-y sha: doc_id: 337720 cord_uid: kmwft059 nan reproductive health services, which overwhelmed the judicial system, and resulted in a 75% increase in maternal mortality [2] . while health care systems face mounting pressure and resource strains due to covid-19, this must not be the time to divert resources away from essential services that support and protect women who have experienced or are experiencing ipv [2] . we have learned many lessons in the global effort to end hiv/aids, including that emerging epidemics such as covid-19 exacerbate and exploit existing inequities of gender, gender identity, ethnicity, sexuality, income, age, and ability, and disproportionately affect those at the margins [7] . moreover, we have seen that the social, psychological and economic impacts of the pandemic will be felt for long after the peak of infections have subsided [8] . women living with hiv disproportionately experience multiple intersecting inequities including high levels of ipv, food insecurity and unstable housing [7, 9] , thus there is a particular concern that 'stay home' regulations may be both unattainable and unsafe for many women living with hiv [7, 9] . global evidence suggests that up to 86% of women living with hiv have experienced some form of gender-based violence in their lifetime [10] . in the us, estimates suggest that the lifetime prevalence of ipv among women living with hiv is double (~ 55%) that of women not living with hiv [11] . this is concerning, as experiences of ipv among women living with hiv have been associated with barriers to hiv care, including lower levels of treatment adherence, and a reduced likelihood of achieving viral suppression [12, 13] . previous research has also highlighted the negative mental and physical health impacts of ipv experiences among women living with hiv [10] . furthermore, recent canadian data suggests that historical experiences of severe * kalysha closson kalyshaclosson@gmail.com ipv, where women have reported lifetime histories of multiple forms (e.g. sexual, physical and emotional) of ipv, may increase women living with hiv's likelihood of dying [14] . a growing body of literature suggests that experiences of violence may alter women's important immune mediators, thereby increasing one's risk to stis, hiv, as well as women living with hiv's ability to suppress hiv in the body [15, 16] . thus, the negative physical health effects of ipv among women living with and at risk of hiv may be in part due to biological responses to violence and trauma. current 'stay home' regulations coupled with increased household and economic stressors, as well as elevated fear of acquiring covid-19 during this time may give rise to opportunities for heightened surveillance and control for abusive partners [1, 17] . as hiv care, research participation, and workplace settings are being transitioned to virtual and telephone-based methods, women living with hiv experiencing violence are less able to connect to critical social and protective networks [18] . as such, necessary social distancing measures have the potential to impact the rates and consequences of ipv, increasing social isolation and mental health concerns, which taken together can hinder women living with hiv's access to, and use of, hiv treatment and violence support, further than they already experience [9, 17] . as part of social distancing requirements in many settings, routine hiv care is being offered via telemedicine [7, 19] . for women who may not have disclosed their hiv status to their partners, this form of care provision, although necessary during these times, may not be a feasible option for many women living with hiv. virtual care provides greater opportunity for disclosing women's hiv status, reduces women's ability to disclose experiences of violence to providers for fear of their abuser overhearing, thus increasing their risk of further experiences of violence [9] . our previous research has found that among a sample of women living with hiv in british columbia, canada, 59% experienced some form of ipv in their lifetime, of those only 12.4% sought violence support [14] . more recent data from the canadian hiv women's sexual and reproductive health cohort study (chiwos), showed 80% of participants reported experiencing violence as an adult, 42% of whom reported seeking help for violence. among chiwos participants who reported seeking help, the most commonly accessed form of help to cope with violence was through health care providers [20] . in other low-income settings, where telemedicine is unlikely to occur, women living with hiv may still struggle to access medication, as their normal collection points, often far from their communities, are no longer accessible, and male partner surveillance of their movement may have increased. as the health care system, and hiv care, may be the first line of violence support for many women living with hiv, during these unprecedented times, it is especially important that hiv care providers take a women-centred trauma-aware approach, which is led by the needs and priorities of their patients [21, 22] . this can help to ensure privacy and address concerns regarding limited mobility and access to medication. there is limited research on how to support women living with hiv experiencing ipv [23] , and the best approaches are particularly unclear during the covid-19 restrictions. future research can learn from the years of community efforts aimed at reducing ipv and hiv [24] [25] [26] , including ensuring efforts are grounded in community-based responses allowing for the meaningful participation of women in all their diversities [7, 9] . research is needed to examine how the covid-19 pandemic is resulting in increased experiences of ipv, the impact of ipv during covid-19 on women living with hiv, and crucially how best to respond to, and prevent, ipv for all women, and women living with hiv in particular. as social distancing measures limit access to supports, such as family, friends, and health care provides, that help women living with hiv cope with experiences of violence and histories of trauma, research is needed to understand the unique ways in which women living with hiv have developed resilience and coping strategies during covid-19 restrictions and how these can be best supported. these results can inform future strategies to reduce experiences of ipv during emergency situations and public health crises [23] . this research will be critical to supporting women living with hiv's healing in the aftermath of the covd-19 pandemic. however, this research will not be easy, and will come with many challenges. for example, virtual data collection while confinement measures are in place may lead to communication being discovered by abusers and thus increase women's risk of violence [23] . and in the global south, virtual technologies, connectivity and data are often incredibly limited. additional methods to reach those most at risk of violence, including women who use drugs, who are unstably housed, and who live in areas of conflict, need to be explored [18] . as innovative technologies and responses are rolled out, there is a critical need for additional guidance on how to safely collect such data in these circumstances [23] . we have limited evidence on the impact of covid-19 on people living with hiv [27] , and how covid-19 and relevant responses to the pandemic impact experiences of ipv. the intersections of the co-occurring pandemics of covid-19 and ipv are critical to the health and wellbeing of women living with hiv. as we continue to practice important social distancing measures to reduce the spread of covid-19, it is vital that efforts are implemented to protect those most vulnerable to the virus and the associated adverse consequences of the public health response. this will include continued and accelerated advocacy for stronger judicial and government policies that ensure the protection of all women, including those living with hiv, who may be at particularly high risk of experiencing violence during global covid-19 lockdowns. by placing women's safety at the center of the covid-19 response, we can recommit to the global goals of ending both aids and gender-based violence. a new covid-19 crisis: domestic abuse rises worldwide. the new york times lessons never learned: crisis and gender-based violence domestic violence cases surge during covid-19 epidemic sixth alarming trends in us domestic violence during the covid-19 pandemic global affairs canada-the equity fund: transforming the way we support women's organizations and movements working to advance women's rights and gender equality global affairs canada. canada launches new feminist international assistance policy the burden of covid-19 in people living with hiv: a syndemic perspective editorial: managing the march of covid-19: lessons from the hiv and aids epidemic covid-19: increased risk to the mental health and safety of women living with hiv in south africa violence enough already": findings from a global participatory survey among women living with hiv psychological trauma and ptsd in hiv-positive women: a meta-analysis intimate partner violence and engagement in hiv care and treatment among women: a systematic review and meta-analysis impact of intimate partner violence on clinic attendance, viral suppression and cd4 cell count of women living with hiv in an urban clinic setting severe intimate partner violence is associated with all-cause mortality among women living with hiv chronic immune barrier dysregulation among women with a history of violence victimization impact of chronic sexual abuse and depression on inflammation and wound healing in the female reproductive tract of hiv-uninfected and hiv-infected women physical distancing in covid-19 may exacerbate experiences of social isolation among people living with hiv violence against women during covid-19 pandemic restrictions covid-19 pandemic disrupts hiv continuum of care and prevention: implications for research and practice concerning community-based organizations and frontline providers help-seeking after experiences of violence among women living with hiv in canada: what are we missing? 28th annual canadian association for hiv research (cahr) annual conference. sk, canada: saskatoon the syndemic illness of hiv and trauma: implications for a trauma-informed model of care women-specific hiv/ aids services: identifying and defining the components of holistic service delivery for women living with hiv/aids united nations women, world health organization. violence against women and girls data collection during covid-19: united nations women gender and sexuality: emerging perspectives from the heterosexual epidemic in south africa and implications for hiv risk and prevention effective hiv prevention requires gender-transformative work with men violence against women: an emerging health problem key: cord-340489-yo3cp5vs authors: nan title: kapitel 13 infektionskrankheiten date: 2008-12-31 journal: innere medizin doi: 10.1016/b978-3-437-42831-9.10013-0 sha: doc_id: 340489 cord_uid: yo3cp5vs zur orientierung infektionskrankheiten werden durch pathogene verursacht, die sich im wirt vermehren: ektoparasiten, helminthen, protozoen, pilze, bakterien, viren, prionen. infektionskrankheiten können alle organe bzw. organsysteme befallen. entstehung und verlauf werden durch faktoren beeinflusst, die sich grob einteilen lassen in erregerund wirtsfaktoren. die kenntnis und richtige einschätzung dieser faktoren sind entscheidend für diagnostik und therapie dieser erkrankungen. oft ist die unterscheidung zwischen infektiösen und nichtinfektiösen erkrankungen mit einer entzündlichen komponente schwer. andererseits können z. b. bei patienten mit immundefekten symptome oder zeichen trotz bestehender infektion fehlen. hautveränderungen als symptom viele infektionskrankheiten zeigen eine mitbeteiligung der haut, mit fokalen läsionen bei bakterieller endokarditis oder als exanthem bzw. enanthem bei viruserkrankungen. hautveränderungen können pathognomonisch sein, so bei meningokokkensepsis und den damit verbundenen petechialen blutungen und später großflächigen ekchymosen. hautveränderungen, auch im zeitlichen verlauf, können wichtige differentialdiagnostische hinweise liefern. der erste schritt ist die beurteilung des klinischen zustandes (› abb. 13.1). bei kritisch kranken patienten muss zunächst parallel zu supportiven maßnahmen eine rasche empirische antiinfektive therapie erwogen und ggf. begonnen werden. die diagnostik kann aber meist ohne zeitverlust in den ablauf integriert werden. infektionskrankheiten, die einen kritischen zustand eines nicht immundefizienten patienten verursachen, sind vor allem bakterielle sepsis, meningitis und bakterielle pneumonie, aber auch malaria tropica. anamnese hier sind mögliche erregerexpositionen, der zeitliche ablauf der erkrankung und die prädisposition des wirts zu beachten. • nicht für alle infektionen ist eine besondere exposition eruierbar (› tab. 13 .2). kontakte zu anderen erkrankten, reiseanamnese, nahrungsaufnahme, berufsanamnese, freizeitbeschäftigungen, tierkontakte inklusive insektenstiche, vorherige erkrankungen und deren therapie, medikamenten-, drogen-sowie sexualanamnese müssen berücksichtigt werden. • der zeitliche ablauf der krankheitsentwicklung muss geklärt werden. nahezu alle infektionserreger haben charakteristische zeitintervalle zwischen exposition und erkrankung (inkubationszeiten). so sind bakterielle erreger durch inkubationszeiten von einigen tagen gekennzeichnet, während viruserkrankungen meist inkubationszeiten von einigen wochen haben (viele ausnahmen!). zusätzlich treten bei vielen erregern saisonale häufungen auf. • die prädisposition des wirts als dritte komponente umfasst die frühere anamnese (speziell infektionen, vorgenommene impfungen), andere erkrankungen oder organschädigungen und die beurteilung des klinischen status, z. b. die integrität von haut und schleimhäuten. spezifische untersuchungen blutkulturen müssen bei allen kritisch kranken, bei allen systemisch kranken und/oder fiebernden patienten entnommen werden. hierbei sind eine rasche und frühe entnahme (vor antibiotikagabe), ausreichende blutmenge pro flasche (mind. 5-8 ml je nach system), eine ausreichende zahl von blutkulturen (mindestens zwei, je als aerob-anaerobes paar von blutkulturen) und sterile abnahme wichtig. die weitere diagnostik richtet sich nach den symptomen oder befunden, z. b. sputum-, urin-, abstrichuntersuchungen untersuchungen von organpunktaten. bei allen untersuchungen ist auf entnahmetechnik, aufbewahrung und richtigen transport zu achten. sputum z. b. muss vor der ersten gabe von antibiotika entnommen und innerhalb weniger stunden aufgearbeitet werden, ansonsten sind pathogene nicht mehr nachweisbar. der rationale einsatz von serologischen untersuchungen wie auch die aufbewahrung von serumproben zur späteren untersuchung von initial-und rekonvaleszentenserum kann zur diagnostik sinnvoll sein wie auch spezielle verfahren z. b. polymerasekettenreaktion-untersuchung einer biopsie. wirts-und pathogenitätsfaktoren beeinflussen entstehung und ablauf von infektionskrankheiten. wirtsfaktoren lassen sich einteilen in unspezifische (angeborene) und spezifische (erworbene, › tab. 13.3). zu den unspezifischen faktoren gehören barrieremechanismen von haut und schleimhäuten, aber auch mechanismen, mit denen erreger aktiv bekämpft werden können. spezifische funktionen des immunsystems sind gegen einzelne erreger gerichtet. beide systeme weisen eine vielzahl von interaktionen auf. mit pathogenität wird die eigenschaft eines mikroorganismus bezeichnet, eine erkrankung auslösen zu können. virulenz ist der grad der pathogenität innerhalb einer spezies. notwendige bedingungen für die pathogenität eines erregers sind: 1. in gewebe oder zellen anhaften oder eindringen zu können, 2. im körper zur replikation fähig zu sein. epithelbarrieren und -läsionen haut und schleimhaut haben mehrere barrieren, um ein eindringen von pathogenen zu erschweren oder vermeiden. dazu gehören neben den anatomischen auch chemische barrieren, z. b. die fettschicht der epidermis, aber auch antibakterielle substanzen, wie das in mukosalen sekreten vorhandene lysozym. monozyten und makrophagen, granulozyten und natural-killer(nk)-zellen sind die wichtigsten zellpopulationen dieses systems. die zellen des monozyten-makrophagen-systems und granulozyten weisen moleküle auf, mit denen erregerspezifische strukturen (z. b. lipopolysaccharide, bestimmte dna-sequenzen oder doppelsträngige rna) erkannt werden können. diese strukturen werden als pathogen-assoziierte molekulare pattern (pamp) bezeichnet. ein beispiel für solche rezeptoren sind die nach einer homologie mit einem rezeptor der drosophila-fliege bezeichneten tlr (toll-like-rezeptoren). die bindung von erregerspezifischen strukturen an diese moleküle führt zur raschen aktivierung einer entzündungskaskade. natural-killer-zellen sind lymphozyten, die durch antigen-antikörper-komplexe oder durch zellen, die keine mhc-(major-histocompatibility-complex)-moleküle auf der oberfläche exprimieren, aktiviert werden können. die proteine des komplementsystems können nach aktivierung im blut zirkulierende erreger lysieren. störungen des komplementsystems führen zu infektionen mit polysaccharidbekapselten erregern, z. b. pneumokokken und meningokokken. als akute-phase-reaktion wird die produktion von proteinen und peptiden bezeichnet, die nach einer infektion oder entzündungsreaktion abläuft. involviert sind makrophagen, die vor allem interleukin (il-1), tnf-α, interferon-α, il-6 und eine reihe von prostaglandinen und arachidonsäuremetaboliten produzieren. eine kooperation dieser beiden systeme findet bei jeder lokalen entzündungsreaktion statt. aktivierte granulozyten und makrophagen produzieren chemotaktische substanzen, aktivieren adhäsionsmoleküle in den lokalen endothelien und sorgen so für eine verstärke einwanderung von entzündungszellen (› abb. 13 defekte des unspezifischen immunsystems • verletzungen von epithelien begünstigen die invasion von erregern, z. b. nach zytostatischer therapie mit abschilferung der intestinalen schleimhaut. der aktive partikeltransport des flimmerepithels des respirationstraktes ist bei rauchern und bei patienten mit zystischer fibrose gestört. in beiden fällen ist die rate von infektionen des respirationstraktes deutlich erhöht. • neutropenie oder funktionsstörungen der granulozyten führen zu häufigen bakteriellen erkrankungen und invasiven mykosen. • das fehlen von natural-killer-zellen führt u. a. zu schweren infektionen mit herpesviren. die wesentlichen mechanismen sind die bildung von antikörpern durch b-zellen sowie spezifischer t-effektorzellen. beide zellsysteme haben eine hohe genetische plastizität und können eine hohe zahl spezifischer antigene erkennen. t-lymphozyten repräsentieren die spezifische zelluläre immunität. die wichtigsten funktionellen gruppen sind t-helferzellen und zytotoxische t-zellen. damit antigene durch die effektorzellen erkannt werden können, sind eine prozessierung und präsentierung durch zelluläre mechanismen notwendig. präsentation von antigen bakterielle oder virale proteine werden intrazellulär in oligopeptide zerlegt, und über mhc-(major-histocompatibility-complex)-moleküle auf der zelloberfläche präsentiert. "professionelle" antigenpräsentierende zellen nutzen mhc-klasse-ii-, alle anderen zellen mhc-klasse-i-moleküle. t-lymphozyten der kontakt von "naiven" t-helferzellen mit präsentiertem antigen führt zur aktivierung, reifung und klonalen expansion der betreffenden t-helferzellen. diese stimulieren die bildung von spezifischen zytotoxischen t-zellen und immunglobulinproduzierenden b-zellen. die zentrale rolle in der stimulierung der effektorzellen der spezifischen abwehr hat dieser zellgruppe ihren namen gegeben. die eigentlichen effektorzellen sind die zytotoxischen t-zellen. sie erkennen infizierte zellen durch die kombination der mhc-klasse-i-moleküle mit den erregerpeptiden und töten sie ab. die initale phagozytose von erregern und nachfolgende antigenpräsentation durch makrophagen mit der ausbildung einer spezifischen zellulären immunität durch t-lymphozyten ist ein beispiel für das zusammenwirken unspezifischer und spezifischer immunmechanismen. der kontakt des passenden antigens mit dem noch unreifen oberflächenimmunglobulin auf b-lymphoyzten führt zur ausbildung und selektion von plasmazellen, die entweder iga, ige oder igg mit höherer spezifität und avidität produzieren. immunglobuline können erreger (z. b. viren) und toxine neutralisieren und die phagozytose und lyse von erregern erleichtern. das spezifische immunsystem bildet außerdem ein gedächtnis für vorangegangene infektionen und reagiert mit einer besseren abwehr oder sogar immunität bei reexposition. beim erstkontakt mit dem erreger kommt es nach rascher aktivierung, reifung und expansion von t-und b-lymphozyten auch zur bildung von gedächtniszellen beider gruppen. defekte des spezifischen immunsystems können angeboren (agammaglobulinämie, kombiniertes immundefektsyndrom) und erworben (aids) sein. bei immunglobulinmangel kommt es vor allem zu infektionen mit polysaccharidbekapselten erregern, bei störungen des t-zellulären systems zu schweren infektionen z. b. mit intrazellulären erregern (z. b. toxoplasma gondii und herpesviren). viren sind obligat intrazelluläre erreger, die keinen eigenen stoffwechsel besitzen. zur replikation sind sie auf zelleigene enzyme angewiesen. zwei bedingungen müssen für virale pathogenität erfüllt sein: 1. das virus muss eine oberflächenstruktur besitzen, mit der es an eine zielzelle binden und dann eindringen kann. 2. durch die replikation in der zelle muss entweder eine störung der zellfunktion oder eine immunreaktion auf die infektion erfolgen. ein pathogenitätsmechanismus ist die möglichkeit, eine latente infektion zu erzeugen. so besitzen z. b. humane herpesviren die fähigkeit, das genom in einer inaktiven, aber reaktivierbaren form in zellen einzubauen. die mechanismen, die das gleichgewicht zwischen latenz und produktiver infektion steuern, sind nur unvollständig bekannt. die einfache kultivierung klonaler populationen und die manipulation von umgebungsbedingungen ermöglichen die untersuchung bakterieller pathogenitätsfaktoren. zusätzlich haben sequenzierung von bakteriellen genen und deren gezielte manipulation das wissen über pathogenitätsmechanismen entscheidend vermehrt. genetische regulation von pathogenitätsfaktoren neben der chromosomalen form kann dna als plasmid oder phage vorliegen. diese "mobilen" genetischen elemente erlauben eine genetische diversifizierung. chromosomale gene und externe gene können für pathogenitätsfaktoren kodieren, deren expression einer komplizierten regelung unterliegen kann. adhäsion, toxinbildung und immunevasion von bakterien wichtige pathogene mechanismen von bakterien sind adhäsion an epithelien oder die bildung von toxinen und immunevasion: • pili oder fimbrien bei escherichia coli werden nach dem identifizierten gen-p(ap)-pili genannt. die expression dieser pili wird durch umgebungsbedingungen (ph-wert und temperatur) so moduliert, dass sie vor allem in den ableitenden harnwegen produziert werden, wo sie zur besseren adhäsion führen. • häufig ist die produktion von toxinen -ebenso wie die von adhäsionsmechanismen -an plasmide oder phagen gebunden. zur produktion des diphtherietoxins muss der betreffende stamm mit einem phagen infiziert sein. andere pathogene toxine sind exotoxine von staphylococcus aureus und der gruppe-a-streptokokken, die z. b. für die toxic-shock-syndrome verantwortlich sind. • immunevasionsstrategien richten sich gegen abwehrmechanismen. beispiele: neisseria gonorrhoeae und haemophilus influenzae entziehen sich durch iga-spezifische proteasen der vernichtung auf der schleimhaut. legionella pneumophila kann nach beladung durch komplementproteine leichter in zellen eindringen. vor allem keime mit intrazellulärem vermehrungszyklus haben oft die fähigkeit, der phagozytose durch makrophagen zu entgehen. • das patientengut ändert sich mit einem ständig steigenden anteil an intensivpflegepatienten und abwehrgeschwächten patienten. • das repertoire an zur verfügung stehenden chemotherapeutika wird immer größer. • die resistenz von bakterien gegen antibakterielle chemotherapeutika nimmt sowohl quantitativ als auch qualitativ zu. jeder arzt, der eine antibakterielle chemotherapie durchführen will, muss wichtige grundprinzipien beherrschen wie auch in wesentlichen zügen das spektrum der antibakteriellen substanzen kennen. indikationsstellung antibakterielle chemotherapeutika sind ursächlich wirksame medikamente und nicht primär gegen symptome, wie z. b. fieber, gerichtet. die gabe solcher substanzen setzt also eine exakte indikationsstellung voraus, es muss mit sehr hoher wahr-scheinlichkeit eine durch bakterien verursachte infektionskrankheit vorliegen. die indikation wird naturgemäß zunächst klinisch gestellt. hierfür genügen in aller regel anamnese, befunde der klinischen untersuchung sowie einige klinisch-chemische und radiologische zusatzbefunde. gleichzeitig erfolgt die materialentnahme zur mikrobiologischen erregerdiagnose, um dadurch die indikation abzusichern. die therapie wird meist vor erhalt der endgültigen erregerdiagnose und des antibiogramms begonnen. man spricht dann von einer kalkulierten chemotherapie, d.h., es wird eine empirische therapie nur auf basis der klinischen befunde eingeleitet. hieraus sollte es in vielen fällen schon möglich sein, die zu erwartenden erreger einzugrenzen, aber auch die zu erwartende resistenzsituation sowohl generell als auch lokal zu kalkulieren. wenn dann mikrobiologische befunde -erregerdiagnose und antibiogramm -vorliegen, die mit der klinik korrelierbar sind, kann eine gezielte chemotherapie durchgeführt werden. das heißt, die kalkulierte chemotherapie muss überprüft und evtl. geändert werden. für die auswahl der chemotherapeutika müssen klinische, mikrobiologische und pharmakokinetische kriterien herangezogen werden. von seiten der klinik sind eventuelle grundkrankheiten zu berücksichtigen, ferner die infektionslokalisation und die tatsache, ob es sich um eine außerhalb (ambulant) oder innerhalb des krankenhauses (nosokomial) erworbene infektionskrankheit handelt. die zu beachtenden bakteriologischen kriterien betreffen das wirkspektrum der jeweiligen antibiotika und deren aktivität innerhalb dieses spektrums. entscheidend ist auch, ob der wirkeffekt bakterizid (keimabtötend) oder nur bakteriostatisch (proliferationshemmend) ist. eine ganze reihe von pharmakokinetischen eigenschaften der jeweiligen substanzen wie säurestabilität, enterale resorption, art der metabolisierung bzw. elimination, penetration in körperkompartimente und -gewebe beeinflusst ebenfalls in der individuellen klinischen situation die festlegung des chemotherapeutikaregimes. nicht zuletzt spielen toxikologische gesichtspunkte (s. u.) eine wichtige rolle. die durchführung der chemotherapie folgt im prinzip den allgemeinen grundsätzen der internistischen pharmakotherapie (› kap. 3), hier nur die zusätzlich besonderen aspekte der antibiotikatherapie. dosierung die dosis des chemotherapeutikums muss ausreichend hoch sein, um den gewünschten wirkeffekt sicher zu erreichen. dauer es lassen sich keine allgemein gültigen regeln aufstellen. so können unkomplizierte harnwegsinfektionen mit einer einmalgabe eines potenten antibiotikums behandelt werden, während für die therapie einer osteomyelitis eine mehrmonatige therapiedauer erforderlich sein kann. applikationsart die parenterale applikation stellt grundsätzlich den sichersten applikationsweg dar. bei schweren und schwersten infektionsverläufen ist daher dieser weg zumindest bei beginn der therapie immer zu wählen. bei einer umstellung von einer parenteralen auf eine orale therapie (= sequentialtherapie) ist darauf zu achten, ob dies mit der parenteral begonnenen substanz überhaupt möglich ist, d.h., ob sie enteral resorbierbar ist. eine orale folgetherapie mit einem anderen antibiotikum kann nur dann erfolgen, wenn es das gleiche spektrum wie das zuvor verwandte parenterale antibiotikum hat. für die orale chemotherapie ist die compliance des patienten entscheidend. applikationsintervall die pharmakodynamischen eigenschaften (= beziehung von serumspiegel, halbwertszeit und wirkaktivität gemäß minimaler hemmkonzentration) eines antibiotikums entscheiden über einmalgabe, mehrfachgabe oder dauergabe. die kombinationstherapie mit zwei oder mehreren substanzen hat in der kalkulierten chemotherapie zum ziel, ein breiteres spektrum möglicher erreger abzudecken. in der gezielten chemotherapie kann eine synergistische wirkung angestrebt werden, erwiesenermaßen sinnvoll nur für die gabe von β-lactam-antibiotika bzw. glykopeptidantibiotika mit aminoglykosiden bei grampositiven kokken als häufigste angewandte kombination. weitere gründe für die gabe einer kombination liegen vor, wenn die dosiserhöhung einer substanz aus toxikologischen gründen nicht mehr möglich ist oder bei einer mischinfektion mehrere erreger therapiert werden müssen. für die kombinierbarkeit verschiedener antibiotika gibt es keine verbindlichen regeln. "drug-monitoring" antibiotika, die bei nierenfunktionsstörungen schnell kumulieren können, wie aminoglykoside und vancomycin, müssen dann gemäß serumspiegelkontrolle dosiert werden. bezüglich allergischer und toxischer nebenwirkungen unterscheiden sich antibiotika nicht von anderen substanzen. je nach spektrum der potenziellen nebenwirkungen, das für die einzelnen chemotherapeutika sehr unterschiedlich sein kann, müssen entsprechende klinische bzw. laborchemische oder auch funktionelle kontrollen erfolgen. die besonderheiten einer antibakteriellen chemotherapie liegen darin, dass sog. biologische nebenwirkungen (folge der hauptwirkung) auftreten können: • selektion resistenter bakterien. das versagen einer antibakteriellen chemotherapie kann mehrere gründe haben. die häufigste ursache ist die primäre oder sekundäre resistenz der verursachenden bakterien. primäre resistenz bedeutet, dass alle bakterien z. b. einer spezies oder gattung gegenüber einem bestimmten antibiotikum von natur aus resistent sind. sekundäre resistenz beinhaltet, dass ein klon einer primär empfindlichen spezies durch mutation oder akquirierung eines resistenzgens (z. b. auf einem plasmid) resistent wird. unter der persistenz eines erregers versteht man das überleben des erregers am infektionsort während einer antibiotikatherapie. hierzu kommt es, wenn der erreger vorübergehend von der wachstumsphase in eine ruhephase übertritt, z. b. bedingt durch verschiedene physikalisch-chemische ursachen am infektionsort. da die meisten gebräuchlichen chemothera-peutika nur auf proliferierende keime wirken, werden sie nicht eliminiert und können daher nach absetzen der antibiotikatherapie zum rezidiv führen. die ineffektivität einer antibiotikatherapie kann natürlich auch durch einen wechsel des ätiologisch bedeutsamen erregers während der therapie bedingt sein, aber ebenso durch fehler in der durchführung der chemotherapie (s. o.). die prophylaktische gabe von antibakteriellen chemotherapeutika hat nur wenige, eingeschränkte indikationsgebiete! hierzu gehört z. b. die perioperative antibiotikagabe zur verhinderung von postoperativen wundinfektionen und septikämien, deren sinn bei bestimmten operativen eingriffen erwiesen ist. in seltenen fällen kann eine expositionsprophylaxe mit antibiotika durchgeführt werden, akzeptiert sind hier die pertussis-und die meningokokkenmeningitis-prophylaxe bei besonders gefährdeten personen, wenn in deren umgebung ein erkrankungsfall aufgetreten ist. heute steht eine große anzahl von antibakteriellen chemotherapeutika aus verschiedensten substanzgruppen zur klinischpraktischen anwendung zur verfügung. › tabelle 13.4 gibt einen orientierenden überblick über die unterschiedlichen substanzgruppen mit beispielen von einzelsubstanzen. hieraus lassen sich in geraffter form das antibakterielle wirkspektrum, wichtige pharmakologische eigenschaften und bedeutende potenzielle nebenwirkungen ablesen. diese klassifizierung folgt klinischen anwendungsgesichtspunkten und nur z. t. der exakten chemischen einteilung. wegen der großen anzahl der zur verfügung stehenden chemotherapeutika musste dabei eine auswahl erfolgen, die sich an deren praktischer bedeutung orientiert. der infektiologisch nicht spezialisierte arzt sollte sich auf ein standardrepertoire von wenigen substanzen beschränken, bei deren therapeutischem einsatz er dann eigene erfahrungen gewinnt. z u s a m m e n f a s s u n g • es steht außer zweifel, dass eine antivirale chemotherapie möglich und die gabe mancher substanzen in bestimmten klinischen situationen bereits absolut indiziert ist. • eine vielzahl von substanzen wird schon seit jahrzehnten regelmäßig in vielen laboratorien auf antivirale wirkung geprüft. aids hat zur verstärkten suche und für erheblich mehr publizität gesorgt. die wirkprinzipien werden im labor und z. t. bereits in klinischen studien anhand vieler substanzen unterschiedlicher chemischer natur untersucht. in der tat stößt z. b. die planung ausreichend kontrollierter studien zur antiviralen therapie bei aids selbst in den usa bereits auf das problem des mangels an studienfähigen patienten. th. mertens die künftigen entwicklungen in der antiviralen chemotherapie betreffen sowohl die charakterisierung neuer ziele (engl.: targets) für antivirale interventionen und die entwicklung antiviral wirksamer substanzen als auch die durch studien begründbaren empfehlungen für die anwendung vorhandener therapeutika. viren unterscheiden sich hinsichtlich struktur und vermehrungsweise grundsätzlich von allen anderen infektionserregern. sie sind für ihre vermehrung vollständig auf die energiegewinnung und syntheseleistung ihrer wirtszelle angewiesen. voraussetzung für eine antivirale chemotherapie ist somit, dass moleküle und biochemische prozesse identifiziert werden, die nur in virusinfizierten zellen vorkommen. der begriff virusselektivität beschreibt die fähigkeit einer substanz, die virusvermehrung zu hemmen, ohne die wirtszelle zu schädigen. viele viren haben strategien entwickelt, um im einmal infizierten organismus zu persistieren. in diesen fällen kann es zu infektionszuständen ohne virusvermehrung kommen (z. b. latenz der herpesviren). da alle bislang verfügbaren antiviralen substanzen im vermehrungszyklus angreifen, entziehen sich persistierende infektionen ohne virusvermehrung derzeit einer antiviralen therapie. die verfügbaren therapeutika haben ein begrenztes wirkspektrum, und es gibt bislang keine "breitbandmedikamente". antivirale therapie setzt somit eine virustypdiagnose voraus. bei akuten viruserkrankungen entscheidet darüber hinaus ein frühzeitiger therapiebeginn über den erfolg, was eine rasche diagnosestellung notwendig macht. bei patienten mit schwerster angeborener (scid), erworbener (aids) oder iatrogener (transplantation) immundefizienz ist es häufig trotz adäquater antiviraler therapie nicht möglich, die virusvermehrung zu beenden, solange es nicht zu einer verbesserung der immunsituation kommt. therapieindikation, -beginn und -dauer allgemeine regeln zur therapie sollten nur auf der grundlage klinischer studien festlegt werden (evidence-based). wesentliche allgemeine kriterien, die berücksichtigt werden müssen, sind die immunsituation des patienten, das risiko schwerer erkrankung, folgeerkrankung oder chronifizierung, die vermeidung unerwünschter arzneimittelwirkungen (uaw) und die vermeidung von resistenzentwicklung. die probleme mögen folgende fragen verdeutlichen, deren antworten z. t. in letzter zeit gegeben wurden (s. u.): muss eine akute hepatitis b oder c therapiert werden? welches ist der optimale zeitpunkt zum beginn einer antiretroviralen therapie bei hiv-infektion? wie soll man hiv-infizierte schwangere behandeln? müssen windpockenerkrankungen oder eine gingivostomatitis herpetica behandelt werden? bei schwerst immunsupprimierten wird zur verhinderung lebensbedrohlicher virusinfektionen vielfach eine antivirale prophylaxe durchgeführt. diese medikamentengabe vor beginn einer aktiven infektion führt natürlich bei etlichen patienten zur unnötigen gabe teils toxischer substanzen. der begriff der präemptiven therapie bezeichnet eine antivirale behandlung nach virologischer diagnose einer aktiven infektion ohne vorliegen von symptomen und ist abzugrenzen von der therapie einer infektionskrankheit. die therapieentscheidung erfordert den nachweis des vorteils einer vorgehensweise für die jeweilige patientengruppe. die optimale dauer der therapie ist in vielen fällen noch nicht durch studien bestimmt worden. resistenzvermittelnde mutationen treten bei jeder virusvermehrung spontan auf. bei längerfristiger anwendung antiviraler chemotherapeutika und damit vor allem bei den erheblich immunsupprimierten patienten mit langdauernder massiver virusvermehrung kann es dann relativ rasch zur selektion resistenter viruspopulationen kommen. diese virusvarianten besitzen mutationen in der viralen polymerase (rt bei hiv), der viralen kinase (herpesviren) oder anderen viralen genen, welche für zielstrukturen der antiviralen substanzen kodieren. voraussetzung für die selektion der spontan auftretenden resistenten virusvarianten ist somit virusvermehrung unter dem selektionsdruck einer antiviralen substanz. klinisch relevante virusresistenzen gegen nukleosidanaloga treten nach bisherigen erfahrungen bei kurzzeitiger therapie bzw. therapie immungesunder patienten nicht auf. vielmehr ist resistenz meist ein problem der langzeittherapie (> 1-2 monate), wenn es nicht gelingt, die produktive virusinfektion durch die therapie zu stoppen (hsv, cmv, hiv). resistenz-vermittelnde mutationen bedingen manchmal einen vermehrungsnachteil für das mutierte virus gegenüber dem wildtyp, wenn der selektionsdruck entfällt. in einigen fällen sind resistente virusvarianten (herpesviren, hiv) weniger pathogen als die wildviren. dies ist aber leider keinesfalls die regel. aufgabe der kliniker ist es, therapieversagen anhand klarer kriterien zu definieren, und den virologen obliegt es, standardisierte tests zur raschen phänotypischen oder genotypischen resistenztestung von viren bereitzustellen. manche zunächst wenig gravierende oder asymptomatische virusinfektionen können folgeerkrankungen auslösen (z. b. immunpathogenese), bei denen die viren dann keine entscheidende rolle mehr spielen und somit eine antivirale therapie zu spät kommt. ansatzpunkte für antivirale substanzen › tabelle 13.5 zeigt, dass die hemmung der virusvermehrung doch an vielen stellen möglich ist. diese frühesten vorgänge bei jeder virusinfektion, die bindung der viren an ihre wirtszelle und bei umhüllten viren die fusion mit der äußeren wirtszellmembran, lassen sich experimentell durch blockade der verantwortlichen rezeptorstrukturen auf seiten der viren oder der zellen hemmen. auch spezifische antikörper wirken auf dieser stufe der infektion. die molekularen vorgänge bei diesen frühen prozessen der infektion sind äußerst komplex und erfordern z. b. bei hiv etliche regulierte strukturelle veränderungen des viralen rezeptors. die hemmung der fusion ist bei hiv durch peptidische fusionsinhibitoren (t20), die an rezeptorstrukturen des virus binden, bereits möglich. auch die hemmung der korezeptorbindung ist denkbar. grund eines durch die strukturproteine gebildeten tiefen oberflächeneinschnittes (canyon) einlagern. dadurch wird die bindung an den zellrezeptor behindert und die zur freigabe der nukleinsäure notwendige, bei einigen virustypen ph-abhängige endosomale, intrazelluläre desintegration der viralen proteinhülle verhindert. bei einigen picornaviren wird die rezeptorbindung wenig behindert, aber das viruskapsid doch so stabilisiert, dass kein uncoating stattfinden kann. es gibt resistenz gegen diese substanzen, aber erstaunlicherweise sogar virusmutanten, die nur noch in anwesenheit dieser substanzen vermehrungsfähig sind. die faszination dieser entdeckung bestand auch darin, dass es plötzlich möglich wurde, aufgrund der kenntnis der molekularen struktur-wirkungs-beziehung antiviral wirksame moleküle sozusagen am reißbrett zu entwerfen (› tab. 13.5) . therapeutisch einsetzbare substanzen erstes und bislang einziges für die systemische therapie verfügbares medikament, das nach diesem mechanismus die picornavirus-replikation hemmt, ist pleconaril. einer der beiden wirkmechanismen von adamantanderivaten gegen influenza-a-viren beruht ebenfalls auf einer hemmung der freisetzung des ribonukleoproteins aus intrazytoplasmatischen vesikeln und des uncoatings durch blockade eines m2-virusprotein-abhängigen ionenkanals. therapeutisch einsetzbare substanzen amantadin und rimantadin stehen seit vielen jahren für die systemische therapie von influenza-a-virus-infektionen zur verfügung. die biologische besonderheit der retroviren besteht darin, dass, beginnend mit dem eindringen des viruscores (nukleokapsid) in die zelle und weiter nach der freisetzung des diploiden einzelsträngigen viralen rna-genoms, zuerst eine doppelsträngige dna hergestellt werden muss. dies geschieht in einem komplexen syntheseprozess über den zwischenzustand eines rna-dna-hybridmoleküls. zwei moleküle der hierfür notwendigen reversen transkriptase (rt) werden bei hiv im viruspartikel mitgebracht. neben der polymerasefunktion besitzt die rt in einer zweiten domäne noch eine enzymatische rnase-h-aktivität, die für die entfernung des rna-stranges vom rna-dna-hybridmolekül erforderlich ist. diese doppelsträngige dna-kopie der viralen rna wird danach als sog. provirales genom kovalent in das wirtszellgenom integriert. hierfür ist ebenfalls ein viruspartikelassoziiertes enzym, die integrase, erforderlich. substanzen zur hemmung der integration der proviralen dna eines retrovirus in das wirtszellgenom (hiv) befinden sich in der klinischen prüfung (integrasehemmer) und werden künftig möglicherweise ein weiteres standbein der antiretroviralen therapie bilden. therapeutisch einsetzbare substanzen die reverse transkriptase (rt) von hiv ist das zielmolekül für die meisten antiretroviralen medikamente. diese werden nach ihrer chemischen struktur unterteilt in nukleosidanaloge rt-hemmer und nicht nukleosidanaloge rt-hemmer (nnrti). eingriffsmöglichkeiten in virusspezifische funktionen ergeben sich während der transkription der viralen genetischen information. vorwiegend typ-i-interferone (ifn) hemmen die replikation verschiedener viren in unterschiedlichem ausmaß, wobei einer der vielfältigen mechanismen (s. u.) in der degradation viraler mrna besteht. einer der antiviralen wirkmechanismen von ribavirin beruht auf der hemmung der mrna einiger viren. therapeutisch einsetzbare substanzen verschiedene humane αund β-interferone stehen für die systemische therapie (chronische hepatitis-b-virus-und hepatitis-c-virus-infektion) und auch topische therapie (papillomaviren) zur verfügung. das relativ breit wirksame nukleosidanalogon ribavirin wird als kombinationstherapeutikum bei chronischer hcv-infektion und zur monotherapie bei rsv-und parainfluenzavirusinfektionen schwer kranker kinder, aber auch bei lassavirusinfektionen eingesetzt. abhängig von der art des vom virus in die wirtszelle eingeschleusten genoms (einzelstrang-rna/dna, doppelstrang-rna/dna) und des zur virusvermehrung erforderlichen genetischen informationsflusses bedarf es besonderer enzyme, die entweder vom virus -im partikel verpackt -mitgebracht (viruspartikelassoziiert, s. o. bei hiv) oder in der infizierten zelle synthetisiert werden (viruskodiert). beispiel für ein viruskodiertes enzym, welches in uninfizierten zellen nicht vorkommt, ist die rna-abhängige rna-polymerase der picornaviren, deren hemmung die wirkung mancher substanzen erklärt (2-[α-hydroxybenzyl]-benzimidazol, enviroxime). viele andere viruskodierte polymerasen von dna-viren unterscheiden sich von zellulären isoenzymen hinsichtlich der akzeptanz und bindung von nukleosidanaloga so weit, dass virusselektive nukleosidanaloga möglich sind. therapeutisch einsetzbare substanzen die vier derzeit gegen herpesviren einsetzbaren nukleosidanaloga, das na-phosphonat cidofovir und auch das pyrophosphatanalogon foscarnet hemmen letztlich alle die viruskodierten polymerasen. die blockierung viraler mrna durch kurze synthetische "antisense"-oligonukleotide ist eine vom konzept her sehr elegante und naturgemäß spezifische möglichkeit der hemmung der viruskodierten proteinsynthese. probleme bereiten die auswahl der geeigneten sequenzen und die chemische modifikation der oligonukleotide, die die aufnahme in die zelle ermöglichen müssen und die stabilisierung in der zelle bei erhaltener wirksamkeit sicherstellen müssen. interferone können die translationsinitiation hemmen. therapeutisch einsetzbare substanzen eingang in die therapie der zytomegalievirusretinitis hat ein intraokulär zu applizierendes antisense-phosphothioat gefunden. zur topischen therapie von herpes-simplex-virus-infektionen stehen ältere nukleosidanaloga zur verfügung mit geringerer virusselektivität. bekanntestes beispiel sind die proteasehemmer zur kombinationstherapie der hiv-infektion. der wirkmechanismus dieser substanzen beruht auf der hemmung der posttranslationalen spaltung der retroviralen gag-(gruppenspezifisches antigen) und gag-pro-pol-polyproteine durch hemmung der homodimeren "aspartatprotease" des virus. es werden unreife, nichtinfektiöse viruspartikel gebildet. auch andere posttranslational nötige modifikationen viraler proteine, z. b. glykosylierung, könnten ein ziel antiviraler substanzen sein. an dieser stelle im replikationszyklus greifen die neuen neuraminidasehemmer ein, die in der lage sind, die korrekte ausschleusung von influenzaviren zu inhibieren. während ihrer untersuchungen zur bereits bekannten interferenz von virusinfektionen entdeckten alick issacs und jean lindenmann 1957 einen übertragbaren virushemmenden faktor, den sie interferon nannten. in einem schlüsselexperiment stellten sie fest, dass zellen, die mit uv-inaktivierten influenzaviren behandelt worden waren, etwas in das gewebekulturmedium abgaben, das die infektion weiterer zellen verhinder-te. in den folgenden 50 jahren der erforschung dieses phänomens wurde ein gewaltiges netzwerk von interaktoren und interaktionen aufgedeckt. trotz vieler erkenntnisse besteht auch heute noch längst kein vollständiges bild aller zusammenhänge und wirkungen. die antivirale wirkung ist dabei nur eine von vielen, und sie ist eine folge regulatorischer funktionen der interferone in der zelle. als folge des schrittweisen erkenntniszuwachses ist auch die nomenklatur schwierig und teilweise redundant. ifn gehören nach heutiger nomenklatur zu den zytokinen. zur unterscheidung der interferone gibt › tabelle 13.6 eine übersicht. alle ifn sind relativ kleine moleküle, die in ihrer reifen form aus 165-172 aminosäuren bestehen. für die antivirale therapie spielen derzeit nur die typ-i-ifn, und hier die α-ifn, eine wesentliche rolle. die induktion kann auf verschiedenen wegen erfolgen. virusinfektionen, vor allem durch rna-viren, führen zur raschen induktion der ifn, die nach wenigen stunden wieder beendet wird. die induktion erfolgt über mehrere positiv regulatorische domänen, aber auch durch negative regulation, die zur verminderung der repressorproteine und einer gesteigerten ifn-gen-expression führt. die ifn werden von den zellen, in denen sie gebildet wurden, freigesetzt. natürliche α-ifn werden von lymphozyten, monozyten, makrophagen und einigen zelllinien gebildet. quelle für β-ifn sind fibroblasten und einige epitheliale zellen. mittlerweile werden die zur antiviralen therapie eingesetzten ifn meist als rekombinante ifn gentechnisch hergestellt. die ifn binden über spezifische zellrezeptoren an die zellen, in denen sie ihre antivirale und andere wirkungen entfalten. diese ifn-rezeptoren sind bekannt, und ihre expression unterliegt wiederum regulatorischen prozessen. viele (> 100) proteine werden durch ifn in den zellen reguliert, von denen etliche auch in die antivirale wirkung eingebunden sind. die stimulation der no-synthetase führt zur no-bildung und damit zu einer eher unspezifischen antiviralen wirkung hemmung der virusreifung ifn aktivieren eine gykosyltransferase, wodurch einerseits die notwendige posttranslationale modifikation mancher viraler proteine gehemmt und andererseits der normale ausschleusungsprozess (budding) behindert wird. bei systemischer anwendung gibt es z. t. erhebliche nebenwirkungen: neben den erwähnten zielen antiviraler therapeutika sind viele weitere denkbar. hinzuweisen ist in diesem zusammenhang auf die möglichkeit der hemmung regulatorischer proteine (z. b. tat oder rev bei hiv) oder auf die hemmung der genomreifung bei herpesviren und die genomverpackung bei im kern replizierenden umhüllten viren. die substanz wird parenteral und oral eingesetzt, auch prophylaktisch bei immunsuppression. die orale bioverfügbarkeit von acv ist allerdings nicht gut und unsicher, so dass bei schweren erkrankungen immer eine i.v. therapie angezeigt ist. die therapeutisch wirksame dosierung liegt bei dem wesentlich langsamer replizierenden vzv deutlich höher als bei hsv, was möglicherweise an der kurzen intrazellulären halbwertszeit von acv-triphosphat liegt. valaciclovir (valacv) ist ein valinester des acv. die substanz wird oral wesentlich besser resorbiert, so dass eine orale bioverfügbarkeit von ca. 60% erreicht wird. valacv wird bei der resorption und bei der ersten leberpassage praktisch vollständig in die wirksame substanz acv umgewandelt. mit einführung des valacv ist es möglich geworden, acv-serumkonzentrationen durch orale valacv-gabe zu erhalten, die ansonsten nur durch i.v. applikation von acv erreicht werden können. resistenzentwicklung acv-resistente hsv-mutanten mit mutationen/deletionen im thymidinkinase(tk)-gen und/ oder polymerase-gen können isoliert werden und sind bei immunsupprimierten u.u. klinisch relevant. tk-minus-mutanten sind nicht neuropathogen, allerdings sind resistente hsv durchaus nicht immer tk-minus-mutanten. in der regel führen tk-mutationen zu kreuzresistenz gegenüber anderen nukleosidanaloga, die durch die tk aktiviert werden müssen (s. u. und ganciclovir). resistenz aufgrund von polymerasemutationen ist seltener, führt aber meist zu breiter kreuzresistenz gegenüber nukleosidanaloga (s. u.). bvdu wird ebenfalls selektiv durch die viralen thymidinkinasen von hsv-1 und vzv zu monophosphat und diphosphat phosphoryliert. die phosphorylierung zu diphosphat kann von der hsv-2-tk nicht geleistet werden, da deren thymidylatkinase-aktivität wesentlich geringer ist, was die geringe wirksamkeit von bvdu gegenüber hsv-2 erklärt. die wirksamkeit von bvdu bei vzv-infektionen (varizellen und zoster) immunkompromittierter patienten ist durchaus sehr gut und vergleichbar der von i.v. verabreichtem aciclovir, jedoch fällt die nutzen-risiko-betrachtung insgesamt auch bei vzv-therapie zu gunsten von aciclovir aus, da bvdu eher mutagen zu sein scheint und nicht zusammen mit 5-fluorouracil (zytostatikum) gegeben werden darf. penciclovir (pcv) pcv ist strukturell dem ganciclovir sehr ähnlich und ebenfalls oral sehr schlecht resorbierbar im gegensatz zu famciclovir (fcv), einer oral sehr gut resorbierbaren substanz (bioverfügbarkeit bis > 70%). nach resorption wird fcv rasch und vollständig in pcv, das wirksame pro-drug, umgewandelt. pcv wird ähnlich wie acv durch die thymidinkinasen von hsv und vzv phosphoryliert. wesentliche indikationen genitale hsv-primärinfektionen sollten möglichst frühzeitig systemisch behandelt werden, auch mit dem ziel, möglicherweise die spätere rezidivhäufigkeit zu verringern. ähnliches gilt auch für die primäre gingivostomatitis herpetica. rekurrierende mukokutane hsv-infektionen können je nach beschwerden und beeinträchtigung, vor allem bei genitalen manifestationen (u.u. patientengesteuert), systemisch behandelt werden. in besonderen fällen mit häufigen genitalen rekurrenzen (6-10 pro jahr) oder mit schwerer psychischer beeinträchtigung kann eine suppressionsbehandlung durchgeführt werden. nach absetzen der therapie treten erneut rekurrenzen auf. topisch kann mit verschiedenen nukleosidanaloga (s. o.) behandelt werden. bei hautmanifestationen ist zwischenzeitliches betupfen der läsionen mit äther oder alkohol sinnvoll. bei schweren systemischen infektionen (enzephalitis, neugeborenensepsis) mit hsv oder vzv oder bei infektionen erheblich immunsupprimierter mit diesen viren muss mit einer sofortigen i.v. acv-therapie begonnen werden. das behandlungsergebnis hängt entscheidend von einem frühzeitigen behandlungsbeginn ab. liegt zum zeitpunkt der geburt eine hsv-infektion im geburtskanal der mutter vor, so besteht die gefahr einer konnatalen infektion des neugeborenen mit der folge einer unbehandelt oft tödlich verlaufenden hsv-sepsis (› kap. 13.5.1). in diesen fällen kann nach heutiger kenntnis eine therapie der schwangeren zur vermeidung einer schnittentbindung durchaus erwogen werden. ebenso muss die möglichkeit der antiviralen therapie in das optimale management der perinatalen vzv-infektion einbezogen werden. bei vzv-exposition eines seronegativen immunsupprimierten patienten (meist kinder) ist die sofortige gabe eines varizellen-hyperimmunglobulins (0,2 ml/kg körpergewicht) indiziert, ggf. in kombination mit antiviraler therapie. bei vzv-manifestationen bei immunsupprimierten patienten (auch zoster) ist eine antivirale therapie indiziert. es gibt durchaus gute argumente für eine generelle antivirale therapie bei varizellen, jedoch wird sie in der praxis kaum durchgeführt. in den letzten jahren hat sich gezeigt, dass acv bei cmv-erkrankungen therapeutisch nicht einsetzbar ist, dass jedoch bei prophylaktischer gabe an erheblich immunsupprimierte transplantatempfänger auch eine gewisse prophylaktische wirkung gegen cmv-erkrankungen nach endogener reaktivierung vorhanden ist. die substanz kann nur i.v. und topisch angewendet werden. pfa wird systemisch bei cmv-erkrankungen immunsupprimierter eingesetzt, wenn eine gcv-resistenz vorliegt, und stellt dann eine alternative zu cidofovir dar. pfa kann topisch erfolgreich bei herpes labialis und genitalis eingesetzt werden. bei den anderen humanen herpesviren sind z. t. gute effekte der oben genannten substanzen in vitro beschrieben worden. in klinischen studien konnte durch anwendung von acv bei ebv-infektionen auch die virusausscheidung deutlich vermindert werden, ein wesentlicher einfluss auf den krankheitsverlauf ließ sich nicht erreichen. dies gilt ebenfalls für die übrigen humanen herpesviren und hängt wohl auch mit der pathogenese der jeweiligen erkrankungen zusammen. das humane hbv ist ein sehr kleines dna-virus mit einer teilweise doppelsträngigen zirkulären dna von ca. 3200 bp. im partikel ist eine polymerase verpackt, die den während der replikation notwendigen, für ein dna-virus sehr ungewöhnlichen schritt einer reversen transkription ermöglicht. dies geschieht an einer prägenomischen rna. angesichts dieser biologischen ähnlichkeiten mit einem retrovirus und auch der homologien der hbv-polymerase mit der reversen transkriptase von retroviren ist es nicht erstaunlich, dass gegen retroviren wirksame substanzen in vitro und in vivo auch gegen hbv wirksam sind. hauptindikation für die antivirale therapie ist heute die chronische hepatitis b wegen der spätkomplikationen: leberzirrhose und hepatozelluläres karzinom. interferone natürliche und auch rekombinant hergestellte humane α-interferone inhibieren die hbv-replikation sehr effektiv und waren für etliche jahre einzige standardtherapeutika bei der behandlung der chronischen hepatitis b. dies war auch die erste indikation, bei der interferone in großem umfang zur antiviralen therapie eingesetzt wurden. virologisch und histopathologisch findet sich bei etwa einem drittel der patienten, die 6 monate lang dreimal wöchentlich 3 mio. ie α-interferone erhielten, eine deutliche besserung der chronischen hbv-infektion. die hbv-dna-konzentrationen im blut nehmen signifikant ab oder fallen -auch in abhängigkeit von der empfindlichkeit der eingesetzten nachweismethodikunter die nachweisgrenze ab. bei etwa 40% der patienten kam es zur normalisierung der transaminasen und zum therapiebedingten verlust von hbeag. leider persistiert dieser positive effekt nach beendigung der interferontherapie nur bei wenigen der ursprünglich erfolgreich behandelten patienten. andererseits machen die bekannten nebeneffekte der therapie (s. o.) eine wirkliche dauertherapie unmöglich. lamivudin (3tc) das nukleosidanalogon 3tc, welches zunächst in der hiv-therapie eingesetzt wurde, hat sich auch bei der hbv-therapie bewährt. nach einjähriger therapie mit täglich 100 mg zeigten in einer studie 60% der patienten eine histologisch nachweisbare verbesserung der hepatitis und sogar 70% eine anhaltende normalisierung der transaminasen (alt). nach fortgesetzter 2-jähriger therapie fanden sich bei einem drittel der behandelten antikörper gegen hbeag. 3tc führt nach lebertransplantation aufgrund einer hbv-induzierten zirrhose zu einer reduktion der neuinfektionen des transplantates, wobei die standardprophylaxe die fortgesetzte gabe von anti-hbs-haltigen hyperimmunglobulinpräparaten ist. verschiedene resistenzvermittelnde mutationen treten in der hbv-polymerase in sehr ähnlichen, konservierten bereichen wie bei hiv in abhängigkeit von der therapiedauer auf. die auswirkungen auf den grad der resistenz einerseits und die verbleibende vermehrungsfähigkeit der mutanten andererseits sind wie bei hiv unterschiedlich. neue nukleosidanaloga (entecavir) finden derzeit mit erfolg eingang in die therapie der chronischen hbv-infektion. langzeittherapie scheint die erfolge zu verbessern, solange keine resistenzen auftreten. es liegt nahe, dass alle in der erprobung befindlichen antiretroviralen nukleosidanaloga auch hinsichtlich ihrer hbv-wirksamkeit und kreuzresistenzen geprüft werden. hcv-infektionen stellen eine besondere therapeutische herausforderung dar, weil ein sehr hoher anteil (75-85%) der infizierten eine chronische infektion entwickelt, die dann nach jahren eines relativ symptomarmen verlaufs plötzlich in eine leberzirrhose und auch ein karzinom übergehen kann. der zunächst klinisch eher milde verlauf führt dazu, dass die therapiebereitschaft der patienten in den asymptomatischen phasen häufig nicht allzu groß ist. bei hcv handelt es sich um ein völlig anderes virus als bei hbv, um ein einzelsträngiges rna-virus mit plusstrang-polarität. das genom dieses flavivirus umfasst ca. 9000 nukleotide. ähnlich wie bei picornaviren ist das primäre translationsprodukt ein polyprotein, das bei hcv posttranslational in zehn struktur-und nicht-struktur-proteine gespalten wird. eines der nicht-struktur-proteine, das ns5a, ist offenbar für die interferonempfindlichkeit des virus verantwortlich. bei hcv unterscheidet man 6 genotypen mit verschiedenen subtypen. diese genotypen α sind geographisch unterschiedlich verteilt. die erfolgsrate einer α-interferon-therapie ist vom genotyp abhängig. in deutschland ist mit ca. 60% der genotyp 1 (subtypen a und b) am häufigsten vertreten, welcher leider die geringste ansprechrate bei α-interferon-therapie aufweist. ähnlich wie bei hiv ist die viruspopulation in einem patienten genetisch nicht einheitlich, sondern stellt ebenfalls eine quasi-spezies dar. das ausmaß der heterogenität der viruspopulation vor therapie und die veränderung unter therapie scheinen prognostische bedeutung zu besitzen. α-interferone die zunächst angewendete 6-monatige monotherapie mit dreimal 3 mio. ie α-interferon pro woche erbrachte je nach studie und bei einschluss aller genotypen nur bei etwas über 10% der patienten einen anhaltenden erfolg. dieser war definiert als mindestens 6 monate nach therapieende anhaltende normalisierung der transaminasen und verschwinden der hcv-rna aus dem blut. in den folgenden jahren konnte durch steigerung der α-interferon-dosis, durch tägliche gaben und verdoppelung der therapiedauer der erfolg verbessert werden. peg-gekoppelte interferone und kombinationstherapie mit ribavirin zwei weitere therapeutische neuerungen, einerseits die anwendung von polyethylengekoppeltem α-interferon 2a (sog. pegyliertes interferon) und andererseits die kombinationstherapie mit dem nukleosidanalogon ribavirin, konnten die ergebnisse unabhängig voneinander nochmals deutlich verbessern. durch anwendung von peg-α-ifn 2a wird bei nur einmal wöchentlicher gabe (180 mg) ein wesentlich gleichmäßigerer ifn-spiegel erreicht. der therapieerfolg liegt bei anwendung von peg-α-ifn 2a bei etwa 30% (genotyp 1) und 40% (alle genotypen) mit vergleichbaren nebenwirkungen wie nach α-ifn-therapie. die ergebnisse mit der kombinationstherapie α-ifn + ribavirin liegen in der gleichen größenordnung. auch hier lassen sich durch dosissteigerungen und verlängerte therapie verbesserungen erreichen. interessant ist die tatsache, dass frühere studien mit einer ribavirin-monotherapie zwar eine verbesserung der leberhistologie und der transaminasen gezeigt haben, aber keine verminderung der viruslast im blut. die naheliegende kombination von peg-ifn und ribavirin hat die ergebnisse weiter verbessert. eine reihe weiterer kombinationen befindet sich in der erprobung. die langzeitprognose der erfolgreich behandelten patienten ist gut und mittlerweile scheint sogar klar zu sein, dass auch chronisch hcv-infizierte patienten mit leberzirrhose, abhängig vom hcv-genotyp, gemessen an virologischen, klinischen und auch histologischen parametern, von der antiviralen therapie profitieren. therapiebestimmende faktoren derzeit sind 3 retroviren bekannt, die für die pathogenese von erkrankungen des menschen bedeutsam sind: • beide erreger von aids, hiv-1 und hiv-2 • htlv-1 als erreger der adulten t-zell-leukämie und der tropischen spastischen paraparese. zielmoleküle der meisten verfügbaren antiretroviralen therapeutika sind 2 partikelgebundene viruskodierte enzyme, die reverse transkriptase (rt) und die protease. die substanzen lassen sich einteilen in nukleosidanaloge reverse-transkriptase-hemmer, nicht nukleosidanaloge reverse-transkriptase-hemmer (nnrti) und proteasehemmer. seit der erstzulassung des ersten rt-hemmers azt in den usa im jahr 1987 sind mehr als 20 medikamente in deutschland zugelassen worden. zanamivir muss i.v. oder als aerosol verabreicht werden, wohingegen das analoge "pro-drug" oseltamivir oral verabreicht werden kann. da die beiden substanzen an unterschiedlichen stellen der funktionellen domäne der neuraminidase binden, besteht keine absolute kreuzresistenz zwischen beiden pharmaka. h5n1-infizierte patienten sind teilweise mit erfolg behandelt worden. neben den etablierten antiviralen therapeutika und therapieindikationen gibt es andere viren, bei denen die therapie am menschen erprobt wurde, aber noch eher experimentellen charakter hat. die therapie von enterovirus-und rhinovirusinfektionen ist seit mehreren jahrzehnten gegenstand wissenschaftlicher forschung und auch von therapieversuchen mit chemischen substanzen, antikörpern und interferonen. eine gruppe von sog. win-substanzen hemmt durch spezifische bindung an das viruskapsid die adsorption des virus an die zielzelle und das intrazelluläre uncoating. in den usa ist ein vertreter dieser substanzen, das pleconaril, klinisch erprobt worden. es ist gegen viele picornaviren (95%) wirksam und hat sich bei der behandlung der aseptischen meningitis und respiratorischer infektionen in kontrollierten studien bewährt. ob pleconaril bei der enterovirusmyokarditis eingesetzt werden kann, ist noch nicht klar. derzeit laufen bei dieser indikation versuche mit interferontherapie. adenoviren sind weltweit verbreitet und besitzen ein erhebliches pathogenes potenzial. insbesondere bei systemischen infektionen immunsupprimierter stellen sie ein schwieriges the-rapeutisches problem dar. zwei in vitro gegen verschiedene adenovirustypen wirksame substanzen, die für andere indikationen zugelassen sind, werden zur experimentellen therapie eingesetzt: cidofovir und ribavirin. für beide substanzen gibt es positive kasuistische mitteilungen, aber der therapeutische wert beider substanzen ist derzeit noch nicht klar. wie bereits erwähnt, zeichnet sich das nukleosidanalogon ribavirin durch ein relativ breites wirkungsspektrum aus. bereits vor knapp 20 jahren wurde es zur behandlung von rsv-infektionen der lunge bei schwer kranken intensivpflichtigen kindern zugelassen. in studien konnte bei aerosolanwendung eine senkung der letalität nachgewiesen werden. auch die intravenöse anwendung ist mit erfolg möglich, und derzeit wird die kombination von ribavirin mit einer etwa gleich wirksamen antikörpergabe evaluiert. auch beim masernvirus, einem weiteren paramyxovirus, ist über einzelne erfolge bei der behandlung von pneumonien immunsupprimierter mit ribavirin berichtet worden. interessanterweise ist ribavirin auch mit gutem erfolg bei schweren hämorrhagischen lassavirusinfektionen eingesetzt worden und gilt als therapie der wahl. auch bei dem verwandten, in südamerika vorkommenden junin-virus konnte die letalität von ca. 20 auf 2% gesenkt werden. leider ist die substanz nicht ausreichend liquorgängig, um einen therapeutischen effekt bei zns-manifestationen zu erreichen. bei der durch jc-virus hervorgerufenen progressiven multifokalen leukoenzephalopathie (pml) wurden bei gabe von cidofovir, aber auch α-interferon klinische besserungen beobachtet. eine reihe mehr oder weniger spezifisch wirksamer substanzen (auch interferone) ist für die topische therapie von papillomen erprobt worden. in jüngster zeit konnte in einer ersten studie gezeigt werden, dass auch hier eine topische applikation von cidofovir wirksam ist. die zunächst mit großen erwartungen durchgeführten studien zur behandlung der juvenilen larynxpapillomatose mit interferonen haben nicht zufrieden gestellt. diagnostik die diagnose einer katheterassoziierten infektion ist häufig schwierig. zur definitiven diagnose einer katheterassoziierten infektion ist der nachweis desselben erregers von der katheterspitze und aus der blutkultur notwendig. bei liegendem katheter kann die diagnose mit der sogenannten "differential time to positivity" (dttp) gestellt werden: im falle einer katheterassoziierten infektion wird eine aus dem katheter entnommene blutkultur mindestens 2 h früher positiv als eine gleichzeitig aus einer peripheren vene entnommene probe. als klinische hinweise können eine entzündung an der einstichstelle, fehlende andere ausgangsherde für eine bakteriämie sowie der nachweis typischer erreger, z. b. staphylokokken, angesehen werden. therapie es werden antibiotika verabreicht und der katheter entfernt. bei implantierten verweilkathetern ist vor der entfernung je nach vorliegendem pathogen ein antibiotischer behandlungsversuch gerechtfertigt. die wichtigste allgemeine maßnahme zur verhinderung nosokomialer infektionen ist das händewaschen. besonders in bereichen mit erhöhtem risiko, wie z. b. in infektions-, intensivund hämatologisch-onkologischen stationen, müssen vor und nach jedem patientenkontakt die hände gewaschen werden, 13 .3 syndrome und spezifische probleme 13 um eine nosokomiale ausbreitung von erregern zu vermeiden. daneben sind bei einigen erkrankungen spezielle maßnahmen erforderlich (s. o.). desinfektionen von medizinischen geräten sind heute durch gesetzliche bestimmungen und hygienevorschriften so geregelt, dass bei richtiger anwendung hierdurch keine erreger übertragen werden können. die liegedauer von intravasalen oder urinkathetern sollte möglichst kurz sein, d.h., jeder katheter, der nicht unbedingt benötigt wird, sollte entfernt werden. wo immer möglich, sollten therapieregime bevorzugt werden, bei denen medikamente oral statt intravenös gegeben werden. infektionen bei immunsupprimierten patienten sind häufig, verlaufen atypisch und können zu schweren komplikationen führen. art der immunsuppression und ausprägung und qualität der immunitätsfaktoren bestimmen spektrum und verlauf von infektionen wesentlich und sind für diagnostisches und therapeutisches vorgehen entscheidend. praxisfall ii eine 41-jährige frau wird wegen einer akuten myeloischen leukämie mit einer hochdosischemotherapie behandelt. am tag 10 nach beginn der chemotherapie sind die leukozyten auf < 500/μl gefallen, sie klagt über kopfschmerzen und wirkt apathisch. nach einer halben stunde steigt die temperatur auf 39,7 °c an, blutkulturen werden abgenommen und eine empirische antibiotikatherapie begonnen. der klinische zustand bessert sich rasch, die patientin ist 24 h nach deren beginn fieberfrei. zu diesem zeitpunkt ist e. coli als erreger in der blutkultur isoliert und identifziert worden. die empirische therapie ist adäquat und wird für weitere 8 tage • prognostisch ungünstigste kategorie: patienten mit lungeninfiltraten. nur bei 20-30% aller patienten finden sich positive blutkulturen, die eine gezielte antibiotische therapie ermöglichen. pilzinfektionen sind oft schwer zu diagnostizieren, da kulturelle verfahren wenig sensitiv sind. als nachweismethoden dienen heute in erster linie blutkulturen für candida-und die hochauflösende computertomografie des thorax sowie der galactomannan-test im serum für aspergillus-infektionen. therapie die behandlung eines neutropenischen patienten mit fieber muss unmittelbar nach auftreten der klinischen symptome, vor dem eintreffen mikrobiologischer befunde erfolgen. sie ist deshalb empirisch und kann später, bei erfolgter erregeridentifizierung, modifiziert werden. • initialtherapie und frühe eskalation: als primäre therapie kommen eine kombination aus einem breitspektrum-(ureido-)penicillin mit einem β-lactamase-hemmer, ein pseudomonas-wirksames cephalosporin oder ein carbapenem in frage. tritt nach 3-4 tagen keine entfieberung auf, sollte eine erneute diagnostik insbesondere mit hinblick auf pilzinfektionen erfolgen. • antimykotische therapie: wegen der gefahr von invasiven pilzinfektionen wird bei neutropenie (insbesondere bei patienten mit pulmonalen infiltraten) frühzeitig, spätestens jedoch nach 6-7 fiebertagen eine antimykotische therapie eingesetzt. zur empirischen therapie eignen sich caspofungin oder liposomales amphotericin b, beim nachweis von lungeninfiltraten ist voriconazol mittel der wahl. • fokussanierung: sie ist in der regel nur bei lokalisierten hautinfektionen möglich oder auch sinnvoll. die chirurgische therapie einer neutropenischen kolitis ist nur bei bildung von intraabdominellen abszessen oder einer offenen perforation sinnvoll, ansonsten erfolgt die therapie konservativ. prophylaxe wegen der hohen inzidenz von infektionen bei neutropenischen patienten werden verschiedene maßnahmen zur infektionsprophylaxe angewandt. diese maßnahmen zielen auf eine verminderung der endogenen keimflora und vermeidung der exposition gegenüber pathogenen organismen. • innerhalb einiger wochen erfolgt offenbar durch die immunantwort eine partielle kontrolle der hiv-replikation. die virämie nimmt ab und stellt sich auf ein individuell unterschiedlich hohes niveau ein. die höhe der virämie in dieser phase (messbar durch die quantitative bestimmung der hiv-rna) ist maßgeblicher parameter der weiteren prognose. patienten mit niedriger hiv-rna (< 10 000 genomkopien/ml blut), haben eine deutlich längere überlebenszeit als solche mit höheren werten. obwohl in dieser phase (klinische latenzphase) meist keine beschwerden vorhanden sind, besteht eine ungeheure dynamik der hiv-replikation, die schließlich zur erschöpfung des immunsystems führt. pro tag werden ca. 10 10 virionen produziert. über 99% werden dabei in den cd4 + -lymphozyten gebildet, die infolge der infektion zerstört werden. es kommt so zum stetigen abfall der t-helferzell-zahl im blut, der sich meist über viele jahre hinzieht. das verhältnis von cd4 + -zellen zu cd8 + -zellen (suppressor-und zytotoxische t-zellen) kehrt sich um (normalerweise cd4 + : cd8 + > 1). wenn die zahl der cd4 + -zellen unter eine kritische schwelle von 200/μl blut sinkt, kommt es zum auftreten von aids-typischen opportunistischen infektionen. bereits vorher können die patienten symptome aufweisen (z. b. oraler soor), die auf einen nahen zusammenbruch des immunsystems hindeuten. die hiv-infektion führt zur unspezifischen stimulation des humoralen immunsystems. dies äußert sich in einer vermehrten bildung von immunglobulinen. neben der infektion lymphatischer zellen werden auch frühzeitig langlebige makrophagen und gliazellen des zns befallen. diese zellen spielen für die dynamik der hiv-infektion keine so große rolle wie die cd4 + -lymphozyten, sind aber therapeutisch schwerer erreichbar und bereiten daher probleme. symptome je nach stadium der erkrankung treten unterschiedliche symptome auf. • akutes retrovirales syndrom: bei bis zu 50% der infizierten kommt es wenige wochen nach der ansteckung zum akuten krankheitsbild, dem sog. akuten retroviralen syndrom. wegen der klinischen ähnlichkeit mit der mononukleose wird das krankheitsbild auch als "mononukleoseähnliches syndrom" bezeichnet. typische symptome sind fieber, nachtschweiß, allgemeines krankheitsgefühl, lymphknotenschwellungen, pharyngitis und exantheme. vereinzelt treten auch schwere neurologische erkrankungen auf (z. b. guillain-barré-syndrom). während dieses akuten stadiums kommt es zum deutlichen abfall der t-helferzell-zahl, die hiv-rna im blut und damit auch die infektiosität ist sehr hoch. nach einigen tagen bis wochen bilden sich diese klinischen veränderungen wieder zurück. • asymptomatisches stadium (klinische latenz): die meisten hiv-infizierten haben über mehrere jahre keine beschwerden, die hiv-infektion wird in diesem stadium oft nur durch zufall entdeckt. als klinisches symptom können generalisierte lymphknotenschwellungen vorhanden sein (daher die frühere bezeichnung "lymphadenopathiesyndrom"). bei relativ konstantem wert der hiv-rna im plasma findet sich ein unterschiedlich rascher abfall der cd4 + -zeilen. • symptomatisches stadium: kennzeichnend ist die zunehmende immunschwäche, die sich im auftreten von opportunistischen infektionen äußert (cdc-kategorien b und c). die zahl der cd4 + -zellen ist stark abgefallen, es findet sich meist ein hoher wert der hiv-rna im plasma. die schweren aids-definierenden erkrankungen treten meist dann auf, wenn die cd4 + -zell-zahlen < 200/μl gesunken sind (› kap. 13.4.2). abb. 13.7 hiv im elektronenmikroskopischen bild. gut erkennbar ist die virushülle (pfeil) mit den knopfartig erscheinenden oberflächenantigenen, die an den cd4-rezeptor anbinden können, ferner das zylinderförmige viruskapsid (doppelpfeil), das aus dem hauptcoreprotein p24 aufgebaut ist (aufnahme: h. gelderblom, berlin). klassifikation die derzeitig gültige klassifikation kommt von den amerikanischen centers for disease control (cdc-klassifikation) und wurde zuletzt 1993 revidiert (› tab. 13.10). sie führt die aids-definierenden erkrankungen auf. ferner gibt sie eine stadieneinteilung der hiv-infektion an, die sich an klinischen und immunologischen parametern orientiert. alle patienten, die eine klinische aids-definition erfüllen, werden in die kategorie c eingestuft (› tab. 13.11). virologische diagnostik die diagnostik der hiv-infektion erfolgt meist durch nachweis virusspezifischer antikörper. als screening-test bei verdacht auf hiv-infektion dient ein eli-sa mit sehr hoher sensitivität und spezifität je > 99%. bei positivem elisa muss ein bestätigungstest erfolgen. meist ist dies ein westernblot, ggf. auch ein immunfluoreszenztest. jeder hiv-test muss nach aufklärung und mit dem einverständnis des patienten erfolgen und bei positivem ausfall durch eine 2. blutentnahme und erneute untersuchung bestätigt werden. zwischen infektion und bildung messbarer antikörper vergehen einige wochen (diagnostisches fenster). 3-6 monate nach infektion weisen fast alle infizierten antikörper auf. in der frühen phase vor einsetzen der antikörperbildung ist also bei negativem hiv-antikörpertest eine übertragung der infektion möglich. in besonderen fällen, wenn eine sichere frühzeitige entdeckung der infektion notwendig ist, kann ein direkter nachweis der viralen rna oder proviralen dna durch pcr erfolgen. diese wird z. b. bei kindern hiv-infizierter mütter eingesetzt. mit dieser oder anderen diagnostischen verfahren (nasba = "nucleic acid squence-based amplification"; bdns = "branch-chain dns) kann ferner ein quantitativer nachweis der hiv-rna (viruslast) im blut erfolgen. dieser test spielt heute eine sehr große rolle für die beurteilung der prognose und als kontrolle der antiretroviralen therapie. immunologische diagnostik die zahl der cd4-lymphozyten im blut stellt den entscheidenden immunologischen parameter für die verlaufsbeurteilung der hiv-infektion dar. die bestimmung erfolgt mittels der facs-methode. begleitende diagnostik durch die hiv-infektion können einige sekundäre laborveränderungen hervorgerufen werden. häufig finden sich anämie, leukozytopenie, thrombozytopenie und erhöhte immunglobuline. viele infizierte haben zusätzliche infektionen. besonders nach hepatitis b und c, lues, toxoplasmose und zytomegalievirus-infektion muss gesucht werden. differenzierungsmaßnahme akute hiv-infektion: klinische kategorien ( • hemmung der protease • hemmung der integration. mittlerweile stehen mehr als 20 substanzen aus verschiedenen klassen für die therapie zur verfügung (› tab. 13.12), medikamente mit weiteren neuen therapieprinzipien (ccr5-antagonisten, integrasehemmer, maturationshemmer) werden derzeit klinisch erforscht. die therapie der hiv-infektion erfolgt heute immer als kombinationstherapie. diese wird auch als "hochaktive antiretrovirale therapie" (haart) bezeichnet. typischerweise besteht sie aus der kombination von 2 nrti mit einem proteasehemmer (pi) oder einem nnrti. durch keine bisher bekannte therapie wird die vollständige elimination von hiv erreicht. daher muss eine einmal begonnene und effektive antiretrovirale therapie unbegrenzt fortgeführt werden, da es sonst erneut zur ungehemmten virusvermehrung kommt. aus unterschiedlicher motivation ist in den letzten jahren die auswirkung von therapiepausen untersucht worden. die erhofften positiven effekte (verbesserung der immunantwort gegen hiv) traten hierunter nicht ein, dagegen konnte ein vermehrtes risiko für schwerwiegende komplikationen gezeigt werden. therapiepausen sind daher nicht zu empfehlen. wiegen. bei einer cd4 + -zahl von > 500/μl wird deshalb meist zunächst abgewartet. eine gesicherte behandlungsindikation besteht für alle patienten mit einer symptomatischen hiv-infektion unabhängig von der zahl der cd4 + -zellen und der hiv-rna. eine besondere herausforderung ist die behandlung hiv-infizierter schwangerer frauen. im ersten trimenon sollte wegen des möglichen teratogenen potenzials keine antiretrovirale therapie erfolgen bzw. eine begonnene therapie ausgesetzt werden. für eine antiretrovirale therapie mit zidovudin ab der 14. schwangerschaftswoche bis zur entbindung mit anschließender 6-wöchiger nachbehandlung des kindes ist eine verringerung der vertikalen hiv-übertragung nachgewiesen. viele frauen werden heute auch in der schwangerschaft mit einer kombinationstherapie behandelt, wobei bisher kaum daten zur fruchtschädigung durch einzelne medikamente vorliegen. wegen seiner teratogenität ist efavirenz auf jeden fall kontraindiziert. therapieziele ziele einer antiretroviralen therapie sind die lebensverlängerung und eine besserung vorhandener symptome und der lebensqualität. bei asymptomatischen patienten können nur die viruslast und der immunstatus als parameter für die wirksamkeit einer therapie herangezogen werden. es sollte heute eine absenkung der hiv-rna unter die nachweisgrenze ultrasensitiver tests (< 50 kopien/ml) angestrebt werden. je nach höhe der ausgangsviruslast wird dieses ziel meist nach 3-6 monaten erreicht. durch eine effektive antiretrovirale therapie steigt die zahl der cd4 + -zellen an. normalwerte werden jedoch meist nur von patienten mit relativ guten ausgangswerten erreicht. probleme der antiretroviralen therapie diese therapie war zunächst mit erheblichen einschränkungen und belastungen behaftet ( von der akuten hiv-infektion bis zum auftreten von aids vergehen bei unbehandelten im durchschnitt ca. 10 jahre. die mittlere zeitspanne von der diagnose aids bis zum tod beträgt dann knapp 2 jahre. ein geringer anteil aller hiv-infizierten (< 5%) zeigt auch nach mehr als 10-jähriger dauer der infektion keine anzeichen eines immundefektes (long-term non-progressor). es ist bisher nicht bekannt, ob diese personen jemals an aids erkranken werden. bei allen anderen führt die hiv-infektion unbehandelt unweigerlich zur ausbildung von aids und zum tod. durch die antiretrovirale kombinationstherapie hat sich die prognose der hiv-infektion dramatisch verbessert. die meisten können damit ein normales leben führen, und die prognose ist günstig. die mittlere lebenserwartung eines patienten unter antiretroviraler therpaie lässt sich heute noch nicht sicher angeben, dürfte sich aber der normalen lebenserwartung annähern. da die hiv-infektion zur unheilbaren, tödlichen erkrankung führt und eine schutzimpfung nicht zur verfügung steht, kommt der prävention eine zentrale rolle zu. das risiko einer sexuellen übertragung der hiv-infektion kann durch vermeidung riskanter sexualpraktiken vermindert werden. die konsequente benutzung von kondomen ist eine entscheidende maßnahme zur verminderung des übertragungsrisikos. bei drogenabhängigen konzentrieren sich präventive strategien auf die suchttherapie sowie auf die kontrollierte verabreichung von ersatzdrogen ("methadon-programm"). eine weitere präventive maßnahme ist die ausgabe steriler spritzen an drogenabhängige. allgemeine vorsichtsmaßnahmen angehörige medizinischer berufe sind beim umgang mit hiv-patienten einer infektionsgefahr ausgesetzt. ein relevantes infektionsrisiko existiert allerdings nur beim kontakt mit infiziertem blut. die höchste gefahr besteht bei stichverletzungen mit hohlnadeln, die blut enthalten: in 0,2-0,5% kommt es zur übertragung von hiv. daher müssen unbedingt vorsichtsmaßnahmen eingehalten werden. am wichtigsten ist die vermeidung von prozeduren, die ein hohes verletzungsrisiko beinhalten ("re-capping" von kanülen!). scharfe gegenstände müssen immer aus dem unmittelbaren arbeitsbereich entfernt und sofort in spezielle container entsorgt werden. da eine infektion prinzipiell auch über schleimhäute und verletzte haut erfolgen kann, müssen in allen situationen, in denen blutkontakt möglich ist, schutzhandschuhe getragen werden. vorgehen bei nadelstichverletzung ist es trotz vorsichtsmaßnahmen zur nadelstichverletzung gekommen, müssen sofort folgende maßnahmen ergriffen werden: blutung anregen und die möglichst gespreizte wunde mit einem desinfektionsmittel auf alkoholbasis gründlich desinfizieren (ca. 4 min). bei hautkontakt ebenfalls desinfizieren oder gründlich mit seife und viel wasser waschen. handelt es sich um eine verletzung mit hohem risiko (injektion von größeren mengen blut, intramuskuläre verletzung mit großlumiger nadel), sollte eine antiretrovirale kombinationstherapie rasch begonnen (optimal innerhalb von 1-2 h nach verletzung) und für 28 tage durchgeführt werden. bei verletzungen mit geringerem risiko (z. b. subkutane verletzung) sollte eine individuelle beurteilung und beratung durch jemanden mit spezieller erfahrung in diesem bereich erfolgen. wichtig sind in allen situationen, in denen ein infektionsrisiko aufgetreten ist, eine psychologische betreuung der betroffenen personen und die exakte dokumentation des unfallhergangs einschließlich der hiv-tests (zeitpunkte 0, 6 wochen, 3 und 6 monate), damit ansprüche des betroffenen im falle einer infektion gewahrt bleiben. als folge der durch hiv ausgelösten immunschwäche kommt es zum auftreten so genannter opportunistischer infektionen. typisch für viele opportunistische erreger ist, dass sie weit verbreitet sind und nach einer primärinfektion, die bereits vor der hiv-infektion stattfindet, zu latenten infektionen führen. diese erreger werden erst durch die immunschwäche zu pathogenen (daher die bezeichnung opportunistisch). alle organe können von diesen infektionen betroffen werden. am häufigsten treten erkrankungen der haut, der mundhöhle, des gastrointestinaltraktes, der lunge, des auges und des nervensystems auf. einige der opportunistischen infektionen kommen bereits vor, wenn die cd4 + -zellen noch nicht maximal erniedrigt sind (zwischen 100 und 200/μl). beispiele hierfür sind die candida-ösophagitis und die pneumocystis-carinii-pneumonie. andere erkrankungen sind charakteristisch für das endstadium der immundefizienz (cd4 + -zelien < 50/μl). neben infektionen treten auch verschiedene tumoren gehäuft auf (kaposi-sarkom, non-hodgkin-lymphome). ii ein 30-jähriger, der bis dahin nie krank war, stellt sich in der notaufnahme vor. seit 3 wochen bemerkt er fieber, das in den letzten wochen täglich 39 °c überschreitet. er klagt über quälenden reizhusten mit wenig auswurf, der auch nachts sehr heftig sei. in den letzten wochen habe er 7 kg gewicht abgenommen (von 60 auf 53 kg). bis zum vortag habe er gearbeitet, was ihm in den letzten tagen sehr schwer gefallen sei. bei nachfrage gibt er an, homosexuelle kontakte zu haben, ein hiv-test sei nie durchgeführt worden. bei der untersuchung fallen eine tachypnoe (30/min) und eine ausgeprägte periphere zyanose auf. auskultation und perkussion sind unauffällig. im mund besteht ein soor. zervikal, inguinal und axillär lassen sich vergrößerte lymphknoten (bis 1,5 cm) tasten. ansonsten keine auffälligkeiten. labor: hb 10,5 g/dl; leukozyten 2800/μl; ldh 680 u/l; in der arteriellen blutgasanalyse po 2 52 mmhg, pco 2 20 mmhg, ph 7,41; sonst keine weiteren auffälligkeiten. röntgen-thorax: ausgeprägte interstitielle infiltrationen in beiden lungenflügeln, z. t. auch alveoläre verschattungen. klinische diagnose: interstitielle pneumonie, dringender verdacht auf pneumocystis-carinii-pneumonie. therapie: sofortige behandlung mit hochdosiertem cotrimoxazol und mit ceftriaxon; ferner gabe von prednison. symptomatisch verabreichung von o 2 per nasensonde, behandlung des soors mit amphotericin-b-suspension. weiterführende diagnostik: hiv-serologie im elisa und westernblot positiv; hiv-rna im plasma 820 000 copies/ml; cd4 + -lymphozyten 10/μl, cd8 + -lymphozyten 400/μl; in der bronchoalveolären lavage nachweis von pneumocystis carinii. verlauf: allmählicher rückgang der klinischen symptomatik; nach diagnosesicherung der pcp absetzen von ceftriaxon und behandlung mit co-trimoxazol über 3 wochen. danach einleitung einer antiretroviralen therapie, fortführung der co-trimoxazol-gabe in prophylaktischer dosierung und entlassung in deutlich gebessertem zustand. ii das spektrum der hautveränderungen im rahmen der hiv-infektion umfasst: • infektiöse veränderungen • allergische reaktionen • sog. idiopathische hauterkrankungen. häufig sind herpes-simplex-virus-infektionen, die als harmlose infektionen, aber auch als schwere, chronische ulzerationen imponieren können. varicella-zoster-virus-reaktivierungen (gürtelrose) treten typischerweise schon in frühen stadien der hiv-infektion auf und erstrecken sich häufig über mehrere dermatome. die behandlung der herpesvirusinfektionen kann mit aciclovir, valaciclovir, famciclovir oder brivudin erfolgen. eine andere häufige virusinfektion sind die dellwarzen (mollusca contagiosa), die sehr charakteristisch sind und oft ausgedehnte körperareale befallen (› kap. 13.5). im analbereich kommen gehäuft feigwarzen (condylomata acuminata) vor. die infektionen mit herpes-simplex-und varicella-zoster-virus werden mit aciclovir behandelt. bei dellwarzen und feigwarzen müssen kürettagen angewendet werden. bakterielle infektionskrankheiten, die bei hiv-infizierten vermehrt diagnostiziert werden, umfassen pyodermien, lues und bazilläre angiomatose. letztere wird durch die erst kürzlich entdeckten erreger bartonella henselae und quintana verursacht und äußert sich in form rötlicher, papulöser hautveränderungen. es kann ferner ein disseminierter organbefall vorkommen. die therapie erfolgt mit penicillin (lues), mit staphylokokken-wirksamen antibiotika (pyodermie) und mit makroliden (bazilläre angiomatose). allergische reaktionen und andere hauterkrankungen neben allergischen reaktionen treten gehäuft psoriasis vulgaris, seborrhoisches ekzem, xerodermie und papulöse dermatitiden auf. außer durch dermatologische lokalbehandlung bessern sich diese erkrankungen oft durch verbesserung der immunsituation im rahmen einer erfolgreichen antiretroviralen therapie. die symptomatik reicht von asymptomatischen veränderungen bis zu schmerzhaften läsionen, die eine nahrungsaufnahme fast unmöglich machen (therapie siehe oben). erkrankungen des gastrointestinaltraktes sind häufig bei fortgeschrittener hiv-infektion. im folgenden sind die häufigsten opportunistischen erkrankungen aufgeführt. hinzu kommen einige seltene infektionen und andere erkrankungen, die nicht spezifisch für die hiv-infektion sind. häufigste aids-definierende opportunistische infektion des gastrointestinaltraktes. symptome dysphagie und retrosternale schmerzen, meist auch candida-beläge in der mundhöhle. diagnostik ösophagoskopie mit dem makroskopischen bild weißer schleimhautbeläge und mikrobiologischer oder histologischer nachweis von candida. therapie fluconazol. nach erfolgreicher behandlung treten häufig rezidive auf. dann ist eine prophylaktische behandlung mit fluconazol indiziert. bei den seltenen fällen einer fluconazol-resistenz wird voriconazol eingesetzt. diese infektionen treten meist bei sehr weit fortgeschrittenem immundefekt (cd4 + -zellen < 50/μl) auf und betreffen v. a. gastrointestinaltrakt und auge. erkrankungen des gastrointestinaltraktes durch cmv können in allen abschnitten vorkommen: als ösophagitis, gastritis, enteritis, kolitis und als proktitis. symptome die ösophagitis verursacht eine dysphagie. bei der cmv-gastritis stehen oberbauchschmerzen im vordergrund. die cmv-enterokolitis manifestiert sich mit durchfällen und bauchschmerzen. bei der cmv-proktitis sind defäkationsschmerzen und blutbeimengungen im stuhl die führenden symptome. diagnostik endoskopische untersuchung mit biopsie. makroskopisch sieht man eine entzündung der schleimhaut und ulzerationen, die wie ausgestanzt wirken. histologisch ist der nachweis von intranukleären einschlusskörperchen in vergrößerten zellen (eulenaugen-zellen) typisch. die abgrenzung von anderen viralen infektionen ist durch die immunhistologie (nachweis viraler antigene) möglich. der nachweis des cmv-pp65-antigens im blut und die cmv-pcr aus dem blut geben hinweise auf die diagnose. therapie zurzeit sind 3 substanzen verfügbar: ganciclovir, cidofovir und foscarnet. sie müssen intravenös verabreicht werden und sind in ihrer wirksamkeit vergleichbar. unterschiede bestehen in den nebenwirkungen: ganciclovir ist hämatotoxisch (leukopenie, anämie), während bei foscarnet und cidofovir die nephrotoxizität im vordergrund steht. in schweren fällen können ganciclovir und foscarnet kombiniert gegeben werden kryptosporidien sind protozoen, die bei tieren und bei menschen durchfallerkrankungen auslösen können. sie werden vor allem durch kontaminiertes wasser übertragen. bei immunkompetenten kommt es zu einer milden, selbstlimitierten diarrhö. symptome hiv-patienten mit ausgeprägter immunschwäche erkranken an schwersten wässrigen durchfällen, die den patienten häufig tag und nacht quälen. infolgedessen kommt es zur auszehrung ("wasting syndrom"). diagnostik die kryptosporidien finden sich an der oberfläche des darmepithels und können dort histologisch nachgewiesen werden. nachweis von oozysten im stuhl, darstellbar mit spezialfärbungen. therapie nicht bekannt. besserung erfolgt meist mit der einleitung einer antiretroviralen therapie. symptomatische maßnahmen (loperamid, opiumtinktur) müssen häufig angewandt werden. mikrosporidien sind obligat intrazelluläre, sporenbildende protozoen. von den mehr als 1000 spezies sind bisher 5 als menschenpathogen bekannt. 2 spezies stehen bei hiv-infektion im vordergrund: enterocytozoon bieneusi infiziert darm und gallengänge. die symptome sind von denen bei der kryptosporidien-infektion nicht unterscheidbar. encephalitozoon verursacht eine allgemeininfektion. die diagnose einer mikrosporidiose erfordert spezielle techniken (fluoreszenztest) und ist aus stuhl oder abstrichen und sekreten möglich. die speziesdifferenzierung ist durch elektronenmikroskopie oder pcr möglich und von therapeuti-scher bedeutung: während encephalitozoon auf eine therapie mit albendazol anspricht, ist für enterocytozoon bieneusi keine wirksame therapie bekannt. das wasting-syndrom ist definiert durch eine gewichtsabnahme von mehr als 10% des ausgangsgewichtes, verbunden mit anhaltendem fieber oder chronischer diarrhö ohne erregernachweis. diagnostik behandelbare opportunistische infektionen müssen ausgeschlossen werden. therapie die antiretrovirale therapie führt meist zur besserung. wie alle opportunistischen infektionen sind lungenmanifestationen in den letzten jahren seltener geworden. nach wie vor stellt die pneumocystis-pneumonie als akut lebensbedrohliche infektion häufig die erstmanifestation von aids dar. die tuberkulose ist weltweit die häufigste todesursache hiv-infizierter menschen. epidemiologie weltweit ist die tuberkulose mit abstand die häufigste opportunistische infektion im rahmen der hiv-infektion. ihre größte verbreitung hat sie in den unterentwickelten ländern. aber auch in südeuropa kommt sie sehr häufig vor. an tuberkulose ist besonders zu denken bei drogenabhängigen und bei patienten aus ländern der dritten welt. die lunge ist meist betroffen, doch handelt es sich bei der tuberkulose hiv-infizierter oft um ein disseminiertes krankheitsbild mit befall unterschiedlichster organe. symptome unspezifisch mit fieber, nachtschweiß, gewichtsabnahme und husten. röntgenologisch finden sich an der lunge typische verläufe im sinne einer reaktivierung mit kavernösen veränderungen und atypische verläufe mit flächenhaften infiltraten und mediastinalen lymphknotenschwellungen. diagnostik eine tuberkulose muss bei allen pulmonalen infiltraten bei hiv-infizierten patienten ausgeschlossen werden. die definitive diagnose wird mit der sputumuntersuchung durch den nachweis säurefester stäbchen gestellt, evtl. ferner untersuchung des magensekrets oder bronchoskopie. zur unterscheidung von ubiquitären mykobakteriosen (mycobacterium-avium-komplex) ist eine mikrobiologische differenzierung nötig. therapie dieselben substanzen wie bei nicht-hiv-infizierten patienten (› kap. 10.5) meist über insgesamt 6 monate, bei komplizierten fällen auch länger. das ansprechen auf die therapie ist allgemein gut. multiresistente tuberkuloseerreger kamen in einigen amerikanischen großstädten bei hiv-infizierten gehäuft und mit sehr hoher letalität vor. in deutschland sind solche infektionen bisher nicht aufgetreten. wichtigste maßnahme zur verhütung von resistenzbildungen ist eine konsequente und effektive therapie. hervorgerufen durch den ubiquitär vorkommenden pilz (früher als protozoon klassifiziert) pneumocystis jiroveci (früher pneumocystis carinii, daher die abkürzung pcp). bereits im kindesalter besteht fast hundertprozentige durchseuchung, so dass das risiko einer erkrankung nur vom ausmaß des immundefektes abhängt. ohne prophylaktische maßnahmen beträgt die inzidenz für patienten mit cd4 + -zellen < 100/μl ca. diagnostik ein klinischer verdacht auf pcp muss sofort weiter abgeklärt werden. bei der körperlichen untersuchung finden sich häufig zyanose und tachypnoe. der auskultationsbefund ist aber typischerweise normal. im röntgenbild des thorax sieht man eine interstitielle zeichnungsvermehrung, in schweren fällen können auch alveoläre verschattungen auftreten (› abb. 13.10). obligate blutuntersuchungen sind die bestimmung der ldh (erhöht) und eine arterielle blutgasanalyse (erniedrigung des po 2 ), da beide parameter prognostische aussagekraft haben. die leukozyten sind meist nicht vermehrt. bei patienten mit den aufgeführten diagnostischen kriterien kann die klinische diagnose einer pcp gestellt werden. die definitive diagnose wird durch den erregernachweis (immunfluoreszenz oder andere färbetechniken) aus der bronchoalveolären lavage (bal) gestellt. therapie bei verdacht auf pcp muss sofort eine therapie eingeleitet werden. dies steht einer späteren diagnosesicherung nach wenigen tagen nicht im wege. mittel der wahl ist co-trimoxazol in hoher dosierung (20 mg trimethoprim, 100 mg sulfamethoxazol/kg kg und pro tag). die therapiedauer beträgt normalerweise 3 wochen. bei schweren unverträglichkeitserscheinungen (allergie) muss auf intravenös verabreichtes pentamidin ausgewichen werden. eine pcp mit arteriellem ausgangs-po 2 von 70 mmhg oder weniger muss zusätzlich mit prednison (2-mal 50 mg/d) behandelt werden, da so die letalität vermindert wird. unbehandelt ist eine pcp immer letal. auch bei optimaler behandlung handelt es sich um eine ernste erkrankung. ungünstige prognostische parameter sind eine ausgeprägte erniedrigung des po 2 und eine starke erhöhung der ldh. patienten mit beatmungspflichtiger respiratorischer insuffizienz haben eine schlechte prognose. bakterielle pneumonien treten schon bei einer helferzellzahl über 200/μl auf, werden aber mit zunehmendem immundefekt häufiger. drogenabhängige sind öfter betroffen als homosexuelle patienten. häufigste erreger sind pneumokokken, haemophilus influenzae, staphylokokken und gramnegative erreger. symptome wie bei pneumonien bei immunkompetenten personen (› kap. 10.4.1). diagnostik von der pcp ist die bakterielle pneumonie bei typischem verlauf durch raschen beginn, purulentes sputum, positiven auskultationsbefund und durch die röntgenuntersuchung abzugrenzen. es gibt aber häufig atypische verläufe, die eine klinische unterscheidung unmöglich machen. therapie bei leichtem verlauf mit cephalosporin der 2. generation bzw. ampicillin plus lactamasehemmer, bei schwerem verlauf kombinationstherapie mit cephalosporinen der 3. generation und einem makrolid oder mit einem chinolon mit pneumokokken-wirksamkeit (moxifloxacin, levofloxacin). bis zum ausschluss einer pcp sollte bei schweren pneumonien auch co-trimoxazol gegeben werden. pneumonien durch pilze kommen insgesamt selten bei hiv-infizierten vor. aspergillus-pneumonien treten im endstadium des immundefektes auf und haben eine schlechte prognose. infektionen mit kryptokokken manifestieren sich gelegentlich an der lunge, meist jedoch erst bei weiterer disseminierung als fungämie oder meningitis. andere pulmonale pilzinfektionen sind in europa sehr selten. candida-pneumonien werden bei hiv-infizierten nicht beobachtet. virale pneumonien sind ebenso sehr selten. zytomegalievirus wird zwar häufig in der lunge nachgewiesen, es handelt sich aber fast immer um eine infektion ohne krankheitswert. symptome des zentralen und des peripheren nervensystems treten im rahmen der hiv-infektion sehr häufig mit sehr vielgestaltigen ursachen auf. neurologische symptome können durch hiv selbst, durch opportunistische erkrankungen oder als unerwünschte wirkungen therapeutischer maßnahmen auftreten. die differentialdiagnose ist daher sehr schwierig. ca. 50% der bevölkerung sind mit dem protozoon toxoplasma gondii infiziert. als opportunistische infektion im rahmen der hiv-infektion tritt die toxoxplasmose aber nur bei schwer eingeschränktem immunstatus auf (cd4 + -zellen < 100/μl). als zeichen der früher erfolgten infektion finden sich antikörper im serum. bei fehlenden igg-antikörpern ist eine toxoplasmose sehr unwahrscheinlich. symptome die zerebrale toxoplasmose äußert sich in fieber, kopfschmerzen, neurologischen ausfällen und epileptischen anfällen. diagnostik nachweis von typischen veränderungen in der kernspintomografie oder computertomografie des schädels möglich (› abb. 13.11). therapie pyrimethamin und sulfadiazin. bilden sich die veränderungen hierunter zurück, ist die diagnose bestätigt. eine prophylaxe mit verminderter dosis ist wegen hoher rezidivgefahr anzuschließen. wenn sich die erkrankung unter der therapie nicht bessert, sollte eine stereotaktisch gewonnene biopsie erfolgen zum ausschluss anderer erkrankungen (z. b. lymphom). durch den hefepilz cryptococcus neoformans kann eine meningitis ausgelöst werden. diese infektion kommt v. a. in afrika und den usa häufig vor, bei uns ist sie seltener. symptome kopfschmerzen und fieber, oft über wochen progredient. diagnostik bestimmung des kryptokokken-antigens im serum mit sehr hoher sensitivität. zum ausschluss einer anderen meningitisform muss eine punktion des liquor cerebrospinalis durchgeführt werden, in dem sich die kryptokokken durch ein tuschepräparat nachweisen und kulturell anzüchten lassen. therapie amphotericin b und flucytosin und zusätzlich evtl. fluconazol. ca. 1/3 der erwachsenen patienten und 2/3 der kinder weisen symptome einer hiv-enzephalopathie auf. sie wird vermutlich durch direkte infektion des zns mit hiv verursacht und führt zu psychomotorischen störungen unterschiedlichen schweregrades. symptome das klinische bild ist sehr variabel. meist stehen konzentrations-und gedächtnisstörungen bis hin zur ausgeprägten demenz im vordergrund, aber auch epileptische anfälle und wesensveränderungen kommen vor. diagnostik nur durch den ausschluss anderer zns-manifestationen möglich. im liquor finden sich nur unspezifische veränderungen, in der kernspintomografie kann eine hirnatrophie erkennbar sein. therapie antiretrovirale behandlung. dabei gibt es verschiedenartige verläufe von der vollständigen rückbildung bis hin zur weiteren progredienz der veränderungen. die progressive multifokale leukenzephalopathie (pml) ist eine meist innerhalb von wochen bis monaten zum tode führende erkrankung des zns, die durch polyoma-viren (jc-virus) ausgelöst wird. charakteristisch sind zunehmende neurologische störungen bei meist erhaltenem bewusstsein und ausgedehnte läsionen im marklager, die sich am besten in der kernspintomographie darstellen. die diagnose kann durch pcr aus dem liquor oder durch hirnbiopsie gestellt werden. unter hochaktiver antiretroviraler therapie kommt es bei einem teil der patienten zur besserung. die cmv-enzephalitis ist eine meist rasch progredient verlaufende zerebrale infektion, die klinisch nicht von anderen enzephalitiden zu unterscheiden ist. die diagnose kann durch eine pcr aus dem liquor untermauert werden, die therapie erfolgt mit foscarnet oder ganciclovir. das zerebrale lymphom ist eine opportunistische erkrankung im endstadium der hiv-infektion. die diagnose kann heute mit hoher wahrscheinlichkeit durch den nachweis von ebv-dna im liquor cerebrospinalis mittels pcr erfolgen. eine definitive sicherung ist nur durch hirnbiopsie möglich. kurzfristige therapieerfolge können mit dexamethason erzielt werden. eine systemische chemotherapie ist nicht wirksam, und auch eine bestrahlung führt meist nur zur sehr kurzfristigen remission. bei der polyneuropathie handelt es sich um eine erkrankung, die sehr häufig in späten stadien der hiv-infektion auftritt. neben einer direkten neuropathischen wirkung des hiv kommen toxische wirkungen von medikamenten ursächlich in frage. die diagnose wird meist klinisch gestellt, elektroneurografische untersuchungen können ggf. zusätzlich erfolgen, ist die polyneuropathie medikamentös ausgelöst, kommt es nach absetzen zur besserung. andernfalls bestehen die beschwerden meist fort. die therapie ist symptomatisch (gabapentin, amitriptylin, carbamazepin). neurotoxische substanzen (d4t, ddi) müssen abgesetzt werden. die zytomegalievirus(cmv)-retinitis tritt bei patienten mit sehr schwerem immundefekt auf (cd4 + -zell-zahlen unter 50/μl). symptome verschwommenes sehen und sehminderung. unbehandelt führt die erkrankung zur erblindung. diagnostik spiegelung des augenhintergrundes mit charakteristischem befund. therapie intravenöse infusionen von ganciclovir, foscarnet oder cidofovir werden eingesetzt. viele der infektionen, die bei aids-patienten auftreten, verlaufen als disseminierte infektionen. dies gilt auch für einen teil der oben beschriebenen erkrankungen (z. b. cmv-infektion, tuberkulose). im folgenden werden diejenigen infektionen vorgestellt, die primär als generalisierte erkrankung durch den erregernachweis im blut diagnostiziert werden. in der regel manifestieren sich diese infektionen als sepsis, d.h mit klinischen symptomen (fieber, tachykardie, tachypnoe) und nachweis von bakterien im blut (bakteriämie). oftmals treten bakteriämien im zusammenhang mit intravenös platzierten kathetern auf. hier spielen vor allem staphylokokken, aber auch gramnegative keime wie pseudomonas aeruginosa eine rolle. pneumokokken-bakteriämien kommen im zusammenhang mit pneumonien vor. eine aids-definierende, selten auftretende komplikation ist die rezidivierende salmonellen-sepsis. diagnostik die blutkultur ist entscheidende maßnahme. therapie die behandlung erfolgt jeweils mit wirksamen antibiotika (antibiogramm). prognose neben der rechtzeitigen diagnose und der antibiotika-sensitivität des erregers ist der allgemeinzustand des patienten ausschlaggebend. mycobacterium avium und intracellulare bilden den mycobacterium-avium-komplex und kommen ubiquitär in der umwelt vor. bei immunkompetenten verursachen sie nur ganz selten infektionen. dagegen ist die disseminierte mac-infektion eine der schwersten infektionen bei hiv-patienten mit hochgradigem immundefekt (cd4 + -zellen < 50/μl). die aufnahme des erregers erfolgt entweder über den magen-darm-trakt oder über die lunge. hier kommt es zunächst zur kolonisierung und im weiteren verlauf zur disseminierung. der nachweis des erregers aus sputum oder stuhl beweist noch keine disseminierte infektion. symptome die disseminierte infektion äußert sich durch fieber, nachtschweiß, gewichtsabnahme, durchfälle, lymphknotenschwellungen, bauchschmerzen und eine hepatosplenomegalie. diagnostik die laborwerte zeigen meist eine anämie und eine erhöhung der alkalischen phosphatase. die diagnose lässt sich durch die anzüchtung des erregers aus der blutkultur oder anderen sterilen materialien (knochenmark, lymphknoten, leber) sichern. im gegensatz zur tuberkulose sind lungeninfiltrate durch mac selten. therapie auf die herkömmlichen antituberkulotika kann man nicht zurückgreifen, da der erreger gegen die meisten dieser mittel primär resistent ist. am wirksamsten ist eine kombination aus clarithromycin und ethambutol (eventuell zusätzlich mit rifabutin). als hiv-assoziierte tumoren (kategorie cdc c) werden das kaposi-sarkom (s. o.), das non-hodgkin-lymphom und das invasive zervixkarzinom gezählt. non-hodgkin-lymphome (nhl) treten bei ca. 5-10% aller aids-patienten auf. histologisch handelt es sich meist um hochmaligne b-zell-lymphome. ein disseminierter und extranodaler befall liegt häufig vor. symptome entsprechend dem befallsmuster: lymphknotenschwellungen und allgemeinbeschwerden (fieber, nachtschweiß) sind häufig; bei knochenmarkbefall kommt es zur panzytopenie, bei befall des magen-darm-traktes zu bauchschmerzen und gewichtsabnahme. im labor findet sich oft eine erhöhung der ldh. bei zns-befall kommt es zum auftreten neurologischer herdsymptome (anfälle, lähmungen). eine besonderheit ist das auftreten primärer zerebraler lymphome, die immer durch ebstein-barr-virus (ebv) ausgelöst sind (diagnostik durch nachweis von ebv-dna mittels pcr im liquor). therapie durchführung einer standard-chemotherapie (chop-schema: cyclophosphamid, adriamycin, vincristin, prednison). ziel ist heute die komplette remission und heilung auch in fortgeschrittenen stadien und bei fortgeschrittenem immundefekt. hierzu werden zunehmend auch aggressive therapieschemata eingesetzt. prognose nicht gut (heilung in < 50%). maligne tumoren, die durch humane papillomaviren (hpv) induziert werden, wurden bei hiv-patienten gehäuft beobachtet. hierzu zählen das zervixkarzinom der frau und plattenepithelkarzinome der analregion. außerdem wurde über vermehrtes auftreten von hodgkin-lymphomen berichtet. die prophylaxe von infektionen bereits vor deren erstem auftreten (primärprophylaxe) oder nach der ersten episode (sekundärprophylaxe) ist weiterhin eine wichtige aufgabe bei der betreuung hiv-positiver patienten, auch wenn opportunistische infektionen durch die antiretrovirale therapie insgesamt seltener geworden sind. klinisch von bedeutung ist die prophylaxe der pcp bei einer cd4 + -zell-zahl von < 200/μl mit cotrimoxazol. die beste vorbeugung aller opportunistischen infektionen ist eine wirksame antiretrovirale kombinationstherapie (haart). durch die verbesserte funktion des immunsystems ist ein schutz gegen opportunistische infektionen vorhanden. in vielen studien wurde nachgewiesen, dass patienten mit supprimierter viruslast ein sehr niedriges risiko für opportunistische infektionen aufweisen. steigen die cd4-zellen dauerhaft auf > 200/μl an, können meist primär-und sekundärprophylaxen abgesetzt werden. patienten, die trotz antiretroviraler therapie deutlich unter 200 helferzellen/μl bleiben, müssen dagegen weiter prophylaktisch behandelt werden. viren erreichen den zustand der persistierenden infektion durch unterschiedliche strategien. beim herpes-simplex-virus (humanes α-herpesvirus) kommt es z. b. nach primärinfektion an haut oder schleimhäuten mit lokaler virusvermehrung (produktive infektion) zur zunächst ebenso produktiven infektion zugehöriger sensibler ganglienzellen. im ganglion geschieht dann die "umschaltung" in eine latente infektion, die durch fehlende virusproduktion und jegliches fehlen von viruspartikeln gekennzeichnet ist. das virale dna-genom bleibt extrachromosomal bei minimaler expression einzelner gene in den ganglien. durch bestimmte triggermechanismen (sonbei zytozidaler virusinfektion kommt es am ende des virusvermehrungszyklus zum tod der wirtszelle. es gibt persistierende und nicht persistierende viren, bei denen aus der weiterhin vitalen wirtszelle nachkommen-viruspartikel ausgeschleust werden. während die folgen einer zytozidalen infektion für den organismus entsprechend dem "alles-odernichts-prinzip" wesentlich von art und anzahl der direkt zerstörten zellen abhängen, kommt es bei nichtzytozidalen virusinfektionen eher zu störungen der wirtszellregulation (z. b. embryopathie oder onkogenese) oder auch sekundär zur immunpathogenese. viele viren sind in der lage, durch gezielte modulation der wirtszell-genexpression abwehrmechanismen der zelle und des organismus (immunsystem, apoptose) zu unterlaufen (sog. immunevasion). 13 .14 und › abb. 13.13). die größe der einzelnen gruppen ist je nach erreger, prophylaktischen maßnahmen (z. b. impfungen) und therapiemöglichkeiten sehr verschieden. vereinzelt können persistierend mit hepatitis-b-virus infizierte patienten noch nach jahren die infektion beenden (serokonvertieren) und so aus der gruppe der persistierend infizierten in die gruppe der immunen wechseln. aus der gesamtpopulation (gelb) treten individuen nach infektion in die gruppe der akut infizierten (rosa). der weitere verlauf hängt davon ab, ob viren persistenzmechanismen entwickelt haben, und von der fähigkeit des wirts, eine protektive immunantwort zu bilden. die manifestationsrate bestimmt den anteil der erkrankten und die letalität den der verstorbenen. bei verlust der spezifischen immunität aufgrund einer immunsuppression können zuvor immune in die erneut infizierbare gesamtpopulation oder selten sogar bei einigen viren (z. b. hepatitis-b-virus) in die gruppe der persistent infizierten zurückkehren. viele infektionen verlaufen beim immungesunden subklinisch. primärinfektionen und rekrudeszenzen können aber auch vielfältige erkrankungen hervorrufen. patienten mit immundefekten sind durch diese viren besonders bedroht. erstaunlicherweise sind die krankheitsbilder bei primärinfektion und reaktivierung nicht nur abhängig vom ausmaß, sondern auch von der art der immunsuppression. als beispiel sei die cmv-infektion genannt, die beim immunkompetenten meist asymptomatisch verläuft. bei hiv-infizierten patienten tritt cmv dagegen in erster linie als retinitis und gastroenteritis, beim knochenmarktransplantierten patienten als interstitielle pneumonie und beim nierentransplantierten als nephritis mit gefahr der abstoßung auf. herpesvirusinfektionen sind mit ausnahme von epstein-barr-virus (ebv) und hhv-6 bis hhv-8 der therapie durch verschiedene verfügbare antivirale substanzen gut zugänglich. das herpes-b-virus des rhesusaffen ist auch hochpathogen für den menschen (enzephalitis). herpes-simplex-virus typ 1 und 2 (hsv-1, hsv-2) beschreibung und einteilung es handelt sich eigentlich eher um varianten eines serotyps, da serologisch erhebliche kreuzreaktionen bestehen. virusisolate sind relativ leicht typisierbar und inzwischen kann man auch zwischen hsv-1-und hsv-2-antikörpern unterscheiden. epidemiologie die primärinfektion mit hsv-1 findet mit 2 gipfeln in der frühen kindheit und im jungen erwachsenenalter meist oral statt. die durchseuchung erwachsener mit hsv liegt weltweit je nach sozioökonomischer situation bei 40-95%. die primärinfektion (erster kontakt eines organismus mit hsv) kann auch durch sexualkontakt, dann meist mit hsv-2, erfolgen. bei bestehender oraler hsv-1-infektion mit latenz in kranialen ganglien ist die erste infektion im genitale mit hsv-2 keine primärinfektion, sondern eine klinisch milder verlaufende exogene zweitinfektion (initiale infektion). die prävalenz von hsv-2-antikörpern ist bei erwachsenen in verschiedenen kollektiven sehr unterschiedlich, aber stets geringer als bei hsv-1. die virusvermehrung auf der schleimhaut (infektiosität) beginnt vor dem auftreten der symptome, und die virusausscheidung erfolgt durchschnittlich 7-10 tage lang (max. bis 23 tage). im gegensatz zur hsv-1-primärinfektion kommt es bei hsv-2 offenbar auch zur virämie. ätiologie und pathogenese während der virusvermehrung auf der schleimhaut werden bereits frühzeitig auch nervenendigungen infiziert. über axonalen transport ( der chronische, lokal destruierende mukokutane herpes simplex ist eine aids-manifestation; es entstehen z. b. größere perianale ulzera, die in der differentialdiagnose der klassischen venerischen infektionen durch ihre schmerzhaftigkeit von der lues abzugrenzen sind und einen belegten, weichen ulkusgrund aufweisen. die weitaus häufigste hsv-erkrankung ist der rekurrierende orale oder genitale herpes. beide haben beim immungesunden eine gute prognose, können aber zu erheblichen psychischen belastungen, bis hin zum suizid, führen. orale und genitale primärinfektionen können zu schweren erkrankungen führen, die frühzeitig und intensiv systemisch behandelt werden sollten. die rekurrierende ulzerative herpeskeratitis führt häufig zum visusverlust -ggf. mit der notwendigkeit einer hornhauttransplantation. enzephalitis, konnatale infektion und infektionen immunsupprimierter können sehr rasch zu lebensbedrohlichen erkrankungen führen und müssen daher schnellstmöglich intensiv behandelt werden. die virologische diagnose kann sehr schnell gestellt werden, wenn das geeignete material mit geeigneten methoden untersucht wird. in der akuten phase der mononukleose können 5-20% der zirkulierenden b-zellen ebv-infiziert sein (polyklonale transformation). es treten teils heterophile autoantikörper auf, was diagnostisch genutzt wird (paul-bunnell-test). im regelfall werden die ebv-infizierten b-zellen durch das intakte immunsystem (t-zellen) eliminiert, dies gelingt aber nicht vollständig, sondern es verbleiben einige latent infizierte b-zellen mit der möglichkeit der reaktivierung im späteren leben (s. u.). eine assoziation des burkitt-lymphoms mit ebv ist aufgrund molekularbiologischer und seroepidemiologischer daten gesichert. ebenso eindeutig ist der zusammenhang zwischen ebv und dem nasopharynxkarzinom (npc), das endemisch in einigen gegenden afrikas und v. a. in südchina vorkommt. mit zunehmendem alter wird das bild der infektiösen mononukleose (im) häufiger beobachtet: sie geht einher mit fieber, pharyngitis und tonsillitis mit gräulichen belägen, generalisierter oder zervikookzipital betonter lymphadenopathie, exanthem (selten enanthem), hepatitis und splenomegalie. das fieber dauert ca. 7-10 tage an und fällt wieder ab. es besteht eine kutane anergie wie beim morbus boeck, bei fortgeschrittener hiv-infektion und anderen schweren krankheitsbildern (disseminierte tuberkulose). eine restsymptomatik (subfebrile temperaturen, müdigkeit) kann monatelang anhalten. eine produktive ebv-infektion ist häufig als orale haarleukoplakie am seitlichen zungenrand bei aids und anderen schweren immundefekten nachweisbar. chronisch aktive ebv-infektionen mit lang anhaltenden, rezidivierenden organsymptomen wurden mit familiärer häufung beschrieben. es ist bislang unklar, ob in diesen fällen ein genetischer defekt oder eine besondere virusvariante verantwortlich ist. erkrankungen der blutzellen und immunorgane eine massive b-zell-proliferation mit nachfolgender kontrolle durch induzierte spezifische t-zellen gehört zum krankheitsbild der im. beim "duncan-syndrom" sind die patienten aufgrund eines genetischen defektes nicht in der lage, diese proliferation zu kontrollieren. die latente ebv-infektion wird durch nicht produktiv infizierte b-lymphozyten aufrechterhalten, die durch den nachweis von ebna-2 (ebv-nukleäres antigen) charakterisiert sind. proteine, die von latent infizierten zellen gebildet werden können, sind für die rolle des ebv in der entstehung von tumoren verantwortlich. sehr gefürchtet ist das lymphozyten-proliferationssyndrom bei immunsupprimierten nach transplantation. ebv ist in b-zell-lymphomen bei hiv-infektion, nach organtransplantation und beim morbus hodgkin nachzuweisen. vor allem in asien kommt es gelegentlich zu einer ebv-induzierten überschießenden t-zell-aktivierung, die letztlich zum hämophagozytotischen syndrom führt. weitere organbeteiligungen eine beteiligung von ebv an erkrankungen, die von infektionen des lungenepithels im rahmen einer chronisch aktiven ebv-infektion ausgehen, bis hin zur beteiligung an der idiopathischen lungenfibrose ist vielfach diskutiert worden. myokarditiden können bei im auftreten und die bestimmenden beschwerden während der rekonvaleszenz sein. die pro gnose ist insgesamt gut. die benigne hepatitis mit mäßig erhöhten transaminasen ist typisch bei der primären ebv-infektion. vielfach werden chronische ebv-reaktivierungen als ursache von anhaltenden gastrointestinalen beschwerden und gelegentlich auch hepatopathien angenommen. inwieweit ein kausaler zusammenhang besteht, ist aber meist unklar und auch schwer zu klären, da ebv-reaktivierungen auch bei anderen grunderkrankungen vorkommen. besondere klinische bedeutung hat cmv für alle immuninkompetenten (untergewichtige frühgeborene, transplantatempfänger, tumorpatienten, aids-patienten). es existieren befunde, wonach cmv evtl. auch bei immungesunden in zellen der gefäßwände durch modulation der zellulären genexpression veränderungen hervorruft, die zur entstehung der atherosklerose und zur entwicklung der restenose beitragen. bei immundefizienten und immunsupprimierten hängt die schwere der erkrankung vom ausmaß der beeinträchtigung des immunsystems ab. mit zunehmender dysfunktion der t-zellen nehmen cmv-reaktivierungen und persistierende aktive cmv-infektionen zu. diese kündigen sich noch vor der klinischen manifestation einer cmv-erkrankung durch lang anhaltende intermittierende oder kontinuierliche cmv-ausscheidung meist im urin an. asymptomatische infektionen die primärinfektion verläuft bei immungesunden älteren kindern und erwachsenen in ca. 90% asymptomatisch. symptomatische infektionen sind klinisch von einer infektiösen mononukleose nicht zu unterscheiden. endogene reaktivierungen mit virusausscheidung, die von zeit zu zeit in abhängigkeit von der aktuellen immunkontrolle der infektion durch den organismus ablaufen, werden im allgemeinen nicht bemerkt. die infektion verläuft bei reifen neugeborenen auch asymptomatisch, ist aber von einer u.u. langen cmv-ausscheidung begleitet. hno-erkrankungen ca. 8% aller klinisch diagnostizierten mononukleosen sind cmv-bedingt. die klinischen zeichen treten nach einer inkubationszeit von 20-60 tagen auf. der verlauf ist gutartig; neben fieber, lymphadenopathie, pharyngitis bzw. tonsillitis, hepatitis, splenomegalie und exanthem treten selten blutbildveränderungen (leukopenie, relative lymphozytose mit lymphomonozytären reizformen, thrombopenie) und gelegentlich eine parotitis (dd mumps) auf. cmv bei immunsuppression und aids bei aids-patienten war die infektion vor der intensiven antiretroviralen therapie (haart) am häufigsten mit einer cmv-retinitis (› abb. 13.21) assoziiert, gefolgt von gastrointestinalen erkrankungen und enzephalitis. die aktiven cmv-infektionen bei 25-90% der aids-patienten sind meist folge einer cmv-reaktivierung. die in anderen klinischen situationen bedeutsame diagnostische unterscheidung zwischen primärinfektion und reaktivierung spielt daher bei aids-patienten keine rolle. cmv-enzephalitiden wurden v. a. bei aids-patienten vor einführung von haart häufiger beobachtet. auch die interstitielle pneumonie ist eine der typischen cmv-erkrankungen, die meist bei erheblich immunsupprimierten transplantatempfängern auftritt. cmv-bedingte hepatitis ist häufig bei konnataler infektion, aber auch möglich bei virusreaktivierungen bei immunsupprimierten. gastrointestinale infektionen mit typischen, teils blutenden schleimhautulzera waren vor einführung der intensiven antiretroviralen therapie eine häufige erkrankung bei aids-patienten und werden gelegentlich bei anderen risikopatienten gefunden. ulzerationen können in allen abschnitten des gastrointestinaltraktes auftreten, vom ösophagus bis zum enddarm (› abb. 13.22). cmv spielt eine wesentliche rolle bei nierentransplantierten. neben lang anhaltenden asymptomatischen virusausscheidungen mit dem urin kommt es zu nephritiden und transplantatabstoßungen. eine primäre und sekundäre thrombozytopenie, aber auch trizytopenie ist typisches symptom bei konnataler infektion und häufig erstes symptom einer cmv-reaktivierung bei knochenmarktransplantierten patienten. intrauterine und perinatale infektionen das krankheitsbild der konnatalen zytomegalie umfasst die in › tabelle 13.25 angegebenen symptome. hinzu kommen können weitere symptome, so auch zahnbildungsschäden. cmv ist heute hauptursache einer intrauterinen infektion des fetus (0,2-2,2%). etwa 5% der intrauterin infizierten kinder zeigen das typische bild einer konnatalen zytomegalie (cid mit einschluss des zns: letalität bis 20% und häufig bleibende schäden). die prognose dieser kinder ist schlecht (gesamtletalität 11%). spätschäden (neurologische defizite, hörverlust) sind zu erwarten. weitere 5% der intrauterin infizierten haben geringfügige symptome bei der geburt, die prognose ist sehr viel besser, in 10% ist auch hier mit spätschäden zu rechnen (› tab. 13.25). fetale infektionen nach reaktivierter infektion bei der mutter führen sehr selten zu klinischen manifestationen, und wenn, dann mit deutlich schwächer ausgeprägter symptomatik als bei primärinfektionen und ohne spätfolgen. bei untergewichtigen frühgeborenen besteht auch nach postnataler infektion (z. b. durch muttermilch) ein hohes risiko, an einer schweren systemischen cmv-infektion zu erkranken. diagnostik bei verdacht auf eine cmv-pneumonie ist die röntgenologische untersuchung wichtig. für die diagnose einer aktiven cmv-infektion, aber auch für die bestimmung der prognose und für therapieindikation und -kontrolle stehen heute quantitative nachweismethoden für virale antigene und dna zur verfügung. • virusnachweis: die cmv-isolierung aus urin, bronchiallavage, speichel u.ä. ist in humanen fibroblasten möglich, sie braucht im gegensatz zu hsv aber viel länger. hier kann der nachweis von early-virus-antigen -bereits vor dem erscheinen des zytopathischen effekts in der infizierten zellkultur -die diagnostik beschleunigen (shell vial culture). eine typische histopathologische veränderung sind die eulenaugenzellen, deren nachweis zwar sehr spezifisch, aber wenig sensitiv ist (› abb. 13.23). • nachweis viraler antigene: der quantitative nachweis von cmv-pp65-antigen in polymorphkernigen leukozyten eignet sich zur früherkennung einer systemischen cmv-reaktivierung. das pp65-antigen (s. o.) ist v. a. bei systemischen infektionen immunsupprimierter während der phase der antigenämie überwiegend in polymorphkernigen leukozyten (pmnl) und zirkulierenden endothelzellen zu finden und damit von großer diagnostischer bedeutung. • das verfahren zur isolierung von hhv-6 entspricht dem vorgehen beim versuch der retrovirusisolierung. hhv-7 wurde erstmals 1990 beim gesunden isoliert. mittlerweile ist gesichert, dass in allen untersuchten populationen erwachsene zu 40-100% mit hhv-6 und hhv-7 infiziert sind, dass beide viren auch aus gesunden isoliert werden können und die durchseuchung in früher kindheit beginnt. offenbar erfolgt die hhv-7-serokonversion später. die übertragung geschieht sehr effektiv über den speichel: 50-100% aller hhv-6-infizierten scheiden hierüber das virus aus. im übrigen dienen auch hier die lymphozyten als virusreservoir. die geschichte von hhv-6 zeigt einmal mehr, wie vorsichtig man bei der ätiologischen verknüpfung des nachweises eines ubiquitären virus mit spezifischen symptomen oder syndromen sein muss. ätiologie und pathogenese ätiologisch sind hhv-6b und hhv-7 bei kindern verantwortlich für das exanthema subitum (dreitagefieber). ferner gibt es beschreibungen von schwereren krankheitsfällen. denkbar ist, dass, wie bei den anderen herpesviren, bestimmte immunologische voraussetzungen zu besonderer pathogenität führen. die klinische bedeutung von hhv-6a ist noch unklar. bei immunkompetenten kindern ist das dreitagefieber oder exanthema subitum (es) eine der klassischen "kinderkrankheiten": nach dreitägiger fieberphase kommt es gleichzeitig mit der entfieberung zum stammbetonten kleinfleckigen exanthem. das es ist häufiger begleitet von übelkeit, erbrechen und auch durchfall. bei erwachsenen kann es zum mononukleoseähnlichen krankheitsbild mit langer rekonvaleszenz kommen. bei immunsupprimierten kommen neurologische, pulmonale und hämatologische komplikationen vor. diagnostik im labor ist eine leukopenie mit relativer lymphozytose zu erkennen, eine thrombopenie kann ebenfalls vorliegen. • nachweis viraler genome: hhv-6-dna kann während der akuten infektion durch pcr leicht aus lymphozyten und speichel nachgewiesen werden: nach überstehen der primärinfektion geht die zahl der latent infizierten lymphozyten erheblich zurück, so dass die pcr im peripheren blut nur noch in 10% aller fälle ein positives ergebnis zeigt. durch quantitative pcr lässt sich ein reaktivierungsereignis diagnostizieren. • antikörpernachweis: die serodiagnostik ist in ihrer aussage durch die hohe durchseuchung von ca. 80% im 2. lebensjahr eingeschränkt. für die frische infektion kommen daher die igg-serokonversion und der igm-nachweis in frage -jedoch sind viele igm-nachweisverfahren qualitativ nicht zufrieden stellend. • virusisolierung: aus speichel und lymphozyten kann hhv-6 durch kokultivierung mit stimulierten nabelschnurlymphozyten isoliert werden: die anzucht ist auf wändig, gelingt aber auch bei gesunden virusträgern -und hier eher aus speichel als aus peripherem blut. therapie obwohl die wirksamkeit verschiedener nukleosidanaloga in vitro gezeigt werden konnte, gibt es keine guten daten zur klinischen wirksamkeit. symptomatische therapie. therapieversuche mit foscarnet oder nukleosidanaloga sind bei schweren erkrankungen immunsupprimierter patienten u.u. angezeigt. durch die entdeckung von hhv-6 und hhv-7 wurde endlich das alte rätsel des dreitagefiebers gelöst, von dem man schon seit langem annahm, dass es sich um eine infektionserkrankung handeln könnte. beim immungesunden erwachsenen kommt es gelegentlich zu schwereren, lang dauernden, mononukleoseähnlichen erkrankungen. einzelne fulminante hepatitiden wurden beobachtet. hhv-6 kann zu verschiedenen komplikationen bei immunsupprimierten patienten (pneumonie, enzephalitis) führen. beschreibung und einteilung humanes herpesvirus typ 8 (hhv-8) wurde zunächst über pcr als herpesvirusspezifische genetische information in einem aids-assoziierten kaposi-sarkom (ks) entdeckt. es ließ sich dann als freies virus aus hhv-8-assoziierten b-zell-lymphomen isolieren und als gamma-herpesvirus charakterisieren. epidemiologie antikörper gegen hhv-8 sind im elisa bei fast allen kaposi-sarkom-trägern, bei 30% der hiv-positiven homosexuellen und zum geringen prozentsatz bei blutspendern nachweisbar. damit ist hhv-8 offensichtlich nicht so verbreitet wie andere herpesviren. die antikörperprävalenz macht es wahrscheinlich, dass hhv-8 überwiegend durch sexualkontakte übertragen wird. pathogenese gamma-herpesviren wirken potenziell transformierend. hhv-8-genom wird mit der pcr inzwischen auch in kaposi-sarkomen von therapeutisch immunsupprimierten transplantationspatienten, in spontanen kaposi-sarkomen und den relativ seltenen hhv-8-assoziierten body-cavity-lymphomen nachgewiesen. kaposi-sarkome bestehen typischerweise aus einem gemisch proliferierender spindel-und endothelzellendie zur entstehung führenden mechanismen sind noch ungeklärt. enterovirusinfektionen kommen bei uns ganz überwiegend im sommer und herbst vor ("sommergrippe"). die aus-scheidung der enteroviren beginnt 2-3 tage nach infektion. sie kann einige tage lang oral erfolgen und für mehrere wochen fäkal. bei der übertragung handelt es sich generell um eine enterale "schmutz-schmierinfektion", wobei in den ländern mit hohem hygienestandard die übertragung durch rachensekrete bedeutsamer ist. sehr selten werden bei schweren immundefekten (z. b. bei agammaglobulinämie) dauerausscheider beobachtet. die paralytische poliomyelitis konnte während der letzten 30 jahre in den westlichen industrieländern durch impfung im rahmen des who-eradikationsprogramms drastisch vermindert werden (europa 1951-1955: ca. 50 000 fälle jährlich, deutschland ist seit 1990 frei von infektionen, s. u.). die seroprävalenz der hepatitis-a-antikörper hat in deutschland seit dem zweiten weltkrieg ebenfalls stark abgenommen (kriegsgeneration bis zu 80% seropositiv, studenten heute < 5%); die bedeutung der hepatitis a als reiseerkrankung (entwicklungsländer) nimmt demzufolge zu. das hepatitis-a-virus wird im gegensatz zu anderen picornaviren, die auch oral ausgeschieden werden, nur fäkal und v. a. in der späten inkubationsphase ausgeschieden. pathogenese picornaviridae führen zu unterschiedlich stark ausgeprägter zytozidaler virusvermehrung, also in der regel zu nicht persistierenden infektionen. die partikelproduktion erfolgt zunächst in epithelzellen des nasen-rachen-raums bzw. magen-darm-trakts und in den regionalen lymphknoten und findet erst danach bei einigen picornaviren in typi poliovirusinfektionen (je nach endemischer oder epidemischer situation 1-5%) unterscheidet man 3 verlaufsformen: • bei der abortiven poliomyelitis kommt es nach der inkubationszeit nur zu einer 2-5 tage anhaltenden "grippesymptomatik" (minor illness), wie sie viele enterovirustypen hervorrufen können. nach einer 2-bis 3-tägigen besserung kann es dann zu plötzlicher verschlechterung kommen (hauptkrankheit). • die meningitische poliomyelitis verläuft unter dem bild der prognostisch günstigen aseptischen meningitis, die ebenso durch viele andere enteroviren verursacht werden kann (sehr selten ist die perakute, letal verlaufende enzephalitis). zweiterkrankungen durch rhinoviren sind möglich, verlaufen aber milder. obwohl rhinovirusinfektionen bekanntlich gutartig verlaufen, besitzen sie angesichts der erkrankungshäufigkeit erhebliche ökonomische bedeutung (113 bekannte serotypen und möglichkeit der reinfektion!). jeder mensch macht viele picornavirusinfektionen durch, meist subklinisch oder als milde erkrankung. schwere krankheitsbilder kommen -auch altersabhängig -vor. picornaviren verursachen einige charakteristische erkrankungen und viele uncharakteristische symptome und syndrome. enteroviren und hepatitis-a-virus hinterlassen eine belastbare typenspezifische immunität. bei rhinoviren sind symptomatische reinfektionen bekannt. picornaviren können bei kardiomyopathien und dem juvenilen diabetes mellitus ätiologisch beteiligt sein. viele picornaviren sind leicht isolierbar. die serologie ist wenig aussagekräftig. einige der vielen tierpathogenen picornaviren können den menschen infizieren. beschreibung und einteilung adenoviren sind nackte und sehr umweltresistente ikosaedrische partikel von 70-100 nm durchmesser (› abb. 13.25). sie enthalten doppelsträngige lineare dna. im genus der mastadenoviren gibt es 6 subgenera a-f mit den zunächst serologisch definierten humanpathogenen virustypen 1-51 (hadv 1, … 51). später wurde auch eine genotypische abgrenzung nach homologie der nukleotidsequenz festgelegt. diagnostik je nach manifestation (auge, gastrointestinaltrakt) müssen andere mikrobiologische erreger abgegrenzt werden. die diagnose stützt sich auf virusisolierung und kaum auf die serologie. • virusnachweis: die meisten adenoviren sind aus rachenspülwasser, augenabstrich, stuhl, urin, liquor und anderen proben leicht in zellkulturen zu isolieren. die schwer anzüchtbaren hadv 40 und 41 werden elektronenmikroskopisch oder im antigen-elisa nachgewiesen, der auch schon eine subgenusdiagnose ermöglicht. • nachweis viraler genome: die pcr ermöglicht den nachweis der virus-dna direkt aus klinischen materialien und sogar eine genotypspezifische diagnose. • antikörpernachweis: die serologie (komplementbindungsreaktion, kbr) gestattet die diagnose einer frischen infektion bei nachweis eines antikörperanstiegs, bei gastrointestinalen infektionen kommt es aber nicht immer zu diesem antikörperanstieg. differentialdiagnose picornaviren, aber auch andere respiratorische und gastrointestinale viren. wichtig ist die frühzeitige abgrenzung zur streptokokkentonsillitis. bei schweren adenoviruserkrankungen, v. a. auch bei immunsupprimierten, ist ein therapieversuch mit cidofovir oder ribavirin möglich und indiziert. ribavirin scheint vorwiegend wirksam gegen viren des subgenus c. komplikationen bei angeborenen oder erworbenen immundefekten können adenoviren auch sehr schwere disseminierte infektionen induzieren, die lunge, gastrointestinaltrakt, leber und zns betreffen und fatal verlaufen. adeno-und rotaviren können vereinzelt auch nach ende einer akuten infektion ausgeschieden und bei gesunden nachgewiesen werden, teils gemeinsam mit enteroviren. adenovirusausbrüche auf neugeborenenstationen können sehr schwer, mit hoher letalität verlaufen. • röteln • echo-virus-9-infektion therapie und prophylaxe die therapie mit ribavirin wurde vereinzelt beschrieben und kann bei immundefekten sinnvoll sein. impfung die masern-lebendimpfung, gemäß impfkalender als mumps-masern-röteln-tripelvakzine (mmr) im 12. bis 15. lebensmonat und mit wiederholung im 6. lebensjahr, hat die zahl der masernfälle in deutschland im jahr 1996 auf 520 zurückgehen lassen. der grad der durchimpfung reicht mit 60% aber nicht aus, um die mensch-zu-mensch-übertragung ganz erlöschen zu lassen. das ziel der who, in europa bis zum jahr 2000 die masern auf weniger als 1 erkrankung/100 000 einwohner zu senken und den tod an masern auszurotten, erfordert immunitätsraten von > 95%, die mit einer einmaligen mmr-impfung nicht erreicht werden können. kürzlich ist es auch in deutschland wieder zu größeren masernausbrüchen gekommen. bei masernexposition ungeschützter personen ist ferner die passive immunisierung mit standard-serum-immunglobulin hilfreich (› kap. 13.10). angesichts der pathogenese ist es verständlich, dass die moderne masern-lebendimpfung auch vor der sspe schützt. häufigkeit masernpneumonie (als direkte folge der maserninfektion oder als folge einer bakteriellen superinfektion des geschädigten flimmerepithels) die disseminierung während der inkubation führt bei 50% zur klinisch und labormäßig fassbaren, aber prognostisch günstigen aseptischen meningitis, selten auch zur zerebellaren ataxie. eine enzephalitis ist selten und geht mit psychiatrischen und neurologischen spätschäden einher (verhaltensstörungen, krampfleiden, taubheit, retrobulbärneuritis, hydrozephalus). diagnostik der typische verlauf erleichtert die diagnose. • antikörpernachweis: serologisch lässt sich der antikörperanstieg mit hilfe der kbr nachweisen. die "kbr-antikörper" fallen allerdings 6-12 monate nach erkrankung unter die nachweisgrenze und sind daher zur immunitätsbestimmung ungeeignet. die frage der immunität wird durch nachweis virushüllenspezifischer antikörper im mumps-igg-elisa beantwortet. bei diagnose einer frischen infektion ist die untersuchung auf mumps-igm-antikörper im elisa die methode der wahl. • nachweis viraler genome: rt-pcr weist quantitativ hcv-rna nach und damit die aktive infektion. hcv-spezifische antikörper beweisen eine akute oder chronische, evtl. auch ausgeheilte infektion. der nachweis von hcv-genomen zeigt eine frische infektion, aber auch chronische carrier-zustände mit virusreplikation an; bei ausgeheilten hcv-infektionen wird die pcr negativ. die akute hcv-erkrankung ist meldepflichtig. therapie und prophylaxe therapie der chronischen hcv-infektion durch kombination von pegylierten alpha-interferonen mit nukleosidanaloga (› kap. 15.7.4) . durch untersuchung von blut-und organspendern, ggf. auch von angehörigen von hochrisikogruppen auf hcv-antikörper kann die verbreitung des virus eingeschränkt werden. die symptomatische therapie (analgetika bei arthralgien) ist möglich. impfungen sind nur gegen gelbfieber, fsme und die japanische enzephalitis verfügbar. ein impfstoff gegen dengue-virus müsste alle 4 serotypen erfassen, da teilimmunität gegen nur 1 typ negative auswirkungen (immunenhancement) bei wildvirusinfektion mit einem weiteren serotyp haben kann. komplikationen bei dengue-fieber kommt es v. a. bei sequentieller infektion durch verschiedene serotypen zu schweren krankheitsverläufen. die affenpocken verlaufen beim menschen ähnlich, meist mit viel ausgeprägterer lymphadenopathie. beim ausbruch 1996/1997 in kongo/zaire waren von 511 erkrankungen ca. 80% durch sekundäre mensch-zu-mensch-infektionen verursacht. das virus kann sich offenbar für begrenzte zeit in der fremden spezies mensch ausbreiten. andererseits war die rate an todesfällen unter den infizierten mit 1,5% viel niedriger als die noch in den 80er jahren beobachtete rate von 10%, so dass die who zurzeit eine wiederaufnahme der auch vor affenpocken schützenden vakzinierung ablehnt. haut-und schleimhauterkrankungen das molluscum contagiosum (dellwarze) ist eine harmlose, auf den menschen beschränkte infektion der epidermis, die höchstens kosmetisch bedeutsam ist. nach einer inkubationszeit (1-30 wochen) wachsen meist multiple, wachsfarbene papeln von 3-8 mm durchmesser heran, die bindegewebig gut abgegrenzt sind und nach 2-12 monaten spontan zurückgehen. die voll ausgebildeten knötchen haben zentral eine pore, aus der sämiges, weißliches material ausgepresst werden kann. dieses enthält die elektronenoptisch nachweisbaren viren. sehr häufig erkranken kinder und immunsupprimierte (aids). die übertragung, auch autoinokulation, erfolgt durch direkten kontakt oder durch gemeinsame handtuchnutzung. bei kindern findet man die veränderungen meist im gesicht und an den extremitäten, bei erwachsenen angesichts der sexuellen übertragung am genitale und dessen umgebung. dellwarzen mit längerer persistenz werden mittlerweile häufig bei aids-patienten beobachtet. melkerknotenvirus (kuh) und orf-virus (schaf) sind primär tierische poxviren, mit denen sich andere tierspezies undmeist bei beruflicher exposition -auch menschen infizieren können. kuhpocken-und melkerknotenvirus (beide sind nicht antigenverwandt) werden von tieren durch direkten kontakt auf den menschen übertragen. betroffen sind meist die hände, wobei das kuhpockenvirus vesikuläre veränderungen, das melkerknotenvirus derbe, oft geschwürig zerfallende knoten verursacht. allgemeinsymptome und lymphangitis sind bei den kuhpocken häufiger. diagnostik bei klinischem verdacht kann der erreger leicht elektronenmikroskopisch als quadervirus aus der vesikelflüssigkeit dargestellt werden. vaccinia-, affen-und kuhpockenvirus lassen sich gut auf der chorioallantoismembran anzüchten und differenzieren. der nachweis von dellwarzen bei erwachsenen ist ungewöhnlich und weist auf eine störung der immunabwehr hin; ggf. sollte eine hiv-infektion ausgeschlossen werden. therapie und prophylaxe keine spezifische antivirale therapie bekannt. die pockenimpfung gegen variola major ist nach ausrottung der humanen pocken weltweit ausgesetzt worden. die pocken waren eine der großen menschheitsseuchen und stellen die 1. infektionskrankheit dar, die durch den menschen weltweit ausgerottet wurde. beschreibung und einteilung die ehemalige familie der papovaviridae (› abb. 13.39) wurde in 2 selbstständige virusfamilien aufgeteilt, die papillomaviridae (durchmesser 55 nm, genom 8 kb) und polyomaviridae (durchmesser 45 nm, genom 5 kb). es handelt sich bei beiden um nackte, ikosaedrische partikel mit doppelsträngiger zirkulärer, superhelikaler dna. einige tierische papillomaviren induzieren tumoren, v. a. wenn sie in spezies inokuliert werden, die nicht natürliche wirte sind. epidemiologie die vermehrung der papillomaviren in konventionellen zellkulturen ist nicht möglich und eine typenspezifische serologie war nicht möglich. lange war dagegen bekannt, dass sie übertragbare warzen des menschen verursachen (› abb. 13.40). erst die molekulare genetik ermöglicht pathogenetische untersuchungen und molekulare epidemiologie. die papillomavirustypen sind so als genotypen definiert (< 50% sequenzhomologie = neuer typ). bisher unterscheidet man > 100 hpv-genotypen, die vielfach den krankheitsbildern zugeordnet werden können. polyomaviren sind in form einer latenten infektion bei den meisten menschen vorhanden. pathogenese humane papillomaviren (hpv) verursachen persistierende infektionen. die ätiologische beteiligung bestimmter hpv-typen an der entstehung anogenitaler malignome ist gesichert. die primäre infektion mit den polyomaviren bkv und jcv bleibt meist unerkannt. sie verläuft häufig als milder respiratorischer infekt und führt bei bkv zur latenz in der niere, während das eher neurotrope jcv im zns -weniger ausgeprägt auch in der niere -latent wird. symptome, verlauf und prognose asymptomatische primärinfektionen mit polyomaviren sind die regel und mit papillomaviren sehr häufig. haut-und schleimhauterkrankungen warzen entstehen nach relativ langer inkubationszeit durch produktive virusinfektion mit hpv in den epithelzellen, wobei die virusvermehrung an differenzierung und keratinisierung der zellen gebunden ist. die normalen hautwarzen sind eine selbstlimitierende erkrankung. die seltene, familiär gehäuft auftretende epidermodysplasia verruciformis, assoziiert mit hpv 20 und 36, zeigt beetartig verschiedene warzenformen, die in 30-60% in ein plattenepithelkarzinom übergehen. hno-erkrankungen die juvenile larynxpapillomatose (hpv 6, 11) ist eine hartnäckige und gefürchtete erkrankung, die möglicherweise auf einer infektion im infizierten geburtskanal der mutter beruht. die durch jcv bedingte progressive multifokale leukoenzephalopathie (pml) tritt bei schwer immunsupprimierten (maligne lymphome, v. a. morbus hodgkin, aids, transplantationspatienten) auf und spielt eine rolle in der differentialdiagnose der zerebralen non-hodgkin-lymphome und anderer demyelinisierender erkrankungen (multiple sklerose, lupus erythematodes mit zns-befall). es kommt an mehreren orten zu herden, die meist keine verdrängungserscheinungen verursachen, aber zu großen entmarkungsherden zusammenfließen können. die patienten zeigen zunehmende wesensveränderungen und kognitive störungen, die erkrankung führt 6 monate nach den ersten neurologischen ausfällen zum tode. weitere erkrankungen schwere immundefekte können zur virurie und zystitis durch bkv führen. papillomavirusinfektionen führen zu spitzen kondylomen (hpv 6, 11, 42 u. a.) und intraepithelialen dysplasien der cervix uteri und der vagina (hpv 6, 11, 16). vergleichbare dysplasien sind auch am penis möglich. diagnostik warzen und kondylome werden klinisch leicht erkannt. anders hpv-assoziierte präkanzerosen, die als epitheldysplasien charakteristische zytologische veränderungen im abstrichpräparat ergeben (› abb. 13.41). hier können dna-und rna-hybridisierung hinweisend auf latente oder aktive infektion durch bestimmte hpv-typen sein. eine pml wird zunächst nach kernspintomographie vermutet und virologisch durch jcv-pcr im liquor oder sicherer im biopsat durch pcr oder elektronenmikroskopie diagnostiziert. bkv-infektionen sind häufig mit nierenerkrankungen assoziiert und werden durch pcr leicht und spezifisch im urin nachgewiesen, so dass eine partikelisolierung entbehrlich ist. bei pml kann die niedrig dosierte chemotherapie mit cytosinarabinosid zum rückgang der symptome führen, aber nur bei relativ intakter zellvermittelter immunität. bei transplantierten mit pml ist daher die therapeutische immunsuppression zurückzunehmen -die prognose der pml bleibt insgesamt schlecht. komplikationen die kausale assoziation bestimmter hpv-genotypen (z. b. hpv 16, 18, insgesamt 40 hpv-genotypen von der cervix nachgewiesen) mit weiblichen genitalkarzinomen hat dazu geführt, dass die hpv-diagnostik mehr eingang in die vorsorgeuntersuchung bei der frau gefunden hat. der nachweis von hpv-genotypen der hochrisikogruppe führt zumindest zur engmaschigen kontrolle oder zum aktiven vorgehen bei gleichzeitigen zytologischen veränderungen. die in der westlichen hemisphäre bei pferd, rind und schwein vorkommenden vesiculoviren können als zoonose beim menschen zu grippeähnlichen infekten, myalgien und auf schleimhäuten zu herpetiformen bläschen mit hoher partikelzahl führen. pathogenese das tollwutvirus bleibt nach infektion für stunden bis wochen im bereich der eintrittspforte in der peripherie; es vermehrt sich wahrscheinlich auch in den zellen der quergestreiften muskulatur oder persistiert in makrophagen. dabei kommt es nicht zur nennenswerten protektiven immunantwort. nach eindringen in die peripheren nervenendigungen gelangt es mit dem axoplasmastrom (ca. 3 mm/h) ins zns. nach erreichen des gehirns verursacht es eine enzephalitis, die histologisch (negri-körperchen) nicht sehr ausgeprägt sein muss, und kehrt dann in verschiedene organe in der peripherie "zurück" (z. b. speicheldrüsen) und auch in verschiedene periphere nervenzellen. durch die intrazelluläre entwicklung innerhalb des nervensystems kommt es erst sehr spät zum effektiven kontakt mit dem immunsystem, so dass neutralisierende und diagnostisch verwertbare antikörper in serum und liquor anfangs fehlen können. die inkubationszeit ist umso kürzer (spanne zwischen 7 tagen und mehreren jahren; durchschnitt: 1-2 monate) und die erkrankungswahrscheinlichkeit umso höher, je näher die verletzung am zns liegt (bein: 10%, gesicht: 80%) und je schwerer sie ist. • 2. phase: neurologisch-psychiatrische symptome (verstärkte speichelsekretion, reizbarkeit). "stille wut" mit aufsteigender paralyse, "wilde wut" mit starker unruhe und charakteristischer hydrophobie in 17-80% (muskelspasmen im mund-, rachen-und larynxbereich), anfangs beim versuch zu trinken, später schon bei der visuellen wahrnehmung von wasser oder anderen akustischen und taktilen reizen. die wilde wut verläuft rascher progredient (2-7 tage) als die stille (bis 30 tage). • 3. phase: präfinales koma (3-7 tage). bei intensivmedizinischer versorgung mit beatmung kann der verlauf viel länger sein. inwieweit unterschiedliche virusstämme für verschiedene verläufe verantwortlich sind, ist noch unklar. es gibt 4 berichte über überlebte erkrankungen, wobei alle patienten vorgeimpft waren, so dass es sich eher um impfversagen handelte. die rate tatsächlich erfolgter, aber asymptomatischer infektionen ist nicht bekannt. • virusnachweis: immunfluoreszenznachweis des virusantigens im abdruckpräparat der kornea. postmortale diagnose durch genomnachweis mittels rt-pcr und histopathologisch am gehirn (negri-körperchen) oder durch immunhistologie. die virusisolierung in mäusen und neuroblastomzelllinien aus speichel ist möglich. • nachweis des viralen genoms: über rt-pcr als standardmethode • antikörpernachweis: die serologische diagnose der tollwut (ift, elisa) ist unzuverlässig. therapie und prophylaxe jede tollwutexposition bedeutet lebensgefahr und erfordert beim ungeimpften eine sofortige postexpositionelle, kombinierte aktive und passive immunisierung. nach ausbruch der erkrankung gibt es keine spezifische therapie -die rabies des menschen verläuft tödlich. virostatika zeigten keinen einfluss, doch sind zytokine wie il-12 evtl. interessant. intensivmedizinische maßnahmen wegen hypoxischem zns-ödem und gestörter thermoregulation. bei beruflicher gefährdung (u. a. tierärzte, förster) ist die aktive schutzimpfung indiziert. biss-und kratzwunden mit evtl. tollwutexposition müssen chirurgisch gereinigt, gründlich desinfiziert und mit rabies-immunglobulin umspritzt werden. präventiv lebenswichtig ist die schnelle postexpositionsimpfung (› kap. 13.10) mit inaktivierten vakzinen und bald evtl. gentechnologisch erzeugten impfstoffen. das risiko bei htlv-1-infektion, einen tumor zu entwickeln, liegt bei ca. 1% (5-10% bekommen insgesamt symptome der infektion). die bedeutung von htlv-2 für erkrankungen des menschen ist unklar, obwohl einiges für eine beteiligung bei leukämien spricht. hautmanifestationen im sinne eines kutanen lymphoms sind häufig im rahmen einer adulten t-zell-leukose (atl), an deren entstehung htlv-1 oft beteiligt ist. jedoch ist die inkubationszeit der atl mit 20-30 jahren lang. atl geht einher mit opportunistischen infektionen durch immunsuppression, lymphadenopathie, hepatosplenomegalie, lungeninfiltraten und osteolysen. das zellbild im peripheren blut kann sehr unterschiedlich sein. bei einigen verläuft die erkrankung eher unter dem bild eines lymphoms. htlv-1 ist selten ursache der tropischen spastischen paraparese (langsam fortschreitende myelopathie mit pyramidenbahnzeichen). diagnostik analog zu hiv durch antikörpernachweis und nachweis viraler rna. antikörper treten evtl. erst spät nach infektion auf. die differenzierung zwischen htlv-1 und htlv-2 bedarf manchmal zusätzlicher tests. inwieweit und in welchen ländern blutspender generell auf htlv-1 getestet werden sollten, muss immer wieder aufgrund der epidemiologischen situation geprüft werden. in › abbildung 13.12 (s. 619) ist der verlauf einer infektionskrankheit schematisch auf einer zeitskala durch die begriffe infektion und beginn der erkrankung veranschaulicht. die zeitliche differenz ist die inkubationszeit, die bei vielen infektionskrankheiten ein charakteristisches merkmal darstellt. es wurden frühzeitig erkrankungen des zentralnervensystems beschrieben, bei denen es nicht gelang, ein viruspartikel oder endogene virale nukleotidsequenzen zu identifizieren. heute gilt als sicher: prionen sind erreger von übertragbaren, chronischen, degenerativen, stets letalen erkrankungen des zentralen nervensystems. sie kommen mit ähnlichen erscheinungsformen als subakute enzephalopathien bei menschen und anderen wirbeltieren (rind, schaf, ziege, katze, hirsch, nerz u. a.) vor. beim menschen unterscheidet man folgende krankheitsbilder: • creutzfeldt-jakob-disease (cjd) • neue variante creutzfeldt-jakob-disease (vcjd) • gerstmann-sträussler-scheinker-syndrom (gss) • fatale familiäre insomnie (ffi) • kuru. bei tieren sind hier insbesondere scrapie beim schaf und die bovine spongiforme enzephalopathie (bse) beim rind aufzuführen. m e r k e allen krankheiten ist gemeinsam: • es werden keine entzündlichen prozesse, kein fieber und keine immunantwort beobachtet. • es gibt ein breites spektrum von symptomen, das für das jeweilige krankheitsbild einen charakteristischen schwerpunkt hat. • eine therapie ist gegenwärtig nicht verfügbar, alle erkrankungen führen zum tod. prionen sind nach ansicht der meisten forscher nukleinsäurefreie proteine. der name prion wurde 1982 von stanley prusiner aus der bezeichnung "proteinaceous infectious particles" abgeleitet. die assoziation von prionprotein als wesentlichem bestandteil des infektiösen agens ist zweifelsfrei bewiesen. eine alternative vorstellung geht von einer konzeptionell noch unklaren beteiligung von nukleinsäuren aus, um die existenz von varianten sowie hereditäre aspekte analog zur genetisch determinierten situation etwa in viralen systemen zu erklären. allen bisher bekannten prionen ist gemeinsam, dass es sich um glykosylierte proteine mit ca. 250 aminosäuren, entsprechend molekülmassen von 33-35 kda handelt, die von zellulären genen kodiert werden. transkription und translation sind im gesunden wie im krankhaften zustand unverändert. soweit sequenzdaten vorliegen, handelt es sich um ein evolutionär insgesamt hoch konserviertes molekül insbesondere im bereich der aminosäurepositionen 124-226. die tatsächlich vorhandenen abweichungen in der aminosäuresequenz von prionen verschiedener spezies definieren zusammen mit anderen faktoren (s. u.) die sog. speziesbarriere für eine heterologe infektion. die höhe der übertragungsbarriere ist für sequenzierte prionen im vergleich zueinander zumindest abschätzbar (unterschiede ausgedrückt als zahl der voneinander abweichenden aminosäuren: schaf -rind 7, rind -mensch > 30, maus -mensch 28). das gen für das menschliche prion (prnp) befindet sich auf dem kurzen arm von chromosom 20 und kodiert für ein primäres genprodukt prp c mit 253 aminosäuren. der index c steht für "cellular". das protein trägt am n-und am c-terminus signalsequenzen (22 bzw. 23 aminosäuren), die posttranslational durch zelluläre peptidasen entfernt werden. an das cterminale ende wird anschließend ein gpi-anker (glykosylphosphatidyl-inositol) für die befestigung in der zellmembran angehängt. diese form des prionproteins ist durch zelluläre proteasen leicht abbaubar. im gegensatz dazu lassen sich aus gehirnen von an übertragbarer spongiformer enzephalopathie (tse) erkrankten menschen und tieren isoformen des prionproteins isolieren, die trotz ihrer mit prp c identischen aminosäuresequenz wegen der spezifischen faltung unlöslich und in vitro nur bis auf den c-terminalen rest von 142 aminosäuren (positionen 90 bis 231) abbaubar sind. dieses restmolekül wird auch als prp27-30 bezeichnet und stellt den proteaseresistenten, aber immer noch infektiösen anteil von prp tse dar. die räumliche struktur von prp c enthält nach modellrechnungen drei α-helices und nur geringe β-faltblatt-bereiche, während der nicht spaltbare prp sc -anteil bis zu 30% β-faltblätter und nur einen geringen gehalt an α-helices aufweist. eine klassifizierung der prionen analog oder ähnlich derjenigen der viren gibt es gegenwärtig nicht. sinnvoll ist zurzeit lediglich die unterscheidung aufgrund der betroffenen wirte unter beachtung der tatsache, dass in tiermodellen mehr als 20 verschiedene stämme von prp sc identifizierbar sind, die sich durch die inkubationszeit, den von der krankheit betroffenen bereich der gehirne und das spektrum der klinischen symptome unterscheiden. interessant ist der befund, dass sich verschiedene klinisch definierte phänotypen von cjd verschiedenen fragmentierungsmustern nach unvollständiger proteinase-k-spaltung zuordnen ließen. fragment-und glykosylierungsmuster von cjd und bse lassen nach experimentellen übertragungen auf transgene mäuse eine definition von prionenstämmen zu. insbesondere ergaben sich nach inokulation von wildtypmäusen mit vcjd bzw. bse identische glykosylierungsmuster, d.h., die beiden krankheiten wurden mit hoher wahrscheinlichkeit durch den gleichen prionenstamm hervorgerufen. aggregatbildung und ablagerung der proteaseresistenten prp sc -moleküle werden als pathogenes prinzip angesehen, das mit dem krankheitsbild der spongiformen enzephalopathie assoziiert ist. als mechanismus der aggregation wird spontane autokatalytische bzw. durch prp sc vermittelte umfaltung zellulärer "gesunder" prp c -moleküle in die schwer abbaubaren, aggregierenden tse-prionen angenommen. im gegensatz zu viruserkrankungen kommt es nicht zum einbringen, exprimieren und vervielfältigen eines genetischen programms, sondern zur kumulativen ausbreitung einer strukturform innerhalb einer population bereits bestehender moleküle. die prionenstruktur macht krank! dies ist ein grundsätzlich neues pathogenes prinzip. creutzfeldt-jakob-disease (cjd) cjd ist die am besten bekannte tse-erkrankung, die 1920 von hans g. creutzfeldt bzw. 1921 von alfons jakob beschrieben wurde. gegenwärtig wird sie unter 4 aspekten der entstehung diskutiert als • sporadisch auftretend (spcjd) • genetisch beeinflusst (gcjd) • iatrogen hervorgerufen (icjd) • und neuerdings als variante form (nvcjd), durch aufnahme boviner prionen erzeugt. sporadisch kommt cjd weltweit mit einer inzidenz von etwa 1 fall pro 1 mio. einwohner pro jahr vor. abweichungen resultieren vornehmlich aus der nichtvergleichbarkeit der erhebungsmethoden in den einzelnen ländern. die altersgruppe der 70-bis 80-jährigen ist am häufigsten betroffen. der bisher jüngste patient in deutschland war 23 jahre alt, der älteste 88 jahre, niemals jedoch war ein kind erkrankt. beide geschlechter scheinen gleichermaßen betroffen zu sein. nach dem auftreten erster symptome (kopfschmerz, müdigkeit, schlaf-und appetitlosigkeit, depression) folgt das bild einer rasch voranschreitenden generellen enzephalopathie mit verlust der bewegungskoordination sowie mit demenz. die krankheitsdauer beträgt in etwa 65% der fälle < 6 monate. eine sichere diagnose kann bislang letztlich nur durch neuropathologische untersuchungen gestellt werden. genetisch bedingte creutzfeldt-jakob-krankheit (gcjd) familiäre häufungen von cjd sind bereits in den 30er-jahren des vorigen jahrhunderts beobachtet worden. von zentraler bedeutung scheint der polymorphismus 129 zu sein, der durch das vorkommen der aminosäuren methionin (m) oder valin (v) an der aminosäureposition 129 im prionprotein charakterisiert ist. in england liegt bei 80% der spcjd-fälle homozygotie 129mm vor, im gegensatz zu 40% in der normalbevölkerung. dagegen sind nur 10% der erkrankten heterozygot mv bei einem 50%igen anteil in der normalbevölkerung. alle bekannten nvcjd-fälle sind 129mm-homozygot (s. u.)! die aminosäureposition 129 befindet sich innerhalb des prionmoleküls an einer übergangsstelle zwischen der zweiten α-helix und dem β-faltblatt und könnte daher von wesentlichem einfluss auf die faltung des moleküls sein. das klinische bild wird bezüglich krankheitsbeginn und -dauer stark von der genetischen disposition in bezug auf die codons 129, 178 und 200 geprägt. weitere punktmutationen und insertionen sind ebenfalls von bedeutung. in den familiären fällen ist die inzidenz der erkrankung stark erhöht und geographisch auf bestimmte regionen begrenzt. so findet sich eine jüdische, aus libyen stammende population in israel mit 50-fach häufigerem auftreten von cjd. charakteristisch ist hier der aminosäureaustausch glu200lys. neben der histopathologischen abklärung ist die sequenzierung des prnp-gens zur sicherung der diagnose gcjd erforderlich. iatrogene übertragung erfolgte nach neurochirurgischen eingriffen, durch verwendung unvollständig sterilisierter neurochirurgischer geräte und elektroden, nach transplantationen von kornea und dura mater von verstorbenen sowie nach der verwendung von aus leichen gewonnenem humanem wachstumshormon (hgh) bzw. hypophysen-gonadotropin. das klinische bild entspricht demjenigen von spcjd, in die diagnose ist die krankengeschichte einzubeziehen. im jahr 1995 trat der erste todesfall auf, der einer neuen variante der cjd zuzuordnen ist. bezüglich des krankheitsbildes liegen ähnliche symptome wie bei den anderen formen vor, jedoch sind das niedrige patientenalter (28 als medianes alter für den krankheitsbeginn, gesamtintervall 14-53 jahre) sowie die epidemiologisch wichtige erkenntnis der fast ausschließlichen geographischen beschränkung auf großbritannien hervorzuheben. im mai 2002 waren weltweit 111 fälle bekannt, davon 107 in großbritannien, 3 in frankreich und 1 in irland. klinisch stehen bei krankheitsbeginn hier eher psychiatrische als neurologische symptome im vordergrund, wie depression, angst, erregung, halluzinationen und schmerz, aber auch neuropsychologische auffälligkeiten wie aphasie oder alexie. später kommen die üblichen sensorischen symptome wie ataxie, parese und demenz hinzu. im gegensatz zu spcjd finden sich neuropathologische besonderheiten. es liegen keinerlei hinweise auf familiäre häufungen vor. die übertragung erfolgt mit großer wahrscheinlichkeit durch den genuss von "infektiösen" nahrungsmitteln. durch die normale zubereitung von speisen werden prionen vermutlich nur unvollständig inaktiviert. das auftreten von nvcjd-prionen im gehirn und in den tonsillen ist ein sicheres diagnostisches merkmal. biologische typisierungen von prionen in versuchstieren sind zeitaufwändig und teuer und nicht für diagnostische zwecke geeignet. epidemiologische untersuchungen zeigten allerdings, dass bislang keine risikofaktoren wie berufszugehörigkeit (landwirte, veterinäre, schlachter, abdecker etc), essgewohnheiten oder geographische nähe zu bse-belasteten landwirtschaftlichen betrieben erkennbar sind. diese tse-erkrankung ist mit der inzidenz von einem fall unter 10 mio. einwohnern pro jahr äußerst selten und mit wenigen sporadischen ausnahmen wohl ausschließlich genetisch determiniert. der erbgang ist autosomal-dominant. im vordergrund steht eine punktmutation mit der konsequenz des aminosäureaustausches von prolin durch leucin (pro102leu). hinzu kommen weitere punktmutationen und ein spektrum von oktapeptid-insertionen, die in zusammenwirken mit dem polymorphismus an der position 129 einfluss auf die klinischpathologischen aspekte der amyloidbildung im gehirn haben. erste symptome von gss sind uncharakteristische beschwerden, wie schlafstörungen, psychische veränderungen, gedächtnisverlust, aphasie und alexie, gefolgt von dem spektrum der anderen tse-symptome, die nach völliger dezerebration einige monate bis 2 jahre nach auftreten der ersten symptome zum tode führen. das erkrankungsalter liegt zwischen 30 und 50 jahren. die diagnose erfolgt anhand der neuropathologischen befunde und ggf. durch sequenzanalysen des prnp-gens. gss ist ausschließlich als hereditär anzusehen, die vertikale weitergabe des gss-spezifischen prnp-gens sollte nicht als übertragung eines krankheitserregers bezeichnet werden. es handelt sich um eine äußerst seltene genetisch bedingte erkrankung, die 1986 zuerst bei 5 mitgliedern einer italienischen familie entdeckt wurde. der erbgang ist autosomal-dominant, scheint jedoch nur eingeschränkt penetrant zu sein, da mehrere familienmitglieder die entscheidende prnp-mutation mit der folge des aminosäureaustausches asp178asn aufwiesen, jedoch symptomlos blieben. die gleiche mutation ist auch bei der familiären form der creutzfeldt-jakob-krankheit (gcjd) von zentraler bedeutung, was zu intensiver diskussion der beiden klinisch-pathologisch sehr unterschiedlichen situationen geführt hat. auch hier ist das codon 129 von bedeutung. das zentrale klinische bild der ffi ist geprägt durch einen stark gestörten schlafrhythmus und entsprechende veränderungen in eeg-schlafmustern und endokrinen zirkadianen stoffwechselleistungen. die erkrankung tritt zwischen dem 40. und 60. lebensjahr auf und führt nach 7-18 monaten zum tode. nach zunächst uncharakteristischen stadien liefert die neuropathologische untersuchung astrogliose, vakuolenbildung und amyloidablagerungen. kuru ist der klassische fall einer horizontal übertragenen spongiformen enzephalopathie. sie wurde zuerst 1957 von gajdusek und zigas beschrieben als eine degenerative krankheit des zentralnervensystems in isolierten populationen in neuguinea. seit dem verbot des dort praktizierten rituellen kannibalismus ende der 50er jahre ist die erkrankung im verschwinden begriffen und heute praktisch ausgelöscht. homozygotie für methionin an der codonposition 129 des prnp-gens ist charakteristisch für die erkrankung, die mit hoher wahrscheinlichkeit durch die horizontale übertragung von infektiösem material eines an spontaner creutzfeldt-jakob-krankheit verstorbenen entstanden und durch die kannibalistischen beerdigungsriten epidemisch verbreitet wurde. die infektion ist vermutlich über den intestinaltrakt verlaufen. eindringen der kuru-prionen durch verletzungen während des eröffnens des leichnams und damit verbundene hautkontaminationen sowie konjunktivale und nasale schmierinfektionen sind als übertragungswege ebenso denkbar. die krankheit beginnt mit unspezifischen beschwerden und führt nach neurologischen ausfällen mit ataxie, schweren lähmungen, damit verbundener unterernährung und letztlich völliger motorischer unfähigkeit zum tode. im fall von iatrogener cjd, kuru und nvcjd ist die übertragbarkeit von infektiösen prionen sehr wahrscheinlich bzw. nachgewiesen. seit jahrzehnten wird tiermehl weltweit als zuschlagstoff in der tierfütterung eingesetzt. in großbritannien wurden ende der 70er-bis anfang der 80er jahre in verschiedenen produktionsanlagen unterschiedliche änderungen des herstellungsprozesses vorgenommen, die offensichtlich eine minderung der inaktivierungseffizienz zur folge hatten. heute wird unter dem eindruck der bse-epidemie eine 20-minütige erhitzung auf 133 °c bei 3 bar überdruck als norm gefordert. zur inaktivierung von prionen an chirurgischen instrumenten, die nicht autoklavierbar sind, wird eine einstündige behandlung mit natronlauge oder natriumhypochlorid empfohlen. um risiken inadäquater dekontaminierung zu vermeiden, wird die benutzung von lediglich einmal zu verwendendem material empfohlen. mit zunehmendem verständnis der pathogenitätsmechanismen ergeben sich hinweise auf mögliche therapiestrategien. so ist es denkbar, in den umwandlungsprozess der prp c -konformation direkt einzugreifen. behinderung von eintritt in den wirtsorganismus und transport von prp sc in das zns ist eine weitere möglichkeit. im fall tierischer erkrankungen wären genetische und züchterische maßnahmen denkbar, etwa die aufzucht von tieren, die von individuen abstammen, die künstlich negativ homozygot für das priongen (prp -/-) gemacht wurden. diese tiere sind nicht infizierbar, da sie selbst keine zellulären prionen synthetisieren können, die dann nach dem eindringen von prp sc in die pathogene konformation umgefaltet werden könnten. da die natürliche funktion des genproduktes des zellulären prp-gens und damit die folgen seines verlustes jedoch nicht bekannt sind, ist dieser weg risikoreich und vorerst nicht gangbar; beim menschen ist er sowieso ausgeschlossen. die konventionelle züchtung nicht erkrankender schafe ist gelungen und hat wohl dazu geführt, dass mittlerweile england, neuseeland und australien scrapie-frei sind. von der invasiven mykose ist die systemische mykose abzugrenzen, die nur infolge hämatogener streuung auftritt. invasive mykosen werden durch etwa 100 verschiedene pilzarten hervorgerufen. sie betreffen nahezu ausschließlich abwehrgeschwächte patienten. prädisponierende faktoren sind abdominalchirurgie, zentrale venenkatheter, störung der normalen flora durch breitspektrum-antibiotika, herabsetzung der abwehr durch kortikosteroide, immunsuppressiva und zytostatika, hiv-infektion, diabetes mellitus, transplantation solider organe oder allogener stammzellen. epidemiologie die meisten pilze leben saprophytär. einige können aber in geringer zahl auf haut und schleimhäuten sowie im darmtrakt vorhanden sein, ohne krankheitserscheinungen hervorzurufen. bei einem pilznachweis in entsprechenden materialien stellt sich daher oft die frage nach ihrer relevanz als erreger. liegen keine der oben genannten risikofaktoren vor, so ist der nachweis von hefepilzen im stuhl klinisch nicht relevant. ätiologie und pathogenese die adhärenz der pilze an die wirtszellen ist notwendige bedingung für eine infektion. sie wird durch wechselwirkungen zwischen kohlenhydrat-und proteinstrukturen der pilzzellwand und der wirtszelle verursacht. es können zell-und gewebsschädigende sekretorische proteasen und phospholipasen gebildet werden. außerdem spielen spezielle morphologische eigenschaften der pilze eine rolle, wie z. b. das "switching", der übergang von der sprosspilzform in die hyphenform bei dimorphen pilzen. für die wirtsabwehr gegen die meisten opportunistischen pilze -insbesondere bei den am häufigsten vorkommenden gattungen candida und aspergillus -sind zahl und funktion der neutrophilen entscheidend. makrophagen und monozyten wird zunehmend bedeutung beigemessen, während die t-zell-vermittelte immunität hauptsächlich für die abwehr der obligat pathogenen pilze und von cryptococcus neoformans relevant ist. diagnostik das klinische bild der meisten pilzinfektionen ist uncharakteristisch, damit ist der erregernachweis besonders bedeutend. beweisend ist die kultur aus physiologisch sterilem material (blut, liquor, biospie) oder die histologie an paraffinschnitten. in allen materialien lassen sich pilze mit optischen aufhellern (blankophoren), giemsa-färbung oder gram-färbung (grampositiv) nachweisen. für die genaue identifizierung der pilze ist die kulturelle anzüchtung erforderlich. hierfür werden als selektive medien z. b. sabouraud-agar und chrom-agar verwendet. als weitere diagnostische möglichkeiten gibt es für einige pilze antigennachweise, z. b. galactomannan für aspergillus und β-1,3-d-glucan für candida, aspergillus und andere. serologische untersuchungsverfahren weisen spezifische antikörper nach und sind generell weniger verlässlich. molekularbiologische nukleinsäurenachweise sind nicht ausreichend standardisiert und der kultur in der identifizierung des erregers unterlegen. therapie zur therapie invasiver pilzinfektionen stehen 6 substanzklassen zur verfügung, deren charakteristika in › tabelle 13.34 wiedergegeben sind. aus der gruppe der sprosspilze kommen krankheitserreger vor allem in der gattung candida vor. trichosporon und blastoschizomyces sind sehr viel seltener. candida verursacht bei schleimhautbefall weißliche beläge, den soor (engl.: thrush). dieser kann bei vorliegen von risikofaktoren zu invasiven infektionen (organbefall, fungämie) führen. eine weitere sprosspilzart, cryptococcus neoformans, verursacht nach einem flüchtigen lungeninfiltrat eine meningoenzephalitis bei abwehrgeschwächten, z. b. hiv-infizierten patienten. praxisfall ii ein 76-jähriger befindet sich zur diabeteseinstellung eher zufällig im krankenhaus, klagt über plötzlich einsetzende bauchschmerzen und wird bewusstlos. es liegt ein rupturiertes bauchaortenaneurysma vor, das notfallmäßig operiert wird. seit 7 tagen liegt er beatmet auf der intensivstation, ein perioperativ diagnostiziertes akutes nierenversagen erfordert die dialyse über einen shaldon-katheter. er fiebert auf, blutkulturen werden abgenommen, eine breitspektrumantibiotika-therapie beginnt. drei tage diagnostik im ct der lunge und des kopfes werden uncharakteristische raumforderungen gesehen, die in verbindung mit der grunderkrankung an diese differentialdiagnose denken lassen sollten. eine histologische sicherung ist zwingend erforderlich. therapie neben der radikalen und ggf. wiederholten chirurgischen sanierung besteht die medikamentöse therapie der wahl in liposomalem amphotericin b. die empfehlungen zur dosierung gehen auseinander: 3-10 mg/kg. ist der patient diabetiker, dann wird sich die nierenfunktion unter dieser therapie sehr bald verschlechtern. einzige alternative ist posaconazol 4 × 200 mg. die therapiedauer kann nicht genau abgegrenzt werden, beträgt aber zumindest 1-2 jahre. nach ende der therapie ist eine regelmäßige nachsorge nötig. verlauf und prognose der verlauf hängt von der erfolgreichen behandlung der grundkrankheit und damit im falle eines diabetes von der adhärenz des patienten ab. wird ein diabetes optimal therapiert, kann die infektion überlebt werden. die gesamtsterblichkeit beträgt 30-70%. nach 1-2 jahren kann unter engmaschiger kontrolle eine therapiepause versucht werden. • malaria tertiana und quartana: bei p. vivax-, p. ovale-und p. malariae-infektionen kommt es ungefähr 1 woche nach krankheitsausbruch zur synchronisation der parasitenvermehrung im blut, d.h., die parasiten wachsen synchron heran und zerstören gleichzeitig ihre wirtserythrozyten (p. vivax und p. ovale an jedem 2., p. malariae an jedem 3. tag). diese synchronisation bedingt die regelmäßigen und charakteristischen fieberanfälle. p. vivax und p. ovale hinterlassen sog. hypnozoiten in der leber, die monate und jahre später zu rezidiven führen können. • malaria tropica: p. falciparum neigt nicht zur synchronisierten vermehrung. eine weitere wichtige besonderheit der malaria tropica ist die veränderung der erythrozytenoberfläche durch die heranwachsenden formen von p. falciparum. die befallenen roten blutkörperchen gewinnen dadurch eine besondere affinität zum gefäßendothel. vor allem in den kapillaren bleiben sie am endothel "kleben" (sequestration) und verstopfen sie. die folge sind hypoxie und metabolitenstau im abhängigen gewebebezirk (› abb. 13.56). diese einzigartige eigenschaft der tropica-parasiten bedingt die gefährlichkeit der malaria tropica, die infolge der zunehmenden ischämie in wichtigen organen (gehirn, lunge, niere, herz) innerhalb weniger tage zum tod führen kann. • erysipel: pathognomonisches a-streptokokken-krankheitsbild, das auch tiefere hautschichten betrifft. beginnt mit lokalisiertem erythem mit schwellung, das sich rasch ausbreitet und klar vom normalen umgebenden gewebe abgrenzbar ist begleitet von hohem fieber, schüttelfrost und allgemeinem toxischem krankheitsgefühl. im gesichtsbereich ist es meist selbstlimitierend, bei anderer lokalisation kann es nur durch gezielte therapie geheilt werden. • akutes rheumatisches fieber: nach durchgemachter streptokokkenpharyngitis mit einer latenzzeit von ca. 18-20 tagen. es kommt zu fieber, schmerzhaften schwellungen der großen und mittleren gelenke und zur pankarditis (v. a. als endokarditis: endocarditis verrucosa). nach längerer latenzzeit evtl. syndrom im zns-bereich (chorea minor). andere spätfolgen sind: erythema nodosum und erythema anulare rheumaticum. man führt den gesamtkomplex des rheumatischen fiebers auf eine immunpathogenese zurück. nur bestimmte m-typen von a-streptokokken verursachen diese folgeerkrankung, solche stämme kommen zurzeit bei uns nicht autochthon vor. häufiger betroffen sind (bei uns meist türkische) patienten aus mediterranem gebiet. • akute glomerulonephritis: tritt auch nach hautinfektionen auf. gute prognose v. a. bei kindern. diagnostik einige der erkrankungen (z. b. erysipel und scharlach) sind schon klinisch pathognomonisch. wichtigste mikrobiologisch-diagnostische maßnahme ist der erregernachweis aus blutkultur und abstrich-und punktionsmaterialien. im verlauf stellt man meist serologisch eine titerbewegung der antikörper gegen streptolysin o (aso-, asl-titer) und/oder bei antikörpern gegen dnase b (adb-, streptodornase-titer) fest. faustregel: der asl-titer steigt v. a. bei infektionen im respirationstrakt, der adb-titer meist bei hauterkrankungen an. asl spielt in der diagnostik des rheumatischen fiebers nur eine geringe rolle, adb hat in der diagnostik der akuten glomerulonephritis größere bedeutung. die adb-werte können hier extrem hoch ansteigen. differentialdiagnose extrem hohe asl-bzw. adb-werte treten auch bei plasmozytomen mit entsprechender antikörperspezifität auf. differentialdiagnostisch kommen bei den pyogenen a-streptokokken-infektionen v. a. infektionen durch s. aureus in frage. dies gilt auch für das toxic shock syndrome. beim erysipel muss, v. a. bei immunsupprimierten, eine aeromonasspp.-ätiologie ausgeschlossen werden (cave: andere antibiotikatherapie!). bei streptokokken-folgeerkrankungen müssen auch autoimmunerkrankungen in die differentialdiagnose einbezogen werden. therapie penicillin g als mittel der wahl. dosierung und dauer hängen von manifestation und klinischer fulminanz ab. bei der streptokokkenpharyngitis reicht eine orale therapie über 10 tage (tagesdosis bei 6-12 mega), bei fulminanter sepsis sind tagesdosen bis zu 40 mega nötig, alternativ evtl. cephalosporine. bei penicillinallergie sind makrolidantibiotika bzw. vancomycin alternativen. bei einigen krankheitsbildern (z. b. phlegmone, fasciitis necroticans) sind ferner schnelle und offensive chirurgische maßnahmen nötig. bei streptokokken-folgeerkrankungen spielt auch die antiphlogistische behandlung eine wichtige rolle und evtl. kortikosteroidgabe. ferner ist eine rezidivprophylaxe mit penicillin oder erythromycin für mind. 1-2 jahre indiziert. bei den übrigen a-streptokokken-erkrankungen keine spezifischen prophylaktischen maßnahmen, was auch für die expositionsprophylaxe mit antibiotika bei scharlachausbrüchen gilt. hämolysierende streptokokken der serologischen gruppe b verursachen v. a. peripartale infektionen bei neugeborenen, bis 48 h post partum eine sepsis und 8-14 tage post partum meningitis. zunehmend findet man sie auch bei pyogenen infektionen geriatrischer patienten. hämolysierende streptokokken der gruppen c und g verursachen auch pharyngitis oder wundinfektion. systemisch-septische infektionen fast ausschließlich bei abwehrgeschwächten patienten. vergrünende bzw. nicht hämolysierende streptokokken haben ihren natürlichen standort im oropharynx des menschen, s. bovis im darm von mensch und tier. sie werden als orale streptokokken bezeichnet. sie verursachen: • karies und parodontitis • nativklappen-("endocarditis lenta") und spät-prothesenendokarditis (› kap. 7.9.1). bei nachweis von s. bovis in der blutkultur muss eine kolonerkrankung (karzinom, divertikulitis) ausgeschlossen werden! diagnostik erregernachweis in entsprechenden materialien. pneumokokken sterben wegen ihres starken autolysesystems auf dem transport rasch ab. wichtig sind -vor allem bei pneumonie -blutkulturen. in der meningitisdiagnostik spielen der mikroskopische erreger-und der spezifische antigennachweis (kapselpolysaccharid) eine zunehmende rolle in der spezifischen schnelldiagnostik. der antigennachweis aus sputum oder trachealsekret ist oft unspezifisch. therapie penicillin g. andernorts schon sehr häufig (spanien!) isolierte penicillin-g-resistente pneumokokken wurden bei uns bisher nur selten gefunden. stämme mit nur mäßiger empfindlichkeit gegen penicilline sind auch bei uns häufiger. eine resistenztestung ist daher durchzuführen. alternative substanzen: cephalosporine der 3. generation (z. b. cefotaxim) oder carbapeneme. es besteht die möglichkeit zur aktiven immunisierung. die vakzine beinhaltet die wichtigsten, vor allem bei septischen verlaufsformen vorkommenden kapseltypen. die impfung ist regelimpfung nach stiko für kinder und ältere menschen. epidemiologie natürlicher standort ist der darm von mensch und tier. der infektionsweg ist endogen-hämatogen nach translokation aus dem darm, auch exogene schmutz-schmierinfektionen sind möglich. klinische bilder e. faecalis ist erreger akuter harnwegsinfektionen und adnexitiden (innerhalb einer mischinfektion) der frau. von größerer bedeutung ist die enterokokkenendokarditis (ca. 10%). ferner spielen enterokokken eine rolle als wundinfektionserreger. diagnostik erregernachweis in entsprechenden materialien, v. a. in der blutkultur (endokarditis). • enterokokken-harnwegsinfektionen: aminopenicilline • enterokokkenendokarditis: auch alternativ mezlocillin. grundsätzlich immer kombinierte behandlung mit gentamicin in den ersten 2 wochen. die therapiedauer liegt bei 4-6 wochen. bei penicillinallergie oder bei stämmen mit "high-level"-resistenz (> 2000 mg/l mhk) gegen gentamicin gabe von glykopeptiden (v. a. teicoplanin). gegen glykopeptidresistente enterokokken wirken linezolid und daptomycin. die wichtigsten sind meningokokken und gonokokken der gattung neisseria. branhamella catarrhalis (früher neisseria catarrhalis, kokkoide stäbchen) gehört zur physiologischen rachenflora, kann aber infektionen des oberen und unteren respirationstrakts verursachen. moraxella-und kingella-arten (kokkoide stäb-chen) werden mitunter als opportunistische infektionserreger isoliert. selten werden veillonellen (anaerob) aus pyogenen mischinfektionen isoliert. man unterscheidet die zyklischen allgemeininfektionen typhus und paratyphus von den mit übelkeit, erbrechen und durchfällen einhergehenden enteritissalmonellosen. die übertragung erfolgt überwiegend auf oralem infektionsweg. definition die gattung salmonella hat einen sehr großen arten-bzw. serovarreichtum. es handelt sich um gramnegative stäbchen aus der familie der enterobakterien. nach heute gültiger, aber umstrittener exakter taxonomie ist der einzige humanmedizinisch bedeutende vertreter die spezies salmonella enterica bzw. die subspezies salmonella enterica subspecies enterica mit einer großen vielfalt an serovaren (zur abgrenzung von speziesnamen werden die serovare mit großbuchstaben gekennzeichnet). unter klinischen gesichtspunkten hat sich folgende einteilung bewährt: s. typhi (die abgekürzte form von s. enterica subsp. enterica serovar typhi) ist klassischer erreger des typhus, s. paratyphi (s. enterica subsp. enterica serovar paratyphi) der erreger des paratyphus, unterteilt in die gruppen a, b und c. wichtigste erreger von enteritissalmonellosen sind s. typhimurium (s. enterica subsp. enterica serovar typhimurium) und s. enteritidis (s. enterica subsp. enterica serovar enteritidis). die übrigen enteritissalmonellen werden aufgrund ihrer oberflächenantigene (o-gruppen) und geißelantigene (h-gruppen) typisiert. sie werden aufgrund der antigenformel benannt oder mit einem speziellen namen, der meist nach dem ersten nachweisort erfolgt (z. b. s. coeln). diese repräsentieren dann serovare und keine (!) spezies oder subspezies. diagnostik bei entsprechender reiseanamnese und typischem fieberverlauf lässt sich häufig klinisch die verdachtsdiagnose stellen. richtungweisend sind das auftreten von roseolen, eine leukopenie, das fehlen eosinophiler leukozyten im differentialblutbild mehrerer blutausstriche und eine relative bradykardie. die kulturelle erregerdiagnose gelingt im sehr frühen krankheitsstadium evtl. noch im stuhl, meist aber in der 1. und 2. krankheitswoche nur in der blutkultur, zur erhöhung der sensitivität sollten multiple blutkulturen abgenommen werden. die kulturelle untersuchung von knochenmark ist besonders sensitiv. am ende der 2. krankheitswoche lässt sich der erreger meist wieder aus dem stuhl isolieren. der antikörpernachweis ist für die akutdiagnostik wenig hilfreich. wegen der schwierigen isolierungsbedingungen (selektivnährböden nötig!) kann dies mehrere tage erfordern. ab ende der 1. bzw. beginn der 2. krankheitswoche kommt es zur messbaren antikörperbildung, hohe titer werden ab der 3. woche erreicht. dann kann die diagnose serologisch (widal-reaktion) bestätigt werden. bei sehr früh begonnener antibiotischer therapie kann der antikörpernachweis negativ bleiben. zur differentialdiagnose siehe auch › tabelle diagnostik erregernachweis aus stuhl, operationsmaterial und evtl. aus der blutkultur. antikörpernachweisverfahren (mikroagglutination, elisa, westernblot) spielen in der diagnostik der akuten enterokolitiden keine wesentliche rolle. bei subakut oder chronisch verlaufenden pseudoappendizitisformen und postinfektionssyndromen sind sie dagegen ausschlaggebend. therapie meist selbstlimitierende erkrankung, keine antibakterielle chemotherapie nötig. bei schwerem oder septischem verlauf behandlung mit tetrazyklinen, chinolonen, co-trimoxazol oder aminoglykosiden. definition erkrankungen durch y. pseudotuberculosis sind zooanthroponosen. ein fäkal-oraler infektionsweg wird angenommen, infektionsquelle sind infizierte tiere oder kontaminierte tierische nahrungsmittel. eine besonderheit scheint die affinität von y. pseudotuberculosis zum lymphatischen gewebe im abdominellen bereich zu sein. wichtigste klinische manifestation ist die pseudoappendizitis (mesenteriale lymphadenitis, akute terminale ileitis), die ihren häufigkeitsgipfel in der altersgruppe von 6-14 jahren hat. symptome fieber und schmerzen im rechten unterbauch. extrem selten septischer verlauf, dann v. a. bei stark abwehrgeschwächten patienten. begleitende krankheitserscheinungen sind reaktive arthritis und erythema nodosum. diagnostik erregernachweis aus schleimhautbiopsien, operationsmaterial, seltener aus stuhl. die serologische diagnostik (titerverlauf im mikroagglutinationstest) spielt jedoch eine wichtige rolle. therapie meist keine antibakterielle chemotherapie erforderlich. nur bei schwerem septischem verlauf antibiotikatherapie mit ampicillin, tetrazyklinen oder aminoglykosiden. ätiologie eine der ältesten und gefährlichsten zooanthroponosen, hervorgerufen durch y. pestis. nagetiere, v. a. ratten, sind wichtigstes erregerreservoir, flöhe die wichtigsten vektoren. epidemiologie kommt in europa nicht mehr vor. sporadische fälle werden aus dem nördlichen und südlichen afrika, aus den usa, südamerika und weiten teilen asiens berichtet. jährlich werden weltweit mehrere tausend krankheitsfälle gemeldet. wegen der relativ kurzen inkubationszeit sind auch touristisch eingeschleppte fälle extrem selten (zur meldepflicht s. ifsg nach § 6 abs. 1 nr. 1). • beulenpest: befall der regionalen lymphknoten im abflussbereich der bissstelle des infizierten flohs mit schwerer allgemeinsymptomatik • lungenpest: aerogene übertragung von mensch zu mensch mit hoher letalität. therapie gut behandelbar mit antibiotika (gentamicin oder doxycyclin). die pest verursachte in den letzten jahren nur selten größere epidemien und hat viel von ihrem früheren schrecken verloren. definition die familie der enterobacteriaceae zeichnet sich durch großen artenreichtum aus. viele gattungen besitzen auch humanmedizinische bedeutung. während den salmonellen, shigellen und yersinien überwiegend spezielle krankheitsentitäten zugeordnet werden können, ist dies für die meisten anderen gattungen nicht möglich. daher werden diese enterobacteriaceae-arten als fakultativ pathogene erreger gesondert betrachtet (› tab. 13 in speziellen fällen (z. b. zur sicheren diagnose der lues connata) ist der nachweis spezifischer igm-antikörper in der indirekten immunfluoreszenz erforderlich. bei neurosyphilis wird eine autochthone intrathekale anti-treponemen-antikörperproduktion nachgewiesen. therapie bedeutsam sind frühzeitige diagnose und therapiebeginn im 1., spätestens im 2. stadium. therapie der wahl ist penicillin g oder ceftriaxon, bei penicillinallergie doxycyclin oder azithromycin. zur vermeidung der frühen neurosyphilis muss die therapie hoch dosiert (procain-penicillin g, 2,4 mio. e i.m.) für 14 tage durchgeführt werden, um ausreichend hohe liquorspiegel zu erreichen. bei patienten mit gleichzeitiger hiv-infektion, bei tertiärer syphilis und bei neurosyphilis wird 2 × 10 mio. e penicillin i.v., alternativ 1 × 2 g ceftriaxon für 14 tage verabreicht. der verlauf der erkrankung und vor allem der effekt der therapie müssen durch regelmäßige serologische aktivitätskontrollen (vdrl-test, cardiolipin-kbr) überprüft werden. prophylaxe eine impfung existiert nicht. eindämmung der übertragungswege durch möglichst lückenlose aufklärung und überwachung der risikogruppen. abb. 13.74 syphilis. a) primäraffekt an der glans penis. b) luesexanthem im stadium ii an den fußsohlen. c) alopecia areata, ebenfalls stadium ii. infektionsschutzgesetz: kommentar und vorschriftensammlung . kohlhammer dolin: principles and practice of infectious diseases, 5 th edn ., 2 vols klinische infektiologie . urban & fischer just-nübling: antibiotikatherapie, 11 anthrax antibiotic associated colitis bacillary dysentery bartonellenerkrankungen botulism botulismus brucellosis chlamydial diseases cholera coagulase-negative staphylococci diphtherie enteric e . coli infections enteric fever enteritissalmonellose enterococci epidemic typhus erysipeloid fakultativ pathogene enterobacteracea gas gangrene/edema gonorrhoe gonorrhoea group a streptococci haem . influenzaerkrankungen hwi infektionen durch staphylokokken infektionen durch streptokokken legionärskrankheit legionellosis legionnaires' disease leprosy leptospirose leptospirosis listeriose-pneumokokken listeriosis lyme-borreliose-stadien lyme-borreliosis lyme disease meningococcal meningitis/sepsis mykoplasmenübertragung nocardiosis oculo-genital infections ornithose ornithosis pertussis pint plague pontiac fever pneumococci pseudomonas psittacosis q-fieber relapsing fever rickettsien rickettsial diseases salmonella foodborne disease salmonellose shigellose staphylococcal diseases streptococcal/enterococcal diseases swine erysipelas verfügbare zubereitungsformen aktivimpfstoffe enthalten impfantigene zur aktivierung des immunsystems mit erregerspezifischer bei einzelnen impfungen sind antikörpergrenzwerte für den optimalen impfschutz bekannt. jedoch kann nicht immer von antikörperkonzentration auf effektiven, sicheren schutz vor infektion geschlossen werden • zellvermittelte immunantwort: routinemäßig schwer zu testen. t-lymphozyten haben für die induktion humoraler antikörperantworten (z. b. gegen proteinantigene) große bedeutung und tragen zur bildung des immunologischen gedächtnisses (memory-zellen) bei. der effekt t-zell-abhängiger impfungen lässt sich z. b. durch bestimmung der masern-, mumps-und röteln-antikörper nach mmr-lebendimpfung abschätzen bei der aktiven immunisierung zum aufbau eines dauerhaften impfschutzes unterscheidet man: • lebendimpfstoffe: vermehrungsfähige, attenuierte (abgeschwächte vollkeim-impfstoff) oder in mehr oder minder reiner form immunologisch relevante antigene des erregers (spalt-impfstoff, extrakt-impfstoff, toxoid-impfstoff, subunitvakzine) durch geeignete spenderkontrolle, herstellungsverfahren von infektionskrankheiten hergestellt. beispiel: humanisierter antikörper palivizumab, zum schutz von frühgeborenen unter bestimmten indikationen (z. b. chronische lungenkrankheit) vor der infektion mit dem respiratory syncytial virus (rsv) stuhl kontaminierte) wunden, verletzungen mit gewebszertrümmerung und reduzierter sauerstoffversorgung oder eindringen von fremdkörpern jede auffrischimpfung mit td sollte anlass sein, eine mögliche indikation einer pertussis-impfung zu überprüfen und ggf. einen kombinationsimpfstoff (tdap) einzusetzen im allgemeinen werden 250 ie verabreicht, die dosis kann auf 500 ie erhöht werden; tig wird simultan mit td/t-impfstoff angewendet dosis), wenn seit der letzten impfung mehr als 10 jahre vergangen sind wenn seit der letzten impfung mehr als 5 jahre vergangen sind quelle: rki: empfehlungen der ständigen impfkommission (stiko) am robert-koch-institut/stand: juli ein patient stellt sich 8 tage nach durchführung des 4. zyklus einer ambulanten chemotherapie nach dem chop-r-schema im 14-tägigen abstand mit fieber in der notaufnahme vor. bei der untersuchung des blutbildes zeigt sich eine leukozytenzahl von 600/μl. a. welche untersuchungen werden neben einer ausführlichen körperlichen untersuchung veranlasst? wie wird der patient initial behandelt? seine körperliche leistungsfähigkeit habe abgenommen, vor allem beim treppensteigen komme er leicht "aus der puste". er wisse seit 1,5 jahren, dass er hiv-positiv sei, habe sich aber bislang nicht weiter untersuchen lassen heute habe er deswegen kaum noch etwas zu sich nehmen können. bei der körperlichen untersuchung fällt ihnen auf, dass der rachen des patienten weiße beläge aufweist, die teilweise abstreifbar sind. am hals finden sich beidseits mehrere, bis zu 2 cm im durchmesser große indolente lymphknoten, von denen der patient angibt, dass diese schon seit 1 jahr unverändert bestünden ihr verdacht bestätigt sich. was empfehlen sie dem patienten? welche sind die 3 wichtigsten antimykotika-substanzklassen? welche candida-spezies sind immer oder häufig gegen fluconazol resistent? werden sprosspilzinfektionen endogen oder exogen erworben? was ist die pathogenetische bedeutung der besiedlung der unteren atemwege mit candida species? ist die candidapneumonie eine häufige erkrankung? wie lange muss die therapie einer candidämie durchgeführt werden? 14. werden fadenpilzinfektionen endogen oder exogen erworben? welche 3 erkrankungen der lunge durch aspergillus species werden unterschieden? ein junger mann berichtet, dass er seit 4 wochen unter leibschmerzen und durchfällen, gelegentlich auch unter obstipation leide. die beschwerden seien das erste mal aufgetreten, als er noch in brasilien war, wo er für seine ethnologische doktorarbeit bei urwaldindianern material gesammelt habe. auf befragen gibt er an welche frage ist zunächst zu stellen? b. welche untersuchungen werden durchgeführt? c. wie wird der patient behandelt? d. was wird dem patienten gesagt? er gibt an, er habe seit 4 tagen fieber bis 38,9 °c, außerdem kopfschmerzen, inappetenz, abdominelle schmerzen und übelkeit. er habe keine malariaprophylaxe eingenommen, da man ihm gesagt habe, dass im januar wegen der tiefen nachttemperaturen in nordindien kein malariarisiko bestehe sie gibt an, sich seit gestern stark unwohl gefühlt zu haben, wie in letzter zeit schon öfter während ihrer regelblutung. im büro heute morgen habe sie dann begonnen stark zu schwitzen, verbunden mit hitze-kälte-wallungen, schließlich sei ihr "schwarz" vor augen geworden. befunde der orientierenden untersuchung: rr 80/60 liegend, puls 112, temperatur 39,8 °c rektal. a. wie lautet ihre verdachtsdiagnose? b. welche untersuchungen veranlassen sie zunächst? c. welche therapeutischen maßnahmen sind angezeigt? d die e.-granulosus-larve bildet in der regel eine mit flüssigkeit gefüllte zyste (› abb. 13 .62), die langsam verdrängend wächst und einen durchmesser von 20 cm und mehr erreichen kann: zystische echinokokkose.im gegensatz dazu bildet die e.-multilocularis-larve komplizierte, schwammartige, gallertig gefüllte schläuche und hohlräume von wenigen millimetern durchmesser: alveoläre echinokokkose. der parasit wächst infiltrativ destruktiv, ähnlich wie ein bösartiger tumor; metastasierungen in alle organe können vorkommen.symptome für beide echinokokkosen werden inkubationszeiten von mehreren jahren, bei der alveolären echinokokkose sogar von mehr als 10 jahren angenommen. die symptome sind von der ausdehnung des organbefalls und von der wachstumsgeschwindigkeit des parasiten abhängig. häufig sind oberbauchschmerzen, tastbarer tumor im bereich der leber mit verdrängungsgefühl oder schmerzen, seltener gallenstau und aszites. oft werden die zysten auch nur zufällig entdeckt. bei lungenbefall kommt es zu hämoptyse, atelektasen und bronchiektasen. nicht selten sind allergische reaktionen an haut und schleimhäuten, gelegentlich asthma bronchiale.diagnostik für die diagnostik der beiden echinokokkoseformen sind vor allem sonographie und computertomographie von großer bedeutung. werden zystische veränderungen in der leber oder lunge festgestellt, muss durch anwendung serologischer verfahren versucht werden, eine diagnose zu stellen. werden zwei antikörperbestimmungen unterschiedlichen aufbaus nebeneinander verwandt, so lässt sich in den meisten fällen eine klärung erreichen. die alveoläre echinokokkose lässt bei den verschiedenen bildgebenden verfahren in der regel keine zystische struktur erkennen, zentrale nekrosen im parasitengewebe können diese allerdings vortäuschen. verkalkungen sind häufig. und eiweißerhöhung; sepsis: leukozytose mit linksverschiebung, crp-, procalcitoninerhöhung) wider. therapie entscheidend ist die frühzeitige penicillin-g-therapie in hoher dosierung (20-40 mega/tag). alternativen (vor allem bei penicillinallergie) sind carbapeneme oder cephalosporine der 3. generation. die therapie dauert mind. [10] [11] [12] [13] [14] tage; von anfang an durchgeführte liquorkontrollen müssen keimfrei sein. prophylaxe eine präventive schutzimpfung für die serotypen a und c zur eindämmung von großepidemien ist möglich (ausnahme: säuglinge). gegen neisseria meningitidis typ b gibt es bisher keine impfung. expositionsprophylaxe mit rifampicin wird personen empfohlen, die intensiven kontakt zu einem erkrankten hatten (familie, kindertagesstätten). die erkrankung ist meldepflichtig (s. ifsg nach § 6 abs. 1 nr. 1). verursacht durch neisseria gonorrhoeae (gonokokken), ist sie die häufigste bakterielle geschlechtskrankheit. außerhalb der genitalschleimhaut sterben die bakterien relativ schnell ab. therapie bei erwachsenen gabe von penicillin oder chinolonen (evtl. nur eine dosis!), bei kindern cephalosporine der 3. generation. diphtherie definition erreger ist corynebacterium diphtheriae, und zwar nur diphtherietoxin-bildende stämme. die übertragung erfolgt durch tröpfcheninfektion oder direkten kontakt mit erkrankten oder gesunden keimträgern. epidemiologie 1975 gab es in deutschland zum bisher letzten mal gruppenerkrankungen bzw. kleinepidemien. einzelfälle (meist eingeschleppt aus epidemiegebieten, zurzeit russland, ukraine) kommen immer wieder vor. da bei uns der impfschutz bei älteren jugendlichen und erwachsenen (nicht durchgeführte wiederimpfungen) durch die aktive impfung stark abnimmt, ist ein epidemieartiges auftreten der diphtherie jederzeit möglich.manifest erkranken ca. 20% der infizierten. pathogenese pathogenetisch entscheidend ist das diphtherietoxin, bestehend aus den untereinheiten a (letal zytotoxisch) und b (vermittelt zelleinschleusung) und kodiert von einem prophagen.das toxin wird am ort der infektion produziert und gelangt per continuitatem oder hämatogen zu anderen gewebsbereichen bzw. organen. rachendiphtherie hauptsächliche manifestation mit rötung und schwellung von rachenschleimhaut bzw. tonsillen und schwerem allgemeinem krankheitsgefühl nach bis zu 5 tagen inkubationszeit. hohes fieber ist selten, dann oft zeichen einer primär-toxischen diphtherie. im weiteren verlauf bilden sich nach wenigen stunden weiße beläge auf der schleimhaut, aus denen die bräunlichen pseudomembranen aus fibrin, entzündungszellen und nekrotischen epithelzellen gebildet werden (› abb. 13 .70), die fest auf den wundflächen haften. eine instrumentelle ablösung führt daher zu blutungen (wichtiges diagnostisches kriterium!). von diesen pseudomembranen kann ein sehr charakteristischer fötid-süßlicher geruch ausgehen. die zugehörigen regionären lymphknoten sind deutlich geschwollen. klinischer höhepunkt nach 4-5 tagen. dieser kann im sekundär-toxischen verlauf mit spätkomplikationen, aber auch unter entfieberung in die hei lungsphase übergehen. selten hämatogene metastasierung der erreger.abb. 13 .69 meningokokken (pfeile) im liquor, umgeben von granulozyten. (aus: thomas, 1986 ). • nasendiphtherie (mit eitrig-blutiger sekretion), augendiphtherie (konjunktivitis) und nabeldiphtherie: vorrangig bei säuglingen definition grampositive fadenbakterien, die in verzweigten geflechten wachsen (strahlenpilze). man unterscheidet anaerobe (gattung actinomyces und arachnia) von aerob wachsenden aktinomyzeten (gattung nocardia). nokardien leben im erdboden, die anaeroben aktinomyzeten auf der menschlichen oropharyngealschleimhaut. therapie nach entnahme von material zur mikrobiologischen diagnostik kalkulierte therapie mit antibiotika mit wirksamkeit gegenüber enterobakterien. häufig wird hierzu ein breitspektrum-β-lactam (z. b. piperacillin oder drittgenerations-cephalosporin), ein fluorchinolon oder ein carbapenem eingesetzt (› kap. 13.2.1). grundlage für eine gezielte chemotherapie ist das ergebnis der antibiotikaresistenzprüfung. weltweit wird bei enterobakterien eine zunehmende resistenzentwicklung beobachtet, von der neben älteren substanzen (ampicillin, co-trimoxazol) zunehmend auch fluorchinolone (v. a. bei e. coli) betroffen sind. hinzu kommt das zunehmende auftreten von stämmen, die "extended spectrum"-β-lactamasen (esbls) bilden, die zur resistenz gegenüber allen β-lactam-antibiotika mit ausnahme der carbapeneme führen. besondere therapiemaßnahmen bei septischem krankheitsverlauf (z. b. gabe von antikörpern, kortikosteroiden oder aktiviertem protein c) und supportive therapiemaßnahmen werden an anderer stelle beschrieben. prävention bisher keine spezifischen maßnahmen. die verhinderung nosokomialer infektionen durch fakultativ pathogene enterobacteriaceae beruht v. a. auf präventiven hospitalhygienischen maßnahmen und dem rationalen umgang mit antibiotika. bestimmte darmpathogene e.-coli-stämme können enteritiden und kolitiden verursachen (› tab. 13 symptome 2-phasiger verlauf nach 1-bis 2-wöchiger inkubationszeit mit charakteristischen, wochenlang anhaltenden hustenanfällen im 2. stadium.diagnostik pcr-basierte nachweisverfahren zum nachweis des pathogenetisch bedeutsamen, von b. pertussis produzierten pertussis-toxins stehen im vordergrund. andere verfahren zum nachweis von b. pertussis (direkte immunfluoreszenz, kultureller erregernachweis, serologischer antikörpernachweis) sind heute von geringerer bedeutung.therapie chemotherapie mit erythromycin: geringer einfluss auf den krankheitsverlauf, verkürzt aber die erregerausscheidungsdauer. nach durchgemachter erkrankung besteht eine lang dauernde, aber nicht unbedingt lebenslange immunität.die aktive impfung erfolgt nicht mehr mit dem klassischen "ganzzell"-impfstoff, sondern mit einer "azellulären" pertussis-vakzine mit hoher effektivität und geringen nebenwirkungen (› kap. 13.10). therapie wie bei ornithose. die mögliche ätiopathogenetische bedeutung von c. pneumoniae bei der entstehung arteriosklerotischer gefäßerkrankungen und folgekrankheiten wie z. b. khk und herzinfarkt wird kontrovers diskutiert. eine rolle in der kopathogenese (entzündung!) erscheint möglich, eine monospezifische bedeutung sehr unwahrscheinlich. auf jeden fall rechtfertigt die bisher vorliegende studienevidenz keine spezifischen therapeutischen konsequenzen (antibiotikatherapie). die serovare l1-l3 von c. trachomatis sind für die klassische geschlechtskrankheit lymphogranuloma venereum verantwortlich mit manifestation im genitalbereich und den benachbarten lymphknoten.die serovare a-c von c. trachomatis, die weltweit aber vorrangig in warmen gebieten vorkommen, verursachen die in stadien fortschreitende keratokonjunktivitis, das trachom, die häufigste einzelursache für blindheit weltweit.natürliche habitate der serovare d-k von c. trachomatis sind die zervix die frau und die urethra des mannes. infektionen erfolgen daher stets von dort aus, perinatal oder durch geschlechtsverkehr.klinische bilder krankheitsbilder bei infektionen mit den serovaren d-k sind v. a. infektionen des genitaltrakts: beim mann nichtgonorrhoische und postgonorrhoische urethritis. symptome sind dysurie, urethralschmerzen und -ausfluss. komplizierend kann eine prostatitis bzw. epididymitis hinzukommen.bei der frau verlaufen infektionen oft symptomlos bzw. als urethral-oder dysuriesyndrom, mehr durch unpässlichkeit denn als richtige krankheit gekennzeichnet. daraus kann eine adnexitis bis zum tuboovarialabszess entstehen. ausgehend von ersterer kann sich eine perihepatitis (fitz-hugh-curtis-syndrom) entwickeln mit entsprechender oberbauchsymptomatik. als folge der adnexitis kann es durch verwachsungen zum tubenverschluss mit sterilität bzw. zur extrauteringravidität kommen. nach perinataler infektion kann es zur typischen chlamydienerkrankung des neugeborenen kommen, der sog. einschluss(körperchen)konjunktivitis. die entsprechende erkrankung des erwachsenen (schwimmbadkonjunktivitis) ist heute viel seltener (chlordesinfektion der schwimmbäder).bei schwer immunsupprimierten ist eine c.-trachomatis-pneumonie möglich.als komplikation nach einer c.-trachomatis-infektion gilt eine reaktive arthritis bei bevorzugter betroffenheit von hla-b27-trägern. therapie chemotherapie bei erwachsenen mit tetrazyklinen oder chinolonen (evtl. partnerbehandlung!), bei kindern mit makroliden. synonym: rickettsiosen definition rickettsia species sind kokkoide gramnegative stäbchenbakterien. die bis vor kurzem auch hierzu gerechneten coxiella species werden aufgrund neuer molekulargenetischer untersuchungen heute mit den legionellen in einer eigenen ordnung geführt. dies gilt auch für die ehrlichia species, die mit 2 anderen gattungen die familie anaplasmatacea bilden, und deren zellwand kein lipopolysaccharid und peptitglykan, sondern cholesterin enthält. alle haben einen obligat intrazellulären lebenszyklus und werden mit ausnahme von coxiella bumetii durch arthropoden übertragen. diagnostik pcr-verfahren und serologische tests. an mögliche doppelinfektionen denken (borreliose, fsme; zecken!). therapie tetrazykline, vor allem doxycyclin, und rifampicin. bartonellen sind gramnegative pleomorphe stäbchenbakterien, die früher z. t. als gattung rochalimaea in der familie rickettsiaceae geführt wurden. sie werden heute in einer eigenständigen familie (bartonellaceae) als gattung bartonella eingeordnet. im gegensatz zu den klassischen rickettsien (s. o.) können sie auf spezialmedien in etwa 1 woche kulturell angezüchtet werden. die pathogenese von bartonella-infektionen ist noch weitgehend unbekannt. eine seltene, aber gefährliche manifestation ist die neuroretinitis mit akutem mono-oder bilateralem visusverlust mit papillitis, retinaler vaskulitis und/oder makulaödem. eine weitere wichtige manifestation ist die bazilläre angiomatose mit sepsis, die nur bei immundefekten (hiv!) auftritt. charakteristisch sind generalisierte gefäßproliferationen an haut und schleimhäuten, kombiniert mit septischen zuständen.eine seltene manifestation ist die bazilläre peliosis, gekennzeichnet durch zystische, mit blut gefüllte läsionen in leber und milz.diagnostik mikrobiologisch durch mikroskopischen und kulturellen erregernachweis im spezialverfahren. wichtiger sind immunfluoreszenz-und elisa-verfahren und auch die pcr.therapie tetrazykline und makrolide. impfindikationen und -empfehlungen hängen von folgenden zielen ab:• ausrottung eines erregers (z. b. pocken, aktuell: poliomyelitis (who))• herdenimmunität: manche erreger können in einer bevölkerung nicht mehr epidemisch auftreten, wenn ein bestimmter mindestanteil der bevölkerung ausreichend immun ist.• individualschutz. die impfpolitik eines landes hängt von den epidemiologischen verhältnissen, der verfügbarkeit von impfstoffen und der impfstrategie ab. in deutschland gibt es von der ständigen impfkommission (stiko) öffentliche empfehlungen, d.h. definitionen von regel-oder standardimpfungen. eine gesetzliche impfpflicht besteht hier nicht. allgemein empfohlene standardimpfungen sollen nach einem von der stiko jährlich aktualisierten impfplan bereits im frühen säuglingsalter (ab dem vollendeten 2. lebensmonat; › tab. 13 für aktualisierungen siehe auch www.rki.de * abstände zwischen den impfungen mindestens 4 wochen; abstand zwischen vorletzter und letzter impfung mindestens 6 monate ** generelle impfung gegen pneumokokken für säuglinge und kleinkinder bis zum vollendeten 2. lebensjahr mit einem pneumokokken-konjugatimpfstoff; standardimpfung für personen ≥ 60 mit polysaccharid-impfstoff und wiederimpfung im abstand von 6 jahren *** mindestabstand zwischen den impfungen 4 wochen **** jährlich mit dem von der who empfohlenen aktuellen impfstoff ***** jeweils 10 jahre nach der letzten vorangegangenen dosis meningokokken (serogruppe c). bei erwachsenen sind auffrischimpfungen gegen tetanus und diphtherie in abständen von 10 jahren vorgesehen. für alle erwachsenen nach vollendetem 60. lebensjahr wird eine standardimpfung gegen influenza und pneumokokken empfohlen, aber selten umgesetzt. für personen ohne individuelles risiko, wird in deutschland z. b. bei reisen in länder mit endemischem auftreten der poliomyelitis eine routinemäßige auffrischung der poliomyelitisimpfung nach dem 18. lebensjahr nicht mehr empfohlen.bei den nach § 20 abs. 3 des infektionsschutzgesetzes (ifsg) öffentlich empfohlenen impfungen ist eine kostenübernahme durch die krankenkassen nicht automatisch gegeben, jedoch werden die kosten für diese schutzimpfungen in der regel nach verhandlungen über die umsetzung der stiko-empfehlungen von den verschiedenen kostenträgern übernommen (kassenleistungen nach § 23 abs. 9 sgb v). bei anerkanntem impfschaden nach einer "öffentlich empfohlenen" impfung werden die kosten zur entschädigung und versorgung durch die bundesländer übernommen.neben den standardimpfungen (regelimpfungen) und den zugehörigen auffrischimpfungen gibt es indikationsimpfungen für risikogruppen (z. b. bei bestimmten grunderkrankungen, individuell erhöhtem expositions-bzw. beruflich erhöhtem risiko). wichtig ist die kenntnis der indikationen für reiseimpfungen (z. b. cholera, fsme, gelbfieber, hepatitiden a und b, influenza, meningokokken, tollwut, typhus). die in den stiko-empfehlungen mit r gekennzeichneten reiseimpfungen werden nicht von den krankenkassen übernommen. indikationen bei den regelimpfungen ist die indikation generell gestellt. indikationsimpfungen erfolgen zum individualschutz prä-oder postexpositionell. als domäne der postexpositionellen impfung wird üblicherweise die passive immunisierung empfänglicher (nicht immuner) angesehen, z. b. standardimmunglobulingabe nach masernexposition bei schwangeren, oder hyperimmunglobulingabe nach varizellen-exposition bei immunsupprimierten oder schwangeren. postexpositionelle aktive impfungen (inkubationsimpfungen) bei immungesunden sind möglich und werden allein (z. b. masern, hepatitis a) oder als kombinierte aktiv-/passivimmunisierungen praktiziert (z. b. tollwut, hepatitis b, tetanus, ggf. hepatitis a). die verbreitete furcht vor inkubationsimpfungen hat ihre wurzeln weniger in der immunologie als in der sorge um schadenersatzansprüche bei trotz impfung schwer verlaufender bzw. nicht vermeidbarer erkrankung. impfabstände zeitliche abstände zwischen impfungen mit totimpfstoffen sind nicht erforderlich. lebendimpfungen müssen simultan oder mit 4-wöchigem abstand verabreicht werden. die empfehlungen zu zeitlichen abständen zwischen auffrischimpfungen sind sinnvolle richtschnur für individuelle impfentscheidungen. eine begonnene, aber nicht vollständig durchgeführte grundimmunisierung kann jederzeit fortgeführt und muss nicht von neuem begonnen werden ("jede impfung zählt."). zur konkreten planung und verschreibung von impfungen siehe produktinformationen der hersteller und gültige rote liste (› tab. 13 impfstoffe sinnvoll und anzustreben sind polyvalente extrakt-impfstoffe, die gereinigte kapselpolysaccharide von möglichst vielen infektionsrelevanten kapseltypen enthalten (› tab. 13.46). so gibt es eine polysaccharidvakzine aus 23 verschiedenen polysaccharidantigenen der ca. 90 bekannten pneumokokkentypen. polysaccharid-impfstoffe führen jedoch bei kindern < 2 jahren zu keiner ausreichenden immunantwort. zur bekämpfung schwerer systemischer pneumokokkeninfektionen war daher die entwicklung von pneumokokken-protein-konjugat-vakzinen ein großer fortschritt, da mit einer bei diesen konjugaten gegebenen t-zell-abhängigen immunisierung auch säuglinge effektiv gegen pneumokokken geschützt werden können. indikationen nach eindeutigen erfolgen in den usa wurde ein 7-valenter pneumokokken-konjugat-impfstoff in deutschland zunächst für die höchstgefährdeten frühgeborenen zugelassen. im juli 2006 wurde dieser erstmals als standard für alle kinder < 2 jahren empfohlen (› tab. 13.44). die verabrei-chung des pneumokokken-polysaccharid-impfstoffes (immunantwort t-zell-unabhängig) an personen > 60 jahre ist seit jahren standard. im sinne einer indikationsimpfung sollte bei kindern und erwachsenen mit erhöhter morbidität und mortalität durch pneumokokken eine immunisierung durchgeführt werden. besonders gefährdet sind patienten mit folgenden grunderkrankungen: 1. angeborene oder erworbene immundefekte mit t-und/ oder b-zellulärer restfunktion: -hypogammaglobulinämie, komplementdefekte -asplenie oder nach splenektomie -krankheiten der blutbildenden organe -zustand nach organtransplantation -sichelzellenanämie -hiv-infektion -neoplastische erkrankungen. 2. chronische krankheiten:-herz-kreislauf-krankheiten -krankheiten der atmungsorgane -diabetes mellitus und andere stoffwechselkrankheiten -chronische nierenkrankheiten -neurologische krankheiten -liquorfistel. durchführen der impfung säuglinge sollen die pneumokokken-konjugat-impfung parallel zu den anderen standardimpfungen nach vollendetem 2., 3. und 4. lebensmonat ebenso wie die 4. impfung ab vollendetem 11. lebensmonat erhalten. bei ca. 90% der vollständig immunisierten kinder in den usa lag eine schützende immunantwort gegen alle 7 verabreichten serotypen vor. auch in europa ist trotz der epidemiologischen verbreitung unterschiedlicher serotypen ein erheblicher rückgang von invasiven pneumokokken-infektionen durch die generelle einführung der konjugatvakzine zu erwarten. nebenwirkungen insgesamt gute verträglichkeit der konjugat-impfung, bisher keine berichteten bleibenden schäden. vereinzelt wurden fieberkrämpfe infolge eines raschen temperaturanstieges beobachtet. lokale schmerzen an der injektionsstelle wurden von ca. 20% der geimpften beklagt. der polysaccharidimpfstoff gegen pneumokokken ist wegen seiner zum teil erheblichen lokalen nebenwirkungen (schmerzen, schwellung) vor allem bei auffrischimpfungen in zu kurzen intervallen bekannt, gilt aber als effektiv und sicher. impfstoff von den in deutschland relevanten serogruppen a, b und c können nur a und c durch einen impfstoff erfasst werden. eine impfstoffentwicklung gegen die häufigste serogruppe b war wegen einer strukturähnlichkeit des kapselpolysaccharids mit der n-acetyl-neuraminsäure in gehirnzellen und dadurch bedingter immuntoleranz bisher nicht erfolgreich (› kap. 13.9.3). jedoch wurde in den letzten jahren u. a. in großbritannien ein meningokokken-konjugat-impfstoff gegen die serogruppe c erprobt und so eine deutliche reduktion schwerer meningokokken-typ-c-infektionen erzielt. indikationen die stiko hat im juli 2006 erstmals die impfung aller kinder zu beginn des 2. lebensjahres mit einer einmaligen meningokokken-typ-c-konjugat-gabe empfohlen. die umsetzung in der pädiatrischen praxis und der erhoffte epidemiologische erfolg mit verminderung der lebensbedrohlichen meningokokken-infektionen sind kritisch zu verfolgen. indikation zur meningokokkenimpfung gegen die serogruppen a, c, w135 und y mit einem quadrivalenten polysaccharidimpfstoff besteht bei:• besonderer gesundheitlicher gefährdung (angeborene oder erworbene immundefekte mit t-und/oder b-zellulärer restfunktion, z. b. komplementdefekte, hypogammaglobulinämie, asplenie)• gefährdetem laborpersonal • reisen in endemiegebiete (entwicklungshelfer, medizinisches personal, pilger). nebenwirkungen insgesamt gute verträglichkeit, gelegentlich lokalreaktionen, selten fieber. impfstoff der impfstoff (kühlkettenversand!) wird in embryonierten hühnereiern hergestellt und enthält daher hühnereiweiß (cave: hühnereiweißallergie!). indikationen die indikationsimpfung ist von einigen afrikanischen ländern für reisen in endemiegebiete vorgeschrieben. für reisende nach asien, die aus endemiegebieten einreisen wollen, besteht ebenfalls impf-oder quarantänezwang. die hinweise der who zu gelbfieber-infektionsgebieten sind zu beachten.die gelbfieberimpfung darf nur in von den gesundheitsbehörden zugelassenen gelbfieber-impfstellen durchgeführt werden. die impfung von kindern < 6 monaten gilt als kontraindiziert. schwangere dürfen, besonders im 1. trimenon nur bei strenger indikationsstellung geimpft werden. eine allergie gegen hühnereiweiß stellt eine kontraindikation dar, evtl. kann bei verdacht darauf durch die intrakutane gabe von 0,1 ml des lebendimpfstoffes vorgetestet werden. der impfschutz ist hervorragend (100%) und hält wahrscheinlich lebenslang an. das internationale impfzertifikat ist jedoch nur 10 jahre gültig, d.h., bei reisen in entsprechende länder ist eine wiederimpfung nach 10 jahren nötig, um den rechtlichen vorschriften zu genügen.neben der bekämpfung der vektoren (insekten) ist die impfung der einzige schutz vor gelbfieberepidemien und urbanem gelbfieber. nebenwirkungen sehr gute verträglichkeit, gelegentlich lokale rötungen, vereinzelt kurzfristige grippeähnliche symptome am 4.-6. tag nach der impfung. impfstoff die derzeit in deutschland zugelassenen impfstoffe zur oralen oder parenteralen applikation vermitteln eine schutzrate von 60-90% (kein schutz gegen paratyphusinfektionen) für 1-3 jahre. indikationen• reisen in endemiegebiete • beruflicher umgang mit infizierten oder dem erreger.kontraindikationen diese sind für den oralen lebendimpfstoff gegeben bei:• darminfektionen zum zeitpunkt der impfung • antibiotikaeinnahmen vor dem 3. tag nach beendigung der impfung • kindern im 1. lebenshalbjahr (kapsel).geimpfte scheiden für einige tage den impfstamm aus. die gleichzeitige einnahme von malariaprophylaxe, antibiotika oder laxanzien kann den impfschutz beeinträchtigen. nebenwirkungen ausgezeichnete verträglichkeit, gelegentlich leichte gastrointestinale beschwerden oder kopf-und gliederschmerzen nach den einnahmen. • reisen (entwicklungshelfer) in endemiegebiete, die indikation sollte von fall zu fall, je nach art der reise, gestellt werden.• bei choleraepidemien.durchführen der impfung die derzeit in deutschland zugelassene impfung erfolgt subkutan mit altersabhängiger dosis in form von 2 impfungen im abstand von 1-2 wochen. bei fortbestehender exposition erfolgen auffrischimpfungen im abstand von 3-6 monaten. dauer und schutzwirkung der zugelassenen choleraimpfung sind begrenzt: der schutz beträgt 40-80% für nur ca. 3 monate. daher wurde sie von der who aus den internationalen gesundheitsvorschriften im reiseverkehr herausgenommen.nebenwirkungen heftige lokale beschwerden (rötung, schwellung, schmerzhaftigkeit) sind häufig, systemische reaktionen mit fieber, kopfschmerzen, gastrointestinalen beschwerden selten. die indikationen ergeben sich aus reiseziel, aktuellen epidemiologischen bedingungen und hygienischen verhältnissen. für die zeitplanung ist entscheidend, ob lebendimpfungen und evtl. nachzuholende grundimmunisierungen oder auffrischimpfungen nötig sind (› tab. 13.49).man kann und soll bei vielen grundimmunisierungen und genug zeit bei der reiseplanung die injektionstermine der totimpfstoffe entflechten. die 3. injektion der grundimmunisierung z. b. gegen hepatitis b kann nach der rückkehr oder im reiseland erfolgen. key: cord-346424-gfccstoz authors: friedrich, brian m; murray, james l; li, guangyu; sheng, jinsong; hodge, thomas w; rubin, donald h; o'brien, william a; ferguson, monique r title: a functional role for adam10 in human immunodeficiency virus type-1 replication date: 2011-05-11 journal: retrovirology doi: 10.1186/1742-4690-8-32 sha: doc_id: 346424 cord_uid: gfccstoz background: gene trap insertional mutagenesis was used as a high-throughput approach to discover cellular genes participating in viral infection by screening libraries of cells selected for survival from lytic infection with a variety of viruses. cells harboring a disrupted adam10 (a disintegrin and metalloprotease 10) allele survived reovirus infection, and subsequently adam10 was shown by rna interference to be important for replication of hiv-1. results: silencing adam10 expression with small interfering rna (sirna) 48 hours before infection significantly inhibited hiv-1 replication in primary human monocyte-derived macrophages and in cd4(+ )cell lines. in agreement, adam10 over-expression significantly increased hiv-1 replication. adam10 down-regulation did not inhibit viral reverse transcription, indicating that viral entry and uncoating are also independent of adam10 expression. integration of hiv-1 cdna was reduced in adam10 down-regulated cells; however, concomitant 2-ltr circle formation was not detected, suggesting that hiv-1 does not enter the nucleus. further, adam10 silencing inhibited downstream reporter gene expression and viral protein translation. interestingly, we found that while the metalloprotease domain of adam10 is not required for hiv-1 replication, adam15 and γ-secretase (which proteolytically release the extracellular and intracellular domains of adam10 from the plasma membrane, respectively) do support productive infection. conclusions: we propose that adam10 facilitates replication at the level of nuclear trafficking. collectively, our data support a model whereby adam10 is cleaved by adam15 and γ-secretase and that the adam10 intracellular domain directly facilitates hiv-1 nuclear trafficking. thus, adam10 represents a novel cellular target class for development of antiretroviral drugs. cell homeostasis and ordered proliferation require the interaction of cellular elements that can be assigned to functional pathways. while cells have partial redundancy and regulated expression of components of important cellular pathways, simple pathogens such as viruses appear to be restricted in their interactions. based upon the hypothesis that disruption of specific cellular proteins would still allow cell and host survival but restrict or inhibit pathogen replication, we have randomly disrupted cellular genes with an insertional mutagen and selected for candidate genes whose inactivation allows cell survival following lytic infection. previously, we reported this strategy was successful in the discovery of several critical host genes, including components of the igf-ii pathway for reovirus and rab9 for marburg virus, validating the initial hypothesis [1] [2] [3] . moreover, we reasoned that viruses evolved from common ancestral archetypes might exhibit conserved viral-host protein-protein interactions. thus, we tested whether candidate genes discovered had broad capability to facilitate replication of viruses from other families and found that disruption of the rab9 pathway also limited the replication of ebola virus, measles virus, and hiv-1 [1] . hiv-1 replication requires the assistance of multiple host cell functions for productive infection and several participating cellular factors have been identified. recent large-scale sirna screens have revealed hundreds of host factors that participate in a broad array of cellular functions and implicate new pathways in the hiv-1 life cycle [4] [5] [6] [7] [8] . host cell encoded factors are required during every step of virus replication, with the possible exception of initiation of reverse transcription [9] [10] [11] [12] . we identified a disintegrin and metalloprotease 10 (adam10) in a gene trap library selected for resistance to lytic infection with reovirus and subsequently found that adam10 expression is critical for hiv-1 replication. adam10 is a cellular metalloprotease that activates numerous and diverse cellular proteins via proteolytic cleavage. in addition to its metalloprotease domain, it also contains a disintegrin domain, an egflike domain, a cysteine-rich domain, a transmembrane domain, and a cytoplasmic domain [13] . adam10 is required in notch signaling during embryogenesis [14] . it also shares some functions with adam17 in the cleavage and release of surface bound tnf-α, e-cadherin, and other proteins [15] [16] [17] [18] [19] [20] . previous studies have indicated that adam10 is found in both the cellular and nuclear membranes [21, 22] . it has been shown that a released intracellular fragment (icf) of adam10 is capable of translocating into the nucleus and is potentially important in the nuclear transport of the androgen receptor [21] . tousseyn and colleagues have shown that this nuclear entry of adam10 is dependent upon sequential proteolytic modification, and demonstrated that the ectodomain of adam10 is first shed by either adam9 or adam15 and the intracellular domain is subsequently cleaved by γ-secretase, releasing the icf [23] . in studies reported herein, it was found that transfecting cells with adam10 small interfering rna (sirna) dramatically inhibited replication of x4 and r5 hiv-1 strains, both in primary human monocyte-derived macrophages and in cd4 + cell lines. moreover, our data indicate that adam10 is critical for post-entry hiv-1 replication events occurring during nuclear trafficking or nuclear entry in human monocyte-derived macrophages and in cd4 + cell lines, and is dependent upon its proteolytic modification. furthermore, we show that adam15 and γ-secretase are also required for hiv-1 replication, suggesting that the adam10 intracellular domain (icd) is required for nuclear trafficking of hiv-1 to the nucleus. we have applied gene trap insertional mutagenesis [1] [2] [3] 24] as a high throughput genetic screen to aid in the discovery of novel genes critical for viral replication. cellular alleles are randomly inactivated, and cells surviving an otherwise toxic viral infection harbor a mutated gene, whose wild type counterpart is potentially utilized in the viral life cycle [1, 3] . to identify targets for broad-spectrum viral inhibition, we determined whether candidate genes implicated in gene trap studies with unrelated viruses serve a functional role in hiv-1 replication. small interfering rna (sirna) was used to knockdown expression of candidate genes, and the effect on hiv infection was determined by assaying hiv-1 p24 production. hela cells modified to stably express cd4 and ccr5 (tzm-bl cells) were screened with sirnas targeting genes trapped with reovirus, influenza a, or marburg virus 48 h prior to infection with lav (x4-tropic). treatment of tzm-bl cells with sirna specific for adam10 inhibited hiv-1 replication~90% (n = 4, data not shown). we also observed that sirna targeting erbb2ip did not affect hiv-1 replication, and thus was also used in these studies as a negative control. to confirm the requirement of adam10 in more physiologically relevant primary cells, human blood-derived macrophages were transfected with sirnas targeting adam10, cd4, or with a scrambled sequence control sirna, and then infected with the r5 hiv-1 strain sf162. figure 1a shows that adam10 silencing effectively inhibited r5-tropic hiv-1 replication when human monocyte-derived macrophages were transfected with sirnas 48 h prior to infection. adam10 sirna inhibition of hiv-1 was similar to that seen with sirna directed against cd4, the primary cellular receptor for hiv-1 [25] . interestingly, adam10 silencing also inhibited replication of the x4-tropic lav strain in cd4 + tzm-bl cells ( figure 1b ). adam17 is a related metalloprotease which shares partial (but not complete) substrate specificity with adam10 [15] [16] [17] [18] [19] [20] , and has been shown to mediate sars-cov envelope shedding [26] . accordingly, the role of adam17 expression in hiv-1 replication was studied by knockdown of expression with rnai in tzm-bl cells; however, adam17 silencing did not significantly inhibit viral replication. these results indicate that adam10, but not adam17, serves a specific role in the viral lifecycle. adam10 silencing for one week does not affect macrophage viability or function adam10 sirna transfectants were confirmed to have significant reductions in adam10 protein expression by both flow cytometry in cell lines 48 h after sirna transfection ( figure 2a ) and western blot analysis in primary macrophages 48 h after sirna transfection ( figure 2b ). kinetics of adam10 mrna down-regulation by sirna was measured using real time pcr in u373-magi-ccr5 cells ( figure 2c ) and primary human macrophages ( figure 2d ). to determine the viability of sirna-transfected macrophages, cytotoxicity was assayed using gapdh coupled to 3-phosphoglyceric phosphokinase and measuring atp [27] . the sirna transfections resulted in no significant adverse cytotoxic effect (p < 0.01), although dose-dependent cell death was observed when cells were treated with chelerythrine, a protein kinase c inhibitor (data not shown). in addition, adam10 sirna-transfected macrophages displayed phagocytic function similar to macrophages transfected with scrambled sirna, as determined by comparing the phagocytosis of captured bodipy beads [28] (data not shown). to determine whether adam10 is required for entry or hiv-1 reverse transcription, small non-genomic dna was isolated from control-and adam10 sirna-transfected macrophages at 48 h post-infection for quantification by real-time pcr. previous studies demonstrated that kinetics of reverse transcription is slower in macrophages than in lymphoid cells, and full-length hiv-1 reverse transcripts are not generated until 36-48 h after infection in macrophages [29, 30] . thus, cdna levels detected at 48 hours post-infection should be reflective of only a single replication cycle. as shown in figure 3a , adam10 silencing did not affect detection of full length hiv-1 cdna, whereas viral dna formation was not detected in cells treated with the reverse transcription inhibitor azt. these data demonstrate that adam10 expression is not required for hiv-1 entry and completion of reverse transcription. to determine if adam10 is required for proviral dna integration, genomic and small non-genomic cdna was isolated from cells at various time points post-infection. if hiv-1 cdna enters the nucleus but does not integrate into the host cell chromosome, then the viral cdna circularizes to form a 2-ltr circle [31, 32] , which can be quantified using real-time pcr. integration is quantified by using one primer directed against hiv-1 and another primer directed against alu, a common repetitive sequence found in the human genome. knockdown of adam10 significantly reduces the amount of integrated hiv-1 cdna in both macrophages ( figure 3b ) and u373-magi-ccr5 cells ( figure 3c ). small non-genomic dna was isolated from cells after infection to quantify formation of 2-ltr circles. as shown in figure 3d , 2-ltr circle formation was not observed in macrophages treated with sirna to adam10, whereas when cells were treated with the integrase inhibitor, raltegravir, 2-ltr circles are detected [33] . inhibition of both hiv-1 integration and 2-ltr circle formation by adam10 sirna indicates that while hiv-1 cdna is efficiently generated, it is not efficiently translocated into the nucleus of adam10 down-regulated cells. to confirm that adam10 function is not required for hiv-1 replication events following integration, u373-magi-ccr5 cells were used in reporter gene assays to gauge the effect of adam10 silencing on tat function. tat function was measured by β-galactosidase (β-gal) activity expressed from a stably integrated hiv-ltr-βgal construct [34] . as shown in figure 4a , tat activity was robust in erbb2ip control sirna-transfected and hiv-1 infected, but not transfected (virus only), cells at 72 h. tat activity in adam10 or cd4 sirna-transfectants at 72 h post-infection was similar to the background levels seen at day 0. however, tat activity was unaffected by adam10 silencing when u373-magi-ccr5 cells were transfected with a plasmid encoding recombinant tat ( figure 4b ), indicating that adam10 does not directly activate tat and that adam10 affects virus replication prior to tat transcription or translation. in agreement, western blots revealed that production of the viral env and p24 proteins were significantly inhibited between days 4-7 post-infection in primary macrophages following adam10 silencing (data not shown). additionally, the role of adam10 was studied in tzm-bl cells transfected with a plasmid-based molecular clone (pnl4-3) or infected with the corresponding hiv-nl4-3 virus. although replication of hiv-nl4-3 was dramatically inhibited in adam10 sirna-transfectants, adam10 was not required in the plasmid-based system ( figure 4c ). the pnl4-3 plasmid has a 15 kb insert that includes a full-length proviral clone and one to two kb of flanking cellular sequence outside both the 5' and 3' ltr and very efficiently directs hiv gene expression following transfection, independent of plasmid integration. thus, the plasmid serves as a surrogate for proviral integration, bypassing the normal early events of the viral life cycle ( figure 4d ). additionally, we used a u1 cell line, which contains two integrated copies of the hiv-1 proviral genome, and can be induced to produce progeny virus following treatment with a phorbol ester [35, 36] . in order to amplify hiv cdna with these full length primers, two template-switching events and continuous 5'ltr and gag sequences must be present on either strand, which is the last event to occur during hiv reverse transcription [79] . (b, c) integration of hiv was significantly lower in adam10 down-regulated (b) primary human macrophages and (c) u373-magi-ccr5 cells than in control erbb2ip down-regulated cells following infection with hiv-sf162. genomic dna was used to quantitate integrated hiv cdna using real-time pcr using primers specific for integrated hiv cdna. (d) 2-ltr circle formation in adam10 down-regulated macrophages was significantly less than that seen in macrophages treated with the integrase inhibitor raltegravir, and was similar to infected but untreated cells. formation of 2-ltr circles was quantitated by real-time pcr [80] . (no inf = no infection, ral = raltegravir, scr = scrambled sirna, **p < 0.01). down-regulation of adam10 had no effect on production of hiv-1 in these cells (data not shown). these data indicate that adam10 supports virus replication prior to gene transcription. taken together, these data suggest that the function of adam10 in hiv-1 replication is bracketed between the levels of nuclear trafficking and nuclear entry. a functional adam10 metalloprotease is not required for hiv-1 replication to determine whether over-expression of adam10 increases hiv-1 replication and infection, we obtained a human adam10 plasmid from dr. stefan lichtenthaler (lmu munich, germany). as shown in figure 5a , over-expression of adam10 resulted in increased hiv-1 replication. the metalloprotease domain potentially responsible for this increase was further investigated. adam10 e384a plasmid contains a single inactivating point mutation in the metalloprotease domain rendering the metalloprotease domain inactive [37] . adam10 e384a and wild type (wt) adam10 plasmids were transfected into u373-magi-ccr5 cells 48 h prior to infection with hiv-sf162 (moi = 0.1). as shown in figure 5b , over-expression of adam10 e384a showed an increase in hiv-1 replication very similar to that seen with wt adam10 over-expression, suggesting that the metalloprotease domain is not the critical domain in adam10 supporting hiv-1 infection. in addition, tissue inhibitors of metalloproteases (timps) 1 and 3, which have been shown to inhibit adam10 metalloprotease activity [38] , had no effect on hiv-1 replication in human macrophages ( figure 5c, d) . this indicates that the adam10 metalloprotease domain is not functionally required for hiv-1 replication. both g-secretase and adam15 are required for hiv-1 replication to determine if the intracellular domain of adam10 plays a role in hiv-1 replication, we independently inhibited the two necessary proteolytic steps that free this fragment. adam9 and adam15 were shown to cleave the ectodomain of adam10 while γ-secretase has been shown to cleave and release the adam10 intracellular domain (icd) [23] . once released, the adam10 icd can then translocate to the nucleus or peri-nuclear region [21, 23] . to determine whether adam9 and/or adam15 were required for hiv-1 replication, cells were transfected with sirnas directed against either adam9 or adam15 mrna prior to infection. as shown in figure 6a , adam15 sirna significantly reduced hiv-1 replication, comparable to the level of replication seen with knockdown of adam10, whereas adam9 knockdown had no effect on hiv-1 replication. next, we studied the role of γ-secretase, a multi-subunit complex, containing presenilin, nicastrin, anterior pharynx-defective 1 (aph-1), and presenilin enhancer protein 2 (psen) [39] , in hiv-1 replication. γsecretase contains either the presenilin-1 (p1) or presenilin-2 (p2) isoform, which contributes to the substrate specificity of the enzyme [40] . to determine if γ-secretase is required for hiv-1 replication, sirna targeting different components of γ-secretase was used to inhibit the enzyme. as shown in figure 6b , sirna targeting p2, nicastrin, and psen all significantly decreased hiv-1 replication in u373 cells. however, p1 sirna did not affect hiv-1 replication. these data show a specific role of presenilin-2, and not presenilin-1, in hiv-1 replication. additionally, we used specific γ-secretase inhibitors, l-685,458 and dapt [41, 42] . cytotoxicity assays were performed to determine optimal, sub-toxic concentrations for either inhibitor ( figure 6c ). figure 6d shows that adding 10 μm of either l-685,458 or dapt to u373 cells 24 h prior to infection and during infection, significantly decreased hiv-1 replication as compared to dmso-only treatment and infection only controls. these findings confirm that γ-secretase is required for hiv-1 replication. taken together, both adam15 and γ-secretase facilitate hiv-1 replication, consistent with their roles in the release of the adam10 intracellular domain. we utilized gene-entrapment of diploid cell lines for our initial selection of candidate genes associated with cell survival following lytic virus selection. several possible outcomes may result, including haploid insufficiency, complete loss of expression from a vector inserted into a dominant allele [43] [44] [45] , or dominant negative effects due to truncated translational products [46, 47] . furthermore, sirna can be used as a confirmatory step across a wide variety of cell types and viruses, once a candidate gene is identified, as we reported for hiv-1 infection [1] . in this study, we identified adam10 by gene trap insertional mutagenesis as a disrupted gene in cells surviving cytolytic reovirus infection, and we demonstrated the importance of adam10 expression at a post-entry step in hiv-1 replication. we also show that overexpression of adam10 increases hiv-1 replication. interestingly, in previous studies solely using sirna or shrna to identify cellular proteins required for hiv-1 replication [4] [5] [6] [7] , brass et al. had also identified adam10 as a required cellular gene [4] . importantly, these studies show that adam10 silencing inhibits hiv-1 in primary human macrophages, which are more relevant to human disease than tissue culture adapted cell lines [48] . macrophages and cd4 + lymphocytes are the predominant cell types infected with hiv-1 clinically, and the importance of adam10 in hiv-1 replication in primary human macrophages supports a role of adam10 in hiv-1 pathogenesis. to determine the precise step in hiv-1 replication in which adam10 participates, we inhibited various processes and queried for viral products that define steps up to and including virus expression from chromosomally integrated viral dna. our data supports a role for adam10 at a step in virus replication prior to integration. hiv-1 must enter the cell and be partially disassembled prior to reverse transcription of viral cdna, and these steps are not inhibited with knockdown of adam10. it was found that adam10 silencing resulted in a failure of viral cdna to integrate, as measured by real time pcr. moreover, hiv-1 2-ltr circles did not accumulate in the nucleus, which occurs after virus enters the nuclear membrane but cannot integrate. it is known that 2-ltr circles accumulate when integration is inhibited with specific integrase enzyme inhibitors [33] . furthermore, adam10 silencing did not affect tat-dependent proviral gene expression as assessed in studies using a plasmid expressing tat and tat-dependent β-galactosidase expression. using pnl4-3, a plasmid containing the hiv-1 genome, knockdown of adam10 did not limit virus transcription, consistent with its role prior to viral transcription from integrated proviral dna. our data are supportive of an important role for adam10 in hiv-1 replication at a step following reverse transcription but prior to hiv-1 integration, likely at the level of nuclear trafficking. a role for adam10 during nuclear trafficking is in concert with known cellular roles for this complex protein. the protein has several known extracellular domains, which include a metalloprotease domain, an integrin binding domain, and a cysteine rich region. a recent study has shown adam10 to be essential for cell entry of plasmodium falciparum due to its interaction with the malaria pfsub2 enzyme [49] . adams function in the proteolytic release of many transmembrane cell surface cytokines, growth factors, receptors, and adhesion proteins, a process known as ectodomain shedding. adam10 is known to cleave over 20 cell surface proteins [15] [16] [17] [18] [19] [20] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] . most known adam10 substrates are involved in cellular adhesion, including ephrin-a2 (efna2), axl, fractalkine (cx3cl1), cxcl16, e-and n-cadherin (chd1 and 2), the γ-protocadherins c3 and b4, ncam, chl1, lag-3, cd23, cd44, cd46, and desmoglein-2 (dsg2). however, while there is known promotion of trans-infection of hiv-1 secondary to interaction with the adhesion molecules, c-type lectins dc-sign and dc-signr [69, 70] , the data presented above do not support a role in cell entry for adam10. surprisingly, the metalloprotease function was not required for hiv-1 replication. more recently, activity has been attributed to the 6 kda fragment released from the carboxy-terminus. this fragment is released from the intracellular domain following sequential proteolytic digestion. adam9 and -15 have been shown to be responsible for releasing the adam10 ectodomain, while presenilin/γ-secretase has been shown to be responsible for the proteolytic release of the adam10 intracellular domain from the plasma membrane, whereupon it localizes to the nucleus [23] . cleavage and release of the adam10 ectodomain are required for the intracellular domain to be subsequently released. we demonstrate that both adam15 and γ-secretase are required for hiv-1 replication, which strongly suggests the intracellular domain of adam10 is critical for hiv-1 replication. we did not find adam9 to be required for hiv-1 replication in our assays. whether this is unique to the cell line used in our assays, as adam10 can be alternatively spliced, or rather that adam15 is specifically required by hiv-1, requires further study. adam10 has a role in androgen receptor nuclear translocation and has been shown to translocate to the nuclear and the perinuclear region during prostate cancer pathogenesis and progression [21] . combined with our data showing that adam10 functions during nuclear trafficking or nuclear entry, we suggest that the intracellular domain may either function to promote trafficking of hiv-1 pic to the nucleus (figure 7) , or serve a scaffolding role during pic assembly. the relationship between adam10 intracellular domain and hiv-1 pic needs further study. it is possible that the icd directly interacts with hiv-1 viral proteins or nucleic acid, or it is essential for another host component that traffics the pic through the nuclear pore. studies are ongoing to determine its precise role in hiv-1 entry into the nucleus. it is intriguing to note that adam10 is not the only adam protein that has dual functionality, as cousin et al. have found that the nuclear translocation of the adam13 intracellular domain is required for gene expression and neural crest cell migration [71] . whether this class of proteins participates in the replicative cycle for other virus may deserve further study. these studies utilized both primary human macrophages and tissue culture adapted cell lines that have been extensively used in the study of hiv-1. primary macrophages with knockdown of adam10 were viable and functionally active, thereby raising the possibility that inhibition of adam10 processing or targeting the intracellular fragment could lead to new set of potential therapeutic targets. monocyte-derived human macrophages were prepared from leukopaks obtained from the university of texas medical branch blood bank (galveston, tx). peripheral blood mononuclear cells were recovered from leukopaks by ficoll-hypaque density centrifugation and were purified by adherence to plastic, as previously described [72] . the following cell lines were obtained from the nih aids research and reference reagent program, division of aids, niaid, nih: cd4/ccr5/cxcr4 + tzm-bl hela cells from dr. john c. kappes, dr. xiaoyun wu and tranzyme inc. [73] ; u373-magi-ccr5 cells (contributed by dr. michael emerman and dr. adam geballe), are a cell line derived from a glioblastoma that has been modified by stable transfection of ltr-β-galactosidase which is trans-activated by hiv tat in relation to the level of virus replication [74] . u373-magi-ccr5 cells also express cd4 and human chemokine receptor ccr5 to enable infection by hiv r5 strains and were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fbs, 0.2 mg/ml g418, 0.1 mg/ml hygromycin, and 1.0 μg/ ml puromycin [75] . rat intestinal epithelial 1 (rie-1) cells were maintained in dmem supplemented with 10% fbs, penicillin, and streptomycin. primary r5 viruses hiv-sf162 [76] and hiv-sx [72] were purchased from the virology core primers and probes for real time pcr were custom ordered from sigma-aldrich. full genomic dna was isolated from monocyte-derived macrophages or cell lines using the qiagen blood and tissue kit. small nongenomic dna, such as reverse transcribed viral cdna and 2-ltr circles were isolated using a qiagen miniprep kit. to detect full length hiv dna, primers m667 (5'-ggc taa cta ggg aac cca ctg-3') and m661 (5'-cct gcg tcg aga gag ctc ctc tgg-3') [79] , along with probe mh603 (5'-(fam)-aca cta ctt gaa gca ctc aag gca agc ttt-(tamra)-3') [80] were used. the primers used to amplify hiv-1 cdna span the primer binding site (pbs); the only time there is contiguous dna on both sides of the pbs is when synthesis of both full strands of viral dna is completed [29, 79] . to identify 2-ltr circle formation, primers mh535 (5'-aac tag gga acc cac tgc tta ag-3') and mh536 (5'-tcc aca gat caa gga tat ctt gtc-3') were used with the mh603 probe [80] . for integration, a two-step pcr reaction was performed. for the initial pcr amplification, alu forward, (5'-gcc tcc caa agt gct ggg att aca g-3'); and hiv-1 gag reverse, (5'-gct ctc gca ccc atc tct ctc c-3') primers were used [81] . the second step real-time pcr used the primers ltr forward, (5'-gcc tca ata aag ctt gcc ttg a-3'); and ltr reverse, (5'-tcc aca ctg act aaa agg gtc tga-3'); and ltr probe (5'-fam-gcg agt gcc cgt ctg ttg tgt gac tct ggt aac tag ctc gc-dbh1-3') [81] . serial dilutions of purified hiv-sx plasmids were used to generate a standard curve to calculate the total concentration of hiv-1 cdna in each sample, which is expressed as total fentograms (fg) of cdna. total mrna was isolated from sirna-transfected cells and primary human macrophages using rneasy mini kits (qiagen, inc., valencia, ca). adam10 specific primers and probe were purchased from applied biosytems (carlsbad, ca). all reactions were performed using applied biosystems taqman universal master mix and run using an applied biosystems 7500 fast real time pcr system and 7500 fast system software [33] . silencing of target genes was determined by normalizing target gene expression to gapdh expression (n = 3). adam10 protein expression was assessed using an analytical flow cytometer in control or adam10 sirna-transfected tzm -bl cells after 48 h. adam10 expression in sirna transfectants was determined by detaching cells in pbs/2 mm edta and staining cells using either a mouse anti-human adam10 antibody (r&d systems, minneapolis, mn) or an igg1 isotype control (southern biotech, birmingham, al), followed by incubation with a phycoerythrin-conjugated rabbit anti-mouse secondary antibody (jackson immunoresearch, west grove, pa). to determine the effect of silencing cellular genes on hiv-1 replication, tzm-bl cells or primary human macrophages were transfected with sirna smartpools for 48 h prior to infection. tzm-bl cells were seeded into t25 flasks (750,000 cells/flask) and transfected as described above. subsequently, cells were infected overnight with hiv lav (moi = 1) and then seeded into duplicate t75 flasks (1.5 million cells per flask). p24 production was assayed from culture supernatants 3 days post-inoculation using the p24 antigen elisa system (beckman/coulter/immunotech, brea, ca) following the manufacturer's protocol. macrophages were infected in 24-well plates with hiv-sf162, and p24 production was assayed from cell lysates and culture supernatants 4 and 7 days, respectively, post-infection using p24 capture elisa (immunodiagnostics, woburn, ma). b-galactosidase assays u373-magi-ccr5 cells were transfected with adam10 or control sirnas for 48 h, and tat was expressed by either infection with hiv-sf162 or transfection with a pcdna3-hiv tat101-flag vector (nih research and reference reagent program) overnight. at various time points following infection or transfection, cells were lysed and analyzed for β-galactosidase activity using the beta-glo assay system (promega, madison, wi) and a bio-tek clarity microplate luminometer (pcdna3-hiv tat101 flag transfections) or dynex mlx luminometer (sf162 infections). tzm-bl cells were seeded in 24 well plates at 2 × 10 4 cells/well, and transfected with 800 ng hiv-sx plasmid dna using lipofectamine ltx transfection reagent (invitrogen) along with plus reagent (invitrogen) using the one tube protocol according to manufacturer's specifications. after three days incubation, p24 was measured by using a p24 capture elisa (see above). transfection of adam10 plasmids (wild type and e384a) was performed using lipofectamine 2000 (invitrogen) according to manufacturer's protocols. adam10 e384a has a point mutation in its metalloprotease domain, rendering it inactive. u373-magi-ccr5 cells were plated in 12-well plates. after plasmid transfection, cells were incubated for 48 h and were then infected with hiv-sf162 (moi = 0.1). forty-eight hours after infection, a β-galactosidase assay was used to measure infection in the cells as described above. the toxicity of sirna treatment was measured by acella-tox bioluminescence cytotoxicity assay (cell technology inc, mountain view, ca), which detects secreted glyceraldehyde-3-phosphate dehydrogenase (gapdh) in cells with diminished membrane integrity. values for released gapdh were normalized to cellular gapdh levels. all statistics were performed using a two-tailed, paired student's t-test (*p < 0.05, **p < 0.01). rab9 gtpase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus discovery of mammalian genes that participate in virus infection mutations in the igf-ii pathway that confer resistance to lytic reovirus infection identification of host proteins required for hiv infection through a functional genomic screen host cell factors in hiv replication: meta-analysis of genome-wide studies global analysis of host-pathogen interactions that regulate early-stage hiv-1 replication genome-scale rnai screen for host factors required for hiv replication a genome-wide short hairpin rna screening of jurkat t-cells for human proteins contributing to productive hiv-1 replication concerted integration of retrovirus-like dna by human immunodeficiency virus type 1 integrase the molecular biology of human immunodeficiency virus type 1 infection human genes other than cd4 facilitate hiv-1 infection of murine cells reverse transcription takes place within extracellular hiv-1 virions: potential biological significance the adams: signalling scissors in the tumour microenvironment selective use of adam10 and adam17 in activation of notch1 signaling proteomic identification of desmoglein 2 and activated leukocyte cell adhesion molecule as substrates of adam17 and adam10 by difference gel electrophoresis proteolytic processing of delta-like 1 by adam proteases adam10-mediated cleavage of l1 adhesion molecule at the cell surface and in released membrane vesicles metalloproteases regulate t-cell proliferation and effector function via lag-3 metalloproteinase inhibitors for the disintegrin-like metalloproteinases adam10 and adam17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules the disintegrins adam10 and tace contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein nuclear translocation of adam-10 contributes to the pathogenesis and progression of human prostate cancer expression of the disintegrin metalloprotease, adam-10, in prostate cancer and its regulation by dihydrotestosterone, insulin-like growth factor i, and epidermal growth factor in the prostate cancer cell model lncap adam10, the rate-limiting protease of regulated intramembrane proteolysis of notch and other proteins, is processed by adams-9, adams-15, and the gammasecretase cellular genetics of host susceptibility and resistance to virus infection the cd4 (t4) antigen is an essential component of the receptor for the aids retrovirus tumor necrosis factor-alpha convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensinconverting enzyme-2 (ace2) nk sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method cd44 is a phagocytic receptor kinetics of human immunodeficiency virus type 1 reverse transcription in blood mononuclear phagocytes are slowed by limitations of nucleotide precursors the kinetics of human immunodeficiency virus reverse transcription are slower in primary human macrophages than in a lymphoid cell line human immunodeficiency virus type 1 2-ltr circles reside in a nucleoprotein complex which is different from the preintegration complex circularization of human immunodeficiency virus type 1 dna in vitro quantitative pcr used to assess hiv-1 integration and 2-ltr circle formation in human macrophages, peripheral blood lymphocytes and a cd4+ cell line human immunodeficiency virus type 1 coreceptors participate in postentry stages in the virus replication cycle and function in simian immunodeficiency virus infection cytokine-induced expression of hiv-1 in a chronically infected promonocyte cell line characterization of a promonocyte clone chronically infected with hiv and inducible by 13-phorbol-12-myristate acetate a disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an alzheimer disease mouse model the in vitro activity of adam-10 is inhibited by timp-1 and timp-3 nicastrin, presenilin, aph-1, and pen-2 form active gamma-secretase complexes in mitochondria presenilin-1 and presenilin-2 exhibit distinct yet overlapping gamma-secretase activities the notch signaling inhibitor dapt down-regulates cdk5 activity and modulates the distribution of neuronal cytoskeletal proteins l-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gamma-secretase activity simultaneous quantitative and allele-specific expression analysis with real competitive pcr sex-specific parent-of-origin allelic expression in the mouse brain allele-specific gene expression is widespread across the genome and biological processes ohki m: t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene biallelic and heterozygous point mutations in the runt domain of the aml1/pebp2alphab gene associated with myeloblastic leukemias potential role for cd63 in ccr5-mediated human immunodeficiency virus type 1 infection of macrophages proteome analysis of plasmodium falciparum extracellular secretory antigens at asexual blood stages reveals a cohort of proteins with possible roles in immune modulation and signaling soluble axl is generated by adam10-dependent cleavage and associates with gas6 in mouse serum adam10-mediated release of complement membrane cofactor protein during apoptosis of epithelial cells regulated cleavage of a contactmediated axon repellent negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of nf-kappab ligand metalloprotease-induced ectodomain shedding of neural cell adhesion molecule (ncam) the disintegrin-like metalloproteinase adam10 is involved in constitutive cleavage of friedrich et al cx3cl1 (fractalkine) and regulates cx3cl1-mediated cell-cell adhesion constitutive and regulated alpha-secretase cleavage of alzheimer's amyloid precursor protein by a disintegrin metalloprotease identification of adam10 as a major source of her2 ectodomain sheddase activity in her2 overexpressing breast cancer cells janus kinase 2 influences growth hormone receptor metalloproteolysis adam10 mediates e-cadherin shedding and regulates epithelial cell-cell adhesion, migration, and beta-catenin translocation cell-matrix interaction via cd44 is independently regulated by different metalloproteinases activated in response to extracellular ca(2+) influx and pkc activation regulated adam10-dependent ectodomain shedding of gammaprotocadherin c3 modulates cell-cell adhesion adam10 cleavage of n-cadherin and regulation of cell-cell adhesion and beta-catenin nuclear signalling hydrogen peroxide and endothelin-1 are novel activators of betacellulin ectodomain shedding adam10 is a principal 'sheddase' of the low-affinity immunoglobulin e receptor cd23 the metalloprotease kuzbanian (adam10) mediates the transactivation of egf receptor by g protein-coupled receptors cytoplasmic relaxation of active eph controls ephrin shedding by adam10 lrp1 shedding in human brain: roles of adam10 and adam17 regulated intramembrane proteolysis of bri2 (itm2b) by adam10 and sppl2a/ sppl2b dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances transinfection of t cells dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans translocation of the cytoplasmic domain of adam13 to the nucleus is essential for calpain8-a expression and cranial neural crest cell migration hiv-1 tropism for mononuclear phagocytes can be determined by regions of gp120 outside the cd4-binding domain identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research cofactor requirement for human immunodeficiency virus type 1 entry into a cd4-expressing human cell line visualization of the intracellular behavior of hiv in living cells macrophage and t cell-line tropisms of hiv-1 are determined by specific regions of the envelope gp120 gene production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone isolation of a tlymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) hiv-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure a quantitative assay for hiv dna integration in vivo a sensitive, quantitative assay for human immunodeficiency virus type 1 integration submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by public health service small business innovation research (sbir) grant ai084705 from division of aids, national institute of allergy and infectious diseases. this work was also partly supported by public health service grant hl088999 from the national heart, lung, and blood institute. we would like to thank merck & co., inc. for generously providing raltegravir and dr. stefan lichtenthaler (lmu munich, germany) for generously providing both adam10 wt and adam10 e384a plasmids. we thank edward siwak, ph.d., associate director of virology core facility, center for aids research at baylor college of medicine, houston, tx for providing hiv-sx and hiv-sf162. d.h.r. was supported by the department of veterans affairs and gifts from maggie chassman, buisson foundation, zirus, and the public health service. key: cord-317037-1qydcc5e authors: kumar, asit; kodidela, sunitha; tadrous, erene; cory, theodore james; walker, crystal martin; smith, amber marie; mukherjee, ahona; kumar, santosh title: extracellular vesicles in viral replication and pathogenesis and their potential role in therapeutic intervention date: 2020-08-13 journal: viruses doi: 10.3390/v12080887 sha: doc_id: 317037 cord_uid: 1qydcc5e extracellular vesicles (evs) have shown their potential as a carrier of molecular information, and they have been involved in physiological functions and diseases caused by viral infections. virus-infected cells secrete various lipid-bound vesicles, including endosome pathway-derived exosomes and microvesicles/microparticles that are released from the plasma membrane. they are released via a direct outward budding and fission of plasma membrane blebs into the extracellular space to either facilitate virus propagation or regulate the immune responses. moreover, evs generated by virus-infected cells can incorporate virulence factors including viral protein and viral genetic material, and thus can resemble noninfectious viruses. interactions of evs with recipient cells have been shown to activate signaling pathways that may contribute to a sustained cellular response towards viral infections. evs, by utilizing a complex set of cargos, can play a regulatory role in viral infection, both by facilitating and suppressing the infection. ev-based antiviral and antiretroviral drug delivery approaches provide an opportunity for targeted drug delivery. in this review, we summarize the literature on evs, their associated involvement in transmission in viral infections, and potential therapeutic implications. cells mediate intercellular communication and modulation of immune responses through shedding and release of extracellular vesicles (evs) [1] . these evs are diverse and originate from plasma membrane and endosomes and include exosomes, micro-vesicles (mvs, also known as microparticles), and apoptotic bodies. they are categorized based on their biogenesis, release pathways, size, content, and function [2] . evs shed from plasma membranes are generally referred to as mvs [3] [4] [5] , while vesicles that are generated by inward budding of endosomes to form multivesicular bodies (mvbs) that fuse with the plasma membrane, and release into the extracellular environment, are known as exosomes [6, 7] ; whereas, cells undergoing apoptosis can release vesicles or cell filaments exclusively from the plasma membrane, called apoptotic bodies [8, 9] . depending on their biogenesis pathway and cellular origin, exosomes are vesicles of endocytic origin and their size usually ranges from 30-120 nm [33] . exosomal markers include tetraspanins (tspan29 and tspan30, escrt components, and tsg101). the invasion of the plasma membrane inwards forms the early endosome and the limiting membrane of the later endosome sprouts further to form the mvbs. mvbs are characterized by the invagination of the inner body membrane, which results in the formation of intraluminal vesicles (ilvs) [34] . during this process, cytoplasmic components and certain peripheral proteins are integrated into them. the ilvs accumulated in the mvb lumen have two routes. one is to diffuse with the lysosomes, which causes the contents of the vesicles to degrade, and the other is fusion with the cytoplasmic membrane and release of the vesicles to the extracellular space by exocytosis, referred as "exosomes" [35] . loading of biological cargos into ilvs involves the endosomal sorting complexes required for transport (escrt) complexes (escrt-0, -i, -ii, -iii and the vps4) and other accessory proteins such as alix/pdcd6ip, tsg101, hrs, etc. [36, 37] . in addition to escrt, other mechanisms can also produce exosomes of certain biochemical components. for instance, in some cells production of exosomes requires lipid ceramide and neutral sphingomyelinase [38] , an enzyme that converts sphingomyelin to ceramide, and related proteins including phospholipase d2 that hydrolyzes phosphatidylcholine into phosphatidic acid and dgk alpha [39, 40] . another mechanism of exosome release relies on small gtpases such as rab27a/b [41] , rab 7, 11, 31 , and 35 in some cells, or soluble n-ethylmaleimide-sensitive factor attachment protein receptor (snare) family proteins like ykt6 [42, 43] , vesicle-associated membrane protein 7 (vamp7) [44, 45] , cd9, and cd63. these proteins are involved in exosome biogenesis and are commonly used as markers unlike exosomes and microvesicles, which are released during normal cellular processes, apoptotic bodies are formed only during programmed cell death [73, 74] . apoptotic bodies' size ranges from 500-4000 nm. during apoptosis, the cell undergoes morphological changes and shrinks to a smaller size with densely packed cytoplasm and other organelles, and eventually their nucleus disintegrates [75] . further, the cells form blebs on its surface and disintegrate into small fragments called apoptotic bodies. these are characterized by the presence of organelles within the vesicles [73] and are cleared from the body by phagocytosis by specific mechanisms [76, 77] . the most commonly used identifiers of apoptotic bodies are annexin v, thrombospondin, and c3b [78] . limited knowledge exists in the literature regarding the role of apoptotic bodies in cell-cell communication during viral infection and their contribution to viral pathogenesis. to understand their possible role and function in intercellular communication, numerous in-depth studies are warranted in the future. uptake of ev seems to depend on the type of recipient cell, its physiological state, and recognition of ligands or receptors on the recipient cell and evs. cells broadly internalize evs either by fusion with the plasma membrane or via endocytosis. internalization of evs by recipient cells occurs by various mechanisms of endocytosis including clathrin-dependent and clathrin-independent mechanisms such as caveolin-mediated uptake, macro-pinocytosis, phagocytosis, and lipid raft-mediated internalization [79, 80] . ev uptake is an energy-dependent process [79] . neurons internalize oligodendrocyte-derived exosomes by clathrin-mediated endocytosis [81] , whereas microglia internalize exosomes by micropinocytosis [82] . epithelial cells internalize exosomes by caveola-dependent endocytosis [83] , while dendritic cells internalize evs through lipid raft domains [84] . different methods are employed to detect ev uptake, among which the most used viruses 2020, 12, 887 4 of 23 method is the use of fluorescent lipid membrane dyes to stain ev membranes. examples of such dyes include pkh67, pkh26, rhodamine b, dii, and did [79, 80, 85, 86] . the internalization of evs by recipient cells can be measured using methods such as flow cytometry and confocal microscopy [86, 87] . evs and viruses are highly heterogeneous in size, structure, and biogenesis, and therefore they cause apparent difficulties in distinguishing and separating evs from viruses. even though evs and viruses overlap in size and biophysical properties, evs far outnumber high-titer viruses during infection [88] . in the past decade, a multitude of isolation and purification methods for evs and virus particles have been developed. differential centrifugation/ultracentrifugation (uc) technique is widely used for the isolation of evs from cell cultures' media and biological fluids that contain viruses [89] . although this technique is considered as the gold standard of ev isolation, it often coprecipitates with proteins and lipoproteins that can affect sample purity and may interfere with downstream analysis [90, 91] , limiting its use in hospital settings. this limitation can be overcome by including multiple isolations and characterization techniques such as antibody-based immunoaffinity purification, tangential flow filtration (tff), and nano-flow cytometry (nfcm) [92] [93] [94] [95] . however, each of these methods has its limitations, which need to be considered before planning ev isolation and purification. for instance, ev isolation using the antibody-based immunoaffinity purification method provides a refined ev population but is limited by the sample volume and amount of final product [96] . moreover, the expression level of ev markers such as cd9, cd63, and cd81 can vary depending on the ev origin and physiological condition, requiring a combination of markers to be used. compared to uc, the tff method can be effective in obtaining ev-enriched formulations from a large volume of samples. however, tff is likely to cost higher than conventional ev isolation methods. further studies are required to explore the utilization of tff for clinical studies [92] . due to limitations associated with isolation procedures, and lack of a standardized isolation process, a validated good manufacturing practice (gmp)-compliant procedure is desperately needed. bari et al. employed conditioned media from mesenchymal stem/stromal cells for the secretome/ev isolation. a key aspect of their study is a large-scale secretome or ev isolation process using uc and tff that complies with gmp, which allows standardized and pharmaceutical grade products suitable for clinical applications [97, 98] . the use of nfcm is reported as a new benchmark for quality assessment of evs. phenotyping of single particles is possible through nfcm using immunofluorescent labeling of evs [94] . however, the limitations in resolution and detection varied depending on the criteria used to define the ev populations based on markers [99] that have excluded many researchers widely utilizing this technique. besides, an nfcm based method can be challenging to develop and to validate ev characterization, given the specific ev population measurement and due to the lack of standard guidelines for handling and analyzing a variety of samples with appropriate normative controls in nfcm. li k et al. have developed an approach termed cushioned-density gradient ultracentrifugation (c-dguc), a variant of ultracentrifugation, for ev refinement [100] . in this approach, samples were processed through a density gradient cushion such as iodixanol (optiprep™) and centrifugal force to maximizes ev recovery followed by density gradient ultracentrifugation steps that eventually provide high-purity purification of evs by effectively removing protein aggregates. however, evs can lose integrity while isolated from a fixed density range [101] . polyethylene glycol (peg) precipitation followed by iodixanol density separation has recently become a useful method to pull down evs, viruses, and proteins or protein-rna aggregates within a sample, followed by an additional centrifugation step. this method results in a significantly higher yield of evs in comparison to the conventional uc method [102] . the contents of evs vary greatly depending upon the condition of the parent cell. thus, apart from characterizing the vesicles, identifying these contents reveals a breadth of information regarding the parent cells. the international society for extracellular vesicles (isev) 2018 guidelines should be followed when isolating evs from cells or plasma/biological fluids for drug encapsulation. the most pragmatic approach appears to be viruses 2020, 12, 887 5 of 23 the isolation of evs using a commercial kit and size exclusion chromatography (sec; also known as gel filtration) methods followed by microfiltration of samples using filters with pore diameters of 0.1, 0.22, or 0.45 µm depending on the size of vesicles required. in sec, evs are separated from other material according to differences in sizes (hydrodynamic radii) [103] that gives this technique the upper edge over conventional methods and can be effectively used for a variety of complex biological samples such as body fluid, blood/plasma, urine, and breast milk [104] [105] [106] [107] . isolation of high-purity evs from samples containing virions is challenging since both evs and some viruses, in this case, retroviruses, are similar in size. as of now, no validated protocol is available to specifically separate evs from virions that are similar in size and carry the same markers [32] . however, a study has demonstrated that defective viruses could be separated from naturally occurring viruses based on differences in buoyant densities [108] . evs loaded with drugs to treat viral diseases require them to target majorly infected cells or tissues. when considering evs as personalized therapeutic carriers, surface engineering of evs is required that can be performed using covalent and noncovalent modification [109] [110] [111] . it is important to optimize the method of isolation for evs for drug loading on a case-to-case basis. upon loading drugs to these evs, the evs can be further separated using a sucrose gradient that utilizes iodixanol and characters each fraction for loading efficiency and total loading. the ev fractions with optimally loaded drugs can be further characterized by their size, shape, and marker proteins for further use. evs released by virus-infected cells can incorporate protein molecules, derived from viral genes involved in viral assembly. delivery of the ev-associated virulence molecules affects recipient cells by rendering them particularly vulnerable to viral infection (table 1) . moreover, incorporating viral proteins can trigger cell death of non-participating immune cells [112] that would contribute to the heavy loss of immune cells during the early stages of viral infection or low viral load. intercellular transfer of viral proteins and viral cell surface receptors by evs not only facilitates evasion of the host's immune response by suppressing antibody production in lymphocytes but also makes immune cells susceptible to viral infection [32, 113] . however, while evidence indicates that evs can, directly and indirectly, mediate the antiviral response, their role in regulating immune response is not yet fully elucidated in vivo. hiv-infected cell-derived exosomes carrying negative regulatory factor (nef) induces apoptosis in t-lymphocytes; nef-transfected microglia-released nef+exosomes reduce the blood-brain barrier (bbb) integrity [112, 114] chemokines and receptors ccr5, cxcr4, mcp-1 facilitate the entry of hiv [115] proinflammatory markers il-6, tnf-β, il-8 hiv-infected cells derived exosome containing tar rna plays a role in the increase of il-6 and tnf-β in macrophages. hiv-infected u1 macrophages upon cigarette smoke condensate (csc) treatment enhanced the packaging of il-6 in evs; il-8 served as a biomarker for hiv patients with altered immune function due to alcohol and tobacco abuse [20, 116, 117] host protein apobec3g inhibit replication of viral infectivity factor (vif) -deficient and wild-type hiv-1 in recipient cells [118] mirna vmir-88 and vmir-99 triggers endosomal toll-like receptor (tlr) 8 and nuclear factor-κb (nf-κb) signaling, stimulating the release of tnfα by delivering ev to bystander macrophages, and may contribute to chronic immune activation [119] oxidative stress factors cellular markers cyp (1a1, 1b1, and 2a6), sod1, cat gfap induce hiv replication. hiv-infected u1 macrophages upon csc treatment promotes differential packaging of cyps and aoes in evs increased levels of glial fibrillary acidic protein (gfap) in plasma evs from hiv subjects can serve as a potential biomarker [116, 120, 121] contribute to viral immune-evasion and act in concert to promote tumor development through the interaction with multiple cellular proteins [122, 123] mirna mir-9, -20b, and let-7b cancer-associated, cellular pathways targeted by these mirnas. induce tumorigenesis through the effect of these micrornas on their targets [124] mir-222 plays a role in cervical carcinogenesis, notably through the downregulation of p27 and phosphatase and tensin homolog deleted on chromosome 10 (pten) [125] mir-7-5p favors cell proliferation [126] mir-92a-3p possesses anti-apoptotic properties [ hbv viral proteins large s, core and p proteins hepatocytes secreted exosomes participate in virus replication [142] viral mirnas hbv-mir-3 represses viral protein production and hbv replication [143] htlv-1 viral proteins gp61, tax, and hbz increase cell-to-cell contact and promote a potential increase in viral spread [144] zika viral genetic material and protein rna and zikv-e evs derived from infected c6/36 cells promote infection and activation of monocytes with enhanced tnf-α mrna expression. [145] in hiv, evs are thought to play an important role in disease progression through multiple mechanisms. viral components may be packaged in evs, which can then be delivered to uninfected cells, modulating the systemic inflammatory status. for instance, hiv-infected cell-derived exosomes carry viral protein nef that induces apoptosis in immune cells and reduces the blood-brain barrier (bbb) integrity to spread viral infection in the brain [112, 114] . it has been shown that evs released during hiv infection are heterogeneous including size variability. a study has shown that treatment-naïve people living with hiv/aids (plwha) contain evs larger in size and numbers compared to plwha who were either virally suppressed, elite controllers, or healthy controls [146] . additionally, cd4 counts and the abundance of evs in the blood were inversely correlated, with low cd4 counts associated with more abundant evs. interestingly, there was no relationship between cd4 counts and ev size. both size and abundance were also inversely correlated with neutrophils and platelet counts, as well as the cd4/cd8 ratio, all of which are markers of disease progression [146] . this suggests that evs may function as a biomarker for hiv disease progression. other studies have observed similar findings. in cells treated with antiretroviral drugs (arvs), increases in relative ev production has been observed [102] , along with decreased loading of genomic, but not non-coding, rna into evs from cells, which were treated with arvs, as opposed to untreated cells. additionally, treatment with interferon-alpha increased the packaging of viral rna into evs. the authors suggest that this occurs because arv or interferon prevents the release of viral particles from cells, which then allows for viral rna to be packaged into evs due to the increased presence of viral rna in the cell. in addition to viral rna, a variety of molecules, e.g., viral & host proteins, cellular markers, mirna, inflammatory molecules such as oxidative stress markers, chemokines and cytokines can also be packaged into evs [20, [115] [116] [117] 119, 121] . a study showed that the viral envelop (env) protein can be packaged into evs from infected cells [147] . the env-containing evs can increase susceptibility to viral infection in cell culture experiments, and depletion of env-containing evs showed decreased susceptibility to viral infection. altered levels of proteins in plasma evs are often described upon viral infection. for example, various examples of significantly altered expression of proteins, and markers associated with cellular stress, have been reported in plasma evs derived from hiv and htlv-1 infected patients. however, the mechanism of specific packaging of these proteins and markers in evs and their role in intercellular communication was not elucidated [148, 149] . blood plasma can be considered as disease biomarkers since it contains glycoproteins and cellular markers carried in evs [150] . dysregulation of cytokines and chemokines is often associated with hiv infection and subsequently contribute to the viral pathogenesis [20, 151, 152] . moreover, the use of substances such as alcohol, tobacco, and drugs is prevalent among hiv-infected individuals [153] [154] [155] [156] . circulating inflammatory cytokines have been found to be elevated in hiv-positive substance users [117, 151, 157, 158] . in prior studies, we demonstrated that exosomes derived from hiv-infected monocytes/macrophage cells exert a protective effect against cytotoxicity and viral replication in hiv-infected macrophages. however, exosomes derived from hiv-infected cells lost their protective capacity that could be due to the selective packaging of cytochrome p450 (cyps) and antioxidant enzyme (aoe) mrnas in exosomes [21] . similar to the previous study, exposure to cigarette smoke condensate (csc) increased the packaging of cytokines, especially il-6 and cyps (1a1 and 1b1) in evs isolated from hiv-infected u1 macrophages [116] . conversely, ev packaging of aoes (sod-1 and catalase) decreased in hiv-infected u1 macrophages more than in uninfected u937 macrophages [116] . recently, our group showed that the astrocytic and neuronal-specific markers (gfap and l1cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco [121] . human cytidine deaminase apobec3g (a3g) can be packaged in evs and inhibit hiv replication with its potential dna-editing activity [118] . hpv-infected cells release evs that make other cells more susceptible to infection as they deliver proteins that affect viral expression, and subsequently tumor development [19, 122, 159] . to enhance protein delivery and hpv replication, hpv-infected cells hijack ev signaling pathways to control the quantitative and qualitative release of evs from hpv-infected cells [123, [159] [160] [161] . as tumor genes and proteins are persistently expressed from evs, this contributes to hpv cancer cell growth [122] , thereby making the signaling pathways of evs harmful to the host. the oxidative stress released from hpv-infected cells into evs should also be considered detrimental to the host as this stress has the potential to induce viral replication of other viruses such as hiv-1 [120] . to make matters more complex, the signaling pathways of evs are not limited to increased hpv replication as the release of evs can also promote an adaptive immune response that becomes beneficial to the host [30] . for example, in the setting of hpv replication and tumor progression, evs have prompted immune activation in head and neck cancers and are being considered as biomarkers for improved clinical outcomes [162] [163] [164] [165] . besides, endogenously engineered evs are being considered as a novel method to deliver anti-hpv immunotherapy [166] , thus making them yet another way to improve clinical outcomes. unique mirna signatures were found in evs released from cervical cancer affected cells that were associated with hpv status [124] [125] [126] [127] 167] . during influenza virus infection, evs carrying host mirna or viral epitopes are thought to be integral to antigen transfer, reducing virus spread, and immune regulation [168] . for example, influenza virus hemagglutinin (ha) epitopes enclosed within exosomes on mhcii molecules have been shown to improve the efficiency of antigen delivery to immune cells [169] . further, exosomal-like vesicles carrying mucin molecules such as muc1, muc4, and muc16 can bind sialic acids and neutralize influenza viruses [128] , which may help reduce virus dissemination. virus replication can also be blocked by some highly upregulated exosomal mirnas, such as the type i interferon-inducing hsa-mir-1975 and mir-483-3p [129, 130] . also, these evs excite other proinflammatory cytokines, such as il-6, tnf-α, and ifn-β [129, 170] , although their efficacy may be dependent on cell source, maturity, and mhc molecules. macrophages have been shown to produce thousands of proteins within exosomal vesicles in response to influenza infection. these evs included a variety of host factors, including cytokines and proteins involved in copper metabolism and autophagy [171] . interestingly, proinflammatory cytokines from macrophages and dendritic cells were suppressed by vaccine-induced evs (e.g., mir-451a, mir-5100, or mir-7704) [172] . although much of the current work has focused on single influenza virus strains, important strain specific ev dynamics have begun to be identified. in one study, nearly half of exosomal mirnas were conserved between h1n1 and h7n7 infection in a549 cells [173] . of the differentially expressed evs, they were >10-fold during infection with the highly pathogenic h7n7 than with uninfected samples. a better understanding of these dynamics and temporal-and strain-specific differences could provide important insight into pathogenicity and pinpoint new therapeutic and universal influenza vaccine targets. hcv belongs to a family of human virus called flaviviridae characterized by positive-sense single-stranded rna that encodes precursor polyprotein that is cleaved into three structural proteins comprising of core protein p22 with envelope glycoprotein e1 & e2, and seven non-structural proteins that play a role in viral pathogenesis [131, 134] . the chronic viral infection leads to hepatic inflammation that is associated with increased production of pro-inflammatory cytokines and chemokines from liver residential immune cells and immune cells recruited to the liver [174] . evs are observed as major modifiers of cellular crosstalk between hcv-infected hepatocytes & immune cells [174] . in hcv pathogenesis evs act as a double edge power by: (1) delivering vireo-independent hcv rna and (2) obtaining antiviral immune responses [174] . the cellular vesicular pathway is exploited by hcv to congregate and release viral particles. this happens by releasing vesicles containing envelope glycoprotein e1 & e2, entire hcv genome & viral particles. when the vesicles containing these components enter the target cells, this helps to establish infection [175] . in systemic alteration of an immune response, major regulators commonly known as specifically enriched micro rnas (mirnas) are delivered by evs. these are loaded into evs and are involved in post-transcriptional regulation of gene expression, which is known to be influential for hcv replication [176, 177] . this confirms that evs have peculiar mirna expression isolated from the sera of chronic hcv patients. exosomes derived from hcv infected cells are responsible for developing infection to other uninfected cells. these exosomes carried viral rna in complex with mir-122, ago2, and hsp90 that support virus replication [133] . evs, isolated from sera of patients with acute or chronic hcv or interferon-stimulated macrophage cultures, mediate inhibitory effects on hcv replication [178] . in co-culture models, the immunoregulatory effects of evs were assessed on the replication of hcv. stimulation with type i & ii interferon n, which is a fast but short-lasting ev-derived antiviral, leads to the production of macrophages by secreting various cytokines resulting in innate immunity. thus, hcv replication in macrophages derives ev-mediated long-lasting inhibitory effects [178] . evs released by hcv infected cells contain viral rna that might trigger plasmacytoid dendritic cells to produce ifnα [132] . the emergence of the life-threatening "atypical pneumonia" caused by severe acute respiratory syndrome coronavirus (sars-cov) in the early 21st century has led to renewed interest in coronaviruses [179] . coronaviruses belong to the family of rna viruses and possess the largest genome among them. similar to other viruses, their genome contains essential genes encoded for open reading frames 1a and 1b (orf1ab), and viral structural proteins, which are required for virus replication, transcription, and virus assembly [180] . a newly emerged coronavirus disease in 2019 (covid-19) is caused by a novel severe acute respiratory syndrome coronavirus-2 (sars-cov-2). sars-cov-2 infection spread within a few months after the first outbreak reported in december 2019 in china, which later became a worldwide crisis. with high morbidity, the disease is often characterized by an atypical severe pulmonary pneumonia [181, 182] . the novel sars-cov-2 is closely related to sars-cov-1 coronavirus responsible for the sars outbreak that emerged in late 2002 in china. its subsequent worldwide spread had caused 8096 cases and 774 deaths by july 2003 [183] . sars-cov-2 infections, which has already infected >18 million people and caused the death of~700,000 people world-wide, are presently occurring and represent an ongoing threat to public health. 399 out of 1590 cases in china reported having at least one comorbidity [184] . the risk of serious adverse outcomes of covid-19 is especially pronounced in patients with comorbidities such as hypertension, diabetes, kidney, and cardiovascular diseases [184, 185] . sars-cov encodes four structural proteins; spike glycoprotein (s), nucleocapsid protein (n), membrane protein (m) & small envelope glycoprotein (e) & several nonstructural proteins of unknown functions [186] . sars-cov-2 spike (s) glycoprotein interacts with angiotensin-converting enzyme 2 (ace-2), the same receptor used by sars-cov to enter the target cells, in particular lung alveolar epithelial cells [187] . it has been demonstrated that evs released by sars-cov-2 infected lung epithelial cells contain viral rna fragments that were subsequently detected in the cardiomyocytes, suggesting viral rna transmission via evs [188] . sars-cov-2 is a positive-stranded rna virus in an envelope with a genome of 29,727 nucleotides [189] . the spike protein s of sars-cov-2 (sars-s) facilitates the viral fusion that can be triggered following the fusion-mediated conformational changes in the target cell receptor that mediates the entry of the virus into the target cells. once inside the cell, a virus may utilize the exosome secretion pathway to enhance its pathogenesis and viral spread [188] . to find a vaccine against sars-cov-2, researchers performed exosome-based research, where they constructed chimeric s protein of the sars by replacing cytoplasmic and transmembrane domains of sars-s with g protein of the vesicular stomatitis virus. this chimeric s-protein was readily expressed on the cell surface, allowed entry of pseudotyped retroviral vectors, and was incorporated into exosomes. subsequently, chimeric s protein-containing exosomes have been tested as a novel protein for vaccine immunogenicity against sars-cov in mouse models [135] . recently, preclinical studies have uncovered a therapeutic role of msc-derived secretome or evs in lung regeneration [190] , which could offer a new therapeutic approach in treating severe covid-19 infection [191, 192] . intravenous transplantation of ace2-negative mesenchymal stem cells (mscs) promoted recovery of patients from severe covid-19 [193] , thus supporting the hypothesis that binding of sars-s protein through ace2 expressed on msc-derived small evs could limit the viral infection through competitively inhibit the binding of sars-s to ace2 expressed on alveolar type ii cells [194] . epstein-barr virus (ebv) is one of the herpes viruses that hijack its host evs. ebv infected cells release evs that contain ebv-coding/non-coding mirnas and transfer it to uninfected cells including b lymphocytes and epithelial cells [83, 195] . the transfer of ebv-coding mirnas to b lymphocytes, especially the akata-lymphoblastoid cell lines-derived evs, causes inflammatory responses of monocytes/macrophages and induces severe lymphoproliferative disease (lpd) [195] . ebv viral reactivation was recently detected in co-cultured latently ebv-infected bl cells in response to the transfer of evs that contain epithelium-specific mirnas from oropharyngeal epithelial cells [83] . ebv-infected cells can transfer non-coding rnas such as bart and bhrf1 mirnas via evs to the target cells. upon entry, mirnas can be directed to cellular sites of mirna-mediated gene repression, causing repression of their target genes cxcl11 and lmp1 [136] . ebv-infected nasopharyngeal carcinoma cells release evs containing galectin-9 protein that interacts with the tim3 membrane receptor and induces apoptosis in t cells [137] . similarly, exosomes released by these cells convey the viral protein latent membrane protein 1 (lmp1) that provoke intrinsic t-cell inhibitory activity and thus modulate immune response mechanisms [138] . herpes simplex virus 1 (hsv-1) is another herpes virus that hijacked its host evs. hsv-1-infected cells release evs with different components based on their stage in the infection cycle [49] . early in the lytic cycle, hsv-1 proteins cause remodeling to evs' cargos, which in turn cause virion egress from infected cells to uninfected cells [49] . hsv-1 evs contain coding and non-coding rnas and more importantly immune components, such as the stimulator of interferon genes (sting) [196] . a recent study demonstrated that sting-containing evs play an important role in inhibiting viral replication during the lytic cycle, as well as inhibiting viral gene expression during the latent stage [141] . another recent report illustrated that mir-h28 and mir-h29 are being expressed late in the virus infection cycle and transferred to uninfected cells via evs [140] ; mirna-28 induces the formation of gamma interferon (ifn-γ) which blocks viral replication in uninfected cells but not in infected cells [197] . ifn-γ loaded evs maximize viral transmission between individuals by diminishing the spread from infected cells to uninfected cells [197] . a study reported that hsv-1 encoded glycoprotein b (gb) modulates the immune response by manipulating the mhc class ii processing pathway by diverting human leukocyte antigen-dr (hla-dr) molecules into the exosome pathway [139] . an ev vaccine for the hepatitis b virus (hbv) is currently under investigation. as in most of the viruses, evs carry hbv viral proteins such as large s, core and p proteins which participate in viral replication [142] . they also play many roles in hbv infection; they are responsible for hbv replication, innate immune response during infection, a biomarker for its diagnosis, and development of a possible vaccine [198, 199] . a recent study elucidated that unmodified evs can be attractive coadjutants to hepatitis b recombinant antigen (hbsag), because it triggers the healthy mice immune response due to an increased ifn-γ concentration and accelerates the production of igg antibodies [200] . hepg2.2.15 cells with integrated hbv genome release evs containing hbv-mir-3 which represses viral protein production and hbv replication [143] . moreover, the study elucidated that engineered evs that are loaded with exosome-anchoring protein nef mutant (nefmut) and hbv core protein can induce cytotoxic t lymphocyte (ctl) immunization in animals for hbv infection [201] . on the one hand, evs are responsible for infection transfer from one cell to another. on the other hand, evs are also responsible for antiviral response initiation by inducing the uninfected cells' immune response [197, 202] . due to their abilities to activate the innate and adaptive immune response, evs can be the future pathway for the treatment of many viral infections. so far, viruses that impair their host immune response such as human t-lymphotropic virus (htlv-1) only use their host's evs to use viral proteins such as gp61, tax, and hbz to increase cell-to-cell contact and promote a potential increase in viral infection [144] . htlv-1 evs were found to contain a protein called tax that is implicated with the dysregulation of the recipient cells' immune response [144, 202] . interestingly, there are viruses that not only hijack host evs, but also boost the production of evs such as in zika virus (zikv). evs released from zikv-infected (c6/36) cells carry viral rna and zikv-e protein that can trigger monocyte activation to induce mrna expression of tnf-α [145] . zikv-infected cells have incrementation in their neutral sphingomyelinase (nsmase)-2/smpd3 gene expression and activity, which provokes the production and excretion of evs in neurons. treatment of zikv requires the hindrance of ev production through the inhibition of smpd3s in neurons to prevent further neuronal death and virus spreading [203] . with the introduction of antiretroviral therapy (art), the morbidity and mortality associated with hiv infection have drastically reduced [204] . however, due to the presence of latent reservoirs and inadequate drug concentration in the central nervous system (cns), the virus continues to replicate and causes a wide range of cns pathologies, including hiv-associated neurocognitive disorders (hand) [205] . therefore, new drug delivery systems that facilitate drug passage across the bbb to effectively suppress the virus in cns, with minimal/tolerable neurotoxicity need to be developed. evs can be used as a potential drug delivery system as they can cross the bbb [206, 207] with less immunogenicity. further, in preclinical studies, ev-based drug delivery platforms have been shown to carry therapeutic small molecules across the bbb to help alleviate multiple cns diseases, including parkinson's disease and brain cancer [208] [209] [210] . evs that can be used as a drug delivery platform are mainly derived from exosomes that linked to an endolysosomal pathway. exosomes released from dendritic cells are considered vaccine candidates for immunotherapy in diseases such as cancer. these exosomes can be further taken up by dendritic cells leading to a presentation of mhc-i or peptide complexes [211] [212] [213] . arvs can be loaded into evs to deliver them across the bbb to achieve viral suppression in the cns [214] . since the autoclaved exosomes show intrinsic stability at a physiological temperature [215] , sterile drug-loaded evs can be formulated. large scale production of ev drug formulation can be achieved using an endogenous drug-loading method that uses cells to release evs with target drugs encapsulated in vitro. evs with encapsulated drugs are capable of targeting the diseased cell or tissue, with targeting characteristics [110] . this inherent feature could be used to deliver drugs selectively to their intended targets while abrogating off-target side effects. virus-targeting antiviral drugs can include protease inhibitors (pis), integrase inhibitors, nucleoside and nucleotide reverse transcriptase inhibitors, and nonnucleoside reverse-transcriptase inhibitors. an ev-based drug delivery platform with either hiv pis alone or in combination with ritonavir is used as a pharmaco-enhancer or second line of therapy for the treatment of hiv [214] . evs can also be used as a vehicle for delivery of crispr-associated endonuclease (cas9) and potentially as the guide rna (grna) to target nucleotide sequences within viral genomes [216, 217] . another therapeutic use of evs is vaccination against infectious diseases and viral infection. ev-mediated delivery of mrna encoding pathogenic proteins required for viral infection might be a vaccine candidate that can induce t helper 1 (th1)-type immune responses and cell-mediated immunity, without the need to attenuate and inactivate pathogenic viruses or bacteria [216, 218] . for the ongoing pandemic of covid-19, anti-hiv pis, other pis, or other antiviral and antibacterial drugs can either be encapsulated in evs derived from various cell lines using endogenous loading technique or from the plasma of patients using exogenous loading method for personalized therapy [214] . repurposing fda-approved antiviral drugs using evs could be a fast way to get tested through clinical trials. although the clinical research done on evs seem promising for therapeutic application, several factors must be considered before translating evs into clinics. at present, available ev isolation methods, such as ultracentrifugation, density gradient centrifugation, precipitation, size exclusion chromatography, affinity, and novel microfluidic techniques are not sensitive enough to distinguish evs subpopulation due to lack of specificity, physical and chemical biomarkers [219] ; therefore, a high level of standardization is required to compare ev protocols and results used across different laboratories before the adoption of ev therapy to various clinical applications. also, evs' pharmacokinetics, half-life, and plasma stability, as well as the interaction of encapsulated drugs with ev components, ev-targeting, and immune clearance of evs, are other limitations that need to be overcome before realizing the clinical applications of evs in drug delivery. a growing body of evidence suggests that virus-infected cells produce evs, encapsulated with viral proteins and parts of viral genetic material, and in some cases they carry the full infectious viral genome that facilitates viral infection and mediates immune responses ( figure 1) . notably, evs can enhance viral infection by: (1) mediating transfer of chemokine co-receptors or cell surface proteins to null-target cells that do not express endogenous viral co-receptors; (2) helping viruses to evade the host immune system; (3) transferring of viral components (viral proteins and rnas) to recipient cells, which induce cytotoxic effects on infected cells, leading to progressive loss of immune cells resulting from the apoptosis of uninfected bystander cells. here, we aimed to shed light on how evs potentially impact infection and the pathogenesis of various viruses. we also evaluated the potential utilization of evs in antiviral and antiretroviral therapy, and in drug delivery. characterizing evs from virus-infected cells and their functional analyses could aid not only in the understanding of the mechanisms of viral infection but also in the utilization of evs as a delivery system for therapeutic agents. overcome before realizing the clinical applications of evs in drug delivery. a growing body of evidence suggests that virus-infected cells produce evs, encapsulated with viral proteins and parts of viral genetic material, and in some cases they carry the full infectious viral genome that facilitates viral infection and mediates immune responses ( figure 1) . notably, evs can enhance viral infection by: (1) mediating transfer of chemokine co-receptors or cell surface proteins to null-target cells that do not express endogenous viral co-receptors; (2) helping viruses to evade the host immune system; (3) transferring of viral components (viral proteins and rnas) to recipient cells, which induce cytotoxic effects on infected cells, leading to progressive loss of immune cells resulting from the apoptosis of uninfected bystander cells. here, we aimed to shed light on how evs potentially impact infection and the pathogenesis of various viruses. we also evaluated the potential utilization of evs in antiviral and antiretroviral therapy, and in drug delivery. characterizing evs from virusinfected cells and their functional analyses could aid not only in the understanding of the mechanisms of viral infection but also in the utilization of evs as a delivery system for therapeutic agents. funding: this study is partially supported by the funding opportunity from the national institute of health (da047178 and ai139088). funding: this study is partially supported by the funding opportunity from the national institute of health (da047178 and ai139088). regulation of immune responses by extracellular vesicles regulation of immune responses by extracellular vesicles a novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure plasma membrane lipid domains as platforms for vesicle biogenesis and shedding? sphingomyelin encrypts tissue factor: atp-induced activation of a-smase leads to tissue factor decryption and microvesicle shedding biology and biogenesis of shed microvesicles a novel exosome biogenesis mechanism: multivesicular structures budding and rupturing at the plasma membrane syntenin-alix exosome biogenesis and budding into multivesicular bodies are controlled by arf6 and pld2 apoptotic-cell-derived membrane microparticles and ifn-α induce an inflammatory immune response the process of ultrastructural changes from nuclei to apoptotic body exosome-mediated transfer of mrnas and micrornas is a novel mechanism of genetic exchange between cells cytotoxic necrotizing factor type 1 delivered by outer membrane vesicles of uropathogenic escherichia coli attenuates polymorphonuclear leukocyte antimicrobial activity and chemotaxis human tumor virus utilizes exosomes for intercellular communication malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system neutral sphingomyelinase inhibition alleviates lps-induced microglia activation and neuroinflammation after experimental traumatic brain injury microglial-derived microparticles mediate neuroinflammation after traumatic brain injury hiv-1 infection-induced apoptotic microparticles inhibit human dcs via cd44 localization of the epstein-barr virus protein lmp 1 to exosomes the role of exosomal transport of viral agents in persistent hiv pathogenesis human papillomavirus e6 and e7 oncoproteins affect the expression of cancer-related micrornas: additional evidence in hpv-induced tumorigenesis exosomes from hiv-1-infected cells stimulate production of pro-inflammatory cytokines through trans-activating response (tar) rna monocyte-derived exosomes upon exposure to cigarette smoke condensate alter their characteristics and show protective effect against cytotoxicity and hiv-1 replication extracellular vesicles exploit viral entry routes for cargo delivery. microbiol classification, functions, and clinical relevance of extracellular vesicles exosomes and other extracellular vesicles in hpv transmission and carcinogenesis binding and fusion of extracellular vesicles to the plasma membrane of their cell targets exosome uptake through clathrin-mediated endocytosis and macropinocytosis and mediating mir-21 delivery endocytosis of extracellular vesicles and release of their cargo from endosomes hepatitis a virus structural protein px interacts with alix and promotes the secretion of virions and foreign proteins through exosome-like vesicles persistent and transient replication of full-length hepatitis c virus genomes in cell culture the role of extracellular vesicles in viral infection and transmission extracellular vesicles-biogenesis, composition, function, uptake and therapeutic applications extracellular vesicles and viruses: are they close relatives? exosomes are fingerprints of originating cells: potential biomarkers for ovarian cancer molecular mechanism to recruit galectin-3 into multivesicular bodies for polarized exosomal secretion microautophagy of cytosolic proteins by late endosomes pdcd6ip, encoding a regulator of the escrt complex, is mutated in microcephaly ceramide triggers budding of exosome vesicles into multivesicular endosomes biogenesis of extracellular vesicles (ev): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies sphingolipid metabolism and neutral sphingomyelinases rab27a and rab27b control different steps of the exosome secretion pathway the mechanisms of vesicle budding and fusion active wnt proteins are secreted on exosomes current knowledge on exosome biogenesis and release ti-vamp/vamp7 and vamp3/cellubrevin: two v-snare proteins involved in specific steps of the autophagy/multivesicular body pathways tetraspanins in extracellular vesicle formation and function modulation of b-cell exosome proteins by gamma herpesvirus infection exosomes derived from hiv-1-infected cells contain trans-activation response element rna herpesviruses hijack host exosomes for viral pathogenesis exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral rna and proteins to the vertebrate neuronal cells dengue-3 virus entry into vero cells: role of clathrin-mediated endocytosis in the outcome of infection blockade of dengue virus entry into myeloid cells by endocytic inhibitors in the presence or absence of antibodies infectious entry of west nile virus occurs through a clathrin-mediated endocytic pathway flavivirus internalization is regulated by a size-dependent endocytic pathway biology of zika virus infection in human skin cells exosome-associated hepatitis c virus in cell cultures and patient plasma the ambiguous roles of extracellular vesicles in hiv replication and higher-order oligomerization targets plasma membrane proteins and hiv gag to exosomes the trojan exosome hypothesis endosomes, exosomes and trojan viruses hiv-1 buds predominantly at the plasma membrane of primary human macrophages cd45 immunoaffinity depletion of vesicles from jurkat t cells demonstrates that exosomes contain cd45: no evidence for a distinct exosome/hiv-1 budding pathway exosomes and retroviruses: the chicken or the egg? hiv-1 is budded from cd4+ t lymphocytes independently of exosomes isolation of exosomes from whole blood by integrating acoustics and microfluidics extracellular vesicles as protagonists of diabetic cardiovascular pathology. front. cardiovasc arf6-regulated shedding of tumor cell-derived plasma membrane microvesicles phospholipase d and phosphatidic acid in the biogenesis and cargo loading of extracellular vesicles comprehensive landscape of extracellular vesicle-derived rnas in cancer initiation, progression, metastasis and cancer immunology follicular dendritic cells carry mhc class ii-expressing microvesicles at their surface rapid secretion of interleukin-1beta by microvesicle shedding shedding microvesicles: artefacts no more apoptosis: a review of programmed cell death apoptosis: controlled demolition at the cellular level apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics clearance of apoptotic cells by phagocytes thrombospondin cooperates with cd36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis annexin v-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure routes and mechanisms of extracellular vesicle uptake dynamics of exosome internalization and trafficking neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis exosomes derived from epstein-barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells capture and transfer of hiv-1 particles by mature dendritic cells converges with the exosome-dissemination pathway endocytosis, intracellular sorting, and processing of exosomes by dendritic cells visualizing of the cellular uptake and intracellular trafficking of exosomes by live-cell microscopy monitoring extracellular vesicle cargo active uptake by imaging flow cytometry modern techniques for the isolation of extracellular vesicles and viruses isolation of extracellular vesicles: determining the correct approach (review) extracellular vesicle isolation: present and future progress in exosome isolation techniques tangential flow filtration for highly efficient concentration of extracellular vesicles from large volumes of immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from biofluids quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography isolation of extracellular vesicles: general methodologies and latest trends pilot production of mesenchymal stem/stromal freeze-dried secretome for cell-free regenerative nanomedicine: a validated gmp-compliant process freeze-dried and gmp-compliant pharmaceuticals containing exosomes for acellular mesenchymal stromal cell immunomodulant therapy cushioned-density gradient ultracentrifugation (c-dguc): a refined and high performance method for the isolation, characterization, and use of exosomes a comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum antiretroviral drugs alter the content of extracellular vesicles from hiv-1-infected cells methods of isolating extracellular vesicles impact down-stream analyses of their cargoes isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods ready-made chromatography columns for extracellular vesicle isolation from plasma size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography defective friend spleen focus-forming virus: interfering properties and isolation free from standard leukemia-inducing helper virus re-engineering extracellular vesicles as smart nanoscale therapeutics extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking surface engineering of extracellular vesicles through chemical and biological strategies hiv nef is secreted in exosomes and triggers apoptosis in bystander cd4+ t cells hiv-1 evades virus-specific igg2 and iga responses by targeting systemic and intestinal b cells via long-range intercellular conduits microglia-derived hiv nef+ exosome impairment of the blood-brain barrier is treatable by nanomedicine-based delivery of nef peptides inhibition of human immunodeficiency virus replication by a dual ccr5/cxcr4 antagonist differential packaging of inflammatory cytokines/chemokines and oxidative stress modulators in u937 and u1 macrophages-derived extracellular vesicles upon exposure to tobacco constituents cytokine profiling of exosomes derived from the plasma of hiv-infected alcohol drinkers and cigarette smokers exosomes packaging apobec3g confer human immunodeficiency virus resistance to recipient cells novel hiv-1 mirnas stimulate tnfα release in human macrophages via tlr8 signaling pathway extracellular vesicles from human papilloma virus-infected cervical cancer cells enhance hiv-1 replication in differentiated u1 cell line circulatory astrocyte and neuronal evs as potential biomarkers of neurological dysfunction in hiv-infected subjects and alcohol/tobacco users dependence of intracellular and exosomal micrornas on viral e6/e7 oncogene expression in hpv-positive tumor cells exosomes from breast cancer cells can convert adipose tissue-derived mesenchymal stem cells into myofibroblast-like cells human papillomavirus and carcinogenesis: novel mechanisms of cell communication involving extracellular vesicles microrna-222 promotes the proliferation and migration of cervical cancer cells mir-92a family and their target genes in tumorigenesis and metastasis characterization of exosome-like vesicles released from human tracheobronchial ciliated epithelium: a possible role in innate defense lung-derived exosomal mir-483-3p regulates the innate immune response to influenza virus infection exosome-delivered and y rna-derived small rna suppresses influenza virus replication hepatitis c virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies short-range exosomal transfer of viral rna from infected cells to plasmacytoid dendritic cells triggers innate immunity exosomes from hepatitis c infected patients transmit hcv infection and contain replication competent viral rna in complex with ago2-mir122-hsp90 infectious hepatitis c virus pseudo-particles containing functional e1-e2 envelope protein complexes exosomal vaccines containing the s protein of the sars coronavirus induce high levels of neutralizing antibodies functional delivery of viral mirnas via exosomes blood diffusion and th1-suppressive effects of galectin-9-containing exosomes released by epstein-barr virus-infected nasopharyngeal carcinoma cells exosomes released by ebv-infected nasopharyngeal carcinoma cells convey the viral latent membrane protein 1 and the immunomodulatory protein galectin 9 the herpes simplex virus-1 encoded glycoprotein b diverts hla-dr into the exosome pathway mir-h28 and mir-h29 expressed late in productive infection are exported and restrict hsv-1 replication and spread in recipient cells extracellular vesicles released by herpes simplex virus 1-infected cells block virus replication in recipient cells in a sting-dependent manner label-free proteomic analysis of exosomes derived from inducible hepatitis b virus-replicating hepad38 cell line hepatitis b virus-encoded microrna controls viral replication htlv-1 extracellular vesicles promote cell-to-cell contact participation of extracellular vesicles from zika-virus-infected mosquito cells in the modification of naïve cells' behavior by mediating cell-to-cell transmission of viral elements elevated abundance, size, and microrna content of plasma extracellular vesicles in viremic hiv-1+ patients extracellular vesicles carry hiv env and facilitate hiv infection of human lymphoid tissue proteomic profiling of exosomes derived from plasma of hiv-infected alcohol drinkers and cigarette smokers proteomic analysis of plasma extracellular vesicles reveals mitochondrial stress upon htlv-1 infection simultaneous enrichment of plasma extracellular vesicles and glycoproteome for studying disease biomarkers inflammatory cytokines and mortality in a cohort of hiv-infected adults with alcohol problems the role of cytokines in the pathogenesis and treatment of hiv infection tobacco smoking and alcohol drinking among hiv infected people using antiretroviral therapy poly-tobacco use among hiv-positive smokers: implications for smoking cessation efforts global epidemiology of injecting drug use and hiv among people who inject drugs: a systematic review smoking, alcohol and illicit drug use effects on survival in hiv-positive persons cocaine alters cytokine profiles in hiv-1-infected african american individuals in the drexelmed hiv/aids genetic analysis cohort hiv research network alcohol use among hiv-infected persons in care: results of a multi-site survey silencing of human papillomavirus (hpv) e6/e7 oncogene expression affects both the contents and the amounts of extracellular microvesicles released from hpv-positive cancer cells exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts human papillomavirus 16 e6 and e7 oncoprotein expression alters microrna expression in extracellular vesicles molecular and functional profiles of exosomes from hpv(+) and hpv(-) head and neck cancer cell lines the interplay between hpv and host immunity in head and neck squamous cell carcinoma pd-1-expressing tumor-infiltrating t cells are a favorable prognostic biomarker in hpv-associated head and neck cancer tumor infiltrating cd8+ and foxp3+ lymphocytes correlate to clinical outcome and human papillomavirus (hpv) status in tonsillar cancer anti-cancer vaccine for hpv-associated neoplasms: focus on a therapeutic hpv vaccine based on a novel tumor antigen delivery method using endogenously engineered exosomes extracellular vesicle microrna cargo is correlated with hpv status in oropharyngeal carcinoma host micrornas and exosomes that modulate influenza virus infection exosome-driven antigen transfer for mhc class ii presentation facilitated by the receptor binding activity of influenza hemagglutinin direct exosome stimulation of peripheral humant cells detected by elispot proteomic and bioinformatic characterization of extracellular vesicles released from human macrophages upon influenza a virus infection microrna-451a in extracellular, blood-resident vesicles attenuates macrophage and dendritic cell responses to influenza whole-virus vaccine integrated analysis of microrna-mrna expression in a549 cells infected with influenza a viruses (iavs) from different host species mechanisms underlying hepatitis c virus-associated hepatic fibrosis the role of extracellular vesicles as allies of hiv, hcv and sars viruses extracellular rna in viral-host interactions: thinking outside the cell toll-like receptor 3-activated macrophages confer anti-hcv activity to hepatocytes through exosomes macrophage-derived extracellular vesicles induce long-lasting immunity against hepatitis c virus which is blunted by coronavirus in severe acute respiratory syndrome (sars) genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding covid-19: what has been learned and to be learned about the novel coronavirus disease evaluation and treatment coronavirus (covid-19) comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis prevalence of comorbidities among individuals with covid-19: a rapid review of current literature coronavirus envelope protein: current knowledge structure, function, and antigenicity of the sars-cov-2 spike glycoprotein detection of viral rna fragments in human ipsc-cardiomyocytes following treatment with extracellular vesicles from sars-cov-2 coding-sequence-overexpressing lung epithelial cells from sars to mers, thrusting coronaviruses into the spotlight mesenchymal stem/stromal cell secretome for lung regeneration: the long way through "pharmaceuticalization" for the best formulation mesenchymal stromal cell secretome for severe covid-19 infections: premises for the therapeutic use mesenchymal stromal cells and their secreted extracellular vesicles as therapeutic tools for covid-19 pneumonia? transplantation of ace2-mesenchymal stem cells improves the outcome of patients decoy ace2-expressing extracellular vesicles that competitively bind sars-cov-2 as a possible covid-19 therapy role of exosomes as a proinflammatory mediator in the development of ebv-associated lymphoma extracellular vesicles during herpes simplex virus type 1 infection: an inquire herpes simplex virus 1 microrna mir-h28 exported to uninfected cells in exosomes restricts cell-to-cell virus spread by inducing gamma interferon mrna transmission of hbv dna mediated by ceramide-triggered extracellular vesicles exosomes mediate hepatitis b virus (hbv) transmission and nk-cell dysfunction exosomes as adjuvants for the recombinant hepatitis b antigen: first report dna vectors generating engineered exosomes potential ctl vaccine candidates against aids, hepatitis b, and tumors exosomes in viral disease exosomes mediate zika virus transmission through smpd3 neutral sphingomyelinase in cortical neurons global mortality and morbidity of hiv/aids the international bank for reconstruction and development/the world bank hiv-associated neurocognitive disorder (hand) and the prospect of brain-penetrating protease inhibitors for antiretroviral treatment elucidation of exosome migration across the blood-brain barrier model in vitro in vivo evidence for the contribution of peripheral circulating inflammatory exosomes to neuroinflammation extracellular vesicles as drug delivery vehicles to the central nervous system exosomes as drug delivery vehicles for parkinson's disease therapy delivery of sirna to the mouse brain by systemic injection of targeted exosomes exosomes as potent cell-free peptide-based vaccine. ii. exosomes in cpg adjuvants efficiently prime naive tc1 lymphocytes leading to tumor rejection vaccination of metastatic melanoma patients with autologous dendritic cell (dc) derived-exosomes: results of thefirst phase i clinical trial exosomes as potent cell-free peptide-based vaccine. i. dendritic cell-derived exosomes transfer functional mhc class i/peptide complexes to dendritic cells repurposing antiviral protease inhibitors using extracellular vesicles for potential therapy of covid-19 hot evs-how temperature affects extracellular vesicles rna delivery by extracellular vesicles in mammalian cells and its applications the crispr/cas9 genome editing methodology as a weapon against human viruses therapeutic applications of extracellular vesicles: clinical promise and open questions extracellular vesicles move toward use in clinical laboratories this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors are grateful to kelli anne gerth (ph.d. student; university of tennessee health science center) for critical reading and editing of the manuscript. the authors declare no conflict of interest.acknowledgments: the authors are grateful to kelli anne gerth (ph.d. student; university of tennessee health science center) for critical reading and editing of the manuscript. the authors declare no conflict of interest. key: cord-301506-q2a5aogo authors: sun, xinhua; lu, fan; wu, zunyou; poundstone, katharine; zeng, gang; xu, peng; zhang, dapeng; liu, kangmai; liau, adrian title: evolution of information-driven hiv/aids policies in china date: 2010-12-24 journal: int j epidemiol doi: 10.1093/ije/dyq217 sha: doc_id: 301506 cord_uid: q2a5aogo background as china continues to commit to universal access to hiv/aids prevention, treatment and care services, its hiv/aids policies have become increasingly information driven. we review china’s key national-level hiv/aids policies and discuss policy gaps and challenges ahead. methods we conducted a desk review of key national-level policies that have had a major impact on china’s hiv/aids epidemic, and examined recent epidemiological data relevant to china’s hiv response. results national-level policies that have had a major impact on china’s hiv/aids response include: ‘four frees and one care’; 5-year action plans; and hiv/aids regulation. these landmark policies have facilitated massive scaling up of services over the past decade. for example, the number of drug users provided with methadone maintenance treatment significantly increased from 8116 in 2005 to 241 975 in 2009; almost a 30-fold increase. the ‘four frees and one care’ policy has increased the number of people living with aids on anti-retroviral treatment from some 100 patients in 2003 to over 80 000 in 2009. however, stigma and discrimination remains major obstacles for people living with hiv/aids trying to access services. conclusions china’s current national policies are increasingly information driven and responsive to changes in the epidemic. however, gaps remain in policy implementation, and new policies are needed to meet emerging challenges. in april 2010, the state council of the people's republic of china officially announced the lifting of its travel ban on people living with hiv/aids wishing to enter the country. 1 this ban was implemented 420 years ago as one of china's first key policies for hiv/aids control and prevention. 2 the announcement reflects china's current approach to hiv/aids: pragmatic solutions to a changing epidemic based on sound scientific information. china has made significant progress in its hiv response over the past decade. china now has each of the 'three ones' 3 recommended by the united nations program on hiv/aids (unaids) as key elements supporting a results-based hiv response, ( table 1 ). in addition to the 'three ones', china has put into effect key laws that have created an enabling environment for china's national hiv response, such as the 2006 hiv/aids prevention and treatment regulations. moving forward, china faces an array of new and ongoing challenges that will need to be addressed in its new 5-year action plan for the containment and control of hiv/aids . we review key national policies that have guided china's hiv response at various stages. we also discuss gaps in policy implementation, and challenges ahead as china continues its efforts to achieve universal access to hiv prevention, treatment and care services. we performed a desk review of all hiv/aids policies issued by the chinese government since the beginning of the epidemic. we limited our review to nationallevel policies that have been put in place or revoked over the past 10 years. we also reviewed available data relevant to selected policies. data were collected from government reports and papers published in peer-reviewed journals. an overview of aids policies issued against specific hiv/aids epidemic phases are summarized in table 2 . the chinese government has issued hundreds of hiv/aids policies over the past 25 years. we focused on national-level policies that have had a significant impact on china's hiv/aids response. the landmark policies include: the implementation and revocation of china's travel ban on people living with hiv/aids; the blood donation law of 1998; china's 5-year action plans for the containment and control of hiv/aids; the 'four frees and one care' policy to improve access to treatment and care services; the creation of high-risk behavioural intervention outreach teams; and the 2006 aids regulation. these key policies were put in place during different phases of the hiv/aids epidemic in china (figure 1 ). in june 1985, the first hiv-infected patient, a us citizen, was diagnosed and reported in china. later that same year, four haemophiliacs were confirmed to have been infected with hiv due to the use of imported blood products. 24 by 1988, a total of seven provinces, municipalities and regions had reported 22 hiv/aids cases; most of them were foreigners or chinese residents returning from overseas. the chinese government issued a series of early policies focused mainly on blood product safety, case identification and preventing the spread of hiv from infected foreigners entering the country. 4 the first hiv/aids policy document entitled 'the joint notice on preventing hiv from entering china by restricting import of blood products' was issued on 17 september 1984. 5 in 1986, the ministry of health classified hiv/aids as a class b infectious disease notifiable through the national infectious disease reporting system. 6 to prevent hiv-infected foreigners from entering into china, a series of policies were issued. foreign students and research scholars visiting and living in china for 412 months had to submit to hiv testing procedures 1 month after entry into the country. other foreigners residing or living in china for 412 months had to provide health certificates and were asked to specify if they were infected with hiv/ aids. foreigners found to be hiv infected at chinese cross-border and customs checkpoints were forced to leave the country and were placed under quarantine before departure. 2, [25] [26] [27] to enforce this between late 1994 and early 1996, outbreaks of hiv infection were identified among commercial plasma donors in regions of central and eastern china. 11, 12 the scale of transmission and the resulting devastation led to one of the worst tragedies of the global hiv pandemic. early small-scale surveys indicated that the epidemic was serious, but the true scope of transmission was largely unknown until 1996, when a large-scale survey was carried out among plasma donors in fuyuan, anhui province, showing an hiv prevalence of 12.5%. 12 to respond to this crisis, china's blood donation law was drafted in 1996 and enacted in 1998. the most important components of the law highlight voluntary donation and prohibit repeated commercial donation. at same time, nationwide blood collection facilities were established and manual collection of blood or plasma was prohibited. since since 1995, hiv spread more quickly throughout china. 10 by 1998, all 31 provinces, municipalities and autonomous regions in mainland china reported hiv cases. 10 to respond to the changing hiv/aids epidemic, several key ministries, including health, finance, public security, justice, and the development commission, met to discuss instituting supportive policies for condom promotion, needle exchange and methadone maintenance programmes. the language of early documents was carefully selected to avoid condoning 'social evils', such as prostitution and drug use. terms such as condom social marketing, needle social marketing and community-clinic-based therapeutic treatment for drug users were used to describe hiv prevention measures that were incorporated into china's first 5-year action plan (2001-05). 16 china's first 5-year action plan was a policy milestone in terms of supporting effective policies for condom promotion, methadone maintenance and needle exchange. one of most important policy directions laid out in the first 5-year action plan was pilot testing of harm reduction strategies, including methadone maintenance treatment (mmt) and needle exchange programmes. 30, 31 however, implementation of the plan was not adequately budgeted, weakening its impact, particularly in the first 3 years, between 2001 and 2003. after the severe acute respiratory syndrome outbreak in 2003, public health rose to the top of china's policy agenda, and funding for the hiv/aids 5-year action plan increased. 18 china's second 5-year action plan (2006-10) was drafted in a more supportive political environment in which public health was given a higher priority. first, there was much stronger political commitment and financial commitment for controlling hiv/aids from chinese central government. in 2003, a new administration led by president hu jintao, premier wen jiabao and vice premier and the then health minister wu yi put the implementation of evidence-based hiv policies high on the national agenda. 18 secondly, china's 'four frees and one care' policy to increase access to anti-retroviral therapy (art) was announced in late 2003 and had greatly facilitated implementation of hiv prevention, treatment and care and support. the second 5-year action plan set more specific and ambitious targets, particularly for prevention programmes for marginalized groups, such as drug users, female sex workers and men who sex with men. for example, one of the 2010 targets was for hiv prevention programmes to achieve 490% coverage of the 'floating population' (such as migrant workers) and people with high-risk behaviours. another target was set to establish mmt clinics in counties and cities with more than 500 drug users registered with the public security bureau, and for 570% of opiate users to receive mmt services. this target has greatly facilitated the scaling up of the mmt programme in china; for example, the number of drug users receiving methadone increased from 8116 in 2005 to 241 975 in 2009, almost a 30-fold increase. 32 other targets included: achieving 50% coverage of needle and syringe exchange programmes; reducing the needle/syringe sharing rate among idus to <20%; increasing the proportion of people with high-risk behaviour who have basic hiv knowledge to at least 90%; increasing the condom usage rate to 590% among high-risk groups. 20 the plan also set specific targets for treating aids patients. some of these targets included: at least 80% of aids patients satisfying the treatment criteria should have received art by 2010; mother-to-child transmission prevention services should be available in 590% of cities and counties; and more than 90% of hiv-positive mothers should have received prevention of mother-to-child transmission (pmtct) services. 20 the evaluation of the second 5-year action plan will take place next year. china's 2010 ungass country progress report 33 suggested that china's aids programme is approaching specific targets set in its second 5-year plan. the 'four frees and one care' policy the massive outbreaks of hiv infection that occurred in central china among paid plasma donors around the mid-1990s led to a huge demand for hiv/aids treatment and care services as more and more people became ill and died. at the united nations high-level special meeting in september 2003, the chinese government announced five commitments in the fight against hiv/aids, later known as the 'four frees and one care' policy. these commitments included: (1) free anti-retroviral drugs to aids patients who are rural residents or people without insurance living in urban areas; (2) free voluntary counselling and testing; (3) free drugs to hiv-infected pregnant women to prevent mother-to-child transmission, and hiv testing of newborn babies; china. first, it resulted in a significant increase in the number of people being tested for hiv 34 . before 2003, less than 22 000 newly identified hiv infections were reported each year. after 2004, over 44 000 newly identified hiv infections were reported each year ( figure 1) . secondly, it resulted in a significant increase in the number of aids patients receiving free anti-retroviral treatment; for example, increased from 100 in 2003 to over 80 000 in 2009, 22 and it helped reduce aids mortality among those on art. 35 thirdly, it increased the number of pregnant women screened for hiv infection and receiving pmtct services. fourthly, all aids orphans have been taken care of by either relatives, or neighbour or local government social welfare programmes. the 'four frees and one care' policy has also significantly reduced stigma in local communities, particularly in areas where the epidemic was driven by contaminated plasma collection. 36 high-risk behavioural intervention outreach teams in 2004, as the primary mode of hiv transmission began to shift from injecting drug use to high-risk sexual behaviours, the chinese ministry of health announced the establishment of high-risk behavioural intervention teams at all levels of the public health system. 37 the primary function of these teams is to conduct an outreach programme among sex workers, men who have sex with men, and migrant labourers to reduce the risk of hiv sexual transmission among these high-risk groups. intervention teams have increased the reach of hiv prevention programmes. on average, these teams have reached about 460 000 female sex workers and 120 000 men who sex with men per month. 38 national hiv/aids regulation in early 2006, the chinese government issued the national hiv/aids regulation to define the roles and responsibilities of government, civil society and people living with hiv/aids. this was the first law in china to highlight protection of human rights of people living with hiv/aids, including the right to marry, to access health-care services, to enjoy equal employment opportunities and to receive schooling. the regulation has laid a legal base for effective but sensitive prevention measures, such as condom promotion, mmt and needle exchanges. however, implementation of hiv/aids regulation varies in different articles and in different places. the most significant gap relates to stigma-related rights for receiving medical services and employment. on 27 august 2010, the first case of a law suit concerning hiv-related stigma for employment was reported and has generated great discussion and concern. 39 the most frequent cases of discrimination that people living with hiv/aids face are when seeking medical care in clinical settings. there is still a long way to go to achieve the goal of zero stigmas in chinese society, though hiv/aids regulation is in place. china now has national laws, policies, action plans, surveillance systems and monitoring and evaluation systems in place to support its hiv/aids response, as well as a government body with the authority to coordinate different sectors. hiv/aids policies and action plans are responsive to the increasing wealth of epidemiologic data available, and a massive scaling up of services has taken place over the past decade. though efforts have been made to achieve universal access to prevention, treatment, care and support services, a number of important gaps exist in the implementation of china's hiv/aids policies. first, despite the increased coverage of hiv testing services, too many people remain unaware of their hiv status. by the end of 2009, there were 326 000 cumulative cases of hiv/aids reported, and an estimated 740 000 people living with hiv/aids in china. 40 this means that less than half of the people living with hiv/aids are aware of their hiv status, and hence unable to receive needed hiv prevention, treatment and care services. secondly, aids mortality remains a major problem despite expanded art programmes because too many people are receiving hiv testing services after they have already progressed to aids. deaths related to hiv/aids increased from 5544 in 2007 to 9748 in 2008 and 12 287 in 2009. 23, 41, 42 the majority of reported deaths occurred among people who had progressed to aids by the time they were tested for hiv, and hence many missed the opportunity to use life-saving art. thirdly, many people remain unwilling to be tested for hiv. after china launched its mass screening campaign in 2004-05, it was presumed that most of people infected with hiv via plasma donation or transfusion were already identified. however, there have been between 1136 and 1614 hiv cases and 2295-3003 aids cases newly identified and reported each year between 2007 and 2009. 23, 41, 42 fourthly, important gaps remain in the implementation of national policies at the provincial and sub-provincial levels. some local governments do not fully implement national hiv/aids policies. 37, 38 interventions among high-risk groups often lack sufficient coverage, depth and frequency to have an impact on the course of the epidemic. fifthly, in some places, the rate of consistent condom use is low among sex workers and their clients. sixthly, coverage of pmtct services and art programmes remain too low, and the quality of services needs to be improved. ii10 international journal of epidemiology seventhly, the involvement of civil society needs to be increased. non-governmental organizations alone lack the necessary capacity and experience to respond to the hiv/aids epidemic, but they can play a vital role in the hiv response. enterprises and individuals have often limited understanding of the importance of hiv/aids and are not fully motivated to participate in hiv/aids activities. new policies are needed to achieve the goals of universal access and respond to the changing dynamics of china's hiv epidemic. new policy areas of special emphasis in china's new 5-year action plan should include reducing stigma and discrimination, encouraging greater civil society participation, hiv routine testing, partner notification, management of opportunistic infections and co-infections with tuberculosis and hepatitis, and treatment of the mobile population. recent data have shown that hiv transmission is gradually shifting away from being caused by injecting drug use and more by sexual contact. in 2007, the proportion of estimated hiv infections associated with sexual transmission exceeded that of injecting drug use. 43 hiv prevalence among men who have sex with men is increasing rapidly. between 2005 and 2009, the proportion of hiv infections associated with male-to-male sexual transmission increased from 0.4 to 8%, representing a 20-fold increase. to address these challenges, the strategies and policies for hiv/aids response at the grass-roots level are being explored and strengthened in several ways. policies and mechanisms that encourage grass-roots governmental department support are being promoted. the participation of grass-roots social and civil society groups is encouraged. improvements are being made on the social security system, grass-roots health-service system, and basic health-care service provision and drug supply. 40,43 plans and models of hiv/aids interventions that are appropriate to local communities are being developed. accessibility and coverage of testing, interventions, treatment, pmtct, care and support and other services are being enhanced. community-based models with multisectoral accountability are being established. sustainable hiv/aids services are being promoted. developing evidence-based decision making processes and policies and strategies is vital to the success of china's future hiv response. as the epidemic shifts towards being caused increased sexual transmission, china will continue to develop and improve its information-driven policy response to hiv/aids. empirically based scientific information will dictate which policies will be effective and sufficient to turn the tide of the hiv epidemic. greater emphasis may need to be placed on communitybased and multisectoral involvement as a comprehensive response to the hiv/aids epidemic. sound policy decisions will be enacted as china works to ensure universal access to hiv/aids prevention, treatment, care and support services. conflicts of interest: none declared. landmark national-level aids policies, e.g., 'four frees and one care', have facilitated massive scaling up of prevention, treatment and care services over the past decade in china. china's current national policies are increasingly information driven and responsive to changes in the epidemic. gaps remain in policy implementation, and new policies are needed to meet emerging challenges. rules for implementation of the law of the people's republic of china on control of the entry and exit of aliens. beijing: the state council of china rules for implementation of the law of the people's republic of china on control of the entry and exit of aliens three ones'-principles for the coordination of national aids responses report on strengthening surveillance to prevent imported cases of hiv/aids. beijing: ministry of health of china ministry of health of china. notice on banning import of blood products such as factor viii. beijing: ministry of health of china ministry of health of china. notice on strengthening hiv/ aids management. beijing: ministry of health of china identification of hiv infection among drug users in china cohort study of hiv infection among drug users in ruili, longchuan and luxi of yunnan province, china ministry of health of china, ministry of public security of china. notice on providing compulsory testing and treatment of sexual transmitted diseases for sex workers and their clients. beijing: ministry of health of china the hiv/aids epidemic in china: history, current strategies and future challenges hiv-1 infection in commercial plasma donors in china prevalence of hiv infection among former commercial plasma donors in rural eastern china epidemiological research cases in china. beijing: people's medical publishing house state development planning commission, ministry of science and technology, ministry of finance. china's medium and long-term plan on prevention and control of hiv/aids. beijing: chinese ministry of health working duty in hiv/aids prevention and control for related ministries, committees, administrations and social groups china's action plan for reducing and preventing the spread of hiv/aids (2001 -2005). beijing: state council office state council of the people's republic china. notice on strengthening aids prevention, treatment and care programs the evolution of china's response to hiv/ aids regulations on aids prevention and treatment. beijing: state council office action plan on hiv/aids prevention and containment ministry of health of china, ministry of public security of china, ministry of justice of china. notice on hiv screening of inmates of prisons and detoxification centers. beijing: ministry of health of china beijing: ministry of health of china annual report of hiv/aids/std prevention, treatment and care in china in state education commission of china, ministry of health of china. notice on performing hiv test for foreign students. beijing: state education commission of china, ministry of health of china ministry of health of china, ministry of public security of china. provisions concerning the provision of health certificate by foreigners seeking entry. beijing: ministry of health of china ministry of health of china, ministry of foreign affairs of china, ministry of public security of china, et al. several provisions concerning hiv surveillance and management. beijing: ministry of health development of a unified web-based national hiv/aids information system in china doctor did not know blood donation law causing 19 patients infected with hiv in bei-an, heilongjiang rapid scale up of harm reduction in china effectiveness of first eight methadone maintenance treatment clinics in china scaling up the national methadone maintenance treatment program in china: achievements and challenges ministry of health of the people's republic of china public health. hiv testing in china five-year outcomes of the china national free antiretroviral treatment program china cipra project 2 team. understanding hiv-related stigma and discrimination in a 'blameless' population announcement on establishing high risk behavioral intervention team among all level of centers for disease control and prevention in china scaling up prevention programs to reduce the sexual transmission of hiv in china the first law suit to aids discrimination in china ii12 international journal of epidemiology 40 ministry of health of china, unaids, who. the estimation of hiv/aids in china in 2009 annual report of hiv/aids/std prevention, treatment and care in china in annual report of hiv/aids/std prevention, treatment and care in china in state council aids working committee office, un theme group on aids in china. a joint assessment of hiv/aids prevention, treatment and care in china key: cord-330698-9t24jo8s authors: wurdinger, thomas; gatson, natosha n.; balaj, leonora; kaur, balveen; breakefield, xandra o.; pegtel, d. michiel title: extracellular vesicles and their convergence with viral pathways date: 2012-07-25 journal: adv virol doi: 10.1155/2012/767694 sha: doc_id: 330698 cord_uid: 9t24jo8s extracellular vesicles (microvesicles), such as exosomes and shed microvesicles, contain a variety of molecules including proteins, lipids, and nucleic acids. microvesicles appear mostly to originate from multivesicular bodies or to bud from the plasma membrane. here, we review the convergence of microvesicle biogenesis and aspects of viral assembly and release pathways. herpesviruses and retroviruses, amongst others, recruit several elements from the microvesicle biogenesis pathways for functional virus release. in addition, noninfectious pleiotropic virus-like vesicles can be released, containing viral and cellular components. we highlight the heterogeneity of microvesicle function during viral infection, addressing microvesicles that can either block or enhance infection, or cause immune dysregulation through bystander action in the immune system. finally, endogenous retrovirus and retrotransposon elements deposited in our genomes millions of years ago can be released from cells within microvesicles, suggestive of a viral origin of the microvesicle system or perhaps of an evolutionary conserved system of virus-vesicle codependence. more research is needed to further elucidate the complex function of the various microvesicles produced during viral infection, possibly revealing new therapeutic intervention strategies. a wide variety of vesicles are actively released from living cells into the extracellular space with their contents reflecting the cellular composition and physiologic state (for review see [1] [2] [3] ). over the years, the different types of extracellular vesicles have been given a variety of names, including exosomes, shed microvesicles, ectosomes, microparticles, virosomes, viruslike particles, and oncosomes. the distinguishing features of each of the vesicle subtypes and the correct nomenclature are currently under intense study. here, we will refer to them under the general term, microvesicles. microvesicles carry rna [mrna, microrna (mirna), and noncoding sequences], cdna and genomic sequences, and a large component of proteins and lipids (see reviews above, as well as [4, 5] ). upon release these microvesicles can move within the extracellular space and are either taken up by neighboring cells or degraded. they can also enter adjoining bodily fluids, such as the systemic circulation and travel to distant sites. in fact, they have been found in abundance in blood (serum and plasma), urine, breast milk, sweat, saliva, ascites fluid, and cerebral spinal fluid (csf) [3] [4] [5] [6] [7] . at least two distinct release mechanisms for microvesicles have been described for two subtypes: (1) exosomes-derived from the multivesicular body (mvb) and (2) shed microvesicles-derived from the plasma membrane. interestingly, both mechanisms have considerable overlap with virus release and biogenesis (summarized in figure 1 and further discussed below). exosomes range from 30 to 100 nm in diameter and are generated by inward budding of the lumen of internal vesicular compartments derived from endosomes [8] . as vesicles accumulate within these endosome-derived compartments, they are referred to collectively as mvbs. these mvbs can either be targeted for degradation through the lysosomal pathway, or they can fuse with the plasma membrane releasing their interior vesicles into the extracellular space. the exact mechanism and kinetics of these fusion and release events are not fully elucidated and may vary among different cell types [9] . for example, depletion of hrs (an escrt-0 component) led to a decrease in exosome secretion in dendritic cells that were stimulated to release with ovalbumin and a calcium ionophore [10] . oligodendrocytes on the other hand seem to secrete exosomes by a mechanism that is escrt independent and ceramide dependent [11] . exosome release by hela cells has been found to involve rab27a/b [12] , and p53 is reported to play a role in exosome release in a nonsmall cell lung cancer cell line [13] . rab11 has also been shown to be involved in the release of exosomes from mvbs by acting in the tethering/docking of mvbs to the plasma membrane to promote homotypic fusion, in the presence of calcium [14] . in addition, tbc1d10a-c, a rab35 inhibitor, led to intracellular accumulation of endosomal vesicles and impaired exosome secretion [15] . shed microvesicles are released by outward budding directly from the plasma membrane and tend to be larger (>100 nm in diameter) and more heterogeneous in size [16, 17] . moreover, this release process is likely controlled by localized cytoskeleton dynamics, with small cytoplasmic membrane-covered protrusions detaching and being released into the extracellular space [18] by an activated gtpase, arf6 [19] . interestingly, recent observations indicate that virus-independent budding from the plasma membrane can be mediated by endosome to plasma membrane relocation of tsg101, a prominent member of the escrt-i complex, frequently noted as an exosome marker [20] . this type of budding is topologically identical to both the inward budding of the limiting membrane of mvbs and viral assembly at the plasma membrane, in that the outer surface of the plasma membrane is on the outer surface of the microvesicle. in fact, certain tumor cells shed retrovirallike vesicles, which can be abundant because of increased transcription of endogenous retroviral sequences [17, 21] , resulting from overall hypomethylation of the genome [22] . in general it seems that the clear distinctions between viruses and microvesicles based on composition and function are fading although they can be separated from vesicles released during the later stages of programmed cell death since these latter vesicles, referred to as apoptotic blebs [2] , are even larger in size [23] . the role of microvesicles in intercellular communication is currently receiving much attention. upon release from the donor cell, the microvesicles can either be taken up by neighboring cells or travel through bodily fluids for cargo delivery into recipient cells at distant sites. although many details are missing, cellular uptake of some microvesicles appears to depend, at least in part, on specific ligand-receptor recognition [24] , and can be mediated by direct fusion of the microvesicles with the plasma membrane or by endocytotic uptake of the microvesicles. for example, quah et al. [25] have shown that bystander naïve b cells are rapidly activated by acquiring the antigen from activated b cells through microvesicle-mediated membrane transfer. in a similar way cd41 is transferred from platelets to endothelial and tumor cells, resulting in increased proadhesive properties of the recipient cells [26, 27] . microvesicles also shuttle mrna between cells and influence the physiological state of the recipient cell, as well as the cellular response to external stress stimuli [28] . in addition, mirnas are transferred by exosomes [6, 29, 30] . for instance, mir-146a was shown to be transferred into recipient prostate cancer cells leading to the inhibition of their proliferation [31] , and recently mirnas which can modulate the immune response were detected in exosomes in breast milk [32] . furthermore, retrotransposon sequences are particularly enriched in tumor microvesicles, and tumor-derived human endogenous retroviral (herv) sequences can be transferred to normal human umbilical vein endothelial cells (huvecs) via microvesicles resulting in a prolonged increase in herv-k mrna levels [17] . this suggests that tumor cells transfer these mobile genetic elements via microvesicles to neighboring normal cells thereby modulating their genotype and phenotype. virology 3 microvesicular shedding of cellular membrane components and the release of internal endosomal-derived exosomes are important for cellular communication and modulation of immune responses [9, [54] [55] [56] [57] (table 1) . while release of microvesicles has been extensively investigated, recently the challenge has been to uncover the specific mechanisms that guide protein sorting and complexing into shed microvesicles and exosomes in various cell types. cells have been reported to secrete highly specified microvesicles after infectious exposure or under various cell activation conditions [5, 54, 56, 58] . through the packaging and transfer of functional proteins, mrna/mirna, and other cytosolic components, microvesicles have been found to be beneficial either to the host cell or to the infectious agent [37, 43] . virus-infected cells proved useful in early studies to elucidate the role of microvesicular shedding in intercellular communication [55, 56] . amongst the most extensively studied viruses with respect to microvesicles are herpes simplex virus (hsv), human immunodeficiency virus (hiv), and the tumorigenic herpes virus, epstein-barr virus (ebv). each virus possesses unique properties that afford protection from immune attack. here, we outline the important immune modulatory steps involved in virus-induced microvesicle sorting and release in these and other related viruses. preservation of the virus depends on microvesicle release of infected cells. microvesicles released by infected cells contain specific components of the cell and the virus, many of which facilitate the ability of virions to persist in a hostile antiviral immune environment [44, 55, 56, 58] . depending on the virus type, and, in some cases, the stage in the viral cycle, intercellular processes are well orchestrated to produce specific cellular and immune outcomes [56] : (1) evading the host immune system, (2) invasion, (3) replication, and (4) persistence (summarized in part in figure 2 and further discussed below). during primary viral infection, humoral and cell-mediated host immune responses such as production of neutralizing antibodies and cytotoxic t-cell attack on infected cells are employed to contribute to viral destruction. early evasion strategies adopted by viruses interfere with complete elimination of the virus, allowing it to persist. during hsv-1 infection the release of microvesicles, formerly known as l-particles containing viral tegument proteins and glycoproteins, can prime surrounding cells for productive infection and reduce immune rejection [48] [49] [50] . such virus-like vesicles lack both the viral capsid and dna and are thereby incapable of producing a replication-infective cycle in the cells on their own [49] [50] [51] . however, some of the viral tegument proteins contained within them are immediate early transcription factors that can produce rapid transcriptional activation of later arriving intact virions [48, 52] . another evasion strategy observed for hsv-1 is targeting of the mhcii molecule processing pathway by viral envelope glycoprotein b (gb) [37] . antigen-presenting cells (apcs) routinely sort the mhcii surface receptor hla-dr to mhcii compartments for processing. the primary role of this pathway is to present [37, [48] [49] [50] [51] [52] [53] peptide antigens to the immune system in order to elicit or suppress t-(helper) cell responses that stimulate b-cell production of antigen-specific antibodies [37] . hsv-1 gb couples with hla-dr, causing sorting through the exosome pathway as opposed to presentation on the cell surface. complexing of gb-dr effectively hijacks the cellular antigen presenting machinery, preventing further peptide loading and, in addition, increasing microvesicle production [37, 53] . this final step releases additional gb-dr complexes into the host immune microenvironment, promoting resistance of viruses to immune attack, and in some cases producing bystander t-cell tolergenicity or anergy [37, 53] . in the case of hiv, microvesicle packaging and spread of the virusencoded nef protein impairs proper endocytosis of the immature mhcii/invariant chain, antibody class switching, and lysosomal degradation of viral peptides allowing hiv virus to evade immune recognition [37, 38] . ebv, human cytomegalovirus (cmv) and hepatitis c virus (hcv) have also found means to evade immune responses by exploiting microvesicles, as discussed below. exosomes and shed microvesicles can both incorporate elements from the cell, as well as from the intruding virion [54] . upon circulation of these microvesicles, they encounter and enter susceptible cells and can sensitize them to viral infection thus increasing systemic spread of the virus to naïve cells. in the case of the human cmv, microvesicles released by infected cells present the c-type lectin family molecule expressed on dendritic cells-used in capture and internalization of pathogens-in complex with the cmv glycoprotein b. this complex can be subsequently distributed to other cells by microvesicles, thereby increasing the susceptibility of these cells to cmv [59] . a similar mechanism is found in the case of hcv. in hcv-positive patients, the cellular membrane protein cd81 associates with one of the hcv envelope glycoproteins, e2. extracellular release of the e2-cd81 complexes within microvesicles allows for increased virus-fusing ability and infectivity of previously naïve cells [60] . microvesicles bearing the e2-cd81 complex and containing hcv rna are of notable importance as they have been reported to be infectious even in the presence of neutralizing antibodies [60] . interestingly, hcv has been shown to release three phenotypically distinct types of microvesicles having variable infectivity from high to low [60] . however, differential release of these microvesicles during hcv pathogenesis remains to be elucidated. infection. conversely, microvesicular release can contribute to viral attack by the host immune system. for example, in early invasion steps of cmv, cmv antigens are transferred from infected epithelial cells (ecs) via ec-derived microvesicles to apcs [43] . these apcs are not detected as infected cells but are rendered more susceptible to infection with subsequent encounters with the virus [43] . while this is a primary infectious viral invasion and replication strategy, inadvertently transferred apcs bearing cmv antigens in transplanted organs serve as markers to the host immune systems to target nonself tissue. harboring of these susceptible apcs by the immune-compromised host and continued microvesicular shedding increases t-cell surveillance and influx into the grafted tissues, thereby exacerbating allograft rejection [43] . microvesicles can also promote the innate immune response to viruses, for example, as observed for hiv whereby transfer of a particular antiviral cytidine deaminase via exosomes inhibits hiv replication [61] . in addition, virus-like vesicles can be used as a vaccination strategy, and recently chimeric virus-like vesicles were engineered using a mixture of coronavirus and influenza proteins functioning as a potential severe acute respiratory syndrome (sars) virus vaccine [62] . viruses can use various microvesicle transport mechanisms as a survival strategy, while in other cases the host immune system can utilize microvesicles for cell signaling and host protection. microvesicles can directly activate or suppress cellular responses, induce or facilitate infection, and transfer material to improve or hinder host immune recognition [9] . these same strategies can be exploited in the development of virus-based therapies. oncolytic viruses armed with therapeutic genes are currently being evaluated for safety and efficacy for cancer therapy [63] [64] [65] . it would be of interest to determine whether microvesicles can alter the efficacy of oncolytic viruses, and other types of viral gene delivery vectors. recent work shows that microvesicles can be loaded with adenoassociated viral (aavs) vectors for more efficient gene delivery [66] , opening a new window into the microvesicle therapeutics field. several human pathogenic viruses are known for their ability to lie dormant in the host immune system, of which hsv and ebv are perhaps the best known examples. in the case of hsv this is due to the ability of the virus to enter a latent state in the nucleus of sensory neurons during which it expresses no viral antigens and does not disturb the physiology of the neurons. in latency a single transcript is generated which encodes a precursor for four distinct hsv, mirnas which act to suppress virus replication [67] . for human herpesvirus 4 (hhv4), better known as ebv, this is largely due to incomplete eradication of the virus after early primary infection. gamma herpesviruses, including ebv, have developed a variety of strategies to exploit host-cell regulatory pathways that lead to a permanent infection of their host. when these pathways are deregulated, what is usually an undamaging herpes infection can predispose to diseaseincluding encephalitis, autoimmunity, and cancer [68] . it was recently demonstrated that ebv exploits the endosomalexosomal pathway by balancing intracellular signaling in infected b cells [69] and controlling epigenetic changes in uninfected neighboring cells via microvesicles [30] . enveloped viruses of the herpes virus family, such as human cmv (hcmv/hhv5) and ebv, depend on the interaction with cellular endosomal membrane systems for replication [70] . interestingly, mature hhv-6 virions are released together with internal vesicles through mvbs by the cellular endosomal-exosomal pathway [71] . thus, many herpesviruses generally seem to exploit endosomal pathways and microvesicles for virus production, release, and immune evasion. however, the finding that viruses such as ebv modulate host-cellular pathways that are not directly involved in virus production needs further investigation. being the first human tumor virus identified, ebv is in many aspects an extraordinarily benign pathogen and is best known as the causative agent of "kissing disease" or infectious mononucleosis. it is estimated that over 90% of the world population is persistently infected with ebv. the ebv life cycle begins by exchange through saliva and ebv virions that seem to preferentially infect naïve resting b cells in secondary lymphoid organs, such as the tonsils. occasionally isolated epithelial cells also become infected and presumably sustain lytic replication [72] , which is required for viral shedding into the saliva for transmission to new hosts [73] . to reach its near universal prevalence without harming the host, ebv and related persistent herpesviruses have evolved complex strategies encouraging immune recognition in proliferative (potentially oncogenic) stages of its life cycle, while elegantly avoiding the immune recognition at other stages by "going into hiding" [74] . upon initial infection at the mantle zone of germinal centers (gcs), the newly infected naïve b cells undergo multiple differentiation stages and tight interactions with surrounding stroma and t cells [75] . interestingly, ebv facilitates these essential interactions for the maturation of b cells, for instance, by upregulation of crucial gc reaction-associated proteins, such as gp183 [76] . this integral part of the ebv life cycle (i.e., mimicking a gc-type reaction) requires tight growth regulation in a specific ebv latency gene expression program (latency iii) and promotes rapid growth and proliferation of these infected cells through nfκb activation. this strategy in expanding the infected pool of b cells without the need for lytic replication may be advantageous under normal conditions but raises the chances of turning-on malignant growth if the viral latency programs are not properly controlled. indeed, if these cells do not progress further into memory cells by shutting down this growth program, they can remain in the proliferative phase and give rise to ebvpositive lymphomas which can kill the host, thus, restricting further viral propagation and spread [77] . in addition, ebv infection at this stage may also predispose to autoimmunity as inappropriate survival signals may interfere with negative selection of self-reactive b cells. of note, immune-suppressed individuals are at increased risk for developing ebv-driven lymphomas, reflecting the importance of a lifelong potent anti-ebv t-cell response [78] . the ability of ebv to persist despite such vigorous t-cell responses indicates that ebv can escape from the adaptive immune system and may do so in part by exploiting the endosomal-exosomal pathway through the secretion of t-cell inhibitory exosomes [44] [45] [46] . when secreted by ebv-positive tumors, these exosomes carry immune-evasive proteins including the viral protein lmp1 [79] and high amounts of galectin 9 that cause massive apoptosis of ebv-specific cd4+ t-cells via specific interaction with t-cell immunoglobulin mucin-3 (tim-3), which can negatively regulate th1 t cell and macrophage activation. the inhibition of anti-ebv immune responses is believed to promote the progression of ebv-positive malignancies, such as hodgkin's disease (hd) [46] and nasopharyngeal carcinoma (npc) [80] . vallhov et al. [81] studied the interaction between exosomes secreted by ebv-driven lymphoblastoid cell lines (lcls) and peripheral blood b cells proliferating in vitro. lcls are 95% latent, but a small proportion of cells is in a lytic stage. exosome-cell interactions could be inhibited by specific antibodies against gp350 the major envelope protein of ebv or cd21 on b cells, indicating an interaction between cd21 on b cells and the gp350 on exosomes [81] . these specific exosome-cell interactions may be exploited for exosome-based anticancer therapies, for example, in delivering the cd154 protein to leukemic b blast cells rendering them immunogenic to t cells [82] . in addition to proteins, it is now clear that microvesicles from many cell types carry and transport functional rna molecules. ebv was the first virus discovered to encode its own small regulatory mirnas [83] . ebv encodes a staggering 44 viral mirna species, derived from two major gene clusters on the viral genome, which have an important role in ebv persistence [84] . next generation sequencing indicates that these ebv-encoded mirnas make up a large fraction (20-25%) of the total cellular mirna in ebv-infected cells, encompassing 300+ different mirna species [85] . similar results were found in the mirna profile of exosomes from ebv-driven lcl cells (pegtel et al., unpublished results) . this is consistent with the idea that viral mirnas manipulate gene regulation in host cellular pathways and also exploit the exosomal mirna communication pathways. indeed, the discovery of ebv-encoded regulatory mir-nas (ebv-mirnas) residing within the lumen of exosomes indicated a novel mechanism by which exosomes can exert inhibitory effects, namely, by translational repression of target genes in noninfected recipient cells via exosomal ebv mirnas [30] . earlier studies in mice had suggested that intact exosomes from ebv-infected cells had strong physiological effects in vivo, consistent with the idea that the luminal content of exosomes is biologically significant, apart from the proteins and lipids that make up their surface [86] . subsequent studies demonstrated that ebv-infected cancer ecs also secrete ebv-mirnas, presumably within exosomes [87] . due to the lack of an accurate in vivo model for human ebv infection it is difficult to investigate the mechanism controlling release of ebv-mirnas through exosomes and to determine whether this contributes to viral persistence in healthy infected individuals. however, ebv-encoded mir-nas are transported from infected b cells to noninfected (ebv-dna negative) t cells and monocytes, supporting the idea of horizontal mirna transfer in humans. thus, viral mirnas in exosomes may contribute to sustain persistent virus infection by delivery of such mirnas into noninfected responding t cells leading to their inactivation (anergy) [45] or destruction [44] . this is consistent with recent data suggesting that exosomes efficiently transport mirnas through the immunological synapse during interactions of t cells with apcs [47] , similar to what is known concerning antigen exchange [88] . studies are underway to establish whether ebv exploits these specialized intercellular contacts for efficient posttranscriptional control in neighboring responding immune cells as a possible mechanism for immune escape. hiv [56, [89] [90] [91] has been a discussion topic in the microvesicle field for many years. not only has it been hypothesized that hiv itself may have microvesicle features, but microvesicles also have been described to have immune modulatory functions on hiv-infected cells and to expand the infectivity of hiv. in 2003 gould et al. [92] postulated that hiv-an enveloped retrovirus-hijacks the microvesicle system to benefit its own assembly and subsequent exit. interestingly, 6 advances in virology inhibitors were identified that blocked the budding of both shed microvesicles and hiv particles [93] . in addition, peptides were identified that prevented interactions of hiv nef protein-a key protein in the hiv life cyclewith mortalin, a cellular heat shock protein, and resulted in inhibition of the release of hiv and nef-containing microvesicles [94] . careful analysis, however, has indicated that although hiv exploits certain proteins that also play a role in exosome formation via the mvb [95] , hiv assembly does not necessarily use the same logistics system as do exosomes. importantly, it has been established that hiv budding occurs mostly at the plasma membrane and not from within the mvb [96] [97] [98] [99] . interestingly, hiv recruits members of the mvb escrt complex for proper hiv budding from the plasma membrane [98] [99] [100] [101] [102] . while in cd4+ t cells hiv release appears to be independent of exosomes [103] , in monocyte-derived macrophages hiv can bud into endosomes [102, 104] . however, several studies highlight that hiv-1 budding also in macrophages occurs primary at the plasma membrane [105] [106] [107] . thus, the controversy about the site of productive virus assembly in macrophages mostly favors the plasma membrane. hiv release in dendritic cells may be triggered by signals similar to those for exosome release [102, 108, 109] , and secretion of hiv from endocytic compartments in dendritic cells can result in hiv release upon interaction with t cells [110, 111] . however, these endocytic compartments were also described to be connected with the extracellular space [112, 113] and suggested to be invaginated domains distinct from classical endocytic vesicles [114] . moreover, microvesicle release from t cells treated with ceramide inhibitors was not affected by such treatment [111] , as previously reported for hiv-1 [115] . however, both viruses and microvesicles produced from ceramide-deficient cells failed to be captured by mature dendritic cells [111] . therefore, more research is warranted on the specific sites of hiv assembly in particular cell types, and to what extent the endosomal compartments play a role in the hiv life cycle, as well as the possible convergence of hiv and shed microvesicle pathways. it seems likely that hiv has simply adapted to use certain host factors for different exit modalities, and that these may vary among different types of cells, as well as under different conditions. it will be of continuing interest to further study the retroviral family, including the endogenous retroviruses, in order to determine whether the microvesicle cargo systems are perhaps a remnant of previous retroviral infections that happened earlier in evolution-and elements of which are now used in an opportunistic setting by retroviruses, such as hiv [56, [89] [90] [91] 102] . this overlap in pathways and the consequence of using overlapping machinery for release can result in phenotypic similarities between microvesicles and retroviruses and potentially interfere with anti-hiv strategies. for instance, hiv released from t cells has similar glycome properties as t-cell microvesicles, arguing for a common origin and indicating phenotypic similarity [116] . more research in the convergence of microvesicle and hiv pathways may improve our understanding of these processes and propel the development of new antiviral drugs directed against hiv. the role of microvesicles during hiv infection has not yet been extensively studied, but they appear to be involved in both hiv infectivity enhancement and resistance depending on the cells of origin. microvesicles derived from hivinfected cells have been reported to contain hiv ccr5 coreceptors, allowing for enhanced hiv infection of other cells [34] . moreover, microvesicles from megakaryocytes and platelets contain cxcr4 and upon transference confer susceptibility to cells normally resistant to hiv infection [35, 117] . in addition, during hiv replication the hiv nef protein can alter the exosomal pathway by increasing the number of intracellular vesicles and mvbs [118] [119] [120] [121] . hiv nef-induced microvesicle release from infected and noninfected cells [39, 40] can induce apoptosis in cd4+ t cells [41] and convey resistance to hiv infection [61] . the transfer of nef or other viral components through microvesicles may represent an important mechanism for immune evasion by viruses. in addition, exosomes can contain apobec3g, a cytidine deaminase that is part of the cellular antiviral system against retroviruses, which upon transference to recipient cells via exosomes can inhibit hiv replication [61] . while cd45, cd86, and mhc class ii molecules have been found in microvesicles from hivinfected cells [42] , possibly serving to silence the immune response, microvesicles derived from cd8+ t cells can act to suppress hiv replication [33] . moreover, exosomes in association with hiv derived from dendritic cells significantly enhance hiv infection of cd4+ t cells [36] . in conclusion, microvesicles from hiv-infected cells as well as from noninfected cells play an important role in hiv replication and dissemination. therefore, interference with microvesicle-mediated signaling could possibly be harnessed to halt hiv infection. retrotransposon elements such as line, alu, and human endogenous retroviruses (hervs) make up about 45% of the human genome and have played an important role in genome evolution [122] . these viral-like elements infected germ cells in the human genome millions of years ago and then became a stable part of the inherited genetic material. although most line elements are inactive, a number of active ones remain and are able to "jump" to new locations in the genome, contributing to genomic instability [123] . these events can have important effects on our genome, for example, by inactivating genes, altering gene expression and facilitating random insertion of new cdna copies in the genome, as in integration of pseudogenes [124] . many tumor cells also release retroviral-like microvesicles that contain active retrotransposon sequences, such as herv-k [125] . recently, tumor-derived microvesicles have been shown to be enriched in retrotransposon elements such as line1, alu, and herv-k [17] . furthermore, herv-k was transferred through microvesicles to normal huvecs, which then showed an increase in herv-k levels 12 hours following exposure to tumor microvesicles. in addition, the mouse retroviral rna vl30 is packaged in retrovirus vectors by mouse packaging cell lines and transferred to human advances in virology 7 cells infected with those vectors [126] . the mouse vl30 has several stop codons in the regions encoding for genes such as gag, pol and env, thereby inhibiting its ability to encode functional proteins [126] . however, transfer of the vl30 mrna together with tissue factor (tf) to human melanoma cells served to induce their metastatic potential. this change in phenotype apparently occurs through formation of a complex with the protein-associated splicing factor (psf) protein which represses transcription of an insulin-like growth factor-1 (igf-1) inducible gene, with dissociation of this complex allowing transcription to proceed [126] . three of the 11 human genes affected by vl30 mrna were oncogenic, suggesting that the transfer of retroviral rna sequences can have catastrophic effects on recipient cells. song et al. [126] have identified human retrotransposon sequences that are >90% identical to the mouse vl-30 suggesting human vl-30 transferred through microvesicles could have similar effects on transcription [126] . long interspersed elements (lines)-most notably l1-comprise about 17% of the human genome. several studies indicate that a subset of l1 elements is still actively expanding in the number of sequences within the human genome by retrotransposition. this active subpopulation, termed transcriptionally active (ta), is approximately 2 million years old, and it has high levels of insertional polymorphism in the human population [127, 128] . some of these new insertions may be intolerable and lethal and therefore eliminated; others may result in phenotypically tolerable disease, such as in coffin-lowry syndrome and choroideremia [129] [130] [131] , while still others have been associated with the induction of cancer, for example, lung cancer [132] . the high level of polymorphism of l1 elements indicates that they continue to have profound effects on the human genome, and recent evidence suggests that microvesicles may be a potential route of delivery for these elements [17] . this microvesicle-mediated trojan horse-like [92] transferance of transposons could perhaps allow for a stealthy dissemination of retrotransposons, especially in a tumor setting, avoiding immune-recognition, and achieving "long distance" delivery. hervs also entered the human genome millions of years ago and comprise about 8% of the human genome. they consist of gag, pol, and env sequences, flanked by two long terminal repeats [133] . most of these sequences are now silent because of acquired mutations and deletions over the course of evolution, but herv-k113 can produce intact, albeit noninfectious, retroviral particles [134] . some of these sequences are still transcriptionally active and are associated with diseases, such as lymphoma and breast cancer [21, 135] . in cancer, hypomethylation of the genome seems to predominantly affect retrotransposon sequences (perhaps because they are highly abundant in the human genome), allowing increased transcription, especially in the case of the most recent entrants, which also happen to be the elements with the most intact coding potential [136] . indeed retroviral-like microvesicles have been found in cancer patients, notably those with lymphomas [21] , breast cancer [137] , and teratomas [138] . as expected, these patients also had high levels of reverse transcriptase, and viral gag and env proteins and rna in the tumor cells and retrovirus-like microvesicles released from them into the circulation [21] . tumor microvesicles from cultured tumor cells also have been shown to be enriched in retrotransposon rna, dna, and reverse transcriptase, suggesting that a subpopulation of these microvesicles may indeed be of retroviral origin [19] . in summary, this review deals with how extracellular vesicles-such as exosomes and shed microvesicles-share pathways with the assembly and release of retrotransposon elements and viruses. in figure 1 we summarize how herpesviruses such as ebv and hsv, originate from the nucleus and can merge with microvesicle pathways. several proteins used for exosome production are used by herpesviruses for functional release. also, the convergence of these pathways may explain the observations of virus-like particles, which can be exosomes or shed microvesicles containing viral proteins or nucleic acids. similar observations have been made for retroviruses and retrotransposon elements with circulating microvesicles containing retrotransposon rna found in some cancer patients. it remains to be investigated to what extent exosomes and shed microvesicles are remnants of previous retroviral colonization. in this review we note the observations of retroviral as well as retrotransposon elements in microvesicles, perhaps allowing further dissemination of such nucleic acid sequences. the use of microvesicle pathway elements by viruses such as hiv may be suggestive of an intricate coevolution of different endogenous and exogenous (retro)virus subtypes. viruses not only use microvesicle pathways for their own assembly and release but are also capable of exploiting the highly complex microvesicle communication system in an intercellular setting as simplified in figure 2 . during viral infection microvesicles can have various effects on different types of cells, either limiting viral infection or enhancing it. thus, a picture is emerging that viruses and microvesicles are codependent pleiotropic entities. more research is needed into the differential functions of different subtypes of microvesicles and their cross-talk in relation to the immune response and outcome of viral infection. shedding microvesicles: artefacts no more exosomes: extracellular organelles important in intercellular communication exosomes-vesicular carriers for intercellular communication brain tumor microvesicles: insights into intercellular communication in the nervous system the multifaceted exosome: biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities glioblastoma microvesicles transport rna and proteins that promote tumour growth and provide diagnostic biomarkers human saliva, plasma and breast milk exosomes contain rna: uptake by macrophages molecular characterization of dendritic cell-derived exosomes: selective accumulation of the heat shock protein hsc73 membrane vesicles as conveyors of immune responses exosome secretion of dendritic cells is regulated by hrs, an escrt-0 protein ceramide triggers budding of exosome vesicles into multivesicular endosomes rab27a and rab27b control different steps of the exosome secretion pathway the regulation of exosome secretion: a novel function of the p53 protein rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner regulation of exosome secretion by rab35 and its gtpase-activating proteins tbc1d10a-c intercellular transfer of the oncogenic receptor egfrviii by microvesicles derived from tumour cells tumour microvesicles contain retrotransposon elements and amplified oncogene sequences mechanisms for the formation of membranous nanostructures in cell-to-cell communication arf6-regulated shedding of tumor cell-derived plasma membrane microvesicles formation and release of arrestin domain-containing protein 1-mediated microvesicles (armms) at plasma membrane by recruitment of tsg101 protein human endogenous retrovirus k (hml-2) elements in the plasma of people with lymphoma and breast cancer hypomethylation of retrotransposable elements correlates with genomic instability in non-small cell lung cancer apoptotic bodies from endothelial cells enhance the number and initiate the differentiation of human endothelial progenitor cells in vitro platelet-derived microvesicles transfer tissue factor to monocytes but not to neutrophils bystander b cells rapidly acquire antigen receptors from activated b cells by membrane transfer modulation of monocyte-endothelial cell interactions by platelet microparticles platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment exosomes communicate protective messages during oxidative stress; possible role of exosomal shuttle rna exosome-mediated transfer of mrnas and micrornas is a novel mechanism of genetic exchange between cells functional delivery of viral mirnas via exosomes unraveling the mystery of cancer by secretory microrna: horizontal microrna transfer between living cells immune-related micrornas are abundant in breast milk exosomes noncytotoxic suppression of human immunodeficiency virus type 1 transcription by exosomes secreted from cd8 + t cells transfer of the chemokine receptor ccr5 between cells by membranederived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection plateletand megakaryocyte-derived microparticles transfer cxcr4 receptor to cxcr4-null cells and make them susceptible to infection by x4-hiv immature dendritic cell-derived exosomes can mediate hiv-1 trans infection the herpes simplex virus-1 encoded glycoprotein b diverts hla-dr into the exosome pathway hiv-1 evades virus-specific igg2 and iga responses by targeting systemic and intestinal b cells via long-range intercellular conduits massive secretion by t cells is caused by hiv nef in infected cells and by nef transfer to bystander cells genetic characterization of hiv type 1 nef-induced vesicle secretion hiv nef is secreted in exosomes and triggers apoptosis in bystander cd4 + t cells differential incorporation of cd45, cd80 (b7-1), cd86 (b7-2), and major histocompatibility complex class i and ii molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation cytomegalovirusinfected human endothelial cells can stimulate allogeneic cd4 + memory t cells by releasing antigenic exosomes blood diffusion and th1-suppressive effects of galectin-9-containing exosomes released by epstein-barr virus-infected nasopharyngeal carcinoma cells localization of the epstein-barr virus protein lmp 1 to exosomes galectin-1 mediated suppression of epstein-barr virus-specific t-cell immunity in classic hodgkin lymphoma unidirectional transfer of microrna-loaded exosomes from t cells to antigen-presenting cells the effect of herpes simplex virus type 1 l-particles on virus entry, replication, and the infectivity of naked herpesvirus dna comprehensive characterization of extracellular herpes simplex virus type 1 virions noninfectious l-particles supply functions which can facilitate infection by hsv-1 assembly of enveloped tegument structures (l particles) can occur independently of virion maturation in herpes simplex virus type 1-infected cells functional roles of the tegument proteins of herpes simplex virus type 1 herpes simplex virus type 1 targets the mhc class ii processing pathway for immune evasion exosomes and other microvesicles in infection biology: organelles with unanticipated phenotypes membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles microvesicles and viral infection b lymphocytes secrete antigen-presenting vesicles exosomal sorting of the cytoplasmic domain of bovine leukemia virus tm env protein pivotal advance: the promotion of soluble dc-sign release by inflammatory signals and its enhancement of cytomegalovirus-mediated cis-infection of myeloid dendritic cells association of hepatitis c virus envelope proteins with exosomes exosomes packaging apobec3g confer human immunodeficiency virus resistance to recipient cells chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix 1 efficiently form viruslike particles (vlps) that protect mice against challenge with sars-cov enhanced antitumor efficacy of vasculostatin (vstat120) expressing oncolytic hsv-1 antitumor efficacy of 34.5enve: a transcriptionally retargeted and vstat120-expressing oncolytic virus oncolytic viruses microvesicleassociated aav vector as a novel gene delivery system micrornas expressed by herpes simplex virus 1 during latent infection regulate viral mrnas viral mirnas exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion lmp1 association with cd63 in endosomes and secretion via exosomes limits constitutive nf-κb activation budding events in herpesvirus morphogenesis human herpesvirus-6 induces mvb formation, and virus egress occurs by an exosomal release pathway epstein-barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers the dynamics of ebv shedding implicate a central role for epithelial cells in amplifying viral output germinal center b cells latently infected with epstein-barr virus proliferate extensively but do not increase in number the intersection of epstein-barr virus with the germinal center ebi2 mediates b cell segregation between the outer and centre follicle persistence of the epstein-barr virus and the origins of associated lymphomas cellular responses to viral infection in humans: lessons from epstein-barr virus direct immunosuppressive effects of ebv-encoded latent membrane protein 1 functional inactivation of ebv-specific t-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy exosomes containing glycoprotein 350 released by ebv-transformed b cells selectively target b cells through cd21 and block ebv infection in vitro ebv-gp350 confers b-cell tropism to tailored exosomes is a neo-antigen in normal and malignant b cells-a new option for the treatment of b-cll identification of virus-encoded micrornas epstein-barr virus micrornas are evolutionarily conserved and differentially expressed the viral and cellular microrna targetome in lymphoblastoid cell lines exosomes derived from il-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis human tumor virus utilizes exosomes for intercellular communication polarized secretion of lysosomes at the b cell synapse couples antigen extraction to processing and presentation exosomes and retroviruses: the chicken or the egg? hiv and mature dendritic cells: trojan exosomes riding the trojan horse? human and nonhuman primate lentiviral infection and autoimmunity the trojan exosome hypothesis identification of an inhibitory budding signal that blocks the release of hiv particles and exosome/microvesicle proteins secretion modification region-derived peptide disrupts hiv-1 nef 's interaction with mortalin and blocks virus and nef exosome release human immunodeficiency virus type 1 gag engages the bro1 domain of alix/aip1 through the nucleocapsid higher-order oligomerization targets plasma membrane proteins and hiv gag to exosomes exosomes and hiv gag bud from endosome-like domains of the t cell plasma membrane visualizing hiv-1 assembly dynamics of escrt protein recruitment during retroviral assembly live-cell visualization of dynamics of hiv budding site interactions with an escrt component the cell biology of hiv-1 virion genesis endosomes, exosomes and trojan viruses hiv-1 is budded from cd4 + t lymphocytes independently of exosomes evidence that hiv budding in primary macrophages occurs through the exosome release pathway plasma membrane is the site of productive hiv-1 particle assembly hiv-1 buds predominantly at the plasma membrane of primary human macrophages hiv-1 assembly in macrophages in macrophages, hiv-1 assembles into an intracellular plasma membrane domain containing the tetraspanins cd81, cd9, and cd53 lysosome-related organelles: a view from immunity and pigmentation dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection capture and transfer of hiv-1 particles by mature dendritic cells converges with the exosome-dissemination pathway in vitro derived dendritic cells trans-infect cd4 t cells primarily with surface-bound hiv-1 virions the achilles heel of the trojan horse model of hiv-1 trans-infection hiv traffics through a specialized, surface-accessible intracellular compartment during trans-infection of t cells by mature dendritic cells the hiv lipidome: a raft with an unusual composition hiv-1 and microvesicles from t cells share a common glycome, arguing for a common origin enhanced activation of platelets with abnormal release of rantes in human immunodeficiency virus type 1 infection human immunodeficiency virus-1 nef expression induces intracellular accumulation of multivesicular bodies and major histocompatibility complex class ii complexes: potential role of phosphatidylinositol 3-kinase interactions between nef and aip1 proliferate multivesicular bodies and facilitate egress of hiv-1 nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of hiv-1 infectivity nef proteins encoded by human and simian immunodeficiency viruses induce the accumulation of endosomes and lysosomes in human t cells retrotransposons revisited: the restraint and rehabilitation of parasites great exaptations human line retrotransposons generate processed pseudogenes human endogenous retrovirus family herv-k(hml-2) rna transcripts are selectively packaged into retroviral particles produced by the human germ cell tumor line tera-1 and originate mainly from a provirus on chromosome 22q11.21 binding of mouse vl30 retrotransposon rna to psf protein induces genes repressed by psf: effects on steroidogenesis and oncogenesis l1 (line-1) retrotransposon evolution and amplification in recent human history identification and characterization of novel polymorphic line-1 insertions through comparison of two human genome sequence assemblies intronic l1 insertion and f268s, novel mutations in rps6ka3 (rsk2) causing coffin-lowry syndrome novel types of mutation in the choroideremia (chm) gene: a full-length l1 insertion and an intronic mutation advances in virology activating a cryptic exon retrotransposable elements and human disease natural mutagenesis of human genomes by endogenous retrotransposons endogenous retroviruses in the human genome sequence human endogenous retrovirus herv-k113 is capable of producing intact viral particles transcription of herv-e and herv-erelated sequences in malignant and non-malignant human haematopoietic cells cpg methylation directly regulates transcriptional activity of the human endogenous retrovirus family herv-k(hml retrovirus-like particles released from the human breast cancer cell line t47-d display type b-and c-related endogenous retroviral sequences induction of retrovirus particles in human testicular tumor (tera-1) cell cultures: an electron microscopic study the authors thank suzanne mcdavitt for help with the editorial process. t. wurdinger is financially supported by nwo-vidi, d. m. pegtel by nwo-veni, x. o. breakefield by nih/nci grants ca069246 and ca141150, and l. balaj by the huygens scholarship nl. key: cord-287348-00yaxpkp authors: martinez, maria jose abad; olmo, luis miguel bedoya del; benito, paulina bermejo title: antiviral activities of polysaccharides from natural sources date: 2005-12-31 journal: studies in natural products chemistry doi: 10.1016/s1572-5995(05)80038-9 sha: doc_id: 287348 cord_uid: 00yaxpkp abstract the ever increasing resistance of human pathogens to current anti-infective agents is a serious medical problem, leading to the need to develop novel antibiotic prototype molecules. in the case of viruses, the search for antiviral agents involves additional difficulties, particularly due to the nature of the infectious viral agents. thus, many compounds that may cause the death of viruses are also very likely to injure the host cell that harbours them. natural products are increasingly appreciated as leads for drug discovery and development. screening studies have been carried out in order to find antiviral agents from natural sources, and the occurrence of antiviral activity in extracts of plants, marine organisms and fungi is frequent. the evidence indicates that there may be numerous potentially useful antiviral phytochemicals in nature, waiting to be evaluated and exploited. in addition, other plants, not previously utilized medicinally, may also reveal antivirals. among natural antiviral agents, recent investigations have reconsidered the interest of phyto-polysaccharides, which act as potent inhibitors of different viruses. this chapter will illustrate a variety of antiviral polysaccharides from natural sources since 1990, with the aim of making this matter more accessible to drug development infectious diseases account for one-third of all deaths worldwide. although the last decade has yielded significant advances in the treatment of infectious diseases, new therapies for viral, fungal, bacterial and parasitic infections are needed [1] . viruses are responsible for a large proportion of the morbidity and mortahty experienced worldwide. these infectious agents consist of a core genome of nucleic acid, dna or rna (nucleoid) contained in a protein shell (capsid) and sometimes surrounded by a lipoprotein membrane (envelope). some genera of viruses are known to cause human infections and include the following: adeno, pox and herpesviruses: herpes simplex virus type 1 and 2, varicella-zoster, cytomegalovirus, epstein-barr virus; papillomaviruses (papillomas, cancer); parvoviruses (erythema infectiosum); dna-viruses (e.g., hepatitis b); (+/-) rna viruses: reo-and rotavirus; (-) rna viruses: influenza, parainfluenza, measles, respiratory syncytial, vesicular stomatitis, rabies virus and togaviruses (rubella, yellow fever); retroviruses (e.g., human immunodeficiency virus, the causative agent of acquired immunodeficiency syndrome); and other several rna viruses such as arenaviruses (lymphocytic choriomeningitis), bunyaviruses (encephalitis), coronaviruses (upper respiratory infections) and picornaviruses (poliomyelitis, diarrhoea). most successful anti-infective agents inhibit essential steps in metabolic processes required by the pathogen but absent in the host. hov^ever, viruses cannot replicate independently. they must enter cells and use the cellular energy-generating, dna-or rnareplicating and protein-synthesizing pathways of the host to achieve viral replication. for this reason, in the case of viruses the search for antiviral agents is very hard since metabolic differences are not available: viruses utilize the host's protein synthesis leaving only some aspects of nucleic acid synthesis and macromolecule processing distinct from the host's metabolism. the identification of a retrovirus, human immunodeficiency virus, as the causative agent of acquired immunodeficiency syndrome, the steadily increasing incidence of various viral infections caused by viruses in immunodeficient patients, e,g., herpes simplex virus, varicella-zoster virus, cytomegalovirus and epstein-barr virus, the increasing evidence for the pathogenic role of a number of viruses, e.g., human papilloma virus in the pathogenesis of primary hepatocellular carcinoma, and the socioeconomic impact of viral infections of the respiratory (influenza, adeno-, corona-and rhinoviruses) and gastrointestinal tract (rotaviruses) have all been important factors in boosting the search for new antiviral agents and new modalities of antiviral chemotherapy. although many compounds with potent antiviral activity in cell cultures and in experimental animals have been detected, at present only several molecules and a-interferon have been approved by the health authorities for therapy of viral infections in humans. among these antiviral substances are several natural compounds isolated from plants used in traditional medicine, including polysaccharides [2] [3] [4] . polysaccharides (ps) constitute a stmcturally diverse class of biological macromolecules with a wide range of physicochemical properties, which are the basis for different applications in the broad field of pharmacy and medicine [5] [6] [7] [8] , several hundred natural ps are currently known and provide one of the richest and oldest reservoirs of structurally and functionally diverse biopolymers. ps constitute important members of the family of industrial water-soluble polymers. besides the classical applications of these biopolymers in industry and pharmaceutical practice, the relatively new field of pharmacologically active polymers will be discussed. ps from plants and other natural sources have long been known to exert antitumour, immunomodulatory, anticoagulant and other types of biological activity, including antiviral activity. the importance of the plant kingdom as a source of new antiviral substances will be illustrated by presenting a survey on natural-derived antiviral ps. in recent years, natural and synthetic polymers of a carbohydrate nature were proved to be selective inhibitors of several enveloped viruses, including such human pathogens as human immunodeficiency virus, herpes simplex virus, human cytomegalovirus, influenza virus and respiratory syncytial virus. recent efforts have been directed at the development of polysaccharidic molecules as selective inhibitors of different classes of viruses [9, 10] . in this search, the following molecular targets were envisaged: for dna viruses in general, the viral dna polymerase; for herpes simplex virus and varicella-zoster virus, the viral dna polymerase via a specific phosphorylation by the viral 2'deoxythymidine kinase; for (+/-) rna and (-) rna viruses, sadenosylhomocysteine hydrolase, a key enzyme in transmethylation reactions required for the maturation of viral mrna; for retroviruses, reverse transcriptase as an initiator of virus replication and/or cell transformation; and for several enveloped viruses (e.g., retro-, herpes-and rhabdoviruses), virus adsorption to the outer cell membrane. this review deals with antiviral ps compiled from several sources during the past decade. under the respective titles, we offer some brief descriptions of the most important global viral infections that are caused either by individual viruses or by groups of related viruses. they are restricted to those generally considered important in human and veterinary medicine and about which reports on active antiviral ps are recorded in the literature. acquired immunodeficiency syndrome (aids) is a pandemic immunosuppressive disease which results in life-threatening opportunistic infections and malignancies. in 1996, the number of aids infections was 23 million and 1.5 million people (half of them in africa) were killed by this disease worldwide. in order to combat human immunodeficiency virus (hiv), the causative agent of aids, enormous amounts of time and money have been dedicated to research into compounds which can be developed as therapeutic agents. hiv is a lentivirus which replicates within critical cells of the immune system, particularly cd4 t-cells and monocyte/macrophages, leading to a progressive loss of helper t-cells and profound immunosuppression. this condition is known as aids. without treatment, the cd4 t-lymphocytes of an infected patient are reduced markedly so that the patient is susceptible to a wide range of opportunistic infections, and ultimately dies. since this retrovirus, designated hiv, has been clearly identified as the primary cause of this disease, numerous compounds, also including plant-derived substances, have been evaluated for their inhibitory effects on hiv replication in vitro. furthermore, many plant products are being used by patients with aids in some countries without any scientific proof that they possess anti-hiv activity. several reviews on natural products for the chemotherapy of hiv infections have been recorded in the literature [11] [12] [13] [14] [15] [16] [17] [18] [19] . carbohydrates were among the natural molecules capable of treating hiv infection. various ps, including sulphated ps, have been found to be anti-hiv active substances of many antivirally active plant extracts, all of which are medicinal plants used in traditional medicine as anti-infectives. for example, a ps (mar-10) was isolated from the aqueous extract of the plant hyssop officinalis l., which was found to inhibit hiv-1 replication as demonstrated by the inhibition of hiv-1 p24 antigen and syncytia formation [20] . from aspalathos linearis burm., a popular medicinal plant in south africa, europe and japan, a ps with strong anti-hiv activity was isolated [21, 22] . this compound almost completely inhibited the binding of hiv-1 to mt-4 cells. the major sugar components of this purified ps were found to be glucose, galactose, mannose and xylose. premanathan et al [23, 24] investigated the ps extracted from rizophora apiculata blume and rizophora mucronata poir. in cell culture systems for their activity against human and simian immunodeficiency viruses (siv), both compounds inhibited hiv-1, hiv-2 or siv strains in various cell cultures and assay systems. it was found that these ps consisted mainly of neutral sugars and uronic acids. in a screening programe of thai plants for anti-hiv activity, it was found that 114 extracts of 81 species belonging to 32 families showed activity [25] . of the effective samples, the extracts of merremia peltata (l.) merrill were the most active. most of the anti-hiv agents were found to be anionic ps. furthermore, sulphated derivatives of paramylon, a (1-3)-p-d-glucan from euglena gracilis klebs. significantly inhibited the cytopathic effect of hiv-1 and hiv-2 [26] , while two naturally occurring ps isolated from the tropical climbing shrub ipomoea cairica (l.) sweet were found to strongly inhibit replication of hiv-1 in vitro [27] . according to de clercq [28] , the replicative cycle of hiv comprises several steps that could be considered adequate targets for chemotherapeutical intervention: virus entry, viral adsorption, virus-cell fusion, reverse (rna -> dna) transcription, proviral dna integration, viral (dna -> e^a) transcription (transactivation), viral (mrna -> protein) translation, virus release, viral assembly, budding and maturation. the most notable difference between hiv replication and other retroviruses is the intricate control of hiv gene expression by viral and cellular factors [29] . possible mechanisms by which hiv kills infected cells include the formulation of multinucleate syncytia, cytopathic components within the virions themselves, and interaction between viral envelope proteins and the cd4 molecule on the cell surface. three hiv enzymes are essential to the life cycle of the virus: hiv reverse transcriptase is crucial for viral replication; hiv protease processes viral polyproteins into fonctional enzymes and structural proteins, thereby facilitating maturation and infectivity of the virion particles; hiv integrase mediates hiv integration into the host chromosome. the chemotherapeutic strategies have therefore been focused on the development of inhibitors of these retroviral enzymes. recently, it has been reported that biological membranes arising from hiv-induced cell fusion, as well as syncytium formation between infected and non-infected cells are interesting approaches in research for a new hiv therapy [30] . natural sulphated ps such as dextran sulphate, pentosan sulphate, chondroitin sulphate and heparin have long been recognized as effective anti-hiv agents [31] [32] [33] [34] [35] . these compounds target the interaction between the viral envelope glycoprotein gpl20 and the cd4 receptor and, as a consequence, they inhibit not only vims adsorption to the cells but also virus-induced syncytium (giant cells) formation [36] [37] [38] [39] [40] [41] [42] . the inhibitory effect of dextran sulphate and its congeners on viral binding, viral replication and syncytium formation appears to be mediated by a specific interaction with the v3 region of gpl20. in addition, sulphated ps may also directly interfere with the binding of hiv particles to the heparin sulphate proteoglycan of the cell surface, whether or not this process occurs independently of, or cooperatively with, the viral envelope cd4 receptor interaction. this class of antiviral agents exhibits several unique properties that are not shared by other currently known antiviral agents: remarkable broadspectrum antiviral activity against hiv-1, hiv-2 and a series of other enveloped viruses; the ability to inhibit syncytium formation between hiv-infected and normal cd4 t-lymphocytes, a mechanism that drastically enhances hiv infectivity and a low induction of viral drugresistance [43] . it should be noted that sulphated ps owe their anti-hiv activity to the presence of the sulphate groups, which in turn are responsible for the inhibition of virus-cell binding. dextran is a highmolecular-weight ps consisting predominantly of a (1 -> 6) linked dglucose units. dextrans can differ in chain length and in degree of branching. the branching occurs via a (1 ^3) and a (1 -> 4) branch points. dextran fractions of different average molecular weight were obtained by partial hydrolysis. these different fractions were sulphated to obtain dextran sulphates of different molecular weight. busso and resnick [44] investigated the effects of three molecular weight ranges of dextran sulphate on five different hiv isolates. the higher molecular weight range of dextran sulphate showed the most potent activity as assessed by a quantitative syncytium formation assay. however, dextran sulphate is poorly adsorbed after oral administration and upon intravenous administration produces thrombocytopenia, so that there is little, if any, evidence for the in vivo efficacy of this compound and more generally of sulphated ps against hiv infection [45] . however, double-blind placebo-controlled clinical pilot trials in advanced hiv disease are being conducted with these compounds [46] . in summary, these compounds offer great promise as candidate drugs for the chemotherapy of hiv infections, and novel sulphated ps which have proved to be potent and selective inhibitors of hiv in vitro are being chemically synthesized [47] [48] [49] [50] [51] [52] . on the other hand, although several groups have reported that sulphated ps are potent and selective inhibitors of hiv, their therapeutic application is however limited by their anticoagulant activity. in view of possible improvements in therapeutic potential, a number of heparin derivatives with reduced anticoagulant activity were studied for their inhibitory activity in an hiv-dependent syncytium formation assay [53] . the most pronounced anti-hiv activity was observed with 30% succinylated standard heparin and 100% succinylated low molecular weight heparin. similarly, and in order to increase the ratio of anti-hiv activity to anticoagulant activity, selectively substituted sulphated ps with oh and/or cooh groups were chemically prepared [54, 55] . this group comprises a family of heterogeneous carbohydrates composed of long, unbranched ps modified, for example, by sulphations and acetylations. the acetylated derivatives are more active as inhibitors of hiv-induced giant-cell formation. furthermore, several soluble derivatized dextrans with different percentages of carbomethyl, benzylamide and sulphonate/sulphate groups were also evaluated for possible inhibitory effects on hiv-1 infection, and from the results obtained, their use as anti-hiv therapeutic agents can be proposed [56] . other natural sources of anti-hiv sulphated ps include algae and other marine organisms. sulphated ps have been found in a variety of marine animals, plants and microorganisms [57, 58] . aqueous extracts of many marine invertebrates have exhibited some activity in the national cancer institute's primary screen for anti-hiv cytopathicity [59] . carrageenans and fucoidan are sulphated ps extracted from red seaweed and brown algae, which have shown potent inhibitory activity against different viruses including hiv. for example, fucoidan, a complex sulphated ps from the alga fucus vesiculosus l. was found to inhibit hiv in vitro, presumably through a direct interaction of the ps with the hiv binding site of the target cells [60] . the aqueous-soluble extracts from this alga, a common littoral alga of the coast of the northern atlantic, the pacific ocean and the baltic sea, inhibited the activity of hiv reverse transcriptase enzyme as well as hiv-induced syncytium formation. beress et al [61] reported a new procedure for the isolation of the anti-hiv ps from this marine alga. a novel sulphated ps named calcium spirulan has been isolated as an antiviral principle from spirulina platensis (nordst.) geitl., a blue-green alga growing in some african and central and south american lakes rich in salts [62] [63] [64] [65] . chemical analyses of calcium spirulan suggest that it contains rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid and galacturonic acid. other sulphated ps from marine sources have demonstrated antiviral activities against hiv, blocking viral replication in cell culture without any toxicity to the host cells. examples include sulphated cell-wall ps from the mediterranean red alga asparagopsis armata harvey [66] , from the marine microalga cochlodinium polykrikoides blooms [67] , fiicose, the main component sugar of the ps isolated from the brown alga sargasum horneri (turner) c. agardh [68] and sargasum muticum (yendo) fensholt. [69] , sulphated ps from brazilian marine algae such as laminaria abyssalis ab joly & ec oliveira [70] , and from tropical marine algae of the codiaceae family [71] , and new sulphated p-galactans from the marine clam species meretrix petechialis lamarck [72] . other natural sources of anti-hiv sulphated ps include fiingi such as ganoderma lucidum (fr.) karst. [73] and lentinus edodes (lem.) mycelia [74] . inhibition of hiv-reverse transcriptases (hiv-rt) is currently considered a usefiil approach in the prophylaxis and intervention of aids and natural products have not as yet been extensively explored as inhibitors of this enzyme [75, 76] . furthermore, the mechanisms of action of sulphated ps may not only be due to the inhibition of virus cell adhesion, but also to the inhibition of hiv-rt [13, 15] . herpes infections are ubiquitous. approximately, 16-35%, 40-80% and >90% of the population are sero-positive for, or infected by herpes simplex virus type i (hsv-1), herpes simplex virus type ii (hsv-2) and varicella-zoster virus, respectively. more alarmingly, over the past decade, the incidence and severity of infections caused by hsv have increased due to the growth in number of immunocompromised patients produced by aggressive chemotherapy regimens, expanded organ transplantation and a greater occurrence of hiv infections. unfortunately, prolonged therapies with acyclovir, the most successful antiherpetic drug, have resulted in some undesirable complications and also induced the emergence of drug-resistant viruses. with this change in disease pattern, and the increase in drug use frequency, acyclovir-resistant hsv infections have emerged. infections with hsv range from simple cold sores and fever blisters to severe central nervous system disorders. development of effective antiviral medications has made prompt recognition important in primary care practice. appropiate therapy can significantly reduce both the medical and psychosocial ramifications of herpes infections and can greatly improve the quality of life of many patients [77] . thus, there is an urgent need for novel anti-hsv agents, especially those with a different mode of action from acyclovir. for the past decades, in addition to a variety of synthetic antiviral drugs with different molecular targets, a large number of phytochemicals have been recognized to control infections caused by viruses belonging to the herpesviridae family. ethnopharmacological screenings of medicinal plants from all over the world have led to the selection of several extracts which are active against herpes viruses. among natural antiviral agents, recent investigations have revived the interest in phyto-ps, which act as potent inhibitors of different enveloped viruses, including members of herpesviridae. for example, a neutral ps extracted from sclerotium glucanicum halleck., namely scleroglucan, revealed promising antiviral activity [78] . these ps are composed of a p-1,3-linked glucopyranose backbone with single p-l,6-linked glucopyranosyl branches every third subunit. the blockage on hsv infection takes place during the very early phases of the viral multiplication cycle, since the greatest inhibitory effect occurred when it was added during the attachment step. as a result of the intensive search for antiviral substances from medicinal plants, antiviral activity against hsv was found in extracts from cedrela tubiflora bertoni. leaves [79] , from prunella vulgaris l., a perennial plant commonly found in china, the british isles and europe [80] and from trichilia glabra l. leaves [81] . phytochemical studies indicate that these plants contain anionic ps as active constituents which may inhibit hsv by competing for cell receptors as well as by some unknown mechanisms after the virus has penetrated the cells. furthermore, the in vitro antiviral activity demonstrated by extracts of the medicinal plant achyrocline flaccida (weinm.) d.c. on hsv-1 is exerted early during viral replication, essentially during viral adsorption to host cells [82] . a bioguided purification process indicated that negatively charged ps were responsible for this antiviral activity. among natural antiviral agents of a carbohydrate nature, recent investigations have also revived interest in sulphated phyto-ps such as carrageenans and fucoidan from seaweed. sea algae represent a very interesting source of potential antiviral compounds, particularly the watersoluble sulphated ps which are abundant constituents of the cell wall. these compounds act as potent inhibitors of different enveloped viruses, including members of herpesviridae, and their activity is linked to the anionic features of the molecule which hinder the attachment of viral particles to host cells. for example, carrageenans are sulphated galactans that can be extracted from certain red seaweeds. they comprise a broad range of structures and can be divided into two families: the k-family, defined by the presence of a c4-sulphate group on the p-d-unit, and the x-family, characterized by the sulphate on c2 and constituted by all the variations of the ^.-structures [83] . investigations have detected antiviral activity in a significant number of algae from various marine environments such as california, united kingdom, the mediterranean, india and japan [84] . in the course of a screening study on the biological properties of natural marine products, diverse carrageenans isolated from the red seaweed gigartina skottsbergii setch. et gard. have proved to be potent inhibitors of hsv-1 and -2 in vitro [85] . mode of action studies are consistent with the carrageenans having a major effect on the attachment of virus to the cells. adsorption of hsv-1 is primarily mediated by the envelope glycoprotein gc, which binds to heparan sulphate residues present on the proteoglycans on the surface of target cells. antiherpetic activity was directly correlated to the amount of a-d-galactose 2,6-disulphate residues in the natural carrageenans. carrageenans were also isolated from chilean samples of stenogramme interrupta (c. ag.) mont., a red seaweed from california [86] . in the course of screening for antiviral properties in sulphated ps isolated from marine algae of the south american coast, carlucci et ah [87] reported the antiviral activity of mannans and xylogalactans isolated from the red seaweed nothogenia fastigiata turner (lam.), and a sulphated galactan from pterocladia capillacea (gmelim) barnet & thuset. hsv-1 and -2 were the most sensitive viruses of these compounds due to an inhibition of virus binding. structural analysis of two xylomannans extracted from nothogenia fastigiata was carried out [88, 89] . the results are consistent with the general pattern previously reported for other xylomannans of the same system, a(l -> 3)-linked dmannans 2-and 6-sulphated and with single stubs of p(l -> 2)-linked dxylose, but one of the new samples contains a significant amount of 2,6disulphated units. the presence of sulphate groups in the molecule was essential to the antiviral properties of these ps [90] . the antiviral activity of sulphated ps is known to increase with molecular weight or the degree of sulphation. however, sulphated ps are generally endowed with anticoagulant properties that may hamper their usefulness as antiviral drugs. duarte et al [91] reported the inhibitory effect of sulphated galactans from the marine alga bostrychia montagriei harvey. the results of the activated partial thromboplastin time assay and the thrombine time assay indicated that three natural sulphated ps have very low anticoagulant activity, confirming that there is no relation between the antiviral and anticoagulant properties. anti-hsv sulphated ps have also been isolated from other seaweeds such as rhamnan sulphate from monostroma latissimum wittrock [92] , and calcium spirulan from the blue-green alga spirulina platensis [62] . lee et al. [93] initiated studies into the structure-activity relationships of calcium spirulan. calcium ion binding with the anionic part of the molecule was replaced with various metal cations. replacement of the calcium ion with sodium and potassium ions maintained anti-hsv-1 activity while divalent and trivalent cations reduced the activity. the cell wall sulphated ps of the red microalga porphyridium spp. honey exhibited impressive antiviral activity against hsv-1 and -2 both in vitro (cell culture) and in vivo (rats and rabbits) [94] . it seems that this ps is able to inhibit viral infection by preventing adsorption of the virus into the host cells and/or by inhibiting the production of new viral particles inside the host cells [95] . novel fiican sulphates with anti-hsv activity were also isolated from the hot water extract of an edible brown alga sargassum horneri [96] and from the brown seaweed leathesia difformis (linnaeus) areschong [97] . other natural sources of anti-hsv ps include fimgi, e.g., various protein bound ps isolated from ganoderma lucidum, a basidiomycetous fungus used to treat human diseases in oriental folk medicine [98] . the possible mode of antiviral activity of these ps seems to be related to their binding with hsv-specific glycoproteins responsible for attachment and penetration, impeding the complex interactions of viruses with cell plasma membranes [99] [100] [101] . furthermore, different semisynthetic ps were evaluated for their inhibitory effect on in vitro replication of hsv-1 and -2 [102] [103] [104] . some neutral and negatively charged carbohydrates were able to inhibit viral infection by interfering mainly with the adsorption process. the in vitro and in vivo effects of sulphated ps were also investigated against the pseudorabies virus (suid hsv-1), the most notable of which is porcine hsv which has a well-documented history of epizootic infections, especially in europe [105] . in vitro experiments revealed that sulphated ps significantly reduced the number of lytic plaques, but in vivo only heparin protected mice against the pseudorabies virus. in terms of their biological and pathogenetic properties, the herpes viruses fall naturally into several subfamily groupings, including cytomegalovirus (cmv), although detailed classification is at present premature. nevertheless the cmv clearly constitute a group of their own with internal consistency. human cmv is, together with hsv-1, one of the agents responsible for opportunistic infections in hiv-infected people. as can be seen in the present review, sulphated ps show antiviral activitiy against enveloped viruses in vitro. it has been suggested that these antiviral mechanisms depend on their polyanionic properties, which interact with the positively charged amino acid sequence of viral envelope glycoproteins. natural sulphated ps can act as potent inhibitors of cmv. the cell wall mucilages of seaweeds are known to be rich in sulphated ps with potential anti-cmv activity. recently, blinkova^/a/. [106] reviewed information on spiridina platensis, a blue-green alga with diverse biological activity. preparations obtained from this alga have been found to be active against several enveloped viruses, including cmv. it was revealed that calcium spirulan, a sulphated ps isolated from spirulina platensis, selectively inhibited the penetration of the virus into host cells [62] . retention of molecular conformation by chelation of calcium ion with sulphate groups may be indispensable to its antiviral effect. the antiviral activity of a polysaccharidic fraction obtained from another red seaweed, nothogenia fastigiata, was also reported [107] . its mode of action against cmv could be ascribed to an inhibitory effect on virus adsorption. other natural sources of sulphated ps include brown algae. a sulphated polysaccharide with potent anti-cmv activity was isolated from the brown alga sargassum horneri [68] . time-of-addition experiments suggested that it inhibited not only the initial stages of viral infection, such as attachment to and penetration into host cells, but also later replication stages after virus penetration. fractions of fiicoidan, another polysaccharide isolated from the brown seaweed leathesia difformis were also found to be selective antiviral agents against human cmv [97] . these compounds offer great promise as candidate drugs for the chemotherapy of cmv infections, and novel sulphated ps are being chemically prepared [108] . ps from terrestrial plants have also been reported as anti-cmv agents. for example, ps from three plant species. astragalus brachycentrus d. c, astragalus echidnaeformis sirjaev and sterculia urens roxb., which are devoid of in vitro antiviral activity, were evaluated in mouse models of murine cmv infections [109] . treatment with the compounds needed to be started one day prior to virus inoculation for maximum protective benefit. treatments starting after virus inoculation were ineffective. the mannose-specific plant ps from the orchid species cymbidium hybrid cym., epipactis helleborine (l.) crantz. and listera ovata (l.) r.br. svenska are potent and selective inhibitors of human cmv in vitro [110] . they presumably interact at the level of virion fusion with the target cell. papillomaviruses are members of the papopavirus family and are distinguished from other members by virtue of their association with a variety of warts in different parts of the body. some types have been implicated in genital warts and cervical carcinomas, while others seem to be associated with other distinctive warts elsewhere. no generally effective control is available, although potentially dangerous lesions can be removed by cryosurgery or laser treatment. however, some medicinal plant preparations have been reported to be beneficial; conceivably these may work by promoting healing or stimulating immune responses, rather than directly inhibiting the virus. natural high-molecular weight sulphated or sulphonated ps, such as cellulose sulphate and dextran sulphate, may be useftil non-toxic microbicidal compounds that are active against a variety of sexuallytransmitted disease agents, including bovine papillomavirus type 1 and human papilloma virus type 11 and type 40 [111] . hepatitis b virus (hbv) is widespread throughout human populations, specially in asia and africa, and it has been estimated that over 200 million carriers exist, some of whom are eventually expected to develop liver carcinoma or cirrhosis. hbv shows a strict tropism for liver hepatocytes in which it displays a protected replication with resultant foci of liver necrosis. the virus is a member of the hepadnaviridae, along with several other species, and it replicates by a mechanism which appears to be unique to this family. in contrast, hepatitis a virus is a picornavirus and the hepatitis d agent appears to be a viroid-like rna enclosed within a hepatitis b capsid, and consequently depends upon its association with the hbv for its spread and survival. control may be effected by passive immunization (with hyperimmune globulin) or by various types of vaccines which are currently being developed and improved. specific chemotherapy has not been consistently successful, but in some countries (e.g., india and china), plant extracts have provided some success. among natural antiviral agents of carbohydrate nature, sulphated ps such as x and k carrageenans showed a potent inhibitory effect on the replication of hbv in the human hepatoma cell line plc/prf/5 [112] . the two types of carrageenans resulted in concentration-dependent reduction of hbv-antigen expression and hbv infectivity, emerging as promising candidates for chemotherapy of acute hepatitis. human rotavirus (hrv), a member of the reoviridae, is a non-enveloped virus which is the major etiologic agent of severe dehydrating gastroenteritis in children worldwide. for the treatment of rotavirus gastroenteritis, intravenous fluid administration has been used successfully for dehydration from diarrhoea. however, in the case of severe inpatients and immunocompromised hosts who are suffering from prolonged diarrhoea and fever, virus-specific treatment will be necessary if possible. several compounds, biomaterials and plant extracts have been found to be inhibitory for hrv of some species in vitro. for example, extracts of stevia rebaudiana bertoni, a family member of chrysanthemum originating from paraguay in south america, inhibited the replication of all four serotypes of hrv in vitro [113] . binding assays with radiolabeled purified viruses indicated that the inhibitory mechanism of this plant extracts is the blockage of virus binding. the inhibitory components of stevia rebaudiana were found to be heterogeneous anionic ps with different ion charges. influenza continues to have a significant impact on public health. annually, 20,000 deaths and 100,000 hospitalizations are attributed to "flu" in the united states alone. airborne transmission, facile viral mutation, vaccine shortages, and actual and perceived side effects and limitations of both vaccines and prophylactic drugs contribute to the drive for new therapies and preventive medicines for influenza. research during the last fifteen years has elucidated many of the mechanisms by which the influenza virus invades, captures and mobilizes the replication capabilities of a host cell, providing new targets in the search for antiviral treatments. for the past decades, besides a variety of synthetic antiviral drugs with different molecular targets, a large number of phytochemicals have been recognized to control infections caused by the influenza virus. serkedjieva et al [114] reported the antiviral activity from a lyophilized infusion from flowers of sambucus nigra l., aerial parts of hypericum perforatum l. and roots of saponaria officinalis l. the preparations contain ps which could be responsible for their antiviral properties. reports on the anti-influenza virus activity of natural ps are recorded in the literature. from a pine cone extract of pinus parviflora sieb. et zucc, an acidic polysaccharide was isolated [115, 116] . this compound showed significant inhibition of both the viral protein synthesis in infected cells and virion-associated rna-dependent rna polymerase activity. in animal models in vivo, most mice inoculated intranasally or intracerebrally with lethal doses of the influenza virus a/wsn/33 died within 12 days. however, the infectivity of the virus that had been preincubated with a polysaccharide prepared from cones of pinus parviflora was significantly reduced [117] . in 1991, sakagami et a/. [118] reviewed the potential antiviral efficacy of natural polysaccharidic-related materials isolated from the pine cone extracts. sulphated ps, potent antiviral agents, were also evaluated in vitro as inhibitors of influenza virus replication [119] . the fact that the sulphated ps are inhibitory to some myxoviruses and retroviruses but not to others seems to depend on the composition of the amino acid sequences of the viral envelope glycoproteins that are involved in virus-cell binding and fiision [120] . as can be seen in the present review, other natural sources of antiviral sulphated ps include the marine environment. sulphated and nonsulphated ps with anti-influenza activity have been isolated from marine sources such as the green marine alga ulva lactuca l. [121] , the japanese sea alga crenomytilus grayanus dunkei. [122, 123] , the blue-green alga spirulina platensis [106] , the red seaweed nothogenia fastigiata [107] and the marine microalga cochlodinium polykrikoides [67] . novel marine ps which have proved to be potent and selective inhibitors of the influenza virus in vitro are being chemically synthesized [124, 125] . the measles virus (mv) is a paramyxovirus which has been associated with several chronic diseases. since the advent of mass immunization campaigns, the incidence of measles has decreased dramatically in many developed countries. this is not the case elsewhere, however, where the virus still takes its toll in large numbers, especially in malnourished or immunocompromised individuals. secondary bacterial infections are prevalent in these populations. although most infections are relatively short-lived and innocuous in healthy individuals, encephalitis can develop in approximately 0.1% of cases; hence vaccination is desirable. following respiratory infection, the virus readily invades local lymphoid tissues and gains access to the blood, from where it disseminates widely in the body. there has not been much demand for chemotherapy; vaccination seems to be the choice. no really effective antiviral has been evaluated, although some plant compounds have been shown to inhibit mv replication effectively, such as ps from the blue-green alga spirulina platensis [62] . rabies has long been recognized as a scourge of livestock with occasional and often fatal intrusions into humans and their pets. initially the virus gains access to muscle, sensory organs or skin, and there replicates in local unmyelinated (sensory) nerve fibers. many layers of epithelial cells are susceptible to the virus but the principal target is the unprotected neuron. the brain is where most damage is done, resulting in the familiar psychomotor disturbances. in addition, the virus spreads into salivary glands (by axoplasmic flow again), from where it is secreted from the asinar mucous cells and transmitted to the victim. the objective of much research on the rabies virus has been the development of better vaccines and therapeutic measures, some of them from natural sources. for example, different natural polymeric carbohydrates inhibited rabies virus infection in chicken-embryo-related cells by interfering with the virus adsorption process [126] . the charge density and the polymeric backbone of the molecules seem to play a role in influencing the antiviral properties, whereas other features such as the sugar moieties do not appear to be relevant. rubella is the sole member of the rudivirus genus in the togavirus family. the virus is transmitted by the respiratory route, and multiplies in upper respiratory tissues from where it gains access to the blood, accompanied sometimes by a characteristic rash and other symptoms. the infection may be more severe in pregnant women. vaccination programmes are in place in many countries, and the belief is that the virus will eventually be brought under control. although there seems to be no apparent role or need for specific antiviral therapy, reports on the antirubella activity of natural products and extracts have been reported [57] . for example, the natural carbohydrate scleroglucan, three sulphate derivatives and dextran sulphate were investigated for their inhibitory effect on rubella virus infection on vero cells [127] . the results indicated that ps blocked a step in virus replication subsequent to virus attachment, such as internalisation and/or uncoating. bunyavirus sandfly fever sicilian virus (sfsv) is a phlebovirus, a member of the bunyaviridae family, which cause acute, incapaciting but self-limiting diseases in man, and which have had a major impact in europe, the middle east and africa. sfsv is a virus closely related to other viruses which are considerably more pathogenic both in humans and animals, such as the african swine fever virus (asfv) and the crimean-congo haemorrhagic fever virus (cchfv). the asfv is a highly contagious bunyavirus infection of pigs which is clinically indistinguishable from the unrelated hog cholera virus. since 1957, epidemics have been recorded intermittently in several european countries bordering the mediterranean, in the caribbean, and in brazil, and more recently in other european countries and parts of africa. although pigs appear to be the only species infected naturally, the virus can be adapted to grow in a variety of other cell types and experimentally infects goats and rabbits. in view of this, it seems worthwhile to consider the possibility of its natural persistence in animals other than swine and man. transmission of asfv usually occurs through the respiratory route. following inhalation, the virus invades and attacks local lymph nodes and the endothelium of blood vessels, with the resuh that high titers of virus circulate in the blood and lymph, and eventually in various secretory fluids. attempts have been made to produce vaccines, but without much success. in any case, the virus mutates frequently in the wild and crossprotection of animals is not complete. since the virion contains several enzyme activities, and the dna can probably code for more in the infected cell, the possibility of specific chemotherapy, along the lines of antiherpes chemicals, should be profitable. several studies have been undertaken to develop drugs which could be used for treatment of fever infections caused by viruses belonging to the bunyaviridae family, including natural ps [128, 129] . antiviral activity was estimated by the reduction of the cytophatic effect of sfsv on infected vero cells. several ps, such as carrageenans, fucoidan, dextran sulphate and pentosan polysulphate caused a concentration-dependent reduction in the virus yield. fabregas et al [130] screened several extracts from marine microalgae for in vitro inhibition of the replication of asfv. that this inhibition could be due to sulphated ps was suggested because the same pattern of viral inhibition was obtained by using exocellular extracts from microalgae enriched in these compounds and/or dextran sulphate of high molecular weight. other natural ps should be considered for the potential treatment of significant human infections caused by bunyaviruses. the mouse model of punta toro virus, a phlebovirus member of the bunyaviridae family, is a model for studying the treatment of rift valley fever infection. tragacanthin ps from astragalus brachycentrus and astragalus echidnaeformis plants caused reduction in mortality, liver infection scores, liver and spleen virus titers, and serum transaminases in treated mice [131] . poliomyelitis is not the scourge that it once was, thanks to improved hygienic practices which have effectively blocked the spread of wild-type polioviruses, especially in developed countries, and thanks to the widespread use of vaccines. the viruses, like other enteroviruses, are transmitted by the fecal-oral route; following ingestion, they replicate in various cell types in the pharynx and small intestine (they are quite stable to stomach acids) and gain access to the circulation. from there, they disseminate and occasionally gain access to the central nervous system, where they may produce the characteristic lesions leading to poliomyelitis. control of virus spread accompanied by vaccination continues to provide the best means of alleviating poliomyelitis. the use of antivirals seems to have less application than for many other viral infections, although the poliovirus continues to serve as a useful laboratory model for antiviral screening. the plant kingdom and seaweeds may be a source of potentially useful and interesting antiviral compounds against polioviruses. for example, anti-poliovirus activity was detected in a polysaccharidic-rich fraction from extracts of several korean seaweeds [132] , and from the leaves of the meliaceae tree trichilia glabra [81] . other compounds of carbohydrate nature with antiviral activity against other viruses have been isolated from natural sources. several compounds belonging to different classes of sulphated ps were evaluated for their inhibitory effects on the replication of the arenavirus junin and tacaribe in vero cells [133, 107] . very potent and selective inhibitors were the sulphated ps dextran sulphate, ^.-carrageenan, fucoidan, heparin and pentosan polysulphate. examples also include aloe polymannose, a high mannose purified from the aloe barbadensis miller plant, which showed activity in enhancing antibody titers against coxsackievirus b3-induced myocarditis in murine models of the disease [134] , and fungal ps which are active in vivo in sendai virus infection [135] . although relatively little work has been done on natural antivirals against plant viruses, several reports concerning antiviral activity against plant virus infection have been recorded; for example, yeast mannans with antiviral activity against the tobacco mosaic virus infection in tobacco plants [136] , and lichenan ps from iceland moss which exhibited antiviral activity against the potato viruses [8] . jacquir immune defic syndr hum retrovirol the technical assistance of ms. brooke-turner is gratefully acknowledged. key: cord-300793-tuq8z6gm authors: weiss, robin a; mcmichael, anthony j title: social and environmental risk factors in the emergence of infectious diseases date: 2004 journal: nat med doi: 10.1038/nm1150 sha: doc_id: 300793 cord_uid: tuq8z6gm fifty years ago, the age-old scourge of infectious disease was receding in the developed world in response to improved public health measures, while the advent of antibiotics, better vaccines, insecticides and improved surveillance held the promise of eradicating residual problems. by the late twentieth century, however, an increase in the emergence and re-emergence of infectious diseases was evident in many parts of the world. this upturn looms as the fourth major transition in human–microbe relationships since the advent of agriculture around 10,000 years ago. about 30 new diseases have been identified, including legionnaires' disease, human immunodeficiency virus (hiv)/acquired immune deficiency syndrome (aids), hepatitis c, bovine spongiform encephalopathy (bse)/variant creutzfeldt-jakob disease (vcjd), nipah virus, several viral hemorrhagic fevers and, most recently, severe acute respiratory syndrome (sars) and avian influenza. the emergence of these diseases, and resurgence of old ones like tuberculosis and cholera, reflects various changes in human ecology: rural-to-urban migration resulting in high-density peri-urban slums; increasing long-distance mobility and trade; the social disruption of war and conflict; changes in personal behavior; and, increasingly, human-induced global changes, including widespread forest clearance and climate change. political ignorance, denial and obduracy (as with hiv/aids) further compound the risks. the use and misuse of medical technology also pose risks, such as drug-resistant microbes and contaminated equipment or biological medicines. a better understanding of the evolving social dynamics of emerging infectious diseases ought to help us to anticipate and hopefully ameliorate current and future risks. popular writing on emerging infectious diseases resounds with dire warnings about the threat of modern 'plagues' and losing the 'war against microbes.' this adversarial language obscures the fact that most of the microbial world is either neutral toward, or supportive of, human well-being and survival. indeed, we would not survive long without commensal microbes such as the beneficial strains of escherichia coli in our gut. that aside, the study of emerging infections is more than a passing fad. the recent rate of identification of such infections, the impact of the sars outbreak, the devastation caused by aids, and the ever-present threat of a new influenza pandemic indicate that we cannot control our disease destiny. nor are emerging infections unique to humans; the irish potato famine in 1845 and the english foot-and-mouth disease epidemic in 2001 underscore the consequences for human societies of disease emergence in crops and livestock. emerging infectious diseases in humans comprise the following: first, established diseases undergoing increased incidence or geographic spread, for example, tuberculosis and dengue fever; second, newly discovered infections causing known diseases, for example, hepatitis c and helicobacter pylori; and third, newly emerged diseases, for example, hiv/aids and sars. this perspective will discuss the human ecology of both the (apparently) new and re-emerging diseases. interest in infectious disease has itself recently re-emerged. in 1972, burnet and white commented, "the most likely forecast about the future of infectious disease is that it will be very dull. there may be some wholly unexpected emergence of a new and dangerous infectious disease, but nothing of the sort has marked the past fifty years" 1 . today, we may criticize the short-sightedness of our mentors' generation, yet in demographic terms they were essentially correct because the proportion of deaths from infectious disease has fallen throughout the twentieth century 2,3 (fig. 1) . humankind currently faces neither apocalyptic extinction nor even a population reduction such as occurred in europe during the black death of the fourteenth century. rather, overpopulation in relation to environmental resources remains a more pressing problem in many developing countries, where poor economic and social conditions go hand-in-hand with infectious disease. in industrializing countries during the nineteenth century, a major reduction in enteric infections was achieved by separating drinking water from sewage-an environmental change that probably saved more lives than all the twentieth century vaccines and antibiotics together. today, however, the growth of shanty towns without sanitation around the megalopolis cities of asia, africa and south america is recreating similar conditions, and in the past 40 years cholera has made a remarkable re-emergence through its longest ever (seventh) pandemic 4 . in most countries, life expectancy has risen over the past 50 years 5 (fig. 2) . the most important exception is those regions where hiv infection is rife. moreover, during the past 15 years, falling living standards in some african countries and the breakdown of public health infrastructure in ex-soviet nations has aided the re-emergence of transmissible diseases like tuberculosis 4,6 . further, severe outbreaks such as the 1918-1919 influenza a pandemic temporarily reversed the decline of deaths caused by infectious disease. the 50 million estimated deaths from that pandemic 7 represented about 2% of the global population at that time, and is twice as many as the cumulative aids mortality of the past 20 years. the next influenza pandemic may be just around the corner, and may spread even faster 8 , if access to appropriate vaccines and drug treatment is not available 9, 10 . for other newly emerging infections that make headlines, such as sars, ebola or vcjd, it is important to keep a sense of demographic proportion. placing these emerging infections on a 'richter' scale of human mortality (box 1) shows that they elicit scarcely detectable minor tremors in numbers of fatalities -despite the fear they invoke. we do not know, however, which one might leap to the top of the scale like hiv has done; indeed, it may be a completely unknown agent, as the sars coronavirus was two years ago. a major challenge is to predict which infection presages the next big quake, hopefully allowing preventive action. like any other animal or plant species, humans have been prone to infection by pathogens throughout their evolutionary history. such ancient infections by helminth and protozoan parasites, bacteria, fungi and viruses are endemic, eliciting a range of effects from a heavy burden of disease (e.g., malaria) to being essentially commensal in immunocompetent hosts (e.g., most types of herpesvirus and papilloma virus). other infections depend on an animal reservoir for their maintenance; their infection of humans may be pathogenic, but it has little part in the evolving ecology of the microbe or parasite. an estimated 61% of the 1,415 species of infectious organisms known to be pathogenic in humans are transmitted by animals 11 , for which the human represents a dead-end host. occasionally, however, a zoonotic infection adapts to human-to-human transmission and diversifies away from its animal origin. epidemic diseases are generally caused by infections that are directly transmissible between humans. hiv is a recent example of a long line of human infections initiated by a switch of host species, stretching back to the origins of measles and smallpox. free-living microbes may also find a human niche that suits their lifestyle, such as the lung for legionella pneumophila and the gut for vibrio cholerae. legionnaires' disease, first recognized in philadelphia in 1976, is the environmental equivalent of a zoonosis. it is seldom passed directly from person to person but it was human ingenuity in designing warm, aerated, humid 'artificial lungs' called air-conditioning systems that allowed the microbe to proliferate and become an opportunistic colonizer of the human lung. cholera, which was unknown beyond the ganges delta before it spread widely in asia and the middle east during the period 1815-1825, at around that time horizontally acquired a toxin gene and other factors in a genetic package that helped it to colonize the gut; the resultant diarrhea aids dispersal of the microbe 12, 13 . human society has undergone a series of major transitions that has affected our pattern of infectious disease acquisition and dissemination 4 . these transitions illustrate the interrelationship between environmental, social and behavioral influences on the emergence and subsequent spread of infectious disease. some infections were acquired when our australopithecine ancestors left their arboreal habitat to live in the savannah. this ecological change included exposure to new species of mosquito and tick as vectors for infection. after the emergence of homo sapiens, the eventual migration of neolithic hunter-gatherers out of africa 50,000 to 100,000 years ago exposed them to new infections in distant regions. the first major transition of prehistoric/early historic times gave rise to the epidemic, or 'crowd,' infections. this change must have started in the millennia following the advent of agriculture-from around 10,000 years ago-as agriculturally based society developed larger, denser populations. the domestication of livestock and the rich dividends available in human settlements to other animals (e.g., rodents, dogs and various insects) provided further opportunities for pathogens to move between species. sometimes such a pathogen (or a mutant strain thereof) would have been well suited to humans as a new host species, and, if human numbers were adequate, could therefore persist indefinitely as a human infection. thus, measles emerged about 7,000 years ago, probably from rindepest of cattle, and diverged to become an exclusively human infection when population size and density became sufficient to maintain the virus without an animal reservoir. similarly, smallpox became epidemic about 4,000 years ago, possibly evolving from camelpox, its closest phylogenetic relative. the next two transitions were primarily to do with great extensions in the spread of infectious diseases, entering distant populations as 'new infections.' thus, the second historical transition occurred in classical times as large eurasian civilizations came into commercial and military contact. they inadvertently exchanged their pools of infections, and vectors such as rats and fleas, across the mediterranean basin, the middle east, india and china. the plague of athens in 430 bce during the peloponnesian war vividly described by thucidides may represent the first report of typhus. this rickettsial infection is transmitted from rats to humans and thence among louse-ridden humans. typhus frequently accompanies human conflict and deprivation, as seen in a recent outbreak among rwandan refugees in burundi 14 . the justinian plague of 542 ce devastated the eastern mediterranean region and probably extended as far as china 15 like the black death 800 years later (and both are attributable to yersinia pestis 16 ). the third historical transition accompanied the era of worldwide exploration and colonization by europeans from circa 1500 ce onward. a contemporary account by one of hernan cortes' fellow conquistadors, bernal diaz, recalls that they might well have failed to overthrow the mighty aztec empire had they not been aided by a raging epidemic. this was possibly a combination of smallpox and measles, both wholly unknown to the new world population. curiously, the columbian exchange was unidirectional regarding infectious diseases; the one contentious possible exception being syphilis. the new world is believed to have had substantially fewer human zoonotic infections 15, 17 , and vector-borne infections like chagas' disease did not travel in the absence of an appropriate vector. two centuries later, captain cook unwittingly repeated the decimation of indigenous peoples through syphilis, measles and tuberculosis in many of the pacific islands, whereas lord jeffrey amherst deliberately attempted to spread smallpox among 'hostile' native americans, one of the better documented cases of germ warfare 18 . the transmission dynamics of infections in naive populations is markedly different from those in which the majority of adults are immune 19 . onboard the beagle, charles darwin observed with his customary acuity, "wherever the values are approximate global death rates for the year 2003, taken from the world health organization (who) and other sources. hbv and hcv, hepatitis b and c viruses; rsv, respiratory syncytial virus; hpv, human papilloma viruses; vcjd, variant creutzfeldt-jakob disease. two major, novel causes of mortality top the list: cigarette smoking and hiv infection; they emerged in the twentieth century and continue to increase in many developing countries. among the chronic and re-emerging infections, malaria and tuberculosis are near the top, so it becomes apparent why there is a need for the global fund for malaria, tuberculosis and aids. accidental injuries, particularly road deaths, continue to rise, with 85% occurring in developing countries 54 . although 2003 was the year of the sars outbreak 52,55 , less than 1,000 people actually died as a result of sars coronavirus infection despite the collateral damage to daily life, psychological wellbeing and economic activity in the affected cities. this richter scale represents a snapshot in time. twenty years ago, hiv was three logs further down the scale, whereas polio was three logs higher. fifty years ago, malaria was finally eradicated from europe, where it had formerly been widespread, including in england (shakespeare's 'ague'). bacterial respiratory diseases used to have a more important role in human mortality and, despite concern over multi-resistance to antibiotics 40, 56 , the situation is considerably better than in the era before the advent of antibiotics. common bacterial infections of childhood, such as diphtheria and whooping cough, have become rarities in the developed world, largely through vaccination. viral diseases have similarly been reduced. thanks to effective immunization policies of the who, smallpox was eradicated in 1977; polio and measles viruses, which have no animal reservoir, may soon be eliminated in the same way. the european has trod, death seems to pursue the aboriginal …most of the diseases have been introduced by ships and what renders this fact remarkable is that there might be no appearance of the disease among the crew which conveyed this destructive importation." today we are living through the fourth historical transition of globalization. urbanization, dense and usually impoverished peri-urban settlements, social upheaval, air travel, long-distance trade, technological developments, land clearance and climate change all influence the risks of infectious disease emergence and spread. although some of the apparent increase in infectious disease may be attributable to better diagnostic methods and surveillance, there seems little doubt that more incidents are occurring, and have the potential to spread more widely than 50 years ago, as outbreaks and spread of infections like nipah virus and sars would not have passed unnoticed. as humans encroach further into previously uncultivated environments, new contacts between wild fauna and humans and their livestock increases the risk of cross-species infection 20 . this process will only diminish as wild species become rarer and eventually endangered, like the great apes today. an example of such contact followed the establishment of piggeries close to the tropical forest in northern malaysia, where, in 1998, the nipah virus first crossed over from fruit bats (flying foxes, pteropus spp.) to pigs and thence to pig farmers 21 . destruction of natural forest has also encouraged fruit bats to relocate nearer human habitation, like the large colony in the botanic gardens in the heart of sydney. indeed, in 1997, hendra, a related paramyxovirus of australian fruit bats 22 , fatally infected a veterinarian examining a sick horse. rodents continue to be sources of re-emerging infections, as witnessed in the 1990s with hantaviruses in the united states. rodentborne hantavirus is prevalent in agricultural systems in south america and east asia, in arid grasslands in north america and elsewhere. in mid-1993, an unexpected outbreak of acute, sometimes fatal, respiratory disease occurred in humans in the southwestern united states 23 . this 'hantavirus pulmonary syndrome' was caused by a previously unrecognized virus, maintained primarily within the native deermouse, and transmitted through excreta. the 1991-1992 el niño event, with unseasonal heavy summer rains and a proliferation of piñon nuts, hugely amplified local rodent populations which led to the 1993 outbreak 23, 24 . in south america, there have been several outbreaks of hantavirus and arenavirus infections linked to forest clearance and the growth of rodent populations in the new grasslands 4 . habitat destruction is not the only cause of increased human infection, however. dengue virus is extending its range and prevalence because its mosquito vector breeds rapidly in the urban environment 25 . in the united states, nature conservation and increased woodland in the eastern states has led to the emergence of lyme disease. this disease is caused by a tick-borne spirochete and the presence of tick-infested deer near suburban homes leads to ticks residing on bushes adjacent to baseball diamonds and gardens. intensification of production of meat and meat products has led to new infections 26 . most notorious is vcjd in the uk arising from consumption of contaminated food products of cattle affected by bse 27 . bse, or 'mad cow disease,' emerged in british cattle in 1986 because of industrialized cannibalism, whereby rendered neural tissue and bone meal from slaughtered cattle were recycled into cattle feed, as well as into pies and hamburgers for human consumption 28 . originally, infectious prions from scrapie in sheep were the suspected source, but it now seems more likely that it arose from a bovine with sporadic prion disease. the extent of the human epidemic remains unclear. although natural transmission is unsustainable (r 0 < 1 in both cattle and humans), there are concerns that vcjd might be transmissible through blood transfusions 29 . without effective diagnostic tests for presymptomatic vcjd infection, this situation is extremely unfortunate. other recently emergent food-borne infections include e. coli o157:h7, which is harmless to cattle but toxic to humans, and salmonella enteriditis in chickens. better hygiene in abattoirs, butchers and domestic kitchens can greatly reduce the incidence of infection. in theory, closed and intensive farming of a single species should reduce the risk of cross-species infection (fig. 3) . but it also allows large-scale epidemics to emerge, as seen recently for avian influenza strains in southeast asia and the netherlands 8, 30 . ancient dietary taboos, such as those of hindus, muslims and jews regarding pork as unclean, doubtless had their roots in protection from infectious disease. today, an increasing demand for consumption of exotic and wild animals raises new risks of infectious diseases such as sars (box 2). changing patterns of human behavior and ecology affect two distinct steps in the emergence of new infectious disease. the first is an increased opportunity for animal-to-human infection to occur owing to greater exposure, which may be necessary but not sufficient to lead to the emergence of a new human infection. the second step is the opportunity for onward transmission once a person has become infected. for each novel epidemic, such as the 1918-1919 influenza pandemic or aids, there are probably thousands of failed transfers. some infections simply do not take in the new host. innate hostspecific restrictions on viral replication have recently become evident for primate lentiviruses 31 , which may explain why certain species that harbor simian immunodeficiency virus, but not others more commonly in contact with humans, gave rise to hiv-1 and hiv-2. even in the case of hiv-1, only one pedigree of three independent chimpanzee-tohuman crossover events 32 has given rise to the aids pandemic, whereas the other two smolder as poorly transmissible infections. fatal pathogenesis is not necessarily coupled with infectiousness 12 , which is evident for h5n1 avian influenza in humans 9 . but genetic reassortment between avian and human influenza viruses could easily give rise to a new, rapidly spreading strain 8 . a poorly infectious pathogen may not spread at all from the index case, as is usual with rabies, or may only infect close contacts and soon peter out, as seen with lassa fever and ebola virus. sars nearly became self-sustaining but was brought under control. some of the most insidious infections are those with long, silent incubation periods during which the person is infectious. these emerge surreptitiously so that when the new disease is eventually recognized, as aids was in 1981, the infection has already spread far beyond control. like the ships of centuries past, the speed of modern air travel works wonders for the dispersal of infectious diseases. sars was eventually constrained by quarantine and strict adherence to infection control guidelines in hospitals, but not before it quickly traveled from guangzhong to hong kong and on to toronto. if ebola broke out in a city with a busy international airport, it might also travel across continents in a similar manner. brockmann 33 has modeled how rapidly such infections can move once they reach a major airport hub; closing the hubs becomes an immediate imperative. we cannot be sure what the initial vector was for the arrival of west nile virus into north america in 1999: a migratory bird blown off course, an infected human with a valid air ticket or a stowaway mosquito on a similar flight. whatever the means of entry and early colonization of crows in new york, it has taken less than four years to reach the pacific coast 25 . thus, west nile virus has found a new reservoir in american birds, just as yellow fever virus reached new world primates 350 years earlier. microbes frequently capitalize on situations of ecological, biological and social disturbance. biologically weakened and vulnerable populations-especially if also socially disordered and living in circumstances of privation, unhygienic conditions and close contact-are susceptible to microbial colonization. the severity of the bubonic plague (black death) in mid-fourteenth-century europe seems to have reflected the nutritional and impoverishment consequences of several preceding decades of unusually cold and wet weather with crop failures compounding the incipient destabilization of the hierarchical feudal system. many of the rapid and marked changes in human social ecology in recent decades have altered the probabilities of infectious disease emergence and transmission. these changes include increases in population size and density, urbanization, persistent poverty (especially in the expanding peri-urban slums), the increased number and movement of political, economic and environmental refugees, conflict and warfare. political ignorance, denial and obduracy often compound the risk of infectious disease transmission-as has been tragically observed with hiv/aids in parts of africa, where widespread poverty, a culture of female disempowerment and political instability further "if there is any conceivable way a germ can travel from one species to another, some microbe will find it," wrote william mcneill in his classic text plagues and peoples 15 . for millennia, small farmsteads accommodated mixed species living closely with humans-goats, pigs, cattle, ducks, geese, chickens and perhaps a water buffalo or a donkey-and exchanged infections. when species are raised separately but are sold together, the opportunity for cross-infection moves from the farm to the marketplace. the 1997 outbreak of avian influenza in hong kong occurred in mixed markets, where live chickens, quail and ducks were stacked together in close quarters with humans. the h5n1 virus that emerged may have been derived by recombination between those of different avian hosts 8 . after 1997, mixed species were separated into different areas of the markets. but this year's h5n1 virus is spreading among intensively reared chickens across southeast asia. the increasing predilection for meat of exotic species has exacerbated the risk of exposure to infections not previously encountered, and this situation probably triggered the sars epidemic 55 . although we are still not sure of the natural reservoir species of sars coronavirus, the live markets and restaurants in guangzhong sold small carnivores, and several species of civet cat, racoon dog and ferret badger captured in china, laos, vietnam and thailand, were brought into close proximity 57 . clearly, some of the palm civet cats were infected with sars-related viruses, but it is less clear whether they represent the original source species. there is a danger in incriminating the wrong species; if the true reservoir resides in the rodent prey of these carnivores, then culling the predators may be counterproductive. stopping the exotic meat trade altogether would seem to be a simple solution to prevent the reappearance of sars, but once the taste for it has been established, that may prove no more practical than attempting to prohibit the tobacco trade. in africa, bushmeat also poses a serious problem for emerging infectious diseases, as well as for nature conservation. sick animals may be more easily captured. for example, 21 human deaths owing to ebola virus infection ensued from the butchering of a single chimpanzee 58 . hiv has crossed from chimpanzees to humans on at least three occasions, and a higher number of zoonotic events from sooty mangabeys are indicated for hiv-2 (ref. 32). whether these cross-species infections arose from butchering the animals or from keeping them as pets is unknown, but a recent survey of primate hunters in africa showed that they are susceptible, like handlers of primates in captivity, to infection (though not disease) from foamy retroviruses 59 . the escalating intercontinental trade in exotic pets can lead to unexpected infectious disease outbreaks. the united states has only recently imposed more stringent regulations and quarantine following cases of monkeypox in humans and in prairie dogs introduced by rodents imported from africa as pets 60 . exacerbate the problem 34, 35 . but we have little understanding of why the prevalence of hiv infection varies so greatly between cities in sub-saharan africa 36 . the urban environment has only recently become the dominant human habitat. urbanism typically leads to a breakdown in traditional family and social structures, and entails greater personal mobility and extended and changeable social networks. these features, along with access to modern contraception, have facilitated a diversity of sexual contacts and, hence, the spread of sexually transmitted diseases 37 . this risk is further amplified by the growth in sex tourism in today's internationally mobile world, which capitalizes on the desperation and ignorance of poverty, combined with exploitative behaviors, in developing countries. more generally, cities often function as highways for 'microbial traffic' 38 . rapid urbanization boosts certain well established infectious diseases, such as childhood pneumonia, diarrhea, tuberculosis and dengue, and facilitates dissemination of various 'emerging' diseases-as occurred for sars in the high-rise housing of hong kong. crowded and dilapidated public housing can potentiate infectious disease transmission through drug abuse and sexually transmitted infections 39 . technological advances in medicine and public health can also inadvertently promote the emergence and spread of infectious disease. it has become commonplace to quip that you go to the hospital at the peril of acquiring an intractable nosocomial infection such as methicillin-resistant staphylococcus aureus 40 , and such infections killed around 40 times as many people as sars did in 2003 (box 1). multidrug-resistant tuberculosis has also become a major problem, and, paradoxically, regions with health programs that reduced wildtype tuberculosis strains can develop into 'hot zones' for multidrugresistant tuberculosis 41 . by far the most effective medical vector of infectious disease has been the syringe and needle. drucker et al. 42 have charted the massive increase in the use of injecting equipment over the past 100 years. individuals with hemophilia treated with pooled clotting factors became almost universally infected with hepatitis b and c viruses before diagnostic screening tests were developed. over 20% of such affected individuals also became infected with hiv 43 , and more recently, transmission of west nile virus by blood transfusion and by organ transplantation has been reported 44, 45 . the use of contaminated needles among intravenous drug users has had similar consequences. infectious diseases have also been amplified by the use of nonsterile medical injections in developing countries 42 . egypt has the highest prevalence of hepatitis c infection in the world because of the use and reuse of syringes and needles in an earlier public health campaign to reduce bilharzia by medication given by injection. the transmission of cjd through contaminated surgical instruments is another example of iatrogenic spread of infection 29 . biological medicines produced from animal-cell substrates present an inherent potential hazard for introducing new infections. great care must be taken to ensure that live attenuated vaccines grown in animal cells or eggs are devoid of pathogens ; for example, several early batches of live and inactivated polio vaccine unwittingly contained live sv40 virus, a polyoma virus of macaques. after sv40 was discovered in 1960, polio vaccine production shifted to virus propagation in primary kidney cells of african green monkeys. these cultures were free of sv40 but possibly contained sivagm, a relative of hiv that fortunately does not infect humans 31 . the irony of the sv40 story is that the united states food and drug administration prohibited the use of well known, permanent cell lines demonstrably free of adventitious infectious agents, for fear that such immortalized cells might exert oncogenic properties on the vaccine. there is no epidemiological evidence of increased tumor incidence in those populations who are known to have received sv40-contaminated polio vaccine. but there have been a number of recent claims of an association of sv40 dna sequences in a variety of human malignancies 46 , although these findings remain controversial 47 . the ultimate medical means of introducing animal viruses into humans is xenotransplantation. the implantation of animal cells or tissues into immunosuppressed individuals seems to be a perfectly designed way to encourage cross-species infection. it is astonishing that trials were started without much thought about the consequences for potentially emerging pathogens, for example, porcine retroviruses 48 . the generation of genetically modified knockout or transgenic animals to prevent hyperacute rejection of donor tissues may exacerbate the infection hazard 49, 50 . happily, there is no evidence so far of retrovirus infection in individuals who were exposed to living pig cells 50 , and clinical xenotransplantation is now stringently regulated; so it seems all the more extraordinary that cellular therapies with fetal lamb cells and extracts continue to be practiced with impunity in alternative medicine clinics in europe and the far east. novel infectious diseases can emerge in any part of the world at any time. hiv and ebola came out of africa, avian influenza and sars from china, nipah virus from malaysia, bse/vcjd from the uk and hantavirus pulmonary syndrome from the americas. it is difficult to predict what new disease will come next or where it will appear, but changing ecological conditions and novel human-animal contacts will be useful clues as to which horizons require scanning with most scrutiny. we must expect the unexpected. as a codicil, another factor that needs to be taken into account is the potential impact of the hiv pandemic on the emergence of other infectious diseases 51 . we already know that persons with aids act as 'superspreaders' of tuberculosis, and we can only speculate what course the sars outbreak might have taken had someone incubating the disease flown to durban rather than toronto 52 . people with aids may persistently harbor infections that would otherwise be transient, and this could hamper the eradication of measles and polio. multivalent pneumococcus vaccines are ineffective in hiv-infected people with cd4 + lymphocyte levels below 200/µl, whereas live 'attenuated' vaccines such as vaccinia can cause virulent disease in the immunocompromised host. immunodeficient persons living at high density could also be the seed-bed for microorganisms that are initially ill adapted to human infection to evolve into transmissible human pathogens. thus, an infection from a zoonotic or environmental source-for example, the mycobacterium avium intracellulare complex-could conceivably emerge as the tuberculosis of the twenty-first century, although direct transmission between individuals with aids of such opportunistic infections have not been documented so far. we shall give girolamo frascatoro the last word on emerging and re-emerging infectious diseases by quoting from his treatise de contagione, published almost 450 years ago, "there will come yet other new and unusual ailments in the course of time. and this disease [syphilis] will pass way, but it later will be born again and be seen by our descendents." we are grateful to m. e. chamberland, h. w. jaffe and s. leff for critically reading the manuscript. natural history of infectious disease human frontiers, environments and disease. past patterns, uncertain futures the challenge of emerging and re-emerging infectious diseases environmental and social influences on emerging infectious diseases: past, present and future mortality trends and setbacks: global convergence or divergence? betrayal of trust: the collapse of global public health (hyperion updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic molecular constraints to interspecies transmission of viral pathogens influenza: old and new threats antivirals and antiviral strategies risk factors for human disease emergence virulence and pathogenesis the cambridge world history of human disease jail fever (epidemic typhus) outbreak in burundi plagues and peoples (penguin genotyping, orientalis-like yersinia pestis, and plague pandemics germs and steel. a short history of everybody for the last 13,000 years smallpox and the indians in the american colonies infectious diseases of humans: dynamics and control unhealthy landscapes: policy recommendations on land use change and infectious disease emergence fatal encephalitis due to nipah virus among pig-farmers in malaysia newly discovered viruses of flying foxes the hantavirus epidemic in the southwest: rodent population dynamics and the implications for transmission of hantavirus-associated adult respiratory distress syndrome (hards) in the four corners region climatic and environmental patterns associated with hantavirus pulmonary syndrome, four corners region, united states emerging flaviviruses: the spread and resurgence of japanese encephalitis, west nile and dengue viruses deaths due to unknown foodborne agents a new variant of creutzfeldt-jakob disease in the uk how the cows turned mad transfusion transmission of vcjd: a crisis avoided transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands in defense of the cell: trim5α interception of mammalian retroviruses aids as a zoonosis: scientific and public health implications dynamics of epidemics spread across airline networks rethinking the african aids epidemic ecological and individual level analysis of risk factors for hiv infection in four urban populations in sub-saharan africa with different levels of hiv infection sexual behaviour in britain: partnerships, practices, and hiv risk behaviours factors in the emergence of infectious diseases urban unfinished business methicillin-resistant staphylococcus aureus in europe modeling the emergence of the 'hot zones': tuberculosis and the amplification dynamics of drug resistance the injection century: massive unsterile injections and the emergence of human pathogens mortality before and after hiv infection in the complete uk population of haemophiliacs transmission of west nile virus through blood transfusion in the united states in 2002 transmission of west nile virus from an organ donor to four transplant recipients cell and molecular biology of simian virus 40: implications for human infections and disease sv40 in human cancers-an endless tale? infection of human cells by an endogenous retrovirus of pigs transgenic pigs and virus adaptation reduced sensitivity to human serum inactivation of enveloped viruses produced by pig cells transgenic for human cd55 or deficient for the galactosyl-α(1-3) galactosyl epitope gulliver's travels in hivland what have we learnt from sars? mission now possible for aids fund research on preventing road traffic injuries in developing countries is needed severe acute respiratory syndrome antimicrobial resistance worldwide: causes, challenges and responses animal origins of sars coronavirus: possible links with the international trade in small carnivores zoonoses and haemorrhagic fever. in safety of biological products prepared from mammalian cell culture naturally acquired simian retrovirus infections in central african hunters human monkeypox: an emerging zoonosis the authors declare that they have no competing financial interests. key: cord-293653-u2qrxq6t authors: watashi, koichi; shimotohno, kunitada title: cyclophilin and viruses: cyclophilin as a cofactor for viral infection and possible anti-viral target date: 2007-02-05 journal: drug target insights doi: nan sha: doc_id: 293653 cord_uid: u2qrxq6t cyclophilin (cyp) is a peptidyl prolyl cis/trans isomerase, catalyzing the cis-trans isomerization of proline residues in proteins. cyp plays key roles in several different aspects of cellular physiology including the immune response, transcription, mitochondrial function, cell death, and chemotaxis. in addition to these cellular events, a number of reports demonstrated that cyp plays a critical role in the life cycle of viruses, especially human immunodeficiency virus (hiv) and hepatitis c virus (hcv). these two viruses are significant causes of morbidity and mortality worldwide, but current therapies are often insufficient. cyp may provide a novel therapeutic target for the management and/or cure of these diseases, in particular hcv. cyclophilin (cyp) and fk506 binding protein (fkbp) are peptidyl-prolyl cis-trans isomerases (ppiases), enzymes that catalyze the cis-trans interconversion of peptide bonds amino terminal to proline residues (fischer et al. 1989; harding et al. 1989; takahashi, 1999; takahashi et al. 1989) . cyp and fkbp are originally identifi ed as cellular factors that bind csa and fk506, respectively, both of which are immunosuppressants used clinically for the prevention of graft rejection following organ transplantation (handschumacher et al. 1984; harding et al. 1989) . therefore, these ppiases are also called immunophilin. the action of ppiases leads to changes in protein conformation (takahashi, 1999) , but the binding of csa and fk506 to cyp and fkbp, respectively, inhibits the activity of these enzymes (fischer et al. 1989; rosen et al. 1990; takahashi et al. 1989 ). however, the inhibition of ppiase activity by csa and fk506 is an insuffi cient requirement for their immunosuppressive function (bierer et al. 1990; schreiber, 1991) . the csa/cyp or fk506/fkbp complex, subsequently interacts with and inhibits calcineurin (cn), a phosphatase involved in the activation of the transcription factor nf-at. proper nf-at function is essential for the generation of a productive t cell response (clipstone and crabtree, 1992; fruman et al. 1992; liu et al. 1991) . in the absence of immunosuppressants, cn dephosphorylates cytoplasmic nf-at, leading to nf-at nuclear translocation and transactivation of downstream genes participating in the immune response (liu et al. 1992; mccaffrey et al. 1993) . csa and fk506 prevent the dephosphorylation and subsequent nuclear translocation of nf-at leading to immunosuppression. more than 10 cyp subtypes are found in mammals (table 1 ). the subcellular localization of cyps varies. cypa is primarily found in the cytoplasm, while cypb, cypd, cype, and ranbp2 are distributed in the endoplasmic reticulum (er), mitochondria, nucleus, and nuclear pore, respectively. members of the cyp family play roles in a variety of cellular processes including the immune response, transcription, mitochondrial function, cell death, and chemotaxis, as described below. while a number of cyp family members have been ideitifi ed, intensive functional analysis has been performed on only a few including cypa, cypb, cypd, and cyp40. cypa is the most abundant cyp subtype found in the cells (waldmeier et al. 2003) , and it is the primary factor mediating the immunosuppressive effects of csa (colgan et al. 2005) . however, even in the absence of csa, cypa plays an important role in regulating the immune responses as seen in cypa-defi cient mice. cypa-knockout mice have an "allergic" phenotype with increased serum igg1 and ige levels and tissue infi ltration by mononuclear cells, eosinophils, and mast cells (colgan et al. 2004 ), related to increased and dysregulated activity of th2 cd4+ t cells. in cypa-knockout cells, interleukin-2 tyrosine kinase (itk), a signaling molecule crucial for the development of a th2 response, is constitutively activated. itk is a member of the tec family of sh2/sh3-containing tyrosine kinases, and it participates in the signal transduction cascade leading to t cell activation. cypa can bind itk, and this negatively regulates itk activity (brazin et al. 2002) . thus, cypa plays a suppressive role in the development of cd4+ t cell responses through its interaction with itk. other studies have reported several non-immune system roles for cypa. cypa interacts with apoptosis-inducing factor (aif) and promotes aif-mediated chromatinolysis during apoptosis (cande et al. 2004) . additionally, cypa interacts with membrane-bound guanylate cyclase-a (gc-a), a receptor for atrial natriuretic factor (anf) ). gc-a and anf are involved in cardiovascular homeostasis, and cypa appears to function as an endogenous inhibitor of gc-a activation by competing for anf binding. further interactions of cypa with prolactin receptor (syed et al. 2003 ) and transcription factor yy1 (yang et al. 1995) have been observed, but the exact role of cypa in these processes remains unclear. cypa was also observed to bind dna in a zinc-dependent manner in a mouse macrophage cell line (krummrei et al. 1995) . however, the best-characterized role identified for cypa is not in normal cellular physiology, but rather as co-factor during the human immunodefi ciency virus-1 (hiv-1) viral life cycle (see below). cypb was originally identifi ed as a cyp family member bearing a signal sequence leading to the er lumen or the secretory pathway (price et al. 1991) , but the specifi c function of cypb is poorly understood. a yeast two-hybrid screening using cypb as a bait identified an interaction with calcium-signal modulating cyclophilin ligand (caml) (bram and crabtree, 1994) . caml is located on the cytoplasmic face of the er membrane (holloway and bram, 1998) . caml participates in calcium signal transduction pathway and it is essential for peripheral t cell development (tran et al. 2005) . however, the importance of cypb binding to caml function remains unknown. cypb also enhances prolactin-driven cell proliferation (rycyzyn et al. 2000) and promotes the nuclear retrotranslocation of prolactin through a direct interaction. additionally, cypb potentiates prolactin-induced stat5 transactivation by promoting the dissociation of pias3, a stat5 repressor (rycyzyn and clevenger, 2002) . cypb can also associate with interferon regulatory factor (irf)-3 (obata et al. 2005) . extracellular cypb can bind platelets (allain et al. 1999 ) and yokoyama et al. 1995 this initiates a transmembranous infl ux of calcium ion, kinase activation, and platelet adhesion to collagen. accumulating evidence suggests that cyps, in particular cypa and cypb, can mediate intercellular communication similar to cytokines. cyps are secreted from cells in response to infl ammatory stimuli or oxidative stress (jin et al. 2000; seko et al. 2004; sherry et al. 1992; xu et al. 1992) and they can act as potent chemoattractants for neutrophils (sherry et al. 1992) , eosinophils (xu et al. 1992) , and t cells (allain et al. 2002) . cypa and cypb are recognized by the cell surface receptor cd147, and cyp binding leads to erk activation and chemotaxis (pushkarsky et al. 2001; yurchenko et al. 2001; yurchenko et al. 2002) . cypd plays a critical role in mitochondrial function and cell death (tanveer et al. 1996) . during ischemia-induced necrosis, e.g. following a heart attack or stroke, the accumulation of calcium and increase of reactive oxygen species (ros) trigger the opening of a pore in the inner mitochondrial membrane, known as the membrane permeability transition pore (mptp) (halestrap, 1999) . calcium overload and ros induce a conformational change in adenine nucleotide translocase (ant), a key component regulating the opening of mptp at the inner mitochondrial membrane. the opening of mptp leads to mitochondrial swelling, rupture of the outer membrane, and the release of small molecules (waldmeier et al. 2003) . cypd is located within the matrix of the mitochondria and it binds ant facilitating its conformational change (crompton et al. 1998; woodfi eld et al. 1998 ). in cypd-knockout cells, necrosis induced by calcium and ros was decreased, but apoptotic cell death induced by cytokines or anticancer agents was unaffected (baines et al. 2005; nakagawa et al. 2005) . cypd-knockout mice also experienced reduced cardiac injury following reperfusion after ischemia. thus, cypd is a key molecule involved in the cell death process. cyp40 regulates the activity of steroid receptors (srs) (duina et al. 1996; owens-grillo et al. 1995; ratajczak et al. 1993) . srs including the glucocorticoid receptor, estrogen receptor, androgen receptor and progesterone receptor are nuclear hormone receptors that exert transcriptional activity following steroid ligand binding and nuclear translocation. in the absence of steroid ligands, srs form complexes with heat shock protein 90 (hsp90) together with the immunophilins cyp40, fkbp51, or fkbp52 in the cytoplasm. these immunophilins control sr activity by increasing receptor avidity for hormone ligands through ppiase-dependent conformational changes. upon hormone binding, this sr/hsp90/ immunophilin complex dissociates, leaving homodimeric sr, which then translocates into the nucleus to transactivate downstream genes. although there are some reports on other cyp subtypes (table 1 ), the precise functions and signifi cances of them are largely unknown. as described above, cyps play essential roles in diverse cellular processes. interestingly, several viruses have evolved to use cyps during their life cycles. in particular, cyps are demonstrated to be involved in the proliferation of hiv-1 and hepatitis c virus (hcv). other viruses using cyps during their life cycle include vaccinia virus (vv), vesicular stomatitis virus (vsv), and sars-coronavirus. a signifi cant role of cyp in vv replication was fi rst identifi ed through the analysis of several csa analogs. the ability of cyclosporins to suppress vv replication correlated with the inhibition of cyp function (damaso and moussatche, 1998) . vv infection stabilizes cypa, leading to the accumulation of cypa (castro et al. 2003) . in vv infected cells, cypa relocalizes to the peripheral region of the nucleus, colocalizing with sites of virus production. cypa is incorporated into viral particles and is located in the viral core. cypa interacted with the nucleocapsid protein of vsv (bose et al. 2003) , and, like vv, cypa is incorporated into vsv viral particles. although the binding and incorporation of cypa occurred beyond the virus serotypes, the functional role of cypa in the viral life cycle appears to be straindependent. inhibition of cyp activity by csa reduced primary transcription of vsv-new jersey (vsv-nj) but not vsv-indiana (vsv-ind) serotype, and cypa activity was required for the replication of vsv-nj to a greater extent than vsv-ind. the authors suggest that differential requirements of cypa are likely the results of evolutionary pressure during lineage development. the nucleocapsid protein (np) of severe acute respiratory syndrome coronavirus (sars-cov) binds cypa (luo et al. 2004) , and another group reported that cypa is incorporated into sars-cov particles (chen et al. 2005) . extracellular cypa binds cd147 on the cell surface, and treatment with a peptide that blocks cd147 binding inhibits viral infection. thus, cypa may be involved in sars-cov invasion into host cells through interaction with np and cd147, respectively. cypa plays an important role in the viral life cycle of hiv-1. in 1993, cypa was found to interact with hiv-1 gag (luban et al. 1993) , and in 1994, cypa was reportedly incorporated into viral particles (franke et al. 1994; thali et al. 1994) . a gene targeting study demonstrated that only cypa among cyp subtypes was essential for hiv-1 proliferation (braaten and luban, 2001) . within the hiv-1 life cycle, cypa plays multiple roles through different interaction partners, including an early step prior to reverse transcription (braaten et al. 1996; mlynar et al. 1997; steinkasserer et al.1995) . although cypa is incorporated into virions through binding to the ca domain of the gag polyprotein (franke et al. 1994; ott et al. 1995; thali et al. 1994 ), this incorporation is not required for viral infection. instead, target cell expressed cypa is important for productive infection and viral replication (hatziioannou et al. 2005; sokolskaja et al. 2004 ). it has been known for several decades that host cells express different restriction factors to prevent infection by certain retroviruses (cullen, 2003) , and several recent studies have suggested that cypa modulates sensitivity to such a restriction factor early in the hiv-1 life cycle prior to reverse transcription. trim5α is a host restriction factor originally identifi ed using expression cloning that recognizes ca limiting retrovirus proliferation (stremlau et al. 2004 ). towers et al. showed that cypa regulates the activity of a host restriction factor (towers et al. 2003) . disruption of cypa-ca binding by introducing of point mutation into ca or treating human cells with csa decreases hiv-1 infectivity. conversely, the loss of cypa-ca binding greatly enhanced hiv-1 infectivity in simian cells (berthoux et al. 2005; kootstra et al. 2003; sayah et al. 2004) . from the results, the hypothesis was proposed by luban et al. that ca binding by cypa prevented normal antiviral effects mediated by trim5α during hiv-1 infection of human cells, but this same interaction mediated hiv-1 restriction in nonhuman primate cells (sokolskaja et al. 2006; luban, in press ). both the mechanism of trim5α restriction of hiv-1 and the modulation of ca recognition by cypa remain unclear, and further studies are clearly needed to resolve these important issues in the hiv-1 life cycle and the host response to hiv-1 infection. cypa may be important for other aspects of hiv-1 infection. cypa interacts with cd147 (pushkarsky et al. 2001) , heparans (saphire et al. 1999) , vpr (zander et al. 2003) , and envelope glycoprotein gp120 (endrich and gehring, 1998) , although their relevances of the interactions should be further verifi ed. hcv is a major causative agent of chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (liang et al. 1993) . hcv infection is a serious health problem affecting approximately 170 million individuals worldwide (poynard et al. 2003) . the current standard therapy for hcv is restricted to interferon (ifn) or pegylated-ifn either alone or in combination with ribavirin. because treatment with these agents, however, fails to produce sustained virus elimination in about one half of the patients (di bisceglie et al. 2002) , alternative and effective strategies to combat against hcv are greatly needed. hcv encodes a single polypeptide that is cleaved by host and hcv-encoded protease including ns3 to generate a set of functional proteins. its genome is replicated by the hcv-encoded rna-dependent rna polymerase (rdrp) ns5b. both of these proteins, ns3 and ns5b, are essential for hcv genome replication and are possible targets for the development of anti-hcv therapeutics (di bisceglie et al. 2002) . small molecule compounds targeting ns3 and ns5b have been developed, and their effi cacy has been examined in clinical trials (di bisceglie et al. 2002) . in addition to these viral enzymes, host cell factors are required for viral replication, and these may provide other options for the development of novel anti-viral agents. disrupting the function of host cell derived factors is particularly appealing as the mutation rate of host proteins is much less than that of viral encoded proteins and should less give rise to drug-resistant viruses. however, while some host factors required for viral genome replication have been identifi ed, other host proteins need to be identifi ed to develop optimal anti-viral therapies with few side effects. at this time, only a limited number of host proteins have been found to be involved in hcv genome replication with biological relevance. hvap-33 is one of snare family proteins that regulate vesicle biogenesis, protein sorting, and membrane fusion. hvap-33 binds hcv ns5a and ns5b, and this interaction regulates the presence of hcv proteins in the subcellular compartment performing viral genome replication (evans et al. 2004; gao et al. 2004; tu et al. 1999) . fbl2 is a member of the f-box protein family, involved in the ubiquitination pathway. fbl2 is geranylgeranylated in cells , and associates with ns5a in a geranylgeranylation-dependent manner to regulate hcv genome replication. however, the mechanism of action of fbl2 in hcv genome replication is not known. additionally, we recently found that cypb is a cofactor for hcv replication in host cells, and this may represent a new target for anti-hcv therapeutics (watashi et al. 2005) . we will fi rst discuss the role of cypb in hcv genome replication followed by the therapeutic implications of this discovery in hcv treatment. we identifi ed csa as an anti-hcv agent using a hcv replicon system, a cell culture system supporting hcv genome replication (lohmann et al. 1999) , and csa inhibits hcv genome replication as potently as ifnα (watashi et al. 2003) . since that time, several groups have made similar observations (firpi et al. 2006; nakagawa et al. 2004; paeshuyse et al. 2006) . as shown in fig.1a , cellular treatment with 1 µg/ml csa decreases hcv rna levels by approximately 1/500 (watashi et al. 2003) . csa also reduces the expression of hcv-encoded proteins to undetectable levels (fig. 1b) . in contrast, fk506 has no effect on the production of hcv rna or proteins ( fig. 1a and b ). the differences between csa and fk506 suggest that csa prevents viral genome replication independently of cn, an effector (copy/pg total rna) amount of hcv rna figure. 1 csa suppresses hcv genome replication. (a) hcv rna was quantifi ed in total rna isolated from hcv replicon-bearing cells treated with various concentrations of csa, fk506, or ifnα for 7 days. the amount of hcv rna per 1 pg total rna was plotted against the concentration of csa (µg/ml), fk506 (µg/ml), or ifnα (× 100 iu/ml). (b) the expression of hcv ns5a and protein disulfi de isomerase (pdi) as a cellular protein was examined in the hcv replicon-bearing cells treated without (control) or with 100 iu/ml ifnα, 1 µg/ml csa, or 1 µg/ml fk506 for 7 days. common to both csa and fk506 mediated immunosuppression. and the anti-hcv effects of csa are mediated by pathway(s) distinct from those of ifnα (watashi et al. 2003 ). the ability of csa to inhibit hcv genome replication correlates with the inhibition of cyp activity (watashi et al. 2005) . moreover, an alternative cyp inhibitor, sanglifehrin, also decreases the levels of hcv rna. thus, the inhibition of cyp activity is essential for the anti-hcv effect of csa, and this strongly suggests that cyp plays a direct, important role in hcv genome replication. moreover, the specifi c knockdown of cypb by rnai reduced hcv rna titer, but knockdown of cypa, cypc, cype, or cyph had no effect on hcv replication activity. these data indicate that cypb plays a critical role in hcv genome replication. the effects of cypb on hcv genome replication are mediated through a direct interaction with ns5b as demonstrated in both in vitro and in cells (watashi et al. 2005) (fig. 2a) . cypb do not bind any other hcv proteins involved in viral replication. ns5b binds hcv genome rna in order to function as a rdrp. cypb but not cypa promotes the rna binding activity of ns5b and stimulates hcv genome replication in cells ( fig. 2a) . this functional support by cypb to ns5b is essential for the effi cient replication of the hcv genome, and csa blocks the interaction of cypb with ns5b, leading to reduced rna binding (fig. 2b ). thus, cypb serves as a cellular cofactor for hcv genome replication. the anti-hcv activity of csa analogs correlates with their ability to inhibit cyp function (watashi et al. 2005) . the dissociation of cypb and ns5b greatly reduces the extent of hcv genome replication. these observations suggest that the inhibition of cypb may represent a novel therapeutic strategy against hcv. this possibility has been examined by two reports using stronger cyp inhibitors than csa. paeshuyse et al. used the csa analog debio-025 to inhibit cyp activity (paeshuyse et al. 2006) , and this compound inhibited hcv replication 10-fold more potently than csa. the authors speculated that debio-025 might be an attractive drug candidate for the treatment of individuals with hcv/hiv coinfection because csa derivatives should also inhibit hiv-1 replication. we used the non-immunosuppressive csa derivative nim811 to target cyp ishii et al. 2006) . nim811 inhibits cyp enzymatic activity two-fold more than csa , and this increased inhibition correlates with greater suppression of hcv genome replication than csa, especially at lower doses. cotreatment of cells with nim811 and ifnα led to a synergistic anti-hcv effect at higher doses of nim811. treatment of nim811 for three weeks eliminated hcv rna from host cells to under detectable level. because the immunosuppression in patients during a viral infection is undesirable, these non-immunosuppressive variants of csa that inhibit cyp activity are likely to offer great promise for the treatment of patients with chronic hcv infection. cyps are cellular ppiases that catalyze conformational changes in proteins, but the role(s) and substrates for this protein family in cells are not well-characterized. however, discoveries of the signifi cance of cyp in life cycles for several viruses make this protein virologically notable and present novel therapeutic anti-viral targets. further analyses of the role of cyps in viral life cycles should reveal novel functions for these proteins as well as provide mechanistic insight into possible therapeutic targets. cyclophilin b binding to platelets supports calcium-dependent adhesion to collagen interaction with glycosaminoglycans is required for cyclophilin b to trigger integrin-mediated adhesion of peripheral blood t lymphocytes to extracellular matrix loss of cyclophilin d reveals a critical role for mitochondrial permeability transition in cell death the cyclophilin multigene family of peptidyl-prolyl isomerases. characterization of three separate human isoforms cyclophilin a is required for trim5{alpha}-mediated resistance to hiv-1 in old world monkey cells probing immunosuppressant action with a nonnatural immunophilin ligand requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype cyclophilin a is required for an early step in the life cycle of human immunodefi ciency virus type 1 before the initiation of reverse transcription cyclophilin a regulates hiv-1 infectivity, as demonstrated by gene targeting in human t cells calcium signalling in t cells stimulated by a cyclophilin b-binding protein regulation of the tyrosine kinase itk by the peptidyl-prolyl isomerase cyclophilin a aif and cyclophilin a cooperate in apoptosis-associated chromatinolysis redistribution of cyclophilin a to viral factories during vaccinia virus infection and its incorporation into mature particles function of hab18g/cd147 in invasion of host cells by severe acute respiratory syndrome coronavirus cyclophilin a functions as an endogenous inhibitor for membrane-bound guanylate cyclase-a. hypertension identifi cation of calcineurin as a key signalling enzyme in t-lymphocyte activation cyclophilin a regulates tcr signal strength in cd4+ t cells via a proline-directed conformational switch in itk cyclophilin a-defi cient mice are resistant to immunosuppression by cyclosporine cyclophilin-d binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore hiv-1 infection: fooling the gatekeeper inhibition of vaccinia virus replication by cyclosporin a analogues correlates with their affi nity for cellular cyclophilins new therapeutic strategies for hepatitis c a cyclophilin function in hsp90-dependent signal transduction the v3 loop of human immunodefi ciency virus type-1 envelope protein is a high-affi nity ligand for immunophilins present in human blood phosphorylation of hepatitis c virus nonstructural protein 5a modulates its protein interactions and viral rna replication cyclosporine suppresses hepatitis c virus in vitro and increases the chance of a sustained virological response after liver transplantation cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins specifi c incorporation of cyclophilin a into hiv-1 virions two cytoplasmic candidates for immunophilin action are revealed by affi nity for a new cyclophilin: one in the presence and one in the absence of csa calcineurin phosphatase activity in t lymphocytes is inhibited by fk 506 and cyclosporin a interactions between viral nonstructural proteins and host protein hvap-33 mediate the formation of hepatitis c virus rna replication complex on lipid raft evaluation of the anti-hepatitis c virus effects of cyclophilin inhibitors, cyclosporin a and nim811 the mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury cyclophilin: a specifi c cytosolic binding protein for cyclosporin a a receptor for the immunosuppressant fk506 is a cis-trans peptidyl-prolyl isomerase cyclophilin interactions with incoming human immunodefi ciency virus type 1 capsids with opposing effects on infectivity in human cells co-localization of calcium-modulating cyclophilin ligand with intracellular calcium pools a new cyclophilin and the human homologues of yeast prp3 and prp4 form a complex associated with u4/u6 snrnps diverse effects of cyclosporine on hepatitis c virus strain replication a novel leucine-rich repeat protein (lrr-1): potential involvement in 4-1bb-mediated signal transduction cyclophilin a is a secreted growth factor induced by oxidative stress isolation and characterization of a 40-kda cyclophilin-related protein abrogation of postentry restriction of hiv-1-based lentiviral vector transduction in simian cells cyclophilin-a is a zinc-dependent dna binding protein in macrophages viral pathogenesis of hepatocellular carcinoma in the united states cloning, expression, and purifi cation of human cyclophilin in escherichia coli and assessment of the catalytic role of cysteines by sitedirected mutagenesis inhibition of t cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity calcineurin is a common target of cyclophilin-cyclosporin a and fkbp-fk506 complexes replication of subgenomic hepatitis c virus rnas in a hepatoma cell line human immunodefi ciency virus type 1 gag protein binds to cyclophilins a and b cyclophilin a, trim5 and resistance to hiv-1 infection nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a nf-atp, a t lymphocyte dna-binding protein that is a target for calcineurin and immunosuppressive drugs a nuclear rna-binding cyclophilin in human t cells the non-immunosuppressive cyclosporin a analogue sdz nim 811 inhibits cyclophilin a incorporation into virions and virus replication in human immunodefi ciency virus type 1-infected primary and growth-arrested t cells specifi c inhibition of hepatitis c virus replication by cyclosporin a drug target insights cyclophilin d-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death rs cyclophilins: identifi cation of an nk-tr1-related cyclophilin role of cyclophilin b in activation of interferon regulatory factor-3 analysis and localization of cyclophilin a found in the virions of human immunodefi ciency virus type 1 mn strain the cyclosporin a-binding immunophilin cyp-40 and the fk506-binding immunophilin hsp56 bind to a common site on hsp90 and exist in independent cytosolic heterocomplexes with the untransformed glucocorticoid receptor cloning, expression and chromosomal mapping of a novel cyclophilin-related gene (ppil1) from human fetal brain the non-immunosuppressive cyclosporin debio-025 is a potent inhibitor of hepatitis c virus replication in vitro viral hepatitis c human cyclophilin b: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence cd147 facilitates hiv-1 infection by interacting with virus-associated cyclophilin a the cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (fkbp59) inhibition of fkbp rotamase activity by immunosuppressant fk506: twisted amide surrogate inhibition of human immunodefi ciency virus type 1 replication by sdz nim 811, a nonimmunosuppressive cyclosporine analog the intranuclear prolactin/cyclophilin b complex as a transcriptional inducer role of cyclophilin b in prolactin signal transduction and nuclear retrotranslocation host cyclophilin a mediates hiv-1 attachment to target cells via heparans cyclophilin a retrotransposition into trim5 explains owl monkey resistance to hiv-1 chemistry and biology of the immunophilins and their immunosuppressive ligands. science hypoxia followed by reoxygenation induces secretion of cyclophilin a from cultured rat cardiac myocytes identifi cation of cyclophilin as a proinfl ammatory secretory product of lipopolysaccharide-activated macrophages target cell cyclophilin a modulates human immunodefi ciency virus type 1 infectivity cyclophilin a, trim5, and innate immunity to hiv-1 mode of action of sdz nim 811, a nonimmunosuppressive cyclosporin a analog with activity against human immunodefi ciency virus type 1 (hiv-1): interference with early and late events in hiv-1 replication the cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys a novel and functional interaction between cyclophilin a and prolactin receptor pharmacodynamics of ciclosporin a (cyclosporine) peptidyl-prolyl cis-trans isomerase is the cyclosporin a-binding protein cyclophilin involvement of cyclophilin d in the activation of a mitochondrial pore by ca2+ and oxidant stress functional association of cyclophilin a with hiv-1 virions cyclophilin a modulates the sensitivity of hiv-1 to host restriction factors caml is a p56lck-interacting protein that is required for thymocyte development hepatitis c virus rna polymerase and ns5a complex with a snare-like protein cyclophilin d as a drug target identifi cation of a nuclear-specifi c cyclophilin which interacts with the proteinase inhibitor eglin c identifi cation of fbl2 as a geranylgeranylated cellular protein required for hepatitis c virus rna replication cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes cyclophilin b is a functional regulator of hepatitis c virus rna polymerase drug target insights direct demonstration of a specifi c interaction between cyclophilin-d and the adenine nucleotide translocase confi rms their role in the mitochondrial permeability transition leukocyte chemotactic activity of cyclophilin cyclophilin a and fkbp12 interact with yy1 and alter its transcriptional activity a giant nucleopore protein that binds ran/tc4 cd147 is a signaling receptor for cyclophilin b active site residues of cyclophilin a are crucial for its signaling activity via cd147 cyclophilin a interacts with hiv-1 vpr and is required for its functional expression molecular cloning, structure and expression of a novel nuclear rna-binding cyclophilin-like gene (ppil4) from human fetal brain molecular cloning and characterization of a novel peptidylprolyl isomerase (cyclophilin)-like gene (ppil3) from human fetal brain key: cord-311559-vkb7a4cm authors: kanwugu, osman n.; adadi, parise title: hiv/sars‐cov‐2 coinfection: a global perspective date: 2020-07-28 journal: j med virol doi: 10.1002/jmv.26321 sha: doc_id: 311559 cord_uid: vkb7a4cm since its first appearance in wuhan, china, severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) has rapidly spread throughout the world and has become a global pandemic. several medical comorbidities have been identified as risk factors for coronavirus disease 2019 (covid‐19). however, it remains unclear whether people living with human immunodefeciency virus (plwh) are at an increased risk of covid‐19 and severe disease manifestation, with controversial suggestion that hiv‐infected individuals could be protected from severe covid‐19 by means of antiretroviral therapy or hiv‐related immunosuppression. several cases of coinfection with hiv and sars‐cov‐2 have been reported from different parts of the globe. this review seeks to provide a holistic overview of sars‐cov‐2 infection in plwh. coronavirus disease 2019 (covid-19) is a potentially fatal respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a newly identified coronavirus, which was first recognized in december 2019 in wuhan, hubei province, china and has since rapidly spread to over 200 countries/territories/areas and have been declared a global pandemic by the world health organization (who). 1 as at the time of writing (3 june 2020), the total confirmed cases were 6 383 805 with 2 732 976 recoveries and a staggering 380 384 deaths have been officially reported globally. 2 the clinical spectrum of covid-19 is broad, and while most people with covid-19 develop only mild or uncomplicated illness, especially in the early phase of illness, 14% to 26% of infected persons develop severe disease that requires hospitalization and oxygen support, with some even requiring admission to an intensive care unit. 3, 4 organ dysfunction, particularly progressive respiratory failure, heart and kidney injuries, is associated with the highest rates of mortality. 5, 6 clinical evidence has shown that disease severity and mortality are associated with older age and underlying comorbidities, such as diabetes, hypertension, and cardiovascular disease (cvd). 4, 6, 7 in line with this, one of the puzzling questions in the wake of this pandemic is "does hiv infection increases the risk of getting and disease severity of covid-19?." this is important, especially in africa, as the region accounts for more than 70% of the global burden of human immunodefeciency virus (hiv) infection. 8 generally, people living with hiv (plwh) are perceived to be at high risk of contracting sars-cov-2, even though currently no specific information about the risk of covid-19 in people with hiv is available. at the end of 2018, it was estimated that 37.9 million people globally were living with hiv with 20.6 million (54%) in the eastern and southern africa region, 5.9 million in asia and the pacific region, 5.0 million in western and central africa, and 2.2 million in western and central europe and north america region. 9 in an attempt to halt the spread of covid-19, governments across the globe are shutting cities down, restricting movements and encouraging residents to stay indoors. beyond the unprecedented disruption of lives, the covid-19 pandemic has severely interrupted hiv care delivery among several other health care services globally as attention, resources and personnel have been diverted to the fight against covid-19. [10] [11] [12] it is estimated that about 19% of hiv-infected patients were unable to get antiretroviral medications or therapy (art) refills due to the osman n. kanwugu and parise adadi contributed equally to this work. pandemic. in addition, there have been reports that several hiv/ aids prevention and control centers globally have been converted to covid-19 treatment centers, which denies hiv patients of their art. [13] [14] [15] this has left a greater proportion of the hiv community in a venerable state, considering that they require regular medication to maintain good health. despite the potentially poor prognosis for most patients within this category when infected with sars-cov-2, data on hiv/sars-cov-2 co-infection is still scarce. herein we summarize the global instances of sars-cov-2/hiv coinfections. the covid-19 pandemic, as well as measures taken by governments across the world, to minimize its spread has triggered unintended consequences in terms of hiv testing and care. 16 quarantine and treatment facility. 18 in the hubei province of china, about 64.15% of hiv patients could not have access to their art due to the measures imitated to curb the spread of the virus. 19 as gokengin et al reported, antiretrovirals are purchased and distributed via designated clinics 20 thus, patients living outside the perimeter could not have access to their art. the situation is further aggravated by shortage of medication as medical consignments are stuck in procurement systems in other countries with no further supplies able to come in. 21 the pandemic has as well compromised the psychological and emotional wellbeing of plwh. shiau et al 22 reported that many hiv patient being managed via telephone have indicated that they are extremely stressed, anxious, and unable to sleep. a recent survey in china, 19 revealed that 28.93% of the respondents hoped they had some social and psychological support. therefore, these psychosocial issues have to be addressed to avoid exacerbating adverse medical consequence among plwh. 22 2 | methods scopus, web of science, pubmed and google scholar were searched for relevant peer-reviewed publications from december 2019 to 3 june 2020, using the following combination of terms: ("hiv" and "covid-19"), ("immunodeficiency" and "covid-19") and ("hiv" and "coronavirus"). the search was limited to only publications in english. publications with information on hiv/sars-cov-2 co-infection were manually sorted out and included in this study. websites of relevant organizations including who, cdc, and usaids were also reviewed for additional information. data on coinfection cases were extracted and entered into microsoft excel. statistical analyses were carried out using ibm spss statistics version 25. the first case of hiv/sars-cov-2 coinfection was reported in wuhan, china, the terminus a quo of the pandemic. subsequent cases of coinfection have been reported in uk, usa, spain, italy, germany and other countries (table 1) . interestingly, only two cases of coinfection have been reported in the whole of the africa continent, notwithstanding the fact that south africa which is at present the epicenter of the covid-19 pandemic in africa 1 and home to over close to 8 million plwh, the largest hiv epidemic in the world. 23 the situation of africa is probably not because less plwh have actually contracted covid-19, but more likely due to unpublished data, taking into account the uncoordinated and poor data collection and management in hospitals and health centers, lack of enthusiasm and ability of most health professionals to conduct research and prepare manuscripts for publication as well as poor collaboration between researchers and health professionals. the region currently accounts for just a little of 1% of global health publications 24 with a significant part being championed by researchers from high income countries. 25 nonetheless, 378 hiv/sars-cov-2 coinfection cases have so far been reported globally with a majority originating from uk (101 cases) and usa (122 cases). the high number of coinfection cases from these countries however does not particularly suggest any increased risk of covid-19 among plwh in them. it is worth noting that studies characterizing a larger population of patients with covid-19 originated from these countries, and hence the high reconsidering reports from china, 32-34 japan, 35 spain, 36 cases. 26, 27 the low proportion of plwh among patients with covid-19 should, however, be interpreted with caution as it could be as a result unyielding commitment to safety precautions (including wearing of nose masks, hand hygiene, social distancing, etc.) by individuals with hiv to limit their exposure to the sars-cov-2, bearing in mind their compromised immune system and the fact that even before covid-19 they were at risk of a broad range of infections, including respiratory tract infections, 23 rather than protection afforded by hiv or art. it could also be that less of the hiv population is being screened for covid-19, looking at the fact some are not even enthused to visit treatment centers and clinics for their art refills. 18 in general, 214 of a total 334 coinfection cases were uncomplicated (ie, mild and moderate) cases while the remaining 35.9% were classified as either severe or critical (complicated), requiring oxygen therapy and/or admission to intensive care unit ( figure 2 ). among subjects with known outcomes (closed cases; n = 300) 82.3% had recovered while the remaining (53 patients) died, thus giving an overall case-fatality rate of 14% among plwh, which is more than 2 times higher than the current rate among the global population. 1 however, just like the country-specific case-fatality ratio of covid-19 in the general population, the case-fatality rate among plwh differ from one country to another, ranging from as high as 27.7% in uk to 0% in china (figure 3 ). similar to uk, the results of the analysis indicated usa also has a high case-fatality ratio (13.9%) among its hiv population which is 2.4 time higher than that of the general population (5.8%). 39 among the top five countries in terms of number of coinfection cases reported, the case-fatality ratio among plwh in spain (3.6%) and italy (4.3%) are lower than that recorded for the general population, 11.3% and 14.4% respectively. 39 characteristics of the healthcare system, among others. 39 nonetheless, it should be noted that majority of the cases included in this study (particularly from uk and usa) were hospitalized patients and as such the high case-fatality rates recorded might not be particularly peculiar to hiv/sars-cov-2 coinfection since as much as 26% (in uk) 26 and 21% (in usa) 27 we acknowledge the selfless efforts of healthcare professionals and all other essential service providers in the fight against covid-19. the authors declare that there are no conflict of interests. world health organization. coronavirus disease 2019 (covid-19): situation reports johns hopkins coronavirus resource center epidemiological and clinical predictors of covid-19 clinical management of severe acute respiratory infection (sari) when covid-19 disease is suspected: interim guidance clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china clinical characteristics of 25 death cases with covid-19: a retrospective review of medical records in a single medical center risk factors associated with clinical outcomes in 323 covid-19 hospitalized patients in wuhan, china hiv and sars-cov-2 co-infection: a case report from uganda the joint united nations programme on hiv/aids (unaids). global hiv & aids statistics -2020 fact sheet what does the covid-19 pandemic mean for hiv, tuberculosis, and malaria control challenges to hiv care and psychological health during the covid-19 pandemic among people living with hiv in china covid-19 pandemic disrupts hiv continuum of care and prevention: implications for research and practice concerning community-based organizations and frontline providers disruption to hiv treatment in africa during covid-19 pandemic could double hiv deaths, modelling studies warn hiv services take a backseat to covid-19 in russia living with hiv in the time of covid-19: a glimpse of hope public health, health systems and palliation planning for covid-19 on an exponential timeline hiv care in times of the covid-19 crisis-where are we now in central and eastern europe? lockdown fears for key populations a rapid assessment of the impact of the new coronavirus pneumonia epidemic on the health needs of hiv-infected patients hiv care in central and eastern europe: how close are we to the target? crisis for people living with hiv in indonesia the burden of covid-19 in people living with hiv: a syndemic perspective potential impact of sars-cov-2 infection in hiv-positive patients in south africa strengthening health research capacity in sub-saharan africa: mapping the 2012-2017 landscape of externally funded international postgraduate training at institutions in the region building research capacity in africa: equity and global health collaborations features of 20 133 uk patients in hospital with covid-19 using the isaric who clinical characterisation protocol: prospective observational cohort study presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area clinical features and outcomes of hiv patients with coronavirus disease 2019 description of covid-19 in hiv-infected individuals: a single-centre sars-cov-2 co-infected patients in istanbul, turkey hospitalized patients with covid-19 and hiv: a case series clinical characteristics of refractory covid-19 pneumonia in wuhan, china clinical characteristics of 145 patients with corona virus disease 2019 (covid-19 epidemiologic and clinical characteristics of 26 cases of covid-19 arising from patient-to-patient transmission in liaocheng, china clinical course of 2019 novel coronavirus disease (covid-19) in individuals present during the outbreak on the diamond princess cruise ship covid-19 in patients with hiv: clinical case series clinical characteristics and outcomes in people living with hiv hospitalized for covid-19 comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis mortality analyses 4 cases: hiv and sars-cov-2 co-infection in patients from long island clinical features and outcome of hiv/ sars-cov-2 co-infected patients in the bronx sars-cov-2: recent reports on antiviral therapies based on lopinavir/ritonavir, darunavir/ umifenovir, hydroxychloroquine, remdesivir, favipiravir and other drugs for the treatment of the new coronavirus could hiv infection alter the clinical course of sars-cov-2 infection? when less is better t people living with hiv at higher risk for developing severe coronavirus disease 2019 (covid-19)? aids patient care stds infection of severe acute respiratory syndrome coronavirus 2 in a patient with acquired immunodeficiency syndrome covid-19 in patients with hiv recovery from covid-19 following hepatitis c, human immunodeficiency virus infection, and liver transplantation recovery from covid-19 in two patients with coexisted hiv infection computed tomography imaging of an hiv-infected patient one case of coronavirus disease 2019 (covid-19) in a patient co-infected by hiv with a low cd4(+) t-cell count early virus clearance and delayed antibody response in a case of covid-19 with a history of co-infection with hiv-1 and hcv co-infection of sars-cov-2 and hiv in a patient in wuhan city severe sars-cov-2 pneumonia in a 58-year-old patient with hiv: a clinical case report from the republic of cyprus covid-19 in people living with human immunodeficiency virus: a case series of 33 patients a case of sars-cov-2 infection in an untreated hiv patient in tokyo, japan a case of hiv and sars-cov-2 co-infection in singapore hiv and sars-cov-2 co-infection: the diagnostic challenges of dual pandemics covid-19 in an hivpositive kidney transplant recipient a case series of five people living with hiv hospitalized with covid-19 in covid-19 in a patient with hiv infection predisposition or protection? covid-19 in a patient on lvad support with hiv/aids. jacc case rep covid-19 pneumonia in patients with hiv-a case series hiv/sars-cov-2 coinfection: a global perspective key: cord-312194-1jiaghrb authors: brondani, m.; donnelly, l. title: the hiv and sars-cov-2 parallel in dentistry from the perspectives of the oral health care team date: 2020-09-18 journal: jdr clin trans res doi: 10.1177/2380084420961089 sha: doc_id: 312194 cord_uid: 1jiaghrb objectives: the aim of this study was to unravel the professional and social consequences of covid-19 as compared with the aids pandemic according to oral health care providers, staff, and administrators. methods: an exploratory qualitative inquiry via at-a-distance, semistructured interviews engaged a purposefully recruited sample of oral health care team workers in british columbia. interviews took place between april 20 and may 15, 2020; they were audio recorded, transcribed verbatim, and deidentified for interactive thematic analysis. an inductive process of coding was used to identify themes, subthemes, and categories of information. results: forty-five interviews were conducted with 18 dentists, 12 dental hygienists, 6 certified dental assistants, and 9 administrators; 22 were females. interviews each lasted an average of 48 min. after the transcripts were coded, 3 subthemes emerged: 1) personal protective equipment and universal precautions as commonsense approaches to care during both pandemics; 2) an (un)collapsed world in terms of global lockdowns; and 3) social unrest in terms of the potential for stigma and discrimination caused by both pandemics. these subthemes made up the covid-19–aids parallel theme. conclusion: this study explored the extent to which the current covid-19 pandemic is leading to professional and social consequences when a parallel is drawn with the aids pandemic. this is the first qualitative study that identifies the potential social unrest of the pandemic from the perspective of oral health care providers and administrators. future studies should include other providers across canada, as well the patients receiving oral health care during this pandemic. knowledge transfer statement: the covid-19 pandemic has unraveled potential societal implications in a parallel to the hiv/aids era from the perspectives of oral health care providers and their staff. such implications are changing the way that oral health care is delivered; it may also be leading to social unrest in the form of stigma and discrimination. this study discusses some of these implications from the perspective of oral health care providers and administrators. although virus outbreaks continue to emerge and threaten public health across the globe, the current novel coronavirus disease 2019 pandemic caused by sars-cov-2 (severe acute respiratory syndrome coronavirus 2; zhu et al. 2020) is perhaps the only infection in modern history to bring oral health care to a halt, probably because of its route of transmission via respiratory and saliva droplets (proffitt 2020) . covid-19 also seems to be bringing sweeping changes to personal protective equipment (ppe), universal precautions (ups; mccarthy 2000) , and the way that oral health care is delivered via new clinical protocols and procedures that minimize or do not generate aerosols (coulthard 2020) . these changes can be compared with the modifications that hiv (human immunodeficiency virus)-implicated in the development of aids (acquired immunodeficiency syndrome)-brought to the practice of oral health care back in the 1980s (depaola 2003) . although the current ppe and ups were introduced in dentistry as a response to the hiv outbreak, the hiv/aids era brought to the surface a much more grim reality: the stigma and discrimination (mawar et al. 2005) still faced by many individuals around the world and in canada when accessing oral heath care brondani, phillips, et al. 2016; jessani et al. 2019) . similarly, stigma is currently faced by certain populations due to sars-cov-2 (hanasoge et al. 2020; logie and turan 2020; nature 2020) . however, the extent to which covid-19 has affected access to professional oral health care and led to societal implications from the perspectives of providers and administrators remains unknown. the main objective of this study was to unravel the potential professional and social consequences of covid-19 according to oral health care providers, staff, and administrators in british columbia, canada. of note, the study presented herein is part of a larger qualitative project entitled "structural preparedness during the covid-19 pandemic and the provision of urgent oral health care." although that project was not designed from the outset to explore the covid-19-aids parallel, the issue frequently emerged during interviews with members of the oral health care team and is reported accordingly. the university of british columbia's behavioural research ethics board approved this study (h20-01147). we employed a qualitative inquiry method via individual interviews with dentists, dental hygienists, certified dental assistants, and staff (e.g., administrators and front desk personnel) from across british columbia between april 20 and may 15, 2020. participants were purposefully informed about the study via an email distributed to a provincewide professional list and through snowball sampling via word of mouth. inclusion criteria covered any of the target audience that was unemployed (e.g., offices or practices were closed) or continued to work to treat dental emergencies after the curtailment of oral health care services in british columbia on march 16, 2020 (bc dental association 2020). inclusion criteria targeted participants of any gender and with various years of experience; however, we attempted to establish a somewhat even representation for professional roles (e.g., dentists, dental hygienists), gender, and years of experience in that role (e.g., <10 y and >10 y) whenever possible. participants contacted the first author, who then shared information about the study, the interview process, and the informed consent. the interviews where scheduled in the order in which the emails were received, and they were conducted at a distance via phone, zoom video communication, or microsoft skype platform at a day and time convenient for the participant, given the recommendations for physical distancing during the pandemic. interviews were conducted by one of the authors or by a hired research assistant; interviewers were calibrated by interviewing the first 2 participants in a group format to refine the interview guide. interview questions included but were not limited to the following: 1) "what do you know about the covid-19 outbreak?" 2) "what do you know about the transmission of the virus?" 3) "why is this pandemic relevant to oral health care?" 4) "what do you understand by being prepared to provide oral health care during the pandemic?" while we did not plan to ask questions about hiv/aids in particular, the nature of qualitative inquiry allowed us to probe for that information after participants willingly compared the 2 pandemics from various perspectives. it is important to note that participants used "hiv" and "aids" interchangeably, even though hiv is the virus implicated in the development of aids and not all patients carrying hiv develop aids. a total of 18 dentists (7 females), 12 dental hygienists (11 females), 6 certified dental assistants (all females), and 9 administrators (5 females) participated, ensuring saturation of the information with 45 interviews. interviews lasted an average of 48 min, led to 51 h of audio recordings, and generated >650 pages of transcripts. participants received a $150 honorarium in appreciation for their time. demographic data on gender, profession, work location, and years of practice were documented for information only. audio recordings were transcribed verbatim, deidentified in the order in which they were completed, and analyzed interactively; transcripts were explored for codes and themes concomitantly with the interviews (smith 2005) . between april 29 and may 21, 2020, the 2 authors independently analyzed 22 transcripts each after 1 round of interactive coding of the first transcript to increase rigor of the audit trial. as the authors independently coded and conducted the thematic analyses of different transcripts, they met constantly via conference calls to discuss the codes, categories, and themes identified for consensus. figure 1 presents an example of this coding process in 1 excerpt from 1 of the transcripts, as we have done previously (brondani et al. 2007; donnelly et al. 2016; feng et al. 2018) . as shown in the figure, "coding" refers to an inductive process of identifying specific ideas or labels in the form of a word or words (e.g., "type of virus," "originated in china," "lockdowns") within sentences or excerpts from the transcripts. similar and related codes are then grouped to represent a specific category or subtheme (e.g., "knowledge"). related categories or subthemes are clustered to represent a main theme, including "origin of the virus." this study opted for an inductive coding process grounded on the qualitative data, rather than a deductive process based on a predefined set of codes, so that important ideas were not overlooked (smith 2005) . from the 45 interviews, 33 participants discussed hiv/aids in one way or another, which allowed us to identify 3 subthemes: 1) ppe, ups, and common sense; 2) an (un)collapsed world; and 3) social unrest. these 3 subthemes were identified under the covid-19-aids parallel as the main theme and are presented in figure 2 . to contextualize the themes, the professional role, work location, and years of experience are added to the excerpts from the transcripts when applicable. across all the interviews, there was no question that the current practice of dentistry and dental hygiene could not be envisioned without the use of protective gloves and masks as a minimum: "we take the necessary precautions to promote a safe environment for us and our patients by removing gloves and masks after every appointment" (dental hygienist with 15 y of practice in the interior of british columbia). specifically, 21 participants took the opportunity during their interview to draw parallels between hiv and sars-cov-2 in terms of ppe and ups. for a certified dental assistant working for >30 y in the same office in northern british columbia, barehand dentistry was the norm until the arrival of hiv: when i first started we didn't use masks or gloves. but that all changedwe were all tested for hiv. and we were very mindful of that and that was when we started wearing glasses, and masks and gloves. now you would never consider working on somebody without any of it. the idea of pre-hiv era barehand dentistry was highlighted by 9 other participants. for 7 other participants, including a dentist who was practicing in vancouver island in the 1980s, the parallel between covid-19 and aids surfaced when commenting on the sterilization of instruments: i think it's just like when hiv/aids happened in the 80s. we used to disinfect our hand instruments with cavicide, and there was not a big deal. then [autoclaves] became mandatory. now [with covid-19] i think it's going to definitely be changes in a lot of the sterilization again. in the words of a dentist who worked on vancouver island for 4 decades, "i remember in the 80s we used to wash any linen, apron, or towel when we were concerned about hiv because we didn't have enough knowledge. we washed them in the washing machine under extremely hot water." however, for many participants, unrest around covid-19 was related to the way that the virus can be transmitted during an appointment: "if before we were worried about hiv and blood, now we will be much more concerned with aerosols and droplets, and they are everywhere, every time care is provided" (certified dental assistant working in vancouver for >20 y). this unrest seems to be predominantly influenced by a lack of a full understanding about transmissibility and the perceived impact of the virus on the cost and time associated with minimizing or eliminating the risk of transmission within a dental setting. all interviewees acknowledged that covid-19 has affected their lives in one way or another. according to a senior administrator working for almost 30 y at a community dental clinic in the greater vancouver, "this covid-19 pandemic is impacting both the demand and supply sides of dental care, and has also major implications in the lives of our patients who might have lost their jobs, and locked at home, and are now struggling to feed their families." in fact, the lockdown experienced by many cities and countries worldwide due to the covid-19 pandemic was questioned by a dental hygienist who started working in the 1980s in the greater vancouver area, as she recalled what happened when hiv/aids emerged: hiv has infected millions of people and if you remember back then, nothing shut down because of aids. the whole world didn't shut down, right. i mean, it was definitely awful, but it wasn't like the whole world pandemic that we now have with covid-19. while considering the lockdown, 12 participants, including a junior dentist <6 y into practice in northern british columbia, brought up the issue of travel bans: "it seems intuitive to close the border if you want to prevent the spread of a disease and contain the infection. but at what cost? i'm not sure it is even working the way it should." a similar yet unparalleled reality was drawn among the sars outbreak in the early 21st century, hiv, and covid-19 according to a senior dental hygienist: for one, canada did not do well with sars because there was just not enough understanding, there wasn't enough response. with hiv, we just treated everybody as infected. but now there is a huge earthquake in our lives that this is causing. one of the participating dentists who started practicing in a small city in british columbia in the earlier hiv pandemic mentioned the issue of transmission of the virus when drawing an (un)parallel between covid-19 and hiv: i do not think they are comparable, no. what comes to mind is that hiv is [a] blood-and other body fluids-borne disease. it is almost impossible to get in the dental office compared to covid-19 through saliva droplets, which is everywhere. for 17 other participants, the relative unrest around covid-19 will likely be transient as it was for hiv, and some of the changes might compose a new norm for years to come. the comparison between covid-19 and hiv also surfaced in terms of profiling and stereotyping those who might have the disease, as mentioned by 6 participants. the following dialogue between the interviewer and a junior practicing dentist in the interior of british columbia for 8 y exemplifies this: for numerous participants, the professional duty remains to make all patients comfortable in the dental chair regardless of who they are or what they have. for a dental hygienist with >35 y of experience working in the vancouver area, it was a matter of welcoming all patients: "with aids i felt confident and i wanted to welcome all patients. . . . i didn't want them to feel stigmatized, and the same with covid patients now." we were also told, "we just treat everybody the same way. [it] is that simple, and it should also be the same now" (dental hygienist working on vancouver island since 1985). the protocol of doubling gloves and masks during the hiv outbreak might come back during the covid-19 pandemic according to a senior dentist working for >30 y in the interior of british columbia. he told us that covid-19 "is going to bring back that piece of protocol [with] the double gloving during aids." another (un)parallel was related to the way that the viruses implicated in the development of those diseases are transmitted. we were told by a dentist with 38 y of experience in vancouver that hiv, it is of low [transmission], if any, in a dental office, with our current universal precautions. as a blood-borne disease, it is much more difficult to get in the dental office, for example, compared to this one that is much easier via saliva droplets and aerosols. according to a young practicing dentist, the almost identical parallel between covid-19 and hiv/aids was based allegedly on the origin of the viruses: this information is coming from everywhere. what i know is that is about a virus, allegedly from a live animal meat market in wuhan, china. isn't that the same history behind hiv back in the 80s, from slaughtering monkeys in the [african] jungles, that was transmitted to us? our main objective with this qualitative inquiry was to unravel the potential professional and social implications of the covid-19 pandemic according to members of oral health care teams from across british columbia, canada. figure 2 shows the intertwined relationship of the covid-19-aids parallel with 3 subthemes identified from 33 transcripts: 1) ppe, ups, and common sense (from barehand dentistry to education and awareness); 2) an (un)collapsed world (from lockdowns to a new reality); and 3) social unrest (from unknown to welcoming patients). these 3 subthemes might overlap in meaning and relevance and are mapped in relation to the main theme indicated via single solid arrows. subtheme are composed of their respective codes. while some of these codes are linked with the subtheme via solid double arrows to denote a mutable relationship (e.g., an [un]collapsed world and "lockdown"), others are linked within themselves to portray interdependency (e.g., barehand dentistry with "awareness and education" and "shift in protective equipment"). the dashed double arrow shows a reversible relationship between codes, such as "virus transmissibility" and "lockdown": as new information and a better understanding about the transmission of the virus emerge, some lockdowns may be eased. as acknowledged by many participants, the arrival of hiv at a time when dentistry was practiced mostly barehanded introduced the concept of ppe and ups (mccarthy 2000) as providers became more aware and educated about infection control (depaola 2003; mccarthy 2000) . ppe and ups are now part of a commonsense approach to infection control that treats every patient and one's bodily fluids as infectious for any blood-borne pathogen, even when the patient is unaware of the infection or is asymptomatic. hiv transmissibility is now widely known, and the development of aids has been successfully curtailed by medications (hulla and montaner 2013) , which is not yet the case for covid-19 as of july 2020 (rabby 2020; sanders et al. 2020) . participants also discussed the fact that awareness and education around hiv/ aids helped them to better understand the risks of transmission of that disease in the context of a dental office. the same can be said about covid-19, as new information about disease transmission can only increase professional knowledge and improve practice skills in providing care to patients who are sars-cov-2 positive . unlike covid-19, the aids era did not cause a global collapse and lockdowns, as the diseases have very different transmissibility pathways, via respiratory droplets (zhu et al. 2020 ) and bodily fluids (shaw and hunder 2012) , respectively. although transmission seems to be heightened by a potential airborne transmission route relevant for the spread of sars-cov-2 (setti et al. 2020) , travel bans and restrictions have been widely criticized (petersen et al. 2020) , given that they failed to effectively affect the epidemic's trajectory (chinazzi et al. 2020 ) when used at random without careful risk assessment (devi 2020). however, unlike the travel bans for individuals who are covid-19 positive that are being lifted <4 mo after the initial outbreak, >40 countries still impose travel restrictions on those living with hiv almost 4 decades after the first cases were reported in north america (unaids 2019). in fact, it has been only 10 y since the united states lifted a 22-y restriction on immigration and travel for those living with hiv/aids (winston and beckwith 2011) . these few months of social/physical distancing, self-isolation, and travel restrictions due to covid-19 have already decreased the workforce across all economic sectors, have caused job loss levels not seen since the 1930s great depression, and have created economic crises and recessions globally (nicola et al. 2020) . although physical distancing interventions seemed to reduce the covid-19 incidence across many countries (islam et al. 2020) , there is a harsh social reality emerging. as voiced by at least 6 participants, the covid-19 pandemic is leading to a number of hate crimes and to anti-asian sentiment, as china is being blamed for the outbreak (chen and trinh 2020; schild et al. 2020) . the way that the covid-19 pandemic is fueling racism and discrimination against asian individuals is unsettlingly, similar to the constant prejudice that gay men still face in association with hiv/aids (mawar et al. 2005) , particularly when accessing oral health care donnelly et al. 2016; jessani et al. 2019) . such stigma, discrimination, and prejudice are a constant for certain ethnicities and religious groups that have been experiencing xenophobia (suleman et al. 2018; hanasoge et al. 2020) and anti-semitism (kogan 2017) . as advocated by chen and trinh (2020) , "if we can unite to overcome a pandemic of epic proportions, certainly we can also confront the socioracial issues [from it]." the idea brought forward by 1 participant to double glove when treating patients with covid-19 does not ease the experience of stigma, and it is probably unjustified given the transmission route of sars-cov-2. despite double-gloving techniques being suggested as an effective means of infection control for high-risk medical surgical procedures involving patients who may have blood-borne infections (padhye et al. 2011; lipson et al. 2018) , some patients who are hiv positive may feel stigmatized if they see the dentist overusing this measure (patel et al. 2015) . additionally, in terms of the origin of both viruses, participants made reference to the fact that sars-cov-2 and hiv are zoonotic in nature (sharp and hahn 2011) , albeit from different animals and a consequence of cross-species infections. however, the extent to which the parallels regarding the origin of the 2 diseases are implicated in the current social unrest discussed by our participants remains unknown. as voiced by the participants of this study, the covid-19 pandemic has unraveled potential societal implications in a parallel to hiv/aids. such implications are likely changing the way that oral health care is delivered for the time being, and it may be leading to social unrest in the form of stigma and discrimination. saturation helped to achieve rigor in this qualitative study, as no new information was provided on the issue under investigation after 45 interviews (saunders et al. 2018) . rigor was also attained by conducting member checking at different stages of the study, where participants were given the opportunity to read their transcripts and/or the thematic analysis and/or the final report (birt et al. 2016) ; however, only 2 participants provided member checking on the thematic analysis, and they did not suggest any changes. our study has several limitations. the focus on british columbia may prevent generalization despite the data reaching saturation. this qualitative research was also highly contextual, as it took place within western canada in a specific period during a worldwide pandemic and might not be readily duplicated. the remote mode of the interviews might have been impersonal when compared with the commonly employed face-toface method and might have prevented us from assessing essential nonverbal cues; yet, it gave us the opportunity to engage with participants from across the province without the need to travel. the honorarium paid to the participants may have attracted those who might have expressed their ideas in a more socially desirable manner. however, the interviewers let the participants be at ease and openly discuss their thoughts. although we tallied the number of times that hiv-related information, codes, and subthemes were assigned within the transcripts, we did not perform any statistical evaluation of these frequencies, nor did we interpret such frequency as a sign of relevance or importance necessarily. future studies are necessary to include the opinions of a larger group of oral health care providers in british columbia and across canada, given the interprovincial differences in professional regulations and population composition. the perspectives of the patients themselves should be sought, particularly around the social unrest experienced as recipients of oral health care during this pandemic. last, follow-up inquiries should explore the roots of covid-19 stigma, how prior destigmatizing interventions can contribute to tackle the discrimination related to sars-cov-19, and how different stigmatizing conditions influence one another. this study explored the extent to which the current covid-19 pandemic is leading to consequences, socially and in terms of professional practice, when a parallel is drawn with the aids pandemic, according to the views of oral health care providers and administrators. the interactive thematic analysis revealed the major theme as the covid-19-aids parallel with 3 subthemes: common sense around ppe and ups, a world that is collapsed in some ways but not others, and a potential social unrest surfacing in terms stereotyping certain patients. this is the first qualitative study that identifies the potential implications of the pandemic when compared with the hiv/aids era to the practice of oral health care. future studies should include oral health care providers across canada, as well as the patients receiving oral health care during this pandemic. m. brondani, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; l. donnelly, contributed to data analysis and interpretation, critically revised the manuscript. both authors gave final approval and agree to be accountable for all aspects of the work. covid-19 information for dental patients member checking: a tool to enhance trustworthiness or merely a nod to validation? elders' assessment of an evolving model of oral health i'm not hiv positive, i'm undetectable": community forum views of people living with hiv/aids and issues of self-stigma stigma experienced by people living with hiv when accessing oral care anti-asian sentiment in the united states-covid-19 and history the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak dentistry and coronavirus (covid-19)-moral decision-making managing the care of patients infected with bloodborne diseases travel restrictions hampering covid-19 response stigma experiences in marginalized people living with hiv seeking health services and resources in canada evaluating point-of-care hiv screening in dental hygiene education settings: patient, faculty, and student perspectives visibility challenges for asian scientists hiv treatment as prevention: the key to an aids-free generation physical distancing interventions and incidence of coronavirus disease 2019: natural experiment in 149 countries dental care utilization: patterns and predictors in persons living with hiv in british columbia anti-semitism and xenophobia covid-19: the need for continuous medical education and training practice and attitudes regarding double gloving among staff surgeons and surgical trainees how do we balance tensions between covid-19 public health responses and stigma mitigation? learning from hiv research the third phase of hiv pandemic: social consequences of hiv/aids stigma and discrimination and future needs universal precautions stop the coronavirus stigma now the socio-economic implications of the coronavirus and covid-19 pandemic: a review efficacy of double gloving technique in major and minor oral surgical procedures: a prospective study hiv-related stigma in the dental setting: a qualitative study covid-19 travel restrictions and the international health regulations-call for an open debate on easing of travel restrictions what will be the new normal for the dental industry? current drugs with potential for treatment of covid-19: a literature review pharmacologic treatments for coronavirus disease 2019 (covid-19): a review saturation in qualitative research: exploring its conceptualization and operationalization go eat a bat, chang!" an early look on the emergence of sinophobic behavior on web communities in the face of covid-19 airborne transmission route of covod-19: why 2 meters/6 feet of inter-personal distance could not be enough origins of hiv and the aids pandemic hiv transmission. cold spring harb perspect med interactive qualitative analysis: a systems method for qualitative research xenophobia as a determinant of health: an integrative review no end to aids without respecting human rights the impact of removing the immigration ban on hivinfected persons a novel coronavirus from patients with pneumonia in china the authors are in deep gratitude to all 45 participants who engaged into a conversation around their ideas, struggles, concerns, and life-changing experiences. the authors acknowledge the other members of the research team: drs. fernanda almeida, kavita mathu-muju, hsingchi von bergmann, and tala maragha and the 2 trainees, denise cua and melody shayanfar. a special thanks goes to dr. angela tether for editorial contributions. the authors confirm that this study is original and has not been submitted for publication elsewhere, and they approve all aspects of the research, including this article. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: funding for this study was obtained via the genome bc rapid response funding for covid-19 research and innovation project (cov092). key: cord-281941-97t45w73 authors: zhou, daijun; mei, qiang; li, jintao; he, haiyang title: cyclophilin a and viral infections date: 2012-08-10 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2012.07.024 sha: doc_id: 281941 cord_uid: 97t45w73 abstract cyclophilin a (cypa) is a peptidyl-prolyl cis/trans isomerase originally identified as the target of the immunosuppressive drug cyclosporine a. a number of reports have demonstrated that cypa plays a critical role in the successful replication of viruses such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), hepatitis b virus (hbv), etc. however, recent studies demonstrated that cypa also possesses a repressive effect on the replication of some viruses like influenza a virus and rotavirus. moreover, cypa could also regulate host ifn-i response to viral infections. together, these evidences showed diverse roles of cypa in viral infection. cypa belongs to a family of highly conserved and ubiquitous proteins known as cyclophilins (cyp). this family possesses peptidyl-prolyl cis/trans isomerase activity [1] and acts as an acceleration factor in protein folding and assembly. till now more than 10 members of cyp subtypes have been found in mammals, including cypa, cypb, cypc, cypd, cype, cyp40, ranbp2, etc. among them, cypa is the most abundant subtype and is widely distributed in almost all tissues. in addition to its role in protein folding and assembly, the ppiase activity of cypa has recently been demonstrated to have other roles, including intracellular trafficking, signal transduction, transcription regulation, cell cycle regulation, and stress response [2] . cypa is commonly believed to be an important immune molecule. cypa was originally identified as the target of the immunosuppressive drug cyclosporine a (csa), which is used clinically for the prevention of graft rejection after organ transplantation [3] . in mammals, csa-cypa complex binds to and inhibits calcineurin (cn), prevents the dephosphorylation and nuclear translocation of nf-at, and at last leads to immunosuppression [4] . cypa plays a vital role in regulating immune response. cypaknockout mice have an ''allergic'' phenotype with increased serum igg1 and ige levels and tissue infiltration by mononuclear cells, eosinophils and mast cells. this phenotype is related to increased and dysregulated activity of th 2 cd4 + t cells [5] . interleukin-2 tyrosine kinase (itk) is a member of the tec family of sh 2 /sh 3 -containing tyrosine kinases and participates in the signal transduction cascade leading to t cell activation [6, 7] . cypa binds itk and negatively regulates its activity. in cypa-knockout cells, itk is constitutively activated. thus, cypa plays a suppressive role in the development of cd4 + t cell responses through its interaction with itk. in recent years many studies showed that cypa was involved in the pathogenesis of viral infection [8] , cardiovascular disease [9] and cancer [10] . for example, cypa expression can be upregulated in viral infections and correlates with final result of infection [11, 12] . cypa plays a critical role in the successful replication of viruses such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), hepatitis b virus (hbv), etc. however, cypa possesses a repressive effect on the replication of some viruses like influenza a virus and rotavirus. in this review we focused our interest on these diverse roles of cypa in viral infections. moreover, the emerging function of cypa on regulating host ifn-i response to viral infections was also discussed. cypa gene polymorphisms have been demonstrated to influence susceptibility to hiv-1 and disease progression. a1604g and c1650g in the promoter region of the cypa gene might affect cypa expression levels and thus affect host susceptibility to hiv-1 and disease progression [13] . furthermore, the a1650g polymorphism in the regulatory region of cypa gene may also be associated with protection from hiv-1 infection in participants of the amsterdam cohort studies on hiv-1 infection and aids [14] . hiv-1 replication was decreased in human cd4 + t cells when cypa was knocked out [15] . these studies suggest cypa may play an important role in promoting hiv infection. the efficient incorporation of cypa into the hiv-1 virion is mediated through the direct binding of the prolyl peptide bond, located in a proline-rich loop of the fourth and fifth helices of the hiv-1 ca, and the active sites of cypa in the context of gag polyprotein [16] . importantly, the proline residue at position 90 (p 90 ) and the glycine residue at position 89 (g 89 ), which are both highly conserved among hiv-1 gag polyproteins, appear to be important for this interaction [17] . cypa is packaged into hiv-1 virions during viral replication at a molar ratio of 1:10 cypa/ca [18] . disruption of its incorporation by gag mutations or by treatment with csa attenuated the infectivity of progeny viruses [19] . therefore, packaging of host cypa into hiv particles is an important step in hiv morphogenesis and essential for hiv replication. the viral protein r (vpr) of hiv-1 is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the pre-integration complex into the nucleus and the induction of g2 host cell cycle arrest [20] . the investigation of conformational heterogeneity of the proline residues in the n-terminus of vpr suggested a functional interaction between vpr and a host cypa [21] . csa blocked expression of vpr without affecting vpr transcription, intracellular transport, or virus incorporation. similarly, vpr expression is also reduced in hiv-1 infected cypa-knockout t cells [22] . in the absence of cypa activity, the vpr-mediated cell cycle arrest is completely lost in hiv-1-infected t cells. however, other study showed an opposite opinion that the interaction with cypa is not essential for the induction of g2 cell cycle arrest by vpr [23] . nef is another hiv accessory protein. it is believed that optimal hiv-1 infectivity requires the presence of both nef and cypa during virion assembly and these factors facilitate a step in the viral life cycle between penetration and reverse transcription. a genetic dissection study of the relative contributions of nef and the cyclophilin a-gag interaction to hiv-1 infectivity demonstrated that nef was not required for incorporation of cypa into hiv-1 virions and vice versa. surprisingly, cypa-deficient virions remained sensitive to inhibition by csa, in a manner that was strongly dependent on the presence of a functional nef gene [24] . cypa-nef fusion protein enhanced the infectivity of nef-defective hiv-1 particles and was specifically incorporated into the virions via association with gag during particle assembly [25] . together, these results demonstrate that nef and cypa may act independently but complementary to render hiv-1 particles fully infectious. cd147 has been identified as the main signaling receptor for cypa on human leukocytes [26] . cd147-cypa interaction may regulate an early step in hiv-1 replication [27] . cypa-cd147 interaction might induce ma phosphorylation to regulate the detachment of the rt complex from the membrane or promote transition from the step of hemifusion to complete fusion, allowing liberation of the rt complex into the cytoplasm [28] . on the other hand it is proposed that cypa-cd147 interaction might indirectly affect ca conformation leading to destabilization of the ca shell [29] . cypa mediates hiv-1 attachment to target cells via heparans, and heparans facilitate cypa-cd147 interaction by first binding cypa and then presenting it to cd147 [30] . therefore, cypa-cd147 interaction may be downstream of cypa-heparan interaction. moreover, the facilitative effect of cypa-cd147 interaction on hiv-1 replica-tion is signaling-independent but probably through specific events mediated by the cytoplasmic domain of cd147 [31] . similar to hiv-1, there were several evidences that cypa is also required for hcv replication. knockdown of endogenous cypa significantly hampers hcv rna replication and viral protein expression [32] and mutation of the residues that reside in the hydrophobic pocket of cypa (histidine in position 126 and arginine in position 55) failed to restore hcv replication [33] . cypa increased the affinity of the polymerase to viral rna by interacting with the ns5b and enhanced hcv replication [34] . these data are in accordance with the finding that the isomerase pocket of cypa serves as a binding site for ns5b polymerase. these findings indicate that cypa enhances hcv replication by catalyzing the comformational change of ns5b. interestingly, recent studies suggest that cypa inhibition may also act on the hcv ns5a protein. ns5a is anchored to the cytoplasmic side of endoplasmic reticulum (er) membrane via an amphipathic n-terminal a-helix [35] and is composed of three cytoplasmic domains: d1 (residues 27-213), d2 (residues 250-342) and d3 (residues 356-447). although sequence conservation of ns5a-d2 and -d3 in hcv genotypes is significantly lower than d1, it was reported that ns5a-d2 from the hcv jfh1 strain was the substrate for the ppiase activities of cypa [36] . in addition, mutations in d2 and d3 of ns5a that interacts with cypa could significantly influence viral rna replication and thus confer resistance to cyclophilin inhibitor [37] . furthermore, verdegem et al. [38] revealed that cypa has in vitro ppiase activity toward some, but not all of the peptidyl-prolyl bonds in ns5a-d3. this finding provided novel insights into the structure of ns5a-d3 and suggested that the interaction with cypa might involve more than one specific ns5a domain. besides the role as a cis/trans prolyl isomerase, cypa has also been implicated in cholesterol transport. it has been shown that a complex consisting of cypa, heat-shock protein 56, cyclophilin 40 and caveolin transports newly synthesized cholesterol from the endoplasmic reticulum to caveolae [39] . hcv depends on cholesterol and any disturbance in cholesterol level or distribution decreases its replication [40] . furthermore, cypa has been involved in transcription regulation and over-expression of cypa was shown to interact with sin3a/rdp3 hdac complexes and regulate hdac-dependent gene silencing in yeast [41] . the orthologous complex msin3a/histone deacetylase 2 (hdac2) was further found to modulate the transcription of the myc target gene, carbamoyl-phosphate synthase-aspartate carbamoyltransferasedihydroorotase (cad) in humans [42] . cad is a crucial enzyme in the pyrimidine de novo biosynthesis that hcv replication is known to depend on [43] . so it is possible that cypa might play some important indirect role in hcv infection. the hbv genome encodes the envelope proteins, the core protein, the polymerase protein and a transactivating x protein [44] . hbv produces three envelope glycoproteins collectively known as hbv surface antigen (hbsag), including the large (lhbs), middle (mhbs), and small (shbs) surface proteins [45] . among them, the shbs protein is predominant and is an important viral component that reacts with the immune system of the host. cypa, as a target protein interacting with shbs proteins, was found to be markedly decreased not only in the liver of hbsag transgenic mice, but also in an hbsag expressing cell line. however, when supernatants from transfected cells expressing hbsag were assayed for cypa, the amount of cypa was found to be higher than that from the controls, suggesting that the decrease of cypa in hbsag positive cells was due to increased secretion of cypa [46] . since both shbs and cypa are secreted via the vesicular secretion pathway [47] , the interaction between shbs and cypa may be directly or indirectly bridged by some cellular components. it is likely that cypa binds to shbs and is secreted along with hbsag particles. under physiological conditions, cypa is an intracellular protein; however, during inflammation, cypa can be secreted [48] . therefore, the expression of hbsag could be speculated to affect cellular functions similar to that of inflammation, resulting in secretion of cypa from hbsag-positive hepatocytes. in addition, the decrease of cypa observed in hbsag expressing cell lines may also affect protein unfolding and contribute to the pathogenesis of hbv infections as in hiv [49] . matrix protein (m1) is the most abundant structural protein that plays a crucial role in the replication, assembly and budding of influenza a virus [50, 51] . cypa was found in the core of the influenza virion [52] and was up-regulated upon infection by avian h9n2 influenza virus in ags cells (a human gastric carcinoma cell line) [53] . liu et al. [12, 54] revealed that cypa interacted with the m1 protein both in vitro and in vivo and affected the early stage of viral replication. over-expression of cypa restricted influenza a virus replication, while the depletion of endogenous cypa resulted in enhanced production of influenza a virus. in addition, the infectivity of influenza virus increased in the absence of cypa. cypa had no effect on viral genome replication or transcription and it also did not impair the nuclear export of viral mrnas. however, it was found that cypa could significantly enhance the degradation of m1 protein through the ubiquitin/proteasome-dependent pathway, indicating that cypa restricts influenza virus replication through accelerating degradation of the m1 protein. notably, the cypa r55a mutation could still bind to the m1 protein and inhibit influenza virus replication, suggesting that the restrictive effect of cypa on influenza virus infection was independent of its ppiase activity. cypa is also associated with the life cycle of other viruses including vaccinia virus (vv), vesicular stomatitis virus (vsv), and severe acute respiratory syndrome coronavirus (sars-cov) [55] . vv infection led to an impressive increase in cypa stability whereas in vv infected cells, cypa relocalized to the peripheral region of the nucleus, colocalizing with sites of virus production. cypa was then incorporated into the virus particle during morphogenesis and localized specifically in the core [1] . in addition, cypa has been found associated with vsv, a negative stranded rna virus. cypa interacts with the nucleocapsid protein of vsv and, like vv, incorporated into vsv viral particles where it likely acts as a chaperone for the nucleocapsid protein that wraps the genomic rna to produce the functional template for transcription [56] . cypa was also reported to regulate sars-cov replication through binding to the nucleocapsid protein and being incorporated into particles [57, 58] . rotavirus (rv) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. our study showed that rv infection triggered a temporal increase of cypa protein. in rv infection, cypa was recruited to the viroplasm of rv in ma104 cells and herein interacted with rv structural protein vp2 to repress rv infectivity to ma104 cells and inhibit rv reproduction through its ppiase activity (unpublished data). it has been shown that cypa is excluded from wild-type simian immunodeficiency virus (sivagm) particles but is efficiently packaged into vif-deficient sivagm virions. the presence of cypa in vifdefective sivagm was correlated with reduced viral replication. infection of cypa-knockout jurkat cells or treatment with cyclosporine a eliminated the vif-sensitive inhibition and resulted in replication profiles similar for wild-type and vif-deficient sivagm [59] . therefore, cypa was predicted as a novel vif-sensitive antiviral factor that may limit zoonotic transmission of sivagm or hiv. dendritic cells have a central function in the host defence, linking innate immunity to microbes to activate pathogen-specific adaptive immunity. recently it has been reported that cypa in dendritic cells could recognize the newly synthesized hiv-1 ca domain and subsequently prompt a ifn-i response through activation of irf3, which is dependent on the ppiase activity of cypa. thus it is concluded that cypa functions as a cell-intrinsic sensor of viral infection able to recognize the newly synthesized viral capsid proteins [60] . in our study we also found that cypa was required for the host ifn-i response in rv infection of ma104 cells, however, this was not dependent on ppiase activity but on the jnk signaling pathway [61] . redistribution of cyclophilin a to viral factories during vaccinia virus infection and its incorporation into mature particles acetylation regulates cyclophilin a catalysis, immunosuppression and hiv isomerization pro-and anti-cancer effects of immunosuppressive agents used in organ transplantation the cyclophilins cyclophilin a-deficient mice are resistant to immunosuppression by cyclosporine regulation of the tyrosine kinase itk by the peptidyl-prolyl isomerase cyclophilin a mechanistic insight into the role of transitionstate stabilization in cyclophilin a cyclophilin and viruses: cyclophilin as a cofactor for viral infection and possible anti-viral target cyclophilin a: promising new target in cardiovascular therapy cyclophilin a: potential functions and therapeutic target for human cancer cyclophilin a interacts with influenza a virus m1 protein and impairs the early stage of the viral replication cyclophilin a restricts influenza a virus replication through degradation of the m1 protein polymorphisms in the regulatory region of the cyclophilin a gene influence the susceptibility for hiv-1 infection cyclophilin a regulates hiv-1 infectivity, as demonstrated by gene targeting in human t cells regulatory polymorphisms in the cyclophilin a gene, ppia, accelerate progression to aids absconding with the chaperone: essential cyclophilin-gag interaction in hiv-1 virions catalysis of cis/trans isomerization in native hiv-1 capsid by human cyclophilin a contribution of cyclophilin a to determination of simian immunodeficiency virus tropism: a progress update vpr and its interactions with cellular proteins structural characterization of the hiv-1 vpr n terminus: evidence of cis/trans-proline isomerism cyclophilin a interacts with hiv-1 vpr and is required for its functional expression induction of g2 arrest and binding to cyclophilin a are independent phenotypes of human immunodeficiency virus type 1 vpr mechanistic independence of nef and cyclophilin a enhancement of human immunodeficiency virus type 1 infectivity nef enhances hiv-1 infectivity via association with the virus assembly complex preferential chemotaxis of activated human cd4+ t cells by extracellular cyclophilin a dealing with the family: cd147 interactions with cyclophilins cd147 facilitates hiv-1 infection by interacting with virus-associated cyclophilin a, proc. nat. acad. sci human immunodeficiency virus type 1 gag protein binds to cyclophilins a and b host cyclophilin a mediates hiv-1 attachment to target cells via heparans cd147 stimulates hiv-1 infection in a signal-independent fashion the isomerase active site of cyclophilin a is critical for hepatitis c virus replication cyclophilin inhibitors for the treatment of hcv infection critical role of cyclophilin a and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis c virus replication complex structure and function of the membrane anchor domain of hepatitis c virus nonstructural protein 5a hepatitis c virus ns5a protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins a and b identification of residues required for rna replication in domains ii and iii of the hepatitis c virus ns5a protein domain 3 of ns5a protein from the hepatitis c virus has intrinsic {alpha}-helical propensity and is a substrate of cyclophilin a annexin 2-caveolin 1 complex is a target of ezetimibe and regulates intestinal cholesterol transport multiple cyclophilins involved in different cellular pathways mediate hcv replication cyclophilin a and ess1 interact with and regulate silencing by the sin3-crpd3 histone deacetylase msin3a/histone deacetylase 2-and prmt5-containing brg1 complex is involved in transcriptional repression of the myc target gene cad dynamics of subgenomic hepatitis c virus replicon rna levels in huh-7 cells after exposure to nucleoside antimetabolites the hepatitis b virus and common mutants entry of hepatitis b virus: mechanism and new therapeutic target hepatitis b virus (hbv) surface antigen interacts with and promotes cyclophilin a secretion: possible link to pathogenesis of hbv infection hepatitis b virus subviral envelope particle morphogenesis and intracellular trafficking extracellular cyclophilins contribute to the regulation of inflammatory responses proteomic analysis of hepatitis b surface antigen positive transgenic mouse liver and decrease of cyclophilin a restriction of viral replication by mutation of the influenza virus matrix protein identification of hsc70 as an influenza virus matrix protein (m1) binding factor involved in the virus life cycle cellular proteins in influenza virus particles proteomics analysis of differential expression of cellular proteins in response to avian h9n2 virus infection in human cells cyclophilin a interacts with influenza a virus m1 protein and impairs the early stage of the viral replication cd147/ emmprin acts as a functional entry receptor for measles virus on epithelial cells requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a function of hab18g/cd147 in invasion of host cells by severe acute respiratory syndrome coronavirus vif counteracts a cyclophilin a-imposed inhibition of simian immunodeficiency viruses in human cells a cryptic sensor for hiv-1 activates antiviral innate immunity in dendritic cells cyclophilin a inhibits rotavirus replication by facilitating host ifn-i production this work was supported by grants from national natural science foundation of china (no. 81102233) and natural science foundation project of cq cstc (no. cstc2011jja10002). key: cord-304188-1nm1tbig authors: moody, m. anthony title: modulation of hiv-1 immunity by adjuvants date: 2014-04-10 journal: curr opin hiv aids doi: 10.1097/coh.0000000000000052 sha: doc_id: 304188 cord_uid: 1nm1tbig purpose of review: to summarize the role of adjuvants in eliciting desirable antibody responses against hiv-1 with particular emphasis on both historical context and recent developments. recent findings: increased understanding of the role of pattern recognition receptors such as toll-like receptors in recruiting and directing the immune system has increased the variety of adjuvant formulations being tested in animal models and humans. across all vaccine platforms, adjuvant formulations have been shown to enhance desirable immune responses such as higher antibody titers and increased functional activity. although no vaccine formulation has yet succeeded in eliciting broad neutralizing antibodies against hiv-1, the ability of adjuvants to direct the immune response to immunogens suggests they will be critically important in any successful hiv-1 vaccine. summary: the parallel development of adjuvants along with better hiv-1 immunogens will be needed for a successful aids vaccine. additional comparative testing will be required to determine the optimal adjuvant and immunogen regimen that can elicit antibody responses capable of blocking hiv-1 transmission. many hurdles remain for the development of a globally deployable hiv-1 vaccine. elicitation of a durable immune response that can prevent hiv-1 infection or disease will likely require the use of an adjuvant for some or all immunizations. at present in the usa there are only two licensed adjuvants, although other adjuvanted vaccines are licensed in other parts of the world, and many more have been tested in human and animal trials. this review will highlight recent work in adjuvant development for hiv-1 vaccines with particular emphasis on antibody responses. the word 'adjuvant' derives from the french adjuvant, which itself derives from the latin adjuvate that can be translated to 'helper'. the term was first used in a modern vaccine context by gaston ramon of institut pasteur in a series of papers in the 1920s (e.g., [1 && ,2,3 && ]) that established the use of adjuvants for eliciting high-titer antitoxin responses. since that time, many compounds and formulations have been tested for their ability to adjuvant a vaccine response, with the development of new adjuvants paralleling an increased understanding of pattern recognition receptors (prrs) and their role in recruiting and directing the immune system. an adjuvant is a compound, formulation, preparation, or delivery system that enhances or modifies the immunogenicity of the primary antigen in a vaccine. adjuvants perform this function in a variety of ways, but nearly all involve the triggering of prrs to stimulate the innate and adaptive arms of the immune system. this is accomplished in one of two ways -through the incorporation of active compounds in a vaccine formulation (e.g., formulating a protein immunogen in a liposome containing a tlr4 agonist) or by incorporating elements in the vaccine that result in the production of immune stimulants (e.g., addition of plasmids expressing cytokines in a dna vaccine regimen). these distinctions are not absolute, and some formulations incorporate elements of both approaches. the development of adjuvants has accelerated in the last 25 years and has to some degree paralleled the development of hiv-1 vaccine candidates. during that time, a number of excellent reviews have been published [1 && ,2,3 && ,4-8] that the reader may find useful. this review will focus on the historical context of adjuvant development since the discovery of hiv-1, recent developments, and finally will highlight the lack of comparative data currently available. shortly after the discovery of hiv-1, then secretary of health and human services margaret heckler held a 1984 press conference in which she predicted that vaccine trials against hiv-1 would be possible within 2 years [9] . the first vaccine trial began in 1986 [10, 11] , and was followed by a series of attempts to develop an effective hiv-1 vaccine. early vaccine studies focused on leveraging strategies that had been successful for other vaccines including virus inactivation [12] [13] [14] and subunit immunogens [15] along with novel strategies such as recombinant viral constructs [11] . although early subunit vaccine candidates were immunogenic [16] , none of the follow-up efficacy trials showed protection [17, 18] . concurrent with the development of vaccine candidates, numerous animal and human studies compared available adjuvants in head-to-head trials. no clearly superior regimen was identified, likely because of the lack of a consistent immunogen across trials along with differing immunization schemes and different outcome measures. for example, mannhalter et al. [19] in 1991 reported the immunization of chimpanzees with a recombinant envelope (env) gp160 using alum, a water-inoil emulsion (termed lipid-based adjuvant), or alum plus deoxycholate. t-cell responses were best for the lipid-based adjuvant and were shown to last for months after the final immunization; antibody responses were not reported. niedrig et al. [12] reported in 1993 on another group of chimpanzees immunized with formaldehyde-inactivated hiv-1 adjuvanted with alum, freund's incomplete adjuvant (an oil-in-water emulsion), or with a zinc hydroxide/lecithin-based adjuvant; in this study, antibody titers were best with the lecithin-based adjuvant, although proliferation and antibodydependent cell-mediated cytotoxicity (adcc) responses were similar between lecithin and alum arms. levi et al. [20] reported in 1993 a comparison in rabbits of alum, iscom, iscomatrix, muramyl dipeptide (mdp), and freund's complete adjuvant with a recombinant gp160 as the immunogen. antibody titers were highest with freund's complete adjuvant and mdp. during the same time period, numerous mouse studies were published and nearly all demonstrated that one adjuvant was superior. these and other head-to-head studies of vaccines are shown in table 1 [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . vaccine candidates deemed the most promising advanced to phase i and phase ii human trials. these studies tested proteins, peptides, and recombinant poxvirus vectors [31] , and although none of the candidates produced overwhelming immunity, the vaccines were generally safe and well tolerated. without a stronger candidate available, a controversial decision was made to pursue a phase iii trial of poxvirus prime-gp120 boost vaccine strategy. the proposal had detractors [32] and supporters [33] , and ultimately demonstrated a modest and shortlived degree of efficacy [34, 35] . the adjuvant used in that trial was alum, the only us food and drug administration (fda)-approved adjuvant at that time. studies are now being considered to examine the same immunization regimen using more potent adjuvants to see whether protection can be enhanced or prolonged. the remainder of this review will address more recent developments in adjuvant research. dna vaccines are attractive for eliciting cd8 þ t-cell responses, as protein production and antigen processing can occur without the need for an infectious vector. dna vaccines are generally not as potent at eliciting antibody responses, although evidence suggests that dna vaccines can prime for subsequent protein boosts [7, 8] . numerous studies have reported the ability of immune modulators to provide an adjuvant effect for dna vaccines [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] ; most of these studies were performed in mice and few compared more than one regimen against an unadjuvanted control. there are no studies a wide variety of adjuvants have been tested for hiv-1 vaccines, but recent comparative data are limited. there are no 'universal' adjuvants, but adjuvants must be selected based on the desired response and carefully paired with the immunogen being tested. heterologous adjuvant strategies may be needed to balance efficacy and side-effects. comparing all available dna-encoded adjuvants, but a few smaller-scale studies have been reported. for example, testing of a series of dna adjuvants in mice suggested that one of the tested adjuvants was superior [e.g., granulocyte/macrophage colony stimulating factor (gm-csf) [50] ], but studies in primates showed a more modest benefit [40] . work is ongoing, but in the absence of a systematic study, at present, it is not clear whether any dna-encoded adjuvant is superior in eliciting desirable immunity for an hiv-1 vaccine. some studies have examined the effect of adding compounds to dna vaccines without having them encoded in a vector. mycobacterial extracts have been shown to enhance t-cell and antibody responses in mice [51] as have tlr9 agonists [52] . liposomes with mannan as a delivery vehicle for a dna vaccine enhanced fecal iga responses and altered subclass responses in mice [53] . another toll-like receptor (tlr) agonist, imiquimod, applied topically adjuvanted a dna vaccine in mice, although the effect was similar to that of gm-csf [54] . as with dna-encoded adjuvant molecules, it is unclear which of these strategies is superior. recent studies have suggested that physical adjuvants may be beneficial for dna vaccines. electrical current as an adjuvant has been tested in mice [55] , rhesus macaques [56, 57] , and humans [58 & ]. the results suggest that electroporation alone is as effective as dna-encoded adjuvants, although side-effects were higher in electroporation groups [58 & ]. electroporation almost certainly acts by increasing uptake of vaccine dna into cells and through minor tissue damage that stimulates damage-associated prrs that recruit an inflammatory response. further testing will be needed to determine whether electroporation can be implemented so as to reduce side-effects yet remain effective. immune stimulatory molecules can be encoded in viral or bacterial vectors that have sufficient room in their genomes (e.g., poxviruses, mycobacteria). as with dna vaccines, studies have tested different adjuvants with mixed results. for poxvirus vectors, cytokines [59, 60] , soluble cd40 ligand [61] , and cd252 [62] have been tested in mice and each enhanced immune responses compared with controls. similar strategies have been tested for other viral vectors (e.g., rhabdovirus [63] ). adjuvants can also be added with the vector but not encoded by it. for example, soluble cd40 ligand added to a dna-prime/poxvirus-boost strategy enhanced t-cell responses though the effect on antibody was variable [64] . it remains to be seen if any of these strategies will ultimately prove useful for human trials. whether other adjuvant formulations can enhance recombinant vectors is under investigation. naito et al. [65] demonstrated in a mouse model that tethering of liposomes to a poxvirus vector overcame previous immunity and could stimulate humoral and cellular immunity. many adjuvants, such as oil-in-water emulsions, can disrupt lipid membranes and so would be considered inappropriate for enveloped replicating vectors like poxviruses. in addition, as replicating vectors stimulate the immune system by the transient infection they cause, it is not clear that an adjuvant that is only transiently present at the site of injection would be useful. future studies will be needed to clarify these questions. for hiv-1 vaccine studies, the greatest variety of adjuvants have been tested for subunit/recombinant protein immunogens. as noted above, a large number of head-to-head trials were performed prior to 2000 (table 1 ), but since that time, few large-scale direct comparisons have been published. older adjuvants continue to be explored to define those parameters critical for efficacy. alum is one of the most commonly employed adjuvants because of its long history of use in humans and the relative ease for regulatory approval; for this reason, research to optimize its utility is ongoing. hansen et al. [66 & ] showed that the ability of alum to adsorb an env protein was important for immunogenicity, but that binding too tightly reduced immune responses after immunization. dorosko et al. showed that alternative methods of delivering alum can direct the immune response; injection of an alum-based peptide immunogen in the region of the supramammary lymph node of goats resulted in antibody secretion into colostrum [67] . novel adjuvants continue to be studied in animal models. lipid-based adjuvants like the as0x series have been shown to stimulate strong antibody responses in guinea pigs, although responses were similar to those elicited by an oil-in-water emulsion adjuvant [68] . one of the adjuvants in this series, as01b, elicited high-titered antibodies in rhesus macaques [69] and was also used in a human hiv-1 clinical trial wherein it generated antibody and t-cell responses [70] . another adjuvant in that series, as02a, also elicited immune responses in humans [71] , but which of the adjuvants in this series is the best for an hiv-1 vaccine is not yet established. oil-in-water emulsions as adjuvants have been used for many years, and include mineral oil-based formulations (e.g., freund's adjuvant) and more modern squalene-based preparations. they have also proved to be useful platforms for exploring the addition of immune stimulants and other compounds. tlr agonists like cpg oligodeoxynucleotides mixed with the squalene-based adjuvant mf59 appeared to enhance the adjuvant effect [72] . the addition of carbopol to mf59 enhanced immunogenicity in rabbits to levels comparable with complete freund's adjuvant, likely because of the slower release of the immunogen [73] . more recently, we reported that combinations of tlr ligands in a different squalene-based oil-in-water emulsion stimulated higher titers of antibodies and a greater breadth of functional responses, and that the combination of tlr7/8 and tlr9 agonists was optimal in rhesus macaques [74] . other adjuvant formulations have been studied as well. liposomes formulated with a modified polyethylene glycol elicited durable antibody responses to an env gp41 peptide; the proposed mechanism was persistence of the modified liposomes leading to a prolonged immune response [75] . compounds derived from pathogens have also shown promise in initial studies. a protein derived from the worm onchocerca volvulus enhanced antibody responses in mice [76] . there have been multiple human trials with env protein immunogens combined with different adjuvant formulations (table 2) [16] [17] [18] 34, 71, [77] [78] [79] [80] . unfortunately, comparative data are lacking, especially head-to-head comparisons of adjuvants using the same immunogen and dosing schedule. the use of adjuvants in humans demonstrates promise; for example, the as02a adjuvant formulated with a env gp120 immunogen was able to elicit similar titers of antibodies despite a 20-fold difference in the high and low immunogen dose groups, suggesting that the adjuvant might have a dose-sparing effect [71] . additional studies will be needed to determine the best adjuvant-immunogen combinations for future large-scale trials. for eliciting mucosal responses, cholera toxin (ct) and other bacterial products have been extensively tested in animal models. ct combined with env gp120 elicited mucosal iga in rhesus macaques [81] ; other studies in rhesus (albeit with a different form of ct) have elicited more mixed responses [82] . ct has also been used to direct responses to the mucosa by combining it with agents that enhance retention at the mucosal surface, and has permitted dose sparing [83] . in addition, modified ct combined with cytokines were able to elicit mucosal antibodies to a peptide immunogen when given to cynomolgus monkeys [84] , suggesting that promising combinations identified by other vaccine strategies (e.g., dna vaccination) might be useful for mucosal immunization. other mucosal strategies are being investigated. interestingly, intranasal cytokines appear to be as effective as ct in eliciting mucosal antibodies in mice [85] . a soybean oil nanoemulsion delivered intranasally with env gp120 elicited iga responses [86] . other bacterial products, like mycoplasmaderived lipopeptides, have been shown to adjuvant vaccines in mice [87, 88] . cranage et al. [89] demonstrated that env gp140 administered with carbopol intravaginally resulted in better mucosal responses than systemic immunization. in mice, thymic stromal lymphopoietin has been shown to elicit mucosal antibody at levels similar to ct [90] . finally, a strategy employing microneedles combined with a tlr4 agonist was able to elicit strong antibody responses including vaginal iga in mice [91 & ]. it is possible that a successful hiv-1 vaccine strategy may involve heterologous immunizations as was used in the rv144 alvac-prime/aidsvaxboost trial [34] . such strategies continue to be investigated in animal models. a regimen containing peptides adjuvanted with imiquimod and an oilin-water emulsion was able to prime for viral vector boosts in rhesus macaques, eliciting strong t-cell and modest antibody responses [92] . similarly, an alphavirus-based particulate vaccine prime combined with a protein boost using mf59 in rhesus macaques resulted in a better response that was also somewhat protective against infectious challenge [93] . side-effect considerations may also drive heterologous prime-boost regimens. a study in rabbits using an oil-in-water emulsion for the prime and alum for the boost showed that antibody responses were highest with the mixed regimen [94] . the authors suggested that the regimen could be used to overcome the undesirable side effects of strongly adjuvanted vaccines by using less reactive adjuvants in subsequent steps. as edelman and tacket [6] aptly stated in 1990, 'the best adjuvant will never correct the choice of the wrong epitope.' for now, the aids vaccine field has not identified the best immunogen(s) and so work continues to find a strategy that will elicit durable and broad protection against infection. the work to find an optimal adjuvant strategy will continue to parallel these efforts. at present, the data to drive rational choices of adjuvants for an aids vaccine are lacking. this is partly because of the lack of a robust immunogen, but it is also because of the paucity of comparative data being published. in the last decade, few headto-head comparisons of adjuvant formulations using the same hiv-1 immunogen have been reported, especially when compared with the first 20 years of the aids pandemic (table 1) . a partial reason for this is that adjuvants are not licensed by themselves but only as part of the licensure of a vaccine product. that is an entirely appropriate regulatory hurdle, but it does mean that an adjuvant licensed or on track for licensure combined with a vaccine for a non-hiv pathogen could be put at risk. if an adjuvant is found to be superior for an hiv-1 vaccine candidate, by definition other adjuvants will be inferior for that hiv-1 vaccine. this does not mean that an adjuvant inferior for an hiv-1 vaccine is inferior for all other vaccines, nor would it render a licensed vaccine ineffective. however, it would create a perception that one company's adjuvant is 'better' than the others, putting vaccines using the 'inferior' adjuvants at risk. given that the vaccine market is small compared with blockbuster drugs [95] , companies appear to be appropriately reluctant to put their investments at risk. in addition, other hurdles face adjuvant development. as preventive measures, vaccines should be well tolerated for the general population and ideally cause no side-effects to anyone. as vaccines require stimulation of the immune system, establishing a balance between stimulation and side-effects is paramount (fig. 1) . however, no medical intervention is without risk and it is likely that a successful vaccine will cause some degree of side-effects in some recipients, and it will be important to determine the level of acceptable risk that balances with vaccine efficacy. public judgment of acceptable risk will depend on vaccine efficacy, that is, a highly effective vaccine against a present threat that has some side-effects will likely be more acceptable than a vaccine that is less effective or is against a pathogen perceived to be less of a threat. until an effective hiv-1 vaccine is available, work to find better adjuvants should continue. a wide variety of adjuvant formulations are available to enhance the response to hiv-1 immunogens and exciting new work suggests that formulations with better balances between safety and efficacy may be possible. however, there is much work remaining to determine the optimal adjuvant immunogen combination that will be effective in controlling the aids pandemic. the views expressed by m.a.m. are entirely his own and do not necessarily reflect those of duke university or the us government. conflicts of interest m.a.m. is supported by the center for hiv/aids vaccine immunology-immunogen discovery grant (chavi-id; grant um1 ai100645). there are no conflicts of interest. papers of particular interest, published within the annual period of review, have been highlighted as: immune stimulation systemic inflammation safety fewer side effects ? figure 1. adjuvant activity balance. adjuvants need to be both well tolerated and effective, but the ability to stimulate an immune response is often associated with side effects. further investigation will be needed to determine whether efficacy can be achieved with a low side-effect profile or if some degree of inflammation will be necessary. key roles of adjuvants in modern vaccines this is an excellent review that contains timeline of adjuvant development and overview of mechanisms of adjuvant action. 2. barouch dh, letvin nl, seder ra. the role of cytokine dnas as vaccine adjuvants for optimizing cellular immune responses vaccine adjuvants: putting innate immunity to work this is an excellent review of adjuvants and the role of prrs in triggering the immune system. good figures and tables; has a useful summary about the kinds of innate immune cells recruited by different stimulatory triggers novel adjuvants for b cell immune responses vaccine adjuvants: mode of action cytokines as adjuvants for improving anti-hiv responses enhancement of human immunodeficiency virus type 1-dna vaccine potency through incorporation of t-helper 1 molecular adjuvants a reflection on hiv/aids research after 25 years a group specific anamnestic immune reaction against hiv-1 induced by a candidate vaccine against aids immunization against aids in humans immune response of chimpanzees after immunization with the inactivated whole immunodeficiency virus (hiv-1), three different adjuvants and challenge fusion-competent vaccines: broad neutralization of primary isolates of hiv immune response to immunostimulatory complexes (iscoms) prepared from human immunodeficiency virus type 1 (hiv-1) or the hiv-1 external envelope glycoprotein (gp120) safety and immunogenicity of a fully glycosylated recombinant gp160 human immunodeficiency virus type 1 vaccine in subjects at low risk of infection. national institute of allergy and infectious diseases aids vaccine evaluation group network bangkok vaccine evaluation group: randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 hiv-1 vaccine among injection drug users in rgp120 hiv vaccine study group: placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent hiv-1 infection immunization of chimpanzees with the hiv-1 glycoprotein gp160 induces long-lasting t-cell memory effects of adjuvants and multiple antigen peptides on humoral and cellular immune responses to gp160 of hiv-1 high-titer hiv-1 neutralizing antibody response of rhesus macaques to gp160 and env peptides saponin adjuvant enhancement of antigen-specific immune responses to an experimental hiv-1 vaccine adjuvant effect of liposomes and adamantylamide dipeptide on antigenicity of entrapped synthetic peptide derived from hiv-1 transmembrane region glycoprotein gp41 comparison of 24 different adjuvants for inactivated hiv-2 split whole virus as antigen in mice. induction of titres of binding antibodies and toxicity of the formulations candidate hiv type 1 multideterminant cluster peptide-p18mn vaccine constructs elicit type 1 helper t cells, cytotoxic t cells, and neutralizing antibody, all using the same adjuvant immunization development of a single-shot subunit vaccine for hiv-1.3. effect of adjuvant and immunization schedule on the duration of the humoral immune response to recombinant mn gp120 immunogenicity of hiv-1lai gp160 and env peptides in squirrel monkey saimiri sciureus using alumine and experimental adjuvants comparison of nucleic acid and protein immunization for induction of antibodies specific for hiv-1 gp120 adjuvant is required when using env lipopeptide construct to induce hiv type 1-specific neutralizing antibody responses in mice in vivo comparison of immunity generated by nucleic acid-, mf59-, and iscom-formulated human immunodeficiency virus type 1 vaccines in rhesus macaques: evidence for viral clearance safety profile of phase i and ii preventive hiv type 1 envelope vaccination: experience of the niaid aids vaccine evaluation group public health. a sound rationale needed for phase iii hiv-1 vaccine trials support for the rv144 hiv vaccine trial: new series vaccination with alvac and aidsvax to prevent hiv-1 infection in thailand risk behaviour and time as covariates for efficacy of the hiv vaccine regimen alvac-hiv (vcp1521) and aidsvax b/e: a posthoc analysis of the thai phase 3 efficacy trial rv 144 intranasal administration of human immunodeficiency virus type-1 (hiv-1) dna vaccine with interleukin-2 expression plasmid enhances cell-mediated immunity against hiv-1 il-15 expression plasmid enhances cell-mediated immunity induced by an hiv-1 dna vaccine immunization of rantes expression plasmid with a dna vaccine enhances hiv-1-specific immunity multimeric soluble cd40 ligand and gitr ligand as adjuvants for human immunodeficiency virus dna vaccines studies on gm-csf dna as an adjuvant for neutralizing ab elicited by a dna/mva immunodeficiency virus vaccine novel codon-optimized gm-csf gene as an adjuvant to enhance the immunity of a dna vaccine against hiv-1 gag enhancement of immune responses to an hiv gp120 dna vaccine by fusion to tnf alpha cdna augmenting the immunogenicity of dna vaccines: role of plasmid-encoded flt-3 ligand, as a molecular adjuvant in genetic vaccination macrophage inflammatory protein-1alpha (mip-1alpha) expression plasmid enhances dna vaccine-induced immune response against hiv-1 soluble multitrimeric tnf superfamily ligand adjuvants enhance immune responses to a hiv-1 gag dna vaccine interferon regulatory factor-1 acts as a powerful adjuvant in the dna based vaccination enhancement of immune responses to an hiv env dna vaccine by a c-terminal segment of listeriolysin o il-15 as memory t-cell adjuvant for topical hiv-1 dermavir vaccine potent cd4þ t cell responses elicited by a bicistronic hiv-1 dna vaccine expressing gp120 and gm-csf comparative ability of various plasmidbased cytokines and chemokines to adjuvant the activity of hiv plasmid dna vaccines enhancement of hiv-1 dna vaccine immunogenicity by bcg-psn, a novel adjuvant augmentation of hiv-1 subtype c vaccine constructs induced immune response in mice by cpg motif 1826-odn hiv-1-specific cell-mediated immune responses induced by dna vaccination were enhanced by mannan-coated liposomes and inhibited by antiinterferon-gamma antibody topical delivery of imiquimod to a mouse model as a novel adjuvant for human immunodeficiency virus (hiv) dna recruitment of antigen-presenting cells to the site of inoculation and augmentation of human immunodeficiency virus type 1 dna vaccine immunogenicity by in vivo electroporation high antibody and cellular responses induced to hiv-1 clade c envelope following dna vaccines delivered by electroporation combined effects of il-12 and electroporation enhances the potency of dna vaccination in macaques safety and comparative immunogenicity of an hiv-1 dna vaccine in combination with plasmid interleukin 12 and impact of intramuscular electroporation for delivery only t-cell immunogenicity data are reported, but demonstrates that dna vaccination with electroporation is effective for eliciting responses in humans improving recombinant mva immune responses: potentiation of the immune responses to hiv-1 with mva and dna vectors expressing env and the cytokines il-12 and ifn-gamma il-12 delivery from recombinant vaccinia virus attenuates the vector and enhances the cellular immune response against hiv-1 env in a dose-dependent manner cd40l expressed from the canarypox vector, alvac, can boost immunogenicity of hiv-1 canarypox vaccine in mice and enhance the in vitro expansion of viral specific cd8þ t cell memory responses from hiv-1-infected and hiv-1-uninfected individuals the adjuvancy of ox40 ligand (cd252) on an hiv-1 canarypox vaccine interferon-beta expressed by a rabies virus-based hiv-1 vaccine vector serves as a molecular adjuvant and decreases pathogenicity multimeric soluble cd40 ligand (scd40l) efficiently enhances hiv specific cellular immune responses during dna prime and boost with attenuated poxvirus vectors mva and nyvac expressing hiv antigens oral vaccination with modified vaccinia virus ankara attached covalently to tmpeg-modified cationic liposomes overcomes preexisting poxvirus immunity from recombinant vaccinia immunization effect of the strength of adsorption of hiv 1 sf162dv2gp140 to aluminum-containing adjuvants on the immune response a demonstration of the 'goldilocks phenomenon' in which binding to alum must be neither too weak nor too strong for optimal immunogenicity induction of hiv-1 mpr(649-684)-specific iga and igg antibodies in caprine colostrum using a peptide-based vaccine characterization of antibody responses elicited by human immunodeficiency virus type 1 primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants human immunodeficiency virus type 1 env trimer immunization of macaques and impact of priming with viral vector or stabilized core protein strong and persistent cd4þ t-cell response in healthy adults immunized with a candidate hiv-1 vaccine containing gp120, nef and tat antigens formulated in three adjuvant systems durable hiv-1 antibody and t-cell responses elicited by an adjuvanted multiprotein recombinant vaccine in uninfected human volunteers neutralizing antibody responses to subtype b and c adjuvanted hiv envelope protein vaccination in rabbits mixed adjuvant formulations reveal a new combination that elicit antibody response comparable to freund's adjuvants tlr-7/8 and 9 agonists cooperate to enhance hiv-1 envelope antibody responses in rhesus macaques adjuvanticity of stealth liposomes on the immunogenicity of synthetic gp41 epitope of hiv-1 rov-asp-1, a recombinant secreted protein of the helminth onchocerca volvulus, is a potent adjuvant for inducing antibodies to ovalbumin, hiv-1 polypeptide and sars-cov peptide antigens safety and immunogenicity of a genetically engineered human immunodeficiency virus vaccine safety and immunogenicity of env 2-3, a human immunodeficiency virus type 1 candidate vaccine, in combination with a novel adjuvant, mtp-pe/mf59. niaid aids vaccine evaluation group a phase i trial in hiv negative healthy volunteers evaluating the effect of potent adjuvants on immunogenicity of a recombinant gp120w61d derived from dual tropic r5x4 hiv-1ach320 phase i/ii study of a candidate vaccine designed against the b and e subtypes of hiv-1 a novel adjuvant for mucosal immunity to hiv-1 gp120 in nonhuman primates cta1-dd adjuvant promotes strong immunity against human immunodeficiency virus type 1 envelope glycoproteins following mucosal immunization capric acid and hydroxypropylmethylcellulose increase the immunogenicity of nasally administered peptide vaccines a comparative evaluation of nasal and parenteral vaccine adjuvants to elicit systemic and mucosal hiv-1 peptidespecific humoral immune responses in cynomolgus macaques cytokines as adjuvants for the induction of antihuman immunodeficiency virus peptide immunoglobulin g (igg) and iga antibodies in serum and mucosal secretions after nasal immunization nasal immunization with a recombinant hiv gp120 and nanoemulsion adjuvant produces th1 polarized responses and neutralizing antibodies to primary hiv type 1 isolates efficient mucosal delivery of the hiv-1 tat protein using the synthetic lipopeptide malp-2 as adjuvant efficient systemic and mucosal responses against the hiv-1 tat protein by prime/boost vaccination using the lipopeptide malp-2 as adjuvant antibody responses after intravaginal immunisation with trimeric hiv-1 cn54 clade c gp140 in carbopol gel are augmented by systemic priming or boosting with an adjuvanted formulation thymic stromal lymphopoietin (tslp) acts as a potent mucosal adjuvant for hiv-1 gp140 vaccination in mice microneedle mediated intradermal delivery of adjuvanted recombinant hiv-1 cn54gp140 effectively primes mucosal boost inoculations this is an interesting study combining microneedles and adjuvants, and can be applied to skin or mucosal surfaces to elicit an immune response prime-boost regimens with adjuvanted synthetic long peptides elicit t cells and antibodies to conserved regions of hiv-1 in macaques antibody-mediated protection against mucosal simian-human immunodeficiency virus challenge of macaques immunized with alphavirus replicon particles and boosted with trimeric envelope glycoprotein in mf59 adjuvant a prime-boost regime that combines montanide isa720 and alhydrogel to induce antibodies against the hiv-1 derived multiepitope polypeptide tab9 bumps on the vaccine road key: cord-340619-3tjquzx8 authors: menghua, wu; xin, zheng; jianwei, liu; yu, zhang; qinwei, yao title: case report: one case of coronavirus disease 2019 (covid-19) in a patient co-infected by hiv with a normal cd4(+) t cell count date: 2020-07-23 journal: aids res ther doi: 10.1186/s12981-020-00301-3 sha: doc_id: 340619 cord_uid: 3tjquzx8 background: the covid-19 has been a severe pandemic all around the world. nowadays the patient with co-infection of hiv and sars-cov-2 was rarely reported. here we reported a special case with hiv and sars-cov-2 co-infection, which showed a prolonged viral shedding duration. case presentation: the patient was infected with hiv 8 years ago through sexual transmission and had the normal cd4(+)t cell count. she was found sars-cov-2 positive using real-time polymerase chain reaction (rt-pcr) during the epidemic. most importantly, the patient had a prolonged viral shedding duration of sars-cov-2 about 28 days. conclusion: the viral shedding duration may be prolonged in people living with hiv. the 14 days isolation strategy might not be long enough for them. the isolation or discharge of these patients needs further confirmation for preventing epidemics. covid-19 is a novel virus disease with over 7250,000 confirmed cases worldwide [1] . and the knowledge regarding epidemiology and clinical presentation has been evolving gradually in the past months since the initial identification. in the general population, the reported case fatality rate ranged from 1.2 to 11.9% in different countries [2, 3] . xu et al. [4] described that 113 patients had persistently positive pcrs results for at least 21 days. and lu et al. [5] also reported a maximum 20 days of prolonged viral clearance period. here we reported a case of hiv and sars-cov-2 coinfection who had a prolonged viral shedding duration about 28 days. a 49-years old female diagnosed with hiv infection 8 years ago under regular art (anti-retroviral therapy) went to our clinic for fatigue (day 1 of illness). he got a fever (day 4) with a maximum temperature of 38 ℃, accompanied by pharyngeal pain. the patient showed chills on day 5. considering the clinical symptoms, the sputum sample was collected for rt-pcr assay of sars-cov-2 and the chest computed tomography (ct) was performed. previous medical history included syphilis, which was cured. the art is efavirenz 600 mg, zidovudine 300 mg, and lamivudine 150 mg. after that, she continued the art regularly. although the nadir cd4 + t cell count was 224, a recent test was normal. the hiv viral load remained undetectable from 2013 (figs. 1, 2) . the ct result showed ground glass dense shadow and cord shadow under the pleura of the lateral segment of the middle lobe and dorsal-base segment of the lower lobe of the right lung (fig. 3) . meantime, he was treated with cefuroxime and traditional chinese medicine (lianqin oral solution and lianhua qingwen capsule). at that time, the result of rt-pcr for sars-cov-2 was negative. but the symptoms were not relieved. we considered the possibility of false-negative to the rt-pcr result [6] . so, we had a re-check of rt-pcr for sars-cov-2 on day 7. the test result on day 7 turned positive, and the patient was diagnosed with covid-19 (moderate type). according to the chinese covid-19 treatment guideline at that time [7] , on day 8, we changed the cefuroxime and traditional chinese medicine to interferon atomization (5 million bid), ribavirin (150 mg tid), and abidol (200 mg tid) for antiviral treatment. meanwhile, the moxifloxacin (400 mg qd) was given to the patient for preventing bacterial infection. on day 12, the temperature of the patient returned to normal. the symptoms of the patient alleviated completely, and the result of the ct scan on day 15 was also back to normal (fig. 2) . we consistently tested the rt-pcr for covid-19 on day 19, day 25, and day 31 to 34, but all the results remained positive. the rt-pcr for covid-19 turned negative for the first time on the day 35. meantime, the igm antibody for sars-cov-2 on day 35 was positive. then we tested the rt-pcr and igm for sars-cov-2 every 3 days. the rt-pcr for sars-cov-2 remained negative, while the igm antibody for nowadays, the covid-19 has been a worldwide epidemic disease. as an epidemic disease, viral shedding duration is the key to disease control. some studies found asymptomatic people who were still carrying the virus after isolation for 14 days [8] . the prolonged viral shedding duration in the general population has been reported by several studies and the prolonged viral shedding duration reported ranges from 21 to 45 days [4, 9] . but there are few studies about the viral shedding duration in suspicious immunocompromised patients. at present, there are several cases of patients in immune suppressive status after organ transplantation. huang reported two patients with covid-19 who had undergone transplantation, one of whom had bone marrow transplantation, and the other had kidney transplantation. these two patients finally transferred to ards (acute respiratory distress syndrome), and eventually died after [10] . among the immunocompromised patients, zhang et al. [11] reported a patient with kidney transplantation who had a prolonged viral shedding duration for 63 days. this is the first report of a patient co-infected with hiv and sars-cov-2 who showed a prolonged viral shedding duration. even though not all people living with hiv are immunocompromised, especially those under art with an undetectable hiv viral and normal cd4 count, these people may still be vulnerable to viral infection or subsequent bacterial pneumonia than the general population. further studies and data collection are needed for this. in our case, the patient had a history of fever and had ct findings of viral pneumonia. covid-19 was diagnosed by the positive result of rt-pcr. and the viral shedding duration lasted about 28 days. xu et al. [4] concluded the risk factors of prolonged viral rna shedding in covid-19 patients: male sex, delayed admission to hospital after illness onset, and invasive mechanical ventilation. these risk factors cannot explain the prolonged duration of the patient in our report. qin et al. [12] reported that the total number of b cells, t cells, and nk cells decreased significantly in patients with covid-19. and the sum of lymphocytes of the severe group dropped more significantly than the moderate group. so, the infection of sars-cov-2 might be dramatically for the people living with hiv. on the one hand, the immune system might be impaired after the infection of sars-cov-2 by the depletion of t lymphocytes [12] . on the other hand, during the chronic phase of hiv infection, generalized immune activation, and systemic cd4 + t lymphocyte depletion occur. fortunately, the cd4 + t cell count of our case was normal from 2016 to now, which might be the reason for not causing severe pneumonia. but the prolonged viral shedding duration. the paradox between the prolonged viral shedding duration and moderate clinical course might be due to the impaired cellular function despite normal cd4 + t cell count in people living with hiv [13] . lu et al. [5] revealed that prolonged viral rna shedding in children was associated with symptomatic infection, fever, pneumonia, and lymphocyte count less than 2.0 × 10 9 /l. the lymphocyte count of our case was also less than 2.0 × 10 9 /l, which might be a co-factor for the prolonged viral shedding duration. at last, there are no specific antiviral drugs for sars-cov-2. this patient was treated with ribavirin and abidol, which may not inhibit the replication of sars-cov-2 effectively. the viral shedding duration may be prolonged in people living with hiv. the 14 days isolation strategy might not be long enough for them. the isolation or discharge of these patients needs further confirmation for preventing epidemics. who coronavirus disease situation reports covid-19 outbreak: an overview clinical features of patients infected with 2019 novel coronavirus in wuhan factors associated with prolonged viral rna shedding in patients with covid-19 symptomatic infection is associated with prolonged duration of viral shedding in mild coronavirus disease 2019: a retrospective study of 110 children in wuhan correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (full version) the enlightenment from two cases of asymptomatic infection with sars-cov-2: is it safe after 14 days of isolation? prolonged viral rna shedding duration in covid-19 covid-19 in post-transplantation patients-report of two cases viral shedding prolongation in a kidney transplant patient with covid-19 pneumonia dysregulation of immune response in patients with covid-19 in wuhan, china. clin infect dis reconstitution of peripheral t cells by tissue-derived ccr4 + central memory cells following hiv-1 antiretroviral therapy publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we appreciated li li for revising the grammar errors in the manuscript. all authors analyzed and interpreted the patient data. wm, zx were major contributors in writing the manuscript. wm and zx were the first co-authors. all authors read and approved the final manuscript. not applicable. all data generated or analyzed during this study are included in this published article. informed consent was obtained in this case and protocols were approved by the human ethics committee of beijing you'an hospital. not applicable. the authors declare that they have no competing interests.received: 7 may 2020 accepted: 7 july 2020 key: cord-303408-coesfldm authors: konstantinova, pavlina; ter brake, olivier; haasnoot, joost; de haan, peter; berkhout, ben title: trans-inhibition of hiv-1 by a long hairpin rna expressed within the viral genome date: 2007-03-01 journal: retrovirology doi: 10.1186/1742-4690-4-15 sha: doc_id: 303408 cord_uid: coesfldm background: human immunodeficiency virus type 1 (hiv-1) can be inhibited by means of rna silencing or interference (rnai) using synthetic short interfering rnas (sirnas) or gene constructs encoding short hairpin rnas (shrnas) or long hairpin rnas (lhrnas). the use of sirna and shrna as antiviral therapeutic is limited because of the emergence of viral escape mutants. this problem is theoretically prevented by intracellular expression of lhrnas generating multiple sirnas that target the virus simultaneously, thus reducing the chance of viral escape. however, gene constructs encoding lhrna molecules face problems with delivery to the right cells in an infected individual. in order to solve this problem, we constructed an hiv-1 variant with a 300 bp long hairpin structure in the 3' part of the genome corresponding to the nef gene (hiv-lhnef). results: intriguingly, hiv-lhnef potently inhibited wild-type hiv-1 production in trans. however, hiv-lhnef demonstrated a severe production and replication defect, which we were able to solve by selecting spontaneous virus variants with truncated hairpin structures. although these escape variants lost the ability to trans-inhibit hiv-1, they effectively outgrew the wild-type virus in competition experiments in supt1 cells. conclusion: expression of the lhnef hairpin within the hiv-1 genome results in potent trans-inhibition of wild-type hiv-1. although the mechanism of trans-inhibition is currently unknown, it remains of interest to study the molecular details because the observed effect is extremely potent. this may have implications for the development of virus strains to be used as live-attenuated virus vaccines. rna interference (rnai) has been used to inhibit the replication of a wide range of viruses including the human immunodeficiency virus type 1 (hiv-1), hepatitis c virus (hcv), hepatitis b virus (hbv), dengue virus, poliovirus, influenza virus a, coronaviruses, herpesviruses, and picornaviruses [1, 2] . due to its sequence specificity, rnai is a potentially selective method for intracellular immuniza-tion against hiv-1 infection. rnai-mediated suppression of hiv-1 replication has been accomplished by synthetic small interfering rnas (sirnas) in a transient manner [3] [4] [5] [6] and by shrna expression vectors in stably transfected cells [7] [8] [9] . despite potent inhibition, the use of both approaches as therapeutic antiviral is limited because of the rapid emergence of hiv-1 escape mutants [9] [10] [11] . strategies to reduce the chance of viral escape include the simultaneous use of multiple sirnas [12, 13] , the intracellular expression of a second generation of escape-anticipating shrnas [14] , or microrna-based double-stranded rnas (mirnas), which do not require perfect sequence complementarity for inhibition [15, 16] . an alternative method to inhibit hiv-1 is the use of gene constructs encoding hiv-1-specific long hairpin rnas (lhrnas, transcripts folding an extended hairpin structure) or long double-stranded rnas (dsrnas, two complementary transcripts that form an extended duplex). these molecules should yield multiple effective sirnas upon intracellular processing [6, 17, 18] . however, lhrna approaches raise concerns about induction of the dsrnatriggered interferon (ifn) response. others and we have shown that endogenously expressed lhrna and dsrna can inhibit hiv-1 production without induction of the innate antiviral response [18] [19] [20] [21] . in fact, most reports on ifn induction by long dsrnas in mammalian cells are based on transfection of cells with in vitro synthesized dsrnas [22, 23] . apparently, endogenously produced dsrna is less active than exogenous dsrna in inducing the ifn response. several antiviral approaches using extended lhrna and long dsrna molecules have been reported in plant and insect cells that lack the innate antiviral ifn response. transient expression of dna constructs encoding virusspecific dsrna in plant protoplasts or insect cells partially protects the cells from infection by the homologous virus [23, 24] . stable expression of such constructs renders the cells resistant to infection [25, 26] . lhrna can inhibit hiv-1 production under certain conditions, without induction of the ifn response [6, 17, 18] . ideally, a single lhrna should generate multiple effective sirnas upon intracellular processing, providing more durable inhibition of hiv-1 than a single shrna. an additional advantage of lhrna inhibitors is that it does not require pre-determination of the optimal shrnas and corresponding hiv-1 target sequences because multiple effective sirnas will be produced. a potential disadvantage of the use of lhrna as therapeutic is that the generation of multiple sirnas will be more likely to cause off-target effects. we have previously reported strong inhibition of hiv-1 production using gene constructs encoding hiv-specific lhrnas and dsrna in transient transfection assays [18] . an alternative for stable expression of shrnas is a conditionally replicating hiv-1-based virus, which was previously used by us to deliver an antiviral shrna cassette into hiv-1 susceptible target cells [27] . in the current study, we constructed the hiv-lhnef variant, which contains a 300 bp extended hairpin structure at the 3' genome position of the nef gene of the otherwise wild-type hiv-1. we tested this hiv-lhnef for its capacity to inhibit the production of wild-type virus. intriguingly, hiv-lhnef potently inhibited wild-type hiv-1 production in trans. however, hiv-lhnef demonstrated a severe production and replication defect, which we were able to solve by selecting spontaneous, escape viruses. we previously described the construction of the hiv-lhnef virus variant [28] , which contains a 300 bp extended hairpin structure (fig. 1a) . the hairpin structure is present in the full-length genomic rna and all spliced mrnas. this structure induced a severe virus production and replication defect. similarly, we were not able to obtain stable expression of the lhrna inhibitor from a lentiviral vector (results not shown), probably due to problems in reverse transcription of the excessively stable hairpin structure [29] . the presence of the lhnef insert resulted in a dramatic drop of the viral transduction titer, possibly also due to self-targeting of nef/ltr sequences in the vector genome. unlike the non-replicating lentiviral vector, the hiv-lhnef virus could generate spontaneous variants by evolution. indeed, replicating virus variants could be selected with a severely truncated lhnef hairpin structure [28] . these escape variants are listed in figure 1b , with the number of basepairs in the remaining hairpin structure in their name. for instance, as44 has 44 remaining basepairs, producing a hairpin of intermediate stability (δg = -84.7 kcal/mol). we now set out to test these variants in further detail, e.g. for their ability to inhibit wild-type hiv-1 in trans. hek293t cells were transfected with the wild type hiv-1 construct or hiv-lhnef. we measured ca-p24 in the culture supernatant as a measure of virus production 2 days post-transfection. whereas wild-type hiv-1 produced high ca-p24 levels, no virus production was detected for hiv-lhnef ( fig. 2a, upper panel) . this loss of virus production is due to the presence of the lhnef hairpin because no such effect was scored for several control constructs: hiv-1 with a cmv insertion in sense and antisense orientation (hiv-cmv and hiv-ascmv) and the nefdeleted hiv-1 construct r1 ( fig. 2a) . notably, hiv-ascmv did not produce high ca-p24 values probably due to promoter interference from the strong cmv promoter. we next co-transfected equal amounts of wild-type hiv-1 the hiv-lhnef rna genome encodes an extended hairpin that is truncated by virus evolution figure 1 the hiv-lhnef rna genome encodes an extended hairpin that is truncated by virus evolution. (a) the proviral dna genome, highlighting the insertion of the antisense asnef fragment (original nef coordinates +8544 to +8844). the primers tta1 and cn1 used to amplify the inverted repeat region are indicated. the inverted repeat (thick arrows) results in the formation of a 300 bp hairpin structure in the rna genome of hiv-lhnef (middle panel). this hairpin encompasses the polypurine tract (ppt). the truncated hairpin structure that emerged by virus evolution is shown in the lower panel. (b) hiv-lhnef escape variants were previously described in detail [28] . the name as (antisense) reflects the number of basepairs in the remaining rna hairpin. δg is the thermodynamic stability of the remaining perfectly basepaired stem segment. the replication column shows the replication capacity of the viruses. with hiv-lhnef, hiv-cmv, hiv-ascmv or r1 ( fig. 2a , lower panel). interestingly, only hiv-lhnef was able to potently inhibit wild-type hiv-1 production in trans. the level of inhibition is comparable to that obtained in a cotransfection with the highly effective shnef inhibitor [9] , which we used as a positive control. we next titrated the hiv-lhnef construct to test if the inhibitory effect is concentration-dependent (fig. 2b ). we measured 92% inhibition of wild-type virus production when mixed 2:1 with the hiv-lhnef inhibitory construct. even at a 7:1 ratio, hiv-1 production was reduced by 61%. the mechanism of this potent inhibition is currently unknown. although hiv-lhnef may be a potently interfering construct, a major problem is that it does not replicate. we studied the replication potential of hiv-lhnef and the as escape variants in pbmc (fig. 3) . as a positive control, the nef-positive wild-type hiv-1 construct was used. as shown previously, hiv-lhnef did not replicate to detectable levels. the escape variant as44, with the intermediate length hairpin, replicated only marginally. all other as variants replicated efficiently (results are summarized in figure 1b ), although these variants reached maximal ca-p24 values at least 1 log lower than that of wild-type hiv-1. this phenotypic difference can be explained by the nefminus genotype of these viral strains as the accessory nef protein contributes to efficient virus replication in primary cells [30, 31] . we next transfected the plasmid constructs encoding wildtype hiv-1, hiv-lhnef and the as escape variants in hek293t cells. these cells do not support hiv-1 replication, but produce virus particles upon dna transfection. virus production was measured by ca-p24 elisa in the culture supernatant at three days post-transfection. unlike the original hiv-lhnef mutant, which demonstrated a severe ca-p24 production defect, all as variants efficiently produced virus (fig. 4a ). this result demonstrates that truncation of the lhnef hairpin overcomes the production and replication defect. we next tested if the as variants are able to inhibit wild-type hiv-1 in trans in the co-transfection assay. compared to the effective hiv-lhnef inhibitor, all as escape variants had lost the ability to actively inhibit wild-type virus production (fig. 4b ). even the poorly replicating as44 variant lost the capacity to inhibit hiv-1 in trans. thus, the capacity of hiv-lhnef to inhibit hiv-1 correlates with its replication defect. as a more sensitive assay for possible trans-inhibition, we tested the individual as variants in a direct competition with wild-type hiv-1 in pbmc. cells were infected with an equimolar mixture of the two viruses (based on ca-p24) that were produced in hek293t cells, and virus was passaged to fresh cells at the peak of infection. virus replication was monitored by ca-p24 elisa and visual inspection for syncytia. cellular dna was extracted at several times post-infection and the proviral nef region was pcr amplified with primers tta1 and cn1 (see fig. 1 ). since the pcr products will differ in size for wild-type hiv-1 and each as variant, both competitors can be detected in the same sample by subsequent agarose gel electrophoresis of the dna fragments. the outgrowth of a particular virus was verified by cloning and sequencing of the pcr products from the last time-point sample (results not shown). wild-type hiv-1 effectively outcompeted all as mutants in pbmc. an example of the competition between hiv-1 and the as19 variant is provided in figure 5a . both viruses are detected in the culture at day 5, but we observed a gradual increase in the intensity of the larger pcr product. a single pcr product was observed at day 58, indicating that hiv-1 predominated the culture and thus effectively outcompeted as19. the same result was obtained when hiv-1 and as19 were mixed in a 1:10 ratio (fig. 5b) . the competition results for all as variants are summarized in table 1 and shown in additional file 1. the as variants are likely to be less replication competent than wild-type hiv-1 due to the nef-minus genotype, but the remnant hairpin structure may also impose a negative effect on the replication capacity. we therefore included the unrelated nef-minus mutant r1 with a 106 nt deletion in nef, but without a hairpin structure [9] . r1 produces ca-p24 levels comparable to wild-type hiv-1 and cannot inhibit viral production in trans ( fig. 2 and 4) . we performed competition experiments between hiv-1 and r1, but also with as19 and r1. hiv-1 also outcompetes the alternative nef-minus r1 mutant (fig. 5c ). as19 and r1 co-existed in the culture up to 62 days post-infection (fig. 5c ), indicating that both nef-minus viruses have very similar replication fitness. we repeated the competition experiments in the supt1 t cell line. because the nef protein has no impact on viral replication fitness in t cell lines [30] , this system may allow a more sensitive screen for trans-inhibition of wildtype hiv-1 by the hairpin-containing as variants. in fact, all as mutants outcompeted wild-type hiv-1, as illustrated for the as19 variant (fig. 6a) . the results for all as variants are summarized in table 1 and shown in additional files 1, 2 and 3. intriguingly, variant r1 co-existed with the wild-type virus for 62 days (fig. 6b ). this result confirms that the nef function is not important in t cell lines. however, because the as variants were able to outcompete hiv-1, it raises the interesting possibility of hairpin-mediated (trans) inhibition by these as variants. perhaps even more surprisingly, as19 and r1 co-existed for the length of the competition experiment. we speculate that the deletion mutant r1 may lack target sequences for trans-inhibition by as19. we have previously described strong inhibition of hiv-1 by rnai-inducing expression vectors encoding shrna or lhrna molecules directed against viral genes [9, 13, 14, 18] . one possibility is that the mechanism of hiv-1 inhibition by hiv-lhnef or the as mutants could be rnai-related. one of the hallmarks of rnai is its sequence-specificity. we therefore tested if hiv-lhnef could inhibit the luc-nef reporter, in which a 250 nt nef target sequence was placed downstream of the photinus luciferase gene [11] . as a positive control, we showed that hiv-lhnef induced a dramatic decrease of wild-type hiv-1 production in trans, comparable in efficiency with the highly effective rnai-inducers shgag and shnef [13] (fig. 7a) . however, no sequence-specific inhibition of the luc-nef reporter was obtained with hiv-lhnef (fig. 7b) . the shgag molecule serves as a negative control in this experiment, and shnef as a potent positive control. in these transient transfection experiments a renilla luciferase expression plasmid was included, which provides a control for transfection efficiency and possible aspecific effects. renilla luciferase expression was similar in all transfections (results not shown), indicating that hivfigure 3 virus replication of hiv-lhnef and the escape variants in pbmc. cells (5 × 10 6 ) were transfected with 10 μg of the proviral dna constructs and ca-p24 production was measured in the culture supernatant at several days post-transfection for up to 4 weeks. half of the culture medium was replaced with fresh complete rpmi medium with il-2 every 4 days and freshly activated pbmc (2 × 10 6 ) were added at every 2 nd addition. the experiment has been repeated five times for as19 and two times for the other variants. in this study, we show that hiv-1 expressing an excessively stable 300 bp lhnef hairpin potently inhibits wildtype hiv-1 in trans. however, hiv-lhnef did not replicate to detectable levels in hiv-1 target cells, probably because steps of its replication cycle are affected by the hairpin insertion, e.g. rna splicing, rna nuclear export or mrna translation. moreover, hiv-lhnef may cause degradation of its own mrna due to processing by the rnai machinery. we postulate that the lhnef hairpin may induce an antiviral response against wild-type hiv-1 either through a sequence-specific rnai mechanism or through aberrant hiv-1 transcripts lacking the 3' non-coding region and polya tail that induce a potent hiv-1-specific rna silencing response in cells. tests with reporter constructs do not support a sequence-specific rnai effect, although the effect induced by lhnef is hiv-1 specific. the mechanism of trans-inhibition is currently unknown, but it remains of interest to study the molecular details because the observed inhibition is extremely potent. we are currently dissecting the mechanism of inhibition by testing hiv-lhnef variants that lack important rna signals (tar, polya, pas, pbs, dis, sd, psi). although hiv-lhnef is a potent inhibitor of wild-type hiv-1, its inability to replicate precluded long-term inhibition experiments. after prolonged culturing of hiv-lhnef, replicating variants emerged through recombination events that introduce large truncations in the lhnef hairpin structure and therefore deletions in the nef gene. interestingly, all as variants inherit a part of the hairpin structure in their genome, there were no perfect deletions of the entire lhnef region. one could speculate that the remaining secondary rna structures are actively selected because they render the viral genome less susceptible for degradation by the rnai machinery [11] . nef represents a pathogenicity factor that disorders adaptive immunity by down regulating cd4 and mhc-1 receptors, by inhibiting t-cell chemotaxis, and by inducing apoptosis in bystander t-cells, and hence plays a major role in the destruction of the host immune system [32, 33] . it has been suggested that any therapeutic intervention aimed at either completely blocking or at least partially reducing the expression of nef during hiv-1 infection would likely enhance the ability of the immune system to fight hiv infection [34] . humans infected with nef-defective hiv-1 strains show low viral loads and no or very slow disease progression and represent long-term non-progressors or long-term slow-progressors [31, 35] . moreover, macaques vaccinated with a siv strain that only lacks nef are better protected against superinfection than macaques vaccinated with a siv strain lacking the three accessory genes nef, vpr and vpx [36] . the cross-protection conferred by the attenuated siv strains appears not to be based on stimulation of the adaptive immune system, but on other (unknown) mechanisms [37] . in this study, we demonstrate effective outgrowth of the nef-minus as escape variants in competitions with wild-type hiv-1. this result was obtained in the supt1 t cell line, which does not provide a clear nef-phenotype. we think that this outcompetition is related to the presence of the genomic hairpin structure, because no such effect was observed for the control nef-deletion virus r1. these in vitro results may relate to the superior protection with nef-deleted viruses in vivo. successful expression of a shrna from a replicating viral vector has been shown for rous sarcoma virus (rsv) in avian cells [38] . we previously described inhibition of hiv-1 replication by a conditional-live hiv-rtta virus that expresses a shrna against the nef gene [27] . this approach is especially suitable for targeting cells that are susceptible to hiv-1 infection. however, hiv-1 escape variants will emerge rapidly under shnef pressure [9] . expression of multiple shrnas or a single lhrna from such a vector could prevent viral escape because multiple precise mutations should occur. use of a murine leukemia virus (mlv) can also be used for efficient and stable delivery of anti-hiv-1 shrna [39] . however, mlv can replicate only in actively dividing cells, which limits its application as a therapeutic virus. one ideal property of replicating hiv-1-based viral vectors is that they specifically target hiv-1 susceptible cells. the replication-competent as escape variants lost the trans-inhibitory properties of hiv-lhnef, but effectively outcompeted wild-type hiv-1 in t cells. it is important to asses what is the minimum length of a hairpin that can mediate trans-inhibition of wild type hiv-1. we have previously shown that a 19 nt shrna expressed from conditionally replicating hiv-1-based virus, can inhibit viral replication [27] . any hairpin longer than that should in theory mediate trans-inhibition of hiv-1. further research is needed to see if we can design constructs that are replication competent, yet remain a potent trans-inhibitor of hiv-1. for example, a lhrna variant with g-u wobbles could be designed, which will destabilize the rna structure and therefore stimulate viral replication. such an approach using 90-100 bp lhrna molecules has been suggested as a means for intracellular immunisation virus competition experiments in the supt1 t cell line luc-nef + against hbv and hcv, without evoking non-specific ifn responses [19, 40] . such constructs could form the basis for an attenuated virus vaccine and anti-hiv therapeutic virus in one. the full-length molecular hiv-1 clone lai [41] (accession number af33819.3) was used to produce wild-type virus. hiv-lhnef has a 93 nt deletion in the 5' region of nef and a 300 bp inverted repeat within the nef gene (fig. 1) . the construction of the hiv-lhnef mutant and the generation of molecular clones of hiv-lhnef escape virus variants as44, as19, as15, as11 and as8 has been described previously [28] . the hiv-1 mutant r1 has a 106 nt deletion in nef (+8513 to +8619) and has been described previously [9] . hiv-cmv and hiv-ascmv were constructed by inserting the cmv promoter in sense or antisense orientation at position +8069 of the lai genome. psuper-shgag and psuper-shnef with the h1 polymerase iii promoter have been described previously [13] . nucleotide numbers refer to the position on the genomic hiv-1 rna transcript, with +1 being the capped g residue. plasmid pgl3-nef (luc-nef), in which 250 nt from the nef gene was placed downstream of the photinus luciferase reporter gene has been described previously [11] . for cellular dna isolation, cells were pelleted for 4 min at 4000 rpm and solubilized in 150 μl lysis buffer (10 mm tris-hcl ph 8.0, 1 mm edta, 0.5 % tween 20) and 200 μg/ml proteinase k (roche) at 56°c for 1 h and at 95°c for 10 min. proviral dna sequences were pcr amplified from 5 μl cellular lysate using the 5' env primer tta1-ad (+8269 to +8289, aca gcc ata gca gta gct gag) and 3' u5 primer cn1 (+9283 to +9253, ggt ctg agg gat ctc tag tta cca gag tc) (fig. 1 [42] . human embryonic kidney (hek) 293t cells were grown as a monolayer in dmem (invitrogen) supplemented with 10% fcs, minimal essential medium nonessential amino acids, penicillin (100 u/ml) and streptomycin (100 μg/ml) at 37°c and 5% co 2 . one day before transfection, cells were trypsinized, resuspended in dmem and seeded in 24-well plates at a density of 1.5 × 10 5 cells per well. cells were co-transfected with 100 -500 ng dna with lipofectamine 2000 (invitrogen) according to the manufacturer's instructions. 1 ng prl plasmid (promega), expressing renilla luciferase (rl) from the cmv promoter, was added as an internal control for cell viability and transfection efficiency. in all transfection experiments we have controlled for the dna input by adding pbluescript (promega) to reach identical dna concentrations. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats by standard ficoll-hypaque density centrifugation, activated with phytohemagglutinin (3 μg/ ml), and cultured in complete rpmi 1640 medium (invitrogen) with il-2 (100 u/ml). cells (5 × 10 6 ) were transfected with 10 μg of the proviral dna by electroporation in 250 μl rpmi with 20% fcs in 0.4-cm cuvettes at 250 v and 960 μf, and 1 × 10 6 fresh cells and 5 ml complete rpmi with il-2 were added afterwards. pbmcs were infected with a fixed amount (5 ng ca-p24) of 293t-produced virus and maintained for up to 4 weeks. half of the culture medium was replaced every 4 days by fresh complete rpmi 1640 medium with il-2 and freshly activated pbmc (2 × 10 6 ) were added at every 2 nd addition. the human t cell line supt1 was cultured in rpmi 1640 medium supplemented with 10% fetal calf serum (fcs) (hybond), penicillin (100 u/ml) and streptomycin (100 μg/ml) at 37°c and 5% co 2 . cells were cultured in 25 cm 2 flasks and split 1 to 10 twice a week. cells were infected with equal amounts (5 ng ca-p24) of 293t-produced virus. when hiv-induced cytopathic effects were observed, virus replication was maintained by passage of the cell-free culture supernatant onto uninfected supt1 cells. at each passage, cell and supernatant samples were stored at -70°c. virus spread was followed by ca-p24 elisa on the culture supernatant as described previously [43] and renilla expression was measured with the renilla luciferase assay system (promega). virus competition experiments in supt1 and pbmc. the composition of the wild type hiv-1 and the as escape mutants was followed by pcr across the nef region with primers tta1 and cn1 (see fig. 1 ). click here for file [http://www.biomedcentral.com/content/supplementary/1742-4690-4-15-s1.ppt] inhibition of virus replication by rna interference the interplay between virus infection and the cellular rna interference machinery inhibition of hiv-1 infection by small interfering rna-mediated rna interference modulation of hiv-1 replication by rna interference sirna-directed inhibition of hiv-1 infection prevention of hiv-1 infection in human peripheral blood mononuclear cells by specific rna interference bispecific short hairpin sirna constructs targeted to cd4, cxcr4, and ccr5 confer hiv-1 resistance enhanced gene silencing of hiv-1 specific sirna using microrna designed hairpins human immunodeficiency virus type 1 escapes from rna interference-mediated inhibition human immunodeficiency virus type 1 escape from rna interference hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome rna interference as an antiviral approach: targeting hiv-1 silencing of hiv-1 with rna interference: a multiple shrna approach a novel approach for inhibition of hiv-1 by rna interference: counteracting viral escape with a second generation of sirnas changes in microrna expression profiles in hiv-1-transfected human cells targets for human encoded micrornas in hiv genes doublestranded nef rna interferes with human immunodeficiency virus type 1 replication inhibition of human immunodeficiency virus type 1 by rna interference using long-hairpin rna escape from the interferon response associated with rna interference using vectors that encode long modified hairpin-rna analysis of double-stranded rna-induced apoptosis pathways using interferon-response noninducible small interfering rna expression vector library effective suppression of human immunodeficiency virus type 1 through a combination of short-or long-hairpin rnas targeting essential sequences for retroviral integration rna interference is mediated by 21-and 22-nucleotide rnas inhibition of viral gene expression and replication in mosquito cells by dsrna-triggered rna interference dissecting rna silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense rna rna silencing of dengue virus type 2 replication in transformed c6/36 mosquito cells transcribing an inverted-repeat rna derived from the virus genome a conditionally replicating hiv-based vector that stably expresses an antiviral shrna against hiv-1 replication hairpin-induced trna-mediated (hitme) recombination in hiv-1 stabilization of the u5-leader stem in the hiv-1 rna genome affects initiation and elongation of reverse transcription natural hiv-1 nef accelerates virus replication in primary human lymphocytes genomic structure of an attenuated quasi species of hiv-1 from blood transfusion donor and recipients hiv-1 nef intersects the macrophage cd40l signalling pathway to promote resting-cell infection hiv-1 nef enhances both membrane expression and virion incorporation of env products. a model for the nef-dependent increase of hiv-1 infectivity lentivirus vectors expressing short hairpin rnas against the u3-overlapping region of hiv nef inhibit hiv replication and infectivity in primary macrophages deacon n: characterization of three nef-defective human immunodeficiency virus type 1 strains associated with long-term nonprogression. australian long-term nonprogressor study group live attenuated aids vaccines: hazards and hopes vaccination with live attenuated simian immunodeficiency virus for 21 days protects against superinfection delivery of short hairpin rna sequences by using a replication-competent avian retroviral vector stable integration of a functional shrna expression cassette into the murine leukemia virus genome specific inhibition of hbv replication in vitro and in vivo with expressed long hairpin rna changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of hiv-1lai, hiv-1mal, and hiv-1eli mfold web server for nucleic acid folding and hybridization prediction functional differences between the long terminal repeat transcriptional promoters of hiv-1 subtypes a through g hiv-1 rna research in the berkhout lab is sponsored by zonmw (vici grant), nwo-cw (top grant) and senter-novem (ts grant with viruvation). the authors thank stephan heynen for performing ca-p24 elisa and nienke westerink for providing the pbmc. graphic quantification of the relative viral abundance in supt1 and pbmc. the density of the pcr products from figures 5 and 6 key: cord-284128-3obc5k5u authors: ahmed, ali; dujaili, juman; sandhu, anisha kaur; hashmi, furqan khurshid title: concerns of hiv-positive migrant workers in covid-19 pandemic: a call for action date: 2020-09-08 journal: journal of global health doi: 10.7189/jogh.10.020342 sha: doc_id: 284128 cord_uid: 3obc5k5u nan t he unaids set a goal to eradicate the acquired immunodeficiency syndrome (aids) epidemic worldwide by 2030 [1] . the global coronavirus disease 2019 (covid-19) pandemic is deemed to pose a greater risk to patients with chronic diseases, including those that are affected by human immunodeficiency virus hiv/aids [2] . during this time, it is crucial to properly identify hiv/aids patients to ensure that they continue to receive timely and equitable access to health care and health support as they are increasingly vulnerable to covid-19 consequences [2] . covid-19 emerged in china at the end of 2019 spreading rapidly to infect over 213 countries and territories worldwide resulting in more than 11 500 000 confirmed cases and 500 000 deaths as of 6th july 2020 [3] . while it has been realized that patients with chronic conditions require additional health support to mitigate risk of covid-19, it is also important to recognise the vulnerability of chronically-ill migrant populations whose health issues are often neglected [2] . this is especially of concern in hiv/aids-infected migrant populations who avoid deportation and are considered illegal as immigration laws in various countries do not permit carriers of hiv infection to work or stay regardless of their legal status [4, 5] . reports have confirmed that russia, middle-east and south-east asian countries usually deport migrant workers who test positive for hiv [6] . reports have confirmed that until 2014, approximately 51 000 hiv-positive patients have been deported back to pakistan from the gulf countries [7] . in a bid to escape deportation, these hiv positive migrants are often forced to obtain antiretroviral therapy (art) illegally through exploitation of informal channels, friends or family to continue treating their condition [6, 8] . in other cases, these migrants are forced to buy expensively priced drugs (or in desperation, share the highly sought treatment with other migrants in order to afford it) [8] . additionally, access to health care has been challenging during the covid-19 pandemic with governments globally opting to curb the spread of the virus by introducing different national measures including lockdown or movement control, self-isolation and social distancing [2] . the lockdown and quarantine measures taken by most countries have been daunting for its hiv/aids-infected migrant population (legal or illegal) many of whom have been forced into unemployment and are unsure how to access appropriate health support, obtain essential medications or treatment [6] . not only do these migrants face difficulties in accessing public and health services they may have depended on previously, but they also may be living in inadequate accommodation where they are unable to observe safe and appropriate social distancing and hygiene protocols [5] . migrants living with hiv/aids often feel reluctant to inform their employers of their condition due to the social stigma associated with being a hiv/aids patient [4] . hence, they continue to work, exposing themselves and others to risk [4] . despite the increased risk of contracting covid-19, hiv-infected migrants in the uk tend not to be eligible for financial employment assistance though they may be in unstable employment. hence, they face increasing difficulties in earning or receiving sufficient funds to afford art medication [4, 9] . ensuring a migrants' good health is critical for the countries in which they originate from and move to [10] however, reports globally show that migrants are often subject to health inequalities due to lack of access to adequate health care as a result of neglect, exclusion, discrimination, unfavourable employment conditions causing ill health, inadequate living conditions, financial constraints or lack of appropriate legal status [10] . studies conducted in australia and europe respectively show that migrants are behind in fulfilling the unaids 90-90-90 target (a strategy where 90% of hiv patients should be aware of their hiv status, receive sustained antiretroviral therapy and achieve viral suppression by the year 2020) and more prone to being lost to follow-up art treatment when compared to non-migrant population [11, 12] . access to health care is also affected by a divide in the skill level of employment that migrants are hired for [10] . highly-skilled professional foreigners, often referred to as 'expatriates', tend to experience better health care access when compared to migrants working in low-skilled employment [10] . the pressures of social adjustment and poor health literacy also increase the migrant workers vulnerability to deteriorating health in times of a pandemic as they navigate through the difficulties of integrating into a foreign culture and language, while accepting the loss of their traditional values, beliefs and support systems [13] . overall, these factors further expose a highly-vulnerable migrant population suffering from hiv/aids to the threat of contracting the covid-19 virus [8] . hiv/aids patients are advised to observe precautions similar to general recommendations for cov-id-19 such as hand washing, cough and sneezing etiquettes, and social distancing [14] . they should also be assured with a 30-day supply of art treatment as well as adequate supply of appropriate medications to treat other acute or chronic illnesses or addictions [14] . while sufficient evidence is not available at present to link hiv/ aids patients undergoing art treatment to a higher susceptibility of covid-19 virus, nonetheless individuals with advanced-stage or poorly regulated hiv (low cd4 tlymphocytes count and high viral load) are at increased risk of infection, health complications and therefore, stand an increased chance of contracting covid-19 [14] . studies have shown that migrants seldom bring infections that pose a threat to the population of the host country [13] . in the past, outbreaks of hepatitis a, b, hiv and tuberculosis have occurred in non-endemic regions due to a lack of timely treatment provision to refugees [13] . a study conducted by mendelsohn et al. in malaysia compared the adherence of art between refugees and the host population. it has been noted that, if asylum seekers are included in primary care, the high levels of compliance achieved (equal to those in the domestic population) have decreased onward hiv transmission [15] . in short, most migrants (whether legal or illegal) lack adequate health entitlements and face health inequalities that prevent them from accessing health care in a safe, appropriate and fair manner [13] . some countries, such as thailand and spain have ensured equal access to health care for all legal and illegal who need to issue advisory to all the countries for timely access of antiretroviral therapy to hiv positive migrants regardless of their legal status. photo: from the authors' own collection, used with permission. migrants in accordance with the human rights framework [13] . the world health organization (who) should provide guidelines to all countries with hiv/aids infected migrants (whether legal or illegal) to adhere to so the migrant population continues to receive fair, assured and uninterrupted supply of art treatment during the covid-19 pandemic to maintain their immunity, health and decrease risk of co-vid-19 contraction. this will ensure another step is taken in a positive direction for the who to fulfil its slogan of "health for all". funding: we declare we received no direct or indirect funding from any institution or organization for this viewpoint. authorship contributions: aa conceived the idea, aks, fkh, and aa retrieved the data, did write up of viewpoint and finally jd reviewed and provided her inputs. all authors approved the final version of manuscript. competing interest: all authors completed the icmje form (available upon request from the corresponding author) and declare no conflict of interest. what is required to end the aids epidemic as a public health threat by 2030? the cost and impact of the fast-track approach redefining vulnerability in the era of covid-19 countries where covid-19 has spread migrants with hiv of concern in covid-19 era infectious diseases and migrant worker health in singapore: a receiving country's perspective i was waiting to die': in russia, hiv+ migrants fear death and deportation 51,000 migrants deported from gulf states due to hiv labour migrants in central asia deserve our attention migrants living with hiv during the coronavirus (covid-19) pandemic 2020 breaking down the barriers: understanding migrant workers' access to healthcare in malaysia gaps in the hiv diagnosis and care cascade for migrants in australia effect of legal status on the early treatment outcomes of migrants beginning combined antiretroviral therapy at an outpatient clinic in milan, italy healthcare is not universal if undocumented migrants are excluded is forced migration a barrier to treatment success? similar hiv treatment outcomes among refugees and a surrounding host community in kuala lumpur key: cord-278174-znc99yos authors: ramsey, glenn title: managing recalls and withdrawals of blood components date: 2004-01-31 journal: transfusion medicine reviews doi: 10.1016/j.tmrv.2003.10.005 sha: doc_id: 278174 cord_uid: znc99yos abstract donor centers are issuing a growing number of recalls and market withdrawals to hospital transfusion services about blood components. more than 1 in 2,000 units were recalled in the late 1990s in the united states. the most common reason for these notices from donor centers is postdonation donor information. most of these units had been transfused, and many present a “risk of a risk” (ie, a problem might have been present that might have affected the recipient). a few regulations and standards address recalls in general terms, but transfusion services generally have wide discretion in the management of specific common recall problems. the food and drug administration (fda) is now including posttransfusion evaluations in its guidelines for emerging infectious threats to the blood supply. we suggest that hospital transfusion services should have standard operating procedures for managing recalls and that the hospital transfusion committee and the quality management program should provide local input or oversight. using the fda’s categories of donor center biological product deviations, we provide recommendations to consider for when to notify the recipient’s physician, after postdonation information is received about a previously transfused blood component. more study of this important everyday issue in transfusion medicine is highly desirable. b lood components are regulated as drugs. however, because they are derived directly from humans, they will always be subject to biological variation. their unit-by-unit production, testing, storage, distribution, and record keeping are also more complex than other medications, leading to further opportunities for deviations from the expected. the us food and drug administration's (fda) current good manufacturing practices extend to after manufacturing so that, if problems are found with the finished drug, measures must be taken to correct the product or prevent consequences to patients if possible. over the past 10 to 15 years, the stricter application of these principles to blood components has led to a growing number of recalls and withdrawals by blood suppliers, as well as a concomitant increase in the numbers of notices sent to transfusion services about blood products they have received. furthermore, because platelets and red blood cells have a short shelf life and because hospitals do not keep a large reserve of blood components in storage, most of these notices about nonconforming blood products are often received after the units had been transfused. in retrospect, many of these recalled units presented a "risk of a risk" (ie, that the problem might have affected the patient if it had actually been present but whether it was truly present is often unknown). the lookback requirements for tracing units from donors later found to have human immunodeficiency virus (hiv) and hepatitis c virus (hcv) infections have been formalized by the fda in rules and guidances. 1,2 (a revised rule for hiv and hcv lookbacks was proposed in 2000 but not finalized as of this writing. 3 ) hcv and hiv lookbacks have been discussed elsewhere 4,5 and will not be covered in depth here. in contrast to hiv and hcv lookbacks, other types of notices about blood products have few specific formal rules or guidelines as to how they should be handled by the transfusion service. the purpose of this review is to discuss the management of recalls and withdrawals of blood components. "recall is an effective method of removing or correcting consumer products that are in violation of laws administered by the food and drug administration" (title 21, code of federal regulations (cfr), section 7.40 (21 cfr 7.40)). the fda classifies recalls into 3 categories, from the most serious, class i, to the least serious, class iii ( table 1 ). all recalls under fda jurisdiction are published on line in the weekly fda enforcement report. 6 there is a lag time of weeks to months from the recall to publication, and the enforcement report does not say when the original problem occurred. the fda says recalls are "voluntary" by the manufacturer, although conducting recalls is required, and the fda has the power to initiate recalls if the manufacturer does not act as required. recalls can include public warnings in serious situations (21 cfr 7.42). in 1 blood component recall involving improper donor testing in a large centralized laboratory, several blood centers who had used that laboratory made community media announcements and advertisements notifying transfusion recipients to seek testing if desired. 7 market withdrawals are also defined in table 1 . the fda has stated that withdrawals because of problems beyond the control of the manufacturer may be classified as withdrawals. for example, the us nationwide removal of tylenol (mcneil-ppc, ft washington, pa) from stores because of fatal cyanide tampering in chicago in 1982 was classified as a market withdrawal not a recall. 8 most notices about blood components involving postdonation donor information are now categorized as market withdrawals. market withdrawals are not published by the fda so their magnitude is unknown. ramsey and sherman 9 analyzed recalls of blood components published by the fda from 1990 through 1997. during this 8-year period, recalls were announced for nearly a quarter-million blood components, comprising about 1 in 700 units available to hospitals. seventy-six percent were in class iii recalls, 24% were of class ii, and only 12 units were designated in class i (because of viral or bacterial infection). three fourths of the recalled units involved incorrect or incomplete testing for syphilis or viral infection. the next largest categories were for donors with reactive or previously reactive infectious disease tests, which involved 10% of recalled units. nearly 90% of recalled units were included in a small number (22) of large recalls of over 1,000 units each. by 1998, the final year examined, published blood component recalls had shifted away from incorrect or incomplete infectious-disease testing (down to 20% of units) and toward donors with previously reactive infectious-disease tests (51% of units). 10 also in contrast to 1990-1997, two thirds of the recalled units were included in a growing number of smaller recalls of under 1,000 removal or correction of a marketed product that the fda considers to be in violation of the law it administers and against which the agency would initiate legal action, eg, seizure. recall classification for use of, or exposure to, a violative product: class i: reasonable probability [of] serious adverse health consequences or death. class ii: may cause temporary or medically reversible adverse health consequences or where the probability of serious adverse health consequences is remote. class iii: not likely to cause adverse health consequences. market withdrawal: removal or correction of a distributed product which involves a minor violation that would not be subject to legal action by the fda or which involves no violation, eg, normal stock rotation practices, routine equipment adjustments and repairs. units each. about 10,000 blood components were recalled in the 1998 enforcement reports. when compared with the total annual blood components distributed in the united states, the risk of a unit being recalled after issue was about 1 in 2,000. another gauge of the types of problems found with blood components comes from the recent requirement for reporting biological product deviations (bpds) to the fda. by definition, bpds involve products that had been issued but that were later found to have safety, potency, or labeling problems. bpds should include all problems that lead to recalls or market withdrawals. the fda has summarized the first full fiscal year (fy 2002) of bpds reported from licensed blood facilities. 11 their statistics are presented as the number of reports, not the number of units involved, so the overall frequency of blood components involved is unknown. one bpd for incorrect testing could involve many units, whereas bpds for donor suitability are often reported on a unit-by-unit basis. however, the categories and numbers of bpds offer a useful fda-defined framework for categorizing problems found in blood components currently. nearly 22,000 bpds were reported from licensed facilities (excluding plasma centers) in fy 2002. (reports which actually did not need to be reported were excluded.) seventy-six percent were for postdonation information concerning donor high-risk behavior and history. the most common categories here, in order, were travel to malaria and creutzfeldt-jacob risk areas, cancer, tattoos, disease or surgery, deferring medications, and intravenous drug use. the next largest type of bpd, in 10% of cases, was for quality control and distribution problems, such as clotted or hemolyzed units or segments, inappropriate product release (eg, unacceptable quality control, outdated), incorrect temperature, and failure to quarantine after another problem was found. the rest of the donor-center bpds were in donor screening (6.0%), labeling (4.3%), routine testing (1.2%), component preparation (1.2%), collection (0.7%), miscellaneous (0.6%), donor deferral (0.2%), and viral testing (0.2%). the most common items within each of these areas were as follows: donor screening, deferring history, but not deferred; labeling, incorrect autologous donor tag; routine testing, incorrect rh typing; component preparation, incorrect temperature; collection, bacterial contamination; miscellaneous, hcv lookback deviation; donor deferral, previous deferral for history; and infectious disease testing, incorrect syphilis testing. incorrect infectious disease testing, previously the most common category of recalled units in the 1990s, was the least common category of bpds in fy 2002. this may reflect the current widespread use of large, dedicated centralized testing laboratories for donors. the fda has detailed regulations and support documents for the conduct of recalls by the manufacturer. 21 cfr 7.3 defines recalls, and 21 cfr 7.40-7.59 describe the manufacturer's obligations and the fda's processes for monitoring and assessing recalls. two publications by the fda's office of regulatory affairs provide details for their inspectors about how to inspect recall operations: the investigation operations manual, chapter 8, and the regulatory procedures manual, chapter 7. 12,13 these 2 publications are on line at www.fda.gov/ora. the fda may conduct effectiveness checks, which are follow-up surveys of consignees such as transfusion services, to verify that recalls are carried out appropriately by the manufacturer. in contrast, the fda provides much less general information about the response to recalls. one line in the cfr is addressed to consignees: "responsibility of recipient. consignees that receive a recall communication should immediately carry out the instructions set forth by the recalling firm and, where necessary, extend the recall to its consignees in accordance with . . . this section (21 cfr 7.49 [d])." therefore, if the hospital transfusion service has shipped the recalled component, or part of it, to another facility, it should conduct a recall to the second facility. for example, some hospital transfusion services send source plasma to a manufacturer so the source plasma buyer should be notified if the original unit is recalled. if the transfusion service is notified about an unsuitable product, but then issues it inadvertently, then a bpd report would be required. hiv and hcv lookbacks have been referred to previously (table 2) . [1] [2] [3] [4] [5] donors with reactive tests for hepatitis b virus (hbv) and human t-cell lymphotropic viruses (htlv) i and ii were ad-dressed by the fda in a 1996 recommendation 14 (now listed under blood memos) and a 1997 guidance for htlv. 15 the fda recommended withdrawing in-dated components from donors with hbv and htlv markers but stated that they were not recommending consignee notification for the purpose of recipient notification. in our own practice, we perform lookback on units from donors with confirmed hepatitis b surface antigen (hbsag) or anti-htlv (see management of specific problems), but this is not required by the fda. recent fda guidances about emerging infection issues have included provisions about posttransfusion actions. in the january 2002 guidance 16 on creutzfeldt-jakob disease (cjd), the fda stated that notifications about donors deferred for travel, bovine insulin, united kingdom transfusion, or a single family member with cjd were intended only for product removal, and not for notification of recipients. for other more direct donor connections to cjd, including actual donor cjd subsequent to transfusion, "consignee notification could enable the consignee to inform the physician . . . so that recipient tracing and medically appropriate notification and counseling may be performed at the discretion of health care providers." there is an ongoing study by the us centers for disease control and prevention (cdc) and the national blood data resource center to investigate transfusion recipients of blood from subsequently diagnosed cjd patients. 17 in this approved study, the recipients are not notified, but national deaths are monitored to see whether any of the recipients die of cjd. as of august 2002, 331 transfusion recipients with 1,325 person years of follow-up had been studied. a similar study is being conducted in great britain for recipients of blood from donors with later variant cjd (vcjd). 18 no secondary cases of cjd or vcjd have been reported to date. if a notice were received in the united states about a blood component from a donor with later cjd, the cdc or the national blood data resource center should be contacted. in the may 2003 west nile virus (wnv) guidance, 19 if a donor has a medical diagnosis of wnv, then other units from ϫ14 days before to ϩ28 days after the onset of illness should be traced for consideration of notifying the recipients' physicians. if the donor is the suspected likely source of another wnv transfusion case, then other units from that donor collected from ϫ28 days to ϩ28 days from the infectious donation should also be traced and the recipients' physicians notified. "however, in cases when a donor is potentially associated with a case of transmission of wnv, but the epidemiological investigation has not established the specific donor as a likely source of transmission of wnv, we are not recommending notification of the transfusion service." this is slightly misworded because units from recent donors associated with a transfused wnv patient should be sought for precautionary quarantine and retrieval from the transfusion service, but the implication is that the recipients' physicians need not be notified if the donor is not the likely source. wnv nucleic acid testing (nat) began in the us and canada in the summer of 2003. when a donor tests reactive by wnv nat, the fda has not specified at this writing whether to use a 14-day lookback period (as per donor wnv illness) or a 28-day period (as per donor transmission of wnv) for recent donations. the december 2002 guidance on smallpox vaccination and blood donation 20 addressed postvaccination blood collections. if a donor should have been ineligible, but his/her units already have been transfused, then "we recommend that medical directors consider the need for prompt record tracing and, as appropriate, notification of the treating physicians or notification of prior recipients of the affected blood and blood components previously collected from that donor." the april 2003 fda guidance 21 on severe acute respiratory syndrome (sars) gave recommendations for lookback investigation. if a blood product has been transfused from a donor who should have been ineligible at the time of donation, then "we recommend that the establishments consider notifying the treating physician of those recipients about the post donation information, including whether the donor developed suspected sars." donors are deferred for 28 days after recovering from suspected sars or for 14 days after exposure to a person with sars or travel to sars-risk areas. however, the guidance states that if the donor is symptom free for more than 14 days after exposure, then product retrieval and quarantine (and thus presumably notification of the treating physician) are not necessary. in the june 2003 draft guidance on donor syph-ilis testing, 22 lookback, quarantine, and consignee notification are not recommended for previous units from donors with later syphilis or a confirmed syphilis test. the recent fda draft guidance on xenotransplantation 23 and on anthrax 24 call for withdrawal of blood components inadvertently collected from donors with these unusual exposures. the anthrax guidance has recommendations on when to notify the recipient's physician after transfusion of a suspect unit. in the 22nd edition of the standards of the american association of blood banks (aabb), effective november 2003, chapter 7 is on deviations, nonconformances, and complications. 25 standard 7.0 requires policies, processes, procedures and defined responsibility for detecting, investigating, and reviewing deviations. standard 7.1 and its subsections call for nonconforming products to be evaluated, traced, segregated if still present, and prevented from unintended use. blood banks and transfusion services must have processes for identification, quarantine, retrieval, and recall. nonconforming products that already have been released must be evaluated for quality, and when quality may have been affected, the nonconformance shall be reported to the customer. (the "customer" is defined elsewhere as the receiver of a product or service, either another organization or another department within the same organization. in this context of nonconforming products, the "customer" does not refer to the patient who received the product.) records of product nonconformances and actions about them must be maintained for 5 years (reference standards 6.2a and 6.2c). the transfusion medicine checklist of the laboratory accreditation program of the college of american pathologists (december 2002 edition) 26 has 2 questions touching on some aspects of recalls and notifications. trm.42120 asks if there is a procedure to identify and quarantine all previous components from donors who now test repeatedly reactive in viral marker screening tests. trm.42170 asks for a "procedure consistent with [medicare] and fda regulations/guidelines for notification and counseling of recipients who have been transfused with a potentially infectious blood component." the commentary for this question refers to federal requirements for notifying recipients about subsequent confirmed positive infections in their donors. the main intent of the question is to require hiv and hcv lookback. however, the mention of guidelines in the question may be construed to include other recent fda guidelines as discussed earlier. some hospitals collect blood products, and some blood collection centers have transfusion services. the previously mentioned regulations and standards apply to those facilities as well (ie, notices should be transmitted from the collection arm to the transfusion arm of the same establishment when necessary). within the extant regulations and standards summarized previously, the transfusion service has broad latitude on how to manage recalls and notifications from blood suppliers, other than hiv and hcv lookback. the remainder of this article offers recommendations based on our experience. however, these are only recommendations, and others may choose to use different approaches. given the paucity of literature about this important everyday area of activity in transfusion medicine, we hope the following will be helpful in providing a framework for others to organize their programs as suited for their hospitals. future study, analysis, and discourse on the management and outcomes of recalls and notifications will be very helpful. in particular, the yield of problem investigations and the medical benefit of these notices for transfusion recipients deserve more examination. at a minimum, we would suggest that recall management processes include the following key elements: 1. have a standard operating procedure. however one chooses to manage recalls and notifications, and in however much detail desired, a procedure is a prerequisite for instructing staff in these key elements. 2. act immediately to quarantine and discard, or return, blood components as instructed by the supplier. time is of the essence when a notice is received. immediate action should be taken to quarantine the unit and prevent release. is a recalled unit of plasma being thawed in the waterbath? in case a large number of units is involved, the transfusion service staff should have round-the-clock ac-cess to facsimile or secure electronic mail to facilitate transfer of information and prevent transcription errors from verbal messages. for laboratories with blood bank computer systems, both computer and physical quarantine must be done, although computer quarantine may be done first to expedite prompt blockage of issue. as noted previously, if a unit is erroneously released after a notice is received to quarantine it, then a biological product deviation report must be sent to the fda. 3. review and determine the medical implications of units already transfused. this is further discussed later. 4. keep records of all notices and actions as required (eg, 5 years in aabb standards). 25 telephone instructions should be logged and obeyed, but also documented pending written follow-up. record systems should include the capability to track investigations in progress. transfusion services may want to consider means to search recall records by unit number or patient or the ability to tag the unit or patient laboratory record that a recall has occurred. large hospitals may find a confidential database useful. transfusion services may wish to review their general strategies for oversight, management, and record keeping with the hospital's risk management and/or legal counsel. although most of these problems occur before the hospital receives the units, there could be potential medicolegal implications for the hospital and the physician. for example, it may be advisable to consider these investigations as a subcategory of the hospital's overall incident management program for quality improvement or at least to bring serious problems to this forum. the transfusion committee or its local equivalent may wish to provide general oversight for the recall management process. there are several advantages to performing recall investigations under the aegis of the transfusion committee. this educates key physicians in the range of problems found with blood components after transfusion. it provides the medical staff's representatives a forum to review and approve the general and specific features of the procedure. the transfusion committee also provides a logical venue to bring all or selected recalls into the hospital quality improvement program. some other resources of expertise in the hospital may be helpful in certain problems. the infection control office can provide advice on transfusions, which may have posed a serious infection risk in hindsight. potentially, this consultation could include immediate measures such as baseline patient infectious disease testing (eg, a donor calls back to report recent previously unsuspected exposure to hiv) or antimicrobial therapy (eg, a platelet culture becomes positive after the product was already transfused). for perplexing conundrums in posttransfusion problems, the hospital ethics committee might provide a useful forum for discussion. the hospital public relations office should be informed when a problem could be of concern to the news media and the public, such as a large recall of blood products in the community. many transfusion recipients have died of their underlying illness by the time a notice arrives about one of their blood components. in an hiv lookback, their next of kin must be notified. however, for other problems, no further investigation is needed. table 3 gives suggestions for whether to inform a recipient's physician about a problem transfusion. the list of problems is adapted from the fda's categories of donor-center bpds, with some additions. our general approach is that if a blood component might have posed a tangible infectious or other risk, then the patient's physician should evaluate whether the patient may have been affected. on the other hand, if the problem with the product did not pose a tangible risk to the patient, then the transfusion service physician, with the oversight of the transfusion committee if desired, can exercise informed medical judgment to not notify the recipient's physician. blood suppliers should provide adequate information for the transfusion service to evaluate the problem and counsel the physician if needed. without violating the donor's confidentiality, the transfusion service may seek further information from the blood supplier if the first notice is insufficient for a decision. many physicians are not familiar with the details of when and why blood donors should be deferred. when the patient's physician is informed about a (table 4 and text). hiv or hcv nucleic acid tests (nat) have short seroconversion windows, but postdonation nats have not been incorporated yet into fda rules and guidances on hiv or hcv lookbacks. [1] [2] [3] problem with a nonconforming blood component, some background information is often useful. for recurring notices such as malaria-area travel, a form letter may be convenient. for most routine notices, we have not required follow-up information from the physician. however, for sensitive issues, the transfusion service physician may offer assistance in patient counseling if desired. when there is concern about the possibility of infection risk from the donor, testing of the donor and/or patient may be indicated. when a donor is deferred for a risk factor before donation, no testing is done at that time. from the collection facility's standpoint, this may discourage test seeking by ineligible donors. unfortunately for the transfusion services, which have previously received blood components from that donor, the current infection status of the donor is thus left unknown. if the donor has been tested since the donation in question, the last date of testing should be included in the notification or be sought by the transfusion service. in serious donor risks, such as known hiv exposure, the transfusion service may ask the collection facility to seek donor testing if feasible. seroconversion window information is helpful for counseling and donor testing after exposure or for recipients after transfusion. if the donor has tested negative after the seroconversion period of the test in question has elapsed, then donor infection at the time of the donation can be ruled out. table 4 shows seroconversion window periods for viral tests required in blood donors. the cdc recommendation for hiv antibody testing after needlestick exposure is 6 months, 29 that is more conservative than the table figures. in the 1996 hiv lookback rule and the 2000 proposed look-back rule for hiv and hcv, 1,3 the fda required 12 months before a negative antibody test to rule out the need for lookback in prior donations. nat for hiv and hcv has much shorter window periods than antibody testing. the cdc recommends 4 to 6 weeks of follow-up testing for hcv rna after needlestick exposure. 29 nat has not yet been factored into fda lookback rules and guidances. blood centers have greatly reduced infectious disease testing errors and problems that predominated in recalls of the early and mid-1990s. today's challenge is the donor who does not reveal a deferring risk factor or condition. an anonymous survey of blood donors for risk behaviors revealed that 1.9% had a deferrable risk at the time of their donation (1.7% after testing and confidential unit exclusion). 30 efforts have been made to reshape screening questions to improve their comprehension by donors. 31 computer-assisted interviews may offer donors a more comfortable way to reveal risk factors. 32 because many current donor risk factors are based on international travel, another area for consideration is making information more widely accessible for travelers and their physicians about when not to donate blood. for example, the cdc's key international travel health publication, the "yellow book," does not tell physicians and travelers about when to avoid blood donation, and for how long. 33 likewise, when blood donor deferrals began for sars-area travel, this was not included in cdc information pages for travelers. 34 more publicity about the consequences of travel on blood donation might reduce recall rates for geographic donor risks. in today's regulatory climate, hospital transfusion services receive numerous recalls and market withdrawals from their blood suppliers. hospitals should have procedures for managing the quarantine, medical evaluation, and records of these recalls. the transfusion committee may provide oversight for local preferences about when to inform the recipient's physician. more study of the medical importance of recalls for transfused patients is needed. because the predominant reason for notices about blood products is postdonation information about the donor, this is an important area for quality improvement by blood suppliers. new mea-sures to improve donor understanding and communication about deferring information could help reduce blood component recalls and withdrawals. guidance for industry: current good manufacturing practice for blood and blood components: (1) quarantine and disposition of units from prior collections from donors with repeatedly reactive screening test for antibody to hepatitis c virus (anti-hcv); (2) supplemental testing, and the notification of consignees and blood recipients of donor test results for anti-hcv risk of human immunodeficiency virus (hiv) transmission by blood products before the implementation of hiv antibody screening food and drug administration (weekly) nordenberg t: recalls: fda, industry cooperate to protect consumers blood component recalls in the united states recommendations for the quarantine and disposition of units from prior collections from donors with repeatedly reactive screening tests for hepatitis b virus (hbv) guidance for industry: revised preventive measures to reduce the possible risk of transmission of creutzfeldt-jakob disease (cjd) and variant creutzfeldt-jakob disease (vcjd) by blood and blood products abstr) 19. guidance for industry: revised recommendations for the assessment of donor suitability and blood and blood product safety in cases of known or suspected west nile virus infection draft guidance for industry: precautionary measures to reduce the possible risk of transmission of zoonoses by blood and blood products from xenotransplantation product recipients and their intimate contacts updated us public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis blooddonor perceptions of health history screening with a computerassisted self-administered interview available at: www.cdc.gov/ncidod/sars/travel_advice. htm key: cord-285603-f4572w5m authors: ortega, joseph t.; serrano, maria luisa; jastrzebska, beata title: class a g protein-coupled receptor antagonist famotidine as a therapeutic alternative against sars-cov2: an in silico analysis date: 2020-06-24 journal: biomolecules doi: 10.3390/biom10060954 sha: doc_id: 285603 cord_uid: f4572w5m the pandemic associated with severe acute respiratory syndrome coronavirus type 2 (sars-cov2) and its disease named covid-19 challenged the scientific community to discover effective therapeutic solutions in a short period. repurposing existing drugs is one viable approach that emphasizes speed during these urgent times. famotidine, a class a g protein-coupled receptor antagonist used for the treatment of gastroesophageal reflux was recently identified in an in silico screening. additionally, a recent retrospective clinical report showed that the treatment with famotidine provided a good outcome in patients infected with sars-cov2. a clinical trial testing effectiveness of famotidine in combination with hydroxychloroquine is currently ongoing in the united states (us). in the 1990s, famotidine was described as an antiviral agent against human immunodeficiency virus (hiv). interestingly, some hiv protease inhibitors are presently being used against sars-cov2. however, it is not clear if famotidine could be effective against sars-cov2. thus, by using a computational analysis, we aimed to examine if the antiviral effect of famotidine could be related to the inhibition of proteases involved in the virus replication. our results showed that famotidine could interact within the catalytic site of the three proteases associated with sars-cov2 replication. however, weak binding affinity of famotidine to these proteases suggests that a successful famotidine therapy could likely be achieved only in combination with other antiviral drugs. finally, analysis of famotidine’s pharmacokinetic parameters indicated that its effect against sars-cov2 infection could be reached only upon intravenous administration. this work will contribute to the pharmacological knowledge of famotidine as an antiviral agent against sars-cov2. the severe acute respiratory syndrome coronavirus type 2 (sars-cov2) pandemic known as covid-19 has affected more than 6 million people worldwide as of june 2020 [1] . with an original epicenter in the wuhan city in china, reported in december 2019, the virus spread all around the world in less than four months [2] . since the outbreak began, several advances were made in the therapeutic management of sars-cov2 infection [3] . all these advances are associated with a better understanding of the viral pharmacological targets and the clinical outcomes of the applied treatments. we focused our interest on the proteases associated with the replication cycle of sars-cov2. the replication cycle of sars-cov2 involves two viral proteases, the main protease (3clpro) and papain-like protease (plpro), and one human host protease, transmembrane serine protease type-ii (tmprss2) [4] [5] [6] [7] . the viral currently available viral protease inhibitors. in addition, we analyzed the pharmacokinetic profiles of these drugs to predict their successful use as anti-sars-cov2 therapeutics. the homology structural model of human tmprss2 (np_005647.3) was built by using the tools of the swiss-model modeling server and the deepview/swiss-pdbviewer 4.01 software [21] . the best model for tmprss2 was obtained using the crystal structure of serine protease hepsin with an inhibitor bound (protein data bank (pdb) identifier (id): 5ce1) as a template. hydrogen atoms were added and partial charges were assigned for the energy refinement. the obtained model was subjected to molecular dynamic (md) simulations using namd 2.12 [22] , as described in ortega et al. [23] using the charmm force field [24] and gasteiger charges. the obtained structure represents the lowest energy frame of the md simulations. the quality of the model was validated via prosaweb [25] and procheck programs [26] . the coordinates for the sars-cov2 main protease, papain-like protease, and hiv protease were obtained from the protein data bank, pdb ids 6lu7, 6wuc, and 3lzs, respectively. the pdb files were optimized by removing co-crystallized molecules and all crystallographic water molecules before being used for further computational analysis. hydrogens were added and partial charges were assigned to all atoms. the obtained pdb files for each protein were further submitted to restrain the molecular mechanics refinement. all molecular dynamic simulations described in this study were performed with namd 2.12 [22] and vegazz 3.1.0.21 software [24, 27] as described in ortega et al. [23, 28] . for each protein, the substrate-binding site located in the catalytic site was identified by using the achilles blind docking server [29] with a respective ligand. the three-dimensional (3d) structure of each inhibitor was obtained from pubchem. molecular docking was performed with vina/vegazz 3.1.0.21 with 30 iterations for each compound. results were prioritized according to the predicted binding free energy in kcal/mol. the results collected from the docking simulation were visualized via the biovia discovery studio visualizer 17.2.0 software. a comprehensive analysis of physicochemical descriptors, parameters related to administration, distribution, metabolism, and elimination (adme), drug-like nature, and medicinal chemistry for famotidine and other protease inhibitors was carried out using swissadme tools [30] . these tools were accessed through the website at http://www.swissadme.ch. moreover, a two-dimensional (2d) representation for the chemical structures of all drugs examined in this report are shown in table 1 . table 1 . chemical structure of the compounds evaluated as protease inhibitors. chemical structure the viral and host proteases play a pivotal role in the sars-cov2 replication. the viral sars-cov2 genome codifies two proteases: the main protease (3clpro) and papain-like protease (plpro). both enzymes were studied as possible targets for antiviral drugs [9, 10] . as reported, hiv inhibitors such as lopinavir and darunavir could block the hiv main protease [14] , while antivirals such as ribavirin, naphthalene derivative, and phenylthioacetic acid derivatives block the plpro [31] . however, it is not clear whether these compounds could be used in combating the sars-cov2 infection. thus, despite testing well-known antiviral drugs, the search for new anti-sars-cov2 therapeutics is urgently required. a drug-repurposing strategy provides an opportunity to discover new effects for already existing fda-approved drugs, and it promises the shortest time to begin a clinical trial [15] . retrospective clinical studies showed that famotidine, a histamine type 2 receptor antagonist, could improve the clinical outcome in patients diagnosed with covid-19 [32] . in addition, recent computational analysis of therapeutic targets for sars-cov2 reported that famotidine could be a hit compound. [33] . thus, we hypothesized that famotidine can inhibit the proteases involved in the viral replication. to gain a deeper understanding if famotidine indeed can interact with the catalytic site of these enzymes, we performed detailed in silico analysis. the main protease, also named chymotrypsin-like protease (3clpro), of sars-cov2 shares~95% homology with 3clpro of sars-cov. structurally, these proteases are also very similar, showing only subtle differences (root median squared of 2.75, in the 306 cα atoms) [34] . moreover, both enzymes functionally belong to a cysteine protease group. the sars-cov2 main protease has a binding site with a catalytic dyad formed by his41 and cys145, and several subsites designated as s1 (his163, glu166, cys145, gly143, his172, and phe140), s2 (cys145, his41, and thr25), s3 (met165, met49, and his41), s4 (met165 and glu166), and s5 (gln189, met165, and glu166) ( figure 1a ). the main protease is responsible for the proteolysis of pre-proteins associated with the viral replication machinery such as rna-dependent rna polymerase, a helicase, and an exoribonuclease, among others. prior reports showed that anti-hiv drug lopinavir could block the activity of both sars-cov and sars-cov2 proteases [16, 35, 36] . thus, in this work, we used a priori docking with lopinavir to define the substrate-binding site in the sars-cov2 main protease [14, 37] . after determining the binding site coordinates, we analyzed the binding pattern and binding free energy of famotidine to the main sars-cov2 protease. the results obtained for famotidine were compared with two hiv protease inhibitors, lopinavir and darunavir (table 2 ). both lopinavir and darunavir showed lower binding free energy (−9.0 kcal/mol and −8.3 kcal/mol, respectively) as compared to famotidine (−6.4 kcal/mol), which is likely related to the chemical features of each inhibitor. nevertheless, each compound (famotidine, lopinavir, and darunavir) could interact with the key residues, his41 and cys145, related to the catalytic activity of the main protease ( figure 1b -d). lopinavir and darunavir were able to produce hydrogen bonds, π-π, and π-sulfur interactions, with hys41 and cys145 residues of the catalytic center and with residues of the subsites s1, s3, and s5 (met49, his54, his163, met165, pro168, arg188, gln189, and thr190) ( figure 1c ,d, respectively). famotidine adopted a distinct binding mode as compared to lopinavir and darunavir. it also produced hydrogen bonds, π-π, and π-sulfur interactions with residues of the catalytic center (hys41 and cys145), but it interacted with different residues of the subsites s1, s2, and s4 (thr24, thr25, hys41, thr45, ser46, phe140, leu141, ser144, cys145, hys163, and glu166) ( figure 1b ). the detailed interaction patterns of lopinavir, darunavir, and famotidine with the main protease are shown in the 2d diagrams in figure 1 . the papain-like protease (plpro) is also a cysteine protease. however, in contrast to the main protease, plpro possesses an asp/his/cys catalytic triad within the substrate-binding site critical for its catalytic activity ( figure 2a ; the active site is shown in blue and the catalytic triad is shown in yellow) [31] . these residues (asp286, his275, and cys111) are located within two main domains of plpro. cys111 is located within the central domain, which is mostly composed of α-helices, while his275 and asp286 are located in the c-terminal region, which is composed of a β-sheet [31] . plpro is responsible for the cleavages of the n-terminus of the polyprotein to release nsp1, nsp2, and nsp3, non-structural proteins associated with the viral replication [38] . furthermore, plpro also has other functions such as de-ubiquitination and de-isgylation (isg: interferon-stimulated gene) related to antagonizing the host's innate immunity. compounds described as inhibitors of the sars-cov plpro could potentially inhibit the activity of the sars-cov2 plpro [39] . thus, we performed the molecular docking analysis to determine the binding free energies and the binding pattern of such compounds to the sars-cov2 plpro, and we compared the results to those obtained for famotidine. firstly, using the crystal structure of plpro (pdb id: 6wuc) and castp 3.0 software, we performed topological analysis of the substrate-binding pocket of the sars-cov2 plpro [28] . this pocket exhibits a solvent-accessible area of~60 å and it involves the following residues: trp106, asn109, cys111, tyr112, leu162, gly163, arg166, met208, ser245, ala246, tyr264, gly271, his272, tyr273, thr301, and asp302. next, we performed a priori docking using a common antiviral drug ribavirin as a ligand to confirm our analysis and to define the binding site coordinates. then, we performed molecular docking of famotidine, ribavirin, and two other compounds described as the sars-cov plpro inhibitors ( figure 2b -e, respectively; table 3 ). our results indicated that all the evaluated compounds could interact in the substrate-binding site via several electrostatic interactions and hydrogen bonds. ribavirin mainly interacted with the following residues: asp164, ser245, ala246, tyr264, tyr273, thr301, and asp302 via van der waals interactions, hydrogen bonds, salt bridges, sulfur-x, and π-alkyl interactions. the binding free energy of ribavirin to the sars-cov2 plpro was −6.1 kcal/mol, while the sars-cov inhibitors showed slightly lower binding free energy (−6.4 kcal/mol). the main interactions of these inhibitors with the sars-cov2 plpro involved the following residues: leu162, asp164, pro248 tyr264, tyr273, and thr301. among the analyzed compounds, famotidine exhibited the highest binding free energy (the lowest affinity) of −5.0 kcal/mol. the main interactions between famotidine and the sars-cov2 plpro within the binding site involved the following residues: asp164, arg166, ser245, ala246, pro247, pro248, and tyr273, and they occurred mostly via hydrogen bonds. all the interactions between the sars-cov2 plpro and the inhibitors evaluated in this work are shown in the 2d diagrams in figure 2 . table 3 . binding free energies calculated for famotidine and other protease inhibitors upon docking to the sars-cov2 papain-like protease. pubmed id binding free energy (kcal/mol) biomolecules 2020, 10, 954 8 of 22 the viral replication cycle of sars-cov2 starts with an interaction between the viral spike protein and the angiotensin-converting enzyme 2 (ace2) receptor expressed on the surface of the target host [40] . then, proteolytic priming occurs in the viral spike protein, allowing the exposure of the fusion motive related to the endosome formation that allows the release of the viral rna into the host cytosol [7, 41] . the human host protease tmprss2 is involved in this processing of sars-cov2 [7] . as reported, protease inhibitors derived from benzoic acid such as nafamostat and camostat showed an antiviral effect against sars-cov2 in vitro by blocking tmprss2 protease activity [7, 42] . this protein contains an intracellular domain (residues 1 to 84), transmembrane spanning domain (residues 84-106), low-density lipoprotein receptor domain (ldlra: residues 133-147), and two extracellular domains. to date, there is no crystal structure solved for tmprss2. thus, we developed a homology model by using a crystal structure of a serine protease hepsin (pdb id: 5ce1) as a template. the homology model of tmprss2 showed two extracellular domains: the cysteine-rich domain (crd) (residues 148-242) and the serine protease domain (spd) (residues 255-489), with the presence of ser441 as a catalytic residue ( figure 3a ). the quality of our homology model was validated using the validation server, and it is depicted with the ramachandran and the prosa plots shown in figure 3b and c. the ramachandran plot denotes the phi and psi angles of each residue that are related to its structural configuration and indicates if these residues are in an energetically favorable conformation. the assessment of the ramachandran plot indicates a good overall geometry for the model, with~98% of the residues in the most favored regions. on the other hand, the prosa plot contains the z-scores of all experimentally determined protein chains in the current pdbs. in this plot, groups of structures from different sources (x-ray, nmr) are distinguished by different colors. our model has a z-score of −8.7 for the overall quality (black dot denoted with yellow circle), which is within the range of scores typically found for native proteins of similar size. thus, this model is suitable for molecular docking calculations. in our molecular docking analysis, we used antivirals nafamostat and camostat as positive controls ( figure 4c ,d, respectively). both compounds interacted within the active site of tmprss2 with the following residues: his296, leu419, asp435, ser436, cys437, trp461, gly462, gly464, and cys465 via electrostatic interactions and hydrogens bonds. the binding free energy for nafamostat was −7.4 kcal/mol and that for camostat was −6.4 kcal/mol (table 4 ). famotidine interacted with his296, asp435, gly439, ser436, ser441, and gly464 within the catalytic site mostly via hydrogen bonds ( figure 4b ). the binding free energy for famotidine (−5.8 kcal/mol) ( table 4 ) was higher than for nafamostat and camostat, indicating its lower affinity to tmprss2 as compared to two other drugs. the detailed interactions between tmprss2 and the inhibitors evaluated in this work are shown in the 2d diagrams in figure 4 . several reports published in the 1990s showed that class a gpcr antagonists, including famotidine, could block hiv replication in vitro [43, 44] . however, the exact mechanism related to famotidine inhibition was never determined, mainly due to the approval of more potent hiv inhibitors. interestingly, hiv has a protease that plays a pivotal role in virus replication by processing the viral polyprotein into mature and functional proteins [45] . the hiv protease is an aspartyl protease and, together with the reverse transcriptase and the integrase, they are the main targets for hiv therapy [46] . there are several drugs approved by the fda as hiv protease inhibitors. for example, lopinavir and darunavir are commonly used as anti-hiv therapeutics. as described earlier in this report, these two antiviral drugs could bind to the main protease of sars-cov2 [42] . we hypothesized that the earlier observed antiviral effect of famotidine against hiv could be related to its interaction with the hiv protease. in order to evaluate this hypothesis, we performed molecular docking of famotidine to the hiv protease. for this analysis, we used the crystal structure of lopinavir-bound hiv protease (pdb id: 2q5k) and of darunavir-bound hiv protease (pdb id: 4ll3) ( figure 5c ,d, respectively). our results indicated that both hiv inhibitors have lower binding free energy (higher affinity) (−10.4 kcal/mol and −10.5 kcal/mol, respectively) than famotidine (−6.4 kcal/mol) ( table 5 ). the hiv inhibitors produced more electrostatic interactions like π-π stacking and π-alkyl interactions with the active site of hiv protease than famotidine. nevertheless, famotidine via hydrogen bonds was able to interact with several residues within the active site, including gly27, which plays a critical role in the structural geometry of the active site ( figure 5b ) [47] . the binding poses and the main interactions with the hiv protease for all the examined inhibitors are shown in figure 5 . all drugs examined in this work could interact with their targets to produce an inhibitory effect. thus, pharmacodynamically, these drugs should act as inhibitors of the respective proteases in sars-cov2. however, often, the results obtained by in silico analyses and in vitro experiments cannot be reproduced in vivo, likely due to drug pharmacokinetic parameters that cannot be fully assessed in the early, experimental stage. in order to gain a deeper understanding if the pharmacokinetic parameters of the sars-cov2 protease inhibitors could be related to positive outcomes in the therapy, we analyzed the adme parameters of famotidine and compared with several known antiviral drugs such as ribavirin, lopinavir, and nafamostat, which were evaluated against sars-cov2. the adme parameters were determined by using the tools available on the swissadme server. the assessment of "drug likeness" in pharmaceutical research is associate with lipinski's "rule of five" that established guidelines for the characteristic of permeable compounds through biological membranes. swissadme used six physicochemical parameter derivatives from lipinski's "rule of five" represented as radar plots of molecule properties. the red area represents "good" property space for oral bioavailability, and the red bold line represents values of calculated properties of the analyzed molecule. when a compound satisfies the "rule of five", it has molecular properties similar to those of typical bioavailable drugs. however, there are some exceptions from the "rule of five" for certain drug classes (i.e., antibiotics, antifungals, vitamins, and cardiac glycosides). all the analyzed drugs are fda-approved and their pharmacokinetics parameters are well known. in figure 6 , we summarize the main adme parameters in the radar representations for each drug property, including lipophilicity, size, polarity, solubility, saturation, and flexibility. interestingly, only ribavirin and famotidine exhibited all the properties characteristic for orally available drugs. of note, famotidine is more polar than ribavirin. all examined compounds except lopinavir have low predicted absorption in the gastrointestinal tract. the water solubility of a drug is related to its absorption, formulation, and administration pathway. thus, we evaluated the water solubility of lopinavir, nafamostat, ribavirin, and famotidine by using three predictors. the swissadme server uses two topological methods to predict aqueous solubility, the esol (estimated solubility) model and the model adapted from ali et al. [48, 49] . the third predictor for solubility was developed by silicos-it. all predicted values are shown as decimal logarithms of the molar solubility in water (log s). the values obtained for each inhibitor are shown in table 6 . ribavirin and famotidine have higher water solubility with logs (esl) of −0.21 and −1.25, respectively than lopinavir and nafamostat (logs (esl) of −6.64 and −3.4, respectively). lopinavir showed the lowest water solubility among the four analyzed drugs. interestingly, lopinavir has a very flexible structure and larger size as compared to the other three inhibitors. on the other hand, nafamostat has a higher number of unsaturated bonds than the other analyzed drugs. both the size and the high content of unsaturated bonds are likely related to the lower solubility of lopinavir and nafamostat. all the analyzed drugs are fda-approved and their pharmacokinetics parameters are well known. in figure 6 , we summarize the main adme parameters in the radar representations for each drug property, including lipophilicity, size, polarity, solubility, saturation, and flexibility. interestingly, only ribavirin and famotidine exhibited all the properties characteristic for orally available drugs. of note, famotidine is more polar than ribavirin. all examined compounds except lopinavir have low predicted absorption in the gastrointestinal tract. figure 6 . the analysis of pharmacokinetic parameters of protease inhibitors. chemical structures and administration, distribution, metabolism, and elimination (adme) parameters for famotidine, ribavirin, lopinavir, and nafamostat, drugs that were evaluated as sars-cov2 inhibitors, are shown. figure 6 . the analysis of pharmacokinetic parameters of protease inhibitors. chemical structures and administration, distribution, metabolism, and elimination (adme) parameters for famotidine, ribavirin, lopinavir, and nafamostat, drugs that were evaluated as sars-cov2 inhibitors, are shown. the colored zone is a suitable physicochemical space for oral bioavailability obtained using the swissadme software. the compounds were scored based on the structure and whether these parameters fit into the values established for each indicator related to oral bioavailability. lipo (lipophility), size (size), polar (polarity), insolu (insolubility), insatu (instauration), and flex (flexibility). the 2d structures of each drug are shown with the specific atoms represented as follows: carbon in gray, oxygen in red, nitrogen in blue, and sulfur in yellow. in the late stage of the disease, the sars-cov2 replication occurs mainly in the lungs. thus, in order to produce a positive effect, an antiviral compound needs to reach the pulmonary tissue. high gastrointestinal absorption of a drug would be advantageous for fast tissue distribution. in fact, fda-approved drugs currently used for treatment of sars-cov2 such as chloroquine, camostat, and lopinavir have high gastrointestinal absorption. however, in the case of lopinavir, reaching the pulmonary tissue could be delayed due to its high volume of distribution, which is associated with its tight binding to plasma proteins [50] [51] [52] [53] . on the other hand, famotidine has low gastrointestinal absorption; however, due to its high polarity, it could be given intravenously. the intravenous route of drug administration increases the probability of reaching the tissue target. thus, if administered intravenously, famotidine has a better chance to be a successful anti-sars-cov2 drug in the late stage of infection than lopinavir. thus, in order to develop efficient anti-sars-cov2 therapy, the pharmacokinetic parameters associated with drug absorption, metabolism, and distribution need to be carefully taken into account. initially, sars-cov2 antiviral treatment strategies were adopted based on the prior clinical approaches used for combating the viral respiratory infections, mainly the sars-cov infection. these strategies included treatments with interferon-alpha, lopinavir/ritonavir combination, ribavirin, and chloroquine [54] [55] [56] . however, the outcomes of these treatments were not as good as expected, mainly due to low viral clearance and side effects. thus, multiple research groups currently focus on basic research approaches using in silico and in vitro screenings to quickly find new therapeutic alternatives [57, 58] . in a pandemic situation, time is crucial. thus, adopting already existing drugs for identifying new functions of these drugs, known as drug repurposing, could decrease the time and cost of developing new therapy [15] . the screening of large libraries of compounds already approved by the fda against the main targets in sars-cov2 can assure an accelerated start to clinical trials [59] . the sars-cov2 replication cycle offers several steps that could be used as molecular targets for antiviral drugs. the most evaluated are viral entry, the main protease, the papain-like protease, and the rna-depended rna polymerase [60] [61] [62] . the computational and in vitro analyses of these targets and specific antiviral drugs are ongoing and some were already reported [33] . based on the results of these studies, some clinical trials of antiviral drugs against sars-cov2 are currently in progress. in this study, by using computational analyses, we examined the therapeutic potential of famotidine as an anti-sars-cov2 drug. structurally, famotidine is a member of 1,3-thiazoles, a sulfonamide, and a member of guanidines. this histamine type 2 receptor antagonist was never tested as an antiviral agent against coronavirus. however, as reported in the 1990s, famotidine could inhibit hiv replication in vitro, but its mechanism of action was never clarified [43, 44] . recently, in silico analysis of the sars-cov2 targets and their potential new inhibitors suggested that famotidine could possibly target the sars-cov2 main protease [33] . thus, in this study, we analyzed if famotidine could also inhibit other proteases involved in the viral replication and, thus, similarly to ribavirin, act as a broad-antiviral agent. ribavirin, a purine nucleoside analogue demonstrated efficacy against variety of dna and rna viral infections [33, 63] . the overwhelming success of ribavirin is largely related to its excellent performance in synergy with standard or pegylated interferon-alpha in chronic hepatitis c virus (hcv) infection [64, 65] . our in silico analysis revealed that famotidine can interact with the sars-cov2 main protease with a binding free energy of −6.4. moreover, it could also interact with two other proteases involved in sars-cov2 replication, the viral plpro and human host tmprss2. however, the binding free energies for these proteases (−5.0 and −5.8, respectively) were higher (lower affinities) than for the main protease. nevertheless, these results indicate that famotidine could bind non-specifically to all proteases involved in virus replication with rather low binding affinity. thus, it seems that, similarly to ribavirin, the best therapeutic effect of famotidine could be achieved in combination therapy with other antiviral drugs [64, 65] . the binding affinity of famotidine to the viral and host proteases could be related to its chemical structure that lacks multiple aromatic rings as compared with other inhibitors, thus showing higher binding free energy (tables 1-4) . these aromatic rings produce electrostatic interactions such as π-π interactions and π-alkyl interactions with the residues in the catalytic site of the enzyme that stabilize the compound and decrease the binding free energy. the molecular docking score functions are used to assign a binding free energy value that could be in relation to the binding efficacy of the molecule to the active site. in this work, we refer to this efficacy as an affinity for the binding site. however, further in vitro assays are necessary to validate this data and to establish a correlation between the affinity values and experimental drug binding constants for each enzyme. nevertheless, the high binding free energy of famotidine reflects its low target specificity. thus, to enhance the potential positive outcome of famotidine therapy, it is highly likely that it has to be administered together with other antiviral drugs. the combination of famotidine and hydroxychloroquine could be a valid approach to treat sars-cov2 infection. hydroxychloroquine elevates the ph of acidic intracellular organelles, such as endosomes and lysosomes, essential for membrane fusion. thus, this drug could inhibit endosome-mediated viral entry [66, 67] . in addition, ph elevation impairs enzymes involved in protein posttranslational modifications. indeed, hydroxychloroquine could inhibit sars-cov cellular entry through changing the glycosylation pattern of ace2 receptor and spike protein [68] . thus, hydroxychloroquine is a non-specific antiviral drug that could improve the effectiveness of other antiviral drugs when administered simultaneously. multiple studies evaluating a combination of hydroxychloroquine with other antiviral drugs are ongoing (nct04370782, nct04374019, nct04373044). for example, a small open-labeled, non-randomized clinical trial from france, evaluating hydroxychloroquine, demonstrated its positive effect in combination with azithromycin [69] . however, larger randomized controlled clinical trials are needed to attain deeper knowledge about the positive effect of these drugs combination. another clinical trial evaluating hydroxychloroquine in combination with camostat recently began in germany (nct04338906). a combination of these two drugs should produce a synergistic effect by inhibiting two main mechanisms associated with the early stage of viral entry. a combination therapy of hydroxychloroquine with famotidine could possibly achieve synergy by blocking both the early infection stage with hydroxychloroquine and the late stage with famotidine. the analysis of pharmacokinetic parameters of famotidine revealed that the anti-sars-cov2 effect of this drug could likely be achieved only if famotidine is administered intravenously. famotidine exhibits low gastrointestinal absorption and it has a low volume of distribution; thus, if given orally, it would most likely not be able to reach the target respiratory pathway to produce an antiviral effect. therefore, drugs with low gastrointestinal absorption and low volume of distribution such as famotidine and nafamostat should be given via intravenous injection in the clinical setup. on the other hand, compounds such as hydroxychloroquine or ribavirin that have high gastrointestinal absorption and high volume of distribution [53] should achieve therapeutic effect upon oral administration. unexpectedly, anti-hiv drug lopinavir, despite its high gastrointestinal absorption, proved to be ineffective against sars-cov2 infection. the poor outcome of lopinavir treatment could be related to other pharmacokinetic parameters of this drug such as high metabolism and low bioavailability [17, 53] . although the volume of distribution of lopinavir upon its oral administration is reasonably small (~16.9 l) [53] , this drug tightly binds to plasma proteins (over 90%) [53] . thus, its plasma concentration of free drug is likely too low to reach the pulmonary tissue to target sars-cov2. thus, in order to develop successful effective antiviral therapy targeting sars-cov2, the pharmacokinetic parameters should be carefully taken into consideration. altogether, in this study, we showed that famotidine could be used as an antiviral agent against sars-cov2, targeting proteases involved in the virus replication, mostly the main protease, as well as the viral plpro and human host tmprss2. based on our analysis, it is unlikely that famotidine would produce a strong antiviral effect. it is rather expected that famotidine should be administered together with other antiviral drugs in order to produce synergistic effect to combat sars-cov2 infection. although our results obtained using in silico analysis need further experimental validation, the potential of famotidine as an inhibitor of proteases implicated in viral replication is already supported by the published data obtained from retrospective studies, which showed the beneficial effect of famotidine against sars-cov2. in addition, one clinical trial aiming to evaluate the safety and effectiveness of famotidine against sars-cov2 is already ongoing. however, taking into account the pharmacokinetic parameters of famotidine, we predict that it could only be effective as an anti-sars-cov2 drug when given intravenously in the clinical health facility by healthcare providers. a review of coronavirus disease-2019 (covid-19) a review of sars-cov-2 and the ongoing clinical trials covid-19: review of epidemiology and potential treatments against expanding our understanding of the role polyprotein conformation plays in the coronavirus life cycle drug development and medicinal chemistry efforts toward sars-coronavirus and covid-19 therapeutics sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a sars-cov-2 protein interaction map reveals targets for drug repurposing the viral, epidemiologic, clinical characteristics and potential therapy options for covid-19: a review an update on the status of covid-19: a comprehensive review prediction of the sars-cov-2 (2019-ncov) 3c-like protease (3cl (pro)) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates molecular mechanism of evolution and human infection with sars-cov-2. viruses designing of improved drugs for covid-19: crystal structure of sars-cov-2 main protease m(pro) unrevealing sequence and structural features of novel coronavirus using in silico approaches: the main protease as molecular target drug repurposing: progress, challenges and recommendations expanding the frontiers of existing antiviral drugs: possible effects of hiv-1 protease inhibitors against sars and avian influenza a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 review of early and emerging options. open forum infect. dis. 2020, 7, ofaa105 efficacy of chloroquine and hydroxychloroquine in the treatment of covid-19 new and future drug development for gastroesophageal reflux disease the swiss-model workspace: a web-based environment for protein structure homology modelling scalable molecular dynamics with namd antiviral activity of flavonoids present in aerial parts of marcetia taxifolia against hepatitis b virus, poliovirus, and herpes simplex virus in vitro charmm general force field: a force field for drug-like molecules compatible with the charmm all-atom additive biological force fields prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins procheck-a program to check the stereochemical quality of protein structures vega-an open platform to develop chemo-bio-informatics applications, using plug-in architecture and script programming the retinoid and non-retinoid ligands of the rod visual g protein-coupled receptor high-throughput parallel blind virtual screening using bindsurf swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds famotidine use is associated with improved clinical outcomes in hospitalized covid-19 patients: a retrospective cohort study analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods structure-based design of antiviral drug candidates targeting the sars-cov-2 main protease treatment of severe acute respiratory syndrome case of the index patient who caused tertiary transmission of covid-19 infection in korea: the application of lopinavir/ritonavir for the treatment of covid-19 infected pneumonia monitored by quantitative rt-pcr ul-haq, z. identification of chymotrypsin-like protease inhibitors of sars-cov-2 via integrated computational approach activity profiling of sars-cov-2-plpro protease provides structural framework for anti-covid-19 drug design potential inhibitors against papain-like protease of novel coronavirus (sars-cov-2) from fda approved drugs role of changes in sars-cov-2 spike protein in the interaction with the human ace2 receptor: an in silico analysis severe acute respiratory syndrome coronavirus 2 (sars-cov-2): an overview of viral structure and host response candidate drugs against sars-cov-2 and covid-19 the effect of histamine type 2 receptor antagonists on human immunodeficiency virus (hiv) replication: identification of a new class of antiviral agents effect of the oral anti-ulcer agent, cimetidine, on hiv type 1 replication the structural biology of hiv-1: mechanistic and therapeutic insights hiv-1 antiretroviral drug therapy. cold spring harb interactions of different inhibitors with active-site aspartyl residues of hiv-1 protease and possible relevance to pepsin estimating aqueous solubility directly from molecular structure revisiting the general solubility equation: in silico prediction of aqueous solubility incorporating the effect of topographical polar surface area plasma protein binding: from discovery to development plasma protein binding in drug discovery and development pharmacokinetic-pharmacodynamic analysis of lopinavir-ritonavir in combination with efavirenz and two nucleoside reverse transcriptase inhibitors in extensively pretreated human immunodeficiency virus-infected patients covid-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics the first 75 days of novel coronavirus (sars-cov-2) outbreak: recent advances, prevention, and treatment coronavirus disease 2019 (covid-19): current status and future perspectives network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 structural elucidation of sars-cov-2 vital proteins: computational methods reveal potential drug candidates against main protease, nsp12 polymerase and nsp13 helicase rapid identification of potential inhibitors of sars-cov-2 main protease by deep docking of 1.3 billion compounds drug targets for corona virus: a systematic review potential interventions for novel coronavirus in china: a systematic review research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases mechanism of action of ribavirin in the treatment of chronic hepatitis c treating viral hepatitis c: efficacy, side effects, and complications targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases chloroquine is a potent inhibitor of sars coronavirus infection and spread hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial key: cord-297612-swc2pitd authors: nosyk, bohdan; armstrong, wendy s; del rio, carlos title: contact tracing for covid-19: an opportunity to reduce health disparities and end the hiv/aids epidemic in the us date: 2020-04-27 journal: clin infect dis doi: 10.1093/cid/ciaa501 sha: doc_id: 297612 cord_uid: swc2pitd sars-cov2 testing and contact tracing have been proposed as critical components of a safe and effective covid-19 public health strategy. we argue that covid-19 contact tracing may provide a unique opportunity to also conduct widespread hiv testing, among other health promotion activities. m a n u s c r i p t massive sars-cov2 testing and contact tracing at a scale and speed never before seen have been proposed as critical components of a covid-19 public health strategy that could, in theory, safely allow us to relax social distancing measures and begin to bring back the world we left behind before a cure or effective vaccine is delivered. one of a number of challenges this strategy faces is that contact tracing is extremely labor intensive. deployed in sexuallytransmitted infections such as hiv, a recent study from new york reported costs of over $500 per contact interviewed with 85% of state-level costs attributable to personnel [1] . for coronavirus these efforts are simpler but the volume of contacts-an average of 36 per positive case [2] pose substantial logistic challenges. digital applications and other technological supports may help, though it appears inevitable that a large labor force will be needed. already, massachusetts has begun hiring to fill their state's projected need for 1,000 contact tracing workers [3] . health canada has an open call for a volunteer workforce to do contact tracing and other related tasks [4] . south korea employed a massive workforce of public health workers at the peak of the epidemic [5] . the value of a such a public health workforce extends beyond contact tracing for covid-19 and could lead to progress fighting many other health conditions. programs in african communities have combined hiv testing with screening for infectious diseases like tuberculosis and malaria as well as non-communicable diseases like hypertension and diabetes [6] . using this model, we can take this opportunity to scale-up testing for infectious diseases as well as noncommunicable diseases and by doing so improve community health. as sars-cov-2 testing is evolving with, not only serological but saliva testing, similar approaches could be taken for optout hiv, hcv and hemoglobin a1c testing for which finger stick methods are already available. this will allow us to begin to address the unacceptable health disparities that have existed for many of these conditions and, not surprisingly, also occur in covid-19 [7, 8] . aside from the potentially profound health benefits of a combination implementation approach, pairing covid-19 contact tracing with testing for hiv may serve to offset the immense costs of such an approach. it is well-established that hiv testing provides outstanding value and can even be cost-saving in the long-term in high-prevalence populations and settings [9] . simply learning of a new hiv infection is known to change behavior, with meta-analyses estimating an 80% reduction in sexual activity with partners of negative or unknown hiv status [10] , and starting arvs with subsequent viral suppression stops hiv transmission [11] . testing focused on the geographic regions with the highest rates of new diagnoses and further targeted using phylogenetics to identify the largest and fastest-growing clusters of infection, has a c c e p t e d m a n u s c r i p t been positioned as a key pillar of the united states' 'ending the hiv epidemic' strategy [12] . these efforts will be familiar to public health leaders in the us -anthony fauci was an architect of the strategy [13] , which was motivated by the profound successes in the african continent, engineered in part by the president's emergency plan for aids relief (pepfar) and headed by deborah birx [14] . yet testing remains one weak link in our efforts to end the hiv epidemic, in part because of persistent stigma and because the communities at greatest risk often have limited access to healthcare and/or are young and without established healthcare. we [bn, cdr] have recently written that for six of the largest us cities comprising nearly 1 in 4 people living with hiv/aids in the us, implementing a wide range of interventions to diagnose, treat and protect against infection at even near-ideal levels would fall short of the ehe targets [15] . we excluded contact tracing because of the relatively limited experimental evidence supporting its effectiveness in hiv and the uncertainty regarding the potential scale this type of effort could actually reach. contact tracing for hiv is difficult to implement and may be profoundly threatening to already marginalized individuals. covid-19 contact tracing may provide a unique opportunity to also conduct widespread hiv testing with modified contact tracing that could be acceptable and important for ending the hiv epidemic. though the task is indeed monumental, the necessary temporary labor force is readily available. were implemented [16, 17] . some project the unprecedented stimulus packages and protections for workers may not return the hardest-hit industries to pre-covid levels, and high unemployment may linger. job loss is a major life stressor and is associated with declines in psychological and physical well-being along with a host of other negative social effects [18] . creating jobs, even if only for a limited term, is a necessary government intervention to not only kickstart the economy but also to mitigate further growth in the concurrent epidemic of loss and despair [19, 20] . mobilizing this labor force in the covid-19 response will allow communities to take meaningful action to protect themselves, an act which in itself may have transformative benefits over the long-term. hiv is known to disproportionately impact urban african american and hispanic/latinx communities [21, 22] as do other chronic health conditions such as diabetes and hypertension. now covid-19 shows the all too familiar progression into vulnerable populations. early cases were seen primarily in international travellers, but currently african american and a c c e p t e d m a n u s c r i p t latinx populations are disproportionately impacted. a public health workforce with point of care screening for these conditions as well could make meaningful strides to reduce the widespread disparities inherent in health. furthermore testing to promote health allows for stigma-free messaging and broad acceptance. testing and contact tracing for covid-19 will provide an entry into social networks in communities where risk factors for conditions like hiv or noncommunicable diseases may be shared. it is imperative however that contact tracing does not increase stigma and discrimination of minority populations and thus the development of a workforce dedicated to culturally competent contact tracing, point of care testing and overall health promotion [23] has to be a priority in order to seize this opportunity. the speed with which covid-19 contact tracing must be conducted is on a different scale, though more intensive efforts can be triaged to specialized staff. the greatest effort howeverand valueis in testing. of course, these potential benefits hinge on the rapid development of antibody testing technology, not to mention sufficient availability of personal protective equipment, and the public health system's ability to rapidly train and deploy the massive influx of temporary workers that would be needed. aside from the devastating death toll, the potential long-term effects of covid-19 on the global economy are severe and may ultimately prove to be greater than any other period in living memory. difficult decisions regarding how and when to restart the economy lie ahead, but the balance sheet can include the long-term benefits of widespread hiv testing, screening for chronic diseases and potentially narrowing health disparities. though it may feel like a distant priority, now more than ever, as the official language of the plan goes, we have unprecedented opportunity to end the hiv epidemic in america. accomplishing this and expanding the impact to other health conditions is an opportunity we must not let go to waste. notes: this study was funded by the national institutes of health/national institute on drug abuse grant no. r01-da-041747 to emory center for aids research and emory vteu. the authors have no conflicts of interest to declare. cost analysis and performance assessment of partner services for human immunodeficiency virus and sexually transmitted diseases the efficacy of contact tracing for the containment of the 2019 novel coronavirus (covid19) coronavirus disease-19: summary of 2,370 contact investigations of the first 30 cases in the republic of korea. osong public health res perspect hiv testing and treatment with the use of a community health approach in rural africa covid-19 and african americans failing another national stress test on health disparities localized economic modeling study group. the impact of localized implementation: determining the cost-effectiveness of hiv prevention and care interventions across six united states cities meta-analysis of high-risk sexual behavior in persons aware and unaware they are infected with hiv in the united states-implications for hiv prevention programs hptn 052 study team. prevention of hiv-1 infection with early antiretroviral therapy ending the hiv epidemic: a plan for the united states at 15 years. lancet localized hiv modeling study group. ending the hiv the usa: an economic modelling study in six cities mortality and morbidity in the 21 st century the epidemic of despair among white americans: trends in the leading causes of premature death trends in racial/ethnic disparities of new aids diagnoses in the united states what lessons it might teach us? community engagement in hiv research addressing hiv criminalization: science confronts ignorance and bias a c c e p t e d m a n u s c r i p t key: cord-292740-b4cdj96q authors: wahid, braira; ali, amjad; idrees, muhammad; rafique, shazia title: immunotherapeutic strategies for sexually transmitted viral infections: hiv, hsv and hpv date: 2016-08-03 journal: cell immunol doi: 10.1016/j.cellimm.2016.08.001 sha: doc_id: 292740 cord_uid: b4cdj96q more than 1 million sexually transmitted infections (stis) are acquired each day globally. etiotropic drugs cannot effectively control infectious diseases therefore, there is a dire need to explore alternative strategies especially those based on the regulation of immune system. the review discusses all rational approaches to develop better understanding towards immunotherapeutic strategies based on modulation of immune system in an attempt to curb the elevating risk of infectious diseases such as hiv, hpv and hsv because of their high prevalence. development of monoclonal antibodies, vaccines and several other immune based treatments are promising alternative strategies that are offering new opportunities to eradicate pathogens. dendritic practice to prevent and treat diseases by boosting, suppressing or stimulating immunity is known as immunotherapy. though, antimicrobial drugs have developed rapidly in recent past but these drugs are of narrow spectrum and target certain groups of microbes leading to an increased resistance. such hindrances in treatment compel scientists to investigate new ways of developing effective and cost-effective treatments especially immunotherapies [1] . the alarming increase in prevalence of immunocompromised people besides booming antimicrobial resistance and lack of novel antimicrobial agents form the basis of development of immunotherapeutic strategies. frequently used immune-based therapies against infectious diseases span the use of cytokines and growth factors to boost natural immunity, increase effector cell response to restrain infectious diseases, introduction of antibodies against different organisms, use of monoclonal antibodies, hyperimmuneglobulins, cytokines, different interleukins and interferon [2, 3] . the 20th century witnessed remarkable discoveries, including antimicrobial agents that changed the face of medical practice [4] . the use of antibiotics and preventive vaccines led to a decline in major endemics in industrialized countries and, to a lesser extent, in developing countries. however, pathogens developed resistance to antimicrobial agents in both developing and developed countries (fig. 1) [5, 6] . during the last century, infectious diseases have been the leading reason of morbidity and mortality in developing world. likewise, millions people have lost their lives in different cities of europe due to bacterial and viral infections [7] . in 1800, edward fig. 1 . evolution of infectious diseases and microbial resistance [7] . jenner reached to the first milestone in controlling infections when he demonstrated immunization by inoculating humans with material extracted from cowpox lesions [8] . the basis of immune-based therapy dates back to 1890, during which emil von behring and shibasabura kitasato introduced serum therapy and successfully cured children infected with diphtheria using horse anti-sera [9] . whenever pathogen gains entry into the host, immune mediators are released to activate the immune system. this initial response controls the infection by stimulating phagocytic effector cells such as macrophages and neutrophils to eliminate infectious agents. modulation of immune response is more reasonable approach to control infections as compared to antimicrobial chemotherapy [10] . immunotherapy against infectious diseases includes modification of antigen-specific response (e.g., by using interferons), mobilization of immune response against pathogens (e.g., by using cytokine inhibitors and cytokines) and minimization of endorgan damage based on use of nonspecific anti-inflammatory agents (e.g., use of steroids) ( table 1 ) [2] . active immunotherapy has been proved effective against acute infectious diseases and the efficacy depend upon the use of suitable target antigens, optimization of antigenic peptide interaction with antigen presenting cells and t-cells, and concurrent blockage of negative regulatory mechanisms which inhibit immunotherapeutic impacts [11] . there are several different types of immune responses evolved qualitatively to avoid infections e.g., different subsets of t helper cells (e.g., th1, th2 and th17), tfh (follicular helper t cells) [12] that secrete il-21 (interleukin-21) and differentiates b cells to generate memory b cells [13] . in addition, memory t cells consist of distinct populations of effector memory cells and central memory cells both having distinct effector function along with homing capacity [14] . the mammalian immune system includes innate and adaptive components, which cooperate to protect the host against microbial infections. innate immunity sense microbes using patternrecognition receptors [15] , like c-type lectin-like receptors [16] , toll-like receptors [15] , cytosolic nod-like receptors [17] , and rig-i-like receptors [18] , which trigger the activation of adaptive immune response and antimicrobial responses. the adaptive immune system, in turn, activates innate effector mechanisms in an antigen-specific manner. several different subsets of functionally distinct dendritic cells are also present and it has now been proved that pattern-recognition receptors and dendritic cells determine the quality and magnitude of acquired immunity [19, 20] . foreign agent or microbe is integrated by dendritic cells and translated to antigen specific b and t cells to boost the strength, persistence, and quality of the adaptive immune response [19] . passive immunization was introduced in 1891, when a boy with diphtheria was injected with diphtheria antitoxin that cured him. anti-diphtheria serum because of its favourable results got public acceptance in all parts of europe during 1894 [21] . serum therapy had been used to control bacterial infections such as neisseria meningitidis and streptococcus pneumoniae since 20th century [22] but this practice was dumped in 1940s due to toxicity associated heterologous sera whereas the recent advancements such as development of monoclonal antibodies provide new insights for elimination of infectious organisms [23] . passive immunotherapy is based on administration of immunoglobulins which is either combined with a toxic counterpart molecule or involve adoptive migration of an activated immune cell effector component to act against host's neoplasm. cellular therapy includes adoptive transfer of specifically sensitized cytotoxic t lymphocytes and nonspecifically activated lak (lymphocyte-activated killer) cells [24] . passive immuno-therapies include use of table 1 immunotherapeutic strategies against infections. mobilization of immunity using vaccines inactivated vaccines contain killed microbes that stimulate body's immunity live attenuated vaccines contain live pathogens with reduced virulence toxoid vaccines consists of pathogen's toxin (poison) that has been made harmless but elicits immune response in host subunit vaccines contain only antigenic part instead of entire microbe conjugate vaccine is a combination of poor (polysaccharides) antigen and carrier protein belonging to same pathogen in dna vaccination, genetic material is directly injected into living host that efficiently elicits humoral and cellular immunity to intravenous immunoglobulin therapy is being used in different clinical trials as therapeutic or prophylactic agent for infectious diseases and various studies have proved it a promising alternative strategy to control infections. intravenous immunoglobulin preparations are made up of igg type antibodies which recruit viruses into lymphoid organs where they are presented to b and t cells leading to an activation of immune response [5, 25] (fig. 2) . immunocompromised patients are more susceptible to chronic infections and a study demonstrated that when patients with primary immunodeficiencies were treated with double amount of immunoglobulin (two times of standard dose), a significant decrease in severity and frequency of infection was observed [26] . treatment of patients of west nile virus encephalitis with intravenous immunoglobulins containing high titres of antibody has been found to be effective [27] . intravenous immunoglobulin therapy can also clear viremia and re-establish cytokine balance in patients suffering from acute pv-b19 infection such as chronic fatigue syndrome. cytomegalovirus and parvovirus b19 virus are the cause of glomerulopathy in transplanted kidneys but introduction of intravenous immunoglobulin preparation before transplantation eliminates the risk of viruses [28, 29] . intravenous immunoglobulin prepared from individuals exposed to hpv, hiv and hsv or any other outbreak may emerge as beneficial adjunct therapy. injection of monoclonal antibodies is another attractive technique of passive immunotherapy that triggers recruitment of lymphocytes and activation of complement system. most monoclonal antibodies that have been developed so far are for immunological disorders and cancer treatment whereas, monoclonal antibodies against infectious diseases are still in progress [30] . the recently developed monoclonal antibodies against different strains of influenza virus have been raised the possibility of mitigating infectious epidemics [31] . the only approved monoclonal antibody palivizumab (synagis) is leading to development of 2nd and 3rd generation monoclonal antibodies astrazeneca/medimmune against respiratory syncytial virus infection in infants [32] . another study has suggested that clinical trial of monoclonal antibodies against hendra and nipah viruses in animals exhibited convincing results [33, 34] . likewise, antibody based treatment of ebola, hiv, and hpv virus is under clinical trials [35] . polyclonal antibodies are more efficient and provide better protection against infections because of their ability to target different antigenic polymorphisms and serotypes. it provides an excellent opportunity to ameliorate the prognosis of emerging infectious diseases and neglected tropical diseases as well. additionally, polyclonal antibodies can simultaneously target variety of epitopes and kill pathogens. symphogen introduced effective methods to develop antigen-specific recombinant human polyclonal antibodies i.e., symplex tm and sympress tm techniques against infectious diseases and cancers. sympress platform uses mammalian cell lines that express high level of single antibody molecule to develop reproducible and robust polyclonal antibodies. polyclonal antibodies have been efficaciously applied to intoxication, envenomation and rabies. rabbit anti-rabies virus glycoprotein g polyclonal antibody (catalog number: csb-pa14899a0rb) induces the endocytosis of virion because this polyclonal antibody cause attachment of the virus to host cellular receptors. this fusion triggered by acidic ph of endosome leads to conformational changes in the glycoprotein trimer. this transmembrane glycoprotein is the viral attachment protein that promotes uptake of virus by infected cells, and acts as target of the host humoral immune response to infection [36, 37] . only two individuals developed rabies out of total 7660 filipino recipients of f (ab 0 )2 equine rabies immunoglobulin (erig), (favirab, sanofi pasteur, lyon, france). as per who guidelines none of them had given post-exposure prophylaxis (pep) strictly [38, 39] . there are limited therapeutic options available against viruses therefore polyclonal serum therapy will very soon be emerged as potent and effective technique against viral infections based on several researches and clinical studies that have demonstrated the effectiveness of animal-derived polyclonal antibody based treatments targeting different venoms, toxins and infections [40] . currently, immunoglobulin therapy is targeting hpa1 (highly pathogenic avian influenza) viruses e.g., h7n9 and h5n1 are new targets for polyclonal f(ab 0 )2 immunoglobulin therapy. in mice, in vivo proof-of-concept studies of equine polyclonal f(ab 0 )2 to hpai h5n1 (highly pathogenic avian influenze-h5n1) have been found to prevent infection after an intranasal hpai h5n1 attack [41] . according to another study, both prior (prophylaxis) or post (therapeutic) administration of four pepsin digested immunoglobulin h5n1 avian influenza equine f(ab 0 )2 preparations exhibited cytopathic effect against h5n1 infected cultured mdck (madin-darby canine kidney) cells and provided protection to mice [42] . in another study, 100% survival rate was observed in influenza-infected-mice that was intravenously infused with high dose 320 lg) of anti-influenza igg [43] . no serious adverse events were observed in phase 1 clinical trial carried out in 21-40 years old, 16 healthy human males who were injected with polyclonal f(ab 0 )2 to hpai h5n1 [44] . likewise, many animal based studies have demonstrated the clinical efficacy of polyclonal antibody therapy against various neglected tropical viral diseases. unimmunized hamsters injected with west nile virus-immune hamster antisera (1 h before and 24 h after a west nile virus challenge) were protected from life-threatening west nile virus infection [45] . decrease in disease and deaths were noticed when marburg and ebola filoviruses challenged non-human primates were injected with marburg and ebola filoviruses specific polyclonal antibodies obtained from non-human primates who had survived flavoviruses exposure [46] . likewise, similar results were obtained when ebola infected mice, guinea pigs, and monkeys were injected with polyclonal sera acquired from ebola immunized mice [47, 48] . equine f (ab 0 )2 have been found effective in both hamsters and mice infected with sars-cov infection because a significant reduction in lung viral titres was observed compared to controls [49] . i) immunoglobulin therapy acts as broad spectrum antimicrobial therapy active against all classes of infectious organisms and can be developed against any kind of pathogen. human body is able to generate antibodies against all existing pathogens. antibodies encoded by variable gene elements are assembled because combination of diverse gene elements in the germline increases the possibility of antibody production against large number of antigens. induction of somatic mutations antibody genes leads to production of more diverse, more specific and high affinity antibodies [50] . ii) therapeutic antibody development does not only target extracellular pathogens it has been reported that monoclonal antibodies are also effective against intracellular pathogens e.g., there are some intracellular viruses which can be neutralized by iga monoclonal antibodies [51] . iii) introduction of antibodies bring about antimicrobial action using different mechanisms such as antibody directed cellular toxicity, toxin and viral neutralization, opsonization, obstruction of microbial attachment and complement activation [52] . iv) pharmacokinetics of immunoglobulin isotype igg demonstrated it as an effective antimicrobial agent because of half life of 20 days [53] and good tissue penetration power [54] . the half life of murine monoclonal antibodies is shorter in humans and this usually triggers human antibody response. humanized monoclonal antibodies and human-mouse chimeric antibodies are synthetic antibody preparations made up of human antibody protein sequences that maintain antigen binding region present on heterologous antibody and have longer half-lives [55] . adoptive immunotherapy is the extraction and ex-vivo activation of native immune cells of patients followed by intravenous injection into human body to target infections [56] . it is an effective and efficient technique to build up immunity against viruses. t cells are depleted and progressively lose their function during cancer and chronic infections therefore, effective t cell responses can curb tumors and viral infections hence, techniques that either provide functional t cells based on adoptive immunotherapy or restore endogenous immune responses are being explored. cd8+t cells play key role in cancer and viral infections whereas but their function is supported by cd4+ t cells in addition to this, cd4+ t cells trigger optimal b-cell responses and boost up innate immunity. therefore, adoptive immunoglobulin strategies based on exploitation of cd4+ t cells alone or in association with cd8+t cells, are effective against cancer and chronic infections [57, 58] . t-cell immunotherapy can be ameliorated by following strategies: culture conditions for ex vivo t-cell expansion to produce t cells with desirable phenotype. novel types of antigen presenting cells and cytokine combinations are able enough to produce functional t cells of peculiar phenotypes. i) t cells are genetically modified for generation of t cells with high affinity towards desired antigen (by introducing chimeric antigen receptor along with specific t-cell receptor) or with specific characters (for example, t cells deficient in hiv co-receptors can be produced in hiv-infected patients). ii) adoptive immunotherapy can be combined with other immunotherapies to reconstitute in vivo function as well as expansion of the transferred t cells, to control inhibitory signals and to prevent exhaustion of the transferred t cells [57] . a study demonstrated that virus specific t cells produced in response to antigen presenting cells along with unmanipulated t cells are helpful in treatment of viral infections such as cytomegalovirus, hiv, ebola virus and adenoviruses [59, 60] . it is anticipated that adoptive immunotherapies that impede inhibitory pathways (such as pd-1 pathway) in association with adoptive t-cell therapy will lead to the long-term maintenance of effective immune responses to eliminate cancer and chronic infections [57] . human immunodeficiency virus (hiv-1) is lentiretrovirus that causes acquired immuno deficiency syndrome (aids) and destroys immune system by interacting with variety of different body cells. this virus is transmitted vertically, sexually and through blood. hiv infection is associated with progressive exhaustion of cd4+ t cells due to their enhanced destruction and diminished production [61] . with cd4+ t cells, hiv replication leads to cell death, syncytium formation and persistent infection thus creating reservoirs for the virus in many cells and tissues [58] . about 78 million people have suffered from hiv virus and 39 million have died since the beginning of this epidemic [62] . it is a major cause of morbidity and mortality in developing world. in 2002, the global prevalence of hiv was 31 million which was increased to 35ã�3 million in 2012. about 1.5 million deaths were reported in 2013 but the incidence is now decreasing due to expanded access to antiretroviral therapy [61] . since last many years patients of hiv have been treated with cart (combined antiretroviral therapy) i.e., use of several antiretroviral drugs which suppress the replication of virus but virus is not completely eradicated therefore, trends are now shifting towards practice of immune based treatments. other limitations associated with combined antiretroviral therapy are transmission of drug resistant hiv strains, emergence of multidrug resistance and cart does not always restore normal cd4+ t cell counts [63] . more than 30 million people are suffering from hiv type-1, initially this condition was considered lethal whereas, the combination of immune-based therapeutics results in longer life span in infected persons, decline viremia, limit hiv replication, inhibit disease progression, and trigger cytotoxic t lymphocyte mediated clearance of infected cells. human immunodeficiency virus-1 impedes activity of cd4+ t lymphocytes leading to exhaustion of these cells and progressive immunodeficiency. within initial three months of infection, high concentration of antibodies is developed against different viral proteins in hiv-1 infected patients and recent studies unfolded that antibodies, cd8+ t cell activity, and cd4+ helper responses lead to control of hiv virus. there are several different immunotherapeutic approaches against hiv infection that are currently being studied [64, 65] . there are several novel strategies that are being explored to discover preventive and therapeutic vaccines for prevention of hiv-1 infection. current therapeutic vaccine approaches include administration of single or multiple antigens of hiv as dna, autologous dendritic cells, inactivated whole hiv particle depleted of gp120 and viral vectors like poxviruses (alvac-hiv, vcp1433, vcp1452, fowlpox, mva) and adenovirus (ad5) [66] . to date, rv144 is the only vaccine that has shown some degree of efficacy [67] . modest efficacy against hiv acquisition was observed in thai phase iii clinical trial of rv144. antibody responses against hiv-1 gp120 envelope (env) were observed in plasma obtained from hiv-1uninfected individuals administered with alvac-hiv (vcp1521) prime and aidsvax b/e boost. peptide microarray analysis from six hiv-1 subtypes and group m consensus exhibited that vaccination triggered antibody responses to the v2 loop or second variable of gp120 of multiple subtypes. v2 responses were further evaluated by elisa and surface plasmon resonance using linear and cyclic v2 loop peptides. antibody responses against cyclic v2 at 2 weeks postimmunization were noticed in about 97% of vaccinated individuals. rv144 vaccination triggered antibodies that targeted a region of the second loop consisting of conserved epitopes. early transmission events of hiv-1 involve second loop interactions and supports the evidence that in rv144 anti-v2 antibodies may contribute to viral inhibition [68] . during 1980s and 1990s preventative vaccine directed against hiv-1 infection was developed using 20 different recombinant envelope proteins belonging to different strains, anticipating the production of neutralizing antibodies for hiv. two recombinant gp120 vaccines bivalent subtype b/e and bivalent subtype b/b were tested in phase 3 but none of these two vaccines proved efficacious [69] . both vaccines triggered production of neutralizing and binding antibodies, but neutralizing antibodies were restricted to the strain used in the vaccine [70] , the narrow neutralizing response is due to deletion and auto-reactivity of precursor b cells which induce the development of broadly reactive neutralizing antibodies [71] . post hoc examination exhibited that individuals carrying high concentration of binding and blocking antibodies may develop considerable level of protection from acquisition [72] . non-efficacy of recombinant envelope vaccines shifted the focus towards development of immune response having crossstrain breadth. early viral control is markedly influenced by breadth and magnitude of early cd8+ t-cells therefore, ctlbased vaccines or cytotoxic t lymphocyte vaccines were developed primarily to target post-infection viremia, and prevention of hiv acquisition was anticipated. cytotoxic t lymphocyte responses against hiv proteins are induced by inserting hiv genes into recombinant viral vectors and shuttling these genes into class i antigen-presenting pathway [73] . replication defective recombinant adenovirus 5 vector with hiv-1 clade b nef/gag/pol inserts was the first t-cell vaccine that underwent clinical efficacy trials and exhibited significant increase in cd8+ t cell but these cd8+ immune responses targeted the variable but not the conserved regions of virus. therefore, an issue of immune t cell breadth same as neutralizing antibody breadth was still there [74] . however, the tolerability, safety, and efficacy of conserved region ad5 based vaccine has been registered under clinicaltrials.gov nct01151319. researchers designed distinctive t-cell immunogen hivconsv different from functionally conserved regions of the hiv-1 proteome that encountered body's immunity using heterologous prime-boost combination of non-replicating poxvirus and non-replicating simian (chimpanzee) adenovirus chadv-63, modified vaccinia virus ankara, and plasmid dna (chad63 vaccine). administration of chadv63.hivconsv combined with other vaccines elicited high frequencies of hiv-1-specific t cells capable of inhibiting hiv-1 replication and exhibited good tolerability and safety together with high immunogenicity [75] . this is another t-cell based approach in which adenovirus vector 5 is primed with dna. to overcome t-cell and antibody breadth problem, different strains belonging to all major hiv-1 clade were used. the dna vaccine (0, 1, 2 months) was a blend of six plasmids expressing env proteins from clades a, b, and c whereas, gag, pol, and nef from clade b followed by an adenovirus 5 vector boost during (6th month) leading to expression of env glycoproteins from clades a, b, and c along with gag-pol fusion protein from clade b [76] . clinical study demonstrated cd4+ t cells response towards hiv-1 envelope, neutralizing antibodies and triggered antibodies towards gp41 and hiv gp120 [77] . rv144 (2004-2009) provided sound proof of vaccine reducing hiv acquisition exhibiting 60.5% efficacy at 1 year followed by 31.2% after 3.5 years with alvac-hiv (vcp1521) that is canarypox vector prime which expresses clade e env along with clade b gag and pro (0, 1, 3, 6 months) and protein boosts in association of alum adjuvant, aids-vax1 clades b/e gp120 (3, 6 months). v2 region of hiv-1 is susceptible to be targeted by protective antibodies associated with vaccine efficacy of the rv144 regimen [78] . immediately after demonstration of rv144 results, pox protein public-private partnership (p5) collaborated to discover poxprotein regimen for sub-saharan africa. the immunologic response observed in people of south africa was similar to responses observed in thailand. alvac vector with clade c env insert was designed by p5 to construct a bivalent clade c recombinant gp120. currently, these vaccines with two adjuvants aso1b and mf591 are under clinical trials in south african region to boost immunity. the results of hvtn 100, phase 1/2 study, alvac/gp120/ are pending and scheduled for efficacy evaluation trial in late 2016 [79] . efficacy trials of six candidate vaccines vax004, vax003, step, phambili, rv144, and hvtn505 proved rv144 as the only hiv-1 vaccine trial that is effective against hiv acquisition. the antibodies are produced in response to v1v2 region of gp120 specifically. igg1 and igg3 subclass mediates adcc (antibody-dependent cell-mediated cytotoxicity) and accord protection against hiv-1 acquisition [80] [81] [82] . therapeutic vaccines are developed to ameliorate immune response in order to ameliorate hiv infection. to date, there is not even single therapeutic hiv vaccine that has been approved by fda. instead, therapeutic vaccines directed against hiv infections are under clinical trials to evaluate the efficacy. the italian national aids center is developing a vaccine directed against hiv-1 tat (transactivator of transcription) protein that is a virulence factor and plays a significant role in hiv gene expression, progression and transmission of disease. tat-specific antibodies may prevent hiv acquisition and transmission. phase 1 study has proved tat vaccination as immunogenic and safe. phase ii trial proposed that tat had induced restoration of cd4+ and cd8+ t cell numbers, and memory cells. considerable reduction in hiv-1 load in blood was observed in response to tat vaccine. phase 3 trials are being studied currently. forty-eight long-term hiv-1 infected people with suppressed viral loads because of antiretroviral therapy (cart) were injected with tat oyi vaccine preparation. the clinical outcome of this phase i/iia trial showed decrease in the extent of hiv rna rebound after cart interruption. this study shows that tat vaccine protects and activates hiv-infected cell in vitro [83] . combined treatment of tat oyi vaccine and cart has found to increase cd4+ t-cell numbers. phase iii studies that has been conducted in south africa (trial registration clinicaltrials.gov nct01513135) unfolded that tat vaccination induces cross-clade neutralizing anti-tat antibodies in patients with different infecting viruses and belonging to different genetic backgrounds [84] . adoptive t cell therapy is transfer of cd4+, cd8+ or autologous antigen specific t lymphocyte cells to hiv infected patients [85] . hiv specific ctl (cytotoxic t lymphocyte) response plays an important role in control of hiv infection. researchers are finding novel approaches to ameliorate hiv-specific cytotoxic t lymphocyte response in an attempt to achieve long-term viral clearance. a research study based on hematopoietic stem progenitor cell (hspc) approach reported the use of protective car (chimeric antigen receptor) to engineer immunity in hiv infected patients. car-modified hspcs cells (chimeric antigen receptor modified hematopoietic stem progenitor cell) differentiate into functional t cells along with natural killer cells which were resistant to hiv infection leading to cessation of hiv replication. therefore, it has been proved that stem cell-based gene therapy with chimeric antigen receptor (car) is an effective and feasible strategy to treat chronic hiv infection [86] . modification of human hspcs (hematopoietic stem progenitor cells) with an hiv-specific cd4f car can differentiate hiv specific t cells and cells of other lineages that are able to decrease viral loads in vivo. this hspc-based approach that use car has been demonstrated safe and feasible in mice whereas, human based multiple hspc-based gene therapy that aims at protecting cells from hiv infection are currently under trials [clinicaltrials.gov identifiers: nct01177059, nct00569985, nct01961063, nct01734850]. the potential toxicities and adverse events related to the use of the cd4f car have not been studied yet therefore, future research must address this subject [86] . the special features present in cd4-10-17b car have made it a suitable candidate for genetic modification of t cells from hiv-1-infected individuals. the broad reactivity to genetically diverse hiv-1 isolates, minimal immunogenic potential, freedom from hiv entry receptor activity, and high potency of virus suppression all speak to the value of this car design [87] . hiv infection is associated with cytokine production. such cytokines influence viral replication and regulate immune system thus, contribute towards the progression of acquired immunodeficiency syndrome. several cytokines involved in immune regulation exert opposite effects, like some stimulate cellular immune function and others induce production of antibodies. aids is characterized by diverse disturbances and imbalances in the regulation of cytokine expression. different cytokines affect expression and replication of hiv differently [88] . production of t-helper type 1 (th1) cytokines e.g., antiviral interferon-c, interleukin-2 (il-2) are decreased whereas t-helper type 2 (th2) cytokines e.g., interleukin-1, il4, il6, il8, il10, tumor necrosis factor are increased. ifn-a, ifn-b and il-16, which inhibit hiv-1 replication in t cells and mdm (monocyte derived macrophages), and il-10 and il-13, which inhibit hiv-1 in monocyte derived macrophage act as hiv-suppressor cytokines. tnf-a, tnf-p, il-1 and il-6, which trigger hiv-1 replication in mdm and t cells, macrophagecolony stimulating factor, which stimulates hiv-1 in mdm and il2, il-7 and il-15 which provoke hiv-1 in t cells act as hivinductive cytokines. ifn-c, il-4 and granulocyte-macrophage colony-stimulating factor act as bifunctional cytokines because of both inhibitory and stimulatory impacts on hiv-1 infection [89] [90] [91] . therefore, cytokine therapy may suggest new ways to prevent progression to aids. hiv-1 patients experience significant decline in cd4 + t cells but il-2 therapy has been shown to improve cd4 + counts [92] the findings of this study were in contrast to previous studies which suggested that il-2 had no supplementary benefits in hiv-1 patients [93] . levy et al. have also demonstrated considerable rise in cd4 cells in hiv-1 infected individuals administered with il-2 in combination with highly active antiretroviral therapy. naã¯ve and memory cd4 cells count, natural killer cells, lymphocyte expression of cd25 and cd28 was higher in il-2 group compared to controls [94] . administration of il-7 in hiv-1 infected individuals led to expansion of cd4 + , cd8 + t cells, and il-7 receptor alpha chain cd127 + . rhil-7 amplify the numbers of naã¯ve and central memory t cells [95] . marked decrease in the production of virus-induced interferon (ifn)-a [96] and interferon-a producing cells (ipcs) has been observed in hiv patients. this study suggested a direct link of interferon-a producing cells (ipcs) with control of hiv replication [97] . phase ii/iii study showed lower rate of hiv progression in 40 of 122 ifn-a vaccine recipients [98] . another study involving 34 patients of asymptomatic hiv infection administered with ifn-a showed decrease in hiv load. of the 32 study patients followed after study (range, 5-33 months), no patients in the ifn-a group developed an aids compared with 5 patients in the placebo group [99] . several researchers have characterized and isolated neutralizing antibodies to target hiv-1 thus, introducing new ways in passive immunization. vaccine trials in animal models has demonstrated that hiv-1 neutralizing antibodies are potent enough to suppress hiv infection [100] . vrc01 is human monoclonal antibody which targets cd4 binding site of human immunodeficiency virus gp120 [101] . likewise, other monoclonal antibodies such as pgt121 [102] , 3bnc117 [103] , r1c7, a4f6, r5c4, r5f6 [104] ccr5 [103] and 10-1074 [105] have been proved effective in suppression of hiv infection in animal models. monoclonal antibody a32 acts as a potent mediator of adcc (antibody dependent cellular toxicity) activity and plays an important role in preventing hiv acquisition [106] . accumulating evidences suggest that administration of monoclonal antibodies to humanized mice resulted in suppression of viral load. likewise, injection of cocktail of hiv-1-specific monoclonal antibodies, as well as the single glycan-dependent monoclonal antibody pgt121 caused dramatic decline of plasma viraemia in rhesus monkey chronically infected with simian-humanimmuno-deficiency virus [107] . human monoclonal antibody f105 has successfully passed through phase i clinical trials (clinicaltrials.gov identifier: nct00001105). f105 binds with cd4 binding site of hiv-1 gp120 and neutralizes laboratory and clinical hiv isolates [108] . recently another antibody 3bnc117 has been reported to boost up humoral immunity against hiv-1 in animals and humans [109] . human based follow up clinical studies and the complete evaluation of therapeutic potential of these monoclonal antibodies is currently under way. number of dendritic cells in blood reduces in case of hiv-1 infection and this can be reversed by dendritic cell therapy [110] . several evidences suggest that development of dendritic cell based vaccines is a promising approach against hiv-1 infection. a significant decrease in viral load had been observed when hiv-1 infected individuals were treated with dendritic cell based vaccination [111] . dendritic cell based therapy controls hiv replication by eliciting t-cell response [112] . therefore, therapeutic vaccination based on monocyte-derived dendritic cells is feasible, safe and effective to control hiv infection [113] . vitamin d3 binding protein or serum gc protein acts as precursor of principal macrophage activating factor (maf). maf precursor activity or serum gc protein is either lost or diminished in hiv infected individuals because of deglycosylation of gc protein by alpha-n-acetylgalactosaminidase produced by hiv-infected cells. thus, in hiv infected patients macrophages with deglycosylated gc protein are inactivated and cause immunsuppression. infusion of gcmaf into hiv infected individuals is an effective immunization strategy because it leads to complete elimination of virus [114] . human papillomaviruses (hpvs) are dna viruses which cause human neoplasias like warts and cancers. about 40 out of more than 100 human papillomavirus types infect anogenital region hpv infection is most prevalent sexually transmitted infection [115] . the most common types worldwide are hpv-16, hpv-18, hpv-52, hpv-31, hpv-45 and hpv-58 [116, 117] . hpv can cause number of cancers such as cervical cancer [118] , oropharyngeal cancer, anal cancer, vulval, vaginal and penile cancer [119] . there is no drug available that directly eliminates hpv but treatments are available for hpv associated health problems like gential warts, cervical change, cervical cancer etc. scientists are looking for new opportunities to develop immune-based treatments to reduce the hpv associated cancer upsurge. 11.14. immuno-based treatment of hpv besides chemotherapy, surgery or radiation, immunotherapeutic approaches and vaccines are an exciting addition to control precancerous diseases and cancer. clinical and epidemiological data reveal that induction of t-cell responses correlates with clearance of hpv-associated lesions, induction of th1-biased immune responses are known to be crucial for immunotherapy, hpv regulatory proteins e6 and e7 that are targeted as viral antigens and development of more potent th-1 directed vaccine platform form the basis of hpv immune-based treatment especially efficacious vaccine development. viruses like particles (vlp) of hpv are used as vaccine directed against hpv associated cancer by activating natural killer cells which in association with dendritic cells induce immune response against viral infections and tumors. in the presence of hpv-vlp, natural killer cells increase maturation of dendritic cells by upregulating cd86, hla-dr and il-12p70. an increase in secretion of ifn-c and cytotoxic activity against hpv ameliorates function of natural killer cell. therefore, virus-like particle vaccine has been proved as best candidate to control hpv-associated malignancies [120] . food and drug administration has approved three prophylactic vaccines gardasil (quadrivalent hpv), gardasil-9, and cervarix, which are highly immunogenic and reduce the risk of hpv infections [121] . vaccines formulation is based on use of viruslike particles originated from l1 proteins that resemble hpv but cannot multiply due to absence of genetic material [122] . hpv vaccines are potent enough to eradicate malignant tumors and preexisting lesions by inducing cellular immunity directed against hpv-infected cells which express early viral proteins like e6 and e7 [123] . the vaccines raise the titer of serum immunoglobulin g antibody directed against different hpv types, secreted in cervico-vaginal region or discharged from micro-abrasions in epithelium directed against different hpv types [124] . gardasil ã� is human papillomavirus quadrivalent recombinant vaccine composed of virus-like particles obtained from l1 capsid proteins belonging to hpv type 6, 11, 16 and 18. the vaccine was manufactured by merck & co and approved in 2006. this vaccine is effective against genital warts and precancerous lesions, vaginal pre-cancer and cancer, vulvar and cervical cancer in young women and adolescents. individuals are treated with intramascular injection of three dose regimen. this vaccine proved highly immunogenic instigating persistent and high-hpv antibody titer. phase iii trials has proved the efficacy of gardasil in young women and male and female adolescents. gardasil 9 is a second generation of merck's cervical cancer vaccine that has been approved by food and drug administration that will protect against anal, vaginal, and cervical cancers. the vaccine is effective against cancers associated with hpv types 16, 18, 31, 33, 45, 52 , and 58 and genital warts associated with hpv types 6 and 11 [125] . a 3rd generation vaccine is still needed to achieve complete protection against all hpv types which cause cervical cancers [126, 127] . cervarix tm manufactured by glaxosmithkline is bivalent l1 virus-like particle vaccine effective against hpv types 16 and 18 both responsible for 70% of all cervical cancers [128] . this vaccine was developed by using insect cells infected with recombinant baculovirus and an adjuvant aso4 consisting of alum combined with a tlr4 ligand, mpl (3-odesacyl-4 0 -monophosphoryl lipid a). the efficacy trials have demonstrated that vaccines are immunogenic, 90.4% efficacious and increase the neutralizing antibody titer thus providing protection against cin2 (cervical intraepithelial neoplasia) lesions and cancers caused by hpv16 and hpv18 [129] . dendritic cell based vaccination is another novel therapeutic paradigm to cure hpv-associated cancers. dendritic cells are recognized as potent antigen presenting cells and lead to induction of t cell responses in vitro and in vivo providing new ways to treat several human malignancies. autologous dendritic cell loaded with hpv16/18 e7 proteins may stimulate t and b cell responses in patients unresponsive to standard treatments. dc based vaccine is efficacious in only those cancer patients who are immunocompetent or at early stages of disease and have low tumor burden [130, 131] . several other therapeutic cancer vaccines mainly targeting hpv oncoproteins e6 and e7 are under clinical trials that stimulate t cell immune response against tumor specific antigen, thereby triggering the immunity to target cancer cells such as adxs11-001 vaccine directed against hpv e7 protein to cure anal and cervical cancer [132] , dna construct ino-9012 that triggers production of interleukins to treat cervical cancer [133] , vgx-3100 against hpv type 16 and 18 [134] , tvgv-1 vaccine against hpv associated cervical pre-cancer, pngvl4a/e7 (detox)/hsp70 dna vaccine provide protection against hpv-16 cervical intraepithelial neoplasia (table 2 ) [135] . immunomodulators include both immunosuppressive and immunostimulatory agents that trigger secretion of cytokines from macrophages (il-12, ifn-12, tnf-a) leading to increased th1 response, antibody production in response to improved antigen presentation by dendritic cells, and cell-mediated immunity which is being used clinically to cure viral infections like herpes simplex virus, human papillomavirus and cancerous lesions in immunocompromised individuals [136] . pd-1 antibodies nivolumab (opdivo ã� ) and pembrolizumab (keytruda ã� ) against vaginal, vulvar and cervical cancer [137, 138] , anti-pd-l1 antibody durvalumab (medi4736) [139] in combination with tremelimumab against six cancer including cervical cancer and anti-ctla-4 antibody ipilimumab (yervoy ã� ) [140] against cervical cancer are under clinical trials. the development of monoclonal antibodies is an emerging therapeutic strategy to cure cancer and viral infections because of low toxicity, high specificity and activation of immune system. a study elucidated that monoclonal antibodies 1g10.1c and 2c5.1c, ae3 and ag7 may form the basis of effective development of immunotherapy against hpv infections [141, 142] . several other monoclonal antibodies are under clinical trials such as bevacizumab (avastin ã� ) which is humanized anti-vascular endothelial growth factor monoclonal antibody against cervical and ovarian cancer [143, 144] , humax ã� -tf-adc [145] and immu-132 are antibody drug conjugates which are being studied in phase i/ii trial in an attempt to cure advanced cancers including cervical cancers [146] . immunotherapeutic treatment based on infusion of cytokines and insertion of immunostimulatory genes in the tumor cell table 2 different forms of hpv therapeutic vaccines that are under clinical trials. genome followed by cytokine based vaccination represents novel approach for therapy of hpv associated cancers. researchers have recently proposed that tumor cells caused by hpv-16 can be genetically modified with dna encoding immunostimulatory molecules specifically cytokines (il-2, il-12, gm-csf) used for vaccination, and impede tumor growth. in order to ameliorate the antigen presentation in tumor bearing patients, dendritic cell-based vaccines loaded with hybrids of the dendritic and tumor cells or hpv 16 e6/e7 dna have also been successfully employed. these encouraging approaches are still being studied [147] . another study suggested that targeting hpv e6 and e7 oncoprotein with adoptive t-cell therapy could be an efficacious strategy against hpv-related cancers such as vagina, penis, vulva, cervical, anal, and oropharynx [148] . herpes simplex viruses are enveloped, double stranded dna viruses having two serotypes: hsv-1 infecting orofacial region and hsv-2 in the genital region [149] . infections caused by herpes simplex virus are prevalent worldwide. significant rate of neonatal morbidity and mortality is attributed to herpes labialis caused by hsv-1 [150] and herpes vulvovaginitis caused by hsv-2 [151] . incidence rate of hsv infections in neonates is 1 per 3000-20,000 live births [152] . hsv cause number of infections such as labialis, conjunctivitis/keratitis, gingivostomatitis, herpetic whitlow, eczema herpeticum, herpes gladiatorum, encephalitis, balanitis, urethritis, vulvovaginitis, and external dysuria [153] . hsv infections can be treated with antiviral agents like acyclovir [154] but drug therapy is associated with toxic side effects, emergence of drug resistance strains, narrow spectrum, and drug treatment is effective only during initial stages of infection [155] . people infected with hsv-1 infection are at lesser risk of acquiring it again but they can still be infected with hsv-2 genital infection. hsv-2 also increases the risk of hiv infection [156] . the immunobiology of herpes simplex virus associated infections is complicated. both humoral and cell mediated immunity are of paramount importance in immunologic responses against hsv infections, due to plethora of evolutionary changes virus evades the immune system, maintains the latency and cause intermittent reactivation of disease. these facts limit the utility of passive immunization to control hsv infections. therefore, the subject of development of immunotherapeutic strategies against hsv infections is still being studied and need more research in the coming episodes. the development of efficacious therapeutic and prophylactic vaccine against herpes virus infections has proven complicated due to complex life cycle (latency) of herpes simplex and poorly understood mechanism of immune control at primary and recurrent stage of disease. previous studies have described that activated innate immunity and virus-specific t helper 1 (th1) cytokines (like gamma interferon) prevent recurrent disease. whereas, regulatory (suppressor) t cells and th2 cytokines (e.g., interleukin-10 [il-10]) down regulate this immune profile thereby, allowing the establishment of recurrent disease and replication of reactivated virus. therefore, an efficacious vaccine must stimulate th1 immunity and be defective in th2 cytokine production especially il-10 [157] . gylcoprotiens gd and gb activate cd4+ t cells and icp27 activate cd8+ t cells [158] . these facts form the basis of development of herpes simplex virus type 2 (hsv-2) glycoprotein-d-subunit vaccine with adjuvant alum and 3-odeacylated-monophosphoryl lipid a which induce helper t cells th1. the results of clinical trials phase i/ii exhibited that glycoprotein d vaccine was efficacious against genital herpes in hsv-1 and hsv-2 sero-negative females whereas, not effective in men and those women who were hsv-1 sero-positive and hsv-2 seroneagtive [159] . another study suggested secreted glycoprotein g of hsv-2 as highly efficacious novel agent for development of prophylactic vaccine to control hsv-2 associated infections [160] . in an experiment carried out in guinea pigs, recombinant hsv-1 glycoproteins gb and gd formulated with an adjuvant were used as immunotherapeutic agents in order to control recurrent genital herpes and resulting increase in antibody titer supported the fact that gbgd immunotherapy could be effective against hsv associated genital infections [161] . an attenuated virus r7020 has been designed and provided immunogenicity against infections caused by hsv-1 and hsv-2 in mice and guinea pigs [162] . however, the area of hsv vaccine development needs further research. though the mechanism of antibody treatment in hsv-1 or hsv-2 infections has not completely elucidated but passive immunization via antibodies is emerging as promising technology for controlling hsv infections however, experimental study in humans have yet to be performed [152] . several evidences have suggested that passively transferred serum hyperimmune may effectively inhibit hsv-1 and hsv-2 spread and significantly reduce the severity of infection caused by these viruses [163] . in vitro studies have defined several mechanisms, including antibody-dependent cell-mediated cytotoxicity whereby antibody may participate in the destruction of virus infected cells [164] . monoclonal antibodies hc1 and hd1 directed against hsv-1 glycoproteins gc and gd had been tested because of their ability to passively immunize mice in order to target acute virusinduced neurological disease and the study later on revealed that passive immunization of monoclonal antibody in mouse decreased the severity of disease and pathogen spread [165] . likewise, nine other monoclonal antibodies aiming against hps glycoproteins gb, gc, gd, ge had also been evaluated in mice and lead to blockage of hsv virus dissemination [166] . monoclonal antibody hu2c holds promise for future development as a novel approach for the treatment of hsv infections. humanized monoclonal antibody mab hu2c were administered in mice and resulted in inhibition of cell-to-cell viral transmission a key mechanism by which hsv-1/2 escapes humoral immune surveillance. mab hu2c was found to neutralize hsv fully independent of complement or/and recruit immune effector cell in a highly efficient manner [167, 168] . these features guarantee the clinical development of mab hu2c for treatment of hsv infections in drug-resistant and immunocompromised patients. components of innate immunity such as interferon, natural killer cells, and macrophages provide protection against hsv infections [169] but, t cells specifically ctls (cd8+ cytotoxic t cells) are dominant determinants of protective immunity [170] . fusion protein tgd-il-2 consisting of human interleukin-2 and truncated hsv-1 glycoprotein d has been proved as efficacious immunotherapeutic agent that can elicit immune system against hsv associated genital infection [171] . another experimental protocol demonstrated the role of dendritic cells in generation of protective immunity [172, 173] . adoptive transfer of virus-specific cytotoxic t lymphocytes has proven efficacious and safe at preventing and controlling viral infections, but there is room to explore expansion and activation protocols utilized to generate virus-specific ctl (cytotoxic t lymphocyte) lines in minimum possible time [174] . successful outcomes of car-t cells provide strong hope for control of infectious diseases. it is anticipated that immunizations with promising clinical outcomes will be available for myriad of infectious diseases and cancers. development of polyclonal and monoclonal antibody therapies for severe neglected tropical widespread diseases is currently under investigation. clinical data and pre-clinical studies highlight the potential of immunotherapies against hpv, hsv, and hiv thus, providing baseline data for future research. infectious diseases are major cause of morbidity and mortality thus, posing serious threats to lives. drug therapy is associated with serious consequences such as emergence of drug-resistant strains and toxic side-effects. therefore, trends are now shifting towards immune-based therapies that reawaken and harness the power of immune system to provide long-lasting protection. therapeutic vaccines, adoptive t cell therapy, gm-csf therapy, cytokine therapy and monoclonal antibodies are being explored and getting popular for treatment of hiv and hpv associated infections in immunocompromised hosts. although, the specific immunization strategy for hsv has not yet been developed but the overall progress made by researchers has increased understanding towards complexity of infections caused by hsv-1 and hsv-2 and described lot of issues that can now be explored and addressed. one hundred years of immunotherapy: review of the first landmark studies immunotherapies in infectious diseases immunotherapy for infectious diseases world health organization, antimicrobial resistance global report on surveillance intravenous immunoglobulin for infectious diseases: back to the pre-antibiotic and passive prophylaxis era? epidemiology of drug resistance. implications for a postantimicrobial era immunotherapy in clinical medicine: historical perspective and current status edward jenner and the history of smallpox and vaccination immunoinformatics: an integrated scenario innate immune recognition immunotherapy: past, present and future differentiation of effector cd4 t cell populations follicular helper cd4 t cells (tfh) central memory and effector memory t cell subsets: function, generation, and maintenance the role of pattern-recognition receptors in innate immunity: update on toll-like receptors signalling through c-type lectin receptors: shaping immune responses how the noninflammasome nlrs function in the innate immune system recognition of viruses by cytoplasmic sensors translating innate immunity into immunological memory: implications for vaccine development dendritic cells in vivo: a key target for a new vaccine science conformations of immunoglobulin hypervariable regions serum therapy revisited: animal models of infection and development of passive antibody therapy antibody-based therapies for emerging infectious diseases new aspects of immunotherapy of leptomeningeal metastasis control of early viral and bacterial distribution and disease by natural antibodies the effect of two different dosages of intravenous immunoglobulin on the incidence of recurrent infections in patients with primary hypogammaglobulinemia: a randomized, double-blind, multicenter crossover trial treatment of west nile virus encephalitis with intravenous immunoglobulin treatment of parvovirus b-19 (pv b-19) infection allows for successful kidney transplantation without disease recurrence successful intravenous immunoglobulin therapy in 3 cases of parvovirus b19-associated chronic fatigue syndrome monoclonal antibodies for treating infectious diseases universal epitopes of influenza virus hemagglutinins? erratum to motavizumab a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge the ebola epidemic crystallizes the potential of passive antibody therapy for infectious diseases the role of site-specific n-glycosylation in secretion of soluble forms of rabies virus glycoprotein n-linked glycosylation of rabies virus glycoprotein. individual sequons differ in their glycosylation efficiencies and influence on cell surface expression rabies post-exposure prophylaxis in the philippines: health status of patients having received purified equine f (ab 0 ) 2 fragment rabies immunoglobulin (favirab) guide for rabies pre and post exposure prophylaxis in humans benefits of using heterologous polyclonal antibodies and potential application to new and undertreated infectious pathogens specific polyclonal f (ab 0 ) 2 neutralize a large panel of highly pathogenic avian influenza a viruses (h5n1) and control infection in mice cross clade prophylactic and therapeutic efficacy of polyvalent equine immunoglobulin f (ab 0 ) 2 against highly pathogenic avian influenza h5n1 in mice evaluation of anti-influenza efficiency of polyclonal igg antibodies specific to the ectodomain of m2 protein of influenza a virus by passive immunization of mice safety, potential efficacy, and pharmacokinetics of specific polyclonal immunoglobulin f (ab 0 ) 2 fragments against avian influenza a (h5n1) in healthy volunteers: a single-centre, randomised, double-blind, placebo-controlled, phase 1 study nipah virus: vaccination and passive protection studies in a hamster model postexposure antibody prophylaxis protects nonhuman primates from filovirus disease passive transfer of antibodies protects immunocompetent and immunodeficient mice against lethal ebola virus infection without complete inhibition of viral replication evaluation of immune globulin and recombinant interferon-a2b for treatment of experimental ebola virus infections inhibition of infection caused by severe acute respiratory syndrome-associated coronavirus by equine neutralizing antibody in aged mice the role of somatic hypermutation in the generation of antibody diversity intracellular neutralization of virus by immunoglobulin a antibodies principles and practice of infectious diseases immunologic and pharmacologic concepts of monoclonal antibodies problems of delivery of monoclonal antibodies passive immunotherapeutic strategies for the treatment of malignant gliomas cd4 t-cell immunotherapy for chronic viral infections and cancer pathogenesis of human immunodeficiency virus infection adoptive cellular immunotherapy for viral diseases principles for adoptive t cell therapy of human viral diseases hiv infection: epidemiology, pathogenesis, treatment, and prevention world health organization, global health observatory (gho) data current status and challenges of antiretroviral research and therapy an unexpected journey: how cancer immunotherapy has paved the way for an hiv-1 cure immunologic control of hiv-1 approaches to preventative and therapeutic hiv vaccines recent update in hiv vaccine development the thai phase iii hiv type 1 vaccine trial (rv144) regimen induces antibodies that target conserved regions within the v2 loop of gp120 randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 hiv-1 vaccine among injection drug users in immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1 redemption of autoreactive b cells correlation between immunologic responses to a recombinant glycoprotein 120 vaccine and incidence of hiv-1 infection in a phase 3 hiv-1 preventive vaccine trial challenges in the design of a t cell vaccine in the context of hiv-1 diversity mapping hiv-1 vaccine induced t-cell responses: bias towards less-conserved regions and potential impact on vaccine efficacy in the step study safety and tolerability of conserved region vaccines vectored by plasmid dna, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type 1-uninfected adults in a randomized, single-blind phase i trial immune and genetic correlates of vaccine protection against mucosal infection by siv in monkeys a phase iia randomized clinical trial of a multiclade hiv-1 dna prime followed by a multiclade rad5 hiv-1 vaccine boost in healthy adults (hvtn204) vaccination with alvac and aidsvax to prevent hiv-1 infection in thailand hvtn 097: evaluation of the rv144 vaccine regimen in hiv uninfected south african adults letter to the editor on: the rv144 vaccine regimen was not associated with enhancement of infection immune correlates of vaccine protection against hiv-1 acquisition review of efficacy trials of hiv-1/aids vaccines and regulatory lessons learned: a review from a regulatory perspective hiv-1 tat immunization restores immune homeostasis and attacks the haart-resistant blood hiv dna: results of a randomized phase ii exploratory clinical trial hiv-tat immunization induces cross-clade neutralizing antibodies and cd4+ t cell increases in antiretroviral-treated south african volunteers: a randomized phase ii clinical trial ex vivo expansion of hiv type 1-specific cytolytic t cells from hiv type 1-seropositive subjects hiv-specific immunity derived from chimeric antigen receptorengineered stem cells novel cd4-based bispecific chimeric antigen receptor designed for enhanced anti-hiv potency and absence of hiv entry receptor activity seminars in virology cytokines and hiv-1: interactions and clinical implications cytokine-based therapies for hiv infection the role of cytokines in the pathogenesis and treatment of hiv infection in vivo expansion of naive and activated cd4+ cd25+ foxp3+ regulatory t cell populations in interleukin-2-treated hiv patients interruption of antiretroviral therapy initiated during primary hiv-1 infection: impact of a therapeutic vaccination strategy combined with interleukin (il)-2 compared with il-2 alone in the anrs 095 randomized study effects of interleukin-2 therapy combined with highly active antiretroviral therapy on immune restoration in hiv-1 infection: a randomized controlled trial il-7 administration drives t cell-cycle entry and expansion in hiv-1 infection decreased interferon-a production in hiv-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors depletion of circulating natural type 1 interferonproducing cells in hiv-infected aids patients active anti-interferon-a immunization: a european-israeli, randomized, double-blind, placebo-controlled clinical trial in 242 hiv-1-infected patients (the euris study) interferon-a in patients with asymptomatic human immunodeficiency virus (hiv) infection: a randomized, placebo-controlled trial antibodies in hiv-1 vaccine development and therapy mechanism of neutralization by the broadly neutralizing hiv-1 monoclonal antibody vrc01 broadly neutralizing antibody pgt121 allosterically modulates cd4 binding via recognition of the hiv-1 gp120 v3 base and multiple surrounding glycans viraemia suppressed in hiv-1-infected humans by broadly neutralizing antibody 3bnc117 monoclonal antibodies against human immunodeficiency virus (hiv) type 2 core proteins: cross-reactivity with hiv type 1 and simian immunodeficiency virus hiv therapy by a combination of broadly neutralizing antibodies in humanized mice an hiv-1 gp120 envelope human monoclonal antibody that recognizes a c1 conformational epitope mediates potent antibody-dependent cellular cytotoxicity (adcc) activity and defines a common adcc epitope in human hiv-1 serum therapeutic efficacy of potent neutralizing hiv-1-specific monoclonal antibodies in shiv-infected rhesus monkeys pharmacokinetics of f105, a human monoclonal antibody, in persons infected with human immunodeficiency virus type 1 hiv-1 therapy with monoclonal antibody 3bnc117 elicits host immune responses against hiv-1 dendritic cell numbers in the blood of hiv-1 infected patients before and after changes in antiretroviral therapy therapeutic dendritic-cell vaccine for chronic hiv-1 infection a dendritic cell-based vaccine elicits t cell responses associated with control of hiv-1 replication a therapeutic dendritic cell-based vaccine for hiv-1 infection retracted: immunotherapy of hivinfected patients with gc protein-derived macrophage activating factor (gcmaf) human papillomavirus: a hidden epidemic in the united states cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings prevalence of hpv infection among men: a systematic review of the literature condom use and the risk of genital human papillomavirus infection in young women the burden of hpv-related cancers natural killer and dendritic cells collaborate in the immune response induced by the vaccine against uterine cervical cancer preclinical refinements of a broadly protective vlp-based hpv vaccine targeting the minor capsid protein the impact of quadrivalent human papillomavirus (hpv; types 6, 11, 16, and 18) l1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine hpv types in generally hpv-naive women aged 16-26 years therapeutic human papillomavirus vaccines: current clinical trials and future directions immune responses to human papillomavirus fda approves new upgraded gardasil 9 gardasil-9: a global survey of projected efficacy gardasil 9 joins the fight against cervix cancer classification of papillomaviruses efficacy of a prophylactic adjuvanted bivalent l1 virus-likeparticle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase iii double-blind, randomised controlled trial advances in dendritic cell-based therapeutic vaccines for cervical cancer hpv16/18 e7-pulsed dendritic cell vaccination in cervical cancer patients with recurrent disease refractory to standard treatment modalities adxs11-001 immunotherapy targeting hpv-e7: final results from a phase 2 study in indian women with recurrent cervical cancer immunotherapy with vgx-3100 (hpv16 and hpv18 plasmids)+ ino-9012 (dna encoding il-12) in human papillomavirus (hpv) associated head and neck squamous cell carcinoma (hnscca): interim safety and immunogenicity results safety, efficacy, and immunogenicity of vgx-3100, a therapeutic synthetic dna vaccine targeting human papillomavirus 16 and 18 e6 and e7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial current state in the development of candidate therapeutic hpv vaccines topical immunomodulators-progress towards treating inflammation, infection, and cancer adxs-hpv immunotherapy promising for anal cancer pd-1 and pd-l1 expression in 1599 gynecological malignancies-implications for immunotherapy phase ii, randomized, open-label study of durvalumab (medi4736) or tremelimumab monotherapy, or durvalumab+ tremelimumab, in patients with recurrent or metastatic (r/m) squamous cell carcinoma of the head and neck (scchn) a phase 1/2 study of ipilimumab in women with metastatic or recurrent hpv-related cervical carcinoma: a study of the princess margaret and chicago n01 consortia generation and characterization of neutralizing monoclonal antibodies against baculo-expressed hpv 16 vlps characterization of two new monoclonal antibodies against human papillomavirus type 16 l1 protein phase ii trial of bevacizumab in the treatment of persistent or recurrent squamous cell carcinoma of the cervix: a gynecologic oncology group study incorporation of bevacizumab in the primary treatment of ovarian cancer a phase i, first-in-human study to evaluate the tolerability, pharmacokinetics and preliminary efficacy of humax-tissue factor-adc (tf-adc) in patients with solid tumors cancer immunotherapy highlights from the 2014 asco meeting genetically modified cellular vaccines for therapy of human papilloma virus type 16 (hpv 16)-associated tumours adoptive t-cell therapy is a promising salvage approach for advanced or recurrent metastatic cervical cancer proportion of herpes simplex virus (hsv) type 1 and type 2 among genital and extragenital hsv isolates depressed specific cell-mediated immunity to herpes simplex virus type 1 in patients with recurrent herpes labialis hsv-2 specific serology should be offered routinely to antenatal patients neonatal herpes simplex infection herpes simplex virus treatment of herpes simplex virus infections with topical antiviral agents antiviral therapy of hsv-1 and-2 association between cervical shedding of herpes simplex virus and hiv-1 herpes simplex virus type 2 vaccines: new ground for optimism? herpes simplex virus type 1 glycoproteins gb, gc and gd are major targets for cd4 t-lymphocyte cytotoxicity in hla-dr expressing human epidermal keratinocytes glycoprotein-d-adjuvant vaccine to prevent genital herpes vaccination with the secreted glycoprotein g of herpes simplex virus 2 induces protective immunity after genital infection herpes simplex virus glycoprotein immunotherapy of recurrent genital herpes: factors influencing efficacy the use of a genetically engineered herpes simplex virus (r7020) with ionizing radiation for experimental hepatoma passive immunization in experimental herpesvirus hominis infection of newborn mice human polymorphonuclear leucocytes as mediators of antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells use of monoclonal antibody directed against herpes simplex virus glycoproteins to protect mice against acute virusinduced neurological disease passive immunization with monoclonal antibodies against herpes simplex virus glycoproteins protects mice against herpetic ocular disease prevention of herpes simplex virus induced stromal keratitis by a glycoprotein b-specific monoclonal antibody overcoming drug-resistant herpes simplex virus (hsv) infection by a humanized antibody augmentation of natural immune defence mechanisms and therapeutic potential of a mismatched double-stranded polynucleotide in cutaneous herpes simplex virus type 2 infection t lymphocytes are required for protection of the vaginal mucosae and sensory ganglia of immune mice against reinfection with herpes simplex virus type 2 immunotherapy of acute and recurrent herpes simplex virus type 2 infection with an adjuvant-free form of recombinant glycoprotein dinterleukin-2 fusion protein antigenic specificities of human cd4+ t-cell clones recovered from recurrent genital herpes simplex virus type 2 lesions immune responses to herpes simplex virus in guinea pigs (footpad model) and mice immunized with vaccinia virus recombinants containing herpes simplex virus glycoprotein d human herpesviruses: biology, therapy, and immunoprophylaxis the authors declared that they have no conflict of interest. key: cord-302530-pp6bl941 authors: gale, paul title: how virus size and attachment parameters affect the temperature sensitivity of virus binding to host cells: predictions of a thermodynamic model for arboviruses and hiv date: 2020-03-12 journal: microb risk anal doi: 10.1016/j.mran.2020.100104 sha: doc_id: 302530 cord_uid: pp6bl941 virus binding to host cells involves specific interactions between viral (glyco)proteins (gp) and host cell surface receptors (cr) (protein or sialic acid (sa)). the magnitude of the enthalpy of association changes with temperature according to the change in heat capacity (δc(p)) on gp/cr binding, being little affected for avian influenza virus (aiv) haemagglutinin (ha) binding to sa (δc(p) = 0 kj/mol/k) but greatly affected for hiv gp120 binding to cd4 receptor (δc(p) = −5.0 kj/mol/k). a thermodynamic model developed here predicts that values of δc(p) from 0 to ~−2.0 kj/mol/k have relatively little impact on the temperature sensitivity of the number of mosquito midgut cells with bound arbovirus, while intermediate values of δc(p) of ~−3.0 kj/mol/k give a peak binding at a temperature of ~20 °c as observed experimentally for western equine encephalitis virus. more negative values of δc(p) greatly decrease arbovirus binding at temperatures below ~20 °c. thus to promote transmission at low temperatures, arboviruses may benefit from δc(p) ~ 0 kj/mol/k as for ha/sa and it is interesting that bluetongue virus binds to sa in midge midguts. large negative values of δc(p) as for hiv gp120:cd4 diminish binding at 37 °c. of greater importance, however, is the decrease in entropy of the whole virus (δs(a_immob)) on its immobilisation on the host cell surface. δs(a_immob) presents a repulsive force which the enthalpy-driven gp/cr interactions weakened at higher temperatures struggle to overcome. δs(a_immob) is more negative (less favourable) for larger diameter viruses which therefore show diminished binding at higher temperatures than smaller viruses. it is proposed that small size phenotype through a less negative δs(a_immob) is selected for viruses infecting warmer hosts thus explaining the observation that virion volume decreases with increasing host temperature from 0 °c to 40 °c in the case of dsdna viruses. compared to arboviruses which also infect warm-blooded vertebrates, hiv is large at 134 nm diameter and thus would have a large negative δs(a_immob) which would diminish its binding at human body temperature. it is proposed that prior non-specific binding of hiv through attachment factors takes much of the entropy loss for δs(a_immob) so enhancing subsequent specific gp120:cd4 binding at 37 °c. this is consistent with the observation that hiv attachment factors are not essential but augment infection. antiviral therapies should focus on increasing virion size, for example through binding of zinc oxide nanoparticles to herpes simplex virus, hence making δs(a_immob) more negative, and thus reducing binding affinity at 37 °c. some viruses infect hosts over a range of temperatures depending on the host species. most notable are the arboviruses which may infect the arthropod vector over a range of ambient temperatures from~15°c to >30°c (mullens et al., 2004) , and then infect a vertebrate host at temperatures from 37°c up to 44°c in the case of west nile virus (wnv) in infected american crows (corvus brachyrhynchos) (kinney et al., 2006) . birds generally have higher body temperatures than mammals (brault 2009) and the avian influenza virus (aiv) when jumping from birds to humans has to adapt to a temperature drop of 8°c since the temperature in the human upper respiratory tract is 33°c compared to~41°c in the avian intestinal tract which is the site of replication of avian viruses (de graaf and fouchier, 2014) . bats have higher core body temperatures than primates (up to 42.1°c for phyllostomus hastatus which is an omnivorous bat from south america) when flying (o'shea et al., 2014) . the core body temperature during flight of the insectivorous free-tailed bat (mops condylurus) which may have been the origin of the 2013/15 severe zaire ebolavirus (ebov) outbreak in west africa (saez et al., 2015) is 40.5°c ± 1°c (o'shea et al., 2014) . thus ebov which infects a range of vertebrate species from bats to primates and deer may have to infect mammalian hosts over a range of temperatures. similarly nipah virus on jumping from fruit bats to pigs and humans (daszak et al., 2013) may experience a small fall in temperature. furthermore, cross-species transmission of viruses to bats (and other mammals) from invertebrates may occur with more regularity than has been appreciated (bennett et al., 2018; leendertz 2016) , and bennett et al. (2018) have suggested that arthropods may host many "bat-associated" viruses that have defied detection in bats themselves (e.g. ebov). depending on the ambient temperature, viruses in arthropods would experience a 9°c to 15°c temperature increase on being ingested by a bat at 41°c (gale 2017) and this could affect the binding affinity of the virus to its host cell depending on the thermodynamics of virus binding as is discussed here. in contrast other viruses only infect related host species, and in effect are maintained at similar temperatures. for example, simian immunodeficiency viruses (siv) infect 36 different nonhuman primate species in sub-saharan africa and sivs from chimpanzees (pan troglodytes) have crossed species barriers on multiple occasions generating human immunodeficiency virus (hiv) types 1 and 2 (sharp et al., 2005) . thus the main source of temperature variation experienced by siv and hiv would be the effect of the circadian rhythm and the slight rise in temperature due to infection of the host which gives a range of 36°c to 38°c in the case of rhesus monkeys (huitron-resendiz et al., 2007) . viruses bind to host cells through interactions between a virus surface (glyco)protein (gp) and a host cell receptor (cr). the cr molecule on the cell surface may be a protein as in the case of hiv (myszka et al., 2000) , ebov (yuan et al., 2015) , hendra virus (xu et al., 2012) and mers-cov (lu et al., 2013) or alternatively is a sialic acid (sa) on a glycan as in the case of aiv (de graaf and fouchier, 2014; fei et al., 2015) and arboviruses such as bluetongue virus (btv) (zhang et al., 2010) . the thermodynamics of virus binding in terms of the changes in enthalpy (δh a_receptor_t ) and entropy (δs a_receptor_t ) on the specific interaction of the gp with the cr receptor at temperature t during binding of virus to the host cell surface has been set out previously (gale 2018 (gale , 2019 . the interactions between the hiv gp and its cd4 cellular receptor (myszka et al., 2000; dey et al., 2007) and between the aiv haemagglutinin (ha) and its α2,3-sa or α2,6-sa receptors (fei et al., 2015) are enthalpy driven, i.e. large negative values of δh a_receptor_t overcome unfavourable values of δs a_receptor_t . also the binding of vesicular stomatitis virus to phospholipid bilayers is enthalpy driven (carneio et al. 2002) with very large negative values of δh a_receptor_t . temperature may impose constraints on viruses' jumping the species barrier through its effect on the binding affinity of gp to cr. there may also be constraints on the activities of viral replication proteins such as the aiv polymerase which showed a significantly higher activity at 33°c than 37°c (ngai et al., 2013) . only virus binding is considered here. thus according to the van't hoff isochore, the binding affinity for a virus to its host cell at temperature t as represented by the association constant k a_virus_t would be greatly diminished for enthalpydriven gp/cr interactions at higher temperatures compared to lower temperatures depending on the magnitude of δh a_receptor_t (gale 2019) . indeed, assuming δh a_receptor_t is constant over the temperature range, the more negative δh a_receptor_t is in magnitude, the weaker the binding at higher temperatures. this presents a paradox if the k a_virus_t falls to less than~10 14 m −1 in that higher temperatures would greatly diminish infectivity (gale 2019) . while the values of δh a_receptor_t and δs a_receptor_t may be constant over the biological temperature range for some gp/cr systems, e.g. aiv haemagglutinin (ha) binding to sialic acid (sa) residues (fei et al., 2015) they may change considerably in magnitude over the biologically relevant temperature range particularly for protein:protein systems as in the case of hiv gp120 binding to cd4 (myszka et al., 2000) (fig. 1) . the effect of this on k a_virus_t is considered in this work. the change in δh a_receptor_t with temperature is defined by the difference in heat capacities (c p ) of the gp/cr "product" complex and the free gp/free host cr "reactants" and is represented by δc p which is the slope of the lines in fig. 1 and typically ranges from~0 kj/mol/k to −5.0 kj/mol/k depending on the system. the previous thermodynamic treatment of virus binding (gale 2019) identified the entropy change (δs a_immob ) on immobilisation of the whole virus particle on the host cell surface as a key parameter for which there are currently no data. δs a_immob would be expected to be large and negative in magnitude due to the decrease in the absolute entropy on immobilisation of a large molecular entity such as a virion. a recent analysis has shown that virion volume (and genome length) for dsdna viruses decreases by about 55-fold as the temperature of occurrence (i.e. host) increases from 0 to 40°c (nifong and gillooly 2016) . the temperature of occurrence in these dsdna viruses ranges from near zero for those inhabiting polar environments to over 40°c for those inhabiting endothermic vertebrates. nifong and gillooly (2016) suggest that this could reflect smaller viruses being able to replicate more quickly (due to more compact genomes) perhaps together with energetic constraints imposed by their hosts. however from the thermodynamic perspective, the decrease in volume and hence mass and radius of the virus will make δs a_immob less negative for a smaller virus than for a larger virus. it is shown here how a less negative δs a_immob increases binding of the virus at the relatively high mammalian body temperatures and it is proposed that thermodynamic binding constraints may give smaller viruses a selective advantage for infecting hosts at the higher body temperatures of mammals and birds. in this respect, the large size of the hiv virion may present a constraint to binding at human body temperatures compared to the much smaller arboviruses for example. in the case of hiv, the gp component of the gp/cr interaction is the envelope protein (env) which is a trimer of gp120 monomers. each hiv fig. 1 . variation in δh a_receptor_t as a function of temperature as reported for hiv gp120 monomer binding to cd4 (○) (myszka et al., 2000) and aiv ha monomer binding to soluble α2-6 sialyllactose (δ) and α2-3 sialyllactose (□) (fei et al., 2015) . the slopes of the lines represent the δc p values which are summarised in table 3 . env trimer interacts with one cd4 molecule (chuang et al., 2017; liu et al., 2017a) . this is different from the aiv ha trimer in which each monomer in the trimer interacts with one glycan cr (de graaf and fouchier, 2014; fei et al., 2015) . initial attachment of the hiv virion to the host cd4 + t cell surface is relatively nonspecific (wilen et al., 2012) with hiv env interacting with negatively charged host cell surface heparan sulphate proteoglycans, or with pattern recognition receptors. according to wilen et al. (2012) , non-specific hiv attachment to the host cell via any of these factors likely brings hiv env into close proximity with the viral receptor cd4 and the crr5 coreceptor, so increasing the efficiency of infection, although the physiologic role of non-specific attachment in vivo remains unclear. here it is suggested that non-specific binding helps to overcome the thermodynamic entropy constraint of immobilisation of a large virus on binding at human body temperature by taking some of the entropy loss in δs a_immob prior to specific hiv env:cd4 binding. this would be consistent with the fact that non-specific attachment factors differ from receptors in that they are not essential, although they augment infection in vitro (wilen et al., 2012) . a review of the literature has not found any results for the effect of temperature on the binding of hiv to cd4 + t cells with which to test or validate the hiv thermodynamic binding model developed here. however, frey et al. (1995) reported the binding of cells expressing hiv gp on their surface to cd4 + t cells over the temperature range of 0°c to 42°c. it is known that increasing host membrane fluidity at higher temperature allows effective recruitment of more cr molecules to bind the hiv virion (harada et al., 2004) such that the number, n, of gp/cr contacts increases with temperature (frey et al., 1995) , and that increasing n can overcome both the effect of temperature on decreasing the gp/cr binding affinity and the effect of a large negative δs a_immob (gale 2019) . the thermodynamic hiv binding model here is therefore modified to accommodate increasing n with temperature and so attempt to reproduce the results of frey et al. (1995) . the success of the model here is in predicting the subsequent decrease in binding reported by frey et al. (1995) at higher temperatures due to δs a_immob . here six case studies are presented to illustrate the effects of the magnitude of n, δc p and δs a_immob on the temperature sensitivity of virus binding. these are:-1 the predicted effect of the magnitude of δc p on the temperature dependence of dengue virus (denv) transmission by aedes albopictus; 2 the predicted effect of the magnitude of δc p on the temperature peak observed for the specific binding of western equine encephalitis virus (weev) to susceptible culex tarsalis brush border fragments (bbfs); 3 a large negative δc p for a protein:protein gp/cr system as represented by hiv gp120:cd4 diminishes host cell binding at human body temperature; 4 a large virus diameter as for the hiv virion diminishes host cell binding at the higher temperature of the human body through the large negative δs a_immob ; 5 non-specific attachment factors may partially overcome the unfavourable δs a_immob and hence enhance specific hiv binding through env:cd4 interactions at human body temperature; and 6 increasing the number, n, of gp/cr contacts with temperature to reproduce the data of frey et al. (1995) for binding of hiv envexpressing cells to cells expressing cd4. it may be thought there is little point in modelling hiv binding with temperature because hiv only infects human hosts at~37°c. however, the model here suggests mammalian temperature may be a constraint on hiv binding (due to the large negative δc p and δs a_immob ) in effect exposing a potential weakness in the hiv infection process which could be exploited for antiviral therapy. the implications of making δs a_immob more negative are considered here as a novel approach to developing antiviral therapies with reference to antoine et al. (2012) using zinc oxide nanoparticles for preventing infection by herpes simplex virus type 2 (hsv-2). 2.1. the change in heat capacity (δc p ) on binding of virus gp to cr receptors on the host cell the change in enthalpy (h) with temperature (t) at constant pressure is the heat capacity, c p . (eq. 10) integrating with respect to temperature relates the absolute enthalpy (h t ) at temperature t to that (h t0 ) at a given reference temperature t 0 through c p according to eq. (1) (see table 1 ). the attachment of a virus to its host cell is driven by the n individual interactions between gp and cr made on binding as represented by the dynamic equilibrium in eq. (2). the change in enthalpy for gp binding to cr at temperature t, δh a_receptor_t , is given by eq. (3). substituting eq. (1) into eq. (3) for the enthalpies for the gp.cr complex (h t (gp.cr)), the free gp (h t (gp)) and the free cr (h t (cr)) relates the enthalpy change for gp binding to cr at temperature t, δh a_receptor_t , to that (δh a_receptor_t0 ) measured experimentally at temperature t 0 (du et al., 2016) according to eq. (4) where δc p is the change in heat capacity defined as the difference between the heat capacities of the gp.cr complex and the sum of the heat capacities of the free gp and cr molecules as given by eq. (5). similarly the entropy change (δs a_receptor_t ) for gp binding to cr at temperature t is related to that (δs a_receptor_t0 ) measured experimentally at temperature t 0 (du et al., 2016) according to eq. (6). modelling the effect of temperature on δh a_virus_t and δs a_virus_t and hence k a_virus_t δh a_virus_t and δs a_virus_t are the changes in enthalpy and entropy respectively on binding of virus to host cell at temperature t. substituting the terms for δh a_virus_t (eq. (7)) and δs a_virus_t (eq. (8)) into eq. (9) expresses δg a_virus_t in terms of n, δs a_immob , δh a_receptor_t and δs a_receptor_t (eq. (10)) where δg a_virus_t is the change in gibbs free energy on association of virus with a host cell at temperature t and δs a_immob is the change in entropy on immobilisation of the whole virus on the cell surface (gale 2018; 2019) . for this work it is assumed that the change in heat capacity on binding of a virus to a host cell is determined by the change in heat capacity on interaction of gp with cr according to eq. (5) and that δs a_immob in eq. (10) is unaffected. in effect therefore the heat capacity of the "bulk" (i.e. non-gp/cr) of the bound virus/host cell is the same as the sum of the heat capacities for the free virus and free host cell. this is acceptable because a change in heat capacity is related to a change in conformation of the molecular components (myszka et al., 2000) and the conformation of the bulk of the host cell and the virus would be unaffected by attachment. indeed, δs a_immob is mainly related to changes in rotational and translational mobility of the virus particle as a whole (see below) and does not involve the conformational changes in gp and cr which are accommodated in the δs a_receptor_t term (gale 2019) . since the heat capacity of the "bulk" of the bound virus/ host cell is the same as the sum of the heat capacities for the free virus and free host cell, δc p for the bulk equals zero, and according to eq. (6) therefore, δs a_immob itself at temperature t (calculated as δs a_immob_t = δs a_immob_t0 + δc p .ln(t/t 0 )) would be unaffected by temperature and hence constant as assumed in the models here. 2.2.2. the effect of the magnitude of δc p on virus binding affinity at temperature t substituting δh a_receptor_t and δs a_receptor_t from eqs. (4) and (6) respectively into eq. (10) gives eq. (11) from which k a_virus_t is calculated according to eq. (12) where r is the ideal gas constant. comparing the predicted effect on k a_virus_t of the magnitude of δc p in case studies 1, 2 and 3 requires a reference temperature, t 0 , to be defined at which the plots of k a_virus_t intercept. values of k a_virus_t intercept at t = t 0 because the n.δc p (t -t 0 + t.ln(t/t 0 )) term in eq. (11) equals zero. therefore for the purpose of demonstrating the effect of δc p on the temperature sensitivity of k a_virus_t , t 0 needs to be chosen to be well above or below the temperature range of interest. it should be noted that the choice of reference temperature is artificial and is only used here out of necessity to demonstrate the effect of different δc p values while fixing the k a_virus_t intercept temperature. indeed a given gp/cr system has its own natural δc p value which cannot be altered and is measured experimentally (myszka et al., 2000; fei et al., 2015) . the t 0 temperature of 37°c is appropriate to study arthropod vector competence for arbovirus transmission over the 10 to 35°c temperature range in case studies 1 and 2 as used previously for the arthropod vector competence model (gale 2019) . however, since 37°c is the human body temperature at which hiv binds to host cells, 37°c is not a good choice for t 0 in case study 3 for which t 0 is therefore set to 4°c. the reference temperature is 37°c in case studies 4, 5 and 6 because the experimentally measured value of δc p for hiv gp120:cd4 binding ( fig. 1) is used together with δh a_receptor_t0 and δs a_receptor_t0 values measured experimentally at a temperature t 0 of 37°c (myszka et al., 2000) , i.e. real data are used. 2.3. case study 1: the predicted effect of the magnitude of δc p on the temperature dependence of denv transmission by aedes albopictus the approach for modelling arbovirus transmission efficiency by the arthropod vector has been fully described previously (gale 2019) and is summarised here for the purpose of the models in figs. 2 and 3. the strength of gp/cr binding at temperature t is often expressed as the dissociation constant, k d_receptor_t , for which smaller values indicate stronger binding (xiong et al., 2013) . the thermodynamic model is based on an enthalpy-driven gp/cr interaction with a k d_receptor_t0 of 10 −3 m at 37°c (t 0 ) as used previously (gale 2019) to represent the interaction between an arbovirus gp and a sa glycan (cr) on the brush border surface of the epithelial cells lining the arthropod midgut. the parameters δh a_receptor_t0 and δs a_receptor_t0 at t 0 = 37°c for denv transmission by aedes albopictus together with n and δs a_immob are set out in table 2 and values of k a_virus_t over the temperature range 10-37°c are calculated for the δc p values in table 3 using δg a_virus_t from eq. (11) in eq. (12). the arbovirus challenge dose is 10 5 virions and the midgut volume is 10 −6 dm 3 giving a virus concentration [v total ] = 1.7 × 10 −13 m. the fraction (f ct ) of arthropod midgut cells with bound arbovirus at temperature t is calculated from k a_virus_t using eq. (13) assuming the concentration of free virus ([v free ])~[v total ]. the number, c.v t , of midgut cells with bound arbovirus at temperature t is calculated by multiplying f ct by the total number of midgut cells (c total = 1000) (eq. (14)) as described previously (gale 2019). the probability, p completet , of an arthropod midgut cell with bound virus successfully leading to infection of the arthropod salivary glands and completion of virogenesis within in the lifetime of the arthropod at temperature t is calculated using the arrhenius equation (eq. (15)) where p complete283 is the probability of this happening at 10°c (283 k) and e a is the activation energy of the rate-limiting step in virogenesis ( table 2 ). the probability, p transmissiont , of arbovirus transmission by the arthropod (i.e. successful infection of the arthropod salivary glands after oral exposure) at temperature t given c.v t midgut cells have bound virus is given by eq. (16). the magnitude of δc p could explain the temperature peak observed in the specific binding of weev to susceptible culex tarsalis bbfs the value of δc p in eq. (11) with the gp/cr binding parameters for denv in table 2 was optimised so that c.v t as a function of temperature best approximated the specific binding affinity of weev to bbfs from susceptible culex tarsalis mosquitoes as determined by houk et al. (1990) and shown in fig. 3 . 2.5. case study 3: a large negative δc p for a protein:protein gp/cr system as represented by hiv gp120:cd4 diminishes host cell binding at higher temperatures 2.5.1. estimation of n for hiv yang et al. (2005) using defective hiv gp120 monomers concluded that a single env trimer is sufficient to mediate the entry of one virion, but that all three gp120 monomers in that trimer must be active. klasse (2012) has proposed that a realistic model allows both a minimum requirement of two gp120 monomers with an increment in function by the third. this is consistent with the cryo-electron microscopy (em) structure of the env glycoprotein trimer (liu et al., 2017a) showing that in each trimer, one gp120 monomer makes a primary contact with the bound cd4 cr molecule while a second makes a partial (table 3) with model parameters for arbovirus binding to mosquito midgut cells in table 2 and δs a_immob = 0 j/ mol/k. experimental data (■) in c) and d) for denv transmission efficiency by aedes albopictus mosquitoes from liu et al. (2017a) . (table 3) with model parameters for arbovirus binding to mosquito midgut cells in table 2 and δs a_immob = 0 j/mol/k. experimental data (■) in b) show specific binding (as the percentage of total virus bound) of western equine encephalitis virus (weev) to brush border fragments from susceptible culex tarsalis mosquitoes as determined by houk et al. (1990) . contact (called the quaternary contact reflecting protein structure nomenclature) and the third gp120 monomer makes no direct contact with the cd4 at all. for the purpose of the model here, n represents the number of env trimers on each hiv virion that interact with a cd4 cr during binding. brandenberg et al. (2015) demonstrate that divergent hiv strains differ in their stoichiometry of entry and require between 1 and 7 env trimers, with most strains depending on 2 to 3 env trimers to complete infection. here it is assumed that n = 3 env:cd4 interactions for hiv binding although the results of brandenberg et al. (2015) do not separate cell surface binding itself from the subsequent hiv/cell fusion in the entry process. if it is later found that cell surface binding only requires one env:cd4 interaction for example, while more are required for the membrane fusion, then the model here for c.v t should be reparameterised with n = 1 although the number of env:ccr5 and nonspecific env interactions with attachment factors (see below) should also be built into the model. it should be noted that as for the arbovirus model, it is assumed that the n = 3 env:cd4 interactions act independently and not co-operatively in binding. this is reflected in eqs. (7) and (8) where δh a_receptor_t and δs a_receptor_t act additively. the env trimer binds a single soluble cd4 with a k d_receptor_t (25°c) of 1.4 × 10 −9 m (chuang et al., 2017) which is 15-fold higher than that for the gp120 monomer:cd4 interaction estimated at 25°c from the data of myszka et al. (2000) in table 4 . to the author's knowledge there are no data for δh a_receptor_t and δs a_receptor_t for env trimer binding to cd4. therefore the values for δh a_receptor_t0 and δs a_receptor_t0 for hiv gp120 monomer:cd4 binding at 37°c from myszka et al. (2000) are used for env trimer binding to a single cd4 in the hiv model in fig. 4a and b as set out in table 4 . dey et al. (2007) reported values at 37°c of −287.9 kj/mol and -786.9 j/mol/k for δh a_receptor_t and δs a_receptor_t respectively for the wildtype hiv-1 yu2 gp120 monomer binding to soluble cd4. the more negative δs a_receptor_t value for gp120 monomer:cd4 binding from dey et al. (2007) reduces the binding affinity for gp120:cd4 by nine-fold (k d_receptor_t = 4.2 × 10 −8 m) at 37°c compared to that of 4.75 × 10 −9 m from myszka et al. (2000) (table 4) . this difference may simply reflect the different strains of hiv-1 used, namely yu2 by dey et al. (2007) and wd61 by myszka et al. (2000) . the hiv gp120 monomer:cd4 binding data of myszka et al. (2000) are chosen here over those of dey et al. (2007) because myszka et al. (2000) present the variation in δh a_receptor_t with temperature as shown in fig. 1 together with information on δc p which dey et al. (2007) do not consider. it is therefore assumed here that δc p = −5.02 kj/mol/k for the env trimer:cd4 interaction as reported by myszka et al. (2000) for the gp120 monomer:cd4 interaction (table 3) . values of δs a_receptor_t are not documented by myszka et al. (2000) other than that at 37°c (table 4 ) and values at lower temperatures are therefore calculated from that at t 0 = 37°c according to eq. (6) using δc p = −5.02 kj/mol/k for hiv gp120:cd4 (table 3) . some reassurance that eq. (6) is appropriate is that the value of δs a_receptor_t of −493 j/mol/k predicted at t = 25°c by eq. (6) is very close to that of −473 j/mol/k calculated from the -t. δs a_receptor_t value reported by myszka et al. (2000) at 25°c (table 4) . predicted values for δh a_receptor_t and δs a_receptor_t for hiv env:cd4 binding at 4°c are set out in table 4 and are used as δh a_receptor_t0 and δs a_receptor_t0 respectively for the hiv model in fig. 4c and d with the reference temperature, t 0 , set to 4°c to assess the effect of varying δc p on the temperature sensitivity of k a_virus_t and c.v t up to and above human body temperature. the 2.5 to 97.5 percentile range for the number of cd4 + t cells in humans is 448 to 1611 cells per mm 3 (10 −6 dm 3 ) of blood with a mean of 919 cells per mm 3 (thakar et al., 2011) . the model here takes 1 mm 3 myszka et al. (2000) ) at 37°c calculated from eq. (4) using δc p = −5.02 kj/mol/k (table 3) with t 0 = 310 k (37°c). d calculated from eq. (6) using δc p = −5.02 kj/mol/k (table 3) with t 0 = 310 k (37°c). e estimated from values for δh a_receptor_t and -tδs a_receptor_t presented in fig. 4 of myszka et al. (2000) for gp120 binding to cd4 at 25°c. f in good agreement with δs a_receptor_t value at t = 298 k (25°c) of −492.8 j/mol/k as calculated from eq. (6) using δc p = −5.02 kj/mol/k (table 3) with t 0 = 310 k (37°c) and δs a_receptor_t0 = −691.04 j/mol/k (see b). of blood with c total = 10 3 cd4 + t cells and introduces v total = 10 5 hiv virions. dividing 10 5 hiv virions in 10 −6 dm 3 by the avogadro number gives [v total ] = 1.7 × 10 −13 m. the advantage of using a high v total that greatly exceeds c total is that as previously demonstrated by gale (2018) not only may the fraction, f ct , of cd4 + t cells with bound hiv be calculated from eq. (13) with [v free ]~[v total ] but also any complications of handling stochastic effects at low virus doses are avoided. it is recognised that the hiv concentration at 10 5 hiv virions per mm 3 used in this model is 10-fold to 100-fold higher than that expected in a recipient person's blood present in the capillaries lining the skin at the wound site where semen or blood from an infected source were introduced since maximum human plasma and human semen hiv loadings are in the region of 13,000 copies per mm 3 and 1800 copies per mm 3 respectively (gupta et al., 1997) . this is not important, however, because the purpose of the models here in figs. 4-6 is not to produce a realistic dose-response but to explore the effect of changing δc p , δs a_immob , n and temperature on the trend in the predicted number, c.v t , of host cd4 + t cells with bound virus. indeed, the overall conclusions of the model in terms of the effect of temperature on the number of cd4 + t cells with bound virus will still be representative and the interpretation applicable to all doses of viruses as there is no cooperation between viruses in this model (gale 2018) . furthermore, the levels of siv in rhesus monkey blood plasma were in the region of 2.1 × 10 8 virions per ml (huitron-resendiz et al., 2007) which is 2.1 × 10 5 virions per mm 3 and representative of the 10 5 used for [v total ] in the model here. c.v t is calculated by multiplying f ct by c total (eq. (14)). 2.6. case study 4: a large virus diameter such as for the hiv virion diminishes host cell binding at the higher temperatures of the human body through the large negative δs a_immob 2.6.1. estimation of δs a_immob for a virus there are currently no data available on the magnitude of δs a_immob which was first defined through development of a thermodynamic model (gale 2019). xiong et al. (2013) reported a k d_virus of 10 −15 m for aiv where k d_virus is the dissociation constant for virus from the host cell and is the inverse of k a_virus_t . according to yang et al. (2005) hiv virions bearing influenza a virus ha required 8 or 9 ha trimers for virus entry. this is equivalent to n = 24 to 27 ha monomers each of k d_receptor = 10 −3 m (xiong et al., 2013) . it is not clear whether the 8 or 9 aiv ha trimers are required for cell binding or membrane fusion in yang et al. (2005) . however, if n = 24 ha monomers are required for aiv binding to the host cell to give a k a_virus_t of 10 15 m −1 then δs a_immob equals −1091 j/mol/k from eq. (17) (derived in gale 2019). this is in the same order of magnitude as assumed for δs a_immob on the basis of virus molecular weight (gale 2019) . it must be emphasized that the estimate of 8 or 9 ha trimers in yang et al. (2005) is based on a number of assumptions that may not hold, for example whereas each infection of a cell is a quantal, all-or-nothing event, the infectivity of a virion could span a wide spectrum of propensities and each mathematical model could have different virological interpretations (see klasse 2012) . however, in the absence of any data for δs a_immob the value of n from yang et al. (2005) at least provides a theoretical approach to estimate it from eq. (17) for this "proof of concept" model. it was shown previously (gale 2019 ) that δs a_immob is the sum of the changes in translational entropy (δs trans ) and rotational entropy (δs rot ) of the whole virus on binding. according to mammen et al. (1998) δs trans ∝ ln(mass) and δs rot ∝ ln(i x x i y x i z ) where i x , i y and i z are the moments of inertia about the three principle axes of rotation. since mass, m, is proportional to the volume and i = mr 2 where r is the radius of the virus, it may be shown that the decrease in entropy on immobilisation (δs a_immob ) of a spherical virus can be expressed as eq. (18). examples of diameters for arboviruses are~50 nm for wnv (mukhopadhyay et al., 2003) and denv, 66 nm for eastern equine encephalitis virus (hasan et al., 2018) with btv being slightly larger at 85 nm (nason et al., 2004) . in contrast, hiv is much larger with a mean diameter of 134 nm in the case of mature hiv virions containing a single core (briggs et al., 2003) . the problem with eq. (18) is that fig. 4 . temperature variation of a) and c) k a_virus_t and b) and d) number, c.v t , of cd4 + t cells per mm 3 blood with bound hiv virions in hiv model for δc p = 0 (solid line), −1.97 (dotted line) and −5.02 (dashed line) kj/mol/ k (table 3 ) and thermodynamic parameters for n = 3 hiv env:cd4 interactions in table 4 at reference temperature a) and b) t 0 = 37°c; c) and d) t 0 = 4°c. δs a_immob = −400 j/mol/k. proportionality constants in units of j/mol/k are needed probably based on r as in the sackur-tetrode equation and the natural logarithm terms should be dimensionless such that the units of radius cancel out. however for the purpose of representing the effect of increasing the radius of the virus on δs a_immob eq. (18) is used with the virus radius in units of nm. thus from eq. (18), the magnitude of δs a_immob for a virus of diameter 134 nm is 18.6% more negative than that for a virus of diameter 66 nm. to illustrate the effect of increasing virus size on the number of cd4 + t cells in 1 mm 3 of blood with bound virus, c.v t is calculated for the hiv model using values of δs a_immob which decrease in steps of 18.6% from −240 j/mol/k (i.e. to −285 j/mol/k, −337 j/ mol/k and −400 j/mol/k). 2.7. case study 5: hiv attachment factors may partially overcome the unfavourable δs a_immob and enhance specific virus binding through env/cd4 interactions at human body temperature 2.7.1. modelling the effect of non-specific binding of hiv to host cd4 + t cells prior to specific env:cd4 interaction the term δs a_immob represents the decrease in entropy of the virus on going from a freely rotating entity to being immobilised in a specific orientation on the host cell surface through n = 3 specific env:cd4 interactions. thermodynamically this could be accomplished in two consecutive steps whereby the virus first binds non-specifically to attachment proteins on the host cell surface with an entropy change of δs a_non_specific , and then rolls into place driven by n = 3 specific env:cd4 interactions resulting in a further entropy loss (δs a_specific ) as the virus particle takes up a specific orientation ready for viral entry. in effect δs a_immob is the sum of two components, namely δs a_non_specific and δs a_specific according to eq. (19) . it should be noted that non-specific binding to hiv attachment factors would not increase δs a_immob to zero because there are multiple ways a virus could bind non-specifically compared to the one way in which it binds to the cd4 receptors through a specific interaction between env and cd4, that is the δs a_specific term is always negative i.e. <0 j/mol/k and δs a_non_specific is always less negative than δs a_immob . prior attachment through non-specific binding would realise the δs a_non_specific loss before specific env:cd4 binding so that specific binding involves the δs a_specific term alone. the value of δs a_immob is estimated to be −1091 j/mol/k (see above) and for the purpose of demonstration here δs a_immob is replaced by δs a_specific in eq. (11) which is set to values of −400, −337, −285 and −240 j/mol/k in effect representing the effect of prior non-specific binding eliminating δs a_non_specific values of −691, −754, −806 and −851 j/mol/k respectively according to eq. (19). 2.8. case study 6: increasing the number, n, of gp/cr contacts with temperature to reproduce the data of frey et al. (1995) two outputs of the model are demonstrated. the first output builds on the model in case study 5 with a δs a_specific of −206 j/mol/k to accommodate prior non-specific binding to attachment factors having eliminated the δs a_non_specific of~−900 j/mol/k. the model assumes n = 3 env:cd4 interactions where n in eq. (11) is increased from n = 1 to n = 3 with temperature between 25°c and 27°c (fig. 6a) to represent the increase in contacts reported by frey et al. (1995) . the second output attempts to include non-specific binding and also ccr5 co-receptor binding and a δs a_immob of −1110 j/mol/k is used to represent the free virus binding (i.e. prior to non-specific binding). kuhmann et al. (2000) demonstrated that approximately four to six ccr5 molecules assemble around the hiv virion to form a complex needed for infection. the model here assumes n = 2 non-specific env interactions with attachment factors followed by n = 4 env:ccr5 interactions according to kuhmann et al. (2000) and finally n = 3 env:cd4 interactions based on brandenberg et al. (2015) to form a complex for binding with n = 9 contacts in total which increase from 1 to 9 with temperature as shown in fig. 6b . doranz et al. (1999) estimated the k d_receptor_t of interaction between ccr5 and hiv gp120 to be 4 × 10 −9 m. this is remarkably similar to the k d_receptor_t for the interaction between cd4 and hiv env of 4.75 × 10 −9 m at 37°c (table 4 ) and the same values for δh a_receptor_t0 and δs a_receptor_t0 not only for the four hiv env:ccr5 interactions but also for the two nonspecific interactions are therefore used as for the three hiv env:cd4 interactions in table 4 with the reference temperature, t 0 at 37°c. published values of δc p for protein/protein binding and for aiv ha binding to sa glycans vary in magnitude (table 3) . for the binding of hiv gp120 to cd4 which is a protein-protein interaction, the magnitude of δh a_receptor_t becomes more negative as the temperature increases i.e. δc p is negative (fig. 1) . similarly, negative values of δc p were reported for other protein-protein systems, namely the binding of e3 and e1 proteins, respectively, in the assembly of the enzyme pyruvate dehydrogenase (jung et al., 2002) , although with δc p values less negative than that for hiv gp120:cd4 (table 3 ). in contrast for aiv ha binding to the sa glycan the magnitude of δc p is not significantly different from zero with δh a_receptor_t constant over the temperature range 15°c to 25°c at least for binding of both α2-3 and α2-6 sialyllactose (fig. 1) . values of k a_virus_t predicted over the temperature range of 10°c to 37°c using different values of δc p intercept at the reference temperature (t 0 ) of 37°c (fig. 2a) with k a_virus_t = 10 12 m − 1 as expected for n = 4 gp/cr interactions each of k d_receptor_t0 of 10 −3 m (eq. (17)). with δc p = 0 kj/mol/k as for aiv ha/sa, k a_virus_t increases with decreasing temperature according to the van't hoff isochore. in contrast with δc p = −5.02 kj/mol/k as for hiv gp120:cd4, k a_virus_t peaks at 26°c and decreases rapidly at lower temperatures. for the simulated denv/ae. albopictus model used here, small negative values of δc p (−2.0 kj/mol/k to 0 kj/mol/k) have little effect on the probability of transmission at low temperatures (fig. 2c,d) . this is because in the model here with δc p in the range of-2.0 kj/mol/k to 0 kj/mol/k, k a_virus_t is high at >10 14 m − 1 at low temperatures ( fig. 2a) and thus c.v t is little affected until higher temperatures (fig. 2b) at which k a_virus_t falls below 10 14 m − 1 (fig. 2a) . large negative values of δc p (−5.0 kj/mol/k) cause c.v t to peak rapidly falling at lower temperatures (fig. 2b ). this has a large effect on the predicted probability of transmission at lower temperatures with p transmissiont 44-fold lower at 10°c (fig. 2d) . it is interesting to note that δc p has little effect on p transmissiont over the range of temperature (23°c to 32°c) at which laboratory vector competence experiments (liu et al., 2017b) are typically conducted (fig. 2c) . 3.3. case study 2: the magnitude of δc p could explain the temperature peak observed in the specific binding of weev to susceptible culex tarsalis bbfs with a δc p = −3.0 kj/mol/k the magnitude of k a_virus_t between 0 and 40°c is relatively constant compare to those with δc p = 0 kj/mol/ k and −5.02 kj/mol/k (fig. 3a) varying by only 40-fold and peaking at 1.62 × 13 13 m − 1 at 18°c. however, this variation in k a_virus_t between 10 12 and 10 13 m − 1 is optimal for affecting the number of midgut cells with bound virus (fig. 3b ). this is because k a_virus_t is high enough such that a significant proportion of midgut cells have bound virus over the 0°c to 40°c temperature range but not high enough to exceed the 10 14 m − 1 above which all the midgut cells are saturated with virus. the variation in c.v t with temperature with δc p = −3.0 kj/mol/k shows some similarity to the percentage variation in the amount of weev bound to bbfs from susceptible culex tarsalis mosquitoes as measured experimentally by houk et al. (1990) as represented by the symbols in fig. 3b . according to houk et al. (1990) the k a_virus_t at 20°c for weev binding to bbfs from susceptible culex tarsalis mosquitoes is 2.2 × 10 11 m − 1 . this is within two orders of magnitude of the value of 1.57 × 10 13 m − 1 predicted by the model here with δc p = −3.0 kj/ mol/k (fig. 3a) . 3.4. case study 3: a large negative δc p for a protein:protein gp/cr system as represented by hiv gp120:cd4 diminishes host cell binding at higher temperature 3.4.1. reference temperature, t 0 , equals 37°c k a_virus_t values for hiv binding and c.v t values for the number of cd4 + t cells in 1 mm 3 of blood with bound hiv as a function of temperature are shown in fig. 4a and b respectively for different δc p values with t 0 = 37°c. the k a_virus_t and c.v t values in fig. 4a and b respectively assume n = 3 env:cd4 contacts with thermodynamic parameters in table 4 and δs a_immob = −400 j/mol/k. the large negative value of δc p of −5.02 kj/mol/k reported for gp120:cd4 (table 3 ) diminishes binding at higher temperatures compared to δc p of 0 kj/mol/k. thus from fig. 4b , only half of the 1000 cells have bound virus at 12°c with δc p = −5.02 kj/mol/k while with δc p = 0 kj/mol/k half the cells still have bound virus at a temperature of 6°c higher (18°c). with δc p = −5.02 kj/mol/k, c.v t is <1 cell (i.e. no virus bound) at 22°c while with δc p = 0 kj/mol/k the slightly higher temperature of 24°c is required to decrease c.v t to <1 cell (fig. 4b) . the three lines representing k a_virus_t for different values of δc p in fig. 4a intercept at 37°c (t 0 ) and would do for c.v t in fig. 4b if extrapolated to 37°c. to demonstrate how the magnitude of δc p affects k a_virus_t at temperatures of 37°c representing mammalian body temperature, the reference temperature is set to 4°c such that the three plots for k a_virus_t intercept at 4°c in fig. 4c with much lower k a_virus_t values predicted at 37°c with a large negative δc p than for a system with δc p = 0 kj/mol/k. the effect of δc p on the number of cells with bound virus at higher temperatures is considerable with c.v t reduced to 1 cell at 22°c with δc p = −5.02 kj/mol/k compared to 31°c with δc p = 0 kj/mol/k (fig. 4d) . it should be noted that the dashed lines in fig. 4c and d are the same as those in fig. 4a and b respectively being based on experimental data for hiv gp120:cd4 (fig. 1) . the purpose of the dotted and solid lines is merely to illustrate the effect of less negative δc p increasing the binding at higher temperatures for the purpose of understanding. 3.5. case study 4: a large virus diameter as for the hiv virion diminishes host cell binding at the higher temperature of the human body through the large negative δs a_immob from eq. (18), δs a_immob becomes more negative as the diameter of the virus increases. the value of k a_virus_t decreases with increasing temperature in fig. 5a while more negative values of δs a_immob merely shift each parallel curve to lower k a_virus_t values as expected from eq. (17) such that k a_virus_t falls below 10 14 m − 1 at progressively lower temperatures. values of c.v t with δs a_immob decreasing from −240 j/ mol/k to −400 j/mol/k in steps of 18.6% are plotted in fig. 5b and show that the number of cells with bound virus falls to <1 at progressively lower temperature as δs a_immob becomes more negative through increasing the virus diameter. the temperature at which c.v t falls to <1 is predicted to increase from 22°c to 43°c as δs a_immob increases in magnitude from −400 j/mol/k to −240 j/mol/k. the conclusion from fig. 5a and b is that smaller viruses with less negative δs a_immob bind more strongly at higher temperatures than larger viruses. only with δs a_immob as high as −240 j/mol/k representing smaller viruses do more than 30% of the 1000 cells have bound virus at the human body temperature of 37°c according to the model in fig. 5b . this is shown in fig. 5c where nearly all the cells have bound virus at 10°c irrespective of δs a_immob while at higher temperatures fewer cells have bound virus as δs a_immob becomes more negative. 3.6. case study 5: hiv attachment factors may partially overcome the unfavourable δs a_immob and enhance specific virus binding through env/cd4 interactions at human body temperature thus increasing δs a_specific for specific env:cd4 binding (as δs a_immob in eq. (11)) from −400 j/mol/k to −240 j/mol/k. fig. 5b shows that the number of cd4 + t cells in a 1 mm 3 vol of blood with bound hiv falls from 1000 to <1 over a temperature range of~10°c. the temperature at which fewer than half the t cells in the 1 mm 3 vol of blood have bound hiv virus increases with increasing δs a_specific from 12.5°c for δs a_specific = −400 j/mol/k to 36.5°c for δs a_specific = −240 j/mol/k (fig. 5b) . indeed once the δs a_specific is >−200 j/mol/k, 98% of the 1000 t cells have bound hiv at 37°c (not shown). with δs a_specific <−285 j/mol/k virus is not specifically bound to any of the 1000 t cells at temperatures above 37°c. it is concluded that the effect of making δs a_immob less unfavourable through prior non-specific binding taking some of the entropy loss enhances subsequent specific binding of hiv through env:cd4 interactions at higher temperatures. fig. 5c shows that at 37°c increasing δs a_specific from −400 to −240 j/mol/k increases the number of cd4 + t cells with hiv bound through specific env/cd4 interactions in a 1 mm 3 vol of blood from 2 × 10 −6 to 307. it should be noted that 2 × 10 −6 cells in 1 mm 3 is equivalent to 1 cell in 500 cm 3 of blood. 3.7. case study 6: increasing the number, n, of gp/cr contacts with temperature to reproduce the data of frey et al. (1995) cells expressing hiv env on their surface can bind and fuse with cells expressing cd4 cr (frey et al., 1995) . frey et al. (1995) reported a three to four fold increase in cell binding at temperatures above 25°c with a substantial decrease in binding at temperatures above 37°c (fig. 6) , the latter being predicted by the thermodynamic model in fig. 5b . frey et al. (1995) attribute the increase at 25°c to the additional adhesion molecules which are brought into contact to increase the avidity of binding at higher temperatures. the thermodynamic model for hiv binding with δs a_immob of −206 j/mol/k reproduces the increase in predicted c.v t in fig. 6a by increasing n from 1 to 3 over the temperature range 25°c and 27°c to reflect the effect of temperature facilitating multiple hiv adhesion/binding sites due to increased membrane fluidity (frey et al., 1995; harada et al., 2004) . increasing n from 1 to 9 over the temperature range 19°c and 27°c gives a sharp increase in c.v t at 26°c (fig. 6b) with δs a_immob of −1110 j/mol/k. the subsequent sharp fall in c.v t at temperatures above 37°c is due to the large negative magnitude of δs a_immob (−206 j/mol/k in fig. 6a and -1110 j/mol/k in fig. 6b ) relative to n such that the diminished gp/cr affinity at higher temperatures is not sufficient to maintain binding even with n = 9 gp/cr interactions in fig. 6b . the decrease in gp/cr binding affinity with temperature for hiv env:cd4 is apparent from the k a_virus_t values in fig. 5a and the increase in the values of k d_receptor_t for the temperatures 4°c, 25°c and 37°c in table 4 . previously a thermodynamic model for the effect of temperature on arthropod vector competence was developed (gale 2019) based on the parameters δh a_receptor_t , δs a_receptor_t , δs a_immob and n, together with the kinetic parameters, p complete283 and e a (table 2 ). this paper furthers that model by allowing the change in heat capacity, δc p , on gp/cr binding to be included. this is important because it defines the effect of temperature on δh a_receptor_t (eq. (4)) and δs a_receptor_t (eq. (6)) and hence k a_virus_t . of potentially greater importance, however, is the parameter δs a_immob first formally identified in the thermodynamic analysis of virus binding (gale 2019) . thus it is proposed here that the value of δs a_immob is not only important in explaining the observation that dsdna viruses infecting warmer hosts are generally smaller in volume than those infecting colder hosts (nifong and gillooly 2016) but also may contribute to explaining the need for attachment factors to allow binding of a large virus such as hiv at the relatively high human body temperature of 37°c. this has implications for the design of novel antiviral therapies. the mechanistic model developed here for hiv goes as far as cell binding and viral membrane fusion (harrison 2015) could be added as the next stage to complete the cell entry process. calculations using eq. (17) on the basis of limited available data suggest δs a_immob~− 1100 j/mol/k for hiv although it is acknowledged above that there is considerable uncertainty in the absence of definitive experimental data. it is possible that statistical thermodynamics approaches analogous to the sackur-tetrode equation for the entropy of a gas may be developed to estimate δs a_immob theoretically. the large size of the hiv virion (134 nm mean diameter) compared to a typical arbovirus (50 to 70 nm diameter) is consistent with a more negative value of δs a_immob providing justification for the more negative δs a_immob values used here for the hiv models in figs. 4-6 compared to that used for the arbovirus models in figs. 2 and 3. figs. 2-5 show the variation of k a_virus_t and c.v t with temperature as predicted by models assuming c total = 10 3 host cells in a 10 −6 dm 3 volume (i.e. mosquito midgut or human blood sample) challenged with v total = 10 5 virions. previously it was shown that if k a_virus_t >~10 14 m −1 then nearly all the host cells have bound virus at such a high virus dose (gale 2018; 2019) . thus the k a_virus_t plots and corresponding c.v t plots are best interpreted in terms of the temperature ranges over which k a_virus_t <~10 14 m −1 . for the arbovirus models in figs. 2 and 3 , the main conclusion regarding the change in heat capacity on gp/cr binding is that as δc p becomes more negative so a peak temperature for k a_virus_t becomes apparent such that binding affinity falls at lower and higher temperatures, directly affecting c.v t when k a_virus_t falls below 10 14 m −1 . values of δc p from 0 to~−2.0 kj/mol/k have relatively little impact on the temperature sensitivity of the number, c.v t , of mosquito midgut cells with bound virus (fig. 2b) , while intermediate values of δc p of fig. 5 . predicted effect of δs a_immob on the temperature sensitivity of a) k a_virus_t and b) the number of host cd4 + t cells, c.v t , with bound hiv virions in a system comprising c total = 10 3 cd4 + host t cells in a 1 mm 3 vol of blood with a challenge dose of 10 5 hiv virions. the values of δh a_receptor_t0 and δs a_receptor_t0 at t 0 = 37°c for specific hiv env:cd4 binding are given in table 4 with n t= 3 env:cd4 specific interactions and δc p = −5.02 kj/mol/k (table 3) ~−3.0 kj/mol/k give a peak binding at a temperature of~20°c as observed experimentally for weev binding to bbfs from susceptible mosquito midguts in fig. 3b . more negative values of δc p at −5.0 kj/ mol/k greatly diminish arbovirus binding affinity at temperatures below~20°c (fig. 2b ) which in turn diminishes the transmission efficiency of the arbovirus at temperatures below 20°c compared to gp/ cr systems with δc p in the range of −2.0 to 0 kj/mol/k (fig. 2d) . in evolutionary terms, to optimise arbovirus infectivity with n = 4 gp/cr interactions over a range of temperatures, gp/cr interactions with small δc p would be selected as for aiv ha/sa binding (table 3) . it is proposed here that to maintain some transmission efficiency at lower temperatures, arboviruses may use sa binding interactions as shown for btv coat protein vp2 (zhang et al., 2010) for which δc p may approximate 0 kj/mol/k as in the case of aiv ha/sa (table 3 ). this would facilitate arboviruses' expanding their range into temperate regions of the world with climate change. with the reference temperature of t 0 = 37°c, c.v t falls rapidly at temperatures above 20°c in the arbovirus model here with n = 4 gp/ cr interactions irrespective of the magnitude of δc p (fig. 2b) as the k a_virus_t values fall below 10 14 m − 1 and converge at 10 12 m − 1 at 37°c (fig. 2a) . this fall is observed experimentally as the temperature is increased from 20°c to 40°c (fig. 3b) for weev binding to bbfs from susceptible mosquito midguts as reported by houk et al. (1990) . 4.4. the large negative δc p for hiv gp120:cd4 diminishes binding at human body temperature in the hiv model the δc p for hiv gp120:cd4 binding is well documented unlike that for arbovirus binding and the values of δh a_receptor_t have been reported over the temperature range of 12°c to 44°c (fig. 1) . the results with varying δc p in fig. 4 are therefore hypothetical but illustrate how the large negative δc p for the hiv env:cd4 interaction diminishes binding at the higher temperatures of mammals and birds compared to a system such as ha/sa for which δc p = 0 kj/mol/k. thus, a less negative δc p increases the number of cells with bound virus at higher temperatures, albeit more markedly for the reference temperature t 0 = 4°c (fig. 4d ) than for t 0 = 37°c (fig. 4b) . in the absence of data for δc p for hiv env:cd4 binding, the value for the gp120 monomer:cd4 interaction is used for the hiv models in figs. 4-6. evidence for a large negative δc p for hiv env:cd4 binding is that cryo-em demonstrates considerable conformational shifts (liu et al., 2017a) . a negative value of δs a_immob presents a repulsive force between the virus and the host cell in effect preventing virus binding and with n = 0 gp/cr contacts, k a_virus_t is < 1 m − 1 according to eq. (17) (gale 2019) . increasing the value of n is one way for the virus to overcome a large negative value of δs a_immob (gale 2019). thus 15 to 18 gp/cr contacts of k d_receptor_t0 = 10 −3 m (37°c) were sufficient to overcome a δs a_immob of −750 j/mol/k such that k a_virus_t > 10 14 m − 1 (fig. 3 of gale (2019) ). this is apparent from fig. 6a where n = 3 gp/cr contacts can overcome a δs a_immob of −206 j/mol/k while n = 9 gp/cr contacts are required to overcome the more negative δs a_immob of −1110 j/mol/k in fig. 6b . even for δs a_immob = 0 j/mol/k as in figs. 2a and 3a, the magnitude of k a_virus_t decreases with increasing temperature (albeit after a peak in the case of δc p < −2.0 kj/mol/k). this is due to the reduced affinity of the enthalpy-driven gp/cr interactions at higher temperatures. making δs a_immob more negative shifts the k a_virus_t curve to lower values (fig. 5a ) thus exacerbating the effect of higher temperature on reducing binding. as k a_virus_t falls below 10 14 m − 1 at higher temperatures, so c.v t falls (fig. 5b) . the magnitude of δs a_immob merely positions the c.v t curve relative to the temperature scale with less negative values of δs a_immob shifting the curve to higher temperatures ( fig. 5b) such that high c.v t values are maintained at progressively higher temperatures as δs a_immob becomes less negative (fig. 5c) . the thermodynamic model here suggests that a large negative δc p for hiv env:cd4 diminishes binding at higher temperatures. however, there may be molecular constraints such that a large negative δc p value is a direct consequence of a molecular mechanism involving a large conformational change that serves a purpose for cell entry/fusion (harrison 2015) such that a negligible δc p value may not be achievable. furthermore, a large negative δs a_immob imposes additional constraints on virus binding at higher temperatures exacerbating the reduced strength of the enthalpy-driven gp/cr interactions at higher temperatures. therefore other mechanisms may be needed by the virus to achieve binding at 37°c. these could include more gp/cr contacts at higher temperature (frey et al., 1995; harada et al., 2004 ). an alternative strategy evolved by viruses could be to make δs a_immob itself less negative such that virus binding is maintained at the higher body temperatures of mammals and birds. two possible evolutionary strategies are suggested here which could make δs a_immob less negative. the first is decreasing the size of the virus and the second is the use of nonfig. 6 . experimental binding of hiv env-expressing cells to cells expressing cd4 as reported by frey et al. (1995) increases three to four fold at 25°c (cross). the number, c.v t , of cd4 + t cells with bound hiv virions per mm 3 blood predicted by the thermodynamic model (circle) using eq. (11) with the number, n, of gp/cr contacts increasing according to the dotted line with temperature in a) from 1 to 3 between 25 and 27°c; and in b) from 1 to 9 between 19°c and 27°c. the values of δh a_receptor_t0 and δs a_receptor_t0 at t 0 = 37°c for non-specific attachment factor binding and for hiv env:ccr5 co-receptor binding are as those for specific hiv env:cd4 binding in table 4 with δc p = −5.02 kj/mol/k (table 3) . δs a_immob = a) −206 j/mol/k to represent prior elimination of δs a_non_specific through binding of virus through nonspecific attachment; and b) −1,110 j/mol/k to represent free virus binding. specific attachment factors to randomly trap the virus on the cell surface prior to specific gp/cr binding. decreasing the size of a virus makes δs a_immob less negative (eq. (18) ) such that smaller viruses bind more strongly at the higher temperatures of mammals and birds (fig. 5) . it is suggested that binding to cells at the higher temperatures of avian and mammalian hosts selects for viruses of smaller diameter through a less negative δs a_immob . this could explain the decrease in volume of viruses with increasing host/environment temperature as reported by nifong and gillooly (2016) for dsdna viruses. thus the average diameters of dsdna viruses decreased from 182 nm at a host/environment temperature of 10°c down to 74 nm at 37°c according to the best fit line of nifong and gillooly (2016) suggesting the hiv virion at 134 nm mean diameter is almost double the size for a virus infecting at avian/mammalian host temperature. it is interesting to note that thermophilic bacteria adapted to high temperature may be subject to selection favouring smaller cell size. this may aid attachment to biofilms which provide some protection against temperature stress. it is proposed here that by taking the entropy loss δs a_non_specific in δs a_immob that non-specific binding to attachment factors facilitates subsequent specific hiv env binding to cd4 receptors on the host cd4 + t cell at human body temperature. thus the shift in the k a_virus_t curve to higher values with a less negative δs a_immob in fig. 5a is consistent with hiv attachment factors augmenting infection (wilen et al., 2012) . indeed with n = 3 env:cd4 contacts, significant binding of hiv to host cells is only achieved at 37°c if δs a_immob is increased to > −280 j/ mol/k (fig. 5c) . δs a_immob is estimated here to be −1091 j/mol/k for hiv. thus the prior non-specific binding of hiv would need to realise a δs a_non_specific of −810 j/mol/k for subsequent effective env:cd4 binding at human body temperature according to the model such that δs a_non_specific in eq. (19) takes~75% of the entropy loss in δs a_immob . wilen et al. (2012) also report that hiv attachment factors are not essential. again this is consistent with the model here when it is noted that the c.v t value of <1 cd4 + t cell with bound virus in fig. 5b is not a threshold and merely represents 1 mm 3 of blood. thus with δs a_immob = −348 j/mol/k, c.v t = 1 × 10 −3 at 37°c (fig. 5c ). this represents one cd4 + t cell with bound hiv in 1000 × 1 mm 3 vol of blood, i.e. in 1 cm 3 of blood. decreasing δs a_immob further to −405 j/ mol/k gives c.v t = 1 × 10 −6 at 37°c representing one cd4 + t cell with bound hiv in 1 litre of blood. the model here suggests human body temperature may be a constraint on hiv binding (due to the large negative values of both δc p and δs a_immob ) in effect exposing a potential weakness in the hiv infection strategy which could be exploited for antiviral therapy. figure 9 of antoine et al. (2012) shows a herpes simplex virus type 2 (hsv-2) bound to a tetrahedral nanoparticle of zinc oxide (zno) called a zinc oxide tetrapod (znot). the mechanism behind the ability of znots to prevent, neutralize or reduce hsv-2 infection relies on their ability to bind the hsv-2 virions (antoine et al., 2012) . the znot being some 40 µm in size is much larger than the attached virion which could still attach to a host cell surface in terms of spatial considerations. however, the magnitude of δs a_immob for an hsv-2 virion bound to a znot nanoparticle would be very large and negative, indeed much more negative than for δs a_immob for a free hsv-2 virion. thus according to fig. 5c , binding of hsv-2/znot to host cells would be greatly diminished particularly at the human body temperature due to the very large negative δs a_immob . although δs a_immob changes logarithmically (i.e. slowly) with virus size according to eq. (18), the number, c.v t , of cells with bound virus is very sensitive to changes in δs a_immob at 37°c (fig. 5c) . it is proposed that antiviral research should take into account and even focus on making the magnitude of δs a_immob more negative in addition to looking at conventional approaches to block the gp/cr interaction. the advantage of focusing on δs a_immob is that only one znot nanoparticle has to bind to a virus to markedly affect δs a_immob , while a large proportion of the gp molecules on the virus have to be bound with inhibitors, including neutralising antibodies, to block virus binding (klasse 2012) and hence eliminate δh a_receptor_t0 in eq. (11). the temperature dependence of virus diffusion in mucus (erickson et al., 2015) could also be considered not only in mechanistic dose-response models for viral infection at mucosal epithelial membranes in the intestine (gale 2018) but also in terms of therapeutics when combined with the thermodynamic approach developed here. normal, acidic cervicovaginal mucus greatly hinders the movement of virions of hsv and hiv, whereas mucus that is neutralized by semen provides a much less effective barrier against the same virions (erickson et al., 2015) . thus binding of the hsv or hiv virion to a znot particle may not only decrease its diffusion coefficient in mucus but also make the magnitude of δs a_immob more negative hence reducing the binding affinity of those virions which do make it through the mucus to the epithelial cell surface at human body temperature. it is assumed for the purpose of the "proof of concept" model here that the thermodynamic parameters for the hiv gp120 monomer:cd4 interaction in myszka et al. (2000) can be used for hiv env trimer binding to a single cd4 in the absence of data. although the k d_receptor_t value for the env trimer:cd4 interaction (1.4 × 10 −9 m, chuang et al., 2017) is 15-fold higher than that for the gp120 monomer:cd4 interaction (0.9 × 10 −10 m, table 4 ) at 25°c, this is close enough to support using the gp120 monomer:cd4 data, particularly as k d_receptor_t values for the gp120 monomer:cd4 interaction vary nine-fold between two hiv strains (see above). the predicted values of k a_virus_t increase with decreasing temperature according the hiv model in fig. 5a . this is unexpected on the basis of the δh a_receptor_t values reported by myszka et al. (2000) for gp120:cd4 which become less negative with decreasing temperature (fig. 1) . however, according to eq. (6) the predicted δs a_receptor_t term becomes more favourable (less negative) with decreasing temperature and it is therefore the entropy term that facilitates the tighter binding of gp120 to cd4 at lower temperatures. unfortunately myszka et al. (2000) only give the full range of values for δh a_receptor_t (fig. 1) with no data for δs a_receptor_t over the range of temperature. however, myszka et al. (2000) do give some graphical data from which δs a_receptor_t at 25°c can be estimated and it is reassuring this δs a_receptor_t is much less negative at −473 j/mol/k than that of −691 j/mol/k measured at 37°c and furthermore is actually less negative than the value of −492.8 j/mol/k predicted for 25°c by eq. (6) ( table 4) and used in the model in fig. 5 . this is strong experimental evidence that warming to 37°c and above does indeed give a significant reduction in the binding affinity of env to cd4 as borne out by the 52-fold increase in the k d_receptor_t value for gp120:cd4 on warming from 25°c to 37°c as calculated from the actual data of myszka et al. (2000) (table 4 ). furthermore it suggests that the actual increase in k a_virus_t on cooling from 37°c down to 25°c may be greater than that predicted by the model in fig. 5a . the decrease in binding affinity with increasing temperature for hiv env:cd4 is also supported by experimental data of moore and klasse (1992) which showed that soluble cd4 dissociated from hiv virions at temperatures above 35°c. similarly doranz et al. (1999) demonstrated that gp120 bound to cd4 + t cells began to dissociate at temperatures above 45°c. the data of frey et al. (1995) in fig. 6 offer further validation of the model in that the binding of hiv env-expressing cells to cells expressing cd4 decreases substantially at 37°c as predicted by the model. the env trimer is stable up to at least 60°c (chuang et al., 2017) suggesting it is not denaturation of the env trimer itself that is responsible for the decreased binding at higher temperatures. however, care has to be taken in using eqs. (4) and (6) to predict δh a_receptor_t and δs a_receptor_t respectively down to very low temperatures. thus although the predicted value of k d_receptor_t for gp120:cd4 at 4°c is 3700-fold lower than that at 37°c (table 4) , frey et al. (1995) cite some studies which suggested weaker binding of soluble cd4 to hiv virions at 4°c compared to 37°c. without actual binding data for hiv over the full temperature range, it cannot be ruled out that k a_virus_t does not exhibit a peak with temperature as for the weev binding in fig. 3b . indeed, binding of 125 ilabelled gp120 to t cells peaked at 16°c with binding at 4°c much lower than that at 37°c (frey et al., 1995) . the observed increase between 4°c and 16°c is unlikely to be due to increased fluidity of the membrane allowing recruitment of more cd4 molecules because unlike the virus, each gp120 only binds a single cr molecule. dimitrov et al. (1992) showed that the activation energy for binding of soluble cd4 to cells expressing hiv env changed at 18°c suggesting 18°c is a transition temperature. this could explain the peak in binding of gp120 at 16°c reported by frey et al. (1995) . thus extrapolation of δh a_receptor_t and δs a_receptor_t through eqs. (4) and (6), respectively to temperatures below 18°c may not be justified in the case of hiv binding. it should be remembered that hiv only infects humans at 37°c and therefore the lower temperature predictions are more of academic interest, although temperature trends may give an insight into weaknesses in the hiv infection strategy as suggested here. it is only the 37°c line in fig. 5c which is important for hiv and it is reassuring to note that this is based on actual experimental data for gp120 monomer:cd4 binding measured at that temperature. the lower temperature lines in fig. 5c are of more general interest, for example in explaining the observed relationship (nifong and gillooly 2016) between larger virus volume and lower host temperature. individual virions of the same virus may vary both in size (briggs et al., 2003) and in the case of some rna viruses in the actual base sequence of the genome giving a spectrum of mutants. the infection process may represent a bottleneck such that only one or two viral variants in the exposure dose successfully establish infection (bull et al., 2011) . thus in the case of norovirus, only minor variants at frequencies as low as 0.01% were successfully transmitted to establish a new infection (bull et al., 2012) . the work here suggests that the actual size of the virion itself may present an additional constraint particularly at mammalian body temperatures. thus although the average diameter for hiv virions with a single core is 134 nm, individual virions display a broad range of diameters extending from 120 to 200 nm with a skewed distribution (briggs et al., 2003) . furthermore while the majority of hiv virions contained a single core, almost a third contained two or more cores. the mean diameter of the hiv virions containing a single core (134 ± 11 nm, n = 89) was significantly smaller than the mean diameter of virions containing two cores (158 ± 16 nm, n = 43) (briggs et al., 2003) . thus the magnitude of δs a_immob may vary between individual hiv virions within the same exposure dose. the key conclusion from fig. 5c is that the number of cd4 + t cells with bound virus is highly sensitive to the magnitude of δs a_immob with just a 4.5% increase in the magnitude of δs a_immob from -290 to -277 j/mol/k for example increasing c.v t by five-fold from 1 to 5 cd4 + t cells per mm 3 of blood at 37°c. thus smaller diameter viruses in a challenge does may have an advantage in binding and hence initiating infection at mammalian body temperatures. this also raises the question of the magnitude of δs a_immob for an aggregate containing several virions of the same virus and in the case of ebov, for the long filaments linking multiple genome copies (beniac et al., 2012) . it has previously been proposed that fusing multiple genome copies into the same filament in the case of ebov takes some of the entropy loss for δs a_immob prior to binding to the cell surface (gale 2017) such that aggregation can be viewed as a cooperative effect in the infection process. the effect of aggregation and virus size on the magnitude of δs a_immob according to eq. (18) needs to be investigated and understood to further the work here. from a thermodynamic perspective, the affinity of virus binding to a host cell is greatly decreased at the higher body temperatures of mammals and birds such that temperature can be considered a constraint to be overcome. the relationship between binding affinity and temperature can be markedly affected by the change in heat capacity (δc p ) on virus binding through its effect on the enthalpy and entropy of the gp/cr interaction. specifically large negative values of δc p reduce binding at both low and high temperatures giving a peak binding at a medium temperature as has been observed experimentally in binding of weev to mosquito midguts. very large negative values of δc p for protein/protein gp/cr interactions are predicted to diminish arbovirus transmission at low temperatures compared to the ha/sa glycan interaction for which δc p is negligible. thus selecting for sa glycan interactions as reported for btv binding may be a mechanism to allow transmission at the lower temperatures experienced by arboviruses infecting arthropods in temperate regions of the world. the magnitude of δs a_immob is currently unknown but is predicted to be large and negative and to be more negative for larger viruses such as hiv (mean diameter 134 nm) compared to the smaller arboviruses (diameter 50 to 70 nm). small virion diameter through a less negative δs a_immob , favours virus binding at higher temperatures giving smaller viruses a selective advantage for infecting hosts at the higher body temperatures of mammals and birds. this is in agreement with the published observational findings that virus volume decreases with increasing host temperature in the case of dsdna viruses. in the case of hiv, the unfavourable effect at human body temperature of the large negative δs a_immob value due to the large size of the virus may be overcome by non-specific binding. it is proposed here that non-specific binding of hiv as the virus first randomly attaches to a host cd4 + t cell surface takes a large proportion (represented by δs a_non_specific ) of the entropy loss of δs a_immob such that hiv can then bind specifically through env:cd4 interactions at the relatively high temperature of the human body. the randomly attached virus "rolls into place" driven by n = 3 specific env:cd4 interactions resulting in a further entropy loss (δs a_specific ) as the virus particle takes up a specific orientation ready for viral entry. non-specific attachment factors are all the more important for a gp/cr system such as the hiv env:cd4 interaction for which δc p is large and negative and diminishes binding at higher temperatures. it is concluded that experimental data on δs a_immob should be obtained and antiviral therapeutic strategies, for example using zinc oxide nanoparticles for herpes simplex virus or neutralising antibodies to block virus binding, should focus not only on blocking the cr-binding sites on the gp molecules themselves but also on targeting δs a_immob by increasing the size of the virus itself and so minimising host cell binding affinity at human body temperatures according to the model here. bacterial cells are larger than viruses and δs a_immob would be expected to be more negative. targeting δs a_immob may also have application as an alternative approach to antibiotics for bacterial pathogens when bacterial pathogenesis depends on attachment to cells. however, it should be noted that δs a_immob decreases logarithmically (i.e. slowly) with increasing size which may limit the practicability to bacteria. prophylactic, therapeutic and neutralizing effects of zinc oxide tetrapod structures against herpes simplex virus type-2 infection the organisation of ebola virus reveals a capacity for extensive modular polyploidy diverse rna viruses of arthropod origin in the blood of fruit bats suggest a link between bat and arthropod viromes different infectivity of hiv-1 strains is linked to number of envelope trimers required for entry changing patterns of west nile virus transmission: altered vector competence and host susceptibility structural organization of authentic, mature hiv-1 virions and cores sequential bottlenecks drive viral evolution in early acute hepatitis c virus infection membrane recognition by vesicular stomatitis virus involves enthalphy-driven protein-lipid interactions structure-based design of a soluble prefusion-closed hiv-1 env trimer with reduced cd4 affinity and improved immunogenicity interdisciplinary approaches to understanding disease emergence: the past, present, and future drivers of nipah virus emergence role of receptor binding specificity in influenza a virus transmission and pathogenesis characterization of human immunodeficiency virus type 1 monomeric and trimeric gp120 glycoproteins stabilized in the cd4-bound state: antigenicity, biophysics, and immunogenicity kinetics of soluble cd4 binding to cells expressing human immunodeficiency virus type 1 envelope glycoprotein use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events insights into protein-ligand interactions: mechanisms, models, and methods predicting first traversal times for virions and nanoparticles in mucus with slowed diffusion characterization of receptor binding profiles of influenza a viruses using an ellipsometry-based label-free glycan microarray assay platform temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 could bat cell temperature and filovirus filament length explain the emergence of ebola virus in mammals? predictions of a thermodynamic model using thermodynamic parameters to calibrate a mechanistic dose-response for infection of a host by a virus. microbial risk anal towards a thermodynamic mechanistic model for the effect of temperature on arthropod vector competence for transmission of arbovirus high viral load in semen of human immunodeficiency virus type 1-infected men at all stages of disease and its reduction by therapy with protease and nonnucleoside reverse transcriptase inhibitors adsorption and infectivity of human immunodeficiency virus type 1 are modified by the fluidity of the plasma membrane for multiple-site binding viral membrane fusion. virology 479-480 cryo-em structures of eastern equine encephalitis virus reveal mechanisms of virus disassembly and antibody neutralization binding of western equine encephalomyelitis virus to brush border fragments isolated from mesenteronal epithelial cells of mosquitoes effects of simian immunodeficiency virus on the circadian rhythms of body temperature and gross locomotor activity thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of bacillus stearothermophilus avian virulence and thermostable replication of the north american strain of west nile virus the molecular basis of hiv entry cooperation of multiple ccr5 coreceptors is required for infections by human immunodeficiency virus type 1 testing new hypotheses regarding ebolavirus reservoirs. viruses 8 quaternary contact in the initial interaction of cd4 with the hiv-1 envelope trimer temperature increase enhances aedes albopictus competence to transmit dengue virus molecular basis of binding between novel human coronavirus mers-cov and its cellular receptor cd26 polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors thermodynamic and kinetic analysis of scd4 binding to hiv-1 virions and of gp120 dissociation structure of west nile virus environmental effects on vector competence and virogenesis of bluetongue virus in culicoides interpreting laboratory data in a field context energetics of the hiv gp120-cd4 binding reaction interactions between the inner and outer capsids of bluetongue virus replication and transcription activities of ribonucleoprotein complexes reconstituted from avian h5n1, h1n1pdm09 and h3n2 influenza a viruses temperature effects on virion volume and genome length in dsdna viruses bat flight and zoonotic viruses investigating the zoonotic origin of the west african ebola epidemic simian immunodeficiency virus infection of chimpanzees establishment of reference cd4+ t cell values for adult indian population receptor binding by a ferret-transmissible h5 avian influenza new insights into the hendra virus attachment and entry process from structures of the virus g glycoprotein and its complex with ephrin-b2 stoichiometry of envelope glycoprotein trimers in the entry of human immunodeficiency virus type 1 tim-1 acts a dual-attachment receptor for ebolavirus by interacting directly with viral gp and the ps on the viral envelope bluetongue virus coat protein vp2 contains sialic acid-binding domains, and vp5 resembles enveloped virus fusion proteins i thank the anonymous reviewer who helped me with the hiv binding data and greatly improved this manuscript. i wrote the whole of this paper, did all the work and developed all the ideas. i have done this work in my spare time at home with no funding. i greatly enjoy doing this sort of paper. the views expressed in this paper are those of the author and not necessarily those of any organisations. none declared. key: cord-292830-gcfx1095 authors: ianevski, aleksandr; zusinaite, eva; kuivanen, suvi; strand, mårten; lysvand, hilde; teppor, mona; kakkola, laura; paavilainen, henrik; laajala, mira; kallio-kokko, hannimari; valkonen, miia; kantele, anu; telling, kaidi; lutsar, irja; letjuka, pille; metelitsa, natalja; oksenych, valentyn; bjørås, magnar; nordbø, svein arne; dumpis, uga; vitkauskiene, astra; öhrmalm, christina; bondeson, kåre; bergqvist, anders; aittokallio, tero; cox, rebecca j.; evander, magnus; hukkanen, veijo; marjomaki, varpu; julkunen, ilkka; vapalahti, olli; tenson, tanel; merits, andres; kainov, denis title: novel activities of safe-in-human broad-spectrum antiviral agents date: 2018-04-23 journal: antiviral res doi: 10.1016/j.antiviral.2018.04.016 sha: doc_id: 292830 cord_uid: gcfx1095 according to the who, there is an urgent need for better control of viral diseases. re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. we tested 55 of these compounds against eight different rna and dna viruses. we found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rift valley fever virus. thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases. dozens of viruses, such as fluav, hsv-1, vzv, cmv and nov, constantly infect human population and represent substantial public health and economic burden (dalys and collaborators, 2017; disease et al., 2017) . emerging and re-emerging viruses, such as ebov, marv, lasv, chikv, zikv, denv, rvfv, mers-and sars-cov, surface from natural reservoirs approximately one each year and also represent global threats (howard and fletcher, 2012; who, 2015) . according to who, there is an urgent need for better control of these viruses, including drug-resistant and vaccine immunity escaping viral strains (bekerman and einav, 2015; de clercq and li, 2016) . antiviral drugs and vaccines are the most powerful tools to combat viral diseases. most drugs and vaccines, however, selectively target a single virus, thereby providing a "one drug-one bug" solution. by contrast, broad-spectrum antivirals (bsas) can cover multiple viruses and genotypes and reduce the likelihood of development of resistance. therefore, some bsas can be used for rapid management of new or drug-resistant viral strains, for treatment of viral co-infections reducing therapy complexity, as well as a first-line treatment or the prophylaxis of acute virus infections. thus, to overcome time and cost issues associated with the development of virus-specific drugs and vaccines, the development of bsas should be prioritized (bekerman and einav, 2015) . nucleotide and nucleoside analogues are excellent examples of bsas. they inhibit transcription and/or replication of different rna and dna viruses (de clercq, 2015) . in particular, valaciclovir inhibits replication of different herpesviruses and hbv (laube et al., 2004; vere hodge and field, 2013) . cidofovir and its lipid conjugate brincidofovir also inhibit replication of dsdna viruses, such as herpesviruses, adv, bkv, and hpv (andrei et al., 2015) . ribavirin blocks viral rna synthesis of fluav, hcv and rsv (hong and cameron, 2002) . favipiravir and bcx4430 also inhibit replication of different rna viruses (mckimm-breschkin et al., 2018) . however, viruses are able to develop resistance to some of these nucleotide and nucleoside analogues. other examples of bsa agents include inhibitors of cellular pathways, which are exploited by different viruses for efficient viral replication (debing et al., 2015) . these agents overcome the problem of antiviral drug resistance. for example, lipid-lowering statins (atorvastatin, lovastatin, simvastatin, and fluvastatin) inhibit cellular hmg-coa reductase and attenuate replication of some enveloped viruses (bernal et al., 2017; enserink, 2005) . anti-malaria quinolones (chloroquine and hydroxychloroquine) inhibit acidification of endosomes, which is an essential process for uncoating of ssrna viruses (al-bari, 2017 ). anticancer kinase inhibitors dasatinib, imatinib, gefitinib, nilotinib, erlotinib and sunitinib impair intracellular viral trafficking and exert bsa effects (bekerman et al., 2017; schor and einav, 2018) . the anti-duchenne muscular dystrophy agent, alisporivir, targets cellular cyclophilin and inhibits the folding of hcv, hiv, mers-and sars-cov proteins, and, therefore, prevents formation of infectious virus particles (boldescu et al., 2017; de wilde et al., 2017; soriano et al., 2011) . thus, both host-directed antivirals and nucleotide/nucleoside analogues could possess bsa activity. here, we hypothesised that some of the identified safe-in-human bsas could possess novel antiviral activities and, therefore, could be used for treatment of many different viral infections. to prove this hypothesis, we reviewed safe-in-man approved, investigational and experimental antiviral agents. we identified 59 compounds that target at least three viral diseases. we tested 55 of the 59 compounds against 8 different viruses and found novel activities for 7 of these agents. we conclude that the spectrum of antiviral activities for existing bsa agents could be expanded towards other viral diseases. information on the viruses and associated human diseases is summarized in table s1 . information on approved, investigational and experimental safe-in-human antivirals is summarized in tables s2-s4 . this information was extracted from drugbank (2018), clinical trials websites (2018) and pubmed. information on 59 approved, investigational, and experimental antivirals, which target ≥3 viral diseases, is summarized in table s5 . eye diagrams and interaction network plots were created with javascript library d3.js v4 (2018). a structural similarity plot for the drugs was constructed and visualized using a c-spade web application (ravikumar et al., 2017) . the compounds used in this study, their suppliers and catalogue numbers are summarized in table s6 . to obtain 10 mm stock solutions compounds were dissolved in 100% dimethyl sulfoxide (dmso, sigma-aldrich) or milli-q water. the solutions were stored at −80°c until use. bhk-21 cells (baby hamster kidney fibroblasts) were grown in glasgow's minimal essential medium (gmem) containing 7.5% fetal bovine serum (fbs; gibco, paisley, uk), 2% tryptose phosphate broth (tpb), 200 mm hepes, 100 u/ml penicillin and 0.1 mg/ml streptomycin (penstrep, lonza basel, switzerland). ach-2 cells, a model for chronic hiv-1 infection, which possesses a single integrated copy of the provirus hiv-1 strain lai (nih aids reagent program), were grown in rpmi-1640 medium supplemented with 10% fbs and penstrep. madin-darby canine kidney epithelial (mdck) cells, human embryonic kidney cells (hek293t) and african green monkey kidney epithelial cells (vero) were grown in dulbecco modified eagle's medium (dmem; sigma-aldrich, st. louis, mo, usa) supplemented with 2 mm l-glutamine (lonza; basel, switzerland), 50 u/ml penstrep and 10% fbs. human telomerase reverse transcriptase-immortalized retinal pigment (rpe) cells were grown in dmem-f12 medium supplemented with 50 u/ml penstrep, 2 mm l-glutamine, 10% fbs, and 0.25% sodium bicarbonate (sigma-aldrich). tzm-bl cells were grown in dmem supplemented with 10% heat-inactivated fbs and penstrep. human lung adenocarcinoma epithelial cells, a549, were cultured in dmem containing 0.75 g/l nahco3, 20 mm hepes (euroclone, milan, italy), penstrep, and 5% fetal bovine serum (fbs, gibco) at 37°c. all cell lines were grown in humidified incubator at 37°c in the presence of 5% co 2. human influenza a/wsn/33(h1n1) virus was generated using eight-plasmid reverse genetics system in hek293 and vero-e6 cells, as described previously (hoffmann et al., 2000) . ev1 strain was propagated in a monolayer of vero cells, as described earlier (myllynen et al., 2016) . hsv-1 was amplified in vero cells, as described previously (nygardas et al., 2013) . zikv fb-gwuh-2016 strain was cultured in vero e6 cells, as described earlier (driggers et al., 2016) . for production of hiv-1, 6 × 10 6 ach-2 cells were seeded in 10 ml of full culture media, and hiv-1 production was induced by the addition of 100 nm phorbol 12-myristate 13-acetate (viira et al., 2016) . the induced cells were incubated for 48 h, and subsequently, the hiv-1 containing media was collected and filtered. the amount of hiv-1 in the stock was estimated by quantification of p24 protein in the media. quantity of p24 was measured using a reference recombinant purified p24 protein and anti-p24-elisa, which was developed in-house. chikv-2sg-nanoluc strain was generated from icdna clone of picres1 representing lr2006opy1 strain belonging to east/central/ south african genotype (utt et al., 2016) . rrv-2sg-nanoluc strain derived from rrv-t48 strain (jupille et al., 2011) . sequence encoding nanoluc protein was placed between non-structural and structural regions of chikv or rrv genomes under the control of native subgenomic promoters of the viruses. to ensure expression of viral structural proteins, a copy of subgenomic promoter (residues −77 to +69 for chikv and residues −77 to +49 for rrv, positions given with respect to start site of subgenomic rna) was inserted immediately downstream of sequence encoding for nanoluc. recombinant rvfv expressing the far-red fluorescent protein katushka instead of the deleted nss protein (rrvfvδnss::katushka) was used in this study (islam et al., 2018) . the virus stocks were stored at −80°c. all the experiments with viruses were performed in compliance with the guidelines of the national authorities using appropriate biosafety laboratories under appropriate ethical and safety approvals. fluav virus was titrated in mdck cells using plaque assay as described previously (denisova et al., 2012) . ev1 titers were determined by plaque assay on a549 cells, as described earlier (marjomaki et al., 2002) . zikv titers were determined by plaque assay on vero-e6 cells, as described earlier (kuivanen et al., 2017) . hsv-1 titers were determined by plaque titration in vero cells in the presence of human immunoglobulin g (20 μg/ml), as described earlier (paavilainen et al., 2016) . chikv-2sg-nanoluc and rrv-2sg-nanoluc were titrated in bhk-21 cells using plaque assay, as described previously (oo et al., 2018; taylor et al., 2016) . for testing the viability and death of compound-treated mock-, fluav-, ev1-, chikv-2sg-nanoluc-, rrv-2sg-nanoluc-, zikv-and hsv1-infected cells, approximately 4 × 10 4 rpe cells were seeded in each well of a 96-well plate. the cells were grown for 24 h in cell growth medium. the media was replaced with virus growth medium (vgm) containing 0.2% bsa, 2 mm l-glutamine, 0.35% nahco3, and 1 μg/ml l-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (tpck)-trypsin (sigma-aldrich) in dmem-f12. in the case of zikv, the media was replaced with dmem-f12 medium supplemented with 50 u/ ml penstrep, 2 mm l-glutamine, 2% fbs, and 0.25% sodium bicarbonate. the compounds were added to the cells in three-fold dilutions at seven different concentrations starting from 30 μm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. when virus induced a cytopathic effect in cells (typically 24-72 hpi), celltox green express cytotoxicity reagent (ctxg, 1:2000 dilution in the assay well, promega, madison, wi, usa) was added in vgm and the fluorescence was measured with a pherastar fs plate reader (bmg labtech, ortenberg, germany) or hidex sense microplate reader (hidex oy, turku, finland). the media was removed from the cells and stored at −80°c. celltiter-glo viability reagent (ctg, promega, madison, wi, usa) containing firefly luciferase and luciferin was added (30 μl per well). the luminescence was measured with a pherastar fs plate reader. for testing virus titers in compound-treated and non-treated fluav-, ev1-, zikv-and hsv1-infected cells the media was collected, serially diluted in pbs, and added to mdck (fluav), a549 (ev1), and vero-e6 (zikv and hsv-1) cells. the media was changed, and the cells were overlaid with plaque assay media. the cells were fixed, and viral titers were calculated. the titers were expressed as plaque-forming units per ml (pfu/ml). the presence of chikv-2sg-nanoluc and rrv-2sg-nanoluc in the media from non-or drug-treated rpe cells was evaluated by passaging the viruses in fresh rpe cells. the viruses expressed nano-luciferase from viral promoter, which was measured from lysed cells 8 h post infection using renilla luciferase assay (promega, madison, wi, usa). for testing compound toxicity and efficacy against hiv-1, approximately 4 × 10 4 tzm-bl cells were seeded in each well of a 96-well plate. tzm-bl cells express firefly luciferase under control of hiv-1 ltr promoter allowing quantitation of the viral infection (tat-protein expression by integrated hiv-1 provirus) using firefly luciferase assay. the cells were grown for 24 h in cell growth medium. compounds were added to the cells in three-fold dilutions at seven different concentrations starting from 30 μm. no compounds were added to the control wells. the cells were infected with hiv-1 (media that contained 30 ng of hiv-1 p24 was used per well) or mock. at 48 hpi, celltox green express cytotoxicity reagent (ctxg, 1:2000 dilution in the assay well) was added and the fluorescence was measured with a pherastar fs plate reader. the media was removed from the cells, the cells were lysed, and firefly luciferase activity was measured using the luciferase assay system (promega, madison, wi, usa) and pherastar fs plate reader. to determine the efficacy of compounds against rvfv, the fluorescent intensity of individual infectious cell foci was quantified. briefly, a549 cells (1 × 10 4 /well) were grown in 96-well black plates with transparent bottoms (greiner bio-one international). compounds were serially diluted in dmso in two-fold steps from 100 μm to 0.39 μm and mixed with 3000 pfu of rrvfvδnss::katushka virus in a total volume of 100 μl medium (moi, 0.3). the final concentration of dmso in the assay was 1%. the growth medium was removed from the wells and 100 μl of compound and virus mixture was added to the cells for each compound concentration. at 16 hpi the medium was removed, and cells were fixed with 3% paraformaldehyde (pfa) for 1 h and washed with phosphate-buffered saline (pbs). 300 μl pbs was added to each well and the plate was analyzed in the trophos plate runner hd (trophos, roche group) to count the number of virus infected cells per well, by identifying all individual cells expressing the far-red fluorescent protein katushka (islam et al., 2018) . the toxicity of compounds was analysed using ctg assay. the half-maximal cytotoxic concentration (cc50) and the halfmaximal effective concentration (ec50) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version 7.0a (graphpad software la jolla, ca, usa) or using hill curve fit with sigmastat 4.0 software (spss inc., chicago, il, usa). the relative effectiveness of drugs were quantified as the selectivity indexes (si = cc50/ec50). there is limited information available on approved, investigational and experimental bsas. to identify all potential bsas, we reviewed approved, investigational and experimental safe-in-human antiviral agents in drug bank, clinical trials websites and pubmed, respectively. first, we searched for drugs, which have been approved for treatment of viral diseases in human. by excluding vaccines and discontinued drugs, we identified 86 active antiviral substances. these compounds target 30 viral, 6 human and 3 unknown factors and inhibit 17 viruses (table s2 ). the approved antivirals include 5 neutralizing antibodies, 3 interferons, 1 antisense oligonucleotide, and 76 small molecules. only 9 of the 76 molecules target viruses or host factors associated with ≥3 viral diseases. fig. 1 shows bsas and other approved antiviral drugs linked to viral and host targets through viruses they inhibit. next, we reviewed all investigational antivirals and their safety profiles. by excluding vaccines from the search parameters, we found 116 active compounds. these compounds target 42 viral, 29 human and 7 unknown factors and inhibit 19 viruses (table s3 ). the drug candidates include 2 neutralizing antibodies, 6 antisense oligonucleotides, 5 interferons, 1 enzyme and 100 small molecules. only 4 of the 100 small molecules target ≥3 viral diseases. fig. 2 shows these and other investigational antiviral agents linked to cellular and host factors through targeted viruses. next, we searched for drugs, which have been approved for treatment of non-viral diseases, but for which antiviral activity has been reported. we found 156 active compounds, which are all small-molecules. these compounds target 32 viral, 87 human and 3 unknown factors, and inhibit 53 human viruses (table s4) . thirty-nine of the 155 antiviral agents target ≥3 viral diseases. fig. 3 shows these and other experimental antiviral agents connected to their host and viral targets through viruses they inhibit. altogether, we identified 339 antiviral agents with available safety information in humans. these agents target 109 host, 76 viral and 13 unknown factors and inhibit 58 different viruses, belonging to 20 viral families. fifty-nine agents have bsa activity (table s5 ). these 59 agents target 55 of the 58 reported viruses (except andv, emcv and rabv) (fig. s1 ). structural analysis of bsa revealed that several drugs (such as, acyclovir, famciclovir and valacyclovir; imatinib and disatinib; minocyclin and doxycycline; ritonavir and lopinavir; hydroxychloroquine and chloroquine; esomeprazole and omeprazole) have similar scaffolds (fig. s2) . these drugs target a limited number of host and viral factors (fig. s3 ). in particular, statins (fluvastatin, lovastatin and simvastatin) target human hmg-coa; dalbavancin, oritavancin, teicoplanin and telavancin target human cyp3a4; acyclovir, valacyclovir, famciclovir, azacitidine, brincidofovir, cidofovir, foscarnet, trifluridine, and vidarabine inhibit viral dna polymerases; bcx4430, favipiravir and ribavirin inhibit viral rna polymerases; and lamivudine and tenofovir disoproxil inhibit viral reverse transcriptases. we hypothesized that approved, investigational and experimental antiviral agents, which target ≥3 viral diseases, could inhibit other viral infections. as a proof of concept, we tested 55 of the 59 identified bsas against fluav, ev1, chikv, rrv and hsv-1 infections in rpe cells (alisporivir, cyt107, sunitinib and thymalfasin were excluded; table s6 ). we also tested 52 of the 59 bsas against zikv infection in rpe cells (alisporivir, cyt107, sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded). we evaluated viability and death of virus/mock-infected cells using ctg and ctxg assays, respectively. the ctg assay quantifies atp, an indicator of metabolically active living cells, whereas ctxg assay uses fluorescent cyanine dye that stains the dna of dead cells. after initial screening we found several hits, which kept infected cells alive or rescued infected cells from virus-mediated death. these hits are gemcitabine, gefitinib and vibarabine (fluav); gemcitabine, pirlindole dibucaine, fluoxetine and dalbavancin (ev1); gemcitabine, imatinib, ivermectin, lopinavir, lovastatin, ezetimibe, fluoxetine, bcx4430, chloroquine and hydroxychloroquine (zikv); chloroquine and mycophenolic acid (chikv); chloroquine, mycophenolic acid, dibucaine and itraconazole (rrv); as well as 5-azacitidine, gemcitabine, trifluridine and vidarabine (hsv-1). we repeated the ctg and ctxg assays with selected compounds and titrated fluav, ev1, zikv and hsv-1 produced in drug-treated and non-treated cells. the presence of chikv and rrv viruses in the media collected from non-or drug-treated rpe cells was evaluated by infecting fresh rpe cells and measuring reporter protein expression from viral promoter (nanoluciferase activity). these experiments confirmed antiviral activity of gemcitabine against fluav (fig. s4) ; gemcitabine, pirlindole, dibucaine, fluoxetine, and dalbavancin against ev1 (fig. s5) ; gemcitabine, lopinavir, ezetimibe, bcx4430, chloroquine, and hydroxychloroquine against zikv (fig. s6) ; chloroquine and mycophenolic acid against chikv (fig. s7) ; mycophenolic acid against rrv (fig. s8) ; and gemcitabine against hsv-1 (fig. s9) . importantly, dalbavancin and ezetimibe demonstrated novel antiviral activities against ev1 and zikv, respectively. in particular, they rescued infected rpe cells from virus-mediated cell death, and lowered production of infectious virus particles without detectable cytotoxicity (table 1) . we also examined toxicity and antiviral activity of 55 of the 59 bsa agents (excluding alisporivir, cyt107, sunitinib and thymalfasin) against hiv-1 infection in tzm-bl cells. our primary screen identified ezetimibe, minocycline and rapamycin as anti-hiv-1 agents. validation experiment confirmed all three hits (fig. s10, table 1 ). interestingly, ezetimibe is a novel inhibitor, whereas minocycline and rapamycin are known anti-hiv-1 agents (heredia et al., 2015; singh et al., 2014) . in addition, we examined antiviral activity and toxicity of 53 of the 59 bsa agents (alisporivir, cyt107, sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded) against rvfv expressing the far-red fluorescent protein katushka in a549 cells. our screen identified azacitidine, bortezamibe, cyclosporine, doxycycline, ezetimibe, fluoxetine, gefitinibe, minocycline, oritavancin, ritonavir and topotecan. azacitidine, cyclosporine, minocycline, oritavancin, ritonavirand and bortezamib remained after excluding compounds with si < 2 ( fig. s11; table 1 ). interestingly, azacitidine, cyclosporine, minocycline, oritavancin and ritonavirand are novel, whereas bortezamib is a known inhibitor of rvfv infection (keck et al., 2015) . thus, we tested several known bsa agents against (−)ssrna, (+) ssrna, ssrna-rt and dsdna viruses and identified novel activities for dalbavancin against ev1, ezetimibe against zikv and hiv-1, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rvfv. fig. 4 shows known, validated and novel interactions between bsas and viruses. several approved and investigational agents, as well as safe in man chemical probes, were discovered for potential treatment of various viral diseases (bekerman and einav, 2015; de clercq, 2015; de clercq and li, 2016) . re-purposing such therapeutics from one viral disease to another could save resources and time needed for development of novel drugs. in this study, we tested 55 approved, investigational and experimental bsa agents against fluav, rvfv, ev1, zikv, chikv, rrv, hiv-1 and hsv-1 in vitro. we identified novel antiviral activities for dalbavancin (against ev1), ezetimibe (against hiv-1 and zikv), azacitidine, cyclosporine, minocycline, oritavancin and ritonavir (against rvfv) (fig. 4) . dalbavancin is a lipoglycopeptide antibiotic, which is approved by the fda for the treatment of acute bacterial skin infections caused by staphylococcus aureus and streptococcus pyogenes. dalbavancin also inhibits mers-cov and sars-cov infections. it binds cathepsin l in the late endosomes/lysosomes and blocks the entry of ebov (zhou et al., 2016) . our study demonstrates that it also inhibits replication of ev1, which also enters the cells via an endocytic route (krieger et al., 2013) . ezetimibe is an fda-approved medication that lowers plasma cholesterol by decreasing its absorption in the small intestine. ezetimibe also inhibits hbv and hdv infections by impairing viral entry mediated by pres1-specific receptor hntcp (blanchet et al., 2014; lempp and urban, 2014; monrroy-bravo et al., 2016) . our study shows that ezetimibe also inhibited hiv-1 and zikv infections. however, the entry of these viruses into the cell is mediated by other receptors (hamel et al., 2015; wilen et al., 2012) . most probably, the anti-hiv-1, ev1, as well as hcv action of ezetimibe is associated with depletion of cholesterol which is required for entry of these viruses into the host cells (sainz et al., 2012) . importantly, this drug was successfully tested in combination with antiretroviral therapeutics to lower cholesterol levels in serum of hiv-infected patients (saeedi et al., 2015; wohl et al., 2008) . the question remains whether ezetimibe alone reduces hiv titers in these patients. azacitidine is a chemical analogue of cytidine, which is used in the treatment of myelodysplastic syndrome. it also inhibits fluav, adv, hiv-1 and hiv-2 replication by blocking viral rna or dna synthesis (beach et al., 2014; rawson et al., 2016) . cyclosporine is an immunosuppressive agent used for the treatment of rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome and keratoconjunctivitis sicca. in addition, cyclosporine is used to prevent graft rejection in organ transplant recipients. cyclosporine also inhibits hcv, fluav, wnv and zikv replication through blocking interaction of cellular cyclophilins with viral proteins and attenuating viral rna synthesis (barrows et al., 2016; firpi et al., 2006; qing et al., 2009) . minocycline is a broad-spectrum antibiotic and antiviral agent, which possesses activity against denv, hiv-1 and wnv (leela et al., 2016; quick et al., 2017; singh et al., 2014) . oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections (zhou et al., 2016) . ritonavir is an antiretroviral medication. it has also antiviral activity against mers-cov (chan et al., 2015) . we showed that azacitidine, cyclosporine, minocycline, oritavancin and ritonavir are active against rvfv. we also confirmed previously reported activities of chloroquine against chikv and zikv, mycophenolic acid against chikv and rrv, fluoxetine, pirlindole and dibucaine against ev1, bcx4430, lopinavir and hydroxichloroquine against zikv, as well as gemcitabine against ev1, fluav, zikv and hsv-1 (cao et al., 2017; delogu and de lamballerie, 2011; delvecchio et al., 2016; denisova et al., 2012; julander et al., 2017; kang et al., 2015; khan et al., 2011; kuivanen et al., 2017; lee et al., 2017; soderholm et al., 2016; ulferts et al., 2016; yuan et al., 2017) (fig. 4) . the number of compounds with novel and confirmed antiviral properties may have been higher if we had used other cell lines, other viruses and viral strains, concentration ranges and purity of compounds, as well as endpoint measurements. also testing other compounds, which target < 3 viral diseases, could reveal novel antiviral properties of these compounds and increase the number of potential bsas. for conducting this properly, a harmonized antiviral bioactivity data annotation, standardization, curation, and intra-resource integration is needed. excellent antiviral profiles from cell-line-based assays might not be reflected in vivo because systemic mechanisms may compensate the blocked target effect. therefore, the identification of bsa targets that are essential for viral replication but redundant for the cell is critical for reducing putative toxicities associated with blocking cellular pathways. thus, follow-up mechanistic studies and in vivo experiments are needed to validate our in vitro results. in conclusion, repositioning of safe-in-man agents from one viral disease to another could play a pivotal role in development of broadly acting antivirals. our study demonstrated the potential value of such approach with some examples, such as dalbavancin, ezetimibe, azacitidine, cyclosporine, minocycline, oritavancin and ritonavir, confirming a principle that "rich becomes richer". effective bsa treatment may shortly become available, pending the results of further pre-clinical studies and clinical trials. the most effective and tolerable compounds will expand the available therapeutics for the treatment of viral diseases. some of these compounds could be used as first-line therapeutics to combat emerging and re-emerging viral threats, having global impact by improving preparedness and the protection of the general population from viral epidemics and pandemics. the authors declare no financial and non-financial competing interests. half-maximal cytotoxic concentration (cc50), the half-maximal effective concentration (ec50) and minimal selectivity indexes (si) for selected broad-spectrum antivirals. ctxg -celltox green express cytotoxicity assay, ctg -celltiter-glo viability assay, otherplaque assay or reporter gene expression assay, n.a.not available. fig. 4 . the interaction network between 55 viruses and 59 bsas, which are safe in man. drug-like shapes represent antiviral agents. blue spheres represent viruses. the diameter of spheres corresponds to the number of interactions between the viruses and the drugs. novel interactions between bsas and viruses are shown in red, validatedin blue, and known -in grey. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases insights into the mechanism of action of cidofovir and other acyclic nucleoside phosphonates against polyoma-and papillomaviruses and non-viral induced neoplasia a screen of fda-approved drugs for inhibitors of zika virus infection novel inhibitors of human immunodeficiency virus type 2 infectivity infectious disease. combating emerging viral threats anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects statins in hiv-infected patients: potential beneficial effects and clinical use use of fda approved therapeutics with hntcp metabolic inhibitory properties to impair the hdv lifecycle broad-spectrum agents for flaviviral infections: dengue, zika and beyond inhibition of autophagy limits vertical transmission of zika virus in pregnant mice treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset clinical trial resources global, regional, and national disability-adjusted life-years (dalys) for 333 diseases and injuries and healthy life expectancy (hale) for 195 countries and territories, 1990-2016: a systematic analysis for the global burden of disease study curious (old and new) antiviral nucleoside analogues with intriguing therapeutic potential approved antiviral drugs over the past 50 years alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model the future of antivirals: broad-spectrum inhibitors chikungunya disease and chloroquine treatment chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the global burden of disease study zika virus infection with prolonged maternal viremia and fetal brain abnormalities infectious disease. old drugs losing effectiveness against flu; could statins fill gap? cyclosporine suppresses hepatitis c virus in vitro and increases the chance of a sustained virological response after liver transplantation biology of zika virus infection in human skin cells targeting of mtor catalytic site inhibits multiple steps of the hiv-1 lifecycle and suppresses hiv-1 viremia in humanized mice a dna transfection system for generation of influenza a virus from eight plasmids pleiotropic mechanisms of ribavirin antiviral activities emerging virus diseases: can we ever expect the unexpected? anti-rift valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broadacting antiviral compound efficacy of the broad-spectrum antiviral compound bcx4430 against zika virus in cell culture and in a mouse model mutations in nsp1 and pe2 are critical determinants of ross river virus-induced musculoskeletal inflammatory disease in a mouse model synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses characterizing the effect of bortezomib on rift valley fever virus multiplication cellular impdh enzyme activity is a potential target for the inhibition of chikungunya virus replication and virus induced apoptosis in cultured mammalian cells echovirus 1 entry into polarized caco-2 cells depends on dynamin, cholesterol, and cellular factors associated with macropinocytosis obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism valaciclovir for chronic hepatitis b virus infection after lung transplantation gemcitabine, a broadspectrum antiviral drug, suppresses enterovirus infections through innate immunity induced by the inhibition of pyrimidine biosynthesis and nucleotide depletion drug repurposing of minocycline against dengue virus infection inhibitors of hepatitis b virus attachment and entry internalization of echovirus 1 in caveolae prevention and treatment of respiratory viral infections: presentations on antivirals, traditional therapies and host-directed interventions at the 5th isirv antiviral group conference effect of ezetimibe in hcv viral load after liver transplantation a novel open and infectious form of echovirus 1 a herpes simplex virus-derived replicative vector expressing lif limits experimental demyelinating disease and modulates autoimmunity deciphering the potential of baicalin as an antiviral agent for chikungunya virus infection inhibition of clinical pathogenic herpes simplex virus 1 strains with enzymatically created sirna pools cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral ns5 protein minocycline has anti-inflammatory effects and reduces cytotoxicity in an ex vivo spinal cord slice culture model of west nile virus infection c-spade: a web-tool for interactive analysis and visualization of drug screening experiments through compound-specific bioactivity dendrograms synergistic reduction of hiv-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase lipid lowering efficacy and safety of ezetimibe combined with rosuvastatin compared with titrating rosuvastatin monotherapy in hiv-positive patients identification of the niemann-pick c1-like 1 cholesterol absorption receptor as a new hepatitis c virus entry factor repurposing of kinase inhibitors as broad-spectrum antiviral drugs minocycline attenuates hiv-1 infection and suppresses chronic immune activation in humanized nod/ltsz-scidil-2rgamma(null) mice immuno-modulating properties of saliphenylhalamide, sns-032, obatoclax, and gemcitabine directly acting antivirals against hepatitis c virus effects of an in-frame deletion of the 6k gene locus from the genome of ross river virus screening of a library of fda-approved drugs identifies several enterovirus replication inhibitors that target viral protein 2c versatile transreplication systems for chikungunya virus allow functional analysis and tagging of every replicase protein antiviral agents for herpes simplex virus design, discovery, modelling, synthesis, and biological evaluation of novel and small, low toxicity s-triazine derivatives as hiv-1 non-nucleoside reverse transcriptase inhibitors who publishes list of top emerging diseases likely to cause major epidemics hiv: cell binding and entry. cold spring harb ezetimibe alone reduces low-density lipoprotein cholesterol in hiv-infected patients receiving combination antiretroviral therapy structure-based discovery of clinically approved drugs as zika virus ns2b-ns3 protease inhibitors that potently inhibit zika virus infection in vitro and in vivo glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) this study was supported by the european regional development fund, the mobilitas pluss project mobtt39 (to denis e. kainov), jane and aatos erkko foundation grant #170046 (to the vejo hukkanen). we thank qiuwei abdullah pan from the department of gastroenterology and hepatology, erasmus mc, university medical center rotterdam for advice. we thank ritva kajander for the hsv-1 culture. supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.antiviral.2018.04.016. key: cord-292286-ygomb3oi authors: zakaryan, hovakim; arabyan, erik; oo, adrian; zandi, keivan title: flavonoids: promising natural compounds against viral infections date: 2017-05-25 journal: arch virol doi: 10.1007/s00705-017-3417-y sha: doc_id: 292286 cord_uid: ygomb3oi flavonoids are widely distributed as secondary metabolites produced by plants and play important roles in plant physiology, having a variety of potential biological benefits such as antioxidant, anti-inflammatory, anticancer, antibacterial, antifungal and antiviral activity. different flavonoids have been investigated for their potential antiviral activities and several of them exhibited significant antiviral properties in in vitro and even in vivo studies. this review summarizes the evidence for antiviral activity of different flavonoids, highlighting, where investigated, the cellular and molecular mechanisms of action on viruses. we also present future perspectives on therapeutic applications of flavonoids against viral infections. throughout human history, thousands of biologically active plants have been identified and used in medicine. virtually all cultures around the world continue to rely on medicinal plants for primary health care. according to the world health organization report, about 80% of the world's population depend on medicinal plants to satisfy their health requirements [30] . furthermore, there are currently hundreds of modern drugs based on active compounds isolated from plants. plants have the ability to produce a wide range of compounds including flavonoids, phytoalexins, lignans, and tannins, which are responsible for key functions in plant growth and development. flavonoids or polyphenolics comprise the largest group of secondary metabolites found in vegetables, fruits, seeds, nuts, spices, stems as well as in red wine and tea (table 1 ) [88] . these compounds are synthesized in response to various abiotic stress conditions such as ultraviolet radiation and play an important role as defense agents against plant pathogens and insects [9, 84] . the first evidence of a biological activity of flavonoids was reported by albert szent-gyorgyii in 1938, who showed that citrus peel flavonoids prevent capillary bleeding and fragility associated with scurvy [109] . since then, a broad spectrum of biological activities such as anti-inflammatory, antioxidant, antibacterial, antiviral, anticancer, and neuroprotective has been described for flavonoids [40, 53, 65, 95, 137] . research for antiviral agents isolated from plants started in 1950s, when the activity of 288 plants against influenza a virus was evaluated in embryonated eggs [14] . during the last 60 years, several plants and plant-derived compounds with antiviral properties were identified. in this article, we review the results of both in vitro and in vivo experiments demonstrating the antiviral activity of flavonoids, especially focusing on those classes of flavonoids that have been extensively investigated. there are now more than 6000 varieties of flavonoids that have been structurally identified [35] . all these compounds comprise a flavan nucleus and a fifteen-carbon skeleton consisting of two benzene rings (a-and b-rings, as shown in fig. 1 ) connected via a heterocyclic pyrene ring (c-ring, as shown in fig. 1 ). flavonoids are divided into several classes such as anthocyanidins, flavones, flavonols, flavanones, flavan, isoflavanoids, biflavanoids, etc (table 1 ) [24] . the various classes of flavonoids differ in the level of oxidation and pattern of substitution of the pyrene ring, whereas individual compounds within the classes differ in the pattern of substitution of benzene rings. while in flavonoids the b-ring links to the c-ring at the c2 position, the b-ring of isoflavonoids is substituted at position c3 (fig. 1 ). biflavonoids comprise of two identical or non-identical flavonoid units conjoined through an alkyl-or alkoxybased linker (fig. 1) . in plants, flavonoids generally occur as aglycones, glycosides and methylated derivatives. they are biosynthesized through the phenylpropanoid pathway, transforming phenylalanine into 4-coumaroyl-coa, which then enters the flavonoid biosynthesis pathway [32] . depending on the plant species, a group of enzymes, such as hydroxylases and reductases, modify the basic flavonoid skeleton, resulting in the different flavonoid classes. finally, transferases modify the flavonoid skeleton with sugar, methyl groups and acyl moieties. these modifications alter the solubility and reactivity of flavonoids [6] . a large body of evidence supports the role of light in the regulation of flavonoid biosynthesis [156] . flavones constitute a major class in the flavonoid family based on a 2-phenyl-1-benzopyran-4-one backbone. natural flavones include apigenin, baicalein, chrysin, luteolin, scutellarein, tangeritin, wogonin and 6-hydroxyflavone. the antiviral activity of flavones is known from the 1990s, when it was showed that the simultaneous application of apigenin with acyclovir resulted in an enhanced antiviral effect on herpes simplex virus types 1 and 2 (hsv-1 and hsv-2) in cell culture [92] . apigenin is most commonly isolated in abundance from the family asteraceae. the organic and aqueous extracts from asteraceae plants with apigenin as a major compound were found to be active against hsv-1, poliovirus type 2 and hepatitis c virus (hcv) [85, 127] . apigenin isolated from sweet basil (ocimum basilicum) showed a potent antiviral activity against adenoviruses (adv) and hepatitis b virus in vitro [17] . besides these dna viruses, apigenin was found to exert antiviral effect against african swine fever virus (asfv), by suppressing the viral protein synthesis and reducing the asfv yield by 3 log [46]. apigenin is also active against rna viruses. for picronaviruses, it has been shown that apigenin is able to inhibit viral protein synthesis through suppressing viral ires activity [82, 107] . furthermore, apigenin affects enterovirus-71 (ev71) translation by disrupting viral rna association with trans-acting factors regulating ev71 translation [153] . shibata et al. [115] showed that apigenin has antiviral effect on hcv through the reduction of mature microrna122, a liver-specific microrna which positively regulates hcv replication. among flavones, baicalein and luteolin have been also extensively investigated with respect to their antiviral activity. baicalein significantly reduced the levels of human cytomegalovirus (hcmv) early and late proteins, as well as viral dna synthesis, although it had no effect on viral polymerase activity [23, 31] . baicalein impaired avian influenza h5n1 virus replication in both human lung epithelial cells and monocyte-derived macrophages by interfering with neuraminidase activity [116] . other studies showed that oral administration of baicalein to balb/c mice infected with influenza h1n1 virus decreased the lung virus titer and increased the mean time to death [139] . similar effects were recorded on mice infected with sendai virus [28] . these inhibitory effects in vivo were mediated by serum baicalin, a metabolite of baicalein which has a glucose residue [26] . baicalin alone exerts its anti-influenza activity by modulating the function of ns1 protein, which down-regulates ifn induction [99] . further studies indicated that baicalin can directly induce ifn-c production in human cd4 ? t cells and cd8 ? t cells and act as a potent inducer of ifn-c during influenza virus infection [19] . recently, novel baicalein analogs with b-rings substituted with bromine atoms demonstrated extremely potent activity against influenza h1n1 tamiflu-resistant virus, indicating that baicalein and its analogs can be favorable alternatives in the management of tamiflu-resistant viruses [21] . in vitro replication of hiv-1 was suppressed by baicalin when infected cells were treated during the early stage of the virus replication cycle [66] . hiv-1 envelope protein was found to be the target site of baicalin's antiviral action via the interference of interactions between the virus structural protein and specific host immune cells [75] . baicalein and baicalin were also investigated against dengue virus (denv). they exerted a significant virucidal effect on extracellular viral particles and interfered with different steps of denv-2 replication [91, 148, 150] . in silico studies revealed that baicalein has strong binding affinity with denv ns3/ns2b protein (-7.5 kcal/mol), and baicalin may interact closely with the virus ns5 protein at a binding affinity of -8.6 kcal/mol [47] . for baicalin, computational studies also showed a high binding affinity (-9.8 kcal/mol) against chikungunya virus (chikv) nsp3 protein, suggesting that baicalin can potentially interfere with chikv infection [114] . it was found that luteolin has antiviral effect on hiv-1 reactivation by blocking both clade b-and c-tat-driven ltr transactivation [87] . luteolin also showed significant inhibition of epstein-barr virus (ebv) reactivation in cells [133] ; it suppressed the activities of the immediate-early genes zta and rta by deregulating transcription factor sp1 binding. xu et al. [142] tested 400 highly purified natural compounds for inhibition of ev71 and coxsackievirus a16 infections and found that luteolin exhibited the most potent inhibition through disruption of viral rna replication. besides these antiviral activities, luteolin or luteolin-rich fractions showed antiviral effects against severe acute respiratory syndrome coronavirus (sars-cov), rhesus rotavirus, chikv and japanese encephalitis virus (jev) [33, 67, 94, 146] . flavonols are characterized by a 3-hydroxy-2-phenylchromen-4-one backbone. among flavonols the antiviral effect of quercetin was the most extensively investigated. early in vivo studies showed that oral treatment with quercetin protected mice from lethal mengo virus [44, 125] . furthermore, an enhanced protection was observed when quercetin was administered in combination with murine type i interferon (ifn) [125] . quercetin also demonstrated a dose-dependent antiviral activity against poliovirus type 1, hsv-1, hsv-2, and respiratory syncytial virus (rsv) in cell cultures [60, 83] . epimedium koreanum nakai, which contains quercetin as the major active component, has been shown to induce secretion of type i ifn, reducing the replication of hsv, newcastle disease virus (ndv), vesicular stomatitis virus (vsv) in vitro, as well as influenza a subtypes (h1n1, h5n2, h7n3 and h9n2) in vivo [18] . hung et al. [51] have suggested possible mechanisms whereby quercetin may exert its anti-hsv activity. they revealed that quercetin inhibits the infection of hsv-1, hsv-2 and acyclovirresistant hsv-1 mainly by blocking viral binding and penetration to the host cell. they also reported that quercetin suppresses nf-jb activation, which is essential for hsv gene expression. recent investigations also pointed out the antiviral activity of quercetin against a wide spectrum of influenza virus strains. it interacts with influenza hemagglutinin protein, thereby inhibiting viralcell fusion [136] . in addition, in silico analysis revealed that quercetin may be a potential inhibitor of the neuraminidase of influenza a h1n1 and h7n9 viruses [79, 80] . molecular docking analysis also found that quercetin may interact with hcv ns3 helicase, ns5b polymerase and p7 proteins [34, 86] . these results correlate with experimental studies showing the anti-hcv activity of quercetin through inhibition of ns3 helicase and heat shock proteins [4, 81] . besides these viruses, the inhibitory activity of quercetin and its derivatives have been reported for other viruses, including advs, arthropod-borne mayaro virus, porcine reproductive and respiratory syndrome virus, canine distemper virus, jev, denv-2, porcine epidemic diarrhea virus, and equid herpesvirus 1 [11, 16, 27, 38, 41, 59, 118, 149] . quercetin also possesses anti-rhinoviral effects by inhibiting endocytosis, transcription of the viral genome and viral protein synthesis [37] . in mice infected with rhinovirus, quercetin treatment decreased viral replication and attenuated virusinduced airway cholinergic hyper-responsiveness [37] . kaempferol is another flavonol extracted from different medicinal herbs. kaempferol and its derivatives bearing acyl substituents have shown inhibitory activity against hcmv [89] . kaempferol derivatives isolated from ficus benjamina leaves were more effective against hsv-1 and hsv-2 than their aglycon form [145] . kaempferol derivatives with rhamnose residue turned out to be potent inhibitors of the 3a channel of coronavirus, which is involved in the mechanism of virus release [112] . one of the kaempferol derivative, kaempferol 3-o-a-lrhamnopyranoside, obtained from zanthoxylum piperitum was shown to significantly inhibit the replication of influenza a virus in vitro [45] . behbahani et al. found that kaempferol and kaempferol-7-o-glucoside have strong hiv-1 reverse transcriptase inhibitory activity [5] . these compounds exerted their effects, at a concentration of 100 lg/ml, on the early stage of hiv replication in target cells. recently, kaempferol-3,7-bisrhamnoside isolated from chinese medicinal taxillus sutchuenensis was shown to have potent in vitro activity on hcv ns3 protease function [144] . antiviral activity of kaempferol on the influenza viruses h1n1 and h9n2 were mentioned in a study conducted by a group of researchers in south korea. mechanistic and structural studies suggested that the compound acts on the virus neuraminidase protein and specific functional groups are responsible for kaempferol's efficacy [57] . a study comparing the antiviral activities of kaempferol and an isoflavone, daidzein, showed that kaempferol exerted more potent inhibitory activities on jev replication and protein expression, than daidzein. jev's frameshift site rna (fsrna) has been proposed as the target site for kaempferol's inhibitory activity against this flavivirus [152] . seo et al. conducted a study comparing the potency of different classes of flavonoids against two rna viruses, namely murine norovirus and feline calicivirus. their findings demonstrated that, among the flavonoids tested, kaempferol exhibited the most potent inhibitory activity against these two viruses [113] . there are number of other flavonols and derivatives acting as antivirals. for example, sulfated rutin, which is modified from glycoside rutin, demonstrated significant activity against different hiv-1 isolates [123] . this compound inhibited hiv-1 infection by blocking viral entry and virus-cell fusion, likely by interacting with hiv-1 envelope glycoproteins. rutin at 200 lm concentration was shown to inhibit ev71 infection by suppressing the activation of mek1-erk signal pathway, which is required for ev71 replication of [129] . rutin and fisetin also inhibited the replication of ev-a71 by affecting the enzymatic activity of the 3c protease [76] . fisetin treatment caused a dose-dependent decrease in the production of chikv nonstructural proteins and inhibition of viral infection [73] . moreover, zandi et al. showed that denv-2 rna copy number was significantly reduced following addition of fisetin to infected cells [149] . yu et al. found that myricetin may serve as chemical inhibitor of sarscoronavirus because it affects the atpase activity of the viral helicase [147] . flavans are characterized by a 2-phenyl-3,4-dihydro-2hchromene skeleton. these compounds include flavan-3-ols, flavan-4-ols and flavan-3,4-diols. among flavan-3-ols, the antiviral activity of catechin and its derivatives epicatechin, epicatechin gallate, epigallocatechin (egc), and epigallocatechin gallate (egcg), which are found in tea, has been largely investigated [122] . among different viruses studied as potential targets, influenza virus has received the most attention after an initial report by nakayama et al. showing that tea catechins, particularly egcg, are able to bind to the haemagglutinin of influenza virus, preventing its adsorption to madin-darby canine kidney cells [98] . furthermore, it has been suggested that egcg may be able to damage the physical properties of the viral envelope, resulting in the inhibition of hemifusion events between influenza virus and the cellular membrane [66] . recently, colpitts and schang reported that egcg competes with sialic acid for binding to influenza a virus, thereby blocking the primary low-affinity attachment to cells [22] . another tea catechin, egc, exerted the inhibitory effect on the acidification of endosomes and lysosomes, thereby reducing viral entry via clathrin-mediated endocytosis [52]. a structure-function relationship analysis of tea catechins revealed the important role of the 3-gallolyl group of the catechin skeleton for its antiviral activity [120] . the results also showed that modification of the 3-hydroxyl position significantly affected the antiviral activity. catechin derivatives containing carbon chains at 3-hydroxyl position demonstrated potent anti-influenza activity in vitro and in ovo [121] . several reports have demonstrated that tea catechins have an antiviral effect against hiv infection. among tea catechins, egcg is the most effective because it exerts its antiviral effect throughout several steps of the hiv-1 life cycle. it directly binds to cd4 molecules with consequent inhibition of gp120 binding, an envelope protein of hiv-1 [62, 134] . these studies identified trp69, arg59 and phe43 of cd4 as potential sites for interaction with the galloyl moiety of egcg. the same residues are involved in interaction with viral gp120 [135] . furthermore, early studies from nakane and ono showed that egcg and ecg were effective at inhibiting hiv-1 reverse transcriptase in vitro [96, 97] . tillekeratne et al. modified the molecular structure of egcg to determine the minimum structural characteristics necessary for hiv-1 reverse transcriptase inhibition [124] . in their study, the gallate ester moiety was found to be important for inhibition. besides these effects, egcg has the ability to reduce viral production in chronically infected monocytoid cells [143] . the inhibitory effect was increased by approximately 25%, when egcg was modified with lyposomes. tea catechins are also effective against herpesviruses. egcg has been shown to block ebv lytic cycle by inhibiting expression of viral genes including rta, zta and ea-d [13] . further studies indicated that one of the mechanisms by which egcg may inhibit ebv lytic cycle involves the suppression of mek/erk1/2 and pi3-k/akt signaling pathways, which are involved in the ebv lytic cycle cascade [78] . isaacs et al. found that egcg can inactivate hsv virions by binding to the envelope glycoproteins gb and gd, which are essential for hsv infectivity [54] . the egcg digallate dimers theasinensin a, p2, and theaflavin-3,3'-digallate inactivated hsv-1 and hsv-2 more effectively than did monomeric egcg [55]. these dimers are stable at vaginal ph, indicating their potential to be antiviral agents against hsv infections. the inhibitory effect of green tea extracts against hbv has been reported [140] . in hepg2.117 cells, egcg inhibited hbv replication through impairing hbv replicative intermediates of dna synthesis, thereby reducing the production of hbv covalently closed circular dna [48] . in contrast, huang et al. found that egcg decreased hbv entry into immortalized human primary hepatocytes by more than 80% but had no effect on hbv genome replication [50]. furthermore, egcg is able to enhance lysosomal acidification, which is an unfavorable condition for hbv replication [155] . besides these viruses, egcg has been found to exert antiviral activity against hcv by preventing the attachment of the virus to the cell surface and suppressing rna replication steps [8, 15] . a recent study also showed inhibitory activity of egcg against another flavivirus, zika virus (zikv): in this study, foci forming unit reduction assays were performed to evaluate the antiviral activity of egcg on zikv at different stages of virus replication. foci observed showed more than 90% inhibition when the cells were treated with egcg during virus entry [10] . similarly, egcg is able to block chikv attachment to target cells, but has no effect on other stages of infection [132] . naringenin, which belongs to the flavanones class, has been shown to reduce the replication of a neurovirulent strain of sindbis virus in vitro [102] . it also reduced sindbis virus-and semliki forest virus-induced cytopathic effect in virus yield experiments [105] . interestingly, naringin, the glycoside form of naringenin did not have anti-sindbis virus activity, indicating that the rutinose moiety of this flavanone blocks its antiviral effect. naringenin is also able to block the assembly of intracellular hcv particles and long-term treatment leads to 1.4 log reduction in hcv [39, 64] . the alphavirus chikv was effectively inhibited when infected vero cells were treated with naringenin at the post-entry stage. in the same study, hesperetin, another flavanone which is found richly in citrus fruits, was found to exert most potent anti-chikv effect during the virus intracellular replication, with an ic 50 of 8.5 lm [1] . molecular docking and molecular dynamics studies by oo et al. also revealed strong and stable interactions between hesperetin and chikv nonstructural protein 2 (nsp2) as well as non-structural protein 3 (nsp3), suggesting that these proteins may be the target of hesperetin's anti-chikv activity [101] . genistein is an isoflavonoid found in a number of plants including soybeans and fava beans. as a tyrosine kinase inhibitor, genistein reduced bovine herpesvirus type 1 and new world arenavirus pichinde replication, by preventing the phosphorylation of viral proteins [2, 126] . kinase inhibitor cocktails containing genistein displayed a broadspectrum antiviral activity against arenaviruses and filoviruses [68] . genistein was shown to inhibit hiv infection of resting cd4 t cells and macrophages through interference with hiv-mediated actin dynamics [42] . furthermore, it may act against hiv ion channel since it has the ability to block the viral vpu protein, which is believed to form a cation-permeable ion channel in infected cells [110] . genistein also exerted its antiviral effects on the replication of hsv-1, hsv-2, and avian leucosis virus subgroup j, by inhibiting virus transcription [3, 106] . the antiviral activity of other flavonoids is presented in table 2 . in spite of the wide range of biological health benefits which flavonoids possess, in addition to their high availability in humans' daily diets, there are challenges ahead for researchers before these natural compounds can be applied as therapeutic options in the clinical setting. bioavailability, defined by the us food and drug administration as ''the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action'', has been the main stumbling block to further advances in the potential use of flavonoids in the medical community. intake of metabolic derivatives of flavonoids from various food sources leads to relatively large differences in the final amount being successfully absorbed and utilized by humans [71] . factors such as molecular sizes, glycosylation, esterification, lipophilicity, interactions with the enteric microorganisms, pka, and other metabolic conjugations along the alimentary tract, affect the absorption and bioavailability of flavonoids in humans [49, 56, 69, 90, 93, 111, 133] . hence, efforts in enhancing the bioavailability of flavonoids upon intake by humans are vitally necessary in order to develop these natural compounds into potential antiviral drugs. the following are a few examples of efforts being carried out to tackle this issue which can be used as platforms for further successes in the future. in the past, researchers have looked into alternative methods to improve the compounds' solubility or to switch the site of absorption in the gut, with the aim of enhancing their bioavailability. a structural modification to hesperetin-7-glucoside, which resulted in a change in site of absorption from the large to the small intestine, has successfully yielded a higher plasma level of hesperetin in healthy subjects [100] . wang et al. [130] formulated a way to increase the oral bioavailability of flavonols extracted from sea buckthorn, by forming a phospholipid complex via solvent evaporation method. relative to the parent compounds, oral bioavailability of the tested flavonols was 172% -242% higher when the phospholipid complex was administered into rats [130] . flavonoids loaded in engineered nanoparticles have also been tested for their bioavailability following oral consumption. improved stability of catechin and egcg in chitosan nanoparticles have been shown to result in a higher rate of intestinal absorption [29] . poly (d, l-lactide) (pla) nanoparticles and polymeric micelles contributed to a more sustainable release of quercetin, which has poor bioavailability and undergoes substantial first-pass metabolism, as well as of the poorly absorbed apigenin [70, 124, 151] . self-microemulsifying drug delivery system (smdds) is another technology which has been used to overcome the problem of low bioavailability of hydrophobic molecules. upon entering the lumen of the intestine, an oil-in-water microemulsion containing the drug will be formed. the microemulsion increases the intestinal absorption of the drug or compound by avoiding the dissolution process [60, 108] . puerarin, an isoflavone isolated from the root of pueraria lobata, exhibited 2.6-fold higher bioavailability when prepared using smdds [154] . however, it is worth noting that while the bioavailability of flavonoids can be increased via different methodologies, it is vital that their biological efficacies are not affected, but maintained or enhanced. for instance, phosphorylated icariin has been found to inhibit duck hepatitis virus a more effectively than the parent compound [138] . isorhamnetin is a methylated flavonol derived from the structure of quercetin. dayem et al. investigated the antiviral potency of isorhamnetin against influenza a h1n1 virus and discovered that the methyl group on the b ring enhances its antiviral activity compared with the other tested flavonoids [25] . the efficacy of isorhamnetin against influenza virus was also shown when in vivo and in ovo models were tested [25] . improvement in bioavailability will definitely enhance the efficacy of different biological effects of all classes of flavonoids. hence, in addition to discovering the hidden potentials of flavonoids, scientists should also aim to identify ways to increase the amount of flavonoids available for the health benefits of human beings. natural compounds have been the center of attention among researchers working in various fields, including those related with antiviral drug development, due to their high availability and low side effects. the phytochemicals flavonoids, which are abundantly found in our daily diets of fruits and vegetables, have been actively studied as potential therapeutic options against viruses of different taxa in the past decade. numerous positive findings have been reported on the in vitro efficacy of flavonoids, but less promising results have been obtained for most compounds in in vivo studies. multiple factors contributed to this scenario, and in vivo studies must be prioritized by researchers. it is well-known that flavonoids possess enormous potential to be included in the daily prescriptions by physicians treating illnesses ranging from infectious and oncogenic to inflammatory and chronic degenerative diseases. however, it is time for researchers worldwide to take the initiative in making these compounds a success not only in the in vitro stage of research, but also in animal models, as well as in subsequent clinical studies. biochemistry and mechanistic studies on the flavonoids' inhibitory activities can improve our understanding of how these natural compounds work and, on the other hand, identify the stumbling block that is hindering further improvements in flavonoids antiviral research. (7) inhibition of chikungunya virus replication by hesperetin and naringenin effect of genistein on replication of bovine herpesvirus type 1 antiherpes evaluation of soybean isoflavonoids suppression of hepatitis c virus by the flavonoid quercetin is mediated by inhibition of ns3 protease activity in vitro anti-hiv-1 activities of kaempferol and kaempferol-7-o-glucoside isolated from securigera securidaca glycosyltransferases: managers of small molecules structural basis of inhibitor specificity of the human protooncogene proviral insertion site in moloney murine leukemia virus (pim-1) kinase epigallocatechin-3-gallate is a new inhibitor of hepatitis c virus entry flavonoids and melanins: a common strategy across two kingdoms the green tea molecule egcg inhibits zika virus entry in vitro inhibition of canine distemper virus by flavonoids and phenolic acids: implications of structural differences for antiviral design effect of naringenin, hesperetin and their glycosides forms on the replication of the 17d strain of yellow fever virus inhibition of epstein-barr virus lytic cycle by (-)-epigallocatechin gallate the action of plant extracts on a bacteriophage of pseudomonas pyocyanea and on influenza a virus epigallocatechin-3-gallate inhibits the replication cycle of hepatitis c virus in vitro antiviral activities of caesalpinia pulcherrima and its related flavonoids antiviral activities of extracts and selected pure constituents of ocimum basilicum epimedium koreanum nakai displays broad spectrum of antiviral activity in vitro and in vivo by inducing cellular antiviral state role of baicalin in anti-influenza virus a as a potent inducer of ifngamma inhibitory effects of flavonoids on moloney murine leukemia virus reverse transcriptase activity synthesis and anti-influenza activities of novel baicalein analogs a small molecule inhibits virion attachment to heparan sulfate-or sialic acid-containing glycans eight flavonoids and their potential as inhibitors of human cytomegalovirus replication the chemistry and biological effects of flavonoids and phenolic acids antiviral effect of methylated flavonol isorhamnetin against influenza antiviral activity of baicalin against influenza a (h1n1/ h3n2) virus in cell culture and in mice and its inhibition of neuraminidase quercetin and quercetin 3-o-glycosides from bauhinia longifolia (bong.) steud. show anti-mayaro virus activity effects of baicalein on sendai virus in vivo are linked to serum baicalin and its inhibition of hemagglutinin-neuraminidase chitosan nanoparticles enhance the intestinal absorption of the green tea catechins (?)-catechin and (-)-epigallocatechin gallate the growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety human cytomegalovirus-inhibitory flavonoids: studies on antiviral activity and mechanism of action flavonoids: biosynthesis, biological functions, and biotechnological applications antiviral activity ofl uteolin against japanese encephalitis virus docking studies of pakistani hcv ns3 helicase: a possible antiviral drug target structure and function of enzymes involved in the biosynthesis of phenylpropanoids inhibition of infectivity of potato virus x by flavonoids quercetin inhibits rhinovirus replication in vitro and in vivo inhibition of hsp70 reduces porcine reproductive and respiratory syndrome virus replication in vitro naringenin inhibits the assembly and long-term production of infectious hepatitis c virus particles through a ppar-mediated mechanism polyphenols as mitochondria-targeted anticancer drugs in vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1 genistein interferes with sdf-1-and hiv-mediated actin dynamics and inhibits hiv infection of resting cd4 t cells anti-hepatitis b virus activity of wogonin in vitro and in vivo effect of quercetin on the course of mengo virus infection in immunodeficient and normal mice. a histologic study antiviral effect of flavonol glycosides isolated from the leaf of zanthoxylum piperitum on influenza virus antiviral activity of flavonoids 2547 anti-h1n1 virus, cytotoxic and nrf2 activation activities of chemical constituents from scutellaria baicalensis antiviral activity of baicalein and quercetin against the japanese encephalitis virus development of self-microemulsifying drug delivery systems (smedds) for oral bioavailability enhancement of simvastatin in beagle dogs antiviral effect of flavonoids on human viruses epigallocatechin gallate, the main component of tea polyphenol, binds to cd4 and interferes with gp120 binding development of chemical inhibitors of the sars coronavirus: viral helicase as a potential target divergent antiviral effects of bioflavonoids on the hepatitis c virus life cycle fisetin: a dietary antioxidant for health promotion inhibition of influenza virus internalization by (-)-epigallocatechin-3-gallate an evaluation of the inhibitory effects against rotavirus infection of edible plant extracts inhibition of lassa virus and ebola virus infection in host cells treated with the kinase inhibitors genistein and tyrphostin chemistry and biological activities of flavonoids: an overview development of biodegradable nanoparticles for delivery of quercetin updated knowledge about polyphenols: functions, bioavailability, metabolism, and health antiviral activity of selected flavonoids against chikungunya virus -o-arylmethylgalangin as a novel scaffold for anti-hcv agents flavonoid baicalin inhibits hiv-1 infection at the level of viral entry fisetin and rutin as 3c protease inhibitors of enterovirus a71 structure-activity relationship of flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities epigallocatechin-3-gallate inhibition of epstein-barr virus spontaneous lytic infection involves erk1/2 and pi3-k/akt signaling in ebv-positive cells computational screen and experimental validation of anti-influenza effects of quercetin and chlorogenic acid from traditional chinese medicine molecular docking of potential inhibitors for influenza h7n9 quercetin: bioflavonoids as part of interferon-free hepatitis c therapy? apigenin inhibits enterovirus 71 replication through suppressing viral ires activity and modulating cellular jnk pathway antiherpetic activities of flavonoids against herpes simplex virus type 1 (hsv-1) and type 2 (hsv-2) in vitro phenolic acids act as signaling molecules in plant-microbe symbioses identification and evaluation of antihepatitis c virus phytochemicals from eclipta alba computational docking study of p7 ion channel from hcv genotype 3 and genotype 4 and its interaction with natural compounds a flavonoid, luteolin, cripples hiv-1 by abrogation of tat function the effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer evaluation of the antiviral activity of kaempferol and its glycosides against human cytomegalovirus metabolomics view on gut microbiome modulation by polyphenol-rich foods baicalin, a metabolite of baicalein with antiviral activity against dengue virus combined effects of flavonoids and acyclovir against herpesviruses in cell cultures absorption, excretion and metabolite profiling of methyl-, glucuronyl-, glucosyland sulpho-conjugates of quercetin in human plasma and urine after ingestion of onions anti-chikungunya activity ofl uteolin and apigenin rich fraction from cynodon dactylon neuroprotective effects of chrysin: from chemistry to medicine differential inhibition of hiv-reverse transcriptase and various dna and rna polymerases by somecatechin derivatives differential inhibitory effects of some catechin derivatives on the activities of human immunodeficiency virus reverse transcriptase and cellular deoxyribonucleic and ribonucleic acid polymerases inhibition of the infectivity of influenza virus by tea polyphenols antiviral activity of baicalin against influenza virus h1n1-pdm09 is due to modulation of ns1-mediated cellular innate immune responses bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, double-blind, crossover trial silico study on anti-chikungunya virus activity of hesperetin anti-sindbis activity of flavanones hesperetin and naringenin anti-hiv-1 activity of flavonoid myricetin on hiv-1 infection in a dual-chamber in vitro model plant derived compounds having activity against p388 and l1210 leukemia cells inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays genistein inhibits the replication of avian leucosis virus subgroup j in df-1 cells apigenin restricts fmdv infection and inhibits viral ires driven translational activity anti-inflammatory effect of quercetin-loaded microemulsion in the airways allergic inflammatory model in mice drugs of natural origin genistein as antiviral drug against hiv ion channel absorption and metabolism of polyphenols in the gut and impact on health kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus comparison of the antiviral activity of flavonoids antiviral activity of flavonoids against murine norovirus and feline calicivirus computational approach towards exploring potential anti-chikungunya activity of selectedflavonoids the flavonoid apigenin inhibits hepatitis c virus replication by decreasing mature microrna122 levels differential antiviral and anti-inflammatory mechanisms of the flavonoids biochanin a and baicaleinin h5n1 influenza a virus-infected cells antiviral activity of chrysin derivatives against coxsackievirus b3 in vitro and in vivo quercetin 7-rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species silymarin efficacy against influenza a virus replication antiviral effect of catechins in green tea on influenza virus biological evaluation of anti-influenza viral activity of semi-synthetic catechin derivatives tea catechins as a potential alternative anti-infectious agent in vitro anti-hiv and -hsv activity and safety of sodium rutin sulfate as a microbicide candidate simplified catechin-gallate inhibitors of hiv-1 reverse transcriptase synergistic action of quercetin and murine alpha/beta interferon in the treatment of mengo virus infection in mice genistein treatment of cells inhibits arenavirus infection in vitro antiviral activity of plant extracts from asteraceae medicinal plants multiple effects of silymarin on the hepatitis c virus lifecycle saururus chinensis (lour.) baill blocks enterovirus 71 infection by hijacking mek1-erk signaling pathway a phospholipid complex to improve the oral bioavailability of flavonoids antienterovirus 71 effects of chrysin and its phosphate ester the green tea catechin, epigallocatechin gallate inhibits chikungunya virus infection colonic metabolites of berry polyphenols: the missing link to biological activity? epigallocatechin gallate, the main polyphenol in green tea, binds to the t-cell receptor, cd4: potential for hiv-1 therapy kinetic and structural analysis of mutant cd4 receptors that are defective in hiv gp120binding quercetin as an antiviral agent inhibits influenza a virus (iav) entry. viruses biological activities of polyphenols from grapes determine the structure of phosphorylated modification of icariin and its antiviral activity against duck hepatitis virus a inhibitory effects of baicalein on the influenza virus in vivo is determined by baicalin in the serum green tea extract and its major component epigallocatechin gallate inhibits hepatitis b virus in vitro tangeretin from citrus reticulate inhibits respiratory syncytial virus replication and associated inflammation in vivo identification of luteolin as enterovirus 71 and coxsackievirus a16 inhibitors through reporter viruses and cell viability-based screening inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (hiv-1) activity of compounds from taxillus sutchuenensis as inhibitors of hcv ns3 serine protease potent antiviral flavone glycosides from ficus benjamina leaves small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 extract of scutellaria baicalensis inhibits dengue virus replication antiviral activity of four types of bioflavonoid against dengue virus type-2 novel antiviral activity of baicalein against dengue virus preparation and in vitro evaluation of apigenin-loaded polymeric micelles anti-japanese-encephalitis-viral effects of kaempferol and daidzin and their rna-binding characteristics apigenin inhibits enterovirus-71 infection by disrupting viral rna association with trans-acting factors characterization and evaluation of self-microemulsifying sustained-release pellet formulation of puerarin for oral delivery epigallocatechin-3-gallate opposes hbv-induced incomplete autophagy by enhancing lysosomal acidification, which is unfavorable for hbv replication light-controlled flavonoid biosynthesis in fruits acknowledgements the authors acknowledge all reviewers whose detailed comments improved the review and apologize to those authors whose contribution to flavonoids antiviral research may have been inadvertently missed. ethical standards this study is supported by the ra mes state committee of science, in the frames of the research projects 15rf-081 and 16yr-1f064. all authors declare that they have no conflict of interest. this article does not contain any studies with human participants or animals performed by any of the authors. key: cord-271188-ewlxy5po authors: liu, wei; zhang, shuping; nekhai, sergei; liu, sijin title: depriving iron supply to the virus represents a promising adjuvant therapeutic against viral survival date: 2020-04-20 journal: curr clin microbiol rep doi: 10.1007/s40588-020-00140-w sha: doc_id: 271188 cord_uid: ewlxy5po purpose of the review: the ongoing outbreak of novel coronavirus pneumonia (covid-19) caused by the 2019 novel coronavirus (sars-cov-2) in china is lifting widespread concerns. thus, therapeutic options are urgently needed, and will be discussed in this review. recent findings: iron-containing enzymes are required for viruses most likely including coronaviruses (covs) to complete their replication process. moreover, poor prognosis occurred in the conditions of iron overload for patients upon infections of viruses. thus, limiting iron represents a promising adjuvant strategy in treating viral infection through oral uptake or venous injection of iron chelators, or through the manipulation of the key iron regulators. for example, treatment with iron chelator deferiprone has been shown to prolong the survival of acquired immunodeficiency syndrome (aids) patients. increasing intracellular iron efflux via increasing iron exporter ferroportin expression also exhibits antiviral effect on human immunodeficiency virus (hiv). the implications of other metals besides iron are also briefly discussed. summary: for even though we know little about iron regulation in covid-19 patients thus far, it could be deduced from other viral infections that iron chelation might be an alternative beneficial adjuvant in treating covid-19. the ongoing outbreak of pneumonia caused by sars-cov-2 in china is eliciting widespread concerns, especially as the virus was recently shown to spread from human to human [1] . this epidemic is calling for national and international attention to develop effective therapeutics including selective vaccines. nonetheless, no specific therapeutic is yet available, leaving the patients to rely on general and supportive therapies, including oxygen supply, broad-spectrum antiviral medicines (e.g., interferon-α), glucocorticoid, and human serum albumin (hsa) [2] . most recently, lopinavir/ritonavir, an approved anti-hiv drug, has been recommended for treatment of sars-cov-2 infection [3] . remdesivir, a novel nucleotide analogue prodrug in development for treating ebola virus and middle east respiratory syndrome (mers) diseases, has also been reported to relieve the pneumonia symptoms for the first case of sars-cov-2 infection, as reported in the usa [4] . although the drugs are showing a promising efficacy, additional therapeutic options should be explored and considered when taking into account the increasing number of sars-cov-2 cases. wei liu and shuping zhang contributed equally to this work. this article is part of the topical collection on virology * sergei nekhai snekhai@howard.edu since iron is involved in many fundamental biological processes including dna/rna synthesis and atp generation, viruses essentially rely on iron to replicate in host cells [5] . thereby, there is an active competition between the virus and the host in fighting for iron. clinical data have indicated that poor prognosis is related to the condition of iron overload observed in patients with infection of hepatitis b/c (hbv/ hcv) viruses [6] [7] [8] . for even though we know little about iron regulations in sars-cov-2 patients, some clues could be obtained from other viral infections (fig. 1) . for example, iron supplementation was shown to increase the mortality in hivinfected patients, irrespective of the severity of anemia, suggesting a detrimental role of excess iron in hiv infection [9] . hiv-1 replication is dependent on host cell enzymes, some of which are involved in transcription, viral mrna translation, and viral assembly that require iron [5] . hiv-1 viral load dropped dramatically in a hemochromatosis patient who underwent venesection, suggesting an iron deprivationmediated control of hiv-1 replication [10] . to this end, iron depletion can have marked anti-hiv effect. hiv-1 transcription and replication were inhibited by number of iron chelators including 2-hydroxy-1naphthylaldehyde benzoyl hydrazine (311) and icl670 (also known as deferasirox or exjade) [11] , 2-benzoylpyridine 4allyl-3-thiosemicarbazone (bp4at) and 2-benzoylpyridine 4ethyl-3-thiosemicarbazone (bp4et) [12] , and ppyet and ppyat [13] . in a recent study, ex vivo inhibition of hiv-1 in peripheral blood mononuclear cells (pbmcs) obtained from patients with sickle cell disease (scd) was linked to the increased expression of ferroportin and reduced intracellular iron levels [14] . thus, we can consider the potential of iron chelation as an alternative beneficial adjuvant in treating sars-cov-2 infection that we discuss below. covs represent the largest group of coronaviridae, belonging to the nidovirales order, whereby positive-sense and singlestranded rna is enveloped inside. apart from covs, torovirinae is the other subfamily of coronaviridae. covs are further subdivided into 4 genera: α-cov, β-cov, γ-cov, and δ-covon the basis of their phylogenetic clustering. sars-cov-2 that causes covid-19 belongs to the β group [15, 16] . covs are named after their "crown-like" appearance observed under electron microscopy with club-shaped peplomers projecting from their surface. covs are non-segmented positive-sense rna viruses containing approximately 30 kilobase (kb) genomes, characterized by the organization of 5′-leader-utr-replicase-s (spike)-e (envelope)-m (membrane)-n (nucleocapsid)-3′utr-poly (a) tail with accessory genes interspersed within the structural genes at the 3′ end of the genome [17] . to penetrate host cells, covs can use various cell surface molecules as their receptors, preferentially ectoenzymes, e.g., aminopeptidase n (apn), angiotensin-converting enzyme 2 (ace2), and dipeptidyl peptidase 4 (dpp4) [18] . the sars-cov-2 is the newly discovered member of the coronavirus family. based on the most recent results of genome sequencing, most contigs of sars-cov-2 match to the fig. 1 life cycle of coronaviruses under iron replete and deficiency conditions. a sufficient intracellular iron levels support coronavirus replication, whereas b iron deficiency undermines its replication process by interfering with viral transcription, translation, assembly, and exocytosis. covs enter into host cells via binding to various receptors and disassemble to release viral genome and nucleocapsid. transcription and translation of viral genes yield viral genomic rna and structure proteins (e.g., s, e, m, and n). after further processing and assembly in endoplasmic reticulum (er) and golgi, new covs are constructed. finally, the new formed virions are exocytosed by fusing with viruscontaining vesicles. the whole process of viral replication requires iron-containing enzymes and consumes abundant atp. iron is a critical participant for mitochondria to produce atp. in short, adequate iron enables the virus to complete its replication process, and otherwise iron deficiency impairs this process genome of the lineage b from β-cov and show more than 85% identity with the genome of a bat severe acute respiratory syndrome (sars)-like cov (bat-sl-covzc45, mg772933.1) [19] . furthermore, it was found that sars-cov-2 share 96% identical at the whole-genome level to a bat cov [20] . despite the sequence diversity with sars-cov s-protein, sars-cov-2 s-protein shares the similar receptor binding domain (rbd) domain as the sars/sars-like cov, in support of the strong interaction of sars-cov-2 with human ace2 molecules [21] . more than 80% of ace2 receptors are expressed in a small population of type ii alveolar cells (at2) [22] , suggesting that at2 cells could be the target cells of sars-cov-2. after binding and fusion with the host cells, covs disassemble to liberate the inside contents of the virion into the cytoplasm, including the nucleocapsid and viral rna. components of the replication-transcription complex are firstly translated [23] . in addition, abundant sub-genomic negative-sense rnas are produced. then, the viral structural proteins (s, e, and m) are translated, inserted into the endoplasmic reticulum (er), and transported to the er-golgi intermediate compartment. after accumulation of adequate viral genomic rna and structural proteins, the n protein and genomic rna assemble in cytoplasm to form the helical nucleocapsid. subsequently, the s, e, and m proteins delivered to the budding compartment interact with nucleocapsid to constitute the assembled virus. finally, the virus is released from the golgi and exocytosed to the extracellular compartment from the host cells by fusing with virion-containing vesicles [24] (fig. 1 ). for the host, iron is an essential trace element necessary for many fundamental enzymatic and non-enzymatic reactions and diverse physiological processes, such as mitochondrial function including atp generation, dna/rna synthesis and repair, and cell survival/ferroptosis [25] . iron is also essential for viral replication. in the context of hiv-1 infection, iron is involved in several key steps of virus replication. in the reverse transcription of viral rna into dna, the required dntps are generated by rnrs which are an iron-dependent proteins [26] . nf-κb can be activated by iron via generating reactive oxygen species (ros) [27] . iкb kinase activation depends on iron efflux [28] [29] [30] , which increases nf-кb levels and contributes to the activation of hiv-1 promoter [31] . nuclear export of new transcribed viral rna is also irondependent [32] . finally, an iron-binding atpase, atp binding cassette subfamily e member 1 (abce1), is involved in the assembly of the gag capsid proteins into mature hiv-1 virions [33] . atp hydrolysis is necessary for the unwinding activity of helicases of sars-cov and mers-cov during the viral replication [34, 35] . iron is an important component of the complexes i, ii, iii, and iv as well as cytochromes, which participate in the oxidative phosphorylation in mitochondria to conduct electron transportation in maintaining mitochondrial functions and atp synthesis [36] . treatment with deferiprone (dfp) induces apoptosis in hiv-1-infected cells through mitochondrial membrane depolarization, leading to permanent elimination of infected cells in culture [37] . iron metabolism in host is fine-tuned through regulation of iron absorption in the intestine, iron storage in the liver and spleen, iron transport in blood, iron utilization (mainly in bone marrow for erythropoiesis), and iron recycling by macrophages. iron is absorbed by duodenal enterocytes and released into plasma by an iron exporter protein, ferroportin, which is expressed on the basolateral side of the duodenal enterocytes. ferroportin is also expressed in macrophages and essentially governs iron release and recycling [38] . the systemic iron homeostasis is fundamentally orchestrated by the hepcidinferroportin axis. hepcidin is mainly expressed and secreted by hepatocytes, and can bind to its sole receptor, ferroportin. after binding to hepcidin, ferroportin is internalized and degraded, leading to the inhibition of iron absorption from the duodenum and reduction of iron release from macrophages [39] . the cellular iron uptake is primarily mediated by the interaction between iron-bound transferrin and transferrin re-ceptor1 (tfr1) [40] . hepcidin expression could be regulated by systemic iron availability (iron deficiency and iron overload), inflammatory cytokines (il-6 and il-1β), bone morphogenetic proteins (bmp2 and bmp6), and erythropoietic signals (erfe, gdf15, and twsg1) [40] . sars patients exhibited increased amounts of proinflammatory cytokines in serum including il-1β, il-6, and ifn-γ coupled to the pulmonary inflammation and extensive lung damage [41] . infection with sars-cov-2 also leads to the increased levels of il1-β, ifn-γ, ip10 (interferon-inducible protein 10), and mcp1 (monocyte chemotactic protein 1), likely inducing t-helper-1 cell response [3] . thus, induction of these cytokines could supposedly promote hepcidin production and lead to iron sequestration in macrophages, which warrants future investigations. of note, macrophages are presumed to be infected by sars-cov-2 [42] . thus, increased iron storage will most likely favor viral replication inside macrophages. furthermore, viruses can manipulate other iron-related proteins to facilitate their replication and propagation. in the context of human cytomegalovirus (hcmv) infection, homeostatic iron regulator protein (hfe), a competitor of tfr1 to bind to transferrin, is degraded after binding by us2 protein, leading to cellular iron overload [43] . in macrophages infected by hiv-1, the interaction of nef protein and hfe also induces cellular iron overload [44] . plus, tfr1 is also used as the receptor during the entry of several types of viruses [45, 46] . iron dependence of viral replication and modulation of host iron metabolism by viruses, as discussed above, signifies the importance of cellular iron homeostasis in viral life cycle and incites the development of iron chelation strategy in treating viral infections. currently, there are two promising strategies to deplete iron. the first strategy is to deplete iron directly by iron chelators which have strong and selective affinity with iron ions [47, 48] . some of these iron chelators have been approved by u.s. food and drug administration for clinical use, such as deferoxamine (dfo, desferal®), deferiprone (dfp, ferriprox®), and deferasirox (icl670, exjade®) [49] . iron chelators can bind free iron or remove iron from ironcontaining proteins [48] . treatment with higher doses of dfp has been shown to prolong the survival of aids patients after hiv-1 infection [50] . increasing evidence suggests that iron chelators can target hiv-1 replication. the enzymatic activity of ribonucleotide reductase 2 involved in reverse transcription, which contains non-heme iron, is inhibited by dfo and 311 [51] . bp4at, bp4et, phenyl-1-pyridin-2yl-ethanone (ppy)-based iron chelators (ppyet and ppyat) inhibit hiv-1 transcription by decreasing cdk2 and cdk9 activities, and by upregulating iκbα expression and downregulating nuclear nf-κb [12, 13] . topical fungicide ciclopirox and the iron chelator dfp inhibit hiv-1 gene expression at the level of transcription initiation by interfering with the hypusine modification of eif5α [32] . patients treated with dfp unveiled strong hiv-1 rna decline while on-drug and also for up to 8 weeks off-drug without viral rebound [52] . dfo and dfp inhibited hiv-1 replication in human pbmcs and macrophages but the inhibition is attributed to a decrease in cell proliferation [53] . similar to dfo and dfp, oral uptake of bidentate iron chelators, cp502 and cp511 inhibit hiv-1 replication by reducing cellular proliferation [54] . host cell enlargement induced by viruses, e.g., hcmv, could be inhibited by iron chelators through inhibiting mitochondrial activity and macromolecular synthesis [55] . nonetheless, these iron chelators may be scrutinized for their antiviral activity against sars-cov-2. the second strategy is to deplete cellular iron through regulating the gene expression involved in iron metabolism. hiv-1 reverse transcription and transcription was suppressed in pbmcs obtained from scd patients due to increased expression of ferroportin and therefore lowered intracellular iron [14] . on the other hand, hepcidin agonists, such as minihepcidin and thiazolidinone derivatives, can reduce systemic iron levels by compromising the function of ferroportin [56] . nonetheless, the possible applications of agonists to target the hepcidin-ferroportin axis or other iron-related genes in order to achieve antiviral effects still need further exploration, and more efforts are thus urgently needed. apart from iron, cumulative evidence has manifested that other metals (e.g., calcium, zinc, and magnesium) are also involved in the replication process of covs. the entry of covs into host cells is mediated by the viral s protein. under this context, previous studies have demonstrated that calcium is indispensable for sars-cov s-mediated fusion [57] . the replication of sars-cov genome requires rnadependent rna polymerase (rdrp) to synthesize descendant rnas from a rna template, which sternly relies on magnesium (mn 2+ ) for its activity [58] . in the meantime, sars-cov rna dimers, a prerequisite for ribosomal frameshifting, are assembled through "kissing" loop-loop interactions. nonetheless, to reach more stable formation of loop-loop kissing complex, the presence of mn 2+ appears to be necessary [59] . regarding zinc (zn 2+ ), the binding of zn 2+ ions to the metal-binding domain (mbd) of sars-cov helicase is essential for its enzymatic activity [60] . additionally, the maturation of covs requires papain-like protease (pl pro ), which could cleave the nonstructural polyproteins (pp1a and pp1ab). however, devoid of zn 2+ ions, the stability of the tertiary structure of sars-cov pl pro is compromised with diminished activity [61] . moreover, mers-cov pl pro bears a folded structure and potent proteolytic and deubiquitinating activities upon binding with endogenous metal ions [62] . to this end, the above findings collectively suggest that disturbing the viral metal dependence would presumably exhibit antiviral effects. literally, versatile metal-oriented therapeutics besides iron chelators have been searched to target diseased conditions for centuries. for instance, bismuth compounds have been used clinically as medicines to treat various gastrointestinal diseases. bismuth (bi 3+ ) ions strikingly compete with zn 2+ ions for the mbd of helicase, leading to compromised enzyme activities and severe deficiencies in viral replication [63] . as metal chelators, aryl diketoacids (adk) have been verified to inhibit enzymes, such as hiv-1 integrase and hcv rdrp, in that adks function to repress the ntpase/helicase activities of sars-cov by mimicking bi 3+ ions [64] . mercury (hg 2+ ) ions and zn 2+ ions act to inhibit viral proteases, such as 3clike protease (3cl pro ) of norovirus, pl pro of sars-cov, hcmv protease, and hcv ns3 protease [62] . of note, the 3cl pro plays a vital role in viral protein maturation for sars-cov. in fact, their metal-conjugated compounds, including phenylmercuric acetate (pma), toluene-3,4-dithiolato zinc (tdt), and nethyl-n-phenyldithiocarbamic acid zinc (epdtc), elicit great inhibition on sars-cov 3cl pro . as a pharmaceutical excipient, pma is widely used in parenteral and topical pharmaceutical formulations. further, zinc acetate is added as a supplement to the drug in treating wilson's disease [65] . zn 2+ ions could directly impair viral rna synthesis, due to its strong suppression on both the replication and transcription complexes [66] . as summarized above, an array of other metals besides iron incredibly account for the functions and activities of enzymes involved in covs' replication, which underpins rational metal-oriented therapeutic development against covs. iron is crucial for both the host and the pathogen. iron supply is required for the replication of many viruses, most likely including covs, and viruses rely on intracellular iron for their propagation. emerging studies indicate that iron manipulation, such as iron chelation, is a promising adjuvant therapy in treating viral infection. while the emerging viral infection by sars-cov-2 is much less understood compared with hiv-1 or sars-cov and mers-cov, based on the previous studies, it is plausible that deprivation of iron supply to the virus could serve as a beneficial adjuvant in treating the sars-cov-2 infection, with the prerequisite of adequate understandings on one's iron status, such as serum iron and ferritin levels, and globin content. meanwhile, other metaloriented therapeutics could also be reasonably conceived for the antiviral purpose. authors' contributions wei liu: participation in drafting and figure preparation. shuping zhang: original draft preparation. sergei nekhai: participation in revision of the submitted article, funding acquisition. sijin liu: conception and design of the study, review and editing, funding acquisition. funding information this work was supported by the international collaboration key grant from the chinese academy of sciences (grant number: 121311kysb20190010). dr. nekhai's research was funded by nih research grants (1p50hl118006, 1r01hl125005, 1um1ai26617 and u54md007597), and the district of columbia center for aids research grant (p30ai087714). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. data availability not applicable. conflict of interests the authors declare that they have no competing interests. ethics approval not applicable. human and animal rights and informed consent not applicable. code availability not applicable. abbreviations 311, 2-hydroxy-1-naphthylaldehyde benzoyl hydrazine; 3cl pro , 3c-like protease; abce1, atp binding cassette subfamily e member 1; ace, angiotensin-converting enzyme 2; adk, aryl diketoacids; aids, acquired immunodeficiency syndrome; apn, aminopeptidase n; at2, small population of type ii alveolar cells; bmp, bone morphogenetic proteins; bp4at, 2-benzoylpyridine 4-allyl-3thiosemicarbazone; bp4et, 2-benzoylpyridine 4-ethyl-3thiosemicarbazone; covid-19, novel coronavirus pneumonia; covs, coronaviruses; dfo, deferoxamine; dfp, deferiprone; dpp4, dipeptidyl-peptidase 4.; e, envelope; epdtc, nethyl-nphenyldithiocarbamic acid zinc; er, endoplasmic reticulum; hcmv, human cytomegalovirus; hfe, homeostatic iron regulator protein; hiv, human immunodeficiency virus; hsa, human serum albumin; ip10, interferon-inducible protein 10; m, membrane; mbd, metal-binding domain; mcp1, monocyte chemotactic protein 1; mers, middle east respiratory syndrome; n, nucleocapsid; pbmc, peripheral blood mononuclear cells; pl pro , papain-like protease; pma, phenylmercuric acetate; ppy, phenyl-1-pyridin-2yl-ethanone; rdrp, rna-dependent rna polymerase; ros, reactive oxygen species; s, spike; sars, severe acute respiratory syndrome; sars-cov-2, the 2019 novel coronavirus; scd, sickle cell disease; tdt, toluene-3,4-dithiolato zinc; tfr1, transferrin receptor1 a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster diagnosis and clinical management of 2019 novel coronavirus infection: an operational recommendation of peking union medical college hospital (v2.0) clinical features of patients infected with 2019 novel coronavirus in wuhan first case of 2019 novel coronavirus in the united states viral infection and iron metabolism annulling a dangerous liaison: vaccination strategies against aids and tuberculosis iron, haemochromatosis and thalassaemia as risk factors for fibrosis in hepatitis c virus infection oxidative stress stimulates proliferation and invasiveness of hepatic stellate cells via a mmp2-mediated mechanism anemia, iron deficiency, and iron supplementation in relation to mortality among hiv-infected patients receiving highly active antiretroviral therapy in tanzania does venesection reduce hiv viral load in patients with hereditary haemochromatosis? iron chelators icl670 and 311 inhibit hiv-1 transcription iron chelators of the di-2-pyridylketone thiosemicarbazone and 2-benzoylpyridine thiosemicarbazone series inhibit hiv-1 transcription: identification of novel cellular targetsiron, cyclin-dependent kinase (cdk) 2, and cdk9 phenyl-1-pyridin-2yl-ethanone-based iron chelators increase ikappab-alpha expression, modulate cdk2 and cdk9 activities, and inhibit hiv-1 transcription increased iron export by ferroportin induces restriction of hiv-1 infection in sickle cell disease transmission routes of 2019-ncov and controls in dental practice genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding emerging coronaviruses: genome structure, replication, and pathogenesis coronaviruses: an overview of their replication and pathogenesis a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease sars-beginning to understand a new virus iron-sulfur clusters in nucleic acid metabolism: varying roles of ancient cofactors intracellular chelation of iron by bipyridyl inhibits dna virus replication: ribonucleotide reductase maturation as a probe of intracellular iron pools signaling role of intracellular iron in nf-κb activation iron causes interactions of tak1, p21ras, and phosphatidylinositol 3-kinase in caveolae to activate ikappab kinase in hepatic macrophages iron-dependent activation of nf-kappab in kupffer cells: a priming mechanism for alcoholic liver disease signaling role of iron in nf-kappa b activation in hepatic macrophages a compilation of cellular transcription factor interactions with the hiv-1 ltr promoter inhibition of hiv-1 gene expression by ciclopirox and deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5a identification of a host protein essential for assembly of immature hiv-1 capsids delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp13 upon atp hydrolysis biochemical characterization of middle east respiratory syndrome coronavirus helicase. msphere metalloproteins containing cytochrome, iron-sulfur, or copper redox centers drug-induced reactivation of apoptosis abrogates hiv-1 infection molecular control of iron transport hepcidin: a promising therapeutic target for iron disorders: a systematic review two to tango: regulation of mammalian iron metabolism plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars a single viral protein hcmv us2 affects antigen presentation and intracellular iron homeostasis by degradation of classical hla class i and hfe molecules hiv-1 nef down-regulates the hemochromatosis protein hfe, manipulating cellular iron homeostasis canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells transferrin receptor 1 is a cellular receptor for new world hemorrhagic fever arenaviruses desferrioxamine-caffeine shows improved efficacy in chelating iron and depleting cancer stem cells exjade(r) (deferasirox, icl670) in the treatment of chronic iron overload associated with blood transfusion dose of desferrioxamine and evolution of hiv-1 infection in thalassaemic patients inhibition of malignant cell growth by 311, a novel iron chelator of the pyridoxal isonicotinoyl hydrazone class: effect on the r2 subunit of ribonucleotide reductase drug-based lead discovery: the novel ablative antiretroviral profile of deferiprone in hiv-1-infected cells and in hiv-infected treatment-naive subjects of a double-blind, placebocontrolled, randomized exploratory trial inhibition of human immunodeficiency virus type 1 replication in human mononuclear blood cells by the iron chelators deferoxamine, deferiprone, and bleomycin human immunodeficiency virus type 1 replication inhibition by the bidentate iron chelators cp502 and cp511 is caused by proliferation inhibition and the onset of apoptosis human cytomegalovirusinduced host cell enlargement is iron dependent new thiazolidinones reduce iron overload in mouse models of hereditary hemochromatosis and beta-thalassemia the sars-cov fusion peptide forms an extended bipartite fusion platform that perturbs membrane order in a calcium-dependent manner biochemical characterization of a recombinant sars coronavirus nsp12 rna-dependent rna polymerase capable of copying viral rna templates rna dimerization plays a role in ribosomal frameshifting of the sars coronavirus a complex zinc finger controls the enzymatic activities of nidovirus helicases thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus structural basis of mercury-and zinc-conjugated complexes as sars-cov 3c-like protease inhibitors bismuth complexes inhibit the sars coronavirus aryl diketoacids (adk) selectively inhibit duplex dna-unwinding activity of sars coronavirus ntpase/helicase evaluation of metal-conjugated compounds as inhibitors of 3cl protease of sars-cov zn(2+) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-353012-rxhi8wd2 authors: zhou, nan; pan, ting; zhang, junsong; li, qianwen; zhang, xue; bai, chuan; huang, feng; peng, tao; zhang, jianhua; liu, chao; tao, liang; zhang, hui title: glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) date: 2016-03-07 journal: journal of biological chemistry doi: 10.1074/jbc.m116.716100 sha: doc_id: 353012 cord_uid: rxhi8wd2 ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. the outbreaks of ebola viruses in 2014 represented the most serious ebola epidemics in history and greatly threatened public health worldwide. the development of additional effective anti-ebola therapeutic agents is therefore quite urgent. in this study, via high throughput screening of food and drug administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of ebola envelope pseudotyped viruses into the cytoplasm. furthermore, teicoplanin also has an inhibitory effect on transcriptionand replication-competent virus-like particles, with an ic(50) as low as 330 nm. comparative analysis further demonstrated that teicoplanin is able to block the entry of middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars) envelope pseudotyped viruses as well. teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of ebola, mers, and sars viruses. mechanistic studies showed that teicoplanin blocks ebola virus entry by specifically inhibiting the activity of cathepsin l, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin l-dependent viruses. notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of ebola, mers, and sars virus infection. their identification, the method of high throughput screening of clinically approved drugs, which could be applied immediately in the clinic, is a reasonable approach. in this study, we identified teicoplanin and several other glycopeptide antibiotics as ebola virus entry inhibitors with high efficiency and low cytotoxicity, providing a promising means to effect the prophylaxis and treatment of ebola virus infection. cell culture-hek293t, a549, hela, huh7.5.1, and madin-darby canine kidney cell lines were maintained in dulbecco's modified eagle's medium (gibco) with 10% fetal calf serum (gibco), 100 units/ml penicillin, and 100 g/ml streptomycin (gibco) at 37°c and 5% co 2 . thp-1 cell lines were maintained in rpmi1640 medium (gibco) with 10% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin at 37°c and 5% co 2 . primary human umbilical vein endothelial cells were maintained in human endothelial-sfm (gibco) with 30 ng/ml endothelial cell growth supplement (merck millipore), 20 ng/ml recombinant human fgf basic (146 amino acids) protein (r&d systems), 20% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin at 37°c and 5% co 2 . plasmids-gp sequence of zaire ebov-2014 was chemically synthesized and inserted into pcdna3.1 plasmid. the phivluciferase and pcmv-vsv-g plasmids were obtained from addgene, and the pcmv-⌬r8.2 plasmid was kindly provided by dr. trono (27) . the p4cis plasmid that encodes a renilla luciferase reporter, vp40, gp and vp24, the pcaggs-np, pcaggs-vp35, pcaggs-vp30, pcaggs-l, pcaggs-t7, and pcaggs-tim1 plasmids were produced as described previously (28) . viruses-pseudotyped viruses were produced by the co-transfection of phiv-luciferase, pcmv-⌬r8.2, and different envelope plasmids into hek293t cells that were 90% confluent in a 10-cm plate with lipofectamine 2000 by following the manufacturer's instructions (invitrogen). the amounts of plasmids were listed as follows: hiv-luc/zaire ebov-gp(2014) pseudotyped viruses: 4.5 g of phiv-luciferase, 4.5 g of pcmv-⌬r8.2, and 7.65 g of pcdna3.1-zaire ebov-gp(2014); hiv-luc/vsv-g pseudotyped viruses: 4.5 g of phiv-luciferase, 4.5 g of pcmv-⌬r8.2, and 2.7 g of pcmv-vsv-g; hiv-luc/sars-cov-s pseudotyped viruses: 6.5 g of phiv-luciferase, 8 g of pcmv-⌬r8.2, and 20 g of pcdna3.1-sars-cov-s; and hivluc/mers-cov-s pseudotyped viruses: 4.5 g of phiv-luciferase, 4.5 g of pcmv-⌬r8.2, and 10 g of pcdna3.1-mers-cov-s. after 48 h, the supernatants that contain the pseudotyped viruses were collected and filtered through a 0.45-m pore-size filter (pall) and then stored at ϫ80°c until use. ebola transcription-and replication-competent virus-like particles (trvlps) were produced by the co-transfection of 250 ng of p4cis plasmids that encode a renilla luciferase reporter gene, vp40, gp, and vp24, 250 ng of pcaggs-t7, 125 ng of pcaggs-np, 125 ng of pcaggs-vp35, 75 ng of pcaggs-vp30, and 1000 ng of pcaggs-l plasmids into hek293t cells that were 50% confluent in a 6-well plate with lipofectamine 2000 (invitrogen). after 24 h, the medium was discarded, and the cells were incubated with fresh medium for 48 h. then the supernatants that contain ebola transcription-and replication-competent virus-like particles were collected and filtered through a 0.45-m pore-size filter (pall) and stored at ϫ80°c until use. high throughput screening of fda-approved drug library-high throughput screening of fda-approved drug library (topscience) was conducted in 96-well plates with 50 m compounds per well. hek293t cells were incubated with compounds at 37°c for 1 h and then infected with 100 l of p24-normalized (5 ng) hiv-luc/zaire ebov-gp (2014) pseudotyped viruses containing 10 g/ml polybrene. after 12 h, the medium was discarded, and the cells were washed briefly with pbs and incubated with fresh medium for 48 h. the intracellular luciferase activity was examined with glomax 96 microplate luminometer (promega), and the compounds that have the effects of more than 50% inhibition on the luciferase activity were selected for the secondary screening, which was executed with both hiv-luc/zaire ebov-gp (2014) and hiv-luc/ vsv-g pseudotyped viruses in a similar procedure. cell viability assay-hek293t cells were seeded in a 96-well plate (2 ϫ 10 4 per well). after 24 h, the cells were incubated with teicoplanin at different concentrations at 37°c for 48 h. the cell viability then was determined by the celltiter 96 aqueous one solution cell proliferation assay (promega). time-of-addition assay-hek293t cells were seeded in a 96-well plate (2 ϫ 10 4 per well). twenty four hours later, the cells were infected with hiv-luc/zaire ebov-gp (2014) pseudotyped viruses and incubated with 50 m teicoplanin at 0, 2, 4, or 8 h post-infection. after 12 h, the medium was discarded, and the cells were washed briefly with pbs and incubated with fresh medium for 48 h. then the intracellular luciferase activity was determined. the hiv-luc/vsv-g pseudotyped viruses were used as the controls for specificity. virion entry or uptake assay-hek293t cells were seeded in a 6-well plate (2 ϫ 10 5 per well). after 24 h, the cells were incubated with teicoplanin at various concentrations at 37°c for 1 h and then infected with p24-normalized (100 ng) hivluc/zaire ebov-gp (2014) or hiv-luc/vsv-g pseudotyped viruses per well. for virion entry assay, after 6 h, the cells were washed twice with pbs and incubated with 0.25% trypsin at 37°c for 2 min to remove the viruses that adhered to the cell surfaces. the cells were then collected and lysed, and the intracellular amount of hiv-1 p24 was then measured by elisa. for virion uptake assay, after 0.5, 1, or 2 h, the cells were washed twice with pbs and incubated with 0.25% trypsin at 37°c for 2 min to remove the viruses that adhered to the cell surfaces. the cells were then collected and lysed, and the intracellular amount of hiv-1 p24 was then measured by elisa. viral entry inhibition on various cell types-primary human umbilical vein endothelial cells, a549 cells, and hela cells were seeded in a 96-well plate (2 ϫ 10 4 per well). after 24 h, the cells were incubated with teicoplanin at various concentrations at 37°c for 1 h. the cells were then infected with hiv-luc/zaire ebov-gp (2014) pseudotyped viruses. after 12 h, the medium was discarded, and the cells were washed briefly with pbs and incubated with fresh medium for 48 h. then the intracellular luciferase activity was measured. the thp-1 cells were plated in a 48-well format (1.25 ϫ 10 5 per well) and incubated with teicoplanin at various concentrations at 37°c for 1 h. the cells were then infected with hiv-luc/zaire ebov-gp (2014) pseudotyped viruses. after 12 h, the supernatants were removed by centrifugation at 300 ϫ g for 10 min, and the cells were suspended and cultured with rpmi 1640 medium at 37°c for 48 h. the intracellular luciferase activity was determined. hek293t cells were seeded in a 96-well plate (2 ϫ 10 4 per well). after 24 h, the cells were incubated with teicoplanin at gradient concentrations at 37°c for 1 h. the cells were then infected with hiv-luc/sars-cov-s or hiv-luc/mers-cov-s pseudotyped viruses. after 12 h, the medium was discarded, and the cells were washed briefly with pbs and incubated with fresh medium for 48 h. the intracellular luciferase activity was then measured. for the ebola trvlps entry inhibition assay, hek293t cells were seeded in a 96-well plate (2 ϫ 10 4 per well). after 24 h, in the pre-transfection experiment, the cells were pre-transfected with 12.5 ng of pcaggs-np, 12.5 ng of pcaggs-vp35, 7.5 ng of pcaggs-vp30, 100 ng of pcaggs-l, 25 ng of pcaggs-t7, and 25 ng of pcaggs-tim1 plasmids with lipofectamine 2000 according to the supplier's protocol (invitrogen). after 24 h, the medium was discarded, and the cells were incubated with teicoplanin with the gradient concentrations at 37°c for 1 h. in the non-pre-transfection experiment, hek293t cells were directly incubated with teicoplanin with the gradient concentrations at 37°c for 1 h without the pre-transfections of the above plasmids. the cells were then infected with ebola trvlps. after 12 h, the medium was discarded, and the cells were washed briefly with pbs and incubated with fresh medium for 48 h. then the intracellular luciferase activity was measured. compound/virus or compound/cell pre-incubation assay-p24-normalized (50 ng) hiv-luc/zaire ebov-gp (2014) pseudotyped viruses were incubated with 50 m teicoplanin in a 96-well plate at 37°c for 12 h. then the compound/virus mixtures were transferred into a microcon 30-kda centrifugal filter device (millipore) and centrifuged (7000 ϫ g) at 4°c for 15 min. afterward the fresh medium was twice added onto the filter device to wash the compound/virus mixtures. then 0.5 ml of dmem was used to suspend the compound/virus mixtures, and the filter device was centrifuged in reverse (500 ϫ g) at 4°c for 5 min, and the solution was collected and used to infect hek293t cells after hiv-1 p24 normalization. after 48 h, the intracellular luciferase activity was measured. for compound/ cell pre-treatment assays, hek293t cells were seeded in a 96-well plate (2 ϫ 10 4 per well). after 24 h, the cells were incubated with teicoplanin at various concentrations at 37°c for 12 h. the medium was discarded, and the cells were twice washed with pbs and incubated with fresh medium. afterward the cells were infected with hiv-luc/zaire ebov-gp (2014) pseudotyped viruses. twelve hours later, the medium was dis-carded, and the cells were washed briefly with pbs and incubated with fresh medium for 48 h. the intracellular luciferase activity was then measured. sirna transfection, rna isolation, reverse transcription, and quantitative real time-pcr-hek293t cells were seeded in a 96-well plate (1 ϫ 10 4 per well). after 24 h, the cells were transfected with sirnas at a final concentration of 200 nm via lipofectamine rnaimax according to the supplier's protocol (invitrogen). forty eight hours later, the cells were lysed by trizol reagent (invitrogen), and the isolation of total rnas was conducted according to the supplier's protocol (invitrogen). the rnas were reverse-transcripted by primescript rt reagent kit (takara), and the quantitative rt-pcrs were conducted on a bio-rad cfx96 real time-pcr detection system (bio-rad) with sybr premix ex taq (takara). after the initial denaturation of cdna was performed at 95°c for 5 min, 40 cycles of the procedures (10 s of denaturation at 95°c, 30 s of annealing at 60°c, and 30 s of extension at 72°c) were performed with the primers for human gapdh, actb, and a variety of target genes. the smart pools of sirnas were obtained from ribobio. dextran uptake assay and immunofluorescence assay-the procedures previously described were followed with minor modifications (29) . briefly, hek293t cells were incubated with 50 m teicoplanin for 12 h. then the cells were incubated with 1 mg/ml dextran-alexa fluor 568 (m r 10,000) (thermo fisher scientific) for 2 h in 1% serum-containing dmem. the cells were washed twice with pbs and incubated with 4% polyformaldehyde at room temperature for 10 min. the cells were washed twice with pbs and incubated with 0.1% triton x-100 at room temperature for 10 min, followed by incubation with pbs containing 5% bovine serum albumin at room temperature for 1 h. afterward the cells were washed with pbs and incubated with rabbit anti-lamp1 antibodies (proteintech) at room temperature for 1 h. the cells were washed with pbs containing 0.1% tween 20 three times and incubated with dylight488-conjugated goat anti-rabbit igg (abcam) at room temperature for 45 min, followed by washing with pbs containing 0.1% tween 20 three times, and incubated with 5 g/ml dapi at room temperature for 5 min. the cells were then washed again with pbs containing 0.1% tween 20 three times. images were obtained by zeiss lsm780 confocal microscopy using zeiss zfn software, and the co-localization of dextran and lamp1 was analyzed from 20 fields (ն5 cells per field) per independent experiment. cathepsin l enzymatic inhibition assay-two protocols previously described were followed with minor modifications (30, 31) . briefly, hek293t cells were seeded in a black 96-well clinically approved drugs screened 1600 clinically approved drugs that inhibit the entrances of hiv-luc/zaire ebov-gp(2014) pseudotype viruses a 133 clinically approved drugs that inhibit the entrances of hiv-luc/vsv-g pseudotype viruses 131 clinically approved drugs that specifically inhibit the entrances of hiv-luc/zaire ebov-gp (2014) high throughput screening of ebola virus entry inhibitors-we initiated our screening procedure by generating hiv-luc/ zaire ebov-gp (2014) pseudotyped viruses. the viruses were allowed to infect hek293t cells in the presence of a 1600member fda-approved drug library. compounds that inhibited virus luciferase activity were identified as the initial hits. as shown in table 1 , we identified 133 hits that could inhibit the entry of hiv-luc/zaire ebov-gp (2014) pseudotyped viruses. to exclude the hits that only inhibited early events of the hiv-1 life cycle and to identify ebov-gp-specific drugs, hiv-luc/ vsv-g pseudotyped viruses bearing vesicular stomatitis virus (vsv) glycoproteins were used for secondary screening of the initial hit compounds. finally, two drugs that act specifically as ebola virus entry inhibitors were identified, teicoplanin and amiodarone (fig. 1a) . teicoplanin specifically inhibits the entry of ebola viruses-teicoplanin is a glycopeptide antibiotic that includes five major components based on different side chains (fig. 1b) . it demon-strated an ic 50 of 0.34 m for its inhibitory effect on hiv-luc/ zaire ebov-gp (2014) pseudotyped viruses (fig. 1c) . the cytotoxicity of teicoplanin was also determined using a cell viability assay, and its cc 50 was greater than 500 m (fig. 1d ). to further confirm whether teicoplanin acts as an ebola virus entry inhibitor, a time-of-addition assay was conducted. the data showed that teicoplanin represses the entry of ebola viruses at the early stage of ebola virus infection (fig. 1e ). in addition, a virion entry assay also demonstrated that teicoplanin inhibits the entry of hiv-luc/zaire ebov-gp (2014) pseudotyped viruses in a dose-dependent manner. however, teicoplanin does not repress the entry of hiv-luc/vsv-g pseudotyped viruses (fig. 1f) . ebola viruses can infect a wide range of host cells, including vascular endothelial cells, epithelial cells, and monocytes, which account for the pathogenesis observed in ebola-infected patients (32, 33) . thus, it was important to examine whether teicoplanin could repress the entry of ebola viruses into different types of cells. the data showed that teicoplanin effectively represses virus entry into primary human umbilical vein endothelial cells ( fig. 2a) , human epithelial cell lines such as a549 (fig. 2b) and hela cells (fig. 2c) , and the human acute monocytic leukemia thp-1 cell line (fig. 2d) . teicoplanin targets the host cells rather than the cell-free viral particles-to elucidate the molecular mechanisms of the anti-ebola virus activity of teicoplanin, it is necessary to determine whether the target of teicoplanin is located directly on the virus itself. the compound virus pre-incubation assay demonstrated that when teicoplanin was pre-incubated with ebola/ hiv pseudotyped viruses and then filtered and washed away, the inhibitory effect of the antibiotic on ebola virus entry did not occur (fig. 3a) . however, the compound cell pre-treatment assay showed that when the host cells were pre-treated with teicoplanin and subsequently washed to remove the drug, its inhibitory effect on ebola/hiv pseudotyped viruses remained (fig. 3b) . together, these data indicated that the target of teicoplanin is located on the host cells. teicoplanin does not block cell receptors-to clarify the molecular mechanism of teicoplanin action, we tested whether teicoplanin inhibits the entry of other viruses able to utilize similar intracellular transport routes as those employed by ebola viruses. it has been reported that both ebola viruses and sars coronaviruses (sars-covs) need to be transported to the endo/lysosomes to release their genomes (fig. 4a) (18) . therefore, hiv-luc/sars-cov-s pseudotyped viruses that bear the s proteins of sars-covs were generated and used to test whether their entry could be repressed by teicoplanin. the data indicated that teicoplanin also inhibits the entry of sars-covs (fig. 4b) . given that niemann-pick c1 (npc1) and angiotensin-converting enzyme 2 (ace2) represent the cell receptors of ebola viruses and sars-covs, respectively (16, 34, 35) , and that teicoplanin inhibits both of these viruses, it is necessary to clarify whether ebola viruses and sars-covs share cell receptor affinity. following depletion of the expression of npc1 and ace2 in hek293t cells using sirnas, the cells were infected with ebola or sars-cov pseudotyped viruses. the results demonstrated that the knockdown of npc1 affected the infectivity of ebola but not that of sars-cov pseudotyped viruses, whereas the converse was observed following ace2 knockdown (fig. 4c ). this indicates that these two viruses are each associated with specific receptors and also excludes the possibility that teicoplanin inhibits the interaction between the viruses and their cell receptors or the events following receptor binding. taken together, these data led us to hypothesize that the target of teicoplanin would be the host factor(s) which is (are) required for both ebola and sars-cov infection. considering that interferon-inducible transmembrane proteins (ifitms) represent the first line of anti-viral defense of the cells, we first examined the effect of teicoplanin on the expression of ifitms. however, the data showed that teicoplanin did not induce the expression of ifitms (data not shown). teicoplanin has no effect on hops complexes-we further examined the host factors that are required for both ebola viruses and sars-covs but not vsvs using sirnas. through genome-wide haploid genetic screening, several host factors essential for ebola viral infection have been identified (16) . accordingly, we infected cells with ebola-, sars-cov-, or vsv-pseudotyped hiv-1 viruses after sirna-mediated knockdown of the expression of 13 individual host factors. we found that cathepsin l (ctsl), vps11, vps18, vps33a, vps39, and vps41 are required for both ebola and sars-cov infection (fig. 5b ) rather than vsv infection. these results imply that the host factors might be the targets of teicoplanin. vps11, vps18, vps33a, vps39, and vps41 are components of hops complexes, which mediate the homotypic fusion between late endosomes and the heterotypic fusion between late endosomes and lysosomes (29, 36, 37) . to investigate whether teicoplanin affects the transport of ebola viruses by inhibiting hops complex function and subsequently disturbing endosome maturation and fusion between the late endosomes and the lysosomes, a dextran uptake assay was conducted (fig. 5c ). we found that within 2 h of uptake, the dextran-alexa fluor 568 could be transported into lysosome-associated membrane protein 1 (lamp1)-positive lysosomes. the co-localization coefficient of dextran and lamp1 was influenced by sirna knockdown of vps39 and vps41, whereas teicoplanin did not exert any effect (fig. 5d) . these data indicated that teicoplanin does not inhibit the transport of dextran-alexa fluor 568. therefore, it is unlikely that teicoplanin inhibits the entry of ebola viruses by affecting hops complexes. in addition, we conducted virion uptake assays to demonstrate that teicoplanin does not inhibit both ebola-and vsv-pseudotyped virion uptake at 0.5, 1, and 2 h post-infection (fig. 5, e and f) . these data indicated that teicoplanin did not affect the endocytosis of ebola-and vsvpseudotyped virions, at least the early phase. in addition, these data also indicated that, except hops complexes, teicoplanin has no effect on the other host factors that are involved in the process of virion uptake. teicoplanin directly inhibits the enzymatic activity of cathepsin l-after excluding the possibility that the machinery for vesicle transport is involved in the inhibitory effect of teicoplanin, we assumed that the enzymes in the late endosome/ lysosome could be the target of teicoplanin. the proteolysis of glycoprotein by cathepsin l has been reported to be required for the membrane fusion of both ebola viruses and sars-covs (31) . in contract, cathepsin b is required for ebola virus infection but not sars-cov infection (17, 31) . as such, we hypothesized that cathepsin l rather than cathepsin b could be the target for teicoplanin. to this end, we examined whether teicoplanin can inhibit the enzymatic activity of cathepsin l (fig. 6a) . two assays to measure the cathepsin l enzymatic activity were performed (30, 31) . we showed that teicoplanin potently inhibits the activity of cathepsin l in a dose-dependent manner (fig. 6, b and e) . considering that the inhibitory dose of teicoplanin on the activity of cathepsin l is higher than that required for ebola virus infection inhibition, a cell viability assay was performed to confirm that the inhibitory effect is not due to cytotoxicity (fig. 6c) . in addition, a comparative analysis of the inhibitory dose of teicoplanin and that of previously reported cathepsin l inhibitors, z-phe-tyr(t-bu)-diazomethyl ketones, on ebola virus infection was conducted. we demonstrated that both compounds can inhibit ebola virus infection at low doses. the ic 50 dose of teicoplanin required to inhibit the ebola entry is approximately four times more than that of z-phe-tyr(t-bu)diazomethyl ketones (fig. 6d and table 2 ). similarly, the ic 50 dose of teicoplanin required to inhibit the enzymatic activity of cathepsin l was also approximately four times greater (fig. 6, b and e, and table 2 ). these data indicated that the high doses of teicoplanin determined to be required for the inhibition of cathepsin l enzymatic activity could be due to the relatively low sensitivity of the cathepsin l activity assay. these data clearly demonstrate the consistency between the inhibition of virus entry and the inhibition of cathepsin l activity and further support our conclusion that the target molecule of teicoplanin is cathepsin l. ebola trvlp system (28) , which can simulate the life cycle of wild-type ebola viruses to a large extent, was applied to investigate whether teicoplanin and its glycopeptide antibiotic homologs dalbavancin, oritavancin, telavancin, and vancomycin can also inhibit the entry of ebola trvlps. accordingly, the p4cis plasmid encoding renilla luciferase, vp40, gp, and vp24 was transfected into hek293t cells along with plasmids expressing t7 rna polymerase, np, vp35, vp30, and l viral proteins to produce ebola trvlps (fig. 7a) . when the target cells were also pre-transfected with np, vp35, vp30, l, t7, and tim1 plasmids, the transcription and replication of the ebola virus minigenome were actively stimulated to produce infectious ebola trvlps. the ic 50 value of teicoplanin on ebola trvlp entry was ϳ390 nm under these conditions (fig. 7b) . additionally, when the target cells were not pre-transfected with the above plasmids, the ebola viral minigenomes were only weakly tranafter washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. the ic 50 was calculated using graphpad prism software. c, for the non-pre-transfection experiment, hek293t cells were incubated with teicoplanin at various concentrations at 37°c for 1 h without pre-transfection of the above plasmids. the methods used for conducting ebola trvlps infection and measuring the intracellular luciferase activity were the same as those described in b. d and e, ic 50 values of dalbavancin on the entry of ebola trvlps were determined under pre-transfection and non-pre-transfection conditions, respectively. f and g, ic 50 values of oritavancin on the entry of ebola trvlps were calculated using pre-transfection or non-pre-transfection conditions, respectively. h and i, ic 50 values of telavancin on the entry of ebola trvlps were determined under pre-transfection and non-pre-transfection conditions, respectively. j and k, effect of vancomycin on the entry of ebola trvlps was determined using pre-transfection or non-pre-transfection conditions, respectively. the results are representative of at least three independent experiments. the bars show the mean values ϯ s.d. (error bars). the p value was determined by a student's t test. n.s., not significant. scribed and replicated with minimal production of infectious trvlps. the ic 50 of teicoplanin on the entry of ebola trvlps was ϳ330 nm under the non-pre-transfection conditions (fig. 7c ). in addition, we also examined whether the homologs of teicoplanin can inhibit ebola trvlp entry. the data demonstrated that the ic 50 values of dalbavancin, oritavancin, and telavancin on ebola trvlp entry were ϳ1.61, 1.73, and 1.89 m, respectively, under pre-transfection conditions (fig. 7, d, f, and h) and ϳ2.77, 2.13, and 2.30 m, respectively, under non-pretransfection conditions (fig. 7, e, g, and i) . however, we found that vancomycin did not inhibit ebola trvlp entry under either condition (fig. 7, j and k) . the cc 50 values of dalbavancin, oritavancin, telavancin, and vancomycin were also determined (data not shown). these data indicated that these homologs might share similar structures that are indispensable for the inhibitory effects on ebola trvlp entry. however, vancomycin would not be expected to have similar structures (fig. 9) . our data demonstrated that teicoplanin inhibits cathepsin l enzymatic activity and that cathepsin l is also essential for mers-cov and sars-cov entry (31, 38) , we therefore hypothesized that teicoplanin and its homologs could also inhibit the entry of mers-covs and sars-covs. the data illustrated that the ic 50 values of teicoplanin, dalbavancin, oritavancin, and telavancin on mers-cov entry were ϳ0.63, 2.99, 2.12, and 3.24 m respectively (fig. 8, a-d) . however, vancomycin did not inhibit the mers-cov entry (fig. 8i) . additionally, the ic 50 values of teicoplanin, dalbavancin, oritavancin, and telavancin on the entry of sars-covs were ϳ3.76, 9.64, 4.96, and 3.45 m, respectively (fig. 8, e-h) . however, vancomycin did not inhibit the entry of sars-covs (fig. 8j) . these data indicated that glycopeptide antibiotics with the exception of vancomycin exhibit broad antiviral activity because of their inhibitory effects on cathepsin l. in our study, we performed high throughput screening of a clinically approved drug library and identified that teicoplanin not only inhibits the entry of ebola/hiv-1 pseudotyped viruses but also of transcription-and replication-competent trvlps. the ic 50 value of teicoplanin on the entry of ebola trvlps is as low as 330 nm, whereas the cc 50 of teicoplanin is over 500 m. in addition, teicoplanin inhibits the entry of ebola viruses into different types of host cells, including primary human umbilical vein endothelial cells, a549 cells, hela cells, and thp-1 cells. it also inhibits the entry of mers-cov/hiv-1 and sars-cov/ hiv-1 pseudotyped viruses. teicoplanin is a glycopeptide antibiotic isolated from actinoplanes teichomyceticus. teicoplanin contains five major components (39, 40) that can form complexes with the c-terminal l-lys-d-ala-d-ala subunits of lipid ii peptidoglycan precursors. teicoplanin binding of lipid ii inhibits their transglycosylation and transpeptidation, leading to disturbed cell wall synthesis in gram-positive bacteria (41) . recently, teicoplanin was reported to inhibit ebola pseudovirus infection in cell culture, which is consistent with our observations. teicoplanin was also demonstrated to have an effect on a common component used by both enveloped ebola viruses and human respiratory syncytial viruses but not by non-enveloped viruses (26) . however, the mechanism by which teicoplanin inhibits the entry of ebola remained unresolved. to elucidate the molecular mechanisms underlying this process, we compared the entry of ebola viruses and sars-covs and the effect of teicoplanin thereon. teicoplanin represses the entry of both ebola viruses and sars-covs, which require transport to the endo/lysosomes to deliver their genomes. because several host factors have been reported to be essential for ebola and sars-cov virus infection, the identification of the common host factors that are required for the entry of both ebola viruses and sars-covs would likely be helpful to clarify the target of teifigure 9 . chemical structures of glycopeptide antibiotics. teicoplanin, dalbavancin, oritavancin, and telavancin, which inhibit the entry of ebola trvlps, mers-covs, and sars-covs, contain key hydrophobic groups. however, vancomycin, which does not exert antiviral activity, does not contain these groups. coplanin. our data demonstrated that ctsl, vps11, vps18, vps33a, vps39, and vps41 are all indispensable for the entry of both ebola viruses and sars-covs (fig. 5b) . the elucidation via a dextran uptake assay that teicoplanin does not affect the functions of hops complexes suggested that the target of teicoplanin was cathepsin l. in support of this, the results of two cathepsin l enzymatic activity assays indicated that teicoplanin indeed inhibits the activity of cathepsin l, which can explain why teicoplanin inhibited both the entry of ebola viruses and human respiratory syncytial viruses as shown in a previous study (26) because cathepsin l is required for the infection mechanism of both (17, 42) . considering that cathepsin l is also required for the entry of mers-covs, teicoplanin thus represents a broad virus entry inhibitor. in addition, the inhibitory effects of teicoplanin and its derivatives on hiv-1, hcv, influenza viruses, flaviviruses, fipv, and sars-covs have also been reported (43) (44) (45) (46) (47) (48) (49) , further supporting that the antiviral target for glycopeptide antibiotics is a common host factor. as teicoplanin can bind lipid iis, we hypothesized that it might interact with the enzymatic domains of cathepsin l and block their functions similar to the reported inhibitory effect of antimicrobial peptide ll-37 on cathepsin l (50). the specific binding sites of teicoplanin on cathepsin l need to be further investigated by molecular docking, amino acid mutation, and surface plasmon resonance to confirm this hypothesis. we also examined the effects of the teicoplanin homologs dalbavancin, oritavancin, telavancin, and vancomycin on the entry of ebola trvlps, mers-cov/hiv-1, and sars-cov/ hiv-1 pseudotyped viruses. the data demonstrated that dalbavancin, oritavancin, and telavancin also inhibit the entry of mers-cov/hiv-1 and sars-cov/hiv-1 pseudotyped viruses. however, vancomycin did not repress their infection. by comparing the structures of these compounds, we found that all the glycopeptide antibiotics that inhibit ebola trvlp, mers-cov/hiv-1, and sars-cov/hiv-1 pseudotyped virus entry contain hydrophobic groups at the amidogen domains of their aminosaccharides. these groups might play an important role in the interactions between the glycopeptide antibiotics and cathepsin l. in contrast, vancomycin lacks the hydrophobic groups, which might be the reason why it does not inhibit these viral infections (fig. 9) . for the treatment of gram-positive bacterial infections in the clinic, teicoplanin can be administered intravenously or intramuscularly once daily following an initial loading dose, which is convenient for outpatient therapy (51) . compared with vancomycin, which was the first discovered glycopeptide antibiotic, teicoplanin is better tolerated with lower nephrotoxicity (52) . the summary of product characteristics for teicoplanin in 2014 shows that, after the completion of the loading dose regimens for most gram-positive bacterial infections, the serum concentrations of teicoplanin are at least 15 mg/liter (8.78 m), which is ϳ27, 14, and 2 times higher than the ic 50 values of teicoplanin against the entry of ebola viruses, mers-cov/hiv-1, and sars-cov/hiv-1 pseudotyped viruses, respectively. as the toxicity of glycopeptide antibiotics is quite low, we propose that glycopeptide antibiotics might be used in the clinic for ebola/mers-cov/sars-cov infection, espe-cially in the case of emergency requirements during outbreaks of these severe viral infections. author contributions-all listed authors contributed to this work and reviewed the manuscript. n. z. and t. pan designed the experiments and performed most of these experiments. j. s. z., x. z., f. h., t. peng, l. t., and j. h. z. performed the different kinds of virusrelated experiments. q. w. l., c. b., and c. l. carried out the high throughput screening of the food and drug administration-approved drug library. n. z. and h. z. contributed to the idea generation, experimental design, and manuscript preparation and conceived the project. filoviridae: a taxonomic home for marburg and ebola viruses world health organization (1978) ebola haemorrhagic fever in zaire world health organization (1978) ebola haemorrhagic fever in sudan preliminary report: isolation of ebola virus from monkeys imported to u isolation and partial characterisation of a new strain of ebola virus newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans folate receptor-␣ is a cofactor for cellular entry by marburg and ebola viruses lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus t-cell immunoglobulin and mucin domain 1 (tim-1) is a receptor for zaire ebolavirus and lake victoria marburgvirus role of ext1 and glycosaminoglycans in the early stage of filovirus entry tyro3 familymediated cell entry of ebola and marburg viruses downregulation of ␤1 integrins by ebola virus glycoprotein: implication for virus entry cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner ebola virus entry requires the cholesterol transporter niemann-pick c1 endosomal proteolysis of the ebola virus glycoprotein is necessary for infection ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc1ϩ endolysosomes is a rate-defining step a monovalent chimpanzee adenovirus ebola vaccine-preliminary report a recombinant vesicular stomatitis virus ebola vaccine-preliminary report reversion of advanced ebola virus disease in nonhuman primates with zmapp postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-ofconcept study protection against filovirus diseases by a novel broad-spectrum nucleoside analog bcx4430 a broad-spectrum antiviral targeting entry of enveloped viruses the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry teicoplanin inhibits ebola pseudovirus infection in cell culture multiply attenuated lentiviral vector achieves efficient gene delivery in vivo a novel life cycle modeling system for ebola virus shows a genome length-dependent role of vp24 in virus infectivity the hops proteins hvps41 and hvps39 are required for homotypic and heterotypic late endosome fusion cathepsin l and cathepsin b mediate reovirus disassembly in murine fibroblast cells inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry an analysis of features of pathogenesis in two animal models of ebola virus infection pathogenesis of ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virusinduced cytolysis of endothelial cells small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus molecular architecture of the multisubunit homotypic fusion and vacuole protein sorting (hops) tethering complex a ypt/rab effector complex containing the sec1 homolog vps33p is required for homotypic vacuole fusion middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 teichomycins, new antibiotics from actinoplanes teichomyceticus nov. sp. i. description of the producer strain, fermentation studies and biological properties teichomycins, new antibiotics from actinoplanes teichomyceticus nov. sp. iv. separation and characterization of the components of teichomycin (teicoplanin) binding of the glycopeptide antibiotic teicoplanin to d-alanyl-d-alanine-agarose: the effect of micellar aggregates preventing cleavage of the respiratory syncytial virus attachment protein in vero cells rescues the infectivity of progeny virus for primary human airway cultures antiretroviral activity of semisynthetic derivatives of glycopeptide antibiotics polycyclic peptide and glycopeptide antibiotics and their derivatives as inhibitors of hiv entry semisynthetic teicoplanin derivatives as new influenza virus binding inhibitors: synthesis and antiviral studies teicoplanin therapy leading to a significant decrease in viral load in a patient with chronic hepatitis c inhibition of hepatitis c virus replication by semi-synthetic derivatives of glycopeptide antibiotics an analog of the antibiotic teicoplanin prevents flavivirus entry in vitro inhibition of feline (fipv) and human (sars) coronavirus by semisynthetic derivatives of glycopeptide antibiotics antimicrobial peptide ll-37 is both a substrate of cathepsins s and k and a selective inhibitor of cathepsin l teicoplanin. a reappraisal of its antimicrobial activity, pharmacokinetic properties and therapeutic efficacy teicoplanin versus vancomycin for proven or suspected infection key: cord-297125-la20vi9j authors: brower, jennifer l. title: the threat and response to infectious diseases (revised) date: 2016-08-01 journal: microb ecol doi: 10.1007/s00248-016-0806-9 sha: doc_id: 297125 cord_uid: la20vi9j the threat from microorganisms is complex, and the approaches for reducing the challenges the world is facing are also multifaceted, but a combination approach including several simple steps can make a difference and reduce morbidity and mortality and the economic cost of fighting infectious diseases. this paper discusses the continually evolving infectious disease landscape, contributing factors in the rise of the threat, reasons for optimism, and the policies, technologies, actions, and institutions that might be harnessed to further reduce the dangers introduced by pathogens. it builds upon and updates the work of other authors that have recognized the dangers of emerging and re-emerging pathogens and have explored and documented potential solutions. in just the past year, the united states has been bombarded with headlines on the dangers of infectious diseases: "hiv 'epidemic' triggered by needle-sharing hits scott county, indiana [1] ;" "american with ebola now in critical condition [2] ;" "seasonal flu vaccine even less effective than thought: cdc [3] ;" "'superbug' outbreak at california hospital, more than 160 exposed [4] ;" "deadly cre bugs linked to hard to clean medical scopes [5] ;" "painful virus [chikungunya] sweeps central america, gains a toehold in u.s. [6] . " the ebola outbreak that began in 2014 and the measles outbreak initiated at disney world in particular brought the threat of "exotic" infectious diseases back to the american and global consciousness. this coupled with the fact that the most commonly circulating strains of the influenza a virus h3n2 drifted [7] from that used in the 2014-2015 influenza vaccines serve as reminders that the threat from microorganisms is continuously evolving and is persistent. the threat of emerging and re-emerging pathogens has been discussed in the scientific literature, the medical community, by policy makers, and the general public over the past 70 years, but much of the discussion was among directly affected populations and their caregivers. general interest flourished after a series of events in the 1990s and early 2000s. in 1992, a report by russian general kuntsevich followed by boris yelstin's decree in april of that year to end all offensive biological weapons programs revealed that the former soviet union had an extensive biowarfare program and that facilities and expertise still existed which would enable russia to unleash deadly pathogens on the world [8] . in 1996 when shoko asahara, the spiritual leader of a japanese religious cult, was arraigned, the magnitude of the organization's attempts to deploy anthrax in 1993 was exposed [9] . in october 2001, the united states was transfixed by the first bioterrorism attacks on its own soil: envelopes containing bacillus anthracis spores were sent through the mail to targets ranging from media companies to government officials [10] . five people died and thousands were treated with prophylactic antibiotics. the attacks and other attempted and planned attacks, along with widely publicized outbreaks such as west nile virus in 1999 [11] and severe acute respiratory syndrome (sars) in 2003 [12] , brought the topic of infectious disease to the forefront. in addition, more incessant threats such as influenza and lower respiratory infections continue to kill and cause economic harm through lost productivity and hospitalizations. furthermore, zoonotic diseases such as salmonella and listeria, which represent more than two-thirds of emerging and re-emerging diseases [13] , raise the visibility of the economic and human and animal health issues caused by pathogens. in april 2015, the sabra dipping company voluntarily recalled about 30,000 cases of hummus potentially contaminated with listeria monocytogenes. at the same time, blue bell recalled nearly 30 products also similarly contaminated. while there were no known casualties as a result of the sabra contamination, authorities in kansas and texas reported that three deaths in each state might be attributed to the blue bell incident [14] . the threat from microorganisms is complex, and the approaches for lowering the challenges the world is facing are also multifaceted, but several simple steps can make a difference. this paper will discuss the emerging infectious disease landscape, contributing factors in rise of the threat, reasons for optimism, and the actions, policies, technology, and institutions that might be harnessed to further reduce the dangers introduced by pathogens. it builds upon the work of other authors who have recognized the dangers of emerging and re-emerging pathogens and have explored and documented potential solutions [15] [16] [17] . microorganisms pose health and economic threats and may pose a strategic threat if a large percentage of the population is overcome or if the potential transmission of infectious diseases across borders causes an increase in tension among state allies or enemies. one organism alone, clostridium difficile, is estimated to cost the united states between $1 and $3 billion per year [18] , with its primary impact on american children [19] . initially identified in the early 1900s as a commensal organism in the digestive tract, c. difficile infection (cdi) has only been recognized as a significant threat to pediatric health over the last decade [19] . the threat to both children and adults is global. infections since 2003 have become more common, more acute, less treatable by standard therapy, and more likely to reoccur [20] . initially, the c. difficile infections were associated with the use of the antibiotic clindamycin, but fluoroquinolones and cephalosporins are currently the more likely cause of disturbed gut microbiota, which increasingly lead to colonization with ribotype 027, a severe variant of c. difficile [20] . according to the centers for disease control and prevention (cdc), emerging infectious diseases are those "whose incidence in humans has increased in the past two decades or threatens to increase in the near future [21] ." while there may be debate about the specifics, for the purposes of this article, re-emerging and emerging diseases are distinguished as follows: re-emerging diseases are those that were known to impact humans or animals in the past and were thought to be brought under control with zero or few infections in the past several decades. these include infections resulting from changes or evolution of existing organisms and changes in the geographic distribution of an organism or populations affected by the organisms. previously unrecognized (in the past several decades) infections are considered emerging. according to this definition, c. difficile would be considered an emerging pathogen as its dangers were not recognized when it was first identified. other outbreaks and trends of concern include the following: tuberculosis (tb), while no longer among the 10 leading causes of death in 2012, was still among the 15 leading causes, killing over 900,000 people in 2012 [22] . in the united states, while overall tb incidence is decreasing, it is still a large problem for foreign-born residents and for the homeless population at a cost of nearly $ 50 million per year [23] . lyme disease caused by the spirochete borrelia burgdorferi was recently recognized as an epidemic. the disease is difficult to diagnose, causes long-term disability if untreated, and may impact as many as 1000,000 people in the united states [24] . more than 40 % of lyme disease patients continued to exhibit symptoms after six months, and for 10 % of infected people, symptoms continued for more than three years [25] . the spread of diseases such as multidrug resistance acinetobacter in at-risk populations is also of increasing concern. "within the last 15 years, members of the bacterial genus acinetobacter have risen from relative obscurity to be among the most important sources of hospital-acquired infections. the driving force for this has been the remarkable ability of these organisms to acquire antibiotic resistance determinants, with some strains now showing resistance to every antibiotic in clinical use [26] ." acinetobacter resistance to drugs such as imipenem and ampicillin/sulbactam increased 25 % from 2003 to 2008 [26] . leptospirosis, one of the most widely distributed zoonotic diseases worldwide, is an emerging public health concern particularly in large urban centers of developing countries [27] . it is also important in the united states in humans, pets, and wildlife. experts believe incidence in humans is underreported, but the cdc estimates that 100-200 leptospirosis cases occur annually with approximately half of those in hawaii [28] . in 1998, 775 triathletes in illinois were exposed to leptospirosis of which 110 became symptomatic [28] , representing the largest human outbreak in the united states. recently, cases in pets have caused concern in california [29], michigan [30], and florida [31] . more than a quarter of the tested deer population in michigan was infected with the disease [30] . west nile virus (wnv) is another zoonotic disease of concern, and the us population and health practitioners have become more aware of this disease over the past decade. birds carry the virus, which is then transmitted by mosquitoes to humans, horses, and other mammals. disease symptoms range from fever to neurological complications, such as encephalitis or meningitis. mortality is observed mostly in older and immunocompromised individuals. in 1999, wnv was introduced to the united states, and its range soon extended across north america [32] . not only is the number of wnv outbreaks increasing but also novel strains are emerging, which display higher virulence. wnv has also developed sophisticated avoidance mechanisms to avoid its elimination [33] . noroviruses are the leading cause of foodborne disease outbreaks worldwide and may soon eclipse rotaviruses as the most common cause of severe childhood gastroenteritis, because rotavirus vaccine use is becoming more prevalent [34] . norovirus rapidly undergoes genetic mutations and recombinations so that new epidemic strains are constantly evolving. although norovirus infection is generally not fatal, infections in children, the elderly, and the immunocompromised can cause morbidity and even death. research into a vaccine or treatment has been impeded by the lack of a cell culture or small animal model. however, vaccines based on norovirus capsid protein virus-like particles show potential and may become broadly available through transgenic expression in plants [34] . vibrio vulnificus, a common gram-negative bacterium in warm coastal waters globally, is an emerging pathogen [35, 36] . up to 30 million vulnerable americans are at risk when consuming raw or improperly prepared seafood tainted with v. vulnificus which can cause primary septicemia [36] . additionally, all individuals are at risk of serious wound infection that may lead to secondary septicemia [37, 38] . even with antibiotic treatment, half of patients may die from primary septicemia and a quarter from secondary [36, 37] . other environmental organisms of concern include the waterborne pathogen that causes legionnaires' disease, legionella bacterium; naegleria fowleri, which causes amebic meningoencephalitis; other mycobacterium (hospital environment) such as mycobacterium abscessus and m.massiliense in lung disease; the mosquitoborne chikungunya virus and the tickborne bourbon virus. in addition to causing acute illness, research has uncovered links between infectious diseases and cancer. in one study by wu et al. [39] , researchers found measurable differences in fecal microbiota between healthy individuals and those with colorectal cancer as determined by pyrosequencing of the 16s rrna gene v3 region. as early as 1981, researchers found that hepatitis b surface antigen (hbsag) carriers had a greater incidence of primary hepatocellular carcinoma (phc) than among non-carriers [40] . the list of emerging and re-emerging pathogens could fill up a tome. these organisms vary in virulence and distribution, but all of them share common characteristics in that the incidence or virulence or both are increasing and humans must find methods of preventing, detecting, and treating them. to combat infectious disease, it is important to understand the factors that are working to increase the occurrence and severity of infections. human behavior has a large impact on the creation of environments where microorganism can evolve and mutate. these changes can sometimes make organisms more infectious and/ or virulent. examples include the following: antibiotics in the environment through overuse and misuse; changes in sexual norms; patterns of drug use and incarceration; global climate change; human incursion into new environments; and changing patterns of human interactions with wild and domesticated animals; expanding travel patterns; vaccination avoidance; and population concentrations in large cities. recent cases are used to illustrate how differences in human behavior have modified the threat from bacteria and viruses. the problem of antibiotic resistance is threefold: there has been a rise in the number or identification of resistant bacterial strains; the pipeline for the development of new medicines to treat infection dried up significantly over the past 20 years; and the most significant problem is the lack of stewardship of existing antimicrobials. these issues have led to a reduction in the efficacy and number of responses available to physicians and their patients. the biological processes that lead to resistance are extremely complicated and not fully understood, resulting in sometimes limited progress in the control and treatment of resistant microorganisms and the diseases they cause [41] despite recognition of the problem nearly a century ago. davies and davies [41] compiled a list of "suberbugs," which have increased pathogenicity and are more impervious to treatment. their list includes the following: multidrug-resistant (mdr) m. tuberculosis; nosocomial (hospital-linked) infections with acinetobacter baumannii, burkholderia cepacia, campylobacter jejuni, citrobacter freundii, clostridium difficile, enterobacter spp., enterococcus faecium, enterococcus faecalis, escherichia coli, haemophilus influenzae, klebsiella pneumoniae, proteus mirabilis, pseudomonas aeruginosa, salmonella spp., serratia spp., staphylococcus aureus, staphylococcus epidermidis, stenotrophomonas maltophilia, and streptococcus pneumoniae. their list does not include the new delhi metallo-beta-lactamase-1 (ndm-1) resistant strains discussed below. as the authors point out, in addition to the direct human toll, treatment is often more costly [42] when resistant organisms are involved. in fact, the issue has become so acute that new terms have developed over the past decades: microorganisms that are pan-drug resistant (pdr) or extremely drug resistant (xdr). one of the most widely dispersed antibiotic resistant organisms is m. tuberculosis. worldwide, this organism is often resistant to multiple drugs, and in 2009, completely drug-resistant forms of tuberculosis were reported in citizens of four countries: afghanistan, azerbaijan, iraq, and iran [43] . in many organisms, such as enteric bacteria which are acquired both in community and hospital settings, resistance (often to β-lactam antibiotics in this case) spreads through horizontal gene transfer on plasmids; however, there have been no documented cases of this in tuberculosis, where all resistance occurs by spontaneous mutation [40] . multidrug resistant pseudomnas aeruginosa is also of concern as it is deadly and widespread [44, 45] . m. tuberculosis is one example of the multitudes of resistant organisms. other widespread and dangerous bugs include staphylococcus aureus (66.4 per 1000 inpatient prevalence rate in 2010 [46] ) and c. difficile (in 183 us hospitals in 2011, c. difficile was the most commonly reported pathogen causing 12.1 % of health careassociated infections and staphylococcus aureus caused the second highest percentage, 10.1 %. klebsiella pneumoniae and klebsiella oxytoca 9.9 % and escherichia coli 9.3 % followed closely behind [47] ). at a single hospital in 2010 and 2011, resistant acinetobacter baumannii infected 13.5 % of patients who were not previously infected [48] . infections with resistant organisms are harder to control; standard treatments are less effective; illness and hospital stays are longer; and mortality is higher. gram-positive organisms resistant to antibiotics were the first concern, but resistance in gramnegative organisms emerged: gram-negative bacteria resistance increases faster than in gram-positive bacteria [49] , and there are fewer antibiotics in the pipeline that work against gram-negative bacteria [50] . cosgrove et al. [51] performed a meta-analysis of studies published between 1980 and 2000 on the impact of methicillin resistance on mortality. these studies included nearly 4000 patients, a third of whom were infected with methicillin resistant staphylococcus aureus (mrsa). mortality was significantly lower in the group infected with susceptible bacteria. in another study, cosgrove's group found that mrsa bacteremia also increased median length of hospital stay by almost 30 % and not surprisingly (given the longer stay), increased hospital charges from an average of $19,212 to $26,424 [52] . a prospective study found similar results in hemodialysis patients at the duke university hospital [53] as did a study on orthopedic patients [54] . vancomycin-resistant enterococci (vre) [55] and enterobacter species resistant to third generation cephalosporins [56] showed a similar trend; however, penicillin-and cephalosporin-resistant streptococcus pneumonia results were dissimilar, and the authors surmised that this might be due to the specific use of vancomycin [51] . chemicals in daily use may also change microorganism susceptibility to antimicrobial agents. for instance, it has been regularly demonstrated in the laboratory that resistance to triclosan, an antimicrobial agent used in many household products including hand sanitizer, and crossresistance to antimicrobials increases with use of triclosan containing products; however these results have not yet been observed in the community. based on the available evidence, the risk of potential antimicrobial resistance outweighs the benefit of widespread triclosan use in antimicrobial soaps [57] . resistance is not something that can be conquered: bacteria with their relatively short lifespans can mutate quickly; however, with knowledge of the 20,000 resistance genes of 400 types [41] , it may be possible to stay one step ahead of resistance and find new ways to treat bacterial infections. other changes that have impacted infectious disease distribution and prevalence are changes in sexual norms, drug use, and incarceration. needle sharing itself can spread infections, and the use of drugs can affect sexual and risk taking behavior which can put people in jeopardy [58] . while homophobia is decreasing in the united states and worldwide, homophobia has been one of the major social determinants of infection particularly with hiv/ aids and other sexually transmitted diseases. for example, men sleeping with men accounted for 53 % of new hiv infections in 2008 [59] . historical legal restrictions, which are now being relaxed in this decade, had ostracized gay people, limiting their self-identification and therefore efforts to target gay communities for education and prevention as well as diagnosis and treatment efforts. injecting drug users account for 12 % of new hiv infections often due to inadequate access to sterile needles and syringes and addiction treatment programs [59] . as noted below, drug use also changes behavior which also leads to increased transmission. drug use and incarceration patterns go largely hand-in-hand. in part because of the united states hard line on drug use, united states incarceration rates are the highest in the world with minorities accounting for a disproportionate percent of the prison population. incarceration rates disrupt community and sexual relationships and compound poverty issues, amplifying the exposure of communities and individuals to hiv infection and other infections [59] . in a second example, methamphetamine use has been shown to affect a person's judgment and may lead to unsafe behaviors such as reduced condom use, multiple partners, and increased drug injection. methamphetamines also increase physical susceptibility because their use dries mucosa intensifying chafing and abrasions, which, in turn, allow microorganisms to enter the body during sexual and other activity [58, 60] . aquaculture contributes to the pollution of rivers, bays, and even our oceans with antibiotics and antibiotic resistance genes (args). from china to the united states, antibiotics and args have been found in surface water of all types. for example, in the coastal water of the bohai bay, china, fluoroquinolones, macrolides, sulfonamides, tetracyclines and chloramphenicoles, and polypeptides were found at concentrations up to several micrograms per liter with higher concentrations where human activity was concentrated [61] . in a review, comparing aquaculture and land animal production with the respect to type, mechanism, and quantity of antibiotic resistance, done, venkatesan, and halden [62] found that aquaculture was similar to terrestrial agriculture in terms of the resistance mechanisms, that 39 antibiotics used in aquaculture are important in human health, and that pathogens isolated from the farmed fish were resistant to multiple antibiotics. due to improper use and disposal of antibiotics, the presence of antibiotic-resistant organisms and genes in natural waterbodies, wastewater, and treated municipal water has been widely demonstrated and reviewed [63] [64] [65] [66] . without additional treatment, this water is commonly used on crops; humans and animals then consume the products, and serious outbreaks have occurred that are difficult to treat because the microorganisms do not respond to commonly used antibiotics [63] [64] [65] [66] . pruden et al. [66] found concerning levels of args in colorado (united states) dairy lagoon water, irrigation ditch water, river sediments, treated drinking water, and recycled wastewater. ramsden et al. [67] similarly found antibacterial resistance in municipal wastewater treatment plants. zuccato et al. [68] discovered that the concentrations of atenolol, bezafibrate, clofibric acid, cyclophosphamide, diazepam, erythromycin, furosemide, lincomycin, oleandomycin, ranitidine, salbutamol, spiramycin, and tylosin were in the nanogram per liter range in river or drinking water or river sediments in several sites in italy. munir et al. [69] examined the presence of antibiotic-resistant genes and bacteria in several types of wastewater effluents in michigan and found that advanced water treatment systems such as membrane bioreactors were significantly more effective than conventional wastewater treatment at removing the tetracycline-resistant gene teto and sulfonamide-resistant gene (sul-i) as well as tetracycline and sulfonamide-resistant bacteria. anaerobic digestion and lime stabilization treatment of wastewater was more effective than the conventional dewatering and gravity thickening methods for removing antibiotic-resistant genes and bacteria [69] . burch et al. [70] were able to significantly reduce the concentrations of the args tet(a), tet(w), and erm(b) using conventional wastewater treatment (aerobic); however, removal of inti1 required batch treatment, while the others required relatively long-term semi-continuous treatment. tet(x) increased in concentration. according to the world bank, nearly 70 million travelers visited the united states and approximately one billion people traveled globally in 2012 [71] . the incidence of tuberculosis in the united states is largely due to foreign visitors and citizens and residents born in other countries [23] . another, travel related resistance threat emerged in the united states in 2010 when three patients were reported to have the gene for new delhi metallo-beta-lactamase (ndm-1), an enzyme that destroys beta-lactam antibiotics including commonly used penicillins, cephalosporins, and carbapenems. the first case was reported in india in 2009 [72] , and to date, india and pakistan have reported the most instances of ndm-1, but the gene is spreading globally, and cases have now been detected in many countries, including great britain, canada, sweden, australia, japan, and the united states. antibiotics are widely used in india and some researchers [73] have demonstrated that overuse of carbapenems led to the development of ndm-1 [71] . research also points to medical tourism as a cause [74] [75] [76] [77] . ndm-1 is a newly identified problem, only recognized since about december 2009 in the medical literature, but it is only one example of diseases transmitted through medical tourism which is defined as travel to a country to get medical care that is not available or is more expensive in one's own country. precise data on the economic value and the number of patients seeking medical procedures are not easily available. in 2009, smith et al. [75] estimated that approximately four million patients crossed borders seeking treatment. in 2011, guidelines to unify definitions of medical tourism and methodologies for reporting its extent were published and accuracy of the types and amounts of medical tourism may improve in the near future [76] . a greater potential threat is related to the increasing travel of immunocompromised patients. lortholary et al. [77] illuminated the fact that as more and more people are living with hiv, having organ transplants, using immunodilators, or suffering from diabetes, more individuals are infected when traveling. the authors suggested preparations and responses to prevent severe illnesses when traveling. infections spread within the united states from travel as well. for example, during the period from 2004 through 2011, 96 cryptococcus gattii infections were reported to the cdc. c. gattii, an environmental fungus typically prevalent in tropical and sub-tropical regions, can cause an uncommon infection of the lungs and/or the central nervous system in those who inhale the fungus. more than 86 % of the cryptococcosis cases occurred in people who had traveled to the pacific northwest. the infection was fatal for 33 % of the patients [78] . many factors have reduced the number of new antibiotics approved in the united states each year as well as reduced domestic production including demanding food and drug administration (fda) regulations, the cost and time to market of development, the consolidation in the pharmaceutical industry, and the lack of financial impetus to produce and distribute antibiotics, which are generally used on a one-off basis versus drugs used to treat chronic conditions such as statins, viagra, and allergy medications. in a may 2012 speech, janet woodcock, the director of the center for drug evaluation and research (cder), acknowledged that new antibiotics were not sufficient to address growing antibiotic resistance and that fda's approach to approval was a significant factor [79] . the fda introduced new regulations for clinical trials at the beginning of the twenty-first century, which led to a cooling of antimicrobial development in the pharmaceutical industry [80] . first, the newly required approach doubled the cost of phase iii clinical trials, already a substantial barrier for development. in phase iii, it is expected that testing will include pairs of relatively large (usually >750 total subjects per study) groups of people conducted for the selected pathogen at the relevant body location(s). this has become challenging as new antibiotics focus on particular pathogens including resistant pathogens, making it difficult to enroll large numbers of patients [81] . in part because of the cost of the new regulations, eli lilly, bristol-myer squib, glaxo smithkline, proctor and gamble, roche, and wyeth left the development business [7, 79] . in addition, while the amount of antibiotics prescribed has continued to grow, the market value has not changed and was estimated at $32 billion in 2010 [79] as compared to a $19.7 billion market in 2012 for statins alone [82] . companies are getting out of the market because the regulatory burden is high, antimicrobials are typically used for short periods of time, public pressure is building to lower use, and the medicines are often subject to price controls outside of the united states [79, 83] . while development has slowed, in the past 15 years, 22 new antibiotics have been brought to market. two approved more recently, fidaxomicin and bedaquiline, have new modes of action. fidaxomicin was shown to effectively treat c. difficile [84] . because the financial incentives are few, much antibiotic production has been outsourced from the united states to india, china, and other countries where labor, raw material, and energy costs are lower [85] . in fact, it has been more than 10 years since the active ingredient for penicillin was last manufactured in the united states. this presents a significant strategic problem for the united states in the case of an outbreak, particularly during times of conflict or worldwide scarcity. global climate change is increasingly accepted as causing extreme, unusual weather patterns [86] . changing weather patterns can impact the presence of infectious agents in many ways. for instance, in may and june 1993, an initially unidentified disease killed ten people in the four corners region of arizona and new mexico. at the outset, 70 % of the patients died of the infection, and after the medical staff developed enhanced protocols, the death rate was only reduced to 40 %. scientists isolated a hantavirus [87] , and later, researchers determined that an unusually wet spring led to increased rodent carrier density which in turn impacted human infection rates; however, these factors alone are not enough to explain persistent hantavirus infection in the southwestern united states [88] . ecosystem changes and human interactions with the environment may increase the transmission of infectious disease [89] . for instance, three studies found robust correlations between the threat to humans from west nile virus and low bird diversity in the united states [89] [90] [91] . the spread of emerging infectious diseases among animals has significant human health and economic costs. zoonotic diseases kill more than two million people per year and transmission occurs from both wild and domesticated animals [92] . halsby et al. [93] reviewed the english literature with respect to infectious diseases caused by pet store animals and found 57 discussions of infections related to pet shops. the most commonly observed diseases were salmonellosis and psittacosis: other diseases such as tularemia were also identified. the human animal interaction has impacted civilization throughout history. according to daszak et al. [94] , "parallels between human and wildlife emerging infectious diseases (eids) extend to early human colonization of the globe and the dissemination of exotic pathogens. in the same way that spanish conquistadors introduced smallpox and measles to the americas, the movement of domestic and other animals during colonization introduced their own suite of pathogens. the african rinderpest panzootic of the late 1880s and 1890s is a paradigm for the introduction, spread, and impact of virulent exotic pathogens on wildlife populations. this highly pathogenic morbillivirus disease, enzootic to asia, was introduced into africa in 1889. the panzootic front traveled 5000 km in 10 years, reaching the cape of good hope by 1897, extirpating more than 90 % of kenya's buffalo population and causing secondary effects on predator populations and local extinctions of the tsetse fly." more recently, bovine tuberculosis, while responsible for only 22 cases of human tuberculosis in the uk, prompted the slaughter of tens of thousands of cattle in the first decade of the twenty-first century [95] . in 2015, throughout the united states, domestic poultry and wild birds have been suffering from a highly pathogenic strain of avian influenza (hpai) h5 [96] . through june 17, 2015, more than 48 million birds were put to death. the cost of the government response is tagged at $500 million primarily to fund the work of 3400 staffers and contractors [97] . on the commercial side, analysts used economic models and found that for a million dollars in direct losses there are $1.8 million in overall economic losses. in mid-may direct losses in poultry production were estimated at $113 million leading to overall losses of more than $300 million [98] . transmission to humans in the united states has not been detected, although related viruses have caused serious illness and death around the world [97] . typically, people have focused on wildlife diseases that affect human health and agriculture. recently, researchers, policy makers, and others have begun to pay attention to wildlife infectious diseases, because a number of endangered species including birds, amphibians, and invertebrates [99] are impacted [94] . human's changing relationship with the environment "deforestation and ensuing changes in land use, human settlement, commercial development, road construction, water control systems (dams, canals, irrigation systems, reservoirs), and climate, singly, and in combination have been accompanied by global increases in morbidity and mortality from emergent parasitic disease [100] ." lyme disease is a prime example of how human destruction of the environment (forests) can lead directly to increased risk for disease exposure. allan, keesing, and ostfeld [101] found that as forest patch size decreased ioxdes nymphal infection prevalence and nymphal density with increased, resulting in a noticeable rise in the density of infected nymphs and concluded that habitat fragmentation affects human health. as humans change or destroy the local environment, they tend to interact with or disturb wildlife populations, creating further instances for exposure to infectious diseases. goldberg et al. [102] found increased rates of interspecific gastrointestinal bacterial exchange between people and nonhuman primates when humans visited chimpanzee and ape habitats. chimpanzees carried antibiotic-resistant bacteria although there had never been treatment with antibiotics. many of the factors discussed above coexist to increase the threat from microorganisms. the antivaccine lobby, especially in the united states, has led to a significant decline in the vaccination rates of infants and children, particularly among specific demographics despite the overwhelming success of vaccines in the fight against vaccine-preventable diseases. for instance, in 1950 (pre-vaccine), 319,124 cases of measles were reported with 468 mortalities. in 2011, there were 211 cases of measles but no deaths. similarly in 1950, 5796 cases of diphtheria were reported resulting in 410 deaths. in 2011, there were no reported cases of diphtheria [103] . up to two % of parents in the united states refuse vaccination completely for their children with up to 19 % more who are cautious or elect to delay vaccination [103] . the reduction in vaccination coverage is typically attributed to the lack of perceived threat due to the success of vaccination, combined with false medical research and media reporting [103] . the reduction in vaccination rates has resulted in the highest number of cases of measles in the united states since it was declared eliminated in 2000 [104] . while native measles has been eliminated in canada, several measles cases are imported each year by international travelers and due to inadequate vaccination, these cases often lead to secondary spread. in the first five months of 2014, 103 cases in five provinces from 21 known importations occurred through infected travelers arrived from the philippines, india, the united states, thailand, pakistan, italy, and the netherlands [105] . travel patterns in canada are exemplary of much of the world. in 12 years, international travel (excluding travel to the united states) more than doubled from 4.5 million to 11 million trips [106, 107] . if the antivaccine trend does not abate, and in conjunction with widespread global travel, the threat from diseases once thought under control may pose a significant threat to the population. influenza outbreaks kill and hospitalize more than 100,000 americans each year. the predominant strategy in the united states is to encourage all eligible populations to get vaccinated; however, for the 2014-2015 flu season, more than half of influenza a (h3n2) viruses had drifted from the h3n2 vaccine virus. this mismatch leads to decreased vaccine effectiveness [7] . it may also discourage individuals from getting the flu vaccine in the future. population growth, urbanization, and travel along with deterioration in public health infrastructure have contributed to the resurgence of infectious diseases. dengue fever provides a prime example of the intersection of the triad. while dengue viruses were dispersed throughout the tropics in the first half of the twentieth century, epidemics were infrequent because urban populations were comparatively small, and the viruses and mosquito vectors were transported on ships versus the air transport of today. the travel of both goods and people during world war ii set the stage for the spread of dengue fever. in the post war era with unparalleled urban growth and travel, serious epidemics occurred more frequently. scarcely 40 years later, dengue hemorrhagic fever became a principal cause of hospitalization and mortality in the pediatric population throughout southeast asia [108] . with respect to the intentional use of microorganisms as a weapon, the united states and the world have an outmoded threat-view focused on soviet era biological weapons, but travel, medicine abuse, and the lack of a us capability to approve and manufacture new antimicrobial and antiviral agents have changed many dimensions of the threat as discussed above. with the dissolution of the soviet union, the fact that the us biological weapons program ended decades ago, and the intellectual, medical, manufacturing, and weaponization knowledge needed to start a bioweapons program, the threat from naturally occurring organisms is far greater than the threat of bioterrorism or biowarfare in 2015. the threats of infectious diseases dwarf that of terrorism and other asymmetric threats to human life. approximately three million people died in 2012 due to lower respiratory infections [22] , and infectious diseases are the major cause of death of children under five. "the most important pathogens are rotavirus for diarrhea and pneumococcus for lower respiratory infections [109] ." however, there is hope that new antibiotics will be identified and developed. recent research such as that performed by ling et al. [110] found new ways to identify antibiotics [111] in the environment and companies are beginning to invest again. under the direction of dr. kim lewis, ling and colleagues identified teixobactin. to do this, the team used the novel screening method to examine 10,000 strains. in both in vitro and in vivo tests, teixobactin was demonstrated to be operative, without major side effects, against the organisms that cause common illnesses such as pneumonia, tuberculosis, and staph infection, diseases which sicken more one million americans yearly. while teixobactin was effective against diseases of public health concern, it was ineffective against gram-negative bacteria. teixobactin binds on several targets triggering cell wall break down. the ability to bind on multiple sites lessens the chance of early teixobactin resistance. in addition to developing the new antibiotic, the researchers commercialized the screening technology, which can examine organisms that cannot typically be cultured in the lab [111] . researchers are also developing techniques to enhance the impact of probiotics in fighting infections and other diseases such as cancer [112] . while recent events bring the threat of microorganisms to the forefront of the public mind, the work of doctors, researchers, public health professionals, and other experts have continued unabated for decades. these attempts include scientific, technological, policy, and commercial attempts to reduce or eliminate the deaths and other losses caused by pathogens. to a large extent, these efforts have succeeded. in 1900, the average lifespan in the united states was 46.3 for men and 48.3 for women, and one of the predominant causes of death was infectious disease. by the end of the century, lifespan had increased to 73.8 for men and 79.5 for women [113] . in 1848, infectious diseases accounted for more than half of all deaths: in 1971, this percentage was reduced tenfold [114] . the increases in life expectancy have been distributed across the world, although some areas have benefitted more than others from breakthroughs in sanitation, nutrition, and medical advances. one of the primary contributors to the reduction in the death rate was the reduction of infant deaths due to infectious diseases. prior to the mid-1930s, infectious disease played the predominant role in infant mortality with half of the 100 (out of 1000) infant deaths due to pathogens [115, 116] . by 2014, the united states infant mortality rate had decreased to 6.1 per 1000 live births [117] . also in the united states, in the midnineteenth century, foodborne and waterborne diseases such as typhoid, cholera, and dysentery resulted in 214 deaths per 100,000. these diseases were eliminated in the united states by the early 1970s [114] . one noteworthy exception to the steady progress in increased life expectancy is due to an infectious disease: hiv/aids decreased life expectancy dramatically in parts of africa over the past 30 years [118] . the leading causes of death and illness have shifted from infectious and parasitic diseases to noncommunicable diseases and chronic conditions. with the introduction of widespread antibiotics [119, 120] in the 1940s and antivirals in the late 1980s [121] , a new era of public health was ushered in, and the death rate due to infectious diseases accounted for less than 20 % of mortality worldwide [22] ; however, the optimism was short lived. even before there was prevalent proof that bacteria could quickly evolve to thwart antibiotics, evidence indicates that bacteria exhibit resistance in nature even without human pressure [122] ; however, mechanisms of resistance impacting disease treatment were first noticed in the late 1930s with regards to the use of sulfonamides [41] . due to overuse, underuse, and incorrect disposal, antibiotic resistance has become a worldwide threat to public health [41] . in addition, the cost and difficulty in developing new antibiotics has stunted the pipeline. finally, environmental [89] , behavioral, and other physical and cultural changes have fostered situations where new pathogens can emerge and old enemies reemerge or spread to new locations. global climate change is altering where species thrive, and more localized or temporary changes modify infectious disease risk to humans as well. while ndm-1 strains are difficult to treat, many of them remain sensitive to an older, seldom used antibiotic, colistin, or aztreonam [123, 124] 85 years, clinical trial number n c t 0 1 7 0 6 3 6 7 ; a n d s a f e t y, to l e r a b i l i t y, a n d immunogenicity study of a clostridium difficile toxoid vaccine in healthy adult volunteers, clinical trial number nct00127803 (a total of 15 studies were found on www. clinicaltrials.gov when searching for 'c. difficile vaccine [125] .' improvements are needed in dosage and timing to achieve high level immunity, however the investment required is large with estimates ranging from $500,000, 000 to $1000,000,000 [126] to take a vaccine or antibody, respectively, through clinical trials. until a vaccine is developed, antibiotics will be used to treat infections. fidaxomicin, the first new antibiotic approved by the fda to treat cdi was approved in may 2011. it was shown to be as effective as oral vancomycin, previously the only fdaapproved therapy for mild-to-moderately severe cdi. vancomycin is expensive and resistance in enterococci is a concern. oral metronidazole has been used by the medical community off label (it was approved for the treatment of certain anaerobic bacteria and parasites); however, relapse was observed in a quarter of patients within a month following treatment. fidaxomicin, in addition to being as effective as standard treatment, is a narrow spectrum antibiotic, allowing patients to maintain healthy native gut microbiota [127, 128] . on a larger scale, according to the world health organization (who), hiv mortality was reduced from 1.7 million in 2000 to 1.5 million in 2012, and diarrhea fell from one of the top five causes of death to number seven, with a similar number of deaths to hiv/aid in 2012 [22] . tuberculosis distribution has declined since the turn of the century, in part because of the reach of the who's directly observed therapy short-course strategy and the implementation of the stop tb partnership plan [129] . malaria cases and mortality has been meaningfully reduced by over 670 cases and four million people respectively over the 12 years between 2001 and 2013 through the use of artemisinin-based drugs, distribution of insecticide-treated bed nets, and indoor residual spraying of insecticide [130] . this demonstrates that research, infrastructure, and other health-based investments have improved prevention and response to infectious diseases. all of this comes at a cost: between 2012 and 2014 governments including the united states, the uk, australia, canada, france, and germany and large non-profits and international institutions such as the gates foundation and the global fund contributed more than $32 billion to the fight against hiv/ aids and nearly $20 billion for international maternal and child health, which is in large part funding for vaccination [131] . in addition, president obama has recognized that infectious diseases pose a national security threat. on september 16, 2014 , in his weekly address [132] , the president stated, "so this is an epidemic that is not just a threat to regional security-it's a potential threat to global security if these countries break down, if their economies break down, if people panic. that has profound effects on all of us, even if we are not directly contracting the disease. and that's why, two months ago, i directed my team to make this a national security priority." because the challenges of new and re-emerging infections are complicated, a combination of science and technological advances, policy initiatives, and cooperative institutions are required. to make a significant difference, the united states and other countries must invest in technology and have systems capable of making these advancements available to those who need them, build technology development, and public health infrastructure; put in place policies and institutions that encourage these investments both in the public and private sectors. the success of programs such as the malaria initiative that combine these approaches is self-evident, but more needs to be done. an illustrative, but not complete, discussion of recent and additional proposals/initiatives is below. the united states, other countries, states, and international institutions have taken many steps to combat the threat. below are many of the important efforts and characteristics needed for resilience to the microbial threat. most importantly, it is critical to have a well-defined leader who is responsible for directing and monitoring progress as well as communicating risks. in president obama's september 2014 executive order [133] , he directed the "national security council staff, in collaboration with the office of science and technology policy, the domestic policy council, and the office of management and budget to coordinate the development and implementation of federal government policies to combat antibiotic-resistant bacteria [133] ." the president also created both a task force and an advisory council; however, he did not put a single individual in charge. identifying and developing a central, qualified, trusted person in charge of coordinating the investments in research, infrastructure and outreach; policies to incentivize behaviors to improve medicine development, infection control in medical and community settings; and communicate risks and responses in a directed and trusted manner at the federal government level, will enhance accountability and the likelihood of success. during times of low or chronic threat (e.g., flu season), the named person can develop a trusted relationship with the public, the medical and public health communities, the pharmaceutical industry, the defense department, international peers, and others involved in infectious disease response and defense. this is particularly difficult in diverse countries with divided political parties. a history of purposeful and innocent ethical lapses and scientific mistakes have contributed to a lack of trust such as the inaccurate flu vaccine in the 2014-2015 season and the confusing messages from the texas hospital and the cdc on ebola in 2015. when a man traveled from africa and came down with a high fever and other symptoms, he was sent home by the hospital with antibiotics for two days [134]: ebola was not well diagnosed in texas. one of the last trusted public health officials was the surgeon general under ronald reagan, dr. c. edward koop. by the time he stepped down in 1989, he had become a household name, a rare distinction for a public health administrator. "dr. koop issued emphatic warnings about the dangers of smoking, and he almost single-handedly pushed the government into taking a more aggressive stand against aids [135] ." dr. anthony fauci, director of the united states national institutes for allergy and infectious disease, has been a source of trusted and accurate infectious disease related information recently with regards to the ebola outbreak of 2014. fauci is a natural leader for the us infectious disease/public health message, "he is someone who is really trusted by all the different organizations and people surrounding the aids challenge, ranging from the scientific community, the academic community and the activist community," according to louis sullivan, m.d., secretary of health and human services during the first bush administration and president emeritus of morehouse school of medicine in atlanta. "i don't know of anyone as broadly accepted by all those disparate groups [136] ." the head of the cdc can also be a valuable spokesperson, but the cdc may have lost some of the public's trust during the ebola crisis [137] . to centralize response, president obama appointed rob klain as the ebola coordinator. he was neither a doctor nor a scientist, and he left the job after six months, while ebola was still spreading in africa. while additional capability was developed at medical centers in the united states under klain's tenure, there were few noticeable signs of progress; he was not open to the media [138] ; and likely as a result, was not embraced by the public. if the president chose a well-respected individual with healthcare and pharmaceutical industry expertise to serve in the white house to coordinate policies, funding and messages from nih, the cdc, the department of defense, the state department, state public health agencies, and other national and international institutions involved in the chain of prevention, detection, and treatment of infectious disease, it would be optimal. critical manufacturing capabilities have moved overseas, particularly to india and china. the us government could provide tax and other incentives and clear policies for approval for drugs, biologics, and manufacturing facilities to get manufacturing of key ingredients back to the united states. this would allow a faster and more certain response in times of emergency and the allow the government to initiate emergency medicine production under president obama's march 2012 executive order [139] -national defense resources preparedness for manufacturing and distribution of medicines during times of crisis and the defense production act of 1950 as amended [140] . international institutions are making significant efforts in preventing, detecting and responding to infectious diseases, and the continued work and support through the who, un, nato, the pan american health organization, the g20, the cdc global health initiative, and other domestic and international bodies will improve international surveillance, reporting, prevention, and response. mechanisms for early reporting would avoid punishment such as travel bans for acknowledgement of dangerous infectious diseases within countries' borders. in addition, leaders in the united states would work to develop trusted relationships with peers in other countries. with more us foreign aid directed towards building public health infrastructure, the funds would have the primary impact of bolstering response and reducing transmission and casualties from infectious diseases within a country and secondary impacts of stabilizing societies (studies have shown that countries with healthy populations are more stable [141] ). these outcomes would result in a safer and more secure world as there would be reduced disease transmission across borders. there are many existing global and domestic health initiatives such as the following: [142] . lessons learned from this work can be utilized to further the goals of improving prevention and response to infectious disease. a research and response focus on diseases we encounter in the modern era as opposed to an emphasis on old soviet threats (unless the intelligence community identifies specific threats in the areas of bioterrorism and biowarfare) would enhance prevention and response capacities and funnel limited resources to current health and disease issues. preparations for naturally occurring outbreaks will not only prevent deaths year to year, but will also help exercise countries to fight intentionally introduced diseases by developing policies, procedures, infrastructure, and new technologies that foster quick innovation and therefore response to any microorganism, natural or manmade. each day, there are technological advances for preventing and combatting infectious diseases in addition to the progress specifically in medical research. for instance, adoption of advanced wastewater treatment systems can reduce exposure to antibiotics and args. this can be accomplished by tax incentives and partial payment by the federal government when wastewater treatment systems are replaced and advanced systems are used. in 2014, $109 billion federal dollars were spent on water utilities (water supply or treatment) accounting for approximately one quarter of public infrastructure spending [143] . state and local governments spent $208 billion for the operation and maintenance of infrastructure double the spending on capital improvements ($112 billion). "although state and local governments rely primarily on their own revenues to purchase capital, federal grants also are an important source of funds. since 1960, federal grants have accounted for one-third or more of the capital spending on infrastructure by states and localities. that share was considerably larger from the mid-1970s through the mid-1980s as a result of federal support for water utilities after passage of the clean water act in 1972 [143] ." a renewal of this investment, with a focus on improving water treatment to remove antibiotics, args and other pollutants and destroying resistant organisms, would expand the positive results. regulations limiting the concentrations of antibiotics and args in treated municipal water, if enacted, in concert with meaningful financial penalties for those violating these standards, may significantly reduce the risk of population exposure. this can be difficult because the source of the contamination is often hard to identify. current antiviral drugs have several disadvantages including their specificity, toxicity and expense. researchers at the charles draper stark laboratories developed draco (double-stranded rna activated caspase oligomerizer). in lab-grown cells, draco killed 15 different viruses, including ones that cause the common cold, influenza, polio and dengue fever with minimal effects on healthy cells [144] ; however, there is still much work to be done before this drug can be fda approved and used by the general public. vectored vaccines use a live-vaccine made with a partial pathogen. they have been developed against sars-cov and demonstrated in mice, but the safety of vesicular stomatitis virus vaccine (vsv) in humans requires further research. newcastle disease virus, a host range-restricted virus, has been developed as a vaccine vector for intranasal immunization against emerging pathogens [145] . science informs advances in drug development. for instance, authors reviewed a variety of genome sequence and gene knockout data for acinetobacter spp., with a focus on the critical systems to find the most appropriate sequences to target for therapies [146] . this is just one early example in the explosive field of bioinformatics. in 2004, in recognition of the importance of bioinformatics as a tool to diagnose and develop therapeutics for infectious diseases, the national institute of allergy and infectious diseases established four bioinformatics resource centers (brcs) to collect, store, and share bioinformatics information on bacteria, viruses, eukaryotic pathogens, and invertebrate vectors of human pathogens. as with the factors involved in the rise of the threat the responses are interrelated. the fda is, and must continue to, evolve its policies and regulations in the approval process so that research can proceed to the stage where drugs and biologics are ready for human use. this is discussed in more detail in the policies section below. because infectious diseases do not respect borders, it is in the strategic interest of the united states, the european union, and other countries with developed public health systems to invest in global public health infrastructure. this requires both a long-term investment as well as an acute response capability. president obama recognized both of these in the fall of 2014. first on september 24, 2014 at the global health summit, president obama discussed long-term capacity building: "we, collectively, have not invested adequately in the public health capacity of developing countries." "this speaks to a central question of our global age-whether we will solve our problems together, in a spirit of mutual interest and mutual respect, or whether we descend into the destructive rivalries of the past. when nations find common ground, not simply based on power, but on principle, then we can make enormous progress. [147] " a few weeks later, president obama discussed the acute strategic needs, "as i have said from the start of this [ebola] outbreak, i consider this a top national security priority. this is not a matter of charity-although obviously the humanitarian toll in countries that are affected in west africa is extraordinarily significant. this is an issue about our safety [148] ." the president also signed the executive order on combating antibiotic-resistant bacteria in september of 2014 [133] . recent outbreaks of diseases thought banished from the united states demonstrate the need for full vaccination. several communities resist vaccination, and incentives to vaccinate will increase population safety and prevent those who cannot be vaccinated from coming down with vaccinepreventable diseases. one common incentive is the requirement to be vaccinated to enter public school. waivers can be sought, but to boost the vaccination rates, state and local governments can reduce the numbers of exemptions provided. mississippi has already followed this course, and it has the highest vaccination rates in the united states. other potential policies include requiring exemption forms to be filed yearly; requiring parents to complete an education component; and requiring private as well as public school children to be vaccinated [149] . several states are implementing one or more related measures. while only four states do not recognize a religious exemption from vaccinations, 33 states do not allow exemptions for personal reasons (all states allow exemptions for medical reasons). in part due to the 2015 measles outbreak, on july 1 of 2016 california will eliminate all non-medical vaccine exemptions. pennsylvania is also pondering eliminating personal exemptions. colorado has made the exemption process more burdensome [150] . dina fine maron of scientific american [151] suggested the following common sense approach: improved education and communication, sustain and enhance immunization outreach, maintain vigilance and rapidly contain imported infections. anthony fauci proposed partnerships, among government, industry, and academia to develop additional timely solutions to the threat of new and resurgent infectious diseases [152] . one example of a successful academia-industry partnership is the response to the hiv/aids epidemic. aids was first recognized in the early 1980s and the death rate steadily increased through the mid-1990s when it was recognized as a worldwide epidemic. research at and collaboration among academic institutions (including wayne state university) and investment by the public and private sectors (burroughs wellcome which later became glaxosmithkline) led to the development of the antiretroviral treatments used today. the partnerships transformed a deadly infection into a principally chronic disease within two decades [153] [154] [155] . partnerships now work to ensure prevention, testing, distribution of anti-hiv/aids drugs and treatment worldwide. over the years fda has introduced innovations for the development and approval of pharmaceuticals including fast track, parallel track, orphan drugs, surrogate endpoints, noninferiority [156] . according to the fda guidance [156] , a non-inferiority (ni) study is used to demonstrate that the degree of inferiority of the drug being tested as compared to the control (an already approved drug) is less than the noninferiority margin. recently, to facilitate the development of biopharmaceuticals, a cross-industry group, including members from astra zeneca, university of texas medical school houston and smaller pharmaceutical companies, proposed a tiered evidence-based regulatory approach. in this approach tier a is the typical large phase iil approach and tier d is equivalent to the animal rule, which states that "for drugs developed to ameliorate or prevent serious or life threatening conditions caused by exposure to lethal or permanently disabling toxic substances, when human efficacy studies are not ethical and field trials are not feasible, fda may grant marketing approval based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is reasonably likely to produce clinical benefit in humans [157] ." tiers b and c rely heavily on preclinical data and combined animal and human pharmacokinetic and pharmacodynamic (pk-pd) data fully integrated into a limited clinical program [158] . in the c. difficile study discussed above, suggestions for prevention include: limit contact, limit inappropriate antibiotic usage, and increase surface cleaning. handwashing with soap from dispensers with sealed refills instead of open refillable dispensers can lower the risk of infection [159] and is just one example of a common sense technique to prevent the spread of many bacterial infections. another common sense response is increased monitoring. cryptosporidium parvum did not appear to pose a risk until 400,000 people became ill, and approximately 100 people died of cryptosporidiosis in milwaukee's water service area in 1993. today, regulators and public health scientists are trying to identify microbes that pose a similar risk in the future. if these microbial contaminants occur in raw water supplies, they may need monitoring and treatment prior to these waters entering the potable water distribution system. the contaminant candidate list (ccl) developed by the united states environmental protection agency outlines a series of biological contaminants of concern that are not currently regulated but may pose a threat. should these contaminants move from the ccl to a regulatory framework, water supply utilities will incur added monitoring and testing of their water supply sources, and potentially added monitoring and treatment costs in their operations, but safety will likely increase as a result of these expenditures. the article discusses many of the problems and solutions due to emerging pathogens with a focus on the impact and response in the united states. these challenges are exacerbated in less well-off countries with poor sanitation, lack of access to preventative health care, unstable governments, or weak public health infrastructure. awareness is key, and this and other articles are working to spread the message. the threat from emerging diseases is continuously evolving as evidenced by the recent appearance of the zika virus. while the virus itself was isolated from the zika forest in uganda in the first half of the twentieth century, it did not begin to take a serious human toll until 2015 when it traveled from the pacific islands to brazil [160] : it is now considered a global threat, with its vector, the aedes species mosquito living on all continents [161] . there have been more than one million cases in brazil, and researchers noticed a surge in fetal microcephaly, a small head size for gestational age and sex indicating issues with brain growth, in zika-prone locations [160] . it is now widely accepted that maternal infection with zika can lead to serious consequences for a fetus. for most infected, the effects will be minimal, but in addition to the fetal effects guillain-barre increases have been associated with zika infections. reliable diagnosis is not yet widely available, but reverse-transcriptase polymerase chain reaction (rt-pcr) testing of serum in the first seven days after symptom onset or igm-capture enzyme-linked immunosorbent assay (mac-elisa) analysis of samples are the most promising methods [158] . animal models for further research, therapeutics and vaccines are required [160] to stop the negative impacts of the disease since the vector is widespread and difficult if not impossible to eradicate. the general growing awareness of the threat posed by infectious disease because of travel, urbanization and all of the other factors described above combined with the serious consequences, primarily for pregnant mothers and their fetuses led to one of the fastest global responses to an infectious disease in the history of humankind. on april 6, 2016, president obama announced that he would direct $589 million in federal dollars remaining from the fund to fight ebola to fight the zika virus. the money will primarily be used for cdc and nih research on the virus, its role in birth defects, and vaccines for prevention. funds will also go to the formation of cdc response teams. this funding falls short of the $1.9 billion in emergency money president obama initially requested, and the shortfall is likely to delay a complete, effective response. internationally, the who designed and disseminated a global strategic response framework and joint operations plan, which can be accessed at http://www.who. int/emergencies/zika-virus/response/en/ . compare this to the response to polio, an enterovirus that causes few symptoms in the vast majority of cases, but can cause paralysis and even death in 1-2 % of cases. though poliovirus circulated in the population for hundreds of years, it did not reach epidemic propositions until the early 1900s. it took nearly 100 years to develop a vaccine and implement widespread vaccination so that in 1994 polio was eradicated in the western hemisphere. polio is now endemic in only three countries: afghanistan, nigeria, and pakistan. more recently effective prevention and treatment options for hiv/aids did not take hold for decades. this timeline is now significantly reduced. research is already underway on vaccines for zika as well as prevention through vector control. we do not know exactly which microorganism will become the next virulent threat, but surveillance and monitoring, robust public health and research infrastructures, policies to encourage the approval of treatments and vaccines, and openness and communication will allow for the quickest responses possible to any emerging, currently unknown threat. hiv 'epidemic' triggered by needle-sharing hits scott county american who contracted ebola now in critical condition flu vaccine less effective than expected: cdc superbug' outbreak at california hospital, more than 160 exposed deadly cre bugs linked to hard to clean medical scopes. deadly cre bugs linked to hard to clean medical scopes painful virus [chikungunya] sweeps central america cdc health advisory regarding the potential for circulation of drifted influenza a (h3n2) viruses the soviet union, russia, and the biological and toxin weapons convention. the nonproliferation review bacillus anthracis bioterrorism incident investigation of bioterrorism-related anthrax, united states, 2001: epidemiologic findings origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china future vaccines for a globalized world first blue bell ice cream, now sabra hummuswhy all the food recalls lately? the perpetual challenge of infectious disease global trends in emerging infectious diseases emerging infections: microbial threats to health in the united states health care costs and mortality associated with nosocomial diarrea due to clostridium difficile clostridium difficile: an emerging pathogen in children narrative review: the new epidemic of clostridium difficile-associated enteric disease the top 10 causes of death: fact sheet n°310 trends in tuberculosis-united states lyme disease: call for a "manhattan project" to combat the epidemic self-reported lyme disease diagnosis, treatment, and recovery: results from multidrug-resistant acinetobacter baumannii: an emerging pathogen among older adults in community hospitals and nursing homes review; leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune venegas a, fresno health officials warn of leptospirosis outbreak among dogs the most widespread animal-to-human disease in the ecological correlates of risk and incidence of west nile virus in the united states west nile virus: the complex biology of an emerging pathogen noroviruses: the leading cause of gastroenteritis worldwide molecular pathogenesis of vibrio vulnificus vibrio vulnificus: disease and pathogenesis wound infections caused by vibrio vulnificus and other marine bacteria cutaneous injury and vibrio vulnificus infection dysbiosis signature of fecal microbiota in colorectal cancer patients hepatocellular carcinoma and hepatitis b virus: a prospective study of 22707 men in taiwan origins and evolution of antibiotic resistance. microbiol impact of antibiotic resistance on clinical outcomes and the cost of care emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drugresistant tuberculosis or totally drug-resistant strains in iran pseudomonas aeruginosa: resistance to the max genetic markers of widespread extensively drug-resistant pseudomonas aeruginosa high-risk clones national prevalence of methicillin-resistant staphylococcus aureus in inpatients at united states health care facilities multistate point-prevalence survey of health care-associated infections surveillance cultures growing carbapenem-resistant acinetobacter baumannii predict the development of clinical infections: a retrospective cohort study antimicrobial resistance in europe and its potential impact on empirical therapy epidemiology of methicillinresistant staphylococci in europe comparison of mortality associated with methicillin-resistant and methicillin-susceptible staphylococcus aureus bacteremia: a meta-analysis the impact of methicillin resistance in staphylococcus aureus bacteremia on patient outcomes: mortality, length of stay, and hospital charges costs and outcomes among hemodialysis-dependent patients with methicillin-resistant or methicillin-susceptible staphylococcus aureus bacteremia adverse clinical and economic outcomes attributable to methicillin resistance among patients with staphylococcus aureus surgical site infection a nationwide, multicenter, case-control study comparing risk factors, treatment, and outcome for vancomycin-resistant and -susceptible enterococcal bacteremia health and economic outcomes of the emergence of thirdgeneration cephalosporin resistance in enterobacter species efficacy of triclosan as an antimicrobial hand soap and its potential impact on antimicrobial resistance: a focused review amphetamine use is associated with increased hiv incidence among men who have sex with men in san francisco structural interventions for hiv prevention in the united states association of methamphetamine use during sex with risky sexual behaviors and hiv infection among non-injection drug users occurrence and distribution of antibiotics in coastal water of the bohai bay, china: impacts of river discharge and aquaculture activities does the recent growth of aquaculture create antibiotic resistance threats different from those associated with land animal production in agriculture? potential ecological and human health impacts of antibiotics and antibiotic-resistant bacteria from wastewater treatment plants detection and fate of antibiotic resistant bacteria in wastewater treatment plants: a review urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review antibiotic resistance genes as emerging contaminants: studies in northern colorado phenotypic and genotypic analysis of bacteria isolated from three municipal wastewater treatment plants on tetracycline-amended and ciprofloxacin-amended growth media presence of therapeutic drugs in the environment release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in michigan aerobic digestion reduces the quantity of antibiotic resistance genes in residual municipal wastewater solids world bank. international tourism, number of arrivals emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study trends in antibiotic use among outpatients in the perils of medical tourism: ndm-1-positive escherichia coli causing febrile neutropenia in a medical tourist trade in health-related services the globalization of healthcare: implications of medical tourism for the infectious disease clinician fungal infections in immunocompromised travelers cryptococcus gattii in the united states: clinical aspects of infection with an emerging pathogen the fda reboot of antibiotic development the united states food and drug administration and the end of antibiotics the evolution of the regulatory framework for antibacterial agents why is big pharma getting out of antibacterial drug discovery? slides: generics to push statin revenues down by $7b. cardiovascular business fidaxomicin versus vancomycin for clostridium difficile infection industrial production of β-lactam antibiotics evidence linking arctic amplification to extreme weather in mid-latitudes genetic identification of a hantavirus associated with an outbreak of acute respiratory illness the ecology and evolutionary history of an emergent disease: hantavirus pulmonary syndrome impacts of biodiversity on the emergence and transmission of infectious diseases increased avian diversity is associated with lower incidence of human west nile infection: observation of the dilution effect avian diversity and west nile virus: testing associations between biodiversity and infectious disease risk mapping of poverty and likely zoonoses hotspots healthy animals, healthy people: zoonosis risk from animal contact in pet shops, a systematic review of the literature emerging infectious diseases of wildlife-threats to biodiversity tuberculosis in humans and animals: are we a threat to each other? cdc (2015) h5 viruses in the united states human food safety not likely threatened by costly avian flu economic impact of avian influenza infectious disease, endangerment, and extinction effects of environmental change on emerging parasitic diseases patterns of gastrointestinal bacterial exchange between chimpanzees and humans involved in research and tourism in western uganda promoting childhood immunizations measles activity in canada working group on measles elimination (2004) measles elimination in canada measles surveillance in canada: trends for dengue, urbanization and globalization: the unholy trinity of the 21st century global, regional and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the global burden of disease study a new antibiotic kills pathogens without detectable resistance use of ichip for high-throughput in situ cultivation of "uncultivable" microbial species new frontiers in probiotic research the 1918 influenza epidemic's effects on sex differentials in mortality in the united states the determinants of mortality infant mortality decline in the late 19th and early 20th centuries: the role of market milk state mortality data accessed 1 department of health and human services, and world health organization (2010) global health and aging on the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of b. influenzae penicillin: the medicine with the greatest impact on therapeutic outcomes antiretroviral drugs. j. c l i n the multifaceted roles of antibiotics and antibiotic resistance in nature bactericidal activity of multiple combinations of tigecycline and colistin against ndm-1-producing enterobacteriaceae new delhi metallo-βlactamase (ndm-1): an update the potential value of clostridium difficile vaccine: an economic computer simulation model clostridium difficile infection in the twenty-first century postantibiotic effect of fidaxomicin and its major metabolite, op-1118, against clostridium difficile the effectiveness of educational interventions for health professionals in direct observed therapy and the directly observed therapy short-course strategy: a systematic review protocol part3.pdf?ua=1. accessed 9 sources and focus of health development assistance 1990-2014 remarks by the president on the ebola outbreak cdc (2015) cdc and texas health department confirm first ebola case diagnosed in the dies at 96 new york times anthony fauci: unfinished business aids, bioterrorism and the evolving legacy of anthony fauci a reflective cdc director talks about how his agency is handling ebola executive order-national defense resources preparedness the defense production act of 1950-as amended why health is important to u.s. foreign policy. a milbank foundation report world health organization maximizing positive synergies collaborative group (2009) an assessment of interactions between global health initiatives and country health systems cbo (2015) public spending on transportation and water infrastruct u r e broad-spectrum antiviral therapeutics newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens essential biological processes of an emerging pathogen: dna replication, transcription, and cell division in acinetobacter spp remarks by president obama in address to the united nations general assembly remarks by the president after meeting on ebola mississippi has no religious exemption for vaccines-and hasn't seen measles in over two decades nearly all states allow religious exemptions for vaccinations. pew research center how to get more parents to vaccinate their kids emerging and reemerging infectious diseases: the perpetual challenge how academia and the pharmaceutical industry can work together: the president's lecture, annual meeting of the jerome horwitz, azt creator, dies at 93 guidance for industry antibacterial drug products: use of noninferiority trials product development under the animal rule guidance for industry a comprehensive regulatory framework to address the unmet need for new antibacterial treatments bacterial hand contamination and transfer after use of contaminated bulk-soap-refillable dispensers zika virus the global distribution of the arbovirus vectors aedes aegypti and ae albopictus acknowledgments dr. ralph mitchell inspired me to look at the world in a new way, from the perspective of the tiny organisms that make the world what it is, but also threaten that world. key: cord-299762-qr6kbwuo authors: fok, jelle anthony; mayer, clemens title: genetic code expansion strategies for vaccine development date: 2020-06-30 journal: chembiochem doi: 10.1002/cbic.202000343 sha: doc_id: 299762 cord_uid: qr6kbwuo by providing long‐term protection against infectious diseases vaccinations have significantly reduced death and morbidity world‐wide. in the 21 st century, (bio)technological advances have paved the way for developing prophylactic vaccines that are safer and more effective as well as enabled the use of vaccines as therapeutics to treat human diseases. here, we provide a focused review on the utility of genetic code expansion as an emerging tool for the development of vaccines. specifically, we discuss how the incorporation of immunogenic non‐canonical amino acids can aid in eliciting immune responses against adverse self‐proteins and highlight the potential of an expanded genetic code for the construction of replication‐incompetent viruses. we close the review by discussing future prospects and remaining challenges for the application of these approaches in the development of both prophylactic and therapeutic vaccines in the near future. the routine vaccination of large populations has been the single most effective medical intervention to reduce death and morbidity in the 20 th century. [1] for example, pioneering work on vaccines has resulted in the eradication of small pox [2] and the reduction of polio cases from 350,000 in 1988 to 66 in 2019. [3] today, routine vaccinations continue to protect millions each year against a growing number of infectious diseases. nevertheless, recent and ongoing pandemics such as those caused by h1n1 influenza a virus, [4] human immunodeficiency virus (hiv) [5] and severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [6] serve as a stark reminder that advances in vaccine development remain highly relevant. such efforts will also be crucial for creating more efficient vaccines that protect against infectious diseases, such as malaria or tuberculosis, [7] and aid efforts in which vaccines are not used prophylactically but as the primary form of treatment for a disease (=immunotherapy). [8] scientific and (bio)technological innovations have repeatedly enabled the development of effective vaccines. [9] many of the techniques for vaccine production and development that have been in use for decades (e.g. serial passaging in vitro or through abnormal hosts) [10] continue to remain relevant today. however, new approaches based on technologies that have become available over the past decades are being explored for the production of safe and effective vaccines. [11] here we focus on genetic code expansion [12] as an innovative tool for the development of prophylactic and therapeutic vaccines. with a broad chemical biology audience in mind, we first provide accessible introductions to immunology and relevant genetic code expansion strategies, before discussing protein-based vaccines, live-attenuated viruses and providing a general outlook on the utility of an expanded genetic code for the development of vaccines in the future. prophylactic vaccinations aim to provide long-term protection against pathogens (i.e. immunological memory) through activation of the adaptive immune system. [13] thus, vaccines function by inducing the production of effector cells or molecules that are capable of neutralizing pathogens by controlling their replication or inactivating their toxic components. depending on the type of vaccine, mode of delivery and additives used, the exact mechanisms by which protection is achieved will differ. [14] here we focus on the induction of a t cell-dependent humoral immune response, that is the secretion of specific antibodies and the formation of memory b cells against pathogen-specific antigens upon immunization. [15] for an in-depth review of more complex pathways that result in the secretion of antibodies in mucosal membranes (mucosal immunity) or the production of cytotoxic t lymphocytes (cell-mediated immunity), the reader is referred to specialized literature. [16] overall, the main steps (numbered 1-15 in fig.1 ) toward acquiring vaccine-induced humoral immunity are: (1-4) stimulation of the innate immune system, leading to local inflammation and transport of antigens to the draining lymph nodes (dlns); (5-12) activation of antigen specific t and b cells, upon which the latter undergoes affinity maturation and differentiation into antibody-producing plasma b cells; and (13-15) antibody secretion in the blood (and tissue) and build-up of immunological memory. [13, 15] stimulation of the innate immune system is triggered by (1) injecting a vaccine that contains a mixture of weakened pathogens or pathogen-specific antigen(s) and immune response-boosting additives (=adjuvants). the resulting local chembiochem this article is protected by copyright. all rights reserved. inflammation leads, amongst others, to recruitment of dendritic cells, which take up the antigens and are subsequently activated (2). the activated dendritic cells undergo changes in their morphology and surface receptors, facilitating the presentation of antigens (3) and inducing their migration to the dlns (4) . in response to these antigen-presenting cells entering the dlns, naïve antigen-specific cd4 + t cells undergo activation and differentiate to helper t cells (5) (6) . in parallel, activation takes place of naïve b cells that feature surface antibodies with (weak) affinity for antigens drained from the site of infection, triggering a multistep process that results in the display of antigens on the surface of activated b cells (7) . subsequently, the interaction between antigen-presenting activated b cells and antigen-specific helper t cells (8) induces b-cell proliferation and their differentiation into plasma b cells (9). the low-affinity antibodies secreted by these plasma cells typically appear in the bloodstream within a few days after the immunization (10). in a slower process, the interaction between activated helper t and b cells also triggers the generation of a geminal center (11) , where b cells undergo massive clonal expansion and affinity maturation (12). in this fine-tuning process, b cells displaying high affinity antibodies are selected and subsequently differentiate either into memory b cells (13) or to long lived plasma cells. the latter enter the bloodstream and tissues where they secrete large amounts of high affinity antibodies (14) . lastly, a few of these plasma cells migrate toward survival niches (15) , which are mostly located in the bone marrow, from which they provide long-term production of antibodies. depending on the type of vaccine and the immunological memory generated, prevention against infectious diseases can be short-term or last lifelong. [17] among the different types of vaccines available, weakened live pathogens often elicit a robust immune response, although safety concerns can limit their applicability (e.g. for immunocompromised patients). [18] conversely, vaccines based on inactivated pathogens are safer, but often require repeated administration of booster doses to prolong protection. similar trade-offs between safety and efficiency are encountered for well-defined vaccines based on subunits of pathogens (i.e. proteins or polysaccharides) [19] or those applied for therapeutic applications in immunotherapy. [8] as mentioned before, biotechnological innovations play a key role in overcoming these limitations, with this review focusing on the utility of genetic code expansion strategies for the development of improved prophylactic and therapeutic vaccines. genetic code expansion is an umbrella term for strategies that enable the ribosomal incorporation of non-canonical amino acids (ncaas) into peptides or proteins of interest. these strategies facilitate ncaa incorporation either through the global reassignment of sense codons or the suppression of nonsense codons (i.e. stop or quadruplet codons). [12, 20] the latter strategy has proven particularly powerful, as it allows for site-selective incorporation of the non-native building block. faithful and efficient expansion of the genetic code by suppression of in-frame nonsense codons in vivo requires three principal components: (1) a metabolically-stable, non-toxic ncaa that is readily taken up by the target organism (=bioavailability); (2) a reassigned nonsense codon in the gene of interest, with the amber stop codon (uag) or quadruplet codons being the preferred choices; and (3) an orthogonal translation system (ots), consisting of an aminoacyl trna synthetase (aars)/trna pair, which exclusively recognizes the ncaa. in this context, orthogonality refers to the fact that none of the ots components interact with endogenous amino acids, aarss or trnas and vice versa. engineering otss for expanding the genetic code of prokaryotes and eukaryotes typically starts from existing aars/trna pairs from archaea, which are poorly recognized by prokaryotic and eukaryotic translation components, thereby providing an initial level of orthogonality. [21] next, the trna anticodon is mutated to the desired sequence (e.g. cua for the reverse-complement of the uag amber stop codon) and, if necessary, its orthogonality can be improved by consecutive rounds of positive (and negative) selection. lastly, the substrate binding pocket of the aars is subjected to site-directed mutagenesis and variants are selected that allow the incorporation of the ncaa of interest. these engineering efforts typically result in selective and efficient otss that load a ncaa onto an orthogonal trna, with the charged trnas subsequently being recruited to the ribosome, where in-frame uag stop codons in mrnas can be suppressed (fig. 2) . although low read-through efficiencies typically result in limited protein yields, the suppression of nonsense codons has enabled the incorporation of more than 150 ncaas into proteins of interest in a variety of organisms. [12] for the majority, these efforts have focused on introducing ncaas harboring functional groups that enable site-selective protein modification and/or aid in elucidating, altering or regulating protein function. [22] more recently, ncaas featuring uniquely-reactive side chains have also been employed to select cyclic peptides with novel ring architectures, design enzymes with new-to-nature reactivities and create organisms whose lives are dependent on these non-native building blocks (=synthetic auxotrophy). [23] in this minireview, we add to this growing list of applications that repurpose otss by highlighting their utility for vaccine development. the immune system of mammals is finely tuned to discriminate between endogenous (self) proteins and those from potentially pathogenic foreign entities (nonself). [24] tolerance to self-proteins is primarily achieved through the inactivation of self-reactive b and t cells during their development, a process that is crucial to prevent misguided attacks of the immune system. [25] the consequences of inadvertently breaking this self-tolerance is readily apparent by the adverse effects of >80 known autoimmune diseases. [26] nevertheless, breaking self-tolerance can also be desirable, for example for cancer immunotherapy, [8] or for treating diseases caused the by adverse actions of self-proteins, such as chronic inflammations and osteoporosis. however, eliciting strong effector mechanisms (e.g. neutralizing antibodies) against adverse self-proteins remains challenging. intentionally breaking self-tolerance typically relies on immunization with modified selfproteins to elicit antibodies that are selective for both the modified and the parent self-protein. [27] while such cross-reactive immune responses to adverse self-proteins have been induced by introducing foreign t-helper cell epitopes into chimeric antigens or by extensive chemical derivatization of self-antigens, the design of effective immunotherapeutics remains a slow process. in contrast to nonselective chemical modification or the introduction of epitopes which can disrupt protein folding, genetic code expansion offers a controlled means to modify therapeutically-relevant proteins. for breaking self-tolerance, nitrated or sulfonated ncaas are of particular interest. in addition of these oxidative stress-induced modifications in self-proteins being associated with the onset of a number of autoimmune diseases, [28] nitroaryl groups have also been used as highly immunogenic haptensmolecules that induce a strong immune response when attached to proteins. [29] while not fully understood, the molecular basis for recognition of nitroaryl groups by the immune system is typically attributed to the interactions of the electron-deficient π-systems with tryptophan and tyrosine residues that are common in b-or t-cell receptors. thus, the ncaas p-nitro-l-phenylalanine (pno2phe), 3-nitro-l-tyrosine (3no2tyr) and p-sulfo-l-phenylalanine (so3tyr) can be considered immunogenic amino acids (fig. 3a) . indeed, injection of proteins of interests featuring these immunogenic ncaas has proven a surprisingly straightforward strategy to produce high-titer of antibodies that were cross-reactive to the wild-type protein (fig. 3b ). here, we will focus on studies performed on the murine tumor necrosis factor-α (mtnf-α) and the murine receptor activator of nuclear factor-κ b ligand (mrankl). tnf-α is a multifunctional cytokine that plays an important role in the acute phase of inflammation or infection and has been identified as a clinical target for treatment of multiple inflammatory diseases, such as crohn's disease and rheumatoid arthritis. [30] to elucidate its role and function in these processes, studying murine tnf-α in mouse models has proven particularly useful. for example, mtnf-α-deficient mice are viable and do not show an apparent phenotype, thus indicating that immunization against this self-protein would not have adverse effects. [31] to test the protective effect of eliciting immunological memory against mtnf-α, mice are typically subjected to a lipopolysaccharide (lps) challenge. the injection of bacterial lpss triggers the production and subsequent release of high-levels of mtnf-α, which induces a potentially lethal septic shock. [32] however, the presence of cross-reactive antibodies against the self-protein in challenged mice results in the breakdown of released mtnf-α, thereby decreasing the severity of the induced shock. [33] to test the utility of genetic code expansion for eliciting antibodies against self-proteins, schultz and coworkers produced a series of mtnf-α variants, in which surface-exposed residues were mutated to immunogenic amino acids (fig. 3d ). [34] immunization of mice with these variants yielded antibodies against the mutant protein, whilein accordance with selftoleranceattempts at immunization with wild-type mtnf-α or with variants featuring somatic mutations failed to elicit a significant immune response. notably, the ability of ncaa-bearing mtnf-α variants to generate cross-reactive antibodies against the self-protein upon immunization was dependent on the site of mutation (fig. 3d) . specifically, variants featuring immunogenic ncaas at positions lys11 or tyr86 gave rise to high levels of antibodies cross-reactive to wild-type mtnf-α. encouragingly, the antibody titers in mice were found to be sustained at >80% for at least 40 weeks after immunization, signifying the potential for inducing persistent immunity against the self-protein. lastly, the protective potential of immunization with ncaa-bearing mtnf-α variants was evaluated by the previously outlined lps challenge model. while mice that received sham injections or vaccines containing wild-type protein succumbed quickly after the challenge (survival rate of 12.5% after 3 days), immunization with pno2phe 11 -or pno2phe 86 -mtnf-α provided substantial levels of protection ( fig 3e) . a survival rate of >87.5% for these variants , immunization with an immunogenic ncaa-bearing self-protein results in the generation of antibodies that show cross-reactivity for the self-protein (c). d: structural representation of mtnf-α (pdb: 2tnf). α-carbons of solvent-exposed residues that were targeted for ncaa incorporation are shown as spheres with color representing the effectiveness of these mutations to elicit cross-reactive antibodies. e: summary of survival rates of mice challenged by lps injection after immunization with relevant mtnf-α variants. f: structural representation of mrankl (pdb: 1jtz). α-carbons of solvent-exposed residues that were targeted for ncaa incorporation are shown as spheres with color representing the effectiveness of these mutations to elicit cross-reactive antibodies (color code same as in panel d). g: representative timeline for immunization of mice against ovx-induced bone loss and cia. chembiochem this article is protected by copyright. all rights reserved. attested on the ability to elicit cross-reactive antibodies that are effective in vivo. a follow-up study by ramirez-montagut and coworkers aimed to shed light on the mechanism by which incorporation of immunogenic ncaas into mtnf-α results in the formation of antibodies with cross-reactivity for wild-type proteins. [35] one key insight this study revealed was that mice with different genetic backgrounds responded differently to the immunization with modified proteins. for example, while pno2phe 11 mtnf-α was immunogenic in c57bl/6 mice (see above), the same mutant protein failed to elicit a significant antibody response in fvb/n mice. one marked difference with respect to immune response between these two mouse strains is located in genes that encode for major histocompatibility complex (mhc) class ii molecules. in the humoral immune response (see introduction), these molecules are responsible for antigen-presentation by dendritic or b cells, thus facilitating interaction of those cells with naïve and antigen-specific cd4 + t cells. [13, 15, 36] class ii mhc genes are known to be highly polymorphic and certain genetic variations in these genes are linked to predisposition for autoimmune disease and responsiveness to immunization. [37] critically, vaccination with pno2phe 11 mtnf-α proved only effective in mice that featured the h-2 b haplotype, while mice with a different haplotype or featuring mhc class ii molecules with only three amino acids mutations responded weakly or not at all to the same immunization regime. isolated cd4 + t cells from h-2 b mice immunized with pno2phe 11 mtnf-α proved to undergo activation when presented with peptide antigens that featured pno2phe but failed to do so in response to the wild-type peptide epitope. in comparison cd4 + t cells isolated from mice featuring a different haplotype or mutated mhc-class ii molecules did not undergo activation in presence of either the wild-type or pno2phecontaining peptide epitope. together, these results are consistent with (1) the negative selection of autoreactive t-cells in the thymus [38] and (2) ncaa-incorporation generating modified (neo-)epitopes that can activate t cells, thus promoting a t celldependent immune response. therefore, a plausible mechanism for immunization with proteins featuring immunogenic ncaas involves the activation of cd4 + t cells by these neo-epitopes being presented on mhc class ii molecules on b cells, triggering clonal proliferation of antigen-specific b cells and the subsequent production of antibodies that are cross-reactive to the wild-type protein. lastly, immunization with mtnf-α variants featuring 3no2tyr or so3phe in position lys11 proved to follow the same mechanism. this observation is notable as these ncaas are naturally-occurring posttranslational modifications, which have been linked to the onset of a number of inflammatory diseases, autoimmune disorders and are believed to contribute to oncogenic signaling. [28] using genetic code expansion strategies to produce homogeneous proteins featuring these posttranslational modifications could therefore prove valuable in efforts aimed at deciphering the biological consequences of formation of 3no2tyr or so3phe-bearing neo-epitopes. the tnf cytokine family member rankl is another self-protein for which raising cross-reactive antibodies is of clinical interest. existing in both membrane-bound and soluble form, rankl is an essential factor in the activation of osteoclasts, which are cells responsible for breaking down the tissue in bones (=bone resorption). [39] as such rankl is a therapeutic target for diseases in which patients suffer from bone erosion caused by an imbalance in bone remodeling, such as in osteoporosis (op) and rheumatoid arthritis (ra). [40] indeed, the recombinant monoclonal antibody denosumab, which prevents the activation of osteoclasts by binding to rankl, is currently employed for the treatment of these diseases. [41] while effective, the repeated injection of denosumab is also costly, preventing a more widespread use. in principle, eliciting cross-reactive antibodies against rankl by immunization with ncaa-bearing variants could provide a cheaper alternative for the treatment of op and ra. [42] to enable such developments, tao et al. produced pno2phecontainig variants of murine rankl (85% sequence identity to human rankl) and tested their ability to elicit cross-reactive antibodies for the self-protein following injection into mice. [43] as it was the case for mtnf-α, the immune response generated was dependent on the site of mutation (fig. 3f) . from the four positions evaluated, mutations of tyr234 and tyr240 gave rise to high titers of cross-reactive antibodies that persisted in mice for at least 24 weeks. the protective potential of pno2phe 234 -mrankl injection was evaluated in mouse models for op and ra. while bone loss induced in female mice by ovariectomy (ovx) is a common model for op, [44] injection of chicken type ii collagen results in collageninduced arthritis (cia), which shares several pathological features with ra. [45] as anticipated, repeated injection of pno2phe 234 -mrankl (fig. 3g ) reduced the severity of symptoms both in ovx-induced op and cia mice, while immunization with the selfprotein did not have a significant effect on the progression of either disease. [43, 46] specifically, ovx mice immunized with the mutant protein showed significantly reduced bone loss in microcomputed tomography analysis. similarly, in the cia model, immunization with the pno2phe 234 -mrankl did not delay the onset of disease, but the severity of clinical symptoms and joint damage was significantly decreased when compared to nonimmunized mice. together, these results provide proof-ofconcept for the potential of applying immunization with pno2phebearing proteins as a potential alternative treatment for ranklinduced and related diseases in humans. traditionally, viral vaccines have come in either of two forms: as inactivated whole virus particles or live-attenuated viruses (lavs). [13] inactivation of viruses is straightforward by heat or the use of formaldehyde and essentially renders replicating viruses into inert antigens able to elicit an immune response. lavs, on the other hand, contain viruses that remain infective and able to undergo limited replication upon injection, but have been sufficiently weakened to not cause damage. [47] given that lavs retain some degree of infectivity, they typically elicit a stronger immune response than fully inactivated viruses particles. as such, lav-based vaccines often provide long-term protection without booster doses and in some cases can be administered orally. [17] however, as lavs maintain their ability to replicate, they have a low chance to revert to virulence, and as a result cause the disease they are meant to prevent. [48] 10.1002/cbic.202000343 chembiochem this article is protected by copyright. all rights reserved. attenuation of viruses has long been (and often remains) an empirical process in which an infectious virus is serially passaged through cell cultures, abnormal hosts and/or below normal human body temperature (i.e. cold-adapted attenuation). [9a] the virus gradually weakens as a result of adapting to these new environments, thereby lowering its virulence and ability to infect and spread in the intended host. with the advent of molecular biology, more rational approaches toward virus attenuation have become available. [49] for example, genes encoding for proteins involved in replication or assembly can be deleted from the viral genome or rendered inactive by mutation. while resulting viruses are per se replication-incompetent their production remains possible in cell-lines that express genes encoding for the missing or dysfunctional proteins. more recently, viral replication has also been controlled by installing rare codons into viral genomes to slow the translation of viral proteins (=codon deoptimization) and by the introduction of micrornas and zinc finger nucleases that selectively break down viral rna. [11,19,29a] nevertheless, lavs created by these means share some of the safety concerns associated with those based on viruses attenuated through empirical approaches. additionally, they often elicit a weaker immune response than their progenitors and their applicability can be limited to certain classes of viruses (e.g. rna viruses or nonintegrating dna viruses). in principle, making viral replication dependent on the incorporation of ncaas is a promising strategy for creating lavbased vaccines. instead of deleting or inactivating viral genes, nonsense codons are introduced as premature terminationcodons (ptcs) at permissive sites throughout the viral genome (fig. 4a) . [23] introduction of ptcs results in incomplete translation of viral proteins, thus rendering the virus replication-incompetent. however, these viruses can be produced in cell lines which express an ots that permits readthrough of the ptc, when its cognate ncaa is supplied. the advantages of this strategy are three-fold: (1) ptc viruses produced in these transgenic cell lines feature minimal structural deviations when compared to their progenitor viruses, which is desired for attaining a high level of infectivity and a strong immune response; (2) safety of the ptc virus can be enhanced by incorporating multiple nonsense codons throughout the viral genome, making reversion to a replication-competent state by mutation less likely; and (3) the introduction of stop-codons to generate lavs is possible on the dna and rna level and therefore widely applicable to different classes of viruses. [50] in the following sections, we highlight the utility of ptc-harboring viruses for controlling the replication of hiv-1 and for creating prophylactic and therapeutic vaccines for influenza a. since hiv was identified as the causal agent for aids in 1983, [51] major progress in the prevention and treatment of hiv/aids has been made. however, despite continuous efforts, it is estimated that as of 2018 more than 37 million people are living with the virus, with 1.7 million people being newly infected that year. [52] while the development of a successful vaccine is presumably the only means to control this ongoing pandemic, this task has proven challenging. [53] experimental vaccines that have been developed either do not provide sufficient levels of protection (e.g, around 30% in the rv144 efficacy trial) [54] or are not safe enough to be tested in clinical trials. for example, while deleting the nef gene in simian immunodeficiency virus resulted in a highly effective lav vaccine (>95% protection in macaques) [55] , it also showed pathogenic potential in animal models. [56] as such, applying new technologies for the development of safe and effective hiv vaccines continues to be highly relevant. toward this end, guo and coworkers were the first to apply genetic code expansion for the construction of replicationincompetent, ptc-harboring hiv-1. [50a] in a trial-and-error approach they installed uag nonsense codons at permissive sites into a number of hiv-1 genes and tested viral assembly and infectivity of the resulting ptc viruses. three structurally similar tyrosine analogues were tested for the production of ptccontaining hiv-1 in engineered cell lines featuring matching otss. while p-iodo-l-phenylalanine was not effectively incorporated in viral proteins and p-acetyl-l-phenylalanine yielded non-infectious variants, only p-azido-l-phenylalanine (pazphe, fig. 4b ) displayed the desired high suppression efficiency and gave rise to infectious viruses. in addition of ptc-virus production varying with the choice of ncaa, the viral protein targeted and the site of mutation proved also crucial, which in part could be ascribed to the complex nature of hiv-1 gene splicing and protein chembiochem this article is protected by copyright. all rights reserved. processing. [57] while ptc mutations at various sites in the hiv-1 genome resulted in the pazphe-dependent production of mature hiv-1 in transgenic cells, the resulting virions often lacked the ability to infect potential host cells (fig. 4c) . encouragingly though, variants featuring either one stop codon at position tyr59 in the gene coding for the hiv-1 protease (pr) or two ptc mutations at positions trp36 and gln127 in the group-specific antigen (gag) protein yielded infectious viruses in presence of pazphe in transgenic cells. while this study provided proof-of-concept for the creation of a live ptc viruses, the chance of these hiv-1 strains to regain functional replication by mutation of the installed nonsense codon(s) to sense codon(s) raises considerable safety concerns for their potential application as lav vaccines. to decrease the likelihood of ptc hiv-1 variants to revert to a virulent state, the use of a nonsense quadruplet codon to encode for a ncaa was explored. [58] for this, an ots adapted to decode uaga codons with nε-(tert-butoxycarbonyl)-l-lysine (nε-boclys, fig. 4b ) in e. coli could be repurposed for the incorporation of this ncaa in mammalian (hek 293t) cells. [59] to avoid mistranslation of five in-frame uag-a sequences in the hiv-1 genome, which could interfere with viral assembly, guo and coworkers created an hiv-1 variant that lacked these sequences and displayed replication and infectivity levels comparable to the parent strain. introducing nε-boclys by suppression of uaga codons at the previously identified positions tyr59 in pr or trp36 in gag gave rise to infectious hiv-1 virions, although at significantly lower levels than the parent variant. a theoreticallylower escape frequency of these hiv-1 variants results from the fact that reversion of the +1-frameshift caused by the incorporation of the quadruplet codon requires a deletion event, which is rare compared to simple point mutations that can revert uag-harboring ptc viruses. [60] lastly, another point of concern for replication-incompetent ptc-harboring viruses is that immunization with a single infection cycle hiv virus would likely result in an insufficient immune response. [61] to clear this hurdle, guo et al. generated a ptcharboring hiv-1 mutant, for which genes encoding for the ots for decoding uag with nε-boclys were inserted at a permissive site in the viral genome. [62] consequently, the replication of this mutant could be controlled in conventional cell lines by simply supplementing nε-boclys into the media (fig. 4d) . unfortunately, the infectivity of the resulting ptc-harboring virus was significantly lower than for the parent hiv-1, presumably due to the low expression levels of the ots components. nevertheless, this type of ptc-harboring virus could potentially allow for controlling hiv-1 replication by co-administering the ncaa, thus keeping the virus infective until a sufficiently strong immune response is elicited. yearly, worldwide influenza outbreaks result on average in about 3-5 million cases of severe illness and >300,000 deaths. [63] rendering the influenza viruses responsible for this loss of life into avirulent vaccines while maintaining sufficient infectivity remains challenging. [64] attenuated influenza viruses do in principle match these criteria, but immune escape due to antigenic drift and shift adds to the challenges associated with creating effective vaccines against seasonal influenza viruses. [65] with the aim to provide a more robust alternative to available attenuated vaccines, zhuo and coworkers set out to create ptc-harboring influenza a strains that could reproduce exclusively in transgenic cell lines and subsequently be used for prophylactic and therapeutic vaccination. [50b] to allow for the production of ptc-harboring influenza a viruses, a hek293t cell line was constructed with an ots for nε-2-azidoethyloxycarbonyl-l-lysine (nε-azelys, fig. 4e ). transfection of this packaging cell line with the genome of an influenza strain featuring a uag codon at a randomly-chosen site (asp101 in the nucleoprotein, np) indeed resulted in the reproduction and correct packaging of influenza a virions (fig. 4f) . critically, no virus production was observed in a conventional cell line upon infection with the progeny virus, which contrasts results obtained for cold-adapted live attenuated influenza vaccine (caiv) and codon-deoptimized influenza a viruses which both showed residual infectivity and reproductivity. [66] moreover, introduction of ptcs proved a general strategy, as for eight different influenza proteins at least one site for mutation was identified that resulted in high packaging and propagation efficiency. aware of mutation of the uag codon being a straightforward escape mechanism for ptc-harboring viruses, influenza a strains harboring 1 to 8 nonsense codons in different viral proteins were constructed (fig. 4f) . these viruses retained good packaging efficiencies (at least 67% compared to the wildtype), while showing escape frequencies over 20 passages ranging from 10 -2 to 10 -9 for variants featuring one nonsense codon to <10 -11 (below the detection limit in the assay) for those harboring more than three stop-codons. to evaluate the in vivo safety of ptc-harboring influenza a strains, ptc-4a was selected as none of its four mutations were in envelope genes, thus providing wild-type-like representation of surface antigens. following injection of ptc-4a at 100,000-times the median lethal dose (ld50) of the parent influenza strain, no mice succumbed or showed any symptoms associated with influenza infection (i.e. body weight loss or other health issues). moreover, lower viral titers were elicited after injection of ptc-4a not only compared to the wild-type virus, but also to a caiv, further underling its overall safety in vivo. similarly, when guinea pigs inoculated with ptc-4a were caged together with noninfected ones, no transmission was detected, which again contrasts results obtained for wild-type viruses or caiv, where transmission was readily observed. thus, these experiments demonstrate the overall safety of ptc-harboring influenza viruses. besides showing high levels of safety, ptc-4a was also able to elicit a strong immune response when injected into mice, ferrets or guinea pigs. in fact, this replication-incompetent influenza a virus not only gave rise to robust antibodies in serum (humoral immunity), but also to high level of secretory immunoglobulin a in the lungs (mucosal immunity) and cytotoxic t lymphocytes (cell-mediated immunity). notably, the levels for all three elicited immune effectors were boosted significantly upon a second injection of ptc-4a and overall comparable to those levels elicited by caiv. furthermore, the protective efficacy of ptc-4a virus was high: when challenged with 50 times the ld50 of the wild-type virus, all mice survived after immunization even with just one dose of ptc-4a, with injection of two doses preventing any symptoms associated with influenza infection. the strong immune response elicited provided also significant protection, when mice were challenged with antigenically distant influenza strains. such heterologous protection was ascribed to surface and internal antigens that are highly conserved among 10.1002/cbic.202000343 chembiochem this article is protected by copyright. all rights reserved. distant influenza strains and to a broad protective effect of influenza-specific of cytotoxic t-lymphocytes. [67] lastly, somewhat counterintuitively, co-injection of ptc-4a and wild-type influenza a into mice resulted in reduction of viral titers when measured over the following days. this inhibition of the wild-type virus by ptc-4a cannot be ascribed to acquired immunity, but results from attenuation by genetic reassortment between the ptc-harboring virus and the wild-type. [65, 68] upon infecting the same host cell, genes are being exchanged between the two strains and progeny viruses can become replication incompetent when they are packaged with at least one ptcharboring gene (fig. 5) . sequencing analysis of propagated viruses confirmed the creation of an assortment of replicationincompetent ptc-harboring influenza strains. notably, the attenuation effect was directly proportional to the number of ptcs introduced, with a variant featuring four or more uag stop codons essentially preventing proliferation of wild-type influenza in vitro. combined, these experiments indicate that viruses harboring multiple ptcs could not only find use in the prophylactic prevention of viral diseases, but also applied as therapeutic vaccines for the neutralization of replicating viruses. the proof-of-concept studies highlighted in this review attest on the future applicability of genetic code expansion strategies for the development of protein-and lav-based vaccines. for the former, the incorporation of ncaas such as pno2phe into selfproteins provides a straightforward means to break self-tolerance and elicit a robust humoral immune response. similarly, the production of lavs by introduction of ptcs has proven effective for containing viral replication in vitro and in vivo. the precise control over the site and number of introduced ncaas into non-immunogenic proteins makes suppression of nonsense codons a promising strategy for creating protein-based vaccines. their potential for inducing immunity against selfproteins makes the technique a promising tool for creating soughtafter therapeutic vaccines against inflammatory diseases and different types of cancer. [69] in addition to the development of vaccines against self-proteins, this strategy could potentially find application for eliciting immune response against pathogenassociated proteins that are not or weakly immunogenic. while the results obtained for self-proteins are promising, the fact that the formation of cross-reactive antibodies was dependent on the genetic background of mice in genes encoding for mhc class-ii molecules is problematic. the genetic variability of mhc genes in the population [70] and the difficulty of understanding and predicting t-cell specificity for particular peptides [71] are important hurdles to overcome for these protein-based vaccines to become applicable for treating human diseases. therefore, more systematic studies on reliably identifying possible site(s) of modification in proteins and the development of otss for more immunogenic ncaas, such as nε-dinitrophenylacetyl-l-lysine) [72] and three halotyrosine residues [73] could prove valuable to for furthering the development of immunogenic self-proteins. lastly, the ability to generate homogenous self-proteins that feature naturallyoccurring, stress-induced posttranslational modifications could provide useful insights for elucidating mechanisms of activation and progression of autoimmune disorders as well as cancer. this information could further unveil which modifications generate an immunogenic epitope and therefore expand the applicability of incorporating ncaas for the development of vaccines. creating lavs by making viral replication dependent on the presence of an ots and a matching ncaa could be widely applicable to develop vaccines for different viruses. especially for viruses for which attenuation using conventional methods is challenging (e.g. herpes simplex virus), [74] a straightforward means to create replication-incompetent variants should be of particular interest. thus far however, approaches for identifying suitable incorporation sites for ncaas have largely been trial-anderror and their effect on virus assembly and infectivity was not always straightforward to interpret. complex processing of viral genomes and proteins leading up to assembly of full virions (e.g for retroviruses) complicates finding permissive sites for ptc incorporation. [75] thus, other virus classes likely provide better targets for development of ptc-harboring lavs, as demonstrated for influenza a. in addition to further exploring the consequences of incorporating ptcs into viral genomes for the generation of infective virions, the resulting lavs must also undergo a rigorous validation with respect to their safety in vivo. intriguingly, the incorporation of ptcs into genomes might also be applicable for the development of vaccines against persistent infectious diseases caused by bacterial pathogens. specifically, multiple studies have recently succeeded to make bacterial survival strictly dependent on the presence of a ncaa and a matching ots (synthetic biocontainment). [23, 76] adapting these biocontainment strategies to bacterial pathogensnote that the ncaa incorporation in mycobacterium tuberculosis has previously been reported [77] could pave the way toward the creation of infective pathogens that can only replicate in engineered cell lines. lastly, to prevent reversion to replicating pathogens in host by mutation of introduced nonsense codons, essential enzymes of the pathogens could be engineered to make their function depend on the incorporation a ncaa. [78] overall, we are confident that future studies will continue to highlight and further progress the utility of genetic code expansion for vaccine development. 889-893; b) s. plotkin the vaccine book plotkin's vaccines handbook of cell signaling 4376-4385; b) accepted manuscript chembiochem this article is protected by copyright. all rights reserved encyclopedia of microbiology accepted manuscript chembiochem this article is protected by copyright. all rights reserved key: cord-295062-8rl4kswe authors: marsh, mark; helenius, ari title: virus entry: open sesame date: 2006-02-24 journal: cell doi: 10.1016/j.cell.2006.02.007 sha: doc_id: 295062 cord_uid: 8rl4kswe detailed information about the replication cycle of viruses and their interactions with host organisms is required to develop strategies to stop them. cell biology studies, live-cell imaging, and systems biology have started to illuminate the multiple and subtly different pathways that animal viruses use to enter host cells. these insights are revolutionizing our understanding of endocytosis and the movement of vesicles within cells. in addition, such insights reveal new targets for attacking viruses before they can usurp the host-cell machinery for replication. viruses are obligatory intracellular parasites; therefore, their replication (and the pathogenic consequences of infection) depends critically on the ability to transmit their genomes from infected to noninfected host organisms and from infected to uninfected cells. the small size, structural simplicity, and lack of any metabolic or motile activities severely limit the types of processes that virus particles can themselves undertake to promote the transfer process. as passive, inert particles, they have evolved to exploit the behavior and the physiology of their hosts. at the cell level, this is manifested in the activation of endogenous cellular responses that provide assistance to viruses so that they can cross membranes and other barriers and deliver their genes into the cytosol or the nucleus. recent studies indicate that cells offer a variety of endocytosis, trafficking, and sorting mechanisms that animal viruses can take. moreover, viruses have become valuable tools to study some of these processes. here, we review the general concepts in virus entry and discuss some of the emerging issues. virus particles as devices for targeted gene transfer a viral particle is composed of nucleic acids (rna or dna), protein, and, in the case of enveloped viruses, membrane lipids. the proteins include structural components, such as capsid proteins; matrix proteins; membrane glycoproteins; and, in many cases, accessory proteins such as reverse transcriptases, rna polymerases, kinases, and proteases. one or more shells of protein protect the rna or dna genome in the so-called capsid, which is often helical or icosahedral. enveloped viruses have, in addition, a lipid bilayer membrane that serves to protect the capsid and the genome and operates as a ''transport vesicle'' during cell-to-cell transmission. transmission involves three main stages: the assembly of virus particles in infected cells, their release to the extracellular space, and entry into a new cell. nonenveloped virus particles and the capsids of enveloped viruses are assembled in the cytosol or the nucleus of the infected cell. the lipid bilayer membrane (envelope) of enveloped viruses is acquired during a budding process through a cellular membrane. with few exceptions, the key proteins in the capsid and the envelope are encoded by the viral genome. given that the components of the virus particle are often synthesized in different parts of the cell, the assembly of a particle is a remarkable example of coordinated molecular sorting. the assembly processes involve the establishment of a complex network of specific interactions that bring the relevant viral components together into a particle with precise stoichiometry and geometry and with the exclusion of most cellular components. that all this is possible with the limited genetic information contained in viral genomes is impressive. it serves as a testament to the viruses' ingenious structural designs and the generous assistance of the cell. enveloped particles leave the infected cell inconspicuously by budding and secretion. nonenveloped viruses are usually thought to undergo release through cell lysis, but some may escape by secretory mechanisms after budding into membrane bound compartments and then losing their membrane (altenburg et al., 1980) . others may subvert cellular autophagy pathways to gain access to exocytic organelles (jackson et al., 2005) . the particles released from cells are stable structures crosslinked by networks of intermolecular interactions. they are resistant to the stresses encountered in the extracellular space during transmission from cell to cell and host to host. however, at the same time, the assembly and maturation program has made sure that the particles can fall apart at a moment's notice during entry into a new cell. the reversibility of assembly is important to liberate the genome in infectious form and release the accessory proteins. many of the stabilizing interactions in the particle must be undone: the envelope must be shed, the capsid opened, and the nucleic acids decondensed. to make this possible, the entire virus particle or specific proteins in it are locked in metastable conformations and poised to undergo major conformational changes when triggered by appropriate cues during entry . the uncoating program is thus built into the virion during assembly, allowing major transformations in the particle without the need for external energy. like assembly, entry and uncoating typically occur in several tightly controlled, consecutive steps. these steps, shown in figure 1 for a virus that enters via endocytosis and moves to the nucleus (e.g., an adeno-or polyomavirus), start with events at the cell surface and end with the decondensation of the genome at the site of replication. as the virus progresses in its entry program, it undergoes changes that lead to events such as penetration, capsid destabilization, and uncoating of the genome. many of these changes result from conformational alterations in metastable viral structures. they are triggered by receptor binding, exposure to low ph, reentry into a reducing environment, enzyme-induced covalent modifications, and other cellular cues (earp et al., 2005; harrison, 2005; hogle, 2002; smith and helenius, 2004) . viruses exploit such signals not only to induce changes in the particle and dissociation of protein subunits but also to coordinate movement from one compartment and location in the cell to another, ensuring that each step in the uncoating program occurs at the right point in the sequence, at the right time, and in the right place. although the incoming viruses depend on cues from the cell, the cell also responds to signals induced by the virus. simply by binding to surface receptors, and perhaps by clustering them, many viruses ''knock at the door'' of the cell by activating cellular signaling cascades. this is often essential because it results in the local activation of ligandtriggered processes that viruses require for entry, such as caveolar/raft endocytosis, clathrin-coat assembly, and actin-cortex dissociation. this aspect of virus entry is complex but increasingly central for understanding the infection process. in order to infect, viruses must first bind to the cell surface. the surface structures that they bind to are of two general types depending on the functional consequences of the interaction. attachment factors serve to bind the particles and thus help to concentrate viruses on the cell surface. such interactions can be relatively nonspecific. often they involve interactions with heparan sulfate or other carbohydrate structures on the cell surface (ugolini et al., 1999; vlasak et al., 2005; young, 2001) . unlike attachment factors, virus receptors actively promote entry. they can do so by initiating conformational changes in the virus particle, by activating signaling pathways, and by promoting endocytic internalization. often the receptors accompany the virus into the cell during endocytic uptake and may then play a role intracellularly in the penetration reaction. given that the interactions are usually highly specific, the presence of receptors determines to a large degree which cell types and species can be infected. these interactions and the molecules involved are of crucial importance for understanding not only the mechanisms of entry but also the biology of infection and pathogenesis. in whether enveloped or nonenveloped, many viruses depend on the host cell's endocytic pathways for entry. they follow a multistep entry and uncoating program that allows them to move from the cell periphery to the perinuclear space. in this example, the virus proceeds to deliver its uncoated genome into the nucleoplasm. the interaction between the virus and the host cell starts with virus binding to attachment factors and receptors on the cell surface, followed by lateral movement of the virus-receptor complexes and the induction of signals that result in the endocytic internalization of the virus particle. after vesicular trafficking and delivery into the lumen of endosomes, caveosomes, or the er, a change in the virus conformation is induced by cellular cues. this alteration results in the penetration of the virus or its capsids through the vacuole membrane into the cytosolic compartment. enveloped viruses use membrane fusion for penetration, whereas nonenveloped viruses induce lysis or pore formation. after targeting and transport along microtubules, the virus or the capsid binds, as in this example, to the nuclear pore complex, undergoes a final conversion, and releases the viral genome into the nucleus. the details in the entry program vary for different viruses and cell types, but many of the key steps shown here are general. recent years, hundreds of attachment factors and receptors for different viruses have been identified, resulting in a large body of valuable information (young, 2001) . the crystal structures of several viruses and viral proteins bound to receptors have also been solved (kwong et al., 1998; rossmann et al., 2002; skehel and wiley, 2000; stewart et al., 2003) . within the scope of this review, it is not possible to discuss the attachment factors and receptors in depth; however, it is important to emphasize that the list encompasses a wide variety of different proteins, lipids, and carbohydrates. included are ion transporters, adhesion factors, signaling proteins, and a variety of other cell-surface receptors. proteoglycans and glycolipids are commonly used as virus receptors or attachment factors (young, 2001) . moreover, influenza viruses and some paramyxoviruses have spike glycoproteins with lectin domains that bind to sialic acid, and the major coat protein of polyomaviruses, vp1, binds to the glycan moiety of specific gangliosides (skehel and wiley, 2000; stehle and harrison, 1997; tsai et al., 2003) . although individual interactions between viruses and their receptors are specific, they are often of low affinity. however, the avidity increase resulting from multiple receptor binding sites on virus particles often guarantees nearly irreversible binding. moreover, multisite binding is likely to cluster receptor proteins, which in turn may activate signaling pathways and/or recruitment to endocytic structures (see below). numerous viruses are known to use more than one type of receptor, either in parallel or in series. the consecutive use of cd4 and a chemokine receptor by the human immunodeficiency virus (hiv type 1) is a well-studied example of the latter. in this case, the two receptors are needed to induce major conformational changes in the hiv envelope protein that initiate membrane fusion (berger et al., 1999) . interestingly, hiv-1 can also bind to glycosylceramides and heparan sulfate, interactions that may facilitate the initial recruitment of virus to susceptible cells (long et al., 1994; ugolini et al., 1999) . moreover, the presence of specific glycosphingolipids in the target cell membrane can enhance cd4/coreceptor-dependent fusion (puri et al., 1998) . viruses, in particular those that undergo rapid mutation, are also known to be able to switch receptors (byrnes and griffin, 2000; klimstra et al., 1998) or adapt to use alternative receptors when the primary receptor is absent (vlasak et al., 2005) . this is one of the risks posed by the current avian influenza epidemic: the avian hemagglutinin glycoprotein may, through mutations, evolve to interact more potently with glycoconjugates on human cells. the entry of coxsackie b virus provides an interesting example of how viruses can exploit the different properties of cell-surface receptors to bring about stepwise entry. in this case, the host cells are epithelial cells that grow in tight monolayers. coxsackie b viruses are human picornaviruses that cause meningitis and myocarditis. they are simple, nonenveloped rna viruses that replicate in the cytosol and do not need low ph for penetration. they share an essential receptor molecule with adenovi-ruses, a glycoprotein called car (the coxsackie and adenovirus receptor) (zhang and bergelson, 2005) . in epithelial cells, car is a component of tight junctions and is therefore not accessible to incoming viruses from the apical side. a recent study addressed the question of how the virus gains access to its receptor, i.e., how it breaches the epithelial barrier (coyne and bergelson, 2006) . the starting point for the study was the observation that many coxsackie b strains interact with a coreceptor present on the apical surface of epithelial cells, the decayaccelerating factor (daf, a gpi-anchored protein) (shieh and bergelson, 2002) . virus binding leads to crosslinking of daf molecules, inclusion into lipid rafts, and activation of the tyrosine kinase c-abl (coyne and bergelson, 2006) . once activated, c-abl was found to activate the rhofamily small gtpase rac, which in turn induced a reorganization of the actin cytoskeleton and promoted transfer of bound viruses from the apical surface to the tight-junction region of the cell. this made it possible for the virus to associate with car and for car to induce a conformational change in the virus particle, an obligatory step in picornavirus uncoating and entry (hogle, 2002) . to release its rna into the cytoplasm, the modified particles were then internalized by caveolar endocytosis activated by phosphorylation of tyr14 in caveolin-1 by fyn, a member of the src family of nonreceptor tyrosine kinases. the detailed route taken by the virus inside the cell remains unclear at this point, but the final release of the viral rna into the cytosol is likely to occur in the endoplasmic reticulum (er). this study elegantly demonstrates how a virus exploits multiple cellular receptors and functions to overcome the obstacles it encounters during entry into cells. in this particular cellular context, the coreceptor is needed to overcome the inaccessibility of the main receptor. as discussed below, the signaling cascades are often important in both entry steps and downstream activation of the cytoskeleton and endocytic machinery. it is apparent that many viruses make use of the cell's signaling pathways during entry. this was first recognized for adenoviruses, which use car as a primary receptor and integrins as coreceptors (li et al., 1998; nemerow and stewart, 1999) . nemerow and coworkers demonstrated that the interaction between adenovirus pentons (protein complexes which together with the hexons make up the adenovirus capsid) and integrins activates phosphatidylinositol 3-kinase (pi(3)k), which in turn activates rac and cdc42, resulting in the polymerization of actin-and clathrin-mediated endocytosis of the virus. activation of many different signaling pathways has since been described with the involvement of a variety of factors, including serine/threonine, tyrosine, and pi kinases; phosphatases; and a variety of small gtpases (including arf, rab, and rho family members) (greber, 2002; pelkmans et al., 2005) . viruses use signaling activities to induce changes in the cell that promote viral entry and early cytoplasmic events, as well as to optimize later processes in the replication cycle. initially, the viruses need to make their presence on the cell surface known so that the cell can launch an endocytic response to bring them in. for example, the internalization of sv40 by caveolar/raft endocytosis is regulated by at least five different kinases . inhibition of tyrosine kinases in particular blocks internalization and dramatically reduces infection (chen and norkin, 1999; pelkmans et al., 2002) . in other cases, a virus may need to induce lateral movement along the membrane. this can be seen very dramatically when viruses such as murine leukemia virus and hiv-1 bind to filopodia and proceed to ''surf'' on the outside of these structures toward the cell body (lehmann et al., 2005) . as discussed above, lateral movement is also needed for coxsackie b viruses to reach the car receptor in epithelial cells, and this movement requires activation of c-abl following virus-induced crosslinking of daf (coyne and bergelson, 2006) . the signals can be generated in several ways. the viruses may activate cellular signaling molecules directly by using them as receptors. this may be why so many viruses bind integrins. viruses may also induce signaling by clustering specific cell-surface proteins or lipids. interestingly, a number of viruses use gpi-anchored proteins and gangliosides, which are only associated with the outer leaflet of the plasma membrane, as their receptors. that this often leads to activation of tyrosine kinases on the cytosolic side may be related to the fact that the gpianchored proteins and gangliosides become lipid-raft associated when clustered (coyne and bergelson, 2006; parton, 1994; parton and richards, 2003; pelkmans et al., 2002; sharma et al., 2004) . being dually acylated, some src-family kinases (though not src itself) are also enriched in lipid-raft microdomains, but on the cytoplasmic surface of the membrane. accordingly, there is increasing evidence that many viruses associate with lipid rafts to initiate intracellular signaling (coyne and bergelson, 2006; damm et al., 2005; ono and freed, 2005) . the transfer of the genome and accessory proteins through the barrier of a cellular membrane into the cytosol is called penetration. for enveloped viruses, penetration involves membrane fusion, and, for nonenveloped viruses, it involves pore formation or membrane lysis. the molecular mechanisms of viral membrane fusion are beginning to be understood in increasingly fine detail but will not be considered here (earp et al., 2005; harrison, 2005; kielian and rey, 2006) . for the most part, the penetration mechanisms of nonenveloped viruses are less well understood. however, recent single-particle cryo-em analysis of polioviruses bound to receptor-containing membranes has started to provide detailed information on the structural changes involved in picornavirus penetration and the organization of poliovirus-induced membrane pores through which the viral rna is believed to enter the cytoplasm (bubeck et al., 2005) . the cellular membrane penetrated during virus entry is either the plasma membrane or the limiting membrane of an intracellular organelle, the lumen of which viruses reach after endocytosis. in all known cases, the viruses or their capsids penetrate first into the cytosol. most rna viruses (though not all) replicate in the cytosol, often in contact with specific organelles (salonen et al., 2005) . with the exception of poxviruses and iridoviruses, dna viruses and their capsids are subsequently transported to the nucleus for replication. it has been known for a long time that certain enveloped viruses such as herpes simplex virus 1 (hsv-1); sendai virus; and many retroviruses, including hiv, have ph-independent fusion proteins and can therefore penetrate into cells by fusing directly with the plasma membrane. it is generally assumed that fusion events at the plasma membrane lead to productive infection, although this is difficult to prove because virus particles are also continuously endocytosed. among nonenveloped viruses, several families, including some picornaviruses (such as polio) and polyomaviruses, do not require low ph for penetration. while many of these are known to require endocytosis for penetration, it has been argued that some may penetrate directly through the plasma membrane, though this remains contentious (hogle, 2002) . however, if we view the whole spectrum of viruses, the majority do need endocytic internalization for penetration and productive infection, most likely because endocytosis offers real advantages. viruses that are ferried into cells inside endocytic vesicles can move deep into the cytoplasm, bypassing many of the barriers associated with the membrane cortex and cytosolic crowding (marsh and bron, 1997) and exploiting the molecular motors that are normally recruited to endocytic vesicles (dohner and sodeik, 2005) . a dependence on low ph for penetration allows viruses to use the decreasing ph of endocytic organelles as a cue to activate the penetration reactions and allows viral escape to the cytoplasm at specific locations or before the virus is delivered to the hydrolytic lysosomes (helenius et al., 1980) . in addition, no viral components remain on the cell surface after penetration for detection by the host's immune defenses. of the endocytic pathways taken by viruses, the most commonly used is the clathrin-mediated endocytic route ( figure 2c and figures 3a and 3b ). it transports incoming viruses together with their receptors into early and late endosomes. clathrin-mediated endocytosis is a continuous process, and, for virus entry, it is usually rapid and efficient (marsh and helenius, 1989) . the incoming viruses are often exposed to the acidic milieu of endosomes within minutes after internalization, and many respond to the ph drop by undergoing changes that lead to penetration. depending on the ph threshold, the site of penetration is either the early (ph 6.5 to 6.0) or the late endosome (ph 6.0 to 5.5). in some cases, such as for ebola virus, sars coronavirus, and the nonenveloped mammalian reoviruses, acidic ph alone is not sufficient to induce fusion, and proteolytic cleavages in viral proteins by acid-dependent endosomal proteases, in particular cathepsins l and b, are needed to trigger the change to the penetration-competent state (chandran et al., 2005; ebert et al., 2002; simmons et al., 2005) . for avian leukosis virus, both interaction of the viral envelope protein with a specific receptor (damico et al., 1998; hernandez et al., 1997) and low ph (mothes et al., 2000) are required for fusion. single-particle tracking of influenza virus in cultured simian kidney epithelial cells shows that, although 60% of the particles enter via clathrin-coated pits, 40% use a clathrin-independent pathway (rust et al., 2004) ( figure 2b) . surprisingly, of those that use the clathrindependent pathway, only 5% associate with preexisting clathrin-coated domains in the membrane; the majority enter via coated pits that assemble underneath the surface bound viral particles. this implies induction of transmembrane signaling by the receptor bound virus particles. a similar induction of coated-pit formation has also been observed for reovirus and semliki forest virus (sfv) (ehrlich et al., 2004; a. vonderheit and a.h., unpublished data) ; whether this represents recruitment of clathrincoat components to the clustered cytoplasmic domains of viral receptor proteins or a more complex signaling cascade leading to clathrin recruitment is unclear. endocytic clathrin-coated vesicles deliver their contents to early endosomes ( figure 2c ). these organelles, though morphologically complex, have usually been considered to be a homogeneous population. however, there is increasing evidence that viruses and other endocytosed cargo are selectively targeted to specific populations of endosomes (kirkham et al., 2005) . for example, the early in mammalian cells, many different mechanisms are available for the endocytic internalization of virus particles. some of these mechanisms, such as clathrin-mediated endocytosis, are ongoing, whereas others, such as caveolae, are ligand and cargo induced. currently, there is evidence for six pathways. (a) macropinocytosis is involved in the entry of adenoviruses. (b) a clathrin-independent pathway from the plasma membrane has been shown to exist for influenza virus and arenaviruses. (c) the clathrin-mediated pathway is the most commonly observed uptake pathway for viruses. the viruses are transported via early endosomes to late endosomes and eventually to lysosomes. (d) the caveolar pathway is one of several closely related, cholesterol-dependent pathways that bring viruses including sv40, coxsackie b, mouse polyoma, and echo 1 to caveosomes, from which many of them continue, by a second vesicle transport step, to the er. (e) a cholesterol-dependent endocytic pathway devoid of clathrin and caveolin-1, used by polyomavirus and sv40. (f) a pathway similar to (d) except dependent on dynamin-2. it is used by echo virus 1. depending on the virus and cell type, penetration reactions occur in five locations: the plasma membrane, early and late endosomes, caveosomes, and the er. note that the additional endocytic mechanism of phagocytosis also operates in many cells but has not as yet been linked to virus entry and is not included here. endosomes that receive the incoming influenza viruses belong to a population that rapidly relocates the viruses, by microtubule-mediated transport, to the perinuclear region, where penetration occurs by fusion (lakadamyali et al., 2003) . sfv fuses soon after delivery to rab5 and eea1-positive early endosomes, but the fused virus is then transported in rab7-positive carriers to late endosomes (vonderheit and helenius, 2005) . there is also a recent observation that vesicular stomatitis virus (vsv) may fuse with the internal membrane vesicles of multivesicular endosomes and that these internal membranes subsequently fuse with the limiting membrane of late endosomes to release the viral rna to the cytoplasm (le blanc et al., 2005) . in this case, penetration would involve two membrane fusion events, the first mediated by the viral envelope protein and the second by an as yet uncharacterized cellular mechanism. interestingly, the endocytic uptake and penetration of anthrax toxin also appears to involve a twostep process, the first involving interaction of the toxin with the internal membranes of a multivesicular endosome and the second a back fusion of these membranes with the endosome-limiting membrane (abrami et al., 2004) . the complexity of the clathrin-mediated endocytic pathway is illustrated by the increasing number of alternative cofactors, adaptors, and tethering proteins that are involved in the formation of clathrin-coated pits and vesicles (robinson, 2004) . using small interfering rna (sirna) silencing screens, more than 90 different kinases have recently been shown to regulate the clathrin-mediated internalization and early steps of infection of hela cells by vsv . these kinases belong to many different classes, including regulators of the cytoskeleton, cell cycle, cell growth, and membrane trafficking. they either increase or decrease the efficiency of vsv entry and early steps in the infection cycle. many of the kinases were shown to have a direct effect on the clathrin-mediated endocytosis of other ligands. that some viruses use clathrin-independent pathways for endocytosis and infection is now widely accepted. the best studied of these are the caveolar/raft pathways first observed for sv40, a simple nonenveloped dna virus (anderson et al., 1996; kartenbeck et al., 1989; stang et al., 1997) . the caveolar/raft pathways, of which there are at least three related variants ( figures 2d-2f ), seem to specialize in the internalization of lipids including cholesterol, gpi-anchored proteins, and components of cholesterolrich microdomains (lipid rafts). caveolae are also involved in the transcytosis of serum components in endothelial cells, and many cargo molecules of these pathways seem to have a role in signaling. the capsids of sv40 and the related polyomavirus are composed of 72 homopentameric vp1 protein units that resemble cholera toxin b chain pentamers (stehle et al., 1996; stehle and harrison, 1997) . like cholera toxin, these viruses bind to the sugar moiety of gangliosides and enter cells via caveolar/raft pathways that are dependent on cholesterol ( figures 2d and 2e ) and the activation of tyrosine-kinase signaling cascades (anderson et al., 1996; pelkmans et al., 2001; smith et al., 2003a; stang et al., 1997; tsai et al., 2003) . activation involves tyrosine kinases, pi kinases, and raft lipids. dynamin 2, actin, caveolin-1, and rho gtpases are also involved, depending on the virus and the cell type (pelkmans et al., 2002) . internalization occurs via caveolae that are activated for longdistance transport in the cytosol or via small vesicles that lack caveolin-1 (damm et al., 2005; tagawa et al., 2005) ( figures 3c and 3d) . the caveolar/raft pathway takes the majority of internalized viruses first to ph-neutral organelles in the cytoplasm called caveosomes ( figure 2d ). following a second activation step, the virions are then transported by caveolinfree, microtubule-dependent vesicles trafficking to the er, where penetration occurs (pelkmans et al., 2001) . a recent study suggests that a lumenal er protein, erp29, a thioredoxin homolog without thiol oxidoreductase activity, facilitates penetration of polyomavirus by exposing the hydrophobic, c-terminal arm of vp1 (magnuson et al., 2005) . in the case of sv40, we have found that the redox conditions in the er, as well as two thiol oxidoreductases, pdi and erp57, are important for sv40 uncoating and infection (m. schelhaas, l. pelkmans, and a.h., unpublished data) . since sirna silencing of derlin 1, a protein linked to the reverse translocation of substrates for er-associated protein degradation (erad), inhibits infection, it is likely that components of the erad pathway play a central role in sv40 penetration from the er. interestingly, a similar mechanism may explain how the cholera toxin a chains penetrate into the cytosol (tsai et al., 2002) . rnai silencing screens with the entire human kinome have demonstrated that, in hela cells, the caveolar/raft pathways that mediate sv40 entry depend on some 80 different kinases. the set of kinases show only partial overlap with those that regulate the clathrin-mediated entry of vsv . interestingly, many of the kinases involved in sv40 entry are known to have functions in integrin signaling and actin regulation. other viruses that use caveolar/raft endocytosis include echo 1, a picornavirus that binds to integrins, and coxsackie b in human caco-2 epithelial cells (coyne and bergelson, 2006; pietiainen et al., 2005) . for echo 1 virus, caveolar/raft uptake and entry require signaling that involves protein kinase c, and penetration seems to occur in caveosomes without the involvement of the er (pietiainen et al., 2004; upla et al., 2004) . in addition to these pathways, there is increasing evidence for clathrin-independent pathways of virus entry into the endosomal system ( figure 2b ). such a pathway has been observed for the arenavirus lymphocytic choriomeningitis virus (lcmv) and, as already mentioned, for a fraction of incoming influenza viruses (borrow and oldstone, 1994; rust et al., 2004; sieczkarski and whittaker, 2002) . the molecular mechanisms involved in the pathway (or pathways) are not understood. by contrast, following binding to car and av integrin coreceptors, adenovirus type 2 (ad2) activates an actin-dependent process classified as macropinocytosis (figure 2a) , but virus endocytosis occurs via clathrin-coated pits. inhibition of macropinocytosis inhibits virus entry, but how exactly this endocytic process influences virus penetration is unclear (meier et al., 2002) . although the formation of primary vesicles displays great heterogeneity, the subsequent steps inside the cell involve either endosomes or caveosomes and require cholesterol (imelli et al., 2004) . that these two organelle classes are not entirely independent of each other is shown by their regulation through a small subset of common kinases and by the observation that cargo can be transferred between them . together with bacterial toxins such as cholera, anthrax, and shiga toxin, viruses are emerging as valuable ligands for charting the various ligand-inducible pathways of endocytosis. the spectrum of pathways is often redundant and cell-type specific. differences in sensitivity to inhibitors, expression of dominant-negative mutants, and livecell microscopy show that the pathways differ in their kinetics and their dependence on host-cell kinases; dynamins; rac-, rab-, and arf-family gtpases; actin and tubulin; and cholesterol. the pathways are also likely to be physiologically regulated (e.g., the upregulation of compensatory pathways when clathrin-mediated endocytosis is inhibited; damke et al., 1995) . this may be one reason why viruses such as sv40 and influenza can use several different pathways, enabling them to infect a wide range of cells under various conditions. the question arises as to whether the pathways that have been identified in tissue culture systems, in some cases under unusual experimental conditions, occur in the relevant cells in vivo or whether there are additional endocytic entry mechanisms still to be discovered. it will take some effort to define how many different endocytic pathways operate in cells and their normal functional activities. further analysis using sirna screens, with larger sirna libraries and other viruses, may allow the identification of unique sets of proteins that are required for the entry of viruses that use a specific endocytic mechanism, thus providing a genetic fingerprint for each pathway. such analyses should demonstrate whether these different pathways involve distinct molecular mechanisms or whether overlapping sets of proteins mediate several variants of essentially a single endocytic mechanism. the existence of ph-independent fusion proteins has an important consequence for some viruses. when these proteins are expressed on the cell surface, they can allow fusion of the infected cell with neighboring noninfected cells expressing appropriate receptors. this leads to formation of heterokaryons, or syncytia, and potentially allows for the dissemination of the virus without the formation of mature virus particles. nevertheless, in the majority of cases, the transfer of viral genomes from cell to cell appears to occur through the formation of virus particles that are released from infected cells and use the mechanisms described above to enter new uninfected hosts. interestingly, the herpes virus varicella-zoster (vzv) appears to use cell-free viruses to spread from human to human but direct cell-to-cell transfer, without formation of infectious virions, to spread within an infected host (chen et al., 2004) . most studies of viral entry are, by necessity, conducted in tissue culture systems using cell lines that differ from the cells that are the normal targets for infection in vivo. although these systems have contributed greatly to our understanding of virus entry, they do not fully replicate the in vivo scenario, and, as a consequence, crucial aspects of entry mechanisms may be missed. in experimental systems, the entry stage of infection is usually studied by adding cell-free virus particles to cells. often the efficiency of entry is low, and experimenters frequently enhance virus adsorption by including charged polymers, such as polybrene, in the medium or by gently centrifuging virus particles onto cells (o'doherty et al., 2000) . viruses that have adapted to growing in tissue culture cells can show an enhanced tendency to use proteoglycans for initial recruitment to cell surfaces (de haan et al., 2005; vlasak et al., 2005) . indeed, hiv-1 strains adapted to grow in t cell lines appear to bind to cells via proteoglycans before engaging cd4 and coreceptors (ugolini et al., 1999) . together this suggests that the events involved in virus adsorption and entry in tissue culture may be different from those that occur in vivo. a number of recent studies suggest why this is the case. transmission of viruses from cell to cell in epithelial cells, for example, may occur through the basolateral domains, where a virus released from one cell can be effectively deposited on adjacent cells, minimizing the events associated with virus recruitment to the cell surface. vaccinia virus (vv) provides an extreme example. vv is produced in several forms, of which the intracellular mature virus (imv) and extracellular enveloped virus (eev) are capable of infecting target cells. the eev is initially assembled with three membranes and is released to the cell surface when the outer membrane fuses with the plasma membrane to produce an extracellular double-membraned particle. the eev remains bound to the surface of the producer cell and induces the polymerization of cytoplasmic actin comets that push the virus around on the surface of the cell, often at the tips of filopodium-like projections (smith et al., 2003b; wolffe et al., 1998) (see figure 4 and greber and way, 2006 [this issue of cell] ). in confluent cultures, and presumably in vivo as well, these projections push viruses into neighboring cells and enhance cell-to-cell transfer. deletion of viral proteins involved in transmembrane signaling and actin nucleation reduces the viral plaque size in tissue culture and pathogenesis in vivo (smith et al., 2003b; wolffe et al., 1998) . recently, structures termed infectious or virological synapses have been increasingly implicated in cell-tocell virus transfer. initially described for the transfer of the human t cell leukemia virus type 1 (htlv-1) from infected to uninfected t cells, these areas of intimate contact between the infected and uninfected cells were recognized to have features in common with immunological synapses (igakura et al., 2003) . in contrast to t cell killing or antigen presentation, virological-synapse formation between t cells does not require major histocompatibility antigens (jolly and sattentau, 2004) . the synapse provides a domain where virus assembly can be focused to release particles efficiently to a target cell. similar means of trans-fer have been described for herpes viruses (johnson and huber, 2002) and for the t cell to t cell transfer of hiv (jolly and sattentau, 2004) . a particularly intriguing example of infectious synapse function is that described for dendritic cells. for many years it has been recognized that a highly efficient way of infecting t cells with hiv in culture is to first present the virus to dendritic cells (cameron et al., 1992) . hiv infection of dendritic cells can occur but is generally regarded as being inefficient. recent studies have suggested an alternative mode of interaction whereby viruses may be captured by c type lectins (geijtenbeek et al., 2000; turville et al., 2002) , and perhaps other receptors involved in antigen acquisition, and internalized into endocytic vesicles without infecting the dendritic cell. this virus can be regurgitated when dendritic cells interact with cd4-positive t cells and is able to infect the t cell in trans through infectious or virological synapses-a process that mimics the normal course of antigen presentation (mcdonald et al., 2003) . viruses can be harbored in endocytic vesicles for hours, if not days, without losing infectivity, potentially allowing the dendritic cells in vivo to undergo maturation and relocation from peripheral tissue, where they would initially encounter viruses, to lymphoid organs. the endocytic compartment in which endocytosed viruses are sequestered is morphologically complex and bears some similarity to the compartments in macrophages where hiv assembles (garcia et al., 2005) . although trans-infection has been documented in tissue culture, it remains unclear whether it operates in the same manner in vivo where, for hiv at least, infection of dendritic cells and the production of viruses in infected dendritic cells may be more relevant (turville et al., 2004) . regardless, the transfer of viruses through infectious synapses is likely to operate in both scenarios. similar dendritic-cell-mediated trans-infection routes have been proposed for a number of other viruses, including herpes, filo-, and flaviviruses (van kooyk and geijtenbeek, 2003) . thus, these viruses have adapted to exploit a loophole in the defense strategies ranged against them by effectively using the cell's endocytosis and/or secretory mechanisms to allow their release to be temporally and spatially coordinated to focus infectious viruses on target cells. it is likely that similar strategies are used in various situations by different viruses; in particular, following reactivation of latent viruses, the transfer of hsv and varicella-zoster virus (chicken pox) from infected neurones to epithelial cells is likely to exploit specialized cell junctions (johnson and huber, 2002) . thus, although the full life cycle of a virus requires assembly, dissemination by release from cells, and entry into new targets, the time during which viruses are genuinely cell free may be minimal. these mechanisms may limit the extent to which a virus is exposed to the humoral immune system (johnson and huber, 2002) and may also explain, for example, why, in hiv patients, infected t cells harbor multiple proviral genomes, suggestive of cells having undergone multiple, reasonably synchronous infection events (jung et al., 2002) . many viruses only infect a restricted range of cells, limiting their species tropism and cell tropism. frequently, the restriction on infection is due to the absence of appropriate receptors. with retroviruses, however, a number of other mechanisms to limit viral replication have been discovered, and many of these operate at the level of entry (towers and goff, 2003) . for hiv in particular, several mechanisms have now been described through which virus replication can be limited independently of receptor expression. cellular rna-editing enzymes can be packaged into virus particles and can introduce mutations in the viral genome during the reverse-transcription event that occurs shortly after virus entry. hiv-1 uses its vif protein to overcome this restriction by preventing packaging of the editing enzymes into virus particles (sheehy et al., 2002) . in another example, the so-called lv1 (or ref1) restriction that prevents hiv-1 replication in old world monkeys is mediated by a homo-oligomeric protein called trim 5a (stremlau et al., 2004; towers, 2005) . trim 5a appears to bind to an exposed loop on the capsid protein that becomes available when the capsid is released to the cytoplasm after fusion (ylinen et al., 2005) . trim 5a appears to prevent uncoating of the incoming particles by crosslinking the capsid protein subunits, though in some cases it may operate after reverse transcription (ylinen et al., 2005) . the fv1 restriction seen with murine leukemia viruses operates after reverse transcription and prevents nuclear entry (towers et al., 2000) . interestingly, the fv1 restriction maps to an endogenous retroviral gag gene (best et al., 1996) , suggesting that interaction between incoming capsids and an endogenous gag protein prevents infection. in another form of restriction, now termed lv2, some strains of hiv-1 and hiv-2 fail to replicate efficiently in hela cells. the phenotype maps to specific amino acids in both env and capsids. for the env-mediated restriction, at least, the failure to efficiently infect cells appears to be related to virus delivery to a raft-mediated endocytic pathway, and experimental manipulations that disrupt raftmediated endocytosis overcome the restriction, as does diverting the virus to a clathrin-mediated pathway by pseudotyping the virus with the vsv-g protein (marchant et al., 2005; schmitz et al., 2004) . thus, not only must viruses find their way to the correct pathway for infection, but diversion to an alternative pathway, or blind alley, can limit the potential for subsequent infection and replication. other mechanisms through which the mode of penetration determines the outcome of subsequent events in virus entry appear to operate for other related viruses (kim et al., 2001) . among the many challenges for the future is the need to identify exactly how many endocytic mechanisms contribute to virus entry and to determine which viruses use these pathways. rnai screens similar to those described by pelkmans et al. (2005) will allow a systematic approach to these questions with the possibility that specific routes can be defined by the requirement for a unique set of genes and their encoded proteins. thus, each endocytic mechanism might be given a unique genetic fingerprint. understanding these processes may allow specific cellular pathways or molecular machines to be targeted pharmacologically to inhibit the entry of any virus that uses the route for infection. such an approach may offer the advantage that it will be more difficult for a virus to find a way around the block by mutation. it will also be important to show that the pathways identified in tissue culture experiments operate in vivo during a normal infection. such experiments will be difficult, especially with human viruses, but inhibitors identified through tissue culture experiments may make it possible to address these issues. at the level of cells and virus particles, developments in morphological techniques from high-end light microscopy to electron microscopy are allowing the events involved in virus entry to be analyzed with increased spatial and temporal resolution. single-particle tracking of virus particles containing components labeled with different fluorochromes or fluorescent proteins is allowing specific events to be followed in remarkable detail. for some viruses with high infectious-unit-to-particle ratios, such as sfv, such studies provide direct information on entry. more careful attention to the preparation and storage of other viruses may allow similar morphological experiments to be interpreted with the confidence that the events observed represent the behavior of infectious particles. the ability to use microscopes in high-throughput screens for inhibitors of virus entry or to determine key components in the entry pathway will have a far-reaching impact on our understanding of virus entry. together, these studies should provide detailed insights to the cellular mechanisms of endocytosis. the identification of natural ligands will also be key in establishing the biological relevance of these pathways. if we revisit this topic in 5 years' time, we will have a very much more detailed picture of virus entry. viruses will continue to hold crucial cards in our attempts to understand cells and their pathogens. acknowledgments m.m. is supported by the uk medical research council. a.h. is supported by the swiss national research foundation, the european union fifth framework (eurogenedrug), and the eth zurich. we thank annegret pelchen-matthews, stefan moese, peter rottier, varpu marjom㤠ki, urs greber, and thomas kershaw for critical comments on the manuscript, and we apologize to colleagues whose work has not been directly cited due to space limitations. membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway ultrastructural study of rotavirus replication in cultured cells bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae chemokine receptors as hiv-1 coreceptors: roles in viral entry, tropism, and disease positional cloning of the mouse retrovirus restriction gene fv1 mechanism of lymphocytic choriomeningitis virus entry into cells the structure of the poliovirus 135s cell entry intermediate at 10-angstrom resolution reveals the location of an externalized polypeptide that binds to membranes large-plaque mutants of sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to cd4+ t cells endosomal proteolysis of the ebola virus glycoprotein is necessary for infection mannose 6-phosphate receptor dependence of varicella zoster virus infection in vitro and in the epidermis during varicella and zoster extracellular simian virus 40 transmits a signal that promotes virus enclosure within caveolae virus-induced abl and fyn kinase signals permit coxsakievirus entry through epithelial tight junctions receptor-triggered membrane association of a model retroviral glycoprotein clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin clathrin-and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae murine coronavirus with an extended host range uses heparan sulfate as an entry receptor the role of the cytoskeleton during viral infection the many mechanisms of viral membrane fusion proteins cathepsin l and cathepsin b mediate reovirus disassembly in murine fibroblast cells endocytosis by random initiation and stabilization of clathrin-coated pits hiv-1 trafficking to the dendritic cell-t-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances trans-infection of t cells signalling in viral entry a superhighway to virus infection mechanism of membrane fusion by viral envelope proteins on the entry of semliki forest virus into bhk-21 cells activation of a retroviral membrane fusion protein: soluble receptor-induced liposome binding of the alsv envelope glycoprotein poliovirus cell entry: common structural themes in viral cell entry pathways spread of htlv-i between lymphocytes by virus-induced polarization of the cytoskeleton cholesterol is required for endocytosis and endosomal escape of adenovirus type 2 subversion of cellular autophagosomal machinery by rna viruses directed egress of animal viruses promotes cell-to-cell spread retroviral spread by induction of virological synapses multiply infected spleen cells in hiv patients endocytosis of simian virus 40 into the endoplasmic reticulum virus membrane-fusion proteins: more than one way to make a hairpin use of helper-free replication-defective simian immunodeficiency virus-based vectors to study macrophage and t tropism: evidence for distinct levels of restriction in primary macrophages and a t-cell line ultrastructural identification of uncoated caveolin-independent early endocytic vehicles adaptation of sindbis virus to bhk cells selects for use of heparan sulfate as an attachment receptor structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody visualizing infection of individual influenza viruses endosome-to-cytosol transport of viral nucleocapsids actin-and myosin-driven movement of viruses along filopodia precedes their entry into cells adenovirus endocytosis requires actin cytoskeleton reorganization mediated by rho family gtpases characterization of human immunodeficiency virus type 1 gp120 binding to liposomes containing galactosylceramide erp29 triggers a conformational change in polyomavirus to stimulate membrane binding an envelope-determined, ph-independent endocytic route of viral entry determines the susceptibility of human immunodeficiency virus type 1 (hiv-1) and hiv-2 to lv2 restriction sfv infection in cho cells: cell-type specific restrictions to productive virus entry at the cell surface virus entry into animal cells recruitment of hiv and its receptors to dendritic cell-t cell junctions adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein role of alpha(v) integrins in adenovirus cell entry and gene delivery. microbiol human immunodeficiency virus type 1 spinoculation enhances infection through virus binding role of lipid rafts in virus replication ultrastructural localization of gangliosides; gm1 is concentrated in caveolae lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the er local actin polymerization and dynamin recruitment in sv40-induced internalization of caveolae caveolinstabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic genome-wide analysis of human kinases in clathrin-and caveolae/raft-mediated endocytosis echovirus 1 endocytosis into caveosomes requires lipid rafts, dynamin ii, and signaling events viral entry, lipid rafts and caveosomes the neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a cd4-dependent cxcr4-utilizing hiv type 1 envelope glycoprotein adaptable adaptors for coated vesicles picornavirus-receptor interactions assembly of endocytic machinery around individual influenza viruses during viral entry viral rna replication in association with cellular membranes lv2, a novel postentry restriction, is mediated by both capsid and envelope selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein interaction with decay-accelerating factor facilitates coxsackievirus b infection of polarized epithelial cells influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry receptor binding and membrane fusion in virus entry: the influenza hemagglutinin how viruses enter animal cells ganglioside-dependent cell attachment and endocytosis of murine polyomavirus-like particles vaccinia virus motility major histocompatibility complex class i molecules mediate association of sv40 with caveolae high-resolution structure of a polyomavirus vp1-oligosaccharide complex: implications for assembly and receptor binding the structure of simian virus 40 refined at 3.1 a resolution virus maturation: dynamics and mechanism of a stabilizing structural transition that leads to infectivity structural basis of nonenveloped virus cell entry the cytoplasmic body component trim5al-pha restricts hiv-1 infection in old world monkeys assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters a conserved mechanism of retrovirus restriction in mammals control of viral infectivity by tripartite motif proteins. hum post-entry restriction of retroviral infections retro-translocation of proteins from the endoplasmic reticulum into the cytosol gangliosides are receptors for murine polyoma virus and sv40 diversity of receptors binding hiv on dendritic cell subsets immunodeficiency virus uptake, turnover, and 2-phase transfer in human dendritic cells hiv-1 attachment: another look clustering induces a lateral redistribution of alpha 2 beta 1 integrin from membrane rafts to caveolae and subsequent protein kinase c-dependent internalization dc-sign: escape mechanism for pathogens human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment rab7 associates with early endosomes to mediate sorting and transport of semliki forest virus to late endosomes role for the vaccinia virus a36r outer envelope protein in the formation of virus-tipped actin-containing microvilli and cell-to-cell virus spread differential restriction of human immunodeficiency virus type 2 and simian immunodeficiency virus sivmac by trim5alpha alleles virus entry and uncoating adenovirus receptors key: cord-291577-nf80kih2 authors: baluku, joseph baruch; mwebaza, shem; ingabire, gloria; nsereko, chris; muwanga, moses title: hiv and sars‐cov‐2 co‐infection: a case report from uganda date: 2020-05-21 journal: j med virol doi: 10.1002/jmv.26044 sha: doc_id: 291577 cord_uid: nf80kih2 there are no reports of severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) and hiv co‐infection from sub‐saharan africa where 70% of people living with hiv are found. we report a case of hiv/sars‐cov‐2 co‐infection from uganda. a 34 year old hiv‐positive female on antiretroviral therapy (tenofovir disoproxil fumarate, lamivudine and efavirenz) for 5 years, tested positive for sars‐cov‐2, the causative agent for coronavirus disease 19 (covid‐19). she was asymptomatic at presentation but subsequently developed headache, chest pain, diarrhoea, anorexia and fatigue on day 3 of isolation without cough, fever or shortness of breath. her cd4 count was 965 cells/mm(3), the hiv viral load was undetectable (<1,000 cells/mm(3)) and other laboratory work up was normal. she was successfully managed with hydroxychloroquine and broad spectrum antibiotics, and was discharged after 24 days. this case demonstrates an atypical clinical presentation of covid – 19 in an hiv infected patient without other co‐morbidity. this article is protected by copyright. all rights reserved. laboratory work up was normal. she was successfully managed with hydroxychloroquine and broad spectrum antibiotics, and was discharged after 24 days. this case demonstrates an atypical clinical presentation of covid 19 in an hiv infected patient without other co-morbidity. keywords: hiv, covid-19, sars-cov-2, uganda to the editor, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of the coronavirus disease 2019 (covid19) , has been reported in over 3.6 million individuals worldwide including at least 35,000 people in africa as of 7 th may 2020 (1) . fever, cough, fatigue and shortness of breath are the predominant clinical manifestations of covid-19, with the disease taking on a severe course in 25% of individuals (2) . advanced age (>60 years) and having a chronic underlying condition have been reported to be associated with severe disease, lack of clinical improvement and mortality among patients with covid -19 (3). sub-saharan africa accounts for more than 70% of the global burden of hiv infection, and the co-infection with sars-cov-2 among hiv-infected patients is inevitable in this region, albeit with yet to be known clinical consequences (4) . there are few case reports on hiv/sars-cov-2 co-infection. from wuhan, china, zhu et al., reported a severe case of a newly diagnosed hiv/sars-cov-2 co-infected male with diabetes who presented with fever, hypoxemia, lymphopenia and chest computed tomography (ct) abnormalities, who was managed on oxygen therapy, the hiv antiviral agent lopinavir/ritonavir, moxifloxacin, gamma-globulin and methyl prednisone (5) . this article is protected by copyright. all rights reserved. blanco et al. also reported 5 cases of hiv/sars-cov-2 co-infection -of whom 4 were virologically suppressed on antiretroviral therapy (art) -from spain, who invariably presented with cough and fever (6) . two of these had other co-morbidities while one was newly diagnosed with advanced hiv (cd4 of 13 cells/mm 3 this article is protected by copyright. all rights reserved. there was no wasting, lymphadenopathy or pallor and her temperature was 36.4 degrees celsius ( o c) (normal). she had a blood pressure of 110/80 millimetres of mercury (mmhg) and a pulse rate of 84 beats per minute (b/m), both of which were normal. the respiratory exam was significant for tachypnea (a respiratory rate of 26 breaths per minute (breaths/min)) with normal oxygen saturation (spo 2 ) of 96% on ambient air. there was no respiratory distress, and auscultation of the chest was normal. on day 3, she cd4+ t-lymphocyte percentage measured on day 12 were 965 cells/mm 3 and 40.1% respectively. the hiv viral load performed (hiv-1 rt -pcr) on a dry blood spot was <1,000 copies/ml, that is, below the threshold of detection. the urine lipoarabinomannan, for tuberculosis, was negative. a repeat fbc on day 11 was normal. all symptoms had resolved by day 12. the respiratory rate was 16 b/min, the pulse rate was 80 beats/min, and she had a blood pressure of 126/88 mmhg. throughout the admission period, her spo 2 was >96% on ambient air and the this is the first case report of hiv/sars-cov-2 co-infection from sub-saharan africa. this case report highlights important clinical aspects of the co-infection. the patient did not have fever or cough, the two commonest symptoms of covid -19 (8) . rather, she developed gastrointestinal symptoms that occur in only 12% of patients with mild covid -19 (9) . at the moment, it is unclear whether hiv-infected patients are likely to present with the less common symptoms of covid -19 although "atypical" ct scan findings have been reported in an hiv-infected patient (10) . in a case series from spain, all hiv/sars-cov-2 co-infected patients had cough and fever (6) . however, unlike the patient reported in this case report, they had other comorbid conditions, with one having concurrent pneumocystis jiroveci pneumonia (pjp). one similarity to this case report is that 4 of the 5 patients this article is protected by copyright. all rights reserved. in the case series also presented with headache. also, similar to this patient, the hiv co-infected patient who had good adherence to art and a suppressed viral load, presented with weakness and non-bloody diarrhoea with no significant clinical signs and laboratory abnormalities in a case series from turkey (11) the presentation with chest pain, tachypnea, normal auscultation findings and tachycardia raises the possibility of alternative diagnoses that could mimic covid -19. specifically, pjp and pulmonary embolism can present in a similar manner, and have been reported among patients with covid -19 (6, 12) . in this patient, pjp was unlikely owing to an apparently normal cd4 count, normal oxygen saturation and the lack of fever. the lack of a ct scan at the treatment site precluded ct angiogram imaging to rule out pulmonary embolism. a chest x-ray was not performed for this patient either. however, its diagnostic accuracy for covid -19, pjp and pulmonary embolism is low (13) . elsewhere, imaging findings among hiv/sars-cov-2 co-infected findings include normal imaging findings, basal interstitial infiltrates, ground glass appearance and pleural effusion, all of which can occur with pulmonary embolism (6, 11, 14) . a high index of clinical suspicion for concurrent chest pathology due to other disease among hiv/sars-cov-2 co-infected is needed. this article is protected by copyright. all rights reserved. save for the elevated alp, the laboratory work up of this patient was unremarkable. the elevated alp, a marker of bone resorption due to osteoblastic activity, is most likely caused by tenofovir rather than a unique feature of hiv/sars-cov-2 co-infection (15) . optimal treatments for hiv/sars-cov-2 co-infection are unknown. the successful use of hydroxychloriquine, quinolones, and azithromycin has also been reported by other case reports (5, 6) . larger studies are needed to evaluate the benefits and risks of hydroxychloroquine with or without antibiotics among hiv/sars-cov-2 co-infected. the optimal art among hiv patients with covid -19 is not established yet. this patient had been taking tenofovir, which has been showed to be effective against sars-cov-2 by binding rna polymerase (16) . the epicos trial (nct04334928) will this article is protected by copyright. all rights reserved. hopefully provide evidence for the role of co-administration of hydroxychloroquine with tenofovir. the benefit of lopinavir/ritonavir combination has been questioned among patients with severe covid -19 (17) . the hiv/sars-cov-2 co-infected cases in literature took on a varied clinical course raging from mild to severe, although cure was realised in most of the cases (5, 6, 10, 11) . it is unclear whether hiv infection is protective against severe covid -19 due to lack of a hyperactive immune response (18) . the patient presented herein had a normal cd4 count and seems to have experienced a moderate course of disease. sensitive prognostic scores for low resource settings are needed to predict clinical course of covid-19 among hiv patients (19) . in conclusion, hiv/sars-cov-2 co-infection is likely to become a common phenomenon in countries with a high hiv prevalence. there is need for larger prospective studies to characterise clinical manifestations of covid -19 among hiv patients and determine optimal treatments, including appropriate art regimens. low resource settings need to build diagnostic capacity for covid -19 "mimics" and prognostic biomarkers. world health organisation. coronavirus disease (covid-19): situation report 108 clinical characteristics of coronavirus disease 2019 (covid-19) in china: a systematic review and meta-analysis risk factors for disease severity, unimprovement, and mortality in covid-19 patients in wuhan, china hiv infection and aids in sub-saharan africa: current status, challenges and opportunities co-infection of sars-cov-2 and hiv in a patient in wuhan city covid-19 in patients with hiv: clinical case series early virus clearance and delayed antibody response in a case of coronavirus disease 2019 (covid-19) with a history of coinfection with human immunodeficiency virus type 1 and hepatitis c virus clinical presentation of covid-19: a systematic review focusing on upper airway symptoms gastrointestinal manifestations of sars-cov-2 infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis computed tomography imaging of an hiv-infected patient with coronavirus disease 2019 (covid-19) hiv/sars-cov-2 co-infected patients in istanbul, turkey acute pulmonary embolism associated with covid-19 pneumonia detected by pulmonary ct angiography frequency and distribution of chest radiographic findings in covid-19 positive patients chest radiographs in acute pulmonary embolism. results from the international cooperative pulmonary embolism registry tenofovir use is associated with an increase in serum alkaline phosphatase in the swiss hiv cohort study sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 could hiv infection alter the clinical course of sars-cov-2 infection? when less is better covid-19 severity scoring tool for low resourced settings we acknowledge the clinical care team at entebbe regional referral hospital for their dedication to the care of covid -19 patients in uganda. the authors declare no conflict of interest. none.this article is protected by copyright. all rights reserved. key: cord-291534-c6cjxq07 authors: gwyer findlay, emily; currie, silke m.; davidson, donald j. title: cationic host defence peptides: potential as antiviral therapeutics date: 2013-05-07 journal: biodrugs doi: 10.1007/s40259-013-0039-0 sha: doc_id: 291534 cord_uid: c6cjxq07 there is a pressing need to develop new antiviral treatments; of the 60 drugs currently available, half are aimed at hiv-1 and the remainder target only a further six viruses. this demand has led to the emergence of possible peptide therapies, with 15 currently in clinical trials. advancements in understanding the antiviral potential of naturally occurring host defence peptides highlights the potential of a whole new class of molecules to be considered as antiviral therapeutics. cationic host defence peptides, such as defensins and cathelicidins, are important components of innate immunity with antimicrobial and immunomodulatory capabilities. in recent years they have also been shown to be natural, broad-spectrum antivirals against both enveloped and non-enveloped viruses, including hiv-1, influenza virus, respiratory syncytial virus and herpes simplex virus. here we review the antiviral properties of several families of these host peptides and their potential to inform the design of novel therapeutics. abstract there is a pressing need to develop new antiviral treatments; of the 60 drugs currently available, half are aimed at hiv-1 and the remainder target only a further six viruses. this demand has led to the emergence of possible peptide therapies, with 15 currently in clinical trials. advancements in understanding the antiviral potential of naturally occurring host defence peptides highlights the potential of a whole new class of molecules to be considered as antiviral therapeutics. cationic host defence peptides, such as defensins and cathelicidins, are important components of innate immunity with antimicrobial and immunomodulatory capabilities. in recent years they have also been shown to be natural, broad-spectrum antivirals against both enveloped and non-enveloped viruses, including hiv-1, influenza virus, respiratory syncytial virus and herpes simplex virus. here we review the antiviral properties of several families of these host peptides and their potential to inform the design of novel therapeutics. viral diseases are a leading cause of morbidity and mortality worldwide, particularly of children [1] , and yet development of effective therapies is slow. in particular, progress is hampered by the fact that the majority of antiviral drugs are specific for only one virus. current approaches are expensive, require rapid identification of the virus before therapy and, at the initial stages of development, involve enormous redundancy of research effort. this also results in efforts being concentrated on a few viruses; of the 60 antiviral drugs that have so far been approved by the us food and drug administration (fda), almost half target hiv-1; the remaining half are used for the treatment of hepatitis b virus (hbv), herpes simplex virus (hsv), varicella-zoster virus (vzv), cytomegalovirus (cmv), influenza (iav) and hepatitis c virus (hcv) infections [2] . these factors, in combination with the rate of development of drug resistance, mean that there is an urgent need for new broader-spectrum intervention strategies. it is therefore exciting that in recent years a new class of antiviral therapeutic peptides are emerging, with 15 peptidebased intervention strategies against viruses currently in various stages of clinical trials [3] . peptide-based strategies are proposed to be cost-effective, with peptides having low molecular weights, rapid elimination following treatment, and low levels of side effects [3] . an exciting current area of advancement is in understanding the antiviral properties of naturally occurring cationic host defence peptides (chdps) and the capacity of this to inform the design of novel synthetic antiviral analogues. in this review we will give an overview of the antiviral activities of chdps and consider their potential in the development of broad-spectrum antiviral therapeutics. defence against infection is emphasised by their conservation across plants, insects, reptiles, birds and fungi [7] . in mammals the two major families of chdps are defensins and cathelicidins. defensins are cationic, amphipathic peptides generated from prepropeptides via proteolysis and are categorised within three subfamilies: a, b and h. defensins were first characterised as ''natural peptide antibiotics'' with the discovery of a-defensins from the granules of neutrophils [8] . the a-defensin family has been identified in a range of higher eukaryotes (including primates, mice, rats, guinea pigs and rabbits) and comprises six distinct peptides in humans (human neutrophil peptides (hnp) 1-4 and human defensins (hd) 5-6), expressed from five defa genes [9] . all have a triple-stranded b-sheet core stabilised by three intramolecular disulphide bonds, and are made first as a prepropeptide which is proteolytically cleaved to the active form [10] . in the case of hd5 and hd6 the key protease is trypsin. hnp1-4 are produced mainly by neutrophils, where they comprise 5-7 % of total neutrophil protein [11] , and neutrophil precursors in the bone marrow [12] . hnp1-3 are also described in nk cells, b cells, cd t cells, macrophages and immature dendritic cells [13] , but can be acquired from neutrophils [14] . the release of active hnps from neutrophil azurophilic granules can be induced by a range of stimuli, including chemokines, fc gamma receptor cross linking, pma and tlr stimulation [13] . in contrast, hd5 and 6 are expressed by paneth cells of the small intestine [15, 16] and epithelial cells of the female genital tract [12, 17, 18] . interestingly, although mice express a large number of intestinal a-defensins (cryptdins) [19] , in contrast to humans they do not express a-defensins in their neutrophils [20, 21] . a-defensins have well-described broad spectrum antimicrobial activity against both gram-positive and -negative organisms in vitro [22] , with cationicity and hydrophobicity being shown to be key determinants of these properties [9, 23] . their cationic charge is proposed to enable interaction with the net negative charge on the surface of the gram-negative bacteria and the teichoic acids of the gram-positive organisms, while their amphipathic structure enables insertion into and disruption of the bacterial membranes, leading to lysis of the cells. in addition, various a-defensins are described as having additional, non-microbicidal properties, including chemotaxis for effector cells of the innate and adaptive immune systems [24, 25] , inhibition of macrophage pro-inflammatory cytokines [26] , modulation of the intestinal microbiome [27] and the formation of protective peptide nanonets [28] . the b-defensin family contains more than 30 members in humans and more than 50 in mice, and are widely expressed across many species, in particular being the only defensins found in birds and with close homologues present in snakes, platypus and sea anemones [29] . they are also triple-stranded b-sheet proteins, but differ from the a-family members in the organisation and arrangement of the three disulphide bonds [10] . the most well characterised human b-defensins are human b-defensin (hbd) 1-3, expressed by epithelial cells [30] , monocytes, macrophages and macrophage-derived dc [31] . hbd1 is encoded by defb1 and is expressed constitutively [30] , whereas expression of hbd2 (defb4a) and hbd3 (defb103a) are up-regulated in response to various inflammatory stimuli, including microbes [32] , tlr and nod proteins [33] and pro-inflammatory cytokines [34] . b-defensins are also described as having broad-spectrum antimicrobial activity in vitro [7] , with potency varying in different family members. interestingly, the weak microbicidal properties of hbd1 were recently shown to be greatly enhanced upon reduction of its disulphide bonds [35] . although relatively mild phenotypes were found in mouse models deficient in defb1 [36, 37] , redundancy in this multi-gene family may be responsible for this observation. in addition b-defensins are described as having a range of immunomodulatory properties, including chemotaxis of cd4 ? memory t cells, macrophages and immature dendritic cells [38] , enhancement of wound healing [39] and the modulation of inflammatory cytokine responses (with the capacity both to promote and to suppress inflammation in different settings) [40] . h-defensins are circular octadecapeptides, the only circular peptides of mammalian origin [41] , formed by the splicing of two nonapeptides, each of which contributes three cysteines to a series of disulphide bonds in the mature peptide [42] . three h-defensins have been found in the leukocytes of rhesus macaques and named rtd1-3, but although six rna transcripts homologous to rtd are found in the human bone marrow they contain a premature stop codon preventing their expression [42, 43] . however, an artificially constructed peptide based on these pseudogenes, called retrocyclin, has been studied with respect to its function and therapeutic potential and shown to kill escherichia coli in the same way as a-defensins, by permeabilising its membrane [44] . the cathelicidin family is quite distinct from defensins; cathelicidins are defined by a conserved cathelin domain and with a variable c-terminal region, which is proteolytically cleaved to produce a mature functional peptide, with a range of structural forms in different family members [45] . in contrast to the extensive defensin family, humans (and mice, rats and rabbits) express a single cathelicidin, whereas multiple cathelicidins are found in other species (e.g. protegrins in pigs). the sole human cathelicidin, human cationic antimicrobial peptide of 18 kda (hcap-18; encoded by the camp gene), is cleaved by proteinase 3 into its active form, ll-37, which is a cationic, amphipathic peptide of 4.5 kda with an a-helical structure [46, 47] . hcap-18 is stored in neutrophil-specific granules, inducible in epithelial cells, macrophages and other leukocytes to a lesser extent, and detectable in a range of body fluids, including airway surface liquid, plasma, urine, breast milk and sweat [48] . it is up-regulated in response to infectious and inflammatory signals [49] and wounding [50] and its expression can be increased by vitamin d metabolites [51] and compounds such as butyrate [52] . ll-37 has well-documented antibacterial potential; however, when studied in the presence of physiological concentrations of cations or serum, ll-37 has high minimum inhibitory concentrations compared to levels described in vivo [6] . mice deficient in mcramp (encoded by the camp gene, the orthologue of camp) have increased susceptibility to infections in multiple systems, including the skin, intestinal tract and lung [53] [54] [55] . although these models clearly demonstrate the critical role for cathelicidin in host defence against infection, it remains unclear to what extent this is due to microbicidal or modulatory properties of the peptide. ll-37 has been shown to have a broad range of immunomodulatory and inflammomodulatory properties [6] . these include chemotactic activity for neutrophils, monocytes and t cells [56] , modulation of cytokine production [57] , effects on dendritic cell differentiation and function [58, 59] , promotion of wound healing [60] and angiogenesis [61] , and modulation of cell death [62, 63] . although the field of antimicrobial peptide research has been dominated by evaluation of the antibacterial activities of these peptides, early studies evaluating the antiviral potential of human a-defensins showed promise and have been followed by an increasing level of research interest (fig. 1 ). the first paper detailing an antiviral role was published 27 years ago, describing inhibition of a number of viruses including hsv types 1 and 2, cytomegalovirus and vesicular stomatitis virus by hnp1 in vitro [64] . in particular, it demonstrated direct antiviral activity of hnp1 against hsv-1 in a temperature-and ph-dependent manner, inhibited by serum, but interestingly less sensitive to the inhibitory effects of cations than the more broadly studied antibacterial properties. hnp1-4, hd5 and hd6 have subsequently been found to be active against hsv-2 (up to approximately 1 log decrease at 50 lg/ml) by preventing viral binding to either glycoprotein b (gb2) or heparan sulphate (the primary receptor for hsv) [65] . gb2 binding capacity was found to be primarily determined by peptide sequence rather than net cationic charge [66] , with lectin-like properties likely to be key to the glycoprotein binding functions [67] . interestingly, those defensins (hnp1-3 and hbd5) which bound viral envelope gb were also found to be effective at preventing infection if added after viral entry, even up to 8 h post-infection (with hnp1 or hd5), suggesting additional later stage effects on viral replication [65] . furthermore, cumulative effects were observed with repeated application of peptide, reducing infection with hsv by greater than 7 logs at 100 lg/ml. these features make a-defensins of considerable interest as potential exogenous vaginal microbicides, with a proof of concept study in mice demonstrating protection against hsv following gel-based application of hd5 [65] . a-defensins have been demonstrated to directly inactivate hiv-1, with the observation of reduced cytopathogenicity in a cd4 ? t cell line [68] . although confusion existed following the retraction of a paper proposing that adefensins were the key component of the cd8 antiviral factor caf [69, 70] , it is worth noting that the retraction related to the source of the a-defensins (probably ''imported'' into cd8 cells from co-cultured cells), and the anti-hiv-1 properties of the peptides were not called into question. indeed, the potential of a-defensins to treat hiv was reinforced when it was found that breast milk a-defensin concentration is significantly associated with a decreased risk of intrapartum and postnatal hiv transmission [71] . recent studies demonstrate that multiple steps in the entry of hiv-1 virus into cells are disrupted by hnp1 [72] . this peptide has been shown to bind to cd4 and to the env glycoprotein on the virus, thus inducing the down-regulation of cd4 and cxcr4 and blocking interaction of env with the co-receptors. by targeting particular conformations of env it also inhibited late fusion steps [72] . hnp1-3 can bind to cd4 and hiv-1 gp120 with high affinity; however, hnp4, which is a much weaker binder, is a more potent inhibitor, meaning this aspect of direct inhibition is not, currently, entirely clear [73] . mechanistic studies have shown that hnp1-3 can also inhibit steps following reverse transcription and integration by inhibiting pkc activity; pkc is important for hiv replication as it upregulates transcription through nf-jb activation and tat phosphorylation, as well as regulating fusion and assembly of the virions [74] . it is worth noting that in these studies the direct effects on the virus particles occurred only in the absence of serum; in its presence, these mechanisms were inhibited and effects are instead on the host cells, resulting in inhibited replication of the virus [69, [74] [75] [76] . defensins can also stimulate an antiviral state in cells by promoting secretion of chemokines [76] . in macrophages this upregulation of chemokines also contributes to inhibition of hiv through competition for receptors [76] . a potential issue with using defensins as a topical antiviral treatment is that, despite chdps (including defensins) being required for in vitro anti-hiv activity of vaginal fluid from healthy women, defensins may also cause immune activation and subsequent loss of cd4 ? t cells by apoptosis, and may indirectly enhance hiv transmission [77] . in particular, it is known that hnp2 and hbd2 are both chemotactic for dc and induce infiltration of dc into cultures of hpv-transformed keratinocytes and subsequent immune activation [78] . in addition, hd5 and 6, which are produced by cervico-vaginal epithelial cells [18] and present at up to 50 lg/ml in the vaginal fluid of healthy women [79] , enhance hiv infectivity in vitro by promoting the virus attachment to target cells [18, 79] . thus, the balance of these effects remains to be determined. a-defensins are also found in high concentrations in the inflamed lung, generating interest in their potential activities against respiratory viruses. hnp1-3 are the most abundant antimicrobial peptides in airway fluid [80] and are up-regulated further following infection or inflammation [81] as they are produced by both immigrating neutrophils and airway epithelial cells [82] . daher et al. [64] first described antiviral effects of hnp1 against the wsn strain of iav in vitro, showing a fairly modest approximately 0.5 log reduction at 50 lg/ml. this property of hnp1 and 2 was later confirmed in other strains of iav (with decreased infectivity of approximately 1 log at 10 lg/ml in a fluorescent focus assay of infectivity). the peptides were shown to have no activity in a haemagglutinin inhibition assay [83] , but to induce aggregation of iav [84] . interestingly hnp aggregation of the pr8 viral strain was much higher than that of the phil82 strain, which has greater surface glycosylation [84] [85] [86] , and neutralising activity of hnps was greater against pr8 than against phil82 [83] , suggesting the reduced carbohydrate attachments on the envelope proteins allow greater interaction with the defensins. however, direct effects of hnp1 on iav viral particles were found to have no impact on viral growth in infected cultures [87] . maximal antiviral effects in this study (1-3 logs at 5-25 lg/ml hnp1 in a range of cell lines) required interaction of hnp1 with the eukaryotic cells before infection and the continued presence of peptide in the culture system. nevertheless, in contrast to pretreatment of virus, pretreatment of the cells did induce some protection. this suggests the predominant mechanism of action is cell-mediated, and has been proposed to be a consequence of hnp-mediated inhibition of pkc activity (essential for endosomal trafficking of the iav) [87, 88] . the capacity of a-defensin to protect against iav was lost in its linearised analogue [87] . in addition to effects on iav infection of epithelial cells, hnp1 and 2 at approximately 40 lg/ml (but not hnp3, hbd2 or 3) can enhance the uptake of iav by neutrophils, following pre-incubation of either the cells or the virus with the defensins [84] . however, this modulation of neutrophil phagocytic capacity was also observed using bacteria, and was not virus specific. these properties of a-defensins suggest potential as templates for novel therapeutics. however, their ability to bind surfactant protein d (sp-d) may be of concern, having mixed competitive or co-operative, and iav straindependent effects on the antiviral activities of sp-d [83] . hnps (but not b-defensins) can bind and precipitate bronchoalveolar lavage (bal) fluid sp-d [83, 89] and may account for sp-d depletion from the lung in diseases with chronic neutrophilic inflammation. it was originally suggested that a-defensins had no activity against non-enveloped viruses, on the basis of the absence of effects against echovirus type ii and reovirus type 3 [64] . however, more recent work has demonstrated inhibition of non-enveloped viruses such as adenovirus, human papillomavirus (hpv) [90] [91] [92] and bk virus [93] . the mechanisms underpinning these antiviral effects against non-enveloped viruses appear to be distinct from those targeting enveloped viruses. a study of hpv (utilising pseudovirus particles) found normal binding, uncoating and internalisation in the presence of a-defensins; however, the peptides prevented the virion from escaping the endocytic vesicles [92] . the antiviral activity was observed using hnp1-4 (and the cathelicidin ll-37), was maximal using hd5, but was not observed using hd6. the sensitivity of adenoviruses to a-defensin-mediated neutralisation is serotype-dependent [90, 94] . hnp1 and hd5 have been shown to inhibit human adenovirus infection in both lung and conjunctival epithelial cells by inhibiting an early step in viral entry [90, 91, 95, 96] . two arginine residues on one face of hd5 were found to be critical for antiviral activity, with arg-28 necessary for killing of both adv and hpv and arg-9 for adv only [97] . viral aggregation is not sufficient for neutralisation and binding of a-defensin to the adenoviral virus capsid appears to be critical, preventing uncoating in the cell and hence entry of the viral genome into the nucleus [97, 98] . in contrast, the antiviral effects of hnp1 and hd5 on the bk polyomavirus appear to primarily relate to viral aggregation preventing receptor binding on the host cells [93] . [100] . similarly, human rhinovirus (hrv) replication in human bronchial epithelial cells induces nf-jb-dependent hbd2 and 3 expression (but not hbd1) [101, 102] , whereas respiratory syncytial virus (rsv) can induce hbd2 in an nf-jb-dependent, but ifn type 1-independent manner in human lung epithelial cells [103] . the extent to which these innate responses are functionally effective against the viral pathogens is starting to be elucidated (fig. 2 ). in contrast to the effects of a-defensins, only one of the bdefensins tested (hbd3) had appreciable effects against hsv [65] . hbd3 was found to inhibit hsv-2 infection of human cervical epithelial cells (by approximately 1 log at 10 lg/ml) by interfering with the viral binding and penetration processes by binding both gb and heparan sulphate. in contrast, hbd1 and hbd2 had low affinity to gb and cellular glycosaminoglycans, and were not able to reduce hsv-2 infectivity. however, only hd5 was applied in vivo in this study [65] . hiv-1 infection of epithelial cells induces hbd2 and 3 expression in vitro [100] and in buccal mucosal cells in vivo [104] , and both inhibit hiv transmission through multiple mechanisms [100, 105] . both down-regulate the co-receptor cxcr4 expression (but not ccr5) on the surface of cd4 ? t cells via an increase in internalisation [106] . in addition, there are both direct effects on the virions in a concentration-dependent manner [100] and on intracellular, post-viral entry inhibition [105] . however, in large-scale studies, copy number variation of total b-defensin gene number positively correlates with hiv load in ethiopian and tanzanian patients [107] . the authors suggest that the chemoattractant nature of bdefensins may bring the target th17 cells into mucosal sites where they can be readily infected by virus. other work, however, has recently shown that exposed but seronegative individuals have much higher hbd2 and 3 copy numbers in oral mucosa (but not vaginal mucosa) than healthy controls, indicating a role for these defensins in combating infection [108] . although not normally regarded as inducible, expression of hbd1 (but not hbd2, 3 or 4) has been observed in primary human blood-derived plasmacytoid dc and monocytes following infection with iav [109] . in contrast, an early decrease in expression of this peptide was found in iav-infected epithelial cell lines [109] . recombinant hbd1 was found to have antiviral effects in vitro (approximately 1.5 log decrease at 50 lg/ml). however, despite evidence of a significant defect in host defence against iav in defb1-deficient mice, this did not extend to differences in viral load, suggesting a primary role of immunomodulatory properties in vivo [109] . iav has been found to up-regulate expression of murine defensins mbd3 and mbd4 in upper and lower airways, as well as transcription of the genes encoding mbd1 and mbd2 in the lung [99] , also suggesting protective roles for these peptides. recombinant mbd2 [110] and mbd3 [111] protected mdck cells against infection with iav pr8 strain (up to approximately 1 log decrease at 100 lg/ml); this protection was effective during binding or internalisation of the virus, but not after viral entry. furthermore, repeated intranasal administration of recombinant mbd2 (2 mg/kg; optimal when premixed with virus before infection) or intravenous delivery of recombinant mbd3 (10 mg/kg) was found to be protective in murine lethal infection models [110, 111] . the latter study also suggested that the effects may relate to immunomodulatory properties, with systemic mbd3 treatment up-regulating ifn-c and il-12 and reducing levels of tnf. thus, although b-defensins can inhibit influenza virus infectivity (albeit less potently than the a-defensins or ll-37) [89] , immunomodulatory properties, perhaps also including up-regulation of iav uptake by neutrophils [84] , may prove to be key to their protective function against this virus in vivo and future therapeutic developments. in addition to effects on iav, evidence has been found for b-defensin activity against rsv, another important respiratory virus, for which no effective vaccine or antiviral treatments exist. in studies using the a549 human lung cell line, hbd2 was identified as a component of an nf-jbdependent, ifn-a/b-independent antiviral response [103] . epithelial cell hbd2 production was induced in response to rsv replication and also in response to the tnf secreted by infected epithelial cells. hbd2, but not hbd1, was found to protect against rsv infection (approximately 2 log decrease at 4 lg/ml), by blocking viral cellular entry [103] . destabilisation of the viral envelope was proposed to occur upon contact with soluble hbd2 in solution, or following exposure to plasma membrane-associated hbd2 during cell entry. in addition to lung epithelial cells, myeloid cells can produce high levels of tnf in response to rsv infection [112] . this has the potential to up-regulate hbd2 expression in the airway lumen and might consequently modulate protection against rsv infection and limit virus spread. interestingly mbd4 (but not mbd3; both considered homologues to hbd2) was found to be upregulated in vivo in the murine lung in response to rsv infection [103] . hbd3 has been proposed to have antiviral activity against vaccinia virus [113] . however, hnp1, hbd1 and hbd2 are unable to neutralise the virus [114] . pre-exposure of virus to synthetic hbd3 for 24 h decreased infection of the bsc-1 monkey kidney cell line, shown both by reduced levels of dna-dependent rna polymerase expression and plaque formation (approximately 1 log decrease at 10 lm) [113] , although the mechanism remains to be elucidated. up-regulation of hbd3 expression was observed in response to vaccinia virus infection in primary human keratinocytes, but this could be inhibited by il-4 and il-13. interestingly, these cytokines are associated with pathology in atopic dermatitis, a condition in which b-defensin expression is reduced [115] and patients are at risk of developing eczema vaccinatum caused by vaccinia virus. circular h-defensins were identified in the leukocytes and bone marrow of macaques (rhesus h-defensins; rtd) [116] and discovered to have effective antibacterial activity. humans were found to have at least six h-defensin genes (deft genes) [117] , but none produce a translated protein, owing to insertion of a premature stop codon. putative ancestral human h-defensins, named retrocyclins (rc), were developed and their antimicrobial properties were tested [117] . in addition to activity against pseudomonas aeruginosa, e. coli, listeria monocytogenes and staphylococcus aureus, antiviral potential has also been described (fig. 3) . both rhesus h-defensins and retrocyclins (including rtd3, rc1 and rc2) have been found to inhibit hsv-1 and hsv-2 infection of human cervical epithelial cell lines following pre-incubation of virus and peptide [66] . however, rc2 was found to have no direct virucidal properties [118] , but to be active irrespective of pre-incubation by blocking attachment and cell penetration of hsv [66] . this activity resulted from peptide binding to gb2, in a manner dependent on the presence of sialic acid and carbohydrate moieties in the glycoprotein's o-and n-linked glycans. prophylactic application of rc2 in a murine hsv-mediated ocular keratitis model demonstrated the capacity of rc2 to modestly reduce viral titres in vivo and reduce blepharitis, corneal vascularization and stromal disease. however, rc2 had no effect upon disease pathology when applied post-infection [118] . initial studies on retrocyclins found no direct inactivation of hiv-1, but demonstrated strong inhibition of proviral dna formation and protection of primary human cd4 ? t cells from t-and m-tropic hiv-1 strains in vitro [117, 119] . these observations led to a significant body of work evaluating retrocyclins as hiv treatments. rc1 has been characterised as a lectin, which protects peripheral blood [117] . this peptide prevents viral entry into cells [42, 119] , blocking formation of the 6-helix bundle required for fusion, by binding to hiv gp120 and cellular cd4 through interactions with their o-and nlinked sugars [120] . activity of many of the initial retrocyclins produced was inhibited by serum, minimising usefulness in serum-containing anatomical compartments [42] . however, analogues, each differing from rc1 by a single amino acid substitution, show greater potential as topical microbicides [121] . of these, rc100 was found to inhibit primary cd4 ? t cell infection by hiv-1 similarly to rc1, but was active in the presence of vaginal fluid, and rc101 is significantly more potent against hiv than rc1; both have potential as topical microbicides [122] . interestingly, the rate of escape mutations of rc100-treated hiv was low; treatment altered sites in hr1 and hr2 of gp410 but these only reduced susceptibility by 10-fold, compared to 10,000-20,000-fold for ccr5 blockade [122] . using an organ culture model, rc101 was found to block transmission of two strains of hiv-1 across cervical mucosa. antiviral activity was retained in the presence of semen and vaginal fluid, there was no cytotoxicity to cervical tissue and, importantly, rc101 did not induce a pro-inflammatory response, with no chemotactic activity for immune cells [123] . in addition, topical intra-vaginal rc101 application in pigtailed macaques was found to be safe and well tolerated, with peptide retained in the cervical and vaginal tissue for up to 4 days post-application, no changes in vaginal ph observed and minimal effects on commensal microbiota [124] . the application of retrocyclins to pathogenic respiratory viruses has also been evaluated. expression of recombinant rc2 in both mdck cells and chicken embryos has been shown to inhibit replication of an h5n1 influenza virus [125] . in addition, rc1, rc2 and rc101 all had antiviral activities in studies using mdck and a549 cell lines (approximately 1 log decrease at 20 lg/ml against iav phil82, but less effective against pr8 strain) [89] . the retrocyclins were more effective than hbd1 and hbd2, with potency equivalent to hnp1-3. this study noted the capacity of the rc peptides to induce aggregation of the virus and increase viral uptake by neutrophils and macrophages [89] . rc2 was also found to inhibit influenza a/x31 infection of cell lines in vitro by blocking the haemagglutinin-mediated fusion of viral and endosomal membranes [85] . the inhibition of hemifusion formation was shown to be a lectin property which involved cross linking and immobilising surface glycoproteins and blocking the protein displacement required to bridge the bilayers for fusion. rc2 was only effective when given before viral internalisation, but had antiviral properties even when only applied to the host cell membrane. interestingly, despite the capacity to bind sp-d, retrocyclins increased, rather than inhibited, the antiviral activity of sp-d [89] . this is in contrast to the a-defensins [83] , and promising for therapeutic use at mucosal sites. to further improve the antiviral activity of synthetic retrocyclins against influenza and simplify their structure, a series of analogues containing 13-14 amino acids, termed hapivirins and diprovirins, were designed [126] . some of these analogues, including hpv1, 8, 11-15, 17, 19 and dpv 13, 16, 1623 and 1632 proved to be more effective than rc2 and rc101 against iav phil82 (h3n2) and pr-8 (h1n1). mechanistically analogous to the retrocyclins, these peptides were found to be active before viral internalisation, induce viral aggregation, opsonise virus, and have an additive effect with sp-d. in addition, hapivirins and diprovirins were shown to inhibit iav-induced macrophage tnf production, adding immunomodulatory mechanism to their therapeutic potential. antiviral activity of retrocyclins has also been reported against sars virus, a coronavirus which infects the alveolar epithelial cells and induces rapid severe lung pathology [127, 128] . in a mouse model of sars infection, rtd-1 treatment increased survival from 25 to 100 %. interestingly, this was in the absence of any impact on viral titres. instead the cytokine profile of the infected animals was modified, with increased early (day 2) production of il-6 and gm-csf, but decreased il-1a, il-1b, il-6, mip1a, and il-12p40 by day 4. these data highlight the possibility that novel peptide therapeutics may be productively targeted at modulation of the inflammatory response to infection, rather than developed for virucidal properties per se. such an approach may prove to have broad-spectrum applicability and lends further support to the potential for these peptides as novel immunomodulatory antiviral agents. cathelicidins have been widely studied with regard to their antibacterial properties and broad array of immunomodulatory activities [6] . although known to be up-regulated in inflammation and released by neutrophils, their roles in defence against viral infection and properties as antiviral agents are less well understood (fig. 4 ). hcap-18 is expressed in human epididymal epithelium and is present at high concentrations in seminal plasma [129] . it is also expressed in cervico-vaginal secretions and upregulated in participants with bacterial sexually transmitted infections [130] . cervico-vaginal ll-37 levels in hivnegative individuals who were in hiv sero-discordant relationships were also found to be greatest in those whose hiv-positive partners had the highest viral load [131] . ll-37 has been shown to inhibit the replication of a range of hiv-1 isolates in primary cd4 ? t cells [132] . this occurred in a manner that was independent of change in expression of any hiv-1 receptors in these cells. a recent study demonstrated that ll-37 was capable of a dosedependent suppression of hiv reverse transcriptase activity [133] . this study also demonstrated that this function was retained by a 16 amino acid fragment of ll-37 (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) , with implications for production of smaller synthetic analogues. thus, epithelial expression of ll-37 has been proposed to contribute to local protection against hiv-1 infection. however, although the cationic peptide fraction of cervico-vaginal secretions was found to have hiv-1neutralising activity, which could be enhanced by addition of recombinant ll-37, this property did not correlate with levels of endogenous ll-37 detected [131] . in addition, in one study ll-37 levels were independently associated with increased hiv acquisition, although both observations might be the result of a high prevalence of sexually transmitted infections in these individuals [130] . therefore, the in vivo significance of ll-37 in hiv remains unclear. we recently demonstrated that ll-37 has antiviral effects against iav, both in vitro and in vivo [134] . both human and murine cathelicidins had antiviral activity when preincubated with influenza viruses in vitro [approximately 1 log decrease at 10 lg/ml against a/pr/8/34 (h1n1), but somewhat less effective against a/udorn/307/72 (h3n2)]. in addition, ll-37 has recently been shown to bind iav, without aggregating or affecting haemagglutination activity, and to have maximal effects in vitro when pre-incubated with virus (although delayed addition of peptide or treatment of the cells, with washing before infection also has some antiviral effects) [135] . surprisingly, ll-37 was found not to alter the binding or initial uptake of virus by cells, but peptide-mediated disruption of viral membranes (shown by electron microscopy) was proposed to affect viral propagation or survival downstream of this [135] . we demonstrated that aerosolised therapeutic administration of either ll-37 or mcramp (given every day for 1 week, with an additional pre-infection dose) provided protection against infection in a mouse model [134] . peptide-treated mice showed increased survival and decreased weight loss compared to control infected animals, and were similarly protected to those treated with zanamavir (a neuraminidase inhibitor currently used therapeutically in humans). the cathelicidin-treated mice showed some decrease in viral loads, but more striking reductions in lung cytokines (in particular gm-csf, il-1b, kc and ccl5), again suggesting the possibility of a key immunomodulatory roles in the antiviral efficacy of such peptides. scrambled control peptides did not have these effects but interestingly the d-enantiomers did, indicating the actions of ll-37 are not solely related to charge and are likely not reliant on specific receptor interactions. the mechanisms by which cathelicidins modulate inflammation in iav infection remain unclear, but may relate to the observation that ll-37 can modulate tolllike receptor signalling [136] . induction of rapid antiviral responses depends, at least in part, on tlr recognition of the viral genome, with ssrna and dsrna viruses recognised by tlr7/8 and tlr3 [137] . ll-37 complexed with self dna and rna has been shown to induce tlr7-, tlr8-and tlr9-dependent inflammatory responses to otherwise non-immunostimulatory nucleic acids [59, 138] . in addition, ll-37 has been proposed to enhance [139, 140] or inhibit [141] tlr3-dependent responses to viral rna or synthetic mimics. these modulatory properties may well prove to underpin the in vivo effects. the expression of ll-37/hcap18 by airway epithelial cells can be induced in vitro by infection with rsv [142] , in a manner that is significantly enhanced by the 1,25oh metabolite of vitamin d. in addition, a recent study found that median serum hcap-18 levels are significantly lower in children with rsv bronchiolitis than in children with bronchiolitis caused by human rhinovirus [143] . furthermore, rsv-infected children with hcap-18 levels lower than the median are more likely to be hospitalised for prolonged periods than those with hcap-18 levels above the median. these findings suggest an important antiviral role for ll-37 in host innate immune response against rsv. in keeping with this hypothesis, we have recently demonstrated that ll-37 exhibits effective, dose-dependent and timing-specific anti-rsv activity in vitro in a number of cell lines [144] . these data indicate that therapeutic use of cathelicidin or strategies to up-regulate cathelicidin expression in vivo (particularly during the winter when low vitamin d levels may lead to diminished cathelicidin expression) may be protective against rsv infections. individuals with atopic dermatitis (ad) have low levels of ll-37 expression and increased susceptibility to skin infections, in contrast to those with psoriasis, who have high levels of ll-37 expression and are less prone to skin infections, despite similar disruption to skin barrier function [145] . the low levels of ll-37 expression in ad are proposed to be important in the susceptibility to eczema vaccinatum, a disseminated viral skin infection that follows inoculation with vaccinia virus (vv). ll-37 expression was induced in response to vv in normal and psoriatic skin biopsies, but not those from ad skin [146] . both ll-37 and mcramp have been shown to have antiviral activity against vaccinia virus in vitro (approximately 1 log decrease at 25 lm) [114] . these peptides were shown to damage the integrity of the double-layered viral envelope [114] , by removing the outer membrane [147] . in addition, camp-/-mice were found to develop significantly more pox skin lesions following infection with vv, demonstrating the antiviral effects of cathelicidins in vivo [114] . interestingly these effects against vv were found to be specific to cathelicidins, with hnp1, hbd1 and hbd2 unable to neutralise the virus [114] . this implies that the different chdps may have different (if sometimes overlapping) targets in order to successfully deal with the enormously wide range of human pathogens. the antiviral activities of host defence peptides have been somewhat neglected until recently, in contrast to the study of their antibacterial effects. yet, as discussed in this review, a strong basis exists for future evaluation of the physiological roles of chdps as key components of innate antiviral host defences and to develop synthetic analogues as novel antiviral therapeutics. the precise mechanisms involved clearly vary in a peptide-and virus-specific manner, from direct effects on the viruses through to primarily immunomodulatory properties. these need to be examined in detail and tested in vivo against specific viral infections. such research is expected to reveal therapeutic strategies that range from simple administration of more potent antiviral and/or immunomodulatory analogues, to therapeutic measures to enhance natural levels of expression, replace down-regulated peptides or provide peptide at an earlier, critical ''tipping point'' stage in infection. although some peptides may prove to be effective when administered after infection is established, the optimal use of other peptide therapeutics may be prophylactic administration in high-risk populations (e.g. infants and elderly in an outbreak of a novel influenza strain for which a vaccine is not available). challenges remain in optimising peptide therapeutics to develop the shortest, cheapest analogues that are stable and function at lower concentrations, in physiological environments, without cytotoxic effects or the generation of resistance. in this regard, the h-defensins appear particularly exciting as potential topical anti-hiv agents. they are well tolerated, certain members of the family appear to function in the presence of serum, they rapidly and directly kill hiv-1 without inducing large-scale immune activation and escape variants are infrequent. engineering of peptides is a simple process, which enables development of more suitable compounds. the difference in the antiviral capabilities and rc 50 values of hnp1 and 3, despite them differing by only a single amino acid, hints at what is possible. the engineering of rc101 from rc1 (again a single amino acid change) leading to significantly higher anti-hiv-1 activity also suggests that further enhancements should be possible. likewise, linear, disulphide bond-free defensins have been generated [148] , which retain potent antimicrobial activity. the development of linear peptides would be significantly easier than recreating their tertiary structure. however, the stability of such peptides against protease degradation remains uncertain and loss of antiviral properties following linearisation has been described in some instances [18, 94] , highlighting the need for further research. thus, in an era of increasing concern about the resurgent threats of infectious diseases, a very limited repertoire of antiviral drugs and fears about the rapidity with which new viruses can spread globally, peptide therapeutics have potential that is clearly worth pursuing. acknowledgments djd is supported by a mrc senior non-clinical research fellowship (g1002046). smc is supported by a university of edinburgh college of medicine and veterinary medicine studentship. the authors have no conflicts of interest that are directly relevant to the content of this article. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. the exclusive right to any commercial use of the article is with springer. global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since antivirals: current state of the art avppred: collection and prediction of highly effective antiviral peptides cationic host defence peptides: multifaceted role in immune modulation and inflammation a re-evaluation of the role of host defence peptides in mammalian immunity antimicrobial peptides and innate immunity. progress in inflammation research antimicrobial peptides of multicellular organisms natural peptide antibiotics of human neutrophils alpha-defensins in human innate immunity defensins: antimicrobial peptides of innate immunity theta-defensins: cyclic peptides with endless potential defensins in viral infections neutrophil-derived defensins as modulators of innate immune function macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens paneth cells of the human small intestine express an antimicrobial peptide gene human enteric defensins. gene structure and developmental expression gene expression, immunolocalization, and secretion of human defensin-5 in human female reproductive tract neisseria gonorrhoeae-induced human defensins 5 and 6 increase hiv infectivity: role in enhanced transmission paneth cell defensins: endogenous peptide components of intestinal host defense mouse neutrophils lack defensins strain-specific polymorphisms in paneth cell alpha-defensins of c57bl/6 mice and evidence of vestigial myeloid alpha-defensin pseudogenes antibacterial activity and specificity of the six human {alpha}-defensins antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? monocyte-chemotactic activity of defensins from human neutrophils human neutrophil defensins selectively chemoattract naive t and immature dendritic cells dying and necrotic neutrophils are anti-inflammatory secondary to the release of alpha-defensins enteric defensins are essential regulators of intestinal microbial ecology human alpha-defensin 6 promotes mucosal innate immunity through self-assembled peptide nanonets the changing of the guard: molecular diversity and rapid evolution of beta-defensins widespread expression of betadefensin hbd-1 in human secretory glands and epithelial cells nibbering ph. expression of beta-defensin 1 and 2 mrna by human monocytes, macrophages and dendritic cells nf-kappab-and ap-1-mediated induction of human beta defensin-2 in intestinal epithelial cells by escherichia coli nissle 1917: a novel effect of a probiotic bacterium nod2/card15 mediates induction of the antimicrobial peptide human beta-defensin-2 multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1 characterization of the mouse beta defensin 1, defb1, mutant mouse model beta-defensin 1 contributes to pulmonary innate immunity in mice beta-defensins: linking innate and adaptive immunity through dendritic and t cell ccr6 human beta-defensin-3 promotes wound healing in infected diabetic wounds beta-defensins: multifunctional modulators of infection, inflammation and more? mammalian defensins in the antimicrobial immune response activity of alpha-and theta-defensins against primary isolates of hiv-1 evolution of primate thetadefensins: a serpentine path to a sweet tooth microbicidal properties and cytocidal selectivity of rhesus macaque theta defensins cathelicidins, multifunctional peptides of the innate immunity the human antibacterial cathelicidin, hcap-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils human cathelicidin, hcap-18, is processed to the antimicrobial peptide ll-37 by extracellular cleavage with proteinase 3 immunomodulatory properties of defensins and cathelicidins the expression of the gene coding for the antibacterial peptide ll-37 is induced in human keratinocytes during inflammatory disorders cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group a streptococcus cutting edge: 1,25-dihydroxyvitamin d3 is a direct inducer of antimicrobial peptide gene expression induction of the human cathelicidin ll-37 as a novel treatment against bacterial infections innate antimicrobial peptide protects the skin from invasive bacterial infection cathelicidin mediates innate intestinal defense against colonization with epithelial adherent bacterial pathogens cathelicidin-related antimicrobial peptide is required for effective lung mucosal immunity in gram-negative bacterial pneumonia ll-37, the neutrophil granule-and epithelial cellderived cathelicidin, utilizes formyl peptide receptor-like 1 (fprl1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells the human antimicrobial peptide ll-37 is a multifunctional modulator of innate immune responses the cationic antimicrobial peptide ll-37 modulates dendritic cell differentiation and dendritic cellinduced t cell polarization plasmacytoid dendritic cells sense self-dna coupled with antimicrobial peptide antimicrobial protein hcap18/ll-37 is highly expressed in breast cancer and is a putative growth factor for epithelial cells an angiogenic role for the human peptide antibiotic ll-37/hcap-18 secondary necrosis of apoptotic neutrophils induced by the human cathelicidin ll-37 is not proinflammatory to phagocytosing macrophages the human cathelicidin ll-37 preferentially promotes apoptosis of infected airway epithelium direct inactivation of viruses by human granulocyte defensins human alpha-and beta-defensins block multiple steps in herpes simplex virus infection theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry multivalent binding of carbohydrates by the human alpha-defensin, hd5 defensins inhibit hiv replication in vitro contribution of human alpha-defensin 1, 2, and 3 to the anti-hiv-1 activity of cd8 antiviral factor retraction of an interpretation alpha-defensins in the prevention of hiv transmission among breastfed infants multifaceted mechanisms of hiv-1 entry inhibition by human alpha-defensin human neutrophil alpha-defensin 4 inhibits hiv-1 infection in vitro dual role of alpha-defensin-1 in anti-hiv-1 innate immunity alphadefensins block the early steps of hiv-1 infection: interference with the binding of gp120 to cd4 alpha-defensins inhibit hiv infection of macrophages through upregulation of cc-chemokines contribution of immune activation to the pathogenesis and transmission of human immunodeficiency virus type 1 infection defensins induce the recruitment of dendritic cells in cervical human papillomavirus-associated (pre)neoplastic lesions formed in vitro and transplanted in vivo human defensins 5 and 6 enhance hiv-1 infectivity through promoting hiv attachment antibacterial peptides in bronchoalveolar lavage fluid elevated concentrations of defensins in bronchoalveolar lavage fluid in diffuse panbronchiolitis production of beta-defensins by human airway epithelia innate defense against influenza a virus: activity of human neutrophil defensins and interactions of defensins with surfactant protein d human neutrophil defensins increase neutrophil uptake of influenza a virus and bacteria and modify virus-induced respiratory burst responses carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins the antigenic structure of the influenza virus a/pr/8/34 hemagglutinin (h1 subtype) alpha-defensin inhibits influenza virus replication by cell-mediated mechanism(s) role of protein kinase c betaii in influenza virus entry via late endosomes interactions of alpha-, beta-, and theta-defensins with influenza a virus and surfactant protein d mechanism of adenovirus neutralization by human alpha-defensins epithelial defensins impair adenoviral infection: implication for adenovirus-mediated gene therapy human alpha-defensins block papillomavirus infection human alpha-defensins inhibit bk virus infection by aggregating virions and blocking binding to host cells insight into the mechanisms of adenovirus capsid disassembly from studies of defensin neutralization human alpha-defensin 1 (hnp-1) inhibits adenoviral infection in vitro adenovirus-directed ocular innate immunity: the role of conjunctival defensin-like chemokines (ip-10, i-tac) and phagocytic human defensin-alpha critical determinants of human alpha-defensin 5 activity against nonenveloped viruses direct evidence from single-cell analysis that human {alpha}-defensins block adenovirus uncoating to neutralize infection enhanced expression of murine beta-defensins (mbd-1, -2,-3, and -4) in upper and lower airway mucosa of influenza virus infected mice human epithelial beta-defensins 2 and 3 inhibit hiv-1 replication rhinovirus increases human beta-defensin-2 and -3 mrna expression in cultured bronchial epithelial cells human rhinovirus infection induces airway epithelial cell production of human beta-defensin 2 both in vitro and in vivo role of human beta-defensin-2 during tumor necrosis factor-alpha/nf-kappab-mediated innate antiviral response against human respiratory syncytial virus oral human beta-defensin 2 in hiv-infected subjects with long-term use of antiretroviral therapy human betadefensins suppress human immunodeficiency virus infection: potential role in mucosal protection cutting edge: human beta defensin 3-a novel antagonist of the hiv-1 coreceptor cxcr4 beta-defensin genomic copy number is associated with hiv load and immune reconstitution in subsaharan africans increased levels of human betadefensins mrna in sexually hiv-1 exposed but uninfected individuals modulation of human beta-defensin-1 (hbd-1) in plasmacytoid dendritic cells (pdc), monocytes, and epithelial cells by influenza virus, herpes simplex virus, and sendai virus and its possible role in innate immunity recombinant mouse beta-defensin 2 inhibits infection by influenza a virus by blocking its entry antiviral activity of recombinant mouse beta-defensin 3 against influenza a virus in vitro and in vivo cytokine (tumor necrosis factor, il-6, and il-8) production by respiratory syncytial virus-infected human alveolar macrophages antiviral activity of human beta-defensin 3 against vaccinia virus selective killing of vaccinia virus by ll-37: implications for eczema vaccinatum cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes a cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins retrocyclin: a primate peptide that protects cells from infection by t-and m-tropic strains of hiv-1 evaluation of a theta-defensin in a murine model of herpes simplex virus type 1 keratitis. invest ophthalmol vis sci the theta-defensin, retrocyclin, inhibits hiv-1 entry theta-defensins prevent hiv-1 env-mediated fusion by binding gp41 and blocking 6-helix bundle formation rc-101, a retrocyclin-1 analogue with enhanced activity against primary hiv type 1 isolates hiv-1 adapts to a retrocyclin with cationic amino acid substitutions that reduce fusion efficiency of gp41 retrocyclin rc-101 blocks hiv-1 transmission across cervical mucosa in an organ culture the formulated microbicide rc-101 was safe and antivirally active following intravaginal application in pigtailed macaques retrocyclin 2: a new therapy against avian influenza h5n1 virus in vivo and vitro hapivirins and diprovirins: novel theta-defensin analogs with potent activity against influenza a virus host-pathogen interactions during coronavirus infection of primary alveolar epithelial cells rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease the human cationic antimicrobial protein (hcap-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa levels of innate immune factors in genital fluids: association of alpha defensins and ll-37 with genital infections and increased hiv acquisition hiv-neutralizing activity of cationic polypeptides in cervicovaginal secretions of women in hiv-serodiscordant relationships the antimicrobial peptide ll-37 inhibits hiv-1 replication effects of cathelicidin and its fragments on three key enzymes of hiv-1 antiviral activity and increased host defense against influenza infection elicited by the human cathelicidin ll-37 the human cathelicidin ll-37 inhibits influenza a viruses through a mechanism distinct from that of surfactant protein d or defensins modulation of the tlr-mediated inflammatory response by the endogenous human host defense peptide ll-37 toll-like receptors and viruses: induction of innate antiviral immune responses self-rna-antimicrobial peptide complexes activate human dendritic cells through tlr7 and tlr8 ll37 and cationic peptides enhance tlr3 signaling by viral double-stranded rnas low concentrations of ll-37 alter il-8 production by keratinocytes and bronchial epithelial cells in response to proinflammatory stimuli antimicrobial peptides inhibit polyinosinic-polycytidylic acid-induced immune responses respiratory epithelial cells convert inactive vitamin d to its active form: potential effects on host defense serum cathelicidin level is associated with viral etiology and severity of bronchiolitis the human cathelicidin ll-37 has antiviral properties against respiratory syncytial virus antimicrobial peptides and the skin immune defense system cytokine milieu of atopic dermatitis skin subverts the innate immune response to vaccinia virus a carpet-based mechanism for direct antimicrobial peptide activity against vaccinia virus membranes antibacterial activity of human neutrophil defensin hnp-1 analogs without cysteines key: cord-274688-cr1rvy8u authors: jewell, britta l; mudimu, edinah; stover, john; ten brink, debra; phillips, andrew n; smith, jennifer a; martin-hughes, rowan; teng, yu; glaubius, robert; mahiane, severin guy; bansi-matharu, loveleen; taramusi, isaac; chagoma, newton; morrison, michelle; doherty, meg; marsh, kimberly; bershteyn, anna; hallett, timothy b; kelly, sherrie l title: potential effects of disruption to hiv programmes in sub-saharan africa caused by covid-19: results from multiple mathematical models date: 2020-08-06 journal: lancet hiv doi: 10.1016/s2352-3018(20)30211-3 sha: doc_id: 274688 cord_uid: cr1rvy8u background: the covid-19 pandemic could lead to disruptions to provision of hiv services for people living with hiv and those at risk of acquiring hiv in sub-saharan africa, where unaids estimated that more than two-thirds of the approximately 38 million people living with hiv resided in 2018. we aimed to predict the potential effects of such disruptions on hiv-related deaths and new infections in sub-saharan africa. methods: in this modelling study, we used five well described models of hiv epidemics (goals, optima hiv, hiv synthesis, an imperial college london model, and epidemiological modeling software [emod]) to estimate the effect of various potential disruptions to hiv prevention, testing, and treatment services on hiv-related deaths and new infections in sub-saharan africa lasting 6 months over 1 year from april 1, 2020. we considered scenarios in which disruptions affected 20%, 50%, and 100% of the population. findings: a 6-month interruption of supply of antiretroviral therapy (art) drugs across 50% of the population of people living with hiv who are on treatment would be expected to lead to a 1·63 times (median across models; range 1·39–1·87) increase in hiv-related deaths over a 1-year period compared with no disruption. in sub-saharan africa, this increase amounts to a median excess of hiv deaths, across all model estimates, of 296 000 (range 229 023–420 000) if such a high level of disruption occurred. interruption of art would increase mother-to-child transmission of hiv by approximately 1·6 times. although an interruption in the supply of art drugs would have the largest impact of any potential disruptions, effects of poorer clinical care due to overstretched health facilities, interruptions of supply of other drugs such as co-trimoxazole, and suspension of hiv testing would all have a substantial effect on population-level mortality (up to a 1·06 times increase in hiv-related deaths over a 1-year period due to disruptions affecting 50% of the population compared with no disruption). interruption to condom supplies and peer education would make populations more susceptible to increases in hiv incidence, although physical distancing measures could lead to reductions in risky sexual behaviour (up to 1·19 times increase in new hiv infections over a 1-year period if 50% of people are affected). interpretation: during the covid-19 pandemic, the primary priority for governments, donors, suppliers, and communities should focus on maintaining uninterrupted supply of art drugs for people with hiv to avoid additional hiv-related deaths. the provision of other hiv prevention measures is also important to prevent any increase in hiv incidence. funding: bill & melinda gates foundation. increase in death rate in people with aids diseases due to overstretched health system 1.00 (1. see footnotes to table 2 in main paper. table a2 . predicted average relative annual increase in hiv mortality and incidence in 5 years from 1 april 2020 in countries in sub-saharan africa that would result from a 6-month disruption of specific hiv services for 50% of the population, with 95% uncertainty bounds. assuming no change in sexual behaviour associated with the period of disruption. for adults and children, except where stated. individual national estimates were generated, and respective values aggregated for the two subregions (eastern and southern africa and western and central africa) and for sub-saharan africa, with estimates extrapolated to represent regional burden of people living with hiv for each region. the impact on drug resistance as a result of this disruption and additional covid-19-related deaths among people living with hiv (plhiv) were not considered. the hiv-related mortality rate was applied to those on antiretroviral therapy (art) who were removed from art due to this disruption following empirical observations from the smart study group (1). cd4 counts declined rapidly as a result, and gradually returned to pre-art levels after interruption. once art coverage was resumed following the disruption, mortality rate for those on art were reapplied. times for cd4 progression used in the optima hiv model are listed in table a4 . the figure below illustrates the plhiv on art and their subsequent cd4 count in the baseline versus the no art for 6-month scenario in 2020 for an example country. for art disruption, this is for the default "off art" mortality rate, cd4 counts dropped even more dramatically following the parameter informed by the smart study (1), as used herein. the default off art mortality rate (see table a2 ) was applied for generating cd4 counts shown in figure a1 ; however, mortality rate off art used in this study followed smart study findings, which would have showed even more dramatic cd4 reductions particularly in 2021. figure a2 illustrates the difference in progression of cd4 counts among adults living with hiv either on or off art for an example country in sub-saharan africa. the hiv-related death rate for people living with hiv who were on art, but who stopped treatment due to potential disruption on the antiretroviral (arv) drug supply as a result of the covid-19 pandemic was modeled to represent findings from the smart study (1). the smart trial showed that immune recovery during arv therapy tended to be rapidly lost after interruption of treatment, with a median loss of 187 cd4 cells/mm 3 hiv-related mortality off art this below is from the appendix to reference 1 describing the viral load and cd4 count changes during art interruption. https://www.thelancet.com/cms/10.1016/s2352-3018(19)30400-x/attachment/3398d3d8-5988-4171-aa74-2f68ce877725/mmc1.pdf risk of aids death is related to the most recent cd4 count, viral load and age, as described in that appendix. viral load returns to previous maximum viral load (vmax) in 3 months and adopts natural history changes thereafter. cd4 rate of decline returns to natural history changes (i.e. those in art naïve patients) after 9 months, unless the count remains > 200 above the cd4 nadir rate of cd4 count decline depends on current viral load. c(t) is the cd4 count at time t, cmin(t) is the cd4 count nadir measured by time t and cc(t-1) is the change in cd4 count from t-1 to t. this is broadly based on evidence from a number of analyses of the effects of art interruption (1-5 below) the imperial college london model is a deterministic, compartmental model of hiv transmission and progression, and has been described previously [1, 2] . briefly, the model is stratified by age, sex, behavioural risk group (including partner change rate, condom use, and frequency of sex acts), and male circumcision status, with population growth in accordance with country-specific projections. hiv this analysis simulates the effect of 3-and 6-month interruptions of different hiv services starting mid-2020 as a result of the covid-19 pandemic. in all scenarios of interruptions to hiv services, there is assumed to be no change to sexual contact rates, but a scenario of a 10% reduction in sexual contacts for all risk groups is modelled separately. each type of disruption is assumed to occur independently so as to illustrate the individual effect of the respective disruption. however, in reality, multiple disruptions might occur simultaneously. results are most sensitive to the assumption about mortality for hiv-infected individuals who stop art. our results are therefore presented using three different estimates of mortality, with the central result using the 'medium' assumption in supplementary table 1 . the smart trial found a 3% risk at 12 months of either death of an opportunistic infection for those with interrupted art [3] . this also implies a mean survival time approximately equivalent to that for hivpositive persons who have never been on art [4] . upper bound 0.44% 5.28% this is the hypothetical worst-case scenario in which many persons deteriorate more rapidly. the emod model source code, parameter definitions and ranges, and description/documentation can be found at https://github.com/institutefordiseasemodeling/emod. analyses are based upon a model calibrated to the hiv epidemic in south africa and described elsewhere. [1] [2] [3] [4] briefly, the model is parameterized with epidemiologic data including population size, fertility, mortality, voluntary male circumcision coverage, health seeking and sexual behaviour. south africa data on age and sex specific hiv prevalence, art coverage, population size and hiv incidence were used to calibrate the model. calibration was performed using a parallel simultaneous perturbation optimization (pspo) algorithm. 5 roulette resampling in proportion to the likelihood of each simulation was used to select 250 model parameter sets. the model's fit to adult hiv prevalence and number receiving art are shown in figure a5 (gray lines: individual simulations; blue line: average of 250 simulations; black dots/lines: data from hiv surveys). to estimate the number of excess deaths in countries and regions throughout sub-saharan africa, we assumed that the number of deaths caused by a 6-month interruption in art was proportional to the number of people virally suppressed on art in a given country or region. a limitation of this assumption is that it does not take into account regional differences in the vulnerability of plhiv with viral load suppression to death from an art interruption, which could arise due to differences in the age distribution, cd4 count, health and nutritional status, and exposure to infections such as tuberculosis. data used to estimate mortality by country and region are shown in supplementary table 1 table a10 . estimation of excess hiv deaths in 1 year due to 6 months interruption of art with 95% uncertainty bounds. updated assessment of risks and benefits of dolutegravir versus efavirenz in new antiretroviral treatment initiators in sub-saharan africa: modelling to inform treatment guidelines interruption of haart in hiv clinical practice. results from the icona study interruption and discontinuation of hart in the macs reasons for modification and discontinuation of antiretrovirals: results from a single treatment centre safety of long-term interruption of successful antiretroviral therapy: the athena cohort study predictors of initial cd4 decline after antiretroviral treatment interruption in the smart study the new role of antiretrovirals in combination hiv prevention: a mathematical modelling analysis maximising hiv prevention by balancing the opportunities of today with the promises of tomorrow: a modelling study the strategies for management of antiretroviral therapy (smart) study group, cd4+ count-guided interruption of antiretroviral treatment time from hiv seroconversion to death: a collaborative analysis of eight studies in six low and middle-income countries before highly active antiretroviral therapy implementation and applications of emod, an individual-based multi-disease modeling platform sexual partnership age pairings and risk of hiv acquisition in rural south africa targeting and vaccine durability are key for population-level impact and cost-effectiveness of a pox-protein hiv vaccine regimen in south africa the future of a partially effective hiv vaccine: assessing limitations at the population level application of a second-order stochastic optimization algorithm for fitting stochastic epidemiological models key: cord-324984-ojrpsdt9 authors: ji, xingyue; li, zhuorong title: medicinal chemistry strategies toward host targeting antiviral agents date: 2020-02-14 journal: med res rev doi: 10.1002/med.21664 sha: doc_id: 324984 cord_uid: ojrpsdt9 direct‐acting antiviral agents (daas) represent a class of drugs targeting viral proteins and have been demonstrated to be very successful in combating viral infections in clinic. however, daas suffer from several inherent limitations, including narrow‐spectrum antiviral profiles and liability to drug resistance, and hence there are still unmet needs in the treatment of viral infections. in comparison, host targeting antivirals (htas) target host factors for antiviral treatment. since host proteins are probably broadly required for various viral infections, htas are not only perceived, but also demonstrated to exhibit broad‐spectrum antiviral activities. in addition, host proteins are not under the genetic control of viral genome, and hence htas possess much higher genetic barrier to drug resistance as compared with daas. in recent years, much progress has been made to the development of htas with the approval of chemokine receptor type 5 antagonist maraviroc for human immunodeficiency virus treatment and more in the pipeline for other viral infections. in this review, we summarize various host proteins as antiviral targets from a medicinal chemistry prospective. challenges and issues associated with htas are also discussed. viral infections still represent a major global public health problem. although hundreds of viruses are known to be pathogenic, only less than 10 of them can be treated in clinic with available antiviral drugs. for some highly pathogenic virus such as zika (zikv), ebola (ebov), severe acute respiratory syndrome (sars), as well as many others, there is still no effective drugs on market against them. most of the antiviral drugs approved are designed to target viral proteins to inhibit viral infections, and they are named as direct-acting antiviral agents (daas). 1 daas have been demonstrated to be very successful in clinic to combat viral infections, and are generally considered as very safe for human use because most of the targeted viral proteins have no human homologs. however, viral polymerases, one of the hottest targets for antiviral design, do share some structural similarity with their human counterparts, especially at the active sites, which is the major underlying reason for the toxicity observed with nucleoside-based antivirals. in addition, viral proteins do not normally share structural similarity among different species or even genotypes of virus, and hence one antiviral agent targeting a specific viral protein can hardly impart the same inhibitory effects against the other virus. consequently, the antiviral drugs available on market can barely be employed to treat newly emerging virus, and the magnitude of this problem is further exacerbated by the lack of broad-spectrum antiviral drugs on market. the other inherent limitation of daas is that daas, in most cases, have low genetic barrier to drug resistance because they act directly on viral proteins, and the resulted selective pressure will facilitate the mutations of virus during replication, which will make the virus refractory to the treatment of daas. 2 altogether, there still exist great unmet needs for the treatment of viral infections, and new antivirals with broad-spectrum antiviral activities as well as new mechanism of action are highly demanded. it is widely known that viruses are unable to complete their replication without the help of the host. they will hijack various host proteins or pathways throughout their replication cycle to facilitate their replication, and hence the inhibition or knockdown of such host proteins will block viral replication. therefore, host proteins as such are potential antiviral targets for drug development. the host targeting antivirals (htas) are complementary to the daas, and are superior to daas from several aspects. chiefly, a specific host protein might play crucial roles in the replication of several types of viruses, and thereby its inhibition will yield broad-spectrum antiviral activities. htas as such are likely to be effective against newly emerging virus, because they may also leverage on the same host protein for replication. for example, heat shock protein 90 (hsp90), a host chaperone responsible for the folding, assembly, and maturation of endogenous proteins, is also found to be crucial for maturation of many viral proteins, and therefore, hsp90 inhibitors are endowed with broadspectrum antiviral activities. 3 altogether, targeting host factors is a very promising strategy with possibility to address the critical challenges faced with the daas. in this review, we summarize the recent advances made in htas from a medicinal chemistry standpoint, and the host targets are generally classified into three different categories based on the development stage of their corresponding inhibitors/modulators, namely the ones which reached food and drug administration (fda) approval, that have entered clinical trials and those in preclinical studies. the focus is mainly placed on the smallmolecule based htas, and the antibody or small interfering rna (sirna)-based strategy will not be included. for several well-studied host targets with quite a lot of known inhibitors, because each one can stand alone as an independent review, and this review is not intended to be comprehensive, so for such targets, only a selection of representative inhibitors will be presented to discuss related issues. it should be noted that chemokine receptor type 5 (ccr5) antagonist but not chemokine receptor type 4 (cxcr4) antagonist has reached fda approval. however, cxcr4 is discussed in this section because it is closely related to ccr5. the chemokine receptors cxcr4 and ccr5 belong to the superfamily of g-protein-coupled receptors with seven transmembrane domains, and both are involved in the entrance of human immunodeficiency virus (hiv) virus. 4 the glycoprotein 120 (gp120) on the surface of hiv envelop will bind to the cd4 receptor on the surface of t lymphocytes, leading to the conformation change of gp120. the gp120 will then bind to coreceptor cxcr4, ccr5, or both to facilitate the entrance of hiv virus. 5 hiv-1 virus can thus be broadly classified into three types based on the coreceptor tropism, namely the x4-, r5-, and dual-tropic viruses. the feasibility of targeting cxcr4 or ccr5 for hiv therapy is supported by the fact that ccr5 mutations confer the host with resistance to hiv infection. 6 ample studies have demonstrated that cxcr4 antagonists can block hiv-1 infection through cxcr4. 7, 8 generally, cxcr4 antagonist can be divided into two types: peptide and nonpeptide based. the peptide-based antagonists were designed using the natural chemokines of cxcr4 as template, such as stromal cell-derived factor 1α (sdf-1α) and viral macrophage inflammatory protein ii (vmip-ii), both of which can bind to and then activate cxcr4 to initiate downstream signaling pathway. for example, peptide v1 (1; figure 1 ) was derived from the first 1 to 21 residues of vmip-ii, and it can block the interaction between cxcr4 and hiv-1, and thereby inhibit the entry of both x4-and dual-tropic hiv-1 virus. interestingly, replacement of all the residues in 1 with all d-amino acid lead to much more potent antiviral activities. 9 due to the inherent limitation with the peptide-based antagonist, several nonpeptide based antagonists have been developed. amd3100 (2) was discovered by random screening, and it inhibited hiv-1 infection by disrupting the interaction between cxcr4 and hiv-1. 10 encouragingly, it showed antiviral efficacy in x4 hiv-1-infected patients in a phase ii clinical trial. however, compound 2 lacks oral bioavailability due to its high positive charge, and cardiotoxicity was also reported in the clinical trial. 11 the structural optimization identified amd3465 (3) with just one macrocyclic structure retained. this compound retained all the biological profiles of 2, and it showed improved antiviral activity (ic 50 : 1-10 nm). it also inhibited the replication of drug-resistant virus strain (zidovudine). 12 amd070 (4), a noncyclam derivative, was discovered by anormed as a new cxcr4 antagonist. it showed much improved oral bioavailability (rat: 20%; dog: 80%), but hepatotoxicity was observed in preclinical studies. 13 further structural optimization led to gsk812397 (5) without the basic sidechain. it showed potent anti-hiv-1 activity with an ic 50 value of 1.5 nm, and it did not show any detectable toxicity (>1000 nm) in in vitro assay. in addition, 5 also presented acceptable pharmacokinetic profiles with the oral bioavailability in the range between 10% and 21% among different species. 14 further structural modification with constraining conformation (6) and/or opening up the tetrahydroquinoline ring (7) resulted in a series of new derivatives with retained anti-hiv activities. 15, 16 however, none of these compounds have been advanced into clinical trials. although compound 2, also known as plerixafor, was approved by the fda for autologous transplantation in patients with non-hodgkin's lymphoma or multiple myeloma, 17 no cxcr4 antagonist has been approved for the treatment of hiv infections. the underlying reasons are at least twofold: one major concern with the cxcr4 antagonist is the toxicity issue, especially that cxcr4 18, 19 or sdf-1α 20 knockout mice die prenatally with multiple neurological, cardiac/vascular, and hematopoietic defects. such potential adverse outcomes are exacerbated in the case of hiv treatment, wherein a life-long treatment is required; the other minor issue with these cxcr4 antagonists is the lack of oral bioavailability. iv injection is not practical in the case of hiv treatment, because this will definitely hurt the compliance of the patients. in comparison, the development of ccr5 antagonists for hiv treatment is much more successful. one ccr5 antagonist maraviroc (8; figure 2 ) has been approved by fda as hiv entry inhibitor in 2007. 21 with a long plasma half-life (15-23 hours), 22 8 is administrated orally once daily. the clinical data showed that 8 is well tolerated in patients and it can significantly repress the viral load in r5-infected haart-treatment experienced patients. moreover, it is active against 200 clinically derived hiv-1 envelope-recombinant pseudoviruses, with 100 of them being resistant to existing drug classes, demonstrating the merits of htas. 23 in the clinic, it is primarily recommended for the patients infected with r5-tropic hiv virus, and a tropism test (although not approved by fda) is highly required to determine the viral tropism population within the patient before the treatment to ensure treatment success. the first generation of the test (known as trofile) failed to distinguish r5 from r5/x4 mixed tropism, resulting in significant drug failure in clinic in r5/x4-infected patients. 24 therefore, 8 is just designated as a backup regimen, despite that a new reliable and cost-efficient tropism test is available in clinic now. interestingly, the combination of 8 with raltegravir/tenofovir/emtricitabine led to faster reduction of 2-long term repeat (2-ltr + ) newly infected cells and recovery of cd4 + t-cell counts after 48 weeks of treatment, 25 and 8 in combination with reverse transcriptase inhibitors is being tested in clinic for preexposure prophylaxis. 26 however, the use of 8 is accompanied with severe side effects, and in some cases even life-threatening conditions such as hepatotoxicity and heart attack were observed. to mitigate these limitations, the second generation of ccr5 antagonists have been developed. two clinical candidates vicriviroc (9) 27 and aplaviroc (10) 28 have made to phase ii trials. however, the studies were halted f i g u r e 2 the chemical structures of representative chemokine receptor type 5 antagonists due to insufficient efficacy and observed hepatotoxicity for 9 and 10, respectively. much of the subsequent medicinal chemistry effort is devoted to the structural modification toward 8 to 10, aiming to improve either efficacy and/or adme profiles. for example, pfizer discovered compound 8's structural analogue pf-232798 (11), 29 which showed not only potent anti-hiv activity (ic 50 : 2.0 nm) and moderate herg inhibition (ic 50 : 12 μm), but also superior oral bioavailability as compared to 8. in addition, compound 11 is also active against maraviroc-resistant hiv-1 isolate strain cc185. the data from phase-ii trials demonstrated its superior safety in patients with no adverse effects observed at a dosage up to 250 mg. however, no further data were disclosed after that. 30 gsk 163929 (12) with a 4,4-disubstituted piperidine scaffold exhibited potent anti-hiv activity and excellent pharmacokinetics (pk) profiles, but it did not progress to clinic trials due to toxicity concerns. 31 the other two representative new scaffolds derived from compound 8 are monocyclic piperidine amides and cyclic and acyclic urea-piperidines. some representative candidate compounds from these two series are shown in figure 2 . among these compounds, only tak-220 (13) was progressed to phase-i clinical trials. 32 however, no further update about the status of compound 13 has been reported. inspired by the scaffold of 9 and 10, several new ccr5 antagonists have been devised as depicted in figure 2 . 33 the most promising antagonist among this series is the one developed by incyte based on the structure of 10. incb9471 (16) showed potent antiviral activity against r5 hiv-1 strains at ic 50 values in sub-nanomolar range. in addition, it presented excellent pk profiles with oral bioavailability being 100% and 95% in rat and dog, respectively. 34 as such, 16 has been advanced to phase ii trials, and the data showed that it was well-tolerated in humans. however, its clinical studies were halted due to business issues. besides the chemotypes described above, ccr5 antagonists with miscellaneous scaffolds have also been discovered, yet none of them have made to clinical trials. 35 targeting cxcr4 or ccr5 alone for hiv treatment possesses several downsides. chiefly, a tropism test is required before the treatment, and tropism shifts are frequently observed in both treatment-experienced and treatment-naive hiv-infected patients. therefore, a dual antagonist against both cxcr4 and ccr5 would be perfect to mitigate these limitations. the first dual-tropic inhibitor amd3451 (17; figure 3 ) with n-pyridinylmethyl cyclam scaffold was discovered in 2004. it was moderately active against not only x4-and r5-tropic hiv strains but also dual -tropic hiv strains with ic 50 in the range of 1 to 30 μm. 36 although 17 is not potent enough for further development, it established the proof of concept of targeting cxcr4 and ccr5 simultaneously for hiv-1 treatment. ribavirin (18; figure 4 ) is an approved antiviral drug used in clinic to treat respiratory syncytial virus (rsv), hepatitis c virus (hcv) infection and viral hemorrhagic fever. although its specific antiviral mechanism of action f i g u r e 3 the chemical structure of a dual-tropic inhibitor amd3451 the chemical structures of 18 and 19 ji and li | 5 remains largely uncertain, it is widely accepted that it elicits its antiviral efficacy via modulating host pathways. for example, 18 showed moderate inhibitory potency against inosine-5′-monophosphate dehydrogenase (impdh) with a k i value of around 250 nm, which is believed to be highly involved in the replication of various viruses (see below). in addition, several other mechanism of actions have also been proposed and evidenced: (a) direct inhibition of rna polymerase by converting 18 to its triphosphate form to competitively bind to the nucleotides binding site in rna polymerase 37, 38 ; (b) 18 can act as a mutagen by inserting into the viral rna to push the virus beyond the threshold of error catastrophe 39, 40 ; (c) 18 shows immunomodulatory effect of shifting a th2 response in favor of a th1 phenotype, which helps to clear virus infections. 41 although the combination of 18 and interferon 2α (ifn-2α) used to be the soc for hcv treatment, it is notorious for several severe side effects. one major adverse effect associated with the use of 18 is hemolytic anemia. 42 to alleviate this unwanted effect, taribavirin (19) was developed as the prodrug of 18. ideally, 19 can be metabolized to 18 mainly in the liver to target hcv-infected hepatocytes, and hence the distribution of 18 within red blood cells will be significantly decreased, and thereby the development of hemolytic anemia will be subsequently eliminated. indeed, the clinical data from phase iii trials revealed that patients receiving 19 (fixed dosage 600 mg, bid) and ifn showed significantly lower rates of anemia as compared to the ones in 18 (1000-1200 mg) and ifn group (5.3% vs 24%). 43 however, the sustained virologic response (svr) rates for 19 and 18 group are 38% and 52%, respectively, failing to demonstrate the noninferiority end point for efficacy. the viser-2 trials also failed to meet the noninferiority end points. 44 with the approval of several daas such as sofosbuvir and ledipasvir, the treatment of hcv in clinic has been significantly revolutionized, and several both ribavirin (rbv)-and ifn-free regimens have been approved with better efficacy and safety profiles as compared to the old soc. initially, it was expected that the hcv treatment would not benefit much from the combination of rbv with other daas due to the safety concern associated with rbv. however, very interestingly, it has been found recently that rbv remained an indispensable component for the optimal treatment for some difficult-to-cure subgroups of hcv patients. 45 for example, in a phase 3 c-edge studies, the combination of rbv with elbasvir/grazoprevir achieved much higher svr ( [3/5] , respectively) as compared to the group without rbv in patients infected with genotype 4 hcv. 46 in addition, rbv increased the barrier to resistance, especially in patients receiving daas with low barriers to resistance. importantly, the combination of rbv with daas showed much improved safety and tolerability as compared with the combination with ifn, and the frequency and severity of anemia is significantly reduced with an adverse effect-induced discontinuation rate of less than 3%. 45 nevertheless, the last generations hcv daas (including glecaprevir/pibrentasvir, sofosbuvir/velpatasvir, sofosbuvir/velpatasvir/ voxilaprevir) are highly effective in most cases without any need to use rbv. cyclophilins are a family of cellular peptidyl-prolyl cis-trans isomerases (ppiase) and are involved in many cellular processes. the important roles cyclophilin a (cypa) plays during hcv replication were found by an unexpected clinical finding. cyclosporin a (csa; 20; figure 5 ), which shows high binding affinity to cypa, is an approved immunosuppressive drug. in a clinical trial for csa's therapeutic potential against hepatitis-associated inflammation, it was unexpectedly found that a significantly more potent antiviral response was observed in the combination group of csa and ifn-α2b as compared with the ifn-α2b alone group. 47 the function of cypa in the replication of hcv was subsequently confirmed in vitro. 48 the main cellular function of cyps is to convert the conformation (trans-to cis-form) of prolines in the protein, which is essential for trafficking of many proteins and forming protein complexes, and these functions are also indispensable for the replication of hcv. therefore, it is anticipated that the cypa inhibitors will show anti-hcv activity. although 20 is an approved drug, its main indication is suppressing immunoreaction after organ transplantation, which is an unwanted "side-effect" for antivirals. fortunately, the immnosuppressive effect of 20 is attributed to the inhibition of calcineurin (cn) but not cypa, and hence it is feasible to eliminate the immnosuppressive effect while retaining the anti-hcv activity by making new csa analogues. nim811 (21) is one of such analogues with a methyl-isoleucine at position 4 of csa. it showed anti-hcv activity in vitro with an ic 50 value of 0.12 μm, whereas its immunosuppressive effect was completely eliminated. 49 the phase i clinical trial showed that 21 is well-tolerated with no obvious adverse effects observed. however, in a subsequent double-blind, placebo-controlled study, the monotherapy with 21 failed to yield significant viral load reduction in genotype 1 hcv-infected patients. the underlying reason is attributed to a relatively low trough concentration (0.47 μm) at a dosage of 600 mg (bid), which is lower than the ic 50 value (1.5 μm) of 21 against hcv in the presence of serum, and the dosage elevation did not result in proportional exposure. 50 therefore, the clinical trial with 21 is not continued. alisporivir (22) is a more potent nonimmunosuppressive csa analogue with an anti-hcv ic 50 values of 0.045 and 0.33 μm in the absence and presence of serum, respectively. in addition, it is much more difficult to develop resistance against 22 both in vitro and in patients, as compared to other daas. 51 in phase ii trial, 22 was studied in combination with rbv as an ifn-free regimen in genotype 2 and 3 patients, and about half of the patients receiving 22 (800 mg, qd) plus rbv, and one-third of those receiving 22 only (1000 mg qd) were cured with a svr. 52 however, unfortunately, in a pivotal phase iii trial designed to study the combination of 22 with rbv and peg-ifn-α2a in treatment-naive, genotype 1 hcv patients, six cases of severe pancreatitis along with one death were reported, and the clinical trial with 22 was then put on hold. it is worth noting that all the severe side effects were observed in the triple-therapy aim including rbv and peg-ifn-α2a, both of which are notorious for their adverse effects, while the monotherapy with 22 or the ifn-free regimen did not result in the same side effects. 53 to mitigate the observed side effects with 21 and 22, the third generation of csa derivatives have been potent anti-hcv activity in vitro with an ec 50 and ec 90 value of 0.1 and 0.3 μm, respectively, and exhibited synergistic effect with ifn-α and additive effect with rbv. it also showed low potential for drug-drug interaction with no obvious induction on the major cytochrome p450 enzymes 1a2, 2b6, and 3a4. in addition, 23 also showed acceptable pk profiles with an oral bioavailability of around 20% in rat and monkey. 54 as such, 23 has been advanced into clinical trials. in patients with genotype 1 hcv infection, 900 mg/day of 23 achieved a decline in plasma viremia by 2.2log 10 after 15 days. 55 it is interesting to note that 23 showed totally different side effects from those of 21 and 22, indicating that the adverse effects may not be associated with the inhibition of cyps, but are likely resulted from the off-target effects of individual inhibitors. non-csa based cyps inhibitors have also been discovered. sanlifehins (sfa) including sanlifehin a (24), b (25), c (26) , and d (27) are a class of natural occurring polyketides isolated from the soil bacterium streptomyces sp. strain a92-308110. sfas were identified as cyps inhibitors with stronger potency as compared to csa derivatives, particular 25, of which the inhibitory potency against all cyps was 30-to 50-fold more potent than 20. it also showed much more potent antiviral activity in vitro with an ec 50 value of 70 nm against hcv genotype 1b. interestingly, albeit slightly less potent as compared to against the wild type, 24, 26, and 27 retained inhibitory effect against csa-resistant huh 9 to 13 subgenomic replicon with ec 50 values ranging from 3.3 to 6.8 μm. 56 however, the pk studies revealed that sfa suffered from poor water solubility (<25 μm) and poor oral bioavailability (<4%). moreover, sfa possessed undesirable immunosuppressive activity via an unknown mechanism. 57 structural modifications have been made to 24, and it was revealed that only the macrocyclic moiety was essential for the cyps inhibition, and modification on the sidechain had little effect on the binding affinity. 58 removal of the spirolactam moiety on the sidechain of 24 only led to the loss of immunosuppressive activity but not the cyps inhibition. such structure-activity relationships are very important for further optimization of sfa as anti-hcv agents ( figure 6 ). both csa and sfa derivatives are macrocyclic molecules with large molecular size, and as such both suffered from some limitations, including poor cell membrane permeability, high risk of drug-drug interactions and off-target toxicity, and synthetic inaccessibility for structural optimization and manufacturing. in 2016, a new family of nonpeptide based small-molecule cyps inhibitors have been designed using fragment-based strategy. 59 the crystal structure of cypa indicated that its ppiase catalytic site consisted of hydrophobic, aromatic, and polar residues, next to the catalytic site is a deep pocket called gatekeeper site, which might contribute to the substrate binding specificity ( figure 7) . a total of 34 409 fragments were docked into these two sites, and several fragments were identified to bind to these two sites separately. eventually, fragment 28 and 29 from each binding site were it has been well-established that cyps are involved in a broad range of viral infections, including hiv-1, 60 influenza virus, 61 hbv, 62 sars coronavirus, 63 human cytomegalovirus (hcmv), 64 papillomavirus, 65 and nidovirus, 66 among others. therefore, it can be envisioned that cyps inhibitors should exhibit broad-spectrum antiviral activity against these viruses. indeed, cyps inhibitors were reported to show broad-spectrum antiviral activities. 67, 68 consequently, the development of cyps inhibitors could benefit the treatment of a variety of viral infections, possibly including the newly emerging unidentified viral infections. it is widely known that virus will hijack a wide range of host factors to facilitate its replication. meanwhile, the host has also evolved an innate immune system to counteract viral infections. compounds with the capacity to mediate the host antiviral pathways are expected to confer broad-spectrum antiviral activity, and nitazoxanide (32; figure 9 ) is such a compound that regulates several host antiviral pathways to convey broad-spectrum antiviral profiles. 32 is originally an fda approved drug for the treatment of diarrhea caused by cryptosporidium parvum and gastrointestinal disorders as the most frequently observed side effects. it also showed no effect on cardiac repolarization in a clinical trial for cardiac safety. 69 in recent studies, 32 was revealed as a promising antiviral agent against a wide range of pathogenic viruses including influenza virus, 70 hbv, 71 hcv, 71 ebov, 72 denv, 73 jev, 74 hiv, 75 and zikv, 76 among others with ic 50 values ranging from 0.06 to 1.0 μg/ml 77, 78 its mechanism studies revealed that multiple host antiviral pathways were involved, and as such 32 is widely known as a polypharmacology antiviral agent. 32 activated protein kinase r (pkr), which plays vital roles in innate immune system. the activation of pkr leads to the phosphorylation of eukaryotic initiation factor 2α, an important host restriction factor against viral replications. 79 hbv viral protein hbx was found to interact with host protein damage-specific dna-binding protein 1 (ddb1) to promote transcription of covalently closed circular dna (cccdna) and degradation of a host restriction factor chromosomes 5/6 (smc5/6). 80 32 was reported to disrupt the interaction between hbx and ddb1 to block the transcription from cccdna. 81 in addition, 32 was also able to activate cellular antiviral response and induce the expression of a subset of ifn-stimulated genes, especially interferon regulatory factor 1, 82 which is known to block the replication of a wide range of viruses. 83 other host targeting mechanisms are also involved in the broad-spectrum antiviral profiles of 32. 72 since 32 has been safely used in clinic for many years, it has been advanced into clinical trials for the treatment of several viral infections. the most advanced one is the treatment of acute uncomplicated influenza with 32. in a phase 2b/3 trial, significant reductions in tci50 viral titer and alleviation in symptoms were observed in 32 (600 mg, twice daily) group as compared to the placebo. no resistance was identified in the influenza virus collected from the patients receiving 32, and no adverse effect on humoral immune response was reported. 84 a large global phase 3 trial is being conducted. several clinical trials of 32 for the treatment of other viral infections are also ongoing. in a pilot clinical trial of 32 for the treatment of chronic hbv, the serum hbv dna of eight of nine patients became undetectable after 4 to 20 weeks of treatment with 32, and more importantly, three out of nine subjects became hbeag negative, which is a rare outcome for current standard of care. this proof-of-concept study presented 32 as a very promising drug to achieve a hbv cure. 85 32 was also tested in clinical trials against hcv infections, and it showed very pronounced efficacy either as monotherapy or in combination with ifn-α and rbv. for example, administration of 500 mg 32 twice a day for 48 weeks with 180 μg of ifn-α once weekly from week 13 to 48 achieved a svr of 79% in patients infected with chronic hepatitis c (chc) genotype 4, as compared to 50% for ifn-α and rbv. however, the development of 32 as an anti-hcv drug was not further pursued due to the approval of several new daas, despite that 32 has much higher genetic barrier to resistance as compared to daas. interestingly, 32 is considered as a prodrug because it is rapidly hydrolyzed to tizoxanide (33) in the plasma with a half-life of only 6 minutes. compound 33 showed broad-spectrum antiviral activity against a panel of viruses both in vitro and in vivo. to obtain new candidate compounds with improved potency and safety profiles, structural modifications were made to 32 primarily on the phenyl ring a and the thiazole moiety. it was revealed that electron-withdrawing group such as nitro and chloro group at c-5 position was favorable for the antiviral activity, and replacement of the thiazole ring with phenyl ring retained the activity. the structural optimization led to the f i g u r e 9 the chemical structures of nitazoxanide analogues identification of a candidate compound rm-5038 (34) with a chloro group at the c-5 position, which showed comparable activity to that of 32 with ec 50 values in the submicromolar range. 34 was considered to be superior as compared to 32 due to the replacement of the potential cytotoxicity nitro group in 32. 32 also suffers from poor systemic exposure after oral administration, and it is primarily biodistributed in the gastric intestinal tract, and excreted via urine and faeces. for antiviral therapy, it is preferred to increase the systemic exposure of the active drug. for such purpose, amino acid based prodrugs for 33 were devised, 86 and they showed much improved aqueous solubility as compared to the parent drug. as such, prodrug 35 exhibited significantly improved pk profiles with oral bioavailability being around 20% in rats, as compared to 2.8% and 0 for 32 and 33, respectively. in addition, 35 also showed preferable safety profiles in laboratory animals, with a no observed adverse effect level being 25 mg/kg/day for a consecutive 28 days in beagle dogs, and it did not present any obvious toxicity in rats after a single oral dosage of 300 mg/kg, and only minor toxicity on central nervous system and respiratory system was observed at a single dosage of 1000 mg/kg. all these pk results present 35 as a very promising candidate compound for further development, and it is now undergoing phase i clinical trial. α-glucosidase is an enzyme removing glucose units from n-linked glycans attached to a nascent glycoprotein, which is essential for proper folding and functions of many glycoproteins. most viral envelope glycoproteins contain n-linked glycans, and α-glucosidase (especially endoplasmic reticulum [er] α-glucosidase) is highly involved in their proper folding and maturation. therefore, the inhibition of er α-glucosidase would yield broad-spectrum antiviral activity. indeed, er α-glucosidase inhibitors showed pronounced antiviral activity against a series of enveloped viruses both in vitro and in vivo including hiv, 87 hcv, 88 human coronavirus, 89 influenza a virus, 90 and denv. 91 although α-glucosidase is critical in the proper folding of viral envelop glycoproteins, it is less important to host cells, and the host cells can well-tolerate the complete shutdown of these er α-glucosidase. 92 moreover, several glucosidase inhibitors are being used in clinic for treating type ii diabetes and gaucher disease. consequently, targeting α-glucosidase for antiviral therapy would not raise a red flag on toxicity issues. to date, a wide range of α-glucosidase inhibitors have been discovered, 93 among which the iminosugars are the most promising inhibitors as antiviral agents. it is widely accepted that the antiviral profiles of iminosugars is attributed to the inhibition of er α-glucosidases (i and ii). the natural occurring iminosugars 1-deoxynojirimycin (dnj; 36; figure 10 ) and castanospermine (37; figure 10 ) has been used as starting points for further modifications. the modifications to 36 were primarily made at the amino position by introducing an alkyl chain. the n-butylated dnj 38 has been advanced to clinical trials for hiv treatment. in a phase ii study, although 38 showed the chemical structures of iminosugars ji and li | 11 some efficacy on viraemia, it failed to maintain a serum concentration needed to inhibit hiv replication in vitro, 87 so the clinical trials of 38 for hiv treatment have been discontinued. the other dnj analogue mon-dnj (39) also showed broad-spectrum antiviral activity, and it has recently been tested in human against denv infection. in the phase i trial, 39 has been demonstrated to be well-tolerated in human with no severe side effects observed after a single dosage of 1000 mg, and the pk data indicated a low interindividual variability and good linearity over a wide range of dosage (nct02061358). in a recent study, 39 has been tested in a proof-of-concept non-human primate trial against ebov infections. however, 39 failed to yield any survival benefit to macaques infected with ebov-makona, despite that 39 showed definitive antiviral activity against ebov in vitro. 94 celgosivir (40), a prodrug of 37, has also been tested in human against dengue fever. in a phase ib, placebo-controlled study, 40 failed to meet the primary end point in patients infected with uncomplicated dengue fever. however, in a mouse model study, it was confirmed that the dosing regime was crucial for the efficacy, 95 and therefore, a four-time daily dosing regime is planned for a phase ii trial (nct02569827). 40 was also studied for the treatment of patients infected with genotype i hcv. in a phase ii trial, 40 only showed a moderate antiviral effect as a monotherapy, but exhibited synergistic effects with ifn-based therapies. 88 however, further development of 40 as anti-hcv agents were discontinued due to inferior efficacy as compared with other approved daas. although the results from the clinical trials of 39 and 40 demonstrated the safety of iminosugars, they are all limited by poor pk profiles and insufficient efficacy. to address these limitations, further structural modifications were made to 36 by introducing various substituted alkyl sidechain, and several analogues were identified as potent antiviral activity against bovine viral diarrhea virus (bvdv), tacaribe virus, and denv with ec 50 values in the submicromolar range, which is hundreds fold more potent than 38. in addition, these compounds showed much superior pk profiles, especially compound 42, which had an oral bioavailability of 94%. most importantly, they all demonstrated significant protective effects in both ebov and bvdv infected mice models, and the in vivo glycan analysis also indicated significant er α-glucosidase suppression in the compounds treatment group. 96 it should be noted that the inhibition of other glucosidase such as intestinal glycosidase will lead to unwanted side effects. to avoid such side effects, a prodrug of compound 42 was designed by masking the free hydroxyl group by acylation. the tetrabutyrate prodrug 43 exhibited preferred stability toward simulated gastric and intestinal fluid, and yet was readily converted to the parent drug in the plasma and liver of mice. in a cell-based assay, compound 43 showed inhibitory activity against ebov with ec 50 value of 15.6 μm, which is slightly less potent as compared with the parent compound (4.1 μm). compound 43 also showed much improved overall drug exposure after either oral or intravenous administration to mice. 97 another strategy to alleviate the side effects of iminosugars is to design more specific inhibitors toward er α-glucosidase. in 2016, the crystal structure of er α-glucosidase ii in complex with 36 and 39 has been successfully resolved, which provided significant insights into the interactions between inhibitors and the active site of the enzyme, laying a firm foundation for the structure-based drug design of more specific inhibitors. 98 3.4 | inosine-5′-monophosphate dehydrogenase impdh is an enzyme catalyzing the conversion of inosine monophosphate (imp) to xanthosine monophosphate, which is a critical step in the de novo biosynthesis of guanine nucleotides. 99 inhibition of impdh will lead to the decrease in the intracellular guanosine-5'-triphosphate (gtp) level, which will disrupt the gene synthesis of both dna and rna virus, and thereby inhibit viral replication. therefore, impdh inhibitors are expected to show broadspectrum antiviral activities. indeed, impdh inhibitors exhibited broad-spectrum antiviral activities against both dna and rna virus in vitro. 100 the approved non-nucleoside-based impdh inhibitors include mycophenolic acid (44) , its prodrug mycophenolate mofetil (45) and mizoribine (46; figure 11 ), and all of them were approved for prevention of organ transplant rejection, but not for antiviral therapy, nevertheless, all these inhibitors were reported to inhibit the replication of a wide range of virus in vitro and even in vivo. for example, 44, an uncompetitive impdh inhibitor with respect to imp and nad+, is active against flaviviruses, paramyxoviruses, orthopoxviruses, avian reoviruses, and denvs with ec 50 values in low micromolar range, and its antiviral potency is closely correlated to the intracellular gtp levels, indicating the involvement of impdh inhibition in its antiviral mechanism. 101,102 however, one major limitation with the application of 44 is the presence of a phenolic hydroxyl group, which is prone to glycosylation for excretion, and thereby limits its efficacy. to overcome this limitation, a series of phenyloxazoles were developed by the vertex group and others. the representative compound from this series is vx-497 (47) , which shows high affinity to impdh with a k i value of 10 nm. it shows potent antiviral activity against a wide range of dna and rna virus including hbv, hcmv, rsv, and murine encephalomyocarditis virus, hcv, zikv, and ebov, 103 among others with ic 50 values ranging from low micromolar to submicromolar levels, and its antiviral activity can be reversed by the addition of guanosine, indicating that its antiviral mechanism is resulted from impdh inhibition. some other vx-497 derivatives also exhibited similar antiviral profiles. 104, 105 in light of the successful application of rbv in clinic for hcv treatment, as a more potent impdh inhibitor and antiviral agent as compared to rbv, 100 other mechanisms are involved in the antiviral action of rbv. in addition, rbv has to be used in combination with ifn-alpha or other anti-hcv agents in clinic, and the monotherapy with rbv failed to yield any antiviral efficacy in patients. 108 although quite a few other impdh inhibitors are under development, they are all intended for other indications. consequently, special attentions should be paid to this issue in pursuit of impdh inhibitors as antiviral agents. kinase represents a huge family of host proteins, and have been successfully targeted by a myriad of smallmolecule inhibitors for the treatment of cancers and inflammatory diseases in clinic. mounting evidence have shown that viruses hijack a variety of human kinases throughout the entire viral life cycle to facilitate their replications, and some kinases are broadly required. [109] [110] [111] therefore, it can be anticipated that the inhibition of such kinases would lead to the disruption of a broad spectrum of viral replications. since tremendous kinase inhibitors have been approved in clinic for treating cancer and inflammatory diseases, and more are under development in the pipelines, increasing efforts have been devoted to repurposing approved kinase inhibitors as broad-spectrum antiviral agents. in this section, only one type of kinases along with their respective inhibitors are picked for the discussion of related issues, and the selection of these examples is not based on the "importance" of the articles, but rather whether there are appropriate issues to discuss. the numb-associated kinases (naks) constitute a diverse family of ser/thr kinases with a broad range of cellular functions. naks have been found to play key roles in a diverse range of human diseases ranging from parkinson's and prostate cancer to viral infections. host kinases adaptor protein 2 (ap2)-associated protein kinase 1 (aak1) and cyclin g-associated kinase (gak), two kinases from this family, are found to regulate intracellular trafficking of a variety of viruses including denv and ebov. two approved kinase inhibitors sunitinib (49) and erlotinib (50) were found to potently inhibit aak1 and gak, respectively, and both are reported to exhibit potent broad-spectrum antiviral activity in vitro. in addition, the combination of 49 and 50 showed very pronounced protective effects against morbidity and mortality in denv and ebov infection mouse models. 112 a cocktail treatment containing 49 and 50 against ebov infection is being investigated in clinic trial, but no clinical data have been disclosed yet (nct02380625). gak and aak1 were also reported to regulate the binding of ap2m1 to hcv core protein, which is essential to the hcv assembly. inhibitors of gak and aak1 disrupted the interaction between ap2m1 and core, and thus inhibited hcv replication with ec 50 values ranging from 0.15 to 1.8 μm. 113 although 49 and 50 exhibited potent inhibitory activity against gak and aak1, both of which are known as pankinase inhibitors, and thus raise off-target toxicity concerns when used as antiviral agents. in 2015, herdewijn et al developed a series of highly selective inhibitors against gak. the crystal structure of gak in complex with one of these inhibitors has been resolved, showing that inhibitors as such behaved as classic type i adenosine triphosphate (atp)-competitive kinase inhibitors. these compounds showed pronounced anti-hcv activity in vitro with ec 50 in the low micromolar range. 114 further structure optimization lead to the identification of compound 51 with a k d value of 8.9 nm, which is endowed with broad-spectrum antiviral activity against a panel of viruses including denv, ebov, chikv with ec 50 values in the low micromolar range. the mechanism of action studies confirmed that the inhibition of gak is an important target underlying the broad-spectrum antiviral activity of compound 51. 115, 116 the other chemotype of gak inhibitor is quin(az)oline, 117 ,118 yet their antiviral activity was not probed. many other kinases are also involved and play essential roles in different life cycle of various viruses, and their corresponding kinase inhibitors show broad-spectrum antiviral activities against a wide range of viruses both in vitro and in vivo. [109] [110] [111] since host kinase is not under the genetic control of virus, kinase inhibitors for antiviral treatment have much higher genetic barrier to resistance. for example, denv developed resistance against the viral ns4b inhibitor sdm25n after eight passages, yet it remained sensitive to the treatment of 49 and 50 under the same conditions. 119 despite with advantages of broad-spectrum antiviral profiles and higher genetic barrier to resistance, there still remain concerns or limitations for repurposing kinase inhibitors as antiviral agents. chiefly, toxicity is the major concern associated with kinase inhibitor, because most of kinases (if not all) play essential roles to regulate the cellular functions. however, it should be noted that, in most cases, the duration of antiviral therapy can be as short as several weeks or even several days, and hence the incidence of severe side effects can be significantly decreased. in addition, the potential toxicity can be further diminished by operating in a well-defined therapeutic window. since most of the kinase inhibitors are designed to target the atp binding site, which is highly conserved among different kinase families, so nearly all kinase inhibitors possess cross inhibitory activity against other kinases, which could be problematic. first, targeting multiple kinase may result in off-target side effects; second, it is challenging to understand the mechanism underlying the antiviral action of these inhibitors because multiple kinases are involved. however, one could also argue that pan-kinase inhibition is favorable for antiviral therapy in that the off-target kinase(s) may also contribute to the viral replication, and targeting multiple kinases simultaneously may result in not only synergetic antiviral activity but also high genetic barrier to resistance as well. altogether, targeting kinases represent a promising strategy for the development of antiviral agents, especially the ones with broad-spectrum antiviral profiles and high genetic barrier to resistance. sodium taurocholate cotransporting polypeptide (ntcp) is expressed on the hepatic basolateral membranes specifically and functions as a cotransporter for bile acids and sodium ions. recently, ntcp has been identified as hbv/hdv infection receptor via interacting with hbv large surface protein, and the silence of ntcp inhibited hbv and hdv infections. 120 therefore, the inhibition of ntcp would block the entry of hbv/hdv virus, and the subsequent formation of the persistent viral reservoir: cccdna will also be halted, which cannot be achieved by current therapy. 121 were also significantly declined in all myrb groups but not the control tdf group, 122 demonstrating superiority by targeting ntcp (figure 12 ). the other discovered ntcp inhibitors include fda approved drugs (ie, csa, 123 ezetimibe, and ritonavir, 124 etc), fasiglifam, 125 oxysterol, 126 vanitaracin a, 127 nti007, 128 and among others. 129 the inhibition of ntcp by these inhibitors normally will result in the loss of transporter function of ntcp, leading to the inhibition of bile acid uptake, which might cause unwanted adverse effects. interestingly, shimura et al 130 identified several csa f i g u r e 1 2 the chemical structures of a subset of gak and aak1 inhibitors. aaak1, ap2-associated protein kinase 1; gak, cyclin g-associated kinase derivatives with anti-hbv activity in vitro via direct interaction with ntcp to inhibit viral attachment. these compounds inhibited multiple hbv genotypes including one clinically relevant nucleoside analog-resistant hbv isolate. importantly, they did not compromise the transporter function of ntcp. two analogs scy446 (56) and scy450 (57; figure 13 ) also did not show meaningful inhibition against cn, and therefore, these two compounds did not show any unwanted immunosuppressive effects. taken together, these results showed that the inhibition of viral attachment via ntcp can be functionally separated from the bile acid uptake, and future efforts should be dedicated to new ntcp inhibitors with transporter function retained to eliminate unwanted adverse effects. farnesoid x receptor (fxr) belongs to the nuclear receptor superfamily and plays regulatory roles in the metabolism of bile acid, lipid, and glucose. recently, it was identified as a proviral host factor for hbv virus. 131 silence of fxr by small hairpin rna resulted in significant decrease in the levels of cccdna, pregenomic-and precore-rnas, secreted relaxed circular dna (rcdna), and hbsag by 40%-70%, and the same effect was observed with the treatment of a fxr agonist gw4064 (58; figure 14 ). importantly, in c3h/hen adult mice infected with a recombinant aav2/8-hbv vector, gw4064 (50 mg/kg/d) significantly suppressed the rcdna and hbsag titers (mean rcdna variation: −0.52 and −0.93log10; mean hbsag variation −0.89 vs −0.73log10, at day 21 and 28, respectively). 132 interestingly, 58 together with other fxr agonists such as way362450 (59), fexaramine (60), and chenodeoxycholic acid (61) also showed inhibitory effect against hcv replications in huh7.5 cells with ic 50 values ranging from 0.75 to 7.03 μm. the mechanism of action studies showed that compound 58's anti-hcv activity is fxr-dependent. it has been reported that fxr agonist downregulated the expression of srib, which is a coreceptor for hcv entrance. as expected, the treatment of 92 also dose-dependently decreased the level of sr-bi. in addition, 58 also presented synergistic anti-hcv activity with other approved daas, and it remained sensitive against other daa-resistant hcv mutations, suggesting the advantages of targeting host factors. 133 encouragingly, one specific fxr agonist eyp001 (structure not disclosed) has successfully entered clinical trial for hbv treatment, and the data from phase i trial showed that eyp001 is very well-tolerated in humans with no treatment-related discontinuations or severe side effects. the pk of eyp001 was linear up to 500 mg with t max and t 1/2 being 2.3-2.5 and 1.4-2.4 hours, respectively. 134 the phase ii trial of eyp001 is ongoing. f i g u r e 1 3 the chemical structures of a subset of sodium taurocholate cotransporting polypeptide inhibitors. the r group in 56 and 57 was not specified in the original paper 130 diacylglycerol acyltransferases (dgats) are the key enzymes catalyzing the biosynthesis of endogenous triglycerides, which are necessary for the biogenesis of lipid droplets in the liver. it has been reported that lipid droplets are the major site for hcv particle assembly and production, and dgats, especially dgat-1, play vital roles during hcv infection. dgat-1 forms a complex with nonstructural protein 5a (ns5a) and core protein to enhance the interaction of the latter two, and the trafficking of ns5a to lipid droplets is also highly dependent on dgat-1's activity. 135 the silence of dgat-1 significantly impaired hcv entry. 136 therefore, dgat-1 could serve as a viable host target for anti-hcv agents development. one specific dgat-1 inhibitor pradigastat (62; figure 15 ) developed by novartis significantly decreased the level of hcv rna in cell supernatant at a concentration of 10 μm, indicating that compound 62 inhibited the assembly or release of virions. since compound 62 is in clinical development for the treatment of dyslipidemia, and it has demonstrated convinced efficacy and safety profiles. 137 therefore, it was directly tested in patients with genotype 1 or 3 hcv infections for safety and efficacy. however, disappointedly, 14 days of treatment with compound 62 failed to afford any significant deduction in serum hcv rna levels in either gt1 or gt3 patients, and thus the trial was terminated. 138 since the pk studies showed that the predicted concentration of compound 62 in liver is approximately 10 to 20 μm based on the plasma concentration on day 8 and 14, which is much higher than the concentration needed for dgat-1 inhibition (ic 50 : 66 nm), so the lack of efficacy is not due to pk problem. the other possibility is that hcv virus hijacked other compensate pathway to facilitate the assembly and release while dgat-1 activity is inhibited in vivo, or dgat-1 plays a nonenzymatic roles during hcv replication, so the inhibition of its enzymatic activity would not yield any inhibitory activity against hcv replication. hsp90 is a highly conserved chaperone protein that assists the maturation of its clientele proteins. there are four the viral entry and intracellular trafficking stage, hsp90 is reported to be critical for the intracellular translocation of viral proteins as well as other host factors critical for viral replications. for example, hsp90 is required for the nuclear translocation of ebv and hsv-1 dna polymerase and 139,140 rna-dependent rna polymerase of influenza virus. 141 to facilitate viral gene expression, hsp90 also activate several host signaling pathways. for instance, hsp90 is upregulated to activate akt and nuclear factor κb for viral gene expression upon hcmv infection. 142 as a chaperone protein, hsp90 is indispensable for the maturation, accurate folding, and maintenance of stability of various proteins including viral proteins. for example, hsp90 facilitates the accurate folding of ns5a of hcv virus in the replication complex to promote viral replication. 143 it can also help to maintain the stability of various viral proteins, including polymerases of vsv, 144 chikv, 145 and rsv, 146 and ribonucleoprotein complex 147 to facilitate viral genome replications. in addition, hsp90 is also involved in the formation of viral capsid. hsp90 can maintain the stability of capsid precursor p1 protein of poliovirus, 147 and it can also increase affinity between core protein dimers to facilitate hbv capsid formation. 148 market for unknown reasons. 64 was shown to inhibit rsv replication in an in vivo model of well-differentiated primary human airway epithelial cells at concentration as low as 1.9 nm. moreover, despite extensive replication in the presence of 64, no resistance against 64 was observed even after 17 passages, which is in direct contrast to previously reported rsv inhibitor. 155 similarly, 63 was reported to inhibit the replication of poliovirus both in vitro and in vivo, and no resistance against 63 was detected after 10 passages, despite that poliovirus is feature with rapid replication rate and high mutation frequency. 156 and inhibited nucleocapsid egress from the nucleus. 159 many other hsp90 inhibitors have also shown definitive antiviral activities against a panel of virus strains. 160, 161 although around 17 hsp90 inhibitors are under development in clinical trials, they are all for cancer indication and none is being tested for antiviral purpose albeit with well-established preclinical results. the major concern associated with the application of hsp90 inhibitors for antiviral treatment is the toxicity. most of the hsp90 inhibitors in clinical trials are accompanied with some severe side effects including cardiotoxicity, gastrointestinal toxicity, and/or ocular toxicity amongst other side effects. [162] [163] [164] it is now widely accepted that these unwanted toxicities are mainly attributed to paninhibition of hsp90 isoforms. for example, the observed cardiotoxicity mainly resulted from the inhibition hsp90α, which is responsible for the maturation of herg channel. 165 therefore, hsp90 isoform-selective inhibitors are expected to side-step some detrimental toxicity observed with pan-inhibitors. indeed, several hsp90 isoform-selective inhibitors against grp94 166, 167 (72) (73) and hsp90β 168 (71) have been successfully developed with much better safety profiles. these inhibitors were devised for cancer treatment, and their antiviral profiles were not investigated. very recently, two grp94 selective inhibitors 72 and 73 were found to inhibit the replication of denv and zikv with ic 50 values in the low nanomolar range, and it was further confirmed that grp94 was essential for the replication of denv and zikv. 169 therefore, it can be deduced that grp94 might be indispensable for the replications of other viruses as well. it can be envisioned that antiviral therapy with hsp90 inhibitors can benefit from hsp90 isoform inhibition, and isoform-selective show broad-spectrum antiviral activities. as compared to hsp90 inhibitors, hsp70 inhibitors are under developed, and the development of hsp70 inhibitors is faced with several challenges: hsp70 has a high affinity toward adp, and the conformation state of hsp70 makes the atp binding site less accessible. 178 therefore, it is very hard to design inhibitors targeting the atp binding site. 179 due to high sequence similarity between hsp70i and hsc70, most of the available inhibitors are unable to discriminate between those two isoforms. shown in figure 10 are a selected subset of hsp70 inhibitors, and all those inhibitors are intended for cancer indication. mkt-077 (76; figure 17 ) is the most advanced one, which has entered phase i clinical trial for cancer treatment. however, its clinical trial was halted due to severe renal dysfunction observed in patients. 180 albeit with highly structural similarity toward 80. 182 in addition, 80 suppressed the inflammation response associated with dengue fever. more importantly, no resistance to 80 was detected even after 10 passages, while significant resistance was observed with a viral ns5 inhibitor under the same conditions, highlighting the advantages of htas over daas in terms of resistance selection. 80 and its analogues have also exhibited broadspectrum antiviral profiles with inhibitory activity against hcv, 183 zikv, 184 west nile, and japanese encephalitis viruses. 182 the hsp70i is at relatively low level in unstressed cell, while its expression is significantly induced upon f i g u r e 1 7 the chemical structures of hsp70 inhibitors and downregulators, hsp, heat shock protein viral infections, indicating an integral role of hsp70i in viral infections. to achieve hsp70 isoform inhibition, several inhibitors targeting an allosteric site on hsp70 were elegantly designed with selectivity toward hsp70i. 185, 186 hs-72 (81) is one of such inhibitors, and it was shown to inhibit the entry of denv mainly by disrupting the association of hsp70i with the denv receptor complex. 187 in comparison, hsc70 selective inhibitors have not been precedented. however, in contrast to direct inhibition, hsc70 downregulators have been reported with broadspectrum antiviral activities. for example, imb-dm122 (82), an analogue derived from natural compound oxymatrine, was discovered as a hsc70 downregulator. the half-life of hsc70 messenger rna (mrna) was reduced by 78% followed by the treatment of 82 (500 μg/ml) in huh7.5 cells. as such, 82 is effective to inhibit hcv replication at a concentration of 125 μg/ml. in addition, 82 is well-tolerated in mice with no obvious toxicity at a single dosage of 1000 mg/kg (intraperitoneal [ip]). 188 intensive structural modifications were further made to the sidechain and/or substituent at the nitrogen position of 82, yielding several analogues with ic 50 s against hcv in the low micromolar range (ie, 83). [189] [190] [191] the oxymatrine analogues were also shown to inhibit both wild-type and lamivudine-resistant hbv infection via downregulating hsc70 expression with excellent safety profile in mice (ld 50 = 750 mg/kg, oral administration). 192 the activity against other virus was also reported with oxymatrine analogues. 193, 194 the other naturally occurring hsc70 downregulator is lycorine (84) . 121 it was reported to decrease the hsc70 mrna level does-dependently with definitive anti-hcv activity in vitro. the structural optimization led to several derivatives with ic 50 values ranging from low micromolar to submicromolar levels. the sar study showed that the double bond between c3 and c4 and the basic nitrogen at n-5 position are crucial to the anti-hcv activity. 195 interestingly, its naturally occurring cousin lycoricidine (85) possessed much more potent inhibitory effect against hcv with an ec 50 value of 0.55 nm, and the mechanism of action studies revealed that downregulation of hsc70 expression at least partially account for the observed antiviral activity. 196 although these compounds are confirmed to downregulate hsc70 to exert their antiviral efficacy, their respective physical binding protein(s) remain to be clarified, but it can be deduced that their physical binding partner(s) must be host factor(s). host cells have developed an innate immune system to act as the first-line defense against invading virus. in addition, a series of intracellular restriction factors are also expressed endogenously to counteract viral infections, 197 and apolipoprotein b mrna-editing enzyme catalytic polypeptide-like 3g (apobec3g, a3g) is one such factor, which possesses the capacity to restrict the replication of a panel of viruses including hiv-1, hbv, hcv, and ev71 among others via different mechanisms. a3g is known as a cytidine deaminase, catalyzing the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. its anti-hiv-1 activity is closely associated with its deaminase function, by which consistent mutation from dg to da in the positive strand of viral dna is frequently observed. 198 the high level of g to a mutation is attributed to the c to u transitions occurred on the complementary negative-strand dna by the cytidine deaminase activity of a3g. introduction of deoxyuracils in the proviral dna can ultimately lead to its cleavage and degradation by specific ap endonucleases. additionally, high percentages of g to a mutation in the hiv-1 genome will result in the loss of functions of viral proteins. 199 interestingly, the antiviral activity of a3g against other virus is independent of its deaminase activity. for example, a3g is reported to inhibit hcv replications by binding to the c-terminus of hcv ns3 protein to reduce its helicase activity, which is essential for hcv replication. 200, 201 a3g was also reported to inhibit hbv replication both in vitro and in vivo, but its anti-hbv mechanism is not related to its deaminase activity, because a3g did not yield any g to a hypermutation within the hbv genome, and the catalytically inactive a3g derivatives were able to result in the same magnitude of hbv inhibition as compared with its wild-type counterpart. 202, 203 although host cells are endowed with antiviral restriction factors such as a3g, viruses are so cunning that they developed their own mechanism to evade host innate immune system. for example, hiv-1 expresses a viral protein ji and li vif, which binds to a3g and form an ubiquitin ligase complex with cullin 5 (cul5), elongin b/c (elob/c), and cbfβ, leading to the ubiquitination and subsequent proteasomal degradation of a3g. 204 in the cases of hbv and hcv infections, endogenous a3g is also eliminated by some unknown mechanisms. therefore, agents either disrupting the formation of ubiquitin complex, stabilizing a3g or inducing the expression of a3g are expected to demonstrate antiviral activity. in 2008, chen et al identified two compounds imb-26 (86; figure 18 ) and imb-35 (87) , which directly binds to a3g and disrupted its interaction with vif, and therefore rescue a3g from vif-mediated degradation. both compounds showed a3g-dependent anti-hiv-1 activity in nonpermissive h9 cells with ec 50 values in the low nanomolar range. moreover, no cytotoxicity was observed at a concentration of around 4 μm, indicating a therapeutic index of greater than 200, and the ld 50 value for 87 is as high as greater than 1000 mg/kg (ip). 205 86 also showed strong antiviral activity against hcv in vitro via stabilizing intracellular a3g. 206 due to the presence of a bromo substituent at the α position of amide, which is highly reactive as a alkylation agent, structural modifications were made to 86, and it turned out that the bromo is not essential for the antiviral activities. most of the synthesized derivatives showed potent antiviral activity against hcv and ev71 virus with ic 50 values ranging from 0.57 to 80 μm. 207, 208 since the amide linkage is liable to hydrolysis, a methylene group was inserted between the carbonyl and amino group. however, such modification resulted in complete loss of activity. 207 to further address this issue, a ring formation strategy was employed to generate compound with 2-aryl-isoindolin-1-ones scaffold (88) . these analogues showed much-enhanced stability toward hydrolysis, and most importantly, the anti-ev71 activity was retained with ic 50 in the low micromolar range. 209 interestingly, the derivatives of 86 also showed definitive antiviral activity against both wild-type and tamiflu resistant influenza virus in vitro with ic 50 values in the low micromolar range, 210 despite the fact that a3g does not yield any inhibitory activity against influenza virus replication, 211 indicating that other a3g-independent antiviral mechanism must be involved. vif, and both inhibited viral replication via a3g pathway. the immunoprecipitation experiment confirmed that neither compound disrupted the interaction between vif and a3g, and thus inhibited the ubiquitination of a3g. in addition, neither compound impaired 20s proteasome activity, suggesting that recovery of a3g is not simply caused by inhibiting the general proteasome activities. the specific mechanism(s) underlying the recovery of a3g need further clarification. 215 both compounds also showed high cytotoxicity toward 293t cells with ic 50 values being 30 and 50 μm, respectively, necessitating further structural optimization. benzimidazole derivatives were also identified as potent anti-hiv-1 agents via stabilizing a3g. 216 the structural modifications led to the identification of compound 93 with an anti-hiv-1 ic 50 value of 3.4 nm in h9 cells, and no toxicity in mice was observed in 2 weeks after the ip injection of 1000 mg/kg of compound 93. the mechanism of action studies showed that this type of compounds stabilized a3g via disrupting the interaction between vif and eloc, which is essential for the vif-mediated a3g degradation. 217 in 2012, zuo et al 218 confirming its a3g-dependent antiviral mechanism. the coimmunoprecipitation and computational analysis suggested that 94 directly bound to eloc at the interface of eloc-vif interaction to suppress vif activity. the sar studies concluded that both the naphthoyl and benzoyl group were essential for the activity, and modifications made to the ester group were well-tolerated. 219 the abovementioned antiviral strategy is based on the recovery of a3g to inhibit viral infection. very interestingly, it was reasonably argued that hiv-1 virus might actually benefit from sub-lethal levels of a3g-induced mutation, which might contribute to the high mutation rate of hiv-1 virus and the capacity of the virus to escape innate immune system and evolve resistance against antiretroviral drugs. therefore, current antiretroviral treatment regime may benefit from the inhibition of a3g deaminase activity. 220 however, a proof of concept study needs to be established to support such hypothesis. bone marrow stromal cell antigen 2 (bst-2), also known as tetherin or cd317, is a potent ifn-induced antiviral molecule inhibiting the release of various enveloped virus particles from infected cells, including filoviruses, 221, 222 arenaviruses, 223 paramyxovirus, 223 γ-herpesviruses, 224 and among others. 225 the broad-spectrum antiviral profiles of bst-2 is attributed to its ability to target a common feature shared by these viruses: host cell-derived lipid bilayer. since the target of the bst-2 is not encoded by viral genome, so the virus cannot mutate the viral protein to evade the antiviral activity of bst-2. however, viruses have evolved other mechanisms to counteract the action of bst-2 by expressing different viral proteins to physically bind to bst-2, and thereby block the interaction between bst-2 and its target. such proteins include hiv-1 vpu, hiv-2 env, siv env, siv nef, kshv k5, and the ebov glycoprotein. for example, hiv-1 vpu, a type i integral membrane protein, can either induce the ubiquitination of bst-2 for degradation or downregulate the cell-surface bst-2 level by sequestering the de novo synthesized bst-2 away from the plasma membrane to counteract its antiviral activity. 226 therefore, it can be envisioned that disruption of the interaction between bst-2 and these viral proteins or stabilization of bst-2 could yield antiviral agents. zhang et al 227 developed a cell-based high-throughput enzyme-linked immunosorbent assay for the quantification of cell-surface bst-2 levels, 227 and a lead compound imb-la (95; figure 19 ) was identified to inhibit vpu-mediated bst-2 degradation and recover the expression of bst-2 at the cell surface. 228 human dead-box polypeptide 3, an atpase/rna helicase, was founded to be involved in a variety of cellular biogenesis process, including cellular differentiation, cell-cycle regulation, apoptosis, and cell survival. recently, it has also been identified as an essential host factors for the replication of both dna and rna virus, including hiv, 231 hcv, 232 denv, 233 and wnv, 234 among others. although the exact mechanism(s) ddx-3 employed to facilitate viral replication is yet to be elucidated, ddx-3 has been proposed as a very promising host target for broad-spectrum antiviral agent development. the crystal structure of ddx-3 in complex with amp has been resolved, 235 and a pharmacophore has been proposed for virtual screening, 236 value of 10 μm. 241 in a follow-up study, the structure-based drug design strategy was employed to devise new inhibitors with more potent activity. the structural modification was mainly made to the two phenyl rings to form more interactions with the other two unexplored binding pockets within the rna-binding site, and one inhibitor which showed much more potent antibudding against filoviruses, arenavirus, and rhabdovirus vlps. for example, both compounds inhibited egress of lfv-z vlps by more than 10-fold at a concentration of 1 μm, and as expected, showed no inhibitory activity against a ppxy mutant and budding defective virus, confirming the antibudding mechanism via inhibition of ppxy and nedd4 interaction. in addition, no cytotoxicity toward hek293t cells was observed for both compounds at concentrations tested (1 μm). 242 in a follow-up study, an intensive structural modifications were made to the lead compound 102, and several more potent analogues were identified with nanomolar activity in the antibudding assay. for example, compound 103 can achieve more than 90% inhibition of li et al identified a natural compound cajanine (105; figure 22 ) as a potent hcv inhibitor via a cell-based phenotype-screening assay with an ic 50 value of 3.12 μm. 244, 245 the structural-activity relationships study revealed that both the hydrophobic benzene ring (106) and prenyl group (107) were not essential for the anti-hcv activity, and several derivatives with more potent anti-hcv activity (ic 50 < 1 μm) were also obtained. the mechanism of action study demonstrated that 105 and its derivatives showed no inhibitory effect against hcv viral proteins, and yet led to the downregulation of a host target chondroitin sulfate n-acetylgalactosaminyltransferase 1 (csgalnact-1), which is a key enzyme responsible for the initiation of chondroitin sulfate chain. csgalnact-1 was shown to play a key role during the replication of hcv virus, and its expression was elevated upon hcv infection. knockdown of csgalnact-1 by sirna led to significant suppression of hcv replications. it is interesting to note that 105 showed no effect on the mrna level of csgalnact-1, and the downregulation of csgalnact-1 protein level was recovered by a general protease inhibitor mg132, indicating that cajanine promoted the degradation of csgalnact-1 by proteasome pathway. in consistency with such host targeting mechanism, 105 showed the same magnitude of inhibitory effect against both wild-type and drug resistant hcv virus strains, and it also showed very pronounced synergistic effects with other daas to inhibit hcv replications, indicating that 105 and its derivatives are worthy of further studies. although it is still unclear whether csgalnact-1 is broadly required for the replication of other viruses, our unpublished data showed that 105 and its derivatives also possessed inhibitory effect against other virus such as influenza virus, hiv-1, hbv, and coxsackie b virus. although it is ascertained solidly that 105 inhibits hcv replication via downregulation of csgalnact-1 protein, it remains unclear how 105 leads to the csgalnact-1 degradation, and which protein(s) 105 binds to physically. answering these types of questions would definitely provide new host targets for the development of new antiviral agents. throughout a drug repurposing campaign, estrogen receptor α (erα) inhibitor tamoxifen (108; figure 23 ) was identified as an hcv inhibitor. the known targets of 108 include erα, p-glycoprotein, calmodulin, and protein kinase c, and so forth. the inhibitors against p-glycoprotein, calmodulin and protein kinase c failed to inhibit hcv f i g u r e 2 2 the chemical structure of cajanine along with its synthesized analogues replication, excluding the possibility of these proteins as the anti-hcv targets for 108. a specific sirna against erα significantly suppressed hcv rna in replicon-containing cells, and transient transfection with erα (but not erβ) expression plasmids also augmented hcv replications. altogether, these results confirmed the important roles erα played during hcv replication. the subsequent binding assay showed that domain c of erα physically bound to ns5b but not ns3, ns4b, and ns5a, and such interaction is essential for the hcv genome replications. the selective estrogen receptor modulators (serms) toremifene (109) and clomiphene (110) were also reported to inhibited several other viruses such as hiv-1, 246 ebov, 247 and hsv. 248 however, the mechanism studies have excluded erα as potential antiviral targets, and the observed broad-spectrum antiviral property for serms resulted from the inhibition of other host factors, including protein kinase c (hiv-1) 249 and chloride channel (hsv-1). 248 these host proteins together with erα represent promising targets for the development of novel antiviral agents to combat against drug resistance and newly emerging unknown viruses. quite a few other host proteins or pathways have also been validated as feasible antiviral targets in vitro and even in laboratory animals, such as lipid biosynthesis and metabolism pathways, 250, 251 pparα, 252 and hnf4α, 253 among others. 121, [254] [255] [256] table 1 summarized the host targets included in this review along with the development stages of their respective inhibitors/modulators. it should be noted that the focus of this review is just on small-molecule based htas, and other validated antiviral host targets with only antibody or sirna-based modulators were not included. it is reasonable to anticipate that such host proteins can also be targeted by small-molecule modulators to convey antiviral activity. notably, the antiviral host targets studied only account for a very small portion of the host factors confirmed to be essential for viral replications. in 2016, ammari et al 257 released a database for hostpathogen interactions, with which one can search the known host-pathogen interactions by inputting either the genes, proteins or the viruses. it can be speculated that most of the recorded host-virus interactions must play critical roles during viral replications, and thus could serve as new targets for the development of htas. daas have shown great success in combating viral infections in clinic, and they are generally safe to use because they directly target viral proteins, which lack homologs in human. however, daas also suffer from several inherent limitations due to its viral protein targeting nature: viral proteins varied among different species and even different genotypes, and thus daa targeting one specific viral protein is unlikely to exhibit inhibitory effect against viral proteins from other viral species or variants. therefore, daas are normally narrow-spectrum antiviral agents, and this is also the underlying reason for the lack of effective antiviral drugs against newly emerging viruses; the other major downside for daas is they are prone to cause drug resistance, particular among rna viruses with very high frequency of replication errors. hta agents perfectly complement to daas in regard to narrow-spectrum activity although a large number of host factors are known to play vital roles throughout the whole life cycle of virus, only very few of them were explored as antiviral targets. this is primarily attributed to several concerns raised by htas. chiefly, targeting a host protein may potentially lead to unwanted toxicity issues, because the target protein may be indispensable for some cellular functions. however, this may not be intrinsically true for all the host proteins, and many host genes are known to be nonessential for cellular functions. therefore, the inhibition of such host proteins will be well-tolerated, and the on-target based toxicity can be minimized. even though the target host protein is essential for some cellular functions, the on-target based toxicity can be mitigated in many ways. first, it csgalnact-1 cajanine hcv, influenza, and hiv preclinical ns5b-erα tamoxifen hcv preclinical is known that the expression level or the activity of many cellular proteins are elevated to facilitate viral replications. therefore, it is possible to knockdown the target protein to a level, at which the viral replication can be blocked while the normal cellular functions can still be maintained. second, most of the time, alternative pathways or proteins exist for a cellular function, and hence the inhibition of one of these proteins or pathways may lead to the blockage of viral replication, but not the cellular function. third, the on-target based toxicity is closely associated with the duration of treatment, and the treatment of most of viral infections only lasts for several weeks or even days. therefore, the toxicity concern raised by targeting a host factor for virus treatment can be further alleviated. it is worth noting that quite a few approved drugs targeting host factors for other indications are considered to be generally safe to use in clinic for years. consequently, toxicity issue is something that one should pay attention to, but not something used to bias against host target antiviral agents. the other major challenge faced with the development of htas is that the antiviral phenotype observed with htas is merely in vitro artifact in some cases, and the correlation between in vitro and in vivo or clinical efficacy is very poor as in the case of development of impdh inhibitors as anti-hcv agents. the other example is the repurposement of statins as anti-hcv drugs. statins showed very pronounced inhibitory effects against hcv replication in vitro, but yield unsatisfactory outcomes in clinic trials. the lack of reliable predictive in vitro models indeed increased the attrition rates in the development of htas. however, despite with these challenges, development of htas can be highly rewarding because htas can potentially address the unmet needs in the treatment of viral infections. the work from the ji lab is financially supported by the national natural science foundation of china (81773608) recent advancement of direct-acting antiviral agents (daas) in hepatitis c therapy the competitive binding between inhibitors and substrates of hcv ns3/4a protease: a general mechanism of drug resistance hsp90: a promising broad-spectrum antiviral drug target the hiv coreceptors cxcr4 and ccr5 are differentially expressed and regulated on human t lymphocytes hiv: cell binding and entry. cold spring harb perspect med the geographic spread of the ccr5 delta32 hiv-resistance allele ccr5 receptor antagonists in preclinical to phase ii clinical development for treatment of hiv targeting chemokine receptor cxcr4 for treatment of hiv-1 infection, tumor progression, and metastasis exploring the stereochemistry of cxcr4-peptide recognition and inhibiting hiv-1 entry with d-peptides derived from chemokines amd3100, a small molecule inhibitor of hiv-1 entry via the cxcr4 co-receptor the bicyclam amd3100 story amd3465, a monomacrocyclic cxcr4 antagonist and potent hiv entry inhibitor multiple-dose escalation study of the safety, pharmacokinetics, and biologic activity of oral amd070, a selective cxcr4 receptor inhibitor, in human subjects blockade of x4-tropic hiv-1 cellular entry by gsk812397, a potent noncompetitive cxcr4 receptor antagonist synthesis of a novel tricyclic 1,2,3,4,4a,5,6,10b-octahydro-1,10-phenanthroline ring system and cxcr4 antagonists with potent activity against hiv-1 design of novel cxcr4 antagonists that are potent inhibitors of t-tropic (x4) hiv-1 replication plerixafor: in patients with non-hodgkin's lymphoma or multiple myeloma function of the chemokine receptor cxcr4 in haematopoiesis and in cerebellar development the chemokine receptor cxcr4 is essential for vascularization of the gastrointestinal tract defects of b-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the cxc chemokine pbsf/sdf-1 ccr5 inhibitors: emerging promising hiv therapeutic strategy efficacy of short-term monotherapy with maraviroc, a new ccr5 antagonist, in patients infected with hiv-1 chemokine control of west nile virus infection virological and immunological response to antiretroviral regimens containing maraviroc in hiv type 1-infected patients in clinical practice: role of different tropism testing results and of concomitant treatments intensification of a raltegravir-based regimen with maraviroc in early hiv-1 infection maraviroc and reverse transcriptase inhibitors combinations as potential preexposure prophylaxis candidates (s)-methyl-1-piperazinyl]-4-methylpiperidine (sch-417690/sch-d), a potent, highly selective, and orally bioavailable ccr5 antagonist pharmacokinetics and short-term safety of 873140, a novel ccr5 antagonist, in healthy adult subjects an imidazopiperidine series of ccr5 antagonists for the treatment of hiv: the discovery of n-{(1s)-1-(3-fluorophenyl)-3-[(3-endo)-3-(5-isobutyryl-2-methyl-4,5,6,7-tetrahydro-1h-imidazo[4, 5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]propyl}acetamide (pf-232798) is pf-232798 a possible successor to maraviroc? novel 4,4-disubstituted piperidine-based c-c chemokine receptor-5 inhibitors with high potency against human immunodeficiency virus-1 and an improved human ether-a-go-go related gene (herg) profile discovery of a piperidine-4-carboxamide ccr5 antagonist (tak-220) with highly potent anti-hiv-1 activity design, synthesis, and biological evaluation of novel 2-methylpiperazine derivatives as potent ccr5 antagonists discovery of incb9471, a potent, selective, and orally bioavailable ccr5 antagonist with potent anti-hiv-1 activity ccr5 receptor antagonists in preclinical to phase ii clinical development for treatment of hiv inhibition of human immunodeficiency virus replication by a dual ccr5/cxcr4 antagonist inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate molecular analysis of the inhibitory effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro polymerase reaction rna virus error catastrophe: direct molecular test by using ribavirin the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen the antiviral compound ribavirin modulates the t helper (th) 1/th2 subset balance in hepatitis b and c virus-specific immune responses ribavirin-induced anemia in hepatitis c virus patients undergoing combination therapy a phase iii study of the safety and efficacy of viramidine versus ribavirin in treatment-naive patients with chronic hepatitis c: viser1 results safety and efficacy of viramidine versus ribavirin in viser2: randomized, doubleblind study in therapy-naive hepatitis c patients ribavirin revisited in the era of direct-acting antiviral therapy for hepatitis c virus infection efficacy and safety of grazoprevir/elbasvir+/− rbv for 12 or 16 weeks in patients with hcv g1, g4 or g6 infection who previously failed peginterferon/rbv: c-edge treatment-experienced combined interferon alpha2b and cyclosporin a in the treatment of chronic hepatitis c: controlled trial specific inhibition of hepatitis c virus replication by cyclosporin a nim811, a cyclophilin inhibitor, exhibits potent in vitro activity against hepatitis c virus alone or in combination with alpha interferon safety, pharmacokinetics, and antiviral activity of the cyclophilin inhibitor nim811 alone or in combination with pegylated interferon in hcv-infected patients receiving 14 days of therapy alisporivir-a host-targeting antiviral, provides low viral breakthrough rate and high barrier to resistance in hcv genotype 1 treatment-naïve patients in the phase iib essential study alisporivir plus ribavirin is highly effective as interferon-free or interferon-add-on regimen in previously untreated hcv-g2 or g3 patients: svr12 results from vital-1 phase 2b study interferon (ifn)-free alisporivir (deb025) treatment in the vital-1 study has a more beneficial overall safety profile vs ifn-containing treatment scy-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis c virus rna replication in vitro the cyclophilin inhibitor scy-635 suppresses viral replication and induces endogenous interferons in patients with chronic hcv genotype 1 infection preclinical characterization of naturally occurring polyketide cyclophilin inhibitors from the sanglifehrin family sanglifehrin a, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action sanglifehrin−cyclophilin interaction: degradation work, synthetic macrocyclic analogues, x-ray crystal structure, and binding data fragment-based discovery of a new family of non-peptidic smallmolecule cyclophilin inhibitors with potent antiviral activities catalysis of cis/trans isomerization in native hiv-1 capsid by human cyclophilin a cyclophilin a interacts with influenza a virus m1 protein and impairs the early stage of the viral replication activation and inhibition of cellular calcium and tyrosine kinase signaling pathways identify targets of the hbx protein involved in hepatitis b virus replication nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a cyclophilin a is required for efficient human cytomegalovirus dna replication and reactivation target cell cyclophilins facilitate human papillomavirus type 16 infection cyclophilins and cyclophilin inhibitors in nidovirus replication p0890: novel cyclophilin inhibitor cpi-431-32 shows broad spectrum antiviral activity by blocking replication of hcv, hbv and hiv-1 viruses discovery of cyclosporine a and its analogs as broad-spectrum anti-influenza drugs with a high in vitro genetic barrier of drug resistance analyzing the relationship of qt interval and exposure to nitazoxanide, a prospective candidate for influenza antiviral therapy-a formal tqt study synergistic effect of nitazoxanide with neuraminidase inhibitors against influenza a viruses in vitro tizoxanide and other thiazolides are potent inhibitors of hepatitis b virus and hepatitis c virus replication the fda-approved oral drug nitazoxanide amplifies host antiviral responses and inhibits ebola virus nitazoxanide: a first-in-class broad-spectrum antiviral agent comparative quantitative monitoring of rabbit haemorrhagic disease viruses in rabbit kittens systematic identification of synergistic drug pairs targeting hiv pediatric drug nitazoxanide: a potential choice for control of zika nitazoxanide inhibits paramyxovirus replication by targeting the fusion protein folding: role of glycoprotein-specific thiol oxidoreductase erp57 nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus the anti-hepatitis c agent nitazoxanide induces phosphorylation of eukaryotic initiation factor 2α via protein kinase activated by double-stranded rna activation hepatitis b virus x protein identifies the smc5/6 complex as a host restriction factor inhibition of hbv transcription from cccdna with nitazoxanide by targeting the hbx-ddb1 interaction nitazoxanide inhibits human norovirus replication and synergizes with ribavirin by activation of cellular antiviral response interferon regulatory factor-1 (irf-1) is involved in the induction of phosphatidylserine receptor (psr) in response to dsrna virus infection and contributes to apoptotic cell clearance in chse-214 cell effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial a pilot clinical trial of nitazoxanide in the treatment of chronic hepatitis b synthesis and pre-clinical studies of new amino-acid ester thiazolide prodrugs the safety and efficacy of combination n-butyl-deoxynojirimycin (sc-48334) and zidovudine in patients with hiv-1 infection and 200-500 cd4 cells/mm3 an alpha-glucosidase i inhibitor for the potential treatment of hcv infection inhibition of endoplasmic reticulum-resident glucosidases impairs severe acute respiratory syndrome coronavirus and human coronavirus nl63 spike protein-mediated entry by altering the glycan processing of angiotensin i-converting enzyme 2 in vivo therapeutic protection against influenza a (h1n1) oseltamivir-sensitive and resistant viruses by the iminosugar uv-4 dengue virus evolution under a host-targeted antiviral glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum synthetic heterocyclic candidates as promising α-glucosidase inhibitors: an overview assessment of the potential for host-targeted iminosugars uv-4 and uv-5 activity against filovirus infections in vitro and in vivo dose-and schedule-dependent protective efficacy of celgosivir in a lethal mouse model for dengue virus infection informs dosing regimen for a proof of concept clinical trial small molecule inhibitors of er α-glucosidases are active against multiple hemorrhagic fever viruses ester prodrugs of ihvr-19029 with enhanced oral exposure and prevention of gastrointestinal glucosidase interaction structures of mammalian er α-glucosidase ii capture the binding modes of broadspectrum iminosugar antivirals impdh as a biological probe for rna antiviral drug discovery: synthesis, enzymology, molecular docking, and antiviral activity of new ribonucleosides with surrogate bases. nucleosides nucleotides nucleic acids broad-spectrum antiviral activity of the imp dehydrogenase inhibitor vx-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase antiviral activity and mode of action studies of ribavirin and mycophenolic acid against orthopoxviruses in vitro an impdh inhibitor, suppresses replication of zika virus and other emerging viral pathogens synthesis and antiviral activity of a novel class of (5-oxazolyl)phenyl amines synthesis and broad-spectrum antiviral activity of some novel benzoheterocyclic amine compounds a randomized, double-blind, placebo-controlled dose-escalation trial of merimepodib (vx-497) and interferon-alpha in previously untreated patients with chronic hepatitis c merimepodib, pegylated interferon, and ribavirin in genotype 1 chronic hepatitis c pegylated interferon and ribavirin nonresponders ribavirin as therapy for chronic hepatitis c. a randomized, doubleblind, placebo-controlled trial repurposing of kinase inhibitors as broad-spectrum antiviral drugs repurposing kinase inhibitors as antiviral agents to control influenza a virus replication new connections: kinase inhibitors as antivirals anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects identification and targeting of an interaction between a tyrosine motif within hepatitis c virus core protein and ap2m1 essential for viral assembly selective inhibitors of cyclin g associated kinase (gak) as anti-hepatitis c agents optimization of isothiazolo[4,3-b]pyridine-based inhibitors of cyclin g associated kinase (gak) with broad-spectrum antiviral activity 3-b]pyridines as inhibitors of cyclin g associated kinase: synthesis, structure-activity relationship studies and antiviral activity identification and optimization of 4-anilinoquinolines as inhibitors of cyclin g associated kinase sgc-gak-1: a chemical probe for cyclin g associated kinase (gak) anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus targeting hepatitis b virus cccdna by crispr/cas9 nuclease efficiently inhibits viral replication final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of myrcludex b in combination with tenofovir in patients with chronic hbv/hdv co-infection cyclosporin a inhibits hepatitis b and hepatitis d virus entry by cyclophilinindependent interference with the ntcp receptor use of fda approved therapeutics with hntcp metabolic inhibitory properties to impair the hdv lifecycle inhibitory effect of fasiglifam on hepatitis b virus infections through suppression of the sodium taurocholate cotransporting polypeptide evaluation and identification of hepatitis b virus entry inhibitors using hepg2 cells overexpressing a membrane transporter ntcp isolation and structure of vanitaracin a, a novel anti-hepatitis b virus compound from talaromyces sp design and synthesis of a novel candidate compound nti-007 targeting sodium taurocholate cotransporting polypeptide [ntcp]-apoa1-hbx-beclin1-mediated autophagic pathway in hbv therapy concept of viral inhibitors via ntcp cyclosporin derivatives inhibit hepatitis b virus entry without interfering with ntcp transporter activity epigallocatechin gallate inhibits hepatitis b virus via farnesoid x receptor alpha farnesoid x receptor-alpha is a proviral host factor for hepatitis b virus that is inhibited by ligands in vitro and in vivo farnesoid x receptor agonist gw4064 indirectly inhibits hcv entry into cells via down-regulating scavenger receptor class b type i the selective fxr agonist eyp001 is well tolerated in healthy subjects and has additive anti-hbv effect with nucleoside analogues in heparg cells diacylglycerol acyltransferase-1 localizes hepatitis c virus ns5a protein to lipid droplets and enhances ns5a interaction with the viral capsid core hepatitis c virus entry is impaired by claudin-1 downregulation in diacylglycerol acyltransferase-1-deficient cells effect of the dgat1 inhibitor pradigastat on triglyceride and apob48 levels in patients with familial chylomicronemia syndrome a diacylglycerol transferase 1 inhibitor is a potent hepatitis c antiviral in vitro but not in patients in a randomized clinical trial herpes simplex virus type 1 dna polymerase requires the mammalian chaperone hsp90 for proper localization to the nucleus nuclear transport of epstein-barr virus dna polymerase is dependent on the bmrf1 polymerase processivity factor and molecular chaperone hsp90 involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits geldanamycin, a potent and specific inhibitor of hsp90, inhibits gene expression and replication of human cytomegalovirus human butyrate-induced transcript 1 interacts with hepatitis c virus ns5a and regulates viral replication antiviral activity and rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses chikungunya virus nsp3 & nsp4 interacts with hsp-90 to promote virus replication: hsp-90 inhibitors reduce chikv infection and inflammation in vivo hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus molecular chaperone hsp90 is a therapeutic target for noroviruses heat shock protein 90 facilitates formation of the hbv capsid via interacting with the hbv core protein dimers hsp90, an unlikely ally in the war on cancer geldanamycin, a ligand of heat shock protein 90, inhibits herpes simplex virus type 2 replication both in vitro and in vivo geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro inhibition of heat-shock protein 90 reduces ebola virus replication heat shock protein 90 controls hiv-1 reactivation from latency hsp90 inhibitors reduce influenza virus replication in cell culture hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance a novel class of geldanamycin derivatives as hcv replication inhibitors targeting on hsp90: synthesis, structure-activity relationships and anti-hcv activity in gs4.3 replicon cells synthesis and biological evaluation of heat-shock protein 90 inhibitors: geldanamycin derivatives with broad antiviral activities hsp90 inhibitor at-533 blocks hsv-1 nuclear egress and assembly inhibition of heat-shock protein 90 reduces ebola virus replication inhibition of hsp90 attenuates porcine reproductive and respiratory syndrome virus production in vitro hsp90 molecular chaperone inhibitors: are we there yet? heat shock protein 90: inhibitors in clinical trials targeting the molecular chaperone heat shock protein 90 (hsp90): lessons learned and future directions the herg channel is dependent upon the hsp90alpha isoform for maturation and trafficking paralog-selective hsp90 inhibitors define tumor-specific regulation of her2 development of a grp94 inhibitor structure-guided design of an hsp90beta n-terminal isoform-selective inhibitor small molecule grp94 inhibitors block dengue and zika virus replication involvement of endoplasmic reticulum chaperones in the folding of hepatitis c virus glycoproteins molecular chaperone grp78/bip interacts with the large surface protein of hepatitis b virus in vitro and in vivo posttranslational folding of vesicular stomatitis virus g protein in the er: involvement of noncovalent and covalent complexes folding, interaction with grp78-bip, assembly, and transport of the human immunodeficiency virus type 1 envelope protein heat shock protein 70 inhibits the activity of influenza a virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo heat shock cognate protein 70 is involved in rotavirus cell entry in vivo and in vitro association of hsc70 with polyomavirus capsid proteins the heat shock cognate protein 70 is associated with hepatitis c virus particles and modulates virus infectivity allosteric opening of the polypeptide-binding site when an hsp70 binds atp atpases as drug targets: insights from heat shock proteins 70 and 90 a phase i and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog mkt 077 phase i trial of the selective mitochondrial toxin mkt077 in chemoresistant solid tumours defining hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection allosteric heat shock protein 70 inhibitors block hepatitis c virus assembly heat shock protein 70 (hsp70) mediates zika virus entry, replication, and egress from host cells identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70 heat shock protein 70 inhibitors. 2. 2,5'-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70 an inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating hsp70 as a host antiviral target small molecular compounds that inhibit hepatitis c virus replication through destabilizing heat shock cognate 70 messenger rna evolution of matrinic ethanol derivatives as anti-hcv agents from matrine skeleton synthesis and biological evaluation of sophocarpinic acid derivatives as anti-hcv agents synthesis, structure−activity relationship and biological evaluation of novel n-substituted matrinic acid derivatives as host heat-stress cognate 70 (hsc70) down-regulators design and synthesis of oxymatrine analogues overcoming drug resistance in hepatitis b virus through targeting host heat stress cognate 70 antiviral effect of matrine against human enterovirus 71 sar evolution and discovery of benzenesulfonyl matrinanes as a novel class of potential coxsakievirus inhibitors design, synthesis and structure-activity relationship optimization of lycorine derivatives for hcv inhibition evaluation of anti-hcv activity and sar study of (+)-lycoricidine through targeting of host heat-stress cognate 70 (hsc70) zinc finger antiviral protein inhibits coxsackievirus b3 virus replication and protects against viral myocarditis hypermutation of hiv-1 dna in the absence of the vif protein single-strand specificity of apobec3g accounts for minus-strand deamination of the hiv genome host apobec3g protein inhibits hcv replication through direct binding at ns3 host apolipoprotein b messenger rna-editing enzyme catalytic polypeptide-like 3g is an innate defensive factor and drug target against hepatitis c virus inhibition of hepatitis b virus replication by apobec3g inhibition of hepatitis b virus replication by apobec3g in vitro and in vivo ubiquitination of apobec3 proteins by the vif-cullin5-elonginb-elonginc complex small molecular compounds inhibit hiv-1 replication through specifically stabilizing apobec3g host apolipoprotein b messenger rna-editing enzyme catalytic polypeptide-like 3g is an innate defensive factor and drug target against hepatitis c virus synthesis and antiviral activity of a series of novel n-phenylbenzamide and n-phenylacetophenone compounds as anti-hcv and anti-ev71 agents synthesis and antiviral activity of n-phenylbenzamide derivatives, a novel class of enterovirus 71 inhibitors synthesis and broad antiviral activity of novel 2-aryl-isoindolin-1-ones towards diverse enterovirus a71 clinical isolates synthesis and antiviral activity of substituted bisaryl amide compounds as novel influenza virus inhibitors high level expression of the anti-retroviral protein apobec3g is induced by influenza a virus but does not confer antiviral activity small-molecule inhibition of hiv-1 vif synthesis and structure-activity relationship studies of hiv-1 virion infectivity factor (vif) inhibitors that block viral replication design, synthesis, and biological evaluation of 2-amino-n-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide derivatives as potent hiv-1 vif inhibitors small molecules that inhibit vif-induced degradation of apobec3g development of benzimidazole derivatives to inhibit hiv-1 replication through protecting apobec3g protein identification of an hiv-1 replication inhibitor which rescues host restriction factor apobec3g in vif-apobec3g complex small-molecule inhibition of human immunodeficiency virus type 1 replication by targeting the interaction between vif and elonginc indolizine derivatives as hiv-1 vif-elonginc interaction inhibitors first-in-class small molecule inhibitors of the single-strand dna cytosine deaminase apobec3g broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein infectious lassa virus, but not filoviruses, is restricted by bst-2/tetherin molecular mechanism of bst2/tetherin downregulation by k5/mir2 of kaposi's sarcoma-associated herpesvirus is tetherin a true antiviral: the influenza a virus controversy antagonism of cd317 restriction of human immunodeficiency virus type 1 (hiv-1) particle release and depletion of cd317 are separable activities of hiv-1 vpu high-throughput assay to identify inhibitors of vpu-mediated down-regulation of cell surface bst-2 a small molecule compound imb-la inhibits hiv-1 infection by preventing viral vpu from antagonizing the host restriction factor bst-2 2-thio-6-azauridine inhibits vpu mediated bst-2 degradation a novel peptide to disrupt the interaction of bst-2 and vpu requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function ddx3 dead-box rna helicase is required for hepatitis c virus rna replication strategies for development of dengue virus inhibitors p-body components lsm1, gw182, ddx3, ddx6 and xrn1 are recruited to wnv replication sites and positively regulate viral replication crystal structure of conserved domains 1 and 2 of the human dead-box helicase ddx3x in complex with the mononucleotide amp pharmacophore modeling and molecular docking led to the discovery of inhibitors of human immunodeficiency virus-1 replication targeting the human cellular aspartic acid−glutamic acid−alanine−aspartic acid box polypeptide 3 toward the discovery of novel anti-hiv drugs. second-generation inhibitors of the cellular atpase ddx3 with improved anti-hiv activity: synthesis, structure-activity relationship analysis, cytotoxicity studies, and target validation targeting ddx3 with a small molecule inhibitor for lung cancer therapy ketorolac salt is a newly discovered ddx3 inhibitor to treat oral cancer targeting the human dead-box polypeptide 3 (ddx3) rna helicase as a novel strategy to inhibit viral replication discovery of the first small molecule inhibitor of human ddx3 specifically designed to target the rna binding site: towards the next generation hiv-1 inhibitors small-molecule probes targeting the viral ppxy-host nedd4 interface block egress of a broad range of rna viruses quinoxaline-based inhibitors of ebola and marburg vp40 egress design and synthesis of cajanine analogues against hepatitis c virus through downregulating host chondroitin sulfate n-acetylgalactosaminyltransferase 1 total synthesis of cajanine and its antiproliferative activity against human hepatoma cells ligands of the antiestrogen-binding site are able to inhibit virion production of human immunodeficiency virus 1-infected lymphocytes fda-approved selective estrogen receptor modulators inhibit ebola virus infection inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and nppb effect of tamoxifen on regulation of viral replication and human immunodeficiency virus (hiv) long terminal repeat-directed transcription in cells chronically infected with hiv-1 different anti-hcv profiles of statins and their potential for combination therapy with interferon identification of the niemann-pick c1-like 1 cholesterol absorption receptor as a new hepatitis c virus entry factor naringenin inhibits the assembly and long-term production of infectious hepatitis c virus particles through a ppar-mediated mechanism effects of bezafibrate in patients with chronic hepatitis c virus infection: combination with interferon and ribavirin herpes simplex virus type 1 abrogates the antiviral activity of ch25h via its virion host shutoff protein ubiquitin c-terminal hydrolase-l3 promotes interferon antiviral activity by stabilizing type i-interferon receptor dauricine combined with clindamycin inhibits severe pneumonia co-infected by influenza virus h5n1 and streptococcus pneumoniae in vitro and in vivo through nf-kappab signaling pathway hpidb 2.0: a curated database for host-pathogen interactions he got both his bachelor's and master's degrees in chemistry from the university of science and technology of beijing in 2006 and 2008, respectively. thereafter, he moved to peking union medical college institute of medicinal biotechnology, and received his phd degree in medicinal chemistry under the supervision of professor zhuorong li in 2011. after that, he took an assistant professor position in the same institution, and he worked as a postdoctoral fellow in dr binghe wang's lab from chinese academy of medical sciences and peking union medical college. she got her bachelor's degree at peking medical school in 1984, then she moved to peking union medical college and received her master's degree in medicinal chemistry in 1988. her research interests include the development of anti-infection, antiosteoporosis, and anticancer drugs. she has published over 150 peer-reviewed scientific papers and over 30 patents medicinal chemistry strategies toward host targeting antiviral agents key: cord-281367-qm5a5c4b authors: des jarlais, don c; johnston, patrick; friedmann, patricia; kling, ryan; liu, wei; ngu, doan; chen, yi; hoang, tran v; donghua, meng; van, ly k; tung, nguyen d; binh, kieu t; hammett, theodore m title: patterns of hiv prevalence among injecting drug users in the cross-border area of lang son province, vietnam, and ning ming county, guangxi province, china date: 2005-08-24 journal: bmc public health doi: 10.1186/1471-2458-5-89 sha: doc_id: 281367 cord_uid: qm5a5c4b background: to assess patterns of injecting drug use and hiv prevalence among injecting drug users (idus) in an international border area along a major heroin trans-shipment route. methods: cross-sectional surveys of idus in 5 sites in lang son province, vietnam (n = 348) and 3 sites in ning ming county, guangxi province, china (n = 308). respondents were recruited through peer referral ("snowball") methods in both countries, and also from officially recorded lists of idus in vietnam. a risk behavior questionnaire was administered and hiv counseling and testing conducted. results: participants in both countries were largely male, in their 20s, and unmarried. a majority of subjects in both countries were members of ethnic minority groups. there were strong geographic gradients for length of drug injecting and for hiv seroprevalence. both mean years injecting and hiv seroprevalence declined from the vietnamese site farthest from the border to the chinese site farthest from the border. 10.6% of participants in china and 24.5% of participants in vietnam reported crossing the international border in the 6 months prior to interview. crossing the border by idus was associated with (1) distance from the border, (2) being a member of an ethnic minority group, and (3) being hiv seropositive among chinese participants. conclusion: reducing the international spread of hiv among idus will require programs at the global, regional, national, and "local cross border" levels. at the local cross border level, the programs should be coordinated on both sides of the border and on a sufficient scale that idus will be able to readily obtain clean injection equipment on the other side of the border as well as in their country of residence. both injecting drug use and hiv among injecting drug users (idus) have become major international public health problems. hiv infection has been reported among idus in over 100 countries [1] . travel by idus, particularly along drug distribution routes, appears to be a major mechanism for the spread of both injecting drug use and hiv among idus. hiv spread north [2] and south [3] from new york city along the east coast in the u.s. stimson [4] , and beyrer and colleagues [5] have reconstructed the spread of hiv among idus in south east asia. beyrer et al. used molecular epidemiology (mapping the different subtypes of hiv) in their reconstruction. a recent study by kato and colleagues (2001) found patterns of hiv genetic subtyping consistent with cross-border transmission either from vietnam to china or from china to vietnam. while these regional and country level analyses have great value in understanding the worldwide spread of hiv among idus, they have important limitations with respect to hiv prevention efforts. reducing hiv spread by attempting to disrupt regional and country drug distribution routes may have the unintended consequence of displacing distribution to new routes, leading to additional spread of injecting drug use and hiv among idus. successful prevention efforts will be greatly facilitated by more detailed understanding of the spread of injecting drug use and transmission of hiv among idus within smaller geographic areas. understanding of hiv transmission across international borders is particularly important, as few hiv prevention programs are coordinated across such borders. we present here data on injecting drug use and hiv among idus in the adjacent border provinces of lang son, vietnam and guangxi, china. hiv among idus was noted in this area in 1996 [6] and since then there has been substantial transmission among idus in both provinces. the present situation shows a clear geographic pattern, with the potential for additional spread across the border between the provinces and within each of the provinces. the data reported here were collected as part of baseline surveys of idus conducted before implementation of a cross-border hiv prevention intervention in lang son province, vietnam and-ning ming county, guangxi province, vietnam [7] . figure 1 shows a map of the area, with the project sites -in lang son province and in ning ming county. there is considerable official and entirely legal movement of commercial goods across the border in both directions. there are also semi-official and informal crossing points and pathways through the hills that permit local residents to cross the border with little or no regulation. crossing the border is a regular aspect of life for many people who live near the border, for example, to attend market days in the larger villages. drug dealers cross the border to sell drugs and drug users also cross to obtain drugs of higher purity and at better prices. the frequency of border crossing can vary, influenced by current price and purity of heroin, the ebb and flow of law enforcement activity and, factors such as the outbreak of severe acute respiratory syndrome (sars) in china. there is also substantial trade and migratory employment in the region. lang son city, aidian, and puzhai (near pingxiang) are bustling centers of legitimate cross-border trade, as well as drug trafficking and sex work. many people cross the border daily and seasonally to find work and many are employed as porters in the cross-border trade. the region is home to many ethnic minority groups (e.g., zhuang, tay, nung), some of whom live on both sides of the border (for example, the zhuang in china and the tay in vietnam are the same ethnic group but are known different names in the two countries). there is frequent intermarriage across the border, although this might be illegal and might lead to loss of nationality. kinship ties, like migratory employment and trade, result in additional cross-border movement. data collection methods for the idu surveys reported on here were essentially parallel in ning ming county and lang son province, with some variation in the community-based subject recruitment strategies used. the availability of large known drug use gathering places and of officially registered idus in lang son permitted greater use of probability-based methods in vietnam, while there was more reliance on peer recruitment in china. in ning ming county, a modified snowball/peer recruitment technique was used. the project peer educators sent recruiting letters to idus they knew personally, inviting them to come to a project center and participate in the survey. the idus who came to project centers for interviews were encouraged to recruit 2-3 additional participants. the research participants received 20 chinese yuan (approximately $2.50) for the interview, 5 yuan for each additional male respondent recruited, and 10 yuan for each additional woman respondent recruited. the eligibility criteria were a minimum of 18 years of age and recent (in the past 6 months) drug injection. approximately one-half of the sample was based on individuals initially selected from the lists of known idus in the project sites. the other half was based on participants initially selected from idus present at gathering or shooting places mapped by project staff as part of the initial project implementation. for the half of the sample based initially on registered lists, 10 clusters of 25 individuals each were selected by probability proportional to size (pps) from the lists of idus in each commune. then four idus were picked at random from each selected cluster and these referred others until the quota for the commune was reached. for the portion of the sample selected initially at idu gathering or shooting places, sample quotas for these places were determined by pps based on the numbers of individuals observed at these places during the mapping phase. the interview team then revisited the selected places and chose four individuals at random from among those present at each place at that time (who were not necessarily those present during the mapping phase). the vietnamese participants were paid 30,000 vietnamese dong (approximately us$2) for participating in the interview and hiv test. in vietnam, an oral informed consent was obtained for participation in the study, with the interviewer certifying that oral consent had been obtained. this procedure was requested by the institutional review board of the national aids standing bureau in order to provide more assurance of confidentiality to prospective participants. in china, standard signed informed consents were obtained from all participants. unique codes were constructed for geographic setting and map of project sites caoloc town each participant based on numeric date of birth and several letters representing, for example, the first letter of the mother's family name. (construction of the record number was slightly different in the two countries.) the objective was to have a unique identifier that the participant could readily reconstruct if he or she lost the project participation card. a structured instrument was used for the interviews, based on version 2b of the questionnaire being used in the world health organization's drug injection study, phase ii [8] . trained interviewers, primarily staff of the local health departments, conducted the interviews. the questionnaire covered demographics, drug use, injection and sexual risk behavior, hiv testing history, hiv and hepatitis knowledge, and cross-border travel patterns. a question on the number of times the subject had crossed the border in the 6 months prior to the interview was included. there are many factors which could influence the "ease/ difficulty" in crossing an international border, including distance to the border, cost of transportation, time needed to reach the border, and the need to have official papers for crossing. it was not practical to measure all such factors. instead, used simple physical distance (in kilometers) to the nearest border point. this gave five distance categories in vietnam and three distance categories in china. the baseline survey was conducted in july 2002 in vietnam and between july and september 2002 in china. the survey included hiv antibody testing. participants were given pre-test counseling and post-test counseling at local health centers. blood was drawn at the time of the interviews by trained phlebotomists from local health departments. participants were given a card with their unique identifier and returned to the local health center to receive their test results using this identification number. indeed, they could only retrieve their results by using this number since blood samples were not otherwise labeled. in china, testing was by double elisa (vironostika hiv-uni-form, organon (holland)) with confirmation of initial hiv-positive results by western blot (genelabs diagnostics). all testing was conducted at the laboratory of the guangxi center for hiv/aids prevention and control in nanning. in vietnam, testing was performed at the laboratory of the lang son provincial health services using the serodia sfd screening test (biorad {france}) and double elisa (genescreen, biorad (france); vironostika, organon [9] ). this is the official protocol of the ministry of health in vietnam and the lang son laboratory is authorized to provide hiv testing according to this protocol by the ministry of health. data were entered and data sets were prepared in epiinfo, hiv prevalence against distance to border. the study was reviewed and approved by the institutional review boards (irbs) of the following institutions: guangxi center for hiv/aids prevention and control, the national aids standing bureau of vietnam, abt associates inc., and beth israel medical center. table 1 shows selected demographic characteristics of the idu subjects recruited in china and vietnam. in both provinces, the subjects were primarily young males who had never been married. although there are known to be female idus on both sides of the border, the project has had difficulty inducing women to participate in the interventions and in recruiting them for the surveys. over twothirds of the subjects in china and one-half in vietnam belonged to ethnic minority groups (primarily zhuang in china and tay and nung in vietnam). the ethnic minority subjects tended to live closer to the border. table 2 in this report, we present data on injecting drug use and hiv infection among idus in lang son province, vietnam and ning ming county, guangxi province, china from a baseline survey conducted before implementation of a peer-based cross-border hiv prevention intervention. several limitations should be noted. first, we were working with cross-sectional data, where longitudinal data from the initial spread of injecting drug use in the area would have certainly been preferable. second, there are measurement and sample size limitations. it would have been helpful to have a measure of ease of travel to the border rather than simple physical distance to the border. also it would have been helpful to have sufficiently large sample sizes so that the relationship between being a member of an ethnic minority group and being hiv seropositive could be examined within individual geographic sites. despite these limitations, there are very clear patterns in the data. there are similar gradients for mean length of injecting history and baseline hiv prevalence running in descending order from the vietnamese site farthest from the border to the chinese site farthest from the border. these patterns are consistent with the theory that both the practice of drug injection and the prevalence of hiv infection among idus spread from northern vietnam to southern china along a major heroin trans-shipment route [5] . the patterns in our data suggest that, in some circumstances, it may be possible to reconstruct histories of the diffusion of injecting drug use and hiv among idus using cross-sectional data. there is clearly a potential for further cross-border transmission of hiv in both directions. our discussions with the peer educators in the cross-border hiv prevention project suggest three primary reasons for these idus crossing the border: 1. obtaining higher quality/lower priced drugs, 2. avoiding police pressure on drug injectors, which can be unpredictably variable and involves largescale periodic crackdowns, and 3. personal factors, such as migratory employment, commerce, and family ties. it would appear to be very difficult to reduce these reasons for idus crossing the border. because of the possibility of arrest, idus who cross the border are unlikely to carry needles and syringes with them, even if they are crossing the border at places without any supervision or inspection. prevention of risky injections among border crossing idus will require very good supplies of sterile injection equipment on both sides. if idus who crosses the border cannot readily access sterile injection equipment on both sides of the border, then their fellow idus will need to have sufficient supplies of sterile injection equipment for use by themselves and the idus who cross the border. this will require large-scale safer injection programs on both sides of the border. the majority of subjects in this study belong to ethnic minority groups, primarily zhuang in china and tay and nung in vietnam. ethnic minority idus were also overrepresented among the border crossers. the issues of ethnic minority status, injecting drug use, and hiv infection deserve much more research and policy development. ethnic minority idus are more likely to be infected with hiv in many places, from african-american and latino/a idus in new york city [10] to roma in eastern europe [11] to first nations in vancouver [12] to vietnamese in australia [13] to manipuris in india [14] . as noted above, there was a strong relationship between ethnic minority status and hiv serpositivity in ning ming (or = 5.08 (95% ci 1.41,18.26, p = .013) [15] . social stigmatization of ethnic minority communities may make them more vulnerable to illicit drug use, including injecting drug use. employment discrimination against ethnic minority communities may increase the extent to which drug distribution occurs in these communities, and to which drugs are transported by minority community members. persons belonging to ethnic minority groups also may have important factors facilitating international travel, such as social support systems and persons that speak the same language on the other side of international borders. the data presented here illustrate many of the factors in the international diffusion of hiv among idus at modest geographic scale. (there is a total distance of 71 kilometers between the two most distant sites in the study). these include gradients of length of injecting drug use and hiv seroprevalence across the international border, border crossing by idus and its association with hiv infection for those crossing from china into vietnam, overrepresentation of ethnic minority persons among the border crossers. both injecting drug use and hiv among idus are already well established among idus on the vietnamese side of the border and injecting drug use is well established on the chinese side of the border. hiv is present among idus on the chinese side of the border, but at lower seroprevalence levels than in lang son. there are multiple reasons that people cross the border in this area, and it would not appear to be possible to stop idus from crossing the border or from injecting drugs across the border. thus, hiv prevention goals must include increasing the safety of injections among border crossers (as well as reducing risk behavior among the idus who do not cross the border). this will require coordinated hiv prevention that increases the likelihood that idus will inject safely on both sides of the border. such a program has been implemented in lang son and guangxi provinces. it includes peer outreach, increased access to sterile injection equipment through syringe distribution and exchange and a pharmacy voucher program. idus may exchange used injection equipment for new needles/ syringes or for vouchers that can be redeemed at local pharmacies for needles/syringes, sterile water, and condoms. idus may also directly receive new needles/ syringes or pharmacy vouchers even if they do not return used equipment. the program also includes large-scale collection and safe disposal of used needles/syringes, general community education about drugs and hiv, and social support for people living with hiv or aids [7] . reducing the international transmission of hiv among injecting drug users will require programs at the global, regional, national, and "local cross-border" levels. the local cross border programs will need to be coordinated on both sides of the border and on a sufficient scale that idus who cross the border will be able to readily obtain clean injection equipment on the other side of the border. the cross-border hiv prevention project currently being implemented in lang son province and ning ming county, guangxi offers an example of how such a coordinated approach can be implemented. evaluation data being collected in lang son and ning ming will be used to gauge the effectiveness of the interventions. global estimate of injecting drug use hiv seroprevalence among connecticut intravenous drug users in 1986-87: race/ethnicity as a risk factor for hiv seropositivity risk for htlv-iii exposure and aids among parenteral drug abusers in new jersey reconstruction of sub-regional diffusion of hiv infection among injecting drug users in south-east asia: implications for prevention overland heroin trafficking routes and hiv-1 spread in south and southeast asia a rapid assessment of hiv/ aids situation and vulnerabilities related to drug use in lang son development and implementation of a cross-border hiv prevention intervention for injection drug users in ning ming county drug injecting and hiv infection: global dimensions and local responses risk, power and the possibility of pleasure: young women and safer sex informed altruism" and "partner restriction" in the reduction of hiv infection in injecting drug users entering detoxification treatment le romes durvar" (god hits whom he chooses; the roma gets hit twice). an exploration of drug use and hiv risks among the roma of central and eastern europe risk factors for elevated hiv incidence among aboriginal injection drug users in vancouver crofts n: hiv, ethnicity and travel: hiv infection in vietnamese au with injection drug use organization naidsc: combating hiv/aids in india des jarlais dc: correlates of hiv status among injection drug users in a border region of southern china and northern vietnam the authors gratefully acknowledge all of the health department and clinic staff, other public officials, peer educators, and pharmacists in ning ming county, lang son province and guangxi province who are participating in and supporting this project. we would also like to acknowledge the support of the national institutes on drug abuse, u.s. national institutes of health. grant number 1r01da1470301. the author(s) declare that they have no competing interests. th conceived of the study, the study design and coordination and assisted in the drafting of the manuscript. dcd participated in the design of the study and drafted and edited the manuscript. th and dcd supervised the data analysis.pf, pj and rk performed the statistical analysis and participated in its design and coordination and participated in the writing and review of drafts of the manuscript.wl, yc & dm supervised the implementation of the project and data collection and processing for the chinese sites, and participated in the writing and review of drafts of the manuscript. dn, tvh, lkv, ndt & ktb supervised the implementation of the project and data collection and processing for the vietnamese sites, and participated in the writing and review of drafts of the manuscript.all authors read and approved the final manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/5/89/prepub key: cord-340389-0fybiybv authors: fahrioglu, umut; ergoren, mahmut cerkez; mocan, gamze title: ccr5-δ32 gene variant frequency in the turkish cypriot population date: 2020-07-31 journal: braz j microbiol doi: 10.1007/s42770-020-00352-8 sha: doc_id: 340389 cord_uid: 0fybiybv recent unaids reports (december 2019) indicate that 37.9 million people have been affected by hiv infection around the globe in 2018, of which 1.7 million are cited as new infections. human immunodeficiency virus-1 (hiv-1) requires both the cd4 receptor, as the primary receptor, and a chemokine co-receptor to gain entry into the cell. in addition to the wt allele for c–c motif chemokine receptor 5 (ccr5-wt), there is another allele with a 32 bp deletion in the protein coding region (ccr5-δ32). individuals who are homozygous for the mutant allele are resistant towards m-tropic hiv infections. in the current study, we aimed to determine the ccr5-δ32 allele frequency in the turkish cypriot population with 326 subjects, 141 men (43.1%) and 185 (56.9%) women. the region of the ccr5 gene containing the δ32 deletion was amplified using flanking primers. the ccr5 gene δ32 allele frequency was calculated at 3% and only observed in heterozygous individuals. we hope that our current publication could be a point of dialog between the physicians, the government officials and the public set up a more modern and well-structured hiv screening program in an effort to control and hopefully eliminate hiv from the turkish cypriot population. human immunodeficiency virus (hiv) is a virus from the family retroviridae, genus lentivirus. hiv epidemic is one of the worst epidemics of the modern times. by destroying the cd4-positive t lymphocytes, hiv is able to make the body lose its ability to fight infection and disease due to an extremely low cd4 cell count. this condition is known as acquired immunodeficiency syndrome (aids). according to the latest unaids data from the 2019 factsheet and the who statistics, 37.9 million people were affected by hiv infection around the globe in 2018, of which 1.7 million are new infections. unfortunately, by the end of june 2019, only 24.5 million (23.3 million according to who) people have been reported to have access to antiretroviral therapy (art) according to unaids, which is a greatly improved number compared to the 8 million in 2010 [1] [2] [3] [4] [5] . according to the recent speech by the executive director of unaids, nearly 350,000 people have died of aids just in the first 6 months of 2020 [6] . despite the improved access to art, drug-resistant hiv is present in individuals with or without treatment, making hiv suppression without drugs an urgent necessity [7] . hiv-1 requires both the cd4 receptor, as the primary receptor, and a chemokine co-receptor to gain entry into the cell [8] . there are two different tropisms of hiv-1 depending on the type of chemokine co-receptor they utilize for attachment. macrophage-tropic (m-tropic) hiv-1 is more likely to be present in early infections, preferring primary cultures of macrophages, and uses c-c motif chemokine receptor 5 (ccr5) chemokine co-receptors for attachment. on the other hand, the t cell-tropic (t-tropic) strain, which requires the c-x-c motif chemokine receptor 4 (cxcr4) chemokine co-receptors for attachment, will appear in late stage infections, preferring primary cultures of cd4 + t cells and established t cell lines, and will cause a faster decline in the number of cd4-positive t cells [9] [10] [11] . the cxcr4 and the ccr5 are both members of the g protein coupled receptor (gpcr) family, with seven transmembrane domains that are structurally similar to each other. both receptors have chemokines as their ligands and play a role in multiple cellular processes such as development, angiogenesis, immune response, and leukocyte trafficking. more specifically, the ccr5 receptor has the cc (or β) chemokines as their ligands [9, 12, 13] . ccr5 wt (ccr5-wt) protein is 352 amino acids long, with 7 membrane spanning regions, 3 extracellular domains, and 3 cytoplasmic domains. one of the other functions of this protein is to target leukocytes to the site of inflammation. in addition to the wt allele, there is another allele with a 32 bp deletion in the protein-coding region (ccr5-δ32). the shorter protein encoded by this allele could not be observed on the cell surface [10] . the wt nucleotide sequence of the protein-coding region and the resulting protein sequence are shown in fig. 1 together with the location of the deletion on the nucleotide sequence. the 32 base pair deletion will lead to a frameshift and therefore cause a much shorter protein. individuals who are homozygous for the mutant allele are resistant towards hiv infections. however, this resistance is not absolute and is only for the m-tropic hiv strains. these individuals will not have the same resistance towards the t-tropic hiv strains [8, 10, 14] . additionally, people who are heterozygous for the ccr5-δ32 allele will show a slower progression to aids when compared to homozygous wt individuals [1] . the ccr5 knock-out mice developed normally. however, the mice had difficulty in clearing listeria infections and partial macrophage dysfunction. the ccr5 may also play an important role in graft-versus-host disease through its downmodulation of the t cell-dependent immune response. as ccr5 has a role in decreasing the t cell-related immune response, absence of ccr5 may increase the chance for graftversus-host disease. this connection remains unclear. individuals who are homozygous mutant may also have a higher chance of death as a result of west nile virus infections [9, 10] . ccr5-δ32 first came to light in 2009, in what is referred to as the "berlin patient". stem cell transplant using ccr5-δ32/ccr5-δ32 cells was performed to treat leukemia in this patient. following the transplant, the hiv infection in the blood and the bone marrow became undetectable without the use of antiretroviral treatment. a second similar case was mentioned in literature in 2019. recently, a paper detailing the ccr5-δ32 allele frequency in 87 countries has been published. dkms (germany, poland, and uk) which collects samples from potential hematopoietic stem cell donors has implemented the genotyping routine to newly registered donors [7, 8, 10] . new techniques are being developed to knockdown ccr5 expression by gene therapy with the help of zinc-finger nuclease (zfn), crispr/cas9, and transcription activator-like effector nuclease (talen) systems [15] . yu et al. have created a double-knockout system for both the cxcr4 and ccr5 genes in the circulating cd4+ cells using the crispr/cas9 system which could potentially lead to more functional hiv prevention [16] . in light of the fig. 1 the nucleotide sequence and the protein sequence for ccr5. the blue-highlighted region shows the 32-base deletion and the orange shows the corresponding amino acids that will be deleted recent covid-19 pandemic, it has also come to our attention that ccr5 cytokine receptor is upregulated in covid-19 patients and is emerging as a possible target in clinical trials for covid-19 treatment [17] . unaids data for cyprus from 2017 states that individuals, from all age groups, living with hiv in cyprus, are less than 1000. the number of people living with hiv has been increasing gradually since 1990. however, there has been a decrease in the aids-related deaths since 2014. these statistics probably only reflect the republic of cyprus and do not include the turkish cypriots living separately on the north side of the island. according to the ministry of health statistics for the turkish cypriots, since 1997, there have been a total of 68 cases reported. all of these are turkish cypriots citizens, since if a non-turkish cypriot is detected to be hiv positive in north cyprus, it is grounds for deportation. thirteen new cases have been identified in 2017 and 2018 in north cyprus. the highest number of cases detected was in 2014 and 2015, with 11 and 10 cases, respectively. only five of the turkish cypriot hiv-positive cases are female [18, 19] . in the current study, we aimed to determine the ccr5-δ32 allele frequency in the turkish cypriot population. we hope that this study will be a valuable addition to the literature and will also help the health authorities from the public health perspective. neither the greek cypriots nor the turkish cypriot populations were part of the extensive study by solloch et al. [10] . however, a greek population only study was reported in 1997 [20] . the study population is made up of 326 turkish cypriots, of which 141 are males and 185 are females. the volunteered subjects have been defined as turkish cypriots who have been living on the island at least for the past three generations. furthermore, due to the small size of population, the subjects which are related, such as first degree relatives, were eliminated from the study. there were no other restrictions set up for the study population. an ethical approval for the study was obtained from the near east university scientific research ethics committee (ydu/2019/68-791). informed consent was obtained from each participant. blood samples collected in tubes containing ethylenediaminetetraacetate (edta from participants. qiaamp dna blood mini kit (qiagen inc., valencia, ca, usa) was used for genomic dna extraction. the region of the ccr5 gene containing the δ32 deletion was amplified using the following flanking primers: 5′-caaaaagaaggtcttcattacacc-3′and 5′-cctgtgcctcttcttctcatttcg-3′. the expected fragments from the wt and the δ32 allele were 189 and 157 bp, respectively (fig. 2) . the pcr reactions were prepared using the 2x pcr master mix by thermo scientific (k0171) with the final primer concentration at 20 pmols for each primer. the dna amount used was around 10 ng. the pcr protocol was the same as angelis et al. [14] . a homozygous wt individual will only display the 189 bp band, a heterozygous individual will display both the 189 and the 157 bp band, and a homozygous mutant individual will only display the 157 bp band. fragments obtained from pcr were separated in 3% agarose gels which contained ethidium bromide for visualization. by using uvtreated solutions, designated pipettes and pipette tips, a class ii laminar hood and dna/dnase-free plasticware and reagents, the risk of contamination was decreased to a minimum. the hardy-weinberg equilibrium (hwe) was evaluated by the goodness-of-fit χ 2 test to calculate genotype distributions and allele frequencies, where a p < 0.05 was considered to indicate significant disequilibrium. the graphpad prism software was used (graphpad software, inc., san diego, ca, usa) to perform for the data analysis. hwe exact test was performed using the website https://www.cog-genomics.org/ software/stats. the subjects were made up of 326 turkish cypriots of which 141 are men (43.1%) and 185 (56.9%) are women. genotype distributions and allele frequencies of the ccr5 gene δ32 variant are shown in table 1 . distribution of the ccr5δ32 genotype was in agreement with the hardy-weinberg equilibrium (p = 0.587, x 2 = 0.293). the p value using the hwe exact test was 0.618. the calculated ccr5 gene δ32 allele frequency was 3% and only the allele was observed in heterozygous individuals. therefore, the allele frequency of the wild-type ccr5 allele was 97%. no ccr5δ32 homozygous turkish cypriots were detected in the studied cohort. ccr5 delta 32 mutation has been studied extensively in many ethnic groups and the allele can be seen more frequently in northern european populations. the allele frequency decreases as you move south and east. the lowest allele frequency is seen in south and south east asia and the sub-saharan africa. ccr5-δ32 allele is thought to have arisen from a single origin. it is thought that the vikings might be responsible for dissipating the mutant allele. the more you move away from the old viking lands, the lower you see the frequency. different selective pressures in different populations such as smallpox might have made it possible to see higher frequencies in europe [10, 14, 21] . only 2-3% of the white population is ccr5-δ32/ccr5-δ32. no obvious phenotype is observed in these individuals [9] . up to 87 countries have been analyzed with regards to their ccr5-δ32 allele frequency. cyprus was not one of those countries. the statistics given by the unaids website do not include turkish cypriots as they live separately. also, the previous publication by christodoulou only provides the data for greek cypriots [18, 20] . in order to contribute to the ccr5-δ32 allele frequency literature in a time where the ccr5 delta 32 is becoming a hot topic again and to provide a much-needed statistic for the turkish cypriot population, 326 turkish cypriots were studied to determine their ccr5-δ32 allele status. this would also provide valuable information from the public health standpoint and help set up a more structured approach to hiv testing. a total of 326 people provided us with a good sample number considering the small population of turkish cypriots (approx. 286,000) [22] . distribution of the ccr5δ32 allele was in agreement with the hardy-weinberg equilibrium (p = 0.587, x 2 = 0.293) in the turkish cypriot population. the p value using the hwe exact test was 0.618. the two p values were in agreement with each other. the ccr5 gene δ32 allele frequency was calculated to be 3% and only observed in heterozygous individuals. this number is also close to the 2.9% reported for the greek cypriot population [20] . no individuals homozygous for the ccr5δ32 allele were detected. perhaps a much larger test population would have made it more likely detect a homozygous mutant individual. failure to detect a homozygous mutant individual in other studies as well would indicate how small the allele frequency is for the ccr5δ32 allele in reality (observed) as compared to the expected. the allele frequencies of neighboring countries such as turkey, greece, egypt, israel, syria, and lebanon (fig. 3 ) are approximately 3%, 5%, 2%, 10%, 3%, and 2%, respectively [10, 23, 24] . the turkish allele frequency has also been cited around 6% in earlier studies, which is higher than what solloch has reported [10, 21, 25] . therefore, it is safe to say that the allele frequency of the turkish cypriots is compatible with the neighboring countries. when we look at other european mediterranean countries such as spain, italy, and france (8%, 6%, and 10% respectively), we can see that the turkish cypriot population allele frequency of 3% would follow the rule of going down as we move southward and eastward. turkish cypriot population is a relatively isolated population from the rest of the world as there are restrictions on travel to and from the north of the island. the population that most interacts with the turkish cypriots is the turkish population and their allele frequency is 3-6%. the turkish cypriot and greek cypriot populations have physically lived separately between 1974 and 2003. after 2003, with the opening of the border between wt wild type, f frequency the two sides, despite being able to interact with each other, interethnic marriages and relationships are still at a very low number (commonly known stigma). there are no official statistics available on the issue as this is a very political and contentious issue. however, as a document by the interpeace and 'cyprus 2015' initiative states, the greek cypriot population does not look too favorably towards interethnic marriages even to other non-orthodox christians [26] . turkish cypriot population has only had 68 hiv/aids cases since 1997 and 2017 with only 5 deaths. for 2018, there are some conflicting numbers being announced for hivpositive cases. one source indicates 129 cases whereas the other one indicates only 27 cases. both of these numbers are obtained from news articles and cannot be found on any official publication. one of the sources is citing the 2018 hiv analysis report and the other one is citing a source from the medical board. according to the medical board sources, the 2019 number for the hiv cases is 74 [27, 28] . north cyprus is a big tourist destination and is also home for many international students from many different countries including some african countries where the hiv infection rate may be very high. the same article is pointing out that the hiv-positive report rate may be low in north cyprus due to couple of reasons. one of the reasons is the stigma attached to being hiv positive in such a small population whereas the second reason is the fear of being deported for foreigners. this may be dangerous as it may cause the disease to advance and be passed on to other people [27] . as the results of our study suggest, most of the turkish cypriot population is at greater risk of hiv infection and faster disease progression due to a very low frequency of the δ32 allele. people are avoiding testing due to the stigma attached to hiv/aids in addition to the threat of deportation from the country if you are a foreigner. by keeping the greater risk in mind and using studies like ours, a dialog with health authorities must begin in order to develop a more structured and up-to-date strategy for testing and preventing hiv with the hopes of eliminating hiv/aids from the turkish cypriot population. another component of preventing hiv is a more serious dialog between the physicians, the government officials, and the public to better face the challenges posed by hiv to the public. ignoring the problem, stigmatizing the issue, and not having a policy on the issue are not an option if we want to take hiv/aids seriously. additionally, having such statistics available for the turkish cypriot population would also create a more complete outlook of the region. even though turkish cypriot administration is not internationally recognized, diseases such as hiv know no boundaries or politics. our current publication could serve as a starting point of dialog for this issue and a warning for people to take hiv infection more seriously. authors' contributions all authors contributed to the study conception and design. material preparation, data collection, and analysis were performed by umut fahrioğlu, mahmut çerkez ergören, and gamze mocan. the first draft of the manuscript was written by umut fahrioğlu and mahmut çerkez ergören and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. funding information this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the research was performed at the near east university, desam laboratories. data availability all data generated or analyzed during this study are included in this published article. conflict of interest the authors declare that they have no conflict of interest. ethics approval ethical approval for the study was obtained from the near east university scientific research ethics committee (ydu/2019/ 68-791). consent to participate informed consent was obtained from all individual participants included in the study. code availability not applicable. abbreviations aids, acquired immunodeficiency syndrome; art, antiretroviral therapy; ccr5, c-c motif chemokine receptor 5; cxcr4, c-x-c motif chemokine receptor 4; gpcr, g protein coupled receptor; hiv, human immunodeficiency virus; m-tropic, macrophagetropic; talen, transcription activator-like effector nuclease; t-tropic, t cell tropic; zfn, zinc-finger nuclease distribution of the mutated delta 32 allele of ccr5 co-receptor gene in iranian population who | data and statistics international committee on taxonomy of viruses (ictv) acquired immune deficiency syndrome (aids) unaids (2020) executive director speech hiv-1 remission following ccr5δ32/δ32 haematopoietic stem-cell transplantation long-term control of hiv by ccr5 delta32/ delta32 stem-cell transplantation the biology of ccr5 and cxcr4 frequencies of gene variant ccr5-δ32 in 87 countries based on next-generation sequencing of 1.3 million individuals sampled from 3 national dkms donor centers the cell tropism of human immunodeficiency virus type 1 determines the kinetics of plasma viremia in scid mice reconstituted with human peripheral blood leukocytes ginocchio cc genotyping of the ccr5 chemokine receptor by isothermal nasba amplification and differential probe hybridization c-c chemokine receptor type five (ccr5): an emerging target for the control of hiv infection ccr5 genotypes and progression to hiv disease in perinatally infected children ccr5 targeted cell therapy for hiv and prevention of viral escape simultaneous knockout of cxcr4 and ccr5 genes in cd4+ t cells via crispr/cas9 confers resistance to both x4-and r5-tropic human immunodeficiency virus type 1 infection disruption of the ccl5/rantes-ccr5 pathway restores immune homeostasis and reduces plasma viral load in critical covid-19 hiv çağımızın hastalığıdır ve bu konuda bilgilenmek önemlidir low frequency of ccr5delta32 allele among greeks in cyprus frequencies of 32 base pair deletion of the (delta 32) allele of the ccr5 hiv-1 co-receptor gene in caucasians: a comparative analysis northern cyprus districts, major towns & villages -population statistics, maps, charts hiv-1 co-receptor ccr5 and ccr2 mutations among greeks implication of hmox1 and ccr5 genotypes on clinical phenotype of egyptian patients with sickle cell anemia the δ ccr5 mutation conferring protection against hiv-1 in caucasian populations has a single and recent origin in northeastern europe solving the cyprus problem : hopes and fears bakan fikri ataoğlu'na sunuldu… -yakın doğu üniversitesi i neu publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-331673-xv1tcugl authors: reina, giacomo; peng, shiyuan; jacquemin, lucas; andrade, andrés felipe; bianco, alberto title: hard nanomaterials in time of viral pandemics date: 2020-07-15 journal: acs nano doi: 10.1021/acsnano.0c04117 sha: doc_id: 331673 cord_uid: xv1tcugl [image: see text] the sars-cov-2 pandemic has spread worldwide during 2020, setting up an uncertain start of this decade. the measures to contain infection taken by many governments have been extremely severe by imposing home lockdown and industrial production shutdown, making this the biggest crisis since the second world war. additionally, the continuous colonization of wild natural lands may touch unknown virus reservoirs, causing the spread of epidemics. apart from sars-cov-2, the recent history has seen the spread of several viral pandemics such as h2n2 and h3n3 flu, hiv, and sars, while mers and ebola viruses are considered still in a prepandemic phase. hard nanomaterials (hnms) have been recently used as antimicrobial agents, potentially being next-generation drugs to fight viral infections. hnms can block infection at early (disinfection, entrance inhibition) and middle (inside the host cells) stages and are also able to mitigate the immune response. this review is focused on the application of hnms as antiviral agents. in particular, mechanisms of actions, biological outputs, and limitations for each hnm will be systematically presented and analyzed from a material chemistry point-of-view. the antiviral activity will be discussed in the context of the different pandemic viruses. we acknowledge that hnm antiviral research is still at its early stage, however, we believe that this field will rapidly blossom in the next period. t he current emergence caused by sars-cov-2 is dramatically changing the everyday life of all of us. due to the high globalization, new viruses can spread all over the world much faster than ever, infecting the communities worldwide. the current technologies and measurements are able to sensibly slow down the infection spread. however, their cost is tremendously high, impacting the healthcare systems and causing the shutdown of industries and the lockdown of the population. the impact of sars-cov-2 on the worldwide economy is estimated to be a 2−3% decline, making this the biggest crisis since the world wars. 1 a possible vaccine is hopefully expected to come in 1−2 years, originating a buffer period pretty much uncertain for many people. vaccination is the only way known to accelerate the flock immunity without causing further death by this pandemic. contemporary history has seen the spread of other viral pandemics such as h2n2 flu (1956−1958) , h3n3 flu (1968), hiv (peak reached between 2005 and 2012), sars (2009), while mers (2012 to now) and ebola (1975 to now) viruses are in a prepandemic phase. the continuous colonization of wild nature lands may touch unknown virus reservoirs causing the spread of contagious epidemics. due to these facts, there is a clear urgency in the development of viral treatments to avoid the risk of new pandemics. 2 in particular, the possibility to have smart antiviral tools able to efficiently disinfect surfaces, block the viral spreading, enhance the survival of infected people, and boost immunization are highly desirable. in particular, more investments in the next years are expected in the antiviral research. hard nanomaterials (hnms) have been extensively studied for many types of applications including drug delivery, bioimaging, and biosensing. 3−5 the use of nanomaterials in biomedical research is highly developed, reaching in some cases clinical approval. 6 despite that, nanomaterials have been mainly developed for cancer therapy, while scarce attention has been spent on their application in viral infections. 7 the continuous virology research has more and more increased into viral replication machinery, allowing the preparation and rationalization of more sophisticated vaccine formulations and viral inhibitors. the use of hnms may be one of the keys to provide more effective biomedical agents with a wide spectrum of activity in viral pandemics. 8 an increasing number of reports describe how hnms can be successfully applied to block viral spread. hnms can be used at different stages of viral infection: blocking viral entry, hampering interaction with infected host cells, and modulating immune responses. due to their core composition (e.g., metal oxides, noble metals), hnms can have an important antiviral activity inactivating some specific proteins of the capsid or dysregulating radical homeostasis in the virus particles. additionally, surface functionalization can sensibly increase hnm antiviral activity, enabling the mimicking of host cells or enhancing the targeting efficiency. 9 in this review, we will critically analyze different strategies for the application of hnms as antiviral agents. the rational and the synthetic strategies will be highlighted and correlated to different relevant examples. particular attention will be paid on the mechanisms of antiviral action and on hnm applicability and efficacy. for the sake of clarity, this review is divided into three parts, namely: blocking viral entry, antiviral activity in host cells, and stimulation of immune system. we have focused on the different stages of infection. in the section dedicated to blocking viral entry, the application of hnms for surface disinfection and inactivation of the virus prior to interaction with host cells will be described. subsequently, the interaction of hnms after internalization into host cells will be addressed, stressing their antiviral delivery features and their activity in viral replication blockage, leading to host cell survival. then, activation of immune response induced by hnms, triggering the innate and the adaptive (e.g., nanovaccines) immunity, will be presented. the different adopted strategies will be correlated to the nanomaterial core (e.g., composition, size, shape) and surface (e.g., chemistry, surface charge) properties. finally, limits (e.g., unknown long-term toxicity), advantages (e.g., high and wide spectrum virucidal activity), and perspectives will be discussed with particular attention to the applications in viral pandemics (e.g., hiv, sars, and influenza viruses). this review is addressed to material and biomaterials scientists who are interested in antiviral research. we acknowledge that application of hnms as antiviral agents is still in the early stages; however, we believe that the research on this topic is going to grow soon. thus, with this contribution we genuinely hope to inspire researchers in the preparation of smart and efficient hnm antiviral agents. the last decades have been characterized by increasing investigation on viruses. in particular, their surface charge, protein composition, and host cell entry mechanism have been elucidated, allowing to formulate the first-generation wide spectrum antivirals. blocking the viral entry is one of the most common known antimicrobial procedure to stop infections at the early stage. in this context, antiviral materials have been used for surface disinfection or for epidemic limitation in humans and animals. due to their high surface to volume ratio, composition, and tunable surface chemistry, hnms are now more and more studied as powerful agents in blocking viral entry. as for other biological interactions, the attachment and entry of viruses into host cells are mediated by multivalent interactions between the surface of the virus and cell surface receptors. 10 nanomaterials can display multivalency that makes them able to compete with the host cells on virus attachment, limiting their infectivity. the mechanism of antiviral actions relies on the inactivation of the capsid proteins. as a matter of fact, hnms have been used for: (1) blocking target proteins for viral entry, (2) capsid protein oxidation, (3) mimicking cell surface, and (4) mechanical rupture of viruses ( figure 1 ). these strategies target proteins and mechanisms of entry common in most of the viruses, thus allowing the preparation of wide spectrum antiviral agents. in this section, the application of different hnms as powerful inhibitors of viral entry will be discussed. noble nanoparticles. noble nanoparticles (nps), made of gold and silver, are attractive as antiviral agents for their surface functionalization versatility and their capacity to cleave disulfide bonds. their use in disinfection has been extensively studied for different types of viruses. the morphology and the size of the nps play a crucial role in their ability to efficiently interact with the capsids and in their toxicity for the organism. these nanomaterials are characterized by a very large specific surface area (inversely proportional to the particle diameter). as the particle size becomes smaller and smaller, the percentage of surface atoms increases, creating many unsaturated bonds due to lack of neighboring atoms. as a consequence, agnps and aunps have unstable atoms with high surface energy. this kind of structure provides a lot of contact adsorption sites and reaction points for further modifications. these chemical features allow to easily combine surface np atoms with other atoms through chemical bonds. besides the composition of the metal core, several studies have pointed out the importance of the control of the surface chemistry. the surface groups can: (1) stabilize nps in the biological media, (2) insert targeting agents, and (3) enhance the circulation time inside the body. antiviral efficiency can be also enhanced by the multivalency effect, where highly branched ligands are used to locally augment the local concentration of the targeting molecules. in this section the main strategies and results for silver (agnps) and gold (aunps) nanoparticles in blocking viral entry will be critically discussed. silver nanoparticles. many studies have shown that naked agnps have a good effect on the control and prevention of a variety of viral diseases (table 1) . however, the antiviral mechanism of nanosilver is still unclear. the antiviral action is associated with the following mechanisms: nanosilver can prevent the virus from entering the host cells and inhibit the virus from binding to the cell receptor, thereby stopping the virus from infecting the targeted cells. agnps may be able to bind the viral surface protein and inhibit the interaction between the virus and the cell membrane receptors (figure 2 , left). however, it has been also reported that agnps can inactivate the virus through denaturation of surface proteins containing cysteine and methionine residues present on the viral capsid, in a similar way reported for bacteria. for example, agnps smaller than 10 nm were shown to interact with the sulfur-bearing residues of gp120 glycoprotein knobs distributed on the lipid membrane of hiv-1 virus, preventing the virus from binding to cd4 receptor site on the host cells, thus inhibiting the viral infection. 11 by means of a viral adsorption assay, it was shown that the agnp mechanism of anti-hiv action is based on the inhibition of the initial stages of the hiv-1 cycle. to demonstrate that the antiviral effect of agnps is due to the particle structure rather than to silver ions present in solution, the antiviral activity of silver sulfadiazine (agsd) and silver nitrate (known antibacterial silver salts) was evaluated. both salts showed a much lower therapeutic index than agnps in vitro, indicating that silver ions themselves are less efficient. 12 these results point out that the antiviral efficacy is not only related to the dose of ag + ions present in solution but is also regulated by different other parameters (e.g., size, charge, and surface functionalization) associated with the nanosize dimension. for instance, in the case of herpesviridae and paramyxoviridae viruses (both enveloped viruses with embedded viral-encoded glycoproteins), agnps can effectively reduce their infectivity, by blocking the interaction between the viral particles and the host cells with an antiviral activity strictly dependent on the size and ζ potential of the agnps. as a general observation, it was reported that smaller nanoparticles have better antiviral effect. this effect was associated with the increase of the surface area, where smaller-sized agnps could bind more efficiently to the viral particles exerting a higher antiviral activity. 13 another study reported the impairment of peste des petits ruminants virus (pprv) replication after incubating infectious viral particles with agnps, which did not exhibit any virucidal effect even up to 900 μg/ml. this result suggested that the anti-pprv activity of the agnps is due to the inhibitory effect 13 alternatively, nanosilver can be combined with viral nucleic acids to change the capsid structure, affect the replication of viral genetic material, and make the virus inactive. for example, tem analyses have shown that nps can cause a change of the structure of the ad3 virus from a hexahedral shape to an irregular shape, destroying its fibers and capsid proteins, leading to inhibition of the virus from binding to the host cells and destroying the dna structure, preventing adenoviral infection. 15 nanosilver can also bind directly to the doublestranded dna of hepatitis b virus to inhibit its replication. 16 in other studies, it has been demonstrated that silver ions released from nanosilver can directly damage the viruses. based in this property, an interesting application has been proposed. agnps were used as a coating on polyurethane condoms, (hsv) . the hypothesized mechanism is that silver ions are transferred directly from oxidized nps to biological targets, such as viral membrane proteins gp120 and gp41. in addition, a small amount of silver ion is also released from the coated contraceptives to improve the antiviral level. 17 although the studies on naked agnps to reduce viral infectivity have shown their potential as broad-spectrum antiviral agents, the understanding of the specific antiviral action mechanism still needs to be elucidated in depth. many studies have shown that the antiviral performance of naked agnps is related to their size, and smaller nanoparticles have better antiviral activities. 16 in addition to particle size, the antiviral action of agnp morphology has also attracted interest to fight against coronavirus. agnps and two types of silver nanowires were able to significantly cause an inhibitory effect on coronavirus transmissible gastroenteritis (tgev)-induced host cell infection and tgev replication. the mechanism is likely based on a direct interaction of agnps with tgev surface proteins (e.g., tgev glycoproteins) to inhibit the beginning of viral infection. it is possible that agnps and ag nanowires alter the structure of some surface proteins of tgev and then inhibit their recognition and adhesion to the cellular receptor papn. 18 although the potential of agnps as antiviral agents has been commonly recognized, unfortunately, their wide biological applications are limited by the risks of self-aggregation and environmental pollution. silver ions can be released from the surface of agnps and potentially pollute the environment, and their agglomeration into bulkier particles or fibers may change their biological characteristics, diminishing the antiviral effect. in several cases, it has been reported that naked agnps may affect human health. 25 therefore, research and development of agnps whose surface is modified or stabilized by protecting molecular layers is an urgent need to overcome these problems ( table 2) . poly(n-vinyl-2-pyrrolidone) (pvp) is the most commonly used stabilizer of agnps. the pvp-coated agnps are able to inhibit the activities of hiv-1, herpes simplex 2 virus (hsv-2), and respiratory syncytial virus (rsv). 11, 26, 27 but compared to foamy carbon, small-sized pvp and bsacoated agnps showed poor antiviral activity to the hiv-1 virus. 11 for rsv, pvp-coated agnps have a specific binding capacity to the viral surface, evidencing a regular spatial arrangement and a clear interaction with g-protein. 26 in addition, to improve the stability of agnps, their surface modification with antiviral drugs was proved to reduce the drug resistance caused by the drugs administered alone. tannic acid-modified agnps showed good antiviral effects on hsv-2 infection in vitro and in vivo. the viral infection was inhibited only when these nps directly interacted with hsv-2 virions. indeed, the pretreatment of host cells with such agnps did inhibit the entry of hsv-2. due to the high affinity of tannins to proteins and sugars, tannic acid can bind glycoproteins on the surface of viruses to make them inert, impairing glycoprotein function and preventing viruses from attaching and entering host cells. 28 the surface modification can also exert a synergistic antiviral effect. agnps decorated with polyphosphonium-oligochitosan (pqpoc) exhibited moderate to excellent antiviral activity against hav, nov, and coxb 4 . in addition, agnps could interact with the virion glycoproteins and prevent viral attachment and penetration. pqpoc can also serve as an effective virus inhibitor by blocking the interaction of the targeted virus with the host through the electrostatic interaction between the cationic polymers and the negatively charged binding sites of the virus. 29 surface-modified agnps can also prevent viral infection by competitive adsorption on host cells. the process of infection of cells by herpes simplex virus type 1 (hsv-1) involves the interaction between viral envelope glycoproteins and heparan sulfate (hs) on cell surface. therefore, researchers designed agnps capped with mercaptoethanesulfonate (ag-mes) to compete with the cellular hs through the sulfonate end groups, thereby blocking the virus from entering the cells. 30 a few years ago, it was shown that curcumin could prevent the replication and the budding of rsv, 31 but the disadvantage of poor solubility and low bioavailability limited its clinical application. 32 curcumin was used as a reducing and capping agent to prepare stable curcumin agnps (cagnps) under physiological conditions. cagnps could reduce cytopathic effects induced by rsv and showed efficient antiviral activity against infection by directly inactivating the virus prior to entry into the host cells. its antiviral effect was higher than curcumin alone or unmodified agnps ( figure 3 ). 33 alternatively, zhu et al. prepared agnps surface-modified with oseltamivir, amantadine, and zanamivir (ag@otv, 34 ag@am, 35 and ag@znv 36 ), by chemical methods. the results showed that these nanoparticles can directly interact with the virions, resulting in viral function damages. overall different studies have reported the capacity of agnps to block viral entry. however, there is not a concerted antiviral mechanism, but their activity differs from case to case, based on viral particle adsorption, capsid structure alteration, or surface protein denaturation. for agnps, the antiviral activity can be associated with different parameters including size, shape, surface charge, and functionalization but also to the topical release of silver ions able to disturb the viral cycle replication. as described before, bare agnps can be used as disinfectant agents, however their use in biological media is acs nano www.acsnano.org review limited by their low colloidal stability and potential cytotoxicity. surface functionalization can alleviate cytotoxicity, but it can also mask the nanoparticle surface, reducing their affinity for viral particles, thus reducing agnp antiviral activity. for these reasons, agnps at the moment could find application mainly for surface disinfection and for topical administration. further studies are needed to prepare safer agnp formulations for systemic administration. in particular, the clarification of the antiviral mechanisms and the use of surface functional groups able to stabilize agnps in biological fluids without affecting their prominent antiviral activity are probably the most important challenges to tackle. gold nanoparticles. compared to agnps, aunps exhibit reduced toxicity on healthy cells, making them more attractive for in vivo and clinical applications. 38 indeed, aunps have been successfully tested as inhibitors of viral entry into the host cells. aunps interact with hemagglutinin (ha), where au is able to oxidize the disulfide bond of this glycoprotein causing its inactivation, thus impeding the membrane fusion of the virus with host cells. targeting ha has emerged as an alternative strategy to the actual therapies (e.g., matrix protein 2 and neuramidase), especially to pandemic viruses that show an accelerated mutation speed of their surface proteins, hence a resistance to conventional treatments increasing their infectivity and mortality. 38 this strategy has been applied to influenza (e.g., h1n1, hcv) and herpes viruses. 39−44 the activity of aunps is proportional to the surface area exposed. as a consequence, the size and the morphology of these metal nps play a substantial role in their antiviral activity. recently, kim et al. have reported that porous aunps are able to inhibit influenza a infection more efficiently than nonporous aunps. 39 this effect has been associated with the higher surface area of the porous material that favors their interaction with capsids and thus increases their antiviral activity ( figure 4 ). besides the per se antiviral activity, aunp surface modifications have been developed in order to enhance their overall therapeutic benefits. the engineering of tailored aunps with selected ligands has allowed the preparation of efficient antiviral nanoagents. the target ligands can be introduced directly during the particle synthesis via ligand exchange reactions or ligand modifications. for instance, direct reduction of gold ions in the presence of gallic acid produced homogeneous aunps able to sensibly reduce herpes simplex virus infection in vitro. 40 compared to free ligand nps, functionalized aunps benefit from the multivalency effect and higher circulation times, decreasing the needed therapeutic concentrations. 39 functionalized aunps can present organic groups that mimic host cell surfaces or other specific molecular patterns that selectively target the virus. normally, negative charges are used to mimic cell surfaces and favor the interaction between the particles and the capsid. in particular, sulfonates and organic sulfates have been used for their capacity to attract the virus via capsid protein interaction and block the ha activity. 41 aunps functionalized with sulfonates showed an increasing inhibition of influenza a compared to the nanoparticles capped with succinic acid. 42 this study also demonstrated that there is not a correlation between the negative charge and the antiviral activity, but instead the inhibition depends mainly on the organic groups used. thiolcapped aunps also displayed powerful inactivation of bovine viral diarrhea virus in vitro. 43 multivalency has been exploited in more complex systems using dendrons as capping agents. this strategy allows to generate higher concentrations of the target ligand in close proximity to the aunps and to increase the binding efficiency of the nanoparticles to the capsid. the driving force of the antiviral efficiency relies on the concentration of the targeting agent onto the particles. sulfonated dendrons were grafted to aunps via a sulfide bond and tested for hiv inhibition. 44 the results showed that acs nano www.acsnano.org review the decorated aunps exerted a higher affinity to the virus. additionally, comparing aunps functionalized with different generation dendrons, those with a third generation displayed the highest inhibition performance with an ic 50 below 0.1 μmol/ml, thus making them attractive for in vivo translation. it is worth noting that the inhibition efficiency is strictly dependent on the available sulfonate groups present on the surface of the nps, making crucial a thorough characterization of the material. 44 the size of the aunps clearly plays an important role in the concentration of targeting ligands exposed per particle. 45 indeed, too big nps have a limited surface area, while too small would not allow an efficient grafting of the dendrons due to steric hindrance. for instance, it has been shown that dendron-functionalized aunps showed a size-dependent antiviral activity for influenza virus, where 14 nm particles exhibited a higher efficiency than 2 nm aunps. this has been associated with the low functionalization grade of the small nanoparticles and to the inappropriate spatial distribution of the interacting ligand/receptor pairs. the development of viral proteomics has profoundly transformed the antiviral and disinfection strategies. in particular, small molecules and peptides able to target and block the viral biochemical machinery have been developed. however, despite these efforts into the drug design, many of these molecules suffer from poor biological effect, low concentration in the diseased areas, and undesired side effects. in this context, aunps have been coupled to biologically inactive small molecules to create biologically active multivalent aunp therapeutics. a bright example has been reported by bowman et al., where the authors functionalized aunps with sdc-1721, a small membrane fusion inhibitor of hiv. 46 the results demonstrated that, while pure sdc-1721 has low activity, functionalized aunps are able to inhibit hiv replication at μm concentrations. similar results have been reported using targeting peptides. in particular, it was evidenced that the functionalized aunps can sensibly reduce the ic 50 up to 2 orders of magnitude compared to pure peptides. 47 preliminary results in vivo confirmed the biosafety of the aunps. 48 nanoparticles generating reactive oxygen species. one of the main advantages of using nps compared to oxidized metals relies on the slow release of ions and clusters from these particles, leading to an enhancement of the antiviral activity. additionally, the use of metal nps containing cu or fe in ionic form catalyzes the generation of radicals via fenton and fenton-like reactions oxidizing the capsid proteins and consequently blocking the viral infection at early stage. for instance, copper ions (derived from sulfates or iodide salts) have been widely used as antiviral agents because of their activity on several kinds of enveloped and non-enveloped viruses including influenza virus, 49−51 herpes simplex virus 52−54 and hepatitis a virus. 55 their mechanism of action relies on the formation of cu + ions (from soluble salts or nanoparticles) that generate hydroxyl radicals. 56 the use of metallic copper nanostructures in the form of particles or sheets has shown only a moderate efficiency due to the low concentration and low release of cu + . 56 for these reasons, cu + salts, where the copper ions are readily present in their active monocationic form, have been favored. in particular, cui nanoparticles (stable at room temperature) have been extensively studied for deactivation of feline calicivirus 56 and h1n1 pandemic influenza virus. 57 however, the use of copper salts at high concentrations can irreversibly alter reactive oxygen species (ros) homeostasis of healthy cells, provoking a general toxicity for the organism, limiting their applications to disinfection. 56 nanostructured cuprous and cupric oxides have been also extensively employed as antiviral agents for in vitro applications. for instance, cuprous oxide nanoparticles (cuonps) were successfully employed against hepatitis c. 58 in particular, it was found that these nps exerted a favorable antiviral activity with no cytotoxic effects. cuonps target the binding and entry step of viral infection to hepatic cells ( figure 5 ). similar results were reported on the use of cuonps against hsv-1, however without any profound investigation on the antiviral mechanism. 59 alternatively, zinc salts have been successfully used as antimicrobial agents from research up to clinical trials for viral warts. 60, 61 more recently, zno nanoparticles (znonps) were developed for the treatment of hsv-2. znonps were prepared with a tetrapod morphology. 62 the results showed that they can mimic cell surface interacting with the hs present on the viral capsid. additionally, these particles have been used for photocatalysis showing to efficiently destroy the viral proteins upon uv irradiation. 62 besides all these interesting examples, in vivo applications are still needed to validate this therapeutic modality. due to the generation of high levels of ros, the toxicity of copper nanoparticles has been widely debated. the antiviral activity of copper nanoparticles is generally associated with the release of cu + ions in solution, thus the leakage of cytotoxic cationic species can be modulated by surface functionalization before in vitro and in vivo applications. on the other side, the use of nanomaterials generating ros can find applications in textile and surface coating. the general broad virucidal efficiency of copper oxide nanoparticles shown for h1n1 pandemic influenza 57 should be tested on sars-cov-2 and might be used for improving mask protection efficiency. carbon nanomaterials. due to their diversity, versatility, and tunable surface chemistry, carbon nanomaterials have been attractive for several types of applications. in particular, the past decade has seen a tremendous raise in the preparation of performant carbon-based nanomaterials in the antiviral field. fullerene and its derivatives are the most studied carbon nanomaterials for their virucidal activity. due to the lack of solubility of pristine fullerene, functionalization strategies have been developed to prepare water-soluble drugs. investigations in the biomedical field evidenced the membranotropic capacity of fullerene derivatives. 63 by modulating shape and functions, fullerene derivatives have been shown to possess antiviral properties through inhibition of viral entry and blockage of viral replication. from these results, the attention has been directed also to other carbon nanomaterials. in particular, functional carbon dots (cds) and graphene oxide (go) have been investigated for their ability to block viral entry into host cells. glycofullerenes. the emerging of mortal viruses, like ebola or zika, and the lack of suitable treatments led the academic and the industrial communities to look for alternative therapeutic routes. most of these pathogens are rna enveloped viruses, and they share common infection mechanisms that can be targeted for the preparation of wide small spacer between core c 60 and surrounding fullerenes. b large spacer between core c 60 and surrounding fullerenes. www.acsnano.org review spectrum antivirals. the external surface of the envelope of these viruses is covered by glycans that tightly interact with lectin receptors on host cells. 64 this strong interaction allows the attachment of the virions to the cells, followed by internalization and infection. blocking lectin receptors is a general strategy used to stop viral infection at an early stage. fullerenes have been widely investigated as antiviral molecules, drug carriers, or tissue scaffolds. 65 fullerene applications have been recently extended to the design of mannosylated derivatives to block the entry of viral particles into host cells. mannose, due to the high affinity with lectin receptors, competes with the virus in the interaction with the host cells. for example, one of the targets is the inhibition of viral particles through the interaction of mannose with the dendritic cell-specific icam-grabbing non-integrin (dc-sign). dc-sign receptors mediate the interactions between dcs and t cells. 66, 67 to exploit these characteristics, mannose was combined with fullerene in the design of the so-called glycofullerenes to study their capacity to inhibit ebola, dengue, and other pathogens. for this purpose, different glycofullerenes were synthesized by changing the number of mannose units (from 12 to 36) and the spacers between the fullerene moieties and by varying steric hindrance in order to obtain a library of molecules. 66 the synthetic route is composed of three steps based on "click chemistry": (1) assembly of glycodendrons by cu(i)-catalyzed azide−alkyne cycloaddition (cuaac), (2) synthesis of alkynesubstituted bingel-hirsch hexakis-adducts, and (3) the coupling between the last two products again by cuaac. to increase the number of mannose moieties up to 36, the glycodendron core was changed from malonate to trialkynyl pentaerythritol. in order to compare the different derivatives, in vitro studies were performed. jurkat cells (lymphocyte t cd4 immortalized cells) expressing dc-sign were used to prove the inhibition capacity of the glycofullerenes on viral infection of ebola ( figure 6 , route a). the study revealed an ic 50 in the μm range for the 12 mannose fullerene, a lower efficiency with the 36 mannose fullerene with a short spacer (peg, with 2 ethylene oxide units), while a nanomolar ic 50 was achieved with 36 mannose fullerenes with a longer spacer (peg, with 3 ethylene oxide units) ( table 3 ). this first proofof-concept study was then expanded, aiming to obtain a better antiviral activity by increasing the valence and inserting longer and flexible spacers. 68 based on these studies, another class of multivalent fullerene dendrimers was then designed. a fast and controlled synthetic route was developed to achieve giant globular multivalent fullerenes, containing hundreds of functional groups. the first study was performed with tridecafullerenes containing 120 mannoses. 69 the molecular structure is composed of 12 hexakis c 60 surrounding a c 60 core ( figure 6 , route b). compared to the previous study, an ic 50 3 orders of magnitude lower was measured on the inhibition of ebola virus (table 3) . 66, 68 in order to present more carbohydrates at the periphery of the dendrimer, a trialkynyl pentaerythritol derivative allowed to afford a tridecafullerene with 360 carbohydrates. in this case, the molecule was synthesized with a c 60 tridecafullerene bearing α(1,2)mannobioside. 70 the use of this disaccharide was already investigated, showing an increase of affinity with dc-sign receptors by a factor of 3−4. 71 the synthetic strategy exploited also the use of strainpromoted copper-free cycloaddition of azides to alkynes (spaac) for the coupling of the core fullerene to the surrounding fullerenes. spaac allows an easier purification avoiding the removal of cytotoxic copper ions. the inhibition performance of this molecule was studied in vitro with viral pseudoparticles of dengue and zika. the comparison was made between 360 and 120 disaccharides tridecafullerenes and 36 disaccharide monofullerene. the results highlighted a picomolar ic 50 inhibition on both zika and dengue models for the 360 disaccharide glycofullerene ( table 3) . the ability to inhibit other types of viruses allows the use of glycofullerenes as broad spectrum antiviral drugs. moreover, the negligible toxicity to other cells proved the biocompatibility of these molecules. following an alternative strategy, a supramolecular assembly of monodisperse glycofullerenes, leading to the formation of micelles, was achieved and tested. 72 these micelles present a uniform and spherical shape ( figure 6 , route c). the aggregation synthetic route is faster compared to a controlled synthesis of giant glycofullerenes, but it might suffer from low reproducibility and batch-to-batch differences between each formulation. this self-assembled c 60 functionalized with 6 or 12 mannoses exposes a large amount of carbohydrate at the surface, leading to an inhibition of ebola virus in the nanomolar range (ic 50 of 424 nm for six mannoses and 196 nm for 12 mannoses, respectively, table 3 ). further in vitro studies evidenced again a good biocompatibility of these glycofullerenes. while there are no in vivo studies yet, these promising results enlarge the panel of molecules in the fight against new emerging viruses. functionalized fullerenes have been used for their ability to compete with viral particles through lectin receptors in host cells. there has been a tremendous advancement in the functionalization of fullerenes leading to the preparation of derivatives with a high amount of mannose, capable to enhance the multivalency effect and thus to increase the therapeutic outcome. first, glycofullerenes do not have an intrinsic virucidal activity. they can reduce the infectivity, but they are not able to completely inactivate the virus. second, the mechanism of action of glycofullerenes relies on their interaction with host cells and not with viral particles. thus, for a therapeutic application, they should be injected at different time points, ensuring that the local concentration is therapeutically relevant to prevent the virus from invading the host cells. in addition, glycofullerenes can be internalized into host cells losing their viral "shield" activity. on the other hand, the well-developed surface chemistry of glycofullerenes can be used for other key receptors involved in viral entry. for instance, in the case of the current sars-cov-2 pandemic, a similar click chemistry strategy can be used to anchor ligands recognized by human lung ace2 receptors and so inhibiting viral entry. 73 other carbon nanomaterials. alongside fullerenes, other carbon nanomaterials (nms) have been scrutinized for their ability to block viral entry. cds and go are the most known and studied carbon nms with marked antiviral properties. cds are zero-dimensional carbon nanoparticles. they are generally produced via hydrothermal decomposition of carbon containing "low-cost" precursors. the use of cds in the biomedical field has been encouraged by their easy preparation, low toxicity, fluorescence properties, and easy surface functionalization. pristine cds have shown moderate viral blocking activity for hiv infection in vitro. 74 this has been associated with the surface of the material rich in carboxylic and hydroxyl groups prone to form noncovalent acs nano www.acsnano.org review interaction with viral membranes. moreover, due to the complexity of the biological systems these nonspecific interactions could not be so effective in vivo, likely reducing the antiviral efficacy. therapeutic targeting molecules can be grafted onto a cd surface to enhance their antiviral activity. in this context, the design of multifunctional cd platforms can be obtained through two different strategies. the first consists in a single-step reaction that foresees the insertion of the therapeutic molecule directly into the step of preparation. target molecules are decomposed with the other precursors, generating the desired functional cds. this protocol is fast and efficient, however the drug loading as well as its activity are hard to estimate. indeed, the hydrothermal treatment can alter the chemical structure of the active molecule, thus vanishing its therapeutic effect. for these reasons, the reaction conditions must be carefully controlled. 75 the second method is a twostep reaction and implies the postfunctionalization via amide formation on the surface of the cds rich in carboxylic groups. this strategy offers a better chemical control, but the yield and the drug loading may not be quantitative and high, respectively. different functionalized cds were prepared to hamper host cell viral entry. for instance, benzoxazine (a low water-soluble antiviral agent) was incorporated into the cd structure during their preparation (figure 7) . the as-prepared cds showed a broad spectrum viral blocking capacity in vitro for enveloped (e.g., japanese encephalitis virus, dengue virus, and zika virus) and non-enveloped viruses (e.g., porcine parvovirus and adenovirus-associated virus). 76 these positive results were explained by the efficient binding and deactivation induced by the multivalent effect of the cds to the viral particles amino-functionalized cds were also tested for the treatment of human norovirus. in this study, cds were functionalized with 2,2′-(ethylenedioxy)bis(ethylamine) (eda) and 3ethoxypropylamine (epa) via amide bond formation. 77 these nms exerted a good viral blockage. in particular, epafunctionalized cds were able to inhibit 100% of viral infection at concentration of 2 μg/ml, while in the case of cds prepared with the other amines, 80% of inhibition was reported. these effects have been associated with the higher positive charge of cd-eda compared to cd-epa. another surface group used for viral targeting is boronic acid (ba), which can bind glycosylated surfaces forming boronic esters. this strategy was successfully adopted to treat hiv where the boronic groups, linked to different nanoparticles (e.g., silica nanoparticles and nanodiamonds) can target gp120 receptors on the viral envelope inhibiting the infection. 78 another recent study proposed the use of cd functionalized with phenylboronic acid for prevention of hiv infection. 74 the functional materials showed good inhibition properties compared to nonfunctionalized cds by preventing the binding to the target cell in vitro. overall, the use of cds for stopping host cell viral entrance has shown good results in vitro. however, there is a lack of proofs in vivo limiting their applications to surface disinfection or masks. in addition, most of the in vitro studies foresee first the contact of the cds with the viral particles and then their incubation with host cells. deeper investigations should be performed adding the nms at other time points (for instance in infected cells) to understand if the antiviral activity is maintained. in addition, cds have been successfully used for photodynamic therapy (generating radicals upon light irradiation) in cancer treatment. the same approach may be used to combat viral infections, where the antiviral activity induced by the surface modification can be sensibly enhanced by ros generation under irradiation. graphene materials, and in particular go and reduced go (rgo), have been used for different biomedical applications including drug delivery, biosensing, and tissue engineering. 5 go platforms have shown also interesting antimicrobial activity. 79 regarding viral infection, go was used to block the virus entrance in host cells. go and rgo can be considered as two-dimensional materials that contain hydrophilic and hydrophobic domains allowing to adsorb many biological molecules including nucleic acids and proteins. go showed low interaction with viruses, however its surface functionalization with target molecules can sensibly enhance its affinity for the viral particles. additionally, go can be used as photothermal agent (generation of heat by nir irradiation) or photodynamic therapy (using visible light irradiation) inactivating the capsids by local thermal shock or by radical formation during irradiation, respectively. the use of phototherapies may significantly augment the antiviral properties of the materials. however, we must keep in mind that these therapeutic modalities can be applied only to disinfection, since the radical/heat production may be harmful for the healthy tissues in vivo. photodynamic therapy has been successfully exploited using bacteriophage ms2 as a model virus. 80 in this study, go was functionalized with an aptamer recognized by the viral surface. the results showed that this functionalization is able to enhance the binding efficiency of the ms2 capsids onto the go surface compared to nonfunctionalized go. subsequently, irradiation in the visible light was able to disinfect the solution, while nonfunctionalized go showed much less activity due to the lack of adsorption. despite these interesting results, this pioneer work remains at an early research stage since the use of high light dose (300 w for 10−140 min) and the lack of material recovery and reuse make its application for surface disinfection difficult. go can be also used as a platform to link antiviral agents. encouraging results were reported using go with hypericin for the treatment of a recently appeared duck reovirus. 81 more recently, deokar et al. reported an original rgo-based multifunctional platform for hsv-1 treatment. 82 in this work, the authors functionalized the material with organic sulfate groups and iron oxide magnetic nanoparticles (fenps). the rgo functionalized with the sulfate is able to mimic the host cell surface and to bind hsv-1. subsequently, the viral particles captured onto the rgo-fenp surface can be concentrated via magnetic precipitation and destroyed via photothermal therapy. this approach is highly efficient for disinfection with low energy (1.6 w/cm 2 for 7 min) and cost effectiveness. hs is a common entry receptor in various types of viruses (e.g., herpes viruses, human papillomavirus, dengue virus). 83 the use of organic sulfate-functionalized graphene sheets mimicking hs, like go and rgo, has been already explored. however, it is worth noting that these nms are prone to strongly adsorb proteins in culture environments (coronation), likely inhibiting their antiviral efficacy. 84 high loadings of sulfate groups were introduced onto rgo using polyglycerol sulfate. 85, 86 this approach has been used for inhibition of orthopoxvirus, pseudorabies virus, and african swine fever virus in vitro. 85, 86 graphene has also been used as antiviral material. polysulfates and fatty amines were grafted onto graphene surface via triazine chemistry for the treatment of herpes simplex virus. 87 this strategy promotes the synergy between the electrostatic and hydrophobic interactions, showing incredibly high inhibition efficacy. overall, graphene materials have shown a good capacity to block host cell viral entry. disinfection with graphene family materials is also promising, offering the possibility to couple high viral binding with phototreatments. regarding the go and rgo activity in cellular environments, different parameters must be considered such as protein coronation, blood circulation time, and activity in vivo. so far, the use of sulfonic groups introduced via diazonium salt decomposition has been largely privileged. we take this opportunity to encourage future studies using other targeting groups (e.g., boronic acids) and grafting methods (e.g., epoxide ring opening or hydroxyl esterification reactions). mechanical disruption of the capsid. the most direct way to suppress viruses and stop the spreading of viral infection is to inactivate them before the attachment to the host cells, by binding to the acceptor proteins. 88 one of the most conserved targets of viral attachment ligands is the heparan sulfate proteoglycan (hspg), previously mentioned. hspgs are expressed on the surface of almost all eukaryotic cell types, and many viruses like hiv-1, hsv, human papilloma virus (hpv) exploit hspgs as the target of their viral attachment ligands. bearing in mind this behavior, different studies have used hspg-mimicking materials to target this type of virus−cell interaction and to achieve broad spectrum efficacy. for example, in one key study, aunps were functionalized with mercaptoethanesulfonate (mes) based on its mimicry of hs (au-mes nps). au-mes nps were shown to interfere with viral attachment, viral entry, and cell-to-cell spreading. 89 the importance of the polyvalent interactions with the virus makes these nps a good candidate for antiviral therapy. however, au-mes nps presented virustatic activity, meaning that upon dilution of the nps, the virus recovers its infectivity due to the reversibility of the cell-virion interaction. this problem was solved by stellacci and colleagues who developed nps coated with mercapto-1-undecanesulfonate (mus) ligands (au-mus nps). the long aliphatic and flexible linkers provide stronger associations with the viral particles compared to au-mes nps, leading to local distortions and eventually inducing a global deformation and breaking of the capsid that inactivates its contagion irreversibly (figure 8 ). 90 these au-mus nps were tested against different hspgdependent viruses, showing a high viricidal activity over hsv, hpv, and rsv ( figure 9a) . furthermore, the activity of the au-mus nps was studied in vivo using mice infected with rsv, indicating that the material can prevent pulmonary dissemination of the infection and showing potential use as medically relevant virucidal drugs to fight viral infections. more recently, the concept was further extended to cyclodextrins modified with mercapto-1-undecanesulfonate, proposing this system as a broad spectrum virucidal macromolecule. 91 besides au-nps, a similar mechanism of disruption was studied with other materials like graphene or go. in a study, in which the toxicity of graphene was evaluated theoretically, it was also shown that graphene nanosheets can interrupt the hydrophobic protein−protein interaction, which is essential to biological functions. 92 this feature was attributed to the hydrophobic nature of graphene. thus, it seems energetically favorable for graphene to slide between the interface of two proteins in contact, due to hydrophobic interactions. in another study inspired by this behavior, the authors performed molecular dynamics simulations of graphene nanosheets in the proximity of the surface of the ebola viral matrix protein vp40 showing that the nanosheets can break the hydrophobic interactions in vp40, a key protein for the replication and stability of ebola virus (figure 8 ). 93 these findings suggest that graphene nanosheets might have potential antiviral activity against ebola; however, there is a lack of experimental evidence that corroborates this mechanism of disruption. on the other hand, go was tested experimentally against pseudorabies virus and porcine epidemic diarrhea virus (pedv), showing significant decrease in the infectivity. 94 it was found that the negatively charged surface of go is important for the adsorption of the virus, whose surface is positively charged, and that go could directly interact with the viral particles and destroy their structures due to the sharp edges of the material (figures 8 and 9b) . the mechanical disruption of the capsid is a peculiar antiviral mechanism associated with some nms. in particular, the use of specific sulfonates able to mimic heparan sulfate can be also used to target sars-cov-2 infection. 95 blocking the viral entry, via liquid/surface disinfection or once in the body, is a powerful strategy to hamper early stage viral contagions. on the other hand, when infections have already spread and reached middle and late stages, alternative pharmacological strategies are required. the study of the viral pathogenesis and machinery inside host cells has allowed the preparation of different drugs. due to the present pandemic, such antiviral drugs are now of top interest in the scientific and medical communities. so far, few antiviral drugs are clinically available, and their mechanisms of action consist on the inhibition of reverse transcriptase (hiv, hepatitis), dna inhibition of polymerase (herpes, hiv), inhibition of protease (hiv), blockage of ion channels (influenza), and inhibition of neuramidase (hiv, influenza, hepatitis). however, these drugs suffer from moderate to severe side effects. additionally, rapid mutations in the viral machinery make them resistant to the treatments, making the control and the stop of the infection challenging. the use of hnms in drug delivery has shown several advantages. first, hnms can increase drug solubility and its circulation time. additionally, they can be functionalized with targeting molecules able to direct the drug to the desired organs and so avoiding side effects and reducing the dosage. more recently, new antiviral mechanisms were discovered. in particular, it was found that different types of hnms are able to change the ros homeostasis in infected host cells, stopping the viral replication and preserving the cell survival. in this section both drug delivery materials and hnms with ros modulation properties will be critically presented ( figure 10) . nanomaterials for drug delivery. different hnms have been widely tested for drug delivery applications. the advantages of this strategy are several including enhance drug solubility and the possibility of targeting and multivalent effects. as a potential broad spectral antiviral agent, agnps can prevent the virus from adsorbing to the host cell in the early stage of infection and thus show strong antiviral activity. after the cells are infected by the virus, there are ways to inhibit cell apoptosis (figure 2, right) . for example, lv et al. studied the anti-tgev activity of agnps in swine testicle cells and explored the possible mechanism of agnp inhibition of tgev infection-induced apoptosis. 18 the results showed that these agnps are able to decrease cell apoptosis through the activation of p38/mitochondria-caspase-3 signaling. 18 although the use of agnps has shown good antiviral action to further improve the therapeutic effects and reduce both side effects and drug resistance, the way of binding drugs or genes and other therapeutic agents to agnps has received particular attention. it has been observed that agnps inhibit the activities of neuraminidase and hemagglutinin, preventing the h1n1 influenza virus from attaching to host cells. at the same time, the potential molecular mechanisms revealed that caspase-3-mediated apoptosis was inhibited by ros generation. agnps modified with polyethylenimine (pei) can bind sirna. ag@pei@sirna exhibited superior abilities for enhanced cellular uptake and blocking ev71 virus infection acs nano www.acsnano.org review and significantly decreased the apoptotic cell population, which prevented the spread of ev71 virus. 37 in addition to drugs and sirna, neutralizing antibodies in combination with agnps were developed. the results demonstrated that there is an additive effect between the antibody and agnps when combined against cell-associated hiv-1 infection in vitro. 96 the membranotropic properties of fullerenes were widely exploited. for example, pristine c 60 , after accumulation at the cell membrane, can translocate into the cytoplasm by crossing the membrane through multiple energy-dependent pathways despite its hydrophobic character. 97 fullerenes can be made water dispersible using surfactants, sonication or first dissolving them in appropriate organic solvents (dmso). the use of fullerenes as inhibitors of viruses started in 1993 with a study focused on hiv infection. 98 this work revealed the interaction between c 60 derivatives and hiv protease through molecular modeling and experimental verification. hiv protease (hiv-pr) is involved in the mechanism of replication in the maturation of hiv virion, cleaving newly synthesized proteins. the active site of hiv protease has a cavity of 10 å closed to the diameter of c 60 cage. 99 molecular docking and experiments on hiv-pr catalytic activity revealed a blockage of the active site of the enzyme by van der waals interactions leading to an antiviral effect. the following studies were based on a structure−activity relationship between functionalized fullerenes and hiv-pr with the aim to increase the antiviral activity. the introduction of pyrrolidinium salts onto c 60 was tested against the activity of hiv-1 strain. 100 in a second study, c 60 bearing two ammonium groups was applied against the activity of hiv-1 and hiv-2 strains. 101 the results showed the importance of having two moieties in a precise position on the fullerene cage and the influence of the charge of the different salts sensibly increasing its antiviral activity. further investigations using different functional groups (e.g., amino acid derivatives) were explored. c 60 functionalized with aminobutyric acid and aminocaproic acid was able to inhibit hiv viral replication at subnanomolar concentrations. 63 in another work, anionic and cationic pyrrolidinium salts and amino acid functionalized fullerene derivatives were used for the inhibition of hiv reverse transcriptase (hiv-rt). fullerene compounds were compared to nevirapine, an available drug against hiv-rt, revealing a better inhibition compared to the pure drug. 102 the antiviral property of carboxylated fullerenes was confirmed by another study. 103 results obtained in vitro on cem cell line showed low toxicity and a submicromolar ec 50 against hiv-1 and hiv-2 strain viral replication. several mechanisms of hiv protease inhibitors have been hypothesized and simulated. some studies investigated the action of fullerene derivatives on viral replication cycle and the virus maturation ( figure 11 ). 98, 104 for the latter, it was suggested an impairment due to a strong interaction between fullerene and the immature capsid. 105 other rna viruses share ways of viral replication similar to hiv. 106 c 60 functionalized with an amino acid derivative was investigated against hepatitis c rna polymerase (hcv-rp). 102 this essential enzyme for viral replication was inhibited in a submicromolar range, similarly to benzo-1,2,4thiadiazine, a potent specific inhibitor of hcv-rp. another publication highlighted the effect of a c 70 poly(carboxylic acid) derivative on different strains of influenza virus (e.g., a, h1n1, h3n2, and b). 107 the inhibition was comparable to tamiflu, rimantadine, ribavirin, and amantadine, but the mechanism of action remains still unclear. these data emphasize that fullerenes c 60 or c 70 can be potent universal antiviral drugs for rna enveloped viruses. however, clear elucidations of the mechanisms of action and in vivo studies are still a missing point. due to their high surface/weight ratio and capacity to pass through cell membranes, carbon nanotubes (cnts) have been extensively explored for drug and gene delivery applications. 108 cnts have been also successfully used for the delivery of antiviral agents. compared to pristine materials, oxidized cnts (ox-cnts) showed an inhibitory activity for hiv viruses per se. 109 this effect has been associated with the oxygenated groups that increase the hydrophilicity and the colloidal stability of the material. the antiviral efficiency has been correlated to the ox-cnt interaction with host cells. 109 however, it is not clear how and what kind of mechanism blocks the viral machinery in host cells. different anchoring strategies have been explored to link antiviral drugs onto cnt surface. for instance, ox-cnts were covalently linked to 2amino-3-nitro-1-(3,5-dimethylbenzyl)-aniline (chi360) and n-(2-aminophenyl-3-nitro-)-3,5-dimethylbenzenesulfonamide (chi415), two active non-nucleoside reverse transcriptase inhibitors for hiv treatment. 109 following the covalent conjugation, only a moderate antiviral effect was observed compared to pure drugs, indicating that most probably the nm cell trafficking played a key role on the virucidal activity. 109 in another study, ox-cnts functionalized with cyclodextrin were used for the delivery of acyclovir (a prodrug inhibitor of the viral dna polymerases) for the treatment of hsv-1. preliminary results showed that when acyclovir was delivered via the nanotubes, the viral antireplicative effect was higher than the free drug. 110 more recently, a similar approach was applied to herpes virus using cyclodextrin and pei-functionalized cnts for co-delivery of cidofovir and plasmid dna. however, the antiviral effect of the materials was not explained, and the transfection effect was not satisfactory. 111 functionalized cnts have been also used for delivery of ribavirin in vivo using grass carp as an animal model for the study of grass carp reovirus. ox-cnts were first functionalized via amidation with bsa, and then ribavirin was covalently bound to the protein via esterification (figure 12) . 112 in vivo tests demonstrated that, when ribovirin was shuttled by ox-cnts, the antiviral efficiency was significantly increased without any evident toxicity and no significant changes in ros-generating enzymatic activities. the use of cnts in drug delivery is however still controversial. 113 graphene-based materials have been limitedly studied as antiviral drug delivery carriers. only a few examples can be found in the literature. graphene quantum dots (gqds) were used for drug delivery of chi360 and chi415 and tested in vitro against hiv similarly to cnts. 109 both the prepared gqd-chi360 and gqd-chi415 showed a high antiviral activity once into host cells, with low toxicity. go has been also used for the delivery of dnazyme into hepatic cells allowing to block the hepatitis c infection. this specific dna single strand is able to recognize the viral mrna and to silence its expression. 114 overall, carbon nms proved to be interesting carriers for antiviral drugs. however, several questions need to be answered before their safe application as antiviral materials. different reports have demonstrated that pristine cnts display relevant toxicity for healthy cells, but by oxidation of the tubes, the side effects can be sensibly reduced. 109 in addition, more investigations should be performed on cnt toxicity. these nanocarriers indeed are not "innocent delivery agents", but they play a key role in drug internalization pathway and in host cell machinery that might be averse to the expected therapeutic effects. 109 so far, cnt application in biological systems has been studied for 20 years. however, due to their possible toxicity, their real application in clinics seems to be steeper and difficult to achieve. 112 materials tuning reactive oxygen species. ros homeostasis in infected cells has been studied for both rna and dna viruses. for instance, it was shown that infection of mice with influenza a decreased the concentration of lung glutathione and the antioxidant vitamin c, providing evidence that the viral infection was associated with oxidative stress in vitro as well as in vivo. 115 similarly, in hiv infection, induced oxidative stress in host t cells and high concentrations of antioxidants are able to slow down the cell-to-cell viral spreading. 115 the increase of ros concentration is a common process in most of the viral infections. however, the mechanism of radical generation is different from case to case. several proofs suggest that modulating ros homeostasis in infected cells can slow down or block the infection. 116 hnms have been shown to be powerful allies against viral infections. in particular, metal or metal oxide nms, once internalized, can regulate the radical production into infected host cells. in this scenario, the nms can work following two different mechanisms: (1) enhancing radical activity or (2) quenching ros inside cell compartments. in the first case, metal oxide nps are able to convert superoxide ions into more reactive hydroxyl radical species via fenton or fenton-like reactions. the excess of superoxide ions is able to oxidize the viral proteins and the genetic material and therefore efficiently block the infection. this approach has been reported using zno nps. 117, 118 in these studies, zno nps have been successfully applied for the treatment of h1n1 influenza virus and herpes simplex virus. the preliminary results showed that pegylated zno particles were able to efficiently reduce the viral infection with ic 50 similar to acyclovir. more importantly, toxicity of zno was modulated by functionalization with peg, which allowed a higher colloidal stability and a more controlled release of zn 2+ ions to catalyze ros formation. indeed, ros (e.g., superoxide and hydroxyl radicals) produced by the nps should be highly reactive and should not only damage the exogenous biological molecules but also attack different cell compartments. overproduction of ros may reduce virus spread but also induce cell death. interestingly, this approach is applicable only at the early infection stage (after 1 h incubation of the host cells with a virus), but it loses its activity at later time points. these 117, 118 this specific mechanism of action restricts zno nps to an application only at the early stage of infections. however, the same approach with other metals able to induce fenton or fenton-like reactions (e.g., fe, cu, and mn) has not been reported yet. the choice of proper capping agents may allow to control the metal ion release and thus tune the ros-mediated antiviral activity. we would like to take an advantage here to suggest the growth of the antiviral research in this direction. another successful approach relies on the reduction of ros concentration in host cells. ros scavenging is able to alleviate the toxicity of the infection enhancing cell viability, giving time to start its endogenous antiviral mechanisms. so, this approach may both block infection and ensure host cell survival. in this context, selenium nps (senps) have been extensively studied for their antiviral activity. the mechanism of action of these nps relies on the quenching of the radicals into host cells due to the infection, stopping the mitochondria depolarization and the consequent apoptotic cascade. 119 additionally, senps can also adsorb onto the viral capsid sensibly reducing their infectivity. senps can be prepared via classical mixing of selenium salt precursors in the presence of a reducing agent. more recently, senps have been instead biosynthesized from actinobacteria showing good stability and capacity to inhibit dengue virus in vitro. 120 moreover, senps were used to carry different antiviral drugs including zanamivir, 121 oseltamivir, 122 amantadine, 123 and ribavirin. 119 their functionalization with the desired drug can be easily achieved adding the molecule during their synthesis through the se ion controlled reduction. these nms have been applied for the treatment of h1n1 virus. notably, senps with ribavirin (administered via intranasal absorption every 24 h for 3 days) showed that infected mice had much less alveolar collapse and perivascular and peribronchiolar edema, compared to the group challenged with the virus (figure 13 ). 119 due to their efficacy and low toxicity, senps can be considered a useful material for the treatment of other viral diseases including sars-cov-2. as a matter of fact, oxidative stress as well as chronic inflammation may contribute to the aggravation of the covid-19 symptoms and to the general spread of the infection. 124 the use of senps could eventually alleviate the toxicity of infected patients, giving time for the immune system to react against the contagion. however, the lack of preclinical studies on senps, together the scarce knowledge of their biosafety and long-term toxicity, still remain the main challenges to tackle for clinical translation. the vast majority of the studies show that human survival to viral attack is based on the stimulation and response of our immune system. when a virus enters and starts infecting tissues, the body reacts and triggers a strong immune response to overcome the pathogen invasion and spread. normally, two different immunological reactions take place. the oxidative stress induced by infection causes the activation of the inflammasome through upregulation of pro-inflammatory cytokines such as il-1β, pro-il-18, and nlrp3. 122 excessive upregulation of this mechanism leads to cell damage and eventually to pyroptosis activated by caspases. 122 it was also shown that generation of radicals is able to depolarize mitochondria, hence affecting host cell respiration and inducing ros-mediated apoptosis at the late stage of infection. 123 besides, activation of pro-inflammatory cytokines can alert the immune system blocking the infection (the innate acs nano www.acsnano.org review immune response). 122 the second immune response is specific (the adaptive immune response). immune cells are trained to attack the virus (cellular response), while specific antibodies are produced by b cells (humoral response). in the last years, hnms were proved to tune the immune responses, demonstrating to be a possible alternative against viral infection. in this section the most relevant strategies for the activation of innate and adaptive immune responses using nms will be described ( figure 14) . innate immune response. innate immune response is the first response that takes place in the presence of any type of infection. during this early stage, interferon stimulating genes (isgs) are upregulated in the infected cells. 125 this selfdefense mechanism slows down the viral replication and alerts sentinel immune cells that start producing proinflammatory cytokines and trigger inflammation. 125 however, many viruses are able to escape this complex mechanism, retarding the immune response and spreading the infection. the interaction of hnms with the immune system has been more and more studied. in the case of antiviral hnms, many examples can be found in the literature where the nms not only slow down the infection but also tune the innate immune response. certain hmns can display an intrinsic immune stimulation. we have already mentioned above that in the early stage of infection, agnps mainly prevent the virus from entering the host cell through the interaction with the external capsid, but in vitro cellular experiments lack to understand the complex interaction with primary immune cells. recent studies have shown that agnps can potentially induce the expression of genes involved in innate and adaptive immunity-associated pathways, which are known to play crucial role in immune regulation. for example, toll-like receptor 7 can be upregulated by agnps after 24 h, by recognizing the singlestranded rna of the viruses and regulating the antiviral immune response. 126 another study showed that in rsvinfected mice treated with agnps, the particles reduced the production of pro-inflammatory tnf-α and il-6 cytokines, but potentiated the anti-rsv activity of neutrophils in an experimental mouse model. 127 however, activation of agnps in the reduction of rsv has been noted only when the nm was intranasally inoculated together with the virus, and no results have been reported on the use of agnps administrated on infected mice. in the case of influenza virus infection of lung epithelial cells, it was found that agnps targeted infected lung epithelial cells and reduced viral replication, by preventing autophagy. however, the blockage of the autophagic flux by agnps does not inhibit viral replication in already infected cells. therefore, agnps are more suitable as viral preventive agents due to their pro-inflammatory response rather than drugs. 128 more recently, agnps were combined with graphene materials and exploited as antiviral material for the treatment of porcine reproductive and respiratory syndrome virus (prrsv). 129 go-agnps were able to clump the virus diminishing its fusion with cell membrane. additionally, once go-agnps were internalized in host cells, they stimulated the isgs that blocked viral budding and its diffusion to other cells in vitro (figure 15 ). 129 similarly, cds used for the treatment of rsv and prrsv were able to activate the innate immune response via an upregulation of isgs in vitro. 130 gold nanorods were applied to boost the innate immune response against rsv in vivo. 131 interestingly, it was shown that these nanorods (when intranasally administered with rsv) were not only able to activate the isgs but also to tune the production of pro-and anti-inflammatory cytokines, resulting in the blocking of the infection with a reduced pulmonary inflammation. 131 as drug carriers, hnms can also affect the internalization pathways, modulate drug efficacy, and the immune cell responses. for instance, it was found that the isoprinosine immunomodulatory antiviral drug displays a much higher antiviral efficacy in vivo when delivered with cnts than as a pure drug (in acs nano www.acsnano.org review zebrafish larvae food administration), probably due to a better cellular uptake and the anti-inflammatory properties of the nanotubes. 132 fullerenes also exhibit immunomodulation properties through the release of cytokines by bovine alveolar macrophages. 133 a study explored the influence of functionalization of c 60 with molecules presenting different surface charges like hydroxyl groups and amino acids. 134 the study concluded that negative charges upregulated tnf-α up-secretion in raw 264.7 macrophages, but highly positively or negatively charged surfaces increased cell toxicity. the upregulation of the cytokine tnf-α is a proof that fullerene can promote cellular immune response. despite these preliminary results, the actual mechanisms of interaction between hnms and the immune system are still at early stage of understanding. we must consider that the immune response needs to be proportional to the infection grade. if on one side nanosized immuno-boosters can alert more efficiently the sentinel cells, the use of hnms that trigger an exaggerated immune response can promote excessive inflammation, damaging healthy cells and promoting uncontrolled side effects. in the particular context of sars-cov-2, the use of agnps, fullerenes, or other pro-inflammatory hnms at the middle and late infection stage may cause an aggravation of the symptoms due to already diffused inflammation. in particular, most of the studies showed the ability to reduce infection when hnms were first incubated with the pathogenic virus, thus limiting their potential use in the early stage infection. 124 the formulation of hnms able to both alert the immune system (e.g., upregulating cytokine) and control lung inflammation (e.g., ros scavenger) even after the early stage infection may be a possible strategy for treatments against sars viruses. adaptive immune response. adaptive immune response is the specific response that the immune system exerts against pathogens. this mechanism is particularly active toward viral infections where the immune system produces specialized lymphocytes (to fight the virus), called memory b cells (to be effective in case of new infections) and antibodies (corresponding to the humoral response). 135 the stimulation of adaptive responses in case of specific infections can be induced artificially through the introduction of attenuated pathogens, stimulating the production of specific antibodies. this is the principle of vaccination, which is the most common procedure for immunization of large areas of population against many kinds of lethal viruses. 135 besides, the use of viral proteins as antigens in the vaccine formulation leads to neutralizing antibodies, but, due to the low immunogenicity of isolated proteins, does not always stimulate sufficiently the immune system to reach total protection. more recently, nanotechnology has been applied to develop more efficient vaccines (e.g., nanovaccines). the use of nanostructures with a size similar to virus (virus-like nanoparticles) sensibly enhances the response helping to reach immunity. 135 hnms can adsorb viral particles and present them to the immune system. 136, 137 this method of vaccination has been successfully applied in vivo for the challenge of herpes simplex 2 virus. hsv-2 starts its spreading in vaginal tissues and then diffuses to the neurons causing death in mice. zno nps (teardrop morphology), after vaginal inoculation with hsv-2, are able not only to prevent viral cell adhesion but also to expose viral antigens to t cells and dcs, leading to immunization. the preclinical trials acs nano www.acsnano.org review against hsv-2 showed a survival to infection higher than 90%. this approach highlights the possibility to couple cell mimicking nms to other co-adjuvants for the formulation of large spectrum nanovaccines ( figure 16 ). 136, 137 hnms have been also applied to the delivery of antigens, exposing them to the immune system. fullerenes were found as suitable carriers for the delivery of drugs or nucleic acids. 138 functionalized fullerene can also self-assemble into virus-sized nps. 139 investigated as vaccines in cancer immune therapy, 140 polyhydroxy fullerenes (called fullerenols) display interesting properties for antiviral therapy, based on their capacity to selfassemble into virus-like particles (vlps) and so to enhance the immunogenicity of the antigens. 141 the great advantage of this strategy relies on the easy encapsulation process during the self-assembly making fullerenols versatile for the formulation of different kinds of vaccines. these vlps were investigated against hiv-1 141 and hepatitis c viruses. 142 compared to conventional protein-or peptide-based vaccines intended to induce antigen-specific adaptive immune responses, dna vaccines are more stable, cost-effective, easy to manufacture, and safe in handling. 143 however, dna vaccines have the disadvantage of being poorly immunogenic. 144 fullerenol vlps allow to avoid the use of other adjuvants. in the case of a vaccine against hiv-1, fullerenol vlps penetrated easily into the cells resulting in an enhancement of dna transfection. this was proved in a study using fullerenol encapsulating dna encoding the hiv-1 envelope protein gp145 ( figure 17 ). 141 in vitro assays were performed in human embryonic kidney cells line (hek293) showing good transfection ability. following various immunization routes (e.g., activation of toll-like receptor signaling or effector memory t cell immune response), fullerenol vlps can induce an innate and a cellular immunity. a similar study was performed for hepatitis c using the hcv recombinant protein as antigen, 142 confirming the potential efficacy of using fullerenols as antiviral vaccines. nevertheless, these results require further mechanistic investigations. indeed, in vitro studies also evidenced a suppressive effect of acquired immune response of c 60 pyrrolidine tris-acid and fullerenol c 60 (oh) 36 . 145 the fullerenol had a dose-dependent effect on t cell receptormediated activation and antibody production by b cells under anti-cd40/il-4 stimulation. however, the molecular mechanism is still unknown. other hnms including aunps (two subcutaneous injections in guinea pigs) and nanodiamonds (three subcutaneous injections in balb/c mice) were explored as carriers of viral proteins for immunization of swine transmissible gastroenteritis virus and h7n9 influenza, respectively, with good preliminary results. 146, 147 in these cases, the vaccine formulation relies on the adsorption of the viral antigen onto the surface of the nanoparticles. however, an effective vaccination depends on several factors. first of all, the size of the nps plays a key role on the immune system. for instance, size-dependent vaccination efficacy has been reported in mice immunization against the foot-and-mouth disease virus (intraperitoneal and subcutaneous injection, every 7 days for 7 weeks) using aunps as antigen carriers. in this study, the most effective activity to stimulate the immune system was exerted by particles with a diameter in the range of 8 nm. 148 both smaller or bigger particles evidenced a drop-off of the immunization effect. this aspect cannot be ascribed to the antigen concentration, but must be associated only to the acs nano www.acsnano.org review nanoparticle size; however, the mechanism of interaction remains unknown. the selected antigen plays also a crucial role in the preparation of wide spectrum vaccines. for instance, in the case of influenza, two major membrane glycoproteins, hemagglutinin and neuraminidase, are generally used as antigens. however, the antibodies produced by this vaccination strategy are selective to the dominant epitope which has a low effectiveness or is totally ineffective against other epitopes or other kinds of influenza viruses. m2 (a viral protein responsible for the budding and scission of the influenza virus) is commonly expressed in different types of influenza viruses with a high rate of conservation but with low antigenicity. it has been shown that aunps functionalized with m2e protein have a high immunization capacity in comparison to the antigen alone. mice immunized with aunps (two intranasal injections), and then challenged, showed a survival rate higher than 90% to california-h1n1pdm, victoria-h3n2, and vietnam-h5n1 infections. 149 this strategy shows that hnms can be used to boost the immune response of low immunogenic molecules, providing a wide spectrum vaccination potential. unfortunately, it has not been determined if the budding process in sars-cov-2 is mediated by viral proteins or via the host cell's endosomal sorting complex, thus more research on the sars-cov-2 viral machinery is highly desirable. all these approaches are based on the capacity of the nanoparticles to adsorb the antigens and expose them to the immune system in the appropriate conformation to produce the neutralizing and protective antibodies. however, the adsorption is not an easy process to control. for instance, aunps have been ineffective in the immunization against sars-cov, when s viral proteins were used as antigens. 150 in particular, the immunization with the protein alone was more efficient than when it was adsorbed onto the aunp surface. this failure was associated with a conformational change or denaturation of the antigen, which did not lead to the production of the specific antibody. 150 covalent chemistry strategies offer a higher control on the antigen quantification with higher reproducibility and possibility to bind different groups onto the surface of hnms. for example, calcium phosphate nanoparticles (capnps) were successfully covalently functionalized with hen egg lysozyme as model antigen showing an immunization 100 times higher in vivo compared to antigens alone using (one subdermal injection in mice). 151 a similar approach was used with iron oxide nps using mannose (to target dcs) and hepatitis b antigen showing good immunological activity in vitro (two subdermal injections in mice at 14 days distance). 152 more recently, other types of vaccination strategies have been applied using hnms. a smart example has been reported using multifunctional capnps on herpes virus. in this study, capnps have been covalently functionalized with alum/mpl as the adjuvant and two peptides as antigens selected via reverse vaccination. the nm stimulated the immune system generating highly efficient antibodies able to block cell-to-cell infection of herpes virus in vivo (three intramuscular injections in mice every 14 days), increasing the survival rate of immunized mice to 100% against the controls (20% survival, figure 18 ). 153 the recent history has shown the spread of different viral pandemics such as h1n1 flu, hiv, and sars. nowadays, the sars-cov-2 pandemic global lockdown has profoundly changed the daily life of most humans, causing uncertainty in short and middle time perspectives. in this context, the scientific community has responded to protect the population by studying new vaccines and disinfection methods to be applied in the near future. despite this tough work, a sars-cov-2 vaccination will hopefully be available in 1−2 years, making this epidemic transient period gloomy with increased instability. the study of more effective vaccines and the production of a wide range antiviral agents is nowadays an extremely hot topic. hnms comprise a family of materials that share a nanostructured hard core and a tunable surface chemistry. in this contribution, we have methodically reviewed different hnms for antiviral properties. hnms can have antiviral properties per se, blocking the viral replication and diffusion, or their antiviral properties can be tailored, playing with surface chemistry. hnms can be used to block viral entry and arrest infection at the early stage. the mechanisms rely on different actions including breaking of capsid disulfide bonds (e.g., noble metal nanoparticles), capsid oxidation (e.g., cuonps), mimicking of cell surface (e.g., carbon nms), or mechanical disruption (e.g., aunps or graphene). surface functionalization additionally confers a higher specificity and pharmacological activity toward the targeted virus. in particular, the high local concentration of ligands on functionalized hnm surface imparts a high multivalent effect, enhancing the viral trapping efficiency of nms. interestingly, hnms that show an intrinsic antiviral activity can further enhance their antiviral efficacy via surface functionalization. some antiviral hnms are good photosensitizers (e.g., cuonps) or exert photothermal activity (e.g., carbon nms), thus their antiviral activity can be trigged by light stimulation. more importantly, most of the settled strategies target common viral entry mechanisms and can be adopted to fight a wide viral spectrum. hnms have been also applied as antiviral agents by their interaction with host cells. hnms are able to block viral replication machinery in host cells (e.g., cnts and fullerenes), inhibiting endogenous enzyme activity. additionally, hnms can be explored for the delivery of antiviral molecules, showing a better antiviral activity and reducing side effects in vitro and in vivo. some hnms can also regulate the ros homeostasis of host cells, reduce apoptosis, and enhance host cell survival during the infection. finally, we have reviewed the role of hnms on immunity. in particular, hnms can stimulate the innate immune response, mainly inducing overexpression of interferon and cytokines. this effect can alert sentinel cells and generally warn the immune system of the infection. hnms can be also used for the activation of the adaptive immune response, foreseeing vaccination. due to the similar size of a virus, functionalized hnms with antiviral molecules (virus-like particles) can enhance their immunogenicity. certainly a huge effort should be done for translation of the research into clinics. indeed, hnm applications as antivirals are still in the early phase of research. several challenges still need to be tackled before their safe use. at the current stage, some hnms have been approved only for surface disinfection. for instance, cunps have been used in filters for the preparation of highly efficient broad spectrum antiviral masks. 50 some hnms have been already clinically approved. for example, fenps were approved for imaging and as a drug to treat iron deficiency anemia in adult patients with chronic kidney disease, while aunps are in clinical trials for the treatment of prostate cancer (photothermal therapy). 154, 155 however, at the moment no clinical trials are running for the use of hnms as antiviral agents. in fact, there are still several concerns on the applications of hnms in drug formulations. compared to molecules, where mainly concentration and exposure routes are concerned, solving hnm toxicity issues is much more complicated. composition, size, shape, and surface functionalization must be considered to respond to the requirement for safety regulations. additionally, interaction on hnms with the immune system must be better elucidated. in particular, the activation of the immune system and complement activation-related pseudoallergy must be taken into account. 156 for instance, ferumoxytol (a fenp-based drug) has been reported to generate severe anaphylactic reactions in humans, 18 of which were fatal. 156 this is due to the possible interaction of hnms with mast cells, provoking their degranulation/activation and release of histamine even at the first exposure (pseudoallergic-mediated hypersensitivity). besides, the mechanism of activation of these cells is still unknown, although it was found that it depends on the hnm composition, size, and surface chemistry and on the corona formation. 156 on the other hand, it has been demonstrated that hnms can travel to the draining lymph nodes, targeting resident dendritic cells and macrophages. therefore, they are able to interact with antigen presenting cells to stimulate innate and adaptive immune responses. 156 we believe that a joint venture of different chemists, materials scientists, virologists, toxicologists, and medical doctors can push forward the preparation and safe application of hnms in this field, hoping to prevent and eventually block the rise of new viral pandemics. alberto bianco − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france; orcid.org/0000-0002-1090-296x; email: a.bianco@ibmc-cnrs.unistra.fr giacomo reina − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france; orcid.org/0000-0002-8147-3162; email: g.reina@ibmc-cnrs.unistra.fr hard nanomaterial, nonpolymeric organic or inorganic materials with sizes comprised between 1 and 100 nm; antiviral, medical term used for any agent or drug altering virus integrity or process involved in viral infection disease; viral infection, process by which viruses invade the body through multiple pathways and multiply in susceptible host cells; viral pathogenesis, approach in biomedical research to understand the process by which a viral infection leads to disease, including mechanism of infection into the host (e.g., viral entry, viral replication) and factors that affect this mechanism (e.g., virus susceptibility to host defenses); membranotropism, the ability of an organism or an agent to interact with biological barriers; immunogenicity, capacity of an exogenous substance or material to trigger an immune response in humans and other animals or to induce a humoral and/or cellular immune responses; multifunctional platform, material modified with different functionalities to achieve a variety of combined treatments aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 recent advancements in two-dimensional nanomaterials for drug delivery metal-based nanomaterials in biomedical applications: antimicrobial activity and cytotoxicity aspects promises, facts and challenges for graphene in biomedical applications mediated tumor targeting using ultrasmall-hybrid nanoparticles: from animal to human with theranostic aguix nanoparticles. theranostics 2020 nanotherapeutic anti-influenza solutions: current knowledge and future challenges nanoparticles: alternatives against drug-resistant pathogenic microbes an overview of functional nanoparticles as novel emerging antiviral therapeutic agents high efficiency of functional carbon nanodots as entry inhibitors of herpes simplex virus type 1 interaction of silver nanoparticles with hiv-1 silver nanoparticles impair peste des petits ruminants virus replication silver nanoparticles inhibit vaccinia virus infection by preventing viral entry through a macropinocytosis-dependent mechanism metal nanoparticles: the protective nanoshield against virus infection inhibitory effects of silver nanoparticles against adenovirus type 3 in vitro silver nanoparticles inhibit hepatitis b virus replication inactivation of microbial infectiousness by silver nanoparticles-coated condom: a new approach to inhibit hiv-and hsv-transmitted infection mode of antiviral action of silver nanoparticles against hiv-1 antiviral activity of mycosynthesized silver nanoparticles against herpes simplex virus and human parainfluenza virus type 3 inhibitory effects of silver nanoparticles on h1n1 influenza a virus in vitro a preliminary assessment of silver nanoparticle inhibition of monkeypox virus plaque formation interaction of silver nanoparticles with tacaribe virus cytotoxicity and antiviral activity of electrochemical − synthesized silver nanoparticles against poliovirus organic-coated silver nanoparticles in biological and environmental conditions: fate, stability and toxicity silver nanoparticles inhibit replication of respiratory syncytial virus inhibition effect of silver nanoparticles on herpes simplex virus 2. gmr tannic acid modified silver nanoparticles show antiviral activity in herpes simplex virus type 2 infection polyphosphonium-oligochitosans decorated with nanosilver as new prospective inhibitors for common human enteric viruses curcumin prevents replication of respiratory syncytial virus and the epithelial responses to it in human nasal epithelial cells bioavailability of curcumin: problems and promises curcumin modified silver nanoparticles for highly efficient inhibition of respiratory syncytial virus infection silver nanoparticle based codelivery of oseltamivir to inhibit the activity of the h1n1 influenza virus through ros-mediated signaling pathways reversal of h1n1 influenza virus-induced apoptosis by silver nanoparticles functionalized with amantadine the inhibition of h1n1 influenza virus-induced apoptosis by silver nanoparticles functionalized with zanamivir delivery of vp1 sirna to inhibit the ev71 virus using functionalized silver nanoparticles through ros-mediated signaling pathways changes in in vitro susceptibility of influenza a h3n2 viruses to a neuraminidase inhibitor drug during evolution in the human host porous gold nanoparticles for attenuating infectivity of influenza a virus highly monodispersed gold nanoparticles synthesis and inhibition of herpes simplex virus infections receptor binding and membrane fusion in virus entry: the influenza hemagglutinin effective multi-strain inhibition of influenza virus by anionic gold nanoparticles self-assembled gold nanoparticles for in vitro inhibition of bovine viral diarrhea virus as surrogate model for hcv inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles inhibition of hiv fusion with multivalent gold nanoparticles targeting cell surface hiv-1 env protein to suppress infectious virus formation novel gold nanorod-based hr1 peptide inhibitor for middle east respiratory syndrome coronavirus inactivation of influenza a virus on copper versus stainless steel surfaces a novel anti-influenza copper oxide containing respiratory face mask inactivation and morphological changes of avian influenza virus by copper ions inactivation of hsv-2 by ascorbate-cu(ii) and its protecting evaluation in cf-1 mice against encephalitis mechanism of copper-mediated inactivation of herpes simplex virus in vitro effect of ascorbic acid on infectivity of herpesviruses and paramyxoviruses disinfection of human enteric viruses in water by copper and silver in combination with low levels of chlorine investigation of the antiviral properties of copper iodide nanoparticles against feline calicivirus novel antiviral characteristics of nanosized copper(i) iodide particles showing inactivation activity against 2009 pandemic h1n1 influenza virus antiviral activity of cuprous oxide nanoparticles against hepatitis c virus in vitro inhibition of herpes simplex virus type 1 by copper oxide nanoparticles topical zinc sulphate solution for treatment of viral warts topical zinc oxide vs. salicylic acid-lactic acid combination in the treatment of warts therapeutic and neutralizing effects of zinc oxide tetrapod structures against herpes simplex virus type-2 infection nanobionics of pharmacologically active derivatives of fullerene c 60 orexin activation counteracts decreases in nonexercise activity thermogenesis (neat) caused by high-fat diet biomedical engineers get to revisit an old friend glyconanoparticles for intervening in hiv gp120 carbohydrate-mediated processes fullerene sugar balls: a new class of biologically active fullerene synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of ebola virus infection synthesis of highly efficient multivalent disaccharide/[60]fullerene nanoballs for emergent viruses multiple modes of binding enhance the affinity of dc-sign for high mannose n-linked glycans found on viral glycoproteins antiviral activity of self-assembled glycodendro[60]fullerene monoadducts searching therapeutic strategy of new coronavirus pneumonia from angiotensin-converting enzyme 2: the target of covid-19 and sars-cov design of boronic acid-attributed carbon dots on inhibits hiv-1 entry glycyrrhizic-acid-based carbon dots with high antiviral activity by multisite inhibition mechanisms benzoxazine monomer derived carbon dots as a broad-spectrum agent to block viral infectivity szunerits, s. phenylboronic-acid-modified nanoparticles: potential antiviral therapeutics covalently synthesized graphene oxide-aptamer nanosheets for efficient visible-light photocatalysis of nucleic acids and proteins of viruses hypericin-loaded graphene oxide protects ducks against a novel duck reovirus graphene-based "hot plate" for the capture and destruction of the herpes simplex virus type 1 heparan sulfate proteoglycans and viral attachment: true receptors or adaptation bias? viruses herpes simplex virus type-1 attachment inhibition by functionalized graphene oxide highly efficient multivalent 2d nanosystems for inhibition of orthopoxvirus particles polyvalent 2d entry inhibitors for pseudorabies and african swine fever virus functionalized nanographene sheets with high antiviral activity through synergistic electrostatic and hydrophobic interactions inhibition of hsv-1 attachment, entry, and cell-to-cell spread by functionalized multivalent gold nanoparticles broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism potential toxicity of graphene to cell functions via disrupting protein−protein interactions graphene-vp40 interactions and potential disruption of the ebola virus matrix filaments antiviral activity of graphene oxide: how sharp edged structure and charge matter sars-cov-2 spike protein binds heparan sulfate in a length-and sequence-dependent manner. biorxiv use of silver nanoparticles increased inhibition of cell-associated hiv-1 infection by neutralizing antibodies developed against hiv-1 envelope proteins fullerene-biomolecule conjugates and their biomedicinal applications inhibition of the hiv-1 protease by fullerene derivatives: model building studies and experimental verification fullerene derivatives: an attractive tool for biological applications synthesis and anti-hiv properties of new water-soluble bis-functionalized[60]fullerene derivatives anti-hiv properties of cationic fullerene derivatives human immunodeficiency virus-reverse transcriptase inhibition and hepatitis c virus rna-dependent rna polymerase inhibition activities of fullerene derivatives chlorofullerene c60cl6: a precursor for straightforward preparation of highly water-soluble polycarboxylic fullerene derivatives active against hiv fullerene derivatives strongly inhibit hiv-1 replication by affecting virus maturation without impairing protease activity a new family of fullerene derivatives: fullerene-curcumin conjugates for biological and photovoltaic applications rna virus replication complexes synthesis and antiviral activity of highly water-soluble polycarboxylic derivatives of [70]fullerene carbon nanotubes as an advanced drug and gene delivery nanosystem synthesis and anti-hiv activity of carboxylated and drug-conjugated multi-walled carbon nanotubes grassi, g. β-cyclodextrin-grafted on multiwalled carbon nanotubes as versatile nanoplatform for entrapment of guanine-based drugs intracellular trafficking and therapeutic outcome of multiwalled carbon nanotubes modified with cyclodextrins and polyethylenimine carbon nanotube-based nanocarrier loaded with ribavirin against grass carp reovirus banning carbon nanotubes would be scientifically unjustified and damaging to innovation deoxyribozyme-loaded nano-graphene oxide for simultaneous sensing and silencing of the hepatitis c virus gene in liver cells oxidative stress during viral infection: a review an absence of reactive oxygen species improves the resolution of lung influenza infection polyethylene glycol-coated zinc oxide nanoparticle: an efficient nanoweapon to fight against herpes simplex virus type 1 inhibition of h1n1 influenza virus infection by zinc oxide nanoparticles: another emerging application of nanomedicine restriction of h1n1 influenza virus infection by selenium nanoparticles loaded with ribavirin via resisting caspase-3 apoptotic pathway anti-oxidant, wound healing, cytotoxic and anti-viral activities inhibition of h1n1 influenza virus by selenium nanoparticles loaded with zanamivir through p38 and jnk signaling pathways inhibitory activity of selenium nanoparticles functionalized with oseltamivir on h1n1 influenza virus inhibition of h1n1 influenza virus-induced apoptosis by functionalized selenium nanoparticles with amantadine through ros-mediated akt signaling pathways oxidative stress as key player in severe acute respiratory syndrome coronavirus (sars-cov) infection interferon-induced ifit proteins: their role in viral pathogenesis nano-sized zinc oxide and silver, but not titanium dioxide, induce innate and adaptive immunity and antiviral response in differentiated thp-1 cells antiviral and immunomodulatory activity of silver nanoparticles in experimental rsv infection silver nanoparticles impair retinoic acid-inducible gene i-mediated mitochondrial antiviral immunity by blocking the autophagic flux in lung epithelial cells antiviral activity of graphene oxide-silver nanocomposites by preventing viral entry and activation of the antiviral innate immune response carbon dots as inhibitors of virus by activation of type i interferon response gold nanorods inhibit respiratory syncytial virus by stimulating the innate immune response anti-betanodavirus activity of isoprinosine and improved efficacy using carbon nanotubes based drug delivery system in vitro effects of fullerene c60 and fullerene black on immuno-functions of macrophages cytotoxicity and tnf-α secretion in raw264.7 macrophages exposed to different fullerene derivatives nanovaccine: a novel approach in immunization an intra-vaginal zinc oxide tetrapod nanoparticles (zoten) and genital herpesvirus cocktail can provide a novel platform for live virus vaccine intravaginal zinc oxide tetrapod nanoparticles as novel immunoprotective agents against genital herpes fullerene c60 as a multifunctional system for drug and gene delivery role of nanotechnology in hiv/aids vaccine development functional nanomaterials can optimize the efficacy of vaccines morphologically virus-like fullerenol nanoparticles act as the dual-functional nanoadjuvant for hiv-1 nanoparticles promoting both humoral and cellular immune responses to hcv recombinant proteins dna vaccineshow far from clinical use? dendritic cell delivery of plasmid dna: applications for controlled genetic immunization potential suppressive effects of two c 60 fullerene derivatives on acquired immunity prospects for the use of spherical gold nanoparticles in immunization nanodiamond enhances immune responses in mice against recombinant ha/ h7n9 assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide consensus m2e peptide conjugated to gold nanoparticles confers protection against h1n1, h3n2 and h5n1 influenza a viruses gold nanoparticle-adjuvanted s protein induces a strong antigen-specific igg response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs nanoparticle-based b-cell targeting vaccines: tailoring of humoral immune responses by functionalization with different tlr-ligands hbs antigen and mannose loading on the surface of iron oxide nanoparticles in order to immuno-targeting: fabrication, characterization, cellular and humoral immunoassay induction of herpes simplex virus type 1 cell-to-cell spread inhibiting antibodies by a calcium phosphate nanoparticle-based vaccine progress in nanomedicine: approved and investigational nanodrugs gold nanoshell-localized photothermal ablation of prostate tumors in a clinical pilot device study engineered nanomaterials and type i allergic hypersensitivity reactions shiyuan peng − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france lucas jacquemin − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france andreś felipe andrade − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france complete contact information is available at: https://pubs.acs.org/10.1021/acsnano.0c04117 the authors declare no competing financial interest. the authors gratefully acknowledge the financial support from the eu graphene flagship project (no. 881603). this work was partly supported the agence nationale de la recherche (anr) through the labex project chemistry of complex systems (anr-10-labx-0026_csc). we wish to acknowledge the centre national de la recherche scientifique (cnrs) and the international center for frontier research in chemistry (icfrc). s.p. is indebted to the chinese scholarship council for supporting her ph.d. internship as a visiting ph.d. student. a.f.a. wish to thanks the eur csc graduate school (strasbourg, france) for supporting his master studies. key: cord-306701-hs9cfdsu authors: gona, philimon n.; gona, clara m.; ballout, suha; rao, sowmya r.; kimokoti, ruth; mapoma, chabila c.; mokdad, ali h. title: burden and changes in hiv/aids morbidity and mortality in southern africa development community countries, 1990–2017 date: 2020-06-05 journal: bmc public health doi: 10.1186/s12889-020-08988-9 sha: doc_id: 306701 cord_uid: hs9cfdsu background: the 16 southern africa development community (sadc) countries remain the epicentre of the hiv/aids epidemic with the largest number of people living with hiv/aids. anti-retroviral treatment (art) has improved survival and prevention of mother-to-child transmission (pmtct) of hiv, but the disease remains a serious cause of mortality. we conducted a descriptive epidemiological analysis of hiv/aids burden for the 16 sadc countries using secondary data from the global burden of diseases, injuries and risk factor (gbd) study. methods: the gbd study is a systematic, scientific effort by the institute for health metrics and evaluation (ihme) to quantify the comparative magnitude of health loss due to diseases, injuries, and risk factors by age, sex, and geographies for specific points in time. we analyzed the following outcomes: mortality, years of life lost (ylls), years lived with disability (ylds), and disability-adjusted life-years (dalys) due to hiv/aids for sadc. input data for gbd was extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service utilisation, disease notifications, and other sources. countryand cause-specific hiv/aids-related death rates were calculated using the cause of death ensemble model (codem) and spatiotemporal gaussian process regression (st-gpr). deaths were multiplied by standard life expectancy at each age-group to calculate ylls. cause-specific mortality was estimated using a bayesian meta-regression modelling tool, dismod-mr. prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases to calculate ylds. crude and age-adjusted rates per 100,000 population and changes between 1990 and 2017 were determined for each country. results: in 2017, hiv/aids caused 336,175 deaths overall in sadc countries, and more than 20 million dalys. this corresponds to a 3-fold increase from 113,631 deaths (6,915,170 dalys) in 1990. the five leading countries with the proportion of deaths attributable to hiv/aids in 2017 were botswana at the top with 28.7% (95% ui; 23.7–35.2), followed by south africa 28.5% (25.8–31.6), lesotho, 25.1% (21.2–30.4), eswatini 24.8% (21.3–28.6), and mozambique 24.2% (20.6–29.3). the five countries had relative attributable deaths that were at least 14 times greater than the global burden of 1.7% (1.6–1.8). similar patterns were observed with ylds, ylls, and dalys. comoros, seychelles and mauritius were on the lower end, with attributable proportions less than 1%, below the global proportion. conclusions: great progress in reducing hiv/aids burden has been achieved since the peak but more needs to be done. the post-2005 decline is attributed to pmtct of hiv, resources provided through the us president’s emergency plan for aids relief (pepfar), and behavioural change. the five countries with the highest burden of hiv/aids as measured by proportion of death attributed to hiv/aids and age-standardized mortaility rate were botswana, south africa, lesotho, eswatini, and mozambique. sadc countries should cooperate, work with donors, and embrace the un fast-track approach, which calls for frontloading investment from domestic or other sources to prevent and treat hiv/aids. robust tracking, testing, and early treatment are required, as well as refinement of individual treatment strategies for transient individuals in the region. we sought to determine hiv/aids related morbidity and mortality trends from 1990 to 2017. we assessed morbidity and mortality in the 16 sadc countries using a descriptive epidemiological analysis of hiv/aids burden based on secondary data from gbd study in 1990, 2005, 2010 , and 2017. we used secondary data from the gbd study. examining time trends of hiv/aids morbidity and hiv/aids mortality enable comparisons across the 16 countries to understand the changing burden facing the sadc population to support policy and programmatic development in the region. the united nations (un) fast-track or "90-90-90" approach to combatting the worldwide hiv/aids epidemic calls for 90% of people living with hiv knowing their status, 90% of people who know their status receiving treatment, and 90% of people on hiv/aids treatment having a suppressed viral load by 2020 [1, 2] . the second phase of the approach calls for upgrading the framework to 95-95-95 by 2030 [1] . hiv/aids-related deaths more than halved since the peak in 2004. in 2017, approximately 940,000 people died from the disease worldwide, compared to 1.9 million in 2004 and 1.4 million in 2010 [3] . in 2017, approximately 1.8 million new hiv infections occurred, compared to 3.4 million in 1996 [3] . a better understanding of the long-term trends in hiv/aids-related morbidity and mortality is needed to enable continued improvements on the impact of ongoing hiv/aids treatment programs [4, 5] . sub-saharan africa (ssa), with more than 1 billion people, is the epicenter of the hiv/aids pandemic. the 16 southern african development community (sadc) countries comprise ground-zero of the pandemic, with prevalence in eight countries exceeding 10% in 2015 [6] . while incidence has progressively declined since the mid-1990s, hiv/aids morbidity and mortality nonetheless continued to increase (see fig. 1 right panel) , reaching a peak in 2005 [3, 7] . between 2000 and 2015 incidence of hiv declined by 35% worldwide. there was an estimated 34% fewer hiv/aids-related deaths in ssa in 2014 versus 2000 [8] . despite the gains sadc countries have the highest morbidity of hiv/aids, with approximately 26 million people living with the disease in 2015 [9] . of all people living with hiv/aids worldwide at the peak of the epidemic 2009, 34% resided in ten sadc countries, making hiv/aids the leading cause of death [6] . hiv/ aids-related mortality in southern ssa increased from being ranked 6th in 1990 to 1st in 2017; in eastern ssa countries the ranking increased from 7th to 3rd [3, 9] . .while great progress has been achieved since 2005 to 2013, with a decline in, the number of hiv/aids-related deaths globally by 39%, sadc countries accounted for nearly 3 in 4 of all people dying from hiv/aids-related causes in 2013 [10] . hiv/aids, therefore, remains a massive public health threat in the region. timely and robust evidence on mortality and trends are essential to informing policy and goal setting, program evaluation, and decision-making. such assessment is an essential starting point for informed health policy debate to measure progress in achieving the 2030 united nations (un) health-related strategic development goals (sdgs) [3, 11, 12] and un fast-track approaches "90-90-90" and "95-95-95". the gbd is a systematic, scientific effort by the institute for health metrics and evaluation (ihme) to quantify the comparative magnitude of health loss due to diseases, injuries, and risk factors by age, sex, and geographies for specific points in time. the gbd study estimates country-specific incidence, prevalence, mortality, years of life lost (ylls), years lived with disability (ylds), and disability-adjusted life-years (dalys) due to diseases such as hiv/aids. input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service utilization, disease notifications, and other sources. cause-specific crude and age-standardized death rates per 100,000 population were obtained from the cause of death ensemble model (codem) and spatiotemporal gaussian process regression (st-gpr). deaths were multiplied by standard life expectancy at each 5-year age-group to calculate ylls. cause-specific mortality was estimated using a bayesian meta-regression modelling tool, dismod-mr. prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases to calculate ylds [4]. ylls were calculated using the product of age-specific life expectancy from the reference life table used in the gbd study. ylds were calculated as a product of the prevalence of hiv/aids and the disability weights used to quantify health levels associated hiv/aids [13, 14] . case definition for hiv/aids used in the gbd 2017 and comprehensive details for the methodology and modeling processes for hiv/aids are provided in supplementary appendix 1, page 47 www.thelancet.com/ journals/lancet/article/piis0140-6736(18)32203-7/full-text#seccestitle540 [1, 4] . all gbd estimates adhere to the 14 guidelines on accurate and transparent health estimate reporting (gather). gather recommends making available statistical code, details on why some sources are used and others are not, and how primary data are adjusted. methodology underlying distinct differences in estimation among unaids, who and ihme are provided [3] . .the hiv/aids-related outcomes were assessed for each country in sadc, a regional economic community whose aim is to increase regional socioeconomic integration to achieve greater economic growth and poverty alleviation. levels of development and poverty, social service delivery, and economic performance vary greatly. nine of the 16 countries were classified in 2017 as either low or low-middle income. only mauritius seychelles and south africa were classified as high-middle income. the ability for each country to respond to the high burden of hiv/aids also varies considerably. sadc aims to strengthen economic cooperation and integration, providing for cross-border investment and trade, and free movement of goods and services across borders [3, 9] . the gbd results tool was used to extract sex-pooled age-standardized morbidity and mortality rates per 100, 000 population for years 1990, 2005, 2010, and 2017. (available at http://ghdx.healthdata.org/gbd-results-tool). to facilitate comparison of hiv/aids outcomes of morbidity and mortality across countries, time, age-groups, and sex, the institute for health metrics and evaluation (ihme) improved previously established metrics like prevalence and incidence. how long do people live with hiv/aids is assessed using hiv/aids-specific mortality rates and hiv/aids-specific years ylls. what causes people to get sick is assessed hiv/aids-specific ylds which reflect the amount of time in a year that people live with a condition accounting for the severity of that condition. adding together ylls and ylds yields dalys. to facilitate comparisons across sadc countries and eliminate potential confounding by age, outcomes are presented as age-standardized rates per 100,000 population i.e., the average of the age-specific hiv/aids rates weighted by country-specific proportions of a standard population in the corresponding age groups [2, 4, 5, 15] . expected rates (e) were determined using a linear equation with the country's socio-demographic index (sdi) in 2017 used as a linear predictor [16] . the sdi, which ranges from 0 to 1 is a summary measure of where a location is on the spectrum of socio-demographic development. the index is calculated from the geometric mean of three rescaled components: total fertility rate of women under 25 years of age, lag-distributed income per capita, and average educational attainment in the population > 15 years., we calculated the observed-to-expected (o/e) rate ratio. uncertainty for each outcome was quantified using uncertainty intervals (uis) based on 1000 bootstrap draws from the posterior distribution [16, 17] . uis were determined by the 25th and 975th ordered values of the posterior distribution of the 1000 draws, and point estimates were computed from the mean of the draws. changes over time were considered statistically significant when the 95% ui of the percentage change did not cross zero [3] . gbd uses the joint united nations program on hiv and aids (unaids) estimates as inputs in their modeling ensemble [8, 18] . for example, pediatric hiv/aids mortality estimates in gbd were produced with the cd4-countspecific mortality and progression parameters developed by unaids [19] . each iteration of gbd re-analyses the entire time series by use of newly available data sources from across all estimation years and continually improved methods. new data and modelling approaches effectively improve model validity and decrease uncertainty from various sources with the consequence that estimates for a given cause, location, and year might differ between gbd iterations and unaids. statistical, analytical, processing, and estimation code used to generate the gbd results are available on their website: http://ghdx.healthdata.org hiv/aids morbidity and mortality remain major public health problems in sadc countries. nearly all new infections in 2016 worldwide occurred in just 12 countries, four sadc countries, i.e., mozambique, zimbabwe, zambia, and tanzania. between 2000 and 2017, hiv incidence worldwide declined by 39%, and hiv/aids mortality declined by 38%, but corresponding declines in sadc countries during the same period were 49 and 55%, respectively. the five leading countries with the proportion deaths attributable to hiv/aids in 2017 were botswana at the top with 28 the five leading countries with the greatest agestandardized mortality rate per 100,000 population in 2017 were lesotho 336.6 (296.7-395.8) at the top, followed by table 2 ). these five countries had agestandardized mortality exceeding 15-fold the global mortality rate of 12.1(11.5-12.9). while the 95% uis for eswatini, south africa, mozambique, botswana overlap, there is no substantial difference in the rates for these countries, but the rate for lesotho is substantially higher than that for the other four since the 95% uis do not overlap. seychelles, mauritius and comoros had mortality rate lower than the global rate per 100,000. heterogeneity in rates between countries in 2017 was high, with rate ratio between comoros, the country with the lowest age-standardized mortality rate (0.3(0.0-2.0)), and lesotho, the country with the highest age-standardized mortality rate (336.6((296.7-395.8) nearly 1300-fold higher. looking back in time, in 1990, 2005, and 2010, botswana and eswatini had consistently the highest age-standardized mortality rates. zimbabwe dropped out of the top 5 in 2017 (table 2) . (table 5) . the map on fig. 2 shows the annual percent changes in hiv-associated mortality for males and females from for each metric, the sdi, a measure of where the country is on the spectrum of development based on income, (table 3) , ylds (table 4 ) and dalys (table 5) . to gain better perspective on the ylls percentage for sadc countries, the corresponding ylls percentages were 75.6 and 66.7% in the 34 member states of the organisation for economic co-operation and development (oecd) and the 28 member states of the european union (eu) (30-40% deficit), respectively. figure 3 displays the ratio of ylls to ylds as proportions of dalys attributable to hiv/aids in sadc countries, worldwide, oecd, and eu 2017. more dramatically from fig. 3 , and highlighting the heterogeneity of the changes in the burden among rich and poor countries, the levels of ylls proportions of dalys due to hiv/ aids in sadc countries in 2017 were equal to the 1990 levels in oecd and eu countries, several years before the advent and widespread use of highly active antiretroviral treatment (haart). botswana, south africa, lesotho, eswatini, mozambique, and namibia all had increasing (worsening) burden, with aroc ranging from + 1.2% in botswana to + 26.3% in madagascar, suggesting that in these countries, the burden of hiv/aids has not abated, but has worsened compared to the levels in 1990. (fig. 4 ). we analyzed mortality and morbidity due to hiv/aids in 16 sadc countries between 1990 and 2017 using estimates from the gbd study. the five leading countries with the proportion deaths attributable to hiv/aids in 2017 were botswana, south africa, lesotho, eswatini, and mozambique, also had the highest age-standardized mortality, yll, yld rates. botswana, eswatini, and lesotho were among the top five countries with highest mortality and morbidity in 1990, 2005, 2010, and 2017. comoros, seychelles, mauritius and madagascar had the lowest rates in 2017. double-digit increasing slopes in aroc (%) observed in 10 countries is worrisome. indicating significant risk that the progress made in slowing the hiv epidemic could be reversed without a continued robust investment in health. while the negative aroc (%) in four countries, drc, tanzania, zimbabwe, and zambia is encouraging, the aroc (%) observed in madagascar, south africa, and angola are concerning. while most sadc countries, except for comoros, seychelles, madagascar and mauritius had morbidity and mortality rates in 2017 greater than the global rate, there was substantial heterogeneity among the countries. the disparity in rates, measured using rate ratios, between the lowest rates observed in comoros, and highest rates observed in lesotho exceeded 1300-fold in 2017 suggesting that sadc countries are on very diverse trajectories regarding the burden of hiv/aids. while art has extended life for most people living with hiv, it is sobering that two-thirds of hiv/aids-related deaths in lmics occurred in individuals not on art [20] . loss to follow-up from care and defaulting, especially for first-line treatment, significantly affect survivability. ideally, when one defaults on the first line, the next step would be initiation into the second line, which because of cost, is out of reach for most rural communities in the sadc, thereby compromising survival of patients [21, 22] . our most poignant finding is that the ratio of hiv/ aids-related ylls/dalys of 93.6% for oecds in 1990 (fig. 3 , first bar) is nearly equivalent but smaller, at 92.0%, than the hiv/aids-related ratio of ylls/dalys in sadc countries nearly three decades later in 2017 (fig. 4, sadc bar) [23]. it is astounding that the 2017 ratio for sadc was lower than the ratio experienced in oecd member states 30 years prior, a period during which there was no widespread use of art or secondary prophylaxis against opportunistic infections? despite the advent of potent art, sadc still lags by almost 30 years demonstrating the uneven progress that has been achieved in different regions. notably, unaids endorsed the concept of "undetectable" = "untransmittable" based on strong scientific evidence that hiv is not sexually transmitted from people living with hiv/aids to their hiv-negative partner if the partner hiv-positive continues to take effective art and is virally suppressed [24] [25] [26] [27] . having many infected people not on treatment increases the risks for infection to the general population. ensuring those who are infected are virally suppressed is a powerful tool to improve survival for those infected and prevent new infections. it has been argued that hiv/aids has remained a massive public health threat, but global financing has plateaued, domestic health spending has stayed low among high-burden countries, and the disease incidence has not declined as quickly in younger as in older populations [20] . eswatini, botswana, and lesotho had among the highest mortality rates in the world before the downward shift of the world epidemic since 2005, suggesting that the extremely high rates during the peak in 2005 continue to drive the epidemic decades later. the 2017 mortality rates in eswatini and lesotho remain among the highest in the world, exceeding 200 more than a decade after the global decline. our study showed hiv/ aids caused more ylls than ylds at all times, underscoring that in sadc countries survival following hiv infection is very short. it is desirable to decrease the proportion of ylls contributing to dalys so that patients live longer. one likely explanation of the relatively small proportion of ylds to dalys in sadc is people with hiv/aids present late for care after the onset of opportunistic infections, underscoring the need for early and periodic testing for hiv while their health is still intact. strategies should be developed to ensure that more people who are unaware of their hiv status are tested and if necessary linked to care immediately. health systems in the sadc region need improvement to help lengthen the lives of individuals with the disease and convert the burden of hiv/aids into mostly ylds rather than ylls. premature mortality, measured using ylls, is indicative of failure of healthcare management of hiv/aids cases in the region to convert the burden of hiv/aids into mostly ylds, therefore extending the lives of the hiv/aids-affected individuals. countries with better healthcare access and quality index have the potential to reduce future burden [28] . we detected huge disparities between the observed mortality compared to that expected based on the country's level of sdi. accordingly, sadc countries are relatively underperforming with respect to the expected reduction in disease burden compared to other countries of similar sdi. at current rates of decline in the burden of hiv/aids, sadc countries might not meet the sdgs target for the disease and are far from the unaids goal of ending aids by 2030 [11] . our study suggests that sadc countries have made some progress, but hiv/aids mortality and morbidity rates are still unacceptably high. while the global mortality and morbidity rates in 2017 were approximately doubled compared to 1990 levels, sadc countries such as south africa had 2017 rate that was 114-times, angola 40-times, and mozambique 10-times the 1990 rate, increases pointing to a cascade of orders of magnitude. in the sadc region, most people with hiv/aids are reliant on medications provided by sources outside of the region. individuals between 15 and 49 years of age, the peak years of economic production, are the most affected by the epidemic [10] . our findings, therefore, imply that sadc countries are economically and socially vulnerable. the number of people who do not know their hiv status is of concern. a pregnant woman with untreated hiv has up to a 45% chance of transmitting the virus to the baby. if the woman and their baby receive antiretroviral treatment, that risk drops to 1% [10, 29] . for hiv infection to become a rare occurrence, sadc countries should coordinate efforts to reduce new hiv infections, increasing access to hiv/aids treatment and care, particularly to religious minorities that discourage contact of their members with the healthcare system [30] [31] [32] . government health spending is a primary source of funding in the health sector across the world, but in ssa, only about a third of all health spending is sourced from the government [20] . in southern africa, public funding for healthcare grew by only 4.5% each year between 1995 and 2015 [33] . keeping the coverage of aids-related services at 2016 levels would lead to an increase in the burden of hiv/aids in almost all sadc countries. art in sadc countries is available through "cost-free" programs funded by the global fund, pepfar, and corresponding governments [24, 25] . the heavily donor-funded art programs have been a success story, but there is uncertainty about their long-term sustainability [34] . pepfar was the largest donor, providing $4.9 billion in 2016, followed by the global fund: with contributions from the uk ($645.6 million), france ($242.4 million), the netherlands ($214.2 million), and germany ($182.0 million). the us government recently proposed a 6% reduction in pepfar and global fund assistance [1, 34] . any reduction in funding could have significant impact on hiv prevention and treatment, as most of the countries are dependent on these organizations for most of their hiv/aids programming budgets [12] . sadc countries should try to ramp up their domestic financing programs in order to reduce dependency on these other organizations/countries and be able to sustain the programs. the un fast-track framework advocates for frontloading resources required for full implementation of basic programs by investing $35.6 billion in lmics in 2020. by 2030, the investment amount would drop to $32.8 billion, in the process averting nearly 28 million new hiv infections and 21 million aids-related deaths [1, 35, 36] . for fast-track goals to be successful in marking a transition toward ending the hiv/aids epidemic, sustained and intensified regional commitment by sadc countries together with the un and the african union over the next decade will be required. our study is subject to a few previously described limitations regarding the estimation of hiv/aids burden [3, 4] . firstly, our study estimated mortality with hiv/aids as the underlying cause of death without accounting for deaths from other non-communicable causes among people with hiv/aids. secondly, national-level estimates may obscure substantial heterogeneity at sub-national level. thirdly, we had no access to traditional risk factors that influence transmission of hiv/aids such as presence of other sexually transmitted infections, stage of infection, male circumcision, and use of art and pre-exposure prophylaxis (prep), therefore we could not explore the importance of these factors. fourthly, sdi was used as a linear predictor to estimate expected rates in 2017, yet the sdi does not always exhibit a linear association with all causes of death including hiv/aids-related deaths. fifthly, time lags in available data, absence of data from specific regions, age groups, or time periods, or unreliability in the data that are available or for geographical areas with the highest hiv/aids-related mortality can affect the precision of estimations. sixth, because gbd results for hiv/aids are a combination of data and estimation, lags in data reporting mean that estimates for the most recent years rely more on the modelling process, as do estimates for locations with low levels of data completeness. despite these limitations, this study gives an insight on the disparities in morbidity and mortality within the sadc region. efforts are underway to collect data at local levels to further reveal the granularity of estimates to reveal nuances hidden by aggregated data. this higher resolution will aid governments in focusing their efforts in regions with higher burden. for better resolution and to illuminate geographic inequality in hiv/aids burden, future analyses should use spatially resolved data at a 5 × 5-kilometer grid level. community-level estimates can help identify where interventions and health policies will have the greatest impact by targeting the most vulnerable individuals. in the absence of a preventive vaccine, at current rates of decline in the burden of hiv/aids, sadc countries will not meet the un's health-related sdgs by 2030 or achieve the unaids goal of ending aids by 2030 [11, 37] . our study should help to inform decisions about policy and programs aiming to improve resource allocation and track accountability. sadc countries need to continue to ensure access and adherence to art and strengthen behavioral interventions to prevent new infections. early testing should be encouraged, perhaps rewarded, in order to link individuals testing positive to care early, when their immune systems are still strong, potentially increasing ylds while reducing ylls and preventing new infections. eliminating hiv/aids will take sustained coordination across multiple health and social sectors in the region, along with adequate funding and supportive public policies. governments in sadc countries should plan strategically as a block in efforts to eliminate the hiv/aids epidemic. the double-digit increasing slopes in aroc (%) observed in 10 countries indicate significant risk that the progress made in slowing the hiv epidemic could be reversed without a continued robust investment in health. it is unacceptable for governments to outsource the huge financial undertaking to outside forces. governments should take responsibility for their people by making hiv/aids funding a priority. hiv/aids programming, including funding for art manufacture, procurement, and distribution across the region, should comprise a significant proportion of the national budgets. sadc should expand its mission to include increasing domestic funding, collaborative licensing, and procurement and manufacture of art. rather than importing hiv/aids medications from abroad, local manufacturing and distribution of art would guarantee seamless supply of the medications for all people in need. for this strategy to be effective, the sadc countries should gaurantee access to medications for people in transit and and make sure they receive care upon return. the coronavirus disease 2019 (covid-19) pandemic disrupted and put the world on edge forcing governments to implement, social distancing, and community containment, city lockdowns or traffic controls, measures which disrupted the continuum of hiv/aids care because of restricted hospital visits, sadc government and community partners should collaborate to sustain hiv service provision for people living with hiv/aids to avoid disruption of routine hiv services. strategies such as dispensing art in 3-6-month doses to meet the needs of people living with hiv and reduce facility visits would reduce disruption [38] . while our study looked at epidemiological data on the burden of hiv/aids in the sadc countries, gbd data does not address issues surrounding the economic impact of hiv, such as healthcare and occupational perspectives. healthcare costs of hiv/aids and the occupational situation of people living with hiv/aids need to be discussed. in high-income countries, the trends indicate that an increasing proportion of the intermediate-age hiv-positive population will age prematurely, experiencing high rates of cardiovascular disease events, cancers, and neurocognitive impairment [39] and becoming frailer. regarding occupational perspectives, the decreased life expectancy of hivpositive persons may prompt this population to retire early from the labor market [40, 41] . strategies should be developed to alleviate poverty, improve economic and financial opportunities for people with hiv/aids, and improve infrastructures to empower individuals with hiv/aids to continue with productive economic activity. gbd results are detailed and carefully researched using transparent methods but they are estimated and rely on many assumptions. to minimize the need for extrapolation, more primary data are needed from all countries where data accuracy and reliability can be poor. in nearly four decades the hiv/aids epidemic has changed dramatically as the virus has rapidly spread to all geographic regions. globally, significant progress has been made in improving diagnosis and access to treatment. however, if hiv/aids-related mortality continues at current level in sadc, none of the countries will reach the sdg target of ending the epidemic by 2030. the downward trajectories observed elsewhere have been sluggish in the sadc regions. there is a need to strengthen existing strategies and create new ones to help end the disparity and help keep hiv/aids on a steeper downward trajectory. education about hiv transmission and prevention and testing and immediate treatment of individuals who test positive, should be implemented and maintained and funded. health ministries should increase efforts to ensure that accessible, affordable and stigma-free testing and treatment, including better access to viral load testing, is available to all people living with hiv/aids [24]. additionally, pharmaceutical interventions like pre-and post-exposure prophylaxis which have changed prevention and treatment protocols for hiv/aids in other regions have not been fully implemented in sadc. sadc countries are facing challenges in meeting hiv/aids-related sdg targets; however, opportunity remains to take actions to accelerate progress by adopting regional multi-sectoral commitments and investments to help attain the sdgs for hiv by 2030, or achieve the unaids goal of ending aids by 2030 [11, 37] . regional coordination among sadc countries will further promote the attainment of sdgs. who validates elimination of mother-to-child transmission of hiv and syphilis in cuba implementing primary healthcare-based pmtct interventions: operational perspectives from muhima cohort analysis (rwanda) global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study disease and injury incidence and prevalence collaborators. global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study global, regional, and national disability-adjusted life-years (dalys) for 359 diseases and injuries and healthy life expectancy (hale) for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study global, regional, and national incidence, prevalence, and mortality of hiv, 1980-2017, and forecasts to 2030, for 195 countries and territories: a systematic analysis for the global burden of diseases, injuries, and risk factors study cause of death collaborators. global, regional, and national agesex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the global burden of disease study institute for health metrics and evaluation (ihme) guidelines for accurate and transparent health estimates reporting: the gather statement metric partnerships: global burden of disease estimates within the world bank, the world health organization and the institute for health metrics and evaluation hiv development assistance and adult mortality in africa global burden of disease collaborative network. global burden of disease study 2017 (gbd 2017) disability weights. seattle: institute for health metrics and evaluation (ihme) disability weights for the global burden of disease 2013 study ): the international bank for reconstruction and development /the world bank mortality collaborators. global, regional, and national agesex-specific mortality and life expectancy, 1950-2017: a systematic analysis for the global burden of disease study an integrative meta-regression framework for descriptive epidemiology unaids reference group on estimates modelling and projections. improved methods and assumptions for estimation of the hiv/aids epidemic and its impact: recommendations of the unaids reference group on estimates, modelling and projections producing hiv estimates: from global advocacy to country planning and impact measurement, global health action survival of people on antiretroviral treatment in zambia: a retrospective cohort analysis of hiv clients on art southern african development community. sadc hiv and aids strategic framework global, regional, and national incidence and mortality for hiv, tuberculosis, and malaria during 1990-2013: a systematic analysis for the global burden of disease study institute for health metrics and evaluation (ihme) what is required to end the aids epidemic as a public health threat by 2030? the cost and impact of the fast-track approach pepfar funding and reduction in hiv infection rates in 12 focus sub-saharan african countries: a quantitative analysis side effects' are 'central effects' that challenge retention in hiv treatment programs in six sub-saharan african countries: a multicountry qualitative study prevention of hiv-1 infection with early antiretroviral therapy sexual activity without condoms and risk of hiv transmission in sero-different couples when the hivpositive partner is using suppressive antiretroviral therapy hiv transmission in male serodiscordant couples in australia, thailand and brazil institute for health metrics and evaluation. financing global health 2017: funding universal health coverage and the unfinished hiv/aids agenda. seattle: institute for health metrics and evaluation healthcare access and quality collaborators. health access and quality based o mortality from causes amenable to personal healthcare in 195 countries and territories, 1990-2015: a novel analysis from the global burden of disease study what will become of me if they take this away? unaids: fast-track: ending the aids epidemic by 2030 donor government funding for hiv in low-and middle-income countries in 2016 undetectable=untransmittable-public health and hiv viral load suppression tang w maintaining hiv care during the covid-19 pandemic lancet hiv 2020 published online a systematic review and meta-analysis of studies evaluating the performance and operational characteristics of dual point-of-care tests for hiv and syphilis trends and drivers of government health spending in sub-saharan africa economic impact of hiv in the highly active antiretroviral therapy era -reflections looking forward publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations none of the other authors has competing financial interests. all authors report no conflicts.authors' contributions png conceptualized the study, had access to raw data, analyzed data, wrote the first draft of the manuscript, and interpreted the data. cmg, sb, ccm, and rk, contributed to the clinical, epidemiological, policy implications sections, and strengthened the intellectual content and recommendations of the study. srr co-wrote the first draft of the paper strengthened the intellectual content of the study. ahm supervised the development of the study, critiqued earlier drafts, and shaped the overall interpretation in relation to previous related studies. the authors read and approved the final manuscript. bill & melinda gates foundation. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit the manuscript. data that support the findings of this study are available at: table 1 : http://ghdx.healthdata.org/gbd-results-tool?params=gbd-api-2017permalink/088010c34dc209c6b1667f763c2626f2 tables 2, 3, 4, 5: http://ghdx.healthdata.org/gbd-results-tool?params=gbdapi-2017-permalink/02aea83bf5cff055c91deb613a168a4b, or by request from the authors.ethics approval and consent to participate not applicable. not applicable. none.author details key: cord-277818-8w15dz20 authors: jaichenco, andre l.; lima, luciana cavalcanti title: infectious disease considerations for the operating room date: 2018-02-09 journal: a practice of anesthesia for infants and children doi: 10.1016/b978-0-323-42974-0.00050-1 sha: doc_id: 277818 cord_uid: 8w15dz20 the risk of infection transmission by anesthesia providers in their work area environment is reviewed. the dynamics of transmission and the strategies for preventing infection transmission in health care institutions are discussed. anesthesiologists have long been patient safety advocates and have taken on increasing responsibility for preventing health care–associated infections. anesthesia providers practice in a nonsterile environment within the operating room and have an impact on bacterial transmission and infection rates. understanding the characteristics of transmission elements provides the practicing anesthesiologist with methods to protect susceptible patients and themselves to avoid spreading infection. it is vital to have in place proper systems to remove contaminated air to minimize the risk of airborne pathogens being transmitted by children. preoperative patient skin and other bacterial reservoir decontamination and hand hygiene by anesthesia providers reduces contamination of the work area and iv access ports. hand hygiene is a well-known and effective solution to the problem of bacterial transmission within and across patients and is considered the most important and cost-effective individual intervention in the prevention of health care–associated infections in children and health care providers compliance with the current “5 moments” world health organization guidelines could make a major inroad into reducing provider hand and workspace contamination. surgical antimicrobial prophylaxis is an essential tool to reduce the risk of postoperative infections, and the anesthesia team plays a central role in ensuring the proper timing of drug administration. protocols, although effective, require continuous feedback and revision. the presence of a susceptible host is an important element in the chain of infection that paradoxically results from advances in current medical therapies and technology (e.g., children undergoing organ transplantation or chemotherapy, or extremely premature neonates) and the presence of children with diseases that compromise their immune systems (e.g., aids, tuberculosis, malnutrition, or burns). the organism may enter the host through the skin, mucous membranes, lungs, gastrointestinal tract, genitourinary tract, or the bloodstream via iv solutions, after laryngoscopy, or from surgical wounds. organisms may also infect the individual because of work accidents with cutting or piercing devices. the development of the rabbit hole that is the perioperative environment is not well understood by the majority of our general pediatric colleagues. similarly, the rabbit hole of the primary care clinic or the pediatric inpatient ward is not well understood by the majority of our anesthesiology colleagues. a pediatric patient may repeatedly enter the rabbit hole over the course of a hospital admission, a journey fraught with dangers of airway mishaps, respiratory and/or cardiac arrests, hemorrhage, profound anxiety and stress experienced by the young patient and his or her family, as well as infection risks. 1 anesthesiologists have long been patient safety advocates. it is not surprising that anesthesia providers in the 21st century have taken on increasing responsibility for preventing health careassociated infections (hais), including surgical site infections (ssis). anesthesia providers practice in a nonsterile environment within the operating room (or) and frequently contact areas of the patient known to have a high rate of contamination such as the axilla, nares, and pharynx. there are two recognized but poorly implemented interventions: preoperative patient skin and other bacterial reservoir decontamination and hand hygiene by anesthesia providers. 2 anesthesia providers have an impact on bacterial transmission and infection rates. specifically, anesthesiologists are known to contaminate their work environment within the or. contamination of the work environment includes contamination of intravenous (iv) access ports. without encouragement, anesthesiologists perform hand hygiene less frequently than once per hour during a case, but with reminders, the rate of hand hygiene is more frequent. improved hand hygiene reduces contamination of the work area and iv access ports from 32% to 8%, which in turn significantly reduces hais. 3, 4 the transmission of infection depends on the presence of three interconnected elements: a causative agent, a source, and a mode of transmission ( fig. 50.1 ). understanding the characteristics of each element provides the practicing anesthesiologist with methods to protect susceptible patients and themselves to avoid spreading infection. there has always been concern about the transmission of infectious agents to the patient from the anesthesiologist and vice versa. 5 in addition, there are many sites within the hospital environment where moist or desiccated organic material with the membranes. droplets remain suspended for only a short duration and distance from the source, but this may be affected by temperature, humidity, force of expulsion, and air currents. larger particle sizes contact the mucosa of the upper airway, whereas aerosols are capable of penetrating into the lower respiratory tract. infectious agents vary in their affinity for receptors in different regions of the respiratory tract. 9,10 when a person coughs, the exhaled air may reach a speed of up to 965 km/hour (600 mph). 11 however, because the droplets are relatively large, they tend to descend quickly and remain suspended in the air for a very brief period, thus obviating the need for special handling procedures for the or air. examples of droplet-borne diseases include influenza, respiratory syncytial virus (rsv), severe acute respiratory syndrome (sars), diphtheria, haemophilus influenzae, neisseria meningitidis, mumps, pertussis, rhinovirus, rubella, and ebola. droplet precautions include communication of infectious risk infection is influenced by the host defense mechanisms that may be classified as either nonspecific or specific: ■ nonspecific defense mechanisms include the skin, mucous membranes, secretions, excretions, enzymes, inflammatory responses, genetic factors, hormonal responses, nutritional status, behavior patterns, and the presence of other diseases. ■ specific defense mechanisms or immunity may occur because of exposure to an infectious agent (antibody formation) or through placental transfer of antibodies; artificial defenses may be acquired through vaccines, toxoids, or exogenously administered immunoglobulins. microorganisms are transmitted in the hospital environment through a number of different routes; the same microorganism may also be transmitted via more than one route. in the or, the three main routes of transmission are through the air and by direct and indirect contact. airborne infections that may infect susceptible hosts are transmitted via two mechanisms: droplets and droplet nuclei. droplet contamination is considered a direct transmission of organisms because there is a direct transfer of microorganisms from the colonized or infected person to the host. this generally occurs with particles whose diameters are greater than 5 µm that are expelled from an individual's mouth or nose, mainly during sneezing, coughing, talking, or during procedures such as suction, laryngoscopy, and bronchoscopy ( fig. 50.2 ). transmission occurs when the microorganism-containing droplets, expelled or shed by the infected person (source), are propelled a short distance (usually not exceeding 60 cm or about 2 feet through the air) and deposited on the host's conjunctivae or oral or nasal mucous droplet nuclei result from the evaporation of droplets while suspended in the air. unlike droplets, the nuclei have an outer layer of desiccated organic material and a very small diameter (1-5 µm) and remain suspended in air indefinitely. the microorganisms contained within these nuclei may be spread by air drafts over great distances, depending on the environmental conditions (dry and cold atmosphere, with limited or no exposure to sunlight favoring the spread). 12 in contrast to droplets, which are deposited on mucous membranes, droplet nuclei may enter the susceptible host by inhalation; examples of droplet nuclei-borne diseases include tuberculosis, varicella, and measles, zoster, smallpox, sars, and middle eastern respiratory syndrome. 10 direct and indirect contacts are the most significant and frequent methods of hospital infection transmission. this type of disease transmission involves direct physical contact between two individuals. the physical transfer of microorganisms from an infected or colonized person to a susceptible host may occur from child to health care provider or from health care provider to child during professional practice (e.g., venous cannulation, laryngoscopy, burn care, or suction of secretions). health care providers working in the or may be exposed to skin contamination by body fluids. this is an issue of grave concern because of the potential exposure of health care providers to patients with unrecognized infections, especially hepatitis b virus (hbv), hepatitis c virus (hcv), and human immunodeficiency virus (hiv). hepatitis b is a highly infectious virus that requires a small amount of blood (10 −7 -10 −9 ml) to transmit the disease. the incidence of skin contamination of anesthesiologists and related personnel by blood and saliva is substantial. one study examined 270 anesthetic procedures during 7 consecutive days. the blood of 35 patients (14%) contaminated the skin of 65 anesthesiologists in 46 incidents. of these contamination events, 28 (61%) occurred during venous cannulation. of anesthesiologists who had been contaminated by blood, 5 of 65 (8%) had cuts in the skin of their hands. 13 the importance of this observation is that seroconversion of health care providers has been reported after skin contamination by infected blood from hiv carriers 14 and hbv infection after blood splashing into health care workers' (hcws') eyes. 15 scabies, pediculosis, and herpes simplex are among the diseases most frequently transmitted by direct contact. [16] [17] [18] [19] [20] [21] [22] [23] meticulous hand washing before and after every patient contact and routine use of barriers such as gloves and eye protection are essential basic methods for protecting ourselves even during routine procedures such as starting an iv line or performing laryngoscopy. 4 indirect contact involves the transmission of microorganisms from a source (animate or inanimate) to a susceptible host by means of a vehicle (e.g., an intermediary object) contaminated by body fluids. tables 50.2 and 50. 3 provide examples of diseases associated with bodily fluids to which hcws may be exposed. the vehicle for transmission may be the hands of a health care provider who is not wearing gloves or a provider who fails to wash his or her hands after providing care to a child. 3, [24] [25] [26] this type of contact can also come from health care providers who touch (with or without gloves) contaminated monitoring or other patient care devices (e.g., blood pressure cuffs, stethoscopes, electrocardiographic cables, or ventilation systems [respirators, corrugated tubes, y-pieces, valves]) that are used without proper cleaning or disinfection between each use. [27] [28] [29] knowledge about the transmission of the spread of bacteria from patients to hcws' hands and to the hospital environment ( fig. 50 .3) has driven many interventions that have reduced patient risks for developing hais. 30 disease transmitted blood hbv, hiv, hcv, cmv, ebv, nanbh seminal fluid hiv, hbv, cmv vaginal discharge hiv, hbv, cmv saliva and sputum hsv, tb, cmv, respiratory diseases cerebrospinal fluid encephalopathic organisms (see table 50 30 characterization of the transmission dynamics of frequently encountered gram-negative bacteria in the anesthesia work area environment 32 demonstrates that the spread follows an epidemiologic pattern similar to that seen in icus and inpatient wards: from patient, to environment and hcws' hands, and to other patients ( fig. 50.4) . in this report, provider hands were less likely to serve as a transmitter of infection than contaminated environmental or patient skin surfaces. these findings have clinical implications for the risk of colonization and subsequent hcis-for example, ssis. this calls attention to the need to develop and enforce strict hand hygiene guidelines for personnel who are providing anesthesia care, but more importantly the need to increase compliance with environmental disinfection of the or (between cases and terminal cleaning), and to study further the directions of the spread of pathogens in the or and anesthesia work areas. this study unequivocally underscores our need to improve cleaning procedures in the or and equipment surfaces to reduce infection risk. there are also reports of equipment, fomites, and drugs (mainly propofol) that have resulted in hospital-acquired infections. 20, [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] propofol is widely used for both inpatient and outpatient anesthesia. this hypnotic agent is a nutrient-rich drug. it is hypothesized that propofol increases bacterial contamination of iv stopcocks and may compromise safety of iv tubing sets when continued to be used after propofol anesthesia. there is a covert incidence of iv stopcock bacterial contamination during anesthesia that is aggravated by the prior presence of propofol. propofol may increase the risk for postoperative infection because of bacterial growth in iv stopcock dead spaces. 52 other facets that may also contribute to infection include the following: ■ up to 40% of anesthetic equipment in direct or indirect contact with a child (blood pressure cuffs, cables, oximeters, laryngoscopes, monitors, respirator settings, and horizontal and vertical surfaces) may be contaminated with blood because of inadequate cleansing procedures between uses. 6,7,27,53,54 ■ in some institutions, up to 8% of the bain circuits that were reused without previous sterilization were contaminated. 55 ■ contamination of syringe contents has occurred with glass particles during ampule opening, which in turn may compromise the sterility of the contents, presumably because of the passage of bacteria contained on glass particles into the solution. 56-58 ■ iv tubing has both blood contamination as well as contamination by blood from syringes used to inject medications. this can occur with the absence of visible blood reflux in the tubing or syringe. simply replacing the needle on a syringe that will be reused is ineffective in preventing cross-infection; it is essential to not use the same syringe in multiple patients. 59 ■ refilling both glass and plastic syringes several times has also been shown to result in contamination of the contents; single use is therefore recommended. 59 studies on vancomycin-resistant enterococci established the importance of a domino effect of contamination in intensive care units (icus) and inpatient wards: spread of vancomycin-resistant enterococci that colonize patients' gastrointestinal tracts ("rectal carriage"), to patients' skin, to the hospital environment, to hands of hcws, and then to other patients. the skin contamination of patients with enteric organisms inspired the rather graphic description, the patient's "fecal patina." 31 also referred to as a "stool veneer," this coating with enteric organisms is limited not only to patients' skin but also extends to surfaces in the surrounding environment that are touched, and thereby contaminated, by patients and by hcws. the environmental contamination spreads out from the patient in a target-like concentric pattern, with the densest contamination closest to the rectum of patients who have rectal carriage of the problem bacteria. this interplay among the blood of a patient in an advanced disease stage or with a higher hiv viral load; a deep percutaneous injury; a procedure wherein the sharp was in the vein or artery of an infected source patient; an injury with a hollow-bore, blood-filled needle; and limited or delayed access to postexposure prophylaxis. after exposure, the risk of infection varies for specific bloodborne pathogens. for hbv, if the source patient has active hbv and the hcp do not already have immunity, the risk for infection after percutaneous injury is between 1% and 30%. if the source patient has active hcv, the risk of hepatitis c transmission is approximately 1.8% (range 0%-7%) after a percutaneous injury. if the source patient has hiv infection, the risk of hiv transmission is approximately 0.3% after a percutaneous exposure and 0.09% after a mucous membrane exposure. the risk of hiv transmission for an exposure with nonintact skin has not been determined and is estimated to be less than the risk after a mucous membrane exposure. 78 anesthesia staff lacking hbv protective antibodies are at great risk for acquiring the disease. 79, 80 these infection rates underscore the need for the use of "safe" needles and the need to advocate the use of "needleless" systems even though they are significantly more expensive. 81, 82 this also emphasizes the need for meticulous handling and disposal of needles and other sharp instruments, as well as the use of special "sharps boxes" designed to minimize accidental needlesticks (e.g., "mailbox"-type boxes that do not allow the hand to enter the disposal area). [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] the u.s. centers for disease control and prevention (cdc) has estimated that in the united states there are approximately 385,000 cutting and piercing accidents annually among hcp in hospitals; 25% of these occur in the or. 77 however, the actual prevalence is thought to be much greater, because many of these events are unreported. the distribution of these accidents among anesthesiologists is shown in fig. 50 .5a; the distribution of the items most frequently associated with cutting and piercing injuries in health care providers is shown in fig. 50 .5b. should such an accident occur (e.g., needle puncture, exposure to nonintact skin, or mucous membrane ■ needles that have been used for spinal or epidural anesthesia were contaminated with coagulase-negative staphylococci (15.7%), yeasts (1.5%), enterococci (0.8%), pneumococci (0.8%), and micrococci (0.8%), suggesting that there may be needle contamination despite standard skin preparation and cleansing. 68 it is unclear whether these skin organisms can be transmitted and cause an infection during administration of a neuraxial block. ■ blood and saliva frequently contaminate the skin of anesthetic personnel during routine anesthetic practice. 13 ■ violations of contemporary guidelines for preventing infections (e.g., hand washing, wearing gloves, surgical masks, ocular protection, scrubs, or syringe reuse) by anesthesiologists are frequent. 4 anesthesia staff are aware that they work in a potentially infectious environment, but they commonly do not adopt appropriate protective measures to reduce infections in both themselves and their patients (11%-99%). 27,69-71 percutaneous contamination from a cutting or piercing accident is the most effective means to transmit bloodborne pathogens. evidence suggests that this is the main route of hiv, hbv, and hcv infection, [72] [73] [74] especially if the injury is caused by hollow-bore needles that were used to draw blood or establish iv access. 75, 76 over 20 other bloodborne pathogens have been transmitted by this means, including those causing herpes, malaria, and tuberculosis. 77 the risk of exposure to blood and bloodborne pathogens is greater for health care personnel (hcp) than for people who do not work around blood. an exposure to infected blood, tissue, or other potentially infectious body fluids can occur by percutaneous injury or contact with mucous membrane or nonintact skin. the risk of infection after an exposure depends on a number of variables and appears to be greater with exposure to a larger quantity of blood or other infectious fluid; prolonged or extensive exposure of nonintact skin or mucous membrane to blood or other infectious fluid or concentrated virus in a laboratory setting; exposure to institutional administrative measures aimed at developing, implementing, and monitoring specifically designed accident prevention policies and procedures are important for reducing and preventing transmission of infectious agents in health care centers. to this end, centers should consider the following: 77,99,100 ■ include infection control as a major goal in the organizational mission statement and implement safety programs, both for patients and hcws. ■ provide sufficient administrative and financial support to carry out this mission. ■ provide sufficient administrative and financial support for the microbiology laboratory and implement an infection surveillance plan, especially for postsurgical infections. ■ establish a multidiscipline cross-functional team (e.g., a team manager, an epidemiologist, a representative from industrial health, and a person trained in quality control) to identify health and safety issues within the institution, analyze trends, assess outcomes, implement interventions, and make recommendations to other members of the organization. ■ provide sufficient administrative and financial support to develop and implement education programs for health care providers, patients, and their families. one positive example of such education is that anesthesiologists who have read the cdc's universal precaution guidelines for the prevention of occupational transmission of hiv and hbv have developed better hygienic practices. 69 ■ provide hcws with hepatitis a and b vaccine and document that an appropriate immunologic response was achieved. provide hepatitis a and b immune globulins (haig, hbig) for those exposed who do not have established immunity. 8 ■ provide a health care service for employees for counseling and postexposure prophylaxis should an exposure to hiv occur. 101 , there are now specific recommendations regarding immediate assessment of risk, assessment of the exposure source (chart review, inform the patient that an accident has occurred and ask permission to determine hbv, hcv, and hiv serologic status), and rapid initiation of appropriate antiviral treatment of the hcw. 82 it is advised to obtain as much information regarding the patient as possible-if the patient is known-to (1) obtain a sample of blood from the patient for determination of potential carrier state (table 50 .4) and (2) report to the health service for immediate institution of prophylaxis and follow-up ( air is delivered to each or from the ceiling, with downward movement toward several exhaust or return ducts near the floor. this design helps provide steady movement of clean air through the breathing and working zones. the aia has specific guidelines for the location of outside fresh air inlets to minimize contamination from exhaust systems and noxious fumes. a greater air inflow rate and a larger air-inlet area are desirable for contaminant control, but these approaches are detrimental to the thermal comfort of the staff and patient. 108 the aia recommends an air-change rate in an or of 20 to 25 air changes per hour (ach) for ceiling heights between 9 feet wound contamination in the or is the result of the patient's skin flora and bacteria shed on airborne particles from the or personnel. room ventilation affects the distribution of these airborne particles in four ways: total ventilation (dilution), air distribution (directional airflow), room pressurization (filtration barrier), and filtration (contaminant removal). as the air flows of the room increase, the greater the dilutional effect on airborne particles. balancing this phenomenon is important because while increased flow increases the effectiveness of air exchange, the resultant turbulent flow increases microbial distribution throughout the room. low-velocity unidirectional flow minimizes the spread of microbes in the room. directional flow can be inward, from the outside into the or (negative pressure), or outward, from the or to the outside (positive pressure). negative-pressure ventilation is used for highly infective rooms in the hospital (e.g., isolation rooms for tuberculosis patients), and positive-pressure ventilation is used for protective environments (e.g., ors and pep step 1: treat exposure site • use soap and water to wash areas exposed to potentially infectious fluids as soon as possible after exposure. • flush exposed mucous membranes with water. • flush exposed eyes with water or saline solution. • do not apply caustic agents, or inject antiseptics or disinfectants into the wound. step 2: report and document standard precautions 117 assume that any person or patient is potentially infected or colonized by microorganisms that could be transmitted and cause an infectious process. standard precautions must be implemented with all patients and include the following: ■ universal precautions-blood and body fluid precautions, developed to reduce bloodborne pathogen transmission ■ body substance isolation, designed to reduce the risk of pathogen transmission by moist body substances standard precautions are used to reduce the transmission of all infectious agents from one person to another, thus protecting health care providers and children against exposure to the most common microorganisms. standard precautions are implemented for any contact with blood and body fluids, secretions, and excretions (except sweat), whether or not they contain visible blood, as well as for any contact with nonintact skin, mucous membranes, and intact skin that is visibly soiled with blood and/ or body fluids. prevention is primary. all hcps should be familiar with standard precautions: wash hands frequently and thoroughly before and after patient care; use personal protective equipment: gloves, gowns, boots, shoe covers, eyewear, masks, and shields, as appropriate for the patient care situation; gloves must be worn when any kind of venous or arterial access is being performed; use sharps with caution: plan ahead (use sharps in a safe environment with a sharps container nearby), dispose of used sharps in puncture-proof receptacles immediately after use, do not recap needles, and use safety devices if available. all hcps should be vaccinated with the hepatitis b vaccine series and should undergo testing for hbsab response after completion of the series to document adequate protection. employees who have not gone through the vaccination series previously should be offered the hepatitis b series through their employer at no cost. 118 summaries of standard precautions, droplet precautions, airborne precautions, and contact precautions are available on line. 100, [119] [120] [121] hand washing overall hand hygiene compliance across health care providers remains less than 50%, with anesthesia providers identified as a particularly noncompliant group (one study found a compliance rate of only 23%). 122 bacterial contamination of anesthesia providers has been directly linked to high-risk bacterial transmission events to iv stopcocks and 30-day postoperative infections. 24 the vast majority of ssis are caused by staphylococcus aureus. transmission of specific staphylococcal phenotypes within and between patients is a major contributor to ssis and hais. 3, 4, 123 the role of anesthesia-provider hand contamination in transmission of enterococcus to the workstation and patient biome is concerning, even though it was not associated with actual infection, because of rising rates of antibiotic-resistant organisms and the observation that enterococcus is becoming a more prevalent pathogen. 3, 124 two approaches are indicated: improved methods of patient reservoir decontamination and more effective and frequent decontamination of provider hands. hand hygiene is a well-known and effective solution to the problem of bacterial transmission within and across patients. compliance with the current "5 moments" world health organization guidelines could make a major inroad into reducing provider hand and workspace contamination. one study found that only 20% of anesthesia providers demonstrated complete knowledge regarding who hand hygiene guidelines. 125 failure of providers to recognize prior contact with the environment and prior contact with the patient as hand hygiene opportunities contributed to this low percentage. several cognitive factors were associated (2.74 meters) and 12 feet (3.66 meters). some controversy exists between engineers and clinicians over the need for laminar airflow ventilation in the or to further minimize airborne infection. careful mathematical analyses of airflow suggest that laminar airflow is not necessary. clinical studies are confirmatory. similarly, the use of ultraviolet light to cleanse the room air is no longer recommended. 109 table 50 .8 shows the 2003 healthcare infection control practices advisory committee and cdc general recommendations for ventilation system specifications for the or. 12 children with tuberculosis require special consideration because of the high risk of occupational transmission of mycobacterium tuberculosis, 110, 111 especially after the emergence of multidrug-resistant strains (table 50.9 ). an easy preventive measure is to screen all children before coming to the or to determine recent exposure to infectious disease such as measles, mumps, rubella, and chickenpox because these infections can pose a significant risk to hcws and patients, especially those who are immunocompromised. 73, 106 another potential source for airborne spread of pathogens is through the anesthesia circuit; this may be reduced by the use of circuit filters. however, at present there are no regulatory requirements to use such devices, and performance characteristics vary widely. 28 • pep should be initiated within 2 hours of the exposure. • the eficacy of pep initiation is thought to diminish after 24 to 36 hours following an exposure. • if the fourth-generation combination hiv ag/ab assay is used to test the source patient, hiv follow-up testing can be completed 4 months after exposure. hand washing is considered the most important and costeffective individual intervention in the prevention of hais in children and health care providers. 126 its importance in medical practice had not been universally accepted, despite the pioneering work by oliver wendell holmes 127 (1843) and ignaz semmelweis 128 with a reduced risk of incomplete knowledge, including providers responding positively to washing their hands after contact with the environment, disinfecting their environment during patient care, believing that they can influence their colleagues, and intending to adhere to guidelines. these results suggest that anesthesia providers have knowledge deficits pertaining to opportunity-based hand hygiene in the intraoperative arena 125 hiv-positive class 1: asymptomatic hiv infection or known low viral load (e.g., <1500 ribonucleic acid copies/ml). hiv-positive class 2: symptomatic hiv infection, acquired immunodeficiency syndrome, acute seroconversion, or known high viral load. if drug resistance is a concern, obtain expert consultation. initiation of pep should be delayed pending expert consultation, and because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. the recommendation "consider pep" indicates that pep is optional; a decision to initiate pep should be based on a discussion between the exposed person and the treating clinician regarding the risks versus benefits of pep. g if pep is offered and administered and the source is later determined to be hiv-negative, pep should be discontinued. recommendations for surgical hand preparation are as follows: remove rings, wristwatch, and bracelets before beginning surgical hand preparation (ii); artificial nails are prohibited (ib); sinks should be designed to reduce the risk of splashes (ii); if hands are visibly soiled, wash hands with plain soap before surgical hand preparation (ii); remove debris from underneath fingernails using a nail cleaner, preferably under running water (ii); brushes are not recommended for surgical hand preparation (ib); surgical hand antisepsis should be performed using either a suitable antimicrobial soap or suitable alcohol-based handrub, preferably with a product ensuring sustained activity, before donning sterile gloves (ib); if the quality of water is not assured in the operating theatre, surgical hand antisepsis using an alcohol-based handrub is recommended before donning sterile gloves when performing surgical procedures (ii); when performing surgical hand antisepsis using an antimicrobial soap, scrub hands and forearms for the length of time recommended by the manufacturer, typically 2-5 minutes. long scrub times (e.g., 10 minutes) are not necessary (ib); when using an alcohol-based surgical handrub product with sustained activity, follow the manufacturer's instructions for application times. apply the product to dry hands only (ib); do not combine surgical hand scrub and surgical handrub with alcohol-based products sequentially (ii); when using an alcoholbased handrub, use sufficient product to keep hands and forearms wet with the handrub throughout the surgical hand preparation procedure (ib); after application of the alcohol-based handrub as recommended, allow hands and forearms to dry thoroughly before donning sterile gloves (ib). at present, alcohol-based handrubs are the only known means for rapidly and effectively inactivating a wide array of potentially harmful microorganisms on hands. the who recommends alcohol-based handrubs based on the following factors: evidencebased, intrinsic advantages of fast-acting and broad-spectrum microbicidal activity with a minimal risk of generating resistance to antimicrobial agents; suitability for use in resource-limited or remote areas with lack of accessibility to sinks or other facilities for hand hygiene (including clean water, towels, and so on); capacity to promote improved compliance with hand hygiene by making the process faster and more convenient; economic benefit by reducing annual costs for hand hygiene, representing approximately 1% of extra costs generated by an hci; minimization of risks from adverse events because of increased safety associated with better acceptability and tolerance than other products. 118 after hand washing, it is very important to dry the hands properly with appropriate paper towels, hot air flow, or both, because the level of pathogen transmission from a hcw's hands to a patient is greatly increased if the hands are wet. 141 sterile cloth towels are most frequently used in ors to dry wet hands after surgical hand antisepsis. several methods of drying have been tested without significant differences between techniques. 118 transmission may also occur from patients' wet sites, such as groins or armpits, or when a hcw gets his or her hands wet when opening parenteral solutions. it is critical for health institutions to establish written procedures and protocols to support adherence to the recommended hand hygiene practices. wearing clean or sterile gloves while caring for children is an effective means of reducing hais. gloves remain a supplementary barrier to infection that should not replace proper hand hygiene. more frequent (on average, 22 opportunities per patient-hour). the greatest adherence rate (59%) was observed in pediatrics, where the average intensity of patient care was smaller than elsewhere (on average, 8 opportunities per patient-hour). the results suggest that full adherence to guidelines is unrealistic and that easy access to hand hygiene at the point of patient care, (i.e., in particular, alcohol-based handrubbing) could help improve adherence to hand hygiene. perceived barriers to adherence with hand hygiene practice recommendations include skin irritation caused by hand hygiene agents, inaccessible hand hygiene supplies, interference with hcw-patient relationships, patient needs perceived as a priority over hand hygiene, wearing of gloves, forgetfulness, lack of knowledge of guidelines, insufficient time for hand hygiene, high workload and understaffing, and the lack of scientific information showing a definitive impact of improved hand hygiene on hai rates. lack of knowledge of guidelines for hand hygiene, lack of recognition of hand hygiene opportunities during patient care, and lack of awareness of the risk of cross-transmission of pathogens are barriers to good hand hygiene practices. furthermore, some hcws believed that they washed their hands when necessary even when observations indicated that they did not. the risk of pathogen transmission via the hands is proportional to the power of the number of times a child is touched. 140 table 50 .10 presents 1. wash hands with soap and water when visibly dirty or visibly soiled with blood or other body fluids (ib) or after using the toilet (ii). 2. if exposure to potencial spore-forming pathogens is strongly suspected or proven, including outbreaks of clostridium difficile, hand washing with soap and water is the preferred means (ib). 3. use an alcohol-based handrub as the preferred means for routine hand antisepsis in all other clinical situations described in terms 4(a) to 4(f) listed below, if hands are not visibly soiled (ia). if alcohol-based handrub is not obtainable, wash hands with soap and water (ib). 4. perform hand hygiene: a. before and after touching the patient (ib); b. before handling an invasive device for patient care regardless of whether or not gloves are used (ib); c. after contact with body fluids or excretions, mucous membranes, non-intact skin, or wound dressings (ia); d. if moving from a contaminated body site to another body site during care of the same patient (ib); e. after contact with inanimate surfaces and objects (including medical equipment) in the immediate vicinity of the patient (ib); f. after removing sterile (ii) or nonsterile gloves (ib). 5. before handling medication or preparing food, perform hand hygiene using an alcohol-based handrub or wash hands with either plain or antimicrobial soap and water (ib). 6. soap and alcohol-based handrub should not be used concomitantly (ii). gloves protect patients by reducing health care provider hand contamination and the subsequent transmission of pathogens to other children, provided the gloves are changed after providing care to each child. additionally, when the use of gloves is combined with cdc standard precautions, they protect the health care provider against exposure to bloodborne infections or infections transmitted by any other body fluids, such as excretions, secretions (except sweat), mucous membranes, and nonintact skin. examination gloves are single-use and usually nonsterile. sterile surgical gloves are required for surgical interventions. some nonsurgical care procedures, such as central vascular catheter insertion, also require surgical glove use. in addition to their sterile properties, these gloves have characteristics of thickness, elasticity, and strength that differ from other medical gloves. the use of gloves in situations when their use is not indicated represents a waste of resources without necessarily reducing crosstransmission. the wide-ranging recommendations for glove use have led to very frequent and inappropriate use. indications for gloving and glove removal are shown in table 50 .11. situations that require and that do not require glove use are presented in fig. 50 .6. ranked consensus recommendations for the use of gloves, categorized according to the cdc/hicpac system, include the following 118, 142, 143 : ■ wear gloves in case of contact with blood or any other potentially infecting body fluid, such as excretions, secretions (except sweat), mucous membranes, and nonintact skin (ic). ■ remove the gloves immediately after providing care to a child. staff should not wear the same pair of gloves to take care of more than one child, nor should they touch the surfaces of any equipment, monitoring devices, or even light switches. ■ alcohol-based handrub dispensers and clean glove boxes (at least two sizes) should be in place near every patient care site (e.g., on top of every anesthesia cart, medication cart, or in the nursing station). ■ disposable gloves should not be washed, resterilized, or disinfected (ib). if gloves are reused, appropriate reprocessing methods should be in place to ensure the physical integrity of the gloves and their full decontamination (ii). ■ sterile gloves are much more expensive than clean, disposable gloves and should be used only for certain procedures, such as when hands are in contact with normally sterile body areas or when inserting intravascular or urinary catheters. clean gloves should be used during any other procedure, including wound dressing. ■ latex-free gloves should be worn when caring for children at risk for latex allergy. surgical antimicrobial prophylaxis is an essential tool to reduce the risk of postoperative infections, and the anesthesia team plays a central role in ensuring the proper timing of drug administration. 147, 148 the aim of the perioperative administration of antibiotics is to obtain plasma and tissue drug concentrations exceeding the minimal inhibitory concentration of those organisms most likely to cause an infection. this will reduce the microbial load of the intraoperative contamination; it is not the intent to cover all possible pathogens, because this can lead to the selection of drug-resistant bacteria. there have been few studies regarding the effectiveness of prophylactic guidelines for prevention of ssis in children. currently, prophylactic antibiotic guidelines exist for certain subsets of the pediatric surgical population, but there are no global recommendations, and the guidelines that exist are mostly based on studies from adults or from expert opinion. a retrospective study suggested that the appropriate use of antibiotic prophylaxis was a vital modifiable risk factor and may be the easiest factor to influence. primary failure to administer the correct dose of antibiotics at the appropriate time resulted in an almost 2-fold increase in the risk of developing an ssi. the importance of correct antibiotic usage and dosing plays a major role in decreasing risk of ssis in children. recommendations are provided for adult (age ≥19 years) and pediatric (age 1-18 years) patients. the guidelines do not specifically address newborn (premature and full-term) infants (table 50. although pediatric-specific prophylaxis data are sparse, available data have been evaluated for specific procedures. selection of antimicrobial prophylactic agents mirrors that in adult guidelines, with the agents of choice being first-and second-generation cephalosporins, reserving the use of vancomycin for patients with documented β-lactam allergies. while the use of a penicillin with a β-lactamase inhibitor in combination with cefazolin or vancomycin and gentamicin has also been studied in pediatric patients, the number of patients included in these evaluations remains small. as with adults, there is little evidence supporting the use of vancomycin, alone or in combination with other antimicrobials, for routine perioperative antimicrobial prophylaxis in institutions that have a high prevalence of methicillin-resistant s. aureus (mrsa). vancomycin may be considered in children known to be colonized with mrsa and decreases mrsa infections. 150 mupirocin is effective in children colonized with mrsa, but choice, alternative antibiotics should be administered to those children at risk of anaphylaxis to β-lactams, based on their history or diagnostic tests (e.g., skin testing). however, the incidence of severe allergic reactions to first-generation cephalosporins in children with reported allergy to penicillin is rare (but not zero) 158, 159 ; furthermore, skin testing does not reliably predict the likelihood of adverse reactions to cephalosporins in those with reported allergy to penicillin. [160] [161] [162] there is no evidence of any risk of cross-reactivity between penicillin and second-and thirdgeneration cephalosporins. for the most part, "allergies" to oral antibiotics that appear on children's charts (rash, vomiting, gastrointestinal disturbances) are reactions to the additives in the antibiotic formulation, including food dyes, fillers, and other compounds, or a manifestation of the underlying infection. iv administration of small test doses of the pure antibiotic in a fully monitored (and anesthetized) child will determine whether the child is at risk for an allergic reaction to the antibiotic. in the case of surgical procedures where antibiotic prophylaxis is mainly directed at gram-positive cocci, children who are truly allergic to β-lactams (cephalosporins) should receive either vancomycin or clindamycin. 163 however, in those children where the history is consistent with either an ige-mediated penicillin allergy (urticaria, angioedema, anaphylaxis, bronchospasm) or a severe non-igemediated reaction (interstitial nephritis, toxic epidermal necrolysis, hemolytic anemia, or stevens-johnson syndrome) it is advisable to switch out the cefazolin. cross-sensitivity occurs when the r1 side chains of the penicillins and cephalosporins are similar, which perhaps surprisingly is not the case with cefazolin. cephalosporins with r1 side chains similar to penicillins include cephalexin, cefaclor, and cefadroxil. the risk associated with use of first-or second-generation cephalosporins with dissimilar side chains, or third-or fourth-generation cephalosporins, "appears to be very low in patients with mild-to-moderate reactions to penicillin g, ampicillin, or amoxicillin. dismissing cefazolin use when there is a vague history of any penicillin allergy should be reconsidered." 155 indications for prophylactic antibiotics surgical wounds are classified into four categories (table 50 .13). the use of antibiotic prophylaxis for postoperative infections is well established for clean-contaminated procedures. within the clean category, prophylaxis has been traditionally reserved for surgical procedures involving a foreign body implantation or for any surgical procedure where an ssi would be catastrophic (e.g., cardiac surgery or neurosurgical procedures). however, there is evidence that postoperative infections resulting from procedures not involving prosthetic elements are underreported; estimates show that more than 50% of all complications occur after the patient is discharged and are thus unrecognized by the surgical team. therefore antibiotic prophylaxis is also recommended for certain procedures, such as herniorrhaphy. 164, 165 the direct and indirect costs of these complications may not affect the hospital budget; however, they represent a substantial cost for the community at large. in the case of contaminated or dirty procedures, bacterial contamination or infection is established before the procedure begins. accordingly, the perioperative administration of antibiotics is a therapeutic, not a prophylactic, measure. the use of antibiotics in children has implications not only for the response to the current treatment but also to future treatments. thus all medical professionals are jointly responsible for the rational use of antibiotics. protocols, although effective, require continuous feedback on their acceptance and ssi results. 166 no surgical protocol can replace there are limited data supporting its use perioperatively. 151, 152 most recommendations for adults are the same for pediatric patients. dosing recommendations in pediatric patients are limited and have been extrapolated from adult data; therefore nearly all pediatric recommendations are based on expert opinion. pediatric efficacy data are few. fluoroquinolones should not be routinely used for surgical prophylaxis in pediatric patients because of the potential for toxicity in this population. the same principle of preoperative dosing within 60 minutes before incision has been applied to pediatric patients. additional intraoperative dosing may be needed if the duration of the procedure exceeds two half-lives of the antimicrobial agent or there is excessive blood loss during the procedure. as with adult patients, single-dose prophylaxis is usually sufficient. if antimicrobial prophylaxis is continued postoperatively, the duration should be less than 24 hours, regardless of the presence of intravascular catheters or indwelling drains. there are sufficient pharmacokinetic studies of most agents to recommend pediatric dosages that provide adequate systemic exposure and, presumably, efficacy comparable to that demonstrated in adults. therefore the pediatric doses recommended in guidelines are based largely on pharmacokinetic data and the extrapolation of adult efficacy data to pediatric patients. because few clinical trials have been conducted in pediatric surgical patients, strength of evidence criteria have not been applied to these recommendations. with few exceptions (e.g., aminoglycoside dosages), pediatric doses should not exceed the maximum adult recommended dosages. generally, if a dose is calculated on a milligram-per-kilogram basis for children weighing more than 40 kg, the calculated dosage will likely exceed the maximum recommended dose for adults; adult dosages should therefore be used for larger children. 153 the timing of antibiotic prophylaxis the 2013 revised policy paper on prophylactic antibiotics developed jointly by the american society of health-system pharmacists (ashp), the infectious disease society of america, the surgical infection society, and the society for healthcare epidemiology of america states: successful prophylaxis requires the delivery of the antimicrobial to the operative site before contamination occurs. thus, the antimicrobial agent should be administered at such a time to provide serum and tissue concentrations exceeding the minimum inhibitory concentration (mic) for the probable organisms associated with the procedure, at the time of incision, and for the duration of the procedure. 154 current evidence suggests that for most β-lactams, a bolus dose at 15 to 45 minutes before incision is ideal and provides maximum interstitial fluid concentrations at the time of initial bacterial seeding (see table 50 .12). because diffusion distances from capillary to pathogen are greater in obese patients, for this patient subset initiating antibiotic infusion 30 minutes or longer before incision is warranted on theoretical grounds. 155 the initial β-lactam bolus dose should be followed by additional doses at every 1 to 2 half-lives per the ashp guidelines. 154 the use of a ssi prevention bundle in pediatric patients improves compliance with preincision antibiotic administration and decreases the ssi infection rate. 156 allergy to β-lactams several studies have shown that the true incidence of allergy to antibiotics is less than that reflected in medical charts. 157 for surgical procedures where cephalosporins are the prophylaxis of the judgment of the medical professional; clinical reasoning must be tailored to the individual circumstances. finally, children with congenital heart disease and a subgroup of those with repaired congenital heart disease may require bacterial endocarditis prophylaxis (see also tables 16.2 and 16. 3). 167 preventing the transmission of pathogenic microbes during anesthesia infection control and anesthesia: lessons learned from the toronto sars outbreak fecal patina in the anesthesia work area intensive care unit environments and the fecal patina: a simple problem? transmission dynamics of gram-negative bacterial pathogens in the anesthesia work area serratia marcescens bacteremia traced to an infused narcotic postoperative infections traced to contamination of an intravenous anesthetic, propofol staphylococcus aureus bloodstream infections among patients undergoing electro-convulsive therapy traced to breaks in infection control and possible extrinsic contamination by propofol postsurgical candida albicans infections associated with an extrinsically contaminated intravenous anesthetic agent occupationally acquired infections in health care workers. part ii occupationally acquired infections in health care workers. part i transmission of hepatitis c virus by a cardiac surgeon transmission of infection by gastrointestinal endoscopy and bronchoscopy nosocomial transmission of multidrug-resistant mycobacterium tuberculosis. a risk to patients and health care workers disease transmission by inefficiently sanitized anesthetizing apparatus contamination, disinfection, and cross-colonization: are hospital surfaces reservoirs for nosocomial infection? pseudomonas aeruginosa respiratory tract infection acquired from a contaminated anesthesia machine pseudomonas aeruginosa cross-infection due to contaminated respiratory apparatus patient-to-patient transmission of hiv in private surgical consulting rooms hepatitis b virus transmission associated with a multiple-dose vial in a hemodialysis unit fine-particle humidifiers. source of pseudomonas aeruginosa infections in a respiratory-disease unit pseudomonas aeruginosa epidemic traced to delivery-room resuscitators an alternative strategy for infection control of anesthesia breathing circuits: a laboratory assessment of the pall hme filter nosocomial contamination of laryngoscope handles: challenging current guidelines leaving more than your fingerprint on the intravenous line: a prospective study references all boats rise with the tide bacterial reservoirs in the operating room transmission of pathogenic bacterial organisms in the anesthesia work area reduction in intraoperative bacterial contamination of peripheral intravenous tubing through the use of a novel device anaesthetics and inhalers blood contamination of anesthesia equipment and monitoring equipment the routine wearing of gloves: impact on the frequency of needlestick and percutaneous injury and on surface contamination in the operating room infection control in the outpatient setting infection prevention in anesthesia practice: a tool to assess risk and compliance safe infection control practices for protection of prevention of airborne exposure during endotracheal intubation guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) blood contamination of anaesthetic and related staff update: human immunodeficiency virus infections in health-care workers exposed to blood of infected patients the eyes as a portal of entry for hepatitis and other infectious diseases molluscum contagiosum the epidemiology of molluscum contagiosum in children nosocomial outbreak of scabies in a hospital in spain an outbreak of scabies in a teaching hospital: lessons learned herpes simplex cross infection in the operating room herpetic whitlow infection in a general pediatrician-an occupational hazard herpetic whitlow: an infectious occupational hazard hand contamination of anesthesia providers is an important risk factor for intraoperative bacterial transmission reducing perioperative infection is as simple as washing your hands hand hygiene in the intensive care unit hygienic practices of consultant anaesthetists: a survey in the north-west region of the uk joint working party of the hospital infection society and the surgical infection study group workbook for designing, implementing and evaluating a sharps injury prevention program pep steps: a quick guide to postexposure prophylaxis in the health care setting. mountain plains aids education and training center the risk of needlestick injuries and needlesticktransmitted diseases in the practice of anesthesiology percutaneous injuries in anesthesia personnel sharp truth: health care workers remain at risk of bloodborne infection universal treatment success among healthcare workers diagnosed with occupationally acquired acute hepatitis c prevention of needle-stick injury. efficacy of a safeguarded intravenous cannula strategies for preventing sharps injuries in the operating room preventing transmission of blood-borne pathogens: a compelling argument for effective device-selection strategies accidental needlesticks in the phlebotomy service of the department of laboratory medicine and pathology at mayo clinic rochester don't get stuck with unsafe needles. instead, get involved in needle device selection update on needlestick and sharps injuries: the needle stick safety and prevention act of 2000 multicenter study of contaminated percutaneous injuries in anesthesia personnel needle injuries among pediatric housestaff physicians in new york city device-specific sharps injury and usage rates: an analysis by hospital department needlestick injuries among health care workers. a literature review sharps injuries among hospital support personnel prevalence of safer needle devices and factors associated with their adoption: results of a national hospital survey effect of implementing safety-engineered devices on percutaneous injury epidemiology a review of sharps injuries and preventative strategies preventing needlestick injuries among healthcare workers: a who-icn collaboration sharps disposal in the ed: simple techniques and equipment monografía-manual para el desarrollo de un programa de prevención de infecciones de sitio quirúrgico, escuela de salud pública prevención de infección de sitio quirúrgico y seguridad del paciente en pre, intra y postquirúrgico, instituto nacional de epidemiología documento de consenso on propofol anesthesia and implications of stopcock contamination recombinant factor viii for the treatment of previously untreated patients with hemophilia a. safety, efficacy, and development of inhibitors kogenate previously untreated patient study group a simple, cost-effective method of preventing laryngoscope handle contamination contamination and resterilization of the bain circuit glass particle contamination: influence of aspiration methods and ampule types glass particle contamination in single-dose ampules drug contamination from opening glass ampules a microbiological study of the contamination of the syringes used in anaesthesia practice anesthesiologists should not give iv medications with common syringe bacterial growth in ropivacaine hydrochloride ropivacaine 0.1% with sufentanil 1 microg/ml inhibits in vitro growth of pseudomonas aeruginosa and does not promote multiplication of staphylococcus aureus growth of staphylococcus aureus in four intravenous anesthetics propofol, but not thiopental, supports the growth of candida albicans growth of microorganisms in propofol, thiopental, and a 1 : 1 mixture of propofol and thiopental outbreak of severe sepsis due to contaminated propofol: lessons to learn infectious disease risk associated with contaminated propofol anesthesia bacterial contamination of needles used for spinal and epidural anaesthesia preventing perioperative transmission of infection: a survey of anesthesiology practice accidental needlesticks: do anesthesiologists practice proper infection control precautions? anesthesia practice-a vector of infection? letter: hepatitis-b antigen on environmental surfaces susceptibility of healthcare workers to measles, mumps rubella and varicella lessons learned: protection of healthcare workers from infectious disease risks guidelines for prevention of transmission of human immunodeficiency virus and hepatitis b virus to health-care and public-safety workers the dynamics of enterococcus transmission from bacterial reservoirs commonly encountered by anesthesia providers hand hygiene knowledge and perceptions among anesthesia providers infection control-a problem for patient safety classic pages in obstetrics and gynecology. oliver wendell holmes. the contagiousness of puerperal fever the etiology, concept, and prophylaxis of childbed fever puerperal sepsis hand washing and hand disinfection: more than your mother taught you effectiveness of gloves in the prevention of hand carriage of vancomycin-resistant enterococcus species by health care workers after patient care quantification of hand hygiene compliance in anesthesia providers at a tertiary care center in northern india apic guideline for handwashing and hand antisepsis in health care settings compliance with handwashing in a teaching hospital improving compliance with hand hygiene in hospitals effectiveness of a hospital-wide programme to improve compliance with hand hygiene hand hygiene: improved standards and practice for hospital care improving adherence to hand hygiene practice: a multidisciplinary approach guideline for hand hygiene in health-care settings. recommendations of the healthcare infection control practices advisory committee and the hipac/shea/apic/idsa hand hygiene task force preliminary analysis of the transmission dynamics of nosocomial infections: stochastic and management effects touch contamination levels during anaesthetic procedures and their relationship to hand hygiene procedures: a clinical audit guideline for isolation precautions in hospitals. the hospital infection control practices advisory committee how-to guide: improving hand hygiene. a guide for improving practices among health care workers bacterial contamination of the hands of hospital staff during routine patient care anatomy of a defective barrier: sequential glove leak detection in a surgical and dental environment examination gloves as barriers to hand contamination in clinical practice prevention of central venous catheter-related bloodstream infections using non-technologic strategies benchmarking for prevention: the centers for disease control and prevention's national nosocomial infections surveillance (nnis) system experience public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis varicella and paediatric staff: current practice and vaccine cost-effectiveness varicella serological status of healthcare workers as a guide to whom to test or immunize controlling varicella in the healthcare setting: the cost effectiveness of using varicella vaccine in healthcare workers varicella vaccination for healthcare workers at a university hospital: an analysis of costs and benefits a survey of policies at children's hospitals regarding immunity of healthcare workers: are physicians protected? prevention and control of varicella-zoster infections in healthcare facilities predicting transient particle transport in enclosed environments with the combined computational fluid dynamics and markov chain method room ventilation systems. operating room design manual occupational transmission of tuberculosis: implications for anesthesiologists occupational transmission of mycobacterium tuberculosis to health care workers in a university hospital in the bacterial and viral filtration performance of breathing system filters prevention of cross contamination, patient to anesthesia apparatus to patient, using filters heat and moisture exchangers and breathing system filters: their use in anaesthesia and intensive care. part 1-history, principles and efficiency heat and moisture exchangers and breathing system filters: their use in anaesthesia and intensive care. part 2-practical use, including problems, and their use with paediatric patients hidden hazards and dangers associated with the use of hme/filters in breathing circuits. their effect on toxic metabolite production, pulse oximetry and airway resistance who guidelines on hand hygiene in health care: first global patient safety challenge clean care is safer care water-assisted liposuction for body contouring and lipoharvesting: safety and efficacy in 41 consecutive patients world health organization. practical guidelines for infection control in health care facilities. searo regional publication no. 41 hand hygiene among physicians: performance, beliefs, and perceptions multiple reservoirs contribute to intraoperative bacterial transmission retrospective evaluation of antimicrobial prophylaxis in prevention of surgical site infection in the pediatric population effects of controlled perioperative antimicrobial prophylaxis on infectious outcomes in pediatric cardiac surgery role of decolonization in a comprehensive strategy to reduce methicillin-resistant staphylococcus aureus infections in the neonatal intensive care unit: an observational cohort study immediate control of a methicillinresistant staphylococcus aureus outbreak in a neonatal intensive care unit clinical practice guidelines for antimicrobial prophylaxis in surgery clinical practice guidelines for antimicrobial prophylaxis in surgery administration of parenteral prophylactic beta-lactam antibiotics in 2014: a review reducing surgical site infections at a pediatric academic medical center drug allergies in the surgical population is there cross-reactivity between penicillins and cephalosporins? a review of evidence supporting the american academy of pediatrics recommendation for prescribing cephalosporin antibiotics for penicillin-allergic patients practical aspects of choosing an antibiotic for patients with a reported allergy to an antibiotic hypersensitivity reactions to beta-lactam antibiotics anaphylactic shock due to cefuroxime in a patient taking penicillin prophylaxis antimicrobial prophylaxis for surgery: an advisory statement from the national surgical infection prevention project comprehensive surveillance of surgical wound infections in outpatient and inpatient surgery community surveillance of complications after hernia surgery quality in pediatric anesthesia prevention of bacterial endocarditis. recommendations by the american heart association key: cord-284409-xiyeceib authors: prabakaran, ponraj; chen, weizao; dimitrov, dimiter s. title: the antibody germline/maturation hypothesis, elicitation of broadly neutralizing antibodies against hiv-1 and cord blood igm repertoires date: 2014-08-28 journal: front immunol doi: 10.3389/fimmu.2014.00398 sha: doc_id: 284409 cord_uid: xiyeceib we have previously observed that all known potent broadly neutralizing antibodies (bnabs) against hiv-1 are highly divergent from their putative germline predecessors in contrast to bnabs against viruses causing acute infections such as henipaviruses and sars cov, which are much less divergent from their germline counterparts. consequently, we have hypothesized that germline antibodies may not bind to the hiv-1 envelope glycoprotein (env) because they are so different compared to the highly somatically mutated hiv-1-specific bnabs. we have further hypothesized that the immunogenicity of highly conserved epitopes on the hiv-1 envelope glycoproteins (envs) may be reduced or eliminated by their very weak or absent interactions with germline antibodies and immune responses leading to the elicitation of bnabs may not be initiated and/or sustained. even if such responses are initiated, the maturation pathways are so extraordinarily complex that prolonged periods of time may be required for elicitation of bnabs with defined unique sequences. we provided the initial evidence supporting this antibody germline/maturation hypothesis, which prompted a number of studies to design vaccine immunogens that could bind putative germline predecessors of known bnabs and to explore complex b cell lineages. however, guiding the immune system through the exceptionally complex antibody maturation pathways to elicit known bnabs remains a major challenge. here, we discuss studies exploring the antibody germline/maturation hypothesis as related to elicitation of bnabs against hiv-1 and present our recent data demonstrating the existence of germline-like precursors of vrc01 antibodies in a human cord blood igm library. elicitation of broadly neutralizing antibodies (bnabs) targeting the hiv-1 envelope glycoproteins (envs), the key to an effective hiv-1 vaccine, remains elusive. previous studies have demonstrated several properties of the hiv-1 envs that could limit their ability to elicit bnabs. these include protection of the conserved structures by variable loops (1) (2) (3) , remarkable genetic diversity (4), a glycan shield (5) , steric occlusion (6) , and conformational masking (7) . until 6 years ago, only a handful of bnabs, including b12, 2g12, 2f5, and 4e10, were known. although the structural and functional studies of those bnabs revealed some important neutralization epitopes (8) , such bnabs have not been successfully elicited by any vaccination approach. in 2007, we first noted that in hiv-1 specific bnabs the number of amino acid mutations from their closest corresponding germline sequences was significantly higher than that of bnabs against the sars cov coronavirus, and hendra and nipah viruses, which cause self-limiting acute infections (9) . using a large nonimmune igm library, we identified several hiv-1 env specific antibodies and found that they had fewer somatic mutations than the hiv-1 bnabs, as well as limited neutralizing activity (9) . these findings indicated that elicitation of hiv-1 bnabs would require far more extensive maturation processes than those needed to generate the bnabs against the sars cov and henipaviruses. so, we have suggested that the difficulty of eliciting these bnabs may be due, at least in part, to the complex and prolonged maturation pathways required for the development of bnabs against the hiv-1, which can take long time (10) . we thus speculated that this may represent another significant challenge in the development of effective hiv-1 vaccines. we quantified the number of mutations in human monoclonal antibodies (mabs) that we selected from phage libraries generated from an hiv-1-infected patient with a known time of infection (10) . we calculated the number of amino acid mutations per heavy chain v gene, and defined it as antibody somatic mutational diversification (asmd). we compared the extent and dynamics of the asmd between hiv-1-specific mabs and a panel of sars covspecific mabs. our experiments based on the asmd predicted that elicitation of hiv-1-specific bnabs would take at least 3 years. an illustrative mathematical model using the asmd rate based on www.frontiersin.org an exponential time dependent function suggested that a much longer time would be needed for the required maturation, unless somatic diversification had already been initiated from an intermediate antibody. thus, all these initial studies corroborated our hypothesis that the infrequent occurrence or absence of bnabs in hiv-1-infected patients could be due, at least in part, to the lack or limited availability of b cell receptors that rapidly mature into bnabs. therefore, we suggested that appropriate immunization protocols of long duration need to be developed using the knowledge gained from the exploration of antibody maturation pathways in humans (10) . from the striking observation that all known potent hiv-1 bnabs are highly divergent from their putative germline predecessors in contrast to bnabs against henipaviruses and sars cov coronavirus, we hypothesized that, since the germline antibodies are so different compared to the highly somatically mutated hiv-1 bnabs, they may not bind to the env. this led us to the hypothesis that the immunogenicity of the highly conserved epitopes on the hiv-1 native envelope glycoproteins (envs) is reduced or eliminated by their very weak or absent interactions with germline antibodies, which could not initiate and/or sustain immune responses leading to elicitation of bnabs: even if immune responses are initiated and sustained, the maturation pathways are so complex that help and long times may be needed for their elicitation. to test our antibody germline/maturation hypothesis, we designed germline-like antibodies corresponding to the known bnabs b12, 2g12, 2f5, x5, m44, and m46 (the latter three antibodies were discovered in our laboratory and possess hiv-1 cross-reactivity with moderate neutralizing activities) and evaluated them for binding to envs (11) . we found that while germline-like x5, m44, and m46 bound to envs with relatively high affinity, the germline-like precursors of b12, 2g12, and 2f5 failed to bind envs in an elisa assay although their corresponding mature bnabs bound strongly. these results provided initial evidence that the env structures containing conserved epitopes might not initiate humoral responses due to limited or absent binding to the germline precursors of bnabs. these germline precursors may also be of limited availability as recently reported (12) . following that initial study, we expanded our investigation to different variants of the two different antibodies (b12 and x5) including their closest germline counterparts and several germline-like intermediates (13) . the experiments showed that b12 intermediate antibodies neutralized only some hiv-1 isolates with relatively weak potency. in contrast, intermediates of x5 neutralized a subset of the tested hiv-1 isolates with efficiencies comparable to those of the matured x5. these results helped explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of cd4-induced (cd4i) antibodies in hiv-1-infected patients (x5 is a cd4i antibody) as well as the maturation pathway of x5. in the case of b12, germlinelike intermediates along the maturation pathway were shown to not only bind some envs but also human self antigens, suggesting that antigens other than the envs could help guide the immune system through the b12 maturation pathway. therefore, we proposed a conceptually new vaccination approach, in which it is critical to identify primary immunogens that bind to the germline antibodies that are predecessors of bnabs. if needed, these immunogens should be combined with secondary immunogens that recognize intermediate and/or matured antibodies to guide the immune system through the prolonged, complex maturation pathways (14) . in this respect, we envisioned that the knowledge of human antibodyomes would become indispensable to elucidate the origin, diversity, and maturation pathways of bnabs and discover germline-like intermediates of bnabs that could provide a basis for the design of novel hiv-1 vaccine immunogens (14, 15) . in recent years, several groups have reported a number of new bnabs that were identified from multiple hiv-1 infected individuals using designed novel antigen baits and advanced technologies implemented in isolating human mabs and high-throughput sequencing (16) . particularly, haynes, kwong, stamatatos, scheif et al. have dealt with a large amount of data delineating structural, genetic determinants, and maturation pathways of different bnabs. these studies not only confirmed our previous findings that the envs fail to engage germline versions of bnabs but also suggested possible holes in b cell repertoires and demonstrated the implications of our antibody germline/maturation hypothesis for finding germline-like precursors, intermediates as well as for designing immunogens that could potentially bind to such bnab intermediates. in this report, we discuss the recent advancements in hiv-1 vaccine research in the context of the antibody germline/maturation hypothesis, and highlight critical factors to be considered when exploring germline-like precursors and intermediates of bnabs. we also report for the first time using 454 sequencing data analysis of a human cord blood igm library to identify putative germline precursors of the heavy and light chains of vrc01-like antibodies. these naturally occurring cord blood-derived vrc01-like heavy and light chains may be useful as putative templates for designing novel vaccine immunogens that can lead to the elicitation of vrc01-like antibodies and for understanding the maturation pathways of this bnab. still there are major challenges to be overcome. new empirical and semiempirical approaches could be successful; recently, new paradigms were discussed that could better fit our increased knowledge of hiv immunopathology and which could possibly be more helpful in guiding future vaccine research than did past unsuccessful approaches (17) . dna isolation, amplification, and 454 sequencing of the human cord blood igm library were previously described in detail (18, 19) . for quality control, we trimmed the 454 sequence reads and retained only sequences with lengths of more than 300 nucleotides, covering the entire antibody variable domains consisting of all three complementarity determining regions (cdrs) along with framework regions (frs). we used imgt/highv-quest for immunogenetic and statistical analyses (20) . the output results from the imgt/highv-quest analysis were stored in a local postgresql database, and structured query language (sql) was used to retrieve the data for further analysis. statistical calculations were carried out using jmp10® statistical software (sas institute, cary, nc, usa). antibody sequences from ighv1-2 and igk3-11 lineages were retrieved from our local antibodyome database consisting of immunogenetic data derived from 454 sequencing of the human cord blood igm library using sql statements. amino acid sequence identities between each of the selected lineage sequences from the 454 sequence data and pertinent germline sequences were calculated based on the pairwise alignment using local blast as implemented in bioedit v7.0.9 (21) . phylogenetic analysis was carried out using the archaeopteryx software (22) . our earlier observation of the extensive maturation of hiv-1 bnabs in contrast to those against some viruses causing acute infections led to the antibody germline/maturation hypothesis (9-11, 13, 14) . according to this hypothesis, it is critical to identify immunogens that would bind to germline and/or intermediate antibodies of bnabs, as well as the exploration of antibodyomes could be useful for identifying such immunogens (14) . figure 1 describes the timeline involving some of the key developments in current hiv-1 vaccine research focused on antibody germline-like intermediates and maturation pathways of bnabs. major research efforts in this direction were spearheaded by deep sequencing and structural biology studies of vrc01-like and other cd4-binding site (cd4b) antibodies from hiv-1-infected individuals. these studies delineated possible maturation pathways of such antibodies with high levels of somatic mutations and convergence in antibody recognition (23, 24) . both studies revealed that the putative germline precursors of these antibodies had weak or no apparent affinity for env, and acquisition of a large number of somatic mutations were needed for the breadth and potency of these antibodies. these studies also explored antibody diversity and found many intermediates of similar lineages of the heavy chain genes from the two ighv families vh1-2 and vh1-46 that paired with different light chain genes. thus, analysis of the vrc01-related antibodyome from hiv-1 infected patients revealed b cell maturation pathways that may help guide the vaccine-induced elicitation of such antibodies. however, if we could find germline-like intermediates of such bnabs from a naïve antibody repertoire, then www.frontiersin.org potential vaccine immunogens developed based on those templates would stimulate an adequate b cell immune response in healthy humans. to this end, we identified vrc01-like intermediate antibodies from a naïve antibody library of human cord blood, which is presented later in the text. we previously analyzed the igm repertoires of healthy individuals and identified several intermediates of b12 from the vh1-3 gene family (15) . sequence analysis of 28,925 unique sequences from the igm repertoires revealed a cdrh3 with a length (20 amino acids) and sequence similar (50%) to that of the b12 cdrh3, but the v gene associated with that cdrh3 was found to be hv4-b (15) . this finding indicates that long cdrh3s may not be a limiting factor for the development of bnabs (25) although long cdrh3 motifs with certain amino acid preferences and/or associations with particular heavy or light chain families favoring polyreactivity may not be undermined. stamatatos and coworkers have conducted experiments screening a large panel of recombinant envs for binding to the germline predecessors of b12, nih45-46, and 3bnc60 to test how env immunogens interact with the predicted germline versions of known bnabs (26) . they found that the mature bnabs reacted with diverse envs but the corresponding germline antibodies did not. they examined in detail the germline b12 and its chimeric forms -either the germline heavy chain paired with the mature light chain and vice versa -to test whether they could interact with any of the recombinant envs derived from clade a, b, and c viruses. among all the recombinant envs tested, at least one env (qh0692) was found to bind a b12 chimera with a mature heavy chain. however, this chimera failed to mediate calcium mobilization, indicating no bcr activation via bcr-antigen engagement. in other studies, they found that the elimination of certain conserved glycosylation sites on envs led to the binding of germline versions of vrc01 and nih45-46 and bcr activation (27) but that the modified envs did not interact with pg9 and 447-52d germlines (28) . haynes and coworkers have succeeded in finding envs capable of engaging the germline versions of a cd4bs bnab, ch103, while studying the co-evolution of the antibody in an hiv-1 infected patient (29) . they found that ch103 is less mutated than most other cd4bs bnabs, and importantly that the unmutated common ancestor of the ch103 lineage avidly bound the transmitted/founder hiv-1 envs. this finding suggests that early founder envs could bind optimally to the germline and intermediate versions of ch103, and therefore, are promising vaccine immunogens, representing an important step forward in hiv-1 vaccine development. similarly, the maturation pathway of the potent v1v2-directed hiv-neutralizing antibody, cap256-vrc26, has been described, in which a germline-like intermediate with a 35-amino acid residue long cdrh3 was shown to bind and neutralize the superinfecting virus weakly, but did not bind or neutralize heterologous viruses (30) . these results suggest that the cap256-vrc26 lineage could be initiated by using a rare superinfecting-virus-like v1v2 env. in another successful effort in identifying an env that could engage the germline versions of bnabs, scheif and coworkers devised a computation-guided approach combined with in vitro screening to engineer a gp120 outer domain. the designed protein not only bound to multiple vrc01-class bnabs and their germline precursors but also activated b cells expressing diverse intermediates of the bnabs (31) . therefore, priming with the protein and subsequent boosting with more native immunogens could help induce early somatic mutations and the ultimate elicitation of vrc01-class bnabs. interestingly, nussenzweig and coworkers' study showed that somatic mutations of the frs and insertions of some bnabs are required for their broad and potent hiv-1 neutralizing activity (32) . based on structural information, they made different germline versions of vrc01, nih45-46, 12a21, and 3bnc117, and found that mutations in frs were also essential for binding, breadth, and potency of most bnabs. this suggested that certain framework mutations could be critical and should be preserved for designing the intermediates of such bnabs. several other studies mining the hiv-1 infected donors' antibodyomes (33-35) revealed putative intermediates of bnabs. many of them with lower levels of somatic hyper mutations could bind to selective envs; for example, intermediates of pgt121-134 were able to preferentially bind native envs relative to monomeric gp120 (36) . we also identified 2f5-like antibodies (m66 and m66.6) with much fewer mutations than 2f5 and suggested their use as a model system for elicitation of such antibodies (37, 38) . all these newly discovered bnabs raise the hopes for effective hiv-1 vaccine development as they reveal characteristic features of bnabs that could help us understand the immunological basis critical for their production and also serve as templates for rational vaccine design. therefore, the focus has been dramatically shifted to explore and overcome the immunological hurdles associated with the elicitation of bnabs, namely, extensive somatic mutations of bnabs. major challenges remain in identification of intermediates with a minimal number of mutations, and appropriate env immunogens that would bind such intermediates and activate bcrs, which can lead to the maturation of the intermediate antibodies to bnabs. recently, new paradigms that better fit our increased knowledge of hiv immunopathology and which may be more helpful in guiding future vaccine research than did past unsuccessful approaches were discussed (17) . we previously characterized the human cord blood cell-derived igm antibodies using 454 sequencing to study gene diversity and somatic mutations (19) . naïve germline antibody repertoires, particularly from babies, may be quite unique for understanding the b cell maturation pathways, as they can also mount an immune response against hiv-1 as recently found (39) . our earlier gene usage analysis of the cord blood igm repertoire showed the biased ighv gene usages (19) as similar to adult igm repertoires (40) . however, we already noted that the ighv1-2 gene usage was significantly higher in the cord blood igm repertoire, i.e., an overall contribution of 20% as compared to 8% in adult igm repertoires. this suggested that the cord blood igm repertoire may be advantageous for the exploration of the ighv1-2*02 lineages when studying germline precursors and intermediates of vrc01 heavy chain. a total of 5,624 heavy chain and 1,096 light chain sequences of ighv1-2 and igkv3-11 lineages, respectively, were used to frontiers in immunology | hiv and aids select the top 10 sequences as closest intermediates for vrc01 in each heavy and light chain categories by using local blast searching. we performed phylogenetic analysis of the selected sequences to identify genetic relationships among vrc01-like intermediates of heavy (figure 2a) and light (figure 2b ) chains. we found two of the antibody heavy chains, hwav6 and jhedt, which were 100% identical to the ighv1-2*02 germline sequence. remarkably, their cdrh3 sequences had the same length (14 amino acids) as that of the vrc01 heavy chain. for these 10 heavy chain sequences, the cdrh3 lengths ranged from 8 to 16 amino acids with sequence variations at the junctions. one of the germline sequences, jhedt, had a point mutation at cys100tyr (kabat numbering) of cdrh3 that exactly mimicked the residue tyr100 of cdrh3 in vrc01. the residue tyr100 at cdrh3 of vrc01 is most likely contributed by the ighd3-16*02 germline with a point mutation cys100tyr. the other heavy chain sequence i76at, which was the closest to vrc01 heavy chain, also had the same mutation at cys100tyr. one of the other germline sequences, hwav6, had trp100b (kabat numbering) of cdrh3 that exactly mimicked the residue trp100b of cdrh3 in vrc01. intriguingly, the trp100b residue is a junctional amino acid of the cdrh3 in germline hwav6, and it exactly replicates the trp100b junctional residue of cdrh3 in vrc01. this suggests a possible maturation mechanism involved in the vrc01-like intermediates where junctional amino acids could determine the maturation pathway far preceding the somatic hypermutation required for affinity maturation (41) . most of the closest ighv genes, 8 out of 10 shown in the figure 2a , have at least one mutation in the v region, and www.frontiersin.org two sequences, g2w0t and gd60c, have two mutations at each of the cdrh1. the pre-existing amino acid mutations found in the v region and cdrh3 sequence information may inform the design of heavy chain germline-like precursors and intermediates, and help naturally reconstruct the b cell clonal lineages in the maturation pathways of vrc01. light chain recognition of envs by vrc01 and vrc01-related antibodies has been studied in detail using structural and 454 sequencing data (33) . the vrc01 light chain commonly uses the igkv1-33 lineage and has a characteristic five amino acid long cdrl3 and a distinctive two amino acid deletion in cdr l1. therefore, we selected the igkv1-33 lineage sequences with five amino acid length cdrl3s, but no sequences were found with a two amino acid deletion in cdr l1 ( figure 2b ). all of them had either framework or cdr mutations or both. four of them had a point mutation at cdrl1 and seven of them had a point mutation at cdrl3. the structural basis for germline gene usage of vrc01-related antibodies targeting the cd4bs has been previously described (42) , which revealed a set of signature features for these antibodies that were verified by mutagenesis. these signature features explained the origin of the igvh1-2 gene and antibody resistance for some env sequences. we found that characteristic residues including the trp100b of heavy chains were conserved while light chains did not have any characteristic residues as reported previously (42) . however, other pre-existing amino acid mutations in light chains could have implications for the vrc01-related intermediates with a characteristic cdrl3 of five amino acid length. we analyzed the amino acid length distributions of cdrh3 and cdrl3 sequences that were of vrc01 origins, namely, ighv1-2 and igkv3-11 for heavy and light chains, respectively, as derived from the human cord blood igm library (figure 3) . the cdrh3 lengths ranged from 4 to 27 amino acids, indicating high cdr3 length diversity (figure 3a ). vrc01 has a cdrh3 length of 14 amino acids, which is shorter than those of most other anti-hiv-1 antibodies (25) . the lcdr3 lengths ranged from 4 to 14 amino acids ( figure 3b) . the cdrl3 of vrc01 has a characteristic length of five amino acids with a mature genetic signature (33) . analysis of the human cord blood igm repertoire showed only a fraction of such light chains with a shorter length of five amino acids ( figure 3b) respectively. these plots show that there are position specific variations in the cdrh1 and cdrh2 regions of ighv1-2 genes. these could indicate possible ighv1-2 specific pre-existing amino acid mutations in cdrh1 and cdrh2, as observed in several naïve antibody heavy chain sequences, which could inform the design of germline precursors and intermediates of vrc01-like antibodies. we previously observed that the v-d-j rearrangement patterns occurred at different frequencies with 1,430 v-d-j combinations in a human cord blood igm repertoire (19) . figure 4a shows the v-d-j diversity associated with ighv1-2 gene sequences using a bubble plot for comparison with different d and j genes. the vrc01 heavy chain uses ighd3-16 and ighj2 genes to recombine with ighv1-2. however, other vrc01-related antibodies exhibit a skewed usage of ighj genes although at least three different ighj genes (ighj1, ighj2, and ighj4) are involved (23) . as the human cord blood igm library has a large functional v-d-j diversity, it can be used to identify potential vrc01-like heavy chain germline precursors and intermediates. in jawed vertebrates the expressed heavy chains may use any of the six ighd reading frames (rfs); however, rf1 is thought to be the preferred one as it mostly encodes tyrosine and glycine. the remaining five rfs encode either hydrophobic or charged amino acids, but the use of inverted rf1, rf2, and rf3 are discouraged. preferential usage of ighd rfs has been long implicated in b cell development and antigen-specific antibody production (43) (44) (45) , and selected based upon its amino acid content (46) . genetic control of igdh rf preference over the regulation of repertoire development has been recognized (47) . here, we have analyzed the productive ighd rf usages in a human cord blood igm library. frequency distribution of rfs is plotted using a pie chart as depicted in figure 4b . we noted that there were not any highly restricted usages of the ighd rfs although some preferential usages depending on the ighd genes were found. this clearly indicates that ighd rfs diversity could add more diverse amino acid contents leading to enormous cdrh3 diversity. it may also be possible that intermediates with different rf choices play a critical role in selecting certain maturation pathways efficiently. the antibody germline/maturation hypothesis led to a paradigm shift in the design of immunogens for bnab elicitation, as well as the realization of the importance of the complexity of the bnab maturation pathways, and exploration of human antibodyomes (14) . in fact, human antibodyome exploration is also promising for other fields of science and medicine (14, 48) . this antibodyome approach is now a major direction of research in the hiv-1 vaccine field (16, 49) . an important goal is to precisely identify naturally occurring germline-like precursors and intermediates of bnabs that could help designing novel immunogens, which could activate the corresponding bcrs and drive the immune system to produce bnabs within a short period of time. we presented an approach using a human cord blood igm library to identify putative germline precursors and intermediates of vrc01-like heavy and light chains, which could be useful in reconstructing the b cell clonal lineages in the maturation pathways of vrc01-related bnabs. this method has the potential to help in the identification of naturally occurring germline-like precursors and intermediates of any known bnab and in the development immunogens based on hiv-1 envs (50) and peptides (51) , as well as non-hiv-1 molecules (12) . however, major challenges remain and new paradigms that better fit our increased knowledge of hiv immunopathology could possibly be more helpful in guiding future vaccine research than did past unsuccessful approaches (17) . we thank the laboratory of molecular technology of saic-frederick, inc., for providing roche 454 sequencing service. we thank tina ju for critically reading the manuscript. this research was supported by the intramural research program of the nih, national cancer institute, center for cancer research, the intramural aids targeted antiviral program (iatap) of the nih and by federal funds from the nih, national cancer institute, under contract nos. no1-co-12400 and hhsn261200800001e. the www.frontiersin.org the antigenic structure of the hiv gp120 envelope glycoprotein crystal structure of a soluble cleaved hiv-1 envelope trimer cryo-em structure of a fully glycosylated soluble cleaved hiv-1 envelope trimer viral sequence diversity: challenges for aids vaccine designs antibody neutralization and escape by hiv-1 examination of the contributions of size and avidity to the neutralization mechanisms of the anti-hiv antibodies b12 and 4e10 hiv-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites hiv vaccine design and the neutralizing antibody problem extensive maturation of cross-reactive hiv-1 neutralizing antibodies but not of neutralizing antibodies against the sars cov, nipah and hendra viruses all known cross reactive hiv-1 neutralizing antibodies are highly divergent from germline and their elicitation may require prolonged periods of time germline-like predecessors of broadly neutralizing antibodies lack measurable binding to hiv-1 envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens identification of non-hiv immunogens that bind to germline b12 predecessors and elicit cross-reactive neutralizing hiv-1 antibodies maturation pathways of cross-reactive hiv-1 neutralizing antibodies therapeutic antibodies, vaccines and antibodyomes origin, diversity, and maturation of human antiviral antibodies analyzed by high-throughput sequencing hiv-1 neutralizing antibodies: understanding nature's pathways paradigm changes and the future of hiv vaccine research: a summary of a workshop held in baltimore on 20 construction of a large naive human phage-displayed fab library through one-step cloning expressed antibody repertoires in human cord blood cells: 454 sequencing and imgt/highv-quest analysis of germline gene usage, junctional diversity, and somatic mutations imgt((r)) tools for the nucleotide analysis of immunoglobulin (ig) and t cell receptor (tr) bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt phyloxml: xml for evolutionary biology and comparative genomics focused evolution of hiv-1 neutralizing antibodies revealed by structures and deep sequencing sequence and structural convergence of broad and potent hiv antibodies that mimic cd4 binding immunologic basis for long hcdr3s in broadly neutralizing antibodies against hiv-1 recombinant hiv envelope proteins fail to engage germline versions of anti-cd4bs bnabs engineering hiv envelope protein to activate germline b cell receptors of broadly neutralizing anti-cd4 binding site antibodies diverse recombinant hiv-1 envs fail to activate b cells expressing the germline b cell receptors of the broadly neutralizing anti-hiv-1 antibodies pg9 and 447-52d co-evolution of a broadly neutralizing hiv-1 antibody and founder virus developmental pathway for potent v1v2-directed hiv-neutralizing antibodies rational hiv immunogen design to target specific germline b cell receptors somatic mutations of the immunoglobulin framework are generally required for broad and potent hiv-1 neutralization multidonor analysis reveals structural elements, genetic determinants, and maturation pathway for hiv-1 neutralization by vrc01-class antibodies mining the antibodyome for hiv-1-neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains de novo identification of vrc01 class hiv-1-neutralizing antibodies by next-generation sequencing of b-cell transcripts the effects of somatic hypermutation on neutralization and binding in the pgt121 family of broadly neutralizing hiv antibodies cross-reactive hiv-1-neutralizing human monoclonal antibodies identified from a patient with 2f5-like antibodies structural basis for hiv-1 neutralization by 2f5-like antibodies m66 and m66.6 early development of broadly neutralizing antibodies in hiv-1-infected infants precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire junctional amino acids determine the maturation pathway of an antibody structural basis for germ-line gene usage of a potent class of antibodies targeting the cd4-binding site of hiv-1 gp120 forced usage of positively charged amino acids in immunoglobulin cdr-h3 impairs b cell development and antibody production preferential use of dh reading frame 2 alters b cell development and antigen-specific antibody production b cell development regulated by gene rearrangement: arrest of maturation by membrane-bound d mu protein and selection of dh element reading frames the restricted dh gene reading frame usage in the expressed human antibody repertoire is selected based upon its amino acid content regulation of repertoire development through genetic control of dh reading frame preference the promise and challenge of high-throughput sequencing of the antibody repertoire b-cell-lineage immunogen design in vaccine development with hiv-1 as a case study immunogen design for hiv-1 and influenza recognition of synthetic glycopeptides by hiv-1 broadly neutralizing antibodies and their unmutated ancestors content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. the guest associate editor marc h. v. van regenmortel declares that, despite having collaborated with the author dimiter s. dimitrov, the review process was handled objectively and no conflict of interest exists. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-300642-c7adeis1 authors: lai, andrew sh; lai, kar neng title: viral nephropathy date: 2006 journal: nat clin pract nephrol doi: 10.1038/ncpneph0166 sha: doc_id: 300642 cord_uid: c7adeis1 viral infections can cause many glomerular diseases. the diagnostic criteria for virus-related nephropathy include detailed clinical and laboratory data, and tissue molecular analysis. several mechanisms are involved in the pathogenesis of virus-related nephropathy, including tropism of the virus in the kidney, induction of abnormal immune complexes, direct cytopathogenic effects, and multiorgan failure. hepatitis b virus is associated with membranous nephropathy and mesangiocapillary glomerulonephritis in endemic areas. hepatitis c virus causes various forms of glomerulonephritis, including cryoglobulinemia-mediated glomerulonephritis. infection with hiv is associated with a collapsing focal segmental glomerulosclerosis, a distinctive disease that affects mainly africans and african americans. in the course of hiv infection, other types of immune complex glomerulonephritis can occur, most frequently in whites. recent reports indicate a role for parvovirus b19 in 'idiopathic' collapsing focal segmental glomerulosclerosis. both hantaviruses, and coronaviruses associated with severe acute respiratory syndrome, can lead to acute renal failure. renal biopsy followed by appropriate serological and molecular testing is essential for defining virus-related glomerular lesions and guiding prognostic and therapeutic evaluation. the pathogenetic links between viral infection and renal disease are often difficult to establish. the criteria for proving causality are complex and include (besides recognition of the clinical syndrome) serological diagnosis, identification of specific viral antigenemia, and detection in glomerular structures of viral antigens and host antibodies. according to koch's postulates, the etiological link should be confirmed by complete cure following eradication of the virus, but this is not always possible. the concentration of viral antigens is higher in tissue than in the circulation, where antigens are complexed with specific autoantibodies. virological and molecular analy sis of pathologic tissues by in situ hybridiza tion, polymerase chain reaction and ultra structural analysis led to successful detection and identifica tion of the virus. it should be noted that tubular uptake of viral particles is common and does not necessarily establish an etiological link with renal disease. improvement of the renal disease concomitant with clearance of the suspected antigen, or recurrence of glomerulonephritis following reinfection, are additional clinical criteria. different mechanisms are operative in different viral nephropathies (box 1). in acute glomerulonephritis, direct viral infection of the glomerulus induces proliferative changes following release of cytokines. 1 the nephropathy is reversible in most cases if the virus is rapidly cleared. in chronic forms of glomerulonephritis, persistent viral infection provides continuous antigenic stimulation, resulting in antibody production and formation of immune complexes. studies indicate a role in the disease pathogenesis for these immune complexes, which can be derived from the circulation or formed in situ. 2,3 viral proteins cause inflammatory renal diseases via synthesis of various mediators that can cause sclerosis and worsen glomerulopathy. 4, 5 viral infections can cause many glomerular diseases. the diagnostic criteria for virus-related nephropathy include detailed clinical and laboratory data, and tissue molecular analysis. several mechanisms are involved in the pathogenesis of virus-related nephropathy, including tropism of the virus in the kidney, induction of abnormal immune complexes, direct cytopathogenic effects, and multiorgan failure. hepatitis b virus is associated with membranous nephropathy and mesangiocapillary glomerulonephritis in endemic areas. hepatitis c virus causes various forms of glomerulonephritis, including cryoglobulinemia-mediated glomerulonephritis. infection with hiv is associated with a collapsing focal segmental glomerulosclerosis, a distinctive disease that affects mainly africans and african americans. in the course of hiv infection, other types of immune complex glomerulonephritis can occur, most frequently in whites. recent reports indicate a role for parvovirus b19 in 'idiopathic' collapsing focal segmental glomerulosclerosis. both hantaviruses, and coronaviruses associated with severe acute respiratory syndrome, can lead to acute renal failure. renal biopsy followed by appropriate serological and molecular testing is essential for defining virus-related glomerular lesions and guiding prognostic and therapeutic evaluation. a direct cytopathic effect of viral proteins has also been postulated. 6 in hepatitis c virus (hcv)-induced mesangiocapillary glomerulonephritis (mcgn), production of circulating cryo globulins is induced as an abnormal host response to infection. cryoglobulins are either type ii or type iii. at least two classes of immuno globulins are involved, one of which is polyclonal. 7 in acute renal failure associated with infection by hantavirus or severe acute respiratory syndrome coronavirus, the pathogenetic mechanisms of interstitial nephritis, disseminated intravascular coagulopathy, and multiorgan failure-rather than formation of immune complexes-are predominant. box 2 lists the viruses that are known to induce renal diseases. globally, the most frequent and well recognized virus-related glomerulonephropathies are those associated with hepatitis b virus (hbv), in which formation of immune complexes is important. hcv is the etiological agent of cryoglobulinemiarelated mcgn in most cases. infection with hiv can induce a broad spectrum of glomerular lesions via multiple pathogenic mechanisms. parvovirus b19 (pvb19) is associated with non-hiv collapsing glomerulopathy, 8 idiopathic focal segmental glomerulosclerosis (fsgs), 9 and immune complex glomerulonephritis. 10 polyoma bk virus and hantavirus most frequently cause tubulointerstitial damage; occasionally, virus is simultaneously localized to the glomerulus. a rare or speculative role in glomerulonephritides is currently attributed to other viruses, such as those causing yellow fever, 11 mumps, 12 measles, 13 varicella, 14 and herpes. 15 hbv is a hepatotropic, double-stranded dna virus of the hepadnaviridae family. hbv itself is not cytopathic; hepatitis develops as a result of the host's immune reaction to infected hepatocytes. hbv uses reverse transcriptase to transcribe rna into dna. unlike retroviruses, however, hbv dna is not integrated into host cell dna during replication. after an hbv particle binds to and enters a hepatocyte, hbv dna enters the cell's nucleus and is converted into covalently closed circular dna. this highly stable genetic material acts as the intermediate template for transcription of rna copies. this pregenomic messenger rna is transported to the cytoplasm. it has dual functions: acting as a template for synthesis of new hbv dna, and carrying genetic information to direct synthesis of viral proteins. today, an estimated 350-400 million people worldwide are infected with hbv. in endemic areas, transmission is usually vertical-that is, from infected mother to child. horizontal transmission occurs via direct contact with blood (e.g. during blood transfusions) or mucous membranes (e.g. during sexual contact), or via the percutaneous route upon contact with blood or body fluids (e.g. during intravenous drug use and needle sharing). familial clustering of the virus occurs in some regions. the reported prevalence of hbv-associated nephropathy closely parallels the geographic patterns of prevalence of hbv. 16 frequently reported in asian populations 17 and in children, 18 particularly male children. 16 by contrast, mesangial proliferative forms with iga deposits seem to be most common in adults. 19 three types of glomerulonephritis with pathologic characteristics similar to the human subtypes have been described in woodchucks chronically infected with hepatitis virus. 20 as in humans, the membranous pattern of injury most frequently affects young woodchucks, whereas the mesangial proliferative pattern of injury tends to affect older animals. the male : female ratio of affected woodchucks was significantly greater than that of the chronic carrier population. 20 in most reports, diagnosis of hbv-asso ciated glomerulonephritis has been based on persistence of circulating hbv or hbv dna, absence of other causative agents, and presence of hbvspecific antigen(s) or viral genome in the glomerulus. one major difference between the human and woodchuck studies is that the hepatitis b e antigen (hbeag) system has not been characterized in the latter. in clinical practice, regression of pathology following viral eradication is not easy to demonstrate because of ethical concerns relating to repeat renal biopsies in humans subsequent to clinical remission. as such, the diagnosis of hbv-associated renal disease usually relies heavily upon detection of hbv-specific antigen(s) in glomeruli. laboratory testing for diagnosis and assessment of response to treatment should include standard liver biochemistries (serum alanine aminotransferase, γ-glutamyltransferase, and bilirubin levels), and hbv serologies (hepatitis b surface antigen, hbeag, anti-hepatitis b e, and anti-hepatitis b core antigen antibodies). hbeag is present in 80% of patients, who might also have high titers of anti-hepatitis b core antigen. 21 subjects with biochemical hepatitis should be tested for circulating hbv dna 22 and undergo liver biopsy. an α-fetoprotein assay could be an important adjunct. 23 serum c3 and c4 levels can be low in 20-50% of patients. hbv-related membranous nephropathy tends to manifest slightly differently in pediatric and adult patients. in children, there is a strong male preponderance, and the most frequent presentation is nephrotic syndrome, microscopic hematuria, and normal or mildly impaired renal function. 24 pediatric chronic hbv carriers often do not have overt liver disease, and transaminase levels are usually normal. in adults, proteinuria or the nephrotic syndrome are the most common manifestations. adult male predominance is less obvious than in pediatric populations. adults are more likely than children to have hypertension, renal dysfunction, and clinical evidence of liver disease. the prognosis of hbv-associated membranous nephropathy in children is favorable. stable renal function and high rates of spontaneous remission have been reported in several geographical areas in which disease prevalence is high. by contrast, adults with hbvassociated membranous nephro pathy typically develop progressive disease. in hong kong, up to 29% of patients had progressive renal failure, and another 10% developed terminal uremia over 5 years. 21 the prognosis is even worse for patients with nephrotic-range proteinuria and abnormal liver function tests at presentation. over 50% of these patients require renal replacement therapy within 3 years. 25 vertical transmission is associated with poorer outcomes than horizontal transmission, as is endemic versus sporadic infection. 21,26 box 2 viral infections that cause nephropathy. as mentioned above, hbv infection can also cause mcgn (with or without cryoglobulinemia), mesangial proliferative glomerulonephritis, and igan. 19,27 polyarteritis nodosa has been reported in some patients with hbv and might respond to treatment with corticosteroids and interferon-α. 28 occasional concomitance of the pathologic subtypes can lead to 'double' glomerulopathies. for instance, membranous nephropathy and igan have been reported to coexist in an hbv carrier. 29 unlike affected children, who have a high rate of spontaneous remission, 30 adults with hbvassociated membranous nephropathy typically develop progressive disease. 21 various management strategies have been tried, but an ideal agent is yet to be found. treatment for hbvassociated renal disease should ideally achieve the following objectives: (i) amelioration of nephrotic syndrome and its complications; (ii) preservation of renal function; (iii) normalization of liver function and prevention of hepatic complications of hbv; and (iv) permanent eradication of hbv. because of the involvement of immune complexes in the disease, immunosuppressive therapy-similar to that used in the idiopathic form of the disease-was once fashionable. corticosteroids were reported to provide symptomatic relief in isolated cases. the contemporary view, however, is that steroid and cytotoxic agents can cause deleterious hepatic flares or even fatal decompensation by enhancing viral replication when the drugs are withdrawn. 31 another approach is treatment with an antiviral agent. interferon-α is a naturally occurring cytokine produced by b lymphocytes, null lymphocytes, and macrophages that exerts antiviral, antiproliferative and immuno modulatory effects. while reportedly useful in children, 32 interferon-α has produced mixed results in adults with hbv-associated membranous nephropathy. 21, 26 introduction of the nucleoside analog lamivudine has revolutionized the treatment of chronic hbv infection. 33 lamivudine is the (-)-enantiomer of 3'-thiacytidine. this analog inhibits dna synthesis by terminating the nascent proviral dna chain through inter ference with the reverse transcriptase activity of hbv. in children and adults with hbv-asso ciated membranous nephropathy, lamivudine has been anecdotally reported to induce remission of nephrotic syndrome and to suppress viral replication. 24, 34 in a recent analysis comparing 10 adult nephrotic patients with hbv-related membranous nephropathy who received lamivudine with 12 matched historical control subjects who presented in the pre-lamivudine era, lamivudine significantly improved proteinuria, aminotransferase levels, and renal outcome over a 3-year period. 25 randomized studies in a larger cohort of patients are needed to prove this effect. a potential limitation of prolonged treatment with lamivudine is emergence of drugresistant virus strains resulting from induction and selection of hbv variants with mutations at the tyrosine-methionine-aspartate-aspartate (ymdd) motif of dna polymerase. one agent that might be useful in lamivudine-resistant cases is adefovir dipivoxil, an acyclic nucleotide analog that is effective against both lamivudineresistant hbv mutants and wild-type hbv. 35 this agent does have nephrotoxic potential, and there are no clinical data on its efficacy in hbvrelated membranous nephro pathy that does not respond to lamivudine treatment. data do indicate, however, that the recommended dose of 10 mg adefovir dipivoxil is associated with a relatively low risk of nephro toxicity. 36 while awaiting an ideal agent for treatment of hbv-associated glomerulopathy, active immuniza tion remains the most effective means of immunoprophylaxis. 37 in taiwan, active immunization of all newborns since 1984 has led to a dramatic (10-fold) decline in the incidence of neonatal hbv infection and its sequelae. 38 in the us, universal vaccination of infants began in 1991, and a 67% reduction in hbv infection was recorded 10 years later. in 2003, the who recommended that all countries establish universal hbv immunization programs for infants and adolescents. 39 hcv is a small rna virus in the flaviviridae family. evolution of hcv has been characterized by the emergence of six major genotypes (based on sequence homology) and more than 50 subtypes. to date, around 170-200 million individuals worldwide are estimated by the who to be chronically infected with hcv. although viral replication is primarily confined to the liver, a variety of extrahepatic disease manifestations are associated with hcv infection. the principal renal manifestation of hcv infection is mcgn type i, usually in the context of type ii (mixed) cryoglobulinemia. 40 the prevalence of mcgn in hcv type ii cryoglobulinemia is approximately 30%. mcgn is occasionally observed in patients with hepatitis c in the absence of cryoglobulinemia. 40 type ii mcgn (e.g. dense deposit disease) has not been described in association with hcv infection. two immunologic features of hcv might underlie predisposition to extrahepatic disease manifestations. first, hcv is known to evade immune elimination, leading to chronic infection and accumulation of circulating immune complexes. mcgn associated with hcv infection might result from this phenomenon. second, hcv stimulates production of monoclonal rheumatoid factors. this feature causes type ii cryoglobulinemia, which accounts for most symptomatic cryoglobulinemic vasculitis. although this manifestation occurs relatively infrequently, as do all the extrahepatic disease manifestations, it accounts for much of the increased morbidity and mortality that accompanies the disease. between 35% and 90% of hcv-infected patients have been reported to have mixed cryoglobulinemia. 41, 42 prevalence increases with duration of hepatitis. it should be noted, however, that the prevalence of mixed cryo globulinemia has not been determined in popula tions of unselected hcv-infected patients. frank symptomatic cryoglobulinemia affects 1% or less of patients and is usually associated with high levels of rheumatoid factor and cryo globulins. testing of unselected patients with cryo globulinemia has shown that up to 90% have anti-hcv antibody. type i mcgn has long been regarded as idiopathic, but a consider able proportion of patients has concomitant chronic hcv infection. the exact proportion of patients with type i mcgn who are anti-hcv-antibody-positive is unknown. the true prevalence of mcgn without detectable cryoglobulinemia is difficult to assess. such cases might represent a subclinical form of cryoglobulinemia in which circulating cryoglobulins have not been detected by standard laboratory techniques. further, production of igm antibodies with anti-igg activity might induce formation of immune complexes that lack cryoprecipitable properties. 43 finally, these patients might develop detectable circulating cryoglobulinemia only later in the course of the disease. 44 laboratory testing coupled with renal biopsy establishes the diagnosis of hcv-related mcgn. most patients will have anti-hcv antibody, as well as hcv rna, in serum. serum transaminase levels are elevated in 70% of patients. cryoglobulins are detected in 50-70% of patients. serum electrophoresis and immunofixation detects type ii (mixed) cryo globulins. monoclonal rheumatoid factor, almost in variably an igmκ, is a distinguishing feature of cryo globulinemic glomerulonephritis. the amount of cryoglobulins, usually measured as a cryocrit, varies between patients and with time in a given patient (range 2-70%). κ light chains are also commonly present in the urine. the serum complement pattern, which does not change greatly with clinical disease activity, is also discriminative. characteristically, early complement components (c4 and c1q) and ch50 are present at very low or undetectable levels in these patients, whereas the c3 level tends to remain normal or is only slightly depressed. renal disease associated with hcv is rare in children. the typical age of disease onset is the fifth or sixth decade of life after longstanding infection, often in association with mild subclinical liver disease. patients might have other symptoms of cryoglobulinemia, such as palpable purpura and arthralgias. renal manifestations include nephrotic (20%) or non-nephrotic proteinuria and microscopic hematuria. 45 acute nephritic syndrome is the presenting feature in about a quarter of cases. renal insufficiency, frequently of mild severity, occurs in about half of patients. over 80% of patients have refractory hypertension at presentation, which might be responsible for a considerable number of cardiovascular deaths. the clinical course of hcv-associated renal disease can vary dramatically. this disease does not frequently progress to uremia, despite the persistence of urinary abnormalities in the majority of patients. when such progression does occur, it tends to be in males and those of older age. according to an italian series, around 15% of patients eventually require dialysis. 46 other forms of glomerular injury, including membranous nephropathy, fsgs, mesangial proliferative glomerulonephritis, and crescentic glomerulonephritis, have been reported in hcv carriers as individual case reports and small series. notably, membranous nephropathy in hcv carriers is characterized by the absence of cryoglobulin and male predominance. 47 in general, therapy can be directed at two levels: removal of cryoglobulins by plasmapheresis; and inhibition of cryoglobulin synthesis by attenuating immune responses (using steroids or cytotoxic agents) or suppressing viral replication (using interferon and ribavirin). before the association between hcv and cryo globulinemic mcgn was established, steroids and cyclophosphamide were the mainstays of treatment. our awareness of this link has facilitated a more rational approach to management of this condition. controlled trials have shown that antiviral therapy with interferon-α is asso ciated with improvements in systemic symptoms of immune complex disease. unfortunately, posttherapy relapse occurs in a large pro portion of patients, particularly when interferon monotherapy is administered in short courses. introduction of combination therapy with interferon-α2b plus ribavirin was an important milestone in the treatment of chronic hepatitis c. 48 this cocktail has also produced favorable results in mixed cryo globulinemia, although non-responses and relapses after initial improvements still occur. 49 the introduction of pegylated forms of interferon (peginterferon) in 2000 was another breakthrough in treatment of chronic hepatitis c. recent data on peginterferon and ribavirin combination therapy are encouraging. 50 an increased rate of treatment failure in carriers of hcv genotype 1 has been recognized. 51 observational studies support the effectiveness of peginterferon and ribavirin combination therapy in hcv-associated cryoglobulinemic mcgn. one therapeutic drawback is the hemolytic effect that complicates ribavirin therapy, particularly in patients with functional renal impairment. this difficulty has been overcome by adjusting the dose according to glomerular filtration rate instead of body weight alone, and utilizing recombinant erythropoietin to combat anemia. post-treatment renal biopsy showed histological improvement in two of three patients who received combination therapy for 12 months. 52 the wide spectrum of glomerulopathies occurring in the course of hiv infection can be classified into four groups. the first group is the classical hiv-associated nephropathy (hivan), a distinct entity with histological features of fsgs with tuft collapse or, more rarely, mesangial hyperplasia. hivan seems to be related to a direct effect of hiv or viral proteins on renal epithelium. 53 the second group is a diffuse proliferative-mesangiocapillary or lupuslike glomerulonephritis, with predominantly mesangial immune deposits, also known as hiv immune-complex-mediated disease. 54 this group also includes other immune-complex-mediated glomerulonephritides with more-heterogeneous histological features. the third group (which includes immunotactoid glomerulonephritis) is heterogeneous with regard to glomerular lesions and pathogenic mechanisms, some of which are still undefined. the true role of hiv infection in glomerulopathies of this type is also uncertain. the final group includes hiv-associated thrombotic microangiopathy/hemolytic uremic syndrome, in which hiv is the main, but not sole, etiological factor. ethnic/geographic background is an important determinant of the type of glomerulopathy associated with hiv; for example, collapsing fsgs is prevalent in patients of african descent. the fsgs variant of hivan is the most commonly reported chronic renal disease associated with hiv infection. hivan affects up to 10% of hiv-infected patients of african descent-mainly males at risk of drug abuse, and often african americans. glomerular changes associated with this variant are capillary wall collapse of varying severity, with widening of bowman's space. visceral epi thelial cells undergo hyperplasia and hypertrophy, and develop protein inclusions in their swollen cytoplasm surrounding the collapsed lobules. sclerosis affects segments of capillary tuft or the whole glomerular surface. tubular cells might undergo degenerative changes, necrosis or flattening. large dense casts can develop in dilated tubules. detection of hiv rna in renal tissue from patients with hivan, and of hiv dna in patients with and without nephropathy, 55 raises the question of whether renal cells can be infected in vivo (tubular epithelial cells can be infected in vitro). 4 renal uptake of viral gene products might induce transactivation of host lai and lai may 2006 vol 2 no 5 www.nature.com/clinicalpractice/neph genes. in renal cells, hiv proteins can cause apoptosis, 4 phenotypic modifications, 56 and subsequent tubulointerstitial fibrosis. clinical manifestations of hivan with fsgs are nephrotic-range proteinuria and renal insufficiency. hypertension and edema are uncommon. in overt cases, ultrasonography typically reveals enlarged, highly echogenic kidneys, which probably develop in response to microcystic tubular dilatation. before effective antiretroviral treatment was available, clinical progression was rapid. intensive antiretroviral treatment delays progression. 57 pathology of the hiv-associated disease mediated by immune complexes resembles lupus nephropathy. the clinical presentation is nephrotic syndrome with microscopic hematuria. progression to renal failure occurs, but more slowly than in hivan. patients often have hcv coinfection, but hiv seems to have the prevailing role. viral antigen has been detected in glomeruli, and antibodies eluted from the kidney react with hiv antigens in circulating immune complexes (iga-p24 antigen, igg-p24 and igg-gp120). in cases of hiv-related igan, circulating immune complexes containing iga idiotype antibodies have been detected. 58 most patients with hiv-associated thrombotic microangiopathy/hemolytic uremic syndrome present with acute renal failure, microscopic hematuria, and non-nephrotic proteinuria. multiorgan involvement is frequent and prognosis is poor, with a high rate of mortality. multifactorial etiologies encompass drugs, neoplasia, lymphoma and infection. pvb19 has been associated with acute glomerulonephritis. typical life stage of onset is the second or third decade. patients present with mild proteinuria and microhematuria, with low levels of serum complement 3. the pathology of this disease is characterized by endo capillary glomerulonephritis, mcgn, or both, with sub endothelial deposits. pvb19 capsid protein is found in the glomeruli. 10 spontaneous recovery is the norm. pvb19 is also associated with collapsing glomerulopathy. prevalence of pvb19 dna in renal biopsies (78%) and peripheral blood (87%) is significantly higher in patients with collapsing glomerulopathy than in those with other nephropathies. 10 glomerular and tubular infection with pvb19 might trigger collapsing glomerulopathy, but only in patients with immune defects and a racial pre disposition (african descent). 8 most intriguingly, tanawattanacharoen and coworkers 9 detected pvb19 dna in 80% of patients with 'idiopathic' fsgs, and frequently also in controls. these results possibly reflect the presence of latent dna from past infection. failure to localize pvb19 nucleic acid within kidney is evidence against ongoing, high-level viral replication. hantaviruses are responsible for 'hemorrhagic fever with renal syndrome' , an acute inter stitial nephritis resulting from direct vascular injury of renal tissue. 59 the severe form leads to acute renal failure in 50% of cases. less severe forms occur in nonendemic areas, and present primarily as fever, hepatitis, and mild renal impairment. in most cases, glomeruli are not affected and the pathology is tubulointerstitial. isolated cases with immune complex glomerular disease have been described, in association with diffuse proliferative glomerulonephritis and complete recovery after remission of the systemic clinical syndrome. 60 six percent of patients suffering from severe acute respiratory distress syndrome had acute renal impairment. 61 despite detection of viral dna in the urine, there was no evidence of viral tropism of the kidney. the pathology is exclusively tubulointerstitial nephritis. the mechanism of disease is probably related to multiorgan failure, rhabdomyolysis, and hemodynamic disturbance. renal infection with bk virus affects kidney allograft recipients, leading to renal dysfunction and sometimes graft loss. 62 more rarely, acute interstitial nephritis is observed in immunocompromised patients. 63 hepatitis a virus infection can present as acute post-infectious glomerulopnephritis with pathology resembling that of igan. 64 more commonly, hepatitis a virus induces acute renal failure secondary to acute fulminant hepatitis. 65 acute renal failure complicating other viral infections, such as epstein-barr virus 66 and dengue fever, 67 is related to multiorgan failure, rhabdomyolysis, and hepatorenal syndrome. diverse mechanisms of glomerular and tubulointerstitial injury and heterogeneous clinicopathologic patterns underlie the relationship between viral infection and glomerular disease. the etiological role of some viruses is still un defined. molecular biology techniques are vital in elucidating the precise location and role of viruses in the pathogenesis of virus-related nephropathy. treatment of hepatitis b virus-associated membranous nephropathy with recombinant alphainterferon a one-year trial of lamivudine for chronic hepatitis b: asia hepatitis lamivudine study group hbv associated nephrotic syndrome: resolution with oral lamivudine adefovir dipivoxil for the treatment of lamivudine-resistant hepatitis b mutants adefovir dipivoxil in chronic hepatitis b infection clinical practice: prevention of hepatitis b with the hepatitis b vaccine universal hepatitis b vaccination in taiwan and the incidence of hepatocellular carcinoma in children: taiwan childhood hepatoma study group safety of immunisation and adverse events following vaccination against hepatitis b membranoproliferative glomerulonephritis associated with hepatitis c virus infection immunological disorders in c virus chronic active hepatitis: a prospective casecontrol study hepatitis c, cryoglobulinemia, and cirrhosis: a meta-analysis secondary membranoproliferative glomerulonephritis hepatitis c virus infection and acute or chronic glomerulonephritis: an epidemiological and clinical appraisal renal involvement in hepatitis c infection: cryoglobulinemic glomerulonephritis long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis membranous glomerulonephritis associated with hepatitis c virus infection: case report and literature review randomised trial of interferon α2b plus ribavirin for 48 weeks or for 24 weeks versus interferon α2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis c virus: international hepatitis interventional therapy group (ihit) treatment of refractory, symptomatic, hepatitis c virus related mixed cryoglobulinemia with ribavirin and interferon-alpha peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection peginterferon-α2a and ribavirin combination therapy in chronic hepatitis c: a randomized study of treatment duration and ribavirin dose hepatitis c virus-related cryoglobulinemic glomerulonephritis: long-term remission after antiviral therapy renal complications of human immunodeficiency virus type 1 hiv-associated immunemediated renal disease renal epithelium is a previously unrecognized site of hiv-1 infection the dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and hiv-associated nephropathy cohort study of the treatment of severe hiv-associated nephropathy with corticosteroids brief report: idiotypic iga nephropathy in patients with human immunodeficiency virus infection mechanism of disease in hemorragic fever with renal syndrome different pathohistological presentations of acute renal involvement in hantan virus infection: report of two cases acute renal impairment in coronavirus-associated severe acute respiratory syndrome bk-virus nephropathy in renal transplants-tubular necrosis, mhc-class ii expression and rejection in a puzzling game bk virus renal infection in a patient with the acquired immunodeficiency syndrome iga-dominant glomerulonephritis associated with hepatitis a renal failure in otherwise uncomplicated acute viral hepatitis epstein-barr virus-associated acute renal failure: diagnosis, treatment, and followup clinical characteristics and risk factors for concurrent bacteremia in adults with dengue hemorrhagic fever some of the authors' work cited in this review was supported by the l & t charitable fund and indocafe. the authors declared they have no competing interests. key: cord-266294-ua22udlc authors: koch, oliver; sheehy, susanne; sargent, catherine; democratis, jane; abbas, sarah; schiefermueller, jurgen; angus, brian j. title: 29 antiviral drugs date: 2010-12-31 journal: side effects of drugs annual doi: 10.1016/s0378-6080(10)32029-0 sha: doc_id: 266294 cord_uid: ua22udlc publisher summary this chapter discusses the adverse effects of antiviral drugs used against cytomegalovirus, herpesviruses, hepatitis viruses, against hiv, and against influenza viruses. the cidofovir, drug active against cytomegalovirus, has been associated with bronchiolitis obliterans. aciclovir and valaciclovir has been reported with renal insufficiency. adefovir , a drug active against hepatitis viruses, is associated with the fall in creatinine clearance in patients with lamivudine-resistant hbe antigen (hbeag)negative disease. drugs active against hiv are comprehensively reviewed as in combination, nucleoside analogue reverse transcriptase inhibitors, nucleoside analogue reverse transcriptase inhibitors, and protease inhibitors. in a randomized controlled trial of indinavir, saquinavir and lopinavir in combination with low-dose ritonavir in 656 patients, median total cholesterol increased by 0.5 mmol/l in the patients with the highest minimum drug plasma concentrations. in patients with aids-associated aids dementia complex taking optimal stable background antiretroviral therapy including either abacavir or placebo, there was significantly more nausea in those who took abacavir. respiratory cidofovir has been used to treat adenovirus pneumonia in four pediatric lung transplant recipients (1 c ). two developed bronchiolitis obliterans, one of whom underwent retransplantation with a good outcome. the other developed tracheomalacia and required continuous low-flow oxygen and positive pressure ventilation through a tracheostomy. urinary tract of six hemopoietic stem cell transplant recipients, who were given cidofovir 5 mg/kg/week for 2 weeks, then on alternate weeks for a minimum of four (range 1-7) doses, three developed adenovirus hepatitis, two had colitis and one had nephritis (2 c ). all had cd34þ selected grafts and/or graft-versus-host disease and all had a cd4 count under 100 â 10 6 /l. the patients were pre-hydrated and brian j. angus antiviral drugs and given probenecid and intravenous immunoglobulin 1-2 g/kg/week followed by 0.5 mg/kg on alternate weeks for a minimum of four doses. two patients died of infection and four responded. none required withdrawal of treatment because of adverse effects. baseline creatinine was slightly raised (167 µmol/l) in the patient with hepatitis and improved with cidofovir. most of the patients had transient increases in creatinine as the number of doses increased. cidofovir 5 mg/kg/week or 1 mg/kg on alternate days adjusted to creatinine clear ance was used to treat 11 hemopoietic stem cell transplant recipients with invasive ade noviral infection, including three with pneu monia, one with hepatitis, four with hemorrhagic colitis and three with hemor rhagic cystitis (3 c ). all had concomitant intravenous immunoglobulin, and those with colitis had in addition one or more of oral immunoglobulin, ribavirin, or donor lymphocyte infusion. they varied in their degree of immunocompromise and the pre sence of graft-versus-host disease and some had leukocyte-depleted allografts. six died. there was nephrotoxicity in two cases. in a retrospective study of 19 recipients of hemopoietic stem cell transplants, mean age 42 years, who were given a mean of 4.5 (range 3-6) weekly doses of cidofovir 1 mg/ kg without probenecid for bk virus-asso ciated hemorrhagic cystitis, 84% had graft versus-host disease and were taking corti costeroids at the time of diagnosis (4 c ). there was no significant rise in creatinine in 14; 5 had renal insufficiency, but in all cases there were other potential causes. in only 3 cases could cidofovir have played a role. in those with normal renal function, 80-100% of the cidofovir was recovered from the urine, raising the possibility of therapeutic intravesicular administration. aciclovir psychiatric hemodialysis has been successfully used to treat an aciclovir induced psychosis (5 a ). • a 70-year-old man with rectal carcinoma and end-stage renal failure on hemodialysis was given intravenous aciclovir. he developed delirium, visual and auditory hallucinations, disorientation in place and time, and impaired recent memory. he recovered fully after 3 consecutive days of hemodialysis. urinary tract reports of renal insuffi ciency associated with the use of intravenous aciclovir continue to appear (6 a ). in a retrospective review of 126 children there was no effect of dose per kilogram, age, or sex on nephrotoxicity (7 c ). the predictors of aciclovir nephrotoxicity were the concomitant use of nephrotoxic drugs and impaired glomerular filtration rate (gfr) at baseline. fetotoxicity it has been suggested that maternal use of aciclovir may be associated with necrotizing enterocolitis in the neonate (8 a ). immunologic an acute allergic reaction to valaciclovir has been described (9 a ). • a 50-year-old woman developed general malaise, diffuse pruritus, angioedema of the hands and feet, and reduced consciousness and faintness within 5 minutes of taking valaciclovir 500 mg for genital herpes. skin prick tests for aciclovir were positive. urinary tract as with aciclovir, renal insufficiency has been reported with valaciclovir (10 a ) • a 73-year-old japanese man with cardio myopathy and chronic renal disease was admitted with anuria, systemic edema, and renal dysfunction after taking valaciclovir 1 g tds 5 days for herpes zoster infection with a non-steroidal anti-inflammatory drug. he was briefly dialysis-dependent but made a full recovery. adefovir (sed-15, 35; seda-30, 344; observational studies in patients with lamivudine-resistant hbe antigen (hbeag) negative disease, adefovir 10 mg alone (n = 14) or in combination with lamivudine (n = 28) caused a fall in creatinine clearance requiring a modification in adefovir dose in two patients on combined therapy; both had underlying liver cirrhosis (11 c ). one developed reduced liver function and biopsy showed steatohepatitis. one on adefovir monotherapy developed gastric cancer. five on combined therapy with underlying cirrhosis developed hepatocellular carcinoma, but this was not statistically significant. adefovir 10 mg/day in combination with lamivudine 300 mg/day has been used in 11 patients with recurrent hepatitis b virus infec tion after liver transplant (12 c ). all patients with chronic hepatitis b pre-transplant underwent a period of preoperative treat ment and peri-operative prophylaxis with lamivudine and hepatitis b immunoglobulin with or without adefovir if lamivudine-resis tant. no patient had adverse effects necessi tating drug withdrawal, but one required a dosage adjustment because of a rise in creati nine from 106 to 150 µmol/l. three had a recurrence of hepatocellular carcinoma, two of whom died 12 and 14 months after starting adefovir therapy and one of whom continued to be positive for hepatitis b virus dna. drug-drug interactions entacavir the interaction of entacavir 1 mg/day with adefovir 10 mg/day has been studied in a fixed-sequence crossover study in 26 heal thy adults (13 c ). the results suggested that combination therapy can be safely adminis tered without the need for dosage adjust ment of either drug. there was headache in nine subjects and dysmenorrhea in two when they took entecavir alone. see 'drugs active against influenza viruses: ion channel inhibitors', below. drug-drug interactions adefovir the interaction of entacavir 1 mg/day with ade fovir 10 mg/day has been studied in a fixedsequence crossover study in 26 healthy adults (13 c ). the results suggested that com bination therapy can be safely administered without the need for dosage adjustment of either drug. there was headache in nine subjects and dysmenorrhea in two when they took entecavir alone. lamivudine (sed-15, 1989 , seda-30, 344; seda-31, 480) observational studies in 33 treatmentnaive hbeag-positive children who took lamivudine and high-dose interferon alpha 2a combination therapy, flu-like symptoms and anorexia were the commonest adverse effects (90 and 76%); weight loss, nausea, vomiting, arthralgia, and loss of hair were also noted (14 c ) . in an open study of the pharmacokinetics of lamivudine in 12 patients receiving peri toneal dialysis, eye redness (n = 2) and diar rhea (n = 2) were the commonest adverse events (15 c ). comparative studies in a double-blind, phase iii, randomized, controlled trial of lamivudine 100 mg/day (n = 687) versus tel bivudine 600 mg/day (n = 680), creatine kinase activity was raised in patients receiv ing lamivudine (3.1%) and telbivudine (7.5%) and fell spontaneously during drug treatment to grade 2 or lower in 74% of those taking lamivudine and 67% of those taking telbivudine (16 c ). grade 3 or 4 rises in transaminases during treatment were more frequent with lamivudine than with telbivudine. observational studies high-dose ribavirin during an outbreak of severe acute respira tory syndrome in toronto was associated with a high rate of adverse events: anemia (odds ratio [or] = 3.0; 99% confidence interval [ci] = 1.5, 6.1), hypomagnesemia (or = 21; 99% ci = 5.8, 73), and bradycar dia (or = 2.3; 99% ci = 1.0, 5.1) (17 c ). the risks of anemia, hypomagnesemia, and bra dycardia attributable to ribavirin were 27%, 45%, and 17% respectively. the authors concluded that the use of high-dose ribavirin is appropriate only for the treatment of infectious diseases for which ribavirin has proven clinical efficacy, or in the context of a clinical trial. they further stated that ribavirin should not be used empirically for the treatment of viral syndromes of unknown origin. skin occasional rashes in areas of drug contact and conjunctival irritation occurred when aerosolized ribavirin was used for 10 months in an infant with immunodeficiency (18 a ). musculoskeletal in a double-blind, phase iii, randomized, controlled trial of lami vudine 100 mg/day (n = 687) versus telbivu dine 600 mg/day (n = 680), raised creatine kinase activity was more common in patients who took telbivudine and it fell sponta neously during drug treatment to grade 2 or lower in 67% of those who took telbivu dine and 74% of those who took lamivu dine. myopathy (characterized by muscle pain, weakness, and moderately raised crea tine kinase activity before and during treat ment) was reported in one patient after telbivudine therapy for 11 months. when telbivudine was withdrawn, the creatine kinase activity normalized within 1 month and the symptoms resolved over 9-12 months (16 c ). cardiovascular syncope occurred in a young man after he took tenofovir, emtricitabine and nevirapine for primary human immunodeficiency virus-1 (hiv-1) infection for 6 weeks and resolved after withdrawal of the antiretroviral drugs (19 a ). metabolism the hemochromatosis gene polymorphism hfe 187c> g and possibly mitochondrial haplogroup j gave relative protection against lipoatrophy during antiretroviral drug therapy in a trial in which 96 patients were randomized to didanosine þ stavudine or zidovudine þ lamivudine, combined with efavirenz and/ or nelfinavir in aids clinical trials group (actg) 384 sub-study a5005s (20 c ). stavudine had a less favorable effect on lipid profile and caused more lipoatrophy than abacavir (38% versus 4.8%) in a ran domized, open trial, stratified by viral load and cd4 cell count, in which 237 adults with hiv infection were assigned to stavudine (n = 122) or abacavir (n = 115), both com bined with lamivudine and efavirenz (21 c ). there were dose-related increases in total cholesterol, ldl cholesterol, and triglycerides in an open study in 56 seronegative volun teers, with persistent increases 2 weeks after withdrawal (22 c ). in a randomized controlled trial of indina vir, saquinavir and lopinavir in combination with low-dose ritonavir in 656 patients, med ian total cholesterol increased by 0.5 mmol/l in the patients with the highest minimum drug plasma concentrations (23 a ). gastrointestinal in a retrospective obser vational study of highly active antiretroviral therapy (haart), 27 of 50 patients who took indinavir in combination with zidovudine and lamivudine developed nausea and were significantly more likely to stop taking the treatment than those who were taking zidovudine þ lamivudine þ tenofovir (24 c ). in a long-term follow-up study of 200 asymptomatic hiv-positive participants for 157 weeks taking a combination of indinavir, lamivudine, and zidovudine, 40 stopped treat ment because of adverse effects, of which nausea (69%), diarrhea (37%), and abdom inal pain (28%) were the most common (25 c ). the most common adverse events in 21 of 151 patients in a prospective study of once-daily saquinavir þ ritonavir and two nucleoside reverse transcriptase inhibitors (nrtis) were abdominal discomfort, diar rhea, and vomiting (26 c ). similar findings were seen by other investigators (27 a ). liver in 199 hiv/hepatitis c co-infected patients, failure to achieve a sustained viral response, nrti therapy, didanosine, and stavudine were significantly associated with worsening of hepatic fibrosis in 34 (17%) (28 c ). after multivariate analysis, didanosine (or = 3.34; 95% ci = 1.39, 7.96) and failure to have a sustained viral response (or = 9.05; 95% ci = 2.06, 40) remained significantly associated with worsening of fibrosis. in a retrospective study of 868 hiv-posi tive subjects (94% men), first-line therapy was efavirenz, lamivudine, and zidovudine; women of child-bearing potential were given nevirapine instead of efavirenz. an efavirenz-based regimen was used in 825 and 39 received a nevirapine-based regimen (29 c ) . during the first year 48 subjects took isoniazid prophylaxis and 214 received antituberculosis therapy (2 months of rifam picin, isoniazid, pyrazinamide, and etham butol followed by 4 months of rifampicin and isoniazid). of a random sample of 133 tested, 17% were hepatitis b surface anti gen (hbsag)-positive. there was grade 2 or worse hepatotoxicity in 97 subjects (11%) and 40 had a first episode of grade 3 or 4 hepatotoxicity. antituberculosis therapy (adjusted hazard ratio [hr] = 8.5; 95% ci = 2.7, 27) and hbsag (adjusted hr = 3.0; 95% ci = 1.3, 7.0) were strongly associated with hepatotoxicity. however, hepatotoxicity had little impact on symp toms, the need for hospitalization and the need for a change in antiretroviral drug regimen. the use of isoniazid preven tive therapy during antiretroviral drug therapy did not increase the risk of hepatotoxicity. three adults developed nodular regen eration of the liver while taking haart regimens containing atazanavir, fosampre navir, and indinavir (30 a ). pancreas in the large eurosida study there was no association between an increased incidence of pancreatitis and cumulative exposure to antiretroviral drugs generally, or to didanosine and stavudine in particular (31 c ). there were 43 (9 presumptive) pancreatic events in 9678 individuals during 33 742 person-years (1.27 per 1000 person-years). the incidences among those with no, 2 or less, and over 2 years of exposure to antiretroviral drugs, including stavudine and didanosine, were 1.24, 1.73, and 0.78 per 1000 person-years, respectively. in multivariate analysis, higher baseline cd4 cell counts were associated with a reduced risk of pancreatitis. urinary tract of 445 hiv-positive patients who started to take tenofovir, 51 (11%) had reduced renal function (32 c ). multivariate analysis showed a significant association between reduced renal function and concurrent use of amprenavir and di danosine, age over 50 years and lower baseline weight. there was an association between concomitant use of tenofovir and amprenavir and reduced kidney function in 441 patients (or = 3.5). acute interstitial nephritis was seen on renal biopsy in three hiv-positive patients taking amprenavir þ tenofovir; it resolved after drug withdrawal (33 a ). in a randomized controlled trial of in dinavir, saquinavir, and lopinavir in combi nation with low-dose ritonavir in 656 patients, there was no apparent dose-related association with renal adverse events (23 c ). immunologic an allergic reaction with possible cross-reactivity to didanosine and tenofovir has been reported (34 a ). • a 25-year-old woman tested positive for hiv-1 and was given a once-a-day regimen including tenofovir plus emtricitabine and efavirenz. after 10 days she developed a diffuse rash with fever and glossitis. efavirenz was withdrawn but her conditions worsened over the next 3 days, so both tenofovir and emtrici tabine were withdrawn, with dramatic resolu tion of symptoms within 24 hours. after 10 days she was given zidovudine plus didanosine and ritonavir-boosted fosamprenavir. after 1 week she again developed a diffuse rash and the treatment was withdrawn. she restarted zidovudine and didanosine 7 days later and within a few days the rash appeared again. under observation she was given zidovudine and atazanavir and after 8 days lamivudine and low-dose ritonavir. she remained well, except for mild hyperbilirubinemia, during the next 5 months. fetotoxicity there is equivocal evidence of a relation between in utero nrti exposure and mitochondrial dysfunction in hivnegative children born to hiv-infected women. in 1037 hiv-negative children, possible cases with unexplained signs of mitochondrial dysfunction according to the enquête perinatale française criteria were identified in a retrospective review (35 c ). associations between possible mitochondrial dysfunction and both overall in utero nrti exposure and the trimester of first in utero nrti exposure were estimated by exact logistic regression. cases (n = 20) were significantly more likely to be boys and to be born in earlier years than non-cases (n = 1017). there was no association between overall in utero nrti exposure and mitochondrial dysfunction. in unadjusted models there were higher odds of first in utero exposure in the third trimester to lamivudine (or = 3.76 versus unexposed; 95% ci = 1.09, 12) and to zidovudine þ lamivudine (or = 3.29 versus unexposed; 95% ci = 0.96, 10) among cases than noncases. when adjusted for year of birth, the odds of first exposure in the third trimester to lamivudine (or = 11; 95% ci = 1.9, 76) and zidovudine þ lamivudine (or = 9.8; 95% ci = 1.7, 72) were significantly higher among cases than non-cases. incomplete data precluded control of possible confounding by maternal viral load and psychoactive drug use. reports of renal toxicity with the combination of tenofovir and didanosine in children suggest that this combination should be avoided (36 a ). combination of saquinavir with darunavir þ ritonavir is currently not recommended as plasma concentrations of darunavir are increased (37 c ). buprenorphine atazanavir and atazanavir þ ritonavir both resulted in increased metabolite concentrations of buprenorphine, and dosage reduction of buprenorphine is recommended; there was no change in the concentrations of the protease inhibitors (38 c ). warfarin in two patients who were taking a non-nucleoside reverse transcriptase inhibitor (nnrti), nevirapine, or a protease inhibitor, nelfinavir or lopinavir þ ritonavir, and two nucleoside analogues, high doses of warfarin were required to maintain therapeutic inrs (39 a ). warfarin has two enantiomers, r-warfarin and s-warfarin, which are substrates of cyp3a4 (r-warfarin), cyp1a2 (r-warfarin), and cyp2c9 (s-warfarin gastrointestinal in patients with aidsassociated dementia adc taking optimal stable background antiretroviral therapy including either abacavir or placebo there was significantly more nausea in those who took abacavir (40 c ). immunologic three patients developed painful lymphadenopathy shortly after starting to take abacavir, mimicking immune reconstitution syndrome (41 a ). they also had a fever and a rash and all were hla-b*5701-positive. they recovered after withdrawal of abacavir. abacavir hypersensitivity occurred in a 36-year-old man after he switched from a twice-daily to a once-daily regimen (42 a ). in a 52-year-old woman, abacavir hyper sensitivity presented as acute fibrinous and organizing pneumonia, with dyspnea, hypoxia, and bilateral infiltrates (43 a ). susceptibility factors genetic a sequence variation in the hiv reverse transcriptase codon 245 has been associated with host hla-b*5701 in 392 hiv-infected, antiretroviral drug-naive adults, and the relation between the codon 245 variation and premature abacavir withdrawal was investigated in 982 treated individuals (44 c ). only one of 24 subjects with b*5701 harbored virus with the clade b 'wild-type' amino acid 245v, compared with 278 of 368 who did not have b*5701. the sensitivity and specificity of codon 245 substitutions for predicting hla-b*5701 were 96% and 75% respectively, and the positive and negative predictive values were 20% and 99.6% respectively. the authors argued that the reverse transcriptase codon 245 could be adopted as a simple, low-cost screening method to identify individuals who could be safely treated with abacavir when detection of hla-b*5701 is not rapidly and easily available. drug-drug interactions alcohol three patients had possible reactions to alcohol while taking abacavir (45 a ). one had a disulfiram-like reaction (nausea, facial flushing, tachycardia) repeatedly on rechallenge with alcohol. another described a feeling of being drunk after small amounts of alcohol. a third had malaise after increasing his alcohol intake. the authors suggested that abacavir might inhibit alcohol dehydrogenase. urinary tract fanconi syndrome and nephrogenic diabetes insipidus associated with didanosine have been reported (46 a ). • a 40-year-old man developed polydipsia, poly uria, fatigue, and weight loss after taking dida nosine, lamivudine and boosted atazanavir for 2 years. he had hypophosphatemia, hypouricemia, hyperchloremic metabolic acidosis with a normal anion gap, normoglycemic glycosuria, and low-molecular-weight protei nuria. the plasma antidiuretic hormone (adh) concentration was high at 4.8 pg/ml (reference range 1.4-4.4 pmol/l). the didano sine was replaced with another protease inhi bitor and the other medications remained unchanged. he improved slowly. drug-drug interactions ganciclovir it has been suggested that ganciclovir and its prodrug valganciclovir inhibit purine nucleoside phosphorylase (pnp) in a similar manner to tenofovir and increase didanosine concentrations, reducing its efficacy (47 a ). • a 68-year-old woman with hiv and cyto megalovirus enteritis was given valganciclovir, lamivudine, didanosine, and lopinavir þ ritona vir. after 3 months, her viral load fell to less than 50 copies/ml and the cd4þ cell count was 317 â 10 6 /l. over the next 9 months her viral load remained suppressed, but the cd4þ cell count fell to 83 â 10 6 /l and she had symptoms of didanosine toxicity. didanosine was replaced with abacavir, leading to complete recovery of the cd4þ cell count and resolu tion of symptoms. reduction of the dosage of didanosine or substitution with an alternative antiretro viral drug may be necessary when ganciclo vir is used. metabolism lipodystrophy tended to occur more often in 58 hiv/hepatitis c co-infected patients who had severe weight loss than in 111 other patients (26% versus 18%), and patients who had persistent weight loss over 5% for 24 weeks after the completion of anti-hepatitis c virus (hcv) therapy were more likely to be taking a stavudine-based antiretroviral therapy (48 c ). in another study patients with lipoatrophy had higher drug exposure to stavudine than controls (49 c ). this was reflected in the higher geometric concentration ratios (0.978 and 0.741 respectively) and a higher percentage of ratios over 1.0, representing a drug concen tration above the normal population curve (46% versus 23%). in addition, the duration of stavudine therapy was independently asso ciated with lipoatrophy. in a multivariate ana lysis, both duration of stavudine therapy and a concentration ratio over 1.0 independently correlated with lipoatrophy. changes in body habitus occur when sta vudine is withdrawn. in 574 hiv-positive women who stopped taking stavudine for over 2.25 years, there were significantly smaller reductions in hip and thigh circum ferences compared with the reductions that occurred at 1-2.25 years after stavudine withdrawal (50 c ). stavudine reduces insulin sensitivity and causes mitochondrial toxicity in healthy sub jects. in 16 participants without a personal or family history of diabetes who were randomized to stavudine 30-40 mg bd or placebo for 1 month, insulin sensitivity was significantly reduced by stavudine (51 c ). in addition, muscle biopsies in those who took stavudine showed significant reductions in mtdna/nuclear dna, but there were no changes in placebo-treated subjects. p mag netic resonance spectroscopy studies of mito chondrial function correlated with measures of insulin sensitivity. in 125 patients in a retrospective study, symptomatic hyperlactatemia in 114 (91%) was associated with stavudine median dura tion 13 months (52 c ). nine patients (7.2%) died; those who died had a higher mean lactate concentration (8.0 versus 5.1 mmol/l) and mean alt activity (164 versus 48 u/l) at the time of diagnosis than those who sur vived. those who died had a lower mean weight than those who survived (48 versus 59 kg). by logistic regression, mortality was associated with patients whose body weight was under 45 kg (or = 9.1; 95% ci = 1.6, 53) and whose serum lactate was over 10 mmol/l (or = 20; 95% ci = 2.6, 159). dosage regimens a meta-analysis of clinical trials conducted before and after regulatory approval of stavudine has shown that a dosage of 30 mg bd has equivalent antiviral efficacy, with some evidence of lower rates of peripheral neuropathy and lipoatrophy, to the standard dosage of 40 mg bd (53 m ). it has been suggested that this is the most appropriate dose in resourcelimited settings (54 m ). reducing the dosage of stavudine by one-half increased fat mtdna and bone density and decreased lactate concentrations in a study of 24 patients already taking a standard dose (55 c hematologic hemoglobin a2 can rise in hiv-infected patients, possibly because of therapy (56 c ). in cross-sectional and cohort studies, hemoglobin a2 was often raised in untreated patients, but a further rise during treatment was specifically attributable to zidovudine. the concentration of hemoglobin a2 may be high enough to lead to a misdiagnosis of beta-thalassemia. genotoxicity micronucleated reticulocyte frequencies have been measured as a mar ker of chromosomal damage in 16 hivinfected mother-infant pairs, of whom 13 women had taken prenatal zidovudine and 3 antiretroviral drugs without zidovudine (57 c ). all the infants received zidovudine for 6 weeks. venous blood was obtained from women at delivery and from infants at 1-3 days, 4-6 weeks, and 4-6 months of life; cord blood was collected immediately after delivery. ten cord blood samples (controls) were obtained from infants of hiv-negative women who did not receive antiretroviral therapy. there were 10-fold increases in micronucleated reticulocytes in women and infants who received zidovudine-containing antiretroviral therapy prenatally and no increases in the other women and infants. micronucleated reticulocytes in the zidovudine-exposed neonates fell over the first 6 months of life to values comparable to cord blood controls. the authors concluded that transplacental zidovudine exposure is genotoxic in humans and they recommended long-term monitoring of zidovudine-exposed infants. teratogenicity a possible association between first trimester exposure to zidovudine and an increased risk of hypospadias based on one cohort study has been reported (58 c ). among 2527 live births to 2353 women, defects were identified in 90 babies (3.56 defects per 100 live births). the rate of defects was 3.19 per 100 live births with first-trimester antiretroviral drug exposure, 3.54 per 100 live births with exposure later in pregnancy, and 4.05 of 100 live births with no antiretroviral drug use. only genital abnormalities, specifically hypospadias, were significantly increased among babies born to women with firsttrimester exposure to antiretroviral drugs (7 of 382 male live births) compared with the two other groups (2 of 892 male live births). logistic regression suggested that use of zidovudine in the first trimester was associated with hypospadias (adjusted or = 11; 95% ci = 2.1, 54). pregnancy in a pharmacokinetic study of three doses of zidovudine 300 mg 3-hourly in pregnancy in six subjects, plasma zidovudine concentrations were substantially lower than previously reported during continuous intravenous therapy (59 a ). in another study in four women who took an initial 600 mg dose followed by two 400 mg doses 3-hourly, the zidovudine auc and concentrations increased approximately in proportion to the increase in dose but varied 6-7 times (60 a ). in both cohorts, the pharmacokinetic results suggested erratic absorption. combined exposure to zidovudine plus co-trimoxazole caused a clinically signifi cant suppression of humoral immune responses to influenza immunization in 23 hiv-positive patients with cd4 counts above 350 â 10 6 /l (61 c ). ribavirin ribavirin did not inhibit the formation of zidovudine triphosphate in peripheral blood monocytes in 14 patients over 8 weeks (62 c urinary tract in a pilot, open, noncomparative add-on study, in which patients who had failed treatment with at least two thymidine-associated resistance mutations were given tenofovir 600 mg/day for 4 weeks in addition to their current failing antiretroviral regimen (n = 10), one developed de fanconi syndrome at week 2 and two developed grade one hypopho sphatemia (63 c ). in a multicenter, observational, retro spective study of 733 hiv-positive patients taking tenofovir, 85 patients (11.6%) stopped taking tenofovir because of adverse events, including nausea, vomiting, diarrhea, and headache. there was no association between any abnormal basal analytical parameter and a greater probability of stop ping treatment (64 c ). in a single-center, cross-sectional evalua tion of b 2 microglobinuria as a marker of proximal renal tubule damage in 92 hivinfected children, tenofovir was significantly associated with very high abnormal values (65 c in an evaluation of the effectiveness and adverse effects of a simplification regimen with tenofovir, lamivudine, and efavirenz in 154 haart-experienced hiv-1-infected subjects with sustained viral suppression, 9 had psychiatric adverse effects related to efavirenz, leading to drug withdrawal in most cases; the symptoms included nightmares, insomnia, nervousness, and anxiety (69 c ). metabolism actg study 5095 was a ran domized, placebo-controlled, double-blind study designed to compare three protease inhibitor-sparing antiretroviral drug regi mens (zidovudine þ lamivudine þ abacavir; zidovudine þ lamivudine þ efavirenz; zidovudine þ lamivudine þ abacavir þ efa virenz) in the initial treatment of hiv-1 infection in 1147 subjects (70 c ). there were modest rises in serum triglycerides, ldl cholesterol, and hdl cholesterol in the two efavirenz-containing arms com pared with the triple-nucleoside arm. urinary tract a renal calculus that occurred in a 47-year-old man taking efavirenz contained efavirenz metabolites (60%) and about 40% of unspecified proteins (71 a ). this was a between-the eyes adverse effect of type 1a, definitively implicating efavirenz (72 h ). breasts in cohort study from cambodia, 2 of 343 patients developed gynecomastia while taking a regimen containing efavirenz (73 c ). susceptibility factors genetic efavirenz is metabolized by cyp2b6. the pharmacokinetics of efavirenz have been studied in 71 children with a g-to-t polymorphism at position 516 of the cyp2b6 gene, which affected its oral clearance (74 c ). children with the t/t genotype had a slower oral clearance rate than those with the g/t genotype and the g/g genotype. the fastest clearance was found in children under 5 years of age with the g/g genotype. the association between efavirenz-induced psychosis and a genetic polymorphism in cyp2b6 has been reported in a child (75 a ). • a 12-year-old hiv-positive girl taking lopinavir 400 mg bd, ritonavir 100 mg bd, stavudine 30 mg bd, didanosine 250 mg/day, and efavirenz 600 mg/day developed an overt psychosis. her serum efavirenz concentration was 7-8 times higher than expected and she had a heterozygous gene polymorphism encoding for the cyp2b6 isoenzyme, which has previously been associated with reduced clearance of efavirenz. the psychotic symptoms resolved gradually after withdrawal of efavirenz. antimalarial drugs two healthy volunteers who took amodiaquine plus artesunate and efavirenz had significant asymptomatic rises in liver transaminases, which did not occur in the absence of efavirenz (76 c ). voriconazole in a randomized, placebocontrolled, two-period, multiple-dose within-group, fixed-dose sequence study of the interaction of voriconazole 200 mg bd with efavirenz 400 mg/day in healthy men, repeated doses of efavirenz substantially reduced the steady-state mean auc and c max of voriconazole by 80% and 66% respectively (77 c ). repeated therapeutic doses of voriconazole moderately increased the steady-state mean auc and c max of efavirenz by 43% and 37% respectively. when voriconazole was co-administered with efavirenz, the incidence of adverse events was similar to that with efavirenz alone. liver hepatotoxicity is a major problem with nevirapine (78 r ). the frequency of large increases in liver enzymes in patients taking efavirenz is 1-8%, whereas in patients taking nevirapine it is 4-18%. a warning about the increased risk of hepatotoxicity in antiretroviral-naive patients who start to take nevirapine-containing combination antiretroviral therapy has been issued based on cd4 cut-off values and sex. however, it is unclear whether this higher risk also applies to stable virolo gically suppressed patients. a meta-analysis of four published randomized studies in 410 patients, including virologically suppressed patients who switched to nevirapine-con taining regimens with a follow-up of at least 3 months, has shown that the risks of hepatotoxicity within the first 3 months were 2% and 4% in those with low and high cd4 counts, respectively, with a com bined or of 1.5 (95% ci = 0.4, 5) (79 m ). the risk of hepatotoxicity at any time dur ing the study was similar in the groups, with a combined hr of 0.8 (95% ci = 0.3, 2.5). the authors concluded that virologically suppressed patients who switch to nevira pine do not have a significantly higher risk of hepatotoxicity or rash when stratified by sex and cd4 cell count. the aim of a retrospective study was to determine whether these recommendations are of use in preventing adverse effects (80 c ). drug-naive patients (n = 142) who started treatment with nevirapine were divided into two groups: those with high or low cd4 counts (n = 61 and 81 respec tively). there were rashes in 4 patients in the high-cd4 group and in 12 of those in the low-cd4 group and hepatotoxicity in 3 and 5 patients respectively. the authors concluded that the advice not to use nevir apine in drug-naive patients at increased risk of toxicity on the basis of sex and cd4 cell count does not seem to be useful in preventing adverse effects. in a retrospective study, 582 patients (72% men) received 744 nevirapine-based haart regimens (81 c ). during 10% of treatments, there were grade 3 or greater increases in transaminase activities, an overall incidence rate of 5.3 cases per 100 person-years. this led to treatment with drawal in 3.9% of cases. in a retrospective study of over 1000 pregnant women taking nevirapine-contain ing regimens, 93% started or continued nevirapine during the first and second trimesters (82 ca ). concurrent chronic hepatobiliary disorders slightly increased the likelihood of hepatotoxicity. of seven patients who had liver dysfunction, six pre viously had had hepatitis c and gall blad der disease. skin widespread vitiligo after erythroderma has been attributed to nevirapine (83 a ). • a 34-year-old man developed erythroderma, a high fever and hepatitis after taking nevirapine for 1 month. there was jaundice and a confluent macular rash on the arms, legs, trunk, and face. histology showed a parakeratotic epidermis with focal spongiosis, necrotic keratinocytes and vacuolar degeneration of the basal layer, together with moderate edema and a perivascular mononuclear cell infiltrate, with some eosinophils in the upper dermis. the lesions faded on withdrawal of nevirapine and administration of oral glucocorticoids, but took around 6 months to finally subside. • a 12-year-old girl took a nevirapine containing regimen for 4 months and had recurrent episodes of fever, cough, sore throat, nausea, and vomiting 2-4 weeks apart. a chest x-ray showed mild bronchial wall thickening with left lower lobe atelectasis or a mild infiltrate. her temperature was 40°c and she had a tachycardia of 145/minute. she had a generalized maculopapular confluent blanching rash, but no target lesions or blisters, conjunctival injection, nasal congestion, a hyperemic posterior pharynx, and several bilateral non-tender cervical lymph nodes. the liver was enlarged by 4 cm but the spleen was not palpable. the white blood cell count was 9.7 â 10 9 /l, with a marked eosinophilia (31%). she was successfully treated with intravenous immunoglobulin and antiretroviral drug withdrawal. nevirapine-induced stevens-johnson syn drome has been reported as having been misdiagnosed as viral keratitis (85 a ). immunologic in a retrospective study of trough plasma concentrations of nevirapine and five oxidative metabolites in 1357 patients with rashes or liver function abnormalities during the first 6 weeks of treatment and controls matched for glucocorticoid use, cd4 cell count, sex, race, and hepatitis b/c status, 49 casecontrol pairs were studied (86 c ). women had significantly greater exposure than men to nevirapine and four of the five metabolites at week 2, but the plasma concentrations were comparable by week 4. there were no strong relationships between plasma concentrations of nevirapine or any of its five metabolites and case-defining events. the authors commented that systemic exposure to 12-hydroxynevirapine and 4-carboxynevirapine, hypothesized to be reactive intermediates for immunemediated adverse reactions, were comparable between the cases and controls and were comparable in proportion to nevirapine exposure. pregnancy in a retrospective, five-center comparison in hiv-1-infected women who took nevirapine as part of combination antiretroviral therapy during pregnancy, 15 of 235 eligible women (6.4%) developed a rash and 8 (3.4%) developed hepatotoxicity, including 4 with a co-existent rash, giving a combined incidence of 19 potential cases of nevirapine toxicity during pregnancy (8.1%) (87 c ). alternative causes of rash or hepatotoxicity were suspected in 7 cases, and only 10 mothers (5.8%) stopped taking nevirapine. of the 170 women who started taking nevirapine during pregnancy, 13 (7.6%) developed a rash and 8 (4.7%) hepatotoxicity. only 2 of 65 women (3.1%) with nevirapine exposure before pregnancy had had a rash. susceptibility factors genetic hla typing and demographic and immunological susceptibility factors for reactions to nevirapine and efavirenz have been studied in 21 hiv-positive patients with rashes (88 c ). isolated rashes were significantly associated with hla-drb101. there were no cases of liver toxicity nor any association with the percentage of cd4 cells. in 326 hiv-1-positive individuals, 309 of whom were japanese, 42% of those who had hypersensitivity reactions to nevirapine had hla-cw8, compared with only 10% of the others and 9-14% of the general japa nese population (89 c ). in the former group, four patients, including one who developed hepatotoxicity, had hla-cw*0801 and one had hla-cw*0803. among the others, three patients had hla-cw*0801. there were no significant differences in the fre quencies of other hla alleles between the two groups. drug dosage regimens nevirapine has a long half-life and could be given once a day, but the risk of rashes and concerns over liver toxicity preclude the routine use of once-daily dosing. however, tolerance to high concentrations of nevirapine can develop when doses are increased slowly during the start of therapy. it is therefore theoretically possible that the benefits of once-daily dosing could be achieved without excess toxicity by switching to once-daily nevirapine after several months of twice-daily administration (90 r ). however, in the daufin study, a twicedaily regimen of zidovudine 300 mg þ lami vudine 150 mg þ nevirapine 200 mg was compared with a once-daily regimen of lamivudine 300 mg þ tenofovir 245 mg þ nevirapine 400 mg (91 c ). the study was stopped after early virological failure was observed in 8 of 36 once-daily patients. resistance mutations accumulated during treatment, with high rates of k65r muta tions and severe nnrti resistance profiles indicative of continuing viral replication caused by suboptimal nevirapine plasma trough concentrations, possibly due to non adherence. non-b-subtype infection (sub types a and c not stated) was observed in 4 of 10 patients with virological failure. the authors suggested that once-daily dosing can be introduced after induction of viral suppression has been achieved with a twice-daily regimen. co-administration of nevirapine 200 mg/day with fluconazole 400 mg/day results in markedly increased trough plasma nevirapine concentrations compared with nevirapine alone (92 c ). in a retrospective study in 112 patients who were given nevirapine-based therapy with or without fluconazole 200 or 400 mg/day, mean nevirapine concentrations were 6.5 mg/l without fluconazole and 11.4 with fluconazole. one patient taking fluco nazole developed hepatitis. six of those who did not take fluconazole developed nevira pine-related rashes. there were no differ ences in 36-week antiviral efficacy between the two groups. liver rises in serum unconjugated bilirubin concentrations were reported in the pivotal phase iii trial. the mechanism is thought to be direct inhibition of bilirubin conjugation by competitive inhibition of udp glucuronosyltransferase. patients with polymorphisms in the ugt 1a1 gene are more likely to develop hyperbilirubinemia (100 r ). urinary tract indinavir causes nephro lithiasis and renal impairment as a result of crystallization in the urinary tract and resultant inflammation (101 a ). continuation of the drug with some improvement in renal function is possible with drug concentration monitoring. co-factors such as concomitant co-trimoxazole therapy and environmental temperature increase the risk (100 r ). using indinavir þ ritonavir at the lower doses of 400 and 100 mg bd seems to reduce these adverse effects. hematuria and flank pain each occurred in 38 patients in a study of asymptomatic hiv-infected individuals who took indina vir in a long-term follow-up study over 157 weeks (25 c ). hair and nails paronychia, alopecia, curling of straight hair, and ingrowing toenails have all been attributed to indinavir (102 r ). pregnancy in a study of 16 pregnant women taking indinavir, two women and eight infants developed hepatotoxicity and had increased concentrations of indinavir, suggesting increased intestinal/hepatic cyp3a activity during pregnancy (103 a ). metabolism of 111 pregnant women 15 taking nelfinavir developed gestational diabetes compared with none taking zidovudine monotherapy and two of 43 taking nrtis and nnrtis; the risk of gestational diabetes was increased in those with hepatitis c or who had begun haart before pregnancy (104 c ). nails paronychia has been reported in a case report occurring during nelfinavir treatment (105 a ). drug-drug interactions fluticasone cush ing's syndrome and adrenal suppression can be caused if protease inhibitors increase systemic glucocorticoid concentrations. iatrogenic cushing's syndrome has been attributed to ritonavir by an interaction with fluticasone (106 a ). • a 16-year-old girl who had taken various antiretroviral drugs eventually took stavudine, lamivudine, and ritonavir and then used inhaled fluticasone þ salmeterol for bronchiectasis. after 3 months she had excessive weight gain, increased appetite, fatigue, facial edema, marked acne, stretch marks on her limbs and abdomen, hypercholesterolemia, hypertriglyceridemia and amenorrhea. cushing's syndrome was attributed to an interaction of fluticasone with ritonavir, which was changed to efavirenz. there was a gradual improvement within 30-60 days, with reduced edema and stretch marks and return of menstruation. a similar case involved a 14-year-old girl (107 a ). observational studies in the aspire 1 study, 7 of 17 healthy volunteers who took saquinavir þ ritonavir for 3 months developed grade 3 gastrointestinal adverse effects and seven had hyperbilirubinemia (108 c ). in the aspire 2 study there was hyperbilirubinemia in 8 of 16 healthy volunteers. tipranavir is a non-peptide protease inhibitor approved for use in patients with resistant strains of hiv (109 r , 110 m , 111 r ). its pharmacokinetics, efficacy, and adverse effects in children and adolescents have been reviewed (112 r ) . it can be used in combination with ritonavir. observational studies in a 24-week multi-center, double-blind, randomized, dose-finding trial of ritonavir-boosted tipra navir in 216 patients, the most common adverse events were diarrhea, nausea, vomiting, fatigue, and headache (113 c ). hematologic tipranavir boosted with ritonavir caused an increased risk of bleed ing in 3 of 30 hiv-infected patients with hemophilia (114 c ). intracranial hemorrhage has been reported in a patient taking tipranavir (115 a ). liver tipranavir is associated with an excess of grade 3/4 rises in liver enzyme compared with other ritonavir-boosted protease inhibitors (116 r ). pancreas acute pancreatitis associated with hypertriglyceridemia has been reported in a patient taking tipranavir þ ritonavir (117 a ). • a 42-year-old man taking tenofovir 300 mg/day, trizivir (zidovudine, lamivudine, and abacavir) one tablet bd, and tipranavir 500 mg bd þ ritonavir 200 mg bd drank six standard alcoholic drinks 2 days before admission and developed marked tenderness in the epigastric region and a raised serum lipase at 113 iu/l (reference range below 65 iu/l). an abdominal computed tomography (ct) scan showed pancreatic edema with peripancreatic fluid consistent with acute pancreatitis. ultrasonography showed a mildly dilated common bile duct with no evidence of cholelithiasis and ct cholangiography showed no evidence of gall-stone pancreatitis. the presumed diagnosis was alcohol-induced pancreatitis. he was managed conservatively by withdrawal of medications, intravenous fluids, and slow resumption of oral intake. however, within 12 days he again developed severe epigastric pain. he denied further alcohol use. the serum concentration of triglycerides was 99 mmol/l, and retrospective testing of blood samples taken during the earlier illness also showed marked hypertriglyceridemia. tipranavir þ ritonavir was withdrawn, efavirenz given, and tenofovir and trizivir continued. his triglyceride concentrations fell to 10.3 mmol/l 3 weeks later and 4.8 mmol/l 12 months later without specific intervention. skin porphyria cutanea tarda has been reported after the introduction of tipranavir þ ritonavir to a backbone of tenofovir and lamivudine (118 a ). • a 59-year-old heterosexual woman switched to tipranavir-containing therapy after virological failure. after 5 days she developed a rash accompanied by nausea, vomiting, malaise, and hyperamylasemia. all antiretroviral drugs were withdrawn, she improved, and treatment was restarted. although the lesions were resolving, several blisters appeared on her hands, mainly on the fingers, along with itching and skin fragility on her arms. aciclovir was ineffective and 1 month later the itch and blisters worsened. urine concentrations of protoporphyrin and coproporphyrin were about 100 and 10 times higher than normal. drug resistance virological response rates in tipranavir-treated individuals were reduced when isolates with substitutions at amino acid positions i13, v32, m36, i47, q58, d60, v82, or i84 were present at baseline (119 c ). in addition, virological response rates to tipranavir were reduced when the number of baseline protease inhibitor mutations was five or more. individuals who took tipranavir without concomitant enfuvirtide and had five or more baseline protease inhibitor mutations began to lose antiviral response at weeks 4-8. however, individuals taking enfuvirtide with tipranavir achieved more than 1.5 log 10 reductions in viral load from baseline at 24 weeks, even if they had five or more baseline protease inhibitor mutations. virological response rates to tipranavir were reduced when the baseline phenotype for tipranavir had a greater than threefold shift in the 50% effective concentration (ec 50 ) from reference. the most common protease inhibitor mutations that were associated with virological failure were l10i/v/s, i13v, l33v/i/f, m36v/i/l v82t, v82l, and i84v. drug-drug interactions interactions with tipranavir þ ritonavir have been reviewed (120 r ). tipranavir is metabolized by cyp3a and tipranavir þ ritonavir in vitro inhibits cyp1a2, cyp2c9, cyp2c19, cyp2d6, and cyp3a and induces glucuronidase and p glycoprotein. inhibitors ritonavir-boosted tipranavir, alone and in combination with comparator protease inhibitors, has been studied in 315 hiv-infected patients (121 c psychiatric oseltamivir-induced worsening of delirium has been reported in an 83-year old man, whose symptoms resolved 2 days after withdrawal (122 a ). japanese authorities advised against using oseltamivir in adolescents aged 10-19 years after two suicides during 2007 and more than 100 reports of neuropsychiatric events identified during post-marketing sur veillance, including delirium, convulsions, and encephalitis (123 r ). however, it is not clear whether these events were due to the influenza or the drug; in phase iii trials, there were similar incidences of neurologi cal and psychiatric events in both treated and untreated patients (124 r ). of 1113 patients enrolled in a japanese neuramini dase inhibitor treatment study, 11 had neuropsychiatric symptoms, in 4 cases before the start of treatment (125 c ). the us food and drug administration (fda) and european medicines agency (ema) have advised doctors to monitor patients for abnormal behavior throughout treat ment (123 r ). gastrointestinal acute hemorrhagic colitis has been associated with oral oseltamivir in a 61-year-old man, who developed abdominal pain, diarrhea and hematochezia after taking two doses of oseltamivir (126 a ). in a systematic review of three trials of neuraminidase inhibitors for preventing and treating influenza in 1500 children, 977 of whom had laboratory-confirmed influenza, those who took oseltamivir had vomiting more often than untreated children but withdrawal was rarely required (127 m ). drug resistance resistance occurs in under 1% of healthy adults but occurs more often in children, from 5.5% up to 18% in one study, although a lower dosage regimen was used in that study. resistance is often seen among immunocompromised patients. it was reported in two of eight patients infected with h5n1 and was associated with a fatal outcome. resistance is associated with loss of fitness (128 r ). in 2008 widespread resistance emerged in h1n1 influenza, but it remains to be seen whether its circulation is sustained after the emergence of oseltamivir-sensitive swine vh1n1. susceptibility factors genetic there was no evidence of a difference in auc 1!20 of oseltamivir or its active metabolite oseltamivir carboxylate between 14 japanese subjects and 14 caucasian subjects, or between children aged 1-2 years old and adults (129 c ). systematic reviews a meta-analysis of the adverse effects of zanamivir in children showed that it was no worse than placebo (127 m comparative studies in a randomized controlled trial in patients with chronic hepatitis c treated with interferon-alfa 2a alone (n = 53), with amantadine 100 mg bd (n = 111), with ribavirin (n = 106) or with amantadine þ ribavirin (n = 108), there was a sustained virological response in 13%, 6%, 18%, and 22% respectively (130 c ). this was statistically different between interferon þ amantadine and triple therapy but not between interferon þ ribavirin and triple therapy. the spectra and frequencies of adverse effects were similar in all four treatment arms. however, six patients withdrew because of adverse effects, three of them in an arm containing amantadine. in two groups of non-responders with hcv genotype 1 chronic infection taking interferon and ribavirin, with or without amantadine 200 mg/day, viral load fell more markedly in the group taking triple therapy including amantadine, but the response rates at the end of treatment were not signifi cantly different (131 c ). although analysis of the viral ion channel p7 showed selective pressure during therapy, no specific resi dues appeared to be linked to the effect of amantadine on the virus. the authors sug gested that this implied that the antiviral effect of amantadine is non-specific and related to reduced endosomal acidification and therefore reduced transport of hepatitis c by a ph-dependent pathway. in a multicenter study of 75 non-respon ders with chronic hepatitis c randomized to interferon monotherapy (n = 26), dual ther apy with ribavirin (n = 24) and triple therapy with additional amantadine 200 mg/day (n = 25), amantadine did not increase the frequency or severity of adverse effects (132 c ). in 22 patients with chronic hepatitis c genotype 1b, with high viral loads treated with interferon-beta for 4 weeks followed by interferon-alfa 2b, ribavirin, and amantadine 150 mg/day for 22 weeks, there was a sustained virological response in 7. only one patient had to stop taking amantadine, because of the adverse effect of light-headedness (133 c ). in 15 renal transplant recipients with chronic hepatitis c, doses of ribavirin monotherapy (n = 7) or ribavirin þ amantadine (n = 8) were adjusted according to creatinine clearance (134 c ). there was no difference between treatment groups with respect to liver enzymes, hepatitis c viremia, liver his tology, or renal function. anemia, the main adverse effect, was most notable in those with a creatinine clearance below 50 ml/ minute. other adverse effects included leukopenia, mood disorders and profuse sweating. only one of those who received combination therapy were still taking aman tadine after 12 months; two completed treatment but with ribavirin alone. four of those taking monotherapy completed treat ment. neither regimen was clearly superior to no treatment, but the study was small and probably underpowered. sensory systems in a post-marketing surveillance study of patients with a new diagnosis of corneal disease and new prescriptions for amantadine over 2 years, 36 (0.27%) of 13 137 patients developed fuchs dystrophy (corneal edema) (135 c ). the relative risk of corneal edema was 1.7 (95% ci = 1.1, 2.8); in 12 patients (0.09%) the diagnosis was made in the first month. drug resistance amantadine is unreliable in the management of influenza because of the emergence of widespread resistance. this is notable in the h3n2 subtype and most h5n1 subtypes, but some seasonal h1n1 remains sensitive to amantadine (128 r treatment of adenovirus pneumonia with cidofovir in pediatric lung transplant recipients infections in t-cell-depleted allogeneic hematopoietic stem cell transplantation: high mortality in the era of cidofovir low-dose cidofo vir treatment of bk virus-associated hemor rhagic cystitis in recipients of hematopoietic stem cell transplant acyclovir-induced neuropsychosis success fully recovered after immediate hemodialy sis in an end-stage renal disease patient acute renal failure caused by intravenous acyclovir for disseminated var icella zoster virus infection determi nants of aciclovir-induced nephrotoxicity in children entérocolite ulcéronécrosante chez un nouveau-né à terme. rôle de l'acy clovir? [necrotizing enterocolitis in a full term infant. is acyclovir involved? immediate allergy from valacyclovir oliguric acute renal failure following oral valacyclovir therapy adding-on versus switching-to adefovir therapy in lamivudine-resistant hbeag negative chronic hepatitis b adefovir dipivoxil therapy in liver transplant recipients for recurrence of hepatitis b virus infection despite lamivudine plus hepatitis b immu noglobulin prophylaxis absence of a pharmacokinetic interaction between entecavir and adefovir lamivudine and high-dose interferon alpha 2a combination treatment in naive hbeag-positive immunoactive chronic hepatitis b in children: an east mediterra nean center's experience pharmacokinetics of lamivu dine in subjects receiving peritoneal dialysis in end-stage renal failure telbivu dine versus lamivudine in patients with chronic hepatitis b adverse events associated with high-dose ribavirin: evidence from the toronto out break of severe acute respiratory syndrome long-term therapy with aerosolized riba virin for parainfluenza 3 virus respiratory tract infection in an infant with severe com bined immunodeficiency syncope as a prob able side effect to combination antiretro viral therapy initiated during primary hiv 1 infection kallianpur araids clinical trials group 384 and a5005s study teams. hemochromatosis gene poly morphisms, mitochondrial haplogroups, and peripheral lipoatrophy during anti retroviral therapy del río labcde (aba cavir vs. d4t (stavudine) plus efavirenz) study team. less lipoatrophy and better lipid profile with abacavir as compared to stavudine: 96-week results of a randomized study antiretroviral drug levels and interactions affect lipid, lipoprotein, and glucose metabolism in hiv-1 seronegative subjects: a pharmacoki netic-pharmacodynamic analysis maxcmin1 and 2 trial groups. phar macokinetics of two randomized trials evaluating the safety and efficacy of indina vir, saquinavir and lopinavir in combination with low-dose ritonavir: the maxcmin1 and 2 trials assess ment of adverse events associated with anti retroviral regimens for postexposure prophylaxis for occupational and nonoccu pational exposures to prevent transmission of human immunodeficiency virus for the protocol 060 study groupefficacy, safety, and tolerability of long-term combination antiretroviral therapy in asymptomatic treatment-naive adults with early hiv infection clinical and pharmacokinetic data support once-daily low-dose boosted saquinavir (1,200 milli grams saquinavir with 100 milligrams ritonavir) in treatment-naive or limited protease inhibitor-experienced human immunodeficiency virus-infected patients dose reduction effective in alleviating symptoms of saquinavir toxicity french national agency for research on aids; viral hepa titis-hc02-ribavic study team. progres sion of fibrosis in hiv and hepatitis c virus-coinfected patients treated with inter feron plus ribavirin-based therapy: analysis of risk factors hepato toxicity in an african antiretroviral therapy cohort: the effect of tuberculosis and hepa titis b nodular regenerative hyperplasia of liver as a consequence of art the role of antire troviral therapy in the incidence of pancrea titis in hiv-positive individuals in the eurosida study amprenavir and di danosine are associated with declining kid ney function among patients receiving tenofovir acute interstitial nephritis of hiv-positive patients under atazanavir and tenofovir therapy in a retrospective analysis of kidney biopsies possible allergic cross-reaction to didanosine and tenofovir in an hiv-1-infected woman seage 3rd gr. in utero nucleoside reverse transcriptase inhibitor exposure and signs of possible mitochondrial dysfunc tion in hiv-uninfected children adverse events experi enced by three children taking tenofovir and didanosine in combination phar macokinetic interaction between darunavir and saquinavir in hiv-negative volunteers interaction between buprenor phine and atazanavir or atazanavir/ritona vir pos sible antiretroviral therapy-warfarin drug interaction factors in aids dementia com plex trial design: results and lessons from the abacavir trial b*5701-associated abacavir hypersensitivity on the basis of sequence variation in hiv reverse transcriptase are disulfiram-like reactions associated with abacavir-containing antiretroviral regi mens in clinical practice? fanconi syndrome and nephrogenic diabetes insipidus associated with didanosine ther apy in hiv infection: a case report and literature review cd4þ cell count decline despite hiv suppression: a prob able didanosine-valganciclovir interaction severe weight loss in hiv/hcv-coinfected patients trea ted with interferon plus ribavirin: incidence and risk factors stavudine plasma concentrations and lipoatrophy hernández-mora dine discontinuation to anthropometric mg, novoa sr, quintana fb, lahoz jg, changes among hiv-infected women complicating the abacavir hypersensitivity reaction abacavir hypersensitiv ity reaction after switching from the twicedaily to the once-daily formulation acute fibrinous and organizing pneumonia as a rare presen tation of abacavir hypersensitivity reaction a simple screening approach to reduce effects of a nucleoside reverse transcriptase inhibitor, stavudine, on glucose disposal and mitochondrial func tion in muscle of healthy adults risk factors for mortality in symptomatic hyperlactatemia among hiv-infected patients receiving antiretroviral therapy in a resource-limited setting systematic review of clinical trials evaluating low doses of stavudine as part of antiretroviral treat ment safety of stavudine in the treatment of hiv infection with a special focus on resource-limited settings blunted humoral response to influenza vaccination in patients exposed to zidovu dine plus trimethoprim-sulfamethoxazole aids clinical trials group 5092s study team. pharmacokinetic evaluation of the effects of ribavirin on zidovudine triphosphate formation: actg 5092s study team efficacy and safety of tenofovir double-dose in treatment-experi enced hiv-infected patients: the teno plus study castillo muñoz a, toxicity in hiv-infected subjects: a rando-baños roldán u, artacho criado s. analisis mized, controlled study increased haemoglobin a2 percentage in hiv infection: disease or treatment? elevated frequencies of micronucleated erythrocytes in infants exposed to zidovudine in utero and postpartum to pre vent mother-to-child transmission of hiv assessment of birth defects according to maternal therapy among infants in the women and infants de las causas y factores predictivos de discon tinuacion del tratamiento con tenofovir en pacientes vih pretratados increased beta-2 microglobulinuria in human immunodeficiency virus-1-infected children and adolescents treated with tenofovir neuropsychiatric side effects of efavirenz therapy pharmacokinetics of oral zidovudine administered during labour: a preliminary study teratogenicity risk of antiretro viral therapy in pregnancy efficacy and safety of a once daily regimen with efavirenz, lami vudine, and didanosine, with and without food, as initial therapy for hiv infection: the eladi study effectiveness and safety of simplification therapy with once-daily tenofovir, lamivu dine, and efavirenz in hiv-1-infected patients with undetectable plasma viral load on haart metabolic effects of protease inhibitor-spar ing antiretroviral regimens given as initial treatment of hiv-1 infection (aids clin ical trials group study a5095) efavir enz urolithiasis anecdotes that provide definitive evidence positive outcomes of haart at 24 months in hiv-infected patients in cambodia efavirenz pharmacokinetics in hiv-1-infected chil dren are associated with cyp2b6-g516t polymorphism psychosis in a 12-year-old hiv-positive girl with an increased serum concentration of efavirenz hepatotoxicity due to a drug interaction between amodia quine plus artesunate and efavirenz pharmacokinetic interaction between voriconazole and efavirenz at steady state in healthy male subjects liver toxicity induced by non-nucleoside reverse tran scrptase inhibitors hepatotoxicity of nevirapine in virologically suppressed patients according to gender and cd4 cell counts risk of side effects associated with the use of nevirapine in treatment-naive patients, with respect to gender and cd4 cell count reasons for disconti nuation of nevirapine-containing haart: results from an unselected population of a large clinical cohort safety issues about nevirapine administration in hiv-infected pregnant women widespread viti ligo after erythroderma caused by nevira pine in a patient with aids nevira pine-associated rash with eosinophilia and systemic symptoms in a child with human immunodeficiency virus infection nevirapine induced stevens-johnson syndrome in an hiv patient case-control exploration of relationships between early rash or liver toxicity and plasma concentra tions of nevirapine and primary metabo lites safety of nevirapine in pregnancy hla-drb1*01 associated with cutaneous hypersensitivity induced by nevirapine and efavirenz hla-cw8 primarily associated with hypersensitivity to nevirapine once-daily nevirapine dosing: a pharmacokinetics, efficacy and safety review once-daily dosing of nevirapine in haart plasma nevirapine levels, adverse events and efficacy of antiretroviral therapy among hiv-infected patients concurrently receiving nevirapine-based antiretroviral therapy and fluconazole phar macokinetics of darunavir (tmc114) and atazanavir during coadministration in hivnegative, healthy volunteers alo pecia associated with ritonavir-boosted ata zanavir therapy pharmacokinetic interaction between rifampicin and ritonavir-boosted atazanavir in hiv-infected patients increased incidence of cutaneous mycoses after haart initiation: a benign form of immune reconstitution disease? fosamprenavir calcium plus ritonavir for hiv infection experience of indinavir/ritonavir 400/ 100 mg twice-daily highly active antiretro viral therapy-containing regimen in hiv-1 infected patients in bamako, mali: the nogoma study lack of indinavir-associated nephrological complications in hivinfected adults (predominantly women) with high indinavir plasma concentration in abidjan indinavir: the forgotten hivprotease inhibitor. does it still have a role? néphropathie tubulo-interstitielle associée a l'indinavir dermatologic adverse effects of antiretroviral therapy: recognition and management pharmacokinetics and safety of indinavir in human immunodefi ciency virus-infected pregnant women obste tric and perinatal complications in hivinfected women. analysis of a cohort of 167 pregnancies between paronychia in an hiv-infected patient under nelfinavir ther apy iatrogenic cushing's syndrome in a adolescent with aidss on ritonavir and inhaled fluticasone. case report and literature review cushing syndrome and severe adrenal suppression caused by fluti casone and protease inhibitor combination in an hiv-infected adolescent pharmacokinetics of saquinavir with atazanavir or low-dose ritonavir administered once daily (aspire i) or twice daily (aspire ii) in seronegative volunteers tipranavir: a new option for the treatment of drug-resistant hiv infection tipranavir: the first nonpeptidic protease inhibitor for the treatment of protease resistance tipranavir: a review of its use in the management of hiv infec tion tipra navir: a new protease inhibitor for the pediatric population efficacy and safety of three doses of tipranavir boosted with ritonavir in treatment-experienced hiv type-1 infected patients increased risk of bleeding with the use of tipranavir boosted with ritonavir in haemophilic patients intracranial haemorrhage possibly related to tipranavir in an hiv-1 patient with cryptococcal meningitis tipranavir: a new protease inhibitor for the treatment of antiretroviral-experienced hiv-infected patients acute pancreatitis caused by tipranavir/ritonavir-induced hypertriglycer idaemia porphyria cutanea tarda in an hiv-1-infected patient after the initiation of tipranavir/ritonavir: case report food and drug administration analysis of tipranavir clini cal resistance in hiv-1-infected treatmentexperienced patients mechanisms of pharmacokinetic and pharmacodynamic drug interactions associated with ritonavir enhanced tipranavir pharmacokinetics, safety, and efficacy of tipranavir boosted with ritonavir alone or in combination with other boosted protease inhibitors as part of optimized combination antiretroviral therapy in highly treatmentexperienced patients oseltamivir-induced delirium in a geriatric patient tamiflu and neuropsychiatric disturbance in adolescents the role of oseltamivir in the treatment and prevention of influenza in children a comparison of the effectiveness of zanamivir and oseltami vir for the treatment of influenza a and b acute hemorrhagic colitis associated with oral administration of osel tamivir for the treatment of influenza a symmonds-abrahams m. neur aminidase inhibitors for preventing and treating influenza in children antivirals for influenza similarity in pharmacokinetics of oseltami vir and oseltamivir carboxylate in japanese and caucasian subjects induction doses of interferon alpha-2a in combination with ribavirin and/ or amantadine for the treatment of chronic hepatitis c in non-responders to interferon monotherapy: a randomized trial hepatitis c virus p7 membrane protein 553 quasispecies variability in chronically infected patients treated with interferon and ribavirin, with or without amantadine a randomized trial of induction doses of interferon alone or in combination with ribavirin or ribavirin plus amantadine for treatment of nonresponder patients with chronic hepatitis c triple therapy of initial high-dose interferon with ribavirin and amantadine for patients with chronic hepatitis c combination therapy with ribavirin and amantadine in renal trans plant patients with chronic hepatitis c virus infection is not superior to ribavirin alone postmarketing sur veillance of corneal edema, fuchs dystrophy, and amantadine use in the veterans health administration key: cord-271948-iq29xqrn authors: obeng, billal musah; bonney, evelyn yayra; asamoah-akuoko, lucy; nii-trebi, nicholas israel; mawuli, gifty; abana, christopher zaab-yen; sagoe, kwamena william coleman title: transmitted drug resistance mutations and subtype diversity amongst hiv-1 sero-positive voluntary blood donors in accra, ghana date: 2020-07-24 journal: virol j doi: 10.1186/s12985-020-01386-y sha: doc_id: 271948 cord_uid: iq29xqrn background: detection of hiv-1 transmitted drug resistance (tdr) and subtype diversity (sd) are public health strategies to assess current hiv-1 regimen and ensure effective therapeutic outcomes of antiretroviral therapy (art) among hiv-1 patients. globally, limited data exist on tdr and sd among blood donors. in this study, drug resistance mutations (drms) and sd amongst hiv-1 sero-positive blood donors in accra, ghana were characterized. methods: purposive sampling method was used to collect 81 hiv sero-positive blood samples from the southern area blood center and confirmed by inno-lia as hiv-1 and/or hiv-2. viral rna was only extracted from plasma samples confirmed as hiv-1 positive. complementary dna (cdna) was synthesized using the rna as a template and subsequently amplified by nested pcr with specific primers. the expected products were verified, purified and sequenced. neighbour-joining tree with the kimura’s 2-parameter distances was generated with the rt sequences using molecular evolutionary genetic analysis version 6.0 (mega 6.0). results: out of the 81 plasma samples, 60 (74%) were confirmed as hiv-1 sero-positive by inno-lia hivi/ii score kit with no hiv-2 and dual hiv-1/2 infections. the remaining samples, 21 (26%) were confirmed as hiv sero-negative. of the 60 confirmed positive samples, (32) 53% and (28) 47% were successfully amplified in the rt and pr genes respectively. nucleotide sequencing of amplified samples revealed the presence of major drug resistance mutations in two (2) samples; e138a in one sample and another with k65r. hiv-1 subtypes including subtypes a, b, crf02_ag and crf09_cpx were found. conclusion: this study found major drug resistance mutations, e138a and k65r in the rt gene that confer high level resistance to most nnrtis and nrti respectively. crf02_ag was most predominant, the recorded percentage of subtype b and the evolutionary relationship inferred by phylogenetic analysis may suggest possible subtype importation. however, a more prospective and detailed analysis is needed to establish this phenomenon. the data obtained would inform the selection of drugs for art initiation to maximize therapeutic options in drug-naïve hiv-1 patients in ghana. the hiv global burden is estimated at 36.9 million infected persons with approximately 25 million living in sub-saharan africa [1] . hiv-1 infection was first detected in ghana in 1986 and has since been responsible for numerous deaths of both children and adults [2] . currently, the prevalence of hiv-1 in ghana is estimated to be 1.7% in 2017 [3] . a major therapeutic intervention to the hiv pandemic has been the introduction and access to antiretroviral therapy (art) [4] . this has reduced hiv-related morbidity and mortality rates and increased life expectancy of infected individuals [5] . however, therapeutic success of these arts is reduced as a result of emergence of hiv-1 drms, viral sd and spontaneously generated polymorphisms due to immune pressure in patients receiving arts [6] . the absence of genotypic drug resistance monitoring may lead to patients harboring viruses with drm which could be transmitted to new hosts, a phenomenon described as transmitted drug resistance (tdr) [7] . tdr is an important public health concern due to increased risk of virologic failure when art is initiated [8] . systematic studies have suggested higher rates of tdr (11-23%) in europe, america, and australia, as compared to countries where scale-up of art is ongoing [9] [10] [11] . in these studies, the prevalence of tdr in nucleoside reverse transcriptase inhibitors (nrti), nonnucleotide reverse transcriptase inhibitors (nnrti) and protease inhibitors (p1), were 7.4, 3.4 and 3% respectively. comprehensive care for people living with hiv/aids (plhiv) in ghana started in 2003 with first-line drugs including azidothymidine (azt), stavudine (d4t), lamivudine (3tc), nevirapine (nvp) and efavirenz (efv). over the past decade, these drugs have expanded to include emtricitabine (ftc), tenofovir (tdf), and abacavir (abc). since its inception, art has been scaled up to about 40%of hiv patients [3] . limited data has shown that drm and tdr in naïve plhiv in ghana are low [12] [13] [14] [15] . a threshold survey among pregnant women in the administrative region where art was first introduced in ghana observed a tdr rate of < 5% [14] . voluntary blood donors (vbd) provide a population base which is young and are more likely to have been recently exposed to hiv [16] . in order to understand the transmission dynamics among newly exposed people living with hiv (plhiv), tdr in a cross-section of vbd needs to be assessed. a cross-sectional study using purposive sampling method was conducted at the southern area blood center (sabc), ghana from august 2016 to february 2017. the center is a satellite facility of the national blood service, ghana (nbs). it is mandated to collect, screen, and distribute safe blood to various hospitals and clinics in the southern part of ghana. the nbs recruits vbd using a questionnaire to first assess their behavioral risk factors and health status to ascertain their suitability to donate blood. upon successful recruitment and blood donation, the blood is screened for hbsag, hcv antibodies, hiv and antibodies for treponema pallidum as procedures for safe blood. the test algorithm for hiv-1 at the blood bank is by the detection of p24 antigen using the hiv (ag/ab) 4 thgen (fortress diagnostics limited, antrim, u.k). currently, a more sensitive test tool such as the pcr and/or inno-lia is not employed. ethical approval was obtained from the ethics and protocol review committee of the college of health sciences, university of ghana. approval to select hiv positive samples was also obtained from the nbs. a total of eighty-one (81) voluntarily donated blood samples that were rejected as been hiv sero-positive using the hiv (ag/ab) 4 thgen (fortress diagnostics limited, antrim, u.k) were used. a data extraction sheet was used in obtaining information on age and gender from the donors' records upon approval from the sabc. study numbers were assigned to anonymize the blood samples. plasma was obtained from the sabc and were transported in cold boxes with ice packs to the virology department of nmimr and stored at − 30°c until further processing. a confirmatory test (inno-lia™ hiv-i/ii score, fujirebio, gent, belgium) was done on all plasma samples following manufacturer's protocol. viral rna was extracted using the qiaamp® viral rna mini kit (qiagen, hilden, germany) following manufacturer's protocol. a two-step reverse transcription method was used to generate complementary dna (cdna) of hiv-1 from extracted rna using transcriptor high fidelity cdna synthesis kit (roche diagnostics, mannheim, germany). an initial reaction mix of 2.0 μl random hexamer primer, 2.4 μl nuclease-free water and 7.0 μl of extracted rna were incubated at 65°c for 10 min and immediately placed on ice. a second reaction mix made up of 4.0 μl of 5x high fidelity reverse transcriptase (5x hfrt) buffer, 0.5 μl protector rnaase inhibitor, 2.0 μl deoxynucleotide triphosphates (dntps), 1.0 μl dithiothreitol (dtt) and 1.1 μl transcriptor hfrt enzyme was prepared. an aliquot of 8.6 μl of the second mix was added to the first reaction, thoroughly mixed and incubated at 45°c for 30 min followed by 85°c for 5 min. nested pcr was done to separately amplify the protease (pr) and reverse transcriptase (rt) genes from the cdna synthesized using the expand high fidelity plus pcr kit (roche diagnostics, mannheim, germany) with specific primers and cycling conditions previously published [17] . in the first round, 5.0 μl of 5 × buffer with mgcl 2 , 0.5 μl of dntps, 1.0 μl each of forward and reverse primers, 0.25 μl of expand high fidelity polymerase and 12.25 μl of nuclease free water were added to 5.0 μ l of cdna. in the second round of the pcr, 5.0 μl of 5x buffer with mgcl 2, 0.5 μl dntps, 0.5 μl of each of forward and reverse primers, 0.25 μl expand high fidelity polymerase and 15.25 μl nuclease-free water were added to 3.0 μl of round 1 product. a fragment of 463 base pairs (bp) and 887 bp for the pr and rt genes respectively, were generated and confirmed by agarose gel electrophoresis. purification of nested pcr products was done using qiaquick pcr purification kit (qiagen, hilden, germany) following manufacturer's protocol. purified amplicons were eluted in 50 μl of elution buffer for cycle sequencing. the bigdye terminator v3.1 cycle sequencing kit (applied biosystems, ma, u.s.a) was used to separately sequence the pr and rt genes of hiv-1 using primers and cycling conditions previously published [18] . a total reaction volume of 10 μl comprising of 2 μl each of a primer, bigdye terminator, bigdye terminator buffer, nuclease free water and purified pcr product was used. sequenced products were purified using the agencourt® cleanseq® dye-terminator removal system (agencourt bioscience corporation, u.s.a) following manufacturer's protocol. the purified product was loaded onto the abi 3130xl genetic analyzer (applied biosystems, ma, u.s.a) to generate sequence data for hiv-1 drm and hiv subtype analyses. nucleotide sequences for each sample were assembled to form a contig using seqmanpro 13 (dnastar incorporation, u.s.a). consensus sequence obtained was aligned with an hiv reference sequence (b-hxb2-prt_ 2253-3700) in bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html). sequences were submitted to the stanford university hiv drug resistance database (https:// hivdb.stanford.edu/hivdb/by-sequences/) to assign subtypes detect hiv drug resistance mutations and. the subtypes were confirmed with the los alamos national laboratory hiv database (http://www.hiv.lanl.gov). neighbor-joining tree with the kimura's 2-parameter distances was generated with the rt sequences using molecular evolutionary genetic analysis version 6.0 (mega 6). statistical analysis was done using spss version 23 (armonk, usa) to describe patients' demographics using frequencies and percentages. a total of eighty-one (81) hiv-1 sero-positive blood samples were selected from the blood bank for this study. the hiv status, age and gender characteristics of participants is summarized in table 1 . majority (40%) were between the ages of 26 to 35 years with 46 to 55year group being the least recorded (10%). majority (74%) were males whilst 14% were females. age and gender information for 10 (12%) of the study samples were not available in the sabc donor records. sixty (74%) of the samples were confirmed as hiv-1 positive, twenty-one samples (26%) were found to be negative for hiv-1, hiv-2 or dual hiv-1/2 infection. of the 60 samples confirmed reactive for hiv-1, 28 (47%) and 32 (53%) were successfully amplified for the pr and rt genes respectively. of these, 12/28 (43%) and 20/32 (63%) were successfully sequenced for the pr and rt genes respectively. eight samples had sequences for both the pr and rt genes. one (1) sample had minor drm in the pr gene whilst 1 minor and 2 major drms were found in the rt gene of three (3) samples as shown in table 2 . out of the 12 pr sequences, 1 (8%) was subtype a, 2 (17%) were subtype b and 9 (75%) were crf02_ag. of the 20 rt sequences, 2 (10%) were subtype a, 8 (40%) were subtype b, 9 (45%) were crf02_ag and 1 (5%) was crf09_cpx as indicated in fig. 1 . no major pr-related drm was observed in the samples whilst major rt-related drms in two (2) samples were found; e138a in one sample and another with k65r. drug resistance implications of the mutations found are shown in table 3 . phylogenetic analysis using the neighbour-joining tree with the kimura's 2-parameter distances was generated with the rt sequences using molecular evolutionary genetic analysis tool version 6.0 (mega6). study samples were labelled with coloured squares (red, green, blue and black squares indicating different subtypes) and reference sequences labelled with accession numbers as shown in fig. 2 . this study examined occurrence of drug-resistance mutations in apparently healthy blood donors who tested positive for hiv-1.the proportion of males to females in this study does not concord with hiv-1 trends. the same could be said for the distribution of hiv infection across the age groups. although, the number of females infected with hiv nationwide is more than males [2] , voluntary blood donors are usually males. females are usually disqualified due to relatively lower hemoglobin (hb) levels. this is partly because of monthly blood loss during menstruation and the toll of pregnancy. moreover, it is physically active men usually between the ages of 25-50 years who volunteer to donate blood. thus, these reasons could have accounted for the pattern seen in this study. the predominance of crf02_ag and absence of hiv-2 and dual hiv-1/2 infections agrees with studies reporting hiv-1 infection as the predominant hiv type in ghana and crf02_ag as the main subtype [19, 20] . the twenty (21) samples that were later confirmed to be hiv negative indicate the need for diagnostic methods with higher sensitivity at blood banks to minimize the probability of transfusing infected blood [10] . hiv-1 tdr occurs when recently infected individuals who are not exposed to antiretroviral drugs harbor drug resistant viruses. in this study, hiv-1 tdr is defined as the presence of at least one major hiv-1 drm in a study participant. two (2) major drms were found in two (2) samples sequenced in the rt gene (e138a and k65r) and none in the pr gene. other mutations were also found, which do not confer drug resistance by themselves but only when they occur in combination with other mutations. the accessory and minor mutations found were f77l and l10f in rt gene and pr gene respectively. the e138a mutation is a polymorphic mutation that occurs in an appreciable number of drug-naïve patients such as the population studied and confers resistance to etravirine (etr) and rilpivirine (rpv), which are nnrt is [21, 22] . in a similar study, e138a was found in artnaïve pregnant women attending antenatal care in a teaching hospital in accra, ghana [23] . this mutation, has been shown to reduce susceptibility to etr and rpv by 2-folds. the k65r mutation is found to reduce viral susceptibility to most nrtis by approximately 2fold and rather increase susceptibility to azt and hence reduced viral replication with zidovudine-containing therapy [24] . the findings of this study are consistent with other studies previously conducted among drug naïve infected individuals, in which pi-associated drm were rarely found [23, 25] . generally, low level drms against pis in the ghanaian population could be attributed to the high genetic barrier of protease inhibitors and that the virus would have to mutate several times to develop resistance to such drugs. additionally, sparing use of protease inhibitors reserved for use mostly in second-line regimen while majority of those on treatment are on reverse transcriptase inhibitors may have accounted for this phenomenon. viral sequence subtyping showed crf02_ag as most predominant subtype in the study population. other subtypes found were b, a and crf09_cpx. this result is similar to some studies conducted in west africa [26, 27] and in ghana [19, 25] . the predominance of crf02_ag has been associated with high viral infectivity and productivity, possible replicative fitness and high viral loads which favour viral transmission [19, 20] . subtype b was the next predominant subtype and was relatively frequent compared to previous studies in ghana [25] . subtype b is most predominant in the americas [28, 29] and western europe [30, 31] . the occurrence of hiv-1 subtypes in different geographical areas has been linked to socio-epidemiologic factors such as mobility and migration [32, 33] . these factors, however, cannot be confirmed for this fig. 1 percentage occurrence of hiv-1 subtypes in sequenced pr and rt genes. figure 1 shows hiv-1 subtype patterns in the pr and rt genes. pr and rt sequences were submitted online to the stanford university hiv drug resistance database (hivdb). one (8%) was subtype a, 2 (17%) were subtype b and 9 (75%) were crf02_ag. of the 20 rt sequences, 2 (10%) were subtype a, 8 (40%) were subtype b, 9 (45%) were crf02_ag and 1 (5%) was crf09_cpx. key: pr protease, rt reverse transcriptase, crf circulating recombinant form the study observed low amplification rate of samples. this could be due to low viral loads in some samples. however, it is not entirely the reason for low amplification rate as samples with low viral loads were successfully amplified whilst others with higher loads were not amplified. previous research shows that samples from patients with persistently low viral load could be genotyped by a nested pcr method [18, 24] . the inability to obtain peripheral blood mononuclear cells (pbmc) to amplify alongside the plasma could also account for a lower amplification rate. amplification success with proviral dna from pbmc was found to be relatively higher than viral rna from plasma [22, 26] . genotyping from plasma rna and proviral dna concurrently could have increased amplification success since some plasma samples may be amplified and not their pbmc samples and vice versa. despite these limitations, the study obtained data that is important for hiv management in ghana. this study found major drug resistance mutations, e138a and k65r that respectively confer high level resistance to nnrtis and nrtis. although, crf02_ag was most predominant, the recorded percentage of subtype b and the evolutionary relationship inferred by phylogenetic analysis may suggest possible subtype importation. a more prospective and detailed analysis is needed to establish this phenomenon. the data obtained is useful for the selection of drugs for art initiation to maximize therapeutic outcomes in drug-naïve hiv-1 patients in ghana. continuous surveillance for drug resistance mutations and subtype diversity in population groups is therefore imperative for effective art outcomes. we thank the national blood service, ghana for the samples used in this study. technical support by the hiv genotyping team of noguchi memorial institute for medical research is acknowledged. the hiv-1 rt and pr sequences have been deposited at genbank under accession numbers mn402725-mn402756. the data sets are available with the corresponding author on request. the data sets used and analysed during this study are available with the corresponding author on request. ethics approval and consent to participate the protocol was approved by the ethics and protocol review committee of the college of health sciences, university of ghana (chs-et/m.1 -p 3.2/ 2016-2017). approval to select hiv positive samples was also obtained from the nbs (nbsgrd/160929/01). the nbs has given the consent to publish the results without personal identification. background, projections, impacts, interventions and policy hiv-1 drug resistance-associated mutations among hiv-1 infected drug-naïve antenatal clinic attendees in rural kenya declining morbidity and mortality among patients with human immunodeficiency virus infection: hiv outpatient study investigators the prevalence of antiretroviraldrug resistance in the united states transmitted hiv-1 drug resistance in africa. lusaka: 8th interest workshop effect of transmitted drug resistance on virological and immunological response to initial combination antiretroviral therapy for hiv (eurocoord-chain joint project): a european multicohort study report on the global epidemic the epidemiology of antiretroviral drug resistance among drug-naive hiv-1-infected persons in 10 us cities prevalence of drug-resistant hiv-1 variants in untreated individuals in europe: implications for clinical management, spread programme variability of the human immunodeficiency virus type 1 polymerase gene from treatment naive patients in accra, ghana genotypic diversity and mutation profile of hiv-1 strains in antiretroviral treatment (art) -naïve ghanaian patients and implications for antiretroviral treatment (art) low level of transmitted hiv drug resistance at two hiv care centres in ghana: a threshold survey hiv-1 drug-resistance surveillance among treatment-experienced and treatment-naıve patients after the implementation of antiretroviral therapy in ghana update: active involvement of young people is key to ending the aids epidemic by 2030 improved conditions for extraction and amplificationof human immunodeficiency virus type 1rna from plasma samples with low viral load performance and quality assurance of genotypic drug-resistant testing for human immunodeficiency virus type 1 in japan epidemiologic dominance of hiv-1 subtype crf02_ag in ghana: preliminary virological evidence of increasing association with new infections higher viral load may explain the dominance of crf02_ag in the molecular epidemiology of hiv in ghana impact of hiv-1 reverse transcriptase e138 mutations on rilpivirine drug susceptibility effect of mutations at position e138 in hiv-1 reverse transcriptase on phenotypic susceptibility and virologic response to etravirine occurrence of transmitted hiv-1 drug resistance among drug-naïve pregnant women in selected hiv care centers in ghana intensification of a failing regimen with zidovudine may cause sustained virologic suppression in the presence of re-sensitizing mutations including k65r prevalence and determinants of transmitted antiretroviral drug resistance in hiv-1 infection predominance of crf02-ag and crf06-cpx in niger, west africa the predominance of human immunodeficiency virus type 1 (hiv-1) circulating recombinant form 02 (crf02_ag) in west central africa may be related to its replicative fitness genetic analysis of hiv-1 isolates from brazil reveals the presence of two distinct genotypes genetic diversity of the envelope glycoprotein from human immunodeficiency virus type 1 isolates of african origin hiv-1 subtypes in luxembourg surveillance of hiv-1 subtypes among heterosexuals in england and wales tracking a century of global expansion and evolution of hiv to drive understanding and to combat disease the origin and diversity of the hiv-1 pandemic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations no competing interest exist. key: cord-278156-zd039ohv authors: dumas, fabrice; preira, pascal; salomé, laurence title: membrane organization of virus and target cell plays a role in hiv entry date: 2014-09-01 journal: biochimie doi: 10.1016/j.biochi.2014.08.015 sha: doc_id: 278156 cord_uid: zd039ohv the initial steps of the human immunodeficiency virus (hiv) replication cycle play a crucial role that arbitrates viral tropism and infection efficiency. before the release of its genome into the host cell cytoplasm, viruses operate a complex sequence of events that take place at the plasma membrane of the target cell. the first step is the binding of the hiv protein envelope (env) to the cellular receptor cd4. this triggers conformational changes of the gp120 viral protein that allow its interaction with a co-receptor that can be either ccr5 or cxcr4, defining the tropism of the virus entering the cell. this sequential interaction finally drives the fusion of the viral and host cell membrane or to the endocytosis of the viruses. here, we discuss how the membrane composition and organization of both the virus and the target cell can affect these steps and thus influence the capability of the viruses to infect cells. the entry of the virus into the cell can be decomposed in three main successive steps: 1/the interactions of the viruses with their specific receptors cd4, 2/the interactions of the viruses with coreceptors (ccr5 or cxcr4) and 3 or 3 0 /either the fusion of the membrane of the virus with the one of the infected cell or the endocytosis of the virus (fig. 1 ). the efficiency of each of these steps is influenced by the membrane protein and lipid composition and by their lateral organization and dynamics at the surface of the target cell. it has been shown that hiv attachment to immune cells first occurs via non-specific adhesion molecules that restrict the virus envelope in vicinity of more specific receptors [1, 2] . cd4 is the principal and mandatory receptor. upon binding to cd4 (step1), the viral gp120 protein undergoes a conformational change allowing additional interactions with a co-receptor (step 2). the main parameter that can affect the efficiency of this first step is the density of cd4 receptors that are present at the surface of the target cell [3e5] . this amount varies a lot from cell to cell type e in macrophages cd4 expression levels are 10e20 fold lower than what they are in cd4 þ t cells e but also depends on the activation state of immune cells [6, 7] . for these reasons, there is a huge variability of infection efficacy depending both on the virus strain and the cell type which is infected, rendering difficult the interpretation and the comparisons of experiments carried out in different studies. beyond the expression level of cd4, a second factor that has to be taken into account is the lateral organization of the proteins. it is now well known that the lateral organization of proteins in the plasma membrane of the cells is heterogeneous [8, 9] and might depend on lipid composition [10] . in this context, many laboratories have observed that cd4 proteins are not randomly distributed at the surface of the plasma membrane of cells but rather confined into domains [11e13] . such a clustering may promote hiv binding since viruses could engage several interactions with multiple cd4 confined in a small area. cd4 has been shown to be partially located into lipid rafts [14] . lipid rafts microdomains result from the preferential association between sphingolipids, saturated lipids, sterols and specific proteins in the external leaflet of the plasma membrane. these microdomains are found in a liquid-ordered state (lo) that renders them resistant to solubilization by detergents at low temperature [15e17]. there is a growing amount of evidence that the presence of cholesterol in the target cell is essential for the infection by many viruses. ebola virus [18] , herpes simplex [19, 20] or murine coronavirus [21] all require cholesterol in the target cell membrane. a long controversy started at the beginning of the millennium to know whether hiv preferentially binds to cd4 proteins that are confined into lipid rafts or not. the putative role of lipid rafts first came from the observation that cholesterol depletion impaired hiv infection [22e24]. similar results have also been obtained by using glucosylceramide synthase inhibitors that blocks glycosphingolipid biosynthesis [25] . nevertheless, these approaches present two drawbacks. first, these treatments are very stressful for the cells and one cannot be sure that the observed effects are the result of the sole decrease in cholesterol content. second, it does not reveal at which step lipid rafts might be involved in the infection process (receptor or co-receptor binding, fusion…). to go further in the comprehension of the role of lipid rafts in hiv infection, detergent extractions and optical microscopy studies have been used and produced contradictory results. as an example, one study showed that a cd4 mutant excluded from lipid raft domains membranes did not allow efficient hiv entry [26] , whereas a similar study, using alternative mutants that also prevent cd4 association with lipid rafts, established that cd4 excluded from lipid rafts do support hiv entry [14] . these two studies have been carried out on different cell types (hek and t cells, respectively). these cell lines very likely have different lipid composition and organization. this might explain the discrepancies in the results that have been described above. this also enlightens the crucial role of membrane composition and organization on the mechanisms of infection process. apparent contradictions also arise from works published by the same authors that successively showed that hiv uses lipid raft-colocalized cd4 for productive entry into cells [24] and that cd4 receptor localized to non-raft membrane microdomains supports hiv-1 entry [27] . one hypothesis, that renders these puzzling observations compatible, is to take into consideration that these studies are probing different steps of the infection process (i.e. binding to cd4 receptor, binding to co-receptor or fusion). our proposal is that the lipid raft localization of cd4 is not required for virus binding while the following steps are dependent on the presence of lipid rafts. this hypothesis is reinforced by the 1-binding of cd4 to gp120 leads to exposure of a co-receptor binding site in gp120. 2-binding of gp120 to co-receptor: depending on virus tropism co-receptor can be either ccr5 (r5 tropism) or cxcr4 (x4 tropism). co-receptor binding triggers new conformational changes of gp120, exposing gp41 protein that inserts into the membrane of host cell. 3-fusion: gp41 protein bends back on itself, forming a six helix bundle that bring closer the viral and target cell membranes leading to their fusion. 3'-endocytosis: an alternative pathway to fusion is the entry of virus into its cell host via endocytosis. virus particle then can be degraded or they can fuse with vesicular membrane, releasing virus content into the cytoplasm of the cell. observation that cd4 lateral mobility affects hiv-1 envelope glycoprotein mediated fusion [28] . in other words, once viruses are bound to the cell surface they might migrate to lipid raft domains where the infection can be completed. furthermore, this model is in agreement with the observation that murine leukemia virus "surfing" at the surface of infected cells precedes entry [29] . this shows that the different steps of hiv infection do not occur at the same place rendering necessary the development of dynamic approaches in order to study the time-course of infection process. the major co-receptors of hiv-1 are the chemokine receptors ccr5 and cxcr4. the use of the co-receptors defines the viral tropism of the strain that will infect the cell. viruses using ccr5 as a co-receptor are called r5, those using cxcr4 are named x4 while those that use both are termed r5x4 [30, 31] . r5 viruses prevail during the chronic phase of the infection while the emergence, after several years and for half the patients, of variants capable of using cxcr4 is associated to the rapid progress towards a pathologic state [32] . the mechanisms by which x4 viruses emerge and precipitate disease progression are still unknown. different studies have proposed that the infection process requires the attachment of several gp120 envelope trimers to multiple cd4 and multiple coreceptors [33e36]. considering the weakness of cd4-gp120 interaction [2] , the viruses should have a very short time (less than 0.2 s) to encounter their co-receptors once bound to cd4 [37, 38] . virus entry might thus not only depend on the local membrane density of cd4 but also on that of chemokine receptors. this local density is expected to be favored by the sequestration of ccr5 and/or cxcr4 with cd4 into delimited membrane domains. this is further supported by high-resolution electron microscopy experiments showing that, ccr5, cxcr4 and cd4 are clustered on microvilli of human macrophages and t cells [13] . co-immunoprecipitation studies have shown a constitutive association between cd4 and ccr5 at the surface of t cells, macrophages and monocytes [39] . similar studies performed on hek cells showed the opposite [40] . a constitutive interaction has later been confirmed by fret experiments that also revealed that this interaction takes place outside of lipid raft domains and is reinforced by the viral gp120 protein [41] . another clue of cd4-ccr5 interaction came from the study of the membrane dynamics of these receptors. it has first been observed that cd4 and ccr5 presented different mobility [42] . more interestingly, we have shown that the presence of cd4 strongly affects the dynamics of ccr5. indeed, when ccr5 was expressed alone in hek 293t cells only one diffusing population confined into large domains was detected. this behavior was markedly modified in the presence of cd4 since two diffusing populations of ccr5 were then observed, leading to the conclusion that the interaction of one cd4 with several ccr5 leads to their confinement into smaller membrane domains than in the absence of cd4 [11] . these confinement zones that concentrate both cd4 and ccr5 might constitute platforms that would facilitate the binding (and/or consecutive fusion) of hiv to its target cell. because of the prevalence of r5 virus strain in primo-infections, most of studies concern ccr5 receptor and, comparatively, only few studies have been carried out on cxcr4 protein organization within the membrane of immune cells while many works concerns clinical investigations [43] . as for ccr5, a co-immunoprecipitation study has shown a constitutive association between cd4 and cxcr4 [40] . however, this study has been performed in hek 293t cells transiently expressing cd4 with cxcr4 or ccr5 that gave contradictory results regarding ccr5 [39] as mentioned in the above ccr5 co-receptor chapter. more recent publications, respectively based on the use of cyclodextrin and ceramide modulators, have also shown that cxcr4 proteins are partially localized into lipid rafts domains and that this localization into lipid rafts is required for efficient infection [44, 45] . even though literature can be contradictory in some points, it seems to be clear that receptor and co-receptors of hiv virus are confined into domains that could act as ports of entry for hiv viruses. this is for example illustrated by the fact that statins inhibit both r5 and x4 virus infection by down-regulating the activity of rho gtpases which is expected to impede the clustering of host lipid rafteassociated receptors [48] . the existence of different and independent confinement zones might also give an explanation for r5 prevalence at the expense of x4. for instance one can hypothesize that cd4 is preferentially confined with ccr5 into microdomains different from those containingcxcr4 [22] . this hypothesis is in agreement with the observation that reducing the expression of cd4 decreases the susceptibility of human cells to infection by x4 viruses [46] . furthermore, it has been shown that lowering ccr5 expression level with molecules modulating cholesterol content (lovastatin, mevastatin and simvastatin), favors the infection by x4 viruses [47] . taken together these results raise the question of the lateral distribution and the stoichiometry of the different receptors at the surface of the target cell. recently, johnston and collaborators have developed affinofile cells in which the expression level of both cd4 and ccr5 can be independently modulated [49] . several strains stably expressing various amounts of cxcr4 have been generated; this allows obtaining any ratio of cd4, ccr5 and cxcr4. this tool provides a powerful method to characterize viral entry efficiency as a function of cd4 and ccr5 expression levels [50] . these affinofile cell lines also permitted to demonstrate that several ccr5 conformations (distinguished thanks to specific monoclonal antibodies), exhibiting different localization and g-protein association, might have implications in selective targeting of hiv-1 [51] . this system is also used in our lab to check whether the expression level of receptors and co-receptors can impact the dynamic of each other and the infection efficiency of various virus strains. receptor binding and subsequent association with the coreceptor, lead to conformational changes of the viral trimeric gp120 protein. this induces the exposure of the hydrophobic gp41 peptide which inserts into the host cell membrane. this brings closer the viral and host membranes. the gp41 protein then bends back on itself, forming a six helix bundle [52, 53] . at this stage, the viral membrane and the target cell membrane have bypassed the repulsion forces and are close enough to spontaneously form the fusion pore [54, 55] . the role of lipids in the modulation of this step has been extensively studied and the implication of lipid rafts is now admitted by most of researchers. it has first been shown on large unilamellar vesicles (luv) that sphingomyelin and cholesterol promote gp41 surface aggregation and membrane restructuring [56] . such an aggregation is thought to be important to reach a stoichiometry allowing the fusion to occur. indeed it has been proposed that at least two envelop proteins have to bind cd4 and a co-receptor and that two fusion proteins of each trimer have to insert into the target cell to promote viral entry [57] . the gp41 protein presents an extracellular domain that contains the fusion peptide whose structure strongly depends on its lipid environment. some authors have proposed that a small amino acid sequence (lwyik), within the gp41 protein, constitute a cholesterol-binding domain [58] which is important for hiv infectivity [59] . this sequence presents homology with the cholesterol recognition amino-acid consensus (crac) motifs described by epand [60] . further works realized on model membranes confirmed that the ectodomain membrane proximal region of gp41 was able to bind to cholesterol membrane domains [61] . the binding to such a cholesterol-rich (>30 mol%) zone promotes b-sheet secondary structuration of the fusion peptide that deeply embeds into the host membrane. this deep insertion has been shown to favor fusogenicity [62e64]. beyond these effects on insertion and structuration of the gp41 virus protein, cholesterol might play a role in the membrane fusion itself. the fusion involves the coalescence of viral and hosts cell membranes, accompanied by the mixing of their cytoplasm. this implies that lipids transiently adopt a non-lamellar organization as described by chernomordik and kozlov [65] . such an organization, named "hemifusion stalk", can be stabilized by inverted cone-shaped and/or by cone-shaped lipids, depending on their outer or inner leaflet location. from this point of view, it is interesting to note that hiv membrane is enriched in phopshoinositides [66] that can induce a positive curvature that favor the initiation of fusion while cholesterol might mediate membrane curvature of host cell during fusion [67] . it has to be noticed that this effect of cholesterol is not specific to hiv fusion process and has been observed in other processes such as nuclear membrane formation [68] . other authors have shown that sphingomyelin plays a role in hiv infection. a study based on surface aggregation assays of rhodamine-labeled gp41 subunit demonstrated that sphingomyelin and cholesterol promoted gp41-aggregation [56] . the effect of sphingomyelinase has also been studied by extraction of rafts, virus binding assays and measurements of the dynamics of hiv receptors. the authors observed that sphingomyelinase treatments decreased cd4 lateral mobility and inhibited hiv fusion with the membrane of the target cell. they propose that these effects are due to the clustering of cd4 molecules that prevents co-receptor engagement and hiv fusion [69] . it has to be noticed that the fusion of the viral membrane and the plasma membrane of the target cell does not necessarily conduce to productive infection since the viral content can be degraded inside the target cell. furthermore, lipid mixing of viral and cell membranes have been observed without release of the viral content inside the target cell [70, 71] . this incomplete fusion is an argument in favor of an endocytotic pathway discussed in the following section. although the mechanism of hiv fusion is now well documented (see part 3 of this manuscript), several lines of evidence suggest that hiv entry into their cell host can also occur via endocytosis [55,72e76] . this process has moreover been proposed to be predominant in hiv internalization into macrophages, endothelial cells or epithelial cells [31,77e80] and has also been shown to depend on the cell activation status [81] . this mini-review is focused on the role specifically played by lipids in this process, we invite the reader to refer to the recent review by melikian for a more general discussion of endocytosis versus fusion in hiv entry [71] . kinetic measurement of fusion performed at different temperatures and the use of specific inhibitors pointed out that hiv viruses enter the cells via endocytosis followed by fusion with endosomes [70] . this work also showed that inhibiting endocytosis allowed lipid mixing of viral and cell membrane without conducing to a complete fusion. interestingly, the obtained results were independent of virus tropism but kinetics appeared to depend on the target cell type. this might be due to differences in endosome trafficking, but it is tempting to propose that these variations might be due to differences in lipid composition of these cell lines. as an example, in similar experiments, markosyan and collaborators have proposed that membrane tension might differ between cell types and affect pore formation and growth [82] . a study, based on cholesterol disruption of macrophage membranes, demonstrate that hiv entry into macrophages is dependent on lipid rafts [83] . this has later been confirmed by a study showing that endocytosis of hiv involves rafts [84] . the originality of this work comes from the fact it does not use pharmacological agents that may have non-specific effects. thanks to genetically modified macrophages allowing manipulating the sub-cellular distribution of cd4, these authors have that the entry of hiv into macrophages is dependent on endocytosis through lipid raft containing cd4 [84] . only few works have been carried out to specifically study the role of lipids in hiv endocytosis and it will be interesting to go further in this field. one difficulty to understand hiv infection mechanism comes from the fact that different entry pathways exist and the importance of each being still a subject of controversy. since most of hiv particles are degraded by the cells and only a small fraction of viruses establishes infection, the identification of productive entry pathway remains difficult. however, whatever the mode of entry of viruses, many observations suggest that membrane composition and organization play important roles. cholesterol, sphingomyelin and lipid raft domains seem to be of first importance not only in the infection process but also in virus prevalence [47] . glycosphingolipids and gangliosides have also been reported to facilitate hiv infection such as galactosylceramides [85] or gm1 and gm3 [86, 87] . these lipids might serve as hiv binding sites or regulate dynamic and clustering of cd4, ccr5 and cxcr4. this information has been recently reinforced by the fact that peptides conjugated to lipid comprising sphingomyelin backbones impair early and late hiv-1 membrane fusion [88] . in this context, the development of new original and noninvasive techniques should allow to discriminate the entry pathway of viruses and to decipher the molecular mechanisms involved in the infection process. among them, the single particle tracking technique is promising. it has been used to study the dynamics and the organization of hiv receptors and co-receptors, revealing the existence of subpopulation of cd4 with different behaviors in t-lymphocytes [12] . tracking entire viruses consists in labeling viruses with fluorescent probes and recording their movements with a videomicroscope. this might be useful to study the time-course of infection process. this approach gives information regarding the percentage of aborted binding (i.e. virus that bind to cd4 without encountering co-receptors and finally unbind). it also permits to measure the time-lapse from binding to cell surface until virus and target cell membranes fusion. these two parameters give real time insights onto the efficiency of the infection process. this method will be useful to verify previously obtained data on the behavior of single proteins in a more physiological and relevant context. such an approach has already been successfully used with influenza viruses [89, 90] and might be extended to inactivated hiv viruses that keep their binding and fusion properties [91, 92] . combined with the use of drugs to modulate lipid membrane composition these techniques will permit to understand the mechanisms involved in infection and more specifically the role played by lipids in this process. syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages hiv-1 attachment: another look differences in cd4 dependence for infectivity of laboratory-adapted and primary patient isolates of human immunodeficiency virus type 1 cxcr-4, and ccr-5 dependencies for infections by primary patient and laboratory-adapted isolates of human immunodeficiency virus type 1 effects of ccr5 and cd4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1 variations in cd4 expression by human monocytes and macrophages and their relationships to infection with the human immunodeficiency virus asymmetric hiv-1 co-receptor use and replication in cd4(þ) t lymphocytes is the fluid mosaic (and the accompanying raft hypothesis) a suitable model to describe fundamental features of biological membranes? what may be missing? front membranes are more mosaic than fluid is the protein/lipid hydrophobic matching principle relevant to membrane organization and functions? cd4 interacts constitutively with multiple ccr5 at the plasma membrane of living cells. a fluorescence recovery after photobleaching at variable radii approach single particle tracking reveals two distinct environments for cd4 receptors at the surface of living t lymphocytes ccr5, cxcr4, and cd4 are clustered and closely apposed on microvilli of human macrophages and t cells hiv-1 entry into t-cells is not dependent on cd4 and ccr5 localization to sphingolipid-enriched, detergent-resistant, raft membrane domains lipid rafts as a membrane-organizing principle rafts defined: a report on the keystone symposium on lipid rafts and cell function functional rafts in cell membranes measuring the strength of interaction between the ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection specific association of glycoprotein b with lipid rafts during herpes simplex virus entry campadelli-fiume, {alpha}v{beta}3-integrin routes herpes simplex virus to an entry pathway dependent on cholesterol-rich lipid rafts and dynamin2 murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release segregation of cd4 and cxcr4 into distinct lipid microdomains in t lymphocytes suggests a mechanism for membrane destabilization by human immunodeficiency virus lipid rafts and hiv pathogenesis: host membrane cholesterol is required for infection by hiv type 1 human immunodeficiency virus type 1 uses lipid raft-colocalized cd4 and chemokine receptors for productive entry into cd4(þ) t cells glycosphingolipids promote entry of a broad range of human immunodeficiency virus type 1 isolates into cell lines expressing cd4, cxcr4, and/or ccr5 blocking of hiv-1 infection by targeting cd4 to nonraft membrane domains cd4 receptor localized to non-raft membrane microdomains supports hiv-1 entry identification of a novel raft localization marker in cd4 restricted lateral mobility of plasma membrane cd4 impairs hiv-1 envelope glycoprotein mediated fusion actin-and myosin-driven movement of viruses along filopodia precedes their entry into cells a new classification for hiv-1 evolution of hiv-1 coreceptor usage through interactions with distinct ccr5 and cxcr4 domains correlation of coreceptor usage and disease progression cooperation of multiple ccr5 coreceptors is required for infections by human immunodeficiency virus type 1 hiv requires multiple gp120 molecules for cd4-mediated infection estimating the threshold surface density of gp120-ccr5 complexes necessary for hiv-1 envelope-mediated cell-cell fusion electron tomography of the contact between t cells and siv/ hiv-1: implications for viral entry single-molecule analysis of human immunodeficiency virus type 1 gp120-receptor interactions in living cells monitoring early fusion dynamics of human immunodeficiency virus type 1 at single-molecule resolution constitutive cell surface association between cd4 and ccr5 specific interaction of cxcr4 with cd4 and cd8alpha: functional analysis of the cd4/cxcr4 interaction in the context of hiv-1 envelope glycoprotein-mediated membrane fusion cd4 and ccr5 constitutively interact at the plasma membrane of living cells: a confocal fluorescence resonance energy transfer-based approach mobility of the human immunodeficiency virus (hiv) receptor cd4 and coreceptor ccr5 in living cells: implications for hiv fusion and entry events the puzzling role of cxcr4 in human immunodeficiency virus infection cxcr4-tropic, but not ccr5-tropic, human immunodeficiency virus infection is inhibited by the lipid raft-associated factors, acyclic retinoid analogs, and cholera toxin b subunit raft localization of cxcr4 is primarily required for x4-tropic human immunodeficiency virus type 1 infection coreceptor competition for association with cd4 may change the susceptibility of human cells to infection with t-tropic and macrophagetropic isolates of human immunodeficiency virus type 1 statins disrupt ccr5 and rantes expression levels in cd4þ t lymphocytes in vitro and preferentially decrease infection of r5 versus x4 hiv-1 statins inhibit hiv-1 infection by down-regulating rho activity a quantitative affinity-profiling system that reveals distinct cd4/ccr5 usage patterns among human immunodeficiency virus type 1 and simian immunodeficiency virus strains affinofile profiling: how efficiency of cd4/ccr5 usage impacts the biological and pathogenic phenotype of hiv ccr5 conformations are dynamic and modulated by localization, trafficking and g protein association core structure of gp41 from the hiv envelope glycoprotein atomic structure of the ectodomain from hiv-1 gp41 hiv entry and envelope glycoproteinmediated fusion common principles and intermediates of viral proteinmediated fusion: the hiv-1 paradigm sphingomyelin and cholesterol promote hiv-1 gp41 pretransmembrane sequence surface aggregation and membrane restructuring analysis of the subunit stoichiometries in viral entry identification of a conserved domain of the hiv-1 transmembrane protein gp41 which interacts with cholesteryl groups a conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity cholesterol and the interaction of proteins with membrane domains the influence of cholesterol on the interaction of hiv gp41 membrane proximal region-derived peptides with lipid bilayers hiv gp41 fusion peptide increases membrane ordering in a cholesterol-dependent fashion fusion activity of hiv gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion a strong correlation between fusogenicity and membrane insertion depth of the hiv fusion peptide mechanics of membrane fusion retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides cholesterol mediates membrane curvature during fusion events spatial regulation of membrane fusion controlled by modification of phosphoinositides sphingomyelinase restricts the lateral diffusion of cd4 and inhibits human immunodeficiency virus fusion inhibition of hiv-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion hiv entry: a game of hide-and-fuse? endocytic entry of hiv-1 retrovirus entry by endocytosis and cathepsin proteases hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes est e, endocytosis of hiv: anything goes hiv: cell binding and entry, cold spring harb hiv-1 infects macrophages by exploiting an endocytic route dependent on dynamin, rac1 and pak1 macropinocytosis-like hiv-1 internalization in macrophages is ccr5 dependent and leads to efficient but delayed degradation in endosomal compartments transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis differential hiv-1 endocytosis and susceptibility to virus infection in human macrophages correlate with cell activation status time-resolved imaging of hiv-1 env-mediated lipid and content mixing between a single virion and cell membrane hiv entry in macrophages is dependent on intact lipid rafts the productive entry pathway of hiv-1 in macrophages is dependent on endocytosis through lipid rafts containing cd4 the galactosyl ceramide/sulfatide receptor binding region of hiv-1 gp120 maps to amino acids 206e275 sphingolipids: modulators of hiv-1 infection and pathogenesis specific interaction of hiv-1 and hiv-2 surface envelope glycoproteins with monolayers of galactosylceramide and ganglioside gm3 early and late hiv-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site endocytosis of influenza viruses, microbes infect visualizing infection of individual influenza viruses elimination of retroviral infectivity by n-ethylmaleimide with preservation of functional envelope glycoproteins inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational and functional integrity of virion surface proteins key: cord-287949-243xlmep authors: onovo, a. a.; atobatele, a.; kalaiwo, a.; obanubi, c.; james, e.; gado, p.; odezugo, g.; magaji, d.; ogundehin, d.; russell, m. title: using supervised machine learning and empirical bayesian kriging to reveal correlates and patterns of covid-19 disease outbreak in sub-saharan africa: exploratory data analysis date: 2020-05-02 journal: nan doi: 10.1101/2020.04.27.20082057 sha: doc_id: 287949 cord_uid: 243xlmep introduction: coronavirus disease 2019 (covid-19) is an emerging infectious disease that was first reported in wuhan, china, and has subsequently spread worldwide. knowledge of coronavirus-related risk factors can help countries build more systematic and successful responses to covid-19 disease outbreak. here we used supervised machine learning and empirical bayesian kriging (ebk) techniques to reveal correlates and patterns of covid-19 disease outbreak in sub-saharan africa (ssa). methods: we analyzed time series aggregate data compiled by johns hopkins university on the outbreak of covid-19 disease across ssa. covid-19 data was merged with additional data on socio-demographic and health indicator survey data for 39 of ssa 48 countries that reported confirmed cases and deaths from coronavirus between february 28, 2020 through march 26, 2020. we used supervised machine learning algorithm, lasso for variable selection and statistical inference. ebk was used to also create a raster estimating the spatial distribution of covid-19 disease outbreak. results: the lasso cross-fit partialing out predictive model ascertained seven variables significantly associated with the risk of coronavirus infection (i.e. new hiv infections among pediatric, adolescent, and middle-aged adult plhiv, time (days), pneumococcal conjugate-based vaccine, incidence of malaria and diarrhea treatment). our study indicates, the doubling time in new coronavirus cases was 3 days. the steady three-day decrease in coronavirus outbreak rate of change (roc) from 37% on march 23, 2020 to 23% on march 26, 2020 indicates the positive impact of countries' steps to stymie the outbreak. the interpolated maps show that coronavirus is rising every day and appears to be severely confined in south africa. in the west african region (i.e. burkina faso, ghana, senegal, cotediviore, cameroon, and nigeria), we predict that new cases and deaths from the virus are most likely to increase. interpretation: integrated and efficiently delivered interventions to reduce hiv, pneumonia, malaria and diarrhea, are essential to accelerating global health efforts. scaling up screening and increasing covid-19 testing capacity across ssa countries can help provide better understanding on how the pandemic is progressing and possibly ensure a sustained decline in the roc of coronavirus outbreak. funding: authors were wholly responsible for the costs of data collation and analysis. on jan 30, 2020, who declared the current novel coronavirus disease 2019 epidemic a public health emergency of international concern 3 . as of march 26, 2020, the number of covid-19 cases surpassed 500,000 globally, and the epidemic registered a total of 1,978 cases and 25 deaths in sub-saharan africa (ssa). at this time, 39 of ssa's 48 countries [excluding burundi, comoros, lesotho, sierra leone, south sudan and sao tome and principe] all reported confirmed cases of coronavirus and imported transmission as the major mode of spread. despite aggressive response by ssa countries to stymie the outbreak, the number of reported new cases and deaths continue to increase. as of march 30, 2020, south africa is the only country with more than 1,000 cases, and eight countries: burkina faso, cameroon, cote d'ivoire, senegal, ghana, mauritius and nigeria including south africa account for 80% of ssa's covid-19 disease outbreak ( figure 1 ). as reported by huang et al, 4 patients with covid-19 present primarily with fever, myalgia or fatigue, and dry cough. although most patients are thought to have a favorable prognosis, older patients and those with chronic underlying conditions may have worse outcomes. certain epidemiological features and clinical characteristics of covid-19 have been previously reported 5,6. however, these findings were based on prediction models or studies from america, asia and europe countries, and the use of these findings might be inappropriate for ssa context. the spatial distribution pattern of coronavirus disease outbreak and the risk factors leading to poor clinical outcomes have not been well understood and delineated. in this study, we employed the exploratory data analysis (eda) technique to maximize insights from our study data, uncover patterns and summarize risk factors associated with the coronavirus disease outbreak in ssa. eda is an approach for data analysis that employs a variety of technique for summarizing and visualizing important characteristics of a dataset. this will be the first study to use supervised machine learning algorithm, "least absolute selection shrinkage operator" (lasso) regression as the principle method for variable selection (predict important variables associated with covid-19) and for statistical inference (confirm or establish relationship of risk factors and covid-19 disease); and empirical bayesian kriging (ebk) used to create a raster estimating the temporal spatial distribution of covid-19 disease outbreak in ssa. we collected and analyzed time series aggregate data on the 1,978 confirmed cases and 25 deaths of covid-19 disease outbreak across ssa between february 28, 2020 through march 26, 2020. according to the united nations, ssa consists of all african countries that are fully or partially located south of the sahara. we focused on 48 of africa's 54 countries excluding algeria, djibouti, egypt, libya, morocco and tunisia, in accordance with the world bank lists of countries in ssa. we accessed the covid-19 data resource hub developed by the tableau community and utilized nearreal time data compiled by johns hopkins university (jhu) and includes data from world health organization (who) and the centers for disease control and prevention (cdc). additional data from socio-demographic and health indicator surveys was derived from resources of the world bank, unicef, who and unaids. this additional data was merged with the jhu covid-19 data to establish the dataset that was used for this study. the dataset included the most recent and available data across ssa, and period of the data spanned between 2013 and 2019. for data on health systems performance index, these was derived from the who publication in 2001 entitled "measuring overall health systems performance for 191 countries". the target variable included in the supervised machine learning model was confirmed cases of coronavirus disease. the target variable was log-transformed to control for skewness and ensure effective linear relationship with the independent variables. explanatory or independent variables in the model included total population, gdp per capita, percentage of population with access to electricity, percentage of population with access to basic drinking water, incidence of malaria (per 1,000 population at risk), percentage of men and women aged 15 and over who currently smoke any tobacco product, diarrhea treatment (percent of children under 5 receiving oral rehydration and continued feeding), percentage of infants who received third-dose of pneumococcal conjugate-based vaccine (pcv), incidence of tuberculosis (per 100,000 people), percent out-of-pocket expenditure, life expectancy at birth, health systems performance index, estimated incidence rate (new hiv infection per 1,000 uninfected population, children aged 0-14 years), estimated incidence rate (new hiv infection per 1,000 uninfected population, adolescents aged 10-19 years), hiv prevalence among people aged 15-49 years, transmission classification of covid-19 disease (1=imported, 2=local transmission), income group (1=high income, 2=low income, 3=lower middle income, 4=upper middle income), geocoordinates of ssa countries (latitude and longitude), and time (days) between the first and last reported coronavirus cases. these variables were included in the analyses because recent research on coronavirus has found that biological variables such as pneumonia, malaria, and diarrhea 7 , socio-demographic variables like age 8 , access to comprehensive health care system 9 were associated with the transmission of coronavirus. consequently, other important variables particularly, cancer, diabetes or coronary heart disease (chd) were not available for analysis. we used lasso regression a supervised machine learning method for 1.) prediction and model selection, and 2.) inferential statistics. for prediction and model selection, we used three separate selection models available in statamp v. 16 . these models were cross-validation (cv), adaptive lasso and minimum bayesian information criterion (bic). in running lasso, the dataset was split into two sample groups; group 1 was training datasets we used to select our model, and group 2 our testing datasets we used to test the prediction. we used lasso because of its greater prediction accuracy and increased model interpretability. for the inferential statistics, we used the cross-fit partialing out lasso technique to estimate the coefficients, robust standard errors, p-values and confidence intervals of the specified variables of interest while the other covariates were selected as controls in the model. we georeferenced all confirmed cases and deaths from coronavirus that was reported across 39 countries and performed three analyses. first, we constructed maps of coronavirus infection across three different . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 2, 2020. . timepoints; time 1 (march 8, 2020), time 2 (march 14, 2020) and time 3 (march 26, 2020) and overlaid the coronavirus transmission classification type. second, we used spatial autocorrelation (global moran´s i) statistics to measure the degree, to which coronavirus infection is clustered, dispersed or randomly distributed. the expected value under the null hypothesis is that "there is no pattern of coronavirus infection in selected ssa countries". moran's i values range from -1 indicating perfect dispersion to +1 indicating perfect spatial clustering. a zero moran's i value indicates a random spatial distribution. third, we used ebk technique in the geostatistical analyst tool of arcgis 10.8 software to estimate the distribution of coronavirus across ssa. we analyzed 39 of ssa 48 countries that reported confirmed cases and deaths from coronavirus between february 28, 2020 through march 26, 2020. we plotted a matrix graph to explore the strength and association between 17 continuous variables and covid-19. overall, in figure 2a, the matrix graph showed that a majority, 82% (13/17) of the continuous variables indicated strong to moderate positive linear relationships, while two variables, out-of-pocket expenditure and hiv incidence rate in children aged 0-14 years showed strong negative linear relationships. prevalence of smoking was weakly correlated with coronavirus. approximately 31% (12/39) of the ssa countries had missing information on smoking prevalence and this may have impacted on this variable as evident with the significant chi-square goodness of fit χ2 (1) = 5.77, p= 0.016. all other variables included in the analysis had complete data for almost all countries, however, in few countries where data was missing, this was ≤ 5%. in figure 2b , spearman's rank-order correlation was run to determine the relationship between the two categorical variables and covid-19. there was a moderate, positive correlation between transmission classification and covid-19 disease, (rs (37) = 0.411, p = 0.009), with transmission classification explaining 17% of the variance in covid-19 disease. we calculated time (days) between the first and last reported coronavirus cases by country and plotted a quadrant chart of confirmed covid-19 cases against time. in figure 3 , majority, 32 (82%) of the countries reported imported transmission as the main way coronavirus is spreading χ2 (1) = 16.03, p= 0.0001, while south africa, ghana, senegal, cameroon, kenya, rwanda and liberia indicated local transmission as the source of covid-19 transmission. there was a strong positive correlation between time (days) and covid-19, (rs (37) = 0.599, p = 0.001), with time (days) explaining 36% of the variation in covid-19. the result is suggestive that new cases of coronavirus tend to rise with rise in time (days). cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 2, 2020. clusters were identified and south africa the only country in cluster 5 ( figure 5) . overall, the result revealed an upward trend and similarities in the way coronavirus is presenting across certain countries in ssa. lasso for prediction and model selection all the explanatory variables, 17 continuous and 2 categorical were included as possible contributors to the lasso model evaluating association with coronavirus disease. the rows for all three models were sorted so that the variables with the highest standardized coefficients are listed on top, (table 1a. ). ten explanatory variables with the highest relative importance were simultaneously selected by all the three learning techniques. these were hiv incidence rate among adolescence aged 10-19 years, hiv incidence rate among children aged 0-14 years, time (days), hiv prevalence among persons aged 15-49 years, infants who received third-dose of pneumococcal conjugate-based vaccine, incidence of hiv among individuals aged 15-24 years, incidence of malaria (per 1,000 persons at risk), incidence of tb (per 100,000 people), smoking prevalence (15years and above) and diarrhea treatment (children under-5 years). we accessed the goodness-of-fit over our training sample and testing sample. (table 1b .) show that the adaptive lasso model has the smallest mean square error (mse), 2.72 *10 -28 and the largest r-squared (100%) in the testing datasets. the top ten important variables selected by adaptive lasso was included as variables of interest in the lasso for inference model using the cross-fit partialing out technique. the remaining variables included in the model were trained as controls. the lasso regression for inference established that hiv incidence rate among adolescence aged 10-19 years (p=0.0001), hiv incidence rate among children aged 0-14 years (p=0.001), time (p=0.0001), infants who received third dose of pneumococcal conjugate-based vaccine (p=0001), incidence of hiv among individuals aged 15-24 years, (p=0.02), incidence of malaria (p=0.001) and diarrhea treatment (p=0.002) could statistically significantly predict coronavirus disease outbreak in ssa (table 2) . for better interpretation of results, we exponentiated the coefficients, subtracted the output from 1 and expressed the new coefficients as percentages. the exponentiated values are included in the lasso for inferential statistics table. all seven variables added statistically significantly to the prediction, p < 0.05. the global spatial autocorrelation moran´s i statistics confirmed the random distribution pattern of coronavirus outbreak among ssa countries ( figure 6 figure 7 shows that coronavirus is increasing per day and spreading outwards. coronavirus appears to be severally confined in south africa and increasing in the west african region (i.e. burkina faso, ghana, senegal, cote d'iviore, cameroon, and nigeria). figure 8 shows that coronavirus deaths tend to be concentrated in western african regions relative to central and southern african regions. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 2, 2020. of the estimated 6,000 new hiv infections that occur globally each day, two out of three are in sub-saharan africa 10 . currently in ssa region there is paucity of research on coronavirus and no documented evidence on how coronavirus disease is affecting or behaving in people living with hiv (plhiv). however, global and local hiv organizations and public health experts believe plhiv not on treatment, has cd4 count <200 copies/cell, a recent opportunistic infection and /or not virally suppressed may be at greater risk of coronavirus infection 11 . our study indicated that hiv incident rate among pediatrics aged 0-14 years, adolescents aged 15-19 years and middle-aged adults 15-24 years were good predictors of coronavirus. our research shows that new hiv infections raise the risk of coronavirus infection by 27.3 percent (p=0.001) in pediatrics 0-14 years, 41.9 percent (p=0.0001) in adolescents aged 15-19 years and 23 percent (p=0.02) in middle-aged adults 15-24. this finding collaborates with a recent study by su l et al 12 , which showed that children and youth are also infected and can spread coronavirus. a retrospective study by tang a et al 13 revealed that there had been several and far more severe pediatric cases of covid-19 in wuhan hospitals than reported. pneumonia affects children and families everywhere but is most prevalent in south asia and sub-saharan africa 14 . people who get pneumonia may also have a condition called acute respiratory distress syndrome (ards). it's a disease that comes on quickly after infection from coronavirus and causes breathing problems. the pneumonia vaccine protects against a kind of bacteria (streptococcus pneumoniae), not the coronavirus, but can support overall health, especially in older individuals or people who have a weak immune system 15 . more than three-quarters, 79% (95% ci: 72.5-85.6) of surviving infants 12 to 23 months across ssa received the third dose of pneumococcal conjugate-containing vaccine (pcv). our study indicates that surviving infants who received pcv were good predictors of coronavirus disease. it is evident from our results that increased cases of pneumonia among infants increases risk of infection from coronavirus by 46.8% (p=0.0001). malaria infection is common in sub-saharan africa, and it is often stated that the continent bears over 90 percent of the global p. falciparum burden 16 , and, generally, young children bear the brunt of the mortality burden. in 2017, four ssa countries; nigeria, democratic republic of the congo, mozambique, and uganda accounted for nearly half of all malaria cases worldwide. according to our results, incidence of malaria was 189 per 1,000 persons (iqr:22 -411) across ssa, and study results suggest that for every oneunit increase in the incidence of malaria, the risk of covid-19 disease increases by 36.8% (p=0.0001). diarrheal disease is the second leading cause of death in children under five years old and is responsible for killing around 525,000 children every year. recent studies have demonstrated that there is an association between diarrhea and coronavirus transmission. a study among 200 covid-19 patients at three hospitals in wuhan, china, indicated that among all patients who presented with digestive symptoms (117 patients), about 67 (58%) had diarrhea, and of these, 13 (20%) experienced diarrhea as the first symptom of their illness 17 . in our study, less than half 43% (95%ci: 28-56) of children under 5 with diarrhea received oral rehydration salts (ort) or the recommended homemade fluids across ssa. study result suggests that for every one-unit increase in diarrhea cases among children under-5, the risk of coronavirus increases by 37.5% (p=0.002). . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 2, 2020. . in terms of growth rate, the trend cluster analysis from our study suggests a combination of linear and exponential growth trends across ssa. the trend in new coronavirus cases appears to be doubling in south africa at every time increment compared to the other countries demonstrating an exponential trend. for countries in cluster 1 and in cluster 4 the trend is linear suggesting increases by less than 10 cases per day. countries in cluster 2 and 3 on the average indicated increases by 10 cases or more at every time increment. spontaneous transitions from low (reporting 10 cases on average) to high clusters (reporting more than 10 cases on average) was evident among countries previously in cluster 3 (i.e. cote d'iviore, cameron, nigeria and ghana). in the coming weeks, our study suggests, that countries in cluster 2; burkina faso, ghana, senegal, cote d'iviore, mauritius, cameroon, nigeria, congo (kinshasa) and rwanda are the most likely to present an exponential trend growth similar to south africa in cluster 5. our study indicates, the doubling time in new coronavirus cases reported across ssa is 3 days. covid-19 who surveillance reports indicate that the ssa countries have responded actively to the pandemic since the first case identified on 28 february 2020. many countries are requesting individuals to stay at home in self-quarantine, closing borders and imposing lockdowns and curfews. schools were ordered to close in the nigerian federal capital territory of abuja after only eight cases were confirmed nationwide in the month of february 2020. south africa banned visitors from high-risk countries, closed schools and quickly opened drive-through testing centers in johannesburg. the steady decline in the roc of coronavirus as indicated in our study from 37% on march 23, 2020 to 23% on march 26, 2020 suggests that measures instituted by countries to stem the outbreak is showing signs of future success. this finding fits with a recent modeling study showing that a combination of multiple approaches (i.e. social distancing, home isolation of cases, augmented by school and university closures) has a substantial effect on slowing coronavirus transmission in a short term. on the other hand, the declining roc may be attributed to low testing capacity in ssa. marius gilbert et al, in a modelling study highlighted differences in african countries capacity to respond to the coronavirus outbreak. countries with the highest importation risk (ie, egypt, algeria, and south africa) have moderate to high capacity to respond to outbreaks. countries at moderate risk (ie, nigeria, ethiopia, sudan, angola, tanzania, ghana, and kenya) have variable capacity and high vulnerability 18 . so far in ssa only a handful of countries (i.e. south africa, senegal, ghana and nigeria) have provided official figures on covid-19 testing data. spatial autocorrelation analysis using the global moran's i statistics indicated that coronavirus was spreading randomly across ssa. a major limitation in this study was that geocoordinate data used for this analysis represents locations of ssa countries not necessarily where covid-19 disease was detected, and this may have influenced results. at the time of this analysis, geocoordinates of counties, districts, and/or testing locations for coronavirus were not publicly available. the interpolated maps in our study show that coronavirus is increasing and spreading outwards per day to countries in central africa and the virus appears to be severely confined in south africa and in the west african region. the interpolated maps also suggest that countries in the west african region are most likely to repot increased number of deaths in the coming weeks compared to countries in central and southern africa. in conclusion, lasso was a good fit for variable selection and inference. the lasso regression model indicated that new hiv infections among pediatrics, adolescents and middle-aged adult plhiv, time, pneumococcal conjugate-based vaccine, incidence of malaria and diarrhea treatment could significantly predict coronavirus disease in ssa. this study can be repeated using more detailed patient-level granular data to provide more insight into the essential characteristics of patients diagnosed with covid-19. our study suggests that experiences learned from the hiv epidemic can be applied to the fight against covid-19. as in the aids response, governments should work with communities and civil society organizations to find local solutions, and health interventions delivered in an integrated manner. integrated and efficiently delivered interventions to reduce hiv, pneumonia, malaria, and diarrhea are essential to accelerating global health efforts. lessons learned from the hiv epidemics have shown that restrictive, stigmatizing and punitive measures can lead to significant human rights abuses, with disproportionate effects on already vulnerable communities. they can often undermine epidemic responses, sending people with symptoms underground and failing to address the underlying barriers that people face in attempting to protect their own health and that of their community. an approach that moves away from compulsory restrictions towards a focus on reaching and serving those who are most vulnerable, scaling up screening and testing for those most in need, empowering people with knowledge and tools to protect themselves and others (e.g. for covid-19, increased social/spatial spacing) and the removal of barriers, mirrors the learnings from the hiv response. the consistent three days decline in the roc of coronavirus outbreak from 37% to 23% is suggestive of the positive effect of measures instituted by countries to stymie the outbreak. to gain better understanding on how the pandemic is progressing, and possibly ensure a sustained decline in the roc of coronavirus outbreak, all countries should scale screening and increase their testing capacity and provide detailed and reliable testing data. recent non-randomized control studies have confirmed that hydroxychloroquine and azithromycin have been found to be efficient on sars-cov-2 and reported to be efficient in chinese cov-19 patients. we propose a silver bullet approach to controlling the outbreak. this silver bullet approach here implies the use of presumptive malaria treatment, or the use of mass drug administration (mda)-which have proved useful in some previous emergencies. through mda, all individuals in a targeted population will be given hydroxychloroquine and azithromycinoften at regular intervalsregardless of whether they exhibit symptoms of the disease. aao conceived of the study including design and method. he was the principal in data management, analyzed the data and drafted the article. aa, co, ak, ej, pg, ge, do, dm and mr contributed to reviews of the draft and final version of the manuscript. we declare no competing interests. all data used are publicly available, and sources are cited throughout. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 2, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 2, 2020. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 2, 2020. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 2, 2020. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 2, 2020. . https://doi.org/10.1101/2020.04.27.20082057 doi: medrxiv preprint table 1a ): variables selected using three lasso selection techniques (the model displays an "x" if a variable was selected using any of the three methods, and most important variables are listed first.). . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 2, 2020. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 2, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 2, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 2, 2020. . https://doi.org/10.1101/2020.04.27.20082057 doi: medrxiv preprint . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 2, 2020. . https://doi.org/10.1101/2020.04.27.20082057 doi: medrxiv preprint severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges a review of the 2019 novel coronavirus (covid-19) based on current evidence emergency committee regarding the outbreak of novel coronavirus (covid-19). geneva: who clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china report of the who-china joint mission on coronavirus disease 2019 (covid-19) -china zhongfa zhang & zhongtao gai (2020) the different clinical characteristics of corona virus disease cases between children and their families in china -the character of children with covid-19 2020: comparison of effects of strategies for mitigation and for suppression coronavirus (covid-19) and hiv the different clinical characteristics of corona virus disease cases between children and their families in china -the character of children with covid-19 hundreds of severe pediatric covid-19 infections in wuhan prior to the lockdown world health organization, world health organization pneumococcal infection in adults: burden of disease early origin and recent expansion of plasmodium falciparum diarrhea is first sign of illness for some covid-19 patients preparedness and vulnerability of african countries against importations of covid-19: a modelling study data used for this analysis was obtained from the covid-19 data resource hub established by the tableau community and included near real-time data compiled by johns hopkins university. additional data from key: cord-274663-zyzgk2z3 authors: chang, stewart t.; sova, pavel; peng, xinxia; weiss, jeffrey; law, g. lynn; palermo, robert e.; katze, michael g. title: next-generation sequencing reveals hiv-1-mediated suppression of t cell activation and rna processing and regulation of noncoding rna expression in a cd4(+) t cell line date: 2011-09-20 journal: mbio doi: 10.1128/mbio.00134-11 sha: doc_id: 274663 cord_uid: zyzgk2z3 next-generation sequencing (ngs) enables the highly sensitive measurement of whole transcriptomes. we report the first application to our knowledge of this technology to the analysis of rna from a cd4(+) t cell line infected with intact hiv. we sequenced the total mrna from infected cells and detected differences in the expression of both host and viral mrna. viral reads represented a large portion of the total mapped sequencing reads: approximately 20% at 12 h postinfection (hpi) and 40% at 24 hpi. we also detected a small but significant suppression of t cell activation-related genes at 12 hpi. this suppression persisted and expanded by 24 hpi, providing new possible markers of virus-induced t cell cytopathology. by 24 hpi, the expression of over 50% of detectable host loci was also altered, indicating widespread alteration of host processes, including rna processing, splicing, and transport to an extent not previously reported. in addition, next-generation sequencing provided insights into alternative viral rna splice events and the expression of noncoding rnas, including microrna host genes. t he hallmark of aids is the loss of cd4 ϩ t cells, the primary target of human immunodeficiency virus type 1 (hiv-1) infection. infected cd4 ϩ t cells undergo fundamental changes that eventually result in cell death and the release of new virus particles (reviewed in references 1 and 2). following the uptake of virus, the viral genome is reverse transcribed and integrated into the host genome. the host machinery is then used to produce viral transcripts that are either spliced into smaller transcripts that serve as the template for viral proteins or left unspliced to be incorporated into new virus particles. microarray analyses have shown that infected cells respond to these assaults with gene expression changes in a number of pathways, including apoptosis, cell cycle, cholesterol biosynthesis, and inflammation (3) (4) (5) ; reference 1 and references therein). many of these cellular responses are also reflected at the level of the host organism. for example, gene expression in the lymph nodes of simian immunodeficiency virus (siv)-infected asian pig-tailed macaques (a model of pathogenic hiv infection) reflects strong and sustained type i interferon responses (6) . a sim-ilar initial interferon response is seen in the natural host, african green monkeys, but it eventually subsides and the infection resolves (6) . despite intense investigation into the molecular events following infection in these and other studies, fundamental gaps in knowledge still remain. for instance, the extent to which host and viral gene expression respond to each other is still poorly understood. new technologies enabling the measurement of both host and viral rna may help to address such shortcomings. in this study, we used next-generation sequencing (ngs) to examine changes in the transcriptome of t cells infected with replication-competent hiv. ngs offers a number of benefits for the study of host-pathogen interactions. increased sensitivity and accuracy over microarrays allow important events such as the initial host response to viral replication to be studied in greater detail (7) . also, host and viral rna transcripts can be assayed simultaneously, rather than on separate technical platforms, allowing greater reproducibility and increased confidence in the results. finally, because ngs allows total rna to be assayed, insights are not limited to annotated transcripts or even protein-coding genes. indeed, previous work in our group has shown that many noncoding rna species are differentially expressed in virus-infected cells (8) . we report here the effects of hiv-1 infection on the expression of polyadenylated rnas in a cd4 ϩ t cell line. using ngs, we detected small but significant changes in host gene expression affecting t cell function that coincided with the initiation of viral rna production at 12 h postinfection (hpi). these changes intensified near peak viral replication at 24 hpi when a multitude of other host processes were disrupted as well. in addition, by using ngs, we observed the dramatic expansion of viral mrna expression and detected new viral splice events occurring during viral replication and differential expression of noncoding rna species, including microrna host genes. these findings provide an unprecedented and comprehensive view into the transcriptomelevel changes that occur within t cells infected with replicating hiv. in this study, we investigated changes in host and viral transcription occurring in a t lymphoblast-based model of hiv infection. sup-t1 cells (5 ϫ 10 6 ) were infected in triplicate with hiv-1 strain lai at a multiplicity of infection (moi) of 5 which resulted in near-complete infection at 24 hours postinfection (hpi). the phenotype of infected cells (including appearance, viability, and the amount of viral mrna and protein) was used to guide the selection of samples for ngs. at 12 hpi, viral mrna could be detected in infected cells at appreciable levels (see fig. s1 in the supplemental material). few cells expressed the viral antigen p24 gag, and visually, the cells appeared to be intact. by 24 hpi, high copy numbers of viral mrna were present (fig. s1 ). nearly all cells showed the presence of p24 gag antigen, syncytia were observed, and nonsyncytial cells had increased in volume. by 28 hpi, the effects of viral infection were more pronounced. the amount of viral rna peaked (fig. s1 ), and cell viability decreased. the bulk of cell death occurred between 32 and 48 hpi. on the basis of these observations, we selected two time points to analyze by ngs: 12 and 24 hpi concurrent with the beginning of viral rna accumulation and the occurrence of near-peak rna levels prior to cell death, respectively. we isolated rna from infected and mock-infected replicate cell samples at these two time points, created cdna libraries from polyadenylated rna, and subjected the libraries to analysis by ngs. for each sample, we obtained on average over 21 million 75-nucleotide (nt) reads mapping to either hiv or human genomes. a large number of reads mapped to the viral genome (2.5 to 8 million depending on the time point, see table s1 in the supplemental material). by 12 hpi, viral reads constituted~18% of the total mapped reads, and by 24 hpi, this proportion increased to~38%. reads mapping to the viral genome indicated the presence of rna splicing events. in addition to splicing events involving known splice sites, we also observed evidence of five splicing events involving one or more previously unobserved splice sites (see fig. s2 in the supplemental material). we tested the presence of one of these sites (located 3= of the annotated splice acceptor site 2) by quantitative pcr (qpcr) and observed an amplicon size consistent with the usage of the site (fig. s2a to s2c) . hiv-1 infection results in a small set of differentially expressed host genes at 12 hpi. to characterize the host response to hiv infection, we mapped the remaining nonviral reads to refseq-annotated human gene loci and quantified them as fpkm (fragments per kilobase of exon per million mapped fragments). we selected a set of 34 host genes whose expression spanned a range of values and measured the expression levels directly by qpcr (see fig. s3 in the supplemental material). measurements by ngs showed a high degree of correlation with qpcr results at both time points (r ϭ 0.97 and r ϭ 0.98 at 12 and 24 hpi, respectively), indicating a close match between the two methods. we then identified specific host genes that were differentially expressed (de) during infection. comparing fpkm for each gene in infected cells to time-matched mock-infected cells, we found that a total of 106 genes were de at 12 hpi (representing all fold changes with benjamini-hochberg-adjusted p values of less than 0.05; table 1 ). by 24 hpi, the number of de genes had increased to 5,006 representing over 50% of all 9,992 gene loci detected under stringent criteria (table 1) . closer examination of these values showed that a large proportion of de genes occurred with smallmagnitude changes. in particular, 69% (73 of 106) and 49% (2,465 of 5,006) of the expressed genes exhibited 1-to 1.5-fold changes at 12 and 24 hpi, respectively ( table 1 ). the observed precision of the sequencing-based measurements (fig. s3 ) supported the use of low-fold change thresholds. other values for the statistical threshold were also used, and for all but the most stringent thresholds (when fewer than 10 genes were observed to be up-or downregulated at 12 hpi), a similar predominance of small-magnitude changes was observed (see table s2 in the supplemental material). comparing the directionality of change at both time points, we found that nearly all of the de genes at 12 hpi continued to be regulated in the same manner (i.e., up-or downregulated) at 24 hpi. specifically, 97 of the 106 de genes at 12 hpi were also de at 24 hpi (with benjamini-hochberg-adjusted p ͻ 0.05), and the majority of these (95 of 97) exhibited the same direction of change relative to mock infection, often with increased degree of change (as indicated by more-intense red or green coloration at 24 hpi than at 12 hpi in a heat map of fpkm fold changes, fig. 1 ). this indicated that regulation coincident with the beginning of viral replication at 12 hpi largely persisted and intensified near peak viral replication at 24 hpi, even for initially low fold changes. hiv-1 infection impacts core t cell functionality by 12 hpi. we determined the possible impact of these de genes by analyzing the corresponding annotations (biological processes, molecular functions, and pathways) using the nih david resource. we found that t cell activation and differentiation were the most overrepresented annotations among de host genes at 12 hpi overall ( fig. 2a) . other annotations associated with de genes included protein kinase regulation, dna recombination, and caspase activity regulation ( fig. 2a ). in addition, several of the de genes at 12 hpi encoded transcriptional regulators; these genes include egr1, klf13, and myc. interestingly, four of the nine genes de at 12 hpi but not at 24 hpi also encoded transcriptional regulators (creb3l3, hexim1, znf660, and zkscan4), indicating regulation specific to early viral replication. seven genes contributed to the overrepresentation of t cell activation among de genes at 12 hpi (bcl11b, cd1d, egr1, ikzf1, irf1, rag1, and sox4), six of which were downregulated, indicating a net suppression of t cell activation (as t cell differentiation in fig. 2 ). five t cell activation-related de genes (bcl11b, egr1, irf1, ikzf1, and sox4) encoded transcription factors, some of which had known relevance to hiv infection. for example, the bcl11b gene product binds directly to the hiv-1 sample relative to averaged time-matched mock-infected cell samples. genes were segregated by the direction of change relative to mock infection at 12 hpi, and hierarchical clustering was done within each directional group. genes that were not also de at 24 hpi (purple type) and genes that were also de at 24 hpi but with changed directionality (gold type) are indicated. annotations indicate overrepresented categories in david (fig. 2 ). matches to top-scoring categories in each david annotation cluster (matching numbers in fig. 2 ) (solid black squares) and matches to related categories in the same david cluster as the top-scoring category (solid gray squares) are indicated. reg, regulation. long terminal repeat (ltr) and inhibits ltr expression (9) . bcl11b downregulation may therefore allow more efficient hiv-1 replication. other de genes related to t cell activation not encoding transcription factors also had known relevance to hiv infection. for example, the cd1d product presents lipid antigens on the surface and is directed by hiv-1 nef for internalization and degradation (10) . downregulation of cd1d at the mrna level would further reduce surface expression and facilitate immune evasion. we complemented functional analysis in david by gene set enrichment analysis (gsea). this method does not rely on the prior determination of de genes (e.g., by applying a p value threshold) but instead uses a ranked gene list as input and is therefore well suited for identifying annotations among genes with small-magnitude changes in expression (11) . gsea identified additional genes that were downregulated and contributed to the suppression of t cell activation; these genes include the cd2, cd4, cd7, cd28, and sit1 genes encoding t cell-specific surface molecules (see fig. s4 in the supplemental material). many of the genes associated with t cell activation in david and gsea also had roles in other pathways, indicating possible pleiotropic effects (e.g., adora2a and rag1 which also contribute to caspase activity [ fig. 1]) . hiv-1 induces large-scale changes in transcription by 24 hpi. by 24 hpi, large-scale changes in the transcriptomes of hivinfected cells were evident (table 1 ; see the heatmap in fig. s5a in the supplemental material). consistent with the large number of de genes at 24 hpi, we found that a wide range of biological processes was affected as determined by further analysis using david ( fig. 2b ; complete listing in table s3 in the supplemental material). t cell activation and differentiation continued to be overrepresented and were associated with an increased number of de genes (as lymphocyte differentiation [ fig. 2b] ). other fundamental cellular functions associated with de genes at 24 hpi included dna metabolism, transcription, mrna processing and splicing, translation, protein degradation, and cell cycle control (fig. 2b ). this breadth of functional regulation suggests that hiv-1 infection resulted in the effective transcriptional reprogramming of sup-t1 cells by 24 hpi. suppression of t cell functionality persists and expands at 24 hpi. as was the case at 12 hpi, genes related to t cell activation and differentiation were predominantly downregulated at 24 hpi. all of the t cell activation-related de genes at 12 hpi continued to exhibit the same direction of change at 24 hpi (fig. 1 ). other t cell activation-related genes de at 24 hpi included cd3d, cd3q, and rhoh, all of which encode membrane proteins crucial for t cell receptor function. a network depicting known interactions among proteins encoded by t cell activation-related de genes at 24 hpi showed two highly connected nodes: lck (lymphocytespecific protein kinase) and tp53 (p53), both of which were strongly downregulated at 24 hpi (fig. 3a) . our observed downregulation of tp53 differed from the upregulation observed in previous microarray studies and may indicate a cell type-specific effect (1) . genes involved in other core t cell functions, such as proliferation, survival, and antigen presentation, were also downregulated at 24 hpi. these genes included cd1d, cd28, cxcr4, tnfrsf4, and treml2. tob1 (transducer of erbb2), a negative regulator of t cell activation, proliferation, and interleukin 2 (il-2) production, showed increased expression and may contribute to the downregulation observed in other genes (data not shown). a connection between tob1 expression and hiv-1 infection has not previously been mentioned. overall, the increased number of de genes related to t cell activation indicated increased suppression of this pathway by 24 hpi. ribonucleoprotein complex biogenesis and rna processing. gene sets related to ribonucleoprotein complex biogenesis were the most overrepresented annotations among de genes at 24 hpi (fig. 2b) . ribonucleoproteins contribute to diverse functions within the cell, including microrna synthesis, ribosomal assembly, and translation (12) . this overlap was reflected at the gene level as well. in particular, ribonucleoprotein complex biogenesis shared many de genes at 24 hpi with another top annotation cluster, rna processing. this set of genes encoding the ribonucleoprotein component of the rna processing machinery was almost entirely downregulated and included many members of the heterogeneous nuclear ribonucleoprotein (hnrnp) and small nuclear ribonucleoprotein (snrnp) families. in a network identifying interactions among the proteins encoded by these genes, a number were found to be highly connected (fig. 3b ). one of these was hnrnpa1 whose gene product regulates the splicing of eukaryotic and viral mrna (13) . hnrnpa1 and other hnrnp gene products are known to affect the localization of hiv-1 gag/pol mrna, and their overexpression has previously been shown to reduce hiv-1 replication (fig. 3b) (14) . other de genes at 24 hpi related to ribonucleoprotein biogenesis and rna processing are also known to affect hiv-1 replication directly; these genes include tardbp (tar dna-binding protein 43) whose protein binds the transactivation response (tar) element of integrated hiv-1 dna and represses hiv-1 transcription (15) (16) (17) . downregulation of hnrnpa1, tardbp, and other related genes may therefore allow more efficient hiv-1 replication in sup-t1 cells. rna transport. the importance of host regulation of rna fate was underscored by another cluster of gene sets related to rna transport (fig. 2b) . host factors for rna transport are known to be coopted by hiv-1 to allow the export of unspliced, partially spliced, or fully spliced viral mrnas out of the nucleus (18) . in particular, the host factors crm1 (exportin 1) and ran gtpase are used for the export of unspliced viral mrna, while the host factor nxf1 (nuclear rna export factor 1) facilitates the export of fully spliced viral mrna in a ran-independent manner (19) . in our data set, we observed no change in the expression of the crm1-encoding gene xpo1, but several members of the ran signaling pathway were downregulated, suggesting that the overall export of unspliced viral rna was suppressed (see fig. s5b in the supplemental material; data not shown for xpo1). however, in contrast to other rna transport-related genes, nxf1 and nxf3 were expressed more highly in infected cells, suggesting that hiv infection may have selectively enhanced the export of fully spliced viral rna (fig. s5b) . limited upregulation of host processes at 24 hpi. despite the presence of an almost equal number of up-and downregulated genes at 24 hpi, relatively few gene sets (functions, processes, or pathways) were associated with upregulated genes. when up-and downregulated de genes at 24 hpi were analyzed separately, more annotation clusters were observed among downregulated genes (70 versus 13 among upregulated genes with significance defined by modified fisher's exact test as p ͻ 0.05). a similar result was obtained by gsea (see fig. s6a and s6b in the supplemental material). these results suggested that upregulated genes were distributed across many gene sets with few occurring in particular functions or pathways. among the few upregulated pathways were stress-activated/jnk cascade signaling and ion transport (fig. s6c) . consistent with these observations, hiv-1 nef has been shown to activate jnk signaling, ultimately activating the caspase cascade and triggering cell death (20) . the triggering of apoptosis at 24 hpi is consistent with our observation that infected cells began to die following the 24-hpi time point. hiv-1 infection does not trigger innate sensing at 12 hpi. notably, innate immunity was absent from the processes associated with de genes at 12 hpi, suggesting that hiv-1 infection impaired t cell activation while evading virus-sensing mechanisms ( fig. 2a) . with the exception of irf1, which was downregulated at 12 hpi (fig. 1) , interferon response factors (irfs) (irf2, irf3, irf7, and irf9) showed no significant change in expression at 12 hpi (data not shown). furthermore, specific irf targets, including irf3 target genes ifi35, ifi44, isg15, and isg20, were also not differentially expressed at this time point (21, 22) . irf3 targets were of particular interest, as intact cytoplasmic hiv-1 dna has been shown to activate irf3 in cd4 ϩ t cells (23) . our observed lack of irf3 target gene expression is consistent with previous observations that replicating hiv-1 suppressed irf3 activity (24) . we also examined the levels of inflammationrelated genes previously observed to be differentially regulated in hiv infection studies (1): anxa1, b2m, and cd69 (generally upregulated) and cd53, cd71, il-13, and il-16 (generally downregulated). these genes were also either not expressed at detectable levels or unchanged in expression at 12 hpi (data not shown). innate immunity and the inflammatory response continued to be absent from the significantly upregulated gene sets at 24 hpi (by gsea [see fig. s6a in the supplemental material]). irf1 was more strongly downregulated at 24 hpi than at 12 hpi, but other irf genes (irf2, irf3, irf7, and irf9) continued to show no significant change in expression ( fig. 1 ; data not shown). in a separate experiment, we confirmed that irf1 was downregulated at 24 hpi by qpcr (by an average of 44%; data not shown). other interferon (ifn)-inducible genes were differentially expressed at this time point but in different directions, such as b2m and ifi16 (both downregulated) and ifi30 and isg20 (both upregulated). this lack of concerted change may have offset the detection of interferon response gene sets as up-or downregulated at 24 hpi by gsea. instead, at 24 hpi, the related gene sets of cellular defense and immune response were found to be primarily downregulated (by gsea [fig. s6b] ). several of the downregulated genes associated with these gene sets were also associated with t cell activation; these genes include cd1d, cd2, cd28, rag1, and tnfrsf4 (fig. s6d) . additional immune response-specific genes downregulated at 24 hpi included il4 and il7r, suggesting that hiv-1 may have impaired the ability of infected cells to signal other cells or respond to extracellular cues. by mapping sequencing reads to refseq transcript annotation, we also observed changes in the expression of several non-protein-coding rna species. specifically, reads mapping to refseq transcripts that began with nr were considered noncoding. as before, expression of these transcripts was compared between infected and mock-infected samples. at 12 hpi, only one annotated noncoding rna (nr_003697 encoding c7orf40) was found to be differentially expressed (benjamini-hochberg-adjusted p ͻ 0.05). however, by 24 hpi, 305 annotated noncoding rnas were found to be differentially expressed by the same criterion, with 73 changed by an average of 2-fold or greater (fig. 4a) . several classes of noncoding rnas were represented, including microrna host genes, hypothetical genes and open reading frames (orfs), small nucleolar rnas (snornas), and pseudogenes (fig. 4a) . de pseudogenes often matched their protein-coding counterparts in directionality and degree of regulation, which suggests that regulatory regions were maintained and polyadenylated transcripts were produced (data not shown). we also observed differential expression of four annotated mi-crorna host genes in infected cells, three of which were downregulated (mir17hg, mir142, and mir621), while the remaining one was upregulated (mir518f), all with 2-fold or greater changes. the downregulation of mir17hg (by 2.25-fold) was of particular interest, as mir17hg encodes a cluster of micrornas, including mir-17, -18a, -19a, -19b, -20a, and -92 (fig. 4b) ; in contrast, the other microrna host genes encode only single micrornas. reads mapped to mir17hg 3= of the mature microrna sequences, indicating that the postexcised polyadenylated product may have been detected (fig. 4b) . we also observed a concurrent upregulation of known targets of mir17hgencoded micrornas, including pcaf, a host factor required for tat-induced hiv-1 gene expression (25) . hiv infection and host noncoding rna. in this study, we used ngs to measure all of the polyadenylated rnas in cd4 ϩ t lymphoblasts infected with intact, replication-competent hiv. ngs offers a number of potential benefits to the study of viral infections; among them is the ability to detect non-protein-coding rnas expressed during infection. in a previous study, we used ngs to detect long noncoding rnas in the lungs of severe acute respiratory syndrome (sars) coronavirus (cov)-infected mice (8) . many of these rnas were found to be differentially expressed, and unique signatures of infection could be identified (8) . in this study, we also detected several noncoding rna species in hiv-infected cells that were differentially expressed (de). in most cases, the functions of these noncoding rnas and their relevance to infection remains unknown. one noncoding rna of interest, the microrna host gene mir17hg, encoded a cluster of micrornas and was strongly downregulated during infection. while our method of rna library preparation precluded direct detection of mature micrornas, the ngs data set allowed the expression of both regulators and targets of micrornas to also be observed. for example, we observed a concurrent upregulation of the known target host gene pcaf, a cellular factor required for hiv replication (25) . in contrast to triboulet et al. (25) who observed that pcaf was upregulated at the protein level but not the mrna levels in infected peripheral blood mononuclear cells (pb-mcs), we found that pcaf was upregulated at the mrna level in infected sup-t1 cells. this may indicate alternative modes of pcaf regulation in different cell types. in addition, mir17hgencoded micrornas have hundreds of other candidate targets in the targetscan database (26) . several of these candidates were found to be coexpressed with pcaf in our data set, including klf3 (unpublished data). klf3 encodes a zinc finger transcription factor whose precise function is unknown but whose sequence is located proximally to a single-nucleotide polymorphism strongly associated with hiv-1 plasma levels (27) . we also observed differential expression of factors that may have mediated mir17hg downregulation. for example, o'donnell et al. (28) found that c-myc regulates expression of mir17hg. consistent with that study, we observed that myc and mir17hg were concordantly expressed, as myc was downregulated at 12 and 24 hpi (fig. 1) . myc suppression by hiv-1 may therefore underlie a number of subsequent of transcriptional events. early regulation of host transcription factors. we also observed discordance between the large amount of viral rna present (~20% of total mappable rna) and the limited amount of altered host gene expression at 12 hpi (~1% of detectable gene loci). that is, despite the use of host machinery and the presence of foreign mrna, host transcription remained relatively unchanged. this result suggests that at 12 hpi, viral transcription occurred on top of largely undisturbed transcription of host genes. the host genes that we detected as differentially expressed at 12 hpi showed that t cell activation and differentiation were suppressed in infected cells, likely via the downregulation of t cell activationspecific transcription factors (bcl11b, irf1, ikzf1, and sox4) . the regulation of these and transcription factors specific for other functions (e.g., egr1, klf13, and myc) may explain how hiv initiated replication with minimal disruption to host gene expression at 12 hpi but elicited larger-scale changes later in infection. the suppression of t cell activation that we observed was consistent with previous studies on the response of t cells to infection by chemokine (c-x-c motif) receptor 4 (cxcr4)-tropic versus chemokine (c-c motif) receptor 5 (ccr5)-tropic hiv-1 strains (29) (30) (31) . for example, sirois et al. (31) found that key t cell activation-related genes, including lck, were downregulated at 24 hpi by a cxcr4-tropic strain, whereas these same genes were upregulated by a ccr5-tropic strain. our study utilized the cxcr4-tropic strain lai, and it would be interesting to generate ngs data for a ccr5-tropic virus for comparison. interestingly, our observations of small-magnitude differences in expression were consistent with those of sirois et al. (31) , who found the expression of t cell activation-related genes to be changed by 0.5fold or less using reverse transcription-pcr (rt-pcr). large-scale disruptions to host transcription near peak viral replication. by 24 hpi, extensive reprogramming of the host transcriptome affecting a multitude of pathways had occurred. remarkably, these large-scale changes occurred without concerted upregulation of innate immunity at the gene set level, suggesting that hiv-1 evaded viral sensing mechanisms. by this time point, the most affected pathways related to rna fate determination, including rna processing. while hiv-related rna processing has been studied intensively, the contribution of most host factors remains incompletely defined (32) . modulation of this pathway by the virus may allow the proportion of unspliced, partially spliced, and fully spliced hiv rna to be adjusted depending on the stage of the hiv life cycle. our finding that over 150 genes related to rna processing were differentially expressed suggests that hiv-1 infection results in a more complete modulation of rna processing than previously identified (5, 33) . however, our observed downregulation of rna processing was consistent with results from an earlier study using ngs to investigate changes in cd4 ϩ t lymphoblasts infected with an hiv-based, nonreplication-competent vector at 24 hpi (34) . altered regulation of this and other pathways has been observed using microarrays as well, although in general, we observed changes in greater numbers of genes affecting these pathways using ngs (3, 5, 33, 35, 36) . finally, we compared our results from ngs to other types of data available regarding hiv-infected cells, including proteinprotein interaction (ppi) data and small interfering rna (sirna)-based screens. in an analysis of hiv-related ppi data, van dijk et al. (37) identified sets of human proteins highly connected to host and viral proteins (37) . several proteins related to t cell activation were identified as highly connected; these proteins included cd4, cxcr4, and lck (see table s4 in the supplemental material). downregulation of these members, as we observed, would therefore potentially affect the functions of many other proteins as well. together, these data emphasize the high degree to which hiv-1 suppressed this pathway. in addition, many of the pathways associated with de genes were identified as essential for hiv-1 replication in a recent meta-analysis of sirna-based screen data (38) . these pathways included dna binding, rna splicing, rna export, nuclear transport, and protein complex assembly (table s3) . while the sirna data suggest that hiv-1 ini-tially requires these pathways to be active to establish an infection, our data suggest that hiv-1 also later suppresses and inactivates these pathways during active replication. in conclusion, using ngs, we have reported a number of unique findings in hiv-infected cells: the direct measurement of viral and host rna, the detection of small changes in the abundance of mrna transcripts, the identification of novel viral rna splice events, and the assay of multiple forms of noncoding rnas, including insights into the regulation of micrornas. these observations give additional insights into the panoply of changes that occur in host cells infected with hiv and provide the groundwork for using new sequencing technologies in future studies investigating the host response to viral infection. sup-t1 cells were obtained from american type culture collection (crl-1942) and propagated in rpmi 1640 medium (gibco) supplemented with 10% fetal bovine serum (hyclone), penicillin (100 u/ml), streptomycin (100 g/ml), and glutamax-i. hiv-1 lai strain (catalog no. 2522) was obtained from the nih aids research and reference reagent program (germantown, md) and propagated in sup-t1 cells. u373-magi-cxcr4 cem cells were obtained from m. emerman through the aids research and reference reagent program, and the virus titer in these cells was measured by the protocol of deminie and emerman (39) . typical titers reached 10 7 infectious units per ml. infections were carried out at a multiplicity of infection (moi) of 5 and performed in triplicate. mock-infected samples received sup-t1 cell conditioned medium and were also performed in triplicate. the infectious dose was optimized to achieve 100% infected cells at 24 hpi with 50% cell viability as measured by trypan blue exclusion assay. infected cells were visualized by immunofluorescence assay with rabbit hiv-1 sf2 p24 antiserum kindly provided by biomolecular technologies through the aids research and reference reagent program. rna preparation and next-generation sequencing. total rna was extracted from 5 ϫ 10 6 cells per sample using a mirvana kit (applied biosystems/ambion, austin, tx), and the quality and concentration of the rna were determined by an agilent 2100 bioanalyzer. samples were submitted for sequencing to illuminafasttrack sequencing services (hayward, ca). cdna libraries were generated using illumina mrna-seq kit. the quality and concentration of these libraries were determined by an agilent 2100 bioanalyzer. the libraries were clonally amplified on a cluster generation station using illumina version 4 cluster generation reagents to achieve a target density of approximately 300,000 (300k)/mm 2 in a single channel of a flow cell. the resulting libraries were then sequenced on a genome analyzer iix using illumina version 4.0 sequencing reagents which generated single reads of 75 nucleotides (nt). image analysis, base calling, and error estimation were performed using illumina analysis pipeline (version 2.6). read mapping and transcript quantification. we mapped the 75-nt reads to human ribosomal sequences using the short-read aligner software bowtie to remove potential rrna sequences (40) . we then mapped the remaining unmapped reads to the hiv genome (genbank accession no. k02013) using the gapped aligner software tophat, which predicts hiv splicing junctions and maps intron-spanning reads to known splicing junctions (41) . to quantify transcript expression, we mapped all reads that remained unmapped to the human reference genome (hg19, build grch37, downloaded from ucsc genome browser, http://genome.ucsc .edu) using the gapped aligner software tophat. refseq transcript annotations were supplied to facilitate the mapping of reads spanning known splicing junctions. on the basis of these human genome mapping results, we then estimated the levels of expression at both the transcript and locus levels for refseq-annotated genes using the transcript assembly software cufflinks (42) . read sequences that mapped to more than one genomic location were excluded from expression quantification. for visualization, bam files were generated using tophat and samtools (43) and displayed using the ucsc genome browser. splice site variant detection and testing. the positions of known viral splice sites were found in the literature (44) , and the corresponding sequences were identified in strain pnl4-3 (genbank accession no. af003887). based on sequence identity to strain lai, a list of known lai-specific splice sites was generated and compared to the ngs tophat output. splice patterns involving splice sites sd 1 and sa 2/2* were tested by quantitative pcr (qpcr) using primers psk027f (5= cagggacttg aaagcgaaag 3=; locations 193 to 212 in hivbrucg accession no. k02013) and psk027r (5= tggggcttgttccatctatc 3=; locations 5136 to 5155). quantitative reverse transcription (rt-pcr). rna was reverse transcribed using the quantitect reverse transcription kit (qiagen, valencia, ca). the resulting cdna samples were diluted 50ϫ. abi taqman assays were run for each sample in triplicate (see fig. s3c in the supplemental material). relative expression was calculated using the ⌬⌬c t method with averaged ⌬c t values (where c t stands for threshold cycle) for the oaz1 gene as a calibrator, as the expression of oaz1 did not significantly change between 12 and 24 hpi in the ngs data. intracellular viral rna load was quantified as previously described (45) . relative change of irf1 was determined by qpcr using primers psk1 (forward primer [5= tctg gctttttcctctgagc 3=] and reverse primer [5= atgcttttctgg ggtcactg 3=]). differential expression analysis. to compare transcript expression across different conditions, we first normalized transcript abundances by the following methodology. transcript abundance was quantified as fpkm (fragments per kilobase of exon per million mapped fragments) values estimated by cufflinks. we chose one sample arbitrarily as a reference. distributions of log 2 -transformed fpkm values between the reference and remaining samples were compared by quantile-quantile plots. we determined the scaling factor for each sample as the median difference of the corresponding quantile values of the sample and reference. only genes/transcripts with raw fpkm values of ն1 in all samples were considered in the estimation of scaling factors. we retained those genes/transcripts with nonzero fpkm values in 100% of the samples of at least one biological condition (our detection criterion). an offset of 1 was added to all normalized values to facilitate the comparisons involving one or more fpkm values of zero and to reduce the variability of the log ratios for low expression values. transcripts were mapped to refseq gene loci, resulting in 9,992 loci with detectable reads. the data are available at http://www .viromics.washington.edu or upon request. the normalized expression data were analyzed for differential expression by using linear model methods as implemented in the r package limma (46) . p values were derived from linear model-based t tests between infected and time-matched mock-infected samples. unless otherwise noted, we defined differential expression by benjamini-hochberg-adjusted p values of less than 0.05 based on the assumption that a false discovery rate of 5% provided an acceptable balance of false-positive control and statistical power. fold changes (fc) were derived from comparing the means of these groups, and multiple groupings of fold changes were used (1.0 to 1.5 fc, 1.5 to 2.0 fc, and 2.0ϩ fc) based on previous observed fold change ranges observed in high-throughput and in particular ngs data in virus-infected systems (8) . david and gsea analyses. to identify annotations among de genes, we used the nih david resource (47, 48) . default settings were used in david with go_bp_fat, go_cc_fat, go_mp_fat, bio-carta, and kegg_pathway gene set annotations. complementary analysis was performed using gene set enrichment analysis (gsea) (11) . default settings were used in gsea with gene ontology (go) categories (c5.all.v2.5 gene sets) and biocarta and kegg pathways (c2.all.v2.5 gene sets) and 5,000 gene set-based permutations. leading-edge analysis was performed within gsea to derive genes for hierarchically clustering upand downregulated gene sets. interactions between de genes were identified using ingenuity pathways analysis (ipa) software (ingenuity systems, redwood city, ca). networks were generated within ipa based on direct, literature-curated interactions. subsets of genes were selected for input into ipa based on de gene overlaps with david annotation clusters. heat maps were generated using the r package gplots. comparisons to published networks of protein-protein interaction (ppi) data were made using cytoscape (49) . this work was funded by public health service grants p30da015625 and p51rr000166 from the national institutes of health. we thank matthew thomas and richard green for technical assistance and marcus korth for editorial assistance. we also thank peter sloot and david van dijk for generously sharing expertise related to proteinprotein interaction networks. supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi:10.1128/mbio.00134-11/-/dcsupplemental. microarray data on gene modulation by hiv-1 in immune cells: 2000 -2006 molecular biology of human immunodeficiency virus type 1 large-scale monitoring of host cell gene expression during hiv-1 infection using cdna microarrays functional genomics analyses of differential macaque peripheral blood mononuclear cell infections by human immunodeficiency virus-1 and simian immunodeficiency virus cellular gene expression upon human immunodeficiency virus type 1 infection of cd4(ϩ)-t-cell lines transcriptional profiling in pathogenic and nonpathogenic siv infections reveals significant distinctions in kinetics and tissue compartmentalization a comparison of massively parallel nucleotide sequencing with oligonucleotide microarrays for global transcription profiling unique signatures of long noncoding rna expression in response to virus infection and altered innate immune signaling bcl11b is a general transcriptional repressor of the hiv-1 long terminal repeat in t lymphocytes through recruitment of the nurd complex hiv-1 down-regulates the expression of cd1d via nef gene set enrichment analysis: a knowledgebased approach for interpreting genome-wide expression profiles rna and disease hnrnp a1 controls hiv-1 mrna splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure role of cellular rna processing factors in human immunodeficiency virus type 1 mrna metabolism, replication, and infectivity the multiple roles of tdp-43 in pre-mrna processing and gene expression regulation cloning and characterization of a novel cellular protein, tdp-43, that binds to human immunodeficiency virus type 1 tar dna sequence motifs tdp-43: multiple targets, multiple disease mechanisms? retroviral mrna nuclear export elements regulate protein function and virion assembly nuclear mrna export: insights from virology nef induces apoptosis by activating jnk signaling pathway and inhibits nf-kappab-dependent immune responses in drosophila irf-3-dependent and augmented target genes during viral infection transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes the cytosolic exonuclease trex1 inhibits the innate immune response to human immunodeficiency virus type 1 human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells suppression of microrna-silencing pathway by hiv-1 during virus replication most mammalian mrnas are conserved targets of micrornas distinct genetic loci control plasma hiv-rna and cellular hiv-dna levels in hiv-1 infection: the anrs genome wide association 01 study c-myc-regulated micrornas modulate e2f1 expression r5 and x4 hiv envelopes induce distinct gene expression profiles in primary peripheral blood mononuclear cells differential effects of r5 and x4 human immunodeficiency virus type 1 infection on cd4 ϩ cell proliferation and activation r5 and x4 hiv viruses differentially modulate host gene expression in resting cd4 ϩ t cells regulation of hiv-1 alternative rna splicing and its role in virus replication microarray study reveals that hiv-1 induces rapid type-i interferon-dependent p53 mrna upregulation in human primary cd4 ϩ t cells analysis of hiv-1 expression level and sense of transcription by high-throughput sequencing of the infected cell temporal gene regulation during hiv-1 infection of human cd4 ϩ t cells impact of viral infection on the gene expression profiles of proliferating normal human peripheral blood mononuclear cells infected with hiv type 1 rf identifying potential survival strategies of hiv-1 through virus-host protein interaction networks host cell factors in hiv replication: metaanalysis of genome-wide studies quantitation of virus stocks produced from cloned human immunodeficiency virus dna ultrafast and memory-efficient alignment of short dna sequences to the human genome tophat: discovering splice junctions with rna-seq transcript assembly and quantification by rna-seq reveals unannotated transcripts and isoform switching during cell differentiation the sequence alignment/map format and samtools alternative splicing of human immunodeficiency virus type 1 mrna modulates viral protein expression, replication, and infectivity detection and quantification of human immunodeficiency virus type 1 p24 antigen in dried whole blood and plasma on filter paper stored under various conditions linear models and empirical bayes methods for assessing differential expression in microarray experiments bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists systematic and integrative analysis of large gene lists using david bioinformatics resources cytoscape: a software environment for integrated models of biomolecular interaction networks key: cord-287337-2ljbsia2 authors: ludwig, christine; wagner, ralf title: virus-like particles—universal molecular toolboxes date: 2008-01-04 journal: curr opin biotechnol doi: 10.1016/j.copbio.2007.10.013 sha: doc_id: 287337 cord_uid: 2ljbsia2 virus-like particles (vlps) are highly organised spheres that self-assemble from virus-derived structural antigens. these stable and versatile subviral particles possess excellent adjuvant properties capable of inducing innate and cognate immune responses. commercialised vlp-based vaccines have been successful in protecting humans from hepatitis b virus (hbv) and human papillomavirus (hpv) infection and are currently explored for their potential to combat other infectious diseases and cancer. much insight into vlp-mediated immune stimulation and optimised vlp design has been gained from human immunodeficiency virus (hiv)-derived vlps presenting promising components of current aids vaccine approaches. owing to their unique features, vlps and virosomes, the in vitro-reconstituted vlp counterparts, have recently gained ground in the field of nanobiotechnology as organic templates for the development of new biomaterials. viruses are vehicles of 15-400 nm in size engulfing their genetic information with a protein-based coat built of many spontaneously aggregating homogenous subunits and sometimes surrounded by a lipid bilayer derived from host cellular membranes. viruses have evolved within a wide variety of eukaryotes (animals, plants, protists, fungi such as yeast) as well as prokaryotes (bacteria, archaea), those infecting the latter referred to as phages. according to the respective host spectrum, viruses have developed diverse shapes and replication strategies in order to survive and replicate within a hostile environment. however, not all vehicles assembled from virus components contain nucleic acids that are obligatory to establish productive infection. these virus-like particles (vlps) representing empty shells are nevertheless still capable of entering target cells and are thought to be released from infected cells to entice host immune responses from the infectious viral offspring. this phenomenon has been exhaustively described for cells infected with hepatitis b virus (hbv) that mass produce empty 22-nm particles solely composed of the small hbv-derived surface antigen (hbsag). since these hbsag particles have been isolated and characterised for the first time [1] , fastgrowing knowledge of vlp functions and structure combined with the accomplishments in genetic engineering have revealed manifold opportunities to exploit vlps for various fields of molecular biology. especially their natural immunogenic properties make them attractive candidates for vaccine strategies still ranking first among molecular vlp-based applications. during the past years vlps have also established themselves in other branches of biotechnology taking advantage of their structural stability and tolerance towards manipulation to carry and display heterologous molecules or serve as building blocks for novel nanomaterials. these nearly unlimited possibilities have popularised vlps within the scientific community as one of the most favourite tools of today's synthetic biology. this review will therefore discuss the role of vlps in current vaccine concepts in the light of their distinctive immunogenic potential and highlight recent achievements in the prospering field of virosome and nanobiotechnology. before 1969 all anti-viral vaccines were either based on inactivated viruses (e.g. rabies vaccine), attenuated strains of pathogenic wild-type viruses (yellow fever or polio) or were using closely related but non-pathogenic virus strains to induce a protective immune response against the pathogenic relative-like smallpox vaccine containing the innocuous vaccinia virus. since hbsagbased particles were first discovered in blood samples of hepatitis b patients and were soon developed into a potent vaccine against the cognate virus infection [2] , subviral particles have rapidly found their way into modern concepts of vaccine design. on the basis of their particulate nature ( figure 1a) , vlps provide an inherent advantage over soluble antigens that have been shown to fail in several vaccine approaches owing to weak immunogenicity or instability. since vlps are not infectious and do not replicate, they also represent a safer alternative to attenuated viruses, which have succeeded to protect from yellow fever, polio, measles, mumps and rubella, but have clearly failed in other infections as formerly shown for attenuated versions of human immunodeficiency virus (hiv), which reconverted into pathogenic species over time (discussed in references [3, 4] ). the demands for higher yields of the hbsag-based subviral vaccine than those recovered from blood samples soon turned towards other more efficient production 538 pharmaceutical biotechnology vlp-based strategies for vaccine design. (a) schematic presentation of a virus-like particle composed of self-assembled virus capsid proteins that are engulfed by a host cell-derived lipid membrane with integrated cellular proteins; random packaging of cellular nucleic acids is indicated. (b) vector constructs used to express chimeric hiv vlps; top: vlps assembled from gag or gag-pol proteins present endogenous (hatched box) epitopes; additional foreign epitopes can either be inserted or fused (yellow box); middle: vlp-display of incorporated envelope proteins by coexpression of either autologous (gp120/gp41) or heterologous (e.g. gp64) envelopes, the latter resulting in pseudotyping; bottom: co-stimulatory molecules (cd40l) can be anchored on vlps via a transmembrane domain (tm); cy, cytoplasmic domain. (c) production of vlps in different expression systems. left: infection of highfive tm insect cells with recombinant baculoviruses (bv) expressing respective capsid proteins; vlps budding from insect cells (electron microscopy images) carry bv gp64; right: co-transfection of mammalian 293 t cells with a gag encoding plasmid; co-expression of gp64 results in pseudotyping of gag vlps. p cmv, cytomegalovirus promoter; p sv40, simian virus 40 promoter. systems exploiting the possibilities of recombinant dna technology. by virtue of hbsag to self-assemble into particulate structures, respective particles were efficiently released from recombinant yeast cells allowing high-yield production and safer purification of the vaccine [5] . apart from yeast spheroblasts, other systems have been exploited for vlp expression. the most prevalent ones include the vaccinia virus expression system, transfection of rna-and codon-optimised dna into mammalian cells or infection of insect cells with recombinant baculovirus. vlps designed for mucosal vaccination can also be efficiently produced in gut bacteria such as highly attenuated salmonella or lactobacillus strains [6, 7] . besides, green plants like tobacco, potato or tomato offer an efficient and inexpensive method for vlp production by infection with plant-specific viruses, such as, for example the tobacco mosaic virus [8, 9 ] . of note, plant-produced vlps have been shown to be highly immunogenic upon oral ingestion of vlp-containing plant material [10]. during the past decade, subviral particles of different origins have been considered as potential vaccines for cognate virus infections including human immunodeficiency virus, human papillomavirus (hpv), norwalk virus (nv), rotavirus or parvovirus. a very comprehensive overview of these vlp-based vaccine approaches is given in references [11, 12] . it is noteworthy that vlps assembled from hpv major capsid protein l1 in yeast were capable of inducing protective immune responses against hpv subtypes 16 and 18 causing cervical cancer in humans, thus resulting in a safe, well tolerated and highly immunogenic vaccine that received approval for marketing in 2006 [13] . a competing product produced in the baculo-expression system has been approved recently and claims to be even more efficient concerning long-term immune responses triggered by the potent adjuvant as04 [14] . apart from hbv or hpv, the enduring efforts to develop a safe, prophylactic aids vaccine have called vlp-based strategies into action. early conventional attempts towards developing a protective hiv vaccine on the basis of soluble hiv-1-derived envelope protein gp120 have been rather disappointing. since particulate antigens had been demonstrated to induce better cellular and humoral immune responses than soluble antigens, the detection that hiv-1 pr55gag polyprotein self-assembles into particulate spheres provided a new rationale for generating a gag-based vlp vaccine [15, 16] . gag is today one of the two most common antigens expressed for vaccine approaches, and respective vlps can be efficiently produced in mammalian or insect cells ( figure 1c ). first immunisation analyses with gag vlps yielded strong th1-biased humoral and cellular immune responses in the absence of adjuvants and induced gagspecific cytotoxic t lymphocytes (ctl) in mice and macaques [17, 18] . today it is a widely held belief that both, a strong cell-mediated immune response and crossreactive neutralising antibodies mainly directed towards conserved epitopes of the envelope (env) protein are essential to achieve protection from hiv infection. hence, current approaches are being extended to novel vlp designs addressing both aspects to induce an integrated immune response. a good vaccine has to fulfil certain criteria concerning immunogenic potential, cell or host tropism, route of administration and uptake. in this regard, not all attempts to use vlps built of structural proteins derived from the cognate virus have yet been successful in eliciting effective immune responses. to allow further reconfiguration of the native structures and adaptations in vlp design, the self-assembling antigens such as hiv gag, hbsag and hbcag (hepatitis b core antigen), parvovirus vp2 capsid protein or p1 protein of yeast transposon ty have been extensively studied in order to identify domains dispensable for particle formation to clear a space for insertion of more relevant sequences. in case of hiv-1 gag, parts of the matrix and capsid proteins or the carboxyl-terminal p6 moiety can be deleted without affecting particle assembly and then be replaced by or fused with heterologous sequences either derived from other regions of the cognate virus or from a foreign virus, thereby allowing efficient display of corresponding epitopes in the gag particle context. since multi-epitope vaccines have been shown to be more successful in inducing broad immune responses, gag-based vlps were soon extended to contain larger regions of the virus, and thus present more epitopes to the immune system. in this respect, the discovery that hiv full-length gag-pol precursors comprising additional enzymatic activities were also capable of forming particles was very helpful to develop a second generation of multi-epitope vlps ( figure 1b ). compared with these type-1 vlps, which are capable of eliciting strong ctl responses towards the inserted epitopes, type-2 vlps exposing incorporated envelope proteins on their surface have been more successful in inducing humoral antibody responses (reviewed in detail in reference [19 ] ). aiming towards induction of broadly cross-neutralising antibodies, numerous env-specific modifications have been conducted to achieve either maximal incorporation of env molecules (by truncation or fusion with heterologous transmembrane domains), to improve surface-presentation of native conformations of env (e.g. by trimer-stabilisation) or distinct exposition of conserved gp41 epitopes [19 ,20] . in this context, hiv-env immunogenicity has also been extensively analysed in the monkey model using simian immunodeficiency virus as a carrier resulting in chimeric siv-hiv viruses (shiv) [4] . apart from env-directed approaches, pseudotyping with fusogenic heterologous envelopes has proven to provide additional immunogenic benefit by augmenting vlp uptake via antigen presenting cells (apcs). for example, incorporation of surface glycoprotein from vesicular stomatitis virus (vsv-g) into hiv vlps significantly increased gag-directed antibody and t cell responses in mice and reduced viremia in challenged monkeys [21] . in other cases, where a less broad tissue tropism is needed, the baculovirus-derived envelope glycoprotein gp64 shown to mediate efficient transduction of mouse cells in vivo might provide a less toxic alternative to efficiently pseudotype vlps [22] . last but not least, vlps pseudotyped with heterologous envs can be simply used as platforms to efficiently present the incorporated env proteins to the immune system. as an example, gag vlps pseudotyped with equine herpesvirus type 1 (ehv-1)-derived glycoprotein gp14 elicited protective immune responses against ehv-1 challenge in mice upon intranasal application [23] emphasising the immunogenic potential of particulate antigens. to further enhance the immunogenicity of recombinant gag vlps, they have been equipped with additional costimulatory molecules such as influenza hemagglutinin (ha) or cholera toxin subunit b, both shown to significantly stimulate mucosal immune responses that have to be essentially induced to fend the virus at the gateway. increased stimulation of mucosal cellular immune responses has also been achieved by expression of vlps in gut bacteria such as salmonella as vehicles for the gag gene [6] . furthermore, an induction of env-specific iga and igg responses following intranasal application of gag-env vlps has lately been reported [24] , underlining the impact of the route of administration. quite recently, skountzou et al. [25 ] have demonstrated that the adjuvant properties of chimeric siv vlps could be significantly enhanced in immunised mice when loaded with co-stimulatory molecules gm-csf (granulocyte-macrophage colony-stimulating factor) or cd40 ligand (cd40l). a schematic illustration of the established strategies to construct and equip chimeric vlps is given in figure 1b . eventually, expression of vlps from dna plasmids encoding rna and codon-optimised gag genes or lentiviral vectors has become apparent as an efficient strategy to design highly immunogenic hiv vaccines (reviewed in reference [4] ). vlp antigens expressed from dna-transfected cells are capable of entering into both, the major histocompatibility complex (mhc) class-i and class-ii processing pathways thereby stimulating cd8 + and cd4 + t cell as well as b cell responses. compared with soluble antigens, dna vaccines are easier to produce and deploy the positive effects of live attenuated vaccines to directly stimulate mhc class-i restricted ctl responses. support for this hypothesis has been provided in a recent study published by bellier et al. [26] . the authors demonstrated efficient expression of murine leukemia virus (mlv) gag-env vlps from plasmid dna in vitro and used these plasmo-retrovlps to induce strong specific ctl responses towards displayed t cell epitopes, protecting mice from lethal virus challenge. currently, vlps are an inherent part of most multi-component status quo hiv vaccines, and one promising concept shown to induce broad long-lasting immune responses is a combination of dna/vlp prime followed by booster immunisation with live attenuated vaccinia vectors [27] . we and others have previously summarised the recent achievements on the way towards a potent vlp-based hiv vaccine [3, 4, 19 ] . the enormous convertibility of vlps illustrated by the numerous approaches to reconfigure particulate antigens for vaccines has also been successfully exploited in other areas such as gene therapy. a demonstration for this has been given in a recent study [28 ] , where vlps carrying the hiv cd4 receptor complex and a cytotoxic compound are specifically targeted to hiv-infected envexpressing cells via an inverse fusion process resulting in release of the compound and killing of infected cells. such therapeutic vlps might be useful tools in the future to attack long-time hiv reservoirs. compared with soluble antigens, which need to be coadministered with adjuvants in several booster injections to elicit protective immune responses, vlps are capable of inducing strong cellular and humoral responses as direct immunogens (reviewed in reference [12] ). vlp size appears to be favourable for uptake by dendritic cells (dc) via macropinocytosis and endocytosis that play a central role in activating innate and adaptive immune responses. a growing body of data indicates that gag vlps that per se contain many immunogenic epitopes are capable of stimulating cellular immune responses via both, the mhc class-i and mhc class-ii pathway [19 ] (see also figure 2a ). owing to the repetitive particle structure, uptake of a single vlp feeds thousands of contained epitopes into the processing and presentation machinery of apcs, a process thought to be supported by the fusogenic activity and the lipid nature of vlps. these assumptions are in line with recent findings that pseudotyping of vlps with envelope proteins like vsv-g, thereby improving uptake via receptor-dependent fusion, increases epitope presentation via the exogenous mhc class-i pathway and subsequent ctl induction [21, 29] . in principle, all established vlps proved to be strong stimulators of the innate immune system. the extensive potential of vlps to activate and mature dcs thereby triggering the expansion of numerous populations of immune cells in vivo has recently been diligently investigated by sailaja and et al. [30 ] . however, vlps used in this study were produced in the baculo-expression system. actually, increasing evidence suggests that contaminating components in vlp preparations might play a crucial role in stimulating both, innate and cognate immune responses. the majority of vlp preparations currently applied are derived from infection of insect cells with recombinant baculoviruses (bv), which are popular owing to their power regarding expression of heterologous proteins, their incapability to replicate in mammalian cells, a low cytotoxicity and the absence of a pre-existing immunity in humans. routine purification procedures like crude gradient centrifugation or ultrafiltration do not strictly discriminate between vlps and bvs. as a consequence, vlp preparations are probably enriched by bvs potentially contributing to the adjuvant properties of vlps (illustrated in figure 1c ). indeed, wild-type bvs have been reported to induce strong innate immune responses upon intranasal inoculation capable of protecting mice from lethal challenge with influenza virus [31] . the associated secretion of inflammatory cytokines such as tumour necrosis factor (tnf)-a, interleukins (il)-6 and-12, and type 1 interferons (ifn-a/-b) was shown to be at least partly induced via the toll-like receptor 9 (tlr9)/myd88 signalling pathway known to recognise internalised cpg-rich dna from bacteria [32] . indeed, it has been reported that bv dna probably released into the cytoplasmic compartments containing tlr9 upon gp64-mediated membrane fusion has a cpg content similar to bacteria, which might be regarded as a danger virus-like particles ludwig and wagner 541 signal. however, residual ifn-a release upon bv stimulation in tlr9/myd88 knockout mice indicated that other possible mechanisms might be involved in bvmediated immunogenicity, as has been suggested for the surface glycoprotein gp64 owing to its distinct n-glycosylation pattern. a model for bv-mediated dc stimulation is provided in figure 2a . the adjuvant properties of bv have been corroborated by recent findings showing that bv-induced humoral and ctl responses against co-administered antigens were triggered via ifna and b, which directly mature dcs thereby driving the differentiation of b and t cells into effector and memory cells [33 ] ( figure 2b ). in further support of this hypothesis, bv-produced hiv vlps formerly demonstrated to elicit strong immune responses in balb/c mice [34] were recently shown to mature monocyte-derived dcs associated with significant upregulation of surface maturation markers (cd40, cd80, cd83, cd86, hla-dr) and increased release of th1-and th2-specific cytokines [35] . apart from bv-derived vlp preparations, specific immunomodulatory effects have also been ascribed to yeast-derived particles, which have been shown to enter dcs via mannose recognition resulting in cross-priming of gag-specific cd8 + t cells [36] . in sum these data suggest that the respective system used for vlp production might significantly influence direction and outcome of the induced immune response. albeit the described adjuvant properties of contaminating baculovirus-or yeast-derived components might substantially trigger the overall immunogenicity of vlp preparations, the development of such a vaccine raises fundamental safety concerns considering the associated regulatory complications. basically, advanced procedures for production and notably purification of vlps will be needed to obviate these unforeseeable side effects. lessons learned from combating the well-known viruses like hbv, hpv or hiv are constantly being carried over to newly emerging and yet less intensively studied virus diseases, for which vlp-based strategies might serve as attractive first trial tools to develop a protective vaccine. as an example, vlps assembled from filovirus-derived matrix protein vp40 have been demonstrated to induce neutralizing antibody responses capable of protecting rodents from lethal challenge with ebola or marburg viruses, which cause severe hemorrhagic fever in humans [37, 38] . very recently, vlps assembled from severe acute respiratory syndrome (sars) coronavirus (sars-cov)-derived s, m, and e proteins have been reported to elicit strong humoral and cellular immune responses in mice, thus providing a promising strategy to confine sars in the near future [39] . on the basis of the accumulating knowledge of vlp function and the importance of diligent vlp design to perform in a vaccine setting, old vlp formulations, such as rabbit hemorrhagic disease virus (rhdv) particles, protecting rabbits from the cognate virus infection, are being discovered for new pur-poses. rhdv vlps chemically modified to allow covalent conjugation of peptides and proteins have lately been shown to induce strong humoral and cellular immune responses towards the attached antigens [40] . this illustrates the potential virtue of combining biological and chemical techniques to overcome restrictions in size or accessibility of vlp-inserted epitopes. a second class of stable spheres resembling vlps and with a wide current range of applications is referred to as virosomes, representing preformed virion-like liposomebased complexes with integrated surface glycoproteins for receptor-mediated endocytosis. like vlps, virosomes possess excellent adjuvant properties and have been established for many viruses like hiv, ebv, sendai virus or rabies either for vaccination purposes or delivery of nucleic acids, drugs or heterologous antigens (reviewed in reference [41] ). among all circulating virosome species, the immunopotentiating-reconstituted influenza virosomes (irivs), which are produced by detergent solubilisation of influenza viruses and then reconstituted with influenza envelope proteins ha and neuraminidase are certainly most prevalent [42] . ha stabilises the liposomal particles mainly composed of phospholipids and phosphatidylcholine and binds to sialic acid expressed on apcs, thus allowing receptor-mediated fusion followed by subsequent presentation of integrated antigens via mhc class-i and surface-exposed antigens via mhc class-ii. irivs are part of licensed hepatitis a virus and influenza vaccines and have been shown to efficiently boost humoral and cellular immune responses towards all kinds of integrated antigens such as tumour markers, malariaspecific peptides, leishmania-derived carbohydrates or toxoids [43] [44] [45] [46] [47] . even cytotoxic drugs, like doxorubicin are efficiently delivered to tumour cells by virosomes displaying polyethylene glycol-conjugated receptorspecific antibodies that might pave the way for novel cancer therapy strategies [48] . albeit virosomes have been demonstrated to induce longer lasting immune responses than conventional adjuvants while igniting fewer adverse side effects [49] , their adjuvant properties can be further increased by integration of co-stimulatory molecules as has been demonstrated for an iriv-based cancer vaccine co-assembled with cd40l [50] . alternatively, strong ctl responses demonstrated to be crucial for tumour growth control could be induced by means of recombinant irivs specifically targeted to plasmacytoid dcs [51] . apart from representing antigen delivery and display platforms, virosomes are excellent carriers of nucleic acids that form stable complexes with the liposome spheres. cusi et al. [52] have formerly shown that virosome-mediated targeting of mumps virus dna to apcs induces specific ctl responses suggesting efficient expression and presentation of the encoded mumps antigens. to protect the active dna component from cellular nucleases, de jonge et al. have recently developed a method to encapsulate plasmid dna into virosomes via a short-chain phospholipid [53 ] . another innovative approach has used virosomes to channel therapeutic sirna complexed with cationic lipids into target cells [54] . virosomes are becoming more and more popular in modern vaccine concepts, since they combine the safety and flexibility of subunit vaccines with the biological and immunogenic properties of vlps. however, the barriers between vlps and virosomes as by definition are beginning to melt. recently, a novel species designated immunovirosomes has been developed where moloney leukemia viruses were mixed with liposomes and conjugated with a receptor-directed antibody to allow tissuespecific targeting of a therapeutic antigen [55] . nanoparticles basically encompass all particulate structures ranging between 5 and 100 nm in size. especially the capability of virus-derived nanostructures to assemble into highly organised regular arrays together with their susceptibility to accept a wide range of chemical modifications has offered new perspectives for the usage of vlps in manifold biotechnological processes. wang et al. have formerly exploited the possibility to chemically modify mutant cowpea mosaic virus (cpmv) particles -which are highly stable and offer multiple reactive sites -by exposing sulfhydryl groups in order to attach fluorescent dyes and gold clusters [56] . cpmv was also shown to efficiently self-assemble into monolayers at perfluorodecalin-water interfaces, which can be cross-linked to form a robust membrane [57] . likewise, the tobacco mosaic virus (tmv)-derived capsid protein has proven to be a suitable scaffold for extensive chemical modification allowing assembly of nanobiopolymers (summarised in reference [9 ] . yet another study has described the usage of recombinant m13 phage particles as organic templates to polymerise nanowires as building blocks for semiconductors or magnetic materials [58] . likewise, the need for new nanowire material for the construction of smaller lithium ion batteries has drawn the attention to filamentous m13 phages, which were modified to surfaceexpose tetraglutamate to nucleate metal ions such as the lithium active compound cobalt oxide [59] . in all these approaches, chemically modified vlps maintained their structural integrity that is a prerequisite for vlps to prevail in the future world of nanoscience. on the basis of their flexibility and stability, simple production and distinctive immunogenic properties, vlps offer vast opportunities of application in the fields of vaccine development, gene therapy as well as nanobiotechnology. many lessons have been learned from vlp-based technologies, which will certainly find themselves confronted by new challenges in the near future, such as the demand for innovative biomaterials or potent vaccines for newly emerging diseases. in this light it appears almost ironical that viruses as such may serve a good purpose in the biotechnological era exploiting their weapons to beat them at their own game. particles associated with australia antigen in the sera of patients with leukaemia, down's syndrome and hepatitis ted slavin's blood and the development of hbv vaccine virus-like particles as hiv-1 vaccines virus-like particles: designing an effective aids vaccine human hepatitis b vaccine from recombinant yeast comparative analysis using a mouse model of the immunogenicity of artificial vlp and attenuated salmonella strain carrying a dna-vaccine encoding hiv-1 polyepitope ctl-immunogen production of human papillomavirus type 16 l1 virus-like particles by recombinant lactobacillus casei cells virus-like particles production in green plants virus-like particles: passport to immune recognition females united to unilaterally reduce endo/ectocervical disease (future) i investigators: quadrivalent vaccine against human papillomavirus to prevent anogenital diseases hpv patricia study group: efficacy of a prophylactic adjuvanted bivalent l1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase iii double-blind, randomised controlled trial assembly and release of hiv-1 precursor pr55gag virus-like particles from recombinant baculovirusinfected insect cells studies on processing, particle formation, and immunogenicity of the hiv-1 gag gene product: a possible component of a hiv vaccine induction of a mhc class i-restricted, cd8 positive cytolytic t-cell response by chimeric hiv-1 virus-like particles in vivo: implications on hiv vaccine development priming of strong, broad, and long-lived hiv type 1 p55gag-specific cd8 + cytotoxic t cells after administration of a viruslike particle vaccine in rhesus macaques recombinant hiv-1 pr55gag virus-like particles: potent stimulators of innate and acquired immune responses rational modifications of hiv-1 envelope glycoproteins for immunogen design immunogenicity and efficacy of immunodeficiency virus-like particles pseudotyped with the g protein of vesicular stomatitis virus lentiviral vectors pseudotyped with baculovirus gp64 efficiently transduce mouse cells in vivo and show tropism restriction against hematopoietic cell types in vitro protection against ehv-1 challenge infection in the murine model after vaccination with various formulations of recombinant glycoprotein gp14 (gb) membrane embedded hiv-1 envelope on the surface of a virus-like particle elicits broader immune responses than soluble envelopes in order to enhance the immunogenic properties of chimeric siv gag-env vlps, the authors have incorporated the immunostimulatory molecule cd40l. alternatively, gm-csf was anchored in the vlp membrane by means of glycosylphosphatidylinositol (gpi) dna vaccines encoding retrovirusbased virus-like particles induce efficient immune responses without adjuvant generation and immunogenicity of novel hiv/aids vaccine candidates targeting hiv-1 env/gag-pol-nef antigens of clade c selective elimination of hiv-1-infected cells by env-directed, hiv-1-based virus-like particles yeast-derived human immunodeficiency virus type 1 p55(gag) virus-like particles activate dendritic cells (dcs) and induce perforin expression in gag-specific cd8(+) t cells by cross-presentation of dcs virus-like particles exhibit potential as a pan-filovirus vaccine for both ebola and marburg viral infections ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies immune responses against severe acute respiratory syndrome coronavirus induced by virus-like particles in mice versatile rhdv viruslike particles: incorporation of antigens by genetic modification and chemical conjugation liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs applications of influenza virosomes as a delivery system structureactivity-based design of a synthetic malaria peptide eliciting sporozoite inhibitory antibodies in a virosomal formulation influenza virosomes as an efficient system for adjuvanted vaccine delivery clinical applications of virosomes in cancer immunotherapy optimized malariaantigens delivered by immunostimulating reconstituted influenza virosomes enhancement of the immunogenicity of synthetic carbohydrates by conjugation to virosomes: a leishmaniasis vaccine candidate targeting her-2/neu with antirat neu virosomes for cancer therapy immunogenicity and adverse effects of inactivated virosome versus alum-adsorbed hepatitis a vaccine: a randomized controlled trial immunereconstituted influenza virosome containing cd40l gene enhances the immunological and protective activity of a carcinoembryonic antigen anticancer vaccine virosome-mediated delivery of tumor antigen to plasmacytoid dendritic cells intranasal immunization with mumps virus dna vaccine delivered by influenza virosomes elicits mucosal and systemic immunity cellular gene transfer mediated by influenza virosomes with encapsulated plasmid dna cellular delivery of sirna mediated by fusion-active virosomes changing viral tropism using immunoliposomes alters the stability of gene expression: implications for viral vector design icosahedral virus particles as addressable nanoscale building blocks self-assembly and cross-linking of bionanoparticles at liquid-liquid interfaces virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodes we would like to thank the authors of the reviewed work for sharing their data and providing insight into their ingenious concepts and strategies. the authors state that no conflicts of interest exist. papers of particular interest, published within the annual period of review, have been highlighted as:of special interest of outstanding interest this is the report about an unconventional method that uses hiv vlps to specifically target and eliminate hiv-infected cells. for this purpose, the authors have created a recombinant vlp displaying the cellderived hiv-specific cd4 receptor complex and carrying thymidine kinase (tk) of herpes simplex virus-1, which has proven to be highly cytotoxic under ganciclovir treatment causing chain termination during replication. this tk is delivered by vlps via an inverse fusion process to latently infected cells that express hiv proteins and surface-expose env upon cytokine stimulation, resulting in efficient cell killing. key: cord-309242-ilsupfl8 authors: schuchat, anne; tappero, jordan; blandford, john title: global health and the us centers for disease control and prevention date: 2014-07-02 journal: lancet doi: 10.1016/s0140-6736(14)60570-5 sha: doc_id: 309242 cord_uid: ilsupfl8 nan anne schuchat, jordan tappero, john blandford why is an article about global health included in a special issue on health in the usa? about half the produce that americans consume is cultivated in other countries, 60 million americans travel or work outside the usa, and most patients with measles and tuberculosis in the usa acquired their infection elsewhere. emerging diseases, globalisation of foods and medicines, the rise in antimicrobial resistance, and the ease with which pathogens can be manipulated for good or harm increase each nation's vulnerability and interdependence. the centers for disease control and prevention (cdc) has engaged in global health for more than 60 years. although tackling infectious disease threats has been a constant, the agency has also provided humanitarian assistance in natural disasters, refugee crises, and famines; improved child survival; and strengthened scientifi c measurement of health metrics. cdc's workforce includes more than 300 internationally deployed public health professionals in addition to around 1330 locally employed staff working in about 60 countries (fi gure). cdc's global health strategy aims to: achieve optimum health eff ects; strengthen global health capacity; improve global health security; and strengthen the organisation's capacity. 1 this report describes selected programmes that illustrate these goals. leveraging the strengths of the us-implementing agencies for a whole-of-government approach, the us president's emergency plan for aids relief (pepfar) targets resource-constrained countries that are hardest hit by the hiv epidemic. cdc has played a central role in pepfar since its inception, bringing more than 30 years of experience of work in hiv/aids. cdc staff work with peers in ministries of health and other host country entities to implement eff ective national programmes in hiv care and treatment, tuberculosis-hiv integration, maternal and child health, hiv prevention, and hiv counselling and testing. through pepfar, cdc is also strengthening public health systems through focused investments in local workforce capacity and health systems that are essential to the scale-up and sustainability of priority pepfar programmes and to the strengthening of the broader public health system. for example, growing hiv treatment pro grammes were shown to be positively correlated with increased rates of facility deliveries by non-hiv infected women. 2 in late 2011, president obama and then-secretary of state hillary clinton advanced the vision of an aids-free generation, defi ned as almost complete elimination of vertical transmission of hiv, greatly reduced incidence of sexual transmission of hiv in adults and adolescents, and universal access to antiretroviral therapy (art) to people who become infected. president obama announced in december, 2011, ambitious new targets for priority evidence-based interventions that were to be realised in just 2 years' time: pepfar, in 2013, was committed to directly support 6 million patients receiving treatment, an increase of 50% over the previous target; provision of therapy to 1·5 million pregnant women to prevent vertical infection of hiv; and to cumulatively reach 4·7 million men with voluntary medical male circumcisions. 3, 4 cdc and its partners worked to make these bold targets a reality. pepfar has directly supported 6·7 million patients receiving treatment; provided more than 1·5 million pregnant women with antiretroviral prophylaxis to prevent mother-to-child transmission of hiv during the preceding 2 years; and with cumulative support of 4·2 million medical male circumcisions, was on track to achieve that target by the end of 2013. 5 cdc implemented an innovative approach to prevent mother-to-child transmission of hiv in malawi by working with the ministry of health and local partners. 6 the new approach, called option b+, off ers all pregnant or breastfeeding women infected with hiv lifelong art. 1 year after this approach was implemented in 2011, the number of hiv-positive pregnant and breastfeeding women receiving art increased by more than 700%, from 1257 to 10 663. option b+ not only reduces motherto-child transmission to less than 5% but also maintains the mother's health, provides lifelong reduction of hiv transmission to uninfected sexual partners, and prevents mother-to-child transmission in future pregnancies. other african and caribbean countries are now initiating this approach. although each country must weigh the potential eff ect and costs of option b+ in reaching the goal of elimination of mother-to-child hiv transmission, this approach could overcome barriers such as inadequate immunological testing capacity, substantial distances between antenatal clinic and art-initiation sites, and human resource constraints. 6 further, cdc and the us health resources and services administration initiated the track 1·0 programme in 2004, to rapidly scale up art delivery initially through us-based partners. 7 in its fi rst phase, pepfar was undertaken as an emergency response to provide accelerated access to crucial, lifesaving services and programmes. with pepfar's reauthorisation in 2008, cdc worked to further strengthen programme sustainability, country stewardship, and local implementation of core programmes. cdc worked with ministries of health to transition service delivery of hiv/ aids care and treatment to local stewardship in 13 countries. as of february, 2012, these countries had contracts or awards in place with indigenous partners to establish local country ownership of their art programmes. the transition of treatment programmes to indigenous partners is consistent with cdc's approach across programme areas of working through partner ministries of health and other local entities. 61% of cdc's pepfar programme funding goes through indigenous entities, and this share should continue to grow as cdc provides the technical support and capacity building to maintain continuity and quality of treatment services. cdc's infl uenza programme contributes to strengthened global health security. infl uenza viruses spread rapidly without respect to geographical borders. control of disease caused by these viruses requires accurate laboratory detection, epidemiological response, and development and delivery of appropriately targeted vaccines and other countermeasures. eff orts that focus on seasonal infl uenza strengthen preparedness for pandemics. addressing avian infl uenza has assumed greater priority since emergence of h5n1 8 and more recently h7n9 infl uenza viruses. 9 the cdc is the national infl uenza center for the usa and one of fi ve who international collaborating centers for surveillance, epidemiology, and control of infl uenza. cdc scientists participate in strain selection for seasonal vaccines and prepare candidate vaccine virus strains for vaccine manufacturers. since the emergence of cases of h5n1 avian infl uenza in people more than a decade ago, cdc has expanded support to and collaborations on infl uenza with dozens of countries. after a local outbreak in 1997 in hong kong, 10,11 avian h5n1 infl uenza resurfaced in 2003, initiating a decade of major epizootics aff ecting birds in asia, northern africa, and europe, prompting intensifi ed national, regional, and global responses. 12 to date, animal-to-human transmission has caused nearly 400 human fatalities, and millions of poultry have been culled in aff ected areas. the cdc contributed to a comprehensive us government response to h5n1 avian infl uenza. it established cooperative agreements with ministries of health in more than 30 countries to strengthen capacity for epidemiological response, laboratory detection, and emergency preparedness; seconded staff to who, world animal health organization, and un food and agriculture organization; collaborated on vaccine and diagnostic studies with the us national institutes of health, the food and drug administration, the biomedical research and advanced development authority, academic investigators, and industry; and established a strategic national stockpile of antiviral drugs and other emergency supplies. by april, 2009, when cdc fi rst detected a novel strain of infl uenza a h1n1, cdc had already enhanced capacity for pandemic response. an infl uenza reagent repository facilitated the surge in laboratory testing in the usa and other countries. cdc shipped reagents for the detection of novel 2009 h1n1 infl uenza virus beginning may 1, 2009 . 13 in addition to 1500 kits shipped to public health laboratories in all 50 states, 1400 diagnostic kits were shipped to laboratries in 153 countries. cdc's investments in strengthening countries' surveillance for severe acute respiratory illness (sari) 14 provided recognition of severe disease in many countries caused by the new infl uenza strain and helped to monitor changes in clinical severity that might signal viral mutations of concern. in 2013, the sari surveillance systems monitored the spread of the new middle east respiratory syndrome (mers) coronavirus. on march 31, 2013, chinese authorities reported to who their detection of a novel h7n9 avian infl uenza virus that caused severe pneumonia in three people in shanghai and anhui. 9,15 from oct 1, 2013, to march 12, 2014, 384 human cases and 123 deaths had been reported by china's national health and family planning commission, and the novel virus had been isolated from live bird markets. 16 the chinese rapidly completed whole genome sequencing of several viral isolates, posted their sequence data, closed live bird markets in several cities, and initiated targeted culling of birds. the virus is of low pathogenicity in poultry, and subclinical avian infection has complicated animal control eff orts. although no sustained human-to-human transmission has occurred, it is too early to be certain of the full eff ect that this novel virus will have on human or animal populations. the chinese authorities responded rapidly and shared information about human disease and genetic sequence data openly. 17 the us cdc has collaborated with china's infl uenza scientists and public health authorities for decades. cdc provided substantial technical assistance, several visiting scientists from china completed training and research projects in atlanta, and senior scientists from the us cdc provided onsite technical support in china. a major milestone was achieved in 2010, when china was designated as the fi fth who international collaborating center for reference and research on infl uenza in human beings. china is also the location of one of cdc's global disease detection (gdd) regional centers. at gdd sites, us cdc epidemiology, laboratory, and veterinary staff work closely with counterparts from the ministries of health to strengthen early detection and response to public health threats. china also hosts a field epidemiology training program (fetp), at which a us cdc resident advisor works closely with epidemiology trainees in a 2 year programme modelled after the cdc's elite epidemic intelligence service. in recognition of these broad partnerships, china designates national, provincial, and district public health departments as cdcs. infl uenza events highlight the importance of strengthening global health security worldwide. emerging threats such as avian infl uenza or severe acute respiratory syndrome can occur anywhere. 18 acceleration of improvements in the capacity for each nation to protect its own citizens and detect and report public health threats of international concern promptly to the global community is urgently needed. 19 cdc's experience in haiti addresses how programmes designed for health eff ect can strengthen capacity and improve preparedness for health security threats. on jan 12, 2010, a devastating earthquake struck port-au-prince, haiti, killing an estimated 230 000 people, injuring 300 000 more, displacing more than 20% of the country's 10 million residents, and destroying much of the capital's infrastructure. 20 the public health response benefi ted from cdc's partnership with the ministry of public health and population (mspp), and the administrative and non-governmental organisation implementation platforms established under pepfar in 2002. 20 hiv services recovered to baseline rapidly. 21 yet an urgent need remained to strengthen reportable disease surveillance because of overcrowding and poor living conditions, 22, 23 expand capacity to identify reportable pathogens at the national public health laboratory, 24 and train a public health workforce. the us government committed more than us$3 billion in new funds for post-earthquake relief, recovery, and reconstruction; facilitated mspp's rapid detection of vibrio cholerae within days of its introduction; and contributed to an eff ective emergency response 20 to the worst national cholera epidemic in the modern era. 25 after these back-to-back crises, in early 2011 mspp, cdc, and other organisations identifi ed seven measurable public health eff ect goals to transform health services and disease outcomes. 26 with the aim to achieve an aids-free generation, in 2011, mspp and pepfar-haiti expanded hiv counselling and testing sites for pregnant women, implemented a case management programme for all hiv-infected women to reduce loss to follow-up, and in 2012 adopted the b+ option that provides all hiv-infected pregnant and breastfeeding women with lifelong art irrespective of cd4 cell count. 6, 27 hiv testing of pregnant women has increased by 74%, from 128 540 in 2010, to 223 626 in 2013. 28 the proportion of identifi ed hivinfected pregnant women who received prophylaxis or art increased from 61% to 92% during this period. 28 since the earthquake, overall access to art has nearly doubled; 29, 30 in 2013, more than 75% of all eligible hivinfected haitians were receiving art. 28 from oct 20, 2010, to april 28, 2014, haiti reported 701 961 cases of cholera and 8555 deaths. 31 the inadvertent introduction of cholera, which had not been seen in haiti in more than a century, accounted for 57% of the reported global cholera burden in 2010 and 2011. 25 although the annual case fatality rate has been sustained at the international standard of 1% or less since 2011, elimination will need substantial investment in infrastructure to address inadequate access to improved drinking water (62%) and sanitation (24%). 32 outbreaks, and use data to drive public health policy. cdc has helped to establish 50 fetps that have trained nearly 3000 graduates from 69 countries. the world is more interconnected than ever, and weak links in public health capacity anywhere can have profound eff ects at distant locations. governments have responsibilities to their citizens and other nations to detect problems rapidly, communicate promptly, and respond eff ectively. the cdc focuses on the protection of americans and improvements in the health and capacity of people worldwide through partnerships with ministries of health, other us government agencies, non-governmental organisations, and multilateral organisations. the goal of these eff orts is to improve health and strengthen capacity while striving for a world more secure from emerging threats. as contributed to the overall planning and writing of the manuscript, the literature search, and the development of the fi gure. jt contributed to the writing of the manuscript, the literature search, and the development of the fi gure. jb contributed to the writing of the manuscript and the literature search. we declare no competing interests. human infection with highly pathogenic avian infl uenza a (h5n1) virus: review of clinical issues detecting 2009 pandemic infl uenza a (h1n1) virus infection: availability of diagnostic testing led to rapid pandemic response infl uenza surveillance in 15 countries in africa emergence of avian infl uenza a (h7n9) virus causing severe human illness-china human infections with the emerging avian infl uenza a h7n9 virus from wet market poultry: clinical analysis and characterisation of viral genome global concerns regarding novel infl uenza a (h7n9) virus infections moving forward to 2014: global ihr (2005) implementation international health regulations-what gets measured gets done lessons learned during public health response to cholera epidemic in haiti and the dominican republic recovery of hiv service provision post-earthquake launching a national surveillance system after an earthquake-haiti rapid establishment of an internally displaced persons disease surveillance system after an earthquake-haiti public health in haitichallenges and progress cholera surveillance during the haiti epidemic-the fi rst 2 years cautious optimism on public health progress in post-earthquake haiti prevention of mother-to-child transmission of hiv and the health-related millennium development goals: time for a public health approach unaids. unaids report on the global aids epidemic 2010 unaids. 2013 unaids report on the global aids epidemic ministère de la santé publique et de la population daily reports on the cholera epidemic in haiti the cure for choleraimproving access to safe water and sanitation world health organization, unicef. world health organization/ unicef joint monitoring programme (jmp) for water supply and sanitation pepfar programs linked to more deliveries in health facilities by african women who are not infected with hiv voluntary medical male circumcision: an hiv prevention priority for pepfar progress in voluntary medical male circumcision service provision-kenya shared responsibility-strengthening results for an aids-free generation impact of an innovative approach to prevent mother-to-child transmission of hiv-malawi scale-up of hiv treatment through pepfar: a historic public health achievement isolation of avian infl uenza a(h5n1) viruses from humans-hong kong human infection with a novel avianorigin infl uenza a (h7n9) virus human infl uenza a h5n1 virus related to a highly pathogenic avian infl uenza virus update: isolation of avian infl uenza a (h5n1) viruses from humans-hong kong, 1997-1998 we thank ezra barzilay, sally ezra, barbara marston, and ann moen for assistance with manuscript and fi gure preparation; and harold jaff e and tom frieden for review of the manuscript. the fi ndings and conclusions of this report are those of the authors and do not necessarily refl ect the offi cial position of the centers for disease control and prevention. key: cord-295099-ghc85pf5 authors: sun, zehua; yan, lixin; tang, jiansong; qian, qian; lenberg, jerica; zhu, dandan; liu, wan; wu, kao; wang, yilin; lu, shiqiang title: brief introduction of current technologies in isolation of broadly neutralizing hiv-1 antibodies date: 2018-01-02 journal: virus res doi: 10.1016/j.virusres.2017.10.011 sha: doc_id: 295099 cord_uid: ghc85pf5 hiv/aids has become a worldwide pandemic. before an effective hiv-1 vaccine eliciting broadly neutralizing monoclonal antibodies (bnmabs) is fully developed, passive immunization for prevention and treatment of hiv-1 infection may alleviate the burden caused by the pandemic. among hiv-1 infected individuals, about 20% of them generated cross-reactive neutralizing antibodies two to four years after infection, the details of which could provide knowledge for effective vaccine design. recent progress in techniques for isolation of human broadly neutralizing antibodies has facilitated the study of passive immunization. the isolation and characterization of large panels of potent human broadly neutralizing antibodies has revealed new insights into the principles of antibody-mediated neutralization of hiv. in this paper, we review the current effective techniques in broadly neutralizing antibody isolation. antibody responses to neutralize human immunodeficiency virus-1 (hiv-1) are mediated by direct binding to viral spikes, which are trimers composed of glycoproteins gp120 and gp41 (pincus et al., 2017a; pincus et al., 2017b; blair et al., 2007; morris et al., 2000; micoli et al., 2000; pegu et al., 2017; haynes and mascola, 2017; liao et al., 2004; brodine et al., 2003; ward and wilson, 2017; debnath et al., 1994; moore et al., 1993) . gp120 with the attachment of virus particles to the target cells, and the gp41 engages in virus-cell fusion (durham and chen, 2016; desai et al., 2015; dale et al., 2011; bieniasz et al., 1997; rausch et al., 1990) . while successful in-vitro, recombinant gp120 has failed to generate cross neutralizing antibodies in vivo possibly due to the natural conformation of gp120 (prevost, 2017; wang et al., 2016; doores et al., 2015; doores et al., 2010; zhou et al., 2010; wu et al., 1997; watkins et al., 1996; robert-guroff et al., 1992) . monoclonal antibodies were first generated in mice in 1975 using a hybridoma technique (patke et al., 2017; holzlohner and hanack, 2017; bhatia et al., 2016; pogson et al., 2016; abusneina and gauthier, 2016; milstein, 1999) , and were subsequently accepted globally by manufacturers. now, it is well understood that rodent and murine derived antibodies are immunogenic in humans; when binding to antigens within the human body, the complexes can be recognized as immunogenic agents. to solve this problem, human equivalent forms were generated from rodent or murine antibodies, known as humanized antibodies (pardi et al., 2017; wiehe et al., 2017; rothenberg, 2016; bhowmick et al., 2016; hamanoue et al., 2016) . in recent years, new techniques have evolved to isolate human monoclonal antibodies against hiv-1 directly which offer an advantage of reduced immunogenicity. a panel of hiv-1 specific potent broadly neutralizing antibodies has been isolated in recent decades. the first generation antibodies, 2f5, 2g12 and 4e10, were used in combination for passive immunization (morris et al., 2014; mehandru et al., 2004; armbruster et al., 2004) . a second generation antibody, igg b12, was observed to neutralize numerous virus isolates with ic50 in nm level (1 nm is equal to about 0.15ug/ml) for the first time (li et al., 2017; bunnik et al., 2010; ashish et al., 2010; zwick et al., 2003; sun et al., 2015) . the new generation of potent broadly neutralizing antibodies, including vrc01-class antibodies, nih45-46, pg9/16,10e8, bg18, ioma, 35o22, acs202, can neutralize virus isolates ranging from 73% to 98% with ic50 less than 50 μg/ml, and can neutralize 59%-72% of virus isolates with ic50 less than 1 μg/ml (morales et al., 2016; huang et al., 2012; braibant et al., 2013; thenin et al., 2012; ringe et al., 2012; euler et al., 2011; davenport et al., 2011; pancera et al., 2010; wu et al., 2010; scheid, 2015; scheid et al., 2011) . worth noting, broadly neutralizing antibody 10e8 neutralizes 98% of tested viruses with geometric mean ic50 of 0.22 μg/ml. 10e8 binds to the cell-surface envelope as opposed to phospholipids. the binding of 10e8 and mper reveals a highly conserved gp41-hydrophobic residue and a critical arginine or lysine just before the transmembrane region. the breadth and potency of 10e8 demonstrates a conserved site for hiv neutralization and potential target for hiv vaccine design (huang et al., 2012) . the highly potent vrc01-class broadly neutralizing antibodies (bnabs) individually neutralize up to 90% of hiv-1 isolates. vrc01class antibodies are considered a sub group of bnabs and have already been isolated from several hiv-1 infected patients. they each neutralize different virus isolates through the interaction with hiv gp120 cd4 binding site. vrc01-class bnabs are derived from human vh1-2 gene family, which occupies 2% of the human antibody repertoire. structural studies revealed that these vrc01-class abs interact with gp120 cd4 binding site by mimicking the cd4 molecule; specifically the secondary structure formed by heavy chain structure and cdr3 in light chain. additionally, an important deletion in cdrl1 present in many vrc01class abs can facilitate the interaction with the cd4 binding site and avoid conflict with glycan at asn 276 (n276) on loop d (zhou et al., 2010; georgiev et al., 2014; wu et al., 2015; zhou et al., 2013) . nextgeneration sequencing (ngs)-derived sequencing reveals that vrc01 lineage is comprised of at least six distinct heavy chain clades and five light chain clades. this observation suggests that extraordinary variation in antibody immunity may only occur within a few antibody lineages − or even a single lineage (wu et al., 2015) . generation of an unmutated common ancestor of vrc01-class antibodies could be a potential mechanism for opposing hiv-1. after stimulation by virus and co-evolution with the virus, the unmutant common ancestor has ability to mature to bnabs. human antibodies can be elicited by b cells to eliminate "foreign immunogens" directly or indirectly. some antibodies can be produced to neutralize foreign antigens by various mechanisms, while some nonneutralizing antibodies can perform effector functions such as mediating nk cells via antibody-dependent cellular cytotoxicity (adcc) (liao et al., 2004; lin et al., 1998; thomann et al., 2015; ito et al., 2009; mandi et al., 1982) . serum immunoglobulins produced naturally by human immunity have been applied in passive immunotherapy against many infectious diseases since 1990. but this approach suffers a number of problems. not only have these polyclonal immunoglobulins displayed mixed efficacies, but they also pose the potential threat of bloodborne pathogen transmission. in addition, the resource of serum is limited and expensive. these disadvantages limit the application of polyclonal immunotherapy (berry, 2017; stevens et al., 2016; wang et al., 2007; sili et al., 2003; lang et al., 1993) . recent technology developments have made it possible to isolate human antibodies against hiv-1 from both in vivo and in vitro sources. these improved techniques include display methods, epstein-barr virus (ebv) immortalization, classical hybridoma procedures, and single-cell sorting followed by molecular cloning. 2. techniques to make human monoclonal antibodies in the mid-1970s, hybridoma technology was invented for production of mouse monoclonal antibodies with defined antigen specificities and neutralization activity for application in clinical therapies (kelso et al., 2016; hugwil, 2013; tomita and tsumoto, 2011; martin-lopez et al., 2007; hencsey et al., 1996; honda et al., 1990; köhler and milstein, 1975) . hybridomas can be generated by effective fusion of b cells and partner cells followed by screening of individual antibody producing cells. production of monoclonal antibody in individual hybridoma cells can be easily quantified by surface plasmon resonance imaging (stojanovic et al., 2015) . b cell hybridomas can be an important source for screening of monoclonal antibodies. highthroughput screening is used to characterize mouse igg antibodies; including sub-isotypes, binding activity, and neutralizing activities (liu et al., 2015; szafran et al., 2016) . fluorescent antigen can be used to sort antigen binding hybridoma cells from a mixture as opposed to the traditional way of screening using multi-micro well plate screening and limiting dilution. as an extension of this technology, t cell hybridoma was also generated (krishnan et al., 2015) . however, mouse monoclonal antibodies were shown to be problematic in humans due to their immunogenic properties and low effector function. high immunogenicity prevented their application in humans where prolonged dosing was required. differences in sequence and glycosylation pattern of the fc region make rodent mabs poorly effective in mediating effector functions in human (li et al., 2006) . human b cells proved to be difficult to immortalize using hybridoma technique (li et al., 2006; cafri et al., 2013; park et al., 1997) . the primary issue was that mabs produced by human b cell hybridoma cells could react against selfantigens. in addition, the lack of sufficient maturation outside of the germinal centers and low binding affinities of mabs derived by hybridoma, meant that only 1.5% of screened clones typically reacted to the target antigen. due to these problems, antibody humanization was developed as an alternative to generate human format antibodies from rodent derived antibodies (martin and rees, 2016; choi et al., 2015; olimpieri et al., 2015; safdari et al., 2013; tsurushita et al., 2004; ohtomo et al., 1995) . humanized antibodies are produced by methods of genetic engineering and can potentially reduce the immune response against non-human derived antibodies. this is done by combining the variable (v) region binding domain of a rodent antibody with human antibody constant (c) regions. this kind of chimeric antibody retains the binding specificity with less immunogenicity to humans. in some cases, the chimeric antibodies retain the effects in mediating human complement-dependent cytotoxicity (cdc) or adcc. it is also possible to produce a humanized antibody without creating a chimeric intermediate first. once the precise sequences of the desired cdrs are known, these dna sequences can be directly cloned into antibody expression vectors with human antibody "scaffold". a wide variety of human antibody expressing vectors has been developed using this method. for example, pdr12 vector is a human igg1 antibody expressing vector which was originally used for expression of human hiv-1 potent neutralizing antibody igg b12. antibody vrc01 was cloned into a different cloning vector described previously by tiller et al. (tiller et al., 2008) . another method to immortalize human memory b cells is by epstein-barr virus (ebv) mediated transformation (lee et al., 2011; klein, 1996; straub and zubler, 1989; ohashi et al., 2017; mclaughlin et al., 2017; traggiai et al., 2004) . the c3b, ebv-binding receptor positive b cells can be immortalized to generate antibodies for isolation using single cell sorting, or can be cultured with feeder cells for further screening or cloning. the optimized methodology is to activate b cells using toll-like receptor 9 (tlr9) agonist cpg dna before or during ebv infection, which has been used to isolate human monoclonal antibodies against various pathogenic viruses (katsumura et al., 2012; bass and darke, 2004; sun et al., 2017) . it has also been reported that human b cells can be immortalized by transforming a retrovirus encoding anti-apoptotic factor b cell lymphoma 6 (bcl6) and bcl-xl in the presence of interleukin 21 (il-21) and cd40 ligands (schrader et al., 2012; rydstrom et al., 2010; kusam et al., 2009 ). this process can trigger memory b cells to differentiate into antibody secreting cells. the immortalization of human b cells provides a substantial resource of human b cells for the subsequent screening. after culturing for 1-2 weeks, antibodies can be collected and isolated from the supernatant to perform neutralization against different viruses. immortalized b cells can also be sorted by antigen using flow-based technology, and the heavy and light chain genes of antibodies secreted by immortalized b cells can then be cloned. the famous 2g12, 2f5 and 4e10 antibodies are generated by this method (buchacher et al., 1994) . however, due to the low efficiency of ebv-induced b cell transformation, this method has limitation in application. though optimization methods were studied based on ebv-induced b cell transformation, the efficiency still needs a dramatic improve (sun et al., 2017; lu et al., 2016; kwakkenbos et al., 2016) . some reports show that memory b cells can be cultured short-term, and the supernatant can be used directly for screening of neutralization activity in a high throughput neutralization assay (micro-neutralization). a number of famous neutralizing antibodies have been isolated based on these methods including pg9, pg16, pgt121-128 and 10e8 (huang et al., 2012; ringe et al., 2012; walker et al., 2011; walker et al., 2009; falkowska et al., 2014) . hiv-1 potent neutralizing antibodies pg9 and pg16 are classic antibodies isolated by microneutralization. these were originally isolated from 30,000 activated memory b cells from one clade a infected donor screened from approximately 1800 characterized hiv infected donors. the memory b cells were cultured at clonal density for the purpose of cloning antibody heavy and light chain pair from each culture well. pg9 neutralized 127 out of 162 viral isolates and pg16 neutralized 119 out of 162 viruses with a median potency (ic50) less than 0.2 μg/ml. 10e8 is an anti-gp41 antibody isolated from 16,500 memory b cells, sorted and cultured with il-2, il-21, and cd40-ligand expressing feeder cells. 10e8 neutralized 98% of the tested pseudoviruses with an ic50 below 50 μg/ml, and 72% of the tested viruses with an ic50 values below 1 ug/ml. b cell culture and microneutralization can isolate potent broadly neutralizing hiv-1 antibodies. however, the large scale screening of b cells makes this method labor intensive and costly, in addition to low yield; typically ten antigen-specific clones can be isolated out of ten thousands of b cells ( fig. 1a and b) . in current reports, ebv transformation was optimized with efficiency from 0.1%-2% to 7.8%, which enabled the generation of immortalized b cell libraries. however the efficiency still needs to be improved to enable greater b cell survival (sun et al., 2017) . phage display is a library method which permits screening of antibodies from a large recombinant library (falkowska et al., 2014; boots et al., 1997; chan et al., 1996; aghebati-maleki et al., 2017; rahbarnia et al., 2017; finlay et al., 2017) . antibodies can be displayed in the form of either single chain variable fragments (scfv) or antigen-binding fragments (fab). recent reports show that single domain antibody can also be displayed in phage display system (duarte et al., 2016; kazemi-lomedasht et al., 2016; li et al., 2016; rotman et al., 2015; tang et al., 2013) . many antibodies against different viruses including rabies virus, severe acute respiratory syndrome (sars) virus, ebola virus, yellow fever virus, hepatitis c virus, dengue virus, hepatitis a virus, influenza virus, and hiv, have been successfully isolated using this technology. the principle for phage display technique in isolating antibodies is to construct a phage display recombinant antibody library first. recombinant antibody library can increase the diversity of antibodies in b cell repertoires, which promotes the screening of antibodies with novel properties (graus et al., 1998) . antibody libraries are in general constructed by randomly assembling antibody heavy and light chain variable region, and through gene shuffling of the heavy and light chain to further increase the diversity. in summary, phage display creates greater diversity of antibodies and provides rapid and high throughput way of screening. m43 and m44 are hiv-1 cross-reactive human monoclonal antibodies isolated from a recombinant phage display library by competitive antigen panning (zhang et al., 2012; zhang et al., 2008; zhang et al., 2006; zhang et al., 2004a; zhang et al., 2004b) . m44 is a gp41 specific cross-reactive antibody, and m43 can recognize both gp120 and gp41. b12 is another potent neutralizing antibody isolated by phage display from recombinant antibody libraries, and it is considered one of the most potent neutralizing antibodies isolated by phage display, as it can neutralize about 40% of known hiv-1 isolates (li et al., 2017; mantis et al., 2007; haussner et al., 2017; wu et al., 2009 ). m43, m44 and b12 can be considered standard antibodies isolated by phage display; however the potencies are much lower than antibodies isolated by micro neutralization, such as pg9/16 and/or 10e8. phage display selects antibodies in vitro, and antibodies expressed in phage system may not necessarily represent those in mammalian cells due to the difference in protein folding and post transcriptional modifications. it is difficult to draw direct conclusions and gain an understanding of the natural occurrence of antibody heavy and light chain selection based on phage display alone (zhang et al., 2003) . to avoid these limitations, other display methods have been developed to display antibodies on the surface of yeast or mammalian cells, which can be sorted by flow cytometry according to antigen specificity. this is known as yeast surface display, which has become a powerful engineering tool for displaying recombinant proteins on the surface of saccharomyces cerevisiae via genetic fusion (traxlmayr and shusta, 2017; mei et al., 2017; andreu and del olmo, 2017; sheehan and marasco, 2015; gera et al., 2013; boder et al., 2012) . yeast surface display is a eukaryotic expression system with the capability to induce post translational modifications on recombinant antibodies. each yeast cell expresses fusion recombinant proteins which allows for the application of cell sorting for a specific antigen. yeast display system was not only reported used for isolation of antibodies, but also reported to be used in displaying antigens and other recombinant proteins (srivastava et al., 2013; guo et al., 2015) . there is a panel of yeast display antigen library which has been constructed, including yeast display h1n1 antigen epitope library, and yeast display siv antigen epitope library for epitope mapping. hiv-1 specific antibodies isolated by display techniques are less potent than those isolated by micro neutralization or single b cell sorting and cloning. however in recent studies, phage/yeast display showed great potentials in isolating single domain antibodies from recombinant libraries. single-domain antibodies are considered a separate class of antibodies composed of antibody fragments consisting of a single monomeric antibody domain. single-domain antibodies allow a broad range of applications due to their small molecular mass and size, efficient production, and high affinity. single domain antibodies can be labeled using fluorescent molecules for diagnostic purpose or biotechnological usage. with the conjunction of drug or toxin, single domain antibodies can also be used for therapeutic application. the most well-studied single domain antibodies are antibody heavy-chain variable domains. the dna sequence will be optimized in order to improve the stability. in addition to heavy-chain variable domains, there are other types of single domain antibodies which have been studied, including ch2 antibodies, and light-chain variable domain antibodies, and some other forms of nanobodies (duarte et al., 2016; li et al., 2016; gong et al., 2016; louis et al., 2014; lulf et al., 2014; bouchet et al., 2012; bouchet et al., 2011; boudet et al., 1995) . mammalian cell display is a powerful method for the isolation of antibodies in scfv format or full length igg with high affinity. it has been shown that single chain antibodies can be displayed on the surface of human hek-293t cells and used for affinity maturation (soga et al., 2015; tomimatsu et al., 2013; beerli et al., 2008) . mammalian cell expression system ensures that all of the cellular components will be involved in process of antibody synthesis. isolation of human antibodies by mammalian cell display can benefit from the advantages of mammalian cell expression system.mammalian cell display relies on the transfection of antibody expression vectors. in comparison with the yeast display system, mammalian cell expression system allows multiple recombinant protein/antibody delivering vectors in one cell. thus a further modification, enrichment, and single clone isolation will be needed for panning purposes (fig. 1c ). an important advance in antibody screening and cloning technology is the development of single cell sorting and single cell reverse transcription pcr (ouisse et al., 2017; evans et al., 2015; battye et al., 2000) . human single b cells with different antigen specificities can be sorted by flow cytometer directly, and the original vh and vl pairing of the antibody from single human b cells can be amplified in a high efficient way, with the requirement of relatively few cell numbers. this methodology is quite efficient in obtaining antibody heavy and light chain from extremely rare and highly discrete b cell subpopulations (fig. 1d) . memory b cells and plasma cells are ideal cell types for the purpose of monoclonal antibody cloning due to the specificity of the cell types. this method is always combined with single cell cloning after flow cytometry based single cell sorting to sort the antigen specific b cells, thus accurate probe design for sorting is essential. the gp120 or gp41 proteins are not ideal probes, because they are reactive with many hiv-1 antibodies, including non-neutralizing but strong binding antibodies (wu et al., 2010; wu et al., 2015; zhou et al., 2015) . one successful example of probe design in fishing potent broadly neutralizing hiv-1 monoclonal antibodies is the agents' pair of rsc3 and δrsc3. rsc3 and δrsc3 are computer-assisted designed for sorting of individual b cells expressing cd4bs antibodies. the resurfaced stabilized core 3 (rsc3) is a functional structure core of the cd4 binding site with loop deletion. rsc3 preserved the antigenic structure of the cd4 binding site and eliminated other antigenic regions by substitution with simian immunodeficiency virus (siv) or non-hiv-1 residues. δrsc3 contains one amino acid deletion (at position 371) in rsc3. this single amino acid mutation can knock out the function of cd4 binding site. cd4 binding site antibodies like vrc01 and b12 can bind efficiently with rsc3 but fail to bind with δrsc3. potent broadly neutralizing antibody vrc01 is isolated through the single b cell positive sorting by rsc3 and negative sorting by δrsc3. vrc01 neutralized up to 90% of the tested pseudoviruses with an ic50 below 50 μg/ml, and 70% of the tested viruses with an ic50 values below 1 ug/ml (wu et al., 2010; wu et al., 2015; mccoy and burton, 2017) . bg18, ioma are another two successful bnmabs isolated by reasonable probe design and single cell sorting/cloning (gristick et al., 2016; freund et al., 2017) . n-glycans on the trimeric envelope glycoprotein (env) can be accommodated by broadly neutralizing antibodies. an engineered crystal structures of the hiv-1 env trimer with an exposed native glycan shield of high-mannose and complex-type nz. sun et al. virus research 243 (2018) 75-82 glycans was used to fishing out the bnmabs. ioma, a new cd4-binding site (cd4bs) antibody was thus defined. the heavy chain of ioma derives from vh1-2*02, which is considered the germline gene of vrc01class bnabs, but its light chain lacks the short cdrl3 which defines vrc01-class bnabs. bg18, bg1, and nc37 are three bnabs isolated by single igg+ b cell sorting by using four different fishing agents in four separate sorts: 2cc core, gp140yu2, a 1:1 mixture of gp14092ug37.8 (clade a) + gp140cza79012 (clade c), and bg505 sosip.664 (sok et al., 2014; dey et al., 2009; yang et al., 2000; sanders et al., 2013; kovacs et al., 2012) . antibody bg18 (vh4-4 and vl3-25) targeted the glycan-v3 portion of envelope, can neutralize 64% of viruses in a 118virus panel tested with a geometric mean ic50 of 0.03 mg/ml. antibodies nc37 (vh1-46/1-2, vk3-20) and bg1 showed geometric mean ic50 of 0.3 and 0.67 mg/ml respectively. nc37 can bind to a quaternary trimer epitope, the core of which overlaps with the cd4bs, and bg1 binds to the v1v2 region. antigen baiting and fishing has proven quite successful in isolating hiv-specific human monoclonal igg expressing b cells and igm expressing b cells from infected donors using cleverly engineered antigen probes. flow cytometry based single cell sorting and cloning can also be applied in understanding the natural selection of antibodies by cloning of antibodies in b cells from different infection stage from one donor or b cells in the same stage from different donors. 454 deep sequencing allows for the study of the maturation of a specific antibody population and corresponding b cell subpopulations by the stimulation of virus isolates in progressingstages of infection (morris et al., 2017; banerjee et al., 2017; carter et al., 2015; karlsson hedestam et al., 2017; soto et al., 2016; stramer et al., 2016; zhu et al., 2013; zhu et al., 2012) . the difficulty in eliciting bnabs in immunized animals has been concluded (walker et al., 2011; sanders et al., 2013; sok et al., 2017) . the enormous antigenic diversity of the envelope glycoprotein and the n-linked glycan shield are considered as two major points. recent study shows that success of rapid elicitation of broadly neutralizing antibodies to hiv-1 by immunization in cows. jr-fl gp120 and bg505 sosip are immunized to cows, and strong immune response is observed. and broadly neutralizing antibodies were isolated from peripheral blood mononuclear cells (pbmcs) by sorting with biotinylated bg505 sosip. the broadly neutralizing antibody nc-cow1 displayed a 72% neutralization breadth with a potent median ic50 of 0.028 μg /ml on a 117-virus panel tested.this study gives lights in investigating the generation of antibodies against pathogens that have evolved to avoid human antibody responses in animals. in recent decades, the technology advances have allowed for specific human antibodies to be isolated directly from human b cells and/ or from recombinant libraries. these isolated human monoclonal antibodies can be used in therapy because of their safety, efficiency, specificity and tolerance in humans; thus are a powerful tool in fighting against pathogens. among the developed antibody isolation methods over the last decade, it is difficult to highlight one as the sole preferred method. different methodology has different advantages. display techniques basing on recombinant library can be used for isolating single domain antibodies with enlarged library size. but this technique cannot provide us the information of the original pair of heavy chain and light chain of a specific antibody. single b cell sorting and culturing facilitate the rapid cloning of potent neutralizing antibodies with the requirement of large labor cost. reasonable design of probes and the development of native-like stabilized trimers have greatly improved efficiency in screening of functional envelope-binding b cells. thus it is hard to conclude the best way to generate novel bnabs. combined methods can be used in screening and isolating potent neutralizing antibodies in order to maximize the advantages of different methods. meanwhile, the development of novel methodology of broadly neutralizing antibodies isolation and screening are encouraged to broaden our knowledge and enlarge the recourses of antibody classes. a purposed method basing on single cell sorting and viral neutralization instead of fishing agents should provide a direct and efficient way to minimum the workload. in addition to passive treatment and presentation, another important purpose of isolation and characterization of potent broadly neutralizing antibodies is the functional study of neutralizing epitopes which provides key knowledge for efficient vaccine design. human broadly neutralizing antibodies serve as a potential source of discovering neutralizing epitopes that can be targeted to fight against many infectious diseases and can thus facilitate the design of the vaccines. antibody structure studies can provide new insights into the mechanism of recognition of immune evasion epitopes at the atomic level, and how antibodies mature to capture the virus mutant (morales et al., 2016; wibmer et al., 2017; rujas et al., 2016) . however in recent reports, few immunogen candidates were reported that can elicit potent broadly neutralizing antibodies in several immunization studies. native-like hiv-1 envelope trimers bg505 sosip.664 and b41 sosip.664 are reported to induce neutralizing antibodies against the tier-2 virus in rabbits, even though the potency is much weaker in comparison to the broadly neutralizing antibodies isolated from human long term nonprogressors (sanders et al., 2015; bradley et al., 2016) .pre-stimulation of b cells by heterogenous epitopes or antigens showed improved stimulation of b cells in generating potent neutralizing antibodies (yang et al., 2015) . it has also been reported that pgt121-class germlinetargeting stabilized trimer immunogens primed pgt121-like responses in pgt121 inferred-germline knock in mice, highlighting the hypothesis of prime-boosting and sequential immunization strategy (steichen et al., 2016) . all these observations highlight the neutralizing antibodies' role in hiv-1 prevention. altogether, understanding antibody generation and function mechanisms can help to guide efficient vaccine design and produce effective therapy. not applicable. not applicable. not applicable. no. not applicable. the authors claimed no interest. ammonium ions improve the survival of glutaminestarved hybridoma cells isolation and characterization of anti ror1 single chain fragment variable antibodies using phage display technique development of a new yeast surface display system based on spi1 as an anchor protein passive immunization with the anti-hiv-1 human monoclonal antibody (hmab) 4e10 and the hmab combination 4e10/2f5/2g12 global structure of hiv-1 neutralizing antibody igg1 b12 is asymmetric evaluation of a novel multi-immunogen vaccine strategy for targeting 4e10/10e8 neutralizing epitopes on hiv-1 gp41 membrane proximal external region improved efficiency of ebv transformation of b-lymphocytes single cell sorting and cloning isolation of human monoclonal antibodies by mammalian cell display antibody immunoprophylaxis and immunotherapy for influenza virus infection: utilization of monoclonal or polyclonal antibodies? hybridoma cell-culture and glycan profile dataset at various bioreactor conditions humanized monoclonal antibody alemtuzumab treatment in transplant hiv-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the ccr-5 co-receptor identification and characterization of uk-201844: a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing engineering antibodies by yeast display anti-human immunodeficiency virus type 1 human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries inhibition of the nef regulatory protein of hiv-1 by a singledomain antibody single-domain antibody-sh3 fusions for efficient neutralization of hiv-1 nef functions single peptide and anti-idiotype based immunizations can broaden the antibody response against the variable v3 domain of hiv-1 in mice structural constraints of vaccine-induced tier-2 autologous hiv neutralizing antibodies targeting the receptor-binding site cross-group neutralization of hiv-1 and evidence for conservation of the pg9/pg16 epitopes within divergent groups diverse hiv-1 subtypes and clinical: laboratory and behavioral factors in a recently infected us military cohort generation of human monoclonal antibodies against hiv-1 proteins; electrofusion and epstein-barr virus transformation for peripheral blood lymphocyte immortalization emergence of monoclonal antibody b12-resistant human immunodeficiency virus type 1 variants during natural infection in the absence of humoral or cellular immune pressure production of lacz inducible t cell hybridoma specific for human and mouse hiv-1 neutralizing antibody response and viral genetic diversity characterized with next generation sequencing human recombinant antibodies specific for hepatitis c virus core and envelope e2 peptides from an immune phage display library antibody humanization by structure-based computational protein design cell-to-cell transfer of hiv-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion binding interactions between soluble hiv envelope glycoproteins and quaternary-structure-specific monoclonal antibodies pg9 and pg16 three-dimensional structure-activity analysis of a series of porphyrin derivatives with anti-hiv-1 activity targeted to the v3 loop of the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 fluorescent protein-tagged vpr dissociates from hiv-1 core after viral fusion and rapidly enters the cell nucleus structure-based stabilization of hiv-1 gp120 enhances humoral immune responses to the induced co-receptor binding site a nonself sugar mimic of the hiv glycan shield shows enhanced antigenicity two classes of broadly neutralizing antibodies within a single lineage directed to the high-mannose patch of hiv envelope generation of immunity against pathogens via single-domain antibody-antigen constructs measuring t cell-to-t cell hiv-1 transfer, viral fusion, and infection using flow cytometry activity of broadly neutralizing antibodies: including pg9, pg16, and vrc01, against recently transmitted subtype b hiv-1 variants from early and late in the epidemic assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging broadly neutralizing hiv antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers phage display: a powerful technology for the generation of highspecificity affinity reagents from alternative immune sources coexistence of potent hiv-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller antibodies vrc01 and 10e8 neutralize hiv-1 with high breadth and potency even with ig-framework regions substantially reverted to germline protein selection using yeast surface display specific determination of influenza h7n2 virus based on biotinylated single-domain antibody from a phage-displayed library selection of recombinant anti-hud fab fragments from a phage display antibody library of a lung cancer patient with paraneoplastic encephalomyelitis natively glycosylated hiv-1 env structure reveals new mode for antibody recognition of the cd4-binding site simian immunodeficiency virus infection evades vaccine-elicited antibody responses to v2 region successful treatment with humanized anti-interleukin-6 receptor antibody (tocilizumab) in a case of aa amyloidosis complicated by familial mediterranean fever peptide paratope mimics of the broadly neutralizing hiv-1 antibody b12 the quest for an antibody-based hiv vaccine effect of medium composition on hybridoma growth and antibody production generation of murine monoclonal antibodies by hybridoma technology a human hybrid hybridoma producing a bispecific monoclonal antibody that can target tumor cells for attack by pseudomonas aeruginosa exotoxin a broad and potent neutralization of hiv-1 by a gp41-specific human antibody the meaning of the anti-cancer antibody cln-igg (pritumumab) generated by human x human hybridoma technology against the cyto-skeletal protein: vimentin, in the course of the treatment of malignancy defucosylated anti-ccr4 monoclonal antibody exercises potent adcc-mediated antitumor effect in the novel tumor-bearing humanized nod/shiscid: il-2rgamma(null) mouse model continuous cultures of fused cells secreting antibody of predefined specificity evolution of b cell analysis and env trimer redesign ebv lytic infection enhances transformation of b-lymphocytes infected with ebv in the presence of t-lymphocytes production and characterization of novel camel single domain antibody targeting mouse vascular endothelial growth factor impact on monoclonal antibody production in murine hybridoma cell cultures of adenosine receptor antagonists and phosphodiesterase inhibitors ebv-b cell interactions: immortalization, rescue from apoptosis, tumorigenicity (a short review) hiv-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120 versatility of using major histocompatibility complex class ii dextramers for derivation and characterization of antigen-specific, autoreactive t cell bcl6 cooperates with cd40 stimulation and loss of p53 function to rapidly transform primary b cells corrigendum: generation of stable monoclonal antibodyproducing b cell receptor-positive human memory b cells by genetic programming immunotherapy with human monoclonal antibodies: fragment a specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin microrna signatures associated with immortalization of ebvtransformed lymphoblastoid cell lines and their clinical traits human antibodies for immunotherapy development generated via a human b cell hybridoma technology a single-domain antibody-linked fab bispecific antibody her2-s-fab has potent cytotoxicity against her2-expressing tumor cells structural analysis of the glycosylated intact hiv-1 gp120-b12 antibody complex using hydroxyl radical protein footprinting immunogenicity of constrained monoclonal antibody a32-human immunodeficiency virus (hiv) env gp120 complexes compared to that of recombinant hiv type 1 gp120 envelope glycoproteins effect of interleukin (il)-12 and il-15 on activated natural killer (ank) and antibody-dependent cellular cytotoxicity (adcc) in hiv infection a rapid method to characterize mouse igg antibodies and isolate native antigen binding igg b cell hybridomas binding of hiv-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (fab) and single chain variable fragment (scfv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations generation of immortalized human na#xp#ve b cell libraries by optimized ebv transformation structural basis for the inhibition of hiv-1 nef by a high-affinity binding single-domain antibody effect of human adenovirus on antibody-dependent cellular cytotoxicity (adcc) in chickens inhibition of hiv-1 infectivity and epithelial cell transfer by human monoclonal igg and iga antibodies carrying the b12 v region extracting human antibody sequences from public databases for antibody humanization: high frequency of species assignment errors enhanced monoclonal antibody production in hybridoma cells by lps and anti-migg identification and specificity of broadly neutralizing antibodies against hiv ebv-directed t cell therapeutics for ebv-associated lymphomas neutralization profiles of newly transmitted human immunodeficiency virus type 1 by monoclonal antibodies 2g12: 2f5, and 4e10 application of modified yeast surface display technologies for non-antibody protein engineering requirement of calmodulin binding by hiv-1 gp160 for enhanced fas-mediated apoptosis the hybridoma revolution: an offshoot of basic research towards a structure of the hiv-1 envelope glycoprotein gp120: an immunochemical approach fragments of the v1/v2 domain of hiv-1 glycoprotein 120 engineered for improved binding to the broadly neutralizing pg9 antibody effectiveness of intranasal immunization with hiv-gp160 and an hiv-1 env ctl epitope peptide (e7) in combination with the mucosal adjuvant lt(r192g) mabgel 1: first phase 1 trial of the anti-hiv-1 monoclonal antibodies 2f5, 4e10 and 2g12 as a vaginal microbicide differential antibody responses to conserved hiv-1 neutralizing epitopes in the context of multivalent scaffolds and native-like gp140 trimers reappraisal of ebv in diffuse large b-cell lymphoma (dlbcl): comparative analysis between ebv-positive and −negative dlbcl with ebv-positive bystander cells humanization of mouse ons-m21 antibody with the aid of hybrid variable regions tabhu: tools for antibody humanization antigen-specific single b cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies crystal structure of pg16 and chimeric dissection with somatically related pg9: structure-function analysis of two quaternary-specific antibodies that effectively neutralize hiv-1 administration of nucleoside-modified mrna encoding broadly neutralizing antibody protects humanized mice from hiv-1 challenge generation and characterization of a novel fusion partner cell line for the production of human macrophage hybridoma bisfabs: tools for rapidly screening hybridoma iggs for their activities as bispecific antibodies use of broadly neutralizing antibodies for hiv-1 prevention identification of human anti-hiv gp160 monoclonal antibodies that make effective immunotoxins design and in vivo characterization of immunoconjugates targeting hiv gp160 immunogenomic engineering of a plug-and-(dis)play hybridoma platform influence of the envelope gp120 phe 43 cavity on hiv-1 sensitivity to adcc responses evolution of phage display technology: from discovery to application peptides derived from the cdr3-homologous domain of the cd4 molecule are specific inhibitors of hiv-1 and siv infection, virus-induced cell fusion, and postinfection viral transmission in vitro. implications for the design of small peptide anti-hiv therapeutic agents subtle alteration of residues including nlinked glycans in v2 loop modulate hiv-1 neutralization by pg9 and pg16 monoclonal antibodies cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates fusion of higg1-fc to 111in-anti-amyloid single domain antibody fragment vhh-pa2h prolongs blood residential time in app/ps1 mice but does not increase brain uptake structural basis for broad neutralization of hiv-1 through the molecular recognition of 10e8 helical epitope at the membrane interface cd40 is a potential marker of favorable prognosis in patients with diffuse large b-cell lymphoma treated with immunochemotherapy antibody humanization methods − a review and update a next-generation cleaved, soluble hiv-1 env trimer, bg505 sosip.664 gp140, expresses multiple epitopes for broadly neutralizing but not nonneutralizing antibodies hiv-1 vaccines: hiv-1 neutralizing antibodies induced by native-like envelope trimers sequence and structural convergence of broad and potent hiv antibodies that mimic cd4 binding hiv-specific b cell response in patients with broadly neutralizing serum activity global gene expression changes of in vitro stimulated human transformed germinal centre b cells as surrogate for oncogenic pathway activation in individual aggressive b cell lymphomas phage and yeast display large-scale expansion of dendritic cell-primed polyclonal human cytotoxic t-lymphocyte lines using lymphoblastoid cell lines for adoptive immunotherapy mammalian cell surface display as a novel method for developing engineered lectins with novel characteristics recombinant hiv envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex rapid elicitation of broadly neutralizing antibodies to hiv by immunization in cows developmental pathway of the mper-directed hiv-1-neutralizing antibody 10e8 identification of dominant antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin antigen of pandemic h1n1 influenza virus hiv vaccine design to target germline precursors of glycandependent broadly neutralizing antibodies preserved antiviral adaptive immunity following polyclonal antibody immunotherapy for severe murine influenza infection quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging two human immunodeficiency virus type 2 cases in us blood donors including serologic, molecular, and genomic characterization of an epidemiologically unusual case immortalization of ebv-infected b cells is not influenced by exogenous signals acting on b cell proliferation. effects of mutant el-4 thymoma cells and transforming growth factor-beta reconstitution and characterization of antibody repertoires of hiv-1-infected elite neutralizers isolation and characterization of hiv-1 envelope glycoprotein specific b cell from immortalized human naive b cell library use of hca in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns a human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface naturally occurring substitutions of conserved residues in human immunodeficiency virus type 1 variants of different clades are involved in pg9 and pg16 resistance to neutralization in vitro glycoengineering of igg1 and its effect on fc receptor binding and adcc activity efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning a rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies hybridoma technologies for antibody production an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus directed evolution of protein thermal stability using yeast surface display humanization of a chicken anti-il-12 monoclonal antibody broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target broad neutralization coverage of hiv by multiple highly potent antibodies optimizing adoptive polyclonal t cell immunotherapy of lymphomas: using a chimeric t cell receptor possessing cd28 and cd137 costimulatory domains hiv-1 gp140 epitope recognition is influenced by immunoglobulin dh gene segment sequence the hiv-1 envelope glycoprotein structure: nailing down a moving target resistance of human immunodeficiency virus type 1 to neutralization by natural antisera occurs through single amino acid substitutions that cause changes in antibody binding at multiple sites structure and recognition of a novel hiv-1 gp120-gp41 interface antibody that caused mper exposure through viral escape immunodominance of antibody recognition of the hiv envelope v2 region in ig-humanized mice interaction of chemokine receptor ccr5 with its ligands: multiple domains for hiv-1 gp120 binding and a single domain for chemokine binding mechanism of human immunodeficiency virus type 1 resistance to monoclonal antibody b12 that effectively targets the site of cd4 attachment rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1 maturation and diversity of the vrc01-antibody lineage over 15 years of chronic hiv-1 infection characterization of stable: soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins identification of non-hiv immunogens that bind to germline b12 predecessors and prime for elicitation of cross-clade neutralizing hiv-1 antibodies broadly cross-reactive hiv neutralizing human monoclonal antibody fab selected by sequential antigen panning of a phage display library identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody identification of a novel cd4i human monoclonal antibody fab that neutralizes hiv-1 primary isolates from different clades selection of a novel gp41-specific hiv-1 neutralizing human antibody by competitive antigen panning cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens identification and characterization of a broadly cross-reactive hiv-1 human monoclonal antibody that binds to both gp120 and gp41 structural basis for broad and potent neutralization of hiv-1 by antibody vrc01 multidonor analysis reveals structural elements: genetic determinants, and maturation pathway for hiv-1 neutralization by vrc01-class antibodies structural repertoire of hiv-1-neutralizing antibodies targeting the cd4 supersite in 14 donors somatic populations of pgt135-137 hiv-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatics de novo identification of vrc01 class hiv-1-neutralizing antibodies by next-generation sequencing of b-cell transcripts a novel human antibody against human immunodeficiency virus type 1 gp120 is v1: v2, and v3 loop dependent and helps delimit the epitope of the broadly neutralizing antibody immunoglobulin g1 b12 we wish to thank dr. mei-yun zhang, dr. zhiwei chen (the university of hong kong), and dr. paul zhou (institute pasteur of shanghai cas) for helpful discussion.z. sun et al. virus research 243 (2018) 75-82 key: cord-312167-d16ylykc authors: lazzarin, serena marita; cannizzaro, miryam; russo, tommaso; sangalli, francesca; callea, marcella; colombo, bruno; moiola, lucia; filippi, massimo title: successful treatment of hiv-associated tumefactive demyelinating lesions with corticosteroids and cyclophosphamide: a case report date: 2020-11-03 journal: j neurol doi: 10.1007/s00415-020-10296-6 sha: doc_id: 312167 cord_uid: d16ylykc nan and onconeural antibodies was negative. search for vzv, cmv, ebv, syphilis (tpha), hcv, hbv, m. tuberculosis was negative too. a 4th-generation hiv test was positive, subsequently confirmed by western blotting and polymerase chain reaction, showing plasma hiv-1-rna levels of 87,900 copies/ml; serum cd4 count was 290/µl. additional serological tests for toxoplasmosis and cryptococcosis were negative. combined antiretroviral therapy (cart) with tenofovir alafenamide, emtricitabine and bictegravir was started. histology from stereotaxic biopsy of the right frontal lobe lesion demonstrated several areas of demyelination. pcr for jc virus and toxoplasma gondii on biopsy was negative, as well as mycobacterium tuberculosis complex and acidalcohol-resistant bacilli tests (fig. 1) . neuroradiological and pathological findings were suggestive of tumefactive demyelinating lesions (tdls). intravenous 1-g methylprednisolone was administered for 7 days. two days after the last dose, a follow-up brain mri showed dimensional decrease of both frontal lesions, with disappearance of mass effect; gadolinium enhancement was substantially decreased (fig. 1 ). however, a new small capsulo-lenticular lesion with analogous features was noted. lumbar puncture showed mild lymphocytic pleocytosis (14/ µl), proteinorachia (84 mg/dl) and no oligoclonal bands; csf glucose was 58 mg/dl. csf was negative for infectious agents, including pcr for jc virus; csf hiv-rna count was 90 copies/ml. due to the persistent inflammatory activity observed at brain mri, a single dose of cyclophosphamide (800 mg/m 2 i.v.) was administered, with benefit. at hospital discharge, the patient was oriented, with mild persisting memory deficits and sporadic anomia. at 3-month follow-up mri, a marked reduction of flair hyperintensities and absence of post-contrast enhancement were observed (fig. 1 ). neurological examination was normal, as well as cognitive function. a clinical and neuroradiological 6-month follow-up has been scheduled to assess the stability of patient's condition; [4] , but we cannot even exclude a direct role for hiv in the demyelinating process. the co-occurrence of hiv infection and tdls implies obvious concerns about the treatment of this particular condition. intravenous high-dose steroid treatment was reported to be successful in sparse cases [2, 3] . however, in literature, no data are available for steroid-unresponsive tdls in hiv patients [5] . given the concern to expose an hiv patient to prolonged lymphopenia, we chose cyclophosphamide to induce a quick-onset and short-duration immunosuppressive effect. we planned to administer a pulse regimen of 800 mg/m 2 every 4 weeks. however, due to the sars-cov-2 pandemic, the further doses were not administered. nevertheless, after 5 months from the first dose, we observed a normalization of neurological examination and a significant improvement of neuroradiological findings. in conclusion, in our case quick initiation of cart and cyclophosphamide led to a significant clinical and radiological amelioration, likely secondary to the restoration of para-physiological immune function. based on our experience, a treatment with cyclophosphamide may be a valid alternative in steroid-resistant hiv patients with tdls. author contributions sml contributed to paper conception, data collection, analysis and interpretation, literature review and paper drafting. cm and tr contributed to data collection, analysis and interpretation, literature review and paper drafting. sf, cm, cb and ml contributed to data collection, analysis and critical review. fm contributed to paper conception, supervised data analysis and interpretation, critically reviewed the paper. funding not applicable. ethics approval the ethics committee of san raffaele scientific institute waived the need for ethics approval for publication of case reports. consent to participate written informed consent was obtained from the patient's spouse for the usage of clinical data in anonymized fashion. consent for publication informed consent for publication was obtained verbally from the spouse. the consent was audio-recorded in the presence of an independent witness. fig. 1 (1) serial 1.5-t mri axial brain scans, performed after admission (a), at one (b) and 3 months (c) after the clinical onset of a frontal lobe syndrome due to large tumefactive demyelinating lesions. (2) a-d images of brain parenchyma sample from stereotactic biopsies, haematoxylin and eosin-stained section showing increased cellularity due to the presence of numerous intraparenchymal foamy macrophages (a, ×40); those cells are highlighted by anti-cd163 antibody (b, ×40). kluver-barrera staining shows loss of myelin (c and d, ×20). e-g brain parenchyma sample with perivascular cuff is composed by mononuclear inflammatory cells stained with haematoxylin and eosin (e, ×40); perivascular cuff is made of macrophages immunoreactive for anti-cd163 antibody (f, ×40) and by t lymphocytes highlighted by anti-cd3 antibody (g, ×20). the fab: a frontal assessment battery at bedside tumefactive demyelination in a patient with human immunodeficiency virus tumefactive demyelination-an unusual neurological presentation of hiv hiv and autoimmunity tumefactive demyelinating lesions: a comprehensive review key: cord-285505-8norumv6 authors: vere hodge, r. anthony title: meeting report: 27th international conference on antiviral research, in raleigh, nc, usa date: 2014-09-16 journal: antiviral res doi: 10.1016/j.antiviral.2014.08.009 sha: doc_id: 285505 cord_uid: 8norumv6 the 27th international conference on antiviral research (icar) was held in raleigh, north carolina, usa from may 12 to 16, 2014. this article summarizes the principal invited lectures. john drach (elion award) described the early days of antiviral drugs and their novel modes of action. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept nucleoside analogs. replacing thymine by 5-chlorouracil led to the generation of a new form of escherichia coli. adrian ray (prusoff award) demonstrated how prodrugs can markedly improve both the efficacy and safety of potential drugs. the keynote addresses, by david margolis and myron cohen, tackled two emerging areas of hiv research, to find an hiv “cure” and to prevent hiv transmission, respectively. these topics were discussed further in other presentations – a cure seems to be a distant prospect but there are exciting developments for reducing hiv transmission. tdf-containing vaginal rings and gsk-744, as a long-lasting injection, offer great hope. there were three mini-symposia. although therapy with tdf/ftc gives excellent control of hbv replication, there are only a few patients who achieve a functional cure. myrcludex, an entry inhibitor, is active against both hbv and hdv. the recent progress with hbv replication in cell cultures has transformed the search for new antiviral compounds. the hbv capsid protein has been recognized as key player in hbv dna synthesis. unexpectedly, compounds which enhance capsid formation, markedly reduce hbv dna synthesis. the development of bcx4430, which is active against marburg and ebola viruses, is of great current interest. this article provides an overview of the invited lectures at the 27th international conference on antiviral research, sponsored by the international society for antiviral research (isar), which was held in raleigh, north carolina, usa from may 12 to 16, 2014 . it begins with reports of lectures by the recipients of isar's three major awards, held in memory of gertrude elion, antonín holý and william prusoff. these are followed by brief summaries of the keynote addresses and the three mini-symposia on ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. because this review article simply provides short accounts of oral presentations, it is not generally accompanied by references to the scientific literature. any descriptions of favorable treatment outcomes should not be taken as recommendations for clinical use. 2. gertrude elion memorial award lecture: collaborative antiviral studies for the discovery of drugs to treat cytomegalovirus infections john c. drach, ph.d., university of michigan, ann arbor, michigan, usa (fig. 1) . gertrude b. (trudy) elion was born in new york city and was pleased to work for the burroughs wellcome co. when based in new york but was concerned when it transferred to research triangle park, north carolina, not many miles from this year's meeting site. however, within just a few months she declared that she was ''at home'' in north carolina. she was awarded the nobel prize in physiology or medicine in 1988 for her pioneering work in purine biosynthesis which paved the way for the discovery of drugs to treat organ rejection, cancer and viral diseases. the focus of john's presentation was on the research conducted in his own and his collaborators' laboratories that ultimately led to the invention of three compounds which were discovered to have antiviral activity against human cytomegalovirus (hcmv) and which later entered clinical trials: bdcrb pyranoside (gw275175x) (phase i), maribavir (phases i, ii and iii) and cyclopropavir (phase i). his major collaborators included karen biron, charles shipman, leroy townsend, and jiri zemlicka. to date, there are only five fdaapproved drugs for treatment of hcmv infections: cidofovir, fomivirsen, foscarnet, ganciclovir and valganciclovir. being inspired by the presence of a naturally-occurring 5,6dimethylbenzimidazole nucleotide in vitamin b12, research on benzimidazole nucleosides was initiated by medicinal chemists in the 1950s and '60s. this led to the synthesis of a trichloro analog in townsend's laboratory at the university of utah and later the discovery of its activity against hcmv in john's laboratory. much work, in both their laboratories at the university of michigan, established that it and its 2-bromo analog (bdcrb) have excellent activity against hcmv with very low cytotoxicity. surprisingly, it was found to be inactive against other herpes viruses and it did not need conversion to a triphosphate to be active against hcmv. collaborative studies with karen biron at burroughs wellcome established that, unlike many other anti-virals that inhibit viral dna synthesis such as ganciclovir (gcv), these compounds acted by a novel mechanism, inhibition of viral dna processing. it was the viral resistance studies which revealed the viral targets, pul89 and pul56. these two proteins, with pul104, form a complex known as the terminase which cuts newly synthesised hcmv dna into unit lengths for packaging into virions. although bdcrb had many desirable properties in vitro, it had poor pharmacokinetics in mice and monkeys due to hydrolysis of its glycosidic bond; therefore it was not developed for human use. much additional work in drach's and townsend's laboratories at michigan and by biron's group at burroughs wellcome ultimately led to two potential drug candidates, bdcrb pyranoside and maribavir (fig. 2) . both compounds have excellent activity against hcmv, low toxicity, and excellent pharmacokinetics. clearly, their modes of action differed markedly from that of gcv. quite unexpectedly, they have different mechanisms of action. bdcrb pyranoside has a mechanism of action very similar to its parent compound bdcrb, inhibition of dna processing. in contrast, maribavir inhibits dna synthesis, albeit indirectly. it is a 2isopropylamine derivative of bdcrb except that it has the unnatural l-sugar configuration. its mechanism of action involves inhibition of the viral kinase (pul97), which phosphorylates another viral protein, pul44. phosphorylated pul44 is necessary for viral dna synthesis. thus inhibition of pul97 by maribavir inhibits viral dna synthesis. interestingly, pul97 is also the kinase that activates (phosphorylates) gcv. resistance studies confirmed that a single mutation in ul97, resulting in a mutation in the kinase (leu397arg), was necessary and sufficient for resistance to maribavir. in a further study of resistance, virus already resistant to bdcrb was passaged in increasing concentrations of maribavir and resistant virus was isolated. this strain grew at the same rate as the wild-type virus and was resistant to both bdcrb and maribavir. as expected, resistance to bdcrb was due to known mutations in ul56 and ul89. however, no mutations were found in ul97. further investigation showed that a single base change in ul27 (t1004c) was necessary and sufficient for resistance to maribavir. the role of the encoded protein was then unknown but the amino acid mutation (leu335pro) is in the middle of the protein. similarly, biron's group detected resistance due to mutations in the ul27 gene. further research studies on maribavir have been summarized in previous icar scientific reports. cyclopropavir (cpv, fig. 3 ) was synthesized in the laboratory of jiri zemlicka, karmanos cancer institute, detroit, michigan. it is a guanosine nucleoside analog which is very active against hcmv. unlike the benzimidazole nucleosides, it also inhibits epstein-barr virus (ebv) and human herpesvirus 8 (hhv-8). like gcv, it is phosphorylated by the kinase encoded by ul97. it is more potent in vitro and in vivo than ganciclovir but has a somewhat different pattern of resistance. in one resistant strain, the key mutation formed a stop codon resulting in a truncated pul97 kinase protein. the phosphorylation of cpv by pul97 is more efficient than that of gcv, with a considerably lower k m and higher v max . interestingly, the phosphorylation of cpv to its monophosphate (cpv-mp) by pul97 is stereoselective; only the (+) isomer of cpv-mp is formed. a single enzyme, gmp kinase, phosphorylates cpm-mp to both its di-and triphosphates. in contrast, acyclovir and gcv require additional cellular enzymes to convert their diphosphates to active triphosphates. cyclopropavir is currently in phase i clinical trials for the treatment of hcmv infections. 3. the antonín holý memorial award lecture: from modified nucleoside to a chemically modified genome piet herdewijn, rega institute for medical research, ku leuven, belgium (fig. 4) . the 2013 icar began with a symposium, on the legacy of the late antonín (tony) holý , at which the establishment of a new isar award in medicinal chemistry was announced. the awardee is to be a senior scientist of international stature in medicinal chemistry and who has made innovative contributions impacting antiviral drug discovery or development. piet is, therefore, the first to receive this award. in the late 1970s, the potent activities of bvdu and bvarau against herpes simplex virus type 1 (hsv-1) and varicella zoster virus (vzv) were discovered; this work motivated piet to start antiviral research with the synthesis of carbocyclic bvdu. through to the early 1990s, he synthesized several other nucleoside analogs with bicyclic bases having good activity against hsv-1 and vzv. during the 1990s, emphasis switched to investigating the effect of modifying the sugar ring, in particular the synthesis of six-membered rings containing an oxygen or a double bond. piet showed examples of compounds with activity against hsv-1, hsv-2, vzv and hcmv. back in 1984, erik de clercq showed piet a paper on aids, one of the authors being phil furman. this publication stimulated the search for anti-hiv compounds. many compounds were discovered with potent activities (and good selectivity indices) against hiv. piet worked out the first structure-activity relationships of anti-hiv dideoxy nucleosides. starting in the late 1980s, tony holý synthesised a series of phosphonates. at the 2013 icar, erik de clercq recalled how this work led, ultimately, to tenofovir, which was to become a major success for treating hiv-infected patients. from its first introduction in 2001, its market share has increased to well over 40%. in 2002, having a single-pill regimen was agreed as a way forward to simplify, and thereby enhance, hiv therapy. this led to atripla being approved in 2006, complera in 2011 and stribild in 2012. tenofovir, in its various prodrug forms, is now available in over 130 countries and is distributed widely to the known hivinfected population. in line with this research, piet synthesized phosphonate nucleosides, with a threose sugar moiety, which showed anti-hiv activity in the same range as 9-(2-phosphonylmethoxyethyl) adenine (pmea). piet's work had taken a different pathway. it is possible to link several nucleotides together to form aptamers. for example (fig. 5) , the above antiviral nucleosides, which have a 6-membered ring in place of the natural furanose, could be incorporated into hexitol nucleic acid (hna) aptamers. x-ray studies revealed the structures of hna-rna duplexes and hna-hna duplexes, the latter having a similar overall form to that of an rna-rna duplex with the same base sequence. hna-containing aptamers were shown to be potent and specific inhibitors of trans-activating region (tar)mediated transcription. normally, an hiv encoded protein, transactivator of transcription (tat), binds to cellular factors and to the viral tar rna regulatory element, resulting in a vastly increased rate of transcription of all hiv genes. hna-containing aptamers prevents this interaction and so inhibit hiv replication. it took four years to engineer a polymerase that would utilise hnas to assemble a strand complementary to a dna template. in line with this research, hexitol-modified sirna has shown good activity in an in vivo anti-hbv model. this success stimulated the concept that it may be possible to generate new forms of biologically active dna. in order to pursue this idea, a culture system with twin growth chambers was devised. alternative nutrient media could be fed into the chambers and the culture from one chamber could be used to seed the second chamber, the former culture being removed. in this example, the aim was to replace thymine with 5-chlorouracil ( fig. 6) using escherichia coli. initially, the nutrient contained 10% 5-chlorouracil and 90% thymine. with each cycle, seeding one chamber from the previous one, the proportion of 5-chlorouracil was increased. after 180 days, in which there had been about 4000 generations of e. coli, thymine had been replaced totally by 5-chlorouracil. an interesting outcome was that the alternative base led to a change not only in the genotype but also in the phenotype; the ''new'' e. coli cells were much longer than the original. this is the first example of a dna polymerase being adapted through evolutionary pressure to accept a nucleotide analog, resulting in the generation of a new living organism. prusoff young investigator award lecture: use of nucleotide prodrugs to enhance selectivity of anti-hiv and -hcv agents adrian s. ray, gilead sciences inc., foster city, ca, usa (fig. 7) . adrian started his lecture with photos of william (bill) prusoff and reminisced of his days with bill, raymond schinazi and yung-chi (tommy) cheng. adrian presented examples to illustrate two models of how a prodrug strategy can transform a potential drug into a much improved clinical candidate. in the first, the prodrug alters the distribution of the pharmacologically active nucleotide analog to tissues where viral infection is taking place (on-target) and away from tissues resulting in adverse events (off-target). in the second, the prodrug enables one to select a drug candidate based more directly on the intrinsic properties of the active nucleotide-triphosphate analog via by-passing an inefficient activation (phosphorylation) of the corresponding nucleoside analog. sofosbuvir (sovaldi ò ), a prodrug of 2 0 -f-2 0 -c-meump, was approved in the usa on 6th december, 2013 for treatment of patients with hepatitis c. this is a fine example of a prodrug enhancing the activity of the parent compound. the nucleoside analogue, 2 0 -f-2 0 -c-meu, is poorly active due to restricted phosphorylation to the monophosphate. sofosbuvir, a nucleotide analogue prodrug of 2 0 -f-2 0 -c-meu, delivers the monophosphate into the cell and this is then further phosphorylated efficiently to give high levels of the triphosphate which inhibits hcv rna polymerase. adrian recalled being much impressed by a result reported at the meeting in 2007 of the american association for the study of liver diseases (aasld). in a phase ii monotherapy trial in patients with hcv, at day 3, the viral loads were reduced by log 10 3.2 and log 10 1.1 for vx-950 (1250 mg bid, n=10) and rg-7128 (1500 mg bid, n=8), respectively. however, from day 4 to 13, the polymerase inhibitor (rg-7128) had continued to reduce the viral load, reaching a reduction of log 10 2.7. on the other hand, the protease inhibitor (vx-950) did not give a sustained reduction, with the viral load starting to increase from day 6. at day 13, the viral load was only log 10 2.2 less than baseline. nucleotide analogues have two advantages over other classes of inhibitors. there is a high genetic barrier to resistance selection, due to the hcv rna polymerase being highly specific for its natural substrates and template. this specificity can be altered but only under extreme evolutionary pressure (see section 3). also, nucleotide analogs often have pan-genotype activity because the active site of the hcv ns5b polymerase is so highly conserved. as an example of how prodrugs can impact a discovery program, allowing for more targeted delivery and for the optimization of the intrinsic properties of the triphosphate, adrian presented the history of the gs-6620 program. the c-adenine analogue (2 0 -c-me-4-aza-7,9-dideazaa, c-nuc1) was compared to the corresponding n-nucleoside, mk608. in a genotype1b replicon assay, the ec 50 values were 2.5 lm and 0.08 lm respectively. however, their triphosphates were equally effective against hcv ns5b polymerase (ic 50 values both 0.3 lm). in the replicon system, the triphosphate of the n-nuc (mk608) was formed more efficiently than that of the c-nuc1, thus explaining the lower activity of the c-nuc1. however, in primary human hepatocytes, c-nuc1 was phosphorylated to the triphosphate more efficiently than the n-nuc (mk608). this illustrates the importance of using primary human cells. c-nuc1 seemed to have a benign in vitro toxicity profile, including not inhibiting the mitochondrial dna polymerase-gamma, but it had very significant toxicity in animals. in a collaboration between gilead and craig cameron at pennsylvania state university, the researchers sought to identify the toxicity target(s) for ribonucleotide analogues, including c-nuc1 and others that had been stopped in phase ii trials. these studies showed a correlation between c-nuc1 and the phase ii candidates, r1626, nm283 and bms986094/idx184. all the latter were efficiently incorporated into rna by the mitochondrial rna polymerase (>70% of the corresponding natural nucleotide). the triphosphate of c-nuc1 was also an efficient substrate (22% the rate of atp). in contrast, the active nucleotide analogs, formed by drugs approved for the treatment of hcv, were poor substrates. ribavirin was poorly incorporated (about 5%) and sofosbuvir was below the limit of detection (= 0.02%). more extensive in vitro and cell culture evaluation of the compounds could have saved the expense of taking them into clinical trials. understanding that the mitochondrial rna polymerase is an important target for ribonucleotide toxicity, the gilead team sought analogs that were not incorporated by this polymerase. adding a cn group to the 1 0 position of c-nuc1 did not change its activity as an hcv ns5b polymerase inhibitor (ic 50 0.3 lm) but it did reduce incorporation in the mitochondrial rna assay (<0.02%). however, in the absence of a nucleotide prodrug to bypass the first phosphorylation step, the resulting di-substituted nucleoside analog would not be a drug candidate because it was not efficiently activated in cells. application of a nucleotide prodrug strategy allowed this nucleotide to be pursued further. oral absorption, delivery of the monophosphate into hepatocytes and high hepatic extraction were criteria used as part of the prodrug optimization process. a nucleotide prodrug, gs-464335 (a mixture of diastereoisomers at phosphorous) was well absorbed in dogs (>80%). comparing the pre-hepatic and post-hepatic plasma drug levels, about 80% of the absorbed drug was taken up by the liver. inside cells, gs-464335 was converted to the corresponding monophosphate which was efficiently converted to the triphosphate. at 24 h, the triphosphate levels remained about 2-fold above the ic 90 value. a pure stereoisomer was selected and later named gs-6620. in a phase ii trial (900 mg, bid 5 days), the mean reduction in hcv load was about log 10 1.5. two subjects achieved hcv rna <25 iu/ ml. however, the pharmacokinetics and antiviral responses were highly variable. whereas the activity results were disappointing, clinical proof of concept was observed in terms of safety. gs-6620 did have a markedly improved safety profile relative to c-nuc1, progressing through chronic toxicology studies in rats and dogs at relatively high doses. the story of gs-6620 illustrates both how nucleotide prodrugs enable further progression of candidates and also the complexity of predicting the behavior of nucleotide prodrugs across species. one wonders what cell culture test or animal model may have predicted such variability. when selecting famciclovir as the prodrug for penciclovir, one potential prodrug was rejected because the pharmacokinetics in rats varied widely between individual animals (vere hodge et al., 1989) . a recent publication by adrian and his team highlights the metabolism of gs-6620 by carboxylesterase 2, an enzyme highly expressed in the human small intestine but not uniformly expressed in different animal species, as a possible reason for the highly variable and suboptimal intestinal absorption of gs-6620 in humans (murakami et al., 2014) . the focus of adrian's talk then switched to hiv. over the last 15 or 20 years in north america, the hiv-infected population has been changing, becoming older (now 33% over 50 years old vs <10% in 1995) and more likely to be obese (in every usa state, >20% adults with bmip30). this has led to a shift in the focus of antiretroviral therapy (art), from solely control of hiv replication to now include tolerability in older, possibly obese, patients. the first example given for hiv was how application of a different prodrug strategy can markedly change the distribution even when delivering the same pharmacologically active nucleotide analog. the first approved prodrug of tenofovir (tfv) was tfv disoproxil fumarate (tdf). more recently, tfv alafenamide (taf) has been progressed into clinical development. a key difference in the properties of the two prodrugs is their stability in plasma, with half-lives of 0.4 and 90 min, respectively. even with a short halflife, tdf gave better delivery of tfv into cells, as indicated by the hiv ec 50 values in cell culture assays but there clearly was room for improvement; the ec 50 values for tfv, tdf and taf are 1.2, 0.015 and 0.003 lm respectively. whereas the gain in cell culture ec 50 value may be modest, this is not the only gain. the increased stability of taf allows it to load on-target cells and tissues (e.g., lymph nodes) for a longer period of time resulting in increased lymphoid cell and tissue levels at greatly reduced circulating tfv levels, leading to less exposure to off-target tissues (e.g., kidney). in monotherapy studies after oral dosing with tdf (300 mg) and taf (25 mg), the plasma tfv auc is reduced from 1920 to 268 ng.h/ml respectively whereas the reduction in hiv load from baseline is improved, from log 10 0.97 to log 10 1.46 copies/ml, respectively, reflecting the more efficient delivery of taf to target cells and tissues. clearly the lower dose of taf (25 mg) relative to tdf (300 mg) will give taf a marked advantage when considering combination pill therapy. understanding how marked a difference a prodrug can make from the taf example, adrian went on to describe how a prodrug approach transformed a new nucleotide project in which intrinsic properties of the pharmacologically-active nucleotide analog were optimized. their starting point was gs-2128 (d4api), which had good activity against both wild-type and resistant hiv strains but was an active inhibitor of mitochondrial polymerase-gamma. on comparing the known structures of hiv rt and mitochondrial polymerase-gamma, differences in the 2 0 -binding pocket were noted. this led to gs-9148 in which 2 0 -f was added to gs-2128 (fig. 8) . compared to tfv, gs-9148 was about 3-fold less active against wild-type hiv but maintained better activity against resistant strains (k65r and multiple thymidine analog resistance mutations). most importantly, it was inactive (ic 50 >300 lm) against mitochondrial polymerase gamma. more than 50 prodrugs were synthesized and evaluated in metabolism studies and in dogs (intravenous and oral administration). then the enantiomers were tested separately in dogs. this led to the selection of gs-9131. whereas tfv is efficiently utilised by renal uptake transporters, gs-9148 was poorly taken into the kidney. no adverse renal findings were observed with the prodrug (gs-9131) in 28-day studies in rats, dogs and monkeys at the highest doses tested (300 mg, 20 mg and 30 mg/kg daily, respectively). in summary, this work has given examples of the prodrug approach being used successfully both to increase selectivity (by loading on-target tissues vs off-target tissues) and to increase activity (via by-passing metabolic constraints). adrian presented cases in which a prodrug strategy was able to fulfil the full potential of a selective, active triphosphate analog and enable its further progression as a clinical candidate. the keynote speakers were david margolis and myron cohen (fig. 9) . david margolis, university of north carolina, nc, usa in hiv-infected patients, there is a long-lasting reservoir of hiv in the form of integrated viral dna in resting cd4+ memory cells of the host immune system. therefore, even if it were possible to eliminate 100% of viral replication, a reservoir of hiv would remain. there may be reservoirs in other long-lived cells. to date, there is only one known hiv patient who has been cured of his infection, the ''berlin patient''. he was treated for cancer by chemotherapy followed by a bone-marrow transplant. being ccr5 +/à, the chemotherapy had a greater chance to remove all the ccr5+ve cells. the bone marrow donor was ccr5àve. although this patient continues to have no sign of hiv infection, this is hardly a viable treatment option for most hiv-infected patients. even in subjects with hiv replication well controlled by therapy, 70% have detectable plasma viremia which does not appear to decay over time (at least two years). to improve the sensitivity of the assay for hiv, 4 billion lymphocytes are mixed with antibody attached to magnetic beads. this selects for the cd4+ t cells, about 0.2-1 billion cells. the limit of detection is 1 copy of hiv rna/million cells, limit of quantitation is 10 copies/million cells. to reduce the reservoir of hiv, it was suggested that activation of integrated hiv in resting cd4+ t cells would give renewed hiv rna synthesis and possibly result in cell death either due to viral cytopathic effects or resulting from hiv-specific immune responses. a small clinical trial was set up to test this hypothesis. vorinostat (vor), a clinically approved drug for treating certain cancers, has been shown to bind to the active site of histone deacetylases. after a single dose, there was an increase in hiv rna (1.5 to 5-fold, mean 2.6-fold). of these subjects, 5 elected to continue with multiple doses. from the 11th to 22nd vor dose, acetylation of histones and activation of hiv rna synthesis became refractory to therapy. also, it is not known what proportion of cells, with latent hiv, can be activated. whereas a single vor dose did increase the expression of hiv rna, this is not an effective therapy for removing the hiv reservoir. myron cohen, university of north carolina, nc, usa myron noted that there are 2.5 million new hiv infections each year. in this context, anal sex may be an important factor because just one or a few virions of hiv can be infective; within 3 weeks, there is rapid virus replication throughout the body and latent hiv reservoirs of ''founder virus'' are already formed. although anal sex has been associated with homosexual couples, myron pointed out that it is not uncommon amongst heterosexual couples. although behavioral education should be encouraged, it can never be the whole answer. various approaches to the prevention of hiv transmission are being evaluated. monoclonal antibodies, broad neutralising antibody (bnab) and vaccines may have potential for prevention of transmission, but most progress is being made with dapivirine rings containing tdf. these are designed to stay in the vagina for a month. phase iii trials are ongoing. a long-acting hiv integrase inhibitor, gsk 1265744 (generally known as gsk 744), is administered i.m. once every 3 months; a two-year safety trial will be required. phase i trial has been completed and phase ii trial is being planned. by analogy with tuberculosis therapy, in which the infectious state is disabled prior to a complete cure, one wonders if hiv transmission rates may decrease with effective art use. in 2005, the hiv prevention trials network (hptn) initiated a study (hptn 052) which enrolled 1,763 hiv sero-discordant couples (couples that have one member who is hiv-infected and the other who is hiv-uninfected), mostly (97%) heterosexual couples. the infected partner had to be well enough not to require immediate art. the couples were randomised to have either immediate or delayed art. both groups received the same care including counselling on safe sex practices, free condoms, treatment for sexually transmitted infections and regular hiv testing. in may 2011, it had been announced that there had been 27 hiv transmissions in the delayed art group (877 couples) compared to only 1 in the immediate art group (886 couples), a 96% reduction. in these 28 cases, the hiv strain was linked to the partner. this is the first randomised clinical trial to show that treating an hivinfected individual with art can reduce the risk of hiv transmission to an uninfected partner. even with ''safer-sex'' counselling, there were 60 pregnancies in the delayed art group, despite that group having more incentive for safer-sex. following the announcement of this result, all infected participants were offered art. myron reported the 10th annual review of this study. in the delayed art group, there had been a total of 28 cases of hiv transmission with the hiv strain linked to the partner and 11 cases of unlinked transmission. in the one case of hiv transmission in the immediate art group, infection had been detected at day 85 of the study and further investigation suggested that the infection event was on day 1. clearly, early art is highly beneficial. cdc guidelines now recommend that all hiv infected patients should have art. 6. mini-symposium: hepatitis b virus anna lok, university of michigan, mi, usa the number of people infected with hbv world-wide, as estimated by the who and cdc in 2007, was between 223 and 240 million, but was declining due to vaccination. in the usa, vaccine use has led to a steady decline in the rate of new infections, decreasing from about 10/100,000 residents in the 1980s to about 1/100,00 today. in contrast, the prevalence of chronic hepatitis b among immigrants remains high, with no decreasing trend. when infection is acquired early in life, chronic infection is the norm. high viral load is associated with progression to liver cancer. there are 7 fda-approved drugs to treat chronic hbv infection, including entecavir (etv), emtricitabine (ftc) and tdf. with several years of continuous therapy, hbeag loss is achieved in about 40% of patients but hbsag loss (the ultimate goal, seen as a ''cure'') is still a distant prospect for most patients. however, cirrhosis can be reduced by long-term antiviral treatment. in one tdf trial at 5 years, 344/348 patients had a liver biopsy which showed that 73% of patients had improved fibrosis scores (p2 units) and that most other patients had no worsening. tdf has now been used for 6 years without detecting hbv resistance, making it one of the first line drugs. tdf is generally well tolerated but its rare side effects include nephrotoxicity (see above for a possible switch to taf when it is approved), reduction in bone mineral density and very rarely lactic acidosis. despite the major progress made in hbv therapy, there remain various challenges. one is cost, about $60,000-$72,000 for 5-year tdf therapy. pharmacy claims show that adherence is a problem; doses used are less than doses prescribed. there is a lack of accurate prediction of how hbv disease will progress in individuals. hbv dna can be integrated into the human genome at an early stage of infection. fortunately, the integrated viral dna is usually not the complete viral genome and patients, who achieve hbsag loss, rarely relapse. stefan mehrle, university of heidelberg, germany (stephan urban, head of hepatitis b research group, university of heidelberg, was originally scheduled to give this presentation). some chronic hbv-infected subjects are co-infected with hepatitis delta virus (hdv). this is a defective virus that replicates only in the presence of hbv. current antiviral drugs do not inhibit hdv. recently, heparan sulphate proteoglycan (hspg) has been shown to be essential for binding both hbv and hdv to primary hepatocytes. in 2012, human sodium taurocholate co-transporting polypeptide (hntcp) was identified as a functional receptor for hbv and hdv. hntcp is also designated as a solute carrier protein 10a1 (slc10a1). hntcp was shown to be a binding factor for the pres1 domain of the hbv l envelope protein. this interaction was found to be essential for hbv and hdv infection. whereas hbv replication is poor in cell lines derived from hepatocytes (e.g. hepg2 and huh-7) in which hntcp is usually weakly expressed, hbv replication is possible in primary human hepatocytes. the critical discovery was that over-expression of hntcp in hepg2 or huh-7 cells conferred susceptibility to hbv and hdv infection. myrcludex-b is a lipopeptide derived from amino acid residues 2-48 of the pres1 region of the hbv l protein. because it quickly (within 5 min) targets the liver, it is being developed for liver imaging and for drug targeting. it also acts as an entry inhibitor for hbv and hdv by interrupting binding between the hbv l protein and hntcp. it specifically inhibits hntcp-mediated taurocholate transport but the effect on hbv replication is much greater. myrcludex-b activity has been investigated in vivo using scid mice reconstituted with human hepatocytes. with prophylactic treatment, not one infected hepatocyte was seen. following therapeutic treatment, at week 6 post-infection, there were a few isolated infected cells. after the end of therapy, the infection seems to spread but only to neighboring cells. myrcludex-b has been synthesised on a 100 g scale. toxicology evaluation in 3 chimpanzees has been completed and clinical trials have been initiated. in a phase i trial using a 20 mg dose, myrcludex-b was well tolerated. results of a further phase i trial are due to be reported later this year (2014). a dose-ranging phase ii trial has been started. kyong-mi chang, university of pennsylvania, pa, usa anti-hbs antibodies clearly play a critical role in controlling hbv disease. their presence has been accepted as an indication of an ''effective cure''. however, these antibodies appear late in the disease course and so they must have a limited role in the early stages of the disease. what is the role of t-cell responses? in contrast to other viruses, there is a delayed onset, about 4-8 weeks rather than days. cd4+ t cells regulate the adaptive response, cd8+ t cells attack hbv-infected cells. the national institute of diabetes and digestive and kidney diseases (niddk), part of the usa national institutes of health (nih), is supporting a prospective clinical trial to investigate hbv-specific t cell responses during the course of hbv disease. there are no clear t cell differences relative to hbv genotype. the t cell responses are highest during acute hbv infection. during the chronic phase of hbv disease, t cell responses remain suppressed. in conclusion, there are a lot of players in the immune control of hbv infection but their relative contributions and how they adapt to control hbv replication are still largely unknown. 6.4. diversifying the hepatitis b pipeline: current efforts to explore novel mechanisms andrea (andy) cuconati, institute for hepatitis & virus research, pennsylvania commonwealth institute, pa, usa current hbv therapy using nucleotide anti-virals has been highly effective in controlling the infection but a ''cure'', as defined by hbsag seroconversion, has remained elusive. at best after 5 years, the rate is about 25%. other approaches are needed. myrcludex-b (see section 6.2) is the lead entry inhibitor. nvr-1221, an encapsidation inhibitor, is entering clinical trials. in addition. studies with novel nucleotide analogues are ongoing. the hbv field has been transformed recently by the introduction of cell-based antiviral assays. stefan mehrle (see section 6.2) has been leading the way. the assay read-out will need to be optimized for high-throughput screening (hts) but, already, the assay has shown some ''hits''. a few compounds inhibited encapsidation of viral rna. (the hbv virion contains partly double stranded (ds) dna but the reverse-transcription from rna to dna occurs within the capsid.) within the cell, hbv dna is transported into the nucleus where the viral dna forms covalently closed circles (cccdna). two specific inhibitors of cccdna formation have been found. current nucleotide anti-hbv compounds do give large reductions of hbv dna in plasma but only a minimal reduction in levels of the hbs antigen (about log 10 0.1). in contrast, one ''hit'', hbf-0259 inhibited surface antigen production but not genomic replication. structure-activity-relationship (sar) studies have given the current lead compound, hbv-0215. in conclusion, the cell-based assay, with complete replication of hbv, has markedly improved the screening for anti-hbv compounds although further optimization is still needed to give hts capability. adam zlotnick, university of indiana, in, usa. over the last few years, there has been much progress towards understanding the critical role of the hbv core protein -it is much more than just a protective coat for the genome because it plays a major role in the hbv life cycle. the core protein, being 183 amino acids long, is known as cp 183 . the first 149 amino acids are involved in core assembly whereas the last 34 residues, rich in serines and arginines, bind to rna. phosphorylation of the serines, particularly s155, s162 and s172, is required for specific packaging of full length hbv rna complexed to the polymerase (reverse transcriptase -pregenomic rna; rt-pgrna). this rt-pgrna complex initiates encapsidation. the core consists mainly of cp 183 but also includes other proteins (about 0.5%). adam showed us a computer model of the core, using different colours to highlight the various critical components. inside the core, the area of highest density (highlighted in red) represented the polymerase which was attached to the inner surface of the core. the ''other proteins'' in the core were shown in blue. the current thinking is that the polymerase, initially acting as a reverse transcriptase, is attached to, and guided by, an ''inside railway track''. this enables the polymerase to jump to the other end of the rna to start the reverse transcription into dna and then jump again to the other end to start, but never complete, the replication of the complementary dna strand. the self-assembly of the core is an energetically ''downhill'' process. somewhat surprisingly, it is possible to get mutations in which the core is even more stable but the rt activity is reduced. the phenylpropenamide derivative, at-130, fills a pocket in the core and so stabilizes it, similar to the change in amino acids in the mutants. in the presence of at-130, core assembly occurs faster; hence it is known as a core assembly enhancer (as adam mentioned, not a term much loved by industry, their preference is for core assembly inhibitors). regardless, the whole capsid structure changes. the binding of only a few drug molecules is required to make the core non-functional. it seems that it is easier to find compounds to enhance core assembly than inhibitors. 6.6. targeting cccdna to cure chronic hepatitis b massimo levrero, sapeinza universita' di roma, italy. the current hbv therapies of choice are tdf alone or with etv. these drugs have an extensive safety record with use up to 7 years. however, as for other nucleoside/nucleotide analogs, there is only a limited (about 1 log 10 ) reduction in the levels of hbv cccdna. the half-life of hbv cccdna seems to be long, but is still unknown. hbv replication parallels host gene expression, in that they involve the acetylation of histones, for example h 3 and h 4 . both host transcription factors and viral proteins bind to the cccdna. massimo summarized various assays to study different stages of cccdna during the replication cycle. potentially, these assays would allow the study of various approaches: to reduce or clear cccdna, to silence cccdna or to prevent the formation of new cccdna so that it would eventually be removed by dilution and cell death. for proof-of-concept, known ''epigenetic'' compounds, which act as transcription inhibitors, have shown that cccdna can be silenced. by reducing histone acetylation, the cccdna becomes too compact to allow transcription. this approach mimics, partly, therapy with interferon. this research is still at an early stage. due to time constraints, the next two speakers were asked to present brief summaries. john morrey (utah state university, ut, usa) described four mouse models but all stages of the life cycle of hbv can be studied only in the chimeric mouse model, in which human hepatocytes are used. however, this model lacks the potential to study the immune system and it is very expensive. stephan menne (georgetown university, dc, usa) described the woodchuck model. woodchuck hepatitis virus (whv) resembles the human virus and the disease in animals has many similarities to that in humans. neonatal infection becomes chronic in about 60-75% of cases. these chronic cases have virtually a 100% life-time risk of developing cancer, the time scale being about 1 year of chronic infection, followed by cancer at years 3 to 4. the use of microbicides is an active area of research for the prevention of transmission of hiv. david katz (duke university, nc, usa) described how mathematical models may aid drug product design. for example, if it is assumed that the microbicide gel is 400 microns thick, the epithelium is 200 microns and the stroma (connective tissue) is 3000 microns and if the partition coefficient between gel and epithelium in known, then it is possible to model drug transfer and suggest how various other parameters, for example the size of the subject, may modify drug delivery. it is important that different disciplines work together, for example biophysicists with behavioral scientists. biophysics can help an understanding of complex physical phenomena but human behavior can be both complex and highly variable. ralph baric (university of north carolina, nc, usa) noted that a particular infective agent, for example norovirus (nov), may cause subclinical or serious disease in different individuals. in general, animal models are designed to give consistent outcomes rather than aiming to mimic the genetic diversity found in human subjects. in a collaborative effort, mice from 8 ''founder'' strains, including 3 wild-derived strains, were selected. the 5 founder laboratory strains were all derived ultimately from a single female mouse ca 1900. the susceptibility of the 8 founder strains to severe acute respiratory syndrome coronavirus (sars-cov) differed widely (ld 50 p10 6 -10 2 ). the founder strains were cross-bred. although ca 90% of the genes was equally distributed among the new mouse lines, there were gene combinations not seen previously. after infecting mice from the different founder strains with a constant sars-cov inoculum and measuring virus load at a set time after infection, there was a correlation between virus load and disease (as measured by vascular cuffing). it was possible to relate the effect to chromosomes 3 (27%) and 13 (20%). hopefully, identification of the important genes may be achieved. by keeping the virus inoculum constant, this system better represents the clinical spectrum of disease. when using this system to evaluate a potential vaccine, it was found that mice, under the age of one year, could be protected. however, there was a range of effectiveness, from good protection to inactive. these variations may give a representation of human diversity. angela kashuba, university of north carolina at chapel hill, nc, usa in four clinical studies, truvada [a combination pill containing tdf and emtricitabine (ftc)] was taken once daily to prevent hiv transmission, known as pre-exposure prophylaxis (prep). the adherence rates were unexpectedly poor in all four studies, particularly low in the study including at risk women. for example in one study, ''high adherence'' was defined as subjects taking at least 80% of drug doses and was achieved by only 54% of subjects. possible reasons may have been the apparent risk of side-effects (the long consent form included 7 pages of side-effects) and the perception that the subjects, as individuals, were not particularly at risk of infection by hiv. importantly, the trial did confirm the concept that prep could be effective. there was >90% protection in those subjects generally taking 7 doses/week and there was some protection, albeit much less, in subjects taking 2 doses/week. adherence rates, reported by subjects, were appreciably higher than the rates evidenced by drug blood level measurements taken just before the next dose (i.e. 24 h after previous dose). in an attempt to better understand and model these data, the drug concentrations (tdf/tfv and ftc) in various tissues were measured. the ratio between drug concentrations in blood and tissue samples differed greatly for tdf/tfv, with less variations for ftc. concentration ratios of tdf/tfv were about 50 in rectal tissue but only 0.2 in vaginal tissue. for ftc, the ratios were 2.6 and 1.3, respectively. when considering the possible consequences of missed doses, the time scale for hiv infection is an important factor. it is thought that hiv takes about 1-3 h to reach the epithelial cells. clearly, adherence is a critical factor for efficacy and so a real-time objective method for measuring adherence is urgently needed before further clinical studies are initiated. 7.4. the novel nucleoside analog bcx4430 exhibits broad-spectrum antiviral activity and confers post-exposure protection against ebola and marburg viruses travis k. warren, usamriid, fort detrick, md, usa ebola and marburg viruses are members of the filovirus family. even in recent outbreaks of these diseases, including the current ebola epidemic in west africa, care workers are becoming infected and dying. drugs, which are being investigated for treating these diseases, are progressed under the fda ''animal rule''. bcx4430 is a c-nucleoside adenine analog (fig. 10 ) which is being progressed by biocryst pharmaceuticals inc. (warren et al., 2014) . in cell culture assays, bcx4430 is active against ebola and marburg viruses, (ec 50 ca 1 lm). with bcx4430 at 30 lm, there was no detectable incorporation into host dna or rna. in rats, bcx4430 is efficiently activated (phosphorylated) to the triphosphate. in a primer-extension assay, there is some read-through beyond a single residue of bcx4430, but there is effective chain termination after the first bcx4430 residue where the template has two consecutive uridine residues. bcx4430 has been tested in rodent and nonhuman primate models of marburg hemorrhagic fever. in mice, there was a dose response (30, 20, 3.3 and 1.1 mg/dose, bid) with full protection at the two higher doses (survivors, 100%, 100%, 95% and 83% respectively). in an experiment with dosing starting at different times (4 h pre-infection, 24, 48, 72, 96 and 120 h post-infection vs placebo), the placebo-treated mice died on days 6, 7 and 8 with one survivor (10%). in the treated groups, the percent survival was 80, 100, 80, 100, 100 and 30, respectively. in guinea pigs, bcx4430 (bid) with treatment starting at different times (1 h pre-infection, 24, 48 and 72 h post-infection) there was full protection (100% survival) for the pre-infection and 24 h groups, with reduced efficacy at the later start times. in cynomolgus monkeys, bcx4300 treatment was started at 1, 24 and 48 h post-infection. in the placebo group, all 6 animals died within days 9 to 12. in all the treated groups, virus loads were reduced by more than log 10 3. there was one late death in the 1 h group but the other 17 monkeys survived. various markers of potential organ damage were reduced in all treated groups. encouraged by these results, 14-day toxicology trials have recently been completed without any serious concerns. biocryst is developing bcx4430 under the fda animal rule and indenabling work is ongoing. when asked about viral resistance, travis explained that it is not ethically permissible to create resistant strains of marburg virus, but samples collected from the monkeys are being sequenced to look for mutations indicative of drug resistance. as yet, mitochondrial toxicity has not been examined. had similar bone marrow transplants, initially seemed to have been ''cured'' but hiv was detected after 70 and 200 days, respectively. latent hiv can survive in various long-lived cells for decades, especially in memory t cells. when these cells proliferate, the integrated hiv genome is duplicated as the cell divides and the cells survive so long as hiv remains silent. compounds known to activate all t-cells are too toxic to become a clinical therapy. however, latency-reversing agents (lra) have greater specificity, ideally activating only the integrated hiv, leading to the death of hiv-containing t-cells. there remains another possibility (perhaps a less popular view) that there is continued low rate of hiv replication. two clinical studies have been initiated in subjects with undetectable plasma hiv levels. raltegravir, an hiv integrase inhibitor, was added to the background therapy. latent hiv is mostly integrated into host dna but hiv may also form episomal circular dna. the proportion of the circular form increases with raltegravir treatment. in the two clinical studies, 13/45 and 9/15 subjects, respectively, had detectable hiv circles which then decayed. this implies that some de novo infection of cells is ongoing. on the other hand, art works well, with no evidence of sequence evolution in the hiv circles at 48 weeks. is it possible that raltegravir is inducing a single round of hiv replication, to give an increase in hiv circles? derek sloan, gilead sciences, foster city, ca, usa like vorinostat, (vor), romidepsin (rmd) is a histone deacetylase inhibitor which is used clinically to treat cancer. memory cd4+ t cells were taken from hiv subjects on suppressive art; ex-vivo treatment with rmd (40 nm) induced a 6-fold increase in intracellular hiv rna which persisted for 48 h. in contrast, a much higher concentration of vor (1 lm) gave a 2 to 3-fold lower response which was only transient. rmd also increased levels of extracellular hiv rna and virions. encouragingly, this ex-vivo induction of latent virus was seen at rmd concentrations that are below the levels of drug achieved in humans by clinical doses of rmd. accordingly, in a phase i/ii trial in hiv-infected subjects on art, rmd gave a better and more sustained response than vor. about 1.5% of cells containing hiv provirus were activated. although this is far too low a percentage to eliminate the latent hiv reservoir, it is hoped that combination of such lra, which give improved results in ex-vivo cell assays, may give better clinical efficacy. gilead scientists have started screening for novel lras. ''gs-1'' has been identified as a hit by hts. research on this lead is at a very early stage. gilead workers are also investigating other approaches. for example, gs-9620 is a toll-like receptor 7 (tlr7) agonist and it acts as an immune stimulator. although it is being evaluated in phase ii studies for the treatment of chronic hbv infections, the potential effect on hiv reservoirs is being investigated. in siv-infected monkeys, oral dosing of tlr7 agonist induced the activation of immune effector cells such as cd8+ t cells and nk cells. based on these data, tlr7 agonists are being further investigated for their effect on latent siv reservoirs in monkeys which have good virological suppression. another approach is to use anti-envelope antibodies. broadly neutralising antibodies (bnabs) are very effective in preventing siv infection when the viral load is low but less effective against a high-load virus challenge. in addition, a prophylactic cmv-vector-based siv vaccine was effective in preventing siv infection in rhesus monkeys. this and similar vaccines are being tested in vivo for their effects on the latent siv reservoirs. in summary, lras are able to activate hiv provirus in memory cd4+ t cells and thereby may enhance the recruitment of immune effector cells to destroy provirus-containing cells. however, a ''cure'' for hiv infection is still a distant prospect. furthermore, latent hiv reservoirs are heterogeneous and so a combination of approaches will likely be required. gerardo garcia-lerma, centers for disease control and prevention, atlanta, ga, usa proof-of-concept studies for prep, are mostly conducted in nonhuman primates. these can be used either to model a single highdose infective challenge or repeated low inoculations, about 10-50 tissue culture infective doses (tcid 50 ). since 2005, rhesus macaque models have been used in a long series of investigations. in a study, in which the monkeys were treated daily with either oral tdf or tdf/ftc and given a weekly siv inoculum rectally, tdf/ftc gave a longer delay in infection than did tdf alone. when using the vaginal infection route, tdf/ ftc gave 100% protection. in contrast, there was far less protection in clinical trials -why? one possible reason may have been that women were having the contraceptive injection, depot medroxyprogesterone acetate (dmpa). a study, in macaque monkeys given dmpa, confirmed that dosing with tdv/ftc gave good drug levels in plasma and in vaginal secretions. therefore, this did not explain the poor protection in the clinical trial. the macaque model has been used successfully to investigate various situations that are presented in the clinic. when macaques were co-infected with siv and a bacteria and treated with tdf/ftc for 12 weeks, there was good, but not complete, protection (80%). with ftc-resistant virus, tdf/ftc remained protective. in this case, ftc-resistant virus has increased susceptibility to tdf. with the k65r mutant hiv, there was protection against a low inoculum but only partial protection (ca 50%) against a high inoculum. whereas daily dosing seems to be acceptable for patients living with hiv, another option for prep is desirable. gsk-1265744 (generally known as gsk-744) is an hiv integrase inhibitor. it can be formulated with nano-particles to provide an injectable drug depot. in the macaque model, gsk-744, injected once monthly, gave full protection against repeated rectal and vaginal exposures. because metabolism of gsk-744 is much slower in humans than macaques, it was expected to remain effective in humans for up to three months. a phase i study confirmed that drug levels remained above the predicted effective level with a 20-week dosing interval. a phase ii trial is planned. another approach is to use vaginal rings, which have been in clinical use as contraceptive devices for years. in the macaque model, tdf-containing rings, replaced every 4 weeks, gave full protection. a phase iii trial has just been initiated. another option, elvitegravir (evg) and taf are being evaluated in a biodegradable polymer. although daily dosing with tdf/ftc has not proved sufficiently successful as prep in clinical use, it has proved that prep is an achievable aim and this has encouraged the progression of other options. courtney fletcher, university of nebraska, omaha, ne, usa atripla was the first triple combination pill taken once daily for hiv therapy. it contained tdf, ftc and efavirenz (efv). the macaque model has been used to investigate the differing tissue distributions of these drugs and how viral replication may be continuing wherever the drug concentrations are lowest. there are two approaches: tissue homogenates and tissue cells. tissue homogenates give both the intracellular and extracellular drug amounts. from tissues, mononuclear cells (mncs) are collected and the intracellular drug concentration measured. this approach is preferred by courtney but this option may be constrained by sample size and the drug concentration may be underestimated. for exam-ple, with raltegravir, after the mncs have been washed 3 times, the drug concentration is very low. much higher raltegravir concentrations are found when the mncs are cleaned by a rapid spin through oil. comparing an oil spin and repeated washes, the oil process gives higher drug levels, typically about 50% higher. following initial studies in macaques, a clinical study, in 32 subjects, investigated distribution of the drugs from atripla in peripheral blood mononuclear cells (pbmc) and various tissues (see above). in 12/32 subjects, there are data on the time to reduce hiv load to <48 copies/ml. in plasma, the time was 3-4 months. in lymphoid tissues, there was a much slower rate of hiv decline. also, patient variability was noted, with the faster responders having the higher drug levels. a drug may be absorbed from the gastrointestinal tract either going via the portal vein to the liver and then into blood circulation or via the lymphoid system. blood flow is about 200 times faster than lymphoid flow. when the water/1-octanol partition-coefficient (logp) of a drug is <5, absorption tends to be via the blood route. the prodrug approach can be used to alter absorption or, as for tfv, stability of the prodrugs (tdf and taf) can influence the relative concentration in lymphoid tissues (see above). this year, the three major award lectures exemplified the strength of icar, covering very different areas of research. john drach (elion award) described his journey through the early days of antiviral research, which led to the identification of novel modes of antiviral action that had not been envisaged previously. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept novel nucleoside analogs. the replacement of thymine by 5-chlorouracil led to the generation of a new form of e. coli. i suggest that this work has important implications in conventional antiviral research. with hiv and hcv protease inhibitors, the genetic barrier is limited by the ability of the viral protease and its substrate (the viral polyprotein cleavage sites) to co-mutate so that the virus can become resistant to the antiviral drug. so far, polymerase inhibitors have not suffered the same fate but this work shows that a poor choice of nucleotide analog could result in a resistant virus with a new type of rna in which the drug replaces a natural nucleoside. adrian ray (prusoff award), describing work at gilead, demonstrated how the prodrug concept can markedly improve both the efficacy and safety of potential drugs. their progress with hiv and hcv therapies has been remarkable. the keynote addresses tackled two emerging areas of hiv research. david margolis summarized work aiming to eradicate hiv from infected subjects and myron cohen described current progress with approaches to prevent hiv transmission. i found both these presentations to be informative and stimulating. hiv ''cure'' still seems to be a distant prospect. in contrast, prior to exposure prophylaxis (prep) has been shown to be an achievable aim although the need for daily dosing is a barrier to success. gerardo garcia-lerma described recent progress which is likely to radically change the prospects for therapeutic convenience and success. tdf-containing vaginal rings, which need replacing only once a month, are being evaluated. another exciting prospect is gsk-744 which has been formulated as a long-lasting injection. a phase i trial confirmed that the drug may be administered at 3month intervals. in the absence of a proven hiv vaccine, prep with drugs has become the most promising strategy to reduce hiv infection rates among high-risk populations. this conference also included three interesting mini-symposia: ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. an innovation this year was a session devoted to the european training network, euvirna and introduced by frank van kuppeveld. all the 18 euvirna fellows, who attended icar, gave short presentations at this session. for further information, please see the isar news (24.1) in the september issue of antiviral research for an account by frank van kuppeveld. for many years, the clinical symposium was, for me, a major highlight of icar. in my report for the 2013 icar, i expressed a hope regarding hcv therapy: ''there is the prospect that the first nucleotide analogue will be licensed by the time of our next icar meeting. the combination of a nucleotide analogue and a ns5a inhibitor looks set to transform hcv therapy across all genotypes. as for hiv, single-pill, once-daily regimens are following on quickly''. on 6th december 2013, sofosbuvir (sovaldi ò ) was the first nucleotide analog to be approved in the usa by the food and drug administration (fda) for treatment of patients with hcv. approval by the european union followed soon afterwards, in january 2014. a ns5a inhibitor, ledipasvir, formulated as a single fixed-dose combination pill with sofosbuvir, is progressing quickly through clinical trials. with such remarkable progress being achieved since the 2013 icar, i was disappointed to discover that there was no presentation on this topic at this year's icar. a paper (sofia, 2014) , which was part of a symposium in antiviral research on ''hepatitis c: next steps toward global eradication'', emphasizes recent successes. after completing therapy, a sustained virological response for 12 weeks (svr12) is regarded as a cure for hcv-infected patients. the combination of sofosbuvir/ledipasvir has shown remarkable results in clinical trials, with svr12 in the range 95-100% across genotypes. this combination was well tolerated. a nda for the sofosbuvir/ledipasvir combination pill was submitted recently. i do not recall any previous antiviral trials in which the ''intention-to-treat'' analyses showed 100% success rates. perhaps similar to the hcv symposium in antiviral research, i hope that the 2015 icar, which will be held in rome, will have a mini-symposium which will include an account of this remarkable progress. it would be interesting to have an update on the clinical impact of this combination therapy for hcv and to have an assessment on the prospects for global eradication of hcv. beside this one disappointment, there were many excellent presentations and i would like to add my thanks to the isar officers and conference committee for organizing another interesting and successful icar. metabolism and pharmacokinetics of anti-hepatitis c virus nucleotide prodrug gs-6620 beyond sofosbuvir: what opportunity exists for a better nucleoside/nucleotide to treat hepatitis c? selection of an oral prodrug (brl 42810; famciclovir) for the antiherpesvirus agent brl 39123 protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 acknowledgements i wish to thank all those authors who have kindly provided me with copies of their presentations and for giving me valuable comments. also, i thank the president of isar for asking me to prepare this meeting report. key: cord-300968-dtaasxk1 authors: kliger, yossef; levanon, erez y.; gerber, doron title: from genome to antivirals: sars as a test tube date: 2005-03-01 journal: drug discovery today doi: 10.1016/s1359-6446(04)03320-3 sha: doc_id: 300968 cord_uid: dtaasxk1 abstract the severe acute respiratory syndrome (sars) epidemic brought into the spotlight the need for rapid development of effective anti-viral drugs against newly emerging viruses. researchers have leveraged the 20-year battle against aids into a variety of possible treatments for sars. most prominently, based solely on viral genome information, silencers of viral genes, viral-enzyme blockers and viral-entry inhibitors were suggested as potential therapeutic agents for sars. in particular, inhibitors of viral entry, comprising therapeutic peptides, were based on the recently launched anti-hiv drug enfuvirtide. this could represent one of the most direct routes from genome sequencing to the discovery of antiviral drugs. severe acute respiratory syndrome (sars) is a pulmonary infection that has been identified in multiple outbreaks around the world, emerging initially in guangdong province, china, in november 2002. the number of reported cases increased exponentially and reached 8422, which resulted in 916 deaths by august 2003 (who website). the syndrome is caused by a previously unknown virus -sars-associated coronavirus (sars-cov) [1] [2] [3] . the global sars outbreak has been contained, mainly owing to strict patient isolation and aggressive containment of infected regions, but the virus itself has potential to reappear. this concern is supported by studies reporting a cyclic pattern for other human-infective coronaviruses that attack mainly in the winter, sometimes skipping years, for example, the related human coronavirus, hcov-oc43, which breaks out every two to four years [4] . to date, there are ~36 antiviral drugs, half of which were developed in the past 15 years to treat a single virus, hiv-1. development of these antiviral drugs gained from the advances in molecular and structural biology coupled with advances in medicinal chemistry and in the industrialization of the drug discovery process. the sars epidemic has emphasized the need to develop drugs against emerging viral infections quickly, and demonstrates the usage of genomic technologies in antiviral research. in the past two decades, biology has become an information-driven science as a result of the emergence of genomic technologies and the expansion of the internet that allows analysis of genomic databases at every researcher's desktop. these advanced genomic technologies led to rapid sequencing of sars-cov [5, 6] and, for the first time in history, the sequencing of a genome of an infective agent preceded the understanding of its basic biology and etiology. armed with this genomic information, research groups around the world suggested multidisciplinary approaches to attain anti-sars drugs. in general, physicians tried to relieve the symptoms mainly by modulating parts of the immune system, while vaccinologists began the long process of developing a vaccine against the virus. molecular and structural biologists suggested ways to interfere with the viral life cycle, and these are the focus of this review (figure 1 ). the strategy starts from virus identification, goes through genome sequencing and, hopefully, ends with an antiviral drug. drug development remains a challenge, but the acceleration in drug discovery offered by genome technologies will hopefully enable significant timetable cuts in achieving antiviral medicine. other, more classical anti-viral strategies used to treat sars patients, like the use of interferon, will not be discussed here. the interested reader is referred to [7, 8] . this review includes a retrospective summary of the development of anti-hiv drugs, followed by an appraisal of anti-sars strategies and their applicability for rapid development of antivirals against sars-cov, if it does resurface, and against the next, probably inevitable, viral threat. in the early 1980s, a sudden increase in life-threatening opportunistic infections and kaposi's sarcoma, considered rare until then, was observed among homosexuals and injecting drug users. these infections were attributed to an acquired immunodeficiency syndrome (aids). about two years of extensive studies passed until the human immunodeficiency virus (hiv) was identified as the etiological agent [9, 10] . an additional two years passed before completing the sequencing of its genome [11] . the huge efforts invested in developing the first drug to treat aids patients bore fruit in record speed when the fda approved azidothymidine (azt) in 1987. azt, a nucleoside analog, inhibits the viral reverse transcriptase -an enzyme that is essential for hiv replication. unfortunately, isolates from these patients showed decreased sensitivity after six months of azt administration. the finding that some of these isolates also exhibited cross-resistance to other nucleoside analogs [12] raised further concern. azt and other nucleoside reverse transcriptase inhibitors were still the sole treatment available for almost a decade. the next important milestones were the development of inhibitors against the protease, another essential viral enzyme, and nonnucleoside reverse transcriptase inhibitors, first approved in 1995 and 1997, respectively. while the nucleoside reverse transcriptase inhibitors were discovered by cell culture screening directed towards a specific class of chemical agent, the non-nucleoside reverse transcriptase inhibitors were discovered by hts of large compound libraries. the discovery of hiv protease inhibitors represents one of the best examples of the application of protein structural knowledge to rational drug design. since then, aids patients have been treated with a cocktail of inhibitors against hiv reverse transcriptase and protease. however, despite the unprecedented successes in the therapy of hiv infection, hiv continues to spread, causing more than 14,000 new infections every day, 95% of these in the developing world (who website). a major problem with current aids treatment is the high frequency of hiv mutations, resulting in drug resistance. thus, efforts are being made to develop agents addressing yet untargeted steps in the hiv life cycle. viral-induced fusion between viral and host cell membranes was acknowledged as a the sars-cov life cycle is vulnerable to therapeutic intervention in several places. (1) virus binding to cellular receptors. outside the cell, blocking the interaction of sars-cov with the cellular receptor will prevent the virus from attaching to host cells. co-receptor antagonists will prevent the initiation of the next step. www.drugdiscoverytoday.com useful drug target with the approval of the hiv fusion inhibitor, enfuvirtide (fuzeon), in 2003 [13] . unlike other hiv drugs that are small molecules developed against viral enzymes, enfuvirtide is a peptide that corresponds to a specific segment of the viral envelope protein. importantly, this segment can be directly pinpointed by computational sequence analysis [14, 15] . this strategy seems promising in developing anti-viral therapeutic peptides to other viruses that possess type 1 viral fusion proteins [e.g. measles virus and respiratory syncytial virus (rsv)], which share some structural motifs with hiv. little is known about viral-induced membrane fusion of other viruses that do not share these motifs. viruses can be divided into two groups based on the composition of their outer surface: (a) non-enveloped viruses are enclosed by a protein shell called a capsid; (b) enveloped viruses are surrounded by a membrane 'stolen' from their last host. in order to infect host cells, that is, to inject their genetic material into the cell, enveloped viruses need to overcome both viral and cellular membrane barriers. viral entry of many enveloped viruses, including sars-cov, involves two major steps. first, the virion binds to receptor(s) localized on the surface of its host cell, and second, the viral membrane fuses with the host cell membrane. sars-cov spike glycoprotein, responsible for these two steps, is translated as a large polypeptide that is subsequently cleaved to produce two functional subunits, s1 and s2. s1 is the peripheral protein, which binds to cellular receptor(s), whereas s2 is a type i transmembrane protein that catalyzes the membrane fusion reaction. both steps are crucial for viral infection, and therefore were suggested as targets for antivirals. since the identification of cd4 as the cellular receptor for hiv in 1984 [16, 17] , several therapeutic agents aiming to inhibit the binding of hiv to cd4 were suggested. unfortunately, these efforts have yet to bear fruit. major difficulties that slow down the development of inhibitors for the binding of cd4 to gp120 include: (i) the gp120binding site for cd4 consists largely of a recessed pocket; (ii) antibodies that bind to cd4 antigen are likely to block virus attachment but can be immunosuppressive because they will lead to depletion of cd4 cells. currently, both a recombinant cd4-igg2 fusion protein (pro-542) and a small-molecule (bms-488043) aiming to prevent hiv from attaching to cd4, are in clinical trials. these efforts reflect the motivation of inhibiting the first step in the viral life cycle, that is, the binding of the virus to its host cell. this approach was strengthened when cxcr4 and ccr5 were identified as additional essential cellular receptors for hiv [18, 19] , and with the discovery that ccr5deficient people are resistant to infection by hiv [20] . similar to hiv, binding of the viral spike glycoprotein to some receptor(s) on host cells is the first step in sars-cov infection. blocking the interaction between these receptors and the virus could prevent infection, thus inspiring the search for sars-cov cellular receptors. recently, human angiotensin converting enzyme-related carboxypeptidase (ace2), a type i integral membrane metalloprotease, was identified as a receptor for sars-cov [21] . a soluble form of ace2 and an antibody recognizing sars-cov s1 efficiently neutralized sars-cov in vitro, supporting the speculation that the ace2-binding site of the spike glycoprotein is an attractive target for vaccine and drug development [21, 22] . this is further supported by an ace2 inhibitor, which also inhibits sars-cov infection in vitro [23] . notably, a 193-amino acid fragment of sars-cov s1, which efficiently bound ace2, blocked spike glycoprotein-mediated infection with an ic 50 of less than 10 nm [24] . more recently, a human lung cdna library was screened to identify receptors for sars-cov, revealing that human cd209l can also mediate infection by sars-cov, although it is a much less efficient receptor than ace2. interestingly, cd209l is expressed in human lung in type ii alveolar cells, which are an important target for sars-cov infection [25] . it is still not known whether interactions between ace2 and cd209l play a role in sars-cov infection and pathogenesis. hiv entry involves the binding of the viral envelope glycoproteins (comprising gp120 and gp41, which are the homologous of sars-cov s1 and s2, respectively) to cd4 on the host cell plasma membrane. this induces conformational changes, enabling the n-terminal heptad repeat region (n-hr) of gp41 to be exposed. at this stage, enfuvirtide binds to the n-hr of gp41, hence blocking further conformational changes required for membrane fusion. enfuvirtide is a synthetic peptide inhibitor corresponding to a segment of gp41, known as the c-terminal heptad repeat (c-hr). following the cd4-induced conformational change of gp41, plasma membrane ccr5 (or cxcr4) molecules are recruited to the binding site, and bind to the cd4-envelope complexes. this triggers a highly stable interaction between the c-hr and the n-hr regions of gp41, which drives the membrane fusion reaction to completion. thus, enfuvirtide can no longer inhibit the fusion process [26] . slower engagement of the co-receptor with the cd4-envelope complexes, results in a stronger inhibition by c-hr-derived peptides [27, 28] . furthermore, reduction in ccr5 binding efficiency resulted in slower fusion kinetics and increased sensitivity to enfuvirtide [28, 29] . further support for this model is provided by the finding that ccr5 and cxcr4 antagonists showed strong anti-hiv synergy with enfuvirtide against ccr5-dependent and cxcr4-dependent hiv isolates, respectively [30, 31] . in addition, pro-542 acts in concert with enfuvirtide in virus-cell and cell-cell fusion assay, by triggering formation there are no peptide fusion inhibitors for influenza virus. it is noteworthy that influenza virus uses a different mechanism to enter its host cells: it is first endocytosed into the cell, followed by a ph-dependent fusion between the viral and the endosome membranes. strikingly, it takes only few milliseconds from the time the ph drops in the endosomes until the fusion process is completed [33] [34] [35] [36] . in contrast, the time scale of hiv infection is about 20 minutes, allowing ample time for binding of entry inhibitors [26, 27, 37] . sars-cov entry kinetics resembles that of hiv. at 5 minutes after exposure, the sars-cov lined the plasma membrane of vero cells [38] . fusion and entry of the viral load into the cytoplasm was observed mainly between 15 and 20 minutes [38] . the timescale similarity between hiv and sars-cov fusion process, as opposed to the fast membrane fusion of influenza virus, indicates that entry inhibitors could be successful with sars-cov. despite the lack of sequence similarity between the fusion proteins of hiv-1 and sars-cov. schematic illustration of (a) hiv-1 gp41 and (b) the equivalent s2 protein from the sars-cov. a leucine/isoleucine heptad repeat adjacent to the n-terminus of both proteins appears in red.the c-hr is in green. cysteine residues (purple) confining a loop structure are located between the two heptad repeats. an aromatic residues-rich motif is marked blue, and the transmembrane segment is in orange. a peptide corresponding to the c-hr, which acts as potent inhibitor of hiv-1 entry into the cell, appears in yellow.the helical wheel is a top view of a single strand of a coiled coil. in the wheel projection of the n-hr (c) and c-hr (d) of sars-cov s2 protein, each of the seven positions (a-g) corresponds to the location of an amino acid residue that makes up the coiled coil.the arrows between the seven positions indicate the relative locations of adjacent residues in an amino acid subsequence.the helical wheel demonstrates how a potential antiviral drug can be discovered based solely on sequence information. homology and the difference in length between sars-cov s2 and hiv gp41, homologous regions of the n-hr and c-hr in sars-cov s2 were identified immediately after the sars-cov genome sequence was published ( figure 2) . thus, a similar strategy might be applied to inhibit the entry of sars-cov [39] , (http://www.virology.net/articles/ sars/s2model.html). indeed, preliminary reports revealed anti-sars activity for peptides corresponding to the c-hr of sars-cov s2 protein [40] [41] [42] , and indicated a mode of action similar to that of enfuvirtide [43] [44] [45] [46] . the kinetic similarity of sars-cov and hiv entries suggests a synergism between sars-cov spike glycoprotein inhibitors and agents that block some of its receptors. the role of different cellular receptors in sars-cov entry should be characterized to discover the receptor(s) that trigger conformational changes and transform the spike protein into the stable 'fusogenic' form. antagonists for these receptor(s) could synergize with fusion inhibitors. the synergy between sars fusion inhibitors and ace2 or cd209l antagonists has not yet been investigated. the first step is to determine optimal fusion inhibitors. intriguingly, whereas polar residues disrupt the heptad repeat in the c-hr of hiv-1 gp41, the c-hr of sars-cov s2 has a perfect leucine/isoleucine heptad repeat ( figure 2d ). this could explain why the exact sequence boundaries of the c-hr-derived peptides are crucial for efficient inhibition [41, 42, 44, 47] , as aggregation of the peptides in solution could abolish anti-viral activity. interestingly, two reports demonstrate that n-hr-derived peptides are also active [40, 41] , while others found that only c-hrderived peptides have anti-sars activity [42, 47] . it is noteworthy that the reason for the poor inhibitory activities of n-hr-derived peptides in other viruses is contributed to their tendency to aggregate in solution, suggesting that, similar to the c-hr-derived peptides, the exact sequence boundaries of the n-hr-derived peptides are important. the main advantage of fusion inhibitors is their immediate discovery as they are simply the corresponding fragments of a known protein. however, their drawbacks as therapeutic peptides are lack of oral bioavailability and high production costs. auspiciously, sars is a respiratory syndrome, thus, peptidic fusion inhibitors could be given by inhalation. this approach was applied successfully in rsv-infected mice [48] . to serve as drug targets, viral proteins should fulfill two criteria: (i) they should be essential for the viral life cycle; and (ii) they should exhibit low similarity to host proteins. sars-cov genome analysis was performed to predict its proteome [49] , and three viral enzymes were suggested as targets for drug discovery: the helicase, the rna-dependent rna polymerase and the main protease. these enzymes are crucial for replication, transcription, translation and posttranslational polyprotein processing (box 1). assay development based on these three sars-cov target enzymes was initiated [50] [51] [52] , thus paving the way for highthroughput in vitro screening approaches to identify candidate inhibitors in compound libraries. the traditional and, in many cases, the most cost-efficient way of dealing with viruses has been through vaccines. the logic of vaccine development against sars-cov emerges from the combination of several findings: (i) re-infection with sars-cov causes only mild illness; (ii) sars is fatal mainly to old people who have difficulty in producing good humoral and cellular immune responses; and (iii) the case fatality ratio of sars ranges from 0-50% depending on the age group affected, with an overall reviews box 1 currently, inhibitors target three enzymes, crucial for sars-cov life cycle: helicase: protein-fold recognition methods followed by a biochemical study suggested a dual use of sars-cov helicase in both rna synthesis and cap formation, suggesting new avenues to treat the virus [65, 66] . screening of a compound library by plaque reduction assay resulted in a helicase inhibitor at the low microm range. in vitro assay confirmed sars-cov helicase as a validated drug target [67] . rna-dependent rna polymerase: molecular modeling revealed that sars-cov rna-dependent rna polymerase does not contain a hydrophobic pocket for non-nucleoside inhibitors.this is in contrast with the non-nucleoside inhibitors activity against hiv-1 reverse transcriptase [68] . of the many nucleoside analogues screened, sars-cov most selective nucleoside analogue inhibitor is beta-d-n4hydroxycytidine, albeit at low efficacy (ec90 of 6 microm by virus yield reduction assay) [69] . ribavirin, a broad-spectrum nucleoside analogue efficacious in the treatment of several viral infections, was used in various countries against sars-cov.while ribavirin was promising in vitro [70] , recent reports revealed that ribavirin did not appear to confer any benefit for patients with sars [71, 72] . main protease: sequence similarity was found between the substrate-binding sites of sars-cov main protease and the main protease of related viruses. remarkably, the sars-cov main protease cleaved the porcine coronavirus transmissible gastroenteritis coronavirus (tgev) main protease substrate [73] .this rationalizes screening of known protease inhibitors for anti-sars activity.this approach was reinforced by the crystallization of the sars-cov main protease together with a tgev inhibitor [74] . the findings revealed that homology modeling is often inadequate for the prediction of the mutual orientation of domains in multidomain proteins. however, the tgev-based homology model also shows that a reasonable model of a substrate-binding site can serve to develop useful ideas for inhibitor design that can inspire medicinal chemists to start a synthesis program long before the 3d structure of the target enzyme is experimentally determined (reviewed in [75] ). in parallel, hts of compound libraries identified inhibitors of the sars-cov main protease in the low âµm range [67, 76, 77] .there are a few studies demonstrating that inhibitors of the hiv protease can also inhibit sars-cov, albeit with much lower efficiency [52, 78] . www.drugdiscoverytoday.com estimate of case fatality of 14-15% (who website). thus, most infected individuals recover from sars. furthermore, the success of a vaccine against other mammal-infective coronaviruses is encouraging [53, 54] . modern antiviral vaccine development depends heavily on the viral genome. the availability of the human genome, together with the recent sequencing of the sars-cov genome, largely increases the probability of success of vaccine development. the full-length spike glycoprotein of sars-cov, expressed by vaccinia virus, induces binding and neutralizing antibody and protectively immunizes mice against a subsequent infection with sars-cov [55] . in addition, dna vaccine encoding the spike glycoprotein of the sars-cov induces t-cell and neutralizing antibody responses, as well as protective immunity, in a mouse model [56] . the discovery of rnai raises many hopes regarding antiviral strategies and carries the promise of a shortcut in the drug discovery process. usually, target discovery is followed by exhaustive hts and/or structure-based screening of many thousands of compounds in the hope that some of them will efficiently bind to the target. theoretically, with small-interfering rna (sirna) as a drug, the course from target to drug is much shorter. encouraging results in mice were obtained using an rnaibased therapy against hepatitis b virus (hbv): transfection with plasmids expressing short hairpin rnas (shrnas) homologous to hbv mrnas effectively inhibited replication initiation in cultured cells and mice liver, showing that such an approach could be useful in the treatment of viral diseases [57] . currently, there are attempts to use sirna as anti-sars drugs, but they are still in preliminary in vitro stages [58] [59] [60] . the application of this relatively new technology to therapeutics faces several safety and technical issues, including delivery of the rna molecule into the virus-infected cells and the activation of interferon system [61, 62] . sars-cov reminds us that viral infections are a global threat. it is vital that the scientific community acquire the ability to develop anti-viral therapy promptly. we can be encouraged by the remarkable speed with which the global community acted in a coordinated research effort to investigate sars-cov. immediately after the last nucleotide of the sars-cov genome was verified, the sequence was distributed through the internet to the worldwide scientific community. among the genomicbased approaches that followed, inhibitors of the viralinduced membrane fusion seem the most promising. the mutation rate of sars-cov is much slower than that of hiv-1 and is among the lowest of rna viruses [63, 64] . however, viral resistance will be an obstacle. the solution could lie in the use of a drug cocktail, combining antiviral drugs with different modes of action (e.g. protease and polymerase inhibitors), to lower the chances for drug-resistant viruses to arise. in addition, drug cocktails are beneficial when the optimal dose of a drug, given as a mono-therapy, is toxic -then, combining drugs with distinct modes of action, in sub-optimal doses, might alleviate toxicity issues. moreover, the recent advancement in the understanding of hiv entry into its host cell revealed an opportunity for synergism, based on the molecular mechanism of viral entry. drugs that inhibit the interaction between ccr5 and the cd4-envelope complexes enhance the efficiency of hiv fusion inhibitors by elongating the exposure time of their target site. the timescale similarity of the sars-cov fusion process to that of hiv is encouraging. hopefully, future identification and characterization of sars-cov receptors will open a way for an efficient antiviral strategy, by synergistically combining viral entry inhibitors. within a few months, scientists have managed to leverage the technological advances of the past 20 years of anti-aids research into an unprecedented antiviral campaign against sars (figure 3) . sars served as a test tube for novel approaches developed following the aids epidemic. the current sars epidemic was finally contained, yet quick development of antivirals is still high priority. today, we are closer than ever to achieving therapeutic solutions for a viral epidemic shortly after viral outbreak. a time line comparing key achievements in aids and sars research. effective international collaborations and technological advances greatly accelerated the understanding of viral diseases. it is anticipated that these research achievements will also lead to faster drug discovery and development. a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome an outbreak of coronavirus oc43 respiratory infection in normandy, france the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus current concepts in sars treatment isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) frequent detection and isolation of cytopathic retroviruses (htlv-iii) from patients with aids and at risk for aids nucleic acid structure and expression of the human aids/lymphadenopathy retrovirus zidovudine resistance of human immunodeficiency virus enfuvirtide: the first therapy to inhibit the entry of hiv-1 into host cd4 lymphocytes an iterative method for improved protein structural motif recognition learncoil-vmf: computational evidence for coiled-coil-like motifs in many viral membrane-fusion proteins t-lymphocyte t4 molecule behaves as the receptor for human retrovirus lav the cd4 (t4) antigen is an essential component of the receptor for the aids retrovirus hiv-1 entry cofactor: functional cdna cloning of a seventransmembrane, g protein-coupled receptor cc ckr5: a rantes, mip-1alpha, mip-1beta receptor as a fusion cofactor for macrophage-tropic hiv-1 resistance to hiv-1 infection in caucasian individuals bearing mutant alleles of the ccr-5 chemokine receptor gene angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus the hiv env-mediated fusion reaction hiv-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by cxcr4 in the hiv-1 env-mediated fusion process sensitivity of hiv-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics impact of mutations in the coreceptor binding site on human immunodeficiency virus type 1 fusion, infection, and entry inhibitor sensitivity anti-human immunodeficiency virus interactions of sch-c (sch 351125), a ccr5 antagonist, with other antiretroviral agents in vitro strong in vitro synergy between the fusion inhibitor t-20 and the cxcr4 blocker amd-3100 human immunodeficiency virus type 1 entry inhibitors pro 542 and t-20 are potently synergistic in blocking virus-cell and cell-cell fusion dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events kinetics of influenza hemagglutinin-mediated membrane fusion as a function of technique kinetically differentiating influenza hemagglutinin fusion and hemifusion machines evolution of intermediates of influenza virus hemagglutininmediated fusion revealed by kinetic measurements of pore formation hiv-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation early events of sars coronavirus infection in vero cells cloaked similarity between hiv-1 and sars-cov suggests an anti-sars strategy interaction between heptad repeat 1 and 2 regions in spike protein of sarsassociated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors suppression of sars-cov entry by peptides corresponding to heptad regions on spike glycoprotein following the rule: formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoproteinmediated viral entry structural characterization of the fusion-active complex of severe acute respiratory syndrome (sars) coronavirus crystal structure of sars-cov spike protein fusion core structural characterization of the sars-coronavirus spike s fusion protein core severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeatderived peptides a rhoa-derived peptide inhibits syncytium formation induced by respiratory syncytial virus and parainfluenza virus type 3 unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group 2 lineage mechanisms and enzymes involved in sars coronavirus genome expression the severe acute respiratory syndrome (sars) coronavirus ntpase/helicase belongs to a distinct class of 5â�² to 3â�² viral helicases small molecules targeting severe acute respiratory syndrome human coronavirus immunogenic peptide comprising a mouse hepatitis virus a59 b-cell epitope and an influenza virus t-cell epitope protects against lethal infection field study of bovine coronavirus vaccine enriched with hemagglutinating antigen for winter dysentery in dairy cows a dna vaccine induces sars coronavirus neutralization and protective immunity in mice inhibition of hepatitis b virus in mice by rna interference inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering rna old drugs as lead compounds for a new disease? binding analysis of sars coronavirus main proteinase with hiv, psychotic and parasite drugs inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells activation of the interferon system by short-interfering rnas nucleic-acid therapeutics: basic principles and recent applications characterization of severe acute respiratory syndrome coronavirus genomes in taiwan: molecular epidemiology and genome evolution molecular evolution of the sars coronavirus during the course of the sars epidemic in china mrna cap-1 methyltransferase in the sars genome multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase identification of novel small-molecule inhibitors of severe acute respiratory syndrome-associated coronavirus by chemical genetics molecular model of sars coronavirus polymerase: implications for biochemical functions and drug design inhibition of severe acute respiratory syndrome-associated coronavirus (sarscov) by calpain inhibitors and beta-d-n4-hydroxycytidine in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds investigational use of ribavirin in the treatment of severe acute respiratory syndrome temporal relationship of viral load, ribavirin, interleukin (il)-6, il-8, and clinical progression in patients with severe acute respiratory syndrome coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor utility of homology models in the drug discovery process characterization of sars-cov main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay high-throughput screening identifies inhibitors of the sars coronavirus main proteinase hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus pages 1020-1028) an important literature citation was omitted from the published reference list.the details of this article we thank l. rychlewski, a. wool, e. eisenberg, n. rabbie, a. toporik, m. olshansky and s.g. peisajovich for critical reading of the manuscript. we are also grateful to artist r. lieber for capturing the central idea of the article with her illustration. key: cord-283127-jetmocvk authors: wang, denong; tang, jin; tang, jiulai; wang, lai-xi title: targeting n-glycan cryptic sugar moieties for broad-spectrum virus neutralization: progress in identifying conserved molecular targets in viruses of distinct phylogenetic origins date: 2015-03-12 journal: molecules doi: 10.3390/molecules20034610 sha: doc_id: 283127 cord_uid: jetmocvk identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as hiv-1. however, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. in this study, we characterized two broadly hiv-neutralizing agents, human monoclonal antibody 2g12 and galanthus nivalis lectin (gna), for their viral targeting activities. although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. the former is hiv-1 specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as hiv-1, severe acute respiratory syndrome coronavirus (sars-cov), and human cytomegalovirus (hcmv). in carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. unlike 2g12, which is strictly specific for the high-density man(9)glcnac(2)asn (man9)-clusters, gna recognizes a number of n-glycan cryptic sugar moieties. these include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent glcnac-terminating n-glycan epitopes (tri/m-gn). these findings highlight the potential of n-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the gna-model of glycan-binding warrants focused investigation. developing broad-range virus-neutralizing antibodies (bnabs) requires identifying the immunological targets that are conserved among the targeted viruses and are suitable for antibody targeting. discovery of oligomannosyl moieties as targets of bnabs against hiv-1 has stimulated substantial interest in carbohydrate moieties as vaccine candidates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . the heavy glycosylation of the hiv envelope with oligomannoses appears to be a defense mechanism for the virus to evade host immune recognition of protein-based neutralizing epitopes. however, isolation of a number of glycan-dependent hiv bnabs from hiv-infected individuals, such as 2g12, pg9/pg16, and other potent bnabs [1, 5, 6, 10, 12, 13] , strongly suggests that the "glycan shield" of hiv virions may also display important targets for immunological intervention against viral infections. like hiv-1, virtually all human viruses decorate their virions with carbohydrate moieties. thus, the potential of viral carbohydrates as immunological targets for other viral pathogens warrants exploration. one intriguing question is whether human viruses of distinct phylogenetic origins, such as hiv-1 and sars-cov, may display conserved glycan targets that are suitable for broad virus neutralization. availability of bnabs targeting such conserved glycans is clearly important for improving a biodefensive public health response to unexpected viral epidemics. in response to the 2003 sars epidemic, our team introduced carbohydrate microarrays to study sars-cov-elicited anti-glycan antibodies [14] . in essence, we characterized the carbohydrate-binding activity of the sars-cov-neutralizing antibodies elicited by immunizing horses with an inactivated sars-cov vaccine. in these horse antibodies, we detected unexpected auto-antibody reactivity specific for the carbohydrate moieties of an abundant human serum glycoprotein, asialo-orosomucoid (asor). the targeted carbohydrates are tri-antennary type ii (galβ1→4glcnac) or multi-valent type ii (tri/m-ii) sugar moieties. the n-glycosylation pathway has potential to generate numerous cryptic n-glycans of distinct structural characteristics. the spike glycoprotein of sars-cov has 23 potential n-linked glycosylation sites [15] [16] [17] [18] . ritchie et al. determined the glycan profiles of the spike protein produced by monkey vero-e6 cells [19] . its major glycans include high-mannose (man5-9glcnac2), hybrid, and bi-, tri-and tetra-antennary complex carbohydrates with and without bisecting glcnac and core fucose. interestingly, sialylation was negligible in the spike proteins produced by monkey vero-e6 cells, which led to exposure of terminal galactoses in tri/m-ii glyco-determinants. thus, induction of anti-asor auto-antibodies by inactivated sars-cov can be attributed to the fact that asor and the sars-cov spike glycoprotein commonly express the tri/m-ii cryptic glyco-determinants. of note, this structural glycomics study revealed that sars-cov also expresses the high-mannose series of carbohydrate structures as its major glycan moieties. thus, despite the fact that the two viruses differ in their general glycan profiles, they commonly overexpress oligomannosyl cores of n-glycans. a significant number of virus-neutralizing agents target oligomannosyl moieties. notably, these include mab 2g12 and lectin gna. however, the two probes represent distinct classes of virus-neutralizing agents. the former is "mono-specific" for hiv-1; the latter is "pauci reactive", being a potent neutralizer for several viruses, including at least hiv-1, hcmv, and sars-cov [20] [21] [22] [23] [24] . given the spectrum of anti-virus activities, gna may serve as a model for exploration of broad-spectrum virus-neutralizing epitopes. an essential step toward this goal is to identify the natural ligands of gna that are preserved among gna-targeted viral pathogens, which is the focus of this study. we reasoned that exploring glycan-binding profiles of broadly virus-neutralizing agents may provide key information to uncover the carbohydrate-based viral neutralization epitopes. in the first set of the experiments, we verified that the preparations of gna or 2g12 we utilized recognize corresponding epitopes presented by the native viral antigens. subsequently, we performed a comparative carbohydrate microarray analysis to characterize the glycan-binding profiles of 2g12 and gna and to pinpoint specific glyco-epitopes they recognize. in figure 1a ,b, we examined detection of gna-and 2g12-epitopes in hiv-1 gp120 glycoproteins. to preserve the native glyco-epitopes, we produced two gp120 preparations in hek293 cells, i.e., bal-gp120-man9 and bal-gp120. bal-gp120-man9 was expressed in hek293 cells in the presence of kifunensine (2 µg/ml), a potent α-mannosidase i inhibitor, following the previously described procedures [25, 26] . the use of the α-mannosidase i inhibitor allows the enrichment of high-mannose type (man9) glycoform through blocking further glycosylation processing to complex or hybrid carbohydrates. thus, bal-gp120-man9 was produced to resemble the native envelope spikes of hiv, which are almost entirely coated with oligomannose antigens [27] . these proteins were applied on elisa plates at 1.0 µg/ml, and the glycan staining was performed in comparison with lectins phaseolus vulgaris-l lectin (pha-l) and sambucus nigra i agglutinin (sna-i). with gna and 2g12 targeting oligomannosyl epitopes, pha-l for tri/m-ii cryptic epitopes, and sna-i recognizing α2-6-linked neu5ac residues, this panel of probes monitors expression of layers of cryptic glyco-epitopes as schematically shown in figure 2a . this assay demonstrated that both bal-gp120-man9 ( figure 1a ) and bal-gp120 ( figure 1b ) were strongly positive with gna and 2g12. pha-l was selectively reactive with bal-gp120 but not with bal-gp120-man9. this pha-l-differential staining pattern reflects the effective blockage of biosynthesis of tri/m-ii complex moieties during bal-gp120-man9 production as we initially planned. sna-1 was negative to both glycoproteins under the same staining condition. elisa results were presented without background subtraction in a-c or with background subtraction in d and e. lectins were applied at the concentrations (µg/ml) as specified. the elisa data shown here are representative results of multiple assays. we further examined whether sars-cov expresses the native gna-epitopes and pha-l-epitopes. in figure 1c , the elisa plates were coated with the native sars-cov antigens that were expressed by vero e6 cells. these plates were treated with a disinfecting fixing agent and gamma-irradiated to inactivate infectious virus particles while preserving antigenic structures of sars-cov. lectins were applied at the concentrations specified in the figures. this assay demonstrates that the pha-l-epitopes were readily detected in the sars-cov-coated elisa plates as we previously observed [14] . moreover, it shows that gna is strongly positive with sars-cov. by contrast, other gal/galnac-reactive lectins, including helix pomatia (hpa) and griffonia (bandeiraea) simplicifolia-i (gs-i), were only marginally reactive with sars-cov antigens. hpa is specific for galnac-and o-glcnac-moieties [28] ; gs-i recognizes the α-gal-epitope [29, 30] . the latter is conserved in many microorganisms but is negligible in sars-cov as expected. in figure 1d , we examined whether hcmv expresses oligomannosyl epitopes using gna and concanavalin a (con a) in comparison with other lectins of different specificities. in this experiment, elisa was coated with purified virus (ad169, creative diagnostics, new york, ny, usa) at protein concentration approximately 1.0 µg/ml for lectin-screening. the two anti-mannose agents differ in epitope-binding specificities. gna is specific for the manα1,3man and manα1,6man moieties of oligomannoses; con a recognizes terminal manα1→moieties that are more broadly expressed by mannose-containing antigens. figure 1e illustrates lectin-binding of a synthetic glycoconjugate, man9-klh, which bears both gna-and con-a-glyco-epitopes [31] . inspection of figure 1d readily reveals that the relative binding activity of gna is markedly higher than the con-a activity in the hcmv-elisa assay although the two mannose-specific lectins are similarly reactive with the man9-klh standard in the assay. other lectins of different glycan-binding specificities show certain reactivities with hcmv lysates, which may reflect the glycome complexity of this most intricate human virus [32, 33] , which requires further study. in summary, we verified expression of the native gna-epitopes by three phylogenetically distinct viruses, hiv-1, sars-cov, and hcmv. in the context of a panel of lectins of different specificities, gna was identified to have the highest relative binding activities with sars-cov and hcmv. the ability of gna to bind and neutralize viruses of distinct phylogenetic origins unavoidably raises questions about its glycan-binding specificity and potential cross-reactivity. one plausible explanation is that these targeted viruses commonly express the defined gna-epitopes of manα1,3man and manα1,6man moieties. however, the selective gna-positive staining of hcmv without parallel con a reactivity ( figure 1d ) suggests an alternative possibility, i.e., presence of other carbohydrate moieties that are negative or weakly reactive with con a but strongly reactive with gna. a carbohydrate antigen with such differential reactivity between gna and con a is a yeast-derived phosphomannan polysaccharide (p-man), which is nevertheless not present in the viral glycome [31, 34] . to support exploration of the potential gna glyco-epitopes in this study, we produced a set of comprehensive antigen microarrays, which include a large-panel of carbohydrates, lipids/liposomes, and protein antigens (supplementary table s1 ). key compounds supporting glyco-epitope analysis include the following: (1) orosomucoid (or) (neu5ac), asor (tri/m-ii), and agalacto-or (agor) (tri/m-gn) (figure 2a ). these autoantigens display distinct glyco-epitopes with identical protein carriers. asor is an asialo-derivative of or, and agor is an agalacto-derivative of asor. they are crucial for defining binding-specificities for n-glycan cryptic epitopes, tri/m-ii, and tri/m-gn. (4) phosphomannan (p-man), a yeast polysaccharide. both m5-6-rb and p-man are known to be positive with gna; the former but not the latter also binds to con a. using these microarrays, we characterized gna, pha-l, and 2g12 for their epitope-binding profiles. results are summarized in figures 2 and 3 . figure 2a is a schematic view of structural relationship among autoantigens, or, asor, and agor and the glyco-epitopes they display. figure 2b shows microarray images of pha-l, gna, or 2g12 staining against a panel of autoantigens, including or, asor, agor, man9, (m9)4, which stands for [(man9glcnac2asn)4]n-klh (m9_2g12) conjugates, and m5-6-rb. the two control probes spotted are klh and e. coli k1 polysaccharide. microarray datasets corresponding to figure 2b are shown in figure 2c . as illustrated, each antigen was spotted in triplicate at given concentrations as specified. microarray detections are shown as the mean fluorescent intensities (mfis) of triplicate microspots for the arrays stained with pha-l, gna, and 2g12, respectively. results were compared using overlay plots of the mfis of staining signal (blue bars) versus those of local backgrounds surrounding the antigen microarrays (red bars). figure 2b ,e show that pha-l and 2g12 are specific for asor (tri/m-ii) and (m9)4, respectively, which is expected. however, gna highly and selectively binds to a number of n-glycan cryptic sugar moieties, including man9, (m9)4, m5-6, and agor. gna-binding of agor is a novel observation. given that gna had no binding to asor (tri/m-ii), which only differs from agor (tri/m-gn) by having the terminal galactoses. this result demonstrates, therefore, that gna is most likely specific for the tri/m-gn-glyco-epitopes of agor with exposed terminal glcnac moieties. in figure 3 below, we illustrate the global antigen-binding profiles of gna and 2g12 against the full panel of antigen preparations. microarray results were plotted as the ratio of antigen-specific signals over the corresponding spots' background readings, i.e., gna ag/bg, or 2g12 ag/bg. to assist a comparative analysis of binding profiles between gna and 2g12, datasets were plotted in the same scale in the y-axis. the positive detections labeled with numbers in the graphs are agor (5), man9 (9), (m9)4 (11), m5-6-rb (15) , and p-man in two dilutions (17 and 18) . the whole datasets for these graphs are listed in supplementary table s2 . in this microarray analysis, gna and 2g12 show minimal or no cross-relativities with irrelevant antigens but illustrate strikingly different glycan-binding profiles. 2g12 is "mono-specific" for man9-clusters (9 and 11) as expected. by contrast, gna is "pauci reactive" with a number of glycan targets. its binding to the spotted oligomannose antigens (5, 9, 11, and 15 ) and p-man (17 and 18) can be attributed to the known gna-specificity for recognition of the shared manα1,3man and/or manα1,6man moieties. however, gna-binding to agor, but not to asor or or, indicates that gna also specifically recognize the tri/m-gn-glyco-determinants displayed by agor. supporting to this prediction is the fact that agor, asor, and or are negative to other anti-mannose agents, including lectin con a, mabs 2g12, tm10, and polyclonal antisera that recognize oligomannosyl moieties of varies cluster configurations [31, 35] . molecular mechanisms underlying gna-recognition of the tri/m-gn-determinants of agor are yet to be explored. a high-precision microarray robot (pixsys 5500c, cartesian technologies, irvine, ca, usa) was used to spot antigen preparations onto glass slides pre-coated with nitrocellulose polymer (fast slides; schleicher & schuell, keene, nh, usa) as described [36] . the antigen preparations applied include carbohydrates, proteins/peptides, and liposomes of various compositions as listed in supplementary table s1 . proteins and carbohydrates were dissolved in phosphate-buffered saline (pbs; ph 7.4) and saline (0.9% nacl), respectively. liposome preparations were suspended in saline (0.9% nacl) at the concentrations specified and were printed in triplicate with spot sizes of ~150 µm and at 375-µm intervals, center to center. the printed microarrays were air-dried and stored at room temperature without desiccant before application. immediately before use, the printed microarrays were rinsed with pbs, ph 7.4, with 0.05% (v/v) tween 20 and then blocked by incubating the slides in 1% (w/v) bovine serum albumin (bsa) in pbs containing 0.05% (w/v) nan3 at room temperature (rt) for 30 min. they were then incubated at rt with 2g12 (nih aids reagent program, germantown, md, usa), biotinylated pha-l (pha-l bi ), or gna bi (ey laboratories, inc., san mateo, ca, usa) at an indicated titration in 1% (w/v) bsa in pbs containing 0.05% (w/v) nan3 and 0.05% (v/v) tween 20. the secondary antibodies or streptavidin conjugates applied for microarray staining are specified in the figure legends. the stained slides were rinsed five times with pbs with 0.05% (v/v) tween 20, air-dried at room temperature, and then scanned for fluorescent signals using a scanarray5000a microarray scanner (perkinelmer life science, boston, ma, usa) following the manufacturer's manual. fluorescence intensity values for each array spot and its background were calculated using scanarray express software (perkinelmer life science, boston, ma, usa). sas institute's jmp-genomics software package (cary, nc, usa) was used for further microarray data processing and statistical analysis. in figure 2 , microarray detections are shown as the mean fluorescent intensities (mfis) of triplicate detections captured by scanarray 5000a for the arrays stained with pha-l, gna, or 2g12. analysis of such microarray data in association with visual inspection of the microarray image provided an evaluation of reproducibility and variation of this antigen microarray technology. in figure 3 , microarray results were plotted as the ratio of antigen-specific signal over corresponding spots' background reading, i.e., gna ag/bg, or 2g12 ag/bg. the whole microarray datasets for pha-l, gna, and 2g12 were presented in supplementary table s2 . two hiv-1 gp120 preparations, bal-gp120-man9 and bal-gp120, were produced in hek293 cells as previously described [25, 26] . bal-gp120-man9 was expressed in the presence of the α-mannosidase i inhibitor, kifunensine (2 µg/ml), to enrich high-mannose type glycoforms. a preparation of sucrose density-gradient-purified hcmv (ad169) was obtained from creative diagnostics (ny, ny, usa). an elisa protocol described previously [37] for detection of anti-carbohydrate antibodies was followed with minor modifications. in figure 1a -e, antigen preparations were diluted in 0.1 m sodium bicarbonate buffer solution, ph 9.6, for coating on elisa microplates (nunc, maxisorp, thermo fisher scientific inc., santa clara, ca, usa) followed by blocking using 1% bsa, pbst. in figure 1c , a sars-cov-specific elisa kit (euroimmun ag, lübeck, germany) was used to measure sars-cov-expression of lectin-specific glyco-epitopes. anti-glycan antibodies or biotinylated lectins were pre-titrated in 1% bsa, pbst for elisa. the bound antibodies were revealed by an alkaline phosphatase (ap)-conjugate of goat anti-human igg-fc-specific antibody, and the biotinylated lectins captured were quantified by ap-streptavidin-conjugate. in this pilot study, we examined whether human viruses of distinct phylogenetic origins may express common carbohydrate moieties. using carbohydrate microarrays and elisa-based viral glycan-profiling analysis, we characterized two broadly hiv-neutralizing agents, human monoclonal antibody 2g12 and lectin gna. although these agents were known to target oligomannosyl antigens, they differ strikingly in the spectrum of viruses they effectively neutralize. the former is solely hiv-specific; the latter is broadly reactive with human viruses, including hiv-1, sars-cov, and hcmv, that are phylogenetically and pathogenically distinct. our carbohydrate microarray analyses demonstrate the two probes differ strikingly in glycan-targeting specificities and the spectrum of glyco-epitopes they recognize. although 2g12 is strictly specific for the high-density man9 clusters that decorate the hiv envelope spike, gna recognizes a number of n-glycan cryptic sugar moieties. these include oligomannoses and the previously unrecognized tri/m-gn-glyco-determinants. the latter appear to be the potent natural ligands of gna. molecular mechanisms underlying the gna-model of "pauci reactive" glycan-binding and broad virus-neutralization warrant further investigation. owing to the potential immunogenic activity as a plant-derived lectin, gna is unlikely suitable for anti-virus therapy in vivo. thus, effort must also be made to establish gna-like potent and broadly virus-neutralizing antibodies, especially humanized or fully human mabs that are readily applicable in the front-line biodefense against emerging viral pathogens. supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/03/4610/s1. antibody domain exchange is an immunological solution to carbohydrate cluster recognition rational antibody-based hiv-1 vaccine design: current approaches and future directions the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2g12 recognizes a cluster of alpha1-->2 mannose residues on the outer face of gp120 the mannose-dependent epitope for neutralizing antibody 2g12 on human immunodeficiency virus type 1 glycoprotein gp120 a limited number of antibody specificities mediate broad and potent serum neutralization in selected hiv-1 infected individuals structure and function of broadly reactive antibody pg16 reveal an h3 subdomain that mediates potent neutralization of hiv-1 pgv04, an hiv-1 gp120 cd4 binding site antibody, is broad and potent in neutralization but does not induce conformational changes characteristic of cd4 rapid development of glycan-specific, broad, and potent anti-hiv-1 gp120 neutralizing antibodies in an r5 siv/hiv chimeric virus infected macaque a potent and broad neutralizing antibody recognizes and penetrates the hiv glycan shield broad neutralization coverage of hiv by multiple highly potent antibodies synthetic carbohydrate antigens for hiv vaccine design human monoclonal antibody 2g12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1 structure of hiv-1 gp120 v1/v2 domain with broadly neutralizing antibody pg9 glycan arrays lead to the discovery of autoimmunogenic activity of sars-cov the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome the sars-cov s glycoprotein: expression and functional characterization identification of n-linked carbohydrates from severe acute respiratory syndrome (sars) spike glycoprotein alpha-(1-3)-and alpha-(1-6)-d-mannose-specific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro marked depletion of glycosylation sites in hiv-1 gp120 under selection pressure by the mannose-specific plant lectins of hippeastrum hybrid and galanthus nivalis profile of resistance of human immunodeficiency virus to mannose-specific plant lectins mannose-specific plant lectins from the amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle expression, glycoform characterization, and antibody-binding of hiv-1 v3 glycopeptide domain fused with human igg1-fc inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, hiv-1 specific antibody envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens the lectin helix pomatia agglutinin recognizes o-glcnac containing glycoproteins in human breast cancer the xenograft antigen bound to griffonia simplicifolia lectin 1-b4. x-ray crystal structure of the complex and molecular dynamics characterization of the binding site evolutionary relationship between the natural anti-gal antibody and the gal alpha 1----3gal epitope in primates uncovering cryptic glycan markers in multiple sclerosis (ms) and experimental autoimmune encephalomyelitis (eae) primate cytomegalovirus glycoproteins: lectin-binding properties and sensitivities to glycosidases quantitative temporal viromics: an approach to investigate host-pathogen interaction anti-oligomannose antibodies as potential serum biomarkers of aggressive prostate cancer n-glycan cryptic antigens as active immunological targets in prostate cancer patients carbohydrate antigen microarrays carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors acknowledge the nih aids reagent program for mab 2g12 and the kabat collection of carbohydrate antigens at sri international for a number of carbohydrate antigens that were applied in this study. this work is supported in part by nih grant numbers r56ai108388 d.w. and j.l.t. conceived and designed the experiments; d.w. and j.t. performed the experiments; l.x.w. contributed key reagents/materials and edited the manuscript; d.w. analyzed the data and wrote the paper.abbreviations or (orosomucoid); asor (asialo-orosomucoid); agor (agalacto-orosomucoid); tri/m-ii (tri-antennary and multivalent type ii (galβ1→4glcnac) chain epitopes); tri/m-gn (tri-antennary or multi-valent glcnac-terminating epitopes); gna (galanthus nivalis agglutinin); pha-l (phaseolus vulgaris-l lectin); sna-i (sambucus nigra i agglutinin); hcmv (human cytomegalovirus); hiv-1 (human immunodeficiency virus-1); sars-cov (severe acute respiratory syndrome coronavirus). the authors declare no conflict of interest. key: cord-304794-z2kx314h authors: métifiot, mathieu; amrane, samir; litvak, simon; andreola, marie-line title: g-quadruplexes in viruses: function and potential therapeutic applications date: 2014-11-10 journal: nucleic acids res doi: 10.1093/nar/gku999 sha: doc_id: 304794 cord_uid: z2kx314h g-rich nucleic acids can form non-canonical g-quadruplex structures (g4s) in which four guanines fold in a planar arrangement through hoogsteen hydrogen bonds. although many biochemical and structural studies have focused on dna sequences containing successive, adjacent guanines that spontaneously fold into g4s, evidence for their in vivo relevance has recently begun to accumulate. complete sequencing of the human genome highlighted the presence of ∼300 000 sequences that can potentially form g4s. likewise, the presence of putative g4-sequences has been reported in various viruses genomes [e.g., human immunodeficiency virus (hiv-1), epstein–barr virus (ebv), papillomavirus (hpv)]. many studies have focused on telomeric g4s and how their dynamics are regulated to enable telomere synthesis. moreover, a role for g4s has been proposed in cellular and viral replication, recombination and gene expression control. in parallel, dna aptamers that form g4s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis a virus (hav), ebv, cauliflower mosaic virus (camv), severe acute respiratory syndrome virus (sars), simian virus 40 (sv40)]. here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of g4-forming oligonucleotides as potential antiviral agents and innovative tools. almost a century ago, the ability of guanosine, but not guanine, to form viscous gels was described (1) . fifty years later, x-ray diffraction data clearly showed that the guanosine moieties in these gels were arranged in a tetrameric organization linked by eight hoogsteen hydrogen bonds ( figure 1 ) (2, 3) . these hydrogen bonds differ from the bonds observed in canonical watson-crick pairing and involve the interaction of the n7 group from one guanine with the exocyclic amino group from a neighboring base (figure 1a ). therefore, a g-tetrad or a g-quartet results from planar association between four guanines that are held together by eight hydrogen bonds and coordinated with a central na+ or k+ cation (4) (5) (6) (7) (8) . in addition, nucleoside derivatives were also used to confirm the structural properties of g-quartets (9) (10) (11) (12) (13) (14) . conversely, a g-quadruplex or g4 is formed by nucleic acid sequences (dna or rna) containing g-tracts or gblocks (adjacent runs of guanines) and composed of various numbers of guanines. depending on the nucleotide sequence, the way g4s can be formed presents a high degree of diversity. the core of a g4 is based on stacking between two or more g-tetrads, wherein the guanines can adopt either a syn or an anti glycosidic bond angle conformation. consequently, each of the four g-tracts that form the core of the structure can run in the same or opposite direction with respect to its two neighbors, forming parallel, antiparallel or hybrid core conformations. depending on these orientations, the g-blocks delimit four negatively charged grooves of different sizes: narrow, medium or wide (figure 1b-e) . for intra-molecular structures (figure 1b and c) , the four g-tracts belong to the same oligonucleotide and are attached by linkers with variable nucleotide sequences and lengths. these loops can adopt three different conformations: lateral, diagonal or propeller (figure 1b-d) . the bi-or tetra-molecular g4 structures (figure 1d -e) are assembled from g-tracts belonging to two or four different strands. the g-blocks can also be interrupted by one to seven non-g nucleotides, which result in bulges that protrude from the g4 core ( figure 1e ). in contrast to the almost mono-morphic canonical duplex, these variable structural parameters are directly related to the nucleotide primary sequence. this unique family of globular-shaped nucleic acid structures (figure 1f -j) presents a high level of plasticity that enables various applications (see 'g4s as antiviral agents' section). however, a potential physiological role for g4s has remained controversial for many years. nonetheless, interest in g-quaduplexes has increased over time, with thousands of reports and reviews published on several aspects of g4s, including the biophysical and chemical characteristics as well as biological function in prokaryotes and eukaryotes (5, (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) . with an increasing volume of sequencing data, databases and algorithms have also been developed to enable search and mapping of g4 in a few mammalians as well as hundreds of bacterial species (25) (26) (27) (28) . additionally, g4s present an intrinsic resistance to 'regular' nucleases. however, g4 specific nucleases have been isolated, first in yeast with kem1/sep1 (29) and later in humans with gqn1 (30) . later, a dna binding protein (g4r1/rhau) that can unfold rna/dna quadruplexes was isolated in human cells (31, 32) , which further highlights the biological relevance of g4s. finally, development of g4specific probes such as monoclonal antibodies (33) (34) (35) and small chemical ligands (36) (37) (38) (39) (40) (41) facilitated in vivo studies that support quadruplex formation in cells. taken together, g4s are likely present in several significant genomic regions and may be a key component in important cellular processes (42, 43) . the sequencing of the human genome highlighted the presence of many sequences enriched in guanines that can potentially form quadruplexes [for a review, see (44) ]. in theory, more than 350 000 putative g-tetrads may be formed with loops of 1-7 nucleotides, and over 700 000, with loops of up to 12 nucleotides. however, the genomic dna is dynamic, and quadruplexes, duplexes or other structural forms at those sites will be influenced by chromatin and other dna-binding proteins. thus, g4 formation depends on the cell type and cell cycle, ultimately impacted by environmental conditions and stresses. without clear signaling and tight regulation, extremities present in linear chromosomes could be recognized as damage dna and it would be deleterious for the cell if processed as such by repair mechanisms (45) . telomere length has also been linked to the lifespan of organisms. telomeres are nucleoprotein structures found at the end of chromosomes protecting the genome from instability. this terminal region of chromosomes contains long tracts (several kilobases) of double stranded ttaggg:ccctaa repeats ending with a 3 protrusion of single stranded ttaggg repeats (10-50 repeats) . telomerase is a ribonucleoprotein enzyme that adds ttaggg repeats to the 3 end of dna strands in the telomere regions. human telomerase is a heterodimeric complex composed of (i) telomerase reverse transcriptase (tert), a dna-polymerase rna-dependent, (ii) a singlestranded rna template referred to as telomerase rna component (terc) and (iii) dyskerin [dyskeratosis congenita 1 (dkc1)], a pseudouridine synthase binding terc through the h/aca motif and stabilizing the telomerase complex (46, 47) . accumulated experimental data indicate the presence of g4s in telomeric dna (6, (48) (49) (50) (51) (52) . for example, dna2, a helicase/nuclease that cleaves g4s, is involved in maintaining telomere integrity (53, 54) . altogether, these observations clearly establish that telomeric g4s are crucial structures for regulating telomere maintenance, thus providing a mechanism for controlling cell proliferation (55, 56) . at the 3 ends of telomeres, if the g-rich overhang is longer than four ttaggg repeats (>23 nucleotides), it can fold over itself and form secondary structures, including g4s (figure 2a) . those structures prevent telomere elongation by the telomerase complex (57) . accordingly, small g4-ligands (e.g. telomestatin) and g4-binding proteins (e.g. trf2) show anti-proliferative and potential anti-tumor activities through telomere interference (36, (58) (59) (60) (61) . however, the exact mechanism is more complicated as telomestatin derivatives present telomerase independent activity, most likely through targeting g4s involved in tumor growth elsewhere in the genome (62) (63) (64) . dna replication is highly regulated, which ensures faithful reproduction of genetic information through each cell cycle. in eukaryotes, this process is initiated at thousands of dna replication origins (oris) distributed along each chromosome (65) . initially, a pre-replicative complex (pre-rc) is assembled during late mitosis and the early g1 phase; it evolves during the s phase to a pre-initiation complex through initiator protein activity (e.g., cyclin-dependent kinases, cdc7, cdc45 and minichromosome maintenance complex (mcm) proteins). this step involves unwinding the origin and dna polymerase recruitment. a tissuespecific temporal system exists, and only a few origins undergo 'firing' (replication fork elongation). the remaining origins are dormant backups used under stress (e.g., dna damage and collisions with transcription machinery). studies on organisms ranging from drosophila to humans have highlighted that repeated elements are enriched with g residues (66) . in vitro, origin g-rich repeated element (ogre) sequences form g4s, and point mutations that affect the stability of these g4s also impair origin function. even if a 200-bp cis-regulatory element is necessary for efficient initiation, g4 structure formation at oris might be the key to selecting the firing origins (67, 68) . additionally, g4s formed in the lagging strand can lead to the stalling of the replication fork ( figure 2d ). on the other side, formation of g4s in the leading strand can displaced it from the template and causes polymerase slippage (figure 2d ). such events could participate to the unusual expansions of g4 forming mini-satellite sequences (69) . altered gene expression levels are implicated in many human diseases. g4s have been found in the promoter region of many genes and implicated in transcription and protein translation regulation [for a review, see (70) ]. particularly interesting, the p1 region, which is upstream of the c-myc promoter, is highly sensitive to dnase i and s1 nucleases (nuclease-hypersensitive element or nhe). this purine-rich sequence can form an intramolecular g4 (71) (72) (73) (74) , which structure has been determined by nuclear magnetic resonance (nmr) (75) . this nheiii 1 region controls up to 90% of gene transcription activation, and the g4 acts as a transcriptional repressor element (76, 77) . because interactions between dna-binding proteins and local dna supercoiling can impact the equilibrium between duplex/singlestranded dna (active transcription) and quadruplex dna (silent), c-myc expression and, ultimately, cell proliferation may be modulated. nucleolin (a nucleolar phosphoprotein) and adar1 (a z-dna binding/rna editing protein) both bind the c-myc promoter in vivo and stabilize the g4 structure of the promoter (78, 79) . due to the high therapeutic potential of genomic g4s, studies have also been performed on promoters of other genes implicated in cancer biology. for example, kras is one of the most frequently mutated genes in human cancer. its promoter contains a g-rich nuclease hypersensitive element that is critical for transcription (80) . the polypurine strand forms g4 structures that bind crucial dna repair proteins (e.g., parp-1, ku70) (81) . direct evidence for g4 formation was also reported for the proximal promoter region of the ret proto-oncogene, and targeting this region with a small molecule represses ret proto-oncogene transcription (82) . although many g-rich sequences have been identified in the genome, they enable generation of corresponding grich rnas when the sequences are located in transcription units. ultimately, these rnas can fold into quadruplex structures and impact gene expression (83) . in 5untranslated regions (5 -utrs), g4s can act as translation repressors (84) (85) (86) . g4 structures have also been proposed to regulate translation driven by ires domains in the absence of an mrna cap [for a recent review, see (87) and the references therein]. finally, it has recently been shown that g4 rnas in coding regions can stimulate ribosomal frameshifts in vitro and in cultured cells providing a role for g4 in translational regulation (88, 89) . a major challenge for future studies is to better understand genome instability, cancer and severe neurological/neuromuscular defects origins. repeated sequences, such as polynucleotide repeats (three and above), mini-and mega-satellites, are source of genome instability through either expansion or contraction (90) . however, dna secondary structures associated with those tandem repeats may be crucial elements in the expansion phenomenon. for progressive myoclonus epilepsy type-1 (epm1), expansion of the dodecamer sequence (cgcg 4 cg 4 ) within the cystatin b promoter is thought to depend on g4 formation (91) . in addition to this potential deleterious consequence, g4s have also been implicated in development of specific immune responses. in antigen-activated b cells, class switch recombination promotes deletion of a dna fragment (several kilobases) that joins a new constant region to the variable immunoglobulin chain (92) . these switch regions (s regions) are intronic, g-rich and repetitive sequences that form g-loops upon transcription (figure 2e ). in vivo, class switch recombination depends on the activity of aid (a cytidine deaminase) and muts␣ (msh2/msh6, and con-served dna repair factors involved in mismatch repair). aid promotes creation of u:g mismatches, and muts␣ binds with high affinity the g4 dna formed upon transcription of the s regions. in parallel, pathogens have also developed countermeasures to evade the immune system. the role of gquartets in this evasion is related to antigenic variation. for plasmodium falciparum, g-rich sequences have been found in the upstream region of group b var genes that can form in vitro stable g4s (93) . similarly, a g4-forming sequence (5 -g 3 tg 3 ttg 3 tg 3 ) is located upstream of the pile expression locus in neisseria gonorrhoeae (94) . formation of g4s is necessary for initiating this pilin antigenic variation through recombination, and interactions with reca could facilitate this specialized reaction. overall, immune evasion and immunoglobulin gene class switch recombination in vertebrates are clearly analogous mechanistically (95) . searches for g4s and elucidation of their function in the viruses' genome have mainly focused on the human immunodeficiency virus (hiv), which causes acquired immunodeficiency syndrome (aids). currently, ∼35 million people are infected with hiv worldwide. with over 2 million new infections and ∼1.6 million deaths from aids per year, the pandemic continues to spread. even without a vaccine, development of highly active anti-retroviral therapies have allowed people to live with hiv as a chronic disease (96) . however, viruses within t cells remain fully capable of replicating and infecting other cells if the drug pressure is removed or when resistance emerges. thus, new drugs must be developed to overcome the treatment's genetic barrier. hiv-1 is an rna virus in the lentivirus genus and is part of the retroviridae family. lentiviruses are singlestranded, positive-sense, enveloped rna viruses. hiv-1 particles contain two molecules of genomic rna that are converted into double-stranded dna by the viral reverse transcriptase (rt). the resulting viral dna is then imported into the nucleus and insertion into the cellular dna is catalyzed by the virally encoded integrase (in). once integrated, transcription from the viral promoter at the 5 long terminal repeat generates mrnas that code various viral proteins and genomic rna. alternatively, the provirus may become latent, which allows the virus and its host cell to avoid detection by the immune system. the presence of g4 structures has been highlighted at both rna and dna levels with implications throughout the viral life cycle. retroviral rnas dimerize in the cytoplasm of an infected cell allowing two copies of the genome to be encapsidated in the newly produced virion (97) . while a single copy of the genome is sufficient for viral replication, the second copy is also used during reverse transcription, and the viral rt switches multiple times between the two rna molecules (98, 99) . the strand transfers are partially responsible for the viral variability through production of recombinant molecules. therefore, understanding the mechanisms that drive dimerization and recombination is essential. dimerization is a two-step process that involves sequences upstream of the splice donor site (100, 101) . the sequences involved in initial dimerization and encapsidation partially overlap at the 5 end of the viral genome. one of the sequences is a highly conserved dimer initiation site (dis) that forms a stem loop. a concentration-dependent kissing-loop interaction is initiated from contacts between consecutive guanines (102); the interaction then spreads to the stems. however, this interaction does not seem sufficiently strong to keep the two copies together during reverse transcription. several studies have identified a g-rich sequence that form bi-molecular g4 structures in the gag region of the hiv-1 genome, near the dis (103-105). localization of these g4-forming sequences correlates with recombination hot spots and exhibits an increased rate of template switching that highlights a potential role for these structures (106) . supporting this hypothesis, recombination in the u3 domain is cation-dependent and is lower in the presence of li + , which is a metal ion that fails to stabilize g4s (106). short rna templates from the central region of the hiv-1 genome contain g-rich sequences near the central polypurine tract (cppt) at the 3 end of the pol gene (in coding sequence); this is a region where one of the two primers used for synthesizing the (−) strand dna is produced during reverse transcription. these sequences can form both intramolecular and dimeric g4 structures. moreover, reconstituted systems have confirmed that g4 structures near the cppt facilitate strand transfer and promote template switching by the rt (107) . interestingly, certain elements from the cppt region are involved in forming the cppt flap, which is a region that plays an important role in nuclear entry of the double-stranded dna (108, 109) . taken together, the g-rich regions located at the 5 end of the genome and in the central region are likely maintained in proximity through inter-rna g4 formation with a crucial role in hiv-1 replication. retroviral nucleocapsid proteins (ncp) are multifunctional elements encoded in the gag gene. notably, ncp participate in many retroviral cycle steps by remodeling nucleic acid structures to favor thermodynamically stable conformations. they are referred to as nucleic acid chaperones and interact with nucleic acid phosphodiester backbones through electrostatic interactions thanks to basic residues (especially residues in the n-terminus) [for reviews, see (110, 111) ]. moreover, hiv-1 ncp (ncp7) exhibits sequence-specific binding to runs of gs, ugs or tgs through interactions involving its two zinc fingers (cchc motifs separated by a proline-rich linker). although there is no doubt that ncp7 tightly binds g4 sequences, data in the literature shows that a hydrophobic interaction engaged by the c-terminal zinc finger of the protein may lead to g4 stabilization (112) , while high concentration of ncp7 promotes g4 unfolding (113) . a recent biophysical study using high-speed atomic force microscopy (hs-afm) addressed direct and real-time investigations on the molecular chaperone activity at the single-molecule level (114) . ncp7 can efficiently promote bimolecular g4 formation and is able to anneal the g4 structures. the g4 structure is induced by both unprocessed ncp15 and mature ncp7, which indicates that both proteins may participate in genome recognition, recombination, dimerization and packaging. ncp7 could act through a potential mechanism that involves synaptic g4 intermediates, as illustrated in figure 3 (115) . transcription of the viral dna is performed by the cellular rna polymerase ii from the viral promoter located in the 5 -long terminal repeat (ltr) of the proviral genome. the u3 region (figure 4a) (tss) and close to the tata box (figure 4b ). this sequence overlaps with the so-called minimum promoter, which is composed of three sp1 as well as two nf-kb binding sites and is crucial for transcription initiation. the presence of eight blocks of guanines suggests that this region is a good candidate for g4 formation. two independent biophysical studies based on dmsmediated foot-printing assays or nmr recently evaluated the ability of this 45-nt g-rich sequence to form g4 structures (116, 117) . dna fragments spanning this region (corresponding to two or three sp1 or nf-kb binding sites with oligonucleotides ranging from 19 to 32 bases) were all able to form stable parallel and anti-parallel g4 topologies with melting temperatures ranging from 43 • c to 56 • c in 100-mm kcl solutions. the three models are formed with rather long sequences (up to 32 nucleotides), allowing the formation of long loops of 5 to 12 nucleotides (figure 4 d, e and f). interestingly, a g4 structure was also observed in the nmr study with an anti-parallel g4 core composed of only two tetrads and an additional watson-crick base pair that stacks on top of the upper tetrad (figure 4g) . notably, these four topologies are mutually exclusive, and forming one of these g4s in the promoter will prevent formation of the three alternative conformations. thus, the equilibrium between these forms may play a role in regulating promoter activity. recently, the interaction between the sp1 protein and a fragment of the hiv-1 promoter sequence folded into a g4 was studied (107) . piekna-przybylska et al. used an affinitybased selection approach using biotinylated g4s immobilized on streptavidin-coated magnetic beads. the pull down experiments followed by western blotting revealed that the sp1 protein can bind the hiv-1 promoter sequence when it adopts a g4 conformation. perrone et al. analyzed the effect of point mutations that disrupt the g4 structures formed in the promoter (116) . the wild-type (wt) and mutated ltr promoters were cloned upstream of a firefly gene in a promoter-free plasmid. in hek 293t cells, the promoter activity of the mutated sequences (unable to form g4) was twice as high as the wt ltr. these data suggest that g4s act as repressor elements in the transcriptional activation of hiv-1. therefore, g4 structures might be critical for hiv-1 fitness and represent novel targets for antiviral drug development (see below). a better understanding of the role for g4s in regulating the hiv-1 promoter activity would also shed light on hiv-1 latency and reactivation mechanisms, from which a new landscape may emerge in clinical research to eradicate hiv from reservoirs (118) . a g-rich sequence composed of three conserved clusters has been identified in the reading frame of the negative regulatory factor (nef) (119) . this coding sequence is located at the 3 -end of the genome and overlaps with the 3 -ltr (figure 4a ). isolated nef g-stretches can form g4s in vitro. moreover, their stabilization represses nef expression and decreases viral replication. nef has multiple roles during hiv infection and has been implicated in immune evasion by promoting cd4 and major histocompatibility complex (mhc) molecule down-regulation. importantly, the absence of nef seems to correlate with low viral load and inhibition of disease progression. thus, targeting the g4s located in the nef coding sequence is a new attractive therapeutic opportunity. sv40 is a polyomavirus with a 5-kb, closed circular and double-stranded dna genome that codes for six proteins and includes a non-coding regulatory region (ncrr). this latter regulatory region contains the ori and the encapsidation sequence (ses) but also controls the transcription direction (early versus late transcription). notably, six gc boxes (gggcgg) are present in this region, which can form an unusual quadruplex structure containing a ctetrad stacked between two g-tetrads as determined by nmr (120) . these repeated sequences are binding motifs for sp1 and, therefore, play an important role in early transcription. on the other hand, sv40 genome replication requires the large t-antigen (tag), which is a multifunctional protein that binds the ori, has adenosine triphosphatedependent helicase activity and interacts with cellular proteins such as p53 and rb (121) . interestingly, tag can unwind g4 dna structures (122, 123) ; thus, it might play a crucial role in regulating replication as well as early and late transcription. perylene di-imide derivatives (pdi) stabilize g4 structures and inhibit both the g4 and tag duplex dna helicase activities. hence, pdi provide tools for probing the role of the g4 helicase activity in sv40 replication (123, 124) and introduces new insights into the link between helicases and tumorigenesis (125) or other human genetic diseases (126) . the human papillomaviruses (hpv) family consists of more than 120 viruses; approximately half are sexually transmissible and 15 are considered high risk due to their carcinogenic properties. these viruses are one of the most common sexually transmitted infections and induce cervical cancer. notably, hpv16 and hpv18 contribute to 70% of cervical cancer induced by hpv infections. the hpv genome consists of 8 kb, circular, double-stranded dna and integration into the infected cell induces dramatic genome instability (127) . the open reading frames encode six 'early' proteins (e1, e2, e4, e5, e6 and e7) and two 'late' structural proteins (l1 and l2). the late proteins are major and minor capsids, respectively, and association with 72 l1/l2 heterodimers forms star-shaped capsomeres. however, l1 can self-assemble into 55-60 nanometers, virus-like particles, which can be used in prophylactic hpv vaccines to protect against an initial hpv infection. g-rich regions have been found in hpv genomes, and their potential to fold into a g4 structure has been described (128) . g-rich loci that fulfill the criteria for g4 formation have only been found in eight types of hpv. however, a strong argument for the relevance of g4s in hpv biology is that the viral protein e1 is a helicase that resembles sv40 tag (121) . consequently, e1 may also present a g4 unwinding activity. for hpv52 and hpv58, potential g4-forming sequences are located in the long control region (lcr), which is a regulatory sequence composed of nearly 1 kb, suggesting a potential role in transcription and replication. the presence of g4s in the sequence coding for the l2 protein (hpv57), e1 (hpv32, hpv42) and e4 (hpv3, hpv9, hpv25) suggests that g4 formation may also alter alternative splicing necessary for producing viral proteins from the overlapping open reading frame (orfs). targeting these g4s could potentially serve as a basis for novel antiviral therapies. hodgkin's lymphoma, burkitt's lymphoma and nasopharyngeal carcinoma (129) . in the case of hiv-1 co-infection, ebv is also associated with hairy leukoplakia and central nervous system lymphoma. the virus is ∼120 to 180 nm in diameter and is composed of a 172 kb dna double helix that circularizes upon entry into the nucleus and becomes a viral episome. human herpes viruses are mostly asymptomatic due to latency. the epstein-barr virus encodes a genome maintenance protein (nuclear antigen 1, ebna1), expressed in all ebvassociated malignancies. ebna1 binds g-rich sequences at the viral replication origin, recruits the replication complex and is involved in metaphase chromosome attachment, which insures maintenance throughout mitosis (130) . thus, formation of secondary structures, such as g4s, may play a role in regulating ebv replication. the level of ebna1 synthesized is tightly controlled; it is sufficiently high to maintain viral infection but sufficiently low to avoid immune recognition by the host's virus-specific t cells. regulation occurs during translation due to secondary structures present in the mrna (131) . destabilization of the g4-forming sequence through antisense oligonucleotide annealing increases the translation rate and, consequently, promotes antigen presentation. on the other hand, stabilization of the g4 with a g4 ligand (pyridostatin) decreases ebna1 synthesis and allows immune evasion. interestingly, g4s are similarly observed in mrna for other maintenance genes in the gammaherpesvirus family. these findings suggest that this mode of translational regulation may be more general among proteins that self-regulate synthesis. in addition, one could imagine alternative therapeutic strategies focused on targeting rna structures within viral orfs to interfere with the virus cycle as well as to promote antigen presentation and to stimulate the host immune response. recently, a g4 ligand called quarfloxin (cx-3543) entered phase ii clinical trials. it is capable to suppress myc transcription by inhibiting the interaction between nucleolin and the c-myc g4 motifs (132) . even if cx-3543 failed trials because of bioavailability issues, original anti-cancer applications based on such ligands seem underway (133) . taken together, all these studies suggest that targeting these g4s could potentially serve as a basis for novel antiviral therapies. hence, as described for cellular targets (134) , small ligands that can stabilize the g4 structure may compose a potential new class of therapeutic agents to fight viral infections (62, 135) [for reviews, see (136, 137) ]. recent studies showed that g4 ligands, such as braco-19 and tmpyp4, inhibited hiv-1 replication (116, 119) . exhaustive viral assays demonstrated that braco-19 acts at both reversetranscription and post-integration steps by targeting of the viral promoter (138) . several laboratories are already working to identify g4 ligands that preferentially interact with the pathogen's g4 over cellular g4s. this new antiviral strategy presents many advantages: (i) these compounds target viral dnas and rnas, which are the source of the disease; (ii) high conservation of these targets across subspecies suggests that they are important for viruses and (iii) mutations will likely impact viral fitness, limiting the emergence of resistant strains. nucleic acids have already been validated as therapeutics (139) . the first example is formivirsen (vitravene r ), a phosphorothioate anti-sense oligonucleotide approved from 1998 to 2002 to treat retinitis caused by cytomegalovirus (cmv) (140) . the second is pegaptanib (macugen r ), used in the clinic from 2004 to 2011. this 2 -fluoropyrimidine rna-based aptamer targets vegf to treat neovascular age-related macular degeneration (141) . however, those two drugs have been withdrawn due to the insufficient benefice associated with their use. regarding g4s, certain g4 oligonucleotides present therapeutic potential, including the thrombin-binding aptamer (tba, g 2 t 2 g 2 tgtg 2 t 2 g 2 ) (142, 143) . the most promising g4 to date is as1411 (agro100), a 26 bases oligonucleotide targeting nucleolin with anti-proliferative properties (144) . as1411 recently completed phase ii clinical trials as anticancer drug (nct00740441) with low toxicity, highlighting the high therapeutic potential of g4 (145) . for most applications, g4-forming molecules have been selected through combinatorial methods (e.g. pegaptanib, anti-tba). two key protocols have been developed. first, polynucleotide arrays facilitated rapid detection of molecules with an affinity to the target. then, the development of selex (systematic evolution of ligands by exponential enrichment) facilitates isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecule with high affinity and specificity (146) . these oligonucleotides, 'aptamers', are emerging as a class of molecules that rival antibodies in both therapeutic and diagnostic applications (147, 148) . in this section, we describe a few examples of aptamers specifically selected to interact with viral components and interfere with viral replication. hepatitis a virus (hav) belongs to the picornaviridae family. even without a specific treatment for hav infection, a vaccine has been produced to protect against initial infection. however, only a limited number of countries recommend vaccination because hav is rarely lethal. the hav genome comprises a 7.5-kb single-stranded (+) rna with a 5 -utr and 3 poly(a) tail. it encodes for a single polyprotein, which includes the 3c protease. this enzyme is crucial for the virus because it cleaves the polyprotein into several capsid proteins and non-structural proteins. additionally, the 3c protease binds regulatory structural elements at the 5 -utr, which control viral genome replication. thus, the 3c protease is an attractive target for develop a specific antiviral treatment. recently, hexadeoxyribonucleotides that specifically bind the hav 3c protease were identified through a hexanucleotide array (149) . in vitro experiments showed that the hexanucleotide gggggt (g 5 t) forms a tetramolecular g4, binds the c-terminal domain of hav protease and is a potent protease inhibitor (150) . every year in winter, influenza viruses cause seasonal respiratory disease epidemics (151) . three subtypes have been defined (a, b and c), which depend on the surface proteins (hemagglutinin and neuraminidase). sixteen hemagglutinin (h) and nine neuraminidase (n) variants have been discovered, but only h 1, 2 and 3 as well as n 1 and 2 are commonly found in humans. these viruses belong to the orthomyxoviridae family, which composes enveloped viruses with a single-stranded negative-sense rna. the genome is 10 to 15 kb but is segmented into multiple (7 or 8) molecules. each rna is 0.9 to 2.3 kb and encodes for 1 or 2 proteins. the non-structural protein 1 (ns1) is a 26 kda multifunctional protein and participates in protein-protein and protein-rna interactions (152) . during infection, ns1 interferes with cellular mrna biology (splicing, maturation and translation). as a result, ns1 prevents interferon (ifn) production, which leads to inhibition of the host's innate immunity (153) . after 15 cycles of selex, which employs a 45-base variable sequence, a g4-forming aptamer was selected that presents high affinity for the ns1 rna binding domain (kd ≈ 20 nm) (154) . in a cellular context, this g4 can block ns1 and restore inf production, which results in antiviral activity without cellular toxicity. severe acute respiratory syndrome coronavirus (sars-cov) is an enveloped virus with a single-stranded rna genome ∼30 kb long. it encodes 2 poly-cistronic orfs, which code for 16 non-structural proteins (nsp). since the 2003 outbreak, the three-dimensional structures of several key proteins have been determined (e.g. rdrp, 3c-like protease). the non-structural protein 3 (nsp3) is one component of the viral replicase complex and contains a domain referred to as sars unique domain (sud), which interacts with g4s. thus, g4s are also relevant for the coronavirus and might be involved either in viral replication or host immune evasion (155) . on the other hand, using a se-lex approach with oligonucleotides that harbor a 30 nucleotide variable sequence and following 20 rounds of selection, dna aptamers against the sars-cov helicase were isolated (156) . these aptamers compose two distinct classes, g4 and non-g4 forming sequences, which were determined through circular dichroism and gel electrophoresis. their inhibitory effect on viral replication is being studied with encouraging results. combinatorial approaches have been used to identify several aptamers that target hiv-1. the first rna aptamer isolated using the selex approach was an rna pseudoknot inhibitor of hiv-1 rt (157) . using another procedure, the surf (synthetic unrandomization of randomized fragments) (158) , isis 5320 was selected and exhibited sub-micromolar inhibition of hiv-1. isis 5320 is a phosphorothioate containing dna octamer (t 2 g 4 t 2 ) that forms g4 with anti-hiv properties. more specifically, it inhibited cell-to-cell and virus-to-cell spread of the hiv-1 by interacting with the v3 loop located on the viral glycoprotein gp120 (159) (160) (161) . its g4 structure and phosphorothioate backbone were reported as essential to this inhibition. later studies showed that phosphodiester oligonucleotides containing only g and t inhibit hiv-1 replication and the most potent molecule, gtg 2 tg 3 tg 3 tg 3 t (t30177, ec 50 at ∼100 nm), formed a g4 in vitro (162) (163) (164) . this oligonucleotide and related molecules, such as t30923 (g 3 tg 3 tg 3 tg 3 t, figure 1j ), are potent hiv-1 in inhibitors in vitro. t30177 was the first in inhibitor tested in clinical trials (zintevir tm developed by aronex pharmaceuticals in 1996) (165) . however, the action mechanism in cells is more complex because it also targets viral entry (166) . in attempts to obtain natural-type oligonucleotide, hotoda et al. identified a hexamer (tg 3 ag) also targeting hiv-1 entry through gp120 binding (167) . this 'hotoda's sequence' adopts a tetramolecular g4 structure and submicromolar hiv-1 inhibition was described for derivatives with 5 -end substitutions. once more, the antiviral activity of the molecule was directly linked to its capability to form g4s. later, selex approaches were developed to isolate dna aptamers with high affinity for the rnase h domain of hiv-1 rt. thus, the target protein used was either the isolated rnase h domain (p15) or the functional heterodimer p66/p51 with a counter-selection that used the p51/p51 form (truncated for the rnase h domain) (168, 169) . interestingly, these selections mainly led to aptamers with g-rich sequences capable of forming g4s. some, but not all, of these g4 inhibited the rnase h activity of hiv-1 rt in vitro with an ic 50 in the nm range (168) . surprisingly, these g4 aptamers (93del and 112del) were also potent in inhibitors in vitro with ic 50 values in the range 10-40 nm (170) . this dual inhibition can be explained by the structural similarities between the in active site and rt rnase h domain (171) . the aptamer 93del can form an original dimeric interlocked g4 (figure 1i) , which is stable even at temperatures over 90 • c (172) . through comparing the structures of the g4-forming aptamers that inhibit in, in inhibition likely requires a stack of 6 gtetrads (164) . similar to t30177, 93del is a potent antiviral agent with multimodal inhibition and, in cells, targets the viral entry step, reverse transcription and integration (173) . additional in vitro studies indicated that free 93del and t30923 can enter human cells, including epithelial (hela), hepatic (huh7) and lymphocytes (h9) cells (174) . however, striking differences were observed in the presence of viral particles; hiv-1 strongly stimulates cellular uptake of aptamer (174) . this latter observation opens an opportunity for specific drug delivery to cells that are infected, which may prevent intracellular side effects from g4 off-targeting. nucleic acids research, 2014, vol. 42, no. 20 12361 given the remarkable physical properties of g4s, scientists have engineered innovative tools using g4 folding to control ribozyme activity and constructed probes for state-ofthe-art imagery as well as quantification techniques, among other approaches. as an example, the aforementioned g4 93del was engineered into aptamer beacons to visualize endogenous protein hiv-1 reverse transcriptase in living cells (175) . the following section describes two such innovations related to viruses. hdv is a small, enveloped virus that depends on hepatitis b virus (hbv) for propagation (176) . its genome is a circular, single-stranded (−) rna molecule of 1679 nucleotides. however, due to self-complementarity over ∼70% of its length, it folds into a partially double-stranded molecule (rod-like rna structure). because hdv does not encode for a polymerase, replication of its genome relies on cellular enzymes. upon polymerization following the 'double rolling circle' mechanism, genomic and anti-genomic circular rna is produced with linear polyadenylated mrna that only codes for the hdv protein, delta antigen (hdag). however, hdv also encompasses an 85 nucleotide ribozyme with self-cleavage activity. molecular engineering led to development of a 'g-quartzyme', which is a ribozyme controlled by a g4 structure (177, 178) . stabilization of the g4 at the 3 end by monovalent ions (k + ) activates the ribozyme. cauliflower mosaic virus (camv) is a pararetrovirus that infects plants (179) . its genome is an 8-kb, circular, doublestranded dna molecule produced through reverse transcription of a pre-genomic mrna. therefore, transcription of the genome is a crucial step for two reasons: (i) it is necessary to code for several viral proteins and (ii) it generates 35s rna that is slightly longer than the genome (terminally redundant), which serves as a matrix to replicate the dna genome. because the unique camv promoter, the 35s promoter, is a strong and constitutive promoter, it has been widely used to develop genetically modified organisms (gmos). consequently, there is a growing need to detect gmos in the food industry, and many methods have been developed based on elisa, polymerase chain reaction and other techniques. notably, a fluorescent assay has been engineered based on g4 formation. it involves two specific primers that recognize the 35s promoter. each primer is linked to a double repeat of the human telomeric sequence ttaggg. as a consequence, binding the two primers facilitates inter-strand quadruplex formation. upon adding berberine (a selective g4-binder), a strong fluorescent signal is produced (180) , which facilitates promoter detection. the x-ray structure of g-tetrads was determined in the early 60s; in the 90s, g4s remained an intriguing deviation from the watson-crick canonical structure. however, a remarkable quantity of information has been obtained over the past two decades, which has allowed this field to grow from basic science to clinical application (181, 182) . thus, the presence of g4s in telomeres and oncogenic promoters has opened broad opportunities for understanding and generating new treatments against cancer. the viral world is extensive and includes viruses with replication schemes based on rna (e.g., flaviviruses), dna (e.g., papillomavirus) or intermediates thereof (e.g., retroviruses) and g4s are part of their life cycle. first, they must address the presence of g4s in their hosts, and second, they must also contain g4-forming sequences that regulate important replication steps. several challenges remain to better define the features that provide specific g4 motifs with the ability to function as structural elements. nevertheless, g4 sequences and g4-binders have been identified in some of the most pathogenic viruses; thus, they are attractive targets for controlling viral infections. pdb ids: 2kf8, 2lpw, 2m4p, 1y8d and 2le6. untersuchungenüber die guanylsäure helix formation by guanylic acid the structure of helical 5 -guanosine monophosphate poly(inosinic acid) helices: essential chelation of alkali metal ions in the axial channel guanine quartets telomeric dna dimerizes by formation of guanine tetrads between hairpin loops a phase diagram for sodium and potassium ion control of polymorphism in telomeric dna a k cation-induced conformational switch within a loop spanning segment of a dna quadruplex containing g-g-g-c repeats physicochemical properties of nucleosides 3. gel formation by 8-bromoguanosine physico-chemical properties of nucleosides. 4. gel formation by quanosine and its analogues nucleoside conformations. x. an x-ray fiber diffraction study of the gels of guanine nucleosides nucleoside conformations. xi. solvent effects on optical properties of guanosine and its derivatives in dilute solutions nucleoside conformations. 12. an infrared study of the polymorphism of guanine nucleosides in the solid state nucleoside conformations. xiii. circular dichroism of guanosine gels and the conformation of gpg and poly (g) four-stranded nucleic acid structures 25 years later: from guanosine gels to telomer dna g-quartet structures in telomeric dna comprehensive supramolecular chemistry multistranded dna structures quadruplex structures in nucleic acids thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids biological aspects of dna/rna quadruplexes beyond nucleic acid base pairs: from triads to heptads g-quadruplex dna structures-variations on a theme genome-wide prediction of g4 dna as regulatory motifs: role in escherichia coli global regulation qgrs mapper: a web-based server for predicting g-quadruplexes in nucleotide sequences grsdb: a database of quadruplex forming g-rich sequences in alternatively processed mammalian pre-mrna sequences quadbase: genome-wide database of g4 dna-occurrence and conservation in human, chimpanzee, mouse and rat promoters and 146 microbes extensive selection for the enrichment of g4 dna motifs in transcriptional regulatory regions of warm blooded animals the yeast kem1 gene encodes a nuclease specific for g4 tetraplex dna: implication of in vivo functions for this novel dna structure a human nuclease specific for g4 dna g4 resolvase 1 binds both dna and rna tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex g4-dna and g4-rna resolving activity in hela cell lysates the dexh protein product of the dhx36 gene is the major source of tetramolecular quadruplex g4-dna resolving activity in hela cell lysates selective recognition of a dna g-quadruplex by an engineered antibody detection of g-quadruplex dna in mammalian cells in vitro generated antibodies specific for telomeric guanine-quadruplex dna react with stylonychia lemnae macronuclei visualization and selective chemical targeting of rna g-quadruplex structures in the cytoplasm of human cells quantitative visualization of dna g-quadruplex structures in human cells stabilization of quadruplex dna perturbs telomere replication leading to the activation of an atr-dependent atm signaling pathway small-molecule-induced dna damage identifies alternative dna structures in human genes telomestatin, a novel telomerase inhibitor from streptomyces anulatus telomerase inhibition with a novel g-quadruplex-interactive agent, telomestatin: in vitro and in vivo studies in acute leukemia prevalence of quadruplexes in the human genome structural insights into g-quadruplexes: towards new anticancer drugs existence and consequences of g-quadruplex structures in dna telomeres: protecting chromosomes against genome instability a telomerase component is defective in the human disease dyskeratosis congenita human dyskerin: beyond telomeres all gene-sized dna molecules in four species of hypotrichs have the same terminal sequence and an unusual 3 terminus genomic reorganization in ciliated protozoans telomeric dna oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs an overhanging 3 terminus is a conserved feature of telomeres monovalent cation-induced structure of telomeric dna: the g-quartet model processing of g4 dna by dna2 helicase/nuclease and replication protein a (rpa) provides insights into the mechanism of dna2/rpa substrate recognition mammalian dna2 helicase/nuclease cleaves g-quadruplex dna and is required for telomere integrity telomerase: dr jekyll or mr hyde? telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening g-quadruplex formation at the 3 end of telomere dna inhibits its extension by telomerase, polymerase and unwinding by helicase discovery of g-quadruplex stabilizing ligands through direct elisa of a one-bead-one-compound library recent developments in the chemistry and biology of g-quadruplexes with reference to the dna groove binders an intramolecular g-quadruplex structure is required for binding of telomeric repeat-containing rna to the telomeric protein trf2 novel g-quadruplex stabilizing agents: in-silico approach and dynamics g-quadruplex stabilizer induces m-phase cell cycle arrest reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action telomestatin impairs glioma stem cell survival and growth through the disruption of telomeric g-quadruplex and inhibition of the proto-oncogene, c-myb replication in context: dynamic regulation of dna replication patterns in metazoans new insights into replication origin characteristics in metazoans unraveling cell type-specific and reprogrammable human replication origin signatures associated with g-quadruplex consensus motifs g4 motifs affect origin positioning and efficiency in two vertebrate replicators stimulation of gross chromosomal rearrangements by the human ceb1 and ceb25 minisatellites in saccharomyces cerevisiae depends on g-quadruplexes or cdc13 making sense of g-quadruplex and i-motif functions in oncogene promoters dna tetraplex formation in the control region of c-myc the cationic porphyrin tmpyp4 down-regulates c-myc and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo direct evidence for a g-quadruplex in a promoter region and its targeting with a small molecule to repress c-myc transcription the dynamic character of the g-quadruplex element in the c-myc promoter and modification by tmpyp4 propeller-type parallel-stranded g-quadruplexes in the human c-myc promoter ribonucleoprotein and protein factors bind to an h-dna-forming c-myc dna element: possible regulators of the c-myc gene puf/nm23-h2/ndpk-b transactivates a human c-myc promoter-cat gene via a functional nuclease hypersensitive element identification and characterization of nucleolin as a c-myc g-quadruplex-binding protein novel interaction of the z-dna binding domain of human adar1 with the oncogenic c-myc promoter g-quadruplex chromatin structure of the promoter region of the human c-k-ras gene g-rich oligonucleotide inhibits the binding of a nuclear protein to the ki-ras promoter and strongly reduces cell growth in human carcinoma pancreatic cells formation of pseudosymmetrical g-quadruplex and i-motif structures in the proximal promoter region of the ret oncogene an rna g-quadruplex in the 5 utr of the nras proto-oncogene modulates translation an unusually stable g-quadruplex within the 5 -utr of the mt3 matrix metalloproteinase mrna represses translation in eukaryotic cells a g-quadruplex structure within the 5 -utr of trf2 mrna represses translation in human cells -utr g-quadruplex structures acting as translational repressors 5 -utr rna g-quadruplexes: translation regulation and targeting stimulation of ribosomal frameshifting by rna g-quadruplex structures translational recoding induced by g-rich mrna sequences that form unusual structures repeat instability as the basis for human diseases and as a potential target for therapy tetraplex formation by the progressive myoclonus epilepsy type-1 repeat: implications for instability in the repeat expansion diseases mutsalpha binds to and promotes synapsis of transcriptionally activated immunoglobulin switch regions putative dna g-quadruplex formation within the promoters of plasmodium falciparum var genes reca-binding pile g4 sequence essential for pilin antigenic variation forms monomeric and 5 end-stacked dimeric parallel g-quadruplexes the g4 genome hiv integrase inhibitors: 20-year landmark and challenges cell biology of retroviral rna packaging retroviral recombination and reverse transcription extensive recombination among human immunodeficiency virus type 1 quasispecies makes an important contribution to viral diversity in individual patients moloney murine sarcoma virus genomic rnas dimerize via a two-step process: a concentration-dependent kissing-loop interaction is driven by initial contact between consecutive guanines functional characterization of the dimer linkage structure rna of moloney murine sarcoma virus a loop-loop 'kissing' complex is the essential part of the dimer linkage of genomic hiv-1 rna evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic rna mode of dimerization of hiv-1 genomic rna dimerization of human immunodeficiency virus type 1 rna involves sequences located upstream of the splice donor site hiv-1 nucleocapsid protein increases strand transfer recombination by promoting dimeric g-quartet formation mechanism of hiv-1 rna dimerization in the central region of the genome and significance for viral evolution hiv-1 genome nuclear import is mediated by a central dna flap analysis of the viral elements required in the nuclear import of hiv-1 dna first glimpses at structure-function relationships of the nucleocapsid protein of retroviruses comparative nucleic acid chaperone properties of the nucleocapsid protein ncp7 and tat protein of hiv-1 g-quartets direct assembly of hiv-1 nucleocapsid protein along single-stranded dna unfolding of dna quadruplexes induced by hiv-1 nucleocapsid protein hiv-1 nucleocapsid proteins as molecular chaperones for tetramolecular antiparallel g-quadruplex formation solution structures of all parallel-stranded monomeric and dimeric g-quadruplex scaffolds of the human c-kit2 promoter a dynamic g-quadruplex region regulates the hiv-1 long terminal repeat promoter topology of a dna g-quadruplex structure formed in the hiv-1 promoter: a potential target for anti-hiv drug development u3 region in the hiv-1 genome adopts a g-quadruplex structure in its rna and dna sequence formation of a unique cluster of g-quadruplex structures in the hiv-1 nef coding region: implications for antiviral activity nmr observation of a novel c-tetrad in the structure of the sv40 repeat sequence gggcgg the large tumor antigen: a 'swiss army knife' protein possessing the functions required for the polyomavirus life cycle the sv40 large t-antigen helicase can unwind four stranded dna structures linked by g-quartets real-time investigation of sv40 large t-antigen helicase activity using surface plasmon resonance simian virus 40 large t-antigen g-quadruplex dna helicase inhibition by g-quadruplex dna-interactive agents dna helicases as targets for anti-cancer drugs the bloom's syndrome helicase unwinds g4 dna genome-wide analysis of hpv integration in human cancers reveals recurrent, focal genomic instability human papillomavirus g-quadruplexes spectrum of epstein-barr virus-associated diseases role for g-quadruplex rna binding by epstein-barr virus nuclear antigen 1 in dna replication and metaphase chromosome attachment g-quadruplexes regulate epstein-barr virus-encoded nuclear antigen 1 mrna translation targeting g-quadruplexes in gene promoters: a novel anticancer strategy? g-quadruplexes as potential therapeutic targets for embryonal tumors nucleic acids targeted to drugs: selex against a quadruplex ligand small-molecule interaction with a five-guanine-tract g-quadruplex structure from the human myc promoter g-quadruplexes as targets for drug design g-quadruplexes: targets in anticancer drug design anti-hiv-1 activity of the g-quadruplex ligand braco-19 nucleic acid aptamers: research tools in disease diagnostics and therapeutics pegaptanib, a targeted anti-vegf aptamer for ocular vascular disease selection of single-stranded dna molecules that bind and inhibit human thrombin thrombin binding aptamer, more than a simple aptamer: chemically modified derivatives and biomedical applications agro100 inhibits activation of nuclear factor-kappab (nf-kappab) by forming a complex with nf-kappab essential modulator (nemo) and nucleolin a phase ii trial of as1411 (a novel nucleolin-targeted dna aptamer) in metastatic renal cell carcinoma systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t4 dna polymerase development of aptamer therapeutics aptamers as tools in molecular biology and immunology specific recognition of proteins by array-bound hexanucleotides functional binding of hexanucleotides to 3c protease of hepatitis a virus influenza: evolving strategies in treatment and prevention multiple alignment comparison of the non-structural genes of influenza a viruses a recombinant influenza a virus expressing an rna-binding-defective ns1 protein induces high levels of beta interferon and is attenuated in mice single-stranded dna aptamer that specifically binds to the influenza virus ns1 protein suppresses interferon antagonism the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes differential inhibitory activities and stabilisation of dna aptamers against the sars coronavirus helicase rna pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase rational screening of oligonucleotide combinatorial libraries for drug discovery combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeficiency virus envelope-mediated cell fusion potent and specific inhibition of hiv envelope-mediated cell fusion and virus binding by g quartet-forming oligonucleotide (isis 5320) inhibition of human immunodeficiency virus type 1 infection in scid-hu thy/liv mice by the g-quartet-forming oligonucleotide suppression of human immunodeficiency virus type 1 activity in vitro by oligonucleotides which form intramolecular tetrads inhibition of the human immunodeficiency virus type 1 integrase by guanosine quartet structures hiv-1 integrase inhibitor t30177 forms a stacked dimeric g-quadruplex structure containing bulges hiv-1 in inhibitors: 2010 update and perspectives human immunodeficiency virus glycoprotein gp120 as the primary target for the antiviral action of ar177 (zintevir) biologically active oligodeoxyribonucleotides. 5. 5 -end-substituted d(tgggag) possesses anti-human immunodeficiency virus type 1 activity by forming a g-quadruplex structure dna aptamers selected against the hiv-1 rnase h display in vitro antiviral activity targeting hiv-1 integrase with aptamers selected against the purified rnase h domain of hiv-1 rt dna aptamers derived from hiv-1 rnase h inhibitors are strong anti-integrase agents closely related antiretroviral agents as inhibitors of two hiv-1 enzymes, ribonuclease h and integrase: 'killing two birds with one stone an interlocked dimeric parallel-stranded dna quadruplex: a potent inhibitor of hiv-1 integrase the guanine-quadruplex aptamer 93del inhibits hiv-1 replication ex vivo by interfering with viral entry, reverse transcription and integration cellular uptake of odns in hiv-1 human-infected cells: a role for viral particles in dna delivery? aptamer beacons for visualization of endogenous protein hiv-1 reverse transcriptase in living cells an update on hdv: virology, pathogenesis and treatment potassium ions modulate a g-quadruplex-ribozyme's activity modulating rna structure and catalysis: lessons from small cleaving ribozymes structure and replication of caulimovirus genomes a novel fluorescent biosensor for detection of target dna fragment from the transgene cauliflower mosaic virus 35s promoter quadruplex nucleic acids therapeutic applications of quadruplex nucleic acids weblogo: a sequence logo generator key: cord-291063-de7v4e5s authors: moens, ugo title: silencing viral microrna as a novel antiviral therapy? date: 2009-05-28 journal: j biomed biotechnol doi: 10.1155/2009/419539 sha: doc_id: 291063 cord_uid: de7v4e5s viruses are intracellular parasites that ensure their existence by converting host cells into viral particle producing entities or into hiding places rendering the virus invisible to the host immune system. some viruses may also survive by transforming the infected cell into an immortal tumour cell. micrornas are small non-coding transcripts that function as posttranscriptional regulators of gene expression. viruses encode mirnas that regulate expression of both cellular and viral genes, and contribute to the pathogenic properties of viruses. hence, neutralizing the action of viral mirnas expression by complementary single-stranded oligonucleotides or so-called anti-mirnas may represent a strategy to combat viral infections and viral-induced pathogenesis. this review describes the mirnas encoded by human viruses, and discusses the possible therapeutic applications of anti-mirnas against viral diseases. viruses are common habitants of the human population, where they establish different forms of infection, including an acute, a chronic, or a persistent infection with production of low levels of virions. some viruses can exist in a true latent state in which infectious particles are only produced upon reactivation stimuli. viruses that reside harmlessly in their host can under certain conditions or in immunocompromised persons be responsible for malignant and nonmalignant diseases, which may even lead to the death of the host. a causal role for human polyomaviruses (hpyv), papillomaviruses (hpv), herpesviruses (hhv), hepatitis b virus (hbv), hepatitis c virus (hcv), and human t-cell lymphotropic virus type-i (htlv-i) and cancer is accepted (for recent reviews see [1] [2] [3] [4] [5] [6] [7] ). it is estimated that oncoviruses are associated with 15% of the human cancers [8] , while nonmalignant infections from human immunodeficiency virus (hiv), hbv and hcv alone cause more than 3 million deaths annually worldwide [9] . other viral infections (hiv no included) were responsible for the death of more than 6000 patients in japan in 2006, ∼7000 individuals in the usa in 2005, and 555 people in united kingdom in 2006 according the statistics of the world health organization [10] . thus the pathogenic properties of viruses necessitate the development of efficient antiviral therapies. viruses attempt to create a favorable cellular environment allowing viral replication or survival by establishing a lifelong latent infection through evading the immune system of their hosts. viruses can hide within a cell by restricting their activity to a minimum so as not to conceal their presence to the immune system and at the same time they will also try to avoid apoptosis. for these purposes, viruses have developed different strategies, one of which includes the posttranscriptional regulation of both cellular and viral gene expressions through modulating the host's rna-interference (rnai) machinery. viruses can suppress the rnai pathway by viral microrna (vmirna) targeting cellular or viral transcripts, or by viral proteins (e.g., human immunodeficiency virus tat protein, influenza virus ns1/ns2 protein, ebola vp35 protein, and vaccinia virus e3l protein) or viral rna (adenovirus va transcripts) that counteract the host's rnai machinery (for recent reviews see [11] [12] [13] [14] [15] [16] [17] ). this review summarizes the recent findings on virus-encoded mirnas and their described functions and briefly discusses the potential of antiviral mirna as a novel therapeutic strategy in combating virus infections. anti-mirna oligonucleotides (amos) are chemically modified synthetic oligonucleotides that are complementary to their target sequence and this will silence the action of the target. amos are modified with the dual purpose to stabilize them and to improve their affinity for their targets. one modification is the 2' sugar modification which implies a chemical modification of the 2'-o of the ribose residue (figure 1(a) ). the 2'-o -methyl amos have a methyl group linked to the 2'-o of the ribose residue, while the 2'-o -methoxyethyl amos contain a methoxygroup. this modification provides improved rnase resistance and binding affinity to rna compared to unmodified antioligonucleotides. however, 2'-o -methoxyethyl amos possess a higher affinity and specificity to rna than their 2'-o -methyl amos. other 2' sugar modifications that have been used include 2'-fluor and locked nucleic acid (lna). in lna-modified oligonucleotides, the 2'-ooxygen is bridged to the 4'-position via a methylene linker to form a rigid bicycle, locked into a c3'-end (rna) sugar conformation (figure 1(a) ). lnas give very strong duplex formation with their target sequences and they display excellent mismatch discrimination, hence avoiding off-target effects (for recent reviews see [28] and [29] ). lna injections against mir-122, a cellular mirna involved in lipid metabolism, resulted in efficient and long-lasting decrease in plasma cholesterol in african green monkeys without any evidence for toxicities [30] . a second type of modification is the phosphorothioate backbone which reduces the affinity to the target somewhat, but it confers significant stability to nuclease degradation (figure 1(b) ). a third generation of antisense oligonucleotides are phosphodiamidate morpholino oligomers (pmo) in which the ribose ring is replaced with a morpholine ring. adding an argininerich peptide (rxr) 4 further increased the stability and tissue retention of the pmo (reviewed in [31] ). an additional modification can be made to improve the cellular uptake of the amo. krützfeldt et al. linked a cholesterol moiety to their amos and referred to these anti-mirnas as antagomirs. antagomirs should be >19 nucleotides in length to provide highest efficiency in silencing target mirna [32] [33] [34] . the putative therapeutic potentials of antagomirs were recently demonstrated in treatment of lipid metabolic disease in animals [35] . an alternative class of amos is peptide nucleic acids (pna), which are synthetic oligonucleotides with n-(2-aminoethyl)-glycine replacing the deoxyribose or ribose backbone [36] . a study published in 2008 reported that pna can efficiently block the action of cellular mirnas [37] . finally, another approach in silencing mirna is the use of so-called microrna sponge, a synthetic mrna that contains multiple binding sites for a particular mirna and that is transcribed from a plasmid containing a strong promoter (reviewed in [28] ). in conclusion, different classes of amo have been shown to be efficient in silencing mirna and may be useful therapeutic tools (reviewed in [29, [32] [33] [34] 38] ). samples and they are referred to as kipyv wupyv [41, 42] . this year a novel human polyomavirus, merkel cell polyomavirus (mcpyv), was identified that is associated with merkel cell carcinoma [43] . the hpyv genome can be divided into three functional regions. the early region encodes the early proteins large t-antigen (lt-ag) and small t-antigen (st-ag), while the late region encodes the capsid proteins vp1-vp3 and the regulatory protein agnoprotein. both regions are separated by the noncoding control region that encompasses the origin of replication and the promoter/enhancer sequences for the early and late genes (reviewed in [44] ). the sv40 genome encodes a viral mirna (vmirna) of which both arms are complementary to the early viral mrnas and reduces expression of the early proteins (figure 2(a); table 1 ). cells infected with mutant sv40 lacking this mirna or with wildtype sv40 yielded comparable levels of infectious viruses, but the latter were less sensitive to lysis by cytotoxic t cells and produced less interferon-γ. thus sv40-encoded mirna allows the virus to evade the immune system [45] . the sv40 mirna is conserved in bkv and jcv and both mirnas generated from the precursor hairpin bind to the same target, that is, the early transcripts. the bkv and jcv mirnas serve the same role as sv40 mirna, that is, downregulation of early expression. jcv mirna, mir-j1, was readily detected in brain samples of pml patients, suggesting a biological role of this mirna [46] . the group of sullivan has also identified a mcpyv-encoded mirna, mir-m1, which does not share sequence identity with the known mirnas of the other polyomaviruses. mcpyv mir-m1 is located in the early region (figure 2 (a)) and can downregulate early gene expression. in accordance with sv40, this may allow the virus to evade the immune system. however, mcpyv is associated with merkel cell carcinoma and the viral genome is integrated in these tumours [43] . blocking mir-m1 by, for example, antagomirs will increase the expression of the viral oncoprotein lt-antigen and as such have little beneficial therapeutic effect [47] . expression of a corresponding or other viral-encoded mirna for the other hpyv wu and ki is lacking so far. human papillomaviruses (hpv) are nonenveloped viruses with a circular dsdna genome of approximately 8000 base-pairs. these viruses are associated with benign and malignant lesions of the skin and the genital tract. more than 100 different hpv genotypes have been identified and based on their association with benign warts or cancer, they are classified as low-risk and high-risk variants, respectively [1] . one study with hpv type 31 failed to clone vmirna from virus-infected cells [58] . however, this does not exclude that other strains may encode vmirna. moreover, the expression of vmirna may be regulated in a temporal and spatial manner, so that the experimental conditions for capturing vmirna may be tricky. in addition, the high mutation rate of hpv genome sequences may impede the prediction of the presence of putative vmirna. adenoviruses are naked dsdna viruses that can cause mild respiratory, gastrointestinal, urogenital, and ocular disease. more than 50 serotypes have been described in human. although there is no proof for a causative role in malignancies, adenoviruses can induce cancer in animal models and have been extensively studied to scrutinize viral mechanisms for cellular transformation [59] . adenovirus encodes small noncoding rnas, known as virus-associated rna or vai and vaii rna, which are generated by rna polymerase iii. this noncoding rna plays an important role for viral replication and neutralizes the antiviral action of interferon by blocking the dsrnainduced protein kinase (pkr), which phosphorylates and thereby inactivates the eukaryotic translational initiation factor 2 [60] . a recent study showed that a minor fraction of vai rna is processed by dicer into functional risc-associated ssrna which can act as mirna ( table 1) . blocking of vai-derived small rna by 2'-o -methyl amo complementary to vai decreased virion production [61] [62] [63] . in an attempt to identify potential targets for vai-derived mirna, computational analysis was used. putative targets include genes encoding apoptosis-related protein napor and pkr-activating protein pact. therefore vai rnaderived mirna may help adenovirus to escape the actions of the host defense mechanisms [64] . herpesviruses are enveloped dsdna viruses with a genome size ranging between ∼130 to ∼250 kilobase-pairs. they are divided into three subfamilies denoted α, β, and γ. approximately 130 different herpesviruses have been identified to date, including the human α-herpesviruses herpes simplex virus 1 (hsv-1 or hhv-1), herpes simplex virus 2 (hsv-2 or hhv-2), varicella-zoster virus (vzv or hhv-3), the β-herpesviruses cytomegalovirus (hcmv or hhv-5), hhv-6a, hhv-6b, and hhv-7, the γ-herpesviruses epstein-barr virus (ebv or hhv-4), and kaposi's sarcoma-associated virus (kshv or hhv-8). all human herpesviruses are able to establish latent infections with only a small subset of viral genes expressed (reviewed in [64] ). as will be discussed in the next section, among the viral transcripts that can be detected in latently infected cells are viral-encoded mirnas, which seem to be required to maintain a latent state of infecton, but may also contribute to the pathogenic properties of the virus. herpes simplex virus-1 (hsv-1 or human herpes virus 1; hhv1) infects the majority of the human population, but remains a latent cohabitant in most people. reactivation of the viruses usually results in cold sores, but it can also cause a spectrum of diseases from sightthreatening ocular infections in immunocompetent adults to more severe infections in newborns and immunosuppressed patients (reviewed in [89] ). an important gene in hsv latency is the lat gene. this gene encodes the latency-associated transcript (lat), which does not code for a protein. lat seems to promote cell survival of the infected cells [90] . gupta and coworkers proposed that the antiapoptotic activity of lat was achieved by an mirna-entrapped in the lat (mir-lat), which downregulates the expression of transforming growth factor-β (tgf-β) and smad3. the latter is a mediator of the signalling pathway induced by tgf-β, while tgfβ can prevent cell proliferation and induce cell death [67] . however, a later study revealed that the described mir-lat was not viral encoded, but in fact a cellular mirna expressed in sh-sy-5y cells [67] and the report describing mir-lat was retracted [91] . work by umbach et al. could also not confirm the existence of this mirna in hsv-1 infected sh-sy-5y cells [48] . later, it was shown that the hsv-1 lat exon encodes hsv-1 mir-lat-icp34.5, which can be detected in hsv-1 infected cells [89] . circa 120 base-pairs upstream in this region, a sequence with 77% homology to the hsv-2 mirna mir-i (see further) is present, but no mature mirna was detected in hsv-1 infected cells. however, the existence of this mirna during hsv-1 latency in vivo remains to be confirmed [68] . computational analysis predicted 24-mirna candidates in the hsv-1 genome, 8 of which were conserved in hsv-2, suggesting they may be functional mirnas [69] . the authors confirmed the expression of one mature mirna, designated mir-h1, in hsv-1 infected vero cells, where it was expressed late in productive replication. this mirna is encoded approximately 450 bp upstream of the transcription start site of the lat transcript, but a corresponding sequence is not conserved in the hsv-2. the function of mir-h1 6 journal of biomedicine and biotechnology downregulates the expression of cmv genes involved in its own replication process, for example, transactivators ie72 and ie86; ul120/121; ul114mhc class i-related chain b (micb), a cellular ligand for the activating receptor nkg2d; downregulation of ie-1 mir-ul148d-1 mir-us4-1 mir-us5-1 mir-us5-2 mir-us24 paramyxoviridae (measles virus) 1 predicted [65] remains to be established and no cellular target mrnas were identified [69] . umbach and coworkers detected in addition to mir-h1 five novel viral mirnas in trigeminal ganglia of mice latently infected by hsv-1, as well as in hsv-1-infected vero cells. these mirnas, that is, mir-h1 to mir-h6, are encompassed in the lat locus ( figure 2 (b)). by quantitative rt-pcr, the authors were able to roughly estimate the number of copies of each mirna during productive infection of vero cells. mir-h1 and mir-h6 were expressed at ∼1200 and 300 copies, respectively, while the other mir-hs were present at less than 40 copies per infected cell. in latently infected trigeminal ganglia, much higher levels were monitored with 63 000 copies of mir-h2, 8000 copies of mir-h3, 800 000 copies of mir-h4, 80 000 copies of mir-h5, and 40 000 copies of mir-h6. the large difference in numbers of mirna transcripts in latently infected cells compared to cells with productive hsv-1 infection indicates that mir-hs play an important role in establishing latent hsv-1 infection. indeed, mir-h2 expression diminished the protein levels of icp0, a viral transcriptional activator that promotes viral replication, while mir-h6 inhibits expression of icp4, which is required for expression of most hsv-1 genes during reproductive infection [48] . hsv-2 typically infects the genital region and establishes a lifelong latent infection. the prevalence of latent hsv-2 infection varies between 10-60%. reactivation can cause oro-facial and genital herpes, but hsv-2 infection can also cause encephalitis and neonatal herpes, and forms a risk factor for hiv acquisition [89] . the only detectable viral transcript during hsv-2 latency is the lat, but the molecular function of this transcript remains largely unknown [92] . hsv-2 lat exon encodes an mirna, referred to as mir-i, which is expressed during latent, as well as during acute infection. remarkably, several promoters regulate mir-i expression in different stages of the viral life cycle. this hsv-2 mirna efficiently diminishes the expression of the viral neurovirulence factor icp34.5, a multifunctional protein required for viral replication in neuronal cells in vivo, and with intrinsic neurovirulent properties [93] . thus, mir-i may affect the outcome of infection (latent versus productive) by modulating the protein levels of icp34.5. whether mir-i has other targets remains to be investigated, but an mir-i analogue is also expressed by hsv-1, indicating the importance of this mirna for these viruses [68] . tang and colleagues identified two new hsv-2 mirnas, mir-ii, which includes mir-ii-5p and mir-ii-3p, and mir-iii, both encoded by exon 2 of lat. the expression of mir-i, -ii, and -iii increased during infection of cells, but mir-iii displayed slower kinetics than the two other mirnas. similar to mir-i, mir-ii silences the expression of icp34.5,while mir-iii functionally resembles hsv-i mir-h2 in that it can downregulate the expression of icp0 [70] . the virus establishes a latent infection, but reactivation leads to herpes zoster, commonly referred to as shingles. acute vzv reactivation may lead to post-herpetic neuralgia [94] . no putative mirnas could be predicted in the vzv genome [65, 71] , but experimental studies to unambiguously proof the existence of vzv mirna are lacking. table 1 and figure 2(c) ). three viral mirnas, designated as ul23-5p, mir-ul23-3p, and mir-us24, were identified that are expressed during productive hcmv infection of permissive cells (human foreskin fibroblasts, astrocytoma u373mg cells, retinal pigment epithelial cells, and human microvascular endothelial cells). their putative cellular target genes include genes encoding transcription factors (e.g., hnf3 and tgif2), receptors (e.g., il-18 receptor 1 precursor; cd206), proteins implicated in t-cell activation (ahnak1), in signal transduction (e.g., rab2l), and in biosynthesis of leukotrienes that sustain inflammatory reactions (coactosin-like protein). whether these genes represent bona fide targets as well as the biological relevance of hcmv mirna-mediated silencing of these genes remains elusive [49] . hcmv usually establishes a lifelong persistent or latent state in healthy individuals by ensuring that infected cells avoid immune recognition. the hcmv-encoded mirna mir-ul112-1 seems to play a central role in helping the virus to hide from the host's immune system. this viral mirna targets mrna for mhc class i-related chain b (micb), and to a lesser extend mica. these proteins are cellular ligands for the activating receptor nkg2d, which is expressed on some natural killer (nk) cells, γ/δ t cells, and cd8+ t cells. during cellular stress (such as viral infection) micb is induced, thus activating nk-cells and t cells that can lead to the killing of infected cells. cells infected with mutant virus lacking this mirna were more susceptible to being killed in an nkg2d-dependent manner by nk cells [72] . the mir-ul112-1 also represses the expression of hcmv genes involved in its own replication process, in part by targeting mrna encoding immediate early proteins. one of them is the viral transactivator protein called immediate early 72 (ie72) that regulates the transcription of viral genes required for acute replication [50] . ie72 plays a pivotal role in controlling latency and reactivation. mir-ul-112-1 can thus restrict reactivation of the virus through negative regulation of ie72 expression [50, 95] . two separate studies showed that mir-ul112-1 also inhibited expression of the immediate early protein ie1. murphy et al. detected increased ie1 levels in cells infected with either a virus lacking mir-ul112-1 or with mutations in the seed sequence of the ie1 gene compared to cells infected with wild-type hcmv [73] . grey and coworkers demonstrated that addition of mir-ul112-1 rna prior to infection reduced ie1 protein levels and blocked viral replication [74] . ie1 is a crucial protein to ascertain lytic replication of hcmv, thus downregulation of ie1 may help the virus to establish latency. this strategy may be a common feature for herpesviruses because immediateearly genes may be putative targets for hsv-1 mir-lat, ebv mir-bart15 and mir-bhrf1-3, and kshv mir-k12-6-3p [73] . yet another target for mir-ul112-1 is the viral protein ul114, a homologue of the mammalian dna repair enzyme uracyl-dna glycosylase. ul114 is required for efficient viral dna replication. hence, mir-ul112-1mediated downregulation of ul114 may prevent viral dna replication and favor a latent infection state [65] . taken together, the actions of mir-ul112-1 seem to be associated with latent viral infection, a state which allows the virus to hide from the immune system. ablating expression of this viral-encoded mirnas by amos may therefore force the virus into a lytic cycle and provide the immune system the opportunity to get ride of the viral infection. -lymphocytes in more than 90% of the human population. ebv is associated with infectious mononucleosis and has been implicated in the pathogenesis of several malignancies including burkitt's and hodgkin's lymphomas, posttransplant and t-cell lymphomas, x-linked lymphoproliferative syndrome, nasopharyngeal, and gastric carcinomas [97] . latently ebv-infected cells are classified in stage i, ii, or iii, each of them characterized by distinct ebv gene expression [98] . among the latency stage-specific ebv transcripts are mirnas. more than 20 different ebv mirnas have been identified that are transcribed during latent infection (table 1 ; [25, 51, [75] [76] [77] [78] [79] ). the ebv mirnas are organized in two major clusters within the ebv genome (figure 2(d) ). one cluster resides in the bart (abbreviation for bamhi-a rightward transcripts) region. the bart region gives rise to multispliced transcripts and is highly expressed in ebv-positive cancers and in epithelial tissues, while there is low bart expression in b lymphocytes. the exact function of bart mrnas remains obscure [76] . the intronic region of bart also encodes the mirnas mir-bart1 to mir-bart20. the second region that encodes multiple mirnas is the untranslated region of the gene encoding bhrf1 (bamhi fragment h rightward open reading frame 1), a viral bcl-2 homologue that prevents apoptosis. bhrf1 encompasses the mirnas mir-bhrf-1-1 to mir-bhrf-1-3 [51, 75] . the expressions of ebv-encoded mirnas in clinical samples and computational analysis to predict putative targets were applied to unravel the biological functions of ebv mirnas. these approaches showed that the mir-barts are abundantly expressed in latently infected epithelial cells, nasopharyngeal carcinomas, ebv-associated gastric carcinoma cell lines and tissues, burkitt's lymphomas latency type i, ebv positive primary effusion lymphomas, and diffuse large b-cell lymphomas, but at a significantly lower level in b cells. this corresponds well with the expression pattern of bart multispliced transcripts (see above). higher levels of bhrf1-3 were measured in latency type iii burkitt's lymphomas and in diffuse large b-cell lymphomas [66, 79, [98] [99] [100] . another study demonstrated that induction of ebv replication in latency i-infected cells was associated with increased expression of mir-bhrf1-1, -2, and -3, but expression levels of mir-bart-1 and -2 did not change. on the other hand, induction of ebv replication in latency iii-infected cells did hardly change the expression levels of bhrf1-1, -2, and -3 [98] . these observations suggest that ebv mirnas may be implicated in the oncogenic properties of the virus, but also in regulating its replication. moreover, a precise knowledge of the latency state of ebv and the expression pattern of viral mirnas may improve the successful treatment of ebv infection with amos. the function of most ebv vmirna remains poorly understood, but some targets of ebv mirnas have been recently identified. several mi-barts prevent expression of viral latent infection membrane protein 1 (lmp1) protein (see table 1 ). lmp1 functions as a constitutively active tumour necrosis receptor [101] , and can activate several signalling pathways including nfκb, ap1, jak/stat, mek/erk, and pkc. lmp1 can also interact with p53 and affects cyclins, cyclin-dependent protein kinases (cdk), and the cdk inhibitors p16 and p27 (reviewed in [102] ). furthermore, lmp1 is expressed in all the ebv related malignancies and promotes cellular transformation. its oncogenic property makes lmp1 an attractive target for ebv therapy. interestingly, overexpression of lmp1 results in growthinhibitory and sensitization to apoptosis induced by stress or chemotherapeutic agents ( [76] and references therein). thus amo-mediated neutralization of mi-barts may lead to elevated lmp1 protein levels and render ebv-positive tumour cells more susceptible to chemotherapy. the viral mirna mir-bart2 can inhibit expression of viral dna polymerase balf5 and may thus interfere with viral replication and prevent lytic infection [51, 77] . silencing mir-bart2 could thus allow the virus the complete its life cycle and produce new infectious virus particles, which then could offer the immune system the opportunity to detect and eliminate ebv. using computational prediction programs such as miranda and rnahybrid (reviewed in [103] ) allowed choy and coworkers to envisage the cellular protein p53 up-regulator of apoptosis (puma) as a target for mir-bart5 [78] . the authors demonstrated that puma levels were decreased in cells expressing mir-bart5 compared to cells lacking mir-bart5. in accordance, when mir-bart5 was specifically inhibited with an anti-mir-bart5 oligonucleotide, puma protein levels decreased and apoptosis was triggered. thus, ebv may promote survival of infected epithelial cells by modulating the expression of an apoptotic protein through an mirna-mediated mechanism. this finding may have important implications in the development of anti-ebv agents such as amos directed against mir-bart5. fewer studies have been directed to determine the targets of mir-bhrf1s. mir-bhrf1-2 is involved in the cleavage of bhrf1 rna in the cytoplasm, but the biological relevance remains to be determined [98] . in another study, xia et al. observed that high levels of mir-bhrf1-3 were correlated with low levels cxcl-11, a potent interferon-inducible tcell attracting chemokine. mirna-mediated suppression of cxcl-11 may serve as an immunomodulating mechanism allowing the virus to survive in the host [79] . on the other hand, enhancing cxcl-11 expression in ebv-positive tumours by amos against mir-bhrf1-3 may increase susceptibility of the tumour cells to the immune system. in agreement with this, two recent studies reported antitumour activity for cxcl-11 in animal models [104, 105] . kaposi's sarcoma-associated virus, so named because it was detected in kaposi's sarcoma, belongs to the γ-herpesviruses and is also known as human herpesvirus-8 or hhv-8. hhv-8 is associated with kaposi's sarcoma, as well as with two rare forms of b-cell malignancy: primary effusion lymphoma (pel) and the plasma cell variant of multicentric castleman's disease. like other herpesviruses, kshv can establish a lifelong latent infection characterized by a limited viral gene expression [106] . a total of 17 kshv mirnas encoded by 12 distinct mirna genes have been reported and their sequences are highly conserved between different kshv genomes in pel cell lines and in clinical samples. however, some polymorphism was observed, particularly in mir-k12-5 and mir-k12-9 [80, 107, 108] . the entire kshv mirna cluster resides within an approximately 4 kilobase-pairs region between open reading frames orf k12 (kaposin) and orf 71 (figure 2(e) ). to elucidate the functions of the kshv mirnas, transcriptome analysis was performed from cells stably expressing the mir-k12 cluster. among the differentially expressed genes were genes encoding proteins implicated in proliferation, immune modulation, angiogenesis, and apoptosis. the gene encoding thrombospondin-1 was targeted by all ten kshv mirnas, but especially by mir-k12-1, mir-k12-3-3p, mir-k12-6-3p, and mir-k12-11. thrombospondin-1 possesses antiproliferative and antiangiogenic properties. other transcripts that were reduced corresponded to the genes for osteopontin, s100 calcium binding protein, plasticity related gene 1 product, and integral membrane protein 2a [80, 81] . the mrna for the bcl-2 interacting protein bclaf1 was identified as a target for mir-k12-5. additional inhibition of bclaf1 expression was obtained in the presence of mir-k9, -10a, and -10b. the exact biological relevance is not yet understood, but sirnamediated depletion of bclaf1 enhanced the frequency of spontaneous lytic reactivation of kshv. mirna-mediated reduction of bclaf1 expression would prevent permanent latency of the virus, a type of infection that represents a deadend pathway of viral spreading [108, 109] . a kshv mirna that has gained special interest is mir-k12-11 because its seed sequence, known to be critically important for mrna target recognition is 100% conserved with the cellular mir-155, suggesting that these mirnas may regulate common targets. the exact role of mir-155 remains unclear, but a number of b-cell lymphomas and solid organ tumors overexpress mir-155, while mir-155 transgenic mice develop b lymphomas [110, 111] . work by the groups of skalsky and cullen confirmed that mir-k12-11 indeed is an orthologue of mir-155, and that they target common transcripts [81, 82] . comparing the gene expression profiles in cells stably expressing either mir-155 or mir-k12-11 revealed that they regulate an analogous set of mrnas. the products of these transcripts include proteins involved in b-cell function (e.g., src-like adaptor or sla), innate immunity (e.g., iκb kinase and phosphoinositide-3-kinase), apoptosis (xiap associated factor-1; ldoc1), cell cycle regulation (e.g., fos), and gene expression (e.g., fos and bach1). bach-1 (btb and cnc homolog 1) is a bzip protein that can repress transcription through heterodimerization with the small maf proteins [112] , while c-fos can heterodimerize with the jun proteins to form the ap-1 complex. ap-1 is a multifunctional protein involved in cellular proliferation, transformation, and apoptosis [113] . for a complete list of mir-155/mir-k12-11 regulated genes, the reader is referred to the work of skalsky et al., and of gottwein et al. [81, 82] . treatment of latently-infected kshv with an antagomir against mir-k12-11 enhanced fos protein levels about 2.5-fold compared to untreated cells [81] . computational analyses further revealed seed sequence homology between the viral mirnas kshv mir-k12-6-5p, ebv-bart5, and hcmv ul70-5p with human mirnas mir-15a plus 16, mir-18a/b, and mir-340, respectively [82] . both mir-15a and mir-16 are believed to possess tumour suppressor activity and to induce apoptosis by silencing bcl2 expression, while mir-18 was demonstrated to be oncogenic [52, 114] . kshv-encoded mirnas seem to be crucial both in survival of the virus in its host, but also to play a causal role in viral-associated pathologies. amo-mediated silencing of kshv-encoded mirnas may thus be a strategy to counteract viral infection, but may also undesirably target cellular mirnas with identical seed sequences as the viral mirnas. poxviruses are dsdna viruses that replicate in the cytoplasm and have as such no access to the nuclear proteins involved in the biogenesis of mirna. nevertheless, mirna precursor sequences have been predicted in the genomes of the human poxviruses vaccinia virus and variola virus, but their existence has not been validated [25, 65] . whether the other human poxvirus, molluscum contagiosum virus, encodes mirna remains to be established. hepatitis b virus (hbv), an enveloped virus with a circular partial dsdna genome, persistently infects more than 300 million people worldwide. hbv can cause a spectrum of liver diseases ranging from mild liver dysfunctions to chronic hepatitis, cirrhosis, and hepatocellular carcinoma [53] . this makes efficient anti-hbv therapy highly vital. amo-based vmirna silencing is probably no option since no mirnas could be detected by computational analysis [65] , and expression of hbv mirnas has not been reported so far. one study identified a putative hbv-encoded mirna, but in vivo expression of this hbv mirna was not tested. computational screening for complementary sequences in the 3' untranslated regions of cellular mrna to this hbv mirna did not reveal putative target transcripts [54] . it therefore seems unlike that this is a bona fide viral mirna. hiv is the causative agent of acquired immune deficiency syndrome (aids), and it is estimated that >30 million people worldwide are infected with this virus. two species, hiv-1 and hiv-2, infect humans (for a recent review see [55] ). hiv utilizes reverse transcriptase to convert its ssrna genome into a dsdna provirus. during this process, the 5' and 3' ends of the viral rna genome are converted into long terminal repeats (ltrs). the ltrs play a pivotal regulatory role in establishing, maintaining, and overriding the latent state of the virus [115] . the central domain of the ltr is referred to the r region, which encompasses the (transactivation-response region) tar. tar binds the viral protein tat, a transactivator that plays an important role in the transcriptional activation of the provirus genome (reviewed in [116] ). the tar encodes proven and putative mirnas (figure 2(f) ). klase and coworkers described an mirna encoded by the hiv-1 tar element. this mirna causes hdac-1 to associate with the viral ltr, resulting in diminished viral gene expression. this suggests a role for hiv-1 mirna in maintaining viral latency [83] . in another report, ouellet and colleagues demonstrated the expression of two tar element-derived mirnas by northern blotting, primer extension, and rnase protection assay. the mirna derived from the left arm of the tar stem has been named mir-tar-5p, while the mirna originating from the right arm was designated mir-tar-3p. the latter appears to preferentially accumulate in hiv-positive cells [84] . the biological role of these mirnas remains to be elucidated, but they may contribute to modulating viral and/or cellular gene expression, with a potential impact on viral replication and/or host antiviral defense efficiency. the mir-tar-5p described by ouellet overlaps with the vmirna no. 1, while mir-tar-3p partially overlaps with vmirna no. 5 described by bennasser and coworkers [85] . they predicted by computer-directed analyses 5 pre-mirnas in the hiv-1 genome, which in principle could yield 10 mature mirnas. their expression has not been validated, nor has their biological role been addressed, but deduction of potential target transcripts resulted in the indentification of cellular genes encoding protein kinases, ion channels, proteins involved in protein synthesis and degradation, growth factors, and dna methylation [85] . tar dna of the long terminal repeat of hiv-1 encompasses an antisense rna (hivainr), which encodes hiv proteins, but that can also form a duplex with the 5' end of all sense hiv mrna, enabling the virus to control the expression of its gene [117] . hivainr can potentially code for several mirnas, referred to as haamirnas. putative targets for these mirnas are the mrnas for interleukin (il)-15, il-2 receptor γ chain, human fragile x mental retardation protein (fmrp), and il-1 receptor-associated kinase 1 (irak1). il-15 is important in the regulation of t-cell maturation, development and survival of natural killer cells, and survival of long-lived memory t cells, while the il-2 receptor γ chain is a common component of the receptors for il-2, -4, -7, -9, -15, and -21. aberant expression of this receptor leads to severe t-cell and nk-cell deficiencies. irak1 is a critical signalling mediator of innate immunity. downregulation the expression of il-15, il-2 receptor γ chain, and irak1 by hiv mirna would impair the immune system and favor survival of the virus in the host. fmrp is an rna-binding protein that is implicated in protein synthesis and mirna processing. thus hiv could use haamirna to deregulate the host mirna mechanism to dispose the virus by depleting fmrp ( [56] and references therein.) although the existence of these haamirnas has not been proven, it is tempting to speculate that, in accordance with other viruses, hiv encodes mirnas allowing hiv to survive in the host. a recent study examined the possibility of hiv-2 tar to encode mirnas. two putative mirnas, mir-tar2-5p and mir-tar-3p were identified, but their expression awaits validation [87] . besides tar, other regions of the hiv genome have been shown to contain mirna sequences. the nef gene of hiv-1 is located at the 3' end of the viral genome and is highly expressed during the early stages of virus replication. nef is a multifunctional accessory protein that is important for viral replication, but that also plays a key role in pathogenesis as nef can downmodulate cd4, cd28, and the class i major histocompatibility complex [86] . hiv-1 encodes a nef -derived mirna referred to as mir-n367 (figure 2(f) ). unlike classical mirna, this mirna does not affect gene expression at the post-transcription level, but rather at the transcription level by promoter interference. mir-n367 suppresses hiv-1 promoter activity via a negative responsive element in the 5'-long terminal region and via nef sequences in the 3' untranslated region [57, 118] . future studies are required to elucidate the precise mechanism by which mir-n3667 represses hiv-1 promoter activity. downregulation of nef expression may suppress hiv-1 replication and allow persistently low pathogenic or latent viral infection [57] . as the nef gene is conserved in hiv-2, hiv-2 may also apply a similar mechanism to maintain a low profile in the host. the identity and action of hiv mirnas remains to be scrutinized before amos-based therapy can be considered as anti-hiv drugs. however, computational alignment of the potential hiv-1 mirnas with specific human t-cell mrnas identified potential cellular targets including genes encoding cd4, cd28 and interleukin-2, il-3, and il-12 [119] . viral mirna-caused inhibition of the expression of these proteins seems advantageous for the virus, and therefore counteracting vmirna by amo may help the host to clear hiv infection. htlv-i persistently infects 10-20 million humans worldwide and is the etiological agent for adult t-cell leukaemia [5] . one study reported that t cells persistently infected with htlv-1 did not express viral micrornas [88] , but meticulous studies should be performed to rule out the existence of htlv-i mirna. none of these viral genomes seem to contain putative mirna sequences, except for measles virus and yellow fever virus, which each possesses a single putative mirna. however, the mirna in yellow fever virus could not be validated, while the existence of mirna in measles virus was not tested [25, 65] . intriguingly, the liver-specific mir-122 facilitates the replication of the oncovirus hcv, but the mechanism for this function of mir-122 in hcv replication is still unknown [120, 121] . numerous human diseases are caused by viral infections, but the intimate relation with the host makes the development of antiviral drugs difficult. vaccination has been proven to be very succesful to combat some viral infections, but mutations and diversity of virus strains has hampered the development of efficient vaccines against other viruses. new antiviral treatments are based on drugs that inhibit specific viral activities such as viral proteases or polymerases (for recent reviews see [122, 123] ). viral-encoded mirna that may be implicated in the viral life cycle and the pathogenic properties of the virus offers a novel attractive target for antiviral therapy. silencing the action of viral mirnas may enable the host cell or the immune system to gain control over the virus and even to eliminate the virus. the idea of targeting viral transcripts is not new, and rna interference has been demonstrated to efficiently mediate inhibition of replication of human pathogenic viruses such as hiv-1, hcv, dengue virus, severe acute respiratory syndrome (sars) coronavirus, poliovirus, human rhinovirus, influenza a virus, hepatitis d virus, hbv, hsv-1, hpv, jcv, ebv, and cmv in cell culture (reviewed in [12] ). besides recent studies have proven the potential of this rna interference as antiviral therapy in animal models [124, 125] , and even in clinical trials such as alnylam against respiratory syncytial virus and nuc b1000 against hbv (reviewed in [126, 127] ). however, anti-hiv rna interference studies revealed that escape virus variants could appear which could evade the inhibitory action of sirna [128, 129] . journal of biomedicine and biotechnology the use of amo to neutralize viral mirna adds a new twist to rna interference. amos are easy to produce and relatively cheap, and easy to administer locally (but not systemically). moreover, they possess low toxicity and are highly specific. most viral mirnas identified so far have little homology to each other and to known host cell mirnas (reviewed in [130] ). this reduces the risk of offtarget effects of anti-mirna oligonucleotides and increases the therapeutic potentials of mirna silencing. the mirna silencing action of both lna and antagomirs is sensitive to single nucleotide exchanges. for antagomirs, it was shown that this effect depends on the position of the mismatch. nucleotide substitutions at the very 5' end or in the centre did not prevent downregulation of mir-122 [33] . these data indicate that changes in the 3' end of the antagomir may abrogate its ability to destroy target mirna and should be taken into account when designing and testing antagomirs. another advantage of amos is that they probably can be used against all serotypes of a specific virus. although not meticulously investigated, mirna sequences between different viral strains seem to be conserved because of their importance for the viral life-cycle (see e.g., kshv-encoded mirnas; [80, 107, 108] ). however, polymorphism in viral mirnas has also been observed (see next paragraph). although amos may provide an attractive novel antiviral therapy, practical problems and other pitfalls may hamper the use of them. for example, antagomirs directed against mir-ul-112-1 could drive the virus towards acute replication and disrupted the inhibition of mcib expression, resulting in possible clearance of the virus by the immune system. however, there is a potential risk of severe pathological effects caused by acutely replicating hcmv, especially in immunocompromised patients [50] . another disadvantage of amos may be off-target effects. as mirna do not require full complimentarity to bind their target sequence, it can be imagined that an amo not only binds to its predicted mirna but also to other mirnas and even mrnas. in addition, ssrna oligonucleotides may interact with toll-like receptors 7 and 8 thereby stimulating the immune system. similar side effects have been reported for sirna (reviewed in [126] ). polymorphism in viral-encoded mirnas has been described in viral-infected cell lines and in clinical samples. for instance, mir-k12-5 of different kshv isolates contains mutations, which can affect maturation and biological activity of this mirna [25, 100, 107] . thus the mirna may not be expressed, in which case the amo will have no effect or the amo may not bind because of the mutations in its target mirna. another problem facing the use of amos in antiviral therapy is that the expression of a specific gene may be regulated by several viral mirnas. for example, translation of the transcript of thebachgene is prevented by three kshv viral mirnas: mir-k12-11, mirna-k12-1, and mir-k12-6. thus the effectiveness of an amo against, for example, mir-12-11 can be compromised. indeed, treatment of latently infected kshv virus with a specific antagomir against mir-k12-12 alone only modestly increased the amount of bach protein [81] . a cocktail of different amos directed against distinct viral mirna may help to overcome this problem. so far, such studies are lacking, but a recent study successfully applied sponge mirna to silence hbv transcripts. an expression vector encoding multiple mirnas targeting hbv hbsag mrna strongly reduced the expression of this protein [131] . another challenge is to improve in vivo delivery of the amos to viral infected cells and obtain long-lasting action of the antagomirs. aerosol delivery devices similar to the ones used for delivery of asthma therapeutics could be used for respiratory viruses [15] . other delivery strategies include intravenal or systemic injection, viral vectors, and lipid-and polymerbased vehicles [131] [132] [133] [134] . recently, sustained inhibition of hcv replication in cell-culture was obtained when celldegradable multilayered polyelectrolyte film (mpf) coated with sirna was delivered to infected cells. by this approach, a single regime of mpf-mediated sirna treatment was sufficient to inhibit hcv replication for 12 days. moreover, mpf-mediated delivery of sirna also protected uninfected cells from hcv infection. another advantage is the very low toxicity of mpf [135] . these promising observations in cell culture put mpf-based delivery of amos forward as an efficient antiviral tool. another limitation of anti-mirnas is the site of application. studies with antagomirs against mir-16 in mice revealed that when injected into tail veins, antagomirs were incapable of silencing mir-16, whereas local injection into the mouse cortex efficiently induced degradation of the target mirna [33] . another drawback of the use of amos is that the chemical modification can exert antiproliferative or other off-target effects such as been demonstrated for the phosphorothioate backbone, which can associate with cellular proteins [29] . antisense oligonucleotides such as lna and pmo have proven to efficiently inhibit rna and dna virus replication in cell culture and animal models, without toxicity for the cell or animal. however, these pmo were directed against viral protein-encoding mrna, and studies of pmo-mediated silencing of viral mirna have not been reported so far (see e.g., [31, [136] [137] [138] ). future viral mirna research is faced with important challenges before amos may enter the clinic. our comprehension on the functions of viral mirna and the interplay between viral infection and cellular mirna expression is just beginning to emerge. studies aimed at the identification of viral mirna and elucidation of their functions should be pursued. difficulties facing computational-based prediction are false positives, but also the shortcoming to detect genuine mirnas. moreover confirmation of expression of mirna by, for example, northern blot may fail to monitor mirna. expression levels of vmirna may be cell-specific, for example, ebv mir-bhfr1-2 had considerably lower expression levels in jijoye cells than in b95-8 cells [7] . dose-and time-dependent studies are required to determine the optimal therapeutic regime. such pharmacokinetics and pharmacodynamic studies are largely lacking [133] , but recent in studies in mice revealed that a single cerebral or tail-vein injection of 240 mg/kg body mass anti-mir122 had a silencing effect for at least 8 days and as long as 23 days in the tissues examined [32, 33] . in another study, a five-week regime of two intraperitonal or subcutaneous injection a week with different concentrations of anti-mir-122 clearly silenced mir-122 and no untoward effects were observed [35] . despite the obstacles facing antiviral mirna silencing as viral therapy, the coming years will certainly see the daylight of intensified research and even the initiation of clinical trials. papillomaviruses and cancer: from basic studies to clinical application immune evasion by kaposi's sarcoma-associated herpesvirus dna tumor viruses and human cancer epstein-barr virus infection in humans: from harmless to life endangering virus-lymphocyte interactions human t-cell leukaemia virus type 1 (htlv-1) infectivity and cellular transformation human oncogenic viruses: hepatitis b and hepatitis c viruses and their role in hepatocarcinogenesis human polyomaviruses and cancer: expanding repertoire connecting viruses to cancer: how research moves from association to causation the challenge of emerging and re-emerging infectious diseases viruses and micrornas the interplay between virus infection and the cellular rna interference machinery riscy business: micrornas, pathogenesis, and viruses rna interference against viruses: strike and counterstrike rnai suppressors encoded by pathogenic human viruses viral and cellular messenger rna targets of viral micrornas cellular versus viral micrornas in host-virus interaction micrornas and the regulation of glucose and lipid metabolism a developmental view of microrna function micrornas: new regulators of immune cell development and function posttranscriptional control of neuronal development by microrna networks mirbase: tools for microrna genomics the functions of animal micrornas micrornas: genomics, biogenesis, mechanism, and function viral and cellular micrornas as determinants of viral pathogenesis and immunity principles of microrna-target recognition combinatorial microrna target predictions emerging role of micrornas in disease pathogenesis and strategies for therapeutic modulation inhibition of microrna with antisense oligonucleotides lna-mediated microrna silencing in non-human primates oligonucleotide antiviral therapeutics: antisense and rna interference for highly pathogenic rna viruses silencing of micrornas in vivo with 'antagomirs specificity, duplex degradation and subcellular localization of antagomirs regulation of microrna by antagomirs: a new class of pharmacological antagonists for the specific regulation of gene function? mir-122 regulation of lipid metabolism revealed by in vivo antisense targeting pna hybridizes to complementary oligonucleotides obeying the watson-crick hydrogen-bonding rules mir-122 targeting with lna/2 -o-methyl oligonucleotide mixmers, peptide nucleic acids (pna), and pna-peptide conjugates anti-mirna oligonucleotides (amos): ammunition to target mirnas implicated in human disease? human polyomaviruses: molecular mechanisms for transformation and their association with cancers the role of polyomaviruses in human disease identification of a third human polyomavirus identification of a novel polyomavirus from patients with acute respiratory tract infections clonal integration of a polyomavirus in human merkel cell carcinoma molecular biology of bk virus and clinical aspects of bk virus renal infection sv40-encoded micrornas regulate viral gene expression and reduce susceptibility to cytotoxic t cells evolutionarily conserved function of a viral microrna merkel cell polyomavirus encodes a microrna with the ability to autoregulate viral gene expression micrornas expressed by herpes simplex virus 1 during latent infection regulate viral mrnas human cytomegalovirus expresses novel micrornas during productive viral infection identification and function of human cytomegalovirus micrornas identification of virus-encoded micrornas a microrna polycistron as a potential human oncogene hepadnaviruses hbv-encoded microrna candidate and its target 25 years of hiv-1 research-progress and perspectives rna silencing and hiv: a hypothesis for the etiology of the severe combined immunodeficiency induced by the virus hiv-1 nef suppression by virally encoded microrna human papillomavirus genotype 31 does not express detectable microrna levels during latent or productive virus replication adenoviruses structure, function and evolution of adenovirus virus-associated rnas suppression of rna interference by adenovirus virus-associated rna adenovirus virus-associated rna is processed to functional interfering rnas involved in virus production sequence-specific interference by small rnas derived from adenovirus vai rna the family herpesviridae: a brief introduction identification of micrornas of the herpesvirus family epstein-barr virus micrornas are evolutionarily conserved and differentially expressed anti-apoptotic function of a microrna encoded by the hsv-1 latency-associated transcript an acutely and latently expressed herpes simplex virus 2 viral microrna inhibits expression of icp34.5, a viral neurovirulence factor prediction and identification of herpes simplex virus 1-encoded micrornas novel less-abundant viral mirnas encoded by herpes simplex virus 2 latencyassociated transcript and their roles in regulating icp34.5 and icp0 mrnas the roles of micrornas in mammalian virus infection host immune system gene targeting by a viral mirna suppression of immediate-early viral gene expression by herpesvirus-coded micrornas: implications for latency a human cytomegalovirus-encoded microrna regulates expression of multiple viral genes involved in replication a combined computational and microarray-based approach identifies novel micrornas encoded by human gammaherpesviruses modulation of lmp1 protein expression by ebv-encoded micrornas epstein-barr virusencoded microrna mir-bart2 down-regulates the viral dna polymerase balf5 an epstein-barr virus-encoded microrna targets puma to promote host cell survival ebv micrornas in primary lymphomas and targeting of cxcl-11 by ebv-mir-bhrf1-3 identification of cellular genes targeted by kshv-encoded microrna a viral microrna functions as an orthologue of cellular mir-155 kaposi's sarcoma-associated herpesvirus encodes an ortholog of mir-155 hiv-1 tar element is processed by dicer to yield a viral micro-rna involved in chromatin remodeling of the viral ltr identification of functional micrornas released through asymmetrical processing of hiv-1 tar element hiv-1 encoded candidate micro-rnas and their cellular targets hiv-1 nef: at the crossroads the hiv-2 tar rna domain as a potential source of viral-encoded mirna. a reconnaissance study analysis of the interaction of primate retroviruses with the human rna interference machinery herpes simplex viruses virology: micro mystery solution anti-apoptotic function of a microrna encoded by the hsv-1 latency-associated transcript genital herpes hsv-1 icp34.5 confers neurovirulence by targeting the beclin 1 autophagy protein varicellazoster virus the functions of herpesvirus-encoded micrornas small rnas and large dna viruses epstein-barr virus: 40 years on epstein-barr virus bhrf1 microand stable rnas during latency iii and after induction of replication expression of viral micrornas in epstein-barr virus-associated gastric carcinoma epstein-barr virus bart micrornas are produced from a large intron prior to splicing regulation of intracellular signalling by the terminal membrane proteins of members of the gammaherpesvirinae mechanisms by which viral proteins of human dna tumor viruses hijack the cell cycle a guide through present computational approaches for the identification of mammalian microrna targets vaccination with ifn-inducible t cell α chemoattractant (itac) genemodified tumor cell attenuates disseminated metastases of circulating tumor cells the cxcr3 targeting chemokine cxcl11 has potent antitumor activity in vivo involving attraction of cd8 + t lymphocytes but not inhibition of angiogenesis kaposi's sarcoma-associated virus conservation of viraly encoded micrornas in kaposi sarcoma-associated herpesvirus in primary effusion lymphoma cell lines and in patients with kaposi sarcoma or multicentric castleman disease micrornas of kaposi's sarcoma-associated herpes virus tandem array-based expression screens identify host mrna targets of virus-encoded micrornas microrna signatures in human cancers pre-b cell proliferation and lymphoblastic leukemia/high-grade lymphoma in eμ-mir155 transgenic mice heme regulates the dynamic exchange of bach1 and nf-e2-related factors in the maf transcription factor network ap-1 as a regulator of cell life and death mir-15 and mir-16 induce apoptosis by targeting bcl2 are viral-encoded micrornas mediating latent hiv-1 infection? a new paradigm in eukaryotic biology: hiv tat and the control of transcriptional elongation human immunodeficiency virus-type 1 ltr dna contains an intrinsic gene producing antisense rna and protein products regulation of human immunodeficiency virus 1 transcription by nef microrna hiv may produce inhibitory micrornas (mirnas) that block production of cd28, cd4 and some interleukins molecular biology: modulation of hepatitis c virus rna abundance by a liver-specific microrna proceedings of the national academy of sciences of the united states of america viral protease inhibitors antiviral agents acting as dna or rna chain terminators t cell-specific sirna delivery suppresses hiv-1 infection in humanized mice exploring gene-deleted adenoviral vectors for delivery of short hairpin rnas and reduction of hepatitis b virus infection in mice the versatility of oligonucleotides as potential therapeutics rna-interference therapy for hbv infection enters phase i clinical trial hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome overcoming hiv-1 resistance to rna interference the properties and functions of virus encoded microrna, sirna, and other small noncoding rnas small rnas: delivering the future lentivirusmediated antagomir expression for specific inhibition of mirna function antisense, rnai, and gene silencing strategies for therapy: mission possible or impossible? vector design for liver-specific expression of multiple interfering rnas that target hepatitis b virus transcripts sustained delivery of sirnas targeting viral infection by cell-degradable multilayered polyelectrolyte films inhibition of replication and transcription activator and latencyassociated nuclear antigen of kaposi's sarcoma-associated herpesvirus by morpholino oligomers lna derivatives of a kissing aptamer targeted to the transactivating responsive rna element of hiv-1 controlling morpholino experiments: don't stop making antisense key: cord-324690-82qsirnk authors: dieffenbach, carl w; fauci, anthony s title: the search for an hiv vaccine, the journey continues date: 2020-05-16 journal: j int aids soc doi: 10.1002/jia2.25506 sha: doc_id: 324690 cord_uid: 82qsirnk nan as we commemorate hiv vaccine awareness day, we honour the thousands of study participants and the research teams that have played essential roles in the completed and ongoing vaccine clinical trials. we also recognize the scientists who have spent years dedicated to creating the tools and technologies to end the global hiv epidemic. on this vaccine awareness day, we must acknowledge that the hiv vaccine research field is quickly approaching a critical crossroads. this past decade has been spent launching improved vaccine concepts and immune-based strategies into trials. despite these advances, a safe and durably effective hiv vaccine has eluded us. in this regard, we must evaluate the current state of the field and make appropriate adjustments to help speed us to our goal. while this quest for a vaccine at times may feel extremely difficult, as evidenced by the recent disappointing results of a phase iii efficacy trial, hvtn 702, described below, we must steadfastly move forward to address critical research gaps and unanswered questions. as we reflect upon the status of hiv vaccine research, we would be remiss if we did not mention the current global pandemic caused by sars-cov-2. it is important to acknowledge the global response to the covid-19 epidemic and to cite the range of vaccine platforms being used for anti-sars-cov-2 candidate vaccines that have their origin in hiv vaccinology. the hiv vaccine field is clearly assisting in addressing this newly emergent pandemic and the national institute of allergy and infectious diseases (niaid) is fostering these essential collaborations. for hiv vaccine awareness day in 2020, we emphatically state that finding safe, effective and durable vaccines for hiv and covid-19 are niaid's top priorities. the world must have both. based upon the challenges that hiv biology presents: global sequence diversity, integration of the viral genome into the host cells and long duration of the symptom free period of infection as well as the fact that no single individual has been spontaneously cured, it is widely accepted that a vaccine must trigger responses that are qualitatively different from the immune responses to natural infection. after the step trial (also referred to as hvtn 502 or merck v520-023) was halted in 2007, vaccine candidates that fulfilled these criteria have been prioritized [1, 2] . over the past decade, three different vaccine approaches have been implemented, possible correlates of protection identified, and two have moved through clinical evaluation to advanced clinical trials. the first of these was based upon the correlates of risk that were generated from analysis of the rv144 trial conducted in thailand, which in 2009, reported modest levels of efficacy [3, 4] . rather than perform a direct repeat of rv144 in thailand, where the epidemic is driven by viruses from clades ae and b, a clade c-specific regimen was developed for testing in southern africa. when evaluated in the hvtn100 trial, the relevant correlate responses were detected and shown to be longer lasting than those of rv144 [5] [6] [7] . with the go-no-go criteria for advancement met, the hvtn702 study moved ahead. however, no protection was observed, and the trial was terminated due to futility. while the field is disappointed with this outcome, it is important to understand the possible reasons for why this occurred. first, there were significant differences in the overall incidence between the study cohorts in hvtn702 and rv144 that could have overwhelmed a modestly protective response. additionally, it is conceivable that the correlates observed in rv144 are specific to the clades and population in thailand and not transferable to the southern africa epidemic. the remaining two vaccine concepts have reproducibly demonstrated efficacy in non-human primate studies. the most advanced concept is currently being evaluated in two studies, imbokodo (hvtn705/hpx2008) in women in southern africa and mosaico (hvtn706/hpx3002) in men and transgender persons who have sex with men in the americas and europe [8] . analysis of the correlates of protection seen in the non-human primate studies point to qualitatively different responses than those observed in rv144, and the trials are evaluating in silico designed immunogens to present the most globally conserved hiv sequences to trigger quantitatively superior cd8 + t cell responses [8, 9] . the third approach, still in preclinical studies, is an siv vaccine delivered via the rhesus cytomegalovirus (rhcmv) vector platform. this strategy has been shown by picker and colleagues to protect 50 percent of monkeys from sustained infection [10] . efforts are currently underway to transition to a human cmv vector platform with phase 1 clinical trials expected in 2021. although humbled by the outcome of hvtn702, we must continue to explore novel methods of inducing or providing protective immunity against hiv infection. one of the most exciting developments in hiv research has been the ability to isolate and produce broadly neutralizing monoclonal antibodies (bnabs) that neutralize nearly all of circulating virus strains from all clades [11, 12] . these human antibodies prevent acquisition of shiv infection, in a dose dependent manner, in animal challenge models and when combinations of these antibodies are used in people living with hiv, strong antiviral activity has been demonstrated. the antibody mediated protection (amp) trials are currently evaluating vrc01, the cd4 binding site targeted bnab, to determine the ability of this single antibody to prevent hiv infection in women in southern africa and msm and transgender persons in the americas [13] . the amp studies have not been impacted by covid-19, and results from the amp studies are expected in fall of 2020. if protection is observed in the amp trials, an important correlate for future vaccine studies will be established. in the meantime, efforts are underway to define immunogens that specifically trigger neutralizing antibodies by targeting five specific epitopes, which are unique sites of vulnerability on the hiv env trimer glycoprotein [12, 14] . the goal is to create a discrete set of immunogens that can reproducibly elicit the production of neutralizing antibody responses against two or more of these target sites. the collaborative hiv immunogen project [12] is seeking to prospectively design and select the optimal combination of immunogens to be evaluated for the induction of broad neutralization of a range of hiv isolates. as we reflect on hiv vaccine awareness day and recent developments in the vaccine field, it is clear that much work remains to be done. going forward we must continue to engage community to ensure a shared understanding of our common goals. while we pursue a safe and effective hiv vaccine, we are also seeking ways of improving chemoprophylaxis. we remain optimistic that effective interventions that people will reliably use can be developed and brought to scale. on hiv vaccine awareness day, let us pause and reflect on how far we have come while continuing to focus on completing the work needed to achieve an effective vaccine. the authors collaboratively conceived the content of the paper. cwd wrote the first draft and asf edited the manuscript into this final form. the authors thank the trial participants, community members, activists and researchers who have so willingly participated in the challenging work of hiv vaccine discovery and development. the authors also thank robert gulakowski, thomas liang, mary marovich and sarah read for their careful review and comments on this manuscript. overview of step and phambili trial results: two phase iib test-of-concept studies investigating the efficacy of mrk adenovirus type 5 gag/pol/nef subtype b hiv vaccine hiv vaccine research: the way forward vaccination with alvac and aidsvax to prevent hiv-1 infection in thailand immune-correlates analysis of an hiv-1 vaccine efficacy trial subtype c alvac-hiv and bivalent subtype c gp120/mf59 hiv-1 vaccine in low-risk, hiv-uninfected, south african adults: a phase 1/2 trial safety and immune responses after a 12-month booster in healthy hiv-uninfected adults in hvtn 100 in south africa: a randomized double-blind placebocontrolled trial of alvac-hiv (vcp2438) and bivalent subtype c gp120/mf59 vaccines immune correlates of the thai rv144 hiv vaccine regimen in south africa evaluation of a mosaic hiv-1 vaccine in a multicentre, randomised, double-blind, placebo-controlled, phase 1/2a clinical trial (approach) and in rhesus monkeys (nhp 13-19) increased valency of conserved-mosaic vaccines enhances the breadth and depth of epitope recognition a live-attenuated rhcmv/siv vaccine shows long-term efficacy against heterologous siv challenge use of broadly neutralizing antibodies for hiv-1 prevention multiple roles for hiv broadly neutralizing antibodies safety, pharmacokinetics and neutralization of the broadly neutralizing hiv-1 human monoclonal antibody vrc01 in healthy adults hiv-1 neutralizing antibody signatures and application to epitope-targeted vaccine design key: cord-287286-4l963z2q authors: green, victoria a.; munshi, saif u.; marakalala, mohlopheni j.; mourão, marina m. title: molecular mechanisms of viral infection and propagation: an overview of the second advanced summer school in africa date: 2010-07-28 journal: iubmb life doi: 10.1002/iub.364 sha: doc_id: 287286 cord_uid: 4l963z2q nan viral replication is dependent on a host; with their small genomes, viruses need to hijack host cellular machinery to complete their life cycle. the coevolution of intimate virus-host relationships has led to many viruses being able to successfully propagate without a significant detrimental effect to the host. however, in some instances, viral infection causes disease. the molecular mechanisms underlying virus-associated disease were the focus of an advanced summer school in africa held in hermanus, south africa, from 6 to 14 march 2010. assembled in the coastal town of hermanus were 45 participants, primarily phd students and postdocs from less developed countries, and 18 speakers, leaders in their respective fields, from around the world (fig. 1 ). the school, the second of its kind in africa was sponsored by the international union of biochemistry and molecular biology (iubmb), the federation of european biochemical societies (febs), the federation of african societies of biochemistry and molecular biology, the united nations educational scientific and cultural organization, and the international centre for genetic engineering and biotechnology (icgeb). the course was honored by the presence of brian clark, professor of biostructural chemistry at the university of aarhus, denmark. prof clark is a renowned scientist who participated in the discovery of the initiation codon for protein synthesis (1) (2) (3) (4) and the first crystallization of a trna (5) . today, prof clark studies the molecular and cellular mechanisms of ageing using a systems biology approach. his presence was particularly significant at the summer school since he has been involved in the organization of several similar events during his tenure as president of the iubmb and febs, most notably the spetses summer school. prof clark stressed the importance of such meetings for the development, not just of young scientists, but of science itself. the main themes of discussion at the summer school were: 1) why viral infection can lead to cancer; 2) how a greater understanding of the mechanisms underpinning human immunodeficiency virus (hiv) propagation can inform new antiviral strategies; 3) the abilities of viruses to evade the immune system and the obstacles to the development of effective vaccines; and, 4) the potential afforded by viruses as research tools. the importance of host factors became apparent in the discussion of all these topics, and how viral research has informed our general knowledge of cell biology. this report serves to summarise the findings presented at the summer school. until peyton rous discovered the first tumor virus in 1911, viruses were viewed as peculiar infectious agents capable of inducing cancer in animals, but not in humans. however, it is now well-established that many different human viruses, including human papilloma virus (hpv), epstein-barr virus (ebv), kaposi's sarcoma-associated herpes virus (kshv), human tcell leukemia virus-1 (htlv-1), hepatitis b virus (hbv), hepatitis c virus (hcv), and merkel cell polyoma virus (mcv) are the etiological agents of human cancers, encompassing at least 15-20% of all tumors worldwide (6) . hence, tumor virology was a focus of this summer school. dr lawrence banks from the icgeb, trieste, provided an overview of hpv and the role of viral proteins in the transformation of cervical epithelial cells. almost all (99.7%) cervical cancers contain hpv dna, usually types 16 and 18 (7) . despite this statistic, cancer induction is not part of the 'normal' hpv life cycle. only in rare cases where there has been a lack of immune clearance leading to persistent infection, along with additional changes in the cell, does cancer develop. banks described how hpv e6 and e7 are the major viral oncoproteins involved in the development of cervical cancer. the hpv life cycle is critically dependent on the differentiation of epithelial cells: there is usually no dna replication, other than in cells of the basement layer, but hpv e6 and e7 induce a pseudo-s phase, creating a permissive environment for viral replication (8) . in addition, e6 and e7 target cellular substrates for proteasome-mediated degradation, notably the tumor suppressors p53 and prb, respectively (9, 10) . hpv dna exists in an episomal form but may be found integrated in malignant cells. integration often involves loss of viral dna. indeed, malignant cells often display no viral replication but maintain expression of e6 and e7 (11) , further emphasizing the roles of these proteins in cancer progression. but why do only a few hpv infections result in malignancy? this issue was tackled by dr john doorbar, national institute for medical research, london. hpv-associated cancers are more predominant in some tissues than others: there are 500,000 cases of cervical cancer reported each year. in contrast, hpv-associated penile cancers are rare (40,000 cases per year), despite no evidence of a disparity in primary infection rates. this implies a difference in the propensity of hpv to form neoplastic lesions at different sites. doorbar speculates that this is due to a graded ability of hpv to replicate in different tissues. as further evidence, hpv-1 causes warts but no lesions at mucosal sites. doorbar believes that the transformation zone in the cervix may be particularly susceptible to neoplasia formation. the exact host factors responsible for the variation in hpv replication ability are unresolved. doorbar's group is also investigating the molecular basis of the different outcomes resulting from hpv infection. hpv gene expression occurs in an ordered manner as the infected cells move through the epithelial tissue. doorbar described how the host protein mini chromosome maintenance protein 7 (mcm7) could be used as a marker for cell proliferation and, thus, as a surrogate for e6/7 activity. mcm7 was expressed in the lower layers of hpv lesions, as shown by laser capture microscopy, and the viral protein e4 was abundantly expressed in the upper layers (12) . interestingly, doorbar's group have shown that high-grade squamous intraepithelial lesions (hsils) are associated with increased mcm7 expression in upper epithelial layers with almost no e4 expression (12) , revealing that viral expression patterns reflect the severity of disease. in addition, proliferation of the basal cells does not stop when the basement layer is complete in high-risk lesions, in contrast to low-risk lesions. the increased basal layer proliferation is correlated with betacatenin activity and e6 levels (13, 14) . what drives increased e6 activity in some infections is unknown: wound healing, episomal copy number, dna methylation patterns, and host genetic background could all contribute and require further investigation. nevertheless, these observations have the potential to tremendously improve existing cervical screening practice, which relies heavily on cytology. though in vitro studies are invaluable for identifying candidate molecules involved in pathogenesis, conformation of their role often requires an in vivo approach. dr paul f. lambert, university of wisconsin medical school, described transgenic mouse models of hpv where the human keratin promoter, hk14, was used to drive expression of hpv genes in stratified squamous epithelia (15) . previous studies had shown that expression of hpv genes was insufficient to cause cervical cancer in mice (15) . lambert's group hypothesized that estrogen was a cofactor for cervical cancer. they generated hk14-hpv16-estrogen receptor transgenic mice, which did develop cervical cancer (16) (17) (18) . this may account for observations that [5 years of contraceptive pill usage and pregnancy increases the risk of women developing cervical cancer. hpv is not just associated with cancer of the cervix, however. prof iqbal parker of the icgeb, cape town, showed that papilloma viruses typically considered low-risk could be involved in fatal cancers of the oesophagus. parker's group found that about 40% of oesophageal cancer (oc) patients had integrated hpv-11 and -39 dna in their tumors (19) . brush biopsies of healthy people confirmed that hpv was enriched in oc patients. however, smoking, alcoholic home brew consumption and cooking on indoor fires in conjunction with certain sulpho-transferase alleles were also risk factors associated with the development of oc in africa (20) . whether hpv infection is an early or initiation event in oc development requires further investigation. the molecular alterations that direct progression from productive infection to hsils in hpv infection are not fully understood. hpv oncoproteins alone are not capable of transformation, as shown in human keratinocytes in vitro (21) and murine cervical epithelia in vivo (15) . rather, papilloma virus-mediated oncogenesis requires supplementary genetic changes that occur over time following the initial infection. whilst it is clear that integration of viral dna into the host genome is crucial to hpv-induced tumor development (22, 23) , whether this is a cause or consequence of wider-spread chromosome instability is not clear. despite the activity of various oncogenic hpv proteins, viral dna integration and cancer development does not often follow. the import of the host, including an ability to regulate viral gene expression in different tissues and to mount an effective immune response, is becoming increasingly apparent in determining the molecular basis of hpv-associated tumor progression. dr ethel cesarman, weill cornell medical college, described human herpes viruses and focused on kshv, also known as human herpes virus 8 (hhv8). kshv causes a cancer often found in immunosuppressed individuals, such as those suffering from aids, including karposi's sarcoma primary effusion lymphoma and multicentric castleman's disease. there is [50% kshv seroprevalence in sub-saharan africa but, as with hpv, the majority of individuals present with no disease. kshv contains up to 90 orf with at least 15 accessory genes. among the accessory genes identified to date, orf74, named kshv g protein-coupled receptor (kshv-gpcr), has become a major focus of investigation. though it is expressed in a very small number of ks lesion spindle cells (24) , it is known to be involved in cell transformation through induction of angiogenic cytokine expression (25) . for example, vascular endothelial growth factor is one cytokine secreted from cells expressing kshv-gpcr, which can induce proliferation and angiogenesis in ks tissue (26) . following the observation that proteins expressed by lymphomagenic viruses during latent infection activate nf-jb, cesarman's group searched for a viral protein whose activity correlated with nf-jb. they identified vflip, an homologue of cflip proteins, and showed that anti-vflip sirnas induced apoptosis in kshv-positive cells only (27) . work is continuing to develop drugs that are vflip-specific, in the hope that these will prove effective anti-kshv agents. during his lectures, dr ingemar ernberg, of the karolinska institute in stockholm, recoined the acronym ebv as ''every bodies virus'' because [95% of humans are infected from childhood, though infections are typically subclinical. a member of the hhv family, ebv was initially isolated from a burkitt's lymphoma cell line (28) , though it was later found associated with a number of different tumors, such as undifferentiated nasopharyngeal carcinoma, hodgkin's disease, nasal t cell lymphoma and gastric carcinoma. ernberg described the four programs of latent viral gene expression (l0-l3) and how all 12 viral genes are expressed in l3, which is associated with cell proliferation (29) . therefore, ernberg's group looked for the signal controlling the switch from l1 to l3. they showed that in l1, there was low expression of the viral gene ebna1, but high levels of the cellular transcription factor oct2, and vice versa in l3. work is ongoing into deciphering the reason for a decrease in oct2 levels that is concomitant with a switch to the l3 program of gene expression. hopefully this will shed light on why ebv set points vary between individuals and, further, what differs between the b cells of those who appear immune to ebv infection and the majority of the population who are susceptible. human t-cell leukemia virus-1 dr aluisio segurado, university of são paulo, described htlv-1, a retrovirus that can result in adult t cell leukemia/ lymphoma (atll) and htlv-1-associated myelopathy (ham/ tsp). it is estimated that 15 to 20 million individuals are infected with htlv-1 worldwide, yet the vast majority remain clinically asymptomatic-only 2-6% develop atll (30) . the reason for the low frequency of htlv-1-associated disease is unknown but segurado hypothesises that host genetics and environmental factors play a role, rather than infection with different strains of htlv. tax (p40tax) is one of the htlv-1 proteins that acts as a transcription activator in atll (31) . however, the low incidence rate and the long latency period prior to development of atll suggests that, in addition to viral infection, accumulation of mutations in host genes is required for cellular transformation in vivo. among the hepatitis viruses, hbv and hcv cause chronic infection leading to the development of cirrhosis and hepatocellular carcinoma (hcc). hbv is a dna virus that integrates into the host genome. dr shahid jameel, icgeb, new delhi, described how this integration deregulates expression of the viral protein hbx, which is able to induce hcc, either alone or in synergy with different cellular proteins (32) . jameel also discussed tumorigenesis induced by hcv: chronic immune-mediated inflammation and associated oxidative chromosomal dna damage probably play a role. interactions of viral proteins with prb and p53 may also predispose to carcinogenesis (33) . although not all the viruses discussed induce the same pathway to cancer, some parallels can be drawn. a requirement for viral oncogenes indicates that the viruses play a crucial role in progression to malignancy. both rna and dna tumor viruses promote growth of infected tissue by activation of cellular oncogenes and inactivation of tumor suppressor genes. interestingly, many cellular oncogenes and tumor suppressor genes (e.g., p53, prb) were identified through studies of rna and dna tumor viruses, respectively. after infection, these viruses can establish either latent (hpv, ebv, kshv, htlv-1) or chronic (hbv, hcv) infection. activation from latent infection may result from changes in the cell environment, an accumulation of viral stress on the cell and/or a decreased ability of the host to maintain latency or clear infection. such activation signals may be dependent on the host's genetic make-up (34) and epigenetic factors (35) . understanding why some individuals infected with tumor viruses do not develop cancer remains critical. it is anticipated that continuing research into what distinguishes 'normal' viral life cycles from the life cycles in malignant tissues, and the changes in host factors accompanying transformation, will address this problem. so far it has been demonstrated that changes in viral gene expression may accompany the development of high-risk lesions (e.g., hpv, ebv). but why are such changes occurring at a low frequency in infected populations? are these changes in gene expression a result of a virus-host interaction disruption? answering these questions requires a great deal more research, but certainly the speakers at the summer school are on course to do just that. a detailed understanding of the molecular mechanisms governing virus infection and propagation is crucial to the development of antiviral strategies through identification of critical processes and drug targets. this was best exemplified at the summer school by three speakers who are elucidating complimentary stages of the hiv life cycle: ariberto fassati, university college london, discussed nuclear import, alessandro marcello, icgeb, trieste, focused on integration and the spatial and temporal regulation of hiv-1 gene expression, and hans-georg kräusslich, universität heidelberg, dealt with assembly and maturation. although existing highly active antiretroviral therapy (haart) can significantly reduce hiv-related illness, the emergence of drug resistant strains, toxic side effects and their ineffectiveness in latently infected cells has ensured that the development of novel hiv-1 therapies remains an important objective. lentiviruses, such as hiv-1, have the ability to infect terminally differentiated nondividing cells and, therefore, require nuclear import. however, the hiv-1 reverse transcription complex (rtc) is larger than the nuclear pore diffusion size limit. in addition, the rtc is enriched for nucleic acids and so must overcome the hydrophobic exclusion of nuclear pore complexes and a concentration gradient of dna to enter the nucleus. the manner in which hiv-1 resolves this dilemma is worth investigating as nuclear import is critical to hiv-1 transmission and aids pathogenesis. dr a. fassati is doing just that and his group has identified some novel mechanisms by which hiv-1 enters the nucleus. a number of viral elements have been shown to play a role in nuclear import e.g., the cppt element, a nuclear localization signal within matrix and the viral proteins integrase (in) and vpr. fassati's group was interested in identifying the host nuclear transport receptor responsible for hiv-1 import. they used purified rtc complexes in primary macrophages to show that importin 7 (imp7) is involved (36) , an import receptor for rna-and dna-binding ribosomal and histone proteins, respectively. further work revealed that the role of imp7 was hivspecific, which correlated with its ability to bind hiv in (37) . rnai-mediated knockdown of imp7 decreased import of dna, but not rna, indicating that reverse transcription is not a requirement for nuclear import (37) . the dna import-function of imp7 is likely hijacked by hiv-1 to facilitate import of the hiv-1 dna genome, although it remains unclear whether the rtc remains intact throughout the nuclear import process. fassati's group continued to search for other nuclear import pathways. they used nucleic acid dye-labeled rtcs and cells treated with digitonin (to permeabilise the plasma, but not the nuclear, membrane) to monitor nuclear import in the presence of various cytosolic extracts. the almost homogenous fraction capable of inducing nuclear import of hiv-1 rtcs was found to contain trnas (38) . further, they showed that nuclear import of at least some species of trnas occurs in uninfected cells (38) -a previously undescribed cellular pathway of unknown function that hiv-1 appears to exploit. trnas are present in hiv-1 virions and it is speculated that the t-arm promotes nuclear import of the rtc via associations with host factors, which have yet to be identified. fassati's work not only describes novel import pathways for hiv-1, and thus, identifies potential drug targets, it has also enhanced our knowledge of cellular biology through identification of the trna nuclear import pathway. latent viral reservoirs are established early during hiv infection, which prevents eradication of the virus, as they are not susceptible to antiretroviral treatment. in addition, latent proviral dna can be reactivated to replenish viral loads on interruption of treatment. therefore, understanding the molecular mechanisms governing latency and reactivation of viral expression is crucial to developing strategies aimed at complete eradication of the virus. to this end, dr a. marcello described his work into the impact of chromatin and chromosome territories on the control of hiv-1 transcription and latency. marcello has shown that the histone methyltransferase suv39h1, the chromodomain-containing protein hp1c and histone h3k9 trimethylation is enriched at transcriptionally silent proviral dna (39) . rnai targeting hp1c can alleviate the chromatin repression on hiv-1 gene expression in pbmcs from hiv-1-infected donors (39) . whilst providing insight into the mechanisms underlying hiv-1 latency, the question remains as to how some cells come to be latently infected, with repressed chromatin architecture at the proviral ltr, given the propensity for hiv-1 to integrate into active transcriptional units. the nucleus is a highly dynamic and organised entity and it is now widely accepted that active transcription is enriched at the nuclear periphery. marcello's group examined the latently infected cell line, j-lat a1 cells developed by jordan, et al (40), by fish and showed that along with activated viral transcription occurring at the nuclear periphery, the latent provirus was located at the periphery too (41) . chromatin conformation capture (3c) analysis revealed an association of the latent provirus with a pericentromeric region of chromosome 12 in trans, which was lost on reactivation of transcription (41) . although it cannot be ruled out that latency is a result of a rare integration event into an inactive gene at the nuclear periphery, marcello's work supports a model in which integration into an active gene and clonal expansion of the activated t cell population is followed by a number of t cells becoming quiescent memory cells by chance, a transition that is accompanied by the repression of the provirus through epigenetic mechanisms. disruption of this repressive chromatin in an hiv-specific manner is an ongoing challenge. the host cell is a crowded environment, so assembling virions need to form stable structures in the producing cell; but the virion needs to be rapidly destabilised upon entry into a new cell. different viruses have evolved different solutions to this assembly-disassembly paradox. in hiv, the solution is provided by the gag polyprotein. initially, a stable, immature virion with a spherical, capsid shell composed of gag is formed (42) . during maturation, sequential cleavage of the gag polyprotein by protease (pr) (43) results in a rearrangement of the virion to form a mature, infectious virus with a conical capsid, composed solely of the capsid (ca) domains of gag, despite no change in the overall size of the virion (44) . dr h. g. kräusslich provided an overview of his work detailing the mechanisms of virus release and the immature to mature virion transition. cryo-em studies revealed that following proteolytic maturation of gag, the mature capsid contains 1,000-1,500 ca proteins assembled into a hexameric lattice (45) . a completely spherical immature capsid was predicted to contain 5,000 gag polyproteins, more than double the number present in the mature core. subsequent cryoelectron tomography studies demonstrated that, in fact, gag is arranged in an incomplete spherical hexagonal lattice which, in addition, contains holes (46) . the ''holes'' may serve to alleviate the strain imparted by the lattice curvature, as there is no evidence of pentameric defects, as observed in the mature core. the differing arrangement of gag and its derivatives in the immature and mature stages may indicate a disassembly of the immature shell, prior to assembly of the mature core. the extent of disassembly is unclear, but such a process would be an attractive drug target. if the immature virus contains an incomplete shell, how is the virus released from the cell? previously, it was thought that gag assembled at the plasma membrane into spherical shells that resulted in membrane budding, with subsequent cleavage of the resulting thin membrane tether by the cellular endosomal sorting complex required for transport (escrt) machinery (47) (48) (49) (50) . kräusslich's group demonstrated that almost complete gag spheres were only present at late-budding sites where functional escrt was absent (51) . along with their observations that the immature shell is composed of an incomplete gag sphere, they proposed a novel model for the release of hiv-1 virions: budding is initiated by gag assembly with escrt recruited early to drive release (51) . the process can be, therefore, considered a kinetic competition: should assembly occur faster than escrt recruitment, release is incomplete. using total internal reflection fluorescence (tirf) microscopy, kräusslich's group was able to confirm that vps4, an escrt-associated atpase, appears in bursts at sites of hiv-1 particle production early in gag assembly (52) . kräusslich furthered understanding of the role of another host factor, cd137/tetherin, in hiv-1 release. known to be an hiv-1 restriction factor, cd137 is downregulated by the hiv accessory protein vpu (53) . however, rodent and mouse cd137 potently inhibits hiv-1 release through its resistance to vpu (54) . this has consequences for the development of small animal models of hiv infection. the work of kräusslich, marcello, and fassati reveals that continued efforts into discerning the molecular mechanisms of hiv-1 replication facilitates development of effective antivirals, an approach applicable to all pathogenic viruses. the immune system is important in curtailing the detrimental effects of hpv infection: more lesions are observed in severe combined immunodeficiency (scid) patients (55) . further, detection of hpv dna in women is age-dependent (56) , which may be due to changes in the immune system. dr j. doorbar described how the immune system struggles to clear hpv: langerhans cells are not very effective as viral proteins are expressed at low levels in the lower epithelial layers. in addition, hpv proteins e6 and e7 downregulate cytokines and interferons, respectively (57) . e5 is also thought to play an immunosuppressive role through inhibition of major histocompatability (mhc) maturation (58) . hence, without intervention, hpv-associated warts and verrucas can take months to disappear. it is also this delay in immune clearance of the virus that may predispose individuals to cancer. fortunately, an hpv vaccine is available. whilst effective, the vaccine protects only against the two most predominant high-risk types, so that it is 75-80% protective (59) . nevertheless, widespread administration of this vaccine would greatly reduce the cervical cancer health burden. fear of influenza pandemic has enveloped the world over recent years. prof j. l. virelizier, pasteur institute, paris, addressed the immune response to this virus. typically, humans successfully eradicate influenza, following the production of antibodies, although the virus is transferred to another individual prior to antibody production. the fear of a pandemic is born out of concern for a gene reassortment giving rise to a virus that is able to both infect humans and cause fatality. antibodies to the envelope protein haemagglutinin (ha) are protective against influenza (60) . however, the virus is able to avoid the immune system through antigenic drift and periodic antigenic shift, the latter having the potential to cause pandem-ics (61) . passive transfer of polyclonal antibodies specific to influenza pr8 ha protected against homologous pr8 virus infection in mice, but transfer of cross-reactive anti-fm1 ha antibodies did not (62, 63) . this highlights the problem that influenza protection requires strain-specific, not cross-reactive, antibodies, which needs to be taken under consideration in vaccine development. virelizier also discussed the relevance of lymphocyte memory to the antiviral immune response. he described the original antigenic sin phenomenon, in which immunisation with one variant and boosting with another recalls not just cross-reactive antibodies but also antibodies to variant-specific determinants (64, 65) . for example, immunisation with ha strain 1 (h1) will not protect against another strain e.g., h3, but will improve the primary response to another h1 variant years later. this relates to the dual function of b cells as both antibody producers and antigen presenting cells: immunisation primes t cells, via b cell antigen presentation, not just b cell antibody production. thus, following primary exposure to another variant, t cell help facilitates a more rapid antibody response, including to newly encountered epitopes (66) . this is important in vaccine administration practices as it emphasises the importance of vaccinating not just the older members of society but also the young, so as to afford them lymphocyte memory. severe acute respiratory syndrome (sars) was first reported in asia in 2003 and within a few months the virus had spread to more than 24 countries in asia, north america, south america, and europe (67) . prof luis enjuanes, consejo superior de investigaciones cientificas (csic), madrid, gave a detailed lecture on his development of a sars-cov vaccine. his group has demonstrated that recombinant virus rsars-cov-de protected balb/c mice from fatal respiratory disease when challenged with sars-cov, and partly protected mice expressing the viral receptor, hace2 (68) . hamsters immunised with rsars-cov-de showed decreased lung inflammation and clinical illness on sars-cov challenge (69) . the recombinant strain also induced antivirus t cell and antibody responses (68) . work is ongoing to elucidate the role of e, but differential gene expression studies implicate regulation of the unfolded-protein stress response (70) . whilst highly promising, the vaccine is not yet considered safe for humans, particularly given the prevalence of coronaviruses and the risk of recombination. existing vaccine production involves chemical attenuation, but this also carries safety concerns, as inactivation can be incomplete. the use of rsars-cov-de, rather than wild type sars-cov, in chemical attenuation could improve the safety of vaccine production, without compromising immunogenicity. hiv has infected more than 60 million people worldwide, mostly in the developing world, and nearly half of these indi-viduals have died (71) . the epidemic is driven, in part, by the exquisite ability of hiv to evade the immune system. dr s. jameel discussed various mechanisms used by hiv to escape the host immune system. the multifunctional hiv accessory protein nef optimises the cellular environment for viral replication via associations with a number of host factors. it has been shown that nef is able to downregulate expression of major histocompatability molecule (mhc)-i and -ii and the mhcii-binding protein cd4 (72) . jameel's group has also identified a role of nef in the downregulation of costimulatory molecules cd80 and cd86 (73) . their ongoing work is investigating the potential role of another hiv accessory protein, vpu, in decreasing antigen presentation through interactions with the mhcii invariant chain, cd74 (74) . given the ability of hiv to evade the immune system so successfully, considerable efforts are being devoted to the development of a vaccine. despite a decade of research, an effective vaccine remains elusive. three characteristics of hiv infection point to the difficulties associated with developing an effective vaccine: 1) persistent replication, 2) cellular and humoral immunity escape, and 3) immunosuppression. neutralising antibodies have been shown to provide protection in primate models (75) . dr jeffrey dorfman, icgeb, cape town, described how he believed a successful hiv-1 vaccine would elicit broadly neutralising antibodies (bnabs). dorfman is hoping to identify novel bnabs by screening sera with a pseudovirus neutralisation assay. such bnabs will be useful for the identification of epitopes that will inform vaccine design. identification of bnabs is hampered, however, by technical difficulties associated with their isolation and production, particularly as they are rare. dorfman has developed a novel method for their production, which involves fusion of a pbmc with a myeloma (76) , sidestepping the requirement for ebv-mediated transformation. prof carolyn williamson, university of cape town, discussed the genetic bottleneck of hiv transmission, i.e., only a single, or very few, viruses establish infection (77) . interestingly, these strains appear more neutralisation-sensitive, which bodes well for bnab-inducing vaccines. whether there is an infectious advantage in mucosal sites for a less stable viral envelope that is more likely to expose conserved epitopes is unclear. although nabs are the basis of protection for most antiviral vaccines, there are concerns over their efficacy in preventing hiv-1 infection. the bnab response would have to be sufficiently strong and rapid at mucosal sites to prevent any infection, even low-level. this is because bnabs are unable to neutralise a virus transmitted through a virological synapse, due to steric hindrance (78) . in addition, any virus that does not contain the epitope will be rapidly selected for. therefore, no matter how broad the nabs are it is likely that a vaccine would need to elicit a number of bnabs to increase its protectiveness. nevertheless, bnabs are the only way of conferring sterilising immunity and, as such, are still the holy grail of hiv vaccine design. other scientists believe an effective hiv-1 vaccine will elicit cytotoxic t lymphocyte (ctl) responses. however, prof j. l. virelizier described how there is no precedent that ctls, in any animal against any virus, kill virus-infected cells by a cytotoxic mechanism. conversely, williamson reported on studies revealing a correlation between specific hla alleles and the breadth of hiv gag-specific t lymphocyte responses with the control of viral replication (79) , although there have been conflicting results (80) . and, of course, correlation is not causation. that is not to say that t cell immunity is not protective: ctls injected into a mouse liver expressing an hbv transgene resulted in decreased viral replication (81) . histology revealed that the protection was not conferred through a cytotoxic mechanism, rather through production of tnfa, as replication was restored in the presence of an anti-tnfa antibody (81) . t cell activation can also confer protection against hiv through production of chemokines. rantes and mip1a and sdf1 are the host ligands for hiv coreceptors ccr5 and cxcr4, respectively (82) . these chemokines can block hiv entry because they compete with hiv for the receptors. virelizier described the ability of truncated rantes 9-68 to inhibit hiv infection, whilst no longer acting as an agonist, as truncation prevents signaling through g proteins (83) . there are reports, however, that hiv can use other coreceptors, including ccr3, strl33/bonzo/tymstr, and bob (84), complicating the use of chemokines or their mimics as a therapy. in addition, chemokines will suffer the same limitations of bnabs at the virological synapse. although t cell activation can be antiviral (through the production of interferons, chemokines, and tnfa), it may be a double-edged sword: virelizier proposed the hypothesis that t cell activation contributes to persistent replication, through nf-jb signaling initiating hiv transcription. nevertheless, given the help that t cells can provide to b cells and antibody production, it is likely that an ideal hiv vaccine would elicit both humoral and cellular immunity. there is a school of thought that a vaccine is not required to prevent hiv-related deaths at all. hiv-infected patients die as a result of their immunosuppression. conversely, primates are able to survive with host-specific siv infection indefinitely because their immune systems are not destroyed. virelizier speculates, therefore, that research should be focused on combating hiv-mediated immunosuppression. in summary, understanding the immune response to viral pathogens is critical, to counteract viral evasion of immune responses and to discern what responses are protective to inform vaccine design. this will enhance our ability to induce effective antiviral immune responses, reducing the probability of disease. beyond the enormous health concern that arose with the sars epidemics and the major economic losses caused by this family of viruses, coronaviruses (covs) have the potential to be promising delivery vectors for vaccine development and gene therapy (85) . a highlight of the course was a presentation by dr l. enjuanes, on the mechanism of transcription used by the coronaviridae family. he emphasised the importance of rna chaperones, which are transcribed by the viral genome and are essential for efficient cov replication (86) . enjuanes' group identified a cov transcriptional enhancer, based on a long distance rna-rna interaction in the transmissible gastroenteritis coronavirus (tgev) (87) . the additional discovery of transcription regulatory sequences permitted genetic manipulation to improve gene expression. as well as enhancing basic knowledge of cov biology, these findings facilitated development of a safe and successful cov-based delivery vector. advantages covs possess over other viruses as expression vectors include: 1) the possibility of spike protein manipulation, to engineer virus tropism (88, 89) ; 2) the replication of the rna genome in the cytoplasm, side-stepping potential problems associated with integration (90); 3) the existence of nonpathogenic strains that infect a wide range of species of health and economic importance; 4) the ability to carry large genomes (27-30 kb) , which could favor the introduction of extensive foreign genes (91); and 5) the availability of cdna clones derived from infectious strains (92, 93) . two expression systems have been developed based on covs: one is a helper-dependent expression system, where the production of large amounts of heterologous antigen (2-8 lg/ 10 6 cells) has been achieved and synthesis has been retained for around 10 viral passages (85) . in addition, there are the single genome cov vectors, which can either be constructed by target recombination or by using an infectious cov cdna clone derived from the tgev genome. this system has been shown to express a foreign gene for at least 20 passages (94, 95) , demonstrating its high stability. ongoing research is exploring the possibility of manipulating the species and tissue tropism of these cov expression systems. enjuanes' work reveals that these cov vectors are flexible, robust tools with the potential to aid vaccine production and gene therapy. virus-associated disease may arise for a number of reasons: a virus replicating in a suboptimal host or tissue, the emergence of a new, more pathogenic variant, an accumulation of detrimental effects when an infection clearance fails, or an enhanced susceptibility of the host. research into the molecular basis of virus infection and propagation will enhance our understanding of virus-associated disease and inform new antiviral strategies and vaccine design. in addition, ways in which viruses can be harnessed for our own means will emerge. participants at the summer school learnt a great deal about how to approach their work from the top-quality research presented. the school embodied not just good science, but also a great atmosphere (fig. 2) , epitomised by the inclusion of every-one in the birthday celebrations of speakers l. enjuanes and j. l. virelizier. informal interaction throughout, and plenty of time allocated for discussion, ensured participants gained invaluable input from lecturers. allowing all participants to present their work at the school further stimulated discussion and has led to new collaborations; an opportunity that would not have been afforded to the developing country scientists without the summer school. a prerequisite stage in the initiation of polypeptide chains n-formyl-methionyl-sigma-ribonucleic acid and chain initiation in protein biosynthesis. polypeptide synthesis directed by a bacteriophage ribonucleic acid in a cell-free system how proteins start coding response of n-fromylmethionyl-srna to uug specific codon-anticodon interaction of an initiator-trna fragment viruses associated with human cancer molecular biology of human papillomavirus infection and cervical cancer recombinant retroviruses encoding human papillomavirus type 18 e6 and e7 genes stimulate proliferation and delay differentiation of human keratinocytes early after infection the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 reversible repression of papillomavirus oncogene expression in cervical carcinoma cells: consequences for the phenotype and e6-p53 and e7-prb interactions integration of the hpv16 genome does not invariably result in high levels of viral oncogene transcripts organization of human papillomavirus productive cycle during neoplastic progression provides a basis for selection of diagnostic markers downregulation of e-cadherin is closely associated with progression of cervical intraepithelial neoplasia (cin), but not with high-risk human papillomavirus (hpv) or disease outcome in cervical cancer hpv16 e6 augments wnt signaling in an e6ap-dependent manner human keratin 14 driven hpv 16 e6/e7 transgenic mice exhibit hyperkeratinosis estrogen contributes to the onset, persistence, and malignant progression of cervical cancer in a human papillomavirus-transgenic mouse model loss synergizes with estrogen and papillomaviral oncogenes to induce cervical and breast cancers requirement for estrogen receptor alpha in a mouse model for human papillomavirus-associated cervical cancer human papillomavirus associated with oesophageal cancer oesophageal cancer in africa hpv16 e6 and e7 proteins cooperate to immortalize human foreskin keratinocytes integration of human papillomavirus type 16 into cellular dna of cervical carcinoma: preferential deletion of the e2 gene and invariable retention of the long control region and the e6/e7 open reading frames presence of episomal and integrated human papillomavirus dna sequences in cervical carcinoma expression of the open reading frame 74 (g-protein-coupled receptor) gene of kaposi's sarcoma (ks)-associated herpesvirus: implications for ks pathogenesis the kaposi's sarcoma-associated herpes virus g protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1alpha endothelial infection with kshv genes in vivo reveals that vgpcr initiates kaposi's sarcomagenesis and can promote the tumorigenic potential of viral latent genes kshv vflip is essential for the survival of infected lymphoma cells reflections on epstein-barr virus: some recently resolved old uncertainties epstein-barr virus and oncogenesis: from latent genes to tumours human t-cell leukemia virus type i and adult t-cell leukemia the px protein of htlv-i is a transcriptional activator of its long terminal repeats the hepatitis b virus x gene potentiates c-mycinduced liver oncogenesis in transgenic mice tumor suppressors, chromosomal instability, and hepatitis c virus-associated liver cancer hcv tumor promoting effect is dependent on host genetic background epigenetic changes in virus-associated human cancers nuclear import of hiv-1 intracellular reverse transcription complexes is mediated by importin 7 hiv-1 exploits importin 7 to maximize nuclear import of its dna genome trnas promote nuclear import of hiv-1 intracellular reverse transcription complexes suv39h1 and hp1gamma are responsible for chromatin-mediated hiv-1 transcriptional silencing and post-integration latency hiv reproducibly establishes a latent infection after acute infection of t cells in vitro transcriptional competence of the integrated hiv-1 provirus at the nuclear periphery cryo-electron microscopy reveals ordered domains in the immature hiv-1 particle sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual gag polyprotein cleavage sites structural organization of authentic, mature hiv-1 virions and cores the stoichiometry of gag protein in hiv-1 structure and assembly of immature hiv tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding aip1/alix is a binding partner for hiv-1 p6 and eiav p9 functioning in virus budding pr55(gag) the protein network of hiv budding three-dimensional analysis of budding sites and released virus suggests a revised model for hiv-1 morphogenesis dynamics of hiv-1 assembly and release tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu hiv-1 antagonism of cd317 is species specific and involves vpu-mediated proteasomal degradation of the restriction factor severe cutaneous papillomavirus disease after haemopoietic stem-cell transplantation in patients with severe combined immune deficiency caused by common gammac cytokine receptor subunit or jak-3 deficiency prevalence of hpv in cytomorphologically normal cervical smears, as determined by the polymerase chain reaction, is age-dependent papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and nf-kappab-responsive genes in cervical keratinocytes the e5 protein of human papillomavirus type 16 perturbs mhc class ii antigen maturation in human foreskin keratinocytes treated with interferon-gamma human papillomavirus vaccines development and application of reference antisera against 15 hemagglutinin subtypes of influenza virus by dna vaccination of chickens avian influenza and human health host defenses against influenza virus: the role of anti-hemagglutinin antibody rationale for treating human influenza infections by passive transfer of specific antibodies disquisitions on original antigenic sin. ii. proof in lower creatures disquisitions of original antigenic sin. i. evidence in man pathogenic epitopes, heterologous immunity and vaccine design summary of probable sars cases with onset of illness from 1 immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in e protein protects against lethal respiratory disease a live attenuated severe acute respiratory syndrome coronavirus is immunogenic and efficacious in golden syrian hamsters the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles years of hiv biology of the hiv nef protein. indian the nef protein of hiv-1 induces loss of cell surface costimulatory molecules cd80 and cd86 in apcs human immunodeficiency virus type 1 vpu protein interacts with cd74 and modulates major histocompatibility complex class ii presentation protection of macaques against vaginal transmission of a pathogenic hiv-1/siv chimeric virus by passive infusion of neutralizing antibodies multiple myeloma: methods and protocols hiv molecular epidemiology: transmission and adaptation to human populations avoiding the void: cell-to-cell spread of human viruses challenges in the development of an hiv-1 vaccine viral evolution and challenges in the development of hiv vaccines cytotoxic t lymphocytes inhibit hepatitis b virus gene expression by a noncytolytic mechanism in transgenic mice chemokines as natural hiv antagonists synthetic full-length and truncated rantes inhibit hiv-1 infection of primary macrophages blocking hiv co-receptors by chemokines coronavirus derived expression systems role of rna chaperones in virus replication identification of a coronavirus transcription enhancer two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier the molecular biology of coronaviruses reverse genetics of the largest rna viruses engineering the largest rna virus genome as an infectious bacterial artificial chromosome infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity the authors thank dr samantha barichievy and laurence eastham for constructive review of the manuscript. key: cord-285151-zynor0b2 authors: eisenhut, michael title: neopterin in diagnosis and monitoring of infectious diseases date: 2013-12-08 journal: j biomark doi: 10.1155/2013/196432 sha: doc_id: 285151 cord_uid: zynor0b2 neopterin is produced by activated monocytes, macrophages, and dendritic cells upon stimulation by interferon gamma produced by t-lymphocytes. quantification of neopterin in body fluids has been achieved by standard high-performance liquid chromatography, radioimmunoassays, and enzyme-linked immunosorbent assays. neopterin levels predict hiv-related mortality more efficiently than clinical manifestations. successful highly active antiretroviral therapy is associated with a decrease in neopterin levels. elevated neopterin levels were associated with hepatitis by hepatitis a, b, and c viruses. serum neopterin levels were found to be a predictor of response to treatment of chronic hcv infection with pegylated interferon combined with ribavirin. neopterin levels of patients with pulmonary tuberculosis were found to be higher in patients with more extensive radiological changes. elimination of blood donors with elevated neopterin levels to reduce risk of transmission of infections with known and unknown viral pathogens has been undertaken. neopterin measurement is hereby more cost effective but less sensitive than screening using polymerase chain reaction based assays. in conclusion neopterin is a nonspecific marker of activated t-helper cell 1 dominated immune response. it may be a useful marker for monitoring of infectious disease activity during treatment and for more accurate estimation of extent of disease and prognosis. neopterin was first isolated from larvae of bees, in worker bees and in royal jelly in 1963, and subsequently from human urine by sakurai and goto in 1967 [1] . neopterin or 2-amino-4-hydroxy-6-(d-erythro-1 ,2 ,3trihydroxypropyl)-pteridine is produced from guanosine triphosphate via guanosine triphosphate cyclohydrolase i (gtpch i) by activated monocytes, macrophages, dendritic cells, and endothelial cells and to a lesser extent in renal epithelial cells, fibroblasts, and vascular smooth muscle cells upon stimulation mainly by interferon gamma and to a lesser extent by interferon alpha and beta with its release being enhanced by tumor necrosis factor [2, 3] . gtpch i mrna expression is synergistically and independently induced by interferon gamma through the jak2/stat pathway of nuclear transcription regulation and through tnf by the nf-kappab pathway (see figure 1 ) [4] . release in response to cytokines released by t-lymphocytes and natural killer cells make neopterin an indicator of activation of cell mediated immunity including release by infections associated with activation of t-lymphocytes and natural killer cells, malignancies, autoimmune diseases, rejection of transplanted organs, and atherosclerosis. at its first isolation in the 1960s neopterin was detected in the pupae of bees by anion exchange chromatography followed by paper chromatography [1] . in the seventies gas chromatographic-massfragmentographic methods were described allowing measurement in urine. subsequently detection and quantification of neopterin succeeded in serum, urine, and other body fluids using standard high pressure and by reverse-phase high-performance liquid chromatography with fluorescence detection. later simpler radioimmunoassays and more recently enzymelinked immunosorbent assays have been developed which are suitable for large numbers of samples [1] . semiquantitative measurement with a dipstick system using polyclonal antineopterin antibodies has been validated and may be suitable for bedside testing and in the setting of developing countries [5] . levels have been observed, which correlate with the activity of disease. this was first described in 1979 [6] and subsequently neopterin elevations were noted in infections with hepatitis viruses, epstein-barr, cytomegalo, measles, mumps, varicella zoster, rubella, and influenza viruses [1, [7] [8] [9] [10] [11] . elevated neopterin levels in body fluids were found at the end of the incubation period before the onset of clinical symptoms. the highest neopterin levels occur just before specific antibodies against the virus become detectable, which is about two to four weeks after onset of increased neopterin production. in acute varicella zoster virus infection peak neopterin levels were observed at the end of the appearance of the rash and in measles virus infection one to three days after appearance of the rash [12, 13] . immunisation with live viruses, for example, measles, mumps and rubella and virus vaccine, resulted in a significant increase of neopterin independent of presence of any symptoms. in measles vaccination neopterin levels were observed to rise at a median of 5 days after vaccination about 7 days before the appearance of antibodies [13] . these investigations point to a future application of measurements neopterin as a correlate of a successful vaccination. neopterin should be investigated as a marker to evaluate protective efficacy of vaccines stimulating cell mediated immunity against mycobacterial, parasitic, or viral diseases. the magnitude of the elicited neopterin levels could be put into relationship to incidence of the disease immunised against the population of immunised children. serum neopterin levels were also found to be significantly elevated in symptomatic dengue virus infections with levels higher than in measles and influenza virus disease [14] . levels correlated with duration of fever and severity of disease [14, 15] . investigations into the physiological functions of neopterin in viral infections revealed that it is able to delay the development of the cytopathic effect of coxsackie b5 virus in hep-2 cells [16] . a proposed mechanism is the stimulation of inducible nitric oxide synthase expression leading to an increase in nitric oxide production. other mechanisms include the induction of the translocation of the nuclear factor-kappa b to the nucleus. infection. testing of 328 samples of 29 hiv infected individuals found that 44/68 (64.7%) of samples, which were hiv-1 rna and p24 antigen positive had elevated neopterin levels (>10 nmol/l). 6/216 (2.8%) samples, which were both hiv-1 and p24 antigen negative had elevated neopterin levels [17] . neopterin levels were also found to be significantly elevated in hiv-2 infection compared to controls [18] . studies investigated markers of immune activation for their usefulness as prognostic markers in hiv infection and showed an increase of neopterin levels in people with hiv infection compared to patients without hiv infection [19] [20] [21] [22] . neopterin levels hereby were found to increase early in the course of hiv infection preceeding cd4+-t-cell decline and clinical manifestations of aids [23, 24] . plasma neopterin levels were found to correlate with plasma hiv viral load [25] . neopterin levels were found to predict hiv related mortality [26, 27] . a retrospective study compared 2 microglobulin, immunoglobulin a, g, and m, adenosine deaminase, and neopterin levels above normal range as predictors of clinical or immunological deterioration in 256 patients with hiv infection. changes in 2 microglobulin levels showed the greatest sensitivity to detect worsening (43%) with neopterin slightly less sensitive (41.9%) followed by immunoglobulin levels (26.8-35 .2%) and adenosine deaminase levels with 21.8% having the lowest sensitivity [28] . marker for viral load to monitor response to antiretroviral treatment. in a land mark study the effects of dual reverse transcriptase inhibitor (rt) therapy and highly active antiretroviral therapy (haart) on neopterin levels in patients with hiv infection were compared to hiv uninfected controls, hiv infected patients not on treatment, and patients who had stopped treatment [22] . rt inhibitor treatment decreased circulating levels of neopterin (mean of 15.6 for treated versus a mean of 22.3 ng/ml for untreated hiv patients, < 0.04). haart decreased neopterin levels significantly further. this confirmed results of a previous study on the effects of haart on neopterin levels [29] . neopterin levels in patients who discontinued haart became similar to untreated hiv patients. neopterin may be a particularly useful surrogate marker for monitoring of control of hiv replication in settings in developing countries where hiv rna viral load measurement is not available and may be a cheaper alternative particularly if semiquantitative dip stick tests are used for urine samples [5] . longitudinal serial measurements in the same individual could overcome difficulties with interpretation in settings where chronic parasitic (malaria) or bacterial schistosoma mansoni praziquantel blood serum levels normalized on treatment [43, 44] (tuberculosis) infections may elevate the baseline neopterin level and could allow monitoring of response to antiretroviral treatment in the absence of resistance testing and provide means to monitor compliance in the outpatient setting (see table 1 for list of diseases in which neopterin levels have been used to monitor treatment response). hepatitis. the first study investigating the role of neopterin in specific forms of viral hepatitis tested urinary levels in patients with hepatitis a, hepatitis b, and non-a, non-b hepatitis virus infection [9] . the authors noted that in 51 patients with acute viral hepatitis 49 patients had elevated urinary neopterin levels with the highest levels found in patients with acute hepatitis a. while all patients with active hepatitis b had elevated neopterin levels, 49/62 hbsag carriers (77%) had normal urinary neopterin levels. the authors noted that neopterin levels were not a reflection of hepatocellular damage as 3 patients with alcoholic hepatitis had normal urinary neopterin levels. in order to address the question whether neopterin is a useful marker for early detection of viral infection in donated blood products before seroconversion, one study investigated neopterin levels in anti-hcv-negative specimens, which were hcv rna and hcv core antigen positive. the investigators found that 8/217 (3.7%) had elevated neopterin levels (>10 nmol/l). 4/115 (3.5%) specimens positive for hbv dna had elevated neopterin levels [17] . in 106 patients with thalassemia major receiving multiple blood transfusion significantly more patients with histologically proven chronic hepatitis (19/21 were anti hcv antibody positive) had elevated blood neopterin levels compared to patients with siderosis of the liver [45] . alanine aminotransferase levels in hcv infected persons correlated significantly with neopterin levels. serum neopterin levels were found to be a useful predictor of response to treatment of chronic hcv infection with pegylated interferon combined with ribavirin. neopterin concentrations were evaluated in 260 hcv patients treated by pegylated interferon combined with ribavirin. mean and median pretreatment neopterin concentrations were lower in patients with sustained virological response than in nonresponders. the rate of response was twofold higher among patients with pretreatment neopterin levels <16 nmol/l than in patients with neopterin levels ≥16 nmol/l, even after controlling for hcv genotype status [38] . a recent study investigated specifically whether serum neopterin levels can discriminate between patients with replicative ( = 30) and nonreplicative ( = 25) hbv carriage [46] . replicative hbv carriage was defined as hbv dna >5 pg/ml by hybrid capture system. neopterin levels had a mean of 14.5 nmol/l in replicative versus 8.8 nmol/l in nonreplicative hbv carriers ( < 0.05). this result was not reproducible in another study, which found that in patients with replicative hbv infection ( = 30) mean serum neopterin level was 24.73 nmol/l and in nonreplicative hbv ( = 30) 14.8 nmol/l a difference, which was not statistically significant [47] . this may have been due to large standard deviations and small numbers in groups. a more recent investigation found that in chronic hepatitis the mean ± sd serum neopterin levels were 14.2 ± 5.6 nmol/l, 20.3 ± 7.9 nmol/l in patients with liver cirrhosis and 5.2 ± 1.4 nmol/l in the control group. serum neopterin levels were significantly higher in patients with chronic hepatitis ( = 0.005) and cirrhosis patients ( = 0.008) than in control subjects. cirrhotic patients had significantly higher serum neopterin levels than patients with chronic hepatitis ( = 0.004). there was a positive correlation between serum neopterin levels and alanine aminotransferase levels in patients with chronic hepatitis ( = 0.41, = 0.004) and cirrhotic patients ( = 0.39, = 0.005). positive correlations were detected between serum neopterin levels and inflammatory score in patients with chronic hepatitis ( = 0.51, = 0.003) and cirrhotic patients ( = 0.49, = 0.001) [48] . infections. neopterin has been investigated as a marker to distinguish viral from bacterial lower respiratory tract infections. the investigators found that serum neopterin levels were elevated (>10 nmol/l) in 96% of patients with viral lrti. the median serum neopterin concentration was almost 2-fold higher in the viral lrti group than bacterial lrti patients (30.5 versus 18.7 nmol/l) and 5-fold higher than those in healthy controls. the specificity for correct identification of viral lrti was 69.5% for a cut-off of >15 nmol/l [49] . serial monitoring of serum neopterin levels in patients with severe acute respiratory syndrome (sars) associated virus revealed that all ( = 129) investigated patients had elevated neopterin levels by day 9 [50] . duration of pyrexia in sars patients correlated positively with neopterin levels. patients on steroid therapy had significantly lower neopterin levels. measurement of neopterin in isolation and in relationship to other inflammatory markers like procalcitonin and c-reactive protein were investigated for discriminatory power between viral and bacterial lower respiratory tract infections. investigators used the crp/neopterin ratio (c/n ratio) to discriminate viral and bacterial etiology of respiratory tract infections. in a study conducted in hong kong sera obtained on the day of hospitalization for lrti from 139 patients with confirmed bacterial etiology and 128 patients with viral etiology were examined. a further 146 sera from healthy chinese subjects with no infection were included as controls. the area under the receiver operating characteristic (roc) curve (area under curve [auc]) for distinguishing bacterial from viral infections was 0.838 for crp and 0.770 for pct. the auc for distinguishing viral from bacterial infections was 0.832 for neopterin. when the markers were used in combination, auc of roc of the c/n ratio was 0.857, whereas (crp × pct)/neopterin was 0.856 [49] . in a subsequently reported study the median of the c/n ratio was 10 times higher in patients with bacterial aetiology than with viral aetiology (12.5 versus 1.2 mg/nmol; < 0.0001) and 42 times higher than those in healthy subjects (12.5 versus 0.3 mg/nmol; < 0.0001). the area under the receiver operator characteristic curve for the c/n ratio was 0.840. a cut-off value of "c/n ratio >3" for ruling in/out bacterial/viral infection yielded optimal sensitivity and specificity of 79.5% and 81.5%, respectively [51] . early studies showed elevated neopterin levels in cerebrospinal fluid (csf) of patients with aseptic meningitis and herpes simplex and measles virus encephalomyelitis [52] [53] [54] . csf levels of neopterin seem to reflect intrathecal production by microglia as pterins have a low permeability across the blood brain barrier with a serum-to-csf distribution at a quotient of 1/40 [55] . it has recently been established that normal csf neopterin is brain-derived. the interindividual variation of csf neopterin in healthy adults was found not to depend on serum neopterin concentration variation (coefficient of variation, cv-csf = 9.7% < cv-serum = 24.5%). additionally individual normal csf neopterin concentrations were found to be invariant to the variation of the albumin quotient, qalb; that is, csf neopterin does not derive from leptomeninges [56] . patients with viral meningitis had elevated csf neopterin levels compared to healthy controls but normal serum levels [54] . csf neopterin levels correlated hereby with csf monocytic cell count. patients with various forms of encephalitis including those caused by herpes simplex virus, varicella zoster virus, and tick borne encephalitis virus had significantly elevated csf neopterin levels compared to controls and higher levels than in patients with viral meningitis without overlap of levels in the two conditions. in hiv infection there was a clear relationship between the severity of aids-related dementia and csf neopterin levels [12, [30] [31] [32] . higher csf hiv viral loads were associated with higher csf neopterin levels [33] . after commencement of combination antiretroviral therapy (art), csf neopterin decreased markedly but remained slightly above normal levels in a substantial number of patients despite several years of receiving art [32, [34] [35] [36] . even patients with systemic virological failure exhibit a substantial reduction of csf neopterin concentrations, though above that of virologically suppressed patients [37] . in patients on combination art, the lowest csf neopterin levels have been found in patients with the lowest csf viral loads (<2.5 copies/ml) [32] . no significant difference in csf neopterin concentrations was found between those treated with protease inhibitor-and nonnucleoside reverse transcriptase based regimens in combination with 2 nucleoside analogues [57] . this would support the idea that viral replication within or close to the csf, at least to some extent, is partly driving the inflammatory response. it has also been suggested that an inflammatory response, once triggered, may lead to a self-sustaining state of cellular activation as has been seen in patients with herpes simplex virus type-1 encephalitis [58] . findings in this study are consistent with these reports. hiv rna levels measured in csf or plasma were not significantly associated with csf neopterin trajectories. in addition, all study participants had experienced virologic control to the limit of standard detection as a result of their treatment and csf neopterin levels were the only factor strongly associated with subsequent decay rates and the ultimate set-point levels [32] . patients with bacterial infections with species other than mycobacteria showed significantly lower urinary neopterin levels compared to patients with viral infections in one study [59] but no statistically significant difference in a more recent study [60] . within the group of bacterial infections it was shown that patients with symptoms for at least 5 days had significantly higher neopterin concentrations than patients with acute illness. this applied particularly to bacterial pneumonia. patients with urinary tract infections were found to have similar levels to patients with viral infections with data on urinary neopterin concentrations but not serum concentrations. thus it remains unclear whether local production of neopterin takes place in urinary tract infections and serum neopterin would stay low. there was no significant difference in neopterin levels between patients with febrile neutropenia and underlying haematological and oncological conditions and gram-negative versus gram-positive infections [61] . in patients on an intensive care unit with sepsis journal of biomarkers 5 and septic shock urinary neopterin/creatinine ratios were found to be significantly higher compared to patients with other forms of systemic inflammatory responses syndromes [62] and serum neopterin levels were higher in nonsurvivors compared to survivors of sepsis and multiorgan failure scores correlated with neopterin levels [63] [64] [65] [66] . in this context it was however noted that neopterin levels correlated negatively with reduced renal function reflecting renal failure causing a reduced excretion of neopterin. future studies could correct for reduced excretion due to reduced renal function by calculation of the serum neopterin/creatinine ratio. investigations on critically ill patients on intensive care units evaluated neopterin levels as tool to discriminate patients with systemic inflammatory response syndrome with and without infectious etiology. neopterin levels were found to have a specificity of 78% for discriminating infectious and noninfectious etiology of critical illness [66] . bacterial meningitis was associated with both elevated serum and csf neopterin levels compared to controls [55] . in lyme neuroborreliosis-a late complication of infection by the tick-born spirochete borrelia burgdorferi-high neopterin concentrations were found in csf of patients, whereas serum neopterin levels were not markedly increased, confirming intrathecal neopterin production [67] . infection with treponema pallidum subsp. pallidum (syphilis) was not associated with elevated neopterin levels [18] . in melioidosis by pseudomonas pseudomallei neopterin concentrations were found to be significantly higher than controls [12] . in brucellosis neopterin levels were with a mean 52.5 mmol/ml significantly higher than healthy controls and patients with tuberculosis [68] . in leprosy caused by mycobacterium leprae 75% of patients with tuberculoid and lepromatous leprosy presented with elevated urinary neopterin excretion [69] . on the basis of in vitro and in vivo data showing that macrophages release neopterin in response to stimulation by t-lymphocytes [70, 71] fuchs et al. [39] investigated urinary neopterin levels by hplc in 55 patients with culture confirmed pulmonary tuberculosis and compared them with 417 normal controls. 83% of patients had levels above the upper tolerance limit (containing with 95% probability 97.5% of healthy controls). neopterin levels were higher than age and gender matched controls for every extent of pulmonary disease and correlated with its extent. the correlation with extent of pulmonary tuberculosis was also demonstrated for serum levels and levels found in bronchioalveolar lavage fluid [72] . subsequent studies showed higher levels of neopterin in serum and pleural effusions of patients with pulmonary tuberculosis compared to controls [73, 74] . peripheral blood mononuclear cells (pbmnc) from tuberculosis patients showed a significantly higher spontaneous production of neopterin. stimulation with phytohaemagglutinin or purified protein derivative did not yield higher neopterin production in pbmnc of patients with tuberculosis showing that it is not the production capacity for neopterin which is different [74] . elevated serum neopterin levels were also found in hiv infected patients with tuberculosis and decreased significantly on antituberculous treatment [40] . a relapse of tuberculosis was in 2 cases characterized by increase in neopterin levels. further studies compared neopterin levels in urine, serum, and bronchoalveolar lavage fluid and found that they correlate significantly in patients with tuberculosis [72, 75] . the elevation of neopterin in patients with tuberculosis was more pronounced in urine than in serum or bronchoalveolar lavage [75] . diseases. patients with pulmonary tuberculosis had significantly higher urinary neopterin levels compared to patients with lung cancer or pneumonia with more than twice the concentration reported in adults [74] . pleural fluid neopterin levels were investigated for its ability to differentiate between tuberculous and malignant pleural effusion and were found to be significantly higher in patients with pleural tuberculosis but performance characteristics including receiver-operating characteristics curve analysis was inferior to adenosine deaminase [76] . waiting for the results of susceptibility tests to select an effective antituberculosis drug regimen often causes a delay in effective treatment which can be disastrous, especially in children. because the x-ray changes tend to resolve very slowly and may get even worse after starting therapy because of a paradoxical reaction due to immune-reconstitution even in otherwise immune-competent patients, other than clinical status there is no reliable parameter to reflect success or failure of the drug regimen [77] . urinary neopterin levels declined on twice weekly measurements in all monitored patients with pulmonary tuberculosis on treatment and fell to below tolerance limits within 10 weeks of treatment in 6/10 patients [39] . measurement of serum neopterin levels in patients on treatment for microbiologically confirmed pulmonary tuberculosis confirmed this observation and showed a significant decline of levels to near normal levels within 6 months of treatment [41] . in the context of emerging multiple drug resistance and difficulties in monitoring compliance and drug absorption neopterin needs to be explored as a tool for monitoring of success in treatment of mycobacterium tuberculosis infection. it may also help to distinguish active from latent disease. people with hiv-m. tuberculosis coinfection with active tuberculosis responded with a reduction of plasma neopterin to antituberculotic treatment but neopterin levels remained above the baseline levels of hiv negative tuberculosis patients and levels were higher in patients with lower cd4 count [78] . the first study of neopterin levels in parasitic infections included measurements of urinary neopterin by hplc in patients with plasmodium falciparum and vivax infections including patients with low grade parasitemia [7] . all patients had elevated urinary neopterin levels compared to uninfected controls to a level of 664 to 5189 micromol neopterin/mol creatinine. levels in patients treated with quinine sulphate and levels in untreated patients were not significantly different. a subsequent detailed interventional study provided data on urinary neopterin levels in volunteers experimentally infected with plasmodium falciparum [79] . serial monitoring revealed that urinary neopterin levels were not elevated until peripheral blood parasite densities had increased through 3 to 4 cycles of intraerythrocytic schizogony. a sharp rise in urinary neopterin was detectable at the beginning of day 14 after infection. there was an increase one day after onset of fever. in one patient a urinary neopterin increase was noted without the occurrence of fever. neopterin production in falciparum malaria seems to be a direct effect of plasmodial antigens on monocytes/macrophages. in vitro studies showed that the monocytic cell line u937 could be stimulated to produce neopterin with lysates of plasmodium falciparum parasitized human erythrocytes and recombinant p. falciparum proteins [80] . at a cut-off point of 10.0 ng/ml, neopterin had a positive and negative predictive value of 0.38 and 0.98 for detection of severe falciparum malaria [81] . chloroquine treatment was followed by a reduction of urinary neopterin levels. when clinical disease resolved within 3-7 days of treatment, neopterin levels normalized rapidly [42] . neopterin levels in nonimmune patients and young children were higher than were those of semiimmune individuals. csf (csf) neopterin levels were investigated for ability to discriminate between different stages of cerebral trypanosoma (t.) brucei (b.) gambiense infection. in an investigation of 512 t. b. gambiense patients originating from angola, chad, and the democratic republic of the congo csf igm and neopterin were the best in discriminating between the two stages (s1 and s2) of disease with 86.4% and 84.1% specificity, respectively, at 100% sensitivity. when a validation cohort (412 patients) was tested, neopterin (14.3 nmol/l) correctly classified 88% of s1 and s2 patients, confirming its high staging power [82] . serum neopterin was also assessed as a disease marker in human schistosoma mansoni infection and levels were found to reflect the extent of hepatic involvement with higher levels found in patients with hepatomegaly. treatment with praziquantel led to a normalisation of serum neopterin levels as a result of a reduction of egg induced immunopathology [43, 44, 83] . the detection of new blood borne viruses including hiv and non-a non-b hepatitis viruses led to investigations into new ways of excluding transmission of blood borne viruses by transfusion of blood products. the government of tirol in austria introduced routine measurement of neopterin levels in all donated blood in 1986. a cut-off of 10 nmol/l was used and led to the exclusion of 1.6% of donors (total number of donors = 76587). the most common cause of elevated levels was in 123 (67%) of cases a viral respiratory tract infection. 6 donors with elevated neopterin had an acute toxoplasmosis and 4 had hiv infection or non-a-non b-hepatitis [84] . in another study 5.26% of 1767 donations with increased neopterin levels were positive for cmv igm indicating acute infection. 0.3% of patients with low neopterin levels had cmv igm. seroconversion was detected in 10 patients with initially elevated neopterin levels on a second serum sample indicating that neopterin may precede the appearance of cmv antibodies by 2-4 weeks [85] . a further study of austrian blood donors showed that neopterin levels were significantly higher in early compared to late infection or carrier state. all early infections (seroconversions) had elevated neopterin levels while only 17% of late and carrier states [86] . a recent study found that using a neopterin elisa 61% of cmv dna-positive samples had elevated neopterin levels [87] . with regard to other viruses 5.5% of donors with above normal neopterin had epstein-barr virus igm generating an almost threefold greater chance of acute ebv infection in donors with increased neopterin (odds ratio: 2.85 (95% confidence interval, 1.5-5.6). with regard to parvovirus b19 infection 73/1060 (6.9%) donors were found to be seropositive for parvovirus b19 igm [88] . a later study by the same group found no hpv dna positive results amongst 1200 patients with normal neopterin levels [89] . an investigation of the association of neopterin levels with chronic hepatitis c virus infection revealed that significantly more patients with elevated neopterin levels and hcv antibodies were hcv pcr positive for hcv rna (odds ratio: 3.76 = 0.002) [90] . neopterin is a nonspecific marker of activated cell mediated immunity involving release of interferon gamma. neopterin may be a useful marker for more accurate estimation of extent of disease and hence prognosis. knowledge of all potential causes of its elevation can overcome problems with reduced specificity in a patient known to have a specific infectious disease. longitudinal serial measurements in the same individual could overcome difficulties with interpretation in settings where chronic parasitic (malaria) or bacterial (tuberculosis) infections may elevate the baseline neopterin level and could allow monitoring of response to antiretroviral, antituberculous, and antiparasitic treatment in the absence of resistance testing and provide means to monitor compliance in the outpatient setting (see table 1 ). this is particularly important in the current context of emerging multiple drug resistance of hiv and mycobacterium tuberculosis. neopterin for which high quality elisa systems to measure urine and blood levels are commercially available is an underused marker in clinical practice and is suitable for introduction into the routine clinical laboratory practice. the author declares that there is no conflict of interests regarding the publication of this paper. neopterin measurement in clinical diagnosis potential role of immune system activation-associated production of neopterin derivatives in humans cytokine-stimulated gtp cyclohydrolase i expression in endothelial cells requires coordinated activation of nuclear factor-b and stat1/stat3 simple dipstick assay for semi-quantitative detection of neopterin in sera erhoehte ausscheidung von neopterin im harn von patienten mit malignen tumoren und mit viruserkrankungen urinary neopterin is elevated in patients with malaria clinical presentation of cmv infection in solid organ transplant recipients and its impact on graft rejection and neopterin excretion urinary neopterin levels in acute viral hepatitis immune activation during measles: beta 2-microglobulin in plasma and cerebrospinal fluid in complicated and uncomplicated disease neopterin levels during acute rubella in children the role of neopterin as a monitor of cellular immune activation in transplantation, inflammatory, infectious, and malignant diseases neopterin excretion during incubation period. clinical manifestation and reconvalescence of viral infection detection of serum neopterin for early assessment of dengue virus infection a preliminary study of neopterin as a potential marker for severe dengue virus infection influence of neopterin and 7,8-dihydroneopterin on the replication of coxsackie type b5 and influenza a viruses neopterin levels during the early phase of human immunodeficiency virus, hepatitis c virus, or hepatitis b virus infection immune stimulation by syphilis and malaria in hiv-2-infected and uninfected villagers in west africa markers predicting progression of human immunodeficiency virus-related disease prognostic significance of plasma markers of immune activation, hiv viral load and cd4 t-cell measurements the prognostic significance in hiv infection of immune activation represented by cell surface antigen and plasma activation marker changes highly active antiretroviral therapy (haart) and circulating markers of immune activation: specific effect of haart on neopterin serum neopterin changes in hiv-infected subjects: indicator of significant pathology, cd4 t cell changes, and the development of aids increased immune activation precedes the inflection point of cd4 t cells and the increased serum virus load in human immunodeficiency virus infection predicting clinical progression or death in subjects with early-stage human immunodeficiency virus (hiv) infection: a comparative analysis of quantification of hiv rna, soluble tumor necrosis factor type ii receptors, neopterin, and 2 -microglobulin serum neopterin level predicts hiv-related mortality but not progression to aids or development of neurological disease in gay men and parenteral drug users are plasma biomarkers of immune activation predictive of hiv progression: a longitudinal comparison and analyses in hiv-1 and hiv-2 infections? 2 -microglobulin and immunoglobulins are more useful markers of disease progression in hiv than neopterin and adenosine deaminase reduction of viral load and immune complex load on cd4+ lymphocytes as a consequence of highly active antiretroviral treatment (haart) in hivinfected hemophilia patients neopterin concentrations in cerebrospinal fluid and serum of individuals infected with hiv-1 levels of human immunodeficiency virus type 1 rna in cerebrospinal fluid correlate with aids dementia stage csf neopterin decay characteristics after initiation of antiretroviral therapy central nervous system immune activation characterizes primary human immunodeficiency virus 1 infection even in participants with minimal cerebrospinal fluid viral burden continuing intrathecal immunoactivation despite two years of effective antiretroviral therapy against hiv-1 infection cerebrospinal fluid neopterin: an informative biomarker of central nervous system immune activation in hiv-1 infection immune activation of the central nervous system is still present after >4 years of effective highly active antiretroviral therapy treatment benefit on cerebrospinal fluid hiv-1 levels in the setting of systemic virological suppression and failure neopterin as a marker of response to antiviral therapy in hepatitis c virus patients neopterin as an index of immune response in patients with tuberculosis neopterin, 2 -microglobulin, and acute phase proteins in hiv-1-seropositive and -seronegative zambian patients with tuberculosis serum interleukin-2 and neopterin levels as useful markers for treatment of active pulmonary tuberculosis neopterin as marker for activation of cellular immunity: immunologic basis and clinical application liver involvement in human schistosomiasis mansoni. regression of immunological and biochemical disease markers after specific treatment praziquantel in the treatment of hepatosplenic schistosomiasis: biochemical disease markers indicate deceleration of fibrogenesis and diminution of portal flow obstruction neopterin as a marker of c hepatitis in thalassaemia major serum neopterin levels in patients with replicative and nonreplicative hbv carriers serum neopterin levels in patients with hbv infection at various stages serum neopterin levels in children with hepatitis-b-related chronic liver disease and its relationship to disease severity value of serum procalcitonin, neopterin, and c-reactive protein in differentiating bacterial from viral etiologies in patients presenting with lower respiratory tract infections serum neopterin for early assessment of severity of severe acute respiratory syndrome diagnostic utility of crp to neopterin ratio in patients with acute respiratory tract infections intrathecal production of neopterin in aseptic meningo-encephalitis and multiple sclerosis immune activation during measles: interferon-and neopterin in plasma and cerebrospinal fluid in complicated and uncomplicated disease role of il-6 and neopterin in the pathogenesis of herpetic encephalitis cerebrospinal fluid neopterin concentrations in central nervous system infection cerebrospinal fluid neopterin is brain-derived and not associated with blood-csf barrier dysfunction in noninflammatory affective and schizophrenic spectrum disorders persistent intrathecal immune activation in hiv-1-infected individuals on antiretroviral therapy persistent intrathecal immune activation in patients with herpes simplex encephalitis value of urinary neopterin in the differential diagnosis of bacterial and viral infections evaluation of procalcitonin and neopterin level in serum of patients with acute bacterial infection evaluation of procalcitonin, neopterin, c-reactive protein, il-6 and il-8 as a diagnostic marker of infection in patients with febrile neutropenia neopterin as a prognostic biomarker in intensive care unit patients the value of neopterin and procalcitonin in patients with sepsis d-erythroneopterin plasma levels in intensive care patients with and without septic complications course of immune activation markers in patients after severe multiple trauma procalcitonin and neopterin as indicators of infection in critically ill patients neopterin production and tryptophan degradation in acute lyme neuroborreliosis versus late lyme encephalopathy assessment of diagnostic enzyme-linked immunosorbent assay kit and serological markers in human brucellosis is neopterin-a marker of cell mediated immune response, helpful in classifying leprosy immune responseassociated production of neopterin. release from macrophages primarily under control of interferon neopterin as a new biochemical marker for diagnosis of allograft rejection. experience based upon evaluation of 100 consecutive cases bal neopterin. a novel marker for cell-mediated immunity in patients with pulmonary tuberculosis and lung cancer neopterin in tuberculous and neoplastic pleural fluids neopterin as a marker for cell-mediated immunity in patients with pulmonary tuberculosis urinary neopterin measurement as a non-invasive diagnostic method in pulmonary tuberculosis pleural fluid neopterin levels in tuberculous pleurisy neopterin levels and pulmonary tuberculosis in infants incomplete immunological recovery following anti-tuberculosis treatment in hiv-infected individuals with active tuberculosis urinary neopterin in volunteers experimentally infected with plasmodium falciparum malaria antigene stimulate neopterin secretion by pbmc and u937 celts neopterin and procalcitonin are suitable biomarkers for exclusion of severe plasmodium falciparum disease at the initial clinical assessment of travellers with imported malaria csf neopterin as marker of the meningo-encephalitic stage of trypanosoma brucei gambiense sleeping sickness liver involvement in human schistosomiasis mansoni. assessment by immunological and biochemical markers serum-neopterinbestimmung zur zusaetzlichen sicherung der bluttransfusion neopterin screening and acute cytomegalovirus infections in blood donors acute cytomegalovirus infections in blood donors are indicated by increased serum neopterin concentrations high prevalence of cytomegalovirus dna in plasma samples of blood donors in connection with seroconversion increased prevalence of igm antibodies to epstein-barr virus and parvovirus b19 in blood donations with above-normal neopterin concentration human parvovirus b19 detection in asymptomatic blood donors: association with increased neopterin concentrations association between chronic hepatitis c virus infection and increased neopterin concentrations in blood donations key: cord-306266-8qdrshz3 authors: scully, crispian title: respiratory medicine date: 2014-06-25 journal: scully's medical problems in dentistry doi: 10.1016/b978-0-7020-5401-3.00015-1 sha: doc_id: 306266 cord_uid: 8qdrshz3 ●. upper respiratory infections are commonplace, especially in young people, and are often contagious; ●. lower respiratory infections are often contagious and some are potentially fatal; ●. asthma is common and may be life-threatening; ●. chronic obstructive pulmonary disease is common and disabling; ●. tuberculosis worldwide is an important infection, affecting people with hiv/aids or malnutrition particularly; ●. lung cancer is common and usually has a poor prognosis. • upper respiratory infections are commonplace, especially in young people, and are often contagious the respiratory tract consists of the upper respiratory tract (urtnose, paranasal sinuses, pharynx and larynx; discussed in ch. 14) and the lower respiratory tract (lrt): the respiratory airways (trachea, bronchi and bronchioles) and lungs (respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli), discussed in this chapter. protective mechanisms in the respiratory tracts include a mucociliary lining. particles or pathogens are trapped in the mucus and driven by ciliary action (the ciliary elevator) to the pharynx. mucociliary trans port declines with age but any effect on clinical infection has not been proved. lymphoid tissues of the waldeyer ring (adenoids, palatine and lingual tonsils) are important in developing an immune response to pathogens. however, the best respiratory defence mechanism is the cough reflex, the components of which include cough receptors, affer ent nerves, the cough centre, and efferent nerves and effector muscles. impairment of any of these -as may be seen in older patients or those with conditions associated with lowered consciousness (e.g. sedative use and neurological disease) -can weaken protection. dysphagia or impaired oesophageal motility may exacerbate the tendency to aspi rate foreign material. the alveolar defence mechanisms include mac rophages, immunocytes, surfactant, phospholipids, immunoglobulin g (igg), ige, secretory iga, complement components and factor b; many immune defects manifest with recurrent respiratory infections. lung function is vital to gas exchange -the blood absorbs oxygen and releases carbon dioxide. normal gas exchange requires adequate alveolar ventilation, normal ventilation/blood flow relationships and adequate alveolar-capillary membrane surface area. breathing (ven tilation) depends on respiratory drive, which reacts to the respiratory load. this process requires work and results in gas exchange. oxygen is transported in combination with haemoglobin in erythro cytes and a small amount dissolved in plasma. the oxyhaemoglobin dissociation curve is sigmoidal; once the oxygen saturation falls below 95%, the amount of o 2 transported to the tissues and brain falls rapidly. high temperatures, acidosis, raised co 2 and raised 2,3 diphosphoglycerate (2,3dpg) levels encourage oxygen offloading, whereas fetal haemoglobin and carboxyhaemoglobin have the con trary effect. chronic hypoxaemia (e.g. at high altitudes) stimulates release of erythropoietin from the kidneys, with a rise in red cell pro duction, and raised 2,3dpg. athletes have abused erythropoietin to gain competitive advantage (ch. 33). the most common lrt disorders are asthma and chronic obstructive pulmonary disease (copd). respiratory disorders are common, and are often caused or aggravated by tobacco smoking. they may significantly affect general anaesthesia (ga) and conscious sedation (cs), since they are often a contraindica tion to use of benzodiazepines, opioids, ga agents and other respira tory depressants. impaired gas exchange leads to laboured breathing and can cause significant incapacity. features include cough, sputum production, wheeze, dyspnoea, chest pain, cyanosis, fingerclubbing ( fig. 15.1) , use of accessory muscles of respiration with indrawing of the intercostal spaces (hyperinflation), and abnormalities in chest shape, movements, respiratory rate and breath sounds. cough may be a feature of any respiratory problem but, if chronic, may herald serious disease -for example, copd, cancer or infec tion such as tuberculosis. mucoid or mucopurulent sputum is often a feature ( fig. 15 .2); purulent sputum indicates acute bronchitis, bronchiectasis or lung abscess. blood (haemoptysis) or bloodstained sputum, though common in acute infections (especially in preexisting copd), bronchiectasis and pulmonary embolism, may herald an even more serious condition -for example, possibly one due to carcinoma or tuberculosis. wheezing is caused by airways obstruction and is a typical sign of asthma or copd. breathlessness (dyspnoea) is distress ing, and may be caused by respiratory or cardiovascular disease, or by anaemia, and is particularly ominous if it persists at rest. excessive resistive load, such as in asthma, copd and cystic fibro sis, impairs airflow. elastic load increases because of, for example, interstitial fibrosis, muscle paralysis and obesity. diagnosis of respiratory disorders is from the clinical features sup ported by imaging (especially chest radiography). spiral computed tomography (ct) can now scan the lungs in a quick 20-30second breathhold and therefore, instead of producing a stack of individual ct slices, which may be misaligned due to patient movement or breathing in between slices, provides highresolution three dimensional images. respiratory function tests can measure individual components of the respiratory process. spirometry is the basic screening test for assess ing mechanical load problems, the quantification involving determi nation of the vital capacity (vc) -slow vital capacity (svc) and/or forced vital capacity (fvc) -and the speed of maximal expiratory flow (mef; fig. 15 .3). in health, about 75% of a normalsized vc is expelled in 1 second (fev 1 ). the peak flow meter, which measures the peak expiratory flow rate (pefr; the earliest portion of forced expiration), is a simple measure of airflow obstruction, when the fev 1 is a much smaller fraction of the vc. in lung restriction, the diminished vc can be mostly expelled in about 1 second. serial meas urements (e.g. in asthma) provide valuable information about disease progress. the reversibility of airways obstruction is usually assessed by spirometry before and after use of a bronchodilator agent. arterial blood gas analysis yields considerable information about gas exchange efficiency. arterial hypoxaemia in adults is defined as pao 2 below 10.7 kpa breathing room air, although it is not usually treated as clinically important unless below 8 kpa, when oxygen saturation will be 90% or less (table 15 .1). arterial carbon dioxide tension (paco 2 ) is used as an inversely pro portional index of 'effective' alveolar ventilation. hence, a high paco 2 is taken to indicate poor alveolar ventilation. alveolar hypoventila tion (raised paco 2 ) with a normal ph probably represents a primary ventilatory change present long enough for renal mechanisms to compensate, as in chronic ventilatory failure. ventilation/blood flow relationships are most simply assessed by considering the size of the difference between the amounts of oxygen and carbon dioxide in the blood and in the air; the differences are small if the lungs are work ing efficiently. disparity between ventilation/blood flow ratios results in abnormally wide differences -and then alveolar-arterial po 2 and arterial-alveolar pco 2 gradients will be abnormal. alveolar capillary surface area is assessed by measuring the uptake of carbon monoxide -usually abnormal in diffuse interstitial inflam matory and fibrotic processes and in emphysema. assessing bronchial reactivity and the exercise response can help evaluate breathlessness. simple exercise testing provides information about overall fitness and the appropriateness of cardiorespiratory responses. radionuclide lung scanning, blood gas analysis and sputum cytology or culture are sometimes needed in addition. management can include oxygen administration by mask or nasal cannula (figs 15.4 and 15.5) . lrt disorders can cause significant incapacity and are often a con traindication to ga, and even to cs. asthma is common, affecting 2-5% of the overall population; it is on the increase, particularly in childhood, with a frequency of up to 20% in some highincome countries. asthma usually begins in childhood or early adult life; about half the patients with asthma develop it before age 10 years. bronchial hyperreactivity causes reversible airway obstruction from smooth muscle constriction (bronchospasm), mucosal oedema and mucus hypersecretion. there are two main types, extrinsic (allergic) and intrinsic asthma (table 15 .2). extrinsic (allergic) asthma, the main childhood type, may be pre cipitated by allergens in animal dander, feathers or hair, drugs (e.g. nonsteroidal antiinflammatory drugs [nsaids] and some antibiot ics), food (e.g. eggs, fish, fruit, milk, nuts), house dust (mite allergens) or moulds. patients frequently have or develop other allergic diseases, such as eczema, hay fever and drug sensitivities. extrinsic asthma is associated with ige overproduction on allergen exposure, and release of mast cell mediators (histamine, leukotrienes, prostaglandins, bradykinin and platelet activating factor), which cause bronchospasm and oedema. about 75% of asthmatic children lose their asthma or improve by adulthood. intrinsic asthma is usually of adult onset and not aller gic, but appears rather to be related to mast cell instability and airway hyperresponsivity. triggers include emotional stress, gastro oesophageal reflux or vagally mediated responses. either type of asthma can be triggered by: infections (especially viral, mycoplasmal or fungal); irritating fumes (e.g. traffic or cigarette smoke); exercise (possibly due to cold air); weather changes; emotional stress; foods (e.g. nuts, shellfish, strawberries or milk) or additives (such as tartrazine); and drugs (e.g. aspirin and other nsaids, beta blockers and angiotensinconverting enzyme inhibitors [aceis]). in wellcontrolled patients with asthma, clinical features may be absent. during an asthmatic episode, symptoms may include dysp noea, cough and paroxysmal expiratory wheeziness with laboured expiration. the frequency and severity of attacks vary widely between individuals (table 15 .3). patients may become distressed, anxious and tachycardic, have reduced chest expansion and be using accessory respiratory muscles to increase their ventilatory effort. nasal polyps are common, especially in aspirinsensitive asthmatics. children with asthma initially suffer from repeated 'colds' with cough, malaise and fever, often at night. asthma is typically diagnosed when the patient has more than one of the following -wheeze, cough, difficulty breathing and chest tightness -particularly if these are frequent and recurrent; are worse at night and in the early morning; occur in response to, or are worse after, exercise or other triggers, such as exposure to pets, cold or damp air, or with emotions or laughter; or occur without an association with colds. there is often: ■ a personal history of atopic disorder ■ a family history of atopic disorder and/or asthma ■ widespread wheeze, heard on chest auscultation ■ a history of improvement in symptoms or lung function in response to adequate therapy. a prolonged asthmatic attack, which is refractory to treatment, may lead to lifethreatening status asthmaticus (persisting for more than 24 hours). failure of the patient to complete a sentence, indrawing of the intercostal muscles, a rapid pulse, a silent chest and signs of exhaustion are suggestive of impending respiratory arrest. diagnosis of asthma is from the clinical history and presentation, based on recognizing a characteristic pattern of episodic symptoms in the absence of an alternative explanation. investigations include a chest radiograph (to exclude other diagnoses, such as a pneumo thorax), spirometry (serial pefr), skin tests and blood examination (usually eosinophilia, raised total ige and specific ige antibody concentrations, which may help identify allergens). occasionally, a histamine or methacholine challenge is used if the diagnosis is unclear. in children with an intermediate probability of asthma, who can perform spirometry and have evidence of airways obstruction, assess the change in fev 1 or pefr in response to an inhaled bronchodilator (reversibility) and/or the response to a trial of treatment for a speci fied period; if there is significant reversibility, or if a treatment trial is beneficial, a diagnosis of asthma is probable. management includes patient education, smoking cessation advice, avoidance of identifiable irritants and allergens, and use of drugs. home use of peak flow meters allows patients to monitor progress and detect any deterioration that may require urgent modification of treatment. treatment should be based on the amount by which peak flow is reduced (a pefr diary should be kept). drugs used for asthma management (table 15 .4) include oxygen, shortacting β 2 agonists (sabas; such as salbutamol), corticosteroids, leukotriene receptor antagonists and omalizumab (a recombinant humanized monoclonal antiige antibody that reduces the antigen specific ige). inhaled longacting β 2 agonists (labas) may be needed ( fig. 15.6 ). deaths from asthma are usually a result of failure to recognize dete rioration or reluctance to use corticosteroids. other factors that have been studied include: ■ air pollution -there is an association between air pollution and aggravation of existing asthma ■ allergen avoidance -there is no consistent evidence of benefit ■ breast-feeding -there is evidence of a protective effect in relation to early asthma ■ electrolytes -there is no consistent evidence of benefit ■ fish oils and fatty acid -there is no consistent evidence of benefit ■ house dust mites -measures to reduce the numbers of house dust mites do not affect asthma severity ■ immunotherapy -allergenspecific immunotherapy is beneficial in allergic asthma ■ microbial exposure -there is insufficient evidence to indicate that the use of probiotics in pregnancy reduces the incidence of childhood asthma ■ modified milk formulae -there is no consistent evidence of benefit pets -there are no controlled trials on the benefits of removing pets from the home ■ tobacco -exposure to cigarette smoke adversely affects quality of life, lung function, need for rescue medications and longterm control with inhaled steroids. there is an association between maternal smoking and an increased risk of infant wheeze ■ weight reduction -there is an association between increasing body mass index and symptoms of asthma. elective dental care should be deferred in severe asthmatics until they are in a better phase; this can be advised by the patient's general practitioner. asthmatic patients should be asked to bring their usual medica tion with them when coming for dental treatment. local anaesthe sia (la) is best used; occasional patients may react to the sulphites present as preservatives in vasoconstrictorcontaining la, so it may be better, where possible, to avoid solutions containing vasoconstric tor. adrenaline (epinephrine) may theoretically enhance the risk of arrhythmias with betaagonists and is contraindicated in patients using theophylline, as it may precipitate arrhythmias. relative analgesia with nitrous oxide and oxygen is preferable to intravenous sedation and gives more immediate control. sedatives in general are better avoided as, in an acute asthmatic attack, even ben zodiazepines can precipitate respiratory failure. ga is best avoided, as it may be complicated by hypoxia and hyper capnia, which can cause pulmonary oedema even if cardiac function is normal, and cardiac failure if there is cardiac disease. the risk of post operative lung collapse or pneumothorax is also increased. halothane or, better, enflurane, isoflurane, desflurane and sevoflurane are the preferred anaesthetics, but ketamine may be useful in children. allergy to penicillin may be more frequent in asthmatics. drugs to be avoided, since they may precipitate an asthmatic attack (see later), include those listed in box 15.1. acute asthmatic attacks may also occasionally be precipitated by anxiety; it is important to attempt to lessen fear of dental treatment by gentle handling and reassurance. even routine dental treatment can trigger a clinically significant decline in lung function in approximately 15% of asthmatics. acute asthmatic attacks are usually selflimiting or respond to the patient's usual medication, such as a betaagonist inhaler, but status asthmaticus is a potentially fatal emergency (ch. 1). there may be complications caused by the antiasthmatic drugs (table 15 .5). gastrooesophageal reflux is not uncommon, with occasional tooth erosion. periodontal inflammation is greater in asthmatics than in those without respiratory disease. persons using steroid inhalers may develop oropharyngeal candidosis or, occasionally, angina bullosa haemorrhagica. guidelines on the management of asthma may be found at: http://www.sign.ac.uk/guidelines/fulltext/101/index.html, http:// www.nice.org.uk/guidance/qualitystandards/indevelopment/asthma. jsp and http://www.britthoracic.org.uk/portals/0/guidelines/ asthmaguidelines/qrg101%202011.pdf (all accessed 30 september 2013). churg-strauss syndrome (css) is a rare, potentially fatal, systemic vasculitis similar to polyarteritis nodosa (pan), characterized by severe asthmalike attacks with peripheral eosinophilia, and intravas cular and extravascular granuloma formation with eosinophil infiltra tion and skin lesions in 70%. cardiopulmonary involvement is the main cause of death. css is diagnosed if at least 4 of the 6 criteria listed in box 15.2 are positive. the 5year survival of untreated css is 25%. combination treatment with cyclophosphamide and prednisolone (prednisone) provides a 5year survival of 50%. management problems relating to patients with css may include res piratory impairment and corticosteroid treatment (ch. 6). chronic obstructive pulmonary disease (copd; chronic obstructive airways disease, coad) is a common, chronic, slowly progressive, irre versible disease (most frequently a combination of chronic bronchitis and emphysema), characterized by breathlessness and wheeze (airways obstruction), cough and sputum. chronic bronchitis is defined as the excessive production of mucus and persistent cough with sputum production, daily for more than 3 months in a year over more than 2 consecutive years. it leads to production of excessive, viscous mucus, which is ineffectively cleared from the airway, obstructs and stag nates, and becomes infected, usually with streptococcus pneumoniae, moraxella catarrhalis and haemophilus influenzae. patchy areas of alveolar collapse can result. emphysema is dilatation of air spaces dis tal to the terminal bronchioles with destruction of alveoli, reducing the alveolar surface area available for respiratory exchange. copd is now the preferred term for conditions with airflow obstruction because of a combination of airway and parenchymal damage; patients were previ ously diagnosed as having chronic bronchitis or emphysema. copd is characterized by airflow obstruction -defined as an fev 1 / fvc ratio reduced to less than 0.7. if fev 1 is 80% or more, a diagno sis of copd should only be made if there are respiratory symptoms (e.g. dyspnoea or cough). the airflow obstruction is not fully revers ible, does not change significantly over months, and is usually progres sive in the long term. the most important causes of copd include cigarette smoking, environmental pollution, dusts, chemicals or occupational exposures to various substances. exposure to smoke from home cooking or heating fuels may contribute. deficiency of the antiproteolytic enzyme alpha1antitrypsin is a rare cause of emphysema. there is often significant airflow obstruction before the person is aware of it and so copd typically remains undiagnosed until patients are in their fifties. differentiation from asthma is important (table 15 .6). a diagnosis of copd should be considered in patients over the age of 35 who have a risk factor (e.g. smoking) and exertional breath lessness, chronic cough, regular sputum production, frequent winter 'bronchitis' or wheeze. clinical judgment is based on history, physical examination, confirmation of airflow obstruction using spirometry (postbronchodilator spirometry) and assessment of the severity of dyspnoea (tables 15.7 and 15.8). copd is characterized by breathlessness and wheeze (airways obstruction), cough and an early morning mucoid sputum production. to investigate symptoms that seem disproportionate to spirometric impairment progressive dyspnoea, low oxygen saturation, carbon dioxide accumu lation (hypercapnia) and metabolic acidosis mean that patients may ultimately become dyspnoeic at rest ('respiratory cripples'), especially when recumbent (orthopnoea), and eventually develop respiratory failure, pulmonary hypertension, right ventricular hypertrophy and rightsided heart failure (cor pulmonale). two clinical patterns of copd are recognized: ■ 'pink puffers' -patients with emphysema who manage to maintain normal blood gases by hyperventilation, and are always breathless but not cyanosed; rather they are pink from vasodilatation ■ 'blue bloaters' -patients with chronic bronchitis who lose their co 2 drive, fail to maintain adequate ventilation and become both hypercapnic and hypoxic with central cyanosis, cor pulmonale and oedema (for these patients, the respiratory drive is from the low po 2 and thus oxygen administration is contraindicated) (table 15 .9). the diagnosis of copd is based upon clinical history and presen tation. investigations include a chest radiograph (which may show hyperinflated lung fields with loss of vascular markings); arterial blood gases (which should be measured if pulse oximetry shows oxygen satu ration less than 92%); spirometry; and lung function tests. fev1 is reduced in all cases (fev 1 of less than 40% signifies severe copd) and the flow-volume curve shows a typical pattern, with reduced flow rates at mid and lowerlung volumes. a ratio of fev 1 :fvc of less than 70% confirms airways obstruction. patients with copd and their family should be educated about the disease, and about required lifestyle changes and medication. nondrug therapy includes: stopping smoking (nicotine replacement therapy or bupropion may help); exercise by pulmonary rehabilitationof proven benefit; weight loss (improves exercise tolerance); and vaccination (pneumococcal and influenza vaccines). drug therapy includes shortacting bronchodilators (anticholinergic drugs [ipra tropium bromide]) and β 2 agonists (salbutamol) to treat the reversible component of airway disease; corticosteroids (inhaled or systemic); and antibiotics (amoxicillin, trimethoprim or tetracycline). mucolytics, such as carbocisteine, reduce acute exacerbations by almost onethird. longterm oxygen therapy (ltot) reduces mortality. people with stable copd who remain breathless or have exacerba tions, despite using shortacting bronchodilators, should be offered the following as maintenance therapy: ■ if fev 1 is 50% of predicted or more: use either a longacting β 2 agonist (laba) or longacting muscarinic antagonist (lama). ■ if fev 1 is less than 50% predicted: either a laba with an inhaled corticosteroid (ics) in a combination inhaler, or a lama. offer a lama in addition to a laba plus ics to people with copd who remain breathless or have exacerbations, despite taking laba plus ics, irrespective of their fev 1 . provide pulmonary rehabilitation for all who need it; noninvasive ventilation (niv) is the treatment of choice for persistent hyper capnic ventilatory failure during exacerbations not responding to medical therapy. the frequency of exacerbations should be reduced by appropriate use of inhaled corticosteroids and bronchodilators, and vaccinations. bronchodilators (shortacting β 2 agonists [saba] and shortacting muscarinic antagonists [sama]) should be the initial empirical treat ment for the relief of breathlessness and exercise limitation. ics have potential adverse effects (including nonfatal pneumonia) in people with copd. offer a oncedaily lama in preference to fourtimes daily sama to people with stable copd who remain breathless or have exacerbations, despite using shortacting bronchodilators as required, and in whom a decision has been made to commence regular maintenance bronchodilator therapy with a muscarinic antagonist (see above). most patients -whatever their age -are able to acquire and main tain an adequate inhaler technique. bronchodilators are usually best administered using a handheld inhaler device (including a spacer device if appropriate). patients with distressing or disabling dyspnoea, despite maximal therapy using inhalers, should be considered for nebulizer therapy. they should be offered a choice between a face mask and a mouth piece to administer their nebulized therapy, unless the drug specifically requires a mouthpiece (for example, anticholinergic drugs). some patients with advanced copd may require maintenance oral corticosteroids when these cannot be withdrawn following an exacer bation. these individuals should be monitored for the development of osteoporosis and given appropriate prophylaxis. theophylline should only be used after a trial of saba and laba, and only to those who are unable to use inhaled therapy, as there is a need to monitor plasma levels and interactions. the dose of theo phylline prescribed should be reduced at the time of an exacerbation if macrolide or fluoroquinolone antibiotics (or other drugs known to interact) are given. there is insufficient evidence to recommend prophylactic antibiotic therapy in the management of stable copd. mucolytic drug therapy should be considered in patients with a chronic cough productive of sputum. if patients remain symptomatic on monotherapy, their treatment should be intensified by combining therapies from different drug classes, such as: ■ β 2 agonist and theophylline ■ anticholinergic and theophylline. inappropriate oxygen therapy in people with copd may depress respiration. ltot is indicated in patients with copd who have a pao 2 of less than 7.3 kpa when sta ble, or a pao 2 greater than 7.3 kpa and less than 8 kpa when stable, and one of: secondary polycythaemia, nocturnal hypoxaemia (oxygen saturation of arterial blood [sao 2 ] of less than 90% for more than 30% of the time), peripheral oedema or pulmonary hypertension. to reap the benefits of ltot, patients should breathe supplemental oxygen for at least 15 hours per day. to ensure that all those eligible for ltot are identified, pulse oximetry should be available in all health care settings. the assessment of patients for ltot should comprise the measurement of arterial blood gases on two occasions at least 3 weeks apart in patients who have a confident diagnosis of copd, who are receiving optimum medical management and whose copd is stable. patients should be warned about the risks of fire and explosion and told not to smoke when using oxygen. ambulatory oxygen therapy should be considered in patients on ltot who wish to continue oxygen therapy outside the home, and who have exercise desaturation, are shown to have an improvement in exercise capacity and/or dyspnoea with oxy gen, and are motivated to use oxygen. adequately treated patients with chronic hypercapnic respiratory failure who have required assisted ventilation during an exacerbation, or who are hypercapnic or acidotic on ltot, should be referred to a specialist centre for consideration of longterm niv. advanced emphysema is occasionally treated with sur gery -excision of large acquired bullae or, rarely, lung transplantation. patients with copd who need dental care can be classified as follows: ■ patients at low risk -experience dyspnoea on effort but have normal blood gas levels. these patients can receive all dental treatment with minor modifications. ■ patients at moderate risk -experience dyspnoea on effort, are chronically treated with bronchodilators or recently with corticosteroids, and pao 2 lowered. a medical consultation is advised to determine the level of control of the disease before any dental treatment. ■ patients at high risk -have symptomatic copd that may be end stage and poorly responsive to treatment. with these patients, a medical consultation is essential before any dental treatment is carried out. patients with copd are best treated in an upright position at midmorning or early afternoon, since they may become increasingly dyspnoeic if laid supine. it may be difficult to use a rubber dam, as some patients are mouthbreathers and not able to tolerate the additional obstruction. la is preferred for dental treatment, but bilateral mandibular or palatal injections should be avoided. patients with copd should be given relative analgesia only if absolutely necessary, and only in hospital after full preoperative assessment. cs with diazepam and midazolam should not be used, as benzodiazepines are respiratory depressants. patients should be given ga only if absolutely necessary, and intravenous barbiturates are contraindicated. secretions reduce airway patency and, if lightly anaesthetized, the patient may cough and contaminate other areas of the lung. postoperative respiratory complications are more prevalent in patients with preexisting lung diseases, especially after prolonged operations and if there has been no preoperative preparation. the most important single factor in preoperative care is cessation of smok ing for at least 1 week preoperatively. respiratory infections must also be eradicated; sputum should first be sent for culture and sensitivity, but antimicrobials such as amoxicillin should be started without await ing results. the medical management of copd should be optimized prior to surgery. the ultimate clinical decision about whether or not to proceed with surgery should rest with a consultant anaesthetist and consultant surgeon, taking account of comorbidities, functional status of the patient and necessity for the surgery. composite assessment tools, such as the american society of anesthesiologists (asa) scoring system, and not just lung function, are the best criteria for the assessment of patients with copd before surgery. those taking corticosteroids should be treated with appropri ate precautions (ch. 6). interactions of theophylline with other drugs, such as adrenaline (epinephrine), erythromycin, clindamycin, azithro mycin, clarithromycin or ciprofloxacin, may result in dangerously high levels of theophylline. ipratropium can cause dry mouth. guidelines for the management of copd may be found at: http:// publications.nice.org.uk/chronicobstructivepulmonarydisease cg101 (accessed 30 september 2013). respiratory viruses usually spread by touch or airborne transmission and the very small particles (2-0.2 micrometres) can avoid the upper respiratory tract defences and the mucociliary elevator to reach the lung alveoli. a range of viruses can cause lower respiratory tract infections (lrtis ; table 15 .10). some viruses (e.g. influenza and respiratory syncytial) can spread from the upper to the lower respira tory tract via infection of the respiratory epithelium and can lead to bacterial superinfection and pneumonitis (pneumonia). mycoplasmal (atypical) pneumonia and tuberculosis (tb) may be direct infections. epidemics of a potentially fatal severe acute respiratory syndrome (sars) have been caused by a coronavirus that originated in china and spread worldwide; h5n1 bird influenza also arose as an epidemic; and a similar epidemic, but of swine influenza (h1n1), emanated from mexico (see later). bacterial infections, such as pneumonia or lung abscess, can also result from material aspirated into the lungs, and are usually unilat eral. those who aspirate more than others have, as a result, more frequent lrti and this is seen in alcohol and other drug abusers, as well as comatose patients. exogenous penetration and contamination of the lung can result from trauma (e.g. a stab wound or road traffic accident) or surgery. entamoeba histolytica can occasionally cause pneumonia -by direct extension from an amoebic liver abscess (table 15 .11). patients with endocarditis, or septic pelvic or jugular thrombo phlebitis, may experience lrti acquired haematogenously and then it is often bilateral. immunocompromised persons (e.g. those with human immunode ficiency virus/acquired immune deficiency syndrome [hiv/aids] and transplant recipients) and people with bronchiectasis or cystic fibrosis are also susceptible to respiratory infections by a range of opportun istic microbes. pneumocystis jiroveci (p. carinii), for example, is a com mon cause of potentially fatal pneumonia in immunocompromised patients -especially those with hiv/aids (chs 20 and 21). clinical features of lrti vary according to the part of the respiratory tract mainly affected: ■ bronchiolitis causes rapid respiration, wheezing, fever and dyspnoea -but is restricted mainly to infants. ■ bronchitis causes cough, wheezing and sometimes dyspnoea. ■ pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. antimicrobial therapy is indicated, particularly for pneumonia. antivirals have not been highly effective. oxygen may be needed. pneumococcal vaccine is indicated for older people. the majority of lrtis are severe illnesses, and are contraindications to all but emergency dental treatment. ga is hazardous and absolutely contraindicated. dental treatment should be deferred until recovery, or be limited to pain relief. influenza is mainly a communitybased infection transmitted in house holds and communities. healthcareassociated influenza infections can arise in any healthcare setting, most commonly when influenza is also circulating in the community. influenza is a contagious disease caused by influenza virus types a, b or c. type a has two main subtypes (h1n1 and h3n2); it causes most of the widespread influenza epidemics and can occasionally be fatal. type b viruses generally cause regional outbreaks of moderate severity, and type c viruses are of minor significance. a person can spread influenza starting 1 day before they feel sick and for another 3-7 days after symptoms start. influenza can be pre vented or ameliorated by vaccination each autumn; this is especially indicated for older people and those with cardiorespiratory disease. influenza attacks virtually the whole respiratory tract; symptoms appear suddenly after 1-4 days and include fever, sore throat, nasal congestion, headache, tiredness, dry cough and muscle pains (myalgia). most people recover in 1-2 weeks but infection can be lifethreatening, mainly because primary influenzal viral pneumonia can lead to sec ondary bacterial pneumonia or can exacerbate underlying conditions (e.g. pulmonary or cardiac disease). the old and very young, and those with chronic disorders, are more likely to suffer complications, such as pneumonia, bronchitis, sinusitis or otitis media. influenza has also been followed by depression, encephalopathy, myocarditis, myositis, pericarditis, reye syndrome and transverse myelitis. rest, maintenance of fluid intake, analgesics, antipyretics, and avoid ance of alcohol and tobacco help relieve symptoms. aspirin must never be given to children under the age of 16 years who have 'flulike symptoms, and particularly fever, as this can cause reye syndrome. zanamivir (an antiviral that works against influenza types a and b) can shorten the symptoms by approximately 1 day, if treatment is started during the first 2 days of illness. other antiviral drugs include amantadine, oseltamivir and rimantadine; they may be helpful but their use is restricted mainly to immunocompromised persons, since they can cause adverse effects. influenza can be a severe contagious illness so all but emergency den tal treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. influenza type a subtype h5n1 can cause an illness known as 'avian influenza' or 'bird 'flu' in birds, humans and many other animal spe cies. hpai a(h5n1) -'highly pathogenic avian influenza virus of type a of subtype h5n1' -is the causative agent and is enzootic in many bird populations, especially in southeast asia. it has spread globally and resulted in the deaths of over 100 people and the slaughter of mil lions of chickens. a vaccine that could provide protection (prepandrix) has been cleared for use in the european union. h5n7 is a more recent emergent infection, similar in many respects. swine influenza is common in pigs in the midwestern united states, mexico, canada, south america, europe (including the uk, sweden and italy), kenya, china, taiwan, japan and other parts of eastern asia. transmission of swine influenza virus from pigs to humans is not com mon, but can produce symptoms similar to those of influenza. a 2009 outbreak in humans ('swine 'flu') was due to an apparently new strain of h1n1 arising from a reassortment produced from strains of human, avian and swine viruses. it can pass from human to human. antiviral agents such as oseltamivir may help. vaccines are now available. an outbreak of a lifethreatening febrile respiratory infection appeared in 2003, originating from guangdong, china, and was named severe acute respiratory syndrome (sars). caused by a newly recognized coronavirus (sarsassociated coronavirus, sarscov), sars spread via close contact to many countries across the world. according to the world health organization, 8437 people worldwide became sick with sars during the course of the first recognized outbreak and 813 died. the incubation period of 2-7 days is followed by a high fever (above 38.0°c), malaise, headache and myalgia. some people also experience mild upper respiratory symptoms and, after 2-7 days, lower respiratory signs -a dry cough and dyspnoea, potentially progressing to hypox aemia. sars can cause a pneumonia with a mortality approaching 10%, particularly in older or immunocompromised people. artificial ventilation has been needed in 10-20% of cases. antiviral agents, such as oseltamivir or ribavirin, may help. inactivated vaccines, virally and bacterially vectored vaccines, recombinant protein and dna vaccines, as well as attenuated vaccines, are under development. sars is a severe illness, and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. for all contact with suspect sars patients, careful hand hygiene is important, including handwashing with soap and water; if hands are not visibly soiled, alcoholbased handrubs may be used as an alternative to handwashing. if a suspected sars patient is admitted to hospital, infection control personnel should be notified immediately. infection control measures (www.cdc.gov/ncidod/hip/iso lat/isolat.htm; accessed 30 september 2013) should include standard precautions (e.g. hand hygiene): healthcare personnel should wear eye protection for all patient contact; contact precautions (e.g. gown and gloves for contact with the patient or their environment); and airborne precautions (e.g. an isolation room with negative pressure relative to the surrounding area and use of an n95 filtering disposable respirator for persons entering the room). pneumonia is classed as 'primary' if it occurs in a previously healthy individual, and is usually lobar; it is called 'secondary' if it follows some other disorder, such as previous viral respiratory infections, aspir ation of foreign material, lung disease (bronchiectasis or carcinoma), depressed immunity (e.g. alcoholism or immunosuppression), or aspir ation of oral bacteria ( pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. complications can include lung abscess or empyema (pus in pleural cavity). it is important to avoid alcohol and tobacco, but use analgesics and antipyretics to relieve the symptoms. broadspectrum antimicrobi als given promptly and empirically usually include a macrolide (azithromycin, clarithromycin or erythromycin), quinolone (moxiflox acin, gatifloxacin or levofloxacin), or doxycycline for outpatients. for in patients, cefuroxime or ceftriaxone plus a macrolide is used. prophylaxis includes immunization against influenza and pneumococci. pneumonia is a severe illness and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. ventilatorassociated pneumonia (vap) is discussed later. legionellosis is a bacterial respiratory infection caused by one of the family legionellaceae, gramnegative aerobic bacilli, ubiquitous in water and soil but particularly preferring warm aquatic environments. the term legionnaire's disease was coined as a result of an outbreak of the previously unrecognized respiratory disease in an american legion meeting in philadelphia in 1976, but it is now recognized worldwide, many infections being contracted during travel abroad, particularly to spain, turkey and some other mediterranean areas. legionella bacteria can be found in natural freshwater environments, usually in insufficient numbers to cause disease. legionella grow best in warm water, as in hot tubs, cooling towers, hot water tanks, large plumbing systems, or the airconditioning systems of large buildings. though there are over 30 legionellaceae, most infections are caused by legionella pneumophila. disease is contracted by inhalation of contaminated mist or vapour, mainly (approximately 46%) through aerosolization of infected water in airconditioning systems, hotwater systems, humidifiers, nebulizers, showers and spa pools. outbreaks have mostly been linked to aerosol sources in the community, cruise ships and hotels, with the most likely sources being whirlpool spas, air conditioning units in large buildings, potable (drinking) water systems, and water used for bathing. risk factors include: ■ exposure to: recent travel with an overnight stay outside of the home (outbreaks of travelassociated legionellosis are infrequently identified but more than 20% of cases are thought to be associated with recent travel) whirlpool spas recent repairs or maintenance work on domestic plumbing ■ systemic illhealth: alcohol use chronic kidney disease diabetes immune defects liver disease malignancy smoking. illness mainly affects males over 45, smokers, heavy drinkers, older people and the immunocompromised. also vulnerable are travellers, especially middleaged and older tourists, and conference or business groups, possibly because of tiredness or age. many young people have been exposed to infection and become seropositive, but remained healthy. there is no evidence of persontoperson transmission of legionellosis. legionellosis manifests as one of two clinical syndromes (table 15 .14). legionnaire's disease is typically a lobular type of pneumonia, which can be fatal but is fortunately rare; infection can range from discrete patches of inflammation and consolidation to involvement of whole lobes. pontiac fever is milder and usually subsides rapidly, often with out treatment. people who should be tested for legionnaire's disease include those with pneumonia in the following groups: because legionella is commonly found in the environment, clinical isolates are necessary to interpret the findings of an environmental investigation. diagnosis can be by rapid urine molecular testing for l. pneumophila antigen, and culture of respiratory secretions on selective media. sensitivity and specificity of the diagnostic tests are shown in table 15 .15. pontiac fever is a selflimited illness; most cases recover within 1 week and few benefit from antibiotic treatment. overall mortality in legionnaire's disease may be as high as 10%, and over 25% in older people and up to 80% in the immunocompromised. erythromycin is standard treatment; cephalosporin is an alternative. legionella species are present in roughly twothirds of potable water samples collected from domestic and institutional taps and drinking fountains, and from a similar percentage of dental units, but water from these dental units often has higher bacterial concentrations (ch. 31). there are reports of legionella infections in dental unit water lines, and antibodies and occasionally frank infection demonstrated in dental staff; at least one patient appears to have contracted and died from infection emanating from a dental practice. prevention is crucial, involving (ch. general aspects tuberculosis (tb) , an infection caused by mycobacteria, affects approxi mately onethird of the world's population (1.5 billion people); it is a major global health problem, some 2 million people dying from it annu ally. tb disproportionately affects the poorest persons in both high income and developing countries. in highincome countries, most human tb arises from mycobacterium tuberculosis, transmitted from person to person through the air. tb usually affects the lungs initially (pulmonary tb) but can also involve brain, kidneys, spine and other parts. from victorian times to about the second world war, mycobacterium bovis infection from infected cows' milk (bovine or btb) was a major cause of morbidity and mortality; it was clinically and pathologically indistin guishable from infection caused by m. tuberculosis. cattletesting and a slaughter programme became compulsory in 1950 and, by the 1980s, the incidence of tb in cattle had been substantially reduced. tuberculosis from m. bovis in cows' milk was virtually eliminated in highincome countries by the tuberculin testing of cattle and pasteurization of milk. in the developing world, many cattle still have tb, and btb is still seen. btb has also increased in highincome countries over the last two dec ades and an infection rate of up to 38% in badgers -and transmission to cattle -may explain this. tb is not spread by touch or by drinking glasses, dishes, sheets or clothing. it is usually transmitted by infected sputum, typically from close contacts such as family members, but is unlikely to be transmit ted between normal social contacts. tb can present an occupational risk to healthcare professionals, including dental staff. one outbreak of drugresistant tb in new york involved at least 357 patients, most of whom contracted tb in one of 11 hospitals; nearly 90% of the patients were also hivpositive, and most were young males of hispanic or african heritage. tb has been transmitted between pas sengers during longhaul airline flights. the risk of transmitting tb though air circulation is now low because the highefficiency particu late air (hepa) filters on newer commercial aircraft are of the same type as those used in hospital respiratory isolation rooms; indeed, the number of times air is cleaned each hour exceeds the recommendation for hospital isolation rooms. subsaharan africa has the highest rates of active tb per capita, driven primarily by the hiv epidemic. the absolute number of cases is highest in asia, with india and china having the great est burden of disease globally. in the usa and most western european countries, the majority of cases occur in foreignborn residents and recent immigrants from countries in which tubercu losis is endemic. immunocompromised people -such as diabetics and severely immuno deficient patients, like those with hiv/aids (about 30% of south africans with hiv/aids also have tb) -and patients in prisons or institutions are at risk. tb also mainly affects medically neglected persons, such as vagrants, alcoholics, intravenous drug abusers or older homeless people. the main groups at increased risk for infection therefore include people who are resourcepoor or immunoincompetent, especially: tb in developing countries is particularly widespread and is increasing, the highest rises in incidence being in southeast asia, subsaharan africa and eastern europe. in highincome countries, the incidence is also rising, probably because of worsening social deprivation, homelessness, immigration, hiv infection and intra venous drug abuse. it is now as common in london as in the devel oping world, and is seen especially in immigrants, such as those from the indian subcontinent, africa and south asia. this increase appears to be a result of the development of tb disease in individu als who may have been infected for some time and of new infections acquired in the uk, or as a result of travel to other countries where tb is common. london accounted for the highest proportion of cases in the uk in 2011 (39%), followed by the west midlands region (11%); 74% of these were born outside the uk and mainly originated from south asia and subsaharan africa. in 2011, there was a rise in the number of tb cases compared to 2010, as well as an increase in drug resistance. more information on tb, including statistics, can be found at: http:// www.hpa.org.uk/publications/infectiousdiseases/tuberculosis/ and http://www.tbfacts.org/tbstatistics.html (both accessed 30 september 2013). initial infection with tb is usually subclinical. about 10% of those infected develop overt disease; of these, half will manifest within 5 years (primary tb), while the remainder will develop postprimary disease. inhaled mycobacteria may cause subpleural lesions (primary lesion) and lesions in the regional lymph nodes (primary complex). body defences usually localize the mycobacteria, though these remain viable; infected persons are not obviously ill and are unlikely to know they are infected (latent ; table 15 .16). latent tb infection (ltbi) usu ally becomes active only after many years, if body defences become weakened (box 15.3). however, active tb can develop shortly after mycobacteria enter the body, if body defences are impaired such as in ageing, drug or alcohol abuse, or hiv/aids. also, in massive infec tions, acute active tb can result, typically causing a chronic productive cough, haemoptysis, weight loss, night sweats and fever. erythema nodosum may be associated. extrapulmonary tb is less common; it may appear as glandular involvement in the neck or elsewhere, and is less infectious than pulmonary tb. lymph node tb may lead to lymphadenopathy, caseation of the nodes and pressure symptoms -for example, on the bronchi. postprimary tb follows reactivation of an old primary pulmonary lesion and results in features ranging from a chronic fibrotic lesion to fulminating tuberculous pneumonia. the pulmonary lesions may extend and lead to a pleural effusion. reactivation or progression of primary tb may also result in widespread haematogenous dissemina tion of mycobacteria -'miliary tb'. multiple lesions may involve the central nervous system, bones, joints, and cardiovascular, gastrointes tinal and genitourinary systems. clinical presentation in tb is thus variable, depending on the extent of spread and the organs involved. as it frequently passes unrecog nized for so long, the mortality is high. similar illnesses to tb may also be caused by atypical (nontuber culous) mycobacteria, such as m. avium complex (mac; see below). the diagnosis of tb is suggested by the history and confirmed by physical examination, a massively raised erythrocyte sedimentation rate (esr), positive tuberculin skin tests (tsts; mantoux or heaf test for a delayed hypersensitivity reaction to protein from m. tuberculosis [purified protein derivative; ppd]) and chest imaging. hypersensitivity develops with 2-8 weeks of infection and can be detected by conversion of the tst from negative to positive, but tsts are neither 100% sensi tive nor specific. a positive mantoux reaction indicates previous immu nization (bcg; bacille calmette-guérin -live attenuated m. bovis) or current infection -not necessarily disease. chest radiography may show scarring and hilar lymphadenopathy. computed tomography (ct) may show areas of calcification or highlight a tuberculous abscess. smears and culture of sputum, blood, laryngeal swabs, bronchoalveolar lavage, gastric aspirates or pleural fluid may be tested for mycobacteria. polymerase chain reaction (pcr) techniques have greatly acceler ated the diagnosis and speciation, though ziehl-neelsen, auramine or rhodamine microbial stains are still used. the mycobacteria growth indicator tube (mgit) system gives results as early as 3-14 days. blood assay for m. tuberculosis (bamt) may be positive by interferongamma release assay (igra). some 15% of people over 65 years have a positive igra. the igra can be used in place of (but not in addition to) tst. igras measure the immune reactivity to m. tuberculosis. white blood cells from most persons that have been infected with m. tuberculosis will release interferongamma (ifnγ) when mixed with m. tuberculosis antigens. a positive test result sug gests that m. tuberculosis infection is likely; a negative result suggests that infection is unlikely. latent infection (ltbi) can be diagnosed with either a tuberculin skin test or an igra (more specific). igra gives a result within 24 hours and should be used biological therapy is given, such as for rheumatoid arthritis or inflammatory bowel disease. prior bcg vacci nation does not cause a falsepositive igra test result. more informa tion on the igra is available at: http://www.cdc.gov/tb/publications/ factsheets/testing/igra.htm (accessed 30 september 2013). active tb is diagnosed by sputum microscopy and culture in liquid medium with subsequent drugsusceptibility testing. nucleic acid people who should be tested for tb include those who have symp toms, those who have had close daytoday contact with active tb disease (family member, friend or coworker), those who have hiv infection or aids, those with lowered immunity, those who are required to for employment or school, and those about to be treated with biological agents. the top priority of tb control programmes is to identify and give complete treatment to all patients with active disease. tb is a notifi able disease and contact tracing is an important aspect of limiting spread. treatment with antibiotics is indicated for people who are sick with tb, those infected but not sick, and those who are close contacts of infectious tb cases. treatment for 'symptomatic sputumpositive' patients, which should be instituted as soon as possible, is combination chemotherapy, usually isoniazid plus rifampicin plus pyrazinamide or ethambutol for 2 months, with continuation of daily isoniazid and rifampicin for a further 4 months. treatment for 'asymptomatic' patients who are believed to have been infected by contacts, but are not unwell, includes isoniazid for 6 months or isoniazid and rifampicin for 3 months. rifapentine is a longacting rifampicin used once weekly. fluoroquinolones (moxifloxacin) may also act against tb. there may be resistance to one or more than one antibiotic. currently, given the potential risk of drugresistant tb being present, treatment is usually started with isoniazid, rifampicin, pyrazinamide and ethambutol (or a quinolone such as gatifloxacin or moxifloxacin) for 2 months, then isoniazid and rifampicin for 4 months. all antituberculous drugs (table 15 .17) have potentially serious adverse effects and require careful monitoring. if patient compliance is considered to be poor, directly observed therapy (dot), where drugs are dispensed by and taken in the presence of a healthcare profes sional, may be indicated. new drugs are on the horizon. immunization using bcg is advocated for schoolchildren, highrisk individuals and healthcare professionals -although its efficacy has been questioned. new vaccines are in development. chemoprophylaxis with isoniazid and rifampicin is indicated in a number of situations (box 15.4) . tb can become resistant to the drugs used to treat it particularly when the drugs are misused or mismanaged. this may occur, for example, when: in some developing countries, approximately 10% of cases are multi ple antibioticresistant; this is termed multidrugresistant tuberculosis (mdrtb); in the uk, only a small minority currently fall into this category but the number of cases is increasing. mdrtb is defined as resistance to rifampicin and isoniazid; it may be atypical in presenta tion and the infection disseminates. more than 4% of people with tb worldwide have mdrtb, and eastern europe has a high prevalence. mdrtb is seen mainly in people with hiv/aids and in hiv/aids and in africans. bedaquiline, is a new antitubercular agent the first active agent against tuberculosis to be registered since 1963. extensively drugresistant tuberculosis (xdrtb) is a rare type of mdrtb, not only resistant to isoniazid and rifampin, but also to any fluoroquinolone and at least one of three injectable secondline drugs (i.e. amikacin, kanamycin, or capreomycin). xdrtb is of special concern for immunocompromised people (e.g. with hiv/aids), who are more likely to develop tb, and have a higher risk of death if they do develop it. xdrtb is most often encountered in people from eastern europe, russia and africa. it has been transmitted in healthcare facilities and is now seen worldwide. it is essentially untreatable, though capreomycin has been used effectively to treat mdrtb in hivpositive individuals. totally drugresistant tb was reported initially in 2007-2009 in india, iran and italy; it is spreading, despite denials, and is most disquieting. chronic ulcers, usually on the tongue dorsum, are the main oral manifestation of tb. they result from coughing of infected sputum from pulmonary tb, including in hivinfected persons with tb, but are rare and such cases (usually middleaged males) may result from neglect of symptoms or default from treatment. occasionally, the diagnosis is made from biopsy of an ulcer after granulomas are seen microscopically. acidfast bacilli are rarely seen in oral biopsies, even with the help of special stains, so unfixed material should also be sent for culture if possible. tuberculous cervical lymphadenopathy is the next most common form of the infection and is particularly com mon among those from south asia. most tb lymphadenitis is pain less, with several enlarged, matted nodes, but systemic symptoms are present only in a minority and only about 15% have pulmonary mani festations on radiography (fig. 15.7) . diagnosis relies on tuberculin testing, which can be positive in both tuberculous and non tuberculous mycobacterial cervical lymphadenitis. any person with lymphadenop athy and recent conversion from a negative to positive tuberculin test should be suspected of having mycobacterial infection, and this should prompt biopsy (e.g. fineneedle aspiration biopsy) for culture or histo logical confirmation. pcr will improve diagnosis, as culture must wait 4-8 weeks for a result. oral complications of antitubercular therapy are rare, but rifabutin and rifampicin can cause red saliva. pulmonary tb is of high infectivity, as shown by cases of tuber culous infection of extraction sockets and cervical lymphadenitis in 15 patients treated by an infected member of staff at a dental clinic. dental staff who themselves were hivpositive, working in a dental clinic for hivinfected persons in new york, have died from tb con tracted occupationally. transmission of mdrtb between two dental workers may have occurred in an hiv dental clinic. infection control is thus important, so staff with tb are usually precluded from their occupation until treated. management of a patient with tb depends upon the level of poten tial infectivity (table 15.18) . patients with open pulmonary tb are con tagious, and dental treatment is thus best deferred until the infection has been treated. treatment with appropriate drugs for 2 weeks drasti cally reduces the infectivity of patients with pulmonary tb. if patients with open pulmonary tb must be given dental treatment, special pre cautions should be used to prevent the release of mycobacteria into the air, to remove any that are present and to stop their inhalation by other persons. reduction of splatter and aerosols, by minimizing cough ing and avoiding ultrasonic instruments, and use of a rubber dam, are important. improved ventilation, ultraviolet germicidal light, new masks and personal respirators, and other personal protective devices, such as hepa filters, are indicated ( fig. 15.8) . mycobacteria are very resistant to disinfectants, so that heat sterilization must be used. la is safe and satisfactory. relative analgesia is contraindicated because of the risk of contamination of the apparatus. ga is also contraindicated for dental treatment because of the risk of contamina tion of the anaesthetic apparatus and because of impaired pulmonary function. aminoglycosides, such as streptomycin, enhance the activity of some neuromuscular blocking drugs and in large doses may alone cause a myasthenic syndrome. possible drug interactions are shown in table 15 .19. other factors, such as alcoholism or intravenous drug use (ch. 34), hepatitis (ch. 9) or hiv disease (ch. 20), may also influence dental management. mycobacteria other than tuberculosis (mott) are widely distributed in water, soil, animals and humans, and rarely cause disease. severe mott infections have been seen, however, in individuals predisposed because of defects in the interleukin12 (il12) and interferongamma (ifngamma) pathways. mycobacterium abscessus, a bacterium found in water, soil and dust, has been known to contaminate medications and products, including medical devices. healthcareassociated m. abscessus can cause a vari ety of infections, usually of the skin, but it can also cause lung infec tions in persons with various chronic lung diseases and is increasingly recognized as an opportunistic pathogen in cystic fibrosis (cf) patients persontoperson transmission of atypical mycobacteria is not important in acquisition of infection, except for skin infections. on rare occasions, mott skin infections have followed tattooing with contaminated tattoo inks. many people become infected with and har bour mott in their respiratory secretions without any symptoms or evidence of disease. individuals with respiratory disease from mott do not readily infect others and, therefore, do not need to be isolated. mott are generally not infectious to others. infection with m. abscessus is usually caused by injections of con taminated substances or by invasive medical procedures employing contaminated equipment or material. infection can also occur after accidental injury where the wound is contaminated by soil. there is very little risk of transmission from person to person. mac complex, m. scrofulaceum and m. kansasii are possible causes of tuberculous cervical lymphadenitis. mac may also infect the lungs (similar to tb), skin or lymph nodes. lung disease is also caused occasionally by m. kansasii, mainly in middleaged and older persons with underlying chronic lung conditions. m. fortuitum and m. chelonae may cause skin and wound infections and abscesses, frequently associated with trauma or surgery. m. marinum may cause 'swimming pool granuloma', a nodular lesion that may ulcerate, usually on an extremity. m. ulcerans may produce chronic ulcerative skin lesions, usually of an extremity. m. abscessus skin infections present with swollen and/ or painful areas that are usually red, warm and tender to the touch, and which can also develop into boils or pustules. other features of m. abscessus infection are fever, chills, muscle aches and malaise. cervical lymphadenitis due to mac, m. scrofulaceum and m. kansasii may affect otherwise healthy young children, most commonly pre school females who have unilateral cervical lymphadenopathy, typically in the submandibular or jugulodigastric nodes, and they may form a 'cold abscess'. mott is the usual cause in children under 12 years but tb is more common in older patients. absence of fever or tuber culosis, a positive tuberculin test and failed response to conventional antimicrobials are highly suggestive of mott, but definitive diagnosis is by smear, culture or pcr of biopsy material obtained by fineneedle aspiration or removal of nodes. treatment is based on results of laboratory testing, which should identify the appropriate antibiotic. preventive treatment of close contacts of persons with disease caused by mott is not needed. most mott are resistant to standard antitubercular medication and, though it is possible that clarithromycin or clofazimine may have some effect, excision of affected nodes is the usual recommended therapy. water from dental units may contain mott species; mycobacterial proliferation in biofilms may explain the extent of this contamination (ch. 31). aspiration syndromes are conditions in which foreign substances are inhaled into the lungs and which can have consequences ranging from asphyxia to infection and lung abscess. dental restorations or frag ments of teeth, plaque, gastric contents and other materials may be aspirated, especially if material enters the pharynx, and particularly if the cough reflex is impaired for any reason. most commonly, aspiration syndromes involve oral or gastric contents associated with gastrooesophageal reflux disease (gord), swallowing dysfunction (ch. 7), neurological disorders and structural abnormalities, such as a pharyngeal pouch. cricopharyngeal dys function involves cricopharyngeal muscle spasm or achalasia of the superior oesophageal sphincter, and can be seen in infants who have a normal sucking reflex but have incoordination during swallowing, pos sibly secondary to delayed development or cerebral palsy. anatomical disorders, such as cleft palate, pharyngeal pouch, oesophageal atresia, tracheooesophageal fistula, duodenal obstruction or malrotation, and motility disorders, such as achalasia, may have an aspiration risk. infirm older patients are also at risk of aspiration, especially if they are bedbound or have neurological disorders. isolated superior laryngeal nerve damage, vocal cord paralysis, cerebral palsy, muscular dystrophy and riley-day syndrome (familial dysautonomia) are all associated with increased risk of aspiration. ventilatorassociated pneumonia (vap), as defined by the centers for disease control and prevention (cdc), is present when the chest radiograph shows new or progressive infiltrate, consolidation, cavitation or pleural effusion in conjunction with either new onset of purulent sputum or change in character of sputum, and an organism isolated from blood, or the isolation of an aetiological agent from a specimen obtained via suction aspiration through an endotracheal or tracheostomy tube. the major route for acquiring endemic vap is oropharyngeal colo nization by endogenous flora or by exogenously acquired pathogens from intensive care units. vap is the most commonly reported health careacquired infection in patients receiving mechanical ventilation, with prevalence rates consistently in the 10-20% range. mortality rates in vap are at least double those in patients without vap, ranging from 24% to 85% when the infection is caused by a multidrugresistant gramnegative pathogen. the healthcare infection control practices advisory committee of the cdc has developed guidelines for the prevention of vap. these include strategies aimed at preventing aspiration of contaminated oral or gastric material (e.g. raising the head of the bed and draining subglottic secretions), and interventions to alter bacterial coloniza tion of stomach (e.g. stress ulcer prophylaxis and selective digestive decontamination) and mouth. oral hygiene, suctioning and the provi sion of moisture to lips and oral mucosa, plus toothbrushing, may be important in prevention of vap. there are also strategies for manag ing ventilator circuits (e.g. replacement of ventilator circuits, use of closed rather than open suction, and use of heat moisture exchange as opposed to heated circuit technology). lung abscess is a localized infection leading to cavitation and necro sis. while some cases result from aspiration of foreign material, most develop from pneumonia caused by infection with staph. aureus or klebsiella pneumoniae. bronchial obstruction by carcinoma is another important cause. symptoms resemble those of suppurative pneumonia. there is a risk of infection spreading locally or leading, via septicaemia, to a brain abscess. diagnosis rests mainly on the chest radiograph, which may sometimes show cavitation or a fluid level. antimicrobial chemotherapy, postural drainage and relief by bronchoscopy of any obstruction are indicated. a wellrecognized cause of lung abscess is inhalation of a tooth or fragment, a restoration or rarely, an endodontic instrument. when undertaking endodontics or cementing restorations, such as inlays or crowns, a rubber dam or other protective device should always be used to avoid the danger of inhalation. lung abscesses may also result from aspiration of oral bacteria, particularly anaerobes, especially in infirm older patients or those who are intubated. the other main dangers in dentistry are with ga, particularly if an inadequate throat pack has been used. patients who inhale tooth frag ments or dental instruments must have chest radiographs (lateral and posteroanterior) and, if necessary, bronchoscopy. loeffler syndrome appears to be an allergic reaction, usually to the parasitic worm ascaris lumbricoides, or drugs such as sulphonamides. it manifests with pulmonary infiltrates (and abnormal chest radio graph) and eosinophilia (eosinophilic pneumonia). the disease usually clears spontaneously. sarcoidosis, so named because skin lesions resembled a sarcoma, is a multisystem granulomatous disorder, seen most commonly in young adult females in northern europe, especially in people of african heritage. the aetiology is unclear but propionibacterium acnes and p. granulosum have been implicated and associations have been reported with exposure to inorganic particles, insecticides, moulds and occupations such as firefighting and metalworking. serum sam ples contain antibodies directed against mycobacterium tuberculosis antigens. sarcoidosis is associated with hladrb1 and dqb1, and a butyrophilinlike 2 (btnl2) gene on chromosome 6. thelper 1 (th1) cells release il2 and ifnγ, and augment macrophage tumour necrosis factor alpha (tnfα) release. cd25 regulatory t cells cause a limited impairment of cellmediated immune responses (partial anergy) but no obvious special susceptibility to infection. sarcoidosis affects the thorax in 90%, but has protean manifestations and can involve virtually any tissue (table 15 .20). sarcoid most typi cally causes löfgren syndrome (fever, bilateral hilar lymphadenopathy, arthralgia and erythema nodosum, especially around the ankles; figs 15.9 and 15.10). other common presentations may include pulmonary infiltration and impaired respiratory efficiency, with cough and dyspnoea in severe cases, or acute uveitis, which can progress to blindness. susceptibility to lymphomas has been suggested but not confirmed. because of its vague and protean manifestations, sarcoidosis is under diagnosed. in the presence of suggestive clinical features, helpful investigations include: chest radiography (enlarged hilar lymph nodes); raised serum angiotensinconverting enzyme (sace ; table 15 .21) in acute disease (this is insensitive, nonspecific and a poor guide to therapy); positive gallium67 citrate or gadolinium or positron emis sion tomography (pet) scans; labial salivary gland or transbronchial biopsy (for histological evidence of noncaseating epithelioid cell granulomas) -except in löfgren syndrome, which is a classical clini cal diagnosis. 18 fdeoxyglucose pet is helpful in identifying sites for biopsy. nonspecific findings may include mild anaemia, leukopenia, eosinophilia, hypergammaglobulinaemia, raised esr and low serum albumin. hypercalcaemia is common because of extrarenal produc tion of active vitamin d and can result in renal damage. alkaline phosphatase, 5'nucleotidase, lysozyme and adenosine deaminase levels are raised in hepatic sarcoidosis. evidence of impaired delayed hypersensitivity reactions to some antigens may be useful. kveim skin tests are not now used. half the patients with sarcoidosis remit within 3 years and about 66% remit by 10 years. patients with only minor symptoms usually need no treatment but corticosteroids, sometimes with azathioprine, methotrexate, tetracyclines, hydroxychloroquine, infliximab or etaner cept, are given if there is active organ disease (ocular disease, progres sive lung disease, hypercalcaemia or cerebral involvement). biopsy of the minor salivary glands frequently shows noncaseating granulomas and association with other features of sarcoidosis, par ticularly hilar lymphadenopathy. this is an important diagnostic find ing that may obviate more invasive procedures. sarcoidosis can involve any of the oral tissues but has a predilection for salivary glands. asymptomatic swelling of the parotid glands or cervical nodes, and less frequently the lips, may accompany systemic disease. superficial or deepseated red submucosal nodules may develop intraorally and on the lips. nontender, wellcircumscribed, brownishred or violaceous nodules with superficial ulceration have also been reported. the oral and lip lesions may occasionally precede systemic involvement. there is enlargement of the major salivary glands in about 6% of cases; some have xerostomia, and the association of salivary and lacri mal gland enlargement with fever and uveitis is known as uveoparotid fever (heerfordt syndrome). salivary swelling may also be seen with out other features of heerfordt syndrome. the salivary gland swellings usually resolve on treatment of sarcoidosis but this may take up to 3 years. facial palsy and other cranial neuropathies may be seen. there is also an association with sjögren syndrome, when ssa and ssb serum autoantibodies are found. rarely there is an association of thyroiditis with addison disease, sjögren syndrome and sarcoidosis (tass syndrome). there is a group of patients who have histologi cal features of sarcoid in one or more sites in the mouth, such as the gingivae, but no systemic manifestations. a few of these patients may ultimately develop other more or less systematized disease but the majority probably have isolated lesions. such cases, where no exog enous cause for the granulomatous reaction can be found, are regarded as having 'sarcoidlike' reactions (orofacial granulomatosis) and treat ment is unnecessary. however, patients should be kept under observa tion for as long as possible. management of patients with systemic sarcoidosis may include con sideration of respiratory impairment, uveitis and visual impairment, renal disease, jaundice or corticosteroid treatment. la is safe and satisfactory. cs is contraindicated if there is any res piratory impairment. ga should only be given in hospital. lung cancer is the most common cancer in highincome countries in males and most frequently affects adult urban cigarettesmokers. bronchogenic carcinoma accounts for 95% of all primary lung cancer and has also become increasingly common in women (because of increased tobacco use), to the extent that the mortality rate for the two sexes has become almost equal. metastases from cancers elsewhere are also frequently found in the lungs. recurrent cough, haemoptysis, dyspnoea, chest pain and recurrent chest infections are the predominant features. local infiltration may cause pleural effusion, lesions of the cervical sympathetic chain (horner syndrome), brachial neuritis, recurrent laryngeal nerve palsy or obstruction of the superior vena cava with facial cyanosis and oedema (superior vena cava syndrome). there are many nonmetastatic extrapulmonary effects of bron chogenic (or other) carcinomas -for example, weight loss, anorexia, fingerclubbing, neuromyopathies, thromboses (thrombophlebitis migrans), muscle weakness, various skin manifestations and ectopic hormone production (of antidiuretic hormone, adrenocorticotropic hormone, parathyroid hormone and thyroidstimulating hormone). metastases from bronchogenic cancer are common and typically form in the brain (which may manifest with headache, epilepsy, hemi plegia or visual disturbances), liver (hepatomegaly, jaundice or ascites) or bone (pain, swelling or pathological fracture). the diagnosis is based on history and physical examination, supported by radiography, ct and magnetic resonance imaging (mri), sputum cytology, bronchoscopy and biopsy. spiral ct appears to detect tumours at an early stage. the overall 5year survival rate is only 8%. radiotherapy is the most common treatment. only some 25% of patients are suitable for surgery but, even then, the 5year survival is only about 25%. chemotherapy has been disappointing, except in smallcell carcinomas. dental treatment under la should be uncomplicated. cs should preferably be avoided. ga is a matter for specialist management in hospital, as patients often have impaired respiratory function, espe cially after lobectomy or pneumonectomy. this, along with any muscle weakness (myasthenic syndrome, eaton-lambert syndrome) that can make the patient unduly sensitive to the action of muscle relaxants, makes ga hazardous. oral cancer may be associated with lung cancer, and vice versa, or develop at a later stage (ch. 22). such synchronous or metachronous primary tumours must always be ruled out. metastases can occasionally affect the orofacial region and cause enlargement of the lower cervical lymph nodes, epulislike softtissue swellings or labial hypoaesthesia or paraesthesia in the jaw. soft palate pigmentation is a rare early oral manifestation. lung cancer is a fairly common cause of death in dental techni cians, but it is unknown whether this is due to smoking alone or to dust inhalation. cystic fibrosis (cf) is one of the most common fatal hereditary dis orders. inherited as an autosomal recessive trait, with an incidence of about 1 in 2000 births, it is the most common inherited error of metabolism and is seen mainly in people of european descent. the gene responsible is on chromosome 7q. cf is caused by defects in the cystic fibrosis transmembrane conductance regulator (cftr), a protein that appears to be part of a cyclic adenosine monophosphate (camp)regulated chloride channel, regulating cl − and na + transport across epithelial membranes, and ion channels and intracellular fluid flow in sweat, digestive and mucus glands. the basic defect in cf is abnormal chloride ion transport across the cell membrane of nearly all exocrine glands. the blockage of salt and water movement into and out of cells results in the cells that line the lungs, pancreas and other organs producing abnormally thick, sticky mucus that can obstruct the airways and various glands, especially in the respiratory tract and pancreas. involved glands (lungs, pancreas, intestinal glands, intrahepatic bile ducts, gallbladder, submaxillary and sweat glands) may become obstructed by this viscid or solid eosino philic material. recurrent respiratory infections result in a persistent productive cough and bronchiectasis, with the lungs becoming infected with a variety of organisms including staph. aureus, haemophilus influenzae, pseudomonas aeruginosa, strep. pneumoniae, burkholderia cepacia, and sometimes mycoses or mycobacteria. mycobacterium abscessus is a nontuberculous mycobacterium increasingly recognized as an opportunistic pathogen in cf patients. viral infections, such as mea sles, can have severe sequelae. pancreatic duct obstruction leads to pancreatic insufficiency, with malabsorption and bulky, frequent, foulsmelling, fatty stools. gallstones, diabetes, cirrhosis and pancreatitis may result. sinusitis is very common. growth is frequently stunted. the mutations can also cause con genital bilateral absence of the vas deferens, so fertility is impaired in most males with cf. in women, fertility may be impaired by viscid cervical secretions, but many women have carried pregnancies to term. most patients have a high concentration of sodium in their sweat (also reflected in the saliva); a sweat test showing sodium and chloride values of more than 60 mmol/l is considered positive, between 40 and 60 mmol/l equivocal, and less than 40 mmol/l negative. physiotherapy and postural drainage are crucially important. clearance of sputum is helped by water aerosols and bronchodila tors (terbutaline or salbutamol), but mucolytics such as carbocisteine, methyl cysteine and dornase alfa are of questionable effectiveness. treatment with ivacaftor, a cftr potentiator, improves chloride transport through the ion channel. amoxicillin and flucloxacillin are effective prophylactic antimicrobi als and may be given by aerosol. vaccination against measles, whoop ing cough and influenza is important. a low fat intake, adequate vitamins and oral pancreatic enzyme replacement (pancreatin) are also necessary. doublelung or heart-lung transplantation may eventually become necessary. sinusitis is very common; most cf patients have recurrent sinusitis and nasal polyps. the major salivary glands may enlarge and hyposali vation sometimes occurs. the lowfat, highcarbohydrate diet and dry mouth may predispose to caries. enamel hypoplasia and black stain may be seen, and both dental development and eruption are delayed. tetracycline staining of the teeth was common but should rarely be seen now. pancreatin may cause oral ulceration if held in the mouth. la is satisfactory but cs is usually contraindicated because of poor respiratory function. ga is contraindicated if respiratory function is poor. lung disease, such as bronchiectasis, liver disease and diabetes, may complicate treatment. bronchiectasis is dilatation and distortion of the bronchi. causes include: ■ congenital defects, which should be considered in all patients include cystic fibrosis, kartagener syndrome, alpha1antitrypsin deficiency, collagen defects (e.g. marfan syndrome) there is no identifiable underlying cause in about 50% of adults and 25% of children. the damaged and dilated bronchi lose their ciliated epithelium and therefore mucus tends to pool, causing recurrent lrtis, typically with strep. pneumoniae, haemophilus influenzae or pseudomonas aeruginosa. overproduction of sputum, which is purulent during exacerbations, a cough (especially during exercise or when lying down) and finger clubbing are typical features, with recurrent episodes of bronchitis, pneumonia and pleurisy. haemoptysis is not uncommon. in advanced bronchiectasis, chest pain, dyspnoea, cyanosis and respiratory failure may develop. complications may include cerebral abscess and amyloid disease. chest radiography and pulmonary function tests are required. high resolution ct (hrct) is useful. postural drainage is important. antimicrobials, such as amoxicillin, cephalosporins or ciprofloxacin, are given for acute exacerbations and for longterm maintenance treatment. ga should be avoided where possible and is contraindicated in acute phases. workers exposed to airborne particles may develop pulmonary disease (pneumoconiosis), which ranges from benign (e.g. siderosis) to malig nant, as in mesothelioma from asbestosis (see appendix 15.1), but any pneumoconiosis can cause significant incapacity. ga may be contraindicated; the physician should be contacted before treatment. berylliosis may be a hazard in some dental technical laboratories, when lung cancer is more frequent. respiratory complications following surgical operations under ga include segmental or lobar pulmonary collapse and infection. they are more common after abdominal surgery or if there is preexistent respiratory disease or smoking (see also ch. 3), and can be signifi cantly reduced by smoking cessation, preoperative physiotherapy and bronchodilators, such as salbutamol. if postoperative pulmonary infection develops, sputum should be sent for culture, and physiotherapy and antibiotics should be given. the common microbial causes are strep. pneumoniae and haemophilus influenza; in this case, suitable antibiotics include amoxicillin and erythromycin. hospital infections may include other microorganisms, such as mrsa, klebsiella, pseudomonas and other gramnegative bacteria. inhalation (aspiration) of gastric contents can cause pulmonary oedema and may be fatal (mendelson syndrome); it is most likely if a ga is given to a patient who has a stomach that is not empty, has a hiatus hernia or is in the last trimester of pregnancy. prevention is by ensuring the stomach is empty preoperatively; if it is not, an anaes thetist should pass an endotracheal tube. antacids or an h2receptor blocker, such as cimetidine or ranitidine, may be given by mouth pre operatively to lower gastric acidity. if gastric contents are aspirated, the pharynx and larynx must be carefully sucked out. systemic corticosteroids have been recommended but probably do not reduce the mortality. respiratory distress in premature infants may be caused by immaturity of surfactantproducing cells, when the alveoli fail to expand fully; this necessitates endotracheal intubation for many weeks. it may, in turn, result in midface hypoplasia, palatal grooving or clefting, or defects in the primary dentition. the same oral effects may be seen with prolonged use of orogastric feeding tubes. the degree to which subsequent growth corrects these deformations is currently unknown, though the palatal grooves typically regress by the age of 2 years. using soft endotracheal tubes does not obviate this problem and, at present, the best means of avoiding palatal grooving appears to be the use of an intraoral acrylic plate to stabilize the tube and protect the palate. acute respiratory distress syndrome (ards) is a sequel to several types of pulmonary injury and some infections, including those with oral viridans streptococci. patients with endstage pulmonary disease are considered for potential transplantation, usually using a lung from a braindead organ donor. a combination of ciclosporin, azathioprine and glucocorticoids is usu ally given for lifelong immunosuppression to prevent a tcell, alloim mune rejection response. inhaled nitric oxide modulates pulmonary vascular tone via smooth muscle relaxation and can improve ventilation/perfusion matching and oxygenation in diseased lungs. early graft failure following lung transplantation has been described by various investi gators as reimplantation oedema, reperfusion oedema, primary graft failure or allograft dysfunction. pathologically, this entity is diffuse alveolar damage. see also chapter 35. a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for 6 months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. cardiopulmonary transplantation (heart and lung transplantation) is the simultaneous surgical replacement of the heart and lungs in patients with endstage cardiac and pulmonary disease, with organs from a cadaveric donor. all transplant recipients require lifelong immunosuppression to pre vent a tcell, alloimmune rejection response. see also chapter 35. a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for 6 months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. national institutes of health: national institute of allergy and infectious diseases healthcare infection control practices advisory committee guideline for the prevention of healthcare associated pneumonia nosocomial pneumonia: state of the science extensively drugresistant tuberculosis as a cause of death in patients coinfected with tuberculosis and hiv in a rural area of south africa a review of the possible role of oral and dental colonization on the occurrence of health careassociated pneumonia: underappreciated risk and a call for interventions reducing ventilatorassociated pneumonia through advanced oraldental care: a 48month study apic infection control and applied epidemiology: principles and practice sepp. ventilatorassociated pneumonia and oral care: a successful quality improvement project guidelines for preventing the transmission of mycobacterium tuberculosis in healthcare settings a randomized trial of dental brushing for preventing ventilatorassociated pneumonia pneumonia associated with a dental unit waterline the pathogenesis of ventilatorassociated pneumonia: its relevance to developing effective strategies for prevention aspects of human disease 32 chronic obstructive pulmonary disease (copd) aspects of human disease 31 in vitro antibacterial activities of oral care products against ventilatorassociated pneumonia pathogens the impact of a simple, lowcost oral care protocol on ventilatorassociated pneumonia rates in a surgical intensive care unit intermittent suction of oral secretions before each positional change may reduce ventilatorassociated pneumonia: a pilot study current trends and newer concepts on diagnosis, management and prevention of respiratory tract infections key: cord-302321-6x7hyald authors: qiao, shan; li, zhenlong; weissman, sharon; li, xiaoming; olatosi, bankole; davis, christal; mansaray, ali b. title: disparity in hiv service interruption in the outbreak of covid-19 in south carolina date: 2020-08-27 journal: aids behav doi: 10.1007/s10461-020-03013-x sha: doc_id: 302321 cord_uid: 6x7hyald to examine hiv service interruptions during the coivd-19 outbreak in south carolina (sc) and identify geospatial and socioeconomic correlates of such interruptions, we collected qualitative, geospatial, and quantitative data from 27 ryan white hiv clinics in sc in march, 2020. hiv service interruptions were categorized (none, minimal, partial, and complete interruption) and analyzed for geospatial heterogeneity. nearly 56% of the hiv clinics were partially interrupted and 26% were completely closed. geospatial heterogeneity of service interruption existed but did not exactly overlap with the geospatial pattern of covid-19 outbreak. the percentage of uninsured in the service catchment areas was significantly correlated with hiv service interruption (f = 3.987, p = .02). this mixed-method study demonstrated the disparity of hiv service interruptions in the covid-19 in sc and suggested a contribution of existing socioeconomic gaps to this disparity. these findings may inform the resources allocation and future strategies to respond to public health emergencies. the outbreak of coronavirus disease 2019 (covid19) , caused by the severe acute respiratory syndrome coronavirus (sars-cov-2), is a rapidly evolving global public health crisis [1] . on march 11, 2020 , the world health organization characterized covid-19 as a pandemic [2] . as of august 5, 2020, infection cases exceeded 18,614,177 across 216 countries and areas, with over 702,642 deaths [3] . these numbers continue to rise daily as the virus continues to spread globally. people living with hiv (plwh) have been recognized as a medically and socially vulnerable population during the covid-19 pandemic [4] . hiv-related services have been unavoidably interrupted and impacted. on march 18, 2020 , the cdc posted specific guideline to address plwh's concerns and questions related to their covid-19 risk and prevention. on march 20, 2020, the nih office of aids research also provided interim guideline related to covid-19 and plwh. however, there have been many challenges in the implementation of these guidelines, particularly in the areas of healthcare delivery and plwh's linkage to care and retention in care [5, 6] . during the unprecedented covid-19 outbreak, many health facilities providing hiv prevention, treatment and care services had to change their schedules and operation modalities to adapt to the challenges imposed by covid-19 and its prevention and control measures (social distancing, travel restrictions, stay at home orders, etc.). routine, nonurgent or elective health care visits and procedures were cancelled or delayed. supportive services such as face-toface counselling, housing services and outreach services were temporarily suspended. in addition, patients avoided accessing health care, even for urgent concerns, due to fear of covid-19 exposure [7, 8] . in order to improve healthcare delivery and provide sustainable quality care to plwh during the covid-19 pandemic and future pandemics, we need a better understanding of the nature and scope of hiv treatment service interruption and their correlates. existing literature suggests a health disparity in covid-19 prevalence and related clinical outcomes (e.g., complications, mortality) in the united states. for example, one recent spatial analysis on covid-19 epidemic reported an uneven impact on incidence and mortality of covid-19 in the united states [9] . the study showed positive correlations of covid-19 incidence and mortality rates with socioeconomic factors [9] . however, no empirical studies have examined if there is a spatial disparity in the hiv service interruption and if existing social determinants of health influence the pattern of hiv service interruption, particularly in southern states such as south carolina. south carolina (sc) has been heavily hit by the covid-19. the first case of covid-19 was reported in sc on march 6, 2020 [10] . on april 2, 2020, sc department of health and environmental control (dhec) announced that the virus had spread to all 46 counties in the state [11] . as of august 5, 2020, covid-19 cases exceeded 94,831 with over 1819 deaths in sc [10] . therefore, using mixed-method data, the current study aims to (1) examine the interruption of hiv service delivery in sc during covid-19 outbreak; (2) demonstrate the pattern of this service interruption by geolocation; and (3) identify any existing health disparity factors (e.g., socioeconomic status) that were associated with the hiv service interruption. sc is one of the seven states targeted by the "ending the hiv epidemic (ethe): a plan for america" [12] campaign due to its high rural hiv burden [12] . with an hiv infection rate of 15.8 per 100,000 people, sc ranked 11th among all 50 states in 2018, with an estimate of 20,166 plwh [13] . african americans and men who have sex with men (msm) are disproportionately affected by hiv/aids in sc [13] . by 2018, there were 27 clinics in sc funded by ryan white hiv/aids program [14] . the ryan white hiv/aids programs are federally funded programs to provide comprehensive hiv primary medical care, essential support services, and medications for low-income plwh who are uninsured and underserved [15] . according to the program profile, it was estimated that 11,369 (56%) plwh were served by the ryan white clinics in sc in 2018 [16] . qualitative information was extracted from the program process reports of 27 ryan white hiv clinics (grantees of ryan white hiv/aids programs) across 46 counties in sc in march, 2020. these reports were submitted to the sc dhec based on the clinics' status of operation and hiv-related service delivery during the covid-19 outbreak. geolocations and zip codes of the hiv clinics were retrieved for geospatial analysis. based on the information provided by the hiv clinics, we identified the service catchment areas of the clinics (i.e., counties these hiv clinics served). we then extracted data from various sources for each county's relevant characteristics, such as number of plwh per 100,000 in 2018 (via aidsvu, https ://aidsv u.org), number of covid-19 cases per 100,000 as of june 13, 2020 (via sc dhec, https ://scdhe c.gov), and socioeconomic status (ses) indicators in 2016 (via us census bureau, https ://censu s.gov/). the ses indicators included poverty (percent of population living below federal poverty line) [17] , high school education (percent of population with a high school degree or equivalent) [18] , median household income [17] , gini coefficient (a measure of income inequality with 0 reflecting complete equality and 1 reflecting complete inequality) [19] , and percent of population without health insurance [20] . the current study was approved by the institutional research board at university of south carolina. based on qualitative data regarding the status of operation at hiv clinics, a medical expert with over 20 years experiences in both hiv-related research and clinic care initially categorized hiv service interruption during the covid-19 outbreak into four types: complete interruption, partial interruption, minimal interruption, and no interruption. specifically, there were four criteria for assessing the hiv service interruption including clinic operation hours (e.g., whether clinics are open for most of weekdays), hiv service coverage (e.g., whether clinics maintain most of existing services), telehealth use (e.g., whether clinics use telehealth in service delivery), and healthcare providers' availability (e.g., whether most of healthcare providers, especially medical case managers can still work in their original capacity). the clinics that failed in meeting the four criteria were considered as "complete interruption", failed in any two or three as "partial interruption", failed in any one of the criteria as "minimal interruption", and those met all the four criteria as "no interruption". another team member reviewed the data and confirmed the initial categorization using the same criteria. we analyzed the qualitative data based on grounded theory [21] . five themes occurred regarding hiv service interruption, including operation days and time, protection measures, service coverage, the use of telehealth and mobile applications (apps), and healthcare provider availability. we then mapped the pattern of hiv service interruptions using the geographic information system (gis) to indicate geospatial distribution. for quantitative data, we developed the county-level indicators (e.g., ses, covid-19 cases, and hiv cases) for each hiv clinic based on its service catchment area. specifically, each of the five ses indicators, number of covid-19 cases per 100,000 people, and number of plwh per 100,000 people were averaged among counties that each clinic served. it should be noted that there may be overlap among some clinics in catchment area with some counties being served by more than one clinic. in such cases, indicators of those counties were used by each serving clinic. anova was used to examine the associations between these countylevel indicators and types of service interruption. one hiv clinic that provided state-wide hiv service was removed from the county-level geospatial analysis and quantitative analysis since its service catchment area was not limited to any specific county. among the 27 hiv clinics in the current study, two clinics (7.4%) were assessed as "no interruption", three (11.1%) as "minimal interruption", 15 clinics (55.6%) as "partial interruption", and seven clinics (25.9%) as "complete interruption". the qualitative information regarding hiv service interruption during the outbreak of covid-19 covered five domains (see sample quotes in table 1 ). in terms of operation time, only one clinic had their main site open for normal hours. most of hiv clinics reduced their office hours, limited office visits, and/or limited face-to-face appointments. seven clinics were closed with only staff checking emails daily. clinics adapted various strategies in adjusting operation hours. some canceled evening hours. some adjusted their schedule, providing different types of services at different dates and hours. all the clinics adapted specific measures to protect patients, visitors, healthcare providers, and staff from covid-19 infection. common strategies included requiring temperature and symptom screening for all visitors entering into the building, asking all the people in the offices and building to wear face masks, and taking precaution to maintain social distancing in offices. one clinic mentioned that they provided covid-19 testing at their sites. one clinic highlighted that their patients were screened and triaged prior to entering into clinic. another clinic reported that they had sufficient stock to provide masks to both clinic staff and visiting patients. due to the closure and reduced operation hours, hivrelated services were interrupted at many clinics in terms of scope and delivery modality. two hiv clinics reported that they had to discontinue home visits and support groups. several clinics suspended walk-in services. one clinic mentioned that their dental offices were only seeing patients for emergency visits. prevention services were available by appointment only and hiv testing services were provided at alternative sites. however, most of clinics still accepted new referrals for hiv treatment and primary care, and they also delivered medical care, case management services, pharmacy services and lab services over telephone and/or in the offices when needed. one clinic reported that they also provided curbside/drive through covid-19 testing on-site and partnering with sc dhec to do covid-19 testing in the community. two clinics also assisted patients with medication pick-up/delivery, food cards, personal pantry and housing services/support as needed. telehealth and mobile apps were widely used in hiv clinics for making appointments and delivering hiv prevention, treatment, and care services. one clinic also introduced an on-line request system for rent, food, and clothing assistance. for the clinics where regular operation hours remained unchanged, telehealth was also provided as an option for patients. one clinic reported, "we have virtual visit capabilities for all patients with regular follow-up appointments if patients have computer and internet access at home. for ryan white patients, medical case managers (mcms) will bring laptop and hot spot to the patient's home, get them online with provider and step out of the room". during the covid-19 outbreak, most healthcare providers and staff had flexible working schedules. policy regarding remote working and return-to-work varied across clinics and also changed along with the rapidly evolving pandemic. most of clinics reported that the providers and staff were working from home using virtual private network (vpn) for access to provide enterprise (ryan white data reporting " "restricting face-to-face visits, restricting patients only to building." "closed for 14 days; m is checking and responding to emails remotely" "currently, offices are operating on normal hours with some limited closures for evening hours based on staffing per day." "as of may 18, 2020 new horizon family health services will begin seeing patients in the office setting with continued reduced hours of operation." "office is closed. working remotely." "staff in office for services monday, wednesday, friday 9a-1p. all staff available 8:30-5 by telephone. using telehealth for most medical appointments, otherwise tuesday 3p-7p and thursday 10a-2p. testing on thursday 3-7pm and 1st and 3rd saturday 10-2 pm. limited in office visits. …telephone messages checked 4 times/day. on call staff available 24/7." "limited office appointments, all mcm's are working in the office full-time as of monday, june 1, 2020." protection measures (for healthcare workers, staff and visitors) "[***] has resume our regularly work schedule with taking precautions and practicing social distances in the office." "all staff are screened daily when entering the building and wearing masks. patients entering the building are screened and also wearing masks." "all patients are required to wear a mask when entering the facility." "patients/clients/employees are required to wear masks in all locations & facilities. we have sufficient stock to provide masks to those without." "patients are covid-19 screened & triaged prior to entrance into clinic." "we request patients to practice social distancing, self-social isolation, and wear a mask while in clinic for appointments." service provided "we are providing curbside/drive thru covid-19 testing on-site and partnering with dhec to test within the community. we continue to provide medical care, behavioral health and dental services using telehealth and in person visits. mcms are providing case management services over the phone and in the office when needed. we are assisting with food cards, personal pantry and housing services/support as needed. prevention is providing [hiv] testing services on-site" "we have also resume(d) testing and have giving appointments if a person calls prior but will not turn away walk-ins…. we are accepting new referral for hiv treatment and primary care." "prevention services continue to be available by appt only; however, they began providing testing services at alternative sites on june 1." "patients who must have face-to-face appointment with provider we are still open." "are limiting patients coming to the office and exploring other options such as the healow app and telehealth visits. our dental offices are only seeing patients for emergency visits at this time." "newly diagnosed patients are being seen in clinic. patients requiring sick visits are also being seen." "appointments are encouraged, however walk in care will be available on a limited basis. all pharmacy services also available. ryan white program is fully staffed and routine clinic hours are in place. patients can be seen with an appointment for id as well as lab services." "introduced online request systems for rent, food, and clothing assistance; online scheduling for tax assistance; no change to hiv connected services at this point other than requesting partners not drop off applications in person." "medications are being delivered as needed. any of the mcm's can be reached via telephone and email." "we are still accepting new referrals and will maintain adequate staffing. we have discontinued support groups and home visits but all other services remain the same." "staff is in the office conducting client appointments by telephone. we are accepting referrals and scheduling/completing intakes & appts for emergency financial assistance and providing supportive services as needed (e.g. medication pick up/delivery, food banks). we've temporarily suspending home visits & support groups." system). in some clinics, mcms were rotating "in office" at least 2 days a week, providing services by appointment only. one clinic reported that their mcms reached out to clients who had not picked up medicines and offered medication delivery. since june 2020, more providers and staff have returned to the office. one clinic staff said, "decisions going forward will be made daily/weekly as to our re-opening with full staffing." the county-level geospatial pattern of the hiv service interruption among all hiv clinics (except the one that provided state-wide service), along with the geospatial pattern of covid-19 cases in sc is shown in fig. 1 . generally, most of the service catchment areas were moderately affected by the covid-19 outbreak in terms of confirmed covid-19 cases per capita. in the upstate area no clinic reported "complete interruption" of hiv services. geospatial heterogeneity in terms of hiv service interruption existed. however, the geospatial pattern of hiv service interruption did not exactly overlap with the density of covid-19 cases at county level. the results of descriptive analysis and anova analysis are shown in table 2 . among the service catchment areas of the hiv clinics, the mean rate of plwh in 2018 and the mean rate of confirmed covid-19 cases as of june 13, 2020 was 429 and 416 per 100,000, respectively. the median household income averaged $43,124 (sd = 5367). the mean of gini coefficients was .46. the percentage of uninsured population was 13.5% on average (statewide: 10.5%) [13] . among these counties, the percentage of people in the county living in poverty was approximately 20% (statewide: 15.3%) [13] . on average, about 83% of population had graduated from high school. anova analysis did not detect significant difference by hiv service interruption types in terms of ses indicators except the percentage of uninsured population. the geospatial distribution pattern of plwh was similar to the distribution pattern of confirmed covid-19 cases. hiv service interruption pattern did not consistently correspond with the density of covid-19 cases. while the clinics that reported no interruption in services were in areas with the lowest rate of covid-19 cases, note: all the identifiable information (e.g., names or locations) were removed in the quotes to protect the identity of the respondents sample quotes from the clinical reports telehealth and apps availability "we are still doing some telehealth and telephone visit as needed." "patients are offered telehealth appts & medication refill appts are also being offered via telehealth and telephone." "we have virtual visit capabilities for all patients with regular follow-up appointments if patients have computer and internet access at home. for ryan white patients, mcms will bring laptop and hot spot to the patient's home, get them online with provider and step out of the room…. we are using the polycom bridge system for virtual visits." "continuing to utilize telehealth options versus in-person visits where possible." "utilizing healow as much as possible. " healthcare provider availability "effective june 8, 2020: mcms working from home using remote desktop to access pe, email etc. and telephone extensions forwarded to their respective cell phones; mcms are rotating working "in office" at least two days a week (7 per day) providing services by appointment only. medical providers practicing telehealth as appropriate and seeing patients face-to-face as necessary. contract id provider seeing patients (face to face) in ad office, while 2 part-time providers are practicing telemedicine…. clinical cm "in office" to assist provider(s) 4 days/wk." "mcms are reaching out to clients who haven't picked up meds and offering medication delivery upon request. housing case manager & mcms continue to provide housing and referral services. our housing facility operating as usual; behavioral health provider returns to office full-time this week & continues to offer services by teleconference and face to face." "no office appts for mcm, all mcms working remotely from home, they vpns obtained from dhec for access to pe." "effective june 1st more staff will be called back into the office." "approximately half of the staff are back in the office.others are still working from home remotely to contact our patients for appointments and any immediate needs. " "most providers are now back to seeing patients in clinic along with virtual visits." "the front office will be open on a reduced schedule ah is the office manager on duty. this is to facilitate case management staff and medication pick up and other essential support services for clients. decisions going forward will be made daily/weekly as to our re-opening with full staffing. all staff available via email/phone we are working via remote." some clinics that reported minimal interruptions were in areas with the highest rate of covid-19 cases. the percentage of uninsured population was significantly different by hiv service interruption type (f = 3.987, p = .02), indicating a potential correlation between health insurance coverage and the hiv service interruption. post-hoc pairwise comparison (data not shown) suggested that the service catchment areas with no interruption in services had a lower percentage of uninsured than those areas with minimal service interruption, partial service interruption, and complete service interruption, although not all of these differences reached statistical significance. using mixed-method data collected from 27 hiv clinics in sc, the current study examined hiv service interruptions in sc during the covid-19 outbreak. we demonstrated geospatial pattern of the hiv service interruption, and explored the potential socioeconomic correlates with the service interruption. of our sample, nearly 82% of the hiv clinics were either partially interrupted (56%) or completely closed (26%) during the covid-19 outbreak. the covid-19 disrupted regular operation routine, reduced office hours, and decreased the availability of healthcare providers and staff in hiv clinics. although most of the clinics still provided core hiv services (e.g., medicine refilling, hiv testing), they had to suspend or cancel faceto-face counseling and social support group. geospatial heterogeneity in terms of hiv service interruption existed. however, the geospatial pattern of hiv service interruption did not exactly overlap with the density of confirmed covid-19 cases in the service catchment area. the areas with a higher insurance coverage (i.e., a lower percentage of uninsured population) tended to show less hiv service interruption. telehealth and mobile applications played an important role in retaining hiv services and delivering other supportive services during the covid-19 outbreak. our findings provide further empirical evidence for the value and need of telehealth as a convenient and inexpensive care option during disasters and public health crisis [22] [23] [24] . however, our qualitative study suggested that not all hiv clinics were able to make a timely shift to a telehealth system. four of the hiv clinics did not have capacity to support virtual visit for patients. in addition, many patients who did not have computer or internet access at home might not be able to benefit from telehealth, leading to a widening disparity in care. further implementation science studies are needed to explore the facilitators and barriers for scaling-up telehealth in responding to covid-19 pandemic and other public health emergencies. the geospatial distribution of plwh largely overlapped with the geospatial distribution of confirmed covid-19 cases through june 2020. this result suggests the importance to integrate the efforts of responding dual epidemics of hiv and covid-19. some hiv clinics reported that they provided covid-19 testing for their communities in partnership with sc dhec. these practices are consistent with the recent recommendation in the literature to take advantage of covid-19 contact tracing as a great opportunity for a more coverage of hiv testing [25] . future studies on needs assessment and infrastructure evaluation are warranted to explore the feasibility and acceptability of providing integrated covid-19 and hiv testing and services for local communities based on existing hiv testing program or infrastructures available at hiv clinics. our findings suggested a big variation across sc in extent of hiv service interruption with a range from no interruption to complete interruption of services. most of hiv clinics had adapted flexible strategies to deal with the crisis caused by covid-19, but they still showed difference in operational hours, healthcare provider availability, and scope of hiv services during the outbreak. this gap in responding to the public health crisis could not be fully explained by the covid-19 itself. existing socioeconomic disparities at county level might contribute to the gap in health service delivering. for example, the percentage of uninsured population in service catchment area was related to the service interruption of hiv clinics. we may need to further examine the characteristics of hiv clinics in terms of their financial situation, human resources, and infrastructure of telehealth to explore if and how existing variation in health infrastructure is associated with disparity of hiv service interruption. the disparity of hiv service interruption during a public health crisis such as covid-19 is an important issue for the healthcare systems and policy makers to consider as such interruption may further amplify the disparities in hiv treatment cascade in settings with a significant hiv burden or among various vulnerable populations. in the global context, there might be 500,000 additional deaths due to a 6-month interruption of art in africa [26] . in the united states, a boston health center reported that disruptions in pre-exposure prophylaxis (prep) care were more prominent among vulnerable population [27] . in maryland, a comprehensive patient and family centered hiv primary care was interrupted among racial, sexual and gender minority youth living with hiv, which might impede their medicine adherence and clinical improvement [28] . to prevent the further widening of the disparities in existing hiv care system, strengthening and empowering community partners of hiv clinics could be essential for more effective and accountable responses to public health emergencies [29] [30] [31] . the current study is subject to several limitations. first, qualitative data was collected within 2 months and reports of the hiv clinics were obtained in different dates. giving the rapidly evolving covid-19 pandemic, the information we collected might lag behind the changes made by the clinics in response to the pandemic. second, we were not able to quantify the hiv service interruption based on existing qualitative information. although we have identified five domains to assess service interruptions, lack of complete and systematic assessment prevented us from developing a numeric index to measure hiv service interruption. the categorization of hiv clinics by a medical expert and other team member might introduce subjective bias. third, we only included the ryan white hiv clinics in the analysis because of the availability of program reports. given the large state-wide coverage of ryan white programs in providing hiv service, the findings may serve as a good presentation of the hiv service interruption issues in sc. fourth, the service catchment areas of the hiv clinics were not mutually exclusive. thus, one county might be included in the calculation of factor variables for more than one hiv clinics. however, since the analysis was based on clinic level, we do not believe that the inclusion of same county level data for different clinics would pose a serious threat to the internal validity of the clinic-level analysis. finally, the small sample size of the quantitative study limited the power to detect statistically significant correlates. despite these limitations, the current study is one of the first efforts to examine the disparity of hiv service interruption and identified the socioeconomic correlates of this disparity. our findings show that the covid-19 pandemic added additional burdens to hiv care system in sc. a rapid shift to telehealth system and integration of hiv and covid-19 care might be effective strategies to respond to this public health crisis. however, existing socioeconomic disparity might contribute to the gap in hiv service interruption, leaving plwh in low ses communities more vulnerable. we call for more attentions to this disparity in hiv service delivery in both clinics' capacity building and the federal or state support to hiv service programs in fighting the covid-19 pandemic. world health organization. coronavirus disease (covid-19) pandemic 2020 world health organization. who director-general's opening remarks at the media briefing on covid-19-11 world health organization. coronavirus disease (covid-19) situation dashboard people of any age with underlying medical conditions 2020 peopl e-with-medic al-condi tions .html?cdc_aa_refva l=https %3a%2f%2fwww .cdc.gov%2fcor onavi rus%2f201 9-ncov%2fnee d-extra -preca ution s%2fgro ups-at-highe r-risk.html. 5. centers for disease control and prevention. covid-19: what people with hiv should know 2020 interim guidance for covid-19 and persons with hiv 2020 keep it going if you can": hiv service provision for priority populations during the covid-19 pandemic in hiv care continuum and covid-19 outcomes among people living with hiv during the covid-19 pandemic spatial disparities in coronavirus incidence and mortality in the united states: an ecological analysis as of south carolina department of health and environment control. sc testing data & projections (covid-19) covid-19 reported in all 46 counties across sc, 4 cases in laurens county ending the hiv epidemic: a plan for america an epidemiologic profile of hiv and aids in south carolina %20par t%20b%20ser vice%20pro vider s%20110 92018 .pdf. part c health resources and services administration what are the demographic characteristics of ryan white hiv/aids program clients in south carolina united states census bureau. small area income and poverty estimates. table 1: 2916 poverty adn median income estimates: counties table dp02: educational attainment for the population 25 years and over: counties american community survey 5-year estimates small area health insurance estimates, 2016: health insurance coverage status by age, race, hispanic origin, sex and income for counties and states grounded theory methodology in-person health care as option b virtually perfect? telemedicine for covid-19 the role of telehealth in the medical response to disasters contact tracing for covid-19: an opportunity to reduce health disparities and end the hiv/ aids epidemic in the us the potential impact of the covid-19 epidemic on hiv, tb and malaria in low-and middle-income countries living with hiv in the time of covid-19: a glimpse of hope addressing health inequities exacerbated by covid-19 among youth with hiv: expanding our toolkit covid-19 pandemic disrupts hiv continuum of care and prevention: implications for research and practice concerning community-based organizations and frontline providers prioritizing community partners and community hiv workers in the covid-19 pandemic the covid-19 and hiv: a tale of two pandemics key: cord-333655-lylt7qld authors: van breedam, wander; pöhlmann, stefan; favoreel, herman w.; de groot, raoul j.; nauwynck, hans j. title: bitter‐sweet symphony: glycan–lectin interactions in virus biology date: 2013-12-06 journal: fems microbiol rev doi: 10.1111/1574-6976.12052 sha: doc_id: 333655 cord_uid: lylt7qld glycans are carbohydrate modifications typically found on proteins or lipids, and can act as ligands for glycan‐binding proteins called lectins. glycans and lectins play crucial roles in the function of cells and organs, and in the immune system of animals and humans. viral pathogens use glycans and lectins that are encoded by their own or the host genome for their replication and spread. recent advances in glycobiological research indicate that glycans and lectins mediate key interactions at the virus‐host interface, controlling viral spread and/or activation of the immune system. this review reflects on glycan–lectin interactions in the context of viral infection and antiviral immunity. a short introduction illustrates the nature of glycans and lectins, and conveys the basic principles of their interactions. subsequently, examples are discussed highlighting specific glycan–lectin interactions and how they affect the progress of viral infections, either benefiting the host or the virus. moreover, glycan and lectin variability and their potential biological consequences are discussed. finally, the review outlines how recent advances in the glycan–lectin field might be transformed into promising new approaches to antiviral therapy. many emerging and re-emerging viral diseases in animals and humans pose significant global health problems for which novel antiviral measures are in urgent demand. in general, rational design of new prophylactic and curative antiviral strategies requires a detailed knowledge of the viral infection mechanism and the host's innate and adaptive immune defense. historically, cell biological and microbiological research was mainly focussed on the nucleic acid and protein level. however, over the last few decades it has become clear that also glycans account to a great extent for the structural and functional diversity displayed by animal and human cells and their pathogens. at this moment, glycobiology is one of the most rapidly expanding disciplines in biology. a rapidly evolving array of powerful, novel techniques for the analysis of glycan structure and function (glycomics) has prompted many scientists to apply these tools to the field of virology, and this glycovirological approach has yielded a wealth of information on the various glycobiological aspects of viral infection and antiviral immunity. particularly fascinating in this context are the many distinct glycan-lectin interactions that may occur during viral infection of a host. virion-associated glycans often serve as ligands for specific host lectins. conversely, the glycan portions of host glycoconjugates function as receptors for various viruses that employ viral lectins for host cell entry. this review reflects on glycan-lectin interactions in the context of viral infection and antiviral immunity. following a general introduction on glycan and lectin biology, specific glycan-lectin interactions are highlighted and discussed within the larger framework of viral infection and immunity. distinction is made between interactions that benefit the host and interactions that benefit the virus. in addition, different factors that contribute to glycan and lectin variationand that consequently affect glycan-lectin interactionsare explored and various approaches to modulate specific glycan-lectin interactions in antiviral therapies are briefly discussed. glycans like nucleic acids, proteins and lipids, glycans are essential components of the animal cell and organism. the term 'glycan' refers to the carbohydrate portion of glycoproteins and glycolipids typically found at cell surfaces, in extracellular matrices and in cell secretions. unlike nucleic acid and protein synthesis, the biosynthesis of glycans is not a template-driven process. instead, glycosylation depends on the concerted action of different glycosyltransferase, glycosidase, and other enzymes. several important glycan types are exclusively assembled by the enzyme sets present in the endoplasmic reticulum (er) and the golgi network: glycan/glycoconjugate synthesis is typically initiated in the er or early golgi and gradual processing and diversificationor 'maturation'occurs as these molecules move further through the different enzyme-equipped compartments of the secretory pathway. the variability inherent to glycan synthesis and maturation forms the basis of the considerable diversity and complexity of the glycan repertoires found on animal glycoconjugates (varki et al., 2009; taylor & drickamer, 2011) . according to the basic glycan 'core' structure, the type of molecule the glycans are linked with and the type of the linkage, glycans and glycoconjugates can be categorized in different classes. two major classes of proteinlinked glycosylation are the n-and o-linked glycans. n-linked glycans are covalently linked to the nitrogen atoms of specific amino acid (aa) residues (typically asparagine) via an n-glycosidic bond. n-acetylglucosamine-to-asparagine (glcnacb-asn) type glycans represent the most common form of n-linked protein glycosylation (weerapana & imperiali, 2006; varki et al., 2009; larkin & imperiali, 2011; schwarz & aebi, 2011; taylor & drickamer, 2011) . o-linked glycans are covalently attached to the oxygen atoms of specific amino acid residues (typically serine or threonine) via an o-glycosidic bond. a common o-glycan typeand potentially the one most studiedis the n-acetylgalactosamine-to-serine/threonine (galnaca-ser/thr) type or mucin-type o-glycan. other (nonmucin) o-glycan types include a-linked o-mannose, a-linked o-fucose, b-linked o-xylose, b-linked o-glcnac (n-acetylglucosamine), a-/b-linked o-galactose, and a-/b-linked o-glucose glycans (van den steen et al., 1998; peter-katalinic, 2005; varki et al., 2009; jensen et al., 2010; gill et al., 2011; taylor & drickamer, 2011) . interestingly, various proteins are also modified with glycosaminoglycans (gags), linear polysaccharide chains composed of repeated disaccharide subunits consisting of a uronic acid/galactose residue and an amino sugar. glycosaminoglycan-carrying glycoproteins are generally referred to as proteoglycans (prydz & dalen, 2000; varki et al., 2009; taylor & drickamer, 2011) . figure 1 illustrates the general structure and classification of some common types of protein glycosylation. similarly as for protein-linked glycosylation, different types of lipid-linked glycosylation can be discerned: the glycosphingolipids (varki et al., 2009; taylor & drickamer, 2011; yu et al., 2011) and the glycophospholipid anchors or glycosylphosphatidylinositol (gpi) anchors (paulick & bertozzi, 2008; varki et al., fig. 1 . classification and basic structure of common types of protein-linked glycosylation. (a) glcnacb-asn type n-linked glycans are covalently attached to the amide nitrogen atoms of asn side chains and are almost exclusively found on asn residues within the sequence asn-x-ser/thr, in which x can be any amino acid except pro. the nature of the glycan structures that decorate the common glycan corethe glycan part shown in a dashed boxdictates classification of n-linked glycans as high-mannose type, hybrid type or complex type glycans, examples of which are shown in the panel. (b) galnaca-ser/thr type o-linked glycans have a galnac residue a-linked to the oxygen atom of the hydroxyl group of ser or thr residues. unlike for glcnacb-asn type n-linked protein glycosylation, there are no clear amino acid motifs that mark these o-linked glycosylation sites. a single galnac residue linked to the ser/thr is termed the 'tn antigen'. depending on the basic structure of the glycan core, more complex (extended) o-linked glycans are categorized into different 'core types'. cores 1-4 are the most common core structures, but also other core types exist. the tn antigen and examples of extended core 1, 2, 3, and 4 o-glycans are shown in the panel. the distinct glycan cores are shown in dashed boxes. (c) glycosaminoglycans (gags) are linear polysaccharide chains composed of repeated disaccharide subunits of a uronic acid/galactose residue and an amino sugar. glycosaminoglycans are classified as hyaluronan (ha), heparan sulfate/heparin (hs), chondroitin sulfate (cs), dermatan sulfate (ds), or keratan sulfate (ks), depending on the structure of their basic disaccharide subunits (shown in square brackets) and further modification (e.g. sulfation at different positions) of the glycan chain. with exception of hyaluronan, all major glycosaminoglycan types are sulfated and occur covalently linked to proteins. hs, cs, and ds are found on ser-linked xylose residues. although no unambiguous consensus sequence for xylosylation exists, the ser attachment site is consistently flanked by a gly residue at its carboxyterminal side. as depicted in the figure, heparan sulfate and heparin have the same basic structure. although they share a common biosynthesis, heparin generally undergoes more extensive sulfation and epimerization of uronic acid to iduronic acid. moreover, heparin is synthesized only in connective tissue mast cells as part of serglycin proteoglycans, whereas heparan sulfate is synthesized in virtually all mammalian cells. ks is found on asn-linked n-glycan core structures (ks i) or ser/thr-linked o-glycan core 2 structures (ks ii). capping or further modification of the glycosaminoglycan chainssulfation exceptedis not depicted (adapted from varki et al., 2009 ). 2009; taylor & drickamer, 2011) represent important glycolipid classes. their basic structure is introduced in fig. 2 . despite their different core structures and linkages to carrier molecules, distinct glycan types can still share conserved structural characteristics as they often follow partially overlapping biosynthetic pathways. although some glycan features may be exclusively found in one specific glycan class, many (sub)terminal glycan modifications can be found in different glycan classes. common (sub)terminal modifications include poly-n-acetyllactosamine chains, abh and lewis histo-blood group antigens (hbga), and sialic acids in different linkages (varki et al., 2009; taylor & drickamer, 2011) . consequently, the glycan moieties of glycoproteins and glycolipids often have more in common than one would expect based on their core structure. apart from the different glycan types introduced here, several other forms of glycosylation exist. however, an in-depth discussion of these is beyond the scope of this review. for more detailed information on glycan structure and biosynthesis, readers may refer to recent reference works on this topic (varki et al., 2009; taylor & drickamer, 2011) . not only the animal and human hosts, but also their pathogens can benefit from the fine-tuned cellular biosynthetic pathways that govern glycosylation. this is most obvious in the case of obligatory intracellular pathogens such as viruses. glycans form an important part of the surface of many viruses. as the glycosylation of viral components is facilitated by the cellular glycosylation machinery, viral glycosylation is similar to that of the host. however, important differences have been noted: viral glycoproteins are often more heavily glycosylated than their cellular counterparts and the composition of individual glycan chains can diverge greatly between virus and host. the biological basis of this variability is further classification and basic structure of major types of lipid-linked glycosylation. (a) glycosphingolipids consist of a hydrophilic glycan moiety linked to a hydrophobic sphingolipid. in higher animals, a ceramide lipid molecule is initially modified with a b-linked glucose or galactose residue, after which further extension and modification of the glycan moiety can occur. extension to larger glycan chains is common on ceramide-linked glucose residues, whereas further glycan extension on ceramide-linked galactose residues is more rare. depending on their glycan core structure, glycosphingolipids are classified in 'series'. the figure depicts a number of glycosphingolipid core structures. the key features that characterize each series are shown in dashed boxes. core structures can be further modified with sialic acids or sulfate groups, which allows subclassification of glycosphingolipids as neutral (lacking charged carbohydrates or ionic groups), sialylated or sulfated. (b) glycosylphosphatidylinositol (gpi) anchors are found in association with certain membrane proteins and serve as linkers between the protein and the lipid membrane. glycosylphosphatidylinositol anchors have a common core structure comprising ethanolamine-po 4 -6mana1-2mana1-6mana1-4glcna1-6myo-inositol-1-po 4 -lipid. differential derivatization of this common core structure through lipid remodeling and modification of the glycan moiety can cause significant glycosylphosphatidylinositol anchor heterogeneity. the protein is linked to the glycosylphosphatidylinositol anchor via an amide linkage between the c-terminal carboxyl group of the protein and the amino group of phosphatidylethanolamine (adapted from varki et al., 2009). situated later in this review (see 'glycan and lectin variation at the virus level'). in line with the similarity between viral and host glycosylation, many of the basic functions covered by glycans in normal animal and human physiology and in viral infection biologywhich is intrinsically linked to the biology of the hostare essentially the same. glycans play important structural roles and are for instance implicated in protein folding and solubility, protease resistance, and masking of highly immunogenic protein stretches ('glycan shielding') (varki et al., 2009; taylor & drickamer, 2011) . alternatively, glycans can also have nonstructural roles and take part in specific recognition events, in which they usually interact with complementary glycan-binding proteins called lectins (varki et al., 2009; taylor & drickamer, 2011) . lectins may simply be defined as carbohydrate-binding proteins, although some definitions are more restrictive and exclude mono-/oligo-saccharide transport proteins, enzymes, glycan-specific antibodies, and even glycosaminoglycan-binding proteins (elgavish & shaanan, 1997; weis, 1997; loris, 2002; varki et al., 2009; gabius et al., 2011) . according to the more strict definitions, glycosaminoglycan-binding proteins and lectins are distinguished based on different factors, including their ligand range, the structural basis of their glycan recognition, and their conservation (varki et al., 2009) . in general, glycosaminoglycan-binding proteins interact with negatively charged glycosaminoglycans via clusters of positively charged aa residues andwith exception of hyaluronan-binding proteins, which seem to share an evolutionarily conserved fold do not appear to be evolutionarily related to each other (varki et al., 2009) . in contrast, most strict sense lectins belong to protein families with defined 'carbohydrate recognition domains' (crd). the crds within a lectin family share structural and functional properties and selectively recognize specific portions of n-glycans, o-glycans, or glycolipids (sometimes also glycosaminoglycans) (varki et al., 2009) . although some crds can efficiently bind monosaccharides, other crds show no apparent affinity for monosaccharides and favor oligosaccharide ligands. the latter crd type often has a preference for ligands with specific linkages between the monosaccharide subunits, as these linkages determine the 3d structure of the glycan ligand and therefore the portions of the glycan that are available for interaction with the crd. interactions between a single crd and a single mono-/ oligo-saccharide (affinity) are often weak, and strong interactions are usually the result of multivalent binding, i.e. the interaction of multiple crds with multiple ligands (avidity). whereas some lectins contain multiple crds that can participate in ligand binding, others contain only a single crd and rely on clustering of individual lectin molecules for high avidity binding. clustering of crds does not only allow stronger interactions with ligands, but also contributes to the specificity/selectivity of interactions at the multivalent level. the relative spacing of the crds allows highly avid, multivalent binding to specific saccharide ligands in a certain density and particular presentation. ultimately, the avidity of lectins for specific glycoconjugates depends on the structure, multivalency, and density of glycans on these molecules (elgavish & shaanan, 1997; weis, 1997; loris, 2002; varki et al., 2009; gabius et al., 2011) . animal lectins are typically expressed in a cell-and/or tissue-specific manner. they are involved in many different biological processes, including glycoprotein trafficking, cell adhesion and signaling, and their expression is usually tightly regulated (varki et al., 2009) . particularly striking is the great number of membrane-associated and soluble lectins that are linked with host immunity. immune system lectins are involved in intercellular communication, positive and/or negative regulation of activation, regulation of inflammation, disposal of damaged and apoptotic cells, etc. several of these lectins have also been identified as 'pattern recognition receptors' (prrs), which act as molecular sensors for pathogens and endogenous stress signals and often trigger specific immune reactions/mechanisms in response to their detection (gordon, 2002; janeway & medzhitov, 2002; cambi & figdor, 2003; mcgreal et al., 2004; cambi et al., 2005; mcgreal et al., 2005; crocker et al., 2007; crocker & redelinghuys, 2008; van kooyk & rabinovich, 2008; garcia-vallejo & van kooyk, 2009; geijtenbeek & gringhuis, 2009; sato et al., 2009; bottazzi et al., 2010; dam & brewer, 2010; kumagai & akira, 2010; svajger et al., 2010; davicino et al., 2011; osorio & reis e sousa, 2011; sancho & reis e sousa, 2012) . the capacity of many immune system lectins to couple glycan recognition events with specific signaling and/or effector functions gives them a key regulatory position in the immune system. figure 3 gives a schematic overview of different types of animal lectins that are considered in this review. lectins play a pivotal role in different aspects of the physiology, including the immune defense against (viral) pathogens. however, it has become apparent that several viruses exploit host lectins to promote their spread. in addition, many viruses encode lectins for the recognition of and infectious entry into target cells. the following section explores how glycan-lectin interactions shape the virus-host interplay, mainly focussing on glycan-lectin interactions directly involving infectious virions. glycan-lectin interactions that benefit the host lectins cover essential roles in the animal host's immune defense. importantly, several membrane-associated host (immune system) lectins act as pathogen recognition molecules: they can bind pathogens and activate signaling mechanisms or capture pathogens for subsequent degradation and presentation to cells of the adaptive immune system (e.g. mhcii-restricted presentation of antigens to t cells), resulting in the induction of a pathogen-specific adaptive immune response. alternatively, pathogens attached to such cell surface lectins may also be directly presented to neighboring immune cells in trans, a process that seems especially significant at sites with a high density of immune cells (e.g. the lymph nodes). hence, binding of a viral pathogen to membrane-associated (immune system) lectins can lead to its clearance and degradation. a prominent example is the interaction between the human immunodeficiency virus type 1 (hiv-1) and human langerin, a c-type lectin mainly expressed on langerhans cells (de witte et al., 2007; . de witte et al. (2007) reported that hiv-1 interacts with langerin via high-mannose glycans on the gp120 envelope protein and is subsequently internalized into birbeck granules, leading to virus degradation (de witte et al., 2007) . also dc-sign, a mannose-binding c-type lectin mainly expressed on dendritic cells (dcs), can bind and internalize hiv-1 virions for degradation and promotes mhcii-restricted as well as exogenous mhci-restricted presentation of hiv-1 antigens (moris et al., 2004 (moris et al., , 2006 . similarly, various other membrane-associated host lectins can aid as prrs in the defense against viral pathogens. however, growing evidence illustrates that many of these lectins, including dc-sign, are also abused by viruses to gain access to their target cells and facilitate viral spread, as is discussed further below. in contrast to the dual role played by different membrane-associated host lectins, soluble host lectins have mainly been associated with protection against viral infection. several soluble host lectins have been reported to aid in neutralization and clearance of various viral pathogens. table 1 gives an overview of membrane-associated and soluble host lectins that are linked with the host's defense against different viruses. current experimental data strongly implicate these host lectins in the defense against the listed viral pathogens and do not attribute explicit proviral effects to the host lectinunlike for the lectin-virus pairs listed further in table 2 . the basic principles of soluble lectin-mediated antiviral protection are most easily conveyed using some specific, wellcharacterized examples. for instance, various studies point out an important role of surfactant protein a (sp-a), surfactant protein d (sp-d), and mannose-binding lectin (mbl) in the defense against influenza a virus (iav) infection. sp-a, sp-d, and mbl are all soluble c-type lectins and share a similar basic structure: lectin monomersconsisting of an n-terminal cysteine-rich domain, a collagen-like domain, a coiled coil neck domain, and a c-terminal crdassemble into trimers which, under physiologic conditions, further multimerize via their n-termini to form typical cruciform-(sp-d) or bouquet-(sp-a and fig. 3 . schematic overview of different types of membrane-associated (a) and soluble (b) animal lectins that are considered in this review. the lectin domains are highlighted and listed in the key. c-type lectin/c-type lectin domain: lectins are classified as c-type lectins based on their ca 2+ -dependency and shared primary structure. in the c-type crd, a ca 2+ ion is directly involved in carbohydrate binding by making coordination bonds to both the crd surface and key hydroxyl groups of the carbohydrate. the c-type lectin family contains both membraneassociated (a.1) and soluble (b.1) lectins. the collectins are soluble c-type lectins characterized by the presence of collagen-like domains. r-type lectin domain: this term refers to a crd that is structurally similar to the crd in ricin, a toxin found in the plant ricinus communis. i-type lectin/ i-type lectin domain: i-type lectins are glycan-binding proteins that belong to the ig superfamily, but are not antibodies or t-cell receptors. the 'sialic acid-binding ig-like lectin (siglec)' family of membrane-associated lectins is currently the only well-characterized group of i-type lectins (a.2). ficolin: ficolins (b.2) are soluble lectins characterized by the presence of collagen-like domains and fibrinogen-like globular domains with a lectin activity. galectin/s-type lectin (domain): galectins (b.3) are soluble lectins that typically bind b-galactose-containing glycoconjugates and show primary structural homology in their crds. galectins were initially referred to as s-type lectins to reflect their sulfhydryl dependency, the presence of cysteine residues and their solubility; however, at present, not all identified galectins fit this initial description anymore. pentraxin/pentraxin domain: pentraxins (b.4) are characterized by the presence of pentraxin domains, which contain an eight amino acid long conserved 'pentraxin signature' (hxcxs/twxs, where x is any amino acid) and display an l-type (legume-type) lectin fold. sap is a soluble lectin that requires ca 2+ ions for carbohydrate ligand binding (adapted from fujita, 2002; varki et al., 2009; bottazzi et al., 2010) . (van de wetering et al., 2004; veldhuizen et al., 2011) . despite their similar structure, these lectins show distinct glycan ligand specificities (veldhuizen et al., 2011) and also their interaction with iav and their effects on infection and spread appear to differ. studies using distinct iav isolates indicate that sp-d and mbl bind mannose-rich glycans on the viral surface glycoproteins hemagglutinin and neuraminidase (a viral lectin and a viral glycosidase, involved in iav entry and release; see discussion on viral lectins) through their crds (malhotra et al., 1994; reading et al., 1997; kase et al., 1999; hartshorn et al., 2000; hillaire et al., 2011) . although sp-a may interact with some iav isolates in a similar manner (malhotra et al., 1994) , binding of this molecule to most of the iav variants tested to date appears not to involve the lectin activity of sp-a; in contrast, virus binding depends on the interaction of the viral hemagglutinin with a sialylated n-glycan on the sp-a crd (hartshorn et al., 1994; benne et al., 1995 benne et al., , 1997 hartshorn et al., 1997; van eijk et al., 2003; mikerov et al., 2008) . in other words: sp-d and mbl binding depend on viral glycosylation, whereas sp-a binding mainly depends on the specificity of the hemagglutinin, and this is mirrored in the spectrum of iav variants these lectins can effectively bind (hartshorn et al., 1994; malhotra et al., 1994; benne et al., 1995 benne et al., , 1997 hartshorn et al., 1997; reading et al., 1997; kase et al., 1999; hartshorn et al., 2000; van eijk et al., 2003; mikerov et al., 2008; hillaire et al., 2011) . interestingly, the porcine variant of sp-d appears to combine the above-mentioned iav-binding functionalities: this molecule may not only bind iav virions through interaction of its crd with high mannose glycans on the virion surface (similar to other sp-d molecules), but also through interaction of a sialylated glycan on the lateral surface of its crd with the iav hemagglutinin (similar to sp-a) and can therefore bind to a broader array of iav variants (van eijk et al., 2002 (van eijk et al., , 2003 (van eijk et al., , 2004 hillaire et al., 2011) . in vitro assays show that sp-a and sp-d can directly neutralize iav infectivity (benne et al., 1995; reading et al., 1997; hartshorn et al., 2000; van eijk et al., 2003; hawgood et al., 2004; hillaire et al., 2011) . both sp-a and sp-d inhibit the viral hemagglutinating activity that is required for iav attachment to target cells (hartshorn et al., 1994; malhotra et al., 1994; benne et al., 1995; hartshorn et al., 1996 hartshorn et al., , 1997 hartshorn et al., , 2000 van eijk et al., 2002 van eijk et al., , 2003 van eijk et al., , 2004 mikerov et al., 2008; hillaire et al., 2011) and sp-d was reported to inhibit the viral neuraminidase (reading et al., 1997; hillaire et al., 2011) . interestingly, both lectins also induce viral aggregation (hartshorn et al., 1994 (hartshorn et al., , 1996 (hartshorn et al., , 1997 van eijk et al., 2003) and function as potent opsonins. sp-a for instance was identified as an opsonin for iav phagocytosis by alveolar macrophages (benne et al., 1997) . moreover, sp-a and sp-d were shown to enhance iav binding to neutrophils (hartshorn et al., 1994 (hartshorn et al., , 1996 (hartshorn et al., , 1997 van eijk et al., 2003) and sp-d-iav complexes were found to internalize upon attachment to neutrophils (hartshorn et al., 1997; van eijk et al., 2003) . pre-incubation of iav with sp-a or sp-d also enhances the virus-induced h 2 o 2 responses in neutrophils (hartshorn et al., 1994 (hartshorn et al., , 1996 (hartshorn et al., , 1997 van eijk et al., 2003) and pre-incubation of the virus with sp-d can protect neutrophils from iav-induced deactivation (hartshorn et al., 1994 (hartshorn et al., , 1996 (hartshorn et al., , 1997 . mbl can counteract iav by roughly the same mechanisms as sp-d (hartshorn et al., 1993; anders et al., 1994; malhotra et al., 1994; hartshorn et al., 1996 hartshorn et al., , 1997 reading et al., 1997; kase et al., 1999) , although its ability to activate the complement cascade expands its capabilities (anders et al., 1994; reading et al., 1995; chang et al., 2010) . ligand binding by mbl (or alternatively ficolins) can lead to activation of mbl-associated serine proteases (masps) and initiate the complement cascade via the so-called lectin pathway (blue et al., 2004; bottazzi et al., 2010) . complement deposition on a virus may interfere directly with crucial steps in the viral infection process (e.g. receptor binding), but can also trigger complement receptor-mediated uptake of the pathogen into immune cells. in addition, for enveloped viruses, complement activation may result in membrane attack complex (mac) formation on the viral envelope and subsequent virolysis (blue et al., 2004; bottazzi et al., 2010) . in line with the available in vitro data, recent work with sp-a-, sp-d-, and mbl-knockout mice also confirmed the antiviral potential of these soluble lectins in vivo (levine et al., 2001 (levine et al., , 2002 zhang et al., 2002; li et al., 2002; hawgood et al., 2004; levine et al., 2004; kingma et al., 2006; chang et al., 2010) . noteworthy caveats regarding these in vivo studies are, however, that direct antiviral effects of these lectins can be hard to uncouple from othere.g. immune-regulatoryeffects and that mice do not represent natural hosts for iav. another interesting example of antiviral activity mediated by soluble host lectins was recently reported for nipah virus (niv): the physiologic, homodimeric form of the soluble lectin galectin-1 can inhibit niv envelope protein-mediated membrane fusion (levroney et al., 2005; garner et al., 2010) . niv encodes two viral membrane although capture of a virus by these lectins may have certain antiviral effects or promote the specific immunity against this pathogen, current experimental data suggest that the listed viruses may also employ these lectins to promote viral infection, spread or persistence. references in table 2 are listed in supporting information, data s2. dimt: discussed in main text. glycoproteinsthe attachment protein niv-g and the fusion protein niv-fthat mediate viral entry and direct the endothelial cell syncytia formation typically associated with niv infection (levroney et al., 2005; garner et al., 2010; lee & ataman, 2011) . binding of niv-g to cell surface receptors induces a conformational change in niv-f, thereby activating its fusogenic activity (levroney et al., 2005; garner et al., 2010; lee & ataman, 2011) . galectin-1 associates with glycans on the niv envelope proteins and interferes with the membrane fusion process in multiple ways (levroney et al., 2005; garner et al., 2010) . not only does galectin-1 binding directly inhibit the crucial conformational change in niv-f associated with membrane fusion, it also reduces the lateral mobility of niv-f and niv-g in the lipid membrane and consequently counteracts the physical separation of niv-f and niv-g that is essential for this conformation change (garner et al., 2010) . moreover, galectin-1 binding impedes endocytosis and maturation of the niv-f precursor niv-f 0 in infected cells (garner et al., 2010) , further illustrating the capacity of this lectin to block different stages of the viral infection/ replication process. in sum, soluble host lectins can counteract viral infection in various ways: they may directly neutralize virus by destabilizing or aggregating virions, interfere with crucial steps in the viral infection process (e.g. entry), and/or opsonize virus to facilitate uptake and degradation. upon virus binding, some soluble lectins can trigger complement deposition on the virus, which may inhibit viral infection, enhance viral uptake via complement receptors and subsequent degradation in immune cells, and/or cause virolysis. considering the above, it is clear that lectins contribute significantly to the host's antiviral defense. host lectins are involved in neutralization and clearance of free virus, immune regulation, andalthough not extensively discussed in this overviewthe detection and clearance of virus-infected cells. figure 4 illustrates how membraneassociated and soluble host lectins can aid in antiviral defense. like their animal hosts, also viruses can benefit from interactions between glycans and glycan-binding proteins. for instance, many viruses, including hiv-1, herpes simplex virus-1, and dengue virus, have been shown to interact with glycosaminoglycan molecules present on target cells (patel et al., 1993; chen et al., 1997; krusat & streckert, 1997; summerford & samulski, 1998; dechecchi et al., 2000 dechecchi et al., , 2001 vanderheijden et al., 2001; delputte et al., 2002; trybala et al., 2002) . viral association with glycosaminoglycans is usually attributed to charge-based attractions between clusters of positively charged aa residues on the virion surface and the negatively charged glycosaminoglycan chains. the interaction with glycosaminoglycans often constitutes the first contact between a virus and its target cell and typically increases infection efficiency. however, it has been documented for several viruses that the ability to interact with glycosaminoglycans can result from adaptation to growth in cell culture (sa-carvalho et al., 1997; klimstra et al., 1998; hulst et al., 2000; mandl et al., 2001) . clearly, use of primary virus isolates is of crucial importance when assessing the occurrence and relevance of such interactions in vivo. aside from potential glycosaminoglycan-binding capacity, several viruses are endowed with a true lectin activity: they carry virally encoded lectins on their surface and use these as keys to gain entry into their target cells (fig. 5a ). similar to animal lectins, such virally encoded lectins often possess characteristic glycan binding regions ('glycan binding pockets') and recognize specific portions of protein-and lipid-linked glycans. the hemagglutinin protein of iav is generally regarded as the prototype of a viral lectin. hemagglutinin is a membrane glycoprotein that forms noncovalently linked fig. 4 . schematic overview of how membrane-associated (a) and soluble (b) host lectins are implicated in antiviral defense. (a.1) binding of virion-associated glycans with membrane-associated host lectins can lead to virus uptake, degradation, and presentation of viral antigens to cells of the adaptive immune system. binding may trigger specific signaling that promotes an effective antiviral immunity. (a.2) binding of virionassociated glycans with membrane-associated host lectins may promote direct presentation of the virus to immune cells in trans. binding may trigger specific signaling that promotes an effective antiviral immunity. (b.1) binding of soluble host lectins to virion-associated glycans may interfere directly with viral infection by destabilizing virions, blocking interaction of the virus with its receptors or interfering with other crucial steps in the infection process (e.g. membrane fusion). soluble host lectins may also aggregate virions, which often negatively impacts viral infectivity (not depicted). (b.2) soluble host lectins can act as opsonins: lectin binding to virion-associated glycans may facilitate viral uptake in immune cells via lectin receptors, leading to viral degradation and potential presentation of viral antigens to cells of the adaptive immune system. lectin binding may also trigger complement deposition on the virus (through the lectin pathway) and facilitate viral uptake via complement receptors. (b.3) detection of virion-associated glycans by soluble host lectins may trigger complement deposition on the virus (through the lectin pathway), which may directly inhibit viral infection and/or elicit lysis of the (enveloped) virus. homotrimers in the (viral) membrane and is responsible for both virus attachment and penetration (skehel & wiley, 2000; harrison, 2008; gamblin & skehel, 2010) . the mature hemagglutinin protein consists of two disulfide-linked subunits, termed ha1 and ha2 (skehel & wiley, 2000; harrison, 2008; gamblin & skehel, 2010) . the ha1 subunit forms the globular 'head' region of hemagglutinin that covers the lectin function of this molecule: sialic acid-binding pockets in the membrane distal part of ha1 allow interaction with sialic acidcontaining receptors on target cells (skehel & wiley, 2000; harrison, 2008; gamblin & skehel, 2010) . variation in the ha1 subunit determines the affinity and specificity (e.g. a2-3vs. a2-6-linked sialic acids) of this molecule (skehel & wiley, 2000; gamblin & skehel, 2010) . as seen for animal lectins, also hemagglutinin binds with its glycan counterparts with a relatively low affinity and efficient virus attachment and entry depends on the interaction of multiple hemagglutinin molecules with multiple sialic acid-containing receptors (skehel & wiley, 2000; gamblin & skehel, 2010) . the stalk-like ha2 subunit of hemagglutinin mediates the ph-dependent fusion process upon internalization of the iav virion in the endosomal compartment of the target cell (skehel & wiley, 2000; harrison, 2008; gamblin & skehel, 2010) . similar to iav, various other enveloped [e.g. influenza b and c viruses (nakada et al., 1984; herrler et al., 1985a; rogers et al., 1986; vlasak et al., 1987; herrler et al., 1988; herrler & klenk, 1991; rosenthal et al., 1998; lamb & krug, 2001; suzuki & nei, 2002; wang et al., 2007b; wang et al., 2008a) , mumps virus (bowden et al., 2010; harrison et al., 2010; chang & dutch, 2012) ] as well as nonenveloped [e.g. murine norovirus (taube et al., 2009 (taube et al., , 2012 , feline calicivirus (stuart & brown, 2007) , and rhesus rotavirus (dormitzer et al., 2002) , among others (taube et al., 2010) ] viruses employ sialic acid-binding viral lectins to infect target cells. importantly however, viral lectins with a different glycan specificity have also been identified. for instance, many virusesincluding human noroviruses (estes et al., 2006; le pendu et al., 2006; cao et al., 2007; bu et al., 2008; choi et al., 2008; donaldson et al., 2008; shirato, 2011) , rabbit hemorrhagic disease viruses (ruvoen-clouet et al., 2000; rademacher et al., 2008; guillon et al., 2009; nystrom et al., 2011) , and the rhesus monkey tulane virus (farkas et al., 2010) were found to interact with specific hbga types. although a broad spectrum of viruses has evolved to use viral lectins to secure efficient target cell infection, the use of viral lectins for cellular attachment comes with a price. the glycan receptors for viral lectins are not necessarily target cell-specific and, whereas low affinity/avidity interactions may be reversible, high affinity/avidity binding of viral lectins to nontarget cell-associated glycoconjugates ('decoy receptors') can prevent the virus from efficiently targeting susceptible host cells. in line with this, it was shown for iav that interaction of the viral lectin hemagglutinin with soluble, sialylated host glycoproteins e.g. sp-a (cfr. supra) or a2-macroglobulincan interfere with the viral hemagglutinating activity that is crucial for receptor binding (rogers et al., 1983; pritchett & paulson, 1989; ryan-poirier & kawaoka, 1991; matrosovich et al., 1992; ryan-poirier & kawaoka, 1993; hartshorn et al., 1994; malhotra et al., 1994; benne et al., 1995; gimsa et al., 1996; benne et al., 1997; hartshorn et al., 1997; matrosovich et al., 1998; van eijk et al., 2003; mikerov et al., 2008; chen et al., 2010; cwach et al., 2012) . although the presence of 'high avidity' glycan decoys invariably puts a strain on viral infection efficiency, this burden may be lighter on viruses that are equipped with a receptor destroying enzyme (rde) that matches the specificity of the viral lectin. intriguingly, the best-known examples in this context are again influenza viruses. as situated above, both influenza a and b viruses display hemagglutinin proteins on their surface, which bind to sialic acids displayed on the host cell surface and mediate ph-dependent fusion of the viral membrane with the host cell membrane (skehel & wiley, 2000; lamb & krug, 2001; wang et al., 2007b; wang et al., 2008a; harrison, 2008; gamblin & skehel, 2010 ). an rde activity was mapped to another viral membrane glycoprotein, designated neuraminidase (gottschalk, 1957; colman, 1994; fig. 5. (a) illustrates how viral lectins promote target cell infection. (b) shows how many viruses that employ viral lectins also benefit from a matching receptor-destroying enzyme (rde) activity, which provides a counterweight against (high avidity) lectin activity. (a) interaction of viral lectins with glycosylated receptors on a target cell promotes viral entry and infection (attachment/internalization/fusion, depending on specific virus biology). (b) although they clearly benefit the virus, the use of (high avidity) viral lectins comes with a price. for instance, viral lectin activity can cause virions to aggregate (b.1) and can impair efficient release of newly formed virions from (glycosylated) infected cells (b.2). moreover, binding of viral lectins to nontarget cell-associated glycoconjugates (decoy receptors) can prevent the virus from efficiently targeting susceptible host cells (b.3). intriguingly, several lectin-carrying viruses are also equipped with an rde that matches the specificity of the viral lectin and provides a counterweight against lectin-mediated glycan binding. in fact, for viruses equipped with both viral lectins and rdes, a functional balance between these molecules appears to be an important determinant of the viral (replicative) fitness. lamb & krug, 2001; gamblin & skehel, 2010) . this enzyme removes sialic acid moieties from glycoproteins and glycolipids by catalyzing the hydrolysis of the a-ketosidic linkage to the subterminal sugar residue and consequently destroys potential receptors for the viral hemagglutinin (gottschalk, 1957; colman, 1994; lamb & krug, 2001; gamblin & skehel, 2010) . viral use of an enzyme that can actually destroy receptors for the virus may seem peculiar at first. importantly, however, the neuraminidase activity can prevent virions from aggregating via hemagglutinin-sialic acid interactions, promotes efficient release of newly formed virions from (sialic acidcarrying) infected cells, and provides a counterweight to the interaction of hemagglutinin molecules with nontarget cell-associated glycans: neuraminidase-mediated removal of sialic acids from decoy receptors prevents virions from establishing high-avidity interactions with these glycoconjugates and may even provide an escape route for virions after hemagglutinin-mediated binding to nontarget cellassociated glycans (colman, 1994; suzuki et al., 1994; gimsa et al., 1996; barrere et al., 1997; gamblin & skehel, 2010) . conceivably, a functional balance between the hemagglutinin and neuraminidase activities is an important determinant of the (replicative) fitness of iav variants in vivo. using iav as a paradigm, fig. 5b illustrates how viral rde activity can balance the high avidity of viral lectins where favorable and consequently improve viral infection efficiency. in contrast to influenza a and b viruses, other viruses combine both lectin and rde functions in one protein complex. for example, the viral membranes of mumps virus, newcastle disease virus, sendai virus, and human parainfluenza virus 3 and 5 are studded with hemagglutinin-neuraminidase proteins (bowden et al., 2010; harrison et al., 2010; chang & dutch, 2012) . another example is the influenza c virus, which carries the hemagglutination, rde and fusion protein functions in one single envelope protein named the hemagglutininesterase-fusion protein (nakada et al., 1984; herrler et al., 1985a, b; rogers et al., 1986; vlasak et al., 1987; herrler et al., 1988; schauer et al., 1988; herrler & klenk, 1991; rosenthal et al., 1998; pekosz & lamb, 1999; lamb & krug, 2001; suzuki & nei, 2002) . whereas the rde of influenza a and b viruses is a neuraminidase, which cleaves off entire sialic acid residues, the rde of influenza c functions as a sialate-o-acetylesterase and cleaves off specific o-acetyl groups (herrler et al., 1985b; vlasak et al., 1987; herrler et al., 1988; schauer et al., 1988; pekosz & lamb, 1999) . a similar situation is seen for certain coronaviruses and toroviruses that carry an accessory protein called hemagglutinin-esterase on their surface (de groot, 2006) . as the name implies, functional hemagglutinin-esterase proteins combine a hemagglutinating activity with a sialate-o-acetylesterase activity (de groot, 2006) . interestingly, for some coronaviruses, the hemagglutinin-esterase protein is not the only envelope protein endowed with a lectin activity. for example, the spike proteins of bovine coronavirus and human coronavirus oc43 have been shown to be potent sialic acid-binding lectins kunkel & herrler, 1993) . also the spike protein of the transmissible gastroenteritis coronavirus of pigs has been shown to possess such a lectin activity (schultze et al., 1996; krempl et al., 1997 krempl et al., , 2000 schwegmann-wessels et al., 2011) . however, transmissible gastroenteritis coronavirus has no hemagglutinin-esterase protein that serves as rde to counteract the hemagglutinating activity of the spike protein. viruses do not only benefit from virally encoded lectins, but can also use host lectins to their advantage. paradoxically, many of the host lectins that are exploited by viruses form part of the immune system. although capture of viral pathogens by these lectins may have certain antiviral effects or promote the specific immunity against this pathogen (cfr. supra), many viruses can also employ such interactions to promote efficient infection and spread or to facilitate persistence (table 2) . virus binding to membrane-associated lectins can lead to concentration of virions at the cell surface and can facilitate infection of target cells. in many cases, host lectins appear to function as true portals for viral entry: the virus binds to the lectin, which drives subsequent internalization of the virus into specific cellular compartments from which the virus can initiate the next stage of infection. however, it has also been shown that lectins present on non-target cells may facilitate infection of target cells, a process called trans-infection. many membrane-associated (immune system) lectins also participate in specific signaling pathways and engagement of such lectins by a virus may modulate both viral infection and the immune response in favor of the pathogen. an immune system lectin that can be used as a paradigm in this context is dc-sign. this molecule is mainly expressed on dendritic cells (dcs), but expression on distinct other cell-typesincluding macrophages, b lymphocytes, platelets, and (immortalized) podocyteshas also been described (geijtenbeek et al., 2000a, b; soilleux et al., 2002; granelli-piperno et al., 2005; gurney et al., 2005; chaipan et al., 2006; rappocciolo et al., 2006; mikulak et al., 2010; svajger et al., 2010) . prototypic dc-sign molecules consist of a c-terminal c-type crd, a neck region made up of 7 and a half 23 aa residue repeats, a transmembrane domain, and a cytoplasmic domain containing motifs involved in receptor internalization and signaling (svajger et al., 2010; tsegaye & pohlmann, 2010) . lectin monomers typically multimerize via their neck regions to form tetramers, which in turn organize in nanoclusters on the cell membrane (svajger et al., 2010; tsegaye & pohlmann, 2010; manzo et al., 2012) . dc-sign functions as a receptor for various ligands. the t cell-expressed molecule icam-3 is probably its most prominent endogenous ligand: dc-sign binds icam-3 on the t cell surface and thereby contributes to the transient, nonantigen-specific interaction of dc with t cells that is required for efficient screening of mhcii-peptide complexes and eventual t cell priming (geijtenbeek et al., 2000a; svajger et al., 2010) . moreover, dc-sign has been implicated in various other processes, including dc differentiation, migration, and antigen capture (svajger et al., 2010) . over the last decade, it has become apparent that dc-sign interacts with a wide variety of viral pathogens. a textbook example of a virus that recruits dc-sign is hiv-1 (lekkerkerker et al., 2006; wu & kewalramani, 2006; piguet & steinman, 2007; tsegaye & pohlmann, 2010; da silva et al., 2011; van der vlist et al., 2011) . dc-sign can bind the enveloped hiv-1 particle and mainly recognizes mannose-rich glycans on the viral envelope glycoprotein gp120 (curtis et al., 1992; geijtenbeek et al., 2000a, b; feinberg et al., 2001; hong et al., 2002; lin et al., 2003; su et al., 2004; hong et al., 2007) . the efficiency of hiv-1 capture by dc-sign has been linked to receptor density. experiments in 293 t-rex cells using an inducible dc-sign expression system have shown that high surface expression levels of dc-sign correlate with optimal binding of hiv-1 particles, and that lowering the dc-sign expression levels can significantly reduce the efficiency of hiv-1 binding . these data suggest that the high dc-sign surface expression levels on certain (immature) dc subsets are compatible with optimal capture of hiv-1 virions, whereas lower dc-sign expression levels on b lymphocytes and especially platelets may be mirrored in a less efficient hiv-1 capture (baribaud et al., 2002; boukour et al., 2006; chaipan et al., 2006; rappocciolo et al., 2006) . nevertheless, studies have shown that also b lymphocytes and platelets can effectively bind hiv-1 virions via dc-sign (boukour et al., 2006; chaipan et al., 2006; rappocciolo et al., 2006) . evidently, the interaction between hiv-1 and dc-sign is also critically dependent on the viral glycome. recent research has shown that virion-associated gp120 of peripheral blood mononuclear cell (pbmc)grown virus predominantly carries oligomannose n-glycans (doores et al., 2010; bonomelli et al., 2011) , which constitute optimal ligands for dc-sign (van liempt et al., 2006) . nevertheless, virus originating from different host cell types can display a different glycosylation profile, and this may modulate the efficiency of dc-sign recruitment (lin et al., 2003) . binding of hiv-1 to dc-sign can entail both negative and positive effects for the virus. in dc-signexpressing antigen-presenting cells, most of the dc-sign-captured virions appear to be internalized into the endolysosomal pathway and rapidly degraded (moris et al., 2004; turville et al., 2004) . in line with this, dc-sign was found to promote mhcii-restricted presentation of hiv-1 antigens (moris et al., 2006) . intriguingly however, in cells that co-express cd4 and ccr5/ cxcr4 (hiv-1 receptor and co-receptors, respectively), dc-sign expression also facilitates hiv-1 fusion: dc-sign efficiently captures and concentrates viral particles at the cell surface, and binding of this lectin to the hiv-1 envelope protein appears to increase exposure of the cd4 binding site nobile et al., 2005; burleigh et al., 2006; hijazi et al., 2011) . although dc-sign-mediated enhancement of hiv-1 fusion may promote mhci-restricted presentation of hiv-1 antigen (proteasome and tap-dependent pathway) and activation of cytotoxic t lymphocytes (moris et al., 2004) , enhanced hiv fusion inevitably leads to more efficient infection. indeed, several studies confirm that dc-sign facilitates productive (cis-) infection in dc-signexpressing cells that also co-express cd4 and ccr5/ cxcr4 nobile et al., 2005; burleigh et al., 2006; hijazi et al., 2011) . dc-sign has also been implicated in hiv-1 transinfection. an initial study showed that dc-sign can efficiently capture hiv-1 particles and transfer them to adjacent target t cells, without the need for productive infection of the dc-sign-expressing cell (geijtenbeek et al., 2000b) . subsequent studies on the subject reported dc-sign-mediated internalization of infectious hiv-1 virions into low ph nonlysosomal compartments, and advocated that the virus traffics in intracellular compartments towards the zone of t cell contact, where it is released into the infectious synapse (i.e. the contact zone between the virus-loaded cell and the target t cell) (kwon et al., 2002; mcdonald et al., 2003) . dc-captured hiv-1 virions as well as t-cellexpressed cd4 and ccr5/cxcr4 were found to concentrate at the dc-t-cell interface, rendering it an ideal micro-environment for efficient infection of target t cells (mcdonald et al., 2003) . moreover, it was postulated that dc-sign-mediated capture of hiv-1 virions temporarily protects them from degradation and preserves viral infectivity (geijtenbeek et al., 2000b; kwon et al., 2002) . the above data supporting dc-sign-mediated capture, uptake, intracellular transport and ultimately transfer of intact hiv-1 particles from dcs to target t cells were united in the 'trojan horse model' of mucosal hiv-1 transmission. this model posits that submucosal dcs capture and internalize hiv-1 virions via dc-sign and, by homing to the lymph nodes, provide a means of transport for the virus to a compartment rich in target cells. the virus-loaded dcs then interact with cd4 + t cells and the virions are transferred to the target t cell via the infectious synapse, ultimately resulting in efficient target cell infection (geijtenbeek et al., 2000b; baribaud et al., 2001; sewell & price, 2001) . however, recent research is not always in line with this initial model and has challenged several of its key features. cavrois et al. (2007) reported that hiv-1 trans-infection does not require intracellular virus trafficking, but primarily depends on cell surface-associated virions that reach the infectious synapses via transport on the cell surface (cavrois et al., 2007) . in line with this, yu et al. (2008) reported that hiv-1 traffics towards the infectious synapse through a specialized, surface-accessible intracellular compartment (yu et al., 2008) . in addition, reports stating that virus capture by dc-sign mainly leads to virus internalization into the endolysosomal pathway and subsequent degradation (moris et al., 2004; turville et al., 2004; moris et al., 2006) and that dc-sign-mediated trans-infection can only occur within the first hours after virus attachment (turville et al., 2004) seem to downplay the importance of dc-sign-mediated trans-infection for efficient hiv-1 infection and spread. these and other data counter the theory that hiv-1 capture by dcs preserves viral infectivity, and suggest that the presenceand transferof infectious virus at later time points may be ascribed to productive dc infection (turville et al., 2004; nobile et al., 2005; burleigh et al., 2006; wang et al., 2007a) . it is also noteworthy that, whereas initial studies identified dc-sign as the main factor involved in hiv-1 capture and transmission by dcs (geijtenbeek et al., 2000b) , recent studies also implicate other lectins in this process (turville et al., 2002; izquierdo-useros et al., 2012) or even conclude that dc-sign is not involved in dc-mediated hiv-1 trans-infection (boggiano et al., 2007) . differences in virus strains, cell types, and experimental setup might in part explain these conflicting data. intriguingly, other recent work indicates that dc-sign-expressing b lymphocytes and platelets may effectively capture and transfer infectious hiv-1 via dc-sign (boukour et al., 2006; chaipan et al., 2006; rappocciolo et al., 2006) , potentially implicating these cells/cell fragments in hiv-1 dissemination in infected patients, although recent work suggests that platelets might negatively regulate viral spread by secretion of cxcl4 (auerbach et al., 2012; tsegaye et al., 2013) . clearly, further research is necessary to allow a better understanding of dc-sign-mediated hiv-1 trans-infection and its relevance for viral infection and spread in vivo. importantly, the role of dc-sign in hiv-1 infection appears not to be restricted to purely physical capture of virions for subsequent degradation or cis-or transinfection. recruitment of dc-sign by hiv-1 also triggers signal transduction that modulates immune responses and infection of dcs and adjacent target cells more indirectly. for example, hodges et al. (2007) reported that binding of hiv-1 to dc-sign compromises dc maturation and primes these cells for trans-infection: upregulation of cd86 and mhcii is suppressed, whereas synapse formation between dcs and cd4 + t cells is promoted (hodges et al., 2007) . moreover, hiv-1 binding to dc-sign was shown to activate cdc42 and promote formation of membrane extensions that facilitate hiv-1 transfer to cd4 + lymphocytes (nikolic et al., 2011) . other work by gringhuis et al. (2007 gringhuis et al. ( , 2010 showed that binding of hiv-1 to dc-sign triggers raf-1 dependent signaling, which modulates toll-like receptor (tlr)-elicited signals to induce synthesis of fulllength hiv transcripts as well as production of the immunosuppressive cytokine il-10 ( gringhuis et al., (2007 gringhuis et al., ( , 2010 . in general, the data discussed above suggest that dc-sign recruitment by hiv-1 might affect viral infection and transmission, as well as the host defense against this pathogen in several ways. over the last decade, dc-sign has become a prototype for lectin-mediated cis-and trans-infection and has been implicated in the infection process of various viruses, including hiv, dengue virus, ebola virus, and iav (see table 2 ). importantly, however, dc-sign is not the only host lectin that is (ab)used by viruses to promote target cell infection or avoid immune recognition and clearance. various other membrane-associated host lectins seem to be exploited by virusesin ways similar to dc-signto aid cis-infection, trans-infection and/or viral persistence. analysis of recent literature suggests that membrane-associated host lectins may constitute weak links in the host's defense against viral pathogens (table 2) . although generally implicated in antiviral defense, soluble host lectins may also support viral infection (table 2) . for example, galectin-1 has been proposed to promote hiv-1 infection (ouellet et al., 2005; mercier et al., 2008; st-pierre et al., 2011; sato et al., 2012) . in vitro experiments pointed out that galectin-1 can enhance hiv-1 infection of different cell typesincluding human lymphoid cell lines, pbmc, cd4 + t lymphocytes, and monocyte-derived macrophages (ouellet et al., 2005; mercier et al., 2008) and increase hiv-1 infection in an ex vivo lymphoid tissue model (ouellet et al., 2005) . further experiments showed that galectin-1 accelerates virion binding to the target cell surface, probably by crosslinking viral and cellular glycans (ouellet et al., 2005; mercier et al., 2008) . a more recent study confirmed these findings and showed that galectin-1 binds to clusters of n-linked glycans on the viral gp120 envelope protein in a b-galactoside-dependent manner (st-pierre et al., 2011) . data from the same study identify the hiv-1 receptor cd4 as a ligand for galectin-1 and suggest that galectin-1 can cross-link gp120 and cd4 (st-pierre et al., 2011) . in sum, it appears that the dimeric lectin galectin-1 can enhance hiv-1 infection efficiency by cross-linking viral and host cell glycans and thereby promoting firmer adhesion of the virus to the target cell surface and facilitating virus-receptor interactions (ouellet et al., 2005; mercier et al., 2008; st-pierre et al., 2011; sato et al., 2012) . some studies have also attributed proviral effects to the collectins mbl, sp-a, and sp-d. for some viruses, it was reported thatunder certain conditionsviral recognition by collectins may enhance cis-or trans-infection (hickling et al., 2000; sano et al., 2003; gaiha et al., 2008; brudner et al., 2013; madsen et al., 2013) . it is conceivable that these collectins can bind the virus and subsequently associate with collectin receptors on the surface of target/ transmitting cells, thereby concentrating virions at the cell surface and facilitating infection or viral transfer. nevertheless, involvement of other mechanisms (e.g. collectinmediated cross-linking of virus-and host cell-displayed glycans, cfr. the galectin-1-hiv-1 example described above) can currently not be excluded. further research is necessary to elucidate the biology behind the potential proviral effects of collectins and to estimate the occurrence and relevance of these events in an in vivo context. figure 6 gives a schematic overview of how membraneassociated and soluble host lectins can be implicated in interactions that benefit the virus and facilitate viral infection and spread. as glycan-lectin interactions often represent key events in viral infection and/or antiviral immunity, variation in glycan or lectin expression and structureeither at the host or at the virus levelmay significantly shift the balance between host and pathogen. a basic insight into the nature and origin of this variability is therefore germane to a proper understanding of glycan-lectin interactions in the context of viral infection biology and immunology. glycan formation is a very complex and versatile biosynthetic process. in contrast to the primary amino acid sequences of proteins, glycan structures are not directly encoded in the host genome. instead, they are synthesized in a step-wise manner via the concerted action of various host-encoded glycosyltransferase, glycosidase, and other enzymes. the availability of these glycoenzymes, the availability of precursor molecules and the accessibility of specific glycosylation sites govern (the efficiency of) glycan addition and modification and hence determine glycan variability. the genetic make-up of the host evidently has a major impact on glycosylation, but also other host-related factors can have pronounced effects. recent research has shown that different cell types within a host can assemble radically different glycomes (roth, 1996; haslam et al., 2008) and that factors such as the activation (comelli et al., 2006; bax et al., 2007; haslam et al., 2008) or infection (lanteri et al., 2003) status of a cell can significantly influence glycosylation processes. clearly, various biological factors contribute to the high glycan heterogeneity that is seen for many animal glycoconjugates. although host glycan variation may influence virtually all viral infections in several ways, its potential impact is probably most evident for viruses that are equipped with viral lectins. a notable example in this context are the noroviruses, a major cause of nonbacterial gastroenteritis in humans. it is well known that the viral capsids of most human noroviruses display an affinity for hbgas, structurally related but highly polymorphic carbohydrate structures found on proteins and lipids of epithelial cells in the gastrointestinal and respiratory tract, on the surfaces of red blood cells and as free antigens in body fluids such as saliva, blood, and intestinal contents (bu et al., 2008; choi et al., 2008; shirato, 2011) . different noroviruses display distinct hbga specificities and can be categorized according to the (range of) hbga structures they preferentially bind shirato, 2011) . human hbga synthesis is controlled by various enzymes, including the glycosyltransferase enzymes encoded in the abo, fut2, and fut3 gene loci. the presence of variant (functional or nonfunctional) alleles at these and other relevant gene loci is a key determinant of hbga phenotype, as it controls which abh and lewis antigens an individual can synthesize (le pendu et al., 2006; shirato, 2011) . although it is still unclear whether they function as primary receptors for noroviruses, current data indicate that hbgas are important determinants of the noroviral tissue specificity. moreover, several studies have established a link between hbga geno-/ phenotype and individual susceptibility to (clinical) infection with specific norovirus variants: hbga phenotypes matching the specificity of the viral lectin correlate with a higher risk of (clinical) infection, whereas nonmatching hbga phenotypes correlate with relative resistance (le pendu et al., 2006; shirato, 2011) . several studies have revealed significant heterogeneity relating to animal lectins. lectin expression is governed by various genetic and nongenetic (e.g. hormone balance, immune status) factors. importantly, gene polymorphisms that affect protein expression and/or functionality have been described for several animal lectins, including mbl and dc-sign. for mbl, mutations in the promoter region of the mbl2 gene were found to affect protein expression levels, probably by influencing binding of transcription factors (eisen & minchinton, 2003; dommett et al., 2006; heitzeneder et al., 2012) . moreover, specific polymorphisms in mbl2 exon 1, encoding the collagen-like domain of mbl, appear to hinder correct and stable oligomerization of mbl protein chains and impede efficient ligand binding and activation of the lectin complement pathway (eisen & minchinton, 2003; dommett et al., 2006; heitzeneder et al., 2012) . several studies suggest a correlation between mbl deficiency and susceptibility to hiv infection, but conflicting data have been reported and further research is clearly necessary to corroborate this link (eisen & minchinton, 2003; dommett et al., 2006; heitzeneder et al., 2012) . similar findings have been recorded for dc-sign. polymorphisms in the promoter region of the dc-signencoding cd209 gene can affect protein expression levels and have been linked with altered susceptibility to and/or altered disease progression after infection with several viral pathogens, including hiv-1 and dengue virus (martin et al., 2004; sakuntabhai et al., 2005; koizumi et al., 2007; selvaraj et al., 2009; wang et al., 2011; boily-larouche et al., 2012) . moreover, distinct gene polymorphisms in the dc-sign-encoding region as well as alternative splicing events give rise to different isoforms of the protein, ranging from variants containing single nucleotide polymorphisms (snps) to variants with truncated lectin domains, variable numbers of 23-aa-residue repeats in the neck domain, alternative cytoplasmic domains or a lacking transmembrane region (mummidi et al., 2001; liu et al., 2004; serrano-gomez et al., 2008; boily-larouche et al., 2012) . information on the expression and biological activity of most of these dc-sign variants is rather limited. recently, however, boily-larouche et al. (2012) reported that naturally occurring genetic variants of dc-sign, carrying specific snps in the neck domain-encoding exon 4, have an enhanced capacity to capture and transfer hiv-1 virions to cd4 + t lymphocytes. moreover, liu et al. (2004) described differ-ent neck domain length variants of dc-signcarrying variable numbers of 23-aa-residue repeats in the neck regionand correlated neck domain length heterozygosity with a reduced risk of hiv-1 infection. recent experimental data provide evidence that naturally occurring dc-sign neck domain variants can differ in multimerization competence in the cell membrane and display altered glycan binding capacity (serrano-gomez et al., 2008) . moreover, the presence of such neck domain variants appears to modulate multimerization of the prototypic dc-sign molecule (serrano-gomez et al., 2008) . the fact that neck domain variation may influence the presence, stability, and functionality of dc-sign multimers on the cell surface can provide a molecular explanation for the link between dc-sign polymorphisms and susceptibility to hiv-1 and other pathogens, although further research is needed to substantiate this (serrano-gomez et al., 2008) . although viruses rely on the host cell machinery for glycoconjugate synthesis, viral glycosylation profiles can significantly differ from the standard glycosylation profile of their host cell. for instance, it is well known that viral glycoproteins are often more heavily glycosylated than host glycoproteins, and that also the nature of their glycan modifications can significantly differ. a prototypic example in this context is the gp120 glycoprotein of hiv-1. the hiv-1 envelope is studded with trimers of noncovalently associated gp120/gp41 heterodimers . gp120 is one of the most heavily n-glycosylated proteins in nature: it contains more than 20 n-linked glycosylation sites, and n-glycans account for about half of its molecular weight (zhu et al., 2000; wei et al., 2003; pantophlet & burton, 2006; scanlan et al., 2007) . intriguingly, whereas mammalian glycoproteins typically carry mainly complex type n-glycans, this is not the case for the viral gp120 glycoprotein. recent reasearch has shown that virion-associated gp120 of pbmc-grown virusas opposed to recombinantly expressed monomeric gp120predominantly carries oligomannose n-glycans (doores et al., 2010; bonomelli et al., 2011) . the synthesis of this unusual glycosylation profile appears to be partially directed by the structure of the gp120/gp41 spike itself: the presence of a dense n-glycan cluster in gp120, combined with the steric consequences of gp120/gp41 trimerization, seems to hinder further processing of (normally transient) biosynthetic glycan intermediates by er and golgi a-mannosidases, ultimately yielding hiv-1 virions with oligomannose-enriched gp120 glycoproteins (zhu et al., 2000; doores et al., 2010; eggink et al., 2010; bonomelli et al., 2011) . clearly, although viral glycosylation is critically dependent on the glycosylation machinery of the host cell, the genetic and structural background of a virus can have a decisive influence in this process. importantly, viral infection itself may also have strong repercussions on the glycosylation biology of a host cell. considering the restricted glycosylation enzyme and precursor availabilities, it is conceivable that overexpression of viral glycoproteins in an infected target cell can result in an increased glycan heterogeneity of both viral and cellular glycoconjugates. moreover, viruses may also actively modify the host and viral glycome by modulating the expression of host cell glycoenzymes (hiraiwa et al., 1997; cebulla et al., 2000; hiraiwa et al., 2003) or via expression of virally encoded glycoenzymes in infected cells (jackson et al., 1999; willer et al., 1999; nash et al., 2000; sujino et al., 2000; vanderplasschen et al., 2000; markine-goriaynoff et al., 2003 , 2004a . an additional source of glycan variation can be discerned for viruses that can infect multiple cell types, or even different host species. for instance, it is well known that hiv can productively infect multiple cell types, and that hiv glycosylation is cell type-dependent (liedtke et al., 1994; willey et al., 1996; liedtke et al., 1997; lin et al., 2003) . cell type-dependent glycosylation differences for hiv have been shown to impact viral interaction with and trans-infection via dc-sign (lin et al., 2003) , as well as viral sensitivity to antibody neutralization (willey et al., 1996) . another, particularly fascinating example in this context is dengue virus (dv). dv is a mosquito-borne flavivirus that can replicate in mosquitos as well as in humans (navarro-sanchez et al., 2003; dejnirattisai et al., 2011) . in humans, immature skin dcs are considered the primary target cell for the virus after a mosquito bite, and dc-sign is believed to be the main dv receptor on these cells (navarro-sanchez et al., 2003; dejnirattisai et al., 2011) . in a recent study, it was shown that insect cell-derived dv can efficiently infect dcs, whereas dc-derived dv is not able to reinfect dcs (dejnirattisai et al., 2011) . similarly, insect cell-derived dv could efficiently bind and infect a dc-sign-expessing cell line, whereas this was not the case for dcderived dv (dejnirattisai et al., 2011) . finally, it was found that insect cell-derived dv predominantly contains high-/pauci-mannose-type n-glycans, whereas dcderived virus contains only complex type n-glycans (dejnirattisai et al., 2011) . projected against the background of dv infection, these data outline a tentative model of the first stages of dv infection in humans: during dv replication in mosquito cells, newly formed dv virions obtain mannose-rich glycans. upon viral transfer to a human host, these virions efficiently infect immature skin dcs via dc-sign. importantly, dv replication in skin dcs yields virions with complex type n-glycans, thus creating a 'glycan mismatch' with dc-sign. due to this mismatch, newly synthesized dc-derived virus will not readily reinfect dcs via dc-sign, but preferentially infect other potential host cells via other receptors (dejnirattisai et al., 2011) . although much more research is needed to verify this tentative model, it elegantly illustrates how cell type-dependent glycan variability may impact a viral infection process. another notable factor to be considered in the context of virus-related variability is the rapid evolution of many viral pathogens. this seems especially significant for rna viruses, as these viruses generally evolve more rapidly than dna viruses due to factors inherent to their biology and infection strategy (belshaw et al., 2008; holmes, 2009; lauring & andino, 2010) . the higher mutation frequency of many rna viruses directly implies a higher chance for addition or deletion of putative glycosylation sites. as has been shown for iav, acquisition or deletion of glycosylation sites may affect crucial steps in the viral infection/replication process (e.g. receptor binding, fusion, release of newly formed virions) (ohuchi et al., 1997; wagner et al., 2000; tsuchiya et al., 2002; kim & park, 2012) , alter the capacity of the virus to avoid induction of/recognition by virus-specific antibodies (glycan shielding) wei et al., 2010; wanzeck et al., 2011; kim & park, 2012; job et al., 2013; sun et al., 2013) , and modulate viral interaction with various immune system lectins (reading et al., 2007; vigerust et al., 2007; reading et al., 2009; tate et al., 2011a, b) . clearly, mutational changes in the viral glycome may affect the virus-host interactome in various ways. ultimately, the net benefit of a specific glycome change will determine if a glycosylation variant may become dominant in the virus population. however, not only the viral glycosylation status, but also the affinity and specificity of viral lectins for specific glycoconjugates may change as a result of mutations. for instance, ample data show that amino acid changes at specific sites of the iav hemagglutinin protein can significantly alter its affinity and/or specificity for particular sialic acid-containing receptorsa factor that is crucial for the virus to infect new host species (skehel & wiley, 2000; wagner et al., 2002; suzuki, 2005; gamblin & skehel, 2010) . finally, functional alterations in the viral rde due to mutations may also have a strong impact on the interaction of viral lectins with host cell glycans. in the case of iav, balanced lectin and rde functions appear to be crucial for efficient viral replication. for iav variants that are well adapted to a certain host species, the substrate specificity and activity of the neuraminidase generally match the ligand specificity and affinity of the hemagglutinin . disruption of this balancefor instance due to reassortment or transmission to a new host speciesoften results in a decreased replicative fitness . interestingly, however, the virus may overcome this hurdle and evolve towards replicative competence by selecting for compensatory mutations in hemagglutinin and/or neuraminidase that restore the functional balance between these molecules . considering these data on iav, it is conceivable that the balance between viral lectin and rde is also an important determinant of the replicative fitness of several other viruses. on a related note, glycan-lectin interactions in virus biology are typically studied using a limited number of (prototypic) virus variants. although the information obtained in these studies can often be extrapolated to include other virus variants, there are important exceptions. for iav, for instance, it is well documented that different virus variants can carry hemagglutinin lectins with distinct glycan ligand specificities and therefore associate with distinct spectra of (decoy) receptors. moreover, iav variants can display different glycan arrays on their surface, which has been shown to modulate viral infection, glycan shielding, and recognition by various immune system lectins (cfr. supra). the fact that the specific genetic make-up of a virus determines specificity/ affinity of viral lectins, co-directs viral glycosylation, etc., and that this can be mirrored in a distinct virus-host interactome remains an important issue in glycovirology. in sum, an intricate web of glycan-lectin interactions can modulate viral infection, and host and virus inherent variability in glycans and lectins adds a further layer of complexity to this matter. considering the pivotal roles of glycan-lectin interactions in many viral infections, interfering with these interactions seems an attractive strategy in the combat against these pathogens. conversely, strategies that promote recognition of viruses by specific immune system lectinsinvolved in viral inhibition and clearancemay also prove useful in antiviral therapies. several possibilities have been and are currently being explored. perhaps the most obvious strategy to modulate glycanlectin binding is the use of molecules that can physically interfere with these interactions. glycan decoys (e.g. carbohydrate-containing drugs, sugar analogs/glycomimetics) or carbohydrate-binding agents (cbas) may be used in antiviral therapies to directly block key glycan-lectin interactions at the side of the lectin and at the side of the glycan, respectively. binding of glycan decoys with a specific lectin can inhibit binding of other ligands by this lectin. for instance, it has been shown that multivalent mannosecontaining molecules or mannose-based glycomimetics can compromise binding of hiv-1 gp120 with dc-sign (wang et al., 2008b; luallen et al., 2009; martinez-avila et al., 2009b; becer et al., 2010) and inhibit dc-sign-mediated trans-infection of cd4 + t lymphocytes (martinez-avila et al., 2009a; sattin et al., 2010; berzi et al., 2012) . likewise, sialic acid-containing glycan decoys and sialic acid analogs are being evaluated for their capacity to block the sialic acid binding site of iav hemagglutinin to inhibit interaction of the virus with sialic acid-containing receptor molecules on the surface of target cells (landers et al., 2002; matrosovich & klenk, 2003; matsubara et al., 2010; papp et al., 2010 papp et al., , 2011 . alternatively, binding of cbas to glycans displayed on the virion surface can inhibit viral cis-or trans-infection via host cell lectins. this is elegantly exemplified in several recent studies, showing that mannose-as well as n-acetylglucosamine-specific cbas can effectively prevent dc-sign-mediated hiv-1 capture and subsequent transmission to t lymphocytes balzarini et al., 2007a; bertaux et al., 2007; huskens et al., 2010; hoorelbeke et al., 2011; alexandre et al., 2012) . although cba binding to host cell-associated glycans may inhibit viruses that employ viral lectins, the potential of this strategy for antiviral therapy remains virtually unexplored. it is noteworthy that the antiviral activity of cbas or glycan decoys that bind to virion surfaces is not necessarily limited to direct inhibition of crucial glycan-lectin interactions, as they can mask greater portions of the virus and interfere with other crucial (including non-glycanlectin) interactions or steps in the infection process. moreover, recent research on hiv-1 highlights the antiviral potential of cbas from yet another angle. although the heavily glycosylated hiv-1 gp120 protein generally provides multiple ligands for mannose-and glcnac-specific cbas, prolonged cba pressure selects for hiv-1 variants with multiple n-glycosylation site deletions in the gp120 protein that are less sensitive to cba-mediated neutralization (balzarini et al., 2004 (balzarini et al., , 2005a witvrouw et al., 2005; balzarini et al., 2006; balzarini, 2007b, c; balzarini et al., 2007b; huskens et al., 2007) . interestingly, however, deletion of n-glycosylation sites can also increase the immunogenicity of the virus and weaken the glycan shield that protects the virus from recognition by virus-specific antibodies and other (nonlectin) immune receptors (botarelli et al., 1991; back et al., 1994; reitter et al., 1998; bolmstedt et al., 2001; kang et al., 2005) , and may decrease the efficiency of hiv-1 trans-infection via immune system lectins like dc-sign (hong et al., 2007; liao et al., 2011) . in addition, it appears that accumulation of mutations under cba pressure is often paralleled by a significant reduction of the viral fitness, which is obviously advantageous in the context of an antiviral treatment (balzarini et al., 2005a; balzarini, 2007b, c) . for further information on cbas and their potential in antiviral therapy, readers may refer to recent expert reviews on this topic (balzarini, 2007c; francois & balzarini, 2012) . although the use of glycan decoys and cbas may seem the most intuitive strategy to interfere directly with glycan-lectin interaction, other options exist as well. for instance, similar to glycan decoys and cbas, lectin-or glycoconjugate-specific immunoglobulins may be used to block specific interactions. evidently, the increasing availability of such 'direct' modulators of glycan-lectin interaction is mirrored in an increasing number of potential antiviral applications. apart from direct modulation, also strategies that influence glycan-lectin interactions more indirectly can be employed. in fact, many of the molecules used to examine glycan-lectin interactions in vitro suggest themselves as potential therapeutics. one approach to indirectly govern glycan-lectin interaction is via the use of drugs that alter the host and/or viral glycome. glycosidases and other enzymes may be used to alter the glycan portions of fully formed and matured glycoconjugates. alternatively, various drugs may be employed to directly modify glycan synthesis: glycoconjugates produced in the presence of such molecules will obtain aberrant glycosylation, which may promote or annihilate their interaction with specific lectins. promising results in glycovirological research have highlighted the antiviral potential of such compounds. for instance, ample data indicate that sialidases may be used to counteract infections where sialic acids play important roles as cellular receptors for viral lectins [e.g. iav binds to sialic acid receptors on the airway epithelium (skehel & wiley, 2000; malakhov et al., 2006; belser et al., 2007; harrison, 2008; chan et al., 2009; triana-baltzer et al., 2009a , 2011 or as viral ligands for host lectins that serve as portals for viral entry [e.g. sialic acids on the porcine reproductive and respiratory syndrome virus bind the macrophage-specific entry mediator sialoadhesin (delputte & nauwynck, 2004; delputte et al., 2007; van breedam et al., 2010a, b) ]. sialidase treatment may also enhance recognition of viral glycoconjugates by mannose-specific immune system lectins that can limit viral infection: in vitro experiments have shown that enzymatic removal of sialic acids from the hiv-1 virion surface can significantly enhance virus binding and neutralization by mbl (hart et al., 2002 (hart et al., , 2003 . in line with this, production of hiv-1 in the presence of the golgi a1-2-mannosidase i inhibitor 1-deoxymannojirimycinwhich blocks the biosynthesis of complex-type, sialylated oligosaccharidesincreased susceptibility of the virus to mbl-mediated neutralization (hart et al., 2003) . interestingly, 1-deoxymannojirimycin treatment of hiv-1-infected cells was also shown to potentiate the antiviral effects of mannose-specific plant cbas towards the newly produced hiv-1 virions (balzarini, 2007a) . these and other examples illustrate the potential of these molecules as antiviral drugs. considering the various structural and nonstructural roles of glycans, it is clear that glycome modulation can also have effects beyond the alteration of glycan-lectin binding events. for instance, interfering with host cell glycosylation processes using specific inhibitors may inhibit assembly of infectious virions (leavitt et al., 1977; katz et al., 1980; pizer et al., 1980; herrler & compans, 1983; montefiori et al., 1988; pal et al., 1989; mehta et al., 1998; dwek et al., 2002; wu et al., 2002; durantel et al., 2007; lazar et al., 2007; scanlan et al., 2007; durantel, 2009; merry & astrautsova, 2010) . moreover, glycome modulation may significantly alter the capacity of the virus to evade recognition by virus-specific antibodies and b-and t-cell receptors via glycan shielding (botarelli et al., 1991; back et al., 1994; willey et al., 1996; reitter et al., 1998; bolmstedt et al., 2001; kang et al., 2005; aguilar et al., 2006; wang et al., 2009; francica et al., 2010; kobayashi & suzuki, 2012) . clearly, glycome-modifying drugs can counteract viruses in various ways and constitute versatile tools in the control of viral infection. also other strategies that can indirectly influence glycan-lectin interactions are certainly worth exploring. for instance, drugs that alter host or viral lectin expression (e.g. cytokines or rnai) may prove useful in antiviral strategies (ochiel et al., 2010; relloso et al., 2002; ge et al., 2003; arrighi et al., 2004; ge et al., 2004; nair et al., 2005; yagi et al., 2010; raza et al., 2011) . furthermore, patients infected with a virus that employs both viral lectins and rdes may also benefit from treatment with rde-specific inhibitors, as these can alter the balance between viral lectin and rde activity which is often crucial for efficient viral replication and spread. notable examples in this context are the several neuraminidase inhibitors that have been used successfully for the treatment of iav infections (kim et al., 1999; lew et al., 2000; roberts, 2001; garman & laver, 2004; alymova et al., 2005; von itzstein, 2007; von itzstein & thomson, 2009; gamblin & skehel, 2010; ikematsu & kawai, 2011) . in sum, drugs that modulate glycan-lectin interactionseither directly or indirectlycan be powerful instruments in the combat against viral pathogens. however, the antiviral strategies suggested above can also have drawbacks. intensive use of antiviral therapeutics may elicit rapid selection of drug-resistant virus variants. another possible drawback relates to the potential off-target effects of these therapies: therapeutics aimed at influencing specific glycan-lectin interactions that play key roles in viral infection processes may also affect general host glycosylation, lectin expression or glycan-lectin interactions that are crucial for normal functioning of the host and its immune defense. it is also conceivable that such drugs, despite their antiviral effects, may benefit the virus in some ways. for instance, although a glycome-modifying drug may promote viral recognition by specific immune system lectins that aid in viral clearance, it may also promote viral interaction with host lectins that can aid in cis-or transinfection. ultimately, the potential of specific agents as antiviral drugs depends on their net (antiviral) effects in vivo. other potential disadvantages concern the pharmacokinetic properties of specific drugs. for instance, if glycan decoys or cbas used in antiviral therapy show a broad reactivity and can respectively bind with multiple (nontarget) lectins or glycans in the host, it is possible that much of the antiviral effect is lost. also, when using peptidic cbas that are not native to the host, an antibody response might be mounted against these components, leading to neutralization and/or faster clearance of the active compound. in spite of these and other potential pitfalls, it is clear that glycan decoys and cbas, as well as various indirect approaches to modulate glycan-lectin interaction, show potential for the treatment of diverse viral infections, either or not in combination with other antiviral strategies. a good understanding of relevant glycan-lectin interactions facilitates specific targeting of these binding events and can help to minimize possible off-target effects and to reduce the risk of drug resistance. glycans and lectins cover crucial roles in virus biology and their interplay often shapes the virus-host interaction. in general, the nature of the glycan, the lectin, and the specific conditions under which their interaction occurs determines the outcome of a specific binding event and directs the virus to a certain fate. based on current knowledge, it is clear that viral lectins generally facilitate viral infection and spread. on the other hand, although it may seem intuitive that host lectin prrs and other immune system lectins exclusively act in defense of the host, there is ample evidence that contradicts this. although many host lectins are involved in the induction of an efficacious immune response against viral pathogens, many viruses can also abuse these lectins to promote infection and spread. intriguingly, analysis of the many studies regarding the role of host lectins in viral infections suggests that soluble host lectins tend to be associated with antiviral activity, whereas membrane-associated host lectins seem to play a more dubious role and are often implicated in pro-as well as antiviral mechanisms (tables 1 & 2) . it is however noteworthy that the information provided in this manuscript reflects current views on glycan-lectin interactions in virus biology, and that future research may alter our understanding and interpretation of specific interactions. the fact that (aspects of) viral glycobiology may change during virus-host co-evolution even advocates periodic re-evaluation of specific glycan-lectin interactions. the biology of glycans and lectins is complex and has long been poorly accessible to virologists and other scientists outside this field. this situation is changing with the emergence of international glycomics consortia (e.g. consortium for functional glycomics), which can provide state-of-the-art techniques and expertise to analyze and interpret virologically/immunologically relevant glycanlectin interactions. still, there are specific pitfalls associated with glycovirological research. in vitro experiments need to be designed and interpreted considering key issues like glycan and lectin variability, the cell-type dependency of host and virus glycosylation and the influence of the lectin-expressing cell type on the final outcome of a glycan-lectin interaction. in addition, the results of in vitro experiments must ultimately be compared withand re-interpreted in the context ofdata obtained in animal models. in fact, our current understanding of specific glycan-lectin interactions in viral infection is mostly based on in vitro experiments, underlining the need for experimental validation of these results in the context of the infected host. in the context of viral infections, many different (lectin-dependent or -independent) interactions and processes take place simultaneously, resulting in a complex network of virus-host factor interaction, signaling, and effector mechanisms, the net effect of which may benefit the host or the virus. many of the interactions taking place are not yet well defined and probably more are still unknown. key to combating viral disease is to make these black boxes more transparent. synergisms between different branches of life sciences are essential to sustain and advance our knowledge in this important field of research. n-glycans on nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry the lectins griffithsin, cyanovirin-n and scytovirin inhibit hiv-1 binding to the dc-sign receptor and transfer to cd4(+) cells neuraminidase inhibitors as antiviral agents complement-dependent neutralization of influenza virus by a serum mannose-binding lectin lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells identification of the platelet-derived chemokine cxcl4/pf-4 as a broad-spectrum hiv-1 inhibitor an n-glycan within the human immunodeficiency virus type 1 gp120 v3 loop affects virus neutralization the alpha(1,2)-mannosidase i inhibitor 1-deoxymannojirimycin potentiates the antiviral activity of carbohydrate-binding agents against wild-type and mutant hiv-1 strains containing glycan deletions in gp120 carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses? targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy profile of resistance of human immunodeficiency virus to mannose-specific plant lectins carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp120: a new therapeutic concept to hit the achilles heel of hiv marked depletion of glycosylation sites in hiv-1 gp120 under selection pressure by the mannose-specific plant lectins of hippeastrum hybrid and galanthus nivalis mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type 1 strains with n-glycan deletions in their gp120 envelopes carbohydrate-binding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign)-directed hiv-1 transmission to t lymphocytes pradimicin a, a carbohydrate-binding nonpeptidic lead compound for treatment of infections with viruses with highly glycosylated envelopes, such as human immunodeficiency virus pradimicin s, a highly soluble nonpeptidic small-size carbohydrate-binding antibiotic, is an anti-hiv drug lead for both microbicidal and systemic use the role of dc-sign and dc-signr in hiv and siv attachment, infection, and transmission quantitative expression and virus transmission analysis of dc-sign on monocyte-derived dendritic cells a novel synthetic reversible inhibitor of sialidase efficiently blocks secondary but not primary influenza virus infection of mdck cells in culture dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins high-affinity glycopolymer binding to human dc-sign and disruption of dc-sign interactions with hiv envelope glycoprotein das181, a novel sialidase fusion protein, protects mice from lethal avian influenza h5n1 virus infection pacing a small cage: mutation and rna viruses interactions of surfactant protein a with influenza a viruses: binding and neutralization surfactant protein a, but not surfactant protein d, is an opsonin for influenza a virus phagocytosis by rat alveolar macrophages entry of hepatitis c virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions a glycomimetic compound inhibits dc-sign-mediated hiv infection in cellular and cervical explant models the relevance of complement to virus biology dendritic cell-mediated trans-enhancement of human immunodeficiency virus type 1 infectivity is independent of dc-sign naturally-occurring genetic variants in human dc-sign increase hiv-1 capture, cell-transfer and risk of mother-to-child transmission enhanced immunogenicity of a human immunodeficiency virus type 1 env dna vaccine by manipulating n-glycosylation signals. effects of elimination of the v3 n306 glycan the glycan shield of hiv is predominantly oligomannose independently of production system or viral clade n-glycosylation of hiv-gp120 may constrain recognition by t lymphocytes an integrated view of humoral innate immunity: pentraxins as a paradigm lentivirus degradation and dc-sign expression by human platelets and megakaryocytes shared paramyxoviral glycoprotein architecture is adapted for diverse attachment strategies lectin-dependent enhancement of ebola virus infection via soluble and transmembrane c-type lectin receptors structural basis for the receptor binding specificity of norwalk virus infection of dendritic cells (dcs), not dc-sign-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to t cells dual function of c-type lectin-like receptors in the immune system how c-type lectins detect pathogens structural basis for the recognition of blood group trisaccharides by norovirus in vitro derived dendritic cells trans-infect cd4 t cells primarily with surface-bound hiv-1 virions cytomegalovirus induces sialyl lewis (x) and lewis(x) on human endothelial cells dc-sign and clec-2 mediate human immunodeficiency virus type 1 capture by platelets das181 inhibits h5n1 influenza virus infection of human lung tissues paramyxovirus fusion and entry: multiple paths to a common end lack of the pattern recognition molecule mannose-binding lectin increases susceptibility to influenza a virus infection dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate the essentiality of alpha-2-macroglobulin in human salivary innate immunity against new h1n1 swine origin influenza a virus atomic resolution structural characterization of recognition of histo-blood group antigens by norwalk virus influenza virus neuraminidase: structure, antibodies, and inhibitors activation of murine cd4+ and cd8+ t lymphocytes leads to dramatic remodeling of n-linked glycans siglecs as positive and negative regulators of the immune system siglecs and their roles in the immune system envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens specificity and affinity of sialic acid binding by the rhesus rotavirus vp8* core celgosivir, an alpha-glucosidase i inhibitor for the potential treatment of hcv infection glucosidase inhibitors as antiviral agents for hepatitis b and c targeting glycosylation as a therapeutic approach lack of complex n-glycans on hiv-1 envelope glycoproteins preserves protein conformation and entry function impact of mannose-binding lectin on susceptibility to infectious diseases lectin-carbohydrate interactions: different folds, common recognition principles noroviruses everywhere: has something changed? genetic diversity and histo-blood group antigen interactions of rhesus enteric caliciviruses structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein potential of carbohydrate-binding agents as therapeutics against enveloped viruses evolution of the lectin-complement pathway and its role in innate immunity from lectin structure to functional glycomics: principles of the sugar code surfactant protein a binds to hiv and inhibits direct infection of cd4+ cells, but enhances dendritic cell-mediated viral transfer influenza hemagglutinin and neuraminidase membrane glycoproteins endogenous ligands for c-type lectin receptors: the true regulators of immune homeostasis controlling influenza by inhibiting the virus's neuraminidase endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription use of sirnas to prevent and treat influenza virus infection signalling through c-type lectin receptors: shaping immune responses identification of dc-sign, a novel dendritic cell-specific icam-3 receptor that supports primary immune responses dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances trans-infection of t cells location, location, location: new insights into o-galnac protein glycosylation two evolutionary strategies of influenza viruses to escape host non-specific inhibitors: alteration of hemagglutinin or neuraminidase specificity pattern recognition receptors: doubling up for the innate immune response neuraminidase: the specific enzyme of influenza virus and vibrio cholerae dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin/cd209 is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction c-type lectin dc-sign modulates toll-like receptor signaling via kinase-dependent acetylation of transcription factor nf-kappab hiv-1 exploits innate signaling by tlr8 and dc-sign for productive infection of dendritic cells association between expression of the h histo-blood group antigen, alpha1,2fucosyltransferases polymorphism of wild rabbits, and sensitivity to rabbit hemorrhagic disease virus binding and transfer of human immunodeficiency virus by dc-sign+ cells in human rectal mucosa viral membrane fusion paramyxovirus assembly and budding: building particles that transmit infections high mannose glycans and sialic acid on gp120 regulate binding of mannose-binding lectin (mbl) to hiv type 1 glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type 1 binding and neutralization by mannose-binding lectin human mannose-binding protein functions as an opsonin for influenza a viruses evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses neutrophil deactivation by influenza a viruses: mechanisms of protection after viral opsonization with collectins and hemagglutination-inhibiting antibodies mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins mechanism of binding of surfactant protein d to influenza a viruses: importance of binding to haemagglutinin to antiviral activity characterizing the glycome of the mammalian immune system pulmonary collectins modulate strain-specific influenza a virus infection and host responses mannan-binding lectin deficiency -good news, bad news, doesn't matter? posttranslational modification and intracellular transport of mumps virus glycoproteins structure and function of the hef glycoprotein of influenza c virus neuraminic acid is involved in the binding of influenza c virus to erythrocytes the receptor-destroying enzyme of influenza c virus is neuraminate-o-acetylesterase the glycoprotein of influenza c virus is the haemagglutinin, esterase and fusion factor 9-o-acetylated sialic acid, a receptor determinant for influenza c virus and coronaviruses lung surfactant protein a provides a route of entry for respiratory syncytial virus into host cells dc-sign increases the affinity of hiv-1 envelope glycoprotein interaction with cd4 assessment of the antiviral properties of recombinant porcine sp-d against various influenza a viruses in vitro human t-cell leukemia virus-1 encoded tax protein transactivates alpha 1->3 fucosyltransferase fuc-t vii, which synthesizes sialyl lewis x, a selectin ligand expressed on adult t-cell leukemia cells transactivation of the fucosyltransferase vii gene by human t-cell leukemia virus type 1 tax through a variant camp-responsive element activation of the lectin dc-sign induces an immature dendritic cell phenotype triggering rho-gtpase activity required for hiv-1 replication the evolution and emergence of rna viruses human immunodeficiency virus envelope (gp120) binding to dc-sign and primary dendritic cells is carbohydrate dependent but does not involve 2g12 or cyanovirin binding sites: implications for structural analyses of gp120-dc-sign binding identification of the optimal dc-sign binding site on human immunodeficiency virus type 1 gp120 differences in the mannose oligomer specificities of the closely related lectins from galanthus nivalis and zea mays strongly determine their eventual anti-hiv activity norovirus and histo-blood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple binding patterns passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein e(rns) resistance of hiv-1 to the broadly hiv-1-neutralizing, anti-carbohydrate antibody 2g12 microvirin, a novel alpha (1,2)-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv-1 activity comparable with that of cyanovirin-n but a much higher safety profile laninamivir octanoate: a new long-acting neuraminidase inhibitor for the treatment of influenza siglec-1 is a novel dendritic cell receptor that mediates hiv-1 trans-infection through recognition of viral membrane gangliosides myxoma virus encodes an alpha2,3-sialyltransferase that enhances virulence innate immune recognition mucin-type o-glycosylation-putting the pieces together addition of glycosylation to influenza a virus hemagglutinin modulates antibody-mediated recognition of h1n1 2009 pandemic viruses modified hiv envelope proteins with enhanced binding to neutralizing monoclonal antibodies human mannan-binding lectin inhibits the infection of influenza a virus without complement antiviral activity of tunicamycin on herpes simplex virus n-linked glycosylation in the hemagglutinin of influenza a viruses neuraminidase inhibitors as anti-influenza virus agents correction of pulmonary abnormalities in sftpdà/à mice requires the collagenous domain of surfactant protein d adaptation of sindbis virus to bhk cells selects for use of heparan sulfate as an attachment receptor evidence for n-glycan shielding of antigenic sites during evolution of human influenza a virus hemagglutinin rantes -28g delays and dc-sign -139c enhances aids progression in hiv type 1-infected japanese hemophiliacs point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants heparin-dependent attachment of respiratory syncytial virus (rsv) to host cells identification and functions of pattern-recognition receptors structural and functional analysis of the surface protein of human coronavirus oc43 dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection orthomyxoviridae: the viruses and their replication prevention of influenza pneumonitis by sialic acid-conjugated dendritic polymers altered t cell surface glycosylation in hiv-1 infection results in increased susceptibility to galectin-1-induced cell death the expanding horizons of asparagine-linked glycosylation quasispecies theory and the behavior of rna viruses treatment of hepatitis b virus-infected cells with alpha-glucosidase inhibitors results in production of virions with altered molecular composition and infectivity mendelian resistance to human norovirus infections tunicamycin inhibits glycosylation and multiplication of sindbis and vesicular stomatitis viruses modes of paramyxovirus fusion: a henipavirus perspective cis expression of dc-sign allows for more efficient entry of human and simian immunodeficiency viruses via cd4 and a coreceptor viral piracy: hiv-1 targets dendritic cells for transmission surfactant protein d enhances clearance of influenza a virus from the lung in vivo absence of sp-a modulates innate and adaptive defense responses to pulmonary influenza infection surfactant protein-d enhances phagocytosis and pulmonary clearance of respiratory syncytial virus novel innate immune functions for galectin-1: galectin-1 inhibits cell fusion by nipah virus envelope glycoproteins and augments dendritic cell secretion of proinflammatory cytokines discovery and development of gs 4104 (oseltamivir): an orally active influenza neuraminidase inhibitor surfactant protein-a-deficient mice display an exaggerated early inflammatory response to a beta-resistant strain of influenza a virus identification of the dc-sign-interactive domains on the envelope glycoprotein of hiv-1 crf07_bc oligosaccharide profiles of hiv-2 external envelope glycoprotein: dependence on host cells and virus isolates host-cell-specific glycosylation of hiv-2 envelope glycoprotein differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr analysis of genetic polymorphisms in ccr5, ccr2, stromal cell-derived factor-1, rantes, and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin in seronegative individuals repeatedly exposed to hiv-1 principles of structures of animal and plant lectins a yeast glycoprotein shows high-affinity binding to the broadly neutralizing human immunodeficiency virus antibody 2g12 and inhibits gp120 interactions with 2g12 and dc-sign surfactant protein d modulates hiv infection of both t-cells and dendritic cells sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection binding of human collectins (sp-a and mbp) to influenza virus adaptation of tick-borne encephalitis virus to bhk-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo the neck region of the c-type lectin dc-sign regulates its surface spatiotemporal organization and virus-binding capacity on antigen-presenting cells the core 2 beta-1,6-n-acetylglucosaminyltransferase-mucin encoded by bovine herpesvirus 4 was acquired from an ancestor of the african buffalo glycosyltransferases encoded by viruses the core 2 beta-1,6-n-acetylglucosaminyltransferase-m encoded by bovine herpesvirus 4 is not essential for virus replication despite contributing to post-translational modifications of structural proteins association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type 1 infection multivalent manno-glyconanoparticles inhibit dc-sign-mediated hiv-1 trans-infection of human t cells gold manno-glyconanoparticles: multivalent systems to block hiv-1 gp120 binding to the lectin dc-sign natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding influenza viruses differ in recognition of 4-o-acetyl substitution of sialic acid receptor determinant molecular mechanisms of serum resistance of human influenza h3n2 virus and their involvement in virus adaptation in a new host sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenza therapy recruitment of hiv and its receptors to dendritic cell-t cell junctions divergent roles for c-type lectins expressed by cells of the innate immune system ligand recognition by antigen-presenting cell c-type lectin receptors alpha-glucosidase inhibitors as potential broad based anti-viral agents galectin-1 promotes hiv-1 infectivity in macrophages through stabilization of viral adsorption alternative approaches to antiviral treatments: focusing on glycosylation as a target for antiviral therapy inhibition of hemagglutination activity of influenza a viruses by sp-a1 and sp-a2 variants expressed in cho cells dc-specific icam-3-grabbing nonintegrin mediates internalization of hiv-1 into human podocytes role of protein n-glycosylation in pathogenesis of human immunodeficiency virus type 1 dc-sign promotes exogenous mhc-i-restricted hiv-1 antigen presentation dendritic cells and hiv-specific cd4+ t cells: hiv antigen presentation, t-cell activation, and viral transfer extensive repertoire of membrane-bound and soluble dendritic cell-specific icam-3-grabbing nonintegrin 1 (dc-sign1) and dc-sign2 isoforms. inter-individual variation in expression of dc-sign transcripts rnai-directed inhibition of dc-sign by dendritic cells: prospects for hiv-1 therapy influenza c virus hemagglutinin: comparison with influenza a and b virus hemagglutinins post-translational modification of the myxoma-virus anti-inflammatory serpin serp-1 by a virally encoded sialyltransferase dendritic-cell-specific icam3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses hiv-1 activates cdc42 and induces membrane extensions in immature dendritic cells to facilitate cell-to-cell virus propagation covert human immunodeficiency virus replication in dendritic cells and in dc-sign-expressing cells promotes long-term transmission to lymphocytes histo-blood group antigens act as attachment factors of rabbit hemorrhagic disease virus infection in a virus strain-dependent manner uterine epithelial cell regulation of dc-sign expression inhibits transmitted/founder hiv-1 trans infection by immature dendritic cells regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety c (2011) myeloid c-type lectin receptors in pathogen recognition and host defense galectin-1 acts as a soluble host factor that promotes hiv-1 infectivity through stabilization of virus attachment to host cells role of oligosaccharides in the processing and maturation of envelope glycoproteins of human immunodeficiency virus type 1 gp120: target for neutralizing hiv-1 antibodies inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles inhibition of influenza virus activity by multivalent glycoarchitectures with matched sizes cell-surface heparan sulfate proteoglycan mediates hiv-1 infection of t-cell lines the glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins cell surface expression of biologically active influenza c virus hef glycoprotein expressed from cdna methods in enzymology: o-glycosylation of proteins the interaction of hiv with dendritic cells: outcomes and pathways effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production dc-sign interactions with human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus basis for the potent inhibition of influenza virus infection by equine and guinea pig alpha 2-macroglobulin synthesis and sorting of proteoglycans nmr experiments reveal the molecular basis of receptor recognition by a calicivirus dc-sign on b lymphocytes is required for transmission of hiv-1 to t lymphocytes selection of predicted sirna as potential antiviral therapeutic agent against influenza virus a serum mannose-binding lectin mediates complement-dependent lysis of influenza virus-infected cells collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice glycosylation as a target for recognition of influenza viruses by the innate immune system loss of a single n-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice a role for carbohydrates in immune evasion in aids dc-sign (cd209) expression is il-4 dependent and is negatively regulated by ifn, tgf-beta, and anti-inflammatory agents anti-influenza drugs and neuraminidase inhibitors differential sensitivity of human, avian, and equine influenza a viruses to a glycoprotein inhibitor of infection: selection of receptor specific variants influenza c virus uses 9-o-acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus protein glycosylation in the endoplasmic reticulum and the golgi apparatus and cell type-specificity of cell surface glycoconjugate expression: analysis by the protein a-gold and lectin-gold techniques binding of rabbit hemorrhagic disease virus to antigens of the abh histo-blood group family distinct glycoprotein inhibitors of influenza a virus in different animal sera alpha 2-macroglobulin is the major neutralizing inhibitor of influenza a virus in pig serum tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle a variant in the cd209 promoter is associated with severity of dengue disease signaling by myeloid c-type lectin receptors in immunity and homeostasis lactoferrin and surfactant protein a exhibit distinct binding specificity to f protein and differently modulate respiratory syncytial virus infection galectins in innate immunity: dual functions of host soluble beta-galactoside-binding lectins as damage-associated molecular patterns (damps) and as receptors for pathogen-associated molecular patterns (pamps) glycans, galectins, and hiv-1 infection inhibition of dc-sign-mediated hiv infection by a linear trimannoside mimic in a tetravalent presentation exploiting the defensive sugars of hiv-1 for drug and vaccine design isolation and characterization of sialate 9 (4)-o-acetylesterase from influenza c virus the s protein of bovine coronavirus is a hemagglutinin recognizing 9-o-acetylated sialic acid as a receptor determinant transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity mechanisms and principles of n-linked protein glycosylation the sialic acid binding activity of the s protein facilitates infection by porcine transmissible gastroenteritis coronavirus cd209 gene polymorphisms in south indian hiv and hiv-tb patients structural requirements for multimerization of the pathogen receptor dendritic cell-specific icam3-grabbing non-integrin (cd209) on the cell surface dendritic cells and transmission of hiv-1 norovirus and histo-blood group antigens receptor binding and membrane fusion in virus entry: the influenza hemagglutinin constitutive and induced expression of dc-sign on dendritic cell and macrophage subpopulations in situ and in vitro host-soluble galectin-1 promotes hiv-1 replication through a direct interaction with glycans of viral gp120 and host cd4 alpha2,6-linked sialic acid acts as a receptor for feline calicivirus dc-sign binds to hiv-1 glycoprotein 120 in a distinct but overlapping fashion compared with icam-2 and icam-3 a novel viral alpha2,3-sialyltransferase (v-st3gal i): transfer of sialic acid to fucosylated acceptors membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions n-linked glycosylation of the hemagglutinin protein influences virulence and antigenicity of the 1918 pandemic and seasonal h1n1 influenza a viruses sialobiology of influenza: molecular mechanism of host range variation of influenza viruses origin and evolution of influenza virus hemagglutinin genes sialoglycoproteins that bind influenza a virus and resist viral neuraminidase in different animal sera c-type lectin dc-sign: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity norovirus-host interaction: implications for disease control and prevention specific sites of n-linked glycosylation on the hemagglutinin of h1n1 subtype influenza a virus determine sensitivity to inhibitors of the innate immune system and virulence in mice glycosylation of the hemagglutinin modulates the sensitivity of h3n2 influenza viruses to innate proteins in airway secretions and virulence in mice ganglioside-linked terminal sialic acid moieties on murine macrophages function as attachment receptors for murine noroviruses glycosphingolipids as receptors for non-enveloped viruses murine noroviruses bind glycolipid and glycoprotein attachment receptors in a strain-dependent manner introduction to glycobiology belshe rb & fang f (2009a) inhibition of neuraminidase inhibitor-resistant influenza virus by das181, a novel sialidase fusion protein novel pandemic influenza a(h1n1) viruses are potently inhibited by das181, a sialidase fusion protein das181, a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis phenotypic and genotypic characterization of influenza virus mutants selected with the sialidase fusion protein das181 glycosaminoglycan-binding ability is a feature of wild-type strains of herpes simplex virus type 1 the multiple facets of hiv attachment to dendritic cell lectins platelet activation suppresses hiv-1 infection of t cells effect of addition of new oligosaccharide chains to the globular head of influenza a/ h2n2 virus haemagglutinin on the intracellular transport and biological activities of the molecule diversity of receptors binding hiv on dendritic cell subsets immunodeficiency virus uptake, turnover, and 2-phase transfer in human dendritic cells the m/gp(5) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner concepts and principles of o-linked glycosylation langerin functions as an antiviral receptor on langerhans cells innate signaling in hiv-1 infection of dendritic cells porcine surfactant protein d is n-glycosylated in its carbohydrate recognition domain and is assembled into differently charged oligomers porcine pulmonary collectins show distinct interactions with influenza a viruses: role of the n-linked oligosaccharides in the carbohydrate recognition domain interactions of influenza a virus with sialic acids present on porcine surfactant protein d specificity of dc-sign for mannose-and fucose-containing glycans effects of heparin on the entry of porcine reproductive and respiratory syndrome virus into alveolar macrophages a multipotential beta-1,6-n-acetylglucosaminyltransferase is encoded by bovine herpesvirus type 4 the carbohydrate recognition domain of collectins n-linked glycosylation attenuates h3n2 influenza viruses the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities the war against influenza: discovery and development of sialidase inhibitors anti-influenza drugs: the development of sialidase inhibitors interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics functional balance between haemagglutinin and neuraminidase in influenza virus infections functionally distinct transmission of human immunodeficiency virus type 1 mediated by immature and mature dendritic cells structural basis for receptor specificity of influenza b virus hemagglutinin crystal structure of unliganded influenza b virus hemagglutinin targeting the carbohydrates on hiv-1: interaction of oligomannose dendrons with human monoclonal antibody 2g12 and dc-sign glycans on influenza hemagglutinin affect receptor binding and immune response a/g polymorphism is associated with dengue hemorrhagic fever and correlated to dc-sign expression and immune augmentation glycan shielding of the influenza virus hemagglutinin contributes to immunopathology in mice asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems antibody neutralization and escape by hiv-1 influenza viruses: role of glycans in viral evolution and vaccine design cell-surface carbohydrate recognition by animal and viral lectins molecular architectures of trimeric siv and hiv-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure the complete genome sequence of shope (rabbit) fibroma virus differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary t-lymphocyte and macrophage cultures resistance of human immunodeficiency virus type 1 to the high-mannose binding agents cyanovirin n and concanavalin a dendritic-cell interactions with hiv: infection and viral dissemination antiviral effects of an iminosugar derivative on flavivirus infections inhibition of dc-sign-mediated transmission of human immunodeficiency virus type 1 by toll-like receptor 3 signalling in breast milk macrophages hiv traffics through a specialized, surface-accessible intracellular compartment during trans-infection of t cells by mature dendritic cells structures, biosynthesis, and functions of gangliosides-an overview complementation of pulmonary abnormalities in sp-d(à/à) mice with an sp-d/conglutinin fusion protein mass spectrometric characterization of the glycosylation pattern of hiv-gp120 expressed in cho cells the authors thank leslie bosseler for critical reading of the manuscript and assistance in creating the figures. the authors apologize to all colleagues whose work has not been cited due to space limitations. key: cord-276870-gxtvlji7 authors: bobrowski, tesia; melo-filho, cleber c.; korn, daniel; alves, vinicius m.; popov, konstantin i.; auerbach, scott; schmitt, charles; moorman, nathaniel j.; muratov, eugene n.; tropsha, alexander title: learning from history: do not flatten the curve of antiviral research! date: 2020-07-15 journal: drug discov today doi: 10.1016/j.drudis.2020.07.008 sha: doc_id: 276870 cord_uid: gxtvlji7 here, we explore the dynamics of the response of the scientific community to several epidemics, including coronavirus 2019 (covid-19), as assessed by the numbers of clinical trials, publications, and level of research funding over time. all six prior epidemics studied [bird flu, severe acute respiratory syndrome (sars), swine flu, middle east respiratory syndrome (mers), ebola, and zika] were characterized by an initial spike of research response that flattened shortly thereafter. unfortunately, no antiviral medications have been discovered to date as treatments for any of these diseases. by contrast, the hiv/aids pandemic has garnered consistent research investment since it began and resulted in drugs being developed within 7 years of its start date, with many more to follow. we argue that, to develop effective treatments for covid-19 and be prepared for future epidemics, long-term, consistent investment in antiviral research is needed. from time immemorial, infectious diseases have ravage q6 d mankind. only 100 years ago, tb was still one of the top three leading causes of death in the usa [1] . fortunately, science advanced dramatically during the 20th century, changing the ways in which our society treats infectious diseases. current preventative vaccines and drugs are catered to treat long-lasting and/or chronic infections or infectious diseases that recur annually or on a regular basis, such as hiv, tb, alexander tropsha is a k.h. lee distinguished professor and associate dean for data science at the unc eshelman school of pharmacy, unc-chapel hill. professor tropsha was awarded a phd in chemical enzymology in 1986 from moscow state university. his research interests are in the areas of computer-assisted drug design, computational toxicology, cheminformatics, (nano)materials informatics, and structural bioinformatics. his has authored 250 peer-reviewed scientific papers, book chapters, and co-edited two monographs. his research has been supported by multiple grants from the nih, nsf, epa, dod, foundations, and private companies. hepatitis c, influenza, and so on. however, the major viral disease outbreaks that have plagued society over the past two decades do not follow this pattern. in fact, they have shown that the scientific community is not adequately prepared to offer or rapidly develop effective treatments when an outbreak happens [2] . as a consequence, all countries have to adopt nontherapeutics measures to slow the progression of the epidemic and 'flatten the curve' to limit the burden of the disease on the healthcare system and allow better support to severely ill patients [3] . a recent study in the new england journal of medicine [4] estimated that the yearly cost of a pandemic could amount to us$60 billion dollars being spent worldwide on treatment, control, and prevention efforts. as seen in the current outbreak of the sars coronavirus 2 (sars-cov-2), this cost might be even higher because of restrictions on international trade and travel, closing of businesses, prohibition of large gatherings of people, and other social-distancing strategies [5] . although these measures do help curb the spread of the disease, they have potentially devastating consequences: it was initially predicted that a large volume of cases over a short period of time would result in the usa having barely enough masks to last even 2 weeks into the pandemic [6] . there might also be other social and political ramifications: from a quick glance at the google trends data [7] , one can see that, on super tuesday in the usa in 2020, the popularity of google searches for 'coronavirus' was nearly three times that for super tuesday. also, because of prohibitions on gatherings of more than 25-50 people in many cities and states, people might have stayed away from the total number of clinical trials launched per outbreak as the function of time during the first 24 weeks after the outbreak start date. the start date of each epidemic is defined as the date when authorities, such as the who, started listing data on the number of cases. time is normalized for each outbreak according to this start date (week 0). we used the deposition dates and numbers of clinical trials as recorded in clinicaltrials.gov. abbreviations: covid-19, coronavirus 2019; mers, middle east respiratory syndrome; sars, severe acute respiratory syndrome. polls out of fear of contracting the virus and might continue to do so in the future, possibly influencing the outcome of the 2020 us election [8, 9] . the democratization of access to the internet has also facilitated the access of the general public to information through mediums such as major media outlets and even formal and informal data analytics [10, 11] . for instance, johns hopkins university hosts a popular, regularly updated map of the reported cases of covid-19 around the world [12] using data from the chinese centers for disease control and who situation reports. the new mantra, 'flatten the curve,' is also representative of the newfound exposure of the public to data science and analysis in response to the covid-19 pandemic [13] . likewise, the rapid growth of global communications systems has allowed media, government, and scientists alike to quickly access and share a large amount of data. this real-time sharing of information has been unprecedented. although it permits governments to respond rapidly to epidemics through the dissemination of prevention and control methods, it can also facilitate public panic by stoking existing fears about an epidemic [14, 15] . this rapid exchange of information applies to scientific data and publications as well: with increased access to the internet, the response of the scientific community has been enriched. we have observed an increasing number of articles being published for successive outbreaks in both peer-reviewed journals and various arxiv (see glossary) preprint servers over the past 20 years. over the past few months, both peer-reviewed journals [16, 17] and arxiv preprint [18, 19] servers have been overpopulated with reports on known drugs or clinical candidates with possible anti-sars-cov-2 activity identified by computational approaches. however, despite many experimental and clinical studies, no effective drugs or treatments have emerged to treat the previous six epidemics of bird flu, sars, swine flu, mers, ebola, and zika as well as, thus far, covid-19. this observation begs the question of whether the rapidity and bulk of immediate responses to epidemics are sufficient to enable the development of effective treatments. in this study, we investigated historical data for seven major disease outbreaks of the past two decades: bird flu (h5n1), sars, swine flu (h1n1), mers, ebola fever, zika fever, and covid-19. we assessed the response of the scientific community to these outbreaks over time, in addition to how effective that response was in producing vaccines and small-molecule antiviral drugs. to this end, we analyzed the number of publications, clinical trials, funding levels, and google trends data from the start of these epidemics until the present day. we observed that there has been little success in combatting outbreaks effectively while they were occurring, let alone after they have passed. by contrast, we also observed that these trends were different for hiv/aids, which has received continuous and uninterrupted attention from researchers around the world and for which multiple targeted therapies have indeed emerged. we expect this analysis to provide insights as to how to better mobilize both federal agencies and scientists to find treatments for covid-19 as well as other future outbreaks. weeks after outbreak bird flu drug discovery today the evolution of the number of publications during the first 24 weeks after their respective outbreak start dates. the start date of epidemic is defined as the date when authorities, such as the who, started listing data on the number of cases. time is normalized for each outbreak according to this start date (week 0). the data on publications include both peer-reviewed papers and preprints. the data were obtained from pubmed, biorxiv, medrxiv, arxiv, and chemrxiv. abbreviations: covid-19, coronavirus 2019; mers, middle east respiratory syndrome; sars, severe acute respiratory syndrome. www.drugdiscoverytoday.com 3 we evaluated the number of publications (in both peer-reviewed journals and arxiv preprint servers) and the number of clinical trials performed over the course of the epidemic to estimate the engagement and success of the scientific community in response to the seven major outbreaks of the past two decades: bird flu, sars, swine flu, mers, ebola, zika, and covid-19. these metrics indicate the velocity with which the scientific community mobilizes to seek solutions to remedy an outbreak, as well as how this velocity correlates with other metrics, such as the number of confirmed cases and the number of people who have died from the disease. in addition, we evaluated the number of unique molecules and/or treatments being tested in clinical trials, because many were replicates of each other. first, we looked at the response of the scientific community on weekly timescale (figs 1 and 2) . we examined the number of clinical trials (fig. 1 ) and publications (fig. 2 ) over the first 24 weeks of each outbreak, with week 0 corresponding to the time point when the federal authorities or the who first started reporting the data on the epidemic (for covid-19, december 31, 2019 is considered the start date). on this standardized timescale, the number of clinical trials launched for covid-19 greatly outnumbered that of any of the previous epidemics; the growth rate of publications on covid-19 was also the highest. one can also see that, for more recent epidemics, such as ebola and zika, more clinical trials were launched during the first 24 weeks of the epidemic than had been the case for previous epidemics, such as bird flu and mers (fig. 1) . the only exception to this general observation was swine flu, which is an anomaly because h1n1 flu strains had been researched extensively before the start of the outbreak in 2009 as a result of the spanish flu pandemic of 1918 and other past h1n1 epidemics [20, 21] . this is in stark contrast to ebola virus and zika virus, which had caused smaller-scale outbreaks previously, yet had little to no information available on how to treat them [22, 23] . equally valuable is to compare the rate of response of the scientific community to the rate of epidemic growth. as a case study, we chose to compare data on sars to that of sars-cov-2 (covid-19). interestingly, the number of total publications for sars over time roughly followed the same trend as the number of cases, with an offset by a short time period of less than a month (fig. 3) . likewise, rapid spikes in the number of cases correspond to slightly offset spikes in the number of publications on sars by around the same period (16 days). in comparison to sars, covid-19 does not show clear spikes in the number of publications or clinical trials corresponding to peaks and dips in the number of new cases or deaths (fig. 4) . instead, the trends in the number of publications and clinical trials appear to follow the trends in the number of cases and/or deaths. indeed, there is a correlation (r = 0.98) between the rate at which the virus spreads throughout the population and intensity of research on covid-19, as measured by the number of publications. although looking for causality in these relationships is nonsensical, the number of new peer-reviewed publications appears to follow roughly the same logarithmic curve as the number of new cases recorded for covid-19, whereas the rate of new publications in nonpeer-reviewed arxiv preprints appears to exceed even that of new covid-19 cases (fig. 4) . the number of covid-19 preprints in arxiv servers surpassed the number of covid-19 papers in peer-reviewed journals in early march, highlighting the rise of online journals and preprint servers, as well as reflecting the shrinking period of time between the original observations and respective publications. this is also in direct contrast to sars: more papers were published at a faster rate during this pandemic than in the sars epidemic beginning in 2003. for example, eight peer-reviewed papers on covid-19 had been published by the time there were 555 cases of the disease (january 22, 2020), whereas sars only had one paper published by the time there were 1804 total cases (april 3, 2003) . however, it is necessary to contextualize these observations in terms of the scientific output, not just by the rate of response. in this regard, we observed that the large number of studies conducted on sars notwithstanding, no us food and drug administration (fda)-approved drug or vaccine to treat the disease has been developed in the 17-year period since the outbreak began (in 2003). it is of particular interest to look at the evolution of both the number of publications and the amount of research funding from the beginning of each outbreak to-date (fig. 5 ). this analysis shows, perhaps not unexpectedly, that, following the spike of the research interest in the initial phases of each epidemic, progressively fewer research papers were published for previous outbreaks after they ended. this trend probably reflects the lack of special funding for such research and, consequently, the lack of successful therapeutic development against the respective diseases. indeed, fluctuations in research funding for each epidemic appear to follow the same spike-like trend as research publications (fig. 5) . this lack of research interest and/or funding outside of periods when these outbreaks are occurring is probably partially responsible for the current situation: no approved anticoronaviral medications exist and, as such, the world is frantically looking to repurpose existing drugs, such as chloroquine and hydroxychloroquine, which is premature and, according to at least some reports, could be potentially harmful to patients [24, 25] . in addition to quantifying the response of the scientific community to these epidemics, we also collected google trends data on the principal search terms representing each of these diseases. google trends data do not represent the number of google searches during a given time period but rather anonymized, aggregated, and normalized information about the relative proportion of searches on google for a given search term, region, and time period [7] . this normalization protects against places with drug discovery today volume 00, number 00 july 2020 reviews larger populations being weighted more heavily. a value of 100 corresponds to the maximum search interest in a topic for the given time and location, whereas a value of 50 corresponds to the search term being half as popular as it was at its peak. google trends as a whole serves as a fairly representative data source for the public perception of different news topics, indicating how interested people appear to be in a given topic over a specific period of time, with rapid peaks in search interest indicating a sudden increase in interest in a topic. first, we gathered and standardized google trends data for each of the epidemics based on their start dates and relative time periods (no google trends data are available before january 2004; thus, some timepoints for bird flu and sars were not available). when observing the first 30 months of the outbreaks, the response of the public to the epidemic appeared to peak during the first 8 months (fig. 6 ). for diseases that had been previously studied, such as swine flu and ebola hemorrhagic fever, there was already some search interest before the start date of the epidemic and, thus, the relative search interest in these diseases was higher during the earlier months of the epidemic. these outbreaks were shorter lived than some of the other diseases, such as mers (infection continues today, although cases peaked in early 2014) [26] , the relative search interest in which peaked an anomalous 20 months into the outbreak. covid-19 is interesting in that, although representing a novel virus, the number of relative searches peaked relatively close to those for all the previously studied illnesses (mentioned earlier) and is following an increasing exponential trend (fig. 6 ). the number of cases of covid-19 worldwide is increasing in a nearly exponential fashion, with more people infected worldwide than in any of these previous epidemics besides h1n1 (swine flu). additionally, for all diseases previously mentioned, their respective search interests peaked during the years when the epidemic was most severe (fig. 6) , so we should expect that the search interest for covid-19 will steadily decrease when the pandemic begins to die down. however, as noted earlier, although we should expect such evolution of the general public interest, research into understanding this disease and developing powerful therapeutics should continue unabated. before the outbreak of sars-cov-2, previous studies had forecasted the re-emergence of a sars-like betacoronavirus [27] . the potential for sustained transmission of sars-cov-2 or for the emergence of another novel betacoronavirus is alarming but has not been sufficient previously to garner any substantial drug discovery or vaccine efforts. these transmission dynamics and this potential for wide-scale, future pandemics makes covid-19 distinct from the six previous epidemics examined earlier. thus, the response to the hiv pandemic, which began in 1981, is also a worthwhile comparison to the current covid-19 response. the change in the number of cases, clinical trials, and publications for hiv is shown in fig. 7 , with marked time points delineating when novel anti-hiv drugs were approved by the fda. it is generally agreed that hiv can now be managed thanks to the powerful pharmacotherapies developed since the pandemic began during the early 1980s. this success is predicated on the significant and constant increase in federal funding for hiv over the course of the epidemic, rising from just a few hundred thousand dollars in financial year (fy) 1982 to more than us$34.8 billion in fy 2019 [28] . this support has allowed the research community to continue to study the disease, accounting for an average of >6000 papers published per year over the past 5 years, as annotated in pubm q7 ed (box 1). azidothymidine, better known as azt, was the first drug approved in the usa to treat hiv and was tested in clinical trials and approved for use in patients in 1987, 6 years after the pandemic began [29] . azt was originally synthesized for use as an anticancer agent [29] and was then repurposed against hiv. given the toxicity of azt and the rapid evolution of drug resistance [30], new compounds targeting various aspects of viral replication were eventually designed, encompassing different classes of antiretroviral drug, such as non-nucleoside/nucleotide reverse-transcriptase inhibitors, protease inhibitors, integrase inhibitors, and fusion inhibitors [30] . currently, combinations of these different classes of antiretroviral drug are used in what is known as antiretroviral therapy (art) to prevent the development of drug resistance. to date, 22 distinct medications and 22 combination therapies have been approved by the fda, most during the early 2000s [30]. the first nonrepurposed drugs approved by the fda were lamivudine (a non-nucleoside reverse transcriptase inhibitor) and saquinavir (a protease inhibitor), both approved in 1995, nearly 15 years after the start of the pandemic [31] . later during the pandemic, new drugs were developed in a streamlined process where lead compounds active against hiv in vitro were structurally modified to improve their efficacy and lower their cytotoxicity [32] . high-throughput screening (hts) campaigns have also proven useful in identifying existing compounds with promise against hiv [33] [34] [35] . hiv serves as a prime example of how scientists in academia, government, and industry can combine efforts to combat a common threat. even though it took 5 years to name the virus that caused the disease and 6 years to approve the first medication, cumulative scientific efforts ultimately paid off in the form of a diversity of treatment regimens available and a major improvement in life expectancy in most parts of the world [36] . although sars-cov-2 might continue to circulate in humans for some time, it differs from hiv in that it has an airborne transmission route and a lower mutation rate because of polymerase proofreading abilities [27, 37] . the latter is especially important to consider when developing therapies, vaccines, and antiviral medications. a lower mutation rate means that there is a higher probability of new treatments working for lengthier periods of time. it is also important to consider whether an infection is chronic or if recovered individuals can become sick again, examples being hiv and influenza, respectively. epidemics last longer or can recur if the pathogen at hand meets these criteria, thus allowing more time for drug discovery to have a tangible output, such as art for hiv and annual vaccines for influenza. infection with sars-cov-2 is not known to be chronic and neither is it known definitively whether secondary infection can occur [38] , but this or a similar virus is predicted to eventually reemerge, meaning that drugs developed and tested now will be useful for future epidemics [23] . the sustained transmission of hiv within the human population, coupled with its extraordinary evolutionary rate, has provided a lasting incentive for pharmaceutical companies to continue identifying new antiviral medications to treat hiv [30] . similarly, the expectation of another covid-19 pandemic motivates rapid drug discovery efforts now for sars-cov-2. there are multiple efforts to identify existing drugs that could be repurposed to combat sars-cov-2 [39] . additionally, modern computational techniques, such as quantitative structure-activity relationship (qsar) modeling, molecular docking, and machine-learning approaches are being used now in covid-19 drug discovery efforts [17, 40, 41] . it should be expected that achieving success in developing covid-19 therapies and preparing for future epidemics will require a substantial, lasting, and well-funded research effort. the sheer amount of resources at our disposal and the velocity of response of the scientific community so far, compared with the successful response to hiv, suggests that the development of drugs to treat covid-19 in the coming years is attainable. in recent years, there has been a noticeable uptick in the response of the scientific community to outbreaks. we have observed that the ebola (2014-2016) and zika (2015-2016) outbreaks had more publications and clinical trials performed in a shorter time period than for previous outbreaks (figs 1 and 2) . however, the response of the scientific community to the covid-19 outbreak represents the most rapid response yet, with an unprecedented number of clinical trials and publications both in peer-reviewed journals and preprint servers. however, clinical and research efforts in response to past epidemics have failed to yield therapies during or after epidemic period; traditionally, it has taken years for effective drugs and vaccines to be developed and approved, much past the point at which they could be clinically useful and/or could be tested in clinical trials. the speed of the response of the scientific community to major outbreaks has increased over the past two decades matching the increasing accessibility of information via the internet, but the outcome of this response has not changed. despite the presence of various intergovernmental agencies, such as the who and the united nations, individual nations have traditionally responded to large-scale epidemics in a disjointed fashion, and they desperately lack a pre-established, adequate, continuous, and centralized effort even within their own countries, let alone, internationally [23, 42] . in the usa in particular, initial measures to predict or surveil disease outbreaks have been alarmingly either defunded or downsized over this same period in which the output of publications and clinical trials has increased [43, 44] . as seen in the data presented in this study, the viruses that had already been studied before their respective epidemics garnered more publications and clinical trials in a shorter period of time than those that were understudied previously. zika virus and ebola virus had both been discovered long before they caused significant morbidity and mortality around the world; yet, they were not studied extensively, and thus, the correlated response of the scientific community was inefficient [23] . had the scientific community had a consistent stream of funding to conduct research on coronaviruses that have hit humankind twice before the current pandemic, there might have been the prospect of pharmacotherapy on the near horizon [23] . when preparing for possible future epidemics, it is better to be safe than sorry; for example, the us government has a stockpile of tecovirimat, a drug used to treat smallpox, in the event of an act of bioterrorism [45] . this drug was developed solely to prevent a future outbreak, given that smallpox was eradicated decades ago because of global vaccination efforts. similar prescient efforts are underway to prevent the next pandemic, such as the rapidly emerging antiviral drug discovery initiative (readdi). this initiative is aiming to create and stockpile a novel broad-spectrum antiviral drug in preparation for the next pandemic [46] . the history of hiv and aids shows clearly that steady interest and robust investment in the study of the disease have yielded the desired fruits. although the speed of response to covid-19 by both the research community and the public is unprecedented, the world needs to pay more attention to infectious diseases before they have the opportunity to cause lasting economic, social, and physical damage to people around the globe [47] . we hope that the data and their analyses presented in this article will stimulate both the funding agencies (governmental, private, etc.) and the scientific community to maintain their interest in searching for an efficient treatment for covid-19, given the comparison with previous, large-scale epidemics. given the unprecedented increase in clinical trials and publications, the growing public interest in the disease, and the looming threat of a future outbreak, it is unlikely that these trends will die down in the coming months and years. vaccine development for covid-19 is occurring at an unprecedented pace: on average, vaccine development takes 10 years, but there is a hope to have a vaccine available for emergency use by early 2021 [48] . however, global research efforts need to become more focused if we are to combat this pandemic effectively. we believe that the ongoing research and clinical trials should be a product of international and intergovernmental collaboration driven by knowledge discovery and artificial intelligence approaches as applied to data from both past and present epidemics. finally, substantial funding on the part of federal and state agencies, along with private foundations, is necessary to support massive research efforts to find a cure or a vaccine more quickly than in the past, and to stay prepared for future outbreaks of viral diseases. it is not enough to say that there are more resources available at our disposal now than ever; it is a matter of using these resources effectively. the historical response to hiv sets a precedent for success in the fight against emerging infectious diseases. we shall use this historical precedent and past failings as a guide for the current battle against covid-19 and all future battles against other, imminent outbreaks. achievements in public health the cost and challenge of vaccine development for emerging and emergent infectious diseases the benefits and costs of using social distanci q8 ng to flatten the curve for covid-19 the neglected dimension of global security -a framework for countering infectious-disease crises coronavirus social-distancing forces painful choices on small businesses forecasting covid-19 impact on hospital bed-days, icu-days, ventilator-days and deaths by us state in the next 4 months what is google trends data-and what does it mean covid-19 likely to weigh on u.s. election turnout, outcomes coronavirus will change the world permanently. here's how. politico mag jhu (2020) covid-19 dashboard by the center for systems science and engineering worldometer (2020) coronavirus cases statistics and charts an interactive web-based dashboard to track covid-19 in real time coronavirus: why you must act now coronavirus: how media coverage of epidemics often stokes fear and panic misinformation making a disease outbreak worse: outcomes compared for influenza, monkeypox, and norovirus in silico screening of chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus rapid identification of potential inhibitors of sars-cov-2 main protease by deep docking of 1.3 billion compounds. mol a data-driven drug repositioning framework discovered a potential therapeutic agent targeting covid-19 computational models identify several fda approved or experimental drugs as putative agents against sars-cov-2 multiple reassortment events in the evolutionary history of h1n1 influenza a virus since 1918 the domestic and international impacts of the 2009-h1n1 influenza a pandemic: global challenges zika virus: history, emergence, biology, and prospects for control integrating clinical research into epidemic response: the ebola experience chloroquine and hydroxychloroquine in covid-19 advance of promising targets and agents against covid-19 in china comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea a sars-like cluster of circulating bat coronaviruses shows potential for human emergence federal funding for hiv/aids: trends over time a timeline of hiv and aids. hivgov 30 aidsinfo (2018) fda-approved hiv medicines the application of structural optimization strategies in drug design of hiv nnrtis easy-hit: hiv full-replication technology for broad discovery of multiple classes of hiv inhibitors evaluating the efficacy and safety of bromhexine hydrochloride tablets combined with standard treatment/ standard treatment in patients with suspected and mild novel coronavirus pneumonia (covid-19) covid-19 ring-based prevention trial with lopinavir/ritonavir pre-exposure prophylaxis for sars-coronavirus-2 hiv epidemiology and the effects of antiviral therapy on longterm consequences viral evolution and the emergence of sars coronavirus immunity passports' in the context of covid-19 structure of mpro from covid-19 virus and discovery of its inhibitors artificial intelligence and machine learning to fight covid-19 mapping the landscape of artificial intelligence applications against improving health and reducing poverty trump disbanded nsc pandemic unit that experts had praised cdc to cut by 80 percent efforts to prevent global disease outbreak tecovirimat for the treatment of smallpox disease antimicrobial division advisory committee meeting. fda readdi: rapidly emerging antiviral drug discovery initiative. readdi responding to covid-19 -a once-in-a-century pandemic? the covid-19 vaccine development landscape the authors wish to thank d. adalsteinsson and p. schultz for multiple discussions of the capabilities of datagraph software used to create the figures, and kennie merz for the suggestion to add the analysis of the hiv/aids pandemic. the authors also acknowledge support from the national institute of health (grant 1u01ca207160) and biomedical data translator initiative of national center for advancing translational sciences, national institute of health (grants ot3tr002020, ot2r002514). key: cord-284523-lknyehsa authors: da mata, élida cleyse gomes; mourão, caroline barbosa farias; rangel, marisa; schwartz, elisabeth ferroni title: antiviral activity of animal venom peptides and related compounds date: 2017-01-06 journal: j venom anim toxins incl trop dis doi: 10.1186/s40409-016-0089-0 sha: doc_id: 284523 cord_uid: lknyehsa viruses exhibit rapid mutational capacity to trick and infect host cells, sometimes assisted through virus-coded peptides that counteract host cellular immune defense. although a large number of compounds have been identified as inhibiting various viral infections and disease progression, it is urgent to achieve the discovery of more effective agents. furthermore, proportionally to the great variety of diseases caused by viruses, very few viral vaccines are available, and not all are efficient. thus, new antiviral substances obtained from natural products have been prospected, including those derived from venomous animals. venoms are complex mixtures of hundreds of molecules, mostly peptides, that present a large array of biological activities and evolved to putatively target the biochemical machinery of different pathogens or host cellular structures. in addition, non-venomous compounds, such as some body fluids of invertebrate organisms, exhibit antiviral activity. this review provides a panorama of peptides described from animal venoms that present antiviral activity, thereby reinforcing them as important tools for the development of new therapeutic drugs. considering the most common pathologies in humans and other animals, cardiovascular and infectious diseases and cancer are among the leading causes of deaths. the cultural and educational background of affected people largely influences the prevention and treatment of human diseases; nevertheless, the availability of new drugs contributes greatly to mitigating diseases. more than 200 viruses are known to cause human diseases [1, 2] . some of them present high public health importance, such as cytomegalovirus (cmv), epstein-barr virus (ebv), hepatitis b and c viruses (hbv and hcv, respectively), herpes simplex virus (hsv), human immunodeficiency virus (hiv), rabies virus and ebola virus. the most recent worldwide estimates presented by the world health organization (who) reported 1.5 million deaths caused by hiv in 2012, 400 million people living with hepatitis b or c, 80% of liver cancer deaths caused by hepatitis viruses, 500 thousand cases of cervical cancer caused by hpv infection, and over 250 thousand cervical cancer deaths each year [3] . the very few antiviral drugs commercially available can induce severe and considerable adverse effects, especially to those patients receiving lifelong treatment for diseases such as hiv. furthermore, viruses possess rapid mutational capacity to trick and infect host cells. all these facts together have propelled the prospection for new antiviral drugs, particularly from natural products, as they constitute more than 25% of the new drug prototypes approved in the last decades [4] . among sources of natural products, animal venoms have revealed a great potential for drug discovery [5] [6] [7] , and despite the harmful action mechanism of animal venoms, most of them have components holding potential medicinal properties to cure diseases. it is widely reported in the literature that animal venoms are rich sources of antimicrobial substances, and contain a vast array of active biological compounds with distinct chemical structures [8] . thus, antimicrobial peptides (amps)a diversified group of peptides that exert essential function in the innate immune host response, when invaded by pathogenic organisms, such as bacteria, fungi and virusare considered the first line of defense of many organisms, including plants, insects, bacteria and vertebrates [9, 10] . some peptides exhibit direct virucidal activity; others disturb attachment of virus particles to the cell membrane surface or interfere with the virus replication. because of the limited efficiency of commonly used drugs and emerging resistance of viruses, antiviral peptides may have the potential for development as putative therapeutic agents [11] . in addition to their reduced market availability, the collateral effects and toxicity of the synthetic antiviral drugs have triggered an expanded search for natural compounds displaying antiviral activities [12, 13] . any compound to be utilized as an antiviral should comply with the virus pathways during the cellular infectious cycle. initially, any rna or dna virus, enveloped or not, expresses glycoproteins that are responsible for the interaction with surface molecules, receptors, usually glycosylated proteins, integrated in the host cell membrane. at this step, any potential antiviral candidate must compete for the cell receptor by inhibiting the virus attachment to the cell membrane, thereby aborting the viral infection. other candidates may act intracellularly by interacting with the virion capsid to prevent its decapsidation; therefore, the viral nucleic acid would not be freed and transcribed. concerning retroviruses, the antiviral candidates can act by inhibiting (i) the viral reverse transcriptase activity; (ii) the pre-integration complex, thus avoiding the transport of circular viral dna to the nucleus; (iii) and also by inhibiting the action of the viral integrase, which would not allow the viral dna to integrate into the cellular chromosome. the proviral dna, after transcription, is transduced into a polyprotein that requires the viral protease in order to generate small proteins to assemble the viral capsid. in this manner, an antiviral compound could inhibit the viral protease by blocking the retroviral morphogenesis ( fig. 1) [14] . some retroviral proteins play a major role in the pathogenesis, by down regulation of cd4 and mhc molecules of the host cell, driving them to the proteasome for degradation. if supposed antiviral candidates target these viral proteins, hiv-1 nef, tat and vpr, their actions can be restrained. all the mentioned mechanisms are directly performed by retroviral molecules [15] , but other mechanisms could also be triggered, such as those involved in the innate immune system, e.g. (i) the induction of toll-like receptor expression, that interacts fig. 1 action mechanism of animal venom peptides or derivatives at different retrovirus replication cycle phases. (1) the chtx and scyllatoxin-based mimetics, such as cd4m33, inhibit the attachment of the viral glycoprotein (gp120) to the host cell receptor cd4. (1a) the peptides cecropin a, magainin 2, papuamide a, dermaseptin ds4, caerins 1.1 and 1.9 and maculation 1.1 disintegrate the viral envelope. (1b and 1c) the peptides cd4m33, bmkn2, kn2-7, polyphemusin, tachyplesin, immunokine and p3bv obstruct the interaction of the viral gp 120 to the cxcr4 and ccr5 co-receptors. ( 2) the peptides miramides a-h inhibit the fusion of the viral envelope to the host cell membrane. (3) the peptides melittin, didemnis a, b and c interfere with the reverse transcription process, aborting the synthesis of double-stranded viral dna. (6) the peptides hecate and tvs-lao act in the post-translation process, in the cleavage of the gag/pol protein precursor thus interfering in the assembly of the viral capsid and in the organization of the polymerase complex with viral nucleic acid, or (ii) production of cytokines that stimulate the action of t cytotoxic cells, and nk cells, and even host cell expression of the major histocompatibility complex molecules, in order to present viral peptides to the other cells of the immune system [16] . furthermore, antiviral compounds may activate innate restriction factors coded by the host cell [17] . the viral dna integration in the host cell chromosome represents the major problem to be overcome in a retroviral infection. until now, there is no available drug capable of completely clearing the virus from the host [18] . furthermore, silent retroviral infection is hidden at anatomical sites that are difficult to reach by drugs, such as the gut-associated lymphoid tissues, lymph nodes and central nervous system. infected cells, including macrophages, are quiescent in these tissues and it is not known when they will activate and release new viral progenies. another challenge for an antiviral candidate is posed by the mutation rate of viral genes, mainly among rna virus, due to the polymerase synthesis error. this is much more intriguing among retroviruses, as the initial virion genome, maintained in quiescent cells in "sanctuary niche", are distinct, mutated from each round of cell infection. thus, in each cycle of viral infection, the hijacked cell produces a growing number of recombinant new virions [19] . the arachnid venoms, utilized as a tool for defense and attack, by killing or immobilizing their prey for feeding or their possible competitors and predators, are composed of a rich molecular diversity and complex mixture, with an intricate protein and peptide expression by mechanisms of gene regulation still under investigation [20, 21] . scorpion venoms have been exhaustively studied, mainly due to the clinical effects after envenomation in humans, which sometimes lead to death [22] . paradoxically, biotechnological applications are devised by the increased understanding of the action mechanisms of venom components, and therefore, many research works deal with the generation of new drugs based on the structure and function of molecules found in these venoms [23] [24] [25] . with the rapid increase in the number of characterized scorpion venom compounds, many new drug candidates have been identified as potential medicines to deal with emerging medical global threats [8, 20] . in scorpions the biologically active peptides are classified as disulfidebridged peptides (dbps) and non-disulfide-bridged peptides (ndbps) [26, 27] , with the former being the main components of scorpion venoms, responsible for the neurotoxic symptoms and signs observed during scorpionism. usually these dbps target the ion channels of excitable and non-excitable cell membranes. these properties make these molecules interesting prototypes of drugs for the treatment of diverse diseases, particularly those affecting the neural system [8] . in relation to the activity of scorpion venom compounds against retroviruses, such as hiv/siv, it has been reported that some dbps can bind to hiv gp120 glycoprotein due to molecular mimicry of lentiviruses host cell cd4 + receptor. as a result, they abolish the gp120-cd4 interaction, which is essential to initiate the conformational changes in the viral envelope that trigger viral entry into host cells [28] . these cd4 mimetic scorpion toxins contain about 30 amino acid residues, with three or four disulfide bridges, characterized by the cysteine-stabilized α/β motif (cs-α/β), in which a β-turn between the two β-strands in these peptides resembles the cdr 2 loop of cd4. both charybdotoxin (chtx) and scyllatoxin, isolated from leiurus quinquestriatus hebraeus venom, present the cs-α/β motif and are capable of blocking k + channels [29] [30] [31] [32] . these toxins have been used effectively as molecular scaffolds for gp120-cd4 interaction assays [28, 33, 34] . since the amino acid residues phe 43 and arg 59 of cd4 were shown to be critical for cd4 binding to gp120, equivalent amino acid residues were added to the new compounds. examples of mimetic peptides using chtx as a scaffold include cd4m and txm1, with 33 and 32 amino acid residues, respectively [33, 35] . among the main modifications, the cd4 cdr 2 loop sequence 40 qgsf 43 was inserted in the equivalent position of the β-turn of chtx. thus, phe 28 of cd4m, or phe 27 of txm1, would function as phe 43 in cd4. the remaining sequence is similar between the two analogs, except in two positions: arg 20 in txm1 (arg 25 in chtx) is replaced by lys in cd4m, and txm1 has a gly 1 as the n-terminal residue in place of val 1 -ser 2 residues in cd4m. thus, the charged n-terminus of the gly 1 residue in txm1 is in a position similar to that of the charged side-chain of arg 59 in cd4 [33] . cd4m was able to inhibit gp120 binding to cd4 with an ic 50 value of 20 μm [35] . likewise, txm1 also competed with cd4 for gp120 binding, besides causing a cd4-like enhancement in gp120 binding to the antibody 17b [33] . subsequently, other cd4 mimetics exhibiting gp120 affinity were successfully generated by phage epitope randomization of the β-turn loop in a chtx-based scaffold [28] . as to scyllatoxin scaffold-based mimetics, a 27-amino acid residue miniprotein named cd4m3 was constructed, which inhibited cd4 binding to gp120 with an ic 50 value of 40 μm [34] . structural and functional analysis performed with cd4m3 suggested additional mutations that, once incorporated in the new compound (cd4m9), caused an increased affinity for gp120, with ic 50 values of 0.1-1.0 μm, depending on the viral strains. additionally, cd4m9 inhibited infection of cd4 + cells by different hiv-1 strains [34] . its β-turn sequence ( 20 agsf 23 ) is similar to that of txm1. after that, based on cd4m9 structural analysis, a potent mimetic with bona fide cd4-like properties was synthesized [36] . denominated cd4m33, it inhibited cd4-gp120 binding in different viral strains with 4.0-7.5 nm ic 50 , with these values being comparable to those obtained with cd4. cdm33 also inhibited hiv-1 cell-cell fusion and infection of cells expressing cd4 and either the ccr5 or cxcr4 co-receptors at similar concentrations to cd4 [36] . its three dimensional structure was further analyzed in complex with gp120 [37] . then, another analog was designed, denominated f23, which differs from cd4m33 due to the presence of phe 23 in replacement by biphenylalanine in position 23 (bip 23 ). the authors showed that f23 had higher mimicry of cd4 than cd4m33. in addition, f23 presented increased neutralization against isolates of phylogenetically related primate lentiviruses [37] . the scorpion venom amps belong to ndbps; many of them and their analogs exert strong antiviral activity, as shown in table 1 . some of these compounds act by direct rupture of the viral envelope, thereby decreasing viral infectivity [8] . amps could also prevent or block the virion from entering into the cell by occupying cell receptors utilized by the viral glycoproteins [38] . other amps do not compete with viral glycoproteins to get attached to cell receptors. instead, they can cross the cell lipoprotein membrane and internalize themselves in the cytoplasm and organelles, yielding alterations in the profile of host cells that can enhance the defense against the virus or may also block the expression of viral genes in the host cell, halting viral dissemination to other cells [9] . mucroporin is a cationic 17-amino-acid residue amp isolated from lychas mucronatus venom. one of its derivatives, named mucroporin-m1, has an enhanced net positive charge, and besides having antibacterial activity, presented antiviral activity against measles, sars-cov and influenza h5n1 viruses (table 1) , possibly through a direct interaction with the virus envelope [39] . additionally, it has been shown to reduce the production of hbv antigens and viral dna in cell culture microenvironment and also to hinder hbv infection in mouse models [40] . the molecular mechanism implicated reveals the specific activation of mitogen-activated protein kinases (mapks) leading to down-regulation of hnf4α expression and consequently less binding to the hbv pre-core/core promoter region [40] . mucroporin-m1 also presented anti-hiv-1 activity [38] . an amphipathic α-helical peptide, hp1090, was screened from the cdna library of heterometrus petersii venomous gland. this 13-amino-acid residue ndbp inhibited the hcv infection (table 1) , acting as a viricide against hcv particles and preventing the initiation of hcv infection by permeabilizing the viral envelope and decreasing virus infectivity [41] . also from h. petersii venom gland cdna library, other α-helical ndbps were synthesized. two of them, hp1036 and hp1239, exhibited potent virucidal activity against hsv-1 (table 1 ) [42] . they showed inhibitory effects on multiple steps of the virus replication cycle, caused the destruction of the viral morphology and also entered the infected cells where they reduced viral infectivity. from the cdna library of mesobuthus martensii venom gland, a compound denominated bmkn2with 13 amino acid residueswas cloned and synthesized. based on its sequence, kn2-7 was designed by making ctry2459-h2 hcv 1.08 μg/ml [43] ctry2459-h3 hcv 0.85 μg/ml [43] the substitutions g3k, a4r and s10r, enhancing its net positive charge and α-helix structure [38] . both compounds exerted anti-hiv-1 activity through inhibition of chemokine receptors ccr5-and cxcr4-mediated activities and replication of the viruses, of which kn2-7 was the most potent (table 1 ) [38] . another ndbp, screened from chaerilus tryznai scorpion venom gland, ctry2459, was able to inhibit initial hcv infection in huh7.5.1 cells by inactivating infectious viral particles (table 1 ) [43] . however, due to the low bioavailability of this 13-amino-acid residue peptide, ctry2459 could not suppress an established infection. thus, in order to enhance the helicity, amphiphilicity and endosomal escape of peptides, the authors designed histidine-rich peptides based on a ctry2459 template. denominated ctry2459-h2 and ctry2459-h3, they were more effective against hcv than ctry2459 (table 1) , significantly reducing intracellular viral production. unlike ctry2459, these analogs reduced the viral rna by 40 and 70%, respectively; however, ctry2459 diminished viral infectivity in a manner similar to that of wild-type peptide [43] . recently, the antiviral activities of scorpio maurus palmatus and androctonus australis crude venoms were shown against hcv. they presented ic 50 values of 6.3 ± 1.6 and 88.3 ± 5.8 μg/ml, respectively. s. maurus palmatus venom was considered a good natural source for characterizing new anti-hcv agents targeting the entry step, since it impaired hcv infectivity in cell culture, but not intracellularly, through a virucidal effect. this effect was not inhibited by a metalloprotease inhibitor or heating at 60°c [44] . snake venoms are composed of a mixture of proteins, peptides (90-95%), free amino acids, nucleotides, lipids, carbohydrates and metallic elements coupled to proteins (5%) [45] . some studies have reported the antiviral activity of snake venoms and their components against measles virus, sendai virus, dengue virus (denv), yellow fever virus (yfv) and hiv [46] [47] [48] [49] [50] . thus, snake venoms are sources of promising candidates for new antiviral drugs ( table 2 ). in relation to antiretroviral activity, the benefits of treating a patient with multidrug-resistant hiv with a snake venom preparation in addition to the antiretroviral therapy were demonstrated in clinical practice [51] . the response was a decreased viral load and elevated t cd4 + cell count. the authors suggest that this activity may be related to the presence of some snake venom molecules that are homologous to hiv-1 glycoprotein or proteases [51, 52] . this homology occurs between the 30-40 highly conserved amino acid residues of snake venom neurotoxins long loop and the sequence 164-174 of short segment hiv-1 gp120. as a result, both may compete for the same receptor or binding site and present anti-hiv activity [50] . the sequence homology between hiv gp120 and snake neurotoxins, such as cobratoxin and bungarotoxin, had generated some antiretroviral patents [53] [54] [55] . linking the gp120 fragment to the hiv peptide fusion inhibitors (fragments of gp41 ectodomains) was shown to improve their anti-hiv efficacy [56] . besides structural homology, other action mechanisms of snake venoms against hiv are also discussed in the literature, such as catalytic/inhibitory activity through enzymes, binding interference (receptor/enzyme), and induction/interaction at the membrane level [50] . the l-amino acid oxidases (laaos or laos, ec1.4.3.2), which constitute one of the most studied main components of snake venoms, are oxidoreductase flavoenzymes with molecular masses around 110 to 150 kda and are usually non-covalently linked homodimeric glycoproteins [57, 58] . these compounds are widely distributed in other organisms and play an important role in biological activities such as apoptosis induction, cytotoxicity, inhibition or induction of platelet aggregation, hemorrhaging, hemolysis and edema, as well as anti-hiv, antimicrobial and antiparasitic activities [59] . tsv-lao, characterized from trimeresurus stejnegeri snake venom, seems to be the first snake venom lao reported to present antiviral activity ( table 2 ) [60] . tsv-lao is a glycoprotein with a molecular weight of about 58 kda that also forms homodimers, similarly to laos from other snake venoms. its precursor sequence, obtained by cdna analysis, codes for a polypeptide of 516 amino acid residues, including an 18-amino-acid potential signal peptide that is identical to those of laos from other snake species. tsv-lao inhibited hiv-1 infection and replication in a dose-dependent manner, and seems to act at nanomolar concentrations by inhibiting syncytium formation (ec 50 of 1.5 nm) and hiv-1 p24 antigen expression (ec 50 of 4.1 nm) [60] . additionally, another lao, isolated from bothrops jararaca venom and denominated bjarlaao-i (table 2) , reduced the viral load in cells infected with dengue virus type 3 strain exposed to the toxin in comparison to controls [61] . its cdna-deduced sequence has 484 amino acid residues and is similar to other snake venom laos. these flavoenzymes also produce hydrogen peroxide (h 2 o 2 ) as a free radical, which appears to enhance their antiviral activity [60] . other compounds found in snake venoms that exhibit antiviral activity are the phospholipases a 2 (pla 2 ). among their biological effects, they seem to interact with the host cells and prevent the intracellular release of virus capsid protein, suggesting that they block viral entry into the cells before virion uncoating [7, 49, 62] . the pla 2 isolated from crotalus durissus terrificus venom (pla 2 -cdt, hiv human immunodeficiency virus, hsv herpes simplex virus, iav influenza virus, vsv vesicular stomatitis virus, denv dengue virus. adapted from jenssen et al. [9] and mulder et al. [77] inhibited both denv and yfv in vero e6 cells [48] . this pla 2 is part of crotoxin, a heterodimeric protein composed of two different subunits non-covalently linked: the basic pla 2 (~16.4 kda) and the acidic protein crotapotin (~9.0 kda) [48] . the mechanism proposed for pla 2 -cdt antiviral activity involves the cleavage of the glycerophospholipid virus envelope and protein destabilization on the virion surface, which partially exposes the genomic rna and culminates with viral inactivation, making it unable to access the cell receptor [63] . pla 2 -cdt also showed in vitro activity against hiv (table 2 ) [62, 64] , as well as the snake venom pla 2 s nmmcm iii from naja mossambica mossambica, taipoxin from oxyuranus scutellatus, and nigexine from naja nigricollis [49] . additionally, the pla 2 variants, lys49 and asp49, denominated blk-pla 2 and bld-pla 2 , from bothrops leucurus venom (table 2) , reduced dengue viral rna in cells treated with these compounds, and presented cytotoxic activity against denv-infected cells in vitro [65] . blk-pla 2 and bld-pla 2 have 121 and 122 amino acid residues, respectively, including seven disulfide bonds. another example of the antiviral effect of biomolecules extracted from snake venoms are the metalloprotease inhibitors, which could prevent the production of new hiv particles by inhibiting the viral proteases [50] . in addition, immunokine® (oxo chemie, thailand), an oxidized derivative of the α-toxin extracted from naja siamensis venom (table 2) , has been shown to inhibit infection of lymphocytes by hiv through the chemokine receptors ccr5 and cxcr4 [7, 66] . many reports detail potent antiviral activity of amphibian skin secretions. such skin secretions constitute the amphibians' first line of defense, consisting of their innate immunity. the secretions produced by the anuran skin granular glands have been screened for many biological activities, including antimicrobial, antineoplastic, antiviral, contraceptive and anthelminthic activities [67, 68] . the dermaseptin family of antimicrobial peptides comprise 24-34 amino acids, exhibiting a linear polycationic molecule disposed as an amphiphilic α-helical structure when associated with a lipid cell bilayer. bergaoui et al. [69] described the dermaseptin s 4 , a chemically synthesized 28-amino-acid drug derived from an amphibian skin antimicrobial peptide, exhibiting anti-herpetic activity (hsv type 2), with reduced cytotoxic effects after biochemical modifications of the original peptide. it also reduced in vitro hiv-1 infection of an established cell line, p4-ccr5, expressing cd4, ccr5, and cxcr4 hiv-1 cell receptors and, primary t lymphocytes, being capable of acting on both r5 and x4 tropic hiv-1 virions. upon insertion in the viral envelope, the dermaseptin s 4 disrupts the virion [69] . caerin 1.1, caerin 1.9 and maculatin 1.1, peptides also derived from the skin secretions of the amphibians litoria caerulea, litoria chloris and litoria genimaculata, respectively, completely abolished hiv infection of t cells, after a few minutes of virion exposure to these modified peptides, which disintegrates the viral envelope, preventing viral fusion to the cell membrane. furthermore, these molecules obstructed viral transfection from dendritic cells to t cells. caerin peptides are composed of 25 amino acid residues in their structure, including four central amino acid residues not present in maculatin peptides. in lipid bilayer membranes, these peptides are adjusted to two α-helices, interlinked by a flexible hinge region limited by pro 15 and pro 19 , which determine the disruption of viral envelope and cell membrane [70] . mastoparan is a tetradecapeptide present in wasp (vespula lewisii) venom [71] that forms amphipathic helical structures that insert into lipid bilayers of bacteria, erythrocytes, mast cells and others, forming pores [72, 73] . mastoparan-7, a mastoparan analogue, displayed a wide spectrum of antiviral activity against enveloped viruses of five different families (rhabdoviridae, poxviridae, flaviridae, paramyxoviridae and herpesviridae) in in vitro assays ( table 2 ). structural studies have indicated pore formation by the insertion of the mastoporan amphiphilic α helix into the viral lipidic envelope, causing its disruption [74] . hiv virions usually infect the host cells in the genital mucosae, by infecting macrophages, being denominated m-tropic virus; after migrating to the lymph nodes, they infect t lymphocytes, changing into t-tropic virus [75] . based on the hiv tropism, a phospholipase a 2 from bee venom, bvpla 2 , blocked the replication of both m and t-tropic hiv virions [65] , while a small peptide derived from bvpla 2 , the p3bv, exclusively inhibited the replication of t-tropic virus, behaving as a ligand for the hiv-1 co-receptor cxcr4 [49, 76] (table 2) . amps isolated from invertebrate organisms presented augmented antiviral activity in human diseases. such peptides enclose melittin, cecropin and alloferon molecules [77] (table 2) . melittin, isolated from honey bee (apis mellifera) venom, is an amphipathic peptide composed of 26 amino acid residues, arranged in two α helical segments. inserted in nanoparticles, melittin exhibited virucidal activity against hiv-1 in the vk2 cell line, an epithelial vaginal cell line, and also inhibited hiv infection in tzm-bl reporter cells (hela cell line expressing hiv receptors) [78] [79] [80] . among other antiretroviral mechanisms, melittin complemented the azidovudin reverse transcription inhibition [81, 82] . hecate, an analogue of melittin, selectively reduced the protein biosynthesis of virus-specified glycoproteins b, c, d, and h of the hsv type 1 [83] . the mechanism is similar to the one detected among hiv-1 infected lymphoblastic cells, previously treated with melittin, by the intervention in the processing of the gag/pol protein precursor. therefore, specific intracellular events are targeted by melittin and its derivatives [82, 84] . cecropins, isolated mostly from the hemolymph of infected pupae of the silk moth hyalophora cecropia, but also from other insects, tunicates and ascaris nematodes, are a family of amps, containing 35-37 amino acid residues arranged in two amphiphilic α-helices linked by a gly-pro hinge. synthetic hybrid peptides, namely cecropin a (1-8)-magainin 2 (1-12), exhibited potent antiviral activity by a mechanism mainly based on the compound hydrophobicity and α-helical content, inhibiting the virushost cell fusion [85] (table 2) . alloferon 1 and 2 are peptides constituted of 12-13 amino acid residues, isolated from the hemolymph of the blowfly calliphora vicina. alloferons exert immunomodulatory activities to control infection by the human influenza virus in mice model of lethal pulmonary infection [75] , whereas their derivatives also inhibited in vitro hsv replication in vero cells [86, 87] (table 2 ). these peptides also displayed a relevant role in the innate immunity, being considered prospective peptides for the pharmaceutical industry [88, 89] . sea organisms are also promising sources of antiviral cationic peptides. they present a broad spectrum of antiviral activity, while one single peptide may present activity against different viruses and other pathogens. the promiscuous antifreeze pa-map peptide, which consists of an α-helix composed of 11 amino acid residues, was isolated from the polar fish pleuronectes americanus ( table 2 ). the pa-map exerted antimicrobial activity against bacteria, fungi, neoplastic cells, and also interacted with the viral envelope of the hsv types 1 and 2, inhibiting the infection of susceptible cells [77, [90] [91] [92] . some sponge species contain linear or cyclic bioactive peptides composed of atypical amino acid residues, generating unique structures that are rarely found in terrestrial organisms [90, 93] . these compounds, particularly the cyclic depsipeptides mirabamides a-h, isolated from siliquaria spongia mirabilis and stelletta clavosa, obstruct the hiv-1 virion entry into tzm-bl cells, thus neutralizing the viral glycoprotein fusion for expressing cd4 and ccr5 hiv cell receptors [94, 95] (table 2 ). peptide concentrations between 40 and 140 nm were sufficient to inhibit infection by 50% (ic 50 ). another cyclodepsipeptide, homophymine a, obtained from homophymia sp., conferred 50% cell protection at 75 nm concentration against hiv-1 infection in vitro [96] (table 2) . discovered in the early 1980s, didemnins a, b and c from the caribbean tunicate trididemnum solidum were the first antiviral marine depsipeptides described. didemnins were effective against vaccinia virus, hsv type 1 and 2, coxsackie virus a-21 and equine rhinovirus, presenting strong activity at low doses [97] . furthermore, these peptides were active in in vivo assays in a rat model infected with herpes simplex virus, reducing the skin lesions after topical administration [98] . didemnins inhibit protein, dna and rna synthesis in cells [99, 100] . the protein synthesis inhibition mechanism may be related to the binding of didemnins to the elongation factor 1 alpha (ef-1 alpha) [101] . didemnin b underwent phases i and ii of clinical trials in the 1980s, but presented low selectivity and therapeutic index, as well as toxic side effects [102] . dehydrodidemnin b (aplidin®, pharma mar sa, spain) is currently under phase iii of clinical trials as an anticancer drug against multiple myeloma and t-cell lymphoma [103] . several antiviral peptides and depsipeptides have been described in marine sponges from the genus theonella sp.: koshikamides f and h isolated from t. swinhoei and t. cupola [104] ; papuamides a and b, and theopapuamide a from theonella sp. and t. swinhoei, respectively [105] [106] [107] . all of them inhibited hiv entry into t cells. theopapuamide b was isolated from an indonesian sponge, siliquariaspongia mirabilis, and was also able to inhibit hiv-1 entry into host cells [108] . papuamide a presented antiviral activity not only against hiv-1, but also against vesicular stomatitis virus and amphotropic murine leukemia virus. due to its tyrosine residue and the presence of a hydrophobic tail, the peptide may insert into the viral membrane, causing its rupture [105] . other peptides from marine sponges that inhibit hiv-1 entry into host cells are: callipeltin a, isolated from sponges of the genus callipelta, which displayed antiviral activity with a high selectivity index (29) between the virus and host cells (si ratio 50% cytotoxic dose [cd 50 ]/ed 50 ) [109] ; celebesides a-c from siliquariaspongia mirabilis [108] ; neamphamide a, from neamphius huxleyi, a compound with structural similarities to callipeptins and papuamides that exhibited low toxicity to host cells and a selectivity index above 10 [110] ; and microspinosamide, isolated from sidonops microspinosa [111] . marine arthropod species have also yielded antiviral peptides, tachyplesin and polyphemusin (t140), and shown anti-hiv-1 activity by attachment to the chemokine receptor, cxcr4, which is also the viral t cell co-receptor. hemocytes of horseshoe crabs (tachypleus tridentatus and limulus polyphemus) are an abundant source of tachyplesin and polyphemusin. the tachyplesin consists of 17-18 amino acid residues, primarily arranged in three tandem repeats of a tetrapeptide, hydrophobic amino acid-cys-aromatic amino acid-arg and an amidated c-terminus, while the polyphemusin analog, t140, is composed of 14 amino acid residues, exposing an antiparallel β-sheet conformation stabilized by a disulfide bridge between cys 4 and cys 13 [112, 113] . as a consequence of the scarcity of new families of antiviral drugs, pharmaceutical companies have strengthened their efforts to increase developments of known current drugs, resulting in little or even no improvement to the existing therapies. these new patent protections guarantee the rights to the same stakeholders who are charging high consumer prices due to the lack of competition [114] . at the same time, the growing demand for new drugs and natural therapeutic products is a matter of extreme necessity to face the emergency of multiresistant viral pathogens. more than 45 compounds obtained from vertebrate and invertebrate organisms presented in vitro or in vivo antiviral activity. although none of those has yet been launched on the market as an antiviral drug, they present chemical structures completely different from the current drugs used in therapy, despite acting on similar targets. those compounds may lead to new classes of therapeutic drugs after additional chemical and pharmacological studies. emerging and reemerging viruses of medical relevance challenge health authorities all around the planet. some viral vaccines have taken too long to be designed and approved for human and animal utilization, and even in some cases could not be developed. preventive and curative measures should always be in the hands of health authorities to ensure control of epidemics, such as the recent ebola virus in africa or arboviruses, particularly in brazilrepresented by the dengue, chikungunya and zika virusesor worldwide pandemics, such as influenza and hiv. therefore, prospection, screening and all other phases of biological activity, validation, clinical development of animal peptides represent an essential scientific investment for protecting and perpetuating humankind. human viruses: discovery and emergence outbreaks of ebola virus disease in africa: the beginnings of a tragic saga world health organization (who) antiviral drug discovery: broad-spectrum drugs from nature bufotenine is able to block rabies virus infection in bhk-21 cells synergic effects between ocellatin-f1 and bufotenine on the inhibition of bhk-21 cellular infection by the rabies virus mechanisms of virus resistance and antiviral activity of snake venoms scorpion peptides: potential use for new drug development peptide antimicrobial agents antimicrobial peptides peptides -a new strategy for combating viral infections incidence rate of modifying or discontinuing first combined antiretroviral therapy regimen due to toxicity during the first year of treatment stratified by age aids -an old disease with new challenges illustrations of the hiv life cycle hiv life cycle, innate immunity and autophagy in the central nervous system innate immunity against hiv-1 infection retroviral restriction factors and infectious risk in xenotransplantation targeting hiv transcription: the quest for a functional cure overcoming pharmacologic sanctuaries scorpion venom components as potential candidates for drug development molecular diversity of spider venom epidemiology of scorpionism: a global appraisal chlorotoxin: structure, activity, and potential uses in cancer therapy toxin modulators and blockers of herg k + channels from the stretcher to the pharmacy's shelf: drug leads from medically important brazilian venomous arachnid species scorpion venom components that affect ion-channels function scorpion venom peptides with no disulfide bridges: a review phage randomization in a charybdotoxin scaffold leads to cd4-mimetic recognition motifs that bind hiv-1 envelope through non-aromatic sequences charybdotoxin, a protein inhibitor of single ca 2+ -activated k + channels from mammalian skeletal muscle structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent k + channel purification and characterization of a unique, potent inhibitor of apamin binding from leiurus quinquestriatus hebraeus venom leiurotoxin i (scyllatoxin), a peptide ligand for ca 2+ -activated k + channels. chemical synthesis, radiolabeling, and receptor characterization conformational changes of gp120 in epitopes near the ccr5 binding site are induced by cd4 and a cd4 miniprotein mimetic rational engineering of a miniprotein that reproduces the core of the cd4 site interacting with hiv-1 envelope glycoprotein engineering a cd4 mimetic inhibiting the binding of the human immunodeficiency virus-1 (hiv-1) envelope glycoprotein gp120 to human lymphocyte cd4 by the transfer of a cd4 functional site to a small natural scaffold rational design of a cd4 mimic that inhibits hiv-1 entry and exposes cryptic neutralization epitopes scorpion-toxin mimics of cd4 in complex with human immunodeficiency virus gp120 crystal structures, molecular mimicry, and neutralization breadth anti-hiv-1 activity of a new scorpion venom peptide derivative kn2-7 virucidal activity of a scorpion venom peptide variant mucroporin-m1 against measles, sars-cov and influenza h5n1 viruses mucroporin-m1 inhibits hepatitis b virus replication by activating the mitogen-activated protein kinase (mapk) pathway and down-regulating hnf4α in vitro and in vivo a new natural a-helical peptide from the venom of the scorpion heterometrus petersii kills hcv inhibitory activity and mechanism of two scorpion venom peptides against herpes simplex virus type 1 design of histidine-rich peptides with enhanced bioavailability and inhibitory activity against hepatitis c virus virocidal activity of egyptian scorpion venoms against hepatitis c virus snake venomics. strategy and applications inhibitory potential of crotalus durissus terrificus venom on measles virus growth selective lysis of virus-infected cells by cobra snake cytotoxins: a sendai virus, human erythrocytes, and cytotoxin model crotoxin and phospholipases a 2 from crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses secreted phospholipases a 2 , a new class of hiv inhibitors that block virus entry into host cells hypothesis of snake and insect venoms against human immunodeficiency virus: a review snake venom preparation for drug-resistant human immunodeficiency virus re: snake venom preparation for drug-resistant human immunodeficiency virus modified venom and venom components as antiretroviral agents pan-antiviral peptides for protein kinase inhibition modified venom and venom components as antiretroviral agents peptide fusion inhibitors targeting the hiv-1 gp41: a patent review snake venom l-amino acid oxidases: trends in pharmacology and biochemistry crystal structure of laao from calloselasma rhodostoma with an l-phenylalanine substrate: insights into structure and mechanism past decade study of snake venom l-amino acid oxidase molecular characterization of trimeresurus stejnegeri venom l-amino acid oxidase with potential anti-hiv activity antiviral and antiparasite properties of an l-amino acid oxidase from the da mata et al snake bothrops jararaca: cloning and identification of a complete cdna sequence secreted phospholipases a 2 (spla 2 ): friends or foes? are they actors in antibacterial and anti-hiv resistance? phospholipase a2 isolated from the venom of crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope use of a phospholipase a 2 for the preparation of pharmaceutical and/or cosmetic compositions for the local and/or systematic treatment and/or prevention of diseases and/or processes caused by intraand extracellular pathogens expressing membrane phospholipids molecular characterization of lys49 and asp49 phospholipases a 2 from snake venom and their antiviral activities against dengue virus therapeutic alternatives from venoms and toxins bioactive molecules from amphibian skin: their biological activities with reference to therapeutic potentials for possible drug development host-defense peptides of the skin with therapeutic potential: from hagfish to human in vitro antiviral activity of dermaseptin s 4 and derivatives from amphibian skin against herpes simplex virus type 2 antimicrobial peptides from amphibian skin potently inhibit human immunodeficiency virus infection and transfer of virus from dendritic cells to t cells a new mast cell degranulating peptide "mastoparan" in the venom of vespula lewisii new insight into the mechanism of action of wasp mastoparan peptides: lytic activity and clustering observed with giant vesicles the effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and cd spectroscopy a mastoparan-derived peptide has broad-spectrum antiviral activity against enveloped viruses m-tropic hiv envelope protein gp120 exhibits a different neuropathological profile than t-tropic gp120 in rat striatum a peptide derived from bee venom-secreted phospholipase a 2 inhibits replication of t-cell tropic hiv-1 strains via interaction with the cxcr4 chemokine receptor current scenario of peptide-based drugs: the key roles of cationic antitumor and antiviral peptides nanoparticulate-based contraceptive/anti-hiv. composition and methods cytolytic nanoparticles attenuate hiv-1 infectivity recent advances in developing insect natural products as potential modern day medicines. evid based method and composition for the treatment of mammalian hiv infection three valuable peptides from bee and wasp venoms for therapeutic and biotechnological use: melittin, apamin and mastoparan an amphipathic alpha-helical synthetic peptide analogue of melittin inhibits herpes simplex virus-1 (hsv-1)-induced cell fusion and virus spread influence of amphipathic peptides on the hiv-1 production in persistently infected t lymphoma cells structure-antiviral activity relationships of cecropin a-magainin 2 hybrid peptide and its analogues studies of insect peptides alloferon, any-gs and their analogues. synthesis and antiherpes activity novel analogs of alloferon: synthesis, conformational studies, pro-apoptotic and antiviral activity antiviral and antitumor peptides from insects anti-tumor activity of immunomodulatory peptide alloferon-1 in mouse tumor transplantation model marine peptides: bioactivities and applications structural and functional characterization of a multifunctional alanine-rich peptide analogue from pleuronectes americanus in vivo antimicrobial evaluation of an alanine-rich peptide derived from pleuronectes americanus bioactive peptides from marine sources: pharmacological properties and isolation procedures mirabamides e-h, hiv-inhibitory depsipeptides from the sponge stelletta clavosa mirabamides a-d, depsipeptides from the sponge siliquariaspongia mirabilis that inhibit hiv-1 fusion homophymine a, an anti-hiv cyclodepsipeptide from the sponge homophymia sp didemnins: antiviral and antitumor depsipeptides from a caribbean tunicate didemnins a and b. effectiveness against cutaneous herpes simplex virus in mice biochemical and cellular effects of didemnins a and b mechanism of action of didemnin b, a depsipeptide from the sea gtp-dependent binding of the antiproliferative agent didemnin to elongation factor 1 alpha the discovery and development of marine compounds with pharmaceutical potential an overview of the marine natural products in clinical trials and on the market mutremdamide a and koshikamides c-h, peptide inhibitors of hiv-1 entry from different theonella species characterizing the anti-hiv activity of papuamide a sponges theonella mirabilis and theonella swinhoei collected in papua new guinea insights into an unusual nonribosomal peptide synthetase biosynthesis: identification and characterization of the ge81112 biosynthetic gene cluster celebesides a-c and theopapuamides b-d, depsipeptides from an indonesian sponge that inhibit hiv-1 entry callipeltin a, an anti-hiv cyclic depsipeptide from the new caledonian lithistida sponge callipelta sp neamphamide a, a new hiv-inhibitory depsipeptide from the papua new guinea marine sponge neamphius huxleyi microspinosamide, a new hiv-inhibitory cyclic depsipeptide from the marine sponge sidonops microspinosa a lowmolecular-weight inhibitor against the chemokine receptor cxcr4: a strong anti-hiv peptide t140 natural products with anti-hiv activity from marine organisms panorama patentário dos medicamentos antirretrovirais no brasil antiviral activity of antimicrobial cationic peptides against junin virus and herpes simplex virus evaluation of the inactivation of infectious herpes simplex virus by host-defense peptides the antimicrobial peptide dermaseptin s 4 inhibits hiv-1 infectivity in vitro in vitro antiviral activity of dermaseptins against herpes simplex virus type 1 the authors thank patrícia souza wanderley for fig. 1 authors' contributions ecgm was a major contributor in writing the manuscript. all authors contributed in writing the manuscript, read and approved the final document. the authors declare that they have no competing interests. key: cord-303189-ktl4jw8v authors: coccia, eliana m.; battistini, angela title: early ifn type i response: learning from microbial evasion strategies date: 2015-03-31 journal: seminars in immunology doi: 10.1016/j.smim.2015.03.005 sha: doc_id: 303189 cord_uid: ktl4jw8v abstract type i interferon (ifn) comprises a class of cytokines first discovered more than 50 years ago and initially characterized for their ability to interfere with viral replication and restrict locally viral propagation. as such, their induction downstream of germ-line encoded pattern recognition receptors (prrs) upon recognition of pathogen-associated molecular patterns (pamps) is a hallmark of the host antiviral response. the acknowledgment that several pamps, not just of viral origin, may induce ifn, pinpoints at these molecules as a first line of host defense against a number of invading pathogens. acting in both autocrine and paracrine manner, ifn interferes with viral replication by inducing hundreds of different ifn-stimulated genes with both direct anti-pathogenic as well as immunomodulatory activities, therefore functioning as a bridge between innate and adaptive immunity. on the other hand an inverse interference to escape the ifn system is largely exploited by pathogens through a number of tactics and tricks aimed at evading, inhibiting or manipulating the ifn pathway, that result in progression of infection or establishment of chronic disease. in this review we discuss the interplay between the ifn system and some selected clinically important and challenging viruses and bacteria, highlighting the wide array of pathogen-triggered molecular mechanisms involved in evasion strategies. the ability of the host to respond to invading pathogens relies on the activation of the innate immune system that orchestrates adaptive immune responses for pathogen clearance. in recent years, our understanding of the mechanisms involved in the activation of the innate response has evolved significantly with the identification and characterization of the mammalian system of pathogen recognition. the innate immune system detects the presence of a pathogen through a set of germline-encoded membrane-associated or cytoplasmic receptors, termed pattern recognition receptors (prrs) that are engaged by microbial-derived products named pathogenassociated molecular patterns (pamps). major classes of prr include toll-like receptors (tlrs), nucleotide-binding oligomerization domain (nod)-like receptors (nlr), c-type lectin receptors (clrs), retinoic-acid inducible gene (rig)-i-like receptors (rlrs) and a growing list of cytosolic dna-sensing receptors [1] [2] [3] [4] [5] [6] [7] . upon engagement, these receptors recruit a number of adaptor proteins to signal downstream and activate three major pathways: the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-b), the mitogen-activated protein kinases (mapks) and the ifn regulatory factor (irf) pathway [8, 9] . downstream tlrs and rlrs, type i ifn and pro-inflammatory cytokines are mainly produced, while nlrs predominantly activate inflammasomes, resulting in the release of il-1 and multiple inflammatory cytokines (fig. 1) . based on the structure of their receptors, interferons are broadly classified into three groups, type i, ii and iii ifns. type i ifn comprises the largest ifn class that includes ifn-␣, constituted by several partially homologous genes and ifn-␤, ifn-, ifn-, ifnrepresented by a single gene. the ifn-␣ and ifn-␤ are the best characterized as antiviral and the most broadly expressed [10] [11] [12] and will be referred as ifn-i from now on. the ifn-␥, the only type ii ifn, is released by activated t and nk cells; type iii ifns, which include ifn-1-4, similarly to ifn-i are believed to regulate the antiviral response [13] . according to the most common view, ifn-i exerts primarily an anti-viral action while ifn-ii acts predominantly on macrophages to induce a microbicidal state against ingested intracellular, non-viral pathogens. anti-microbial ifn-i activity is not intrinsic, but mediated, in both autocrine and paracrine manner, by a unique set of induced genes named ifn-stimulated genes (isgs) [10, 11, 14] . secreted ifn-i, indeed, binds to a common heterodimeric ubiquitously expressed receptor composed of ifnar1 and ifnar2 chains, and initiates a signaling cascade that has been characterized in detail and reviewed elsewhere [10] [11] [12] . briefly, canonical ifn-i pathway results in activation of the janus kinase (jak) family (cytoplasmic tyrosine kinases) that, in turn, activate by phosphorylation the signal transducers and activators of transcription (stats) [15] . activated stats complex with irf9 to form the heterocomplex ifn-stimulated gene factor 3 (isgf3), that translocates in the nucleus, binds to upstream sequence elements named ifn-stimulated response elements (isre), and activates the transcription of isgs (fig. 2) . these genes act to promote viral clearance and establish an antiviral state in uninfected bystander cells, or to induce apoptosis and several anti-microbial mechanisms in infected cells. they also stimulate cells at the interface of innate and adaptive immunity, such as macrophages and dendritic cells (dcs) that trigger the adaptive response [16, 17] . the ability to break in innate immunity before the onset of the adaptive response is, thus, crucial for the survival of virtually all mammalian pathogens. on the other side of the coin, an essential part of the early response to pathogens is aimed at limiting their ability to hijack host cellular machinery and evade the ifnmediated antimicrobial mechanisms. nowadays, while it is largely accepted that also bacteria can induce the production of ifn-i, the role of the ifn system in the pathogenesis of bacterial infections can be either detrimental (mainly in intracellular bacterial infections) or protective (mainly in extracellular bacterial infections). the route and tropism of bacterial infection, viral co-infections and last, but not least, the balance between ifn-i and ifn-ii effects are all determinants of the different outcome [18] . the ratio between ifn-i and ii species produced in response to infection, might differ as consequence of the capacity of the pathogen to stimulate specific cell types in the infected tissues. moreover, taking into consideration that several antibacterial ifn-ii-induced genes are stimulated to a much lesser degree by ifn-i, it is quite difficult in vivo to separate the activities strictly dependent on one of the two ifn families. a more reliable view consists of a crosstalk between the two pathways: if the bacterium is able to stimulate ifn-i production, generally it occurs early after the infection as an immediate innate immune response, while ifn-ii intervenes later on when immune cells (such as t cell subsets and nk cells) are activated. also due to this complex picture, while a number of viral evasion strategies have been mechanistically defined so far, studies aimed at characterizing the bacterial components that inhibit the ifn system are only recently starting to be elucidated in molecular details [19] [20] [21] . main strategies that pathogens have evolved to disarm the ifn-i response include: (i) blocking ifn-i production by modifying, curtailing or limiting production of their pamps to make them inaccessible to prrs and/or hitting the components of prrs signaling pathways; (ii) interfering with the ifn-i signaling by inhibiting signal transducers; (iii) blocking or disturbing the action of isgs; (iv) hijacking host proteins or components of the ifn system. each of these strategies involves a number of different molecular mechanisms and the combination of more strategies may be necessary to overcome the ifn-i response by a single pathogen. depending on the nature of the pathogen, these countermeasures that tip the balance toward the pathogen, may result in increased replication or in the establishment of a persistent infection. leaving out strategies that envision ifn-i repression due to a general inhibition of cellular gene expression, reviewed elsewhere [22, 23] , here we summarize recent findings on evasion tricks utilized by pathogens to specifically subvert the ifn system. a special focus is put on few highly pathogenic bacteria and emerging or re-emerging viruses, which represent a major threat to human health due to the lack of effective vaccines and/or therapeutics. for an in-depth coverage of other pathogens and strategies to escape the host immune response, the reader is referred to more comprehensive and specific reviews [19] [20] [21] [23] [24] [25] [26] . recent years have seen major advances in our understanding of the innate response to infectious pathogens and specifically of how pathogen recognition promotes ifn-i release [8, 27, 28] . cytoplasmic and membrane-associated prrs recognize a variety of pathogen components. viral pamps mainly consist of nucleic acids originating from the uncoating of infecting virions, the transcription of viral genomes and the replication of genomic intermediates, in the form of single-stranded (ss) and double-stranded (ds) dna, and ss and ds rna, as well as viral glycoproteins. bacterial pamps include various molecules, ranging from lipoproteins, lipopolysaccharide (lps), flagellin and peptidoglycan to unique bacterial nucleic acid structures, such as cyclic dinucleotides (cdns). multiple endosome-associated tlrs (tlr3, 7, 8 and 9) are specialized in the detection of viral and bacterial nucleic acids. tlr3 recognizes dsrna, tlr7/8 are bound by ssrna and tlr9 by cpgcontaining dna. tlr2, 4 and 13, previously considered sensors only for bacterial components, are now been involved also in recognition of viral ligands and in the induction of ifn-i [29] . all tlrs contain an intracellular domain, the toll-interleukin-1 receptor (tir), which recruits one or more tir-containing adaptor proteins to transmit signals downstream. tlr3 signals through the adaptor tir domaincontaining adaptor inducing ifn-␤ (trif) to activate the two related kinases, inhibitor of kb kinase (ikk)-and tank-binding kinase 1 (tbk1) that mediates activation by phosphorylation of irf3 and irf7. tlr7/8 and tlr9 use, as adaptor, myeloid differentiation primary-response protein 88 (myd88) that then initiates signaling cascades involving il-1r-associated kinases (irak1-4) and tnfrassociated factor (traf) 3/6 proteins, which finally converge at the activation of the ib kinase (ikk) family members ikk-␣, ikk-␤, ikk-and tbk1 responsible for activation of nf-b and irf3/7 [4] . in the cytoplasm, two closely related helicases, retinoic acidinducible gene 1 (rig-i) and melanoma differentiation-associated gene-5 (mda5), recognize dsrna of many replicating viruses in a tlr-independent manner. rig-i preferentially senses short dsrna and ssrna with a 5 -triphosphate (5 -ppp rna), while mda5 recognizes long dsrna and poly i:c [30] [31] [32] . like viral rnas, bacterial rnas can possess 5 -ppp termini and secondary structures that make them rig-i agonists. consistent with this notion, rig-i can act as a sensor of bacterial rna and may help maintain homeostasis to gut microbiota [33, 34] . upon recognition of non-self rna, rig-i and mda5 are recruited to the mitochondrial antiviral signaling protein (mavs; also known as cardif, visa or ips1), which triggers a signaling cascade that leads to the activation of ikk-and tbk1 and, in turn, irf3/7 phosphorylation [35] . stimulator of ifn genes (sting), initially identified as a cytosolic dna sensor, also participates in the rig-i signalling [36] . sting interacts with the adaptor mavs at the mitochondrial associated membranes (mam) facilitating the recruitment of tbk1 and the activation of irf3 [37] . a more general role of sting in innate immune responses, not only limited to its function as adaptor of rna and dna sensors, has been now established [38] . in addition to these rna sensors, viral rnas may also be recognized by effector molecules that are themselves ifn-induced proteins with antiviral functions. these molecules include dsrnaactivated protein kinase (pkr) that binds and is activated by dsrna from viruses and bacteria [39] , ifn-induced tetratricopeptide repeat proteins 1/2/3 (ifit1, ifit2, and ifit3) that, as rig-i, bind 5 -ppp rna and may recognize viral mrnas that lack 2 -omethylation [1, 40] . a growing list of cytosolic dna sensors then recognizes dna from different sources including viruses, bacteria and apoptotic cells [3, 6, 7] . more than ten dna cytosolic receptors have been proposed so far that include dai (dna-dependent activator of irf), rna polymerase-iii, ifn-inducible interferon gamma-interferoninducible protein 16 (ifi16), sting, extrachromosomal histone h2b, leucine rich repeat (in flii) interacting protein (lrrfip1), ku70, deah box protein (dhx) 9 and dhx36, cyclic gmp-amp synthase (c-gas). as other sensors, dna sensor activation results in the production of ifn-i and proinflammatory cytokines and chemokines via the sting-tbk1-irf3 axis. in addition to the activation of irf3-and nf-b-dependent signaling cascades, cytosolic dna can also promote an apoptosis-associated speck-like protein containing a card (asc)-dependent inflammasome-mediated response resulting in the secretion of proinflammatory cytokines [3, 7, 25, 27, 28] . the intracellular nlrs scaffold large signaling complexes to mediate innate immunity and inflammatory responses. they may trigger the assembly of inflammasomes and modulate the nf-b, mapk and irf signaling pathways. in particular, nod1 and nod2 are important for immune detection of intracellular bacterial pathogens and are also involved in a variety of immune homeostatic functions [41] . upon the recognition of bacterial peptidoglycans by nod1 and nod2, receptor-interacting serine-threonine kinase 2 (rip2) is activated via cellular inhibitors of apoptosis 1 and 2 (ciap1 and 2), subsequently leading to ubiquitination of nf-b essential modulator (nemo) and the activation of the proinflammatory nf-b pathway. in parallel, the recognition of muramyl dipeptide present in all peptidoglycans, can also lead to the activation of mapk pathway via rip2, which contributes to cytokine production. for the induction of ifn-i, nod2 activated by viral rna, signals through the mitochondrial mavs independently of rip2. then, some sensors can aggregate with adaptors as apoptosisassociated speck-like protein containing a caspase recruitment domain (asc) and caspase 1 to form multimeric structures named inflammasome [42] (fig. 1) . as first line strategy to face these sensing pathways, pathogens hide detection by modifying their pamps. they may also degrade/inactivate target key signal transduction hubs by counteracting host-induced post-translational modifications required for signaling molecule activity and use concomitant different strategies as better described below and also discussed by thomas kufer and igor brodsky in this issue. we focus here on some members of coronaviruses, flaviviruses, hepaciviruses, filoviruses and retroviruses as well as bacteria whose pamps could in principle activate the ifn system, but that efficiently betray host sensing and effector pathways (fig. 3) . coronaviruses (cov) are large enveloped rna viruses, in the family coronaviridae, of both veterinary and clinical importance. two newly emerging viruses in the family, the severe acute respiratory syndrome cov (sars-cov) and the middle east respiratory syndrome cov (mers-cov), have been recently responsible for severe disease in humans (reviewed in [43] [44] [45] [46] [47] ). a common trait in their pathogenesis is the lack of induction of a robust ifn-i response in infected cells [48, 49] . to do this, cov have developed multiple strategies (recently reviewed in [50, 51] ). rig-i, mda5 and the host isgs ifit1 and 2 are critically involved in sensing of cov infection. however, as first line of hiding from recognition, cov encode several highly conserved nonstructural proteins (nsps) implicated in viral rna capping activity in order to examples of viral and bacterial antagonists that block subvert or exploit the ifn system. pathogens affect at every step the ifn system by multiple mechanisms. sites of intervention by several antagonists are indicated. ifn antagonists may prevent prr recognition by hiding or modifying pamps, may inhibit prr signaling by directly targeting adaptors and signaling effectors, may interfere with the ifn signaling by impairing signaling transducers and may block or disturb the action of isgs. some antagonists have more than one cellular target while others target common signaling molecules, effectively blocking ifn induction from a variety of pamps. see the text for more details and references. mimick n7-and 2 -o-methylated 5 cap structure of cellular mrnas [52] . these viral proteins include a rna-triphosphatase, a guanine-n7-methyltransferase, and a 2 -o-methyltransferase encoded by nsp13, 14 and 16, respectively [50, 53] . consistently, human and mouse cov mutants lacking 2 -o-methyltransferase activity induce higher expression of ifn-i and are highly sensitive to ifn-i effects [54, 55] . sars-cov nsp14 also encode a 3 ,5 exoribonuclease that is involved in rna-proofreading, but probably it also functions in degrading viral pamps, further hiding immune detection [50, 56] . similarly, nsp11 encodes a ribonuclease that may degrade viral pamps [57] . another strategy used to avoid detection is the replication in protected sites as the double membrane vesicles (dmvs) that the virus induces in the host cytoplasm. in the case of sars-cov these membranes contain the replicase complex and the viral genomic rna suggesting that replication occurs in these sites and the generated nucleic acids are soon after shielded [58, 59] . in addition, the highly basic nucleocapsid (n) protein of sars-cov has been reported to directly inhibit ifn-i production induced by both poly(i:c) or sendai virus, by a mechanism that involves steps upstream rig-i. thus, it is conceivable that by binding dsrna, n protein prevents rig-i/mda5 activation [60] . others sars-covencoded proteins, as orf-3b and orf6, may also interfere with the rlr recruitment of the adaptor mavs based on their preferential localization at the mitochondrial membrane even if their mechanism of action is not yet elucidated [61, 62] . the sars-cov membrane (m) protein blocks transcription of ifn-i when stimulated by dsrna or members of the rig-i signaling pathway including rig-i, mavs, ikk-, and tbk1, but it does not influence the transcriptional activity of the ifn promoter when irf3 or irf7 are overexpressed. the physical association of sars-cov m with rig-i, tbk1, ikk-, and traf3 suggests that m protein may prevent the formation of the functional complex with tbk1 thereby inhibiting activation of irf3/irf7 and ifn-i transcription [63] . similarly, the papain-like protease (plp) domain contained in the sars-cov nsp3 protein, an essential component of the viral replicase complex, interacts with the adaptor sting blocking the binding to mavs and the recruitment of the tbk1/irf3 complex [64] [65] [66] . finally, through its deubiquitinating activity, the plp protein removes ubiquitin from several components of the rlr pathway blocking their activation (reviewed in [50, 51, 67, 68] ). recently, two mers-cov accessory proteins, the orf-4a and orf-4b products, have also been identified as immunosuppressive factors. mers-cov 4a is a rna-binding protein that interacts in a mrna-dependent manner with pkr-associated activator (pact), a cellular dsrna-binding protein, which potently stimulates rig-iinduced ifn production by binding to the c-terminal repression domain of rig-i [69] . so, orf-4a inhibits rig-i/mda5 pathway without a direct binding to the sensor, but by perturbing the function of a stimulator of rig-i signaling as pact [70] . the orf-4bencoded accessory protein is also able to inhibit ifn-i induction in vitro, however, the mechanism involved is not yet elucidated [71] . the genus flavivirus, of the flaviridae family, includes west nile virus (wnv), dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), tick-borne encephalitis virus (tbev) and several other viruses all causing serious medical problems in humans [72] [73] [74] [75] . currently, vaccines for humans are available only for yfv, jev, and tbev and no clinically approved antiviral therapy is available for the treatment of flavivirus infections [75] . in particular, denv and wnv are re-emerging as global life-threatening human pathogens [75, 76] . both viruses are sensitive to ifn antiviral effects and the severity of the disease is mostly dependent on the ability to avoid and/or attenuate induction of ifn-i and its effector responses through several viral encoded ifn-antagonists [73, 74] . interestingly, some of these strategies are shared with cov. the most relevant prrs for the detection of wnv and denv products, described so far, are the tlr3, 7, 8 and the rlrs, rig-i/mda-5. suppression of both tlr-and rlr-mediated ifn induction has been shown to be important for viral replication [77, 78] . as cov, flaviviruses contain a cap structure that is generated by a methyltransferase mapped to the n-terminal region of the ns5 protein [74, 79] . through 2 -o-methylation of the viral mrna cap wnv, denv and jev evade ifit1-dependent and -independent mechanisms of host restriction in vitro and in vivo [80, 81] . moreover, to hide their nucleic acids during the replicative cycle, both denv and wnv induce the formation of convoluted membranes in the endoplasmic reticulum (er) and golgi apparatus that envelop the virus replication complex [82, 83] protecting viral nucleic acids from both tlr and rlr recognition, as it occurs along the infection with cov. so far, an antagonism at the level of sensing signaling has been clearly defined only for denv that fails to induce an ifn response in myeloid cells where it replicates, despite other pro-inflammatory cytokines and chemokines are produced [84, 85] . the ability to inhibit ifn-i production is due to the viral ns2b/3 protease that binds and cleaves the adaptor/sensor sting [85] [86] [87] . interestingly, first evidences indicate that the ns2b/3 proteases of other flaviviruses, as jev or yfv, are not able to cleave sting, while the ns4b of yfv can do it [68] . to the same flaviviridae family belongs the hepacivirus genus, distantly related to flaviviruses, that includes hepatitis c virus (hcv) a major cause of chronic liver disease [88, 89] . hcv uses several strategies to efficiently evade innate immunity and this escape is considered the main determinant of viral persistence that leads to a chronic infection in 70-80% of infected people [88, 90] . hcv rna can be sensed by different prrs, namely rlrs, tlrs, nlrs, and, as recently reported, by protein kinase r (pkr). rig-i is the best described sensor for the poly u/uc region located within the 3untranslated region (utr) of the viral rna, along with a 5 -ppp. this region is essential for viral replication and, thus, highly conserved among hcv genotypes. the key viral protein involved in the evasion strategies is the ns3/4a protease, which consists of ns3 and ns4a. the complex is essential for several steps in the viral cycle including viral rna replication, polyprotein processing and viral assembly [91] . taking advantage of its serine protease activity, ns3/4a cleaves the adaptor mavs, preventing its dimerization and downstream signaling [92, 93] . after cleavage, mavs dissociates from the mitochondrial associated endoplasmic reticulum membranes (mam) where upon hcv-induced rig-i activation it is recruited and colocalizes with ikk[94] , thus impairing ifn-i expression [95] . importantly, the cleavage of mavs by the viral protease has been confirmed in patients [96] . to antagonize ifn-i production, ns4b protein instead targets sting. hcv ns4b, indeed, contains a sting homology domain and interacts with sting in the er blocking sting interaction with mavs and tbk1 [97, 98] . even if the exact molecular mechanism involved in ns4b inhibition of sting signaling has not yet been defined in the context of viral infection, ns4b likely cooperates with ns3/4a in targeting the rig-i signaling pathway. interestingly, ns2b/3 and ns4b of other members of the flaviviridae family including denv and yfv as mentioned above also block sting signaling and possess the same sting homology domain, indicating a conserved mechanism of sting antagonisms between flaviviruses and hepaciviruses. intracellularly, both tlr3 and tlr7 have been shown to sense hcv rna, depending on the infected cell type considered [88] . sensing by tlr3 may occur in liver cells, as hepatocytes, and liver resident macrophages kupffer cells, while tlr7 sensing occurs predominantly in plasmacytoid dcs (pdcs) and macrophages. as reported above for the mavs adaptor, the serine protease ns3/4a also cleaves the key adaptor of tlr3, trif [99] , thus preventing an ifn response in productively-infected hepatocytes. in these cells, the hcv-induced mir-21 has been recently reported to be involved in evasion of ifn-i production and stimulation of hcv replication, upon suppression of myd88 and irak1 expression, that is required for the tlr7-mediated sensing of the virus [100] . in macrophages, myd88 signaling is instead targeted by the ns5a protein that, upon the direct binding to the adaptor, impairs the recruitment of irak-1 and cytokine production in response to tlr ligands [101] . interestingly, tlr3 and tlr7 levels are decreased in patients chronically infected with hcv [102, 103] and this has been recently correlated with increased levels of mir-758 [104] . hcv-infected cells, however, can trigger ifn-i production in non-productively-infected pdcs in a cell-cell contact-and tlr7dependent manner depending on the intracellular hcv rna level of cocultured infected cells [105] . this production of ifn-i by pdcs may thus account for the strong ifn-i response observed in the liver of infected people [106] . interestingly, a robust expression of isgs correlated with a decreased response to ifn therapy [107] supporting a pathogenetic role of a high ifn signature in chronically infected individuals where the virus has established a persistent infection. in contrast, in monocytes and macrophages upon clathrinmediated endocytosis and recognition of the virus by tlr7, hcv activates the inflammasome and not ifn-i production in an infection-independent process [108] . an association between tlr-polymorphisms and cytokine production in response to tlr7 agonist in vitro has also been reported, supporting a pathogenic role of tlr7-mediated sensing in immune cells [109] . interestingly, this cell-type dependent stimulation of ifn-i and inflammasome in response to hcv infection is also observed in human immunodeficiency virus type 1 (hiv-1) infection that as hcv can establish a persistent infection (see discussion below). pkr has been shown to sense hcv rna very early in infection even prior to rig-i sensing [110] . pkr is a dsrna binding protein that upon activation phosphorylates the ␣ subunit of the eukaryotic translation initiation factor 2 (eif2 ␣) to inhibit translation of host capped mrna but not of non-capped mrna, as that of hcv. pkr, upon binding hcv rna and independently of its kinase-activity, interacts with mavs to induce the transcription of a number of early isgs including ifn stimulated gene 15 (isg15), but not ifn-i. isg15, in turn, deubiquitinates rig-i inhibiting its functions [110] . by doing so, hcv blocks sensing by pkr and reinforces hcv evasion from rig-1 signaling. amongst rna viruses that, as hcv, can establish a persistent infection, hiv-1, a lentivirus from the retroviridae family, represents a paradigm for its ability to prevent or circumvent the innate immune response mediated by ifn-i. in spite of evidence that a sustained ifn-i response occurs in hiv-infected patients, it fails to clear the infection in the first place and to prevent the early establishment of long-lived hiv-1 reservoirs [111] [112] [113] . hiv-1 has, indeed, developed a number of strategies to block the ifn signaling and the activity of ifn-induced host restriction factors. here, we only briefly summarize these strategies some of which have been only recently discovered, leading to the identification of immune pathways, thus far, unrecognized (as recently reviewed elsewhere [114] [115] [116] [117] [118] [119] [120] ). in the past few years, the knowledge on innate immunity against hiv-1 has evolved enormously with the recent identification and the characterization of the molecular basis of retroviral recognition by prrs [116, 118, 121] . retroviral replication generates several structural and intermediate molecules as ssrna, hybrids rna/dna, ss and ds dna produced upon reverse transcriptase, that are potentially available for recognition by cellular prrs as "non-self". with the exception of pdcs, which produce high levels of ifn-i upon detection of hiv-1 ssrna by tlr7, in all other immune cells ifn-i production is prevented or barely detectable unless viral countermeasures are disabled. in conventional dcs (cdcs), monocytes and resting cd4 + t cells, indeed, hiv-1 sensing is prevented by the restriction factor sterile alpha motif (sam) and the hystidine/aspartic acid (hd) domain-containing protein1 (samhd1) that blocks reverse transcription or directly degrades genomic rna, thus preventing prr recognition [122, 123] . this samhd1mediated restriction is overcome by the viral protein vpx [124, 125] that is a hiv-2 and siv accessory protein absent in hiv-1. this apparent disadvantage is, however, effectively exploited by the virus that maintains a reward by not replicating in myeloid cells and by reducing the impact of ifn-i production in these cells. the block of productive infection in non-cycling cells where samdh1 is active, particularly in cdcs, results in lack of maturation and thus in impairment in priming of naive, hiv-1-specific t cells for optimal anti-hiv-1 immunity [126] . furthermore, in the small fraction of cdcs that become infected, the recognition of hiv-1 genomic rna by tlr8 paradoxically licenses hiv-1 transcription [127] . in this case, productive dc infection allows an increased transmission to t cells while inhibiting ifn-i production. in monocytes instead, recognition of viral rna by tlr8, does not trigger ifn-i production but, as in the case of hcv, leads to the formation of the nlrp3 inflammasome with activation of caspase-1 and il-1␤ production, favoring the establishment of an inflammatory milieu that fuels hiv-1 replication [108, 126] . in contrast, in cells that are target of a productive infection, such as macrophages and t lymphocytes, ifn-i production is prevented by an escape mechanism mediated by the hiv-1 protease that drives rig-i to the lysosomes [128] . the ssdna derived from proviral dna upon rt can, instead, be sensed by the newly identified dna sensor interferon gammainterferon-inducible protein 16 (ifi16) [129] , however, hiv exploits the host cytosolic nuclease 3 repair exonuclease1 (trex1) to digest hiv-1 dna generated during infection that, thus, does not accumulate at levels sufficient to be detected by ifi16, unless trex1 activity is blocked [130] . finally, resting cd4 + t cells are not permissive to virus replication due to the expression of an active samhd1 that, as mentioned before, degrades genomic rna and prevents efficient rt and recognition of ssdna. however, this prevention of sensing may not be complete and partial recognition of rt intermediates by the ifi16 sensor not only leads to the initiation of ifn-i production, but also to the activation of the inflammasome, triggering cell death mechanisms including pyroptosis and apoptosis [131] . finally, the viral capsid exploits two cellular proteins, cyclophilin a and cpsf6, and binds just a right amount of both to allow opening of the capsid and rt process, while preventing sensing of the viral cdna before integration with the following production of ifn-i [132, 133] . overall, viruses as hcv and hiv-1 have evolved nifty strategies to dampen the host innate response in cells where a productive infection may take place, while they induce infection-independent mechanisms in non-permissive cells to facilitate the viral life cycle and promote a chronic inflammation. the genus filoviruses from the filoviridae family are among the most virulent known human pathogens and comprise one species of marburg virus and five species of ebola viruses, including zaire ebolavirus (ebov) that is the most lethal and responsible of the recent severe outbreak of hemorrhagic fevers causing up to 90% mortality in untreated humans [134] . several immunoevasion strategies that result in total impairment of the innate immune system are responsible for most of ebov virulence [135] . a major target of these strategies that are exerted by few viral proteins, is the ifn-i response that controls in vivo filoviruses infection [136] . at the level of viral sensing, the ebov vp35 inhibits rig-i signaling [137, 138] . vp35 binds with high affinity to dsrna and 5 -ppp dsrna in a sequence-independent manner. four crystallographic structures of ebov vp35 rbd/iid from zaire and reston viruses and of marv vp35 rbd/iid have elucidated how vp35 rbd/iid dimers bind to rna strands and how the dimers mimic the rlr shape, hiding the rna recognition site [139, 140] . interestingly, the residues involved in both protein-protein interactions at the rbd/iid dimer interface and involved in dsrna binding are highly conserved among all known ebov and marv species. moreover, all residues that are important for dsrna binding are also crucial for ifn-i inhibition. as the knowledge on the importance of ifn-i in controlling the immunity against bacteria increases, studies aimed at characterizing the evasion mechanisms that these pathogens employ to evade, inhibit, or otherwise manipulate the innate immune response are arising. the mechanisms induced by bacteria for ifn-i expression in different cell types are various and reflect the heterogeneity of host-pathogen interactions established along bacterial infections. while the extracellular bacteria activate ifn signaling mainly through the interaction with molecules present on the cell surface, the intracellular bacteria are recognized as they enter a cell by cell-surface or endosome/phagosome bound receptors or by cytoplasmic pathogen sensors once they escape from these compartments [141] . here, we report only few examples of bacterial evasion mostly related to signaling pathways converging in ifn-i stimulation. a more exhaustive discussion of bacterial evasion strategies from prr-signaling pathways and inflammasome is provided by the contribution of thomas kufer and igor brodsky in this issue. to elude prr recognition many bacterial pathogens, like viruses, have modified the molecular structure of their pamps. lps, an ubiquitous component of gram-negative bacterial cell wall, is a pamp that is recognized by the tlr4/md-2 complex present on the cell surface. some bacterial species have evolved an alternative form of lps resulting in a weak antagonist of tlr4/md-2 signaling. this strategy is utilized by yersinia pestis, the causative agent of pestis infection. the acylation status of lipid a from hexa-to tetra-acylated is reduced when the temperature increases from 21 • c (flea temperature) to 37 • c (human temperature). this alteration renders lipid a less recognizable lps by tlr4 and, following transmission from fleas to humans, contributes predominantly to the virulence of the bacterium [142] . another example is shigella that, after internalization and proliferation within epithelial cells, hypoacylates lipid a to become less visible to the immune system once leave the infected epithelial cells [143] . in addition to lps, other bacterial components that may induce ifn-i, include bacterial nucleic acids and peptidoglycans. these pamps can be recognized outside or inside the host cells, leading to the activation of distinct signaling pathways [141] . intracellularly bacterial rna and dna nucleic acids are recognized by the several intracellular receptors that also sense viral pamps, while bacterial peptidoglycans are detected by nod1 and nod2 system. most of these pathways then converge in the activation of the sting/tbk1/irf3 axis [6] . as viruses, bacteria also encode proteins with enzymatic activity that interfere with the activation of adaptor molecules involved in prr signaling. this is the case of the yersinia pestis virulence factor yopj that, besides being a potent inhibitor of the nf-b and mapk signaling pathways, also inhibits tlr-mediated ifn response. as a deubiquitinating protease, yopj prevents or removes the k63polymerized ubiquitin conjugates, which are required for traf3 and traf6 activation in the signal transduction pathway leading to irf3 activation [144] . similarly, the type iii effector ospi of shigella flexneri inactivates by deamination the e2 ubiquitin ligase ubc13, a factor important for traf6 auto-polyubiquitinylation and activation [145] . another interesting example is represented by the translocated intimin receptor (tir), which is one of the first type iii effector proteins discovered in a/e pathogens including the enteropathogenic e. coli (epec), enterohemorrhagic e. coli o157:h7 (ehec), and citrobacter rodentium. in addition to the role played in the attachment to the host membrane, this factor shares sequence similarities with conserved regions present in the cytoplasmic tails of inhibitory receptors of the host immune system, such as the immunoreceptor tyrosine-based inhibition motifs (itims). tir utilizes these itimlike motifs to mimic an endogenous innate immunoregulatory mechanism. in particular, tir recruits the host src-homologyregion-2-domain-containing phosphatase 1 to the adaptor traf6 and thus prevents polyubiquitinylation and activation of traf6 in the ifn-stimulated pathway [146] . adaptor molecules downstream sensors transmit signals to classical ib kinase complex, including nemo/ikk-␥, tank and to the atypical ikk-related kinases ikk-and tbk1 that trigger activation of nf-b and irfs, respectively [147, 148] . ifn-i production downstream of these signaling pathways depends essentially on the presence and activation of irfs and their contribution changes depending on the cell type considered [9] . the irf family is presently composed of nine mammalian members namely irf1 to 9, coded by distinct but related genes that exert a number of functions in the regulation of innate and adaptive immune responses. the irfs with intrinsic antiviral function include irf3 and irf7 that are essential for the prr-mediated ifn gene transcription, but also induce some ifn effectors in an ifn-independent manner. irf9, as mentioned before, is part of the heterocomplex isgf3 that drives the expression of most isgs including irf1 (fig. 2) . although irf1 is itself an isgs, which affects different aspects of the immune response even independently from ifn-i production [149, 150] , it also represents a positive regulator of ifn-i gene expression in response to specific stimuli in a cell type specific manner [151] . moreover, irf1 plays a crucial role in regulating mavs-dependent signaling from peroxisomes [152] . in this respect, irf1 regulates the transcriptional profile of antiviral genes unique to that induced by ifn-i and cooperatively promotes an effective antiviral program against a broad spectrum of viruses [152, 153] . irf5 is instead specifically involved in inflammatory cytokines induction [9] . given the unique functions exerted by irfs, viruses have evolved strategies aimed at the specific destruction of these transcription factors. with regard to the viruses covered here, most of them inhibit irf activation either indirectly by acting on sensors and elements of the signaling pathway that activate them, as described above, or directly by impairing/hijacking irf activity (fig. 3) . as described above, the sars-cov plp affects the activation of both irf3 and nf-b not directly, as initially suggested [66, 154] , but by targeting rig-i, mavs, traf3 [63] and, as more recently reported, sting [64, 65] . interestingly, this activity is independent from plp protease activity. recently, it has been reported that the plp of mers-cov also suppresses ifn-i transcription by interfering with irf3 phosphorylation and nuclear translocation [155] . as sars-cov, mers-cov plp is a viral deubiquitinating enzyme that acts on both k48-and k63-linked ubiquitination and isg15-linked isgylation, two posttranslational modifications that play important roles in regulating the rig-i and sting/irf3 and nf-b activation [156] . whether the deubiquitination and deisgylation activity of mers-cov plp are directly responsible for inactivation of irf3/nf-b or upstream signaling pathway, it remains unclear. the wnv ns1 protein inhibits the tlr3-induced activation of ifn-i and il-6 transcription through inhibition of nuclear translocation of irf3 and nf-b [157] . recent studies indicate that this effect seems to be dependent on ns1 domains that control viral replication [158] . interestingly, in the draining lymph nodes the protein released predominantly from macrophages and dcs can inhibit the innate immune signaling pathways in uninfected cells and impairs cytokine production in response to infection [159] , thus suggesting that ns1 could also influence the development of the adaptive immune response directed to wnv. the ns2b/3 serine protease of denv, instead, blocks the serine 386 phosphorylation and nuclear translocation of irf3 by directly interacting with ikk-and masking the kinase domain [160] . two tick-borne flaviviruses, lgtv and tbev, have been recently reported to inhibit irf1 independently of their ability to antagonize ifn signaling. in particular, a weak expression of irf1 protein and nuclear localization, without reduction in irf1 mrna expression, was observed in dcs, an early cellular target of infection [161] . several hcv proteins interfere with irf activity. the hcv ns3 protease impairs irf3 activation by blocking the interaction with tbk1 and irf3 [162] . in addition, the ns2 protein inhibits, in a dose-dependent manner, ikk--and especially tbk1-induced irf3 phosphorylation [163] . the basic amino acid region 1 (br1) in the n-terminal region of the core protein is also crucial in inhibiting irf3 dimerization as well as phosphorylation induced by ndv infection and poly (i:c) [164] . interestingly, this domain has been identified as the binding region for a dead box protein the ddx3, which has been recently found to enhance the tbk1/ikk--induced ifn-␤ promoter activity upon binding to the adaptor mavs [165] . the hcv core also decreased the expression levels of ddx3 suggesting that the irf3 inhibition may be mediated by the core effect on ddx3. moreover, through binding to ddx3, the hcv core protein also promotes hcv replication. thus, the core protein appears to switch ddx3 from an ifn-inducing mode to an hcv-replication mode [166] . irf1 expression is, instead, suppressed by the hcv core at the transcriptional level. this event blocks the expression of several antiviral and immunomodulatory genes of both innate and adaptive immunity and, in doing so, facilitates the establishment of hcv persistent infection [167] . in line with this hypothesis, accumulating evidence suggests that hcv also targets dcs to control the host antiviral response and trigger persistence. as wnv ns1, hcv core is, indeed, a secreted protein found in the peripheral blood of patients with chronic infection that may thus affect directly dc functions. in this context, it has been recently reported that the core protein suppresses ifn-i production in response to tlr agonists and to rig-i stimulated by hcv pamp in a cell culture model of pdcs, through the reduced levels of irf7 and of phosphorylated stat1 protein [168] . the effect on irf7 is, however, not direct but probably mediated by the reduced levels of ifn-i production by core-stimulated pdcs. whether or not this also occurs along the natural infection and contributes to hcv persistency remains to be determined. to increase virus replication and establish viral persistence and latency, hiv-1, besides to dismantle or exploit almost all cell intrinsic innate recognition pathways, as discussed above, also directly hits irfs [113, 119, 169] . in t cells, the virion-associated accessory proteins, vif, vpr and vpu, directly target irf3 for ubiquitin-associated proteasome degradation [170] [171] [172] . recently, this vpu effect on irf3 degradation has been, however, challenged and it has been reported that vpu, instead, mediates a partial cleavage of irf3 in a caspase-dependent manner. interestingly, this cleavage produces a c-terminal fragment that can act as a negative regulator of irf3-dependent gene activation [173] . thus, hiv-1, as already reported for several viruses, can also exploit the apoptotic machinery to interfere with irf3 function [174] . in myeloid cells, instead, hiv-1 does not inhibit but, rather, stimulates both irf1 and irf7 expression. irf1 activity is, however, exploited by the virus to induce a distinct subset of isgs that despite displays intrinsic and unique antiviral actions, does not restrict viral infection [175] . irf1 is also induced in hiv-1-productively infected t cells where it may regulate viral promoter activity even in the absence of the viral transactivator tat driving initial transcription of the viral genome [176, 177] . later on, however, when viral replication is mostly accomplished by the viral transactivator, irf1 is sequestered by tat to accelerate proteasomal-mediated irf1 degradation (remoli al and battistini a, unpublished) and to quench irf1 transcriptional activity on target genes [178] . by so doing, tat disarms the unique antiviral response against viral infections that irf1 could exert [153] . a block of ifn transcription in primary cd4 + t cells may also depend on cd3/cd28-mediated activation of ikk-. we, indeed, recently reported that ikk-activation results in a peculiar pattern of irf phosphorylation in t cells, including a splicing isoform of irf3, which may function as an inhibitor of ifn-␤ expression, and phosphorylation of irf1 that blocks its activity on ifn-i promoter [179] . the ebov vp35, in addition to prevent rlr recognition, inhibits ifn-i promoter activation mediated by tbk-1/ikk-overexpression but not by a constitutively active irf3, strongly suggesting a specific inhibition of the kinase activity of tbk-1/ikk-. indeed, vp35 interacts with the tbk-1/ikk-kinase domain and functions as a substitute substrate, thus inhibiting both the kinase activity and the binding of the physiological irf3/7 substrate [180, 181] . notably, mutations of vp35 residues, involved in this ifn antagonism, do not alter the function of vp35 in viral replication and transcription [182] . in dcs, vp35 targets irf7. by interacting with the small ubiquitinlike modifier sumo e2 enzyme ubc9 and the e3 ligase pias1, vp35 promotes irf7 sumoylation, a post-translational modification that prevents irf translocation into the nucleus and, in turn, ifn-i gene transcription. a similar effect of vp35 was also reported for irf3 [183] . this vp35 activity is independent of its ability to recognize dsrna and maps to the n-terminus, which is essential for interactions with both irf7 and pias1. interestingly, sumo modification of irf3/7 is a part of the negative feedback loop of normal ifn-i signaling [184] that is exploited by ebov to weaken host innate immunity. downstream prrs, bacteria mainly target the mapk pathway and the nemo-ikk-nf-b signaling axis, which primarily induces inflammatory cytokines (reviewed in [19] and thomas kuper and igor brodsky in this issue). as an example of bacteria that target the irf pathway. listeria monocytogenes suppresses ifn-i gene induction downstream of tlr-triggered myd88 signaling pathway, acting on irf3. indeed, a mapk phosphatase renders irf3 hypophosphorylated by enhancing the formation of a mapk phosphatase-irf3-tbk-1 ternary complex in response to infection [185] . in fig. 2 is illustrated the ifn-i signaling pathway downstream the ifn-i receptor (ifnar) (a heterodimer of ifnar1 and ifnar2) that is activated upon binding of virus-infected cell-secreted ifn-i, and some isgs, including positive and negative feed-back regulators. most of the viruses here covered and some bacteria also target these pathways (fig. 3) . upstream the jak/stat pathway, the sars-cov 3a promotes serine phosphorylation within the ifnar1 degradation motif and increases ifnar1 ubiquitination [186] . the plp from sars-cov has a complex mechanism of interference with the jak/stat pathway. through its de-ubiquitinase activity upregulates the expression of the ubiquitinating enzyme e2-25k, leading to degradation of the erk kinase that, in turn, interferes with stat1 phosphorylation [187] . the orf6-encoded protein, instead, antagonizes stat1 function by interacting and sequestering in the er components of the nuclear import complex, as karyopherin alpha 2 and karyopherin beta 1. by doing so, orf-6 competes for the binding of the nuclear import complex to stat1, thus inhibiting stat1 nuclear import [188] . however, the majority of these evidences have been obtained by overexpression or stably expression of individual viral components in cell culture, which represents an experimental setting that may not accurately reflect the innate immune signaling occurring during sars-cov infection in vivo. wnv interferes with ifnar complex by promoting phosphorylation-dependent ubiquitination and degradation of ifnar1. this effect is mediated by the hydrophobic ns4a and ns4b proteins that potently induce the unfolded protein response (upr). this pathway is physiologically induced by different stimuli, including the accumulation of misfolded proteins in the er [189, 190] that, as mentioned before, represents the site of flaviviruses replication. activation of the upr pathway inhibits ifn activation and induces a general er stress response, thus facilitating viral replication. the methyltransferase domain of ns5 from langat virus and jev, instead, binds directly to ifnar through its methyltransferase domain and inhibits the activation of kinases associated to the receptor [191, 192] . several wnv non structural proteins, as ns2a, ns2b, ns3, ns4a, ns4b and ns5, have been reported to prevent the phoshorylation of jak1, tyk2 and, as a consequence, the activation of stat1/2 (recently reviewed in [73] ). likewise, expression of denv nonstructural protein ns2a, ns4a, or ns4b proteins impairs the jak/stat signaling pathway by reducing the phosphorylation and nuclear translocation of stat1 [193] . phosphorylation of stat2 is also blocked by denv ns5 through inhibition of jak1 and tyk2 activity [194] . ns5 also binds to the coiled-coil region in the first half of the human stat2 protein and acts as a bridge between ubr-4, a member of the n-recognin family, and stat2 leading to stat2 ubiquitination and proteasomal-mediated degradation [195, 196] . interestingly, only proteolytically-processed ns5 can efficiently mediate stat2 degradation, though both unprocessed and processed ns5 proteins are able to bind stat2. the jev ns5 protein greatly reduces tyk2 and, partially, stat1 phosphorylation, probably through its phosphatase activity [197] . the tbev ns5, instead, blocks stat1 phosphorylation by promoting the association with the pdz membrane protein scribble [198] . by activating the ras/raf/mek pathway, hcv replication has been shown to increase the phosphorylation of a motif contained in the cytoplasmic tail of ifnar1, which is involved in controlling the receptor ubiquitin-dependent endocytosis and attenuation of stat1/2 phosphorylation [199] . several hcv proteins have also been implicated in the regulation of the ifn response pathway interfering directly with the jak/stat signaling. however, contrasting results have been reported that probably stem from the use of different cell lines or different hcv expression/replication systems [200] [201] [202] [203] [204] . the core protein has been reported to upregulate the expression of socs3, thereby inhibiting tyrosine phosphorylation of stat1 [201] , although the decreased stat1 phoshorylation has not been detected in other studies [200, 205] . the hcv core protein, expressed alone, has been reported to directly bind to stat1 and to prevent its phosphorylation and subsequent expression of downstream isgs [206] . the ns5a, similarly, binds and prevents stat1 phosphorylation specifically in hepatocyte-derived cell lines [207] . two strategies are used by ebov vp24 to limit the jak1/tyk2mediated activation of stat1/2 and their subsequent nuclear localization. vp24 binds within the tyrosine-phosphorylated-stat1 binding region located in the c terminus of members of the npi-1 subfamily of karyopherin alpha nuclear localization signal receptors preventing their binding and shuttling to the nucleus of tyrosine-phosphorylated-stat1 [208, 209] . the crystal structure of human kpnalpha5 c terminus in complex with vp24 has been recently resolved and a unique nonclassical nuclear localization signal binding site on kpna5 has been identified. this motif is necessary for binding and efficient nuclear import of tyrosinephosphorylated-stat1 [210] . ebov vp24 can also directly bind stat1; whether the binding occurs with the unphosphorylated or phosphorylated stat1 or both isoforms it is not yet clear [211] . however, also unphosphorylated stat1 enters the nucleus to activate and sustain the expression of a number of ifn-induced immune regulatory genes, which are distinct from those activated by the phosphorylated stat1. thus, ebov vp24 binding and inhibition of either forms of stat1 may be important in the suppression of an antiviral state. notably, in spite of the high similarity of ebov and marv genome organization and high sequence homology between many of the ebov and marv encoded proteins, marv vp24 unlike ebov vp24, does not inhibit jak/stat signaling [212] . this is consistent with the observation that regions important for karyopherin alpha binding are different between these two vp24s [210] . in contrast, tyrosine phosphorylation of jak1 and stat is inhibited by the marv matrix protein vp40 that also inhibits this ifn-ii signaling. interestingly, also jak1-dependent and il-6-induced tyrosine phosphorylation of stat1 and stat3 are targeted by marv vp40 suggesting that marv may globally inhibit jak1dependent cytokine signaling with mechanisms different from that employed by ebov [212] . among the escape mechanisms used by bacteria to evade immune responses, some have been recently reported to target ifn-i signaling molecules. having found that influenza viruses replicate to a higher efficiency in cells co-infected with staphylococcus aureus, warnking et al. [213] demonstrated that an impaired stat1/stat2 dimerization is responsible for a poor induction of isg transcription in spite of an abundant secretion of ifn-i driven by the flu virus infection. similarly, the inhibition of the response to ifn-i by mycobacterium tuberculosis was observed in human macrophages and correlated with mycobacterial pathogenicity [214] . in primary cells and thp-1 cells, indeed, mycobacterium tuberculosis specifically inhibits ifn-i signal transduction pathway by impairing the activation of stat1, while the avirulent mycobacterium bovis bcg fails to do so. alteration in isgf-3 complex formation was instead observed in human macrophages infected with nonpathogenic lactobacillus rhamnosus gg where only stat1 homodimers were found. in contrast, the pathogenic streptococcus pyogenes led to formation of not only stat1 homodimers but also of isgf-3 [215] . based on these finding the authors speculated that the efficient induction of ifn-i production and related transcription factor activation by streptococci would lead to fast and effective immune responses that, however, could play a role in the pathogenesis. although the first antiviral isgs were discovered decades ago, until recently the mechanisms of action was defined for only a limited number of isg-encoded proteins. the renewed interest in the innate immune response to retroviruses with the identification of how several host restriction factors may limit retroviral infection [118] [119] [120] , as well as large-scale functional screening of isgs, have identified genes that coordinately control the infection of a range of rna and dna viruses and have begun to dissect their mechanism of action [153, 216] . interestingly, some of the most potent antiviral effectors reinforce the system by further inducing ifn or isgs. thus, by directly disarming and/or making the use of individual ifn-induced effector proteins, the antiviral effect of host cells may still be attenuated even though ifn-i is induced, and viruses can ensure protection from both autocrine and paracrine effects of secreted ifn-i. here, we will primarily focus on isgs for which viral countermeasures have been identified in the context of viruses covered in the present review. recent reviews report more extensively on isg viral antagonists and specifically on ifn-induced hiv restriction factors that are not covered here [117] [118] [119] [120] [217] [218] [219] . pkr is one of the major effectors of the ifn-i-induced antiviral state. upon activation from binding dsrna molecules via a dsrna binding domain, pkr phosphorylates the translation initiation factor, eif2␣, thus blocking cellular and viral protein synthesis in infected cells. due to its ability to bind dsrna molecules pkr is also a nucleic acid sensor, as mentioned above [220] . viruses have evolved specific mechanisms to inhibit pkr activity or escape its action downstream. these include: the production of small and highly structured rna molecules that prevent the dsrnainduced dimerization and activation of pkr; expression of proteins that bind directly to and inhibit the activity of pkr or pkr activators; proteins that behave as pseudosubstrate and competitive inhibitors of pkr [221] [222] [223] . amongst viruses covered in this review, both antiviral and proviral roles of pkr have been reported for hcv. the hcv ns5a and e2 proteins directly interfere with the antiviral action of pkr. ns5a binds directly to pkr, while the glyco-protein e2 acts as competitive substrate with eif2 ␣ for pkr binding, resulting in inhibition of pkr kinase activity and in increased hcv replication. the full length ires of hcv rna, which is recognized by pkr, may mediate either activation or inhibition of pkr [224] . moreover, ns5a, by binding to various domains of the ires, can alter the activation of pkr [225] . another indirect inhibitory strategy mediated by the hcv ires has been recently reported. upon hcv rna sensing, pkr activates the mavs/traf3/irf3 pathway that, however, does not induce ifn but a set of isgs including isg15. as mentioned, isg15 deubiquitinates rig-i to negatively control the rig-i/mavs pathway and prevent uncontrolled detrimental ifn-i expression in physiological conditions. thus, hcv hijacks a protective cellular pathway to curtail host innate response [110] . through a specific ires-mediated inhibition of eif2␣-dependent translation, the hcv ires also regulates the translational activity of pkr [224] . eif2 ␣ is, indeed, essential to the translation of capped mrna, as those of isgs, while non-capped mrnas, as those of hcv, are translated independently from this factor. a general attenuation of isgs expression by hcv ires can be achieved also through a direct activation of pkr [226] . interestingly, this attenuation of isg expression is observed in acute but not persistent infection, where, instead, a sustained isg expression occurs [206, 227] . other proteins from flaviviruses have been shown to inhibit pkr including the ns2a protein of jev that physically interacts with pkr and blocks its activation in response to several stimuli [228] . ebov vp35 also interferes with the pathway regulated by pkr by blocking and also reversing pkr activation, thereby preventing translational arrest of viral mrnas. this pkr antagonism seems to be functionally different from dsrna binding and irf3 inhibition [229] [230] [231] . ebov vp 35 also associates with pact (pkr-associated activator). this complex abolishes pact interaction with the cterminal domain of rig-i, which is required for full activation of rig-i [232] . as for retroviruses, hiv-1 infection does not activate pkr due to both viral and cellular controls (reviewed in [233] ). in vitro, pkr is activated by low amounts of tar rna, whereas high concentrations inhibit the kinase function. the viral tat protein also counteracts pkr activation by several other mechanisms: it sequesters the activating dsrna; it can act as a substrate homologue of eif2␣ preventing the pkr mediated inhibition of protein synthesis; it prevents pkr auto-phosphorylation and exploits pkr activity to get phosphorylated and increase its binding to tar rna. moreover, hiv-1 replicates only in cells that have high levels of the tar rna binding protein (trbp), a strong inhibitor of pkr activation. interestingly, during hiv-1 infection of lymphocytes, when hiv-1 replicates at high levels, increased amounts of adar1, an ifn-induced rna editing enzyme that binds to pkr to inhibit its activation, have been observed. moreover, pact contributes to pkr dephosphorylation during hiv-1 replication probably due to its binding to adar1 [234] . thus, hiv-1 has evolved to replicate in cells with high levels of trbp, to induce the expression of adar1 and to change the function of pact for pkr inhibition [235] . isg15 is an ubiquitin-like modifier that is induced rapidly by ifn-i and possesses antiviral activity against a number of viruses. isg15 antiviral functions include inhibition of virus release, isgylation of both viral and host proteins and immunomodulatory cytokine-like properties in its unconjugated and secreted form, as recently reviewed [236, 237] . more than 160 host proteins that are isgylated have been identified including irf3, pkr and rig-i. isgylation preferentially targets newly translated proteins and, as a consequence of isgylation, degradation of the target protein is reduced by competition with ubiquitin conjugation [238, 239] . to date, only few viral proteins have been shown to be isgylated and functional characterizations of isg15 conjugation has not been always verified under conditions of endogenous protein expression [237] . moreover, often, isg15-mediated protection might not be a result of direct antagonism of virus replication. as an example, isg15 has been shown to inhibit the release of hiv-1 and ebov. this effect is mediated by an ubiquitin antagonism. isg15 disrupts the ubiquitin-mediated regulation of ebov vp40, necessary to produce budding and release of vp40 vlps [240] , as well as the ubiquitination of the hiv-1 gag protein, which is required for the interaction with the cellular protein tsg101 to mediate hiv-1 budding and release [241] . several viruses have, thus, developed countermeasures against isg15 and/or its conjugation. strategies, identified so far, include viral proteins that bind isg15 or that remove isg15 from target proteins (reviewed in [237] ). sars-cov plp has both deubiquitinating and deisgylating activities [242, 243] . recently, the structural basis of recognition and processing of deubiquitin and isg15 by plp has been reported [244] . interestingly, despite mers-cov encodes a single plp similar to sars-cov, there is little to no sequence conservation among residues important for the deubiquitinating and deisgylating activity, suggesting that mers-cov plp is likely to recognize and process ubiquitin and isg15 substrates differently than sars-cov plp [155, 244] . however, by affecting this post-translational modification, both viruses may modify cellular protein localization, protein activity and stability as well as signal transduction in order to increase viral replication and severity of infection. nevertheless, although these results stem from in vitro overexpression or mutant studies, the direct evidence for isg15 antagonism by these proteins remains to be demonstrated during viral infection. despite the well-characterized role in restricting replication of several viruses, isg15 may actually also promote the replication of specific viruses, as hcv. in hcv infections both anti-viral or proviral effects exerted by isg15 could be related to the net effect of isgylation on the various viral and host proteins targeted by isg15. an antiviral effect has been reported when isg15 could conjugate to hcv ns5a, thereby enhancing the inhibitory effect of ifn-i on hcv replication [245] . in contrast, several groups have reported that isg15 and isgylation promote hcv production in a cell culture model independently of upstream ifn signaling [246, 247] . although counter-intuitive, this finding may be explained by the observed inhibition of ifn-i induction by hcv upon isg15 overexpression that negatively controls the rig-i/mavs pathway at the level of rig-i ubiquitination [110] . a negative regulation of ifn-i expression may also be mediated by usp18, an isg displaying a potent inhibitory effect on the ifn-i pathway, to prevent autoinflammatory consequences of uncontrolled ifn-i production. similarly, usp18 may dampen the detrimental role that the hyperactivation of ifn-i signaling plays in the pathogenesis of some viral and bacterial infections, including hiv-1, hcv and mycobacterium tuberculosis. usp18 is, indeed, stabilized by isg15 in an unconjugated free form [248] . in this respect, the suggested potential role of the isg15/usp18 pathway in hcv persistence is consistent with the observation that increased expression of hepatic isgs before ifn treatment is associated with an absent or poor response in patients chronically infected with hcv [249] . thus, isg15/usp18 pathway might explain the paradox that the preactivation of the endogenous ifn system, while fails to clear the infection, instead, may stimulate hcv production and blunt the effect of exogenous ifn-i. usp18 is similarly induced during some bacterial infections, including salmonella and mycobacterium tuberculosis. the decreased survival of mice that carry a point mutation in usp18 results from higher salmonella load in the spleen and liver, an increased inflammatory response and increased ifn-i signaling. similarly, these usp18 mutant mice are more susceptible to mycobacterium tuberculosis infection and have increased bacterial load in the lung and spleen, elevated inflammatory cytokine production and more severe lung pathology [250] . in line with these findings, the results of dorhoi et al. [251] have shown that ifnar1 deficient mice were protected from death upon aerogenic infection with mycobacterium tuberculosis. moreover, a rather detrimental effect of ifn-i was also found in whole blood of patients with tuberculosis, where a neutrophil-driven, ifn-inducible transcriptional signature was associated with clinical severity [252] [253] [254] . these results thus reveal that some viruses and bacteria, to push replication and persistence, utilize an opposite, but as much as effectual strategy, consisting in enhancing/perpetuating an ifn-i response by targeting negative regulators of ifn-i expression. anti-microbial molecules, such as nitric oxide (no) radicals and reactive oxygen species (ros) mediate the antibacterial properties of ifn-i. the key producers of no is inducible nitric oxide synthase (inos), an enzyme that can be induced by both ifn-i and -ii, although the latter is the conventional inducer that plays a crucial role in fighting the infection of intracellular bacteria [255] . similarly, ifn-i and -ii induce the subunits of the phagocyte nicotinamide adenine dinucleotide phosphate (nadph) oxidase, which generates ros for killing organisms [256] . thus, pathogens have evolved several ways of avoiding ros-and no-mediated killing. in spite of the fact that no data are available, so far, on the strategies exploited by bacteria to contrast the ifn-mediated transcriptional regulation of inos and nadph oxidase, a common theme for successful intracellular pathogens is the ability to avoid the colocalization with these harmful host enzymes. intracellular salmonella, which resides within a specialized membrane compartment called the salmonella-containing vacuole (scv) in macrophages, uses a t3ss called salmonella pathogenicity island 2 (spi2) to mediate protection from no and ros intermediates [257] . intracellular organisms have also developed mechanisms to detoxify and repair no-mediated damage [255] , as well as to avoid the induction of inos activity [258] . given the crucial role played by these antimicrobial enzymes in contrasting bacterial infection, it is likely that strategies pinpointed by pathogens to inhibit the ifn-driven expression of these molecules will be discovered in the nearest future. to effectively resist the continual microbial threat from the environment, vertebrates possess several defense mechanisms including innate and adaptive immunity. as an essential component of the innate immunity, the ifn system constitutes the first line of defense against a number of pathogens to clear an incoming infection and instructing an ensuing adaptive response. successful pathogens have, thus, evolved sophisticated strategies to subvert and/or exploit the host immune system where blocking the ifn response, in the first place, is required to replicate and survive. the elucidation of some of these strategies has led to the identification of several, thus far, poorly recognized features of the innate immune response. in parallel, with the enormous recent advances in the comprehension of the molecular mechanisms of innate immune responses to pathogens, specific processes by which pathogenic microorganisms subvert these innate immune pathways, including the ifn system, is becoming progressively appreciated and it is reasonable to assume that many more will be discovered in the near future. by learning from the anti-immune strategies of pathogens we can, thus, not only identify key pathogen regulators as useful target to exploit to the host advantage, but we can also unveil weaknesses of host defenses and intervene to more precisely tune the immune response. the number and diversity of pathogen strategies for counteracting at each step the ifn system is stupefying. although beyond the scope of this review to discuss all antagonisms in detail, the ones that we have here reported, represent common and recurrent strategies used by a number of pathogens. this is illustrated by the existence of both viral and bacterial examples of pamp modifications as well as of viral and bacterial proteins that share cellular-like domain or cellular-like enzymatic properties that can compete with the host counterparts to dampen their physiological activities. in this respect, common hubs in the signaling pathways downstream pathogen sensors that trigger ifn-i, as few common adaptors or cofactors and transcription factors, are attractive targets of pathogen antagonism. the recognition of the mechanisms involved in microbial countermeasures may have several translation implications. the definition of microbial ability to elude the detection, as the methylation of their rna caps, can suggest strategies to utilize methyltransferase mutants as successful vaccine candidates against a number of different viruses as already suggested for denv [259] . many of the pathogen proteins responsible for ifn-i antagonism are also determinants of virulence and pathogenesis and, as such, they are highly conserved and may, thus, constitute attractive targets for the development of promising therapeutics against various clinically relevant pathogens reducing the bias of resistance mutations. in turn, some of the cellular identified targets of pathogen proteins as well as protein interacting partners might turn out to be new drug targets for treating a range of different diseases that disarme common components of the ifn pathway. in this respect, the resolution of the crystallographic structures of viral antagonists in complex with their different viral and cellular ligands, is then crucial for the rational design of new drugs. the system biology approach and the ability to simultaneously investigate diverse pathways has led to appreciate the interconnection between these pathways also in terms of shared components and stimulation by different pathogens. thus, a single therapeutic strategy could modulate multiple pathways to the host benefit. nevertheless, each approach needs to be complemented with effective treatments that also overcome other concurrent strategies that often the same pathogen put in place. on the other side of the coin, recovery of a full innate response must be finely tuned. ifn-i is, indeed, not always protective but can instead play a pathogenic role as reported for some bacterial and viral infections, where an uncontrolled ifn production is a determinant of disease progression. even in these cases, however, insights in the mechanisms involved in turning an ifn protective response into a pathogenetic one, may be as well relevant for nonpathogen-induced diseases, as autoimmunity and inflammatory diseases. in this context, cell death and inflammasome activation have been described as crucial ifn-i-regulated events exploited by both pathogen and host to get their own advantage. despite inflammasome facilitates pathogen clearance and is beneficial to the host, in some instances, ifn-induced non-canonical nlrp3 inflammasome activation and pyroptosis appear to be detrimental due to excessive cell death, inflammation, and collateral tissue damage in vital organs [260] . so pathogen proteins themselves or modified versions of them could be used as therapeutics working in suppressing inappropriate immune activation. this would be a bright way of hijacking molecules evolved during pathogen adaptation and associated fitness, to shift the balance to the host advantage. similarly, some identified targets of viral proteins in prr signaling pathways might be turned out to be new targets for treating a range of diseases. to find the way to generally induce an ifn response that is protective against a number of different infectious diseases, may be particularly relevant during an outbreak of unknown etiology or during the arising of newly emerging and re-emerging strains. likewise, finding the key to unlock the detrimental outcome of excessive ifn-i production during an infectious disease can open the way to cure autoimmune and inflammatory diseases. the authors declare no competing financial interests. innate immune sensing and signaling of cytosolic nucleic acids signaling by myeloid c-type lectin receptors in immunity and homeostasis immune sensing of dna intracellular toll-like receptors advances in nod-like receptors (nlr) biology the interferon response to intracellular dna: why so many receptors? intracellular sensing of viral dna by the innate immune system pathogen recognition and innate immunity irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors regulation of type i interferon responses interferon-stimulated genes: a complex web of host defenses mechanisms of type-i-and type-ii-interferon-mediated signalling charleston, type i and iii interferon production in response to rna viruses interferome: the database of interferon regulated genes the jak-stat pathway at twenty type i interferons in host defense ifn regulation and functions in myeloid dendritic cells confounding roles for type i interferons during bacterial and viral pathogenesis bacteria fighting back: how pathogens target and subvert the host innate immune system anti-immunology: evasion of the host immune system by bacterial and viral pathogens bacterial subversion of host innate immune pathways inverse interference: how viruses fight the interferon system viral tricks to grid-lock the type i interferon system viral evasion and subversion of patternrecognition receptor signalling cellular sensing of viral dna and viral evasion mechanisms microbial strategies for antagonizing toll-likereceptor signal transduction toll-like receptors and their crosstalk with other innate receptors in infection and immunity a virological view of innate immune recognition microbial sensing by toll-like receptors and intracellular nucleic acid sensors length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 rig-i-mediated antiviral responses to single-stranded rna bearing 5 -phosphates 5 -triphosphate rna is the ligand for rig-i rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids mitochondrial antiviral signaling protein (mavs) monitors commensal bacteria and induces an immune response that prevents experimental colitis identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation mita/sting: a central and multifaceted mediator in innate immune response dsrna-dependent protein kinase pkr and its role in stress, signaling and hcv infection ifit1: a dual sensor and effector molecule that detects non-2 -o methylated viral rna and inhibits its translation nod proteins: regulators of inflammation in health and disease mechanisms of inflammasome activation: recent advances and novel insights advancements in the battle against severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group coronaviruses: important emerging human pathogens from sars to mers: 10 years of research on highly pathogenic human coronaviruses severe acute respiratory syndrome vs. the middle east respiratory syndrome human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential to sense or not to sense viral rna -essentials of coronavirus innate immune evasion sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon ribose 2 -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 coronavirus non-structural protein 16: evasion, attenuation, and possible treatments 2 -o methylation of the viral mrna cap evades host restriction by ifit family members attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2 -o-methyltransferase activity rna 3 -end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp11 was essential for nsp11 to inhibit ifn-beta induction sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment sars-cov nucleocapsid protein antagonizes ifnbeta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response middle east respiratory syndrome coronavirus 4a protein is a double-stranded rnabinding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling flaviviral rnas: weapons and targets in the war between virus and host immune evasion strategies of flaviviruses flavivirus rna cap methyltransferase: structure, function, and inhibition flaviviruses and flavivirus vaccines emerging viral diseases: confronting threats with new technologies innate immunity evasion by dengue virus west nile virus infection and immunity flavivirus methyltransferase: a novel antiviral target 2 -o methylation of the viral mrna cap by west nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo ifit1 inhibits japanese encephalitis virus replication through binding to 5 capped 2 -o unmethylated rna the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex composition and three-dimensional architecture of the dengue virus replication and assembly sites dengue virus inhibits the production of type i interferon in primary human dendritic cells inhibition of the type i interferon response in human dendritic cells by dengue virus infection requires a catalytically active ns2b3 complex denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity activation and evasion of antiviral innate immunity by hepatitis c virus the global burden of hepatitis c interferon-stimulated genes and their role in controlling hepatitis c virus nonstructural protein 3-4a: the swiss army knife of hepatitis c virus cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity mitochondrial-associated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus dissociation of a mavs/ips-1/visa/cardif-ikkepsilon molecular complex from the mitochondrial outer membrane by hepatitis c virus ns3-4a proteolytic cleavage cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system hepatitis c virus ns4b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis c virus ns4b blocks the interaction of sting and tbk1 to evade host innate immunity immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the tolllike receptor 3 adaptor protein trif hcv-induced mir-21 contributes to evasion of host immune system by targeting myd88 and irak1 hepatitis c virus nonstructural protein 5a modulates the toll-like receptor-myd88-dependent signaling pathway in macrophage cell lines expression of toll-like receptors in chronic hepatitis c virus infection distinct toll-like receptor 3 and 7 expression in peripheral blood mononuclear cells from patients with chronic hepatitis c infection hepatitis c virus infection decreases the expression of toll-like receptors 3 and 7 via upregulation of mir-758 plasmacytoid dendritic cells sense hepatitis c virus-infected cells, produce interferon, and inhibit infection recovery, persistence, and sequelae in hepatitis c virus infection: a perspective on long-term outcome interferon-induced gene expression is a stronger predictor of treatment response than il28b genotype in patients with hepatitis c hiv and hcv activate the inflammasome in monocytes and macrophages via endosomal toll-like receptors without induction of type 1 interferon sex-specific association between x-linked toll-like receptor 7 with the outcomes of hepatitis c virus infection hepatitis c virus reveals a novel early control in acute immune response redefining the viral reservoirs that prevent hiv-1 eradication therapeutics for hiv-1 reactivation from latency hiv-1 latency: an update of molecular mechanisms and therapeutic strategies hiv-1, interferon and the interferon regulatory factor system: an interplay between induction, antiviral responses and viral evasion innate antiviral immune signaling, viral evasion and modulation by hiv-1 innate immune recognition of hiv-1 the interface between the innate interferon response and expression of host retroviral restriction factors unmasking immune sensing of retroviruses: interplay between innate sensors and host effectors type i ifn -a blunt spear in fighting hiv-1 infection interactions between hiv-1 and the cellautonomous innate immune system innate immune sensing of hiv-1 by dendritic cells the ribonuclease activity of samhd1 is required for hiv-1 restriction samhd1 host restriction factor: a link with innate immune sensing of retrovirus infection samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx vpx relieves inhibition of hiv-1 infection of macrophages mediated by the samhd1 protein dendritic cells in progression and pathology of hiv infection hiv-1 exploits innate signaling by tlr8 and dc-sign for productive infection of dendritic cells rig-i-mediated antiviral signaling is inhibited in hiv-1 infection by a proteasemediated sequestration of rig-i ifi16 senses dna forms of the lentiviral replication cycle and controls hiv-1 replication lieberman, the cytosolic exonuclease trex1 inhibits the innate immune response to human immunodeficiency virus type 1 cell death by pyroptosis drives cd4 t-cell depletion in hiv-1 infection the capsids of hiv-1 and hiv-2 determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hiv-1 evades innate immune recognition through specific cofactor recruitment transmission dynamics and control of ebola virus disease (evd): a review characterization of host immune responses in ebola virus infections the role of the type i interferon response in the resistance of mice to filovirus infection evasion of interferon responses by ebola and marburg viruses filoviral immune evasion mechanisms marburg virus vp35 can both fully coat the backbone and cap the ends of dsrna for interferon antagonism structural basis for marburg virus vp35-mediated immune evasion mechanisms the yin and yang of type i interferon activity in bacterial infection structural modifications of bacterial lipopolysaccharide that facilitate gram-negative bacteria evasion of host innate immunity intracellular shigella remodels its lps to dampen the innate immune recognition and evade inflammasome activation yopj targets traf proteins to inhibit tlr-mediated nf-kappab, mapk and irf3 signal transduction the shigella flexneri effector ospi deamidates ubc13 to dampen the inflammatory response inhibition of tlr signaling by a bacterial protein containing immunoreceptor tyrosine-based inhibitory motifs triggering the interferon antiviral response through an ikk-related pathway ikkepsilon and tbk1 are essential components of the irf3 signaling pathway regulation of immunity and oncogenesis by the irf transcription factor family interferon regulatory factors in immune cell development and host response to infection evidence for licensing of ifn-gamma-induced ifn regulatory factor 1 transcription factor by myd88 in toll-like receptor-dependent gene induction program peroxisomes are signaling platforms for antiviral innate immunity a diverse range of gene products are effectors of the type i interferon antiviral response regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease immunity by ubiquitylation: a reversible process of modification west nile virus nonstructural protein 1 inhibits tlr3 signal transduction abrogation of tlr3 inhibition by discrete amino acid changes in the c-terminal half of the west nile virus ns1 protein modulation of innate immune signaling by the secreted form of the west nile virus ns1 glycoprotein dengue virus subverts the interferon induction pathway via ns2b/3 protease-ikappab kinase epsilon interaction tick-borne flaviviruses antagonize both irf-1 and type i ifn signaling to inhibit dendritic cell function interaction between the hcv ns3 protein and the host tbk1 protein leads to inhibition of cellular antiviral responses hepatitis c virus ns2 protease inhibits host cell antiviral response by inhibiting ikkepsilon and tbk1 functions impairment of interferon regulatory factor-3 activation by hepatitis c virus core protein basic amino acid region 1 dead/h box 3 (ddx3) helicase binds the rig-i adaptor ips-1 to up-regulate ifn-beta-inducing potential hepatitis c virus core protein abrogates the ddx3 function that enhances ips-1-mediated ifn-beta induction repression of interferon regulatory factor 1 by hepatitis c virus core protein results in inhibition of antiviral and immunomodulatory genes hepatitis c virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor-7 and signal transducer and activator of transcription (stat) 1 protein expression battistini, hiv-1 targeting of ifn regulatory factors human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells hiv-1 accessory proteins vpr and vif modulate antiviral response by targeting irf-3 for degradation vpu-deficient hiv strains stimulate innate immune signaling responses in target cells hiv-1 vpu induces caspase-mediated cleavage of irf3 apoptosis as an hiv strategy to escape immune attack hiv infection of dendritic cells subverts the ifn induction pathway via irf-1 and inhibits type 1 ifn production irf-1 is required for full nf-kappab transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer modulation of human immunodeficiency virus 1 replication by interferon regulatory factors intracellular hiv-1 tat protein represses constitutive lmp2 transcription increasing proteasome activity by interfering with the binding of irf-1 to stat1 ikappab kinase epsilon targets interferon regulatory factor 1 in activated t lymphocytes the ebola virus vp35 protein inhibits activation of interferon regulatory factor 3 ebola virus protein vp35 impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk-1 basic residues within the ebolavirus vp35 protein are required for its viral polymerase cofactor function ebola zaire virus blocks type i interferon production by exploiting the host sumo modification machinery virus infection triggers sumoylation of irf3 and irf7, leading to the negative regulation of type i interferon gene expression beneficial innate signaling interference for antibacterial responses by a toll-like receptor-mediated enhancement of the mkp-irf3 axis the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferoninduced responses through downregulation of extracellular signal-regulated kinase 1-mediated signalling pathways severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane virus-induced unfolded protein response attenuates antiviral defenses via phosphorylation-dependent degradation of the type i interferon receptor a conserved peptide in west nile virus ns4a protein contributes to proteolytic processing and is essential for replication inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns5 as an interferon antagonist identification of residues critical for the interferon antagonist function of langat virus ns5 reveals a role for the rna-dependent rna polymerase domain inhibition of alpha/beta interferon signaling by the ns4b protein of flaviviruses dengue virus ns5 inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription 2 phosphorylation ns5 of dengue virus mediates stat2 binding and degradation dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns5 through a protein tyrosine phosphatase-mediated mechanism tick-borne encephalitis virus ns5 associates with membrane protein scribble and impairs interferon-stimulated jak-stat signalling activation of the ras/raf/mek pathway facilitates hepatitis c virus replication via attenuation of the interferon-jak-stat pathway expression of hepatitis c virus proteins inhibits signal transduction through the jak-stat pathway ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling-3 hcv structural proteins interfere with interferon-alpha jak/stat signalling pathway expression of hcv structural proteins impairs ifn-mediated antiviral response identification of the nonstructural protein 4b of hepatitis c virus as a factor that inhibits the antiviral activity of interferonalpha hepatitis c virus inhibits interferon signaling through up-regulation of protein phosphatase 2a intracellular innate immune cascades and interferon defenses that control hepatitis c virus hcv ns5a inhibits interferon-alpha signaling through suppression of stat1 phosphorylation in hepatocyte-derived cell lines ebola virus vp24 proteins inhibit the interaction of npi-1 subfamily karyopherin alpha proteins with activated stat1 ebolavirus vp24 binding to karyopherins is required for inhibition of interferon signaling ebola virus vp24 targets a unique nls binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated stat1 the ebola virus interferon antagonist vp24 directly binds stat1 and has a novel, pyramidal fold marburg virus evades interferon responses by a mechanism distinct from ebola virus super-infection with staphylococcus aureus inhibits influenza virusinduced type i ifn signaling through impaired stat1-stat2 dimerization inhibition of response to alpha interferon by mycobacterium tuberculosis lactobacilli and streptococci activate nf-kappa b and stat signaling pathways in human macrophages interferon-stimulated genes: roles in viral pathogenesis host restriction factors in retroviral infection: promises in virus-host interaction intrinsic antiviral immunity evolutionary conflicts between viruses and restriction factors shape immunity protein kinase pkr and rna adenosine deaminase adar1: new roles for old players as modulators of the interferon response the dsrna protein kinase pkr: virus and cell control inhibition of pkr by rna and dna viruses activation of the antiviral kinase pkr and viral countermeasures hepatitis c virus controls interferon production through pkr activation regulation of pkr by hcv ires rna: importance of domain ii and ns5a hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation failure of innate and adaptive immune responses in controlling hepatitis c virus infection blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein 2a ebola virus vp35 antagonizes pkr activity through its c-terminal interferon inhibitory domain the vp35 protein of ebola virus inhibits the antiviral effect mediated by double-stranded rna-dependent protein kinase pkr ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling mutual antagonism between the ebola virus vp35 protein and the rig-i activator pact determines infection outcome multiple levels of pkr inhibition during hiv-1 replication the pkr activator, pact, becomes a pkr inhibitor during hiv-1 replication hiv-1 translation and its regulation by cellular factors pkr and pact the antiviral activities of isg15 interferon-induced isg15 pathway: an ongoing virus-host battle positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification the isg15 conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg15 isg15 inhibits ebola vp40 vlp budding in an l-domain-dependent manner by blocking nedd4 ligase activity innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease inhibition of hepatitis c virus replication by ifn-mediated isgylation of hcv-ns5a the interferon stimulated gene 15 functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response isg15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment human intracellular isg15 prevents interferon-alpha/beta overamplification and auto-inflammation activation of endogenous type i ifn signaling contributes to persistent hcv infection contribution of increased isg15, isgylation and deregulated type i ifn signaling in usp18 mutant mice during the course of bacterial infections type i ifn signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics genome-wide expression profiling identifies type 1 interferon response pathways in active tuberculosis an interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis common patterns and disease-related signatures in tuberculosis and sarcoidosis inducible nitric oxide synthase and control of intracellular bacterial pathogens nadph oxidases: an overview from structure to innate immunity-associated pathologies salmonella pathogenicity island 2-dependent evasion of the phagocyte nadph oxidase modulation of inducible nitric oxide synthase expression by the attaching and effacing bacterial pathogen citrobacter rodentium in infected mice rational design of a live attenuated dengue vaccine: 2 -o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques role of type i interferons in inflammasome activation, cell death, and disease during microbial infection we apologize to the many colleagues whose data and influence have been overlooked due to space or our knowledge limitations. a special thank to members of the e.m. coccia's and a. battistini's laboratory for helpful discussion and critical reading of the manuscript and eugenio morassi for preparing drawings. our work is supported in part by grant rf-2010235199 from italian ministry of health (to emc) and from istituto superiore di sanità (to ab). key: cord-286352-uftl1mx5 authors: baril, martin; dulude, dominic; steinberg, sergey v; brakier-gingras, léa title: the frameshift stimulatory signal of human immunodeficiency virus type 1 group o is a pseudoknot date: 2003-08-15 journal: journal of molecular biology doi: 10.1016/s0022-2836(03)00784-8 sha: doc_id: 286352 cord_uid: uftl1mx5 abstract human immunodeficiency virus type 1 (hiv-1) requires a programmed −1 ribosomal frameshift to produce gag–pol, the precursor of its enzymatic activities. this frameshift occurs at a slippery sequence on the viral messenger rna and is stimulated by a specific structure, downstream of the shift site. while in group m, the most abundant hiv-1 group, the frameshift stimulatory signal is an extended bulged stem-loop, we show here, using a combination of mutagenesis and probing studies, that it is a pseudoknot in group o. the mutagenesis and probing studies coupled to an in silico analysis show that group o pseudoknot is a hairpin-type pseudoknot with two coaxially stacked stems of eight base-pairs (stem 1 and stem 2), connected by single-stranded loops of 2nt (loop 1) and 20nt (loop 2). mutations impairing formation of stem 1 or stem 2 of the pseudoknot reduce frameshift efficiency, whereas compensatory changes that allow re-formation of these stems restore the frameshift efficiency to near wild-type level. the difference between the frameshift stimulatory signal of group o and group m supports the hypothesis that these groups originate from a different monkey to human transmission. the frameshift stimulatory signal of human immunodeficiency virus type 1 group o is a pseudoknot introduction human immunodeficiency virus type 1 (hiv-1) is classified into three groups: m (main), that accounts for more than 99% of the worldwide pandemic, n (new or non-m/non-o), that is not clearly defined yet, and o (outlier), that has a genomic sequence differing by 60% from group m. each group is furthermore divided into subtypes based on differences in their env, gag or pol gene sequences. 1 although much less abundant than group m, group o viruses are spread widely in cameroon and equatorial guinea. 2 their resistance to non-nucleoside reverse transcriptase inhibitors active against group m 3 as well as the rapid rate at which they develop resistance to protease inhibitors indicate that they could become an important threat. 4 -6 hiv-1 uses a programmed 2 1 ribosomal frameshift during translation of its full-length messenger rna (mrna) to produce the gag -pol polyprotein, the precursor of the viral enzymes. this programmed event allows a minority of ribosomes to shift the reading frame at a specific sequence before they encounter the stop codon of gag, and to extend the translation to the pol gene, while conventional translation of the same mrna by the majority of ribosomes produces the gag polyprotein, the precursor of the viral structural proteins. 7, 8 the efficiency of the frameshift event controls the gag -pol to gag ratio, which is critical for rna dimerization, particle assembly, replication and viral infectivity. 9 -11 therefore, increasing or decreasing the frameshifting efficiency can interfere with the formation of infectious viral particles. the programmed 2 1 ribosomal frameshift requires two cis-acting elements in the viral mrna: a heptameric slippery sequence where the frameshift takes place (u uuu uua in hiv-1) and a downstream stimulatory signal. in group m, the stimulatory signal was previously assumed to be a simple 11-bp stem capped by a 4-nt loop, 7, 12 although an alternative structure was proposed but without any experimental support. 13 it was recently shown independently by our group 14 and by dinman et al. 15 that the frameshift stimulatory signal is longer than believed and that the sequence downstream of the classic stem-loop contributes to the frameshifting event. using a combination of site-directed mutagenesis and probing experiments, we demonstrated that this signal is an extended bulged stem-loop for subtype b of group m, 14 the subtype that prevails in north america and europe, and also that the same extended bulged stem-loop structure is found in all subtypes of group m (our unpublished results). in this signal, the upper portion of the stem corresponds to the classic stem-loop previously considered as the frameshift stimulatory signal, while the lower portion is formed by a base-pairing between the spacer separating the slippery sequence from the classic stem-loop and a complementary pyrimidine-rich sequence downstream of this stem-loop ( figure 1 ). this structure thus differs from most frameshift stimulatory signals, which were shown to be pseudoknots. 8 , 16 dinman et al. 15 proposed an alternative structure for the frameshift signal of hiv-1 group m. in their model, the pyrimidine-rich sequence downstream of the classic stem-loop interacts with three bases in the loop capping this stem through watson-crick interactions and forms a triplex with 4 bp on top of this stem. however, as discussed by dulude et al., 14 a careful analysis of this model shows that it is highly improbable, a major criticism being that the pyrimidine-rich strand that forms the triplex is in an antiparallel orientation relative to the purine-rich strand of the duplex with which it interacts, an orientation that is sterically not favored. here, the structure of the frameshift stimulatory signal of hiv-1 group o was investigated with a combination of mutagenesis and probing studies. the hiv-1 group o frameshift region or its mutants was inserted at the beginning of the coding sequence of a luciferase reporter gene, such that luciferase expression depends on a 2 1 frameshift. the luciferase activity was then assessed in 293t cultured cells and in vitro, in a rabbit reticulocyte lysate (rrl). the structure of the frameshift stimulatory signal of hiv-1 group o was also analyzed by probing the rna fragment encompassing this region with rnase v 1 . the proposed structure was then assessed by computer modeling. group o is subdivided into two subtypes: mvp5180 and ant70, for which there are seven and one complete pol sequences, respectively. our results show that the frameshift stimulatory signal of hiv-1 group o mvp5180 consists of a pseudoknot, and also, that this pseudoknot promotes a higher frameshift efficiency (and thus a higher gag -pol to gag ratio) than the group m frameshift stimulatory signal. however, sequence differences in subtype ant70 prevent the frameshift stimulatory signal from adopting the same pseudoknot structure as in subtype mvp5180. while the totality of studies on the hiv-1 frameshift have been carried out using group m, the frameshift region of group o has not been examined so far. we decided to investigate whether the frameshift region of hiv-1 group o encompasses the same slippery site and stimulatory signal as group m. as mentioned above, there are seven complete pol sequences for subtype mvp5180 and one complete pol sequence for subtype ant70 (los alamos national laboratory hiv sequence database). 17 we started the analysis of the frameshift region with mvp5180, the most represented subtype of group o. examination of mvp5180 sequences suggests that the slippery sequence in this group o subtype is the same as in group m, while the frameshift stimulatory signal could be predicted to be an 8-bp stem (the thermodynamic stability calculated with m-fold 18 is 2 14.9 kcal/mol), capped by a 10-nt loop and separated from the slippery sequence by an 8-nt spacer. this stem-loop is different from that initially proposed as a frameshift stimulatory signal for group m. moreover, for group o, because of the absence of base complementarity between the spacer region preceding the stemloop and the sequence downstream of this stemloop, the extended bulged stem-loop structure demonstrated for group m 14 cannot form ( figure 1 ). however, examination of the frameshift region sequence in mvp5180 suggests that its frameshift stimulatory signal could form a the slippery site is uuuuuua (underlined). all mutants of subtype mvp5180 were cloned by inserting between the kpn i-bam hi sites of the vector, the pcr product bearing the mutation investigated. for subtype ant70 of group o, the corresponding vector was constructed by exchanging the eco47iii-bam hi fragment with an appropriate oligonucleotide cassette. for the (2 1) constructs, the luciferase sequence is in the 2 1 reading frame relative to the aug initiation codon, so that a 2 1 frameshift is required to produce luciferase. an adenine was added immediately after the slippery sequence (at position 25) for the (0) constructs, so that luciferase is expressed by ribosomes that do not shift the reading frame. (b) sequences of the frameshift region of all constructs used in this study. nucleotides substituted or deleted compared to subtype mvp5180 of group o are underlined or represented by broken lines, respectively. derivatives was inserted between the first and second codon of luc, generating a series of phiv/ o-luc (2 1) constructs ( figure 2 ). these insertions were such that a 2 1 frameshift was required to express the luc reporter protein. an in-frame (0) control construct, in which ribosomes synthesize luc through conventional translation, was also made for each (2 1) frameshift construct examined, by adding an adenine immediately after the slippery sequence. the frameshift efficiency for each construct was measured by dividing the luciferase activity of the (2 1) construct by the sum of that of the (0) and (2 1) constructs, assuming that the same level of frameshift occurs in both (0) and (2 1) constructs. the frameshift efficiency of these constructs was assessed in cultured cells and in vitro. for assays in cultured cells, the different phiv/o-luc (0) and (2 1) constructs were transfected into 293t cells, and luciferase activity was measured in cell extracts 48 hours later. for in vitro assays, luciferase activity was measured after translation in rrl of the hiv/o-luc mrnas generated by transcription with t7 rna polymerase of the stu i-linearized plasmids. figure 3 shows the characteristics of the structure of the different constructs and their frameshift efficiency, and the results are summarized in table 1 . consistent with what was systematically observed in previous reports, the frameshift efficiency was higher in vitro than in cultured cells (see ref. 14 and references therein), and was less sensitive in vitro to the presence of the stimulatory signal, compared to the situation in cultured cells, 19, 20 which was proposed to result from differences between the rate of translation in vitro and in cultured cells. (21) construct, an in-frame (0) control was made to monitor the frameshift efficiency. the numbers between brackets represent the frameshift efficiency of each construct relative to phiv/o-87-luc, which is arbitrarily set at 100%. results are the means of five to six independent experiments. standard error on the mean was inferior or equal to 10%. destabilized or deleted, the frameshift efficiency was reduced by about tenfold in cultured cells and three to fivefold in vitro, compared to phiv/ o-87-luc. however, in phiv/o-1.12-luc, where the re-formation of the stem-loop was possible, the frameshift efficiency was restored to the wild-type level in vitro and in cultured cells. these results confirm the existence of the predicted 8-bp stem-loop structure and its involvement in the frameshift process. since the region downstream of the stem-loop increases frameshifting in the presence of this stem-loop, as shown in figure 3 , these results also support our suggestion that the frameshift stimulatory signal in mvp5180 is more complex than a simple stemloop and could correspond to a pseudoknot structure. characterization of the pseudoknot in the frameshift region of subtype mvp5180 of hiv-1 group o figure 4 (a) shows a standard representation of the hypothetical pseudoknot that we proposed to act as a frameshift stimulatory signal in hiv-1 group o. to demonstrate the existence of this pseudoknot, we made a series of mutations in the loop capping the 8-bp stem, named stem 1, as well as in the downstream complementary sequence predicted to interact with this capping loop and to form stem 2 of the pseudoknot. individually, these mutations impair the formation of stem 2, but when combined, should allow it to re-form ( figure 4) . the frameshift efficiencies of the constructs containing these mutations were assessed in vitro and in cultured cells and are presented in figure 4 (b) (see also table 1 to provide an independent support for the pseudoknot structure in subtype mvp5180 of hiv-1 group o frameshift stimulatory signal, we made a structural probing analysis of an rna fragment encompassing the gag/pol frameshift region of this subtype. enzymatic probing with rnase v 1 , an enzyme that cleaves rna in helical conformation, showed that the regions corresponding to stems 1 and 2 were attacked by the enzyme, with the strongest cleavage sites in stem 1 ( figure 5 ). stem 2 was also sensitive to rnase v 1 , contrasting with loop 2, which was only weakly attacked. the stacking of the two nucleotides in loop 1 likely accounts for its sensitivity to rnase v 1 . altogether, probing experiments are in perfect agreement with the mutagenesis studies, showing that the frameshift stimulatory signal of hiv-1 group o mvp5180 is a pseudoknot. structural analysis of frameshifting pseudoknots and other frameshift stimulatory signals can lead to the identification of conserved motifs and also to a prediction of the mechanism through which frameshift is stimulated. 21 based on the characteristics of their structure, two major classes of frameshifting pseudoknots can be proposed. in the first class, pseudoknots have an unpaired residue (usually an adenine) at the junction of the two stems, allowing a characteristic bent conformation that was proposed to be the conserved motif promoting frameshift stimulation. 22 the pseudoknot of the mouse mammary tumor virus (mmtv), resolved by nmr, 23 and that of the beet western yellow virus (bwyv), resolved by x-ray crystallography, 24 belong to this class. the frameshifting pseudoknots of the second class do not have an unpaired residue at the junction of the coaxially stacked stems. among pseudoknots of this class are those of the simian retrovirus-1 (srv-1), resolved by nmr, 25 and of the infectious bronchitis virus (ibv), which has been extensively studied by mutagenesis and probing studies. 26 -28 no additional structural data on frameshifting pseudoknots with stacked stems are available, making identification of a conserved motif difficult. in addition to these two classes of pseudoknots, the rous sarcoma virus (rsv) has an unusual pseudoknot, where the loop capping stem 1 is partly involved in the formation of stem 2 but contains two additional small sub-stem elements. 29 combining our mutagenesis and probing results with the structural information on 2 1 frameshifting pseudoknots recently provided by crystallographic and nmr studies, we made an in silico modeling of the hiv-1 group o pseudoknot ( figure 6 ). as shown above, this pseudoknot was predicted to be a classic hairpin-type pseudoknot (e.g. with base-pairing in a hairpin loop). it has two 8-bp stems (stem 1 and stem 2) connected by loops of 2 nt (loop 1) and 20 nt (loop 2). although the lengths of the stems and loops make the rna pseudoknot of group o different from other pseudoknots promoting frameshift, these lengths still fit to the general steric requirements that allow the coaxial arrangement of the two stems. 30 among the pseudoknots whose tertiary structure has been resolved, that of the srv-1 rna 25 is the best to be used as a starting conformation to model the structure of the group o rna pseudoknot. in both pseudoknots, there is no intervening nucleotide between the two stems, so that one can presume that, like in the srv-1 rna pseudoknot, there is no kink between the two stems of the group o pseudoknot. also, the lengths of stems 1 and 2 in both molecules differ by only two basepairs while the length of loop 1 differs by only one nucleotide, which necessitates only minimal rearrangements. the only region that is notably different in both pseudoknots is loop 2, which is 8-nt longer in the group o rna than in the srv-1 rna, and is much longer than needed for connecting the two stems, which suggests the existence of additional elements in the secondary structure of the pseudoknot. indeed, visual analysis of the nucleotide sequence of loop 2 revealed the possibility to form an additional 3-bp stem in this loop, immediately after the junction with stem 1 (stem ss in figure 6 ). if this sub-stem is positioned coaxially to stem 1, it would effectively reduce the number of nucleotides in loop 2 down to only eight. modeling such a structure showed that formation of this additional substructure and its coaxial position with respect to stem 1 does not create any problem for the connection of stem 1 and stem 2 with loop 2. formation of this substem would provide an additional stabilizing effect for the whole structure, where most nucleotides of the connector region become involved in h-bonding with the minor groove of stem 1, forming a triplex interaction. although disruption of the putative sub-stem by mutagenesis did not affect frameshift efficiency in our assays (data not shown), it is still possible that it could affect frameshifting with the full-length viral rna, and not with a reporter system. a similar 4-bp stem in loop 2 can also be proposed for the ibv pseudoknot, although a deletion in loop 2, impairing the formation of this putative stem, was found not to affect the frameshift efficiency. 31 the triplex interaction between loop 2 and stem 1 in group o pseudoknot involves an aacaa sequence. this is reminiscent of the situation observed in bwyv 24 and srv-1, 25 where an aacaa sequence present in loop 2 is implicated in loop-helix triplex inter-actions. furthermore, as in srv-1, the cytosine of the aacaa sequence bulges out in our modeling of group o pseudoknot. su et al. 24 and michiels et al. 25 from their studies with bwyv and srv-1, respectively, suggested that the formation of a triplex structure in a pseudoknot that promotes frameshift could be a signal to frameshift recognized by the translating ribosome. our results with hiv-1 group o support this suggestion. subtype ant70 of hiv-1 group o does not contain the same frameshift stimulatory signal as subtype mvp5180 as mentioned above, there is a second subtype for hiv-1 group o, ant70, 1 for which only one complete pol sequence is available. 17 sequence analysis suggests that subtype ant70 uses the same slippery sequence, followed by the same 8-bp stem-loop as in subtype mvp5180. however, sequence differences in the region downstream of this stem-loop prevent formation in ant70 of stem 2 of the pseudoknot present in mvp5180 (see figure 4 (a)). the frameshift efficiency of subtype ant70 was assessed with construct phiv/ o-ant70-luc containing the frameshift region from subtype ant70. it was found to be about half of that of phiv/o-87-luc both in vitro and in cultured cells (figure 4(b) ), which is consistent with the incapacity for this frameshift region to form the pseudoknot found in subtype mvp5180. this does not exclude however the possibility that the frameshift region of subtype ant70 can form another pseudoknot (see discussion). our results show that the frameshift stimulatory signal of subtype mvp5180 of hiv-1 group o is a pseudoknot, where 8 nt of a 10-nt loop capping an 8-bp stem base-pair with a downstream complementary sequence. the presence of this pseudoknot was demonstrated by site-directed mutagenesis and enzymatic probing of the frameshift stimulatory region. among the two distinct classes of 2 1 frameshifting pseudoknots described above, the group o pseudoknot falls in the same class as those of srv-1 25 and ibv, 26 characterized by the absence of any intervening nucleotide between the coaxial stems. interestingly, the frameshift efficiency of the group o pseudoknot is two and four times lower than that of the srv-1 and ibv pseudoknots, respectively. this can result from the difference in the length of stem 1, since shortening this stem from 11 to 10 bp caused an 85% loss of the frameshift efficiency for the ibv pseudoknot. 28 the lower frameshift efficiency observed for the hiv-1 group o pseudoknot can also relate to the absence of a guanosine-rich region in the 5 0 arm of stem 1, which was found to contribute significantly to stimulation of frameshift in ibv, 28 srv-1 32 and mmtv. 33 formation of stem 2 28 and rsv pseudoknots, 29 where destabilization of stem 2 has a less drastic effect than in srv-1 34 and mmtv, 33 for which the frameshift efficiency is reduced by eight to tenfold under these conditions. the use of a pseudoknot as a frameshift stimulatory signal makes hiv-1 group o distinct from group m, where this signal is an extended bulged stem-loop. 14 this observation supports previous phylogenetic analyses according to which the group o and group m clusters of hiv-1 originated from different monkey to human transmissions. 35, 36 since evolution led to the selection of a stimulatory signal in group o different from group m, it likely corresponds to a need specific to group o. here, we found that the frameshift efficiency in cultured cells was nearly 5% for group o mvp5180. when the frameshift region of group o was replaced with the frameshift region of group m subtypes, under conditions ensuring that any difference observed between the frameshift efficiency of group o and that of group m results from the difference in the structure of the stimulatory signal, we found that the frameshift efficiency of group m subtypes was about 2%, half of that of group o (our unpublished results). we can thus propose that the protease of hiv-1 group o viruses could be less active than group m protease, and that group o viruses require a gag -pol to gag ratio higher than that of group m members, so as to incorporate more gag -pol in their virions for an optimal fitness during infection. although there are no data available on the protease activity of group o, this protease is known to contain several secondary mutations that are found together with primary mutations causing resistance to protease inhibitors in variants of group m. 4 -6 moreover, mutations implicated in resistance to protease inhibitors usually reduce the activity of the enzyme in the absence of inhibitors. another possibility to account for the higher frameshift efficiency of group o is that the sequence variations in the frameshift region of group o, compared to group m, reduce the activity of the three peptides encoded by this region, the spacer peptide, p1, the transframe peptide, tfp, and p6. these peptides are important for the formation of infectious viral particles, 37 -39 and a higher production of gag -pol in group o viruses could compensate for a possible decrease in the activity of these peptides. surprisingly, the frameshift region of the other subtype of group o, ant70, also contains an 8-bp stem capped by a 10-nt loop, but cannot form the same pseudoknot as mvp5180, since variations in the sequence downstream this stem-loop impair the formation of stem 2. the level of frameshift efficiency in this subtype, as measured in our reporter system, is about twofold less than in mvp5180. we cannot, however, exclude the possibility that the loop capping stem 1 interacts with another region further downstream to form a pseudoknot that would promote frameshift to a level close to that in mvp5180, and such complementary regions can indeed be found in the viral rna sequence. loop 2 in these putative pseudoknots would be very large, but other viruses, such as the human coronavirus, are known to use a frameshift stimulatory pseudoknot where loop 2 contains more than 160 nt. 40 finally, ribosomal frameshifting in hiv-1 represents an interesting novel target for an antiviral treatment. it is particularly important to develop new anti-hiv agents directed against group o viruses, since these viruses are naturally resistant to non-nucleoside reverse transcriptase inhibitors 3 and bear mutations that could lead to faster resistance against protease inhibitors. 4 -6 the presence of a pseudoknot as a frameshift stimulatory signal in hiv-1 group o suggests that future anti-frameshift agents targeted against this signal must be developed independently for group m and group o members. all the plasmids used in this study were derived from pcdna3.1-luc, originating from pcdna3.1/hygro(þ ) (invitrogen) where the luciferase gene was inserted under control of a cmv and a t7 promoter. 14 an oligonucleotide cassette containing a 5 0 utr region of 55 nt, an initiator aug and, two codons downstream, an eco47iii restriction site, was inserted between restriction sites kpn i and bam hi of pcdna3.1-luc, generating pcdna3.1-5 0 utr-luc, where the insertion precedes the second codon of luciferase. the frameshift region of hiv-1 group o subtypes mvp5180 and ant70 (gen-bank accession nos. l20571 and l20587, respectively) was inserted between the eco47iii and bam hi sites, creating phiv/o-87-luc (21) and phiv/o-ant70-luc (21) constructs ( figure 2 ). these constructs are such that the luciferase coding sequence is in the 21 reading frame relative to the initiator codon, so that only ribosomes that make a 2 1 frameshift produce luciferase, while ribosomes translating in the 0 reading frame terminate translation at a stop codon overlapping codon six of the luc sequence. for each construct, an inframe control plasmid, the (0) construct, was created by inserting an additional adenine just after the slippery sequence of the (2 1) construct. derivatives of phiv/ o-87-luc (deletion and substitution mutants) were created by pcr, by first amplifying mutated dna fragments from phiv/o-87-luc construct with two primers for deletion mutants or mutants with substitutions 3 0 of the stem-loop of the stimulatory signal, and with four primers for mutants with substitutions in the slippery sequence and in the stem-loop, as described by ho et al. 41 the amplified dna fragments were then subcloned between the kpn i and bam hi sites of pcdna3.1-luc, and all constructs were verified by sequencing the entire insert. transfections of hiv-1 group o plasmids in 293t cells were carried using a standard calcium-phosphate precipitation method, 42 with 3 mg of a phiv/o-luc construct and 1.25 mg of pcdna3.1/hygro(þ)/lacz coding for b-galactosidase, as described. 14 for luciferase assays, 1.5 ml of cell extract (600 ml) was added to 50 ml of luciferase assay reagent (promega) and the light emitted was measured with a berthold lumat lb 9507 luminometer. frameshift efficiencies were calculated by dividing the luciferase activity of the (21) construct by the sum of the luciferase activity of the (0) and (2 1) constructs. the b-galactosidase activity was measured with the chlorophenolred-b-galactopyranoside substrate (calbiochem), as described, 43 with aliquots of 10 ml of cell extracts, and used to normalize luciferase activities for variations in transfection efficiency. in vitro transcriptions were carried out essentially as described, 14 using stu i-linearized phiv/o-luc constructs. these rna transcripts (0.2 mg) were translated in 25 ml of rrl (promega) at 30 8c for 15 minutes, a reaction time for which the translation system functions at its maximal rate. the reaction was stopped by addition of edta to a final concentration of 6 mm. luciferase activity was monitored as mentioned above, with 2.5 ml of the translation mixture. frameshift efficiencies were calculated as described above. enzymatic probing of the structure of an rna fragment encompassing the frameshift region of hiv-1 was performed as described with minor modifications. 14, 29 an oligonucleotide cassette containing a t7 promoter followed by the hiv-1 group o gag/pol frameshift region (bases 26 to 87, figure 2 ) was cloned between the nae i and bsp119i sites of pgem w -7zf(2) (promega), generating the recombinant plasmid pgem-hiv/o. the rna transcript, produced by in vitro transcription of the bsp119i-linearized plasmid, was 5 0 -end-labeled with g-32 p using a standard dephosphorylationrephosphorylation method, 44 purified from a 10% (w/v) acrylamide -7 m urea gel and dissolved in 500 mm nh 4 oac, 10 mm mg(oac) 2 , 1 mm edta and 0.1% (w/v) sds. probing with rnase v 1 (in a total volume of 10 ml containing 10 5 cpm of 5 0 -end-labeled rna supplemented with 1 mg of yeast trna) was done at 25 8c for 15 minutes in 10 mm tris-hcl (ph 8), 10 mm mgcl 2 , 100 mm kcl and 0 to 0.01 units of enzyme (ambion). the reaction was stopped by adding an equal volume of formamide gel loading buffer, the sample was heated two minutes at 95 8c and immediately loaded for analysis on a 10% and a 20% polyacrylamide -7 m urea gel. preliminary modeling was done interactively, using insightii/discover package (version 2000, accelrys inc., san diego, ca). the solution structure of the srv-1 rna pseudoknot 25 (pdb identificator 1e95), having six base-pairs in each of the two stems and one and 12 nucleotides in loops 1 and 2, respectively, was used as a starting conformation. the nucleotide sequence of the molecule was changed and additional base-pairs were appended to both stems according to the suggested model of the secondary structure for the rna pseudoknot of group o subtype mvp5180. the unpaired nucleotides of loops 1 and 2 were arranged as in the structure of these regions in the srv-1 rna pseudoknot, providing a reasonable system of h-bonds and basebase stacking interactions. the model was submitted to unrestrained energy minimization using the amber forcefield 30 until an energy minimum was reached. visualizations were done in a silicon graphics o2 computer. the pdb file is available on request. evaluation of hiv type 1 group o isolates: identification of five phylogenetic clusters geographical distribution of hiv-1 group o viruses in africa susceptibility of human immunodeficiency virus type 1 group o isolates to antiretroviral agents: in vitro phenotypic and genotypic analyses phylogenetic analysis of protease and transmembrane region of hiv type 1 group o naturally occurring sequence polymorphisms within hiv type 1 group o protease characterization of ribosomal frameshifting in hiv-1 gag-pol expression structure and function of the stimulatory rnas involved in programmed eukaryotic 21 ribosomal frameshifting. the ribosome overexpression of the hiv-1 gag -pol polyprotein results in intracellular activation of hiv-1 protease and inhibition of assembly and budding of virus-like particles overexpression of the gag -pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production maintenance of the gag/gag -pol ratio is important for human immunodeficiency virus type 1 rna dimerization and viral infectivity direct structural evidence for formation of a stem-loop structure involved in ribosomal frameshifting in human immunodeficiency virus type 1 structure of the autoregulatory pseudoknot within the gene 32 messenger rna of bacteriophages t2 and t6: a model for a possible family of structurally related rna pseudoknots characterization of the frameshift stimulatory signal controlling a programmed 21 ribosomal frameshift in the human immunodeficiency virus type 1 the frameshift signal of hiv-1 involves a potential intramolecular triplex rna structure structure, stability and function of rna pseudoknots involved in stimulating ribosomal frameshifting a compilation and analysis of nucleic acid and amino acid sequences. human retroviruses and aids edit expanded sequence dependence of thermodynamic parameters improves prediction of rna secondary structure a heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mrna secondary structure: demonstration by expression in vivo comparative studies of frameshifting and nonframeshifting rna pseudoknots: a mutational and nmr investigation of pseudoknots derived from the bacteriophage t2 gene 32 mrna and the retroviral gag-pro frameshift site a characteristic bent conformation of rna pseudoknots promotes 21 frameshifting during translation of retroviral rna the structure of an rna pseudoknot that causes efficient frameshifting in mouse mammary tumor virus minor groove rna triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot solution structure of the pseudoknot of srv-1 rna, involved in ribosomal frameshifting characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot evidence for an rna pseudoknot loop-helix interaction essential for efficient 21 ribosomal frameshifting the role of rna pseudoknot stem 1 length in the promotion of efficient 21 ribosomal frameshifting secondary structure and mutational analysis of the ribosomal frameshift signal of rous sarcoma virus mutational analysis of the rna pseudoknot component of a coronavirus ribosomal frameshifting signal analysis of the role of the pseudoknot component in the srv-1 gag-pro ribosomal frameshift signal: loop lengths and stability of the stem regions an rna pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger rna identification and analysis of the pseudoknot-containing gag -pro ribosomal frameshift signal of simian retrovirus-1 origin of hiv-1 in the chimpanzee pan troglodytes troglodytes identification of a new human immunodeficiency virus type 1 distinct from group m and group o proline residues within spacer peptide p1 are important for human immunodeficiency virus type 1 infectivity, protein processing, and genomic rna dimer stability autoprocessing of hiv-1 protease is tightly coupled to protein folding the late domain of human immunodeficiency virus type 1 p6 promotes virus release in a cell typedependent manner ribosomal frameshifting viral rnas site-directed mutagenesis by overlap extension using the polymerase chain reaction transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation a sensitive method for the detection of beta-galactosidase in transfected mammalian cells enzymatic approaches to probing of rna secondary and tertiary structure this study was supported by a grant from the canadian institutes of health research to l.b.-g. and guy lemay and a grant from the canadian institutes for health research to s.v.s. s.v.s. acknowledges fellowships from the canadian institutes of health research and from the fonds de la recherche en santé du québec. we thank guy lemay for his interest in this work. we are grateful to pascal chartrand, gerardo ferbeyre and hugo soudeyns for stimulating discussions and critical reading of this manuscript. key: cord-289274-3g67f8sw authors: tosoni, elena; frasson, ilaria; scalabrin, matteo; perrone, rosalba; butovskaya, elena; nadai, matteo; palù, giorgio; fabris, dan; richter, sara n. title: nucleolin stabilizes g-quadruplex structures folded by the ltr promoter and silences hiv-1 viral transcription date: 2015-10-15 journal: nucleic acids res doi: 10.1093/nar/gkv897 sha: doc_id: 289274 cord_uid: 3g67f8sw folding of the ltr promoter into dynamic g-quadruplex conformations has been shown to suppress its transcriptional activity in hiv-1. here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing g-quadruplex structures. the implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize g-quadruplex structures present in the ltr promoter. nucleolin recognized with high affinity and specificity the majority, but not all the possible g-quadruplexes folded by this sequence. in addition, it displayed greater binding preference towards dna than rna g-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. the interaction translated into stabilization of the ltr g-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both sirnas and a nucleolin binding aptamer greatly increased ltr promoter activity. these data indicate that nucleolin possesses a specific and regulated activity toward the hiv-1 ltr promoter, which is mediated by g-quadruplexes. these observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy. g-quadruplexes (g4s) are nucleic acids secondary structures that may form in single-stranded g-rich dnas and rnas under physiological conditions (1) (2) (3) . four gs bind via hoogsteen-type hydrogen bonds to yield g-quartets that in turn stack on top of each other to form the g4. g4s are highly polymorphic, both in terms of strand stoichiometry (forming both inter-and intramolecular structures) and strand orientation/topology. the presence of k + cations specifically supports g4 formation and stability (4) (5) (6) . in eukaryotes and prokaryotes, g4 dna motifs have been found in telomeres, g-rich micro-and mini-satellites, near promoters, and within the ribosomal dna (rdna) (7) (8) (9) . in the human genome, genes that are near g4 dna motifs fall into specific functional classes; for example, promoters of oncogenes and tumor suppressor genes have particularly high and low g4-forming potential, respectively (10) (11) (12) . human g4 dna motifs have been reported to be associated with recombination prone regions (13) and to show mutational patterns that preserved the potential to form g4 dna structures (9) . rna g4s have been detected in the 5 and 3 -utr and coding regions, in which they act as important regulators of pre-mrna processing (splicing and polyadenylation), rna turnover, mrna targeting and translation (14, 15) . regulatory mechanisms controlled by g4s involve the binding of protein factors that modulate g4 conformation and/or serve as a bridge to recruit additional protein regulators. indeed, g4 binding proteins can be classified into three functional groups: telomere-related proteins, such as the shelterin complex; proteins that unfold the g4 structure, such as the helicase and heterogeneous nuclear ribonucleoprotein families; proteins that stabilize g4s, a large group which includes nucleolin, maz and nucleophosmin (3, (16) (17) (18) . g4 structures and their cognate proteins are key players in numerous essential processes in eukaryotic cells. their misregulation has been associated with a number of relevant human diseases, such as the amyotrophic lateral sclerosis (19) (20) (21) , alzheimer (22) and fragile x syndrome (23) , in which expansion of g4-forming regions has been reported. moreover, mutations in g4-interacting proteins have been linked to genetic diseases, such as the werner syndrome and fanconi anemia (24, 25) . in recent years, new studies have contributed to increase our knowledge of the biological significance of g4s in prokaryotes (26, 27) and viruses (28) . we and other groups have identified functionally significant g4s in the nef coding region (29) and the unique ltr promoter (30) (31) (32) of the human immunodeficiency virus (hiv), the etiologic agent of the acquired immune deficiency syndrome (aids). these studies have shown that g4 folding at the ltr promoter decreased viral transcription with an effect that was augmented by g4 ligands (30, 33) . in this direction, the significance of these structures as focal points of interactions with host and viral factors is supported also by the observation that g4-folded sequences are specifically recognized by various viral proteins, such as the epstein barr virus nuclear antigen 1 (34, 35) and the sars coronavirus unique domain (sud), which occurs exclusively in highly pathogenic strains (36) . for this reason, we decided to pursue the investigation of putative cellular/viral proteins that may be involved in the regulation of the g4 ltr promoter activity in hiv. we employed a concerted approach combining electrophorethic mobility shift assay (emsa) and analysis by electrospray ionization mass spectrometric (esi-ms) to identify possible factors capable of binding the ltr g4 structure. in order to validate the findings, we then tested their stabilizing activity on the g4 fold and evaluated their ability to inhibit ltr-driven transcription in cells. the results provided new insights into the role of the ltr g4 in the viral life cycle, which could pave the way for the possible development of novel therapeutic strategies. all desalted oligonucleotides and aptamers were purchased from sigma-aldrich, milan, italy (supplementary table s1 ). the hiv-1 ltr region was inserted into the promoterless luciferase reporter vector pgl4.10-luc2 (promega italia, milan, italy) to form the pgl4.10-ltr-luc2 vector, as previously reported (30) . the renilla plasmid (p4.74, promega italia, milan, italy) was used as an internal control. the human enhanced green fluorescent proteinnucleolin plasmid (gfp-nucleolin) was purchased from addgene (addgene, cambridge, ma, usa). the pegfp empty vector was used as control (clontech, takara bio, otsu, japan). human embryonic kidney (hek) 293t cells (atcc # crl-3216) were grown in dmem (gibco, thermo fisher scientific, waltham, ma, usa) supplemented with 10% heat-inactivated fetal bovine serum (fbs, gibco, thermo fisher scientific, waltham, ma, usa). jurkat tlymphocytes cells (atcc # tib-152) were grown in rpmi 1640 (gibco, thermo fisher scientific, waltham, ma, usa) supplemented with 10% heat-inactivated fbs. mcf-7 human breast cancer cells (atcc # htb-22) were grown in rpmi 1640 supplemented with 10% heat-inactivated fbs. mcf10a normal human mammary epithelial cells (atcc # crl-10317) were grown in dmem/f12 (gibco, thermo fisher scientific, waltham, ma, usa) supplemented with 10% heat-inactivated fbs and egf (0.02 g/ml), hydrocortisone (0.5 g/ml), cholera toxin (0.1 g/ml), insulin (10 g/ml) (purchased all from sigma-aldrich, milan, italy). all cultures were grown in a humidified incubator maintained at 37 • c with 5% co 2 . oligonucleotides were 5 -end labeled with [␥ -32 p]atp using t4 polynucleotide kinase at 37 • c for 30 min. after dna precipitation, labeled species were resuspended in lithium cacodylate buffer (10 mm, ph 7.4) and kcl 100 mm. the oligonucleotides were denatured for 5 min at 95 • c and gradually cooled to room temperature to achieve proper folding of g-quadruplex structures. protein nuclear extracts of hek 293t and jurkat cells were obtained by using nx-tract kit (sigma-aldrich, milan, italy). recombinant full-length human nucleolin was expressed in hek293 cells and purified as described by the manufacturer (origene technologies, rockville, usa). labeled oligonucleotides (40 nm) were incubated in 20 l of reaction in emsa binding buffer and nuclear extract (0.5 g/l) or purified nucleolin (300 ng) for 2 h at 37 • c. emsa binding buffer composition was: tris-hcl 20 mm, ph 8, kcl 30 mm, mgcl 2 1.5 nm, dtt 1 mm, glycerol 8%, protease inhibitor cocktail (sigma-aldrich, milan, italy) 1%, naf 5 mm, na 3 vo 4 1 mm, poly [di-dc] (sigma-aldrich, milan, italy) 1.25 ng/l. in competition experiments, an excess of cold oligonucleotides was added to the samples and their ability to disrupt the g4 structures was monitored to evaluate binding specificity. after incubation, reaction solutions were loaded onto 5% native polyacrylamide gel in 1× tbe buffer and kcl 100 mm. dna-protein complexes were resolved by running the gel overnight at 27 v at 4 • c. emsa gels were dried using a gel dryer (bio-rad laboratories, milan, italy), free and bound dna molecules were visualized by phosphorimaging (typhoon fla9000, ge healthcare europe, milan, italy) and quantified by imagequant tl software (ge healthcare europe, milan, italy). after the desired complex was located on the gel, the corresponding band was cut and either directly in-gel digested for mass spectrometric (ms) analysis, or further purified by sds-page. extraction was performed in sds-page sample buffer. after 5 min in boiling water, samples were incubated overnight at 37 • c. finally, supernatant was loaded on 12% sds-page. the band of interest was excised after coomassie staining. hek 293t cells were seeded in a 10-cm dish in dmem supplemented with 10% heat-inactivated fbs and incubated overnight. cells were next either mock-transfected or transfected with of pnl4-3 (the reagent was obtained throughout the nih aids reagent program, division of aids, niaid, nih) (37) using calphos tm mammalian transfection kit (clontech, otsu, japan) according to the manufacturer's protocol. after 8 h, cells were washed with pbs 1× and fresh growth medium was added. forty eight hours post-transfection, cells were washed twice with cold pbs and scraped off. after a short centrifugation, pellet was resuspended in total protein extraction buffer (kcl 600 mm, tris-hcl 20 mm, ph 7.8, glycerol 20%, dtt 2 mm, protease inhibitor cocktail). cells were then lysed with three repeated freeze/thaw cycles and supernatant cleared by centrifugation, stored at −80 • c, and subsequently used in emsa assays. bands were treated according to established in-gel digestion protocols. briefly, they were first washed with 50% ch 3 oh and 2.5% acetic acid, dehydrated with ch 3 cn, and then reduced with 30 l of dtt (10 mm in 100 mm nh 4 hco 3 ) for 30 min at room temperature. the excess of dtt was eliminated before treating the bands with 30 l of iodoacetamide (50 mm in 100 mm nh 4 hco 3 ) for 30 min at room temperature in order to alkylate cysteine residues. bands were washed with 100 mm nh 4 hco 3 , dehydrated with ch 3 cn twice, and then digested. a 1 g aliquot of ms-grade trypsin (thermofisher scientific, waltham, ma, usa) in 50 l of 50 mm nh 4 hco 3 was added to the dehydrated bands, followed by incubation on ice for 30 min. the excess of trypsin was eliminated and substituted with 20 l of 50 mm nh 4 hco 3 and the sample was incubated overnight at 37 • c. peptides were extracted twice with 5% formic acid and two more times with 50% ch 3 cn, 5% formic acid. the peptide mixture was further desalted in a silica nanocolumn (polymicro technologies, phoenix, az, usa) packed in house with pinnacle c18 pack material (thermo fisher scientific, waltham, ma, usa). all materials were ms grade purchased from sigma aldrich, st. louis, mo, us except where otherwise indicated. the desalted mixture was finally analyzed by direct infusion electrospray ionization (esi) on a thermo fisher scientific (waltham, ma, usa) ltq-orbitrap velos mass spectrometer. the instrument was calibrated by using a 0.5 mg/ml solution of csi in 50% ch 3 oh, which provided a typical <2 ppm mass accuracy. all analyses were performed in nanoflow mode by utilizing quartz emitters produced in house by using a p2000 laser pipette puller (sutter instruments co., novato, ca, usa). up to 5 l samples were typically loaded onto each emitter by using a gel-loader pipette tip. a stainless steel wire was inserted through the back-end of the emitter to supply an ionizing voltage that ranged between 0.8 and 1.2 kv. bands containing bovine serum albumin (bsa) and empty gel bands were used as positive and negative control, respectively. putative peptides that were not present in blank samples were submitted to tandem mass spectrometric (ms/ms) analysis. these determinations involved isolating the precursor ion of interest in the ltq element of the instrument, activating fragmentation in either the ltq or the c-trap, and performing fragment detection in the orbitrap. the masses of the 50 more intense fragments were employed to perform a mascot database search (38) to identify their parent protein. the matched protein was deemed as being positively identified when two or more peptides provided a mascot score greater than 22. hek 293t protein nuclear extract (0.6 g/l) was incubated with biotinylated ltr oligo g4 folded (600 nm) in 250 l of reaction containing tris-hcl 20 mm, ph 8, kcl 30 mm, mgcl 2 1.5 nm, protease inhibitor cocktail 1%, naf 5 mm, na 3 vo 4 1 mm, poly [di-dc] 1.25 ng/l for 2 h at 37 • c. the binding reaction was followed by incubation (2 h at 37 • c) with 30 l of streptavidin-agarose beads (sigma-aldrich, milan, italy). after pbs washes, proteins were eluted with increasing amount of nacl (0.2 and 1m), and concentrated with amicon ultra 0.5 (merck millipore, germany). beads were collected by brief centrifugation, resuspended in 50 l of laemmli buffer, and finally incubated at 95 • c for 5 min. supernatants were separated on sds-page and analyzed by western blot. oligonucleotides were diluted to 0.1 m in lithium cacodylate buffer (10 mm, ph 7.4) and kcl 100 mm heat denatured for 5 min at 95 • c, and folded in g4 structure at room temperature for 16 h. samples were incubated alone, with purified human nucleolin (530 ng) or bovine serum albumin (bsa, negative control) for 1h at 37 • c. fluorescence melting curves were determined by using a lightcycler ii (roche, milan, italy). after a first equilibration step at 30 • c for 2 min, a stepwise increase of 1 • c every minute for 65 cycles was performed to reach 95 • c. a measurement was completed after each cycle by using 470 nm excitation and 530 nm detection. oligonucleotide melting was monitored by observing 6-carboxyfluorescein (6-fam) emission, which was normalized between 0 and 1. t m was defined as the temperature for which the normalized emission was 0.5. gene-specific pooled sirna trilencer targeting human ncl and a scrambled negative control duplex were purchased from origene (ncl trilencer-27 human sirna, origene technologies, rockville, md, usa). mcf7 cells were transfected with 1, 2 and 4 nm aliquots of human ncl sirna and control sirna by using lipofectamine rnaimax (invitrogen, thermo fisher scientific, waltham, ma, usa) following the manufacturer's instructions. pltr luciferase plasmid and renilla construct were transfected into the same cells 24 h later by using lipofectamine 3000 (invitrogen, thermo fisher scientific, waltham, ma, usa). in the double transfected cells, ltr promoter activity was assessed as firefly luciferase signal, normalized to renilla luciferase activity, by using dual-glo r luciferase assay system (promega italia, milan, italy), according to the manufacturer's directions (30) . depending on the transfected cell line (i.e. either mcf7 or mcf10a), the pgl4.10-ltr-luc2 vector provided signals ranging form 10 3 to 2 × 10 6 luciferase units, as measured by a victor x2 multilabel plate reader (perkin elmer italia, milan, italy). in contrast, the promoterless pgl4.10-luc2 and untransfected cells displayed a background signal lower than 10 luciferase units. all data were acquired in mediumfree pbs. dna aptamers as1411 and the control cro26 were added to cell medium at the time of transfection of nucleic acids research, 2015, vol. 43, no. 18 8887 pgl4.10-ltr-luc2 and p4.74-renilla plasmids and the luciferase signal read 24 h after transfection. each assay was performed in duplicate and each set of experiments was repeated at least three times. immunoblot analysis was performed on cell protein extracts obtained as previously described (39) . protein concentrations were quantified by using the pierce r bca protein assay kit (thermo scientific, rockford, il, usa) and the samples stored at −80 • c. each sample was electrophoresed on 12% sds-page and transferred to a nitrocellulose blotting membrane (amersham tm protan tm, ge healtcare life science, milan, italy) by using transblot sd semi-dry transfer cell (bio-rad laboratories, milan, italy). the membranes were blocked with 5% skim milk in pbst (0.05% tween 20 in pbs). membranes were incubated with the respective primary antibody directed against ncl (rabbit polyclonal c23 (h-250); santa cruz biotechnology, dallas, tx, usa), p24 (rabbit polyclonal; abcam, cambridge, uk), and ␤-actin (mouse monoclonal; sigma-aldrich, milan, italy). after three washes in pbst, membranes were incubated with ecl plex goat-␣-rabbit igg-cy5 or ecl plex goat-␣-mouse igg-cy5 (ge healthcare life sciences, milan, italy). images were captured on the typhoon fla 9000, and quantified by imagequant tl software. taq polymerase stop assay was carried out as previously described (30) . briefly, the 5 -end labeled primer (5 -ggcaaaaagcagctgcttatatgcag-3 ) was annealed to the template (supplementary table s1) in lithium cacodylate buffer in the presence or absence of kcl 100 mm by heating at 95 • c for 5 min and gradually cooling to room temperature. where specified, samples were incubated with purified human nucleolin (61 ng) at 37 • c for 2 h. primer extension was then conducted by using 2 u of amplitaq gold dna polymerase (life technologies, thermo fisher scientific) for 30 min at 37 • c or 47 • c. reactions were stopped by ethanol precipitation. primer extension products were separated on a 15% denaturing gel, and finally visualized by phosphorimaging (typhoon fla 9000). the dna substrate of interest was gel-purified before use and prepared in desalted/lyophilised form. the oligonucleotide was 5 -end-labeled with [␥ -32 p]atp by t4 polynucleotide kinase, purified by using microspin g-25 columns (amersham biosciences, europe), resuspended in lithium cacodylate buffer 10 mm, ph 7.4, kcl 100 mm, heatdenatured and folded. the oligonucleotide (1.23 m) was incubated either alone or with purified human nucleolin (300 ng) in emsa binding buffer for 2 h at 37 • c. sample solutions were then treated with dimethylsulfate (dms, 0.5% in ethanol) for 5 min and stopped by addition of gel loading buffer containing 10% glycerol and ␤-mercaptoethanol. samples were loaded onto 16% native polyacrylamide gels and run until the desired resolution was obtained. dna bands were localized via autoradiography, excised and eluted overnight. the supernatants were recovered, ethanolprecipitated and treated with piperidine 1m for 30 min at 90 • c. samples were dried in a speed-vac, washed with water, dried again and resuspended in formamide gel loading buffer. reaction products were analyzed on 20% denaturing polyacrylamide gels, visualized by phosphorimaging analysis, and quantified by imagequant tl software. spr was performed on the biacore t100 platform (ge healthcare, life science, milan, italy). purified human ncl was immobilized on serie s sensor chip cm5 by amine coupling. immobilization was performed in hepes-nacl running buffer (hepes ph 7. in previous work, we demonstrated that the hiv-1 ltr region can fold at least three different g-quadruplex (g4) structures at positions −92/−48 with respect to the transcription initiation site of the representative hxb2 lai (nc 001802) strain (30) . disruption of g4 by point mutations increased the transcript levels, thus indicating that g4 formation may contribute to the modulation of viral transcription. we reasoned that stabilization and unfolding of g4 at the ltr level are likely regulated by interactions with viral/cellular proteins. in order to test this hypothesis, ltr g4-forming sequences were incubated with nuclear protein extracts. ltr sequences of different lengths were employed to check whether individual g4 structures identified in the ltr promoter displayed different protein binding capabilities. in particular, we assayed sequences that possessed the minimal requirements to fold into an individual g-quadruplex (i.e. only 4 g-tracts: ltr-ii, ltr-iii and ltr-iv) and sequences capable of providing multiple g4s (i.e. more than 4 g-tracts: ltr-ii+iii+iv and ltr-iii+iv) ( figure 1a) . the corresponding 32 p-labeled oligonucleotides were incubated with nuclear extracts derived from two cell lines: jurkat t-lymphocytes that are a model for the natural hiv-1 targets in vivo; human embryonic kidney 293t cells that lack hiv-1 cell receptors and sustain all viral steps with the exception of virion attachment and entry. the latter can however be transfected with the hiv-1 proviral genome to produce fully competent and infectious viral particles, which indicates that their cytoplasmic/nuclear protein makeup is competent to sustain viral replication. samples were analysed onto native polyacrylamide gels to monitor the formation of slower running bands corresponding to oligonucleotide-protein complexes. as shown in figure 1b , all g4 ltr oligonucleotides formed slower running bands. the fact that the patterns obtained from the two nuclear extracts were essentially identical indicated that the selected cell lines contained very similar sets of g4-binding proteins. in particular, a very specific band migrated with the same rate in all g4 oligonucleotide samples (arrow in figure 1b) , thus suggesting that the same protein was able to bind all ltr g4 sequences considered. the intensity of the observed bands indicated that the longer sequence (i.e. ltr-ii+iii+iv) was bound most efficiently, whereas the shorter sequence (i.e. ltr-iv) supported only very modest complex formation. the ltr-ii+iii+iv oligonucleotide was incubated with extracts of hiv-1 producing and non-producing 293t cells to test whether the presence of viral proteins affected in any detectable way the observed emsa profiles ( figure 1c ). the actual presence of viral proteins in the transfected cells was assessed by western blot analysis ( figure 1d ). while viral proteins were well represented in the transfected extract, the emsa determinations revealed no major difference and confirmed that cellular proteins must constitute the major players in ltr g4 binding ( figure 1c) . in previous work, we demonstrated that one-or twonucleotide point mutations in the g-tracts involved in g4 pairing were able to partially or totally disrupt, respectively, g4 folding in the ltr sequence (ltr-m4, ltr-m5, ltr-m4+5), whereas a mutation in the loop was not (ltr-m3", figure 2a ) (30) . based on these observations, we used mutant ltr-ii+iii+iv oligonucleotides to assess the specificity of the observed protein for the g4 structure (figure 2b ). when incubated with the nuclear extracts, only the sequences that retained g4-folding capabilities (i.e. wt and m3") were able to form bands corresponding to the desired complexes (arrow in figure 2b ), whereas the mutants that partially fold or cannot form stable g4s were much less efficiently bound. a scrambled sequence matching the wt base composition did not bind at all (figure 2b and c). it should be noted that both a slower and a faster running bands were present in all samples (see asterisk in figure 2b ), indicative of nucleic acid binding proteins that may not be selective for g4. a competition experiment was performed to confirm the binding specificity of the selected protein, which involved mixing the labeled wt sequence with increasing amounts of unlabeled wt, mutant ltrs or scrambled sequence. as shown in figure 2d and e, only the wt and m3" sequences were able to effectively compete for protein binding, whereas the other oligonucleotides did not decrease the amount of protein bound to wt. interestingly, however, 10fold addition of m4, m5 and m4+5 oligonucleotides increased the amount of protein bound to the wt sequence. in this case, the mutant sequences may compete for binding to proteins that are sequence-but not structure-specific, thus leaving more g4-specific protein available to bind to the g4-folded wt sequence. furthermore, the fact that wt sequences folding g4 or ds structures bound different prog-tracts are shown in bold and are numbered (2-6') above the sequence. in the mutant sequences, the mutated bases are shown in red. the names of the mutant sequences correspond to the g-tracts where g bases have been mutated. scra stands for a sequence where gs have been scrambled to such an extent that no g4 can form. the ability of the mutant sequences to fold into g4 is reported on the right: y = folding, y/n = partial folding, n = no folding (measured as reported in (30) teins confirmed that the observed protein was specific for the g4 conformation ( figure 2f ). with the goal of identifying the protein of interest, the relevant emsa band was excised from the gel and submitted directly to trypsin digestion (see materials and methods). alternatively, the material extracted from the emsa band, which could possibly include multiple co-migrating species, was further purified on a sds gel before trypsin digestion. in either case, the digestion products were subjected to ms/ms analysis and database searching ( figure 3a ) (40, 41) , which provided an excellent match with human nucleolin (ncl) ( table 1 ). the experiment was separately repeated three times to corroborate the results. positive identification was also confirmed by performing emsa analysis of samples that included the g4-folded wt and mutant ltr-ii+iii+iv sequences with either nuclear extracts or purified human ncl. ncl displayed the same binding activity towards the wt and mutant ltr sequences manifested by the unknown protein ( figure 3b ). ncl binding to the wt ltr-ii+iii+iv g4 was concentration dependent (figure 3c and d) . surprisingly, however, two ncl/ltr complex bands were identified ( figure 3b and c). it has been reported that both native and purified ncl display self-cleaving activity and indeed preparations of ncl usually exhibit multiple bands (42) . our purified ncl also showed multiple bands on sds gel, as detected by both coomassie staining and western blot analysis (supplementary figure s1a ). ms analysis of the four bands detected by coomassie staining showed full-length peptide coverage only for the upper major band, whereas coverage of the n-terminal portion was missing in the lower three bands (supplementary figure s1b) . we therefore ascribed the upper and lower bands to the full-length and cleaved forms of ncl, respectively. interestingly, the band obtained from nuclear extracts migrated the fastest and, indeed, the nterminal portion was not observed by ms peptide analysis (table 1 and supplementary figure s1b ). in addition, when the protein bound to ltr g4 in the emsa gel was extracted and analyzed separately on sds gel, it migrated at around 75 kda, corresponding to one of the cleaved forms of ncl (data not shown). therefore, we conclude that the ncl form present in the nuclear extracts corresponds to its cleaved portion lacking the n-terminus. in addition, ncl formed a complex with a scrambled sequence, which displayed a slightly slower migration rate compared to the g4-bound ncl ( figure 3b ). the amount of complex was similar to that afforded by m4 and m5 ltr sequences. binding of ncl to g-rich oligonucleotides has been previously reported (43) and provides an excellent indication that the rna binding domains (rbd) of the purified ncl are indeed active. in analogous fashion, biotinylated wt, m4+5 ltr-ii+iii+iv, and scrambled-sequence oligonucleotides were incubated with nuclear extracts and then added to streptavidin-functionalized agarose beads to facilitate removal of unbound proteins. immobilized proteins were eluted with buffers of increasing ionic strength and identified by western blot analysis ( figure 3e) . ncl was released from the wt sequence when treated with increasing concentrations of nacl or heated to 95 • c in denaturing buffer, whereas it was released from the m4+5 and scrambled sequences at the lowest nacl concentration, consistent with a lower affinity for these non-g4-forming sequences. these data confirm that ncl specifically binds the g4 folded conformation of the ltr-ii+iii+iv sequence. taq polymerase stop assays were performed to assess the stabilization imparted by ncl to the various ltr g4 structures. the wt ltr-ii+iii+iv and m4+5 mutant sequences were extended to include a primer-annealing region and used as templates for a single-cycle taq reaction (supplementary table s1 ). elongation of the wt template was performed at 37 • c or 47 • c in the presence/absence of ncl. in samples containing 100 mm kcl and no ncl, a pausing site corresponding to the most 3 -end g-tract was observed only in the reaction elongated at 37 • c. the pause was not observed at 47 • c, consistent with possible destabilizing effects of temperature on the g4 structure (compare lanes 2 and 5 of figure 4a ). addition of ncl induced more evident pauses at both elongation temperatures (figure 4a, lanes 3 and 6) , which clearly highlighted the stabilizing properties of ncl on g4 conformation. as expected from the inability of the m4+5 mutant to fold g4 structures, no pausing sites were observed regardless the presence of protein ( figure 4a, lanes 7-9) , thus confirming the specificity of ncl binding for full-fledged g4s. at the same time, fret melting assays were also carried out to study the stabilizing effects of ncl on the g4 structures. the assays involved the synthesis of constructs that combined selected g4-forming sequences, such as ltr-ii+iii+iv and the shorter ltr-ii, ltr-iii, ltr-iv, and ltr-iii+iv (figure 2a) , with fam and tamra moieties placed at their 5 -and 3 -ends, respectively. the results showed that ncl conferred the highest stabilization in the series to the ltr-ii+iii+iv construct, followed by ltr-iii+iv. progressively lower stabilization was observed for ltr-iii and ltr-ii, whereas ltr-iv was the least affected in the series ( table 2 ). the negative control bovine serum albumin (bsa) did not afford any detectable stabilization to the selected sequences. to identify the position of putative ncl binding sites onto the dna g4 structures, we performed dimethylsulfate (dms) footprinting. when a complex of ncl with the ltr-ii+iii+iv dna was assessed ( figure 4b ), the methylation pattern revealed that the 3 region of the ltr sequence was protected by protein binding with unique specificity for two g bases in g-tract 4, as highlighted in figure 4c . based on the facts that ncl has been described as a rna-binding protein (44) and that the ltr sequence is present in the u3 region of the hiv-1 genome during the initial infection steps, we tested whether ncl could also bind the rna version of the ltr g4. in this case, ncl was incubated with labeled rna or dna oligonucleotides capable of folding ltr g4s. increasing concentrations of unlabelled rna and dna counterparts were employed to compete for ncl binding. the results clearly showed that the protein was able to bind both types of g4 oligonucleotides (lanes 1 and 6, figure 5a ). however, the dna g4 was consistently able to outcompete the rna version table 1 ). only characteristic b and y ions are indicated (40, 41) . the data matched the sequence of peptide t487-k508 of ncl, which is reported on top with the observed fragments. (b) emsa analysis of the binding of nuclear extract (ne) proteins and purified ncl to the wt and mutant ltr sequences. arrows indicate relevant protein/ltr g4 complex bands. (c) emsa analysis of the binding of increasing amounts of purified ncl to the wt ltr-ii+iii+iv g4. the vertical bar highlights the portion of the gel where the two ncl/ltr g4 complex bands are observed. (d) quantification of the upper and lower ncl/ltr g4 complex bands obtained in the emsa in panel (c). (e) pull-down assay of nuclear extract proteins with wt, mutant g4 ltr-ii+iii+iv and random (rnd) sequences, immobilized on agarose beads. shown is the western blot analysis with an ncl antibody. proteins complexed to the beads-bound ltrs were washed with augmented stringency by increasing the ionic strength of the wash buffer (0.2 and 1 m). the final elution was obtained in denaturing buffer at 95 • c. for protein binding (lanes 7-10, figure 5a and b), whereas the rna was incapable of outcompeting the dna (lanes 2-5, figure 5a and b). we had previously shown that the ltr rna g4s fold into parallel structures (33) , whereas the dna counterparts display rather hybrid-like conformations (30) ; therefore, the preferential binding toward the dna g4 is likely caused by these substantial structural differences between the dna and rna g4 conformations. surface plasmon resonance (spr) analysis was next used to assess the affinity of the purified ncl for the wt ltr-ii+iii+iv g4: a k d of 2.5 ± 0.1 nm was obtained, which indicates an extremely high affinity of the protein for this ltr g4 ( figure 5c ). in contrast, the scrambled oligonucleotide showed no affinity for ncl (supplementary figure s1 ). a luciferase reporter assay was established to explore the downstream biological effects of ncl binding to the ltr promoter. two epithelial breast cell lines were selected for the assay: mcf-7 breast cancer cells and mcf-10a normal breast epithelial cells. the latter inherently express lower amounts of ncl compared to tumor cells (45, 46) . for this reason, the mcf-10a cells were transfected with wt or m4+5 ltr luciferase reporter plasmids, either alone or in the presence of increasing amounts of ncl expression vector. the luciferase signal was measured to determine the level of activation of the ltr promoter. the results showed that the activity of wt ltr decreased to 65% of the control, while that of the m4+5 promoter remained unvaried ( figure 6a ). the mcf7 cell line that overexpresses ncl was employed to perform additional activity assays. in this case, cells were treated with increasing amounts of sirnas designed to target ncl mrna (47) , and then transfected with wt or mutant m4+5 ltr luciferase reporter plasmids. analysis of ncl content showed that the protein was effectively depleted at 1-4 nm sirna, reaching 8% of the initial amount at 4 nm ( figure 6b ). measured by luciferase activity, the effect of ncl depletion on ltr promoter activity was quite astonishing: at 4 nm of sirna, the promoter activity of the wt sequence was 37 folds that of the control while the mutant m4+5 sequence was only 1.5 folds (figure 6c) . as a complementary approach, we tested the effect of the ncl-targeted dna aptamer as1411, which has been reported to bind with high affinity ncl in cells (48) . at 1 m of as1411, we again observed a significant increment of the wt ltr promoter activity to reach 11 times that of the non-treated control while the mutant m4+5 sequence increased of only 2.5 times ( figure 6d ). in contrast, the control sequence cro26 that is complementary to as1411 did not modify ltr promoter activity. these data indicate that the specific binding of ncl to g4 structures in the ltr promoter exerts significant repressive effects on hiv-1 promoter activity. we have identified ncl as a prominent host factor capable of binding with high affinity to the g4 structures present in the ltr promoter of hiv-1. we observed that the specific interaction leads to g4 stabilization and contributes to silence viral transcription. conversely, we also demonstrated that ncl depletion produces extraordinary enhancing effects on ltr promoter activity. these observations are consistent with the multifaceted nucleic acid binding and chaperoning activities attributed to this protein. indeed, ncl is most abundant in the nucleolus, but can be found also in cell membranes and, upon stress stimuli, in the nucleoplasm and cytoplasm, to some extent (49, 50) . among other functions, it is involved in transcription (50) by specific interactions with sequences that can adopt complex secondary structures (44, (51) (52) . it is widely believed that ncl plays a chaperone role by helping the correct folding of complex nucleic acids structures. indeed, ncl has been shown to display a marked preference for both endogenous and exogenous g-rich sequences that can fold into g4 (53) . it has been recently reported that binding of ncl to the endogenous (ggggcc) n hexanucleotide repeat expansion (hre) in c9orf72 is responsible for the initiation of molecular cascades that lead to neurodegenerative diseases (19) . at the promoter level, binding of ncl to g4 structures augments the basal effect of the folded conformation (45, (54) (55) (56) . one of the best documented example of g4-mediated regulation among g4 promoters (57) is that of c-myc, which shows striking similarities with the g4mediated regulation of the hiv-1 ltr promoter reported here and previously by us (30) . both cases involve multiple g-tracts that enable folding into alternative g4 conformations; g4 parallel-like topology (58); at least one g 3 n 1 g 3 motif; binding sites for sp1; silencing effect on promoter activity (59); ncl-binding activity and higher affinity of ncl towards the dna g4 compared to the rna g4 counterpart (45) . in the case of c-myc, it has been shown that the n-terminal of ncl is dispensable for its g4 binding activity (56) . here, we show that ncl can naturally produce cleaved forms that lack the n-terminal but retain full binding capabilities. these observations demonstrate that cells allow the formation of ncl cleaved species that maintain their g4/nucleic acid binding activity. on the other, they suggest that the hiv-1 virus and human host cells have likely evolved identical mechanisms to control transcription at the dna promoter level. the results of our experiments provided valuable insights into the determinants of ncl binding to the various ltr g4-forming structures. the lower binding of ncl to ltr-iv compared to the other g4 structures suggests that the interaction has both conformation-and sequence-dependent characteristics, which in turn implies the fascinating possibility that different g4s in the hiv-1 ltr promoter may exert different functions based on their binding partners. moreover, the greater affinity demonstrated for dna than rna constructs indicates that the interaction is deeply influenced by conformational differences between g4 structures folded by the different types of biopolymers. the facts that the hiv-1 genome consists of rna, that this g4forming sequence is also present in the u3 region of the genome (33) , and that viral rna during the first steps of infection is still present in the cell cytoplasm where ncl levels are low, suggest that the specific binding of ncl to the dna version may represent an essential mechanism for regulating viral transcription. in this direction, it has been shown that ncl is involved in different steps of the hiv-1 life cycle. inhibition of surface ncl by different cellular and synthetic compounds (60-62) affects cell attachment/entry by the virus (63) . in addition, ncl can bind hiv-1 gag protein to promote viral budding (64) , or to enhance gag release (65) . further, ncl involvement in the viral life cycle has been corroborated also by evidence that hiv infection modifies the protein's cellular distribution (66, 67) . the activities performed by ncl in other viruses have also been described in a number of recent papers (68) (69) (70) (71) (72) . for example, binding of ncl to epstein-barr virus (ebv) nuclear antigen 1 (ebna1) modulates viral replication and transcription (73) , and virus-induced relocalization of ncl has been observed in some instances (74, 75) . in this broader context, these reports support our new findings that point toward a significant role played by this protein in hiv-1 replication. the observation that ncl interaction with ltr g4 silences viral transcription is in apparent contrast with the well-known ability of ncl to interact with histone h1, which induces chromatin decondensation (76) that in turn facilitates the passage of the dna polymerases (77) . however, the opposite effect consisting of ncl-mediated repression of dna replication has been also reported (49, (78) (79) (80) , and has been attributed to the interaction of ncl with the dna processing enzyme replication protein a (rpa). in addition, ncl has been reported to recruit a dna helicase that unwinds g4 structures (81) . in both cases, ncl binding to cellular proteins is a transient response to stress stimuli. these observations prompt the intriguing possibility that when ncl is redistributed by the cellular stress imposed by hiv infection (66, 67) , it may be then recruited by the g4 structure of viral promoter to transiently downregulate hiv transcription and enable the virus to prepare for subsequent efficient transcription, when viral proteins, such as tat, take over. alternatively, this interaction may be required as a first switch to viral latency and to recruit proteins that further consolidate latency. the signals that trigger latency are not known at this point and are thus the object of intense studies. however, factors that repress viral transcription at the ltr promoter have been proposed to play a determinant role in latency mechanisms (82, 83) . finally, the very specific nature of nucleolin binding indicates that its viral target must be less prone to mutations. this observation makes the viral g4/nucleolin complex into a very appealing target for the development of antiviral strategies that may afford a different mechanism of action, the possibility of targeting viral latency, and a lower probability of incurring into drug resistance. in conclusion, we have shown that the specific binding of the cellular protein ncl to the ltr promoter regulates viral transcription. this result alone paves the way for the investigation of different regulation mechanisms of hiv-1 transcription/latency, which may lead to new possible targets for the design of specific inhibitors. g-quadruplex structures: in vivo evidence and function formation of parallel four-stranded complexes by guanine-rich motifs in dna and its implications for meiosis g-quadruplex nucleic acids and human disease a sodium-potassium switch in the formation of four-stranded g4-dna neurodegenerative diseases: g-quadruplex poses quadruple threat g-quadruplexes and metal ions gene function correlates with potential for g4 dna formation in the human genome g-quadruplexes in promoters throughout the human genome the disruptive positions in human g-quadruplex motifs are less polymorphic and more conserved than their neutral counterparts g-quadruplex formation within the promoter of the kras proto-oncogene and its effect on transcription conserved elements with potential to form polymorphic g-quadruplex structures in the first intron of human genes evidence of genome-wide g4 dna-mediated gene expression in human cancer cells genome-wide analyses of recombination prone regions predict role of dna structural motif in recombination exploring mrna 3 -utr g-quadruplexes: evidence of roles in both alternative polyadenylation and mrna shortening 5 -utr rna g-quadruplexes: translation regulation and targeting the evolving world of protein-g-quadruplex recognition: a medicinal chemist's perspective the kras promoter responds to myc-associated zinc finger and poly(adp-ribose) polymerase 1 proteins, which recognize a critical quadruplex-forming ga-element hras is silenced by two neighboring g-quadruplexes and activated by maz, a zinc-finger transcription factor with dna unfolding property c9orf72 nucleotide repeat structures initiate molecular cascades of disease g-quadruplex structures contribute to the neuroprotective effects of angiogenin-induced trna fragments characterization of dna g-quadruplex species forming from c9orf72 gc-expanded repeats associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration a g-rich element forms a g-quadruplex and regulates bace1 mrna alternative splicing the fragile x syndrome d(cgg)n nucleotide repeats form a stable tetrahelical structure human werner syndrome dna helicase unwinds tetrahelical structures of the fragile x syndrome repeat sequence d(cgg)n fancj helicase defective in fanconia anemia and breast cancer unwinds g-quadruplex dna to defend genomic stability genome-wide study predicts promoter-g4 dna motifs regulate selective functions in bacteria: radioresistance of d. radiodurans involves g4 dna-mediated regulation the genome-wide distribution of non-b dna motifs is shaped by operon structure and suggests the transcriptional importance of non-b dna structures in escherichia coli g-quadruplexes in viruses: function and potential therapeutic applications formation of a unique cluster of g-quadruplex structures in the hiv-1 nef coding region: implications for antiviral activity a dynamic g-quadruplex region regulates the hiv-1 long terminal repeat promoter topology of a dna g-quadruplex structure formed in the hiv-1 promoter: a potential target for anti-hiv drug development u3 region in the hiv-1 genome adopts a g-quadruplex structure in its rna and dna sequence anti-hiv-1 activity of the g-quadruplex ligand braco-19 g-quadruplexes regulate epstein-barr virus-encoded nuclear antigen 1 mrna translation role for g-quadruplex rna binding by epstein-barr virus nuclear antigen 1 in dna replication and metaphase chromosome attachment the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone probability-based protein identification by searching sequence databases using mass spectrometry data a rapid micropreparation technique for extraction of dna-binding proteins from limiting numbers of mammalian cells proposal for a common nomenclature for sequence ions in mass spectra of peptides contributions of mass spectrometry to peptide and protein structure increased stability of nucleolin in proliferating cells by inhibition of its self-cleaving activity dna binding properties of a 110 kda nucleolar protein localization of nucleolin binding sites on human and mouse pre-ribosomal rna identification and characterization of nucleolin as a c-myc g-quadruplex-binding protein the nucleolin targeting aptamer as1411 destabilizes bcl-2 messenger rna in human breast cancer cells epithelial-mesenchymal transition in human gastric cancer cell lines induced by tnf-alpha-inducing protein of helicobacter pylori antiproliferative activity of g-rich oligonucleotides correlates with protein binding stress-dependent nucleolin mobilization mediated by p53-nucleolin complex formation functions of the histone chaperone nucleolin in diseases two rna-binding domains determine the rna-binding specificity of nucleolin molecular basis of sequence-specific recognition of pre-ribosomal rna by nucleolin agro100 inhibits activation of nuclear factor-kappab (nf-kappab) by forming a complex with nf-kappab essential modulator (nemo) and nucleolin heterogeneous nuclear ribonucleoprotein k and nucleolin as transcriptional activators of the vascular endothelial growth factor promoter through interaction with secondary dna structures a cis-element with mixed g-quadruplex structure of npgpx promoter is essential for nucleolin-mediated transactivation on non-targeting sirna stress the c-terminus of nucleolin promotes the formation of the c-myc g-quadruplex and inhibits c-myc promoter activity targeting myc expression through g-quadruplexes structures, folding patterns, and functions of intramolecular dna g-quadruplexes found in eukaryotic promoter regions direct evidence for a g-quadruplex in a promoter region and its targeting with a small molecule to repress c-myc transcription midkine, a cytokine that inhibits hiv infection by binding to the cell surface expressed nucleolin the anti-hiv pentameric pseudopeptide hb-19 binds the c-terminal end of nucleolin and prevents anchorage of virus particles in the plasma membrane of target cells identification of v3 loop-binding proteins as potential receptors implicated in the binding of hiv particles to cd4(+) cells nucleolin and the packaging signal, psi, promote the budding of human immunodeficiency virus type-1 (hiv-1) tandem immunoprecipitation approach to identify hiv-1 gag associated host factors specific changes in the posttranslational regulation of nucleolin in lymphocytes from patients infected with human immunodeficiency virus intracellular accumulation of cell cycle regulatory proteins and nucleolin re-localization are associated with pre-lethal ultrastructural lesions in circulating t lymphocytes: the hiv-induced cell cycle dysregulation revisited identification of nucleolin as a cellular receptor for human respiratory syncytial virus cell surface nucleolin facilitates enterovirus 71 binding and infection host cell nucleolin is required to maintain the architecture of human cytomegalovirus replication compartments dynamic and nucleolin-dependent localization of human cytomegalovirus ul84 to the periphery of viral replication compartments and nucleoli nucleolin interacts with the dengue virus capsid protein and plays a role in formation of infectious virus particles nucleolin is important for epstein-barr virus nuclear antigen 1-mediated episome binding, maintenance, and transcription nucleolin interacts with the feline calicivirus 3 untranslated region and the protease-polymerase ns6 and ns7 proteins, playing a role in virus replication nucleolin interacts with us11 protein of herpes simplex virus 1 and is involved in its trafficking a major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone h1 nucleolin is a histone chaperone with fact-like activity and assists remodeling of nucleosomes formation of a complex between nucleolin and replication protein a after cell stress prevents initiation of dna replication regulation of dna replication after heat shock by replication protein a-nucleolin interactions novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation nucleolin inhibits g4 oligonucleotide unwinding by werner helicase an ap-1 binding site in the enhancer/core element of the hiv-1 promoter controls the ability of hiv-1 to establish latent infection establishment and molecular mechanisms of hiv-1 latency in t cells key: cord-264699-l8db5gll authors: kino, tomoshige; chrousos, george p. title: virus-mediated modulation of the host endocrine signaling systems: clinical implications date: 2007-06-30 journal: trends in endocrinology & metabolism doi: 10.1016/j.tem.2007.03.003 sha: doc_id: 264699 cord_uid: l8db5gll viruses, which are among the simplest infective pathogens, can produce characteristic endocrine manifestations in infected patients. in addition to the classic modification of the host endocrine system by either direct or indirect destruction of the endocrine organs and/or effects exerted by systemic production of inflammatory and/or stress mediators, recent progress in molecular virology and endocrinology has revealed that virus-encoded molecules might alter the host endocrine-signaling systems by affecting extracellular and/or intracellular signal transduction and hormone sensitivity of host target tissues. here, we provide a brief overview of such viral-mediated modulation of host endocrine signaling systems. we propose that virus-encoded molecules and the signaling systems they influence are potential therapeutic targets for the treatment of disorders that are associated with some viral infections. viruses are among the simplest infective pathogens on earth. viruses cause a variety of pathological conditions of occasionally pandemic proportions, and constitute potential major threats to the human population [1] . even after the development of vaccines, which have been tremendously effective in controlling many viral infections, viruses are still among the most common human pathogens, sometimes causing life-threatening diseases [1] . the latter is evident from recent epidemics, such as acquired immunodeficiency syndrome (aids), caused by the human immunodeficiency virus type-1 (hiv-1), and severe acute respiratory syndrome (sars), caused by the sars corona virus [2, 3] . replication and survival of viruses inside host cells are dependent on the cellular machinery of the host. viruses might usurp the regulation of and change host-cell functions for their benefit. indeed, through molecules encoded by viral genetic material, whose expression is synchronized with specific phases of the viral life cycle, viruses increase the chance of their own replication and survival. virusinduced alterations of host-cell functions cause a broad spectrum of cellular damage, from a transient, mild change of function to neoplastic transformation, apoptosis and necrosis. recent research has revealed that viruses, through the molecules they encode, modulate endocrine signaling systems in the host to cause characteristic changes in both endocrine organs and hormone target tissues. here, we provide a brief overview of recent progress in understanding virus-induced modifications of endocrine signaling systems and of the resultant pathological states in the host. on the basis of this understanding, potential new therapeutic targets for the development of anti-viral disease agents are envisioned. virus-induced modifications of host endocrine systems: the classic view generally, entry of viruses into and infection of host tissues evokes several levels of host reactions that might be associated with pathological manifestations. thus, infection of host tissues might damage infected organs and tissues directly. by contrast, viruses themselves and tissues that are infected with viruses might activate the host immune reaction, leading to the development of inflammatory lesions and/or the destruction of infected tissues [4] . in addition, host immune responses induced against viruses and infected tissues might damage uninfected tissues, partly because of the similarity between viral antigens and those of host cells (molecular mimicry), and because of a systemic, generalized inflammatory reaction that results in dysfunction of multiple organs [5] . viral-induced inflammation and viremia also activate the systemic stress response, in which the hypothalamicpituitary-adrenal (hpa) axis has a major role. secreted glucocorticoids, the end-effectors of the hpa axis, protect peripheral organs from inflammation-induced tissue damage by suppressing an overshoot of the host immune reaction and by rendering tissues resistant to toxic inflammatory agents [6] [7] [8] . thus, viral infections classically induce the host reactions shown in box 1 and figure 1 . examples of classic host responses to viral infections are also described in table 1. both host-and virus-encoded molecules in the regulation of gene expression and function. numerous cell-surface molecules and nuclear hormone receptors (nrs) that transduce the signals of circulating hormones in the cytoplasm and/or the nucleus have been identified and their biological functions have been elucidated. in parallel, it has become evident that some viruses cause pathological changes because they encode molecules that affect host signaling pathways directly [9, 10] and these actions sometimes cause serious clinical problems (table 2) [10, 11] . indeed, viruses have developed some of their unique, highly sophisticated molecules through the mutational selection of protein isoforms that increase the chance of their replication and/or survival inside infected cells, and their propagation from cell-to-cell and host-to-host. these viral molecules regularly influence crucial steps in host signaling systems and, thus, dramatically change cellular functions towards conditions that are beneficial to the replication, survival and/or propagation of the viruses that encode them. sometimes, these viral molecules mimic functions of host proteins. for example, hiv-1-encoded gp120 molecules, which are located on the surface of the viral particle and have a major role in the entry of viruses into target cells, demonstrate amino acid sequence similarity to the growth hormone-releasing hormone (ghrh) receptor of the host and suppress the activation of this receptor by ghrh. this phenomenon might contribute to growth retardation of hiv-1-infected children [12] . this viral molecule is also similar to vasoactive intestinal peptide, which interacts with cd4, a receptor for hiv-1, to facilitate its entry into t cells [13, 14] . the amino acid sequence of the hiv-1 nef protein resembles a portion of the thyroid-stimulating hormone receptor, and this crossreactivity might contribute to the development of grave's disease in hiv-1-infected patients [15] . in addition to their extracellular actions, many viral molecules act inside infected cells to modulate intracellular host signaling systems, including transcriptional regulation of target genes by hormones. these molecules are intended primarily to benefit viral replication, but, frequently, they alter the intracellular milieu, disturbing cellular functions as a 'side-effect' and causing specific, recognizable, pathological conditions [10, 16] . in the following sections, examples of extracellular and intracellular modifications of host endocrine signaling systems caused by viral molecules are discussed. human t-cell leukemia (lymphotrophic) virus type 1 (htlv-1), the single-stranded rna virus of the retroviridae family that specifically infects cd4-positive t cells, is a causative agent of adult t-cell leukemia (atl) and htlv-associated myelopathy [11] . atl patients infected with htlv-1 may also develop hypercalcemia because of aberrant expression of parathyroid hormone (pth)-related protein (pthrp) [17] . pthrp is a 141 amino acid protein that shares significant n-terminal sequence homology box 1. virus-induced alterations of the host endocrine system activation of the hpa axis/stress system indirectly, as a result of a general inflammatory response to the systemic viral infection and secretion of mediators of inflammation with stress systemstimulating activity. damage of virus-infected endocrine cells by viral replication, proliferation and assembly. damage of virus-infected endocrine organs by activation of the immune reaction against these organs. damage against uninfected endocrine organs through an autoimmune mechanism. alteration of hormonal activity and/or hormone secretion by viral gene products. with pth [18] . in humans, pthrp is present in many normal and neoplastic tissues, whereas pth is present only in the parathyroid gland [18] . leukemic cells from atl patients, as well as lymphocytes from asymptomatic htlv-1-positive carriers, contain high levels of pthrp; the secreted protein exerts biological effects that are similar to those of pth because pthrp binds to the pth receptor and stimulates downstream cellular events [18] . hence, pthrp, like pth, increases serum ca 2+ concentrations by stimulating bone turnover and enhancing ca 2+ absorption from the kidney and the gut [18] . accumulating evidence indicates that the htlv-1-encoded tax protein stimulates pthrp synthesis in htlv-1-infected lymphocytes by activating the pthrp promoter [11] . tax is a nuclear phosphoprotein of either 37 or 40 kda that is required for transactivation of the htlv-1 ltr and the transformation of t cells [19] . tax does not bind dna directly but transactivates numerous cellular genes by interacting physically with host transcription factors [19] . tax appears to stimulate the promoter activity of the gene that encodes pthrp by communicating with multiple transcription factors, including activator protein 1 (ap1), ap2, est1 and sp1 [11] . tax forms a ternary complex with est1 and sp1, and their binding to the pthrp promoter strongly stimulates its transcriptional activity [20] . aggravated hypercalcemia, however, is observed most often in end-stage atl when tax expression is relatively low [11] . mice infected experimentally with tax-defective htlv-1 have high levels of pthrp and develop hypercalcemia [21] . thus, as yet unknown viral factors appear to contribute to the development of htlv-1-associated hypercalcemia in addition to tax. alteration of glucocorticoid and insulin sensitivity in hiv-1-infection: implications for aids-related lipodystrophy and insulin-resistance syndrome hiv-1 infection is associated with profound immunosuppression, muscle wasting and, occasionally, the aids-related lipodystrophy and insulin-resistance syndrome. the latter is characterized by a sometimes striking phenotype and marked metabolic disturbances, especially after addition of potent anti-hiv-1 treatment regimens, which have significantly improved patients' life expectancy and allowed sufficient time for the syndrome to develop [10] . patients with this syndrome have a combination of regional lipodystrophy and characteristic redistribution of adipose tissue with accumulation of visceral, breast and nuchal fat and loss of subcutaneous fat [10, 22] . the aids-related lipodystrophy and insulin-resistance syndrome is accompanied by profound dyslipidemia and either carbohydrate intolerance or overt diabetes mellitus [10] . as the phenotypic and metabolic manifestations of the aids-related lipodystrophy and insulin-resistance syndrome are reminiscent of the typical phenotype of chronic glucocorticoid excess or cushing syndrome, this syndrome was referred to initially as a pseudo-cushing state [10] . anti-hiv-1 drugs, such as nucleotide reverse transcriptase and protease inhibitors, appear to contribute to the development of this syndrome, based on the evidence that the majority of aids patients develop this syndrome after taking such compounds [22, 23] . however, some patients develop the characteristic features of the syndrome before treatment, which indicates that either hiv-1 infection itself induces these pathological changes or that it increases the vulnerability of patients to the facilitation of expression of this syndrome by anti-viral drugs [24, 25] . in this context, anti-hiv-1 drugs might exacerbate already present, smoldering or subclinical lipodystrophy and insulin-resistance that is masked by illness-associated wasting, in agreement with the evidence that the drug effects listed above do not explain the complete clinical picture of aids-related lipodystrophy and insulin resistance [10, 26] . because the clinical picture of aids-related lipodystrophy and insulin-resistance syndrome overlaps with that of cushing syndrome, the adrenal function of patients with these syndromes has been examined, however, their hpa axis is normal [27] . moreover, glucocorticoid receptors (grs) are present at normal concentrations on peripheral leukocytes and the affinity of these receptors for dexamethasone is similar to that of controls. therefore, biochemical hypercortisolism is not likely to be a major cause of immunosuppression, muscle wasting and aids-related lipodystrophy and insulin resistance. rather, either localized or tissue-specific hypersensitivity to glucocorticoids might be involved in these phenomena [10] . furthermore, hypersensitivity to glucocorticoids might explain the increased vulnerability of aids patients to hhv-8 infections and the development of kaposi sarcoma [28] . in agreement with these findings, one of the hiv-1 proteins, vpr, which is a 96-amino acid virion-associated accessory protein that has multiple functions (including influencing transcriptional activity and arresting the cell cycle), increases the effects of gr stimulation by several fold, functioning as a nuclear receptor coactivator in cooperation with a host cell coactivator complex containing p300 or its homolog creb-binding protein (cbp) [29] [30] [31] [32] . these proteins regulate many signal transduction cascades mediated by transcription factors, including nrs, crebinding protein (creb), ap-1, nuclear factor-kb (nf--b) and the signal transducers and activators of transcription [33] . p300 and cbp have intrinsic histone acetyltransferase activity and attract other histone acetyltransferase coactivators, such as the p300/cbp-interacting factor (p/caf) and p160-type nuclear receptor coactivator proteins to the transcription initiation site [33] . thus, p300/cbp act as regulatory 'platforms' for the transcription of numerous host genes, regulating transcriptional activity of many trans-acting factors and nrs (figure 2 ). vpr contains a nr coactivator box. this is similar to those on p160 and p300/cbp nr coactivators, potentiating the effect of ligandbound gr and p160. vpr penetrates the cell membrane easily to exert its biological effects even when added in the media surrounding the cells [34] , thus, its effects might extend to tissues that are not infected with hiv-1. another hiv-1 accessory protein, tat, the most potent transactivator of the hiv-1-ltr, also potentiates gr-induced transcriptional activity moderately, possibly through accumulation of the positive-acting transcription elongation factor b (ptefb) complex, which is comprised of the cyclin-dependent kinase 9 and its partner molecule cyclin t, on glucocorticoid-responsive promoters [35] . because tat, like vpr, circulates in blood and exerts its actions as either an auto/paracrine or an endocrine factor by penetrating the cell membrane [36] , it is possible that tat modulates tissue sensitivity to glucocorticoids irrespective of whether a cell is infected with hiv-1. concomitantly with vpr, tat might induce tissue hypersensitivity to glucocorticoids that might contribute to viral proliferation indirectly, by suppressing activity of the host immune system and altering the host metabolic balance, both functions governed by glucocorticoids [10] . vpr reduces tissue sensitivity to insulin by potentiating the actions of glucocorticoids as well as by modulating the transcriptional activity of insulin ( figure 3 ) [37, 38] . insulin uses the forkhead transcription factors (foxos) to control gene induction. baseline, unphosphorylated foxos are active, reside in the nucleus and bind to their responsive sequences in the promoter region of insulin-responsive genes. by contrast, insulin activates akt kinase, which phosphorylates specific serine and threonine residues in foxos to render them inactive [39] . indeed, once foxos are phosphorylated at specific residues, they lose their figure 2 . linearized diagrams of (i) p300, (ii) ctbp1, (iii) vpr, (iv) tat, and (v) e1a and their mutual-interaction domains. numerous transcription factors, transcriptional regulators and viral molecules bind the transcriptional coactivator p300 or its homolog cbp. binding sites of p160 nr coactivators and vpr overlap, and both bind nrs and p300 or cbp. thus, vpr mimics the host p160 nr coactivators and enhances nr transcriptional activity. p300/cbp facilitates attraction of transcription factors, cofactors and general transcription complexes by loosening the histone-dna interaction through acetylation of histone tails by its hat domain. the n-terminal portion of ctbp1 interacts physically with hdac5 and rb, which repress transcription. ctbp1 regulates interaction with its binding partners by sensing nadh levels through its nad + -binding domain. e1a binds to the c-terminal portion of p300 and associates physically with the n-terminal portion of ctbp1 through its c-terminal end. the hat domain of p300 and the nad + -binding domain of ctbp1 are indicated in red. this figure is based on refs [16, 33, 45] . abbreviations: creb, cre-binding protein; hat, histone acetyltransferase; hdac5, histone deacetylases 5; nf-kb, nuclear factor-kb; nad, nicotinamide adenine dinucleotide; nr, nuclear hormone receptor; p/caf, p300/cbp-associating factor; ptefb, positive-acting transcription elongation factor b; rb, retinoblastoma protein; sf-1, steroidogenic factor-1; stat2, signal transducer and activator of transcription 2; tfiib, transcription factor iib. transcriptional activity, by binding to and translocating into the cytoplasm with proteins of the 14-3-3 family [39] . we have found that vpr moderately inhibits insulininduced translocation of foxo3a into the cytoplasm through inhibiting its association with 14-3-3 [37] . based on these in vitro findings, vpr might be a key viral factor that induces lipodystrophy, insulin resistance and hyperlipidemia by interfering with and/or modulating cellular activities, such as transactivation of nrs and insulin [26] . unpublished information indicates that induction of insulin resistance by vpr might be compounded by the ability of the viral protein to interfere with signal transduction by another nr, peroxisome proliferation receptor g. * paget's disease of bone and the measles virus paget's disease of bone is a chronic, focal, skeletal disorder that causes bone deformities and fractures as a result of uncoupled bone remodeling [40] . the primary abnormality of this disease resides in the osteoclasts, which demonstrate increased capacity for bone resorption and hypersensitivity to vitamin d [40] . immunocytochemical studies have shown that osteoclasts from patients with paget's disease of bone contain paramyxoviral-like nuclear inclusions that cross-react with antibodies to measles virus, respiratory syncytial virus and canine distemper virus nucleocapsid antigen [40] . nucleocapsid transcripts of measles virus (mvnp), the negative single-strand rna virus of the paramyxoviridae family, have also been detected in osteoclasts from patients with paget's disease of bone [41] . indeed, forced expression of mvnp in normal osteoclast precursor cells enhances formation of mature osteoclasts that possess many characteristics of those in patients with paget's disease, including increased sensitivity to vitamin d [40] . transgenic expression of mvnp induces bone lesions similar to those found in paget's disease [42] . osteoclasts that express mvnp demonstrate increased levels of tafii-17, which is one of the components of the general transcriptional complexes that are attracted to the ligand-activated vitamin d receptor [40] . thus, it is possible that paget's disease of bone might be caused, in part, by the measles virus, through its encoded molecule mvnp increasing the sensitivity of osteoclasts to vitamin d. possible alteration of endocrine system by adenovirus e1a protein adenoviruses are the double strand dna viruses of the adenoviridae family. this group of viruses causes illness of the respiratory system, such as common cold syndrome, pneumonia, croup and bronchitis, as well as illnesses of other organs, such as gastroenteritis, conjunctivitis and cystitis [43] . adenoviruses encode the e1a protein, which is expressed just after infection and is necessary for the transcriptional regulation of adenovirus-encoded genes [43] . in addition to viral genes, e1a regulates the transcriptional activity of several host genes through interaction with the host transcriptional integrator p300 and its homologous molecule cbp ( figure 2) [33, 44] . in an in vitro system, e1a, in contrast to vpr, blocks the actions of glucocorticoids on the transcriptional activity of genes, producing resistance to glucocorticoids [30] . e1a also interacts with the c-terminal tail-binding protein (ctbp), which functions as a transcriptional repressor of numerous transcription factors by communicating with class ii histone deacetylases and other inhibitory molecules, such as the retinoblastoma protein (rb) (figure 2 ) [45] . e1a suppresses the activity of p300/cbp and ctbp by binding to functionally crucial domains [33, 45] . although there is no supportive clinical evidence, it is possible that adenovirus changes the peripheral action of circulating hormones, such as glucocorticoids and other bioactive molecules that activate nrs and directly regulate the transcriptional activity of their target genes, ultimately contributing to the pathological states observed in adenoviral infection. in 2003, the virus that caused a newly identified illness called sars spread rapidly worldwide, causing almost 800 deaths [3, 46] . sars starts with flu-like symptoms, such as fever, myalgia, somnolence, gastrointestinal symptoms, cough and sore throat, and eventually develops into pneumonia with severe respiratory failure [3, 46] . mechanical ventilation is required in $10-20% of cases and the death rate is almost 10% [3, 46] . the causative virus of this severe respiratory syndrome is the sars coronavirus (sars-cov), a positive single-strand rna virus of the coronaviridae family [47] . subsequently, the angiotensin-converting enzyme 2 (ace2) was identified as a cellular receptor for the entry of this virus [48] . ace2 was first cloned as a homolog of ace in 2000 [49, 50] . although it functions as a carboxypeptidase similar to ace, it has different substrate specificity: ace2 cleaves a single residue from angiotensin i (ang i) and ang ii to generate the inactive forms angiotensin (1-9) and angiotensin (1-7), respectively, whereas ace removes dipeptides from the c-terminus of ang i to produce potent, bioactive ang ii (figure 4 ) [51] . from mouse knockout studies, ace2 is a negative regulator of the rennin-angiotensin system that counterbalances the function of ace [52] . ang ii is a potent stimulator of vascular constriction, na + uptake in the renal tubules, aldosterone secretion from the adrenal glands and vasopressin secretion from the pituitary gland [53] . ang ii also has important roles in lung physiology and pathophysiology [51] . indeed, ace and ang ii function as promoting factors for acute lung injury, whereas ace2 protects against lung injury [51] . as sars-cov infection downregulates ace2 expression markedly in the lung and reduces circulating concentrations of ang ii [54] , it is likely that sars-cov contributes to severe lung disease and other systemic complications through dysregulation of the reninã�angiotensin system. this is in contrast to other coronaviruses that infect the lung epithelium like sars-cov but cause much milder diseases [51] . conclusions: virus-induced modulation of host signaling systems as a new therapeutic target vaccines, which stimulate the host-immune reaction to protect the organism from the entry of viruses, are the most effective, first-line therapeutics against viral infection. this approach has been extremely useful in controlling epidemics, such as those caused by smallpox, poliomyelitis, measles, influenza, varicella and rubella viruses [1] . however, some viruses are resistant to vaccine development by conventional approaches. for example, so far, anti-hiv-1 vaccines have not been sufficiently effective to protect from infection by this virus, despite the tremendous efforts of the scientific community. this is possibly because of the high genetic instability of hiv-1, which changes the immunogenicity [55, 56] . anti-viral agents are used because vaccines are either not available or have failed to protect us. expansion of our understanding of virus-induced modifications of the host signaling systems, such as those discussed above, might prove highly beneficial for the development of new vaccines and novel anti-viral agents, based on knowledge of the potential indispensability of specific viral molecules for the entry, replication, and intracellular and extracellular survival and propagation of these viruses. indeed, this approach has been followed successfully in the development of nucleoside analogs, such as acyclovir for the herpes simplex virus and ganciclovir for cytomegalovirus, which specifically block transcription promoted by virus-encoded and virus-specific dna polymerases [57] . oseltamivir, a neuraminidase inhibitor that blocks the influenza virusencoded neuraminidase, is effective in controlling infection by blocking the release of newly replicated viruses from the infected cells, a step in which this enzyme has a major role [58] . thus, either vaccines or chemical compounds, including nucleotides (e.g. antisense dna and sirna) that inhibit the expression and/or function of specific viral molecules, are promising approaches for the treatment of viral illnesses. indeed, vaccines against hiv-1 tat are under development with promising effects [59] . we envision that future research to further elucidate the effects of viral molecules on the host endocrine signaling systems will improve our ability to intercept viral infection and prevent virus infection-associated diseases that affect endocrine systems. and endocrine target organs. a different view of smallpox and vaccination hiv/aids epidemiology, pathogenesis, prevention, and treatment ace2: from vasopeptidase to sars virus receptor immunology of viral infections molecular mimicry and immune-mediated diseases the hypothalamic-pituitary-adrenal axis and immune-mediated inflammation effectiveness of prolonged glucocorticoid treatment in acute respiratory distress syndrome: the right drug, the right way? immune modulation of the hypothalamicpituitary-adrenal (hpa) axis during viral infection adenovirus e1a: remodelling the host cell, a life or death experience aids-related lipodystrophy/insulin resistance syndrome hypercalcemia, parathyroid hormone-related protein expression and human t-cell leukemia virus infection hiv gp120 inhibits the somatotropic axis: a possible gh-releasing hormone receptor mechanism for the pathogenesis of aids wasting perturbation of in vitro hiv pathogenic effects by peptides showing sequence similarities with the c2 conserved domain of gp120 vasoactive intestinal peptide 1-12: a ligand for the cd4 (t4)/human immunodeficiency virus receptor nucleotide and amino acid homology between the human thyrotropin receptor and the hiv-1 nef protein: identification and functional analysis glucocorticoid and mineralocorticoid resistance/hypersensitivity syndromes constitutive expression of parathyroid hormone-related protein gene in human t cell leukemia virus type 1 (htlv-1) carriers and adult t cell leukemia patients that can be transactivated by htlv-1 tax gene parathyroid hormone and parathyroid hormone-related peptide, and their receptors molecular mechanisms of cellular transformation by htlv-1 tax interaction of human t-cell lymphotropic virus type i tax, ets1, and sp1 in transactivation of the pthrp p2 promoter humoral hypercalcemia of malignancy: severe combined immunodeficient/beige mouse model of adult t-cell ace converts angiotensin i (ang i) into ang ii, which acts as a vasopressor and a mitogen for smooth muscle cells and fibroblasts through the angiotensin 1 (at1) receptor. by contrast, ace2 [53] converts ang i and ang ii into angiotensin (1-9) and angiotensin (1-7), respectively lymphoma independent of human t-cell lymphotropic virus type-1 tax expression metabolic abnormalities and cardiovascular disease risk factors in adults with human immunodeficiency virus infection and lipodystrophy metabolic complications associated with antiretroviral therapy metabolic disorders among hiv-infected patients treated with protease inhibitors: a review hiv-associated lipodystrophy syndrome human immunodeficiency virus type-1 accessory protein vpr: a causative agent of the aids-related insulin resistance/lipodystrophy syndrome? endocrine and metabolic evaluation of human immunodeficiency virus-infected patients with evidence of protease inhibitor-associated lipodystrophy aids-related kaposi's sarcoma: evidence for direct stimulatory effect of glucocorticoid on cell proliferation partner molecules of accessory protein vpr of the human immunodeficiency virus type 1 human immunodeficiency virus type 1 (hiv-1) accessory protein vpr induces transcription of the hiv-1 and glucocorticoid-responsive promoters by binding directly to p300/cbp coactivators the hiv-1 virion-associated protein vpr is a coactivator of the human glucocorticoid receptor hiv-1 protein vpr suppresses il-12 production from human monocytes by enhancing glucocorticoid action: potential implications of vpr coactivator activity for the innate and cellular immunity deficits observed in hiv-1 infection cbp/p300 in cell growth, transformation, and development hiv-1 vpr displays natural proteintransducing properties: implications for viral pathogenesis nuclear receptor coactivator p160 proteins enhance the hiv-1 long terminal repeat promoter by bridging promoter-bound factors and the tat-p-tefb complex tat-mediated delivery of heterologous proteins into cells hiv-1 accessory protein vpr inhibits the effect of insulin on the foxo subfamily of forkhead transcription factors by interfering with their binding to 14-3-3 proteins: potential clinical implications regarding the insulin resistance of hiv-1-infected patients vpr protein of human immunodeficiency virus type 1 binds to 14-3-3 proteins and facilitates complex formation with cdc25c: implications for cell cycle arrest foxo proteins in insulin action and metabolism paget disease of bone measles virus rna detected in paget's disease bone tissue by in situ hybridization expression of measles virus nucleocapsid protein in osteoclasts induces paget's disease-like bone lesions in mice textbook of human virology the multifunctional role of e1a in the transcriptional regulation of creb/cbp-dependent target genes ctbp, an unconventional transcriptional corepressor in development and oncogenesis severe acute respiratory syndrome (sars): development of diagnostics and antivirals a novel coronavirus associated with severe acute respiratory syndrome angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a novel angiotensin-converting enzymerelated carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase angiotensin-converting enzyme 2 in lung diseases angiotensin-converting enzyme 2 is an essential regulator of heart function pleiotropic at1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin ii a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury hiv pathogenesis: knowledge gained after two decades of research hiv vaccines antivirals for the treatment of herpesvirus infections neuraminidase inhibitors for influenza hiv-1 tat-based vaccines: from basic science to clinical trials pituitary-testicular interrelationships in mumps orchitis and other viral infections hypopituitarism as a late complication of hemorrhagic fever herpes simplex virus infections in neonates and early childhood aids/hpa axis therapeutic management of extrahepatic manifestations in patients with chronic hepatitis c virus infection autoimmunity in congenital rubella syndrome molecular mimicry, microbial infection, and autoimmune disease: evolution of the concept infection, thyroid disease, and autoimmunity viral persistence: parameters, mechanisms and future predictions selective inhibition of nuclear steroid receptor function by a protein from a human tumorigenic poxvirus this article was funded by the intramural research program of the national institute of child health and human development, national institutes of health. key: cord-287754-dh6abx2t authors: akkouh, ouafae; ng, tzi bun; singh, senjam sunil; yin, cuiming; dan, xiuli; chan, yau sang; pan, wenliang; cheung, randy chi fai title: lectins with anti-hiv activity: a review date: 2015-01-06 journal: molecules doi: 10.3390/molecules20010648 sha: doc_id: 287754 cord_uid: dh6abx2t lectins including flowering plant lectins, algal lectins, cyanobacterial lectins, actinomycete lectin, worm lectins, and the nonpeptidic lectin mimics pradimicins and benanomicins, exhibit anti-hiv activity. the anti-hiv plant lectins include artocarpus heterophyllus (jacalin) lectin, concanavalin a, galanthus nivalis (snowdrop) agglutinin-related lectins, musa acuminata (banana) lectin, myrianthus holstii lectin, narcissus pseudonarcissus lectin, and urtica diocia agglutinin. the anti-hiv algal lectins comprise boodlea coacta lectin, griffithsin, oscillatoria agardhii agglutinin. the anti-hiv cyanobacterial lectins are cyanovirin-n, scytovirin, microcystis viridis lectin, and microvirin. actinohivin is an anti-hiv actinomycete lectin. the anti-hiv worm lectins include chaetopterus variopedatus polychaete marine worm lectin, serpula vermicularis sea worm lectin, and c-type lectin mermaid from nematode (laxus oneistus). the anti-hiv nonpeptidic lectin mimics comprise pradimicins and benanomicins. their anti-hiv mechanisms are discussed. in the last few decades, africa remains the continent which has been afflicted to the most serious extent by the hiv/aids pandemic, although the number of infections elsewhere, for instance in asia, has also been on the rise [1] . many children die from relatively preventable causes, especially in places where there is a high incidence of hiv infections/aids [2] . the search for plant extracts with anti-hiv activity is continuing [3] . polysaccharides [4] and other compounds [5] have been proposed or used for treatment. lectins have been extensively studied because they possess a variety of potentially exploitable activities. the purpose of this article is to review lectins with anti-hiv activity. lectins are carbohydrate-binding proteins of non-immunoglobulin nature. they are capable of recognition of and reversible binding to complicated glycoconjugates moieties without altering the covalent structure of any of the recognized glycosyl ligands [6] . in nature, lectins are distributed across a wide variety of organisms like algae, fungi, sea corals, higher plants, prokaryotes, invertebrates and vertebrates. they are involved in many biological processes like recognition and binding of carbohydrates, host-pathogen interactions, cell targeting, cell-cell communication and induction of apoptosis, cancer metastasis and differentiation [6] . the antiviral, antifungal and antibacterial activities of lectins have been reported [7, 8] . the intent of this article is to review lectins with anti-hiv activity. the human immunodeficiency virus (hiv) type 1 is an enveloped virus that causes the acquired immunodeficiency syndrome (aids). in humans the condition is manifested by progressive failure of the immune system [5, 9] . it infects viral cells such as t-helper cells, macrophages and dendritic cells [10] . this infection leads to low levels of cd4+ through a number of mechanisms. also, apoptosis of uninfected cells, direct viral killing of infected cells and killing of infected cd4+ cells by cytotoxic lymphocytes occur. the envelope protein complex determines viral tropism and facilitates the membrane fusion process that allows invasion of the viral genome [11] . gp160 is a precursor env gene code that is later processed by a host cell protease to form the cleavage products gp120 and gp41. the envelope protein complex and the gp160 are extensively glycosylated and proteolytically cleaved. when new virus particles come to the host cell, gp120 and gp41 will lie on opposite sides of the virus membrane. gp120 will sit on the outside of the virus particle, forming the virus's spikes and gp41 will sit on the inside of the membrane. each gp41 will be anchored to a gp120 through the membrane [12, 13] . gp120 binds to cd4 receptor on any target cell that has such a receptor and will present to t-lymphocytes and macrophages. this cd4 is associated with spontaneous mutation in the env gene. the presence of cxcr4 is enough for this mutant strain to infect human cells. these strains have seven mutations in the sequence and coding for gp120. it is also proposed that the mutations induce conformational changes in gp120 that allow the virus to directly interact with cxcr4 [14] . prior to binding the host cell, gp120 remains effectively hidden from antibodies. gp120 is buried in the protein and shielded by sugars, which makes neutralization by antibodies very difficult. it is only exposed when in close proximity to a host cell and the space between the viral and host cell membranes is small enough to hinder the binding of antibodies. this is one of the reasons why there is not a vaccine available to hiv [15] . gp41 is non-covalently bound to gp120 because it is buried within the viral envelope. when gp120 binds to cd4, it will change its conformation and gp41 will become exposed, where it can assist in fusion with the host cell [12, 13] . some lectins exhibit significant activity against human immunodeficiency virus (hiv) and other viruses with an envelope. this makes them particularly attractive targets for the development as novel antiviral drugs. cyanovirin-n and griffithsin are examples of lectins that can inhibit hiv and other viruses [7, [16] [17] [18] . virally encoded glycoproteins cover the surfaces of retroviruses such as hiv and many other viruses with an envelope. gp41 and gp120 present on the envelope of hiv, heavily glycosylated with glycans, are estimated to contribute almost 50% of the molecular weight of gp120 [19] [20] [21] . agents that strongly and specifically interact with the glycans may disturb the interaction between the cells of the host and the proteins of the viral envelope [22, 23] . on the viral surface, sugar-binding proteins can crosslink with glycans and prevent other interactions with the co-receptors. antiviral lectins can prevent penetration of the host cells by the viruses. they prevent infection by binding to the sugars that adorn the surface of the envelope of hiv [24] . the majority of other current antiviral therapeutics act through inhibition of the viral life cycle. antiviral lectins are suitable for topical applications and can exhibit lower toxicity than others used for antiviral therapy. the antiviral proteins are often resistant to low ph and high temperatures. also they are odorless, which are properties favorable for potential drugs [19] . examples of lectins that exhibit antiviral activity and bind high-mannose carbohydrates are jacalin [25] , concanavalin a [26] , urtica diocia agglutinin [27] , myrianthus holstii lectin, p. tetragonolobus lectin [28] and narcissus pseudonarcissus lectin [29] . lectins are also derived from marine organisms. the natural antiviral products that exhibit the highest activity among the lectins are cyanovirin-n, scytovirin, microcystis virdis lectin and griffithsin [7, 18, 30, 31] . jacalin is the major protein from the jackfruit artocarpus heterophyllus seeds. artocarpus heterophyllus in the moraceae family is an evergreen tree that is grown in various tropical countries in south and southeast asia. the seed is large and oblong with a brown tegmen and a slim membranous testa. chatterjee et al. [32] , roque-barreira et al. [33] and kondoh et al. [34] , were among the first who identified jacalin around 1980. jacalin had the property of binding human iga with specificity for the iga1 subclass [32] [33] [34] . in the recent years various studies have been carried out on jacalin, revealing its importance as a lectin of diverse applications ranging from the isolation of human iga1 to hiv research. jacalin has been reported to have a molecular mass of 65-66 kda [35] . the seeds of this fruit contain a minor mannose-binding lectin [36] . it is a two-chain lectin consisting of an α-chain of a 133 amino acid residues non-covalently bound to a β-chain of 21 amino acid residues [37] . the glycosylated α-chain has a molecular mass of 15.8 kda [38] . sugars which interact with the lectinic-site of jacalin suppress the binding of jacalin to cd4. these findings give some insight into the mechanism by which jacalin inhibits hiv-1 infection of cd4+ cells [39] . concanavalin a is a carbohydrate-binding protein isolated from canavalia ensiformis or jack bean. it is a member of the legume lectin family and is used for human nutrition or animal fodder [40] . the beans of this plant are mildly toxic and copious consumption should be avoided. concanavalin a was the first lectin that was available on a commercial basis and is widely used in biology and biochemistry to characterize glycoproteins and sugar-containing entities on the surface of various cells [41] . the plant contains large quantities of the enzyme urease. this is a reason that the plant cannot be used in fodder mixtures that contains urea. it will liberate harmful ammonia from urea. because of this, the plant has been investigated as a potential source of the urease enzyme [42] . concanavalin a is a homotetramer with a molecular mass of 26.5 kda [43] . it has 235 amino acid residues and binds specifically to certain structures that are found in several glycoproteins, sugars and glycolipids, especially internal and non-reducing terminal α-d-mannosyl and α-d-glucosyl groups [44] . con a binds to envelope glycoprotein gp120 of hiv-1 and hiv-2 [45] . however, hiv-1 resistant strains have been reported [46] . the detailed mechanism of lectin-mediated neutralization has not been completely elucidated, but there is a minimum of two mechanisms, a direct one and an indirect mechanism to account the efficacy of removal of only a few glycans to render hiv resistant. the direct mechanism is that the mutated n-glycosylation sites harbor the "key target glycans" for lectin-mediated neutralization and that these are removed by the mutations. binding of lectins to these glycans either prevent hiv binding to receptors on the cells or affect alterations of gp120 following binding. the indirect mechanism suggests that the removal of one or more n-glycosylation sites renders n-glycans elsewhere more exposed to carbohydrases in the golgi body and the endoplasmic reticulum. local changes in utilization of glycosylation site can affect the glycosylation setup and protein folding. thus, lectin-binding high-mannose glycans on the wild type are probably more highly processed in the lectin-selected strains, leading to a loss of their affinity for the [46] . gna-related lectins are nearly related in their carbohydrate-binding activities [47] [48] [49] and exhibit significant anti-hiv, anti-hsv and antitumor [50] actions. the most gna-related lectins exhibit mannose-binding activity, while polygonatum cyrtonema lectin [51] and ophiopogon japonicas lectin [52] also display carbohydrate affinity to sialic acid. some agents that interact with viral-envelope glycans may compromise the efficient entry of the virus into its susceptible target cells. they do not interfere with the glycosylation enzymes from the cell, but rather act by directly binding to the intact glycans on the envelope. this kind of carbohydrate-binding agents force the virus to delete a part of its glycan shield so that it can escape drug pressure. the result of this can be the initiation of an immune response against uncovered immunogenic envelope epitopes. the mechanisms of antiviral action of the carbohydrate-binding agents are, firstly, direct antiviral activity by binding to the glycans of the viral envelope and blocking entry of the virus and secondly, an indirect antiviral action. this will result from the progressive creation of deletions in the glycan shield of the envelope. the immune system will be triggered to act against previously hidden immunogenic epitopes of the viral envelope [53] . agglutinins from hippeastrum hybrid and galanthus nivalis inhibited cell-cell fusion between clone69trevenv cells induced to express the viral envelope proteins, gp120/gp41 (env), and highly cd4-positive supt1 cells [54] . the mannose-specific plant lectins hippeastrum hybrid agglutinin, galanthus nivalis agglutinin, narcissus pseudonarcissus agglutinin; cymbidium agglutinin; cyanovirin-n; and n-acetylglucosamine specific urtica dioica agglutinin efficiently abrogate dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign)-directed hiv-1 capture and subsequent transmission to t lymphocytes [55] . the mannose-specific plant lectins from galanthus, hippeastrum, narcissus, epipactis helleborine, and listera ovata, and the n-acetylglucosamine-specific urtica dioica lectin would primarily be targeted at the virus-cell fusion process [56] . banana lectin binds to the glycosylated viral envelope and inhibits cellular entry and thus suppresses hiv-1 infection. its anti-hiv activity is comparable to snowdrop lectin and griffithsin, and to the anti-hiv drugs t-20 and maraviroc [57] . myrianthus holstii is a tree with a large of branches then can grow from 1 to 20 meters in height. it is of the urticaceae family and often has stilt roots up to 60 cm high and a short bole. the plant is harvested from the wild as local source of food and wood. this tree is grown in middle africa, in rainforest, montane forests, at edges or in regrowth and along rivers [58] . myrianthus holstii lectin obtained from the roots by lh-20 chromatography has a molecular mass of 9 kda. this lectin consists of two major constituents with a molecular mass of 9284 and 9300 da, respectively. myrianthus holstii lectin protected cem-ss cells from the cytopathic effects of hiv with an ec50 value of 1.4 μg/ml (150 nm). this one is similar to the urtica diocia agglutinin (ec50 = 105 nm). it inhibited syncytium formation with an ec50 value of 9.8 μg/ml indicating that this lectin reversibly inhibits hiv infection [28] . narcissus pseudonarcissus is also known as wild daffodil or lent lily and belongs to the amaryllidaceae family. this plant produces seeds and is native to western europe. it grows in woods, grassland and rocky ground. it contains a dimeric protein with a molecular mass of 26 kda. it is composed of two 13-kda subunits. it has specificity toward α-linked mannose and is used in characterization of early stages of apoptosis and has mitogenic activity toward human lymphocytes [59] . this lectin agglutinates rabbit erythrocytes but is non-reactive with human erythrocytes [60] . mannose-binding agglutinins from other narcissus species also manifest hiv-1 infection inhibitory activity like that of narcissus pseudonarcissus agglutinin. their hiv-1 infection inhibitory activity does not correlate with their hemagglutinating activity [61] . comparative analyses revealed that the dimer-based super-structure may be important to the anti-hiv property of polygonatum cyrtonema lectin, a novel anti-hiv mannose-binding lectin from galanthus nivalis agglutinin-related lectin family [62] . urtica diocia is a herbaceous perennial flowering plant and is often called stinging nettle or common nettle. it grows in europe, north america, africa and asia and will not grow more than 2 meters in height. it has a long history of use as a source of fiber, food and medicine. urtica diocia agglutinin is a special plant lectin that differs from other plant lectins. the cytopathic effect of hiv has a rate of ec50 = 105 nm [28] and has a small molecular mass of 8.5 kda. this is a t cell mitogen distinguishable from classical t cell lectin mitogens. it is able to discriminate a particular population of cd4+ and cd8+ cells, and can induce an original pattern of t cell cytokine production as well as activation. plant lectin mitogens have been successfully and extensively used in the analysis of events that occur during lymphocyte activation and proliferation [63, 64] . the lectin suppressed entry of hiv-1 into host cells with a half-maximal effective concentration at 8.2 nm. surface plasmon resonance analysis disclosed a high association constant of the lectin with gp120. the lectin binds to viral envelope hemagglutinin against various strains, including a clinical isolate of pandemic h1n1-2009 virus, indicating that it is potentially useful for antiviral therapy [65] . griffithsin is a 13-kda protein with 121 amino acids isolated from the red alga griffithsia. this protein is a highly potent hiv entry inhibitor [18] . it blocks cd4-dependent gp120 binding to the receptor expressing cells. there it will bind to other gp's like gp120, 160 and 41. this will happen in a glycosylation-dependent manner. it inhibits gp120 binding of monoclonal antibody (mab) 2g12, which recognizes a carbohydrate-dependent motif, and (mab) 48d, which binds to cd4-induced epitope. griffithsin also moderately interfered with the binding of gp120 to scd4. the binding of griffithsin to soluble gp120 was not inhibited by fucose, xylose, galactose, n-acetylgalactosamine or sialic acid containing glycoproteins but it was inhibited by mannose, glucose and n-acetylglucosamine. griffithsin binds the terminal mannose residues of surface n-linked glycans on hiv-1, hiv-2, ebola virus, hepatitis c virus, and severe acute respiratory syndrome coronavirus. it means that this is a type of lectin that binds to several viral glycoproteins in a monosaccharide-dependent manner. griffithsin interacts with gp120 leading to exposure of the cd4-binding site cd4bs through binding of the glycan at position 386 [66] . unlike cyanovirin and galanthus nivalis agglutinin, the interaction between griffithsin and gp120 relies on the specific trimeric "sugar tower" including n295 and n448. this was supported by findings from griffithsin env binding experiments [67] . griffithsin does not have mitogenic activity toward human t-cells and does not elicit secretion of proinflammatory cytokines in treated human cell lines. subutaneous injections of griffithsin induce no toxicity other than some splenomegaly and hepatomegaly. serum samples of griffithsin-treated animals display activity against hiv-1-enveloped pseudoviruses in a cell-based neutralization assay. griffithsin suppresses hiv gp120 binding to dc-sign and dc-sign-mediated transfer of hiv-1 to cd4+ t-lymphocytes. it drives gp120 away from the gp120-dc-sign complex. functionally intact carbohydrate binding sites are essential to its optimal action. its dimeric form is paramount to its hiv inhibitory action. griffithsin synergizes with the antiviral drugs enfuvirtide, maraviroc and tenofovir against hiv-1 clade b and clade c isolates in primary peripheral blood mononuclear cells and in cd4+ mt-4 cells. dissimilarity in glycosylation patterns on the viral envelope of clade b and clade c gp120 does not affect the synergism [68] . however, a discrepancy exists between hiv-1 gp120 binding activity and hiv inhibitory activity of the griffithsin variants d30a, d70a, and d112a, suggesting the presence of an anti-hiv mechanism unrelated to simple gp120 binding [69] . griffithsin has three nearly-equivalent mannose binding sites all essential for anti-hiv activity. griffithsin mutants d30a, d112a, and d70a, each with mutation of one of the three sites, demonstrated only a 2-3 fold decrement in binding to immobilized gp120 but 26-or more fold decline in hiv inhibiting ability in single-round hiv infection assays, hiv cytopathicity assays and co-cultivation assays. d70a exhibited superior hiv inhibitory activity despite reduced gp120 binding activity compared with d30a and d112a in some strains, furnishing evidence for absence of a direct correlation between binding to hiv gp120 and hiv inhibition. hence gp120 binding does not necessarily ensue in an antiviral effect; the anti-hiv mechanism of griffithsin action involves not only binding and sterically hindering the activity of gp120, but more importantly to alter the gp120 structure or its oligomeric state as a consequence of the binding of g120 mannose residues, and cross-link gp120 glycans, to render hiv uninfective [69] . griffithsin interacts with gp120 leading to exposure of the cd4-binding site cd4bs through binding of the glycan at position 386 [66] . unlike cyanovirin and galanthus nivalis agglutinin, the interaction between griffithsin and gp120 relies on the specific trimeric "sugar tower" including n295 and n448. this was supported by findings from griffithsin env binding experiments [67] . however, a discrepancy exists between hiv-1 gp120 binding activity and hiv inhibitory activity of the griffithsin variants d30a, d70a, and d112a, suggesting the presence of an anti-hiv mechanism unrelated to simple gp120 binding [69] . griffithsin synergizes with other carbohydrate-binding agents in inhibitory action against hiv-1, hiv-2, and even against hiv-1 strains demonstrating resistance to some carbohydrate-binding agents. the carbohydrate-binding agents tested have different binding patterns on the gp120 envelope and hence no steric hindrance. the observation opens up the possibility of employing combinations of carbohydrate binding agents in topical microbicidal agents to counter hiv transmission [70] . a mannose-binding lectin from the green alga oscillatoria agardhii (oaa) with selectivity for the cluster of á1-2-mannose, its homologue oscillatoria agardhii agglutinin homologue (oaah), and a designed hybrid oaah (opa), inhibit hiv replication, syncytium formation between hiv-1-infected and uninfected t cells, dc-sign-mediated hiv-1 capture and transmission to cd4+ target t cells, infectivity of a variety of hiv-1 and hiv-2 clinical isolates. surface plasmon resonance analysis and flow cytometry revealed that both are competitive inhibitors of the binding of the manα(1-2)manspecific 2g12 monoclonal antibody. the hiv-1 nl4.3 (2g12res), nl4.3 (mvnres) and iiib (grftres) strains were inhibited as well as the wild-type hiv-1 strains. oaa and opa synergize with hippeastrum hybrid agglutinin, and griffithsin [71] . combinations of grft and other cbas showed synergistic activity against hiv-1, hiv-2, and even against certain cba-resistant hiv-1 strains. the cbas tested appear to have distinct binding patterns on the gp120 envelope and therefore do not necessarily compete with each other's glycan binding sites on gp120. as a result, there might be no steric hindrance between two different cbas in their competition for glycan binding (except for the hha/gna combination). these data are encouraging for the use of paired cba combinations in topical microbicide applications (e.g., creams, gels, or intravaginal rings) to prevent hiv transmission [72] . combined nmr and crystallographic results provide structural insights into the mechanism by which oaa specifically recognizes the branched man-9 core, distinctly different from the recognition of the d1 and d3 arms at the non-reducing end of high-mannose carbohydrates by other antiviral lectins [73] . cyanovirin-n is a bacterial protein that is produced by the cyanobacterium nostoc ellipsosporum that shows virucidal activity against several viruses [7, 74] . it is an entry inhibitor of hiv [75] . it was discovered during a screening program in the search for naturally occurring virucidal agents that may be developed into anti-hiv microbicides. this protein consists of a single 101 amino acid chain, which exhibits significant internal sequence duplication. it reveals 13 conservative amino acid changes as well as direct homology between 16 amino acid residues. the molecular mass is approximately 10 kda [7] . it also consists of four cysteines, which can form two intrachain disulfide bonds. the intact disulfide bonds appear to be required for anti-hiv activity. when cleavage of these bonds happens due to β-mercaptoethanol treatment its hiv inhibitory effects were abolished. when stored in solution phase, reduced cyanovirin-n could reform the required disulfide bonds, accompanied by recovery of anti-hiv activity [75] . cyanovirin-n inhibited the in vitro cytopathicity of laboratory strains and several clinical isolates of hiv type 1, 2 and simian immunodeficiency virus. it also effectively prevented cell-to-cell fusion and transmission of hiv from infected cells to uninfected cells. hiv virions that have undergone pretreatment with cyanovirin-n neutralized virus infectivity, but are nontoxic to host cells. the unique virucidal effects arise from its association with the viral envelope glycoprotein 120. cyanovirin-n interferes with critical interactions between viral gp120 and cell surface receptors, which are required for successful virus fusion and entry into cells [7] . linker-cyanovirin-n (a cyanovirin-n derivative with a flexible and hydrophilic linker (gly4ser)3 at the n-terminus), and 10 k peg-ald-lcvn (n-terminal α-amine of n-terminal α-amine of linkercyanovirin-n pegylated to create 10 k peg-aldehyde-linker-cyanovirin-n), are characterized by the specificity and affinity of cyanovirin-n for high mannose n-glycans. linker-cyanovirin-n displayed anti-hiv-1 activity with reduscd cytotoxicity on cat keratinocytes and mt-4 t lymphocytes. 10 k peg-ald-lcvn inactivated hiv-1 with markedly decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. the linker-extended cyanovirin-n and the mono-pegylated derivative are thus promising candidates for the development of an anti-hiv-1 agent [76] . humic acids potentiate the anti-hiv activity of azt, griffithsin, and cyanovirin [77] . cvn specifically recognizes with nanomolar affinity man(9)glcnac(2) and the d1d3 isomer of man(8)glcnac(2) [78] . scytovirin is an antiviral protein isolated from the cyanobacterium scytonema varium. this protein consists of a single 95-amino acid chain with a molecular mass of 9.7 kda and significant internal sequence duplication. it has 10 cysteines forming five intrachain disulfide bonds [30] . [30, 79] . cvn specifically recognizes with nanomolar affinity man(9)glcnac(2) and the d1d3 isomer of man(8)glcnac(2) [80] [81] [82] . microcystis viridis is a unicellular freshwater bloom-forming cyanobacterium. it showed transient hemagglutinating activity in laboratory culture during stationary phase under nonaeration conditions. this lectin is composed of a single 13-kda polypeptide with 113 amino acid residues and two tandemly repeated homologous domains of 54 amino residues. microcystis viridis lectin binds oligomannosides with sub-micromolar affinities and that two novel carbohydrate recognition domains composed of four non-contiguous regions. the residues make numerous intermolecular contacts with their carbohydrate ligands. this lectin inhibits hiv type 1 envelope-mediated cell fusion with an ic50 [83] . the cyanobacterial lectin microvirin is potentially exploitable since it potently inhibits hiv-1 with minimal toxicity. synchronized infectivity assays reveal different kinetics of hiv-1 entry inhibition by microvirin and cyanovirin. synergism demonstrated by combinations of the inhibitors in spite of common specificity for maná(1-2)man terminals indicate recognition of distinctly different glycans and/or glycan conformations on gp120. entry assays utilizing amphotropic viruses disclose the inactivity of microvirin, in contrast to the potent inhibitory activity of cyanovirin. in view of the similarity in the carbohydrate-binding site common to microvirin and cyanovirin, it appears that gp120 is a clustered glycan epitope and multivalent-protein interactions applicable to cyanovirin but not to microvirin are needed for inhibition of certain viruses [84] . actinohivin, an actinomycete lectin, is composed of 114 amino acid residues and contains three segments, all of which display high specific affinity to gp120 due to multivalent interaction of the three sugar-binding pockets with three high-mannose type glycans hmtgs of gp120 via the "cluster effect" of lectin. it exhibits potent in vitro anti-hiv activity [85] . actinohivin suppresses entry of hiv-1 to susceptible cells. specific and potent anti-hiv activity is produced by cooperative binding of three segments of actinohivin to three high mannose-type glycans (hmtgs) of hiv-1 gp120. the anti-hiv activity of actinohivin is enhanced after dimerization due to a rise in the number of hmtg-binding pockets [86] . the 30-kda â-galactose-specific lectin inhibited the cytopathic effects brought about by hiv-1 and the production of viral p24 antigen. its effect at the early stage of viral replication was disclosed by time-of-addition analysis of its anti-hiv-1 activity indicated. the lectin inhibited cell-to-cell fusion of hiv infected and uninfected cells. its suppression of hiv-1 entry into host cells was shown by employing fluorescence-based real-time quantify pcr [87] . the lectin inhibited the production of viral p24 antigen and cytopathic effect induced by hiv with ec50 values of 0.23 and 0.15 μg/ml, respectively [88] . dendritic cells (dcs) play a vital role in hiv-1 transmission; dcs capture assaulting hiv-1 via interaction of the c-type lectin dc-sign with gp120 oligosaccharides and translocate to lymphoid tissues in which transmission of hiv-1 to t cells occurs. it is reasonable to target gp120 for inhibiting interactions with dcs and transmission of hiv-1. the nematode (laxus oneistus) ca 2+ -dependent c-type lectin mermaid, a structural homologue of dc-sign devoid of cytotoxicity, shares the glycan specificity with dc-sign, interacts with high mannose structures on gp120 and prevents hiv-1 binding to dc-sign on dcs. mermaid inhibits dc-sign-mediated hiv-1 transmission from dc to t cells. thus the lectin has potential for development into an anti-hiv-1 agent [89] . pradimicins and benanomicins are non-peptidic lectin mimics which recognize d-mannopyranoside in the presence of calcium ions. they exert antifungal and anti-hiv activities through binding to mannopyranoside-containing glycans of pathogens. pradimicin a, an original member of pradimicins, binds to terminal d-mannopyranoside residues at the non-reducing end of glycans, as well as to internal 6-substituted mannopyranoside residues [90] . mannose binding lectin triggers the complement pathway culminating in pathogen opsonization and phagocytosis. mannose binding lectin deficiency is associated with to hiv transmission and progression of aids. expression of the lectin has been detected in astrocytes, microglia, neurons, and oligodendrocytes of the hiv-1-infected frontal cortex. in hiv encephalitis, monomeric and trimeric mannose binding lectin expression and mcp-1 expression are elevated in the neuronal axons. differential mbl expression was not observed in healthy hiv-negative subjects and normal or gp120 transgenic mice. the findings indicate that mannose binding lectin could cause neuroinflammation and neuronal injury through activating the complement pathway [91] . hiv-1goes into the brain at the early stages of infection and leads to damage and impairment of the central nervous system mannose binding lectin binds to the n-linked mannose residues on gp120, a neurotoxin, and is instrumental in intravesicular packaging of gp120 in neuronal subcellular organelles and subcellular trafficking of gp120 through the endoplasmic reticulum and golgi vesicles.the vesicular complexes were translocated along the microtubule network a functional carbohydrate recognition domain necessary for the actions of mannose binding lectin [92] . in summary, the use of lectins extracted from various sources is a great promise and potential for use in future hiv therapy. latest research has found potent results towards the anti-hiv effects of various lectins as shown in this review. there is still no vaccine against hiv. the n-linked glycans of gp120 v1 protects the v3 region of oligomeric gp120 from potential neutralizing antibodies [93] . because of the inventive immunological escape mechanisms of this virus, additional research should be funded in order to understand these mechanisms. also for the lectins, research should be promoted and funded and aid in the transition to clinical application. the mannose binding lectins actinohivin (ah), cyanovirin-n (cv-n), griffithsin (grft), microcystis viridis lectin (mvl), oscillatoria agardhii agglutinin (oaa), scytovirin (svn), from cyanobacteria and algae display high anti-hiv potencies with nanomolar-picomolar ic50 values. they differ in tertiary and quaternary structures, but contain internal repeats within their primary structures. the number of sequence repeats often corresponds to the number of domains and binding sites in each lectin. there is pronounced amino acid sequence homology and also structural resemblance between the domains. each of the lectins has distinctive characteristics. some are multimeric and/or manifest domain swapping, whereas others are monomeric. monomeric cv-n, oaa, and svn have two sugar binding sites; ah and engineered monomeric grft possess three sites; domain-swapped cv-n and mvl display four sites; and domain-swapped grft has six sugar binding sites. the lectins recognize different epitopes on high mannoses. the number of binding sites on the protein and/or the number of epitopes recognized on man-8/9 correlate with its antiviral activity. they target mannose on gp120 of hiv, inhibiting the conformational change into the active state. the most straightforward delivery mode is introduction into the rectum or vagina, or in multifunctional contraceptive gels or rings, or in situ expression by modified commensal bacteria which inhabit the gut or vagina [94] . engineered vaginal lactobacillus strain has been proposed for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-n to prevent sexual transmission of the virus [95] . grft could be obtained in multigram quantities after extraction from nicotiana benthamiana plants transducted with a tobacco mosaic virus vector expressing the lectin. the employment of lactobacillus jensenii expressing an anti-hiv lectin can curtail the costs of the development of a user-friendly microbicide [96] . the anti-hiv activities of various lectins are summarized in tables 1 and 2 . hiv/aids-related mortality in africa and asia: evidence from indepth health and demographic surveillance system sites cause-specific childhood mortality in africa and asia: evidence from indepth health and demographic surveillance system sites in vitro anti-hiv activity of five selected south african medicinal plant extracts polymeric anti-hiv therapeutics hiv/aids epidemiology, pathogenesis, prevention, and treatment lectins as cell recognition molecules discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development purification and characterization of a mucin specific mycelial lectin from aspergillus gorakhpuresis: application for mitogenic and antimicrobiol activity how does hiv cause aids manipulation of dendritic cell function by viruses robbins basic pathology hiv entry and its inhibition the hiv-1 envelope glycoproteins: fusogens, antigens, and immunogens spontaneous mutations in the env gene of the human immunodeficiency virus type 1 ndk isolate are associated with a cd4-independent entry phenotype access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1 cyanovirin-n binds to gp120 to interfere with cd4-dependent human immunodeficiency virus type 1 virion binding, fusion, and infectivity but does not affect the cd4 binding site on gp120 or soluble cd4-induced conformational changes in gp120 cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans isolation and characterization of griffithsin, a novel hiv-inactivating protein from the red alga griffithsia sp structural studies of algal lectins with anti-hiv activity improved procedure for the construction of neoglycolipids having antigenic and lectin-binding activities, from reducing oligosaccharides a novel in vitro system to generate and study latently hiv-infected long-lived normal cd4+ t-lymphocytes inhibition of hiv entry by carbohydrate-binding proteins proteins that bind high-mannose sugars of the hiv envelope jacalin, a lectin interacting with o-linked sugars and mediating protection of cd4+ cells against hiv-1, binds to the external envelope glycoprotein gp120 inhibition of human immunodeficiency virus infection by the lectin jacalin and by a derived peptide showing a sequence similarity with gp120 effects of succinylated concanavalin a on infectivity and syncytial formation of human immunodeficiency virus the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborine and the (n-acetylglucosamine)-specific plant lectin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro isolation and characterization of myrianthus holstii lectin, a potent hiv-1 inhibitory protein from the plant myrianthus holstii inhibition of herpes simplex, rabies and rubella viruses by lectins with different specificities a potent novel anti-hiv protein from the cultured cyanobacterium scytonema varium the antiviral lectin cyanovirin-n: probing multivalency and glycan recognition through experimental and computational approaches comparative studies of new marker lectins for alkali-labile and alkali-stable carbohydrate chains in glycoproteins jacalin: an iga-binding lectin jacalin, a jackfruit lectin, precipitates iga1 but not iga2 subclass on gel diffusion reaction preparation and x-ray characterization of four new crystal forms of jacalin, a lectin from artocarpus integrifolia activation of t and b cells by a crude extract of artocarpus integrifolia is mediated by a lectin distinct from jacalin a jackfruit (artocarpus heterophyllus) seed-derived lectin of versatile applications in immunobiological research structural and electron-microscopic studies of jacalin from jackfruit (artocarpus integrifolia) show that this lectin is a 65 kda tetramer jacalin, a lectin with anti-hiv-1 properties, and hiv-1 gp120 envelope protein interact with distinct regions of the cd4 molecule in silico analysis of molecular mechanisms of legume lectin-induced apoptosis in cancer cells ultrastructural visualization of surface carbohydrate structures on mycoplasma membranes by concanavalin a canatoxin, a toxic protein from jack beans (canavalia ensiformis), is a variant form of urease (ec 3.5.1.5): biological effects of urease independent of its ureloytic activity non-glycosylated recombinant pro-concanavalin a is active without polypeptide cleavage isolation, physicochemical characterization and carbohydrate-binding specificity of lectins correlation between carbohydrate structures on the envelope glycoprotein gp120 of hiv-1 and hiv-2 and syncytium inhibition with lectins resistance of human immunodeficiency virus type 1 to the high-mannose binding agents cyanovirin n and concanavalin a structural analysis and binding properties of isoforms of tarin, the gna-related lectin from colocasia esculenta induction of apoptosis by polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma l929 cells polygonatum cyrtonema lectin induces apoptosis and autophagy in human melanoma a375 cells through a mitochondria-mediated ros-p38-p53 pathway molecular modeling, docking and dynamics stimulations of gna-related lectins for potential prevention of influenza virus (h1n1) molecular mechanisms of polygonatum cyrtonema lectin-induced apoptosis and autophagy in cancer cells galanthus nivalis agglutinin (gna)-related lectins: traditional proteins, burgeoning drugs? targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy inhibition of hiv-1 env-mediated cell-cell fusion by lectins, peptide t-2, and neutralizing antibodies carbohydrate-binding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign)-directed hiv-1 transmission to t lymphocytes current lead natural products for the chemotherapy of human immunodeficiency virus (hiv) infection a lectin isolated from bananas is a potent inhibitor of hiv replication edible wild plants of tanzania; regional land management unit the potentially insecticidal narcissus pseudonarcissus lectin demonstrates age-related mitogenecity related mannose-specific lectins from different species of the family amaryllidaceae anti-human immunodeficiency virus type 1 (hiv-1) activity of lectins from narcissus species crystal structures of a novel anti-hiv mannose-binding lectrin from polygonatum cyrtonema hua with unique ligand-binding property and super-structure urtica dioica agglutinin: a superantigenic lectin from stinging nettle rhizome early biochemical events in lymphocyte t activation by mitogens boodlea coacta is a potent entry inhibitor of hiv-1 and influenza viruses binding of the mannose-specific lectin, griffithsin, to hiv-1 gp120 exposes the cd4-binding site removal of two high-mannose n-linked glycans on gp120 renders human immunodeficiency virus 1 largely resistant to the carbohydrate-binding agent griffithsin synergistic activity profile of griffithsin in combination with tenofovir, maraviroc and enfuvirtide against hiv-1 clade c the role of individual carbohydrate-binding sites in the function of the potent anti-hiv lectin griffithsin development of vaginal microbicides for the prevention of heterosexual transmission of hiv. hiv/aids in women: an expanding epidemic broad anti-hiv activity of the oscillatoria agardhii agglutinin homologue lectin family combinations of griffithsin with other carbohydrate-binding agents demonstrate superior activity against hiv type 1, hiv type 2, and selected carbohydrate-binding agent-resistant hiv type 1 strains structural basis of the anti-hiv activity of the cyanobacterial oscillatoria agardhii agglutinin primary sequence determination, and disulfide bond structure of cyanovirin-n, an anti-hiv (human immunodeficiency virus) protein from the cyanobacterium nostoc ellipsosporum pegylation of cyanovirin-n, an entry inhibitor of hiv linker-extended native cyanovirin-n facilitates pegylation and potently inhibits hiv-1 by targeting the glycan ligand humic acids (ha) strongly potentiate anti-hiv effects of azt, griffithsin, and cyanovirin the potent anti-hiv protein cyanovirin-n contains two novel carbohydrate binding sites that selectively bind to man8 d1d3 and man9 with nanomolar affinity: implications for binding to the hiv envelope protein gp120 the novel fold of scytovirin reveals a new twist for antiviral entry inhibitors mechanisms of hiv-1 subtype c resistance to grft, cv-n and svn the cyanobacterial lectin scytovirin displays potent in vitro and in vivo activity against zaire ebola virus differential inhibitory effects of cyanovirin-n, griffithsin, and scytovirin on entry mediated by envelopes of gammaretroviruses and deltaretroviruses isolation and characterization of a mannan-binding lectin from the freshwater cyanobacterium (blue-green algae) microcystis viridis solution structure of the monovalent lectin microvirin in complex with man(α)(1-2)man provides a basis for anti-hiv activity with low toxicity mechanism by which the lectin actinohivin blocks hiv infection of target cells the high mannose-type glycan binding lectin actinohivin: dimerization greatly improves anti-hiv activity a β-galactose-specific lectin isolated from the marine worm chaetopterus variopedatus possesses anti-hiv-1 activity a new lectin from the sea worm serpula vermicularis: isolation, characterization and anti-hiv activity c-type lectin mermaid inhibits dendritic cell mediated hiv-1 transmission to cd4+t cells mannose-binding geometry of pradimicin a expression of mannose binding lectin in hiv-1-infected brain: implications for hiv-related neuronal damage and neuroaids intracellular mannose binding lectin mediates subcellular trafficking of hiv-1 gp120 in neurons protection of neutralization epitopes in the v3 loop of oligomeric human immunodeficiency virus type 1 glycoprotein 120 by n-linked oligosaccharides in the v1 region engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-n antiviral lectins as potential hiv microbicides algal lectins as potential hiv microbicide candidates the antiviral protein cyanovirin-n: the current state of its production and applications mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type 1 strains with n-glycan deletions in their gp120 envelopes ,2)-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv-1 activity comparable with that of cyanovirin-n but a much higher safety profile subtype c gp120 and sensitivity to the lectins, griffithsin, cyanovirin-n and scytovirin new carbohydrate specificity and hiv-1 fusion blocking activity of the cyanobacterial protein mvl: nmr, itc and sedimentation equilibrium studies overexpression and purification of scytovirin, a potent, novel anti-hiv protein from the cultured cyanobacterium scytonema varium primary structure and carbohydrate binding specificity of a potent anti-hiv lectin isolated from the filamentous cyanobacterium oscillatoria agardhii griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate scaleable manufacture of hiv-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component potential anti-hiv agents from marine resources: an overview carbohydrate-binding proteins of marine invertebrates we gratefully acknowledge the award of an hmrf research grant (reference no. 12110672) from food and health bureau, the government of hong kong special administrative region. o.a. and t.b.n. were responsible for writing the review and did the final editing of the manuscript. s.s.s., y.s.c. and w.p. prepared the tables. r.c.f.c. assisted in providing references for the manuscript and proofread the manuscript. x.d. and c.y. helped in compilation of references. the authors have declared that no competing interests exist. key: cord-267182-ctvnmjsl authors: mboowa, gerald; sserwadda, ivan; amujal, marion; namatovu, norah title: human genomic loci important in common infectious diseases: role of high-throughput sequencing and genome-wide association studies date: 2018-03-20 journal: can j infect dis med microbiol doi: 10.1155/2018/1875217 sha: doc_id: 267182 cord_uid: ctvnmjsl hiv/aids, tuberculosis (tb), and malaria are 3 major global public health threats that undermine development in many resource-poor settings. recently, the notion that positive selection during epidemics or longer periods of exposure to common infectious diseases may have had a major effect in modifying the constitution of the human genome is being interrogated at a large scale in many populations around the world. this positive selection from infectious diseases increases power to detect associations in genome-wide association studies (gwass). high-throughput sequencing (hts) has transformed both the management of infectious diseases and continues to enable large-scale functional characterization of host resistance/susceptibility alleles and loci; a paradigm shift from single candidate gene studies. application of genome sequencing technologies and genomics has enabled us to interrogate the host-pathogen interface for improving human health. human populations are constantly locked in evolutionary arms races with pathogens; therefore, identification of common infectious disease-associated genomic variants/markers is important in therapeutic, vaccine development, and screening susceptible individuals in a population. this review describes a range of host-pathogen genomic loci that have been associated with disease susceptibility and resistant patterns in the era of hts. we further highlight potential opportunities for these genetic markers. hiv/aids, tuberculosis, and malaria are 3 major global public health threats causing substantial morbidity, mortality, negative socioeconomic impact, and human suffering [1] . whole-genome sequencing (wgs) of hosts and their cognate pathogens has transformed our understanding of the contribution of genomics in infectious disease processes. as the antibiotic era changed our understanding of infections so has the genomic revolution. e host-pathogen coevolutionary "arms race" is a phenomenon that has been described to result from interaction within host innate, adaptive immune responses, exposure to antibiotics, and competition with commensal microbiota [2, 3] . when positive selection in one geographical region causes large allele frequency differences between populations than those expected for neutrally evolving alleles, high frequency of the derived allele (i.e., when a new allele increases to a frequency higher than that expected under genetic drift) [4] and hts technologies are harnessed and used to decipher the nature and dynamics of these signals. e dynamics of host-pathogen interactions (e.g., length of exposure, geographical spread, morbidity, mortality, and cooccurring environmental events) influence the genetic architecture of resistance variants in modern populations [4] . e application of genomic sequencebased approaches to understand the host-pathogen interface continues to provide us with important clues for our survival. human genetic variation is a major determinant of genetic susceptibility to many common infectious diseases [5] . malaria, hiv/aids, and tuberculosis are some of the common infectious diseases in which a range of genetic susceptibilities and resistant conferring loci have been identified using both traditional molecular-based approaches and hts technologies. hts has enabled us to identify genomic signatures from these interactions. ese markers identified through infectious disease genome-wide association studies (gwass) have considerable significances that can be exploited to understand host protective mechanisms against pathogens and identify new molecular targets for diagnostic, prophylactic, and therapeutic interventions (table 1 ). in the past, candidate gene studies have been used to identify disease susceptibility genetic loci for many major human infections. but with the advent of hts approaches, many new loci have been and continue to be identified in diverse populations. a paradigm shift from candidate gene studies to gwas and hts has ushered in a "big data" genomic revolution era enabling us to redefine the genomic architecture of host-pathogen disease susceptibility. both exome and whole-genome sequencing approaches are proving more successful and affordable. host genetics influence clinical course of infectious diseases as well as genetic variation of the pathogens determining their survival in presence of selective pressure from the host and environment (antimicrobials) and identify genetic markers of drugresistant pathogens and parasites. furthermore, hts has given us unprecedented resolution of understanding the role of host genetics to infectious diseases susceptibility. is genomic revolution has generated data informative for understanding the frequency of many genetic traits, including those that cause disease susceptibility in african populations and populations of recent african descent [17] . pathogens have always been a major cause of human mortality, so they impose strong selective pressure on the human genome [18, 19] . hts applied to screening populations of host immune-specific cells and their respective pathogens can highlight the host-pathogen unique genetic signatures important in host-pathogen coevolution, profiling immunological history, pathogen-induced immunodominance genetic patterns, predicting clinical outcomes of common infections (such as hiv/aids disease progression phenotypes like long-term nonprogressors and rapid progressors, as well as highly exposed persistently seronegative group), rapid diagnosis plus screening outbreaks involving risk group 4 highly infectious pathogens, and genetic characterization of live-attenuated vaccine vectors (figures 1(a) and 1(b)). gwass demand recruitment of large well-phenotyped clinical cohorts within appropriate study designs and settings. however, they are very expensive, and therefore few funding agencies are able to finance such studies yet they may offer unique opportunities to unveil genetically important signals. currently, there are a growing number of communicable disease-specific research initiatives that are specifically interested in looking at the stages of the infections and gwas of disease progression. a classic example is the collaborative african genomics network (cafgen), a h3africa-funded consortium probing host genetic factors that are important to the progression of hiv and hiv-tb infection in sub-saharan african children (https://www.h3africa.org/consortium/projects/16projects/89-cafgen). cafgen is specifically looking at both rapid and long-term hiv/aids progression status while infectious diseases account for 15 million deaths per year worldwide, and disproportionately affect the young, elderly people, and the poorest sections of society making them a high priority [20] . e world health organization in 2016 estimated the global mortality for tuberculosis, hiv/aids, and malaria to be at 3.23 million with most deaths occurring in sub-saharan africa. is region has continued to lead in both prevalence and incidence of these major infectious killer diseases [21] . research investment in infectious diseases was poorly matched. data show that funding does not correspond closely with burden [22] . review of findings to date suggests that the genetic architecture of infectious disease susceptibility may be importantly different from that of noninfectious diseases [20] . other authors have extensively reviewed the genetic susceptibility to diseases [4, 5, 13, 16, [18] [19] [20] . e ancient biological "arms race" between microbial pathogens and humans has shaped genetic variation in modern populations, and this has important implications for the growing field of medical genomics [4] . as humans migrated throughout the world, populations encountered distinct pathogens, and natural selection increased the prevalence of alleles that are advantageous in the new ecosystems in both host and pathogens [4] . temporal patterns supporting figure 1 : (a) pipeline for interrogation of pathogen genomes using high-throughput sequencing and computational approaches. dna extraction for hts can be done from either direct clinical specimen of individuals who are suspected to be infected with the disease or from enriched/isolated cultures. quality control and read preprocessing are critical steps in the analysis of datasets generated from highthroughput sequencing technologies. fastqc is an example of a tool for general quality assessment of hts data from all technologies. genomes can be recreated with no prior knowledge using de novo sequence assembly as well as recreating the genome using prior knowledge based on a reference genome-alignment/mapping. e former is necessary for novel genomes and where the sequenced genome differs from reference. sequence data analysis is important in infectious disease outbreak investigations, molecular typing, antimicrobial drug resistance, transmission, surveillance, and microbial evolution. (b) pipeline for interrogation of host genomes using high-throughput sequencing and computational approaches. for a given infectious disease in a population, an appropriate study design is determined and host dna is collected from cases (exposed to pathogen and infected) and controls (exposed to pathogen and uninfected). hts of dna from both cases and control is performed. quality control (qc) procedures vary in different pipelines. ese include qc on individuals for missingness, gender checks, duplicates and cryptic relatedness, population outliers, heterozygosity and inbreeding, qc on snps for missingness, minor allele frequency, and hardy-weinberg equilibrium. many of these are computationally intensive, operationally challenging, and constantly evolving. genome-wide association studies (gwass) involving case-control studies compare the frequencies of common genetic variants, assume an appropriate statistical model, and account for multiple testing correction threshold to identify susceptibility and protective polymorphisms in the population. evidence of host-pathogen coevolution have been reported [50] . common infectious diseases have shown geographical disparities, for example, mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages [51] . molecular epidemiological studies show that, with the exception of sub-saharan africa, almost all hiv-1 subtypes, circulating recombinant forms, and several unique recombinant forms have been detected, but there is a specific geographic distribution pattern for hiv-1 subtypes [52] [53] [54] . hiv diversity plays a central role in the hiv pandemic [55] and has significant implications for diagnosis, vaccine development, and clinical management of patients [56] . high levels of plasmodium falciparum malaria endemicity are common in africa [57] . formal characterization of disease-causing agents has always been fundamental to understanding the evolution of pathogens and the epidemiology of infectious disease. linking this information with temporal, spatial, and clinical data can bring understanding of evolution, geographical spread, and disease associations for the pathogen, providing vital information for identifying sources of infection as well as designing interventions to prevent and treat disease [58] . new sequencing technology has enabled the identification of thousands of single nucleotide polymorphisms in the exome, and many computational and statistical approaches to identify disease association signals have emerged [59] . foremost is a better understanding of disease pathogenesis and resistance in the expectation that this will lead in time to improved interventions such as better drugs or vaccines to prevent or attenuate the great global burden of infectious disease morbidity and mortality. with over 10 million deaths annually from infectious diseases and the threat of new epidemics and pandemics, this is a very high priority [20] . during the past decade, we have also witnessed the emergence of many new pathogens not previously detected in humans, such as the avian influenza virus, severe acute respiratory syndrome (sars), and ebola [60] . now that the scientific community has access to complete genomes of infectious diseases though application of hts, priority should be the dissection of host-pathogen interactions through development of powerful computational tools. hts has profoundly altered our understanding of human diversity and disease. e interaction between hosts and pathogens is a coevolution process which may simply be described as "shooting a moving target." hts and computational modelling tools will offer potential to understand this interaction leading to better vaccine designs and therapeutic targets. sanger dna sequencing is limited in throughput and high cost as compared to hts platforms, which differ in their details but typically follow a similar general paradigm: template preparation, clonal amplification, followed by cyclical rounds of massively parallel sequencing [61] . nanopore-based sequencing approaches such as single molecule, real-time (smrt) sequencing technologies have been developed and consistently produce some of the longest average read lengths compared to hts. smrt sequencing is particularly useful for projects involving de novo assembly of small bacterial and viral genomes as well as large genome finishing [62] . many important sequencing platforms are reviewed in great depth [61, 63] . computational tools are an important integral part of genomics. new computational methods are constantly being developed to collect, process, and extract useful biological information from a variety of samples and complex datasets [64] . e scale and complexity of genomic data is everexpanding, requiring biologists to apply increasingly more sophisticated computational tools in the generation, analyses, interpretation, and storage of this data. e data are generated in different sizes, formats, and structures requiring a wide range of tools to manipulate. despite the level of specialization needed in bioinformatics, it is important that life-scientists have a good understanding of it for a correct experimental design which allows them to reveal the information in a metagenome [64] . hts technologies are generating an astonishing amount of unprecedented information in the history of biology which has spurred a biomedical big data to knowledge (b2d2k) revolution. us, a new exhilarating rapidly evolving scientific field, bioinformatics (biology meets computer programming), has recently emerged and uses novel computational approaches to help solve important biological problems. bioinformatics is a set of activities: data acquisition, database development, data analysis, data integration, and analysis of integrated data. e majority of available bioinformatics software requires some knowledge of the text-based command line of the unix or linux operating systems, allowing custom programming scripts and pipelines to automate data manipulation and analysis in a single step [65] . although bioinformatics tools/software are both "open sources" and commercially available, clinicians have limited bioinformatics knowledge [66] [67] [68] [69] . it is clear that user-friendly bioinformatics pipelines are key to facilitating more widespread use of wgs, with more widespread bioinformatics expertise [65] . positive selection (also known as darwinian selection) in genes and genomes can point to the evolutionary basis for differences among species and among races within a species [70] . many other aspects of human biology not necessarily related to the "branding" of our species, for instance, hostpathogen interactions, reproduction, dietary adaptation, and physical appearance, have also been the substrate of varying levels of positive selection. comparative genetics/genomics studies in recent years have uncovered a growing list of genes that might have experienced positive selection during the evolution of humans and/or primates [71] . microbial genome evolution is shaped by a variety of selective pressures. understanding how these processes occur can help to address important problems in microbiology by explaining observed differences in phenotypes, including virulence and resistance to antibiotics. greater access to whole-genome sequencing provides microbiologists with the opportunity to perform large-scale analyses of selection in novel settings such as within individual hosts [72] . identification of positive selection genetic signatures in the genomes can help us to understand the kinetics and directions of continuing host-pathogen coadaptation and impact on their diagnosis, transmission, fitness, immunogenicity, and pathogenicity. given the potential for strong selective pressure, that genetic programs controlling hostpathogen interactions in humans and other species are littered with signatures of positive selection [73] . e high mortality and widespread impact of malaria has resulted in this disease being the strongest evolutionary selective force in recent human history, and the genes that confer resistance to malaria provide some of the best-known case studies of strong positive selection in modern humans [27] . a number of specific genomic variants including î²-globin locus, g6pd deficiency, duffy, ovalocytosis, abo, and human leukocyte antigen confer resistance to malaria in the human host. elevated frequencies of hemoglobinopathies such as thalassemia and sickle cell disease, which are caused by mutations at the î²-globin locus, are maintained via balancing selection ("the malarial hypothesis") [74] [75] [76] . is hypothesis suggests that some human diseases such as thalassemia are polymorphisms which provide heterozygote advantage because of the trade-offs between the advantages of resistance to malaria and negative effects due to the disease [77] , where the hemoglobin s (hbs) homozygote disadvantage is recompensed through the malaria resistance of the heterozygote (hbas) in regions of malaria endemicity [78, 79] . e â��32 mutation at the ccr5 locus is a wellstudied example of natural selection acting in humans. e mutation is found principally in europe and western asia, with higher frequencies generally in the north. homozygous carriers of the â��32 mutation are resistant to hiv-1 infection because the mutation prevents functional expression of the ccr5 chemokine receptor normally used by hiv-1 to enter cd4+ t cells [80] [81] [82] [83] [84] [85] [86] [87] . host genetic factors play important roles in susceptibility to tuberculosis infection, and different gene polymorphisms in different ethnicity and genetic backgrounds may lead to different effects on tuberculosis risk [88] . polymorphisms in natural resistance-associated macrophage protein 1 (nramp1), toll-like receptor 2 (tlr2), interleukin-6 (il-6), tumor necrosis factor-alpha (tnf-î±), interleukin-1 receptor antagonist (il-1ra), il-10, vitamin d receptor (vdr), dendritic cell-specific icam-3grabbing nonintegrin (dc-sign), monocyte chemoattractant protein-1 (mcp-1), nucleotide oligomerization binding domain 2 (nod2), interferon-gamma (ifn-c), inducible nitric oxide synthase (inos), mannose-binding lectin (mbl), and surfactant proteins a (sp-as) genes have been variably associated with tuberculosis infection, and there is strong evidence indicating that host genetic factors play critical roles in tuberculosis susceptibility, severity, and development among different populations [89] [90] [91] . several nramp1 polymorphisms were significantly associated with ptb in african and asian populations, but not in populations of european descent [92, 93] . gwass are based on the "common disease, common variant" hypothesis, and they have been performed largely using single nucleotide polymorphism (snp) arrays that focus canadian journal of infectious diseases and medical microbiology only on common genetic polymorphisms (for which the minor allele frequency is >5%) [35, [94] [95] [96] . gwas approach has potential to provide candidates for the development of control measures against infectious diseases in humans [13] . for over 50 years, candidate gene studies have been used to identify loci for many major causes of human infectious mortality, including malaria, tuberculosis, and hiv-1 [20] . e first successful gwas was published in 2005 ushering genome-wide approaches that have identified loci in diverse populations. common genetic variants have also been demonstrated to regulate susceptibility/resistance to infectious diseases, for example, the ccr5â��32 polymorphism that modulates hiv/aids disease progression [97] . genome-wide association study approaches are being increasingly utilized to define genetic variants underlying susceptibility to major infectious diseases [34] . infectious diseases follow a series of stages right from acquisition, disease development, rate of progression, convalescence, and asymptomatic carrier state. erefore, every stage will be influenced by one or a set of mutations in a population or individual. different populations will have mutations that affect diseases at different stages. is is where gwas has and will continue to play an important role in identifying these mutations. a recent study suggested that host genetic risk in tb is depended upon the pathogen's genetic background and demonstrated the importance of analyzing the interaction between host and pathogen genomes in tb [98] . studies are exploiting unique designs like extreme phenotype designs to identify complex trait genomic loci, while others have identified genetic associations of infectious diseases by integrating estimation of population admixture events to detect disease susceptibility loci after teasing out different ancestries and allelic, genotypic, or haplotype risk ratios. a genomic database for all mutations identified to be conferring resistance to infectious diseases in different populations is a vital product of more than 10 years of gwas. more studies should be appropriately designed to identify new potential infectious disease resistance-conferring mutations in human hosts. we searched the pubmed database for studies published since january 2005 using the terms "malaria," "tuberculosis," "hiv/aids," "genome-wide association." search terms included combinations of ((disease-query[title] and genome-wide association[title])) where a disease-query was either a communicable or noncommunicable disease. two search restrictions were set: publication date set (from 2005/01/01) and species (humans). figure 2 indicates more gwass carried out in noncommunicable diseases than infectious diseases. ere is an urgent need for a major increase in funding for communicable disease control in the developing world and for more balanced allocation of the resources already provided [22] . genomics and whole-genome sequencing have the capacity to greatly enhance knowledge and understanding of infectious diseases and clinical microbiology [65] . human genetics is an indispensable tool for enhancing the understanding of the molecular basis of many common diseases [99] . over 4,500 snps have been associated with a variety of human traits and complex diseases [59] . we envisage that with the reducing costs, hts and genomics will become an indispensable component of every healthcare system. hts and computational tools offer a potential to stratify populations for risk of infectious disease based on genomic profiling thereby prioritizing interventions such as vaccines and therapeutics to the "most-at-risk" populations since there is no "one-size-fits-all" approach to treating infectious diseases. with the growing number of sequencing facilities on every continent, the future will offer a less costly approach that will integrate a genomic profile in routine patient management, improving management of diseases, and therapeutic development. it is less likely to find a population or individual who carries mutations conferring resistance to an infectious disease at every stage of the infection. different individuals have different mutations that offer resistance to different stages of the infections. a genomic catalogue of these mutations identified through hts, computational tools, and gwas now combined with the rapidly growing genomeediting technology known as crispr/cas9 will enable introduction of an array of disease-stage-specific resistanceconferring mutations. is will offer interventions at all levels of the disease process unlike traditional vaccines. disclosure e views expressed in this publication are those of the authors and not necessarily those of the aas, nepad agency, wellcome trust, or the united kingdom government. e authors declare that they have no conflicts of interest. e global fight against hiv/aids, tuberculosis, and malaria: current status and future perspectives evolution of bacterial pathogens within the human host arms races between and within species natural selection and infectious disease in human populations genetics and genomics of infectious disease susceptibility host determinants of hiv-1 control in african americans e major genetic determinants of hiv-1 control affect hla class i peptide presentation a whole-genome association study of major determinants for host control of hiv-1 genomewide association study of an aids-nonprogression cohort emphasizes the role played by hla genes (anrs genomewide association study 02) common genetic variation and the control of hiv-1 in humans genomewide association study implicates pard3b-based aids restriction multiple-cohort genetic association study reveals cxcr6 as a new chemokine receptor involved in long-term nonprogression to aids genome-wide association study indicates two novel resistance loci for severe malaria genome-wide and fineresolution association analysis of malaria in west africa a genome-wide association study of pulmonary tuberculosis in morocco genome-wide association analyses identifies a susceptibility locus for tuberculosis on chromosome 18q11 genetic variation and adaptation in africa: implications for human evolution and disease from evolutionary genetics to human immunology: how selection shapes host defence genes pathogen-driven selection in the human genome evolution, revolution and heresy in the genetics of infectious disease susceptibility genetics of sub-saharan african human population: implications for hiv/aids, tuberculosis, and malari donor funding priorities for communicable disease control in the developing world e role of genomics in studying genetic susceptibility to infectious disease e genomics and genetics of human infectious disease susceptibility e genetic theory of infectious diseases: a brief history and selected illustrations sickle cell protection from malaria population genetics of malaria resistance in humans host genetic determinants of hiv infection and disease progression in children unravelling the mechanisms of durable control of hiv-1 long term non-progressor (ltnp) hiv infection immunogenetics of hiv disease immunogenetic basis of hiv-1 infection, transmission and disease progression identifying recent adaptations in large-scale genomic data human genetic susceptibility to intracellular pathogens human genetic susceptibility to infectious disease host genomic influences on hiv/aids host genetics of hiv acquisition and viral control genetic susceptibility to infectious diseases: big is beautiful, but will bigger be even better? genetic susceptibility to infectious diseases genetic susceptibility to infectious disease genetic polymorphisms linked to susceptibility to malaria genetic epidemiology of infectious diseases in humans: design of population-based studies a locus at 5q33. 3 confers resistance to tuberculosis in highly susceptible individuals genetic variants that confer resistance to malaria are associated with red blood cell traits in african-americans: an electronic medical record-based genome-wide association study association mapping by pooled sequencing identifies toll 11 as a protective factor against plasmodium falciparum in anopheles gambiae a novel locus of resistance to severe malaria in a region of ancient balancing selection genome-wide association studies and susceptibility to infectious diseases genome-wide association studies for common diseases and complex traits genome-wide association studies in plasmodium species biological and biomedical implications of the co-evolution of pathogens and their hosts mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications estimated global distribution and regional spread of hiv-1 genetic subtypes in the year 2000 global and regional distribution of hiv-1 genetic subtypes and recombinants in 2004 hiv-1 genetic variability and clinical implications large-scale analysis of the prevalence and geographic distribution of hiv-1 non-b variants in the united states a world malaria map: plasmodium falciparum endemicity in 2007 genomic perspectives on the evolution and spread of bacterial pathogens computational and statistical approaches to analyzing variants identified by exome sequencing computational resources in infectious disease: limitations and challenges highthroughput sequencing technologies mind the gap: upgrading genomes with pacific biosciences rs longread sequencing technology coming of age: ten years of next-generation sequencing technologies e road to metagenomics: from microbiology to dna sequencing technologies and bioinformatics whole genome sequencing in clinical and public health microbiology integrating diverse databases into an unified analysis framework: a galaxy approach creating reusable tools from scripts: the galaxy tool factory arg-annot, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes identification of acquired antimicrobial resistance genes rapid detection of positive selection in genes and genomes through variation clusters positive selection on the human genome pairwise diversity and tmrca as potential markers for hiv infection recency mini-review: defense strategies and immunity-related genes e rate of mutation of human genes e sickle-cell and haemoglobin c genes in some african populations e î²-globin recombinational hotspot reduces the effects of strong selection around hbc, a recently arisen mutation providing resistance to malaria resistance to malaria in humans: the impact of strong, recent selection e host genetic diversity in malaria infection suggestions as to quantitative measurement of rates of evolution e geographic spread of the ccr5 î�32 hiv-resistance allele dating the origin of the ccr5-î�32 aids-resistance allele by the coalescence of haplotypes e î� ccr5 mutation conferring protection against hiv-1 in caucasian populations has a single and recent origin in northeastern europe distribution of the ccr5 gene 32-bp deletion in europe global distribution of the ccr5 gene 32-basepair deletion distribution of the ccr5 gene 32-basepair deletion in west europe. a hypothesis about the possible dispersion of the mutation by the vikings in historical times frequencies of 32 base pair deletion of the (î�32) allele of the ccr5 hiv-1 co-receptor gene in caucasians: a comparative analysis more about the viking hypothesis of origin of the î�32 mutation in the ccr5 gene conferring resistance to hiv-1 infection host genetic effect on tuberculosis susceptibility in chinese uyghur nramp1, vdr, hla-drb1, and hla-dqb1 gene polymorphisms in susceptibility to tuberculosis among the chinese kazakh population: a casecontrol study innate immune gene polymorphisms in tuberculosis host genome polymorphisms and tuberculosis infection: what we have to say? slc11a1 (formerly nramp1) gene polymorphisms and tuberculosis susceptibility: a meta-analysis human genetics of tuberculosis: a long and winding road variation across the allele frequency spectrum genetic heterogeneity in human disease uncovering the roles of rare variants in common disease through whole-genome sequencing dual effect of ccr5 î�32 gene deletion in hiv-1-infected patients pathogen lineagebased genome-wide association study identified cd53 as susceptible locus in tuberculosis what did we learn from the genome-wide association study for tuberculosis susceptibility? is work was supported through the deltas africa initiative (grant no. del-15-011) to thrive-2 (the training health researchers into vocational excellence in east africa). e deltas africa initiative is an independent funding scheme of the african academy of sciences' (aas) alliance for accelerating excellence in science in africa (aesa) and supported by the new partnership for africa's development planning and coordinating agency (nepad agency) with funding from the wellcome trust (grant no. 107742/z/15/z) and the uk government. all authors participated in writing the manuscript. ey further reviewed and approved the final manuscript. key: cord-286711-nr6vnl9h authors: nan title: other viruses causing gastroenteritis date: 2003-12-31 journal: perspectives in medical virology doi: 10.1016/s0168-7069(03)09037-2 sha: doc_id: 286711 cord_uid: nr6vnl9h publisher summary besides the viruses producing the majority of human viral gastroenteritis, other viruses infect more rarely but are sometimes able to cause epidemics. in particular, they cause chronic infection in the immunocompromised. some of these viruses discussed in this chapter are toroviruses, picobirnaviruses, enteroviruses, human immunodeficiency virus (hiv), herpesviruses, and coronaviruses. toroviruses make up a genus of the coronaviridae family. they are a well-described cause of diarrhea in calves and horses but may also infect sheep, goats, and pigs. picobirnaviruses are related to members of the birnaviridae family. they are found in the feces of hiv-infected patients with diarrhea more frequently than in hiv-infected patients without diarrhea, but a virus-specific immune response was not measurable. the genus enterovirus is of the picornaviridae family. all enteroviruses infect man via the gastrointestinal tract where they have their first site of replication, probably in lymphoid tissues of the pharynx and gut. hiv, the causative agent of the acquired immunodeficiency syndrome (aids), is a member of the lentivirus genus of the retroviridae family. cytomegalovirus (cmv) and herpes simplex viruses, members of the herpesviridae family, are found as the cause of colitis and esophagitis, mainly in hiv-infected patients. coronavirus is another genus of the coronaviridae family. coronaviruses infect the respiratory and gastrointestinal tracts. they are a recognized cause of the common cold in man. besides the viruses producing the majority of human viral gastroenteritis (sections ii-v), other viruses infect more rarely, but are sometimes able to cause epidemics. in particular, they cause chronic infection in the immunocompromised. torovimses comprise a genus of the coronaviridae family (enjuanes et al., 2000) . they are enveloped and possess a genome of single stranded rna of positive polarity which is approximately 20-25kb in size. the rna is surrounded by nucleoprotein (n) in helical symmetry. the nucleocapsid has a toroid shape (inspiring the name from lat. torus = convex moulding of a column) and is enclosed in an envelope consisting of membrane protein (m) and a lipid bilayer. inserted in the envelope are the surface proteins s (for spike) and he (for haemagglutinin-esterase). the he protein has sequence similarities with corresponding proteins of coronaviruses and influenza c viruses. toroviruses may have acquired this gene by a recombination event in the past. replication is special in that mrna is synthesized from 'core promoters' of a negative stranded rna template and not by fusion of a common leader sequence with subgenomic transcripts as in the coronaviruses (snijder and horzinek, 1995) . toroviruses are a well-described cause of diarrhoea in calves (botv; breda v) and horses (eqtv, berne virus) but may also infect sheep, goats and pigs. humans seem to become infected by a closely related, but distinct virus (hutv) (koopmans and horzinek, 1994; enjuanes et al., 2000) . in cases of human diarrhoea toroviruses have been diagnosed by electron microscopy and eia (koopmans et al., 1993) . in a recent survey in canada of 1365 faeces from children with diarrhoea, rotavirus was found in 32%, adenovirus in 4%, torovirus in 3%, norwalk like viruses in 2%, and astroviruses and sapporo-like viruses each in tess than 1% (waters et al., 2000) . this suggested that toroviruses are not a very frequent, but a consistent cause of diarrhoea in humans (koopmans et al., 1997; waters et al., 2000) . m petric has described the epidemiology of toroviruses (section vi, chapter 1). as molecular tests become available, the true extent of prevalence and incidence of human tv infections will become apparent. picobirnaviruses are related to, but not recognized yet as, members of the birnaviridae family (leong et al., 2000) . they comprise icosahedral, single shelled, non-enveloped particles which contain 2 segments of double stranded rna as their genome. these viruses (diameter 60 nm) occur in three known genera: aquabirnavirus (in fish; type species: infectious pancreatic necrosis virus, ipnv), avibirnavirus (in birds; type species: infectious bursal disease virus, ibdv) and entornobirnaviruses (in drosophila; type species: drosophila x virus (bxv); leong et al., 2000) . picobirnaviruses are only 30-40 nm in diameter and have 2 (or sometimes 3) segments of dsrna as their genome (leite et al., 1990) . they were detected in children with diarrhoea and in several animal species (asymptomatic) (pereira, 1991; ludert et al., 1991; gallimore et al., 1993 gallimore et al., , 1995 . recently they were found in the faeces of hiv-infected patients with diarrhoea more frequently than in hiv-infected patients without diarrhoea (grohmann et al., 1993; giordano et al., 1999) , but a virusspecific immune response was not measurable (grohmann et al., 1993) . the viral genome has recently been cloned and sequenced, and reagents derived from this will help to unravel the epidemiology of picorbirnavirus (rosen et al., 2000) . b i rosen has reviewed aspects of the molecular biology and epidemiology of these viruses (section vi, chapter 2). the genus enterovirus of the picornaviridae family contains a large number of species [polioviruses of types 1-3, coxsackieviruses of groups a (24 types) and b (6 types) and echoviruses (>70 types), melnick, 1996] . enteroviruses consist of an almost featureless capsid of 27-30 nm diameter containing 60 protomers, each possessing 3 surface proteins, vp1, vp2, vp3, and, in most viruses, an internal capsid protein, vp4. the capsid encloses a genome of single stranded rna of positive polarity of 7-8.5 kb length. the rna has a single open reading frame (orf) encoding a polyprotein precursor which is co-and post-translationally cleaved in a complex cascade of events into the 3 to 4 structural and a number of non-structural proteins (for details see racaniello, 2001) . all enteroviruses infect man via the gastrointestinal tract where they have their first site of replication, probably in lymphoid tissues of the pharynx and gut. usually enteroviruses are excreted in the stool for several weeks after infection but can also be isolated easily from the pharynx. after primary replication viruses spread via blood to other organs (nerve, muscle, fatty tissue) where they replicate further. most infections are asymptomatic, but enteroviruses can be the cause of meningitis, meningoencephalitis (poliomyelitis), myocarditis, pleurodynia, conjunctivitis and rashes (melnick, 1996) . in most cases of enterovirus infection there is no diarrhoea. however, some diarrhoea outbreaks have been found to be associated with enterovirus infections; coxsackievirus a1 and echoviruses of types 4, 11, 14, 18, 19 and 22 have been involved (townsend et al., 1982; patel et al. 1985; melnick, 1996) . recently, echoviruses of types 22 and 23 have been removed from the enterovirus genus and, as they are very different in their sequence from all other picornaviridae, been classified in a separate genus parechovirus (king et al., 2000) . the aichivirus is an at present unassigned species in the picornaviridae family (king et al., 2000) , but is proposed as a separate new genus (yamashita et al. 1998) . it is icosahedral, and the capsid has only 3 proteins. virions are stable at ph 3.5. aichivirus grows well in cell culture (bsc-1 and veto cells) and has been shown to be a consistent cause of human gastroenteritis (yamashita et al., 1991 (yamashita et al., , 1993 . it has also been found as a cause of traveller's diarrhoea (yamashita et al., 1995) . its genome has recently been cloned and sequenced (yamashita et al., 1998) , and it is hoped that the epidemiology of this virus will be further clarified with the availability of specific molecular reagents. t yamashita and k sakae have summarised our present knowledge of aichivirus in section vi, chapter 3. hiv, the causative agent of the acquired immunodeficiency syndrome (aids), is a member of the lentivirus genus of the retroviridae family. it infects the cd4 subset of lymphocytes overwhelmingly, but also macrophages, using different sets of receptors/ coreceptors. its replication (for details see freed and martin, 2001) damages the functions of infected cells before they are destroyed. the infection is symptomless for a long time, but then severe, generalised secondary infections appear (caused by other viruses, bacteria, fungi or protozoa) which are generally lethal. there is evidence of extensive hiv infection in gut-associated lymphoid tissue (galt) and also in enterocytes which contribute to the development of chronic diarrhoea in many aids patients (nelson et al., 1988; heise et al., 1991; kotler et al., 1991; rabeneck, 1994) . cytomegalovirus (cmv) and herpes simplex viruses, members of the herpesviridae family, are found as the cause of colitis and oesophagitis, mainly in hiv-infected patients (levinson and bennets, 1985; jacobson and mills, 1988; dieterich and robinson, 1991; theise et al. 1991; mentec et al., 1994; cotte et al., 1996) . with the application of highly active antiretroviral therapy (haart) it has become less urgent or indicated to commence and maintain a specific anti-cmv therapy with ganciclovir (whitcup et al., 1999; pollok, 2001) . coronavirus is another genus of the coronaviridae family (enjuanes et al., 2000) . the particles are 120-160 nm is diameter, enveloped and possess a genome of linear. positive-sense, single stranded rna of 27 -31 kb in size. the genome is surrounded by nucleocapsid protein (n; in helical symmetry) which in turn is contained in an envelope consisting of m protein and a host cell-derived lipid bilayer into which are inserted the surface proteins s (forming spikes), a small membrane protein (e) and the haemagglutinin esterase (he) protein. protein is expressed from rna molecules which are in most cases subgenomic, and all mrnas carry a leader sequence derived from the 5' end of the genome (for details see lai and holmes, 2001) . coronaviruses infect the respiratory and gastrointestinal tract. they are a recognized cause of common cold in man (mcintosh, 1996) . whilst coronavirus infections are firmly associated with gastroenteritis in animals (e.g. transmissible gastroenteritis virus (tgev) of pigs; kim et al., 2001) , their association with gastroenteritis in man has often been claimed, but its significance for human diarrhoeic disease has been controversial and not yet been firmly established (gerna et al., 1985; zhang et al., 1994; siddell and snijder, 1998; . j grant has produced a review of the histopathological findings of viral gastrointestinal tract infections, with particular emphasis on the data obtained from the immunocompromised (section vi, chapter 4). cytomegalovirus dna level on biopsy specimens during treatment of cytomegalovirus gastrointestinal disease cytomagalovirus colitis in aids: presentation in 44 patients and a review of the literature coronaviridae hivs and their replication detection and characterization of bisegmented double stranded rna viruses (picobirnavirus) in human faecal specimes detection and characterization of a novel bisegmented double-stranded rna virus (picobirnavirus) from rabbit faeces human enteric coronaviruses: antigenic relatedness to human coronavirus oc43 and possible etiologic role in viral gastroenteritis diarrhea and enteric emerging viruses in hiv-infected patients enteric viruses and diarrhoea in hiv-infected patients human immunodeficiency virus infection of enterocytes and mononuclear cells in human jejunal mucosa coronaviruses. in: fields' virology serious cytomegalovirus disease in the acquired immunodeficiency syndrome (aids) differential detection of transmissible gastroenteritis virus and epidemic diarrhea virus by duplex rt-pcr picornaviridae association of torovirus with acute and persistent diarrhoea in children toroviruses of animals and humans: a review enzyme-linked immunosorbent assay reactivity of torovirus-like particles in fecal specimens of humans with diarrhea detection, localisation and quantitation of h/v-associated antigens in intestinal biopsies from patients with aids coronaviridae: the viruses and their replication a novel avian virus with trisegmented double stranded rna and further observations on previously described similar viruses with bisegmented genome birnaviridae cytomegalovirus colitis in acquired immunodeficiency -a chronic disease with varying manifestations identification in porcine faeces of a novel virus with a bisegmented double stranded rna genome coronaviruses. in: fields virology, 3 '~ edition enteroviruses: polioviruses, coxsackieviruses, echoviruses and newer enteroviruses cytomegalovirus colitis in hiv-1 infected patients: a prospective research in 55 patients human immunodeficiency virus detected in bowel epithelium from patients with gastrointestinal symptoms an epidemic of acute diarrhoea in sural southern india associated with echovirus type 11 infection double-stranded rna viruses viruses causing diarrhoea in aids aids enteropathy: what's in the name? picornaviridae: the viruses and their replication cloning of human picobirnavirus genomic segments and development of a rt-pcr assay coronaviruses, toroviruses and arteriviruses the molecular biology of toroviruses cytomegalovirus esophagitis in aids diagnosis by endoscopic biopsy outbreak of coxsackie a1 gastroenteritis; a complication of bone marrow transplantation etiology of community-acquired pediatric viral diarrhea: a prospective longitudinal study in hospitals, emergency departments, pediatric practices and child care centres during the winter rotavirns outbreak, 1997 to 1998. the pediatric rotavirus epidemiology study for immunization study group discontinuation of anticytomegalovirus therapy in patients with hiv infection and cytomegalovirus retinitis isolation of cytopathic small round viruses with bsc-1 cells fi'om patients with gastroenteritis prevalence of newly isolated cytopathic small round virus (aichi strain) in japan isolation of cytopathic small round virus (aichi virus) fro pakistani children and japanese travellers from south east asia complete nucleotide sequence and genetic organization of aichi virus, a distinct member of the picornaviridae associated with acute gastroenteritis in humans biological and genetic characterization of haemagglutinating coronavirus isolated from a diarrhoeic child key: cord-297135-mg2qs3b6 authors: smith, kumi; bartlett, nicholas; wang, ning title: a harm reduction paradox: comparing china's policies on needle and syringe exchange and methadone maintenance date: 2012-07-31 journal: international journal of drug policy doi: 10.1016/j.drugpo.2011.09.010 sha: doc_id: 297135 cord_uid: mg2qs3b6 abstract background china has launched methadone maintenance treatment (mmt) and needle and syringe exchange programmes (nsep) as part of the country's hiv prevention strategy amongst injection drug users. mmt is expanding, with backing from multiple government ministries, however, nsep have received less political support and funding. methods semi-structured, serial interviews were conducted with key informants, knowledgeable about china's harm reduction policies. concurrent content analysis allowed for revision of the interview guide throughout the data collection process. this was combined with a systematic analysis of official government policy documents on nsep and mmt, including white papers, legal documents, and policy statements. findings early consensus between public security and public health sectors regarding methadone's dual use in hiv prevention as well as method of drug control created broad institutional support for mmt programmes amongst policy makers. in contrast, nsep were seen as satisfying only the hiv prevention goals of the public health sector, and were perceived as condoning illicit drug use. furthermore, nsep's roots in china, as an experimental collaboration with international groups, created suspicion regarding its role in china's drug control policy. nsep and mmt's distinct paths to policy development are reflected in the complex and occasionally contradictory nature of china's harm reduction strategy. conclusions these discrepancies highlight the need for a more politically sustainable and comprehensive integration of harm reduction projects. recommendations include improved evaluation methods for nesp, nsep-mmt cross-referral system, and stronger nsep advocacy within the non-profit and public health sectors. unsafe drug injection has been the main contributor to hiv transmission in asia, and hiv infection amongst injection drug users (idu) accounts for nearly half of all new infections on the continent (bao & liu, 2009; open society institute, 2008) . recent research shows that idu-driven epidemics may play a key role in 'seeding' generalised transmission of hiv through dual risk groups such as idu who purchase sex or commercial sex workers who inject drugs (saidel et al., 2003) . in 2007 idu made up the single largest risk group amongst cumulative reported hiv/aids cases in china (40.7%); however since 2009 heterosexual transmission is thought to have replaced injection drug use as the predominant mode of transmission, suggesting that this seeding effect may already be taking place (gill & oakie, 2007; grassly et al., 2003) . this underscores the importance of targeted hiv prevention amongst primary risk groups such as idu in order to avert a generalised epidemic in china and the greater asia region. epidemiologists found evidence of an epidemic in china beginning in the mid-1990s; however, it was not until 2002 that the government mobilised resources to combat it. in the aftermath of the panic surrounding china's 2003 severe acute respiratory syndrome (sars) outbreak, public health took on a new significance in government policy (gill & oakie, 2007) , ushering in an era of relative political openness and collaboration with international organisations. the state council aids working committee, an interagency committee formed to streamline the government's aids policy, created an opportunity for a rare partnership between the ministries of health and public security and this resulted in experimentation with progressive strategies including needle and syringe exchange programmes (nsep) and methadone maintenance treatment (mmt) (state council, people's republic of china, 2001 china, , 2006a . the chinese interagency committee's commitment to such programmes has now resulted in a national harm reduction 0955-3959/$ -see front matter © 2011 elsevier b.v. all rights reserved. doi:10.1016/j.drugpo.2011.09.010 programme that is to date more ambitious than many of the countries where these methods originated (hammett et al., 2008; international harm reduction development [ihrd], 2008; lurie & drucker, 1997; marsch, 1998; strathdee, 2004; . however, whereas mmt has enjoyed broad political support and consistent scale-up in china, nsep continues to face obstacles in its regular operations (ihrd, 2008) . contrasting programmatic outcomes between nsep and mmt reveal the pragmatic consensusbuilding process that has made each programme possible. this paper explores the origins, patterns of scale-up, and challenges in operating nsep and mmt as they operate in china, to better understand the nature of government support and its implications for these two harm reduction intervention strategies. it also extends policy recommendations to enhance existing strategies and harmonise hiv and drug misuse related goals shared across sectors of the chinese government. china's mmt programme refers to a national network of clinics providing daily oral doses of methadone to relieve narcotic craving whilst suppressing symptoms associated with opiate withdrawal (ministry of health, 2008) . the method posits that by taking methadone, clients can lessen the frequency of heroin injection and thereby reduce the associated risks of disease transmission through unsafe injection. advocates assert that mmt programmes also indirectly reduce drug-related criminal activity and improve social and employment stability (ihrd, 2008; ministry of health, 2008) . needle and/or syringe exchange as defined by sullivan and wu, refers to "fixed locations including pharmacies, hospitals or designated needle exchange centres, from where idu can obtain clean needles and syringes," and can also include the secondary exchange activities of injection equipment from machines or exchangers/peer leaders who deliver the products to idu in the community (hammett et al., 2007) . some nsep provide additional services such as distribution of cotton, alcohol swabs, cookers; counselling on safer injection, wound care, and referrals to drug treatment programmes (normand, vlahov, & moses, 1995) . the rationale driving nsep is that provision of sterile injection equipment can reduce the occurrence of unsafe equipment sharing that leads to the spread of blood borne diseases (hagen & thiede, 2000) . advocates also point out that nsep can provide drug users in the community with educational and referral services. since the 1990s there has been a consensus amongst the scientific community that nsep can reduce the spread of hiv without increasing drug use (goldstein, 1998; henderson, vlahov, celentano, & strathdee, 2003; lurie & drucker, 1997; national institutes of health, 1997; remis, bruneau, & hankins, 1998; watters, estilo, & clark, 1994; wodak & cooney, 2006) . however, debate on this topic persists in policy circles both in china and in the west. over a two-year period from 2007 to 2009, key informants were invited to take part in interviews. interviewees were from organisations contacted by the first two authors in the course of collecting preliminary data for epidemiological research grants to prevent transmission of blood borne diseases amongst idu. each stakeholder was informed that they could refrain from commenting on sensitive issues and that no personal identifying information would be used in the final report. interviews were conducted by the first author between march 2008 and june 2009 and recorded as shorthand notes, which were later transcribed into a word processing programme to facilitate key term searches. a broad topic guide allowed for greater flexibility to explore a range of issues but helped to maintain a focus on differences between nsep and mmt, roles of different government industries in china's harm reduction programmes, and suggestions for future programmes. prior to intervention-specific questions, definitions of key interview terms were explained to each participant. eighteen key informants representing nine establishments participated in interviews. they included individuals from the national centre for disease control and prevention (beijing, n = 2), government operated mmt clinics (kunming, kaiyuan, beijing; n = 6), independently operated nsep (beijing, kunming, kaiyuan; n = 3), municipal level departments of public security (kunming, n = 2), and mandatory and voluntary detoxification centres (kunming, kaiyuan; n = 5). in addition a web-based search was conducted in both english and chinese for publicly available government documents, including white papers, legal documents, and policy statements. iterations of boolean searches were used involving various combinations of the terms: "drugs," "narcotics," "drug control," "opiate addiction," "medical maintenance treatment," "methadone," "injection drug use," "china," "disease control," and "public health" and "hiv/aids." relevant peer reviewed articles published in both languages were also included after searching pubmed/medline and china academic journals (cnki) databases using the same search terms listed above. a key distinction is the international attention garnered by mmt in the form of publications, recognition from international drug policy groups-director zunyou wu of the chinese national centre for aids/std control & prevention (ncaids) was awarded the international harm reduction association's rolleston award for significant contributions to the field (ihrd, 2008)-and study tours by health experts from russia, vietnam, and malaysia (malinowska-sempruch & bartlett, 2006) . on the other hand, coverage of china's nseps is largely confined to the foreign media (french, 2006; jacobs, 2010; watters et al., 1994) and zunyou wu himself has described nsep as an emergency measure that "will only be used in places where methadone maintenance is not available" (state council, people's republic of china, 2001) . formal political and budgetary support for mmt can be identified in the language of the legal and strategic documents related to china's hiv control strategy. the hiv/aids prevention and care regulations of 2006 for example, urges various departments "to take active and well prepared measures to implement community based drug-maintenance treatment" (state council, people's republic of china, 2006) , and the new drug prohibition law enacted by the ministry of public security (mps) in june 2008 (state council, people's republic of china, 2007) in which mmt is referred to more directly as "medical detoxification," signals unprecedented support within china's public security sector for methadone dispensation as part of china's drug control programme. in contrast, support for nsep has been limited to provincial level statements, and as described by a behaviour change expert at ncaids, has never been codified as a part of national law as it is believed to be too controversial for national public security authorities to support . second, whereas mmt is administered by a centralised national working group under ncaids that includes representatives from the ministry of health (moh), mps, the state food and drug administration (sfda) and the yunnan institute for drug abuse, nsep lacks any such national coordinating mechanism. instead, implementation of nsep is delegated to individual programmes, who consult an ncaids-authored 150 page implementation guide on needle exchange work (national centre for aids/std control & prevention [ncaids], 2007). lastly, whereas mmt clinics are fully funded by central government as part of moh subsidised services, 43% of all funding for nsep in 2004 came from international organisations (wu, sullivan, wang, rotherham-borus, & detels, 2007b) . as a result of greater institutional support for mmt, its programmes are on a much broader scale than nsep. according to statistics from ncaids internal reports as well as published data from the mps, the respective coverage rates amongst nonincarcerated idu for these two programmes was 4.8% and 1.9%, respectively, with an average of 156 idu accessed per mmt clinic and 40 idu per needle exchange station (chinese centre for disease control and prevention [china cdc], 2008; open society institute, 2008) . in 2010 the united nations regional task force on injecting drug use and hiv/aids for asia and the pacific, estimated that coverage rates for mmt had increased to 12.9% whilst the proportion of idus accessing nsp" was only 1.7% (united nations regional task force on injecting drug use and hiv/aids for asia and the pacific (2008)). nsep were introduced in the early 2000 and were spearheaded by international organisations such as the world aids foundation, the ford foundation, australian agency for international development (ausaid), and the uk department for international development (dfid), with the ad hoc support of local authorities (hammett et al., 2003; hammett et al., 2008; ihrd, 2008) . the collective monetary resources and experience provided by these foreign organisations were pivotal in china's early nseps. by leveraging their programme funding and capitalising on the interest in public health following the 2003 sars outbreak, these international organisations successfully forged alliances with health bureaus and police departments in high prevalence provinces including yunnan, guangxi, and xinjiang (hammett et al., 2003; hammett et al., 2005; ihrd, 2008; lurie & drucker, 1997; wu et al., 2007a) . although these programmes were implemented by local cdcs; management, evaluation, and dissemination of the largely positive results was overseen by the international organisations. a study of fifteen nsep in southern china later found that those receiving funding from international agencies had higher rates of needle distribution and collection than those that had not (henderson et al., 2003) . another early determinant of nsep work in china was the existing commercial availability of injection equipment in chinese pharmacies (thomas, 2005) . the fact that most nsep provided free injection equipment to clients conflicted directly with the interests of pharmacies which were an alternative access point. "needle social marketing" projects were developed as a compromise to allow idu to bring used needles in exchange for vouchers for new needles at participating pharmacies, thus allowing nsep funds to subsidise pharmacies for needles they provided to idu. this pharmacy-based intervention strategy also shifted the debate away from the controversy of knowingly providing needles for injecting controlled substances and instead emphasised the opportunities for behavioural intervention created by the peer outreach components of nsep (ihrd, 2008; state council, people's republic of china, 2006b; watters et al., 1994) . it would not be for another four years, however, that ncaids conducted the first scientific evaluation of needle social marketing, in which the largely favourable results were used to inform the development of national guidelines on nsep (ihrd, 2008; state council, people's republic of china, 2006b; . the legislation surrounding needle exchange in china therefore emerged ex post facto, following the demonstrated success of programmes that had operated in the legal void preceding formal recognition. in contrast, the use of methadone for opiate substitution treatment was virtually unheard of in china until 2003 when ncaids decided to launch a pilot programme to investigate its potential as an official hiv control strategy (pang et al., 2007 ; united nations task force on injection drug use and hiv/aids, 2010). legalisation of government provision of a narcotic substance required determined lobbying by proponents to overcome factions opposed to mmt (hammett et al., 2008) . several factors facilitated this, including existing scientific research demonstrating the efficacy of substitution treatment in reducing drug-related crime in addition to reducing hiv transmission (gossop, trakada, steward, & witton, 2005; liu et al., 2007; wu et al., 2007b) , strong advocacy by public health 'champions,' (hammett et al., 2008) and hong kong's relatively successful experience with mmt programmes (newman & whitehill, 1979) . the fact that the scientific literature could demonstrate such programmes' ability to reduce drug-related crime allowed the public security sector to lend its support to mmt in a way they could not for nsep. furthermore inter-ministerial consensus was needed to pilot mmt because as a controlled substance methadone could only be distributed according to special measures dictated by relevant government agencies (ministry of health, ministry of public security of china, & sfda, 2006) . the fact that the most contentious debates surrounding mmt were overcome before piloting began, established an early precedent for policy consensus amongst stakeholders and provided a consistent policy environment for later scale-up. conflicting attitudes between public health and public security authorities regarding harm reduction are hardly unique to china (thomas, 2005) . however, its centralised policy making process and decentralised implementation process make consensus amongst the top leadership uniquely important for advancing new policies. the division of labour amongst the various departments overseeing harm reduction programmes further complicates the implementation process: moh and sfda oversee all blood testing and distribution of drugs and medical supplies. mps operates all mandatory detoxification centres where individuals deemed addicted to drugs serve compulsory "treatment" terms, and the ministry of justice (moj) operates separate detention centres for individuals deemed 'addicts', who serve out longer sentences under the 2008 drug prohibition law state council, people's republic of china, 2007) . diverging goals amongst the responsible government agencies also drive decentralisation: whereas public health practitioners prioritise reducing the risk of infection from blood borne diseases amongst idu, public security authorities are charged with enforcing laws against the sale and use of illicit drugs . as a result public security authorities have a more favourable view of mmt programmes, which are consistent with their stated aims of treating drug addiction as well as reducing demand for illicit drugs and incidence of drug-related crimes. in contrast they feel that nsep address neither of these aims (lurie & drucker, 1997; wu et al., 2007a) . these distinct views are reflected in the organisational structures of each programme. whereas mmt clinics are operated by the government and jointly overseen by moh, mps, and sfda, nsep are implemented solely by district level health bureaus (the local branches of the moh) and non-government organisations (ihrd, 2008) . public security officials admit confusion regarding their stance on nsep; a member of the yunnan provincial public security bureau stated in 2009 that: . . .there is a conflict between the laws that we as public security must follow and the regulation about hiv prevention. there is confusion as to what to follow in practice but it is important at the end to uphold the law (reid & aitken, 2009 ). this confusion has resulted in police forces that tolerate some needle exchange activities whilst simultaneously using these sites to target active users for arrest. nsep staff and participants frequently report being arrested or harassed near or around their exchange stations (souder, 2004) , and as described by key informants working as volunteer or staff at nsep, secondary needle exchangers sometimes experience being tracked by police who follow them to find idu for arrest (cohen & amon, 2008; human rights watch, 2008) . whilst alleged police harassment has been reported by mmt clinic attendants, key informants who work as staff at these clinics described past instances of engaging in negotiations with their counterparts in the local psb to request that police not target their clients for arrest. as several of the staff pointed out, mmt clients are far more defensible to the police because of the undisputed legality of taking methadone; whereas nsep clients are assumed to be active users of heroin, a behaviour which is legally forbidden. the difficulty in tracking the disease status of current idus has contributed to debates regarding the impact of harm reduction interventions on hiv incidence. (degenhardt et al., 2011; grulich & wilson, 2010; ihrd, 2008; montaner et al., 2010; palmateer et al., 2010; reid & aitken, 2009; strathdee, 2004; thomas, 2005) . even in well conducted cohort studies, self-selection bias, differential loss to follow up, and confounding by other prevention programmes, pose significant threats to validity. in addition, the political nature of harm reduction has rendered research funding for such programmes highly unreliable, further hindering effective and long-term evaluation studies that could otherwise justify their cost effectiveness. this is particularly the case in china where respect for scientific evidence and dependence on performance indicators for public official promotions make programme evaluations central to the policy making process (wu et al., 2007b) . the evaluation challenges specific to mmt and nsep reflect the fundamental differences in their operations. according to interviews with key informants at mmt clinics, to ensure the legality of government administered methadone, the national guidelines for mmt in china require the regular monitoring of clinic participants as well as mandatory testing for opiate use and annual disease monitoring for hiv, syphilis and hcv. daily contact between methadone patients and mmt staff, inherent to clinic operations, allows for the collection of large amounts of data necessary for monitoring and evaluation. moreover, routine testing of mmt clinic attendees can yield incidence rates of blood borne disease, which is the gold standard for evaluation of harm reduction programmes, in addition to measures of its impact on the illicit drug trade. a national working group evaluation report estimated that in 2008 alone, 3377 new hiv infections had been averted, 16.5 fewer tonnes of heroin had been consumed and that the illegal trade of heroin was reduced by 607 billion chinese yuan (yin, 2009) . such reports provide a shared platform on which hiv prevention specialists and public security authorities can align their goals. in contrast, national guidelines for nsep in china require tracking of only two indicators: needle return rate (calculated as number of needles collected divided by the number distributed per time period) and the number of idu accessed through outreach activities in a given amount of time (china cdc, 2008) . ideally nsep would also collect information to capture changes in hiv transmission patterns amongst participants, but this would require active tracking of nsep clients and regular hiv testing, a task currently only available through china cdc certified testing sites that require personal identification. self-identification in association with needle exchange activity places participants at risk of arrest and undermines a central nsep tenet of participant anonymity. as a result, existing reports on disease outcomes amongst nsep participants have been limited to cross sectional surveys utilising complex methods (china cdc, 2008; united states centre for disease control [us cdc], 2006) . other variables may proxy for measures of programme efficacy, such as participants' self-reported disease status or aids knowledge, attitudes, and behaviours (kab) however, true leverage over china's policy makers may lie in what is perceived as scientific evidence of reduced hiv transmission without a commensurate rise in illicit drug use. future evaluations of nsep effects on hiv transmission may benefit from alternative observational designs such as ecological studies in which hiv incidence rates in regions with and without nseps can be used to quantify the potential effects of such programmes, though such designs must carefully control for potential confounders that may distort measures of programme effect on the outcome. the ability of local government programmes to generate extrabudgetary revenue can often be a strong predictor of programme survival in china. this is due to the model of local self-reliance adopted during the economic liberalisation of the 1980 in which provincial governments were deliberately cut off from central support as a way to encourage optimisation of regional resources for economic growth (us cdc, 2006) . though an effective economic policy, such models have rendered many social services such as primary healthcare and education unaffordable for many chinese, who must pay ever increasing fees. the fact that mmt clinics generate revenue by charging patients up to 10 rmb (about usd 1.60) per daily dose of methadone is significantly different from nsep which do not provide tangible project returns. whilst both types of programmes currently receive budgetary support from central agencies, a lack of financial sustainability for nsep has not earned it any more support amongst central policy makers or local implementing officials. the most revealing pronouncement to date on the future of harm reduction in china was the second five-year action plan of 2006, which specified target coverage rates for both mmt and nsep by 2011 (state council, people's republic of china, 2006a) . most of the support for harm reduction measures still comes from the national level ministries, who ultimately play only a symbolic role over nsep and mmt programmes and whose participants still face interference from local law enforcement agencies. nonetheless, the new national drug prohibition law passed in 2008 made explicit mention of harm reduction for the first time by specifying "medicinal drug treatment" (a reference to mmt) as an acceptable form of drug treatment, further solidifying crucial support from the public security sector. partly in response to these shifts, experts at ncaids confirmed that the organisation has begun to advocate a complementary scheme in which mmt will be concentrated in urban areas with nsep operating more in remote rural areas. whilst this approach might maximise idu coverage, it misses opportunities to strengthen and improve china's overall harm reduction programme, ideas for which are provided below. referral systems to link idu encountered through nsep outreach efforts have been proposed as a way to integrate the two programmes. the nsep do not require personal identification and can deliver services in the community and are therefore effective at accessing active idu and difficult to reach populations who may for various reasons be ineligible or unwilling to access mmt. a referral system could also encourage harmonisation of data collection systems and improve overall knowledge of idu behaviours. one limitation of an integrated system, however, would be the eligibility for mmt programme participation, which requires that individuals serve at least one term in a compulsory isolated drug treatment centre. since this would limit referral services only to those idu who have ever been arrested, the authors also recommend that mmt programme eligibility be extended to all voluntary participants who are deemed by a licenced physician at the mmt clinic to be suffering from opioid addiction. in addition, public security officials must be forbidden from access to patient information collected by mmt clinics in order to grant patients assurance that their personal security will not be threatened as a result of mmt attendance. in order to conduct evaluation capable of generating political support, distribution of rapid hiv test kits to nsep staff could provide a way for them to obtain data on key biomarkers such as hiv whilst preserving values such as client anonymity. the chinese cdc has not approved these test kits for large scale testing because of inconsistent findings regarding test sensitivity and specificity (us cdc, 2008 , and in order to minimise missed opportunities for post-test counselling and treatment referral, which is standard in the existing vct system. as a method of primary screening and rapid surveillance work, however, oral tests are gaining credibility in the public health community to access mobile or underground populations because they can be conducted on-site and produce immediate test results. in the event that such test kits come into practice amongst nsep in china, however, monitors will need to exercise caution so as not to minimise the self-selection bias that drove the debate over the vancouver needle exchange study (bruneau et al., 1997; schetcher et al., 1999; strathdee et al., 1997) . if nsep attendees are systematically higher risk idu as in the case of the vancouver study, if baseline rates of needle sharing are already high, and if exposure to hiv interventions is considered fixed for the duration of the study, multivariate model derived estimates may fail to control for residual confounding of the relationship between nsep use and hiv status. careful study design and unambiguous interpretation of findings will be essential for meaningful evaluation of nsep. finally, greater advocacy for nsep from supporters within the government and non-government sectors will be critical for building cross-sector support for nsep. pre-requisites will include the development of more independent and participatory civil society organisations working in needle exchange, a formal policy mechanism for nsep, similar to the mmt national working group, and close collaboration between civil society and ngos to advocate for nsep. in 2008 ncaids oversaw the operation of 897 nsep operating in 26 provinces, servicing an estimated 36,000 idu a month. this large-scale programming may signal shifting priorities in public health as practitioners adapt practices to the situation on the ground. such policy changes will play an important role, not only for the expansion of harm reduction programmes in china, but to build institutional capacity for carrying out more communitybased, participatory work for the control and prevention of drug use related hiv transmission. china has already earned international credibility as a world leader in mmt. consideration of the policies suggested could further improve what is already a dynamic and far-reaching harm reduction response to hiv amongst drug users in the world's most populous country. systematic review of hiv and hcv infection among drug users in china high rates of hiv infection among injection drug users participating in needle exchange programmes in montreal: results of a cohort study chinese centre for disease control and prevention health and human rights concerns of drug users in detention in guangxi province mortality among regular or dependent users of heroin and other opioids: a systematic review and meta-analysis of cohort studies china's muslims awake to nexus of next of needles and aids china and hiv -a window of opportunity clinton supports needle exchanges but not funding. washington post reduction in criminal convictions after addiction treatment: 5-year follow-up modelling emerging hiv epidemics: the role of injecting drug use and sexual transmission in the russian federation, china and india is antiretroviral therapy modifying the hiv epidemic? changes in injection risk behaviour associated with participation in the seattle needle-exchange programme development and implementation of a cross-border hiv prevention intervention for injection drug users in ning ming county (guangxi province), china and lang son province law enforcement influences on hiv prevention for injection drug users: observations from a cross-border project in china and vietnam hiv prevention for injection drug users in china and vietnam: policy and research considerations social evils' and harm reduction: the evolving policy environment for human immunodeficiency virus prevention among injection drug users in china and vietnam readiness for cessation of drug use among recent attenders and nonattenders of a needles exchange programme an unbreakable cycle: drug dependency treatment, mandatory confinement and hiv/aids in china's guangxi province new york: international harm reduction development program of china turns drug rehab into a punishing ordeal an evaluation of needle exchange programmes in china an opportunity lost: hiv infections associated with lack of a national needle-exchange programme in the usa who need protecting? rethinking hiv, drugs, and security in the china context. china and eurasia forum quarterly the efficacy of methadone maintenance interventions in reducing illicit opiate use, hiv risk behaviour and criminality: a meta-analysis association of highly active antiretroviral therapy coverage, population viral load, and yearly new hiv diagnoses in british columbia, canada: a population-based study official notice on drug treatment programme for medicinal maintenance treatment programme for opiate addicted individuals, joint document issued by ministry of health, ministry of public security, and state food and drug administration. national centre for aids/std control and prevention double-blind comparison of methadone and placebo maintenance treatment of narcotic addicts in hong kong preventing hiv transmission: the role of sterile needles and bleach countries with injection-driven hiv epidemics evidence for the effectiveness of sterile injecting equipment provision in preventing hepatitis c and human immunodeficiency virus transmission among injecting drug users: a review of reviews effectiveness of first eight methadone maintenance treatment clinics in china advocacy for harm reduction in china: a new era dawns enough sterile syringes to prevent hiv transmission among injection drug users in montreal potential impact of hiv among idu on heterosexual transmission in asian settings: scenarios from the asian epidemic model do needle exchange programmes increase the spread of hiv among injection drug users? an investigation of the vancouver outbreak souder to nih: harm reduction causes harm national narcotics law of the people's republic of china china hiv/aids prevention & control action plan china hiv/aids prevention & control action plan hiv/aids prevention and care regulations alive team letter to nih. drug policy alliance needle exchange is not enough: lessons from the vancouver injecting drug use study rapid scale up of harm reduction in china harm reduction policies and programmes for persons involved in the criminal justice system china country advocacy brief. beijing, china. united states centre for disease control syringe and needle exchange as hiv/aids prevention for injection drug users do needle syringe exchange programmes reduce hiv infection among injection drug users: a comprehensive review of the international evidence. substance use and misuse evaluation of a needle social marketing strategy to control hiv among idus in china evolution of china's response to aids harm reduction in china. presentation at the unaids theme group meeting on key: cord-297257-lzybfwc2 authors: savarino, andrea; shytaj, iart luca title: chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids date: 2015-06-18 journal: retrovirology doi: 10.1186/s12977-015-0178-0 sha: doc_id: 297257 cord_uid: lzybfwc2 the restoration of the immune system prompted by antiretroviral therapy (art) has allowed drastically reducing the mortality and morbidity of hiv infection. however, one main source of clinical concern is the persistence of immune hyperactivation in individuals under art. chronically enhanced levels of t-cell activation are associated with several deleterious effects which lead to faster disease progression and slower cd4(+) t-cell recovery during art. in this article, we discuss the rationale, and review the results, of the use of antimalarial quinolines, such as chloroquine and its derivative hydroxychloroquine, to counteract immune activation in hiv infection. despite the promising results of several pilot trials, the most recent clinical data indicate that antimalarial quinolines are unlikely to exert a marked beneficial effect on immune activation. alternative approaches will likely be required to reproducibly decrease immune activation in the setting of hiv infection. if the quinoline-based strategies should nevertheless be pursued in future studies, particular care must be devoted to the dosage selection, in order to maximize the chances to obtain effective in vivo drug concentrations. the quest for clinical candidates to counteract immune activation has become a "hot topic" in aids research, because hiv infection is characterized by malignant immune hyperactivation which correlates with disease progression and poor response to antiretroviral therapy (art) [1] [2] [3] [4] [5] . moreover, immune hyperactivation is also regarded as a major obstacle to a cure for aids [6] . in the beginning of the millennium, an article authored by one of us launched chloroquine as a tool to inhibit viral replication and the related malignant immune activation associated with some viral diseases [7] . this article sparked a new wave of studies, in that it extended a theory, previously designed for hiv/aids [8] , to other viral diseases characterized by excessive immune activation. as will be discussed below, by accumulating in the acidic organelles, chloroquine exerts both direct antiviral effects on enveloped viruses and decreases activation of several cell types involved in the immune response. chloroquine has since shown promise in preclinical studies (both in vitro and in vivo), as a therapeutic agent against emerging viruses such as mers cov [9] . of note, chloroquine has been indicated as a promising candidate for filovirus treatment [10] , especially during the latest ebola epidemic [11, 12] . in two studies out of three, chloroquine showed antiviral activity in mice at the maximum tolerated dose [10, 13, 14] , thus rendering this drug an interesting agent for further testing of combination anti-ebola therapies. however, the effects of chloroquine and its hydroxyl analogue hydroxychloroquine, on hiv infection, i.e. the initial target for the repurposing of these drugs, have remained controversial. on the one hand, based on the results of some earlier clinical trials, chloroquine/hydroxychloroquine has been recently resuggested as a promising candidate to restrict the hivrelated immune activation [15, 16] . on the other hand, the results from the latest clinical trials indicate that chloroquine/hydroxychloroquine has no beneficial effect on immune activation [17, 18] . we here provide a state of the art of the studies investigating the use of chloroquine/hydroxychloroquine as a therapeutic tool for hiv/aids and suggest the possible biological grounds for the clinical results obtained. moreover, we describe the reasons why our group decided to proceed further with strategies based on another drug, i.e. auranofin, which shares with chloroquine an antirheumatic effect [19] . several reviews have recently been published on immune activation in hiv infection [6, 16, 20, 21] . briefly, immune hyperactivation, commonly measured as the expression levels on peripheral blood lymphocytes of markers such as hla-dr, cd38, or cd69 correlates with, and also predicts, disease progression (reviewed in [22, 23] ). immune activation gradually decreases following therapy initiation [24] and is maintained high in immunological non-responders, who are individuals maintaining low cd4 counts despite prolonged exposure to art [3, 4] . while the initial studies were focused on the relationship between disease progression and activation of cd8 + t-cells [1] , later studies better concluded that there is a broader relationship between disease progression and immune hyperactivation, involving also cd4 + t-cells [5, 25] and innate immunity [26] . immune activation and viral replication are believed to be mutually enhanced in a vicious circle. the virus, recognized by the immune system as non-self, induces immune activation, which, in turn, fuels viral replication by furnishing to the virus material to synthesize the different viral components. for example, lymphocyte activation increases the cytoplasmic levels of deoxyribonucleotides necessary for viral dna synthesis by reverse transcriptase [27] . this vicious circle may still persist in anatomical compartments incompletely penetrated by art. hiv-induced immune activation is not limited to specific immunity, but exerts its effects on innate immunity as well. hiv-1 was shown to activate plasmacytoid dendritic cells (pdcs), which, differently from myeloid dendritic cells (the most potent antigen-presenting cells in the body), induce innate antimicrobial immunity by producing type i interferons ( figure 1 ) [26] . pdcs internalize hiv-1 through viral envelope/cd4 interactions, and the internalized virus activates these cells mainly through toll-like receptor 7 (tlr-7) signaling ( figure 1 ). comparative pathology corroborates the hypothesis that over-stimulation of this pathway may be associated with deleterious effects. sooty mangabeys (cercocebus atys), which can be infected by a simian homolog of hiv (i.e. simian immunodeficiency virus, siv) but do not develop aids, display weak ifn-α production upon stimulation with tlr-7 antagonists [28] . on the contrary, rhesus macaques (macaca mulatta), which do progress to aids, produce high amounts of ifn-α when their pdcs are subjected to the same stimuli [28] . moreover, another species displaying nonpathogenic siv infection, i.e. the african green monkey (chlorocebus aethiops), is characterized by an efficient control of ifn-α production following acute infection [29] . pdcs decrease in peripheral blood during progression to aids, because, upon activation, they migrate to the lymphoid tissue [30] . as a huge number of cells reside in the gut-associated lymphoid tissue (galt), according to the microbial translocation theory, the intestinal mucosa damaged by the consequent inflammation may become permeable to products of the gut microbiome which further enhance hiv-related immune hyperactivation [31, 32] . finally, immune activation is one primary driver of both generation and maintenance of the viral reservoir, which is mainly constituted by latently infected, central and transitional memory cd4 + t-cells (henceforth t cm and t tm , respectively) [33] . also in this case, comparative pathology has provided clues for understanding this phenomenon. it was shown that cd4 + t cm cells from sooty mangabeys express, upon activation, low levels of ccr5, the main coreceptor for virus entry into cells, thus limiting infection of this important cellular compartment [34] . instead, activated t cm cells from aids-developing species, such as humans and rhesus macaques, up-regulate the levels of ccr5 to a higher extent than cells from sooty mangabeys, thus facilitating the generation of a consistent viral reservoir [34] . after these cells become quiescent, viral replication switches off, and latently infected, hiv-reservoir cells proliferate through low-level antigenic stimulation (t cm ) or il-7-driven homeostatic proliferation (t tm ) [33] . both processes are enhanced by generalized immune activation. multiple in vitro effects of chloroquine could support its possible use as a modulator of immune activation in hiv/aids: 1. chloroquine and its hydroxyl analogue hydroxychloroquine were shown in several studies to inhibit hiv-1 replication (reviewed in: [7] ). the effects of these quinolines, mainly due to the induction of a defect in the maturation of the viral envelope glycoprotein gp120 [35, 36] , might mimic the effects of broadly neutralizing antibodies directed against the viral envelope, although the effects of these antibodies are weaker than those directed against the cd4binding site [37] . these effects are additive to those of non-nucleosidic reverse transcriptase inhibitors (nnrtis) and synergistic to those of protease inhibitors (pis) [38] . as quinoline drugs accumulate in lymphoid tissues [39] , they might decrease ongoing viral replication during art in anatomical sanctuaries and, consequently switch off one of the main drivers of immune activation. chloroquine is also an inhibitor of p-glycoprotein (p-gp) and multidrug resistance proteins (mrps) [40, 41] , cell surface glycoproteins which extrude several antiretroviral drugs to the extracellular medium. in line with this evidence, chloroquine was shown to increase the intracellular levels of pis [38] . the effects of chloroquine in com-bination with nrtis are instead controversial: some reported an additive effect [42] , while others did not detect it [43] . the combined effects of chloroquine and integrase inhibitors are as yet unknown. 2. chloroquine accumulates in phagosomes of pdcs and inhibits their hiv-induced activation [44] . it might therefore impact on innate immunity-induced immune hyperactivation. 3. a recent study showed that hydroxychloroquine selectively induces apoptosis in the memory t-cell compartment (cd45ra − cd45ro + ) [45] . as, upon activation, naïve t-cells (cd45ra + cd45ro − ) acquire a cd45ra − cd45ro + phenotype, the "antimemory" effect should limit immune activation (figure 2 ) [46] . there is growing consensus that induction of apoptosis in the memory t-cell compartment might have a detrimental effect on the viral reservoir [47] [48] [49] . in this light, chloroquine/hydroxychloroquine should have an anti-reservoir potential. this view is supported by another recent study which shows that chloroquine sensitizes to apoptosis the latently infected cells upon viral reactivation, likely by removing the anti-apoptotic effect of the virus structural gag gene products [50] . these effects are potentially interesting, since it has been well demonstrated that viral reactivation from latency does not necessarily result in cell death [51] . the macaque aids model is an important tool for preclinical assessment of strategies aimed at treating hiv/ aids [52] . to our knowledge, chloroquine has been tested in this model on two occasions. in a first study, chloroquine (25 mg every other day for 30 days, i.e. a cumulative dosage comparable to that administered to humans with rheumatioid arthritis) was administered to three chinese rhesus macaques infected with the simian hiv-homologue, sivmac 251 [53] . although a decrease in activated pdcs was shown, no effects were seen on viral load and cd4 + and cd8 + t-cell activation (measured as cd38 expression) [53] . as the immune activation set point is established during acute infection [4] , vaccari et al. [54] treated with chloroquine (18.7 mg/day for 112 consecutive days) seven sivmac 251 -infected rhesus macaques during the viral load peak that characterizes acute infection. apart from an unexpected, although transient, increase in the comparison of the susceptibility to chloroquine/hydroxychloroquine and auranofin of the cellular subsets involved in hiv production and persistence. shown in the figure is a schematic depiction of a activation and b differentiation stages of cd4 + t-lymphocytes and their correlation with viral production, latency and viral reactivation. both chloroquine/hydroxychloroquine and auranofin can influence these transitions by exerting a pro-apoptotic effect, the efficacy of which is graphically exemplified by the intensity of the blue color in the corresponding rectangles. efficacy gradients are based on data derived from refs. [45, 48, 50] . expression of interferon-regulated genes (perhaps not population-relevant as possibly driven by only one animal), no significant differences were reported in viral load and t-cell activation and proliferation (measured as expression of cd69 and ki67, respectively) [54] . a trend was however noticed for maintenance of decreased levels of ki67, cd69 and ccr5 in the gut of the chloroquinetreated animals, although the differences with values from the control group did not reach statistical significance. the effect of chloroquine in this simian model in the presence of art is still unknown. chloroquine and hydroxychloroquine have so far been tested in several hiv clinical trials. the results summarized in figure 3 support the hypothesis that the chloroquine/hydroxychloroquine dosage may be an important driver of at least partial clinical success. suppressive effects on immune activation by chloroquine were shown in the trial conducted by murray et al. [55] . however, in this trial, the dosage administered was not the same for all individuals, some of them receiving 500 mg/die instead of 250 mg/die. it is thus possible that the statistical significance of the effects reported in this study was driven by the higher dosage of the drug. this view is supported by a later study which tested chloroquine at 250 mg/die and failed to show any effect of the drug [18] . in two clinical trials conducted in the 1990s, sperber et al. reported suppressive effects on immune activation (measured at that time as il-6 production) and viral load in individuals treated with 800 mg of hydroxychloroquine/day (bioequivalent to 500 mg/day of chloroquine) [56, 57] . the other clinical trials testing hydroxychloroquine at a lower dosage (i.e. 400 mg/day) led to conflicting results. earlier studies [58, 59] and the more recent study of piconi et al. [60] reported significant effects on viral load [58] , cd4 counts [59] , and immune activation. [60] . instead, a more recent clinical trial, randomized and double blind, showed disappointing results, even hinting at possibly deleterious effects of hydroxychloroquine on viral load and cd4 counts [17] . this trial was conducted in the absence of art, and this might explain differences between this study and the study of piconi et al., which was conducted on individuals under art [60] . another trial in art-treated patients is currently ongoing and will provide more information on the effects of hydroxychloroquine (clinicaltrials.gov identifier: nct01232660). the hydroxychloroquine levels show high inter-subject variability and, although individuals receiving the higher hydroxychloroquine dosages (800 and 1,200 mg/day) also showed significantly higher blood levels of the drug than those receiving 400 mg/die, the range of the blood concentrations was in part overlapping in the different dosage groups [61] . chloroquine has similar pharmacokinetics [62] ; therefore, not only the dosage but also individual differences in drug metabolism and distribution may explain the different conclusions of the aforementioned studies. a large clinical trial has recently been completed (clinicaltrials.gov identifier: nct00819390) and its results can help to better represent the response of a population, thus abolishing the bias due to limited sample size. in this trial, however, chloroquine has been tested at 250 mg/day in the absence of art; thus, in light of the results of the aforementioned clinical trials and considerations derived from basic science (see next paragraph), it is not surprising that the preliminary results released so far for this trial (https://clinicaltrials.gov/ct2/ show/nct00819390) do not show any significant effect of chloroquine on immune activation, viral load and cd4 counts. chloroquine/hydroxychloroquine-treated individuals display blood concentrations that are highly variable and only rarely exceed 10 or 20 µm, respectively [61, 62] . therefore, at the steady state levels, these blood concentrations only in part overlap those at which a therapeutic effect is expected. for example, the ec 50 of chloroquine on pbmc proliferation upon activation is, in general, ≥10 µm [63] , and this value can explain the varying results obtained in the different clinical trials, with clearer effects associated with the higher drug dosages. similarly, the pro-apoptotic effect of hydroxychloroquine on the memory t-cells is only moderate at the concentrations reachable in blood, especially in the lower range [45, 61] . the pro-apoptotic effect of chloroquine described by li et al. on latently infected cells upon viral reactivation is instead more marked, although still partial, at the upper range of clinically achievable blood concentrations (5-10 µm) [50] . this effect could therefore be visible in vivo in terms of viral reservoir reduction, but only treating with high chloroquine dosages in the presence of suppressive art. moreover, to maximize the chances to obtain viral reservoir reduction in vivo, chloroquine treatment should be prolonged, as the events of virus reactivation from latency are rather rare (estimated as one event of transition from latency to productive infection every 10 ml of blood each day) [64] . the effect of chloroquine on pdc activation (see figure published clinical studies evaluating the effects of chloroquine/hydroxychloroquine administration, alone or in combination with other drugs, in hiv infected subjects. highlighted in blue, red or white are the studies that have reported a positive, negative, or neutral outcome of the therapy respectively. cq chloroquine, hcq hydroxychloroquine. [65] . in this case, the in vitro effect is in line with the results of two in vivo studies [53, 60] . the use of chloroquine-related compounds with increased potency is yielding promising results in vitro [66] , and it will be interesting to test the best-performing candidates in the simian aids model. the effects of chloroquine/hydroxychloroquine on viral replication have been repeatedly shown in vitro at lower drug levels than those inducing the cellular effects [35, 36, 63, 65] . the blood concentration/ec 50 ratio is however much narrower than those shown by antiretroviral drugs [63] . the antiretroviral effects of chloroquine/hydroxychloroquine may though become visible in anatomical sanctuaries of those individuals treated with pi-containing antiretroviral regimens. in any case, we recommend that chloroquine/hydroxychloroquine be tested at the highest recommended dosages in future hiv clinical trials. alternative/complementary interpretations of the results so far obtained are possible. for example, the effectiveness of the art regimen employed may play a role in determining the magnitude of the effects (if any) observed following chloroquine/hydroxichloroquine addition. the study of piconi et al. [60] , showing some benefit in immunological non responders, may indicate that the effects of chloroquine may be visible only in some subsets of individuals with peculiar immunological characteristics, and that these effects can be hindered when immunologically non homogeneous cohorts are studied. in this regard, larger studies, with cohorts stratified according to immunological responsiveness to art, could provide further information on the effects of chloroquine/hydroxychloroquine. another open question remains the influence of the duration of drug exposure, as it has been shown that chloroquine/hydroxychloroquine has cumulative effects [67] . as a proportion of hiv-infected patients in africa may already be on chloroquine medication to prevent malaria, it might be worth examining the long-term effects of this treatment. in this regard, an ongoing phase iii clinical trial will assess the long-term effects of chloroquine and trimethoprim-sulfamethoxazole phrophylaxis on survival and disease control in hiv-infected individuals with suppressed viral load and good clinical response to art [68] . given the aforementioned problems in the pharmacokinetics of chloroquine/hydroxychloroquine, our group chose to follow a different, yet partly similar, approach to corroborate treatment of hiv/aids. based on the feedback received from basic science studies and clinical trials that have been published throughout the years, we decided to use drugs the desired effects of which be striking in vitro at concentrations lower than the trough plasma concentrations in vivo. we also decided to redirect our research on the basis of the plasma concentrations rather than on whole-blood concentrations (widely used for chloroquine/hydroxychloroquine), because we thought that the former might better mimic the tissue culture concentrations. the drug that we selected is the gold-based compound auranofin, the pharmacodynamics and pharmacokinetics of which are well known, due to its decade-long employment for treatment of rheumatoid arthritis [69] . the main rationale for the use of auranofin in our studies was its ability to target the central/transitional memory cd4 + t-cell compartment ( figure 2 ) [48, 70] , which is known to harbor the main viral reservoir in patients receiving art [33] . auranofin is drastically active at submicromolar (i.e. ≤250 nm) concentrations, which are below those readily achievable in human plasma [71] . the administration of auranofin ultimately led to a reduction of the viral reservoir in art-treated sivmac251-infected macaques [70] . a review on our preclinical studies has recently been published [46] and the reader is addressed to it for further detail. not surprisingly for a drug effective against an autoimmune disease such as rheumatoid arthritis, auranofin may as well be beneficial in terms of reduction of cell activation. in particular, the downregulation of the cd28 molecule induced by auranofin can disrupt the co-stimulatory signal often crucial for lymphocyte activation [48] . moreover, apart from memory cd4 + t-cells, auranofin also targets the memory cd8 + t-cell compartment [48] , i.e. a cellular subset known to be hyperactivated during hiv infection [2] . interestingly, as described for hydroxychloroquine [60] , auranofin was shown to disrupt in various cell lines the tlr-4 signaling [72] , which is activated by bacterial lipopolysaccharides and likely constitutes another source of immune hyperactivation. in vitro data indicate that the impact of auranofin on lymphocyte activation may be mediated, at least in part, by modulation of oxidative stress [48] . of note, the addition of a potent pro-oxidant drug, such as buthionine sulfoximine (bso), increases the potency of auranofin, decreasing phytohemagglutinin-induced activation and expression of the α-chain of the il-2 receptor [73] . this is in line with our preliminary data in sivmac251-infected macaques, in which a combined regimen of art, auranofin and bso induced a functional cure-like condition following suspension of all therapies [74] . these observations provide proof of concept that drastically decreasing immune hyperactivation arrests siv disease progression and turns the virus/immune system balance in favor of the latter. clinical trials will be required to assess the potential of auranofin to decrease immune activation in art-treated subjects. finally, other drugs used or proposed for treatment of rheumatoid arthritis might find a place in the treatment of hiv/aids. for example, the janus kinase inhibitors tofacitinib and ruxolitinib have shown a promising in vitro activity against hiv replication [75] . the ongoing in vivo studies on these compounds could provide an opportunity to analyze the effects of this treatment on viral replication and immune activation. as and ils contributed to the ideas and to the interpretation of the data presented in the manuscript. as and ils contributed to manuscript drafting and preparation of the figures. both authors read and approved the final manuscript. cd8+ lymphocyte activation at human immunodeficiency virus type 1 seroconversion: development of hla-dr+ cd38− cd8+ cells is associated with subsequent stable cd4+ cell levels. the multicenter aids cohort study group increased numbers of primed activated cd8+ cd38+ cd45ro+ t cells predict the decline of cd4+ t cells in hiv-1-infected patients t cell activation is associated with lower cd4+ t cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy immune activation set point during early hiv infection predicts subsequent cd4+ t-cell changes independent of viral load relationship between t cell activation and cd4+ t cell count in hiv-seropositive individuals with undetectable plasma hiv rna levels in the absence of therapy immune activation and hiv persistence: considerations for novel therapeutic interventions effects of chloroquine on viral infections: an old drug against today's diseases? the potential place of chloroquine in the treatment of hiv-1-infected patients screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture a systematic screen of fda-approved drugs for inhibitors of biological threat agents ebola virus (ebov) infection: therapeutic strategies antiviral therapies against ebola and other emerging viral diseases using existing medicines that block virus entry [v1; ref status: awaiting peer review evaluation of ebola virus inhibitors for drug repurposing lack of protection against ebola virus from chloroquine in mice and hamsters immune responses during spontaneous control of hiv and aids: what is the hope for a cure persistent immune activation in chronic hiv infection: do any interventions work? hydroxychloroquine trial team: effects of hydroxychloroquine on immune activation and disease progression among hiv-infected patients not receiving antiretroviral therapy: a randomized controlled trial assessment of chloroquine as a modulator of immune activation to improve cd4 recovery in immune nonresponding hiv-infected patients receiving antiretroviral therapy comparison of phenytoin with auranofin and chloroquine in rheumatoid arthritis-a double blind study immune activation and hiv persistence: implications for curative approaches to hiv infection hiv-associated chronic immune activation residual immune dysregulation syndrome in treated hiv infection biomarkers of immune dysfunction following combination antiretroviral therapy for hiv infection positive effects of combined antiretroviral therapy on cd4+ tcell homeostasis and function in advanced hiv disease shorter survival in advanced human immunodeficiency virus type 1 infection is more closely associated with t lymphocyte activation than with plasma virus burden or virus chemokine coreceptor usage endocytosis of hiv-1 activates plasmacytoid dendritic cells via toll-like receptor-viral rna interactions low levels of deoxynucleotides in peripheral blood lymphocytes: a strategy to inhibit human immunodeficiency virus type 1 replication divergent tlr7 and tlr9 signaling and type i interferon production distinguish pathogenic and nonpathogenic aids virus infections nonpathogenic siv infection of african green monkeys induces a strong but rapidly controlled type i ifn response impaired restoration of plasmacytoid dendritic cells in hiv-1-infected patients with poor cd4 t cell reconstitution is associated with decrease in capacity to produce ifn-alpha but not proinflammatory cytokines residual immune dysregulation syndrome in treated hiv infection microbial translocation in the pathogenesis of hiv infection and aids hiv reservoir size and persistence are driven by t cell survival and homeostatic proliferation low levels of siv infection in sooty mangabey central memory cd4+ t cells are associated with limited ccr5 expression inhibition of human immunodeficiency virus infectivity by chloroquine effect of chloroquine on reducing hiv-1 replication in vitro and the dc-sign mediated transfer of virus to cd4+ t-lymphocytes llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (hiv-1)-neutralizing properties and high affinity for hiv-1 gp120 quinoline antimalarials as investigational drugs for hiv-1/aids: in vitro effects on hiv-1 replication, hiv-1 response to antiretroviral drugs, and intracellular antiretroviral drug concentrations short communication: preferential concentration of hydroxychloroquine in adenoid tissue of hiv-infected subjects the effect of lysosomotropic agents and secretory inhibitors on anthracycline retention and activity in multiple drug-resistant cells reversal of mrp-mediated doxorubicin resistance with quinoline-based drugs antiretroviral treatment influence of quinacrine and chloroquine on the in vitro 3′-azido-3′-deoxythymidine antiretroviral effect chloroquine modulates hiv-1-induced plasmacytoid dendritic cell alpha interferon: implication for t-cell activation hydroxychloroquine preferentially induces apoptosis of cd45ro + effector t cells by inhibiting autophagy: a possible mechanism for therapeutic modulation of t cells a cure for aids: a matter of timing? maintenance of cd4+ t-cell memory and hiv persistence: keeping memory, keeping hiv a candidate anti-hiv reservoir compound, auranofin, exerts a selective 'antimemory' effect by exploiting the baseline oxidative status of lymphocytes plasmodium infection reduces the volume of the viral reservoir in siv-infected rhesus macaques receiving antiretroviral therapy gag-mediated autophagy promotes cd4 t cell survival: a possible mechanism for hiv reservoir persistence. xx international aids conference, towards an hiv cure symposium stimulation of hiv-1-specific cytolytic t lymphocytes facilitates elimination of latent viral reservoir after virus reactivation nonhuman primate models in aids research inhibitory effects of chloroquine on the activation of plasmacytoid dendritic cells in sivmac239-infected chinese rhesus macaques transient increase of interferon-stimulated genes and no clinical benefit by chloroquine treatment during acute simian immunodeficiency virus infection of macaques reduction of immune activation with chloroquine therapy during chronic hiv infection hydroxychloroquine treatment of patients with human immunodeficiency virus type 1 comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type 1 hydroxychloroquine, hydroxycarbamide, and didanosine as economic treatment for hiv-1 hydroxychloroquine, hydroxyurea and didanosine as initial therapy for hiv-infected patients with low viral load: safety, efficacy and resistance profile after 144 weeks hydroxychloroquine drastically reduces immune activation in hiv-infected, antiretroviral therapy-treated immunologic nonresponders hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis chloroquine levels in blood during chronic treatment of patients with rheumatoid arthritis anti-hiv effects of chloroquine: mechanisms of inhibition and spectrum of activity modeling latently infected cell activation: viral and latent reservoir persistence, and viral blips in hiv-infected patients on potent therapy inhibition of human immunodeficiency virus type 1 replication by hydroxychloroquine in t cells and monocytes modulation of hiv-1-induced activation of plasmacytoid dendritic cells by 6-desfluoroquinolones hydroxychloroquine and chloroquine retinopathy: a systematic review evaluating the multifocal electroretinogram as a screening test immune suppression by myeloid cells in hiv infection: new targets for immunotherapy auranofin versus placebo in rheumatoid arthritis gold drug auranofin restricts the viral reservoir in the monkey aids model and induces containment of viral load following art suspension gold levels produced by treatment with auranofin and sodium aurothiomalate auranofin, as an anti-rheumatic gold compound, suppresses lps-induced homodimerization of tlr4 the interaction of auranofin and buthionine sulfoximine blocks activation of human peripheral t lymphocytes investigational treatment suspension and enhanced cell-mediated immunity at rebound followed by drug-free remission of simian aids ruxolitinib and tofacitinib are potent and selective inhibitors of hiv-1 replication and virus reactivation in vitro submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www ils is supported by a fellowship from the "sapienza" university (rome, italy). the istituto superiore di sanità holds a us patent on the use of the auranofin for treatment of hiv/aids. as is the leading inventor of the patent. key: cord-293857-o8rlqsq5 authors: ghosh, arun k.; brindisi, margherita title: organic carbamates in drug design and medicinal chemistry date: 2015-01-07 journal: j med chem doi: 10.1021/jm501371s sha: doc_id: 293857 cord_uid: o8rlqsq5 [image: see text] the carbamate group is a key structural motif in many approved drugs and prodrugs. there is an increasing use of carbamates in medicinal chemistry and many derivatives are specifically designed to make drug–target interactions through their carbamate moiety. in this perspective, we present properties and stabilities of carbamates, reagents and chemical methodologies for the synthesis of carbamates, and recent applications of carbamates in drug design and medicinal chemistry. carbamate-bearing molecules play an important role in modern drug discovery and medicinal chemistry. organic carbamates (or urethanes) are structural elements of many approved therapeutic agents. structurally, the carbamate functionality is related to amide-ester hybrid features and, in general, displays very good chemical and proteolytic stabilities. carbamates are widely utilized as a peptide bond surrogate in medicinal chemistry. this is mainly due to their chemical stability and capability to permeate cell membranes. another unique feature of carbamates is their ability to modulate inter-and intramolecular interactions with the target enzymes or receptors. the carbamate functionality imposes a degree of conformational restriction due to the delocalization of nonbonded electrons on nitrogen into the carboxyl moiety. in addition, the carbamate functionality participates in hydrogen bonding through the carboxyl group and the backbone nh. therefore, substitution on the o-and n-termini of a carbamate offers opportunities for modulation of biological properties and improvement in stability and pharmacokinetic properties. carbamates have been manipulated for use in the design of prodrugs as a means of achieving first-pass and systemic hydrolytic stability. carbamate derivatives are widely represented in agricultural chemicals, such as pesticides, fungicides, and herbicides. they play a major role in the chemical and paint industry as starting materials, intermediates, and solvents. furthermore, organic carbamates serve a very important role as optimum protecting groups for amines and amino acids in organic synthesis and peptide chemistry. in recent years, carbamate derivatives have received much attention due to their application in drug design and discovery. however, there are hardly any reviews on this subject in the literature. in the present perspective, we plan to provide an overview of the leading role of organic carbamates in medicinal chemistry, with particular focus on therapeutic carbamates and carbamate-based prodrugs. in this context, we will highlight the chemical methodologies adopted for the synthesis of these carbamate derivatives. also, we will outline successful designs of organic carbamates, including a variety of cyclic ether-derived carbamates, as suitable amide bond surrogates leading to a wide range of novel organic carbamates as potent hiv-1 protease, βsecretase, serine protease, and cysteine protease inhibitors. this information may be useful in further design of carbamate-based molecules as drugs or prodrugs. peptide-based molecules are an important starting point for drug discovery, especially in the design of enzyme inhibitors. because of their high affinity and specificity toward biological functions, peptide-based molecules also serve as valuable research tools. however, the poor in vivo stability, inadequate pharmacokinetic properties, and low bioavailability have generally limited their broader utility. hence, a variety of peptide mimics are being developed to improve drug-like character along with increased potency, target specificity, and longer duration of action. 1−3 to this end, several classes of peptidomimetics are tailored by replacing the native amide bond with unnatural linkages 4−6 such as retro-amide, 7 urea, 8−12 carbamate, 13 and heterocycles 14,15 as peptide bond surrogates. these functionalities confer metabolic stability toward aminopeptidases, the enzymes involved in the metabolism of peptidelike drugs. the carbamate's emerging role in medicinal chemistry is also due to its chemical stability and to its capability to increase permeability across cellular membranes. these attributes of organic carbamates have been exploited in drug design. as a result, the carbamate motif is becoming the choice for peptide bond surrogates. other uses of carbamates are well-known. particularly, the employment of carbamates in various industries as agrochemicals, in the polymer industry, and also in peptide syntheses. 16−18 in addition, among the various amineprotecting groups, carbamates are commonly used to enhance their chemical stability toward acids, bases, and hydrogenation. 19 one important feature of organic carbamates is represented by the amide resonance. the amide resonance in carbamates has been studied in detail employing both experimental and theoretical methods by estimating the c−n bond rotational barriers. 20−25 the amide resonance in carbamates has been shown to be about 3−4 kcal mol −1 lower than those of amides, owing to the steric and electronic perturbations due to the additional oxygen. 26 three possible resonance structures (a, b, and c, figure 1 ) contribute to the stabilization of the carbamate moiety. carbamate motifs are characterized by a pseudo double bond. this implies the potential deconjugation of the heteroatom-(σ-bond)-carbon-(π-bond)-heteroatom system that restricts the free rotation about the formal single σ-bond. therefore, two isomers, syn and anti, may coexist in carbamates ( figure 2) . 23, 27 although carbamates display close similarity to amides, they show preference for the anti-isomer conformation. 28 the anti rotamer is usually favored by 1.0−1.5 kcal mol −1 for steric and electrostatic reasons with respect to the syn counterpart. 22 in many cases, the energy difference may be close to zero. as a result, those carbamates are found as an approximately 50:50 mixture of syn and anti isomers, as in the case of a number of boc-protected amino acid derivatives. this issue is of key importance since this balanced rotamer equilibria and the low activation energies render carbamates as optimal conformational switches in molecular devices. 23 the influence of the r and r 1 substituents on the free-energy difference between the two conformations has been investigated. beyond steric effects, electronegativity of r 1 must be considered since it may affect the conformation in many ways, including changes in the dipole moment and bond angles. 28 only the anti conformation would be expected in five-, six-, and seven-membered cyclic carbamates. calculations of the dipole moment for the carbamate group support this expectation. 29 solvent, concentration, salts, and ph strongly influence the free energy difference of the syn and anti isomers of carbamates as well. intra-and intermolecular hydrogen bonding may also perturb the syn−anti isomer equilibrium of carbamates. 22, 25, 30 a representative example of hydrogen bonding and concentration dependence was provided by gottlieb, nudelman, and collaborators. 28 the authors took into consideration n-boc-amino acids and their corresponding methyl esters. an unusual abundance of syn-rotamer for n-boc-amino acids was detected. n-boc-amino acid esters give the expected spectra, consistent with previous reports of only a single species being observed at room temperature. concentration-dependent 1 h nmr spectra indicate that the proportion of the syn-rotamers increases with concentration, supporting the existence of an aggregation process. 28 since decreasing temperature is another method for stabilizing oligomerization, nmr experiments were also performed at different temperatures. as expected, when the temperature increases, the favored rotamer switches from syn to anti. overall, the collected data strongly supports the concept that the syn rotamers of n-carbamoylated amino acids form intermolecularly h-bonded species and the oh of the carboxylic acid must be involved in this process, as the corresponding esters do not behave similarly. to explain this phenomenon, the formation of a dimer was suggested ( figure 3 ). support of this hypothesis was provided by adding increasing amounts of acetic acid to a solution of a carbamoylated amino acid ester. as expected, the syn rotamer appeared, and its concentration increased as a function of the amount of acid added. in contrast, addition of acetic acid to a solution of the corresponding carbamoylated amino acid did not affect the anti/syn ratio. in this context, moraczewski and co-workers designed a more effective hydrogen-bonding system that selectively perturbs the syn/anti rotamer equilibrium of a target carbamate group. 22 the authors examined the abilities of acetic acid and 2,6-bis(octylamido)pyridine (3) to perturb the syn/ anti ratio of carbamates 1 and 2 ( figure 4 ). 22 in a cdcl 3 solution, acetic acid moderately stabilizes double hydrogen bonding of the syn rotamer of phenyl carbamate 1 ( figure 4a) , with no relevant effect on the syn/anti ratio for 2pyridyl carbamate 2 ( figure 4b ). in the second case, the carboxylic acid favors donation of a hydrogen bond to the more basic pyridyl nitrogen and forms the complex shown in figure 4b . on the contrary, in the case of the donor−acceptor−donor triad 3, it strongly stabilizes the syn rotamer of 2 ( figure 4d ) over the anti rotamer ( figure 4c ). there is no effect on the syn/anti ratio for 1, presumably because of a steric deterrent to the formation of a hydrogen-bonded complex ( figure 4e ). the carbamate moiety plays a noteworthy role in medicinal chemistry, not only because it is found in drugs but also for its presence in a number of prodrugs. 31 the rate and level of their hydrolysis is a key issue for the duration and intensity of their pharmacological activity. fast hydrolysis of carbamate-bearing drugs may result in weak or shortened activity. on the contrary, carbamate-based prodrugs must undergo extensive hydrolysis at a suitable rate for releasing an active drug and obtaining the expected activity profile. vacondio et al. recently proposed an interesting study in which they compiled a large number of reliable literature data on the metabolic hydrolysis of therapeutic carbamates. 32 the authors were able to exploit the collected data to gain a qualitative relationship between molecular structure and lability to metabolic hydrolysis. a trend was extrapolated, according to which the metabolic lability of carbamates decreased in the following series: aryl-oco-nhalkyl ≫ alkyl-oco-nhalkyl ∼ alkyl-oco-n(alkyl) 2 ≥ alkyl-oco-n(endocyclic) ≥ aryl-oco-n(alkyl) 2 ∼ aryl-oco-n(endocyclic) ≥ alkyl-oco-nharyl ∼ alkyl-oco-nhacyl ≫ alkyl-oco-nh 2 > cyclic carbamates. 32 therefore, carbamates derived from ammonia or aliphatic amines are sufficiently long-lived. an example is represented by cefoxitin (4), a second-generation cephalosporin antibiotic ( figure 5 ). cyclic fiveor six-membered carbamates are quite stable and do not usually undergo metabolic ring opening. the antibacterial agent linezolid (5) is a representative example of this class ( figure 5 ). for these drugs, carbamate hydrolysis is not necessarily the half-lifedetermining metabolic reaction. on the contrary, fatty acid amide hydrolase (faah) inhibitor 6 (urb524) 33 showed significant hydrolysis in buffer at physiological ph after 24 h ( figure 5 ). other representative therapeutic carbamate drugs and prodrugs will be discussed in sections 4 and 5, respectively. organic carbamates play an important role in organic synthesis, especially as subunits of biologically active compounds. accordingly, simple and efficient methods for the synthesis of carbamates are of great interest. a number of methods have been developed for the synthesis of carbamates. 3.1. carbamate synthesis via traditional methods. over the years, a variety of carabamates have been prepared by utilizing the hofmann rearrangement of amides, 34−36 the curtius rearrangement of acyl azides, 37,38 the reductive carbonylation of nitroaromatics, 39 the carbonylation of amines, 40 the reaction of alcohols with isocyanates, 41 and carbon dioxide alkylation. 42−45 the hofmann rearrangement (method i, scheme 1) is wellrecognized as a useful method to convert primary carboxamides to amines or carbamates, characterized by the reduction of one carbon in the structure. 46 much effort has been devoted to the development of modified reagents to optimize the hofmann rearrangement since the classical method for this transformation, involving the use of an alkaline solution of bromine, is unsatisfactory and unreliable. 35 a variety of oxidants and bases have been proposed as modified agents, e.g., iodine(iii) reagents such as phi(oac) 2 , 47 meobr, 48 nbs-ch 3 ona, 49 nbs-koh, 46 lead tetraacetate, 50 and benzyltrimethylammonium tribromide. 51 these modified methods, however, require more than 1 equiv or an excess amount of the oxidizing reagent, which is not very convenient. the curtius rearrangement (method ii, scheme 1) is the thermal decomposition of acyl azides into the isocyanate intermediate. this method is widely employed in the transformation of carboxylic acids into carbamates and ureas. acyl azides are usually prepared from carboxylic acid derivatives such as acyl chlorides, 52,53 mixed anhydrides, 54,55 and hydrazides. 56, 57 subsequent isocyanate intermediates can be trapped by a variety of nucleophiles to provide the carbamate derivatives. the acid chloride method is not suitable for acidsensitive functionalities. one-pot transformations of carboxylic acids into carbamates avoids the isolation of unstable acyl azides. however, protocols involving the use of diphenylphosphoryl azide (dppa) for the one-pot curtius reaction are also characterized by issues related to toxicity and the high boiling point of dppa, which creates difficulties during workup and purification. 58−60 other general methods for carbamate preparation involve the use of the highly toxic phosgene, 61 phosgene derivatives, 62, 63 or isocyanates. 64 significant efforts have been made to find an alternative to the phosgene process. a very attractive substitute for phosgene is carbon dioxide because it is a classic renewable resource (method iii, scheme 1). in addition, its use is also very attractive due to its environmentally benign nature (nontoxic, noncorrosive, and nonflammable). 65 carbon dioxide is wellknown to react rapidly with amines to form carbamic acid ammonium salts. the majority of the approaches in this context rely on the creation of the carbamate anion via the reaction of carbon dioxide and amines, followed by the reaction with electrophiles. nevertheless, since the nucleophilicity of the carbamate anion is lower than that of the amine formed in the equilibrium of the salt formation, the subsequent reaction of the carbamate salts with alkyl halides does not selectively provide urethanes. 44, 66 the formation of carbamates from isocyanates (method iv, scheme 1) is fundamentally important to polyurethane industries. synthetic limitations and toxicity issues, however, are associated with the use of phosgene, the most common route to obtain isocyanates. 64 the readily available alkyl chloroformates are the most frequently used reagents for the preparation of carbamates (method v, scheme 1). however, these reagents display major drawbacks, as a large excess of base and a long reaction time are required in order to gain acceptable reaction efficiency. moreover, excess reagents are not suitable for the synthesis of molecules bearing multiple functionalities in which the chemoselectivity is critical. 67 3.2. carbamate synthesis via activated mixed carbonates. a number of organic carbonates have been developed as low-cost and benign alternatives to the phosgenebased routes for the synthesis of organic carbamates. in this context, several new alkoxycarbonylating agents (7−11) based on mixed carbonates have been developed ( figure 6 ). these methods are often used for the synthesis of carbamates in drug design. 68−72 mixed carbonates with a p-nitrophenyl moiety are frequently used for the preparation of a large range of carbamates. 73−76 for this, p-nitrophenyl chloroformate (7, pnpcocl), when treated with the suitable alcohol in the presence of base, furnishes the corresponding activated carbonates, which have been shown to be useful and effective alkoxycarbonylating reagents for suitable amines (scheme 2). examples of carbamate derivatives are shown in table 1 . several alkoxycarbonylating reagents for amino groups having heterocyclic groups, such as n-hydroxyimide, have scheme 1. traditional synthetic methodologies adopted for the synthesis of carbamates been reported. moreover, the utility and versatility of carbonates and oxalates containing an electron-withdrawing group, such as n-hydroxyimide and benzotriazole derivatives as reagents for various tranformations, have been described. 72, 81, 82 takeda et al. reported that 1-alkoxy[6-(trifluoromethyl)benzotriazolyl]carbonates easily derived from 1,1-bis[6-(trifluoromethyl)benzotriazolyl]carbonate (8, btbc) showed high acylating reactivity toward alcohols as well as amino groups. 83 btbc was prepared from 6-trifluoromethyl-1hydroxybenzotriazole and trichloromethyl chloroformate and purified by washing with dry ether. moreover, it can be stored for several months in a freezer. btbc was allowed to react with primary alcohols in acetonitrile at room temperature to give stable activated carbonates. the carbonates were treated with amines in the presence of 4-dimethylaminopyridine (dmap), providing the corresponding carbamates (scheme 2 and table 2 ). 83 in connection with our research work aimed at synthesizing biologically active polyfunctional molecules for probing enzyme active sites, we required a more general and synthetically reliable method for the synthesis of various carbamate derivatives. in 1991, we described the utility of di(2-pyridyl) carbonate (9, dpc) 84 as an efficient, high-yielding, and convenient alkoxycarbonylation reagent for amines overcoming many of the limitations of existing methodologies. 85 dpc was readily prepared from commercially available 2-hydroxypyridine and triphosgene in the presence of triethylamine and subsequently reacted with the suitable primary or secondary alcohol (e.g., (+)-menthol) to provide a mixed carbonate. alkoxycarbonylation of primary and secondary amines with the mixed carbonates was carried out in the presence of triethylamine and furnished the corresponding carbamates in good yields (scheme 2, method a, and table 3 ). potassium hydride was used in the place of triethylamine in the preparation of the mixed carbonates containing tertiary alcohols (scheme 2 and table 3 ). 85 subsequently, we investigated the scope of n,n′-disuccinimidyl carbonate (10, dsc) 81 promoted alkoxycarbonylation of amines with a host of alcohols under mild conditions. 86 rich and co-workers highlighted the convenience of succinimidylbased mixed carbonates for the high-yielding introduction of a 2-(trimethylsilyl)ethoxycarbonyl (teoc) protecting group to amino acids, without oligopeptide byproduct formation. 87 dsc was found to be a highly effective alkoxycarbonylating reagent for a variety of primary and sterically hindered secondary alcohols. dsc is commercially available, or it can be conveniently prepared from n-hydroxysuccinimide following a procedure tracing out the synthesis of dpc. 85 the ready availability of dsc, the stability of the mixed carbonates, and the mildness of the reaction procedure render this method a reliable route to organic carbamates (scheme 2 and table 4 ). 86 since azides were extensively employed as incipient amines in the context of amino sugar and amino acid syntheses, their conversion into the corresponding carbamate derivatives could provide a novel, effective route for medicinal chemistry applications. in this context, a facile synthetic protocol to transform various azides into the corresponding functionalized journal of medicinal chemistry urethanes in high yields has been developed. 90 in general, mixed carbonates of variously protected alcohols were prepared by reaction of excess dsc or dpc, as described previously. exposure of mixed carbonates to catalytic hydrogenation conditions with azides in the presence of 10% palladium on charcoal in tetrahydrofuran furnished the corresponding carbamates. interestingly, the use of triethylamine as a promoter has a notable effect on the yield and the rate of the alkoxycarbonylation process (scheme 2 and table 5 ). 90 more recently, yoon and co-workers exploited 2-substitutedpyridazin-3(2h)-ones as electrophilic transfer reagents. 91, 92 in particular, the authors investigated the carbonylation potency of phenyl 4,5-dichloro-6-oxopyridazine-1(6h)-carboxylate (11) to amines for the preparation of phenylcarbamates (scheme 2 and table 6 ). compound 11 is stable in air and in organic solvents at high temperature and is prepared easily from cheap and commercially available 4,5-dichloropyridazin-3(2h)-one (12) in the presence of phenylchloroformate and triethylamine (scheme 2). 3.3. recent methodologies for carbamate synthesis. the application of carbon dioxide in organic synthesis has recently attracted much interest. most of the approaches rely on the generation of the carbamate anion via the reaction of carbon dioxide and amines, followed by the reaction with electrophiles, usually alkyl halides. 93−95 in this context, a mild and efficient preparation of alkyl carbamates on solid supports was described by jung et al. 96 amines and anilines were coupled with merrifield's resin through a co 2 linker in the presence of cesium carbonate and tetrabutylammonium iodide (tbai). carbon dioxide was supplied by bubbling it into the reaction suspension, where n,n-dimethylformamide (dmf) was the solvent of choice (scheme 3). 96 the reaction conditions are convenient for purification, and the reactions undergo complete conversions. the method is convenient for the generation of large combinatorial libraries for rapid screening of bioactive molecules. chiral substrates susceptible to racemization have survived the conditions (table 7) . later, these authors reported a one-pot synthesis of n-alkyl carbamates starting from primary amines (scheme 4). 96 carbamates were generated via a three-component coupling of primary amines, co 2 , and an alkyl halide in the presence of cesium carbonate and tbai in anhydrous dmf (scheme 4 and table 8 ). direct n-alkylation of the intermediate carbamate a in the presence of additional cesium carbonate by using a different alkyl halide gave rise to the desired n-alkyl carbamate b (scheme 4). isolation of the intermediate a proved to be unnecessary, offering shortened synthetic sequences. 40 it is interesting to note that tbai helps to minimize the overalkylation of the produced carbamate, presumably by enhancing the rate of co 2 incorporation and/or stabilizing the incipient carbamate anion through conjugation with the tetrabutylammonium cation. 97 sakakura and co-workers reported urethane synthesis by the reaction of dense carbon dioxide with amines and alcohols by a procedure that is not only phosgene-free but also completely halogen-free (scheme 5). 98 dialkyl carbonate synthesis from an alcohol and co 2 is catalyzed by metal complexes such as dialkyl(oxo)tin and dialkyl(dichloro)tin. however, the alcohol conversion is very poor. similarly, the direct reaction of an amine, an alcohol, and carbon dioxide in the presence of dialkyltin compounds produced urethane only in a poor yield. the low conversion observed was attributed by the authors to thermodynamic limitations and catalyst deactivation by coproduced water. in order to overcome this issue, a new reaction system utilizing acetals as a chemical dehydrating agent, with subsequent alcohol regeneration (scheme 5), was developed. in order to obtain urethane in good yields, dense-phase co 2 under high pressure was necessary to lower the major side reactions, namely imine formation from acetone and alkylation of amines by alcohols. however, developing less toxic and more active catalysts based on metals other than tin was required. later, these authors reported novel nickel-based catalytic systems for dehydrative urethane formation from carbon dioxide, amines, and alcohols (scheme 6). 99 interestingly, adding nitrogenbased bidentate ligands efficiently improved the catalytic activity of ni(oac) 2 -based catalysts (scheme 6 and table 9 ). bipyridines and phenanthrolines with strong coordinating abilities (low steric hindrance and high electron densities) were the better choice for obtaining urethanes in high yields. it is important to note that the ni-phenanthroline system is more active and less toxic than dialkyl(oxo)tin under the same reaction conditions. it is also noteworthy that the catalytic activity of the ni(oac) 2 -(4,4′-dimethylbipyridine) system is highly dependent on the ligand/metal ratio (table 9 ). peterson and co-workers proposed a method for rapid sar development of compounds bearing urea or carbamate functionalities (scheme 7). 100 for carbamate formation, an journal of medicinal chemistry amine, in principle, could proceed through the carbamic acid− isocyanate reaction, and subsequent reaction with an alcohol may provide a carbamate product. while this is precedented by an intramolecular reaction variant to produce cyclic carbamates, 101 the desired intermolecular coupling was not fruitful under the proposed reaction conditions. carbamic acids produced from secondary amines, however, did react with alcohols under mitsunobu conditions (dibenzyl azodicarboxylate, dbad, and tributylphosphine) in a dbu-catalyzed reaction with gaseous carbon dioxide, providing the corresponding carbamates (scheme 7 and table 10 ). this reaction did not proceed through the isocyanate intermediate but rather through an s n 2 displacement of the activated alcohol. this hypothesis is supported by the observed inversion of stereochemistry upon conversion of a chiral secondary alcohol to the corresponding carbamate (table 10) . 100 very recently, jiao and co-workers reported a practical, pdcl 2 -catalyzed efficient assembly of organic azides, carbon monoxide, and alcohols for the direct synthesis of carbamates via isocyanate formation and application in situ (scheme 8). 102 mild and neutral reaction conditions and generation of harmless n 2 as the byproduct render this protocol very useful, particularly for the synthesis of bioactive compounds. moreover, the employment of co at atmospheric pressure and the use of a small amount of pdcl 2 catalyst (2 mol %) in the absence of any ligand represent a real alternative to customary carbamate synthetic methods (table 11) . 102 the synthesis of carbamates through the generation of carbamoyl chlorides is not convenient because of the requirement of the toxic phosgene. also, such carbamoyl chlorides are highly reactive, prone to hydrolysis, unstable, and not suitable for long-term storage. for these problems, batey and co-workers identified the use of carbamoylimidazolium salts as convenient n,n′-disubstituted carbamoyl transfer reagents, showing increased reactivity over carbamoylimidazoles as a result of the imidazolium effect (scheme 9). 103−105 these salts are readily prepared by the sequential treatment of secondary amines with n,n′-carbonyldiimidazole (cdi) and iodomethane (scheme 9). authors envisaged that the carbamoylimidazolium salts, while relatively unreactive with alcohols, would react with nucleophlic alkoxides to produce the corresponding carbamates (table 12 ). in the case of phenols, tertiary amines are appropriate bases for the in situ generation of the reactive phenoxides. the lower acidity of aliphatic alcohols presumably prevents the formation of the alkoxide anion, which would serve as the reactive nucleophile. less acidic alcohols react with carbamoylimidazolium after their conversion into more nucleophilic sodium alkoxides (scheme 9). 106 the use of solid-supported reagents has become ubiquitous due to enhanced reactivity and selectivity, milder reaction conditions, convenient work-ups, and decreased solvent waste. the modified hofmann rearrangement, proposed by gogoi et al., is operationally simple, inexpensive and applicable to a variety of aliphatic and aromatic amides for the synthesis of methyl carbamates (scheme 10). 36 kf/al 2 o 3 represents a useful and interesting solid-supported strong base, which replaces organic bases in a variety of reactions. 107 sodium hypochlorite is an inexpensive, convenient, and safe alternative to the currently employed oxidants. 108 this prompted the authors to investigate kf/al 2 o 3 along with naocl as an efficient reagent system for hofmann rearrangement. kf/al 2 o 3 basicity stems from the formation of koh in the initial preparation of the solid-supported material by the reaction of kf with alumina supports. under these highly basic reaction conditions, hypochlorite ion is the predominant form of chlorine, reacting with the amide to form an n-chloroamide, which later undergoes rearrangement to the isocyanate. in the presence of methanol, the isocyanate is rapidly converted into the corresponding methyl carbamate (table 13) . 36 modifications of the curtius rearrangement have also been explored. lebel and co-workers have reported a useful protocol for the preparation of tert-butyl carbamates from the corresponding carboxylic acids. 109 their reaction with di-tert(scheme 11a and table 14) . these authors extended the same methodology to the direct synthesis of carbamates of aromatic amines using aromatic carboxylic acids (scheme 11b and table 14) . 110 in particular, the reaction of a chloroformate or di-tert-butyl dicarbonate and sodium azide with an aromatic carboxylic acid produced the corresponding acyl azide, presumably through the formation of an azidoformate. in contrast to what was observed with aliphatic carboxylic acids, using similar reaction conditions, aromatic carboxylic acids led mainly to the formation of the corresponding tert-butyl ester, likely via the displacement of an azide leaving group with tert-butoxide. this may be ascribed to the higher stability of aromatic acyl azides with respect to their aliphatic counterparts. therefore, for these substrates, the curtius rearrangement can be promoted only at higher temperatures (40 vs 75°c). 110, 111 as mentioned, alkyl chloroformates are the most frequently used reagents for the preparation of carbamates, although the need of an excess amount limits their usefulness. a promising method for preparing carbamates involves the use of a catalytic promoter. 112−115 lately, indium-mediated reactions have gained significant consideration due to the high reactivity and unique properties of indium reagents, among them nontoxicity and inertness toward air and water. 116−118 moreover, pretreatment is not required for activating indium metal. in this context, jang and co-workers developed a simple, efficient, and selective method for synthesizing carbamates from amines, employing a catalytic amount of indium and only an equimolar amount of alkyl chloroformate (scheme 12). 67 the method shows the generality for a wide variety of sterically diverse amines and alcohols and can also be applicable for the selective protection of amino groups under mild conditions (table 15) . arndtsen et al. proposed another application of indium-based reagents for the generation of n-protected amines in a single step (scheme 13 and table 16 ). 119 since organoindium reagents readily transfer their organic groups to an imine carbon, only one-third of an equivalent is required, and the only byproduct is represented by indium trichloride. tetraorganoindium reagents can also be employed in a similar fashion for transferring all four organic groups. therefore, one-fourth of an equivalent of indium is necessary for their reaction with imines. copper(i) chloride (10%) was found to be the most efficient catalyst. sodeoka and colleagues reported the use of 1-alkoxycarbonyl-3-nitro-1,2,4-triazole reagents as useful intermediates for the preparation of carbamates (scheme 14). 121 to achieve a rapid and clean reaction, the features of the leaving group have a key role. an ideal leaving group should have a highly electronwithdrawing element in order to increase the electrophilicity of the carbonyl carbon, and the nucleophilicity should be low to avoid side reactions. it should also be easily separated from the reaction product. 3-nitro-1,2,4-triazole (nt), 120 although showing nucleophilicity, could be easily removed from the table 9 . nickel-catalyzed urethane synthesis from co 2 99 a conversion of amine. b urethane/consumed amide × 100. c ni/l = 1:1. d ni/l = 1:5. reaction due to its insolubility in dichloromethane or chloroform. nt-based reagents have a series of benefits such as high stability, since they can be stored for long periods without decomposition. reactions of these nt reagents with primary and secondary amines proceeded quickly to give the corresponding carbamates in >95% yield (scheme 14a and table 17 ). in contrast to aliphatic amines, aromatic amines were less reactive. however, the addition of triethylamine was found to be effective in promoting the reactions (scheme 14b and table 17 ). 121 the reductive carbonylation of aromatic nitro compounds to the corresponding carbamates has remained a subject of great interest both from mechanistic and application standpoints (scheme 15). in this section, we will briefly mention the methodologies involving the use of an alcohol, although other procedures employing chloroformates have also been recently reported. 122, 123 cheng and collaborators report the use of ru(co) 4 − and ru 3 (co) 12 complexes for the catalysis of this reaction and highlighted the key effect of alcohol on the selectivity of carbamates (table 18) . 124 the results clearly indicate that low selectivity of carbamate is closely related to the ability of the alcohol to reduce nitroarenes to amino derivatives. therefore, the employment of an alcohol that cannot reduce nitroarene greatly increases the selectivity of carbamate. later, the binuclear rhodium complex [(ph 3 p) 4 rh 2 (μ-oh) 2 ]·2c 6 h 6 was employed as an effective catalyst for the reductive carbonylation of nitrobenzenes to carbamate esters (table 18) . 125 palladiumbased catalysts have also been explored (table 18) . 126−128 carbamate synthesis via transfunctionalization of substituted ureas and carbonates in the presence of di-n-butyltin oxide (dbto) as the catalyst was reported by chaudhari and colleagues (scheme 16a and table 19 ). 129 the carbonate reactivity pattern seems to be driven by the leaving group ability of the alkoxides and phenoxide to form the carbamate observed in aminolysis of carbonates. it has been shown that basicity of reacting urea plays a vital role in the catalytic activity of this reaction. indeed, aliphatic ureas show higher reactivity compared to aromatic ureas due to their higher basicity. the basic dbto is supposed to work as a nucleophile by attacking the carbonyl carbon of the carbonate, thus generating the catalytically active species dibutyl alkoxy carbonato tin [a]. 130 use of dialkyl carbonates as environmentally friendly and nontoxic phosgene substitutes in alkoxycarbonylation reactions has also been exploited by porco et al. (scheme 17). 132 particularly, the authors examined the scope of zr(iv)catalyzed carbonate−carbamate exchange processes to prepare carbamates from dialkyl carbonates employing 2-hydroxypyridine (hyp) as a catalytic additive (table 20) . recently, padiya and co-workers reported a useful method for preparing carbamates in an aqueous media (scheme 18). 133 interestingly, they found that 1,1′-carbonyldiimidazole (cdi), although unstable in water, rapidly reacts in aqueous media with amine to give good yields of the corresponding nsubstituted carbonylimidazolide. carbonylimidazolide derived from the primary amine reacts in situ with a nucleophile such as phenol, providing the corresponding carbamate. the product precipitates out from the reaction mixture and can be obtained in high purity by filtration, making the method simple and scalable (table 21) . 133 cdi was also found to mediate the lossen rearrangement, which occurs in the transformation of an activated hydroxamic acid into the corresponding isocyanate (scheme 19). 134 the proposed methodology is experimentally efficient and mild, being characterized by imidazole and co 2 as the only stoichiometric byproducts. this method is a green and unconventional alternative to the curtius and hofmann rearrangements (table 22) . 135 another method based on the lossen rearrangement was recently proposed. 136 the methodology envisaged the reaction of a hydroxamic acid with an alcohol, promoted by 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride; tct) in the presence of an excess of n-methyl morpholine (nmm) (scheme 20 and table 22 ). carbamates are inherent to many fda approved drugs. this structural motif is also a key functionality in numerous medicinal agents with clinical potential. in this section, a series of therapeutic carbamates with a variety of applications is outlined. 4.1. miscellaneous carbamates with clinical relevance. 4.1.1. rivastigmine. rivastigmine (194, figure 7 ) tartrate (exelon, novartis pharma) is a carbamate derivative that reversibly inhibits the metabolism of acetylcholinesterase (ache) and butyrylcholinesterase (buche) preferentially in the central nervous system (cns). it is used for the treatment of mild-to-moderate alzheimer's disease (ad) dementia and dementia due to parkinson's disease. 137, 138 the drug can be administered orally or via a transdermal patch. the transdermal patch reduces side effects such as nausea and vomiting. rivastigmine undergoes extensive metabolism by che-mediated hydrolysis to the decarbamylated metabolite, without involvement of the major cytochrome p450 (cyp450) isozymes. the metabolite may undergo n-demethylation as well as conjugation. the pharmacokinetic half-life of rivastigmine in ad patients is around 1.5 h. when given orally, rivastigmine is well-absorbed, with a bioavailability of about 40% administered as a 3 mg dose. 137, 139 4.1.2. muraglitazar. muraglitazar (195) contains a carbamate functionality. it is a potent, novel nonthiazolidindione peroxisome proliferator-activated receptor dual agonist (pparα/γ) that demonstrated highly efficacious glucose and lipid lowering activities in vivo, along with an excellent adme profile. 140 in a double-blind randomized clinical trial, muraglitazar resulted in a statistically significant improvement in plasma triglyceride, hdl cholesterol, apob, and non-hdl cholesterol concentrations at week 12. muraglitazar reduced triglyceride concentrations to a larger extent than did pioglitazone, regardless of baseline triglyceride levels. muraglitazar and pioglitazone treatment was associated with slight (3−4%) increases in ldl cholesterol. however, muraglitazar development was discontinued due to major adverse cardiovascular side effects. 141 4.1.3. roxifiban. roxifiban (196) is a carbamate derivative with a methyl ester prodrug. it is a potent, nonpeptide antagonist of the glycoprotein iib/iiia receptor. 142, 143 the free acid resulting from roxifiban hydrolysis blocks the binding of fibrinogen to the receptor, thereby inhibiting platelet aggregation and providing a mechanism for antithrombotic mebendazole (199) is a methyl carbamate derivative showing broad-spectrum anthelmintic properties. it demonstrated efficacy in the oral treatment of ascariasis, uncinariasis, oxyuriasis, and trichuriasis. like other benzimidazole anthelmintics, mebendazole's primary mechanism of action is consistent with tubulin binding. 148 mebendazole was discontinued in 2011. 149 4.1.6. flupirtine and retigabine. flupirtine (200) and retigabine (201) are ethyl carbamate derivatives. flupirtine is a centrally acting nonopioid analgesic 150 that was identified within an antiepileptic drug discovery program by the u.s. national institutes of health. the doses used in a small clinical trial exceeded those established for analgesic activity. 151 on the basis of this data, subsequent structural optimization resulted in retigabine. 152 retigabine has anticonvulsant properties that appear to be mediated by opening or activating neuronal voltage-gated potassium channels. flupirtine showed n-methyl-d-aspartate (nmda) receptor antagonist properties. 4.1.7. felbamate. felbamate (202, felbatol, meda pharmaceuticals) is an alkyl carbamate derivative. it is an antiepileptic drug. the mechanism of action of felbamate involves a dual mechanism involving inhibition of n-methyl-d-aspartate (nmda) receptor response and positive modulation of γamino butyric acid subtype a (gaba a ) receptor, thus decreasing neuronal excitation. 153 felbamate is rapidly absorbed (t max = 2−6 h) with an oral bioavailability > 90%. 154 felbamate undergoes moderate metabolism via cyp3a4 and cyp2e1 isoenzymes, which are amenable to inhibition and induction effects. 155, 156 the clinical use of scheme 11. synthesis of carbamates by modified curtius rearrangement it is a nonnucleoside reverse transcriptase inhibitor (nnrti). the drug is used as part of highly active antiretroviral therapy (haart). 157, 158 however, its use is associated with variable treatment response and adverse effects, in most part because of the large differences in pharmacokinetics. 159 cyp2b6 is the main enzyme catalyzing the major clearance mechanism of efavirenz (8-hydroxylation to 8-hydroxyefavirenz) in vivo. 160, 161 4.1.9. zafirlukast. zafirlukast (204, accolate, astrazeneca) is a cyclopentyl n-aryl carbamate derivative. it is a selective and competitive receptor antagonist of the cysteinyl leukotrienes d-4 and e-4, which is indicated for the prophylaxis and treatment of mild-to-moderate persistent and chronic asthma. 162 both o → ch 2 and o → nh bioisosteric analogues of zafirlukast were found to be potent. the carbamate moiety present in zafirlukast provided an excellent in vitro and in vivo profile and high oral bioavailability. 163 zafirlukast undergoes hepatic metabolism, where hydroxylation by cytochrome cyp2c9 is the major biotransformation pathway. the metabolites of zafirlukast do not significantly contribute to its overall activity. 164 4.1.10. mitomycin c. mitomycin c (205, mmc, mutamycin) is a complex carbamate derivative. it is an antitumor antibiotic that was identified in the 1950s in fermentation cultures of the gram-negative bacteria streptomyces caespitosus. 165 mmc is a site-specific, nondistorting dna cross-linking agent. 166, 167 however, recent reports suggest that dna may not be the primary target of the drug. in particular, interaction of mmc with rrna and subsequent inhibition of protein translation has been proposed. 168 mmc is customarily used as a chemotherapeutic agent in the treatment of several types of cancer, such as bladder, colon, and breast cancers. 169 4.2. therapeutic carbamates as hiv protease inhibitors. hiv protease is an aspartic acid protease responsible for the cleavage of the gag−pol polyprotein into functional proteins essential for the production of infections progeny virus. inactivation of hiv-1 protease either by site-directed mutagenesis or by chemical inhibition results in the formation of immature, noninfections virus particles. as a consequence, hiv-1 protease is an attractive target in antiviral therapy. hiv protease is a c 2 -symmetric, 198-amino acid homodimeric aspartyl protease in which each protein subunit contributes one asp-thr-gly motif to the single active site. 170 the x-ray crystallographic analysis of the native protein and subsequent protein−ligand complexes and extensive research programs on other aspartyl proteases, including human renin, 171 provided a path toward accelerated drug discovery programs targeting hiv protease. 172−174 a number of fda-approved hiv protease inhibitor drugs contain an important carbamate functionality. in this section, currently approved protease inhibitor drugs are discussed (figure 8) . 4.2.1. ritonavir. ritonavir (206, norvir, abt-538, a-84538, abbvie, inc.) structure possesses a thiazolyl methyl carbamate functionality. it is a peptidomimetic inhibitor of both the hiv-1 and hiv-2 proteases and was approved by the fda in march 1996. 175 this first-generation protease inhibitor was developed at abbott laboratories. the discovery of ritonavir was based on 50 of 0.025 μm, bioavailability of 78%, and a plasma half-life of 1.2 h. ritonavir has a high molecular weight; however, it showed excellent pharmacokinetic properties. this is possibly due to the increased stability of the thiazole groups to oxidative metabolism and also due to its effect on cytochrome p450 oxidative enzymes. ritonavir is a type ii heme ligand that fits into the cyp3a4 active site cavity and irreversibly binds to the heme iron via the thiazole nitrogen. 176 amprenavir was identified as a potent, orally bioavailable hiv-1 protease inhibitor with a low molecular weight and a mean ic 50 of 12 nm. 177 it is marketed with a twice-a-day dosing format. amprenavir structure bears a stereochemically defined tetrahydrofuranylcarbamate engaging in a weak backbone interaction with the protease. 178, 179 in vitro and in vivo studies have shown that amprenavir is primarily metabolized by cyp3a4, and the two major metabolites result from oxidation of the tetrahydrofuran and aniline moieties. properties. drv also maintains high potency against multidrugresistant hiv-1 strains. the design of drv originated from the backbone binding concept envisaging that an effective protease inhibitor maximizes rich networks of hydrogen-bonding interactions with the backbone atoms throughout the active site of the protease. 185 the bis-thf moiety present in drv was designed based on the x-ray structure of inhibitor-hiv-1 protease complexes. the bis-thf carbamate moiety of drv was found to be essential for enzyme affinity (see figure 14 for details). drv demonstrated exceptional potency against both wild-type hiv isolates and a wide range of resistant variants. 186 hiv-1 variants. in 2008, drv received full approval for the treatment of therapy-naive adults and children. 188 drv is metabolized by the isoenzyme cyp3a4. 189, 190 however, in the presence of a low dose of ritonavir, drv exhibits very good pharmacokinetic properties in patients. 191 metabolism prodrugs are chemically modified forms of the actual pharmacologically active drug that undergo in vivo transformation to release the active drug molecule. this is a wellestablished strategy to improve drug disposition properties (physicochemical, biopharmaceutical, or pharmacokinetic properties) of pharmacologically relevant compounds and thereby increase their drug-like profile. 192, 193 a prodrug strategy helps to overcome a variety of hurdles in drug formulation and delivery such as (i) poor oral absorption and aqueous solubility, (ii) poor lipid solubility, (iii) chemical instability, (iv) rapid presystemic metabolism, (v) toxicity and local irritation, and (vi) lack of site-selective delivery. 193 a functional group on the parent drug may be used to form a chemical bond with the promoiety. generally, the linker should be self-removing or cleavable so that the parent drug can be released spontaneously or under a certain triggering condition, such as the presence of an enzyme or a change in ph. the promoiety coupled to the parent drug provides the ability to improve the drug-like properties or overcome the barriers in delivering the drug to its target cells. 194 carbamates are the esters of carbamic acid, preferentially used in the design of prodrugs as a means of achieving first-pass and systemic hydrolytic stability. carbamates are typically enzymatically more stable than the corresponding esters. they are, in general, more susceptible to hydrolysis than amides. 195 thus, bioconversion of carbamate prodrugs requires esterases for the release of the parent drug. upon hydrolysis, carbamate esters release the parent phenol or alcohol drug and carbamic acid, which, due to its chemical instability, breaks down to the corresponding amine and carbon dioxide. carbamates of primary amines can also fragment into isocyanates and alcohols on treatment with bases, a further potential pathway for metabolic degradation. 192, 195 the oh-catalyzed hydrolysis of these carbamate esters (r′-nhco-or) is strongly dependent on both the pk a of the proton on the leaving group (roh) and the degree of substitution on the nitrogen of the carbamate ester. 196 since phenols have a lower pk a with respect to alcohols, carbamate esters of phenols are generally more chemically labile than those of alcohols. in the case of alcohols, both the n-monosubstituted and n,n-disubstituted carbamates are chemically stable toward hydrolysis. in phenols, n,ndisubstituted carbamates are chemically stable, whereas nmonosubstituted carbamates are the most labile toward chemical hydrolysis. short-lived carbamates have also been used as prodrugs of heteroaromatic amines (e.g., capecitabine, 217) and amidines (lefradafiban (221), dabigatran). 195 5.1. alcohol and phenol carbamate prodrugs. most of the therapeutically relevant carbamate prodrugs have been designed as substrates of specific enzymes. antibody-directed enzyme prodrug therapy (adept) 197, 198 and gene-directed enzyme prodrug therapy (gdept) 199 are new strategies for targeting tumors. carboxypeptidase g2 (cpg2), an enzyme of bacterial origin, has been shown to catalyze the cleavage of an amide, carbamate, or urea linkage between glutamic acid and an on the basis of this specificity, a large number of prodrugs have been designed and synthesized for cpg2. as shown in figure 9 , the prodrug 208 (zd2767p) 200 is activated by hydrolysis at the carbamate bond by cpg2 to the corresponding potent di-iodophenol mustard (209) . 201 208 was found to possess the best profile in terms of enzymatic kinetics, cytotoxicity, and in vivo efficacy. it was selected for clinical development. the half-life (t 1/2 ) of the drug is approximately 2 min, which is enough for diffusion into the tumor cell from the local release site and to minimize peripheral toxicity. 192, 202 irinotecan was designed to deliver camptothecin as a predominant topoisomerase i inhibitor for anticancer therapy. irinotecan hydrochloride salt 210 (cpt-11, camptosar; pfizer) is a parenteral aqueous soluble carbamate prodrug of antineoplastic topoisomerase i inhibitor 211 (sn-38, 7-ethyl-10-hydroxy-camptothecin). the potent antitumor activity of irinotecan is due to rapid formation of active metabolite 211 in vivo (figure 9 ). in this molecule, a dipiperidino ionizable promoiety is linked to the phenol functionality by a carbamate bond, thus improving the overall aqueous solubility. 203−205 the bioconversion back to 211 occurs primarily by human liver microsomal carboxylesterases, ces 1a1 and ces2, which release the ionizable piperidinopiperidine promoiety and 211, the active form of the drug. 205 beyond minimizing the rate of enzymatic hydrolysis of its prodrug, sustained drug action can also be provided by decreasing the rate of drug metabolism. this is the case of bambuterol (212, bambec, astrazeneca), a bis-dimethyl carbamate prodrug of the β2-agonist terbutaline (213), which is used as a bronchodilator in the treatment of asthma. the phenolic moiety of terbutaline is subjected to rapid presystemic metabolism. in bambuterol, protection of this functionality also avoids first-pass intestinal and hepatic metabolism. this prodrug is inactive, however, after oral administration; it is slowly converted to terbutaline, mainly outside the lungs, by a series of hydrolysis and oxidation reactions (maily catalyzed by plasma cholinesterase, pche, and by cyp450, figure 9 ). 206, 207 this allows a once-daily bambuterol treatment with respect to the three daily terbutaline administrations. 208 an n,n′-dimethyl ethylenediamine spacer, used for the evaluation of cyclization-elimination-based prodrugs of phenols 209 and alcohols, 210 has been used for the development of prodrugs as a part of the adept activation strategy. when activated by a specific enzyme, the terminal amino group on the spacer activates and initiates an intramolecular cyclization reaction to eliminate a phenol 211 or alcohol 212 parent drug with parallel release of the cyclized spacer. in one such application, scherren et al. 213 explored paclitaxel-2′-carbamates. this is particularly interesting because a free 2′-hydroxyl group is important for biological activity. in general, carbamate linkages are more stable in vivo than esters and carbonates. since the proteolytic active form of plasmin is located in the tumor, linking a cytotoxic drug to a plasmin substrate may result in tumor-selective delivery. on the basis of this rationale, following plasmin hydrolysis, the spacer is expected to undergo spontaneous cyclization to yield a cyclic urea derivative (imidazolidinone), thereby releasing paclitaxel (214) , as illustrated in scheme 21. 192, 214 5.2. amine and amidine carbamate prodrugs. the amine group is one of the most common functional groups in many approved drugs. amines in drugs can cause physicochemical hurdles that have the potential to limit their safety and effective delivery to desired sites of action. therefore, a variety of prodrugs of amines have been designed to overcome formulation and delivery barriers. the carbamate functionality has been utilized in many prodrug strategies designed for amines. short-lived carbamates are also used as prodrugs of heteroaromatic amines and amidines. 192 gabapentin (216, neurontin; pfizer, figure 10 ) is a structural analogue of γ-aminobutyric acid (gaba). it is marketed as an anticonvulsant and an analgesic agent. gabapentin shows a number of limitations, including saturable absorption, high interpatient variability, lack of dose proportionality, and a short half-life. gabapentin enacarbil (215, horizant, previously known as xp13512) is a carbamate prodrug of gabapentin. the prodrug is benefited by a monocarboxylate transporter type 1 (mct1). mct1 is expressed in all segments of the colon and upper gastrointestinal tract. the prodrug also helps the sodium-dependent multivitamin transporter (smv t), responsible for absorption of multiple essential nutrients. 215, 216 following absorption via these pathways, the prodrug is rapidly converted to gabapentin by nonspecific esterases, mainly in enterocytes and to a lesser extent in the liver. during conversion to gabapentin, each molecule of 215 also generates carbon dioxide, acetaldehyde, and isobutyrate (figure 10 ). 217 the oral bioavailability of 215 was improved from 25 to 84% in monkeys. it showed doseproportional gabapentin exposure in humans. 193 in 2011, xenoport received fda approval (horizant) for the treatment of moderate-to-severe restless legs syndrome. in 2012, horizant was also approved for the management of postherpetic neuralgia (phn) in adults. 218 capecitabine (217, xeloda, roche) was designed to achieve greater selectivity than its active form, 5-fluorouracil (220, 5-fu). 219 it is an orally administered carbamate prodrug of 5-fu, belonging to the fluoropyrimidine carbamate class. it requires a cascade of three enzymes for the bioconversion to the active drug. 220 as shown in figure 10 , the enzymatic bioconversion starts in the liver, where human carboxylesterases 1 and 2 (ces1 and ces2) cleave the carbamate ester bond. 219 intact capecitabine is absorbed in the intestine, and its bioconversion in the liver releases the parent drug. to some extent, its bioconversion proceeds in tumors, thus avoiding any systemic toxicity. in particular, the remaining transformations to 5-fu are catalyzed by cytidine deaminase and thymidine phosphorylase. the latter enzyme is highly enriched in tumors, thus providing selective release of 5-fu in cancer cells. 220, 221 the absorption of capecitabine is evident since 95% of an orally administered dose is recovered in urine and the t max of 5-fu is reached in approximately 1.5−2 h. 193 capecitabine is currently approved as a first line of therapy for colorectal and breast cancers and is also approved for use in combination with other anticancer drugs. 192, 222 alkoxycarbonyl derivatives can serve as useful prodrugs for benzamidines. for example, the methoxycarbonyl methyl ester lefradafiban (221, bibu104, boehringer ingelheim, germany) is effectively converted to the active platelet aggregation inhibitor fradafiban (222, bibu 52) after oral administration. this was revealed by monitoring the plasma concentrations of 222 and by ex vivo platelet aggregation studies. lefradafiban is the orally active prodrug of fradafiban, a glycoprotein iib/iiia receptor antagonist. 192 esterases, but not cyp450-dependent enzymes, are involved in the conversion of lefradafiban to fradafiban in vivo (figure 10 ). 223 over the years, we have developed a series of novel hiv-1 protease inhibitors incorporating cyclic ether-derived carbamates designed based on the x-ray structures of inhibitor-hiv-1 protease complexes. 224, 225 in this endeavor, we have specifically developed stereochemically defined cyclic ether templates, where the cyclic ether oxygen could effectively replace a peptide carbonyl oxygen. the advantage of such replacement is to reduce peptidic features and improve metabolic stability of compounds. these cyclic ligands have been incorporated as carbamate derivatives. the evolution of the carbamate structural template is shown in figure 11 . on the basis of the x-ray crystal structure of saquinavir (223)-bound hiv-1 protease, we first investigated 3-(r)-tetrahydrofuranylglycine so that the 3-(r)-thf ring oxygen would interact with the asp30 nh, similar to the asparagine side chain carbonyl oxygen of saquinavir (compound 224). 226−228 in an effort to reduce molecular weight, the p3 quinoline was removed, and the amide bond was replaced with a carbamate to provide inhibitor 225 with significant reduction of molecular weight (515 da from 670 da). the x-ray crystal structure of 225bound hiv-1 protease revealed that the ring oxygen of the 3-(s)-tetrahydrofuran (3-(s)-thf) is within proximity to form a hydrogen bond with the asp29 nh bond in the s2 subsite. the importance of the carbamate moiety is evident. the carbamate nh forms a hydrogen bond with the backbone carbonyl of gly27, and the carbamate carbonyl functionality makes a tightly bound water-mediated hydrogen bond with the backbone nh's of the flap ile50 and ile50′ in the active site. our further investigation of the 3-(s)-thf in inhibitors containing a hydroxyethylene isostere led to a series of exceptionally potent inhibitors. 178 as shown in table 23 , 3-(s)-thf-containing carbamate drivatives (compounds 226− 231) provided very potent inhibitors in antiviral assays. the potency enhancing effect of 3-(s)-thf carbamate was subsequently demonstrated in inhibitors containing the (r)-(hydroxyethyl)sulfonamide isostere. 229 clinical development of inhibitor 46 (vx476) led to fda approval of amprenavir for the treatment of hiv/aids patients. 179, 230 further development of carbamate-derived novel hiv-1 protease inhibitors is shown in figure 12 . we have designed a variety of inhibitors incorporating cyclic sulfones and bicyclic ligands (figure 12, compounds 232−237) . 230, 231 these ligands were conceived in order to maximize hydrogen-bonding interactions with the protease backbone as well as to fill in the hydrophobic pocket in the s2 subsite. on the basis of the x-ray structure of saquinavir-bound hiv-1 protease, we then designed a fused bicyclic tetrahydrofuran (bis-thf) ligand to form hydrogen bonds with backbone aspartates in the s2 subsite as well as to fill in the hydrophobic site adjacent to the p3-quinoline ring of saquinavir ( figure 12) . 185, 232 an x-ray structural analysis of 236-bound hiv-1 protease revealed that the bis-thf carbamate mimics the majority of p2−p3-amide bonds of saquinavir. a detailed structure−activity study also established that the stereochemistry of the bis-thf ring, and the position of the ring oxygens is critical to potency. with the development of a bis-thf carbamate that could form a network of hydrogen bonds in the s2 subsite of hiv-1 protease, we investigated transition state isosteres that can be functionalized to form hydrogen bonds in the s2′ subsite. our basic hypothesis was to design inhibitors that form a network of hydrogen bonds with the protease backbone atoms throughout the active site of hiv-1 protease, from s2 to s2′ subsites. this backbone binding strategy to combat drug resistance led to the development of a series of very potent carbamate-derived protease inhibitors. 185, 225, 226 as shown in figure 13 , we incorporated the bis-thf ligand in the (r)-hydroxyethylsulfonamide isostere bearing p-methoxysulfonamide as the p2′ ligand so that the methoxy oxygen can interact with aspartate backbone atoms in the s2′ subsite. the resulting inhibitors exhibited notable potency. 233, 234 inhibitor 239 with a (3r,3as,6ar)-bis-thf as the p2 ligand is significantly more potent in an antiviral assay than corresponding inhibitor 238 with an enantiomeric bis-thf ligand. an x-ray structure of 239-bound hiv-1 protease revealed that the carbamate nh formed a hydrogen bond with the backbone gly27 carbonyl group and that carbamate carbonyl of 239 is involved in an interesting tetra-coordinated hydrogen-bonding interaction with the structural water molecule, inhibitor sulfonamide oxygen, and the flap ile 50 nh residues. also, the structure revealed interactions with the backbone atoms in both the s2 and s2′ subsites. 235, 236 further replacement of the p-methoxy group at the s2′ to a p-amino group led to inhibitor 48 ( figure 14) . this inhibitor showed marked enzyme inhibitory activity as well as antiviral activity. an in-depth antiviral study revealed that 48 maintained excellent antiviral activity against multidrug-resistant hiv-1 variants. 237−239 the x-ray structural studies of darunavir-bound hiv-1 protease showed extensive active site interactions ( figure 14) . particularly, it formed a network of hydrogen bonds with the protein backbone throughout the active site. darunavir also exhibited favorable pharmacokinetic properties. subsequently, clinical development led to its fda approval as darunavir for the treatment of hiv/aids patients. 240, 241 the carbamate functionality of darunavir (48) was assembled as shown in scheme 22. (3r , 3 a s , 6 a r ) -3 -hydroxyhexahydrofuro[2,3-b]furan (bis-thf) 47 was treated with disuccinimidyl carbonate to provide activated mixed carbonate 240. reaction of this activated carbonate with hydroxyethylsulfonamide isostere 45 provided darunavir. 242, 243 the backbone binding inhibitor design strategies to combat drug resistance have been further utilized by us and others to advance a number of other preclinical and clinical inhibitors with carbamates. 185, 225 figure 15 shows selected bis-thfderived carbamates (241−244) with marked enzyme and antiviral activities. 244−247 like darunavir, inhibitor-bound x-ray structures of these inhibitors showed a network of hydrogen bonds in both s2 and s2′ subsites of hiv-1 protease. the inhibitor side chains as well as the bis-thf bicyclic framework also effectively filled the hydrophobic pockets in the active site. we have outlined a selected number of cyclic ether-derived carbamates that have been developed based on the backbone table 22 . carbamates from cdi-and tct-mediated lossen rearrangement 135, 136 scheme 20. tct-mediated lossen rearrangement for carbamate synthesis binding concept in figure 16 . 185, 225 particularly, incorporation of these stereochemically defined oxacyclic ligands such as cp-thf, tp-thf, tris-thf, and fluoro-bis-thf provided exceptionally potent inhibitors (51 and 245−249) with clinical potential. 247−252 the importance of the carbamate functionality in these inhibitors is particularly worthy of note. x-ray crystal structures of these inhibitors in complex with hiv-1 protease provided the ligand-binding site interactions responsible for their respective antiviral potency against wild-type and multidrug-resistant viruses. in general, inhibitors are involved in hydrogen-bonding interactions with asp29, asp30, gly27, asp25, asp25′, and asp30′ in the hiv-1 protease active site. furthermore, the ring cycles adequately fill the hydrophobic pockets in the active site. 251 inhibitors the search for an effective treatment for alzheimer's disease (ad) remains a major challenge in medicine. one of the pathological hallmarks of ad is the formation of β-amyloid (aβ) peptides in the cortex of ad patients. aβ-peptides are generated from β-amyloid precursor protein (app) by sequential cleavage by β-secretase (also known as bace1 or memapsin 2) and γ-secretase. due to this central role of aβproduction, both β-secretase and γ-secretase have been implicated as important therapeutic targets for ad intervention. 253,254 as a result, design and synthesis of selective βsecretase and γ-secretase inhibitors have become an intense area of research over the years. 7.1. development of β-secretase inhibitors. following the discovery of β-secretase, the first-generation β-secretase inhibitors were designed and synthesized by ghosh, tang, and co-workers. 255 as shown in figure 17 , utilizing a carbamate derivative of the leu−ala isostere 250, potent pseudopeptide inhibitors 251 and 252 were identified. the x-ray crystal structure of 252-bound β-secretase was determined to provide molecular insight into the ligand binding site interactions. 256 the in-depth structural analysis thus provided critical drug design templates and led to the beginning of structure-based design approaches to peptidomimetic/nonpeptide β-secretase inhibitors. 254, 255 the x-ray structure of 252-bound β-secretase revealed that the p2 asparagine side chain carboxamide nitrogen formed an intermolecular hydrogen bond with the p4 glutamic acid carbonyl group. on the basis of this molecular insight, a number of 14−16membered cycloamide-carbamate-based macrocyclic inhibitors were designed and synthesized. 257 as shown in figure 18 , acyclic carbamate derivatives (253 and 254) were less potent than their corresponding cyclic inhibitors. inhibitor 255, with a 16-membered macrocycle containing a trans-olefin, amide and carbamate functionalities within the macrocycle, showed good β-secretase inhibitory activity. saturated inhibitor 256 is less potent against bace1, but it showed enhanced potency for bace2. x-ray structural studies of inhibitor 256-bound secretase revealed that the carbamate carbonyl forms a hydrogen bond with the gln73 side chain carboxamide residue. interestingly, unsaturated inhibitor 255 showed slight selectivity against memapsin 1 (k i = 31 nm). the design of a selective inhibitor is important for reducing toxicity through off-target effects. particularly, selectivity over other aspartic proteases, such as bace2, pepsin, renin, cathepsin d (cat-d), and cathepsin e, may be important for the reduction of side effects and drug efficiency. 258 on the basis of our detailed structure−activity studies and xray structural analysis, we have designed a variety of highly selective and potent bace1 inhibitors. in this perspective, we will highlight only the development of bace1 inhibitors bearing carbamate functionalities. as shown in figure 19 , inhibitor 257 is a potent bace1 inhibitor. however, it did not show selectivity against bace2 or cat-d. subsequent structure-based design led to the development of selective inhibitors 258 and 259, which contain a pyrazolylmethyl and oxazolymethyl carbamate at the p3 position, respectively. 259 inhibitor 258 showed excellent bace1 potency and selectivity over bace2 and cathepsin d. the x-ray crystal structure of 258-bound β-secretase revealed that the carbamate carbonyl formed a hydrogen bond with the thr-232 backbone nh. also, figure 9 . examples of phenol carbamate prodrugs and their metabolic activation. ethylenediamine spacer and release of paclitaxel the pyrazole nitrogen formed a strong hydrogen bond with the thr-232 side chain hydroxyl group. the p2-sulfonyl functionality formed a number of hydrogen bonds in the s2 subsite as well. on the basis of this molecular insight, oxazole-derived 259 was designed to provide a more stable and selective inhibitor. the synthesis of inhibitors 258 and 259 is outlined in scheme 23. urethanes 263 and 264 were prepared by treatment of 2,5-dimethylpyrazolylmethanol (260) or 2,5dimethyl-4-oxazolemethanol (261) with triphosgene in the presence of triethylamine, followed by l-methionine methyl ester hydrochloride (262) . saponification of the resulting methyl esters provided the corresponding acids. coupling of amine 265 with acids 263 and 264, as described previously, and subsequent oxidation of the sulfides with m-chloroperbenzoic acid furnished inhibitors 258 and 259. 259 freskos and co-workers have reported a series of β-secretase inhibitors that incorporated polar carbamate derivatives as the p2 ligand. 260, 261 this strategy led to improve the cat-d selectivity. it was hypothesized that the s2 subsite of cat-d is more lipophilic and less tolerant of polar groups. as can be seen in figure 20 , benzyl carbamate derivative 266 displayed 6-fold selectivity over cat-d. however, polar 3-pyridylmethyl derivative 267 improved selectivity nearly 90-fold. the corresponding 4-pyridyl methyl compound 268 provided a reduction in selectivity (∼50-fold). 3-(s)-tetrahydrofuranyl carbamate 269 showed a nearly 30-fold selectivity over cat-d. these inhibitors have also shown good to excellent ic 50 values in hek cells. 7.2. development of γ-secretase inhibitors. over the years, many structural classes of potent and selective γ-secretase inhibitors have been reported. a number of inhibitors displayed drug-like properties and also inhibited aβ production in animal models. in this section, we will review inhibitors with carbamate functionality. on the basis of γ-secretase inhibitor 270 (ly-411575), peters and co-workers designed a series of carbamate derivatives of dibenzazepinone as potent and metabolically stable γ-secretase inhibitors. 265 as shown in figure 21 nm. absolute stereochemistry of the active enantiomer was not determined. piperidine carbamate 274 also showed good potency. carbamate derivative 275 displayed a good membrane aβ ic 50 value; however, it showed poor cyp properties. further modification led to compound 276 with good inhibitory activity and improved cyp properties. bergstrom and co-workers reported a series of carbamateappended n-alkyl sulfonamides as γ-secretase inhibitors. 269, 270 figure 23 depicts selected examples that show potent aβ inhibitory activity. sulfonamide derivative 277 was identified as a potent γ-secretase inhibitor. exploration of carbamateappended n-alkylsulfonamides resulted in potent inhibitors such as 278−280. tertiary carbamate 280 showed significant reduction of brain aβ in transgenic mice compared to that of its benzyl derivative. this compound also showed improved brainto-plasma ratio and good absolute brain concentration. hepatitis c virus (hcv) is a bloodborne virus that is found worldwide. there are multiple strains or genotypes of the hcv virus. hcv infections lead to progressive liver damage, cirrhosis, and liver cancer. in recent years, there have been a number of new and effective antiviral drugs developed for the treatment of hepatitis c. these include the development of hcv ns3/4a protease inhibitors and inhibitors hcv ns5a. in this section, carbamate-derived therapeutics will be discussed. 8.1. carbamate-derived serine protease inhibitors. serine proteases are a large family of proteolytic enzymes that play a variety of critical roles in many physiological processes. 271, 272 deregulation of serine proteases has been related to the pathogenesis of diseases such as stroke, inflammation, alzheimer's disease, cancer, and arthritis. therefore, significant research efforts have been focused in the discovery of serine protease inhibitors. the active site of all serine proteases consists of a catalytic triad of ser, his, and asp. the nucleophilic attack by the hydroxyl group of serine at the carbonyl carbon of the scissile bond of the substrate, via general base catalysis by histidine, leads to the tetrahedral transition state. the tetrahedral intermediate ultimately collapses, leading to cleavage products. 273−275 these key active residues are conserved in all serine proteases. x-ray structural studies revealed that these residues are superimposable in the majority of serine proteases. 273, 276 therefore, selectivity over other serine proteases represents a key issue to be taken into consideration during inhibitor design. most early inhibitors acted via a covalent mechanism in which an electrophilic group formed a covalent bond with the serine hydroxyl of the catalytic triad. the electrophilic groups are commonly referred to as serine traps or warheads. however, covalent inhibitors lack selectivity and specificity against other proteases in the same class or clan. the rational design of covalent serine protease inhibitors usually involves the selection of a good substrate to be linked to a serine trap/warhead. chloromethyl ketones, diphenyl phosphonate esters, trifluoromethyl ketones, peptidyl boronic acids, α-ketoheterocycles, and β-lactam derivatives are usually employed as warheads. on the basis of these warheads, a variety of irreversible and reversible covalent serine protease inhibitors were designed. 275 in this section, representative serine protease inhibitors containing a carbamate functionality will be outlined. carbamate derivative 281 (figure 24 ), a diphenyl phosphonate ester containing a cbz group and bearing a single amino acid side chain, showed very good inhibitory activity against human plasma kallikrein, useful for the treatment of hereditary angioedema. 277, 278 thrombin is an attractive therapeutic target for drug development against pulmonary embolism, thrombosis, and related diseases. 279, 280 compound 282 showed good potency and selectivity against human thrombin. it is stable and displayed no activity against acetylcholinesterase and no selectivity over cysteine proteases. peptidyl boronic acid-based thrombin inhibitors were developed by dupont-merck. in particular, n-boc derived inhibitor 283 is a potent inhibitor (k i = 0.004 nm). 281,282 imperiali and co-workers introduced trifluoromethyl ketones as specific serine protease inhibitors, particularly for chymotrypsin and elastase. 283 researchers at astrazeneca designed numerous peptidyl trifluoromethyl ketone derivatives as potent human elastase inhibitors. 284−286 further optimization of features resulted in the development of a number of orally active inhibitors. in particular, methyl carbamate derivative inhibitor 284 was shown to be a very potent inhibitor (k i = 13 nm) with excellent oral bioavailability in laboratory animals. 286, 287 optically pure compound 284 with an (s)-configuration at the p1 isopropyl side chain became a candidate for clinical development for potential treatment of elastase-implicated respiratory diseases. peptidomimetic boronic acid-based hepatitis c virus (hcv) ns3/4a protease inhibitors were designed and synthesized for the treatment of chronic hcv infections. hcv infections can lead to progressive liver damage, cirrhosis, and liver cancer. 288 the ns3/4a serine protease plays a critical role in virus replication and has become an antiviral drug development target. 289, 290 the first specific and potent hcv protease inhibitor with good oral bioavailability was ciluprevir, which contains a carbamate functionality (285, biln 2061, figure 25 ). this noncovalent macrocyclic peptidic inhibitor was the result of a substrate-based approach for the design of active site inhibitors. this inhibitor is very active in enzymatic (ic 50 = 3 nm) and cell-based replicon assays (ic 50 = 1.2 nm) of hcv genotype 1. 291 ciluprevir was later discontinued due to cardiac toxicity in animal models, but its development paved the way to boceprevir (victrelis, schering-plough, approved by fda in may 2011) and telaprevir (vx-950, vertex pharmaceuticals and johnson & johnson). 292, 293 in particular, for the development of boceprevir, the introduction of a ketoamide moiety, together with p2 and p3 optimization, led to inhibitor 286 showing a k i of 66 nm. 294 its x-ray crystal structure in complex with the enzyme also provided insight for further optimization. indeed, a cyclopropylalanine residue was found to be optimal at p1, and the resulting carbamate derivative 287 showed a k i of 15 nm. although inhibitors 286 and 287 displayed good enzyme inhibitory potency, they did not display cellular activity in a subgenomic hcv replicon assay, possibly because of their strong peptidic character. 295 the discovery that an nmethylated leucine at p2 was critical for both enzymatic potency and cellular activity led to the potential of cyclopropylfused proline being envisaged as an optimum, conformationally constrained surrogate for this part of the inhibitor. combination of the p2-optimized ligand with previously optimized p1 and p3 residues provided carbamate derivative 288 with a k i = 3.8 nm and ic 90 = 100 nm. 296 finally, truncation and p1 optimization, by the employment of a cyclobutyl moiety, led to compound 289 (k i = 76 nm), the direct boceprevir ancestor. 297 subsequently, compounds 290 and 291 ( figure 26 ) with a carbamate containing p2 proline core showed very potent inhibitory activity (ic 50 = 2 nm for 290 and 23 nm for 291). 298 similarly, macrocyclic inhibitor 292 with an α-amino cyclic boronate showed good potency (ic 50 = 43 nm). 299 the electron-withdrawing effect of the ester and amide functionalities was also utilized in the design of α-ketoesteror α-ketoamide-derived transition state inhibitors. 300 a range of hcv ns3/4a protease inhibitors were designed and synthesized, incorporating α-ketoamide templates at the scissile site. structure-based design led to a variety of potent acyclic and cyclic inhibitors with ketoamide templates, as exemplified in compounds 293 (ic 50 tripeptidyl α-ketobenzoxazole 295 inhibited human neutrophil elastase (hne) with an ic 50 of 3 nm. the ketooxazolinederived inhibitor 296 displayed very potent activity against hne (ic 50 = 0.6 nm). 304 8.2. hcv ns5a inhibitors. carbamate derivatives also play a key role as inhibitors of hcv ns5a, which represents a new and promising target for hcv therapy. hcv ns5a is a zincbinding phosphoprotein, and its role in the hcv virus life cycle is still not clear. 305 however, it plays a critical role in hcv rna replication. also, it is involved in virion morphogenesis. 306 due to the lack of enzymatic function, inhibitors of this viral-encoded protein have been pursued. 307 researchers at bristol-myers squibb screened a library of compounds for their ability to inhibit hcv rna replication. this led to the identification of a lead compound specifically interfering with rna replication and later proving to inhibit the activity of ns5a protein. subsequent optimization was focused on broadening the genotype specificity and improving pharmacokinetic properties of compounds. symmetry of the molecule played an important role in inhibitory potency. this finally led to the discovery of daclatasvir (297, bms-790052, figure 28 ) a first-in-class inhibitor of the hcv ns5a replication complex. 308 daclatasvir was approved in europe in august, 2014. ledipasvir (298, gs-5885, gilead sciences, figure 28 ) is another carbamate-containing hcv ns5a inhibitor with potent antiviral activity against hcv genotypes 1a and 1b. 309 harvoni, a combination of ledipasvir and sofosbuvir (a nucleotide polymerase inhibitor), was approved by the fda in october 2014 for the treatment of chronic hcv genotype 1 infection. this also represents the first approved regimen that does not require administration with interferon or ribavirin. 310, 311 9. carbamates as cysteine protease inhibitors cysteine proteases, also known as thiol proteases, are proteolytic enzymes responsible for the degradation of proteins. 312 these enzymes are divided into three classes based on their sequence homology: the papain, caspase, and picornaviridae families. the papain family of proteases is the most known and studied. 313 cysteine proteases have been identified in a variety of diverse organisms, such as bacteria, eukaryotic micro-organisms, plants, and animals and are divided into the clans ca, cd, ce, cf, and ch in the merops peptidase database. the largest subfamily among the class of cysteine proteases is the papain-like cysteine proteases, originating from papain as the archetype of the cysteine proteases. clan ca proteases utilize catalytic cys, his, and asn residues that are invariably in this order in the primary sequence of the protease. clan ca, family c1 (papain-family) cysteine proteases are wellcharacterized for many eukaryotic organisms. also, the best characterized plasmodium cysteine proteases, namely, the falcipains, belong to papain-family (clan ca) enzymes. clan cd presents two catalytic residues, his and cys, in sequence; clan ce has a triad formed by his, glu, or asp and cys at the c-terminus; in clan cf, the asparagine residue of the catalytic triad is replaced by a glutamate residue and the catalytic triad is ordered as glu, cys, and his; clan cg has a dyad of two cysteine residues, and clan ch presents a cys, thr, and his triad with the catalytic cysteine at the n-terminus. the proteolytic mechanism involves the formation of a thiolate−imidazolium ion pair, which provides a highly nucleophilic cysteine thiol. over the years, many cysteine protease inhibitors have been designed by appropriately linking electrophilic warheads to the specific recognition sequence of peptide substrates. reversible inhibitor warheads include aldehydes, α-ketoamides, α-ketoesters, and α-ketoacids. these inhibitors interact with the protease active site, forming the tetrahedral intermediate, but are eventually hydrolyzed, regenerating both the enzyme and the inhibitor in an equilibrium reaction. irreversible inhibitors of cysteine proteases include epoxides, aziridines, haloketones, vinyl sulfones, and acyloxymethylketones. these inhibitors inactivate the target through alkylation of the active site cysteine thiol, permanently disabling enzyme function. 273,313,316−318 the occurrence of severe acute respiratory syndrome (sars) in 2003 and the subsequent identification of a novel coronavirus as the etiological agent recognized cysteine proteases sars-cov 3clpro and sars-cov plpro (papainlike protease) as possible targets for drug design. 319, 320 subsequent structure-based design based on a previous inhibitor's x-ray co-crystal structure with the enzyme 321 provided carbamate derivative 299 ( figure 29 ) as a potent sars-cov 3clpro inhibitor (ic 50 = 80 μm). 322 a wide variety of human rhinovirus 3c (hrv 3c) protease inhibitors were developed by the incorporation of α,βunsaturated carbonyl moieties as warheads. hanzlik et al. reported the first hrv 3c protease inhibitors containing a peptide portion and incorporating α,β-unsaturated esters. 323 the peptide parts were selected based on the substrate cleavage site. the representative carbamate-containing inhibitor 300 ( figure 29 ) showed an ic 50 value of 130 nm. human cathepsin k plays a critical role in bone resorption. in an effort to block bone resorption, noncovalent cathepsin k inhibitors were developed. kim et al. provided carbamate derivative 301 ( figure 29 ) as a noncovalent and reversible cathepsin k (ic 50 = 0.01 μm) and l inhibitor (ic 50 = 0.002 μm). 324 glaxowellcome scientists developed carbamate-containing ketoamide-based cathepsin k inhibitors such as 302 ( figure 29 ) (ic 50 = 0.072 nm). 325 starting from a potent ketone-based inhibitor with unsatisfactory drug-like properties, 326,327 incorporation of p 2 −p 3 elements from the ketoamide-based inhibitor 302 led to a hybrid series of ketone-based cathepsin k inhibitors with improved bioavailability, as exemplified in inhibitor 303 ( figure 29 ) (ic 50 = 4 nm). 328 cathepsin s has been suggested for the development of agents against a range of immune disorders. a new class of nonpeptidic and noncovalent cathepsin s inhibitors was reported in 2007. 329 subsequent structural optimization resulted in a very potent and competitive noncovalent carbamate-containing inhibitor 304 ( figure 29 ) (ic 50 = 20 nm). metabolizing enzyme inhibitors carbamates have been employed in the design of serine hydrolase inhibitiors. in this section, we will focus on inhibitors of endocannabinoid metabolizing enzymes, in which the carbamate functionality plays an important role. the endocannabinoid system is known to be a ubiquitous neuromodulatory system with a wide range of action that can be found in every primitive organism. it is composed of cannabinoid receptors (cbrs), endogenous cannabinoids (endocannabinoids, ecs), and the enzymes responsible for their production, transport, and degradation. 330, 331 ecs are a class of signaling lipids, such as n-arachidonoyl ethanolamine (anandamide, aea), oleamide, and 2-arachidonoyl glycerol (2-ag), that exert their biological actions through the interaction with two g-protein coupled receptors, cb1 and cb2. they modulate a range of responses and processes including pain, inflammation, appetite, motility, sleep, thermoregulation, and cognitive and emotional states. 332 the actions of these signaling lipids are rapidly terminated by cellular reuptake and subsequent hydrolysis operated by a number of enzymes. an attractive approach involved the modulation of the ec system and aimed at eliciting the desirable effects of cbrs activation through the pharmacological inactivation of the main endocannabinoid metabolizing enzymes, namely, monoacylglycerol lipase (magl) and α/β-hydrolase domain containing 6 and 12 (abhd6 and abhd12). these three serine hydrolases account for approximately 99% of 2-ag hydrolysis in the cns, 333 whereas fatty acid amide hydrolase (faah) is responsible for aea inactivation. 330 inactivation of these enzymes would elevate the endogenous concentrations of all of its substrate and consequently prolong and potentiate their beneficial effects on pain and anxiety without evoking the classical cb1r agonists side effects (hypomotility, hypothermia, and catalepsy). monoacylglycerol lipase (magl) is the primary enzyme responsible for the hydrolysis of 2-ag in the cns. 333 about 85% of the total 2-ag hydrolysis in the brain is ascribed to magl. magl is a 33 kda membrane enzyme belonging to the superfamily of the serine hydrolases with a catalytic triad represented by ser122, his269, and asp239. 334 it is ubiquitously present in the brain (cortex, hippocampus, cerebellum, thalamu, and striatum), where it localizes to presynaptic terminals, even if lower levels are found in the brainstem and hypothalamus. a concomitant distribution in membranes as well as in the cytosol has been reported. magl shares a common folding motif called the α/β-hydrolase fold. studies in recent years have shown that magl inhibitors elicit antinociceptive, anxiolytic, and antiemetic responses. magl inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. 335 recently, the potential of magl inhibitors for the therapy of fragile x syndrome has been reported. 336 the early discovered magl inhibitors were molecules able to target the cysteine residues present in the active site of the enzyme. later, the research has been focused on the synthesis of compounds covalently binding to ser241 of the catalytic triad. among them, carbamate 305 (urb602, figure 30 ) was the first selective inhibitor of 2-ag degradation, although its potency remained limited (ic 50 = 28 μm on rat brain). 337 selective magl inhibitors bearing a carbamate scaffold were developed by cravatt and coworkers. 338 inhibitor 306 (jzl184, figure 30 ) exhibited selectivity toward faah in vitro (ic 50 = 3.9 nm and 4 μm for human recombinant magl and faah, respectively). 338 more recently, cravatt and co-workers reported a distinct class of o-hexafluoroisopropyl (hfip) carbamates bearing a reactive group that is bioisosteric with endocannabinoid substrates. 339 the representative compound, 307 (kml29, figure 30 , ic 50 = 5.9 nm, human magl), displays excellent potency and in vivo. in comparison to previously described o-aryl carbamates, inhibitor showed enhanced selectivity over faah and other serine hydrolases. 339 abhd6 gene encodes a ∼35 kda protein containing an nterminal transmembrane region followed by a catalytic domain that includes the canonical gxsxg active-site motif of serine hydrolases. abhd6 is a unique and highly conserved enzyme in mammals and is mainly expressed in the brain, liver, kidney, and brown adipose tissue. as a member of the serine hydrolase class, abhd6 is predicted to hydrolyze esters, amides, or thioester bonds in substrates that could include small molecules, lipids, or peptides. although, the full range of substrates regulated by this enzyme in vivo is currently unknown. 340 recent studies have also shown that abhd6 carbamate inhibitors produce anti-inflammatory and neuroprotective effects in a mouse model of traumatic brain injury. 341 among them, optimized inhibitor 308 ( figure 30 ) displayed an ic 50 value of 70 nm and notable selectivity. 342 faah is a membrane-bound enzyme belonging to the amidase family. the analysis of its crystal structure revealed a core composed of a characteristic ser-ser-lys catalytic triad. 343 the catalytic residues of faah are buried deep within the enzyme and are accessible by two narrow channels. the importance of faah was demonstrated by the generation of faah knockout mice. faah −/− mice showed an elevated resting brain concentration of aea and manifested (i) an analgesic phenotype in both the carrageenan model of inflammatory pain and in the formalin model of spontaneous pain, 344 (ii) a reduction in inflammatory responses, 345 and (iii) improvements in slow wave sleep and memory acquisition. 346, 347 the urb class of compounds was the first class of inhibitors identified for faah, and it is well-represented by 309 (urb597, figure 30 , ic 50 = 4.6 nm). 348 the n-(6phenyl)hexylcarbamate analogue 310 (jp83, figure 30 , ic 50 = 14 nm) is another very potent compound representative of the biphenyl series of inhibitors. 349 gattinoni and co-workers developed a series of oxime carbamate inhibitors. compound 311 ( figure 30 , ic 50 = 8 nm) displayed good affinity and selectivity toward faah. 350 more recently, butini et al. developed a new class of potent and selective faah reversible carbamate inhibitors. 351 among them, compound 312 (nf1245, figure 30 , k i = 0.16 nm on mouse brain faah) showed excellent activity. the compound showed impressive selectivity toward all the enzymes and receptors of the endocannabinoid system. 351 in this perspective, the role of carbamates in drug design and medicinal chemistry has been highlighted. in particular, the perspective covers physical properties of carbamates and the development of novel chemical methodologies overcoming the historical safety and toxicity issues related to their preparation. furthermore, the importance of carbamate-derived compounds in medicinal chemistry and their widespread employment as drugs and prodrugs have been discussed. also showcased is the exploitation of organic carbamates in the development of numerous aspartic acid, serine, and cysteine protease inhibitors. we hope that this perspective will stimulate further use of organic carbamate as a structural motif in drug design and medicinal chemistry. the authors declare no competing financial interest. financial support of this research by the national institutes of health (gm53386) and purdue university is gratefully acknowledged. we would like to thank our colleagues anthony tomaine, heather osswald, and anindya sarkar (purdue university) for helpful discussions. ad, alzheimer's disease; cat-d, cathespsin d; faah, fatty acid amide hydrolase; ppar, peroxisome proliferator-activated receptor; gaba, γ-amino butyric acid; haart, highly active antiretroviral therapy; bis-thf, bis-tetrahydrofuran; bace1, beta-site amyloid precursor protein cleaving enzyme 1; mdr, multidrug resistant; nnrti, non-nucleoside reverse transcriptase inhibitors; adept, antibody-directed enzyme prodrug therapy; gdept, gene-directed enzyme prodrug therapy; mct1, monocarboxylate transporter type 1; smvt, sodiumdependent multivitamin transporter; ces1, carboxylesterases 1; magl, monoacylglycerol lipase ■ references unusually low barrier to carbamate c−n rotation amide resonance in thioand seleno-carbamates: a theoretical study role of conjugation in the stabilities and rotational barriers of formamide and thioformamide. an ab initio valence-bond study on the stabilization of the syn-rotamer of amino acid carbamate derivatives by hydrogen bonding the dipole moment and structure of the carbamate group chemical aspects of the restricted rotation of esters, amides, and related compounds hydrolysis in drug and prodrug metabolismchemistry qualitative structuremetabolism relationships in the hydrolysis of carbamates design, synthesis, and structure−activity relationships of alkylcarbamic acid aryl esters, a new class of fatty acid amide hydrolase inhibitors a mild amide to carbamate transformation electrochemically induced hofmann rearrangement an efficient modification of the hofmann rearrangement: synthesis of methyl carbamates hydrazide und azide organischer saüren i azides: their preparation and synthetic uses ruthenium carbonyl catalyzed reductive carbonylation of aromatic nitro-compoundsa selective route to carbamates an efficient one-pot synthesis of n-alkyl carbamates from primary amines using cs 2 co 3 isocyanates from primary amines and carbon-dioxidedehydration of carbamate anions mechanistic aspects of the copolymerization of co 2 and epoxides by soluble zinc bis(phenoxide) catalysts as revealed by their cadmium analogues selective conversion of carbon dioxide to dimethyl carbonate by molecular catalysis catalytic production of urethanes from amines and alkyl halides in supercritical carbon dioxide efficient cs 2 co 3 -promoted solution and solid phase synthesis of carbonates and carbamates in the presence of tbai nature of n-bromosuccinimide in basic-mediathe true oxidizing species in the hofmann rearrangement preparation of methyl carbamates from primary alkylcarboxamides and arylcarboxamides using hypervalent iodine a versatile modification of the hofmann rearrangement preparation of methyl carbamates via a modified hofmann rearrangement oxidative rearrangement of amides with lead tetraacetate oxidation using quaternary ammonium polyhalides 0.1. an efficient method for the hofmann degradation of amides by use of benzyltrimethylammonium tribromide curtius conversion of acids to amines under neutral conditions via an anthrylmethyl carbamate a concise synthesis of tamiflu: third generation route via the diels− alder reaction and the curtius rearrangement -1,3-dienes: preparation from dienoic acids curtius rearrangement and wolff homologation of functionalized peroxides a convenient route to 1-benzyl 3-aminopyrrolidine and 3-aminopiperidine regioselective synthesis of the 3,4-dihydrofuro[3,2-d]pyrimidin-2(1h)-one skeleton: a new class of compound general route to 2,4,5-trisubstituted piperidines from enantiopure β-amino esters. total synthesis of pseudodistomin b triacetate and pseudodistomin f a solid-phase synthesis of n,n′-disubstituted ureas and perhydroimidazo[1,5-a]pyrazines via the curtius rearrangement reliable protocol for the large scale synthesis of diphenylphosphoryl azide (dppa) an improved method for the synthesis of enantiomerically pure aminoacid ester isocyanates a safe and efficient method for preparation of n,n′-unsymmetrically disubstituted ureas utilizing triphosgene an efficient new protocol for the formation of unsymmetrical tri-and tetrasubstituted ureas recent advances in isocyanate chemistry halogen-free process for the conversion of carbon dioxide to urethanes by homogeneous catalysis a direct synthesis of carbamate ester from carbon dioxide, amine and alkyl halide indium-catalyzed reaction for the synthesis of carbamates and carbonates: selective protection of amino groups evolution of a series of peptidoleukotriene antagonists: synthesis and structure/ activity relationships of 1,3,5-substituted indoles and indazoles rational design of peptide-based hiv proteinase inhibitors triphosgene, a crystalline phosgene substitute 2′-carbonyl-bis(3,5-dioxo-4-methyl-1,2,4-oxadiazolidine): a new reagent for the preparation of carbamates and amides, application to the synthesis of dipeptides 1,1′-bis[6-(trifluoromethyl)benzotriazolyl] oxalate (btbo): a new reactive coupling reagent for the synthesis of dipeptides, esters, and thio esters a new method for protecting amines alkoxycarbonylation of .alpha.,.omega.-diamino acids with 2-(trimethylsilyl)ethyl 4-nitrophenyl carbonate solid phase synthesis of hydantoins using a carbamate linker and a novel cyclization/cleavage step prodrugs for amidines: synthesis and anti-pneumocystis carinii activity of carbamates of 2,5-bis(4-amidinophenyl)furan syntheses of fda approved hiv protease inhibitors substituent effects on p2-cyclopentyltetrahydrofuranyl urethanes: design, synthesis, and x-ray studies of potent hiv-1 protease inhibitors synthesis and biological evaluation of novel allophenylnorstatine-based hiv-1 protease inhibitors incorporating high affinity p2-ligands design and synthesis of stereochemically defined novel spirocyclic p2-ligands for hiv-1 protease inhibitors a novel active ester synthesis reagent (n,n′-disuccinimidyl carbonate) β-elimination of βhydroxyamino acids with disuccinimido carbonate a synthesis of a new type of alkoxycarbonylating reagents from 1,1-bis[6-(trifluoromethyl)benzotriazolyl] carbonate (btbc) and their reactions di-2-pyridyl carbonate: a new efficient coupling agent for the direct esterification of carboxylic acids carbonate promoted alkoxycarbonylation of amines: a convenient synthesis of functionalized carbamates dissuccinimidyl carbonate: a useful reagent for alkoxycarbonylation of amines synthesis and evaluation of novel activated mixed carbonate reagents for the introduction of the 2-(trimethylsilyl)ethoxycarbonyl(teoc)-protecting group re-and si-face-selective nitroaldol reactions catalyzed by a rigid chiral quaternary ammonium salt: a highly stereoselective synthesis of the hiv protease inhibitor amprenavir (vertex 478) enantioselective synthesis of cyclopentyltetrahydrofuran (cp-thf), an important high-affinity p2-ligand for hiv-1 protease inhibitors an efficient synthesis of functionalized urethanes from azides n-nitration of secondary amines with 4-chloro-5-methoxy-2-nitropyridazin-3-one (6h)-carboxylate as carbonyl source: facile and selective synthesis of carbamates and ureas under mild conditions preparation and characterization of copper(i) amides a convenient method for the synthesis of carbamate esters from amines and tetraethylammonium hydrogen carbonate palladium-catalyzed generation of o-allylic urethanes and carbonates from amines/alcohols, carbon dioxide, and allylic chlorides cs 2 co 3 -promoted efficient carbonate and carbamate synthesis on solid phase efficient cs 2 co 3 -promoted solution and solid phase synthesis of carbonates and carbamates in the presence of tbai halogen-free process for the conversion of carbon dioxide to urethanes by homogeneous catalysis nickel-catalyzed dehydrative transformation of co 2 to urethanes parallel synthesis of ureas and carbamates from amines and co 2 under mild conditions the mechanism of the mitsunobu reaction and its application to co 2 fixation pdcl 2 catalyzed efficient assembly of organic azides, co, and alcohols under mild conditions: a direct approach to synthesize carbamates an efficient new protocol for the formation of unsymmetrical tri-and tetrasubstituted ureas a new protocol for the formation of carbamates and thiocarbamates using carbamoyl imidazolium salts carbamoylimidazolium salts as diversification reagents: an application to the synthesis of tertiary amides from carboxylic acids carbamoylimidazolium and thiocarbamoylimidazolium salts: novel reagents for the synthesis of ureas, thioureas, carbamates, thiocarbamates and amides kf/al 2 o 3 mediated organic synthesis competing reactions of secondary alcohols with sodium hypochlorite promoted by phase-transfer catalysis boc-protected amines via a mild and efficient one-pot curtius rearrangement curtius rearrangement of aromatic carboxylic acids to access protected anilines and aromatic ureas one-pot curtius rearrangement processes from carboxylic acids an environmentally benign access to carbamates and ureas synthesis of carbamates using yttria−zirconia based lewis acid catalyst group 3 metal (sc, la) triflates as catalysts for the carbomethoxylation of aliphatic amines with dimethylcarbonate under mild conditions synthesis of carbamates from aliphatic amines and dimethyl carbonate catalyzed by acid functional ionic liquids indium in organic synthesis indium-and galliummediated carbon−carbon bond-forming reactions in organic synthesis recent advances in indium-promoted organic reactions general approach to the coupling of organoindium reagents with imines via copper catalysis 1,2,4-triazoles containing nitro groups convenient method for the preparation of carbamates, carbonates, and thiocarbonates ultrasound promoted 'one pot' conversion of nitrocompounds to carbamates facile procedure for the synthesis of n-aryl-n-hydroxy carbamates the role of alcohol in the catalytic reductive carbonylation of nitrobenzenes to carbamates in the presence of rh reductive carbonylation of nitrobenzenes catalyzed by a new binuclear rhodium complex reductive carbonylation of aromatic dinitro compounds with a palladium(phenanthroline)2(triflate)2 catalyst and an aromatic carboxylic acid as cocatalyst the synthesis of carbamate from the reductive carbonylation of nitrobenzene over pd-based catalysts catalytic activity of pdcl 2 complexes with pyridines in nitrobenzene carbonylation carbamate synthesis via transfunctionalization of substituted ureas and carbonates dialkyl and diaryl carbonates by carbonate interchange reaction with dimethyl carbonate investigation of dialkyltin compounds as catalysts for the synthesis of dialkyl carbonates from alkyl carbamates synthesis of carbamates and ureas using zr(iv)-catalyzed exchange processes unprecedented "in water" imidazole carbonylation: paradigm shift for preparation of urea and carbamate introducing catalytic lossen rearrangements: sustainable access to carbamates and amines carbonyldiimidazole-mediated lossen rearrangement cyanuric chloride: an efficient reagent for the lossen rearrangement a review of rivastigmine: a reversible cholinesterase inhibitor rivastigmine for dementia associated with parkinson's disease the tolerability and safety of cholinesterase inhibitors in the treatment of dementia a novel peroxisome proliferator-activated receptor alpha/gamma dual agonist with efficacious glucose and lipid-lowering activities squibb announces discontinuation of development of investigational oral treatment for type 2 diabetes oral antiplatelet, antithrombotic efficacy of dmp 728, a novel platelet gpiib/iiia antagonist discovery of an orally active series of isoxazoline glycoprotein iib/iiia antagonists a synthetic inhibitor of histone deacetylase, ms-27-275, with marked in vivo antitumor activity against human tumors the histone deacetylase inhibitor ms-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21cip1/waf1 1 enantioselective renal excretion of albendazole metabolites in patients with neurocysticercosis treatment of hydatid disease with high oral doses of mebendazole. long-term follow-up of plasma mebendazole levels and drug interactions teva pharmaceuticals product line changes: mebendazole tablets new anticonvulsant drugs synthesis and quantitative structure-activity relationships of anticonvulsant 2,3,6-triaminopyridines a neuropharmacological evaluation of felbamate as a novel anticonvulsant drug interactions with the newer antiepileptic drugs (aeds)part 1: pharmacokinetic and pharmacodynamic interactions between aeds discontinuation of phenytoin and carbamazepine in patients receiving felbamate effects of felbamate on the pharmacokinetics of phenobarbital efavirenzstill first-line king? prospective, randomized, open label trial of efavirenz vs lopinavir/ ritonavir in hiv+ treatment-naive subjects with cd4+<200 cell/mm 3 in mexico efavirenz primary and secondary metabolism in vitro and in vivo: identification of novel metabolic pathways and cytochrome p450 2a6 as the principal catalyst of efavirenz 7-hydroxylation the cytochrome p450 2b6 (cyp2b6) is the main catalyst of efavirenz primary and secondary metabolism: implication for hiv/aids therapy and utility of efavirenz as a substrate marker of cyp2b6 catalytic activity impact of cyp2b6 polymorphism on hepatic efavirenz metabolism in vitro a randomized, doubleblind, placebo-controlled trial of the effect of zafirlukast on upper and lower respiratory responses to cat challenge evolution of a series of peptidoleukotriene antagonists: synthesis and structure/ activity relationships of 1,3,5-substituted indoles and indazoles pharmacokinetic profile of zafirlukast crosslinking of dna by enzymatically or chemically activated mitomycins and porfiromycins, bifunctionally "alkylating" antibiotics sequence preferences of dna interstrand cross-linking agentsimportance of minimal dna structural reorganization in the cross-linking reactions of mechlorethamine, cisplatin, and mitomycin-c dna interstrand cross-linking by 2,5-bis(1-aziridinyl)-1,6-benzoquinonenucleotide-sequence preferences and covalent structure of the dg-to-dg crosslinks at 5′-d(gn(n)c) in synthetic oligonucleotide duplexes mitomycin c inhibits ribosomal rna: a novel cytotoxic mechanism for bioreductive drugs mitomycin c: mechanism of action, usefulness and limitations active human immunodeficiency virus protease is required for viral infectivity structure-based design: from renin to hiv protease three-dimensional structure of aspartyl protease from human immunodeficiency virus hiv-1 conserved folding in retroviral proteases: crystal structure of a synthetic hiv-1 protease x-ray analysis of hiv-1 proteinase at 2.7 a resolution confirms structural homology among retroviral enzymes new drugsreports of new drugs recently approved by the fda. ritonavir structure and mechanism of the complex between cytochrome p4503a4 and ritonavir in vitro antiviral activity of 141w94 (vx-478) in combination with other antiretroviral agents 3-tetrahydrofuran and pyran urethanes as high-affinity p2-ligands for hiv-1 protease inhibitors crystal structure of hiv-1 protease in complex with vx-478, a potent and orally bioavailable inhibitor of the enzyme safety and pharmacokinetics of amprenavir (141w94), a human immunodeficiency virus (hiv) type 1 protease inhibitor, following oral administration of single doses to hiv-infected adults new aza-dipeptide analogues as potent and orally absorbed hiv-1 protease inhibitors: candidates for clinical development bms-232632, a highly potent human immunodeficiency virus protease inhibitor that can be used in combination with other available antiretroviral agents clinical pharmacokinetics and summary of efficacy and tolerability of atazanavir design of hiv protease inhibitors targeting protein backbone: an effective strategy for combating drug resistance novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (pi) uic-94017 (tmc114) with potent activity against multi-pi-resistant human immunodeficiency virus in vitro tmc114, a novel human immunodeficiency virus type 1 protease inhibitor active against protease inhibitor-resistant viruses, including a broad range of clinical isolates fda approves new hiv treatment for patients who do not respond to existing drugs clinical pharmacokinetics of darunavir p-glycoprotein mediates efflux transport of darunavir in human intestinal caco-2 and abcb1 gene-transfected renal llc-pk1 cell lines absorption, metabolism, and excretion of darunavir, a new protease inhibitor, administered alone and with lowdose ritonavir in healthy subjects challenges and rewards prodrugs: design and clinical applications 659−670. (195) florencio zaragoza, d. lead optimization for medicinal chemists: pharmacokinetic properties of functional groups and organic compounds niculescu-duvaz, i. optimization of alkylating agent prodrugs derived from phenol and aniline mustards: a new clinical candidate prodrug (zd2767) for antibody-directed enzyme prodrug therapy (adept) niculescu-duvaz, i. i. antibody-directed enzyme prodrug therapy (adept): a review introduction to the background, principles, and state of the art in suicide gene therapy a phase i trial of antibody directed enzyme prodrug therapy (adept) in patients with advanced colorectal carcinoma or other cea producing tumours zd2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumor regressions in colorectal tumor xenografts a higher yielding synthesis of the clinical prodrug zd2767p using di-protected 4-[n,n-bis(2-hydroxyethyl)amino]-phenyl chloroformate structural insights into cpt-11 activation by mammalian carboxylesterases piperidino)-1-amino]-carbonyloxycamptothecin, by human carboxylesterases ces1a1, ces2, and a newly expressed carboxylesterase isoenzyme bioactivation of the anticancer agent cpt-11 to sn-38 by human hepatic microsomal carboxylesterases and the in vitro assessment of potential drug interactions hydrolysis of 3h-bambuterol, a carbamate prodrug of terbutaline, in blood from humans and laboratory animals in vitro oral bambuterol versus terbutaline in patients with asthma cyclization-activated prodrugs. basic carbamates of 4-hydroxyanisole cyclization-activated prodrugs. basic esters of 5-bromo-2′-deoxyuridine synthesis of self-immolative glucuronide-based prodrugs of a phenol mustard. anti-cancer drug des bioreduction activated prodrugs of camptothecin: molecular design, synthesis, activation mechanism and hypoxia selective cytotoxicity synthesis and biological evaluation of 2′-carbamate-linked and 2′-carbonate-linked prodrugs of paclitaxel: selective activation by the tumor-associated protease plasmin elongated multiple electronic cascade and cyclization spacer systems in activatible anticancer prodrugs for enhanced drug release alpha-isobutanoyloxyethoxy)-carbonyl] aminomethyl)-1-cyclohexane acetic acid], a novel gabapentin prodrug: ii. improved oral bioavailability, dose proportionality, and colonic absorption compared with gabapentin in rats and monkeys ] aminomethyl)-1-cyclohexane acetic acid], a novel gabapentin prodrug: i. design, synthesis, enzymatic conversion to gabapentin, and transport by intestinal solute transporters clinical pharmacokinetics of xp13512, a novel transported prodrug of gabapentin hydrolysis of capecitabine to 5′-deoxy-5-fluorocytidine by human carboxylesterases and inhibition by loperamide design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue rational development of capecitabine capecitabine: a review profound and sustained inhibition of platelet aggregation by fradafiban, a nonpeptide platelet glycoprotein iib/iiia antagonist, and its orally active prodrug, lefradafiban, in men bis-tetrahydrofuran: a privileged ligand for darunavir and a new generation of hiv protease inhibitors that combat drug resistance enhancing protein backbone bindinga fruitful concept for combating drug-resistant hiv crystal structure of an in vivo hiv-1 protease mutant in complex with saquinavir: insights into the mechanisms of drug resistance potent hiv protease inhibitors: the development of tetrahydrofuranylglycines as novel p2-ligands and pyrazine amides as p3-ligands anti-hiv protease inhibitor darunavir inhibitors of hiv-1 protease containing the novel and potent (r)-(hydroxyethyl)sulfonamide isostere design and synthesis of amprenavir, a novel hiv protease inhibitor the development of cyclic sulfolanes as novel and high-affinity p2 ligands for hiv-1 protease inhibitors nonpeptidal p2 ligands for hiv protease inhibitors: structure-based design, synthesis, and biological evaluation structure-based design of non-peptide hiv protease inhibitors potent hiv protease inhibitors incorporating high-affinity p2-ligands and (r)-(hydroxyethylamino)sulfonamide isostere high resolution crystal structures of hiv-1 protease with a potent non-peptide inhibitor (uic-94017) active against multi-drug-resistant clinical strains ultra-high resolution crystal structure of hiv-1 protease mutant reveals two binding sites for clinical inhibitor tmc114 a potent human immunodeficiency virus type 1 protease inhibitor, uic-94003 (tmc-126), and selection of a novel (a28s) mutation in the protease active site potent inhibition of hiv-1 replication by novel nonpeptidyl small molecule inhibitors of protease dimerization design of hiv-1 protease inhibitors active on multidrug-resistant virus darunavir, a conceptually new hiv-1 protease inhibitor for the treatment of drugresistant hiv darunavir, a new pi with dual mechanism: from a novel drug design concept to new hope against drug-resistant hiv stereoselective photochemical 1,3-dioxolane addition to 5-alkoxymethyl-2(5h)-furanone: synthesis of bis-tetrahydrofuranyl ligand for hiv protease inhibitor uic-94017 (tmc-114) synthesis and optical resolution of high affinity p2-ligands for hiv-1 protease inhibitors a novel bistetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (pi), grl-98065, is potent against multiple-pi-resistant human immunodeficiency virus in vitro ultra-potent p1 modified arylsulfonamide hiv protease inhibitors: the discovery of gw0385 suppression of hiv-1 protease inhibitor resistance by phosphonate-mediated solvent anchoring tmc310911, a novel human immunodeficiency virus type 1 protease inhibitor, shows in vitro an improved resistance profile and higher genetic barrier to resistance compared with current protease inhibitors flexible cyclic ethers/polyethers as novel p2-ligands for hiv-1 protease inhibitors: design, synthesis, biological evaluation, and protein-ligand x-ray studies design and synthesis of potent hiv-1 protease inhibitors incorporating hexahydrofuropyranol-derived high affinity p(2) ligands: structure-activity studies and biological evaluation probing multidrug-resistance and protein-ligand interactions with oxatricyclic designed ligands in hiv-1 protease inhibitors grl-04810 and grl-05010, difluoride-containing nonpeptidic hiv-1 protease inhibitors (pis) that inhibit the replication of multi-pi-resistant hiv-1 in vitro and possess favorable lipophilicity that may allow blood-brain barrier penetration the discovery of β-secretase and development toward a clinical inhibitor for ad: an exciting academic collaboration design of potent inhibitors for human brain memapsin 2 (βsecretase) structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor structure-based design of cycloamide-urethane-derived novel inhibitors of human brain memapsin 2 (beta-secretase) cathepsin d is membrane-associated in macrophage endosomes design, synthesis and x-ray structure of protein-ligand complexes: important insight into selectivity of memapsin 2 (beta-secretase) inhibitors design of potent inhibitors of human beta-secretase design of potent inhibitors of human beta-secretase. part 2 chronic treatment with the gammasecretase inhibitor ly-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation studies of abeta pharmacodynamics in the brain, cerebrospinal fluid, and plasma in young (plaque-free) tg2576 mice using the gammasecretase inhibitor n2-[(2s)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-n1-[(7s)-5-methyl-6-oxo-6,7-dihydro-5h-dibenzo quantitative measurement of changes in amyloid-beta(40) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor ly-411575 novel orally active, dibenzazepinone-based gamma-secretase inhibitors design, synthesis, and evaluation of tetrahydroquinoline and pyrrolidine sulfonamide carbamates as gamma-secretase inhibitors discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors 2,6-disubstituted n-arylsulfonyl piperidines as gamma-secretase inhibitors nitrogen-appended n-alkylsulfonamides as inhibitors of gamma-secretase carbamate-appended nalkylsulfonamides as inhibitors of gamma-secretase serine proteases of the human immune system in health and disease proteases: multifunctional enzymes in life and disease irreversible inhibitors of serine, cysteine, and threonine proteases strategies for the inhibition of serine proteases protease inhibitors: current status and future prospects structural basis of substrate specificity in the serine proteases novel amidine-containing peptidyl phosphonates as irreversible inhibitors for blood coagulation and related serine proteases synthesis and kinetic studies of diphenyl 1-(npeptidylamino)alkanephosphonate esters and their biotinylated derivatives as inhibitors of serine proteases and probes for lymphocyte granzymes thrombin inhibitors: a new generation of antithrombotics the current status of coagulation the solution conformation of (d)phe-pro-containing peptides: implications on the activity of ac-(d)phe-pro-boroarg-oh, a potent thrombin inhibitor synthesis of conformationally-restricted boropeptide thrombin inhibitors inhibition of serine proteases by peptidyl fluoromethyl ketones nonpeptidic inhibitors of human leukocyte elastase. 5. design, synthesis, and x-ray crystallography of a series of orally active 5-aminopyrimidin-6-one-containing trifluoromethyl ketones nonpeptidic inhibitors of human leukocyte elastase. 6. design of a potent, intratracheally active, pyridone-based trifluoromethyl ketone discovery and biological activity of orally active peptidyl trifluoromethyl ketone inhibitors of human neutrophil elastase orally active trifluoromethyl ketone inhibitors of human leukocyte elastase clinical consequences of hepatitis c virus infection challenges in modern drug discovery: a case study of boceprevir, an hcv protease inhibitor for the treatment of hepatitis c virus infection hepatitis c virus ns3-4a protease inhibitors: countering viral subversion in vitro and showing promise in the clinic an ns3 protease inhibitor with antiviral effects in humans infected with hepatitis c virus discovery of (1r,5s)-n-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl sch 503034, a mechanism-based inhibitor of hepatitis c virus ns3 protease, suppresses polyprotein maturation and enhances the antiviral activity of alpha interferon in replicon cells hepatitis c virus ns3-4a serine protease inhibitors: sar of p′2 moiety with improved potency depeptidization efforts on p3-p2′ alpha-ketoamide inhibitors of hcv ns3-4a serine protease: effect on hcv replicon activity discovery of sch446211 (sch6): a new ketoamide inhibitor of the hcv ns3 serine protease and hcv subgenomic rna replication discovery of boceprevir, a direct-acting ns3/ 4a protease inhibitor for treatment of chronic hepatitis c infections synthesis and antiviral activity of hcv ns3/4a peptidomimetic boronic acid inhibitors novel macrocyclic hcv ns3 protease inhibitors derived from alpha-amino cyclic boronates inhibition of cathepsin b and papain by peptidyl alpha-keto esters, alpha-keto amides, alpha-diketones, and alpha-keto acids design, synthesis, and biological activity of m-tyrosine-based 16-and 17-membered macrocyclic inhibitors of hepatitis c virus ns3 serine protease in vitro antiviral activity of sch446211 (sch6), a novel inhibitor of the hepatitis c virus ns3 serine protease discovery and structure−activity relationship of p1−p3 ketoamide derived macrocyclic inhibitors of hepatitis c virus ns3 protease synthetic inhibitors of elastase hcv-targeted antivirals: current status and future challenges small molecule inhibitors of the hepatitis c virus-encoded ns5a protein chemical genetics strategy identifies an hcv ns5a inhibitor with a potent clinical effect modeling shows that the ns5a inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis c virus half-life a phase 1, randomized, placebocontrolled, 3-day, dose-ranging study of gs-5885, an ns5a inhibitor, in patients with genotype 1 hepatitis c ledipasvir and sofosbuvir for untreated hcv genotype 1 infection fda approves first combination pill to treat hepatitis c evolutionary lines of cysteine peptidases thiol proteases: inhibitors and potential therapeutic targets review: novel cysteine proteases of the papain family cysteinyl proteinases and their selective inactivation michael acceptors as cysteine protease inhibitors use of cysteine-reactive small molecules in drug discovery for trypanosomal disease identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors structure-based design, synthesis, and biological evaluation of peptidomimetic sars-cov 3clpro inhibitors synthesis and evaluation of peptidyl michael acceptors that inactivate human rhinovirus 3c protease and inhibit virus replication 4-piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin k inhibitors p2−p3 conformationally constrained ketoamide-based inhibitors of cathepsin k azepanone-based inhibitors of human and rat cathepsin k an azepanone-based inhibitor of human cathepsin k with improved oral bioavailability in the rat and the monkey acyclic, orally bioavailable ketone-based cathepsin k inhibitors nonpeptidic, noncovalent inhibitors of the cysteine protease cathepsin s discovery and development of fatty acid amide hydrolase (faah) inhibitors the endocannabinoid system: drug targets, lead compounds, and potential therapeutic applications cannabinoid receptors: where they are and what they do a comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol crystal structure of the human monoacylglycerol lipase, a key actor in endocannabinoid signaling therapeutic potential of monoacylglycerol lipase inhibitors uncoupling of the endocannabinoid signalling complex in a mouse model of fragile x syndrome urb602 inhibits monoacylglycerol lipase and selectively blocks 2-arachidonoylglycerol degradation in intact brain slices selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects highly selective inhibitors of monoacylglycerol lipase bearing a reactive group that is bioisosteric with endocannabinoid substrates discovery and optimization of piperidyl-1,2,3-triazole ureas as potent, selective, and in vivo-active inhibitors of alpha/beta-hydrolase domain containing 6 (abhd6) selective inhibition of alpha/betahydrolase domain 6 attenuates neurodegeneration, alleviates blood brain barrier breakdown, and improves functional recovery in a mouse model of traumatic brain injury a functional proteomic strategy to discover inhibitors for uncharacterized hydrolases structural adaptations in a membrane enzyme that terminates endocannabinoid signaling mice lacking fatty acid amide hydrolase exhibit a cannabinoid receptormediated phenotypic hypoalgesia the endogenous cannabinoid system protects against colonic inflammation characterization of the sleep-wake patterns in mice lacking fatty acid amide hydrolase inhibition of fatty-acid amide hydrolase accelerates acquisition and extinction rates in a spatial memory task. neuropsychopharmacology design, synthesis, and structure-activity relationships of alkylcarbamic acid aryl esters, a new class of fatty acid amide hydrolase inhibitors mechanism of carbamate inactivation of faah: implications for the design of covalent inhibitors and in vivo functional probes for enzymes a new group of oxime carbamates as reversible inhibitors of fatty acid amide hydrolase discovery of potent inhibitors of human and mouse fatty acid amide hydrolases key: cord-297530-7zbvgvk8 authors: kühnert, denise; wu, chieh-hsi; drummond, alexei j. title: phylogenetic and epidemic modeling of rapidly evolving infectious diseases date: 2011-08-31 journal: infect genet evol doi: 10.1016/j.meegid.2011.08.005 sha: doc_id: 297530 cord_uid: 7zbvgvk8 epidemic modeling of infectious diseases has a long history in both theoretical and empirical research. however the recent explosion of genetic data has revealed the rapid rate of evolution that many populations of infectious agents undergo and has underscored the need to consider both evolutionary and ecological processes on the same time scale. mathematical epidemiology has applied dynamical models to study infectious epidemics, but these models have tended not to exploit – or take into account – evolutionary changes and their effect on the ecological processes and population dynamics of the infectious agent. on the other hand, statistical phylogenetics has increasingly been applied to the study of infectious agents. this approach is based on phylogenetics, molecular clocks, genealogy-based population genetics and phylogeography. bayesian markov chain monte carlo and related computational tools have been the primary source of advances in these statistical phylogenetic approaches. recently the first tentative steps have been taken to reconcile these two theoretical approaches. we survey the bayesian phylogenetic approach to epidemic modeling of infection diseases and describe the contrasts it provides to mathematical epidemiology as well as emphasize the significance of the future unification of these two fields. molecular phylogenetics has had a profound impact on the study of infectious diseases, particularly rapidly evolving infectious 1567-1348 ó 2011 elsevier b.v. doi:10.1016/j.meegid.2011.08.005 agents such as rna viruses. it has given insight into the origins, evolutionary history, transmission routes and source populations of epidemic outbreaks and seasonal diseases. one of the key observations about rapidly evolving viruses is that the evolutionary and ecological processes occur on the same time scale ). this is important for two reasons. first, it means that neutral genetic variation can track ecological processes and population dynamics, providing a record of past evolutionary events (e.g., genealogical relationships) and past ecological/population events (geographical spread and changes in population size and structure) that were not directly observed. second, the concomitance of evolutionary and ecological processes leads to their interaction that, when non-trivial, necessitates joint analysis. arguably the most studied infectious disease agent to date has been human immunodeficiency virus (hiv) and it has been the subject of thousands of phylogenetic studies. these have shed light on many aspects of hiv evolutionary biology, epidemiology, origins, phylogeography, transmission dynamics and drug resistance. in fact, the vast body of literature on hiv makes it clear that almost every aspect of the biology of a rapidly evolving pathogen can be better understood in the context of the evolution of the virus. whether it is retracing the zoonotic origins of the hiv pandemic or describing the interplay between the virus population and its host's immune system, a phylogenetic analysis frequently sheds light. although probabilistic modeling approaches to phylogenetics predate sanger sequencing (edwards and cavalli-sforza, 1965) , it was not until the last decade that probabilistic modeling became the dominant approach to phylogeny reconstruction. part of that dominance has been due to the rise of bayesian inference (huelsenbeck et al., 2001) , with its great flexibility in describing prior knowledge, its ability to be applied via the metropolis-hastings algorithm to complex highly parametric models, and the ease with which multiple sources of data can be integrated into a single analysis. the history of probabilistic models of molecular evolution and phylogenetics is a history of gradual refinement; a process of selection of those modeling variations that have the greatest utility in characterizing the ever-growing empirical data. the utility of a new model has been evaluated either by how well it fits the data (formal model comparison or goodness-of-fit tests) or by the new questions that it allows a researcher to ask of the data. in this review we will describe the modern phylogenetic approach to the field of infectious diseases, and particularly with reference to bayesian inference of the phylogenetic epidemiology of rapidly evolving viral pathogens such as hepatitis c virus (hcv), hiv and influenza a virus. the review is separated into two main sections. in section 2 we discuss phylogenetic methods for reconstructing the history of infectious epidemics, including identification of origins, dating of common ancestors, relaxed phylogenetics and coalescent-based population dynamics. in section 3 we review epidemiological models and finish by outlining progress in the development of phylodynamical models that marry statistical phylogenetics with dynamical modeling. the introduction of an efficient means of calculating the probability of a sequence alignment given a phylogenetic tree (known as the phylogenetic likelihood; felsenstein, 1981) heralded the beginning of practical phylogenetic tree reconstruction in a statistical framework. at around the same time the coalescent was introduced: a theory relating the shape of the genealogy of a random sample of individuals to the size of the population from which they came (kingman, 1982 ; see section 2.3 for details). both of these advances have been subsequently developed to the point that, together they enable the estimation of viral evolutionary histories and past population dynamics. bayesian inference brings together the likelihood, pr(djh) (the probability of the data given the model parameters) and the prior, p(h) (the probability of the model parameters prior to seeing the data), so that the posterior probability of the model parameters (h) given the data is: in a standard phylogenetic setting, the probabilistic model parameters include the phylogenetic tree, coalescent times and substitution parameters, and a prior probability distribution over these parameters must be specified. by using kingman's coalescent as a prior density on trees, bayesian inference can be used to simultaneously estimate the phylogeny of the viral sequences and the demographic history of the virus population (drummond et al., 2002 (drummond et al., , 2005 , see box 1). extension of phylogenetic inference methods to accommodate time-stamped sequence data (rambaut, 2000; drummond et al., 2002) and relaxation of the assumption of a strict molecular clock (thorne et al., 1998; kishino et al., 2001; sanderson, 2002; drummond et al., 2006; rannala and yang, 2007) provided sophisticated methods for ancestral divergence time estimation. for virus species that occupy more than one host species (e.g influenza a), models that aim to detect cross-species transmission may provide clues to the origin of a virus strain in a host population (reis et al., 2009 ). when a new epidemic emerges, one of the first goals is to trace it back to its genetic and geographic origin. the reconstruction of phylogenetic trees to infer the evolutionary relationships has been a key tool to uncover the origin of regional epidemics such as those resulting from hiv (gao et al., 1999; santiago et al., 2002) , hcv markov et al., 2009 ) and sars coronavirus (sars-cov) . some studies have also attempted to use phylogenetic trees to draw conclusions about transmission history and geographic spread of viral epidemics (motomura et al., 2003; santiago et al., 2005; gilbert et al., 2007) . however, great care should be taken when coming to conclusions about aspects of the epidemic process that are not explicitly modeled in the reconstruction of the phylogenetic tree and even if they are, the user needs to consider the appropriateness of the underlying model assumptions. one common and straightforward method used to identify the origin of an epidemic involves determining the non-epidemic genotype or lineage most closely related to the epidemic, i.e., the molecular sequences clustered most closely with the epidemic strain on a phylogenetic tree. while the method is intuitive, its success heavily depends on the collected data. the closest simian immunodeficiency virus (siv) relative of hiv-1 is sivcpz (gao et al., 1999; santiago et al., 2002) , which is harbored in chimpanzee sub-species pan troglodytes troglodytes and p.t. schweinfurthii in the form of the respective sub-species specific siv lineages sivcpzptt and sivcpzpts. although sivcpz became the prime candidate for the zoonotic source of hiv-1 as soon as it was identified, alternative sources could not be ruled out due to the paucity of identified chimpanzee infections (vanden haesevelde et al., 1996) . the source of hiv-1 was confirmed much later after the collection of sivcpz from fecal samples of wild p. t. troglodytes apes in the cameroon forest . hiv-1 groups m and n are much more closely related to sequences from the fecal samples than previously identified sivcpz strains. this finding uncovered the distinct origins of hiv-1 group m (pandemic) and group n (non-pandemic) traced to chimpanzee communities of southeastern and central cameroon respectively. the precise geographic identification of these wildlife chimpanzee reservoirs of hiv-1 by phylogenetic techniques provided the crucial evidence that sivcpz gave rise to the hiv/aids pandemic. conversely, if strains sufficiently closely related to the epidemic strain cannot be identified then phylogenetic trees are not able to easily provide answers about origins. for example, there has been much heated debate on the origin of the 1918 h1n1 influenza a pandemic -whether its source was avian, non-human mammalian or even human. the uncertainty mainly stems from the absence of sequences from the immediate ancestral source population of the 1918 virus (gibbs and gibbs, 2006) . a similar, though less severe problem has been encountered with the search for the origin of hiv-1 o group. strains of hiv-1 o group have been revealed to be most closely related to sivgor found in western lowland gorillas (gorilla gorilla gorilla) takehisa et al., 2009 ). however, hiv-o sequences are moderately divergent from the known sivgor sequences and consequently, the route of transmission that has given rise to hiv-1 o group and sivgor is still indeterminate. the interspersion of an emergent viral strain with other strains in a phylogenetic tree is often interpreted as evidence supporting multiple independent viral introductions. for example, hiv lineages are paraphyletic with siv lineages creating several separate clusters of hiv suggesting multiple zoonotic viral transmissions into the human population (santiago et al., 2005; keele et al., 2006) . while it is intuitive that separate clusters of the emergent virus suggest multiple introductions, it is not clear from the number of clusters alone how many independent events are responsible for the observed pattern. incomplete taxon sampling will lead to undercounting. for example, there may exist an unsampled sequence that will split an emergent viral cluster, or an additional unsampled emergent cluster. both scenarios, if detected, would increase the lower bound of the inferred number of events. the number of events could also be incorrectly estimated due to phylogenetic estimation error. finally, in situations where the event is potentially reversible, such as with drug-resistance mutations, e.g., adamantane resistance in h3n2 influenza virus (nelson et al., 2009) , it is quite possible that reversions are also present in the phylogenetic history, and these are not always detectable by a simple parsimony reconstruction, again leading to undercounting. for all these reasons, the applications of bayesian modeling of phylogeography and character evolution on phylogenies is crucial to quantitatively assess the uncertainty generated from these different sources of error (see section 2.4). in contrast to hiv-1, it has been clearly established for almost two decades that the progenitor of hiv-2 is sivsm from sooty mangabey (cerocebus torquatus atys) (hirsch et al., 1989; gao et al., 1992) . it was suggested by (santiago et al., 2005) that the geographic origin of hiv-2 groups a and b are in the eastern sooty mangabey range according to the clear geographic clustering displayed in the phylogenetic tree and branching position of the hiv-2 strains. although this heuristic approach to locating phylogeographic origins is commonly used, it has several disadvantages aside from the sampling error mentioned earlier. first, it relies on strong geographic signals to produce an unambiguous geographic clustering pattern in the trees. second, the lack of a formal statistical framework results in an inability to quantify the associated uncertainty with the geographic estimates. a number of statistical phylogenetic methods aim to reconstruct the migration process by treating geographic locations as another state that evolves down the tree. the states are either discrete (lemey et al., 2009b) , denoted by names of cities or provinces, or continuous represented by the latitude and longitude of the location (biek et al., 2006 (biek et al., , 2007 lemey et al., 2010) . even with comprehensive sampling, using a single phylogenetic tree is insufficient to reflect the complex genetic origin of virus species that undergo recombination or reassortment. reassortment arises when segments of the viral genome come from different viruses, while recombination also requires the genetic material from one source to (break and) join with that from another. these two processes enable the generation of novel combinations from two existing genotypes. moreover, these often large genetic changes may provide the potential for adaptation to a new host species (parrish et al., 2008) . reassortment has played an important role in the evolution of the influenza a virus (lindstrom et al., 2004; holmes et al., 2005; nelson et al., 2008) . evidence for recombination have also been found in dengue (holmes et al., 1999) , hiv, hcv and sars-cov . there are many phylogenetic methods that aim to detect recombination by identifying discordance in the topologies of different parts of the alignment (grassly and holmes, 1997; salminen et al., 1995; lole et al., 1999; smith, 1992; robertson et al., 1995; paraskevis et al., 2005) , which is a potential consequence of recombination. most of these methods use a sliding window approach to compute a summary statistic along the length of sequence. phylogenetic approaches are based on estimating either (i) bootstrap values or (ii) clade posterior probabilities for each window and a sudden change in bootstrap value, clade posterior probability or site percentage identity is an indication of the presence of a breakpoint around the region. other methods explicitly estimate the position of the breakpoint in an alignment, providing access to test the strength of support for recombination (holmes et al., 1999) . finally, some approaches portray the evolutionary history by networks to incorporate horizontal transfer (huson, 1998) or ancestral recombination graphs (bloomquist and suchard, 2010) . as a rule, rna viruses mutate rapidly, so that viruses isolated only a few months apart may exhibit measurable genetic differences (drummond et al., 2003a and references therein) . indeed, the mutation rate of some rna viruses is so high that it can result in evolutionary changes within a host during the course of infection. this is particularly true of long term chronic infections caused by viruses such as hiv and hcv. it is therefore not appropriate to consider the analysis of sequences that have been sampled years apart as if they are contemporaneous. sequence data with this type of temporal structure are called heterochronous and from such data the substitution rate can be estimated and divergence times calibrated to a calendar scale. here, a tree with branch lengths in calendar units is termed a ''time tree''. fig. 1 depicts an example of a serially sampled time tree of a rapidly evolving virus. to account for temporal structure in sequence data, the earliest methods estimated the time scale by estimating a gene tree with unconstrained branch lengths and then performing a linear regression of root-to-tip genetic distance against sampling times (see for review drummond et al., 2003b) . this method was used to provide the first estimate of the time of the most recent common ancestor (t mrca ) of hiv-1 m group, placing it in the 1930s (korber et al., 2000) . despite its simplicity, this method also accurately estimated the age of the oldest hiv sequence sampled in 1959. a maximum likelihood based method (the single rate dated tips (srdt) model; rambaut, 2000) , estimates ancestral divergence times and overall substitution rate on a fixed tree, assuming a strict molecular clock. the srdt model was used to date the most recent common ancestor of hiv-2 subtype a in 1940 ±16 and that of subtype b in 1945 ±14 (lemey et al., 2003) . using the serial coalescent as a tree prior in bayesian coalescent methods (drummond et al., 2002 (drummond et al., , 2005 drummond and rambaut, 2007) allows the time scale to be simultaneously estimated with other phylogenetic and demographic parameters. recently, a relaxed clock bayesian coalescent analysis that included two historical viral samples from 1959 (zr59) and 1960 (drc60) (worobey et al., 2008) , pushed back the estimated t mrca of hiv-1 m group to 1908 hiv-1 m group to (1884 hiv-1 m group to -1924 . besides estimating the time of an epidemic outbreak, it may also be important to know how long the ancestors of the epidemic strain had circulated in the source population prior to the epidemic. this can sometimes be indicated by the length of the branch ancestral to the epidemic clade. in the case of the 2009 swine-origin influenza a virus, the length of the branch leading to 2009 s-oiv strains is estimated to be 9-17 years depending on the viral segment analyzed, suggesting roughly a decade of unsampled diversity (smith et al., 2009) . to estimate the age of the common ancestor of sivsm strains, the t mrca of hiv-2/sivsm has been dated, indicating that the common ancestry prior the zoonosis of hiv-2 group a and b spans only the last few centuries (wertheim and worobey, 2009 ). this does not necessarily indicate that sivsm first arose only centuries ago, just that the common ancestor of all current sivsm may be recent. however, even this conclusion has recently been questioned (worobey et al., 2010) as a result of independent calibration evidence that suggests the t mrca could in fact be greater than 32,000 years ago, leading to debate about the fidelity of the statistical substitution models commonly employed for divergence time dating when the true divergence times are very ancient compared to the sampling interval. as demonstrated by wertheim and pond, 2011 , substitution models that do not take into account the effects of selection can produce underestimated branch lengths leading to much younger age estimates in presence of purifying selection. this will be more problematic for data sets for which the total sampling interval is only a small fraction of the total age of the tree. while incorporating sampling dates provides additional information to phylogenetic inference, it also implies that the reliability of those dates has a heavy impact on the validity of the inference. the h1n1 influenza virus that re-emerged in 1977 was found to have missed decades of evolution and was genetically remarkably similar to the h1n1 1950 virus (nakajima et al., 1978) . it is thus thought to be descended from a strain that was kept frozen in an unknown laboratory for perhaps decades before again becoming a ''wild'' strain again (zimmer and burke, 2009 ). if the missing evolution is not corrected for, analyses including the re-emergent strains produce biased date estimates and increased variances of the t mrca of the re-emergent lineages and across the phylogeny (wertheim, 2010) . in cases where the sampling dates of sequences are contentious or unknown, a method that can handle sequences with unknown dates is required. for example, the leaf-dating method estimates the unknown date or age of a sequence as a parameter, treating it the same way as the age of internal nodes (drummond et al., 2003c; nicholls and gray, 2008; shapiro et al., 2010) . unrealistic sampling dates may also be the result of human error and are thus not recognized prior to an analysis. therefore, diagnostics for unrealistic dates are important to pick up errors in the recorded dates. one possible method is to plot the root-to-tip genetic distance against sampling year if the virus does not display significant departure from constant rate (wertheim, 2010) . another is to check calibrations by dropping each calibration point in turn and re-estimating the date to confirm that the estimated dates are consistent ryder and nicholls, 2011) . early methods that accommodated heterochronous data assumed a strict clock model. however, a comprehensive study of heterochronous rna viral sequences using the srdt model (rambaut, 2000) demonstrated that the majority of the 50 rna viral species studied rejected the constant rate molecular clock hypothesis (jenkins et al., 2002) . the unrooted phylogeny is the other extreme of the scale of rate variability across branches of a phylogenetic tree. neither of them is a realistic representation of the underlying evolutionary process and the reality lies somewhere between the two. this has spawned the development of numerous methods that relax the molecular clock assumption and differ in their assumption of the pattern of rate variation across the branches. the local clock model approach assigns different rates to clades/ regions of the tree. however, without external information, it is difficult to know a priori what is the best partitioning of the tree into local clock models. bayesian model averaging overcomes the challenge of rate assignment by averaging over all possible local clock models , estimating the substitution rates, and the number and position of changes in substitution rate, simultaneously. another category of relaxed clock models is based on 'rate smoothing', including non-parametric rate smoothing (sanderson, 1997) , penalized likelihood (sanderson, 2002) and bayesian autocorrelated relaxed clock methods (thorne et al., 1998; kishino et al., 2001; aris-brosou and yang, 2002; rannala and yang, 2007) . these methods restrict the rates on parent and descendant branches to be similar by penalizing large departures from parent branch rates. hence, rate variation is expected to occur through small and frequent changes. different bayesian autocorrelated clock models differ in the distribution used to model a branch rate given its parent rate (thorne et al., 1998; kishino et al., 2001) . however, analysis of sequence data from influenza a and dengue-4 do not provide any evidence of autocorrelation of branch rates suggesting that autocorrelated models may not be appropriate when analyzing a genealogy of sequences from a single virus species. whereas lineage-effects may be expected to cause autocorrelation of rates (through incremental changes to life-history, metabolic rate et cetera), the gene-specific action of darwinian selection will also cause apparent rate variation among lineages, by producing a general over-dispersion of the molecular clock over the entire phylogeny (takahata, 1987 (takahata, , 1991 . this second source of rate variation among lineages may be better modeled by uncorrelated relaxed clock models , which make no assumption about the autocorrelation of rates between ancestral and descendent branches. published analyses have provided strong evidence supporting the uncorrelated relaxed clock model (e.g., salemi et al., 2008; worobey et al., 2008) over the strict clock model. as well as estimating the age of ancestral divergences, it is also of interest to estimate the time of cross-species transmission if the disease is zoonotic in origin. one method of identifying the time of the host-switch is by applying non-homogeneous substitution models. the motivation of non-homogeneous substitution models is to acknowledge possible differences in pattern of substitution in the virus within different host species, which violates the assumptions of homogeneity and stationarity underlying the standard substitution models. therefore it may be more appropriate to apply different substitution models to different parts of the tree (forsberg and christiansen, 2003) . non-homogeneous substitution models permit the equilibrium frequencies, and hence the model parameters, to change on a branch and all the descendant lineages from the point of change are assumed to have different equilibrium base frequencies to the lineages prior to that point. this technique has been used to suggest that the immediate ancestral population of 1918 influenza a virus resided in a mammalian host (reis et al., 2009 ). however, it does not indicate whether the most recent common ancestor of the swine influenza virus and the 1918 virus resided in humans or other mammals. interpretation of estimated divergence times can be difficult. there may be direct ancestors that are more ancient, but the lineages that would reveal them have not been sampled or did not survive to the present due to processes such as genetic drift. therefore, the estimated t mrca may not answer the question of interest. for epidemics that resulted from a zoonotic transmission, the host switch event is of paramount interest, but estimating the t mrca of the epidemic strain does not directly estimate the time of the transmission, and only serves as a lower bound. likewise, if there have been processes causing a loss of genetic diversity in the past or the sampling is not comprehensive, then the estimated t mrca could be substantially younger than the age of the viral lineage. an obvious example of the former occurs in seasonal influenza due to seasonal population fluctuations and also strong positive darwinian selection caused by immune surveillance (fitch et al., 1991; bush et al., 1999) , leading to rapid lineage turnover and a recent common ancestor of any single-season sample. similarly, the analysis by worobey et al. (2008) shows that the t mrca of hiv-1 group m seems to have been pushed back due to the inclusion of an additional pre-epidemic sample from 1960 which is highly divergent to the 1959 sequence (zr59). in general the inclusion of older samples can increase the estimated age of root by (i) revealing previously unsampled lineages that are outgroup to the t mrca estimated without them, or (ii) simply because more temporal sampling breaks up long internal branches as well as potentially revealing ancient evidence of variants that were assumed modern, resulting in a slower estimated rate and therefore older estimated root height. finally, it is likely that current techniques alone cannot always recover accurate divergence dates in the distant past, as illustrated by recent analyses suggesting a much deeper history of siv (worobey et al., 2010) than previously suggested (sharp et al., 2000; wertheim and worobey, 2009 ). fig. 2 illustrates the problem with three estimated viral time-trees that have vastly different inferred ages of their most recent common ancestor. we would expect the greatest confidence in the inferred age of the human influenza a time-tree where the sample period is a large fraction of the total age of the time tree, and the least confidence in the inferred age of the hepatitis c time-tree in which the sampling period is a small fraction of the inferred age of greater than 1000 years. so, apart from better models of rate variation across lineages (see guindon et al., 2004 , for early steps in this direction), future research in divergence time dating will likely focus on models that more accurately account for purifying selection and its role in maintaining the structure and function of the encoded genes. the impact of darwinian selection is expressed both in distortions of the genealogy (o'fallon, 2010; o'fallon et al., 2010) and the substitution process (e.g., bloom et al., 2007; cartwright et al., 2011) from neutral expectations. consideration of the action of pervasive purifying selection is especially important in viral genomes prone to clonal interference and which are compact, information rich and subject to great levels of functional and structural constraint in their evolutionary trajectories, especially when considering long time periods. beyond that there is also a need for more statistically rigorous methods of incorporating diverse sources of calibration information, such as biogeography, archaeology and paleontological evidence. bayesian statistical frameworks are uniquely suited for this sort of integration of multiple sources of information. genealogy-based population genetics can be used to infer demographic parameters including population size, rate of growth or decline, and population structure. when the characteristic time scale of demographic fluctuations are comparable to the rate of accumulations of substitutions then past population dynamics are ''recorded'' in the substitution patterns of molecular sequences. coalescent theory can therefore be combined with temporal information in heterochronous sequences to uncover past epidemiological events and pinpoint them on a calendar time scale. kingman's coalescent (kingman, 1982) describes the relationship between the coalescent times in a sample genealogy and the population size assuming an idealized wright-fisher population (fisher, 1930; wright, 1931) . the original formulation was for a constant population, but the theory has since been generalized to any deterministically varying function of population size for which the integral r t 1 t 0 nðtþ à1 dt can be computed (griffiths and tavaré, 1994) . parametric models with a pre-defined population function, such as exponential growth, expansion model and logistic growth models can easily be used in a coalescent framework (see fig. 3 and box 1 for details). for example a ''piecewise-logistic'' population model was employed in a bayesian coalescent framework to estimate the population history of hcv genotype 4a infections in egypt . this analysis demonstrated a rapid expansion of hcv in egypt between 1930-1955, consistent with the hypothesis that public health campaigns to administer anti-schistosomiasis injections had caused the expansion of an hcv epidemic in egypt. the coalescent process is highly variable, so sampling multiple unlinked loci (felsenstein, 2006; heled and drummond, 2008) or increasing the temporal spread of sampling times (seo et al., 2002) can both be used to increase the statistical power of coalescentbased methods and improve the precision of estimates of both population size and substitution rate (seo et al., 2002) . however in many virus species, the entire genome acts as a single locus, or undergoes recombination only when the opportunity arises through superinfection. the lack of independent loci therefore places an upper limit on the precision of estimates of population history. in many situations the precise functional form of the population size history is unknown, and simple population growth functions may not adequately describe the population history of interest. non-parametric coalescent methods provide greater flexibility by estimating the population size as a function of time directly from the sequence data and can be used for data exploration to guide the choice of parametric population models for further analysis. these methods first cut the time tree into segments, then estimate the population size of each segment separately according to the coalescent intervals within it. the main differences among these methods are (i) how the population size function is segmented along the tree, (ii) the statistical estimation technique employed and (iii) in bayesian methods, the form of the prior density on the parameters governing the population size function. in the 'classic skyline plot' (pybus et al., 2000) each coalescent interval is treated as a separate segment, so a tree of n taxa has n à 1 population size parameters. however, the true number of population size changes is likely to be substantially fewer, and the generalized skyline plot (strimmer and pybus, 2001) acknowledges this by grouping the intervals according to the small-sample akaike information criterion (aic c ) (burnham and anderson, 2002) . the epidemic history of hiv-2 was investigated using the generalized skyline plot (strimmer and pybus, 2001) , indicating the population size was relatively constant in the early history of hiv-2 subtype a in guinea-bissau, before expanding more recently (lemey et al., 2003) . using this information, the authors then employed a piecewise expansion growth model, to estimate the time of expansion to a range of 1955-1970. while the generalized skyline plot is a good tool for data exploration, and to assist in model selection (e.g., pybus et al., 2003; lemey et al., 2004) , it infers demographic history based on a single input tree and therefore does not account for sampling error produced by phylogenetic reconstruction nor for the intrinsic stochasticity of the coalescent process. this shortcoming is overcome by implementing the skyline plot method in a bayesian statistical [1978, 2005] and represents a significant fraction (%0.32t mrca ) of the overall tree height, but still small enough that the estimated root should be viewed with caution. (c) a phylogeny of human influenza a subtype h3n2: the sampling interval spans 12.2 years [1993.1, 2005.3 ] and represents almost the full height of the tree (%0.94t mrca ), and all divergence times are likely to be quite accurately estimated, since interpolation between many known sample times is inherently less error prone than extrapolation to ancient divergence times. framework, which simultaneously infers the sample genealogy, the substitution parameters and the population size history. further extensions of the generalized skyline plot include modeling the population size by a piecewise-linear function instead of a piecewise-constant population, allowing continuous changes over time rather than sudden jumps. the bayesian skyline plot (drummond et al., 2005) has been used to suggest that the effective population size of hiv-1 group m may have grown at a relatively slower rate in the first half of the twentieth century, followed by much faster growth (worobey et al., 2008) . on a much shorter time scale, the bayesian skyline plot analysis of a dataset collected from a pair of hiv-1 donor and recipient was used to reveal a substantial loss of genetic diversity following virus transmission (edwards et al., 2006) . further analysis with a constant-logistic growth model estimated that more than 99% of the genetic diversity of hiv-1 present in the donor is lost during horizontal transmission. this has important implications as the process underlying the bottleneck determines the viral fitness in the recipient host. one disadvantage of the bayesian skyline plot is that the number of changes in the population size has to be specified by the user a priori and the appropriate number is seldom known. one solution is provided by methods that perform bayesian model averaging on the demographic model utilizing either reversible jump mcmc (opgen-rhein et al., 2005) or bayesian variable selection (heled and drummond, 2008) , and in which case the number of population size changes is a random variable estimated as part of the model. the methods for demographic inference discussed so far assume no subdivision within the population of interest. like changes in the size, population structure can also have an effect on the pattern of the coalescent interval sizes, and thus the reliability of results can be questioned when population structure exists ). in the next section we will discuss approaches to phylogeographic inference, including coalescent approaches to population structure. phylogeography is a field that studies the evolution and dispersal process that has given rise to the observed spatial distribution of population or taxa. phylogeographic methods can be divided into two approaches. the first performs post-tree-reconstruction analysis to answer phylogeographic questions, while the second jointly estimates the phylogeny and phylogeographic parameters of interest. when treating geographic location as discrete states, the former approach has been popular in the past couple of decades. it has the advantage of being less computationally intensive, but the outcome of the analysis depends on the input tree. due to its simplicity, the most popular method for inferring ancestral locations has been maximum parsimony (slatkin and maddison, 1989; swofford, 2003; maddison and maddison, 2005; wallace et al., 2007) , however this method does not allow for any probabilistic assessment of the uncertainty associated with the reconstruction of ancestral locations. a mugration model is a mutation model used to analyze a migration process. a recent study of influenza a h5n1 virus introduced a fully probabilistic 'mugration' approach by modeling the process of geographic movement of viral lineages via a continuous time markov process where the state space consists of the locations from which the sequences have been sampled (lemey et al., 2009b ). this fig. 3 . the underlying wright-fisher population and serially-sampled genealogies from two populations. the first population has a constant population size over the history of the genealogy, while the second population has been exponentially growing. the coalescent likelihood calculates the probability of a genealogy given a particular background population history (e.g., constant or exponentially growing) and can therefore be employed to estimate the population history that best reflects the shape of the co-estimated phylogeny. facilitates the estimation of migration rates between pairs of locations. furthermore, the method estimates ancestral locations for internal nodes in the tree and employs bayesian variable selection (bvs) to infer the dominant migration routes and provide model averaging over uncertainty in the connectivity between different locations (or host populations). this method has helped with the investigation of the influenza a h5n1 origin and the paths of its global spread, and also the reconstruction of the initial spread of the novel h1n1 human influenza a pandemic (lemey et al., 2009b) . however, a shared limitation of models for discrete location states is that ancestral locations are limited to sampled locations. as demonstrated by the analysis of the data set on rabies in dogs in west and central africa, absence of sequences sampled close to the root can hinder the accurate estimation of viral geographic origins (lemey et al., 2009b) . phylogeographic estimation is therefore improved by increasing both the spatial density and the temporal depth of sampling. however, dense geographic sampling leads to large phylogenies and computationally intensive analyses. the structured coalescent (hudson, 1990) can also be employed to study phylogeography. the structured coalescent has also been extended to heterochronous data (ewing et al., 2004) , thus allowing the estimation of migration rates between demes in calendar units. the serial structured coalescent was first applied to an hiv dataset with two demes to study the dynamics of subpopulations within a patient (ewing et al., 2004) , but the same type of inference can be made at the level of the host population. further development of the model allowed for the number of demes to change over time (ewing and rodrigo, 2006a) . migrate (beerli and felsenstein, 2001) also employs the structured coalescent to estimate subpopulation sizes and migration rates in both bayesian and maximum likelihood frameworks and has recently been used to investigate spatial characteristics of viral epidemics (bedford et al., 2010) . additionally, some studies have focused on the effect of ghost demes (beerli, 2004; ewing and rodrigo, 2006b) , however no models explicitly incorporating population structure, heterochronous samples and nonparametric population size history are yet available. one ad hoc solution involves modeling the migration process along the tree in a way that is conditionally independent of the population sizes estimated by the skyline plot (lemey et al., 2009a) . thus, given the tree, the migration process is considered independent of the coalescent prior. however this approach does not capture the interaction between migration and coalescence that is implicit in the structured coalescent, since coalescence rates should depend on the population size of the deme the lineages are in. as we will see in the following section, statistical phylogeography is one area where the unification of phylogenetic and mathematical epidemiological models looks very promising. in some cases it is more appropriate to model the spatial aspect of the samples as a continuous variable. the phylogeography of wildlife host populations have often been modeled in a spatial continuum by using diffusion models, since viral spread and host movement tend to be poorly modeled by a small number of discrete demes. one example is the expansion of geographic range in eastern united states of the raccoon-specific rabies virus (biek et al., 2007; lemey et al., 2010) . brownian diffusion, via the comparative method (felsenstein, 1985; harvey and pagel, 1991) , has also been utilized to model the phylogeography of feline immunodeficiency virus collected from the cougar (puma concolor) population around western montana. the resulting phylogeographic reconstruction was used as proxy for the host demographic history and population structure, due to the predominantly vertical transmission of the virus (biek et al., 2006) . however, one of the assumptions of brownian diffusion is rate homogeneity on all branches. this assumption can be relaxed by extending the concept of relaxed clock models to the diffusion process . simulations show that the relaxed diffusion model has better coverage and statistical efficiency over brownian diffusion when the underlying process of spatial movement resembles an over-dispersed random walk. like their mugration model counterparts, these models ignore the interaction of population density and geographic spread in shaping the sample genealogy. however there has been progress in the development of mathematical theory that extends the coalescent framework to a spatial continuum (barton et al., 2002 (barton et al., , 2010a , although no methods have yet been developed providing inference under these models. box 1: the anatomy of a bayesian coalescent analysis using mcmc bayesian phylogenetic inference by markov chain monte carlo (mcmc) (yang and rannala, 1997; mau et al., 1999) involves the simulation of the joint posterior distribution of substitution model parameters (/) and the phylogenetic tree given the sequence data (d). by restricting the phylogenetic model to time-trees (see fig. 1 ) and coupling the phylogenetic likelihood with a coalescent prior, the parameters (h) of the population history, n h (t), can also be estimated simultaneously by sampling from the posterior probability distribution (drummond et al., 2002) : the term pr(djg,/) is often referred to as the phylogenetic likelihood, and is the probability of the data given the time-tree g and substitution model parameters. it can be computed by the pruning algorithm (felsenstein, 1981) , which efficiently sums over all ancestral sequence states at the internal nodes of the tree. an extension of the likelihood accommodates heterogeneity across sites (yang, 1994) . if the time-tree g relates a heterochronous sample of sequences, then the substitution parameters / also includes the overall substitution rate l, and this can be estimated from the heterochronous data, so that the population history is estimated on a calendar scale. the normalizing constant pr(d) is also known as the partition function or marginal likelihood and its magnitude provides a measure of model support, although its estimation requires advanced mcmc techniques (e.g., thermodynamic integration or transdimensional mcmc). coalescent models come into play when determining the prior density for the time-tree topology and coalescent/ divergence times. the coalescent provides a probability distribution, f g (gjh), conditional on a deterministic model of population size history, n h (t). its parameters (h) can in turn be estimated as hyperparameters. given a time-tree g = {e g ,t} of n contemporaneous samples composed of an edge graph e g and coalescent times t = {t n = 0,t nà1 , . . . ,t 2 ,t 1 } the coalescent density is: the prior distributions f h (h) and f u (/) are usually selected from standard univariate or multivariate distributions. in the previous section we have seen that phylogenetics can be used to infer the date of an outbreak, its source population and the viral transmission history, directly from time-stamped genomic data. whereas phylogenetic models mainly address questions about evolutionary history, dynamical models are often used to make predictions about the future. predictive models are important because they provide the possibility of anticipating certain aspects of the outcome of emerging epidemics and assessing the risk of pandemics, and the potential effects of planned intervention. phylogenetic inference is based on genetic data such as sampled dna sequences from infected hosts. current models using such data to infer information about the past often require simplifying assumptions about the population size e.g., to be constant or to be subject to pure exponential growth. epidemiologists, on the other hand, fit their models to prevalence or incidence data. standard epidemiological models are described by sets of ordinary differential equations tracking the (often non-linear) changes in numbers of susceptible and infected individuals. consequently, the simple prior assumptions for the population sizes (of infected individuals) used in phylogenetics appear inadequate from an ecological perspective. epidemiological models play a major role in deciding which measures of disease control are taken to avoid or stop viral outbreaks. the effects of isolation, vaccination and other measures are estimated through model simulations, serving as a basis for decisions on which public health policies to institute and actions to take. however, knowledge of the phylogenetic history of viral outbreaks can be vital in reconstructing transmission pathways which contributes to effective management and future prevention efforts (e.g., cottam et al., 2008) . the epidemiological and ecological processes determining the diversity of fast evolving rna viruses act on the same time scale as that on which mutations arise and are fixed in the population (holmes, 2004) . this implies that genetic sequence data can provide independent evidence on transmission histories. whereas epidemiological data typically provides information about who was infected and when, it generally does not provide positive evidence about transmission history. thus the combination of these sources of information should open the way to more detailed epidemiological inference, including bayesian estimation of contact networks and transmission histories (welch et al., 2011) . standard epidemiological models are based on flux between host compartments dividing the host population e.g., into susceptible (s), infected (i) and recovered or removed (r) individuals. standard models are termed si, sis and sir. the choice of model is based on the characteristics of the considered disease, the existence of a latent period, immunity after infection et cetera (see box 2) (anderson and may, 1991; keeling and rohani, 2008) . restricting the focus to the time evolution of the number of individuals in each compartment, these models grasp the overall progress of an epidemic. certain disease characteristics require adaptations or extensions of standard models, for example, the inclusion of asymptomatic infections that account for a sampling bias towards symptomatic infections in case the virus of interest does not always cause noticeable symptoms (e.g., aguas et al., 2008) . an important threshold ratio is the basic reproduction ratio r 0 , the expected number of secondary infections caused by one primary infection in a completely susceptible population (diekmann et al., 1990) . based on its value epidemiologists make predictions on the effect of the disease. in classical deterministic epidemiological models, if the basic reproduction ratio is larger than one, an epidemic is expected. box 2: compartmental models for infectious diseases (keeling and rohani, 2008 ) let s, e, i and r be the fractions of susceptible, exposed, infected and recovered/removed individuals in the host population. the left hand side of each equation block gives the model equations, the right hand side the (non-trivial) endemic equilibria, which are only obtainable for r 0 > 1. the basic reproduction ratio r 0 depends on the corresponding model. apart from the si model, the overall population is assumed to be constant, such that the sum of fractions for each model equals one. under the assumption of homogeneous mixing in the population the transmission term bs i can be derived, which determines the total rate of new infections. si model. fatal infections, eventually killing the infected, can be modeled with only two compartments: susceptible and infected. assume a fixed birth rate m and death rate l. the sir model. transmission of the disease to susceptibles leads to a period of illness until recovery, which in turn implies immunity. demography is described by the birth and death rate l and recovery is obtained at rate c; its reciprocal 1/c is the mean infectious period. here, r 0 ¼ b lþc . the last equation is redundant since s + i + r = 1. instead, after infection the individuals go back to the susceptible stage. therefore, the disease can persist even without including newborns in the population. ignoring demography, the dynamics are characterized by coupled differential equations _ s ¼ ci à bsi and _ i = bs i à ci. since s = 1 à i, they can be replaced by one equation. seir model. in order to account for a latent period with assumed average duration 1/r, the sir model can be extended by including exposed individuals composing a fraction e of the population. exposed individuals are infected, but not yet infectious. the differential equations for s (and r) are as in the sir model. dynamics in e and i are described as follows. further models are sirs, seis, msir, mseir, mseirs, etc., where m denotes passively immune infants, allowing for diseases where an individual can be born with a passive immunity from its mother. typically, epidemiologists fit a suitable set of deterministic differential equations to empirical data, often the number of infections or related hospitalizations in a population. consequently, the model can be used to estimate if an epidemic can be kept under control by measures such as (i) vaccination and (ii) antiviral prophylaxis for susceptible individuals, (iii) treatment of infected individuals or (iv) isolation of infected individuals from susceptible individuals. decisions on public health policies are often based on these estimates. the simplest epidemiological models assume homogeneous mixing within a population. in many cases this assumption is not valid. due to host contact dynamics viral infections spread easily within social units such as schools, cities and farms, less so among them. integration of population structure is therefore essential. however, even within subpopulations individual dynamics might differ stochastically (see fig. 4 ). such randomness can be accounted for by considering stochastic models (see e.g., survey by britton, 2010) . before introducing stochastic compartmental models thoroughly, we illustrate them based on a stochastic sir model simulation. we simulate the spread of a virus strain in a population divided into n subpopulations which are connected by comparatively rare migration events. let l ¼ f0; . . . ; n à 1g denote the set of locations. a single infected individual initiates the epidemic in one of the n completely susceptible populations. after an exponentially distributed waiting time one of the following events happens: infection at mass action infection rate b. migration at migration rate m ik for ik 2 l. birth of a susceptible individual at rate l. death of an individual at rate l. fig. 5 shows a realization of the simulated dynamics for n = 3 populations. the epidemic starts in population 1 (blue) and many individuals get infected before the first individuals in population 2 (yellow) and eventually population 3 (red) get infected. let s k , i k and r k be the fractions of individuals in each subpopulation k 2 l. the sum s k + i k + r k equals one for every k 2 l. the deterministic analogue of our model can be described with the following differential equations: however it is important to realize that this set of differential equations cannot capture all of the behaviors of its stochastic counterpart. in fact, starting from a deterministic representation like this, there are multiple stochastic markov processes that exhibit the same deterministic limit, but can potentially have exponentially different behavior in their stochastic properties, such as the time to extinction (e.g., . formally, two distinct sources of variance can be considered in stochastic models of populations (engen et al., 1998) . the first is environmental stochasticity and is often modeled by admitting temporal variation in the parameters of the population model. the second is demographic stochasticity and describes the stochasticity of fluctuations in populations of finite size due to the inherent unpredictability of individual outcomes. to model demographic stochasticity (also known as internal stochasticity; chen and bokka, 2005) in the absence of environmental (external) stochasticity, the time-evolution of an epidemic can be represented by a jump process and its corresponding master equation (gardiner, 2009 ). the master equation describes the time evolution of the probability distribution over the discrete state space. for the closed sir model (kermack and mckendrick, 1927) the master equation for the numbers of individuals in each of the three compartments (n s , n i , n r ) is: _ p n s ;n i ;n r ðtþ ¼ bðn s þ 1þðn i à 1þp n s þ1;n i à1;n r ðtþ ð 4þ þ cðn i þ 1þp n s ;n i þ1;n r à1 ðtþ à ðbn s n i þ cn i þp n s ;n i ;n r ðtþ a single realization of this epidemic jump process is described by a sequence of timed transition events (individual infection or recovery events). in the closed sir model, the waiting or sojourn time between a pair of sequential events is exponentially distributed (i.e., the transition process is memoryless), and thus the process is a continuous-time markov process. stochastic models of this form can also be viewed in terms of their reaction kinetics. for the closed stochastic sir model above the two 'reactions' are infection and recovery: indicating that a susceptible contacts an infectious individual and gets infected at reaction rate b whereas an infected recovers at reaction rate c. more precisely, the time (s) an individual spends in the susceptible and infected compartments are exponentially distributed with rates bi and c, respectively. it is the binary infection reaction that leads to the non-linear dynamics of the system. for stochastic models r 0 > 1 does not necessarily imply an outbreak of the disease. instead, a higher basic reproduction ratio suggests a higher probability of an outbreak, but the precise relationship depends on the specific model considered and the initial condition. algorithms have been developed that allow exact and approximate simulation of coupled reactions such as the closed sir (bartlett, 1957; gillespie, 1976 gillespie, , 2001 ). fig. 6 shows simulated viral outbreaks under a stochastic sir and sis model with r 0 % 2.3 in a population divided into three distinct subpopulations. note that there is no outbreak in (3) although r 0 > 1. deterministic epidemic models can be derived from the underlying jump process, and can represent useful macroscopic laws of motion in the appropriate limit. however such approaches are not adequate for modeling systems in which small numbers of individuals are frequently involved. for a similar reason, it is awkward to reconcile large-limit deterministic models with the small sample genealogies that are obtained with molecular phylogenetic approaches. therefore, stochastic continuous-time discrete-state formulations of epidemic models may be more suited to forming connections between the two disciplines. the forward simulations of a stochastic epidemic model introduced with fig. 5 demonstrate the relationship between epidemic models and genealogies. knowing the exact parameters and resulting dynamics throughout the simulated outbreak, we can build a full transmission history for the outbreak (which is not unique given only the time evolution of the number of infected individuals, since at each event the infected individuals involved are chosen randomly). an infection event in the forward simulation corresponds to a bifurcation in the transmission tree. restricting the full tree to a ''sample genealogy'' that only includes the individuals that were infectious at a specific sampling time yields very different results for different times during the outbreak, which underlines the importance of sampling methods (see e.g., stack et al., 2010) . as we can see in the simulations, virus transmission often depends on spatial structure. the interaction among humans living in the same city, for example, differs from among-city interaction, which is important whenever viral transmission exceeds city borders. there are many other social and spatial units this concept applies to: households, schools, or on a larger scale, regions, countries and continents. in fact, most phylogenetic and epidemiological studies model the dynamics of spatially distributed systems, albeit many of them ignore spatial structure for the sake of simplicity. durrett and levin demonstrate that models ignoring spatial structure yield qualitatively different results than spatial models (durrett and levin, 1994) . phylodynamics is a term used to describe a synthetic approach to the study of rapidly evolving infectious agents that considers the action (and interaction) of both evolutionary and ecological processes. the term phylodynamics was introduced by grenfell et al. (2004) to describe the ''melding of immunodynamics, epidemiology, and evolutionary biology'' that is required to analyse the interacting evolutionary and ecological processes especially of rapidly evolving viruses for which both processes have the same time scale. two distinct pursuits have been labeled phylodynamics by recent studies. the first relies on the idea that ecological processes and population dynamics can effectively be tracked by neutral genetic variation, such that past ecological and population events are ''imprinted'' in genetic variation within populations and can be reconstructed along with the reconstruction of evolutionary history. the idea is sound for truly neutral variation, but the compact genomes of rapidly evolving viruses are not simple recording devices. instead they are packed with functional information and mutations play an active role in population and ecological processes through the action of darwinian selection. hence, the more challenging second phylodynamic pursuit is the analysis of the inevitable interaction of evolutionary and ecological processes that requires the joint analysis of both. we will call the former pursuit phylogenetic epidemiology, and reserve the term phylodynamics for approaches that aspire to model the interaction of ecological and evolutionary processes. the effect of novel mutations on population dynamics through their interaction with the immune system or anti-viral drugs are examples of phylodynamics in this stricter sense. the focus of many studies aspiring to combine population genetic and epidemiological approaches is the basic reproduction ratio r 0 , estimates of which are used to develop containment strategies for emerging pandemics. such estimates can be obtained from phylogenetic analysis, e.g., through estimating population growth rates . another popular way to infer population dynamic information from genomic data is the application of parametric and non-parametric coalescent models (strimmer and pybus, 2001; drummond et al., 2005; minin et al., 2008) . phylogenetic methods can be used to estimate r 0 , which can then be used to investigate transmission patterns and the number of generations of transmission. depending on the distribution of the generation time (i.e., the duration of infectiousness) the relationship between r 0 and the growth rate r of the population can be used to compute the basic reproduction number (wallinga and lipsitch, 2007) . little is known about generation time distributions, the usual approach is to fit the epidemic models to the observed data. wallinga and lipsitch list the resulting equations for r 0 for exponential, normal, or delta distributions of generation time. they show that without knowledge of the generation time distribution an upper bound for the reproductive number can still be estimated. others obtain r 0 estimates based on coalescent theory, as for example (rodrigo et al., 1999) who estimated it in vivo for hiv-1. in a recent study on the influenza a (h1n1) outbreak in 2009 both epidemiological and bayesian coalescent approaches for the computation of r 0 were applied (fraser et al., 2009) . whereas the epidemic approaches gave estimates of 1.4-1.6 for r 0 , the bayesian coalescent approach yielded a posterior median of 1.22. all estimates are larger than one, correctly indicating that the virus spreads successfully, rather than dying out. however, an agedependent heterogeneous epidemic model best fits the data and results in an estimate of r 0 = 1.58. structures determining host interaction are often modeled as contact networks (welch et al., 2011) . the transmission of foot and mouth disease virus is highly dependent on the interaction among farms and the detection of infected farms is essential. a plausible approach is to consider each farm as an individual in a contact network. through phylogenetic analysis of consensus sequences (one sequence for each farm) contacts between farms can be traced in order to find infected but non-detected farms such that contacts between farms can be traced in order to find infected but non-detected farms (cottam et al., 2008) . changes in effective population size estimated through phylogenetic analyses can indicate past changes in population size. therefore, many recent studies infer the demographic history of a virus using bayesian skyline plot models (drummond et al., 2005) . for example, (siebenga et al., 2010) are interested in the epidemic expansion of norovirus gii.4 which they investigate by reconstructing the changes in population structure using bayesian skyline plots. similarly, (hughes et al., 2009 ) explore the heterosexual hiv epidemic in the uk. analyses of the genomic and epidemiological dynamics of human influenza a virus explore the sink-source theory and investigate the spatial connections of a seasonal global epidemic (rambaut et al., 2008; lemey et al., 2009b; bedford et al., 2010) . coalescent theory has also been adapted to fit an epidemic sir model to sequence data (volz et al., 2009 ). frost and volz (2010) provide an overview on how appropriate interpretation of coalescent rates differs among the different population dynamic approaches it is being used with. interpretation of the coalescent-based skyline plots must be made with caution. as opposed to generation times referring to durations of infection in epidemiological theory, for coalescent approaches being applied to infectious diseases the generation times usually describe times between transmission events. accordingly, although prevalence does affect phylogenetic reconstruction through sampling, the population dynamic patterns are mainly determined by incidence (frost and volz, 2010) . one early attempt to integrate dynamical and population genetic models used coupled differential equations and markov chain theory to model the within-host time evolution of viral genetic diversity under basic dynamic models of a persistent infection (kelly et al., 2003) . the main focus was the impact of the dynamical model on the variance in the number of replication cycles, as this is a key determinant of the rate of genetic divergence and thus potential for adaptation. interestingly, the model reveals that multiple cell type infections can decrease viral evolutionary rates and increase the likelihood of persistent infection. genetic diversity within hosts is closely related to between host dynamics: gordo and campos (2007) develop structured population genetic models, explicitly incorporating epidemiological parameters to analyze the relationship between genetic variability and epidemiological factors. a simple sis model is simulated based on two different models of host contact structure, the island model and a scale free contact network. for low clearance rates and low intrahost effective population size, levels of genetic variability turn out to be maximal when transmission levels are intermediate, independent of the host population structure. in a scale free contact network the population consists of many low-connectivity hosts and very few high-connectivity hosts, a common pattern for sexually transmitted diseases (e.g., lloyd and may, 2001; liljeros et al., 2001) . in this setting genetic variation appears to be lower in highly connected than in weakly connected hosts. with their study gordo and campos (2007) underline that an integration of population genetics and epidemiology can have important implications for public health policies. in a deterministic framework day and gandon (2007) model the interaction of evolutionary and ecological processes by coupling sis host dynamics with viral evolution. the interaction of evolution and ecology is incorporated through the fitness of each virus strain. for strain i they define a fitness r i = b i n s à l à v i à c, where b i is the strain-specific transmission rate per susceptible, v i is the strainspecific virulence (determining the increase in mortality rate due to infection), l is the baseline mortality rate and c is the recovery rate. the evolutionary dynamics of strain frequencies are tracked quantitatively and the evolutionary dynamics of strain frequencies are intimately linked with the overall infection dynamics of the host population via the strain-specific virulence and transmission rates. their analysis provides insight into the mechanistic laws of motion connecting genetic evolution with the evolution of virulence and transmission rates. an exceptional feature of influenza viruses is the limited genetic diversity which appears to contradict the viruses' high mutation rate. integrating single virus strain features and host immunity into a stochastic transmission model ferguson et al. (2003) search an explanation for this. although epidemiological factors play a role in limiting influenza diversity, strain-transcendent immunity must be relevant as well. through a phylodynamic analysis of interpandemic influenza in humans koelle et al. (2006) underline the importance of the viral structure for antigenicity and the immune recognition dynamics of influenza epitopes. they consider clusters that contain strains with similar conformations of ha epitopes such that there is high cross-immunity of strains within each cluster. a genotype-phenotype model that implements neutral networks (the clusters) is coupled with an epidemiological transmission model in which the number of susceptible, infected and recovered individuals in each cluster are modeled. model simulations result in time series of infected cases that agree with the typical annual outbreaks in temperate regions and empirical dominance of certain antigenic clusters. according to this model, years in which a formerly dominant cluster is replaced by a new one have the highest numbers of infections. in the following year there are particularly few infec-(1) (3) (4) fig. 6 . simulated viral outbreak under stochastic sir (1-3) and sis (4) model among three populations (denoted by blue, yellow and red curves). the initial condition is a single infected individual in the blue population. in (3) the disease does not break out (numbers of susceptibles in dotted lines and infected in solid lines). (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) tions, presumably due to higher host immunity caused by the previous year's outbreak. thereafter follow ''average'' years until the next cluster-transition occurs, i.e., until another cluster becomes dominant again. another natural explanation of the contradiction between high mutation rates and constant genetic diversity is the fixation of many deleterious mutations that leads to the extinction of the respective strains. recent population genetic models account for population dynamics e.g., in order to enhance the understanding of allele fixation processes and the importance of demographic stochasticity (parsons and quince, 2007; champagnat and lambert, 2007; parsons et al., 2010) . structured models do not only allow for more realistic dynamics, they can also bridge the gap to phylogenetic/-geographic methods since most of them are sample-based, ideally, with each sample representing one infected individual. modeling coupled host-virus dynamics welch et al. (2005) embed an epidemic population model into a branching and coalescent structure, producing a scaled coalescent process that describes the inter-host dynamics given a virus sample genealogy. their simulations show that, for large sample sizes, the model provides accurate estimates of the contact rate and the selection parameter. overall, phylodynamic methods have been developed and proven useful for the analysis of various viruses. however, phylogenetic reconstruction is still quite restricted by coalescent assumptions. an alternative to the coalescent for cases in which sample sizes are big compared to the overall population is the birth-death with incomplete-sampling model (gernhard, 2008; stadler, 2009) , and this framework has recently been extended to include heterochronous data (stadler, 2010) , opening the way for an alternative approach to phylodynamic inference from timestamped virus data. bayesian phylogenetic inference has led to an explosion of analyses of rapidly evolving viruses in recent years. while this explosion has been fruitful in elucidating the manifold variation in origin, transmission routes and evolutionary rates underlying the present diversity of infection agents, there is a nascent field that promises to extend the conceptual reach of molecular sequence data, through a unification of phylogenetics and mathematical epidemiology. this new field of phylodynamics encompasses both inference of classical epidemiological parameters using phylogenetics as well as exciting new approaches that aim to investigate the consequences of the inevitable interaction between evolutionary (mutation, drift, darwinian selection) and ecological (population dynamics and ecological stochasticity) processes. the research being pursued has broader consequences for evolutionary biology and molecular ecology. this interaction of evolution and ecology will occur whenever a population contains genotypes with different intrinsic dynamical properties (e.g., virulence, transmission rates, recovery rates). whereas this condition is almost always met in real populations and frequently definitive in its role in shaping outcomes, the mathematical and theoretically analysis of darwinian selection within epidemiological models is the most challenging and least studied area within the emerging field of phylodynamics. it is thus ripe for future research. in the meantime, it is likely that phylodynamic research will rapidly develop new methods for statistical phylogeography and structured population dynamics. prospects for malaria eradication in sub-saharan africa infectious diseases of humans: dynamics and control effects of models of rate evolution on estimation of divergence dates with special reference to the metazoan 18s ribosomal rna phylogeny measles periodicity and community size neutral evolution in spatially continuous populations a new model for evolution in a spatial continuum a new model for extinction and recolonization in two: dimensions quantifying phylogeography global migration dynamics underlie evolution and persistence of human influenza a (h3n2) effect of unsampled populations on the estimation of population sizes and migration rates between sampled populations maximum likelihood estimation of a migration matrix and effective population sizes in n subpopulations by using a coalescent approach a virus reveals population structure and recent demographic history of its carnivore host a highresolution genetic signature of demographic and spatial expansion in epizootic rabies virus thermodynamics of neutral protein evolution unifying vertical and nonvertical evolution: a stochastic arg-based framework stochastic epidemic models: a survey model selection and multimodel inference: a practical information-theoretic approach predicting the evolution of human influenza a history can matter: non-markovian behavior of ancestral lineages evolution of discrete populations and the canonical diffusion of adaptive dynamics stochastic modeling of nonlinear epidemiology integrating genetic and epidemiological data to determine transmission pathways of foot-and-mouth disease virus applying population-genetic models in theoretical evolutionary epidemiology on the definition and the computation of the basic reproduction ratio r0 in models for infectious-diseases in heterogeneous populations measurably evolving populations inference of viral evolutionary rates from molecular sequences bayesian random local clocks, or one rate to rule them all relaxed phylogenetics and dating with confidence estimating mutation parameters, population history and genealogy simultaneously from temporally spaced sequence data tools for constructing chronologies: crossing disciplinary boundaries beast: bayesian evolutionary analysis by sampling trees bayesian coalescent inference of past population dynamics from molecular sequences extinction times in autocatalytic systems the importance of being discrete (and spatial) a method for cluster analysis population genetic estimation of the loss of genetic diversity during horizontal transmission of hiv-1 demographic and environmental stochasticityconcepts and definitions using temporally spaced sequences to simultaneously estimate migration rates, mutation rate and population sizes in measurably evolving populations coalescent-based estimation of population parameters when the number of demes changes over time estimating population parameters using the structured serial coalescent with bayesian mcmc inference when some demes are hidden evolutionary trees from dna sequences: a maximum likelihood approach phylogenies and the comparative method accuracy of coalescent likelihood estimates: do we need more sites, more sequences, or more loci? ecological and immunological determinants of influenza evolution positive darwinian evolution in human influenza a viruses a codon-based model of host-specific selection in parasites, with an application to the influenza a virus who rapid pandemic assessment collaboration viral phylodynamics and the search for an 'effective number of infections origin of hiv-1 in the chimpanzee pan troglodytes troglodytes human infection by genetically diverse sivsm-related hiv-2 in west africa stochastic methods: a handbook for the natural and social sciences the conditioned reconstructed process molecular virology: was the 1918 pandemic caused by a bird flu the emergence of hiv/aids in the americas and beyond a general method for numerically simulating the stochastic time evolution of coupled chemical reactions approximate accelerated stochastic simulation of chemically reacting systems a likelihood method for the detection of selection and recombination using nucleotide sequences unifying the epidemiological and evolutionary dynamics of pathogens sampling theory for neutral alleles in a varying environment modeling the sitespecific variation of selection patterns along lineages the comparative method in evolutionary biology bayesian inference of population size history from multiple loci an african primate lentivirus (sivsmclosely) related to hiv-2 phylogenetic evidence for recombination in dengue virus the phylogeography of human viruses whole-genome analysis of human influenza a virus reveals multiple persistent lineages and reassortment among recent h3n2 viruses gene genealogies and the coalescent process bayesian inference of phylogeny and its impact on evolutionary biology uk hiv drug resistance collaboration, rambaut, a.uk hiv drug resistance collaboration splitstree: analyzing and visualizing evolutionary data rates of molecular evolution in rna viruses: a quantitative phylogenetic analysis chimpanzee reservoirs of pandemic and nonpandemic hiv-1 modeling infectious diseases in humans and animals linking dynamical and population genetic models of persistent viral infection a contribution to the mathematical theory of infections the coalescent performance of a divergence time estimation method under a probabilistic model of rate evolution epochal evolution shapes the phylodynamics of interpandemic influenza a (h3n2) in humans timing the ancestor of the hiv-1 pandemic strains the molecular population genetics of hiv-1 group o tracing the origin and history of the hiv-2 epidemic bayesian phylogeography finds its roots phylogeography takes a relaxed random walk in continuous space and time reconstructing the initial global spread of a human influenza pandemic: a bayesian spatial-temporal model for the global spread of h1n1pdm bats are natural reservoirs of sars-like coronaviruses animal origins of the severe acute respiratory syndrome coronavirus: insight from ace2-s-protein interactions the web of human sexual contact genetic analysis of human h2n2 and early h3n2 influenza viruses, 1957-1972: evidence for genetic divergence and multiple reassortment events how viruses spread among computers and people full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination sinauer associates phylogeography and molecular epidemiology of hepatitis c virus genotype 2 in africa bayesian phylogenetic inference via markov chain monte carlo methods smooth skyride through a rough skyline: bayesian coalescent-based inference of population dynamics different subtype distributions in two cities in myanmar: evidence for independent clusters of hiv-1 transmission recent human influenza a (h1n1) viruses are closely related genetically to strains isolated in 1950 the origin and global emergence of adamantane resistant a/h3n2 influenza viruses multiple reassortment events in the evolutionary history of h1n1 influenza a virus since 1918 dated ancestral trees from binary trait data and their application to the diversification of languages a method to correct for the effects of purifying selection on genealogical inference a continuous-state coalescent and the impact of weak selection on the structure of gene genealogies inference of demographic history from genealogical trees using reversible jump markov chain monte carlo slidingbayes: exploring recombination using a sliding window approach based on bayesian phylogenetic inference cross-species virus transmission and the emergence of new epidemic diseases fixation in haploid populations exhibiting density dependence i: the non-neutral case some consequences of demographic stochasticity in population genetics genetic history of hepatitis c virus in east asia the epidemic behavior of the hepatitis c virus the epidemiology and iatrogenic transmission of hepatitis c virus in egypt: a bayesian coalescent approach evolutionary analysis of the dynamics of viral infectious disease an integrated framework for the inference of viral population history from reconstructed genealogies estimating the rate of molecular evolution: incorporating noncontemporaneous sequences into maximum likelihood phylogenies the genomic and epidemiological dynamics of human influenza a virus inferring speciation times under an episodic molecular clock using non-homogeneous models of nucleotide substitution to identify host shift events: application to the origin of the 1918 'spanish' influenza pandemic virus recombination in aids viruses coalescent estimates of hiv-1 generation time in vivo missing data in a stochastic dollo model for binary trait data, and its application to the dating of proto-indo-european highresolution molecular epidemiology and evolutionary history of hiv-1 subtypes in albania identification of breakpoints in intergenotypic recombinants of hiv type 1 by bootscanning a nonparametric approach to estimating divergence times in the absence of rate constancy estimating absolute rates of molecular evolution and divergence times: a penalized likelihood approach simian immunodeficiency virus infection in free-ranging sooty mangabeys (cercocebus atys atys) from the tai forest a viral sampling design for testing the molecular clock and for estimating evolutionary rates and divergence times a bayesian phylogenetic method to estimate unknown sequence ages origins and evolution of aids viruses: estimating the time-scale phylodynamic reconstruction reveals norovirus gii.4 epidemic expansions and their molecular determinants a cladistic measure of gene flow inferred from the phylogenies of alleles origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic analyzing the mosaic structure of genes protocols for sampling viral sequences to study epidemic dynamics on incomplete sampling under birth-death models and connections to the sampling-based coalescent sampling-through-time in birth-death trees exploring the demographic history of dna sequences using the generalized skyline plot paup⁄: phylogenetic analysis using parsimony (⁄ and other methods). version 4 on the overdispersed molecular clock statistical models of the overdispersed molecular clock origin and biology of simian immunodeficiency virus in wild-living western gorillas estimating the rate of evolution of the rate of molecular evolution human immunodeficiency viruses: siv infection in wild gorillas sequence analysis of a highly divergent hiv-1-related lentivirus isolated from a wild captured chimpanzee phylodynamics of infectious disease epidemics a statistical phylogeography of influenza a h5n1 how generation intervals shape the relationship between growth rates and reproductive numbers statistical inference to advance network models in epidemiology integrating genealogy and epidemiology: the ancestral infection and selection graph as a model for reconstructing host virus histories the re-emergence of h1n1 influenza virus in 1977: a cautionary tale for estimating divergence times using biologically unrealistic sampling dates purifying selection can obscure the ancient age of viral lineages dating the age of the siv lineages that gave rise to hiv-1 and hiv-2 direct evidence of extensive diversity of hiv-1 in kinshasa by 1960 island biogeography reveals the deep history of siv evolution in mendelian populations maximum likelihood phylogenetic estimation from dna sequences with variable rates over sites: approximate methods bayesian phylogenetic inference using dna sequences: a markov chain monte carlo method historical perspective-emergence of influenza a (h1n1) viruses key: cord-284385-ster02o9 authors: gambichler, thilo; reuther, judith; scheel, christina h; becker, jürgen christian title: on the use of immune checkpoint inhibitors in patients with viral infections including covid-19 date: 2020-07-01 journal: j immunother cancer doi: 10.1136/jitc-2020-001145 sha: doc_id: 284385 cord_uid: ster02o9 the present review summarizes up-to-date evidence addressing the frequently discussed clinical controversies regarding the use of immune checkpoint inhibitors (icis) in cancer patients with viral infections, including aids, hepatitis b and c, progressive multifocal leukoencephalopathy, influenza, and covid-19. in detail, we provide available information on (1) safety regarding the risk of new infections, (2) effects on the outcome of pre-existing infections, (3) whether immunosuppressive drugs used to treat ici-related adverse events affect the risk of infection or virulence of pre-existing infections, (4) whether the use of vaccines in ici-treated patients is considered safe, and (5) whether there are beneficial effects of icis that even qualify them as a therapeutic approach for these viral infections. cancer cells may escape immune surveillance through a variety of mechanisms, including the activation of immune checkpoint pathways that serve to suppress immune responses against tumor cells. immune checkpoint inhibitors (ici) boost antitumor immune responses by interrupting coinhibitory signaling pathways to promote immunemediated killing of tumor cells. the introduction of ici in 2011 for therapy has been a revolutionary milestone in the management of many solid cancers and hematological malignancies (eg, melanoma, merkel cell carcinoma, squamous cell carcinomas, colorectal cancer, renal cell carcinoma, urothelial cancer, hodgkin lymphoma). [1] [2] [3] currently, antibodies targeting three different inhibitory checkpoint proteins are approved as first-line, second-line or third-line treatments for various types of malignancies: cytotoxic t lymphocyte antigen-4 (ctla-4; ipilimumab), programmed cell death protein-1 (pd-1; pembrolizumab, nivolumab, cemiplimab), and programmed cell death protein ligand-1 (pd-l1; durvalumab, atezolizumab, avelumab). [1] [2] [3] [4] full activation of t lymphocytes predominantly depends on several different signals. indeed, t lymphocyte activation is regulated both by costimulators and coinhibitors known as immune checkpoints. 5 antigen-major histocompatibility complex (mhc) and t cell receptor (tcr) binding associated with the activation of costimulatory receptors (ie, cd28) enables t lymphocytes to proliferate, differentiate and migrate toward specific antigens. by contrast, when antigen-mhc and tcr binding is associated with signaling of coinhibitory receptors (ie, ctla-4), t cell activation will be suppressed. ctla-4 is not expressed in naïve t lymphocytes, but is quickly induced on t cell activation. importantly, ctla-4 predominantly regulates the amplitude of t cell activation during the early priming phase in lymphoid organs. 1 3 5 the binding of ctla-4 to b7 proteins is in direct competition with cd28 costimulatory signals, and the ratio between cd28 and ctla-4 binding determines activation of t lymphocytes versus anergy, and represents an important mechanism in the prevention of excessive immune responses. hence, the main task of ctla-4 is to stop autoreactive t lymphocytes at the initial stages of activation, predominantly in lymphoid tissues, to prevent autoimmunity. 5 thus, it is not surprising that it is also expressed on regulatory t cells (tregs). similar to tregs, pd-1 plays an important role in limiting immune responses in peripheral tissues. the interaction of pd-1 with its ligands (pd-l1/2) inhibits t cell proliferation and cytokine secretion mediated by tcrs. 1-3 5 the pd-1 receptor is physiologically expressed by activated t lymphocytes, b lymphocytes, monocytes, natural killer (nk) cells, and tregs. pd-l1 is expressed on several cells, including tumor cells and some host cells such as myeloid, lymphoid and epithelial cells. the interaction between pd-1 and pd-l1 blocks cd8+ open access cytotoxic t cell proliferation and survival, leads to apoptosis of tumor-infiltrating lymphocytes, and promotes differentiation of cd4+ t lymphocytes into tregs. 1 5 6 most cancer cells possess the ability to express inhibitory ligands such as pd-l1, for example, in response to interferons (ifn). this process can limit normal anticancer immune responses, thus assisting in immune escape. hence, ici do not result in killing tumor cells directly but enhance or restore immune responses and endogenous antitumor activity. [1] [2] [3] [4] [5] [6] exhaustion of t lymphocytes is the most important factor contributing to weakened t cell activity against both cancer and infectious agents. notably, t cell exhaustion is a distinguishing feature of many chronic viral infections such as hiv and hepatitis b virus (hbv) infection. indeed, t cell exhaustion was first described in the context of chronic infections. 7 8 in the following, t lymphocytes with a similar phenotype were also detected in the tumor microenvironment. 2 7-9 exhausted t lymphocytes are functionally characterized by a loss of interleukin 2 (il-2) production, impaired proliferation, diminished cytotoxicity, and altered production of proinflammatory cytokines. 2 7-9 moreover, the overexpression of immune checkpoint receptors, including pd-1 and ctla-4, is a characteristic. given the similarities between the immune response to cancer and chronic infections, one may hypothesize that the use of ici should not be harmful for tumor patients with infections or may even provide a benefit. however, as of yet, tumor patients with existing viral infections are excluded from participation in many treatment protocols for ici. 7 8 with respect to acquired infectious diseases during ici treatment, no increased risk was observed in clinical studies. 1-4 7 8 however, ici treatment frequently results in activation of autoreactive t lymphocytes and disturbances in immune tolerance thereby causing autoimmune-like/inflammatory side effects. these are summarized as immunerelated adverse events (iraes) and include autoimmune colitis, pneumonitis, hypophysitis, hepatitis, thyroiditis and so on. [10] [11] [12] since iraes may require immunosuppressive therapy, including high-dose corticosteroids and/or tumor necrosis factor (tnf)-α blockers, the risk of infection or reactivation of chronic or latent viral infections (eg, hbv or hepatitis c virus (hcv)) may be secondarily increased. [10] [11] [12] in this respect, it should also be noted that much of the morbidity of persistent viral diseases is caused by collateral damage caused by the chronic reactive inflammation associated with the inability of viral clearance; both may be boosted by ici therapy. in this review, we present information on the pros and cons of using ici in patients with viral infections including covid-19. antiretroviral therapy has significantly decreased the incidence of aids and thus the mortality of hiv infection. however, complete eradication of hiv with antiviral agents has not yet been achieved, presumably because hiv persists in cellular reservoirs. the major hiv reservoir is a small pool of latently infected resting memory cd4+ lymphocytes carrying an integrated form of the viral genome that lacks the ability to produce viral proteins. 13 in hiv-infected subjects receiving highly active antiretroviral therapy (haart), inhibitory checkpoint proteins such as pd-1 are expressed on persisting infected t cells. indeed, there is a wealth of evidence that high expression of pd-1 on cd4+ lymphocytes clearly correlates with hiv persistence. 8 13 however, different inhibitory checkpoint proteins are differentially expressed by t cell subtypes; for example, pd-1 expression is increased in memory t cells and tregs, whereas ctla-4 is highly expressed in both memory t cells and tregs. 8 14 interestingly, the frequency of pd-1 expression on cd4+ and cd8+ lymphocytes appears to strongly correlate with disease outcome. specifically, in untreated patients with hiv, high pd-1 expression has been shown to correlate with a decrease in cd4+ t lymphocytes during both acute and chronic infection. 7 8 15 similar to pd-1 upregulation, overexpression of ctla-4 on cd4+ t lymphocytes more frequently correlates with progressive disease. 7 8 16 hiv persistence can be reversed by ici in vitro, 7 8 17 and in preclinical animal models, t cell exhaustion is ameliorated by pd-1 blockade. 18 together, the above discussed pathomechanism and preliminary experimental data warrant studies regarding safety and efficacy of ici in hiv-infected patients. interestingly, even though patients living with hiv on haart have a life expectancy similar to the general population, these patients still have an increased risk to develop cancer. 19 20 in this context, initial observations on hiv-positive cancer patients treated with ici are emerging. in a systematic review, of 73 hivinfected patients who received ici therapy for advanced cancer, anti-pd-1 monotherapy was the most frequently employed regimen (n=62). 19 in this cohort, grade iii or higher iraes were observed in 8.6% of patients. hiv remained suppressed in 93% of patients and cd4+ lymphocyte count increased in patients with available pretreatment and post-treatment hiv load and cd4 cell count data, respectively. none of the previous studies reported the occurrence of immune reactivation inflammatory syndrome during ici therapy. 19 similar to the safety profile, efficacy of ici was favorable with an objective response of 63% in kaposi sarcoma, 30% in non-small cell lung carcinoma, and 27% in melanoma. 19 prompted by these encouraging results, phase i and ii trials investigating icis in hiv-infected patients with advanced solid tumors and lymphomas are currently conducted. 21 22 accordingly, a task force formed by the american society of clinical oncology (asco) recently recommended the inclusion of hiv-infected patients in oncology trials, particularly patients with cd4+ counts higher than 350 cells/µl, thus representing a group of patients with intact immunological function and survival comparable with the general population. 23 moreover, data from the first clinical trial investigating the safety, tolerability and pharmacokinetics of ctla-4 inhibition (ipilimumab) open access in patients with chronic hiv infection in the absence of concurrent malignancies were recently reported. 24 although only based on a limited number of patients (n=24), this study did not reveal any safety concerns that would preclude further investigation of using clta-4 inhibition to enhance the immune response against hiv. one patient who developed facial palsy received medium dose prednisone; still, no worsening of his hiv infection was observed. 24 furthermore, in a randomized, doubleblind, placebo-controlled, phase i dose-escalating study testing pd-1 inhibition (nivolumab) in haart-treated hiv-infected adults without concurrent cancer (n=8), even a single, low-dose infusion appeared to enhance hiv-specific immunity. 25 in summary, the exclusion of hiv-infected patients from oncology ici trials appears to be neither supported by evidence from basic research nor early clinical trials. on the contrary, many lines of evidence suggest that ici could be employed to improve hiv-specific immunity and thus contribute to hiv remission or even possible cure strategies. 23 26 27 however, the mechanisms regulating hiv persistence are complex and not yet fully understood, leading to the hypothesis that a combined treatment approach including ici and cytokines will be required to accomplish such complete remissions. 28 hepatitis b and c viral caused hepatitis is one of the leading causes of morbidity and death worldwide. hbv and hcv account for the majority of viral-hepatitis-related mortality, mostly attributable to cirrhosis and hepatocellular carcinoma. to date, it is still a challenge to achieve complete hbv clearance or to prevent hcv relapse once directly acting antiviral treatment regimens failed. 29 one important constraint for the use of ici in patients with concomitant virus hepatitis for treatment of cancer is the possible occurrence of immune-mediated hepatotoxicity, a frequent irae caused by ici. this notion is particularly troublesome, as an immune-mediated hepatitis may pose a significant diagnostic challenge in patients with underlying viral or autoimmune hepatitis. [30] [31] [32] pu et al 33 recently published a comprehensive review on 186 cancer patients with concurrent hbv or hcv infection who had received ici treatment. 33 about 20% of patients developed an increase of hepatic transaminases which was higher than those reported in ici-treated cancer patients without concurrent viral hepatitis. 33 all grade 3 or 4 toxicities were reversible by means of antiviral treatment or corticosteroids without necessitating a discontinuation of ici. importantly, no negative influence on infection status was reported in patients receiving corticosteroids. 33 it should be mentioned, however, that ici should be withhold once an irae is encountered requiring immunosuppressive drugs; but, ici may be resumed once the irae has resolved. importantly, the incidence of other adverse events in this particular patient population was not significantly increased when compared with ici-treated cancer patients without chronic viral hepatitis. based on a recent publication reviewing the available data on the use of ici in cancer patients with hepatitis, it is recommended that all patients scheduled to receive ici should be screened for hbv and hcv, and in patients who are tested positive, prophylactic antiviral treatment is indicated. unfortunately, however, it is currently unclear for how long the prophylactic treatment should be continued. primary prophylaxis should also be considered in patients with chronic hbv infection, if not already on treatment. liver function tests and viral load should routinely be monitored in virus positive patients. 34 35 it is well known that hbv-specific exhaustion of t cells is maintained by ongoing hbv-antigen stimulation. furthermore, pd-l1/2 expression, secretion of immunosuppressive cytokines, for example, il-10 and transforming growth factor (tgf-ß), dysfunction of dendritic cells, enhanced numbers of pd-1+ nk cells, tregs, and myeloidderived suppressor cells negatively influence hbv-specific t cell immunity. 6 8 34 35 similar to hbv infection, chronic hcv infection is also associated with increased pd-1 and tim-3 expression as another marker for t cell exhaustion. 36 37 although direct-acting antiviral regimens for hcv-infected patients have shown great overall success, thereby placing the need for therapeutic alternatives into perspective, the application of ici to treat therapy-resistant chronic hcv infection is appealing. 28 29 36 38 39 gardiner et al 40 recently reported a phase i proof-of-concept trial demonstrating that pd-1 inhibition through nivolumab resulted in prolonged suppression of hcv replication in some patients with chronic infection. based on these encouraging results, further exploration of pd-1 pathway inhibition is warranted for other chronic viral diseases, possibly in combination with direct-acting antiviral regimens. 40 however, similar to the situation for hbv infection, the number of clinical trials assessing ici in chronic hcv infection remain limited. 36 41-43 taken together, ici appears to be safe and effective in cancer patients with concurrent hbv or hcv. thus, hbv and hcv infection should not contraindicate ici. 34 35 even though the risk of virus reactivation and virus-related hepatotoxicity appears to be low, it is recommended that patients with active hbv or hcv should routinely be monitored and treated with antiviral agents if indicated, in particular in patients receiving immunosuppressive medication for ici-induced irae. 33 since pd-1 plays a significant role in the natural history of both hbv-induced and hcv-induced hepatitis, there is a rationale for the use of ici in these conditions. initial studies indicate that anti-pd-1 treatment is safe in chronic hbv and hcv infection, but further trials are needed to determine whether ici can be used to gain hbv long-term remission. 36 progressive multifocal leukoencephalopathy progressive multifocal leukoencephalopathy (pml) is a rare, often lethal disease of the central nervous system caused by the jc polyomavirus (jcv). 44 in general, jcv open access infection is indolent and asymptomatic, but may become symptomatic and fatal in immunocompromised patients, for example, patients infected with hiv, lymphoproliferative malignancies or those undergoing immunosuppressive therapies. 45 46 currently, no proven therapeutic strategy for this disease has been established. based on evidence that t cell exhaustion might affect the course of jcv infections, icis are currently tested for treatment of pml. 47 for example, cortese et al 48 assessed the safety and efficacy of pd-1 inhibition by pembrolizumab with different predisposing conditions (n=8). in this small cohort, five patients achieved a clinical benefit or stabilization of pml and a decrease in jcv viral load. contrasting results were observed in another study, three kidney transplant recipients suffering from pml were treated with nivolumab and all three patients died within 8 weeks with evidence of disease progression. 49 as an explanation for this adverse outcome, 48 the authors speculate that immunosuppressive therapy (ie, calcineurin inhibitors) might have led to persistent t cell dysfunction and lymphopenia. the authors further point out that patients with severe lymphopenia also did not respond favorably to ici treatment. hence, they concluded that ici may be ineffective in such patients. 48 49 in contrast, a number of case reports describe favorable outcomes for patients with pml receiving ici. for example, hoang et al 50 described a biopsy-confirmed pml case in which the pd-1 inhibitor nivolumab seems to have stimulated immune activation resulting in effective disease control in the patient with a concomitant hematological malignancy. [50] [51] [52] together, at present, there are few studies on the safety and/or efficacy of ici in patients with pml. the available studies do not show consistent results which, might be due to the great heterogeneity of predisposing conditions leading to pml. despite these difficulties, the present data suggest that the underlying cause of immunosuppression, pretreatment, and laboratory parameters, such as lymphopenia, have relevant effects on the success of ici therapy of pml. this notion may be extrapolated to ici treatment of cancer in patients with pml. to the best of our knowledge, however, there exist no definite data on patients with pml receiving ici treatment because of coexisting cancer. of course, the use of immunosuppressive comedication for ici-induced iraes is challenging in this particular patient population. thus, prospective studies are necessary to determine whether icis are a safe and effective approach for pml and pml-associated cancers. influenza, a highly contagious respiratory disease, is responsible for a significant economic burden on the healthcare systems. the co-circulating influenza a (subtype: h1n1, h3n2) and b (lineage: victoria, yamagata) viruses cause seasonal epidemics which affect a major part of the global population annually and cause more than 645,000 influenza-associated deaths worldwide. 53 54 since patients with cancer are at higher risk of influenza-associated complications, vaccination, the primary preventive tool against influenza, is recommended. particularly, ici-treated patients produce robust humoral and cellular immune responses. still it is important to note that bersanelli et al 53 reported that the post-vaccination occurrence of the influenza syndrome (fever ≥38°c and the presence of at least one respiratory symptom together with generalized symptoms) was significantly increased in patients with cancer receiving ici. in this study, the lack of efficacy of vaccination was more pronounced among the elderly. 53 importantly, influenza vaccination did not negatively impact the efficacy of the anticancer effects of ici treatment. moreover, the same authors reported that ici-treated cancer patients who received influenza vaccination and/or developed influenza syndrome showed longer overall survival. the effect of immunosuppressive therapy for ici-induced irae on the influenza syndrome was not addressed in these studies. 54 in rare cases, influenza infection and vaccination are associated with the occurrence of organ-specific autoimmune conditions, such as guillain-barré syndrome, a rapid onset muscle weakness caused by the immune system attacking peripheral nerves. the risk of influenza vaccine-induced guillain-barré syndrome is extremely small. notably, however, yuen et al 55 reported a patient with previous post-vaccination guillain-barré syndrome who developed a fatal reactivation following the initiation of ici. indeed, the hypothesis that influenza vaccination may induce adverse immune responses in ici-treated patients is corroborated by results from animal experiments showing increased t cell responses to viral antigens under pd-1 inhibition. 56 moreover, it has been suggested that vaccines may stimulate an overwhelming expansion of autoreactive t lymphocytes which cross-recognize vaccines as well as self-antigens. 57 58 läubli et al 59 reported a small cohort of ici-treated cancer patients that had received influenza vaccinations and subsequently experienced higher rates of iraes than expected (n=23). later studies investigating larger patient cohorts did not confirm such an increase of frequency or severity of iraes in patients on ici who previously received influenza vaccination. 57 60-65 the data of chong et al 57 do not indicate an increase in incidence or severity of irae in patients with cancer on ici who received influenza vaccinations. twenty per cent (75/370) of patients experienced a new irae (any grade), of those 48% received corticosteroids or other immunosuppressants. in the latter subgroup, no negative influence on vaccination outcome was reported. 57 in this context, the study of awadalla et al 65 should be pointed out. they actually demonstrated that the administration of a influenza vaccination was not correlated with an increased risk of subsequent myocarditis among patients on ici. indeed, rates of vaccination were lower among patients who did develop ici-induced myocarditis, and the vaccination was associated with a lower risk of other iraes, in particular ici-induced pneumonitis. 65 unfortunately, there is little data available whether ici would be beneficial in the management of severe influenza cases. in this context, yu et al 66 reported that influenza infection substantially increases the number of highly pd-1 positive innate lymphoid cells in the lungs of mice. anti-pd-1 treatment resulted in reduction of total lung innate lymphoid cells with almost complete loss of pd-1 highly positive cells. hence, they suggested that anti-pd-1 treatment may provide an effective approach for both disease prevention and treatment. 66 in march 2020, the covid-19 outbreak, which is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was officially proclaimed a pandemic by the who. up through end of april, 2020, almost 3 million cases of covid-19 were confirmed with more than 200,000 deaths reported worldwide. 67 covid-19 is predominantly characterized by high fever, dry cough, fatigue, and eventually pneumonia, and can cause death in severe cases. to date, most published data on covid-19 have been generated in china. in 14% of confirmed cases, the course of disease was severe and in 5% critical. the so-called case fatality rate (cfr) was as high as 1%, thereby being much greater than the cfr usually observed in seasonal influenza (approximately 0.1%). however, current infection rates as well as cfr still have to be considered with caution. 68 risk for severe disease and death is strongly associated with older age (in particular >70 years), cardiovascular disease, diabetes, obesity, chronic respiratory disease, hypertension, and cancer. [68] [69] [70] laboratory predictors for severe and fatal disease predominantly include elevated lactate dehydrogenase, pro-calcitonin, and d-dimers, increased serum levels of cytokines il-6, il-10 and tumor necrosis factor-α (tnf-α), as well as decreased lymphocyte counts, particularly cd8+ t and nk cells. [69] [70] [71] [72] indeed, biao et al 71 recently demonstrated that the number of total t lymphocytes in the peripheral blood was significantly decreased in patients with covid-19. this was particularly pronounced in older patients and in patients who needed intensive care unit (icu) treatment. importantly, lymphopenia was negatively associated with patient survival. biao et al [69] [70] [71] also showed that t cell counts of patients who recovered increased, while il-6, il-10 and tnf-α levels decreased. 71 since the cytokine increases were paralleled by a decrease in lymphocytes, diao et al 71 speculated that elevated proinflammatory cytokines may promote the reduction of t lymphocytes in patients with covid-19. however, this observation has to be substantiated in future studies. 71 additionally, as assessed by flow cytometry of peripheral blood, t cells of severely affected patients are characterized by a much higher pd-1 expression than healthy controls. 71 specifically, enhanced pd-1 and tim-3 expression on t cells was observed when patients progressed from prodromal to symptomatic stages, indicating that t lymphocyte exhaustion-similar to other viral infections-is a hallmark of covid-19 as well. the expression of critical inhibitory checkpoint proteins of t cell exhaustion, including pd-1 and tim-3, and the increase of tnf-α, il-10 and other cytokines (which actually all may modulate the expression of pd-1 and tim-3) are likely to mediate t cell lymphopenia in patients with covid-19 through programmed cell death. 73 to date, very limited data are available addressing the clinical outcome of ici-treated cancer patients also suffering from covid-19 infection. [74] [75] [76] [77] the blockade of pd-1+ on cd8+ lymphocytes by ici treatment might be reasonable in patients with covid-19 in order to abrogate functional t cell exhaustion and restore vigorous t lymphocytic cytotoxicity against both the tumor and the viral antigens. however, as discussed by chiappelli et al, 75 this may work only at the initial and intermediate stage when pd-1 expression on cytotoxic t cells ranges between low and medium levels. at the more advanced stage, when pd-1 expression is high on cd8+ t lymphocytes, t lymphocyte exhaustion is likely irreversible, and thus, ici will no longer have an effect. 75 the excess of cytokines is also of great significance with respect to the cytokine release syndrome (crs, 'cytokine storm'), a phenomenon of massive inflammatory reaction, where cytokines (eg, il-6, il-10, tnf-α) are rapidly produced in large amounts in response to infectious agents. 71 similar to sars-cov and the middle east respiratory syndrome (mers-cov), sars-cov-2 is associated with increased amounts of proinflammatory cytokines in the serum, which are suspected to cause pulmonary inflammation and extensive lung damage. 72 however, unlike sars-cov and mers-cov, sars-cov-2 infection appears to be associated with the activation of both t helper 1 (th-1) and th-2 lymphocytes. sars-cov-2 predominantly targets epithelial cells of the respiratory tract, leading to severe alveolar damage. however, covid-19 also shows evidence for changes in the lung stroma, suggesting that also pulmonary fibrosis is induced at some time point. 58 moreover, similar to sars-cov and mers-cov, sars-cov-2 may also pose the risk of autoimmunity due to cross-reactivity of the induced immune reaction to both viral (eg, spike surface proteins) and host protein epitopes. lyons-weiler 58 recently hypothesized that based on homology with human proteins, such pathogenic priming involving autoimmunity might also occur with sars-cov-2. similar to the results of previous sars-cov animal experiments, agrawall et al 78 reported that mice vaccinated against mers-cov developed severe th-2driven immunopathologies in the lung following postvaccination mers-cov challenge. 58 78 given the data existing so far on possible autoimmunity particular in the advanced stage of covid-19, the administration of ici could pose the risk of immune overactivation and aggravation of autoimmune processes. similar to the 'cytokine storm' observed in patients with advanced covid-19, crs has been observed in rare cases as iraes in patients receiving ici. 79 80 furthermore, one of the observed irae of anti-pd-1-based ici is autoimmune pneumonitis, which may occur in up to 5% of all treated patients. [79] [80] [81] patients with non-small open access cell lung cancer treated with ici may even experience autoimmune pneumonitis in up to 20% of cases. 81 since the clinical symptoms and radiographic findings in iraeinduced pneumonitis are similar to those of covid-19 pneumonitis, it may be in some cases difficult to arrive at proper diagnostic conclusions as the basis for appropriate management. the occurrence of iraes frequently necessitates the use of systemic corticosteroids or other immunosuppressive/immunomodulating agents such as mycophenolate acid or tnf-α blockers. whether the use of systemic corticosteroids is harmful in the setting of covid-19 is unclear. the use of glucocorticoids in patients with sars-cov-associated and mers-covassociated pneumonitis is still controversial because of divergent clinical outcomes reported in the existing literature. 82 still, high-dose glucocorticoids are one of the most frequently used adjuncts in acute respiratory distress syndrome, even though the effectiveness of steroids in the management of acute lung injury is ambiguous. 82 this is also true for covid-19. nevertheless, it seems that early and short-time use of low-dose methylprednisolone may be one feasible approach in sars-cov-2-related pneumonitis and respiratory distress syndrome. 83 hence, possible ici-induced iraes could be safely managed with methylprednisolone in the setting of covid-19. based on the observation of cytokine excess (eg, tnf-α), during advanced stages of covid-19, one may speculate that tnf-α blockers such as infliximab may not only be beneficial for the management of ici-induced iraes, but also for covid-19. 84 notably, cytokine il-6 antibodies (eg, tocilizumab) are currently under intense investigation in patients with covid-19. tocilizumab is already approved in the usa to manage crs occurring after chimeric antigen receptor t cell treatment. 82 notably, it has been demonstrated that tocilizumab is effective in the management of iraes occurring in ici-treated patients who are refractory to corticosteroids. 82 furthermore, there is currently an ongoing phase ii trial investigating a combination of tocilizumab with anti-pd-1/ctla-4 therapy in order to diminish iraes in unresectable stage iii or stage iv melanoma patients (nct03999749). 84 a novel approach to ici in cancer and viral infections, particularly covid-19, represents cd200 checkpoint reversal. 85 the cd200 immune checkpoint causes suppression of the secretion of proinflammatory cytokines, that is, il-2 and ifn-ү, and enhances the production of myeloidderived suppressor cells and tregs that are implicated in impaired antitumor and antiviral immune responses. 85 anti-cd200 targeted treatments have shown promising effects in animal models accompanied by downregulation of inhibitory pd-1 receptors. 85 in a murine coronavirus model, the checkpoint inhibitory cd200-cd200r1 system has been demonstrated to downregulate the single strand rna virus sensor toll-like receptor seven in myeloid-derived cells and respective interventions restored ifn-ү levels resulting in enhanced virus elimination. 86 87 together, experience and evidence are growing with regard to the use of ici in patients with the novel infectious disease covid-19. in comparison to chemotherapeutic regimens ici cannot be considered immunosuppressive. hematological iraes caused by ici are very infrequent, with only few cases reported. in a metaanalysis of 9324 patients, the frequency of neutropenia was smaller than 1%. 88 previously reported reactivation of viral infections (ie, cytomegalovirus, hepatitis b) under ici therapy were mostly observed following immunosuppressive treatment of iraes. hence, it does not appear reasonable to assume that patients undergoing ici are at higher risk of becoming infected by sars-cov-2 or other infectious agents compared with patients without ici treatment. 76 on the basis of preliminary basic research data and clinical observations in patients with covid-19, one may assume that ici could safely be employed in cancer patients with a sars-cov-2 infection and even covid-19. as long as there is no clear evidence, however, it remains a caseby-case scenario depending on many factors discussed above, particularly with respect to how advanced the cancer and/or covid-19 are. 76 89 92 notably, ici may even represent an effective approach in the management of covid-19 patients without cancer. 71 73 76 79 additionally, a combination of ici with an anti-il-6 antibody is an attractive approach to reduce the risks of both iraes and possible cytokine excess frequently observed in severe covid-19 cases. 93 a study following this strategy is currently recruiting patients. the objective of this prospective, controlled, randomized, multicenter study is to compare the efficacy of the combined administration of a chloroquine analog, nivolumab, and tocilizumab versus standard of care in patients with advanced or metastatic cancer who have covid-19 and are not eligible to a resuscitation unit ( clinicaltrials. gov: nct04333914). patients will be randomized into two different cohorts: (1) asymptomatic or mild symptoms: chloroquine analog versus nivolumab versus standard of care (1:1:1); (2) moderate/severe symptoms: chloroquine analog versus tocilizumab versus standard of care (1:1:1). it has to be stressed, however, that hydroxychloroquine use has been reported to be ineffective or even associated with higher mortality and therefore should only be considered in the context of well-designed controlled and regulatory approved clinical trials. furthermore, two protocols have been registered at clinicaltrials. gov investigating ici in covid-19 patients without cancer: in a phase ii randomized trial, the protocol corimuno19-nivo will evaluate the efficacy and safety of nivolumab alone versus standard of care in covid-19 patients hospitalized in an icu (nct04343144), and in an open-label, controlled, single-center pilot study, nivolumab will be employed in adult patients with covid-19 aiming to investigate the open access efficacy and safety of nivolumab in relation to viral clearance (nct04356508). based on the data presented in this review and in line with the recommendations of the study group for infections in compromised hosts, we assume that pd-1/ pd-l1-and/or ctla-4-based ici treatment does not seem to independently enhance the risk of infection or cause more virulent course of disease. 27 hence, the above discussed viral infections should not be considered as contraindications per se for patients who are scheduled for or are on ici. over the course of ici treatment, however, supportive immunosuppressive therapies may be required to treat ici-associated iraes, which in turn may increase the risk of new or reactivation of persisting viral infections. hence, physicians caring for patients receiving immunosuppressants for treatment of ici-induced iraes should maintain close surveillance for the occurrence of symptoms or signs suggestive of new infection or worsening of preexisting viral infections. 27 for almost all aforementioned viral infections, there are convincing data that disease-associated t cell exhaustion is a fundamental immune escape mechanism. accordingly, increasing lines of evidence suggest that icis represent not only an effective antitumor treatment regimen in such patients but also a potential approach in the management of the viral infection per se. contributors tg conceived of the review article and collected the literature. tg, jr, and chs provided the extraction and interpretation of scientific data and drafted the initial manuscript. jcb assisted with design and scientific review. all authors contributed to manuscript editing, proofread and have approved the final manuscript. funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. competing interests tg has received speakers and/or advisory board honoraria from bms, sanofi-genzyme, msd, novartis pharma, roche, abbvie, almirall, janssen, lilly, pfizer, pierre fabre, merck-serono, outside the submitted work. jr and chs declare that they have no competing interests. jcb is receiving speaker's bureau honoraria from amgen, pfizer, merck-serono, recordati and sanofi, is a paid consultant/advisory board member for boehringer ingelheim, etherna, in prother, merckserono, pfizer, 4sc and sanofi. his group receives research grants from bristol-myers squibb, merck serono, htg, iqvia, and alcedis. provenance and peer review not commissioned; externally peer reviewed. open access this is an open access article distributed in accordance with the creative commons attribution 4.0 unported (cc by 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. see https:// creativecommons. org/ licenses/ by/ 4. 0/. thilo gambichler http:// orcid. org/ 0000-0001-7862-3695 jürgen christian becker http:// orcid. org/ 0000-0001-9183-653x immune checkpoint inhibitors: recent progress and potential biomarkers programmed cell death-1/programmed cell death ligand-1 checkpoint inhibitors: differences in mechanism of action mechanisms of action and rationale for the use of checkpoint inhibitors in cancer review of indications of fda-approved immune checkpoint inhibitors per nccn guidelines with the level of evidence novel immune checkpoint targets: moving beyond pd-1 and ctla-4 single-cell transcriptomics reveal that pd-1 mediates immune tolerance by regulating proliferation of regulatory t cells immune checkpoints and their inhibition in cancer and infectious diseases immune checkpoint blockade in infectious diseases cd8 t cell exhaustion in chronic infection and cancer: opportunities for interventions immune checkpoint inhibitors in challenging populations tumour-and class-specific patterns of immune-related adverse events of immune checkpoint inhibitors: a systematic review the role of immunosuppressive agents in the management of severe and refractory immune-related adverse events hiv reservoir size and persistence are driven by t cell survival and homeostatic proliferation pd-1 expression on human cd8 t cells depends on both state of differentiation and activation status exhaustion of activated cd8 t cells predicts disease progression in primary hiv-1 infection upregulation of ctla-4 by hiv-specific cd4+ t cells correlates with disease progression and defines a reversible immune dysfunction programmed cell death-1 contributes to the establishment and maintenance of hiv-1 latency role of pd-1 co-inhibitory pathway in hiv infection and potential therapeutic options safety and efficacy of immune checkpoint inhibitor therapy in patients with hiv infection and advanced-stage cancer: a systematic review characteristics of immune checkpoint inhibitors trials associated with inclusion of patients with hiv: a systematic review and meta-analysis a phase ii exploratory study of durvalumab (medi4736) in hiv-1 patients with advanced solid tumors amc 095 (aids malignancy consortium): a phase i study of ipilimumab (ipi) and nivolumab (nivo) in advanced hiv associated solid tumors (st) with expansion cohorts in hiv associated solid tumors and classical hodgkin lymphoma (chl) modernizing clinical trial eligibility criteria: recommendations of the american society of clinical oncology-friends of cancer research hiv working group an open-label, multiple ascending dose study of the anti-ctla-4 antibody ipilimumab in viremic hiv patients clinical trial of the anti-pd-l1 antibody bms-936559 in hiv-1 infected participants on suppressive antiretroviral therapy the era of immune checkpoint therapy: from cancer to viral infection-a mini comment on the 2018 medicine nobel prize escmid study group for infections in compromised hosts (esgich) consensus document on the safety of targeted and biological therapies: an infectious diseases perspective (immune checkpoint inhibitors, cell adhesion inhibitors, sphingosine-1-phosphate receptor modulators and proteasome inhibitors) role of cytokine agonists and immune checkpoint inhibitors toward hiv remission incidence of extrahepatic cancers among individuals with chronic hepatitis b or c virus infection: a nationwide cohort study workup and management of immunemediated hepatobiliary pancreatic toxicities that develop during immune checkpoint inhibitor treatment safety and efficacy of immune checkpoint inhibitors (icis) in cancer patients with hiv, hepatitis b, or hepatitis c viral infection anti-pd-1/pd-l1 therapy for infectious diseases: learning from the cancer paradigm safety and efficacy of immune checkpoint inhibitors in patients with hbv/hcv infection and advanced-stage cancer: a systematic review the safety and efficacy of immune checkpoint inhibitors in patients with advanced cancers and preexisting chronic viral infections (hepatitis b/c, hiv): a review of the available evidence safety and efficacy of immune checkpoint inhibitors in patients with non-small cell lung cancer and hepatitis b or hepatitis c infection overcoming cd8+ t-cell exhaustion in viral hepatitis: lessons from the mouse model and clinical perspectives epigenetic stability of exhausted t cells limits durability of reinvigoration by pd-1 blockade checkpoint inhibitors and therapeutic vaccines for the treatment of chronic hbv infection coexpression of pd-1, 2b4, cd160 and klrg1 on exhausted hcv-specific cd8+ t cells is linked to antigen recognition and t cell differentiation a randomized, double-blind, placebo-controlled assessment of bms-936558, a fully human monoclonal antibody to programmed death-1 (pd-1), in patients with chronic hepatitis c virus infection nivolumab in patients with advanced hepatocellular carcinoma (checkmate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial anti-pd-1 blockade with nivolumab with and without therapeutic vaccination for virally suppressed chronic hepatitis b: a pilot study enhancing virus-specific immunity in vivo by combining therapeutic vaccination and pd-l1 blockade in chronic hepadnaviral infection progressive multifocal leukoencephalopathy and other disorders caused by jc virus: clinical features and pathogenesis analysis of the systemic and intrathecal humoral immune response in progressive multifocal leukoencephalopathy progressive multifocal leukoencephalopathy: current insights can immune checkpoint inhibitors keep jc virus in check? pembrolizumab treatment for progressive multifocal leukoencephalopathy effectiveness of immune checkpoint inhibitors in transplant recipients with progressive multifocal leukoencephalopathy progressive multifocal leukoencephalopathy treated with nivolumab sustained response and rationale of programmed cell death-1-targeting for progressive multifocal leukoencephalopathy increased program cell death-1 expression on t lymphocytes of patients with progressive multifocal leukoencephalopathy influenza vaccine indication during therapy with immune checkpoint inhibitors: a transversal challenge. the invidia study state of the art about influenza vaccination for advanced cancer patients receiving immune checkpoint inhibitors: when common sense is not enough severe relapse of vaccineinduced guillain-barré syndrome after treatment with nivolumab pd-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination safety of inactivated influenza vaccine in cancer patients receiving immune checkpoint inhibitors pathogenic priming likely contributes to serious and critical illness and mortality in covid-19 via autoimmunity influenza vaccination of cancer patients during pd-1 blockade induces serological protection but may raise the risk for immune-related adverse events immune-mediated adverse events following influenza vaccine in cancer patients receiving immune checkpoint inhibitors influenza vaccination in patients with lung cancer receiving anti-programmed death receptor 1 immunotherapy does not induce immune-related adverse events safety of influenza vaccine in patients with cancer receiving pembrolizumab immune-related adverse events in patients with cancer receiving influenza vaccination and immune checkpoint inhibitors immunogenicity of influenza vaccination in patients with cancer receiving immune checkpoint inhibitor influenza vaccination and myocarditis among patients receiving immune checkpoint inhibitors single-cell rna-seq identifies a pd-1 hi ilc progenitor and defines its development pathway covid-19 dashboard by the center for systems science and engineering (csse) at johns hopkins university (jhu) covid-19: risk factors for severe disease and death clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study risk factors for severity and mortality in adult covid-19 inpatients in wuhan reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) clinical features of patients infected with 2019 novel coronavirus in wuhan, china covid-19 immunopathology and immunotherapy a rapid fatal evolution of coronavirus disease-19 in a patient with advanced lung cancer with a long-time response to nivolumab state of the art about influenza vaccination for advanced cancer patients receiving immune checkpoint inhibitors: when common sense is not enough do checkpoint inhibitors compromise the cancer patients' immunity and increase the vulnerability to covid-19 infection? what the oncologist needs to know about covid-19 infection in cancer patients immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus controversies about covid-19 and anticancer treatment with immune checkpoint inhibitors severe cytokine release syndrome in a patient receiving pd-1-directed therapy pneumonitis in non-small cell lung cancer patients receiving immune checkpoint immunotherapy: incidence and risk factors effectiveness of glucocorticoid therapy in patients with severe coronavirus disease 2019: protocol of a randomized controlled trial risk-adapted treatment strategy for covid-19 patients insights from immuno-oncology: the society for immunotherapy of cancer statement on access to il-6-targeting therapies for covid-19 cd200 checkpoint reversal: a novel approach to immunotherapy recommendations for coronavirus infection in rheumatic diseases treated with biologic therapy cd200 receptor controls sex-specific tlr7 responses to viral infection clinical diagnosis and treatment recommendations for immune checkpoint inhibitor-related hematological adverse events covid-19: the use of immunotherapy in metastatic lung cancer the many faces of the anti-covid immune response metastatic melanoma treatment with checkpoint inhibitors in the covid-19 era: experience from an italian skin cancer unit impact of pd-1 blockade on severity of covid-19 in patients with lung cancers adjunct immunotherapies for the management of severely ill covid-19 patients key: cord-276006-mjjnkqv6 authors: jarach, natanel; dodiuk, hanna; kenig, samuel title: polymers in the medical antiviral front-line date: 2020-07-31 journal: polymers (basel) doi: 10.3390/polym12081727 sha: doc_id: 276006 cord_uid: mjjnkqv6 antiviral polymers are part of a major campaign led by the scientific community in recent years. facing this most demanding of campaigns, two main approaches have been undertaken by scientists. first, the classic approach involves the development of relatively small molecules having antiviral properties to serve as drugs. the other approach involves searching for polymers with antiviral properties to be used as prescription medications or viral spread prevention measures. this second approach took two distinct directions. the first, using polymers as antiviral drug-delivery systems, taking advantage of their biodegradable properties. the second, using polymers with antiviral properties for on-contact virus elimination, which will be the focus of this review. anti-viral polymers are obtained by either the addition of small antiviral molecules (such as metal ions) to obtain ion-containing polymers with antiviral properties or the use of polymers composed of an organic backbone and electrically charged moieties like polyanions, such as carboxylate containing polymers, or polycations such as quaternary ammonium containing polymers. other approaches include moieties hybridized by sulphates, carboxylic acids, or amines and/or combining repeating units with a similar chemical structure to common antiviral drugs. furthermore, elevated temperatures appear to increase the anti-viral effect of ions and other functional moieties. viruses are not living organisms per se, but small structures containing only a nucleic acid genome within a mostly protein based protecting membrane. unlike living organisms, viruses must penetrate a living host cells to reproduce and replicate [1] . viruses are usually classified into seven major classes [2] [3] [4] [5] [6] [7] [8] . class i includes viruses with double-stranded dna, like poxvirus, which uses asymmetric transcription to raise their mrna, much like "regular" cells, but depend on the hosting cell's polymerases to transcript their genome. class ii contains single-stranded dna viruses. the dna's polarity in these viruses is equal to their mrna's. this class contains the anelloviridae, circoviridae, parvoviridae, geminiviridae, microviridae, and more. class iii includes viruses with double-stranded rna. these viruses' mrna demonstrates an asymmetric transcription of the virus' genome. this group includes the reoviridae and birnaviridae viruses. class iv consists of viruses with single-stranded rna. these viruses' mrna is the sequence's base identical to their virion rna. these viruses are referred to as "positive-sense single-stranded rna viruses". this group contains togaviridae, astroviridae (causing diarrhea), caliciviridae, flaviviridae (including many different diseases such as hepatitis-c, bovine viral diarrhea virus, and more), picornaviridae (including the rhinoviruses, which causes the "common cold"), arteriviridae, and coronaviridae [including the severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome-related coronavirus (mers-cov), and severe acute as will be described later [50] . ganciclovir and acyclovir are well known commercial antiviral drugs which, though not having any electrical charge, have a well-established effect on some virus polymerase. some other molecules with antiviral effects are 5-(n-ethyl-n-isopropyl)amiloride (eipa), verapamil, and diltiazem. some studies suggest their effect is a result of the ion-blocking effect. others suggested that the viral inhibition is based on interactions with virus proteins [51] . other neutral antiviral molecules are ascorbic and dehydroascorbic acids [52] , triclosan [53] and the camphor imine derivatives. various studies conducted in recent years showed the antiviral potential of the latter, especially against influenza viruses [54] [55] [56] . there are two main approaches to the use of polymers in the antiviral campaign. one includes the use of polymers with only a supportive role, using polymers for antiviral drug delivery. the second includes introducing the polymers in the anti-viral front-line using polymers containing ions and/or metal particles or polymers with antiviral moieties such as amines, ions moieties, carboxylic acid moieties, sulphates, phenols and more. although the first one has been in the focus of most relevant research, only a few examples will be demonstrated related this work, as the second approach is the focus of the current review. chapter 1: chapter 2: the enhancement of effective drug release has intensively occupied researchers in recent years. one route suggested is the use of drug-conjugated bio-degradable polymers as drug-delivery agents. by controlling the degradation rate, the drug release could also be controlled. based on this method, several antiviral-drug-conjugated polymers have been studied over the years. ahmad et al., for example, studied the uses of a novel chitosan/xanthan gum-based hydrogels as an acyclovir (a commercial antiviral drug) delivery. they found that this cross-linked polymer could be used for antiviral drug delivery, while its efficiency is depended on the drug to polymer ratio, ph, loading time, cross-linking density and other controlled parameters [44] . other research groups have also studied antiviral drugs that release polymeric systems and mechanisms. based on their results, it was concluded that the degree of the antiviral effect (i.e., the drug release rate) was dependent on several controllable parameters such as the chemical structure of the polymer, the molecular weight, loading concentrations and the combination with the use of inherently antiviral-polymers [57] [58] [59] . liu et al. also studied the effect of temperature on viruses [60] . they found that tobacco plants gained better viral resistance at 30 • c compared to at 20 • c. haam et al. studied the efficiency of using poly(ethylene glycol)-block-poly(phenylalanine) as a drug delivery system, especially against the influenza a virus [61] . they found that due to the better virus envelope penetrating ability of the polymer compared to the model drugs, the antiviral effect increased. chen et al. studied the use of poly-l-glutamine (pgn) conjugated with zanamivir and demonstrated that this system had an antiviral efficiency against early and late-stage influenza virus infection [62] . antiviral drug delivery has also been studied using metal-organic frameworks (mofs), exploiting both their porous structure and their biodegradable properties. an example of this approach is the work done by gref et al. who studied the use of a mof obtained by reacting fe(iii) and trimesic acid as an anti-hiv azt-tp drug delivery system [63] . other examples are the work of keskin and kızılel about the antiviral drug delivery uses of three different mofs developed by reacting zn 4 o(co) 2 and three different dicarboxylate linkers [64] and the work novio et al. about the uses of iron-catechol-based mofs as antiviral drugs delivery systems [65] . while polymer antibacterial and antimicrobial properties have been well studied over the years, only a few studies were conducted on the antiviral properties of polymers. several proposed approaches are discussed in this paper, as follows. first, polymerization of commercial antiviral drugs and/or polymers grafted with commercial drugs (pro)drugs. second, adding antiviral additives such as metal or ion-particles to achieve an antiviral effect. other approaches are amine-containing polymers and/or polycations, poly(carboxylic acid)s, polyanhydrides and/or polyanions, sulphate and sulphuric acid-containing polymers and hydroxyl-containing polymers, polyphenols, and/or some other non-ionic polymers. in contrast to their use in a supporting role, described above, when used in a front-line role, polymers do not go through any degradation process to accomplish their antiviral effects. "macromolecular (pro)drugs", polymerized commercial antiviral drugs or polymers grafted with commercial drugs as pendant groups, are straightforward examples for polymers with inherent antiviral activity. several studies have been conducted using this approach, such as zelikin and his group about ribavirin [66, 67] . larson also studied the uses of (pro)drugs to overcome immunity against some commercial antiviral drugs. while this approach was found useful in some circumstances, it wasn't an efficient method for all drugs tested [68, 69] , thus efficacy might be attributed to other mechanisms. bovin and associates, synthesized poly(n-2-hydroxyethylacrylamide) (6 sln-paa) by polymerization of the co-monomers neu5acα2-6galβ1-4glcnacβ (6 sln) and polyacrylamide as shown in figure 1 [70] . owing to the 6 sln antiviral properties, the entire co-polymer gains antiviral effect against the influenza virus. an increase attraction between viruses and polymer enhanced the drugs' original antiviral effect. polymers 2020, 12, x for peer review 5 of 32 sulphate and sulphuric acid-containing polymers and hydroxyl-containing polymers, polyphenols, and/or some other non-ionic polymers. in contrast to their use in a supporting role, described above, when used in a front-line role, polymers do not go through any degradation process to accomplish their antiviral effects. "macromolecular (pro)drugs", polymerized commercial antiviral drugs or polymers grafted with commercial drugs as pendant groups, are straightforward examples for polymers with inherent antiviral activity. several studies have been conducted using this approach, such as zelikin and his group about ribavirin [66, 67] . larson also studied the uses of (pro)drugs to overcome immunity against some commercial antiviral drugs. while this approach was found useful in some circumstances, it wasn't an efficient method for all drugs tested [68, 69] , thus efficacy might be attributed to other mechanisms. bovin and associates, synthesized poly(n-2-hydroxyethylacrylamide) (6′sln-paa) by polymerization of the co-monomers neu5acα2-6galβ1-4glcnacβ (6′sln) and polyacrylamide as shown in figure 1 [70] . owing to the 6′sln antiviral properties, the entire co-polymer gains antiviral effect against the influenza virus. an increase attraction between viruses and polymer enhanced the drugs' original antiviral effect. based on a similar principle, carraher et al. studied the effect of two commercial antiviral drugsbased polymers. they found that cisplatin derivatives containing tilorone polymers have an antiviral effect on herpes simplex-1, vaccinia, and varicella zoster and reovirus viruses and a tilorone derivative polymers have also an antiviral effect on both dna and rna viruses [71, 72] . polymers (see figure 2 ) were synthesized using tilorone or tilorone 11,567 (both commercially available) dissolved in distilled water, and later added to a stirred solution of potassium tetrachloroplatinate(ii) in distilled water at room temperature. they also found these two polymers require far lower concentrations to achieve the same antiviral effect, compared with k2ptcl4, acyclovir and/or cisplatin, as shown in figure 2 . based on a similar principle, carraher et al. studied the effect of two commercial antiviral drugs-based polymers. they found that cisplatin derivatives containing tilorone polymers have an antiviral effect on herpes simplex-1, vaccinia, and varicella zoster and reovirus viruses and a tilorone derivative polymers have also an antiviral effect on both dna and rna viruses [71, 72] . polymers (see figure 2 ) were synthesized using tilorone or tilorone 11,567 (both commercially available) dissolved in distilled water, and later added to a stirred solution of potassium tetrachloroplatinate(ii) in distilled water at room temperature. they also found these two polymers require far lower concentrations to achieve the same antiviral effect, compared with k 2 ptcl 4, acyclovir and/or cisplatin, as shown in figure 2 . the addition of metallic nanoparticles to achieve antiviral properties is one of the most studied approaches. an example is the study conducted by rashid et al. who examined the addition of silver nanoparticles to polyaniline. they report that this addition contributed to the polymer antimicrobial and antiviral properties [73] . naka et al. registered a us patent (2007/0169278 a1), based on a similar method, including the addition of metal ions (ag, cu, zn, al, mg, and ca) to textile to obtain an antiviral effect [74] . imai et al. also explored the addition of copper ions to polymers to obtain an antiviral effect. they showed that when adding copper ions to zeolites (cuzeo), the cotton gained antiviral properties against avian influenza virus h5 [75] . silver (ag) particles have an antiviral effect even in their metal form; yamamoto et al. showed that the addition of ag particles to a cotton textile granted it antiviral effect against influenza a and feline calicivirus [76] . moreover, the addition of eagle's minimal essential medium decreased the efficiency of tag particles. elechiguerra et al. [77] and lara et al. [78, 79] also studied the addition of ag particles to polymers. they showed that small ag particles (1-10 nm) added to poly(n-vinyl-2-pyrrolidone), interacted with hiv-1′s gp120 protein causing viral inhibition. shree et al. found that large ag particles (~69 nm) interfere with respiratory syncytial virus cell attachment [80] . rogers et al. showed that the addition of 10-80 nm ag particles to acacia gum (a natural polysaccharide) blocked monkeypox virus interactions with host cells [81] . the same effect on tacaribe virus was reported by hussain et al. [82] . ishihara et al. showed that the antiviral effect of ag-chitosan composites increase with the concentration of ag particles [83] and balagna et al. showed that ag-silica composite nanoparticles coated masks via a sputtered coating method has an antiviral effect on sars-cov-2 virus [84] . romero and his associates experimented with the addition of zinc acetate (zn(ac)2) to carrageenan-based gels. the combination possessed antiviral properties against herpes simplex virus 2 (hsv-2) and simian immunodeficiency virus (sivs) both in vitro and in vivo (tested on mises) [85] . the anti-viral properties of quaternary ammonium has been studied extensively in the literature [37] [38] [39] . lino et al. explored the antimicrobial and antiviral effect of silica nanoparticles coated with didodecyldimethylammonium bromide (ddab) [86] . two silica nanoparticles were tested -snp8 and snp80, with 8 nm diameter and 80 nm diameter respectively, while the snp8 was used as a reference. as illustrated in figure 3 , coating glass slides with snp80 followed by another layer of the addition of metallic nanoparticles to achieve antiviral properties is one of the most studied approaches. an example is the study conducted by rashid et al. who examined the addition of silver nanoparticles to polyaniline. they report that this addition contributed to the polymer antimicrobial and antiviral properties [73] . naka et al. registered a us patent (2007/0169278 a1), based on a similar method, including the addition of metal ions (ag, cu, zn, al, mg, and ca) to textile to obtain an antiviral effect [74] . imai et al. also explored the addition of copper ions to polymers to obtain an antiviral effect. they showed that when adding copper ions to zeolites (cuzeo), the cotton gained antiviral properties against avian influenza virus h5 [75] . silver (ag) particles have an antiviral effect even in their metal form; yamamoto et al. showed that the addition of ag particles to a cotton textile granted it antiviral effect against influenza a and feline calicivirus [76] . moreover, the addition of eagle's minimal essential medium decreased the efficiency of tag particles. elechiguerra et al. [77] and lara et al. [78, 79] also studied the addition of ag particles to polymers. they showed that small ag particles (1-10 nm) added to poly(n-vinyl-2-pyrrolidone), interacted with hiv-1 s gp120 protein causing viral inhibition. shree et al. found that large ag particles (~69 nm) interfere with respiratory syncytial virus cell attachment [80] . rogers et al. showed that the addition of 10-80 nm ag particles to acacia gum (a natural polysaccharide) blocked monkeypox virus interactions with host cells [81] . the same effect on tacaribe virus was reported by hussain et al. [82] . ishihara et al. showed that the antiviral effect of ag-chitosan composites increase with the concentration of ag particles [83] and balagna et al. showed that ag-silica composite nanoparticles coated masks via a sputtered coating method has an antiviral effect on sars-cov-2 virus [84] . romero and his associates experimented with the addition of zinc acetate (zn(ac) 2 ) to carrageenan-based gels. the combination possessed antiviral properties against herpes simplex virus 2 (hsv-2) and simian immunodeficiency virus (sivs) both in vitro and in vivo (tested on mises) [85] . the anti-viral properties of quaternary ammonium has been studied extensively in the literature [37] [38] [39] . lino et al. explored the antimicrobial and antiviral effect of silica nanoparticles coated with didodecyldimethylammonium bromide (ddab) [86] . two silica nanoparticles were tested -snp8 and snp80, with 8 nm diameter and 80 nm diameter respectively, while the snp8 was used as a reference. as illustrated in figure 3 , coating glass slides with snp80 followed by another layer of based on their antiviral effect, some have suggested the use of metallic nanoparticles such as ag, mg, tio2 etc. as coating agents to achieve antiviral properties on polymeric surfaces and fabrics. for example, us patent number 2019/0045793 a1 demonstrated tio2, crystalline silver copper aluminum silicate, alumina and some more metallic-based coatings [87] . the addition of tio2 was also demonstrated by thilagavathi and parthasarathi who also studied the addition of metal-based particles to textiles to obtain an antiviral effect aiming at the production of antiviral surgical gowns. it was found that tio2 particles in nonwoven polyester grants an antiviral effect eliminating hiv, hepatitis b, and hepatitis c viruses (only) under visible light [88] . for further information about the uses of metal/metal oxide nanoparticles in polymeric matrices to obtain antiviral properties, see the review by hilal et al. [89] . another method of antiviral coating was demonstrated by sahin et al. who studied the consequence of adding 3% sodium pentaborate pentahydrate, 0.03% triclosan and 7% glucapon solution to cotton textile [90] . they reported that after 30 min in solution (and after drying it), the textile gained antimicrobial and antiviral properties. the antimicrobial effect was tested against five bacteria (such as escherichia coli, staphylococcus aureus, and salmonella enterica subsp), one yeast (candida albicans), two fungi (aspergillus niger and trichophyton mentagrophytes) and two viruses (adenoid 75 strain of human adenovirus type 5 and chat strain of human poliovirus type 1). the use of organo-metal hybrid polymers was also suggested. one example is polymeric anhydride of magnesium and proteic ammonium phspholinoleates that demonstrated antiviral properties against hsv-1, adenovirus 5 and canine parvovirus, as shown by durán and nunes in their us patent number 5,073,630 [91] . organotin polymers have also been studied as antiviral polymers. frank and her group proposed several organotin polymers with antiviral effects against vaccinia virus and zika virus [92] . those polymers, as shown in figure 4 , were synthesized using organotin dihalides, camphoric acid and lamivudine. the mechanism for the antiviral effect of organotin polymers is described in the study of barot et al. during their study, they figured that organotin materials inhibited the cell proliferation and sister chromatid exchanges, which blocked the replication of the cells. since most viruses depend on the cell replication, these materials have inherent antiviral properties [93] . some other organo-metalhybrid polymers that have been reported as antiviral polymers are bis(cyclopentadienyl)zirconium dichloride and diethylstilbestrol-based polymers [94] . based on their antiviral effect, some have suggested the use of metallic nanoparticles such as ag, mg, tio 2 etc. as coating agents to achieve antiviral properties on polymeric surfaces and fabrics. for example, us patent number 2019/0045793 a1 demonstrated tio 2 , crystalline silver copper aluminum silicate, alumina and some more metallic-based coatings [87] . the addition of tio 2 was also demonstrated by thilagavathi and parthasarathi who also studied the addition of metal-based particles to textiles to obtain an antiviral effect aiming at the production of antiviral surgical gowns. it was found that tio 2 particles in nonwoven polyester grants an antiviral effect eliminating hiv, hepatitis b, and hepatitis c viruses (only) under visible light [88] . for further information about the uses of metal/metal oxide nanoparticles in polymeric matrices to obtain antiviral properties, see the review by hilal et al. [89] . another method of antiviral coating was demonstrated by sahin et al. who studied the consequence of adding 3% sodium pentaborate pentahydrate, 0.03% triclosan and 7% glucapon solution to cotton textile [90] . they reported that after 30 min in solution (and after drying it), the textile gained antimicrobial and antiviral properties. the antimicrobial effect was tested against five bacteria (such as escherichia coli, staphylococcus aureus, and salmonella enterica subsp), one yeast (candida albicans), two fungi (aspergillus niger and trichophyton mentagrophytes) and two viruses (adenoid 75 strain of human adenovirus type 5 and chat strain of human poliovirus type 1). the use of organo-metal hybrid polymers was also suggested. one example is polymeric anhydride of magnesium and proteic ammonium phspholinoleates that demonstrated antiviral properties against hsv-1, adenovirus 5 and canine parvovirus, as shown by durán and nunes in their us patent number 5,073,630 [91] . organotin polymers have also been studied as antiviral polymers. frank and her group proposed several organotin polymers with antiviral effects against vaccinia virus and zika virus [92] . those polymers, as shown in figure 4 , were synthesized using organotin dihalides, camphoric acid and lamivudine. the mechanism for the antiviral effect of organotin polymers is described in the study of barot et al. during their study, they figured that organotin materials inhibited the cell proliferation and sister chromatid exchanges, which blocked the replication of the cells. since most viruses depend on the cell replication, these materials have inherent antiviral properties [93] . some other organo-metalhybrid polymers that have been reported as antiviral polymers are bis(cyclopentadienyl)zirconium dichloride and diethylstilbestrol-based polymers [94] . polyethyleneimine (pei) is one of the most studied amine-containing polymers in the field of antimicrobial polymers. for example, vos et al. studied the antiviral effect of branched (pei) coating over polyether sulphone (pes) membrane [95] . pei coating led to an increase of ≥ 3 log -units (≥99.9%) in ms2 bacteriophages hosting strain salmonella typhimurium wg49 reduction on the membrane surface. as shown in figure 5 , the effect of pei has been examined on glass slides and by using bulk solution with 1.3% pei. besides its inherent antiviral effect, yang et al. considered some modification of pei. they showed that owing to their effect on the ph and ability to block viral entry through electrostatic interactions, mannose derivatives containing pei had antiviral effects [96] . polyethyleneimine (pei) is one of the most studied amine-containing polymers in the field of antimicrobial polymers. for example, vos et al. studied the antiviral effect of branched (pei) coating over polyether sulphone (pes) membrane [95] . pei coating led to an increase of -units (99.9%) in ms2 bacteriophages hosting strain salmonella typhimurium wg49 reduction on the membrane surface. as shown in figure 5 , the effect of pei has been examined on glass slides and by using bulk solution with 1.3% pei. besides its inherent antiviral effect, yang et al. considered some modification of pei. they showed that owing to their effect on the ph and ability to block viral entry through electrostatic interactions, mannose derivatives containing pei had antiviral effects [96] . another study about modified peis was conducted by moeller et al. they examined several modified peis obtained by reacting pei and different cyclic carbonate derivatives [46] . while their study focused on antimicrobial/antibacterial properties, generally, antimicrobial materials also exhibit antiviral properties against enveloped viruses. modified peis were also evaluated by alvarez-lorenzo et al. synthesizing modified pei by polymerization of aziridine and magnetite-silica core-shell particles. it was evident that these polymers inhibited bacteriophage ms2, hsv-1, enveloped viral hemorrhagic septicaemia viruses (vhsv), and nonenveloped infectious pancreatic necrosis virus (ipnv) [97] . polymers 2020, 12, x for peer review 3 of 33 oxide (cu2o), sulphide (cu2s), iodide (cui), and chloride (cucl) have high antiviral efficiency, compared to solid-state silver and cupric compounds [22] . zinc is another example of a metal cation that has been well studied for its in vitro antiviral effect on the viral proteins (at low concentrations) and dna (at high concentrations) [23] . read et al. in their review about the role of zinc in antiviral immunity, pointed out that the zinc concentrations that were needed to achieve the antiviral effect (mm [23] ) are much higher than the physiological concentrations (μm), with some differences in the needed concentrations between different types of viruses [24] . for example, van hemert and his associates demonstrated the inhibition effect of some zinc derivatives over the binding and elongation of coronaviruses' rdrp enzymes [25] . they showed that a combination of 2-320 μm of pyrithione with 2-500 μm of zn(ac)2 has a major effect, with increasing effect at higher concentrations, over sars-cov and eav populations. moreover, they suggested that water-soluble zinc-ionophores might have a higher antiviral effect. hong et al. demonstrated the effect of 60-300 μm zncl2 on the rna polymerase of hepatitis c viruses [26] . takagi et al. also demonstrated the effect of zncl2, but with lower concentrations of 50-150 μm on the replication of hepatitis c viruses [27] . further, 50-200 μm of znso4 has an antiviral effect, as shown by ahlenstiel et al. [28] and by brendel et al. [23] . some other zn salts that showed antiviral effects are pyrrolidine dithiocarbamate (pdtc), zinc gluconate (zn(glu)2) [24, 29] , zinc lactate (zn(lac)2), zinc citrate (cizar), zinc picolinate (zn(pic)2), and zinc aspartate (zn(asp)2) [24] as well as zinc ionophores pyrithione [30] . also, it seems that the antiviral effect of zinc ions is temperaturedepended. while at lower temperatures (4-18 °c) zinc has no significant effect on the virus population, at elevated temperatures (20-25 °c) the antiviral effect is significantly increased [23] . while only a few studies have been conducted on the effect of iron ions, it seems that these ions have some level of antiviral effect, as described in aagripanti et al. work [31] . cobalt (iii) is also a metal ion with antiviral effects [32] . some other metal-based anions showed antiviral properties like nickel [20] , polyoxometalates, polyatomic ions containing three transitional metals such as titanium, vanadium, tungsten, molybdenum, etc. that form clusters with the surrounding oxygen (the transition metal oxyanions linked to each other with sharing oxygen atoms). those anions show antiviral properties by affecting larson studied modified pei composed of n,n-dodecylmethyl-pei that exhibited antiviral effect on hsv-1 and hsv-2 viruses (see also figure 6 ) [98] , influenza a virus [99] and on poliovirus and rotavirus [100] . other modified pei tested by larson was based on n,n-hexylmethyl-pei coated on polyethylene, a surface with antiviral effect eliminating poliovirus and rotavirus [100] . another study about modified peis was conducted by moeller et al. they examined several modified peis obtained by reacting pei and different cyclic carbonate derivatives [46] . while their study focused on antimicrobial/antibacterial properties, generally, antimicrobial materials also exhibit antiviral properties against enveloped viruses. modified peis were also evaluated by alvarez-lorenzo et al. synthesizing modified pei by polymerization of aziridine and magnetite-silica coreshell particles. it was evident that these polymers inhibited bacteriophage ms2, hsv-1, enveloped viral hemorrhagic septicaemia viruses (vhsv), and nonenveloped infectious pancreatic necrosis virus (ipnv) [97] . larson studied modified pei composed of n,n-dodecylmethyl-pei that exhibited antiviral effect on hsv-1 and hsv-2 viruses (see also figure 6 ) [98] , influenza a virus [99] and on poliovirus and rotavirus [100] . other modified pei tested by larson was based on n,n-hexylmethyl-pei coated on polyethylene, a surface with antiviral effect eliminating poliovirus and rotavirus [100] . chitosan has also been studied for its antiviral and antibacterial effects. as been described in chirkov review, chitosan (and chitin) can induce interferon synthesis, which leads to suppression of the virus replication by causing damage to the rna and/or mrna [36] . the chitosan effect was tested chitosan has also been studied for its antiviral and antibacterial effects. as been described in chirkov review, chitosan (and chitin) can induce interferon synthesis, which leads to suppression of the virus replication by causing damage to the rna and/or mrna [36] . the chitosan effect was tested on influenza a, influenza b, alfalfa mosaic virus, bean goldish mosaic virus, peanut stunt virus, tmv, tobacco necrosis virus and some other plant viruses. moreover, some chitosan sulphate derivatives also showed an antiviral effect on hiv-1. another review about the antiviral effect of chitin polymers (i.e., chitin and chitosan) and their monomer (i.e., n,n-diacetylchitobiose dimer and n-acetylglucosamine) was conducted by rogers et al. deducing similar conclusions [101] . lei et al. also studied the antiviral effects of chitosan and found that chitosan obtained from musca domestica l housefl's larvas has an antiviral effect, based on tests conducted on autographa californica multicapsid nucleopolyhedrovirus (acmnpv) and bombyx mori nuclear polyhydrosis virus (bmnpv) [102] . wu et al. studied a combination of cytosinpeptidemycin and chitosan oligo-saccharide, found it has an antiviral effect on tms, most likely due to several mechanisms, such as a suppression effect of the viral rna, an effect on the virus's subcellular localization as well as punctate formation of tmv mp in some plants leaves [103] . as opposed to the above, ishihara found chitosan to be ineffective against the influenza a virus [83] . lembo et al. studied the effect of other amines-containing polymers like poly(amidoamine)s [104] . six polymers labeled as isa1, isa23, agma1, agma1 4 , and agma1 7 were studied and found to exhibit antiviral effects on hsv-1, hsv-2, human cytomegalovirus, human papillomavirus-16, respiratory syncytial virus, human rhinovirus, and vesicular stomatitis virus. other amine-containing polymers were synthesized by xiao et al. by reacting polyhexamethylene guanidine hydrochloride and acrylamide using β-cyclodextrin (cd) with eight active and five active sites. they achieved star polymers with antiviral effect on non-enveloped adenovirus [105] . pitha et al. researched the antiviral effect of amine-containing polymers. they showed that both poly(9-vinyladenine) and poly(l-vinyluracil) exhibited better antiviral properties, compared to commercial antiviral drugs [106] . as forementioned, quaternary ammonium moieties have been studied for their antiviral properties. duizer et al. investigated the antiviral effect of several hyperbranched quaternary ammonium containing polymers on influenza a (an envelope virus) and poliovirus sabin1 (a non-envelop virus) [40] . they coated glass and plastic surfaces with the polymers, and then tested their effect on the respective viruses' populations. to obtain the hyperbranched quaternary ammonium polymers, they reacted in a polymer solution containing polyamine, k 2 co 3 , tert-amylalcohol and heptylbromide. while the envelope containing influenza a virus showed significant decay, the non-enveloped sabin1 showed almost no change. klibanov et al. also studied ammonium hybrid polymers for their antiviral properties on influenza viruses [107] . polymers labeled 1a-c, 2a-c, 3, 4, and 5 (see figure 7) , exhibited an antiviral effect in correlation with remaining cationic polymers attached to the slide surfaces. it was postulated that the antiviral effect happened by contact between the viruses and the polymers. moreover, they postulated the polymers labeled as 1a-c and 2a-c possessed probably some other antiviral mechanisms. ammonium containing phenylene ethynylene based polymers and oligomers have also been studied as antiviral materials by whitten et al. [108] . they tested several polymers and oligomers, as shown in figure 8 , against two model non-enveloped viruses, ms2 and t4 bacteriophages. the first is an rna virus and the latter is a dna one. they discovered that these hybrid polymers caused a ammonium containing phenylene ethynylene based polymers and oligomers have also been studied as antiviral materials by whitten et al. [108] . they tested several polymers and oligomers, as shown in figure 8 , against two model non-enveloped viruses, ms2 and t4 bacteriophages. the first is an rna virus and the latter is a dna one. they discovered that these hybrid polymers caused a partial dissociation of the virus structure in the dark, while under visible light and/or uv, these materials led to photochemical damage to the viruses' capsid protein. ammonium containing phenylene ethynylene based polymers and oligomers have also been studied as antiviral materials by whitten et al. [108] . they tested several polymers and oligomers, as shown in figure 8 , against two model non-enveloped viruses, ms2 and t4 bacteriophages. the first is an rna virus and the latter is a dna one. they discovered that these hybrid polymers caused a partial dissociation of the virus structure in the dark, while under visible light and/or uv, these materials led to photochemical damage to the viruses' capsid protein. larson investigated the antiviral and antimicrobial effects of ammonium containing n,n-dodecylmethyl-polyurethane (synthesis as illustrated in figure 9 ) [109] . it was concluded that this polymer, used as a coating, has an antiviral effect against influenza a viruses (enveloped) but did not affect the non-enveloped poliovirus. larson investigated the antiviral and antimicrobial effects of ammonium containing n,ndodecylmethyl-polyurethane (synthesis as illustrated in figure 9 ) [109] . it was concluded that this polymer, used as a coating, has an antiviral effect against influenza a viruses (enveloped) but did not affect the non-enveloped poliovirus. zhao and associates studied the combined antiviral effect of phosphonium and ammonium, by synthesizing phosphonium-type cationic polyacrylamide as shown in figure 10 [42] . they showed that this copolymer has an antiviral effect against non-enveloped adenovirus. zhao and associates studied the combined antiviral effect of phosphonium and ammonium, by synthesizing phosphonium-type cationic polyacrylamide as shown in figure 10 [42] . they showed that this copolymer has an antiviral effect against non-enveloped adenovirus. oligomeric ammonium-silane based systems and their impact on herpesvirus has been studied by prusty et al. [110] . their results are consistent with other works related to the antiviral effect of ammonium. shuto et al. patented the antiviral (and antimicrobial) properties of quaternary ammonium ion (us patent number 10,550,274b2), antiviral coatings containing acrylic melamine, quaternary ammonium, multi-valent aromatic carboxylic-acid, and phosphoric acid were included in the claims [111] . it should be noted that the coating was tested against influenza-a viruses. quaternary pyridinium containing polymers have also been studied for their antiviral and antimicrobial properties. xiao and xue examined the antiviral effect of quaternary pyridinium containing co-polymers on several influenza viruses (a, pr8, 8, 34) , as demonstrated in figure 11 [35] . oligomeric ammonium-silane based systems and their impact on herpesvirus has been studied by prusty at el [110] . their results are consistent with other works related to the antiviral effect of ammonium. shuto et al. patented the antiviral (and antimicrobial) properties of quaternary ammonium ion (us patent number 10,550,274b2), antiviral coatings containing acrylic melamine, quaternary ammonium, multi-valent aromatic carboxylic-acid, and phosphoric acid were included in the claims [111] . it should be noted that the coating was tested against influenza-a viruses. quaternary pyridinium containing polymers have also been studied for their antiviral and antimicrobial properties. xiao and xue examined the antiviral effect of quaternary pyridinium containing co-polymers on several influenza viruses (a, pr8, 8, 34) , as demonstrated in figure 11 [35]. as shown by muñoz-fernández et al., caprolactam containing polymers also have antiviral properties. they synthesized poly(n-vinyl caprolactam)-nanogels with different levels of crosslinking agents and showed that a polymer with 80 mg of vcl and 4% of bis crosslinking agent inhibited the replication of r5-hiv-1 viruses in cells [112] . it was concluded that the antiviral effect of this polymer is affected by its thermal properties, collapsing to nanoparticles at body temperatures. since copper ions are inherently antiviral, studies have been conducted on polymers conjugated with them. for example, in 2012 gabbay has registered a us patent consisting of adding copper as shown by muñoz-fernández et al., caprolactam containing polymers also have antiviral properties. they synthesized poly(n-vinyl caprolactam)-nanogels with different levels of cross-linking agents and showed that a polymer with 80 mg of vcl and 4% of bis crosslinking agent inhibited the replication of r5-hiv-1 viruses in cells [112] . it was concluded that the antiviral effect of this polymer is affected by its thermal properties, collapsing to nanoparticles at body temperatures. since copper ions are inherently antiviral, studies have been conducted on polymers conjugated with them. for example, in 2012 gabbay has registered a us patent consisting of adding copper particles to some polymeric fibers, such as polyamide 6,6 fibers [113] . moreover, a review on the uses of copper and silver particles has been conducted by sánchez et al. indicating that antiviral activity can be identified in chitosan with green seed extract, polyhydroxybutyrate (phb) with cinnamaldehyde (for more information about the antiviral effect of cinnamaldehyde, see [114] ), poly(lactic acid) (pla) with silver ions, polyhydroxybutyrate valerate (phbv) with silver nanoparticles or with copper ions and more [115] . finkelstein and merigan studied the antiviral effect of several commercial negatively charged carboxylate-based polymers, as illustrated in figure 12 [116] . based on their findings, it was concluded that a higher molecular weight with higher carboxylate free groups led to a higher antiviral effect, although in vivo experiments showed that the polymers were activated only when complexed with organic cations (arginine and poly l-ornithine (plo)). in addition, bounding the carboxylate groups by amidation reduced the antiviral effect to a minimum. they emphasized that, unlike their previous hypothesis, a higher antiviral effect was achieved using non-degradable polymers. finkelstein and merigan studied the antiviral effect of several commercial negatively charged carboxylate-based polymers, as illustrated in figure 12 [116] . based on their findings, it was concluded that a higher molecular weight with higher carboxylate free groups led to a higher antiviral effect, although in vivo experiments showed that the polymers were activated only when complexed with organic cations (arginine and poly l-ornithine (plo)). in addition, bounding the carboxylate groups by amidation reduced the antiviral effect to a minimum. they emphasized that, unlike their previous hypothesis, a higher antiviral effect was achieved using non-degradable polymers. some other polycarboxylates were also studied by loebenstein and stein [117] . by assessing the antiviral effect of poly(ethylene-co-maleic anhydride), poly(acrylic acid), poly(methacrylic acid), poly(vinyl methyl ether-co-maleic anhydride), poly(vinyl methyl ether-co-maleic acid), poly(vinyl methyl ether-co-maleic anhydride-co-methyl ester), poly(styrene-co-maleic anhydride), poly(isobutylene-co-maleic anhydride) and poly( -olefin octadecene-co-maleic anhydride) they understood that these co-polymers had an antiviral effect on tmv only in vivo. regelson and feltz, who studied the antiviral effect of ethylene-maleic anhydride-based-polymers showed, like finkelstein and merigan, that those polymers had an antiviral effect also in vitro [118] . based on the antiviral effect of the malic acid/anhydride derivatives, tsunekuni et al. registered a us patent number 2010/0272668a1 claiming several polymeric fibers containing an olefine-maleic acid copolymer, a styrene-maleic acid copolymer, a vinyl ester-maleic acid copolymer, a vinyl acetatemaleic acid copolymer and a vinyl chloride-maleic acid copolymer [119] . styrene-alt-maleic acid copolymer was also found to inhibit r5 and x4 hiv-1′s infection by krebs et al. [120] . for further information about the effect of maleic anhydride containing polymers on different viruses, see the manuscript by popescu et al. [121] . using the alkyne-azide click chemistry, mata et al. studied the antiviral effect of carbosilanecontaining anionic dendrimers [122] . they found that these negatively charged dendrimers displayed an antiviral effect against hiv-1 viruses and that phosphonate containing dendrimers did not show any antiviral effect. as a result of the similarity to carboxylate, carboxylic acids containing polymers have been suggested as antiviral polymers, as claimed by mandeville and neenan in their us patent number 6,060,235 [123] . humic acid (ha) based phenolic polymers are polyanion polymers exhibiting antiviral activity. helbig et al. studied these polymer systems, by reacting and analyzing the effect of different o-diphenolic compounds on the polymers' antiviral effect on hsv-1 viruses [124] . their results indicate that increasing the number of carboxylic groups and the number of unsaturated moieties in the starting compounds led to an increase in the antiviral activity of the respective some other polycarboxylates were also studied by loebenstein and stein [117] . by assessing the antiviral effect of poly(ethylene-co-maleic anhydride), poly(acrylic acid), poly(methacrylic acid), poly(vinyl methyl ether-co-maleic anhydride), poly(vinyl methyl ether-co-maleic acid), poly(vinyl methyl ether-co-maleic anhydride-co-methyl ester), poly(styrene-co-maleic anhydride), poly(isobutylene-co-maleic anhydride) and poly(α-olefin octadecene-co-maleic anhydride) they understood that these co-polymers had an antiviral effect on tmv only in vivo. regelson and feltz, who studied the antiviral effect of ethylene-maleic anhydride-based-polymers showed, like finkelstein and merigan, that those polymers had an antiviral effect also in vitro [118] . based on the antiviral effect of the malic acid/anhydride derivatives, tsunekuni et al. registered a us patent number 2010/0272668a1 claiming several polymeric fibers containing an olefine-maleic acid copolymer, a styrene-maleic acid copolymer, a vinyl ester-maleic acid copolymer, a vinyl acetate-maleic acid copolymer and a vinyl chloride-maleic acid copolymer [119] . styrene-alt-maleic acid copolymer was also found to inhibit r5 and x4 hiv-1 s infection by krebs et al. [120] . for further information about the effect of maleic anhydride containing polymers on different viruses, see the manuscript by popescu et al. [121] . using the alkyne-azide click chemistry, mata et al. studied the antiviral effect of carbosilanecontaining anionic dendrimers [122] . they found that these negatively charged dendrimers displayed an antiviral effect against hiv-1 viruses and that phosphonate containing dendrimers did not show any antiviral effect. as a result of the similarity to carboxylate, carboxylic acids containing polymers have been suggested as antiviral polymers, as claimed by mandeville and neenan in their us patent number 6,060,235 [123] . humic acid (ha) based phenolic polymers are polyanion polymers exhibiting antiviral activity. helbig et al. studied these polymer systems, by reacting and analyzing the effect of different o-diphenolic compounds on the polymers' antiviral effect on hsv-1 viruses [124] . their results indicate that increasing the number of carboxylic groups and the number of unsaturated moieties in the starting compounds led to an increase in the antiviral activity of the respective polymers. other acid-containing polymers reported as antiviral polymers are poly(lysine), poly(glutamic acid), and poly(acrylic acid) [43] . sano has considered the effect of alginate on tmv [125] , which is a natural anionic polymer, usually obtained from seaweeds [126] . he indicated that alginate had an antiviral effect on tms, demonstrating greater effect with decreasing polymer chains' stiffness [125] . as shown by mooney and lee, alginate also exhibits an antiviral effect on adenovirus [126] . sulphate containing polymers have also been studied for their antiviral properties. görög et al. studied the effect of polyvinyl-alcohol-sulphate (pvas) and polyvinyl-alcohol-sulphate-co-acrylic acid (pavas) as inhibitors for hiv-1, hiv-2, herpesvirus, human cytomegalovirus, vesicular stomatitis virus, respiratory syncytial virus, sindbis virus, semliki forest virus, junin virus, tacaribe virus, and murine sarcoma virus [127] . the two polymers were shown to have an antiviral effect over those viruses but do not affect non-envelop viruses such as reovirus and poliovirus. they also found that the greater the sulphonation degree and/or the greater the molecular weight of the polymers, the higher the antiviral effect became. hayashi et al. explored the antiviral effect of modified calcium spirulan, a sulphated polysaccharide. they established that several modified spirulan have an antiviral property. unlike other studies, non-toxic metal ions have a greater effect compared to copper and silver modified spirulan [128] . patents have also been registered in the field of sulphate containing polymers, like the us patent of munson and tankersley who patented the use of sulphonated naphthalene formaldehyde condensates (snfc) as antiviral materials [129] . the effect of sulphate on several viruses has also been analyzed by schelhaas at el. in their inquiry, they tested the antiviral effect of sulphated glycomimetic oligomers and polymers as described in figure 13 and table 1 [130] . spontak et al. studied the antiviral and antimicrobial properties of poly[tert-butylstyrene-b-(ethylene-alt-propylene)-b-(styrene sulphonate)-b-(ethylene-alt-propylene)-b-tert-butylstyrene] (teset). as already mentioned, they found that the antiviral efficiency of the polymer increases with increasing the degree of sulphonation (in their study, they changed the degree of mol% of the mid-block) [131] . polymers 2020, 12, x for peer review 17 of 32 [76] . polymers 2020, 12, x for peer review 17 of 32 mimura et al. suggested the use of sulphate modified cellulose and branched cellulose as antiviral polymers. they found that cellulose and branched cellulose with a high degree of sulphonation demonstrated an antiviral effect on hiv-1 [132] . neurath et al. also pointed the anti-hiv-1 effect of sulphate modified cellulose as well as a similar effect of buffergel and aryl sulphonates [133] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so3-iimethylformamide complex with curdlan, they obtained antiviral properties against hiv-1 viruses [132] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv-1 [134] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv-1 and hsv-2. they suggested the antiviral effect is due to blocking of virion adsorption to host cells [135] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [136] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these mimura et al. suggested the use of sulphate modified cellulose and branched cellulose as antiviral polymers. they found that cellulose and branched cellulose with a high degree of sulphonation demonstrated an antiviral effect on hiv-1 [132] . neurath et al. also pointed the anti-hiv-1 effect of sulphate modified cellulose as well as a similar effect of buffergel and aryl sulphonates [133] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so3-iimethylformamide complex with curdlan, they obtained antiviral properties against hiv-1 viruses [132] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv-1 [134] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv-1 and hsv-2. they suggested the antiviral effect is due to blocking of virion adsorption to host cells [135] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [136] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these [133] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so3-iimethylformamide complex with curdlan, they obtained antiviral properties against hiv-1 viruses [132] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv-1 [134] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv-1 and hsv-2. they suggested the antiviral effect is due to blocking of virion adsorption to host cells [135] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [136] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these mimura et al. suggested the use of sulphate modified cellulose and branched cellulose as antiviral polymers. they found that cellulose and branched cellulose with a high degree of sulphonation demonstrated an antiviral effect on hiv-1 [132] . neurath et al. also pointed the anti-hiv-1 effect of sulphate modified cellulose as well as a similar effect of buffergel and aryl sulphonates [133] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so3-iimethylformamide complex with curdlan, they obtained antiviral properties against hiv-1 viruses [132] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv-1 [134] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv-1 and hsv-2. they suggested the antiviral effect is due to blocking of virion adsorption to host cells [135] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [136] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these mimura et al. suggested the use of sulphate modified cellulose and branched cellulose as antiviral polymers. they found that cellulose and branched cellulose with a high degree of sulphonation demonstrated an antiviral effect on hiv-1 [132] . neurath et al. also pointed the anti-hiv-1 effect of sulphate modified cellulose as well as a similar effect of buffergel and aryl sulphonates [133] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so3-iimethylformamide complex with curdlan, they obtained antiviral properties against hiv-1 viruses [132] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv-1 [134] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv-1 and hsv-2. they suggested the antiviral effect is due to blocking of virion adsorption to host cells [135] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [136] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these [133] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so 3 -iimethylformamide complex with curdlan, they obtained antiviral properties against hiv-1 viruses [132] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv-1 [134] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv-1 and hsv-2. they suggested the antiviral effect is due to blocking of virion adsorption to host cells [135] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [136] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these polymers on the virus might increase or decrease, depending on the molecular weight [137] . furthermore, the antiviral effect might be attributed to the viral envelope surface protein decapsulation process caused by the polymers. combining both amine and sulphate moieties, sulphated cellobiose-polylysine dendrimers were studied by yoshida et al. [138] . they found that a shorter distance between the terminal sulphate cellobiose units increases the anti-hiv-1 effect. sulphonic acid-containing polymers were studied by clercq et al. in the course of their study, they synthesized four polymers -poly(4-styrene sulphonic acid) (pss), poly(anethole sulphonic acid) (pas), poly(vinyl sulphonic acid) (pvs) and poly(2-acry-lamido-2-methyl-l-propane sulphonic acid) (pamps). they concluded these four polymers exhibited antiviral effect on hiv-1 and hiv-2 in mt-4 cells, using concentrations which were not toxic to the hosting cells. they also reported these polymers inhibited the syncytium formation in co-cultures of molt-4 cells infected with hiv-1 and hiv-2 [139] . pvs was also tested by shimonaski et al. with similar results [140] . cardin et al. examined a sulphonic acid polymer named mdl 101028, a biphenyl disulphonic acid urea copolymer [141] . they found this polymer has an antiviral effect on hiv-1 viruses. sulphites containing polymers also show an antiviral effect, as was demonstrated by christopher et al. in their us patent number 20170304815a1 [142] . they demonstrated a change in the oxidation degree of the sulphite's ions using a catalyst, which led to novel antiviral polymers. several hybrid polymers containing anions moieties and sulphate moieties were demonstrated by orsi et al. [143] . they revealed that dextran sulphate, scleroglucan, and lambda carrageenan have antiviral effects on hsv-1 which is greater than the effect of glyloid sulphate 4324 and locust bean gum. furthermore, these polymers influence hsv-2, with similar effect like dextran sulphate, glyloid sulphate 4324 and lambda carrageenan which is higher than the effect of scleroglucan and glycogen sulphate 4435. finally, they explained that those polymer efficacies were based on their ionic potential. polyphenols are another example of antiviral polymers. as was demonstrated by tempesta in his us patent number 5,211,944, proanthocyanidins-based polymers have antiviral properties against several viruses, including influenza a, b, and c, respiratory syncytial virus and herpes viruses [144] . gilbert et al. also confirmed the antiviral effect of polyphenols. they showed an antiviral effect of sp-303, a natural polyphenolic polymer, on respiratory syncytial and parainfluenza type 3 viruses [145] . due to the antiviral effect of polyphenols, lebrun et al. suggested the use of catechin polyphenol grafted non-woven cellulosic fabrics as bio-based cleaning wipes and filters [146] . in their study, they used laccase enzymatic oxidation to obtain the catechin grafting over the cellulose-based fabrics. these treated fabrics showed antiviral effect (5-log after 1 h) on t4d bacteriophage virus of escherichia coli b. panarin et al. [147] synthesized several co-polymers (as shown in figure 14 ) by reacting 2-dimethylaminoethyl methacrylate and 2-diethylaminoethyl methacrylate with 2-deoxy-2-methacryalamido-d-glucose and n-vinyl-n-methylacetamide with 2-dialkylaminoethyl methacrylate units. they established the antiviral properties of these polymers against influenza a virus subtype h1n1. panarin et al. [147] synthesized several co-polymers (as shown in figure 14 ) by reacting 2dimethylaminoethyl methacrylate and 2-diethylaminoethyl methacrylate with 2-deoxy-2methacryalamido-d-glucose and n-vinyl-n-methylacetamide with 2-dialkylaminoethyl methacrylate units. they established the antiviral properties of these polymers against influenza a virus subtype h1n1. benzophenones were also studied in the field of antiviral polymers. sun et al. examined the antiviral effect of benzophenones particles (see figure 15 ) on poly(vinyl alcohol-co-ethylene)-based membranes [148] . they found that these particles performed under visible light (due to the formation of rnmh·) but could also affect viruses in the dark. however, only non-envelope double-stranded dna viruses were tested. this conclusion points to the potential of these articles as rechargeable antiviral materials. benzophenones were also studied in the field of antiviral polymers. sun et al. examined the antiviral effect of benzophenones particles (see figure 15 ) on poly(vinyl alcohol-co-ethylene)-based membranes [148] . they found that these particles performed under visible light (due to the formation of rnmh·) but could also affect viruses in the dark. however, only non-envelope double-stranded dna viruses were tested. this conclusion points to the potential of these articles as rechargeable antiviral materials. in addition, 6′-fluorinated-aristeromycin analogs were proposed as antiviral materials, owing to their effect on the rna polymerase (rdrp) and the host cell s-adenosyl-l-homocysteine (sah) hydrolase. polymers based on this group have been discussed by jeong et al. as general antiviral polymers, however, some have only limited effect on few types of viruses. the most efficient of these polymers is illustrated in figure 16 , as well as its antiviral test results [149] . in addition, 6 -fluorinated-aristeromycin analogs were proposed as antiviral materials, owing to their effect on the rna polymerase (rdrp) and the host cell s-adenosyl-l-homocysteine (sah) hydrolase. polymers based on this group have been discussed by jeong et al. as general antiviral polymers, however, some have only limited effect on few types of viruses. the most efficient of these polymers is illustrated in figure 16 , as well as its antiviral test results [149] . polymers 2020, 12, x for peer review 21 of 32 tong and sankarakumar suggested the use of imprinted polymers. accordingly, a one-stage mini-emulsion polymerization, as illustrated in figure 17 , could be used to entrap viruses instead of inhibiting their activity [150] . notably, this method is virus-specific, depending on the imprinted polymer matching a specific virus. tong and sankarakumar suggested the use of imprinted polymers. accordingly, a one-stage mini-emulsion polymerization, as illustrated in figure 17 , could be used to entrap viruses instead of inhibiting their activity [150] . notably, this method is virus-specific, depending on the imprinted polymer matching a specific virus. polymers 2020, 12, x for peer review 22 of 32 while most antiviral effects reported in literature stem from materials composition, some report on temperature-dependent antiviral effects. wout et al. studied short-time pasteurization effects on human milk. the process of heating the milk to 72 °c for 16 s is well effective against bovine viral diarrhea, hepatitis a and c viruses, hiv-1 and hiv-2, porcine parvovirus, and pseudorabies viruses [151] . while heating above 44°c may cause damage to most viruses, at lower temperatures, as was shown by rott and scholtissek [152] and by goede et al. [153] , the viral activity might increase when temperatures are increased. similar conclusions were reported by molla et al. while studying the effect of temperature on poliovirus formation and rna synthesis [154] . yamaya et al., however, showed that, above 37 °c, the influenza viruses' replication decrease [155] , while reed showed that for panonychus-citri's nonoccluded virus, the biocidal temperature (above which the virus population is damaged) is 46 °c [156] . chan, et al. showed that for sars-cov, the biocidal temperature is <38 °c [157] and harrison showed that rothamsted tobacco necrosis virus biocidal temperature is even lower than influenza's [158] . therefore, it seems that biocidal temperature differs from virus to virus and that dna viruses tend to be more stable than rna viruses [159] . along with the direct effect of temperatures on some viruses' populations, some researchers have studied the temperature-dependent antiviral effects of ions. for example, brendel et al. showed that the effect of zn 2 ⁺ ions is temperature-dependent increased with increasing temperatures [23] . sánchez et al. studied the antiviral effects of ag nanoparticles on norovirus [160] . they tested this effect at two different temperatures, namely 25 and 37 °c, and found an increased antiviral effect at higher temperatures. kaufman et al., at their investigation about the thermodynamics of the antiviral effect of ions on the interaction between ns3 protein and single-stranded rna, also suggested that increasing the temperature may increase the antiviral effect [161] . bisaillon et al. studied the kinetics of metal ions binding to hepatitis c's rna polymerase and suggested the reaction's kinetics is temperaturedependent; increasing the temperatures caused accelerated binding [20] . surfactin is a surfactant used for antiviral and antimicrobial applications. pauli et al. showed that its antiviral effect on the non-enveloped hsv-1, hsv-2, suid herpes virus type 1 (shv-1), vsv, siv, feline calicivirus (fcv), and murine encephalomyocarditis virus (emcv) increased with increasing temperatures [162] . for shv-1, the efficiency increased linearly with increasing temperature up to 30 °c, above which the rate of inhibition was too fast to measure. hogle et al. showed that the efficiency of drugs that bind to poliovirus also increases with rising temperatures [163] . while most antiviral effects reported in literature stem from materials composition, some report on temperature-dependent antiviral effects. wout et al. studied short-time pasteurization effects on human milk. the process of heating the milk to 72 • c for 16 s is well effective against bovine viral diarrhea, hepatitis a and c viruses, hiv-1 and hiv-2, porcine parvovirus, and pseudorabies viruses [151] . while heating above 44 • c may cause damage to most viruses, at lower temperatures, as was shown by rott and scholtissek [152] and by goede et al. [153] , the viral activity might increase when temperatures are increased. similar conclusions were reported by molla et al. while studying the effect of temperature on poliovirus formation and rna synthesis [154] . yamaya et al., however, showed that, above 37 • c, the influenza viruses' replication decrease [155] , while reed showed that for panonychus-citri's nonoccluded virus, the biocidal temperature (above which the virus population is damaged) is 46 • c [156] . chan, et al. showed that for sars-cov, the biocidal temperature is <38 • c [157] and harrison showed that rothamsted tobacco necrosis virus biocidal temperature is even lower than influenza's [158] . therefore, it seems that biocidal temperature differs from virus to virus and that dna viruses tend to be more stable than rna viruses [159] . along with the direct effect of temperatures on some viruses' populations, some researchers have studied the temperature-dependent antiviral effects of ions. for example, brendel et al. showed that the effect of zn 2+ ions is temperature-dependent increased with increasing temperatures [23] . sánchez et al. studied the antiviral effects of ag nanoparticles on norovirus [160] . they tested this effect at two different temperatures, namely 25 and 37 • c, and found an increased antiviral effect at higher temperatures. kaufman et al., at their investigation about the thermodynamics of the antiviral effect of ions on the interaction between ns3 protein and single-stranded rna, also suggested that increasing the temperature may increase the antiviral effect [161] . bisaillon et al. studied the kinetics of metal ions binding to hepatitis c's rna polymerase and suggested the reaction's kinetics is temperature-dependent; increasing the temperatures caused accelerated binding [20] . surfactin is a surfactant used for antiviral and antimicrobial applications. pauli et al. showed that its antiviral effect on the non-enveloped hsv-1, hsv-2, suid herpes virus type 1 (shv-1), vsv, siv, feline calicivirus (fcv), and murine encephalomyocarditis virus (emcv) increased with increasing temperatures [162] . for shv-1, the efficiency increased linearly with increasing temperature up to 30 • c, above which the rate of inhibition was too fast to measure. hogle et al. showed that the efficiency of drugs that bind to poliovirus also increases with rising temperatures [163] . tannins are natural polyphenols, that have been studied for their antimicrobial and antiviral properties. mileva et al. showed that, much like other antiviral materials, tannins' antiviral effects are temperature-dependent, and are more efficient at 37 • c than at room temperature [164] . the antiviral battle has been the focus of numerous studies over the years. while originally focusing on small molecules based on antiviral drugs, in recent years, researchers started concentrating on hybrid and composite polymers as a promising approach to the global viral problem. the antiviral campaign is waged in two main approaches. the first employing polymers as drug delivery systems based on biodegradable polymers conjugated with antiviral drugs. in this approach, polymers are used in a supporting role, increasing efficacy and half-life of delivered drugs. the second exploiting hybrid and composite polymers. in this approach, polymers are incorporated with metal and/or metal-ions particles like zinc, silver, copper, zirconium, magnesium, tungsten, and more or otherwise hybridized antiviral polymers based on electrically-charged moieties, are employed, whether anionic or anionic, such as carboxylate and/or organic acids/anhydride, or cationic or cationic such as ammonium, phosphonium, and amines. some other approaches studied include sulphate containing polymers, phenol/hydroxyl-containing polymers and organometal polymers such as organotin-based polymers. moreover, some studies have suggested the polymerization of commercial antiviral drugs to achieve a more efficient treatment. lastly, it was shown that some antiviral effects are temperature dependent. most of the reported investigations pointed out that increased temperatures enhance the antiviral impact of ions and the hybrid polymers. thus, a combination of anti-viral polymers along with the ability to increase temperature may be an additional tool to increase antiviral properties. the authors declare no conflict of interest. expression of animal virus genomes polypeptide components of virions, top component and cores of reovirus type 3 structure and classification of viruses the species problem in virology classification and identification of aquatic animal viruses the molecular mechanisms of copper and silver ion disinfection of bacteria and viruses early interactions of hepatitis a virus with cultured cells: viral elution and the effect of ph and calcium ions effect of ionic concentration on the infectivity of a virus of the citrus red mite, panonychus citri the significance of oxidation in chemical inactivation of poliovirus on the cytotoxicity of vitamin c and metal ions: a site-specific fenton mechanism roles of copper and superoxide anion radicals in the radiation-induced inactivation of t7 bacteriophage reversal of the silver inhibition of microorganisms by agar application of silver nanoparticles in viral inhibition: a new hope for antivirals pharmacology and toxicology of heavy metals: silver theory meets experiment: metal ion effects in hcv genomic rna kissing complex formation evidence for a non-catalytic ion-binding site in multiple rna-dependent rna polymerases effect of metal ion binding on the structural stability of the hepatitis c virus rna polymerase multivalent ion effects on electrostatic stability of virus-like nano-shells highly efficient antiviral and antibacterial activities of solid-state cuprous compounds the mechanism of the antiherpetic activity of zinc sulphate the role of zinc in antiviral immunity zn2+ inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture characterization of soluble hepatitis c virus rna-dependent rna polymerase expressed in escherichia coli zinc is a negative regulator of hepatitis c virus rna replication the antiviral role of zinc and metallothioneins in hepatitis c infection effect of treatment with zinc gluconate or zinc acetate on experimental and natural colds antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections virus inactivation by copper or iron ions alone and in the presence of peroxide cobalt complexes as antiviral and antibacterial agents broad spectrum anti-rna virus activities of titanium and vanadium substituted polyoxotungstates in vitro antimicrobial effects of virus block, which contains multiple polyoxometalate compounds, and hygienic effects of virus block-supplemented moist hand towels antibacterial/antiviral property and mechanism of dual-functional quaternized pyridinium-type copolymer. polymers (basel) studies of inhibitory effect of ammonium ions in several virus-tissue culture systems a common mode of antiviral action for ammonium ions and various amines antiviral effects of ferric ammonium citrate different virucidal activities of hyperbranched quaternary ammonium coatings on poliovirus and influenza virus enhanced antioxidant and antifungal activity of chitosan derivatives bearing 6-o-imidazole-based quaternary ammonium salts novel quaternary phosphonium-type cationic polyacrylamide and elucidation of dual-functional antibacterial/antiviral activity synthetic biologically active polymers chitosan/xanthan gum based hydrogels as potential carrier for an antiviral drug: fabrication, characterization, and safety evaluation quaternary ammonium-based biomedical materials: state-of-the-art, toxicological aspects and antimicrobial resistance from multifunctionalized poly(ethylene imine)s toward antimicrobial coatings inactivation of foot-and-mouth disease virus by commercially available disinfectants and cleaners synergistic antiviral effect of xanthates and ionic detergents mechanism of the antiviral activity of new aurintricarboxylic acid analogues nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis b and hepatitis d infection ion transport blockers inhibit human rhinovirus 2 release antiviral effects of ascorbic and dehydroascorbic acids in vitro application of nanotechnology in textile engineering: an overview camphor-based symmetric diimines as inhibitors of influenza virus reproduction discovery of a new class of antiviral compounds: camphor imine derivatives broad range of inhibiting action of novel camphor-based compound with anti-hemagglutinin activity against influenza viruses in vitro and in vivo polymeric films as a promising carrier for bioadhesive drug delivery: development, characterization and optimization. saudi pharm a novel polymeric chlorhexidine delivery device for the treatment of periodontal disease release of antimicrobial and antiviral drugs from methacrylate copolymer system: effect of copolymer molecular weight and drug loading on drug release elevated ambient temperature differentially affects virus resistance in two tobacco species efficient antiviral co-delivery using polymersomes by controlling the surface density of cell-targeting groups for influenza a virus treatment polymer-attached zanamivir inhibits synergistically both early and late stages of influenza virus infection towards an improved anti-hiv activity of nrti via metal-organic frameworks nanoparticles biomedical applications of metal organic frameworks versatile iron-catechol-based nanoscale coordination polymers with antiretroviral ligand functionalization and their use as efficient carriers in hiv/aids therapy macromolecular (pro)drugs in antiviral research macromolecular prodrugs of ribavirin combat side effects and toxicity with no loss of activity of the drug antiviral polymeric drugs and surface coatings conjugating drug candidates to polymeric chains does not necessarily enhance anti-influenza activity polymer-bound 6 sialyl-n-acetyllactosamine protects mice infected by influenza virus antiviral and anticancer activity of cisplatin derivatives of tilorone cisplatin derivatives as antiviral agents antibacterial activity of polyaniline coated silver nanoparticles synthesized from piper betle leaves extract anti-viral fiber, process for producing the fiber, and textile product comprising the fiber inactivation of high and low pathogenic avian influenza virus h5 subtypes by copper ions incorporated in zeolite-textile materials antiviral activity of silver nanoparticles immobilized onto textile fabrics synthesized by radiochemical process interaction of silver nanoparticles with hiv-1 pvp-coated silver nanoparticles block the transmission of cell-free and cell-associated hiv-1 in human cervical culture mode of antiviral action of silver nanoparticles against hiv-1 silver nanoparticles inhibit replication of respiratory syncytial virus a preliminary assessment of silver nanoparticle inhibition of monkeypox virus plaque formation interaction of silver nanoparticles with tacaribe virus antiviral activity of silver nanoparticle/chitosan composites against h1n1 influenza a virus virucidal effect against coronavirus sars-cov-2 of a silver nanocluster/silica composite sputtered coating a modified zinc acetate gel, a potential nonantiretroviral microbicide, is safe and effective against simian-human immunodeficiency virus and herpes simplex virus 2 infection in vivo nanoparticles and surfaces presenting antifungal, antibacterial and antiviral properties coating composition, resin composition and antiviral product development of tri-laminate antiviral surgical gown for liquid barrier protection polymeric membranes incorporated with metal/metal oxide nanoparticles: a comprehensive review developing novel antimicrobial and antiviral textile products polymeric anhydride of magnesium and proteic ammonium phospholinoleate with antiviral, antineoplastic and immunostimulant properties organotin polymers as antiviral agents including inhibition of zika and vaccinia viruses antiviral activity of metal-containing polymers-organotin and cisplatin-like polymers metal-containing and related polymers for biomedical applications virus reduction through microfiltration membranes modified with a cationic polymer for drinking water applications high selectivity, and resistance mitigation antiviral properties of polymeric aziridine-and biguanide-modified core-shell magnetic nanoparticles decreasing herpes simplex viral infectivity in solution by surface-immobilized and suspended n,n-dodecyl,methyl-polyethylenimine biocidal packaging for pharmaceuticals, foods, and other perishables hydrophobic polycationic coatings disinfect poliovirus and rotavirus solutions agricultural uses of chitin polymers antioxidant, antifungal and antiviral activities of chitosan from the larvae of housefly, musca domestica l. food chem novel combined biological antiviral agents cytosinpeptidemycin and chitosan oligosaccharide induced host resistance and changed movement protein subcellular localization of tobacco mosaic virus agmatine-containing poly(amidoamine)s as a novel class of antiviral macromolecules: structural properties and in vitro evaluation of infectivity inhibition tailor-made antimicrobial/antiviral star polymer via atrp of cyclodextrin and guanidine-based macromonomer uptake and fate of water-soluble, nondegradable polymers with antiviral activity in cells and animals polymeric coatings that inactivate both influenza virus and pathogenic bacteria cationic phenylene ethynylene polymers and oligomers exhibit efficient antiviral activity antiviral and antibacterial polyurethanes of various modalities anti-herpesviral effects of a novel broad range anti-microbial quaternary ammonium silane, k21 antimicrobial and antiviral polymeric materials inhibitory effect of cinnamaldehyde, derived from cinnamomi cortex, on the growth of influenza a/pr/8 virus in vitro and in vivo polymers and biopolymers with antiviral activity: potential applications for improving food safety interferon-stimulating and in vivo antiviral effects of various synthetic anionic polymers induced interference by synthetic polyanions with the infection of tobacco mosaic virus ethylene maleic anhydride copolymers as viral inhibitors a styrene-alt -maleic acid copolymer is an effective inhibitor of r5 and x4 human immunodeficiency virus type 1 infection biomedical applications of maleic anhydride copolymers synthesis of anionic carbosilane dendrimers via "click chemistry" and their antiviral properties against hiv antiviral polymers comprising acid functional groups and hydrophobic groups anti-herpes simplex virus type 1 activity of humic acid-like polymers and their o-diphenolic starting compounds antiviral activity of alginate against infection by tobacco mosaic virus alginate: properties and biomedical applications sulphated polymers are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, respiratory syncytial virus, and toga-, arena-, and retroviruses effects of structural modification of calcium spirulan, a sulfated polysaccharide from spirulina platensis, on antiviral activity antiviral sulfonated naphthalene formaldehyde condensation polymers prophylactic antiviral activity of sulfated glycomimetic oligomers and polymers inherently self-sterilizing charged multiblock polymers that kill drug-resistant microbes in minutes synthesis, structure and antiviral activity of sulfates of curdlan and its branched derivatives anti-hiv-1 activity of anionic polymers: a comparative study of candidate microbicides naphthalene sulfonate polymers with cd4-blocking and anti-human immunodeficiency virus type 1 activities structural characterization and antiviral activity of two fucoidans from the brown algae sargassum henslowianum antiviral activity of polysaccharides against infection of tobacco mosaic virus antiviral activity of chondroitin sulfate against infection by tobacco mosaic virus elucidation of anti-hiv mechanism of sulfated cellobiose -polylysine dendrimers sulfonic acid polymers as a new class of human immunodeficiency virus inhibitors antiviral activity of an interferon-inducing synthetic polymer potent inhibition of human immunodeficiency virus by mdl 101028, a novel sulphonic acid polymer inhibition of herpes simplex virus infection by negatively charged and neutral carbohydrate polymers tempesta proanthocyanidin polymers having antiviral activity and methods of obtaining same the antiviral activity of sp-303, a natural polyphenolic polymer, against respiratory syncytial and parainfluenza type 3 viruses in cotton rats antiviral effects of polyphenols: development of bio-based cleaning wipes and filters synthesis and antibacterial and antiviral properties of silver nanocomposites based on water-soluble 2-dialkylaminoethyl methacrylate copolymers daylight-driven rechargeable antibacterial and antiviral nanofibrous membranes for bioprotective applications synthesis, and anti-rna virus activity of 6 -fluorinated-aristeromycin analogues preventing viral infections with polymeric virus catchers: a novel nanotechnological approach to anti-viral therapy antimicrobial and antiviral effect of high-temperature short-time (htst) pasteurization apllied to human milk effect of temperature on the multiplication of an influenza virus effect of temperature on survival and growth of infectious pancreatic necrosis virus effects of temperature and lipophilic agents on poliovirus formation and rna synthesis in a cell-free system effects of high temperature on pandemic and seasonal human influenza viral replication and infection-induced damage in primary human tracheal epithelial cell cultures effects of temperature on virus-host relationships and on activity of the noninclusion virus of citrus red mites, panonychus citri the effects of temperature and relative humidity on the viability of the sars coronavirus studies on the effect of temperature on virus multiplication in inoculated leaves literature review of the effect of salinity on the larger marine algae antiviral properties of silver nanoparticles against norovirus surrogates and their efficacy in coated polyhydroxyalkanoates systems thermodynamic study of the effect of ions on the interaction between dengue virus ns3 helicase and single stranded mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from bacillus subtilis stabilization of poliovirus by capsid-binding antiviral drugs is due to entropic effects abstract tannins as antiviral agents this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord-305195-e41yfo89 authors: rainwater-lovett, kaitlin; rodriguez-barraquer, isabel; moss, william j. title: viral epidemiology: tracking viruses with smartphones and social media date: 2016-02-12 journal: viral pathogenesis doi: 10.1016/b978-0-12-800964-2.00018-5 sha: doc_id: 305195 cord_uid: e41yfo89 the science of epidemiology has been developed over the last 200 years, using traditional methods to describe the distribution of diseases by person, place, and time. however, in the last several decades, a new set of technologies has become available, based on the methods of computer sciences, systems biology, and the extraordinary powers of the internet. technological and analytical advances can enhance traditional epidemiological methods to study the emergence, epidemiology, and transmission dynamics of viruses and associated diseases. social media are increasingly used to detect the emergence and geographic spread of viral disease outbreaks. large-scale population movement can be estimated using satellite imagery and mobile phone use, and fine-scale population movement can be tracked using global positioning system loggers, allowing estimation of transmission pathways and contact patterns at different spatial scales. advances in genomic sequencing and bioinformatics permit more accurate determination of viral evolution and the construction of transmission networks, also at different spatial and temporal scales. phylodynamics links evolutionary and epidemiological processes to better understand viral transmission patterns. more complex and realistic mathematical models of virus transmission within human and animal populations, including detailed agent-based models, are increasingly used to predict transmission patterns and the impact of control interventions such as vaccination and quarantine. in this chapter, we will briefly review traditional epidemiological methods and then describe the new technologies with some examples of their application. the science of epidemiology has been developed over the last 200 years, using traditional methods to describe the distribution of diseases by person, place, and time. however, in the last several decades, a new set of technologies has become available, based on the methods of computer sciences, systems biology, and the extraordinary powers of the internet. technological and analytical advances can enhance traditional epidemiological methods to study the emergence, epidemiology, and transmission dynamics of viruses and associated diseases. social media are increasingly used to detect the emergence and geographic spread of viral disease outbreaks. large-scale population movement can be estimated using satellite imagery and mobile phone use, and fine-scale population movement can be tracked using global positioning system (gps) loggers, allowing estimation of transmission pathways and contact patterns at different spatial scales. advances in genomic sequencing and bioinformatics permit more accurate determination of viral evolution and the construction of transmission networks, also at different spatial and temporal scales. phylodynamics links evolutionary and epidemiological processes to better understand viral transmission patterns. more complex and realistic mathematical models of virus transmission within human and animal populations, including detailed agent-based models, are increasingly used to predict transmission patterns and the impact of control interventions such as vaccination and quarantine. in this chapter, we will briefly review traditional epidemiological methods and then describe the new technologies with some examples of their application. insight into the epidemiology of viral infections long preceded the recognition and characterization of viruses as communicable agents of disease in humans and animals, extending at least as far back as the treatise of abu becr (rhazes) on measles and smallpox in the tenth century. successful efforts to alter the epidemiology of viral infections can be traced to the practice of variolation, the deliberate inoculation of infectious material from persons with smallpox (see chapter on the history of viral pathogenesis). documented use of variolation dates to the fifteenth century in china. edward jenner greatly improved the practice of variolation in 1796 using the less-virulent cowpox virus, establishing the field of vaccinology. an early example of rigorous epidemiological study prior to the discovery of viruses was the work of the danish physician peter panum who investigated an outbreak of measles on the faroe islands in 1846. through careful documentation of clinical cases and contact histories, panum provided evidence of the contagious nature of measles, accurate measurement of the incubation period, and demonstration of the long-term protective immunity conferred by measles. the discovery of viruses as "filterable agents" in the late-nineteenth and early twentieth centuries greatly enhanced the study of viral epidemiology, allowing the characterization of infected individuals, risk factors for infection and disease, and transmission pathways. traditional epidemiological methods measure the distribution of viral infections, diseases, and associated risk factors in populations in terms of person, place, and time using standard measures of disease frequency, study designs, and approaches to causal inference. populations are often defined in terms of target and study populations, and individuals within study populations in terms of exposure and outcome status. the purpose of much traditional epidemiological research is to quantify the strength of association between exposures and outcomes by comparing characteristics of groups of individuals. exposures or risk factors include demographic, social, genetic, and environmental factors, and outcomes include infection or disease. in viral epidemiology, infection status is determined using diagnostic methods to detect viral proteins or nucleic acids, and serologic assays to measure immunologic markers of exposure to viral antigens. infection status can be defined as acute, chronic, or latent. standard measures of disease frequency include incidence, the number of new cases per period of observation (e.g., 1000 person-years), and prevalence, the number of all cases in a defined population and time period. prevalence is a function of both incidence and duration of infection and can increase despite declining incidence, as observed with the introduction of antiretroviral therapy for human immunodeficiency virus (hiv) infection in the united states. although the number of new cases of hiv infection declined, the prevalence of hiv infection increased as treated individuals survived longer. commonly used study designs include, l cross-sectional studies in which individuals are sampled or surveyed for exposure and disease status within a narrow time frame, l cohort studies in which exposed and unexposed individuals are observed over time for the onset of specified outcomes, l case-control studies in which those with and without the outcome (infection or disease) are compared on exposure status, and l clinical trials in which individuals are randomized to an exposure such as a vaccine or drug and observed for the onset of specified outcomes appropriate study design, rigorous adherence to study protocols, and statistical methods are used to address threats to causal inference (i.e., whether observed associations between exposure and outcome are causal), such as bias and confounding. much can be learned about the epidemiology of viral infections using such traditional methods and many examples could be cited to establish the importance of these approaches, including demonstration of the mode of transmission of viruses by mosquitoes (e.g., yellow fever and west nile viruses), the causal relationship between maternal viral infection and fetal abnormalities (e.g., rubella virus and cytomegalovirus), and the role of viruses in the etiology of cancer (e.g., epstein-barr and human papilloma viruses). the epidemiology of communicable infectious diseases is distinguishable from the epidemiology of noncommunicable diseases in that the former must account for "dependent happenings." this term was introduced by ronald ross to capture the fact that infectious agents are transmitted between individuals or from a common source. traditional epidemiological and statistical methods often assume disease events in a population are independent of one another. in infectious disease epidemiology, individuals are defined in terms of susceptible, exposed, infectious, and recovered or immune. key characteristics of viral infections that determine the frequency and timing of transmission, and thus the epidemiology, include the mode of transmission (e.g., respiratory, gastrointestinal, sexual, bloodborne, and vector-borne), whether infection is transient or persistent, and whether immunity is short or long lasting. temporal changes in the transmission dynamics of viral infection can be displayed with epidemic curves, by plotting the number or incidence of new infections over time to demonstrate outbreaks, seasonality, and the response to interventions. key metrics in infectious disease epidemiology that capture the dependent nature of communicable diseases include: (1) the latent period, the average time from infection to the onset of infectiousness; (2) the infectious period, the average duration of infectiousness; (3) the generation time, the average period between infection in one individual and transmission to another; and (4) the basic reproductive number (r 0 ), the average number of new infections initiated by a single infectious individual in a completely susceptible population over the course of that individual's infectious period. if r 0 is larger than one, the number of infected individuals and hence the size of the outbreak will increase. if r 0 is smaller than one, each infectious individual infects on average less than one other individual and the number of infected individuals will decrease and the outbreak ceases. the reproductive number (r) is a function not only of characteristics of the viral pathogen (e.g., mode of transmission), but also the social contact network within which it is transmitted and changes over time in response to a decreasing number of susceptible individuals and control interventions. an important concept related to the interdependence of transmission events is herd immunity, the protection of susceptible individuals against infection in populations with a high proportion of immune individuals because of the low probability of an infectious individual coming in contact with a susceptible individual. the concepts and methods of infectious disease epidemiology provide the tools to understand changes in temporal and spatial patterns of viral infections and the impact of interventions. traditional epidemiological methods provide powerful analytical approaches to measure associations between exposures (risk factors) and outcomes (infection or disease). recent technological advances enhance these methods and permit novel approaches to investigate the emergence, epidemiology, and transmission dynamics of viruses and associated diseases. expanded access to the internet and social media has revolutionized outbreak detection and viral disease surveillance by providing novel sources of data in real time (chunara, 2012) . traditional epidemiologic surveillance systems rely on standardized case definitions, with individual cases typically classified as suspected, probable, or confirmed based on the level of evidence. confirmed cases require laboratory evidence of viral infection. surveillance systems are either active or passive. active surveillance involves the purposeful search for cases within populations whereas passive surveillance relies on routine reporting of cases, typically by health care workers, health care facilities, and laboratories. data acquired through active surveillance are often of higher quality because of better adherence to standardized case definitions and completeness of case ascertainment but are more expensive and resource intensive. however, both active and passive surveillance are prone to delays in data reporting. the major advantage of using the internet and social media to monitor disease activity is that the signal can be detected without the lag associated with traditional surveillance systems. influenza is the most common viral infection for which the internet and social media have been used for disease surveillance because of its high incidence, wide geographic distribution, discrete seasonality, short symptomatic period, and relatively specific set of signs and symptoms. however, the internet and social media have several limitations compared to traditional active and passive surveillance systems and complement rather than replace these methods. these limitations include lack of specificity in the "diagnosis," and waxing and waning interest and attention in social media independent of disease frequency. in 2008, the internet company google developed a webbased tool called google flu trends, for early detection of influenza outbreaks. google flu trends is based on the fact that millions of people use the google search engine each day to obtain health-related information (ginsberg, 2009 ). logs of user key words for pathogens, diseases, symptoms, and treatments, as well as information on user location contained in computer internet protocol (ip) addresses, allow temporal and spatial analyses of trends in search terms ( figure 1 ). early results suggested that google flu trends detected regional outbreaks of influenza 7-10 days before conventional surveillance by the centers for disease control and prevention (carneiro, 2009 ). however, accurate prediction was not as reliable as initially thought, and google estimates did not closely match measured activity during the 2012-2013 influenza season. google now reevaluates estimates using data from traditional surveillance systems (specifically those of the centers for disease control and prevention) to refine model and parameter estimates. these refinements more accurately capture the start of the influenza season, the time of peak influenza virus transmission, and the severity of the influenza season. a similar approach, called google dengue trends, is used to track dengue virus infections by aggregating historical logs of anonymous online google search queries associated with dengue, using the methods developed for google flu trends. early observations suggest google queries are correlated with national-level dengue surveillance data, and this novel data source may have the potential to provide information faster than traditional surveillance systems ( figure 2 ). other internet sources are being explored to enhance viral surveillance. wikipedia is a free, online encyclopedia written collaboratively by users and is one of the most commonly used internet resources since it was started in 2001. as with google searches, the use of disease-specific queries to wikipedia are expected to correlate with disease activity. the number of times specific influenza-related wikipedia sites were accessed provided accurate estimates of influenza-like illnesses in the united states 2 weeks earlier than standard surveillance systems and performed better than google flu trends (mciver, 2014) . similarly, social media data are being evaluated for surveillance purposes. twitter is a free social networking service that enables users to exchange text-based messages of up to 140 characters known as tweets. as with google flu trends, the number of tweets related to influenza activity is correlated with the number of symptomatic individuals. several published studies reported correlations between twitter activity and reported influenza-like illnesses (chew, 2010; signorini, 2011 ; figure 3 ). limitations to using social media, such as twitter, to monitor disease activity are illustrated by the ebola virus outbreak in west africa in early 2014. despite the fact that ebola had not yet occurred in the united states, posts to twitter on ebola rose dramatically, likely in response to intense media coverage and fear. clearly, such tweets could not be interpreted to indicate ebola disease activity in the united states. studies reporting misleading associations, or the lack of correlation between social media and disease activity, are rarely published, providing a cautionary note. while initial efforts using data from the internet for viral disease surveillance offer promising results, concerns have been raised regarding the utility and robustness of these approaches (lazer, 2014) . integration into existing surveillance frameworks will be necessary to maximize the utility of these data streams. the internet allows rapid processing and communication of health-related information, including the aggregation and display of surveillance data for viral infections. traditional surveillance networks can be linked through the internet to allow rapid integration and dissemination of information. information on viral disease outbreaks available through internet postings of health care agencies such as the world health organization (who) and centers for disease control and prevention (cdc), as well as press reports and blogs, can provide data that are more current than traditional surveillance systems. information from these online sources can be made available to a large, global audience. several of the most commonly used surveillance sites report animal as well as human diseases (see sidebar 1 and figure 4 ). mapping spatial patterns of disease and relationships with environmental variables preceded the development of modern epidemiology. the classic example is john snow's hand-drawn map of london cholera cases of 1854. however, routine mapping of health data only became commonplace in the 1990s after desktop geographic information systems became widely available. combined with satellite imagery and remotely sensed environmental and ecological data, spatial mapping of viral infections is a powerful tool for surveillance and epidemiological research. spatial epidemiology is typically used to identify and monitor areas of differential risk. an early example was a large outbreak of st. louis encephalitis virus infection in houston, texas in 1964. spatial analysis showed that the outbreak was concentrated in the city center, with lower incidence at the outskirts. further investigation revealed that the city center was associated with the lowest economic strata, unscreened windows, lack of air-conditioning and pools of standing water, factors facilitating virus transmission. investigation into the spatiotemporal dynamics of viral diseases at smaller spatial scales has become promed, the program for monitoring emerging diseases, is an internet-based reporting system established in 1994 that compiles information on outbreaks of infectious diseases affecting humans, animals, and food plants. promed relies on official announcements, media reports, and local observers, including the network of subscribers. a team of experts screen, review, and investigate reports before posting and often provide commentary. reports are distributed by email to direct subscribers and posted on the promed-mail web site. promed-mail currently reaches over 60,000 subscribers in at least 185 countries. started in 2006 by epidemiologists and software developers at boston children's hospital, healthmap monitors disease outbreaks and provides real-time surveillance of emerging public health threats, including viral infections (figure 4) . healthmap organizes and displays data on disease outbreaks and surveillance using an automated process. data sources include online news aggregators, eyewitness reports, expertcurated discussions and validated official reports. google flu trends http://www.google.org/flutrends google dengue trends http://www.google.org/denguetrends healthmap http://healthmap.org promed http://www.promedmail.org possible with increasing availability of global positioning systems devices and geocoding algorithms. such studies have revealed spatial heterogeneity in the local transmission of some directly (e.g., hiv and influenza) and indirectly (e.g., dengue and chikungunya) transmitted viruses. for example, clustering analyses of the residential locations of people with dengue in bangkok over a 5-year period showed evidence of localized transmission at distances less than 1 km (salje, 2012; figure 5 ). analyses of data from a large population-based cohort of hiv-infected persons in rakai district, uganda revealed strong within-household clustering of prevalent and incident hiv cases as well clustering of prevalent cases up to 500 m (grabowski, 2014) . beyond descriptive applications, mapping spatiotemporal patterns of viral infections can provide fundamental insights into transmission dynamics at different spatial scales. traveling waves from large cities to small towns were shown to drive the spatiotemporal dynamics of measles in england and wales (xia, 2004) . the incidence of dengue hemorrhagic fever across thailand manifested as a traveling wave emanating from bangkok and moving radially at a speed of 148 km/month (cummings, 2004) . insight into the spatial epidemiology of viral infections and associations with environmental risk factors can be greatly enhanced when information on the spatial location of cases is combined with remotely sensed environmental data (rodgers, 2003) . the spatial coordinates of cases can be overlaid on satellite imagery to demonstrate relationships with environmental features-such as bodies of water-and formally analyzed using spatial statistical techniques. satellite sensors that detect reflected visible or infrared radiation provide additional information on temperature, rainfall, humidity, and vegetation among other variables, which are particularly important for the transmission dynamics of vector-borne viral infections. satellite data for epidemiologic analyses are provided by a number of sources such as: (1) earth-observing satellites with high spatial resolution (1-4 m) but low repeat frequencies such as ikonos and landsat satellites; (2) oceanographic and atmospheric satellites such as modis and aster with lower spatial resolution (0.25-1 km) that provide images of the earth surface twice a day; and (3) geostationary weather satellites such as geos with large spatial resolution (1-8 km). the statistical relationships between cases and environmental risk factors can be used to construct risk maps. risk maps display the similarity of environmental features in unsampled locations to environmental features in locations where the disease is measured to be present or absent. spatial analysis of the initial cases of west nile virus infection in new york city in 1999 identified a significant spatial cluster (brownstein, 2002) . using models incorporating measures of vegetation cover from satellite imagery, the risk of west nile virus could be estimated throughout the city. a more recent risk map for west nile virus in suffolk county, new york, was generated with data on vector habitat, landscape, virus activity, and socioeconomic variables derived from publicly available data sets (rochlin, 2011; figure 6 ). population movement plays a crucial role in the spread of viral infections. in the past, quantifying the contribution of movement to viral transmission dynamics at different spatial scales was challenging, due to limited data. as an early example, the impact of restrictions of animal movement on transmission of foot-and-mouth disease in 2001 was estimated, using detailed contact-tracing data from farms in the united kingdom (shirley, 2005) . however, such detailed data are rarely available for patterns of human movement. studies have attempted to model the impact of long-range human movement on the spread of viral diseases using measures such as distance between cities, commuting rates, and data on air travel. this approach has been used to explain regional and interregional spread of influenza viruses. data on air traffic volume, distance between areas, and population sizes have been invoked to describe and predict local and regional spread of chikungunya virus in the americas (tatem, 2012) . new technologies have greatly enhanced the capacity to study the impact of human movement on transmission dynamics of infectious diseases. data from mobile phones and gps loggers can be used to characterize individual movement patterns and the time spent in different locations (figure 7) . individual movement patterns can be overlaid on risk maps to quantify movement to and from areas of high (sources) and low risk (sinks) as well as to estimate potential contact patterns. gps data loggers generated 2.3 million gps data points to track the fine-scale mobility patterns of 582 residents from two neighborhoods in iquitos, peru, to better understand the epidemiology of viral infections (vazquez-prokopec, 2013) . most movement occurred within 1 km of an individual's home. however, potential contacts between individuals were irregular and temporally unstructured, with fewer than half of the tracked participants having a regular, predictable routine. the investigators explored the potential impact of these temporally unstructured daily routines and contact patterns on the simulated spread of influenza virus. the projected outbreak size was 20% larger as a consequence of these unstructured contact patterns, in comparison to scenarios modeling temporally structured contacts. in addition to identifying individual and environmental characteristics associated with temporal and spatial patterns of viral infections, transmission networks are critical drivers of the dynamics of viral infections. analysis of transmission networks defines the host contact structure within which directly transmitted viral infections spread. network theory and analysis are complex subjects with a long history in mathematics and sociology, but have recently been adapted by infectious disease epidemiologists. the epidemiologic study of social networks is facilitated by unique study designs, including snowball sampling or respondent-driven sampling, in which study participants are asked to recruit additional participants among their social contacts. differing sexual contact patterns serve as an example of the importance of contact networks to the understanding of viral epidemiology. concurrent sexual partnerships amplify the spread of hiv compared with serial monogamy. this could partially explain the dramatic differences in the prevalence of hiv in different countries. social networks were shown to affect transmission of the 2009 h1n1 influenza virus, and were responsible for cyclical patterns of transmission between schools, communities, and households. technological advances in quantifying contact patterns, with wearable sensors and the use of viral genetic signatures, have greatly enhanced the ability to understand complex transmission networks. self-reported contact histories and contact tracing are the traditional epidemiological methods to define transmission networks. contact tracing has a long history in public health, particularly in the control of sexually transmitted diseases and tuberculosis, and is critical to the control of outbreaks of viral infections such as the middle east respiratory syndrome coronavirus (mers-cov) and ebola virus. to better understand the nature of human contact patterns, sensor nodes or motes have been used to characterize the frequency and duration of contacts between individuals in settings such as schools and health-care facilities. these technologies offer opportunities to validate and complement data collected using questionnaires and contact diaries. as an example, investigators used wireless sensor network technology to obtain data on social contacts within 3 m for 788 high school students in the united states, enabling construction of the social network within which a respiratory pathogen could be transmitted (salathe, 2010) . the data revealed a high-density network with typical small-world properties, in which a small number of steps link any two individuals. computer simulations of the spread of an influenza-like virus on the weighted contact graph were in good agreement with absentee data collected during the influenza season. analysis of targeted immunization strategies suggested that contact network data can be employed to design targeted vaccination strategies that are significantly more effective than random vaccination. advances in nucleic acid sequencing and bioinformatics have led to major advances in viral epidemiology. population (sanger) sequencing has been the standard method for dna sequencing but is increasingly replaced by deep sequencing in which variants within a viral swarm are distinguished. sequencing allows for the detection of single nucleotide polymorphisms (snps) and nucleotide insertions or deletions ("indels"), analysis of synonymous and nonsynonymous mutations, and phylogenetic analysis (see chapter on virus evolution). sequencing techniques can be applied to both viral and host genomes. snps may be associated with changes in viral pathogenesis, virulence, or drug resistance. molecular techniques applied to pathogens also have been fundamental to the study of the animal origins of many viral infections including hiv and mers. phylogeographic approaches were used to trace the origins of the hiv pandemic to spillover events in central africa (sharp, 2010) . more recently, sequence data were used to track the animal reservoirs of mers-cov associated with the 2014 outbreaks (haagmans, 2014) , and to compare the ebola virus strain circulating in the 2014 west africa outbreak to strains from prior outbreaks (gire, 2014) . epidemiologic studies that probe host genomes can be either candidate gene studies or genome-wide association studies. the goal of these studies is to link specific changes with an increased risk of infection or disease. as an example, a small subset of individuals who failed to acquire hiv infection despite exposure, prompted studies to determine how these individuals differed from those who acquired infection. a 32-base-pair deletion in the human ccr5 gene, now referred to as ccr5-delta 32, accounted for the resistance of these subjects. individuals who are ccr5-delta 32 homozygotes are protected against hiv infection by ccr5tropic hiv strains, while heterozygotes have decreased disease severity. infectious disease epidemiologists are increasingly linking evolutionary, immunologic, and epidemiological processes, a field referred to as phylodynamics voltz, 2013) . because of the high mutation rates of viral pathogens, particularly rna viruses, evolutionary and epidemiological processes take place on a similar timescale (see chapter on virus evolution). according to this framework, phylodynamic processes that determine the degree of viral diversity are a function of host immune selective pressures and epidemiological patterns of transmission ( figure 8 ). intrahost phylodynamic processes begin with molecular characteristics of the virus as well as the host's permissiveness and response to infection. for example, a single amino acid substitution in epstein-barr virus was shown to disrupt antigen presentation by specific human leukocyte antigen polymorphisms (liu, 2014) . this resulted in decreased t-cell receptor recognition and successful viral immune escape. the virus must also induce an "optimal" host immune response to maximize transmission to new hosts. if the virus induces a strong, proinflammatory immune response not balanced by the appropriate anti-inflammatory responses, the host may succumb to the overabundance of inflammation and cannot propagate viral transmission. alternatively, a virus that fails to stimulate an immune response may also replicate uninhibited, overwhelming, and killing the host prior to transmission. selective pressures maximize replication while sustaining transmission between hosts. interhost dynamics are affected by several factors including evolutionary pressures, timescales of infection, viral latent periods, and host population structures. typically, only a small number of virions are transmitted between hosts, creating a genetic bottleneck that limits viral diversity. a virus that mutates to cause highly pathogenic disease but is not transmitted cannot propagate its pathogenicity. cross-immunity between viral strains also precludes the replication of particular viral lineages. influenza vaccine strains require annual changes due to new circulating influenza strains that have escaped immune pressures through high mutation rates and gene re-assortment. the strong selection pressure of cross-immunity is reflected in the short branch lengths in a phylogenetic tree of influenza viruses isolated from infected individuals. thus, the selection of influenza strains for future vaccines is partly determined by cross-immunity to prior circulating strains, because influenza viral strains that circulated in the past may elicit immune protection against currently circulating strains. at the population level, phylodynamic methods have been used to estimate r 0 for hiv and hepatitis c virus, for which reporting and surveillance data are often incomplete (volz, 2013) . phylodynamic and phylogeographic models also have been useful in reconstructing the spatial spread of viruses to reveal hidden patterns of transmission. for example, epidemiological and molecular studies of influenza virus transmission were compared at different spatial scales to highlight the similarities and differences between these data sources (viboud, 2013) . the findings were broadly consistent with large-scale studies of interregional or inter-hemispheric spread in temperate regions with multiple viral introductions resulting in epidemics followed by interepidemic periods driven by seasonal bottlenecks. however, at smaller spatial scalessuch as a country or community-epidemiological studies revealed spatially structured diffusion patterns that were not identified in molecular studies. phylogenetic analyses of gag and env genes were used to assess the spatial dynamics of hiv transmission in rural rakai district, uganda, using data from a cohort of 14,594 individuals residing in 46 communities (grabowski, 2014) . of the 95 phylogenetic clusters identified, almost half comprised two individuals sharing a household. among phylodynamics links evolutionary, immunologic, and epidemiologic processes to explain viral diversity, as shown here for equine influenza virus. for viral evolution, these processes take place on a similar timescale. within host mutations (1) result from an interplay between optimization of viral shedding, immunologic selective pressures and host pathogenicity. transmission bottlenecks and host heterogeneity (2) further determine the population genetic structure of the virus, which in turn influences and is determined by the epidemic dynamics. larger scale spatial dynamics at local, regional, and global levels (3) the remaining clusters, almost three-quarters involved individuals living in different communities, suggesting transmission chains frequently extend beyond local communities in rural uganda. the timescale of infection is also important for viral diversity and transmission dynamics. some viruses are capable of initiating an acute infection that is cleared within days, while other viral infections are chronic and persist for a lifetime. the duration of infection impacts how quickly a virus must be transmitted and has implications for the infectious period and the potential to be transmitted to new hosts. viruses with long latent periods create interhost phylogenetic trees with longer branch lengths. the long duration between infection and transmission permits accumulation of viral changes through many rounds of viral replication before transmission to the next host. examples include hepatitis b virus, hepatitis c virus, and human immunodeficiency virus. availability of computational resources allows widespread use and development of classic approaches to the mathematical modeling of viral transmission dynamics, such as compartmental, metapopulation and network models, to address epidemiologic questions (see chapter on mathematical methods). these models have been used extensively in the study of viral dynamics and to explore the potential impact of control interventions. new sources of high-resolution spatial, temporal, and genetic data create opportunities for models that integrate these data with traditional epidemiological data. such analyses improve estimates of key transmission parameters and understanding of the mechanisms driving virus spread. agent-based models (also known as individual-based models) can now be run using desktop computers, and offer advantages over more traditional mathematical models. because each unit in a population is modeled explicitly in space and time and assigned specific attributes, agent-based models can reproduce the heterogeneity and complexity observed in the real world. more traditional compartmental, differential equation models often require simplifying assumptions that limit applicability. agent-based models have been used to study the spread of viruses in populations as well as the evolution of viruses within and across populations. while agent-based models are intuitive and easy to formulate, these models are often difficult to construct due to the large number of parameters necessary to describe the behavior and interaction between individual units. commercial frameworks that offer large computational power and intuitive user interphases have also become increasingly available. the global epidemic and mobility model (gleam) on the gleamviz platform (www.gleamviz.com), for example, contains extensive data on populations and human mobility, and allows stochastic simulation of the global spread of infectious diseases using user-defined transmission models. our understanding of the epidemiology of viral infections is being revolutionized by the integration of traditional epidemiological information with novel sources of data. l data streams from the internet are promising sources to enhance traditional surveillance but have yet to be fully validated. l molecular data on viral genomic sequences provide unprecedented opportunities to characterize viral transmission pathways. l phylodynamic and phylogeographic models have been used to estimate r 0 , and characterize the spatial spread of viruses. l network analysis reveals hidden patterns of transmission between population subgroups that are not easy to capture with traditional epidemiological methods. l novel analytical and computational resources are playing a key role in integrating information from multiple large data banks. these more comprehensive methods improve our ability to estimate the impact of infection control measures. the combination of traditional and evolving methodologies is closing the gap between epidemiological studies and viral pathogenesis. these developments have laid the foundation for exciting future research that will complement other approaches to the pathogenesis of viral diseases. with these evolving technologies in mind, it is timely to ask: is the world able to control viral diseases more effectively? it is a mixed score card. on the one hand, smallpox has been eradicated and we are on the verge of elimination of wild polioviruses. furthermore, deaths of children under the age of 5 years (which are mainly due to viral and other infectious diseases) have decreased by almost 50% in the last few decades. on the other hand, the aids pandemic continues to rage in low-income countries, with only a slight reduction in the annual incidence of new infections. the united states has not done any better in reducing hiv incidence which has been unchanged for at least 20 years. the 2014-15 ebola pandemic in west africa reflects the limited capacity for dealing with new and emerging viral diseases on a global basis. in conclusion, epidemiological science continues to advance with evolving new technologies, but their application to public health remains a future challenge and opportunity. new technologies for reporting real-time emergent infections google trends: a web-based tool for real-time surveillance of disease outbreaks unifying the epidemiological and evolutionary dynamics of pathogens studying the global distribution of infectious diseases using gis and rs spatial analysis of west nile virus: rapid risk assessment of an introduced vector-borne zoonosis pandemics in the age of twitter: content analysis of tweets during the 2009 h1n1 outbreak travelling waves in the occurrence of dengue haemorrhagic fever in thailand detecting influenza epidemics using search engine query data rakai health sciences program. the role of viral introductions in sustaining community-based hiv epidemics in rural uganda: evidence from spatial clustering, phylogenetics, and egocentric transmission models middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation the parable of google flu: traps in big data analysis wikipedia usage estimates prevalence of influenza-like illness in the united states in near real-time a molecular basis for the interplay between t cells, viral mutants, and human leukocyte antigen micropolymorphism assessing and maximizing the acceptability of global positioning system device use for studying the role of human movement in dengue virus transmission in iquitos predictive mapping of human risk for west nile virus (wnv) based on environmental and socioeconomic factors a high-resolution human contact network for infectious disease transmission revealing the microscale spatial signature of dengue transmission and immunity in an urban population the evolution of hiv-1 and the origin of where diseases and networks collide: lessons to be learnt from a study of the 2001 foot-and-mouth disease epidemic the use of twitter to track levels of disease activity and public concern in the u.s. during the influenza a h1n1 pandemic air travel and vectorborne disease movement usefulness of commercially available gps data-loggers for tracking human movement and exposure to dengue virus using gps technology to quantify human mobility, dynamic contacts and infectious disease dynamics in a resource-poor urban environment contrasting the epidemiological and evolutionary dynamics of influenza spatial transmission viral phylodynamics measles metapopulation dynamics: a gravity model for epidemiological coupling and dynamics key: cord-301349-m4nr3pqx authors: mirza, muhammad usman; saadabadi, atefeh; vanmeert, michiel; salo-ahen, outi m.h.; abdullah, iskandar; claes, sandra; de jonghe, steven; schols, dominique; ahmad, sarfraz; froeyen, matheus title: discovery of hiv entry inhibitors via a hybrid cxcr4 and ccr5 receptor pharmacophore‐based virtual screening approach date: 2020-09-02 journal: eur j pharm sci doi: 10.1016/j.ejps.2020.105537 sha: doc_id: 301349 cord_uid: m4nr3pqx chemokine receptors are key regulators of cell migration in terms of immunity and inflammation. among these, ccr5 and cxcr4 play pivotal roles in cancer metastasis and hiv-1 transmission and infection. they act as essential co-receptors for hiv and furnish a route to the cell entry. in particular, inhibition of either ccr5 or cxcr4 leads very often the virus to shift to a more virulent dual-tropic strain. therefore, dual receptor inhibition might improve the therapeutic strategies against hiv. in this study, we aimed to discover selective ccr5, cxcr4, and dual ccr5/cxcr4 antagonists using both receptorand ligand-based computational methods. we employed this approach to fully incorporate the interaction attributes of the binding pocket together with molecular dynamics (md) simulations and binding free energy calculations. the best hits were evaluated for their anti-hiv-1 activity against cxcr4and ccr5-specific nl4.3 and bal strains. moreover, the ca(2+) mobilization assay was used to evaluate their antagonistic activity. from the 27 tested compounds, three were identified as inhibitors: compounds 27 (ccr5), 6 (cxcr4) and 3 (dual) with ic(50) values ranging from 10.64 to 64.56 μm. the binding mode analysis suggests that the active compounds form a salt bridge with the glutamates and π-stacking interactions with the aromatic side chains binding site residues of the respective co-receptor. the presented hierarchical virtual screening approach provides essential aspects in identifying potential antagonists in terms of selectivity against a specific co-receptor. the compounds having multiple heterocyclic nitrogen atoms proved to be relatively more specific towards cxcr4 inhibition as compared to ccr5. the identified compounds serve as a starting point for further development of hiv entry inhibitors through synthesis and quantitative structure-activity relationship studies. the most effective treatment of patients infected with human immunodeficiency virus 1 (hiv-1) is currently highly active antiretroviral therapy (haart), which utilizes a combination of several drugs targeting viral proteins such as reverse transcriptase, integrase and protease [1, 2] . an alternative approach relies on the multi-targeted inhibition of viral cell entry [3] , which consists of a series of structural events arbitrated by viral and cellular membrane proteins. the interaction between the hiv-1 glycoprotein gp120 and the cellular receptor cd4 is an important initial step in viral entry. it induces a conformational change in gp120 and mediates further interaction with either one of the two co-receptors, chemokine receptor 5 (ccr5) and cxc-chemokine receptor 4 (cxcr4) during viral entry into the host cells [4, 5] . both chemokine receptors belong to the g-protein coupled receptor family and regulate hiv-1 cellular tropism. ccr5-tropic or r5 strains preferentially uses ccr5, whereas cxcr4-tropic or x4 strains preferentially use cxcr4. dual-tropic strains use both co-receptors for their entry in the cd4 + target cells [5] [6] [7] . the chemokine ligand 5 (ccl5) also known as rantes (regulated upon activation, normal t cell expressed, and secreted) is the natural ligand for ccr5 [8] , whereas the cxcchemokine ligand 12 (cxcl12), also known as sdf-1 (stromal cell-derived factor 1) is the natural ligand for cxcr4 [9] . inhibition of either ccr5 or cxcr4 leads hiv to shift to a more virulent dual-tropic strain. therefore dual inhibition of both co-receptors is of paramount importance to improve the therapeutic strategies against hiv infection [10, 11] . although numerous selective ccr5 and cxcr4 antagonists have been reported [12] [13] [14] [15] , only a few ccr5/cxcr4 dual antagonists have been identified so far [3] . ccr5 and cxcr4 share an overall similar architecture, and the overlapping regions of the binding pockets pave the way for the identification of dual co-receptor antagonists [16] . maraviroc received marketing approval by the food and drug administration (fda) as a very potent and selective ccr5 antagonist with a favourable hiv resistance profile. it inhibits rantes binding to a recombinant ccr5 expressed in the human embryonic kidney (hek-293) cell line with an ic 50 value of 5.2 nm [17] . plerixafor (or amd3100) is a selective cxcr4 antagonist that specifically inhibits sdf-1α binding to cxcr4 (ic 50 = 12.5 nm) [18] its close analogue amd3451 (a tetraazamacrocycle) was first reported as a dual cxcr4/ccr5 antagonist [11] . alongside, tetraazamacrocycle-based transition metal complexes were shown to act as dual cxcr4/ccr5 antagonist and were endowed with excellent in vitro activity against hiv [19] . other dual cxcr4/ccr5 antagonists include, peptide-based triazoles analogues [20] , diterpene derivatives (ec 50 0.02 and 0.09 µm against r5 and x4 hiv strains, respectively [21] ), and pyrazole derivatives (ic 50 3.8 and 0.8 µm against ccr5-and cxcr4-utilizing hiv-1 strains) [22] . an analogue of the antiparasitic drug suramin nf279 [23] , the natural penicillixanthone a [24] and a coumarin-based analogue gut-70 [25] are also known as dual ccr5/cxcr4 antagonists (figure 1 ). modern structure-based virtual screening approaches are indispensable in the discovery of novel hits targeting specific proteins and is nowadays a crucial part in the hit-to-leadoptimization phase of drug discovery [26] [27] [28] . this approach resulted in the discovery of various potent antiviral compounds against viruses such as ebola [29] , dengue [30] , zika [31, 32] , sars-coronavirus [33, 34] and influenza [35] . the aim of this study was to discover selective ccr5, cxcr4 and dual ccr5/cxcr4 antagonists based on both receptor-and ligand-based virtual screening methods together with molecular dynamics (md) simulations and binding free energy calculations. the most promising hits were evaluated for cxcr4 and ccr5 antagonism via a ca 2+ mobilization assay using u87.cd4.cxcr4 and u87.cd4.ccr5-transfected cell lines. in addition, their anti-hiv-1 activity was evaluated in tzm-bl cells, using nl4.3 (cxcr4 tropic) and bal (ccr5 tropic) hiv-1 strains. databases of commercially available compounds (molport and interbioscreen databases, ~1.2 million compounds) were used for the virtual screening (vs) and prepared using the ligprep module of maestro v.11 (schrödinger, llc, new york) software. possible protonation states were generated using the opls3 force field [36] at ph 7±2. at most, one stereoisomer was generated per ligand such that the specified chiralities were retained. for the pharmacophorebased screening, the database was converted to the phase database format with the generate phase database module of maestro [37, 38] . the vs was specifically performed on ccr5, based on the x-ray crystal structure of ccr5 complexed with maraviroc (pdb id: 4mbs; a chain) [39] was retrieved from the protein data bank (pdb; www.rcsb.org). the co-crystallized ligands, ions and water molecules were removed from the receptor and all hydrogen atoms, missing loops and side chains were added, and bond orders assigned using the protein preparation wizard of maestro [40] . the protonation states of histidine residues were inspected interactively; his88 and his231 were changed to hie (protonated at nɛ atom) and hip (double protonated), respectively, to optimize the h-bonding network. restrained minimization using the opls3 force field was first applied for hydrogen atoms only and then for heavy atoms until non-hydrogen atoms attained an average root-mean-square deviation (rmsd) of 0.3 å. the top hits from this virtual screening campaign against ccr5 were comparatively docked with cxcr4 in order to discover dual acting ccr5/cxcr4 antagonists. therefore, the x-ray crystal structure of cxcr4 complexed with an isothiourea derivative (it1t) (pdb id: 3odu) [41] was prepared for docking, similarly as described above. in cxcr4, the protonation state of his113 and his281 were changed to hie and hip, respectively. 2.3 receptor-based virtual screening and prime/mm-gbsa binding free energy vs by molecular docking was conducted with the glide module of maestro [42] . the docking site was defined with the receptor grid generation tool of maestro. the enclosing cubic grid was centered at coordinates (150.03, 108. 25, 22.41) and ligand diameter midpoint box was set to 10 å × 10 å × 10 å . the limitation length for ligands to be docked was set to 12 å . a maximum of five poses per ligand were considered. the glide high-throughput virtual screening (htvs) mode was used for the initial docking of the large database after which the top 10% of the hits based on the docking score were chosen and then re-docked using the standard precision (sp) algorithm. subsequently, 10% of the highest-ranked compounds from this step were redocked using the extra-precision (xp) mode and finally, the top 10% of the glide xp score-ranked ligands were selected for the binding free energy calculations with the prime/mm-gbsa module of maestro [43] . the approximate energies were calculated using the vsgb 2.0 solvation model [44] and opls3 force field, first keeping all binding site residues fixed and then allowing the residues within 5 å from the ligand to move. flexible sampling was done by minimization of the complex. for the best-ranked compounds, molecular property prediction was carried out with maestro's qikprop module. besides the lipinski's rule of five (≤ 2 violations), favorable cell permeability (qppcaco > 500 nm s -1 ; qppmdck > 500 nm s -1 ) and acceptable aqueous solubility (-6.5 < qplogs < 0.5), a series of pains filters were applied to eliminate compounds with potential toxicophores. as a second screening approach, pharmacophore modeling was carried out with the phase module of maestro [37, 38] against ccr5. after preparing the maraviroc-ccr5 crystal complex (pdb id: 4mbs) with the protein preparation wizard of maestro, this structure was used for generating the pharmacophore hypothesis. the nɛ nitrogen atom of the triazole ring and the amide nh of maraviroc form h-bonds with the hydroxyl groups of tyr37 and tyr251, respectively ( figure 2 ). in addition, the positively charged nitrogen in the bicyclic ring engages in an ionic interaction with glu283. therefore, a h-bond acceptor (a), a h-bond donor (d) and a positive ionic (p) feature were selected as the pharmacophoric features for the hypothesis (figure 2 ). this pharmacophore model was then used to screen through the commercial ligand database in search of matching ligands. the ligands were scored and ranked by the phase screen score according to how well they superimpose on the features associated with the hypothesis. the previously calculated qikprop properties were then used to filter the best-ranked 3000 hit compounds as described above. the 500 ligands that passed the filter were then docked into the maraviroc binding site with glide using the xp mode and the subsequent prime/mm-gbsa binding free energy calculation according to the abovedescribed protocol. phase generated pharmacophore model. pharmacophoric features: red sphere for hydrogen-bond acceptor (a); light blue sphere for hydrogen-bond donor (d); blue sphere for the positive ionic (p) feature. the arrows are pointing in the direction of the lone pair. ligand atom color code: carbongreen; nitrogenblue; oxygenred; fluorinelight green. all hydrogen atoms except for maraviroc's polar hydrogens have been omitted for clarity. after a careful analysis of the best-ranked virtual hits, the receptor-bound docked poses of the most promising compounds were subjected to md simulation to estimate the stability of the binding complexes. all simulations were performed with the amber 18 simulation package [45] using the same md simulation protocol as described previously [46, 47] . each solvated system was minimized stepwise, followed by the heating and equilibration stages. finally, a production run of 20 ns was performed at 300 k and 1 bar pressure. the time step was set to 2 fs and the trajectory snapshots were saved every 2 ps and analysed using the cpptraj program [48] of amber. moreover, h-bond occupancy was also examined for best compounds over simulation period. the binding free energies (δg total ) of the most promising hit compounds were calculated using the mm-gbsa (molecular mechanics-generalized born surface area) method, implemented in amber 18. the amber ff99sb molecular mechanics (mm) force field [49] was used to estimate energy contributions from the atomic coordinates of the ligand, receptor and the complex in a gaseous phase. the total binding free energy is calculated as a sum of the molecular mechanics binding energy (δe mm ) and solvation free energy (δg sol ) as given below: where, δe mm is further divided into internal energy (δe int ), electrostatic energy (δe ele ), and van der waals energy (δe vdw ), and the total solvation free energy (δg sol ) is contributed by the sum of polar (δg p ) and non-polar (δg np ) components. δg bind is the free energy of binding of the ligand evaluated after entropic calculations, which is (tδs). the mm-gbsa approach has been successfully used in binding free energy calculations [50] of antiviral inhibitors [51, 52] . the selected compounds were purchased from molport (riga lv1011, latvia) in 2 to 5 mg masses. the purity the compounds was greater than 95%. the most promising compounds resulting from vs were evaluated for antiviral activity by a luciferase assay in tzm-bl cells infected with wild type hiv-1 strains nl4.3 (cxcr4-tropic strain, x4) and bal (ccr5-tropic strain, r5). amd3100 (specific cxcr4 antagonist) and maraviroc (specific ccr5 antagonist) were included as best-in-class positive controls. the cellular toxicity of the compounds was also evaluated in tzm-bl cells. the anti-hiv replication assays in tzm-bl cells have been described previously [53] . the ability of the compounds to inhibit the ca 2+ flux induced by cxcl12 in u87.cd4.cxcr4 cells and by rantes/ccl5 in u87.cd4.ccr5 cells was investigated via calcium mobilization assays. intracellular calcium fluxes were measured with the flipr tetra system as described previously [54] . the ic 50 (i.e., the compound concentration that inhibits the cxcl12-induced cxcr4 signaling or ccl5-induced ccr5 signaling by 50%) was calculated for each compound. the well-known antagonists, amd3100 (cxcr4) and maraviroc (ccr5) were used as a positive control. to identify novel ccr5 antagonists, two parallel virtual screening campaigns were carried out using both receptor-and a ligand-based (pharmacophore) approaches. a total of ~1.2 million compounds were pre-filtered using a set of selected criteria: the lipinski rule of five (≤ 2 violations), predicted permeability in caco-2 and mdck cells (> 500 nm/s) [55] , and the predicted ic 50 value for the blockage of human ether-a-go-go related gene potassium ion (herg k + ) channel (qplogherg value set to -5). herg k + inhibition is a well-known safety issue encountered in the medicinal chemistry optimization of various chemokine receptor antagonists, including maraviroc [56] . as a result, the database was reduced to ~30%, which then underwent the subsequent glide docking protocol and the compounds were ranked based on xp docking and prime/mm-gbsa scores. in the pharmacophorebased screening, the top compounds were selected from the pool of the best-ranked 3000 compounds predefined by the pharmacophore hypothesis (as explained in methods). collectively, 137 compounds were selected and interactively visualized for molecular interactions. receptor-based virtual hits included the compounds with the lowest (best) binding affinity values and a good interaction profile with the binding site residues of ccr5. whereas, the main criteria in the pharmacophore-based screening included the potential to establish strong h-bond interaction (salt bridge) with glu283, π-π stacking interaction with trp86, and hydrophobic interactions with tyr108, ile198, and tyr198. furthermore, the visual inspection process contributed to the identification of compounds that revealed significant molecular interactions with the critical binding pocket residues of ccr5 and helped to exclude compounds that showed unrealistic docking conformations. among the top hits, 77 were found to follow the set selection criteria and showed strong interactions throughout the 20-ns md simulations with favourable binding free energies. although some of these compounds exhibited significantly different binding conformations, all top hits established an extensive interaction network with ccr5. these compounds were further investigated by mm-gbsa calculations and h-bond analysis. after a careful post-md inspection, 43 compounds were selected based on the following criteria; (1) overall backbone stability of the protein/ligand complex, (2) electrostatic (δe ele ) and van der waals (δe vdw ) interaction energy, (3) h-bonds occupancy, and (4) binding pocket residual contribution towards ligands. all these 43 hits were comparatively docked at cxcr4 receptor to investigate their potential for dual ccr5/cxcr4 antagonists [3] . as described earlier, both ccr5 and cxcr4 share marked similarities in their overall structures and in their active site geometry. these similarities include the presence of seven transmembrane domains with a high number of proline residues, a c-terminal threonine and serine-rich cytoplasmic region, four extracellular loops with a high number of cysteine residues and an n-terminal extracellular domain. the regions of the binding pockets contain critically important conserved residues which might allow ccr5 and cxcr4 to host the same ligand. based on these evidences, we specifically sorted out potential hits and 27 structurally diverse compounds were purchased and tested for their anti-hiv activity (figure s1) the ca 2+ mobilization assay allowed to evaluate the antagonistic activity (i.e., the potency to inhibit the rantes/ccl5 and sdf-1/cxcl12-induced calcium mobilization) of the selected compounds. both rantes (natural ccr5 ligand) and cxcl12 (natural cxcr4 ligand) produced a robust transient increase of cytosolic ca 2+ flux in u87.cd4.ccr5 and u87.cd4.cxcr4 cells, respectively. among the tested compounds, compounds 3, 6, and 27 showed antagonistic activity and dose-dependently inhibited the intracellular-induced ca 2+ flux ( table 1 ). the structures of these compounds are shown in the values of cc 50 , ec 50 and ic 50 are in micromolar if not otherwise stated. the results represent the mean of four readings. the ability of the compounds to inhibit the cytopathogenic effect induced by nl4.3 (cxcr4-tropic strain, x4) and bal (ccr5-tropic strain, r5) hiv-1 strains was further evaluated in tzm-bl cells ( the calcium mobilization clearly indicated that compounds 3, 6, and 27 bind to the host coreceptors. although compound 27 showed an ic 50 value in the low micromolar range as ccr5 antagonist, no antiviral activity was observed even at the highest tested concentration of 100 μm. the predicted binding modes of compounds 3 (ccr5/cxcr4 antagonist), 6 (cxcr4), and 27 (ccr5) were investigated to obtain an atomic understanding inside the binding pocket. for all three compounds, the md simulation time was further extended from 20 to 50 ns to enhance the investigations on the stability of the complexes and the compounds' binding energy contributions. the statistical correlation was also evaluated between the average amber/mm-gbsa total binding free energy (δg total ) at the co-receptors and the experimental ic 50 values from ca 2+ mobilization assay ( the stability analysis of the top-ranked compounds against ccr5 is diagrammatically illustrated in (figure 4, s2-s3) . overall, all 27 tested compounds occupied the bottom of the ccr5 binding cavity defined by the residues of the transmembrane (tm) helices (1, 2, 3, 4, 5 and 7), and these binding poses were supported by the co-crystallized conformation of maraviroc ( figure 4a) . moreover, the protonated nitrogen of the compounds identified through the pharmacophore-based hypothesis was found in the close vicinity of the negatively charged glu283, and most of these nitrogen atoms engaged in salt bridge interactions. all compounds exhibited excellent binding energies against ccr5 as indicated via the energy plot, where compounds 3, 6, and 27 ranked among the top, albeit no inhibition of ca 2+ flux evoked by rantes was observed for 6 ( figure 4b ). the stability of these compounds inside the deep binding cavity of the corresponding co-receptors revealed convergence in rmsd values ( figure s2) . compound 27 reached an equilibrium value of ~1.5 å (relative to the equilibrated position of the ligand) somewhat late than the others, at around 20 ns, due to its flexible fatty acid side-chain ( figure s2) . compound 6 adopted a favourable conformation inside the cxcr4 binding pocket at around 13 ns and the dual antagonist 3 displayed consistently stable binding at both co-receptors already from the early steps of the simulation (~ 2-3 ns) (figures s2) . the stable rmsd value in the last ~ 30 ns was evident from the stable molecular interactions governed by the ligand inside the respective binding site of the co-receptors. furthermore, the tm helices of cxcr4 and ccr5 also exhibited significant stability as observed from the root-mean-square fluctuations (rmsf < 1 å), although tm5, which is connected to tm4 by the largest extracellular loop ecl2, showed somewhat larger fluctuations (around 2 å) ( figure 4a and s3). the binding cavity of both co-receptors is mostly hydrophobic due to the presence of a relatively large number of nonpolar amino acids. the analysis of the binding interactions demonstrated that the stability of the compounds inside the binding pocket of cxcr4 and ccr5 is mostly governed by π-stacking and h-bond interactions ( figure 5a-d) . for further in-depth understanding of interactions, h-bond occupancy was also recorded (distance ≤ 3 å; angle ≥120°) throughout the 50 ns simulations ( figure 5e ). per-residue decomposition was also performed for the key residues involved binding the ligands. in 27/ccr5 complex, the interactions were mainly governed by multiple h-bond interactions with the binding site residues. compound 27 contains a short flexible fatty acid side-chain (9hydroxynonanoic acid) linked to monic acid by an ester linkage. as evident from the molecular interactions, compound 27 established a network of h-bonds over a period of 50 ns and adopted a more favourable conformation inside the binding cavity ( figure 5a) chain (figure 5a) . the per-residue decomposition analysis revealed favourable binding free energy contributions (< -1.5 kcal/mol) with trp86, tyr108, phe109, tyr148, whereas the interaction with glu283 revealed a positive value due to the desolvation effect. in the cxcr4/6 complex, the phenyl rings on either side of the molecule were fluctuating during the simulation, with especially the terminal phenyl moiety connected to the aliphatic carbon side chain, whereas the central piperazine ring remained stable in its position. as shown in figure 5b , the terminal side-chain benzene established a strong π-π stacking with trp94 and revealed the most favourable energy decomposition value of -4.6 kcal/mol as compared to the other π-π stacking interactions (with tyr108 and phe109). this π-π stacking interaction was found completely missing with trp86 in the 6/ccr5 complex because the molecule was flipped inside the binding cavity of ccr5 (figure s4) , which might explain the lack of activity against ccr5. moreover, the oxygen atom of the side-chain carboxylic acid of glu288 formed a strong and well populated (82.7%) salt-bridge (average distance, 2.47 å) with the protonated nitrogen atom of the piperazine ring of compound 6, which could stabilize the central piperazine ring. glu288 also showed a favourable binding free energy contribution of -3.37 kcal/mol. the dual ccr5/cxcr4 antagonist 3 contains a salicyl alcohol moiety, a chiral β-hydroxyl group, a secondary amine, and an ether-linked aryloxyalkyl sidechain. the nonpolar side chain facilitated binding with the hydrophobic binding cavity of the co-receptors. the protonated nitrogen of secondary amine in 3 retained similar spatial coordinates in both complexes and established well-populated salt-bridge (64.2 and 58.5%; average distance, 2.15 and 2.64 å) with the glutamate residue of ccr5 (glu283) and cxcr4 (glu288) at respective sites ( figure 5c-e) . the salt-bridge with glutamate provided an anchor point for a dual antagonist to extend in both directions. on one side, the aryloxyalkyl tail reached deep into the binding cavity of ccr5, where the terminal phenyl ring formed hydrophobic interactions with tyr37, tyr89, gln280, thr284 and met287 ( figure 5c) . whereas, the phenyl ring formed a π-π interaction with trp252 and established one h-bond with tyr255 (46.4%; average distance, 2.55 å) in cxcr4 ( figure 5d ). on the other side, the two hydroxyl groups of the salicyl alcohol moiety established h-bonds with thr195 of ccr5 (35.1%; average distance, 2.23 å), although no stable h-bond was observed by this group in cxcr4, except an additional stacking interaction with trp94. among the interacting residues, tyr108 (ccr5) and tyr116 (cxcr4) showed favourable interactions, while glu283 (ccr5) and glu288 (cxcr4) revealed a slightly negative value. the ec 50 values of the different compounds against ccr5 and cxcr4 were correlated quantitatively with the amber/mm-gbsa binding energy values derived from the md simulations in order to assess the performance of the calculations. for each md simulated coreceptor complex with the bound ligand, a total of 5000 snapshots (every 4 ps) were generated. the δg total values were calculated at 10 different time scales (0-2 to 0-20 ns) and compared with the experimental ec 50 values. table 3 tabulates average δg total values for ccr5 at different time scales (0-2 to 0-20 ns) of md simulation along with the pec 50 (-log of ec 50 ) values. the quantitative correlation between δg total and experimental pec 50 values at the shortest time scales (0-2 ns and 0-4 ns) was rather poor (r 2 = 0.347 and 0.285), albeit a gradual increase in correlation was observed with larger time scales (> 0-12 ns and above). the initial poor correlation was due to the event of a single conformation of the proteinligand complex, which did not incorporate the flexibility in the beginning of the simulation. this underlying phenomenon was also evident from the correlations obtained from the docking scores (glide xp) alone, which showed poor r 2 value (0.353) ( figure s5) . afterwards, improved correlations were observed with a longer time scales of 0-18 (r 2 = 0.622) and 0-20 ns (r 2 = 0.631) (table 3, figure 6 ). the overall trend of ligand rmsd trajectories of most complexes (except for few) showed stability after ~8 ns (8-20 ns), which is rationalized through the electrostatic and vdw interaction energies. consequently, the plot of δe mm components (δe elec and δe vdw ) with respect to md simulations at various time scales (8-10 to 8-20 ns) were correlated separately with experimental pic 50 values, as shown in figure 6 . ccr5 and cxcr4 are both important co-receptors involved in the hiv-1 entry process [3, 4, 11, 57] . the ccr5 binding cavity is much deeper and open, while the entrance to the active site of cxcr4 is relatively covered having a strong negative charge on the surface as compared to ccr5 [39] . a highly negative surface charge density across the binding site makes cxcr4 atypical among the chemokine receptors and this difference is evident form the structural attributes of the cxcr4-specific inhibitors. despite the substantial difference in the surface electrostatic potential, many compounds have been reported against both coreceptors [11, 19, 22, 58, 59] . comprehensive virtual screening experiments were carried out to investigate the multiple topographies of the ccr5 binding pocket using both receptor-and ligand-based approaches. the top hits from the ccr5 virtual screening campaign were afterwards docked in the binding cavity of cxcr4 to investigate the dual antagonism of these compounds. 27 compounds were selected for biological evaluation, from which three displayed promising activity. compound 3 is salmeterol, a β 2 -adrenergic receptor agonist acting as a dual cxcr4/ccr5 antagonists. compound 6 is the antihistamine drug cinnarizine with cxcr4 antagonistic potency and compound 27 is the antibiotic mupirocin displaying potent ccr5 antagonism. to date, no antiviral activity of these compounds has been reported, except for moderate activity of salmeterol against the dengue virus [60] . although, the pharmacological properties of these compounds have already been explained in the previous studies, very briefly, salmeterol is indicated to improve airflow in chronic obstructive pulmonary disease, and asthma. it falls under the category of long acting β-2 adrenergic receptor inhibitors with an elimination half-life of 5.5 hr when administered via pulmonary route. this long-acting feature of salmeterol is considered associated with its ability to bind with both active and exo-sites of the receptor. a blood maximum concentration of salmeterol is achieved within 15 min of pulmonary administration [61] . cyp3a4 is considered predominantly involved in the metabolism of salmeterol through α-hydroxylation. some frequently known adverse effects of salmeterol may include hyperlactatemia, metabolic acidosis, depression, anxiety, hypophosphatemia, and hypokalemia [62] . although, a 6-month long clinical trial of salmeterol have proved satisfactory in context of safety profile [63] . in context of these findings and keeping in view the serious side-effects of antiretroviral therapy, it could be most likely stated that further development and repurposing salmeterol for hiv treatment could serve a promising approach. cinnarizine is an antihistaminic compound used for motion sickness and vestibular disorders and is also considered as a nootropic agent [64, 65] . it is a long-known calcium channel blocker with slight potency towards certain dopamine receptors [66] . after oral administration, a maximum plasma concentration approaches in nearly 3 hours with elimination half-life of 3 to 4 hours [67] . its six metabolites are known, which are produces via different of cyps [68] . although, cinnarizine has long been used in clinical practice with considerable tolerance but concerns has been raised for its association with drug-induced acute and chronic parkinsonism, linked to its ability to interact with dopamine receptors [69] . on the other hand, long-term clinical trials up to 4-month have demonstrated reasonable safety [70] suggesting its beneficial application for further development as an anti-hiv agent. mupirocin, also known as pseudomonic acid a, is a natural produced by pseudomonas fluorescens that can inhibit bacterial isoleucyl-trna synthetase [71] . it is found effective against many gram-positive bacteria and some gram-negative bacteria. the presence of epoxide residue in mupirocin makes it prone to extensive metabolism quickly converting it into inactive monic acid, which makes its applications limited to topical antibacterial ointment [71] . its elimination half-life ranges between 20 and 40 minutes after an intravenous administration [72] . due to this short half-life and lack of detailed pharmacological data from parenteral or oral administration requires extensive further experimentation for the use of mupirocin against hiv. compounds 3, 6 and 27 showed highly significant amber/mm-gbsa (calculated) binding energy values during screening. through molecular modelling studies, the compounds were found well-fitted inside the binding site of both co-receptors and remained stable throughout the md simulation period. during md simulations, the compounds remained in a close proximity to a negatively charged glutamate residue in the respective co-receptors. molecular interactions of compounds 3 and 6 were in line with the pharmacophore hypothesis and the co-crystalized complexes of ccr5/maraviroc [39] and cxcr4/it1t [41] , respectively. the protonated nitrogen atoms of the tropane group of maraviroc engaged in a salt-bridge interaction (2.78 å) with glu283 of ccr5, while the protonated nitrogen of the imidazothiazole moiety (n1) of it1t showes a salt-bridge interaction (2.8 å) with glu288 of cxcr4. likewise, the piperazine nitrogen of compound 6 and the secondary amine of compound 3 established a consistent salt-bridge interaction over a period of 50 ns with the respective glutamates. the importance of the glutamate residue deep inside the active site in the stabilization of the complexes has been reported previously [73] [74] [75] [76] [77] [78] [79] and, therefore, glu288 of cxcr4 and glu283 of ccr5 are important residues for structure-based drug discovery of cxcr4 and ccr5 antagonists. in addition, both compounds engaged in significant nonpolar interactions with the corresponding hydrophobic residues of the respective co-receptors, and these interactions are supported with previous mutagenesis and modelling studies [39, 41, 80, 81] . compound 27, the most promising compound after receptor-based screening (ic 50 = 10.64 µm), showed a significant h-bond interaction profile with the residues lining the binding pocket of ccr5, as reported also for maraviroc [39, 80] . compound 6 did not inhibit the calcium flux induced by rantes in u87.cd4.ccr5 cells, despite the significant mm-gbsa binding energy with ccr5 (δg total = -56.48 kcal/mol). the difference in electrostatic potential of the entrance of the binding site may provide the reason to this discrepancy, which has long been recognized between r5 and x4 strains [82] , and is supported by numerous mutational studies [83] [84] [85] [86] . ccr5 bears a neutral to positive electrostatic surface at the binding site entrance, with a deep negatively charged binding pocket, which might hinder the cationic piperazine moiety of compound 6 [74] . cxcr4 has a strongly negative surface charge due to a highly negative n-terminus that can favourably accommodate compound 6 [87] . likewise, compounds 15 and 21, having relatively similar electronegative cyclic nitrogen atoms, showed selective anti-hiv activity against x4 hiv-1 strain (ic 50 of 41.13 and 64.33 µm, respectively). furthermore, the binding pocket of the h 2histamine receptor (pdb id: 6bqg), the primary target of cinnarizine [88] , has a similar, negatively charged surface ( figure s6) , which is in agreement with the selective binding of compound 6 with cxcr4. in contrast, compound 27 possesses an overall negative charge [89] and is a potent ccr5 antagonist, lacking activity against cxcr4. per-residue decomposition analysis revealed binding free energy contributions by important binding site residues involved in electrostatic and vdw interactions. among the residues that were found to stabilize the complex formation, glutamate residues produced positive decomposition values for compounds 3 and 27. this was due to the contribution of unfavourable solvation energy by blocking out a sufficient amount of solvent that might stabilize these negatively charged glutamates. however, due to the presence of long carbon chains in 3 and 27 reduced the solvent exposure of glutamate residues inside the binding cavity resulting in a more favourable value. compound 6 showed a fairly negative energy value for glutamate interaction due to the presence of two protonated nitrogen atoms of the piperazine ring, which counteracted the desolvation effect. this more negative per-residue decomposition value of glu288 in cxcr4 is supported by the evidence from taylor et al., [59] . they changed the protonated piperidine ring [22] to a doubly protonated piperazine ring aiming at increasing the electrostatic interactions, hence counteracting the desolvation effect. although the identified compounds were less active against ccr5 and cxcr4 as compared to the positive controls maraviroc and amd300, it should be underlined that these compounds are directly derived from virtual screening approaches without any further optimization. according to the literature, this is the first account of the activities of these compounds against ccr5 and cxcr4. moreover, the structural pharmacophores that are present in compounds 2, 3, 6, and 27 were not observed in the previously reported ccr5 and cxcr4 antagonists. pyrimidine, quinoline, tetrahydroquinoline, guanide, p-xylylenediamine, indole, and cyclic pentapeptide are commonly known pharmacophores as cxcr4 inhibitors [90] . the compounds 3, 6, and 27 possess relatively different structural architectures that do not know resemble with these moieties. this suggests that the discovery of 3, 6, and 27 as anit-hiv drugs could provide and new platform with a unique set of compounds that do not resemble with already known cxcr4 inhibitors. among potent ccr5 inhibitors, maraviroc has 8-azabicyclo[3.2.1]octane and triazole residues, vicriviroc has piprazine, trifluoromethyl, and pyrimidine residues, aplaviroc contain 1,4,9triazaspiro [5.5] undecane and cenicriviroc is composed of biphenyl with fused tetrahydroazocine and imidazole [91] . the only similarity of the compounds reported in this study was found in the form of piprazine in 6, which resembles vicriviroc. additionally, it is worth mentioning that the high selectivity of compound 27 towards ccr5 could serve as a promising starting point for further optimization towards novel and more potent ccr5 antagonists. moreover, it is notable that among the 27 screened compounds, 13 of them displayed antiviral activity against hiv-1 in low to medium micromolar range, which indicates an enrichment rate of approximately 48.14%. hence, this systematic study focuses on an additional argument emphasizing the advantages of virtual screening for discovering new drug candidates. the study outlines the identification of new selective ccr5 and cxcr4 antagonists, as well as the discovery of a dual ccr5/cxcr4 antagonist. the important pitfalls that must be addressed in virtual screening efforts against chemokine receptors is defining an effective scoring method using electrostatic surface potentials towards hit selection and prioritization. the pipeline of receptor-and ligand-based virtual screening, used in this study, proved as a promising tool to find antagonists for the selected targets. the results were validated through specific ca 2+ gpcr signaling assays and via an anti-hiv-1 replication assay using cxcr4and ccr5-tropicnl4.3 and bal strains. quantitative structure activity relationship studies and synthesis of related derivatives could serve as a future direction for anti-hiv discovery. assessing the impact of haart on the incidence of defining and nondefining aids cancers among patients with hiv/aids: a systematic review economic impact of hiv in the highly active antiretroviral therapy era-reflections looking forward ccr5/cxcr4 dual antagonism for the improvement of hiv infection therapy chemokine co-receptor ccr5/cxcr4-dependent modulation of kv2. 1 channel confers acute neuroprotection to hiv-1 glycoprotein gp120 exposure molecular mechanism of hiv-1 entry hiv-1 entry into cd4+ cells is mediated by the chemokine receptor cc-ckr-5 in vivo evolution of hiv-1 co-receptor usage and sensitivity to chemokine-mediated suppression importance of plc-dependent pi3k/akt and ampk signaling in rantes/ccr5 mediated macrophage chemotaxis natural amines inhibit activation of human plasmacytoid dendritic cells through cxcr4 engagement promiscuous drugs as therapeutics for chemokine receptors inhibition of human immunodeficiency virus replication by a dual ccr5/cxcr4 antagonist small molecules anti-hiv therapeutics targeting cxcr4 small molecule hiv entry inhibitors: part ii. attachment and fusion inhibitors recent progress in small molecule ccr5 antagonists as potential hiv-1 entry inhibitors small molecule and peptide-based cxcr4 modulators as therapeutic agents. a patent review for the period from chemokine receptor ccr5: insights into structure, function, and regulation maraviroc (uk-427,857), a potent, orally bioavailable, and selective smallmolecule inhibitor of chemokine receptor ccr5 with broad-spectrum anti-human immunodeficiency virus type 1 activity the amd3100 story: the path to the discovery of a stem cell mobilizer (mozobil) synthesis and evaluation of transition metal complex dual cxcr4/ccr5 antagonists, national spring meeting of the function, bioinformatics, hiv-1 env gp120 structural determinants for peptide triazole dual receptor site antagonism dual role of novel ingenol derivatives from euphorbia tirucalli in hiv replication: inhibition of de novo infection and activation of viral ltr pyrazolo-piperidines exhibit dual inhibition of ccr5/cxcr4 hiv entry and reverse transcriptase p2x1 receptor antagonists inhibit hiv-1 fusion by blocking virus-coreceptor interactions penicillixanthone a, a marine-derived dualcoreceptor antagonist as anti-hiv-1 agent inhibition of hiv-1 replication by a tricyclic coumarin gut-70 in acutely and chronically infected cells a structure-based drug discovery paradigm the compromise of virtual screening and its impact on drug discovery perspectives towards antiviral drug discovery against ebola virus structure-based in silico screening identifies a potent ebolavirus inhibitor from a traditional chinese medicine library antiviral compounds discovered by virtual screening of small− molecule libraries against dengue virus e protein identification of zika virus ns2b-ns3 protease inhibitors by structure-based virtual screening and drug repurposing approaches structure-based discovery of clinically approved drugs as zika virus ns2b-ns3 protease inhibitors that potently inhibit zika virus infection in vitro and in vivo virtual screening identification of novel severe acute respiratory syndrome 3c-like protease inhibitors and in vitro confirmation structural elucidation of sars-cov-2 vital proteins: computational methods reveal potential drug candidates against main protease, nsp12 polymerase and nsp13 helicase ensemble-based virtual screening reveals potential novel antiviral compounds for avian influenza neuraminidase opls3e: extending force field coverage for drug-like small molecules phase: a new engine for pharmacophore perception, 3d qsar model development, and 3d database screening: 1. methodology and preliminary results phase: a novel approach to pharmacophore modeling and 3d database searching structure of the ccr5 chemokine receptor-hiv entry inhibitor maraviroc complex protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments structures of the cxcr4 chemokine gpcr with small-molecule and cyclic peptide antagonists glide: a new approach for rapid, accurate docking and scoring. 2. enrichment factors in database screening a hierarchical approach to all-atom protein loop prediction the vsgb 2.0 model: a next generation energy model for high resolution protein structure modeling inhibition of oncogenic kinases: an in vitro validated computational approach identified potential multi-target anticancer compounds in silico structural elucidation of rna-dependent rna polymerase towards the identification of potential crimean-congo hemorrhagic fever virus inhibitors ptraj and cpptraj: software for processing and analysis of molecular dynamics trajectory data simmerling, ff14sb: improving the accuracy of protein side chain and backbone parameters from ff99sb assessing the performance of the mm/pbsa and mm/gbsa methods. 1. the accuracy of binding free energy calculations based on molecular dynamics simulations molecular dynamics investigation on a series of hiv protease inhibitors: assessing the performance of mm-pbsa and mm-gbsa approaches investigating interactions between hiv-1 gp41 and inhibitors by molecular dynamics simulation and mm-pbsa/gbsa calculations expanding the antiviral spectrum of 3-fluoro-2-(phosphonomethoxy) propyl acyclic nucleoside phosphonates: diamyl aspartate amidate prodrugs comparison of cell-based assays for the identification and evaluation of competitive cxcr4 inhibitors predicting a drug's membrane permeability: a computational model validated with in vitro permeability assay data overcoming herg affinity in the discovery of the ccr5 antagonist maraviroc putative cholesterol-binding sites in human immunodeficiency virus (hiv) coreceptors cxcr4 and ccr5 small-molecule antagonists of ccr5 and cxcr4: a promising new class of anti-hiv-1 drugs design and computational support for the binding stability of a new ccr5/cxcr4 dual tropic inhibitor: computational design of a ccr5/cxcr4 drug n-desmethylclozapine, fluoxetine, and salmeterol inhibit postentry stages of the dengue virus life cycle lactic acidosis following intentional overdose by inhalation of salmeterol and fluticasone effect of long-term salmeterol therapy compared with as-needed albuterol use on airway hyperresponsiveness cinnarizine for prevention of nausea and vomiting during platin chemotherapy central effects of cinnarizine: restricted use in aircrew, aviation, space, and environmental medicine d2 receptor blockade by flunarizine and cinnarizine explains extrapyramidal side effects. a spect study pharmacokinetics of cinnarizine after single and multiple dosing in healthy volunteers the distribution of cinnarizine and its metabolites in the rat flunarizine and cinnarizineinduced parkinsonism: a historical and clinical analysis intermittent claudication: a controlled study in parallel time of the short-term and long-term effects of cinnarizine mupirocin: a topical antibiotic with a unique structure and mechanism of action the clinical development of mupirocin binding of 2-aryl-4-(piperidin-1-yl) butanamines and 1, 3, 4-trisubstituted pyrrolidines to human ccr5: a molecular modeling-guided mutagenesis study of the binding pocket a binding pocket for a small molecule inhibitor of hiv-1 entry within the transmembrane helices of ccr5 activation of ccr5 by chemokines involves an aromatic cluster between transmembrane helices 2 and 3 analysis of binding sites for the new small-molecule ccr5 antagonist tak-220 on human ccr5 analysis of the mechanism by which the small-molecule ccr5 antagonists sch-351125 and sch-350581 inhibit human immunodeficiency virus type 1 entry mapping interaction sites on human chemokine receptors by deep mutational scanning ligand-guided optimization of cxcr4 homology models for virtual screening using a multiple chemotype approach allosteric model of maraviroc binding to cc chemokine receptor 5 (ccr5) pharmacological modulation of chemokine receptor function phenotype-associated sequence variation in the third variable domain of the human immunodeficiency virus type 1 gp120 molecule minimal requirements for the human immunodeficiency virus type 1 v3 domain to support the syncytium-inducing phenotype: analysis by single amino acid substitution structural basis for coreceptor selectivity by the hiv type 1 v3 loop, aids research and human retroviruses changes in and discrepancies between cell tropisms and coreceptor uses of human immunodeficiency virus type 1 induced by single point mutations at the v3 tip of the env protein mutation of asp171 and asp262 of the chemokine receptor cxcr4 impairs its coreceptor function for human immunodeficiency virus-1 entry and abrogates the antagonistic activity of amd3100 cloning of a human seven-transmembrane domain receptor, lestr, that is highly expressed in leukocytes 5-ht2c receptor structures reveal the structural basis of gpcr polypharmacology the bioavailability of mupirocin in nasal secretions in vitro small molecule inhibitors of cxcr4 ccr5 inhibitors: emergence, success, and challenges key: cord-303208-4bui0ioe authors: jarlais, don c des; arasteh, kamyar; gwadz, marya title: increasing hiv prevention and care for injecting drug users date: 2010-02-26 journal: lancet doi: 10.1016/s0140-6736(10)60314-5 sha: doc_id: 303208 cord_uid: 4bui0ioe nan in the lancet today, bradley mathers and colleagues 1 make a heroic eff ort-in fact, a systematic review-to document the coverage (services provided per individual in need of services) for hiv prevention and care for injecting drug users (idus) throughout the world. whilst the problems in obtaining data and in assessing the quality of data that could be obtained were formidable, two conclusions can be safely drawn. first, there is great variation in coverage of hiv-related services for idus across diff erent countries; and second, in much of the world, coverage is clearly inadequate. there is considerable evidence that hiv-prevention programmes for idus, particularly combined programming, in which multiple programmes are provided, can be very eff ective in reducing injecting-related hiv transmission. 2,3 this evidence suggests that the primary global need is not for new interventions to change the behaviour of idus, but for eff ective interventions to change the behaviour of policy makers to make policies and programmes consistent with the evidence base for hiv prevention and care for idus. over the past 25 years, several useful theories have been developed for changing the health behaviour of idus and others at high risk for hiv. it may be time to apply those theories to changing the behaviour of policy makers. the fi rst set of theories are diff usion of innovations and social learning/modelling theories. these theories articulate how new behaviours spread through social systems and how individuals in these systems infl uence each other to either adopt (or resist) new behaviours. 4,5 these theories have been applied in idu interventions developed by many diff erent researchers and have the major advantage of often producing self-sustaining behavioural changes in the idu community. applying these theories to changing the behaviour of national policy makers raises interesting questions. is the policy-making system relatively open or relatively closed to innovations? is the policy-making system highly centralised (one decision centre) or decentralised (multiple decision centres)? who might become the early adopters and serve as models or opinion leaders for others in the system? the answers to such questions would provide guidance about initiating system change. the second theory is contingency management. this theory comes from basic behaviourism theory. 6,7 if you want to increase the frequency with which an individual performs a specifi c behaviour, provide a reward when syringe-exchange programmes in which a drug user is given a new sterile syringe for bringing in a used and potentially contaminated syringe might be the most common example of contingency management to reduce hiv transmission. within behaviour theory, onefor-one reinforcement is typically not the most eff ective schedule for behavioural change. for syringe exchange, the maximum reductions in risk behaviour seem to occur when drug users are provided with suffi cient numbers of syringes to meet their own needs, and they obtain extra syringes which can then be given to peers, leading to additional social reinforcement. syringeexchange programmes typically have many services in addition to basic exchange, 8 which serve as additional reinforcers. who would provide what sort of reward to political leaders for implementing hiv services for idus? the reward of averting large numbers of hiv infections in idus is certainly important, but is not consistent with a primary principle of contingency management: that rewards should be given immediately after performance of the desired behaviour. international donors, however, could require that evidence-based services for idus be included in national plans to address hiv for a country to receive any hiv prevention and treatment resources. the global fund for aids, tuberculosis and malaria has adopted this approach, although, as shown by mathers and colleagues, there is a need for better follow-through to ensure that appropriate resources are actually provided to idus, particularly for antiretroviral therapy. the third theory is psychological framing of decisions. framing refers to setting the psychological context within which a decision will be made. 9, 10 the same person might make diff erent decisions depending on how a question is framed. aids reframed the act of sharing syringes for idus. before aids, sharing was an act of assistance and solidarity; after aids, sharing syringes became an act in which a fatal disease could be transmitted. many policy makers frame hiv prevention for idus in terms of what appears to encourage or condone drug use, and then oppose harm-reduction programmes. within this frame, data showing that such programmes do not lead to increased drug use have had little eff ect in reducing opposition. it might be more eff ective to frame hiv prevention for idus in terms of the health of the community as a whole, and that public health is fundamental to the economic wellbeing of a society. for example, the economic costs of the epidemic of severe acute respiratory syndrome in china were instrumental in convincing the government there that it needed to address aids. 11 what are we learning? literally millions of dollars and euros have been spent on collecting data and evaluating interventions to change the hiv-related behaviours of idus. it is time to establish a mechanism for collecting and systematically analysing data on eff orts to change policies to increase implementation of hiv programmes for idus. hiv continues to spread among idus in many diff erent countries, and the need for scaling up prevention and treatment is urgent. although theorybased policy-implementation interventions need to be adapted to local situations, we suggest that contingency management and framing the issue in community health-economic terms might be the most useful for immediate policy change. long-term sustained eff orts to protect the health of individuals who use both licit and illicit drugs might require that policy makers acquire a basic scientifi c understanding of drug use and addiction, and frame policies toward drug users within a public health and human rights perspective. 12 the vaccine was incorporated into the cdc's vaccines for children program at the same time. some public health professionals speculated that the vaccine's rapid fda approval and lightning-fast inclusion in cdc programmes would be followed by a colossal failure in the delivery system. 2 specifi cally, although the vaccine's greatest potential clearly lies in the benefi ts it could confer on girls who face a high risk of cervical cancer, public health experts anticipated that the vaccine would mostly go to girls at low risk of the disease. the groups of girls more and less likely to benefi t from hpv vaccination can be readily distinguished in the usa. those who are poor and have restricted access to regular pap testing (and the follow-up assessments and treatments that it triggers) are at high risk of invasive cervical cancer. those who are more affl uent and have access to regular pap testing are at low risk of the disease because the test, when properly done, is highly eff ective. 3 cervical cancer burden diff ers greatly between these two groups. in the fairly poor state of mississippi (median annual household income us$36 674), the ageadjusted cervical cancer mortality rate is 3·6 per 100 000. in rhode island, which is a wealthy state (median $55 980), the cervical cancer mortality rate is 50% lower (1·8 per 100 000). were the vaccine to mostly go to girls in states such as rhode island rather than those in mississippi, such a pattern would not only fail to match contingency management and relapse prevention as stimulant abuse treatment interventions syringe exchange, injecting and intranasal drug use choices, values and frames contributions of behavioural decision theory to research in political science community attitudes toward hiv prevention for injection drug users: fi ndings from a cross-border project in southern china and northern vietnam human rights and hiv prevention among drug users key: cord-288982-63ddlh20 authors: peeling, rosanna w. title: diagnostics in a digital age: an opportunity to strengthen health systems and improve health outcomes date: 2015-11-09 journal: int health doi: 10.1093/inthealth/ihv062 sha: doc_id: 288982 cord_uid: 63ddlh20 diagnostics play a critical role in clinical decision making, and in disease control and prevention. rapid point-of-care (poc) tests for infectious diseases can improve access to diagnosis and patient management, but the quality of these tests vary, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. a new generation of poc molecular tests that are highly sensitive and specific, robust and easy to use are now available for deployment in low resource settings. decentralisation of testing outside of the laboratory can put tremendous stress on the healthcare system and presents challenges for training and quality assurance. a feature of many of these poc molecular devices is that they are equipped with data transmission capacities. in a digital age, it is possible to link data from diagnostic laboratories and poc test readers and devices to provide data on testing coverage, disease trends and timely information for early warning of infectious disease outbreaks to inform design or optimisation of disease control and elimination programmes. data connectivity also allows control programmes to monitor the quality of tests and testing, and optimise supply chain management; thus, increasing the efficiency of healthcare systems and improving patient outcomes. 'without diagnostics, medicine is blind.' alain merieux. diagnostics and laboratory services have been labelled as the achilles heel of global health. 1 in the past, low quality diagnostics and ineffective laboratory services resulted in a mistrust of laboratory results, which in turn led to a decreasing demand for laboratory services and a low priority for funding. 2 the 2008 maputo declaration has called on governments, multilateral agencies, development partners, professional associations and academic institutions to address laboratory challenges that limit the scale-up of services for tb, malaria and hiv diagnosis and care. together with the 2010 us centers for disease control and prevention's call to end the neglect of laboratories, this has provided a major milestone on a pathway to place diagnostics and laboratory services as important pillars of a healthcare system. 3, 4 recent outbreaks of influenza, middle east respiratory syndrome coronavirus (mers-cov) and ebola virus disease illustrate the importance of diagnostics in outbreak investigations. [5] [6] [7] with rapid technological innovations in the last 10 years and donor investments in the development of improved diagnostics for infectious diseases of public health importance, it is time to re-examine the achilles heel and explore the promises and challenges of diagnostics in a digital age. accurate diagnostic tests play a key role in clinician trust and patient management. diagnostic testing is traditionally considered as a tool to rule in or rule out a condition or infection when clinical presentation in a patient is non-specific. for diseases such as hiv and other sexually transmitted infections, most infections are asymptomatic but can cause serious long-term consequences. diagnostics are used to screen for infection so that treatment can be given to prevent the development of long-term complications and to interrupt the chain of transmission to sexual partners or to the fetus in the case of pregnant women. a test for the human papillomavirus helps to determine risk of disease progression to cervical cancer. for pathogens that have developed antibiotic resistance, such as neisseria gonorrhoeae, diagnostic testing is used to determine antibiotic susceptibility to ensure treatment efficacy. for hiv patients, a cd4 test result is used to determine if the patient is eligible for antiretroviral treatment and, for those on treatment, a viral load assay is used to determine treatment compliance and drug efficacy. a test of cure is used to determine when treatment can be stopped ( figure 1 ). beyond patient management, diagnostics play a critical role in various aspects of public health through disease prevention and control. diagnostics are critical in surveillance, to monitor trends in disease and antimicrobial resistance and assess the impact of interventions. diagnostic tools 'of sufficient sensitivity and specificity to detect levels of infection that can lead to transmission' were identified as one of the three essential requirements for disease elimination or eradication. 8 who has set eradication or elimination targets for a number of neglected tropical diseases (ntds), including guinea worm, polio, yaws, trachoma, schistosomiasis, lymphatic filariasis, onchocerciasis, human african trypanosomiasis, chagas' disease and visceral leishmaniasis (vl) 9 and the elimination of mother to child transmission (emtct) of hiv and syphilis. mass drug administration (mda) is the main control strategy for many of these diseases, resulting in the need for highly sensitive tests to detect the few remaining cases in areas of low transmission intensity after multiple rounds of mda. highly specific tests are then needed to certify elimination and maintain post-elimination surveillance. there is often little commercial interest in the development of diagnostics that are used mainly for surveillance because of the low return on investment. for a ntd such as schistosomiasis, the chinese national control programme has defined stages for schistosomiasis control as morbidity control (prevalence over 5% as defined by stool examination), infection control (prevalence of 1-5%), transmission control (prevalence lower than 1%), transmission interruption (no case detected in 5 years successively) and elimination (no case detected in the 5e years after transmission was interrupted). diagnostics results are used to inform national and regional control policy, redirect resources and to transition to the next phase of the control and treatment strategy. 10 the who targets for emtct and who/unaids 90-90-90 targets for hiv by 2020 also include diagnostic test coverage as indicators for efficiency of service delivery that help countries with setting a course to have an aids free generation and to end the hiv epidemic by 2030. diagnostic testing is usually performed in laboratories. in countries where the laboratory infrastructure is limited, who advocates for the use of a syndromic approach for patient management whereby patients are treated for all the major causes of that syndrome. this often leads to overtreatment and increases risk for the development of antimicrobial resistance. in the last decade, rapid point-of-care (poc) diagnostic tests fulfilling the assured criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable) have become commercially available and are widely used for infectious diseases such as malaria, hiv and syphilis. [11] [12] [13] [14] [15] these tests have allowed control programmes to increase access to testing, identify those who need to be put on treatment, optimise disease control and save lives. however, the quality of these tests varies, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. a new generation of immunoassays, molecular and nanotechnology platforms has been developed in recent years that can improve patient management and disease surveillance. these platforms have superior performance and make use of optical readers, mobile phones and data transmission capabilities to improve reading of results and data transmission. such technologies provide real-time results to inform patient management decisions. data, linked to precise geographical locations using gps, can be transmitted to a central database to inform disease control programmes or to monitor progress towards elimination. many of these assays can also yield quantitative results to give estimates of pathogen copy number or, in the case of hiv, to monitor treatment compliance or efficacy. these technology platforms can also be used to detect multiple targets from a single specimen. for geographic areas with multiple diseases targeted for elimination, if surveillance is conducted in the same sentinel population, such as children under the age of 10 years, using the same specimen, such as a blood sample, exposure to multiple ntds can be detected on a multiplex platform. this may be a more cost-effective means of conducting post-mda surveillance than collecting specimens and testing for a single ntd at a time, 16, 17 although cost-effectiveness of using these platforms for multiple ntds remains to be demonstrated. increased sensitivity of detection using these advances has made it possible to have tests that detect antibodies from oral fluids. the use of these non-invasive specimens has made it possible to design hiv tests for self-testing or for home use. 18 the availability of self-testing at home has allowed testing to be de-stigmatised and should help countries with achieving the first 90 of the unaids/who 90-90-90 targets. 19 bead-based immunoassays bead-based technologies are more versatile and potentially offer higher sensitivity compared to traditional solid phase immunoassays. 20 instead of using enzymatic amplification of a colorimetric substrate, bead-based assays can use a range of labels, including laser or fluorescence to detect the binding of the secondary antibody to an antigen or antibody target. the beads are in solution, offering more sites for ligand binding compared to a solid phase support. fluorescent intensities can yield quantitative results. protein arrays offer the potential of quantification of the antigen target as a surrogate measure of bacterial, parasite or viral load to inform treatment strategies. detection of multiple targets from the same pathogen can lead to increased efficiency over single-target assays. detection of targets from multiple pathogens using a single specimen can offer substantial cost and sample savings over traditional elisa measurements. the detection platforms are often equipped with data transmission capacities and allows near real-time surveillance of disease trends and monitoring the effect of special interventions. microfluidic devices offer many advantages, such as high throughput, short analysis time, small volume and high sensitivity, which make them an ideal immunoassay format for clinical diagnoses. the current microfluidic immunoassays have limited multiplexing capability compared to flow cytometric assays but are improving. immunoassays developed on a microfluidic platform that reproduce all the steps of a traditional elisa in a miniaturised format are now available. 21 the microfluidic disc can fit into the palm of a hand and is rapid and inexpensive to manufacture. these platforms allow multiplexing, but have only been validated for antibody detection in blood samples. antigen detection from urine or stool samples will require multistep specimen processing and/or purification or concentration before being put into the antigen-antibody reaction within the microfluidic channels. a recent publication showed that it is possible to use the power of smart phones to power a microfluidic immunoassay reaction by plugging the reaction chamber into a smart phone using a dongle. 22 over 80 assays can be run before recharging is necessary. the results are interpreted and displayed on the phone and the data transmitted to a central database if required. a dual test to detect antibodies to hiv and syphilis has been designed on this platform and will have utility not only to improve access to prenatal screening but also to allow surveillance data for the dual elimination of mtct of hiv and syphilis to be available in real time. 23 nanosensor assays a growing number of promising diagnostic tools are based on nanotechnology. the application of nanomaterials to detect host or pathogen biomarkers has the potential to yield ultrasensitive assays. quantum dots are fluorescent semiconductor nanoparticles typically between 10 and 100 atoms in diameter and the technology has been applied to the development of ultrasensitive tests for hiv and other viral infections. 24 it is also possible to use these technologies to combine pathogen detection and speciation with genetic analysis, such as detection of single nucleotide polymorphisms. hence, they can be used for high throughput screening of drug mutation targets. quantum wires are being used in the development of a fully automated diagnostic test for malaria infection, speciation and drug resistance status in less than 20 minutes. 25 these nanosensors can ultimately be configured to a handheld pda-type device or a thin plate about the size of a small business card containing a tiny nanowire sensor. operationally, these tests are simple to use and give answers in less than 30 minutes. they can be used during a doctor's visit or at home by a patient, or early detection of bioterrorism in a community setting. linking mobile connectivity and gps will enable anonymised disease data to be sent to a data cloud for real-time surveillance. advocacy to apply these novel technologies to ntds and antimicrobial resistance monitoring is an urgent priority. this requires precise engineering of nanomaterial surfaces as the interface between the nanomaterial and the specimen is where the reaction occurs. molecular assays offer superior diagnostic performance compared to the limit of detection of immunoassays. in the last two decades, nucleic acid amplification tests (naats) have become the 'gold' or reference standard to which the performance of other diagnostic tests is compared. until recently, they have remained largely laboratory based and are expensive and inaccessible. a number of poc molecular assays, with or without amplification, are now on the horizon that may transform the delivery of health services. 26, 27 the first sample-in/answer-out real-time pcr platform that can be used wherever there is a source of electricity has been introduced for the diagnosis of tb and detection of rifampicin resistance. [28] [29] [30] the genexpert real-time pcr platform has a large menu of test targets; thus, allowing polyvalency to test for different pathogens. it requires minimal onsite expertise and provides a result in less than 2 hours. it has additional advantages of random access and remote quality control. however, the cartridges are expensive and not affordable or sustainable unless subsidised in most high burden countries. to maximise the impact of these novel technologies, it is critical that patient pathways be modified to take advantage of the speed to result and to find cost-efficient means of implementation compared to traditional laboratory testing. 31 isothermal amplification platforms such as helicase dependent amplification (hda), cross priming amplification (cpa), recombinase polymerase amplification (rpa) and loop-mediated amplification (lamp) assays can be engineered into poc naats as there is no requirement for sophisticated equipment to perform thermal cycling. the details of these technologies have been described elsewhere. 27, 32 poc naats based on these technologies have largely been applied to high burden diseases such as hiv and tb, but other technologies, such as lamp, have been developed and evaluated for the diagnosis of vl and hat. in the case of vl, the lamp assay for leishmania donovani is highly sensitive and specific for the diagnosis of vl (using a blood sample), and for the diagnosis of post-kala azar dermal leishmaniasis (pkdl) (using a skin biopsy). 33 diagnosis of pkdl will be important for the elimination agenda since patients with this condition are an important source of infection. in the case of hat, the lamp assay was shown to have a sensitivity of 87.3% (95% ci 80.9 to 91.8%) and a specificity of 92.8% (95% ci 86.4 to 96.3%) compared to a pcr reference standard. 34 advocacy and investments are needed to apply these technologies to the control and elimination of ntds. 35 the promise of diagnostics in a digital age in a digital age, data from social media and poc diagnostics in communities can be used to provide early warning for infectious disease outbreaks, and timely information to inform disease control and elimination programmes. the isense project, an epsrc irc funded project in early warning sensing systems in infectious diseases, aims to create low-cost latent sensing systems to analyse self-reported symptoms on the web, including social networks and micro-blogging sites (twitter) and searches (bing, google). isense will develop a new generation of early warning sensing systems to identify outbreaks of deadly infectious diseases, such as flu, methicillin-resistant staphylococcus aureus (mrsa) and hiv, by linking self-reported symptoms on the web to a new sensor-enabled disease surveillance infrastructure for an early warning sensing system for infectious diseases (http://www.i-sense.org.uk/research). the simplest poc diagnostic tests that are widely used today are rapid diagnostics tests (rdts) in a lateral flow format. these are read with the naked eye, which is subjective and prone to human error and may be further exacerbated by poor lighting in health posts. in addition, rdts lack on-board quality control and are often used in remote areas where health workers receive minimal training. as a result, accuracy of rdts performed in the field can be quite variable. this may adversely affect patient care and the accuracy of the data gathered for surveillance. digital imaging technology in electronic readers and smart phones can be used to capture and interpret rdt results and transmit data. given the large number of brands of rdts for hiv, malaria and syphilis, it would be ideal to have diagnostic test readers that could accommodate a variety of test technologies (e.g., lateral flow, immunofiltration) and formats (e.g., strips, cassettes of various sizes and shapes). a number of rdt readers are already on the market and others are in the pipeline. given the widespread adoption of smart phones in resource-limited settings, rdt readers using this technology have the potential to combine high-resolution test images with the computing capability required to run image analysis software and transmit data. the readers range in price depending on the technical complexity of the instrument and their compatibility with the type of rdts. data collection for the emtct is difficult when testing is highly decentralised and data quality is difficult to verify. to track global progress towards elimination of mtct of hiv and syphilis many challenges need to be addressed. currently, data from antenatal screening need to be manually accessed from individual testing sites. electronic readers for rdts are able to capture and automatically transmit data and may, therefore, serve a critical purpose for strengthening data collection and surveillance in the context of monitoring and evaluation of dual elimination. 36, 37 improving supply chain management real-time data monitoring via electronic readers could help to improve coordination of supply chain management by multiple partners. operational data on stocks, device usage and condition can be uploaded via wi-fi or cellular networks and transmitted to central databases. by linking the data to supply chain management software, stock-outs can potentially be avoided and health system efficiency improved. in recent years, rdts have been introduced to increase hiv and syphilis screening in antenatal clinics, enabling same day testing and treatment of infected mothers to prevent adverse outcomes of pregnancy and fetal loss. the emtct targets to be achieved are 95% of pregnant women access antenatal care, 95% of pregnant women at antenatal care tested for hiv and syphilis, and 95% of those testing positive receive treatment. to achieve these targets, countries will need to have a system to track progress the capacity for ongoing surveillance once the elimination target is achieved. studies have shown that the introduction of rapid rdts for syphilis for prenatal syphilis screening has strengthened health systems by improving access to diagnostics, health worker job satisfaction and reducing stillbirths and neonatal mortality. in peru, the introduction of rdts for prenatal screening has resulted in infected pregnant women being screened and treated in a single visit instead of 5-6 visits over 27 days. 38, 39 challenges of new technologies assuring quality of poc lateral flow tests and devices decentralisation of testing outside of the laboratory can put tremendous stress on the healthcare system and presents challenges for training and quality assurance. when testing is decentralised, programme managers are often unable to monitor testing coverage and quality, making it difficult to identify problems of sub-standard testing and stock-outs in a timely manner. 40 international health for poc molecular devices, requirements for instrument calibration, ongoing maintenance and frequency of failure, power usage and environmental sustainability should also be considered. creation of a central database to collect surveillance data from village antenatal clinics all the way through to national and global databases is needed. this involves obtaining consensus on where to host the data, what data to collect, who has access to the data and finding affordable software. middleware solution that provides 'open access' will allow for rapid coordinated transmission of data to governments. technology is needed to ensure data is collected, transmitted and stored in a way that conforms to ethical and legal standards, maintaining patient privacy and confidentiality. these governance issues may be resolved by ensuring separate levels of access to operational data versus patient data. in addition, data storage should be in-line with country specific regulations. further discussion needs to take place around data ownership. an excellent example of a national database that informs critical programmatic decisions in real time is the kenyan national early infant diagnosis (eid) portal (www.nascop.org/eid), which has now been extended to cover hiv viral load and other diagnostics. this database is the result of a successful public-private partnership that involved the government of kenya ministry of health, usaid/president's emergency plan for aids relief (pepfar), the us cdc, the clinton health access initiative (chai), hewlett packard, safaricom and strathmore university. hewlett packard built a basic data centre at the national aids/std control programme (nascop) and at testing laboratories for early infant diagnosis. safaricom, the largest provider of mobile services in kenya, set up a short code service for sms. strathmore university students built the application. pepfar, cdc and chai rolled out the eid programme countrywide. the result is a national eid portal running on a government data centre-hosting all the testing and program data entered from computers in laboratories equipped with laboratory information systems (web-based applications). over 2000 health facilities that provide eid services have access to all the data and test results over sms, mobile web, web and email in near real time. data analytics include sample transport tracking, volume of testing at each facility, test results, including the number of rejected samples and indeterminate results. results are delivered via electronic and paper means (sms printer, email, courier, web) and real time eid/prevention of mtct programme analytics are available online by facility, and at regional and national levels. the new generation of diagnostics equipped with digital technologies are transforming the field of clinical decision-making and disease control and prevention. rapid poc tests for infectious diseases can improve access to diagnosis and patient management, but the quality of these tests varies, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. electronic readers have the potential to provide fast, accurate, standardised rdt interpretation and real-time data reporting with a huge range of positive functions, including improving quality assurance, supply chain management and providing accurate timely data for surveillance. although countries need to consider data governance and confidentiality issues, data connectivity allows control programmes to monitor the quality of tests and testing, and optimise supply chain management; thus, increasing the efficiency of healthcare systems and improving patient outcomes. author's contribution: rp has undertaken all the duties of authorship and is guarantor of the paper. achilles heel" of global efforts to combat infectious diseases laboratory medicine in africa: a barrier to effective health care the maputo declaration on strengthening of laboratory systems. geneva: world health organization laboratory systems and services are critical in global health: time to end the neglect? severe respiratory disease concurrent with circulation of h1n1 influenza identification of the novel coronavirus in patients with severe acute respiratory syndrome the case for improved diagnostic tools to control ebola virus disease in west africa and how to get there the principles of disease elimination and eradication tools to support policy decisions related to treatment strategies and surveillance of schistosomiasis japonica towards elimination diagnostics for the developing world point-of-care tests for diagnosing infections in the developing world point-of-care diagnostics for global health diagnosing infections -current and anticipated technologies for point-of-care diagnostics and home-based testing point-of-care diagnosis of tuberculosis: past, present and future a diagnostics platform for the integrated mapping, monitoring, and surveillance of neglected tropical diseases: rationale and target product profiles development of a new platform for neglected tropical diseases surveillance hiv self-testing in resource-limited settings: regulatory and policy considerations unaids. 90-90-90 -an ambitious treatment target to help end the aids epidemic bead-based microfluidic immunoassays: the next generation microfluidics-based diagnostics of infectious diseases in the developing world smartphone dongle for simultaneous measurement of hemoglobin concentration and detection of hiv antibodies multisite laboratory evaluation of a dual human immunodeficiency virus (hiv)/syphilis point-of-care rapid test for simultaneous detection of hiv and syphilis infection integrated quantum dot barcode smartphone optical device for wireless multiplexed diagnosis of infected patients plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth emerging technologies in point-of-care molecular diagnostics for resource-limited settings point-of-care nucleic acid testing for infectious diseases feasibility, diagnostic accuracy, and effectiveness of decentralised use of the xpert mtb/rif test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study point-of-care system for detection of mycobacterium tuberculosis and rifampin resistance in sputum samples geneva: unitaid; geneva opportunities and challenges for cost-efficient implementation of new point-of-care diagnostics for hiv and tuberculosis application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis diagnostic accuracy of loopamp trypanosoma brucei detection kit for diagnosis of human african trypanosomiasis in clinical samples multiplex screening for blood-borne viral, bacterial, and protozoan parasites using an openarray platform using electronic readers to monitor progress toward elimination of mother-to-child transmission of hiv and syphilis: an opinion piece elimination of elimination of mother-to-child transmission (emtct) of hiv and syphilis. global guidance on criteria and processes for validation. geneva: world health organization point-of-care tests to strengthen health systems and save newborn lives: the case of syphilis rapid syphilis tests as catalysts for health systems strengthening: a case study from peru the costs of accessible quality assured syphilis diagnostics: informing quality systems for rapid syphilis tests in a tanzanian setting the author would like to acknowledge david mabey and debi boeras in providing helpful comments in the preparation of this manuscript.funding: none. ethical approval: not required. key: cord-285430-o086q2qa authors: gribble, karleen; mathisen, roger; ververs, mija-tesse; coutsoudis, anna title: mistakes from the hiv pandemic should inform the covid-19 response for maternal and newborn care date: 2020-07-25 journal: int breastfeed j doi: 10.1186/s13006-020-00306-8 sha: doc_id: 285430 cord_uid: o086q2qa background: in an effort to prevent infants being infected with sars-cov-2, some governments, professional organisations, and health facilities are instituting policies that isolate newborns from their mothers and otherwise prevent or impede breastfeeding. weighing of risks is necessary in policy development: such policies are risky as was shown in the early response to the hiv pandemic where efforts to prevent mother to child transmission by replacing breastfeeding with infant formula feeding ultimately resulted in more infant deaths. in the covid-19 pandemic, the risk of maternal sars-cov-2 transmission needs to be weighed against the protection skin-to-skin contact, maternal proximity, and breastfeeding affords infants. conclusion: policy makers and practitioners need to learn from the mistakes of the hiv pandemic and not undermine breastfeeding in the covid-19 pandemic. it is clear that in order to maximise infant health and wellbeing, covid-19 policies should support skin-to-skin contact, maternal proximity, and breastfeeding. the care of mothers and infants in the covid-19 pandemic has proven challenging, as policy makers have grappled with how to protect newborns when their mothers are suspected or confirmed as having covid-19. for the general population, isolation of the infected from the uninfected and physical distancing are essential to preventing disease transmission and achieving good health outcomes. however, mothers and infants present a special situation as the risk of mother-to-child transmission of sars-cov-2 needs to be weighed against the protection from infectious diseases and the support for bonding and caregiving provided by close maternal proximity and breastfeeding. in low-, middle-and high-income countries, some policy makers and practitioners appear to have given more weight to the risk of sars-cov-2 transmission than the consequences of maternal separation and reducing breastfeeding. at the most extreme, infants are being isolated from their mothers with suspected or confirmed covid-19 for periods of up to 2 weeks (e.g. [1, 2] ). in some contexts, expressed breastmilk may be provided, while in others even provision of breastmilk is prohibited. more commonly, skin-to-skin contact after birth is being withheld and where infants are permitted to share a room with their mothers, a distance of two meters or a screen separates them (e.g. [3, 4] ). these decisions reduce infant access to breastfeeding and breastmilk and have been made on the basis of very little evidence. a similar weighing against breastfeeding was made in the early response to the hiv pandemic. experience of the hiv pandemic the possibility of mother-to-child transmission of hiv through breastfeeding was first raised in 1985 in a letter published in the lancet [5] . in this case, an australian woman who had been given an hiv contaminated blood transfusion for a postpartum haemorrhage, contracted hiv and her infant was also found to be hiv positive [5] . it was proposed that the mother transmitted hiv to her infant through breastfeeding. shortly thereafter, hiv was isolated in breastmilk [6] . the fear of mother-to-child transmission of hiv through breastmilk led authorities in the usa to quickly respond by recommending that hivpositive mothers should not breastfeed [7] . policy makers in the developing world followed suit and hiv-positive women were supplied with free infant formula despite there being no knowledge of the magnitude of risk of hiv transmission through breastfeeding [8] . replacement infant formula feeding by hiv-positive women was quickly institutionalised in many countries. however, it was not until 1992 that a reliable estimate of hiv transmission through breastmilk was published indicating that after 24 months of breastfeeding 14% of breastfed infants risked contracting hiv [9] . further research highlighted that this was the cumulative risk and not, as supposed, a one-off risk when mothers commenced breastfeeding, and breastfeeding for a shorter period of 6 months carried a risk of about 4% [10] . in addition, if breastfeeding during the first 6 months was exclusive, this risk was even further diminished. it emerged that supporting exclusive breastfeeding maximized infant hiv-free survival [11] . rather than improving infant health, policies to move away from supporting breastfeeding in the hiv pandemic had a devastating impact on infant mortality in many middle-and low-income countries. more infants lost their lives through diarrhoea and pneumonia related to infant formula feeding compared to those who lost their lives through hiv infection [11] . the hard lesson was learnt that an hiv negative dead infant is still dead. the aftermath of these recommendations had serious repercussions that lasted for more than a decade as the fear of hiv transmission and the normalisation of bottle feeding changed infant feeding practices. even in communities that previously had a strong breastfeeding culture, mothers who were hiv-negative opted to infant formula feed with attendant increases in infant morbidity and mortality [11] . while policy development in the hiv pandemic might share some similarities with the covid-19 pandemic, the method of transmission and the impact of infection on infants are quite different. hiv is a blood borne infection that can be transmitted in utero, during birth, and via breastmilk [12] . hiv can cause serious disease, and before the introduction of effective anti-retroviral treatment, mortality rates of those infected with hiv as infants approached half by 3 years of age [13] . in contrast, sars-cov-2 appears to be primarily transmitted through respiratory droplets and contact routes [14] . to date there is no clear evidence that vertical transmission occurs and samples of amniotic fluid, and cord blood taken from mothers with confirmed covid-19 have been found negative for sars-cov-2 [15] . in a small number of cases, viral rna particles have been detected in expressed breastmilk, but no live virus has been found and breastmilk is not thought to be a transmission route [16, 17] . antibodies to sars-cov-2 have been found in breastmilk suggesting that breastfeeding may provide specific protection against the virus [18] . a recent review of infants born to 655 women with covid-19 concluded that it was rare for infants to be infected with sars-cov-2 and that rates of infection are not increased when infants remain proximate to their mothers and breastfeed [17] . children infected with sars-cov-2 are mostly asymptomatic or have mild symptoms [19] . it seems that fever and cough are relatively the most reported symptoms amongst paediatric cases and many children with sars-cov-2 infection might remain undetected. complications and death appear to be rare [19] . as described, hiv and covid-19 are starkly different in terms of transmission method and consequences of transmission. nonetheless, it should be noted that despite the possibility of hiv being transmitted via breastmilk and that, prior to antiretroviral use, infection with hiv was commonly fatal, policies to move away from breastfeeding in the hiv pandemic caused great harm. since there is no evidence that sars-cov-2 is transmitted via breastmilk and covid-19 is rarely serious in infants, the relative harm of policies impeding breastfeeding in the covid-19 pandemic may therefore be even greater than was the case for the hiv pandemic. on 13 march 2020 the who published interim guidance on the clinical management of covid-19, including on the care of newborn infants [20] . this guidance was heavily weighted towards supporting maternal proximity and breastfeeding. it states that women with suspected, probable or confirmed covid-19 should be supported to have their infants placed skin-to-skin with them immediately after birth, initiate breastfeeding within an hour of birth, to keep their infants by their side day and night, and exclusively breastfeed [20] . alongside these practices, mothers should apply infection prevention and control measures by practicing respiratory hygiene, washing their hands before and after contact with their infants, and ensuring that surfaces that they have been in contact with are cleaned and disinfected [20] . where mothers are unable to breastfeed because of illness they are to be assisted to express milk for their infants [20] . if this is not possible, the use of donor human milk should be explored and if this is not available wet nursing or infant formula may be considered. ongoing milk expression and relactation when mothers are well enough is also recommended [20] . infographics to assist in the promotion of the guidance were released by the who, as shown in figs. 1, 2 and 3. the who released updated guidance on the management of covid-19 on 27 may 2020, the recommendations regarding maternal and newborn care were unchanged [21] . the rationale for these recommendations is based on the importance of breastfeeding in preventing infant morbidity and mortality [20] [21] [22] . worldwide, 72% of hospital admissions for diarrhoea and 57% of admissions for respiratory infections can be attributed to a lack of breastfeeding [23] . in low-and middle-income countries, infants who are not breastfed have a mortality rate that is eight times greater than exclusively breastfed infants and globally more than 800, 000 lives could be saved if breastfeeding were universally practiced [23] . even in high-income countries, hospitalisation rates are greatly elevated in non-breastfed infants [24] . the who covid-19 guidance, notes that health service practices should minimise disruption to breastfeeding [21] . however, covid-19 policies that require isolation of mothers and infants and denial of breastmilk prevent infants from having access to breastfeeding. withholding skin-to-skin contact after birth reduces rates of exclusive breastfeeding by one third and impedes maternal bonding and sensitivity [25, 26] . any increase in physical distance between mothers and infants reduces the frequency of breastfeeding and may therefore negatively impact on the establishment of breastfeeding [27] . reduction of breastfeeding not only has an impact on infant physical health, but adversely impacts infant cognitive development [28] . it can also reduce maternal caregiving capacity and so result in increased rates of child maltreatment [29] . maternal deaths due to breast and ovarian cancers as well as type 2 diabetes can also be attributed to short breastfeeding duration practices [28] . policies that impede breastfeeding also enhance the marketing of infant formula as manufacturers can argue that normal prohibition on donations of infant formula do not apply because mothers cannot breastfeed. donations are a powerful marketing tool that are proscribed by the who international code of marketing of breastmilk substitutes and the operational guidance on infant and young child feeding in emergencies [30, 31] . however, in the covid-19 pandemic, infant formula manufacturers and distributors are donating to hospitals, governmental and non-governmental agencies, and individuals worldwide [32] [33] [34] . the negative short and long-term harms of donations in emergencies are well known [35] . at all times, but especially in emergencies, protection and support for breastfeeding needs to be strengthened and not undermined [36] . covid-19 policy makers must learn from past mistakes. in 2010 this journal published a thematic issue on "hiv and infant feeding: lessons learnt and ways ahead" in which moland et al. [37] concluded, "the scientific evidence base warns against hasty dismissal of the evolved benefits of breastfeeding … in future, the global health professional community should be more sceptical of claims about the risks of breastfeeding." we stand now in the future spoken of, facing a new pandemic. with such substantial, well documented evidence of the importance of maternal proximity and breastfeeding for child survival, development, and health as well as the protection that breastfeeding affords mothers, we cannot repeat the mistakes of the hiv pandemic. breastfeeding should not be interrupted because of a fear that sars-cov-2 could be transmitted from mothers to infants during breastfeeding and so harm infants. countries, professional associations, and health facilities should follow the lead of the who, taking heed of the experiences of the past and take care in the covid-19 response to not undermine breastfeeding. approach to the management of covid-19 in pregnancy and the newborn chinese expert consensus on the perinatal and neonatal management for the prevention and control of the 2019 novel coronavirus infection technical guideline: covid-19 infection approach in the perinatal period promgulating the interim guidance for acute respiratory infection caused by the sars-cov-2 virus strain (covid-19) in pregnant women and infants postnatal transmission of aidsassociated retrovirus from mother to infant isolation of aids virus from cell-free breast milk of three healthy virus carriers current trends recommendations for assisting in the prevention of perinatal transmission of human t lymphotropic virus type iii/ lymphadenopathy-associated virus and acquired immunode!ciency syndrome free formula milk for infants of hiv-infected women: blessing or curse? health policy plan risk of human immunodeficiency virus type 1 transmission through breastfeeding international multicentre pooled analysis of late postnatal mother-to-child transmission of hiv-1 infection. ghent international working group on mother-to-child transmission of hiv hiv prevention is not enough: child survival in the context of prevention of mother to child hiv transmission in utero and intra-partum hiv-1 transmission and acute hiv-1 infection during pregnancy: using the bed capture enzyme-immunoassay as a surrogate marker for acute infection mortality after the first year of life among human immunodeficiency virus type 1-infected and uninfected children clinical characteristics of 19 neonates born to mothers with covid-19 vertical transmission of severe acute respiratory syndrome coronavirus 2: a systematic review excretion of sars-cov-2 in human breast milk maternal transmission of sars-cov-2 to the neonate, and possible routes for such transmission: a systematic review and critical analysis antibodies in the breast milk of a maternal woman with covid-19 novel coronavirus infection (covid-19) in children younger than one year: a systematic review of symptoms, management and outcomes clinical management of severe acute respiratory infection (sari) when covid-19 disease is suspected: interim guidance. geneva: world health organization clinical management of covid-19: interim guidance. geneva: world health organization frequently asked questions: breastfeeding and covid-19 for health care workers breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect breastfeeding and infant hospitalisation: analysis of the uk 2010 infant feeding survey early skin-to-skin contact for mothers and their healthy newborn infants skin-to-skin contact the first hour after birth, underlying implications and clinical practice randomised trial of infant sleep location on the postnatal ward the cost of not breastfeeding: global results from a new tool does breastfeeding protect against substantiated child abuse and neglect? a 15-year cohort study infant and young child feeding in emergencies: operational guidance for emergency relief staff and program managers, version 3. oxford: infant and young child feeding in emergencies core group sixty-third world health assembly. infant and young child nutrition. wha 63.23 how companies are exploiting the covid-19 pandemic pretending their marketing is 'humanitarian' and that their products build immunity countries failing to stop harmful marketing of breast-milk substitutes, warn who and unicef: agencies encourage women to continue to breastfeed during the covid-19 pandemic now more than ever, baby friendly facilities must protect formula feeding in emergencies marketing of breast-milk substitutes: national implementation of the international code status report. geneva: world health organization ways ahead: protecting, promoting and supporting breastfeeding in the context of hiv publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. the authors initiated the manuscript via group discussion. all authors contributed to the drafting of the manuscript and revised the manuscript. all authors agreed to the final manuscript and consent to publication in international breastfeeding journal. the authors declare no competing interests. the author(s) read and approved the final manuscript. availability of data and materials not applicable. ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. received: 19 may 2020 accepted: 9 july 2020 key: cord-311366-uodq4foi authors: sanyal, anwesha; shen, chengli; ding, ming; reinhart, todd a.; chen, yue; sankapal, soni; gupta, phalguni title: neisseria gonorrhoeae uses cellular proteins cxcl10 and il8 to enhance hiv‐1 transmission across cervical mucosa date: 2019-04-11 journal: am j reprod immunol doi: 10.1111/aji.13111 sha: doc_id: 311366 cord_uid: uodq4foi problem: neisseria gonorrhoeae (ng) infection has been shown to increase sexual transmission of hiv‐1. however, the mechanism of ng‐induced enhanced hiv‐1 transmission is unknown. methods: (a) the cervical tissues were exposed to ng, and cytokine induction was monitored by measuring cytokine proteins in culture supernatants and cytokine mrnas in tissues. (b) transcription and replication of hiv‐1 in tzm‐bl, u1, and ach2 cells were measured by beta‐gal activity and p24 proteins in the supernatant, respectively. (c) hiv‐1 transmission was assayed in an organ culture system by measuring transmitted hiv‐1 in supernatant and hiv‐1 gag mrna in the tissues. (d) transcriptome analysis was done using second generation sequencing. results: (a) ng induced membrane ruffling of epithelial layer, caused migration of cd3+ cells to the intraepithelial region, and induced high levels of inflammatory cytokines il‐1β and tnf‐α. (b) ng‐induced supernatants (ngis) increased hiv‐1 transcription, induced hiv‐1 from latently infected cells, and increased transmission of hiv‐1 across cervical mucosa. (c) transcriptome analysis of the epithelial layer of the tissues exposed to ng, and hiv‐1 showed significant upregulation of cxcl10 and il8. il‐1β increased the induction of cxcl10 and il‐8 expression in cervical mucosa with a concomitant increase in hiv‐1 transmission. conclusion: we present a model in which il‐1β produced from cervical epithelium during ng exposure increases cxcl10 and il8 in epithelia. this in turn causes upon hiv‐1 infection, the migration of hiv‐1 target cells toward the subepithelium, resulting in increased hiv‐1 transcription in the sub‐mucosa and subsequent enhancement of transmission across cervical mucosa. heterosexual transmission is the most common route of hiv-1 infection in women. 1, 2 a key co-factor in the transmission of hiv-1 in women is the prior existence of bacterial, viral, and parasitic microbes in the cervix that can alter the cervical environment and thereby influence hiv-1 transmission. [3] [4] [5] [6] [7] [8] gonorrhoeae caused by neisseria gonorrhoeae (ng), a gram-negative diplococci, is one of the most severe and common forms of sti 9,10 that has been shown to increase hiv-1 acquisition. [10] [11] [12] the presence of pro-inflammatory cytokines in the vaginal fluid of nginfected women and some cell line-based studies with ng led to the speculation that ng-induced inflammatory cytokines either directly or indirectly could increase hiv-1 transmission. [12] [13] [14] [15] [16] additional mechanisms of ng-induced enhanced hiv-1 transmission that have been suggested include recruitment of increased number of endocervical cd4+ t cells in ng-infected women providing more targets for hiv-1, 17 activation of cd4+ t cells by ng, 18 epithelial tight junction disruption, 19 and increased hiv-1 transcription by ng-secreted proteins. 20 the molecular mechanism by which ng enhances hiv-1/transmission in the female genital tract is still uncertain. part of the uncertainty is due to lack of a suitable ex vivo model that mimics in vivo situation. hiv-1/ng interaction has been mostly studied in in vitro cell culture using cd4+ t cells, endometrial epithelial cells, 15, 18, 21, 22 and immortalized cell lines. 15, 23 however, these cell systems do not accurately reflect situation that occur in human cervix/vaginal tissue. in addition, we do not know the mechanism of hiv-1 transmission through the epithelia of the cervical mucosa, especially when epithelia do not express cd4 and ccr5/ cxcr4. [24] [25] [26] regardless of how hiv-1 crosses the epithelium, hiv-1 exposure to the epithelial layer or epithelial cells has been shown to induce production of cytokines and chemokines which serve as signaling molecules. 27, 28 these signaling molecules may play an important role in hiv-1 transmission by attracting target immune cells to fuel hiv-1 infection in sub-mucosa and hence transmission. 29, 30 here we describe use of a primary cervical tissue-based organ culture model of ng infection that provides the natural cervical tissue architecture observed in cervix of ng-infected women. using this organ culture, we showed that ng exposure to cervical tissues induced epithelial membrane ruffling and inflammatory cytokine response, reminiscent of in vivo situation. furthermore, using this model we have shown that ng induces il-1β from cervical epithelium post-exposure and increases the production of epithelial proteins cxcl10 and il8, two key proteins that may be responsible for hiv-1 transmission, suggesting that increase in cxcl10 and il-8 production in epithelia may be responsible for ng-induced enhanced hiv-1 transmission across cervical mucosa. this study for the first time describes a molecular mechanism of ng-induced enhancement of hiv-1 transmission across cervical mucosa. the study protocol for the procurement of the cervical tissues from patients undergoing hysterectomy was approved by the institutional review board (irb) at the university of pittsburgh. a highly opaque (opa + ) neisseria gonorrhoeae (ng) phenotype with pil±, a clinical isolate from the clinical laboratory at the alleghany county hospital (gift from dr timothy meitzner, university of pittsburgh), was used for all the experiments. this ng strain was routinely grown in 5% co 2 at 37°c on gonococcal medium base (gcb; difco) or in chocolate agar plates (remel) for 18-24 hours. 31 this was then selected for opa-positive colonies by choosing the opaque phenotype when the cultured colonies were observed with oblique light under a dissecting microscope. 32 the working cultures of each bacteria were generated with two to three colonies from each culture types from the plate, suspended in 10% rpmi with the absorbance adjusted to a concentration of 1 × 10 7 cfu/ml for each experiments. before using these resuspended colonies for experiment, they were washed and centrifuged at 800 g for 5 minutes to remove cytokines. hiv-1 bal strain (cat #510 from nih aids reagent program) was used in all experiments. they were grown in phytohaemagglutinin (pha)-stimulated cd8-depleted peripheral blood mononuclear cells as described previously. 33 the virus-containing cell supernatant was filtered using an amicon ultra-15 filter device (millipore, billerica, us) to remove the soluble cytokines. the residual levels of cytokines were tested using msd and were found to be below 10 pg/ml. the control culture supernatant was prepared from uninfected cells in similar fashion. primary cd8-depleted pbmc were prepared by immune-magnetic depletion of cd8+ t cells from peripheral blood mononuclear cells (pbmc). 34 ecto-cervical tissues were collected and processed within 2 hours of surgery as described before. 36 the ecto-cervical punch biopsies (6 mm diameter) were placed into a 12-well transwell (becton dickson, nj, usa) with the epithelial layer facing up and its edges were sealed with 3% agarose at room temperature. ng at concentrations of 1 × 10 7 bacteia/ml or cell-free hiv-1 bal (tcid50 of 10 6 ) was added on the epithelial layer of the tissue in upper chamber depending on the experiments. 36 total cellular rna from cells or homogenized tissues was isolated by rnazol b (tel-test, inc) using the manufacturers protocol, and gapdh was used as a housekeeping gene. the level of pro-inflammatory cytokines, il-1β, il-6, il-8, and tnf-α messenger rna (mrna), was measured in the ng or control media exposed tissues/ cells using rt-pcr with gene-specific primers/probes by real-time rt-pcr using primers and probes as described as described previously. 38 the tissues post-exposure to ng and hiv-1 bal were washed and frozen at −80°c after being embedded in oct (thermo fisher. usa) and then cryo-sectioned (15-30 μm thickness). these cryo-sectioned tissues were then subjected to micro-dissection under microscope. epithelial layer was carefully removed, and rna was extracted from the epithelium with rna-bee™ (tel-test, inc, friendswood, tx). rna was then either used for whole genome transcriptional profile analysis or rt-pcr to confirm the significantly dysregulated genes obtained from the ion torrent analysis. to examine the morphology of the mucosal epithelia, hematoxylin and eosin (h&e) staining of the ecto-cervical tissues was performed. the tissues were washed and embedded in oct and cryo-sectioned into 7 μm thick layers each. they were then stained with hematoxylin and eosin and evaluated as described previously. 39 the thickness of epithelial layers was measured in three representative areas of mucosa from the basement membrane up to the surface using the metamorph software. the images were taken using a evos ® xl core digital imaging system under 20× or 40× objective lenses. to study the effect of ng on the migration of cells toward the epithelial surface, anti-cd3 antibody and control antibody (diluted 1:100) were added to the ng-exposed tissue as well as the control tissues and incubated for 1 hour at room temperature in a moist chamber. these were washed twice for 3 minutes in pbs and hrp polymer conjugate from the superpicture kit for ihc detection (invitrogen #87-9263 was added to the tissue and incubated again for 10 minutes at room temperature in the moist chamber). they were washed twice again for 3 minutes each, and dab chromagen, from the superpicture kit for ihc detection, was added to the tissue and incubated for 10 minutes at room temperature in the moist chamber. then, the slides were washed for 5 minutes in pbs. images were taken with the nikon eclipse e600 microscope using a 20× or 40× oil objective. 40 the ng-exposed tissue as well as control-exposed tissue was calculated. the number of positive cells in the ng-exposed and control-exposed subepithelium regions on the slides was compared and quantitated for migration. 39 human ecto-cervical tissues were exposed to ng for 24 hours and fixed in 2.5% glutaraldehyde for 1 hour at room temperature. the barcoding kits. quantitation of cdna libraries was performed using the ion library quantitation kit (life technologies) to evaluate appropriate template dilution factor for subsequent emulsion pcr and sequencing. this was followed by next-generation sequencing using the ion torrent platform according to manufacturer's protocols ( life technologies, carlsbad, ca). raw sequencing reads were in fastq format. clc genomics bench 7 was used to assess the quality of raw sequencing reads. reads were accepted based on the length (longer than 25 nucleotides) and phred quality score higher than 20. then, the trimmed reads were mapped to homo sapiens (hg19) mrna sequences. to make sure there were sufficient counts for each gene, only genes with mean read counts higher than 10 were retained in the analysis. bioconductor edger in r package was employed to perform the differential expression analysis. compared with control group, the genes with f i g u r e 1 ecto-cervical tissue-based organ culture model to study physical responses and induction of inflammatory cytokines from the cervical tissues in presence of ng. a, transwell with ectocervix tissue surrounded with agarose, ng was added to the apical surface of the tissue, with media at the top and bottom well, and incubated for 24 or 48 h. b, h&e staining on ecto-cervical tissues exposed to ng or control media in the organ culture for 24 h. images were obtained by bright field microscopy (e: epithelium; l: lumen of ecto-cervix; sm: sub-mucosa of the ecto-cervix). magnification for viewing these ecto-cervical tissue sections was 20×. each donor had 2-3 control-exposed and 2-3 ng-exposed biopsies, with 5-10 random images obtained from each biopsy. c, membrane ruffling in the presence of ng characterized by microvilli projection observed in cervical biopsy post-24 h exposure to ng under sem, with a magnification of 20 000× f i g u r e 2 cellular responses induced by ng in cervical tissues and cervical tissue-derived cell lines. cervical tissue biopsies exposed to ng showed elevation in cytokine levels. inflammatory cytokines (a) mrna and (b) protein at 24 and 48 h compared to media control showed high fold changes in il-1β (5-to 10-fold in mrna and 100-to 200-fold in protein) and tnf-α (2-to 5-fold in mrna and 5-to 10-fold in protein). there was a significant increase in the level of il-1β and tnf-α protein in supernatant at both time points though we did not observe a significant increase in tnf-α mrna at 24 h. the (c) mrna profile and (d) secreted cytokine protein profile of the e6/e7 cells upon exposure to ng showed a similar increase in cytokine responses as in the tissues with an significant increase of 3-fold and 5-fold (il-1β) and 5-fold and 10-fold (tnf-α) of cytokines at the mrna (c) and protein levels, respectively. ecto-cervical tissue biopsies exposed to either (e) live ng or heat-killed ng showed no difference in cytokine response. bars represent mean ± sd of three independent experiments with different donors. each donor had 2-3 control-exposed and 2-3 ng-exposed biopsies for each experimental set. experiments with e6/ e7 cells were carried out in triplicates. p ≤ 0.05 was considered to be statistically significant compared to the control tissues for these fold changes analyzed by one sample students t test benjamini-hochberg adjusted false discovery rate (fdr) <0.05 and absolute fold change >2 were considered as significant differential expression. in most of the cases, if not otherwise mentioned, the data were presented as mean ± sd and were plotted using the prism software student's edition. all the analyses were also done using the same software. for analyzing mrna expression levels, parametric single sample t test was used to determine the significance (p ≤ 0.05) for the fold change observed in ng-exposed groups relative to controls. this was used because of the relatively small sample size. to determine significance (p ≤ 0.05) in cell numbers of cd3+ cells between ng-exposed group and controls, a non-parametric paired wilcoxon signed-rank test was used and the data were represented as mean ± se after quantification. a polarized cervical tissue-based organ culture was set up in a 12well transwell system with the epithelial layer of the cervical tissue orientated up as described in methods ( figure 1a ). ng at a concentration of 1 × 10 7 cells/ml in 300 μl was added onto the top of the tissue exposing epithelial layer. following 24 hours of incubation, culture supernatant from the bottom of the wells and the exposed tissues were saved for measurement of soluble cytokines and intracellular cytokines, respectively. we found that the tissues exposed to ng 24 hours were 97% viable compared to tissues exposed to media control as determined by mtt assay 44 as well as microscopic examination of h&e-stained tissue sections under the light microscope ( figure 1b ). scanning electron microscopic (sem) analysis of the epithelial layer of tissues exposed to ng showed signs of epithelial membrane ruffling which is characteristic of the ng infection process in the female cervix ( figure 1c ) and is usually noted in biopsy from patients with cervicitis. we observed no overgrowth of the bacteria on the tissues post-24 hours. twenty percent of the inoculated ng was found to be adherent to the tissues after 24 hours since pro-inflammatory responses to ng infection in cervix are often observed in ng-infected women, we evaluated such responses to ng infection in the cervical tissues in our organ culture. exposure of cervical tissues to ng (3 × 10 6 ) induced high levels of il6, il8, il-1β, and tnf-α at both 24 and 48 hours after ng inoculation, with il-1β and tnf-α being the highest compared to control tissues exposed to media (2500 pg/ml for il-1β and 500 pg/ml for tnf-α in ng exposed compared to 10 pg/ml and 17 pg/ml in control media, respectively). this increase in the cytokine production was noted both at the mrna level (figure 2a the p-value was calculated as significant using unpaired t test of equal variance because of unequal number of biopsy per condition. g, increased localization of cd3+ cells observed in the subepithelium in ng-exposed tissues over cm-exposed tissues. figure is a representative image at 200× magnification. h, quantitation of immuno-stained cd3+ cells showed a statistically significant increase in cd3+ t cells on ng-exposed tissue compared to cm-exposed tissues. p ≤ 0.05 was considered as significant in all cases. for the cd3+ tissue stain, experiments were carried out in 2-3 biopsies from tissues of three donors and non-parametric paired wilcoxon signed-ranked test was used in the analysis of this data (data not shown). to determine whether live ng was required for induction of cytokine response, cervical tissues in the same organ culture were exposed to heat-killed (65°c for 30 minutes) ng for 24 and 48 hours no significant difference in the cytokine levels between the heat-killed and live ng was observed ( figure 2e ), indicating that live ng was not essential for the inflammatory cytokine response. these results implied that outer membrane structures of ng might be sufficient for inducing inflammatory responses. since the epithelial layer exposed to ng seemed to induce proinflammatory cytokines, we sought to determine whether exposure of ng to epithelium per se was sufficient for the induction of these pro-inflammatory responses. for this purpose, the ecto-cervixderived epithelial cell line e6/e7 was evaluated for their ability to induce inflammatory responses upon exposure to ng. like cervical tissues, these epithelial cells, upon exposure to ng, induced a very similar profile of cytokines as observed in the organ culture setup with an increase in the expression of intracellular il-1β, tnf-α, il8, and il6 cytokine mrnas ( figure 2c ) and secreted il-1β and tnf-α cytokine proteins (100-250 pg/ml of il-1β in ng exposed compared to 20-50 pg/ml in control and 100-500 pg/ml of tnf-α in ng exposed compared to 10-50 pg/ml in control). this also demonstrated an average change of 3-fold and 5-fold (il-1β) and 5-fold and 10-fold (tnf-α) of cytokines at the mrna( figure 2c ) and protein levels, respectively ( figure 2d) , as compared to media controls, which was also in line with earlier studies. 46 there was a significant increase in il-1β and tnf-α (24 and 48 hours) both at the mrna and protein level (p ≤ 0.05). f i g u r e 4 transcriptome analysis of cervical epithelium exposed to hiv-1 and ng identified upregulated cellular genes. heat map showing transcriptome analysis of epithelial layer of the cervix using next-generation sequencing of tissue epithelium exposed to (a) hiv-1 (n = 6) and (b) ng (n = 6). ng depicts neisseria numbered 1-6, and hiv-1 is also numbered 1-6. c depicts tissues exposed to control supernatant and numbered as 1-6. figure shows only the six tissues which were subjected to transcriptome analysis and genes which were found to be upregulated. venn diagram showing (c) common genes cxcl10 and il8 expressed by ng and hiv-1 exposure on the cervical epithelium. the expression of the genes in (a) ng-exposed and (b) hiv-1-exposed ecto-cervical epithelia was at least 2-fold difference with false discovery rate (fdr) <0.05 compared to the controls epidemiological studies suggest that it is not the ng microbe per se, but ng-induced cervical milieu may be responsible for increased hiv-1 transmission in women. 18, 47 therefore, we investigated whether culture supernatants from cervical tissues exposed to ng (referred to as ng-induced supernatants, abbreviated as ngis) and reminiscent of ng-induced cervical milieu had any effect on the hiv-1 transcription in a tzm-bl cell-based hiv-1 ltr-driven reporter gene assay. a dose-dependent stimulation of hiv-1 ltr activity was observed with a serial 10-fold dilution of ngis, but not with control conditioned media ( figure 3a ). in contrast, ng bacteria alone did not show any significant activation of hiv-1 ltr in tzm-bl cells ( figure 3b ). tion of replication competent hiv-1 in latently infected ach2 (t cell derived) and u1 (monocytic) cells. 48 ngis activated high levels of hiv-1 from both ach2 ( figure 3c ) and u1 cells ( figure 3d ), compared to cells exposed to control conditioned media. we next examined whether enhancement of hiv-1 replication by ngis in cells can be translated to enhanced hiv-1 transmission across cervical mucosa. for this purpose, we used our standard organ culture for measuring hiv-1 transmission as described previously. 36, 38 undiluted ngis was added onto the top of the epithelial layer of the cervical tissue and pre-incubated for 24 hours, after which hiv-1 bal (tcid 50 of 1 × 10 6 ) was added onto the tissue. the level of hiv-1 p24 in the bottom well was used to monitor hiv-1 transmission across cervical mucosa. results indicate a 55% increase (1.65-fold) in the hiv-1 transmission across the cervical tissues with ngis collected 24 hour after ng exposure and a 350% increase (3.5-fold) in hiv-1 transmission with ngis collected 7 days after exposure to ng with a significant p value of 0.029 and 0.0061, respectively, using an unpaired t test with equal variance. ( figure 3e ) there was also a concomitant increase in intracellular hiv-1 gag mrna in cervical tissues (2-fold with 24-hour ngis and 4-fold with 7-day ngis) compared to control media ( figure 3f ). there have been reports that ng exposure recruits cd4+ t cell, 17, 49 which could increase targets for hiv-1 infection, to the cervices of higher numbers of cd3+ t cells were localized in the intraepithelial layer in the tissue exposed to ng, compared to tissues exposed to control media. during this period, we could not detect macrophages or dendritic cells in this region of the tissue (data not shown). this could be due to relatively short ng exposure (24 hours) the upregulated genes found in the transcriptome analysis was verified using rt-pcr on additional tissues (n = 10). numbers indicate fold changes of degs upon ng exposure on cervical tissues compared to tissues exposed to media control. a all the genes confirmed to be upregulated were statistically significant. a non-parametric paired t test was used for calculating the statistical significance. we have shown above that ng exposure to cervical epithelia induces upregulation of inflammatory cytokine mrnas and secreted cytokine proteins. to determine how ng-induced cytokines may be involved in increased hiv-1 transmission across cervical epithelia, the epithelia exposed to ng and hiv-1 were examined for expression of cellular genes that may be upregulated during exposure to ng and hiv-1. for this purpose, transcriptome analysis using nextgeneration sequencing was performed on cellular rna isolated from micro-dissected epithelial layer of the six tissues exposed to hiv-1 and ng separately. the genes which were found to be differentially regulated with high statistical significance after analysis are shown on the right of the heat map with red showing lower expression and green showing higher expression ( figure 4a,b) . such analysis identified with high statistical significance (p ≤ 0.05) 33 differentially expressed (−3 to 8-fold) genes in ng exposed and 7 differentially expressed (2 to 7-fold) genes in hiv-1 exposed tissues, compared to part of the same tissues exposed to control medium (tables s1 and s2 ). venn diagram analysis of these differentially expressed genes in hiv-1-and ng-exposed epithelia indicated that only two genes cxcl10 and il8 were found to be common upregulated genes between tissues exposed to hiv-1 and ng groups ( figure 4c ). to confirm the rna-seq analysis of ng-exposed tissues, we examined expression of seven genes (cxcl10, cxcl3, cxcl20, tnfa1p6, il8, il6, and il-1β) by rt-pcr in 10 tissues exposed to ng or control supernatant. results shown in table 1 indicate upregulation of these seven genes by more than 5-fold with high significance (p ≤ 0.05) compared to tissues exposed to control supernatant. similarly, rt-pcr analysis of the seven differentially ta b l e 2 fold changes of degs (differentially expressed genes) upregulated upon hiv-1 exposure on cervical tissues compared to tissues exposed to media control the upregulated genes found in the transcriptome analysis was verified using rt-pcr on additional tissues (n = 14). numbers denote fold changes of degs in tissues exposed to hiv-1 compared to tissues exposed to media control. a six genes out of the seven genes were found to be statistically upregulated by rt-pcr. a non-parametric paired t test was used for calculating the statistical significance. (2500 pg/ml) on the cervical tissues increased the cxcl10 and il8 in the tissues at the mrna level. f, il-1β as well as a combination of cxcl10 and il8 increased the hiv-1 transcription in the tissues compared to media exposed control. g, monoclonal antibodies against il-1β (10 μg/ml) in the ngis along with hiv-1 decreased transmission compared to ngis and hiv-1 alone. p ≤ 0.05 was considered as significant, and either parametric t tests or t test unequal variance was used to calculate the p values expressed genes from rna sequence analysis from a total of 14 hiv-1-exposed tissues also showed that six (cxcl10, muc1, il36a, fmo2, il8, and wars) out of seven genes were found to be upregulated (3-to 44-fold) with high statistical significance(p ≤ 0.05) in all tissues compared to tissue exposed to media control (table 2) . a comparison of the rt-pcr data of the transcripts between the ng-and hiv-1-exposed tissues also confirmed that cxcl10 and il-8 were the only two genes common between these two groups that were significantly elevated (11.44 and 4.24 in hiv-1-exposed tissues, respectively, and 9-fold and 29-fold in ng-exposed tissues, respectively). elevated levels of cxcl10 and il8 proteins were also detected in ngis collected at both at 24 hours and 7 days post-exposure of ng ( figure s1b ). to determine the role of cxcl10 and il8 directly in hiv-1 transmission across epithelia, tissues were incubated for 24 hours with il8 and cxcl10 (200 ng/ml and 1000 pg/ml, respectively, at concentrations present in the ngis) and then exposed to hiv-1. figure 5a showed a significant enhancement despite the worldwide problem of increased hiv-1 transmission driven by ng, the molecular mechanism associated with ng-induced models have a few disadvantages to using a full animal model. the cellular circulation system here is limited; therefore, it does not help study the mechanism of migration of cells to different areas of the body. in spite of this, the system is well equipped to study the basic immediate impact of hiv-1 infection and its transmission across the mucosa along with the migration of cells from the basal to the apical end of the tissue explants. 33 another limitation of this study is the small sample size. this is primarily because of the low availability of cervical tissues to work with, and thus, we could not use more than 3 tissues for any experiment. there is also a difference in age, race, and the menstrual cycle among different tissues obtained which may affect the susceptibility to infection. this may be the reason why some tissues transmitted more easily and produced more cytokine response than the others when exposed to hiv-1 or ng. like in our case, one tissue exposed to hiv-1 during the cytokine upregulation study showed very high cytokine response compared to others. this was a more susceptible tissue. the higher sample size (n = 14) in this study ( table 2 ) helped in determining the significance of the study. the authors declare no conflict of interest. this article is a direct contribution from a member of the american academy of microbiology. phalguni gupta https://orcid.org/0000-0003-2427-3700 hiv-1 transmission and acute hiv-1 infection women and hiv in sub-saharan africa vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition the role of bacterial vaginosis and trichomonas in hiv transmission across the female genital tract microbiome in human immunodeficiency virus infection acute sexually transmitted infections increase human immunodeficiency virus type 1 plasma viremia, increase plasma type 2 cytokines, and decrease cd4 cell counts cofactors in malefemale sexual transmission of human immunodeficiency virus type 1 the role of sexually transmitted diseases in hiv transmission neisseria gonorrhoeae and chlamydia trachomatis among human immunodeficiency virus-infected women symptomatic vaginal discharge is a poor predictor of sexually transmitted infections and genital tract inflammation in high-risk women in south africa modulation of hiv transmission by neisseria gonorrhoeae: molecular and immunological aspects the molecular mechanisms used by neisseria gonorrhoeae to initiate infection differ between men and women distinct proinflammatory host responses to neisseria gonorrhoeae infection in immortalized human cervical and vaginal epithelial cells cytokine and antibody responses in women infected with neisseria gonorrhoeae: effects of concomitant infections differential response of primary and immortalized cd4+ t cells to neisseria gonorrhoeae-induced cytokines determines the effect on hiv-1 replication pathogen recognition in the human female reproductive tract: expression of intracellular cytosolic sensors nod1, nod2, rig-1, and mda5 and response to hiv-1 and neisseria gonorrhea increase in endocervical cd4 lymphocytes among women with nonulcerative sexually transmitted diseases neisseria gonorrhoeae enhances hiv-1 infection of primary resting cd4+ t cells through tlr2 activation neisseria gonorrhoeae induced disruption of cell junction complexes in epithelial cells of the human genital tract neisseria gonorrhoeae-derived heptose elicits an innate immune response and drives hiv-1 expression neisseria gonorrhoeae enhances infection of dendritic cells by hiv type 1 t lymphocyte response to neisseria gonorrhoeae porin in individuals with mucosal gonococcal infections induction of hiv-1 long terminal repeat-mediated transcription by neisseria gonorrhoeae inhibiting sexual transmission of hiv-1 infection target cells in vaginal hiv transmission hiv infection of the genital mucosa in women selective up-regulation of cytokine-induced rantes gene expression in lung epithelial cells by overexpression of ikappabr il-1beta, il-6, tnfalpha, fetal fibronectin, and endotoxin in the lower genital tract of pregnant women with bacterial vaginosis targeting early infection to prevent hiv-1 mucosal transmission glycerol monolaurate prevents mucosal siv transmission the laboratory diagnosis of neisseria gonorrhoeae construction and characterization of a derivative of neisseria gonorrhoeae strain ms11 devoid of all opa genes memory cd4(+) t cells are the earliest detectable human immunodeficiency virus type 1 (hiv-1)-infected cells in the female genital mucosal tissue during hiv-1 transmission in an organ culture system a semiquantitative assay for cd8+ tcell-mediated suppression of human immunodeficiency virus type 1 infection dependence of cd8+ t-cell-mediated suppression of hiv type 1 on viral phenotypes and mediation of phenotype-dependent suppression by viral envelope gene and not by beta-chemokines development of an in vitro organ culture model to study transmission of hiv-1 in the female genital tract study of hiv-1 transmission across cervical mucosa to tonsil tissue cells using an organ culture retrocyclin rc-101 blocks hiv-1 transmission across cervical mucosa in an organ culture improved detection of simian immunodeficiency virus rna by in situ hybridization in fixed tissue sections: combined effects of temperatures for tissue fixation and probe hybridization simian immunodeficiency virus infection alters chemokine networks in lung tissues of cynomolgus macaques: association with pneumocystis carinii infection simian immunodeficiency virus infection potently modulates chemokine networks and immune environments in hilar lymph nodes of cynomolgus macaques isolation, characterization, and functional analysis of ferret lymphatic endothelial cells surface pattern differentiation of the epithelial cells of the human uterine ectocervix parameters of human immunodeficiency virus infection of human cervical tissue and inhibition by vaginal virucides inflammatory cytokines produced in response to experimental human gonorrhea response to neisseria gonorrhoeae by cervicovaginal epithelial cells occurs in the absence of toll-like receptor 4-mediated signaling tnf-alpha and tlr agonists increase susceptibility to hiv-1 transmission by human langerhans cells ex vivo analysis of tat function in human immunodeficiency virus type 1-infected low-level-expression cell lines u1 and ach-2 type iv pili of neisseria gonorrhoeae influence the activation of human cd4+ t cells hiv exposure to the epithelia in ectocervical and colon tissues induces inflammatory cytokines without tight junction disruption cxcl10 and trafficking of virus-specific t cells during coronavirus-induced demyelination pivotal role of dendritic cellderived cxcl10 in the retention of t helper cell 1 lymphocytes in secondary lymph nodes dual role of macrophages in tumor growth and angiogenesis fourteen-member macrolides inhibit interleukin-8 release by human eosinophils from atopic donors catechins inhibit cxcl10 production from oncostatin m-stimulated human gingival fibroblasts black tea polyphenol inhibits cxcl10 production in oncostatin m-stimulated human gingival fibroblasts nf-kappab-dependent synergistic regulation of cxcl10 gene expression by il-1beta and ifngamma in human intestinal epithelial cell lines influence of nfkappab inhibitors on il-1beta-induced chemokine cxcl8 and -10 expression levels in intestinal epithelial cell lines: glucocorticoid ineffectiveness and paradoxical effect of pdtc chalcone derivatives: anti-inflammatory potential and molecular targets perspectives human immunodeficiency virus receptor and coreceptor expression on human uterine epithelial cells: regulation of expression during the menstrual cycle and implications for human immunodeficiency virus infection setting the stage: host invasion by hiv neisseria gonorrhoeae co-infection exacerbates vaginal hiv shedding without affecting systemic viral loads in human cd34+ engrafted mice key: cord-338654-ma9ayu80 authors: eaton, lisa a.; kalichman, seth c. title: social and behavioral health responses to covid-19: lessons learned from four decades of an hiv pandemic date: 2020-04-25 journal: j behav med doi: 10.1007/s10865-020-00157-y sha: doc_id: 338654 cord_uid: ma9ayu80 our public health approaches to addressing covid-19 are heavily dependent on social and behavioral change strategies to halt transmissions. to date, biomedical forms of curative and preventative treatments for covid-19 are at best limited. four decades into the hiv epidemic we have learned a considerable amount of information regarding social and behavioral approaches to addressing disease transmission. here we outline broad, scoping lessons learned from the hiv literature tailored to the nature of what we currently know about covid-19. we focus on multiple levels of intervention including intrapersonal, interpersonal, community, and social factors, each of which provide a reference point for understanding and elaborating on social/behavioral lessons learned from hiv prevention and treatment research. the investments in hiv prevention and treatment research far outweigh any infectious disease in the history of public health, that is, until now with the emergence of covid-19. which public health response must rely on behavioral and social factors for disease prevention varies greatly depending on disease transmission factors (e.g., mode of disease transmission, rapid travel and trade patterns, economic and health care infrastructure stability). unfortunately, covid-19, has presented itself as a formidable agent posing multiple unique challenges. as a novel virus with no residual immunity from prior exposure, herd immunity can't be relied upon to halt social networks of disease transmission. covid-19 is highly contagious, with multiple transmissions occurring per one contagious person (reproduction rate) during the early stage of outbreak-a relatively elevated rate compared with many other infectious diseases . infections initially present with symptoms that are mostly indistinguishable from other viral diseases, stalling early detection. furthermore, with no curative option, treatment is focused on symptom management. the current state of covid-19 disease transmission has left our public health approaches to be heavily dependent on social and behavioral change strategies to halt transmissions. although different from the social and behavioral dynamics of transmission and pathogenesis, hiv infection offers lessons learned for those who are embarking on this area of research for preventing the spread of covid-19. almost 40 years after the first cases of aids were identified in the us, and with still no effective vaccine, we have learned a considerable amount about the reliance on social and behavioral approaches to slowing infectious disease. the investments in hiv prevention and treatment research far outweigh any infectious disease in the history of public health, that is, until now with the emergence of covid-19. here we outline broad, scoping lessons learned from the hiv epidemic tailored to the nature of what we currently know about covid-19. as a unifying framework, we posit that the social ecological model of health (hanson et al., abstract our public health approaches to addressing covid-19 are heavily dependent on social and behavioral change strategies to halt transmissions. to date, biomedical forms of curative and preventative treatments for covid-19 are at best limited. four decades into the hiv epidemic we have learned a considerable amount of information regarding social and behavioral approaches to addressing disease transmission. here we outline broad, scoping lessons learned from the hiv literature tailored to the nature of what we currently know about covid-19. we focus on multiple levels of intervention including intrapersonal, interpersonal, community, and social factors, each of which provide a reference point for understanding and elaborating on social/behavioral lessons learned from hiv prevention and treatment research. the investments in hiv prevention and treatment research far outweigh any infectious disease in the history of public health, that is, until now with the emergence of covid-19. with our existing healthcare infrastructure we are currently unable to clinically manage the onslaught of coronavirus disease 2019 . existing vaccine development platforms have rendered an 18-month optimistic timeframe for broad-reaching biomedical approaches to prevention (coreil, 2013) . avenues for prevention and treatment outside of an effective vaccine will, therefore, invariably rely on changing social/behavioral patterns (e.g., physical distancing and selfisolation) for containing the spread of disease. the extent to 2005) may prove useful as it attends to levels of intervention that may serve to frame responses to covid-19. the model has multiple foci, including intrapersonal, interpersonal, community, and social factors, each of which provide a reference point for understanding and elaborating on social/behavioral lessons learned from hiv prevention and treatment research. sustained, individual-level, behavioral change is challenging to achieve. interventions targeting individual-level behavior change have remained a cornerstone of hiv prevention (herbst et al., 2005; crepaz et al., 2007; johnson et al., 2006) . there exist critical threads of similarity in our current covid-19 prevention strategies to efforts aimed at stemming hiv transmission. interventions to alter the behavioral patterns of individuals can have important and substantive impact on risk reduction and disease outcomes, but are likely not sufficient for disease eradication. further, it has long been known in hiv prevention and treatment research that intervention effects to alter behavioral patterns can vary greatly across populations and that sub-populations in greatest need of support for changing behavioral patterns are also most likely to experience barriers to accessing support (faugier & sargeant, 1997) . covid-19 prevention efforts are currently largely dependent on individual-level behavior change, including self-isolation and physical distancing, to an unprecedented scale. we know from hiv prevention and treatment research that information, motivation, and behavioral skills are key to initiating behavior change, but are unlikely sufficient for sustained change if only individual components are addressed (fisher et al., 2006) . changes in behavioral patterns are challenging to maintain overtime and, similar to many vaccines, may rely on booster sessions to support (eaton et al., 2013) . limitations to maintaining altered behavioral patterns are observed across a broad spectrum of health behavior change and rely on multi-level intervention approaches (see below) (summers & curtis, 2020; wiltink et al., 2007) . medical mistrust and conspiracy beliefs undermine datadriven public health interventions. uptake of effective hiv treatment advances has long been stymied by underlying mistrust of health care and conspiracy beliefs regarding the origin of hiv, the existence and characteristics of the virus itself, and the purpose and impact of antiretroviral medications (kalichman, 2009; bogart & bird, 2003; eaton et al., 2017) . mistrust of governmental initiatives to implement public health interventions are largely based on a history of abuse of vulnerable populations (e.g., tuskegee syphilis experiment) (bogart & thorburn, 2005) and racial/ethnic discrimination documented within health care infrastructure (obermeyer et al., 2019) . however, new social influences, primarily driven by more general anti-science and antigovernment forces, are impacting covid-19 containment efforts (dyer, 2020) . social media is used to distribute messages warning that covid-19 does not exist, that it is harmless, that is manmade, that a cure exists and is being withheld by the government, that it is being used to justify an imminent 'police state', etc. (butler, 2020) . these conspiracy-driven messages are all well-documented lines of thought in hiv social science research, and critically, result in harmful outcomes (nattrass, 2012; kalichman, 2017) . we have observed in hiv research that conspiracy beliefs tend to be driven by a rejection of authority (i.e., as a political ideology) or as a psychological state of paranoia (kalichman, 2009 ). most critically, we know that poor political leadership results in highly fertile social foundations for developing conspiracy theories towards public health initiatives (nattrass, 2012) . there is likely no better example of the destructive power of conspiracy thinking on public health than the history of the hiv epidemic in south africa during the presidency of thabo mbeki. it is estimated that 330,000 individuals died due to lack of hiv treatment and 35,000 infants were born with hiv as a result of conspiracy driven public health programing (chigwedere et al., 2008) . the need to combat anti-science rhetoric and conspiracy thinking in the era of covid-19 has been apparent since its first days in right-wing media and anti-vaccine activists' propaganda (depoux et al., 2020; garfin et al., 2020; li et al., 2020; llewellyn, 2020) , and failure to squelch the seeds of mistrust will be paid in the cost of human life. social stigma of hiv is grounded in racism, homophobia, and sexism, and the stigmatization of covid-19 has already occurred. social stigma is complex and omnipresent, and acts as a driver of social hierarchy. the extent to which social stigma has weakened efforts to slow hiv transmission is challenging to quantify, however, it is acknowledged as one of the most formidable social factors working against hiv prevention and treatment (brent, 2016) . stigma in hiv social science literature is viewed as a process where individuals living with hiv experience status loss as a result of their hiv positive status (goffman, 1963) . those who experience stigma are devalued, ostracized, and ignored (goffman, 1963; link & phelan, 2001) . in the case of covid-19, elements of social stigma and social hierarchy are currently unfolding. elderly and individuals with compromised immune systems have elevated morbidity and mortality related to covid-19 relative to other groups. political discussion is rife regarding the social tipping point of protecting populations viewed as most vulnerable (e.g., advocacy for the need to protect all human life (mckinley & goldmacher, 2020) vs advocacy for weighing the value of human life (rodriguez, 2020) ). these actions effectively create an in-group vs out-group mentality, or social hierarchy, and have detrimental impacts on public health measures (nyblade et al., 2009 ). the establishment of in-groups versus out-groups perpetuates the devaluing of out-group members, bolsters disproportionate power and influence of in-group members, and is used as a justification for the mistreatment and disregard of out-group members (link & phelan, 2001) . for example, the use of the phrase "chinese virus" (rogers et al., 2020) for covid-19 serves to establish social dominance, blame, and social delineation, all justifications for acts of discrimination, in this case, against people of asian heritage (person et al., 2004) . approaches to addressing stigma in the context of promoting well-being and disease prevention exist and likely provide relevant tools applicable to addressing covid-19. interventions to address stigma have been developed that target individuals, health care workers, communities, and social figures, which will likely find new purpose in covid-19 (andersson et al., 2020; rao et al., 2019; stangl et al., 2013) . research on the hiv pandemic has long established that hiv co-occurs with multiple intersecting epidemics, creating what singer termed a syndemic (singer & clair, 2003) . individuals experiencing mental health and substance abuse problems, food insecurity, housing instability, and overall social marginalization are the most vulnerable to hiv, and hiv infection perpetuates these co-occurring conditions (robinson et al., 2016; walters et al., 2020; turpin et al., 2020; . while the transmission of covid-19 is, of course, markedly different from hiv, there needs to be heightened concern for communities most afflicted by poverty, high population density, barriers to physical distancing, and limited access to health care and other resources-these factors will amplify vulnerability to covid-19. in turn, we should expect covid-19 to exacerbate mental health problems and substance abuse by cutting off social support, increasing stress, further reducing access to services, and impeding healthcare among individuals living in poverty and possessing limited personal agency and social capital (stein, 2020) . multi-level community interventions yield more robust and sustainable outcomes than single-level efforts to prevent hiv transmission. initial social/behavioral interventions to slow hiv transmission largely prioritized the changing of individual level behavior (johnson et al., 2009 ). the field evolved, however, to acknowledge that individual behavior is heavily influenced by the broader social and structural systems wherein behavior occurs (iom, 1995) , and the need to develop multi-level interventions capable of addressing multiple systems that influence behavior (e.g., health care providers, employment, personal safety, health care access) was prioritized (blankenship et al., 2006; des jarlais, 2000) . in the context of covid-19, the most pressing social/behavioral component to containment appears to be physical distancing and hygiene strategies. social influence models of behavior change have proven effective in changing and sustaining changes in behavior by shifting social norms and reinforcing risk reduction efforts (kelly et al., 1997 (kelly et al., , 2006 latkin et al., 2009) . while these models have relied on face-to-face interactions as the vehicle of change, they have been translated to online platforms that are immediately implementable for sustaining measures to contain and mitigate covid-19 (green et al., 2015; marhefka et al., 2012 marhefka et al., , 2013 marhefka et al., , 2014 young et al., 2014; rice et al., 2012; chiu & young, 2015) . examples of multi-level interventions to impact these practices include messaging to promote social distancing and good hygiene behaviors (individuallevel behavior change, delivered at the community-level), the closing of schools and non-essential business services (structural-level), and the increased availability of sanitizing products (e.g., hand sanitizer, disinfectant wipes) in public spaces (structural-and social-level). multi-level interventions pose multiple assessment and implementation related challenges, but have the potential for high efficacy in altering the course of disease (auerbach, 2009 ). community mobilization for disease prevention. engaging multiple societal sectors to address health, social, or environmental issues is the cornerstone of community or social mobilization (unaids, 1997). as outlined by lippman et al. (2013) critical components of community mobilization include: a defined or shared concern, critical consciousness regarding the concern, organizational structure with links to groups/networks, individual or institutional leadership, collective/shared activities and actions, and social cohesion. across the history of hiv in the us there exist multiple instances of community mobilization efforts beneficially impacting the lives of people affected by hiv. similar to what we are currently observing with covid-19, community mobilization efforts to address hiv have typically focused on changing federal level responses. particularly in the earliest days of the hiv epidemic in the us, community mobilization efforts were ultimately highly effective at challenging and changing the response to hiv at the federal level (unaids, 1997) . but efforts were sustained and hard fought. we are observing similar patterns with covid-19 where communities most affected (e.g. high population density cities and communities initially impacted) are appealing to the broader community for a national level response. hiv emerged as a global threat to public health more than 40 years prior to covid-19, and while the lack of public health preparedness for the current pandemic is well recognized (carinci, 2020) , social and behavioral scientists aiming to contribute to the containment and mitigation of covid-19 will be well-served by the lessons we have learned in hiv prevention and treatment research. preventing the worst care scenarios of covid-19 morbidity and mortality can be achieved through intrapersonal, interpersonal, community, and societal levels of data-driven and well-coordinated interventions. hiv prevention and treatment sciences has not always achieved these goals, but the lessons we have learned should be evaluated for use in the case of covid-19 and for future pandemics. stigma reduction interventions in people living with hiv to improve health-related quality of life transforming social structures and environments to help in hiv prevention structural interventions: concepts, challenges and opportunities for research exploring the relationship of conspiracy beliefs about hiv/aids to sexual behaviors and attitudes among african-american adults are hiv/aids conspiracy beliefs a barrier to hiv prevention among african americans? the value of reducing hiv stigma a fake pandemic": anti-vaxxers are spreading coronavirus conspiracy theories covid-19: preparedness, decentralisation, and the hunt for patient zero estimating the lost benefits of antiretroviral drug use in south africa the relationship between online social network use, sexual risk behaviors, and hiv sero-status among a sample of predominately african american and latino men who have sex with men (msm) social media users effect of community-based voluntary counselling and testing on hiv incidence and social and behavioural outcomes (nimh project accept; hptn 043): a cluster-randomised trial. the lancet global health social and behavioral foundations of public health the efficacy of behavioral interventions in reducing hiv risk sex behaviors and incident sexually transmitted disease in black and hispanic sexually transmitted disease clinic patients in the united states: a meta-analytic review the pandemic of social media panic travels faster than the covid-19 outbreak structural interventions to reduce hiv transmission among injecting drug users trump claims public health warnings on covid-19 are a conspiracy against him a reanalysis of a behavioral intervention to prevent incident hiv infections: including indirect effects in modeling outcomes of project explore stigma and conspiracy beliefs related to preexposure prophylaxis (prep) and interest in using prep among black and white men and transgender women who have sex with men sampling hard to reach populations an information-motivation-behavioral skills model of adherence to antiretroviral therapy the novel coronavirus (covid-2019) outbreak: amplification of public health consequences by media exposure stigma: notes on the management of spoiled identity advantages and disadvantages for receiving internet-based hiv/aids interventions at home or at community-based organizations the injury iceberg: an ecological approach to planning sustainable community safety interventions a meta-analytic review of hiv behavioral interventions for reducing sexual risk behavior of men who have sex with men institute of medicine. assessing the social and behavioral science base for hiv/aids prevention and intervention: workshop summary sexual risk reduction for persons living with hiv: research synthesis of randomized controlled trials behavioral interventions for african americans to reduce sexual risk of hiv: a meta-analysis of randomized controlled trials denying aids: conspiracy theories, pseudoscience, and human tragedy pence, putin, mbeki and their hiv/aidsrelated crimes against humanity: call for social justice and behavioral science advocacy prevention of hiv and sexually transmitted diseases in high risk social networks of young roma (gypsy) men in bulgaria: randomised controlled trial randomised, controlled, community-level hiv-prevention intervention for sexual-risk behaviour among homosexual men in us cities the efficacy of a network intervention to reduce hiv risk behaviors among drug users and risk partners in chiang mai retrospective analysis of the possibility of predicting the covid-19 outbreak from internet searches and social media data conceptualizing stigma conceptualizing community mobilization for hiv prevention: implications for hiv prevention programming in the african context covid-19: how to be careful with trust and expertise on social media effectiveness of healthy relationships video-group-a videoconferencing group intervention for women living with hiv: preliminary findings from a randomized controlled trial interest in, concerns about, and preferences for potential video-group delivery of an effective behavioral intervention among women living with hiv internetbased video-group delivery of healthy relationships-a "prevention with positives" intervention: report on a single group pilot test among women living with hiv how cuomo, once on sidelines, became the politician of the moment the aids conspiracy: science fights back. columbia combating hiv stigma in health care settings: what works dissecting racial bias in an algorithm used to manage the health of populations national center for inectious diseases scot: fear and stigma: the epidemic within the sars outbreak a systematic review of multi-level stigma interventions: state of the science and future directions mobilizing homeless youth for hiv prevention: a social network analysis of the acceptability of a face-to-face and online social networking intervention substance use, mental illness, and familial conflict non-negotiation among hiv-positive african-americans: latent class regression and a new syndemic framework texas' lieutenant governor suggests grandparents are willing to die for us economy trump defends using 'chinese virus' label, ignoring growing criticism. the new york times syndemics and public health: reconceptualizing disease in bio-social context a systematic review of interventions to reduce hivrelated stigma and discrimination from 2002 to 2013: how far have we come covid-19 and rationally layered social distancing novel digital architecture of a "low carb program" for initiating and maintaining long-term sustainable health-promoting behavior change in patients with type 2 diabetes the syndemic threat of food insecurity and hiv testing a syndemic index of psychosocial and structural factors associated with hiv testing among black men community mobilization and aids a syndemic model of exchange sex among hiv-positive men who have sex with men long-term weight loss maintenance after inpatient psychotherapy of severely obese patients based on a randomized study: predictors and maintaining factors of health behavior project hope: online social network changes in an hiv prevention randomized controlled trial for african american and latino men who have sex with men estimation of the reproductive number of novel coronavirus (covid-19) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis key: cord-277417-f71jwdzj authors: geoghegan, jemma l.; holmes, edward c. title: the phylogenomics of evolving virus virulence date: 2018-10-10 journal: nat rev genet doi: 10.1038/s41576-018-0055-5 sha: doc_id: 277417 cord_uid: f71jwdzj how virulence evolves after a virus jumps to a new host species is central to disease emergence. our current understanding of virulence evolution is based on insights drawn from two perspectives that have developed largely independently: long-standing evolutionary theory based on limited real data examples that often lack a genomic basis, and experimental studies of virulence-determining mutations using cell culture or animal models. a more comprehensive understanding of virulence mutations and their evolution can be achieved by bridging the gap between these two research pathways through the phylogenomic analysis of virus genome sequence data as a guide to experimental study. despite many advances in our understanding of virus evolution, how virulence evolves in a virus, particularly following a jump to a new host species, continues to be contentious. will a virus become more or less virulent in a new host? what level of virulence is optimized by natural selection and why? is there any consistent association between host-jumping and virulence such that predictions can be made about how virulence might evolve following emergence? not only will the answers to these questions reveal fundamental aspects of virus biology, but they also may assist in infectious disease management and mitigation, particularly as humans, other animals and plants face a continual threat from emerging viruses. the term 'virulence' has different meanings depending on context, can be assessed in a variety of ways and is often only an operational measure 1 . to be as general as possible, we assume a simple working definition of virulence: the harm caused by pathogen infection, particularly in terms of host morbidity and mortality. virulence is also a complex trait determined by a combination of pathogen, host and environmental factors. although it is obviously necessary to understand all three, we focus on the pathogen (virus) component as this is the most tractable, with the small genomes and rapid replication and evolution of viruses facilitating comparative and experimental studies, and because there is strong evidence for heritable virus genetic variation for virulence 2 . although we purposely focus on the analysis of virus genomes, genome-wide association studies (gwas) promise to open up new ways to explore the evolutionary impact of viral infections on host genomes 3 and hence the intimate interaction between host and virus 4 . another complexity in studies of virulence evolution is that metagenomics is increasingly showing that mixed (that is, polymicrobial) infections are commonplace 5 , and even seemingly healthy hosts can carry multiple microorganisms of the kind often thought to be pathogenic 6, 7 . determining which microorganism is the cause of a particular disease syndrome can be troublesome, and it is possible that overt illness might result from synergistic interactions between multiple microorganisms that overwhelm the host. hence, the model of one pathogen-one disease that has dominated studies of human infections, and implicitly models of virulence evolution, may be overly simplistic. it is also possible that measurements of relative virulence vary between humans and wildlife populations. for example, whereas dengue is considered an important infectious disease of humans, the virulence of dengue virus is low in terms of overall mortality in humans, and infections of equivalent severity may go unnoticed in wildlife, particularly as there is a strong sampling bias towards the most virulent presentations. virulence evolution in viruses has traditionally been studied from one of two separate research paths -the theoretical and the empirical -that have largely been pursued independently. although theory and empiricism have each generated important and parallel insights, they have each been able to paint only a partial picture of virulence evolution. few attempts have been made to bridge this divide 8,9 . there is now a large body of long-standing evolutionary theory that considers what level of virulence maximizes pathogen fitness under variable conditions, such as differing modes of transmission, levels of co-infection, selection pressures, and both within and between hosts 10 . although of great value, a drawback is that this work is unavoidably based on a small number of case studies, the most famous of which is the co-evolution of myxoma virus (myxv) and european rabbits following the release of myxv as a biological control 11 . however, the insights virulence although virulence can be defined in a range of casespecific ways, the simplest definition is the severity or harmfulness of a pathogen. the study of genetic material recovered from microbial (including viral) communities in which genome sequence data are simultaneously generated for all the microorganisms present. the reduction in numbers or elimination of pest organisms through the introduction of a pathogen. from examples such as myxv may be insufficient to adequately inform theoretical models confronted with novel, real-world emergence events. by contrast, empirical studies involve laboratorybased methods to identify the mutations that affect virulence (that is, virulence determinants), usually on the basis of a combination of reverse genetics and cell culture and/or animal models 8, 9, [12] [13] [14] [15] [16] . these studies are often very successful in pinpointing causal mutations (see are not considered in an evolutionary context, their relevance for general theories of virulence evolution is usually ignored. in addition, in vitro methods may not reflect real-world selection pressures, there may be little consideration about how virulence mutations affect inter-host transmission, and animal models commonly differ from the species infected in the field. for example, virulence determinants in myxv identified on the basis of in vitro studies and mouse models have often not been upheld in reverse genetic experiments using the natural rabbit host 17 . similarly, despite the regularity of their use, there has been a long-standing debate over the validity of ferrets as accurate models for human influenza 18 . bridging the gap between the theoretical and empirical approaches would bring a new impetus to studies of virulence evolution. in this review, we outline how this can be achieved within a phylogenomics framework. we show that virus phylogenies are being increasingly used to help identify virulence determinants and that the data obtained can be used to test general theories of virulence evolution. such a phylogenomic approach to studying virulence evolution is timely because of the rapidity with which virus genome sequence data are now being generated, including during ongoing disease outbreaks of emerging viruses [19] [20] [21] , and because of the development of new phylogeny-based methods for studying and visualizing genomic data [22] [23] [24] . however, the success of this approach also requires that phylogenomic data are combined with relevant clinical, epidemiological and experimental metadata so that a direct link can be made between virulence, virus genotype and phenotype, and population fitness. arguably the most interesting context of virulence evolution is following a host jump as this sits at the heart of virus emergence, and the question of how virulence will evolve is commonly asked following the appearance of a new virus or the emergence of an existing virus with altered host range (box 1). to make meaningful inferences, it is important to compare virulence in both the reservoir (that is, donor) and novel (that is, recipient) host species. although this may sound straightforward and is tractable in some cases 25 , in reality it faces a number of difficulties. in many cases, including common infections such as hepatitis c virus 26 , as well as emerging infectious diseases such as zika virus (zikv), the reservoir species is unknown or is at best uncertain. even if a reservoir species is known, we generally know little, if anything, about virulence in that species, and there is likely to be an ascertainment bias towards the most virulent cases. for example, although species of fruit bats appear to be reservoirs for ebola virus (ebov) 27 , little is known about its virulence in these animals 28 , as is true of many wildlife infections. the identification of host species may also change with better sampling, which is invariably poor in wildlife. for example, it was long thought that the canine parvovirus (cpv) that emerged in dogs in the late 1970s had jumped from cats infected by a closely related virus 29 . however, more recent sampling of wild carnivore species has shown this to be incorrect, such that the true reservoir species for cpv is unclear 30 . therefore, it is crucial to understand disease processes, including virulence, in reservoir hosts under natural conditions, which will require more detailed studies of animal ecology. although we currently know little about virulence in reservoir species, comparative data tell us that, on average, low-virulence infections have a greater chance of successfully establishing transmission cycles in humans than viruses with higher mortality 31 . this greater chance is presumably because high virulence requires a greater supply of susceptible hosts during the early stages of emergence. evolutionary biologists have had a long fascination with virulence [32] [33] [34] [35] [36] [37] . because there is a very large literature base on this subject, we necessarily provide only a brief overview here. a straightforward interpretation of virulence evolution is that natural selection will optimize the level of virulence that maximizes pathogen fitness, expressed as the basic reproductive number (r 0 ) 1 , although in reality fitness is shaped by a complex set of host-pathogen interactions 38, 39 . current evolutionary theory tells us that when a virus jumps to a new species, its initial virulence can vary from asymptomatic to highly pathogenic, and precisely where it lies on this virulence spectrum is difficult to predict. however, it is possible that the direction of virulence evolution can be anticipated, at least in part, if the key relationship between virulence and transmissibility, and hence fitness, is understood. importantly, there is also evidence from insect viruses that host phylogeny is able to predict some aspects of virulence evolution following species jumps, with related host species tending to have similar levels of virulence 25 . a commonly stated idea is that there is often an evolutionary trade-off between virulence and transmissibility because intra-host virus replication is necessary to facilitate inter-host transmission but may also lead to disease, and it is impossible for natural selection to optimize all traits simultaneously. in the case of myxv, this trade-off is thought to lead to 'intermediate' virulence grades being selectively advantageous: higher virulence may mean that the rabbit host dies before interhost transmission, whereas lower virulence is selected against because it does not increase virus transmission rates. a similar trade-off model has been proposed to explain the evolution of hiv virulence 40 . however, many doubts have been raised about the general applicability of the trade-off model 35, [41] [42] [43] , virus fitness will be affected by traits other than virulence and transmissibility 39, 41, 44 , contrary results have been observed in experimental studies 45 and relatively little is known about evolutionary trade-offs in nature. for example, in the case of the second virus released as a biocontrol against european rabbits in australia -rabbit haemorrhagic disease virus (rhdv) -there is evidence that virulence has increased through time, probably because virus transmission often occurs through blow flies that feed on animal carcasses, making host death selectively favourable 46 . similarly, experimental studies of plant rna viruses have shown that high virulence does not necessarily impede host adaptation 47 and, in the case of malaria, higher virulence www.nature.com/nrg reverse genetics experimental method used to identify gene function by modifying the sequence of a target gene and analysing its phenotypic consequences. the inference of evolutionary patterns and/or processes through the comparison of whole-genome sequences. depiction of the evolutionary history of a genetically related group of organisms. contains branches and nodes. infectious diseases that have recently appeared in populations or known diseases that are rapidly increasing in incidence or geographic range. (r 0 ). represents the number of secondary infections caused by an infectious host in an entirely susceptible population. occurs when a change in one trait increases fitness but simultaneously reduces fitness because of its impact on another trait, thereby preventing the organism from optimizing both traits. was shown to provide the plasmodium parasites with a competitive advantage within hosts 48 . other factors in addition to evolutionary trade-offs can shape the level of virulence in an emerging virus. for example, 'short-sighted' virulence evolution within a single host may be detrimental for inter-host transmission 49 , and newly emerged 'spillover' infections that have experienced only a limited number of transmission events are likely to have virulence levels that have not yet been optimized for transmissibility by natural selection 50 . accordingly, for spillover infections, ongoing transmission may be largely at the mercy of random drift effects, including the severe population bottlenecks that routinely accompany such events 51 . finally, it is possible that virulence may sometimes simply be a coincidental by-product of selection for another trait or selection for transmission in another species. theory therefore tells us that natural selection can increase or decrease pathogen virulence, depending on the particular combination between host, virus and environment 1, 32, 33, 37, 41, 52, 53 . although providing a useful framework, theory can provide only useful generalities because the relevant factors vary substantially and need to be assessed on a case-by-case basis. virulence evolution could, however, be better understood if its genomic basis were known. phylogenetic studies of viruses, including those that consider whole-genome sequences, are commonplace and are often used to understand a variety of aspects of virus evolution. in particular, virus phylogenies are being increasingly used to understand the evolution of key phenotypic traits such as virulence 9 (and see the examples described below). phylogenomics provides an informative way to help understand virulence evolution and establishes a set of hypotheses that can be tested using appropriate experimental assays 8, 9 . we also believe that phylogenomics provides valuable information on how natural selection acts on virulence and can be used to test general models of virulence evolution, thereby providing a key link between theoretical and empirical approaches to studying virulence evolution. the crux of this approach involves mapping mutations there is no clear understanding on whether emerging viruses become more or less virulent following a jump to a new host. before a host jump, pathogens are likely to have been selected to optimize their virulence for onward transmission in the reservoir host. however, immediately following a host jump, the pathogen is likely to be initially poorly adapted to the new host, which may inhibit successful emergence 135 , and the host is likely to be poorly adapted to the new pathogen 25 . indeed, high host mortality is associated with a reduced likelihood of successful onward transmission in the case of human viruses 31 . there are three possible scenarios for virulence evolution immediately following a host jump (the filled nodes in the figure): • some of the most devastating epidemics have been associated with an increase in virulence following a cross-species transmission event (see the figure, part a). for example, whereas hiv almost invariably progresses to aids if untreated in humans, in african non-human primates, the closely related simian immunodeficiency virus (siv) persists without causing disease 136, 137 or is associated with less overt disease than in humans 138 . similarly, the (deliberate) transfer of myxoma virus (myxv) from the south american tapeti, where it was largely benign, initially resulted in mortality of ~99% in european rabbits 11 . • no apparent change in virulence following a host jump can be seen in h3n8 influenza a virus that jumped from horses to dogs 139, 140 (see the figure, part b). disease is mild in both cases, and there are minimal biological differences between these viruses 141 . similarly, rabies virus (rabv) causes an equally severe, ultimately fatal encephalitis in foxes and dogs and following spillover to humans 142 . • cases of decreased virulence may be largely overlooked because avirulent viruses will often persist in a host population without detection. an example of decreased virulence following a host jump may be that of the infectious haematopoietic necrosis virus (ihnv) that spread from sockeye salmon to rainbow trout in north america and japan (see the figure, part c). upon the initial cross-species transmission event in the 1950s to 1960s, the virus seemed at first to be avirulent in the new host in contrast to the mortality seen in sockeye salmon. since then, however, virulence in some genotypes of ihnv in rainbow trout has increased, itself associated with an increase in transmissibility 143, 144 . for many diseases, however, the extent of changes in virulence upon emergence is not known, in part because little is usually known about disease epidemiology in reservoir hosts, and there is likely to be a sampling bias towards the cases of highest virulence. nature reviews | genetics r e v i e w s volume 19 | december 2018 | 759 the initial and sometimes transient appearance of a pathogen in a new species following a host jump. onto phylogenetic trees of viruses sampled within and/or between disease outbreaks and from reservoir and novel hosts. the phylogenetic location of these changeswhether they fall on shallow or deep nodes (branches) and/or singularly or in parallel -makes it possible to infer, at least in broad terms, the selection pressures acting on virulence mutations and from this infer important aspects of virulence evolution (box 1 ; fig. 1 fig. 1 | phylogenomics of virulence evolution. a | a model phylogeny with virulence determinants mapped to a fairly deep node suggesting that higher virulence has increased virus fitness. b | a model phylogeny with virulence traits mapped to shallow nodes suggesting that higher virulence reduced pathogen fitness so that viruses with these mutations are purged from the population or require compensatory mutations. c | a model phylogeny with a high-virulence mutation arising multiple times independently owing to parallel or convergent evolution. the occurrence of parallel/convergent mutations that occur more frequently than by chance 8 is likely to reflect adaptive evolution (fig. 2) . d | the relationship between virulence, fitness and host jumps. a virus is assumed to be at a fitness peak (high r 0 ), in this case high virulence, in the reservoir host, so that the mutations determining both virulence and host range are expected to be subject to strong purifying selection (for example, a low value of d n /d s ). as the virus emerges in the new recipient host, it will initially be maladapted (that is, reside in a fitness valley) and subject to genetic drift as the population is small. as it adapts to the new host, virulence will be selectively optimized (in this case declining), increasing r 0 and resulting in positive selection (for example, d n /d s > 1, although other measures of selection pressure are available). once the virus becomes adapted to the new host, the virulence determinants are again subject to purifying selection. the greater the fitness of a virulence determinant, the more rapidly it will spread through the virus population and the deeper it will fall on a virus phylogeny (that is, closer to the root of the tree), including on the branch linking reservoir and novel hosts. of particular importance are repeated occurrences of the same mutation falling on deep branches across multiple outbreaks, or multiple cross-species transmission events, as both parallel evolution and convergent evolution can be signatures of adaptive evolution 8,54-57 (fig. 1) . for example, in the case of west nile virus (wnv), a single mutation in the virus helicase protein repeatedly evolved in high-mortality outbreaks in birds, which is indicative of a selective advantage 58 . similarly, the reversion to virulence in oral polio vaccine (opv) strains of poliovirus has been associated with extensive parallel evolution 8 , and parallel evolution was also associated with hostspecific adaptation in experimental studies of crossspecies transmission involving drosophila virus c 59 . because adaptive evolution has been at play, phylo genies of the sequence in question may have a characteristic shape 24 , and the sequences associated with selected branches may also contain genomic signatures indicative of positive selection, such as the rapid fixation of amino acid changes or an increased rate of nonsynonymous to synonymous substitutions per site (ratio d n /d s ) 8, 60 . in the case of frequent parallel or convergent evolution for specific virulence mutations, it is also possible that the amino acid sites involved will have signatures of positive selection, such as an elevated d n /d s (as was the case in wnv; see below). following the same logic, mutations that fall on shallow branches in virus phylogenies (that is, closer to the tips) are present in a smaller proportion of the population and are therefore more likely to be of lower fitness such that they may be removed by purifying selection. hence, virulence-determining mutations that repeatedly fall on tip branches alone are likely to inhibit some other aspect of pathogen fitness, thereby reducing r 0 at the population scale. although this approach has a solid theoretical and empirical basis 61,62 , a complicating factor is that a virulence-determining mutation that has very recently emerged will necessarily fall towards the tips rather than on an internal branch even if it is selectively advantageous. similarly, although popular, d n /d s measures are less robust over short timescales, such as during outbreaks, because mutations may not have reached fixation by positive selection or had time to be purged by purifying selection, and it can be difficult to detect selected mutations that occur only once 63, 64 . approaches to detect positive selection that do not rely on d n /d s , such as those based on tree shape 24 , or tracking mutations that are increasing in frequency compared with those thought to be evolving neutrally 64, 65 , may therefore add analytical power. the phylogenetic mapping of virulence mutations can proceed in two ways depending on the extent of a priori knowledge. in a 'top-down' approach, in which virulence determinants are unknown, a virus phylogeny is inferred, mutations are mapped onto this phylogeny and the mutations on key branches are then identified. such 'key branches' include those directly associated with cross-species transmission events, invasions of new geographic areas, increases in rates of transmission, spikes in morbidity and/or mortality or clear instances of positive selection. the mutations identified in this way are candidates for virulence determinants that can be tested in an appropriate experimental framework 8 . an example of this approach is shown in fig. 2 . the second, 'bottom-up' , approach utilizes existing knowledge of virulence determinants, such as that determined by an experimental study. the putative virulence determinant is then mapped onto the phylogeny, and its phylogenetic location (that is, deep or shallow branch, singular or parallel/convergent evolution) is used to infer how it affects virulence evolution, whether it is associated with reciprocal mutations that reflect evolutionary trade-offs and the selection pressures it faces. although this phylogenomic approach is being increasingly used to identify virulence determinants, and we discuss a number of real data examples below, it can be used to make general statements about the nature of virulence evolution. specifically, a virulence mutation that falls deep in the phylogeny such that it is inherited in all subsequent branches, and one evolving in parallel or with evidence of positive selection, necessarily implies that virulence is selectively advantageous. conversely, a virulence determinant that occurs sporadically on shallow branches and is subject to strong purifying (negative) selection suggests that virulence is not directly beneficial, probably because it inhibits some other component of overall fitness. in such cases, each instance of high virulence may represent an independent and transient evolutionary event. if only a single mutation is associated with a change in virus virulence, as in the case of wnv, then this change in virulence is likely to be selectively advantageous without an evolutionary trade-off with transmissibility, as a reduction in transmissibility would probably need to be compensated for by additional reciprocal mutations located elsewhere in the genome. hence, if multiple mutations fall on a branch associated with a change in virulence, it is possible that some are virulence determinants and the others are associated with evolutionary trade-offs on other traits. although we have described it in terms of emerging viruses, this phylogenomic approach can, in theory, be applied to any system in which a phylogeny can be inferred and in which it is possible to experimentally assess the impact of individual mutations on virulence. similarly, it can be used to study other virological traits associated with disease emergence, particularly host range. for example, the repeated evolution of the same amino acid changes following the cross-species transmission of avian influenza virus to humans strongly suggests that they directly affect host range 66 , and a similar approach has been used to elucidate the nature of the evolutionary arms race between viruses and their hosts 67, 68 . critically, however, the approach described here should also be considered an idealized one that works best when a limited number of genomic mutations act independently to shape virulence. virulence determinants nature reviews | genetics r e v i e w s volume 19 | december 2018 | 761 the transmission of a pathogen from one host species to another. also called hostjumping or host-switching. an evolutionary process by which two or more separate lineages develop identical characteristics independently. the descendants of unrelated ancestors that have evolved similar traits independently. natural selection that leads to advantageous mutations spreading through a population. mutations in coding regions can be either synonymous (which do not change the amino acid; measured as d s ) or non-synonymous (which change the amino acid; measured as d n ). the d n /d s ratio >1 is sometimes used to infer the occurrence of positive selection, although the accuracy of this measure depends on various factors, including the timescale of sampling. natural selection that acts to remove low-fitness (including deleterious) mutations from populations. it is the most common form of natural selection and gives a d n /d s < 1. also called negative selection. may be harder to identify when there are more complex interactions between mutations 9 , which appears to be true of myxv (box 2). although epistasis is likely to be commonplace in rna viruses 69 , little is currently known about whether virulence mutations interact epistatically 70 . similarly, this approach may work best for rna viruses because their constrained genome sizes mean that there are probably a limited number of virulence determinants, increasing the likelihood that they are subject to parallel and/or convergent evolution, and rates of recombination (which complicate phylogenetic relationships) are often fairly low within species 71 . to illustrate how a phylogenomic approach can shed light on the evolution of virus virulence, we now briefly outline a number of cases in which it can be or has been applied. we begin by considering cases in which virulence determinants have been successfully mapped (wnv and avian influenza a virus (aiv)), move on to those in which revealing the mutations that underpin changes in virulence has been more complex (myxv, marek's disease virus (mdv) and hiv) and end by examining virulence evolution in two recent disease outbreaks (ebov and zikv). the evolution of virulence in strains of oral polio vaccine (opv) 8 . opv is an attenuated form of poliovirus that can occasionally revert to a virulent form and cause outbreaks of poliomyelitis. a | phylogenetic analysis of opv strains in nature reveals that some mutations associated with high virulence have experienced more frequent parallel evolution than expected by chance (and occupy well supported nodes) and hence are likely to be seletively favoured 8 . b | computational evolutionary analysis then reveals that this parallel evolution for high virulence is associated with a hypothetical threonine-to-proline (t-to-p) amino acid change that is subject to significant adaptive evolution (which can be detected in a variety of ways) 60, 63 . c | the virulence impact of these mutations is then confirmed in both in vitro (cell culture; part ca) and in vivo (mouse; part cb) experimental studies. in all cases, the red shading signifies increased virulence. the interaction among genes at different loci. the function and evolution of one gene may be dependent on the presence of one or more other genes. analysis in each case has told us about the evolution of virulence in general. in 1999, a new lineage of wnv became the leading cause of arthropod-borne viral encephalitis in humans and horses in north america, spreading from east to west across the continent 72 and causing severe mortality in many bird species, particularly the american crow 73 . phylogenomic analysis revealed that a single thr249pro (t249p) amino acid substitution in the virus ns3 helicase protein was associated with high-virulence wnv outbreaks in corvids on multiple continents. experimental analysis in captive crows then showed that this mutation was sufficient to explain the high fatality rates in american crows, perhaps because it increased the rate of virus replication 58 . wnv therefore provides an important example of where a single genetic switch controls virulence, which is obviously the easiest scenario to detect using a phylogenomic approach. of more general importance was that t249p evolved in parallel and experienced an elevated rate of nonsynonymous change, suggesting that high virulence was selectively favoured in the absence of an evolutionary trade-off as no reciprocal mutations were observed elsewhere in the viral genome 58 the co-evolution of european rabbits (oryctolagus cuniculus) and the myxoma virus (myxv) released as a biological control against them is arguably the most famous case study in virulence evolution. fenner and colleagues 93 were able to identify 'grades' of myxv virulence using laboratory assays in european rabbits. the most lethal grade i strains (shaded red in the figure) were characterized by almost 100% mortality, whereas the most 'attenuated' grade v strains had mortality <50% and longer survival times. grade i strains were released in both australia (starting strain, sls) and europe (starting strain, lausanne) in the early 1950s, and soon after lower virulence viruses began to appear in parallel in both continents, with intermediate virulence grades (for example, grade iii) becoming the most common in the field. although of large importance, the studies of fenner were necessarily limited in that the genetic basis of virulence evolution was unknown. that the trajectory of virulence evolution was the same in both australia and europe meant that the same virulence determinants might be involved on both continents. therefore, it was a surprise when the first large-scale genome comparisons of myxv revealed that virulence grades changed frequently across the virus phylogeny, as depicted in the phylogeny, and that different mutations appeared in the australian and european epidemics (depicted as different filled shapes at branch tips), with no parallel evolution of possible virulence determinants within australia and europe 94, 145 . for example, there were no mutations that were unique to the most attenuated grade v viruses, nor to the highest virulence grade i viruses. hence, there are multiple genetic routes to attenuation or virulence in myxv, such that there has been convergent evolution for phenotype but not genotype, and both attenuating and virulence-restoring mutations have been fixed in australian myxv. therefore, there is no simple way to predict virulence evolution from genome-scale comparisons in the case of myxv and no parallel or convergent evolution to guide experimentation. indeed, experimentally verified virulence determinants in rabbits remain elusive 17 . the ongoing evolution of myxv virulence reflects both mutations acquired in the virus and resistance evolution in the rabbit host 11 . this co-evolutionary process has also resulted in a marked increase in myxv virulence 146 . unlike viruses sampled in the 1950s before the evolution of any host resistance, most virus isolates sampled in the 1990s that have evolved in the face of host resistance induce a lethal immune collapse syndrome with similarities to septic shock 146 . may have been responsible for a substantial proportion for the cross-continent spread of the virus 74 , in which case t249p may have been selected to increase replication (transmissibility) in that species, a coincidental by-product of which was heightened virulence in crows. ever since the emergence of the highly pathogenic h5n1 subtype of influenza virus, there has been concern over whether this influenza virus could establish sustained transmission in humans, in which it causes only sporadic spillover infections at present 75, 76 . more recently, highly pathogenic h7n9 has sporadically infected humans 77 and continues to spread through poultry populations in china 78 , evolving from a low-virulence ancestor 79 . although the true number of human cases, and hence accurate mortality, is difficult to ascertain, it is clear that both h5n1 and h7n9 cause fairly high mortality in humans and could have serious consequences were they to trigger a large-scale human epidemic. this concern has led to attempts to use genomic data to help in pandemic risk assessment 80 . at the virus subtype level, the presence of a run of polybasic amino acids in the hinge region between the ha1 and ha2 subunits that make up the haemagglutinin (ha) protein of influenza virus helps it establish a systemic, and subsequently more serious, infection and thereby acts as a useful marker of high-virulence strains of the h5 and h7 aiv subtypes 79, 81, 82 . this marker makes it relatively easy to distinguish between potentially lowvirulence and high-virulence aivs, although what triggers the evolution of the high-pathogenicity variants in these subtypes is unclear 83 . other individual amino acid changes, affecting a variety of gene functions, have also been proposed as specific virulence determinants for h5n1 (refs 84-86 ) as well as in those viruses that circulate in human populations such as seasonal h3n2 (ref. 87 ), the h1n1 virus responsible for the global pandemic of 1918-1919 (ref. 88 ) (in which host inflammatory and cell death responses to infection appear to play a key role) 89 , and influenza b virus 90 (table 1) . the key unresolved question is how natural selection will shape both virulence and transmissibility if an aivlike h5n1 or h7n9 virus is eventually able to develop sustained transmission in humans. an added complexity is that phylogenomic analyses reveal a consistent set of mutations that distinguish human and avian influenza viruses, although whether these affect host range alone, or both host range and virulence, is unclear 66, 84 . the canonical study of virulence evolution following a species jump is myxv in european rabbits, with a body of classic work undertaken by fenner and colleagues 91-93 (box 2). in both australia and europe, highly virulent strains of myxv were used as a biological control against the european rabbit population, with releases beginning in the early 1950s. in both continents, the same trajectory of virulence evolution was observed: virulence declined from the highly virulent (that is, grade i) release strains to encompass a far wider range of virulence grades, including the most attenuated grade v strains, with strains of 'intermediate' virulence the most commonly sampled in the field. this pattern, reflecting a combination of the virus evolving more attenuated strains and the host developing resistance, fuelled the idea of a trade-off between virulence and transmissibility. sixty years after the initial release of myxv, the first large-scale genomic studies of its spread were performed (box 2). phylogenomic analysis revealed that the virulence phenotype has changed on a regular basis 94 . however, a major surprise was that each change in virulence was associated with a different set of mutations across multiple genes 94, 95 . although which mutations had the greatest impact on virulence is still unclear and requires further experimental analysis, such a phylogenomic pattern indicates that there are multiple routes to achieving the same levels of virulence, including attenuation, such that there has been convergent evolution for phenotype but not genotype. it is likely that this evolutionary flexibility in part reflects the fairly large genome size of myxv (a double-stranded dna virus of ~160,000 bp), which may mean that there is a large number of potential virulence determinants that can interact through epistasis 96 , in turn complicating any phylogenomic analysis. marek's disease virus. whether 'imperfect' (that is, 'leaky') vaccination against infectious disease, in which disease symptoms are reduced but there is less impact on virus replication and transmission, will change the selection pressures acting on the pathogen and affect virulence evolution has been the source of debate 97, 98 . although still contentious, particularly in the case of human disease, there is good evidence that imperfect vaccination has increased virulence in the case of mdv, a dna herpesvirus that poses a major problem to the poultry industry 99 . in the 1960s, the appearance of virulent mdv strains forced the development of the first generation of marek's disease vaccines. however, because these vaccines were imperfect, 'very virulent' mdv began to appear within 10 years, necessitating a second-generation vaccine. this very virulent mdv was followed, more rapidly, by the appearance of 'very virulent plus' mdv, requiring a third-generation vaccine (fig. 3) . imperfect mdv vaccines enhance virulence by elongating the infectious periods and hence transmission potential of virulent strains that would have been removed by natural selection before transmission in the absence of vaccination 99 . although the genomic basis to mdv virulence evolution is currently uncertain, with some causative amino acid changes proposed 100 , initial phylogenomic studies suggest that, as in the case of myxv, there are multiple genetic pathways to high virulence 101 (and which again may reflect the fairly large size of the viral genome). not only does virulence evolution in mdv have important implications for vaccination strategies against other diseases in which vaccine efficacy is fairly low 102 , but it also shows that in some circumstances increased virulence can be selectively advantageous. given the importance of hiv to human health and that it ignited much of the research on disease emergence, it is no surprise that there has been considerable discussion on the evolution of hiv virulence [103] [104] [105] [106] [107] . indeed, it is striking that hiv in humans is markedly www.nature.com/nrg r e v i e w s 764 | december 2018 | volume 19 influenza a viruses are categorized into subtypes on the basis of the diversity in two proteins present in the viral envelope: haemagglutinin (h or ha) and neuraminidase (n or na). more virulent than the closely related viruses that naturally infect non-human primates in africa (box 1). although there have been suggestions that hiv has begun to evolve reduced virulence 108 , discussions of the trajectory of virulence evolution are necessarily complicated by the fact that antiviral therapy has greatly extended life expectancy. hiv virulence is often approximated as the degree of variation in the set point viral load (spvl) that is established soon after initial infection 104 . the higher the spvl, reflecting greater levels of virus replication, then the more rapidly the patient will progress to aids in the absence of antiviral therapy, although other studies have suggested that the replicative capacity of the virus itself is a more informative marker of virulence and is also a direct measure of virus fitness 109 . indeed, some 'controller' individuals are able to control levels of hiv in the absence of antiviral therapy, and it has been shown that this is in part due to infection with viruses of reduced replicative capacity 110 . importantly, viral genetic variation may play a more important role in shaping hiv virulence than host factors, with approximately one-third of the observed variability in spvl assigned to virus factors 111 and only ~13% seemingly due to the host 112 . this observation also implies that spvl, and hence virulence, can be selectively optimized 113 . in support is evidence that spvl, and hence virulence, has declined in some african hiv subtypes, even accounting for the use of antiviral therapy, and that this reflects a trade-off between virulence and transmissibility 114 . importantly, however, despite many studies into the determinants of hiv virulence, the virus genomic mutations responsible for determining spvl are still uncertain and multiple genes may be involved 104 . the difficulty in assigning the genetic determinants of spvl may be in part due to genetic variation across viral populations 111 . for example, heritability in spvl was highest (~60%) between individuals in the swiss hiv cohort, which also represents the most homogenous viral population 113 marek's disease virus (mdv) of chickens present on poultry farms. this imperfect vaccine reduced disease symptoms but did not prevent virus replication, thereby extending the infectious periods, and hence potential for transmission, of virulent strains that would have been removed by natural selection before transmission to a new host in the pre-vaccine era 99 . because of this, 'very virulent' mdv began to appear within 10 years, necessitating the development of a secondgeneration vaccine that was also imperfect. this was followed, in an even shorter period, by the appearance of 'very virulent plus' mdv, requiring a third-generation vaccine. although the genomic basis of mdv virulence is currently unknown, the phylogenies at the bottom of the figure hypothetically assign virulence to multiple causative mutations (as in the case of myxoma virus). the dashed arrows indicate the evolution of viruses to the next virulence grade. the amount of phenotypic variation in a population that is attributable to individual genetic differences. the 2013-2016 outbreak of ebov (makona variant) in west africa was the largest and longest described in humans since the first description of the disease in 1976, with approximately 29,000 cases and some 11,000 deaths. in addition to hindering attempts at disease control, this elongated period of transmission in humans may have resulted in different selection pressures from those faced in the animal reservoir. this outbreak also raised key questions about virulence evolution, particularly whether natural selection would have favoured ebov variants causing higher or lower human case fatality rates had the virus not been stamped out by public health intervention 115 . phylogenetic analysis of ebov during the 2013-2016 outbreak revealed an ala82val (a82v) substitution in the virus glycoprotein to be of particular importance 70, 116 . a82v is notable as it falls on a deep internal branch of the ebov phylogeny, compatible with adaptive evolution, whereas other amino acid changes are associated with individual or only small clusters of sequences. moreover, a82v improves binding to the human npc1 receptor utilized by ebov 117 , which would increase infectivity in humans, at the same time reducing infectivity in cells from the bat reservoir species 70, 116 . intriguingly, the appearance of a82v on the ebov phylogeny is associated with two key epidemiological features: an increase in case numbers and an increase in mortality (fig. 4) . if there was indeed an increase in both ebov transmissibility and virulence, then higher virulence is likely to have directly increased viral fitness, and in the absence of an evolutionary trade-off as only a single substitution was identified. however, these apparent changes in phenotype also coincided with the movement of the virus from guinea to sierra leone, such that any change in case numbers and mortality could in fact be due to a change in epidemiological factors (such as access to health care or differing human demographics and/or transmission networks), and recent studies using animal models suggest that a82v has no direct impact on virulence 118 . zikv is the most recent emerging virus to lead to a major public health scare and is puzzling because a seemingly benign virus suddenly increased in virulence, causing severe neurological disease in humans. before 2007, there were fewer than 20 human cases of zikv reported and all were mild infections restricted to africa and asia 9,119 . consequently, neither the disease caused by zikv nor the molecular determinants of zikv virulence were well characterized, and it is likely that there was systematic under-reporting of infections, including those associated with severe disease. in 2007, the pacific islands reported the first major outbreaks of zikv before the virus spread to the americas in 2014. although the majority of human infections range from asymptomatic to mild, the virus was associated with the neurological guillain-barré syndrome in french polynesia in 2013 (ref. 120 ), and those cases from the americas, particularly brazil, were linked to more severe diseases, including congenital abnormalities such as microcephaly 121 . phylogenetic analysis revealed that the most recent zika epidemics are due to the asian lineage of zikv, rather than the african lineage 122 , and that the virus spread cryptically in brazil for at least a year before its detection 20 . although there are multiple amino acid differences between the african and asian lineages 122, 123 , it has been claimed that those in the asian lineage that spread through the americas may be directly linked to both increased infectivity in aedes aegypti mosquitoes 124 and microcephaly, most notably a ser139asn (s139n) amino acid change in the prm protein 125 . however, there is still considerable uncertainty in this area, with others arguing that viruses from both lineages can cause neurovirulence but that cases often go unreported 9, 126 . hence, the case of zikv highlights the difficulty in assessing virulence evolution within a background of sparse and biased sampling even with phylogenomic data and shows the importance of collecting reliable, real-time epidemiological data even in low-incidence situations. 70, 116 . maps above the phylogeny show the spread of ebov over the timeline of the outbreak in the three affected countries in west africa, where blue-shaded regions correspond to the wild-type virus variant (a82) and redshaded areas correspond to mutated virus variant (v82). it is possible that a82v was also associated with an increase in both ebov case numbers and mortality (that is, virulence) as the outbreak progressed, such that increased virulence is directly selectively advantageous, although this is confounded by epidemiological factors. virulence evolution has been one of the longest-standing issues in evolutionary biology. although a strong body of theory has been developed, there are few cases in which we understand the forces that have shaped particular instances of virulence evolution and even fewer in which we have successfully linked evolutionary theory with individual genomic changes. we believe that a synthesis of experimental studies of virulence determinants and long-standing theory of virulence evolution set within a phylogenomic framework will generate a more comprehensive understanding of virulence evolution. in particular, not only does a phylogenomic approach enable potential virulence determinants to be identified, which is being increasingly used in the case of emerging viruses, but this analysis also sheds light on the models of virulence evolution that have occupied theoreticians for decades. recent advances in real-time genomics during disease outbreaks 127, 128 and the increased demand for precision in public health interventions may help in the development of a new understanding of the evolution of pathogen virulence. we contend that this can be achieved within a phylogenomic framework as long as relevant data are available and strong links are made between genomics, phylogenetics, epidemiology, and experimental studies of virus virulence and fitness. therefore, it is critically important to collect clinical (that is, disease symptoms and severity) and epidemiological (that is, time and place of sampling) metadata concurrently with the sequencing of virus genomes and to sample across a range of clinical syndromes, not just those associated with severe disease. we also stress the value of gathering concurrent and historical data from likely reservoir species as these will provide a more complete insight into virulence evolution and determining the full range of microorganisms that infect a particular species, as well as their interactions, as assigning disease syndromes to individual pathogens may often be difficult. thankfully, advances in metagenomics now make the latter task feasible 6, 7, 129 . similarly, there is a marked lack of good virulence grading schemes among viral infections. although such schemes can sometimes be simplistic, assuming discrete virulence categories that may not exist in nature and incorporating degrees of subjectivity, the case of myxv shows that they are key to considering the relationship between genotype and phenotype that is essential to understanding virulence evolution. finally, it is possible that an increased understanding of virulence evolution drawn from a phylogenomic approach may contribute to new strategies for pathogen control and eradication, and there is a clear potential for this framework to inform and improve the fields of disease management and the biological control of invasive pests. although predicting where and when a new disease might emerge is clearly unfeasible because of the immense complexities involved 54, 130 , predicting the overall trajectory of the virulence evolution of a virus in a novel host may be more achievable. once again, biocontrol presents a compelling example. although controversial [131] [132] [133] , the proposed release of cyprinid herpesvirus 3 (cyhv-3) as a biological control against invasive common carp (cyprinus carpio l.) in australia may present a unique opportunity to follow, in real time, the co-evolution between host and virus at both the genotypic and phenotypic scales. both theory and virus natural history predict that cyhv-3 virulence will decline with time 134 , and it will be interesting and informative to see how any such virulence evolution is manifest in phylogenomic data. this review provides insight into how virulence theory might be united with empirical data and what is needed to better understand the relationship between virulence and transmissibility elevated virulence of an emerging viral genotype as a driver of honeybee loss microbial genome-wide association studies: lessons from human gwas genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis c virus unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach redefining the invertebrate virosphere the evolutionary history of vertebrate rna viruses this study is a powerful example of the analytical approach outlined here. the authors use phylogenetics and experimental study to describe the evolutionary pathways by which opv strains become pathogenic and propose a framework for vaccine design did zika virus mutate to cause severe outbreaks? within-host parasite cooperation and the evolution of virulence myxoma virus and the leporipoxviruses: an evolutionary paradigm molecular determinants of ebola virus virulence in mice the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis the role of vp1 amino acid residue 145 of enterovirus 71 in viral fitness and pathogenesis in a cynomolgus monkey model molecular determinants of infectious pancreatic necrosis virus virulence and cell culture adaptation virulence determinants of pandemic a(h1n1)2009 influenza virus in a mouse model reverse engineering field isolates of myxoma virus demonstrates that some gene disruptions or loss of function do not explain virulence changes observed in the field using ferrets as an animal model for investigating influenza antiviral effectiveness virus genomes reveal factors that spread and sustained the ebola epidemic epidemic establishment and cryptic transmission of zika virus in brazil and the americas the evolution of ebola virus: insights from the 2013-2016 epidemic nextstrain: real-time tracking of pathogen evolution can we read the future from a tree? elife predicting evolution from the shape of genealogical trees this study combines experimental cross-species transmission and phylogenetics to reveal the nature of virulence evolution in a drosophila virus differential infection patterns and recent evolutionary origins of equine hepaciviruses in donkeys fruit bats as reservoirs of ebola virus bat-man disease transmission: zoonotic pathogens from wildlife reservoirs to human populations evolution of canine parvovirus involved loss and gain of feline host range host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species virological factors that increase the transmissibility of emerging human viruses coevolution of hosts and parasites evolution in health and disease challenging the trade-off model for the evolution of virulence: is virulence management feasible? evolution of infectious diseases the evolution of virulence relationship between within-host fitness and virulence in the vesicular stomatitis virus: correlation with partial decoupling this article argues that current formulations of r 0 are too simplistic to understand pathogen evolution and that a more complex variation in hiv-1 set-point viral load: epidemiological analysis and an evolutionary hypothesis virulence evolution and the trade-off hypothesis: history, current state of affairs & the future this paper provides an overview of the theory that there is a trade-off between virulence and transmissibility, highlighting areas in which the theory has been challenged and extended over the past 20 years adaptive virulence evolution: the good old fitness-based approach virulence and transmissibility of pathogens: what is the relationship? size of genetic bottlenecks leading to virus fitness loss is determined by mean initial population fitness virulence evolution of a generalist plant virus in a heterogeneous host system virus biocontrol: grand experiments in disease emergence and evolution high virulence does not necessarily impede viral adaptation to a new host: a case study using a plant rna virus virulence and competitive ability in genetically diverse malaria infections short-sighted evolution and the virulence of pathogenic microorganisms epidemic dynamics at the human-animal interface transmission bottlenecks as determinants of virulence in rapidly evolving pathogens transient virulence of emerging pathogens evolution of virulence in opportunistic pathogens: generalism, plasticity, and control this article discusses what aspects of virus evolution might be predictable, including virulence, and how this can be tested and highlights the importance of parallel evolution as a marker of adaptation mutation driven parallel evolution during viral adaptation convergent evolution of lysozyme sequences? population size changes and selection drive patterns of parallel evolution in a host-virus system a single positively selected west nile viral mutation confers increased avian virogenesis in american crows this study demonstrates that positive selection on a single amino acid substitution within the genome of wnv was responsible for increased virulence in the american crow host shifts result in parallel genetic changes when viruses evolve in closely related species datamonkey 2.0: a modern web application for characterizing selective and other evolutionary processes the population genetics of dn/ds phylogenetic evidence for deleterious mutation load in rna viruses and its contribution to viral evolution the genomic rate of molecular adaptation of the human influenza a virus pathogen population bottlenecks and adaptive landscapes: overcoming the barriers to disease emergence dynamics of molecular evolution in rna virus populations depend on sudden versus gradual environmental change characterization of the 1918 influenza virus polymerase genes ancient adaptive evolution of the primate antiviral dna-editing enzyme apobec3g positive selection of primate trim5alpha identifies a critical species-specific retroviral restriction domain emerging viruses: why they are not jacks of all trades? human adaptation of ebola virus during the west african outbreak why do rna viruses recombine? fluid spatial dynamics of west nile virus in the usa: rapid spread in a permissive host environment nile virus emergence and large-scale declines of north american bird populations globalization, land use, and the invasion of west nile virus h5n1 influenza: a protean pandemic threat are we ready for pandemic influenza? dissemination, divergence and establishment of h7n9 influenza viruses in china genesis and spread of newly emerged highly pathogenic h7n9 avian viruses in mainland china emergence and adaptation of a novel highly pathogenic h7n9 influenza virus in birds and humans from a 2013 human-infecting low pathogenic ancestor viral factors in influenza pandemic risk assessment influenza type a in humans, mammals and birds: determinants of virus virulence, host-range and interspecies transmission influenza: lessons from past pandemics, warnings from current incidents emergence of a highly pathogenic avian influenza virus from a low pathogenic progenitor the potential for respiratory droplet-transmissible a/h5n1 influenza virus to evolve in a mammalian host molecular basis for high virulence of hong kong h5n1 influenza a viruses enhanced virulence of clade 2.3.2.1 highly pathogenic avian influenza a h5n1 viruses in ferrets ns1 protein amino acid changes d189n and v194i affect interferon responses, thermosensitivity, and virulence of circulating h3n2 human influenza a viruses a new influenza virus virulence determinant: the ns1 protein four c-terminal residues modulate pathogenicity this paper presents a comprehensive study of the virulence of the 1918 influenza virus in a mouse model, revealing the importance of virus-host interactions involving a wide set of interacting virus genes a single amino acid in the polymerase acidic protein determines the pathogenicity of influenza b viruses biological control as exemplified by smallpox eradication and myxomatosis a comparison of the virulence for european rabbits (oryctolagus cuniculus) of strains of myxoma virus recovered in the field in australia, europe and america evolutionary history and attenuation of myxoma virus on two continents this article presents the first large-scale phylogenomic analysis of myxv and shows that, despite parallel evolution at the phenotypic (that is, virulence grade) level, different virus mutations were responsible for evolution genome scale evolution of myxoma virus (myxv) reveals host-pathogen adaptation and rapid geographic spread mechanisms of genetic robustness in rna viruses imperfect vaccines and the evolution of pathogen virulence imperfect vaccines and imperfect models this paper presents a clear demonstration that imperfect vaccination was responsible for increased virulence in the case of the mdv of chickens and considers the evolutionary implications of imperfect vaccination in general vlip, a viral lipase homologue, is a virulence factor of marek's disease virus a phylogenomic analysis of marek's disease virus reveals independent paths to virulence in eurasia and north america immunity promotes virulence evolution in a malaria model has the rate of progression to aids changed in recent years? virulence and pathogenesis of hiv-1 infection: an evolutionary perspective this key study of the evolution of hiv virulence focuses on the factors that determine the spvl and shows the importance of virus genetic traits in shaping spvl and hence disease severity temporal trends of initial cd4 cell counts following human immunodeficiency virus seroconversion in italy, 1985-1992. the human immunodeficiency virus italian seroconversion study is the virulence of hiv changing? a meta-analysis of trends in prognostic markers of hiv disease progression and transmission association between the rate of cd4 + t cell decrease and the year of human immunodeficeny virus (hiv) type 1 seroconversion among persons enrolled in the swiss hiv cohort study is hiv-1 evolving to a less virulent form in humans? replicative fitness of transmitted hiv-1 drives acute immune activation, proviral load in memory cd4 + t cells, and disease progression impaired replication capacity of acute/ early viruses in persons who become hiv controllers viral genetic variation accounts for a third of variability in hiv-1 set-point viral load in europe common genetic variation and the control of hiv-1 in humans phylogenetic approach reveals that virus genotype largely determines hiv set-point viral load a transmission-virulence evolutionary trade-off explains attenuation of hiv-1 in uganda can ebola virus evolve to be less virulent in humans? this study uses a combination of phylogenetics and experimental study to demonstrate the importance of the a82v substitution in the ebov glycoprotein to viral infectivity and suggests that a82v has increased virus virulence filovirus receptor npc1 contributes to species-specific patterns of ebolavirus susceptibility in bats recently identified mutations in the ebola virus-makona genome do not alter pathogenicity in animal models zika fever and congenital zika syndrome: an unexpected emerging arboviral disease guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: a case-control study zika virus in the americas -yet another arbovirus threat differential virulence between asian and african lineages of zika virus genomic insights into zika virus emergence and spread evolutionary enhancement of zika virus infectivity in aedes aegypti mosquitoes the results of this study suggest that a mutation in the ns1 protein in the zikv associated with the recent outbreak in the americas causes increased infectivity in aedes aegypti mosquitoes a single mutation in the prm protein of zika virus contributes to fetal microcephaly replication of early and recent zika virus isolates throughout mouse brain development the genomics of emerging pathogens towards a genomicsinformed, real-time, global pathogen surveillance system metatranscriptomics and the evolutionary biology of rna viruses predicting virus emergence amidst evolutionary noise biocontrol of common carp in australia poses risks to biosecurity biocontrol of invasive carp: risks abound safe and effective biocontrol of common carp cyprinid herpesvirus 3 and its evolutionary future as a biological control agent for carp in australia virulence evolution in emerging infectious diseases natural siv hosts: showing aids the door understanding the benign nature of siv infection in natural hosts the results of this study suggest that simian immunodeficiency virus in chimpanzees, which is the ancestor of hiv, increases mortality in these animals transmission of equine influenza virus to dogs influenza virus reservoirs and intermediate hosts: dogs, horses, and new possibilities for influenza virus exposure of humans equine and canine influenza viruses h3n8 viruses show minimal biological differences despite phylogenetic divergence rabies, still neglected after 125 years of vaccination virulence comparisons of infectious hematopoietic necrosis virus u and m genogroups in sockeye slamon and rainbow trout differential virulence mechanisms of infectious hematopoietic necrosis virus in rainbow trout (oncorhynchus mykiss) include host entry and virus replication kinetics the evolution of myxoma virus: genomic and phenotypic characterization of isolates from great britain reveals multiple successful evolutionary pathways distinct from those in australia next step in the ongoing arms race between myxoma virus and wild rabbits in australia is a novel disease phenotype a single-amino-acid substitution in the ns1 protein changes the pathogenicity of h5n1 avian influenza viruses in mice a single amino acid in the stalk region of the h1n1pdm influenza virus ha protein affects viral fusion, stability and infectivity genetic determinants of japanese encephalitis virus vaccine strain sa14-14-2 that govern attenuation of virulence in mice effect of an 88-amino-acid deletion in nsp2 of porcine reproductive and respiratory syndrome virus on virus replication and cytokine responses in vitro the glutamic residue at position 402 in the c-terminus of newcastle disease virus nucleoprotein is critical for the virus low evolutionary rate of infectious pancreatic necrosis virus (ipnv) in italy is associated with reduced virulence in trout characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus m148r and m149r are two virulence factors for myxoma virus pathogenesis in the european rabbit genetic and structural analysis of a virulence determinant in polyomavirus vp1 sheeppox virus kelch-like gene sppv-019 affects virus virulence related links national geographic: leaky vaccines enhance spread of deadlier chicken viruses both authors contributed to all aspects of the manuscript. the authors declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. nature reviews genetics thanks s. alizon and the other, anonymous reviewer(s) for their contribution to the peer review of this work. key: cord-297303-cpajrgba authors: nguyen, annie l.; christensen, christopher; taylor, jeff; brown, brandon title: leaning on community-based participatory research to respond during covid-19 date: 2020-05-14 journal: aids behav doi: 10.1007/s10461-020-02922-1 sha: doc_id: 297303 cord_uid: cpajrgba nan we partnered with the hiv + aging research project, a community-based, nonprofit organization founded as a collaboration between local hiv clinicians and people living with hiv, to study the clinical and psychosocial aspects of aging among long-term hiv survivors. we obtained a patient-centered outcomes research institute (pcori) engagement award, which was meant to build local capacity to conduct research through community-based participatory research (cbpr) approaches. located in palm springs, california (a popular retirement community in the coachella valley with the highest proportion of people aging with hiv in the united states), we have been working over the past five years to prepare stakeholders to conduct innovative and local research. as part of this, we formed a steering committee led by research and community member co-leaders, and including older adults living with hiv, non-medical caregivers of older adults with hiv, community-based organizations that serve people living with hiv, researchers, and healthcare providers. the steering committee meets on a monthly basis, with a core team meeting weekly to be able to rapidly respond to emerging health concerns and priorities. as the covid-19 pandemic arrived, and the committee shifted to remote meetings, the urgency of the pandemic and understanding the unmet needs of the community aging with hiv became apparent. since the committee is responsive to current needs, they shifted their efforts to covid-19 with the idea to conduct a needs and post-traumatic stress disorder (ptsd) assessment survey of older adults living with hiv in the coachella valley. all stakeholders played a role in developing question topics and helped guide the creation of the survey to be most beneficial to people aging with hiv. recognizing the need for a rapid response, we designed the needs assessment as a one-time, anonymous, internet-based survey to assess the impact of covid-19 and mitigation efforts (i.e., shelter-in-place mandates, physical distancing guidelines) on daily needs, mental and physical health needs, and social isolation. we utilized tools from the national institutes of health disaster research response database (www.dr2.nlm.nih.gov), and instruments from researchers engaged in the field of hiv and aging. the initial survey was presented virtually at a steering committee meeting, and extensive feedback was received from community members and other stakeholders. once we reached consensus on questions, we then piloted the survey with 10 participants to identify steps in the outreach protocol or instruments that needed refinement. in the implementation phase of the study, we reached out to local service-based groups in the coachella valley known to assist older adults living with hiv and distributed recruitment emails to their members through their mailing lists. we were able to move from idea germination to survey implementation in less than three weeks. data collection are ongoing but we report some initial, descriptive findings here (n = 35). respondents were predominantly white (91.4%), gay men (91.4%) with a mean age of 63.5 years (sd = 7.6) and a mean of 26.1 years (sd = 8.6) living with hiv. most participants (62.9%) reported that covid-19 impacted their lives "extremely" or "very much" and 60.0% were fearful of getting covid-19 themselves, while 82.9% were worried about friends, family, and loved ones getting covid-19. some participants (20.0%) stated that they were confused about where to obtain true and accurate information about covid-19. frustration (48.6%), boredom (42.9%), sleep disruptions (48.6%), and not getting enough exercise (65.7%) were commonly reported by participants. half (54.3%) reported experiencing anxiety and 22.9% reported feelings of depression. in terms of substance use behaviors, 20.0% reported increases in alcohol use, 14.3% reported increases in marijuana use, and 5.7% reported increases in other substance use during the pandemic. one-third experienced financial challenges because of covid-19, 20.0% experienced difficulty paying for basic needs like food, electricity, and rent, and 34.3% state that they or someone in their household have requested unemployment benefits. however, only 11.4% have received unemployment benefits. some participants (11.4%) reported skipping meals or reducing portions because they were worried they didn't have enough money for food. over a quarter of participants (28.6%) reported that they missed a dose of their hiv medication during the covid-19 pandemic, with the majority stating that they forgot as the reason for missing a dose. several people further explained that covid-19 has been "a disorienting event" and that being "more stressed = more forgetful." on a likert scale ranging from 1 (not at all) to 10 (extremely), participants reported a mean of 6.3 (sd = 2.6) for feeling socially isolated, with 41.3% reporting in the upper range of 8 to 10. our initial findings suggest covid-19 has significant impacts on mental health and exercise behaviors as well as negative implications for financial well-being. importantly for people aging with hiv, covid-19 appears to be causing disruption to medication adherence. in addition to the initial findings presented here, we reiterate the key role of cbpr in making this study possible. the speed at which we were able to respond was due to several factors: (1) our team's existing networks of experts in hiv and aging, (2) an established steering committee with members who are familiar with reviewing and providing feedback about research, (3) relationships that have cultivated trust between academics and community members over time, and (4) a responsive institutional review board. our next step is to provide data in real-time to local service based groups in the coachella valley, so organizations can respond to the emergent needs of older adults living with hiv. at the completion of the study, findings will contribute to understanding the impact of covid-19 on older adults living with hiv with implications for emergency preparedness in order to better prepare for the ongoing threat of covid-19 and the next public health crisis or pandemic. there will not be an immediate end to the covid-19 pandemic and people in the most vulnerable groups, including people aging with hiv, may need to shelter-in-place long after much of their surrounding community is able to return to a semblance of normal life. centers for disease control and prevention website mental health, psychosocial challenges and resilience in older adults living with hiv growing older with hiv/aids: new public health challenges a multidimensional assessment of successful aging among older people living with hiv in palm springs, california. aids res hum retroviruses we would like to thank the members of the harp-ps steering committee for their valuable input, and our plwh community for their continuing altruism and support of this research. we also acknowledge funding from the usc clinical and translational science institute (1ul1tr001855), the patient centered outcomes research institute (4367-ruoc), and nih/nia (k01 ag064986-01) to a.n. these funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord-285443-9y2kkmby authors: pessi, antonello title: cholesterol‐conjugated peptide antivirals: a path to a rapid response to emerging viral diseases date: 2014-10-20 journal: j pept sci doi: 10.1002/psc.2706 sha: doc_id: 285443 cord_uid: 9y2kkmby while it is now possible to identify and genetically fingerprint the causative agents of emerging viral diseases, often with extraordinary speed, suitable therapies cannot be developed with equivalent speed, because drug discovery requires information that goes beyond knowledge of the viral genome. peptides, however, may represent a special opportunity. for all enveloped viruses, fusion between the viral and the target cell membrane is an obligatory step of the life cycle. class i fusion proteins harbor regions with a repeating pattern of amino acids, the heptad repeats (hrs), that play a key role in fusion, and hr‐derived peptides such as enfuvirtide, in clinical use for hiv, can block the process. because of their characteristic sequence pattern, hrs are easily identified in the genome by means of computer programs, providing the sequence of candidate peptide inhibitors directly from genomic information. moreover, a simple chemical modification, the attachment of a cholesterol group, can dramatically increase the antiviral potency of hr‐derived inhibitors and simultaneously improve their pharmacokinetics. further enhancement can be provided by dimerization of the cholesterol‐conjugated peptide. the examples reported so far include inhibitors of retroviruses, paramyxoviruses, orthomyxoviruses, henipaviruses, coronaviruses, and filoviruses. for some of these viruses, in vivo efficacy has been demonstrated in suitable animal models. the combination of bioinformatic lead identification and potency/pharmacokinetics improvement provided by cholesterol conjugation may form the basis for a rapid response strategy, where development of an emergency cholesterol‐conjugated therapeutic would immediately follow the availability of the genetic information of a new enveloped virus. copyright © 2014 european peptide society and john wiley & sons, ltd. the spectrum of infectious diseases that threaten human health is rapidly broadening: the number of previously unknown conditions that have emerged since 1970 exceeds 40, with a new disease discovered on average more than once a year [1, 2] . according to a 2008 world health organization (who) report, between the years 2002 and 2007, a record of 1100 worldwide epidemic events occurred [3] . a comprehensive examination of episodes of emerging infectious disease outbreaks between 1940 and 2004, after controlling for reporting bias, came to the conclusion that the incidence of the events has increased significantly over time [4] . for viral diseases in particular, in addition to the constant fear of a new wave of pandemic influenza, the list of emerging or reemerging viruses includes hiv [5] . at the time when this manuscript was being finalized, the largest and longest outbreak of ebola virus was still spreading [6] . the latter virus, together with nipah virus, hantavirus, lassa virus, and marburg virus, is on the centers for disease control and prevention list of bioterrorism agents. for most of these viruses, the therapeutic options are limited if not completely absent. the best description of the features of an outbreak of a novel virus comes from the sars pandemic of 2002/2003the first major emerging disease threat of the 21st century. in the short period between the who global alert (march 2003) and the who announcement that the pandemic was over (5 july 2003) , sars affected 8098 people, caused 774 deaths, disrupted international travel, and cost huge business losses. sars presented many of the features of the most feared pandemic diseases: a respiratory disease that is transmitted from person to person, with a long asymptomatic incubation period, and symptoms very similar to other commonplace illnesses. at the time of its first appearance, its causative agent (sars-associated coronavirus) was unknown, and there was neither a diagnostic test nor a specific treatment. the international response to the new threat was extremely efficacious, leading to the isolation of sars-cov [7] and sequencing of its genome in the record time of just 2 weeks [8, 9] . the measures that proved efficacious in containing the disease were isolation, quarantine, travel surveillance, increased personal hygiene, and personal protection equipment (masks, gloves, and eyeglasses). as to pharmacological treatments, however, a who expert panel review concluded that it was not possible to determine whether any of the treatments used for sars-infected patients did some benefit and suggested that some treatments might have actually been harmful [10] . as the sars case vividly illustrates, our ability to quickly isolate and genetically fingerprint the causative agent of new viral diseases is not matched by an ability to develop suitable treatments. this is not surprising, because drug discovery typically requires additional information to that simply deriving from knowledge of the viral genome. against this background, peptides may represent a special case, because for enveloped viruses, it is generally possible to identify the sequence of candidate peptide inhibitors directly from genomic information. for all enveloped viruses, fusion between the viral and the target cell membrane represents an obligatory step of the life cycle. interfering with this process is a well-established therapeutic strategy [11] that has led to the development of the peptide fusion inhibitor enfuvirtide (fuzeon ® , also known as t20, roche, basel, switzerland), which is in clinical use for hiv [12] . viral fusion is mediated by specialized proteins, which harbor a structural motif that consists of a repeating pattern of seven amino acids: the heptad repeat (hr). the most prevalent class (class i) of fusion proteins typically has two hr regions: the first one close to the n-terminus (hrn), adjacent to the fusion peptide, and the second one close to the c-terminus (hrc), immediately preceding the transmembrane domain. it is generally accepted that in the key intermediate of viral fusion, the so-called prehairpin intermediate, bridging the viral and cell membranes, the hrn and hrc regions are separated, and the hrn forms a trimeric coiled coil ( figure 1 ). folding of the hrc onto the hrn trimer leads to the formation of a six-helical bundle (6hb), and in this process, the two membranes are brought in close apposition, which leads to their fusion [11] (figure 1 ). peptides corresponding to the sequence of the hr regions can bind to the prehairpin intermediate, prevent its transition to the 6hb, and block fusion (figure 2(a) ). this is the mechanism of inhibition of enfuvirtide [12] , which applies to several other peptides derived from the hr regions of many viruses [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] . the characteristic sequence pattern of the hr provides an important advantage for the development of peptide-based antivirals, because hr can be easily identified through computer programs (e.g. learncoil [30] or multicoil [31, 32] ). this was the case for sars-cov, where hr regions were immediately identified [33] and led to the development of peptide inhibitors [27, 29] , in the absence of any structural information on the fusion protein. this came later and largely confirmed the predictions [34] [35] [36] [37] . this pathway to drug development offers the opportunity to develop a specific antiviral in a very short timeframe. however, not all hr-derived peptides display the same antiviral potency as the hiv fusion inhibitor enfuvirtide. a major factor at play is the difference in fusion kinetics: viruses that unlike hiv transition through the hairpin intermediate very rapidly offer a shorter window of opportunity for peptide inhibitors that, accordingly, show reduced potency [38] [39] [40] . if the peptide lead derived from genomic information has insufficient potency, it is necessary to modify it through peptide engineering strategies that, although often successful [18, [41] [42] [43] [44] [45] [46] [47] [48] [49] , may be time consuming. an alternative approach, which does not require any change in the native hr sequence, exploits the role of cholesterol in viral fusion. cholesterol plays a key role in the fusion and budding of enveloped viruses [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] , with hiv as the most studied case. it has been shown that cholesterol depletion from virus-infected cells suppresses virus production [60] and that depletion of cholesterol from either virions or target cells inhibits virus-cell fusion [61] [62] [63] [64] . hiv is able to influence the cholesterol metabolism of the host cell [65] [66] [67] [68] , and its lipid membrane is highly enriched in cholesterol [69] . moreover, a link has been established between low levels of cholesterol in antigen-presenting cells and the status of long-term nonprogressor, a rare infected individual who controls disease progression in the absence of antiretroviral therapy [70, 71] . the role of cholesterol in viral fusion is related to its role in membrane fluidity and in particular in the formation of lipid rafts [60, [72] [73] [74] [75] [76] . accordingly, the composition of the lipid membrane of hiv is highly enriched in cholesterol and sphingomyelin [69, 74, 75] . moreover, cd4, the primary receptor for hiv, is enriched in lipid rafts [77] . in 2009, ingallinella et al. proposed that addition of a cholesterol group to an hr-derived peptide would augment its affinity for membranes and in particular for the cholesterol-rich lipid rafts. the resulting increase in local concentration at the site of action would translate into enhanced antiviral potency (figure 2(b) ). application of this concept to the hr-derived peptide c34 yielded c34-chol, where the cholesterol group was attached, via a thioether bond, to the side chain of a cys residue added to the c-terminus, with an intervening gly-ser-gly spacer. cholesterol conjugation brought a dramatic improvement of antiviral potency: depending on the viral strain tested, c34-chol was 25-to 100-fold more potent than unconjugated c34 and 50-to 400-fold more potent than enfuvirtide [78] . no such improvement was apparent when cholesterol was substituted with palmitic acid [78] , a lipid that is both a poorer membrane anchor and is not enriched in lipid rafts [79] . numerous examples have since accumulated that cholesterol conjugation can improve the antiviral potency of hr-derived peptides from many enveloped virusesthese include in vitro and in vivo studies (table 1) . cholesterol conjugation has also been successfully applied to a covalent inhibitor derived from c34, where an isothiocyanate group on the side chain of asp 632 targets lys 574 in hrn [80] . interestingly, in line with the hypothesized mechanism of action of cholesterol conjugation, another lipid that is enriched in the hiv lipidome, dihydrosphingosine [69] , also augments potency when conjugated to an hr peptide; in this case, one derived from the hrn instead of the hrc [81] . cholesterol-conjugated peptides show improved pharmacokinetics and are active in vivo cholesterol conjugation also solves an outstanding problem of peptide therapeutics, the short in vivo half-life because of enzyme degradation and rapid clearance [82] . an effective way to improve peptide pharmacokinetics (pk) is conjugation to lipids [82] , which drive binding to serum proteins. the most typical derivatization is with long chain fatty acids, but derivatization with cholesterol has also been explored [83] [84] [85] . in a comparative subcutaneous pk study in mice at the concentration of 3.5 mg/kg, c34 was undetectable in plasma after 6 h, while 130 nm of c34-chol was still detectable 24 h after the injection: a concentration ≈300-fold higher than the ic 90 measured against multiple hiv-1 strains [78] . in another example, a single dose of 2 mg/kg of a cholesterolconjugated inhibitor of nipah virus, administered intraperitoneally to golden hamsters, was sufficient to achieve a plasma concentration >100-fold higher than the in vitro ic 50 for 24 h [86] . a single intramuscular injection of 1.6 mg/kg of a cholesterolconjugated inhibitor of newcastle disease virus (ndv) administered to chickens yielded a plasma concentration of 210 nm at 24 h and ≈120 nm after 48 h, to be compared with the in vitro ic 50 = 8.1 nm and ic 90 = 13 nm [87] . overall, the pk of a cholesterol-conjugated inhibitor is expected to be at least comparable withand likely better thanthe pk of enfuvirtide, which is administered twice daily by subcutaneous injection [88] . one further possibility to potentiate the activity of hr-derived peptide inhibitors is to exploit multimerization, another modification that can be implemented on the native sequence of the peptide. multimerization brings an avidity effect [89] , which effectively reduces the k off of the inhibitor fusion protein complex and increases the potency of fusion [90, 91] and entry [92] inhibitors. the effect of cholesterol conjugation, which increases the k on by facilitating the encounter of the peptide with the viral target protein, is complementary: the two modifications can therefore work in concert. a reagent that can simultaneously dimerize the peptide and install a cholesterol group onto it is described in figure 3 [93] . trimers or higher-order multimers could be prepared by the same strategy, but the maximum benefit of multimerization is achieved at the level of a dimer [90, 91] . the reagent is stable upon prolonged storage at à20°c. notably, the same cysteine-containing precursor peptide used to produce the monomeric cholesterol-conjugated peptide can be reacted with the cholesterol dimer-forming moiety: this enables parallel synthesis of the cholesterol-conjugated monomer and dimer (figure 3) , which can then also be tested in parallel to establish the optimal inhibitor. using the reagent in figure 3 , dimeric cholesterol-conjugated inhibitors were prepared for hiv [93] , nipah [93] , and measles [94] virus (mv), which were more potent and effective than the corresponding cholesterol-conjugated monomers. potentiation of a trimeric hiv peptide inhibitor [91] by cholesterol conjugation [95] has independently been reported by another laboratory. since its discovery in 2009, the scope of cholesterol conjugation has considerably increased, and it now includes examples for retroviruses, paramyxoviruses, coronaviruses, orthomyxoviruses, henipaviruses, and filoviruses (table 1 ). for some of these viruses, in vivo efficacy has been demonstrated in a suitable animal model [86, 87, 94] . for example, porotto et al. showed that once-daily intraperitoneal administration of a cholesterol-conjugated inhibitor of nipah virus to golden hamsters, an established model of infection, effectively prevented what would otherwise be fatal nipah virus encephalitis. prophylactic treatment was 100% efficacious when initiated 2 days before viral challenge and 80% efficacious when initiated concurrently with the infection. notably, in light of the highly lethal nature of this virus, treatment 2 days after infection led to survival of 40% of the animals [86] . the same laboratory used intranasal infection of mv in suckling mice expressing the human slam transgene as a model to evaluate the efficacy of cholesterol-conjugated hr-derived peptides. infection with mv causes a lethal acute neurological syndrome, which was completely prevented, in 100% of the animals, by prophylactic once-daily administration of a cholesterol-conjugated dimer derived from the sequence of the mv hrc domain [94] . li et al. used cholesterol conjugation to improve the potency of their previously described inhibitor of ndv [96] . intramuscular injection of the cholesterol-conjugated peptide 1 day before virus infection, and then every 3 days, protected 70% of the chickens from different serotypes of ndv [87] . moreover, treatment of the animals 2 days after infection resulted in 50% protection [87] . two interesting features of these studies deserve further comment: first, in the aforementioned three studies, the peptide was detectable in the brain 24 h after administration [86, 87, 94] , indicating that cholesterol conjugation may enable penetration of the blood-brain barrier, a difficult feat for drugs in general and for biologics in particular [97] . second, the modest but observable degree of protection observed at 2 days postexposure (40% in the porotto et al. [86] and 50% in the li et al. [87] study) is comparable with the results reported for monoclonal antibodies against ebola virus in macaques (50% efficacy when dosed 2 days after exposure) [98, 99] , including the zmapp antibody cocktail [99] , which is being used as an emergency therapeutic in the latest ebola outbreak [100] . it must be noted though that all the examples reported so far come from viruses featuring class i fusion proteins. while being the most prevalent class, it does not include major pathogens such as hepatitis c virus and dengue virus, both belonging to class ii, and herpes simplex virus belonging to class iii. class i proteins are mainly α-helical and form 6hbs as the postfusion structure, as exemplified by hiv (figure 1 ). class ii fusion proteins are characterized by trimers of hairpins composed of β-structures [101] , with a fusion loop located at the tip of an elongated β-sheet, in place of a fusion peptide upstream of an α-helix. class iii proteins form trimers of hairpins by combining a central α-helical trimeric core, similar to class i proteins, with fusion loops structurally similar to class ii proteins [101] . despite these structural differences, all fusion proteins adopt a hairpin structure [102] that is susceptible to inhibition. for example, although the entry of herpes viruses (featuring a class iii fusion protein) is a complex process, not yet fully clarified, in which multiple glycoproteins are involved, several laboratories have reported that peptides derived from the hr regions of gb and gh of herpes simplex virus type 1, bovine herpes virus type 1, and human cytomegalovirus are able to inhibit infection [103] . for dengue virus, helical domains have been identified in the stem region of e protein (a class ii protein) that are critical for virus assembly and entry [104] . peptide inhibitors derived from this membrane-binding region have been described [105] [106] [107] , one of which inhibits all four virus serotypes [107] . moreover, de novo computational design of peptide inhibitors has been successful [108, 109] . the work by xu et al. in particular [109] indicates how the same computational approach may yield candidate peptide inhibitors for fusion proteins of all classes. given the ubiquitous importance of cholesterol-rich lipid rafts in viral fusion, it is expected that cholesterol conjugation may enhance the antiviral potency also for peptides derived from class ii and iii fusion proteins. however, no data in this regard have yet been reported. the combination of bioinformatic lead identification (lead id) and potency/pk improvement provided by cholesterol conjugation may form the basis for a rapid response strategy to emerging viral diseases (figure 4) . the project would begin immediately after the genome of the new pathogen is made available. the lead id phase would consist in the bioinformatic analysis (e.g. via learncoil [30] or multicoil [31, 32] ) of the newly sequenced genome to identify the hr regions; it could last a few days. as discussed in the previous section, the computational identification of inhibitory peptides for viruses utilizing class ii or iii fusion proteins would be less straightforward, and the number of candidate peptides to be tested likely larger. however, given the high throughput of parallel solid-phase figure 4 . rapid response strategy based on cholesterol-conjugated fusion inhibitors. from the sequenced genome of the emerging virus, the hr regions are identified bioinformatically. this corresponds to the lead identification (lead id) phase. in the lead optimization (lead opt) phase, candidate hr peptides are synthesized in the form of suitable precursors for cholesterol conjugation and reacted with the reagents shown in figure 3 . the cholesterol-conjugated monomer and dimer are tested for antiviral activity. the lead opt cycle continues until a peptide is found potent enough to be moved to large-scale production. peptide synthesis and the modularity of the cholesterol conjugation reaction, the initial number of target sequences should not represent a key limiting factor in the lead optimization (lead opt) phase. in lead opt, candidate peptides would be synthesized in the form of suitable precursors for cholesterol conjugation and reacted with the reagents shown in figure 3 or similar ones. the cholesterol-conjugated monomers and dimers would be tested for antiviral activity. in the logic of off-the-shelf reagents for immediate use, variations of the reagents of figure 3 to optimize the length of the linker joining the monomers might provide considerable increase in potency [89] , as shown by kay et al. [91] . haloacetyl or maleimide-functionalized cores with polyethylene glycol spacers of different length could all be reacted with the same precursor to rapidly select the most potent inhibitor. the lead opt cycle would continue until a peptide was found, potent enough to be moved to large-scale production. with a relatively small number of candidate peptides, and parallel synthesis and conjugation, it is expected that the lead opt phase would be rapid, optimally a few weeks. large-scale production of the candidate peptide therapeutic could begin immediately, because the protocol for lab-scale and large-scale solid-phase peptide synthesis are essentially the same. preclinical evaluation (formulation, animal pk to predict the human dose) could proceed in parallel with the good manufacturing practices batch production. abbreviated preclinical toxicology, in connection with the 'animal rule' [110] , would be justified in an emergency situation. the strategy outlined in the previous section could provide in a very short timeframe a cholesterol-conjugated therapeutic specific for the emerging virus. although not necessarily the optimal drug for the new disease, it would represent a key complement to preventive measures and could enable better control of the initial outbreak. further research efforts may then lead to a substitute therapy with increased efficacy and/or better ease of administration. one could even envisage setting up a core laboratory that routinely runs the lead id and lead opt stages on newly identified viruses of potential concern, in advance of an actual outbreak of disease. the limited resources necessary could be part of the emergency and preparedness response set out by the centers for disease control and prevention (http://emergency.cdc.gov/) or the global alert and response by who (http://www.who.int/csr/en/) for preparing for and responding to public health emergencies. emerging and reemerging diseases: a historical perspective emerging viral diseases the world health report global trends in emerging infectious diseases middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group world struggles to stop ebola identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome sars: systematic review of treatment effects mechanisms of viral membrane fusion and its inhibition design of recombinant protein-based sars-cov entry inhibitors targeting the heptad-repeat regions of the spike protein s2 domain analysis of a peptide inhibitor of paramyxovirus (ndv) fusion using biological assays, nmr, and molecular modeling inhibition of henipavirus fusion and infection by heptad-derived peptides of the nipah virus fusion glycoprotein development of antiviral fusion inhibitors: short modified peptides derived from the transmembrane glycoprotein of feline immunodeficiency virus peptide inhibitors targeting virus-cell fusion in class i enveloped viruses downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency peptides corresponding to the heptad repeat sequence of human parainfluenza virus fusion protein are potent inhibitors of virus infection a synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of sendai virus-cell fusion: an emerging similarity with functional domains of other viruses antiviral activity of membrane fusion inhibitors that target gp40 of the feline immunodeficiency virus envelope protein peptide-based inhibitors of the hiv envelope protein and other class i viral fusion proteins inhibition of ebola virus entry by a c-peptide targeted to endosomes peptides from conserved regions of paramyxovirus fusion (f) proteins are potent inhibitors of viral fusion inhibition of hendra virus fusion inhibition of measles virus infection and fusion with peptides corresponding to the leucine zipper region of the fusion protein severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeat-derived peptides identification of a minimal peptide derived from heptad repeat (hr) 2 of spike protein of sars-cov and combination of hr1-derived peptides as fusion inhibitors interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors learncoil-vmf: computational evidence for coiled-coil-like motifs in many viral membrane-fusion proteins multicoil2: predicting coiled coils and their oligomerization states from sequence in the twilight zone multicoil: a program for predicting two-and three-stranded coiled coils the complete genome sequence of severe acute respiratory syndrome coronavirus strain hku-39849 (hk-39) structural characterization of the sars-coronavirus spike s fusion protein core structures and polymorphic interactions of two heptad-repeat regions of the sars virus s2 protein structural characterization of the fusion-active complex of severe acute respiratory syndrome (sars) coronavirus crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core sensitivity of hiv-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics a quantitative and kinetic fusion protein-triggering assay can discern distinct steps in the nipah virus membrane fusion cascade kinetic dependence of paramyxovirus entry inhibition broad antiviral activity and crystal structure of hiv-1 fusion inhibitor sifuvirtide short-peptide fusion inhibitors with high potency against wild-type and enfuvirtide-resistant hiv-1 design of helical, oligomeric hiv-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus design of a novel hiv-1 fusion inhibitor that displays a minimal interface for binding affinity remodeling of gp41-c34 peptide leads to highly effective inhibitors of the fusion of hiv-1 with target cells inhibition of hiv type 1 infectivity by constrained alpha-helical peptides: implications for the viral fusion mechanism short constrained peptides that inhibit hiv-1 entry hydrocarbon doublestapling remedies the proteolytic instability of a lengthy peptide therapeutic design and evaluation of sifuvirtide, a novel hiv-1 fusion inhibitor lipid rafts are involved in sars-cov entry into vero e6 cells cholesterol enhances mouse hepatitis virus-mediated cell fusion a single point mutation controls the cholesterol dependence of semliki forest virus entry and exit cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry the major cellular sterol regulatory pathway is required for andes virus infection lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles ebola virus entry requires the cholesterol transporter niemann-pick c1 cholesterol dependence of varicella-zoster virion entry into target cells influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion plasma membrane rafts play a critical role in hiv-1 assembly and release membrane raft microdomains mediate lateral assemblies required for hiv-1 infection role for human immunodeficiency virus type 1 membrane cholesterol in viral internalization role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected t cells human immunodeficiency virus impairs reverse cholesterol transport from macrophages nef increases the synthesis of and transports cholesterol to lipid rafts and hiv-1 progeny virions nef mobilizes lipid rafts in macrophages through a pathway that competes with abca1-dependent cholesterol efflux the hiv lipidome: a raft with an unusual composition alterations in cholesterol metabolism restrict hiv-1 trans infection in nonprogressors new clues to understanding hiv nonprogressors: low cholesterol blocks hiv trans infection evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions lipid composition and fluidity of the human immunodeficiency virus lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes role of lipid rafts in virus replication the pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation addition of a cholesterol group to an hiv-1 peptide fusion inhibitor dramatically increases its antiviral potency membrane targeting of lipid modified signal transduction proteins a multi-functional peptide as an hiv-1 entry inhibitor based on self-concentration, recognition, and covalent attachment sphingopeptides: dihydrosphingosine-based fusion inhibitors against wild-type and enfuvirtide-resistant hiv-1 converting peptides into drug leads by lipidation structure-activity and protraction relationship of longacting glucagon-like peptide-1 derivatives: importance of fatty acid length, polarity, and bulkiness glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice dpp-iv-resistant, long-acting oxyntomodulin derivatives inhibition of nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry a cholesterol tag at the n terminus of the relatively broad-spectrum fusion inhibitory peptide targets an earlier stage of fusion glycoprotein activation and increases the peptide's antiviral potency in vivo enfuvirtide: a fusion inhibitor for the treatment of hiv infection dependence of avidity on linker length for a bivalent ligand-bivalent receptor model system potent d-peptide inhibitors of hiv-1 entry design of a potent d-peptide hiv-1 entry inhibitor with a strong barrier to resistance designed oligomers of cyanovirin-n show enhanced hiv neutralization a general strategy to endow natural fusion-protein-derived peptides with potent antiviral activity fatal measles virus infection prevented by brain-penetrant fusion inhibitors design of a modular tetrameric scaffold for the synthesis of membrane-localized d-peptide inhibitors of hiv-1 entry characterisation and evaluation of antiviral recombinant peptides based on the heptad repeat regions of ndv and ibv fusion glycoproteins drug transport across the blood-brain barrier successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies therapeutic intervention of ebola virus infection in rhesus macaques with the mb-003 monoclonal antibody cocktail experimental ebola drugs enter the limelight virus membrane fusion virus membrane-fusion proteins: more than one way to make a hairpin peptide inhibitors against herpes simplex virus infections the helical domains of the stem region of dengue virus envelope protein are involved in both virus assembly and entry peptide inhibitors of dengue-virus entry target a late-stage fusion intermediate peptide inhibitors of flavivirus entry derived from the e protein stem release of dengue virus genome induced by a peptide inhibitor structural optimization and de novo design of dengue virus entry inhibitory peptides computational identification of self-inhibitory peptides from envelope proteins new drug and biological drug products; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible. final rule viral entry inhibitors targeted to the membrane site of action capturing a fusion intermediate of influenza hemagglutinin with a cholesterol-conjugated peptide, a new antiviral strategy for influenza virus c-peptide inhibitors of ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking key: cord-292521-tpb12dkq authors: howard, john; thompson, thomas z.; macarthur, rodger d.; rojiani, amyn m.; white, joseph title: widely disseminated cryptococcosis manifesting in a previously undiagnosed human immunodeficiency virus (hiv)-positive 18-year-old date: 2020-10-12 journal: am j case rep doi: 10.12659/ajcr.924410 sha: doc_id: 292521 cord_uid: tpb12dkq patient: male, 18-year-old final diagnosis: disseminated cryptococcosis in previously undiagnosed hiv symptoms: diarhea • lymphadenopathy medication: — clinical procedure: — specialty: general and internal medicine • pathology objective: unusual clinical course background: after initial infection with hiv, loss of cd4+ t cells progresses along a predictable timeline. the clinical latency stage lasts an average of 10 years, until the cd4+ t cell count falls below 200 cells/ul or the patient develops an aids-defining opportunistic infection/cancer. this report describes an unusual opportunistic infection in a young patient with no prior clinical evidence of hiv infection. case report: an 18-year-old man presented with fever, abdominal pain, and dyspnea for the previous 2 weeks and was symptomatically treated for gastroenteritis. he presented 2 weeks later with extreme fatigue, and a ct scan revealed diffuse lymphadenopathy. he was transferred to a regional hospital, but upon arrival and prior to detailed investigative work-up, he developed cardiac arrest. despite maximal resuscitative efforts, he died approximately 8 h after admission. at autopsy, diffuse lymphadenopathy, splenomegaly, and pulmonary congestion were noted. disseminated cryptococcal infection involving almost every organ system was identified at autopsy. a postmortem hiv-1 antibody test was positive. the cause of death was severe immunodeficiency as a result of advanced hiv infection resulting in disseminated cryptococcal infection, with cerebral edema, herniation, and respiratory failure. conclusions: this patient’s non-specific symptoms in conjunction with his rapid decline made arriving at a correct diagnosis challenging. only during autopsy was the disseminated fungal infection discovered, leading to suspicion of hiv infection. hiv autopsies are not uncommon, but the clinical history is usually known beforehand. this case report highlights the importance of considering hiv-related conditions in patients presenting with this array of symptoms, as well as to alert healthcare providers and staff to the need for increased biosafety precautions. in most cases, following primary infection with hiv, the loss of cd4+ t cells continues over a fairly predictable, extended period of time. the period during which the body's defenses attempt to bring the virus under control, at least to a more stable set point, is defined as the clinical latency stage, which lasts an average of 10 years, until the cd4+ t cell count falls below 200 cells/µl or the patient develop an aids-defining opportunistic infection/cancer. the patient described here had no clinical evidence of hiv infection or other manifestation of the disease. because he remained asymptomatic before presentation, there was no indication of a need for hiv testing. his clinical presentation is highly unusual in that his initial non-specific gastrointestinal symptoms and fatigue did not in any way raise suspicion of hiv infection, and his subsequent diffuse lymphadenopathy was suspected to be lymphoma. the identification of disseminated cryptococcosis at autopsy in the absence of prior clinical evidence of hiv infection led to a postmortem diagnosis of aids. this case may have been acute retroviral syndrome; however, since the patient had no prior testing or evidence of hiv infection, it is impossible to confirm that diagnosis [1] . this case report presents important information by defining the very extensive dissemination of cryptococcosis and discussing the implications for differential diagnosis. additionally, patient care and exposure at autopsy are important considerations in the absence of a prior hiv diagnosis. an 18-year-old african american man presented to the hospital with complaints of fever, nausea, dyspnea, and abdominal pain over the previous 2 weeks. he was subsequently symptomatically treated for gastroenteritis without additional workup, and blood culture, stool analysis, and colonoscopy were not performed. this case occurred before the present covid-19 pandemic. this was his first clinical episode and he otherwise appeared to be healthy, with no evidence of cachexia. he had no history of recent travel, prior surgery, or blood transfusion. there was no social history of sexual activity or preference. he returned 2 weeks later with extreme fatigue to the point of being unable to walk, at which point a ct of his chest, abdomen, and pelvis was ordered, which revealed diffuse lymphadenopathy. a lymphoma was suspected, and the patient was admitted for work-up (day 1). a complete blood count (cbc) was also ordered, which demonstrated a leukocytosis (15 200 cells/µl) with an absolute and relative neutrophilia (90%). soon after admission, before the patient could undergo additional investigative studies including lymph node biopsy or complete pulmonary work-up, he rapidly declined and developed respiratory distress requiring intubation. he was transferred to a regional medical center for a heightened level of care. upon evening arrival, he exhibited altered mental status, prompting a chest x-ray that revealed bilateral hilar adenopathy and right lower-lobe predominant consolidations. subsequent x-rays over the next several hours showed increasing levels of pulmonary edema. another cbc was ordered, which showed a further increase in wbc (16 900 cells/µl), with 93% neutrophils (15 700 cells/µl) and 5% lymphocytes (850 cells/µl), as well as hemoglobin and hematocrit values of 11.5 qm/dl and 36.2%, respectively. a complete metabolic panel revealed elevated ast (147 iu/l) and alt (62 iu/l), as well as extremely high crp (6.40 mg/dl) and procalcitonin (5.33 ng/ml) levels. the following morning (day 2), he had a sharp decline in hemodynamic status and had a cardiac arrest secondary to ventricular tachycardia. he received multiple rounds of advanced cardiac life support over the next 4 h, but died despite maximal resuscitation efforts. an unrestricted autopsy was performed on this generally normal-appearing man, with no evidence of cachexia, no external wounds, rash, icterus, or other obvious pathology. autopsy findings demonstrated diffuse lymphadenopathy observed in the mediastinal, retroperitoneal, mesenteric, and inguinal groups, splenomegaly, and pulmonary congestion. histologic examination of submitted tissues revealed disseminated cryptococcal infection involving the spleen ( figure 1a hiv is spread through contact with certain human body fluids, after which the virus invades and attacks the host's immune system. hiv infects host cd4 + t cells via interaction between an hiv envelope-bound protein called env, and host cell cd4, ccr5, and cxcr4 receptors [2] . the hiv protein env extends from the virion and consists of 2 glycoproteins, gp120 and gp41, which make up the cap and stem, respectively, of env [2] . gp120 is required for binding to host cd4 receptors, while gp41 plays a role in promoting fusion of the viral envelope and host cell plasma membrane after receptor binding [2] . the hiv virus then uses host cell machinery to reproduce, destroying these cells in the process. cd4 + t cells are a vital part of the human immune system; therefore, their destruction by the hiv virus weakens the body's overall immune capabilities. after initial seroconversion due to infection with hiv, progressive loss of cd4+ t cells follows a fairly predictable timeline. there is typically a clinical latency stage, followed by clinical evidence of aids. the clinical latency stage develops as the body's immune response is able to substantially, but not completely, lower the viral load. the clinical latency stage for patients who are not receiving treatment lasts an average of 10 years, until the cd4+ t cell count falls below 100-200 cells/µl. infected individuals not receiving treatment eventually progress to the third stage of hiv infection, known as aids, when their cd4 + t counts drop below 200 cells/µl or they develop an aids-defining opportunistic infection/cancer. the acute hiv infection stage, also known as acute retroviral syndrome (ars), typically presents (in 50% or more of patients) with symptoms 2-4 weeks after seroconversion [1] . symptoms during this stage can last from a few days to several weeks, and fall under 4 broad categories as defined by braun et al. [3] : neurological, constitutional, gastrointestinal, and skin/mucosal (table 1) . typical ars is defined as the presence of a fever plus at least 1 of the 17 most common symptoms associated with ars, or, in the absence of fever, at least 2 of the 17 symptoms listed in table 1 [3] . during the typical presentation of ars, the cd4 + t cell count usually falls rapidly as the virus replicates and may be followed by acute infections or other manifestations. in our patient, both of these possibilities remain viable options. however, because the patient presented to us at the autopsy stage, we are unable to confirm the precise pathogenesis. he was not previously known to be hiv-infected, and was unable to give any specific history to the health care providers. it is possible that the patient had latent hiv infection for many years prior, but was asymptomatic and not cachexic, and developed disseminated cryptococcosis when his immunocompromised status crossed a threshold level with a cd4 + t cell count of less than 100-200 per µl. on average, progression from initial presentation to a cd4 + t cell in this range takes about 10 years. however, higher hiv rna levels are associated with faster decline in cd4 + t cell counts, such that our patient's decline could have occurred in less than 5 years. many individuals in the usa become infected with hiv as early as age 13 years. another possibility is that our patient could have been congenitally infected. while less likely, the possibility also remains that this represents the acute retroviral syndrome presenting 6 weeks or so after initial exposure to hiv. ec state that treatment should proceed in 3 stages: induction, consolidation, and maintenance. induction begins with interval treatments of iv amphotericin b in conjunction with oral flucytosine for a minimum of 2 weeks [7] . after moderate clinical improvement, treatment can move to the consolidation stage, in which fluconazole (400-800 mg daily) is indicated for at least 8 weeks. finally, the dosage of fluconazole can be decreased to a maintenance dose (200 mg/day), which should be taken for at least 1 year [5] . despite advances in both hiv and cryptococcosis treatment, ec remains a major cause of mortality in hiv-positive individuals, with mortality rates for untreated individuals ranging from 22% to 96% at 10-12 weeks, and 100% for untreated cryptococcal meningoencephalitis [8, 9] . cryptococcal meningoencephalitis can result in death by causing cerebellar tonsillar herniation leading to cardiopulmonary collapse, as was seen in our patient. cryptococcal infection is most commonly due to cryptococcus neoformans and cryptococcus gattii. the most common site of infection is the lungs, with macroscopic, tan-to-white nodules that can demonstrate central necrosis. when dissemination occurs, the brain and skin are the most common secondary infection sites. skin lesions can appear as molluscum contagiosum-like raised, umbilicated papules or weeping, ulcerated lesions. brain lesions are often cystic and periventricular. microscopically, immunocompetent patients demonstrate granulomas with multinucleated giant cells containing the fungal organisms. these granulomas can become necrotizing, with surrounding histiocytes. in immunocompromised patients, inflammation is minimal, with expansion within alveolar spaces or production of cryptococcomas in other organs. the fungal organisms are often variable in size, with a thick, refractile-appearing capsule. we present the case of an 18-year-old man with no evident prior clinical disease presenting with symptoms of fever, nausea, dyspnea, and abdominal pain, which were followed 2 weeks later by diffuse lymphadenopathy. he died shortly thereafter and at autopsy showed disseminated cryptococcal infection and hiv positivity. the time from initial presentation to death was only 16 days. the patient's non-specific symptoms in conjunction with his rapid decline made arriving at a correct diagnosis challenging. only during autopsy was the disseminated fungal infection discovered, leading to suspicion of hiv infection. hiv autopsies are not uncommon, but the clinical history is often known beforehand. this case report highlights the importance of considering hiv-related conditions in patients presenting with the discussed array of symptoms. early diagnosis can greatly improve patient outcomes and alert healthcare providers and staff to the need for increased biosafety precautions. clinical suspicion of hiv infection should be high for individuals in at-risk groups, as the presentation can be highly variable or atypical. lack of understanding of acute hiv infection among newly-infected persons-implications for prevention and public health: the nimh multisite acute hiv infection study: ii hiv: cell binding and entry. cold spring harb perspect med frequency and spectrum of unexpected clinical manifestations of primary hiv-1 infection centers for disease control and prevention: aids and opportunistic infections cryptococcosis -guidelines for the prevention and treatment of opportunistic infections in adults and adolescents with report of 12 cases and review of the literature treatment of cryptococcal meningitis in peruvian aids patients using amphotericin b and fluconazole clinical presentation, natural history, and cumulative death rates of 230 adults with primary cryptococcal meningitis in zambian aids patients treated under local conditions long-term mortality and disability in cryptococcal meningitis: a systematic literature review according to the cdc, the most prevalent opportunistic infections for hiv-positive individuals living in the usa are candidiasis of the bronchi, trachea, esophagus, or lungs, coccidioidomycosis, extrapulmonary cryptococcosis, and cytomegalovirus [4] . extrapulmonary cryptococcosis (ec) in immunocompromised hosts can affect any organ in the body, but it most commonly manifests as cryptococcal meningoencephalitis, with associated symptoms including fatigue, headache, altered mentation, stiff neck, and fever [5] . other associated symptoms include skin rash, dyspnea, and cough, which are indicative of disseminated cryptococcal infection [6] . our patient did not have any skin manifestations. the diffuse lymphadenopathy was not investigated because of the unstable condition of the patient and his subsequent cardiac arrest. however, at autopsy, the nodes were seen to be severely involved with cryptococcosis, with no evidence of lymphoma. the guidelines for treatment of none. key: cord-269194-b1wlr3t7 authors: engstrom-melnyk, julia; rodriguez, pedro l.; peraud, olivier; hein, raymond c. title: chapter 5 clinical applications of quantitative real-time pcr in virology date: 2015-12-31 journal: methods in microbiology doi: 10.1016/bs.mim.2015.04.005 sha: doc_id: 269194 cord_uid: b1wlr3t7 abstract since the invention of the polymerase chain reaction (pcr) and discovery of taq polymerase, pcr has become a staple in both research and clinical molecular laboratories. as clinical and diagnostic needs have evolved over the last few decades, demanding greater levels of sensitivity and accuracy, so too has pcr performance. through optimisation, the present-day uses of real-time pcr and quantitative real-time pcr are enumerable. the technique, combined with adoption of automated processes and reduced sample volume requirements, makes it an ideal method in a broad range of clinical applications, especially in virology. complementing serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses, real-time pcr provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points that allows for more timely predictions of therapeutic failures than traditional methods and, lastly, assessing cure rates following targeted therapies. all of these serve vital roles in the continuum of care to enhance patient management. beyond this, quantitative real-time pcr facilitates advancements in the quality of diagnostics by driving consensus management guidelines following standardisation to improve patient outcomes, pushing for disease eradication with assays offering progressively lower limits of detection, and rapidly meeting medical needs in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. processes, small sample volumes and can be utilised in a wide variety of applications, making it the method of choice in today's molecular laboratories. through the aid of fluorescent signalling probes to measure amplification of dna at each pcr cycle, at the point of exponential dna accumulation, real-time pcr is able to provide broader linear dynamic ranges and increased assay performance as determined by sensitivity, specificity, precision, and reproducibility. due to the consistency in signal intensity changes during the exponential growth phase of pcr, it is also easily adaptable for quantitative reporting. however, there are three properties that are uniquely associated with quantitative real-time pcr: quantification, standardisation, and lower limit resulting. the accumulation of fluorescence signal is measured at each pcr cycle of the reaction and the cycle at which this signal exceeds a predetermined background fluorescence threshold during the logarithmic phase of amplification is referred to as the cycle threshold (c t ). the c t value is inversely proportional to the viral copy number in the specimen, and through comparisons of this value to an external calibration curve or an internal quantitation standard, the initial nucleic acid target concentration can be calculated (heid, stevens, livak, & williams, 1996; livak & schmittgen, 2001) . however, accurate quantitation within each sample is hindered when relying solely on an external standard as amplification efficiencies for each individual sample may be variable and inconsistent. by utilising a standard internal reference template, with the rationale that any variable influencing amplification efficiency should example of amplification and detection of target nucleic acid by real-time pcr. affect both template and target similarly, inhibition and amplification effects are compensated for which allows for more accurate quantitation ( figure 2 ). this control can be further enhanced when incorporating an internal reference that utilises the same primer sequence as the target since any potential additional effects on pcr efficiency for each of the two targets is eliminated. thus, the competitive real-time pcr strategy is the most reliable approach for nucleic acid quantitation (diviacco et al., 1992; gilliland, perrin, & bunn, 1990; stieger, demolliere, ahlborn-laake, & mous, 1991; wang, doyle, & mark, 1989; zentilin & giacca, 2007) and is the basis for the majority of present-day virology assays. it is equally important to utilise appropriate quantitation standards, when available, to ensure accurate quantitative results, inter-laboratory correlation, and overall standardisation. standardisation of reported viral loads ensures not only interlaboratory consistency but also high clinical utility of viral load monitoring, sets the foundation for establishing clinical correlations and critical thresholds leading to better management of infections and treatments, and are critical for the development of clinical guidelines (miller et al., 2011) . with the wide availability of assay methods, viral targets, specimen type, and lack of standard reference material (hayden et al., 2012) , viral load variability across laboratories can range significantly, as high as 4.3 log copies/ml . specifically, results from proficiency testing/external quality assessment programmes as well as interlaboratory specimen exchange studies have demonstrated that there is significant variability in quantitative results for assays that lack appropriate standards quantitation of viral target using competitive quantitation standard (qs). the qs compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation viral target in each specimen. the competitive qs contains sequences with identical primer binding sites as the viral target to ensure equivalent amplification efficiency and a unique probe binding region that distinguishes the two amplicons. the competitive qs is added to each specimen at a known copy number and is carried through the subsequent steps of specimen preparation, reverse transcription (when applicable), simultaneous pcr amplification, and detection. viral target concentration in the test specimens is calculated by comparing the viral target signal (solid line) to the qs signal (dashed line) for each specimen and control (a, b) . in the presence of inhibitors, both qs and viral target are equally suppressed and yield accurate viral load calculations (c). 1 introduction (hayden et al., 2008; pang et al., 2009; preiksaitis et al., 2009; wolff, heaney, neuwald, stelrecht, & press, 2009 ). findings such as these reinforce the fact that with this high degree of variability and discrepancy, clinicians are unable to compare test results between two different laboratories and, further, clinically relevant cut-offs set by one test would not apply to results of another . without standardisation, the quality of patient care is dramatically impacted, preventing meaningful inter-laboratory comparison of patient results and influencing disease prevention and management programmes (kraft, armstrong, & caliendo, 2012) . this is especially critical for transplant patients, who may be initially monitored at one institution and then transferred to another for longer-term follow-up receiving results that no longer correlate. therefore, whenever possible, viral load monitoring tests must report results in iu/ml and be fully traceable to the higherorder first who international standard. they must generate highly accurate and reliable results based on a robust calibration methodology and have excellent reproducibility across the dynamic range of the test with demonstrated co-linearity to the who standard. lastly, there exist two distinct end-points with quantitative real-time pcr, which should be of consideration for result interpretation and reporting: the lower limit of detection (llod/lod) and the lower limit of quantitation (lloq/loq). these two limits are assessed differently and are not equivalent in either definition or, in some cases, their assigned values. the lod (also referred to as analytical sensitivity) represents the lowest viral load level at which !95% of tested samples are detected (clsi ep17-a, 2004); theoretically, viral levels at or below the lod are not detected !5% of the time. it differentiates between 'detectable' and 'undetectable' results. the lloq, on the other hand, is the lowest viral level that is within the linear and analytically acceptable range of the assay (clsi ep17-a, 2004) . in other words, the lloq is the lowest point at which an accurate viral load can be assigned and determines which 'detectable' sample will have a reported viral load. a common misconception is that the lod of the assay is the minimum viral level for a 'detected' result but 'undetectable' and 'detectable' viral levels are never differentiated by a single theoretical viral threshold as viral levels less than the lod may still have a high probability of being detected. this probability spans a broad range in which the lower the viral titre, the more likely the 'undetectable' result. ultimately, the statistical probability will favour the 'undetectable' result ( figure 3 ). and because the lloq can be equal or greater than the lod on some viral load assays, it is not unusual for 'detectable but below the loq' (detectable/bloq) result reporting (cobb et al., 2011) . further, the 'detectable/bloq' results should not be inferred that the actual viral concentration of the sample is between the lod and loq. the clinical demand has driven and shaped the evolution of pcr and continues to do so as we gain a greater understanding of the infections we monitor and treat. through the study of the natural history and disease progression attributed to specific viral infections, the need for sensitive, accurate, precise, reproducible, and reliable quantitative measurements of viral levels has become a necessity. with the deeper understanding of the natural history of human immunodeficiency virus (hiv) infections, it is now well understood that progressive immunosuppression and the onset and development of clinical disease are strictly associated with increasing viral burden (furtado, kingsley, & wolinsky, 1995; ho, moudgil, & alam, 1989; mathez et al., 1990; nicholson et al., 1989; schnittman et al., 1990) . thus, quantitative real-time pcr is critical for monitoring patients infected with hiv (hufert et al., 1991; mellors et al., 1995) and those undergoing antiretroviral therapy (art) to ensure viral replication is sufficiently and effectively suppressed and to monitor potential for viral resistance to the medication (dhhs hiv, 2014) . this monitoring and maintained viral suppression is absolutely necessary not only to maintain progression-free survival of hiv-infected patients but also to reduce subsequent hiv transmission (cohen et al., 2011; diffenbach, 2012) . due to the significance of viral load monitoring and maintaining viral suppression, the demand for likelihoods of different test results given different viral concentration. when the viral concentration tends to 0, the proportion of 'target not detected' increases to 1 (dotted line), increasing the likelihood of 'not detected' results. as the concentration tends to lloq (dashed line), the likelihood of 'detected but <lloq' results peaks. when the concentration tends to infinity, the proportion of quantitative results tends to 1 (solid line), resulting in a continual increase in the likelihood of 'detected: quantitative' results. at any concentration, the sum of the three types of reported results is always 100% and throughout the concentration continuum, variations in result reporting exist. as the concentration of viral levels approaches the lloq, near equal likelihood of 'detected: quantitative' and 'detected but <lloq' results are possible, as are 'not detected' results. decreasing concentrations will further shift the likelihoods and increase the chance of 'detected but <lloq' or 'not detected' results. diagram assumes lloq ¼ llod. increasingly more sensitive assays for hiv has driven the innovation of diagnostic tests and continues to push the limits of the lod and loq ever lower (glaubitz et al., 2011; sizmann et al., 2010) . additionally, the amount of viral dna in samples from either hepatitis b virus (hbv) or cytomegalovirus (cmv) chronic carriers is indicative of active viral replication in liver cells and is correlated with liver disease progression (chen, lin, et al., 2006; chen, yang, et al., 2006; emery et al., 2000; humar et al., 1999; humar, kumar, boivin, & caliendo, 2002; iloeje et al., 2006; wursthorn, manns, & wedemeyer, 2008) , highlighting the need for quantitative viral levels in chronic disease monitoring. similarly, hepatitis c virus (hcv) levels have been linked to prognosis and treatment outcomes (trepo, 2000) , treatment response (pearlman & ehleben, 2014; zeuzem et al., 2009) , as well as assessing sustained virologic response (svr), or 'cure', following treatment (pearlman & traub, 2011) . more recently, as new antiviral therapies for hcv treatment rely on targeting specific biological steps of the viral replication cycle, reliable quantitative monitoring of the viral burden at specific time-points to ensure treatment compliance and resistance emergence during treatment is recommended (au & pockros, 2014 ; aasld/idsa/ias-usa, 2014). alongside the changing clinical needs, several instrument and manufacturing innovations have been introduced to meet these newer requirements. the first pcrbased diagnostic test and the first automated system were both introduced in the early 1990s, allowing for improved standardised technique, increased efficiency, and reduction in error and contamination. in 1996, to meet the clinical needs of monitoring patients infected with hiv, the first 'personalised healthcare' diagnostic test was fda approved, that monitored whether art was working and whether a patient was on optimal treatment. entire systems were introduced in the early 2000s that automated the up-front sample preparation step, leading to further reduction of hands-on time, increased reliability and reproducibility. with the introduction of real-time pcr techniques to further enhance assay performance, the first pcr tests that allowed for simultaneous amplification and detection were introduced in 2003. throughout the decade, improvements in assays and instrumentation began pushing the boundary for hiv-1 detection, achieving greater and greater sensitivity, a feat that has proven to be incredibly relevant in understanding risks of viral load rebound and virologic failure (doyle et al., 2012; estevez et al., 2013; pascual-pareja et al., 2010) . more recently, shortly after the release of two new higher-order standards for cmv (first who international standard and nist cmv standard), fully automated real-time quantitative pcr assays were fda approved to meet laboratory and clinical demands for standardisation. not only does the instrumentation reduce assay design variability and inconsistency across laboratories, but also the assays are standardised to the who and report in international units, providing accuracy across the entire dynamic range that is only achieved by calibration and traceability to these higher-order standards. but it is not until these fda-approved tests gain widespread use will inter-laboratory agreement for cmv viral load results truly improve (kraft et al., 2012) . the objective of test innovation is to evolve and adapt to clinical and laboratory needs. as patient outcome gaps are identified, and as we gain greater understanding and insight into the clinical progression of disease and disease management, technological achievements facilitate advancements in quality of diagnostics and patient outcomes. life-threatening illnesses convert to chronic/manageable disease with the aid of viral load monitoring. and pressure exists to develop more precise, accurate, sensitive assays, which, in turn, drives the development of more efficacious drugs. this synergy demonstrates the value of diagnostics: it is an integral part of the patient care continuum. applications of quantitative real-time pcr for virology are extensive. it is especially necessary when antibody seroconversion is delayed after an acute infection and early diagnosis are essential (dibiasi & tyler, 2004; thomson et al., 2009) , in immunocompromised patients that may not have an optimal antibody response (kadmon et al., 2013) , and for the diagnosis of congenital or perinatally acquired viral infections (park, streicher, & rothberg, 1987; young, nelson, & good, 1990) . additionally, it is critical in maintaining a high level of patient care at each stage of the disease and infection (figure 4 ). screening tests are designed with one key parameter in mind: exceptional sensitivity (herman, gill, eng, & fajardo, 2002) . they must ensure that disease is not missed in a population that is generally free from risk to allow for appropriate early intervention, thereby effectively reducing mortality and morbidity. although traditional applications of real-time pcr in the continuum of care and patient management. population-based screening programmes and recommendations do not often utilise nucleic acid testing (nat) for virology targets, relying more on immunoassays and antigen testing, nats and real-time pcr assays are still integral components. donor-eligibility determination ensures that a donor is eligible to donate cells or tissues to be used as human cells, tissues, and cellular and tissue-based products (hct/ps), which can include haematopoietic stem/progenitor cell, organ, semen, and other types of donations. in part, living and cadaveric hct/p donor eligibility is granted if screening shows that the donor is free from risk factors for, and clinical evidence of, infection due to relevant communicable disease agents and diseases such as hiv type 1 and 2, hbv, hcv, and west nile virus (wnv) (fda testing, 2014) . the fda testing recommendations stipulate that hct/ps tests for hiv and hcv may include fda-licensed nat blood donor screening and that, specifically, an fda-licensed nat should be used to assess infection with wnv. despite the fact that these nat tests provide qualitative as opposed to quantitative results, the molecular technology utilised is often real-time pcr due to its enhanced accurate and reliable resulting (fda assays, 2014) . additionally, pre-emptive virology screening post-transplant may reduce subsequent complications and provide a more cost-effective management strategy (evers, 2013) . pre-emptive therapy utilises routine viral screening to initiate therapy at the first indications of viraemia, prior to clinical manifestation of disease. this strategy reduces the overall morbidity and mortality post-transplant compared to strategies in which treatment is initiated at the onset of clinical disease (sch€ onberger et al., 2010). the pre-emptive treatment model utilising routine screening by quantitative realtime pcr of asymptomatic post-transplant patients has been shown to be especially effective for paediatric patients undergoing haematopoietic stem cell transplant, who are uniquely at a high risk of cmv and epstein-barr virus (ebv) infections (evers, 2013) . this strategy and prospective screening utilising a quantitative real-time pcr assay for bk polyoma virus (bkv) is recommended as part of routine posttransplant follow-up of all kidney transplants since early identification and management of bkv infection may prevent future incidence of polyoma virus-associated nephropathy (hirsch et al., 2005; kdigo, 2009) . nats and real-time pcr are also utilised as vital parts of certain screening algorithms. the center for disease control (cdc) currently recommends that hcv rna testing be utilised for anyone who may have been exposed to hcv within the preceding 6 months. in addition, nats would identify active hcv infection among persons who have tested anti-hcv positive or those with an indeterminate antibody test indicating need for referral for further medical evaluation and care (cdc hcv, 2013). viral diagnostic tests are used to determine presence or absence of current or previous infection. because many viral infections present with similar symptoms, accurate diagnosis is critical as each requires unique and vastly different interventions and/or management strategies. historically, diagnosis of viral infections has relied on viral growth in cell culture, immunoassays, antigen assays (elisa), haemagglutination testing, and electron microscopy (krishna & cunnion, 2012) . the introduction of molecular methods including nats and real-time pcr assays has vastly improved viral diagnosis with their superior sensitivity, specificity, and rapid result reporting (emmadi et al., 2011) . nat-based infectious disease testing, providing rapid results, aids in outbreak detection (ebola), genotype identification (hcv), and identification of possible drug resistance (hiv-1), which can lead to rapid clinical therapeutic decisions and early infection control to prevent spread of disease (espy et al., 2006) . viral culture was traditionally considered to be the 'gold standard' for viral diagnosis because of increased sensitivity compared to rapid antigen-testing methods. however, a significant limitation of viral culture was a long time to result, reaching 14 days for cmv (gleaves, smith, shuster, & pearson, 1985) . improvements in culture included the introduction of shell viral culture, which reduced the result turnaround-time (tat) for cmv to 24 h. although considered to be a vast improvement, certain viruses require immediate treatment intervention to prevent life-threatening infection and therefore, a 24-h tat is simply much too long. further, reliance on viral culture for diagnostics testing introduces other pronounced drawbacks, the most noteworthy being that not all routine viruses grow in culture and that virus viability, and thus its culture ability, could be impacted by sample collection, transport conditions, or prior patient treatment. with these limitations in mind, nat testing has tremendous advantages and has replaced culture for most viruses due to its greatly reduced tat (typically less than 8 h) while still retaining high sensitivity. the applications of nat testing, and more specifically, real-time quantitative pcr technology, in viral diagnostics and confirmatory testing continue to expand. the cdc-updated recommendations for hiv testing state that specimens that are reactive on the initial antigen/antibody combination immunoassay and non-reactive or indeterminate on the hiv-1/hiv-2 antibody differentiation immunoassay should be tested with an fda-approved hiv-1 nat test (branson, 2010; branson et al., 2014) . additionally, nats have demonstrated utility in high-risk populations, in which antibody testing alone might miss a considerable percentage of hiv infections that are otherwise detectable by nat virologic tests (priddy et al. 2007; stekler et al., 2009) . particularly, immunoassays for hiv diagnosis are limited by the marked delay between infection and seroconversion, a time when hiv viral levels are at their peak ( figure 5 ). for this reason, nats are the recommended method for diagnosis of hiv during the acute phase of infection (10-50 days post-infection). hiv viral rna is the first marker to manifest itself approximately 10 days post-infection at initiation of the acute phase of infection (lindback et al., 2000) . not until 4-10 days after initial detection of hiv rna do the hivp24 antigen levels rise to detectable levels using fourth-generation immunoassays. this marked delay and the need for rapid diagnosis by the identification of hiv rna is especially critical when an early diagnosis is medically warranted. during the acute phase of the hiv infection, patients typically present with flu-like symptoms, which can often lead to a misdiagnosis. misdiagnosing those at the highest risk of infection such as prison inmates, iv drug users, and men who have sex with men (msm), also increases the risk of further disease spreading (chu & selwyn, 2010) . therefore, the use of a real-time pcr testing method that is able to identify the presence of the hiv virus at this initial stage is critical. additionally, nats are critical for early diagnosis of congenital or perinatally acquired viral infections, especially infants born to hiv-infected mothers (tang & ou, 2012) . the maternal antibodies against hiv can persist in exposed infants for up to 18 months of age, which, in turn, prevents the use of antibody-based assays for early diagnosis of infection. there is a high level of morbidity and mortality during the first 2 years of life for infected infants; therefore, it is of high importance to determine infection status quickly in the exposed infant to implement the appropriate art therapy early (van rossum, fraaij, & de groot, 2002) . in addition to hiv, additional viruses have been demonstrated to exhibit perinatal transmission including respiratory virus, cmv, herpes simplex virus (hsv), varicella zoster virus (vzv), hbv, enterovirus, rotavirus, and human papilloma virus (prober et al., 1987) . for all these, nat testing may serve as a valuable tool for early intervention, especially in this critical neonate population that lacks fully developed immune systems. in addition to their applications for hiv diagnosis, nat testing is also utilised as a diagnostic for hcv. although diagnostic hcv rna real-time pcr tests are predominately used as confirmation of a positive hcv antibody result (cdc hcv, 2012), nat testing is recommended for the confirmation of a non-reactive hcv antibody test for patients suspected of having recent hcv exposure. hcv rna can be detected as early as 2 weeks post-exposure, whereas hcv antibodies are not detectable until 8-12 weeks post-infection (ghany, strader, thomas, & seeff, 2009 ). therefore, similar to strategies outlined for hiv, nat testing is the most suitable for detection and diagnosis during the acute phase of hcv infection. further, immunocompromised patients, those with weakened immune systems can include those that are hiv positive, on immunosuppressive drugs, or undergoing chemotherapy, may lack the ability to generate an appropriate immune response required for an anti-hcv test. in these special circumstances, the cdc recommends considering hcv rna testing for diagnosing viral infections. rapid diagnostic testing is also very important for viruses with high potential to create an epidemic or outbreak. throughout history, influenza has caused many such outbreaks, the most notable being the 1918 spanish flu thought to be responsible for an estimated 50 million deaths (taubenberger & morens, 2006) . in response to the possibility of future outbreaks, the world health organization (who) has established that pcr-based influenza testing is now the first-choice diagnostic test for both humans and animals (who influenza, 2011) . the conversion to molecular tests was based on the fact that these assays are more sensitive and specific for detecting influenza viruses compared to other non-molecular methods. nat methods also have a lower likelihood of false positive or false negative results, and therefore, result interpretation is less impacted by community-based influenza prevalence (cdc influenza, 2014) . the introduction of rapid molecular testing, which can provide results in as little as 15 min, is extremely beneficial especially in hospitals, nursing homes, and chronic care facilities where early influenza identification can prevent outbreaks. the number of molecular-based diagnostic tests has expanded even further with the introduction of tests for wnv, respiratory syncytial virus, hsv, rotavirus, and even more recently, ebola. as development and improvements continue to be made to real-time pcr technology that will reduce both cost and time to result even further, the number of nat diagnostic tests will continue to increase and the applications for real-time pcr in diagnostics will also expand. once a patient is appropriately diagnosed and linked to care, partly through the aid of quantitative real-time pcr, clinicians will often consider several baseline factors, which collectively help to guide therapeutic decision. these decisions are based on a series of questions including: are treatment options available? what treatment regimen should be prescribed? for how long will the patient need to be on therapy? factors influencing these therapeutic decisions include disease complications, patient predisposition, prior treatment experience, viral genotype, viral resistance profile, and host genetic profile, among others. and depending on the viral infection, a quantitative baseline viral load may also serve an important role in helping to guide treatment decision (table 1) . over the past 25 years, pharmaceutical development and clinical trials investigating cutting-edge antiviral treatments have relied heavily on the data generated from pcr-based-and eventually quantitative real-time pcr-based-technology to determine safe and effective drug use (cobb et al., 2011; mackay, arden, & nitsche, 2002) . practice guidelines continue to reference registrational and nonregistrational studies utilising quantitative real-time pcr to guide both clinicians and laboratories in the proper implementation of treatment and testing in order to deliver the most effective personalised care to patients (aasld/idsa/ias-usa, kotton et al., 2013) . because of this extensive co-utilisation of real-time pcr, it is widely accepted as the gold-standard technology for measuring a patient's quantitative viral load before, during, and after the course of treatment. once the decision to initiate treatment for chronic hcv infection is made, several baseline factors are routinely considered (aasld/idsa/ias-usa, 2014). among these are complications like liver fibrosis stage, co-infection with hiv, hepatocellular carcinoma, end-stage liver disease, genetic variations like hcv genotype, subtype and resistance markers, and prior treatment experience and outcome. the association of baseline viral load, measured by quantitative real-time pcr, to chronic hcv treatment outcome has been well documented with earlier therapies (pegylated-interferon plus ribavirin) but, given the lack of alternative therapeutic options, has not been recommended as a therapeutic decision factor (jensen et al., 2006; pawlotsky, 2012) . historically, practice guidelines have recommended the measurement of baseline viral load to serve only as an initial time-point required for effective monitoring of treatment without any prognostic indication (yee, currie, darling, & wright, 2006) . currently, tremendous advancements in the treatment of chronic hcv infection that employ direct-acting antivirals (daas) reported cure rates (as determined by svr) of >90% for even the once most difficult to treat hcv genotype-1 patients, the most predominant in the united states. because of this high potency of these drugs across patient populations and the greater importance of numerous other factors, including hcv genotype and prior treatment experience, in determining the appropriate course of treatment, the most recent aasld/idsa practice guidelines still do not recommend a baseline quantitative viral load as a therapeutic decision factor. however, in the rapidly evolving field of hcv treatment, the recent fda approval of a fixed-dose combination drug consisting of two daas (sofosbuvir and ledipasvir) for the treatment of hcv genotype-1, the manufacturer's drug label now includes a new indication for quantitative real-time pcr. it is indicated that treatment naïve and non-cirrhotic patients with a specific baseline viral load are eligible for shortened therapy, an indication with tremendous implications. according to the prescribing information, patients with a baseline viral load below 6 million iu/ml are eligible to have shorter therapy duration of 8 weeks, much shorter than the 12-or 24-week duration for other patient populations (harvoni, 2014) . this therapeutic decision practice is the first of its kind in treatment of chronic hcv infection and is likely to be a recurring theme as daa manufacturers strive to develop high efficacy regimens requiring shorter treatment durations. additionally, shorter treatment durations are more favourable to patients and payers when considering the cost of achieving svr with daas and may improve patient drug adherence and completion of therapy (hep c online, 2014) . as much as quantitative real-time pcr helped to develop this claim for this particular regimen, this technology will also be employed by numerous laboratories to aid in this part of therapeutic decision. in contrast to chronic infection, treatment of patients presenting in the acute phase of hcv infection, within the first 6 months after exposure, is not recommended by aasld/idsa for patients in whom hcv infection spontaneously clears (aasld/ idsa/ias-usa, 2014). therefore, careful monitoring of hcv rna by a sensitive nucleic acid test is required in order to confirm spontaneous clearance, defined as hcv rna negative at two specific measurements. quantitative and qualitative real-time pcr assays are both widely used for this purpose, given their comparable sensitivity. factors influencing art decision for hiv-infected patients include determination of pregnancy, aids-defining conditions, acute opportunistic infections, low cd4 counts, hiv-associated nephropathy, potential drug interactions, co-infection with hcv or hbv, hiv resistance testing, and prior treatment experience (dhhs hiv, 2014). plasma hiv rna viral load, performed widely by quantitative real-time pcr, is also recommended as a pre-art decision factor specifically for treatment naïve patients. the department of health and human services (dhhs hiv) recommends that only art-naïve patients with a plasma hiv viral load below 100,000 cp/ml can be prescribed various regimen options, which they otherwise should be restricted from taking with higher viral load. this is primarily due to inferior virologic responses in patients with higher viral loads observed in clinical studies (sax et al., 2009) . these clinical trial studies employed quantitative real-time pcr in order to help determine this cut-off and many labs have utilised the same technology to help guide hiv-treating clinicians in this decision. in the case of chronic hbv infection, several studies have shown that hepatitis b 'e' antigen (hbeag) and high levels of hbv dna are independent risk factors for the subsequent development of cirrhosis and hepatocellular carcinoma (chen, lin, et al., 2006; chen, yang, et al., 2006; iloeje et al., 2006) . however, due to the fluctuating nature of chronic hbv infection, the prognostic utility of one high hbv dna level at a single time-point is limited. thus, hbv baseline dna viral load, along with hbeag, alanine aminotransferase (alt) levels, and fibrosis, collectively aids in the decision to treat with antiviral agents as well as which hbv antiviral regimen to choose and duration of treatment (lok & mcmahon, 2009 ). typically, patients with an hbv dna viral load >20,000 iu/ml, signs of liver disease (i.e. high alt levels and/or significant fibrosis), and loss of hbeag are considered for immediate treatment with antivirals, whereas patients <2000 iu/ml are closely monitored for viral load changes prior to treatment. patients who fall in between this range are monitored for persistent viraemia and signs of liver disease before deciding to treat. quantitative real-time pcr, therefore, plays a crucial role in the care of chronic hbv patients who, if not treated at the appropriate time with the appropriate regimen and duration, are at greater risk of liver complications. unlike treatment guidelines for hcv, hiv, and hbv, management of cmv after solid organ transplant is not associated with specific quantitative cmv viral load cutoffs in order to make therapeutic decisions (kotton et al., 2013) . this is partly due to the historical lack of an international standard and varying assay designs, which has led to poor inter-institutional correlation of quantitative nats. in addition, the widespread practice of universal prophylaxis, where cmv antiviral medication is administered to patients early in the post-transplant period and continued for a finite period of time, has diminished the clinical utility of baseline viral loads for making therapeutic decisions. however, with the recent availability of the who cmv international reference standard, the establishment of viral load cut-offs that can be applied to pre-emptive monitoring of patients prior to treatment initiation may soon become more widely accepted . until then, institutions are required to determine their own test performance characteristics and clinical cut-offs. several studies have shown that a low cmv virologic threshold (e.g. detectable viraemia) using quantitative real-time pcr should be used for starting pre-emptive therapy especially in high-risk cases where the organ donor screens positive and the receptor screens negative for cmv serology (atabani et al., 2012; couzi et al., 2012; sun, cacciarelli, wagener, & singh, 2010) . among a variety of baseline risk factors that may indicate longer cmv treatment duration, significant predictive value has been demonstrated with higher baseline viral loads where longer treatment duration may prevent cmv disease relapse (kotton et al., 2013; sia et al., 2000) . clinical trial studies supporting the recent fda approval of a quantitative real-time pcr cmv test calibrated to the who international standard also demonstrated clinical value for baseline testing of patients with cmv disease who are undergoing treatment with the anti-cmv drugs ganciclovir or valganciclovir (razonable et al., 2013) . data from this study suggested that patients with a baseline cmv viral load <18,200 iu/ml are likely to resolve cmv disease more rapidly than those who have a higher baseline viral load. further studies are needed to determine universal thresholds for pre-emptive therapy initiation and predictive value for cmv baseline viral load in defining optimal treatment duration. there exists a clear application for quantitative real-time pcr technology in baseline determination of patients with significant viral infections, and in fact, quantitative viral load determination plays a critical role in therapeutic decision for many other viral infections. high baseline viral load has been shown to correlate with advanced disease during infection with numerous viruses such as bkv, hsv-1, ebv, and adenovirus and may potentiate the need for longer duration therapies in certain scenarios (cincinnati children's hospital medical center, 2012; domingues, lakeman, mayo, & whitley, 1998; gustafson et al., 2008; randhawa et al., 2004) . after the patient's baseline assessment or pre-emptive monitoring suggests if treatment is available, which treatment regimen to choose and perhaps the duration of therapy, the patient can move on to therapeutic administration. quantitative realtime pcr has helped and continues to set the stage for decisions that potentially saves lives, reduces complications, decreases morbidity, and lessens the economic burden to both the patient and the healthcare system. serial measures of viral load serve as an individualised map of a viral infection through the estimation of the amount of virus found within an infected person. tracking viral load in the continuum of care is a vital tool used predominantly to monitor treatment response and its effectiveness, early signs of resistance emergence during therapy of chronic viral infections, and viral activation or reactivation in immunocompromised patients following bone marrow or solid organ transplantation. while the goal of treatment for chronic hcv infection is svr, patients may fail therapy due to non-response, on-treatment breakthrough, or post-treatment relapse ( figure 6 ). the early change in quantitative viral load over time may be predictive of treatment efficacy and a shorten therapy for patients who respond rapidly to treatment (yee et al., 2006) . this 'response-guided therapy' (rgt) is best exemplified during treatment of chronic hcv patients. specifically, the sooner a patient becomes hcv rna undetectable during treatment, the lower the relapse rate when treatment is shortened. conversely, the longer it takes for a patient to become hcv rna undetectable, the longer they need to remain on treatment to limit relapse. however, given the poorer efficacy of earlier regimens, not all patients who received therapy achieved svr. for this reason, 'futility rules' or 'stopping rules' were also developed, which required that failure of a patient to respond (target not detected or viral load cutoff ) by a given time-point indicated the need to immediately discontinue therapy. monitoring hcv viral loads during treatment. despite advances in treatment for hcv patients, failure to achieve svr is still a reality. patients who do not achieve svr fall into four categories: (1) null responders (black line) achieve less than 2-log decrease in hepatitis c viral load upon treatment; (2) partial responders (red line; light grey in the print version) experiences at least a 2-log decrease in hepatitis c viral load during hcv treatment but fail to proceed to an undetectable viral load level; (3) breakthrough patients (orange line; light grey in the print version) have an undetectable hcv viral load, but the virus rebounded during treatment; (4) relapsers (blue line; dark grey in the print version) have had an undetectable hcv viral load, but the virus rebounded after they completed hcv treatment. although these rgt notions were originally developed from observations made during treatment with the older therapies, peg-ifn and ribavirin, rgt was also required during treatment with the much more potent first-generation daas, telaprevir and boceprevir, and stopping rules were put in place during treatment with the second-generation daa, simeprevir (aasld/idsa/ias-usa, 2014; ghany et al., 2009; yee et al., 2006) . newer ifn-free daa regimens targeting hcv, which are better tolerated by patients and by virtue of the targets they inhibit, have a higher barrier to resistance, yield more rapidly declining viral kinetics, and, thus, do not contain treatment indications for rgt in their prescribing information (harvoni, 2014; olysio, 2014; sovaldi, 2013; viekira, 2014) . while rgt was a major driver for regular viral load monitoring during antiviral therapy, it is not the only reason to monitor hcv viral load. in the interval between baseline measurement and assessment of svr, the 2014 aasld/idsa guidelines also include recommendations for monitoring initial response (week 4 on treatment with a repeat at week 6 if detectable) and end of treatment in order to provide an assessment of drug compliance/early efficacy and predict treatment outcomes, respectively (aasld/idsa/ias-usa, 2014). in the most recent revision to these web-based guidelines, it is recommended that an hcv viral load increase of greater than 10-fold on repeat testing at week 6 (or thereafter) should prompt a discontinuation of hcv treatment. many clinicians also closely monitor and report the declining viral loads to their patients in order to demonstrate treatment efficacy, motivating patients to continue treatment and remain adherent to the drug regimen until the next follow-up appointment (fusfeld et al., 2013) . regardless of monitoring during hcv treatment for rgt, adherence/compliance, patient motivation, early treatment efficacy, etc., quantitative real-time pcr is widely used by laboratories due to its sensitivity, accuracy, and reproducibility of each consecutive viral load test. for patients infected with chronic viral infections, such as hiv, the lifelong regimen of highly active art aims to suppress hiv viral levels to near undetectable levels, ensuring progression-free survival (delay or all together prevention of the progression to aids) and reducing potential transmission. alongside monitoring immune function and immunologic efficacy through cd4 t-cell count, hiv viral levels are critical in the clinical evaluation and assessment of hiv-infected patients undergoing art. determining a patient's hiv viral load is indicated prior to entry into care, at the initiation of art, at 2-8 weeks after art initiation, and then typically every 3-4 months while on treatment: (1) to establish a baseline level of hiv viral load; (2) to establish viral response to the therapy to assess the virologic efficacy of art; and (3) to monitor for abnormalities that may be associated with antiretroviral drugs (dhhs hiv, 2014) . the baseline hiv viral load is not only linked to treatment options (sax et al., 2009) but also helps to establish the magnitude of viral load decline after initiation of art and provides prognostic information about the probability of progression to aids or death (marschner et al., 1998; murray, elashoff, iacono-connors, cvetkovich, & struble, 1999; thiebaut et al., 2000) . once treatment is initiated, the goal is to reach and maintain suppressed hiv replication as determined by undetected viral levels utilising highly sensitive nat tests, which is generally achieved within 8-24 weeks after art initiation. the need for sensitive assays to effectively assess viral suppression hinges on the need to suppress hiv replication to the extent that viral evolution and drug resistance mutations do not emerge, which typically do not occur in patients whose hiv rna levels are maintained below the llod of current real-time quantitative pcr assays (kieffer et al., 2004) . due to the introduction of more sensitive real-time pcr assays, which can detect as few as 20 viral copies/ml, natural variability in hiv viral levels over time, even in patients with effective suppression, is much more evident (lima, harrigan, & montaner, 2009; gatanaga et al., 2009; willig et al., 2010) . although controversy exists between the clinical significance of viral loads between llod and <200 copies/ ml, there are reports suggesting that this low-level viraemia is predictive of virologic rebound (doyle et al., 2012; eron et al., 2013; laprise, de pokomandy, baril, dufresne, & trottier, 2013) , virologic failure (estevez et al., 2013) , and indication of drug resistance (taiwo et al., 2010) , signifying the need for highly sensitive assays. viraemic blips, a single detectable hiv viral load (<500 copies/ml) in an otherwise seemingly suppressed patient (figure 7) , however, do not indicate subsequent virologic failure or development of resistance mutations (castro et al., 2013; lee, kieffer, siliciano, & nettles, 2006; nettles et al., 2005) . blips are not unusual (havlir et al., 2001) and appear to be more common in winter, suggesting that host-related and seasonal factors are associated with the occurrence of viraemia (van sighem et al., 2008) . on the other hand, persistent hiv rna levels !200 copies/ml are often evidence of viral evolution and accumulation of drug resistance on-treatment hiv patient monitoring. (a) hiv viral loads will fluctuate as patients are on treatment, and, in most instances, will remain 'undetectable' (at or below dotted line); viral 'blips' are not uncommon and will result in transient 'detectable' and even quantifiable results (above the dashed line). (b) virologic failure will lead to a sustained high-level viral titre that, without intervention, will increase with time. mutations (aleman, soderbarg, visco-comandini, sitbon, & sonnerborg, 2002; karlsson et al., 2004) . once treatment failure is confirmed, immediate intervention is recommended to avoid progressive accumulation of resistance mutations and effective response of new regimen (dhhs hiv, 2014), which is benefited by low hiv rna levels and/or higher cd4 cell counts (eron et al., 2013) , and even a brief interruption in therapy may lead to a rapid increase in hiv rna and a decrease in cd4 cell count and increases the risk of clinical progression (deeks et al., 2001; lawrence et al., 2003) . with the development and administration of newer drugs that target specific biological processes of hiv, routine and clinical monitoring of viral loads using a real-time quantitative pcr assay continues to be critical to predict treatment failure and early emergence of drug resistance mutations, within a timeframe that would increase subsequent treatment success. viral load monitoring is also essential when the recipient of a solid organ transplant is cmv seropositive and the decision is made to initiate treatment only once the cmv levels predictive of disease are reached. this strategy, known as pre-emptive therapy, utilises intensive monitoring for cmv activity by sensitive real-time quantitative pcr methods and short-term antiviral treatment is given only to those with significant viral counts before symptoms occur. cmv is one of the most common opportunistic pathogens that infect solid organ transplant recipients (fishman, 2007) and is associated with increased morbidity and mortality (sagedal et al., 2004; schnitzler et al., 2003) . following primary infection, the virus establishes a lifelong latent infection in several sites of the body and may reactivate in the presence of immunosuppression, such as in transplant recipients. once reactivated, cmv is able to modulate the immune system and is known to be a potent upregulator of alloantigens (razonable, 2008) , increasing the risk of chronic allograft dysfunction (reischig, 2010; sagedal et al., 2002; smith et al., 2010) and acute rejection (sagedal et al., 2004) . pre-emptive therapy reduces the incidence of cmv disease (khoury et al., 2006; reischig et al., 2008) , which has been documented as a serious problem in randomised trials upon completion of universal antiviral prophylaxis therapy (kalil, levitsky, lyden, stoner, & freifeld, 2005; lowance et al., 1999; paya et al., 2004) . long-term studies have demonstrated that patients receiving preemptive therapy, when compared to prophylaxis therapy, were less likely to develop moderate-to-severe kidney scaring and atrophy and significantly better survival of the transplanted organ (reischig et al., 2012) . however, challenges still exist around defining appropriate thresholds to initiate pre-emptive therapy (humar & snydman, 2009) . but with new standardised real-time pcr assays, widespread adoption, and utilisation of these tests, pre-emptive therapy relying in intensive viral load monitoring may become the standard for certain at-risk patients. test of cure, or end of treatment response, is assessed following a given therapeutic regimen for signs of treatment efficacy. in few cases, a quantitative viral load measurement serves as a way to establish a cure rate, but, in others, may only be used as a confirmation of virologic suppression as clinical cure may not yet be possible with current therapies or technical limitations by real-time pcr that limits the overall sensitivity of viral detection. regardless of the clinical utility for measuring a virologic suppression, quantitative real-time pcrs with their current limits of detection and limits of quantitation are valuable tools in measuring low-level viraemia and establishing undetectable viral loads. utilisation of quantitative real-time pcr to assess virologic cure is perhaps best exemplified by treatment of patients with chronic hcv. according to the aasld/ idsa guidelines, patients who have 'undetectable' hcv rna in the serum, when assessed by a sensitive pcr assay, 12 or more weeks after completing treatment, are deemed to have achieved a sustained virologic response . achieving an svr is considered a virologic cure of hcv infection since, in these patients, hepatitis c-related liver injury stops and recurrence of infection is marginal, detected in <1% of patients after 5 years post-treatment (aasld/idsa/ias-usa, 2014; manns et al., 2013) . in agreement with these guidelines, the fda recommendation to pharmaceutical daa manufacturers also stipulates that viral rna clearance at svr-12 be measured in clinical trials using an fda-approved sensitive and specific quantitative hcv rna assay (fda hcv, 2013) . according to prescribing information accompanying the current daas, the threshold of svr-12 is defined as a quantitative threshold of hcv rna <25 iu/ml at 12 weeks after the end of treatment (feld et al., 2014; kowdley et al., 2014; lawitz et al., 2013) . this is somewhat dissimilar to the aasld/idsa guidelines as 'undetected' viral levels are not equivalent to 'detected but below the limit of quantitation' (figure 3 ). but, with the benefit of high sensitivity and reproducibility, quantitative real-time pcr has a clear established role in assessment of hcv virologic cure in both clinical trials and clinical practice and is able to meet the needs for assessing svr. quantitative real-time pcr may also play a critical role in the assessment of cmv disease resolution. the consensus guidelines recommend that two consecutive negative samples be obtained with a minimum treatment course of 2 weeks before treatment is discontinued, which is thought to minimise the risk for development of resistance and disease recurrence (asberg et al., 2009; chou, 2001; sia et al., 2000) . still, some transplant centres may extend treatment (secondary prophylaxis) in patients with compartmentalised disease for as long as necessary to reduce the likelihood of recurrent cmv infection (kotton et al., 2013) . resolving cmv disease has the long-term benefits of reducing mortality, potential allograft rejection, and the risk of bacterial, fungal, or viral opportunistic infections, among many other transplantand non-transplant-specific effects (arthurs et al., 2008; fishman, 2007) . although there is currently no cure for hiv infection, highly sensitive quantitative and qualitative real-time pcr tests targeting total hiv dna and rna have been used in clinical studies for both sterilisation (elimination of hiv-infected cells) and functional (controlled hiv in the absence of art) cures (kibirige, 2013; lewin & rouzioux, 2011) . improvements in real-time pcr technology may lead to profound increases in assay sensitivity and the ability to achieve single-copy detection (1 cp/ml) may lead us to a better understanding of hiv virology and what may be needed therapeutically to achieve a cure (alidjinou, bocket, & hober, 2014) . if therapeutic strategies are one day able to achieve an hiv cure, these highly sensitive tests will no doubt play a key role in the continuum of care for patients and, most importantly, in the confirmation of cure. clinical laboratories have undergone changes to become more efficient and flexible while delivering the same high-quality results. when choosing to implement new testing, even beyond viral targets, laboratories have to consider first and foremost the performance and medical value of the test and then factors such as tat, ease of use, and cost. real-time pcr with its wide dynamic range, high specificity, and high sensitivity is considered the gold standard for the quantification and identification of a variety of targets including bacteria, fungi, viruses, or oncological mutations (klein, 2002) . furthermore, the multiplexing capability of real-time pcr increases the number of targets and information gathered from the same test, further improving laboratory workflow, tat, and costs (deshpande & white, 2012) . while novel technologies have entered clinical laboratories including mass spectrometry and next-generation sequencing, real-time pcr remains a staple and an attractive option for clinical laboratories aiming to create molecular laboratory-developed tests (ldts). in addition, pcr can quickly be adapted to provide a robust test for the identification of emerging disease and molecular testing is now able to reach beyond the clinical laboratory and further enhance healthcare (farrar & wittwer, 2015; foudeh, didar, veres, & tabrizian, 2012) . most molecular tests used in clinical laboratories are fda-approved and commercially available. there are instances, however, when a test may not be available for a specific virus or the sample type and/or clinical indication used by the laboratory differs from those of the fda-approved assay, typically leading a laboratory to design its own pcr-based test or modify existing assays. fda defines an ldt as 'a type of in vitro diagnostic test that is designed, manufactured, and used within a single laboratory' and recognises that 'ldts are important to the continued development of personalised medicine' (fda ldt, 2014) . laboratory developed tests can be grouped into three categories, fda-cleared or approved test that have been modified, tests that are not subject to fda clearance or approval, and tests for which no performance specifications have been provided by the manufacturer (e.g. analytespecific reagents or asrs) (burd, 2010; code of federal regulations, 2009 ). with alternative sample types or applications, fda-approved tests are often modified to fit the testing needs of laboratories, including alternative collection media and sample types or expanded clinical applications. as an example, a recent gap was created in the hcv-screening algorithm for the confirmation of a positive enzyme immunoassay result following the discontinuation of the only fdaapproved confirmatory test (alter, kuhnert, & finelli, 2003) . in response, the cdc published recommendation for the use of fda-approved tests detecting hcv viraemia (cdc hcv, 2013), despite the fact that most of these assays did not have specific claims for confirmatory testing; as a result, several laboratories chose to validate these assays as ldts to meet the screening needs for hcv. additionally, ldts are the only option for the identification of the aetiologic agents of viral infections that can occur in transplant patients, such as ebv, adenovirus, vzv, and bk virus, that often present with non-specific clinical manifestations (razonable, 2011) and for which fda-approved assay options are lacking. ldts are an integral part of molecular laboratory testing. whether created from the ground-up or modified from fda-approved assays, ldts are answering the clinicians' needs for information as an aid for diagnosis or treatment of patients. as with any clinical tests, ldts have to meet the minimum standards set forth by clia prior to report patient results (code of federal regulations, 2009). in july 2014, fda informed congress of the agency's ldt regulatory oversight framework (fda ldt, 2014) . fda aims to address concerns over high-risk ldts with inadequately supported claims, lack of appropriate controls, and falsification of data that may lead to inadequate treatment, possible harm to patients, and unnecessary healthcare cost. presently, there is still a high degree of uncertainty as to what the final regulation scope will be and the possible impact on molecular laboratories will have to be seen. palaeopathology confirmed the truism that humanity, since its inception, has been exposed to genetic and infectious diseases with early documentation of trachoma (8000 b.c.e.), tuberculosis (7000 b.c.e.), and pneumonia (ca. 1150 b.c.e.) (aufderheide & rodreguez-martin, 1998; hershkovitz et al., 2008; roberts & manchester, 1995; webb, 1990) . even today, emerging infectious diseases (eids) continue to appear unpredictably driven by changes in human demographic, land use, and population behaviour (lederberg, hamburg, & smolinski, 2003; sehgal, 2010; taylor, latham, & woolhouse, 2001) . these infections can be classified as either newly emerging/a previously unknown disease or re-emerging infectious diseases/a previously known disease, that reappears after a significant reduction in incidence or elimination (morens & fauci, 2013) . eids are a threat not only to human health but also to global stability and economy. efforts to monitor these eids are in place both at the global level spearheaded by the who and at the national level. in the united states, governmental agencies (department of health and human services, united states agency for international development, department of defense) are supporting activities to detect, assess, and respond to potential outbreaks. specifically, pcr and real-time pcr are easily adaptable to detect nucleic acid targets that are unique to each given pathogen, and as such, they play essential roles in the identification and detection of infectious pathogens and have been routinely used by health organisation agencies during epidemic outbreaks such as severe acute respiratory syndrome (sars), h5n1, h1n1, and ebola (shuaib et al., 2014; who influenza, 2011) . the sars epidemic appeared in november 2002, in the chinese province of guangdong before reaching the adjacent hong kong in 2003 (who sars, 2003 . this sars eventually spread to 26 countries and resulted in more than 8000 cases. in response, the cdc triggered its emergency operations centre and issued a draft genome in april 2003, 33 days after the initial who global alert (cdc sars, 2013) . soon after, real-time quantitative pcr assays were described and put in use for the diagnosis of sars (drosten et al., 2003; peiris et al., 2003) . a host of measures were taken in order to contain this epidemic, and the molecular identification and diagnosis of the infectious agent by pcr played a key role in providing critical information to address the situation and contributed to the care of the patients infected. additionally, the re-emerging 2014 ebola epidemic (cdc ebola, 2014; who ebola, 2014) started in guinea in march of 2014 before spreading to nearby west african countries and eventually reaching the united states and europe (who ebola, 2014) . at the height of the epidemic, fda issued an emergency use authorisation (eua) for the use of the first real-time rt-pcr assay (fda eua, 2014) and less than 4 months later, five additional realtime pcr tests were authorised under an eua (fda eua, 2014) to provide an early diagnosis of the ebola viral disease (cdc ebola, 2014). eids remain a constant and unpredictable threat to human health. the flexibility of real-time pcr technology continues to show how promptly it can be used for the detection of infectious agents. by providing a rapid diagnostic, real-time pcr can help in starting the appropriate treatment right away and maximise the chances of a positive outcome. the goal of point-of-care testing (poct) is to quickly obtain a test result that will be used to implement the appropriate treatment for an improved clinical outcome. by definition, poct is laboratory testing that takes place at or near the site of patients (cap poc, 2013) . the advantages of poct are an improved tat and result availability regardless of normal core laboratory hours, access to care in remote areas, and greater patient involvement. the fight against aids largely contributed to the development of poct devices with viral load capabilities (hong, studer, hang, anderson, & quake, 2004; lee et al., 2010; marcus, anderson, & quake, 2006; tanriverdi, chen, & chen, 2010; unitaid, 2014; vulto et al., 2009) . originally developed to meet the difficult conditions associated with remote places, far from any core laboratory facility often found in the developing world, the design and convenience of a portable poct device with fast turnaround and accurate results extends the reach of healthcare. with this in mind, these poct systems could easily be used in developed nations at hospitals, within clinics a physicians' offices, pharmacies, correction facilities, or mobile health units, to target pathogens that benefit from immediate actionable results, for which not only accurate but also quick results are critical (kiechle & holland, 2009 ). ultimately, test menu available on these platforms will drive its implementation as a complement for the clinical laboratory core testing. the ideal molecular poct system that includes medical value, simplicity, fast tat, and ruggedness remains an ongoing engineering challenge. however, the latest advances in microfluidics are a great example of the potential of these devices and brings the real-time pcr lab-on-a-chip closer to mainstream diagnostic use. this is an exciting time for molecular poct and the upcoming years should bring new systems and perhaps a paradigm change in the world of healthcare. as the needs of the clinicians, laboratory, and patients continue to evolve, so do the applications of molecular diagnostics and pcr. over the past decade, quantitative real-time pcr technology has been increasingly phased into clinical practice and all of the potential present-day applications of real-time pcr-based methods are enumerable. they serve to advance experimental approaches within biological fields, pushing the boundaries of what we know and what we can learn, as well as to diminish empiric medical identification and management of viral diseases. the high sensitivity of the technology has reduced risks of the most commonly transmitted transfusion illnesses and has become an integral part of managing a variety of viral infections by providing pretreatment prognostic information, therapeutic effectiveness through monitoring, and end of treatment response assessment. quantitative real-time pcr complements serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses and are able to predict therapeutic failures sooner than traditional methods, allowing for a more timely management response. real-time pcr assays can be rapidly developed in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. the next few years are likely to see an even further increase in the expansion of the clinical applications of nucleic acid quantification, particularly following bone marrow and solid organ transplantation for which the newest standardised assays may provide an avenue for the development of consensus management guidelines for initiating pre-emptive anti-cmv treatment. further, with the drive towards hiv eradication and complete elimination of the virus from within cells of infected patients, innovations in quantitative real-time pcr assay design will continue to push the boundaries of detection and introduce assays with progressively lower limits of detection. thus, quantitative real-time pcr has and will facilitate advancements in the quality of diagnostics and of what we can achieve in research, medicine, and patient outcomes. recommendations for testing, managing, and treating hepatitis c drug resistance at low viraemia in hiv-1-infected patients with antiretroviral combination therapy quantification of viral dna during hiv-1 infection: a review of relevant clinical uses and laboratory methods guidelines for laboratory testing and result reporting of antibody to hepatitis c virus delayed-onset primary cytomegalovirus disease and the risk of allograft failure and mortality after kidney transplantation long-term outcomes of cmv disease treatment with valganciclovir versus iv ganciclovir in solid organ transplant recipients cytomegalovirus replication kinetics in solid organ transplant recipients managed by preemptive therapy novel therapeutic approaches for hepatitis c the cambridge encyclopedia of human paleopathology the future of hiv testing centers for disease control and prevention and association of public health laboratories. laboratory testing for the diagnosis of hiv infection: updated recommendations validation of laboratory-developed molecular assays for infectious diseases a commutable cytomegalovirus calibrator is required to improve the agreement of viral load values between laboratories point of care testing influence of episodes of intermittent viremia ("blips") on immune responses and viral load rebound in successfully treated hiv-infected patients centers for disease control and prevention centers for disease control and prevention. recommendations for the identification of chronic hepatitis c virus infection among persons born during 1945-1965 testing for hcv infection: an update of guidance for clinicians and laboratorians guidance for clinicians on the use of rt-pcr and other molecular assays for diagnosis of influenza virus infection centers for disease control and prevention past hbv viral load as predictor of mortality and morbidity from hcc and chronic liver disease in a prospective study risk of hepatocellular carcinoma across a biological gradient of serum hepatitis b virus dna level cytomegalovirus drug resistance and clinical implications diagnosis and initial management of acute hiv infection evidence based clinical practice guideline for management of ebv-associated post-transplant lymphoproliferative disease (ptld) in solid organ transplant protocols for determination of limits of detection and limits of quantitation: approved guideline. pennsylvania: clinical and laboratory standards evolution in the sensitivity of quantitative hiv-1 viral load tests health care financing administration, department of health and human services, part 493. laboratory requirements, section 493.1253. standard: establishment and verification of performance specifications prevention of hiv-1 infection with early antiretroviral therapy high incidence of anticytomegalovirus drug resistance among d+r-kidney transplant recipients receiving preemptive therapy virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in hiv-infected patients with detectable viremia multiplexed nucleic acid-based assay for molecular diagnostics of human disease panel on antiretroviral guidelines for adults and adolescents. guidelines for the use of antiretroviral agents in hiv-1-infected adults and adolescents. department of health and human services molecular methods for diagnosis of viral encephalitis preventing hiv transmission through antiretroviral treatmentmediated virologic suppression: aspects of an emerging scientific agenda a novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates application of competitive pcr to cerebrospinal fluid samples from patients with herpes simplex encephalitis plasma hiv-1 rna detection below 50 copies/ml and risk of virologic rebound in patients receiving highly active antiretroviral therapy identification of a novel coronavirus in patients with severe acute respiratory syndrome application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation molecular methods and platforms for infectious disease testing: a review of fda-approved and cleared assays efficacy and safety of raltegravir for treatment of hiv for 5 years in the benchmrk studies: final results of two randomised, placebo-controlled trials. the lancet infectious diseases real-time pcr in clinical microbiology: applications for routine laboratory testing quantification of viral loads lower than 50 copies per milliliter by use of the cobas ampliprep/cobas taqman hiv-1 test, version 2.0, can predict the likelihood of subsequent virological rebound to >50 copies per milliliter pre-emptive virology screening in the pediatric hematopoietic stem cell transplant population: a cost effective analysis extreme pcr: efficient and specific dna amplification in 15-60 seconds complete list of donor screening assays for infectious agents and hiv diagnostic assays emergency use authorizations guidance for industry chronic hepatitis c virus infection: developing direct-acting antiviral drugs for treatment, draft guidance fda laboratory developed tests fda testing hct/p donors for relevant communicable disease agents and diseases treatment of hcv with abt-450/r-ombitasvir and dasabuvir with ribavirin. the new england infection in solid-organ transplant recipients microfluidic designs and techniques using lab-on-a-chip devices for pathogen detection for point-of-care diagnostics changes in the viral mrna expression pattern correlate with a rapid rate of cd4 + t-cell number decline in human immunodeficiency virus type 1-infected individuals assessment of motivating factors associated with the initiation and completion of treatment for chronic hepatitis c virus (hcv) infection detection of hiv type 1 load by the roche cobas taqman assay in patients with viral loads previously undetectable by the roche cobas amplicor monitor aasld practice guidelines, diagnosis, management, and treatment of hepatitis c: an update competitive pcr for quantitation of mrna accuracy to 2nd international hiv-1 rna who standard: assessment of three generations of quantitative hiv-1 rna nucleic acid amplification tests comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in culture specimens quantification of adenovirus dna in unrelated donor hematopoietic stem cell transplant recipients gilead sciences, foster city prevalence and predictive value of intermittent viremia with combination hiv therapy multicenter comparison of different real-time pcr assays for quantitative detection of epstein-barr virus factors contributing to variability of quantitative viral pcr results in proficiency testing samples: a multivariate analysis real time quantitative pcr medications to treat hcv screening for preclinical disease: test and disease characteristics detection and molecular characterization of 9,000-year-old mycobacterium tuberculosis from a neolithic settlement in the eastern mediterranean polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations quantitation of human immunodeficiency virus type 1 in the blood of infected persons detection of specific polymerase chain reaction product by utilizing the 5 0 -3 0 exonuclease activity of thermus aquaticus a nanoliter-scale nucleic acid processor with parallel architecture progression of hiv-1 infection. monitoring of hiv-1 dna in peripheral blood mononuclear cells by pcr clinical utility of quantitative cytomegalovirus viral load determination for predicting cytomegalovirus disease in liver transplant recipients cytomegalovirus (cmv) virus load kinetics to predict recurrent disease in solid-organ transplant patients with cmv disease ast infectious diseases community of practice. cytomegalovirus in solid organ transplant recipients predicting cirrhosis risk based on the level of circulating hepatitis b viral load early identification of hcv genotype 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy polymerase-chain-reaction-based diagnosis of viral pulmonary infections in immunocompromised children meta-analysis: the efficacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients immunologic and virologic evolution during periods of intermittent and persistent low-level viremia kidney disease: improving global outcomes transplant work group. kdigo clinical practice guideline for the care of kidney transplant prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients the use of ultra-sensitive molecular assays in hiv cure-related research point-of-care testing and molecular diagnostics: miniaturization required genotypic analysis of hiv-1 drug resistance at the limit of detection: virus production without evolution in treated adults with undetectable hiv loads quantification using real-time pcr technology: applications and limitations updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation ledipasvir and sofosbuvir for 8 or 12 weeks for chronic hcv without cirrhosis interpreting quantitative cytomegalovirus dna testing: understanding the laboratory perspective role of molecular diagnostics in the management of infectious disease emergencies virologic failure following persistent low-level viremia in a cohort of hiv-positive patients: results from 12 years of observation sofosbuvir for previously untreated chronic hepatitis c infection structured treatment interruption in patients with multidrug-resistant human immunodeficiency virus microbial threats to health: emergence, detection, and response simple amplification-based assay: a nucleic acid-based point-of-care platform for hiv-1 testing hiv-1 viral load blips are of limited clinical significance hiv cure and eradication: how will we get from the laboratory to effective clinical trials? increased reporting of detectable plasma hiv-1 rna levels at the critical threshold of 50 copies per milliliter with the taqman assay in comparison to the amplicor assay viral dynamics in primary hiv-1 infection. karolinska institutet primary hiv infection study group analysis of relative gene expression data using real-time quantitative pcr and the 2 àddct method chronic hepatitis b: update international valacyclovir cytomegalovirus prophylaxis transplantation study group: valacyclovir for the prevention of cytomegalovirus disease after renal transplantation survey and summary: real-time pcr in virology long-term clearance of hepatitis c virus following interferon alpha-2b or peginterferon alpha-2b, alone or in combination with ribavirin parallel picoliter rt-pcr assays using microfluidics use of changes in plasma levels of human immunodeficiency virus type 1 rna to assess the clinical benefit of antiretroviral therapy productive human immunodeficiency virus infection levels correlate with aids-related manifestations in the patient quantitation of hiv-1 rna in plasma predicts outcome after seroconversion roadmap for harmonization of clinical laboratory measurement procedures emerging infectious diseases: threats to human health and global stability specific synthesis of dna in vitro via a polymerasecatalyzed chain reaction the use of plasma hiv rna as a study endpoint in efficacy trials of antiretroviral drugs intermittent hiv-1 viremia (blips) and drug resistance in patients receiving haart serial determinations of hiv-1 titers in hiv-infected homosexual men: association of rising titers with cd4 t cell depletion and progression to aids interlaboratory comparison of cytomegalovirus viral load assays transmission of human immunodeficiency virus from parents to only one dizygotic twin detection of hiv-1 at between 20 and 49 copies per milliliter by the cobas taqman hiv-1 v2.0 assay is associated with higher pretherapy viral load and less time on antiretroviral therapy chronic hepatitis c virus: advances in treatment, promise for the future valganciclovir solid organ transplant study group: efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cytomegalovirus disease in solid organ transplant recipients hepatitis c genotype 1 virus with low viral load and rapid virologic response to peginterferon/ribavirin obviates a protease inhibitor sustained virologic response to antiviral therapy for chronic hepatitis c virus infection: a cure and so much more coronavirus as a possible cause of severe acute respiratory syndrome interlaboratory comparison of epstein-barr virus viral load assays detection of acute hiv infections in an urban hiv counseling and testing population in the united states low risk of herpes simplex virus infections in neonates exposed to the virus at the time of vaginal delivery to mothers with recurrent genital herpes simplex virus infections correlates of quantitative measurement of bk polyomavirus (bkv) dna with clinical course of bkv infection in renal transplant patients cytomeglavirus infection after liver transplantation: current concepts and challenges management of viral infections in solid organ transplant recipients virologic suppression measured by a cytomegalovirus (cmv) dna test calibrated to the world health organization international standard is predictive of cmv disease resolution in transplant recipients cytomegalovirus-associated renal allograft rejection: new challenges for antiviral preventive strategies long-term outcomes of pre-emptive valganciclovir compared with valacyclovir prophylaxis for prevention of cytomegalovirus in renal transplantation valacyclovir prophylaxis versus preemptive valganciclovir therapy to prevent cytomegalovirus disease after renal transplantation the archaeology of disease impact of early cytomegalovirus infection and disease on long-term recipient and kidney graft survival the impact of cytomegalovirus infection and disease on rejection episodes in renal allograft recipients primer-directed enzymatic amplification of dna with a thermostable dna polymerase enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia abacavir-lamivudine versus tenofoviremtricitabine for initial hiv-1 therapy increasing viral burden in cd4 + t cells from patients with human immunodeficiency virus infection reflects rapidly progressive immunosuppression and clinical disease the association of cytomegalovirus sero-pairing with outcomes and costs following cadaveric renal transplantation prior to the introduction of oral ganciclovir cmv prophylaxis prospective, comprehensive, and effective viral monitoring in children undergoing allogeneic hematopoietic stem cell transplantation deforestation and avian infectious diseases ebola virus disease outbreak-nigeria cytomegalovirus (cmv) dna load predicts relapsing cmv infection after solid organ transplantation improved hiv-1 rna quantitation by using a novel dual-target approach subclinical viremia increases risk for chronic allograft injury in pediatric renal transplantation hiv testing in a high-incidence population: is antibody testing alone good enough competitive polymerase chain reaction assay for quantitation of hiv-1 dna and rna preemptive therapy for cytomegalovirus based on real-time measurement of viral load in liver transplant recipients hiv drug resistance evolution during persistent near-target viral suppression past, present and future molecular diagnosis and characterization of human immunodeficiency virus infections. emerging microbes & infections, 1, e19 a rapid and automated sample-to-result hiv load test for near-patient application 1918 influenza: the mother of all pandemics risk factors for human disease emergence clinical progression of hiv-1 infection according to the viral response during the first year of antiretroviral treatment delayed anti-hcv antibody response in hiv-positive men acutely infected with hcv genotype and viral load as prognostic indicators in the treatment of hepatitis c hiv/aids diagnostics technology landscape efficacy of highly active antiretroviral therapy in hiv-1 infected children immunologic, virologic, and clinical consequences of episodes of transient viremia during suppressive combination antiretroviral therapy viekira pak™ (ombitasvir, paritaprevir, and ritonavir tablets a microchip for automated extraction of rna from gram-positive bacteria. transducers quantitation of mrna by the polymerase chain reaction prehistoric eye disease (trachoma?) in australian aborigines world health organization. global alert and response: ebola outbreak world health organization. the use of pcr in the surveillance and diagnosis of influenza world health organization. severe acute respiratory syndrome cost ramifications of increased reporting of detectable plasma hiv-1 rna levels by the roche cobas ampliprep/cobas taqman hiv-1 version 1.0 viral load test multi-site pcr-based cmv viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology natural history: the importance of viral load, liver damage and hcc management and treatment of hepatitis c viral infection: recommendations from the department of veterans affairs hepatitis c resource center program and the national hepatitis c program office discordant human immunodeficiency virus infection in dizygotic twins detected by polymerase chain reaction. the pediatric infectious competitive pcr for precise nucleic acid quantification expert opinion on the treatment of patients with chronic hepatitis c key: cord-270868-4s3q2i6v authors: collins, lauren f.; moran, caitlin a.; oliver, nora t.; moanna, abeer; lahiri, cecile d.; colasanti, jonathan a.; kelley, colleen f.; nguyen, minh l.; marconi, vincent c.; armstrong, wendy s.; ofotokun, ighovwerha; sheth, anandi n. title: clinical characteristics, comorbidities and outcomes among persons with hiv hospitalized with coronavirus disease 2019 in atlanta, georgia date: 2020-10-01 journal: aids doi: 10.1097/qad.0000000000002632 sha: doc_id: 270868 cord_uid: 4s3q2i6v background: there are limited data describing the presenting characteristics and outcomes among us persons with hiv (pwh) requiring hospitalization for coronavirus disease 2019 (covid-19). methods: we performed a case series of all pwh sequentially admitted with covid-19 from 8 march 2020 to 23 april 2020 at three hospitals in atlanta, georgia. sociodemographic, clinical and hiv-associated characteristics were collected. results: of 530 confirmed covid-19 cases hospitalized during this period, 20 occurred among pwh (3.8%). the median age was 57 (q1–q3, 48–62) years, 65% were men, and 85% were non-hispanic black. presenting median symptom duration was 5 (q1–q3, 3–7) days; cough (90%), fever (65%), malaise (60%) and dyspnea (60%) were most common. on admission, 40% of patients required oxygenation support and 65% had an abnormal chest radiograph. median length of hospitalization was 5 (q1–q3, 4–12) days, 30% required intensive care, 15% required intubation, and 15% died. median cd4(+) cell count prior to admission was 425 (q1–q3, 262–815) cells/μl and 90% of patients had hiv-1 rna less than 200 copies/ml. half of the patients had at least five comorbidities; hypertension (70%), dyslipidemia (60%) and diabetes (45%) were most prevalent. all three patients who died had cd4(+) cell count more than 200, hiv suppression and each had a total of five comorbidities. conclusion: the multisite series in the southern united states provides characteristics and early outcomes of hospitalized pwh with covid-19. nearly all patients had controlled hiv and a high comorbidity burden. additional study of covid-19 among pwh is needed to determine the role of age, comorbidities and hiv control in mediating covid-19 presentation and its sequelae. the severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) pandemic continues to spread unabated globally, with more than 1 million confirmed cases reported in the united states. coronavirus disease 2019 (covid-19), a novel pneumonia caused by sars-cov-2, particularly devastates those of older age and with multimorbidity. the centers for disease control and prevention categorizes people with 'poorly controlled hivor aids' at higher risk than the general population for severe illness from covid-19 [1] ; however, supportive data are lacking. the southeast persists as the epicenter of the united states hivepidemic with the majority of new diagnoses, lower rates of virologic suppression and higher rates of deaths from late-stage aids than other parts of the country [2] . to understand how covid-19 may affect persons with hiv (pwh) in the southern united states, a prematurely aging population with a high comorbidity burden [3, 4] , we analyzed cases among hospitalized pwh in atlanta, georgia. pwh admitted with covid-19 at one of three hospitals in atlanta, georgia between 8 march 2020 and 23 april 2020 were recorded. the included hospitals were: first, grady memorial hospital, a 961-bed public safety-net hospital; second, emory university hospital, a 587-bed tertiary academic medical center; and third, the atlanta veterans affairs medical center, a 466-bed hospital serving veterans in the metropolitan and surrounding areas. covid-19 was confirmed by detection of sars-cov-2 by reverse transcription-pcr (rt-pcr) from a respiratory specimen. sociodemographic, clinical and hiv-associated characteristics were manually extracted from the electronic medical record. non-aids comorbidities were included if documented by a healthcare provider in the outpatient setting or during hospitalization. viral suppression was defined as hiv-1 rna less than 200 copies/ml. the research was approved by the emory university institutional review board. of 530 confirmed covid-19 cases admitted during this period, 20 occurred among pwh (3.8%). the median age was 57 (q1-q3, 48-62) years, 65% were men, and 85% were non-hispanic black; 60% were admitted to the public safety-net hospital. although 50% denied recent travel or a known exposure, seven (35%) reported known sick contacts. three of these were nursing home residents, two had ill household contacts, one was recently incarcerated, and one interacted with clients from italy at the workplace in atlanta (table 1) . median symptom duration on admission was 5 (q1-q3, 3-7) days; cough (90%), fever (65%), malaise (60%) and dyspnea (60%) were most common. two patients experienced both anosmia and ageusia. overall, presenting vital sign abnormalities were subtle (table 1) . forty percent of patients required oxygenation support in the emergency department including two patients who were intubated on arrival. absolute lymphocyte count was depressed, and serum creatinine, lactate dehydrogenase, c-reactive protein and d-dimer were all elevated (table 1) . chest radiograph was abnormal in 65% of patients, with 40% showing diffuse multifocal or bibasilar opacities and 15% demonstrating a focal infiltrate. over half (55%) of patients received a therapeutic for sars-cov-2 including open-label hydroxychloroquine þ/à azithromycin or clinical trial enrollment. median length of hospitalization was 5 (q1-q3, 4-12) days, 30% required intensive care, 15% required intubation, and 15% died. one patient remained admitted to date requiring high-flow oxygen support via nasal cannula on hospital day 6. all patients had sars-cov-2 detected by rt-pcr on initial specimen testing (19/20 specimens were collected by nasopharyngeal swab and 1/20 by mini-bronchoalveolar lavage). three patients had repeat testing performed for discharge planning. each individual had two nasopharyngeal swabs obtained several days after their initial test, including one individual who had a negative test result on both repeat tests (obtained on hospital days 18 and 20) and two individuals who had positive test results on both repeat tests (obtained on hospital days 7 and 10, and on hospital days 12 and 14, respectively). fifty-five percent of patients had hiv diagnosed at least 10 years ago. the most frequently reported hiv acquisition risk factor was heterosexual behavior (55%), followed by msm activity (25%) and injection drug use (10%). median absolute cd4 þ cell count and percentage prior to admission were 425 (q1-q3, 262-815) cells/ml and 29% (q1-q3, 21-36), respectively, and 90% of patients had viral suppression (table 1 ). in the 2 years prior to hospitalization, 82% of patients were continuously virologically suppressed over a median of 4.5 (q1-q3, 3.3-5.0) hiv care visits per patient. all 20 patients were prescribed antiretroviral therapy prior to hospitalization, and 95% reported therapy adherence on admission. eighty percent were prescribed an integrase strand transfer inhibitor-containing regimen, most commonly bictegravir/emtricitabine/tenofovir alafenamide (35%) or dolutegravir/abacavir/lamivudine (25%). the burden of non-aids comorbidities was high in this cohort, with 50% of patients having at least five comorbidities, and only 15% with no comorbidities ( table 1 ). the most prevalent comorbidities were hypertension (70%), dyslipidemia (60%), diabetes (45%) and psychiatric illness (40%). nine patients were prescribed an angiotensin-converting enzyme inhibitor or angiotensin ii receptor blocker on their medication list prior to admission. forty percent of patients reported current/former cigarette smoking, 50% currently used alcohol and 20% reported a history of crack/cocaine use. one patient had chronic active hepatitis b virus and five patients had a history of hepatitis c virus, including two who spontaneously cleared and three who achieved sustained virologic response after treatment with directacting antivirals. a summary of demographic, comorbidity, hiv-specific and treatment characteristics for the three patients whom died is provided in table 2 . the multisite series of pwh hospitalized with covid-19 in atlanta is notable for several reasons. the median age of 57 years is 4-6 years younger than reports of non-hiv hospitalized covid-19 us cohorts [5, 6] . half of pwh in this study had at least five comorbidities, an alarming burden that exceeds previous reports of hospitalized covid-19 cases including adults in new york and california [5, 6] and specifically among pwh in spain [7] . among hospitalized pwh in this series, nearly all had cd4 þ cell counts at least 200 and viral suppression (inclusive of all three patients who died, table 2 ), despite the three hospitals' catchment areas treating significant proportions of demographically similar pwh with uncontrolled hiv and aids [8] . it is possible that the premature onset of multimorbidity among pwh considerably impacts sars-cov-2 infection and illness severity, distinct from hiv infection or its associated immunosuppression. the immunologic intersection between sars-cov-2 and chronic hiv infection remains to be examined. clearly, in a subset of people with covid-19, a hyperimmune response to sars-cov-2 leads to severe lung damage and poor outcomes exemplified by elevated systemic levels of proinflammatory cytokines, coagulation dysfunction and dysregulated cellular responses [9] . curiously, we and others [5] have not yet observed a high prevalence of severe covid-19 disease among pwh in general, and particularly not among pwh with advanced immunosuppression, that is, cd4 þ cell count less than 200. further research will be necessary to understand whether the immune response to sars-cov-2 differs among pwh and how multimorbidity, which is common in hiv [3, 4] , influences clinical outcomes of covid-19 among pwh specifically. pwh in the southern united states represent a group particularly vulnerable to covid-19 severity and sequelae, given the confluence of income inequality, housing instability and food insecurity -disproportionate among minorities because of structural racism and discrimination -limiting the ability to adopt optimal 1792 aids 2020, vol 34 no 12 includes chronic obstructive pulmonary disease (n ¼ 3), asthma (n ¼ 1), obstructive sleep apnea (n ¼ 1), pulmonary fibrosis (n ¼ 1). i includes one patient with renal cell carcinoma status post nephrectomy and subsequent deceased-donor kidney transplantation currently on immunosuppression; one patient with prostate cancer who has repeatedly declined treatment; one patient with hodgkin's lymphoma treated with chemotherapy (n ¼ 1). j out of the 9 listed above. infection prevention measures and augmenting comorbidity risk, which in turn confers worse outcomes [2] . the predominance of non-hispanic black men in this cohort reflects the demographic of hiv/aids in atlanta; however, more data are needed to determine the role of racial/ethnic disparities in covid-19 presentation and outcomes, especially as this can be exacerbated in high hiv-burden areas [10] . the indirect effects of the sars-cov-2 pandemic on pwh and those at risk for hiv are not yet fully understood. closure of clinics, reduced access to medications and fewer support services may lead to decreased rates of retention in care and lower rates of virologic suppression, particularly among the most marginalized [11] . in addition, many clinics have scaled back testing for hiv and other sexually transmitted infections, and others have suspended visits for preexposure prophylaxis. the overall impact on the hiv care continuum and the ending the hiv epidemic initiative must be closely monitored. it is anticipated that additional resources are needed to support the unique needs of pwh, including to increase access to sars-cov-2 testing and clinical trials, and more broadly to build socioeconomic and healthcare infrastructure that will enhance hiv-related, comorbidity, mental and sexual health and well being [12] . this report supports dedicated larger study of covid-19 among pwh to determine the role of age, comorbidities, immunocompetency, hiv control and antiretroviral agents in mediating covid-19 presentation and its sequelae. covid-19): people who are at higher risk challenges of reaching 90-90-90 in the southern united states comorbidities in persons with hiv: the lingering challenge the prevalence and burden of non-aids comorbidities among women living with or at-risk for hiv infection in the united states presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area characteristics of hospitalized adults with covid-19 in an integrated healthcare system in california covid-19 in hiv investigators. covid-19 in patients with hiv: clinical case series centers for disease control and prevention. estimated hiv incidence and prevalence in the united states clinical and immunological features of severe and moderate coronavirus disease 2019 characteristics and clinical outcomes of adult patients hospitalized with covid-19 -georgia continuous retention and viral suppression provide further insights into the hiv care continuum compared to the crosssectional hiv care cascade collective call to action for hiv/aids community-based collaborative science in the era of covid-19 we sincerely thank the patients, clinicians and staff at grady memorial hospital, emory university hospital and the atlanta veteran's affairs medical center. we also thank the emory-grady hiv clinical cohort registry for contribution of data.the current work was supported by the emory center for aids research (award number p30-ai-050409) as well as the emory specialized center of research excellence (score) on sex differences (award number u54ag062334; to i.o.). l.f.c. is also supported by the national center for advancing translational sciences (ncats) of the national institutes of health (nih) (award numbers ul1tr002378 and tl1tr002382) and c.a.m. by the ncats of the nih (award number kl2tr002381). c.d.l. is also supported by the national institute of allergy and infectious diseases (niaid) of the nih (award number k23-ai124913). there are no conflicts of interest. key: cord-287853-cob7ur35 authors: sharma, vaneet kumar; sharma, ity; glick, james title: the expanding role of mass spectrometry in the field of vaccine development date: 2018-05-31 journal: mass spectrom rev doi: 10.1002/mas.21571 sha: doc_id: 287853 cord_uid: cob7ur35 biological mass spectrometry has evolved as a core analytical technology in the last decade mainly because of its unparalleled ability to perform qualitative as well as quantitative profiling of enormously complex biological samples with high mass accuracy, sensitivity, selectivity and specificity. mass spectrometry‐based techniques are also routinely used to assess glycosylation and other post‐translational modifications, disulfide bond linkage, and scrambling as well as for the detection of host cell protein contaminants in the field of biopharmaceuticals. the role of mass spectrometry in vaccine development has been very limited but is now expanding as the landscape of global vaccine development is shifting towards the development of recombinant vaccines. in this review, the role of mass spectrometry in vaccine development is presented, some of the ongoing efforts to develop vaccines for diseases with global unmet medical need are discussed and the regulatory challenges of implementing mass spectrometry techniques in a quality control laboratory setting are highlighted. as the target populations for the vaccines are healthy individuals, pregnant women, or infants, vaccine safety is of paramount importance. appropriately, the vaccine landscape is changing from traditional vaccine approaches to cost-effective, highly scalable, and safe recombinant vaccines. 9, 10 using recombinant dna technology, antigens are expressed in yeast, escherichia coli, baculovirus expression vector system (bevs), or mammalian cell lines. [11] [12] [13] recombinant antigens are engineered to mimic the first step of virus attachment to the cell surface which is mediated by specific glycoproteins. the expressed recombinant antigens undergo multiple purification cycles to produce highly purified vaccines. 14, 15 in order for recombinant vaccines to be acceptable to regulatory authorities, in-depth analytical characterization needs to be performed in this review, we focus on the expanding role of mass spectrometry in vaccine development, irrespective of the route of production. we also highlight the regulatory challenges and limitations of mass spectrometry-based techniques which constrain its further implementation as a quality control batch release assay in cgmp manufacturing. the role of mass spectrometry ( alternatively, an o-linked glycan can be formed by linking the oligosaccharide to a serine or threonine residue. glycosylation play a fundamental role in antigen conformation, folding, stability, solubility, and importantly, immune response. 16, 17 plants, yeasts, and non-human cell lines generate glycans that are not compatible and bioactive within human hosts. 18 thus, about 70% of all recombinant glycoproteins are produced in mammalian-based expression systems such as chinese hamster ovary (cho) cells. 19 analytical characterization of a glycoprotein is challenging as there is inherent unpredictability associated with the glycans; they are either macroheterogeneous (potential glycan site in the protein not glycosylated) or microheterogeneous (different glycan structures found on the same site in the expressed protein). glycosylation analysis is performed to understand the nature of structural heterogeneity of glycans, quantify them, and more importantly to determine where glycosylation occurs (site specific analysis). mass spectrometry based techniques are valuable tool for detecting and investigating glycosylation ( figure 2 ). although ms-based glycosylation analysis is not without limitations, the advances in ms instrumentation and glycan analysis software have led to increased resolution, automated identification, quantitative determination, and accurate structural characterization. 16, 20, 21 another critical quality attribute (cqa) of glycoprotein is intramolecular disulfide bonds (s-s linkages); they ensure correct folding, functional activity and stability. incorrect formation of disulfide bonds can cause protein misfolding which tends to promote aggregation which could result in an unwarranted immune response. confirmation of correct disulfide bond formation in the recombinant first study analyzing the site-specific nglycosylation of gp120. this report describes the structural characterization of the expressed gp120. reversed phase hplc of the tryptic digest. peptides collected from rp-hplc were further identified by amino acid analysis (aaa) or nterminal sequencing analysis. leonard et al 38 hiv-1 gp120 expressed in cho cells. mass spectrometric characterization of the glycosylation pattern. of hiv-1 gp120. maldi and nanoesi-lc-ms/ms using a hybrid quadrupole-time-of-flight tandem mass spectrometer (q-tof) was used to assign glycosylation sites. zhu et al 111 hiv gp140 jr-fl and con-s env expressed in cho and hek293 cells. a glycopeptide-based mass mapping approach was used to characterize the glycosylation of two env protein vaccine candidates in a glycosylation site-specific fashion. mass spectrometry based approach was used to map the complete glycosylation profile at every site in eleven hiv-1 env trimers. high-resolution lc-ms/ms was performed using an orbitrap velos pro hybrid mass spectrometer (thermo scientific) equipped with etd and coupled to an acquity uplc system (waters). go et al 49 hiv gp120 monomers of the bg505. sosip. global n-glycan site occupancy of hiv-1 gp120 by metabolic engineering and high-resolution intact mass spectrometry was performed. released n-glycan analysis was carried out using synapt g2si ion mobility mass spectrometer. native highresolution mass spectrometry was performed on q exactive hybrid quadrupole-orbitrap mass spectrometer. as illustrated in the following section and in table 1 , mass spectrometry-based techniques have been used to perform the structural characterization, glycosylation profiling and antigen quantitation during the development of the hiv, influenza, dengue, ebola, meningococcal, and other vaccines. the review also highlights that mass spectrometry-based methods such as glycan analysis has been used to analyze a specific envelope glycoproteins (env) and has broad applicability to any other glycoprotein-based vaccines. the quest for a safe and effective vaccine to protect against human one of the hypotheses being explored for protective immunity against hiv is to induce broadly neutralizing antibodies (bnabs) that target the highly glycosylated hiv-1 virion-associated envelop. 33 a number of approaches are being pursued in the hiv-1 vaccine development field to establish protective immunity, including monomeric gp120 subunits and oligomeric gp140 and trimeric envelope glycoprotein to elicit bnabs. [34] [35] [36] each of these envelop (env) immunogens have multiple exposed epitopes and are heterogeneously glycosylated molecules, with more than 50% of their mass consisting of glycans (∼28 n-linked glycans). it is critically important to monitor this hiv env glycan shield during vaccine development. 37 crispin and collaborators also extensively investigated the native glycosylation profile of envs, in particularly, trimeric immunogen bg505 sosip.664. [50] [51] [52] [53] their studies led to an increased understanding of the glycan shield of the env and it is now largely accepted that to induce bnabs, the hiv vaccine candidate should have a glycan profile similar to the one present on the native env trimers. [54] [55] [56] in a thorough study on trimeric env glycosylation, behrens et al 51 influenza virus is a segmented, enveloped rna virus and is among the ic-idms-mrm method is truly an alternative to the approved srid method as it has dual purpose "potency and content determination", was found to be equivalent to the srid method and can also be used in response to a pandemic influenza threat. 75, 76 additionally, a direct ultra-performance liquid chromatography (uplc)-idms method was reported for the rapid and accurate quantification of influenza na. 77 label-free ms-based methods have also been reported for the simultaneous identification and quantification of ha and na in influenza vaccine with samples analyzed by lc-ms e on a waters synapt g2 mass spectrometer. 78 quantification of proteins by labelfree lc-ms e is a powerful tool, in this method, alternating scans of low collision energy and elevated collision energy during lc-ms analysis to obtain both protein identity and quantity in a single experiment. quantification based on the experimental data showed that the signal intensity was proportional to concentration which allowed for the amount of any protein in the mixture to be estimated. lc-ms e utilizes parallel, multiplex fragmentation where all peptide precursors are simultaneously fragmented throughout the chromatographic the srid assay has been used for over 40 years as a quantitative and potency method throughout the world despite issues regarding variability and availability of standard reagents. thus, even though new methods like hplc and mass spectrometry are being developed, it will take some time for these methods to be adopted worldwide. as the next influenza pandemic cannot be predicted, the health authorities' pandemic preparedness efforts include efforts to ensure expedited availability of pandemic vaccines. methods such as hplc and ms, with their ability to quantitate antigens without standard reference reagents, can become a cornerstone of pandemic influenza preparedness. the viral genus flavivirus, includes dengue virus, yellow fever virus, and and 153 were glycosylated and, predominately, the n-glycan at asn67 was a high mannose-type and at asn153 was mainly a combination of complex-and hybrid-type glycans. 87 this study provided important new insights for the role of glycans in the dengue virus-host cell interactions. in a separate study, accurate quantitation of the expressed four viral particles in the tetravalent dengue vaccine (cyd) was performed using targeted ms in selected reaction monitoring (srm) mode. 88 the study described an orthogonal quantitation strategy (targeted ms in srm mode) and demonstrated that the variability of the ms method was low (between 8% and 17%) and the assay was linear between 6.25 and 200 nmol/l. based on the reported method performance, it could be used to release future batches of the tetravalent dengue vaccine. ebola the data presented demonstrated that the n-glycan patterns were similar between gp 1,2 but o-glycan patterns were remarkably different on gp 1,2 the five ebola viruses. this study should serve as the foundation for future ebola viral entry and immunogenicity studies. 91 to improve on the conventional approaches for absolute quantitation of gp1 in ebola virus-like particles (evlps), an isotope dilution full-scan liquid chromatography-high-resolution mass spectrometry method was developed using an ultimate 3000 hplc and an orbitrap elite hybrid ion trap-orbitrap mass spectrometer. 92 the reported ms quantitation method provided not only a means to rapidly determine evlp batch quality based upon quantitation of antigenic gp1 but also ensured adequate preclinical/clinical dosing. chikungunya is a mosquito-borne viral disease, endemic in africa and southeast asia and has also recently emerged in the caribbean. currently there are no drugs or vaccines available for treatment or prevention. the name "chikungunya" derives from a makonde word meaning "to become contorted," and describes the stooped appearance of sufferers with joint pain (arthralgia ms-based approaches have been used to perform the physicochemical characterization for all of these vaccines. 96 in one of the studies, researchers used lc-ms to characterize glycosylated lysine residues in menveo®. 97 in another study a lc-ms method was used to determine the relative reactivity of lysine residues in crm 197 to determine which of these amino acids were more susceptible to conjugation. 98 lc-ms was also used to quantify the bexsero® vaccine which was the first vaccine developed by reverse vaccinology; a genome-based approach to vaccine development. 99 in the field of vaccines, vera-velasco et al not only quantified the viral f, g and m proteins present in the viral particles but also analyzed the cellular proteomic composition in the niv vaccine candidate. traditionally, viral particles have been described as pure entities carrying only viral-derived proteins but the authors successfully analyzed the ratio between cellular and viral proteins in the niv vaccine candidate using lc-ms/ms. 108 west nile fever is a flavivirus that causes a viral infection typically spread by mosquitoes. to date, no vaccine is available to prevent the west nile infection. partially purified virus-like particles were resolved by sds-page, and the coomassie blue-stained band corresponding to env protein was excised from the gel, destained, and analyzed by ms. microcapillary lc-ms was performed using a lcq deca ion-trap mass spectrometer (thermo finnigan) to identify the presence of env protein in virus-like particles to help in developing a potential vaccine. 109 in should be a supplemental batch release test to ensure that the product is within specifications and without any unwarranted modifications. using ms techniques will ensure improved quality and increased safety for clinical trial participants. in conclusion, the evidence suggests that the emergence of ms-based techniques has advanced the field of vaccine development. history of vaccination vaccines, new opportunities for a new society research and development of new vaccines against infectious diseases recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production. hum vaccines immunother the challenge of emerging and reemerging infectious diseases emerging infectious diseases: threats to human health and global stability establishing a global vaccinedevelopment fund new vaccines against epidemic infectious diseases recombinant subunit vaccines: potentials and constraints recombinant therapeutic protein vaccines virus-like particles as vaccine viral vaccines and their manufacturing cell substrates: new trends and designs in modern vaccinology cell culture technology protein subunit vaccine purification cgmp production and analysis of bg505 sosip. 664, an extensively glycosylated, trimeric hiv-1 envelope glycoprotein vaccine candidate a systematic approach to protein glycosylation analysis: a path through the maze structural mass spectrometry in biologics discovery: advances and future trends impact of host cell line choice on glycan profile therapeutic glycoprotein production in mammalian cells analysis of protein glycosylation by mass spectrometry carbohydrates on proteins: site-specific glycosylation analysis by mass spectrometry recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry the quest for an antibody-based hiv vaccine developing an hiv vaccine vaccination with alvac and aidsvax to prevent hiv-1 infection in thailand new approaches to hiv vaccine development antigenicity and immunogenicity of rv144 vaccine aidsvax clade e envelope immunogen is enhanced by a gp120 n-terminal deletion comparable antigenicity and immunogenicity of oligomeric forms of a novel, acute hiv-1 subtype c gp145 envelope for use in preclinical and clinical vaccine research hiv-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies the hiv glycan shield as a target for broadly neutralizing antibodies assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in chinese hamster ovary cells comparison of hplc/esi-fticr ms versus maldi-tof/tof ms for glycopeptide analysis of a highly glycosylated hiv envelope glycoprotein glycosylation site-specific analysis of hiv envelope proteins (jr-fl and con-s) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopesʼ accessibility glycosylation site-specific analysis of clade c hiv-1 envelope proteins characterization of glycosylation profiles of hiv-1 transmitted/founder envelopes by mass spectrometry methods development for analysis of partially deglycosylated proteins and application to an hiv envelope protein vaccine candidate analysis of the disulfide bond arrangement of the hiv-1 envelope protein con-s gp140 δcfi shows variability in the v1 and v2 regions characterization of host-cell line specific glycosylation profiles of early transmitted/founder hiv-1 gp120 envelope proteins glycosylation and disulfide bond analysis of transiently and stably expressed clade c hiv-1 gp140 trimers in 293t cells identifies disulfide heterogeneity present in both proteins and differences in o-linked glycosylation influences on the design and purification of soluble, recombinant native-like hiv-1 envelope glycoprotein trimers generation and characterization of a bivalent hiv-1 subtype c gp120 protein boost for proof-ofconcept hiv vaccine efficacy trials in southern africa glycosylation benchmark profile for hiv-1 envelope glycoprotein production based on eleven env trimers cell-and protein-directed glycosylation of native cleaved hiv-1 envelope composition and antigenic effects of individual glycan sites of a trimeric hiv-1 envelope glycoprotein molecular architecture of the cleavage-dependent mannose patch on a soluble hiv-1 envelope glycoprotein trimer glycosylation profiling to evaluate glycoprotein immunogens against hiv-1 envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens the glycan shield of hiv is predominantly oligomannose independently of production system or viral clade structural constraints determine the glycosylation of hiv-1 envelope trimers ion mobility mass spectrometry for extracting spectra of n-glycans directly from incubation mixtures following glycan release: application to glycans from engineered glycoforms of intact, folded hiv gp120 maldi-ms/ms with traveling wave ion mobility for the structural analysis of n-linked glycans ion mobility mass spectrometry for ion recovery and clean-up of ms and ms/ms spectra obtained from low abundance viral samples global nglycan site occupancy of hiv-1 gp120 by metabolic engineering and high-resolution intact mass spectrometry structural principles controlling hiv envelope glycosylation global site-specific nglycosylation analysis of hiv envelope glycoprotein comprehensive characterization of reference standard lots of hiv-1 subtype c gp120 proteins for clinical trials in southern african regions cell culture as a substrate for the production of influenza vaccines: memorandum from a who meeting current and emerging cell culture manufacturing technologies for influenza vaccines critical review of current and emerging quantification methods for the development of influenza vaccine candidates influenza vaccine: the challenge of antigenic drift confronting the next pandemic-workshop on lessons learned from potency testing of pandemic (h1n1) 2009 influenza vaccines and considerations for future potency tests application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of 2009 pandemic influenza (h1n1) vaccines at multiple manufacturing sites in china quantification of influenza virus hemagglutinins in complex mixtures using isotope dilution tandem mass spectrometry optimization of digestion parameters for protein quantification simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry quantification of viral proteins of the avian h7 subtype of influenza virus: an isotope dilution mass spectrometry method applicable for producing more rapid vaccines in the case of an influenza pandemic development of influenza a (h7n9) candidate vaccine viruses with improved hemagglutinin antigen yield in eggs quantification of immunoreactive viral influenza proteins by immunoaffinity capture and isotope-dilution liquid chromatography-tandem mass spectrometry immunocapture isotope dilution mass spectrometry in response to a pandemic influenza threat quantification of influenza neuraminidase activity by ultra-high performance liquid chromatography and isotope dilution mass spectrometry label-free mass spectrometry-based quantification of hemagglutinin and neuraminidase in influenza virus preparations and vaccines quantification of proteins by label-free lc-mse absolute quantification of proteins by lcms e a virtue of parallel ms acquisition selective and quantitative detection of influenza virus proteins in commercial vaccines using two-dimensional high-performance liquid chromatography and fluorescence detection approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography, gel electrophoresis and mass spectrometry strain identification of commercial influenza vaccines by mass spectrometry simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by lc-ms e fast and highly selective determination of hemagglutinin content in quadrivalent influenza vaccine by reversed-phase high-performance liquid chromatography method advancing dengue vaccine development site-specific characterization of envelope protein n-glycosylation on sanofi pasteur's tetravalent cyd dengue vaccine absolute quantification of dengue virus serotype 4 chimera vaccine candidate in vero cell culture by targeted mass spectrometry after ebola in west africaunpredictable risks, preventable epidemics identification of n-glycans from ebola virus glycoproteins by matrix-assisted laser desorption/ ionisation time-of-flight and negative ion electrospray tandem mass spectrometry comparison of n-and o-linked glycosylation patterns of ebolavirus glycoproteins development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoprotein in ebola virus-like particle vaccine preparations chikungunya virus: pathophysiology, mechanism, and modeling development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a chikungunya virus-like particle vaccine characterization of nglycosylation profiles from mammalian and insect cell derived chikungunya vlp label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group b outer membrane vesicle vaccine physicochemical characterisation of glycoconjugate vaccines for prevention of meningococcal diseases defined conjugation of glycans to the lysines of crm 197 guided by their reactivity mapping quantification by lc-ms e of outer membrane vesicle proteins of the bexsero ® vaccine the dual role of lipids of the lipoproteins in trumenba, a self-adjuvanting vaccine against meningococcal meningitis b disease robust manufacturing and comprehensive characterization of recombinant hepatitis e viruslike particles in hecolin ® hepatitis e virus: a renewed hope with hecolin molecular and structural characterization of the l1 virus-like particles that are used as vaccine antigens in cervarix™, the as04-adjuvanted hpv-16 and-18 cervical cancer vaccine viruslike particles in vaccine development virus-like particle-based human vaccines: quality assessment based on structural and functional properties status of vaccine research and development of vaccines for nipah virus proteomic composition of nipah virus-like particles production of immunogenic west nile virus-like particles using a herpes simplex virus 1 recombinant vector characterization of human enterovirus71 virus-like particles used for vaccine antigens mass spectrometric characterization of the glycosylation pattern of hiv-gp120 expressed in cho cells glycan profiles of the 27 n-glycosylation sites of the hiv envelope protein cn54gp140 comparative analysis of the glycosylation profiles of membrane-anchored hiv-1 envelope glycoprotein trimers and soluble gp140 mapping the complete glycoproteome of virion-derived hiv-1 gp120 provides insights into broadly neutralizing antibody binding haemagglutinin quantification and identification of influenza a&b strains propagated in per. c6 ® cells: a novel rp-hplc method hplc-based quantification of haemagglutinin in the production of egg-and mdck cell-derived influenza virus seasonal and pandemic vaccines optimization and qualification of a quantitative reversed-phase hplc method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays characterization of a recombinant influenza vaccine candidate using complementary lc-ms methods comparative glycomics analysis of influenza hemagglutinin (h5n1) produced in vaccine relevant cell platforms glycosylation analysis of engineered h3n2 influenza a virus hemagglutinins with sequentially added historically relevant glycosylation sites particle quantification of influenza viruses by high performance liquid chromatography design and expression of a qconcat protein to validate hi3 protein quantification of influenza vaccine antigens evaluation of haemagglutinin content by rp-hplc to generate pandemic influenza vaccine topological n-glycosylation and site-specific n-glycan sulfation of influenza proteins in the highly expressed h1n1 candidate vaccines glycosylation characterization of an influenza h5n7 hemagglutinin series with engineered glycosylation patterns: implications for structurefunction relationships cross reactive material 197 glycoconjugate vaccines contain privileged conjugation sites quality by design approach in the development of an ultra-high-performance liquid chromatography method for bexsero meningococcal group b vaccine proteomics-driven antigen discovery for development of vaccines against gonorrhea production and purification of filovirus glycoproteins in insect and mammalian cell lines key: cord-299754-tgexahwd authors: van tol, sarah; hage, adam; giraldo, maria isabel; bharaj, preeti; rajsbaum, ricardo title: the trimendous role of trims in virus–host interactions date: 2017-08-22 journal: vaccines (basel) doi: 10.3390/vaccines5030023 sha: doc_id: 299754 cord_uid: tgexahwd the innate antiviral response is integral in protecting the host against virus infection. many proteins regulate these signaling pathways including ubiquitin enzymes. the ubiquitin-activating (e1), -conjugating (e2), and -ligating (e3) enzymes work together to link ubiquitin, a small protein, onto other ubiquitin molecules or target proteins to mediate various effector functions. the tripartite motif (trim) protein family is a group of e3 ligases implicated in the regulation of a variety of cellular functions including cell cycle progression, autophagy, and innate immunity. many antiviral signaling pathways, including type-i interferon and nf-κb, are trim-regulated, thus influencing the course of infection. additionally, several trims directly restrict viral replication either through proteasome-mediated degradation of viral proteins or by interfering with different steps of the viral replication cycle. in addition, new studies suggest that trims can exert their effector functions via the synthesis of unconventional polyubiquitin chains, including unanchored (non-covalently attached) polyubiquitin chains. trim-conferred viral inhibition has selected for viruses that encode direct and indirect trim antagonists. furthermore, new evidence suggests that the same antagonists encoded by viruses may hijack trim proteins to directly promote virus replication. here, we describe numerous virus–trim interactions and novel roles of trims during virus infections. eukaryotes are constantly exposed to a variety of pathogens, including viruses. as with other environmental signals, viral invasion triggers tightly regulated intracellular signaling cascades to optimally respond to infection. mammals enact both an innate and an adaptive immune response to identify an infecting pathogen, to clear the foreign agent, and to protect against subsequent invasion. a primary mechanism for fine-tuning molecular pathways is utilization of post-translational modifications. altering the functional proteome influences protein interactions, transcriptional programs, translation, secretion, and cytoskeletal arrangement. a variety of molecules, including phosphates, sugars, lipids, or proteins, can be attached or removed enzymatically to modulate protein function. post-translational modifications thus enable rapid and reversible regulation. under positive selection [42] . this species-specific pattern of positive selection of closely related trims suggests that individual trims play specific antiviral roles. in addition to differential mrna expression upon viral infection, several trim family members are intimately involved in the antiviral response. type-i ifns and other cytokines, such as pro-inflammatory cytokines induced via the nf-κb pathway, have been noted to differentially regulate the expression of a significant population of trims [7, [45] [46] [47] [48] [49] [50] [51] . likewise, trim overexpression influences the transcription of type-i ifn, pro-inflammatory cytokines, and ifn-stimulated genes (isgs) [7, 16] . the roles of trims in viral infection include intrinsic restriction of viral pathogens, positive regulation of immune pathways that promote viral clearance, and negative regulation of antiviral pathways to limit immunopathology [6, 7, 52] . the incorporation of trim antagonists into viral genomes exemplifies the importance of trims in antiviral responses [53] [54] [55] [56] [57] [58] . here, we will focus on the role of trims in the direct and indirect inhibition of viruses and novel mechanisms of viral-mediated antagonism and hijacking of trims. excellent reviews on the roles of trims in autophagy, cancer, and other diseases have been recently published [59] [60] [61] . cells identify pathogen invasion due to the presence of pathogen-associated molecular patterns (pamps) contained in viral components, which are recognized by host pattern recognition receptors (prrs) [62] . examples of viral pamps include some envelope or capsid proteins, viral nucleic acid, or intermediates of genome replication [62] . upon pamp engagement of a prr, a signaling cascade is initiated that relies on post-translational modifications for proper coordination. these modifications include ubiquitination and phosphorylation, which facilitate the assembly of adaptor and enzymatic molecules needed to activate and inactivate transcription factors and other effector molecules [2, 62, 63] . these transitions in the transcriptional profile and functional proteome enable the cell to respond optimally to the pathogen and to communicate (e.g., cytokine secretion) with neighboring cells to limit viral replication and promote clearance. examples of pathways critical in response to viral infection include ifn induction and signaling and nf-κb activation [64] . trim e3 ligases regulate ifn production and signaling as well as nf-κb induction at multiple levels, from prr-mediated pamp recognition to regulation of transcription factors and from promotion of signaling complex assembly to degradation of inhibitors [2, 7] . in this section, we discuss trim regulation of antiviral pathways (summarized in figures 1 and 2 ). viral double-stranded rna (dsrna) or single-stranded rna (ssrna) containing 5'-triphophates produced during virus replication, in the cytoplasm of a host cell, act as a retinoic acid-inducible gene i rig-i-like receptor (rlr) agonist [65] [66] [67] [68] . rig-i and melanoma differentiation-associated protein (mda5), encoded by ddx58 and ifih1, respectively, bind distinct viral rna agonists yet they induce similar downstream antiviral pathways [69] . rlrs are atp-dependent rna helicases that have two n-terminal caspase-activated recruitment domains (cards), a central dead box, and an auto-inhibitory c-terminal domain [70] . the unique pamps recognized by these receptors enable the host to respond to a broader range of pathogens [69] . upon engagement of the pamp with the rlr, a conformational shift exposes the cards, which allows homo-oligomerization and recruitment of the rlrs to their adaptor mitochondrial antiviral signaling protein (mavs) at the mitochondrial outer membrane (mom) [66, [71] [72] [73] . a variety of factors influence the activation of rlrs downstream of pamp recognition including atp hydrolysis [66, 74] , rlr oligomerization [71, 72, 74] , and post-translational modifications [75] [76] [77] [78] [79] . interaction of the n-terminal cards of both the rlrs and mavs induces the adaptor to form prion-like aggregates and exposes domains to recruit critical ring e3 ligases including tumor necrosis factor (tnf) receptor-associated factors (trafs) 3 and 6 [80, 81] . downstream of traf6, the ubd-containing adaptor tgf-β-activated kinase 1(tak1)/mitogen activating protein 3k7 (map3k7)-binding protein (tab) 2/3 recruits the critical kinase tak1 [82] [83] [84] . tak1 auto-phosphorylates to enable the phosphorylation of nf-κb essential modulator (nemo), the regulatory domain of inhibitor of nf-κb (iκb) kinase (ikk) complex, to activate the enzymatic domains of ikkα and β [83, 85] . ikkα and β then phosphorylate iκb, resulting in the recruitment of another e3 ligase, β-trcp (β-transducin repeat containing e3 ubiquitin protein ligase). β-trcp ligates k48-linked ubiquitin to iκb, inducing the proteasome-mediated degradation of the nf-κb inhibitor [63] . once the inhibitor is destroyed, nf-κb is phosphorylated and its nuclear localization sequence is exposed allowing nuclear translocation [63, 86] . inside the nucleus, nf-κb regulates the transcription of a variety of genes including pro-inflammatory cytokines and chemokines, such as pro-il-1β, tnf-α, and il-6, and negative regulators of the pathway to limit an exacerbated inflammatory response [86] . vaccines 2017, 5, 23 5 of 36 another e3 ligase, β-trcp (β-transducin repeat containing e3 ubiquitin protein ligase). β-trcp ligates k48-linked ubiquitin to iκb, inducing the proteasome-mediated degradation of the nf-κb inhibitor [63] . once the inhibitor is destroyed, nf-κb is phosphorylated and its nuclear localization sequence is exposed allowing nuclear translocation [63, 86] . inside the nucleus, nf-κb regulates the transcription of a variety of genes including pro-inflammatory cytokines and chemokines, such as pro-il-1β, tnf-α, and il-6, and negative regulators of the pathway to limit an exacerbated inflammatory response [86] . trims play an integral role in the positive and negative regulation of antiviral pathways. trims can act as pathogen prrs, as is the case for trim21 in the recognition of non-enveloped viruses bound by immunoglobulin (ig). additionally, these trims can regulate the activation of other prrs that recognize viral pathogen-associated molecular patterns (pamps) in the cytosol (ddx41 (dead-box helicase 41), cyclic gmp-amp synthase (cgas), deah-box helicase 33 (dhx33), nucleotide-binding oligomerization domain-containing protein 2 (nod2), retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein (mda5)) and at membrane surfaces (toll-like receptors, tlrs). downstream of the initial pattern recognition, trims also influence the recruitment and interaction of adaptor molecules (stimulator of ifn genes (sting), mitochondrial antiviral signaling protein (mavs), tgf-β-activated kinase 1(tak1)/map3k7-binding protein (tab) 2, myeloid differentiation primary response gene 88 (myd88), tir-domain-containing adapterinducing interferon-β (trif), nf-κb essential modulator (nemo), nucleosome assembly protein (nap-1), and tumor necrosis factor (tnf) receptor-associated factors (traf) family memberassociated nf-κb activator (tank)) and enzymes (traf3, traf6, tak1, inhibitor of nf-κb (iκb) kinase (ikk) α,β,ε, tank binding kinase 1 (tbk1)) to signaling complexes in order to activate transcription factors. this includes ifn regulatory factor (irf)3 and irf7, important in type-i interferon (ifn) signaling, and nf-κb, important in expression of pro-inflammatory genes, which regulate the expression of antiviral effectors. type-i ifn production is critical for an effective antiviral response. trims play an integral role in the positive and negative regulation of antiviral pathways. trims can act as pathogen prrs, as is the case for trim21 in the recognition of non-enveloped viruses bound by immunoglobulin (ig). additionally, these trims can regulate the activation of other prrs that recognize viral pathogen-associated molecular patterns (pamps) in the cytosol (ddx41 (dead-box helicase 41), cyclic gmp-amp synthase (cgas), deah-box helicase 33 (dhx33), nucleotide-binding oligomerization domain-containing protein 2 (nod2), retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein (mda5)) and at membrane surfaces (toll-like receptors, tlrs). downstream of the initial pattern recognition, trims also influence the recruitment and interaction of adaptor molecules (stimulator of ifn genes (sting), mitochondrial antiviral signaling protein (mavs), tgf-β-activated kinase 1(tak1)/map3k7-binding protein (tab) 2, myeloid differentiation primary response gene 88 (myd88), tir-domain-containing adapter-inducing interferon-β (trif), nf-κb essential modulator (nemo), nucleosome assembly protein (nap-1), and tumor necrosis factor (tnf) receptor-associated factors (traf) family member-associated nf-κb activator (tank)) and enzymes (traf3, traf6, tak1, inhibitor of nf-κb (iκb) kinase (ikk) α,β,ε, tank binding kinase 1 (tbk1)) to signaling complexes in order to activate transcription factors. this includes ifn regulatory factor (irf)3 and irf7, important in type-i interferon (ifn) signaling, and nf-κb, important in expression of pro-inflammatory genes, which regulate the expression of antiviral effectors. type-i ifn production is critical for an effective antiviral response. trims in cytokine signaling. downstream of the initial pathogen recognition and induction of pro-inflammatory cytokines, trims can regulate their cytokine signaling pathways through interactions with cytokine receptor adaptors (tab2/3) and enzymatic proteins (ikkα, ikkβ and ikkε) within the signaling complexes, the activity and stability of pathway negative regulators (protein inhibitor of activated stat 3 (pias3), suppressor of cytokine signaling (socs), and influence the transcription of various cytokine-effector genes (nf-κb-induced pro-inflammatory cytokines, signal transducer and activator of transcription (stat)-induced genes, interferon stimulated genes (isgs)) or cytokine signaling regulators (tumor necrosis factor (tnf) receptors (tnfr1/2, and stat-induced genes)). in addition to activation of nf-κb, rlr signaling induces type-i ifn [73] . traf3, in cooperation with nemo, recruits and stabilizes traf family member-associated nf-κb activator (tank) or nucleosome assembly protein (nap1) which are critical in linking tank binding kinase 1 (tbk1), and in some cases inhibitor of kappa light polypeptide gene enhancer in b cells (ikkε), to the mavs signalosome [81, 82] . once activated, tbk1 and/or ikkε phosphorylate the ifn regulatory factor (irf) 3 and irf7 [87, 88] . upon phosphorylation, the irfs homodimerize and translocate to the nucleus where they bind to dna regulatory regions [87, 89] . to induce optimal ifn-β transcription, activated irf3, nf-κb, and ap-1 (activator protein 1) must translocate to the nucleus and bind to their respective regulatory regions of the ifnb1 promoter [64] . the resulting ifn-β is then secreted and signals in a paracrine and autocrine manner. binding of ifn-β to its heterodimeric receptor results in the activation of tyrosine kinases, janus kinase 1 (jak1) and tyrosine kinase 2 (tyk2), which phosphorylate signal transducer and activator of transcription (stat) 1 and stat2. following phosphorylation, stat1 and stat2 heterodimerize and associate with irf9 to form ifn-stimulated gene factor 3 (isgf3) and translocate to the nucleus [87] . within the nucleus, isgf3 binds to genes with an ifn stimulated response element (isre) in their promoter to activate transcription [87] . the resulting proteins expressed from these isgs, such as pkr (protein kinase r), mxa (myxovirus resistance gene a), isg15, and trims, are involved in creating a cellular environment prohibitive to viral entry and replication [87] . as with other immune pathways, isgf3 also promotes the transcription of type-i ifn negative regulators to mitigate deleterious effects [87] . trims play a critical role in both the positive and negative regulation of the rlr pathway to ensure optimal virus restriction while minimizing self-inflicted damage ( figure 1 ). several trims have been shown to positively regulate the receptors rig-i and mda5 [90] [91] [92] . the best characterized example of trim-mediated rig-i activation involves trim25. trim25 ligates k63-linked poly-ub chains onto the n-terminal card at k172, which induces downstream signaling [92] . additionally, trim25 catalyzes the synthesis of unanchored k63-linked poly-ub chains, which facilitate rig-i oligomerization and stabilization [77] . both oligomerization and stabilization of rig-i promotes the interaction of its cards with mavs [93] . adding complexity to this interaction, trim25 k48-linked . trims in cytokine signaling. downstream of the initial pathogen recognition and induction of pro-inflammatory cytokines, trims can regulate their cytokine signaling pathways through interactions with cytokine receptor adaptors (tab2/3) and enzymatic proteins (ikkα, ikkβ and ikkε) within the signaling complexes, the activity and stability of pathway negative regulators (protein inhibitor of activated stat 3 (pias3), suppressor of cytokine signaling (socs), and influence the transcription of various cytokine-effector genes (nf-κb-induced pro-inflammatory cytokines, signal transducer and activator of transcription (stat)-induced genes, interferon stimulated genes (isgs)) or cytokine signaling regulators (tumor necrosis factor (tnf) receptors (tnfr1/2, and stat-induced genes)). in addition to activation of nf-κb, rlr signaling induces type-i ifn [73] . traf3, in cooperation with nemo, recruits and stabilizes traf family member-associated nf-κb activator (tank) or nucleosome assembly protein (nap1) which are critical in linking tank binding kinase 1 (tbk1), and in some cases inhibitor of kappa light polypeptide gene enhancer in b cells (ikkε), to the mavs signalosome [81, 82] . once activated, tbk1 and/or ikkε phosphorylate the ifn regulatory factor (irf) 3 and irf7 [87, 88] . upon phosphorylation, the irfs homodimerize and translocate to the nucleus where they bind to dna regulatory regions [87, 89] . to induce optimal ifn-β transcription, activated irf3, nf-κb, and ap-1 (activator protein 1) must translocate to the nucleus and bind to their respective regulatory regions of the ifnb1 promoter [64] . the resulting ifn-β is then secreted and signals in a paracrine and autocrine manner. binding of ifn-β to its heterodimeric receptor results in the activation of tyrosine kinases, janus kinase 1 (jak1) and tyrosine kinase 2 (tyk2), which phosphorylate signal transducer and activator of transcription (stat) 1 and stat2. following phosphorylation, stat1 and stat2 heterodimerize and associate with irf9 to form ifn-stimulated gene factor 3 (isgf3) and translocate to the nucleus [87] . within the nucleus, isgf3 binds to genes with an ifn stimulated response element (isre) in their promoter to activate transcription [87] . the resulting proteins expressed from these isgs, such as pkr (protein kinase r), mxa (myxovirus resistance gene a), isg15, and trims, are involved in creating a cellular environment prohibitive to viral entry and replication [87] . as with other immune pathways, isgf3 also promotes the transcription of type-i ifn negative regulators to mitigate deleterious effects [87] . trims play a critical role in both the positive and negative regulation of the rlr pathway to ensure optimal virus restriction while minimizing self-inflicted damage ( figure 1 ). several trims have been shown to positively regulate the receptors rig-i and mda5 [90] [91] [92] . the best characterized example of trim-mediated rig-i activation involves trim25. trim25 ligates k63-linked poly-ub chains onto the n-terminal card at k172, which induces downstream signaling [92] . additionally, trim25 catalyzes the synthesis of unanchored k63-linked poly-ub chains, which facilitate rig-i oligomerization and stabilization [77] . both oligomerization and stabilization of rig-i promotes the interaction of its cards with mavs [93] . adding complexity to this interaction, trim25 k48-linked polyubiquitination negatively regulates rlr activation, but the ubiquitin specific protease 15 (usp15) can specifically disassemble these poly-ub chains to stabilize trim25 [94] . trims 4, 13, and 38 have also been implicated in positive regulation of the rig-i pathway. similar to trim25, trim4 also catalyzes the ligation of k63-linked poly-ub chains onto rig-i card [91] . additionally, trim38 functions as an e3 sumo (small ubiquitin-like modifier) ligase and sumoylates both rig-i and mda5 to prevent the ligation of k48-linked poly-ub chains thus stabilizing these prrs [7, 95, 96] . the capacity of multiple trims to activate rig-i suggests that ubiquitination is crucial in rig-i signaling, but the relative contribution of each trim is not well understood. perhaps multiple trims allow for redundancy in the instance that one trim is inhibited or if trims play cell-type specific roles in rlr signaling. recently trim65 was identified as an e3 ligase of mda5. unlike trim25, trim65 ubiquitinates mda5 at the rna helicase domain [90] . the covalent linkage of k63-linked poly-ub onto k743 promotes mda5 oligomerization and downstream activation of irf3 [90] . demonstrating the specificity of mda5 activation, trim65 only promotes the restriction of encephalomyocarditis virus (emcv), a picornavirus, and not vesicular stomatitis virus (vsv), a rhabdovirus [90] . in mouse cells, trim13 was shown to impair mda5-mediated activation of the ifn pathway through an unclarified mechanism [50] . another trim inhibitor of the mavs pathway is trim59, which interacts with evolutionarily conserved signaling intermediate in toll pathways (ecsit) and mavs and subsequently inhibits the transcription of irf3 and nf-κb target genes [97] . although trims have not been identified as rig-i negative regulators, their role in mda5 inhibition suggests there may be unidentified trim-mediated rig-i inhibition. the role of trim25 in the regulation of rlr pathways and/or type-i ifn induction has been shown to be conserved among different species. in fact a diverse range of vertebrates encode rig-regulating trims. in salmonids, trim25, mavs, mda5, and rig-i were induced following infection with an alphavirus although the signaling pathways were not addressed directly [98] . duck trim25 catalyzes the synthesis of unanchored poly-ub chains to activate rig-i [99] . despite lacking lysine 172 in duck rig-i, duck trim25 ubiquitinates rig-i's card domains and promotes rlr signaling [99] . in chicken cells, despite lacking a functional rig-i gene, knockdown of chicken trim25 results in reduced ifn-β upon infection with specific strains of the influenza a virus (iav) [100] , suggesting that trim25 is involved in activation of ifn signaling through a rig-i-independent mechanism, perhaps activation of mda5 or mavs. expression of chicken trim25 is induced after newcastle disease virus (ndv), poly(i:c) treatment, or poly(da:dt) treatment [101] , probably via a type-i ifn signaling-dependent pathway. similar to human trim25, trim27-l stimulates ifn-β production in response to iav infection in ducks [102] . this anti-viral benefit is absent in chickens and turkeys, as they do not carry the trim27-l gene in their trim cluster [102] . however, expression of duck trim27-l and d2card in chicken df1 cell lines was shown to facilitate ifn-β and mx1 expression [102] . this difference may account for the different pathologies in avian species as waterfowl are typically more resistant to some strains of iav as compared to chickens. downstream of rlr activation, a variety of trims promote mavs signaling. trim25 has been implicated in the k48-linked polyubiquitination of mavs, which results in its proteasome-mediated degradation and release of downstream signaling molecules (tbk1, nemo, and possibly traf3) to induce type-i ifn production [103] . recently, trim31 has been described to mediate the k63-linked ubiquitination of mavs at lysines 10, 311, and 461 [104] . this ubiquitination promotes the prion-like aggregation of mavs needed for optimal signaling, exemplified by the decrease in tbk1 and ikkε phosphorylation [104] . in a trim31-deficient murine model, trim31 knock-out mice demonstrated an increased susceptibility to vsv infection and an upregulation in ifn-β production. although rig-i was demonstrated to be required for activation of trim31 function, its role in mda5-mediated signaling was not investigated in-depth [104] . trim14 has been demonstrated to play a crucial role in linking the nf-κb and irf3 branches of rlr signaling [30] . despite lacking a ring domain, trim14 interacts with nemo via its pry-spry domain and promotes k63-linked ubiquitination of nemo, which is critical in recruiting nemo to mavs [30] . the role of trim44 stabilization of mavs has also been characterized [7, 105] . although no trim inhibitors of mavs have been described, screens of trims that inhibit mavs-mediated type-i ifn production may reveal such trims. in addition to regulating rlrs and mavs, trims modulate downstream adaptors and enzymes. several of these trims can likewise mitigate signaling downstream of other prrs that converge on shared molecules such as nemo and tak1, but trims identified to be involved at the level of signaling using rlr induction are described below. recently, the short isoform of trim9 (trim9s) was shown to bridge gsk3β ( glycogen synthase kinase 3 beta) to phosphorylated tbk1 to promote tbk1 oligomerization and activation of irf3 [106] . to facilitate the interaction between gskβ and ptbk1, trim9s must be auto-ubiquitinated [106] . this activation of tbk1-signaling occurs downstream of rlr and sting signaling [107] and biases the immune response toward the type-i ifn pathway while limiting nf-κb-induced transcription [106] . trim11's coiled-coil domain interacts with the coiled-coil domain 2 of tbk1 to prohibit the kinase's interaction with adaptors nap1 or tank [108] . impairing this interaction results in lack of ifn-β production [108] . nap1 is targeted for degradation following trim38-mediated k48-linked poly-ub, which likewise decreases activation of ifn-β [109] . interaction between the arf (adp-ribosylation factor domain) (c-terminus) domain of trim23, and both the coiled-coil 1 and lz domains of nemo, allow trim23 to facilitate k27-linked ubiquitination of nemo [110] . this ubiquitination of nemo facilitates the activation of irf3 and nf-κb signaling downstream of pathogen recognition, but not tnf-α signaling [110] . similar to the described trim9s mechanism, trim26 is able to interact with tbk1 and auto-phosphorylates k27-linked poly-ub chains to bridge tbk1 and nemo [111] . this interaction was demonstrated downstream of mavs signaling and promoted irf3 activation [111] . indirectly, trim68 antagonizes ifn-β transcription [112] . this trim is able to inhibit both toll-like receptor (tlr) and rlr-driven activation of the type-i ifn pathway [112] . several trims are likewise implicated in the inhibition of the nf-κb activation branch of prr signaling. murine specific trim30α negatively regulates the tab2/tab3 complex, which impedes the recruitment and activation of tak1, thus preventing the phosphorylation of nemo and subsequent nf-κb activation [7, 113] . through a different mechanism in the brain, the long isoform of trim9 (trim9l) inhibits β-trcp [33] . perturbation of β-trcp inhibits both canonical and non-canonical nf-κb activation [33] . the trim9l protein sequence includes a degron motif that, when phosphorylated at serine residues 76 and 80, recruits β-trcp [33] . titrating β-trcp from the nf-κb inhibitors prevents their degradation and subsequent pro-inflammatory signaling [33] . several other trims also inhibit nf-κb activation including trim11 [114] , trim29 [115] , and trim39 [116] . the mechanisms for trim11 regulation have not been clearly elucidated, but trim29 targets nemo for degradation in alveolar macrophages [115] and trim39 stabilizes cactin [116] , a nuclear, negative regulator of nf-κb. the bias of trims in the negative regulation of the nf-κb branch of rlr signaling suggests that trims may play a role in promoting the type-i ifn pathway at the cost of nf-κb activation, and further supports the hypothesis that groups of trims may have evolved as part of the antiviral type-i ifn system [48] . however, it is important to note that some studies showing negative regulatory roles of trims have only used overexpression assays with large concentrations of trim expressing vectors, which could lead to artifacts. however, it is now clear that overall trims act at several levels to regulate rlr signaling to balance viral clearance and cell survival. in addition to regulating cytosolic rna-stimulated responses, trims also regulate the pathways following cytosolic dna recognition. in the cytoplasm, the host expresses multiple dna and rna receptors aside from rlrs, including ifi16 (interferon gamma inducible protein 16), cyclic gmp-amp synthase (cgas), and ddx41. the listed double-strand (ds) dna receptors activate the adaptor molecule stimulator of ifn genes (sting) at the endoplasmic reticulum (er) [117] [118] [119] [120] [121] . upon recognition of dsdna, which can result from infection with dna viruses, cgas oligomerizes and catalyzes cyclic dinucleotide (c-gmp-amp) synthesis [95] . c-gmp-amp and other dinucleotides activate sting and induce sting dimerization [95, 119, 120] . consequently, sting recruits tbk1, which phosphorylates irf3 for type-i ifn induction [117] . the dna helicase ddx41 recognizes both cytoplasmic dsdna and cyclic dinucleotides, both of which promote ddx41 activation of sting [121, 122] . five trims are known to influence sting-mediated signaling ( figure 1 ). the pry-spry domain of trim21 interacts with ddx41's helicase domain and catalyzes ubiquitination at lysine residues 9 and 115, targeting the prr for degradation [123] . this degradation pathway occurs in myeloid dendritic cells and restricts type-i ifn induction [123] . the murine-specific trim, trim30α, ubiquitinates sting following herpes simplex virus 1 (hsv-1) and targets the adaptor protein for degradation [124] . both trim32 and trim56 facilitate k63-linked polyubiquitination of sting to promote dimerization and activation of sting-mediated antiviral responses [125, 126] . finally, trim38 sumoylates cgas and sting similar to the sumoylation of rig-i and mda5 [95] . sumoylation inhibits the ligation of k48-linked ubiquitin and results in their stabilization [95] . in addition to regulating cytosolic prr signaling, trims also modulate membrane-bound prrs including toll-like receptors (tlrs) ( figure 1 ). several tlr family members recognize viral pamps. the main tlrs involved in virus recognition include endosome-localized tlrs 3, 7, 8, and 9 [127] . tlr3 recognizes both double-stranded rna and the viral rna mimic poly(i:c), tlr7 and tlr8 recognize single-stranded rna, and tlr9 recognizes cpg [127] . tlr4 is associated mainly with the plasma membrane, although it can also be internalized in endosomes, and can recognize some viral surface antigens. tlrs 4, 7, 8, and 9 signal through iraks (interleukin-1 receptor-associated kinase) 1 and 4, which are recruited via the adaptor molecule myd88 (myeloid differentiation primary response gene 88) [127] . traf6 then re-localizes to the tlr signaling complex to activate tak1, inducing nf-κb [82, 127] similar to the pathway described in the aforementioned rlr section. tlr3 and 4 also signal through iraks 1 and 4, and interact with the adaptor trif (tir-domain-containing adapter-inducing interferon-β), resulting in the activation of traf3. ubiquitination downstream of traf3 induces tbk1-and ikkε-mediated phosphorylation of irf3 and irf7 to induce type-i ifn [127] . trim21's pry-spry domain interacts with irfs, including irf3, 5, and 7, to induce their degradation [128] [129] [130] . although trim21 may promote degradation of irfs downstream of other prrs, the interaction between the two proteins may depend on the induction of a specific tlr pathway. trim38 targets tlr signaling at multiple points. downstream of tlr2, 3, 4, or 7, trim38 targets traf6 for proteasome-mediated degradation following k48-linked polyubiquitination [131] . downstream of tlr3 and tlr4 signaling, trim38 also targets trif and nap1 for degradation [109, 132] . finally, trim56 has been shown to promote tlr3 activation via interaction with trif in a ring ligase-independent manner [133] . perhaps this interaction promotes the stability of trif to facilitate downstream signaling. disruption of adaptor protein availability thus bottlenecks antiviral signaling. nucleotide-binding domain and leucine-rich repeat-containing receptors (nlrs) are another class of cytosolic receptors that recognize both pamps and damage-associated molecular patterns (damps). damps are host-derived molecules that are expressed only after a cell experiences stress and/or damage commonly due to inflammation [134, 135] . upon activation of a nlr, the receptor assembles an inflammasome in cooperation with the adaptor asc to recruit pro-caspases [134, 135] . the nlrp3 (nlr family pyrin domain containing 3) -induced inflammasome promotes the cleavage of pro-caspase 1 to caspase 1, which then promotes a pro-apoptotic response involving the caspase-1-mediated cleavage of pro-il-1β and pro-il-18 to their active forms il-1β and il-18 [134, 135] . the secreted pro-inflammatory cytokines then promote further inflammatory responses, such as pyroptosis, which may be damaging to the host when uncontrolled [134, 135] . in some instances another nlr, nod2, may function as a cytosolic dsrna receptor and converge with the rlr pathway at the level of mavs [136, 137] . at this point only a few trims have been described to interact with nlrs in the control of viral infections. trim33 binds to and ubiquitinates dhx33, a cytosolic dsrna receptor that acts upstream of nlrp3, at lysine k218 to facilitate inflammasome activation [138] . the knockdown of trim33 diminishes the activation of caspase 1 and likewise decreases the release of il-1β and il-18 [138] . the interaction of trim33 with dhx33 relies on the b-box and coiled-coil domains [138] . in contrast, the murine-specific trim30α impairs the nlrp3 inflammasome through an unknown mechanism [139] . another nlrp3 inhibitor identified using a dextran sodium sulfate-induced colitis model is trim31 [140] . the coiled-coil domain of trim31 interacts with the leucine rich and nacht domains of nlrp3 and ligates k48-linked poly-ub chains to target nlrp3 for proteasome-mediated degradation [140] . although the authors did not evaluate virus infection directly, trim31-deficient cells stimulated with poly(i:c) express higher levels of nlrp3 compared to wild-type cells suggesting trim31 may regulate nlrp3 downstream of virus recognition [140] . trim27 is able to promote degradation of nod2 [141] , which may prohibit this receptor from recognizing dna and rna virus infection [136, 137] . the further investigation of trim interactions with nlrs is important for understanding the immune response during bacteria-virus co-infections. several trims are also involved in regulating the cytokine signaling following initial pathogen recognition ( figure 2 ). downstream of tnf-α engagement with its receptor tnfr1, the tak1 complex is recruited to activate nf-κb [63] . trim8 specifically promotes the activation of tnf-α-induced nf-κb signaling through inhibition of the nf-κb nuclear repressor protein inhibitor of activated stat (pias) 3 [142] . the trim8-mediated repression of pias likewise promotes il-6-dependent activation of stat3 [143] . specifically downstream of il-1β and tnf-α signaling in ifn-β-primed cells, trim38 targets tab2 for lysosomal degradation [49, 144] . as described above, the degradation of tab2 inhibits the recruitment and activation of tak1 which blocks pro-inflammatory signaling. zheng and colleagues showed that trim27 catalyzes k48-linked poly-ub at residues k251 and k372 of tbk1 to promote proteasome-mediated degradation downstream of ifn signaling [145] . trim27-tbk1 interactions require the coiled-coil and b-box trim domains [145] . in endothelial cells, trim28 sustains the expression of tnfr1 and tnfr2 to promote the activation of pro-inflammatory pathways [146] . the role of trim28 in the activation of the endothelium may play an important, unexplored role in immune cell trafficking in response to infection. in response to cytokines (i.e., il-12) secreted from activated dendritic cells (dcs), ifn-γ (type-ii ifns) is released from t cells and natural killer cells [147] . after this, type-ii ifn binds its receptor, and the downstream kinases jak1 and jak2 promote stat1 phosphorylation and homodimerization to form gamma activating factor, which translocates to the nucleus to bind gamma-stimulated elements in gene promoters [147] . trim24 inhibits stat1 transcription via binding to the stat1 promoter [148] . in contrast, trim8 is able to destabilize socs-1 (suppressor of cytokine signaling 1), a negative regulator of the ifn-γ signaling pathway, resulting in increased type-ii ifn signaling [149] . the regulation downstream of ifn signaling suggests that this is a negative regulatory mechanism to prevent inflammatory response overactivation. in response to type-i ifns, trim6 catalyzes the formation of k48-linked unanchored poly-ub chains to facilitate the activation of ikkε, which favors isgf3 formation due to stat1 phosphorylation at s708 [11] . this modification increases stat1-stat2 dimerization [150, 151] . this trim6-enahnced activation of ikkε may also play a role in activating ifn-β transcription and translation downstream of rlr activation [11] . trim22 induces the lysosome-mediated destruction of foxo4 and consequently impairs the transcription of ifn-β downstream of tlr3 and rlr signaling [152] . after the innate response to pathogen invasion, the host evolves an adaptive immune response. establishment of an adaptive response requires the presentation of pathogen antigens to t and b lymphocytes. although trim proteins have been mostly studied as regulators of innate immune responses and the role of trims in the regulation of antigen presentation has not been investigated intensively, several studies indicate that trims play an important role in regulating t cell activation. expression of trim22, for example, has been shown to be down-regulated upon cd28/cd2-mediated activation despite being expressed at high levels in resting t cells [153] . in contrast, trim22 expression in t cells is increased following il-2 and il-15 cytokine signaling [154] , both of which act as pro-survival signals. recently, trim24 was shown to influence the expression of th2-type cytokines but not other cd4 + t cell sub-types [155] . as th2 cells predominantly act in allergy-and parasite-induced responses while th1 cells are more important for viral clearance, deregulation of trim24 expression may impact the cd4 + population composition and the capacity of the host to efficiently clear the virus. in the past, trim27 was described as impairing the activation of cd4 + t cells via k48-linked poly-ub of pi3kc2b (phosphatidylinositol 4-phosphate 3-kinase c2 domain-containing subunit beta) [156] . this impairment was observed specifically in cd4 + t cells downstream of t cell receptor (tcr) engagement and not cd8 + t cells [156] . additionally, trim28 depletion in vivo promotes expansion of the th17 population resulting in an autoimmune phenotype [34] . following tcr-mediated activation, trim28 was phosphorylated suggesting it plays a role t cell activation [34] . throughout the lifespan of trim30 knockout mice, the ratio of cd4 + to cd8 + progressively increased and the cd4 + t cells proliferated abnormally [157] . the role of trim30 is intrinsic to cd4 + t cells, because the same defect was observed upon adoptive transfer of knockout cd4 + t cells to wild-type mice [157] . it remains to be seen whether other trims play important roles in adaptive immunity. it is likely that trims have unexplored functions in recognition of antigen presentation by the t cell receptor, and or in differentiation of cd4 + /cd8 + t cells. despite the numerous host evasion mechanisms pathogens employ, a variety of host encoded molecules, such as trims, are able to restrict viruses [2] . in addition to conferring an antiviral state indirectly by regulating cytokine production downstream of prr signaling, trims are capable of restricting the effectiveness of pathogens through direct interactions with viral proteins crucial to their entry, dissemination, or life cycle [6] . the categories of viral restriction include: inhibition of viral transcription, replication or translation, degradation or interference of viral proteins, and impairment of virus entry or exit. we next outline the various means by which hosts deploy trims to counter and clear pathogens. trim5α is an example of a trim functioning as both a direct virus restriction factor as well as a pathogen-recognition receptor ( figure 3 ). in old-world monkeys, such as african green monkeys and rhesus macaques, trim5α enables natural resistance to hiv-1 while new-world monkeys, like owl monkeys, are protected from hiv-1 by fusion of trim5 with cyclophilin a (tcypa) [158] [159] [160] [161] . progress on the topic ranges from revealing the α isoform of trim5 as the restriction factor to discerning the role in restriction of each structural motif [158, 159] . . the role of trims in retrovirus replication. trim5α oligomerization into a hexagonal lattice associates directly with hiv-1 capsids to promote premature uncoating. recognition of hiv-1 capsid by trim5α also triggers nf-κb/ap-1-mediated innate immune signaling via synthesis of unanchored k63-linked poly-ub chains that activate the tak1 kinase. one potential mechanism of trim5αmediated restriction could be involved ubiquitination of trim5α and proteasomal degradation of trim5α-capsid complexes. trim11 mobilizes cellular microtubule formation to prematurely uncoat hiv-1 and facilitate rapid release of the vrna from the viral core, resulting in inhibition of virus replication. trim19 translocates to the cytoplasm and binds daxx to prevent its degradation by the proteasome, allowing for daxx-mediated disruption of hiv-1 reverse transcription (rt). trim22 inhibits sp1, preventing ltr-mediated transcription. the connection between trim5α-mediated restriction and proteasomal-mediated degradation of hiv-1 requires further characterization. however, one potential model may involve improved proliferation of hiv-1-specific cd8 + t cells due to enhanced production of viral peptides from proteasome-mediated degradation of hiv-1 capsids. several components inherent to trim5α play a role in hiv-1 capsid restriction, including dimerization, oligomerization, and ubiquitination [162] [163] [164] . there have been different models proposed for the mechanism of restriction and all have been attributed to the interaction of the spry domain of trim5α with the retrovirus capsid. binding of trim5α to viral cores serves to prematurely uncoat the viral particle and induces early release of the viral genome. elucidation of the trim5α higher order structure has revealed the significant contributions made by each component of the trim5α protein [26, [163] [164] [165] [166] . monomeric trim5α can dimerize in an antiparallel fashion through the coiled-coil domains, thereby generating the most fundamental component needed to bind the viral capsid [163, 167] . once bound to its target, trim5α can form higher-order structures resembling a hexagonal net [164, 165, 168, 169] . trim5α and trim5 variants from several primate species, including rhesus macaques, african green monkeys, and owl monkeys, are capable of forming flexible, hexagonal frameworks encompassing hiv-1 capsid surfaces [164] . the hexagonal nets are formed from trim5α trimers and are dependent on the trim5α's b-box domain [165, 166] . these trimers have been shown to be flexible and may allow for ideal binding of the spry domains on the highly variable hiv-1 capsid [165, 166] . the ability of rhesus macaque trim5α (rhtrim5α) to form higher-order structures upon binding to the capsid also allows the formation of trim5α ring dimers, which enhances its e3-ubiquitin ligase activity and innate anti-hiv-1 activity [29] . as one potential mechanism of rhtrim5α-mediated restriction it was proposed that as the linker 2 regions of the dimer change their conformation, the spry domains that are bound to the hiv-1 capsid in their hexagonal lattice formation disturb the viral structure [163] . the induction of this conformational change disrupts the integrity of the viral core and may be responsible for pre-mature viral uncoating. capsid by trim5α also triggers nf-κb/ap-1-mediated innate immune signaling via synthesis of unanchored k63-linked poly-ub chains that activate the tak1 kinase. one potential mechanism of trim5α-mediated restriction could be involved ubiquitination of trim5α and proteasomal degradation of trim5α-capsid complexes. trim11 mobilizes cellular microtubule formation to prematurely uncoat hiv-1 and facilitate rapid release of the vrna from the viral core, resulting in inhibition of virus replication. trim19 translocates to the cytoplasm and binds daxx to prevent its degradation by the proteasome, allowing for daxx-mediated disruption of hiv-1 reverse transcription (rt). trim22 inhibits sp1, preventing ltr-mediated transcription. the connection between trim5α-mediated restriction and proteasomal-mediated degradation of hiv-1 requires further characterization. however, one potential model may involve improved proliferation of hiv-1-specific cd8 + t cells due to enhanced production of viral peptides from proteasome-mediated degradation of hiv-1 capsids. several components inherent to trim5α play a role in hiv-1 capsid restriction, including dimerization, oligomerization, and ubiquitination [162] [163] [164] . there have been different models proposed for the mechanism of restriction and all have been attributed to the interaction of the spry domain of trim5α with the retrovirus capsid. binding of trim5α to viral cores serves to prematurely uncoat the viral particle and induces early release of the viral genome. elucidation of the trim5α higher order structure has revealed the significant contributions made by each component of the trim5α protein [26, [163] [164] [165] [166] . monomeric trim5α can dimerize in an antiparallel fashion through the coiled-coil domains, thereby generating the most fundamental component needed to bind the viral capsid [163, 167] . once bound to its target, trim5α can form higher-order structures resembling a hexagonal net [164, 165, 168, 169] . trim5α and trim5 variants from several primate species, including rhesus macaques, african green monkeys, and owl monkeys, are capable of forming flexible, hexagonal frameworks encompassing hiv-1 capsid surfaces [164] . the hexagonal nets are formed from trim5α trimers and are dependent on the trim5α's b-box domain [165, 166] . these trimers have been shown to be flexible and may allow for ideal binding of the spry domains on the highly variable hiv-1 capsid [165, 166] . the ability of rhesus macaque trim5α (rhtrim5α) to form higher-order structures upon binding to the capsid also allows the formation of trim5α ring dimers, which enhances its e3-ubiquitin ligase activity and innate anti-hiv-1 activity [29] . as one potential mechanism of rhtrim5α-mediated restriction it was proposed that as the linker 2 regions of the dimer change their conformation, the spry domains that are bound to the hiv-1 capsid in their hexagonal lattice formation disturb the viral structure [163] . the induction of this conformational change disrupts the integrity of the viral core and may be responsible for pre-mature viral uncoating. proteasomal-mediated degradation of viral components is a host restriction strategy characteristic of many trim family proteins involved in host innate immunity. however, the functional role of the proteasome in trim5α-mediated hiv-1 restriction has been difficult to discern and has been subject of debate. early work with proteasomal inhibitors, like mg132, suggested a two phase restriction of hiv-1 by trim5α. in the proteasome-independent phase, trim5α inhibits nuclear entry of the reverse transcription (rt) products [170, 171] . in the second phase, trim5α can inhibit late rt products in the presence of a functional proteasome, suggesting trim5α may utilize the proteasome to disrupt the viral rt complex [170, 171] . while proteasome inhibition can block disassembly and/or degradation of viral core components, it does not appear to rescue infectivity, which has led some investigators to conclude that degradation of capsid by the proteasome is not the mechanism of trim5α-mediated restriction [172] . in addition, thus far no study has described ubiquitination sites on the hiv-1 capsids, which could potentially link degradation of the capsid with proteasomal function. however, additional studies have shown proteasomal involvement in trim5α restriction of hiv-1 by degrading trim5α itself [173] . trim5α's e3 ligase activity can facilitate auto-ubiquitination, thereby signaling for its own destruction by the host proteasomal machinery [36, 173] . these occurrences have led some to speculate on a potential model where trim5α interacts with the hiv-1 capsid leading to auto-ubiquitination, capsid uncoating, and delivery to the proteasome [173] [174] [175] [176] . despite numerous investigations centered around proteasomal involvement, controversy within the literature surrounding the exact model connecting trim5α, hiv-1 cores, and proteasomes remains, and proteasome-dependent and independent mechanism have been proposed [175, 176] . polyubiquitination through the e3 ligase activity of trim5α's ring domain may also be a factor involved in inhibition of retroviral replication [177] . in addition to its restriction activity, trim5α can induce antiviral type-i ifns. multiple trim5 orthologs induce ap-1-mediated innate immune signaling [169, 178] . in the presence of the hiv-1 capsid, the e3 ligase activity of trim5 facilitates the generation of unanchored k63-linked poly-ub chains. these k63-linked chains promote tak1 autophosphorylation and activation, resulting in the induction of ap-1-and nfκb-mediated transcription [169] . collectively, these recent findings serve to better characterize the mechanisms through which trim5α achieves inhibition of hiv-1 replication. the restriction benefits conferred by trim5α can be observed in the progression to disease upon infection with simian immunodeficiency virus (siv) in rhesus macaques. course of infection studies with either a trim5α-sensitive or -resistant strain of siv revealed that inoculation with a trim5α susceptible strain of siv provided higher survival rates for rhesus macaques. also, delayed development of the pathology associated with trim5α-sensitive siv compared to subjects treated with a trim5α-resistant strain was observed [179] . in addition to these benefits, the restriction of the trim5α sensitive siv strain correlated with prolonged maintenance of cd4 + central memory t cells [179] . the preservation of cd4 + central memory t cells has been extensively characterized as important in resisting viremia and generating ctl (cytotoxic t lymphocytes) subsets [180] [181] [182] . the prolonged presence of these cd4 + t cells in the trim5α-susceptible siv-infected treatment may be responsible for the higher survival rate [179] . in spite of this, trim5α escape mutants were present [179] . half of the subjects that received the trim5α-sensitive strain of siv still developed aids, and a third succumbed to infection at a similar time as the trim5α-resistant group [179] . sequencing of the siv capsids from these macaques revealed two mutations in the region encoding the gag protein, which resulted in nonsynonymous substitutions that mimicked the alterations made by the authors to generate their trim5α resistant strain [179] . subversion of trims is a central evolutionary strategy for viral innate immune evasion, as demonstrated by the high mutational rate of retroviruses. although first described as a viral restriction factor against hiv-1 in old-world monkeys, humans and other species are capable of utilizing trim5α to inhibit other retroviruses. human trim5α is incapable of restricting hiv-1, yet a single amino acid mutation in its pry-spry domain can confer resistance to the pathogen [183] . instead, human trim5α can bind to and restrict n tropic mouse leukemia virus (n-mlv) [183] . interestingly, the 13-amino-acid stretch in the pry-spry domain of primate trim5α is under strong positive selection [44] . trim5α paralogues have been identified in bovine [184, 185] , ovine [186] , and piscine [40] species, and have also been shown to restrict retroviruses. overall, trim5α plays a convergent role in the recognition and restriction of retroviruses. aside from its role in non-human primates, trim5α has been described functioning in a cell-type specific manner [187] [188] [189] . in contrast to conventional dc-sign + (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) dcs, langerhans cells (lcs) benefit from the anti-retroviral capabilities of trim5α through an increased activity of the lc autophagocytic components [187] . upon langerin-mediated hiv-1 uptake, the presence of cellular autophagosomes increases as a result of trim5α-directed assembly, leading to targeting of the viral capsid for destruction [187] . further evidence of cell-type specific hiv-1 restriction by trim5α was found in rhesus macaque dcs, which in contrast to macrophages appear to be permissive to hiv-1 infection [188] . the lack of trim5α-mediated restriction in both human and macaque conventional dcs may be due to sumoylation of trim5α, which promotes its sequestration in nuclear bodies [189] . however, this lack of trim5α-mediated restriction in dcs provides an innate immune sensing advantage by allowing recognition of hiv-1 reversed transcribed dna by cgas, resulting in type-i ifn production [189] . it remains to be seen whether hiv-1 can actively promote or enhance sumoylation of trim5α in dcs as mechanism to antagonize type-i ifn production. the ability of trim5α to promote restriction of hiv-1 may go beyond direct intervention of the viral lifecycle. trim5α has also been linked to the activity of other components of cellular innate immunity including antigen presentation to cytotoxic t lymphocytes (ctl). tcypa and rhtrim5α enhanced the ability of cd8 + t cells to identify hiv-1 infected cells, and promoted a hiv-1-specific immune response [174] . the presence of these trim5 orthologs was associated with increased associations between hiv-1 particles and the host proteasome [174] . it is speculated that this may facilitate improved hiv-1-specific ctl development, as amplified peptide concentrations could support enhanced antigen presentation to cd8 + t cells. the mechanism linking direct restriction of viral capsids with heightened cd8 + t cell activation warrants further investigation. a plethora of trim-mediated restriction mechanisms targeting hiv-1 and other retroviruses have been proposed [15] , and have been reviewed previously [6] . more recent reports acknowledged trim11 as a potent host restriction factor of hiv-1 [114, 190] . the mechanisms of trim11-mediated restriction include curbing the amount of viral reverse transcription products allowed to accumulate in the host. through an interaction with the viral capsid-nucleocapsid protein (ca-nc) complexes, trim11 promotes premature uncoating and release of the viral genetic material, reducing transduction efficiency. neither proteasomal nor lysosomal inhibitor treatments recovered viral p24 protein in the pellets of trim11 overexpressing cells, suggesting that ubiquitin-mediated degradation by the proteasome or lysosomal acidification is not required for trim11-mediated uncoating [190] . furthermore, while rhtrim5α-mediated inhibition of hiv-1 is rescued by proteasome inhibitors [170, 171] , trim11-mediated inhibition was not, indicating that trim11 and trim5α restrict hiv-1 by different mechanisms [114, 190] . in contrast, using the microtubule dynamics inhibitors nocodazole and taxol, the authors were able to demonstrate a restoration of hiv-1 capsid levels in cells supplemented with exogenous trim11, suggesting that microtubules may contribute to trim11-mediated hiv-1 restriction [190] . earlier work with the use of nocodazole and taxol in a study examining hiv-1 and trim5α also demonstrated recovered hiv-1 infectivity in the absence of functional microtubules [191] . although the exact mechanism of trim11-mediated restriction of hiv-1 in the aforementioned study has yet to be determined, the authors demonstrated that purified trim11 associated with in vitro assembled hiv-1 capsids [190] , indicating that trim11 interacts directly with the capsid and it probably does not require trim5α or other cellular proteins for promoting untimely capsid uncoating. microtubule involvement in viral uncoating has been implicated as both a host restriction and a viral propagation mechanism [192] [193] [194] [195] . functional microtubules and their associated motor proteins, like dynein, have been suggested as key components in genome release for both iav and hiv-1 [192] [193] [194] [195] . interestingly, in the case of iav, unanchored poly-ub chains contained in the virion are recognized by the host histone deacetylase 6 (hdac6), a component of the aggresome-autophagy pathway, which interacts with dynein and microtubules to promote viral uncoating [192, 195] . it will be interesting to examine whether hiv-1 utilizes a similar mechanism and whether this is mediated by trim11. additionally, several members of the trim family (trims 1, 9, 18, 36, 46, and 67) have been shown to possesses a c-terminal domain motif allowing for association with cytoskeletal elements like microtubules [31] . so far only trim1 (also called mid2) and trim18 (also called mid1) have been shown to be directly involved in microtubule stabilization [196] , and trim1 has been implicated in restriction of n-mlv [159] . whether trim1-mediated restriction is dependent on microtubules, or whether other microtubule-interacting trims may have viral restriction activity by microtubule-dependent mechanisms, remains to be seen. trim-mediated suppression of hiv-1 replication has revealed additional avenues through which trims subdue pathogens. trim22 prevents normal viral transcription events by regulating the effectiveness of the transcription factor sp1 to bind the hiv-1 long terminal repeat (ltr) promoter region [197] . this restriction was independent of trim22's e3 ligase activity and did not involve direct interaction between trim22 and sp1, implying that the observed reduction in hiv-1 ltr-mediated transcription requires additional unspecified factors [197] . trim37 exhibits anti-retroviral functions through interference of hiv-1 infection, replication, and transcription, possibly interfering with dna synthesis [198] . however further investigation into trim-mediated transcriptional inhibition will be required to reveal additional pathways in which trims play a significant role. another example of indirect inhibition of virus replication by trims is illustrated by the promyelocytic leukemia protein (pml)/trim19. pml/trim19 interferes with hiv-1 infection in human and murine fibroblasts through the reduction of reverse transcriptase products [199, 200] . this effect was dependent on two events; the translocation of pml/trim19 from the nucleus to the cytoplasm in the presence of hiv-1 infection, as well as association of the pml/trim19 cytoplasmic bodies (cb) with daxx [201] . pml/trim19 interacts with daxx in a protective fashion in order to prevent its degradation by the proteasome, thereby making pml/trim19 a necessary component for daxx-mediated inhibition of hiv-1 rt [201] . the inhibition of hiv-1 by pml/trim19 and daxx may be cell-type dependent, since studies using different cell types have shown different results [200, 201] . another well characterized trim involved in pathogen recognition is trim21, which has been shown to detect intracellular antibody-opsonized viruses (reviewed previously in [6, 7] ). some immunoglobulin-coated non-enveloped viruses are internalized into the host cell. within most cells, a high affinity antibody receptor, trim21, binds to the highly conserved fc region of virion-bound immunoglobulin (ig)g, igm, or iga [7, 202] , and targets the virus for degradation by the proteasome before the virus can transcribe its genes [203] . trim21's pry-spry domain interacts with fc residues conserved across mammalian species [204] . trim21 binding with an immunoglobulin-virion (ig-v) complex triggers tightly regulated intracellular antibody neutralization and pro-inflammatory pathways [7, 202] . upon engagement of trim21 with the ig-v complex within the cytoplasm, the e2 ube2w (ubiquitin conjugating enzyme e2 w) monoubiquitinates trim21, which promotes ube2n/ube2v2 e2 complex recruitment to trim21 for k63-linked poly-ub [12] . following poly-ub, trim21 is recruited to the proteasome to initiate degradation of the antibody-bound virus. concurrently, with proteasome-mediated degradation, poh1 de-ubiquitinates trim21 [12] . this antibody-dependent intracellular neutralization appears to be limited to non-enveloped dna and rna viruses that can enter the host cytosol with immunoglobulin attached to the virion surface [205] . the de-ubiquitinated trim21 is then able to promote both ifn induction and nf-κb activation [12, 205, 206] , hypothesized to result from the release of the viral genome into the cytoplasm for prr-mediated recognition [207] . however, some antiviral cytokines are induced independent of prrs [207] , but the pathway is not well characterized. in addition, it has also been proposed that recognition of the ig-v complex in the cytoplasm by trim21 triggers the synthesis of unanchored k63-linked poly-ub chains that activate nf-κb, ap-1 and irf3 pathways [205] . notably, when the affinity of trim21 for the fc portion of an antibody is decreased, the viral neutralization efficiency is maintained while the activation of cytokine signaling is diminished [208] . this suggests that the association of trim21 with ig-v complexes is important for triggering antiviral responses. trim21-deficient mice infected with mouse adenovirus 1 experienced lethal disease while wild-type mice were protected, exemplifying the importance of trim21 in viral neutralization in vivo [209] . most likely, these mechanisms are shared in other species due to the highly conserved fc and trim21 interacting residues, however the activity of trim21 as an intracellular antibody receptor in non-human, non-murine models has only been demonstrated in pig cells infected with foot-and-mouth disease virus [210] . trim family proteins can target several components of pathogens ranging from structural elements to factors essential for transcription and replication. a variety of influenza a virus (iav) and influenza b virus (ibv) proteins are targets of trim-mediated inhibition of virus replication (figure 4 ). despite being a target of iav-ns1 (nonstructural protein 1)-dependent ifn antagonism, trim25 interacts with the n-terminus of ibv-ns1 preventing the viral protein's c-terminal component from binding viral rna [211] . preventing ibv-ns1 from interacting with rig-i allows signaling through this rlr pathway to proceed [211] . another trim-mediated anti-iav mechanism is polyubiquitination of iav nucleoprotein (np) by trim22, which leads to np proteasome-mediated degradation and reduced virus replication [212] . the polymerase basic protein 1 (pb1) of iav is also a target of trim-mediated inhibition. pb1 is one of the three components that make up the rna-dependent rna-polymerase responsible for transcribing the eight segments of the iav genome [213] . trim32 facilitates k48-linked polyubiquitination of pb1, resulting in enhanced turnover of this viral subunit, and a reduction in viral titers [214] . aside from targeting pathogen components for proteasomal degradation via ubiquitination, some members of the trim family have been shown to enact their restriction factor capabilities through other means. the trim56 c-terminal domain, rather than its e3 ligase activity, is required to reduce iav and ibv replication. the mechanism trim56 employs to diminish vrna levels of both influenza viruses is currently unknown, although inhibition of translation through direct interaction between trim56 and the vrnas has been proposed [215] . trims have also been reported to mediate restriction against flaviviruses ( figure 5 ). the flavivirus genus comprises more than 70 viruses including a number of important human pathogens such as dengue virus (denv), zika virus (zikv), west nile virus (wnv), tick-borne encephalitis virus (tbev), japanese encephalitis virus (jev), hepatitis c virus (hcv), and yellow fever virus (yfv) [216] . flaviviruses are small enveloped viruses hosting a positive-sense single-stranded rna genome. several flaviviral proteins are associated with viral persistence, immune system evasion, or viral replication [217] . influenza viruses is currently unknown, although inhibition of translation through direct interaction between trim56 and the vrnas has been proposed [215] . trims have also been reported to mediate restriction against flaviviruses ( figure 5 ). the flavivirus genus comprises more than 70 viruses including a number of important human pathogens such as dengue virus (denv), zika virus (zikv), west nile virus (wnv), tick-borne encephalitis virus (tbev), japanese encephalitis virus (jev), hepatitis c virus (hcv), and yellow fever virus (yfv) [216] . flaviviruses are small enveloped viruses hosting a positive-sense single-stranded rna genome. several flaviviral proteins are associated with viral persistence, immune system evasion, or (2) . rna genome is released into the cytoplasm (3). the positive-sense genomic ssrna is translated into a polyprotein, which is cleaved into all structural and non-structural proteins (4). replication takes place at the surface of endoplasmic reticulum in cytoplasmic viral factories (5) . in this step, the trims restrict virus replication, degrading viral proteins such as ns2a in japanese encephalitis virus (jev) by trim52 and viral rna inhibition in jev and dengue virus (denv) by trim56. trim22 and trim79 degrade ns5 protein in hepatitis c virus (hcv) and tick-borne encephalitis virus (tbev), respectively. virus assembly occurs at the endoplasmic reticulum. the virion buds at the endoplasmic reticulum and is transported to the golgi apparatus (6) . the prm protein is cleaved in the golgi, thereby maturing the virion, which is fusion competent (7) . release of new virions by exocytosis (8) . trims and antagonism function also are used by flaviviruses. trim21 inhibits ifn-β production during jev infection. trim23 promotes yellow fever virus replication. denv short noncoding sfrnas bind trim25 to inhibit ifn expression. hcv encodes a nonstructural protein, ns5a, which inhibits the phosphorylation and nuclear translocation of stat1 in the ifn-α2-induced jak/stat pathway via their ifn sensitivitydetermining region [218, 219] . trim14's spry domain specifically interacts with ns5 of hcv and induces ns5a degradation [220] , which is an example of trim-mediated ifn-independent inhibition. trim22 specifically binds the ns5a-d1 protein (domain 1) via its spry domain and the positive-sense genomic ssrna is translated into a polyprotein, which is cleaved into all structural and non-structural proteins (4). replication takes place at the surface of endoplasmic reticulum in cytoplasmic viral factories (5) . in this step, the trims restrict virus replication, degrading viral proteins such as ns2a in japanese encephalitis virus (jev) by trim52 and viral rna inhibition in jev and dengue virus (denv) by trim56. trim22 and trim79 degrade ns5 protein in hepatitis c virus (hcv) and tick-borne encephalitis virus (tbev), respectively. virus assembly occurs at the endoplasmic reticulum. the virion buds at the endoplasmic reticulum and is transported to the golgi apparatus (6) . the prm protein is cleaved in the golgi, thereby maturing the virion, which is fusion competent (7) . release of new virions by exocytosis (8) . trims and antagonism function also are used by flaviviruses. trim21 inhibits ifn-β production during jev infection. trim23 promotes yellow fever virus replication. denv short noncoding sfrnas bind trim25 to inhibit ifn expression. hcv encodes a nonstructural protein, ns5a, which inhibits the phosphorylation and nuclear translocation of stat1 in the ifn-α2-induced jak/stat pathway via their ifn sensitivity-determining region [218, 219] . trim14's spry domain specifically interacts with ns5 of hcv and induces ns5a degradation [220] , which is an example of trim-mediated ifn-independent inhibition. trim22 specifically binds the ns5a-d1 protein (domain 1) via its spry domain and utilizes its e3 ubiquitin ligase activity to target ns5a for removal [221] . wenchun and colleagues have shown that trim52 interacts with the ns2a protein of jev and targets the protein for proteasome-mediated destruction [222] . ns2a is a small, hydrophobic transmembrane protein involved in the virus life cycle and subversion of host antiviral responses [223, 224] , including inhibition of the double-stranded rna-activated protein kinase pkr during jev infection [225] . trim52-dependent inhibition of jev ns2a protein occurs in bhk-21 and 293t cells and is therefore important for restricting jev replication [222] . recent evidence also suggests that the ring and c-terminal domains of trim56 may be important in the restriction of other flaviviruses, including yfv and denv [226] , but the mechanism of action remains elusive. trim56 might modulate post-translational modification of one or more viral proteins and/or host factors to suppress viral replication [227] . although trim56 fails to restrict hcv replication when overexpressed in human hepatoma huh7 cells [228] , trim56 overexpression in hek293 cells support some selectable hcv rna replicons at very low efficiencies [227] . perhaps trim56 facilitates degradation of viral proteins similar to mechanisms observed with trims 14 and 22 against hcv and trim52 against jev. further studies are needed to elucidate the underlying molecular mechanisms of trim56-mediated restriction of denv and yfv. interestingly, trim56 is capable of binding to the protease n pro of bovine diarrheal virus (bvdv), a pestivirus of the flaviviridae family [228] . the restriction of this viral protein is critical considering that n pro is capable of mediating irf3 degradation, thus impairing production of ifn-β [228] . trim56-mediated restriction of bvdv is specific, as closely related viruses are not likewise impaired in their replication [228] . trim79α, also known as trim30-3 or trim30d, is present only in rodents. trim79α is highly expressed in the spleen, lymph node, and bone marrow in a type-i ifn-dependent manner, and is required for effective restriction of tbev replication [229] . trim79α is an important mediator of the innate cellular response to restrict langat virus (a member of the tbev serogroup) infection by targeting the viral rna polymerase and major ifn antagonist, ns5 [229] . ns5 has a methyltransferase and rna-dependent rna polymerase activity that associates with ns3 and ns2b to form the viral replication complex. ns5 inhibits ifn-α/β-dependent responses by preventing jak-stat signaling and thus suppresses ifn-stimulated gene (isg) expression [229] [230] [231] [232] . taylor and colleagues demonstrated that trim79α interacts with ns5 from lgtv and tbev and blocks the replication of these viruses via a lysosomal-targeting mechanism. despite ns5 being the most conserved of the flaviviral proteins, trim79α did not target ns5 from wnv, nor could it inhibit wnv replication [232] . besides flaviviruses, other positive-sense rna viruses have been reported to be inhibited by trim-mediated mechanisms and can occur through both direct and indirect means. for example, trim25 acts as a cofactor for the zinc-finger antiviral protein (zap), a member of the poly(adp-ribose) polymerase family that is known to bind and promote viral rna degradation [233] . trim25 enhances zaps' ability to inhibit sindbis virus translation [233] . however, although trim25 is capable of promoting k48-and k63-linked polyubiquitination of zap isoforms, ubiquitination does not appear to affect zap antiviral activity [233] . it will be of interest to elucidate what other factors may be ubiquitinated by trim25 that could affect zap antiviral activity. trim19-iv has been shown to confer resistance to encephalomyocarditis virus (emcv) hampering viral replication and protein synthesis [234] . this ability was shown to originate from trim19-iv's c-terminus, specifically through an interaction with the viral 3d polymerase (3dpol), leading to nuclear body sequestration of the viral polymerase [234] . additionally, sumoylation of trim19-iv was required to facilitate restriction of emcv [234] . trims play an additional role in restricting dna virus replication. eight different trim proteins (trim5, 6, 11, 14, 25, 26, 31, and 41) were identified to inhibit hepatitis b virus (hbv) [235] . in particular, trim41 was the only trim from this group of eight that specifically reduced both the enhancers i and ii components of hbv. this inhibition of viral transcription required trim41's ring and pry/spry domains, implicating its e3 ligase activity [235] . trim22 is associated with hbv clearance in acutely infected chimpanzees, and possesses anti-hbv activity under physiological conditions [236] . gao and colleagues also reported that trim22 was one of the most strongly induced trim family molecules in human hepatoma hepg2 cells after treatment with ifns [237] . epstein-barr virus (ebv) replication and transcription activator (rta) protein activates ebv lytic genes for proliferation [238] . trim5α promotes the ubiquitination of rta upon interaction, thereby blocking the ebv lytic cycle [238] . the involvement of trim5α in a non-retroviral innate immune response implies trims possess diverse, situation-specific functions that have yet to be characterized. trims may also operate as recognition receptors for other components of host immunity. trim19 isoforms are able to capture varicella-zoster virus nucleoproteins to impede nuclear egress and thus block the release of new virions [239] . comprehension of the multiple roles trim-family proteins play may become critical in discerning the impact that they have on the innate immune response. as we have described, trim family proteins play an important role in innate immunity and counter pathogens [7] . in retaliation to this evolutionary pressure, pathogens have adapted to antagonize trims. mechanisms of viral-mediated trims restriction range from impairment of trim-promoted innate immune signaling complex assembly to direct hindrance of the trim proteins. interfering with trims, either directly or indirectly, undermines their intended function, and enables viruses to gain an early advantage over the host [58] . the following section highlights several recent examples that elucidate viral manipulation of trims. a broad range of rna viruses have evolved effective evasion strategies to manipulate host antiviral immunity. influenza viruses employ well-studied examples of trim antagonism ( figure 4 ). the nonstructural protein 1 (ns1) of influenza a and b virus (iav/ibv) has been characterized as a viral antagonist of host innate immunity through interactions with trim25 [57, 100, 240, 241] . as described above, trim25 plays a critical role in the activation of the rlr pathways [57, 242, 243] . the ns1 protein from iav directly interacts with the coiled-coil domain of trim25 to impede its multimerization [57, 244] . since dimerization is required for trim25 e3 ligase activity, ns1 binding to trim25 leads to impaired ubiquitination of the rig-i and downstream signaling, resulting in a reduced antiviral response [57, 244] . the trim25 interaction with iav ns1 is species-specific. human trim25 interacts with iav strains isolated from many species while chicken trim25 binds only ns1 from avian strains and murine trim25 did not bind any ns1 [100] . riplet, a close relative of trim25, lacks a b-box domain but shares homologous ring and spry domains. its predicted coiled-coil structure also binds ns1 to inhibit rig-i signaling in mice and humans [100] . aside from interacting with trim25, iav ns1 also associates with host rig-i directly [244] . as seen with iav, the n-terminal domain of ibv ns1 is also able to block the lys63-linked ubiquitination of rig-i and subsequent antiviral signaling downstream of the rlr pathway [211] . members of the coronaviridae, flaviviridae, and bunyaviridae families also encode viral proteins that inhibit trim-mediated regulation of rlr signaling. severe acute respiratory syndrome and middle east respiratory syndrome coronavirus (sars/mers-cov) are large, positive-sense single-stranded rna viruses. akin to the influenza virus ns1 protein, the nucleoprotein of sars-cov was demonstrated to interact with the spry domain of trim25, preventing the necessary interaction and subsequent ubiquitination of the rig-i card domains [245] . a similar loss of rig-i-induced ifn-β is achieved when mers-cov nucleoprotein associates with trim25 [245] . for denv, manokaran et al. compared two viral sequences (pr1 and pr-2b) and identified mutations that resulted in the increased production of subgenomic flavivirus non-coding rnas (sfrnas) by the pr-2b strain [55] ( figure 5 ). the pr-2b sfrnas were capable of binding to host trim25 and prevented usp15-mediated deubiquitination [55] , which is crucial for activation of rig-i [94] . this data provides unique molecular insight into the epidemiological fitness of denv, suggesting that denv sfrnas can bind to host proteins to promote viral evasion of innate immunity [55] . the nss protein of severe fever with thrombocytopenia syndrome virus (sftsv), a negative-sense rna virus in the family bunyaviridae, interacts directly with trim25, and indirectly with rig-i and tbk1 to isolate these signaling molecules from associating with mavs [246] . as with the other viral protein-trim25 interactions described above, downstream activation of irf3 and subsequent ifn-β production are impaired [246] . trim25 is a common target of a diverse group of rna viruses, suggesting that other pathogens may also impair trim25-mediated stimulation of the rlr pathway. similar to iav, hiv-1 encodes multiple proteins that restrict trim function ( figure 3 ). for example, hiv-1 vpr protein has been suggested to play a role in trim manipulation [114] . interestingly, protein expression levels of trim11 have shown to be under the control of vpr in a dose-dependent manner, where the presence of trim11 was decreased when the concentration of vpr was low [114] . currently, the mechanism hiv-1 vpr employs to antagonize trim11 is unknown. following infection of human neural precursor cells (hnpcs) with hiv-1, trim32 becomes upregulated, primarily due to the viral trans-activator of transcription protein (tat) [247] . this tat-mediated upregulation of trim32 induces proliferation arrest in hnpcs, eventually leading to neurodegeneration [247] . flaviviruses also encode viral proteins that antagonize trim-mediated innate immunity ( figure 5 ). yfv and denv ns5 protein have 10 amino acid residues on the n-terminus, which are essential for antagonism of type-i ifn signaling [54, 248] . yfv ns5 binds stat2 only after ifn treatment, and appears to inactivate isgf3 within the nucleus [54] . morrison et al. found in denv ns5 a glycine and a threonine residue within the n-terminus that are required for binding with ubr4 (ubiquitin protein ligase e3 component n-recognin 4) to mediate stat2 degradation [249] . ubr4, a member of the n-recognin family, is a potential e3 ligase that recognizes and degrades proteins containing destabilizing n termini. [250] . ubr4 interacts preferentially with proteolytically-processed denv ns5, but not with yfv ns5 or wnv ns5 [249] . although ubr4 does not belong to the trim family, it is possible that trim members may also be involved in stat2 degradation by ns5. for example, trim23 was identified as an essential factor in yfv replication due to its interaction and poly-ub of residue k6 on yfv-ns5, promoting binding with stat2 and inhibition of type-i ifn signaling [54] . another flavivirus, japanese encephalitis virus (jev), induces expression of trim21 in human microglial cells, which attenuates jev-induced antiviral signaling [53] . the study by manocha et al. demonstrated that trim21 overexpression suppressed phosphorylation of irf3 and activation of ifn-β, while silencing trim21 permitted efficient type-i ifn responses in jev-infected human microglial cells [53] . this study provides evidence that jev suppress the ifn-i response due to induction of trim21. finally, we recently showed that nipah virus (niv), a single-stranded negative-sense highly pathogenic rna virus (paramyxoviridae family, genus henipavirus) that causes fatal diseases in humans [251] , can inhibit trim6-mediated type-i ifn responses [56] (figure 6 ). mechanistically, the niv matrix (niv-m) structural protein, which is required for virus assembly and budding [252, 253] , targets trim6 for degradation. interestingly, niv budding requires trafficking of niv-m from the cytoplasm to the nucleus before reaching the cell membrane for virus assembly [252, 253] . the reason for niv-m trafficking to the nucleus is still unclear, however, a lysine residue (k258) in the niv-m bipartite nuclear localization signal that is conserved in divergent henipaviruses and is required for trafficking, is critical for the ifn antagonist function [56] . consistent with this, the matrix proteins of ghana, hendra and cedar viruses were also able to inhibit ifn-β induction [56] . it is currently unknown whether trim6 e3-ligase activity affects niv-m trafficking or whether it directly interferes with niv-replication. niv-m-induced trim6 degradation did not appear to require the proteasome, however. although the precise mechanism of trim6 degradation remains to be elucidated, inhibitors that recover trim6 protein levels could potentially be used as therapeutic drugs against niv infections. these findings highlight the importance of trim6 as an antiviral factor of the type-i ifn system. a variety of dna viruses also encode viral proteins that interfere with trims. alternative methods for subverting trim-induced innate immune responses can be noted through alterations made to host transcription by hepatitis b virus (hbv). the hbv-encoded protein x (hbx) methylates a single cpg in trim22's promoter region [254] . this viral-mediated epigenetic modification blocks ifn-α/γ-induced irf1, a transcriptional activator, from binding the trim22 promoter and consequently facilitates viral proliferation and escape [255] . in epstein-barr virus (ebv), kap1 (krab [kruppel-associated box domain]-associated protein 1)/trim28 regulates activation of the viral lytic cycle [256] . in cells undergoing lytic stage activation, kap1/trim28 becomes phosphorylated at serine residue 824 by ataxia telangiectasia mutated (atm). this post-translational modification impairs kap1/trim28's restriction factor function and allows ebv to transition from latency to lytic stage [256] . additionally, the anti-malarial drug chloroquine acts as an activator for atm by phosphorylating kap1/trim28, which ultimately leads to promotion of the ebv lytic cycle and escape of the virus particles [256] . mutations in gene expression serve as an additional focal point highlighting the role that trims play in a dysfunctional antiviral response. when a population of 765 hbv-infected individuals was screened, chronic hbv infection was found to correlate to a single t to c silent mutation single nucleotide polymorphism (snp) in the trim22 ring domain [257] . although the implications to patient outcomes are considerable, additional investigations into the basic biological mechanisms are warranted to discern the importance of these snps in trim-mediated innate immunity. figure 6 . trim6 is targeted by nipah and ebola viruses to enhance virus replication. ebola virus (ebov) inhibits type-i ifn production by multiple mechanisms. ebov vp35 binds and inhibits rig-i, ikkε, and tbk-1 to inhibit ifn production. trim6 ubiquitinates vp35 on k309 and promotes vp35 activity as the cofactor of the viral polymerase and enhances virus replication. additional unidentified ubiquitination sites of vp35 exist. whether trim6 enhances ebov replication by promoting viral genome replication or viral gene transcription is not known. in nipah virus infection, the viral matrix protein (niv-m) promotes trim6 degradation, resulting in reduced synthesis of k48-linked unanchored polyubiquitin chains, ikkε oligomerization, ikkε-t501 autophosphorylation, irf3 phosphorylation, and reduced ifn induction. these combined losses confer an impaired host-antiviral response. a variety of dna viruses also encode viral proteins that interfere with trims. alternative methods for subverting trim-induced innate immune responses can be noted through alterations made to host transcription by hepatitis b virus (hbv). the hbv-encoded protein x (hbx) methylates a single cpg in trim22's promoter region [254] . this viral-mediated epigenetic modification blocks ifn-α/γ-induced irf1, a transcriptional activator, from binding the trim22 promoter and consequently facilitates viral proliferation and escape [255] . in epstein-barr virus (ebv), kap1 (krab [kruppel-associated box domain]-associated protein 1)/trim28 regulates activation of the viral lytic cycle [256] . in cells undergoing lytic stage activation, kap1/trim28 becomes phosphorylated at serine residue 824 by ataxia telangiectasia mutated (atm). this post-translational modification impairs kap1/trim28's restriction factor function and allows ebv to transition from latency to lytic stage [256] . additionally, the anti-malarial drug chloroquine acts as an activator for atm by phosphorylating kap1/trim28, which ultimately leads to promotion of the ebv lytic cycle and escape of the virus particles [256] . mutations in gene expression serve as an additional focal point highlighting the role that trims play in a dysfunctional antiviral response. when a population of 765 hbv-infected individuals was screened, chronic hbv infection was found to correlate to a single t to c silent mutation single nucleotide polymorphism (snp) in the trim22 ring domain [257] . although the implications to patient outcomes are considerable, additional investigations into the basic biological mechanisms are warranted to discern the importance of these snps in trim-mediated innate immunity. in addition to impairing trim gene expression, dna viruses can mimic host proteins to prohibit trim function. the immediate early protein (icp0) of herpes simplex virus 1 (hsv-1) possesses a ring domain and facilitates the degradation of trim27 through ubiquitination [258] . this destruction of trim27 is facilitated through the host's proteasome [258] , mimicking the numerous instances of trim-mediated restriction. gammaherpesvirus mhv-68 similarly targets trim19 for proteasome-mediated degradation [239] . the ie1 proteins of human cytomegalovirus (hcmv) mimics the coiled-coil domain of trims to recruit and sequester trim19 from nuclear bodies thus impairing the activation of ifn responses [239] . so far, most studies on trims have focused on their roles as antiviral factors by directly restricting virus replication or indirectly by inducing antiviral cytokines, as described above. the fact that trims are targeted by viruses for immune evasion further highlights their important roles in protecting the host against infections. however, whether trims may directly act as host factors required for virus replication, or "pro-viral" factors, has not been addressed. some studies have shown that certain viral antagonists can hijack trims to activate their ifn antagonist activity (e.g., trim23 ubiquitinates yfv-ns5 for antagonism of stat2 function [54] ), but these are indirect effects that provide an advantage to the virus by reducing host antiviral responses. since ubiquitination of viral proteins may positively influence specific steps of the replication cycle, it would not be surprising if trims are involved in directly promoting virus replication by non-degradative ubiquitination of viral proteins. indeed, we recently reported the first example of such a role for trim6 [259] . as described above in section 2.6, trim6 is involved in antiviral type-i ifn responses by catalyzing the synthesis of unanchored k48-linked polyubiquitin chains that activate ikkε (figures 2 and 6 ). further evidence of trim6 as an antiviral factor is highlighted by our findings that the niv can inhibit ifn-i responses by targeting trim6 [56] . furthermore, knockdown of trim6 in lung a549 cell lines and primary human monocyte derived dendritic cells has shown increased replication of multiple viruses, including iav, emcv, and sendai virus (sev), most probably due to reduced type-i ifn responses [11] . in addition, trim6 a549 knockout cells showed reduced ifn responses [259] . unexpectedly, despite a defect in the ifn response, infectious ebola virus (ebov: filoviridae family) replicated less efficiently in trim6 knockout cells as compared to parental wild type (wt) cells. this observation raised the question whether trim6 may be acting as a pro-viral factor or an enhancer of virus replication. in support of this hypothesis, we found that trim6 interacts with ebov-vp35, which is a major viral ifn antagonist by targeting rig-i [260, 261] and the kinases ikkε and tbk-1 [262] . however, since vp35 also plays a critical role as the cofactor of the virus polymerase [263] , trim6 could directly affect polymerase function. mass spectrometry analysis and co-immunoprecipitation assays demonstrated that trim6 ubiquitinates vp35 on k309 [259] , a lysine residue located on its ifn antagonist domain. moreover, minigenome reporter experiments showed that trim6 can enhance vp35-mediated polymerase activity, and this effect requires the e3-ubiquitin ligase activity of trim6. although vp35 is ubiquitinated in multiple (currently unidentified) residues in addition to k309, the trim6-dependent effects on vp35 minigenome activity required an intact k309 residue. vp35 was also able to inhibit rig-i induced trim6-enhanced ifnβ in reporter assays, however the precise mechanism was not elucidated [259] . collectively, these findings suggest that trim6 is a host factor hijacked by ebov-vp35 for both immune evasion and for promoting virus replication via ubiquitination of vp35 ( figure 6 ). future studies will address whether ubiquitination of vp35 regulates virus rna replication or transcription. it remains to be seen if other trims that are targeted by viruses may also directly enhancing virus replication via ubiquitination of viral proteins. autophagy (self-eating) is a highly conserved catabolic mechanism through which eukaryotic cells deliver dispensable, or potentially dangerous, cytoplasmic material to lysosomes for degradation [264] . the process is characterized by the formation of autophagosomes, which sequester the cytoplasmic structures targeted for destruction. autophagy has been linked to a wide range of physiological processes, including cell differentiation and development, the degradation of aberrant structures and turnover of damaged organelles, as well as innate and adaptive immunity [265, 266] . a growing number of studies indicate that several trims are linked to autophagy and recent excellent reviews are available on the emerging roles of trims in autophagy [59, 60, 267, 268] . a good example of the potential role of trims in autophagy and virus infections is rhesus trim5α, which acts both as a regulator of autophagy by providing a platform for the assembly of activated ulk1 and beclin 1 (key components of the autophagy regulatory complexes) and as a receptor for selective autophagy [269, 270] . in its role as an autophagic cargo receptor, trim5α directly recognizes viral capsid sequences via its spry domain [158, [269] [270] [271] . this is an example of selective autophagy in mammalian cells, which could occur via direct substrate recognition by trims and connects autophagy with a role in defense against viral pathogens. the rhesus trim5 can execute precision autophagy of the hiv-1 capsid. in contrast, the weak affinity of human trim5 for the hiv-1 capsid precludes effective precision autophagy. as a consequence, rhesus trim5, but not human trim5, could contribute to defense against hiv-1 through precision autophagy. since this recognition depends on the c-terminal region of trims (e.g., spry domain of trim5α), other types of c-terminal domains on trims could selectively recognize diverse protein targets. thus, trim proteins, as a group, could comprise a class of broad-repertoire, high-fidelity, selective autophagic receptors. given the breadth of the role of trims in various diseases, it will be important to explore precision autophagy-in addition to bulk autophagy-as a therapeutic target against viral infections. the proposed role of trims in autophagy raise questions. for example, how many trims act as autophagic receptors and what are their specific targets? do trim proteins function as hubs connecting different signaling pathways or different systems? how is the autophagic role of trims integrated with the other functions of trims, including regulation of gene expression and pro-inflammatory signaling? what is the interplay between the e3 ligase activity of trims and precision autophagy? it is important to determine inhibitory compounds of trim proteins for their use as therapeutic tools in infectious diseases. however, because some trim proteins have simultaneous dual functions in carcinogenesis and the immune response, it should be considered that putative drugs (inhibitors of some trim proteins) for cancer therapy may affect immunological reactions as a side effect. further detailed analysis of trim proteins is needed for their use as novel therapeutics with minimal side effects. this review highlighted the roles of trims in virus-host interactions. extensive reports detail trim involvement in immune signaling and direct virus restriction. in addition, viral antagonism of trims exemplifies the importance of this protein family in antiviral responses. despite these advancements, many trims have yet to be characterized. additionally, the molecular mechanisms underlying trim-mediated virus restriction or viral protein-mediated trim inhibition are not fully elucidated. the role of trims in regulating poly-ub chain topology is also of interest. in several examples, including trims 5 [169] , 6 [11] , 21 [12] , and 25 [77] , trims facilitate the synthesis of unanchored poly-ub chains. the relative contribution of trims in synthesizing specifically unanchored poly-ub chains, versus covalently linked poly-ub chains, is unclear. classically, the e2 is considered more important in determining poly-ub chain characteristics [4, 5] , but post-translational modifications may influence the decision between covalent and non-covalent linkage [12] as may the choice of the partnering e3. perhaps trims play a unique role in the synthesis of unanchored chains. in the future, evaluating the factors that regulate e2-trim pairing may add an additional layer of complexity to trim-mediated regulation and the ubiquitin code. although one study addressed this question by testing interactions between trims and the e2-conjugases and a few trim-e2 pairs were identified [272] , the complexity of potential transient interactions, and the possibility of cell-type specific expression for the combination of trims and e2-conjugases makes this task a huge challenge. importantly, another question is how poly-ub chains of unconventional linkages may affect virus replication. for example, unanchored poly-ub chains can be incorporated into the iav virion to enable hijacking of the host's aggresomal pathway to facilitate viral replication [192] . the e2 and e3 enzymes responsible for synthesizing these poly-ub chains have not been identified, but perhaps iav hijacks a trim to make these poly-ub chains critical in efficient infection. therefore, unanchored poly-ub chains may have both pro-viral and antiviral functions [195] and elucidating how to tip the balance towards antiviral responses may help develop novel antiviral therapies. additionally, iav replication via host ubiquitin and aggresome systems relies on the host's cytoskeletal network [192] . this may further implicate the role of trims in similar pathways as several trims associate with microtubules [18, 31] . many trims assemble into cytoplasmic bodies, which can be dynamic structures as described with trim32 [35] . the exact role of cytoplasmic bodies is not well characterized. possible trim-cytoplasmic body functions may include facilitation of signaling complex assembly [11] , regulation of active trim solubility, and/or generation of autophagosome-like complexes to target proteins for degradation. viruses can also disrupt or reorganize trim-cytoplasmic bodies, further supporting a role in antiviral responses. understanding how trim sub-cellular localization influences activity will also benefit the field. the applicability of the information garnered from studying trim-virus interactions may facilitate the identification of novel antiviral targets. further studies will advance our understanding of the complexities involved with trim signaling such as isoform-specific roles, cell-type specific activity, cytoplasmic body assembly and function, and post-transcriptional modification-induced trim function. finally, future studies will need to address whether other trims, in addition to trim6, may be hijacked by viruses to directly enhance replication, acting as pro-viral factors. the increasing complexity of the ubiquitin code ubiquitin enzymes in the regulation of immune responses ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions building ubiquitin chains: e2 enzymes at work ube2d3 and ube2n are essential for rig-i-mediated mavs aggregation in antiviral innate immunity the roles of the trim e3-ubiquitin ligase family in innate antiviral immunity intrimsic immunity: positive and negative regulation of immune signaling by tripartite motif proteins ubiquitin acetylation inhibits polyubiquitin chain elongation ubiquitin modifications ubiquitin-binding domains unanchored k48-linked polyubiquitin synthesized by the e3-ubiquitin ligase trim6 stimulates the interferon-ikkepsilon kinase-mediated antiviral response sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of trim21 trim family proteins and their emerging roles in innate immunity trim protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity trim e3 ligases interfere with early and late stages of the retroviral life cycle the e3-ligase trim family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors structural determinants of trim protein function the tripartite motif family identifies cell compartments trim16 acts as an e3 ubiquitin ligase and can heterodimerize with other trim family members solution structure of the rbcc/trim b-box1 domain of human mid1: b-box with a ring functional role of trim e3 ligase oligomerization and regulation of catalytic activity the tripartite motif coiled-coil is an elongated antiparallel hairpin dimer structure and catalytic activation of the trim23 ring e3 ubiquitin ligase structural insights into the trim family of ubiquitin e3 ligases crystal structure of trim20 c-terminal coiled-coil/b30.2 fragment: implications for the recognition of higher order oligomers a b-box 2 surface patch important for trim5α self-association, capsid binding avidity, and retrovirus restriction the trim5alpha b-box 2 domain promotes cooperative binding to the retroviral capsid by mediating higher-order self-association tripartite motif ligases catalyze polyubiquitin chain formation through a cooperative allosteric mechanism ring dimerization links higher-order assembly of trim5alpha to synthesis of k63-linked polyubiquitin trim14 is a mitochondrial adaptor that facilitates retinoic acid-inducible gene-i-like receptor-mediated innate immune response subclassification of the rbcc/trim superfamily reveals a novel motif necessary for microtubule binding genomic analysis of the trim family reveals two groups of genes with distinct evolutionary properties negative regulation of nf-κb activity by brain-specific tripartite motif protein 9 trim28 prevents autoinflammatory t cell development in vivo 14-3-3 proteins sequester a pool of soluble trim32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation rapid turnover and polyubiquitylation of the retroviral restriction factor trim5 the cellular level of trim31, an rbcc protein overexpressed in gastric cancer, is regulated by multiple mechanisms including the ubiquitin-proteasome system genomics and evolution of the trim gene family identification of a genomic reservoir for new trim genes in primate genomes origin and evolution of trim proteins: new insights from the complete trim repertoire of zebrafish and pufferfish an evolutionary screen highlights canonical and noncanonical candidate antiviral genes within the primate trim gene family positive selection of the trim family regulatory region in primate genomes discordant evolution of the adjacent antiretroviral genes trim22 and trim5 in mammals positive selection of primate trim5alpha identifies a critical species-specific retroviral restriction domain human trim gene expression in response to interferons induction of trim22 by ifn-gamma involves jak and pc-plc/pkc, but not mapks and pi3k/akt/mtor pathways expression of the immune regulator tripartite-motif 21 is controlled by ifn regulatory factors type i interferon-dependent and -independent expression of tripartite motif proteins in immune cells trim38 negatively regulates tlr3/4-mediated innate immune and inflammatory responses by two sequential and distinct mechanisms trim13 is a negative regulator of mda5-mediated type i interferon production expression profiling of trim protein family in thp1-derived macrophages following tlr stimulation tripartite-motif proteins and innate immune regulation regulatory role of trim21 in the type-i interferon pathway in japanese encephalitis virus-infected human microglial cells the interferon signaling antagonist function of yellow fever virus ns5 protein is activated by type i interferon dengue subgenomic rna binds trim25 to inhibit interferon expression for epidemiological fitness the matrix protein of nipah virus targets the e3-ubiquitin ligase trim6 to inhibit the ikkepsilon kinase-mediated type-i ifn antiviral response influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways trim family proteins: roles in autophagy, immunity, and carcinogenesis precision autophagy directed by receptor regulators--emerging examples within the trim family trim proteins and diseases pattern recognition receptors and inflammation expanding role of ubiquitination in nf-κb signaling convergence of the nf-κb and irf pathways in the regulation of the innate antiviral response in vivo ligands of mda5 and rig-i in measles virus-infected cells the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses 5'-triphosphate rna is the ligand for rig-i reis e sousa, c. rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates differential roles of mda5 and rig-i helicases in the recognition of rna viruses rig-i-like receptor regulation in virus infection and immunity ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral innate immune response structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf 3 rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner phosphorylation-mediated negative regulation of rig-i antiviral activity the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 ubch8 regulates ubiquitin and isg15 conjugation to rig-i mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response mavs recruits multiple ubiquitin e3 ligases to activate antiviral signaling cascades traf molecules in cell signaling and in human diseases activation of the iκb kinase complex by traf6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain tab2 and tab3 activate the nf-κb pathway through binding to polyubiquitin chains direct activation of protein kinases by unanchored polyubiquitin chains the regulation of nf-κb subunits by phosphorylation. cells type i interferons in infectious disease are the ikks and ikk-related kinases tbk1 and ikk-epsilon similarly activated? primary activation of interferon a and interferon b gene transcription by interferon regulatory factor 3 trim65-catalized ubiquitination is essential for mda5-mediated antiviral innate immunity trim4 modulates type i interferon induction and cellular antiviral response by targeting rig-i for k63-linked ubiquitination trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i the ubiquitin-specific protease usp15 promotes rig-i-mediated antiviral signaling by deubiquitylating trim25 multifaceted roles of trim38 in innate immune and inflammatory responses innate immunity to rna virus is regulated by temporal and reversible sumoylation of rig-i and mda5 trim59 interacts with ecsit and negatively regulates nf-κb and irf-3/7-mediated signal pathways de novo transcriptome analysis shows that sav-3 infection upregulates pattern recognition receptors of the endosomal toll-like and rig-i-like receptor signaling pathways in macrophage/dendritic like to-cells activation of duck rig-i by trim25 is independent of anchored ubiquitin species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein molecular characterization, tissue distribution and expression analysis of trim25 in gallus gallus domesticus duck trim27-l enhances mavs signaling and is absent in chickens and turkeys mavs ubiquitination by the e3 ligase trim25 and degradation by the proteasome is involved in type i interferon production after activation of the antiviral rig-i-like receptors the ubiquitin e3 ligase trim31 promotes aggregation and activation of the signaling adaptor mavs through lys63-linked polyubiquitination novel function of trim44 promotes an antiviral response by stabilizing visa trim9 short isoform preferentially promotes dna and rna virus-induced production of type i interferon by recruiting gsk3β to tbk1 crosstalk between cytoplasmic rig-i and sting sensing pathways trim11 negatively regulates ifnβ production and antiviral activity by targeting tbk1 tripartite motif-containing protein 38 negatively regulates tlr3/4-and rig-i-mediated ifn-β production and antiviral response by targeting nap1 polyubiquitin conjugation to nemo by triparite motif protein 23 (trim23) is critical in antiviral defense autoubiquitination of trim26 links tbk1 to nemo in rlr-mediated innate antiviral immune response trim68 negatively regulates ifn-β production by degrading trk fused gene, a novel driver of ifn-β downstream of anti-viral detection systems trim30 α negatively regulates tlr-mediated nf-kappa b activation by targeting tab2 and tab3 for degradation the human antiviral factor trim11 is under the regulation of hiv-1 vpr identification of a role for trim29 in the control of innate immunity in the respiratory tract trim39 negatively regulates the nfκb-mediated signaling pathway through stabilization of cactin sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity sting is a direct innate immune sensor of cyclic di-gmp structure of sting bound to cyclic di-gmp reveals the mechanism of cyclic dinucleotide recognition by the immune system structural and functional analysis of ddx41: a bispecific immune receptor for dna and cyclic dinucleotide the helicase ddx41 recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response the e3 ubiquitin ligase trim21 negatively regulates the innate immune response to intracellular double-stranded dna trim30α is a negative-feedback regulator of the intracellular dna and dna virus-triggered response by targeting sting the ubiquitin ligase trim56 regulates innate immune responses to intracellular double-stranded dna trim32 protein modulates type i interferon induction and cellular antiviral response by targeting mita/sting protein for k63-linked ubiquitination the critical role of toll-like receptors-from microbial recognition to autoimmunity: a comprehensive review the e3 ubiquitin ligase ro52 negatively regulates ifn-β production post-pathogen recognition by polyubiquitin-mediated degradation of irf3 self protection from anti-viral responses-ro52 promotes degradation of the transcription factor irf7 downstream of the viral toll-like receptors tripartite motif 21 (trim21) differentially regulates the stability of interferon regulatory factor 5 (irf5) isoforms e3 ubiquitin ligase tripartite motif 38 negatively regulates tlr-mediated immune responses by proteasomal degradation of tnf receptor-associated factor 6 in macrophages hung, t. trim38 negatively regulates tlr3-mediated ifn-β signaling by targeting trif for degradation trim56 is an essential component of the tlr3 antiviral signaling pathway mechanisms and functions of inflammasomes activation and regulation of the inflammasomes activation of nucleotide oligomerization domain 2 (nod2) by human cytomegalovirus initiates innate immune responses and restricts virus replication nod1 and nod2 signaling in infection and inflammation the e3 ubiquitin ligase tripartite motif 33 is essential for cytosolic rna-induced nlrp3 inflammasome activation tripartite-motif protein 30 negatively regulates nlrp3 inflammasome activation by modulating reactive oxygen species production the e3 ubiquitin ligase trim31 attenuates nlrp3 inflammasome activation by promoting proteasomal degradation of nlrp3 trim27 negatively regulates nod2 by ubiquitination and proteasomal degradation nucleo-cytoplasmic trafficking of trim8, a novel oncogene, is involved in positive regulation of tnf induced nf-κb pathway trim8 modulates stat3 activity through negative regulation of pias3 trim38 inhibits tnfα-and il-1β-triggered nf-κb activation by mediating lysosome-dependent degradation of tab2/3 siglec1 suppresses antiviral innate immune response by inducing tbk1 degradation via the ubiquitin ligase trim27 tripartite motif-containing 28 bridges endothelial inflammation and angiogenic activity by retaining expression of tnfr-1 and -2 and vegfr2 in endothelial cells interferons and viral infections tripartite motif 24 (trim24/tif1α) tumor suppressor protein is a novel negative regulator of interferon (ifn)/signal transducers and activators of transcription (stat) signaling pathway acting through retinoic acid receptor α (rarα) inhibition trim8/gerp ring finger protein interacts with socs-1 multiple functions of the ikk-related kinase ikkepsilon in interferon-mediated antiviral immunity iκb kinase ε (ikkε) regulates the balance between type i and type ii interferon responses control of foxo4 activity and cell survival by trim22 directs tlr3-stimulated cells toward ifn type i gene induction or apoptosis the interferon-inducible staf50 gene is downregulated during t cell costimulation by cd2 and cd28 regulation of the interferon-inducible p53 target gene trim22 (staf50) in human t lymphocyte activation t-cell-intrinsic tif1α/trim24 regulates il-1r expression on th2 cells and th2 cell-mediated airway allergy tripartite motif containing protein 27 negatively regulates cd4 t cells by ubiquitinating and inhibiting the class ii pi3k-c2β tripartite motif-containing protein 30 modulates tcr-activated proliferation and effector functions in cd4+ t cells the cytoplasmic body component trim5α restricts hiv-1 infection in old world monkeys trim5α protein restricts both hiv-1 and murine leukemia virus a trim5-cyclophilin a fusion protein found in owl monkey kidney cells can restrict hiv-1 cyclophilin a retrotransposition into trim5 explains owl monkey resistance to hiv-1 trim5α requires ube2w to anchor lys63-linked ubiquitin chains and restrict reverse transcription dynamic conformational changes in the rhesus trim5α dimer dictate the potency of hiv-1 restriction primate trim5 proteins form hexagonal nets on hiv-1 capsids mechanism of b-box 2 domain-mediated higher-order assembly of the retroviral restriction factor trim5α crystal structure of the trim5α bbox2 domain from rhesus macaques describes a plastic oligomerisation interface recent insights into the mechanism and consequences of trim5α retroviral restriction hexagonal assembly of a restricting trim5αprotein trim5 is an innate immune sensor for the retrovirus capsid lattice proteasome inhibition reveals that a functional preintegration complex intermediate can be generated during restriction by diverse trim5 proteins proteasome inhibitors uncouple rhesus trim5α restriction of hiv-1 reverse transcription and infection fates of retroviral core components during unrestricted and trim5-restricted infection proteasomal degradation of trim5α during retrovirus restriction nonhuman trim5 variants enhance recognition of hiv-1-infected cells by cd8+ t cells capsid-binding retrovirus restriction factors: discovery, restriction specificity and implications for the development of novel therapeutics hiv-1 capsid recognition, and innate immune signaling trim5α-mediated ubiquitin chain conjugation is required for inhibition of hiv-1 reverse transcription and capsid destabilization trim5 retroviral restriction activity correlates with the ability to induce innate immune signaling trim5α restriction affects clinical outcome and disease progression in simian immunodeficiency virus-infected rhesus macaques preserved cd4+ central memory t cells and survival in vaccinated siv-challenged monkeys defective cd8 t cell memory following acute infection without cd4 t cell help requirement for cd4 t cell help in generating functional cd8 t cell memory a single amino acid change in the spry domain of human trim5αleads to hiv-1 restriction evolution of a cytoplasmic tripartite motif (trim) protein in cows that restricts retroviral infection isolation of an active lv1 gene from cattle indicates that tripartite motif protein-mediated innate immunity to retroviral infection is widespread among mammals ovine trim5α can restrict visna/maedi virus receptor usage dictates hiv-1 restriction by human trim5α in dendritic cell subsets lack of endogenous trim5α-mediated restriction in rhesus macaque dendritic cells endogenous trim5α function is regulated by sumoylation and nuclear sequestration for efficient innate sensing in dendritic cells an hiv-1 capsid binding protein trim11 accelerates viral uncoating functional evidence for the involvement of microtubules and dynein motor complexes in trim5α-mediated restriction of retroviruses influenza a virus uses the aggresome processing machinery for host cell entry hiv-1 uncoating is facilitated by dynein and kinesin 1 cytoplasmic dynein promotes hiv-1 uncoating unanchored ubiquitin in virus uncoating mid1 and mid2 are required for xenopus neural tube closure through the regulation of microtubule organization hiv-1 transcriptional silencing caused by trim22 inhibition of sp1 binding to the viral promoter anti-hiv-1 activity of trim 37 the interferon-induced antiviral protein pml (trim19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion trim19/pml restricts hiv infection in a cell type-dependent manner pml/trim19-dependent inhibition of retroviral reverse-transcription by daxx translocalized iga mediates neutralization and stimulates innate immunity inside infected cells antibodies mediate intracellular immunity through tripartite motif-containing 21 (trim21) trim21 is an igg receptor that is structurally, thermodynamically, and kinetically conserved intracellular antibody-bound pathogens stimulate immune signaling via the fc receptor trim21 simultaneous neutralization and innate immune detection of a replicating virus by trim21 trim21 promotes cgas and rig-i sensing of viral genomes during infection by antibody-opsonized virus trim21 immune signaling is more sensitive to antibody affinity than its neutralization activity intracellular antibody receptor trim21 prevents fatal viral infection swine trim21 restricts fmdv infection via an intracellular neutralization mechanism robust lys63-linked ubiquitination of rig-i promotes cytokine eruption in early influenza b virus infection trim22 inhibits influenza a virus infection by targeting the viral nucleoprotein for degradation structure of influenza a polymerase bound to the viral rna promoter trim32 senses and restricts influenza a virus by ubiquitination of pb1 polymerase the c-terminal tail of trim56 dictates antiviral restriction of influenza a and b viruses by impeding viral rna synthesis historical perspectives on flavivirus research pathogenesis of flavivirus infections: using and abusing the host cell understanding the molecular mechanism(s) of hepatitis c virus (hcv) induced interferon resistance mutations in the nonstructural protein 5a gene and response to interferon in patients with chronic hepatitis c virus 1b infection trim14 inhibits hepatitis c virus infection by spry domain-dependent targeted degradation of the viral ns5a protein interferon α(ifnα)-induced trim22 interrupts hcv replication by ubiquitinating ns5a trim52 inhibits japanese encephalitis virus replication by degrading the viral ns2a dengue virus inhibits α interferon signaling by reducing stat2 expression inhibition of interferon signaling by dengue virus blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein 2a overlapping and distinct molecular determinants dictating the antiviral activities of trim56 against flaviviruses and coronavirus identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus trim56 is a virus-and interferon-inducible e3 ubiquitin ligase that restricts pestivirus infection tick-borne flaviviruses antagonize both irf-1 and type i ifn signaling to inhibit dendritic cell function mechanisms of evasion of the type i interferon antiviral response by flaviviruses inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns5 as an interferon antagonist trim79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral rna polymerase trim25 enhances the antiviral action of zinc-finger antiviral protein (zap) promyelocytic leukemia isoform iv confers resistance to encephalomyocarditis virus via the sequestration of 3d polymerase in nuclear bodies identification and characterization of multiple trim proteins that inhibit hepatitis b virus transcription trim22: a diverse and dynamic antiviral protein tripartite motif-containing 22 inhibits the activity of hepatitis b virus core promoter, which is dependent on nuclear-located ring domain trim5α promotes ubiquitination of rta from epstein-barr virus to attenuate lytic progression emerging role of pml nuclear bodies in innate immune signaling influenza a virus trims the type i interferon response the role of trim25 in development, disease and rna metabolism ubiquitin in influenza virus entry and innate immunity trim25 identification in the chinese goose: gene structure, tissue expression profiles, and antiviral immune responses in vivo and in vitro subcellular localizations of rig-i, trim25, and mavs complexes the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination hijacking of rig-i signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type i interferon responses tripartite containing motif 32 modulates proliferation of human neural precursor cells in hiv-1 neurodegeneration ns5 of dengue virus mediates stat2 binding and degradation dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling a family of mammalian e3 hendra and nipah viruses: different and dangerous evidence for ubiquitin-regulated nuclear and subnuclear trafficking among paramyxovirinae matrix proteins ubiquitin-regulated nuclear-cytoplasmic trafficking of the nipah virus matrix protein is important for viral budding hepatitis b virus x protein: trimming antiviral defences in hepatocytes suppression of interferon-mediated anti-hbv response by single cpg methylation in the 5 -utr of trim22 chloroquine triggers epstein-barr virus replication through phosphorylation of kap1/trim28 in burkitt lymphoma cells tripartite motif-containing 22 gene-364t/c polymorphism associated with hepatitis b virus infection in chinese han population identification of trim27 as a novel degradation target of herpes simplex virus 1 icp0 the host e3-ubiquitin ligase trim6 ubiquitinates the ebola virus vp35 protein and promotes virus replication ebola virus vp35 protein binds double-stranded rna and inhibits α/β interferon production induced by rig-i signaling mutual antagonism between the ebola virus vp35 protein and the rig-i activator pact determines infection outcome ebola virus protein vp35 impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk-1 filovirus replication and transcription. future virol eaten alive: a history of macroautophagy autophagy, immunity, and microbial adaptations autophagy fights disease through cellular self-digestion autophagy in leukocytes and other cells: mechanisms, subsystem organization, selectivity, and links to innate immunity trim-directed selective autophagy regulates immune activation trim proteins regulate autophagy and can target autophagic substrates by direct recognition trim proteins regulate autophagy: trim5 is a selective autophagy receptor mediating hiv-1 restriction specific recognition and accelerated uncoating of retroviral capsids by the trim5α restriction factor functional interactions between ubiquitin e2 enzymes and trim proteins the authors declare no conflict of interest. key: cord-278876-il7g78w1 authors: akkina, ramesh; ellerbrok, heinz; hall, william; hasegawa, hideki; kawaguchi, yasushi; kleanthous, harold; mcsweegan, edward; mercer, natalia; romanowski, victor; sawa, hirofumi; vahlne, anders title: 2016 international meeting of the global virus network date: 2017-03-16 journal: antiviral res doi: 10.1016/j.antiviral.2017.03.005 sha: doc_id: 278876 cord_uid: il7g78w1 the global virus network (gvn) was established in 2011 in order to strengthen research and responses to current viral causes of human disease and to prepare against new viral pandemic threats. there are now 38 gvn centers of excellence and 6 affiliate laboratories in 24 countries. gvn scientists meet annually to learn about each other's current research, address collaborative priorities and plan future programs. the 2016 meeting was held from october 23–25 in hokkaido, japan, in partnership with the japanese society for virology, the national institute of infectious diseases of japan and the research center for zoonosis control of hokkaido university. this report highlights the accomplishments of gvn researchers in many priority areas of medical virology, including the current zika epidemic, infections by human papillomavirus, influenza, ebola, lassa, dengue, hiv, hepatitis c, and chikungunya viruses, and the development of improved diagnostics and new vaccines. the global virus network (gvn) was established in 2011 in order to strengthen research in response to viral causes of human disease and to prepare for new viral pandemic threats (mann, 2011) . the gvn now has 38 centers of excellence and 6 affiliates in 24 countries. network scientists meet annually to address research and collaborative priorities, learn about each member's current work and plan future programs. these international conferences have become critical platforms for the exchange of ideas (for further information, go to http://www.gvn.org). the 2016 international gvn meeting was held from october 23e25 in hokkaido, japan, in partnership with the japanese society for virology (jsv), the national institute of infectious diseases of japan and the research center for zoonosis control of hokkaido university. it brought together directors of gvn centers and their colleagues for two days of scientific presentations and discussions of emerging and re-emerging viral threats. this meeting report highlights accomplishments of gvn researchers in many priority areas of human virology, including the current zika epidemic, infections by human papillomavirus, influenza, ebola, lassa, dengue, hiv, hepatitis c and chikungunya viruses, and the development of improved diagnostics and new vaccines. the sapporo meeting was the first gvn event held jointly with a local national virology society. more than 1000 jsv members had the opportunity to attend scientific sessions, thereby expanding opportunities for collaborative dialogue, particularly with young japanese virologists. the main objectives of the meeting were to present and discuss current findings in medical virology, including advances in research on hiv vaccines and other important retroviruses; provide a framework to encourage collaborations among world experts; and address the gvn's annual strategy for continued development. the gvn was founded because there was no research organization independent of national governments which had the depth of expertise needed to respond promptly to new pandemic viral outbreaks and to prepare for such outbreaks. the gvn was not intended to replace organizations such as the centers for disease control and prevention (cdc) or the world health organization (who), but to complement their activities with independent opinions and expertise. in parallel, a second major goal of the gvn was to advocate for the strengthening of virology as a core discipline in infectious diseases and to help develop the next generation of virologists (dimaio, 2014) . in march 2011, 30 of the world's leading virologists gathered in washington, d.c. to pledge their support for an international coalition of virology institutions, ready to act in times of outbreaks and committed to advancing knowledge of pathogenic viruses. today, the gvn is comprised of 38 centers of excellence and 6 affiliates in 24 countries (fig. 1 ). centers are led by world-class virologists who have expertise in two or three areas of virology, strong publication records, a commitment to building technical and scientific capacities in resource-poor countries, and the readiness to support the gvn infrastructure. albert osterhaus, phd, dvm, director of the research center for emerging infections and zoonoses (riz) at the university of veterinary medicine hannover, germany, a gvn center of excellence, was presented with the robert c. gallo award for scientific excellence and leadership for his pioneering contributions to influenza and coronavirus research as well as his contributions to advancing the gvn mission. osterhaus was cited for his decades-long career, which has included the discovery of more than 50 new human and animal viruses and helping the who to combat outbreaks of mers (middle east respiratory syndrome), sars (severe acute respiratory syndrome), and pandemic influenza. he was also praised as a leader within the gvn for his active participation in international conferences and virology training programs, for making important basic science contributions to medical virology and for translating those contributions into broad public health initiatives. the award is named for robert c. gallo, md, the director of the institute of human virology at the university of maryland at baltimore, and the co-founder and scientific director of the gvn. dr. gallo and colleagues discovered the first human retroviruses, human t-cell leukemia virus (htlv)-1 and -2; and human herpesvirus-6, which was subsequently demonstrated by others to cause roseola (gallo, 2005) . gallo co-discovered the human immunodeficiency virus (hiv) as the cause of aids and together with colleagues discovered one of the first cytokines (il-2). il-2 later became critical to growing continuous in vitro cell-lines, and is now important in many immune therapy protocols. dr. gallo and colleagues also developed the first blood tests for htlv-1 and hiv. robert gallo reviewed recent hiv vaccine trials that used four different methods. the thai-u.s. army trial (rv144) appeared to have given very modest protection in the early months after vaccination, while the others failed to prevent infection or in one case was associated with greater infection. gallo explained that for the last several years, the emphasis has been on induction of antibodies to the hiv envelope component, gp120, rather than to a primary induction of cellular immunity to prevent infection. this approach was based on the logic of trying to prevent integration and establishment of a permanent infection. this effort has mainly evolved around two programs. the first was a detailed analysis of the rv144 results, particularly correlates of protection, which have mainly highlighted antibodies interacting with viv2 gp120 epitopes and epitopes induced by gp120 interactions with cd4, referred to as cd4i abs, which target conserved envelope regions needed for gp120 interaction with ccr5. functional correlations were mainly with fc effector functions rather than neutralizing antibodies (nab). the second major approach is one that is geared toward inducing broad nab, which is a reasonable goal, but one that is not being met, as it is proving far more difficult than supposed. moreover, in studies in nonhuman primates (nhp), as well as the results with rv144, correlation of protection is rare with nab, even when they are generated. gallo also described a candidate immunogen expressed as a chimeric protein of gp120 and cd4, which has shown some efficacy against heterologous challenges in primates, and correlates with cd4i antibodies that have fc effector activity (fouts et al., 2015) . this vaccine is in phase i trials (https://clinicaltrials.gov/show/ nct02756208). like all anti-gp120 antibodies correlating with protection, these antibodies do not persist, and attempts to increase persistence have resulted in hyper-activation of t-cells, which then form more targets for hiv infection. persistent boosting to maintain the antibodies leads to changes in the antibody isotype, which can reduce their efficacy. based on the rv144 trial and recent nhp studies, gallo concluded that two critical basic science problems remain to be solved for the development of a successful antienvelope hiv vaccine: durability of the antibodies and a correct immune balance. yasushi kawaguchi, at the university of tokyo, described cell factors that are involved in a unique nuclear-pore-independent export system for macromolecular complexes in the nucleus, and which may be potential targets for novel anti-herpetic drugs. vesicle-mediated nucleocytoplasmic transport is a unique mechanism for nuclear export of macromolecular complexes in which a complex in the nucleus buds through the inner nuclear membrane to form a vesicle in the perinuclear space (primary envelopment). the vesicle then fuses with the outer nuclear membrane to release the complex into the cytoplasm (de-envelopment) . this type of transport is observed in herpesvirus-infected mammalian cells for export of viral nucleocapsids, but is not common in other types of cells, indicating that herpesviruses may expropriate this mechanism (oda et al., 2016) . however, the cellular mechanism for vesicle-mediated nucleocytoplasmic transport remains largely unknown. he went on to describe recently identified host cell proteins involved in this unique nuclear export system, and how these proteins could be targets for novel anti-herpetic drugs, since this transport system is essential for the life cycle of herpesviruses and is unique in biology. hideki hasegawa, a gvn center director at the national institute of infectious diseases in tokyo, explained that secretory iga antibodies on mucosal surfaces play an important role in protection against influenza virus infection. secretory, polymeric iga antibodies induced by intranasal, inactivated influenza vaccine have higher neutralizing and cross-neutralizing ability against homologous and heterologous influenza viruses compare to monomeric iga antibodies in humans (suzuki et al., 2015) . a method of producing secretory multimeric iga antibodies in vitro was established recently, and hasegawa and colleagues examined the improvement of neutralizing effects by multimerization using monoclonal iga antibodies. the method of in vitro production of multimeric iga antibodies was established by introducing cdna constructs of h, l, and j chains of iga with secretory component (sc) into expi293f cells. this method allowed production of large amounts of recombinant monoclonal polymeric iga antibody. the antibody-coding cdna was isolated and cloned from single plasma cell from the peripheral blood mononuclear cells of a subject previously vaccinated intranasally with inactivated influenza a (h5n1) vaccine. monomeric and multimeric iga antibody specific for influenza virus was prepared, and functional analysisdsuch as elisa and neutralization testsdwas performed. similar to secretory iga antibodies in human nasal washes, the recombinant iga showed that polymerization of the antibody molecule enhanced the neutralizing activity. mass spectrometry analysis found that the antibodies had a molecular weight of 720 kda, suggesting they consisted mostly of the tetramer form. these tetrameric iga antibodies may be clinically useful as mucosal agents for the prevention and treatment of influenza. erica ollmann saphire, a gvn center co-director at the scripps research institute (usa), reported on recent outbreaks of ebola virus disease and other viral diseases and the urgent need for novel therapeutics. monoclonal antibodies offer a promising solution to the lack of currently available therapies, but it is often unclear how to select the most effective antibodies or combinations for optimal therapy (saphire and aman, 2016) . integrated analysis of a large array of antibodies provides a rational basis for the selection of optimized therapeutics, and can lead to rapid understanding of the mechanisms underlying protective efficacy. she described the results of a field-wide analysis of antibodies against ebola virus, a particularly potent antibody against marburg virus, and a novel panel of antibodies against lassa virus. a newly formed international group, the viral hemorrhagic fever immunotherapeutic consortium, aims to galvanize and focus efforts against ebola and similar viruses, using multidisciplinary approaches (saphire et al., 2017) . sharon lewin, a gvn center director at the doherty institute for infection and immunity (australia), reported on hiv cure research (lederman et al., 2016) . hiv persists predominantly in long-lived resting cd4 þ t cells that are found in blood and at greater frequency in tissue. hiv can also persist in specific t-cell subsets enriched in certain tissue sites, including t follicular helper cells in lymph nodes and th17 (ccr6þ) cells in the gastrointestinal tract. long-lived infected macrophages, specifically microglia in the brain or kupffer cells in the liver, may also play a role, though this remains controversial. on art, hiv-specific t cells express multiple exhaustion markers, and these cells are often unable to recognize and kill infected cells due to multiple immune escape mutations that are archived in long lived latently infected cells. furthermore, in some tissue sites such as b-cell follicles in lymph nodes, there is limited penetration of hiv-specific t-cells. lewin described several approaches that are being tested to achieve hiv remission, including reducing latently infected cells through early art or latency reversal, boosting immune clearance, reducing immune activation and homeostatic proliferation, eliminating tissue reservoirs, and gene therapy to make cells resistant to hiv. recently completed clinical trials of latency-reversing agents (lra) in hiv-infected patients on art have included histone deacetylase inhibitors (hdaci) and high-dose disulfiram (delagr everie et al., 2016) . other interventions, including activation of protein kinase c (pkc) with bryostatin and ingenols, are under evaluation. studies of hdaci and disulfiram have demonstrated that hiv transcription can be activated in vivo with varying efficiency, but to date there is no evidence that these interventions alone can clear latently infected cells. in addition, recent in vitro studies have shown that activating transcription with hdaci, but not other lras, may lead to profound changes in hiv rna splicing, therefore limiting potency. other concerns regarding hdaci include short-and long-term toxicity, changes in host gene expression and adverse effects on immune function. more potent and specific lras are necessary and in development for use alone and in combination. additional interventions to promote cell death may be needed. targeting bcl2 or inhibitors of anti-apoptotic proteins (iap) is being evaluated in vitro, and immune-mediated clearance with bispecific antibodies, broadly neutralizing antibodies and t-cell vaccines are in clinical development. however, even if the number of latently infected cells is reduced, long-term immune control will be required through therapeutic vaccination and/or immunomodulation. immune checkpoint blockers, including antibodies to ctla-4 and pd-1 that have recently been licensed for the treatment of melanoma, are potentially attractive tools to enhance hiv-specific t-cells and reverse latency. clinical trials of these agents are now starting in hiv-infected individuals with malignancies. shyamasundran kottilil, at the institute of human virology (usa), described the challenges of current treatments of chronic hepatitis c. eighty-five percent of hcv infections become chronic; 20e30% of those cases will develop cirrhosis, and 5e10% of these patients will progress to hepatocellular carcinoma and/or decompensation of the liver, leading either to death or liver transplantation. in contrast to chronic hepatitis b or hiv infection, chronic hcv infection can be cured with direct-acting antiviral (daa) treatment. "cure" is defined in terms of sustained virologic response (svr), which is the absence of detectable levels of hcv rna in the plasma after 12 or 24 weeks of daa treatment (jazwinski and muir, 2011) . current hcv inhibitors act against the ns3/4 protease (e.g., asunaprivir and grazoprevir), the ns5a protein (e.g., daclatasvir and velpatasvir) and the ns5b polymerase (e.g., sofosbuvir and beclabuvir). treatment with a combination of the two inhibitors, sofosbuvir and velpatasvir, for 12 weeks, will result in a cure of more than 99% of treated patients. still, clinical challenges remain, including treatment of patients with end-stage liver and renal disease owing to metabolism and/or toxicity of the drugs. other challenges include the treatment of hiv/hcv co-infections, injection drug users, pediatric patients and maternal-fetal transmission and pregnancy. estaban domingo, at the centro de biología molecular severo ochoa in spain, presented studies on the population dynamics of hcv after antiviral interventions in infected human hepatoma huh-7.5 cells. unexpectedly, serial passage of hcv in these cells in the absence of antiviral drugs resulted in increased resistance to inhibitors that target viral or cellular proteins, and was associated with a fitness gain of the virus. if daa-resistance mutations become widespread in human populations, alternative drug treatments will be needed. one such drug is favipiravir (furuta et al., 2013) . this is a broad-spectrum antiviral agent active against numerous rna viruses, including influenza, norovirus, rabies, and rift valley fever virus. it acts as a lethal mutagen (de avila et al., 2016) . efficacy and safety trials of favipiravir for treating influenza, as well as ebola virus disease, already have been done. in cell cultures], the efficacy of favipiravir against hcv was found to be comparable to its efficacy against other rna viruses. in hcv-infected huh-7.5 cells, favipiravir demonstrated features typical of lethal mutagenesis. it was concluded that favipiravir offers an alternative for patients chronically infected with hcv. research on biomarkers of progression in human papillomasvirus (hpv)-related cancers was highlighted in this session by franco buonaguro (national cancer institute, italy). buonaguro described persistent infections with high-risk hpvs that are often associated with progression to mucosal cancers in anogenital as well as oropharyngeal areas of the body. he also noted that for cervical cancers, for which screening programs are available, it is difficult to identify the lesions likely to progress to invasive cancers. given that only 1 in 500 hpv16-positive cervical dysplasias will eventually progress further, biomarkers clearly are needed. tumor progression is characterized by increased expression of the viral e6 gene and e6-dependent degradation of p53, and increased expression of e7, which is known to bind and inactivate prb; and integration of viral dna into the host genome with the consequent disruption of the e2 viral gene (annunziata et al., 2012) . molecular markers able to identify viral infections associated with progressing cervical neoplasia are strongly needed for screening and triage. in particular, predictive biomarkers are needed to detect lesions at high risk of recurrence or progression, in order to implement appropriate treatment and to avoid overtreatment of patients with a high probability of regression. to achieve such goals, expression profile analysis of p53-related genes in hpv-16-positive genital carcinomas, along with autologous nontumor tissue, was performed, and significant differences in the expression levels of genes involved in regulation of apoptosis, cell cycle, proliferation and dna repair pathways were identified. moreover, oncogenic driver mutations in critical genes (such as pik3ca and tp53), recurrent chromosomal gain/loss, and oncogene activation by hpv integration have all been recognized to cause deregulation of cell signaling pathways and cancer progression (tornesello et al., 2014) . more recently, analysis of the regulatory regions of human genes encoding for cell proliferation proteins has shown a high frequency of mutations in lesions associated with less oncogenic hpv genotypes, and even in hpv-negative lesions. validation of these candidate biomarkers is currently under way on a larger number of cases, including different grades of hpv-related neoplastic lesions (cin1-3 and invasive cervical cancer). such studies will contribute to the development of new tools for the identification of premalignant lesions at high risk of progression to invasive cervical carcinoma. jonathan gershoni, a gvn center director at tel aviv university (israel), described an analysis of the vast repertoire of antibodies circulating in polyclonal serum (the "igome.") he highlighted how the paratope region of an antibody mirrors the corresponding region of an antigen (e.g., a virus protein) and therefore favors an indirect approach of igome profiling by describing all peptides interacting with the entity of antibodies. the spectrum of antibody specificities is dynamic and varies with age, physiology, and exposure to pathological insults. the igome is an extraordinarily rich source of informationea molecular record of all previous encounters, as well as a status report of current immune activity (weiss-ottolenghi and gershoni, 2014) . the ability to profile antibody specificities of polyclonal serum at very high resolution has been an important and serious challenge, which can now be met by probing, recording, and qualifying the archive of antibodies present in the serum of an individual with a vast library of peptides. gershoni also described "deep panning", a methodology that merges the flexibility of combinatorial phage-display peptide libraries with the power of next-generation sequencing to enable high-resolution/high-throughput interrogation of the igome (ryvkin et al., 2012) . this approach produces heat-maps that characterize the interaction between the immune system and antigens. this method was used to characterize and differentiate sera from hiv-infected individuals versus hcv-infected individuals. by analyzing the immune response to influenza antigens in birds, jonathan showed that this technique could be used to monitor and characterize the efficiency of vaccines, suggesting that igome analysis may become a very useful tool for vaccine development. since the appearance of chikungunya virus (chikv) in the western hemisphere in 2014, the gvn has had a special focus on arboviruses. in 2014, the network sponsored a joint symposium with the american society of tropical medicine and hygiene on "the global spread of chikungunya: epidemiology, evolution, pathogenesis and global needs" and published a special report describing research goals and public health priorities for the outbreak (mcsweegan et al., 2015) . a task force of international experts also was formed to provide a source of expert information and a platform for collaboration. marc lecuit at the gvn center at the institut pasteur in paris described chikv, its geographical spread, the clinical presentation of infection and the development of a mouse model that has allowed pathophysiology studies and the evaluation of novel therapeutics. murine fibroblasts of skeletal muscles, joint capsules and dermis were found to be primary viral targets, and type i ifn-sensing, non-hematopoietic cells were critical for innate immune control. a genome-wide sirna screen identified 156 proviral and 41 antiviral host factors affecting viral replication, which allowed identification of potential drug targets. twenty-one small molecule inhibitors that affect six host pathways have been identified; three showed prophylactic effects in the mouse model. a calmodulin inhibitor (pimozide) and a fatty acid synthesis inhibitor (tofa) showed antiviral effects in mice when used together (karlas et al., 2016) . other experimental data showed that chikv infection does not directly target the placental barrier, therefore vertical transmission from viremic mothers to neonates likely occurs via laborinduced placental breaches. chikv also was found to be a significant cause of cns disease in larger human outbreaks, with the virus detected in affected tissues (g erardin et al., 2016) . scott weaver, chair of a gvn task force on zika virus (zikv), reviewed the sudden emergence of zikv from relative obscurity to global menace. serosurveys have shown its historical incidence in parts of asia and africa (weaver et al., 2016) . a widespread outbreak in the micronesian island of yap in 2007 later expanded to neighboring islands in south pacific. coincident with these outbreaks, a 20-fold higher incidence of guillain-barre syndrome (gbs) was noted. the explosive outbreak in brazil that began in 2015 and has involved millions of people has since spread to 47 other countries and territories in the western hemisphere. the peridomestic mosquito, aedes aegypti, and the invasive aedes albopictus mosquito have been identified as the major insect vectors. surprisingly, sexual transmission of zikv also was confirmed in travelers returning to non-endemic regions. a most disturbing aspect of the recent outbreaks is the high incidence of fetal microcephaly and other birth defects directly related to infection in pregnant women. weaver also discussed important knowledge gaps. when and how should infants be tested for possible congenital zika infection? is microcephaly among some neonates just the tip of the neurological iceberg? why does the overall risk of microcephaly and other birth defects seem to differ in other parts of latin america and the caribbean, compared to brazil? it appears that these new outbreaks in the western hemisphere have not been caused by novel viral strains. instead, the introduction of zikv into naïve populations via rapid global travel, the growth of tropical cities, and changing vector populations seem to have allowed virus amplification and subsequent epidemic spread. prospects for control are dependent on mosquito control, new therapeutics and development of an effective vaccine. vaccine prospects appear good, with about 40 candidates now in development and animal models available for preclinical testing. two dna-based vaccines have entered phase i trials, and an inactivated vaccine is nearing phase i. deploying a vaccine, however, remains dependent on developing an accurate test for zikv. current tests are hampered by antigenic cross-reactivity between zikv and other closely related flaviviruses such as dengue, yellow fever, and st. louis encephalitis, some of which are endemic in the same geographic areas. richard scheuermann, gvn center co-director at the j. craig venter institute (usa), discussed using the influenza research database (ird, fludb.org) and the virus pathogen resource (vipr, viprbrc.org) to identify diagnostic peptide regions in zika virus (zikv) and other flaviviruses. these databases identified 76 and 82 diagnostic amino acid sites from the zikv ns1 and e proteins, respectively, that distinguish it from other flaviviruses, with sensitivity/specificity above 98% (sun et al., 2017) . similar numbers of such diagnostic sites were also identified for other flaviviral species. sliding window analysis revealed several contiguous peptide regions that contain multiple diagnostic sites specific for each of the different flaviviruses. these regions are now being used to develop peptide arrays for the detection of antibodies specific for each of the viruses including zikv. these peptide arrays could have a significant impact on diagnosis and epidemiological analysis of future disease outbreaks. diane griffin at the gvn center at johns hopkins university (usa) noted that insect-borne viruses causing encephalitis have become a global health concern due to their recent expansion into new regions. while the fatality rates vary, many patients recovering from serious episodes of viral encephalitis are likely to have lifelong physical and mental disabilities. viral persistence and immunemediated clearance are important factors in these sequelae. griffin described experiments with sindbis virusda prototype alphavirusdusing a variety of mouse models (griffin, 2016) . infection of neurons in adult mice resulted in non-fatal encephalomyelitis. recovery was mediated through a coordinated effort between antibody to the e2 viral surface protein and ifn-g. while ifn-g signaling leads to clinical disease via pro-inflammatory cytokines, it synergistically helps clear the virus by increasing b-cellattracting chemokine production for recruitment of antibodyproducing cells to the central nervous system (cns). however, the non-cytolytic immune phenomenon does not lead to elimination of intracellular viral rna from long-lived neurons, so that continued suppression of virus and the prevention of reactivation and reemergence in the cns require immune control after recovery. evidence was presented that b and t cells that infiltrated during the acute phase of infection remain there after recovery and continue to exert their protective effect locally. long-term residence of these immune cells in the cns is likely facilitated by the continued presence of viral proteins. arthropods make up the largest phylum of the animal kingdom and are known to harbor and transmit viruses of agricultural, veterinary and medical importance. the true extent of the 'virosphere' of arthropods, however, is largely unknown and needs to be more completely explored. yong-zhen zhang, an investigator at the national institute for communicable disease control and prevention in beijing, china, presented work on newly identified rna viruses from arthropods collected in different regions of china. among recent examples are jingmen tick virus (jmtv), and viruses belonging to a new family, chuviridae (qin et al., 2014) . jtmv has four genomic segments, and, sequence analysis has revealed an unexpected connection between segmented and non-segmented viruses. chuviruses exhibit various genomic organizations, including non-segmented, bi-segmented, and circular genomes. these viruses provided evidence of an evolutionary link between segmented and non-segmented negative strand viruses. overall, many novel viruses representing five new families, have been discovered in china and await further characterization. peter palese, a gvn center director at the icahn school of medicine at mount sinai (usa), described ongoing efforts to design a universal influenza vaccine and to understand the immune responses to such constructs. he said that, despite the availability of fda-approved vaccines and antivirals, seasonal and pandemic influenza remain a serious threat associated with substantial morbidity and mortality. while annual seasonal influenza virus vaccination is effective e albeit underutilized in most countries e a safe, universal vaccine providing broad and long-lasting immunity would represent a major breakthrough. today's licensed vaccines focus on eliciting humoral immunity to the globular head (ha1) of the hemagglutinin (ha) glycoprotein, an antigen subject to antigenic drift, which requires continued surveillance of circulating influenza viruses. since the 2009 h1n1 pandemic, research efforts on universal vaccines have refocused, looking to shift immunity to the more conserved domains of ha, which is expected to cover many more circulating influenza viruses. palese also has developed vaccine constructs that express chimeric hemagglutinins (cha) that result in the redirection of the immune response away from the immunodominant (variant) ha1 head domain towards the much more conserved stalk (ha2) region, and which also express the highly conserved neuraminidase (na) (tran et al., 2016) . this type of immunogen has been demonstrated to be efficacious following vaccination in mice, conferring heterosubtypic protection against virus challenge. the mechanism by which this approach confers antiviral activity suggests that redirecting immunity to the stalk region preferentially supports induction of antibody-dependent cell-mediated cytotoxicity (adcc). data presented showed that broadly neutralizing ha stalk-specific antibodies require fcgr interactions for protection. palese proposed a two-contact model for optimal induction of adcc by influenza virus-specific mabs targeting the stalk regiondand not the globular headdand he noted the importance of the ha binding to effector cells via its sialic acid receptor was required for optimal adcc induction. passive transfer studies in mice further supported the role of h1-specific stalk antibodies and protection against weight loss following virus challenge. this novel technology is being supported through government and industry support, and gmp-quality batches of two cha immunogens (ch5/1n1 & ch8/1n1) that display heterogenous globular heads have been produced for evaluation in the clinic. such studies will make it possible to assess the role of additional effector mechanism in protecting against influenza virus infection and to evaluate the importance of reducing the immunodominance of the variable ha head domain. this type of translational study will bring us a step closer to a universal influenza vaccine. hiroshi kida at hokkaido university in japan reported on the establishment of a library of 2900 avian influenza virus strains isolated from ducks, and reassortant viruses generated in the laboratory, with 144 combinations of the nine na and sixteen ha subtypes (virusdb.czc.hokudai.ac.jp). because influenza a viruses of all known subtypes can perpetuate among migratory ducks and contribute genes to the generation of reassortant viruses in pigs, none of the 144 combinations can be ruled out as possible future pandemic strains. this virus library is kept as a source for vaccine candidates to assure effective preparedness for future pandemics (haredy et al., 2013) . vaccine constructs prepared from h1n1, h5n1, h6n2, h7n7, h7n9 and h9n2 viruses in the library elicited sufficient immune responses to protect chickens, mice, and macaques from challenge with isolates from poultry and humans, suggesting their utility against pandemic influenza. in addition, he noted that current seasonal vaccines prepared by ether or detergent disruption are not sufficiently immunogenic, especially in children and the elderly, and need to be significantly improved. furthermore, methods for controlling pandemic influenza should be based on measures that are used for the control of seasonal influenza. hiroshi noted that inactivated whole virusbased vaccine candidates might provide solutions to the limitations of current vaccines. to accomplish the goal of a new and effective influenza vaccine, a collaborative research group, the all japan collaborating study group for the development of influenza vaccines, was established in april 2015. five japanese manufacturers of influenza vaccines participate in the program. the goals of the study group are: to compare the immunogenicity and safety of whole virus particle vaccine and "split" vaccines; use the results of pre-clinical and clinical trials to revise the standard of influenza vaccines for human use; and determine the optimum route of inoculation and evaluate proprietary adjuvants. massimo palmarini, a gvn center director at the mrc-university of glasgow center for virus research (scotland), reported on studies of bluetongue, a major infectious disease of ruminants, caused by the double-stranded rna virus known as bluetongue virus (btv). studies of btv in sheep offer unique perspectives for understanding the pathogenesis of arboviral diseases, as observations made in the naturally occurring disease can be effectively reproduced in a convenient experimental setting, using the same animal species (caporale et al., 2014) . the clinical outcome of btv infection in sheep is extremely variable, but similar to other arbovirus infections, in that a rapid onset of the antibody response correlates with a more favorable clinical outcome. btv infection of its natural sheep host was used to examine a previously uncharacterized mechanism adopted by an arbovirus to manipulate host immunity at the early stages of infection. btv is transported rapidly via the lymph to the peripheral lymph nodes, where it infects and disrupts follicular dendritic cells (fdc). these cells of stromal origin promote the formation and maintenance of the germinal centers in which b cells differentiate into memory cells and plasma cells, and are also responsible for supporting antibody class-switching and affinity maturation. this work showed that btv hindered b-cell division in germinal centers, resulting in the delayed production of high-affinity and virus-neutralizing antibodies. importantly, the humoral immune response to a second antigen is also hampered in infected sheep. thus, an arbovirus can evade the host antiviral response by inducing an acute immunosuppression. although transient, this immunosuppression occurs at the critical, early stage of infection, when a delayed host humoral immune response likely affects the systemic dissemination of the virus and the clinical outcome of disease (melzi et al., 2016) . ab osterhaus, the gvn center director at the research center for emergin, reviewed the complex relationships among human and animal species that have promoted cross-species transmission, emergence and the eventual evolution of a plethora of human pathogens. changes affecting modern human populations worldwide and their dramatic impact on the global environment have taken domestication, agriculture, urbanization, and industrialization to unprecedented levels. this has created new and global multi-faceted human-animal interfaces, associated with major epidemiological transitions, accompanied by an unexpected surge of emerging and re-emerging human infectious diseases that have their origin in animal reservoirs. until the beginning of the last century, infectious diseases were the major cause of human mortality. around 1900, infections caused about fifty percent of deaths in the western world, but in the following decades, this percentage decreased to less than a few percent. this was largely due to the implementation of public health measures such as the installation of sewers and the development of clean drinking water systems, but also to development of vaccines and antimicrobials. major successes in this regard were the eradication of smallpox and rinderpest through wellorchestrated vaccination campaigns in humans and cattle, respectively. these successes prompted policymakers and scientists to predict that infectious diseases of humankind and of their domestic animals would eventually be brought under control in the industrialized world. paradoxically the following decades confronted the world with an ever-increasing number of emerging or re-emerging infectious diseases, some causing true human or animal pandemics. pathogens spilling over from wildlife reservoirs, either directly or via intermediate hosts, were the basis of most of the outbreaks. striking examples in humans were the emergence of aids from chimpanzees, avian flu from migratory birds, and sars, mers, and ebola virus disease from bat reservoirs. a complex mix of predisposing factors in our globalizing world, linked to major changes in the societal environment and global ecology, collectively created opportunities for viruses and other pathogens to infect and adapt to new animal and human hosts. this paved the way for the unprecedented spread of infections, with dramatic consequences for public and animal health, animal welfare, food supplies, economies, and biodiversity. it is important to realize that, because of the complex and largely interactive nature of the predisposing factors, it is virtually impossible to predict the next pathogen threat, where it will come from and when it will strike. however, a better understanding of the underlying processes may eventually lead to enhanced predictive capacity, improving preparedness for outbreaks in humans and animals. importantly, the increased emergence of viral infections has been largely paralleled by medical, veterinary, technological, and scientific progress. investments to better understanding human-animal interfaces should offer a future head start in the struggle against infectious diseases of humans. ayato takada at the global institution for collaborative research and education at hokkaido university in sapporo, japan, described an ebola virus glycoprotein-specific monoclonal antibody (mab 6d6) that was broadly cross-reactive with all known ebola virus species. this mab recognized the putative epitope in the highly conserved internal fusion loop (ifl) and neutralized infectivity by inhibiting membrane fusion. mab 6d6 may, therefore, have potential as a therapeutic agent in animal models. he also described a novel antibody-dependent enhancement (ade) mechanism in which fc receptor-mediated intracellular signaling increased uptake of ebola virus into cells in vitro (furuyama et al., 2016) . activation of the fc-receptor-mediated signaling pathway was essential for ade of ebola virus infection; this finding provides new insights into mechanisms of ade and the development of treatments for ade-associated diseases. finally, he noted that neutralizing and ade antibodies show balanced effects depending on total antibody concentration, raising theoretical concerns about the use of convalescent human sera or plasma with low antibody titers. therapeutic treatment with convalescent sera with in vitro neutralizing activity was not sufficient to protect against ebola virus infection in nonhuman primates (mire et al., 2016) . it may be that ade antibodies counterbalanced the neutralizing capacity of other antibodies. these laboratory phenomena warrant further evaluation. luc willems, a gvn investigator at gembloux agro-bio tech in belgium, described how bovine delta-retroviral ribonucleic acids induce cell transformation and oncogenesis. among the strategies developed by viruses to escape the host immune response, mechanisms involving viral non-coding rnas have recently been discovered. viruses can express micrornas that directly target cellular transcripts, while others produce long non-coding rnas acting as microrna sponges or epigenetic modulators. these strategies, based on ribonucleic acids, control the cell's fate without eliciting immune responses due to the absence of viral proteins. virally encoded mirnas were first identified in dna viruses. most of them are generated from endonucleolytic cleavage of long viral transcripts. however, retroviruses were initially assumed not to encode mirnas because of the potential self-cleavage of their rna genome (gillet et al., 2016) . to date, four retrovirusesbovine leukemia virus (blv), bovine foamy virus (bfv), avian leucosis virus (alv-j) and simian foamy virus (sfv) e have been shown to express viral mirnas in infected cells. blv naturally infects bovine species to cause a benign lymphocytosis, but approximately 5% of infections lead to b-cell leukemia/lymphoma. remarkably, blv persists and replicates in vivo in the absence of significant levels of viral mrna transcription. in contrast, blv mirnas are highly expressed in infected cells, representing up to 40% of all cellular mirnas. consequently, because ribonucleic acids likely are less immunogenic than viral protein antigens, blv mirnas could alter the cell's fate by escaping immune recognition (gillet et al., 2016) . as with the 2014 appearance of chikv in the western hemisphere, the sudden emergence and dissemination of zikv have driven similar cooperative responses at the gvn, including the formation of a second task force of experts to exchange information and share resources; the organization of a webinar for business leaders to inform them about the virus and prospects for therapies and vaccines (http://www.btsmeetings.com/zika); providing expert information to the news media to ensure accurate reporting; and acquiring a grant from the allergan foundation to establish a bank of acute and convalescent sera from confirmed zikv patients to aid in developing better diagnostics. among other accomplishments, shyamasundran kottilil and colleagues at the institute of human virology in baltimore began a project with the gvn, and funded by the gilead foundation, to develop a training model for physicians using daa treatment for hcv patients in india. members of the htlv-1 task force published a commentary on the need for htlv-1 screening prior to organ transplant, and an agenda for future research (gallo et al., 2016; willems et al., 2017) . other gvn investigators published new research on west nile virus and rabies virus (kobayashi et al., 2016; phongphaew et al., 2017; anindita et al., 2016) . during the past year, the network expanded the number of cooperating research centers by adding the university of miami, emory university and tulane university school of medicine in the usa and the international vaccine institute in south korea (http:// gvn.org/coe/). these centers are expected to strengthen cooperative research on vaccines, antiviral drugs, and emerging viruses such as zikv and ebola virus. among other projects, the gvn staff in baltimore expects to assist with a pilot study of hepatitis b in arunachal pradesh, india. funded by the john martin foundation, the project will screen 30,000 people, provide vaccinations for those who are not infected, and develop a longitudinal cohort to treat patients with chronic hepatitis b infections. members of the gvn task force on htlv-1 expect to publish additional commentaries and reviews during 2017. similarly, a gvn task force report on zikv emergence and research priorities is in preparation. gvn staff also will continue to seek additional patient serum samples for the zikv serum bank based at the university of texas medical branch in galveston, texas. additionally, a project to sequence and conduct phylogenetic analyses of collected zika virus isolates has begun in cooperation with the world reference center for emerging viruses and arboviruses (wrceva), which is funded by the national institute of allergy and infectious diseases (usa). the gvn will host its fourth annual short course on medical virology for young investigators. the short course attracts an international class of 15e20 postdoctoral students and fellows for five days of meetings and lectures with experts in medical virology research and clinical practice. information about the course and registration is available online at http://www.gvn.org. the 2017 international gvn meeting will be in melbourne, australia during september 25e27. it will be co-hosted by the gvn centers of excellence, the institut pasteur (paris) and the peter doherty institute for infection and immunity (melbourne generation of recombinant rabies viruses encoding nanoluc luciferase for antiviral activity assays characterization of the human papillomavirus (hpv) integration sites into genital cancers lethal mutagenesis of hepatitis c virus induced by favipiravir virus and host factors affecting the clinical outcome of bluetongue virus infection ongoing clinical trials of human immunodeficiency virus latency-reversing and immunomodulatory agents is virology dead? mbio balance of cellular and humoral immunity determines the level of protection by hiv vaccines in rhesus macaque models of hiv infection favipiravir (t-705), a novel viral rna polymerase inhibitor fcg-receptor iia-mediated src signaling pathway is essential for the antibody-dependent enhancement of ebola virus infection history of the discoveries of the first human retroviruses: htlv-1 and htlv-2 global virus network's task force on htlv-1 chikungunya virus-associated encephalitis: a cohort study on la r eunion island bovine leukemia virus small noncoding rnas are functional elements that regulate replication and contribute to oncogenesis in vivo alphavirus encephalomyelitis: mechanisms and approaches to prevention of neuronal damage an mdck cell culture-derived formalin-inactivated influenza virus whole-virion vaccine from an influenza virus library confers cross-protective immunity by intranasal administration in mice direct-acting antiviral medications for chronic hepatitis c virus infection a human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs rab8b regulates transport of west nile virus particles from recycling endosomes a cure for hiv infection new organization pledges scientific expertise for viral outbreaks the global virus network: challenging chikungunya follicular dendritic cell disruption as a novel mechanism of virus-induced immunosuppression passive immunotherapy: assessment of convalescent serum against ebola virus makona infection in nonhuman primates the interaction between herpes simplex virus 1 tegument proteins ul51 and ul14 and its role in virion morphogenesis valosin-containing protein (vcp/p97) plays a role in the replication of west nile virus a tick-borne segmented rna virus contains genome segments derived from unsegmented viral ancestors deep panning: steps towards probing the igome feverish quest for ebola immunotherapy: straight or cocktail? how to turn competitors into collaborators comprehensive annotation of mature peptides and genotypes for zika virus relationship of the quaternary structure of human secretory iga to neutralization of influenza virus tp53 and pik3ca gene mutations in adenocarcinoma, squamous cell carcinoma and high-grade intraepithelial neoplasia of the cervix cryo-electron microscopy structures of chimeric hemagglutinin displayed on a universal influenza vaccine candidate zika virus: history, emergence, biology, and prospects for control profiling the igome: meeting the challenge reducing the global burden of htlv-1 infection: an agenda for research and action we thank the participants who presented their data at the 2016 international gvn meeting. we thank the japanese society of virology and hokkaido university organizers for their outstanding management of the meeting and related events, and the japanese hosts: hokkaido university's research center for zoonosis control, the global institution for collaborative research and education (gi-core) global station for zoonosis control (gsz) and the national institute of infectious diseases (niid). support also was provided by numerous sponsors who are noted at http://gvn.org/sapporo_2016. key: cord-275859-ix8du1er authors: mouzakis, kathryn d.; lang, andrew l.; vander meulen, kirk a.; easterday, preston d.; butcher, samuel e. title: hiv-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mrna entrance channel of the ribosome date: 2012-12-15 journal: nucleic acids res doi: 10.1093/nar/gks1254 sha: doc_id: 275859 cord_uid: ix8du1er the human immunodeficiency virus (hiv) requires a programmed −1 ribosomal frameshift for pol gene expression. the hiv frameshift site consists of a heptanucleotide slippery sequence (uuuuuua) followed by a spacer region and a downstream rna stem–loop structure. here we investigate the role of the rna structure in promoting the −1 frameshift. the stem–loop was systematically altered to decouple the contributions of local and overall thermodynamic stability towards frameshift efficiency. no correlation between overall stability and frameshift efficiency is observed. in contrast, there is a strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3–4 bp in the stem–loop, which are predicted to reside at the opening of the mrna entrance channel when the ribosome is paused at the slippery site. insertion or deletions in the spacer region appear to correspondingly change the identity of the base pairs encountered 8 nt downstream of the slippery site. finally, the role of the surrounding genomic secondary structure was investigated and found to have a modest impact on frameshift efficiency, consistent with the hypothesis that the genomic secondary structure attenuates frameshifting by affecting the overall rate of translation. translation is a high-fidelity process in all organisms. failure to maintain reading frame typically results in incorrect protein synthesis and/or early termination. however, a programmed change in reading frame can result in the translation of new proteins, thereby maximizing genomic coding capacity. many retroviruses, including human immunodeficiency virus type 1 (hiv-1) (1) , and some coronaviruses, such as severe acute respiratory syndrome (2) and infectious bronchitis virus (ibv) (3), use a programmed à1 ribosomal frameshift (à1 prf) to control translation levels of their enzymatic proteins (4) (5) (6) (7) . in the retroviruses, the à1 prf site lies between the gag and pol open reading frames (orfs), with pol in the à1 reading frame relative to gag. the gag orf encodes the viral structural proteins, whereas the pol orf encodes the enzymatic proteins. during translation of hiv-1 mrna, the majority of ribosomes terminate at a stop codon at the end of the gag orf, producing the gag polyprotein (2, 8) . however, the hiv à1 prf induces $5% of ribosomes to shift into the à1 reading frame, thus producing the gag-pol polyprotein (1, (9) (10) (11) . the 5% frameshift efficiency determines the ratio of viral proteins produced and is important for viral replication and infectivity (10, (12) (13) (14) (15) . a decrease in frameshift efficiency can inhibit viral replication (16, 17) . the hiv-1 frameshift site is composed of a heptanucleotide slippery sequence (uuuuuua) followed by a downstream rna stem-loop ( figure 1a ). the slippery sequence follows a general xxxyyyz consensus sequence, where x can be any nucleotide (nt) type, y can be a or u and z is not g in eukaryotes (15, 18) . this sequence allows near-cognate and cognate re-pairing of the a-and p-site trna anticodons, respectively, in the à1 reading frame. hiv-1's slippery sequence is especially 'slippery', and in the absence of a downstream structure increases the basal level of ribosomal frameshifting from $0.0001% to 0.1% per codon (9, 19, 20) . however, in order to further stimulate frameshifting to the levels required for viral replication, the slippery site must be followed by a stable rna structure (9, (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (figure 1a) . thus, frameshifting is achieved by the cis coupling of the slippery site and downstream structure (1, (9) (10) (11) 21) . multiple models have been proposed to explain the frameshift mechanism (1, 6, (31) (32) (33) (34) (35) (36) (37) (38) . common among them are the following steps: (i) during translation, the ribosome pauses when the slippery sequence (uuu uua in the 0 frame) is engaged in the ribosomal a-and p-sites (4, 18, 22, 39) . the pause is triggered by the downstream structure's resistance to unwinding. (ii) while paused, $5% of ribosomes slip 1 nt in the 5 0 -direction and continue elongation in the à1 reading frame. the proposed models are differentiated by the exact step at which the frameshift occurs: during aminoacylated-trna accommodation (31) , after accommodation, but before peptidyl transfer (1), after large subunit translocation (5) or after peptidyl transfer due to an incomplete translocation (37) . alternatively, the 'many pathways model' of à1 prf suggests that frameshift efficiency is the sum of frameshift events occurring, each of which could occur at these different points in elongation (36) . an important role of the downstream structure is to induce ribosomal pausing on the slippery sequence, which is necessary but not sufficient to promote efficient levels of frameshifting (40) (41) (42) . interestingly, the pause length does not appear to correlate with frameshift efficiency (40) . interactions with the translational machinery have also been hypothesized to contribute to frameshifting (5, 23, 31, 32, 43) . previous studies have observed general trends between hiv-1 stem-loop thermodynamic stability and frameshift efficiency (30, 44, 45) . however, a quantitative correlation between thermodynamic stability and frameshift efficiency has not been described, and the role of individual base pairs has not been systematically investigated. for frameshift sites with a downstream pseudoknot structure, mechanical stability has been proposed to be a determinant of frameshift efficiency (46) (47) (48) . it has been hypothesized that mechanical tension lowers the energy barrier for frameshifting (5) , where the amount of tension sensed by the ribosome is proportional to the mechanical stability of the translocation barrier (46, 47, (49) (50) (51) . however, a recent study found no correlation between pseudoknot mechanical stability and frameshift efficiency, but instead observed a correlation between frameshifting and the ability to form alternative structures (52) . other factors can modulate the frameshift efficiency, such as translation initiation rates (37, 53) . increased translation initiation rates lead to increased polysome density, which can cause ribosomes to stack at the frameshift site. this in turn affects the rate of mrna refolding during translation and leads to a decrease in overall frameshift efficiency (37, 53) . ribosome stacking can be promoted by rna structure that precedes the frameshift site. studies examining the secondary structure of the hiv-1 genomic rna within capsids have revealed that the frameshift site is part of a conserved three-helix junction (3hj) (54, 55) . it has been hypothesized that the role of this secondary structure is to decrease the rate of translation (54) , which may affect frameshifting by facilitating pausing and inducing ribosome stacking. here, we investigate the role of the hiv-1 rna structure in frameshifting, focusing on elucidating the relationships between frameshift efficiency and (i) the downstream rna stem-loop thermodynamic stability, (ii) spacer length and (iii) surrounding genomic secondary structure. by systematically altering the base pair composition of the stem-loop, we dissect the contributions of global and local thermodynamic stability on frameshifting. these data reveal that the thermodynamic stability of the first 3-4 bp in the stem-loop is a primary determinant of frameshift efficiency. our data further indicate that the base pairs important for frameshifting are located at a distance of 8 nt from the slippery site, which corresponds to the length of the spacer and is consistent with a structural model of the ribosome paused at the frameshift site. finally, we find that the conserved genomic rna secondary structure serves to attenuate the frameshift efficiency, likely by affecting the overall rate of translation. importantly, our study describes the first quantitative and predictive model for frameshift inducing stem-loops, which can be generally applied to many à1 prf viral systems. dna templates used for the dual-luciferase frameshift assay were cloned into a p2luc vector between the rluc and fluc reporter genes. briefly, complementary synthetic oligonucleotides [integrated dna technologies (idt), inc.] with bamh i and sac i compatible ends were cloned into the p2luc vector using the bamh i and sac i sites between the rluc and fluc reporter genes. oligonucleotides comprising the template sequences (supplementary table s1 ) and their complements were phosphorylated, annealed and ligated into the p2luc vector to produce the experimental constructs. this places the fluc gene in the à1 reading frame relative to rluc; analogous to the orientation of the gag and pol genes in the hiv-1 genome. for the spacer mutation constructs (ms13-17), a compensatory number of nts were added or removed downstream of the frameshift site to maintain the appropriate reading frame of the downstream reporter gene. the 'wild-type' (wt) sequence utilized here corresponds to the most frequently occurring sequence found in hiv-1 group m subtype b nl4-3 laboratory strain (56) . positive control sequences and their complements were also cloned into the p2luc vector and have two thymidine residues (supplementary table s1 , bold) in the slippery sequence (supplementary table s1 , underlined) replaced with cytidines, and an additional nt inserted immediately before the sac i complementary sequence (gagct), which places the rluc and fluc genes in-frame. in all constructs, a pml i restriction site was included at the end of the template to allow for run-off transcription after digestion with the pml i enzyme (neb). resultant products were transformed into escherichia coli competent cells (dh5a). plasmid dna was purified from cell cultures (qiagen) and the sequences of all constructs were verified (university of wisconsin-madison biotechnology center). microgram quantities of rna for the frameshift assay were transcribed in vitro using linearized p2luc plasmid dna, purified his6-tagged t7 rna polymerase (10â), 11.25 mm ntps and two units of rnasin plus rnase inhibitor (promega), in 200 ml for 90 min at 37 c. pyrophosphate was pelleted by centrifugation (10 min, 13 200 rpm, room temperature) and rna was phenol/ chloroform extracted. unincorporated ntps and salt were separated from the rna using size-exclusion chromatography [two econo-pac p6 cartridges (bio-rad) in series]. monomeric rna folding was achieved by denaturation at 95 c for 5 min followed by incubation on ice for 30 min. rnas were lyophilized to dryness and resuspended in water to a concentration of 1 mg/ml and stored in aliquots ($25 ml) at à80 c. rna integrity and purity were checked with 1% agarose gel electrophoresis. finally, rnas used for uv spectroscopy were purchased from idt. in vitro frameshift assays were completed with each rna reporter (experimental and positive control) using a rabbit reticulocyte lysate (rrl) system (promega, nuclease treated, l416a). differences from our previously described protocol (56) include the following: translation reactions contained 1.25 mg rna, 10 units of rnasin plus rnase inhibitor (promega, n2615), and 8.75 ml of rrl in 12.5 ml. following a 90-min incubation at 37 c, reactions were quenched with the addition of 0.5 ml 0.156 m edta ph 8.0 (6 mm final), as described previously (28) . for each reporter, a minimum of three independent frameshift assays were completed. each independent assay included six replicate reactions. luminescence was measured using the dual-luciferase reporter assay (promega) as previously described (57) . readings were taken with a veritas microplate luminometer equipped with dual-injectors (turner bio-systems) for 10 s after 25 ml of the respective substrate was injected into the reaction mixture (2-s lag time prior to measurement). ratios of firefly/renilla luminescence were calculated for each of the experimental and control translation reactions. the frameshift efficiency was calculated by taking the ratio of the experimental/control luminescence (firefly/renilla). frameshift efficiencies were averaged and their standard deviations were propagated through to yield a standard error of the mean (sem). for the wt and a subset of the mutant stem-loop (ms) rnas (supplementary table s2 and figure 2 ), rna overall thermodynamic stability, ág global , was measured using uv absorbance at 260 nm as a function of temperature with a cary model 400 bio uv-visible spectrophotometer equipped with a peltier heating accessory and temperature probe. all samples contained 10 mm potassium phosphate buffer, ph 7.0, 2 mm rna, in a volume of 1 ml. for rnas that were too stable to measure ág global under these conditions, urea was added to 4, 6 and 8 m, and the ág global was deduced by extrapolating to 0 m urea as described below. prior to data collection, samples were heated from 20 c to 95 c, at 10 c/min, held at 95 c for 5 min and cooled from 95 c back to 20 c at the same rate to ensure homogenous folding. samples were heated at 1 c/min from 20 c to 95 c. identical traces were obtained by cooling, indicating a lack of hysteresis. a 260 data were collected in 0.5-min intervals and raw data were baseline corrected by subtraction of a 320 values at each temperature. the average hyperchromicity [equation (1) ] and temperature were calculated from four curves and the sem was determined for each average. for rna with a single melting transition, the average hyperchromicity can be fit by equation (2) to measure áh and t m . here, a f and a u are the temperature-dependent a 260 of the folded and unfolded forms of the rna, determined to be linear functions of the temperature. k is given by equation (3), where t is the desired temperature (kelvin, 310k for our calculations) for the ág calculation, and r is the gas constant in units of kcal/(mol â k). with an average t m and áh extrapolated using equation (2), assuming a ácp of zero, the ág global can be calculated using equation (4) . error in ág global is calculated using standard propagation of error (supplementary 'materials and methods' section). average hyperchromicity data for each rna were fit using equation (2) and overall thermodynamic stabilities were calculated using equation (4) (prism 4.3, graphpad). for rnas with melting temperatures approaching or >95 c, a linear extrapolation of ág global versus urea concentration was applied to determine the ág global at 0 m urea (supplementary figure s1) (58) . for all other rnas, determination of ág global at standard buffer conditions was sufficient to produce minimal error in ág global . all the data used to calculate the reported ág global values were established using a minimum of three independently prepared samples at each buffer condition. a small and non-cooperative transition was observed in the 40-50 c range for rnas with less stable terminal base pairs (wt, ms2, ms6, ms7, ms10 and ms12). this transition was not present for rnas with very stable terminal base pairs (e.g. ms1 and ms5) and can be attributed to helical fraying. for all rnas, only the major cooperative unfolding transitions were used in data fitting. starting values were determined by examining the first derivative plots for each set of averages. local stabilities, ág local , for base pairs were calculated using nearest-neighbor parameters at 1 m nacl, 37 c (59-61) (supplementary table s2 ). frameshift efficiency was plotted as a function of overall and local thermodynamic stability. overall and local thermodynamic stabilities were predicted at 1 m nacl. data were fit to a one-phase exponential decay function [equation (5)] (prism 4.3, graphpad). here, the amplitude, k and plateau are variables and x o is used to offset the exponential fit. x o was set to the most negative x value in the data set. errors were determined using a 95% confidence interval. modeling the hiv-1 frameshift site onto the eukaryotic ribosome we utilized a well-established dual-luciferase frameshift assay (57, 67, 68) to quantitatively measure frameshift efficiency in rrl. the sequence of the frameshift site stemloop was varied to dissect the relative contributions of local and overall rna stability on hiv-1 frameshift efficiency. we hypothesized that once the ribosome is paused on the slippery sequence, the thermodynamic stability of the base pairs encountered at the base of the stem-loop should be a critical determinant for frameshifting. after the ribosome has completed one translocation step, it moves away from the slippery site and the reading frame is established. therefore, we further hypothesized that downstream base pairs in the stem-loop should have a much lower impact on frameshifting. to test this hypothesis, 12 ms constructs ( figure 1b) were created using nearest-neighbor parameters (59) (60) (61) 69) to systematically alter the stability of different regions of the stem-loop. we define local stability (ág local ) as the thermodynamic stability of base pairs directly downstream of the spacer ( figure 1a) , as determined by their nearest-neighbor interactions (59) (60) (61) . global stability (ág global ) is defined as the overall thermodynamic stability of the stem-loop. the thermodynamic stabilities (ág global ) were experimentally determined for a subset or rnas (wt, ms1-2, ms5-7, ms10 and ms12) using uv-monitored thermal denaturation (figure 2, supplementary figure s1 and supplementary table s2 ). owing to the extreme stabilities of the structures (25), thermal denaturation curves were measured at low ionic strength (10 mm potassium phosphate buffer) in the presence of varying concentrations of urea and extrapolated back to 0 m urea (58) . results followed the same trend as those predicted from nearest-neighbor parameters (59-61,69) (r 2 = 0.95) ( figure 2 ). as expected, the measured stabilities were lower than the predicted values at 1 m nacl (70) . upon correction of the experimental values to 1 m monovalent ionic strength (71), we observe an excellent agreement (r 2 = 0.95) between experimental and predicted free energies ( figure 2) . indeed, free energy prediction is robust for small, stable rnas with no competing suboptimal folds (20) . frameshift efficiencies for the different rna constructs were measured (figure 3 and table 1 ). increases in the local stability of the first 3 bp resulted in significant increases in frameshift efficiency ( figure 3a, ms1-5 ). in contrast, sequence changes that significantly lowered the local stability of the first 3 bp resulted in decreased frameshift efficiencies ( figure 3a , ms10-12 and table 1 ). no correlation between frameshift efficiency and overall thermodynamic stability is observed ( figure 3b ). instead, we observe a strong correlation (r 2 = 0.88) between frameshift efficiency and local stability of the first 3 bp at the base of the stem-loop using a one-phase exponential decay function ( figure 3c and supplementary table s3 ). the frameshift efficiency for each variant frameshift site can be predicted using the parameters derived from the correlation, and each predicted frameshift efficiency falls within 1 sd of its measured value (data not shown). these results support the hypothesis that the stability of the base pairs at the base of the stem-loop is a primary determinant of frameshift efficiency. extremely stable rna structures can promote longterm ribosomal stalling or 'roadblocking', (35, 72) . in the dual-luciferase assay, roadblocking would result in decreased translation levels of the downstream firefly luciferase reporter gene product. however, the dual-luciferase assay controls for this, as frameshift efficiencies are normalized relative to in-frame control constructs (57, 67, 68) . nevertheless, we asked if differential degrees of roadblocking might occur for our various constructs. the luminescence data reveal a consistent ratio of firefly/renilla activity (data not shown) for all constructs (table 1) . these values were calculated using the luminescence data from the positive control dual-luciferase constructs, where the renilla and firefly genes are in frame and the slippery site is mutated such that the ribosome cannot frameshift. the consistency in the relative expression levels of the reporter genes indicates that roadblocking, if occurring, is uniform for all constructs. it has been hypothesized that during frameshifting, the mechanical force of translocation causes a build-up of tension that is transmitted through the spacer region ( figure 1a ) and sensed at the anticodon-codon level (5, 49) . we therefore investigated the influence of nt deletions and insertion in the spacer region ( figure 4 ). the wt construct was compared to a version with an adenosine insertion that increases the spacer length by 1 nt (ms13) (figure 4) . additionally, we created spacers with a single nt deletion (ms14) and 2-nt deletions (ms15-17) (figure 4 ). the resulting frameshift efficiencies were measured ( figure 4d ). interestingly, ms15 shows a large increase in frameshift efficiency. this cannot be due to the 2-nt deletion, since ms16 and ms17 have the same spacers yet display wt-levels of frameshifting. we hypothesized that the 2-nt deletion in the spacer of ms15 increased frameshift efficiency by altering the base pairs in the stem-loop encountered by the ribosome during frameshifting. in other words, by deleting 2 nt in the spacer, a ribosome footprint may extend 2 nt further into the stem. in support of this hypothesis, a very stable set of base pairs are located 2 nt from the base of the stem (5 0 -ggc-3 0 /5 0 -gcc-3 0 ). in ms16 and ms17, we replaced these base pairs with the less stable base pairs normally found at the base of the stem (5 0 -cug-3 0 /5 0 -cag-3 0 ) ( figure 4c ). indeed, when these changes are made, the frameshifting efficiency is indistinguishable from wt, despite the apparent 2-nt spacer deletion ( figure 4d) . interestingly, the overall stability of ms17 is increased relative to ms16 ( figure 4c ), yet the frameshift efficiency is unaltered ( figure 4d ). these data indicate that changes in the spacer region correspondingly alter the base pairs encountered by the ribosome when it is engaged with the slippery site. when plotting the data from all 18 rna constructs studied (including the ms13-17 rnas) as a function of overall rna stability, no correlation is observed ( figure 4e ). however, we observe a strong correlation between frameshifting and the thermodynamic stability of the first 3-4 bp 8 nt downstream of the slippery site ( figure 4f and g) . conversely, the correlations grow considerably weaker as more base pairs are considered in the analysis (supplementary figure s2) . likewise, no correlation is observed between local stability of base pairs at the top of the stem-loop and frameshift efficiency (supplementary figure s2l) . the observed correlations are exponential functions with baselines of 2-4% frameshifting, which correspond to the lowest observed frameshift efficiencies in the presence of a stem-loop secondary structure downstream of the slippery site. the stem-loop is flanked by a 5 0 -u and 3 0 -g ( figure 1a) , that could potentially form a u-g wobble at the base of the stem. inclusion of this wobble pair in the local stability term produces consistently weaker correlations between frameshift efficiency and local stability (supplementary figure s3) . to further address whether or not this u-g wobble pair can form during frameshifting, we modeled the frameshift site stem-loop and spacer onto the eukaryotic ribosome ( figure 5 ). the spacer was connected to the terminal nt in the a-site, to recapitulate the position of the ribosome when it is engaged on the slippery sequence in the 0 reading frame. the model indicates that the minimal spacer distance between the slippery site and the stem-loop is 7 nt; however, formation of a u-g wobble at the base of the stem is blocked by steric clash with the ribosomal s3 protein ( figure 5b and d). therefore, experimental data and structural modeling support an hiv-1 frameshift site spacer length of 8 nt. within viral capsids, the hiv-1 frameshift site rna is part of a conserved 3hj secondary structure ( figure 6a ) (54, 55) . it has been hypothesized that the role of this secondary structure is to slow down the rate of translation (54) , which in turn may modulate frameshift efficiency. we therefore compared the 3hj secondary structure (3hj wt) to a similar construct with mutations designed to disrupt secondary structure formation in the p1 and p2 helices ( figure 6b , 3hj mut). we observe a significant decrease in frameshift efficiency, from 4.6 ± 0.5% to 2.5 ± 0.4%, when the 3hj secondary structure is present ( figure 6c and table 1 , compare 3hj wt to 3hj mut). as expected, there is no significant difference between the observed frameshifting efficiencies of the 3hj mut and the wt construct used above (4.7 ± 0.5 and 4.6 ± 0.5, respectively). the observed frameshift efficiencies for our 3hj wt and wt reporter constructs in rrl both fall within the range of previously measured frameshifting efficiencies for hiv-1 in vivo, which range from 2% to 5% (9, 15, 21, 24 ). next, we tested the local stability hypothesis in the context of the 3hj secondary structure by increasing the local stability of 2 bp in the p3 helix ( figure 6a ). the 2-bp change (3hj ms1) results in a large, 5-fold increase in frameshift efficiency ( figure 6c and table 1 ). this increase is similar to the 4-fold difference between the ms1 and wt rnas ( figure 3a and table 1 ). we conclude that the 3hj secondary structure indirectly modulates frameshifting, likely by altering the kinetics of translation, as previously hypothesized (54) . this effect must happen prior to frameshifting, as the ribosome disrupts the 3hj secondary structure as it encounters the slippery site. once the ribosome is engaged with the slippery site, local stability is the primary determinant of frameshifting efficiency, as illustrated by comparison of 3hj wt to 3hj ms1 ( figure 5c ). in this work, we report a strong correlation between the thermodynamic stability of the first 3-4 bp at the base of the stem-loop and frameshift efficiency in hiv-1. we therefore hypothesize that the frameshift mechanism involves a thermodynamic block to translocation, determined by the local stability of base pairs positioned directly at the mrna entrance channel. this is in agreement with previous studies investigating antisense-induced frameshifting using either mixed locked nucleic acid/dna (73) or morpholino/rna (38) oligonucleotides. when these oligonucleotides were used to direct antisenseinduced frameshifting, the local stability of the duplex was also critical to frameshift stimulation (38, 73) . in light of the local stability hypothesis, we can re-examine data from prior studies that investigated trends between thermodynamic stability of the hiv-1 stem-loop and frameshift efficiency (30, 44, 45) . indeed, we find that these results are generally consistent with local stability being the primary determinant in frameshift efficiency. for example, bidou et al. mutations in the frameshift site that arise in response to cytotoxic t-cell escape (74) and protease inhibitor resistance (75, 76) are also consistent with our results. prado et al. (74) investigated the frameshift efficiency of four hiv-1 strains with mutations in the frameshift site. in this study, the only mutation that produced a significant change (decrease) in frameshift efficiency was one with a decreased local stability due to disruption of the first base pair in the stem-loop. nijhuis et al. if the local stability of 3-4 bp is sufficient to determine frameshift efficiency, why does the hiv-1 frameshift site stem-loop contain 11 watson-crick base pairs? we can see two possible explanations for this. first, the additional base pairs ensure that the stem-loop has a high probability of folding and cannot be out-competed by suboptimal folds, which can severely impact hiv replication (77) . in the >9100-nt genome, there are a total of only 11 helices that are equal or larger in size (54) . second, the additional base pairs serve to cooperatively stabilize the base pairs at the base of the stem. these effects may explain why severely truncated constructs produce lower frameshift efficiencies compared to stem-loops with identical local stability (ms9 versus wt, ms16 versus ms17). cooperative stabilization of local stability may also explain why severe truncations of a hairpin downstream of the simian retrovirus type-1 (srv-1) slippery site result in lower frameshift efficiency (78) . the observed frameshift efficiencies for the 3hj wt and wt reporter constructs in rrl both fall within the range of previously measured frameshifting efficiencies for hiv-1 in vivo, which range from 2% to 5% (9, 15, 21, 24) . the wide range of observed frameshifting efficiencies in vivo is likely influenced by viral and cellular factors, for example, modulation of translation initiation by the hiv-1 tar rna structure (79) and polysome density (53) . we find that the conserved 3hj secondary structure in the hiv-1 genomic rna (54) causes a significant decrease in frameshift efficiency ( figure 6 ). this observation is consistent with the previous hypothesis that the 3hj secondary structure induces ribosomal pausing (54) . pausing at the upstream secondary structure may promote stacking of consecutive ribosomes (80) , promoting a net decrease in frameshift efficiency because the mrna would have less time to refold between ribosomes. our data support this model and also indicate that local stability has a far greater impact on frameshift efficiency ( figure 6 ). prokaryotic ribosomes use two active mechanisms during translation to unwind rna (49) . in the first mechanism, the ribosomal helicase activity raises the free energy of an encountered base pair by +0.9 kcal/mol (49) . this destabilizes the base pair, which can then be opened by the mechanical force generated by translocation. if the base pair is resistant to this force, tension may be created which is sensed at the codon-anticodon base pairs (31, 32, 43, 49, 51) . because g-c pairs require more force for unwinding (49) , the tension sensed in the decoding center would be proportional to the local rna stability (51) . the mechanical tension may either cause the trnas to slip 1 nt in the 5 0 -direction (5, 31, 32, 36, 49) or, alternatively, cause the ribosome to translocate incompletely by 2 nt instead of 3 nt (37), which would also result in a à1 frameshift. when the hiv-1 frameshift site is modeled onto the eukaryotic ribosome, base pairs critical for frameshifting are positioned at the entrance to the mrna entry channel ( figure 5b and d), in agreement with chemical probing and toeprinting results with a prokaryotic ribosome stalled on the hiv-1 frameshift site (23) . interestingly, the decoding center and the mrna channel are highly conserved between prokaryotes and eukaryotes (65) and a bacterial translation system produces similar levels and changes in frameshift efficiency in response to changes in the hiv-1 stem-loop sequence (11) . our data support an 8-nt spacer length between the slippery site and the stem-loop, as the effect of deletions in the spacer correlates with corresponding changes in stem-loop local stability 8-nt downstream of the slippery site ( figure 4 ). consistent with this idea, deletion of 1 nt in the spacer region of the beet western yellow virus (bwyv) à1 prf site promotes the melting of the first base pair in the downstream structure (81) . if the mrna channel length is maintained, it follows that spacer lengths in all à1 prf sites should be !7 nt in length. yet, some frameshift sites have been drawn with 5-to 6-nt spacers [reviewed in (4, 5) ], including that of bwyv (81) . our data suggest that these frameshift site structures may be partially unwound at the time of frameshifting, in order to accommodate the requisite spacer length and positioning of the slippery sequence in the ribosomal decoding center. unfortunately, there are currently no high-resolution structural views of ribosomes engaged with frameshift site structures. in conjunction with functional studies such as the one presented here, high-resolution structural views will ultimately be required to define the frameshifting mechanism. prior studies have observed relationships between spacer length and à1 prf efficiency in various systems (22, 39, 82, 83) and are consistent with a spacer length of 7-8 nt and local stability being the primary determinant in frameshift efficiency. spacer lengths of 6-8 nt produced the highest level of à1 prf for the antisense oligonucleotides, stem-loop and pseudoknot stimulatory structures (82) . in agreement with our conclusions, these spacers position base pairs with strong local stabilities at the entrance to the mrna channel. hiv-1 group m subtype b is the dominant form of hiv-1 in north and south america, europe, japan, thailand and australia. the less common non-b subtypes (a, c, d, e, f, g, h, j and k) have decreased local stabilities; for example, a frequent c to u mutation in the first base pair of the stem-loop in these subtypes results in formation of a u-g wobble pair in place of a c-g (28, 84) . interestingly, the exponential relationship we observe predicts that such a change would have little effect on frameshift efficiency. for instance, mutants ms10-12 all incorporate u-g wobble pairs at these positions, which significantly destabilize the local stability by +5.5 kcal/mol relative to wt ( figure 1 ). nevertheless, these mutations result in near wt frameshift efficiencies ( figure 3a ), owing to the exponential relationship between local stability and frameshift efficiency (figures 3c, 4f and g). these results are consistent with the observed frameshift efficiencies of the less common subtypes (28) . finally, a randomized trial of hiv patients receiving protease inhibitor therapy examined mutations in the gag-pol frameshift site and found no relationship between overall stability of the stem-loop and virological response (85) . this observation is consistent with our results that show no correlation between overall stability and frameshifting (figures 3b and 4e) . characterization of ribosomal frameshifting in hiv-1 gag-pol expression programmed ribosomal frameshifting in hiv-1 and the sars-cov an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv ribosomal frameshifting on viral rnas frameshifting rna pseudoknots: structure and mechanism programmed translational frameshifting rna pseudoknots and the regulation of protein synthesis structure and function of the stimulatory rnas involved in programmed eukaryotic-1 ribosomal frameshifting a heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1 a reassessment of the response of the bacterial ribosome to the frameshift stimulatory signal of the human immunodeficiency virus type 1 importance of ribosomal frameshifting for human immunodeficiency virus type 1 particle assembly and replication characterization of human immunodeficiency virus type-1 (hiv-1) particles that express protease-reverse transcriptase fusion proteins maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type 1 rna dimerization and viral infectivity the human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy overexpression of the hiv-1 gag-pol polyprotein results in intracellular activation of hiv-1 protease and inhibition of assembly and budding of virus-like particles overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production mutational analysis of the ''slippery-sequence'' component of a coronavirus ribosomal frameshifting signal comparative mutational analysis of cis-acting rna signals for translational frameshifting in hiv-1 and htlv-2 ribosome structure: revisiting the connection between translational accuracy and unconventional decoding translational frameshifting at the gag-pol junction of human immunodeficiency virus type 1 is not increased in infected t-lymphoid cells the sequences of and distance between two cis-acting signals determine the efficiency of ribosomal frameshifting in human immunodeficiency virus type 1 and human t-cell leukemia virus type ii in vivo interaction of the hiv-1 frameshift signal with the ribosome characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1 solution structure and thermodynamic investigation of the hiv-1 frameshift inducing element solution structure of the hiv-1 frameshift inducing stem-loop rna structure of the rna signal essential for translational frameshifting in hiv-1 efficiency of a programmed -1 ribosomal frameshift in the different subtypes of the human immunodeficiency virus type 1 group m human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mrna secondary structure: demonstration by expression in vivo in vivo hiv-1 frameshifting efficiency is directly related to the stability of the stem-loop stimulatory signal the 9-a solution: how mrna pseudoknots promote efficient programmed -1 ribosomal frameshifting a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting the three transfer rnas occupying the a, p and e sites on the ribosome are involved in viral programmed -1 ribosomal frameshift the mechanics of translocation: a molecular ''spring-and-ratchet'' system achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins the many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed à1 ribosomal frameshifting targeting frameshifting in the human immunodeficiency virus mechanisms and implications of programmed translational frameshifting characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot ribosomal pausing at a frameshifter rna pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency ribosomal movement impeded at a pseudoknot required for frameshifting ribosomal pausing during translation of an rna pseudoknot torsional restraint: a new twist on frameshifting pseudoknots analysis of natural variants of the human immunodeficiency virus type 1 gag-pol frameshift stem-loop structure proline residues within spacer peptide p1 are important for human immunodeficiency virus type 1 infectivity, protein processing, and genomic rna dimer stability correlation between mechanical strength of messenger rna pseudoknots and ribosomal frameshifting triplex structures in an rna pseudoknot enhance mechanical stability and increase efficiency of -1 ribosomal frameshifting rna reactions one molecule at a time the ribosome uses two active mechanisms to unwind messenger rna during translation characterization of the mechanical unfolding of rna pseudoknots predicting ribosomal frameshifting efficiency programmed -1 frameshifting efficiency correlates with rna pseudoknot conformational plasticity, not resistance to mechanical unfolding the presence of the tar rna structure alters the programmed -1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (hiv-1) by modifying the rate of translation initiation architecture and secondary structure of an entire hiv-1 rna genome high-throughput shape analysis reveals structures in hiv-1 genomic rna strongly conserved across distinct biological states selection and characterization of small molecules that bind the hiv-1 frameshift site rna programmed ribosomal frameshifting in siv is induced by a highly structured rna stem-loop urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, a-chymotrypsin, and b-lactoglobulin thermodynamic parameters for an expanded nearest-neighbor model for formation of rna duplexes with watson-crick base pairs expanded sequence dependence of thermodynamic parameters improves prediction of rna secondary structure testing the nearest neighbor model for canonical rna base pairs: revision of gu parameters structural aspects of messenger rna reading frame maintenance by the ribosome the structure of the eukaryotic ribosome at 3.0 å resolution the path of messenger rna through the ribosome one core, two shells: bacterial and eukaryotic ribosomes crystal structure of the eukaryotic ribosome a dual-luciferase reporter system for studying recoding signals an in vivo dual-luciferase assay system for studying translational recoding in the yeast saccharomyces cerevisiae rnastructure: software for rna secondary structure prediction and analysis rna secondary structure prediction a unified view of polymer, dumbbell, and oligonucleotide dna nearest-neighbor thermodynamics 2012) mrna pseudoknot structures can act as ribosomal roadblocks stimulation of ribosomal frameshifting by antisense lna functional consequences of human immunodeficiency virus escape from an hla-b*13-restricted cd8+ t-cell epitope in p1 gag protein a novel substrate-based hiv-1 protease inhibitor drug resistance mechanism mutational patterns in the frameshift-regulating site of hiv-1 selected by protease inhibitors opening of the tar hairpin in the hiv-1 genome causes aberrant rna dimerization and packaging stem-loop structures can effectively substitute for an rna pseudoknot in à1 ribosomal frameshifting the 5 0 utr of hiv-1 full-length mrna and the tat viral protein modulate the programmed à1 ribosomal frameshift that generates hiv-1 enzymes ribosome pausing and stacking during translation of a eukaryotic mrna footprinting analysis of bwyv pseudoknot-ribosome complexes spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a u6a heptamer supports a role for messenger rna (mrna) tension in frameshifting identification and analysis of the gag-pol ribosomal frameshift site of feline immunodeficiency virus differential stability of the mrna secondary structures in the frameshift site of various hiv type 1 viruses gag mutations can impact virological response to dual-boosted protease inhibitor combinations in antiretroviral-naive hiv-infected patients the authors thank jordan burke, ashley richie and lauren michael for helpful discussions. they also thank raymond gesteland (university of utah) for the generous gift of the p2luc plasmid dna and prof. a.c. palmenberg and her laboratory (university of wisconsin-madison) for equipment use. conflict of interest statement. none declared. key: cord-284608-ba7wq52t authors: sias, catia; salichos, leonidas; lapa, daniele; del nonno, franca; baiocchini, andrea; capobianchi, maria rosaria; garbuglia, anna rosa title: alpha, beta, gamma human papillomaviruses (hpv) detection with a different sets of primers in oropharyngeal swabs, anal and cervical samples date: 2019-03-04 journal: virol j doi: 10.1186/s12985-019-1132-x sha: doc_id: 284608 cord_uid: ba7wq52t background: recent studies have shown a 13-fold increase of oropharyngeal cancer in the presence of hpv, while α-hpv detection seems to be rare in oral cavity in comparison to anal or cervical district, many novel β and γ types have been isolated in this anatomical site suggesting a wide tropism range. currently, there are no guidelines recommending hpv oral cavity screening as a mandatory test, and it remains unknown which hpv types should be included in hpv screening programs. our goal was to assess hpv prevalence in oropharyngeal, anal, and cervical swabs using different sets of primers,which are able to amplify α, β, γ hpv types. methods: we analysed the presence of hpv dna in oropharyngeal (n = 124), anal (n = 186), cervical specimens (n = 43) from hiv positive and negative patients using fap59/64 and my09/11 primers. all untyped strains were genetically characterized through pcr amplification and direct sequencing of partial l1 region, and the resulting sequences were classified through phylogenetic analysis. results: hpv prevalence was 20.9% in 124 oropharyngeal swab samples, including infections with multiple hpv types (5.6%). hpv prevalence in this anatomical site was significantly associated with serostatus: 63.3%in hiv positive and 36.3% in hiv negative patients (p < 0.05). unclassified types were detected in 6 specimens. in our analysis, we did not observe any difference in hpv (α, β, γ) prevalence between men and women. overall, β species were the most frequently detected 69.7%. when using anal swabs, for hiv positive patients, β genus prevalence was 1% and γ genus was 3.7% including 6 unclassified types. in cervical samples from 43 hiv positive women (18 hpv negative and 25 positive by my09/11 pcr), only one sample was positivite for β(1) species (2.4%) using fap primers. six of the untyped strains clustered with sequences from species 7, 9, 10, 8,12 of γ genus. four sequences remained unclassified. finally, β and γ hpv prevalence was significantly lower than their respective hpv prevalence as identified by the luminex system in all anatomical sites that were analyzed in previous studies. conclusion: this study provides new information about viral isolates present in oropharyngeal site and it will contribute to improve the monitoring of hpv infection. electronic supplementary material: the online version of this article (10.1186/s12985-019-1132-x) contains supplementary material, which is available to authorized users. human papillomavirus (hpv) persistent infections are considered the primary cause of ano-genital cancer, where greater than 99% of cervical cancer and more than 60% of anal cancer contain hpv dna [1, 2] . throughout the past years, several studies have suggested the link between hpv infection and other epithelial cancers, including cutaneous and oropharyngeal cancers. oropharyngeal cancers are often referred to as "head and neck cancer" (hnc), and mainly include squamous-cell carcinoma which occurs in the oral cavity, base of tongue, tonsils, adenoids pharynx and larynx. recent studies on hpv infection in oral exfoliated cells have shown that there is a 13-fold higher risk for oropharyngeal cancer in the presence of virus [3] [4] [5] . the highest prevalence (up to 50%) for these hpv associated cancers has been found in the tonsillar cancers sub-set [6] [7] [8] . since hpv16 presented a global prevalence in oropharyngeal squamous cell carcinoma (opscc) and oral squamous cell carcinoma (oscc) of 40.6 and 14.9% respectively [9] , iarc categorized this genotype as a risk factor with for the pathogenesis of both cancers [10] . additionally, other hpv types have also been linked with these cancers, including hpv18, 31, and 33 [11] including β and ɣ types [4] . furthermore, while α-hpv detection seems to be rare in oral cavity in comparison to anal or cervical districts [12] , many novel β and γ types have been isolated in oral cavity [13] suggesting a wide tropism range of these genera [14] . at the same time, different studies on hpv prevalence provided contradicting result for β and ɣ types not only in oropharyngeal site, but also in cervical and anal anatomical sites [14] [15] [16] [17] [18] [19] . to date, there are no guidelines recommending hpv oral cavity screening as a mandatory test, and no hpv dna test has been approved for hpv detection in oral cavity. in addition, it remains unknown which hpv types should be included in oral cavity screening. agalliou [4] highlighted the link between β, γ hpv types and oral cancers, assessing the presence of hpv types which are not generally detected with commercial assays in cervical cancer screening. even though different methods have been used to detect novel hpv types, the association between new β and γ hpv types and oral cavity cancer has not yet been established. in this study, we analyzed the presence of hpv dna in oral, anal, and cervical specimens collected from hiv positive and hiv negative individuals, living in the same geographic area (regione lazio) by using my09/11 [20, 21] fap59/64 primers [22] . these primers, both targeting highly conserved region within l1 orf, are considered broad range pv primers and allow the detection of the great majority of already known and officially recognized hpv types. my09/11 had been used in α hpv detection in cervical sites, where they showed good sensitivity in alpha hpv types amplification [21] , while fap primers officially recognized hpv from α-pv, β-pv, and ɣ-pv genera, and might detect potentially new types [22] [23] [24] [25] . this property is particularly relevant, since ɣ and β papillomaviruses have been already identified in several anatomic sites [14, 26] . granted that that hpv prevalence is significantly higher among hiv infected people and at multiple anatomic sites [26] [27] [28] [29] we included both hiv positive and hiv negative people in our study in order to also verify whether β and ɣ hpv positivity is influenced by host immune status. this is a retrospective study carried out at laboratory of virology inmi l spallanzani on residual oropharyngeal samples collected for respiratory virus detection, anal and cervical swabs collected for hpv testing in diagnostic routine. the hospital ethics committee approved the protocol. date of birth, date of swabs sampling, hiv serostatus were recorded from institutional database. in hiv positive patients, the most recent cd4 t cell count (± 1 month from the date of sample collection) and hiv rna viral load (± 1 month from the date of sample) with a detection limit of 40cp/ml (abbott molecular inc., des plaines, il, usa) were used to correlate clinical features and hpv positivity. oropharyngeal swabs -oropharyngeal samples were collected as following: the nylon-flocked tip was rotated 3-4-times against right and left buccal mucosa, palatine, tonsils, upper and lower pharynx area. the swab was then plunged and stirred in 1.5 ml dmem medium with streptomycin and ampicillin. specimens were refrigerated within 3-5 h after collection until processing. anal swabs -we retrospectively analysed 186 anal swabs previously tested by my09/11 primers and typed by clart hpv2 clinical array or sanger sequencing (see hpv detection and typing section, below). one hundred samples belonged to hiv positive women (50 hpv positive by my09/11 pcr and 50 negative by my09/11 pcr), 86 anal swabs were collected from men who have sex with men (msm). all msm specimen resulted positive in my09/11 pcr. cervical swabs -a total of 43 cervical swabs (18 hpv negative and 25 hpv positive, assigned by my09/11 pcr) were considered in this study. all samples belonged to hiv positive women. general characteristics of hiv infected patients are presented in additional file 1: table s1 . oropharyngeal swabs were removed from the medium which we divided in two parts: half part was used for detecting respiratory viruses panel (influenza-a, b, rsv, rhinovirus, coronaviruses, metapneumovirus, adenovirus) and the other half was employed in testing hpv dna, having been stored at − 80°c until use. before nucleic acid extraction, all specimens were pre-treated. briefly, 600 μl of clinical material was digested with 20 μl proteinase k solution (qiagen, hilden germany) and lysed with al lysis buffer (qiagen, hilden, germany) at 56°c for 10 min. nucleic acid extraction was done with a magnetic bead-based automated platform (qiasymphony, hilden, germany) in accordance with the manufacturer's instructions. nucleic acids were eluted in 60 μl of ave elution buffer (qiagen, hilden, germany). nucleic acid from anal and cervical swabs were extracted as described elsewhere [30, 31] . samples that were β-globin negative were excluded from the study [32, 33] . ten μl of eluted nucleic acids were employed for evaluating the presence of hpv types by using my09(5'cgtccmarrggawactgatc3') and my11(5'gcmcagggwcataayaatgg3') [20, 21] pcr and faststart dna polymerase (roche diagnostics gmbh, mannheim, germany). the pcr assay conditions were: 95°c for 5 min, then 39 cycles (denaturation 95°c/30 s, annealing 55°c/45 s, and extension 72°c/1 min). one last step for extension was employed at 72°c for 10 min. fifteen μl of the pcr products were mixed with 6 loading solution in 1.8% agarose gel electrophoresis stained by ethidium bromide and run for 30 min at 130 v. hpv genotyping of positive samples was conducted using genomica clinical hpv array (genomica, madrid, spain). clart hpv2 clinical array hpv is able to detect: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, all adequate samples were retested using fap 59 (5'taacwgtiggicayccwtatt3') and fap 64 (5'ccwatatcwvhcatitciccatc3') primers [22] able to detect α, β and γ hpv types. faststart dna polymerase were used in this assay condition: 5 min at 94°c, 40 cycles (denaturation 94°c/30 s, annealing 52°c /45 s, and extension 72°c 1 min) followed by a final extension step at 72°c for 7 min. pcr products were also run on agarose gel. positive samples with fap set of primers and those positive with my09/11 pcr protocol, but negative in genomica typing assay were purified and sequenced on the automated abi prism 3100 instrument, by using a bigdye terminator cycle sequencing kit (applied biosystems, warrington, uk). hpv sequences were aligned using mafft [34] using the l-ins-i method. after visual inspection using seaview [35] we removed sample sequence q1359 and q1017 were excluded from the alignment because they represented mixed infection and the sanger sequence interpretation was not optimal. reference sequences hm999999_hpv147 and gu129016_hpv148 were also excluded from the alignment. then, we further implemented gblocks [36] to remove ambiguous positions using the least stringent options. to infer a maximum likelihood tree, we implemented raxml [37] using a gtrcat model, with 100 bootstrap replicates. tree visualization was achieved using figtree [38] . finally, pairwise similarity matrices were constructed using blast. oropharyngeal swabs-a total of 124 oropharyngeal swabs with β-globin positive signal were considered in this study. hpv prevalence was 20.9% (26/124), including infections with multiple hpv types (7/124, 5.6%) ( table 1 ). my09/11 pcr gave positive results in 18 samples. however, a blast search against genbank indicated that 5 amplified fragments were identical to human sequences (5/124, 4%), thus true hpv positive rate was estimated at 10.5%. out of 13, three samples were identified as α and ten as β types. alpha-types were detected in two hiv positive patients (2/55, 3.6%), and in 1 hiv negative patient. hpv α-types, 16 and 70, were detected in hiv positive subjects (2/55, 3.6%) after being typed by clart hpv2 clinical array, while hpv13 was amplified in hiv negative subject (1/69, 1.4%); it was typed by sanger sequencing. all β hpv types belonged to β 2 species (table 1) , and hpv145 was the most frequent type (4/10), both in hiv positive and hiv negative patients; 20 samples were identified as positive by fap pcr. no specific amplicons were observed. seven fap-detected hpv types were found in samples that also gave positive results with my09/my11 pcr (table 1) . types β 1 were the most represented (n = 13), while one sample was identified as γ type (hpv132, γ 12 ). additionally, one mixed infection (q1017) and 5 untyped hpv strains were also detected. among β 1 species, the most frequent types were hpv5 (n = 5, 38.6%) and hpv19 (n = 5, 38.6%). three multiple infections harboured β 1 and β 2 types, whereas β 2 types and untyped strains were observed in 4 other multiple infections. no multiple infection harboured α with β or γ species (table 1) . overall, β species were most prevalent (n = 23/33, 69.7%). hpv distribution differed significantly when the number of hpv β, γ-types were considered in hiv positive and hiv negative subjects: 21 (21/33, 63.6%) vs 12 (12/33, 36 .3%) respectively (chi square test, p < 0.005). hiv positive people with hpv in oropharyngeal swabs showed a mean cd4 t cell count of 532.3 ± 275.6. among hiv positive patients with detectable hpv, 6/16 had no detectable hiv rna in plasma and the other patients (n = 10) showed a mean rna copies/ml169.0 ± 130.0. hpv prevalence rates were compared for statistical significance, using a chi squared test, in men and women both hiv positive and hiv negative. however, no difference was observed (chi square test, p > 0.05). anal swabs-eighty-six anal swabs from msm resulted positive by my09/11 pcr, and 100 anal swabs from women (50 hpv positive by my09/11 pcr and 50 negative by my09/11 pcr) were retested with fap primers. among msm anal swabs, 15 samples showed single hpv infection by clart array testing, and 71 patients harboured multiple hpv infection with at least one high risk (hr) type. in 61.2% of samples we observed a clinically evident anal pathology, including 7 subjects with anal intraepithelial neoplasia (ain) ii, 46 patients with ain i. among hpv positive women, 18 harboured a single infection (ain ii, n = 0; ain i, n = 5); atypical squamous cells of undetermined significance (asc-us), n = 0; normal, n = 13) and 31 samples showed hpv multiple infection, all with at least 1hr type (62%) (ain 2,n = 0; ain i, n = 9; normal cytology, n = 22). hpv type was undetermined in one sample. among single infection with ain i, one specimen was infected with a low risk (lr)type (hpv81). nineteen anal swab specimens were identified as hpv positive by fap primers detection (female anal swabs, n = 3; male anal swabs, n = 16) ( table 2 ). overall α-type strains were detected in 9 samples with fap primers which were not previously detected by my09/11 primers. all but one α-type strains were also detected with α mucosal hpv multiple infection typed with clart hpv2 clinical array which included at least one hr type. hpv β 2 types (hpv9 and hpv37) were found in two specimens co-infected with α hpv types; 2 specimens harboured γ 10 hpv types (hpv121 and hpv180), both associated with type α hpv multiple infections. all fap untyped strains (n = 6) were found in anal specimens infected by α types with at least one high hr type. considering cytological aspects, 12/19 samples, which resulted positive with fap primer pcr, had ain (i, ii) lesions, while 7/19 samples showed normal cytology (table 2) . among anal swabs from females (n = 100), only 3 samples (3.0%), previously tested my09/11 hpv positive, resulted hpv positive with fap primer (hpv32, α 1 species; hpv114, α 3 species; hpv90, α 14 species). no untyped hpv strains were observed among female samples. overall, β genus prevalence was 1% and γ genus was 3.7% including untyped isolates. mean of cd4 t cell count was 690.7 ± 343.9 cells/μl. overall 170/186 patients (91.4%) were under antiretroviral therapy (art). all patients without art had a cd4 t cell count > 400/ μl. current art use was not associated with risk of β and ɣ infection (chi square test, p > 0.05). cervical swabs-twelve specimens showed a single hpv infection, and 13 had multiple infections by clart hpv2 array; 17/25 samples harboured at least 1 hr genotype. cytology findings were available for 20 specimens already resulted positive by clart hpv2 clinical array: 10 had normal cytology, 6 were asc-us or low grade squamous intraepithelial lesion (lsil), 3 harboured hpv single infections, 3 showed hpv multiple infections, and 4 were high grade squamous intraepithelial lesion (hsil). all my09/11 hpv negative women (n = 18) showed normal cytological findings. eleven/43 cervical samples resulted fap positive (25.6%). however, only 6 hpv types were not previously detected by my09/11 primers. five samples harboured α types (hpv90, n = 3 α 14 ; hpv32, n = 1, α 1 ; hpv68 α 7 , n = 1), and only one specimen gave positivity for β 1 (hpv14) (2.4%). the mean cd4 t cell count was 539 ± 230 cells/μl. ninety-six percent of women were receiving art per who guideline of starting art at cd4t cell count < 350 cell/mm 3 . the woman harboring β hpv strain was under art. she had a cd4 t cell count of 540 cells/μl and hiv rna viral load was not detected. overall cervical and anal β and γ type positivity was not related to immune status as measured by cd4 t cell count (see table 2 ) and art treatment. furthermore, the immune-re constitution could potentially prevent the persistence of β and ɣ types in anal and cervical sites or alternatively, β and ɣ types might show to have a less tropism to these anatomical sites. for the phylogenetic analysis of our untyped hpv samples we used 10 partial cds sampled sequences from major capsid protein l1 fap pcr products and 59 reference sequences. our reference sequences represent 50 classified and 9 untyped hpv sequences (see table 3 ). according to our phylogenetic analysis, 6 of our sampled sequences (q1766, q1644, q760, q337, q656 and q654) clustered with previously classified reference sequences from species 7, 10, 8, 12 and 9 -γ genus. sequences q763, q1234, q2164 and q127 remained unclassified while showing great similarity with unclassified genomic regions af489714 (fams9), kp692119, and af217684.1 respectively (see fig. 1 and table 3 ). in hnc, particularly oropharyngeal cancer, the true prevalence and involvement of hpv in the carcinogenetic process is still unknown. in fact, many studies report low prevalence of hpv obtained with systems used for the determination of hpv in cervical cancer screening (specific for α types), while other authors found higher prevalence when they used a platform able to detect hpv types belonging to genera other than α genus [4] both in oral cavity and in oral cancer [4, 39] . agalliu recently found a correlation between the infection of some β, ɣ hpv types and oscc [4] . furthermore, a recent meta-analysis provided additional evidence of the involvement of β hpv types in the development of scc in immunocompetent individuals [40] . additionally, several mechanistic studies have consistently showed that e6 and e7 from several β hpv types are able to target cellular proteins, such as p53 and prb, and to deregulate fundamental events involved in cellular transformation [41] . thus β and ɣ hpv types detection could be included in a hpv screening test for oral cancer prevention, while a correct and suitable detection of hpv infection is becoming a priority. currently, there are few studies that compare the hpv prevalence obtained using the protocols, which are normally for cervical cancer screening with those obtained using methods that are able to detect hpv types belonging to genera other than α or new ones. in this study, in order to assess the impact of pcr system in hpv prevalence determination, we carried out the detection of hpv with my09/11 primer set, used in several genotyping molecular assays (for example linear array hpv genotyping test, roche molecular system, ca usa; clart hpv2 clinical arrays, genomica, madrid, spain,) and fap primers able to detect α, β, γ hpv types in several specimens. in oropharyngeal swabs samples, my09/ 11 primer set was able to detect 39.4% (13/33) of hpv positive samples, while fap set of primers detected the presence of hpv in 20/33 samples (60.6%). beta hpv was the main genus detected with a prevalence of 69.7% in hpv positive samples. this prevalence was in agreement with bottalico et al. that used fap primer and my09/11 for detecting hpv in oral swabs [14] . hpv5 was the most frequent β 1 type and hpv145 the main type among β 2 species as described by bottalico [14] . hpv38, which was reported as the main β 2 type in forslund study [42] , was absent in our samples. hr, namely hpv16, was observed only in one specimen from an hiv positive subject, whereas hr hpv were frequently observed in bottalico study group [14] . the low frequency of hr could be imputed to immune status of the patients. our patients were under haart therapy and most of them had a cd4 t cell count > 200 /μl. no severe compromise of the immune-system could have limited a persistence of hr in this anatomical site. unfortunately, no information was given in the bottalico paper on patient cd4 t cell count, and this fact did not allow a correct comparison of these results. according to previous data reported by forslund et al. [42] , no difference in hpv prevalence was observed among men and women suggesting that host specific factors could contribute to hpv persistence and neoplasia development. gamma genus represented 21.8% of hpv positive samples. to note, 6/7 γ strains were untyped. phylogenetic analysis revealed that 2 samples (q1766 and q1644) belonged to γ 7 species, while q656 to γ 12 species and q337 to γ 9 species. interestingly, the oral untyped genotypes fell in different genera and cluster with the anal untyped strains, suggesting a specific tropism of these gamma types for oral mucosa. gamma 12 and 7 were the most representative γ species described by bottalico in oral rinse and agalliu in hnc, suggesting their potential involvement in oscc development. unlike forslund and hampras [42, 43] , we did not identified any β 3, β 4, β 5 hpv types. this may be due to the types of biological samples that were used or to differences in the efficiency of extraction methods that were applied, which could have influenced the outcome of pcr [44] . finally, differences in results might also be explained by the geographical distribution of hpv types. in general, differences in hpv β and γ types and their prevalence were observed by the luminex platform. according to him study [43] , among β 1 genotypes, hpv12 and hpv5 were the prevalent genotypes. while, among β 2 strains, hpv38 was the most represented,and it was never detected in our samples. interestingly hr hpv38 was also the main β 2 type observed also in moscicki study, whereas hpv21 represented the main β 1 type [45] . a different pattern of β types could be related to a different cellular input as described in a previous study [46] , where several β hpv types showed essentially increasing prevalence with increasing dna input. beta hpv types 8, 14, 20, 21, 23, 38 , and 92 showed increased prevalence only in higher dna input groups [46] . this may impose compromises in comparisons between studies; for example, our hpv38 negative result could be explained by an insufficient dna input in the pcr assay. further research is needed to establish whether there is an influence between cell number input and hpv β detection in oropharyngeal anatomical site and which cut-off would be suggested to avoid false negative results. possible type-specific differences in sensitivity to cellular input have to be evaluated in wider studies. if they are confirmed, they may be explained by the different sensitivities in detection systems and/or in the fig. 1 a maximum likelihood phylogenetic analysis was inferred on 10 unknown collected samples (bold) and 59 reference sequences using the gtrcat model. out of 10 unknown samples, 6 sequences were clustered within previously classified hpv species lineages. bootstrap supports lower than 70% were excluded from the tree. genbank accession number: mh647655-mh647663 different viral load spectra. we detected 9 α strains in anal site fap primers that had not been typed by genomica, and 2 β 2 types, hpv9 and hpv37, that had not been reported among oropharyngeal swabs. βeta types frequency was sensibly lower to that found by luminex system reported by torres et al. [17] which found 65.6% of β types in anal swabs from hiv positive msm and 59.1% among hiv negative msm. in this study, hpv12 and hpv 107 were the prevalent types among β 1 species, while hpv38 and hpv120 were the most frequently observed β 2 types. among γ genus, the γ 10 species was the most prevalent in both msm hiv positive and -negative groups, while we observed only γ untyped strains. donà reported higher prevalence of β (27.6%) and γ types (29.3%) in a group of msm similar to ours using the luminex system [16] . some hypothesis could put forth to explain these data: i) the luminex system could be much more sensitive than the fap system, or ii) that cross-reactivity could occur in β and γ types detection when α multiple infections are present. in literature, cross-reactivity was also observed among α genotypes. preisler observed cross-reactivity both in lr and hr genotypes using hc2, cobas, and aptima assays, despite what manufacturer claims: about 25% of hpvdna results in primary screening accounting for cross-reactivity, regardless of the assay [47] . to obtain improved analytical and clinical performance, cross-reactivity studies should be focused, since this aspect could influence the effectiveness in a future head neck hpv cancer screening. in cervical swabs the fap primers mainly showed the presence of α genotypes (hpv90, α 14 ; hpv32, α 1 ) which were not detected by clart hpv2 clinical array/ my09-11primers and 1 β 1 type (hpv14). this low prevalence of β types confirms the data reported by bottalico et al. [14] , which found a prevalence of 1% of the β types and 3% of the γ types in the cervical samples, emphasizing a weak β and γ type tropism towards the female genital mucosa. however, the genotypes hpv93 and hpv124, detected by bottalico in cervical specimens, were never observed among our cervical samples, suggesting a different geographic distribution of hpv β and γ type similarly to that described for α genus. conversely, these findings were in disagreement with those obtained by luminex system, as described in previous study [18] . a quantitative detection of the viral load in hair follicles demonstrated that the β genotype copy number was considerably lower than that reported for mucosal high-risk types from α genus in cervical tissue [48] . thus, only comparable viral load detection studies with different methods and the potential for multiple infections detection could explain these differences in prevalence of β types, which are reported in literature. overall, our results confirmed a prevalence of > 20% for hpv strains in the oropharyngeal anatomical district. the genus β and γ were predominant when the analysis was carried out with fap primers. the discrepancy on the prevalence and hpv types reported in other studies [16] [17] [18] 49] seems to be due to the detection system: highest prevalence was always obtained with the luminex system. different sensitivity and specificity of hpv detection methods were also a problem in the determination of α hpv types, as described in a systematic review where approximately 30-60% of all positive results showed discordance [50] . a global proficiency program like labnet for α types in cervical hpv infection surveillance programs should be planned also for α, β and γ hpv type detection in oropharyngeal samples [51] . standardizing methods for oral sample collection and hpv detection would ensure comparability between different detection methods in oral cavity samples [52, 53] . in addition, considering that γ genus has been growing rapidly (currently 98 γ types have been identified) surpassing α and β genera, and that 77% of the new types deposited in the hpv center within 2015 belonged to γ genus [54-56], our data reinforces the relevance of using primer sets able to detect a wide spectrum of hpv strains including β and γ types as well as new genotypes for hpv detection in the oropharyngeal anatomical site. this seems to be a crucial point since the meta genomic approach applied in some analysis [13] should be used with caution, taking in account the possibility of having an overestimation of hpv types [57] and requiring a confirmation of positivity by the sanger method. additional file 1 worldwide burden of cancer attributable to hpv by site, country and hpv type casecontrol study of human papillomavirus and oropharyngeal cancer associations of oral α-, β-, and γ-human papillomavirus types with risk of incident head and neck cancer detection and quantitation of human papillomavirus (hpv) dna in the sera of patients with hpv-associated head and neck squamous cell carcinoma hpv infections and tonsillar carcinoma human papillomavirus (hpv) in head and neck cancer. an association of hpv 16 with squamous cell carcinoma of waldeyer's tonsillar ring incidence of human papillomavirus (hpv) positive tonsillar carcinoma in stockholm, sweden: an epidemic of viral-induced carcinoma? hpv dna, e6/e7 mrna, and p16ink4a detection in head and neck cancers: a systematic review and meta-analysis preventable exposures associated with human cancers prevalence of microsatellite instability, inactivation of mismatch repair genes, p53 mutation, and human papillomavirus infection in korean oral cancer patients oral human papillomavirus in healthy individuals: a systematic review of the literature characterization of three novel human papillomavirus types isolated from oral rinse samples of healthy individuals the oral cavity contains abundant known and novel human papillomaviruses from the betapapillomavirus and gammapapillomavirus genera betapapillomaviruses in the anal canal of hiv positive and hiv negative men who have sex with men beta and gamma human papillomaviruses in the anal canal of hiv-infected and uninfected men who have sex with men prevalence of beta and gamma human papillomaviruses in the anal canal of men who have sex with men is influenced by hiv status cervical infection with cutaneous beta and mucosal alpha papillomaviruses prevalence and epidemiologic profile of oral infection with alpha, beta, and gamma papillomaviruses in an asian chinese population the use of polymerase chain reaction amplification for the detection of genital human papillomaviruses identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms a broad range of human papillomavirus types detected with a general pcr method suitable for analysis of cutaneous tumours and normal skin molecular methods for identification and characterization of novel papillomaviruses diversity of human papillomaviruses in skin lesions multiple oral carcinomas associated with a novel papillomavirus in a dog beta and gamma human papillomaviruses in anal and genital sites among men: prevalence and determinants prevalence and risk factors for anal human papillomavirus infection in human immunodeficiency virus (hiv)-positive and high-risk hiv-negative women relationship between prevalent oral and cervical human papillomavirus infections in human immunodeficiency virus-positive and -negative women high diversity of alpha, beta and gamma human papillomaviruses in genital samples from hiv-negative and hiv-positive heterosexual south african men an anal cancer screening program for msm in italy: prevalence of multiple hpv types and vaccine-targeted infections frequency and multiplicity of human papillomavirus infection in hiv-1 positive women in italy oral human papillomavirus dna detection in hiv-positive men: prevalence, predictors, and co-occurrence at anal site enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform seaview and phylo_win: two graphic tools for sequence alignment and molecular phylogeny selection of conserved blocks from multiple alignments for their use in phylogenetic analysis raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models beta-hpv types in patients with head and neck pathology and in healthy subjects association between β-genus human papillomavirus and cutaneous squamous cell carcinoma in immunocompetent individuals-a metaanalysis the biology of beta human papillomaviruses the nasal mucosa contains a large spectrum of human papillomavirus types from the betapapillomavirus and gammapapillomavirus genera prevalence and concordance of cutaneous beta human papillomavirus infection at mucosal and cutaneous sites analysis of the effect of dna purificationon detection of human papillomavirus in oral rinse samples by pcr prevalence and transmission of beta and gamma human papillomavirus in heterosexual couples prevalence and multiplicity of cutaneous beta papilloma viruses in plucked hairs depend on cellular dna input crossreactivity profiles of hybrid capture ii, cobas, and aptima human papillomavirus assays: split-sample study beta-papillomavirus dna loads in hair follicles of immunocompetent people and organ transplant recipients concordance of beta-papillomavirus across anogenital and oral anatomic sites of men: the him study concordant testing results between various human papillomavirus assays in primary cervical cancer screening: systematic review continuing global improvement in human papillomavirus dna genotyping services: the 2013 and 2014 hpv labnet international proficiency studies detection of oral hpv infection -comparison of two different specimen collection methods and two hpv detection methods oral human papillomavirus infection incidence and clearance: a systematic review of the literature international standardization and classification of human papillomavirus types towards quality and order in human papillomavirus research hpviewer: sensitive and specific genotyping of human papillomavirus in metagenomic dna we are grateful to all member of virology laboratory inmi l. spallanzani for sample collection. we thank dr. mariana badescu for english editing. the authors declare that they have no competing interests. key: cord-282675-s4zmffj3 authors: sagaon-teyssier, luis; kamissoko, aliou; yattassaye, adam; diallo, fodié; rojas castro, daniela; delabre, rosemary; pouradier, fabrice; maradan, gwenaëlle; bourrelly, michel; cissé, mamadou; vidal, laurent; dembélé keïta, bintou; spire, bruno title: assessment of mental health outcomes and associated factors among workers in community-based hiv care centers in the early stage of the covid-19 outbreak in mali date: 2020-10-15 journal: health policy open doi: 10.1016/j.hpopen.2020.100017 sha: doc_id: 282675 cord_uid: s4zmffj3 • malian healthcare workers presented mental disorders in the early stage of covid-19. • nurses were at lower risk of mental health disorders than other worker categories. • women were at greater risk of mental health disorders than men. • a lack of protection equipment and nurses was associated with mental disorders. as of july 14, 2020, the covid-19 case-fatality rate in mali was the fifth highest in the world health organization's (who) african region, and the third highest among west african countries, with deaths representing 5.0% of total cases behind 6.2% and 5.1% in niger and burkina faso, respectively [1] . the first cases in mali were observed on march 25, 2020, over a month after the continent's first case, and 6 and 16 days after the two abovementioned countries, respectively. however, the number of cumulated cases in mali was much higher up to july 14: 2423 cases versus 1037 and 1099 cases in burkina faso and niger, respectively [1] . the lag between africa's first cases and those in mali did not create the same opportunity -in terms of prompt emergency preparation -which the lag in 2014 created during the ebola outbreak. moreover, international aid has slowed down because of covid-19's pandemic status. data for mali indicate that authorities' proactive decisions to contain the disease's spread, including commercial air traffic interruption, bans on mass gatherings and curfews, [2] have been insufficient. furthermore, as observed elsewhere, the public health response is being undermined by poor adherence to social distancing for different reasons [3, 4] . however, the most likely explanation for mali's higher vulnerability and poorer medical outcomes is its inherently weak healthcare system. compared with niger and burkina faso, the shortage of healthcare workers (hcw), health expenditures and infrastructure is more acute in mali [5] . more specifically, the country's dependency on foreign aid, unreliable medical equipment, difficulties to procure drugs [6] , staff attrition [7] , shortage of human resources (hr) [8, 9] and resulting degradation of working conditionsincluding increased workload [10] [11] [12] -all contribute to putting hcw who are on the front line fighting covid-19 under greater stress. added to this is the fact that care for covid-19 is only concentrated in 6 hospitals (5 in bamako and 1 in kati (19 km from bamako)) positioned at the top of the country's 4level healthcare pyramid [13] . the deleterious impact on hcw mental health [14, 15] because of the these structural problems and unpreparedness to contain covid-19 constitutes a serious public health issue [16] . unfortunately, data about mental health among hcw in mali is inexistent. however, the who asserts that increasing terrorism and insecurity in mali since 2012 are two factors that could have mental health implications for the general population [17] , and therefore this would include hcw. lessons learned from previous disease outbreaks in the world, such as sars-cov-1 in 2003, which resulted in severe psychological damage in hcw, highlighted the need to strengthen hcw mental resilience and their preparation for new outbreaks, by providing psychological first aid [18] [19] [20] [21] . despite this, a growing body of literature on the current covid-19 outbreak indicates a high prevalence of mental health disorders (mhd) in hcw, suggesting they were not adequately prepared for the magnitude of this pandemic [22] [23] [24] [25] [26] [27] [28] . studies found that between 20.1 and 64.7% of hcw had depressive symptoms, 35.1 to 51.6% anxiety symptoms, and 18.2 and 38.9% insomnia [25] [26] [27] 29] . mhd were often associated with gender, occupational differences, age [26, 27, 30] , place of work, and poor social support [31] . although structural factors were evoked in some cases as moderators of mhd risk [28] , no study to date has adequately examined their effect on hcw psychological outcomes. the current unprecedented pandemic is expected to have a long-lasting effect on mental health, especially for hcw [32, 33] . various studies all urge specific interventions to mitigate this effect, and to ensure hcw wellbeing during and after the pandemic [34] [35] [36] . training, social support, communication, and effective health equipment procurement are the primary elements suggested [37] [38] [39] [40] [41] [42] [43] . however, most of the abovementioned studies concerned developed countries, and focused primarily on front-line hcw. few studies to date have highlighted the importance of also taking into account non-front-line hcw, including community health workers (chw) and other non-medical staff [39, 44] . indeed, the absence of significant differences in psychological outcomes between these hcw and their front-line counterparts suggests that the risk of mhd during the current outbreak is similar in both groups [24] , especially in low-income countries [21] . furthermore, empirical studies performed to date were all conducted at an advanced stage (understood here as between 2 and 4 months after the outbreak started) in the countries investigated. they did not account for psychological profiles at outbreak onset (i.e., baseline) or in the early stage (understood here as the first 2 weeks). the mental health impact of covid-19 might be more severe on hcw with a fragile baseline profile [19, 35] . accordingly, while assessing mhd as the outbreak develops is vital to provide suitable psychological support, understanding profiles in the early stage is also essential to identify the most vulnerable hcw. covid-19's rapid spread throughout the world raised the question early on of whether african countries, especially those in sub-saharan africa (ssa), were adequately prepared or not [45] [46] [47] [48] . although the extent of the outbreak is currently less dramatic than initially feared, ssa authorities continue to give top priority to its containment, with little attention for preexisting serious public health concerns, especially the fight against malaria, tuberculosis and hiv. the potential interruption of prevention activities and treatment (e.g., antiretroviral treatment (art)) could harm the advances already made by ssa countries in this fight [49, 50] . fortunately, the work of non-governmental organizations (ngos) -which play a crucial role in controlling these diseases in ssa countries -is, at least in part, compensating for this lack of attention. furthermore, lessons learned by ngos from previous outbreaks and epidemics, especially hiv, are invaluable, and must be integrated into the overall response to covid-19 and future outbreaks [2, 51] . ngos providing healthcare services in mali are located at the bottom level of the country's 4-level healthcare pyramid, specifically at the community level. together with public community healthcare centers, they offer basic health services (e.g. essential medicines, maternity room, prevention and promotion of health, etc.) and are the main point of entry into the healthcare system. hcw in these healthcare structures screen for people with health conditions (e.g. people with covid-19 symptoms) requiring referral to structures in the 3 higher levels of care (district, regional, or national) [52,53]. the involvement of some malian ngos in health promotion and care activities has contributed to the achievement of important milestones in the country's response to other epidemics, such as hiv. this is especially true of arcad santé plus, the main malian ngo working on improving access to healthcare for people living with hiv (plwh) and other vulnerable populations since 1994. in december 2019, the number of plwh receiving hiv care in the ngo's 18 healthcare sites -located in 6 of the country's 10 administrative regions -was close to 29 000, or 26% of the plwh in mali. this is a substantial figure when one considers that only 35% of all plwh in mali had access to art in 2016 [54] . the main challenge faced by arcad santé plus in the current context is how to adopt government indications to prevent covid-19 in the workplace. this includes adjusting working hours and adapting hiv care centers' opening hours to reduce patient flow, while guaranteeing continued prevention and care for hiv and other health problems. on april 1, 2020 (6 days after the first covid-19 cases in mali), arcad santé plus launched the covidprev program whose main objective is to reduce the risk of covid-19 infection in its hcw (whether salaried or volunteers) and in plwh frequenting its centers [2] . covidprev's planned reorganization of activities and the continued uncertainty surrounding the outbreak, together with the need to guarantee hiv care-related activities, constitute a double burden for the ngo's workers. although not on the front line in the fight against covid-19, their mental health might be seriously harmed by these supplementary sources of mhd. prospective research is vital for ngos to achieve their objectives as part of national public health strategies. however, to our knowledge, no empirical evidence exists concerning the impact of covid-19 on public health in africa, and especially on the mental health of front-line and non-front-line hcw. arcad santé plus's activities directly related to the delivery of healthcare are performed by doctors, pharmacy doctors, midwives and nurses. activities related to prevention (e.g., health awareness, support for treatment adherence) and social support (e.g., moral, material) are primarily performed by chw (including community mobilizers and navigators) and psychosocial counselors. these two caregiver categories account for a large proportion of arcad santé plus's staff, and are continuously provided training for the promotion of health-, disease-and population-based issues, especially those related to hiv and stis. finally, administrative and logistics personnel ensure that the operation runs as smoothly as possible. in terms of the current covid-19 pandemic, hcw proximity to people -indoors and outdoors -makes them an important vector for disseminating information about covid-19 in order to protect plwh and other vulnerable populations. as one of arcad santé plus's top priorities has always been to protect its hcw from health problems -in order to ensure they can optimally provide hiv prevention and care -just days after the ngo's launch of its covidprev program, we implemented a public health and social sciences action research study aimed at providing the ngo with data about the current mental health state of its workforce, so that it could incorporate targeted measures in its covidprev program to protect its hcw. more specifically, this study explored individual and structural factors associated with depression, anxiety and insomnia in this workforce. data were collected from april 6 to 11, 2020 (i.e., two weeks after the first two covid-19 cases in mali) for hcw (salaried and volunteers) in arcad santé plus's 18 community-based hiv care centers, located in 6 administrative regions in mali (koulikoro, kayes, mopti, ségou, sikasso, gao) and in the capital bamako (fig. 1) . to be eligible, participants had to be at least 18 years old, and planned to work throughout the outbreak. a self-administered questionnaire collected the following information: demographic and socioeconomic data, self-perceived health status, mental health data, and basic covid-19 awareness. in addition, structural factors (characteristics) of the 18 hiv care centers (number of years open, hiv caseload, number of doctors, nurses, etc.) were provided by facility managers. study approval was obtained from the malian ethical committee (n°2020/57/ce/fmos/faph). analyses were performed for three mhd. the 9-item patient health questionnaire (phq-9) was used to assess depression and its severity [55] (total score from 0 to 27). the 7-item generalized anxiety disorder assessment (gad-7) was used to measure anxiety [56] (from 0 to 21). finally, the 7-item insomnia severity index (isi) assessed participant insomnia during the month preceding the survey [57] (from 0 to 28). all three tools were implemented in their validated french version [58] [59] [60] . analyses were performed for the three continuous scores. the following potential individual characteristics to explain individual variability in the outcomes were tested: age (continuous variable); gender (woman=1 vs man=0); marital status: married/cohabitating (=1) or single/separated/divorced/widowed (=0); a three-category variable for the number of financially dependent family members, constructed using the median as the cutoff (none, 1 to 7, and >7); self-perceived health status: "good" (=1) or "very good"/"excellent" (=0); worker type classified into a four-category variable as follows: i) doctors, pharmacy doctors and midwives ii) nurses; iii) chw (including community mobilizers and navigators) and psychosocial counselors; iv) other, including administrative and logistics personnel (secretary, driver, etc.). it is important to note that the worker type variable reflects not only the activity type, but is also a proxy of the worker's education level: higher than high-school for nurses, midwives, pharmacy doctors and doctors; and lower than highschool for the other worker categories. to measure preexisting (i.e., prior to the covid-19 outbreak) serious work-related psychological damage resulting from ethical or moral transgression in the workplace, participants answered the 9item moral injury event scale (mies) [61] . two sub-scores -perceived transgression (ranging from 1 to 36) and perceived betrayal (1 to 18) -were constructed, and specified as continuous variables. finally, basic covid-19 awareness was assessed using unicef's "fact or fiction" 10-question quiz (appendix 1) with a score ranging from 0 (no correct response) to 10 (all responses correct). the following potential structural characteristics (all continuous variables) to explain variability in the outcomes due to differences between hiv care centers were tested: the number of years since the center opened, and 6 other variables assessing the density of personnel (per 100 patients) for 6 worker categories (required to permit separate analyses): i) doctors, ii) pharmacy doctors, iii) midwives, iv) nurses, v) chw and vi) psychosocial counselors. healthcare supply characteristics were tested by constructing dichotomous variables indicating whether the center offered (=1) or not (=0) each of the following 12 services: i) medical consultation for the general population, ii) specific medical consultation for key populations, iii) hiv screening, iv) hiv care for adults and/or pediatric care, v) delivery of arv drugs, vi) biomedical analyses, vii) nursing care, viii) community-based talks and/or distribution of condoms and lubricants, ix) psychological care, x) hiv pre-exposure prophylaxis (prep) delivery, xi) hiv post-exposure prophylaxis (pep) delivery, and xii) social/financial support. equipment characteristics were tested by constructing 5 dichotomous variables indicating whether each center had (=1) or not (=0) the following items: i) an electricity generator, ii) air conditioning, and iii) a refrigerator. with regard to personal protective equipment (ppe), dichotomous variables indicated the availability (=1) or not (=0) of: i) face masks, ii) gowns, iii) safety goggles, and iv) gloves. finally, an indicator recorded potential drug stock-outs during the previous 6 months (between october 2019 and march 2020) (yes=1 or no= 0). descriptive statistics were calculated for the sample. for the phq-9, gad-7 and isi scores, statistics included the proportion of non-zero scores, their mean (standard deviation), and median with interquartile range [iqr] . furthermore, for descriptive purposes only, cutoffs of ≥ 10, ≥ 7, and ≥ 15 were used to distinguish severity for depression, anxiety, and insomnia, respectively [55] [56] [57] [58] [59] . estimations were performed with the outcomes specified as continuous dependent variables. a general mixture model (gmm) with a negative binomial (nb) distribution was used (see appendix 2 for details on the estimation strategy). all statistical analyses were conducted using r software, version 4.0.0 [62] . of the 188 workers identified in arcad santé plus's 18 community-based hiv care centers, 135 were working at the time of data collection and intended to continue working throughout the outbreak (table 1 ) (study population). most were men (60.7%) and median age was 40 years iqr [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] (see table 1 for more details on individual characteristics). with respect to study outcomes, table 1 shows that mean scores were lower than their corresponding variances, justifying the choice to use an nb distribution instead of a poisson distribution (assuming equal mean and variance). phq-9, gad-7 and isi scores indicated high prevalences of depression, anxiety and insomnia symptoms in the study population (71.9%, 73.3% and 77%, respectively). furthermore, 18.5% and 21.5% of all participants had moderate/severe depression and anxiety, respectively, whereas only 2.2% had severe/moderate insomnia. half the hiv care centers opened before 2012 (table 1) , the median years of activity being 8 iqr [7] [8] [9] [10] [11] [12] . in terms of hr availability, median densities (all values per 100 patients) of doctors and nurses were 0.13 (iqr[0.06-0.20]) and 0.10 (iqr[0.02-0.20]), respectively. the most disadvantaged hiv care centers (i.e., the 25% corresponding to the 1 st quartile) had extremely limited hr, especially nurses (fewer than 0.02) and chw (fewer than 0.01), and no psychosocial counselors. the most severe ppe shortages concerned goggles (no center), face masks (only 5 centers) and gowns (only 6 centers). eight centers had experienced drug stock-outs in the 6 months preceding the survey, mostly of arvs and drugs for opportunistic infections (see table 1 for more details on services provided). a constructed using correct responses to unicef's "fact or fiction" quiz (see appendix 1); b presented only for descriptive purposes, as multivariable models were estimated using these scores as continuous variables. c two hiv care centers declared 1 midwife. the density of midwives for these two centers was 0.01 and 0.02 per 100 plwh, respectively, although the median and iqr were equal to 0. d these variables were not tested in the model as they were constant across all 18 hiv care centers (i.e., response was similar for all centers). the lr-test comparing 1-level and 2-level null models concluded that variability in phq-9 (depression), gad-7 (anxiety) and isi (insomnia) scores were due to both individual characteristics and differences in hiv care center characteristics, as indicated by the icc: 33%, 31% and 16% of the total variability, respectively (see bottom of table 3 ). bivariate analysis in table 2 showed that the higher the number of financially-dependent family members, the higher the risk of depression (p=0.037) for those who self-perceived good health status (versus very good/excellent, p=0.029), and for those with a strong perception of work-related moral transgression (p<0.001) and/or betrayal (p=0.010). nurses had the lowest risk of depression (p=0.006). with regard to structural characteristics, medical consultations for the general population (p<0.001) and arv delivery (p=0.036) were associated with a higher risk of depression. in contrast, depression was less likely in hcw in centers with a higher density of nurses (p=0.045) and greater availability of face masks (p=0.039). multivariable analysis (see table 3 ) showed that depression was 60% more likely in women than in men the bivariate analysis for anxiety (table 2) shows that women were at higher risk than men (p=0.027). anxiety was also more likely in participants who perceived they had good health status (versus very good/excellent) (p=0.009). in terms of structural characteristics, face mask availability was associated with a lower risk of anxiety in participants working in centers which provided them (p=0.041). the higher risk of anxiety in women was a tendency in the multivariable model ( 01-1.04) ). face mask availability was associated with a 62% lower risk of anxiety in participants working in centers which provided them (irr: 0.38, 95%ci(0.21-0.67)). furthermore, the risk of anxiety was lower in workers in hiv care centers with a higher density of nurses, decreasing by 95% per additional nurse per 100 plwh (irr: 0.05, 95%ci(0.01-0.62)). sleeping disorders were more frequent in women (p=0.030), in those perceiving good health status (p=0.024), and in those perceiving work-related betrayal (p=0.002) ( table 2 ). in terms of structural characteristics, insomnia was related to face mask availability (p=0.008). multivariable analysis (table 3) confirmed the higher risk of insomnia in women (53%) (irr: 1.53, 95%ci(1.12-2.09)). insomnia also increased by 6% per one-point increase in the perceived workplace betrayal score (irr: 1.06, 95%ci(1.02-1.09)). worker category was related with the risk of insomnia, which was 49% lower in nurses than other hcw (irr: 0.51, 95%ci(0.29-0.93)). finally, the only structural factor associated with insomnia was face mask availability. participants working in care centers where they were available had a 43% lower risk of insomnia (irr: 0.57, 95%ci(0.38-0.86)). mhd prevalence was high in our study population during the early stage (i.e., first 2 weeks) of the covid-19 outbreak in mali. more specifically, of arcad santé plus's 135 non-front-line hcw who participated in the present study, 71.9%, 73.3% and 77% declared at least one symptom of depression, anxiety, and insomnia, respectively. these figures are much higher than those in studies of front-line hcw [27, 29] (61.7%, 51.6% and 38.9%, respectively) performed in countries at an advanced stage (i.e., between 2 to 4 months) after the outbreak. furthermore, these countries had relatively more developed healthcare systems. this result suggests that arcad santé plus's non-front-line hcw might be more vulnerable to deteriorating psychological outcomes as the current covid-19 outbreak progresses and after it ends. the present article demonstrates that mhd in hcw were related not only to individual characteristics, but also to hiv care center characteristics. specifically, the risks of having these conditions were 51%, 62% and 43% lower, respectively, in people working in hiv care centers providing face masks than in people working elsewhere. a lack of masks was also the only structural factor linked to insomnia. furthermore, depression and anxiety were 91% and 95%, respectively, less likely to occur in hcw in hiv care centers with a higher density of nurses. mental health in arcad santé plus's chw in mali seemed to be associated with uncertainty about covid-19 at the beginning of the outbreak, as suggested by the relationship between the (un)availability of face masks and insomnia, depression, and anxiety. however, the risk of the latter two mhd seemed to be also related with preexisting contexts in hiv care centers, such as hr scarcity, especially nurses. the temporary reorganization of activities planned by arcad santé plus as part of its purpose-built covidprev program -including fewer working hours, lower patient flow and a reduction of some services -should attenuate workers' exposure to psychological disorders by reducing their workload [63] . however, it is vital to also include screening for psychological disorders and suitable treatment, as these same changes may themselves lead to psychological problems [64] [65] [66] . in addition, adequate ppe must be guaranteed for hcw, especially face masks, one of the most important tools in stopping the spread of covid-19 [37] . the relationship discovered between the density of nurses in hiv care services and workers' psychological outcomes not only highlights areas for improvement in the management of mental health among arcad santé plus's hcw during the current covid-19 outbreak, but also provides insight into how these workers' performance in hiv-related care could be improved in the short and long terms. our results indicate that managers should investigate whether better reallocation of nursing resources is needed according to hiv caseload, whether more nurses need to be hired, and whether improvements in doctor-nurse task-shifting -an increasingly important care strategy, especially in ssa -is necessary, especially seeing as arcad santé plus continues to expand its offer to include more non-hiv specific health-related services. furthermore, including chw as a full hcw category (albeit voluntary) in all organizational changes is crucial, both during and after the current covid-19 outbreak, as these workers play an essential role in healthcare in ssa [67] [68] [69] . chw account for a large proportion of arcad santé plus's staff (20% of respondents in the present article) and their activities are central to what makes this ngo attractive to people benefitting from its services. they are crucial in reaching key populations and promoting retention in hiv prevention and care programs. their contribution to the introduction of pre-exposure prophylaxis (prep) for hiv among men having sex with men in mali is just one example of this [70] . the role of chw (not just in mali) in connecting the most vulnerable people with healthcare systems during the current covid-19 outbreak highlights the importance of strengthening chw workforces long after this pandemic ends. this observation is not new to africa, a continent where the work of chw during the last decade in the field of hiv has led to successful testing of new task-shifting models and has strengthened the argument for the demedicalization of prevention and care [67] [68] [69] [71] [72] [73] ]. however, the success of any new healthcare service model which incorporates chw as key actors, depends not only on financial sustainability, but also on the capability of the healthcare system to limit staff attrition and to protect them -and all hcw -from potential health problems, including mhd [7] . the abovementioned implications of the relationship between structural factors and hcw mhd are supported by our results for individual factors. apart from nurses, all other worker categoriesincluding chw -were at greater risk of depression and insomnia (respectively, 60% and 49% more than nurses). these results confirm the importance of taking into account non-front-line hcw mental health in related analyses [39, 44] . the present work reflects the higher work-related psychological risk among women in healthcare-related professions observed in the literature [24, 63] . despite their nonfront-line status, this finding could be explained -at least partly -by both the importance which women in general seem to attach to psychosocial support [74] , and occupational exposure related to hiv care delivery [75] [76] [77] . furthermore, our results contribute to the existing literature by demonstrating that work-related factors are not the only source of mhd. factors related to the day-to-day life of hcw were also strongly associated with the risk of depression. indeed, depression was over twice as likely in participants with financially-dependent family members. family responsibilities imply not only a supplementary workload, but also mental efforts that may lead to increased levels of depression [78] . the psychological distress related to medical decisions running counter to hcw morals and ethics is an important mental health dimension. in the present article, this distress was assessed using the moral injury event scale, as suggested by greenberg et al., and walton et al. [41, 44] . more specifically, our results showed that the risks of depression and anxiety were higher in workers who perceived work-related moral transgression, while the risk of insomnia was higher in those perceiving workrelated betrayal. given their non-front-line status in terms of covid-19 care, and the fact that we assessed psychological outcomes during the early stage of the outbreak, this result would seem to be mostly explained by psychological distress linked to their hiv care-related activities. the complex context which these workers are confronted with may also result in their having to take decisions which run counter to their moral and ethical values. the lack of arv delivery during stock-outs, the slowdown in international funding for the treatment of opportunistic infections, and external intimidation because of the services they offer (e.g., counseling for men who have sex with men), are three examples where such decisions might be made. this present article has limitations. first, although the study sample was exhaustive -in that it included hcw in arcad santé plus's 18 healthcare services in mali working in the early stage of the covid-19 outbreak and reporting that they intended to continue to work throughout the outbreak -the sample size was nevertheless small. this meant that the relationship between certain structural factors and individual psychological outcomes could not be measured, as there was insufficient variability within some structures. however, the comparability of our results for individual factors with existing literature demonstrates that implementing suitable techniques -in our case gmm with an nb distribution -helped to overcome the limitations imposed by the small sample size. second, our sample was not representative of the population of malian hcw. despite the difficulties they face, the working conditions of arcad santé plus's non-front-line hcw are relatively less difficult than those of their counterparts in the general malian health system. considering that in mali there is a greater-than-usual shortage of hr (0.036 medical professionals per 100 patients) with respect to arcad santé plus hiv care centers (0.23 per 100 patients, which interestingly matches the minimum recommended by the who [79] ), our results for non-front-line hcw most likely underestimate the current situation regarding mental health in the national healthcare system. finally, the short questionnaire used prevented the collection of more detailed information about participants' working and living conditions. however, the choice to use a short questionnaire was governed by necessity in order to limit desirability bias, and by a desire not to overburden respondents who were already having to adapt to constantly changing circumstances related to covid-19. despite these limitations, the analyses conducted here provide evidence of non-negligible mhd affecting hcw in arcad santé plus's care network in the early stage of the covid-19 outbreak. in light of this action research, results from the first analyses (carried out 1 week after data collection) prompted the ngo to add two new actions to its covidprev program: i) the distribution of a large quantity of basic ppe, including face masks, gloves, and cleaning products; and ii) the drafting and distribution of an information leaflet presenting the current mental health situation of its hcw, in order to promote self-help in this population through the program's specifically developed fora, given governmental restrictions on movement. we aim to conduct further research to investigate whether and to what degree the covid-19 outbreak aggravates mhd in this population. more broadly, the results of the present article provide evidence-based arguments that should be taken into account in malian healthcare policy. irrespective of the covid-19 outbreak, conducting situational research is crucial to understand how and to what extent the physical and mental health of hcw is related to working and living conditions. psychosocial support is a key element in the management of day-to-day work-related activities, and becomes indispensable during serious health shocks such as the current covid-19 outbreak. the long-established trustful relationship between arcad santé plus and users of its hiv prevention and care services is a crucial factor in ensuring the dissemination of key covid-19 messages in mali. indeed, the arrival of this new disease has underlined the huge importance of hcw -front-line and non-front-line -and has placed them at the core of health systems worldwide. however, the outbreak has also revealed weaknesses in integrating non-front-line hcw in the response to covid-19, especially hcw in ngos who perform crucial health-related activities. these people should have been integrated early on after the outbreak, not only as important vectors for information dissemination and prevention, but also as a group whose health and well-being are at stake and need to be protected. one of the main lessons to be learned from previous outbreaks and which the current covid-19 pandemic reminds us of, is that "not being on the front line" does not mean "not needing support to reinforce the front line". the effectiveness of the international response to pandemic outbreaks, and in general the effectiveness of public health strategies at national and local levels, depend on the capacity of hcw to fully and competently perform their duties within the healthcare system. estimations were carried out with the outcomes specified as continuous dependent variables to avoid any loss of information that might result from their dichotomization, that is to say, underestimation of the variability, and reduced power to estimate outcomes' relationships with explanatory variables [80, 81] . however, this choice was methodologically challenging as it required us to implement a model adapted to non-negative dependent variables with skewed distributions and often overdispersed and zero-inflated. given this context, the most suitable method to estimate the associated factors to the outcomes in this article was the general mixture model (gmm) with a negative binomial (nb) distribution [82, 83] . this was preferred to the poisson-type distribution, as indicated by log-likelihood ratio test (lr-test), which rejected the null hypothesis where overdispersion is absent. gmm models have often been shown to be better adapted to non-count data, and a better alternative to tobit and two-part models [83] . the restricted (residual) maximum likelihood estimation method was implemented in order to manage estimations with small samples [84, 85] . for each outcome the estimation strategy consisted in: 1) verifying the pertinence of using a multilevel model. a 2-level null model with random intercepts was estimated and compared with a 1-level null model using the lr-test. this comparison allowed us to verify whether there was any outcome variability arising from differences between hiv care centers. the intra-class correlation coefficient (icc) -adapted to the gmm -was estimated to assess the amount of outcome variability arising from structural differences [86] [87] [88] . single estimations were performed for each explanatory variable at the individual level. each estimation was compared with the null model using the lr-test in order to assess the contribution of the corresponding explanatory variable. eligibility of individual-level variables for inclusion in the multivariable model was based on the following criteria: p-value < 0.2 and/or lr-test p-value < 0.1 (i.e. significant contribution to the model fitting). the step-wise forward selection procedure was implemented and the final multivariable model for individual characteristics chosen on the basis of the aic criterion. this best-fit model for individual factors was used to conduct a bivariate analysis for the structural factors (i.e., hiv care center structural characteristics). more specifically, the same tests and criteria as those for individual factors were used for these structural variables to be eligible for the 2level part of the multivariable model, and also in order to construct the final model. estimated incidence rate ratios (irr), 95% confidence intervals and p-values are presented in the results section. 3) verifying the need to use a zero-inflated model. the final multivariable models (estimated using negative binomial distribution) were re-estimated using zero-inflated negative binomial distribution (zinb). these models were compared using the lr-test in order to verify whether using zinb was necessary or not [89] . who | regional office for africa n the covid-19 response must integrate people living with hiv needs in sub-saharan africa: the case of mali social distancing: how religion, culture and burial ceremony undermine the effort to curb covid-19 in south africa economic considerations for social distancing and behavioral based policies during an epidemic identifying future disease hot spots: infectious disease vulnerability index. rand corporation poor performance of community health workers in kalabo district staff attrition among community health workers in home-based care programmes for people living with hiv and aids in western kenya health workers, quality of care, and child health: simulating the relationships between increases in health staffing and child length focussing on the wellbeing of health care workers in sub-saharan africa overworked? on the relationship between workload and health worker performance working conditions of nurses and absenteeism: is there a relationship? an empirical analysis using national survey of the work and health of nurses perceived working conditions and sickness absence -a four-year follow-up in the food industry. safety and health at actions pour la prévention et la réponse à la maladie à covid-19 (covid-19). mali: ministère de la santé et des affaires sociales occupational moral injury and mental health: systematic review and meta-analysis precarious sleep? nonstandard work, gender, and sleep disturbance in 31 european countries covid-19 threatens health systems in sub-saharan africa: the eye of the crocodile 2020 was sars a mental health catastrophe? prevalence of psychiatric disorders among toronto hospital workers one to two years after the sars outbreak immediate and sustained psychological impact of an emerging infectious disease outbreak on health care workers focus on mental health during the coronavirus (covid-19) pandemic: applying learnings from the past outbreaks covid-19-pandemie: belastungen des medizinischen personals: ein kurzer aktueller review mental health impact of covid-19 pandemic on spanish healthcare workers covid-19 pandemic: looking after the mental health of our healthcare workers generalized anxiety disorder, depressive symptoms and sleep quality during covid-19 outbreak in china: a web-based cross-sectional survey factors associated with mental health outcomes among health care workers exposed to coronavirus disease prevalence of depression, anxiety, and insomnia among healthcare workers during the covid-19 pandemic: a systematic review and meta-analysis covid-19 and mental health: a review of the existing literature depression, anxiety, stress levels of physicians and associated factors in covid-19 pandemics mental health and psychosocial problems of medical health workers during the covid-19 epidemic in china mental health problems faced by healthcare workers due to the covid-19 pandemic-a review battle buddies: rapid deployment of a psychological resilience intervention for healthcare workers during the covid-19 pandemic burnout and somatic symptoms among frontline healthcare professionals at the peak of the italian covid-19 pandemic supporting the well-being of healthcare workers during and after covid-19 covid-19: adverse mental health outcomes for healthcare workers optimal sleep health among frontline healthcare workers during the covid-19 pandemic covid-19: protecting healthcare workers is a priority mitigating the psychological impact of covid-19 on healthcare workers: a digital learning package a multinational, multicentre study on the psychological outcomes and associated physical symptoms amongst healthcare workers during covid-19 outbreak covid-19: a new work-related disease threatening healthcare workers managing mental health challenges faced by healthcare workers during covid-19 pandemic the need of health policy perspective to protect healthcare workers during covid-19 pandemic. a grade rapid review on the n95 respirators effectiveness urgent need to develop evidence-based self-help interventions for mental health of healthcare workers in covid-19 pandemic mental health care for medical staff and affiliated healthcare workers during the covid-19 pandemic is sub-saharan africa prepared for covid-19 preparedness and vulnerability of african countries against importations of covid-19: a modelling study is africa prepared for tackling the covid-19 (sars-cov-2) epidemic. lessons from past outbreaks, ongoing pan-african public health efforts, and implications for the future will covid-19 be a litmus test for post-ebola sub-saharan africa? preparedness is essential for malaria-endemic regions during the covid-19 pandemic symptoms, stress, and hiv-related care among older people living with hiv during the covid-19 pandemic covid-19 in africa: care and protection for frontline healthcare workers the phq-9: validity of a brief depression severity measure a brief measure for assessing generalized anxiety disorder: the gad-7 the insomnia severity index: psychometric indicators to detect insomnia cases and evaluate treatment response are scores on english and french versions of the phq-9 comparable? an assessment of differential item functioning rapid detection of generalized anxiety disorder and major depression in epilepsy: validation of the gad-7 as a complementary tool to the nddi-e in a french sample reliability, factor analysis and internal consistency calculation of the insomnia severity index (isi) in french and in english among lebanese adolescents psychometric evaluation of the moral injury events scale r: a language and environment for statistical computing. r foundation for statistical computing workload, mental health and burnout indicators among female physicians the psychological costs of war: military combat and mental health mental health interventions in the workplace and work outcomes: a best-evidence synthesis of systematicreviews tracing new occupational diseases, an introduction. safety and health at implementation and operational research: a comparison of two task-shifting models of pharmaceutical care in antiretroviral treatment programs in south africa the potential of taskshifting in scaling up services for prevention of mother-to-child transmission of hiv: a time and motion study in dar es salaam the impact of community health worker-led home delivery of antiretroviral therapy on virological suppression: a non-inferiority cluster-randomized health systems trial in dar es salaam reaching a different population of msm in west africa with the integration of prep into a comprehensive prevention package. (cohmsm-prep anrs 12369 -expertise france) engaging community health workers in maternal and newborn care in eastern uganda benefits and limitations of community art groups (cags) in thyolo, malawi: a qualitative study demedicalizing aids prevention and treatment in africa impact of covid-19 outbreak on healthcare workers in italy: results from a national e-survey occupational hazards among healthcare workers in africa: a systematic review occupational exposure to hiv among healthcare workers in pmtct sites in port harcourt occupational exposure to hiv among nurses at a major tertiary hospital: reporting and utilization of post-exposure prophylaxis; a cross-sectional study in the western cape, south africa full-time employed and a family caregiver: a profile of women's workload, effort, and health nursing and midwifery personnel (per 10 000 population) n.d the cost of dichotomising continuous variables dichotomizing continuous predictors in multiple regression: a bad idea the log of gravity estimating poisson pseudo-maximum-likelihood rather than log-linear model of a logtransformed dependent variable small sample methods for multilevel modeling: a colloquial elucidation of reml and the kenward-roger correction getting started with the glmmtmb package n the coefficient of determination r2 and intra-class correlation coefficient from generalized linear mixed-effects models revisited and expanded n.d repeat personal victimization: random effects, event dependence and unexplained heterogeneity reliability of environmental sampling culture results using the negative binomial intraclass correlation coefficient logistic regression using sas: theory and application designed the study. l.s.-t. and a.k. conducted the statistical analyses key: cord-301704-mb2oylqb authors: eapen, paul; cates, jennifer; mundell, rich; palmer, kenneth e.; fuqua, joshua l. title: in preparation for outdoor pharming: griffithsin can be expressed in nicotiana excelsiana and retains activity after storage as silage date: 2020-03-18 journal: front bioeng biotechnol doi: 10.3389/fbioe.2020.00199 sha: doc_id: 301704 cord_uid: mb2oylqb griffithsin is an algae-derived lectin with strong anti-viral activity against hiv and a positive safety profile. multiple clinical studies are investigating griffithsin's utility as topical hiv microbicide. hiv microbicides are an extremely cost-sensitive market and plant-based griffithsin protein expression has the potential to meet those demands. the griffithsin product used in the clinic has been expressed and purified in n. benthamiana, using a tmv-based viral vector system, geneware®. outdoor pharming of biopharmaceuticals would further alleviate startup costs for biotechnology firms and may allow broader product accessibility. therefore, this study assessed expression in a hybrid tobacco line, n. excelsiana, that is susceptible to tmv-based viral vectors and can be grown outdoors. in addition to using this hybrid line we expand on methods for in planta storage of griffithsin in leafy plants by ensiling kilogram quantities of griffithsin. the ensiling process allows year-round biomanufacturing, minimal environmental-controlled storage, and reduces the industry need for multiple growth areas to maintain multi-product manufacturing of plant-based pharmaceuticals. this study shows that griffithsin can be expressed in n. excelsiana and is stable, recoverable, and active from ensiled tissue. these studies can pave the way for future plant-based pharmaceuticals to be expressed and stored in this manner. human immunodeficiency virus (hiv) is one of many diverse enveloped viruses that can cause life-threatening human disease. as the global hiv infected population continues to grow, there has become an urgent need to find new microbicides that can block viral entry into human cells (lusvarghi and bewley, 2016) . while there have been many potential new therapies to treat hiv, significant research has been done on microbicides that can be topically applied to combat hiv. pre-exposure prophylaxis has been shown to be one of the most effective ways to prevent hiv transmission; however, various issues, such as cost, side effects, access, and adherence have led to less than ideal effectiveness of these drugs (abdool karim et al., 2010; marrazzo et al., 2015; vamvaka et al., 2016; marrazzo, 2017) . this is particularly evident in rural and developing regions, where access to these drugs may be difficult or non-existent. hiv is particularly devastating in sub-saharan africa where it accounts for more than 70% of all global cases (kharsany and karim, 2016) . in this region, one in 20 people is hiv + and up to 8 million people do not have access to basic medications to treat hiv. therefore, there is a particular need to develop a highquality stable virucide that is able to be transported to diverse regions in less-than-ideal environmental conditions that would retain activity to combat the virus months or years after it has been manufactured. griffithsin (grft) is a lectin derived from the red alga griffithsia sp. that has been shown to exhibit broad-spectrum antiviral activity against hiv in picomolar concentrations (mori et al., 2005; emau et al., 2007; vamvaka et al., 2016) . grft's antimicrobial activity comes from its ability to bind to viral envelope glycoproteins, specifically to terminal mannoses on oligosaccharides that are present in several enveloped viruses, such as hiv-1, hcv, and sars-cov (o'keefe et al., 2010; barton et al., 2014 barton et al., , 2016 . this binding activity to surface glycoproteins blocks the virus from entering into human cells. in the case of hiv, grft binds to the surface glycoprotein gp120 and blocks the virus from binding to its receptors on host cells (hoelscher et al., 2018) . grft has shown to be a very effective anti-viral against hiv, and a previous study has shown that manufacturing grft at an industrial scale is feasible and easily scalable (fuqua et al., 2015a,b) . in addition, the manufacturing process was assessed and found to have a highly favorable environmental output index while still having almost no risk to health and safety (alam et al., 2018) . to develop grft as an affordable antiviral, mass production at a commercial scale is required for feasibility and low costs to the consumer. this production would ideally be from an easyto-use construct that is easily scalable and able to be stored and transported with minimal cold chain requirements. grft can only be isolated in small quantities from the native algae species, so various studies have looked at whether it is possible to express this protein in other systems (mori et al., 2005) . previous studies have shown that is possible to express this protein in various organisms, such as e. coli (giomarelli et al., 2006) , tobacco (n. benthamiana) (o'keefe et al., 2009; hahn et al., 2014; fuqua et al., 2015a) , and transgenic rice endosperm (vamvaka et al., 2016) . plants have proven to be particularly advantageous for expressing grft because of high yields of properly folded, active protein. in addition, plants are typically regarded as a safe production platform for biopharmaceuticals (hoelscher et al., 2018) . therefore, current research has focused on how to separate manufacturing and processing from plant growth and how to optimize outdoor plant-based pharmaceutical manufacturing methods. many studies have focused on how proteins of interest may be stored without loss of activity for both short-and long-term use (fuqua et al., 2015b; vamvaka et al., 2016; hoelscher et al., 2018) . silaging provides an efficient method of storing plant material for long-term periods by preserving green or wet crops under anaerobic conditions allowing fermentation. silage is typically used for animal feed purposes and allows livestock to be fed in winter when other food sources may not be available. silage is made by harvesting crops, shredding the plant material, and packing the material tightly to purge as much oxygen as possible. silage is typically stored in a silo, which is a typically oxygen limiting container that does not allow oxygen to come into contact with the plant material. this allows the silage to undergo anaerobic fermentation and produces acid to create a hostile environment for micro-organisms that may cause spoilage (driehuis et al., 2018) . while ensiled plant material is common for livestock, ensiling plant material for plant-based pharmaceuticals (pharming) was proposed by hahn et al. (2014) . in the following study we explored the feasibility and practicality of silage as a method of storing grft expressed in n. excelsiana. using silage as a storage method for antiviral proteins would allow large-scale outdoor growth, a minimally environmentally controlled storage option for shipment of proteins, separates upstream and downstream phases of manufacturing, and reduces the industry need for multiple growth areas to maintain a continuous supply of biomass. n. excelsiana is an interspecific hybrid derived from a cross between n. excelsior and n. benthamiana. seed was kindly provided by kentucky bioprocessing (pogue et al., 2010 (pogue et al., , 2014 shamloul et al., 2014) . n. excelsiana plants were infected with a tmv-based virion carrying the rna expression cassette for grft, similar to previous techniques used to express grft in n. benthamiana (o'keefe et al., 2009) . briefly, plants were grown in controlled indoor environment with a 16-h light and 8-h dark cycle in 8 separate batches. batches of n. excelsiana were sown over a sixweek period and inoculated with either 50 or 150 µg per plant of grft-expressing virion from 20 to 31 days after first sown. all plant material above the soil level was harvested 14 days after inoculation and rough chopped. a portion of the rough chopped plant material was used for expression characterization and the remaining material was ensiled. ensiling was accomplished by rough chopping the plant material, wilting or drying, and then packing into silos. plant material was compressed with a pneumatic press to remove most of the air and then sealed to begin the anaerobic fermentation process. the plant material packed silos were then stored at ambient temperature and humidity for the duration of the study, 116-146 days. after a fixed storage duration was completed, the plant material was unpacked and homogenized in a blender with sodium acetate buffer at ph 4. grft was extracted using this ph 4 buffer, heating to 55 • c, and incubating overnight with a bentonite mgcl 2 mixture (fuqua et al., 2015b) . grft activity was assessed using gp120 elisa and sds-page tests. sds-page was used to separate and analyze the proteins after extraction. to 45 µl of each sample, 15 µl of 4x sds loading dye with 2-mercaptoethanol was added. a gel was loaded with the samples and a ladder in 1x xt mes buffer. electrophoresis was at 200 v for 45 min. coomasie stain was poured onto the gel, microwaved for 30 s, and rotated at 60 rpm for 25-30 min. the gel was then rinsed in di water, and the destain was added to the gel. the gel was microwaved for 30 s and rotated at 60 rpm for 30 more minutes. gels were imaged on a kodak image station 4000r pro and densitometry analysis was done using carestream se m software to analyze amount and size of the proteins found in the samples. a standard curve was generated using known concentrations of grft (1, 3, 5, 10 µg) vs. their gel intensity. unknown samples were then fit to the curve and concentrations were calculated. gp120 elisa gp120-binding elisas were used to assess glycan-binding activity of grft. a 96-well plate was coated with 0.1 ml gp120 at 0.25 µg/ml overnight at 4 • c. the plate was then blocked for a minimum of 2 h with a 3% bsa solution in 1x pbs with 0.05% tween-20 (pbs-t) using 0.3 ml per well. the plate was washed with pbs-t, and grft was added to the wells. the grft had a starting concentration of 250 ng/ml and a 2x dilution in 1x pbs was done across the plate. the plate was incubated for 1 h and then washed. the primary antibody was a rabbit anti-grft polyclonal serum diluted 1:10,000 in 1x pbs, 0.1 ml per well for 1 h, and then the plate was washed. the secondary antibody was hrp-conjugated goat anti-rabbit diluted to 1:25,000 in 1x pbs, 0.1 ml per well for 1 h, and then the plate was washed. 0.1 ml of tmb solution was added to the wells, developed for 8 min, and then 1m h 2 so 4 was added to stop the reaction. the plate was read at 450 nm using a plate reader and plotted optical density (od 450 ) vs. log of concentration and a four parameter non-linear regression was used to fit a curve and extrapolate the ec 50 . all statistical analysis was performed using graph pad prism r 6.0. on eight separate occasions n. excelsiana plants were inoculated with griffithsin virion through a high-pressure spray and allowed to grow for 14 days. n. excelsiana plants averaged a per plant biomass yield of 86.15 ± 5.05 g. plant extract samples showed only two bands on sds-page, at approximately 13 and 19 kda. the molecular weights correspond to grft and tmv coat protein, respectively, as previously reported with expression in n. benthamiana (o'keefe et al., 2009; fuqua et al., 2015b) . each of the eight batches were quantified for protein yield and griffithsin expression was determined by sds-page and densitometry ( table 1) . the average expression was 344 ± 129 mg/kg and ranged from 214 to 584 mg/kg. we observed that inoculation of plants less than 26 days post-sow (dps) provides a trend (pvalue of 0.09) toward higher yields (404 ± 155 mg/kg) vs. plants inoculated at or beyond 31 dps (244 ± 38 mg/kg), when analyzed by a two tailed student's t-test. after quantification of grft expression levels a portion of the plant material from each was then directed to the ensiling process. the first three harvests were not ensiled, but were used to evaluate product expression before committing to the ensiling process. once expression in n. excelsiana was established we ensiled the next five harvests in nine different silos after grft expression was quantified. the silos consist of sealed pvc pipes with a one-way valve to release gases from the fermentation process (see figure 1a) . the average amount of plant material that was successfully ensiled was 0.56 kg (range: 0.372-1.0 kg). plant material was allowed to wilt after harvest because of concerns that water content in n. excelsiana was too high to allow successful silage (queiroz et al., 2018) . ensiled material was removed from the silos and the plug or puck -like plant material was homogenized with buffer in a blender (see figures 1b,c) . the tissue homogenate (green juice) was evaluated by sds-page. grft yields were quantified from densitometry (figure 2) . a subset of silage tissues was clarified with heat and bentonite as previously described in fuqua et al. (2015b) to evaluate the implications of silage material on the purification process. ensiling plant material causes a reduction in plant mass over time with an average of 59% reduction in biomass. the average percent recovery of grft from all silos was calculated based upon the tissue mass and expression level of the plant material at the time of ensiling vs. the total grft recovered from the ensiled material. we recovered approximately 62.8% (range: 28.7-97.1%) of the grft with 161 mg/kg after silage (range: 66.7-230.5 mg/kg) (see table 2 ). activity levels of grft were tested after silage against purified grft control samples and showed comparable activity. grft from silos 2-4 were tested in a gp120 elisa and compared to a purified control product (figure 3) . silos 2-4 were chosen because they represent two different n. excelsiana harvests and were ensiled for the longest period of any of our silos, 138 or 146 days. the recovered silage products had an average ec 50 value of 10.01 ± 0.04 ng/ml vs. the control levels of 13.41 ± 2.08 ng/ml. grft has been shown to be a promising new topical microbicide that can neutralize many viruses with extremely small concentrations of the compound by binding to terminal mannose units on oligosaccharides that are present on the surface of viral envelopes. in addition, grft has been shown to be safe and effective in various conditions, both in vivo and in vitro. previous research has shown grft can be expressed in plants, rice, and even in e. coli. additional studies have demonstrated that grft can be stored in rice or in dried plant material for extended periods of time. this study provides evidence that it is possible to scale up current methods of viral-vector based grft manufacturing by expression in n. exclesiana, a cultivar that can be grown outdoors, unlike the conventional production host n. benthamiana (pogue et al., 2010 (pogue et al., , 2014 . we were able to express grft in n. excelsiana with an average of yield of 344 ± 129 mg/kg, which is approximately half of what is normally expressed in n. benthamiana plants. however, the biomass per plant is nearly double an average n. benthamiana plant and therefore, yields nearly the same amount of protein per plant (shamloul et al., 2014) . the expression values and optimization related to the timing of infection were performed in a controlled indoor growth environment, which allows for evaluation relative to past n. benthamiana expression. n. excelsiana has the advantage of being developed for outdoor plant growth and should be able to be used in a more agricultural scale, allowing field production of grft (pogue et al., 2010 (pogue et al., , 2014 . in previous technoeconomic analysis grft production utilities accounted for over 34% of the total cost of goods (alam et al., 2018) . growing outdoors would eliminate some of those costs and simultaneously reduce the capital investment needed to build a plant growth facility. additionally, outdoor production of grft expressing plants may allow local biopharmaceutical manufacturing in areas with the greatest product needs. grft is being developed as an hiv prophylactic and currently the largest market would be in sub-saharan africa. for this indication using existing local infrastructure to express the protein may provide the best mechanism to meet potential product demands (fischer et al., 2013) . we believe that manufacturing grft products using outdoor pharming will have overall lower start-up costs than the same product expressed in plants in indoor controlled growth environments. however, advantages of outdoor pharming may be offset by additional burdens caused by environmental related controls. environmental monitoring of expression vector release will be required and there is the potential for enhanced material processing to remove environmental contaminants. we are proposing to begin piloting outdoor growth experiments to investigate yield and expression parameters that would provide data for revising our technoeconomic model to include outdoor pharming as our source of plant material. the technoeconomic analysis will account for cost advantages of outdoor pharming, figure 2 | sds-page of silage extracts. plant material from silos 1 and 6 was homogenized with 100 mm sodium acetate ph4 buffer, adjusted to ph4, heated to 55 • c for 15 min, and centrifuged. the process samples and resulting clarified extracts were visualized with sds-page (a). there are few protein impurities and minimum grft loss as the material is processed. four concentrations of purified grft are used as a standard and the primary impurity is tmv coat protein. silos 2-5 and 7-9 were unpacked and materials were homogenized with sodium acetate buffer and the resulting homogenate was visualized with sds-page (b). four concentrations of purified grft are used as a standard and the extract yields two primary bands, a ∼19 kda tmv coat protein band and a ∼13 kda grft band. as well as, for additional expenditures and provide an unbiased cost-estimate of grft production. another important potential issue to understand is that to perform year-round manufacturing with an outdoor crop you would need to grow in a climate conducive to continuous growth or identify a cost-effective stable storage solution for your plant material. even in a climate conducive to year-round plant growth, manufacturing yields would be subject to environmental figure 3 | activity of silage extracts in a gp120 elisa. activity from silos 2-4 was assessed through a gp120 elisa and all show a similar ec 50 to the standard purified grft product. fluctuations. by separating production and processing, several growing areas can be potentially planned out, and harvests can be shipped to a central processing or holding facility where they await purification into a final pharmaceutical product. silaging grft has been discussed and the concept was piloted with leaf tissue vacuum-sealing (hahn et al., 2014) . however, the bench scale experiment performed by hahn et al. (2014) does not meet the requirement for a storage method for bulk biomass that would feed a production facility. in this report we discuss methodologies for storing kilogram quantities of grft-expressing biomass in a manner that requires minimal environmental controls and little biomass manipulation. the practice was modeled after current agricultural practices and could be scaled to accommodate field production. grft was recovered from silage stored at ambient temperatures for > 100 days with no observed impact on potency and product recoveries ranging from 28.7 to 97.1%. the high degree of variation in recovery needs to be explored in depth to identify the critical parameter and make the ensiling process more consistent. we hypothesize the most critical aspect to successful and consist ensiling is managing the liquid content in the plant material. the liquid content of the ensiled material impacts the success of the fermentation process and lost liquid during compression of the plant material likely includes some of the protein of interest. drying or wilting the plant material in the field before ensiling may improve the yields by limiting the amount of expressed protein that escapes the silo. additionally, the ensiling process reduces the biomass of the plant material, which increases the concentration of grft. the reduced processing burden have favorable cost and process implications that should be explored by alternative technoeconomic studies (tuse et al., 2014) . the acidification process imparted by ensiling tissue is not likely conducive to the stability of all proteins and seems to degrade the majority of plant proteins present in the tissue. however, grft and tmv coat protein were both stable through the process, but they both have pis below 5.5. interestingly, tmv has been explored as an antigen carrier in many subunit vaccines and may be another target for silage storage (mallajosyula et al., 2014; banik et al., 2015; mccormick et al., 2018) . in the future we will evaluate the infectivity of tmv and the stability of the virion to better understand if it would be suitable for antigen presentation. this study confirms previous reports on the viability of n. excelsiana as a plant expression host (pogue et al., 2010; shamloul et al., 2014) and is the first report of grft being expressed in n. excelsiana. the data reported here necessitates evaluation in the context of outdoor pharming with a technoeconomic analyses as the primary metric to determine viability. the data support silage as a viable and scalable method to store grft for long-term needs. the recovery led to a high yield of grft, and methods for storing the silage can be optimized using our study findings. utilizing silage allows for ease of transportation and storage, as silage is extremely stable and requires relatively small amounts of space relative to the yield. the study may be applied in whole with n. excelsiana expression supported by ensiling techniques or may be more useful independently, with the ensile process used with n. benthamiana. our preliminary evaluation of ensiling n. benthamiana has resulted in similar positive outcomes. in conclusion, this study showed that growing grft expressed in n. excelsiana at an agricultural scale and ensiled for storage is a method that needs to be explored for cost and simplicity advantages to help understand the processes potential. the datasets generated for this study are available on request to the corresponding author. jc performed the griffithsin analytical experiments and data analysis. rm ensiled the plant material and provided guidance on the scalability of silage. pe drafted the manuscript. kp provided scientific guidance on griffithsin and plant viral vectors. jf directed the experiments, performed data analysis, and drafted parts of the manuscript. all authors reviewed and provided corrections to the manuscript. this work was supported by funding from usamrmc/tatrc funding no. w81xwh-10-2-0082-clin1 and by niaid grant no. u19ai113182. effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of hiv infection in women technoeconomic modeling of plant-based griffithsin manufacturing development of a multivalent subunit vaccine against tularemia using tobacco mosaic virus (tmv) based delivery system pharmacokinetics of the antiviral lectin griffithsin administered by different routes indicates multiple potential uses activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models silage review: animal and human health risks from silage griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide commercial aspects of pharmaceutical protein production in plants bulk production of the antiviral lectin griffithsin improving the large scale purification of the hiv microbicide, griffithsin recombinant production of anti-hiv protein, griffithsin, by auto-induction in a fermentor culture a novel and fully scalable agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants highlevel expression of the hiv entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves hiv infection and aids in sub-saharan africa: current status, challenges and opportunities griffithsin: an antiviral lectin with outstanding therapeutic potential single-dose monomeric ha subunit vaccine generates full protection from influenza challenge hiv prevention: opportunities and challenges tenofovir-based preexposure prophylaxis for hiv infection among african women intranasal administration of a two-dose adjuvanted multi-antigen tmv-subunit conjugate vaccine fully protects mice against francisella tularensis lvs challenge isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae scaleable manufacture of hiv-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems production of pharmaceutical grade recombinant native aprotinin and non-oxidized aprotinin variants under greenhouse and field conditions silage review: foodborne pathogens in silage and their mitigation by silage additives optimization and utilization of agrobacterium-mediated transient protein production in nicotiana manufacturing economics of plantmade biologics: case studies in therapeutic and industrial enzymes rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against hiv the authors would like to thank kentucky bioprocessing (owensboro, ky) for growing and providing n. excelsiana for expression studies. conflict of interest: kp and jf are founders of grow biomedicine, llc that is exploring commercialization of griffithsin technologies.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 eapen, cates, mundell, palmer and fuqua. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-274019-dao10kx9 authors: rife, brittany d; mavian, carla; chen, xinguang; ciccozzi, massimo; salemi, marco; min, jae; prosperi, mattia cf title: phylodynamic applications in 21(st) century global infectious disease research date: 2017-05-08 journal: glob health res policy doi: 10.1186/s41256-017-0034-y sha: doc_id: 274019 cord_uid: dao10kx9 background: phylodynamics, the study of the interaction between epidemiological and pathogen evolutionary processes within and among populations, was originally defined in the context of rapidly evolving viruses and used to characterize transmission dynamics. the concept of phylodynamics has evolved since the early 21(st) century, extending its reach to slower-evolving pathogens, including bacteria and fungi, and to the identification of influential factors in disease spread and pathogen population dynamics. results: the phylodynamic approach has now become a fundamental building block for the development of comparative phylogenetic tools capable of incorporating epidemiological surveillance data with molecular sequences into a single statistical framework. these innovative tools have greatly enhanced scientific investigations of the temporal and geographical origins, evolutionary history, and ecological risk factors associated with the growth and spread of viruses such as human immunodeficiency virus (hiv), zika, and dengue and bacteria such as methicillin-resistant staphylococcus aureus. conclusions: capitalizing on an extensive review of the literature, we discuss the evolution of the field of infectious disease epidemiology and recent accomplishments, highlighting the advancements in phylodynamics, as well as the challenges and limitations currently facing researchers studying emerging pathogen epidemics across the globe. electronic supplementary material: the online version of this article (doi:10.1186/s41256-017-0034-y) contains supplementary material, which is available to authorized users. globalization has dramatically changed the way in which pathogens spread among human populations and enter new ecosystems [1, 2] . through migration, travel, trade, and various other channels, humans have and will continue to intentionally or unintentionally introduce new organisms into virgin ecosystems with potentially catastrophic consequences [3] . humans are not the only culprits, however; global climate pattern changes can alter local ecosystems, creating favorable conditions for the rapid spread of previously overlooked or even undiscovered organisms among humans, giving rise to unexpected epidemics [4, 5] . recent years have been marked by global epidemics of ebola, dengue, and zika, derived from pathogens previously restricted to local outbreaks [6] . according to the world health organization, more than one and a half billion people are currently awaiting treatment for neglected tropical diseases with similar potential for global spread, for which we have limited knowledge of etiology and treatment options [7] . this lack of knowledge further limits our ability to investigate the putative role of these pathogens in future epidemics or even pandemics. epidemiological strategies have been and still are the first line of defense against an outbreak or epidemic. despite conventionality, traditional epidemiological methods for the analysis of global infectious diseases are subject to errors from various sources (fig. 1) and are thus often inadequate to investigate the epidemiology of an infectious disease. putative outbreak investigations typically ensue following case notification of one of the diseases recognized by local and global public health organizations. trained investigators subsequently collect data on cases and diagnoses to establish a disease cluster. during active surveillance, more cases may be detected through outreach to healthcare facilities and nearby health departments. relevant case contacts, such as family, friends, and partners, are also sought to provide details on demographics, clinical diagnoses, and other potential risk factors associated with the spread of the disease [8] . however, the lack of infrastructure, trained personnel, and resources in low-and middleincome countries are prohibitive against field epidemiology investigations, as contact tracing and surveillance both require systematic, unbiased, and detailed investigations. the reconstruction and interpretation of transmission networks are often very sensitive to response, selection, and recall biases and are strictly limited by surveillance data collected in many regions with diverse socioeconomic and cultural backgrounds [9] [10] [11] . in addition, even with a highly effective surveillance system, environmental, zoonotic, and vector-borne transmission dynamics confound analysis by shadowing alternative (i.e., not human-to-human) routes of disease acquisition. furthermore, routine analyses of pathogen subtype and drug resistance are conducted only in a subset of developed nations, wherein variation in screening assays and protocols and therapy regimens increases the discordance in surveillance [12, 13] . despite the limitations to traditional infectious disease epidemiology, major advances in study designs and methods for epidemiological data analysis have been made over the past decade for a multifaceted investigation of the complexity of disease at both the individual and population levels [14, 15] . however, many challenges for infectious disease research remain salient in contemporary molecular epidemiology, such as the incorporation of intra-and inter-host pathogen population characteristics as influential factors of transmission. combating current and future emerging pathogens with potential for global spread requires innovative conceptual frameworks, new analytical tools, and advanced training in broad areas of research related to infectious diseases [16] [17] [18] . an expanded multi-disciplinary approach posits advancement in infectious disease epidemiology research and control in an era of economic and health globalization [2, 16, 19, 20] . fortunately, recent developments in phylogenetic methods have made possible the ability to detect evolutionary patterns of a pathogen over a natural timescale (months-years) and allow for researchers to assess the pathogen's ecological history imprinted within the underlying phylogeny. when reconstructed within the coalescent framework, and assuming a clock-like rate of evolution, the evolutionary history of a pathogen can provide valuable information as to the origin and timing of major population changes [21] . phylogenetic methods also provide key information as to the evolution of both genotypic and phenotypic characteristics, such as subtype and drug resistance (fig. 1) . even though phylogenetic methods are also limited in certain areas, such as restriction of analysis to only the infected population, a significant subset of these limitations can be overcome by complementary use of data from surveillance (both disease and syndromic) and monitoring [22] (fig. 1) . by integrating phylogenetic methods with traditional epidemiological methods, researchers are able to infer relationships between surveillance data and patterns in pathogen population dynamics, such as genetic diversity, selective pressure, and spatiotemporal distribution. systematic investigation of these relationships, or phylodynamics [23] , offers a unique perspective on infectious disease epidemiology, enabling researchers to better understand the impact of evolution on, for example, spatiotemporal dispersion among host populations and transmission among network contacts, and vice versa [21, 24] . the study of the interconnectedness of these pathogen characteristics was previously limited by the cost and timescale of the generation of molecular data. recent decades have been characterized by technology with the ability to rapidly generate serial molecular data from identifiable sources for which we can obtain detailed relevant information through epidemiological surveillance, allowing for the merging of phylodynamics and epidemiology, or evolutionary epidemiology [24, 25] . hence, progress in the field of molecular evolution has provided the opportunity for real-time assessment of the patterns associated with local, national, and global outbreaks [26] , cross-species transmission events and characteristics [27] , and the effectiveness of treatment strategies on current [28] and recurring epidemics [29] . these assessments are essential for monitoring outbreaks and predicting/preventing pandemic inception, a good example being the recent study of middle east respiratory syndrome coronavirus global transmission [30] (additional file 1 (video s1)). but has the, field of evolutionary epidemiology quite reached its full potential? in this article, we systematically discuss how the application of phylodynamic methods has and will continue to impact epidemiological research and global public health to understand and control infectious diseases locally and across the globe. in a strict sense, the concept of phylodynamics is anything but new. the phylogenetic tree reconstructed by haeckel in 1876 using phenotypic traits [31] was used to explain the distribution of the earliest humansthe "twelve races of man"-across the globe and the location of the "centre of creation." this incorporation of both spatial information and phylogenetic relationships in the inference of population distributions and diversity among geographical locations is a branch of phylodynamics, often referred to as phylogeography. since then, the progression of genetic sequencing technology as well as geographical information systems (gis) has enabled evolutionary biologists to gain a higher resolution view of infectious disease dynamics. the 21 st century, in particular, has witnessed unparalleled advances in methods and techniques for molecular sequence data generation and analyses. however, the relationship of progress and perfection is far from linear, along with its relationship to navigational ease. for example, phylodynamic inference has transitioned into a highly statistics-focused process with the corresponding challenges, including informative samples that can significantly affect the accuracy of results [32] [33] [34] . several research groups [32, 33] have reviewed and/or demonstrated the impact of neglecting critical quality control steps on obtaining reliable inferences using the recently developed phylodynamic frameworks, particularly with high throughput, or next-generation, sequencing (ngs) data. some important steps include ensuring uniform spatial and temporal sampling [32] , sufficient time duration between consecutive sample collections for observing measurable evolution [33] , coverage of deep sequencing, and consideration of genomic recombination [34] . the reliance on phylodynamic methods for estimating a pathogen's population-level characteristics (e.g., effective population size) and their relationships with epidemiological data suffers from a high costincreasing the number of inference models, and thus parameters associated with these models, requires an even greater increase in the information content, or phylogenetic resolution, of the sequence alignment and associated phenotypic data. low coverage [35] and the presence of organism-or sequencing-mediated recombination [36] , can skew estimates of the evolutionary rate and even impact the underlying tree topology, particularly when dealing with priors in the bayesian statistical framework commonly used for phylodynamic inference. programs such as splitstree [37] can take as input a nucleotide alignment and output a network in which the dual origins of recombinant sequences are displayed in a phylogeneticlike context. however, network-reconstructing programs have difficulty distinguishing actual recombination events from phylogenetic uncertainty, and branch lengths do not usually reflect true evolutionary distances [38] . despite much work ongoing in this area, there are currently no broadly applicable methods that are able to reconstruct phylogenetic network graphs that explicitly depict recombination and allow for phylodynamic inference. although the bayesian framework has shown to be fairly robust with the inclusion of recombinant sequences in large population studies [39] , the inclusion threshold has not been thoroughly investigated and is likely dependent on a number of factors, such as sample size and sequence length. recombinant sequences are thus usually removed prior to analysis; however, the ability to incorporate recombinant sequences is imperative given our knowledge of the role of recombination in virus adaptation [40] , for example. more details on methods that can potentially account for recombination, applicable to a variety of pathogens, are discussed by martin, lemey, & posada [36] . while the traditional realm of phylogenetics has focused on rapidly evolving viruses, the development of whole-genome sequencing (wgs) has made possible the expansion of phylodynamic methods to the analysis of slower-evolving microorganisms, such as bacteria, fungi, and other cell-based pathogens. wgs has widened the range of measurably evolving pathogens, allowing for the identification of sparse, genetically variable sites, referred to as single nucleotide polymorphisms (snps), among populations sampled at different time points. the use of wgs in phylogenetics is highly beneficial not only in resolving relationships for slower-evolving organisms but also in reconstructing a more accurate evolutionary history (phylogeny) of an organism, rather than the genealogy (single gene), which can differ significantly from the phylogeny due to the presence of selective pressure or even genetic composition [41] . however, as with phylodynamic analysis of rapidly evolving viruses, wgs analysis of cell-based pathogens comes with its own challenges, as discussed in detail elsewhere [42] . implementation of phylodynamic and/or phylogeographic analysis has transitioned over the last two decades from maximum likelihood to the bayesian framework. this framework provides a more statistical approach for testing specific evolutionary hypotheses by considering the uncertainty in evolutionary and epidemiological parameter estimation. given surveillance data (e.g., the duration of infection) and the specification of an epidemiological mathematical model, bayesian phylogenetic reconstruction can also be used to estimate epidemiological parameters that might otherwise be difficult to quantify [21] . for example, during the early stage of an epidemic, wherein the pathogen population is growing exponentially, the rate of exponential growth can be estimated from the phylogeny using a coalescent model that describes the waiting time for individual coalescent events of evolutionary lineages. this rate estimate can be combined with knowledge of the duration of infection for a particular pathogen to estimate the basic reproduction number, r 0 (e.g., [43] ), as well as the prevalence of infection and number of infected hosts. transmission dynamics can similarly be inferred following the early exponential growth of the pathogen, during which the pathogen has become endemic. estimation of these parameters is described more thoroughly in volz et al. [21] . with the expansion of phylodynamic methods to global epidemics, theoretical studies have found that inferences of infection dynamics within the coalescent framework are limited by the assumption of a freely mixing population [32] . this assumption is often violated with the inclusion of several isolated geographical areas with single or few pathogen introductions. without considering this factor, population structure within a phylogeny can severely bias inferences of the evolutionary history and associated epidemiological parameters [32, 44] . to overcome this limitation, software packages such as beast (bayesian evolutionary analysis sampling trees) [45] [46] [47] have recently developed algorithms that allow for the integration of coalescent, mathematical, and spatial diffusion models [48] [49] [50] [51] [52] [53] . more importantly, beast readily implements a comparative phylogenetic approach, which incorporates parameterization of phenotypic trait evolution to identify predictors of population dynamics and spatial spread, all of which are estimated/assessed simultaneously during reconstruction of the evolutionary history [54, 55] . statistical evaluation of the risk factors for pathogen population growth and spread can be performed concurrently with the assessment of phylogenetic resolution within the data [54] , discussed above as a challenge to complex phylodynamic analyses. for example, in the absence of strong phylogenetic resolution, bayesian statistics are more sensitive to long-branch attraction bias [56, 57] , wherein rapidly evolving lineages appear to be closely related, regardless of their true evolutionary relationships. this phenomenon, therefore, influences inferences of spatiotemporal spread of the studied pathogen, as well as estimation of the relationship of pathogen population behavior with potential risk factors, such as climate change, host and/or vector distribution, accessibility and so on. the influence of low-resolution molecular data on the reliability of phylodynamic inferences highlights the importance of the implementation of the method described by vrancken et al. [54] , or even a priori estimation of the phylogenetic and temporal resolution (sufficient time between sampling) [58, 59] . unlike other phylogenetic frameworks, bayesian inference enables utilization of prior knowledge in the form of prior distributions (in combination with information provided by the data); however, abuse of prior knowledge is possible and can lead to incorrect conclusions. even within the bayesian school of thought, scientists do not always agree with regard to the specification of prior distributions under certain conditions. the incorporation of prior information is, however, intuitively appealing, as it allows one to rationalize the probability of an estimate based on previous knowledge of the typical behavior of the parameter among populations of the organism under study. but what can we do if we have no knowledge regarding a particular organism or population? this has become a more pertinent issue recently with the increasing rate of discovery, facilitated by ngs, of organisms for which we have limited prior knowledge, such as novel viruses and bacteria, [60] . one of the advantages of the bayesian phylodynamic approach is the ability to test multiple hypotheses regarding the evolution or epidemiological models used to describe infectious disease behavior, but because of the intricate relationship of these models, reliable inferences require testing of all combinations of the individual proposed models. although often neglected due to computational complexity, improved estimates of marginal likelihoods used for statistical model comparison have been demonstrated with less computational effort [61] . additionally, if we know that we know nothing about the parameter in question, then, in fact, we know something. referred to as the "objective bayesian" approach, this ideal allows researchers to alter a normally "subjective" prior to create one that is minimally informative. this term is used because the impact of this type of prior on parameter estimation can be controlled to a minimum, allowing the data to dominate the analytical process and conclusions drawn [62] . although similarly appealing, this approach can be particularly problematic with small datasets [63] or biased datasets, such as the exclusion of potential intermediate sampling locations [27] . the expanding volume of sequence data and increasing efforts to combine epidemiological and laboratory data in open access locations can help to improve evolutionary estimates. additionally, the growing availability of data and collaboration can accelerate our understanding of the emergence and spread of infectious diseases through coordinated efforts by multidisciplinary researchers across various institutions and public health organizations. more detail on the benefits of open access databases and data sharing in the context of phylogenetic epidemiology is reviewed in [64] and [65] . combining pathogen genetic data with host population information (e.g., population density and air traffic) in a statistical framework is critical for the reliable assessment of factors potentially associated with pathogen population dynamics and geographic spread. the comparative phylogenetic approach described above [66] was used recently to identify potential determinants of the dengue virus (denv) introduction to and spread within brazil. results from nunes et al. [67] suggested that for three denv serotypes, the establishment of new lineages in brazil had been occurring within 7 to 10-year intervals since their primary introduction in 1985, most likely from the caribbean. additionally, they observed that aerial transportation of humans and/or vector mosquitoes, rather than distances between geographical locations or mosquito (particularly aedes aegypti) infestation rates, were likely responsible. the study by nunes et al. marked one of the first uses of the comparative phylogenetic approach for vector-borne tropical diseases and implies the need for a similar approach in future studies aimed at investigating transmission patterns of a broad range of emerging vector-borne viruses. for example, this approach will allow researchers to determine if specific universal factors, such as vector species, are predictive of global transmission route or if health policy and prevention strategies tailored specifically to the pathogen, irrespective of the vector, are required for effective control. with the development of molecular clock models for serially sampled data [68] , phylogenetic analyses have helped to uncover the timing of transmission events and epidemiological origins. moreover, when paired with comparative phylogeographic models, researchers have been able to identify risk factors most likely associated with these particular events. since the inception of the zika virus (zikv) pandemic around may of 2015 in brazil [69] , phylogeneticists and epidemiologists have sought to reveal mechanisms by which zikv has spread and the factors fueling the wide geographical leaps. a full-genome phylogeographic analysis of zikv isolates collected during 1968-2002 revealed very intricate spatiotemporal transmission patterns across africa prior to the introduction into asia [70] . from its origin in uganda, two independent transmission events appeared to play a role in the spread of zikv from east africa to the west circa 1920: the first involved the introduction of zikv to cã´te d'ivoire with subsequent spread to senegal, and the second involved the spread of the virus from nigeria to west africa. results from spatiotemporal analysis demonstrated that uganda was the hub of the african epidemic as well as the common ancestor of the malaysian lineages sampled during the 1966 outbreak [70] . following the emergence and rapid spread of zikv in brazil and other south american countries [69] , faria's group sought to further characterize the spatiotemporal dynamics of zikv following introduction into this region [26] . in addition to sequencing data, air traffic data for visitors to brazil from other countries associated with major social events during 2012-2014 were included to test different hypotheses of airline-mediated introduction of zikv in brazil. the results linked the origin of the brazilian epidemic to a single introduction of zikv estimated to occur between may and december 2013, consistent with the confederations cup event, but predating the first reported cases in french polynesia. although these findings are of great value and importance to public health organizations, the authors drew an additional, and similarly valuable conclusion-large-scale patterns in human (and mosquito) mobility extending beyond air traffic data will provide more useful and testable hypotheses about disease emergence and spread than ad hoc hypotheses focused on specific events. this conclusion further supports the proposal for greater availability of epidemiological data among the scientific community. understanding both the rapid spread of the virus throughout south and central america and the caribbean as well as the initial emergence of the virus from the ugandan zika forest in the early 1900s is important for application to the control of future outbreaks, but increasing data may not be the only answer. moreover, several different risk factors are likely responsible for these two migration events. therefore, a more comprehensive approach that allows for the analysis of multiple potential factors and their distinct contribution to independent migration events without the loss of information (i.e., use of data that span the entire evolutionary history) is imperative for fully understanding a global epidemic from beginning to present. a combined approach to understanding the emergence and expansion of an epidemiologically diverse viral population: hiv crf02_ag in the congo river basin although viral spread is often attributed to human mobility [71] , factors such as population growth and accessibility can also play an important role, as with the emergence of human immunodeficiency virus type 1 (hiv-1) group m subtypes a and d in east africa [72] and circulating recombinant form (crf) 02_ag in regions of the congo river basin (crb) [73] . the democratic republic of congo (drc) has been reported to be the source of hiv-1 group m diversity [74] [75] [76] ; however, the epidemiological heterogeneity of crf02_ag within surrounding regions comprising the crb had remained a mystery since its discovery in 1994 [77] , with prevalence ranging from virtual non-existence [78] [79] [80] [81] [82] [83] to accounting for as high as 20% of infections [84] , depending on the geographical location. the region with the highest proportion of crf02_ag infections, cameroon [85, 86] , has been characterized by a rapidly growing infected population (0.5% in 1985 to 6% in 2008 [87] ), of which the majority (60%) is caused by this clade. using both molecular sequence data and unaids surveillance data [88] , the spatiotemporal origin of crf02_ag was estimated to occur in the drc in the early 1970s (1972) (1973) (1974) (1975) , with the rapid viral population growth in cameroon following a chance exportation event out of drc. although similar phylodynamic techniques as described above for other viral species were used to infer the spatial origins of crf02_ag, the timing of the origin of this viral clade was inferred using both coalescent analysis of molecular sequence data and prevalence information [73, 89] . coalescent models allow for estimation of the effective population size (ne), of fundamental importance to infectious disease epidemiology, as it describes the level of genetic diversity within a population over the course of its evolutionary history. during the exponential growth period of an epidemic, the change in ne has been shown to linearly correlate with prevalence of infection [90, 91] and can, therefore, be used to estimate the latter, as mentioned above, but also, when combined, faria et al. [73] were able to show that fitting of ne and prior prevalence data can narrow the uncertainty of the temporal origin estimates by over 29% as compared to coalescent estimates alone. furthermore, surveillance data was recently used during simultaneous phylodynamic coalescent estimation to identify factors associated with ne dynamics throughout the entire evolutionary history of the cameroonian sequences [92] , revealing that changes in ne were more reflective of incidence dynamics rather than prevalence, consistent with previous mathematical modeling [90, 91] . although associations between ne and potentially related factors are frequently assessed, statistical analysis of these has until recently been primarily limited to post hoc examination (e.g., [91, 93] ), which ignores uncertainty in demographic reconstruction, as discussed above. simultaneous implementation of evolutionary reconstruction and estimation of the relationship of covariate data with ne will be available in the newest version of beast v1 [92] . although this tool has obvious implications for global assessment of factors contributing to the growth and dynamics of an epidemic, similar applications of this method to other data sets has suggested that reduced molecular data relative to covariate data may result in an impact of inclusion of the data on ne estimates. this finding posits a potential concern for convenience sequence sampling, as factors that are not responsible but are represented by large amounts of data may influence ne estimates, resulting in unreliable population dynamic inferences. as mentioned above, care is needed to ensure sufficient sampling and an appropriate sampling strategy for reliable reconstruction of the evolutionary and epidemiological history of the infectious organism of interest. traditional phylodynamic analysis applied to nosocomial outbreaks has been successfully used in the past to identify the likely source; however, the inclusion of extensive patient data, such as treatment regimens, admission and discharge dates, and length of stay, can improve not only phylogenetic estimates but also the translation of the interpretation to public health policy. epidemiological and genomic data on methicillin-resistant staphylococcus aureus (mrsa) infections were recently utilized by azarian and colleagues to reconstruct mrsa transmission and to estimate possible community and hospital acquisitions [94] . findings from this study revealed that as high as 70% of the mrsa colonization within the hospital's neonatal intensive care unit (nicu) was acquired within the nicu itself. these findings indicated that current, standard prevention efforts were insufficient in preventing an outbreak, calling for the improvement of current care or alternative implementation strategies. the earlier uses of phylodynamic methods focused primarily on the molecular evolution of rapidly evolving viruses, greatly advancing the fields of virus vaccine and treatment strategies [23] . on the other hand, epidemiological approaches have focused on influential factors related to social, economic, and behavioral patterns. integrating the phylodynamics and epidemiology approaches into a single analytical framework, referred to as evolutionary epidemiology [24, 25] , represents one of the most powerful multi-disciplinary platforms. examples discussed herein of the adoption of an integrative and multifactorial mindset reveal the potential for accelerating our understanding of the emergence and spread of global infectious diseases, presently expanded to include bacterial and other cell-based pathogens. however, although a highly evolved analytical platform and an improved understanding of the translation of molecular evolutionary patterns to infection and transmission dynamics have aided in facilitating this transition, several challenges still remain. the 21 st century has witnessed a major shift in breadth of scientific knowledge at the level of the individual researcher, requiring more focused training (e.g., molecular mechanisms) and greater collaborative efforts; meanwhile, a consensus of commonality and crossdisciplinary understanding is necessary for globalization of not only the economy, but also public health. this kind of understanding can be better achieved through interdisciplinary instruction on the theoretical and application skills related to both phylogenetics and epidemiology during early education. if successfully achieved, this combined training, in addition to access to modern ngs technology, such as handheld sequencers, would increase the mobility of labs and researchers, expanding the concept of lab-based research. mobilized labs would, in turn, reduce our current reliance on few major public health organizations and the impact of limited resources on sampling and surveillance in developing countries. increasing mobility is nevertheless inconsequential without the cooperative sharing of genomic and epidemiological information. although data are typically readily available to the public following peer-reviewed publication, the median review time of manuscripts submitted to, for example, nature is 150 days [95] , this in addition to the time required for thorough analysis of the original data. this timeline seems quite long in retrospect of the 1918 "spanish flu," which spread to one-third of the global population in a relatively brief 12-month period [96] . data sharing prior to publication, even if only among a proportion of consenting institutions, may accelerate the process of dissemination of research findings to public health decision makers and practitioners, and its practice is not entirely unheard of. an excellent example of this type of collaboration is the "nextstrain" project (http://www.next strain.org/). nextstrain is a publicly available repository currently comprised of evolutionary datasets for ebola, zika, and avian and seasonal influenza viruses contributed by research groups from all over the world for the purpose of real-time tracking of viral epidemics. similar projects have also recently developed in other research fields. modeled after the stand up to cancer initiative, the synodos collaborative funded by the children's tumor foundation in partnership with sage bionetworks brings together a consortium of multidisciplinary researchers, who have agreed to the sharing of data and relevant information, as well as results [97] . the ultimate goal of this cooperation is to accelerate the drug discovery process, which is highly applicable to global infectious disease research. without a similar collaborative approach to synodos, the preparedness of the global reaction to rising epidemics is at risk. recent years have been marked by local outbreaks across vast geographical regions within a timespan of months to years. hence, both the rapid dissemination of data and results and the rapid response of government and public health organizations are required for the effective prevention of a global epidemic, or pandemic. additionally, with the type of results, particularly risk factors, that are generated using this multifaceted approach (e.g., both human population and pathogen molecular characteristics), the question then arises of how organizations will actually utilize this information for treatment and prevention strategies. moreover, as the techniques and methods advance, are the infrastructures in place for global cooperation and immediate response following the presentation of a potentially more complex story? although gaps remain in current evolutionary modeling capabilities when used with epidemiological surveillance data, it is only a matter of time before the challenges described herein and elsewhere are met with more realistic models that capture the complexity of infectious disease transmission. furthermore, theoretical research in the field of infectious disease phylodynamics is still growing. consequently, there is a need for a review of the more recently developed methods and techniques and their performance, as well as their application in areas within and outside the realm of infectious disease. for example, in the era of global health, translational genomics, and personalized medicine, the accumulating availability of genetic and clinical data provides the unique opportunity to apply this approach to studies of, e.g., tumor metastasis and chronic infections, which comprise complex transmission dynamics among tissues and/or cell types, not unlike the geographical spread of infectious diseases. globalization and health understanding the development and perception of global health for more effective student education globalization of infectious diseases: the impact of migration the ecology of climate change and infectious diseases global climate change and emerging infectious diseases deciphering emerging zika and dengue viral epidemics: implications for global maternal-child health burden traditional and syndromic surveillance of infectious diseases and pathogens a structural approach to selection bias recall bias in epidemiologic studies response bias, social desirability and dissimulation resistance considerations in sequencing of antiretroviral therapy in low-middle income countries with currently available options surveillance for antimicrobial drug resistance in under-resourced countries new tools to understand transmission dynamics and prevent hiv infections the global one health paradigm: challenges and opportunities for tackling infectious diseases at the human, animal, and environment interface in low-resource settings charting a future for epidemiologic training the role of epidemiology in the era of molecular epidemiology and genomics: summary of the 2013 ajesponsored society of epidemiologic research symposium transforming epidemiology for 21st century medicine and public health the eco-in eco-epidemiology eco-epidemiology: thinking outside the black box viral phylodynamics overview of syndromic surveillance what is syndromic surveillance? mmwr unifying the epidemiological and evolutionary dynamics of pathogens phylogenetic and epidemic modeling of rapidly evolving infectious diseases evolutionary epidemiology: preparing for an age of genomic plenty zika virus in the americas: early epidemiological and genetic findings genomic analysis of the emergence, evolution, and spread of human respiratory rna viruses ebola virus epidemiology, transmission, and evolution during seven months in sierra leone eighteenth century yersinia pestis genomes reveal the long-term persistence of an historical plague focus the global spread of middle east respiratory syndrome: an analysis fusing traditional epidemiological tracing and molecular phylodynamics new york: d. appleton and company the effects of sampling strategy on the quality of reconstruction of viral population dynamics using bayesian skyline family coalescent methods: a simulation study exploring the temporal structure of heterochronous sequences using tempest (formerly path-o-gen) towards a new paradigm linking virus molecular evolution and pathogenesis: experimental design and phylodynamic inference the effect of ambiguous data on phylogenetic estimates obtained by maximum likelihood and bayesian inference analysing recombination in nucleotide sequences application of phylogenetic networks in evolutionary studies a comparison of phylogenetic network methods using computer simulation the early spread and epidemic ignition of hiv-1 in human populations why do rna viruses recombine? improving bayesian population dynamics inference: a coalescent-based model for multiple loci measurably evolving pathogens in the genomic era pandemic potential of a strain of influenza a (h1n1): early findings the confounding effect of population structure on bayesian skyline plot inferences of demographic history bayesian phylogenetics with beauti and the beast 1.7 beast: bayesian evolutionary analysis by sampling trees beast 2: a software platform for bayesian evolutionary analysis bayesian phylogeography finds its roots counting labeled transitions in continuous-time markov models of evolution fast, accurate and simulation-free stochastic mapping phylodynamic inference for structured epidemiological models new routes to phylogeography: a bayesian structured coalescent approximation efficient bayesian inference under the structured coalescent simultaneously estimating evolutionary history and repeated traits phylogenetic signal: applications to viral and host phenotypic evolution generalized linear models for identifying predictors of the evolutionary diffusion of viruses long-branch attraction bias and inconsistency in bayesian phylogenetics on the distributions of bootstrap support and posterior distributions for a star tree iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies how to measure and test phylogenetic signal virus discovery in the 21st century bayesian evolutionary model testing in the phylogenomics era: matching model complexity with computational efficiency the case for objective bayesian analysis analyzing small data sets using bayesian estimation: the case of posttraumatic stress symptoms following mechanical ventilation in burn survivors make data sharing routine to prepare for public health emergencies a code of conduct for data on epidemics unifying viral genetics and human transportation data to predict the global transmission dynamics of human influenza h3n2 air travel is associated with intracontinental spread of dengue virus serotypes 1-3 in brazil bayesian molecular clock dating of species divergences in the genomics era zika: the origin and spread of a mosquito-borne virus molecular evolution of zika virus during its emergence in the 20th century population migration and the spread of types 1 and 2 human immunodeficiency viruses spatial phylodynamics of hiv-1 epidemic emergence in east africa phylodynamics of the hiv-1 crf02_ ag clade in cameroon aids: prehistory of hiv-1 human immunodeficiency virus: phylogeny and the origin of hiv-1 unprecedented degree of human immunodeficiency virus type 1 (hiv-1) group m genetic diversity in the democratic republic of congo suggests that the hiv-1 pandemic originated in central africa coding region of a new hiv type 1 subtype a strain (hiv-1 ibng) from nigeria genetic diversity of hiv type 1 in likasi, southeast of the democratic republic of congo genetic subtypes of hiv type 1 in republic of congo hiv-1 subtypes and recombinants in the republic of congo increasing hiv type 1 polymorphic diversity but no resistance to antiretroviral drugs in untreated patients from central african republic: a 2005 study increase of hiv-1 subtype a in central african republic highly divergent subtypes and new recombinant forms prevail in the hiv/aids epidemic in angola: new insights into the origins of the aids pandemic analysis of partial pol and env sequences indicates a high prevalence of hiv type 1 recombinant strains circulating in gabon the prevalence of diverse hiv-1 strains was stable in cameroonian blood donors from two independent epidemics of hiv in maryland world health organization. who | epidemiological fact sheets on hiv and aids epidemic dynamics revealed in dengue evolution viral phylodynamics and the search for an 'effective number of infections' understanding past population dynamics: bayesian coalescent-based modeling with covariates a high-resolution genetic signature of demographic and spatial expansion in epizootic rabies virus genomic epidemiology of methicillin-resistant staphylococcus aureus in a neonatal intensive care unit does it take too long to publish research? influenza: the mother of all pandemics children's tumor foundation announces historic new initiative in neurofibromatosis research the "virogenesis" project receives funding from the european union's horizon 2020 research and innovation program under grant agreement no. 634650. not applicable.availability of data and materials not applicable. authors' contributions bdr and cm contributed to the writing of the manuscript and creating of figures and additional material. bdr, cm, xc, mc, ms, jm, and mp discussed the contents of the manuscript and contributed to editing and revision. all authors read and approved the final manuscript. no financial or non-financial competing interest existed for any one author during the writing of this manuscript. not applicable. reported studies do not involve human participants, human data or human tissue. submit your next manuscript to biomed central and we will help you at every step: key: cord-283346-0v4b6do2 authors: ansari, abdul wahid; bhatnagar, nupur; dittrich-breiholz, oliver; kracht, michael; schmidt, reinhold e.; heiken, hans title: host chemokine (c-c motif) ligand-2 (ccl2) is differentially regulated in hiv type 1 (hiv-1)-infected individuals date: 2006-08-17 journal: int immunol doi: 10.1093/intimm/dxl078 sha: doc_id: 283346 cord_uid: 0v4b6do2 several cytokines and chemokines including chemokine (c-c motif) ligand-2 (ccl2) are induced in hiv-1 infection. however, the impact of hiv-1 viremia on ccl2 regulation is largely unknown. we utilized a dna oligonucleotide microarray covering 110 inflammatory genes. five genes were induced by at least 2-fold in pbmcs of hiv-1 viremic (>100.000 rna copies ml(−1)) as compared with aviremic (<50 rna copies ml(−1)) individuals. these genes were ccl2, cxc chemokine ligand-10, ifn-γ, gtp-cyclohydrolase-1 and c-c chemokine receptor-1. in addition to microarray data verification by real-time pcr, analysis of independent patient samples revealed a similar expression pattern. ccl2 was the most strongly regulated gene at mrna level and its serum concentration was significantly elevated in viremic compared with aviremic and hiv-1 seronegative controls, indicating a positive correlation between viremia and ccl2. flow cytometric studies demonstrated a higher percentage of ccl2-expressing cd14+ monocytes in viremic compared with aviremic individuals. these results suggest a highly restricted modulation of host inflammatory gene response by hiv. genes up-regulated in the viremic state, in particular ccl2, presumably serve as potential enhancing factors in hiv-1 replication, represented by high viral load in hiv-1 viremic patients. inhibition of increased ccl2 production could provide a new therapeutic intervention in hiv-1 infection. hiv type 1 (hiv-1) infection transforms the host's cellular environment into a favorable niche for its replication and survival (1, 2) . in most instances, an alteration of the cytokine and chemokine network is observed. however, potential effects of viremia on these inflammatory mediators are not fully elucidated. monocytes and macrophages are not only the prime targets for hiv-1 infection but also a source of inflammatory cytokines and chemokines (3) . these key modulators of the immune system play a critical role in recruiting new target cells including cd4+ t cells to the site of infection (4) . in the past few years, significant progress has been made to understand the molecular mechanisms underlying the host-pathogen interaction with the advent of microarray technology (5, 6) . however, until now we do not know the crucial host factors influencing this host-virus relationship. there are reports about general impact of hiv-1 on host cell gene expression both in patient-derived b and cd4+ t cells as well as in in vitro infected cells (7) (8) (9) (10) . however, the role of hiv-1 viremia on host cell cytokine/chemokine expression has not been studied in detail. during hiv-1 infection, a series of cytokines and chemokines is induced which may either support or inhibit viral replication. for example, the c-c chemokines macrophage inflammatory protein (mip)-1a/ccl3, mip-1b/ccl4 and regulated upon activation, normal t cell expressed and secreted (rantes)/ ccl5 inhibit m-tropic hiv-1 infection by competing with the virus for its binding to the co-receptor ccr5 (11) (12) (13) (14) (15) (16) (17) . in contrast, there are reports demonstrating stimulation of hiv-1 replication by the chemokines growth-regulated oncogene-a, cxcl10 and ifn-c inducible protein (ip)-10/cxcl10 (18, 19) . macrophage chemoattractant protein (mcp)-1, also known as ccl2, attracts monocytes, t lymphocytes and nk cells in vitro (20, 21) . ccl2 is expressed by various cell types and is induced after hiv-1 infection of macrophages (22) . ccl2 along with mip-1a, mip-1b and rantes has been demonstrated to increase the replication of t-tropic strains in cd4+ t cells (23) . recently, it has been reported that ccl2 plays a critical role in the neuropathogenesis of hiv-1-associated dementia and protects human neurons and astrocytes from n-methyl-d-aspartate or hiv-tat-induced apoptosis (24) . to investigate the impact of hiv-1 viremia on the host inflammatory cytokine/chemokine network, more specifically on ccl2, we utilized dna microarray approach on pbmc rna derived from hiv-1-infected viremic and aviremic individuals. in this study, we report that hiv-1 viremic patients show an altered expression of key inflammatory cytokines and chemokines as compared with aviremic individuals. in particular, ccl2 is highly regulated both at mrna and protein level, demonstrating a correlation between viremia and host ccl2 response. a total of 10 aviremic and 10 viremic hiv-1 patients were analyzed for gene expression studies. for determination of serum ccl2 concentration, 36 hiv-1 patients were selected based on their viral load (18 aviremic and 18 viremic). clinical characteristics and therapy status of each patient are given in table 1 . in addition to above subjects, a new cohort of patients (n = 16) was recruited for ccl2, cxcl10 and ifn-c gene expression analysis by real-time pcr. for plasma viral load measurement, peripheral blood was drawn in edta-coated tubes and stored at -80°c until use. viral load was detected by standard or ultra-sensitive cobas amplicor hiv-1 monitor tm test version 1.5 (roche diagnostics, mannheim) or by b-dna analyzer system 340 (bayer diagnostics, fernwald) at our center. all subjects except patient number a6, b7, b9 and c13 were on highly active anti-retroviral therapy (haart) at the time of this study. written informed consent from each subject and approval from institutional ethics review board were obtained before this study. heparinized venous blood was drawn from hiv-1-infected viremic individuals maintaining a viral load of >100 000 rna copies ml ÿ1 and from aviremic individuals with undetectable viral load of <50 rna copies ml ÿ1 in their plasma. pbmcs were isolated from these patients by conventional ficoll-hypaque (biochrome ag, berlin) gradient centrifugation. cells were washed with pbs and rna extraction procedure was immediately followed according to manufacturer's protocol of rneasy mini kit (qiagen, hilden, nl, usa). all rna samples were treated with dnasei (rnase-free dnase set, qiagen) to eliminate genomic dna contamination. the microarray used in this study was the first version of the inflammation array (mwg biotech) and contained 110 oligonucleotide probes for inflammatory genes that were previously validated by our laboratory (25) as well as five 'housekeeping' genes. each probe was spotted twice in random order. total rna from pbmc of aviremic and viremic patients as indicated in the legend of table 2 was purified with a qiagen rneasy kit followed by 'on column' dnasei digestion (qiagen). rna was used to prepare cy3-labeled crna by oligo dt-t7-primed double-stranded cdna synthesis (cdna synthesis system, roche) followed by in vitro transcription with t7-polymerase (megascript t7 kit, ambion) as directed by the manufacturers. cdna yield was determined photometrically. equal amounts of crnas derived from~2.5 lg of total rna were hybridized individually to microarrays in preprepared hybridization solution (mwg biotech) at 42°c overnight and then washed sequentially in 23 ssc, 0.1% highly regulated and hiv-1 pathogenesis-associated genes were selected for further verification by real-time pcr. approximately 1-2 lg of pooled rna from each patient group or individual patients were reverse transcribed using oligo(dt) and omniscript reverse transcriptase (qiagen) according to manufacturer's instructions. diluted cdna was used as template and amplified using gene-specific primers (table 3) . primers were designed with the aid of primer express software (pe applied biosystems gmbh, weiterstadt) and synthesized by mwg biotech. the taqman b-actin control reagents (pe applied biosystems) or glyceraldehydes-3phosphate dehydrogenase (gapdh) were standard internal housekeeping controls. all amplifications were performed either with abi prism 7700 sds (pe applied biosystems) or icycler (biorad, munich) using 10 lm concentration of each primer in a final reaction volume of 25 ll in duplicates for 40 cycles using sybr green i chemistry. to obtain a standard curve, transcribed cdna was serially diluted. mrna levels were normalized according to the expression of b-actin/gapdh in the same sample and values obtained for viremic patients expressed relative to those obtained for aviremic patients which were set as 1. fold change in the genes were calculated by 2 ÿddc t method (26) . to determine the level of ccl2 production in hiv-1-infected aviremic and viremic patients, we examined serum of 48 individuals. eighteen viremic and 18 aviremic hiv-1 patients and 12 hiv-1 seronegative healthy controls were included. all aviremic and majority of the viremic patients were receiving anti-retroviral therapy at the time of study. sandwich ccl2 two pools of rna generated from pbmc of three (patients a1-a6, experiment 1) or five (patients b1-b10, experiment 2) or rna isolated from two individual patients (patients c11-c14, experiment 3), respectively, were used to compare inflammatory gene expression in the aviremic with the viremic state. microarray experiments were performed as described in methods. original spot intensity measurements for the group of genes that was consistently up-regulated by at least 2-fold in pbmc of viremic patients and whose signal intensity was at least 3% of the average signal intensity of all genes on that particular array are shown. for comparison, the spot measurements of four 'housekeeping' genes are also shown. identifiers for the genes (refseq accession number) and for the oligonucleotide probes (oligo name of the mwg human inflammation array) are shown. for data on the individual patients (indicated by capital letters followed by numbers) see table 1 . the complete set of dna microarray data can be obtained upon request (kracht.michael@mh-hannover.de). statistical analyses of two groups of subjects were performed with student's t-test. evaluation of three groups was performed with one-way analysis of variance test. all analyses were done using prism 4 (graphpad) software package. p values <0.05 were considered significant. to understand host cellular inflammatory gene response to hiv-1 infection, we examined a larger range of well-known inflammatory genes in 20 patients, whose clinical features are summarized in table 1 . for microarray screening, four pools of rnas were generated representing rna from three and five patients of the aviremic and viremic state, respectively. in addition, rna samples from four individual patients were also analyzed separately by microarray. a total of 110 inflammatory genes were analyzed. as indicated in table 2 , five genes were reproducibly upregulated by at least 2-fold in all experiments. these genes comprised ccl2 (mcp-1), cxcl10 (ip-10), ifn-c, gch1 (metabolic enzyme) and ccr1 (mip-1a receptor). all other genes were either not regulated reproducibly or not expressed at significant levels (data not shown). furthermore, we could not detect genes that were reproducibly down-regulated in viremic patients. differentially expressed genes analyzed by quantitative real-time pcr mrna expression of five induced genes detected by dna microarray was further analyzed by quantitative real-time pcr using gene-specific primers. analysis of rna from pooled samples of experiment 2 (according to table 2 ) confirmed up-regulation of ccl2, cxcl10, ifn-c, gch1 and ccr1 (fig. 1a) . this was also true when re-analyzing rna from four individual patients shown in fig. 1(b) . although a certain degree of variability was observed between the two viremic patients, the overall expression of these genes was clearly higher than in both hiv-1 aviremic individuals. furthermore, increased mrna transcripts were again detected in ccl2, cxcl10 and ifn-c when more rna samples derived from additional aviremic and viremic patients were analyzed individually. significantly higher mrna transcript was observed in viremic (p < 0.05) compared with aviremic in all genes ( fig. 2a) , confirming the consistency with microarray data. ccl2, cxcl10 and ifn-c showed 3-fold higher expression in viremic patients. however, haart status in viremic patients did not affect mrna expression of the analyzed genes. in addition, in vitro hiv-1 iiib infection of healthy donor pbmc resulted in strong induction of ccl2 as measured by real-time pcr (fig. 2b) . these data are in line with previous findings (22, 27) . in general, a t-tropic (x4) strain infects cd4 t cells rather than monocytes but there is a small population of cxcr4expressing monocytes which may become the target cell for x4 viruses, although we cannot formally exclude the possibility of bystander effects mediated by the factors released by cd4 t cell upon hiv-1 iiib engagement. however, there are reports about higher levels of pro-inflammatory mediators such as tumor necrosis factor (tnf)-a, il-6 and il-1b in hiv-1 disease, and these mediators are also known to play a role in enhancing ccl2 expression. of note, ccl2 mrna levels in long-term nonprogressors (n = 2) hiv-1 patients were similar to that of aviremic patients (data not shown). collectively, these findings suggest that a high viremia is associated with a selected set of inflammatory host genes and confers a greater impact on ccl2. to investigate the ccl2 protein level, we performed a ccl2 elisa on sera from 12 healthy donors, from 18 aviremic patients receiving haart, and from 18 viremic patients selected on the basis of high viral load who were receiving haart or not. a significantly higher concentration of ccl2 protein was observed in hiv-1 viremic (p = 0.0008) in comparison with aviremic individuals (fig. 3a) . a positive correlation (r 2 = 0.116 and p = 0.045) between viremia and ccl2 (fig. 3b) was obtained by linear regression analysis. regression analysis of cd4/cd8 t cell ratio versus ccl2 pointed to an inverse correlation (p = 0.060 and r 2 = 0.10) which did not reach statistical significance (data not shown). moreover, additional experiments with intermediate viral load patients ranging between 400 and 100 000 rna copies ml ÿ1 revealed a similar correlation (p = 0.018 and r 2 = 0.122), suggesting that production of ccl2 varies with the viral load (supplementary figure 1 , available at international immunology online). given the fact that viremic patients had elevated concentrations of ccl2 in the serum, it was interesting to know whether the potency of the patient's pbmc to produce ccl2 could be recapitulated in vitro. two-color flow cytometry was performed on previously frozen pbmc samples. in order to obtain uniform conditions, cells were stimulated with pma for 24 h prior to staining. as expected, monocytes were primary producers of ccl2. the dot plot analysis clearly indicated a higher production of ccl2 by cd14+ monocytes derived from viremic patients (fig. 4a ). there was a highly significant increase in the percentage of ccl2-producing monocytes in viremic (p < 0.005) as compared with aviremic patients (fig. 4b) . this was also true when flow cytometry was performed using freshly isolated pmbc of hiv-1 patients (supplementary figure 2 , available at international immunology online). furthermore, we could demonstrate a strong inverse correlation (p = 0.019 and r 2 = 0.515) between cd4+ t cells and percentage of ccl2+ cd14+ monocytes (fig. 4c) . these findings suggest that viremic patients with low cd4 t cell counts have a high ccl2 expression which could be a potential marker of disease progression. however, we could not observe a significant statistical correlation with viral load (data not shown) except a tendency toward a positive correlation. the data presented here are based on a small cohort of patients (n = 10, including six aviremic and four viremic). a possible explanation could be that monocytes are not the sole contributors of ccl2 found in serum, and other cells secrete ccl2 that finally comes into circulation. in contrast to ccl2, we could not detect any significant change (p = 0.211) in the expression of the ccr2, the receptor for ccl2, on patient monocytes (fig. 4d ), indicating that viremia had no significant effect on receptor expression. we analyzed 10 aviremic and 10 viremic hiv-1-infected patients by dna microarray approach. taking into account that blood samples of the patients had to be collected over a longer period of time and to account for variability among individual patients, rna samples were divided into three separate microarray experiments in which rna samples were either pooled or analyzed individually. furthermore, strict filter criteria were applied to identify the genes that were (i) expressed at measurable levels and (ii) regulated in all experiments by at least 2-fold. this analysis revealed a significant effect of viremia on the mrna level of ccl2, cxcl10, ifn-c, gch1 and ccr1. induction of ccl2 and cxcl10 are in line with recent studies on siv-or simian hiv (shiv)-infected macaques model of hiv encephalopathy (28) . to exclude the possibility that the induction of the genes could be the response of one or two patients in the pooled samples, we extended our investigations to a fresh cohort of patients not included in the previous studies using the real-time pcr approach. again a similar expression pattern in ccl2, cxcl10 and ifn-c mrna was observed in viremic as compared with aviremic patients. these results may imply that persistent infection itself, as present in aviremic patients, does not lead to significant upregulation of these inflammatory mediators. only when viral replication is detected, as found in viremic patients, the host inflammatory response leads to an increase in mrna expression of specific inflammatory genes. monocytes from hiv-1 patients have been shown to express more ccl2 when compared with healthy controls (29) . in this regard, our flow cytometric analysis of patient-derived pbmc showed a clear increase in the ccl2-expressing cd14+ monocytes in viremic patients. this increase in the percentage of cd14+ccl2+ monocytes inversely correlates with the cd4+ t cell count. since low cd4+ t cell count is associated with viremia and disease progression, an increased expression of ccl2 in these patients could potentially reflect an additional marker of disease progression. ccl2 is induced in many inflammatory diseases including infection with various bacterial and viral pathogens. production of ccl2 is not specific to hiv-1. several other viruses have been shown to induce ccl2 expression. for example, viruses such as human rhinovirus (30) , respiratory syncytial virus (31) , severe acute respiratory syndrome coronavirus (32) and shiv (33) , the best available primate model of hiv-1 infection, have been reported to induce ccl2 expression. recently, it has been shown that the recruitment of cd4+ t cells by cxcl10 and 11 produced by hiv-1-infected monocyte-derived macrophages and dendritic cells in lymphoid organs contributes to hiv-1 disease (34) . in this regard, in vitro hiv-1 infection of macrophages is already known to activate ccl2 expression and ccl2 may potentially influence hiv-1 pathogenesis (22) . we also observed a similar transcriptional regulation in the ccl2 gene in in vitro hiv-1 infection of pbmc obtained from four healthy volunteers. a very recent study using a mouse model of hiv-1 infection demonstrated ccl2 gene induction in brain tissues of infected animals (35) . these data suggest that hiv-1 viremia alters the transcriptional program of the host cytokine/chemokine network, and this effect is most prominent with respect to ccl2 expression. transcriptional regulation could be translated into production of ccl2 because significantly elevated serum ccl2 concentrations were observed in hiv-1 viremic as compared with aviremic or healthy individuals. furthermore, we could demonstrate a positive correlation between viremia and serum ccl2. interestingly, an inverse correlation of cd4+ t cells was associated with more ccl2-expressing cd14+ monocytes. since ccl2 is a strong lymphocyte chemoattractant, its higher production by host cells upon hiv-1 infection may result in recruitment of more target cells to the site of infection, resulting in higher hiv-1 replication and rapid spreading of the virus (fig. 5) . we can assume that the concentration of ccl2 at the site of infection is much higher than in the periphery; hence, a strong recruitment of ccr2-expressing target cells can be expected. ccr2, the receptor for ccl2, is expressed on the majority of monocytes and to a lesser extent on cd4+ t cells. however, we could not observe any significant effect of viremia on ccr2 expression on these cells, suggesting ccl2 concentration-dependent but surface ccr2-independent recruitment of target cells to the site of infection. higher ccl2 expression might be at least in part mediated by tnf-a since it is a well-known inducer of ccl2, and higher tnf-a levels are associated with viremia as reported in many studies. moreover, prior exposure of monocytes or monocyte-derived macrophages to b-chemokines has already been shown to enhance viral replication (36) . treatment with the hiv-1 protease inhibitor indinavir reduces the plasma ccl2 level in severely immunodeficient hiv-infected individuals (37) . this effect is most likely explained by reduced viral replication due to effective treatment with indinavir. a higher level of ccl2 has been previously reported in hiv-1 patients plasma (38) ; however, there was no discrimination between viremic and aviremic patients. here, we report that ccl2 is regulated not only at the protein but also at the mrna level in hiv-1-infected viremic individuals. the potential role of ccl2 in hiv-1 replication in vitro can be explained in pha blast model of hiv-1 iiib infection. we observed >2-fold increase in the viral titer after 5 days of post-infection (data not shown), which is in line with previous reports (23, 39) . although we are not providing direct evidence for ccl2 in virus replication, the studies presented here strongly suggest that ccl2 is an integral host factor in hiv-1 pathogenesis, supporting the hypothesis that ccl2 presumably could act as a potential enhancer of hiv-1 replication. in summary, our results from gene expression profiling and increased production of ccl2 in hiv-1 viremic individuals suggest that active hiv-1 replication, as manifested in viremic patients, at least in part is mediated by the inflammatory chemokine ccl2. our data show a clear correlation between the viremia and host ccl2 gene response. unraveling the mechanism of increased hiv-1 replication and potential involvement of ccl2 could provide more insight to understand the virus-host relationship, and inhibition of increased ccl2 production may lead to new therapeutic interventions in hiv-1 infection. supplementary figures are available at international immunology online. apc allophycoerythrin ccl2 c-c chemokine ligand-2 ccr1 c-c chemokine receptor-1 cxcl10 cxc chemokine ligand-10 gapdh glyceraldehydes-3-phosphate dehydrogenase gch1 gtp-cyclohydrolase hiv-1 regulatory/accessory genes: keys to unraveling viral and host cell biology host factors and the pathogenesis of hivinduced disease regulation of chemokine/cytokine network during in vitro differentiation and hiv-1 infection of human monocytes: possible importance in the pathogenesis of aids hiv-1 nef mediates lymphocyte chemotaxis and activation by infected macrophages large-scale monitoring of host cell gene expression during hiv-1 infection using cdna microarrays cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays gene expression and viral production in latently infected, resting cd4+ t cells in viremic versus aviremic hiv-infected individuals cellular gene expression upon human immunodeficiency virus type 1 infection of cd4(+)-t-cell lines nef triggers a transcriptional program in t cells imitating single-signal t cell activation and inducing hiv virulence mediators decreased survival of b cells of hiv-viremic patients mediated by altered expression of receptors of the tnf superfamily cc ckr5: a rantes, mip-1alpha, mip-1beta receptor as a fusion cofactor for macrophage-tropic hiv-1 the beta-chemokine receptors ccr3 and ccr5 facilitate infection by primary hiv-1 isolates identification of rantes, mip-1 alpha, and mip-1 beta as the major hiv-suppressive factors produced by cd8+ t cells identification of a major co-receptor for primary isolates of hiv-1 hiv-1 entry into cd4+ cells is mediated by the chemokine receptor cc-ckr-5 mechanisms for the inhibition of hiv replication by interferons-alpha, -beta, and -gamma in primary human macrophages divergent regulation of hiv-1 replication in pbmc of infected individuals by cc chemokines: suppression by rantes, mip-1alpha, and mcp-3, and enhancement by mcp-1 human immunodeficiency virus type 1 (hiv-1)-induced groalpha production stimulates hiv-1 replication in macrophages and t lymphocytes the c-x-c chemokine ip-10 stimulates hiv-1 replication monocyte chemoattractant protein 1 acts as a t-lymphocyte chemoattractant purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line human immunodeficiency virus replication induces monocyte chemotactic protein-1 in human macrophages and u937 promonocytic cells cc-chemokines enhance the replication of t-tropic strains of hiv-1 in cd4(+) t cells: role of signal transduction mcp-1 (ccl2) protects human neurons and astrocytes from nmda or hiv-tat-induced apoptosis disruption of the c-jun-jnk complex by a cell-permeable peptide containing the c-jun delta domain induces apoptosis and affects a distinct set of interleukin-1-induced inflammatory genes analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method chemokines during the early stages of infection in human pbmcs investigations on four host response factors whose expression is enhanced in x4 shiv encephalitis invasive chronic inflammatory monocyte phenotype in subjects with high hiv-1 viral load the role of p38 mapk in rhinovirus-induced monocyte chemoattractant protein-1 production by monocyticlineage cells differential chemokine expression following respiratory virus infection reflects th1-or th2-biased immunopathology modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro simian human immunodeficiency virus-associated pneumonia correlates with increased expression of mcp-1, cxcl10, and viral rna in the lungs of rhesus macaques roles for cxc chemokine ligands 10 and 11 in recruiting cd4+ t cells to hiv-1-infected monocyte-derived macrophages, dendritic cells, and lymph nodes a mouse model for study of systemic hiv-1 infection, antiviral immune responses, and neuroinvasiveness dichotomous effects of beta-chemokines on hiv replication in monocytes and monocyte-derived macrophages change in circulating levels of the chemokines macrophage inflammatory proteins 1 alpha and 11 beta, rantes, monocyte chemotactic protein-1 and interleukin-16 following treatment of severely immunodeficient hiv-infected individuals with indinavir plasma levels of monocyte chemoattractant protein-1 but not those of macrophage inhibitory protein-1alpha and rantes correlate with virus load in human immunodeficiency virus infection endogenous ccl2 (monocyte chemotactic protein-1) modulates human immunodeficiency virus type-1 replication and affects cytoskeleton organization in human monocyte-derived macrophages we are indebted to the patients who participated in this study and the staff of the immunologische ambulanz. this work was supported in part by a grant from the german research foundation (sfb566/a02). a.w.a. and n.b. are supported by fellowships of the md/phd program in 'molecular medicine' and phd program in infection biology at hannover medical school. key: cord-257272-4q52p1pd authors: marsh, mark; pelchen-matthews, annegret; hoxie, james a. title: roles for endocytosis in lentiviral replication date: 1997-01-31 journal: trends in cell biology doi: 10.1016/s0962-8924(97)20038-3 sha: doc_id: 257272 cord_uid: 4q52p1pd endocytosis is essential for the entry of many viruses into cells. the primate lentiviruses [human immunodeficiency virus (hiv) 1 and 2, and the simian immunodeficiency viruses (sivs)], however, use endocytosis in other aspects of their life cycles. here, the authors describe the ways in which the endocytic pathway is used by hiv and siv and discuss the mechanisms through which endocytosis may contribute to the pathogenic properties of these viruses. endocytosis is an essential property of cells. it is also used by many viruses to enter cells' and for immune responses to viral infection dependent on major histocompatibility class ii (mhc-ii). the primate lentiviruses seem not to require endocytosis for entry, but use endocytic pathways for other aspects of their replicative cycle. in this article, we consider the various ways in which endocytosis bears on human immunodeficiency virus (hiv) and simian immunodeficiency virus (siv) infection. we consider the role of endocytosis in virus entry, how endocytosis can modulate the surface expression of the viral receptors and, finally, the endocytic properties of the viral envelope glycoprotein (env). like other retroviruses, lentiviruses consist of a core structure containing the viral genome, surrounded by a lipid membrane bearing multiple copies of the envelope glycoprotein env. entry into cells requires fusion of the viral membrane with a cell membrane in a reaction that is usually dependent on the interaction of env with the principal cellular receptor, cd4 (ref. 2). the fusion reactions for hivs are ph independent' and, in tissue culture, occur at the cell surface without the need for endocytosis3*4. whether endocytosis facilitates entry of these viruses into t cells, monocytes, dendritic cells and other targets in vivo is unclea9, although it is likely that transcytosis provides at least one route for hiv to cross the rectal epithelium6. cell-surface expression of receptor molecules cd4 cell-surface expression can be regulated by endocytosis and potentially alter the susceptibility of a cell to hiv infection. the mechanisms controlling cd4 internalization are now understood in some detail'. cd4 can undergo rapid and efficient endocytosis through clathrin-coated pits and vesicles. this activity is regulated by phosphorylation-dependent endocytosis signals in the cytoplasmic domain of cd4 and, in lymphoid cells, through interaction with p56lck-a src-family protein tyrosine kinase. association with ~56~~~ prevents cd4 entry into endocytic vesicles. phosphorylation of specific serine residues in the cytoplasmic domain of cd4 triggers dissociation from p56lck, thereby removing the restraint to endocytosis. the phosphorylation of cd4 also activates the endocytosis signal that first allows the protein to cluster into clathrin-coated pits, internalize and be delivered to early endosomes. the activated signal also leads to endosomal sorting of internalized cd4 and subsequent delivery of cd4 to late endosomes and/or lysosomes where it is degraded'. in hiv-and siv-infected cells, cd4 cell-surface expression is downmodulated. in the later stages of the infection cycle, downmodulation occurs through the intracellular interaction of cd4 with env (ref. 8) . early in the cycle, however, downmodulation is linked to expression of the viral nef proteina. nef is a small (-27 kda) virally encoded protein that has been shown to interact with a number of cellular proteins including src-family kinases (e.g. ~56~~~), serine ki-nase9 and a number of other cell proteins including endocytosis is essential for the entry of many viruses into cells. the primate lentiviruses furnan immunodeficiency virus (hiv) 1 and 2, and the simian immunodeficiency viruses (sivs)], however, use endocytosis in other aspects of their life cycles. here, the authors describe the ways in which the endocytic pathway is used by hiv and siv and discuss the mechanisms through which endocytosis may contribute to the pathogenic properties of these viruses. the p subunit of coatomerg. the precise cellular function(s) of nef remains to be established, but its effects on cd4 surface expression appear to be due to its ability to influence the endocytic properties of cd4 molecule@. nef-induced cd4 downmodulation is independent of serine phosphorylation, suggesting that nef acts through mechanisms different from those that normally regulate cd4 internalizations. nef may also induce the endocytosis of mhc-i (ref. 10) and thus may influence the extent to which infected cells are recognized by cytotoxic t cells. whether nef downmodulates cd4 and mhc-i expression through similar mechanisms is unclear. while cd4 is necessary in most cases for hiv and siv infection, additional celhrlar molecules are required for virus entryz. recently, several members of the family of seven-iransmembrane-domain (7tm), g-proteincoupled receptors for inflammatory chemokines were identified as co-receptors for various hiv-l strains1',i2. significantly, the chemokine ligands for some of these receptors (rantfs, mip-la and mip-1p) have been implicated in protecting individuals from hiv-l infectioni and preventing some infected individuals from developing aids (ref. 14). these chemokines appear to prevent viral entry, but their mode of action is unknown. they could directly block interaction of env with the co-receptor or they could downmodulate the co-receptors from the cell surface. endocytic modulation has been demonstrated for several related 7tm proteins15,16, and it is likely that endocytosis will also regulate the surface expression of the chemokine receptors and thereby influence viral infection. endocytosis is also used by hiv and siv to regulate the cell-surface expression of env. env is the major virally encoded protein of the virion membrane and is responsible for both the receptor-binding and fusion properties of the virus. the pathway of env synthesis is similar to that described for many viral and cellular membrane proteins (fig. l) , but lentiviral envs may have characteristics relevant to the pathology of these particular viruses. for example, much of the hiv-l env exported from the rough er (ref. 21) . no differences have been detected in the synthesis and transport of the cp-mac and bk28' envs, suggesting that events subsequent to delivery to the plasma membrane account for the different surface expression levels. significantly, the amine acids around tyr723 have features in common with the tyr-containing signals identified in other proteins. moreover, the change is reminiscent of the 'jd' mutation in the low-density lipoprotein receptor (ldl-r), where a similar tyr-to-cys change disrupts the endocytosis signal, causing the receptor to accumulate on the plasma membrane". the bk28 env, expressed in the absence of other siv proteins, is actively internalized from the cell surface. by contrast, the cp-mac env, containing the tyr723cys mutation, is internalized less efficientlyz3. when the cytoplasmic domain of siv env is used to replace the cytoplasmic domain of cd4, the env signal is as efficient as the ldl-r endocytosis signal in mediating endocytosisz3. furthermore, electron microscopy shows that a tyr723-containing env associates with coated pits and is likely, therefore, to use the clathrin-dependent endocytic does not accumulate at the plasma membrane or incorporate into virions. instead. the protein undergoes acid-dependent proteolysis presumably in late endosomes and/or lysosomes". the mechanisms in volved in the transport of env into the endocytic pathway and the significance of these events in the biology of the viruses are unclear. many cellular membrane proteins con tain tyrosine or dileucine-based signals that mediate endocytosis and other sort.. ing eventsis. the cytoplasmic domains of hiv and siv env contain several such1 sequences that could potentially act as sorting signals, and studies in polarized cells indicate that env does contain sorting information 19. sivs cultured in certain human t-cell lines frequently acquire a mutation that deletes -130 amino acid!. from the c-terminus of env, leaving a predicted cytoplasmic domain of -20 amino acids. these short-tailed viruses. have revealed the location of one such sorting signal. cells infected with a variant of siv (cp-mac, derived from the siv bk28 molecular clonezo) express up to 25fold more env on their surfaces than cells infected with bk28 (fig. 2) . the increased expression is due to a tyr-to-cys change at position 723 in the cytoplasmic domain of u-mac em forum when the analogous tyr (position 721) is mutatedzl, although the increase is less (4-5 fold) than that seen for viruses with short cytoplasmic domains. a cd4 chimera containing the full-length sw tail is internalized efficiently, but the endocytosis of this molecule is only slightly reduced by mutations at tyr721, suggesting that the full-length tail contains more than one endocytosis signal (a. pelchen-matthews, j. hoxie and m. marsh, unpublished) . together, these findings indicate that env carries a functional endocytosis signal. an analogous tyrosine is conserved in virtually all the primate lentivirus env sequences (e.g. tyr712 in many strains of hiv-l), and amino acids surrounding this residue also show a high degree of conservation. thus, reports that the hiv-l env also undergoes intemalization2" suggest that this endocytosis activity is an important and conserved feature of primate lentiviruses. indeed, similar signals may be present in other retroviral envelope proteinsz5. env? lentiviruses are believed to bud from the surface of infected cellsz6 and might be expected to direct env to this site. thus, the presence of endocytosis signals that actively clear env from the plasma membrane appears counterintuitive. in fact, the envelope glycoproteins of viruses such as influenza or respiratory syncytia virus are endocytosed poorly and are expressed in abundance on the surface of infected cells. these latter viruses, however, generally cause acute and self-limited infections, whereas the lentiviruses establish chronic and persistent infections. env is highly immunogenic and renders infected cells targets for humoral immune responses. for hiv-1 at least, virus production remains high during the course of the infection despite ongoing immune responses 27,28. thus, as with other persistent viruses, hiv must have evolved mechanisms that permit virus production in the face of this immune attack. although mutations in the ectodomain of env and in other viral proteins may help hiv escape the immune response, it is possible that virus production in an immunologically competent host depends on the regulated expression of env on the plasma membrane. as such, determinants that reduce the surface exposure of env could be advantageous for infected cells and, consequently, virus production. env internalized from the cell surface could also play a role in triggering mhc-ii-dependent cellular immune responses since mhc-ii molecules generally acquire their antigens from endocytic compartmentp. the endocytosis of env may also play a role in protecting newly infected cells. a consequence of viral fusion at the cell surface is that the env of entering virions is inserted into the plasma membrane of the target cell. these exogenously acquired proteins could immediately make the cell a target for immunological attack and/or predispose the cell to fuse with adjacent cd4+ cells. consequently, a signal that would quickly remove these viral proteins might increase the likelihood that an infected cell will survive to produce new virions. a further possibility is that the endocytosis signals in env may assist in coordinating the assembly of new virions. if the plasma membrane is the principal site for hiv and siv budding and cell-surface env is detrimental to the infected cell, it might be advantageous for the surface expression of env to be linked temporally and spatially to the assembly process. such linking could be achieved through the interaction of core components derived from the gag gene with the env cytoplasmic domain2g. this association may prevent internalization of env by interfering with the binding of clathrin adaptor molecules (fig. 3) . recently published evidence indicates that the endocytosis of hiv-l env is reduced when env is expressed together with the hiv gag protein30. thus, competition between viral (gag) and cellular (adaptors) proteins for binding sites in the env cytoplasmic domain will ensure that only those env molecules being incorporated into virions will accumulate at the cell surface. the plasma membrane is not, however, the only site of hiv assembly. there are reports that hivs and siv can bud into intracellular vesicles (see for example . by analogy to other viruses, such as bunya-and corona-virus, these vesicles are often considered to be derived from exocytic compartments26. a recent study has indicated that, in hiv-l-infected forum monocytes, viruses bud primarily into intracellular vesicles (m. moore, pers. commun.). moreover, immunoelectron microscopy has demonstrated that these vesicles are mhc-ii-enriched compartments (miics) the late endosomes and lysosomes implicated in loading mhc-ii with peptides35. how can virus bud into endocytlc organelles? experiments in polarized cells indicate that env directly influences the site of budding. when gag is expressed alone in such cells, gag particles are released into the apical and basal medium, but, when gag and env are expressed together, particles only appear in the basal medium36f37. significantly, the amino acids around tyr723 in bk28, and the corresponding residues in hiv, resemble several well-characterized lysosome-targeting signals1s,38. the signal in env may function, therefore, in both endocytosis and targeting to late endosomes/lysosomes (fig. 1) and, in some cell types at least, may facilitate budding at this location. the infectious potential of virions assembled on intracellular membranes and their relative contribution to the total population of infectious viruses is unknown. however, the recent observation that miics can fuse directly with the plasma membrane3g raises the possibility that the release of intracellular virions might be regulated. furthermore, the assembly and release of virus from miics may be related to the observation that mhc-ii can be found in abundance in hiv membrane@. if endocytosis signals in env are as important as we propose, it might be anticipated that mutations that disrupt these signals would have an impact on virus infection and pathogenesis. experiments in tissueculture systems indicate that siv mutants containing the tyr723cys mutation show a marked increase in their infection kinetics and cytopathic effect21,23. et 01. (1995) 1. acquired immune deficiency syndrome hum /. viral. 61, 629-632 personal subscription to trends in cell biology? see the subscription order form for details importantly, in the siv system, the consequences of mutations that disrupt the endocytosis signals can be permission to cite her unpublished work. we apologize to those authors whose work could not be cited owing to editorial key: cord-264994-j8iawzp8 authors: fitzpatrick, meagan c.; bauch, chris t.; townsend, jeffrey p.; galvani, alison p. title: modelling microbial infection to address global health challenges date: 2019-09-20 journal: nat microbiol doi: 10.1038/s41564-019-0565-8 sha: doc_id: 264994 cord_uid: j8iawzp8 the continued growth of the world’s population and increased interconnectivity heighten the risk that infectious diseases pose for human health worldwide. epidemiological modelling is a tool that can be used to mitigate this risk by predicting disease spread or quantifying the impact of different intervention strategies on disease transmission dynamics. we illustrate how four decades of methodological advances and improved data quality have facilitated the contribution of modelling to address global health challenges, exemplified by models for the hiv crisis, emerging pathogens and pandemic preparedness. throughout, we discuss the importance of designing a model that is appropriate to the research question and the available data. we highlight pitfalls that can arise in model development, validation and interpretation. close collaboration between empiricists and modellers continues to improve the accuracy of predictions and the optimization of models for public health decision-making. m icrobial pathogens are responsible for more than 400 million years of life lost annually across the globe, a higher burden than either cancer or cardiovascular disease 1 . diseases that have long plagued humanity, such as malaria and tuberculosis, continue to impose a staggering toll. recent decades have also witnessed the emergence of new virulent pathogens, including human immunodeficiency virus (hiv), ebola virus, severe acute respiratory syndrome (sars) coronavirus, west nile virus and zika virus. the persistent global threat posed by microbial pathogens arises from the nonlinear mechanisms of disease transmission. that is, as the prevalence of a disease is reduced, the density of immune individuals drops, the density of susceptible individuals rises and disease is more likely to rebound. the resultant temporal trajectories are difficult to predict without considering this nonlinear interplay. for instance, many microbial diseases exhibit periodic spikes in the number of cases that are unexplainable by pathogen natural history or environmental phenomena. by explicitly defining the nonlinear processes underlying infectious disease spread, transmission models illuminate these otherwise opaque systems. forty years ago, nature published a series of papers that launched the modern era of infectious disease modelling 2,3 . since that time, these methodologies have multiplied 4 . transmission models now employ a variety of approaches, ranging from agent-based simulations that represent each individual 5 to compartmental frameworks that group individuals by epidemiological status, such as infectiousness and immunity 2, 3 . accompanying the methodological innovations, however, are challenges regarding selection of appropriate model structures from among the wealth of possibilities 6 . at this anniversary of the publication of these landmark papers 2,3 , we reflect on contributions that transmission modelling has made to infectious disease science and control. through a series of case studies, we illustrate the overarching principles and challenges related to model design. with expanding computational capacity and new types of data, myriad opportunities have opened for transmission modelling to bolster evidence-based policy (box 1) 7, 8 . in all pursuits, modelling is most informative when conducted collaboratively with microbiologists, immunologists and epidemiologists. we offer this perspective as an entry point for non-modelling scientists to understand the power and flexibility of modelling, and as a foundation for the transdisciplinary conversations that bolster the field. even within the same disease system, the ideal model design depends on the specifics of the questions asked. here, we highlight a series of models focused on one of the defining infectious agents of our era: hiv. the virus has challenged science, medicine and public health at every scale, from its deft immune evasion to its death toll of more than 35 million over the last four decades 9 . we describe how clinical needs, research questions and data availability have shaped the design of hiv models across these scales. unless otherwise indicated, the term 'hiv' is inclusive of both hiv-1 and hiv-2. within-host models. at a within-host scale (table 1) , models can be used to simulate cellular interactions, immunological responses and treatment pharmacokinetics 10 . in such simulations, viral dynamics are often modelled using a compartmental structure, with the growth of one population, such as circulating virions, dependent on the size of another population, such as infected cells. for example, a seminal within-host model fit to viral load data by perelson et al. 11 revealed high turnover rates of hiv-1, counter to what was then the prevailing assumption that hiv-1 remained dormant during the asymptomatic 'latency' phase. the corollary to these high rates of viral turnover was that drug resistance would likely evolve rapidly under monotherapy. further analyses of this model indicated that a combination of at least three drugs was necessary to maintain drug sensitivity 12 . once combination therapy did become available, extension of the perelson et al. model demonstrated that the two-phase decline in viral load observed following treatment initiation was attributable to a reservoir of long-lived infected cells 13 . with this insight also came the realization that prolonged treatment would be necessary to suppress viral load. the incorporation of meagan c. fitzpatrick 1,2 , chris t. bauch 3 , jeffrey p. townsend 4,5,6 and alison p. galvani 2,5,6 * the continued growth of the world's population and increased interconnectivity heighten the risk that infectious diseases pose for human health worldwide. epidemiological modelling is a tool that can be used to mitigate this risk by predicting disease spread or quantifying the impact of different intervention strategies on disease transmission dynamics. we illustrate how four decades of methodological advances and improved data quality have facilitated the contribution of modelling to address global health challenges, exemplified by models for the hiv crisis, emerging pathogens and pandemic preparedness. throughout, we discuss the importance of designing a model that is appropriate to the research question and the available data. we highlight pitfalls that can arise in model development, validation and interpretation. close collaboration between empiricists and modellers continues to improve the accuracy of predictions and the optimization of models for public health decision-making. stochasticity into this within-host framework allowed model fitting to 'viral blips'-transient peaks in viral load, even under antiretroviral treatment 14 . analysis of this data-driven stochastic model demonstrated that homeostatic proliferation maintained the infected cell reservoir and produced these viral blips, a finding that was later confirmed experimentally 15, 16 . the implication for clinical care was that intensified antiretroviral treatment would be unable to eliminate the latent reservoir of infected cells as had been hypothesized, sparing patients from potentially fruitless trials with such regimens. individual-based models. whereas the unit of interest for withinhost modelling is an infected cell, the analogous unit for individualbased models is an infected person (table 1) 5, 17, 18 . individual-based models are often used to explore the interplay between disease transmission and individual-level risk factors, such as comorbidities, sexual behaviours and age. such models are capable of incorporating data with individual-level granularity, including those regarding contact patterns, patient treatment cascades and clinical outcomes. individual-based models are uniquely suited for representing overlap in individual-level risk factors and translating the implications of this overlap for public health policy. for example, an individual-based model was recently used to demonstrate that the majority of hiv transmission among people who inject drugs in new york city is attributable to undiagnosed infections 18 . these modelling results underscore the urgency for the city to invest in more comprehensive screening and improved diagnostic practices. population models. most commonly, models are created at the population scale, capturing the spread of a pathogen through a large group (table 1) . at this scale, compartmental models shift in focus from the pathogen to the host. unlike individual-based models, compartmental models will aggregate individuals with a similar epidemiological status. for instance, the archetypical 's-i-r' model separates the entire population of interest into one of three categories: s, susceptible to infection; i, infected and infectious; or r, recovered and protected 19 . in practice, most models will have additional compartments or stratification beyond this simple structure. age stratification is essential when either the disease risk or the intervention is age-specific. as an example, an age-stratified multipathogen model demonstrated that schistosomiasis prevention targeted to zimbabwean schoolchildren could cost-effectively reduce hiv acquisition later in life 20 . this framework was extended to additional countries with a range of age-specific disease prevalence and co-infection rates to assess the potential value of treating schistosomiasis in adults. although adult treatment is not usually considered efficient, the model showed that it could be cost-effective in settings with high hiv prevalence 21 . these models strengthened the investment case for treatment of schistosomiasis, an otherwise neglected tropical disease. network models are also deployed to represent dynamics on the population scale (table 1) . these models impose a structure on contacts between hosts, unlike compartmental models which assume that contacts are random among hosts within a compartment. in a network model, nodes represent individuals and the connections between nodes represent contacts through which infection may spread 22 . sources for network parameterization may include surveys, partner notification services or phylogenetic tracing 23, 24 . as with individual-based models, network models tend to require significant amounts of data to fully parameterize, but various computational and statistical methods have been developed to analyse the impact of uncertain parameter values on model predictions 25 . network models are applied to discern the influence of contact structure on disease transmission and on the effectiveness of targeted intervention strategies. for instance, network models predicted that hiv would spread more quickly through sexual partnerships that are concurrent versus serially monogamous, even if the total numbers of sexual acts and partners remain constant 26 . the study prompted a more rigorous engagement of epidemiologists with sociological data to tailor interventions for specific settings 27 . other network models have focused on the more rapid transmission within clusters of high-risk individuals and slower transmission to lower-risk clusters, a dynamic which explains discrepancies between observed incidence patterns and the expected pattern based on an assumption of homogeneous risks 28 . these studies both illustrate the importance of accounting for network-driven dynamics when individuals are highly aggregated with regards to their risk factors, and when appropriate data for parameterization are available. metapopulation models. metapopulation models represent disease transmission at dual scales, considering not just the interactions of individuals, but also the relationships between groups of individuals, which are typically defined geographically (table 1) . transmission intensity is often higher within groups than across groups, especially when the groups are spatially segregated 29 . one metapopulation model of hiv in mainland china considered there are the three principal objectives of modelling, all of which can inform public health policy. predicting disease spread. models can be used to estimate the infectiousness of a pathogen within a given population. a fundamental concept is that of r 0 , the basic reproduction number, which quantifies the number of infections that would result from a single index case in a susceptible population. r 0 governs the temporal trajectory of an outbreak and the scale of interventions required for its containment. models may be used to infer r 0 as well as forecast changes in r 0 that could drive transitions in epidemic dynamics, such as the shift from sporadic outbreaks to sustained chains of transmission. example: assessing real-time zika risk in texas 90 . selecting among alternative control strategies. simultaneous field trials of multiple infectious disease control options are often infeasible. models can simulate a wide range of control strategies and thus optimize public health policies according to translational objectives and real-world constraints. modelling can also extrapolate from the individual clinical outcomes of interventions or novel therapeutics to the population-level impacts. extrapolating to the population level is essential to evaluate the indirect benefits of interventions, including a reduction in transmission, or unanticipated repercussions, such as evolution of resistance. example: comparing antibiotic 'cycling' versus 'mixing' to minimize the evolution of antimicrobial resistance 107 . hypothesis testing. it is often logistically or ethically infeasible to empirically test scientific hypotheses in the field or experimentally. modelling can identify parsimonious explanations of observed phenomena, including complex outcomes that can arise from the nonlinear processes common in microbiological systems. even simple models can be useful to help us understand dynamics that are common to many microbiological systems through identification of basic mechanisms that apply across a range of infections. by examining a new infectious agent through the lens of previously characterized systems, models provide insight into the ways that a particular microbial infection might follow or break from typical patterns. example: investigating whether individual heterogeneity within social networks significantly impacts disease spread 22 . transmission within and between provinces, driven by the mobility of migrant labourers 30 . the study suggested that hiv prevention resources could be most effectively targeted to provinces with the greatest initial incidence, as rising incidence in other provinces is driven more by migration from the high-burden provinces than by local transmission. given that the chinese provinces with employment opportunities for migrants are also those with the heaviest burden of hiv, migrant workers who acquire hiv often do so in the province where they work. however, government policy requires migrants to return to their home province for treatment. the movement of these workers perpetuates the disease cycle, as new migrants move to fill the vacated jobs and themselves become exposed to elevated hiv risk. these results therefore call for reconsideration of provincial treatment restrictions. multinational models. global policies, such as the treatment goals set by the joint united nations programme on hiv/aids (unaids), have been modelled on a global scale (table 1) by considering the effectiveness of the policies for each nation. for example, a compartmental model was used to evaluate the potential impact of a partially efficacious hiv vaccine on the epidemiological trajectories in 127 countries that together constitute over 99% of the global burden 31 . the model was tailored to each country by fitting to country-specific incidence trends as well as diagnosis, treatment and viral suppression data. this model revealed that, even with efficacy as low as 50%, a hiv vaccine would avert millions of new infections worldwide, irrespective of whether ambitious treatment goals are met. these results identify the synergies between vaccination and treatment-as-prevention, and provide evidence to support continued investment in vaccine development 9, 32 . from the cellular level to the population level, hiv modelling has led to improvements in drug formulations, clinical care and resource allocation. as scientific advances continue to bring pharmaceutical innovations, modelling will remain a useful tool for illuminating transmission dynamics and optimizing public health policy. hiv was not controlled before it became a pandemic, but our response to future outbreaks has the potential to be more timely 33 . when diseases emerge in new settings, such as ebola in west africa and sars in china, modelling can be rapidly deployed to inform and support response efforts (fig. 1) . unfortunately, the urgency of public health decisions during such outbreaks tends to be accompanied by a sparsity of data with which to parameterize, calibrate and validate models. as detailed below, uncertainty analysis-a method of analysing how uncertainty in input parameters translates to uncertainty in model outcome variables-becomes all the more vital in these situations. media attention regarding model predictions is often heightened during outbreaks, ironically at a time when modelling results are apt to be less robust than for well-characterized endemic diseases. we discuss the importance of careful communication regarding model recommendations and associated uncertainty to inform the public without fuelling excessive alarm. despite these challenges, and especially if these challenges can be navigated, the timely assessment of a wide range of intervention scenarios made possible by modelling would be particularly valuable during infectious disease emergencies. ebola virus outbreaks. the 2014 ebola virus outbreak struck a populous region near the border of guinea and sierra leone, sparking a crisis in a resource-constrained area that had no prior experience with the virus. as the caseload mounted and disseminated geographically, it became apparent that the west african outbreak would be unprecedented in its devastation. models were developed to estimate the potential size of the epidemic in the absence of intervention, demonstrating the urgent need for expanded action by the international community [34] [35] [36] , and to calculate the scale of the required investment 37 . initial control efforts included a militarily enforced quarantine of a liberian neighbourhood in which ebola was spreading. modelling analysis in collaboration with the liberian ministry of health demonstrated that the quarantine was ineffective and possibly even counterproductive 38 . connecting the microbiological and population scales, another modelling study integrated within-host viral load data over the course of ebola infection and between-host transmission parameterized by contact-tracing data. the resulting dynamics highlighted the imperative to hospitalize most cases in isolation facilities within four days of symptom onset 39 . these modelling predictions were borne out of empirical observations. early in the outbreak, when the incidence was precipitously growing, the average time to hospitalization in liberia was above six days 40 . as contact tracing improved, the concomitant acceleration in hospitalization was found to be instrumental in turning the tide on the outbreak 40 . in another approach, phylogenetic analysis and transmission modelling were combined to estimate underreporting rates and social clustering of transmission 41 . this study informed public health authorities regarding the optimal scope and targeting of their efforts, which were central to stemming the epidemic. although data can be scarce for emerging pathogens, modellers can exploit similarities with better-characterized disease systems to investigate the potential efficiency of different interventions (box 1). as vaccine candidates became available against ebola, ring vaccination was proposed based on the success of the strategy in eliminating smallpox 42 , another microorganism whose transmission required close contact between individuals and for which peak infectiousness occurs after the appearance of symptoms. compartmental models had suggested parameter combinations for which ring vaccination would be superior to mass vaccination 43 , and methodological advances subsequently allowed for explicit incorporation of contact network data 44 . modelling based on social and healthcare contact networks specific to west africa supported implementation of ring vaccination 45 , and the approach was adopted for the clinical trial of the vaccine 46 . in 2018, two independent outbreaks of ebola erupted in the democratic republic of the congo. during the initial outbreak in équateur province, modellers combined case reports with time series from previous outbreaks to generate projections of final epidemic size that could inform preparedness planning and allocation of resources 47 . ring vaccination was again deployed, this time within two weeks of detecting the outbreak. a spatial model quantified the impact of vaccine on both the ultimate burden and geographic spread of ebola, highlighting how even one week of additional delay would have substantially reduced the ability of vaccination to contain this outbreak 48 . the second outbreak was reported in august in the north kivu province. armed conflict in this region has interfered with the ability of healthcare workers to conduct the necessary contact tracing, vaccination and treatment. as conditions make routine data collection difficult and even dangerous, modelling has the potential to provide crucial insights into the otherwise unobservable characteristics of this outbreak. in contrast to the unexpected emergence of ebola in a new setting, the influenza virus has repeatedly demonstrated its ability to cause pandemics. a pandemic is an event in which a pathogen creates epidemics across the entire globe. the 1918 pandemic killed an estimated 50 million people worldwide 49 , exceeding the combined military and civilian casualties of world war 1. while the 2% case-fatality rate of the 1918 strain was approximately 40 times higher than is typical for influenza 50 , pathogenic strains with case-fatality rates exceeding 50% periodically emerge 51 . modelling has illustrated how repeated zoonotic introductions impose selection for elevated human-to-human transmissibility, which thereby exacerbates the threat of a devastating influenza pandemic 52 . such threats underscore the importance of surveillance systems and preparedness plans, which can be informed by modelling (box 1). transmission models are able to optimize surveillance systems, accelerate outbreak detection and improve forecasting [53] [54] [55] [56] . for example, a spatial model integrating a variety of surveillance data streams and embedded in a user-friendly platform is currently implemented by the texas department of state health services to generate real-time influenza forecasts (http://flu.tacc.utexas.edu/). modelling has also motivated the development of dynamic preparedness plans, which adapt in response to the unfolding events of a pandemic, as models identified that adaptive efforts would be more likely to contain an influenza pandemic than static policies chosen a priori 57 . other pandemic influenza analyses used agestructured compartmental models to study the trade-off between targeting influenza vaccination to groups that transmit many infections but experience relatively low health burdens (for example, schoolchildren) versus groups that transmit fewer infections but experience greater health burdens (for example, the elderly) 58 . such examples illustrate the insights that modelling has provided to the decision makers charged with maintaining readiness against simultaneously rare but catastrophic situations. modelling has also examined the impact of human behaviour, including vaccination decisions and social interactions, on the course of an epidemic. public health interventions are not always sufficient to ensure disease control, as behavioural factors can thwart progress [59] [60] [61] [62] . for example, reports in 1974 of potential neurological side effects from the whole-cell pertussis vaccine led to a steep decline in vaccine uptake throughout the uk, followed by a slow recovery (fig. 2a) 63 . vaccine uptake ebbed and flowed over the next two decades, with higher rates of vaccination in the wake of large pertussis outbreaks (fig. 2b) 61, 63 . compartmental models analysing the interplay between vaccine uptake and disease dynamics confirmed the hypothesis that increases in vaccination were a response to the pertussis infection risk 61 , and showed that incorporating this interplay can improve epidemiological forecasts. network models extending these coupled disease-behaviour analyses types of projection that can be generated include outbreak trajectories, disease burdens and economic impact. d, probabilistic uncertainty analyses convey not only model projections of policy outcomes, but also quantification of confidence in the projections. e, as policies are adopted and the microbiological system is influenced accordingly, the model can be iteratively updated to reflect the shifting status quo, thereby progressively optimizing policies within an evolving system. have illustrated how the perceived risk of vaccination can have greater influence on vaccine uptake than disease incidence 64 . more recently, vaccine refusal has led to the resurgence of measles in the usa 62, 65 . researchers are turning to social media to gather information about attitudes toward vaccines and infectious diseases, and to glean clues about vaccinating behaviour 55, 66, 67 . for instance, signals that vaccine refusal is compromising elimination can be detected months or years in advance of disease resurgence by applying mathematical analysis of tipping points to social media data that have been classified on the basis of sentiment using machine learning algorithms 66 . these and other data science techniques might help public health authorities identify the specific communities that are at increased risk of future outbreaks. on shorter timescales, the near-instantaneous availability of social media data facilitates its integration into models developed for outbreak response 55, 66 . other behavioural factors that have been incorporated into transmission models include attendance at social gatherings, sexual behaviour and commuting patterns-elements which are also often affected by perceived infection risk 59, 68, 69 . antimicrobial resistance. a substantial portion of the increase in human lifespan over the last century is attributable to antibiotics 70 , but the emergence of pathogen strains that are resistant to antimicrobials threatens to reverse these gains. the extensive use and misuse of antibiotics has led to the evolution of multidrug-resistant, extensively drug-resistant and even pan-drug-resistant pathogens across the globe. precariously, this evolution outpaces the development of new antibiotics. mathematical modelling is being used to identify strategies to forestall the emergence and re-emergence of antimicrobial resistance 71, 72 . models are particularly valuable for comparing alternative strategies, such as administration of different antibiotics within the same hospital ward, temporal cycling of antibiotics and combination therapy [73] [74] [75] [76] . high-performance computing now permits the rapid exploration of multidimensional parameter space. models can thereby narrow an array of possible interventions down to a subset likely to have the highest impact or optimize between trade-offs, such as effectiveness and cost (box 1). by contrast, expense, feasibility and ethical considerations may impose more limitations on in vivo investigations (box 1). not only can models identify the optimal strategy for a given parameter set, but they can generate the probability that this intervention remains optimal across variation in the parameters. for example, an optimization routine combined with simulation of hospital-based interventions identified combination therapy as most likely to reduce antibiotic resistance 75 . as a complementary approach, modelling can incorporate economic considerations into these evaluations. a stochastic compartmental model showed that infection control specialists dedicated to promoting hand hygiene in hospitals are cost-effective for limiting the spread of antibiotic resistance 74 . although most models of antibiotic resistance have focused on transmission in healthcare settings, the importance of antibiotic resistance in natural, agricultural and urban settings has been increasingly recognized [77] [78] [79] [80] [81] [82] [83] . for example, a metapopulation model of antimicrobial-resistant clostridium difficile simulated its transmission within and between hospitals, long-term care facilities and the community. this model demonstrated that mitigating risk in the community has the potential to substantially avert hospital-onset cases by decreasing the number of patients with colonization at admission and thereby the transmission within hospitals 84 . this study illustrates how models can consider the entire ecosystem of infection to elucidate dynamics that might not be captured through focus on a single setting. during the initial phase of an outbreak, the predictive power of models is often constrained by data scarcity. this challenge is exacerbated for outbreaks of novel emerging diseases given that our understanding of the disease will rely on the unfolding epidemic (fig. 1) . not only can the absence of data constrain model design, but sparse data requires extensive sensitivity analyses to evaluate the robustness of conclusions. univariate sensitivity analyses, in which individual parameters are varied incrementally above and below a point estimate, can identify which parameters most influence model output (box 1). such comparisons reveal both salient gaps in knowledge and targets for preventing and mitigating the outbreak (box 1) 85 . as an outbreak progresses, each day has the potential to provide more information about the new disease, including its duration of latency, the symptomatic period, infectiousness, transmission modalities, underreporting and the case-fatality rate. however, collecting detailed data to inform each of these parameters can strain resources when they are thinly spread during an emergency response. sensitivity analysis can support clinicians and epidemiologists in prioritizing data collection efforts 86 . parameterization challenges are compounded for complicated disease systems, such as vector-borne diseases. for example, models of zika virus infection span both species and scales, as the disease trajectory is influenced by factors ranging from mosquito seasonality and mosquito abundance down to viral and immunological dynamics within human and mosquito hosts 87, 88 . adding to this complexity, the ecological parameters vary seasonally and geographically-heterogeneities that may be amplified by socioeconomic factors modulating human exposure to infected mosquitoes 89 . in the absence of the high-resolution data that would be ideal to tailor a mosquitodriven disease system to a given setting, uncertainty analysis can unify parameterization from disparate data sources. in contrast to univariate sensitivity analyses, uncertainty analysis simultaneously samples from empirical-or expert-informed distributions for many or all input parameters. collaboration between modellers and disease experts is thus instrumental to ensuring the biological plausibility of these parameter distributions 90, 91 . the uncertainty analysis produces both a central point estimate and a range for each outcome, a combination which can inform stakeholders about the best-case and worst-case scenarios as well as the likelihood that an intervention will be successful [92] [93] [94] . in constructing models and communicating results, there are common pitfalls which can compromise the rigor and impact of the research. a pervasive pitfall is the incorporation of excessive model complexity, particularly through inclusion of more parameters than can be reliably parameterized from data. intuition might suggest that a complex representation of a microbiological system would more closely represent reality. however, the predictive power of a model can be degraded if incorporating additional parameters only marginally improves the fit to data. this tendency results in complicated transmission models that overfit data in much the same way that complicated statistical regressions can overfit data, replicating not only the relevant trends but also the noise in a particular data set. these overfit models thus become less useful for prediction and generalization 6, 95 . to guide appropriate model complexity and parameterization, modellers have used the mathematical theory of information to develop criteria which quantify the balance between realism and simplicity. such criteria penalize additional parameters but reward substantial improvements in fit, thereby identifying the simplest model that can adequately fit the data 61, 96, 97 . these methods can be applied to select among models or alternatively to calculate weighted average predictions across models. in a similar vein, modelling consortiums serve to address uncertainty surrounding model design [98] [99] [100] . in a consortium, several modelling groups develop their models independently, each applying their particular expertise and perspective. for example, consortia of malaria modellers were convened to predict the effectiveness of interventions, including a vaccine candidate 101 and mass drug administration 102 . congruence of output among models engenders confidence that model results are robust. another pitfall concerns the quality of data used to inform the model. incompleteness of data has been an issue since 1766, when daniel bernoulli published a compartmental model of smallpox and acknowledged that more extended analyses would have been possible if the data had been age-stratified 103 . even today, using data to develop models without knowledge of how the data were collected or the limitations of the data can be risky. data collected for an alternative purpose can contain gaps or biases that are acceptable for the original research question, yet lead to incorrect conclusions when incorporated for another purpose in a specific model. in ideal circumstances, modellers would be involved in the design of the original study, ensuring both seamless integration of the results into the model and awareness on the part of the modeller with regard to data limitations. failing that, it is very helpful for modellers to collaborate with scientists familiar with the details of empirical studies on which their results might depend. this lack of familiarity with the biases or incompleteness of data sources may be particularly dangerous in the era of digital data. 'big data hubris' can blind researchers to the limitations of the dataset, such as being a large but unrepresentative sample of the general population, or the alteration of search engine algorithms partway through the data collection process 6 . some of these limitations can be addressed by using digital data as a complement to traditional data sources. in this way, the weakness of one data source (for example, low sample size of traditional surveys or bias in large digital data) can be compensated by the strengths of another data source (for example, balanced representation in small survey versus large scale of digital data). a final pitfall that often arises in the midst of an ongoing outbreak concerns the interpretation of epidemic projections. initial models may assume an absence of intervention as a way to assess the potential consequences of inaction. such projections may contribute to the mobilization of government resources towards control, as was the case during the west african ebola outbreak 35, 37, 38 . in this respect, the projections are intended to make themselves obsolete 104 . in retrospect and without knowledge of the initial purpose of the model, it may appear that the initial predictions were excessively pessimistic 105 . additionally, people living in outbreak zones often change their behaviour to reduce infection risks, thereby mitigating disease spread through, for example, reducing social interactions or increasing vaccine uptake (fig. 2) 59, 61, 66 . thus, risk assessment constitutes a 'moving target' 105 . for example, input parameters estimated from contact tracing early in an outbreak could require adjustments to reflect these behaviour changes and accurately predict subsequent dynamics 106 . the need for proficient communication skills is heightened during an outbreak. this concern is particularly relevant when presenting sensitivity and uncertainty analyses. although predictions at the extreme of sensitivity analyses also tend to be less probable than mid-range projections, there can be a temptation to focus on the most sensational model scenarios. ensuing public pressure on the basis of misunderstood findings can cause unwarranted alarm and trigger counterproductive political decisions. in both publications and media interactions, underscoring the improbability of extreme scenarios explored during sensitivity analysis, as well as how improved interventions turn a predictive model into a counterfactual one, may pre-empt this pitfall 33 . the role for modelling in supporting epidemiologists, public health officials and microbiologists has progressively expanded since the foundational publications forty years ago, in concert with the growing abundance and granularity of data as well as the refinement of quantitative approaches. models have now been developed for virtually every human infectious disease, as well as in many that affect animals and plants, and have been applied across the globe. interdisciplinary collaboration among empiricists, policymakers and modellers facilitates the development of scientifically grounded models for specific settings and generates results that will be actionable in the real world. reciprocally, modelling results may guide the design of experiments and field studies by revealing key gaps in our understanding of microbiological systems. furthermore, modelling is a feasible and cost-effective approach for identifying impactful policies prior to implementation decisions. through all these avenues, epidemiological modelling galvanizes evidence-based action to alleviate disease burden and improve global health. vaccine uptake appears to be entrained by surges in infection incidence. mathematical models can capture the interplay between natural and human dynamics exemplified in this dataset and a wide variety of other study systems. global, regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: a systematic analysis for the global burden of disease study population biology of infectious diseases: part i population biology of infectious diseases: part ii modeling infectious disease dynamics in the complex landscape of global health formalizing the role of agent-based modeling in causal inference and epidemiology big data. the parable of google flu: traps in big data analysis predicting the effectiveness of prevention: a role for epidemiological modeling bridging the gap between evidence and policy for infectious diseases: how models can aid public health decision-making preventing acquisition of hiv is the only path to an aids-free generation multiscale modelling in immunology: a review hiv-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time dynamics of hiv-1 and cd4 + lymphocytes in vivo decay characteristics of hiv-1-infected compartments during combination therapy modeling latently infected cell activation: viral and latent reservoir persistence, and viral blips in hiv-infected patients on potent therapy hiv reservoir size and persistence are driven by t cell survival and homeostatic proliferation modeling the within-host dynamics of hiv infection assessment of epidemic projections using recent hiv survey data in south africa: a validation analysis of ten mathematical models of hiv epidemiology in the antiretroviral therapy era the risk of hiv transmission at each step of the hiv care continuum among people who inject drugs: a modeling study infectious diseases of humans: dynamics and control cost-effectiveness of a community-based intervention for reducing the transmission of schistosoma haematobium and hiv in africa evaluating the potential impact of mass praziquantel administration for hiv prevention in schistosoma haematobium high-risk communities when individual behaviour matters: homogeneous and network models in epidemiology connecting the dots: network data and models in hiv epidemiology detailed transmission network analysis of a large opiate-driven outbreak of hiv infection in the united states bayesian inference of spreading processes on networks concurrent partnerships and the spread of hiv concurrency is more complex than it seems network-related mechanisms may help explain long-term hiv-1 seroprevalence levels that remain high but do not approach population-group saturation metapopulation dynamics of rabies and the efficacy of vaccination predicting the hiv/aids epidemic and measuring the effect of mobility in mainland effectiveness of unaids targets and hiv vaccination across 127 countries an hiv vaccine is essential for ending the hiv/aids pandemic opinion: mathematical models: a key tool for outbreak response ebola virus disease in west africa-the first 9 months of the epidemic and forward projections estimating the future number of cases in the ebola epidemic -liberia and sierra leone impact of bed capacity on spatiotemporal shifts in ebola transmission dynamics and control of ebola virus transmission in montserrado, liberia: a mathematical modelling analysis strategies for containing ebola in west africa effect of ebola progression on transmission and control in liberia interrupting ebola transmission in liberia through community-based initiatives epidemiological and viral genomic sequence analysis of the 2014 ebola outbreak reveals clustered transmission selective epidemiologic control in smallpox eradication emergency response to a smallpox attack: the case for mass vaccination the impact of contact tracing in clustered populations harnessing case isolation and ring vaccination to control ebola efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola ça suffit!) projections of ebola outbreak size and duration with and without vaccine use in équateur ebola vaccination in the democratic republic of the congo emergence and pandemic potential of swine-origin h1n1 influenza virus estimated influenza illnesses, medical visits, hospitalizations, and deaths averted by vaccination in the united states global epidemiology of avian influenza a h5n1 virus infection in humans, 1997-2015: a systematic review of individual case data the role of evolution in the emergence of infectious diseases information technology and global surveillance of cases of 2009 h1n1 influenza optimizing provider recruitment for influenza surveillance networks influenza a (h7n9) and the importance of digital epidemiology disease surveillance on complex social networks optimizing infectious disease interventions during an emerging epidemic optimizing influenza vaccine distribution nine challenges in incorporating the dynamics of behaviour in infectious diseases models evolutionary game theory and social learning can determine how vaccine scares unfold department of health & human services. measles cases and outbreaks the pertussis vaccine controversy in great britain the impact of rare but severe vaccine adverse events on behaviour-disease dynamics: a network model measles outbreak: opposition to vaccine extends well beyond ultra-orthodox jews critical dynamics in population vaccinating behavior digital epidemiology multiscale mobility networks and the spatial spreading of infectious diseases capturing sexual contact patterns in modelling the spread of sexually transmitted infections: evidence using natsal-3 the perpetual challenge of antimicrobial resistance factors affecting the reversal of antimicrobial-drug resistance retrospective evidence for a biological cost of vancomycin resistance determinants in the absence of glycopeptide selective pressures multistrain models predict sequential multidrug treatment strategies to result in less antimicrobial resistance than combination treatment universal or targeted approach to prevent the transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae in intensive care units: a cost-effectiveness analysis modeling antibiotic treatment in hospitals: a systematic approach shows benefits of combination therapy over cycling, mixing, and mono-drug therapies why sensitive bacteria are resistant to hospital infection control call of the wild: antibiotic resistance genes in natural environments antibiotic resistant bacteria are widespread in songbirds across rural and urban environments spatial and temporal distribution of antibiotic resistomes in a peri-urban area is associated significantly with anthropogenic activities impact of point sources on antibiotic resistance genes in the natural environment: a systematic review of the evidence investigating antibiotics, antibiotic resistance genes, and microbial contaminants in groundwater in relation to the proximity of urban areas systematic review: impact of point sources on antibioticresistant bacteria in the natural environment environmental factors influencing the development and spread of antibiotic resistance quantifying transmission of clostridium difficile within and outside healthcare settings optimal cost-effectiveness decisions: the role of the cost-effectiveness acceptability curve (ceac), the cost-effectiveness acceptability frontier (ceaf), and the expected value of perfection information (evpi) probabilistic uncertainty analysis of epidemiological modeling to guide public health intervention policy zika viral dynamics and shedding in rhesus and cynomolgus macaques evaluating vaccination strategies for zika virus in the americas epidemic dengue and dengue hemorrhagic fever at the texas-mexico border: results of a household-based seroepidemiologic survey assessing real-time zika risk in the united states one health approach to cost-effective rabies control in india cost-effectiveness of canine vaccination to prevent human rabies in rural tanzania cost-effectiveness of next-generation vaccines: the case of pertussis optimizing the impact of low-efficacy influenza vaccines reassessing google flu trends data for detection of seasonal and pandemic influenza: a comparative epidemiological study at three geographic scales a new look at the statistical model identification model selection in ecology and evolution direct and indirect effects of rotavirus vaccination: comparing predictions from transmission dynamic models data-driven models to predict the elimination of sleeping sickness in former equateur province of drc learning from multi-model comparisons: collaboration leads to insights, but limitations remain public health impact and cost-effectiveness of the rts, s/as01 malaria vaccine: a systematic comparison of predictions from four mathematical models role of mass drug administration in elimination of plasmodium falciparum malaria: a consensus modelling study bernoulli's epidemiological model revisited ebola: models do more than forecast models overestimate ebola cases coupled contagion dynamics of fear and disease: mathematical and computational explorations ecological theory suggests that antimicrobial cycling will not reduce antimicrobial resistance in hospitals the authors gratefully acknowledge funding from the notsew orm sands foundation (grants to m.c.f., j.p.t. and a.p.g.), the national institutes of health (grant nos. k01 ai141576 and u01 gm087719 to m.c.f. and a.p.g., respectively) and the natural sciences and engineering research council of canada (grant no. rgpin-04210-2014 to c.t.b.). the authors also thank c. wells and a. pandey, both members of the yale center for infectious disease modeling and analysis, for their helpful discussions regarding the hiv and ebola modelling literature. m.c.f. and a.p.g. drafted the initial manuscript. m.c.f., c.t.b., j.p.t. and a.p.g. all critically revised the content. the authors declare no competing interests. correspondence should be addressed to a.p.g.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-265600-lnik974k authors: celerino da silva, ronaldo; segat, ludovica; crovella, sergio title: role of dc-sign and l-sign receptors in hiv-1 vertical transmission date: 2011-01-26 journal: hum immunol doi: 10.1016/j.humimm.2011.01.012 sha: doc_id: 265600 cord_uid: lnik974k the innate immune system acts in the first line of host defense against pathogens. one of the mechanisms used involves the early recognition and uptake of microbes by host professional phagocytes, through pattern recognition receptors (prrs). these prrs bind to conserved microbial ligands expressed by pathogens and initiate both innate and adaptative immune responses. some prrs located on the surface of dendritic cells (dcs) and other cells seem to play an important role in human immunodeficiency virus type 1 (hiv-1) transmission. dendritic cell–specific intercellular adhesion molecule–3 grabbing non-integrin, cd209 (dc-sign) and its homolog, dc-sign-related (dc-signr or l-sign) receptors are pprs able to bind the hiv-1 gp120 envelope protein and, because alterations in their expression patterns also occur, they might play a role in both horizontal and vertical transmission as well as in disseminating the virus within the host. this review aims to explore the involvement of the dc-sign and l-sign receptors in hiv-1 transmission from mother to child. the joint united nations program on hiv-1/aids estimates that 2.1 million children worldwide are infected with human immunodeficiency virus type 1 (hiv-1) [1] and that more than 430,000 children were newly infected in 2008. mother-to-child transmission, also known as vertical transmission, accounts for more than 40% of all hiv-1 infections in children [2] and can occur in three ways [3] [4] [5] : during pregnancy through the placenta (transplacental or intrauterine transmission); during delivery (intrapartum transmission) through amniotic fluids, infected blood and cervical secretions; and during the process of breastfeeding. in addition, some independent factors have been associated with vertical transmission of hiv-1, such as high maternal viral load, low amount of cd4 ϩ t cells, vaginal delivery and lower gestational age [4, 5] . global estimates show that, without specific medication, the rate of transmission of hiv-1 from mother to child is around 15-45% [6] ; using antiretroviral therapies, this percentage has sharply dropped to 2%. however, although prophylactic antiretroviral therapy can reduce mother-to-child transmission to 2%, the limited access to timely diagnosis and drugs in many developing countries reduces the potential impact of this strategy [5] . despite the sharp fall in the rate of viral transmission, a significant percentage of children are still being infected with hiv-1; the mechanisms used by the virus to escape the immune response and infect targets cells of children born to infected mothers treated with antiretroviral therapies still remain to be clarified. a better understanding of the immunologic mechanisms acting at the maternal-fetal interface and of host-pathogen interaction is essential for the development of alternative interventions aimed to prevent viral transmission. this review aims to explore the involvement of the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin, cd209 (dc-sign) and dc-sign-related c-type lectin domain family 4, member m (l-sign) receptors in hiv-1 transmission from mother to child. the innate immune system is the first line of host defense against pathogens; it involves the early recognition and uptake of microbes by host professional phagocytes, such as dendritic cells (dcs) and macrophages, through germline-encoded receptors, known as pattern recognition receptors (prrs) [7] . these proteins bind to conserved microbial ligands expressed by the pathogens, and initiate both innate and adaptative immune responses. prrs are involved in phagocytosis, and antigen presentation could activate intracellular signaling and cytokine secretion. the efficiency of this initial pathogen recognition may have important consequences in the pathogenesis of infectious diseases [8] . some prrs located on the surface of dcs and other cells seem to play an important role in hiv-1 transmission. of particular interest are the dc-sign and its close relative, l-sign (also known as dc-sign related [dc-signr], cd209l or clec4m) receptors [9, 10] . dc-sign and l-sign receptors, two c-type lectins, are long type ii integral membrane proteins [11, 12] that are involved in both innate and adaptive immunity [13] [14] [15] . they present strong dependence on calcium and act as cellular adhesion's receptors and are involved in pathogen recognition [16, 17] . as pathogen-recognition receptors, both these lectins recognize a wide range of microorganisms, some of which have a major impact on public health. for example, dc-sign captures viruses, such as ebola virus [18] , hepatitis c virus [19, 20] , dengue virus [21] , cytomegalovirus [22] , and sars coronavirus (sars-cov) [23, 24] , bacteria such as mycobacterium tuberculosis [25] and helicobacter pylori [26] , and parasites such as leishmania pifanoi [27] . l-sign is able to capture viruses such as ebola virus [18] , hepatitis c virus [19, 20, 28] , and more recently sars-cov [23, 29] , as well as bacteria such as m tuberculosis [30] , and leishmania infantum [27] . both dc-sign and l-sign can recognize and capture human immunodeficiency virus 1 by binding to the gp120 glycoprotein [31] [32] [33] [34] [35] . dc-sign and l-sign receptors are organized into three structurally distinct regions ( fig. 1) : an intracytoplasmatic tail domain responsible for internalization and signal transduction [36] that consists of n-terminal, ll, and yksl motifs and a triacidic group (eea), then a transmembrane domain and finally an extracellular domain, which is further divided into two structures, the neck repeat region and the carbohydrate recognition domain (crd) [17] . the neck repeat region usually consists of 7 full and 1 incomplete tandem repeats of a sequence of 23 highly conserved amino acids, but the number of repeats can vary in the population: the neck repeat region of the l-sign receptor is highly polymorphic (4 -10 repetitions are often found), while the one of dc-sign is less polymorphic (mainly seven repetitions) [12, 17, 37] . the neck repeats region plays a crucial role in tetramerization and supports carbohydrates' recognition, thus directly influencing the receptor's binding affinity to pathogens [8, 17, 36] . the number of tandem-neck repeats determines the multimerization status (fig. 2 ). feinberg et al. [38] showed that the lack of two repeats (five-repeat allele) results in partial dissociation of the final tetramer, whereas the lack of even more than five repeats causes a reduction in the overall stability of the molecule. by using a series of recombinant soluble receptors with different number of repeats, synder et al. [39] showed that the binding affinity to hiv-1 gp120 glycoprotein was affected by the length of the repeats and multimerization status, with tetrameric forms presenting a higher affinity than shorter monomeric forms. the crd, both in dc-sign and l-sign, is flexibly connected to the neck repeat region, allowing a departure from the membrane, which enables the binding of pathogens in a calcium-dependent manner [36] . dc-sign and l-sign belong to the cd209 gene family and probably originated following a gene duplication event [17] . the human genes encoding dc-sign and l-sign map on 19p13.2-3 and extend approximately for 13 kb [35, 40] . in addition, they share the same introns and exons organization (consisting in seven exons and five introns) and the encoded proteins present a high similarity at the amino acid level (77% identity) [17] . the two receptors are characterized by different expression patterns. dc-sign is highly expressed in monocytes and cd34 ϩ monocyte-derived dcs and in subsets of immature and mature dcs in various tissues such as dermis, mucosa, spleen, placenta (specialized macrophages of decidua and hofbauer cells in the chorionic villi), and lung (specialized macrophages in the alveoli) [36, 41, 42] . in contrast, l-sign is not expressed by dcs or monocyte-derived dcs in vitro, and its expression is limited to some tissues, such as lymph nodes (endothelial cells in the subcapsular sinus), liver (sinusoidal endothelial cells), placenta (capillary endothelial cells), lung (alveolar cells and endothelial cells), and intestine (villi capillaries in the lamina propria of the terminal ileum, peyer's patches) [12, 36] . as a specific adhesion receptor, dc-sign mediates the interaction between dcs and t cells by binding with high affinity to icam-3 [9, 11] . the interaction between dc-sign and icam-3 expressed on t cells contributes to a close interaction between dcs and t cells required for an efficient antigen presentation. dc-sign can also facilitate the capture of viral antigens by class i and class ii mhc, leading to activation of specific cd8 ϩ and cd4 ϩ t cells [36] . the interaction between dc-sign and l-sign and the hiv-1 has been already well studied [9, 16, [32] [33] [34] 36, 39, [43] [44] [45] [46] [47] [48] [49] . as mentioned before, dc-sign can bind to the gp120 protein of hiv-1 [41, 42, 50] capturing the virus and possibly increasing hiv-1 transmission [12] . similar to dc-sign, l-sign also captures the hiv-1 virus by binding to the gp120 and promoting the enhancement of t cell infection by hiv-1 in trans [17, 34] ; however, l-sign can also internalize the virus and promote virus degradation in a proteasomedependent manner, possibly directly affecting the outcome of the infection by hiv-1 [5] . the presence of l-sign on the surface of endothelial cells in lymph node sinuses represents an obvious mechanism by which the virus can be transmitted to cd4 ϩ cells that traffic into lymph nodes via the afferent lymphatics. in addition, since l-sign binds to icam-3 and may bind to other cell surface receptors, interactions between t-cells and the endothelial cell surface may occur more frequently, increasing the likelihood of virus transmission [9] . although dc-sign and l-sign are not direct receptors for hiv-1 infection, they work efficiently in the capture of hiv-1 from the periphery, thus facilitating viral transmission to secondary lymphoid organs rich in t cells and increasing the infection of target cd4 ϩ cells [16, 32, 33] . the hiv-1 virus can infect cells through two different paths: the trans-and the cis-infection (fig. 3a, b) . although infection in trans occurs when dc-sign is expressed on a separate cell from the one that becomes infected, the infection in cis may occur when dc-sign is co-expressed with cd4 and chemokine receptor (e.g., ccr5) on a permissive cell type, such as macrophages [16, 36, 40] . co-expression of cd4, ccr5, and cxcr4 receptors with dc-sign receptor has been blamed for the increase in the efficiency of hiv-1 infection, suggesting the involvement of the dc-sign receptor in viral transmission, besides increasing the efficiency of the infection in cis [51] . both dc-sign and l-sign receptors are involved in hiv-1 infection in trans [34] . however, the hiv-1 infection in cis, has only been observed so far for dc-sign receptor. the interaction of dc-sign and l-sign molecules with the hiv-1 occurs through the connection between their crd with the gp120 viral envelope glycoprotein [16, 17] . binding of the gp120 to dc-sign and l-sign molecules may induce a conformational change in the gp120 itself that enables a more efficient interaction with cd4 and/or the chemokine receptor and subsequent membrane fusion with t cells. alternatively, the binding of viral particles to the dcs may increase the probability that entry will occur after binding to the cd4 and co-receptor complex on target cells [10] . the ability of dc-sign and l-sign receptors to capture and transmit the hiv-1 to t cells may largely depend on their membrane organization in rafts or their capability to multimerize [10] . in addition, alternative splicing events can occur in dc-sign e l-sign, leading to the production of a vast repertoire of membrane-bound and soluble isoforms, which may also differently affect the process of hiv-1 transmission [5, 17, 52] . the human placenta is responsible for a close juxtaposition between fetal and maternal blood; however, this apparent barrier is permeable enough to display the hiv-1 from the mother, a fact that leads to fetal exposure to virus [53] . thus, the placenta can play an important role in the transmission of hiv-1 infection. most cases of vertical transmission via uterus occur through of the placenta (fig. 4) , especially during the third trimester of pregnancy [43] . it is estimated that the trans-placental or intrauterine hiv-1 transfer comprise 17-38% of cases of vertical transmission, but little is known about the mechanisms involved in the transmission of the virus [54] . some cell types are pointed out as likely targets for the viral spread, such as macrophages present in the decidua and specialized macrophages present in the placenta (fig. 4) , also known as hofbauer cells [54] . both decidual macrophages and hofbauer cells play important roles in the placental physiology, the firsts promoting the development and control of blood flow and the latter acting in the defense against infectious agents (hofbauer cells) [54] . hofbauer cells, specialized macrophages from human placenta, support infection by hiv-1 both in vitro and in vivo [55] . this ability to support hiv-1 infection is probably associated with the expression of some cellular receptors related to the infection of t lymphocytes, such as cd4, ccr5, cxcr4, and dc-sign, also expressed in these cells [54, 55] . during pregnancy, there is an increased expression of dc-sign by hofbauer cells in the chorionic villi, and this expression has been correlated with increased rates of hiv-1 vertical transmission [54] . thus, dc-sign can enhance the binding of hiv-1 on the surface of hofbauer cells, providing an efficient mechanism by which the virus can be transmitted to other receivers permissible to hiv-1 in trans. thus, one can wonder: how is the contact between native virus-bound infected cells with the fetal cells expressing receptors for hiv-1? the main physical barrier between fetal hofbauer cells and maternal fluids is the wall of the trophoblast cells; however trophoblast cells can express receptors for hiv-1 entry, so they can be infected by the virus [55] . moreover, breaches in the wall of the trophoblasts can originate from spontaneous processes or in consequence of infectious diseases (such as corioamniotite) and be-havioral habits such as smoking and drug use [55, 56] , allowing a direct contact between fetal hofbauer cells dc-sign ϩ cd4 ϩ ccr5 ϩ cxcr4 ϩ and viral particles adsorbed to the maternal decidual macrophages or dcs expressing dc-sign, present in maternal blood [54, 55, 57] . therefore, the contact between maternal and fetal cells through the wall of the trophoblasts, allows the efficient spreading of hiv-1 to fetal cells expressing receptors for viral binding and entry, allowing establishment of the infection [55] . a study has suggested that the mechanism of hiv-1 association with the cells, such as hiv-1 adsorbed to the dc-sign receptor, operates more efficiently in pregnancies where the viremia remains low because of the administration of antiretroviral therapies [54] . why does this happen? some authors explain that binding of viral particles to dc-sign may focus or concentrate the virus particles at the surface of the dc and may thus increase the probability that entry will occur after binding to the cd4 and co-receptor complex on target cells [9] . in addition, the hiv-1 virus can remain viable for several days on the dc-sign-expressing cell, and then can be more efficiently transferred to t cells than to the transfer executed by free cells [10, 16] . for the l-sign receptor, this fact is not observed. in addition, some authors propose three different mechanisms to explain how the hiv-1 virus is transmitted from mother to child, via the placenta. the first mechanism suggests that hofbauer cells infected with hiv-1 or with the virus adsorbed to their cell membrane through receptors, such as dc-sign, may enter the fetus through the umbilical vein [54, 55] . the second mechanism is that hofbauer cell infected by hiv-1 or carrying the virus adsorbed on the surface, remains in situ in the chorionic villi, promoting hiv-1 antigen presentation and subsequent t lymphocytes infection. however, this mechanism seems unlikely, since t lymphocytes are inconspicuous in chorionic villi [54, 55] . the third mechanism argues that hofbauer cells may become infected by hiv-1 and may release infectious viral particles, which may become adsorbed to l-sign on the immediately adjacent placental capillary endothelium. the endothelium may, in turn, mediate infection of hiv-1 receptorpositive t-lymphocytes circulating in the blood. infected t lymphocytes or hofbauer cells either productively infected with hiv-1 or simply with the virus adsorbed to their surface may then travel between the placenta and the fetus in umbilical cord blood [16, 54, 55] . in the intra-and post-partum vertical transmission of hiv-1, the virus is delivered and transmitted because of the contact with amniotic fluid, maternal blood, and cervical secretion (intrapartum) or with breast milk (post-partum) [3, 58] . it has been reported that, in the absence of a prophylactic antiretroviral therapy, the breast milk of infected mothers is responsible for more than 40% of cases of children infected with hiv-1 via vertical transmission [2] [3] [4] 59] . some factors, such as the viral load in the plasma and breast milk may be relevant for vertical transmission of hiv-1 [60] . transmission of the hiv-1 virus from mother to child via breast milk can occur by free virus particles and/or viral particles associated with cells [4] ; in this case, the expression of cellular receptors for recognition and adhesion of pathogens is required. among the cell types involved in the transmission of hiv-1 via breastfeeding, macrophages and mammary epithelial cells should be mentioned. breast milk is the only bodily fluid that contains a large number of macrophages, comprising more than 80% of all cells present in colostrum [61] . expressing ccr5, macrophages are prime targets of the hiv-1 virus, which uses the co-receptors ccr5 for viral entry [4] . in addition, macrophages, derived from peripheral blood monocytes (pbmo), are present in different concentrations throughout lactation, acting as immunoprotective in situ [61] . moreover, it has been reported that macrophages also express dc-sign receptors [61] . in certain situations, the expression levels of dc-sign on macrophages can be quite high, especially when stimulated with interleukin (il)-4, which also promotes reduction in the expression of ccr5 and cxcr4, suggesting the need for changes in local inflammatory th2 dominance for an acceleration of hiv-1 transmission via breastfeeding [59 -61] . local production of il-4 during infectious processes, as in mastitis, can over-regulate the expression of dc-sign on macrophages, suggesting the association of mastitis with high viral load in breast milk and high risk of vertical transmission of the virus [61] . along with the macrophages, mammary epithelial cells may also be infected by hiv-1, through the co-receptor cxcr5 [4] . some studies suggest the possibility of a hiv-1 compartmentalization between blood and milk, suggesting that the virus could be produced in and transmitted by the milk, through the mammary epithelial cells. in this sense, the viruses that derived from mammary epithelial cells can determine the tropism of hiv-1 transmitted to cells located in the gastrointestinal tract [60] . after being introduced in the organism through infected breast milk, the virus reach the mucosa of the upper intestine, where, in the lamina propria, a large pool of lymphocytes expressing ccr5 and cxcr4 facilitate viral replication. the presence of dcs expressing a series of receptors, such as cd4/ccr5, dc-sign, and dc206, has been reported in the human gut [58] . from the mucosa, the virus is systemically spread and produces a profound depletion of cd4 ϩ t cells, with monocytes and macrophages also acting as cellular reservoirs for hiv-1 [3] . the viral entry through the mucosa of the gastrointestinal tract can be mediated by the binding of the dc-sign receptor, expressed on dcs, to the viral gp120 protein [2] . this interaction appears to be more pronounced in the tonsils at the top of the esophagus and intestinal tract [4] . the infectivity of viruses associated with cells and captured by dc-sign is stable even in presence of the acidification process occurring in the gastrointestinal tract, suggesting that the virus bound to dcs through dc-sign is protected from the action of the gastric juice. the fact that the free viral particles loose their infectivity when exposed to acidic environments, suggest that the transmission of the virus to free cells in milk is hampered by gastric juice [60] . however, it is possible that free cells become infected in the oral mucosa and esophagus, where the acidity is not high. studies with raji cells expressing dc-sign, preincubated with pbs and with the hiv-1 virus, showed an efficient viral transfer. however, raji cells expressing dc-sign, incubated with hiv-1 virus and uninfected human milk, showed a significant reduction of the binding of hiv-1 gp120 to dc-sign receptor. this suggests that in human milk some factors that could prevent the interaction between the gp120 and dc-sign receptor exist [4] . similar tests were conducted for l-sign receptor, however, breast milk did not inhibit the interaction between the viral proteins and the receptor, suggesting that l-sign receptor can be used by the virus to increase its infectivity [4] . some studies also report that exclusive feeding with uninfected mothers' milk during the first months of life protects against a variety of infections, including hiv-1, and help fight morbidity and mortality, suggesting that there should be certain components in the human milk that may protect against the transmission of the virus [59] . so, what are these likely factors in breast milk, and how do they act in protection against the infection caused by hiv-1? breast milk is provided with a series of antimicrobial compounds, such as lactoferrin, lysozyme, secretory leukocyte protease inhibitor, lactodifucotetrase, lacto-n-fucopentose i, ii, and iii, and monofucosilacto-n-hexose iii, among others, which are associated with a reduced rate of hiv-1 transmission [2, 59] . some studies attribute this reduction in viral transmission to certain antigens, such as lewis structures, which compete with the gp120 for a binding site in the dc-sign receptor, inhibiting the viral transfer to cd4 ϩ t cells [2] . inhibition of the binding between gp120 and dc-sign receptor is probably due to the size of the compound, which contains many lewis structures that mask the interaction sites [4] . compounds containing lewis structures and present in breast milk were shown to interact with dc-sign, blocking the response of th1 cells and resulting in an increased responsiveness of th2 cells, suggesting that these compounds may influence the immune response by acting as immunomodulatory factors [4] . a constituent of human milk, bile salt-stimulated lipase, a lewis x (le x )-containing glycoprotein secreted by the pancreas as well as by mammary gland, has been shown to inhibit dc-sign binding to hiv and dc-sign-mediated transfer of hiv-1 to cd4 ϩ lymphocytes, by competing with the virus for the binding to dc-sign [62] . the binding of bile salt-stimulated lipase to dc-sign can be prevented using an antibody against le x , thus demonstrating the importance of the le [10] epitope. others constituents of human milk, such as human milk oligosaccharides and muc1 (epithelial mucin), have shown promising results and could be used to develop drugs that inhibit the hiv-1 binding to dc-sign [2, 59] . hong et al. [2] found a reduction of more than 60% in the interaction between gp120 and the receptor protein dc-sign when using human milk oligosaccharides at a concentration of 0.5 g/l, the one usually present in breast milk. additionally, saeland et al. [59] also described the blocking of the interaction between the gp120 and dc-sign receptor in the presence of muc1 factor, present in human milk. because of the blockade, there was the prevention of the virus transmission to cd4 ϩ t cells. blocking dc-sign may be a double-edged sword. it may reduce the entrance of certain viruses, such as hiv-1, but at the same time it may also reduce the ability of the infant's immune system to detect and fight other pathogens [2] . for some genes, susceptibility and/or resistance to certain (infectious but not only) diseases has been associated with gene expression levels and with the presence of gene variations/mutations. can this happen also for dc-sign and l-sign? can variations in the gene encoding dc-sign and l-sign be associated with vertical transmission of hiv-1? to date, except one study regarding the l-sign gene, no other genetic studies trying to associate mutations in the genes encoding for dc-sign and l-sign with the vertical transmission of the hiv-1 have been performed. boily-larouche et al. [5] performed an association study in a well-characterized cohort of 197 hiv-1-infected mothers and their children from zimbabwe, and found that children with two copies of h1 and/or h3 haplotype of l-sign were about 3.6 times more at risk for intrauterine transmission of hiv-1 and 5.7 times at risk for intrapartum transmission. the h1 and h3 haplotypes are characterized by two single nucleotide polymorphisms in the promoter region (p-198a) and the intron 2 (int2-180a) that associate with a reduction of the transcriptional activity. the same study also showed that infants homozygous for the h1 haplotype showed a more than fourfold decrease in the level of placental l-sign transcripts, and in particular of the membrane linked isoforms [5] . a reduced expression of l-sign (especially of the membrane isoforms) in the endothelial cells of capillaries in the placenta may facilitate the binding of hiv-1 to viral entry receptors of endothelial cells, such as ccr5, which can facilitate the migration of maternal hiv-1 across the placental barrier, resulting in intrauterine transmission of hiv-1 [5] . the membrane bound l-sign receptors are responsible for catching the virus. after capture, the virus adhered to l-sign may undergo degradation processes or be presented as antigens. thus, these receptors act to protect the infant against infection by hiv-1 [5] . boily-larouche et al. [5] explain these discoveries with the hypothesis that when the levels of placental l-sign-bound membrane are reduced, virus fails to bind to l-sign and binds preferentially to ccr5 receptors on endothelial cells of capillaries, resulting in loss of integrity of the placental barrier and increase the passage of cells infected by hiv-1 in fetal circulation, leading to vertical transmission. in view of what has been discussed, much evidence exist that the dc-sign and l-sign receptors are involved in the transmission of hiv-1 from mother to child. therefore, the dc-sign and l-sign receptors should be likely targets for the development of new drugs and antiretroviral therapies, to challenge the spread of viral transmission. in addition to this, given that only a few genetic studies have been performed to investigate the possible involvement of dc-sign and l-sign receptors in the genetic mechanisms correlated with vertical transmission of the hiv-1 virus, we believe that more detailed studies aiming to elucidate the role of genetic variants from different worldwide populations in susceptibility and/or resistance to hiv-1 infection are needed. human milk oligosaccharides reduce hiv-gp120 binding to dendritic cell-specific icam3-grabbing nonintegrin dcsigns) macrophage hiv infection and the gastrointestinal tract reservoir component in human milk binds dc-sign and inhibits hiv transfer to cd4 ϩ t lymphocytes functional genetic variants in dc-signr are associated with motherto-child transmission of hiv mother-to-child transmission of hiv: a global perspective the evolution and genetics of innate immunity promoter and neck region length variation of dc-sign is not associated with susceptibility to tuberculosis in tunisian patientes the role of dc-sign and dc-signr in hiv and siv attachment, infection, and transmission dc-sign, a c-type lectin on dendritic cells that unveils many aspects of dendritic cell biology dc-sign: a guide to some mysteries of dendritic cells polymorphic varieants in dc-sign, dc-signr and sdf-1 in high risk seronegative and hiv patients in northern asian indians molecular characterization of dendritic cells operating at the interface of innate of acquired immunity dc-sign and dc-signr genetic diversity among different ethnic populations: potential implications for pathogen recognition and disease susceptibility c-type lectins on dendritic cells: key modulators for the induction of immune responses dc-sign (dendritic cell-specific icam-3 grabbing non-integrin) and dc-sign-related dcsignr): friend or foe? the signs for infection c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans cd209l) and dcsign(cd209) mediate transinfection of liver cells by hepatitis c virus hepatitis c virus glycoproteins interact with dc-sign and dcsignr cd209) mediates dengue virus infection of human dendritic cells human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells helicobacter pylori modulates the t helper cell 1/t helper cell 2 balance through phase-variable interaction between lipopolysaccharide and dcsign role of the c-type lectins dc-sign and l-sign in leishmania interaction with host phagocytes dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein cd209l lsigns) is a receptor for severeacute respiratory syndrome coronavirus identification of the mycobacterial carbohydratestructure that binds the ctype lectins dc-sign, l-sign and signr1 structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr dc-sign, a dendritic cell-specific hiv-binding protein that enhances trans-infection of t cells identification of dc-sign, a novel dendritic cell-specific icam-3 receptor that supports primary immune responses dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans a dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin dcsigns)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv infection dendritic-cell interactions with hiv: infection and viral dissemination impact of polymorphisms in the dc-signr neck domain on the interaction with pathogens extended neck regions stabilize tetramers of the receptors dc-sign and dc-signr characterization of dc-sign/r interaction with human immunodeficiency virus type 1 gp120 and icam molecules favors the receptor's role as an antigen-capturing rather than an adhesion receptor influence of polymorphism in dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-related. dcsignr) gene on hiv trans-infection most dc-signr transcripts at mucosal hiv transmission sites are alternatively spliced isoforms association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type 1 infection dc-sign, a dentritic cell-specific hiv receptor present in placenta that infects t cells in trans-a review dendritic cells and transmission of hiv binding of human immunodeficiency vîrus type 1 to immature dendritic cells can occur independently of dc-sign and mannose binding c-type lectin receptors via a cholesteroldependent pathway dc-sign on b lymphocytes is required for transmission of hiv to t lymphocytes the polymorphisms in dc-signr affect susceptibility to hiv type 1 infection evolution of dc-sign use revealed by fitness studies of r5 hiv variants emerging during aids progression abundant and superficial expression of c-type lectin receptors in ectocervix of women at risk of hiv infection cd209 gene polymorphisms in south indian hiv and hiv-tb patients cis expression of dc-sign allows for more efficient entry of human and simian immunodeficiency viruses via cd4 and a co-receptor extensive repertoire of membrane-bound and soluble dendritic cell-specific icam-3-grabbing nonintegrin 1. dcsigns;1 and dc-sign2 isoforms effect of intrauterine hiv exposure on the frequency and function of uninfected newborns' dendritic cells placental expression of dc-sign may mediate intrauterine vertical transmission of hiv transplacental transmission of hiv: a potential role for hiv binding lectins mechanisms and timing of mother-to-child transmission of hiv vertical transmission of hiv: parameters which might affect infection on the study of in utero transmission of hiv 1 dendritic cells transmit hiv through human small intestinal mucosa muc1 in human milk blocks transmission of human immunodeficiency virus from dendritic cells to t cells transmission of macrophage-tropic hiv by breast-milk macrophages via dc-sign breast milk macrophages spontaneously produce granulocyte-macrophages colonystimulating factor and differentiate into dendritic cells in the presence of exogenous interleukin-4 alone bile salt-stimulated lipase from human milk binds dc-sign and inhibits human immunodeficiency virus type 1 transfer to cd4 ϩ t cells we thank the laboratory of immunopathology keizo asami, the department of genetics, federal university of pernambuco, the graduate program in genetics and molecular biology for supporting physical and scientific, as well as facepe and cnpq, for financial support. s.l. is recipient of a fellowship grant (apq-0020-4.01/08) from facepe key: cord-288440-w7g2agaf authors: jia, rui; ding, shilei; pan, qinghua; liu, shan-lu; qiao, wentao; liang, chen title: the c-terminal sequence of ifitm1 regulates its anti-hiv-1 activity date: 2015-03-04 journal: plos one doi: 10.1371/journal.pone.0118794 sha: doc_id: 288440 cord_uid: w7g2agaf the interferon-inducible transmembrane (ifitm) proteins inhibit a wide range of viruses. we previously reported the inhibition of human immunodeficiency virus type 1 (hiv-1) strain bh10 by human ifitm1, 2 and 3. it is unknown whether other hiv-1 strains are similarly inhibited by ifitms and whether there exists viral countermeasure to overcome ifitm inhibition. we report here that the hiv-1 nl4-3 strain (hiv-1(nl4-3)) is not restricted by ifitm1 and its viral envelope glycoprotein is partly responsible for this insensitivity. however, hiv-1(nl4-3) is profoundly inhibited by an ifitm1 mutant, known as δ(117–125), which is deleted of 9 amino acids at the c-terminus. in contrast to the wild type ifitm1, which does not affect hiv-1 entry, the δ(117–125) mutant diminishes hiv-1(nl4-3) entry by 3-fold. this inhibition correlates with the predominant localization of δ(117–125) to the plasma membrane where hiv-1 entry occurs. in spite of strong conservation of ifitm1 among most species, mouse ifitm1 is 19 amino acids shorter at its c-terminus as compared to human ifitm1 and, like the human ifitm1 mutant δ(117–125), mouse ifitm1 also inhibits hiv-1 entry. this is the first report illustrating the role of viral envelope protein in overcoming ifitm1 restriction. the results also demonstrate the importance of the c-terminal region of ifitm1 in modulating the antiviral function through controlling protein subcellular localization. interferon inhibits virus infection by inducing the expression of hundreds of host genes, known as interferon-stimulated genes (isgs), some of which encode antiviral effectors [1] . among these antiviral proteins are a small protein family called interferon-inducible transmembrane (ifitm) proteins. ifitms have an average length of 130 amino acids, contain two predicted transmembrane domains and a conserved intracellular domain [2, 3] . although ifitm1 was shown to modestly inhibit vesicular stomatitis virus (vsv) and hepatitis c virus (hcv) more than a decade ago [4, 5] , the antiviral function of ifitm1, 2 and 3 was established only when their potent restriction of influenza a virus, west nile virus and dengue virus was reported in a genome-wide functional screen [6] . subsequent studies revealed that many viruses are susceptible to ifitm restriction. these include flaviviruses (west nile virus, dengue virus, and yellow fever virus), filoviruses (marburg virus and ebola virus), sars coronavirus, reovirus, rift valley fever virus, human immunodeficiency virus type 1 (hiv-1), jaagsiekte sheep retrovirus (jsrv), and others [6, 7, 8, 9, 10, 11, 12, 13] (reviewed in [14, 15] ). the in vivo importance of ifitm proteins in antiviral defense is supported by their protection of mice from influenza a virus infection [16, 17, 18] . in support of this, single nucleotide polymorphisms in ifitm3 gene have been shown to associate with pathogenesis severity of influenza a virus infection in humans [16, 19] . humans have five ifitm genes, including ifitm1, ifitm2, ifitm3, ifitm5 and ifitm10. they are clustered within a 26.5kb region on chromosome 11 except for ifitm10 that is located 1.4 mb downstream [2, 20, 21] . ifitm5 has a calcium-binding domain at its c-terminal region and is involved in bone mineralization and maturation [22] . ifitm5 has thus also been named bone restricted ifitm-like protein (bril). ifitm1, ifitm2 and ifitm3 are expressed in a variety of tissues. their expression is stimulated by type i and type ii interferon due to the presence of the interferon stimulation response element (isre) in their promoters [2, 23] . in addition to their roles in antiviral defense, ifitm1, ifitm2 and ifitm3 have also been reported to participate in antiproliferative signaling, cell adhesion, and oncogenesis (reviewed in [21] ). for instance, expression of these three ifitm members is significantly upregulated in colorectal tumor cells concurrent with the activation of the wnt/beta-catenin signaling pathway [24] . the function of ifitm10 is unknown except that it is the most conserved among all ifitm proteins across many eukaryotic species [20, 25] . ifitm1, 2 and 3 proteins differ in their abilities to inhibit different viruses. for example, ifitm3 inhibits influenza a virus more potently than inhibits marburg and ebola viruses; in contrast, these latter two viruses are strongly inhibited by ifitm1 [11] . further, ifitm2 and ifitm3, but not ifitm1, inhibit rift valley fever virus [7] . this different restriction profile is likely a result of the sequence divergence between ifitm1, 2 and 3. ifitm2 and 3 share higher sequence homology between each other than with ifitm1 [21] . in this study, we report that human ifitm1 differentially affects the replication of two closely related hiv-1 strains bh10 and nl4-3 and that the viral envelope glycoprotein is responsible for this differential sensitivity to ifitm1 restriction. interestingly, deleting the c-terminal sequence of ifitm1 allows the mutant to potently inhibit both bh10 and nl4-3 viruses, which highlights the important role of this c-terminal domain in regulating the antiviral function of ifitm1. human ifitm1 inhibits the replication of hiv-1 bh10 but not hiv-1 we have previously shown that ifitm1, 2 and 3 inhibit the replication hiv-1 strain bh10 in supt1 cells [10] . when we tested another hiv-1 strain called nl4-3, ifitm1 did not exhibit any inhibitory effect (fig. 1a) , which was in contrast to the marked suppression of hiv-1 nl4-3 replication by ifitm2 and 3 (s1 fig.) . this observation was further confirmed by the resistance of an hiv-1 nl4-3 -derived virus called nleny1-ires to ifitm1 (fig. 1a) . human ifitm1 has a relatively long c-terminal region than ifitm2 and 3. we previously showed that ifitm1 mutants lacking this c-terminal sequence still strongly inhibit hiv-1 bh10 (fig. 1b and 1c ) [10] . given the resistance of hiv-1 nl4-3 to the wild type human ifitm1, we expected that hiv-1 nl4-3 would also be refractory to the c-terminus truncated ifitm1. surprisingly, ifitm1 mutants δ(117-125), δ(112-125) and δ(108-125) all strongly inhibited hiv-1 nl4-3 replication ( fig. 1b and 1c) . these data suggest that the c-terminal sequence of ifitm1 negatively modulates the anti-hiv-1 function of ifitm1. deleting the c-terminal sequence of ifitm1 leads to inhibition of hiv-1 entry we have previously shown that, in contrast to ifitm2 and 3, human ifitm1 does not affect the entry of hiv-1 bh10 [10] . similarly, no inhibitory effect of ifitm1 on hiv-1 nl4-3 entry was observed ( fig. 2a) . interestingly, the c-terminal truncated ifitm1 mutants, δ(117-125), δ(112-125) and δ(108-125), diminished hiv-1 nl4-3 entry by 2 to 3-fold ( fig. 2a and 2b) . this inhibition appears to be specific to hiv-1, since neither the wt ifitm1 nor its c-terminal truncations affected entry that was mediated by the g protein of vesicular stomatitis virus (vsv) ( fig. 2a and 2b) . in order to understand how the c-terminal sequence of ifitm1 might regulate its ability to impair hiv-1 entry, we examined the cellular localizations of ifitm1 and its c-terminal deletion mutants by immunofluorescence and confocal microscopy. the wild type ifitm1 exhibited a predominant intracellular distribution and showed co-localization with the early endosome marker rab5 (fig. 3a) . removing the last 9 or 14 amino acids from c-terminal sequence re-localized the majority of ifitm1 to the cell periphery (fig. 3a) , indicating that these c-terminus truncated ifitm1 mutants are mostly positioned at the plasma membrane where hiv-1 entry occurs. it was also noteworthy that the δ(18-125) deletion mutant exhibited an intracellular localization similar to the wild type ifitm1 (fig. 3a ), yet δ(18-125) markedly diminished hiv-1 entry (fig. 2 ). this suggests that the δ(18-125) mutant may employ a distinct mechanism to deter hiv-1 entry. we next asked how the c-terminal sequence of ifitm1 modulates protein subcellular distribution. no apparent endocytic sorting signals are present within the sequence 117-qii-qekrgy-125 of ifitm1, which was deleted in δ(117-125). one possibility is that this sequence may serve as a retention signal to sequester ifitm1 in the endoplasmic reticulum (er) or golgi [26] . this hypothesis is refuted by the results showing that blocking endocytosis with the dynamin inhibitor dynasore caused relocation of ifitm1 to the plasma membrane, as opposed to a ifitm1-kdel variant that had the er retention signal kdel inserted at the cterminus and exhibited er localization even in the presence of dynasore (fig. 3b) . these data suggest that ifitm1 is an endocytic protein and that removal of its c-terminal sequence causes its relocation to the plasma membrane where it interferes with hiv-1 entry. mouse ifitm1 diminishes hiv-1 entry ifitm1 orthologs from different species are highly conserved, except for mouse and rat ifitm1 that have shortened c-termini (fig. 4a ). these two ifitm1 proteins are 19 and 16 amino acids shorter at their c-termini compared to the human ifitm1. this prompted us to test whether they would act like c-terminal truncations of human ifitm1 and inhibit hiv-1 deletion mutations are indicated. both the wild type and mutated ifitm1 have a flag tag attached to the n-terminus. levels of the wild type and mutated ifitm1 in stably transduced supt1 cells were examined by western blotting. an amount of 500 μg/ml doxycycline was used to induce ifitm1 expression. levels of tubulin were probed as internal controls. (c) replication of nl4-3 and bh10 in supt1 cells stably expressing ifitm1 mutants δ(117-125), δ(112-125) or δ(108-125). virus production was determined by measuring levels of viral rt activity in culture supernatants. a representative result of three independent infections is shown. fig. 4b show that mouse ifitm1 suppressed hiv-1 replication in supt1 cells. further, mouse ifitm1 markedly decreased the entry of hiv-1 but not the entry that was mediated by vsv g protein ( fig. 4c and 4d ). these data further highlight the importance of the c-terminal sequence of ifitm1 in modulating its antiviral function and also suggest the greater antiviral potential of mouse ifitm1. we next performed mutagenesis to identify which viral protein(s) accounts for the resistance of hiv-1 nl4-3 to human ifitm1. to this end, we generated chimeric viruses by exchanging dna fragments between hiv-1 bh10 and hiv-1 nl4-3 and tested which viral dna fragment(s) bears the genetic information that determines the resistance of hiv-1 nl4-3 to ifitm1 (fig. 5a) . the results showed that exchange of the 2.7kb dna fragment, located between the sali and bamh1 restriction sites (called sb fragment), allowed bh(sb) resistant to ifitm1 and, reciprocally, nl(sb) sensitive to ifitm1 inhibition (fig. 5b ). this sb dna fragment encodes four viral proteins, tat, rev, vpu and env. to further determine which viral protein plays the countering role, we exchanged between hiv-1 bh10 and hiv-1 nl4-3 only the env sequence. the results of infection experiments showed that hiv-1 nl4-3 env enabled hiv-1 bh10 to replicate in ifitm1-expressing cells, whereas inserting the env of hiv-1 bh10 into hiv-1 nl4-3 dramatically reduced virus replication in the presence of ifitm1 (fig. 5c) , supporting the role of nl4-3 env in overcoming ifitm1 inhibition. no significant effect in this regard was observed for the small dna fragment that is located between the sali site and the beginning of env (fig. 5c ). hiv-1 nl4-3 is more efficient than hiv-1 bh10 in cell-to-cell transmission one mechanism behind the ifitm1 inhibition of hiv-1 bh10 is the reduction in gag/p24 levels in the infected cells as well as the diminution in virus production ( fig. 6a and 6b ) [10] . similar defects were also observed for hiv-1 nl4-3 infection of ifitm1-expressing supt1 cells ( fig. 6c and 6d ), which indicates that hiv-1 nl4-3 escapes ifitm1 in the spread infection without the need to restore this decrease in gag/p24 expression. we recently reported that hiv-1 bh10 became resistant to ifitm1 restriction in the spread infection through acquiring mutations in viral env and vpu proteins that together enhance the virus transmission between cells [27] . in order to test whether the same mechanism supports the resistance of hiv-1 nl4-3 to ifitm1, we compared the cell-to-cell transmission efficiency of hiv-1 bh10 and hiv-1 nl4-3 . indeed, hiv-1 nl4-3 was 3-fold more efficient than hiv-1 bh10 in transmitting between supt1 cells (fig. 7a ). this phenotype was readily reversed when the env sequence was exchanged between hiv-1 bh10 and hiv-1 nl4-3 , either by exchanging the sb fragment or the env sequence alone (fig. 7a) . similar observations were made for virus transmission from control supt1 cells to supt1 cells expressing ifitm1 (fig. 7b) . collectively, these data suggest that the env protein of hiv-1 nl4-3 mediates greater cell-to-cell transmission, a property that may allow hiv-1 to escape ifitm1 inhibition under the virus spread infection condition. transfected with plasmid dna expressing the wild type ifitm1 or its variant ifitm1-kdel that has the er retention signal attached to the c-terminus. cells were either treated with dynasore (160 μm) or dmso as control. ifitm1 and ifitm1-kdel were detected by immunostaining with anti-flag antibody. the endogenous calreticulin was stained with anti-calreticulin antibody. nuclei were stained with dapi. images shown represent the major subcellular distribution of each protein. this study was based on the observation that two closely related hiv-1 subtype b strains bh10 and nl4-3 were differentially inhibited by human ifitm1 in virus spread infection. this finding allowed us to identify the viral component(s) in nl4-3 that gave rise to ifitm1 resistance. results of mutagenesis and virus replication studies revealed an essential role of viral env protein in hiv-1 nl4-3 evasion from ifitm1. exchanging the env sequences between bh10 and nl4-3 reversed the susceptibility of the parental strains to ifitm1 inhibition. this role of hiv-1 env protein in determining viral sensitivity to ifitm1 corroborates our recent report showing that hiv-1 bh10 was able to develop resistance to ifitm1 through mutating env and vpu [27] . there are precedents illuminating the function of viral env in overcoming host restriction. one example is the hiv-2 env that antagonizes tetherin [28] . in addition, hiv-1 env has been shown to regulate the establishment of latent infection in resting cd4+ t cells [29] , likely through modulating the functionality of certain cellular pathways [30] . in the case of ifitm1, it remains to determine whether hiv-1 env serves as the viral target of ifitm1 or env acts as a viral antagonist to counter ifitm1. although ifitm1 does not affect the entry of either hiv-1 bh10 or hiv-1 nl4-3 , production of both viruses is diminished in the one-round infection assay, which alludes to inhibition of a post-entry step of hiv-1 infection by ifitm1. to our surprise, in spite of being inhibited to similar degrees in the one-round infection assay, only hiv-1 bh10 , but not hiv-1 nl4-3 , is crippled by ifitm1 in the long-term virus replication assay (fig. 1) . these seemingly conflicting observations could be reconciled by the major difference between the one-round infection and the long-term infection, i.e. the latter infection mode involves virus cell-to-cell transmission that actually dominates hiv-1 transmission over infection by free virus particles [31] . it is thus possible that the superiority of hiv-1 nl4-3 over hiv-1 bh10 in replicating in ifitm1expression supt1 cells, despite that both viruses are equally impaired by ifitm1 in the oneround infection, might be a result of the greater ability of hiv-1 nl4-3 to transmit between cells. indeed, we found that hiv-1 nl4-3 was 3-fold more efficient in transmission from cell to cell than hiv-1 bh10 . hiv-1 nl4-3 has therefore utilized its strong cell-to-cell transmission to compensate for the deficiency in virus production caused by ifitm1. the efficiency of cell-to-cell transmission is determined by viral env protein. inserting the env sequence of hiv-1 bh10 into hiv-1 nl4-3 diminishes cell-to-cell transmission of the parental virus. reciprocally, hiv-1 nl4-3 env protein increases cell-to-cell transmission of hiv-1 bh10 by 3-fold. concurrent with the change in cell-to-cell transmission, this exchange in env sequences also switches the sensitivity of hiv-1 bh10 and hiv-1 nl4-3 to human ifitm1. one possible scenario is that hiv-1 env protein may modulate virus sensitivity to human ifitm1 restriction by virtue of its ability to mediate and regulate virus cell-to-cell transmission. the env sequences of hiv-1 bh10 and hiv-1 nl4-3 differ at 23 amino acid positions. changing each of these 23 amino acids in hiv-1 bh10 env to the counterpart in hiv-1 nl4-3 did not increase the resistance of hiv-1 bh10 to human ifitm1 (data not shown). it can be conceived that two or more of these 23 amino acids in env are required to generate resistance to ifitm1. human ifitm1 does not affect the entry of either hiv-1 bh10 or hiv-1 nl4-3 (fig. 2) [10]. interestingly, ifitm1 gains the ability to inhibit hiv-1 entry when its c-terminal sequence is truncated (fig. 2) . in agreement with this observation, c-terminal truncation enhances the ability of ifitm1 to promote the infection of coronavirus oc43 [32] . this gained function appears to be virus specific, since the truncated ifitm1 does not affect virus entry mediated by vsv g protein (fig. 2) . one possible explanation of this differential inhibition is that hiv-1 and vsv adopt different pathways for entry. hiv-1 entry is ph-independent and occurs at the plasma membrane, whereas vsv enters cells in the endosomes of low ph [33, 34] . in support of this scenario, removal of the last 9 amino acids from the c-terminus of human ifitm1 relocates this protein from intracellular sites to cell periphery (fig. 3) . this cellular locationdependent inhibition phenomenon was previously reported for ifitm3. deleting the endocytic sorting signal 20-yeml-23 in ifitm3 led to redistribution of ifitm3 to the plasma membrane and the consequent loss of inhibition of influenza a virus entry that takes place at late endosomes [35, 36, 37, 38] . in addition to the regulation by their own sequences, subcellular distribution of ifitms can also be modulated by other factors. for example, compared to its intracellular localization in 293t cells as shown in this study, the exogenous ifitm1 has been observed at the plasma membrane in a549 cells [39] . in addition to the cell type-dependent effect, other factors including expression levels and treatment with interferon may also affect where ifitm proteins are localized in cells. it is unclear how the c-terminal sequence modulates the subcellular distribution of ifitm1, especially in light of the recent report showing that ifitm1 c-terminal domain is extracellular [39] . one mechanism by which extracellular/luminal protein domain regulates protein subcellular localization is the er-luminal retention signal [26] . our data rule out the presence of erluminal retention signal within the c-terminal region of ifitm1, because inhibiting endocytosis with the dynamin inhibitor dynasore redistributes ifitm1 to the plasma membrane, but the same treatment fails to do so to an ifitm1 variant with the exogenous er retention signal inserted at the c-terminus (fig. 3b) . alternatively, deleting the c-terminal sequence may change the membrane topology of ifitm1 and, as a result, disturb its subcellular localization. it remains to be determined if other hiv-1 strains are also differentially inhibited by human ifitm1. we suspect that the chance of finding human ifitm1-sensitive hiv-1 strains, either lab-adapted or naturally-existing or circulating, would be low if ifitm1 exerts selection pressure in vivo on hiv-1 replication. since both hiv-1 bh10 and hiv-1 nl4-3 are strains that have been propagated in cultured human t cell lines, they have adapted to the in vitro culture conditions and as a result, they could have lost the original viral sequences that confer resistance to ifitm1, which may have happened to hiv-1 bh10 . we envision that since hiv-1 has never encountered mouse ifitm1, most, if not all, of the hiv-1 strains would be sensitive to the mouse ifitm1 inhibition. work is ongoing to examine the effect of ifitm proteins from different species on other strains of hiv and different lentiviruses. the hiv-1 proviral dna clone hiv-1 nl4-3 was obtained from the nih aids research and reference reagent program. the nleny1-ires dna is a derivative of hiv-1 nl4-3 [40] . the nleny1-ires-es dna is similar to nleny1-ires except for insertion of stop codon at the beginning of env gene and is therefore env-negative [40] . rab5-gfp was a gift from stephen ferguson and michel tremblay. the tet-ifitm1 dna construct has the cdna of human ifitm1 gene inserted into the pretrox-tight-pur vector. the ifitm1 mutants δ(117-125), δ(112-125) and δ(108-125), which have different lengths of the c-terminal sequence (illustrated in fig. 1) , have been previously reported [10] . the er retention signal kdel was added to the c-terminus of ifitm1 by a pcr-based method. the cdna clone of mouse ifitm1 was purchased from atcc, amplified by pcr, and cloned into pretrox-tight-pur to generate tet-miftm1. like all human ifitm1 constructs, a flag tag was added to the n-terminus of mifitm1 to facilitate detection by western blotting. all these dna clones were verified by sequencing. the anti-flag antibody was purchased from sigma, anti-tubulin antibody from santa cruz biotechnology, fitc-conjugated anti-hiv-1 p24 antibody from beckman, anticalreticulin antibody from abcam, and bmqc from invitrogen. the hek293 cells and supt1 cells were originally obtained from atcc. supt1 is a human cd4+ t cell line. they are cultured in rpmi1640 medium supplemented with 10% fetal bovine serum (fbs), 2 mm l-glutamine, 100 u of penicillin/ml and 100 μg of streptomycin/ml. in order to stably transduce supt1 cells, we first prepared retrovirus stocks by transfecting the gp-2 packaging cells (clontech) with the pretrox-tight-pur vector together with the pcmv-vsv-g plasmid (expressing the g protein of vesicular stomatitis virus (vsv)). the viruses were then used to infect supt1 cells together with another retrovirus carrying the rtta activator gene (clontech). the infected supt1 cells were cultured in the rpmi1640 medium supplemented with 10% tetracycline-free fbs (clontech), 2 μg of puromycin/ml and 1 mg g418/ml to select for stably transduced cells. hiv-1 stocks were produced by transfecting human embryonic kidney 293t (hek293t) cells with hiv-1 proviral dna clone using lipofectamine2000 (invitrogen). the culture supernatants were clarified by centrifugation at 3,000 rpm in a beckman bench-top centrifuge at 4°c for 30 min. the viruses were aliquoted, used immediately or stored at -80°c for future use. amount of viruses was determined by measuring viral p24(ca) in elisa. supt1 cells stably transduced with the tet-ifitm retroviral vectors were treated with doxycycline (0.5 mg/ml) before exposure to hiv-1 equivalent to 20 ng viral p24(ca) per 2x10 6 cells. virus growth was monitored by measuring viral reverse transcriptase activity in the supernatants at different time intervals. the experiments were performed as described in [41] . briefly, two virus stocks were first produced by transfecting the hek293t cells using either hiv-1 nl4-3 proviral dna or the nleny1-ires-es dna (env-) and the pcmv-vsv-g dna together with pcmv-blam-vpr. the virus stocks were named hiv-1 nl4-3 /blam-vpr that has hiv-1 envelope protein, and nleny1/vsv-g/blam-vpr that has the vsv-g protein as the envelope, respectively. the amount of viruses in each stock was determined by measuring viral p24(ca) in elisa. the supt1 cell lines were first treated with doxycyline (0.5 mg/ml) for 16 hours, then infected with either hiv-1nl4-3/blam-vpr (an amount equivalent to 100 ng viral p24(ca)) or nleny1/ vsv-g/blam-vpr (an amount equivalent to 50 ng viral p24(ca)) by spinnoculation at 1,800 x g for 45 min at room temperature. following a 2-hour incubation at 37°c, cells were washed twice with co 2 -independent medium, then incubated in the loading solution at room temperature for one hour. the loading solution contained 2 μl of ccf2/am (1 nm) and 8 μl of 0.1% acetic acid (containing 100 mg/ml pluronic-f127surfactant) in 1 ml of co 2 -independent medium. after washing with development media (containing 10 μl of probenecid (250 mm) and 100 μl tetracycline-free fbs in 1 ml co 2 -independent medium, invitrogen), cells were kept in dark at room temperature for 16 hours before being fixed in 1% paraformaldehyde and analyzed by flow cytometry. first, supt1 cells were infected with hiv-1 particles bearing vsv-g protein to enhance infection efficiency. forty hours post infection, the infected cells (designated as donor cells) were washed with complete medium, then mixed with equal number of un-infected supt1 cells (designated as target cells), which had been labeled with a cell tracker bmqc (invitrogen). after 8 hours, the cell mixtures were fixed with 1% paraformaldehyde and permeabilized with 0.1% triton x-100. after staining with fitc-conjugated anti-hiv-1 p24 antibody, the p24positive donor and target cells were scored by flow cytometry. hek293 cells were seeded onto poly-lysine-coated coverslips and transfected with the indicated ifitm1 dna constructs. cells were fixed in 4% paraformaldehyde in phosphate buffered saline for 10 min at room temperature, permeabilized with 0.1% triton x-100 for 10 min, and incubated with blocking buffer containing 3% bsa and 6% skim milk. cells were then stained with indicated primary antibodies (1:100 dilution) for 2 hours at room temperature, followed by staining with fitc or tritc conjugated secondary antibodies (1:100 dilution). the nuclei were stained by dapi. dynasore treatment was performed by incubating cells with 160 μm dynasore for 1 hour at 37°c prior to fixation. images were acquired with leica tcs sp5 laser scanning confocal microscope using 100x objectives with sequential-acquisition setting. all the images were obtained using the laser power and digital gain below saturation at a resolution of 1024 x 1024 pixels (8 bit). imagej software was used for post-acquisition measurement of the fluorescent intensity. supporting information s1 fig. effect of ifitm2 and 3 on the replication of hiv-1 nl4-3 . supt1 cells were treated with doxcycyline to induce the expression ifitm2 and 3 before being exposed to hiv-1 nl4-3 . virus production was determined by measuring viral reverse transcriptase activity in culture supernatants at various time intervals. (eps) conceived and designed the experiments: cl wq. performed the experiments: rj sd qp. analyzed the data: rj sd sll cl. contributed reagents/materials/analysis tools: sll cl. wrote the paper: sll wq cl. interferon-stimulated genes: a complex web of host defenses molecular analysis of a human interferon-inducible gene family small isgs coming forward partial inhibition of vesicular stomatitis virus by the interferon-induced human 9-27 protein interleukin-1 inhibits hepatitis c virus subgenomic rna replication by activation of extracellular regulated kinase pathway the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitm-2 and ifitm-3 but not ifitm-1 restrict rift valley fever virus interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections the ifitm proteins inhibit hiv-1 infection distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus interferon inducible transmembrane protein 3 (ifitm3) restricts reovirus cell entry ifitm proteins restrict viral membrane hemifusion the broad-spectrum antiviral functions of ifit and ifitm proteins ifitms restrict the replication of multiple pathogenic viruses ifitm3 restricts the morbidity and mortality associated with influenza ifitm3 limits the severity of acute influenza in mice enhanced survival of lung tissue-resident memory cd8(+) t cells during infection with influenza virus due to selective expression of ifitm3 interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals the dispanins: a novel gene family of ancient origin that contains 14 human members the small interferon-induced transmembrane genes and proteins bril: a novel bone-specific modulator of mineralization interferome: the database of interferon regulated genes identification of the ifitm family as a new molecular marker in human colorectal tumors evolution of vertebrate interferon inducible transmembrane proteins retention of a type ii surface membrane protein in the endoplasmic reticulum by the lys-asp-glu-leu sequence hiv-1 mutates to evade ifitm1 restriction antagonism to and intracellular sequestration of human tetherin by the human immunodeficiency virus type 2 envelope glycoprotein the hiv envelope but not vsv glycoprotein is capable of mediating hiv latent infection of resting cd4 t cells hiv envelope-cxcr4 signaling activates cofilin to overcome cortical actin restriction in resting cd4 t cells avoiding the void: cell-to-cell spread of human viruses interferon induction of ifitm proteins promotes infection by human coronavirus oc43 hiv entry and envelope glycoprotein-mediated fusion acid-dependent viral entry the n-terminal region of ifitm3 modulates its antiviral activity by regulating ifitm3 cellular localization identification of an endocytic signal essential for the antiviral action of ifitm3 phosphorylation of the antiviral protein interferoninducible transmembrane protein 3 (ifitm3) dually regulates its endocytosis and ubiquitination the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication a membrane topology model for human interferon inducible transmembrane protein 1 dynamics of hiv-1 recombination in its natural target cells a sensitive and specific enzyme-based assay detecting hiv-1 virion fusion in primary t lymphocytes key: cord-257217-f9sdt7ax authors: nunes, marta c.; kuschner, zachary; rabede, zelda; madimabe, richard; van niekerk, nadia; moloi, jackie; kuwanda, locadiah; rossen, john w.; klugman, keith p.; adrian, peter v.; madhi, shabir a. title: clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in hiv-infected and hiv-uninfected south african children date: 2014-02-03 journal: plos one doi: 10.1371/journal.pone.0086448 sha: doc_id: 257217 cord_uid: f9sdt7ax background: advances in molecular diagnostics have implicated newly-discovered respiratory viruses in the pathogenesis of pneumonia. we aimed to determine the prevalence and clinical characteristics of human bocavirus (hbov), human rhinovirus (hrv), polyomavirus-wu (wupyv) and –ki (kipyv) and human coronaviruses (cov)-oc43, -nl63, -hku1 and -229e among children hospitalized with lower respiratory tract infections (lrti). methods: multiplex real-time reverse-transcriptase polymerase chain reaction was undertaken on archived nasopharyngeal aspirates from hiv-infected and –uninfected children (<2 years age) hospitalized for lrti, who had been previously investigated for respiratory syncytial virus, human metapneumovirus, parainfluenza i–iii, adenovirus and influenza a/b. results: at least one of these viruses were identified in 274 (53.0%) of 517 and in 509 (54.0%) of 943 lrti-episodes in hiv-infected and -uninfected children, respectively. human rhinovirus was the most prevalent in hiv-infected (31.7%) and –uninfected children (32.0%), followed by cov-oc43 (12.2%) and hbov (9.5%) in hiv-infected; and by hbov (13.3%) and wupyv (11.9%) in hiv-uninfected children. polyomavirus-ki (8.9% vs. 4.8%; p = 0.002) and cov-oc43 (12.2% vs. 3.6%; p<0.001) were more prevalent in hiv-infected than –uninfected children. combined with previously-tested viruses, respiratory viruses were identified in 60.9% of hiv-infected and 78.3% of hiv-uninfected children. the newly tested viruses were detected at high frequency in association with other respiratory viruses, including previously-investigated viruses (22.8% in hiv-infected and 28.5% in hiv–uninfected children). conclusions: we established that combined with previously-investigated viruses, at least one respiratory virus was identified in the majority of hiv-infected and hiv-uninfected children hospitalized for lrti. the high frequency of viral co-infections illustrates the complexities in attributing causality to specific viruses in the aetiology of lrti and may indicate a synergetic role of viral co-infections in the pathogenesis of childhood lrti. pneumonia is a leading cause of mortality in children under 5 years age worldwide, including in hiv-infected children [1] [2] [3] . the aetiology of childhood pneumonia may include infection with bacteria and/or respiratory viruses. although respiratory viruses are more frequently identified than bacteria in children with pneumonia, this may be biased by lack of availability of sensitive and specific tests for diagnosing bacterial causes of pneumonia [4] . furthermore, respiratory viral infections may heighten the susceptibility to developing a super-imposed bacterial infection resulting in severe pneumonia [5, 6] . traditionally, respiratory viruses that have been associated with lower respiratory tract infections (lrti) include respiratory syncytial virus (rsv), parainfluenza viruses i-iii (piv i-iii), influenza viruses a/b and adenovirus. two human coronaviruses (cov), oc43 (cov-oc43) and 229e (cov-229e) were initially identified as causes of upper respiratory tract infections (urti) in the 1960s using classical culture methods [7, 8] . more recently, advances in molecular diagnostics have resulted in the discovery of other respiratory viruses which have also been associated with lrti. included among these are human metapneumovirus (hmpv) [9] , human bocavirus (hbov) [10] , human coronavirus nl63 (cov-nl63) [11] and hku1 (cov-hku1) [12] and wu and ki polyomaviruses (wupyv, kipyv) [13] [14] [15] . also, human rhinovirus (hrv), which was previously mainly associated with mild urti, has increasingly been implicated in having a role in the pathogenesis of lrti and asthma exacerbations [16, 17] . due to impaired humoral and cell-mediated immunity, hiv infection in children has been described as a risk factor for severe illness and mortality caused by respiratory-viral associated lrti, such as rsv, hmpv and influenza virus [18, 19] . there are, however, limited data on the role of other respiratory viruses, including the more recently-discovered viruses which occur as single or co-infecting pathogens in hiv-infected children hospitalized with lrti, and of these studies, most have small sample sizes [20] [21] [22] . the aim of this study was to identify the prevalence of hbov, hrv, wupyv, kipyv, cov-oc43, cov-nl63, cov-hku1 and cov-229e among hiv-infected and -uninfected children who were hospitalized for lrti using real-time reverse transcriptase-polymerase chain reaction (rt-pcr). the study-cohort had been previously investigated for rsv, influenza a/b, piv i-iii and adenovirus by immunofluorescence assay and hmpv by nested-pcr as described [5, 23] . the main 9-valent pneumococcal conjugated vaccine (pcv9) efficacy trial and subsequent retrospective analysis of study participants were approved by the human research ethics committee (medical) of the university of the witwatersrand. the main study did not have a clinical trials number since it was undertaken prior to registration of clinical trials having been made mandatory. signed informed consent was obtained from the parent/legal guardians of all the study participants as part of the main trial. the ethics committee did not require additional consent for this study. this was a retrospective study of children who participated in a phase iii trial in south africa which investigated the efficacy of a pcv9 as described [5, 24] . briefly, 39836 children recruited from march 1998 to october 2000 were randomized (1:1) to receive 3 doses of either pcv9 or placebo. vaccination occurred at a mean of 6.661.2 (6 standard deviation), 11.262.5, and 15.963.9 weeks of age [24] . hospital-based surveillance for all-cause hospitalization was undertaken at chris hani-baragwanath hospital, the only public hospital in the study community. hospitalized children had their signs and symptoms recorded and underwent hiv testing according to the study protocol [24] . nasopharyngeal aspirates (npa) were obtained for respiratory viral studies from children hospitalized with lrti [5] , and archived from january 2000 onward. in the present study only npa collected from 1 st february 2000 to 31 st january 2002 from children less than 2 years old were analysed. if a child had recurrent lrti hospitalizations, only npa collected at least 28 days apart were included in the analysis. blood cultures were performed with the use of an automated blood-culture system (bact-alert, organon teknika). total nucleic acids were extracted from archived npa using a nuclisens easymag platform (biomerieux), and eluted in a final volume of 60 ml of elution buffer [25] . rna was reverse transcribed with high capacity cdna reverse transcriptase (invitrogen, life technologies) and primed with oligo-dt primers (invitrogen, life technologies). real-time pcr was done in an abi 7500 rt-pcr system (applied biosystems, life technologies), reactions were performed in 20 ml using taqman universal pcr master mix (applied biosystems, life technologies) and the primers and probes listed in table s1 . five duplex rt-pcr reactions, targeting the 8 respiratory viruses, were developed. internal controls (the human genes: ribonucleoprotein and glyceraldehyde-3-phosphate dehydrogenase; or the spiked viruses: lambda and newcastle disease virus) were included to check the efficiency of the extraction step and to detect the presence of pcr inhibitors. positive controls were included in each experiment. the clinical definitions used in this study are the same as previously described [5] . briefly, lrti was defined as any episode with clinical diagnosis of pneumonia or bronchiolitis done by a study physician. children with lrti were categorized as having clinical pneumonia if they had evidence of alveolar consolidation on chest x-ray (cxr-ac) or if they fulfilled the clinical diagnosis of lrti without wheeze on chest auscultation but had rales and/ or bronchial breathing. children were categorized as having bronchiolitis in the presence of wheezing on chest auscultation and in the absence of documented cxr-ac or bronchial breathing on chest wall auscultation. a clinical diagnosis of who severe/very severe pneumonia was made if the child had a cough ,14 days in duration and lower chest wall in-drawing and/or any of the following signs and symptoms of severe pneumonia: feeding difficulties, convulsions, central cyanosis, or encephalopathy. the previously developed respiratory index of severity in children (risc) score was used to compare disease severity between single and multiple viral infections [26] . demographic, clinical and laboratory characteristics at admission were compared between hiv-infected and -uninfected children, chi-square or fisher's exact tests were used to compare the distribution of categorical variables and mean or median of continuous variables were compared by two-tailed student t-test or mann-whitney test, respectively. viral prevalence was compared between hiv groups and clinical and laboratory characteristics were compared between episodes with single and multiple viral detection using multivariate logistic regression models adjusted for study intervention arm (i.e. whether received pcv9 or placebo), year of sampling, detection of a virus previously-tested and age. the results are presented as adjusted odds ratios (aor) with 95% confidence intervals (95% ci) and p-values. a p-value, 0.05 was considered significant. analyses were performed using stata version 11.0 (statacorp, texas, usa). during the follow-up period included in this analysis, there were a total of 2147 hospitalizations for lrti, including 2094 (97.5%) in which npa had been collected. of the initial collected samples 69.7% were available for rt-pcr analysis. the same proportion of samples were available for rt-pcr from pcv9-recipients (69.2%) and placebo-recipients (70.2%; p = 0.621), from hivinfected (78.2% vs. 73.1%; p = 0.130) and hiv-uninfected children (65.5% vs. 68.6%; p = 0.218); children from whom npa were available for rt-pcr testing compared to those in whom samples were unavailable were older (median age: 10 vs. 8 months; p,0.001), were 1.3-fold less likely to have tested positive for one of the previously-tested respiratory viruses (33.3% vs. 42.3%; p,0.001), had a higher prevalence of cyanosis (11.4% vs. 8.1%; p = 0.025), higher evidence of cxr-ac (26.6% vs. 22.1%; p = 0.041), higher c-reactive protein (crp) levels (median: 15 vs. 12 mg/l; p = 0.003) and higher procalcitonin (pct) concentration (median: 0.26 vs. 0.15 ng/ml; p = 0.006); table s2 . there were no other demographic, clinical or laboratory differences observed between the lrti-episodes with npa available versus unavailable for rt-pcr. a total of 517 npa samples from hiv-infected children were analysed by rt-pcr, including 45.0% from pcv9-recipients and 55.0% from placebo-recipients. among hiv-uninfected children, 943 specimens were available for rt-pcr analysis, including 49.5% from pcv9-recipients and 50.5% from placeborecipients. on admission hiv-infected children compared to hiv-uninfected were younger (median age: 9 vs. 11 months; p, 0.001), more frequently presented with cyanosis (23.8% vs. 4.6%; p,0.001), had lower mean oxygen saturation (89.8% vs. 92.2%; p,0.001), had a higher median respiratory rate (54 vs. 48 breaths per minute; p,0.001), were more likely to present as pneumonia (88.8% vs. 49.6%; p,0.001) than bronchiolitis, had higher crp (18 vs. 14 mg/l; p = 0.007) and pct levels (0.47 vs. 0.17 ng/ml; p,0.001), had a longer hospital stay (4 vs. 1 median days; p, 0.001), had a higher case-fatality rate (17.8% vs. 0.95%; p,0.001) and more frequently had bacteraemia (7.8% vs. 2.7; p,0.001); table 2 . of the 1460 specimens analysed, all but 13 (1 from hivinfected and 12 from hiv-uninfected children) were previously tested for hmpv by nested-pcr [23] and 1458 were tested by an immunofluorescence assay for rsv, which if found negative, were further tested for influenza a/b, piv i-iii and adenovirus as described [5] . among hiv-infected children, the rt-pcr viral panel was positive in 274 (53.0%) lrti-episodes for at least one of the newly-tested viruses; table 3 . the prevalence of any of the newlytested respiratory viruses was similar between pcv9-and placeborecipients in hiv-infected children, except for wupyv (11.2% vs. 6.3%; p = 0.047, respectively) and hbov (12.5% vs. 7.0%; p = 0.034, respectively); table s3 . in hiv-infected children hrv was the most frequently detected virus (31.7%) followed by cov-oc43 (12.2%), hbov (9.5%), kipyv (8.9%), wupyv (8.5), cov-nl63 (1.7%) and cov-hku1 (1.4%); table 3 . the newlytested viruses were frequently identified as co-infecting viruses among hiv-infected children, including 49.4% of lrti-episodes associated with hrv; table 4 . the most common viral coinfections with hrv included kipyv (14.6%), wupyv (11.6%), cov-oc43 and hbov (11.0%, each); table 4 . among the 486 children on whom blood culture was done, bacteria were isolated on 38 (7.8%) occasions, 20 (52.6%) of which were associated with concomitant detection of one of the newly-tested viruses and 22 (57.9%) of any of the viruses. in hiv-uninfected children at least one newly-tested virus was detected in 509 (54.0%) lrti-episodes, with hrv also being the most common (32.0%), followed by hbov (13.3%), wupyv (11.9%), kipyv (4.8%), cov-oc43 (3.6%), cov-nl63 (2.6%), cov-hku1 (1.6%) and cov-229e (0.42%); table 3 . comparing hiv-uninfected pcv9 and placebo recipients, differences in the prevalence of newly-tested viruses were evident for kipyv (2.1% vs. 7.4%; p,0.001), cov-hku1 (0.21% vs. 2.9%; p = 0.001), cov-oc43 (2.1% vs. 5.0%; p = 0.017) and hrv (36.2% vs. 27.9%; p = 0.007); table s3 . of the 302 lrti-episodes in which hrv was identified, 51.3% had at least one other virus detected, including 13.6% with rsv, 12.3% with wupyv or hbov and 2.0% (n = 6) with both wupyv and hbov; table 5 . the prevalence of bacteraemia in hiv-uninfected children among those with blood culture results was 2.7% (n = 24/881) of which 12 (50.0%) occurred in the presence of infection with one of the newly-tested viruses and 15 (62.5%) in presence of any of the studied viruses. by multivariate analysis, adjusting for pcv-vaccination status, period of collection and age, single infections with a newly-tested virus were more frequent in hiv-infected (30.2%) than hivuninfected children (25.5%) (adjusted odds ratio [aor] 1.3; p = 0.033); table 3 . also, hiv-infected compared to hivuninfected children had a higher prevalence of kipyv (aor table 1 . number of specimens analysed in the current study and total specimens collected. when compared to lrti-episodes associated with the identification of only a single virus the detection of multiple viruses in hiv-infected children was significantly associated with a higher frequency of bronchial breathing (aor 2.11; p = 0.015) and in hiv-uninfected children with a higher prevalence of cyanosis (aor 2.50; p = 0.008) and wheezing (aor 1.55; p = 0.006); table 6 . no differences were detected in the severity of lrti-episodes with single and multiple viral infections using the risc score previously developed. in both hiv-infected and -uninfected children with bacteria isolated from blood, there were no significant differences in the clinical and laboratory characteristics between children in whom viruses were detected and children without any virus detected. the same was observed restricting the analysis to streptococcus pneumoniae isolation (data not shown). to our knowledge, this study provides the most in-depth analysis of the prevalence of hrv and some of the newlydiscovered respiratory viruses in hiv-infected children. our study identified hrv to be the most frequently detected virus both in hiv-infected and -uninfected children followed by cov-oc43 in hiv-infected and hbov in hiv-uninfected. most cases of hospitalizations associated with single infections overall were noted for hrv and rsv, the rate of co-infections was high in children infected with the other newly-discovered viruses. : previously-tested by immunofluorescence assay. 4 : previously-tested by nested pcr. 5 : including viruses previously-tested by immunofluorescence assay (rsv, influenza a, piv i-iii and adenovirus) and nested-pcr (hmpv). 6 : all multiple infections included at least one newly-tested virus except in 2 hiv-uninfected children. viral co-infections with at least one newly-tested virus in hivinfected children 118 (22.8%) and in hiv-uninfected children 269 (28.5%). 7 : not adjusted for detection of viruses previously-tested. 8 : : previously-tested by immunofluorescence assay. 5 : previously-tested previously by nested-pcr. 6 : including viruses previously-tested by immunofluorescence assay (influenza a, rsv, piv, adenovirus) and nested-pcr (hmpv). 7 : 487 patients had specimens available for culture. bacteria isolated included: hbov with escherichia coli (n = 2) and salmonella sp (n = 1); wupyv with streptococcus pneumoniae (n = 2) and escherichia coli (n = 1); kipyv with streptococcus pneumoniae (n = 3), escherichia coli (n = 3) and haemophilus parainfluenzae (n = 1); cov-nl63 with streptococcus pneumoniae (n = 2); cov-oc43 with escherichia coli (n = 3), salmonella sp (n = 1), streptococcus viridans (n = 1) and other streptococcus (n = 1); hrv with streptococcus pneumoniae (n = 6), escherichia coli (n = 3), streptococcus viridans (n = 1) and citrobacter freundii (n = 1); hmpv with salmonella sp (n = 2); piv with streptococcus viridans (n = 1); influenza a with streptococcus pneumoniae (n = 1) and escherichia coli (n = 1); adenovirus with pseudomonas aeruginosa (n = 1 table 5 . respiratory viruses co-infections in hiv-uninfected children hospitalized with lower respiratory tract infections. : previously-tested by immunofluorescence assay. 5 : previously-tested previously by nested-pcr. 6 : including previously-tested by immunofluorescence assay (influenza a, rsv, piv, adenovirus) and nested-pcr (hmpv). very few viral aetiology studies have been conducted in africa: in a mozambican study of virus-associated acute respiratory infections (ari) in infants with an estimated 3-5% hiv prevalence, the most frequently detected viruses were hrv (26%), influenza (15%) and adenovirus (14%) [27] . a recent study from south africa on children with ari requiring medical attention, 61% (n = 383) being hiv-infected or hiv-seropositive, also reported that hrv was the most frequently detected virus (33%) followed by rsv (30%), piv (8%) and hbov (6%) [21] . in the south african study hospitalized children with respiratory disease had higher hrv detection rates compared to healthy children (36% vs. 19%; p = 0.047) which may indicate a causality effect of hrv in disease [21] . in kenya in older children (5-17 years old), despite hrv being the most commonly detected virus (38%) in patients presenting with ari, its detection rate was similar among asymptomatic controls [28] . indeed two other studies in kenya found that with the exception of rsv, viral detection in the nasopharynx did not have a significant association with pneumonia in hospitalized children under 5 years [29] or under 12 years, [30] compared to children both without symptoms of urti or with respiratory symptoms but not meeting any criteria for pneumonia. it is uncertain as to what the impact of hiv infection on the duration of shedding of these viruses might be. an analysis of serial respiratory samples from otherwise healthy children detected shedding of cov-nl63 for up 21 days [31] , hrv up to 41 days and of hbov up to 44 days [32] . detection of cov-hku1 and cov-229e in respiratory specimens of transplanted children were also reported for at least 38 days and 11 weeks, respectively, possibly suggesting that immunocompromised children may have a prolonged duration of shedding of these viruses [33, 34] . such prolonged shedding in immunocompromised individuals such as hiv-infected children, may result in a greater frequency of coincidental identification of these viruses when investigated for respiratory illness, affect the seasonal occurrence of the viruses as well as potentially present a threat to greater nosocomial transmission of these viruses because of higher incidence of hospitalization of hiv-infected children. the casual relationship between viral dna detection, development of disease and mixed infections is very complex. in the case of hbov serological studies have shown that the presence of viral dna in respiratory samples was not proof of an acute primary infection and that prolonged viral shedding could be an explanation for viral detection at high rates in asymptomatic controls [35] . enrolment of healthy non-hospitalized children and quantification of viral loads may help to elucidate the problem of co-infections and is currently being under-taken in a multi-centre study on aetiology of pneumonia in children [36] . contradictory findings on whether infection with multiple viruses contribute to disease severity in hospitalized children have been reported, with some studies reporting an increased severity among children with viral co-infections [21, 37] and others finding decrease severity associated with co-infections [38] . in some studies no differences were observed [39] . we found that multiple viral infections were associated with bronchial breathing in hiv-infected children and with cyanosis and wheezing in hiv-uninfected children. bacterial co-infections can also increase the severity of viral diseases [40] . furthermore, in children with invasive pneumococcal disease, superimposed viral co-infections are common and may lead to higher mortality rates [41] . of the participants in our study from whom bacterial cultures performed, no differences in clinical and laboratory characteristics were observed between children with or without viral infections. our analysis, however, included pcv9 vaccinees who probably present with less severe lrti cases since it was shown before that pcv9 vaccination was associated with a decrease in lrti viral-associated hospitalizations [5, 23] . we detected a high prevalence of bacterial co-infections with the newly-tested viruses especially in hiv-infected children (8%), what may have contributed to the higher c-reactive protein and procalcitonin levels, the longer hospital stay and the higher mortality in hiv-infected children compared to hiv-uninfected. our high co-infection detection rates are similar to other molecular detection studies of paediatric respiratory samples, with coronaviruses (40-75%) [42, 43] , hbov (up to 78%) [13, 44, 45] and polyomaviruses (68-79%) [46] [47] [48] . our results may, however, have under-estimated the true prevalence of viral co-infections since the previously-studied viruses were tested by immunofluorescence [5] or conventional pcr [23] . although the sensitivity of the conventional methods is reported to be high, comparative studies have shown that rt-pcr is considerably more sensitive for detection of respiratory viruses [21, 49] . moreover the specimens tested by immunofluorescence were initially screened for rsv and only if negative were further tested for influenza a/ b, piv i-iii and adenovirus [5] . limitations of our study include that this was a post-hoc analysis and only 70% of specimens collected during the study period were available for further testing [24] . compared with the specimens available for rt-pcr the specimens unavailable were collected from younger children and were more likely to have tested positive for a previously-studied virus. this selection bias may have also contributed to an under-estimation of the number of co-infections. along the same line rsv-associated lrti are more common among younger children [21, 39] , for whom we had less available samples increasing our uncertainty on the prevalence of rsv coinfections with the newly-tested viruses. an equal proportion of pcv9-and placebo-recipients hospitalized with lrti were investigated in this study and our analyses were adjusted for the initial intervention arm. further detailed analyses on the effect of pcv9 on the incidence of hospitalization associated with the individual newly-tested viruses will be reported on in future. although the development of new diagnostic techniques allows more detailed investigation and has led to the discovery of a number of new putative respiratory pathogens, the role for some of these potential pathogens in respiratory illness remains speculative in the absence of fulfilling koch's postulates for causality. in the present study we demonstrate that at least one respiratory virus was identified in the majority of hiv-infected and hiv-uninfected children hospitalized for lrti. the difficulty in attributing disease causality to specific viruses due to the high frequency of viral co-infection suggests a possible synergy among different pathogens during childhood lrti. table s1 primer and probe sequences for real-time reverse transcriptase-polymerase chain reaction viral detection used in the study. (docx) estimates of world-wide distribution of child deaths from acute respiratory infections global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since the impact of a 9-valent pneumococcal conjugate vaccine on the public health burden of pneumonia in hiv-infected and -uninfected children a role for streptococcus pneumoniae in virusassociated pneumonia respiratory viral and pneumococcal coinfection of the respiratory tract: implications of pneumococcal vaccination a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease a newly discovered human pneumovirus isolated from young children with respiratory tract disease cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a new human coronavirus clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia identification of a third human polyomavirus a newly reported human polyomavirus, ki virus, is present in the respiratory tract of australian children identification of a novel polyomavirus from patients with acute respiratory tract infections the role of rhinovirus in asthma exacerbations rhinovirus viremia in children with respiratory infections differing manifestations of respiratory syncytial virus-associated severe lower respiratory tract infections in human immunodeficiency virus type 1-infected and uninfected children increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodeficiency virus type-1 respiratory viruses in hiv-infected patients with suspected respiratory opportunistic infection contribution of common and recently described respiratory viruses to annual hospitalizations in children in south africa human polyomaviruses, wu and ki in hiv exposed children with acute lower respiratory tract infections in hospitals in south africa pneumococcal coinfection with human metapneumovirus a trial of a 9-valent pneumococcal conjugate vaccine in children with and those without hiv infection evaluation of nuclisens easymag for automated nucleic acid extraction from various clinical specimens development of the respiratory index of severity in children (risc) score among young children with respiratory infections in south africa viral acute respiratory infections among infants visited in a rural hospital of southern mozambique accepted for publication) clinical epidemiology of newly discovered respiratory viruses in hiv-infected and hivuninfected south african children a preliminary study of pneumonia etiology among hospitalized children in kenya viral etiology of severe pneumonia among kenyan infants and children human coronavirus nl63 associated with lower respiratory tract symptoms in early life epidemiology of multiple respiratory viruses in childcare attendees human respiratory coronavirus hku1 versus other coronavirus infections in italian hospitalised patients detection of four human coronaviruses in respiratory infections in children: a one-year study in colorado clinical assessment and improved diagnosis of bocavirus-induced wheezing in children rationale and expectations of the pneumonia etiology research for child health (perch) study the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children the association of newly identified respiratory viruses with lower respiratory tract infections in korean children prevention (2012) severe coinfection with seasonal influenza a (h3n2) virus and staphylococcus aureus-maryland viral coinfections in children with invasive pneumococcal disease clinical disease in children associated with newly described coronavirus subtypes coronavirus causes lower respiratory tract infections less frequently than rsv in hospitalized norwegian children human bocavirus commonly involved in multiple viral airway infections frequent and prolonged shedding of bocavirus in young children attending daycare presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection wu polyomavirus in children with acute lower respiratory tract infections clinical and epidemiologic characterization of wu polyomavirus infection advances in the laboratory diagnosis of viral respiratory disease the authors thank the essential contribution of the members of the vaccine trialist group [24] for their involvement in the original study, all the trial participants, all rmpru staff involved in the study and biomérieux south africa for providing reagents. key: cord-264225-vzcfeh7t authors: talbert-slagle, kristina; atkins, katherine e.; yan, koon-kiu; khurana, ekta; gerstein, mark; bradley, elizabeth h.; berg, david; galvani, alison p.; townsend, jeffrey p. title: cellular superspreaders: an epidemiological perspective on hiv infection inside the body date: 2014-05-08 journal: plos pathog doi: 10.1371/journal.ppat.1004092 sha: doc_id: 264225 cord_uid: vzcfeh7t nan worldwide, more than 250 people become infected with hiv every hour [1] , yet an individual's chance of becoming infected after a single sexual exposure, the predominant mode of hiv transmission, is often lower than one in 100 [2] . when sexually transmitted hiv-1 infection does occur, it is usually initiated by a single virus, called the founder strain, despite the presence of thousands of genetically diverse viral strains in the transmitting partner [3] . here we review evidence from molecular biology and virology suggesting that heterogeneity among cd4+ t cells could yield wide variation in the capability of individual cells to become infected and transmit hiv to other cells. using an epidemiological framework, we suggest that such heterogeneity among cd4+ t cells in the genital mucosa could help explain the low infection-to-exposure ratio and selection of the founder strain after sexual exposure to hiv. during sexual transmission, founder viral strains preferentially infect cd4+ t cells using the ccr5 coreceptor [4, 5] . at the time of initial exposure to hiv, these cd4+ t cells exhibit baseline heterogeneity due to stochasticity in cellular gene expression [6] and dynamic variation in immunological status (activated, resting, etc.) [7] . in addition, because cd4+ t cells are mobile, they are heterogeneously distributed in the genital mucosa, with varying degrees of clustering and contact [8] [9] [10] [11] . in other contexts, it is well-known that heterogeneity among isogeneic cells inside the body can affect many cellular behaviors and outcomes, including infection dynamics [12, 13] . epidemiological analyses of disease outbreaks among people indicate that heterogeneity in the ability of individuals in a population to spread disease can have a significant impact on whether a local outbreak becomes an epidemic [14] . heterogeneity among a population of cd4+ t cells may play a similarly critical role in the establishment and spread of hiv in the genital mucosa after sexual exposure. to quantify the spread of infectious disease, epidemiologists use the basic reproductive number, r 0 , which describes the average number of secondary infections that arise from one infected individual in an otherwise totally susceptible population [15] . the basic reproductive number can be approximated as the product of the following: (1) the average number of susceptible individuals contacted by an infected individual during the infectious period (the ''number of contacts'') and (2) the average probability that a susceptible individual will become infected by a single infected individual during its infectious period (the ''shedding potential''). thus, r 0 &number of contacts |shedding potential: the number of secondary infections caused by a specific individual throughout the time that the individual is infectious is called the ''individual reproductive number'' [14] . for any disease within a given population, there exists a distribution of individual reproductive numbers, of which r 0 is the mean [14] . in populations of homogeneous individuals, the distribution of individual reproductive numbers will be clustered around the population average value of r 0 , and thus, this average value will more accurately predict the likelihood of transmission from each infected to each susceptible individual. if r 0 .1, then an outbreak is likely to become an epidemic, and if r 0 ,1, then an outbreak will not spread beyond a few initially infected individuals [15, 16] . in heterogeneous populations, however, the population average value of r 0 is less predictive of transmission dynamics [14] . for example, in populations with highly right-skewed distributions of individual reproductive numbers, most individuals infect few, if any, others, but a few individuals infect many others. in such populations, there is a high probability that a disease outbreak will not be sustained in the population and will instead go extinct [14] . in some cases, however, those rare individuals in the tail of the distribution with a much higherthan-average individual reproductive number while they are infected, known as ''superspreaders'' [15] , can have a significant impact on whether an outbreak becomes an epidemic or goes extinct. epidemiological outbreak investigations, which track the spread of disease by a technique called contact tracing, have identified the existence of superspreaders in many well-known infectious disease outbreaks, including typhoid fever, measles, smallpox, ebola, and severe acute respiratory syndrome (sars) [14, 17, 18] . these rare individuals often make a significant, sometimes deciding, contribution to the dynamics of disease spread (table 1) . during the 2003 sars outbreak in singapore, for example, the majority of individuals who became infected spread the virus either to no one else or to only one other [14] . five infected individuals, however, were superspreaders, each infecting at least 20 others (figure 1 ) [19] . in this example, r 0 , which is an average population value, did not adequately describe the dynamics of sars because it did not capture the heterogeneity among individuals in their ability to spread disease or the key contribution made by superspreaders to establishment and spread of the virus [14] . for a population of hiv-infected cells inside the body, the basic reproductive number, r 0 (a population average), has been quantified [20] . as with individual humans inside a population, however, empirical evidence indicates that individual human cells inside a population are also heterogeneous [12] , varying in their contact with one another, their ability to become infected (permissivity), and also in whether and to what extent they transmit infectious virus to other cells during their infectious period. such heterogeneity among cd4+ t cells in the genital mucosa of a single individual could generate a skewed distribution in the individual cellular reproductive number, or icrn, in the context of hiv infection. here we review evidence for heterogeneity among cd4+ t cells that could lead to wide variation in icrn and possibly give rise to cellular superspreaders. cd4+ t cells exhibit considerable heterogeneity in activation status (e.g., resting or activated) and expression of surface molecules important for hiv infection (including the hiv coreceptors ccr5 and cxcr4) in human penile [21] , foreskin [22, 23] , cervical [24, 25] , and rectal [26] tissue. in addition, various studies that stain for cd4+ t cells in uninfected genital mucosal tissue, such as cervical tissue [27] and human foreskin [22] , indicate that t cells vary in their spatial distribution and the extent to which they form clusters. cell density and spatial arrangement have been identified as important sources of heterogeneity among cells that can affect virus spread in vitro [8, [28] [29] [30] . indeed, imaging studies exploring the dynamics of virus spread in a model of sexual transmission to female nonhuman primates indicate that virus spreads unevenly among clusters of cells in the endocervix [9] . cells in these clusters tend to be in close proximity, and if cell-tocell transmission is far more efficient than cell-free transmission, as some studies suggest [31] , then a cell that is physically touching its neighbors could generate more secondary infections than a cell that is not close enough to others to transmit virus by direct contact [8] . thus, heterogeneity in cell distribution and clustering inside the body could generate wide variation in the efficiency of virus transmission from cell to cell and in icrn. transmission of virus from an infected to a susceptible cell also depends on a cell's permissivity to productive infection. the level of surface expression of cd4 and ccr5 (the predominant coreceptor utilized during acute infection [32] ) varies widely among cd4+ t cells [25, 33] , even in a single individual [34] , and affects cellular permissivity to hiv [35, 36] . indeed, low expression of cd4 or ccr5 can completely inhibit infection of cd4+ t cells by certain viral strains [37] . a recent multiparameter analysis of hiv entry efficiency at the level of single cells indicated large cell-to-cell variation in expression of cd4, ccr5, and the coreceptor cxcr4, which subsequently influenced permissivity of individual cells to hiv binding and entry [38] . in addition, cd4+ t cells isolated from rectal and cervical tissue exhibit considerable heterogeneity in expression of the surface integrin a4b7, which can specifically bind the v2 loop of the hiv envelope protein gp120 and may improve cell-to-cell spread by activating other cellsurface molecules in the viral synapse [39] . experiments in vitro indicate that hiv preferentially infects cells expressing high levels of ccr5 and that infection can be further enhanced by high levels of surface a4b7 expression in some individuals [40, 25] . heterogeneity among cd4+ t cells in expression of specific cell surface receptors can thus affect permissivity and cell-to-cell transfer of hiv infection in the genital mucosa. permissivity of cells to productive hiv infection can also be affected by intracellular proteins called restriction factors, which block the progress of hiv through the cell [41] . expression of some cellular restriction factors has been shown to vary among different human populations [42] and even between different types of cd4+ t cells within the same individual [43] . notably, an intracellular host restriction factor known as samhd1 (sterile alpha motif [sam] and histidine/aspartic acid [hd] domain-containing protein 1) blocks reverse transcription in resting-but not activated-cd4+ t cells, inhibiting hiv replication. these findings help to explain the inability of resting cd4+ t cells to produce infectious virus [44] . hiv combats the effects of some cellular restriction factors with its own viral proteins, but the success of these proteins in overcoming cellular resistance and facilitating virus production depends on their quantity within the cell, which can also vary depending on viral gene expression levels [41] . together, these data suggest that among cd4+ t cells in the genital mucosa, significant heterogeneity may exist in the number of contacts that become infected by any given infected cell even within the same host, potentially leading to a skewed distribution of icrn. release of infectious viral particles from an infected cd4+ t cell, or shedding potential, can be influenced by many factors in both the cell and the virus. variation in virus gene expression at the level of individual cells has been demonstrated in vivo in a mouse model of cytomegalovirus infection [45] , and heterogeneity among individual cells in the production of virus particles, or virus shedding, has been shown through analysis of cells isolated from simian immunodeficiency virus (siv)-infected nonhuman primates, in which rare activated cd4+ t cells were shown to individually release large quantities of virus [10] . here we focus on variability in gene expression and its potential impact on an individual cell's capability to release infectious virus as a possible source of heterogeneity in shedding potential. stochasticity in cellular gene expression is a common phenomenon [6] and has also been observed in viral gene expression, including hiv. in populations of genetically identical cells infected with hiv, viral genes tended to be expressed at either high or low levels but were also rarely expressed at intermediate levels, varying from cell to cell [46] . the site of virus integration also affects viral gene expression. hiv viral dna preferentially integrates at sites of active cellular gene expression although not at any specific site or in any specific gene [47] [48] [49] . some viral integration events occur at sites of much higher gene expression than others [47] , and gene expression can vary significantly depending on the site of integration, even in genetically identical cells [50] . since the majority of hiv-infected cells in lymph nodes and peripheral blood contain a single virus [51] , while cells in splenic tissue contain one to eight integrated proviruses (mean 3.2) [52] , viral integration site can be an important factor in determining viral gene expression and likely also subsequent virus shedding potential from any given cell. within the population of cd4+ t cells in the genital mucosa, therefore, a wide range of icrn may exist due to stochastic and/or infection-driven variations in viral gene expression and viral particle production, arising from potentially wide heterogeneity in virus shedding potential among infected cells. experiments in a nonhuman primate model of hiv infection have demonstrated that the vast majority of cd4+ t cells in the genital mucosa of a healthy, uninfected individual are resting cells, which outnumber activated cells 70:1 (figure 2a ) [9] . activated cd4+ t cells express higher levels of ccr5 [33] and a4b7 [40] than do resting cells. in addition, experiments in nonhuman primates indicate that infected, activated cells contain five times more viral rna, release 10-fold more viral particles to their surrounding environment, and tend to form larger cell clusters than resting cells [9] . notably, although the nonhuman primate model has long been used to study many facets of hiv infection [53] , the virus used in these experiments, siv, expresses a protein that allows it to productively infect resting cd4+ t cells. in contrast, hiv-1 does not express this protein and is thus unable to generate productive infection in resting cd4+ t cells [54] . resting human cd4+ t cells in the human genital mucosa are therefore even less likely to be able to produce and spread hiv than are resting cd4+ t cells in a nonhuman primate model. together, these data suggest that upon infection with hiv the majority of cd4+ t cells in the genital mucosa would spread the virus to few if any others and that only rare cells would have the capacity to release large quantities of hiv to their nearest neighbors. these rare cells may be activated cd4+ t cells, which have been described as ''amplifiers'' that can cause additional cells to become infected with hiv [10, 55] . such rare cells may exhibit a specific set of traits that facilitate establishment of hiv inside the body. for example, experiments done in vitro indicate that cd4+ th17 cells, which express high levels of ccr5 as well as the chemokine receptor ccr6 and the integrin a4b7, are preferentially targeted during early infection, and analysis of samples from female sex workers who are infected with hiv indicate that cd4+ th17 cells are selectively depleted from figure 1 . contact tracing of sars in singapore showed that most people (gray circles) transmitted the virus to very few others, while a few individuals acted as ''superspreaders,'' infecting many more people than average. patient numbers corresponding to those individuals who were identified as superspreaders are shown. all cases trace back to patient 1 [18] . doi:10.1371/journal.ppat.1004092.g001 the cervix during hiv infection [25, [56] [57] [58] . in addition, given that the majority of hiv infections begin with a single founding strain [59] and most infected cells in peripheral blood and lymph node tissue contain a single copy of hiv dna (although these may or may not be representative of hiv integration in mucosal lymphatic cells) [51] , infection of a single cell has the potential to establish hiv infection inside the genital mucosa after a given exposure. infection of a rare cd4+ t cell with very high icrn could thus be the superspreading event that both establishes hiv infection in the genital mucosa and selects a single founder strain ( figure 2b) . such a founding superspreader infection event would parallel, for example, the dynamics that governed the sars outbreak in singapore, giving rise to an epidemic despite a low individual reproductive number for most individuals. as shown in figure 1 , infection of a single superspreading individual (labeled as ''1'') triggered the sars outbreak in singapore in 2003. a highly skewed distribution of individual reproductive numbers in a population, in which most individuals infect few if any others but a tiny minority are superspreaders, has two important implications when applied to cd4+ t cells in the genital mucosa. first, since the majority of the cells would have a low icrn, most, if not all, of the viral strains that successfully overcome physiological barriers during exposure are likely to infect cells that have an icrn less than one. provided that none of these cells becomes latently infected, which has been shown to occur within days after infection in vitro [60, 61] but has yet to be confirmed in vivo, a ''local outbreak'' inside the body would go extinct. in this case, infection of most cells immediately after sexual exposure would not lead to sustained infection, which could explain the very low infection-toexposure ratio for sexual exposure to hiv. second, such short-lived ''local outbreaks'' of hiv within an individual could still yield a low level of virus production, even if the initial outbreak of hiv infection ultimately goes extinct. among a group of nonhuman primates exposed to a low physiological dose of siv, some animals experienced initial low levels of viral replication and immune response without ever proceeding to full infection or seroconversion, a phenomenon called occult infection [62] [63] [64] . in addition, some hiv-exposed, seronegative humans who continue to engage in high-risk sexual behavior exhibit immunological markers that indicate a prior immune response to hiv infection even though they remain seronegative, supporting the idea that local infections may have occurred in these patients [65] [66] [67] . finally, analysis of the unsuccessful hiv vaccine step trial suggested that a large portion of the exposed individuals may have experienced occult infection, implying that this phenomenon could be more widespread than previously suspected [68] . there is, however, no direct experimental evidence for occult infection in humans. if infection of a rare superspreader cd4+ t cell establishes the founder strain, then the few features of founder viral strains that have been observed might in fact confer a selective advantage during early infection. these features include shorter envelope glycoproteins with fewer n-linked glycosylation sites [69, 70] as well as preferential infection of cd4+ t cells expressing high levels of ccr5. some founder viral strains have also exhibited high affinity for the a4b7 integrin receptor [71, 72] though this has not been universally observed [73, 5] . the founder virus, which more closely resembles the ancestral founder strain than it does the predominant strain in the transmitting partner [74] , may be selected by its ability to infect a subtype of cd4+ t cell: a cellular superspreader. this specific efficiency could explain why hiv founder strains have not shown a consistent infectivity advantage in cd4+ t cells over strains from chronic infection in vitro [5, 75, 76] , as these experiments likely do not fully replicate the cellular heterogeneity of the in vivo environment [32, 77, 78] , where the founder viruses might have an advantage in infecting the rare superspreader cells. here we have applied two key concepts from epidemiology: r 0 , which is the approximated product of number of contacts and shedding potential throughout the infectious period, and individual reproductive number, to suggest that a skewed distribution of individual cellular reproductive number among cd4+ t cells in the genital mucosa gives rise to cellular superspreaders that may drive establishment of hiv infection inside the genital mucosa after sexual transmission. the definition of r 0 provided here implicitly integrates transmission over the time that an index cell was infected, meaning that we have incorporated duration of infectiousness into our overall definition of r 0 . thus, a cd4+ t cell could theoretically become a superspreader either by spreading a large amount of infectious virus to other cells in a short amount of time, or by spreading a smaller amount of virus to other cells for a comparatively longer period of time, or through some combination of the two. though any of these mechanisms are possible in early infection [79] , studies in nonhuman primates suggest that establishment of infection in the genital mucosa typically occurs within 3-7 days after male-to-female sexual exposure [62, 80] . the lifespan of a productively infected cd4+ t cell is on average 2.2 days [81] . thus, in the specific case of hiv infection in the genital mucosal tissue, if infection becomes established via a superspreading event, it is likely to occur within the first few days of exposure and be driven by a relatively short-lived, productively infected cell that generates a much higher-thanaverage number of secondary infections due to a high shedding rate, or a high contact rate, or both. notably, as in other superspreading events, establishment of hiv by infection of a cellular superspreader could occur even if the basic reproductive number for the entire population of susceptible cells is low. if cellular hiv superspreaders do exist, and if they are the cellular culprits driving the establishment of hiv infection inside the body, then the most successful strategy for preventing the infection from becoming established in the body is to block or remove these cells before or shortly after infection in order to drive a local, withinhost outbreak to extinction [13, 82] . such cells may have specific traits, such as high expression of surface receptors including ccr5 and possibly also a4b7, that allow them to be identified and targeted by novel therapies to prevent establishment of infec-tion. several recent studies suggest that th17 cells, a subset of cd4+ t cells, are preferentially infected by early viral strains and selectively depleted from the cervix during hiv infection [58, 25] . we are not aware of in vivo data that explicitly support the existence of cellular superspreaders; nevertheless, the data reviewed here suggest their existence, warranting further empirical research. identification and targeting of cells most likely to become superspreaders could facilitate the development of preexposure or immediate postexposure therapies that could prevent a local outbreak of hiv inside the genital mucosa from becoming a within-host epidemic that spreads throughout the body [9, 83] . in this review, we have applied epidemiological concepts of disease spread specifically to explore unsolved questions regarding establishment of the hiv founder strain and the low infection-to-exposure ratio of infection after sexual transmission. we suggest that these concepts may also be applied more broadly to explain the documented existence of hiv founder strains after transmission via injection drug use and from mother to child [59] since cd4+ t cells in the blood of healthy, uninfected individuals are also heterogeneous, with only a very small subset exhibiting an activated or replicating phenotype [84] . since heterogeneity among cells has been acknowledged as an important factor in a variety of cellular processes, including certain viral infections [12] , we suggest that the epidemiological framework described here may also be applicable to the establishment and spread of other cellular diseases inside the body, including not only infections but perhaps also certain cancers. the impact of cellular heterogeneity may be particularly profound if the distribution of individual cellular reproductive numbers is highly skewed, yielding cellular superspreaders. global report: unaids report on the global aids epidemic 2013. 2013 edition. online: joint united nations programme on hiv/aids heterosexual risk of hiv-1 infection per sexual act: systematic review and meta-analysis of observational studies barriers to mucosal transmission of immunodeficiency viruses high multiplicity infection by hiv-1 in men who have sex with men phenotypic properties of transmitted founder hiv-1 nature, nurture, or chance: stochastic gene expression and its consequences heterogeneity of cd4+ memory t cells: functional modules for tailored immunity the clustering of infected siv cells in lymphatic tissue glycerol monolaurate prevents mucosal siv transmission roles of substrate availability and infection of resting and activated cd4+ t cells in transmission and acute simian immunodeficiency virus infection heterogeneity and plasticity of t helper cells cellular heterogeneity: do differences make a difference? quality and quantity: mucosal cd4+ t cells and hiv susceptibility superspreading and the effect of individual variation on disease emergence infectious diseases of humans: dynamics and control modeling infectious diseases in humans and animals largest measles epidemic in north america in a decade-quebec, canada, 2011: contribution of susceptibility, serendipity, and superspreading events super-spreaders in infectious diseases severe acute respiratory syndrome-singapore estimation of the initial viral growth rate and basic reproductive number during acute hiv-1 infection hiv infection and immune defense of the penis differential compartmentalization of hiv-targeting immune cells in inner and outer foreskin tissue susceptibility to human immunodeficiency virus-1 infection of human foreskin and cervical tissue grown in explant culture hiv-1 sexual transmission: early events of hiv-1 infection of human cervico-vaginal tissue in an optimized ex vivo model characterization of a human cervical cd4+ t cell subset coexpressing multiple markers of hiv susceptibility hiv-1 pathogenesis differs in rectosigmoid and tonsillar tissues infected ex vivo with ccr5-and cxcr4-tropic hiv-1 immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone spatiotemporal dynamics of hiv propagation population context determines cell-to-cell variability in endocytosis and virus infection single-cell analysis of population context advances rnai screening at multiple levels cell-to-cell transmission of viruses hiv-1 transmission biology: selection and characteristics of infecting viruses quantification of cd4, ccr5, and cxcr4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages mechanisms of nonrandom human immunodeficiency virus type 1 infection and double infection: preference in virus entry is important but is not the sole factor differences in cd4 dependence for infectivity of laboratory-adapted and primary patient isolates of human immunodeficiency virus type 1 transmitted/founder and chronic hiv-1 envelope proteins are distinguished by differential utilization of ccr5 a quantitative affinityprofiling system that reveals distinct cd4/ccr5 usage patterns among human immunodeficiency virus type 1 and simian immunodeficiency virus strains an expanded model of hiv cell entry phenotype based on multiparameter single-cell data hiv-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral t cells the integrin alpha4beta7 forms a complex with cell-surface cd4 and defines a t-cell subset that is highly susceptible to infection by hiv-1 the restriction factors of human immunodeficiency virus analysis of human apobec3h haplotypes and anti-human immunodeficiency virus type 1 activity intracellular detection of differential apobec3g, trim5alpha, and ledgf/p75 protein expression in peripheral blood by flow cytometry restrictions to hiv-1 replication in resting cd4(+) t lymphocytes single cell detection of latent cytomegalovirus reactivation in host tissue stochastic gene expression in a lentiviral positive-feedback loop tat fluctuations drive phenotypic diversity hiv-1 integration in the human genome favors active genes and local hotspots retroviral dna integration: aslv, hiv, and mlv show distinct target site preferences transcription start regions in the human genome are favored targets for mlv integration hiv promoter integration site primarily modulates transcriptional burst size rather than frequency single cell analysis of lymph node tissue from hiv-1 infected patients reveals that the majority of cd4+ t-cells contain one hiv-1 dna molecule recombination: multiply infected spleen cells in hiv patients update on animal models for hiv research detection of plasma viremia in human immunodeficiency virus-infected individuals at all clinical stages intravaginal inoculation of rhesus macaques with cell-free simian immunodeficiency virus results in persistent or transient viremia susceptibility of human th17 cells to human immunodeficiency virus and their perturbation during infection memory ccr6+cd4+ t cells are preferential targets for productive hiv type 1 infection regardless of their expression of integrin beta7 preferential hiv infection of ccr6+ th17 cells is associated with higher levels of virus receptor expression and lack of ccr5 ligands hiv transmission. cold spring harb perspect med 2: a006965 a double-fluorescent hiv-1 reporter shows that the majority of integrated hiv-1 is latent shortly after infection determinants of the establishment of human immunodeficiency virus type 1 latency propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus occult systemic infection and persistent simian immunodeficiency virus (siv)-specific cd4(+)-t-cell proliferative responses in rhesus macaques that were transiently viremic after intravaginal inoculation of siv a period of transient viremia and occult infection precedes persistent viremia and antiviral immune responses during multiple low-dose intravaginal simian immunodeficiency virus inoculations epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern thailand. chiang mai heps working group new insights into hiv-1 specific cytotoxic t-lymphocyte responses in exposed, persistently seronegative kenyan sex workers cervical hiv-specific iga in a population of commercial sex workers correlates with repeated exposure but not resistance to hiv hiv vaccine development in the aftermath of the step study: re-focus on occult hiv infection? characterization of glycosylation profiles of hiv-1 transmitted/founder envelopes by mass spectrometry recurrent signature patterns in hiv-1 b clade envelope glycoproteins associated with either early or chronic infections the genotype of early-transmitting hiv gp120s promotes alpha (4) beta(7)-reactivity, revealing alpha (4) beta(7) +/cd4+ t cells as key targets in mucosal transmission hiv-1 envelope replication and alpha4beta7 utilization among newly infected subjects and their corresponding heterosexual partners early infection hiv-1 envelope v1-v2 genotypes do not enhance binding or replication in cells expressing high levels of alpha4beta7 integrin previously transmitted hiv-1 strains are preferentially selected during subsequent sexual transmissions transmitted/founder and chronic subtype c hiv-1 use cd4 and ccr5 receptors with equal efficiency and are not inhibited by blocking the integrin alpha4beta7 application of a case-control study design to investigate genotypic signatures of hiv-1 transmission evaluation of cervical mucosa in transmission bottleneck during acute hiv-1 infection using a cervical tissue-based organ culture generation of transmitted/founder hiv-1 infectious molecular clones and characterization of their replication capacity in cd4 t lymphocytes and monocytederived macrophages stochastic theory of early viral infection: continuous versus burst production of virions sexual transmission and propagation of siv and hiv in resting and activated cd4+ t cells hiv-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time immediate antiviral therapy appears to restrict resting cd4+ cell hiv-1 infection without accelerating the decay of latent infection early events in sexual transmission of hiv and siv and opportunities for interventions circulating cd4(+) and cd8(+) t cells are activated in inflammatory bowel disease and are associated with plasma markers of inflammation the basic reproductive number of ebola and the effects of public health measures: the cases of congo and uganda the reemergence of ebola hemorrhagic fever, democratic republic of the congo an epidemic of measles in southern greenland, 1951; measles in virgin soil. ii. the epidemic proper an explosive point-source measles outbreak in a highly vaccinated population. modes of transmission and risk factors for disease epidemiologic determinants for modeling pneumonic plague outbreaks primary pneumonic plague in mukden, 1946, and report of 39 cases with three recoveries dynamically modeling sars and other newly emerging respiratory illnesses: past, present, and future evidence of airborne transmission of the severe acute respiratory syndrome virus sars reference investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto transmission potential of smallpox in contemporary populations smallpox and its eradication. geneva: world health organization. xvi we thank pradeep uchil, walther mothes, and daniel dimaio for helpful discussions and maggie zhou for contributions to the manuscript revision. key: cord-272051-arz8r204 authors: federico, maurizio title: hiv-protease inhibitors block the replication of both vesicular stomatitis and influenza viruses at an early post-entry replication step date: 2011-08-15 journal: virology doi: 10.1016/j.virol.2011.05.002 sha: doc_id: 272051 cord_uid: arz8r204 the inhibitors of hiv-1 protease (pis) have been designed to block the activity of the viral aspartyl-protease. however, it is now accepted that this family of inhibitors can also affect the activity of cell proteases. since the replication of many virus species requires the activity of host cell proteases, investigating the effects of pis on the life cycle of viruses other than hiv would be of interest. here, the potent inhibition induced by saquinavir and nelfinavir on the replication of both vesicular stomatitis and influenza viruses is described. these are unrelated enveloped rna viruses infecting target cells upon endocytosis and intracellular fusion. the pi-induced inhibition was apparently a consequence of a block at the level of the fusion between viral envelope and endosomal membranes. these findings would open the way towards the therapeutic use of pis against enveloped rna viruses other than hiv. hiv protease inhibitors (pis) are a family of small molecules specifically designed for blocking the activity of the hiv aspartylprotease (for a review, see flexner, 1998) . the pi treatment leads to the block of hiv maturation with consequent release of noninfectious viral particles (kaplan et al., 1993) . pis exert their inhibitory effect by disabling the enzyme before it can cleave the gag-pol polyprotein into its essential products. pis, together with anti-hiv compounds targeting alternative steps of the virus life cycle, are part of the most advanced anti-hiv therapies allowing millions of infected people to co-exist with the virus experiencing a good quality of life. however, it is now clear that pis can also directly or indirectly inhibit the activity of many cell proteases. in particular, pis can inhibit the activity of both 20s (andré et al., 1998) and 26s (pajonk et al., 2002) proteasome subunits, as well as that of caspases (badley, 2005) , with alterations in the susceptibility to apoptosis stimuli. both expression and release of matrixmetalloproteinases (mmps) can be also targeted by pis (bourlier et al., 2005; de barros et al., 2007) with consequences in the extracellular matrix modeling and, more in general, in the cell-cell communication. in addition, pis have been found inhibiting cell signaling pathway involving both nf-κb (dewan et al., 2009 ) and akt (kumar et al., 2009; srirangam et al., 2006) . from a clinical point of view, the pi treatment may induce beneficial effects other than those against hiv, as in the case of kaposi sarcoma regression (sgadari et al., 2002) , but can also have detrimental consequences, as for the dyslipidemia occurring in pi-treated hiv patients (calza et al., 2004) . to enter target cells, enveloped rna viruses fuse their envelope either at the cell surface in a ph-independent way, or upon internalization in intracellular vesicles through a ph-dependent mechanism (steven and spear, 2006) . in this latter case, low ph is required to induce the fusion between viral envelope and endosomal membranes, a phenomenon leading to the release of virion contents into cytoplasm. although the viral fusion process is guided by the presence of viral envelope proteins, the contribution of cell proteins is critical. using rna interference as a tool for gene expression inhibition, it was demonstrated that several cell proteins are involved in the mechanism of entry of many viruses including hiv-1 nguyen et al., 2006) , influenza (hao et al., 2008) , west nile (krishnan et al., 2008) , borna disease (clemente et al., 2010) , human hepatitis c (ng et al., 2007) , and vesicular stomatitis (vsv) (pelkmans et al., 2005) viruses. ideally, these cell products might represent potential antiviral therapeutic targets. here, the pi-induced inhibition of replication of both vsv and influenza virus is described for the first time. this was most likely a consequence of a pi-dependent block of the viral envelope fusion at the endosomes. considering that pis are well tolerated drugs in vivo, and that many relevant human pathogens belong to the family of rna viruses infecting cells through an endocytic pathway, this finding would open the way towards a broader therapeutic use of pis. hiv virions emerging from cells treated with pis remain immature viral particles as a consequence of the block of gag polyprotein cleavage. hence, pis do not affect the amounts of hiv release from infected cells, but dramatically decrease their infectivity. surprisingly enough, however, the hiv-1 release from cells infected with hiv-1 pseudotyped with the envelope protein from vsv (vsv-g) has been found inhibited by pis in single cycle replication assays. in fact, when cem gfp cells, i.e., a human t cd4 + lymphoblastoid cell line expressing gfp under the control of hiv-1 ltrs (gervaix et al., 1997) , were infected with 100 ng hiv-1 cap24 equivalent/10 5 cells of (vsv-g) δenv hiv-1, decreased percentages of hiv-1 expressing cells were observed in ritonavir-treated cells as compared to control conditions (fig. 1a) . the outcome did not change by challenging the cells with doses up to 300 ng hiv-1 cap24 equivalent/10 5 cells (not shown). conversely, and as expected, the pi-treatment had no apparent effects on single cycle replication of non-pseudotyped hiv-1, as tested in pi-treated cultures of cem gfp cells infected with wt hiv-1 (fig. 1b) . in these experiments, the presence of t-20 (i.e., a potent inhibitor of the hiv-1 env-mediated fusion) (kilby et al., 1998) added 16 h after challenge ensured that the wt hiv-1 replication was limited to a single cycle. as anticipated, ritonavir blocked the spread of wt hiv-1 in multiple-cycle replication assay (fig. 1b) . to exclude that the observed effect was a consequence of the inhibition of some yet unidentified function of the hiv-1 protease at an early step of the virus life cycle, the assay was reproduced using a vsv-g pseudotyped, pi-resistant hiv-1 strain (here referred to as pm4). this viral mutant expresses a pi-resistant viral protease as a consequence of the four amino acid substitutions in the protease gene, i.e., m 46 i , l 63 p , v 82 t , and i 84 v (condra et al., 1995) . first, the real resistance to pis of pm4 hiv-1 was checked by evaluating the percentages of cem gfp infected cells 6 days after the infection in the presence or not of ritonavir (fig. 1c) . then, cem gfp cells treated with t-20 were infected with (vsv-g) pm4 hiv-1 in the presence or not of ritonavir. this experimental setting was representative of a singlecycle replication assay since t-20 disabled both hiv-1 env-mediated viral entry and possible cell re-infections. similarly to what observed with (vsv-g) δenv hiv-1 strain, a significant inhibitory effect of ritonavir on the hiv-1 expression was detected in (vsv-g) pm4 challenged cells (fig. 1d ). this strongly suggests that the viral protease activity was not involved in the pi-induced inhibition of (vsv-g) hiv-1 expression. overall, these results can be a consequence of a still unrecognized inhibitory effect of pis on some step of the viral entry driven by vsv-g. next, the possibility that the pi inhibitory effect was operative also against vsv was investigated. to this end, hela cells were pre-treated overnight with different concentrations of six pis, i.e., ritonavir, indinavir, saquinavir, nelfinavir, amprenavir and atazanavir. then, the cells were challenged with 0.2 m.o.i. of vsv. after 1 h of adsorption in a small volume, the viral inoculum was removed, the cells extensively washed, and incubated for additional 8 h in the presence of pis. afterwards, the supernatants were harvested and titrated for the amounts of infectious vsv. as depicted in figs. 2a-b, a strong reduction of vsv replication (more than 2 logs) was observed when the cells were treated with 2.5 μm saquinavir and nelfinavir. the inhibitory effect appeared more potent (up to about 4 logs) by increasing the concentration of these pis to 10 μm, and remained significant (i.e., more than 1 log) by increasing the m.o.i. up until 1 (not shown). no differences were observed when the pis pretreatment was carried out for 2, 4, 6, or 8 h (not shown). as calculated through a wide dose-response curve, the 50% inhibitory concentration (ic 50 ) values of saquinavir and nelfinavir were 1.4 and 1.1 μm, respectively. on the other hand, significant inhibition of viral replication were detected in ritonavir and indinavir treated cells starting to the concentrations of 20-30 μm (fig. 2b) . the ic 50 of ritonavir and saquinavir were 16 and 14 μm, respectively. the idea that pis negatively affect the vsv replication was further strengthened by performing a set of more stringent challenge experiments. in detail, hela cells were pre-treated with optimal concentrations of the most effective pis, and then infected with 2-fold decreasing amounts of vsv starting to m.o.i. 2. the cytopathic effect was monitored 24 h post challenge. the outcome of this assay was scored in terms of the viral dilutions no more able to induce cytopathic effect. notably, in these conditions an about 30-fold reduction of vsv infectivity was observed in cells treated with saquinavir or nelfinavir (fig. 2c ). since the average replication time of vsv is 6-8 h (whelan et al., 2004) , in this experimental setting vsv was expected to complete at least three replication cycles in control cells. then, it was controlled whether the antiviral effect was a consequence of a general cytotoxicity of pis. to this aim, cells were treated with different pi concentrations for 8 h, then labeled with carboxyfluorescein diacetate succinimidyl ester (cfse), and thereafter refed with complete medium in the presence of pis. cfse was expected to be equally distributed upon cell division, then resulting in a halving of the overall cell fluorescence after cell duplication. cytotoxic effects were measured in terms of inhibition of the cell duplication as detectable by the impairment of the halving of the cfse-associated cell fluorescence. fig. 2d reports the mean fluorescence intensities (mfis) measured in cell cultures treated with different concentrations of the pis active against vsv replication. block of cell duplication with cell mortality over 20% has been detected using 100 μm ritonavir, saquinavir, and nelfinavir, while indinavir showed a much lower cytotoxic effect. the 50% cytotoxic concentration (cc 50 ) values of pis ( fig. 2d , insert) were calculated as the doses inhibiting by 50% the fluorescence halving, expectedly a consequence of the block of the duplication in 50% of cells. the therapeutic indexes (i.e., cc 50 divided by ic 50 ) appeared higher than 22 for both saquinavir and nelfinavir. this is suggestive of specific antiviral effects for these pis. taken together, these data reveal a previously unrecognized effect of pis against vsv. in addition, these results support the idea that the pi-induced reduction of (vsv-g) hiv-1 expression is a consequence of the inhibition on some step of the viral entry involving the vsv-g envelope protein. to add relevance to our findings, the possibility that the inhibitory effect of pis applied also to alternative virus species infecting by phdependent fusion was investigated. to this aim, the well characterized influenza virus/mdck cell system was considered. mdck cells were treated overnight with different pis, and then challenged with m.o.i. 0.1 and 0.5 of the pr8 influenza virus. after challenge, the cells were extensively washed, and reseeded in medium without serum in the presence of both pis and tosylamido-2-phenil ethyl chloromethyl ketone (tpck)-treated trypsin. twenty-four h later (i.e., the expected time for the completion of the replication cycle of influenza virus) (sidorenko and reichl, 2004; smith and ribeiro, 2010) , the supernatants were harvested and titrated for the contents of infectious particles. a strong inhibition of the replication of influenza virus (up to 4 logs at the lower m.o.i.) has been detected with 10 μm saquinavir and 5 μm nelfinavir (fig. 3a) . ritonavir inhibited the viral replication less efficiently (fig. 3a) , while indinavir, amprenavir and atazanavir appeared ineffective (not shown). in the interpretation of these data, it should be emphasized that the inhibitory effect of pis cannot be a consequence of a pi-induced impairment of the activation of the hemagglutinin envelope protein (ha) of the influenza virus, which is a step required for productive infection (steinhauer, 1999) . in fact, influenza virus preparations used for challenges were exclusively recovered from the allantoic fluid of embryonated chicken eggs, where ha is cleaved by egg proteases. consistently, tpck-trypsin did not influence the infectivity of the virus preparations in single cycle replication assay (not shown). the cell cytotoxicity of pis active against influenza virus was then evaluated in mdck cells similarly to what above described for hela cells (fig. 3b ). block of cell duplication in the presence of cell mortality over 20% has been detected with 100 μm ritonavir and saquinavir, and with 50 μm nelfinavir. the therapeutic indexes appeared higher than 15 for both saquinavir and nelfinavir. the assay gave similar results when the cells were analyzed 24 h after cfse labeling (not shown). taken together, these data suggest that pis could target the replication of diverse enveloped viruses infecting by a ph-dependent endocytic pathway. next, it was investigated whether the pis most effective against vsv and influenza virus replication counteract also the replication of viruses infecting by a ph-independent mechanism. as a proof of principle, the pis were assayed against the replication of newcastle disease virus (ndv), i.e., a paramixovirus infecting upon fusion at the cell surface. mdck cells were pre-treated with 2.5 to 10 μm saquinavir or nelfinavir, and then infected with 2-fold decreasing amounts of ndv starting to m.o.i. 1. the cytopathic effect was monitored 48 h post challenge. no significant reductions in the ndv infectivity have been noticed in pi-treated as compared to control cell cultures (fig. 4) . these results indicate that pis do not affect the replication of viruses entering by a ph-independent way. the inhibitory effect is operative when pis are co-administered with the infecting virus to identify the virus replication step targeted by pis, the effects of pis added before and/or after viral challenge were evaluated. the assays were carried out using both hela/vsv and mdck/influenza virus systems. in a first set of experiments, the pi treatment was discontinued just before the virus challenge. then, the cells were infected with vsv or influenza virus at m.o.i. of 0.2 and 0.5, respectively. after 1 h of adsorption, the cells were extensively washed and refed in the appropriate medium. in these conditions, no inhibition of the replication of both vsv and influenza virus was detectable (fig. 5a) . alternatively, pis were added soon after the virus adsorption or at different times after the challenge within a timeframe of 8 h (for vsv infection) and 24 h (in the case of challenge with influenza virus), i.e. the respective replication times. the supernatants were harvested 8 h (for vsv) and 24 h (for influenza virus) post-infection, and titrated for the amounts of infectious virus particles. it appeared that pis inhibited the virus replication also when added early after challenge (fig. 5b) . the inhibitory effect dropped shortly in the case of vsv infection, and more gradually when cells were infected with influenza virus. saquinavir and nelfinavir have shown similar inhibitory efficiencies (not shown). these results indicate that the presence of pis at early times after virus challenge is mandatory for the inhibitory effect, consistently with the idea that pis target an early event of the replication cycle of both viruses. vsv, and (vsv-g) hiv-1 in an hiv-1 protease independent manner, further supported the idea that pis would influence some common early replication step, e.g., virus attachment, endocytosis, and/or endosomal fusion. to dissect among these different possibilities, the intracellular fate of challenging virus was followed using gfp-labeled hiv-1-based vlps (muratori et al., 2006) pseudotyped with either wt or fusiondefective vsv-g (fig. 6a) . the fluorescence of these vlps relies on the high incorporation levels of the product of fusion between gfp and a hiv-1 nef mutant acquiring a palmitoylation site at its n-terminus as the consequence of the g to c substitution at the amino acid 3. first, the possible effects of pis on the virus attachment on target cells were investigated. to this end, 10 5 hela cells treated or not with pis were challenged with 500 ng of (wt vsv-g) nef g3c -gfp vlps for 1 h at 4°c. afterwards, the cells were extensively washed and facs analyzed. to control that the cell-associated fluorescence was not a consequence of endocytosed vlps, a part of the cell samples was treated with trypsin. it appeared that the treatment with pis did not significantly affect the cell binding of fluorescent vlps (fig. 6b ). this strongly suggests that pis do not interfere with the binding of viral particles on the cell membrane. next, the influence of pis on virus endocytosis was investigated. to this aim, gfp-fluorescent vlps incorporating a vsv-g mutant (here referred to as fd vsv-g) unable to support the ph-dependent fusion were used. the impaired fusion activity was a consequence of the a to k amino acid substitution at the position 133 (fredericksen and whitt, 1995) . the use of this envelope protein mutant was associated with the trypsin treatment just before the facs analysis to ensure that the cell-associated fluorescent signal exclusively referred to the intracellular accumulation of vlps. hela cells treated or not with pis were challenged with 500 ng hiv-1 cap24 equivalents of (fd vsv-g) nef g3c -gfp vlps/10 5 cells, and incubated for 3 h at 37°c in the presence or not of pis. then, the cells were treated with trypsin, and the cell-associated gfp fluorescence was evaluated by facs analysis. as control, vlp-challenged cells were incubated at 4°c before the trypsin treatment. again, no apparent differences in the cellassociated fluorescence were detected between control and pi-treated cells (fig. 6c) , suggesting that pis have no influence on the efficiency of the endocytosis of viral particles. this conclusion was enforced by the results obtained through an alternative experimental approach where control or pi-treated hela cells were infected with 0.2 pfu/cell of vsv. thirty and 60 min later, the cells were extensively washed, treated with trypsin to leave out non-adsorbed viral particles, lysed, and analyzed by western blot for the presence of vsv-g. no significant reduction of the vsv-g specific signals was detectable at both time points in pi-treated cells as compared to control conditions (fig. 6d) . considering that the neosynthesis of vsv-g starts at least 90 min post-infection (rothman and lodish, 1977) , these results, consistently with the data obtained with fluorescent vlps, suggest that pis do not affect the endocytosis of viral particles. the results from these experiments indicate that the inhibitory effect of pis is not the consequence of impaired attachment and endocytosis of viral particles. next, the possibility that the inhibitory effect of pis was a consequence of a defect in the endosome to cytoplasm virus delivery was investigated. to this end, the differences in the cell-associated fluorescence levels in cells internalizing wt or fd vsv-g pseudotyped nef g3c -gfp vlps were exploited. in fact, using the same experimental conditions described for the endocytosis assay, it was reproducibly observed that, early after vlp challenge, the mean fluorescence intensity (mfi) of cells challenged with (wt vsv-g) vlps was near 2-fold higher than that of cells treated with (fd vsv-g) vlps. more significantly, in the latter condition both percentages of fluorescent cells and mfi heavily dropped 16 h after challenge. on the contrary, both facs parameters appeared only slightly reduced in cells challenged with (wt vsv-g) vlps (fig. 7a ). these differences can be explained by the fact that, due to the inability of fd vsv-g to induce fusion and delivery of the vlp contents into cytoplasm, the nef g3c -gfp molecules incorporated in (fd vsv-g) vlps were addressed to rapid degradation into the endosomal/lysosomal compartment. conversely, the efficient fusion between viral envelope and endosome membranes induced by wt vsv-g allowed the release of nef g3c -gfp molecules into cytoplasm, where they are expected to be degraded with a kinetic much slower than that operating in endosomes/lysosomes. next, pi-treated or untreated hela cells were challenged with 500 ng hiv-1 cap24 equivalent of either (wt vsv-g) or (fd vsv-g) nef g3c -gfp vlps/10 5 cells, and 3 and 16 h later, the cells were treated with trypsin. facs analysis of the cell-associated gfp fluorescence revealed that the pi treatment did not affect the fluorescence levels in cells challenged with (fd vsv-g) nef g3c -gfp vlps at both time points. on the contrary, after 3 h it was observed a relevant reduction in the mfi within pi-treated cells challenged with (wt vsv-g) nef g3c -gfp vlps as compared with untreated cells. more strikingly, a strong decrease of both mfi and percentage of fluorescent cells was detectable in pi-treated cells at 16 h post-challenge (fig. 7b) . as expected, among the different pi tested, saquinavir and nelfinavir produced the strongest inhibitory effect (data not shown). next, dose-response endocytosis assays using different concentrations of nelfinavir were carried out by analyzing the cell-associated fluorescence 16 h after the challenge. the results showed that, within the cells challenged with (wt vsv-g) nef g3c -gfp vlps, the pi treatment reduced both mfi and percentages of fluorescent cells in a dose-dependent manner. at the highest pi concentrations, both parameters reached levels similar to those detectable in cells challenged with (fd-vsv-g) nef g3c -gfp vlps (fig. 7c) . hence, it seemed that the pi treatment diverted the fate of endocytosed (wt vsv-g) towards that of (fd vsv-g) vlps. this strongly suggests that pis negatively affect the fusion of viral envelope with the endosomal membranes. together, these results support the idea that the inhibitory effect of pis acts at the level of the delivery of the viral particles from endosomes to cytoplasm of infected cells. a possible explanation for the apparent block of viral envelope fusion could be that pis may increase the ph in endosomes in a way to inhibit the low ph-dependent conformational changes needed for both vsv-g and influenza ha to switch the fusion process. to test whether pis affect intracellular ph, both hela and mdck cells were labeled with lysosensor green dnd-189. this reagent becomes fluorescent only in acidic intracellular compartments, meanwhile exhibiting decreased fluorescence intensity upon ph increase. thus, it represents a useful reagent for detecting possible ph variations in endosomes. cells were treated overnight with the pi doses most effective against vsv and/or influenza virus replication, then labeled with lysosensor green dnd-189 for 30 min in the presence of pis, and finally analyzed by facs. as control, cells treated with bafilomycin a1, i.e., a powerful inhibitor of the vacuolar atpase proton pump, were also tested. as depicted in fig. 8 , no significant variations in the cellassociated fluorescence have been detected in pi-treated cells as compared with control conditions, indicating that pis do not affect the intracellular ph. these results strongly suggest that the pi-induced block of virus delivery in cytoplasm would not be a consequence of the increase of endosomal ph. viruses hijack cell functions to replicate in host cells. hence, the selective targeting of cell products supporting virus replication could represent a successful antiviral strategy. the results from the here described investigations indicate that drugs designed to counteract the activity of the hiv protease show a strong inhibitory effect against activity(ies) of target cells. the antiviral potency of the most effective pis reached about 4 logs in the reduction of virus yields, with therapeutic indexes higher than 15. the pi-induced effect against vsv appeared comparable in magnitude to that observed in cell treated with type-1 interferons (masters and samuel, 1983) . on the other hand, the potency of pis against the influenza virus replication appeared comparable to that of most potent inhibitors. among these, t-705 (furuta et al., 2002) reduced the viral yield of the most susceptible influenza strain tested of at best 2.7 logs at the concentration of 5 μm upon mdck cell challenge with 0.01 m.o.i. (sleeman et al., 2010) . das181, i.e. a sialidase fusion protein, blocked the influenza virus replication at sub-micromolar concentrations, however when mdck cells were challenged with 0.001-0.005 m.o.i. (triana-baltzer et al., 2009) . conversely, micromolar concentrations of pis induced more than 3 logs of reduction of the influenza virus yield from mdck cells challenged with 0.5 m.o.i. in the challenge experiments where pis were added soon after the adsorption of vsv or influenza virus inocula, the antiviral effect appeared slightly reduced as compared with control conditions where pis were maintained throughout. considering also the results obtained in the endocytosis assays, it is conceivable that this subtle difference could be a consequence of the accession to cytoplasm of a small amount of viral particles during the adsorption time and before the pi treatment. the possibility that pis affect some virion structural component in a way to hinder the process of fusion in endosomes appeared unlikely. in fact, the treatment of 10-fold concentrated vsv or influenza virus preparations with up to 500 μm pis for 30 min before challenge did not produce significant decrease of the viral yields (not shown). however, we cannot formally exclude that pis inhibit some yet unidentified protease activity associated with vsv-g or influenza virions. in this regard, it has been proposed that pis could negatively affect the chymotrypsin-like protease activity of the pa subunit of the rna-directed rna polymerase of influenza virus (savarino, 2005) . furthermore, it was reported that nelfinavir reduces the replication of sars coronavirus (yamamoto et al., 2004) likely as a consequence of the inhibition of the 3c-like viral protease (for a review, see 35). the results from here reported experiments indicate that pis act on early events of viral replication, but not on virus attachment and endocytosis. this latter finding was consistent with previously reported results indicating that pis do not affect the endocytosis of hiv-1 particles in dendritic cells (muratori et al., 2009) . rather, it appeared that pis interfere with the delivery of viral particles from endosomes to cytoplasm. this did not seem to depend on a pi-induced increase of the endosomal ph possibly inhibiting the low phdependent conformational changes of viral envelope proteins required for viral fusion. the concept that the activity of cell proteases is part of the mechanisms underlying the replication of many virus species is widely accepted. for example, the cleavage of ha generated by cell proteases is required for the activation of the viral envelope protein preceding the viral fusion of influenza virus (klenk et al., 1975; lazarowitz and choppin, 1975) . similarly, the activation of envelope proteins of ebola (schornberg et al., 2006) , nipah (pager and dutch, 2005) , and corona viruses (bosch et al., 2008) is regulated by cathepsins, i.e., a family of cell aspartyl-proteases. the identification of host factors, in particular aspartyl-proteases, involved in the here described pi-induced inhibition of viral entry deserves further investigations. the results from functional genetic screens have demonstrated that alternative cell proteases act as co-factors in the replication of different viruses clemente et al., 2010; hao et al., 2008; krishnan et al., 2008; ng et al., 2007; nguyen et al., 2006; pelkmans et al., 2005) . concerning vsv, the comparison of these data with those regarding the cell proteases known to be sensitive to pis identifies both proteasome subunits as possible relevant pi targets (clemente et al., 2010 ). this appears consistent with the recently reported evidence that mg132 (i.e., a proteasome inhibitor) inhibits vsv replication (neznanov et al., 2008) . alternative functional genetic screens revealed that also mmp-21 could be part of the mechanism of vsv replication (clemente et al., 2010) . considering that the activity of mmp-21, like other mmps, might be affected by pis, it would be of interest investigating the role of this mmp in the here described antiviral effect of pis. the here reported findings would open the way towards preclinical assays designed to test the potency of pis against in vivo infections sustained by orthomyxo-and rabies viruses. it will be also of interest extending the investigations on additional pathogenic enveloped viruses infecting by endocytosis. vsv-g pseudotyped hiv-1 preparations were obtained from supernatants of 293t cells 48 h after co-transfection with a cmv immediate-early promoted vsv-g-expressing vector and vectors expressing nl4-3 hiv-1, its δenv derivative, or pm4 hiv-1 (molar ratio 1:5) performed by the lipofectamine 2000-based method (invitrogen). supernatants were clarified and concentrated by ultracentrifugation as described (federico et al., 2001) . virus preparations were titrated by measuring hiv-1 cap24 contents by quantitative enzyme-linked immunosorbent assay (elisa; innogenetic). both preparations and assays of vsv (indiana strain) have been performed basically as described (gresser et al., 1968) . briefly, high titer stocks of vsv have been prepared upon infection of hela cells with at low multiplicity of infection (m.o.i.), i.e. b0.01 plaque forming unit (pfu)/cell. supernatants were harvested 24 h later, clarified, stocked, and frozen. vsv titrations of high titer stocks have been carried out by plaque method. the a/puerto rico (pr)/8/34 h 1 n 1 human influenza virus was grown in 11-day-old embryonated chicken eggs. the allantoic fluid was clarified by centrifugation at 5000×g for 15 min at 4°c. the virus was pelleted by centrifugation at 65,000×g for 1 h at 4°c and resuspended in 1 ml of phosphatebuffered saline. portions of the solution were stored as aliquots at −20°c. influenza preparations were titrated by standard plaque assay on mdck cells. virus titers for these stocks ranged from 1 to 8 × 10 7 pfu/ml. preparations of the hertz strain of ndv were recovered after 48 h of incubation in 9-11-day-old embryonated chicken eggs inoculated in the allantoic cavity. after harvesting, the allantoic fluid was clarified by centrifugation at 10,000×g, and titrated as infectious units/ml through the end-dilution method by assessing the cytopathic effect on mdck cells 48 h post infection. the titer of the viral stock used was 2.1 × 10 7 infectious units/ml. cem gfp cells were grown in roswell park memorial institute (rpmi) medium supplemented with 10% heat-inactivated fetal calf serum (fcs). hela, mdck, 293t and 293/gpr inducible hiv-1 packaging cells (sparacio et al., 2001) were grown in dulbecco's modified eagle's medium plus 10% fcs. infections of cem gfp cells with hiv-1 or pseudotyped derivatives were carried out by spinoculation at 400 × g for 30 min at room temperature (r.t.) using 200 ng and 50 ng cap24 equivalent of hiv-1 and (vsv-g) hiv-1/10 5 cells, respectively. then, virus adsorption was prolonged for additional 2 h at 37°c and, finally, cells were washed and refed with the complete medium. hela cells were infected with vsv by adsorbing the viral inoculum for 1 h at 37°c in a small volume (e.g., 0.1 ml of serum free medium for 2 × 10 5 cells in 12 well plates). thereafter, the cells were extensively washed, and refed with appropriate complete medium. mdck cells were used as targets of pr8 influenza virus. the challenges were carried out as for vsv infection, except that after virus adsorption, cells were refed with medium without serum in the presence of 1 μg/ml of tpck-treated trypsin (worthington biochemical corporation). hela and mdck cells served to titrate infectious vsv and influenza virus in supernatants harvested after challenge experiments. the titrations were carried out by the end-dilution method with triplicate conditions by challenging the cells with 3-fold scaled supernatant dilutions (in the presence of 1 μg/ml tpck-trypsin for influenza virus titrations), and by evaluating the cytopathic effect after 36 h (for vsv) and 72 h (for influenza virus). concentrated virus preparations previously titrated by the plaque assay were used as standards. for ndv infections, mdck cells were challenged with 2-fold decreasing viral dilutions starting to m.o.i. 1, and the cythopatic effect was assessed 48 h later. t-20, ritonavir, indinavir, saquinavir, nelfinavir, amprenavir and atazanavir were obtained from the nih aids research and reference reagent program. both control and pi-treated cells were labeled with 1 μm cfse (molecular probes, invitrogen) following the manufacturer's recommendations. a cell sample was immediately processed to determine the fluorescence levels at the zero time. thereafter, the remainder cell cultures were refed with complete medium and, at the time of completion of one cell duplication (on the average, 12 h for both hela and mdck cells at their logarithmic phase), were harvested, and the fluorescence measured by facs analysis. dead cells were identified upon labeling with 5 μg/ml of propidium iodide (sigma-aldrich). preparation of fluorescent vlps, challenge, and detection assays fluorescent vlps were obtained as previously described (muratori et al., 2009) . briefly, 293/gpr hiv-1 packaging cells were cotransfected with vectors expressing the green-fluorescent protein (gfp) fused at its n-terminus with a g 3 c hiv-1 nef mutant, together with a vector expressing wt or fusion-defective (fd) vsv-g (fredericksen and whitt, 1995) . supernatants were harvested 2 days later, concentrated by ultra-centrifugation on 20% sucrose cushion, and titrated for the hiv-1 cap24 contents. for hela cell challenge, 500 ng cap24 equivalent of fluorescent (vsv-g) hiv-1 vlps/10 5 cells were adsorbed for 1 h at 4 or 37°c in a volume of 0.1 ml in 48 well plates. thereafter, 0.1 ml of complete medium was added and, finally, the cells were extensively washed and analyzed by facs. in the endocytosis assays, the facs analysis was carried out after incubation with trypsin for 15 min at 37°c. both cells and purified vlp preparations were lysed in pbs, 1% triton x-100 in the presence of anti-proteolytic agents. for the preparation of cytoplasmic extracts, whole cell lysates were centrifuged at 6000×g for 10 min at 4°c, and the supernatants frozen at −80°c. aliquots of 200 ng hiv-1 cap24 equivalent of vlps and of 30 μg of total cell proteins were separated in 10% sds-page, and then transferred by electroblotting on nitrocellulose membranes (sartorius ag) for 60 min at 100 v with a bio-rad transblot. nitrocellulose membranes were blocked in 3% bovine serum albumin (bsa) fraction v (sigma) in ttbs/edta (10 mm tris-hcl, ph 7.4; 100 mm nacl; 1 mm edta; 0.1% tween-20) for 30 min at room temperature, then incubated for 1 h at r.t. with specific antibodies diluted in 1% bsa/ ttbs-edta. the following abs served for the revelation of both vlpand cell-associated products: arp 444 sheep anti-nef antiserum from mark harris, university of leeds, leeds, uk; rabbit polyclonal anti-vsv-g abs from immunology consultant laboratories; monoclonal anti human β-actin from amersham pharmacia biotech. immune complexes were detected through horseradish peroxidase-conjugated goat anti-sheep, anti-rabbit (both from calbiochem) and antimouse abs (nen), followed by enhanced chemioluminescence reaction (euroclone). the lysosensor probe dnd-189 (molecular probes, invitrogen) served to detect possible changes in the endosomal ph upon pi treatment. this is an acidotropic probe accumulating in acidic organelles which exhibits decreasing fluorescence upon ph increase. cells pre-treated with pis or, as control, with bafilomycin a1 (sigma-aldrich) were labeled with 1 μm of lysosensor dnd-189 in complete medium for 30 min in the presence of the drugs. afterwards, cells were extensively washed, fixed, and fluorescence evaluated by facs analysis. when appropriate, data are presented as mean + standard deviation values (sd). in some instances, statistical analysis was performed according to paired student's t-test, and confirmed using the non-parametric wilcoxon rank sum test. p-values b0.05 were considered significant. an inhibitor of hiv-1 protease modulates proteasome activity, antigen presentation, and t cell responses in vitro and in vivo effects of hiv protease inhibitors on apoptosis cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide protease inhibitor treatments reveal specific involvement of matrix metalloproteinase-9 in human adipocyte differentiation identification of host proteins required for hiv infection through a functional genomic screen dyslipidaemia associated with antiretroviral therapy in hiv-infected patients identification of host factors involved in borna disease virus cell entry through a small interfering rna functional genetic screen in vivo emergence of hiv-1 variants resistant to multiple protease inhibitors inhibition of human preadipocyte proteasomal activity by hiv protease inhibitors or specific inhibitor lactacystin leads to a defect in adipogenesis, which involves matrix metalloproteinase-9 an hiv protease inhibitor, ritonavir targets the nuclear factor-kappab and inhibits the tumor growth and infiltration of ebvpositive lymphoblastoid b cells hiv-1 nef activates stat1 in human monocytes/macrophages through the release of soluble factors hiv-protease inhibitors vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity vitro and in vivo activities of anti-influenza virus compound t-705 a new reporter cell line to monitor hiv infection and drug susceptibility in vitro effect of repeated inoculation of interferon preparations on infection of mice with encephalomyocarditis virus drosophila rnai screen identifies host genes important for influenza virus replication partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry activation of influenza a viruses by trypsin treatment rna interference screen for human genes associated with west nile virus infection ritonavir blocks akt signaling, activates apoptosis and inhibits migration and invasion in ovarian cancer cells enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion u cells by cloned human leukocyte interferon. i. effect on early and late stages of the viral multiplication cycle generation and characterization of a stable cell population releasing fluorescent hiv-1-based virus like particles in an inducible way human immunodeficiency virus type 1 (hiv-1) protease inhibitors block cell-to-cell hiv-1 endocytosis in dendritic cells different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication identification of host genes involved in hepatitis c virus replication by small interfering rna technology unpaking" human immunodeficiency virus (hiv) replication: using small interfering rna screening to identify novel cofactors and elucidate the role of group i paks in hiv infection cathepsin l is involved in proteolytic processing of the hendra virus fusion protein the human immunodeficiency virus (hiv)-1 protease inhibitor saquinavir inhibits proteasome function and causes apoptosis and radiosensitization in non-hiv-associated human cancer cells genome-wide analysis of human kinases in clathrin-and caveolae/raftmediated endocytosis synchronised transmembrane insertion and glycosylation of a nascent membrane protein expanding the frontiers of existing antiviral drugs: possible effects of hiv-1 protease inhibitors against sars and avian influenza role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein hiv protease inhibitors are potent antiangiogenic molecules and promote regression of kaposi sarcoma structured model of influenza virus replication in mdck cells in vitro antiviral activity of favipiravir (t-705) against drug-resistant influenza and 2009 a(h1n1) viruses modeling the viral dynamics of influenza a virus infection generation of a flexible cell line with regulatable, high-level expression of hiv gag/pol particles capable of packaging hiv-derived vectors effects of hiv protease inhibitor ritonavir on akt-regulated cell proliferation in breast cancer role of hemagglutinin cleavage for the pathogenicity of influenza virus biochemistry. viral glycoproteins and an evolutionary conundrum novel pandemic influenza a(h1n1) viruses are potently inhibited by das181, a sialidase fusion protein transcription and replication of nonsegmented negative-strand rna viruses hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus this work was supported by grants from the aids project of the ministry of health, rome, italy. t-20 and pis were obtained from the nih aids research and reference program. i thank p. borghi, department of cellular biology and neuroscience, istituto superiore di sanità, rome, italy for kindly supplying both vsv and ndv preparations. i also thank a.r. castrucci and a.m. ciccaglione, department of infectious, parasitic and immunomediated diseases, istituto superiore di sanità, rome, italy, for kindly providing influenza virus preparations and the vector expressing fd vsv-g, respectively. i'm indebted to g. fornari luswergh for her excellent editorial assistance. key: cord-282063-tkp1tifx authors: saberi, parya title: research in the time of coronavirus: continuing ongoing studies in the midst of the covid-19 pandemic date: 2020-04-18 journal: aids behav doi: 10.1007/s10461-020-02868-4 sha: doc_id: 282063 cord_uid: tkp1tifx nan a novel human coronavirus called severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was identified in late 2019 in wuhan, china, resulting in a pandemic [1] . due to the need for physical distancing and isolation, universities closed and most medical clinics were ordered to continue with "essential" in-person visits only. in addition to a wave of medical appointment cancellations, many medical research activities, such as participant recruitment, study visits, and outcomes assessments were halted. in light of rapid societal change in response to the pandemic, this paper summarizes our considerations for conducting remote research. the discussion provided here is not exhaustive, but rather a starting place for a fundamental shift in how research is conducted. even without a pandemic, over 19% of trials close without meeting target accrual rates [2] , underscoring the need to investigate new methods for research recruitment. reports from the pew research center show 95%, 90%, and 82% of individuals ages 13-17, 18-29 years, and 30-49, respectively, use some form of social media [3, 4] . this indicates a wide reach for online advertisement and recruitment for research, with the added potential to recruit populations typically considered hard-to-reach [5] . compared to traditional recruitment methods (e.g. print and television), studies using advertisements via social media have been shown to be financially feasible, attract large numbers of individuals, and have condensed recruitment periods. they have also been shown to provide opportunities for connecting with individuals with specific health conditions, living in remote geographic locations, or who may have been difficult to recruit due to stigma or medical mistrust [6, 7] . some examples of social media to advertise and recruit participants include: social networks (such as facebook, instagram, and twitter), dating apps (grindr, scruff, and jack'd), and online listservs for various medical societies or health conditions. the main disadvantage of using social media for study recruitment is the lack of reach to those with limited or no internet access. this can result in reduced generalizability of research findings [7, 8] . researchers concerned about generalizability, however, can supplement online advertisement with traditional methods. another remote recruitment method involves networkbased online referral strategies. one example is respondentdriven sampling for online research which involves recruiting initial participants (i.e. "seeds") who are then asked to recruit friends to enroll in the study, and so on [9] . finally, a host of specialist 0medical research recruiting agencies now exist that tout increased recruitment efficiency. screening (or pre-screening) can be conducted via simple web forms on a study website. this can increase web traffic to the study website, decrease the workload for research staff, and increase a potential participant's trust in the legitimacy of the study (e.g. if the webpage is part of a reputable university with an "edu" domain). the main disadvantage of online screening is that it may either deter some to participate (e.g. if the form is too complicated or lengthy) or increase inappropriate participation (e.g. an individual or bot responding repeatedly to be eventually deemed eligible). one option to minimize these barriers is to include a simple pre-screening form on the study website and to contact the interested individual by phone call or text message to complete the screening process. to ensure certain key study inclusion criteria, participants can be asked to text message photographs of documents. for example, an identification card bearing their name and date of birth to verify age or a photograph of a letter of hiv diagnosis or antiretroviral medication vial bearing their name to verify hiv serostatus [10, 11] . signing consent and medical release forms can also be conducted online using various tools such as docusign or a qualtrics survey [10, 11] . detailed review of the consent process is critical to ensure an individual's understanding of the study. there are numerous ways of providing remote interventions. text messaging has been used frequently for motivational messages, reminders, and ecological momentary assessments [12] [13] [14] . many text messaging platforms have been developed and evaluated for hipaa-compliant communication with participants [15] . telehealth via videochat platforms have also been used for the provision of study interventions [16] . this modality lends itself well to interventions that do not require physical contact but are enhanced by face-to-face communication, such as interventions for mental health, smoking cessation, and medication adherence counseling. participants in prior studies noted high acceptability levels in using videochat for research intervention delivery [17] . benefits included being able to speak more candidly and being less intimidated, and experiencing reduced barriers for research participation (e.g. financial barriers related to travel expenses or time off from work, stigma associated with research participation, and physical disabilities precluding mobility) [17, 18] . in addition to providing interventions, these platforms can be used for one-on-one qualitative interviews and focus groups. they allow for video-or audio-recording of the session (with consent from the participant) which can be used later for interview transcription and analysis. other remote methods of delivering an intervention include mobile health applications [11] , computers-based programs [19] , and the internet [20] . in addition to self-reported outcomes, which may be subject to recall and social desirability biases [21, 22] , objective monitoring of study outcomes can be conducted remotely in numerous ways. for example medication adherence can be evaluated by drug hair concentrations using mail-in hair samples [23] [24] [25] and text messaged photographs of pill counts or refill dates [23] [24] [25] . adherence has been monitored using medication event monitoring system (mems) caps, ingestion sensors, and "wise"-products [26] [27] [28] [29] [30] . some labs provide infectious disease testing services which can examine 4th generation hiv antibody testing using dried blood spot and chlamydia and gonorrhea testing using mail-in swabs, as well as mail-in samples for hepatitis b and c testing [31] [32] [33] . alcohol use can be remotely monitored using bluetooth-enabled breathalyzers that estimate breath alcohol concentration or wrist-worn alcohol biosensors that continuously measure transdermal alcohol content [34] [35] [36] . study assessments can be conducted via online surveys which can be emailed or text messaged to participants. when surveys are conducted online, research has shown less social desirability bias, especially related to sensitive health information [37] . in cases of limited literacy, research staff can read questions to study participants or use survey platforms that include the ability to audio-record questions. qualitative research can also be conducted using telephone, videochat, or remote focus group platforms currently typically used for marketing. study incentives can be provided remotely in numerous ways. for cross-sectional surveys or one-time assessments, provision of e-gift cards is the most convenient remote incentive. for longitudinal research, reloadable debit cards are a convenient method of payment and have been shown to be highly feasible and acceptable by research participants [24] . these debit cards can be mailed to participants without any funds loaded until confirmation of receipt and funds can be transferred from one card to another in case of lost or stolen cards. these are important benefits of reloadable debit cards over mailed gift cards. in the era of pandemics, such sars-cov-2, there is a need to continue research activities, while keeping research participants and staff safe. aligning research activities with remotely-conducted research methodology has the potential benefits of reducing time and cost for conducting the study, improving ease of participation for many individuals, enhancing the generalizability of findings, and increasing the speed of publication of study findings, all while preventing potential viral transmissions to research participants or staff. world health organization. coronavirus disease (covid-2019) situation reports a state-of-the-art conference on implementing evidence in health care-reasons and recommendations social media and technology social media fact sheet: pew research center the intersection of youth, technology, and new media with sexual health: moving the research agenda forward using online social media for recruitment of human immunodeficiency virus-positive participants: a cross-sectional survey the use of facebook in recruiting participants for health research purposes a systematic review advantages and limitations of webbased surveys: evidence from a child mental health survey web-based network sampling -efficiency and efficacy of respondent-driven sampling for online research telehealth and texting intervention to improve hiv care engagement, mental health and substance use outcomes in youth living with hiv: a pilot feasibility and acceptability study protocol wyz: a pilot study protocol for designing and developing a mobile health application for engagement in hiv care and medication adherence in youth and young adults living with hiv meta-analysis on the effect of text message reminders for hiv-related compliance social inequity and structural barriers to completion of ecological momentary assessments for young men who have sex with men and trans women living with hiv in san francisco mobile phone text messaging for promoting adherence to antiretroviral therapy in patients with hiv infection scoping review and evaluation of sms/text messaging platforms for mhealth projects or clinical interventions overcoming technological challenges: lessons learned from a telehealth counseling study. telemed e-health a pilot study to engage and counsel hiv-positive african american youth via telehealth technology use of technology for delivery of mental health and substance use services to youth living with hiv: a mixed-methods perspective computerized counseling reduces hiv-1 viral load and sexual transmission risk: findings from a randomized controlled trial test of a web-based program to improve adherence to hiv medications practical and conceptual challenges in measuring antiretroviral adherence assessing the association between self-report items for hiv pill adherence and biological measures moving antiretroviral adherence assessments to the modern era: correlations among three novel measures of adherence feasibility and acceptability of novel methods to estimate antiretroviral adherence: a longitudinal study novel methods to estimate antiretroviral adherence: protocol for a longitudinal study real-time and wireless assessment of adherence to antiretroviral therapy with co-encapsulated ingestion sensor in hiv-infected patients: a pilot study cognitive behavioural therapy for adherence and depression in patients with hiv: a three-arm randomised controlled trial consortium for ring a biomarkers and biometric measures of adherence to use of arv-based vaginal rings feasibility, performance, and acceptability of the wisebag (tm) for potential monitoring of daily gel applicator use in durban real-time adherence monitoring for hiv antiretroviral therapy innovative approach for enhancing testing of hiv, hepatitis b, and hepatitis c in the general population: protocol for an acceptability and feasibility study evaluation of the 'colli-pee', a first-void urine collection device for self-sampling at home for the detection of sexually transmitted infections, versus a routine clinic-based urine collection in a one-to-one comparison study design: efficacy and acceptability among msm in belgium acceptability and feasibility of self-collecting biological specimens for hiv, sexually transmitted infection, and adherence testing among high-risk populations (project caboodle!): protocol for an exploratory mixed-methods study temporal dynamics of transdermal alcohol concentration measured via new-generation wrist-worn biosensor the feasibility of using smartphones and mobile breathalyzers to monitor alcohol consumpti on among people living with hiv/aids. addiction science & clinical practice wristworn alcohol biosensors: strengths, limitations, and future directions a comparison of web and telephone responses from a national hiv and aids survey acknowledgements special thanks mr. nikolai caswell and ms. albaloo saberi for their help on this paper. research reported in this publication was supported by the california hiv/aids research program (chrp) award number hd15-sf-060 (saberi) and the national institutes of health award numbers r34mh114604 (saberi) and r21mh108414 (saberi). key: cord-279828-es498qul authors: boulle, andrew; davies, mary-ann; hussey, hannah; ismail, muzzammil; morden, erna; vundle, ziyanda; zweigenthal, virginia; mahomed, hassan; paleker, masudah; pienaar, david; tembo, yamanya; lawrence, charlene; isaacs, washiefa; mathema, hlengani; allen, derick; allie, taryn; bam, jamy-lee; buddiga, kasturi; dane, pierre; heekes, alexa; matlapeng, boitumelo; mutemaringa, themba; muzarabani, luckmore; phelanyane, florence; pienaar, rory; rode, catherine; smith, mariette; tiffin, nicki; zinyakatira, nesbert; cragg, carol; marais, frederick; mudaly, vanessa; voget, jacqueline; davids, jody; roodt, francois; van zyl smit, nellis; vermeulen, alda; adams, kevin; audley, gordon; bateman, kathleen; beckwith, peter; bernon, marc; blom, dirk; boloko, linda; botha, jean; boutall, adam; burmeister, sean; cairncross, lydia; calligaro, gregory; coccia, cecilia; corin, chadwin; daroowala, remy; dave, joel a; de bruyn, elsa; de villiers, martin; deetlefs, mimi; dlamini, sipho; du toit, thomas; endres, wilhelm; europa, tarin; fieggan, graham; figaji, anthony; frankenfeld, petro; gatley, elizabeth; gina, phindile; govender, evashan; grobler, rochelle; gule, manqoba vusumuzi; hanekom, christoff; held, michael; heynes, alana; hlatswayo, sabelo; hodkinson, bridget; holtzhausen, jeanette; hoosain, shakeel; jacobs, ashely; kahn, miriam; kahn, thania; khamajeet, arvin; khan, joubin; khan, riaasat; khwitshana, alicia; knight, lauren; kooverjee, sharita; krogscheepers, rene; jacque kruger, jean; kuhn, suzanne; laubscher, kim; lazarus, john; le roux, jacque; lee jones, scott; levin, dion; maartens, gary; majola, thina; manganyi, rodgers; marais, david; marais, suzaan; maritz, francois; maughan, deborah; mazondwa, simthandile; mbanga, luyanda; mbatani, nomonde; mbena, bulewa; meintjes, graeme; mendelson, marc; möller, ernst; moore, allison; ndebele, babalwa; nortje, marc; ntusi, ntobeko; nyengane, funeka; ofoegbu, chima; papavarnavas, nectarios; peter, jonny; pickard, henri; pluke, kent; raubenheimer, peter j; robertson, gordon; rozmiarek, julius; sayed, a; scriba, matthias; sekhukhune, hennie; singh, prasun; smith, elsabe; soldati, vuyolwethu; stek, cari; van den berg, robert; van der merwe, le roux; venter, pieter; vermooten, barbra; viljoen, gerrit; viranna, santhuri; vogel, jonno; vundla, nokubonga; wasserman, sean; zitha, eddy; lomas-marais, vanessa; lombard, annie; stuve, katrin; viljoen, werner; basson, de vries; le roux, sue; linden-mars, ethel; victor, lizanne; wates, mark; zwanepoel, elbe; ebrahim, nabilah; lahri, sa'ad; mnguni, ayanda; crede, thomas; de man, martin; evans, katya; hendrikse, clint; naude, jonathan; parak, moosa; szymanski, patrick; van koningsbruggen, candice; abrahams, riezaah; allwood, brian; botha, christoffel; henndrik botha, matthys; broadhurst, alistair; claasen, dirkie; daniel, che; dawood, riyaadh; du preez, marie; du toit, nicolene; erasmus, kobie; koegelenberg, coenraad f n; gabriel, shiraaz; hugo, susan; jardine, thabiet; johannes, clint; karamchand, sumanth; lalla, usha; langenegger, eduard; louw, eize; mashigo, boitumelo; mhlana, nonte; mnqwazi, chizama; moodley, ashley; moodley, desiree; moolla, saadiq; mowlana, abdurasiet; nortje, andre; olivier, elzanne; parker, arifa; paulsen, chané; prozesky, hans; rood, jacques; sabela, tholakele; schrueder, neshaad; sithole, nokwanda; sithole, sthembiso; taljaard, jantjie j; titus, gideon; van der merwe, tian; van schalkwyk, marije; vazi, luthando; viljoen, abraham j; yazied chothia, mogamat; naidoo, vanessa; alan wallis, lee; abbass, mumtaz; arendse, juanita; armien, rizqa; bailey, rochelle; bello, muideen; carelse, rachel; forgus, sheron; kalawe, nosi; kariem, saadiq; kotze, mariska; lucas, jonathan; mcclaughlin, juanita; murie, kathleen; najjaar, leilah; petersen, liesel; porter, james; shaw, melanie; stapar, dusica; williams, michelle; aldum, linda; berkowitz, natacha; girran, raakhee; lee, kevin; naidoo, lenny; neumuller, caroline; anderson, kim; begg, kerrin; boerlage, lisa; cornell, morna; de waal, renée; dudley, lilian; english, rené; euvrard, jonathan; groenewald, pam; jacob, nisha; jaspan, heather; kalk, emma; levitt, naomi; malaba, thoko; nyakato, patience; patten, gabriela; schneider, helen; shung king, maylene; tsondai, priscilla; van duuren, james; van schaik, nienke; blumberg, lucille; cohen, cheryl; govender, nelesh; jassat, waasila; kufa, tendesayi; mccarthy, kerrigan; morris, lynn; hsiao, nei-yuan; marais, ruan; ambler, jon; ngwenya, olina; osei-yeboah, richard; johnson, leigh; kassanjee, reshma; tamuhla, tsaone title: risk factors for covid-19 death in a population cohort study from the western cape province, south africa date: 2020-08-29 journal: clin infect dis doi: 10.1093/cid/ciaa1198 sha: doc_id: 279828 cord_uid: es498qul background: risk factors for covid-19 death in sub-saharan africa and the effects of hiv and tuberculosis on covid-19 outcomes are unknown. methods: we conducted a population cohort study using linked data from adults attending public sector health facilities in the western cape, south africa. we used cox-proportional hazards models adjusted for age, sex, location and comorbidities to examine the association between hiv, tuberculosis and covid-19 death from 1 march-9 june 2020 among (i) public sector “active patients” (≥1 visit in the 3 years before march 2020), (ii) laboratory-diagnosed covid-19 cases and (iii) hospitalized covid-19 cases. we calculated the standardized mortality ratio (smr) for covid-19 comparing hiv positive vs. negative adults using modelled population estimates. results: among 3,460,932 patients (16% hiv positive), 22,308 were diagnosed with covid-19, of whom 625 died. covid-19 death was associated with male sex, increasing age, diabetes, hypertension and chronic kidney disease. hiv was associated with covid-19 mortality (adjusted hazard ratio [ahr] 2.14; 95% confidence interval [ci] 1.70-2.70), with similar risks across strata of viral load and immunosuppression. current and previous tuberculosis were associated with covid-19 death (ahr [95%ci] 2.70 [1.81-4.04] and 1.51 [1.18-1.93] respectively). the smr for covid-19 death associated with hiv was 2.39 (95%ci 1.96-2.86); population attributable fraction 8.5% (95%ci 6.1-11.1). conclusion: while our findings may over-estimate hivand tuberculosis-associated covid-19 mortality risks due to residual confounding, both hiv and current tuberculosis were independently associated with increased covid-19 mortality. the associations between age, sex and other comorbidities and covid-19 mortality were similar to other settings. the effects of the intersecting pandemics of hiv, tuberculosis and coronavirus disease-19 in sub-saharan africa are unknown. studies to date suggest no increased risk of adverse outcomes in hiv co-infected patients, but these are small studies from europe and north america, often limited to hospitalized patients, and may not be relevant to sub-saharan africa where people living with hiv (plwh) are younger with different comorbidities, frequently including tuberculosis [1] [2] [3] [4] [5] [6] [7] [8] . plwh may experience more severe covid-19 disease due to hiv-related immune suppression, which may be exacerbated by transient immune deficiency from coronaviruses [9, 10] . in support of this hypothesis a large uk cohort study reported increased risk of covid-19 death with immunosuppressive comorbidity, including plwh [8] . two factors may reduce risk of severe covid-19 in plwh, however: dysfunctional immunity may lessen a virus-induced cytokine storm [11, 12] , and some antiretroviral drugs (tenofovir and some protease inhibitors) have in vitro activity against coronaviruses, with better outcomes reported for plwh receiving tenofovir disoproxil fumarate (tdf) vs. other antiretrovirals [12, 13] . tuberculosis may exacerbate covid-19 with impaired immune responses and increased angiotensin converting enzyme 2 receptor expression in respiratory epithelial cells, while covid-19 pneumonia may enhance tuberculosis progression [14] [15] [16] [17] . it is important to establish if hiv and tuberculosis increase risk of covid-19 death so that patients with these conditions can be provided with augmented prevention and potential therapeutic interventions. we used linked data from adults attending public sector health facilities in the western cape province, south africa, to identify factors associated with covid-19 death. 9 we conducted a cohort study using de-identified data from the western cape provincial health data centre (wcphdc) of public sector patients aged ≥20 years with documented sex and not known to have died before march 1, 2020 (before the first diagnosed covid-19 case in south africa, and several weeks before the first documented covid-19 death) and included all follow up through june 9, 2020. the outcome was covid-19 associated death. our main analysis examined risk of covid-19 death in the general population, so all patients were included irrespective of sars-cov-2 testing. the study was approved by the university of cape town and stellenbosch university health research ethics committees and the western cape province department of health. individual informed consent requirement was waived for this secondary analysis of de-identified data. the western cape has nearly 7 million inhabitants, of whom ~520,000 are plwh with >90% of them dependent on public sector health services. the wcphdc has been described in detail [18] . briefly, wcphdc consolidates administrative, laboratory, and pharmacy data from routine electronic clinical information systems used in all public sector health facilities with linkage through a unique identifier. multiple data sources are triangulated to enumerate health conditions such as diabetes mellitus ("diabetes"), hypertension, tuberculosis and hiv, with high or moderate certainty evidence assigned for each inferred condition (supplementary table 1 ). high certainty evidence of hiv comprises a positive hiv diagnostic test and/or hiv-rna test and/or triple antiretroviral therapy (art) and/or registration in the hiv disease management system; moderate certainty is assigned for those with only a cd4 count measure and/or two antiretroviral drugs prescribed (previously used for vertical hiv transmission prevention) and/or icd-10 diagnosis code of hiv. hiv testing coverage is high, as >90% of plwh know their hiv diagnosis [19] . high certainty evidence of tuberculosis comprised laboratory evidence of mycobacterium tuberculosis infection (any anatomical site, using xpert rif/mtb, microscopy, culture) and/or registration on the electronic tuberculosis registers and/or combination tuberculosis treatment and/or admission to a tuberculosis hospital. comorbidities were based on high or moderate certainty evidence but restricted to high certainty evidence in sensitivity analyses. the virologic, immunologic and art status of plwh on march 1, 2020 was categorized, based on most recent measures, as "confirmed virologically suppressed on art" (hiv-rna<1000 copies/ml in last 15 months and art dispensed in last 6 months), "likely virologically suppressed on art" (hiv-rna <1000 copies/ml 15-24 months previously or hiv-rna <1000 copies/ml >24 months previously if art dispensed in last 6 months), "viraemic or immunosuppressed" (hiv-rna >1000 copies/ml in last 15 months or cd4 count <200 cells/µl within 18 months before march 2020) or "unknown". until january 2020, adult first-line art was tdf+emtricitabine/lamivudine+efavirenz, with abacavir replacing tdf for patients with kidney disease; zidovudine+emtricitabine/lamivudine+protease inhibitor was used for second-line art for most patients. dolutegravir was introduced in first and second-line since january 2020. diabetic control was categorized according to glycosylated haemoglobin (hba1c) measurement within the last 2 years as <7% (controlled); 7-8.9% (poorly controlled), ≥9% (uncontrolled). all covid-19 diagnoses were based on a positive sars-cov-2 pcr test. testing was available for all patients with covid-19 symptoms until june 1, 2020; thereafter public sector laboratory testing was restricted to patients requiring admission or aged >55 years or 11 with comorbidities, due to temporary limited testing capacity. hospital admissions and all deaths in sars-cov-2 positive cases are recorded and reviewed daily. we used cox-proportional hazards models adjusted for age, sex and other comorbidities to examine the association between hiv, tuberculosis and covid-19 death among (i) all public sector patients with ≥1 health visit in the 3 years before march 1, 2020 (considered "active patients"), (ii) laboratory-diagnosed covid-19 cases and (iii) hospitalized covid-19 cases. we adjusted for location within cape town vs. rest of the province and subdistrict of residence within cape town to account for geographical variation in infection rates and as a proxy for socio-economic status. patients were censored on date of death if deceased without a covid-19 diagnosis, or on june 9, 2020, whichever was earliest. database closure was 7 days later to allow for death reporting delays. for the analysis of covid-19 death in laboratory-diagnosed cases we included cases diagnosed before june 1, 2020 when testing was available for all patients with covid-19 symptoms, but included all patients diagnosed by june 9, 2020 in sensitivity analysis. the proportional-hazard assumption was assessed with schoenfeld residuals [20] . all analyses were conducted using stata 15.1. we also calculated the standardized mortality ratio (smr) of the actual number of covid-19 deaths in plwh vs. the number that would be expected if plwh had the same risk of covid-19 death as hiv-negative people of the same age and sex. we used data on the age, sex and hiv status of all covid-19 deaths (public and private sector) and the thembisa western cape hiv model to estimate the western cape population size and hiv prevalence, by age and sex, in 2020. [21] we calculated 95% confidence intervals (ci) for the smr using 12 since individual socio-economic status and some comorbidities are not recorded in wcphdc, we calculated e-values to determine the minimum strength of association that an unmeasured confounder (e.g. raised body mass index [bmi] or socio-economic status) would need to have with hiv/ tuberculosis and covid-19 death to fully account for any association between hiv/tuberculosis and covid-19 death [22] . we conducted quantitative bias analysis to assess the impact of potential confounding by obesity on an association between hiv and covid-19 death. among 3,460,932 "active patients" aged ≥20 years on march 1, 2020, 22,308 were diagnosed with covid-19, of whom 625 (2.8%) died (table 1) . among covid-19 cases, 69% were diagnosed and 67% of deaths occurred before the change in testing criteria (june 1). the proportion of men was lower among covid-19 cases vs. non-cases (31% vs. 42%), likely due to initial cases being among essential workers in retail and manufacturing sectors employing predominantly women. the proportion of women peaked (76%) in week 5 of the epidemic, declining thereafter. diabetes and hypertension were common in all patients, with higher prevalence among covid-19 cases than non-cases ( and previous tuberculosis 14% vs. 8%). 13 although the proportion of plwh was similar among surviving and deceased covid-19 cases, a greater proportion of covid-19 deaths were in patients aged <50 years in those with vs. without hiv (39% vs 13%) ( table 2) . a substantial proportion of covid-19 deceased plwh had diabetes (50%) and hypertension (42%), however these conditions were more common in deceased people without hiv (62% for each condition). current and previous tuberculosis were more frequent in plwh irrespective of covid-19 with 14% and 37% of covid-19 deceased cases with hiv having current and/or previous tuberculosis respectively. among all public sector patients, the probability of covid-19 death by 100 days since figure 1) . to assess whether the association between hiv or tuberculosis and covid-19 mortality could be due to residual unmeasured confounding e.g. by socio-economic status, or unrecorded comorbidities, we calculated the e-value for an unmeasured confounder. for among all laboratory diagnosed covid-19 cases, there were 135 deaths among an estimated ~520,000 plwh in the province (260 deaths/million) and 786 deaths among 6.36 million people without hiv (124 deaths/million). the smr for covid-19 mortality in plwh, relative to hiv-negative people was 2.39 (95% ci 1.96-2.86) and the attributable fraction of public and private sector covid-19 deaths due to hiv was 8.5% (95% ci 6.1-11.1). among nearly 3.5 million adults (16% plwh) in south africa we found an approximately two-fold association of covid-19 death with hiv, irrespective of viraemia or immunosuppression prior to the covid-19 episode, and a similar association between covid-19 death and current tuberculosis. among plwh on art, receiving tdf was associated with lower covid-19 mortality compared to other antiretrovirals. while the hivand tuberculosis-associated increased risk of covid-19 death may be over-estimated if there is residual confounding due to socio-economic status or unrecorded comorbidities, our results, supported by sensitivity analyses, demonstrate that plwh and persons with tuberculosis are at increased risk of severe covid-19. nonetheless, despite a high burden of advanced hiv in the province, the attributable fraction of all deaths ascribed to hiv was <10%. while most case series of hiv and sars-cov-2 co-infection have not shown poor outcomes in plwh, [1-3, 5-7] some cohorts of hospitalized plwh with covid-have reported substantial morbidity and mortality including among patients with suppressed viral load on art [23, 24] london have not shown differences in mortality risk [25] [26] [27] , however, the absence of increased mortality risk in hospitalized patients with comorbidities may be explained by selection bias; risk factors for covid-19 death may be attenuated by restricting to the subset of hospitalized patients already at high mortality risk [28] . it is therefore expected that in our analysis the increased risk of death associated with all comorbidities was progressively attenuated when restricting to cases (people with sufficiently severe symptoms to be tested) and hospitalized patients. similar to our findings, several studies have reported a high prevalence of comorbidities among plwh with severe covid-19 [3, 6, 7] . the high prevalence of comorbidities in deceased plwh suggests that the effect of hiv may at least partly be due to an increased risk of comorbidities at younger ages [2, 7] , including those not recorded in wcphdc such as cardiovascular disease. persistent immune dysfunction may also be important in severe covid-19 despite viral suppression; the hazard ratio point estimates for association with covid-19 death were greater in immunosuppressed or viraemic plwh, although the numbers of these patients with covid-19 were small with wide cis. further, cd4 <200 cells/µl during admission was associated with covid-19 death. while this may partly be due to the well-described lymphopenia in severe covid-19 which is prognostic of poor outcomes, about half of patients with low cd4 during admission were either new hiv diagnoses or had previous immunosuppression, viraemia or no recent art [10] . among covid-19 cases in plwh on art, receipt of tdf (vs. other therapies) was associated with reduced covid-19 mortality. however, this association is likely to be over-estimated; in south africa only patients on second-line art or with poor renal function would not be on tdf, and both of these factors may themselves increase mortality. nonetheless, the association remained when adjusting for kidney disease, viral suppression and art duration, and concurs with results from a recently published cohort of plwh on art from spain [13] . we found both current and previous tuberculosis to be associated with covid-19 death, but since current tuberculosis itself causes death, in the absence of autopsy evidence it is difficult to disentangle the effects of covid-19 vs tuberculosis disease on mortality per se [17] . in our study, the overall high prevalence of diabetes in people with and without hiv, high proportion with poor glycaemic control and very elevated risks for covid-19 death for diabetics compared to those reported from other countries are concerning [8] . diabetes is often diagnosed late and/or untreated or poorly controlled in resource-limited settings, and the resulting microvascular disease even in people with good current diabetic control may increase covid-19 mortality [29] . to our knowledge this is the largest report on sars-cov-2 from africa, the largest report on table 1 : characteristics of (i) western cape "active patients" aged ≥20 years in public sector (public sector health care visit in last 3 years before march 1, 2020) according to covid-19 outcome (ii) covid-19 cases in "active patients" and (iii) hospitalized covid-19 cases in "active patients". reference category is hiv negative; restricted to hiv-negative patients and 199 of 601 plwh with cd4 measurement at time of covid-19 diagnosis or admission; adjusted for all other variables listed in this table in a model that included the listed categories of cd4 count instead of the binary variable hiv positive vs negative; the effect o f the other variables on mortality was similar to those presented here. hr hazard ratio; ci confidence interval; hba1c glycosylated haemoglobin; vl viral load; art antiretroviral therapy; mo months; yr years; plwh people living with hiv figure 1 : comparison of adjusted hazard ratios (hr) and 95% confidence intervals (ci) for associations with covid-19 death from cox-proportional hazards models among (i) all public sector patients ≥20 years with a public sector health visit in the previous 3 years (n=3,460,932) (ii) all adult covid-19 cases diagnosed before june 1, 2020 (n=15,203) and (iii) all hospitalized covid-19 cases (n=2, 978). covid-19 in patients with hiv: clinical case series clinical features and outcomes of hiv patients with coronavirus disease 2019 covid-19 in people living with human immunodeficiency virus: a case series of 33 patients covid-19 in patients with hiv a case series of five people living with hiv hospitalized with covid-19 in clinical characteristics and outcomes in people living with hiv hospitalized for covid-19 description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort opensafely: factors associated with covid-19 death in 17 million patients dysregulation of immune response in patients with covid-19 in wuhan, china lymphopenia predicts disease severity of covid-19: a descriptive and predictive study could hiv infection alter the clinical course of sars-cov-2 infection? when less is better why aren't people living with hiv at higher risk for developing severe coronavirus disease 2019 (covid-19)? incidence and severity of covid-19 in hiv-positive persons receiving antiretroviral therapy: a cohort study tuberculosis and type 2 diabetes mellitus: an inflammatory danger signal in the time of covid-19 tuberculosis, covid-19 and migrants: preliminary analysis of deaths occurring in 69 patients from two cohorts active tuberculosis, sequelae and covid-19 co-infection: first cohort of 49 cases active or latent tuberculosis increases susceptibility to covid-19 and disease severity data centre profile: the provincial health data centre of the western cape province, south africa available at: www.thembisa.org 22. vanderweele tj, ding p. sensitivity analysis in observational research: introducing the e-value hospitalized patients with covid-19 and hiv: a case series clinical features and outcome of hiv/sars-cov-2 co-infected patients in the bronx outcomes among hiv-positive patients hospitalized with covid-19 covid-19 and people with hiv infection: outcomes for hospitalized patients comparative outcomes in hospital admissions with covid-19 in people living with hiv and people living without hiv: a retrospective study diabetes mellitus in zambia and the western cape province of south africa: prevalence, risk factors, diagnosis and management reference category for hazard ratio is hiv negative; only included in adjusted analysis; adjusted for all other variables listed in this table in a model that included the listed categories of hiv viral load (vl), antiretroviral therapy (art) and immunosuppression instead of the binary variable hiv positive vs negative; the effect of the other variables on mortality was similar to those presented here. hr hazard ratio; ci confidence interval; hba1c glycosylated haemoglobin; vl viral load; art antiretroviral therapy all public-sector sars-cov-2 cases diagnosed before 1 june2020 key: cord-296309-i1mpov7k authors: houldcroft, charlotte j.; beale, mathew a.; breuer, judith title: clinical and biological insights from viral genome sequencing date: 2017-01-16 journal: nat rev microbiol doi: 10.1038/nrmicro.2016.182 sha: doc_id: 296309 cord_uid: i1mpov7k whole-genome sequencing (wgs) of pathogens is becoming increasingly important not only for basic research but also for clinical science and practice. in virology, wgs is important for the development of novel treatments and vaccines, and for increasing the power of molecular epidemiology and evolutionary genomics. in this opinion article, we suggest that wgs of viruses in a clinical setting will become increasingly important for patient care. we give an overview of different wgs methods that are used in virology and summarize their advantages and disadvantages. although there are only partially addressed technical, financial and ethical issues in regard to the clinical application of viral wgs, this technique provides important insights into virus transmission, evolution and pathogenesis. supplementary information: the online version of this article (doi:10.1038/nrmicro.2016.182) contains supplementary material, which is available to authorized users. since the publication of the first shotgunsequenced genome (cauliflower mosaic virus 1 ), the draft human genome 2 and the first bacterial genomes (haemophilus influenzae 3 and mycoplasma genitalium 4 ) , and enabled by the rapidly decreasing cost of high-throughput sequencing 5 , genomics has changed our understanding of human and pathogen biology. several large projects that aim to systematically analyse microbial genomes have recently been completed or are ongoing (for example, sequencing thousands of microbiomes 6 and fungal genomes 6,7 ); these projects are shaping our knowledge of the genetic variation that is present in pathogen populations, the genetic changes that underlie disease and the diversity of microorganisms with which we share our environment. the methods and data from whole-genome sequencing (wgs), which have been developed through basic scientific research, are increasingly being applied to clinical medicine, involving both humans 8 and pathogens. for example, wgs has been used to identify new routes of transmission of mycobacterium abscessus 9 in healthcare facilities (nosocomial transmission) and to understand neisseria meningitidis epidemics in africa 10 , whereas the sequencing of (table 1) , and the importance of deeply sequencing some viral pathogens. we will also explore two areas in which viral wgs has recently proven its clinical utility: metagenomic sequencing to identify viruses that cause encephalitis (box 1) ; and the role of wgs in molecular epidemiology and public health management of the pan-american zika virus outbreak (box 2) . finally, we will briefly consider the ethical and data analysis challenges that clinical viral wgs presents. for small viruses, such as hiv, influenza virus, hepatitis b virus (hbv) and hepatitis c virus (hcv), the sequencing of partial genomes has been widely used for research, but it also has important clinical applications. one of the main applications and reasons for sequencing viruses is the detection of drug resistance. for example, the management of highly active antiretroviral therapy (haart) for hiv relies on viral sequencing for the detection of drug-resistant variants. haart has substantially improved the survival of patients who have hiv, but successful therapy requires long-term suppression of viral replication with antiretroviral drugs, which may be prevented by impaired host immunity, suboptimal drug penetration in certain tissue compartments and incomplete adherence to therapy 19 . when viral replication continues despite treatment, the high mutation rate of hiv enables resistant variants to develop. it has become standard practice in many parts of the world to sequence the hiv pol gene, which encodes the main viral enzymes, to detect variants that confer resistance to inhibitors of reverse transcriptase, integrase or protease 20 , particularly when patients are first diagnosed and when viral loads indicate treatment failure. sequencing resistant variants has enabled targeted changes in treatment, which has resulted in greater reductions in viral loads than with standard care (undetectable hiv load in 32% versus 14% of patients after six months) 21, 22 . thus, sequencing resistant variants to guide hiv treatment improves disease outcomes. similar approaches have been used to identify resistant variants of hcv 23 , hbv 24 and influenza virus 25 . partial genomes has been used to detect drug resistance in rna viruses, such as influenza virus 11 , and dna viruses, such as human cytomegalovirus (hcmv) 12 . viral genome sequencing is becoming ever more important, especially in clinical research and epidemiology. wgs of pathogens has the advantage of detecting all known drug-resistant variants in a single test, whereas deep sequencing (that is, sequencing at high coverage) can identify low levels of drug-resistant variants to enable intervention before resistance becomes clinically apparent 13, 14 . whole genomes also provide good data with which to identify linked infections for public health and infection control purposes 15, 16 . however, progress in using viral wgs for clinical practice has been slow. by contrast, wgs of bacteria is now well accepted, particularly for tracking outbreaks and for the management of nosocomial transmission of antimicrobial-resistant bacteria 17, 18 . in this opinion article, we will address the challenges and opportunities for making wgs, using modern next-generation sequencing (ngs) methods, standard practice in clinical virology. we will discuss the strengths, weaknesses and technical challenges of different viral wgs methods why sequence whole genomes? limited sequencing of the small number of genes that encode targets of antiviral agents, such as hiv pol, has been the norm in clinical practice. for the detection of a limited number of antiviral-resistant variants, wgs has been too costly and labour-intensive to use compared with sequencing only the specific genes that are targeted by the drugs. however, the increasing number of resistance genes that are located across viral genomes, together with decreasing costs of sequencing and the use of sequence data for transmission studies, are driving a reappraisal of the need for wgs. for example, antiviral treatment for hcv now targets four gene products (ns3, ns4a, ns5a and ns5b), and these pcr reactions (which increases the chance of failure), requires more starting material, is more labour intensive and generally less tractable for diagnostic use 31 . sequencing the whole genome simultaneously captures all resistant variants and removes the need to design and optimize pcr assays for the detection of resistance to new drugs. a good example of this is hcmv, for which wgs can simultaneously capture the genes that encode targets of licensed therapies, such as ul27 (unknown function), ul54 (dna polymerase) and ul97 (serine/ threonine protein kinase), and of newer drugs, such as letermovir, which targets ul56 (terminase complex). this enables comprehensive antiviral-resistance testing in a single test 12 . in addition, wgs can provide information on antigenic epitopes, virus evolution in a patient over time 12 , and evidence of recombination between hcmv strains 32 . wgs can also detect putative novel drug-resistant variants and predict changes to epitopes, although phenotypic testing of variants is required to confirm clinical resistance 33 and to map epitope changes 34 . as pre-existing resistance to antiviral drugs increases (for example, hcv that is resistant to protease inhibitors 35 and hbv that is resistant to nucleoside analogue reverse-transcriptase inhibitors 36 ), wgs will provide the comprehensive resistance data that are required for selecting appropriate treatment. the complete knowledge of all resistant variants can also support novel decisions in clinical management; for example, the identification of extensive genome-wide hcmv drug resistance in a patient supported the decision to treat the individual with autologous cytomegalovirus-specific t cells instead of antiviral drugs 37 . wgs may also better identify transmission events and outbreaks, which is not always possible with sequences of subgenomic fragments. for example, wgs of respiratory syncytial virus (rsv) identified variation outside of the gene that is traditionally used for genotyping, and such information could be used to track outbreaks in households when the genetic variability in single genes is too low for transmission studies 38 . the numerous phylogenetically informative variant sites that can be obtained from full-length or near full-length genomes removes the need for high-quality sequences, which enabled the robust linking of cases of ebola virus infection and public health interventions in real time during the 2015 epidemic 39 . genes encompass more than 50% of the viral genome 26 . individually sequencing each of these genes can be as expensive and time-consuming as wgs 27 . partial-genome sequencing is particularly problematic for large viral genomes, in particular those of the herpesviruses hcmv 12 , varicella zoster virus (vzv) 28 , herpes simplex virus 1 (hsv-1) 29 and hsv-2 (ref. 30 ). these viruses have traditionally been treated with drugs that target the viral thymidine or serine/threonine protein kinases and dna polymerase. however, the increasing number of drugs in development that interact with different proteins that are encoded by viral genes scattered across the genome, means that targeted sequencing for resistance testing is costly, involves more . the routine use of pathogen wgs for diagnostic purposes 40 is likely to have wider clinical and research benefits. for example, zika virus sequences that were generated for epidemiological purposes inform public health decisions 41 . in addition, hiv genomes that were sequenced to identify antiviral-resistant variants have also been used to study virus evolution 42 and viral genetic association with disease, including genotype-phenotype association studies and genome-to-genome association studies, which look for associations between viral genetic variants, host genetic variants and outcomes of infection, such as viral load set point in hiv infection 43, 44 . why do we need deep sequencing? modern methods, which use massively parallel sequencing, enable better examination of diversity and the analysis of virus populations that contain nucleotide variants or haplotypes at low frequencies (less than 50% of the consensus sequence). minority variant analysis is particularly powerful for rna viruses, reversetranscribing dna viruses and retroviruses, because they typically show high diversity, even in a single host. hiv is the classic example; the reverse transcriptase of hiv is error-prone and introduces mutations of x4 or r5x4 genotypes is predictive of maraviroc treatment failure 50 . sub-consensus frequencies of x4-tropic or r5x4-tropic hiv are also important for the success 51 and failure 52 of bone marrow transplants from ccr5-deficient (ccr5-δ32) donors, and this information may influence the decision to stop antiviral therapy in these patients 51 . minority variants and the identification of haplotypes can also be used to detect mixed infections. infections with different hcmv genotypes or super-infections 53 are associated with poor clinical outcomes, and the detection of such mixed infections by wgs might justify more aggressive treatment. sanger sequencing of a virus population can detect minority variants at frequencies between 10% and 40% 54 , whereas ngs can sequence those same pcr amplicons to a much greater depth 55 , and, consequently, capture more of the variability that is present. sensitivity and specificity are specific for the analysed virus and the sequencing method. many studies of drug resistance in hiv that use deep-sequencing of pcr amplicons require minority variants to be present at >1% to decrease the possibility of false-positive results 56, 57 . this may miss drug-resistance mutations at frequencies of 0.1-1% and lead to poor treatment outcome 57 . although a 1-2% frequency threshold (or lower) may be clinically relevant for the detection of drug resistance in hiv, it is less clear whether the same degree of sensitivity is required for monitoring vaccine escape in hbv or drug resistance in herpesviruses (discussed below). large cohorts of patients need to be tested before, during and after treatment 46, 50 to establish thresholds for minority drug-resistant variants 12 and vaccine-escape variants that are clinically relevant for each virus. direct deep sequencing of clinical material, either by shotgun methods or rna-seq methods (so called metagenomic methods), also enables the unbiased detection and diagnosis of pathogens, and provides an alternative to culture, electron microscopy and quantitative pcr (qpcr; see below). sequencing viral nucleic acids, whether from cultures or directly from clinical specimens, is complicated by the presence of contaminating host dna 58 . by contrast, most bacterial sequencing is currently carried out on clinical isolates that are cultured; thus, sample preparation is comparatively straightforward (table 2 and at an extremely high rate (4.1 ± 1.7 × 10 −3 per base per cell) 45 . many closely related, but subtly different, viral variants exist in a single patient. these variants are sometimes described as a quasispecies or a cloud of intra-host virus diversity. the presence of a mixed population of viruses introduces problems for the determination of the true consensus 'majority' sequence, but these minority (non-consensus) variants may also change the clinical phenotype of the virus, and can be used to predict changes in genotype, tropism or drug resistance. for example, a minor variant that confers drug resistance in hiv that is present in only 2.1% of sequencing reads in a patient at baseline can rapidly become the majority (consensus) variant under the selective pressure of drug treatment 46 . similar changes in the frequency of resistance-associated alleles during treatment have been observed for hbv 47 , hcv 48 , hcmv 12 and influenza virus 49 . deep sequencing of viruses is not only required to detect drug resistance, it is also key for genotypically predicting the receptor tropism of hiv, which has treatment implications. hiv can be grouped by its use of cellular co-receptor into r5 (uses cc-chemokine receptor 5 (ccr5)), x4 (uses cxc chemokine receptor 4 (cxcr4)) or r5x4 (dual tropism). maraviroc is a ccr5 antagonist that blocks infection of r5-tropic hiv, but not of x4-tropic and r5x4-tropic hiv. just a 2% frequency for cases of encephalitis of unknown origin, metagenomic techniques are promising diagnostic tools. there are various protocols in use, but the main methods that are used are rna sequencing (rna-seq) and metagenomics. for rna-seq, the total rna, or a subset of rna, is extracted from a sample (for example, cerebrospinal fluid or a brain biopsy), converted to complementary dna (cdna) and sequenced. metagenomics generally describes the same procedure for dna, but may also include simultaneous sequencing of dna and rna through the incorporation of a cdna-synthesis step. rna-seq may improve the detection of pathogenic viruses, as many viruses have rna genomes and viral mrnas in the cerebrospinal fluid (csf) or brain indicate both the presence of the virus and which viral genes are being transcribed. however, dna viruses, which experience low-level transcription, may be poorly detected using rna-seq, and read numbers for dna viruses may be higher in metagenomic datasets 63 . both methods have successfully identified new or known viral pathogens in cases of encephalitis of unknown origin. metagenomics has been used to aid the diagnosis and characterization of enterovirus d68 in cases of acute flaccid paralysis 83 . metagenomics identified herpesviruses in the csf of four patients who had suspected viral meningoencephalitis 136 . rna-seq also identified herpes simplex virus 1 (hsv-1) in a patient with encephalitis, although the use of a dnase i digestion (which was intended to decrease the amount of host nucleic acid) decreased the number of hsv-1 reads 63 . mumps vaccine virus has also been detected in a patient with chronic encephalitis using rna-seq 137 . rna-seq has been very successful in the identification of encephalitis caused by astroviruses 138,139 and coronaviruses 65 . the deaths of three squirrel breeders from encephalitis were linked to a novel squirrel bornavirus, which was identified by separate metagenomic sequencing of dna and rna 62 . metagenomics provides more information about the virus in a sample than pcr alone, which may be important for molecular epidemiology, whereas rna-seq can identify viral sequences and viral gene expression. reviewed in ref. 59 ). currently, genome sequencing of viruses can be achieved by ultra-deep sequencing or through the enrichment for viral nucleic acids before sequencing, either directly or by concentrating virus particles. all of these approaches have their own costs and complexities. three main methods are currently used for viral genome sequencing: metagenomic sequencing, pcr amplicon sequencing and target enrichment sequencing (fig. 1) . metagenomic approaches have been used extensively for pathogen discovery and for the characterization of microbial diversity in environmental and clinical samples 60, 61 . total dna and/or rna, including from the host, bacteria, viruses, fungi and other pathogens, are extracted from a sample, and a library is prepared and sequenced by shotgun sequencing or rna sequencing (rna-seq). box 1 explores the diagnostic applications for metagenomics and rna-seq; for example, in encephalitis of unknown aetiology [62] [63] [64] , for which conventional methods such as pcr are often not diagnostic, metagenomics and rna-seq have detected viral infections 65-67 and other dna (cdna)-amplified fragment length polymorphism (aflp), abbreviated to vidisca), filtration, ultracentrifugation and the depletion of free nucleic acids, which mostly come from the host, have all been tried [74] [75] [76] [77] ; however, these methods may also decrease the total amount of viral nucleic acids so that it is insufficient for preparing a sequencing library. non-specific amplification methods, such as multiple displacement amplification (mda), which make use of random primers and φ29 polymerases, can increase the dna yield. however, these approaches are time consuming, costly, and may increase the risk of biases, errors and contamination, without necessarily improving sensitivity 78, 79 . moreover, the proportion of host reads often remains high 80 . when metagenomic methods are used for pathogen discovery or diagnosis, it is crucial to use appropriate bioinformatic tools and databases that can evaluate whether detected pathogen sequences are likely to be the cause of infection, incidental findings or contaminants. bioinformatic analyses of large metagenomic datasets require high-performance computational resources. the fact that metagenomics requires no prior knowledge of the viral genome, can be considered an advantage 27 as it enables novel viruses to be sequenced without the need for primer or probe design and synthesis. this is particularly relevant for rapid responses to emerging threats, such as zika virus 81 . for virus-associated cancers, metagenomics can inform clinical care, provide information on cancer evolution and generate high-coverage data of integrated virus genomes 69 . however, incidental findings, both in host and microbial sequences, may also present ethical and even diagnostic dilemmas for clinical metagenomics 82 (see below). a recent example involved a cluster of cases of acute flaccid myelitis that were associated with enterovirus d68 (ref. 83 ). the metagenomic data from samples taken from patients showed the presence of alternative pathogens, some of which are treatable, and was debated in formal 84 and informal scientific channels (see omicsomics blogspot article). regulation and reporting frameworks will be important to resolve future issues of this kind. an alternative to metagenomic approaches is to enrich the specific viral genome before sequencing. pcr amplification of viral genetic material using primers that are complementary to causes 68 of encephalitis. in addition, these methods have been used to sequence the whole genome of some viruses, including epstein-barr virus (ebv) 69 and hcv 27 . however, in clinical specimens, the presence of contaminating nucleic acids from the host and commensal microorganisms 58 (table 2) decreases sensitivity. the proportion of reads that match the target virus genome from metagenomic wgs is often low; for example, 0.008% for ebv in the blood of a healthy adult 70 , 0.0003% for lassa virus in clinical samples 71 and 0.3% for zika virus in a sample that was enriched for virus particles through filtration and centrifugation 72 . the read depth is often inadequate to detect resistance 27 and the cost is high. thus, metagenomic sequencing has typically only been carried out on a small number of samples for research purposes 72, 73 . the concentration of virus particles (see the zika virus example above 72 ), depletion of host material and/or sequencing to high read depth can increase the amount of virus sequence, but all of these methods add to the cost. the concentration of virus particles from clinical specimens by antibody-mediated pull-down (for example, virus discovery based on complementary whole-genome sequencing (wgs) of zika virus can help to understand the epidemiology of the recent outbreak in south america, including the origin and spread of the virus, and the connection between the virus and microcephaly. it also informs control measures, such as stopping importation of zika cases or disrupting transmission from a reservoir, and blood safety measures in hospitals. for flaviviruses, such as zika virus, wgs, or at least near whole-genome sequencing, is required to provide molecular epidemiology studies sufficient power 41 . wgs, phylogenetic analysis and molecular clock dating, combined with other epidemiological data, were useful to study the introduction of zika virus to south america 41 . for example, the most recent common ancestor of strains that are circulating in brazil pre-dates the 2014 football world cup, which makes it highly unlikely that this event was responsible for the introduction of the asian-lineage zika virus to south america 41 . wgs is also central for understanding the pathogenesis of zika virus; for example, by trying to identify sequence changes that are associated with microcephaly, as it is currently unclear which genome regions determine pathogenesis. it is likely that numerous whole-genome sequences of zika virus from around the world and from individuals with microcephaly and asymptomatic infection are required to link particular mutations to birth defects. so far, no changes in the zika virus genome have been unambiguously associated with microcephaly 41, 72, 81 . wgs and fragment sequencing were used to identify a case of zika virus transmission through platelet transfusion 140 . this case suggested that asymptomatic donors can transmit the virus to immunocompromised individuals. pcr-based testing had already established the presence of zika virus in the blood supply in a previous outbreak, but no infection was detected in recipients of blood products 141 . based on this new evidence, blood products may need to be screened routinely for zika virus 140 . finally, wgs of zika virus isolates has identified sequence polymorphisms in primer binding sites 142 , which may make pcr-based diagnosis and the quantification of viral load more difficult. this highlights the need to characterize population-level diversity, especially in epidemics in which the locally circulating virus may have diverged from viruses from other locations or time periods. several projects are underway to determine population-level diversity, including the zika in brazil real time analysis (zibra) mobile laboratory project 143 , which uses portable metagenomic sequencing of zika virus and real-time reporting of results 107 . a known nucleotide sequence has been the most common approach for enriching small viral genomes, such as hiv and influenza virus. recent examples of pcr amplicon enrichment followed by wgs include phylogenetic analysis of a measles virus outbreak at the 2010 winter olympics 85 and tracking the recent ebola virus 39 and zika virus (box 2) epidemics. pcr amplicon wgs of norovirus, which has a genome size of 7.5 kb, has been used to understand virus transmission in community 86 and hospital 87 settings, which revealed both independent introductions of the pathogen to the hospital and nosocomial transmission despite measures to control infection 87 . other pcr-based deep-sequencing studies have generated several whole genomes of influenza virus 88 (~13.5 kb), dengue virus 89 (~11 kb) and hcv 90 (9.6 kb). this was feasible because these viruses all have relatively small genomes that require only a few pcr amplicons to assemble whole-genome sequences. however, the pcr products, respectively 86, 87 . for clinical applications this is problematic because of the high laboratory workload that is associated with numerous discrete pcr reactions, the necessity for individually normalizing concentrations of different pcr amplicons before pooling, the increasing probability of reaction failure due to primer mismatch (particularly for highly variable viruses), and the high costs of labour and consumables 94 . therefore, although pcr-based sequencing of viruses as large as 250 kb is technically possible, the proportional relationship between genome size and technical complexity make pcr-based sequencing of viral genomes that are more than 20-50 kb impractical with current technologies, particularly for large multi-sample studies or routine diagnostics. another consideration is that increasing numbers of pcr reactions require a corresponding increase in sample amount, and this is not always possible as clinical specimens are limited. improvements in microfluidic technologies may help to overcome some of these barriers; for example, fluidigm, raindance and other 'droplet' sequencing technologies. microfluidics-based pcr and the pooling of multiple amplicons have been used successfully to sequence several antimicrobial-resistance loci (for example, from the microbiome of pigs) 95 and can also be used for viral genomes, potentially down to the single-genome level 96 . highly variable pathogens, particularly those that have widely divergent genetic lineages or genotypes, such as hcv 97 and norovirus, cause problems for pcr amplification, such as primer amplification 27, 92 and primer mismatches 86 . careful primer design may help to mitigate these problems, but novel variants remain problematic. target enrichment. methods of target enrichment (also known as pull-down, capture or specific enrichment methods) can be used to sequence whole viral genomes directly from clinical samples without the need for prior culture or pcr 98-100 . these methods typically involve small rna or dna probes that are complementary to the pathogen reference sequence (or a panel of reference sequences). unlike specific pcr amplicon-based methods, the reaction can be carried out in a single tube that contains overlapping probes that cover the whole genome. in a hybridization reaction, the probes, which are bound to a solid phase (for example, streptavidin-labelled heterogeneity of rna viruses, such as hcv 27 , norovirus 86 , rabies virus 91 and rsv 38 , may necessitate the use of multiple overlapping sets of primers to ensure the amplification of all genotypes. pcr amplicon sequencing is more successful for wgs from samples that have low virus concentrations than metagenomic methods 27 , although other methods such as target enrichment of viral sequences may work equally well in such samples, as shown for norovirus samples 92 . overlapping pcrs combined with ngs have been used to sequence the whole genomes of larger viruses, such as hcmv 93 , but this method has limited scalability, as many primers and a relatively large amount of starting dna are required 93 . this limits the number of suitable samples that are available and also the genomes that can be studied using this method. for example, 8-19 pcr products were required to amplify the genome of ebola virus 39 , and two studies of norovirus needed 14 and 22 103 and hhv7 (ref. 104 ). the reaction is carried out in a single well and, similarly to microfluidics-based pcr, is amenable to high-throughput automation 102 . the lack of a culture step means that the sequences that are obtained are more representative of original virus rather than cultured virus isolates, and there are fewer mutations than in pcr-amplified templates 69, 100 . the success of this method depends on the available reference sequences for the virus of interest; specificity increases when probes are designed against a larger panel of reference sequences, as this leads to better capture concentrations 27, 69 . with metagenomics, the proportion of sequencing data that map to the pathogen from unenriched clinical samples is small. target enrichment can increase the percentage of on-target viral reads from 0.01% to 80% or more 69 . the improvement in quality and depth of sequence that results enables more samples to be sequenced per run than unenriched metagenomic libraries for equivalent on-target sequencing performance. this improvement also decreases the price of sequencing, although the cost of library preparation is increased. there are alternative approaches for the enrichment of viral reads, including pulsed-field gel electrophoresis (pfge) 105 , which separates large viral genomes from smaller fragments of host dna. enrichment techniques that use degenerate rna or dna probes to capture hundreds of viral species have also been developed; for example, virome capture sequencing (vircapseq) 106 . this method is designed for the detection of both known and novel viruses, although its performance remains to be evaluated. to date, there has been very little direct comparison between the three methods for viral genome sequencing in clinical practice, with only one paper evaluating relative performance for the sequencing of hcv 27 . results from this study, in which three different enrichment protocols, two metagenomic methods and one overlapping pcr method were evaluated, showed that metagenomic methods were the least sensitive, yielded the lowest genome coverage for comparable sequencing effort and were more prone to result in incomplete genome assemblies. the pcr method required repeated amplification and was the most likely to miss mixed infections, but when reactions were successful it resulted in the most consistent read depth, whereas read depth was proportional to virus copy number in metagenomics and target enrichment. pcr generated more incomplete sequences for some hcv genotypes (particularly genotype 2) than metagenomics and target enrichment. target enrichment was the most consistent method to result in full genomes and identical consensus sequences. the ease of library preparation for metagenomic and target enrichment sequencing of hcv was considered a major advantage for clinical applications, but pcr may still be appropriate for samples that have very low viral loads. of the diversity in and between samples. target enrichment is possible despite small mismatches between template and probe; however, whereas pcr amplification requires only knowledge of flanking regions of a target region, target enrichment requires knowledge of the internal sequence to design probes. however, if one probe fails, internal and overlapping regions may still be captured by other probes 69, 100 . target enrichment is not suitable for the characterization of novel viruses that have low homology to known viruses for which metagenomics, and, in some cases, pcr using degenerate primers, which are a mix of similar but variable primers, may be more appropriate. as with all methods, the technique is constrained by the starting virus concentration. although viruses could be sequenced from samples with viral loads as low as 2,000 international units (iu) ml -1 (for hcv) or 2,500 iu ml -1 (for hcmv), there was a reduced depth of coverage in sequencing data at lower virus figure 1 | methods for sequencing viral genomes from clinical specimens. all specimens originally comprise a mix of host (in blue) and pathogen (in red) dna sequences. for pathogens that have rna genomes, rna in the sample is converted into complementary dna (cdna) before pcr and library preparation. direct metagenomic sequencing provides an accurate representation of the sequences in the sample, although at high sequencing and data analysis and storage costs. pcr amplicon sequencing uses many discrete pcr reactions to enrich the viral genome, which increases the workload for large genomes substantially but decreases the costs. target enrichment sequencing uses virus-specific nucleotide probes that are bound to a solid phase, such as beads, to enrich the viral genome in a single reaction, which reduces workload but increases the cost of library preparation compared with pcr. similar results were achieved in a study that compared pcr amplicon sequencing and target enrichment sequencing of norovirus 92 . with target enrichment sequencing, the whole viral genome could be sequenced in all 164 samples, whereas pcr-based capsid sequencing was only possible in 158 out of the 164 samples, owing to low virus titres and pcr primer mismatches, which suggests that target enrichment is more sensitive than pcr for sequencing norovirus and better accommodates between-strain sequence heterogeneity 92 . target enrichment has also been used for samples that have low viral loads and incomplete genome coverage in metagenomic sequencing 107 . both metagenomic and target enrichment sequencing can be used for pathogen genomes of all sizes, whereas pcr-based methods are less suitable for large viral genomes or for non-viral (that is, bacterial, fungal and parasite) genomes. direct comparisons of different methods 27,92 will be important for determining when each method should be used, based on sensitivity and specificity, as well as factors such as cost, scalability and turn-around time, which are particularly important in clinical applications (table 1) . beyond the technical challenges of viral wgs that are mentioned above, there are several other roadblocks that may slow the advance of wgs in the clinic. they may be considered in three groups: ethical issues, including incidental host and microbiological findings; regulatory issues, such as the establishment of standards, good laboratory practice, and sensitivity and specificity thresholds for sequencing; and analytical issues regarding data interpretation and the numerous choices of analysis options. in many clinical tests (for example, magnetic resonance imaging (mri) scans and sequencing of the genomes of patients), there is a risk of detecting a disease association that is not part of the original investigation but might be of clinical importance for the individual or their family. these incidental findings remain a topic of intense medical ethical debate 108 . the risk of incidental findings in pathogen sequencing (for example, the discovery of hiv infection during metagenomic sequencing for other pathogens) is not novel and the solution the issues of sensitivity and contamination are especially important in wgs, because of the risk of both false-negative and false-positive detection of pathogens. highly sensitive sequencing (whether metagenomic, pcr-based or target enrichment-based) may detect low-level contaminating viral nucleic acids 112, 113 . for example, murine leukaemia virus 114, 115 and parvovirus-like sequences 116, 117 are just two of many contaminants that can come from common laboratory reagents, such as nucleic acid extraction columns 118 . as with other highly sensitive technologies, robust laboratory practices and protocols are required to minimize contamination. it is also important to remember that the detection of viral nucleic acid does not necessarily identify the cause of illness, and it is good practice when using ngs methods for the diagnosis of viral infections to confirm the findings with alternative independent methods that do not rely on testing for nucleic acids. for example, in cases of encephalitis of unknown origin, positive ngs findings can be confirmed through immunohistochemical analysis of the affected tissue 65, 119 , or identification of the virus by electron microscopy or tissue culture 82 . the standardization of methods, including bioinformatics, will be key to the success of ngs and wgs in clinical virology. software packages that use a graphical user interface (gui) are preferable to tools that require command-line expertise. strict version control of software and analysis pipelines is required to ensure that results are reproducible, to make best practices easily shareable, and to enable the accreditation of analysis software. however, best-practice analysis methods are continually evolving and the premature standardization of best practices in an overly rigid manner may inhibit innovation. commercialization and regulation may help, as they provide financial and regulatory incentives to ensure that analysis tools and technologies meet clinical needs. finally, the development of well-curated databases that show which variants are truly indicative of drug resistance will be crucial for accurate clinical interpretation. such databases have already been created for hiv 120 , hbv 121, 122 and hcv 123 , but without recognition of their value by funding agencies and corresponding centralized funding to ensure their continued maintenance and upkeep, these databases and associated tools may become swiftly outdated or unusable. in clinical virology laboratories that use multiplex pcrs is to suppress results that have not been requested (j.b., unpublished observations). in the united kingdom, the clinical virologist who interprets the test results is part of the team that manages the patient, and, as such, may decide to discuss an unexpected result with the physician in charge. incidental host genetic findings (for example, the detection of variants that predispose to cancer development) in a pathogen metagenomic analysis are not reported to the individual in the united kingdom, because this is only permissible with the consent of the patient. in regard to both host and virus incidental findings, target enrichment and pcr have the advantage of only providing results about the pathogen of interest. the ethical and privacy concerns that are associated with the presence of host genetic data in publically available metagenomic datasets have been well reviewed 82 and represent a separate challenge. regulatory challenges. regulation, as well as helping to address some of the ethical concerns, is also important in standardizing wgs of viruses. the framework that is required to make viral wgs sufficiently robust and reproducible in clinical practice will come from several areas. the framework of laboratory accreditation and benchmark testing that are already available (for example, clinical laboratory improvement amendments of 1988 (clia) regulations in the usa, or accreditation according to medical laboratory quality and competence standardization criteria for iso 15189) will support the development of viral wgs standards, provided that there is sufficient need and pressure to implement clinical viral wgs. lessons learned from the use of pcr in diagnostics may be useful here, starting with ensuring good clinical laboratory and molecular practices 109, 110 . this will mean including negative samples in every sequencing run to assess contamination thresholds, spiking samples with a known virus to provide a sensitivity threshold, and including positive controls and controls for batch-to-batch variation 111 , all of which will increase sequencing costs and are likely to deter the adoption of pathogen genome sequencing by laboratories that are sequencing only small batches of samples. the centralization of virus wgs can help to ensure the maintenance of adequate standards, the processing of large batches of samples and reducing costs. although there are good reasons for sequencing whole genomes and, in general, for using ngs, if diagnostic or hospital-based laboratories are to be persuaded to transition away from sequencing subgenomic fragments, they need to see the benefit of the additional information for patient care and the practical feasibility of wgs. this includes wgs workflows that are as scalable and automatable as subgenomic fragment sequencing, a suitable regulatory framework and a price for sequencing whole genomes that is competitive with sequencing fragments. currently, the cost of sequencing viral genomes, despite their small size, remains higher than the cost of sequencing subgenomic resistance genes. the cost difference between sequencing a target region and the whole virus genome is largely governed by the size of the genome versus the size and number of target loci. in addition, whole-genome information may provide important additional knowledge, as discussed above. what does the future hold? current ngs technologies that are based around illumina, 454, ion torrent or sanger methodologies all generate short-read data, which presents challenges for haplotype phasing; that is, determining whether genetic variants (whether inter-host or intra-host) occur on the same genetic background (single viral genome or clonal) or on related, highly similar but different genetic backgrounds in the same population (sometimes called a viral swarm or cloud). furthermore, repetitive regions and recombination are more difficult to resolve using short reads owing to problems such as mapping ambiguities. the clinical implications of understanding whether, for example, multidrug-resistant variants occur together on a single viral genome or are distributed between a mixed population of viruses, each with different drug-resistance profiles, are currently unclear. although there are computational tools 124 to help resolve these issues, new technologies can generate longer reads. newer, single-molecule sequencers, such as pacbio (pacific biosciences) and minion (oxford nanopore), are capable of extremely long-read sequencing, and whole viral genomes (for example, viruses that have genomes less than 20 kb in size, such as ebola virus, norovirus and influenza a virus) could theoretically be obtained from and selective depletion of dna with a certain methylation pattern), no similar methods exist, so far, for viral sequencing. viral wgs is of increasing clinical importance for diagnosis, disease management, molecular epidemiology and infection control. there are several methods that are available to achieve wgs of viruses from clinical samples; amplicon sequencing, target enrichment or metagenomics. currently, the choice of method is specific to both the virus and the clinical question. metagenomic sequencing is most appropriate for diagnostic sequencing of unknown or poorly characterized viruses, pcr amplicon sequencing works well for short viral genomes and low diversity in primer binding sites, and target enrichment works for all pathogen sizes but is particularly advantageous for large viruses and for viruses that have diverse but wellcharacterized genomes. two obvious areas of innovation currently exist: methods that can effectively deplete host dna without affecting viral dna, and the further development of long-read technologies to achieve the flexibility and competitive pricing of short-read technologies. new technologies are required to unite the strengths of these different methods and enable healthcare providers to invest in a single technology that is suitable for all viral wgs applications. single reads. minion also has the advantage of being very fast, taking as little as four hours to go from sample receipt to reporting of analysed data 125 128 . results from the better-established pacbio technology are more promising, including a recent report of a mean read length of 12,777 bp for pseudorabies virus 129 , which has a double-stranded dna genome that is around 142 kb in length. 9.2 kb reads have been achieved using pacbio for hcv, although 9.2 kb of the 9.6 kb genome had been pre-amplified by pcr 130 . a drawback of both ngs and single-molecule sequencing is the need for high coverage to minimize the effect of sequencing errors. this is particularly problematic for studies of drug resistance, as drug resistance most frequently results from single-nucleotide mutations or small deletions (1-3 bases), especially in lower-fidelity rna viruses 131 . achieving the high coverage that is necessary to ensure accurate variant typing is challenging when there is a lot of host dna compared with viral sequences, and when the error profile of a technology makes point mutations particularly hard to detect 125 . at the time of writing, minion sequencing (r9 pore chemistry) has raw high quality (so called '2d reads') read error rates of around 5% (j. quick, personal communication), which compares unfavourably with the error rates of other technologies (illumina (<0.1%), ion torrent (~1%), but not pacbio (13% single pass)) 132 , although accuracy can been improved using circular consensus read sequencing 133, 134 . however, combining these long-read technologies with target enrichment provides a potential way forward 127, 135 , as ambiguities can be resolved if sufficient depth of sequence is achieved for the target pathogen, and error rates for all methodologies may be decreased by further technological and analytical improvements. depleting the nucleic acids of the host is an alternative solution, as a higher proportion of virus reads would be recovered from each sequencing run. although there are already solutions in place to achieve this for bacterial sequencing (for example, depletion of human ribosomal rna or mitochondria, the complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by m13mp7 shotgun sequencing initial sequencing and analysis of the human genome whole-genome random sequencing and assembly of haemophilus influenzae rd the minimal gene complement of mycoplasma genitalium technology: the $1,000 genome the human microbiome project mycocosm portal: gearing up for 1000 fungal genomes making a definitive diagnosis: successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease whole-genome sequencing to identify transmission of mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study emergence of a new epidemic neisseria meningitidis serogroup a clone in the african meningitis belt: high-resolution picture of genomic changes that mediate immune evasion genetic makeup of amantadineresistant and oseltamivir-resistant human influenza a/h1n1 viruses detection of low frequency multi-drug resistance and novel putative maribavir resistance in immunocompromised pediatric patients with cytomegalovirus clinical application of wholegenome sequencing to inform treatment for multidrugresistant tuberculosis cases low-abundance drug-resistant viral variants in chronically hiv-infected, antiretroviral treatment-naive patients significantly impact treatment outcomes origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic genomic surveillance elucidates ebola virus origin and transmission during the 2014 outbreak routine use of microbial wholegenome sequencing in diagnostic and public health microbiology microbial sequences benefit health now clinical management of hiv-1 resistance hiv-1 genotypic drug resistance testing: digging deep, reaching wide? drug-resistance genotyping in hiv-1 therapy: the viradapt randomised controlled trial persisting long-term benefit of genotype-guided treatment for hiv-infected patients failing haart. the viradapt study: week 48 follow-up molecular surveillance of hepatitis c molecular diagnosis and treatment of drug-resistant hepatitis b virus next generation sequencing for whole genome analysis and surveillance of influenza a viruses hepatitis c virus resistance to directacting antiviral drugs in interferon-free regimens comparison of next generation sequencing technologies for the comprehensive assessment of full-length hepatitis c viral genomes drug resistance of clinical varicella-zoster virus strains confirmed by recombinant thymidine kinase expression and by targeted resistance mutagenesis of a cloned wild-type isolate de novo assembly of human herpes virus type 1 (hhv-1) genome, mining of noncanonical structures and detection of novel drughepatitis b virus in drug-resistant and drug-naive patients and to detect minor variants in reverse transcriptase and hepatitis b s antigen improved detection of emerging drugresistant mutant cytomegalovirus subpopulations by deep sequencing identification of minority resistance mutations in the hiv-1 integrase coding region using next generation sequencing low-frequency drug resistance in hiv-infected ugandans on antiretroviral treatment is associated with regimen failure direct sequencing and characterization of a clinical isolate of epstein-barr virus from nasopharyngeal carcinoma tissue by using nextgeneration sequencing technology twenty years of bacterial genome sequencing environmental genome shotgun sequencing of the sargasso sea the challenge and potential of metagenomics in the clinic a variegated squirrel bornavirus associated with fatal human encephalitis next-generation sequencing (ngs) in the identification of encephalitis-causing viruses: unexpected detection of human herpesvirus 1 while searching for rna pathogens human ifnar2 deficiency: lessons for antiviral immunity human coronavirus oc43 associated with fatal encephalitis diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing whole-genome sequence analysis reveals the enterovirus d68 isolates during the united states 2014 outbreak mainly belong to a novel clade actionable diagnosis of neuroleptospirosis by next-generation sequencing specific capture and wholegenome sequencing of viruses from clinical samples the genetic diversity of epstein-barr virus in the setting of transplantation relative to nontransplant settings: a feasibility study enhanced methods for unbiased deep sequencing of lassa and ebola rna viruses from clinical and biological samples detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study epstein-barr virus from burkitt lymphoma biopsies from africa and south america share novel lmp-1 promoter and gene variations protocol for metagenomic virus detection in clinical specimens viral metagenomics and blood safety the diagnosis of infectious diseases by whole genome next generation sequencing: a new era is opening autologous antibody capture to enrich immunogenic viruses for viral discovery bias in whole genome amplification: causes and considerations resistance mutations using short-and long-read next generation sequencing technologies antiviral drug resistance in herpesviruses other than cytomegalovirus letermovir and inhibitors of the terminase complex: a promising new class of investigational antiviral drugs against human cytomegalovirus islands of linkage in an ocean of pervasive recombination reveals two-speed evolution of human cytomegalovirus genomes analysis of mutations in the gene encoding cytomegalovirus dna polymerase in a phase 2 clinical trial of brincidofovir prophylaxis epitope mapping of the hemagglutinin molecule of a highly pathogenic h5n1 influenza virus by using monoclonal antibodies detection of a sexually transmitted hepatitis c virus protease inhibitor-resistance variant in a human immunodeficiency virus-infected homosexual man outbreak of infections by hepatitis b virus genotype a and transmission of genetic drug resistance in patients coinfected with hiv-1 in japan novel autologous t-cell therapy for drug-resistant cytomegalovirus disease after lung transplantation local evolutionary patterns of human respiratory syncytial virus derived from whole-genome sequencing real-time, portable genome sequencing for ebola surveillance whole-genome sequencing for routine pathogen surveillance in public health: a population snapshot of invasive staphylococcus aureus in europe zika virus in the americas: early epidemiological and genetic findings evidence of self-sustaining drug resistant hiv-1 lineages among untreated patients in the united kingdom a genome-to-genome analysis of associations between human genetic variation, hiv-1 sequence diversity, and viral control genome-wide association study of hiv whole genome sequences validated using drug resistance extremely high mutation rate of hiv-1 in vivo prevalence and evolution of low frequency hiv drug resistance mutations detected by ultra deep sequencing in patients experiencing first line antiretroviral therapy failure composition and interactions of hepatitis b virus quasispecies defined the virological response during telbivudine therapy resistance-associated ns5a variants of hepatitis c virus are susceptible to interferon-based therapy intrahost dynamics of antiviral resistance in influenza a virus reflect complex patterns of segment linkage, reassortment, and natural selection nextgeneration sequencing to assess hiv tropism long-term control of hiv by ccr5 delta32/delta32 stem-cell transplantation shift of hiv tropism in stem-cell transplantation with ccr5 delta32 mutation mixed cytomegalovirus glycoprotein b genotypes in immunocompromised patients use of massively parallel ultradeep pyrosequencing to characterize the genetic diversity of target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery chicken skin virome analyzed by highthroughput sequencing shows a composition highly different from human skin zika virus associated with microcephaly beyond research: a primer for considerations on using viral metagenomics in the field and clinic a novel outbreak enterovirus d68 strain associated with acute flaccid myelitis cases in the usa (2012-2014): a retrospective cohort study re-analysis of metagenomic sequences from acute flaccid myelitis patients reveals alternatives to enterovirus d68 infection whole-genome sequencing of measles virus genotypes h1 and d8 during outbreaks of infection following the 2010 olympic winter games reveals viral transmission routes deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting next-generation whole genome sequencing identifies the direction of norovirus transmission in linked patients molecular epidemiology and evolution of influenza viruses circulating within european swine between 2009 and 2013 genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity whole genome pyrosequencing of rare hepatitis c virus genotypes enhances subtype classification and identification of naturally occurring drug resistance variants evolutionary trends of european bat lyssavirus type 2 including genetic characterization of finnish strains of human and bat origin 24 years apart norovirus whole genome sequencing by sureselect target enrichment: a robust and sensitive method extensive genome-wide variability of human cytomegalovirus in congenitally infected infants detection of a divergent parainfluenza 4 virus in an adult patient with influenza like illness using next-generation sequencing clusters of antibiotic resistance genes enriched together stay together in swine agriculture dynamic regulation of hiv-1 mrna populations analyzed by single-molecule enrichment and long-read sequencing ve-seq: robust, unbiased enrichment for streamlined detection and whole-genome sequencing of hcv and other highly diverse pathogens enhanced virome sequencing using targeted sequence capture hybridization capture using short pcr products enriches small genomes by capturing flanking sequences (capflank) deep sequencing of viral genomes provides insight into the evolution and pathogenesis of varicella zoster virus and its vaccine in humans mode of virus rescue determines the acquisition of vhs mutations in vp22-negative herpes simplex virus 1 complete genome sequence of pseudorabies virus reference strain nia3 using single-molecule real-time sequencing a method for near full-length amplification and sequencing for six hepatitis c virus genotypes antiviral resistance: mechanisms, clinical significance, and future implications coming of age: ten years of next-generation sequencing technologies inc-seq: accurate single molecule reads using nanopore sequencing a flexible and efficient template format for circular consensus sequencing and snp detection a novel method for the multiplexed target enrichment of minion next generation sequencing libraries using pcr-generated baits detection of virus in csf from the cases with meningoencephalitis by next-generation sequencing deep sequencing reveals persistence of cell-associated mumps vaccine virus in chronic encephalitis astrovirus va1/hmo-c: an increasingly recognized neurotropic pathogen in immunocompromised patients next-generation sequencing for diagnosis and tailored therapy: a case report of astrovirus-associated progressive encephalitis probable transfusiontransmitted zika virus in brazil potential for zika virus transmission through blood transfusion demonstrated during an outbreak in french polynesia complete genome sequences of zika virus strains isolated from the blood of patients in thailand in 2014 and the philippines in mobile real-time surveillance of zika virus in brazil efficient de novo assembly of singlecell bacterial genomes from short-read data sets a method for selectively enriching microbial dna from contaminating vertebrate host dna genome diversity of epstein-barr virus from multiple tumor types and normal infection complete genome sequence of the human herpesvirus 6a strain aj from africa resembles strain gs from north america genome sequence of human herpesvirus 7 strain ucl-1 the genomic sequence of lymphocryptovirus from cynomolgus macaque virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis distinct zika virus lineage in incidental findings of uncertain significance: to know or not to know -that is not the question good laboratory practice when performing molecular amplification assays wide spectra of quality control ch. 3 control for stochastic sampling variation and qualitative sequencing error in next generation sequencing tales from the crypt and coral reef: the successes and challenges of identifying new herpesviruses using metagenomics human cancers and mammalian retroviruses: should we worry about bovine leukemia virus? future virol disease-associated xmrv sequences are consistent with laboratory contamination dna extraction columns contaminated with murine sequences false-positive results in metagenomic virus discovery: a strong case for follow-up diagnosis the perils of pathogen discovery: origin of a novel parvovirus-like hybrid genome traced to nucleic acid extraction spin columns reagent and laboratory contamination can critically impact sequence-based microbiome analyses vision for investigating the microbiology of health and disease rationale and uses of a public hiv drugresistance database hepseq: international public health repository for hepatitis b hepatitis b virus reverse transcriptase sequence variant database for sequence analysis and mutation discovery the los alamos hepatitis c sequence database base-seq: a method for obtaining long viral haplotypes from short sequence reads identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing bacterial and viral identification and differentiation by amplicon sequencing on the minion nanopore sequencer enrichment of long dna fragments from mixed samples for nanopore sequencing rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis the authors thank j. brown the authors declare no competing interests. omicsomics blogspot article: http://omicsomics.blogspot. co.uk/2015/07/leaky-clinical-metagenomics-pipelines.html key: cord-302403-kahi8cbc authors: miller, robert f.; lipman, marc c.i. title: pulmonary infections date: 2009-05-15 journal: clinical respiratory medicine doi: 10.1016/b978-032304825-5.10034-0 sha: doc_id: 302403 cord_uid: kahi8cbc nan it is hard to believe that the first consistent reports of acquired immunodeficiency syndrome (aids) were only 25 years ago. since then, respiratory and general physicians have become accustomed to dealing with an extraordinary range of esoteric and previously unheard of conditions. pneumocystis carinii (now known as pneumocystis jirovecii) pneumonia is frequently part of the differential diagnosis of treatment-nonresponsive pneumonic illness. human immunodeficiency virus (hiv) testing is almost routinely offered as a "rule out" test in clinical cases that defy simple diagnosis. in many parts of the developing world, advanced hiv disease is unfortunately the likeliest reason for seeking medical care. the change this has wrought on people, countries and their economies is huge and depressing. the isolation of hiv from patients with aids in the mid 1980s paved the way for intensive research with the ultimate development of drugs directed against this chronic infection. however, despite such advances, the methods by which hiv infection leads to severe immune dysregulation and clinical disease are still not fully defined. the introduction in 1996, in the developed world, of highly active antiretroviral therapy, also known as combination antiretroviral therapy (cart) hereto referred to as haart, has altered the natural history of this extraordinary condition. before haart, defined as a combination of medications that usually includes at least three potent anti-hiv agents, treatment largely consisted of specific opportunistic infection management and less effective antiretroviral therapy. the clinical consequences of this change are enormous. the relative hazard for development of pneumocystis pneumonia (pcp) in an hivinfected individual has fallen by more than 80%. drug therapy does have a down side. it has significant unwanted effects, as well as major interactions with other medication (e.g., rifamycins used in treating tuberculosis). the profound change in immunity induced by haart may also lead to disease (the immune reconstitution inflammatory syndrome [iris] ). notwithstanding haart, respiratory disease remains an important cause of morbidity and mortality. much of the world cannot afford such medication, and more than two thirds of hiv-infected individuals have at least one respiratory episode during the course of their illness. in the early stages of hiv infection, when patients have relatively preserved immune responses, individuals have the same infections found in the general population, although at a greater incidence. with progressive hiv disease, subjects are at an increased risk of opportunistic disease. for example, the north american prospective study of pulmonary complications of hiv infection (pspc), a multicenter cohort drawn from all hiv risk groups at various stages of immunosuppression, revealed over an 18-month study period that of approximately 1000 subjects who were not using haart: 33% reported an upper respiratory tract infection 16% had an episode of acute bronchitis 5% had acute sinusitis 5% had bacterial pneumonia 4% had pcp develop the immune dysregulation that arises from hiv infection means that bacteria, mycobacteria, fungi, viruses, and protozoa can all cause disease in subjects with advanced infection. table 34 -1 shows the organisms that typically infect the lung in hiv disease. of these, bacterial infections, tuberculosis, and pcp are the most important. in the west, 40% of aids diagnoses are due to pcp. this chapter provides a brief general overview of the epidemiology and pathogenesis of hiv infection before concentrating on hiv and its infectious pulmonary complications. it is reported that by the end of 2006, 39.5 million individuals worldwide had acquired hiv infection (figure 34 -1). of these, more than 40% are thought to have had aids develop (for definition of aids, see tables 34-2 and 34-3, and box 34-1). globally, 4.3 million individuals acquired hiv infection in 2006, and over this time 2.9 million died of aids. the developing world has been most affected. sub-saharan africa is the current epicenter of the pandemic (two thirds of all infections); here, one in five adults is hiv infected. south and southeast asia are responsible for almost a fifth of the estimated hiv global burden. in central-eastern europe and central asia, there are currently 1.7 million hivinfected individuals. in the developed world, north america and western europe account for approximately 1.4 million and 740,000 infections, respectively. most of these are spread through sexual contact, although vertical (mother-to-child) and bloodborne infections are also common. in the developing world, heterosexual transmission is the norm; however, in north america and europe, homosexual and bisexual men constitute the largest group of infected individuals. hiv was first isolated in 1983 from patients with symptoms and signs of immune dysfunction. two subtypes (hiv-1 and hiv-2) have subsequently been identified. hiv-1 (hereafter referred to as hiv) is responsible for most infections, has a more aggressive clinical course, and is the focus of this chapter. hiv is a human retrovirus belonging to the lentivirus family. cell-free or cell-associated hiv infects through attachment of its viral envelope protein (gp120) to the cd4 antigen complex on host cells. the cd4 receptor is found on several cell types, although the t-helper lymphocyte is the main site of hiv infection in the body. hiv gp120 must also bind to a cell surface protein coreceptor called chemokine receptor 5 (ccr5) or to other co-receptors, including cxcr4, depending on the host cell type. polymorphisms in genes for ccr5 may affect disease progression by reducing the ability of hiv to enter and infect cells. however, at a population level, this effect is small. once hiv is inside the cell, it can, by use of the enzyme reverse transcriptase (rna-dependent dna polymerase), transcribe its hiv rna into a dna copy that can translocate to the nucleus and integrate with host cell dna by use of its viral integrase. the virus (as proviral dna) remains latent in many cells until the cell itself becomes activated. this may arise from cytokine or antigen stimulation. the viral genetic material is then transcribed into new rna, which, in the form of a newly created virion, buds from the cell surface and is free to infect other cd4-bearing cells. hiv infection directly attacks the immune system and in particular the t-helper cells that are central to a coordinated immune response. this leads to progressive immune dysfunction and an inability to resist opportunistic disease. the pathogenic process is not well defined, although it is thought that at the time of primary infection, hiv spreads to the lymph nodes, circulating immune cells, and thymus. this is a massive viral infection of the human host; and although there seems to be a relatively potent immune response, in fact, this initial onslaught is so devastating (targeting as it does specific memory t cells responsible for sustaining long-term protective immunity) that the scene is set for progressive immune failure. this occurs through a combination of direct cell killing by hiv replicating within cells, as well as the negative effects of chronic immune activation. ultimately, these lead in most individuals to immune system destruction and dysfunction. this is reflected not only by clinical disease indicating profound immunosuppression but also by a measurable reduction in the circulating absolute cd4 cell count, the percentage of t cells expressing cd4 markers, and in the progressive reduction in cd4/cd8 t-cell ratio. the use of haart, as well as preventative (prophylactic) therapies for opportunistic infections, has changed the clinical picture of hiv disease in countries where these interventions are available. death rates have fallen to one sixth of their previous levels. however, in the absence of such treatments, the median interval between hiv seroconversion and progression to aids in the developed world has been estimated to be 10 years, although rather less in resource-poor countries. almost all individuals have aids develop if untreated, and without haart, 95% of these will die within 5 years. in many parts of the world, the main causes of death in patients with hiv infection include bacterial pneumonia, tuberculosis, and pcp. the course of hiv infection can be divided clinically into several distinct periods: acquisition of the virus seroconversion, with or without a clinical illness (primary hiv infection) clinically silent period, lasting several months to years development of symptoms and signs indicating some degree of immunosuppression aids (where the subject has opportunistic disease implying profound immunosuppression [e.g., pcp]) the time from acquisition of hiv infection to development of detectable antibodies (the "window" period) is usually approximately 6-8 weeks. between 30 and 70% of individuals who become infected will have a seroconversion illness. hiv antibody is normally present within 2-3 weeks of these symptoms, although this can take longer. hiv rna in peripheral blood is detectable before this and is often used to confirm infection. the nonspecific features of primary hiv infection are almost always self-limiting, and typically seroconversion mimics a "flulike" illness or glandular fever. most individuals with primary hiv infection recover from the acute symptoms within 4 weeks. a proportion may have persistent symmetric generalized lymphadenopathy. there is no difference in prognosis in this group compared with asymptomatic hiv-positive individuals. although a proportion of individuals remain completely well without any treatment for an extended period (approximately 20% after 10 years), many hiv-infected individuals have minor symptoms and signs suggesting immune dysfunction. examples of these include new or worsening rashes (including herpes simplex), tiredness, cough, and low-grade anemia. certain clinical symptoms and signs provide important prognostic information. most studies have shown that oral candidiasis and constitutional symptoms (e.g., malaise, idiopathic fever, night sweats, diarrhea, and weight loss) are the strongest clinical predictors of progression to aids. the term aids was created as an epidemiologic tool to capture those conditions that early in the hiv epidemic seemed to suggest significant immune destruction. over time, it has been modified to incorporate the expanding spectrum of recognized diseases affecting immunosuppressed individuals, such as cervical carcinoma and recurrent bacterial pneumonia (see box 34-1). the 1993 centers for disease control and prevention (cdc) classification included an immunologic criterion for aids (cd4 count <200 cells/ml or cd4 percentage <14% of total lymphocytes) regardless of clinical symptoms (see table 34 -3). these data are used to define a point at which the risk of severe opportunistic infection rises dramatically. an example of this can be seen in the multicenter aids cohort study (macs) of homosexual and bisexual men without aids, which found that the incidence of pcp in subjects who did not use prophylaxis rose from 0.5% at 6 months in men with a baseline cd4 count >200 cells/ml to 8.4% in those with a cd4 count <200 cells/ml. apart from cervical carcinoma, aids indicator diseases differ little between men and women. injecting drug users have a high incidence of wasting syndrome, recurrent bacterial pneumonia, and tuberculosis. geographic differences in diseases occur that reflect the opportunistic pathogens present in the local environment (e.g., histoplasmosis or visceral leishmaniasis usually occur only in patients from endemic areas). in the developed world, sexual, racial, and hiv risk factor survival differences after an aids diagnosis mainly arise from variation in ease of access to medical care. it is certainly the case, however, that better treatment outcomes are associated with genuine specialist care provided by people with extensive experience in the field. in countries where haart is available, the spectrum of hiv-related disease has changed. in the eurosida cohort (a pan-european prospective study of hiv infection), between 1994 and 2002, opportunistic infections associated with very low cd4 counts (e.g., cytomegalovirus [cmv] retinitis and mycobacterium avium-intracellulare complex [mac] infection) were observed less frequently over time. malignant disease, such as non-hodgkin lymphoma, increased as an aids-defining event between 1994 and 2004. although death rates have fallen in haart-treated populations, there has been a rise in the proportion of non-aids deaths. in some series, this accounts for most events. causes include liver disease (often caused by viral hepatitis) and cancer, as well as cardiovascular disease and drug-related toxicity. in such circumstances, aids deaths usually occur among patients who have not accessed medical care regularly and who are initially seen with advanced hiv disease. a new manifestation of opportunistic infection has been described in patients commencing haart. the immune reconstitution disease, iris, may cause severe, if temporary, clinical illness as the individual's immunity recovers. patients will appear to develop a relapse of their original (and partially treated) disease. this is often seen in mac infection, tuberculosis, hepatitis b, cmv retinitis, and herpesviral infection. metabolic complications of haart, such as ischemic heart disease and diabetes, are a potential problem in hiv practice in the developed world. a significant number of individuals taking haart also experience drug toxicity. an increasing number of patients are also surviving to manifest symptoms associated with chronic hepatitis b and c infection. hivassociated nephropathy (often with chronic kidney disease) is common in black africans and is a significant cause of long-term morbidity. laboratory markers and clinical symptoms (e.g., oral thrush) can independently reflect the immune changes that lead to serious disease. staging systems have been developed that can predict the risk of progression to severe opportunistic disease (aids). the fall in absolute blood cd4 t-lymphocyte count is the most widely used prognostic marker, although cd4 counts may be affected by a number of factors apart from hiv, including intercurrent infection, cigarette smoking, exercise, time of day, and laboratory variation. the percentage of cd4 cells and ratio of cd4/cd8 cells are more stable measures and may be used if the cd4 absolute counts seem to vary widely from visit to visit. measurement of plasma hiv rna "viral load" provides important prognostic information that can both guide therapy and suggest long-term outcome. it has a particular value in subjects who are clinically well and have high cd4 counts, because it can give some indication of the expected speed of clinical progression. it is clear from the frequency with which hiv-related respiratory disease occurs that the pulmonary immune response is profoundly dysregulated. however, the mechanism underlying this has not been fully explained. in part, this is because the alterations that arise reflect the complex interplay between systemically derived hiv and other circulating antigens trafficking through the pulmonary vasculature, local immune cells, and airborne antigen. most studies investigating the pulmonary immune response have used in vitro cell culture systems that seek to mimic the pulmonary environment or in some cases bronchoscopy and bronchoalveolar lavage (bal) to recover lung cells from infected individuals. until recently, these have been performed on symptomatic patients who required bronchoscopy as a diagnostic procedure. such subjects generally have advanced hiv infection, are often taking a number of different drugs (including antiretrovirals), and may have any number of different pathogens causing their pulmonary disease-which by themselves can influence the immune findings. finally, the question of whether bal fluid truly represents the site of the immune response (the lung parenchyma) remains unclear. for all these reasons, reported data should be interpreted with some care. an individual's risk for respiratory disease is determined by his or her medical history (e.g., receipt of effective preventative or antiretroviral therapy), place of residence and travel history (e.g., the influence of geography on mycobacterial and fungal disease), and state of host immunity. falling blood cd4 counts or high plasma rna "viral loads" increase the chance of respiratory infection, with an increased spectrum of potential organisms responsible for infection in the more immunosuppressed individual. for example, hiv-infected individuals with a cd4 count <200 cells/ml are four times more likely to have one episode of bacterial pneumonia per year than those with higher cd4 cell counts. more exotic organisms are found in subjects with very low cd4 counts. these include bacteria such as rhodococcus equi and nocardia asteroides and fungi such as aspergillus species and penicillium marneffei. just as with p. jirovecii, this reflects the importance of t-cell depletion and macrophage dysfunction in the loss of host immunity (a process that has been confirmed by animal experiments). among hiv-infected patients, injecting drug users are at greatest risk for development of bacterial pneumonia and tuberculosis. individuals who have had previous respiratory episodes (pcp or bacterial pneumonia) seem to be at increased risk of further disease. whether this relates to host or environmental factors is not certain, although it seems likely that structural lung damage and abnormal pulmonary physiology would, in part, contribute to this. this argument is supported by the increased rates of pneumonia in hiv-infected smokers compared with nonsmokers. recent work has shown chronic obstructive pulmonary disease (copd) and lung cancer occur more frequently among hiv-infected individuals compared with the general population. given that a large number of hiv-infected individuals smoke heavily, there is a pressing need to target this population for smoking cessation. this is reinforced by the association demonstrated in some (but not all) studies between smoking and a more rapid progression to first aids illness and death. the presentation mimics bacterial exacerbations of chronic obstructive lung disease; most patients have a productive cough and fever. the pathogens commonly identified are similar to those in the general population (i.e., streptococcus pneumoniae and haemophilus influenzae). however, patients with advanced disease may be infected with pseudomonas aeruginosa or staphylococcus aureus. response to appropriate antibiotic therapy in conventional doses is good, although relapses frequently occur. bronchiectasis is increasingly recognized in hiv-infected patients with advanced hiv disease and low cd4 lymphocyte counts. it probably arises secondary to recurrent bacterial or p. jirovecii infections. the diagnosis is most often made by high-resolution/fine-cut computed tomography (ct) scanning. its prevalence has not been accurately determined, although with improved survival from both opportunistic infections and hiv disease, it is likely that it will be increasingly common in clinical practice. the pathogens isolated in patients with bronchiectasis are those seen in bronchitis. in addition, pseudomonas cepacia and moraxella catarrhalis have been described. community-acquired bacterial pneumonia occurs more frequently in hiv-infected patients than in the general population. it is especially common in hiv-infected injecting drug users. the spectrum of bacterial pathogens is similar to that in non-hiv-infected individuals (see table 34 -1). s. pneumoniae is the most common cause, followed by h. influenzae. hiv-infected individuals with s. pneumoniae pneumonia are frequently bacteremic. in one study, the rate of pneumococcal bacteremia in hiv-infected individuals was 100 times that of an hiv-negative population. more recent work has confirmed this to be the case for all causes of hiv-related bacterial pneumonia. typically, blood cultures have a 40-fold increased pickup rate in hiv-positive patients. the widespread use of haart has led to some decrease in rates of bacterial pneumonia and bacteremia, although they are still considerably higher than those seen in a non-hiv-infected population. bacterial pneumonia has a similar presentation in hivinfected and uninfected individuals. chest radiographs are frequently atypical, mimicking pcp in up to 50% of cases ( figure 34 -2). by contrast, radiographic lobar or segmental consolidation may also be seen in a wide range of bacterial organisms (figure 34 -3); these include s. pneumoniae, p. aeruginosa, h. influenzae, and mycobacterium tuberculosis. pcp may also present with lobar or segmental consolidation. in subjects with more advanced hiv disease and low cd4 lymphocyte counts, p. aeruginosa and s. aureus also cause pneumonia. complications of bacterial pneumonia frequently occur, and pleural effusions are twice as likely in hiv infection (often occurring with s. aureus infection); empyema and intrapulmonary abscess formation are present in up to 10% of patients. inevitably, the mortality rate is high (approximately 10%). nocardia asteroides infection. this has been reported in patients with advanced hiv disease and low cd4 lymphocyte counts. the widespread use of trimethoprim/sulfamethoxazole (tmp/smx) for prophylaxis of pcp may have reduced the incidence of infection. the clinical presentation is often indistinguishable from that of other bacterial infections. chest radiographic appearances may mimic tuberculosis (see later), with upper lobe consolidation, cavitation, interstitial infiltrates, pleural effusion, and hilar lymphadenopathy. the diagnosis is made by identification of the organism in sputum/bal fluid or lung tissue. rhodococcus equi. r. equi usually produces pneumonia in patients who have advanced hiv infection and have been in contact with farm animals or with soil from fields or barns where animals are housed. the presentation is subacute, with 2-3 weeks of cough, dyspnea, fever, and pleuritic chest pain. the chest radiograph typically shows consolidation with cavitation. pleural effusions are common. the diagnosis is usually made by culture of sputum or blood; bronchoscopy with bal or pleural aspiration may be necessary in some cases. bartonella henselae. b. henselae is a gram-negative bacillus that causes bacillary angiomatosis in hiv-infected patients. clinically, the cutaneous lesions may mimic kaposi sarcoma, from which they may be distinguished by demonstration of organisms in tissue with warthin-starry silver stain. bacillary angiomatosis may also infect the lungs, where it produces endobronchial red or violet polypoid angiomatous lesions, which may resemble kaposi sarcoma. biopsy is necessary to confirm a diagnosis. tuberculosis hiv infection is associated with at least a 40-fold increased risk of an individual having active tuberculosis develop compared with noninfected subjects. taken together with its ability to infect both the immunosuppressed and immunocompetent, tuberculosis is perhaps, therefore, the single most important disease associated with hiv infection. it is estimated that there are at least 13 million individuals with hiv-tuberculosis coinfection. as such, tuberculosis is a major cause of hiv-related morbidity and mortality. it is also a major driver in both resource-rich and resource-poor countries for the current overall increase in tuberculosis rates. where hiv infection is endemic, tuberculosis control at a population level is almost impossible if treatment for both infections is not available. in the united kingdom, many centers routinely offer hiv antibody testing to all patients with tuberculosis, regardless of risk factors for hiv infection. in the united states, the cdc now recommends hiv testing as a routine part of health care for all patients aged 13-64 accessing medical services. the advantage of this is that individuals who are found to be hiv infected can be given haart. furthermore, strategies to modify high-risk behavior and reduce ongoing hiv transmission may be offered. active tuberculosis can occur at any stage of hiv infection, and unlike almost every other hiv-related infection, may do so despite effective antiretroviral therapy. in the united states, united kingdom, and most european countries, reporting of tuberculosis in both hiv-infected and non-hiv-infected individuals is mandatory. clinical disease in hiv-infected patients may arise in several different ways: by reactivation of latent tuberculosis, by rapid progression of pulmonary infection, and by reinfection from an exogenous source. pulmonary disease is the most common presentation; and clinical manifestations are related to the level of an individual's cell-mediated immunity. for example, subjects with early hiv disease have clinical features similar to "normal" adult postprimary disease (table 34 -4). symptoms typically include weight loss, fever with sweats, cough, sputum, dyspnea, hemoptysis, and chest pain. these patients may have no clinical features to suggest associated hiv infection. the chest radiograph frequently shows upper lobe consolidation, and cavitary change is common (figure 34-4) . the tuberculin skin test (purified protein derivative [ppd] ) is usually positive, and the likelihood of spontaneously expectorated sputum or bal fluid being smear positive for acid-fast bacilli is high. in individuals with advanced hiv disease (i.e., low cd4 lymphocyte counts and clinically apparent immunosuppression), it may be difficult to diagnose tuberculosis. the clinical presentation here is often with nonspecific symptoms. fever, weight loss, fatigue, and malaise may be mistakenly ascribed to hiv infection itself. in this context, pulmonary tuberculosis is often similar to primary infection, with the chest radiograph showing diffuse or military-type shadowing ( figure 34 -5), hilar or mediastinal lymphadenopathy, or pleural effusion; cavitation is unusual. in up to 10% of patients the chest radiograph may appear normal; in others, the pulmonary infiltrate can be bilateral, diffuse, and interstitial in pattern, thus mimicking pcp. hilar lymphadenopathy and pleural effusion may also be produced by pulmonary kaposi sarcoma or lymphoma, with which m. tuberculosis may coexist. the tuberculin skin test is usually negative, and spontaneously expectorated sputum and bal fluid are often smear negative (but culture positive). in addition to pulmonary tuberculosis, extrapulmonary disease occurs in a high proportion of hiv-infected individuals with low cd4 lymphocyte counts (<150 cells/ml). mycobacteremia and lymph node infection ( figure 34 -6) are common, but involvement of bone marrow, liver, pericardium, meninges, and brain also occurs. evidence of extrapulmonary tuberculosis should be sought in any hiv-infected patient with suspected or confirmed pulmonary tuberculosis, by culture of stool, urine, and blood or bone marrow. traditional solid phase culture and speciation techniques may take 6-10 weeks. liquid culture methods (e.g., bactec, becton dickinson) that detect early growth may provide a diagnosis in only 2-3 weeks. molecular diagnostic tests that use m. tuberculosis genome detection (e.g., by polymerase chain reaction [pcr] ) offer the possibility of yet more rapid diagnosis (within hours), but are not yet in routine clinical use. they are also less useful in primary samples with low bacterial load (e.g., smear negative sputum)-which is often when they will be most needed in hiv-coinfected patients. the recent description of simple, but highly sensitive and specific, methods that use the inoculation of large quantities of, for example, sputum onto microscopic plates with subsequent rapid detection (in days) of both mycobacterial growth and resistance patterns (mods) is of great potential significance. until the results of culture and speciation are known, acidfast bacilli identified in respiratory samples, biopsy tissue, an aspirate, or blood in an hiv-infected individual, regardless of the cd4 lymphocyte count, should be regarded as being m. tuberculosis, and conventional antituberculosis therapy should be commenced. if culture fails to demonstrate m. tuberculosis and instead another mycobacterium (see later) is identified, treatment can be modified. multiple drug-resistant (mdr) tuberculosis-that is, m. tuberculosis that is resistant to isoniazid and rifampicin (rifampin), with or without other drugs, is now an important clinical problem in hiv-infected individuals in the united states, where it is responsible for approximately 3% of all tuberculosis in hiv-infected patients. outbreaks of mdr tuberculosis have occurred in both hiv-infected and non-hiv-infected individuals in the united states in prison facilities, hostels, and hospitals. similar outbreaks have also been documented among hiv-infected patients in europe. inadequate treatment (including case management and supervision of medication) of tuberculosis and poor patient compliance with antituberculosis therapy are the most important risk factors for development of mdr tuberculosis. other cases have arisen because of exogenous reinfection of profoundly immunosuppressed hiv-infected patients who are already receiving treatment for drug-sensitive disease. despite antituberculosis therapy, the median survival in hiv-infected individuals with mdr-tuberculosis was initially only 2-3 months. recently this has improved, largely because of an increased awareness of the condition with early initiation of suitable therapy as determined by drug sensitivity testing. extensively drug-resistant (xdr) tuberculosis-that is, m. tuberculosis resistant to isoniazid and rifampicin (rifampin), plus any fluoroquinolone and one or more of the three injectable second-line drugs (capreomycin, kanamycin and amikacin)-is an increasingly important clinical problem. originally described in south africa in association with hiv infection, xdr tuberculosis has also been identified in most parts of the world. as of march 2007, 35 countries had reported at least one case; although in many places, testing for fluoroquinolone sensitivity is not standard practice; this number may be, in fact, a huge underestimate. what is of concern about xdr tuberculosis is that, despite specific therapy, mortality is high among hiv-infected individuals. the current picture seems to mirror early reports of mdr tuberculosis in hiv infection: in the original south african study from kwazulu natal, survival was less than 3 weeks from the time of receipt of the first sputum sample. mycobacterium avium-intracellulare complex. before the widespread availability of haart, disseminated mac infection developed in up to 50% of hiv-infected patients. it remains a problem in patients with advanced hiv disease not receiving antiretroviral therapy and who have cd4 lymphocyte counts <50 cells/ml. clinical presentation is nonspecific and may be confused with the effects of hiv itself. fever, night sweats, weight loss, anorexia, and malaise are common. anemia, hepatosplenomegaly, abdominal pain, and chronic diarrhea are frequent findings. the diagnosis of disseminated mac infection is based on culture of the organism from blood, bone marrow, lymph node, or liver biopsy specimens. also, mac is frequently identified in bal fluid, sputum, stool, and urine, but detection of the organism at these sites is not diagnostic of disseminated infection. evidence of pulmonary mac infection is not usually obtained from a chest radiograph, which may be negative or show nonspecific infiltrates. rarely, focal consolidation, nodular infiltrates, and apical cavitation (resembling m. tuberculosis) have been reported. mycobacterium kansasii. mycobacterium kansasii is the second most common nontuberculous opportunistic mycobacterial infection in hiv-infected individuals and usually appears late in the course of hiv infection in patients with cd4 lymphocyte counts <100 cells/ml. the most frequent presentation is with fever, cough, and dyspnea. in approximately two thirds of those who have m. kansasii infection, the disease is localized to the lungs; the remainder have disseminated disease that affects bone marrow, lymph node, skin, and lungs. the diagnosis is made by culture of the organism from respiratory secretions or from bone marrow, lymph node aspirate, or skin biopsy. focal upper lobe infiltrates with diffuse interstitial infiltrates are the most common radiographic abnormalities; thin-walled cavitary lesions and hilar adenopathy have also been reported. mycobacterium xenopi. mycobacterium xenopi may occasionally be isolated from sputum or bal fluid samples, but its significance is uncertain. patients have low cd4 counts, and m. xenopi is usually accompanied by isolation of a copathogen, such as p. jirovecii. treatment of the latter condition is associated in most cases with resolution of symptoms. there is some evidence that starting haart prevents disease recurrence, provided there is an adequate immune response. pneumocystis jirovecii pneumonia. the development of pcp is largely related to underlying states of immunosuppression induced by malignancy or treatment thereof, organ transplantation, or hiv infection. in 2007 in the united states, united kingdom, europe, and australasia, pcp is largely seen only in hiv-infected individuals unaware of their serostatus or in those who are intolerant of, or noncompliant with, anti-p. jirovecii prophylaxis and haart. until recently, p. jirovecii was regarded taxonomically as a protozoan, on the basis of its morphology and the lack of response to antifungal agents such as amphotericin b. the organism has now been ascribed to the fungal kingdom. the demonstration of antibodies against p. jirovecii in most healthy children/adults suggests that organisms are acquired in childhood and persist in the lungs in a dormant phase. subsequent immunosuppression (e.g., as a result of hiv infection) allows the fungus to propagate in the lung and cause clinical disease. however, this "latency" hypothesis is challenged by several observations: p. jirovecii cannot be identified in the lungs of immunocompetent individuals. "case clusters" of pcp in health care facilities suggest recent transmission. different genotypes of p. jirovecii are identified in each episode in hiv-infected patients who have recurrent pcp. genotypes of p. jirovecii in patients who have pcp correlate with place of diagnosis and not with their place of birthsuggesting infection has been recently acquired. taken together, these data suggest that pcp arises by reinfection from an exogenous source. the clinical presentation of pcp is nonspecific, with an onset of progressive exertional dyspnea over days or weeks, together with a dry cough with or without expectoration of minimal quantities of mucoid sputum. patients often complain of an inability to take a deep breath, which is not due to pleurisy (table 34-5) . fever is common, yet patients rarely complain of temperatures or sweats. in hiv-infected patients, the presentation is usually more insidious than in patients receiving immunosuppressive therapy, with a median time to diagnosis from onset of symptoms of more than 3 weeks in those with hiv compared with less than 1 week in non-hiv-infected patients. in a small proportion of hiv-positive individuals, the disease course of pcp is fulminant, with an interval of only 5-7 days between onset of symptoms and progression to development of respiratory failure. in others, it may be much more indolent, with respiratory symptoms that worsen almost imperceptibly over several months. rarely, pcp may present without respiratory symptoms as a fever of undetermined origin. clinical examination is usually remarkable only for the absence of physical signs; occasionally, fine, basal, end-inspiratory crackles are audible. features that would suggest an alternative diagnosis include a cough productive of purulent sputum or hemoptysis, chest pain (particularly pleural pain), and signs of focal consolidation or pleural effusion (see table 34 -5). it should be noted that infection with more than one pathogen occurs in almost one fifth of individuals, and thus symptoms may be the product of several agents. the chest radiograph in pcp is typically unremarkable initially. later, diffuse reticular shadowing, especially in the perihilar regions, is seen and may progress to diffuse alveolar consolidation that resembles pulmonary edema if untreated or if the patient is seen late in disease. at this stage, the lung may be massively consolidated and almost airless (figure 34-7) . up to 20% of chest radiographs are atypical, showing lobar consolidation, honeycomb lung, multiple thin-walled cystic air space formation (pneumatoceles), intrapulmonary nodules, cavitary lesions, pneumothorax, and hilar and mediastinal lymphadenopathy. predominantly apical changes, resembling tuberculosis, may occur in patients who have pcp develop having received anti-p. jirovecii prophylaxis with nebulized pentamidine (figure 34 -8). all these radiographic changes chest radiograph early: perihilar "haze" or bilateral interstitial shadowing late: alveolar-interstitial changes or "white out" (marked alveolar consolidation with sparing of apices and costophrenic angles) arterial blood gases pao 2 : early, normal: late, low paco 2 : early, normal or low; late, normal or high are nonspecific, and similar changes occur with other pulmonary pathogens, including pyogenic bacterial, mycobacterial, and fungal infection, as well as kaposi sarcoma and nonspecific interstitial pneumonitis. respiratory symptoms in an immunosuppressed, hiv-infected individual with a negative chest radiograph should not be discounted, because over an interval of 2-3 days radiographic abnormalities may appear. the diagnosis of pcp is made by demonstration of the organism in induced sputum, bal fluid, or lung biopsy material by use of histochemical or immunofluorescence techniques. the early promise of molecular diagnostic techniques has not been borne out. many fungal infections of the lung are confined to specific geographic regions, although with widespread travel, they may present in patients outside these areas. candida, aspergillus, and cryptococcus species are ubiquitous and occur worldwide. in contrast to infections of the oropharynx and esophagus, candidal infection of the trachea, bronchi, and lungs is rare in hiv-infected patients, as are candidemia, disseminated candidiasis, and deep focal candidiasis. the clinical presentation of pulmonary candidal infection has no specific features. chest radiography is equally nonspecific-it may be negative or show patchy infiltrates. isolation of candida from sputum may simply represent colonization and does not mean the patient has candidal pneumonia. indirect evidence may be obtained from positive cultures or rising antibody titers. however, in hivinfected patients, a high antibody titer alone is a less reliable indicator, and antibodies may be absent in proven cases of invasive candidal infection. some correlation occurs between identification of large quantities of candida species in bal fluid and candida species as the cause of pneumonia. definitive diagnosis is made by lung biopsy. by contrast with patients immunosuppressed and rendered neutropenic by systemic chemotherapy, infection with aspergillus species is relatively rare in hiv-positive individuals. risk factors for aspergillosis are neutropenia, which is commonly drug induced (zidovudine or ganciclovir), or patient's receipt of corticosteroids. fever, cough, and dyspnea are the most common presenting symptoms, but pleuritic chest pain and hemoptysis are found in approximately one third of patients. patterns of pulmonary disease include cavitating upper lobe disease, focal radiographic opacities resembling bacterial pneumonia, bilateral diffuse and patchy opacities (nodular or reticular-nodular in pattern), pseudomembranous aspergillosis, which may obstruct the lumen of airways, and tracheobronchitis. diagnosis of pulmonary aspergillosis is made by the identification of fungus in sputum, sputum casts, or bal fluid associated with respiratory tract tissue invasion ( figure 34 -9). the role of antigen testing (such as galactomannan assays), which is commonplace in hematology patients at risk of invasive aspergillus, has not been clearly defined in hiv-infected individuals. infection may present in one of two ways: either as primary cryptococcosis or complicating cryptococcal meningitis as part of disseminated infection with cryptococcemia, pneumonia, and cutaneous disease (umbilicated papules mimicking molluscum contagiosum; figure 34 -10). primary pulmonary cryptococcosis presents in a very nonspecific way and is frequently indistinguishable from other pulmonary infections. in disseminated infection, the presentation is frequently overshadowed by headache, fever, and malaise (caused by meningitis). the duration of onset may range from only a few days to several weeks. examination may reveal skin lesions, lymphadenopathy, and meningism. in the chest, signs may be absent or crackles may be audible. arterial blood gas tensions may be normal or show hypoxemia. the most common abnormality on the chest radiograph is focal or diffuse interstitial infiltrates. less frequently, masses, mediastinal or hilar lymphadenopathy, nodules, and effusion are noted. the diagnosis of cryptococcal pulmonary infection ( figure 34 -11) is made by identification of cryptococcus neoformans (by staining with india ink or mucicarmine, and by culture) in sputum, bal fluid, pleural fluid, or lung biopsy. cryptococcal antigen may be detected in serum by use of the cryptococcal latex agglutination (crag) test. titers are usually high but may be negative in primary pulmonary cryptococcosis, in which case bal fluid (crag) is positive. in patients with disseminated infection, c. neoformans may also be cultured from blood and cerebrospinal fluid. the mortality rate is high in this disseminated form (up to 80%). the endemic mycoses caused by histoplasma capsulatum, coccidioides immitis, and blastomyces dermatitidis are found in hiv-infected patients living in north america (especially the mississippi and ohio river valleys). histoplasmosis is also found in southeast asia, the caribbean islands, and south america. coccidioidomycosis is endemic in the southwest united states (southern california), northern mexico, and in parts of argentina and brazil. blastomycosis has a similar distribution, with an extension north into canada. progressive, disseminated histoplasmosis in patients with hiv typically presents with a subacute onset of fever and weight loss; approximately 50% of patients have mild respiratory symptoms with a nonproductive cough and dyspnea. hepatosplenomegaly is frequently found on examination, and a rash (similar to that produced by cryptococcus species) may be seen. rarely, the presentation may be rapidly fulminant, with clinical features of the sepsis syndrome, including anemia or disseminated intravascular coagulation. the chest radiograph may be unremarkable (in up to one third of patients), although characteristic abnormalities are bilateral, widespread nodules 2-4 mm in size. other radiographic features are nonspecific and include interstitial infiltrates, reticular nodular shadowing, and alveolar consolidation. histoplasmosis may disseminate to the central nervous system and produce meningoencephalitis or mass lesions. the diagnosis is made reliably by identification of the organism in wright-stained peripheral blood or by giemsa staining of bone marrow, lymph node, skin, sputum, bal fluid, or lung tissue. it is important that identification is confirmed by detection of h. capsulatum var. capsulatum polysaccharide antigen by radioimmunoassay, which has a high sensitivity. false-positive results may occur in patients infected with blastomycosis and coccidioides species. tests for histoplasma antibodies by complement fixation or immunodiffusion may be negative in immunosuppressed, hiv-positive patients. the clinical presentation of coccidioidomycosis is variable. the chest radiograph may show focal pulmonary disease with focal alveolar infiltrates, adenopathy, and intrapulmonary cavities or, alternately, diffuse reticular infiltrates. diagnosis is made by isolation of the organism in sputum or bal fluid. disseminated disease is identified by isolating the fungus in blood, urine, or cerebrospinal fluid. serologic tests may also be used for diagnosis. blastomycosis presents in patients who have advanced hiv infection, when cd4 lymphocyte counts are usually less than 200 cells/ml. clinical symptoms include cough, fever, dyspnea, and weight loss. patients may present late in respiratory failure. disseminated disease can occur with both pulmonary and extrapulmonary features. there is frequently multiple involvement of the skin, liver, brain, and meninges. chest radiographic abnormalities include focal pneumonic change, miliary shadowing, or diffuse interstitial infiltrates. diagnosis is made by culture from bal fluid, skin, and blood. in this infection, cytologic or histologic diagnosis is important for early diagnosis, because culture of the organism may take 2-4 weeks. the mortality rate is high in patients with disseminated infection. p. marneffei infection is particularly common in southeast asia. most hiv-infected patients present with disseminated infection and solitary skin or oral mucosal lesions, or with multiple infiltrates in the liver or spleen, or bone marrow (leading to presentation with pancytopenia). pulmonary infection has no specific clinical features, and chest radiographs may be negative or show diffuse, small nodular infiltrates. diagnosis is made by identifying the organism in bone marrow, skin biopsy samples, blood films, or bal fluid. the differential diagnosis of p. marneffei infection includes both pcp and tuberculosis. these occur with equal frequency in hiv-infected and non-hiv-infected patients; however, respiratory complications after influenza infection are increased in patients affected with underlying conditions such as cardiac or pulmonary disease and immunosuppression. in prospective studies of hivinfected patients undergoing bronchoscopy for evaluation of suspected lower respiratory tract disease, the communityacquired respiratory viral infections (i.e., influenza, parainfluenza, respiratory syncytial virus, rhinovirus, coronavirus and adenovirus) are found only rarely, if at all. cmv chronically infects most hiv-infected individuals, and up to 90% of homosexual hiv-infected men shed cmv intermittently in urine, semen, and saliva. clinical disease may be caused by cmv in patients who have advanced hiv infection and cd4 counts <100 cells/ml. chorioretinitis is most frequently encountered, but encephalitis, adrenalitis, esophagitis, and colitis are also seen. frequently, cmv is isolated from bal fluid, being found in 40% of samples from patients with cd4 counts <100 cells/ml. however, the role of cmv in causing disease in this context is unclear (see later). in patients who have cmv as the sole identified pathogen, clinical presentation and chest radiographic abnormalities (usually diffuse interstitial infiltrates) are nonspecific. diagnosis of cmv pneumonitis is made by identifying characteristic intranuclear and intracytoplasmic inclusions, not only in cells in bal fluid but also in lung biopsy specimens (figure 34-12 ). pulmonary involvement with leishmania species may rarely occur as part of the syndrome of visceral leishmaniasis in hiv-infected patients. patients usually have advanced hiv disease with cd4 lymphocyte counts less than 300 cells/ml and present with unexplained fever, splenomegaly, and leukopenia. respiratory symptoms are often absent. diagnosis of visceral leishmaniasis is most often made by staining a splenic or bone marrow aspirate and subsequent culture. occasionally, the parasite is found by chance in a skin or rectal biopsy or bal fluid taken for other purposes. the chest radiograph may be negative or show reticular-nodular infiltrates. toxoplasma gondii infection in patients who have aids usually occurs as a result of reactivation of latent, intracellular protozoa acquired in a primary infection. patients are invariably systemically unwell, with malaise and pyrexia. clinical disease in association with hiv infection is most commonly seen in the central nervous system, where it produces single or multiple abscesses. multisystem infection with t. gondii is uncommon in patients who have hiv infection. toxoplasmic pneumonia is frequently difficult to distinguish from pcp. nonproductive cough and dyspnea are the symptoms most commonly reported. chest radiographic abnormalities include diffuse interstitial infiltrates indistinguishable from those of pcp ( figure 34-13) , as well as micronodular infiltrates, a coarse nodular infiltrate, cavitary change, and lobar consolidation. the diagnosis is made by hematoxylin-eosin or giemsa staining of bal fluid that reveals cysts and trophozoites of t. gondii. staining of bal fluid is not always positive; the diagnostic yield is increased either by staining of transbronchial biopsy material or by performing nucleic acid amplification procedures such as pcr to detect t. gondii dna in bal fluid. the most frequent manifestation of infection with cryptosporidium species in hiv infection is a noninflammatory diarrhea that may be of high volume, intractable, and life threatening. cryptosporidium species may colonize epithelial surfaces, including the trachea and lungs, occasionally resulting in pulmonary infection. most cases of pulmonary cryptosporidiosis have co-pathology such as pcp or bacterial pneumonia; ascertaining the exact role of cryptosporidiosis as the cause of respiratory symptoms may be difficult. diagnosis is made by ziehl-neelsen or auramine-rhodamine staining of bal fluid or transbronchial biopsy specimens. pulmonary microsporidia infection may occur as part of systemic dissemination from gastrointestinal infection with septata intestinalis or encephalitozoon hellem. the organism may be identified by conventional staining in bal fluid. electron microscopy is necessary to distinguish the two species. the nematode strong yloides stercoralis is endemic in warm countries worldwide. in immunosuppressed patients, the organism has an increased ability to reproduce parthenogenetically in the gastrointestinal tract without the need for repeated exposure to new infection-so-called autoinfection. this results in a great increase in worm load and a hyperinfective state ensues; massive acute dissemination with s. stercoralis may occur in the lungs, kidneys, pancreas, and brain. although infection with s. stercoralis is more severe in immunocompromised patients, it is no more common in patients who have hiv infection. presentation with hyperinfection may be with fever, hypotension secondary to bacterial sepsis, or disseminated intravascular coagulation. the clinical features of respiratory s. stercoralis infection are nonspecific. s. stercoralis in sputum or bal fluid (figure 34 -14) may be identified in hiv-positive patients in the absence of symptoms elsewhere; this can predate disseminated infection and, as such, requires prompt treatment. it is apparent from the foregoing discussion that hiv-related pneumonia of any cause may present in a similar manner. a wide range of investigations is available to aid diagnosis. these are listed in table 34 -6. if the subject is producing sputum, it is important to obtain samples for bacterial and mycobacterial detection. in up to one third of cases, these will assist in diagnosis. three samples on consecutive days (preferably either with overnight or early morning production) is the critical first step in the diagnosis of pulmonary tuberculosis. this is considerably easier and safer for health care personnel than obtaining hypertonic saline-induced sputum or bal fluid. blood cultures are also important, because very high rates of bacteremia have been reported in both bacterial and mycobacterial disease (see earlier). a patient who is initially seen with symptoms and signs consistent with pneumonia should have chest radiography and arterial oxygen assessments performed at his or her first consultation. the question at this stage is usually whether this infectious episode is due to bacterial infection, tuberculosis, or pcp. in general, alveolar and interstitial shadowing is taken as evidence for pcp, although important caveats apply. transcutaneous pulse oximetry and arterial blood gas analysis are useful tests for hypoxemia. they can be used to distinguish an alveolar condition (i.e., pcp) from bacterial pneumonia. the alveolitis produces a greater impairment of oxygen transfer (especially during exercise), such that for a given clinical situation there will be more hypoxemia and a wider alveolararterial oxygen gradient (a-ao 2 ) in those with pcp. with pulse oximetry, this manifests as low oxygen saturations at rest that decrease further with exercise. in general, more information can be obtained from arterial blood gas analysis, although this advantage is offset by the need for direct arterial puncture. of patients with pcp, fewer than 10% have a normal pao 2 and a normal a-ao 2 . these measures are sensitive, although not particularly specific for pcp, and similar results may occur with bacterial pneumonia, pulmonary kaposi sarcoma, and m. tuberculosis infection. the diagnostic value of identifying exercise-induced desaturation, measured by transcutaneous oximetry, has been validated only in hiv-infected patients who have pcp and a normal or "near normal" appearance on chest radiographs. the test's value has not been confirmed in patients with abnormal chest radiographs because of pcp or other pathogens. exercise-induced desaturation may persist for many weeks after treatment and recovery from pcp, even in the absence of active pulmonary disease. abnormalities of lung function are well documented with hiv infection. the most common of these relate to tests measuring gas exchange, rather than the size of the conducting airways. in general, an overall reduction in diffusing capacity for carbon monoxide (dlco) occurs at all stages of hiv infection, with the largest changes found in hiv-infected patients with pcp. thus, to some extent, patients who have probable pcp can be differentiated and treatment guided. a normal dlco in an individual who has symptoms but a negative or unchanged chest radiograph makes the diagnosis of pcp extremely unlikely. data from the north american pspc cohort study suggest that individuals with rapid rates of decline in dlco are at an increased risk for development of pcp. recent work suggests that hiv-infected smokers are at increased risk of early-onset emphysema. smoking history must, therefore, be taken into account when assessing a patient's lung function results in the context of possible pcp. high-resolution (fine-cut) ct scanning of the chest may be helpful when the chest radiograph is normal, unchanged, or equivocal. the characteristic appearance of an alveolitis (i.e., areas of ground-glass attenuation through which the pulmonary vessels can be clearly identified) may be present, which indicates active pulmonary disease (figure 34-15 ). this feature, however, is neither sensitive nor specific for pcp, although its sensitivity can be improved if evidence for reticulation and/or small cystic lesions is added to this. hence, a negative test result implies an alternative diagnosis. in the context of an hiv-infected patient who is seen with an acute or subacute pneumonitis, an elevated serum lactate dehydrogenase (ldh) enzyme level is strongly suggestive of pcp. when interpreting such results, it is important to remember that other pulmonary disease processes (e.g., pulmonary embolism, nonspecific pneumonitis, and bacterial and mycobacterial pneumonia) and extrapulmonary disease (castleman disease and lymphoma) may also cause elevations of ldh and may need to be considered in the correct clinical context. from the previous information, it is evident that noninvasive tests cannot reliably distinguish the different infecting agents from each other but may be useful in excluding acute opportunistic disease. thus, the clinician is left with either proceeding to diagnostic lung fluid or tissue sampling (by either induced sputum collection or bronchoscopy and bal with or without transbronchial biopsy table 34-7) or treating an unknown condition empirically. haart has also altered the investigation of respiratory disease. the numbers of invasive procedures performed are falling and tend to be in patients spontaneously expectorated sputum is inadequate for diagnosis of pcp. sputum induction by inhalation of ultrasonically nebulized hypertonic saline may provide a suitable specimen (see table 34 -7). the technique requires close attention to detail and is much less useful when samples are purulent. sputum induction must be carried out away from other immunosuppressed patients and health care workers, ideally in a room with separate negative-pressure ventilation, to reduce the risk of nosocomial transmission of tuberculosis. although very specific (>95%), the sensitivity of induced sputum varies widely (55-90%), and therefore a negative result for p. jirovecii prompts further diagnostic studies. the use of immunofluorescence staining enhances the yield of induced sputum compared with standard cytochemistry. fiberoptic bronchoscopy with bal is commonly used to diagnose hiv-related pulmonary disease. when a good "wedged" sample is obtained, the test has a sensitivity of more than 90% for detection of p. jirovecii (figure 34-16 ). just as with induced sputum, fluorescent staining methods increase the diagnostic yield, which makes it the procedure of choice in most centers. more technically demanding (both of the patient and the operator) than induced sputum collection, bronchoscopy and bal have the advantage that direct inspection of the upper airway and bronchial tree can be performed and, if necessary, biopsies taken. transbronchial biopsies may marginally increase the diagnostic yield of the procedure. this is relevant for the diagnosis of mycobacterial disease, although the relatively high complication rate in hiv-infected individuals (pneumothorax and the possibility of significant pulmonary hemorrhage in up to 10%) outweighs the advantages of the technique for routine purposes. samples of bal fluid are examined for bacteria, mycobacteria, viruses, fungi, and protozoa. inspection of the cellular component may also provide etiologic clues-cooperation of a pathology department with experience in opportunistic infection diagnosis is vital. the drug interactions associated with antiretroviral protease inhibitor (pi) therapy mean that special care should be exercised when sedation is used with either benzodiazepine or opiate drugs. prolonged sedation and life-threatening arrhythmias have been reported. a diagnostic strategy, therefore, includes sputum induction and, if results are nondiagnostic or if the test is unavailable, bronchoscopy and bal. if this does not yield a result, consideration is given to either a repeat bronchoscopy and bal with transbronchial biopsies or surgical biopsy. the latter can be performed as either an open lung or thoracoscopic procedure. surgical biopsy has a high sensitivity. although empirical therapy is usually reserved for the management of presumed bacterial pneumonias, and at first sight may seem unwise when dealing with possible opportunistic infection, in reality pcp is almost invariably a diagnosis of exclusion, and certain clinical and laboratory features may guide the assessment of an hiv-infected individual's risk for this condition. the likelihood that p. jirovecii is the causative organism increases if the subject is not taking effective anti-pneumocystis drug prophylaxis or has a previous medical history with clinical or laboratory features that suggest systemic immunosuppression (i.e., recurrent oral thrush, longstanding fever of unknown cause, clinical aids, or blood cd4 count <200 cells/ml). hence, some centers advocate use of empirical therapy for hiv-infected patients who are seen with symptoms and chest radiographic and blood gas abnormalities typical of mild pcp, without the need for bronchoscopy. invasive measures are reserved for those with an atypical radiographic presentation, those who fail to respond to empirical therapy by day 5, and those who deteriorate at any stage. most clinicians in north america and the united kingdom would seek to obtain a confirmed diagnosis in every case of suspected pcp. in practice, both strategies seem to be equally effective, although a number of caveats should be borne in mind when empirical treatment is given for pcp. patients who have pcp typically take 4-7 days to show clinical signs of improvement, so a bronchoscopically proven diagnosis ensures that the treatment being given is correct, particularly in the first few days of therapy, when it may not be well tolerated. in addition, the diagnosis of pcp has implications for the infected individual, because it may influence the decision to start either haart or anti-pneumocystis prophylaxis. finally, empirical therapy requires the patient to be maximally adherent to treatment, because nonresolution of symptoms may be seen as failure of therapy rather than of compliance. molecular biologic techniques (such as pcr) are increasingly used in the diagnosis of respiratory disease. two examples of this are dna amplification of loci of the p. jirovecii and m. tuberculosis genomes. the advantages of molecular methods are that the diagnosis may be made by use of samples that are more readily obtained than bal fluid (i.e., expectorated sputum or nasopharyngeal secretions) and also that these methods are rapid (the answer may be available within a working day, compared with conventional mycobacterial culture, which may take weeks). despite encouraging results in the research setting (sensitivity and specificity have been reported as 60-100% and 70-100%, respectively), problems persist when these techniques are applied to routine diagnostic samples. these include extraction of nucleic acid from clinical material, cross-contamination with the products of previous assays, and clinical interpretation of a test result. currently, treatment individuals infected with hiv, compared with the non-hivinfected general population, have an increased likelihood of adverse reactions to therapy. this includes tmp/smx (see later) and other antibacterial and antimycobacterial agents. in addition, there are complex drug interactions with other medications, particularly components of haart. before instituting therapy for any infectious complication in an hivinfected individual, it is important to consult with a physician experienced in the care of patients with hiv infection and to seek advice from a specialist pharmacist. the main organisms causing pneumonia in hiv-infected individuals are similar to those found in the general population with community-acquired pneumonia. thus, bacterial pneumonia in hiv-infected patients should be treated in a similar manner to that in hiv-negative individuals, by use of the published american thoracic society (ats) and british thoracic society (bts) guidelines. in addition, expert advice on local antibiotic resistance patterns should be sought from infectious disease or microbiology colleagues, because treatment is usually begun on an empirical basis before the causative organism is identified and antibiotic sensitivities known. the same clinical and laboratory prognostic indices that are described for the general population apply to hiv-infected patients and should be documented on presentation. response to appropriate antibiotic therapy is usually rapid and is similar to that seen in the non-hiv-infected individual. early relapse of infection after successful treatment is well described. those hiv-infected patients who have presumed pcp and are being treated empirically with high-dose tmp/ smx, and who have infection with either s. pneumoniae or h. influenzae rather than p. jirovecii, may also improve. in addition, in those patients who are treated with benzylpenicillin for proven s. pneumoniae pneumonia but do not respond, and penicillin resistance can be discounted as the cause, it is important to consider whether there is a second pathologic process, such as pcp. co-pathogens are reported in up to 20% of cases of pneumonia. before instituting treatment, assessment of the severity of pcp should be performed on the basis of history, findings on examination, arterial blood gas estimations, and chest radiographic abnormalities. patients can then be stratified into those with mild, moderate, or severe disease (table 34-8) . this is important, because some drugs are of unproven benefit and others are known to be ineffective for the treatment of severe disease. in addition, adjuvant glucocorticoid therapy may be given to patients with moderate or severe pneumonia. patients with glucose-6-phosphate dehydrogenase deficiency should not receive tmp/smx, dapsone, or primaquine, because these drugs increase the risk of hemolysis. several drugs are effective in the treatment of pcp. tmp/ smx is the drug of first choice (tables 34-9 and 34-10). overall it is effective in 70-80% of individuals when used as firstline therapy. adverse reactions to tmp/smx are common and usually become apparent between days 6 and 14 of treatment. neutropenia and anemia (in up to 40% of patients), rash and fever (up to 30%), and biochemical abnormalities of liver function (up to 15%) are the most frequent adverse reactions. hematologic toxicity induced by tmp/smx is neither attenuated nor prevented by coadministration of folic or folinic acid. furthermore, the use of these agents may be associated with reduced therapeutic success. during treatment with tmp/ smx, full blood count, liver function, and urea and electrolytes should be monitored at least twice weekly. it is not known why hiv-infected individuals, especially those with higher cd4 counts, have such a high frequency of adverse reactions to tmp/smx. the optimum strategy for an hiv-infected patient who has pcp and who becomes intolerant of high-dose tmp/smx has not been established. many physicians "treat through" minor rash, often adding an antihistamine and a short course of oral prednisolone (30 mg every 24 h, reducing to zero over 5 days). if treatment with tmp/smx fails, or is not tolerated by the patient, several alternative therapies are available (see tables 34-9 and 34-10). the combination of clindamycin and primaquine is widely used for treatment of pcp whatever the severity, although there is no license in the united kingdom or united states for this indication. the combination is as effective as oral tmp/smx and oral trimethoprim-dapsone for the treatment of mild and moderate-severity disease. as a second-line treatment it is effective in up to 90% of patients. methemoglobinemia caused by primaquine occurs in up to 40% of patients. if 15 mg four times daily of primaquine is used, rather than 30 mg four times daily, the likelihood of methemoglobinemia is reduced. diarrhea develops in up to 33% of patients receiving clindamycin. if this occurs, stool samples should be analyzed for the presence of clostridium difficile toxin. this oral combination is as effective as oral tmp/smx and oral clindamycin plus primaquine (see earlier) for treatment of mild and moderate-severity pcp. the combination has not been shown to be effective in patients who have severe pcp. most patients experience methemoglobinemia (caused by dapsone), which is usually asymptomatic. up to one half of patients have mild hyperkalemia (<6.1 mmol/l) caused by trimethoprim. a methotrexate analog, trimetrexate, is given intravenously together with folinic acid "rescue" to protect human cells from trimetrexate-induced toxicity. in patients who have moderate regimen recommended by cdc/ nih/idsa, widely used in usa methylprednisolone iv at 75% of dose given above for prednisolone methylprednisolone prednisolone 1 iv q24h, days 1-3 0.5 iv days 4-6 then 40 g po q24h reducing to 0, days 7-16 nb, none of these regimens for adjuvant glucocorticoid therapy have been compared in prospective clinical trials. to severe disease, trimetrexate-folinic acid is less effective than high-dose tmp/smx, but serious treatment-limiting hematologic toxicity occurs less frequently with trimetrexate-folinic acid. atovaquone is licensed for the treatment of mild and moderate-severity pcp in patients who are intolerant of tmp/ smx. in tablet formulation (no longer available), this drug was less effective but was better tolerated than tmp/smx or intravenous pentamidine for treatment of mild or moderateseverity pcp (see tables 34-9 and 34-10). there are no data from prospective studies that compare the liquid formulation (which has better bioavailability) with other treatment regimens. common adverse reactions include rash, fever, nausea and vomiting, and constipation. absorption of atovaquone is increased if it is taken with food. intravenous pentamidine is now seldom used for the treatment of mild or moderate-severity pcp because of its toxicity. intravenous pentamidine may be used in patients who have severe pcp, despite its toxicity, if other agents have failed (see tables 34-9 and 34-10). nephrotoxicity develops in almost 60% of patients given intravenous pentamidine (indicated by elevation in serum creatinine), leukopenia develops in approximately half, and up to 25% have symptomatic hypotension or nausea and vomiting. hypoglycemia occurs in approximately 20% of patients. given the long half-life of the drug, this may occur up to several days after the discontinuation of treatment. pancreatitis is also a recognized side effect. for patients who have moderate and severe pcp, adjuvant glucocorticoid therapy reduces the risk of respiratory failure by up to half and the risk of death by up to one third (see tables 34-9 and 34-10). glucocorticoids are given to hivinfected patients with confirmed or suspected pcp who have a pao 2 <9.3 kpa (<70 mmhg) or an a-ao 2 of >4.7 kpa (<33 mmhg). oral or intravenous adjunctive therapy is given at the same time as (or within 72 h of starting) specific anti-p. jirovecii therapy. clearly, in some patients treatment is commenced on a presumptive basis, pending confirmation of the diagnosis. in prospective studies, adjuvant glucocorticoids have not been shown to be of benefit in patients with mild pcp. however, it would be difficult to demonstrate this, given that survival in such cases approaches 95% with standard treatment. patients with mild pcp may be treated with oral tmp/smx as outpatients if they are able to manage at home, willing to attend the outpatient clinic for regular review, and that there is clinical and radiographic evidence of recovery. if the patient is intolerant of oral tmp/smx despite clinical recovery, either the treatment is given intravenously or treatment may be changed to oral clindamycin plus primaquine. all patients with moderate and severe pcp should be hospitalized and given intravenous tmp/smx or intravenous clindamycin and oral primaquine (plus adjuvant steroids). patients with moderate or severe disease who show clinical and radiographic response by day 7-10 of therapy may be switched to oral tmp/smx to complete the remaining 14 days of treatment. if the patient has failed to respond within 7-10 days or deteriorates before this time while receiving tmp/smx, then treatment should be changed to clindamycin and primaquine or trimetrexate plus folinic acid. deterioration in a patient who is receiving anti-p. jirovecii therapy may occur for several reasons (table 34-11) . before ascribing deterioration to treatment failure and considering a change in therapy, these alternatives should be evaluated carefully. it is also important to consider treating any co-pathogens present in bal fluid, to perform bronchoscopy if the diagnosis was made empirically, to repeat the procedure, or to carry out open lung biopsy to confirm that the diagnosis is correct. most centers advocate admission to the icu for pcp with respiratory failure and for acute severe deterioration after bronchoscopy. the prognosis for severe pcp in such circumstances has improved over the past decade. this is likely because of a greater understanding of successful general icu management of respiratory failure and acute respiratory distress syndrome (ards) rather than specific improvements in pcp care. factors associated with poor outcome include increasing patient age, need for mechanical ventilation, and development of a pneumothorax. the latter reflects both the association between this complication and pcp, as well as the subsequent difficulty in successful mechanical ventilation of such individuals. the treatment of hiv-related mycobacterial disease is complex. not only do individuals have to take prolonged courses of relatively toxic agents, but also these antimycobacterial drugs have side effects similar to those of other prescribed in the developed world, isoniazid-related peripheral neuropathy is rare in hiv-negative subjects taking pyridoxine. the nucleoside reverse transcriptase inhibitors (rti) didanosine and stavudine, which are now less frequently used in the united states and the united kingdom but which remain a mainstay of haart in the developing world, can also cause a painful peripheral neuropathy. this complication develops in up to 30% of patients if stavudine and isoniazid are coadministered. rash, fever, and biochemical hepatitis are common adverse events with rifamycins, pyrazinamide, and isoniazid (occurring more frequently in patients with tuberculosis who have hiv infection with hepatitis c coinfection). the nonnucleoside rti drugs (e.g., nevirapine) have a similar toxicity profile. if treatment for both hiv and tuberculosis is co-administered, ascribing a cause may be problematic. drug-drug interactions between medications used to treat tuberculosis and hiv infection occur because of their common pathway of metabolism through the hepatic cytochrome p-450 enzyme system. rifampin is a potent inducer of this enzyme (rifabutin less so), which may result in subtherapeutic levels of nonnucleoside rti and pi antiretroviral drugs, with the potential for inadequate suppression of hiv replication and the development of resistance to hiv. in addition, the pi class of antiretroviral drugs inhibits the metabolism of rifamycins, which leads to increases in their plasma concentration and is associated with increased drug toxicity. the nonnucleoside rti drugs are inducers of this enzyme pathway. coadministration of rifabutin with efavirenz requires an increase in the dose of rifabutin to compensate for the increase in its metabolism induced by efavirenz (see later). the optimal duration of treatment of tuberculosis, by use of a rifamycin-based regimen, in a patient who has hiv infection is unknown. current recommendations (joint tuberculosis committee of the bts and the ats/cdc/infectious disease society of north america [idsa]) are to treat tuberculosis in hiv-infected patients in the same way as for the general population (i.e., for 6 months for drug-sensitive pulmonary tb). in addition, ats/cdc/idsa guidelines recommend that treatment be extended to 9 months in those who have cavitation on the original radiograph, continuing signs, or a positive culture after 2 months of therapy. recent work has highlighted the increased risk for development of rifampin monoresistance in hiv-infected individuals on treatment. this is especially so if intermittent regimens are used and may arise from a lack of efficacy of the other drugs present in the combination (e.g., intermittent isoniazid). hence, daily medication regimens are recommended and should be closely supervised in all hiv-positive patients. it should be remembered that although rifabutin is usually given three times a week with ritonavir-boosted protease inhibitors, this seems to achieve adequate rifamycin levels; there have been no reports of this leading to rifamycin resistance in patients who are appropriately adherent. directly observed therapy (dot) is an important, although fairly labor-intensive, strategy that has the support of the world health organization. the best time to start therapy in patients being treated for tuberculosis is unknown. decision analyses show that early treatment with antiretroviral therapy leads to a marked reduction in further opportunistic disease. against this is balanced the risk of needing to discontinue antituberculosis therapy or hiv therapy because of drug toxicity or drug-drug interactions. iris is reported to be more likely if the treatments are started at the same time as each other. pragmatically, delaying the start of antiretroviral therapy simplifies patient management and may reduce or prevent adverse drug reactions and drug-drug interactions and may also reduce the risk of iris. on the basis of current evidence, patients with cd4 counts >200 cells/ml have a low risk of hiv disease progression or death during 6 months of treatment for tuberculosis. in these patients, the cd4 count should be closely monitored, and antiretroviral therapy may be deferred until treatment for tuberculosis is completed. in patients who have cd4 counts from 199-100 cells/ml, many centers currently delay starting antiretroviral therapy until after the first 2 months of treatment for tuberculosis have been completed; patients are given concomitant pcp prophylaxis. in patients who have cd4 counts of <99 cells/ml, antiretroviral therapy is started as soon as possible after beginning treatment for tuberculosis. this is based on evidence that shows a significant short-term risk of hiv disease progression and death in this patient group if antiretroviral therapy is delayed. two options exist for starting antiretroviral therapy in a patient already being treated for tuberculosis. first, the rifampin-based regimen is continued, and antiretroviral therapy is commenced, for example, with a combination of two nucleoside rtis and a non-nucleoside rti, such as efavirenz (if the patient weighs <50 kg, the efavirenz dose is often increased to 800 mg once daily to compensate for rifampin-induced metabolism of efavirenz). alternately, the rifampin is stopped and rifabutin is started: antiretroviral therapy is given, with a combination of two nucleoside rti drugs and either a single ritonavir-boosted pi or a nonnucleoside rti. here the dose of rifabutin is adjusted to take into account the pharmacokinetic effect of the co-administered drug. with a boosted pi, it is usually prescribed at a dose of 150 mg three times weekly and with efavirenz it is increased to 450 mg once a day. before the advent of antiretroviral therapy, it was recognized by tuberculosis physicians that patients who were apparently responding to their antimycobacterial treatment would sometimes have a short period of clinical deterioration develop. this "paradoxical reaction" (in the face of overall treatment response) was seen as an interesting and probable immunebased phenomenon of generally little consequence. the widespread introduction of haart has led to an increased awareness by clinicians of similar, but generally more severe, events in hiv-infected individuals. in the context of hiv, these are termed iris, or immune reconstitution disease. they can present in a number of ways and with a range of opportunistic conditions. perhaps the most common of these is similar to a paradoxical reaction. here, after initiation of antiretroviral therapy in a patient being treated for tuberculosis, for example, there arises the return of the original or the development of new symptoms and signs. these are often of a systemic nature and may be associated with marked radiographic changes. examples of this include fever, dyspnea, lymphadenopathy, effusions, parenchymal pulmonary infiltrates, or expansion of cerebral tuberculomas. this form of iris is seen most frequently with mycobacteria (commonly tuberculosis or mac), fungi (notably, cryptococcus), and viruses (hepatitis and herpes viridae). iris develops in up to one third of hiv-infected patients being treated for tuberculosis when antiretroviral therapy is started. the median onset of tuberculosis-related iris is approximately 4 weeks from beginning antituberculosis treatment or 2 weeks from commencing haart. it seems to be more likely in patients who have disseminated tuberculosis (and hence presumably more antigen present as well as more potential for significant inflammatory reactions) and a lower baseline blood cd4 count. a rapid fall in hiv load, as well as a large increase in cd4 counts in response to haart, may also predict iris. the relationship between early use of haart and low blood cd4 counts suggests that care must be taken when starting antiretrovirals in patients with tb at sites where rapid expansion of an inflammatory mass could be life threatening. examples of this would include cerebral, pericardial, or peritracheal disease (figure 34-17) . it is important to note that iris is currently a diagnosis of exclusion. there is no laboratory test available to assist with this; it should be made only after progressive or (multi) drug-resistant tuberculosis, poor drug adherence (to either antituberculosis or antiretroviral agents) and drug absorption, or an alternative pathologic process have been excluded as an explanation for the presentation. criteria have been drawn up that seek to provide clinical diagnostic criteria (table 34-12) . the mechanism leading to iris is unclear. it is not due to failure of treatment of tuberculosis or to another disease process; if anything, it is most likely to represent an exuberant and uncontrolled response to mycobacterial antigens (from both dead and live organisms). current treatments include nonsteroidal antiinflammatory drugs or glucocorticoids. the latter are undoubtedly effective, although they can lead to hyperglycemia and hypertension. recent preliminary data suggest that the leukotriene receptor antagonist montelukast may be of benefit in iris (this drug is unlicensed for this indication). recurrent aspiration of lymph nodes or effusion may also be needed. although iris is often self-limiting, it may persist for several months. rarely, temporary discontinuation of antiretroviral therapy is required. in this situation there may be precipitous falls in cd4 counts; patients are at risk of other opportunistic infections. attention has also focused on what is possibly more of a concern-the form of iris referred to as "unmasking phenomenon." here, individuals with presumably latent tuberculosis infection who start haart have systemic active (and often infectious) tuberculosis develop within a 3-month period. although it is likely that the patient's disease would have presented in time anyway and that some of the reported cases may, in fact, represent ascertainment bias, the current view is that this is real and represents an adverse effect of haart. given that the people most at risk live in countries with limited facilities for pre-haart screening, this has major implications for antiretroviral therapy roll-out programs in resource-poor areas. combination antimycobacterial therapy by itself does not cure mac infection. a commonly used regimen is oral rifabutin, 300 mg once daily, with oral ethambutol, 15 mg/kg once daily, and oral clarithromycin, 500 mg once daily or every 12 h. if clarithromycin is not used, oral rifabutin, 600 mg once daily, is given-the lower dose adjusting for yet more drug-drug interactions. use of three drugs has no impact on overall outcome, although it reduces the risk of resistance and possibly enhances early mycobacterial killing. in patients severely compromised by symptoms, intravenous amikacin, 7.5 mg/kg once daily for 2-4 weeks, is also given. trough blood levels must be measured to ensure toxic accumulation of amikacin does not occur. fluoroquinolones such as moxifloxacin or levofloxacin may be extremely useful, because they have good antimycobacterial activity with limited side effects. at present, many of these agents are not licensed for this indication. given the concerns over xdr tuberculosis, it is important to ensure that patients are adherent to such treatments, and hence reduce the risk of fluoroquinolone resistance developing. a frequently used regimen includes rifampin, isoniazid, and ethambutol in conventional doses; all drugs are given by mouth. the treatment regimens for fungal infections complicating hiv infection are shown in table 34 -13. after initial treatment of cryptococcal infection, there is a high likelihood of relapse of infection; hence, lifelong secondary preventative therapy is needed unless antiretroviral therapy is commenced and results in sustained improvements in cd4 counts (>250 cells/ml) and suppression of hiv load in peripheral blood. secondary prophylaxis is most often oral fluconazole 200-400 mg four times daily. just as with mycobacterial disease, "late" iris events can occur after months or even years. these should be investigated to exclude active disease and other conditions. oral itraconazole, 200 mg twice daily, is the current treatment of choice. the dose is adjusted to achieve blood trough drug levels that are above the standard lowest effective concentration. there are no data on the impact of antiretroviral therapy on which to base decisions about discontinuation of secondary prophylaxis. treatment of this infection is difficult. after initial treatment with amphotericin b, itraconazole or fluconazole may be given for long-term suppression. the overall prognosis is poor, with a 40% mortality rate despite therapy. there are no data on the impact of antiretroviral therapy on which to base decisions about discontinuation of secondary prophylaxis. oral itraconazole has now replaced amphotericin b as the treatment of choice for p. marneffei infection, apart from the subgroup who are acutely unwell. fluconazole is less effective than itraconazole. after initial treatment, lifelong suppressive therapy with itraconazole is needed. there are no data on the impact of antiretroviral therapy on which to base decisions about discontinuation of secondary prophylaxis. the treatment regimens are shown in table 34 -14. a combination of sulfadiazine and pyrimethamine is the regimen of choice for t. gondii infection. the most frequent dose-limiting side effects are rash and fever. adequate hydration must be maintained to avoid the risk of sulfadiazine crystalluria and obstructive uropathy. alternate regimens are given in table 34 -14. once treatment is completed, lifelong maintenance is necessary to prevent relapse, unless antiretroviral therapy achieves adequate immune restoration (blood cd4 count >250 cells/ml and undetectable hiv load). visceral leishmaniasis is usually treated with liposomal amphotericin b, although this is still associated with a high rate of relapse. second-line therapy (or first-line in resourcepoor environments) is to use sodium stibogluconate (see table 34-14) . phenomena temporally associated with starting haart. this includes, but is not limited to: 1. new or enlarging lymphadenopathy, cold abscesses, or other focal tissue involvement 2. new or worsening central nervous system disease 3. new or worsening radiological features of tuberculosis 4. new or worsening serositis (pleural effusion, ascites, pericardial effusion, or arthritis. 5. new or worsening constitutional symptoms such as fever, night sweats, and/or weight loss 6. retrospective review indicating that a clinical or radiologic deterioration occurred with no change having been made to tuberculosis treatment c. immune restoration, e.g., a rise in cd4 lymphocyte count in response to haart d. a fall in hiv "viral load" in response to haart alternative diagnoses to be excluded progressive underlying infection treatment failure due to drug resistance (mdr or xdr) treatment failure from poor adherence adverse drug reaction another diagnosis coexisting (e.g., non-hodgkin lymphoma) the treatment of choice is ivermectin. risk of treatment failure with thiabendazole in hiv-infected individuals is higher than that in non-hiv-infected patients. cytomegalovirus pneumonitis is treated with intravenous ganciclovir, 5 mg/kg every 12 h, for 14 days. drug-induced neutropenia is managed with granulocyte colony-stimulating factor. some centers use valganciclovir, an oral formulation of ganciclovir, at a dose of 900 mg orally every 12 h, to treat cmv pneumonitis. side effects and their management are as for ganciclovir. there are no data that demonstrate efficacy for cidofovir for treatment of cmv pneumonitis, but this agent is used as second-line therapy in many centers. phosphonoformate (foscarnet) can be used for treatment of cmv endorgan disease (e.g., pneumonitis), although it has an extensive toxicity profile. it is also a moderately effective antiretroviral agent. this effect is occasionally used as an adjunct in controlling nonresponsive viral infections. within the past few years, drug therapy has radically altered the depressingly predictable nature of progressive hiv infection. combinations of specific opportunistic infection prophylaxis and antiretroviral therapy can reduce both the incidence and the mortality associated with common conditions. the observational north american macs cohort demonstrated that the risk of pcp in individuals with blood cd4 counts of <100 cells/ml can be reduced almost fourfold if both specific prophylaxis and haart are taken (from 47% to 13%). however, as common conditions are prevented, so other less treatable illnesses may arise. the initial impact of p. jirovecii prophylaxis was a reduction in the incidence of pcp at the expense of an increase in cases of disseminated mac infection, cmv infection, esophageal candidiasis, and wasting syndrome. new prophylactic therapies targeting those conditions associated with high morbidity and mortality (in particular mac) have further improved survival. it has become apparent that specific infection prophylaxis may also confer protection against other agents. this "crossprophylaxis" is particularly seen with the use of tmp/smx for pneumocystis, which also provides cover against cerebral toxoplasmosis and several common bacterial infections (although not s. pneumoniae) and with macrolides for mac infection, which further reduce the incidence of bacterial disease and also pcp. use of large amounts of antibiotic raises the possibility of future widespread drug resistance. this is clearly of concern, and recent reports suggest that, indeed, in some parts of the world the incidence of pneumococcal tmp/smx resistance is rising. current preventive therapies pertinent to lung disease focus on p. jirovecii, mac, m. tuberculosis, and certain bacteria (table 34-15 ). numerous studies have demonstrated the greatly increased risk in subjects who do not take adequate drug therapy with blood cd4 counts <200 cells/ml. clinical symptoms are also an independent risk factor for pcp, and hence the current guidelines recommend lifelong prophylaxis against p. jirovecii in hiv-infected adults who have had prior pcp, cd4 counts <200 cells/ml, constitutional symptoms (documented oral thrush or fever of unknown cause of <37.8 c that persists for more than 2 weeks), or clinical aids. the importance of secondary prophylaxis (i.e., used after an episode of pcp) becomes clear from historical data, which indicate a 60% risk of relapse in the first 12 months after infection. the increase in systemic and local immunity that occurs with haart has led to several studies evaluating the need for prolonged prophylaxis in individuals with sustained elevations in blood cd4 counts and low hiv rna load. in summary, it seems that both primary and secondary pcp prophylaxis can be discontinued once cd4 counts are >200 cells/ml for more than 3 months. a caveat to this is that the patient should have a low or undetectable hiv rna load, that the cd4 percentage is stable or rising and is >14%, and that the individual plans to continue haart long term with good adherence. the risk of pcp recurrence is real if the cd4 count falls below 200 cells/ml. if this does happen, pcp prophylaxis should be restarted. similar algorithms have been successfully used for all the major infections except tuberculosis. they all rely on an estimation of the general blood cd4 count above which clinical disease is highly unlikely. for example, secondary prophylaxis of mac may be discontinued once the blood cd4 count is consistently >100 cells/ml. this is a general guideline, however, and patients must be assessed on an individual basis. as with treatment strategies, tmp/smx is the drug of choice for prophylaxis (table 3416) . it has the advantages of being highly effective for both primary and secondary prophylaxis (with 1-year risk of pcp while on the drug being 1.5 and 3.5%, respectively). it is cheap, can be taken orally, acts systemically, and provides some cross-prophylaxis against other infections, such as toxoplasmosis, salmonella species, staphylococcus species, and h. influenzae. its main disadvantage is that adverse reactions are common (see earlier), occurring in up to 50% of individuals taking the prophylactic dose. the standard dose of tmp/smx is one double-strength tablet (160 mg trimethoprim, 800 mg sulfamethoxazole) per day. other regimens have been tried; these include one "double-strength" tablet three times weekly and one single-strength tablet per day. in general, when used for primary prophylaxis, these regimens are tolerated well (if not better than the standard) and seem as efficacious as one double-strength tablet per day. the data are less clear on secondary prophylaxis, in which subjects are at a much higher risk of recurrent pcp. attempts to desensitize patients who are intolerant of tmp/ smx have met with some success. in patients who cannot tolerate tmp/smx, dapsone is a safe and inexpensive alternative. it has been studied in a number of trials as both primary and secondary prophylaxis and is effective at an oral dose of 100 mg/day. when combined with pyrimethamine (25 mg three times weekly), it provides a degree of cross-prophylaxis against toxoplasmosis. before starting dapsone, patients are tested for glucose-6-phosphate dehydrogenase deficiency. nebulized pentamidine has largely fallen from use as a prophylactic agent. this is despite it being better tolerated and having a similar efficacy to tmp/smx for primary preventive therapy. however, its breakthrough rate is higher in subjects who have lower cd4 counts (i.e., <100 cells/ml) and in those who take it as secondary prophylaxis. other disadvantages include equipment costs and complexity (alveolar deposition is crucial, and hence the nebulizer system used is important), the risk of transmission of respiratory disease (e.g., tuberculosis) to other patients and staff during the nebulization procedure, an alteration in the clinical presentation of pcp while on pentamidine (increased frequency of radiographic upper zone shadowing, increased incidence of pneumothorax), and a lack of systemic protection against pneumocystis and other infectious agents. there is also an acute bronchoconstriction effect during nebulization. long-term follow-up studies have not demonstrated any significant negative effect on lung function. atovaquone oral suspension is used as a second-line prophylactic agent in subjects intolerant of tmp/smx. it seems to have similar efficacy to dapsone (given together with weekly pyrimethamine), with a reduced incidence of side effects, of which the most frequent are rash, fever, and gastrointestinal disturbance. azithromycin is used in many centers as a third-line prophylactic agent. it is given at a dose of 1250 mg once weekly, and may provide protection against some bacterial infections, as well as mac. a low blood cd4 count (<50 cells/ml) is the current best laboratory predictor of prophylaxis failure. this is not particularly surprising given that the median blood cd4 count of subjects not on prophylaxis who have pcp develop is below 50 cells/ml. persistent fever of unknown cause is an important clinical risk factor for pcp. used as preventive therapy, tmp/ smx significantly reduces the chance for development of pneumocystis. it is, therefore, vital that subjects who are most vulnerable be encouraged to use this drug on a regular basis. the pspc cohort study revealed that 21% of subjects with a cd4 count <200 cells/ml were not receiving any form of pcp prophylaxis. the effective and safe (i.e., replication incompetent) bacterial vaccines that are available would be expected to be widely used to prevent hiv-related disease. in fact, uptake of both pneumococcal and the h. influenzae type b (hib) vaccines is poor (current estimates for the former are at most only 40% of the infected population with the recommended 23-valent vaccine). one reason for this may be that the protection conferred by vaccination (90%) in the general population is not seen in immunosuppressed hiv-infected individuals, reflecting their inability to generate adequate memory b-cell responses (especially those subjects with cd4 counts <200 cells/ml). however, in north america, cdc/idsa recommend the pneumococcal vaccine as a single dose as soon as hiv infection is diagnosed, with a booster at 5 years, or if an individual's blood cd4 count was <200 cells/ml and subsequently increased on haart. several studies show pneumococcal immunization reduces the risk of invasive pneumococcal infection in this population. this does not seem to be the case in a developing world setting, where not only is the 23-valent vaccine ineffective against both invasive and noninvasive pneumococcal disease, but the overall incidence of pneumonia is increased. infection with h. influenzae type b is much less common in hiv-infected adults and, therefore, immunization with hib vaccine is not routinely recommended. there is little evidence to suggest that the high frequency of bacterial infections in the hiv population is related to bacterial colonization. therefore, continuous antibiotics are rarely indicated, although both tmp/smx and the macrolides (clarithromycin and azithromycin) given as long-term prophylaxis for opportunistic infections have been shown to reduce the incidence of bacterial pneumonia, sinusitis/otitis media, and infectious diarrhea. the use of tmp/smx also confers a survival advantage in many studies performed in resource-poor settings. there is little evidence, however, that tmp/smx protects against pneumococcal infection. the interaction between hiv and tuberculosis is of fundamental importance, because the annual risk for the development of clinical tuberculosis in a given individual is estimated to be 5-15% (i.e., similar to a non-hiv-infected subject's lifetime risk). hiv-infected individuals with pulmonary tuberculosis are less likely to be smear positive than their hiv-negative counterparts, although they can still transmit tuberculosis. thus, within a community, tuberculosis prevention involves case finding and treatment of active disease, as well as specific prophylactic drug therapies for those exposed. if possible, hiv-positive subjects should make every effort to avoid encountering tuberculosis (e.g., at work, homeless shelter, health care facility). one of the problems with standard methods of tuberculosis contact tracing in hiv infection is that both tuberculin skin test results and chest radiology may be unreliable. however, in the absence of bacillus calmette-guã©rin (bcg) immunization, a positive ppd (e.g., <5 mm induration with 5 tuberculin units) indicates a greatly increased risk (6-to 23-fold compared with nonanergic, ppd-negative, hiv-infected subjects) of future active disease. the chance that hiv-infected subjects may contract disseminated infection if given bcg means that (having excluded active infection) the only option in these circumstances is to use a preventative drug regimen. options include at least 6 months of isoniazid (together with pyridoxine to prevent peripheral neuropathy). this is safe and well tolerated, although compliance is a problem (especially with regimens longer than 6 months), and dot may need to be instituted (e.g., 900 mg isoniazid twice weekly). there is little evidence to suggest that this single-agent regimen leads to isoniazid resistance, which probably reflects the low mycobacterial load present in such individuals. attempts to shorten the length of treatment for latent infection have produced variable results. recent studies used rifampin and pyrazinamide for short-course prophylaxis (2 months). this was as effective as 12 months of isoniazid in hiv-coinfected individuals, although it was associated with fatal hepatotoxicity (almost exclusively in the hiv-negative population). hence, it is currently out of favor. if used, liver function should be closely monitored, and it is recommended that this regimen not be given to patients with preexisting liver disease (e.g., because of alcohol or viral hepatitis). because rifampin should not be used by subjects taking pis, this may also limit widespread application of the two-drug regimen. the same applies to combinations of isoniazid and rifampin taken for at least 3 months, which are also effective in hivnegative individuals. alternate protocols also exist for subjects thought to be resistant to first-line prophylactic agents. these have not been widely clinically evaluated. it is recommended that hiv-infected subjects who have had close contact with an active case of tuberculosis should also receive prophylaxis. there is little evidence to suggest that anergy confers an increased risk for development of clinical disease. however, patients who have not had bcg, have a negative skin test, and have started haart may benefit from regular skin tests, because some studies suggest that cutaneous responses may return with increasing cd4 counts, and that this may help in identifying newly infected individuals requiring prophylaxis. in populations where the prevalence of tuberculosis is low and bcg may be given during childhood or adolescence (e.g., the united kingdom), the value of ppd testing is more limited. here, an arbitrary cutoff of 10 mm for tuberculin reactions is used to define who should receive preventive therapy. the introduction of immune-based blood tests that can accurately distinguish between bcg vaccination and tuberculosis infection may be helpful when screening for evidence of latent tuberculosis. the two currently available commercial tests use fairly specific cd4-directed mycobacterial antigen responses with consequent production of detectable interferon-g. the need for reasonably intact cd4 function means that they may, in fact, be less useful in hiv infection, especially in those subjects with very low blood cd4 counts, who are possibly at greatest risk of developing active tuberculosis. secondary tuberculosis prophylaxis may be important, because studies indicate a high rate of relapse in endemic areas. here, no specific guidelines exist, although 6 months of isoniazid and rifampin after a full treatment course shows a greatly reduced risk of relapse within the subsequent 2 years. whether this is enough to prevent clinical disease (which may also arise from reinfection in areas of high tuberculosis prevalence) without concomitant antiretroviral therapy is unclear. in the developed world, secondary prophylaxis is usually not recommended. the use of haart also can reduce the risk of tuberculosis in endemic areas. work in south africa indicates that this is most beneficial in patients with advanced disease and leads to a reduction in rr of at least 80%. data from north america indicate that the prevention of disseminated mac infection has an effect on survival (25% reduction in mortality rate in subjects taking clarithromycin). the us guidelines advise prophylaxis with a macrolide (either clarithromycin, 500 mg orally twice per day, or azithromycin, 1250 mg orally, once a week) in all hiv-infected individuals with blood cd4 counts >50 cells/ml. in europe, where the prevalence of disseminated mac infection is probably lower (perhaps because of previous bcg vaccination), this may be less relevant. here, surveillance cultures of blood may be more cost-effective in the at-risk hiv population with low cd4 counts. routine stool and sputum cultures probably do not add much to this strategy, because disseminated mac is much more common than isolated organ disease. single-agent prophylaxis may lead to antibiotic resistance. this does not seem to be reduced by the addition of a second drug (rifabutin) to the prophylactic regimen. the latter is now a second-line prophylactic agent, largely as a result of its rather worse protective effect and its adverse interaction profile with pis. as mentioned earlier, if an individual sustains a rise in cd4 count >100 cells/ml for >6 months, it is safe to discontinue prophylaxis. several clinical and laboratory features have prognostic significance in hiv-infected individuals with pcp (box 34-2). a severity score on the basis of the serum ldh levels, the a-ao 2 , and the percentage of neutrophils in the bal fluid can predict survival reasonably accurately, with the highest scores indicating the worst outcome. other workers have shown that increased age (<50 years) leads to an increased mortality rate-in part as a result of late, "unsuspected" diagnosis. the overall mortality rate from an episode of pcp is approximately 14% and has not changed since the advent of haart. among individuals with access to haart, post-pcp survival has improved. in 1981, the median survival after pcp was 9 months. by 1995, this had risen to 20 months. the introduction of haart has led to a further improvement, with survival in the period up to year 2000 of 40 months. in those without access to haart and/or prophylaxis, survival post-pcp, unfortunately, remains poor. in general, mortality from bacterial respiratory infection in hiv-infected individuals is similar to that seen in the general population. clinical and laboratory markers of disease severity that have been defined in the adult general population (e.g., those described in the ats or the bts guidelines for the management of community-acquired pneumonia in adults) apply to hiv-infected patients. these are confusion, raised respiratory rate, abnormal renal function, and low blood pressure. recurrent pneumonia is common (reported in up to 55% of cases) and may lead to chronic pulmonary disease (see earlier). although tuberculosis normally responds to standard multipledrug therapy, work from africa has highlighted the increased mortality rate in hiv-infected compared with non-hivinfected individuals. a relationship has also been described between mortality and declining blood cd4 count: hivinfected patients with cd4 counts <200 cells/ml have a mortality rate of 10% compared with 4% in those with cd4 counts from 200-499 cells/ml. compared with hiv-infected individuals without tuberculosis, the main effect on mortality is seen in patients with higher cd4 counts (>200 cells/ml), where the relative risk of death is three times that of the nontuberculous population. several case-controlled studies have indicated that in the absence of effective treatments, mac-infected patients have a reduced survival compared with blood cd4 level-matched control subjects (approximately 4 months vs 9 months, respectively). currently available treatment regimens may reduce this difference, although severe anemia seems to be an independent predictor of mortality. the presence of cmv in bal fluid also containing p. jirovecii has been related to outcome (see earlier). the mortality rate at 3 and 6 months after bronchoscopy is greater in those with cmv detected at bronchoscopy. however, cmv recovered as a sole pathogen does not impact on survival. the effect of antiretroviral therapy on opportunistic infections the introduction of haart, together with the wide availability of accurate methods of determining plasma rna viral load, has led to profound changes in both clinical practice and hiv outcome. although it is still the case that respiratory disease remains above non-hiv infected background levels, in particular, bacterial pneumonia, tb, and lung cancer are more common, despite apparently effective haart, in hiv-infected subjects. overall data indicate that clinical progression is rare in subjects who are able to adhere rigorously to at least 95% of their antiretroviral drug regimen. mortality rates have fallen by 80% for almost all conditions, and it seems that a damaged immune system can, to a clinically significant extent, be reconstituted for a period of at least several years. hence, clinicians need to consider not only opportunistic infection or malignancy within their diagnostic workup but also the effects of drug therapy itself. the side effect profile of haart (e.g., metabolic and mitochondrial toxicities, liver damage, and neuropsychiatric disorders), as well as the large number of drug-drug interactions, makes this a very complex area of management. the best example of this is hiv-related tuberculosis. here, not only is there overlapping toxicity and pharmacologic interaction, but iris is common. research is needed to address this area. studies should inform the decision on when to start haart in patients already on antituberculosis medication. other work needs to focus on understanding box 34-2 prognostic factors associated with poor outcome in pneumocystis jirovecii pneumonia on admission patient's age no previous knowledge of hiv status tachypnea (respiratory rate >30/min) second or subsequent episode of pneumocystis jirovecii pneumonia poor oxygenation ã� pao 2 <7.0 kpa (<53 mmhg) or a-ao 2 >4.0 kpa (>30 mmhg) low serum albumin (<35 g/dl) low hemoglobin (<12.0 g/dl) peripheral blood leukocytosis (>10.8 ã� 10 9 /l) elevated serum lactate dehydrogenase levels (>300 iu/l) cd4 count <50 cells/ml marked chest radiographic abnormalitiesdiffuse bilateral interstitial infiltrates with or without alveolar consolidation medical comorbidity (e.g., pregnancy) at bronchoscopy 1. in bronchoalveolar lavage fluid detection of a. copathology cmv bacteria b. neutrophilia (>5%) 2. detection of pulmonary kaposi sarcoma serum lactate dehydrogenase levels that remain elevated development of pneumothorax high apache ii score on admission to the icu need for mechanical ventilation why full pulmonary immunity is not restored. this may reflect abnormalities in the innate immune response, which is currently poorly described in hiv infection. despite the benefits of haart, it is likely that in the long term many patients will progress to severe disease. there is currently little research in this area. research should focus on correlating clinical and laboratory findings. an example of this would be assessing the risk of an individual for development of active tuberculosis. it is clear that much of the excess mortality in hiv-tuberculosis coinfection occurs early in hiv infection. thus, if tests can be devised that indicate who has latent tuberculosis infection (and who is, therefore, most likely to have clinical disease develop), steps can be taken to prevent illness. as discussed previously, immune-based tests have shown promise in immunocompetent individuals with tuberculosis infection. if these can be refined to work consistently in patients with hiv infection at a reasonable cost, there is the possibility of targeting those at risk of future tuberculosis, or of tuberculosis "unmasking" after starting haart. the other role for a test such as this would be in rapid diagnosis of active tuberculosis. it is common to be faced with a patient who has nonspecific symptoms and a wide differential diagnosis. often treatment is multiple and empirical. a quantitative test would help resolve some of these dilemmas by indicating the chance of the condition being caused by a particular disease. an example would be the patient from an endemic tuberculosis area, with low cd4 counts, who has both pulmonary and central nervous system disease. is this tuberculosis, toxoplasmosis, cryptococcosis, or viral or bacterial infection? any such test for tuberculosis would also have to distinguish between the different states of old (treated), old (inactive), old (latent), and active. although not insurmountable, at present, this is not possible. rapid diagnostic assays that assess organism viability are also important. if a clinician can receive early feedback on whether treatment is producing a suitable killing effect, therapy can be tailored to the individual. this enables regimens to be "dose adjusted" as needed and removes the element of concern that is often present when patients are slow to respond. examples of this would be in the treatment of pcp or mycobacterial disease. the frequency of bacterial infection (often recurrent) with its attendant sequelae makes effective strategies for vaccination an important priority. it is uncertain why there is a differential response to vaccination; even in the united states, african americans do not seem to derive the same benefit as whites. this needs further research, together with more emphasis on identifying the local immune response present in the lung in such individuals. bacterial infections may be clinically indistinguishable from other pathogens, and only two thirds of all respiratory infections are formally diagnosed. there is a need for improved methods to assist with this. the use of rapid antigen tests may be one way forward. this is especially so given the high incidence of (potentially fatal) bacteremia present in such populations. for maximum benefit, this needs to use a system that is simple and cheap, and hence suitable to both resourcerich and resource-poor countries. m. tuberculosis is globally the most important hiv-related pathogen. strategies of control and prevention are vital to ensure that millions of people do not become coinfected and that those who are do not go on to have clinical disease develop. rapid diagnostics are critical. the encouraging reports of the simple and cheap method of mods to both diagnose tuberculosis and then provide resistance data in field settings (see earlier) argues for large-scale roll out and evaluation. beyond public health measures, such as dot, fixed-dose combination drugs, case management, and education, research needs to improve on current drug therapy. long-acting preparations such as rifapentine show promise but, as the problem with rifampicin monoresistance demonstrates, there is still much work to be done. for the first time in many years, there are several antimycobacterial drugs that are in various stages of clinical trials. all are promising, and several have novel mechanisms of action. the global alliance and the who "stop tb" campaigns have been crucial in this regard. the fluoroquinolones, moxifloxacin and gatifloxacin, are closest to the market. they are potent drugs with considerable ability both to kill and also sterilize mycobacteria-infected sites. trials of treatment-shortening regimens are ongoing worldwide. vaccination against m. tuberculosis with bcg has understandably not been widely used in an immunosuppressed hiv-infected population. however, a safe vaccine may be the only affordable way of protecting large parts of the world from tuberculosis. so far there seems to be more success in vaccines to either enhance or replace the primary protective effects of bcg. the use of immunotherapy (e.g., with heat-killed mycobacterium vaccae) in combination with chemotherapy has been disappointing in clinical trials. newer methods of diagnosis (e.g., pcr tests on saliva) may prove invaluable for quick and easy disease confirmation, although their applicability to routine samples needs further evaluation. p. jirovecii prophylaxis was the first important hiv treatment widely available. however, despite the efficacy of tmp/smx, compliance remains a problem. regimens that use a gradual increase in dosage when starting prophylaxis may help. one concern with widespread use of prophylaxis is that resistance will start to occur to tmp/smx. reports have indicated that there are mutations in the p. jirovecii dihydropteroate synthase gene that confer resistance. these seem to be increasing over time, although they do not seem to be present in many patients who fail treatment for pcp with tmp/smx. the implications of this are uncertain but could include a greater likelihood of treatment failure and the possibility of worsening patterns of global bacterial drug resistance. hiv-infected populations in the developed world have high rates of smoking. the evidence that this is harmful above those effects seen in the general population continues to accrue. the accelerated course of both obstructive lung disease and cancer, together with the increased risk of respiratory infection in smokers, persuasively argues the case for targeted smoking cessation. that hiv infection and haart have profound (and probably negative) effects on blood lipids and insulin resistance further support the need to reduce smoking rates in this population. it seems that we are starting to see increased rates of cardiovascular disease in this now aging population. the natural history of hiv-related respiratory disease continues to evolve. haart and newer therapeutic strategies have made a significant impact on morbidity and mortality. yet individuals continue to become hiv infected, progress, and die from an ever-expanding range of conditions. p. jirovecii remains the most common aids-defining event in the developed world, whereas m. tuberculosis is globally the most common cause of death. bacterial respiratory infection is not far behind. given the huge number of individuals with hiv infection, the only effective way to manage this disease is to find simple ways of treating hiv itself, and thus contain the worst ravages of this illness. treating opportunistic infections among hiv-infected adults and adolescents revised recommendations for hiv testing of adults, adolescents, and pregnant women in healthcare settings treatment of hiv-related tuberculosis in the era of effective antiretroviral therapy treatment and prophylaxis of pneumocystis carinii pneumonia immune reconstitution disease associated with mycobacterial infections in hiv-infected individuals starting antiretroviral therapy immune reconstitution inflammatory syndrome in hiv guidelines for preventing opportunistic infections among hiv-infected persons-2002 on behalf of the bhiva guidelines writing committee pneumocystis and trypanosoma cruzi: nomenclature and typifications oxford: blackwell scientific aids and respiratory medicine key: cord-295290-hs5ntlok authors: atlan, h.; gersten, m. j.; salk, p. l.; salk, j. title: mechanisms of autoimmunity and aids: prospects for therapeutic intervention date: 1994-12-31 journal: research in immunology doi: 10.1016/s0923-2494(94)80181-9 sha: doc_id: 295290 cord_uid: hs5ntlok summary the network theory of autoimmunity is presented with recent experimental data relevant to the understanding of the pathogenesis of aids. schematically, effector t cells specific for self-antigens exist normally, but their activity is modulated and prevented by networks of regulatory t cells. as a result of mimicry between molecular components of microorganisms and self-antigens, autoimmune disease can be triggered by specific foreign pathogens which alter the state of activity of the network from suppression to activation. conversely, by a procedure known as t-cell vaccination, autologous effector t cells re-injected after in vitro stimulation and attenuation may alter the state of the network from an activation to a suppression. numerous observations are reviewed that support the concept of autoimmune activity in the destruction of non-infected t4 cells. such activity is presumed to be triggered by an antigen of viral origin, the most likely, but not the only one, being the envelope protein gp120. based on this hypothesis, a t-cell vaccination procedure against effector t cells responsible for autoimmunopathic activity in hiv-seropositive patients is proposed, similar to the one known from experimental study of autoimmunity and presently being tested in human autoimmune diseases. its purpose would be to prevent t-cell loss and the onset of immunodeficiency disease in hiv-seropositive patients. apart from its potential therapeutic value, this procedure will have use as a therapeutic test from which insight will be gained about the immunopathogenesis of aids. in previous communications (atlan, 1992 ; atlan et al., 1993) , we have suggested that t-cell vaccination may be applied.to hiv-seropositive patients in order to prevent the development of aids. t-cell vaccination is a well-documented procedure designed as a specific cellular imsubmitted december 9, 1993 , accepted february 22, 1994 (*) to whom correspondence should be sent. present address: service de biophysique, hbpital de i'hdtel dieu, 1 place du parvis de notre dame, 75004 paris, france, munotherapy against autoimmunity (cohen, 1991a) . t-cell vaccination has been effective in a number of experimental autoimmune diseases, and is presently being tested in humans affected by multiple sclerosis (hafler et al., 1992; zhang et al., 1993) and rheumatoid arthritis (van laar et al., 1993) with no toxic effects and encouraging preliminary results. several lines of evidence suggest that development of the acquired immunodeficiency syndrome (aids) in individuals infected with the human immunodeficiency virus (hiv) does not result solely from direct cytopathic effects of the virus (shearer, 1983; ziegler and stites, 1986; klatzmann and montagnier, 1986 ; klatzmann and gluckman, 1986; procaccia et al., 1987; ascher and sheppard, 1988; isaksson et al., 1988 ; kaplan et al., 1988 ; kopelman and zolla-pazner, 1988; schattner, 1988; martinez-a. et al., 1988; bacchetti and moss, 1989; duesberg, 1989 ; schnittman et al., 1989 ; ascher and sheppard, 1990; hoffman et al., 1991; maddox, 1991; hsia and spector, 1991; morrow ec al., 1991) . it is true that more sensitive pcr techniques have shown that the proportion of cd4+ t lymphocytes latently infected with hiv is much higher than originally thought, even during the clinical latency period (embretson et al., 1993) . however, the presence of viral dna in the genome of the cells is not lethal in itself in the absence of activation. therefore, the question remains open concerning the immunopathology of aids. the long and variable interval between infection with hiv and development of immunodeficiency disease (klatzmann and gluckman, 1986; isaksson et al., 1988; kaplan et al., 1988; bachetti and moss, 1989) , clinical and pathological similarities with known autoimmune disorders (procaccia et al., 1987 ; kopelman and zolla-pazner, 1988 ; eat = experimental autoimmune thyroiditis. i aa = adjuvant arthritis. aids = acquired immune deficiency syndrome. cm1 = cell-mediated immunity. cmv = cytomegalovirus. cl-l = cytotoxic t lymphocyte. dth = delayed-type hypersensitivity. eae = autoimmune encephalomyelitis. schattner, 1988; morrow et al., 1991) and the observation of genetic correlations with patterns of hiv disease progression (steel et al., 1988; jeannet et al., 1989; simmonds et al., 1991; puppo et al., 1991; louie et al., 1991) , all suggest more complex pathological processes involving the immune response of the organism to hiv infection. a number of immunopathic mechanisms with several features of autoimmunity have been proposed as contributing to the development of aids. these include cross-reactive recognition of self-mhc and a secondary antiidiotypic response to cd4, to be found in the first large set of references mentioned above, elimination of infected t4 cells by virus-specific, hlarestricted cytotoxic lymphocytes (shearer, 1986; zinkernagel, 1988) , elimination of uninfected t4 cells by immune responses directed against hiv (klatzmann and gluckman, 1986; salk, 1987; lyerly et al., 1987; lanzavecchia et al., 1988; lanzavecchia, 1989 ; siliciano et al., 1988 ; israel-biet et al., 1990 ; morrow et al., 1991) and/or t4 cell antigens (stricker et al., 1987; martinez-a. et al., 1988; kowalski et al., 1989; zarling et al., 1990) , unrestricted t-cell activation by hiv-infected macrophages with consequent immune dysregulation and t-cell death sheppard, 1988, 1990) , superantigen effects of hiv components leading to widespread deletion of t4 clones (imberti et al., 1991) , other non-specific immunosuppressive effects mediated by hiv components including gp120 (weinhold et al., 1989) and the tat gene product (viscidi et al., 1989) and inappropriate activation-induced t-cell death (apoptosis) in mature t4 cells (a process which is normally observed only in immature thymocytes) (ameisen and capron, 1991; terai et al., 1991; montagnier et al., 1991; groux et al., 1992 versus-host and host-versus-graft responses to allogeneic class ii mhc antigens, with consequent immune suppression, have also been suggested (shearer, 1983; klatzmann and gluckman, 1986; moser et al., 1987) . on the basis of observed cross-reactivity between mhc and hiv proteins, it has been suggested that hiv envelope antigens induce a chronic autoreactivity similar to graft-versus-host disease (habeshaw et al., 1992) . more recently, a highly plausible hypothesis on the immunopathology of the development of arc/aids has been proposed (pantaleo et al., 1993) . this hypothesis is based on the observation that large numbers of human immunodeficiency viruses and of hiv-infected cells are present in the lymphoid organs during the clinical latency period, associated with a progressive dysruption of the follicular dendritic cell (fdc) network in the lymph nodes. it is suggested that during the progression of the disease, most of the hiv-infected cd4+ t cells are no longer prevented from circulating in the peripheral blood by being destroyed in the lymph nodes, when the fdc network cannot continue to perform its normal functions of trapping and antigen presentation. then, further activation of large numbers of circulating infected cd4+ t cells would allow for viral replication and cell death responsible for the increased viraemia observed at the late stages of the disease. however, the critical event in this mechanism, namely the destruction of fdc, does not seem to be produced by a direct viral cytopathic effect, but again by an immunopathic response leading to activated cd8 + cytotoxic t lymphocytes (ctl) infiltrating the lymph node follicles. thus, on the one hand, immune responses to hiv infection might be immunoprotective through destruction of hiv-infected cells and neutralization of free virus. on the other hand, however, some aspects of the immune response might be autoimmunopathic and contribute to the development of immunodeficiency through destruction of both infected and uninfected t4 cells, and/or of follicular dendritic cells in the lymph nodes. the rate of tccell decline and hiv disease progression might thus reflect, in part, the balance between immunoprotective and autoimmunopathic facets of the anti-hiv immune response. in the present article, the first section will review the relevant experimental and theoretical background on autoimmunity and t-cell vaccination. recent developments in the network theory of autoimmunity (cohen, 1986; cohen and atlan, 1989; atlan and hoffer-snyder, 1989; cohen and young, 1991; atlan and cohen, 1992; cohen, 1992a cohen, , 1992b shed light on the nature of various autoimmunopathic mechanisms that might be operative in hiv disease and suggest ways in which such autoimmunopathic processes might be prevented or controlled. according to that theory, for example, molecular mimicry between a strong foreign epitope and a self-antigen may lead to the breaking of tolerance to the self-antigen through perturbation of a pre-existing regulatory network. autoimmunopathic destruction of t4 cells might thus result, in this case, from the destabilization of a self-tolerance-maintaining regulatory network by immune responses to hiv components with homologies to antigens normally present on t4 cells. another alternative would be that hiv antigens presented by cd4+ t cells or follicular dendritic cells would have a similar destabilizing effect by activating helpers and cytotoxic cells against those cells. in section ii, we shall examine existing evidence for autoimmunopathicity in hiv disease and discuss how the concepts developed in section i can be applied to a possible immunotherapy of hiv-seropositive patients. several models of how regulatory networks might be related to autoimmunopathicity in hiv disease and implications with respect to potential modes of intervention will be discussed. in section iii, we shall propose a detailed protocol for a first therapeutic test to be implemented. a general discussion will serve as a conclusion. i. -network theory of autoimmunity contrary to the global theories of the idiotypic network, initiated by the pioneering work of jerne (1974) , the network theory of autoimmunity is a local description of interactions between identified cell populations involved in the occurrence of specific autoimmune diseases (cohen and cohen, 1989a) . according to its main concept, autoimmunity is "natural" (horton, 1993) and is present in healthy organisms. it is controlled by a network of regulatory t cells (and possibly lymphokines, b cells, antibodies) which prevents normally existing effector cells reactive to a given selfantigen from being activated. the balance between stimulating and suppressive effects depends on the connection structure of the network, i.e. the nature and the strength of the interactions between the network elements. depending on that structure, the network stabilizes in a normal or pathological state, where the effector cells directly responsible for the disease are activated or inactivated, respectively. however, the connections between the elements can be modified by external antigenic stimuli or internal regulatory signals produced by the network's own temporal evolution (atlan and hoffer-snyder, 1989) . thus, such a network, like neural networks in the central nervous system, is endowed with "cognitive" properties, i.e. self-organizing learning and adaptive capacities (atlan and cohen, 1992; cohen, 1992a cohen, , 1992b . relevant examples of those mechanisms will be discussed further (in section ii, parts 1 b and 2). the network theory of autoimmunity is based on the evidence that effector cells with specificity for self-antigens normally exist in vivo (guilbert et al., 1982; burns et al., 1983 ; cohen and young, 1991) . such effector cells are either prevented or not from exerting autoimmune effects. this depends on the state of activity of other, regulatory, cell populations, mainly (but not solely) antigen-specific and idiotypic-specific (ben-nun et al., 1981a , holoshitz et al., 1983 lider et al., 1987; lider et al., 1988 ; kakimoto et al., 1988; lohse et al., 1989; roubaty et al., 1990) . when this finely tuned regulatory system is appropriately perturbed by the introduction of a foreign antigen, crossreactive with a given self-antigen or interfering with antigen presentation, the ensuing stimuli delivered to the various component cells of the network change the steady state of the network in such a way that expression rather than suppression of autoimmunity occurs. conversely, the regulatory network is enhanced or strengthened with respect to its ability to suppress autoimmunity by exposure to a non-pathogenic form of the relevant effector cells. such enhancement is learned and "memorized" in the structure of the network, so that subsequent exposure either to the pathogenic effector cells or to the potentially immunopathogenie antigen would not produce autoimmunity. this concept is particularly relevant to studies of t-cell vaccination as a prophylactic and therapeutic procedure against autoimmune diseases (ben-nun et al., 1981a; cohen et al., 1985; cohen, 1986 cohen, ,c, 1991a atlan and hoffer-snyder, 1989; roubaty et al., 1990; cohen and young, 1991; elias et al., 1991; beraud, 1991; atlan and cohen, 1992 ; hafler et al., 1992) . studies with experimental autoimmune diseases have provided most of the data underlying the network theory of autoimmunity. the best studied model has been experimental autoimmune encephalomyelitis (eae). this disease, characterized by the acute onset of paralysis in genetically susceptible animals following appropriate inductive stimuli, has been shown to be mediated by t4 effector cells specific for myelin basic protein (mbp) 198 1 b) . spontaneous recovery may occur and is associated with the appearance of suppressor cells which react specifically with idiotypic determinants present on the anti-mbp t4 cells (ben-nun and cohen, 1982; ellerman et al., 1988) or with antigenic determinants on mbp 198 lb) . these and other observations (lider et al., 1988; lohse et al., 1989) have led to the formulation of a model in which the onset, remissions and relapses of eae and other autoimmune diseases can be explained by changes in the interactions among an effector cell and two pairs of regulatory elements: (i) antigen-specific helper and suppressor cells, and (ii) idiotype-specific helper and suppressor cells (cohen and atlan, 1989) . according to this model, once a state of autoimmunity has resulted from a change in the normal balance among the various elements of a regulatory network, which has previously served to suppress an anti-self response, the autoimmune state may be stable and persist even after the agent which precipitated the change is no longer present. alternatively, if the host response to a foreign antigen induces the de nova formation of a regulatory network leading to autoimmunity, then the continued presence of cross-reactive self-epitopes may perpetuate the autoimmunopathic state of the network even after the precipitating antigen has been cleared. other experimental models of autoimmune disease suggest the existence of further levels of complexity in the regulation of autoimmunity. for example, adjuvant arthritis (aa) is an autoimmune disease which may be experimentally induced by injection of killed mycobacferium tuberculosis, which contains an antigenic protein cross-reactive with a joint cartilage proteoglycan . the disease may also be induced by inoculating cells from an anti-m. tuberculosis t4 clone which also recognize the proteoglycan (van eden et al., , 1988 . more recent investigation into the nature and dynamics of the regulatory network in aa suggests that at least one additional component may be involved karin, 1991; atlan and cohen, 1992) . the onset and severity of the autoimmune disease triggered by m. tuberculosis has been correlated with the presence of a population of non-specific cd8+ suppressor cells which downregulate the antiidiotypic regulatory cells to a greater extent than they do the cytolytic effector cells. according to the proposed network model (atlan and cohen, 1992) , this results in a net increase in effector activity. this contrasuppressive, pathogenic response is partially non-specific lohse et al., 1989; karin, 1991) , being directed not only against the specific antiidiotypic regulatory cells, but also against other anticlonotypic regulatory cells. the latter includes cells which control responses to different epitopes of the self-antigen and others which are involved in the regulation of unrelated autoimmune diseases, such as eae. it is thus conceivable that partially non-specific suppressor cells might exert contrasuppressive effects and facilitate the expression of autoimmunopathic res-ponses in other diseases. in this regard, it is interesting to note that a subpopulation of cd8+ suppressor cells acting via release of a soluble inhibitory factor has been observed in association with hiv infection (joly et al., 1989; sadat-sowti et al., 1991) . the pathogenesis of experimental autoimmune thyroiditis (eat) provides another example of additional network complexity. this disorder, which can be produced experimentally by inoculation of cells from a thyroglobulinspecific cytotoxic t-cell clone, may entail dysfunction of an immune regulatory network containing humoral as well as cellular components (roubaty et al., 1990) . in this regard, given the homologies that have been described between the hiv envelope glycoprotein (gp120) and immunoglobulins (bjork, 1991) , it is possible that anti-hiv responses, cross-reactive with selfimmunoglobulins, might non-specifically perturb the humoral arm of a bipartite cellular/humoral regulatory system and thereby contribute to the immunopathogenesis of aids. although the detailed mechanisms of network regulation of autoimmunity may be more complicated than presently understood, an appreciation of the dynamic aspect of regulatory networks is useful when considering means to perturb the system intentionally so as to strengthen or to restore the development of a nonimmunopathic state. network models suggest that vaccination with autoimmune effector cells (in a form or under conditions in which the cells are not capable of exerting pathogenic effects) should elicit antiidiotypic and other regulatory cells which might suppress an autoimmune effector cell response, thereby preventing or ameliorating autoimmune disease. an example of such a model is shown in figure 1 , where a network of effector cells and five populations of regulatory cells account for complicated and paradoxical data on aa (atlan and cohen, 1992) . this example is particularly relevant since it involves the participation of a foreign pathogen (m. tuberculosis) working as an adjuvant in the triggering of autoimmunity. in the framework of the proposed hypothesis on aids, the role of hiv could be similar -as will be detailed below. h. atlan et al. (atlan and cohen, 1992) . full and dotted arrows represent activating and suppressive connections, respectively. higher doses of m. tuberculosis (mt) -a foreign pathogen working as an adjuvant in the onset of aa, probably because of mimicry with the self-antigen -produce more severe forms of the disease. t-cell vaccination with attenuated effector cells prevents the onset of the disease. experimental observations on the state of activity of the different cell populations under different conditions (healthy, diseased, vaccinated) are accounted for by the steady states of the network computed as functions of the connections. t-cell vaccination works as a learning process of the network, following given laws which modify the connections in such a way that further exposure to pathogenic doses of mt do not produce a diseased state (cohen and atlan, 1989; atlan and hoffer-snyder, 1989; atlan and cohen, 1992 ). this concept is consistent with the results of studies using t-cell vaccination in a number of experimental autoimmune diseases. vaccination with subpathogenic doses of effector t cells, or with large numbers of effecters attenuated by irradiation or treatment with glutaraldehyde, mitomycin c, hydrostatic pressure or other agents (cohen, 1986 (cohen, , 1991a lider et al., 1987) , has been shown to elicit antiidiotypic (lider et al., 1987 (lider et al., , 1988 roubaty et al., 1990; elias et al., 1991) and/or antiergotypic (lohse et al., 1989; beraud, 1991) suppressor cells and to be effective in preventing and/or treating eae (ben-nun et al., 1981a; lider et al., 1988; lohse et al., 1989) , aa (holoshitz et al., 1983 ; lider et al., 1987; , eat (roubaty et al., l!m) , collagen-ii-induced arthritis (kakimoto et al., 1988) , spontaneous autoimmune diabetes (elias et al., 1991) and experimental autoimmune uveitis (beraud, 1991) . in humans, clinical trials have been initiated to explore the use of t-cell vaccination for the treatment of some autoimmune diseases with no evidence of toxicity (hafler et al., 1992; zhang et al., 1993; van laar et al., 1993) . in patients treated for multiple sclerosis, it is still too early to make any assessment on possible clinical improvement, because of the slow evolution of the disease. however, as expected from abovementioned previous animal studies on eae (lider et al., 1988; lohse et al., 1989) , an antiidiotypic response has been observed after t-cell vaccination with myelin basic proteinspecific t-cell clones (zhang et al., 1993) . a similar procedure was achieved by a different group (van laar et al., 1993) on patients suffering from rheumatoid arthritis with variable disease duration. on average, a slight decrease in disease activity was observed, with an indication of a clearer improvement in patients with recent onset of the disease. in view of the foregoing, the first question to be asked concerning a given autoimmunopathic mechanism is the nature of the selfantigen involved. a given self-antigen must be identified as the target for autologous ctl and/or autoantibodies. then, the vaccination procedure will be aimed at strengthening deficient regulatory responses capable of suppressing the activity of those ctl, or antibodies. it has previously been suggested that the immunosuppression observed in aids might be in part attributed to an hiv-triggered immunopathic response directed against uninfected as well as infected t4 cells (kiatzmann and montagnier, 1986; klatzmann and gluckman, 1986; salk, 1987; lyerly et al., 1987; lanzavecchia et al., 1988 ; siliciano et al., 1988 ; lanzavecchia, 1989; israel-biet et al., 1990) . in that case, the antigen must be looked for in the membrane of cd4+ t cells themselves. however, as mentioned above, another alternative would be to consider the lymph node fdc as the target for cd8+ ctl activated by the hiv infection. in view of existing experimental evidence, we shall consider the implications of the two possible targets for autoimmunopathic responses -cd4+ t cells and fdc -as far as the identification of the antigen and regulatory network is concerned. from an autoimmunity viewpoint, the simplest assumption would be that the cd4 molecule itself is the self-antigen. however, viral molecules interacting with cd4 must be involved to trigger the immunopathicity against cd4+ t cells. therefore, the hypothesis of cd4+ t cells being the targets of immunopathic responses must be divided into two possible models, for which supporting data from the literature will be reviewed. in the first model, a viral antigen -the most likely being the envelope glycoprotein gp120 -is directly involved and plays the role of a "self''-antigen after it has been incorporated into the cd4+ t-cell membrane. in the second model, a true self-antigen, pre-existing in that membrane, is activated by hiv infection. a) the gp120 antigen model according to this view, autoimmunopathic destruction may occur as a result of anti-gp120 effecters killing both infected t4 cells expressing gp120 among other viral antigens on their membrane, and uninfected t4 cells which have bound free gp120 ( fig. 2a ). that such gp120 binding is likely to occur is supported by the high degree of gp120 shedding after infectious viruses are released (gelderblom et al., 1985; schneider et al., 1986) and by the detection of gp120 in sera of hiv-seropositive individuals (oh et al., 1992) . free gp120 bound by the cd4 molecule might well be internalized and, depending upon the route of metabolism, reexpressed in association with class i (making it a target for classical cd8 + cytotoxic t cells) or class ii mhc molecules. hoffenbach et al. (1989) detected mhc-restricted anti-env cd8+ cytotoxic lymphocytes in both seropositive and seronegative donors. since the frequency of these cells declined in seropositives with clinical deterioration, the authors hypothesized that this response was primarily immunoprotective. however, they also noted that these env-specific cytotoxic cells might be capable of causing autoimmunopathic t6cell destruction through the mediation of bound env or molecular mimicry. siliciano et al. (1988) were able to precisely demonstrate such a potentially autoimmunopathic effect. they detected anti-gp120 cytotoxic cd4+ lymphocytes in normal seronegative individuals which could kill, in an antigen-specific, mhc-restricted manner, uninfected activated autologous t4 cells pulsed with gp120. the presence of anti-gp120 cytotoxic cells in sufficient frequency would be expected, according to the network theory, to induce the formation of a network which would regulate the activity of these effector cells. given the persistence of hiv infection, and therefore the continued presence of hiv antigen, we postulate that the regulatory network established would be configured so as to favour stimulation of the antigp120 response. from the viewpoint of autoimmunity, however, such a network would be ineffective, insofar as it would fail to restrain the effecters of autoimmunopathic destruction. thus, in this model, autoimmunopathicity in aids would result from (i) gp120 bound to uninfected t4 cells coupled with (ii) a regulatory network configured to stimulate, rather than suppress, the anti-gp120 autoimmune effecters. the significance of the autoimmunopathicity would depend upon the relative balance between the protective aspect of the response (destruction of infected t4 cells and consequent reduction in viral proliferation) and the immunopathic aspect of the response (destruction of normal t4 cells and consequent reduction of residual immunological potential). b) the t4-cell membrane self-antigen model the second model is more closely analogous to classical experimental autoimmune disorders. several findings suggest that one (or more) selfantigen might be normally present on the cd4+ cell membrane and that pre-existing suppressed ctl against that antigen would be activated by hiv infection. for example, zarling et al. (1990) have found such ctl in the blood of hivseropositive patients (and not of hiv-infected chimpanzees), capable of killing uninfected cd4+ cells from both hiv-seropositive patients and seronegative controls. israel-biet et al. (1990) have found cd3 + cells in hiv-infected patients able to kill allogeneic ebv-transformed b cells and cd4+ cell blasts from seropositive and from seronegative subjects. hoffenbach et al. (1989) and plata (1989) have observed an unusually high frequency of ctl precursors primed to hiv antigens -and not to other viruses -in the repertoire of normal humans in the absence of hiv infection. as in other instances of autoimmunity triggered by foreign pathogens (van eden et al., , 1988 morrow et al., 1991; karin, 1991; cohen and young, 1991; horton, 1993) , the triggering of a true autoimmune response by an hiv antigen could imply some mimicry between that antigen and the pre-existing self-antigen. class ii mhc antigen has been suggested as being the postulated self-antigen (hoffman et al., 1991) . however, in that case, one might expect aids to be characterized by a significant depletion of b cells and antigen-presenting cells, the primary expressors of mhc class ii antigen, rather than, or in addition to, t6cell depletion. rather, one might hypothesize that t4 cells carry an additional unique marker which is crossreactive with gp120 (perhaps structurally or physiologically associated with cd4 on the t4 cell membrane), either directly or by complexing with mhc molecules. the detection by both hoffenbach et al. (1989) and siliciano et al. (1988) of anti-env specificities in cytotoxic lymphocytes of seronegative individuals might be postulated to be due to the existence of such a cross-reactive self-antigen. the inability of siliciano et al. (1988) to detect killing by such cells of uninfected autologous t4 cells not pulsed with gp120 might be related to a subthreshold level of surface expression of the self-antigen (marker) under the ambient conditions of the assay. in summary, according to the t6cell membrane self-antigen model ( fig. 2b ), effecters specific for this putative cross-reactive selfantigen exist prior to hiv infection; but they are inactive, owing to a dominant suppressive effect exerted by a pre-existing regulatory network. this phenomenon would be similar e.g. to what is observed in experimental aa (van eden et al., ,1988 atlan and cohen, 1992) , where a foreign bacterial antigen in the adjuvant (from m. tuberculosis) is cross-reactive with a joint cartilage self-antigen. the bacterial antigen serves as a strong antigenic stimulus to a pre-existing regulatory network, quantitatively related to the amount injected. in the absence of adjuvant stimulation, the network is normally in a suppressive state, preventing the activity of cytotoxic effector cells against the joint cartilage. similarly, in our tqcell self-antigen model, the pre-existing regulatory network would be destabilized by the strong antigenic stimulation produced by the introduction of (foreign) gp120. as a result, the network would be driven to a new steady state characterized by activated effector cells able to destroy (in viva) uninfected marker-bearing t4 cells, independent of the presence of bound and reexpressed gp120. early in the course of hiv infection, these activated cytotoxic cells are still subject to some network regulation by the suppressor cells. therefore, only a mild form of autoimmune disease, typified by a moderate degree of killing of t4 cells, occurs. with time, however, the network moves into a more pathological state in which the suppressor cell activity is itself suppressed or insufficient. as in other instances of pathological autoimmunity, the progression of the disease may be explained by several mechanisms. for example, after the onset of an active state against the antigen, a progressive increase in the efficiency and diversification of antigen presentation may produce a reinforcement of the antigenic stimulus. such processes of alterations in self-antigen presentation, leading e.g. to the display of previously cryptic self-determinants (lehmann et al., 1993) , may explain the time course of autoimmune diseases, including virus-induced progressive states of autoimmunity, such as chronic active hepatitis (ohtani et al., 1987) and eaelike lesions induced with coronavirus (watanabe et al., 1983) . a "vicious circle of cytokine-driven upregulation of autoantigen presentation" (lehmann et al., 1993) may be produced by crossreactivity with viral antigens, but may also occur in the absence of cross-reactivity, leading to "ongoing t-cell stimulation, further t-cell recruitment and determinant spreading, greater cytokine production, and so forth". this process can be accelerated as intercurrent infectious and non-infectious stresses contribute to an increase in virus and viral antigen production, leading to a heightened activation of the effector and helper regulatory cells. particular intercurrent infections could also precipitate an increase in contrasuppression, as in aa, where the level of contrasuppressor activity varied directly with the dose of adjuvant mycobacterium administered (karin, 199 1; atlan and cohen, 1992) . such a mechanism could contribute to the observation that certain opportunistic infections appear to accelerate the clinical progression of hiv/aids. in any case, as mentioned above, as far as the connection structure of the regulatory network is concerned, this kind of vicious circle would amount to a reinforcement mechanism of learning and distributed memory, well-known in neural network modeling (weisbuch, 1986 ; atlan and hoffer-snyder, 1989; atlan and cohen, 1992) . as a result, the earlier disease-free state of the network is destabilized and the network evolves towards more and more activating pathogenic states. c) common features of the two models; t-cell vaccination against anti-gpl20 ctl as a therapeu tic test in both models (sections la and lb), humoral participation in the onset of hiv-triggered autoimmunopathic response is possible, as in eat. this might be mediated, for example, through the reported homology between a gp120 epitope and immunoglobulins (bjork, 1991) , as well as cell surface proteins with a role in cd4 binding (beretta et al., 1987; golding et al., 1988; young, 1988) , which might result in the perturbation of a possible t-cell/b-cell regulatory network. in any case, the search for anticlonotypic cell populations being activated, and for increased concentrations of different antibodies and cytokines in response to the cell clone or line being used in the vaccination procedure, will in itself provide useful information on the nature of the antigen and the regulatory network. the different reinforcement mechanisms discussed in lb may also be operative in the model of section la since, as mentioned above, they can be triggered after the first immunopathic response has been induced even in the absence of cross-reactivity between pre-existing selfdeterminants and viral antigens. increased efficiency in gp 120 antigen presentation by specific binding to cd4 and direct processing by cd4+ t cells has been reported by lanzavecchia et al. (1988 lanzavecchia et al. ( , 1989 . these authors have found that cd4+ cells can capture soluble gp120 and present it to gpl20-specific mhc class-iirestricted clones. as in other well-established autoimmune disorders, an association between hla haplotype and disease incidence has been reported for development of arc/aids (steel et al., 1988 ; jeannet et al., 1989; simmonds et al., 1991; puppo et al., 1991 ; louie et al., 1991) . such an association would be consistent with either model for the development of autoimmunopathic disease consequent to hiv infection. however, as in other instances of autoimmunity triggered or facilitated by foreign pathogens, the observation of similar syndromes and cellular lesions occurring in the absence of viral infection -idiopathic cd4+ t-lymphocytopenia or "immunodeficiency without virus" (fauci, 1993 ; smith et al., 1993 ; ho et al., 1993) -on a small number of patients with genetic predisposition would be more strongly in favour of the second model. in theory, the two models, although not mutually exclusive, differ significantly in the prospects for "cure" through the therapeutic elimination of virus. in the first case, complete elimination of virus would also remove the source of the "simulated self-antigen", i.e. gp120 bound to t4 cells, and autoimmune destruction of t4 cells would be abrogated. in the second case, however, removal of the inciting cross-reactive foreign antigen, gp120, would not necessarily result in a return of the immunoregulatory network to a predominantly suppressive mode. the new steady state of autoimmunity might be a stable state, sustained by the continued presence of bona fide tcspecific self-antigen. in practical terms, however, the possibility of completely eradicating an established hiv infection is unlikely, at least in the forseeable future. therefore, regardless of which model may prove to be correct, it would be useful to explore adjunctive interventions aimed specifically at controlling the autoimmunopathic destruction of t4 cells. in either case, t-cell vaccination using autologous cloned anti-gp120 cytotoxic cells would be expected to enhance (or induce) the antiidiotypic and other regulatory cells of the network, which would act to downregulate the activity of the anti-gp120/anti-self cytotoxic cells. this should occur whether or not the viral infection is concurrently controlled or eliminated, because the focus of intervention is the autoimmunopathic destruction which occurs as a result of the host response to infection, rather than the virocytopathic destruction which occurs as a result of infection per se. an associated reduction in viral burden by independent means, however, would be expected to synergize in limiting autoimmunopathic destruction by reducing antigenic stimulation to the autoimmunopathic effector cell. to the extent that the autoimmunopathic destruction of t4 cells is a major factor in the development of hiv-associated disease, such t-cell vaccination should serve to enable the hivinfected host to live with, integrate and compensate for the perturbations induced by a persistent viral infection. under these conditions, hiv infection might be expected to come to resemble infection with other persistent viruses, such as ebv, cmv and hsv, which are relatively innocuous in most cases. in addition, from the response to the proposed intervention, it will be possible to test the validity of the hypothesis and to expand it, for example, by looking for in vivo activation of anti-antigp120 suppressor cells and for their tcr specificities. considering the above-mentioned observations on the progressive destruction of fdc (follicular dendritic cells) by ct8+ cytotoxic cells during the latency period (pantaleo et al., 1993) , we are still facing a tissular anti-self immune response. again, the question is raised as to the nature of the antigen which transforms fdc into targets for cd8+ ctl. in view of the observed concomitant accumulation of viruses in the lymph nodes, it is reasonable to assume that cd8+ ctl might be activated by the large numbers of viral antigens presented by fdc. in addition, the antigen presentation function of fdc in the microenvironment of lymphoid organs may be favorable to the onset of a vicious circle or reinforcement of antigen presentation (atlan and hoffer-snyder, 1989; atlan and cohen, 1992; lehmann et al., 1993) , whereby the strength of activation against various antigenic determinants increases continuously, as discussed above. however, contrary to the tccell target models, there is no direct documented experimental indication of possible specific mechanisms responsible for the activation of cd8+ ctl against fdc. therefore, since we do not want to produce an overall suppression of antiviral cellular response, these cd8+ anti-fdc cytotoxic cells should be first characterized by some antigen specificity before a t-cell vaccination procedure against those cells can be tested. in the meantime, the proposed procedure described in the following section might also turn out to be effective in the context of the fdc target model. that would be the case to the extent that anti-gp120 ctl might participate in immunopathic responses involving fdc, as well as in the direct destruction of cd4+ t cells discussed above (sections i and ii/l). in view of the foregoing considerations, we propose that t-cell vaccination be explored first as a strategy for reducing direct autoimmunopathic tccell destruction in hiv infection. the t-cell vaccination technique (cohen, 1991a) would consist of reinjecting cloned autologous effector cytotoxic cells after in vitro activation and either irradiation or treatment with a crosslinking agent (such as glutaraldehyde) to prevent proliferation. thus, although the effector cells are presented to the regulatory network in an immunologically active form, they are both autologous and non-proliferative, hence not pathogenic. reinjection of these cells is intended to launch the network into a new stable state where the regulatory suppressor cells are strongly activated. the resulting change in connectivity of the network would suppress the development of active, pathogenic, effector or helper cells, and thereby suppress autoimmunopathicity. the key to the success of such an approach is identification of the responsible effector cell. as dis-cussed earlier, the working hypothesis is that cytotoxic effecters with specificity for gp120 are responsible for the immunopathic destruction of uninfected t4 cells that culminates in aids. however, it is still possible that autoimmunopathic destruction of t4 cells could be mediated by cytotoxic effecters with specificities directed against hiv antigens other than gp120, against cellular antigens which are crossreactive with hiv antigens other than gp120, or against cellular antigens which are not crossreactive with hiv (e.g. new cd4 epitopes exposed as a result of binding with gp120 (stricker et al., 1987; kowalski et al., 1989) ). the effects of t-cell vaccination with anti-gp120 cytotoxic effecters will be assessed by looking for desired induced modifications of the regulatory network, in the form of in vivo activation of anti-anti-gp120 suppressor cells and antibodies. qualitative and quantitative assessment of such responses will determine whether or not it is useful to pursue studies using effecters with other specificities. in the flow chart presented in table i, we outline a first protocol currently under development. we propose that studies be initiated in a small group of hiv seropositives with comparable viral burdens who have recently begun to manifest a decline in t4 cells. to facilitate the subsequent analysis, it will be preferable to study subjects who have been followed with serial t4 counts for at least 18 months, whose t4 level is above 500, and who, at study entry, demonstrate a significantly negative t4-cell slope. thus, a possible therapeutic effect could be subsequently assessed by looking for a slowdown of that slope. cell-mediated immune responsiveness will be assessed by standard skin test for delayed-type hypersensitivity (dth), since a correlation has been observed between favourable clinical history of hiv-seropositive patients and high dth response . the post-vaccination follow-up might indicate that dth response could be used as a criterion for future patient selection. peripheral blood t lymphocytes will be plated in limiting dilution with irradiated autologous antigen-presenting cells and gpl20. cloning will be performed at the outset to permit calculation table i effectiveness of vaccination ; therapeutic efficacy (*) it is planned to prepare autologous ebv-transformed b cells into which are introduced the gene for gp120 or cd4. this would provide a permanent source of stimulator and target cells for ctl generation and testing, as well as for growth expansion. (**) cd4 antigenicity should also be tested, since anti-cd4 ctl could also be activated through interactions of cd4 with molecules different from gp120. of precursor frequencies prior to therapeutic intervention. responding hiv-uninfected clones will be expanded in vitro and then tested for antigen specificity, cytotoxicity, mhc restriction and cd4/8 phenotype. we suggest that t-cell receptor (tcr) gene usage also be explored, since use of a restricted number of vp genes might permit the future development of a non-autologous and therefore generally applicable preparation (cohen, 199 1 a) . this technique has been used successfully in some instances of experimental autoimmunity (howell et al., 1989; vandenbark et al., 1989; offner et al., 1991) . in eae (owhashi et al., 1988) and eat (texier et al., 1992) , monoclonal antibodies against tcr of antigen-specific effector t-cells produced a protective effect similar to that induced by vaccination against the effector cells themselves. moreover, in eae, tcr specific for the encephalitogenic determinant of mbp use similar va and v chains in two different species, although the m i-! c and antigenic determinants are different (burns el al., 1989) . similarly, in our case, a characterization of conserved tcr structures could dispense the technique with the need for cumbersome autologous preparations on a patient per patient basis. initially, however, autologous antigenspecific cytotoxic clones will be expanded and activated in vitro, followed by irradiation or treatment with a cross-linking agent, prior to reinoculation into the donor. it is anticipated that the effectiveness of vaccination may be evaluated by serial measurements of(i) dth responsiveness, (ii) the amlr (autologous mixed lymphocyte reaction) against the injected t-cell clone, (iii) levels of circulating t cells with idiotypic specifications identical to injected cells (anti-gp120), and (iv) levels of antiidiotypic and antiergotypic t-cell responses (anti-anti-gp120). on the other hand, measures of therapeutic efficacy would be monitored by following peripheral blood t4 cell counts (both absolute and as a percent of total t cells) and clinical indicators of disease progression. measures of static virus burden (hiv-dna pcr) and active virus burden (glycine-dissociated p24 antigen, hiv-rna pcr) might be included to monitor for secondary effects on virus clearance. the major thrust of aids research has been identification of the responsible virus and elucidation of its biology,. in the hope of discovering means by which the virus might be successfully controlled or eliminated. the underlying rationale has been that the virus, hiv, directly causes the disease syndrome known as aids. therefore, most strategies for vaccination have been based on the production of neutralizing antiviral antibodies. however, several lines of evidence indicate that antiviral antibody production could be counterproductive in favouring viral infection by preventing efficient cell-mediated immunity. this suspicion is based on (1) the known antagonism between humoral and cellular response and (2) the observed mimicry between hiv protein gp120 and immunoglobulins as well as self-epitopes in other components of the immune system (beretta el al., 1987; golding et al., 1988; young, 1988; bjork, 1991) which may enable the virus to escape a strong host-immune response (sastry and arlinghaus, 1990) . that is why an alternative strategy was proposed to develop anti-hiv vaccine that would elicit strong cell-mediated immunity (cmi) by virus-specific ctl in the absence of neutralizing antibody production (sastry and arlinghaus, 1990 ; shearer and clerici, 1992; salk et al., 1993) . however, as discussed earlier, it has become apparent that several aspects of hiv infection are difficult to reconcile with the notion that the virus itself is the sole direct and proximal cause of disease. moreover, some of the viral stimulation of ctl activity itself, in as much as it does not succeed in completely eradicating viral infection, may be maintained and diverted as a factor of autoimmunity directed against cd4+ t cells, both infected and non-infected, as well as against fdc in lymphoid organs. in other words, an autoimmunopathic disorder precipitated by hiv infection may be an important proximal cause of hiv-related disease. therefore, in parallel with a vaccine strategy aimed at developing early cm1 to prevent chronic hiv infection and conversion to seropositivity, a therapeutic-prophylactic strategy against these autoimmunopathic disorders is necessary for hiv-infected seropositive patients. the thesis presented above is that a major cause of the development of hiv-related disease is the destruction of t4 cells by anti-gp120 cytotoxic lymphocytes. experimental testing of this thesis is proposed because such cytotoxic lymphocytes have been detected in both seropositive and seronegative individuals. they are postulated to kill via gp120 on the surface of uninfected, as well as infected t4 cells, and also perhaps via a tcspecific self-antigen which is cross-reactive with gp120. discussed within the context of network theory, this becomes a testable hypothesis, since t-cell vaccination with autologous anti-gp120 cytotoxic lymphocytes is expected to specifically abrogate the activity of such immunopathic effector cells. the success of such an approach will depend both upon the relative importance of immunoprotective as compared to autoimmunopathic effects of anti-gp120 effector cells and upon the existence of immunoprotective factors with specificities for other hiv antigens such as p24 and p17. in addition, such direct cytotoxicity of anti-gp120 ctl against cd4+ t cells is certainly not the only possible autoimmunopathic effect which could be triggered by hiv infection. as discussed in the introduction and in section ii (2), one might consider that fdc, rather than or in addition to t4 cells, could be the target of autoimmune mechanisms triggered by hiv infection. in other words, cytotoxic cd8+ cell activity against fdc would be triggered by hiv antigens and would be responsible for the observed destruction of the lymph node reticulum, leading to increased viraemia and tccell loss. if such putative anti-fdc-specific effector cells could be identified in seropositive patients and expanded in vitro, a t-cell vaccination procedure against those cells could be designed and attempted. in any case, a significant consequence of viewing aids in this manner is that it shifts the focus of emphasis from hiv alone to hiv in the context of the particular immune system into which it is introduced. the discovery of new facts and observations has rendered inadequate the classical clonal selection concept of a simple cause-and-effect relationship between exposure to a foreign antigen and the production of specific antibodies against that antigen. rather than a mere system of defence against foreign invaders, the immune system appears today as a complex dynamic network of interacting elements -effector cells, antigen-presenting cells and various regulatory cells -all modulated by genetic factors, cytokines and mutual recognition units. in particular, recent studies on autoimmunity (see e.g., in addition to the above mentioned, pereira et al., 1989 ; coutinho and bandeira, 1989; cohen, 1991b) ) have shown that immune responses at the molecular and cellular level are normally triggered by selfconstituents (self-antigens, mhc molecules, idiotypes etc.), at least as much as by molecules or cells coming from the outer world. thus, as discussed elsewhere (atlan and cohen, 1989a, 1989b) , "problems of complexity, generation of diversity and self-organization have entered the field of immunology" and the immune system has emerged "as an evolving computing network or self-organizing entity -a non-programmed history of encounters with partially random internal and external antigenic stimuli is constantly integrated and serves to modulate and specify the more general initial genetic determinations". this shift in emphasis means that one must focus on the complex interactions between hiv as an incoming stimulus and the immune system as a pre-existing functional network similar to the nervous and neuro-endocrine systems, in a constant process of self-organization. whereas reduction of viral burden by an appropriate combination of chemotherapy, active immunization and passive administration of hyperimmune globulin may be useful and should be pursued, such measures may not be sufficient, and an autoimmunopathic component of the disease may need to be addressed independently. to this end, the goal of therapeutic intervention would be to help the immune system reorganize itself and retain or regain a functional state capable, if need be, of coexisting indefinitely with a persistent viral infection. such a goal is reminiscent of the use of bcg vaccination against tuberculosis in the pre-antibiotic era. drawing on the accumulated insights from other, better understood autoimmune disorders, it has been proposed that t-cell vaccination be investigated in hiv-infected individuals as a means to strengthen the host's immune regulatory network and its consequent control of autoimmunopathicity. its specific implementation has the advantage of not requiring a detailed understanding of mechanisms in order to be effective, since it emerges from the natural history of hiv/aids considered in the light of other autoimmune disorders precipitated by microorganisms. in fact, from the results of its application, it may be possible to learn more about the dynamics of the human immune system exposed to hiv. a more in-depth understanding of these dynamics, coupled with a precise identification of the specificity of the autoimmunopathic effector t-cell clones (by assessment of their tcr variable gene usage), should contribute much to the development of an effective therapeutic and potentially prophylactic vaccine against hivrelated disease. as discussed above, such a t-cell receptor vaccine would be more easily and broadly vaccine basis. applicable than the autologous t-cell implemented on a patient-by-patient par un antigene d'origine virale, le plus probable, mais non le seul, &ant la proteine d'enveloppe gp120. sur la base de cette hypothese, les auteurs proposent d'appliquer une procedure de vaccination par cellules t contre des celhrles effectrices responsables d'une activite autoimmune pathologique chez des sujets seropositifs pour le vih. cette procedure est semblable a celle mise au point chez i'animal sur des modtles experimentaux d'autoimmunitt, actuellement testee chez l'homme sur des maladies autoimmunes. le but recherche est d'empkher la destruction de cellules t et l'installation de deficience immunitaire chez des sujets seropositifs pour le vih. mais en dehors de son efficacite cventuelle, cette procedure presente l'avantage d'un test therapeutique d'oh l'on est en droit d'attendre de nouvelles informations sur la pathologie immunitaire du sida. mats-cl&s: sida, autoimmunitt, lymphocyte t, immunotherapie; reseaux, vaccination par cellules t, immunopathogenese; revue. ceil dysfunction and depletion in aids: the programmed cell death hypothesis aids as immune system activation: a model for pathogenesis. c/in aids as immune system activation. -ii. the panergic amnesia hypothesis t cell vaccination of hiv-seropositives : a therapeutic test for the autoimmune component of aids preface, in "theories of immune networks introduction to immune networks paradoxical effects of suppressor t cells in the onset of adjuvant arthritis: neural network analysis can aids be prevented by t-cell vaccination? immunol simulation of the immune cellular response by small neural networks, in "theories of immune networks incubation period of aids in san francisco vaccination against autoimmune encephalitis with t-lymphocyte lines reactive against myelin basic protein the rapid isolation of clonable antigen-specific t lymphocyte lines capable of mediating autoimmune encephalomyelitis spontaneous remission and acquired resistance to autoimmune encephalomyelitis (eae) are associated with suppression of t-cell reactivity: suppressed eae effector t cells recovered as t-cell lines t-cell vaccination in autoimmune diseases hiv env glycoprotein shares a cross-reacting epitope with a surface protein present on activated human monocytes and involved in antigen presentation hiv-l : seven facets of functional molecular mimicry both rat and mouse tcrs specific for the encephalitogenic determinant of mbp use similar v, and vp chain genes even though the mhc and encephalitogenic determinants being recognized are different isolation of myelin basic protein-reactive t-cell lines from normal human blood regulation of autoimmune disease: physiological and therapeutic natural id-anti-id networks and the immunological homunculus t-cell vaccination and suppression of autoimmune disease physiological basis of t-cell vaccination against autoimmune disease t-cell vaccination in immunological disease the immunological homunculus and autoimmune disease, iq "molecular autoimmunity the cognitive principle challenges clonal selection the cognitive paradigm and the immunological homunculus network regulation of autoimmunity : an automaton model t-lymphocyte clones illuminate pathogenesis and affect therapy of experimental arthritis. arthritis rheum autoimmunity, microbial immunity and the immunological homunculus tolerize one, tolerize them all : tolerance is self-assertion human immunodeficiency virus and acquired immunodeficiency syndrome : correlation but not causation vaccination against autoimmune mouse diabetes with a t-cell epitope of the human 65-kda heat shock protein a suppressor t-lymphocyte cell tine for autoimmune encephalomyelitis analysis of human immunodeficiency virusinfected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell-resolution cd4+ t-lymphocytopenia without hiv infection. no lights, no camera, just facts loss of envelope antigens of htlv-iii/lav, a factor in aids pathogenesis? identification of homologous regions in human immunodeficiency virus i gp41 and human mhc class ii bl domain activation-induced death by apoptosis in cd4+ t cells from human immunodeficiency virus-infected asymptomatic individuals naturally occurring antibodies against nine common antigens in human sera. -i. detection, isolation, and characterization does the hiv envelope induce a chronic graft-versus-hostlike disease? t-cell vaccination in multiple sclerosis: a preliminary report idiopathic cd4+ t-lymphocytopenia. immunodeficiency without evidence of hiv infection unusually high frequencies of hivspecific cytotoxic t lymphocytes in humans an idiotypic network model of aids immunopathogenesis lines of t lymphocytes induce or vaccinate against autoimmune arthritis natural autoimmunity vaccination against experimental allergic encephalomyelitis with t-cell receptor peptides human immunodeficiency virus dna is present in a high percentage of cd4+ lymphocytes of seropositive individuais selective depletion in hiv infection of t cells that bear specific t-cell receptor vp sequences aids two months after primary human immunodeficiency virus infection autoreactive cytotoxicity in hivinfected individuals hla antigens are risk factors for development of aids towards a network theory of the immune system cell-mediated suppression of hiv-specific cytotoxic t lymphocytes isolation of t-cell line capable of protecting mice against collagen-induced arthritis a six-year follow-up of hiv-infected homosexual men with lymphadenopathy : evidence for an increased risk for developing aids after the third year of lymphadenopathy the interactions between mycobacterium tuberculosis and the immune system leading to the development of autoimmune disease hiv infection: facts and hypotheses approaches to aids therapy association of human immunodeficiency virus infection and autoimmune phenomena antibodies to cd4 in individuals infected with human immunodeficiency virus type 1 harming and protecting responses to hiv t cells can present antigens such as hiv gp120 targeted to their own surface molecules determinant spreading and the dynamics of the autoimmune t-cell repertoire therapeutic vaccination against adjuvant arthritis using autoimmune t-lymphocytes treated with hydrostatic pressure anti-idiotypic network induced by t-cell vaccination against experimental autoimmune encephalomyelitis control of experimental autoinimune encephalomyelitis by t cells responding to activated t cells influence of host genotype on progression to aids among hivinfected men human t-cell lymphotropic virus 111s glycoprotein (gp120) bound to cd4 determinants on normal lymphocytes and expressed by infected cells serves as target for immune attack aids research turned upside down immunological consequences of hiv infection: advantage of being low responder casts doubts on vaccine development new insights on the mechanisms of cd4+ lymphocytes depletion in aids aids virus infection and autoimmunity : a perspective of the clinical, immunological, and molecular origins of the autoallergic pathologies associated with hiv disease graft-vs-host reaction limited to a class ii mhc difference results in a selective deficiency in l3t4+ but not in lyt-2+ t helper cell function t-cell receptor peptide therapy triggers autoregulation of experimental encephalomyelitis identification of hiv-l envelope glycoprotein in the serum of aids and arc patients autoantibodies against liver cell membrane detected by enzymelinked immunosorbent assay in acute and chronic liver disease protection from experimental allergic encephalomyelitis conferred by a monoclonal antibody directed against a shared idiotype on rat t-cell receptors specific for myelin basic protein the immunopathogenesis of human immunodeficiency virus infection v-region connectivity in t cell repertoires hiv-specific cytotoxic t lymphocytes igm, igg and iga rheumatoid factors and circulating immune complexes in patients with aids and aids-related complex with serological abnormalities major histocompatibility gene products and human immunodeficiency virus infection prevention of experimental autoimmune thyroiditis through the antiidiotypic network a lectin-binding soluble factor released by cd8+cd57+ lymphocytes from aids patients inhibits t-cell cytotoxicity prospects for the control of aids by immunizing seropositive individuals a strategy for prophylactic vaccination against hiv a novel hiv vaccine strategy human immunodeficiency virus infection and autoimmune phenomena shedding and interspecies type seroreactivity of the envelope glycopolypeptide gp120 of the human immunodeficiency virus the reservoir for hiv-l in human peripheral blood is a t cell that maintains expression of cd4 acquired immune-deficiency syndrome (aids) : a consequence of allogeneic ia-antigen recognition aids: an autoimmune pathologic model for the destruction of a subset of helper t lymphocytes t helper cell immune dysfunction in asymptomatic, hiv-l seropositive individuals: the role of thl-th2 cross-regulation analysis of host-virus interactions in aids with anti-gpl20 t-cell clones : effect of hiv sequence variation and a mechanism for cd4+ cell depletion determinants of hiv disease progression : six-year longitudinal study in the edinburgh haemophilia/hiv cohorts unexplained opportunistic infections and cd4+ t-lymphocytopenia without hiv infection. an investigation of cases in the united states hla haplotype al b8 dr3 as a risk factor for hiv-related disease an aids-related cytotoxic autoantibody reacts with a specific antigen on stimulated cd4+ t cells apoptosis as a mechanism of cell death in cultured t lymphoblasts acutely infected with hiv-l protection from experimental autoimmune thyroiditis conferred by a monoclonal antibody to t-cell receptor from a cytotoxic hybridoma specific for thyroglobulin immunization with a synthetic t-cell receptor vregion peptide protects against experimental autoimmune encephalomyelitis arthritis induced by a t-lymphocyte clone that responds to mycobucterium tuberculosis and to cartilage proteoglycans cloning of the mycobacterial epitope recognized by t lymphocytes in adjuvant arthritis effects of inoculation with attenuated autologous t cells in patients with rheumatoid arthritis inhibition of antigen-induced lymphocyte proliferation by tat protein from hiv-l adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis hiv-l gpl20-mediated immune suppression and lymphocyte destruction in the absence of viral infection networks of automata and biological organization hiv and hla similarity hivinfected humans, but not chimpanzees, have circulating cytotoxic t lymphocytes that lyse uninfected cd4+ cells mhc-restricted depletion of human myetin basic protein-reactive t cells by t cell vaccination hypothesis: aids is an autoimmune disease directed at the immune system and triggered by a lymphocytic retrovirus virus-triggered aids: a t-cellmediated immunopathology? we are grateful to irun r. cohen, rene de vries, maurizio zanetti and joelle nataf for their helpful comments and fruitful discussions.the work of h.a. was supported by an ishaiah horowitz scholarship in residence at the hadassah university hospital, jerusalem. key: cord-293379-c4qdmkw5 authors: weiss, robin a title: hiv and aids: looking ahead date: 2003 journal: nat med doi: 10.1038/nm0703-887 sha: doc_id: 293379 cord_uid: c4qdmkw5 although the future of hiv science is uncertain, we need to reappraise hiv diversity, pathogenesis and immunity. the aids pandemic threatens the success of existing vaccine programs and may accelerate the emergence of new infectious diseases. a pollo conferred on cassandra the gift of clairvoyance but later added the caveat that no-one would listen to her prophesy; thus, her warnings of the fall of troy and the house of agamemnon went unheeded. doctors and scientists are less farsighted, and making predictions exposes us to ridicule in hindsight. not long after the us surgeon general declared that it was "time to close the book on infectious diseases", hiv began to 'cruise' the san francisco bath houses while severe acute respiratory syndrome (sars) lay further in the future. in 1984 margaret heckler, then us secretary for health and human services, declared on behalf of the national institutes of health that "we hope to have a [aids] vaccine ready for testing in about 2 years." how foolhardy it was to accept an invitation from nature medicine to comment on the next 20 years of hiv science! if we could foresee the future of research, then we would surely be doing it now. the other contributors to this issue have made the sensible prognostications, whereas anything i put forward here must be regarded as unsure prediction, idle speculation or even wild conjecture 1 . the pace of hiv science, like all science, is driven by technology with unexpected breakthroughs. without pcr, we would not have accurate measurements of hiv viral load and turnover; without rapid dna sequencing and bioinformatics, we would not have such an exquisite database on hiv genetic variation; and if plant virologists had not been curious to investigate gene silencing, we would not have rna-mediated interference (rnai) as a medical research tool. thus, my message for future progress on hiv is that we ignore non-hiv research at our peril. no doubt this prophesy will fall on deaf ears at the funding agencies, especially those in the charitable sector: the late bernie fields' exhortation 2 to "get back to basics" in hiv science is not part of their mission. our colleagues at the institut pasteur must have felt as frustrated as cassandra when so few heeded their first report 3 in may 1983 on a previously unknown retrovirus that was possibly associated with aids. at that time, 'lymphadenopathy virus' (lav) was just one more candidate agent alongside other animal and human viruses, and alongside an idea that a fungal infection secretes a cyclosporin-like immunosuppressant. but none of us present at the cold spring harbor laboratory meeting on human retroviruses in the fall of 1983 should have failed to be impressed by how the french scientists hardened their evidence. by early 1984, they had detected the virus in individuals with aids, including gay men, two brothers with hemophilia 4 and a heterosexual couple 5 from africa and their child, and had pointed out the similarity of lav to animal lentiviruses on account of its cytopathic effects and morphology 6 . they were accurate in almost every finding, including the only correct interpretation of the open reading frames when the viral genome was cloned and sequenced 7 . hard on the heels of the second french report 4 came news of other hiv isolates from the united states 8, 9 . apart from the overoptimistic claims of a vaccine, early hiv research was extraordinarily fruitful (see accompanying reviews in this issue [10] [11] [12] . propagating hiv in t-cell lines provided ample antigen for diagnostic tests of infection that by 1985 had been translated into kits suitable for the mass screening of blood donors. cd4 was identified as the cellular binding receptor for hiv in 1984. the importance of the regulatory and auxiliary genes began to be elucidated in 1985. azidothymidine (zidovudine) became the first antiretroviral drug to enter clinical trials in 1986. meanwhile, we began to grasp the scale of the aids pandemic unfolding before our eyes: 'slim' disease in africa was indeed aids 13 . figure 1 illustrates both the success of hiv science and the formidable challenges before us; it contrasts the control of mortality from aids in the united states with its exponential rise in sub-saharan africa. but fig. 1a belies the heterogeneity of the african epidemic. as the 'four-city' studies have shown 14 , we still do not know why hiv spread explosively in some places and not in others. fifteen years ago, aids in south africa was seen in a handful of gay white men who had traveled to the united states, but now more than four million south african black men, women and children are infected with hiv. by contrast, it was estimated 15 years ago that about 7% of adults in kinshasa were infected, but this proportion remained stable until recently, despite the imploding infrastructure and human conflict that the democratic republic of congo has suffered. predicting the future of the hiv epidemic will be no easier than interpreting the recent past. epidemiological evidence for the transmission of hiv by sexual and parenteral routes was clear before hiv was identified, and mother-to-child transmission soon after. the modes of transmission remain the same today and seem unlikely to change tomorrow. i previously questioned 1 whether biting insects with large mouthparts might act as 'dirty needles' to transfer hiv passively, given that another lentivirusequine infectious anemia virusis transmitted in this way. but there is no evidence of transmission by insects, and if it were occurring then we would expect to see more children seroconverting before puberty. it was recently postulated 15 that in africa, contaminated syringes and needles are responsible for more hiv transmission than is sexual contact. although the number of infections by unsterile injections may have been underestimated during the pandemic phase of hiv, as well as during the mid-twentieth century 16 , sexual spread is driving the african pandemic 17, 18 . if injection were the main route of hiv infection in africa, as it has been in eastern europe and china, then again we would expect to see more children of hiv-negative parents developing aids. as valdiserri et al. 19 argue in this issue, much has been accomplished in reducing the transmission of hiv and, given politi-cal will, persuasive 'risk' education and sufficient resources, "the science exists to turn the pandemic around." certainly, the continuing spread of disease could be slowed significantly, as has been seen in senegal, thailand and uganda, but whether without an efficacious vaccine we can reduce r 0 to less than onethat is, reduce the mean rate of transmission from one infected person to less than one other personremains speculative. india is currently estimated to have 4 million people infected with hiv (second only to south africa), and this number could rise to 24 million in the next 10 years. perhaps we should not be too pessimistic. people do change their outlook and lifestyle in the face of devastating disease. for example, male circumcision has been identified as a factor that lessens the risk of female-to-male hiv infection in africa 20 . who would have thought a few years ago that men imbued with their traditional social customs would readily come forward to take part in randomized controlled trials of adult circumcision? 21 whereas the 'sexual synapse' is a frequent route of hiv transmission from one person to anotherone that may be blocked specifically by a condom and hopefully one day by vaginal viricidesthe 'immunologi-cal synapse' is a pathway for virus transmission from cell to cell (see accompanying review in this issue) 22 and has been elegantly shown 23 for human t-cell lymphotropic virus type i. we are only just beginning to understand the impact of the multiple delivery of virions from dendritic or other antigen-presenting cells to cd4 + t-helper-cells. i propose that the immunological synapse may account for the recent observation that although only a small proportion of cd4 + t-cells in a lymphoid organ are infected by hiv, these cells contain several proviruses 24 . this type of 'multihit' infection at the cellular level may overcome the saturatable restriction factors of the host cell 25, 26 . packaged rna genomes transcribed from more than one provirus in the same infected cell will assemble into heterozygous virions, which in turn will accelerate genetic recombination 24 and the evolution of drug resistance and immune escape. in the future, we should pay more attention to the comparative pathology of lentiviruses, including hiv-2 (ref. 27) . in this issue, stevenson 11 points to the lessons to be learned from primates that are naturally infected with a high viral load but do not develop disease. he contrasts oncoviruses to hiv and the primate lentiviruses that can infect nondividing macrophages and dendritic cells. i argue further that all lentiviruses are macrophage-tropic, but only some infect lymphocytes (primate and feline immunodeficiency viruses). for example, let us consider maedi-visna virus, which is solely macrophage-tropic. maedi-visna in sheep is the prototypic disease from which lentiviruses derive their name 28 . infected sheep develop a wasting disease and neurodegeneration similar to that seen in humans with aids, but they do not show t-helper-cell immune deficiency. as maedi-visna is remorselessly progressive, with a high rate of mortality, i have argued previously 29 that the infection, activation and apoptosis of t-cells in hiv are epiphenomena alongside the underlying progression of macrophage disease. like the prophecies of cassandra, this view remains unheeded perhaps because it would necessitate a complete reappraisal of both hiv pathogenesis and the inability of the immune system to ultimately control lentivirus infection. such a question has been posed for the katie ris variation observed in rna viruses in general 30 ; for hiv, the answer is probably a lot of each. hiv generates variants at a far greater rate than do other rna viruses such as measles, polio and even influenza 31 (fig. 2) . the rapid radiation of hiv-1 group m into the subtypes or clades that comprise today's pandemic strains and all of their variants could have arisen from a conjunction of two features of hiv: the extraordinarily high level of sustained replication and turnover in vivo 11, 32 and the functional tolerance of amino acid substitutions. it is estimated that humans were first infected with hiv-1 group m about 70 years ago 33 and with hiv-2 about 60 years ago 34 , with the viruses crossing from chimpanzees and sooty mangabeys, respectively 35 . in the early years, hiv-1 radiated out into the different clades that we know today, probably from small founder populations of virus. the regions in which hiv-1 has been present the longest have the most complex array of genotypes (fig. 2) . in the next 20 years the pattern will change, and an increasing number of circulating recombinant forms (crfs) of hiv-1 will become apparent. thus, the neat geographic delineation of subtypesb in the americas, e in thailand, a and d in east africa and c in southern africaare likely to be superseded by crf viruses. indeed, crfs between hiv-1 groups m and o have been described 36 , even though the parental genomes derive from distinct zoonotic events 35 . it is possible that natural recombinants could arise between hiv-1 and hiv-2 now that hiv-1 has spread across west africa, where hiv-2 was already endemic. although there are some constraints to the co-packaging of hiv-1 and hiv-2 rna 37 , it is worth investigating the possibility of hybrids in dually infected persons. does hiv variation matter? although there is no evidence that hiv has evolved in terms of virulence or modes of transmission in the past 20 years, evolution of drug resistance 12 and of immune escape (see accompanying review in this issue 38 and refs. 39,40) clearly occurs under selective pressure. thus, hiv mutation and recombination have a great impact on therapy and vaccine design. i remain to be convinced, however, that the emphasis of vaccine design on the basis of clades is hiv science. efficacious, broadly based vaccines require immunogens representing those regions of the virus that change the least. when these have been identified, clades can be addressed. for humoral immunity, the challenge is not only the immunogen itself but also the access of antibodies to the neutralization targets. given the effect of the n-linked sugars of gp120 on immune escape from hiv 39 , the glycobiology of hiv is back in fashion. will the drop in aids mortality owing to antiretroviral therapy (fig. 1) be maintained so that people on treatment can expect a normal life span? this will depend on the ability of the virus to develop multidrug resistance while remaining fit for transmission. we shall need new drug targets 12 , but a big practical challenge in the future will be to marry good drug therapy to easy adherence, particularly in resourcepoor settings. this means that drug formulation must be designed to optimize appropriate use. even then, if drugs administered to one infected individual are shared among their family or community such that several persons are simultaneously taking suboptimal doses, this could be a recipe for the rapid evolution and spread of multidrug-resistant hiv strains. in the future, i expect that host genetic variation will also have a larger role in hiv science. in addition to identifying individual host factors 26, [41] [42] [43] , whole-genome scanning for pharmacogenomics and for what i call 'infectogenomics' (host genes that affect the virulence of infection) will provide information on how to better manage hiv infection. hiv may have a severe impact on several of the immunization programs for children and adults. inactivated or subunit vaccines may simply be ineffective in people infected with hiv: this has been shown for the pneumococcal polysaccharide vaccine 44 , although antiretroviral therapy may restore responsiveness 45 . with viruses, hiv-positive individuals have the potential to become long-term shedders of what would otherwise be acute, short-lived infections, changing the dynamics of immunization and eradication. thus, the large numbers of children with aids in africa may impede campaigns to eradicate measles and polio and to protect against yellow fever. a recent detailed review 46 on the safety, immunogenicity and effectiveness of vaccines in children with hiv concludes that the benefits currently far outweigh the risks. nevertheless, epidemiological modeling will be needed to see whether one can minimize the untoward effects of hiv on other infectious diseases. another concern is that an 'attenuated' vaccine may itself act as a virulent pathogen in the immunocompromized individual. case reports of severe complications of bacillus calmette-guérin (bcg), measles and polio have been reported, and world health organization (who) guidelines recommend withholding bcg and yellow fever vaccines from symptomatic children infected with hiv 46 . hiv hiv 6 yea 6 yea 10% 10% c c j g d figure 2 the scale of hiv variation. sequence divergence of envelope glycoproteins of hiv (gp120, v2-c5) compared with that of influenza a h3 (ha1). the length of the spokes indicates the degree of divergence with the scale indicated. hiv variation in a single person 6 years after infection (9 genomes analyzed) is similar to that of worldwide influenza a (96 genomes analyzed) in a single year. the greatest degree of variation is in the democratic republic of congo, where hiv first developed and has diversified into subtypes a-k (except for subtype b, prevalent in the west, and subtype e, prevalent in thailand). adapted with permission from ref. 31 . gulliver's travels in hivland aids: time to turn to basic science isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) isolation of new lymphotropic retrovirus from two siblings with haemophilia b, one with aids isolation of human t-lymphotropic retrovirus (lav) from zairian married couple, one with aids, one with prodromes a new type of retrovirus isolated from patients presenting with lymphadenopathy and acquired immune deficiency syndrome: structural and antigenic relatedness with equine infectious anaemia virus nucleotide sequence of the aids virus frequent detection and isolation of cytopathic retroviruses (htlv-iii) from patients with aids and at risk for aids isolation of lymphocytopathic retroviruses from san francisco patients with aids hiv and aids: 20 years of science hiv-1 pathogenesis 20 years of therapy for hiv-1 infection slim disease: a new disease in uganda and its association with htlv-iii infection multicentre study on factors determining differences in rate of spread of hiv in sub-saharan africa: methods and prevalence of hiv infection let it be sexual: how health care transmission of aids in africa was ignored the injection century: massive unsterile injections and the emergence of human pathogens expert group stresses that unsafe sex is primary mode of hiv transmission in epidemiology: sexual transmission of hiv in africa accomplishments in hiv prevention science: implications for stemming the epidemic male circumcision and risk of hiv infection in sub-saharan africa: a systematic review and meta-analysis male circumcision: current epidemiological and field evidence hiv-1 transmission, acute infection, and the quest for strategies to prevent infection spread of htlv-i between lymphocytes by virus-induced polarization of the cytoskeleton multiply infected spleen cells in hiv patients cellular inhibitors with fv1-like activity restrict human and simian immunodeficiency virus tropism restriction of multiple divergent retroviruses by lv1 and ref1 human immunodeficiency virus type 2 cultivation of visna virus in tissue culture lentivirus tropism and pathogenesis are rna viruses adapting or merely changing? evolutionary and immunological implications of contemporary hiv-1 variation modelling viral and immune system dynamics timing the ancestor of the hiv-1 pandemic strains tracing the origin and history of the hiv-2 epidemic aids as a zoonosis: scientific and public health implications human immunodeficiency virus type 1 intergroup (m/o) recombination in cameroon nonreciprocal packaging of human immunodeficiency virus type 1 and type 2 rna: a possible role for the p2 domain of gag in rna encapsidation antibody neutralization and escape by hiv-1 rapid evolution of the neutralizing antibody response to hiv type 1 infection isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein the effect of genetic variation in chemokines and their receptors on hiv transmission and progression to aids global survey of genetic variation in ccr5, rantes, and mip-1α: impact on the epidemiology of the hiv-1 pandemic 23-valent pneumococcal polysaccharide vaccine in hiv-1-infected ugandan adults: double-blind, randomised and placebo controlled trial smallpox vaccination and patients with human immunodeficiency virus infection or acquired immunodeficiency syndrome disseminated vaccinia in a military recruit with human immunodeficiency virus (hiv) disease aids-related malignancies risk of human immunodeficiency virus infection in herpes simplex virus type 2-seropositive persons: a meta-analysis interactions between herpes simplex virus type 2 and human immunodeficiency virus type 1 infection in african women: opportunities for intervention infection with gb virus c and reduced mortality among hiv-infected patients effect of coinfection with gb virus c on survival among patients with hiv infection hiv-1 suppression during acute scrub-typhus infection decrease in human immunodeficiency virus type 1 load during acute dengue fever catastrophic ape decline in western equatorial africa vaccine for aids and ebola virus infection hiv and aids in relation to other pandemics there is much concern over the risk of reintroducing smallpox vaccination to people infected with hiv 47 because disseminated vaccinosis may ensue, as has been reported in an hiv-positive military recruit 48 . aids is characterized by opportunistic infections and by what we may call opportunistic neoplasms 49 . some opportunistic infections in turn exacerbate hiv in a vicious cycle. thus, genital herpes simplex infection is a risk factor for hiv transmission 50 , while hiv increases and prolongs herpes shedding 51 . other concurrent infections may ameliorate the risk or severity of hiv infection, as has been reported for gb virus c 52,53 , scrub-typhus 54 and dengue 55 . elucidating the mechanisms of cross-protection may give us clues to controlling hiv.many opportunistic infections are either zoonoses or come from nonparasitic, freeliving microbes. although they are typically not transmitted between humans, this pattern might change in the future if the opportunistic infections were to hop from one immunocompromised host to another. high-density immunodeficient populations are arguably unique in the annals of host-parasite evolution 1 .thus, viruses, bacteria, fungi and protozoa now have 42 million hiv-infected people in whom to adapt to human parasitism. zoonoses occur naturally, but the prevalence of hiv infection could greatly increase the chances of an infection of animal origin, such as sars, influenza, ebola or nipah viruses, to adapt more rapidly to human transmission. individuals with aids would be the 'superspreaders', as they are for tuberculosis. it is perhaps reassuring that such a horrific situation has not yet occurred, but predictive modeling is required to determine whether it could occur in the future. the aids pandemic compounds the threat from the deliberate or accidental release of new infectious agents. like cassandra, i shall end with a true prophesy that is at the same time optimistic and grim: 20 years from now, the risk of further lentivirus transfer from apes to humans 35 will approach zero because feral chimpanzees will be extinct, thanks to the bushmeat trade and the ebola epidemic 56 , unless the apes are protected by immunization. who would have predicted 20 years ago that we would have a vaccine for ebola before one for hiv 57 key: cord-334010-gxu0refq authors: banerjee, nilotpal; mukhopadhyay, sumi title: viral glycoproteins: biological role and application in diagnosis date: 2016-01-18 journal: virusdisease doi: 10.1007/s13337-015-0293-5 sha: doc_id: 334010 cord_uid: gxu0refq the viruses that infect humans cause a huge global disease burden and produce immense challenge towards healthcare system. glycoproteins are one of the major components of human pathogenic viruses. they have been demonstrated to have important role(s) in infection and immunity. concomitantly high titres of antibodies against these antigenic viral glycoproteins have paved the way for development of novel diagnostics. availability of appropriate biomarkers is necessary for advance diagnosis of infectious diseases especially in case of outbreaks. as human mobilization has increased manifold nowadays, dissemination of infectious agents became quicker that paves the need of rapid diagnostic system. in case of viral infection it is an emergency as virus spreads and mutates very fast. this review encircles the vast arena of viral glycoproteins, their importance in health and disease and their diagnostic applications. being an obligate intracellular parasite [32] , virus is the most deadly microbe to be dealt with. globally it accounts for extremely high morbidity and mortality throughout the age groups of people [3, 62] . thousands of new viral strains are discovered till date affecting people producing a huge global burden of viral infections resulting immense challenge towards healthcare system [9, 20, 48] . with the capability of fast mutation, viruses affect the host cells with new and newer mechanisms. so to detect them at the earliest, there is an extreme need of dynamic diagnostic system. glycans are major components of the outermost surface of viruses. thus, majority of the interactions of viral pathogens with their hosts are influenced by the pattern of glycans and glycan-binding receptors that each expresses. [5, 95, 98] glycans are most complex biomolecules due to extensive branching of carbohydrates, and a variety of glycoproteins have been identified in human viral pathogens. these pathogenic glycans either virus encoded or host derived usually elicit high humoral responses in human body [34] . these virus specific high levels of glycan specific antibodies have been exploited to develop novel diagnostic assays. viral diagnostic tests can be broadly classified into three categories in general. those are direct detection, indirect examination (virus isolation), and by serology. in case of direct detection, the clinical sample is examined directly to identify any presence of virus particles, virus antigen or viral nucleic acids. in case of indirect examination, the sample has to be added into cell culture, eggs or animals to grow the virus in vitro. this is known as virus isolation. serology always constitutes the bulk of the work of any virology laboratory, especially in overpopulated third world countries. serological diagnosis is generally made by detecting titres of antibody in infection [8] . generally, the majority of common viral infections are diagnosed by serology [86] . viruses can be directly detected through electron microscopy. it can also be enumerated by molecular biological techniques like pcr/rtpcr by detecting viral genomes. these techniques are extremely useful but are technically demanding, costly and require skilled personnel. on the other hand, indirect detection by virus isolation is dependent on cell culture techniques. the major problem of cell culture is it takes a long time (up to 4 weeks). also, the sensitivity is poor and depends on many factors, such as the specimen condition and the condition of the cell line. cell cultures are also very susceptible to microbial contamination and toxins present in the specimen. also, many viruses do not grow at all in cell culture e.g. hepatitis b and c, viruses causing diarrhea, parvovirus etc. serology is the mainstream of viral diagnosis [8, 44] . with increase in the growth of sophisticated immunoassay techniques, effective viral immunodiagnostic assays are now available in the market [13, 91, 96] . the detection of structural glycoproteins of viruses or early glycoprotein antigen formation in the host due to viral infection or the quantification of titres of antibodies against viral antigenic glycoprotein is an emerging discipline in viral immunodiagnostics [47] . the detection of these structural glycoproteins of viruses is done by lectins or monoclonal antibodies acting as probe or by measuring the titres of host antibodies against antigenic glycoprotein. there are several good review works on viral glycoproteins. namely, the work of kazuya i.p.j. hidari and takashi suzuki on glycan receptor in influenza virus [43] . yuan et al. [116] worked on receptor glycoprotein interaction in zaire ebola virus (zev). this review attempts to conglomerate the importance of glycoprotein in widely studied viral infection and their application in diagnosis. a fully assembled infectious virus is known as virion. the simplest virions consist of two basic components, namely nucleic acid (single-or double-stranded rna or dna) and a capsid, which is a protein coat, functions as a shell to protect the viral genome from nucleases. this capsid comes into play during infection to attach the virion to specific receptors exposed on the prospective host cell. capsid proteins are coded by the viral genome. due to its limited size, the genome codes for only a few structural proteins (besides non-structural regulatory proteins involved in virus replication). capsids are formed as single or double protein shells and consist of only one or a few structural protein species. therefore, multiple protein copies must self assemble to form the continuous three-dimensional capsid structure [35] . the structural viral proteins are extremely important to the virus, so as to facilitate the transfer of the viral nucleic acid from one host cell to another. the proteins determine the antigenicity of the virus. host's primary immune response is directed against the antigenic determinants of these proteins rather glycoprotein in major cases. there are enveloped viruses and these envelopes are made up of either lipid or glycoprotein. viral envelopes mainly consist of envelope proteins (e), membrane proteins (m) and spike proteins (s) [24] . lipid envelopes are derived from the host cell. whereas the envelope glycoproteins are virus encoded. however, there are sugars attached to the viral glycoproteins which often reflect the host cell that harboured the virus. the surface glycoproteins of an enveloped virus attach the virion to a target host cell by properly interacting with a cellular receptor [22] . structural biological analysis of viral envelope glycoproteins reveals that viruses have wide range of folds to facilitate their attachment with proper host receptors. bowden et al. [10] stated that arenaviridae group of viruses have a/b fold, whereas filoviridae possess 'chalice' of gp1. similarly, paramyxoviridae shows six bladed b propeller and large trimeric haemaglutinin is shown by orthomyxoviridae. glycosylated gp120 trimer is observed in the lentiviruses of retroviridae. viruses exhibit 'semaphorins' which are family of cell surface signalling glycoproteins [19] . these semaphorins binds with cell surface receptors to initiate important physiological processes. these observations are made by recent study of viral glycoproteins by employing macromolecular crystallography [10] . the m and s proteins of the virus are usually rich in n glycosylated proteins, which have been demonstrated as important virulent factor of viruses [98] . thus, e, m and s viral glycoproteins are involved in viral host binding and subsequent virus-host membrane fusion to establish the pathogenesis of the virus. two envelope glycoproteins, namely e1 and e2 develop the viral spike of the virions of flaviviridae family [61] are involved in the engagement with host receptor and conformational change required for membrane fusion (fig. 1c) . studies show that e2 can express independently but e1 is dependent upon e2 in case of hcv. sars coronavirus possess a spike(s) glycoprotein [70] , which itself performs the membrane fusion for the entry of the virion and its fusion with host cell [115] . in case of chikungunya virus, attachment is facilitated by the e2 glycoprotein [89] and fusion mainly by the e1 glycoprotein, thus both the processes are mutually exclusive, whereas in dengue virus, it is carried by the same e protein (http://www.uniprot.org/ uniprot/q8jux5). interestingly, the dengue virus apart from synthesizing the basic capsid, membrane and envelope proteins also produces seven non-structural secretory glycoproteins ns1,2a, 2b, 3, 4a,4b,5 [46] . these proteins are not integrated in the virus but secreted in the host. studies have found heterogeneity in the e glycoprotein of dengue virus [56] . five different glycans are present in this glycoconjugate including mannose, galnac and glcnac, fucose and sialic acid. b cell and t-cell epitopes are predicted in a study by analysing this e glycoprotein [46] . the dengue viral envelope is more ordered than the inner viral core, as the envelope is composed of 90 glycoprotein e dimer icosahedral scaffold [58] . computational studies are there to develop vaccines against dengue virus [4, 97] . there are three glycoproteins present in hiv [1] ; namely gp 120, gp 160 and gp 41 [38] . all these are encoded by the env gene [76] . the hiv envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 n-linked glycans ( fig. 1d ) [73] . experiments proved that the glycan part of the gp 41 protein has important role in the efficient intracellular transport of another glycoprotein gp 160. those gp 160 proteins lack gp41 are arrested in golgi complex after their biosynthesis [27] . zaire ebola virus is the member of filoviridae group, and the glycoproteins (gp) have found to be major pathogenic determinants [24-26, 63, 75, 99] . in the ebola virion gp gene is the 4th gene among total seven genes in the linear gene order. this synthesizes several proteins. among them two are predominant. those are sgp and d-peptide (delta peptide). these two proteins are produced due to a furin cleavage of a precursor pre-sgp protein. the gp is actually a spike protein which is composed of two subunits joined by disulphide linkage gp1-gp2 [92] . the chikungunya virus on the other hand are known to produce 4 non structural glycoproteins (nsp1-4) [91] these nsps have been demonstrated to have important role in keeping the replicase complex of the virus intact in the host as well as to circumvent important host immune responses. chikungunya virus has two envelope proteins, namely e1 and e2 [11] . thus, viral glycoproteins have diverse structure and function. taken together, glycoproteins are important components of the virus structure and each have unique role to establish pathogenesis. [17] . viral glycoproteins have a definite role in their pathogenesis. the primary goal of viral infection is to identify a receptor on the host cell surface and binding with it. subsequently this will pave the way of viral entry into the host cell. in most cases, the first attachment site of the virus is a glycan, either a glycoprotein or a glycolipid. so, glycoproteins play a crucial role in viral pathogenesis. the study of glycoproteins in viral infection is most important to know the disease process as well as to develop antiviral treatments. glycoprotein-receptor interactions also play important roles in pathogen pattern recognition and in the regulatory signals that control the activities of cells of the immune system. the most important cause behind viral infection is that it has evolved to present its own sugars and receptors in a manner that mimics or interferes with host glycan-based immune functions. glycomic studies are ongoing in several viruses. several advanced technologies are there to decipher structural and functional aspects of glycans like glycan microarray [40] , mass spectrometry and nano lc. glycan array represents the actual in vivo interaction in silico. the arrayed multivalent demonstration of polysaccharides mimics the cell surface display. there are two types of carbohydrate microarray. those are polysaccharide and oligosaccharide microarray [53] . natural polysaccharides are randomly immobilized on solid matrices exploiting hydrophobic physical absorption or charge-based interaction. polysaccharide microarrays are useful for comparative antigenicity analyses. being hydrophilic in nature, oligosaccharides need chemical derivatization before arraying. through oligosaccharide microarray we can study structure-activity relationships [52] . microarrays were developed on maleimide-functionalized surfaces using seven thiol-containing synthetic highmannose oligosaccharides for the identification of human immunodeficiency virus (hiv) vaccine candidate antigens [2] . the binding profile reveals that several proteins which interact with gp120 of hiv, like the receptor of the innate immune system known as dc-sign (cd 209). in case of influenza glycans, there are protocols for fluorescent labeling of virus, coupling of virus to a glycan microarray, analysis of a glycan microarray slide experiment, and data interpretation. studies have shown that there are a2, 3-linked sialic acid motif (sa2, 3gal) in avian, equine, and canine species. whereas a2, 6-linked sialic acid motif (sa2, 6gal) is present in humans. saa2, 3gal and saa2,6gal are present in swine, these are causing corresponding host tropism. zhao et al. [117] showed that, association mining results of glycan microarray [2] data with 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds [72] . the study suggest that besides neu5aca2-6galb, human-origin viruses could bind glycans with neu5aca2-8neu5aca2-8neu5ac and neu5gca2-6galb1-4glcnac substructures; galb and glcnacb terminal substructures, without sialic acid branches these were linked with the binding of human, swine, and avian origin viruses. sulfated neu5aca2-3 substructures were associated with the binding of human-and swine-origin viruses. finally, through three-dimensional structure characterization, it has been revealed that the role of glycan chain shapes is more important than that of torsion angles [117] . though characterization of glycoproteins is tough but, through mass spectrometry, it is now easier to identify structural details of complex glycoproteins. mass spectrometry derived glycoproteomics [118] helps us to precisely identify viral and cellular proteins that are functionally, structurally, and dynamically altered during virus infection, but enables us to identify important proteins having active role in the infection pathway. additionally, isolation and purification techniques along with quantitative strategies in conjunction with ms significantly improve its sensitivity to detect low-abundant proteins. with time, more virus and host genomes are being sequenced and ms-based glycoproteomics is becoming a very important tool for virology. a work by barrientos et al. [7] revealed that post translational modification of secretory glycoprotein of zaire ebola virus can be characterized by mass spectrometry. maldi-tof ms (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry) also enables to identify regions susceptible to limited proteolysis in sgp of zev. another work by anastassia et al. [54] shows that 0.1 microgram of viral glycoprotein can be purified by nano-liquid chromatography. after nano-lc the sample is analysed through mass spectrometry. one more work showed that they purified heat shock protein 90 by nanolc-ms of respiratory syncitial virus which have important role in virus particle assembly [54, 80] . so it is evident that using these modern techniques, the biological roles of glycoproteins can be studied more conveniently. virus is a nucleic acid surrounded by proteins. this infective particle is called a virion. in most cases this virion is covered with a fascinating coat composed of glycoproteins through which the virus communicates with its host. the co-evolution of host and virus leads the way of making the glycoprotein coat so fascinating [23] . it is evident that the infectivity of a virus rather of its nucleic acid is fully dependent on its glycoproteins. enveloped viruses generally encode membrane proteins and these special proteins are necessary to mediate the specific binding against host cell ligands. this also directs initial events of membrane fusion and viral internalization. these fascinating envelope proteins are generally glycosylated [15] . the process of glycosylation takes place in the endoplasmic reticulum (er)-golgi complex secretory pathway. the host cell encoded glycosyl-transferase enzyme catalyses the glycosylation. this glycosylation is necessary to make the virion host compatible, which is needed by the virus for its pathogenesis. so glycans present at the envelop proteins acts as immunological barriers to resist evasion by the host immune system [22] . several viruses exhibit different glycoproteins on their surface (table 1 ). hepatitis c virus has two envelope glycoproteins namely e1 and e2 [59, 98] . these two proteins play an important role in viral infectivity and can be used as candidate subunit vaccine [98] . on the other hand, in case of ebola virus there are glycoproteins gp1 and gp2 which causes cell attachment and cell fusion and therefore the main target of the host antibodies. the ebola virus has an rna editing mechanism to regulate these gp 1 and 2 genes which when expressed at high level, disrupts normal cell physiology [24] . in case of hiv-1, the gp-120 protein initiates viral entry to the cd4 cells [16] . recent studies proved that several types of glycans in hiv-1 produce different levels in infectivity. those viruses have more oligomannose and less structural complexity, infects more efficiently. this ultimately proves that mature oligosaccharide structure of the envelope glycans play a pivotal role in the infection process of hiv-1. due to n-linked glycosylation of gp-160 protein in the endoplasmic reticulum, and further folding and cleaving in the golgi complex; the gp-120 protein of the envelope is produced [71] . there are two popular glycoproteins present on the surface of influenza virus namely, haemaglutinin (ha) and neuraminidase (na) [84, 88] . these are the key molecules for the viral infection which binds with sialic acid. initially, ha binds with sialic acid during the initiation of infection. after viral replication na degrades its substrate sialic acid to accelerate release of new viruses [30] . so it is evident from the examples of viruses of diverse family that glycoproteins are the key molecules for a virus to establish an infection within the host and to survive further within the host system. in the study of viral pathogenesis, a special type of glycoprotein, called semaphorin have been established [6] . semaphorins are family of cell surface signaling glycoproteins which binds to the family of plexin glycoprotein cell surface receptors. semaphorins also activate repulsive guidance pathways having active part in axon guidance, immune regulation and activation, and vascular development [57, 93] . semaphorins have eight known classes. among them two are found in invertebrates, five in vertebrates, and the eighth class in viruses which are known as 'viral semaphorins' [19] . the ectodomains of cellular semaphorins contain c-terminal domain elaborations like psi (plexin, semaphorin and integrin) domains, immunoglobulin (ig)-like domains, thrombospondin domains and pdz-domain-binding sites which occassionally attach to the cell-surface. whereas the n-terminal having a plexin-binding sema-domain, is conserved in all cases of virus host cell attachment. the sema-domain is the [18, 43] fusion with host cell membrane sialic acid and attachment [43] 3-5 million cases worldwide [78, 105] sars-cov spike(s) glycoprotein [25, 115] membrane fusion [115] 8422 within the duration of 1st november 2002 to 7th august 2003 occurring worldwide [113, 114] hepatitis c virus e1 and e2 [55, 98] binding to host receptor and conformational change necessary for membrane fusion [98] 130 to 150 million people globally [103, 106] human immunodeficiency virus 1 gp120, gp160, gp41 [16] intracellular transport [16] 35 million globally up to 2013 [83, 104, 108, 112] zaire ebola virus spike protein gp1-gp2 [64] primary host cell activation [64] up to 28th june 2015 total 27,550 cases [107, 110, 111] dengue virus e (dimer) [64] host cell fusion and attachment [64] who reported recently that there are 390 million dengue infections per year globally [109] . presently dengue is endemic in 112 countries [109] . chikungunya virus e1 and e2 [41, 51] host cell binding according to who, this disease occurs mainly in africa, asia and indian sub-continent [102] . [10] have revealed that human sema3a and mouse sema4d semadomains comprises of structurally conserved homodimer of seven-bladed b-propellers [6, 50, 65, 69, 77] . the immune-regulatory semaphorins like sema3a, 4a, 4d, and 7a helps in b cell mediated immunity (sema4d), t cell activation as well as differentiation (sema4a, sema3a, and sema4d), and inflammation (sema7a) [93] . these semaphorins provide a molecular basis for how viruses can optimize their own proteins to override normal physiological interactions. a work by shirato h as 'norovirus and histo-blood group antigens' in the journal jpn j infect dis. (2011;64(2):95-103) describes that norovirus (nov) causes viral gastroenteritis and interestingly bind to histoblood group antigens (hbgas), like abh antigens and lewis antigens. it has been shown epidemiologically that persons with different abh phenotypes are infected with nov strains in a genotype-dependant fashion. an in vitro binding assay using nov virus-like particles (vlps) showed a uniform recognition pattern for type 1 and 2 core structures of histo blood group antigens. nov vlps bind more tightly to type 1 carbohydrates than to type 2. type 1 carbohydrates are found to be expressed at the surface of the small intestine and targeted by nov. this property speaks about nov tissue specificity. so it is evident that glycoproteins perform a major and active role in viral pathogenesis and disease progression. glycoproteins provide tissue tropism to the virus. some virues used to infect the respiratory system whereas some affects the liver. the cause is the type of glycoprotein with which the virus binds to accelerate its invasion. in a study by raska et al. [81] , it has been proved that there are differential glycosylation in viruses like hiv1 depending upon the cells which produce the virus. n-glycosylation of reconbinant gp120 of hiv1 is varied and affected the recognition by serum antibodies. glycosylation of gp120 protein of hiv1 affects its recognition by neutralizing and non neutralizing monoclonal antibodies. this study also says that this glycosylation is cell specific. another study by lin et al. [64] stated that there are c-type lectins expressed on the dendritic cell surface known as dc-sign(dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) or cd 209 and dc-signr which binds to hiv1 and transmit to t cells through the viral envelope env glycoprotein. but interestingly other highly glycosylated viruses failed to interact with dc-signr [64] . lin et al. showed that dc-sign (r) or cd 209 selectively binds with hiv1 env and zaire ebola virus glycoproteins containing more highmannose. by modulating n-glycans on env or glycoprotein during virus production in different primary cells or in the presence of the mannosidase i inhibitor deoxymannojirimycin affected dc-sign(r) infectivity enhancement. they also predict that viruses containing glycoproteins with a high amount of high-mannose n-glycans effectively interact with dc-sign(r), but those viruses having only complex n-glycans cannot effectively react with dc-sign(r). so it is evident that virus-producing cell type is a crucial factor in depicting both n-glycan status and virus interactions with dc-sign(r), which establishes virus tropism and infection within the human body [64] . liu et al. [66] described in their study that sialic acid present on cell surface is essential for human enterovirus d 68 (ev-d68) entry. crystallographic studies showed that ev-d68 with sialylated glycan receptor analogues binds on the viral surface. sialic acid receptor induces a cascade of conformational changes within the virus to secrete a fattyacid-like molecule which regulates the stability of the virus. so, it is evident that binding of virus to a sialic acid receptor and to immunoglobulin-like receptors facilitates viral entry in enteroviruses. glycan based viral immunodiagnostics usually have high sensitivity and specificity. glycoprotein based igm serology was developed for the diagnosis of recent primary rubella virus infections and significant sensitivity and specificity was obtained. similarly, glycoprotein based serology tests to detect antibodies to herpes simplex virus glycoproteins g-1 and g-2, which evoke a type-specific antibody response have also been developed. these tests are used to confirm a diagnosis of genital herpes, and also to establish diagnosis of hsv infection in patients with atypical complaints, to identify asymptomatic carriers, and identify persons at risk for acquiring hsv. glycan based immunodiagnostics have also been developed for the rapid identification of different strains of influenza virus. a novel peptide based elisa which has sensitivity and specificity of 96.55 and 74.4 % respectively is very promising. on the other hand, the main diagnostic challenge related to sars is to diagnose it differently with atypical pneumonia [68, 79] . the key diagnostic tools are immunofluorescent staining, elisa and rt-pcr [67] . but all these techniques are extremely sophisticated and of little use in case of epidemics; especially in the developing world. sars coronavirus possess a spike(s) glycoprotein, which itself performs the membrane fusion for the entry of the virion and its fusion with host cell (fig. 1b) [115] . igg based diagnostics against this s protein has been developed [114] . indirect elisa test has been developed by using recombinant sars 's' protein and the n (nucleoprotein) protein. the sensitivity of sars 's' and 'n' proteins are 100 and 96.7 % respectively whereas the specificity of sars 's' and 'n' are 98.55 and 98.4 % [114] . similarly, as acute hepatitis c infection is asymptomatic, so it is difficult to diagnose early. generally patients come to the clinic with damaged liver. initially patients are screened by anti-hcv antibody test and further confirmed by testing viral rna. there are two glycoproteins e1 and e2 in hcv. standardised pcr system is available in hcv diagnostics but a core antigen test is also in the market. in 1995 tanaka et al. [21] suggested that the hcv core proteins can be used as antigens for the chronic stage. studies for developing algorithms confirms 99.05 % sensitivity in case of rt-pcr whereas 98.10 % for the core antigen test [82] . another novel glycoproteomic serum biomarker has been identified which can diagnose hcv along with progressive liver cirrhosis is wisteria floribunda agglutinin positive mac-2-binding protein (wfa?-m2bp) [29] . the diagnostic threshold for cut-off index values of this protein is 1.435 and 4.615 in hcv negative and hcv positive patients showing progressive liver cirrhosis [39] . in case of hiv1, elisa and pcr are two gold standard tests. type specific conformational epitopes of the gp160 and gp 41 glycoproteins of the hiv envelope are used for the recognition of 'early hiv antibodies' [14] . enzyme immuno assay (eia) and rt-pcr are used in these assays. these tests are very specific but they fail to diagnose early infection [14] . the most challenging part in hiv diagnosis is to diagnose the acute stage and differentiate ''window phase'' patients from the serologically positive patients [87] . popular serological tests fail to diagnose all patients of hiv as they exploit the gag proteins which literally decrease after disease progression [60, 74] . there are three glycoproteins present in hiv; namely gp 120, gp 160 and gp 41. all these are encoded by the env gene [76] . the hiv envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 n-linked glycans (fig. 1d) [73] . experiments proved that the glycan part of the gp 41 protein has important role in the efficient intracellular transport of another glycoprotein gp 160. those gp 160 proteins lack gp41 are arrested in golgi complex after their biosynthesis [27] . as stated above, there are limitations of the popular gag antigen based serological tests which cannot diagnose hiv patients of different clinical stage. but antibodies against precursor gp160 env protein and final env proteins gp 120 and gp 41 can detect all clinical stages of hiv [74] . the sensitivity of current available diagnostic system is 38 % at \7 days, 97 % at 7-41 days and 95 % at 42-93 days [74] the specificity is almost 95 % [90] . in case of zaire ebola virus, initially there was controversy about the role of glycoproteins in the pathogenesis of ebv. but later on, scientific researches proved that the primary host cell activation by the ebv is mediated by gp1-2 [100] . an antigen capture elisa has been developed in zaire ebov using mabs [85] . it has been reported that these tests have both high sensitivity and specificity [85] . similarly, dengue (denv) ns1 is a highly conserved glycoprotein, expressed as both membrane-associated and secreted forms [33, 36, 94] . secreted ns1 has been detected ranging from 2-0.04 lg/ml in the serum of dengue-infected patients during the early stages of the disease. a high ns1 level has been demonstrated to circulate as early as 1 day after onset of symptoms up to early convalescences thus provides an alternative to virus culture or pcr for early dengue diagnosis when igm or igg antibodies are not present yet in dengue infected patients [42, 49] . circulating dengue ns1 in sera can be detected either using elisa assay or lateral flow based rdts [31] . thus, glycan based viral immunodiagnostics or glyco-immunodiagnostics are helpful in early diagnosis of patients with viral infection [28] . current diagnosis scenario in chikungunya is igm and igg based elisa and nucleic acid detection by rt-pcr [12, 51] . but there is no antigen based elisa. this makes the condition crucial as the primary health care providers in the virus affected countries do not have rt-pcr facilities. it is not recommended to maintain rt-pcr facilities in primary health care centre by policy. it is extremely costly and demands expertise. it is not possible to provide such facility in the densely populated tropical countries. as chikungunya causes short duration fever, often the patients are not diagnosed properly. the joint pain generally persists for some days but can be present for a year [102] . there is a study which shows even multi organ failure in chikungunya infected patients [45] . chikv has two envelope glycoproteins, namely e1 and e2 (fig. 1g) [11] . recombinant chikv e1 and e2 glycoprotein based elisa showed a sensitivity of 77.5 and 90 % respectively whereas the specificity for both cases was 100 % [11, 54] highlighting the potential for these two glycoproteins in the diagnosis. viral glycoproteins are integral parts of enveloped viruses and they actively take part in their pathogenesis. exploting glycoproteins, viruses enter into their host and combat with host immune system. recent advances in technology deciphers different role of glycoproteins which are dependent on their structures. different viruses have different mode of pathogenesis and glycoproteins directly takes part in the host binding and entry. during maturation from host cell viruses have viral glycoproteins: biological role and application in diagnosis 7 host glycoproteins on their surface to avoid the immunity of the host. so, to detect viruses and to decide for developing vaccines [37] , glycoproteins always play a key role. antibody production is a prominent feature of the immune response in patients with viral infection, and particular isotypes correlate with resistance or susceptibility to infection [101] . a substantial proportion of the antibodies detected in patients with acute or chronic infections is directed against viral glycan epitopes. as levels of anti glycan antibody are high and specific for each viral infection this permits diagnostic discrimination between the different viral infections. taken together, viral glycoproteins have important functions in pathogenesis and can be exploited to develop viral diagnostics. oligosaccharide and glycoprotein microarrays as tools in hiv glycobiology; glycan-dependent gp120/ protein interactions comparative dynamics, morbidity and mortality burden of pediatric viral respiratory infections in an equatorial city computational prediction and analysis of envelop glycoprotein epitopes of denv-2 and denv-3 pakistani isolates: a first step towards dengue vaccine development glycans in immune recognition and response structure of the semaphorin-3a receptor binding module secreted glycoprotein from live zaire ebolavirus-infected cultures: preparation, structural and biophysical characterization, and thermodynamic stability viral immunodiagnosis different serotypes of dengue virus (denv) imported by polish travellers from dengue endemic areas to poland cells under siege: viral glycoprotein interactions at the cell surface expression and evaluation of chikungunya virus e1 and e2 envelope proteins for serodiagnosis of chikungunya virus infection chikungunya virus infection: an overview mmwr, interpretation and use of the western blot assay for serodiagnosis of human immunodeficiency virus type 1 infections detection of antibodies to human immunodeficiency virus (hiv) that recognize conformational epitopes of glycoproteins 160 and 41 often allows for early diagnosis of hiv infection chikungunya virus envelope glycoproteins of human immunodeficiency virus type 1: profound influences on immune functions hantavirus gn and gc envelope glycoproteins: key structural units for virus cell entry and virus assembly purification of the influenza hemagglutinin glycoprotein and characterization of its carbohydrate components a poxvirus-encoded semaphorin induces cytokine production from monocytes and binds to a novel cellular semaphorin receptor ebola to have a global burden of $5.9 billion by end of detection of hcv core antigen assembly of the coronavirus envelope: homotypic interactions between the m proteins dengue: guidelines for diagnosis, treatment, prevention and control: new edition. geneva: world health organization laboratory diagnosis and diagnostic tests shedding of ebola virus surface glycoprotein is a mechanism of self-regulation of cellular cytotoxicity and has a direct effect on virus infectivity development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein biosynthesis and role of filoviral glycoproteins the glycosylation of human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) is important for the efficient intracellular transport of the envelope precursor gp160 dengue virus type 1 nonstructural glycoprotein ns1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion clinicopathological characteristics and diagnostic performance of wisteria floribunda agglutinin positive mac-2-binding protein as a preoperative serum marker of liver fibrosis in hepatocellular carcinoma influenza hemagglutinin and neuraminidase membrane glycoproteins development and characterization of mouse monoclonal antibodies against monomeric dengue virus non-structural glycoprotein 1 (ns1) university of texas medical branch at galveston dengue vaccines: challenges, development, current status and prospects cellular and humoral immunity to varicella zoster virus glycoproteins in immune and susceptible human subjects human arbovirus infections worldwide dengue viral infections influenza, epidemiology and prevention of vaccine-preventable diseases carbohydrates of human immunodeficiency virus analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: an update for probing virus-glycan interactions using glycan microarrays chikungunya: a bending reality evaluation of a dengue ns1 antigen detection assay sensitivity and specificity for the diagnosis of acute dengue virus infection glycan receptor for influenza virus approaches for the development of rapid serological assays for surveillance and diagnosis of infections caused by zoonotic flaviviruses of the japanese encephalitis virus serocomplex fatal cases of chikungunya virus infection in colombia: diagnostic and treatment challenges development of global consensus of dengue virus envelope glycoprotein for epitopes based vaccine design influenza symptoms and the role of laboratory diagnostics, health professionals, seasonal. laboratory diagnostic procedures for testing for influenza (flu)-cdc microbial evolution and co-adaptation: a tribute to the life and scientific legacies of joshua lederberg: workshop summary a clinico-hematological profile of dengue outbreak among healthcare professionals in a tertiary care hospital of ahmedabad with analysis on economic impact structural basis of semaphorin-plexin signalling unique epitopes recognized by antibodies induced in chikungunya virus-infected nonhuman primates: implications for the study of immunopathology and vaccine development determination of specific antibody responses to the six species of ebola and marburg viruses by multiplexed protein microarrays synthetic gpi array to study antitoxic malaria response proteomic analysis of virus-host interactions in an infectious context using recombinant viruses structure of hepatitis c virus envelope glycoprotein e1 antigenic site 314-324 in complex with antibody igh526 identification of amino acids in the dengue virus type 2 envelope glycoprotein critical to virus infectivity semaphorins command cells to move structure of dengue virus: implications for flavivirus organization, maturation, and fusion functional role of hepatitis c virus chimeric glycoproteins in the infectivity of pseudotyped virus distinct igg recognition patterns during progression of subclinical and clinical infection with lymphadenopathy associated virus/human t lymphotropic virus hcv glycoproteins: assembly of a functional e1-e2 heterodimer high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections diagnosis of ebola haemorrhagic fever by rt-pcr in an epidemic setting viral glycoproteins: biological role and application in diagnosis 9 differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr structural basis of semaphorin-plexin recognition and viral mimicry from sema7a and a39r complexes with plexinc1 structure and inhibition of ev-d68, a virus that causes respiratory illness in children molecular diagnosis of severe acute respiratory syndrome a study of influenza and influenza-related complications among children in a large us health insurance plan database the ligand-binding face of the semaphorins revealed by the high-resolution crystal structure of sema4d molecular diagnosis of severe acute respiratory syndrome conference report: ''functional glycomics in hiv type 1 vaccine design'' workshop report development of monoclonal antibodies to highly pathogenic avian influenza h5n1 virus and their application to diagnostics, prophylaxis, and therapy several n-glycans on the hiv envelope glycoprotein gp120 preferentially locate near disulphide bridges and are required for efficient infectivity and virus transmission antibody response to human immunodeficiency virus in homosexual men: relation of antibody specificity, titer, and isotype to clinical status, severity of immunodeficiency and disease progression ebola virus disease: societal challenges and new treatments human immunodeficiency viruses: molecular virology, pathogenesis, diagnosis and treatment structural basis for semaphorin signalling through the plexin receptor pandemic (h1n1) 2009-update 103. disease outbreak news. world health organization the aetiology, origins, and diagnosis of severe acute respiratory syndrome protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein 90 in virus particle assembly differential glycosylation of envelope gp120 is associated with differential recognition of hiv-1 by virus-specific antibodies and cell infection comparison of two diagnostic algorithms for the identification of patients with hcv viremia using a new hcvantigen test oral sex and hiv transmission structure of influenza hemagglutinin in complex with an inhibitor of membrane fusion laboratory diagnostic systems for ebola and marburg hemorrhagic fevers developed with recombinant proteins glycosylation changes on serum glycoproteins in ovarian cancer may contribute to disease pathogenesis supplementary article, challenges for rapid molecular hiv diagnostics influenza virus neuraminidase: structure and function a single-amino-acid polymorphism in chikungunya virus e2 glycoprotein influences glycosaminoglycan utilization sensitivity and specificity of a qualitative rna detection assay to diagnose hiv infection in young infants diagnostic virology ebola virus pathogenesis: implications for vaccines and therapies semaphorins and their receptors in immune cell interactions characterization of the dengue virus envelope glycoprotein expressed in pichia pastoris types of influenza viruses, seasonal influenza (flu), cdc three types of influenza viruses-cdc biomarkers in japanese encephalitis: a review changes in the plasma lipid profile as a potential predictor of clinical outcome in dengue hemorrhagic fever essentials of glycobiology role of ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages effects of ebola virus glycoproteins on endothelial cell activation and barrier function diagnosis of recent primary rubella virus infections: significance of glycoprotein-based igm serology, igg avidity and immunoblot analysis chikungunya: who fact sheet on chikungunya providing key facts and information on scope of the problem, who is at risk, prevention, who response hepatitis c is a liver disease caused by the hepatitis c virus: the virus can cause both acute and chronic hepatitis infection, ranging in severity from a mild illness lasting a few weeks to a serious aids who fact sheet on hiv/aids with key facts and information on signs and symptoms, transmission, risk factors, testing and counselling, prevention, treatment and who response influenza (seasonal) who fact sheet on influenza: includes key facts, definition, symptoms, transmission, seasonal epidemics, effects, prevention, who response prevention and control of viral hepatitis infection: framework for global action who urgently needed: rapid, sensitive, safe and simple ebola diagnostic tests, the goal of interrupting chains of ebola virus transmission depends heavily on laboratory support who fact sheet dengue and severe dengue provides key facts, definition, provides information on global burden, transmission, characteristics, treatment, prevention and control and who response ebola virus disease, who fact sheet on ebola: key facts, definition, transmission, symptoms, diagnosis, treatment, prevention who launches new guidelines on hiv testing services summary of current situation, literature update and risk assessment-as of 5 summary table of sars cases by country the sars-cov s glycoprotein serological evidence of ebolavirus infection in bats, china association analyses of large-scale glycan microarray data reveal novel hostspecific substructures in influenza a virus binding glycans current approaches on viral infection: proteomics and functional validations viral glycoproteins: biological role and application in diagnosis 11 key: cord-303165-ikepr2p2 authors: tulchinsky, theodore h.; varavikova, elena a. title: expanding the concept of public health date: 2014-10-10 journal: the new public health doi: 10.1016/b978-0-12-415766-8.00002-1 sha: doc_id: 303165 cord_uid: ikepr2p2 ancient societies recognized the needs of sanitation, food safety, workers’ health, and medical care to protect against disease and to promote well-being and civic prosperity. new energies and knowledge since the eighteenth century produced landmark discoveries such as prevention of scurvy and vaccination against smallpox. the biological germ theory and competing miasma theory each proved effective in sanitation, and immunization in control of infectious diseases. non-communicable diseases as the leading causes of mortality have responded to innovative preventive care of health risk factors, smoking, hypertension, obesity, physical inactivity, unhealthful diets, and diabetes mellitus. health promotion proved effective to modern public health in tackling disease origins, individual behavior, and social and economic conditions. the global burden of infectious and non-communicable diseases, aging and chronic illness faces rising costs and still inadequate prevention. the evolution of concepts of public health will have to address these new challenges of population health. the development of public health from its ancient and recent roots, especially in the past several centuries, is a continuing process, with evolutionary and sometimes dramatic leaps forward, and important continuing and new challenges for personal and population health and well-being. everything in the new public health is about preventing avoidable disease, injuries, disabilities, and death while promoting and maximizing a healthy environment and optimal conditions for current and future generations. thus, the new public health addresses overall health policy, resource allocation, as well as the organization, management, and provision of medical care and of health systems in general within a framework of overall social policy and in a community, state, national, transnational, and global context. the study of history (see chapter 1) helps us to understand the process of change, to define where we came from and where we are going. it is vital to recognize and understand change in order to deal with radical transformations in direction that occur as a result of changing demography and epidemiology, new science, evolving best practices in public health and clinical medicine, and above all inequalities in health resulting from societal system failures and social and economic factors. health needs will continue to develop in the context of environmental, demographic and societal adjustments, with knowledge gained from social and physical sciences, practice, and economics. for the coming generations, this is about not only the quality of life, but the survival of society itself. over the past century there have been many definitions of public health and health for all. mostly they represent visions and ideals of societal and global aspirations. this chapter examines the very base of the new public health, which encompasses the classic issues of public health with recognition of the advances made in health promotion and the management of health care systems as integral components of societal efforts to improve the health of populations and of individuals. what follows in succeeding chapters will address the major concepts leading to modern and comprehensive elements of public health. inevitably, concepts of public health continue to evolve and to develop both as a philosophy and as a structured discipline. as a professional field, public health requires specialists trained with knowledge and appreciation of its evolution, scientific advances, concepts, and best practices, old and modern. it demands sophisticated professional and managerial skills, the ability to address a problem, reasoning to define the issues, and to advocate, initiate, develop, and implement new and revised programs. it calls for profoundly humanistic values and a sense of responsibility towards protecting and improving the health of communities and every individual. in the twenty-first century, this set of values was well expressed in the human development index agreed to by 160 nations (box 2.1). public health is a multidimensional field and therefore multidisciplinary in its workforce and organizational needs. it is based on scientific advances and application of best practices as they evolve, and includes many concepts, including holistic health, first established in ancient times. the discussion will return to the diversity of public health throughout this chapter and book many times. in previous centuries, public health was seen primarily as a discipline which studies and implements measures for control of communicable diseases, primarily by sanitation and vaccination. the sanitary revolution, which preceded the development of modern bacteriology, made an enormous contribution to improved health, but many other societal factors including improved nutrition, education, and housing were no less important for population health. maternal and child health, occupational health, and many other aspects of a growing public health network of activities played important roles, as have the physical and social environment and personal habits of living in determining health status. in recent decades recognition of the importance of women's health and health inequalities associated with many high-risk groups in the population have seen both successes and failures in addressing their challenges. male health issues have received less attention, apart from issues associated with specific diseases, or those of healthy military personnel. the scope of public health has changed along with growth of the medical, social, and public health sciences, public expectations, and practical experience. taken together, these have all contributed to changes in the concepts and causes of disease. health systems that fail to adjust to changes in fundamental concepts of public health suffer from immense inequity and burdens of preventable disease, disability, and death. this chapter examines expanding concepts of public health, leading to the development of a new public health. public health has evolved as a multidisciplinary field that includes the use of basic and applied science, education, social sciences, economics, management, and communication skills to promote the welfare of the individual and the community. it is greater than the sum of its component elements and includes the art and politics of the funding and coordination of the wide diversity of community and individual health services. the concept of the interdependence of health in body and in mind has ancient origins. they continue to be fundamental to individuals and societies, and part of the fundamental rights of all humans to have knowledge of healthful lifestyles and to have access to those measures of good health that society alone is able to provide, such as immunization programs, food and drug safety and quality standards, environmental and occupational health, and universal access to high-quality primary and specialty medical and other vital health services. this holistic view of balance and equilibrium may be a renaissance of classical greek and biblical traditions, applied with the broad new knowledge and experience of public health and medical care of the nineteenth, twentieth, and the early years of the twenty-first centuries as change continues to challenge our capacity to adapt. the competing nineteenth-century germ and miasma theories of biological and environmental causation of illness each contributed to the development of sanitation, hygiene, immunization, and understanding of the biological and social determinants of disease and health. they come together in the twenty-first century encompassed in a holistic new public health addressing individual and population health needs. medicine and public health professionals both engage in organization and in direct care services. these all necessitate an understanding of the issues that are included in the new public health, how they evolved, interact, are put together in organizations, and are financed and operated in various parts of the world in order to understand changes going on before our eyes. great success has been achieved in reducing the burden of disease with tools and concepts currently at our disposal. the idea that this is an entitlement for everyone was articulated in the health for all concept of alma-ata in 1978. the health promotion movement emerged in the 1970s and showed dramatically effective results in managing the new human immunodeficiency virus (hiv) pandemic and in tackling smoking and other risk factors for non-communicable diseases (ncds). a health in all policy concept emerged in 2006 promoting the concept that health should be a basic component of all public and private policies to achieve the full potential of public health and eliminate inequalities associated with social and economic conditions. profound changes are taking place in the world population, and public health is crucial to respond accordingly: mass migration to the cities, fewer children, extended life expectancy, and the increase in the population of older people who are subject to more chronic diseases and disabilities in a changing physical, social, and economic climate. health systems are challenged with continuing development of new medical technologies and related reforms in clinical practice, while experiencing strong influences of pharmaceuticals and the medicalization of health, with prevention and health promotion less central in priorities and resource allocation. globalization of health has many meanings: international trade, improving global communications, and economic changes with increasing flows of goods, services, and people. ecological and climate change bring droughts, hurricanes, arctic meltdown, and rising sea levels. globalization also has political effects, with water and food shortages, terrorism, and economic distress affecting billions of people. in terms of health, disease can spread from one part of the world to others, as in pandemics or in a quiet spread such as that of west nile fever moving from its original middle eastern natural habitat to the americas and europe, or severe acute respiratory syndrome (sars), which spread with lightning speed from chinese villages to metropolitan cities such as toronto, canada. it can also mean that the ncds characteristic of the industrialized countries are now recognized as the leading causes of death in low-and middle-income countries, associated with diet, activity levels, and smoking, which are themselves pandemic risk factors. the potential for global action in health can also be dramatic. the eradication of smallpox was a stunning victory for public health. the campaign to eradicate poliomyelitis is succeeding even though the end-stage is fraught with setbacks, and measles elimination has turned out to be more of a challenge than was anticipated a decade ago, with resurgence in countries thought to have it under control. global health policies have also made the achievements of public-private partnerships of great importance, particularly in vaccination and acquired immunodeficiency syndrome (aids) control programs. there have been failures as well, with very limited progress in human resources development of the public health workforce in low-income countries. the new public health is necessarily comprehensive in scope and it will continue to evolve as new technologies and scientific discoveries -biological, genetic, and sociological -reveal more methods of disease control and health promotion. it relates to or encompasses all community and individual activities directed towards improving the environment for health, reducing factors that contribute to the burden of disease, and fostering those factors that relate directly to improved health. its programs range broadly from immunization, health promotion, and child care, to food labeling and fortification, as well as to the assurance of well-managed, accessible health care services. a strong public health system should have adequate preparedness for natural and human-made disasters, as seen in the recent tsunamis, hurricanes, biological or other attacks by terrorists, wars, conflicts, and genocidal terrorism (box 2.2) . the concepts of health promotion and disease prevention are essential and fundamental elements of the new public health. parallel scientific advances in molecular biology, genetics and pharmacogenomics, imaging, information technology, computerization, biotechnology, and nanotechnology hold great promise for improving the productivity of the health care system. advances in technology with more effective and less expensive drug and vaccine development, with improved safety and effectiveness, and fewer adverse reactions, will over time greatly increase efficiency in prevention and treatment modalities. the new public health is important as a conceptual base for training and practice of public health. it links classical topics of public health with adaptation in the organization and financing of personal health services. it involves a changed paradigm of public health to incorporate new advances in political, economic, and social sciences. failure at the political level to appreciate the role of public health in disease control holds back many societies in economic and social development. at the same time, organized public health systems need to work to reduce inequities between and inside countries to ensure equal access to care. it also demands special attention through health promotion activities of all kinds at national and local societal levels to provide access for groups with special risks and needs to medical and community health care with the currently available and newly developing knowledge and technologies. the great gap between available capabilities to prevent and treat disease and actually reaching all in need is still the the mission of the nph is to maximize human health and well-being for individuals and communities, nationally and globally. the methods with which the nph works to achieve this are in keeping with recognized international best practices and scientific advances: 1. societal commitment and sustained efforts to maximize quality of life and health, economic growth with equity for all (health for all and health in all). collaboration between international, national, state, and local health authorities working with public and private sectors to promote health awareness and activities essential for population health. 3. health promotion of knowledge, attitudes, and practices, including legislation and regulation to protect, maintain, and advance individual and community health. 4. universal access to services for prevention and treatment of illness and disability, and promotion of maximum rehabilitation. 5. environmental, biological, occupational, social, and economic factors that endanger health and human life, addressing: (a) physical and mental illness, diseases and infirmity, trauma and injuries (b) local and global sanitation and environmental ecology (c) healthful nutrition and food security including availability, quality, safety, access, and affordability of food products (d) disasters, natural and human-made, including war, terrorism, and genocide (e) population groups at special risk and with specific health needs. 6. promoting links between health protection and personal health services through health policies and health systems management, recognizing economic and quality standards of medical, hospital, and other professional care in health of individuals and populations. 7. training of professional public health workforces and education of all health workers in the principles of ethical best practices of public health and health systems. 8. research and promotion of current best practices: wide application of current international best practices and standards. 9. mobilizing the best available evidence from local and international scientific and epidemiological studies and best practices recognized as contributing to the overall goal. 10. maintaining and promoting equity for individual and community rights to health with high professional and ethical standards. source of great international and internal national inequities. these inequities exist not only between developed and developing countries, but also within transition countries, mid-level developing countries, and those newly emerging with rapid economic development. the historical experience of public health will help to develop the applications of existing and new knowledge and societal commitment to social solidarity in implementation of the new discoveries for every member of the society, despite socioeconomic, ethnic, or other differences. political will and leadership in health, adequate financing, and organization systems in the health setting are crucial to furthering health as an objective with defined targets, supported by well-trained staff for planning, management, and monitoring the population health and functioning of health systems. political leadership and professional support are both indispensable in a world of limited resources, with high public expectations and the growing possibilities of effectiveness of public health programs. well-developed information and knowledge management systems are required to provide the feedback and information needed for good management. it includes responsibilities and coordination at all levels of government. non-governmental organizations (ngos) and participation of a well-informed media and strong professional and consumer organizations also have significant roles in furthering population health. no less important are clear designations of responsibilities of the individual for his or her own health, and of the provider of care for humane, high-quality professional care. the complexities and interacting factors are suggested in figure 2 .1, with the classic host-agent-environment triad. many changes have signaled a need for transformation towards the new public health. religion, although still a major political and policy-making force in many countries, is no longer the central organizing power in most societies. organized societies have evolved from large extended families and tribes to rural societies, cities, regions, and national governments. with the growth of industrialized urban communities, rapid transport, and extensive trade and commerce in multinational economic systems, the health of individuals and communities has become more than just a personal, family, and/or local problem. an individual is not only a citizen of the village, city, or country in which he or she lives, but a citizen of a "global village". the agricultural revolutions and international explorations of the fifteenth to seventeenth centuries that increased food supply and diversity were followed only much later by knowledge of nutrition as a public health issue. the scientific revolution of the seventeenth to nineteenth centuries provided the basics to describe and analyze the spread of disease and the poisonous effects of the industrial revolution, including crowded living conditions and pollution of the environment with serious ecological damage. in the latter part of the twentieth century, a new agricultural "green revolution" had a great impact in reducing human deprivation internationally, yet the full benefits of healthier societies are yet to be realized in the large populations living in abject poverty in sub-saharan africa, south-east asia, and other parts of the world. global water shortages can be addressed with new methods of irrigation, water conservation and the application of genetic sciences to food production, and issues of economics and food security are of great importance to a still growing world population with limited supplies. further, food production capacity can and must be enlarged to meet current food insecurity, rising expectations of developing nations, and population growth. the sciences of agriculture-related fields, including genetic sciences and practical technology, will be vital to human progress in the coming decades. these and other societal changes discussed in chapter 1 have enabled public health to expand its potential and horizons, while developing its pragmatic and scientific base. organized public health in the twentieth century proved effective in reducing the burden of infectious diseases and has contributed to improved quality of life and longevity by many years. in the last half-century, chronic diseases have become the primary causes of morbidity and mortality in the developed countries and increasingly in developing countries. growing scientific and epidemiological knowledge increases the capacity to deal with these diseases. many aspects of public health can only be influenced by the behavior of and risks to the health of individuals. these require interventions that are more complex and relate to societal, environmental, and community standards and expectations as much as to personal lifestyle. the dividing line between communicable and non-communicable diseases changes over time. scientific advances have shown the causation of chronic conditions by infectious agents and their prevention by curing the infection, as in helicobacter pylori and peptic ulcers, and in prevention of cancer of the liver and cervix by immunization for hepatitis b and human papillomavirus (hpv), respectively. chronic diseases have come to the center stage in the "epidemiological transition", as infectious diseases came under increasing control. this, in part, has created a need for reform in the funding and management of health systems due to rapidly rising costs, aging of the population, the rise of obesity and diabetes and other chronic conditions, mushrooming therapeutic technology, and expanding capacity to deal with public health emergencies. reform is also needed in international assistance to help less developed nations build the essential infrastructure to sustain public health in the struggle to combat aids, malaria, tuberculosis (tb), and the major causes of preventable infant, childhood, and motherhood-related deaths. the nearly universal recognition of the rights of people to have access to health care of acceptable quality by international standards is a challenge of political will and leadership backed up by adequate staffing with public health-trained staff and organizations. the challenges of the current global economic crisis are impacting social and health systems around the world. the interconnectedness of managing health systems is part of the new public health. setting the priorities and allocating resources to address these challenges requires public health training and orientation of the professionals and institutions participating in the policy, management, and economics of health systems. conversely, those who manage such institutions are recognizing the need for a wide background in public health training in order to fulfill their responsibilities effectively. concepts such as objectives, targets, priorities, cost-effectiveness, and evaluation have become part of the new public health agenda. an understanding of how these concepts evolved will help the future health provider or manager to cope with the complexities of mixing science, humanity, and effective management of resources to achieve higher standards of health, and to cope with new issues as they develop in the broad scope of the new public health for the twenty-first century, in what breslow called the "third public health era" of long and healthy quality of life (box 2.3). health can be defined from many perspectives, ranging from statistics on mortality, life expectancy, and morbidity rates to idealized versions of human and societal perfection, as in the world health organization's (who's) founding charter. the first public health era -the control of communicable diseases. second public health era -the rise and fall of chronic diseases. third public health era -the development of long and high-quality life. preamble to the constitution of the who, as adopted by the international health conference in new york in 1946 and signed by the representatives of 61 states, entered into force on 7 april 1948, with the widely cited definition: "health is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity". this definition is still important conceptually as an ideal accepted as fundamental to public policy over the years. a more operational definition of health is a state of equilibrium of the person with the biological, physical, and social environment, with the object of maximum functional capability. health is thus seen as a state characterized by anatomical, physiological, and psychological integrity, and an optimal functional capability in the family, work, and societal roles (including coping with associated stresses), a feeling of well-being, and freedom from risk of disease and premature death. deviances in health are referred to as unhealthy and constitute a disease nomenclature. there are many interrelated factors in disease and in their management through what is now called risk reduction. in 1878, claude bernard described the phenomenon of adaptation and adjustment of the internal milieu of the living organism to physiological processes. this concept is fundamental to medicine. it is also central to public health because understanding the spectrum of events and factors between health and disease is basic to the identification of contributory factors affecting the balance towards health, and to seeking the points of potential intervention to reverse the imbalance. as described in chapter 1, from the time of hippocrates and galen, diseases were thought to be due to humors and miasma or emanations from the environment. this was termed the miasma theory, and while without a direct scientific explanation, it was acted upon in the early to mid-nineteenth century and promoted by leading public health theorists including florence nightingale, with practical and successful measures to improve sanitation, housing, and social conditions, and having important results in improving health conditions. the competing germ theory developed by pioneering nineteenthcentury epidemiologists (panum, snow, and budd), scientists (pasteur, cohn, and koch), and practitioners (lister and semmelweiss) led to the science of bacteriology and a revolution in practical public health measures. the combined application of the germ (agent-host-environment) and miasma theories (social and sanitary environment) has been the basis of classic public health, with enormous benefits in the control of infectious and other diseases or harmful conditions. the revolutionary changes occurring since the 1960s have brought about a decline in cardiovascular and cancer mortality, and conceptual changes such as health for all and health in all to bring health issues to all policies at both governmental and individual levels. the concepts of public health advanced with the 1974 marc lalonde health field concept (new perspectives on the health of canadians, 1974) , stating that health was the result of the physical and social environment, lifestyle and personal habits, genetics, as well as organization and provision of medical care. the lalonde report was a key concept leading to ideas advanced at the alma-ata conference on primary care held in 1978 and more explicitly in the development of the basis for health promotion as articulated in the ottawa charter of 1986 on health promotion. this marked the beginning of a whole new aspect of public health, which proved itself in addressing with considerable success the epidemic of hiv and cardiovascular diseases. in the usa, the surgeon general's reports of 1964 on smoking and health, and of 1979 defining health targets as national policy promoted the incorporation of "management by objectives" from the business world applied to the health sector (see chapter 12). this led to healthy people usa 2000 and later versions, and the united nations (un) millennium development goals (mdgs), aimed primarily at the middle-and low-income countries (box 2.4). the identification of infectious causes of cancers of the liver and cervix established a new paradigm in epidemiology, and genetic epidemiology has important potential for public health and clinical medicine. in the basic host-agent-environment paradigm, a harmful agent comes through a sympathetic environment into contact with a susceptible host, causing a specific disease. this idea dominated public health thinking until the midtwentieth century. the host is the person who has or is at risk for a specific disease. the agent is the organism or direct cause of the disease. the environment includes the external factors which influence the host, his or her susceptibility to the agent, and the vector which transmits or carries the agent to the host from the environment. this explains the causation and transmission of many diseases. this paradigm (figure 2. 2), in effect, joins together the contagion and miasma theories of disease causation. a specific agent, a method of transmission, and a susceptible host are involved in an interaction, which are central to the infectivity or severity of the disease. the environment can provide the carrier or vector of an infective (or toxic) agent, and it also contributes factors to host susceptibility; for example, unemployment, poverty, or low education level. the expanded host-agent-environment paradigm widens the definition of each of the three components ( figure 2 .3), in relation to both acute infectious and chronic noninfectious disease epidemiology. in the latter half of the twentieth century, this expanded host-agent-environment paradigm took on added importance in dealing with the complex of factors related to chronic diseases, now the leading causes of disease and premature mortality in the developed world, and increasingly in developing countries. interventions to change host, environmental, or agent factors are the essence of public health. in infectious disease control, the biological agent may be removed by pasteurization of food products or filtration and disinfection (chlorination) of water supplies to prevent transmission of waterborne disease. the host may be altered by immunization to provide immunity to a specific infective organism. the environment may be changed to prevent transmission by destroying the vector or its reservoir of the disease. a combination of these interventions can be used against a specific risk factor, toxic or nutritional deficiency, infectious organism, or disease process. vaccine-preventable diseases may require both routine and special activities to boost herd immunity to protect the individual and the community. for other infectious diseases for which there is no vaccine (e.g., malaria), control involves a broad range of activities including case finding and treatment to improve the individual's health and to reduce the reservoir of the disease in the population, and other measures such as bed nets to reduce exposure of the host to vector mosquitoes, as well as vector control to reduce the mosquito population. tb control requires not only case finding and treatment, but understanding the contributory factors of social conditions, diseases with tb as a secondary condition (substance abuse and aids), agent resistance to treatment, and the inability of patients or carriers to complete treatment without supervision. sexually transmitted infections (stis) which are not controllable by vaccines require a combination of personal behavior change, health education, medical care, and skilled epidemiology. with non-infectious diseases, intervention is even more complex, involving human behavior factors and a wide range of legal, administrative, and educational issues. there may be multiple risk factors, which have a compounding effect in disease causation, and they may be harder to alter than infectious diseases factors. for example, smoking in and of itself is a risk factor for lung cancer, but exposure to asbestos fibers has a compounding effect. preventing exposure to the compounding variables may be easier than smoking cessation. reducing trauma morbidity and mortality is equally problematic. the identification of a single specific cause of a disease is of great scientific and practical value in modern public health, enabling such direct interventions as the use of vaccines or antibiotics to protect or treat individuals from infection by a causative organism, toxin, deficiency condition, or social factor. the cumulative effects of several contributing or risk factors in disease causation are also of great significance in many disease processes, in relation to infectious diseases such as nutritional status as for chronic diseases such as the cardiovascular group. the health of an individual is affected by risk factors intrinsic to that person as well as by external factors. intrinsic factors include the biological ones that the individual inherits and those life habits he or she acquires, such as smoking, overeating, or engaging in other high-risk behaviors. external factors affecting individual health include the environment, the socioeconomic and psychological state of the person, the family, and the society in which he or she lives. education, culture, and religion are also contributory factors to individual and community health. there are factors that relate to health of the individual in which the society or the community can play a direct role. one of these is provision of medical care. another is to ensure that the environment and community services include safety factors that reduce the chance of injury and disease, or include protective measures; for example, fluoridation of a community water supply to improve dental health, and seat-belt or helmet laws to reduce motor vehicle injury and death. these modifying factors may affect the response of the individual or the spread of an epidemic (see chapter 3). an epidemic may also include chronic disease, because common risk factors may cause an excess of cases in a susceptible population group, in comparison to the situation before the risk factor appeared, or in comparison to a group not exposed to the risk factor. these include rapid changes or "epidemics" in such conditions as type 2 diabetes, asthma, cardiovascular diseases, trauma, and other non-infectious disorders. disease is a dynamic process, not only of causation, but also of incubation or gradual development, severity, and the effects of interventions intended to modify outcome. knowledge of the natural history of disease is fundamental to understanding where and with what means intervention can have the greatest chance for successful interruption or change in the disease process for the patient, family, or community. the natural history of a disease is the course of that disease from beginning to end. this includes the factors that relate to its initiation; its clinical course leading up to resolution, cure, continuation, or long-term sequelae (further stages or complications of a disease); and environmental or intrinsic (genetic or lifestyle) factors and their effects at all stages of the disease. the effects of intervention at any stage of the disease are part of the disease process (figure 2 .4). as discussed above, disease occurs in an individual when agent, host, and environment interact to create adverse conditions of health. the agent may be an infectious organism, a chemical exposure, a genetic defect, or a deficiency condition. a form of individual or social behavior, such as reckless driving or risky sexual behavior, may lead to injury or disease. the host may be immune or susceptible as a result of many contributing social and environmental factors. the environment includes the vector, which may be a malaria-bearing mosquito, a contaminated needle shared by drug users, lead-contaminated paint, or an abusive family situation. assuming a natural state of "wellness" -i.e., optimal health or a sense of well-being, function, and absence of disease -a disease process may begin with the onset of a disease, infectious or non-infectious, following a somewhat characteristic pattern of "incubation" described by clinicians and epidemiologists. preclinical or predisposing events may be detected by a clinical history, with determination of risk including possible exposure or presence of other risk factors. interventions, before and during the process, are intended to affect the later course of the disease. the clinical course of a disease, or its laboratory or radiological findings, may be altered by medical or public health intervention, leading to the resolution or continuation of the disease with fewer or less severe secondary sequelae. thus, the intervention becomes part of the natural history of the disease. the natural history of an infectious disease in a population will be affected by the extent of prior vaccination or previous exposure in the community. diseases particular to children are often so because the adult population is immune from previous exposure or vaccinations. measles and diphtheria, primarily childhood diseases, now affect adults to a large extent because they are less protected by naturally acquired immunity or are vulnerable when their immunity wanes naturally or as a result of inadequate vaccination in childhood. in chronic disease management, high costs to the patient and the health system accrue where preventive services or management are inadequate, not yet available, or inaccessible or where there is a failure to apply the necessary interventions. the progress of diabetes to severe complications such as cardiovascular, renal, and ocular disease is delayed or reduced by good management of the condition, with a combination of smoking cessation, diet, exercise, and medications with good medical supervision. the patient with advanced chronic obstructive pulmonary disease or congestive heart failure may be managed well and remain stable with smoking avoidance, careful management of medications, immunizations against influenza and pneumonia, and other prevention-oriented care needs. where these are not applied or if they fail, the patient may require long and expensive medical and hospital care. failure to provide adequate supportive care will show up in ways that are more costly to the health system and will prove more life-threatening to the patient. the goal is to avoid where possible the necessity for tertiary care, substituting tertiary prevention, i.e., supportive rehabilitation to maximum personal function and maintaining a stable functional status. as in an individual, the phenomenon of a disease in a population may follow a course in which many factors interplay, and where interventions affect the natural course of the disease. the epidemiological patterns of an infectious disease can be assessed in their occurrence in the population or their mortality rates, just as they can for individual cases. the classic mid-nineteenth-century description of measles in the faroe islands by panum showed the transmission and the epidemic nature of the disease as well as the protective effect of acquired immunity (see chapter 1). similar, more recent breakthroughs in medical, epidemiological, biological, and social sciences have produced enormous benefit for humankind as discussed throughout this text, with some examples. these include the eradication of smallpox and in the coming years, poliomyelitis, measles, leprosy, and other dreaded diseases known for millennia; the near-elimination of rheumatic heart disease and peptic ulcers in the industrialized countries; vast reduction in mortality from stroke and coronary heart disease (chd); and vaccines (against hepatitis b and hpv) for the prevention of cancers. these and other great achievements of the twentieth and early part of the twenty-first centuries hold great promise for humankind in the coming decades, but great challenges lie ahead as well. the biggest challenge is to bring the benefits of known public health capacity to the poorest population of each country and the poorest populations globally. in developed countries a major challenge is to renew efforts of public health capacity to bear on prevention of chronic conditions such as diabetes and obesity, considered to be at pandemic proportions; and the individual and societal effects of mental diseases. in public health today, fears of a pandemic of avian influenza are based on transmission of avian or other animal-borne (zoonotic) prions or viruses to humans and then their adaptation permitting human-to-human spread. with large numbers of people living in close contact with many animals (wild and domestic fowl), such as in china and south-east asia, and rapid transportation around the world, the potential for global spread of disease is almost without historical precedent. indeed, many human infectious diseases are zoonotic in origin and transferred from natural wildlife reservoirs to humans either directly or via domestic or other wild animals, such as from birds to chickens to humans in avian influenza. monitoring or immunization of domestic animals requires a combination of multidisciplinary zoonotic disease management strategies, public education and awareness, and veterinary public health monitoring and control. rift valley fever, equine encephalitis, and more recently sars and avian influenza associated with bird-borne viral disease which can affect humans, each show the terrible dangers of pandemic diseases. ebola virus is probably sustained between outbreaks among fruit bats, or as recently suggested wild or domestic pigs, and may become a major threat to public health as human case fatality rates decline, meaning that patients and carriers, or genetic drift of the virus with possible airborne transmission, may spread this deadly disease more widely than in the past (see chapter 4). the health of populations, like the health of individuals, depends on societal factors no less than on genetics, personal risk factors, and medical services. social inequalities in health have been understood and documented in public health over the centuries. the chadwick and shattuck reports of 1840-1850 documented the relationship of poverty and bad sanitation, housing, and working conditions with high mortality, and ushered in the idea of social epidemiology. political and social ideologies thought that the welfare state, including universal health care systems of one type or another, would eliminate social and geographic differences in health status and this is in large part true. from the introduction of compulsory health insurance in germany in the 1880s to the failed attempt in the usa at national health insurance in 1995 (see chapters 1, 10 and 13) and the more recent achievements of us president obama in 2010-2011, social reforms to deal with inequalities in health have focused on improving access to medical and hospital care. almost all industrialized countries have developed such systems, and the contribution of these programs to improve health status has been an important part of social progress, especially since world war ii. but even in societies with universal access to health care, people of lower socioeconomic status (ses) suffer higher rates of morbidity and mortality from a wide variety of diseases. the black report (douglas black) in the uk in the early 1980s pointed out that the class v population (unskilled laborers) had twice the total and specific mortality rates of the class i population (professional and business) for virtually all disease categories, ranging from infant mortality to death from cancer. the report was shocking because all britons have had access to the comprehensive national health service (nhs) since its inception in 1948, with access to a complete range of services at no cost at time of service, close relations to their general practitioners, and good access to specialty services. these findings initiated reappraisals of the social factors that had previously been regarded as the academic interests of medical sociologists and anthropologists and marginal to medical care. more recent studies and reviews of regional, ethnic, and socioeconomic differentials in patterns of health care access, morbidity, and mortality indicate that health inequities are present in all societies including the uk, the usa, and others, even with universal health insurance or services. the ottawa charter on health promotion in 1986 placed a new paradigm before the world health community that recognized social and political factors as no less important ion health that traditional medical and sanitary public health measures. these concepts helped the world health community to cope with new problems such as hiv/aidsfor which there was neither a medical cure nor a vaccine to prevent the disease. its control came to depend in the initial decades almost entirely on education and change in lifestyles, until the advent of the antiretroviral drugs in the 1990s. there is still no viable vaccine. although the epidemiology of cardiovascular disease shows the direct relationship of the now classic risk factors of stress, smoking, poor diet, and physical inactivity, differences in mortality from cardiovascular disease between different classes among british civil servants are not entirely explainable by these factors. the differences are also affected by social and economic issues that may relate to the psychological needs of the individual, such as the degree of control people have over their own lives. blue-collar workers have less control over their lives, their working life in particular, than their white-collar counterparts, and have higher rates of chd mortality than higher social classes. other work shows the effects of migration, unemployment, drastic social and political change, and binge drinking, along with protective effects of healthy lifestyle, religiosity, and family support systems in cardiovascular diseases. social conditions affect disease distribution in all societies. in the usa and western europe, tb has re-emerged as a significant public health problem in urban areas partly because of high-risk population groups, owing to poverty and alienation from society, as in the cases of homelessness, drug abuse, and hiv infection. in countries of eastern europe and the former soviet union, the recent rise in tb incidence has resulted from various social and economic factors in the early 1990s, including the large-scale release of prisoners. in both cases, diagnosis and prescription of medication are inadequate, and the community at large becomes at risk because of the development of antibioticresistant strains of tubercle bacillus readily spread by inadequately treated carriers, acting as human vectors. studies of ses and health are applicable and valuable in many settings. in alameda county, california, differences in mortality between black and white population groups in terms of survival from cancer became insignificant when controlled for social class. a 30-year follow-up study of the county population reported that low-income families in california are more likely than those on a higher income to have physical and mental problems that interfere with daily life, contributing to further impoverishment. studies of the association between indicators of ses and recent screening in the usa, australia, finland, and elsewhere showed that lower ses women use less preventive care such as papanicolaou (pap) smears for cervical cancer than women of higher ses, despite having greater risk for cervical cancer. many factors in ses inequalities are involved, including transportation and access to primary care, differences in health insurance coverage, educational levels, poverty, high-risk behaviors, social and emotional distress, feeling a lack of control over one's own life, employment, occupation, and inadequate family or community social support systems. many barriers exist owing to difficulties in access and the lack of availability of free or low-cost medical care, and the absence or limitations of health insurance is a further factor in the socioeconomic gradient. the recognition that health and disease are influenced by many factors, including social inequalities, plays a fundamental role in the new public health paradigm. health care systems need to take into account economic, social, physical, and psychological factors that otherwise will limit the effectiveness of even the best medical care. the health system includes access to competent and responsible primary care as well as by the wider health system, including health promotion, specific prevention and population-based health protection. the paradigm of the host-agent-environment triad (figures 2.2 and 2. 3) is profoundly affected by the wider context. the sociopolitical environment and organized efforts at intervention affect the epidemiological and clinical course of disease of the individual. medical care is essential, as is public health, but the persistent health inequities seen in most regions and countries require societal attention. success or failure in improving the conditions of life for the poor, and other vulnerable "risk groups", affect national or regional health status and health system performance. the health system is meant to reduce the occurrence or bad outcome of disease, either directly by primary prevention or treatment as secondary prevention or by maximum rehabilitation as tertiary prevention, or equally important indirectly by reducing community or individual risk factors. the the effects of social conditions on health can be partly offset by interventions intended to promote healthful conditions; for example, improved sanitation, or through good-quality primary and secondary health services, used efficiently and effectively made available to all. the approaches to preventing disease or its complications may require physical changes in the environment, such as removal of the broad street pump handle to stop the cholera epidemic in london, or altering diets as in goldberger's work on pellagra. some of the great successes of public health have been and continue to be low technology. examples, among many others, include insecticide-impregnated bednets and other vector control measures, oral rehydration solutions, treatment and cure of peptic ulcers, exercise and diet to reduce obesity, hand washing in hospitals (and other health facilities), community health workers, and condoms and circumcision for the prevention of stis, including hiv and cancer of the cervix. the societal context in terms of employment, social security, female education, recreation, family income, cost of living, housing, and homelessness is relevant to the health status of a population. income distribution in a wealthy country may leave a wide gap between the upper and lower socioeconomic groups, which affects health status. the media have great power to sway public perception of health issues by choosing what to publish and the context in which to present information to society. modern media may influence an individual's tendency to overestimate the risk of some health issues while underestimating the risk of others, ultimately influencing health choices, such as occurred with public concern regarding false claims of an association between the measles-mumps-rubella (mmr) vaccine and autism in the uk (see the wakefield effect, chapter 4). the new public health has an intrinsic responsibility for advocacy of improved societal conditions in its mission to promote optimal community health. an ultimate goal of public health is to improve health and to prevent widespread disease occurrence in the population and in an individual. the methods of achieving this are wide and varied. when an objective has been defined in "social justice is a matter of life and death. it affects the way people live, their consequent chance of illness, and their risk of premature death. we watch in wonder as life expectancy and good health continue to increase in parts of the world and in alarm as they fail to improve in others. a girl born today can expect to live for more than 80 years if she is born in some countries -but less than 45 years if she is born in others. within countries there are dramatic differences in health that are closely linked with degrees of social disadvantage. differences of this magnitude, within and between countries, simply should never happen. these inequities in health, avoidable health inequalities arise because of the circumstances in which people grow, live, work, and age, and the systems put in place to deal with illness. the conditions in which people live and die are, in turn, shaped by political, social, and economic forces. social and economic policies have a determining impact on whether a child can grow and develop to its full potential and live a flourishing life, or whether its life will be blighted. increasingly the nature of the health problems rich and poor countries have to solve are converging. the development of a society, rich or poor, can be judged by the quality of its population's health, how fairly health is distributed across the social spectrum, and the degree of protection provided from disadvantage as a result of ill-health." preventing disease, the next step is to identify suitable and feasible methods of achieving it, or a strategy with tactical objectives. this determines the method of operation, course of action, and resources needed to carry it out. the methods of public health are categorized as health promotion, and primary, secondary, and tertiary prevention (box 2.6). health promotion is the process of enabling people and communities to increase control over factors that influence their health, and thereby to improve their health (adapted from the ottawa charter of health promotion, 1986; box 2.7). health promotion is a guiding concept involving activities intended to enhance individual and community health and well-being (box 2.8). it seeks to increase involvement and control by the individual and the community in their own health. it acts to improve health and social welfare, and to reduce specific determinants of diseases and risk factors that adversely affect the health, well-being, and productive capacities of an individual or society, setting targets based on the size of the problem but also the feasibility of successful intervention, in a cost-effective way. this can be through direct contact with the patient or risk group, or act indirectly through changes in the environment, legislation, or public policy. control of aids relies on an array of interventions that promote change in sexual behavior and other contributory risks such as sharing of needles among drug users, screening of blood supply, safe hygienic practices in health care settings, and education of groups at risk such as teenagers, sex workers, migrant workers, and many others. control of aids is also a clinical problem in that patients need antiretroviral therapy (art), but this becomes a management and policy issue for making these drugs available and at an affordable price for the poor countries most affected. this is an example of the challenge and effectiveness of health promotion and the new public health. health promotion is a key element of the new public health and is applicable in the community, the clinic or hospital, and in all other service settings. some health promotion activities are government legislative and box 2.6 modes of prevention l health promotion -fostering national, community, and individual knowledge, attitudes, practices, policies, and standards conducive to good health; promoting legislative, social, or environmental conditions; promoting knowledge and practices for self-care that reduce individual and community risk; and creating a healthful environment. it is directed toward action on the determinants of health. l health protection -activities of official health departments or other agencies empowered to supervise and regulate food hygiene, community and recreational water safety, environmental sanitation, occupational health, drug safety, road safety, emergency preparedness, and many other activities to eliminate or reduce as much as possible risks of adverse consequences to health. l primary prevention -preventing a disease from occurring, e.g., vaccination to prevent infectious diseases, advice to stop smoking to prevent lung cancer. l secondary prevention -making an early diagnosis and giving prompt and effective treatment to stop progress or shorten the duration and prevent complications from an already existing disease process, e.g., screening for hypertension or cancer of cervix and colorectal cancer for early case finding, early care and better outcomes. l tertiary prevention -stopping progress of an already occurring disease, and preventing complications, e.g., in managing diabetes and hypertension to prevent complications; restoring and maintaining optimal function once the disease process has stabilized, e.g., promoting functional rehabilitation after stroke and myocardial infarction with long-term follow-up care. health promotion (hp) is the process of enabling people to increase control over, and to improve their health. hp represents a comprehensive social and political process, and not only embraces actions directed at strengthening the skills and capabilities of individuals. hp also undertakes action directed towards changing social, environmental, and economic conditions so as to alleviate their impact on public and individual health. health promotion is the process of enabling people to increase control over the determinants of health and thereby improve their health. participation is essential to sustain health promotion action. the ottawa charter identifies three basic strategies for health promotion. these are advocacy for health to create the essential conditions for health indicated above; enabling all people to achieve their full health potential; and mediating between the different interests in society in the pursuit of health. these strategies are supported by five priority action areas as outlined in the ottawa charter for health promotion: regulatory interventions such as mandating the use of seat belts in cars, requiring that children be immunized to attend school, declaring that certain basic foods must have essential minerals and vitamins added to prevent nutritional deficiency disorders in vulnerable population groups, and mandating that all newborns should be given prophylactic vitamin k to prevent hemorrhagic disease of the newborn. setting food and drug standards and raising taxes on cigarettes and alcohol to reduce their consumption are also part of health promotion. promoting a healthy lifestyle is a major known obesity-preventive activity. health promotion is provided by organizations and people with varied professional backgrounds working towards common goals of improvement in the health and quality of individual and community life. initiatives may come from government with dedicated allocation of funds to address specific health issues, from donors, or from advocacy or community groups or individuals to promote a specific or general cause in health. raising awareness to inform and motivate people about their own health and lifestyle factors that might put them at risk requires teaching young people about the dangers of sexually transmitted diseases, smoking, and alcohol abuse to reduce risks associated with their social behavior. it might include disseminating information on healthy nutrition; for example, the need for folic acid supplements for women of childbearing age and multiple vitamins for elderly, as well as the elements of a healthy diet, compliance with immunization recommendations, compliance with screening programs, and many others. community and peer group attitudes and standards affect individual behavior. health promotion endeavors to create a climate of knowledge, attitudes, beliefs, and practices that are associated with better health outcomes. international conferences following on from the ottawa charter were held in adelaide in 1988 , sundsvall in 1991 , jakarta in 1999 , mexico in 2000 , bangkok in 2005 , and nairobi in 2009 . the principles of health promotion have been reiterated and have influenced public policy regarding public health as well as the private sector. health promotion has a track record of proven success in numerous public health issues where a biomedical solution was not available. the hiv/aids pandemic from the 1980s until the late 1990s had no medical treatment and control measures relied on screening, education, lifestyle changes, and supportive care. health promotion brought forward multiple interventions, from condom use and distribution, to needle exchanges for intravenous drug users, to male circumcision in high-prevalence african countries. medical treatment was severely limited until art was developed. the success of art also depends on a strong element of health promotion in widening the access to treatment and the success of medications to reduce transmission, most remarkably in reducing maternal-fetal transmission (see chapter 4). similarly, in the battle against cardiovascular diseases, health promotion was an instrumental factor in raising public awareness of the importance of management of hypertension and smoking reduction, dietary restraint, and physical exercise. the success of massive reductions in stroke and chd mortality is as much the result of health promotion as of improved medical care (see chapter 5). the character of public health carries with it a "good cop, bad cop" dichotomy. the "good cop" is persuasive and educational trying to convince people to do the right thing in looking after their own health: diet, exercise, smoking cessation, and others. on the other side, the "bad cop" role is regulatory and punitive. public health has a serious responsibility and role in the enforcement of laws and regulation to protect the public health. some of these are restrictive box 2.8 elements of health promotion 1. address the population as a whole in health-related issues, in everyday life as well as people at risk for specific diseases. 2. direct action to risk factors or causes of illness or death. 3. undertake activist approach to seek out and remedy risk factors in the community that adversely affect health. 4. promote factors that contribute to a better condition of health of the population. 5. initiate actions against health hazards, including communication, education, legislation, fiscal measures, organizational change, community development, and spontaneous local activities. 6. involve public participation in defining problems and deciding on action. 7. advocate relevant environmental, health, and social policy. 8. encourage health professional participation in health education and health advocacy. 9. advocate for health based on human rights and solidarity. 10. invest in sustainable policies, actions, and infrastructure to address the determinants of health. 11. build capacity for policy development, leadership, health promotion practice, knowledge transfer and research, and health literacy. 12. regulate and legislate to ensure a high level of protection from harm and enable equal opportunity for health and well-being for all people. 13. partner and build alliances with public, private, nongovernmental, and international organizations and civil society to create sustainable actions. 14. make the promotion of health central to the global development agenda. of individual rights that may damage other people or are requirements based on strong evidence of benefits to population health. readily accepted are food and drug standards, such as pasteurization of milk, and iodization of salt; requirements to drive on the right-hand side of the road (except in some countries such as the uk), to wear seat belts and for motorcyclists to wear safety helmets; and not smoking in public places. enforcement of these and similar statutory or regulatory requirements is vital in a civil society to protect the public from health hazards and to protect people from harm and exploitation by unscrupulous manufacturers and marketing. cigarette advertising and sponsorship of sports events by tobacco companies are banned in most upper income countries. the use of transfats in food manufacturing and baking is now banned and salt reduction is being promoted and even mandated in many us local authorities to reduce cardiovascular disease. advertising of unhealthy snack foods on children's television programs and during child-watching hours is commonly restricted. banning high-sugar soda drink distribution in schools is a successful intervention to reduce the current child obesity epidemic. melamine use in milk powders and baby formulas, which caused widespread illness and death of infants in china, is now banned and a punishable offence for manufacture or distribution in china and worldwide. examples of this aspect of public health are mentioned throughout this text, especially in chapters 8 and 9 on nutrition, and environmental and occupational health, respectively. the regulatory enforcement function of public health is sometimes controversial and portrayed as interference with individual liberty. fluoridation of community water supplies is an example where aggressive lobby groups opposing this safe and effective public health measure are still common. this is discussed in chapter 7. equally important is the public health policy issue of resource allocation and taxation for health purposes. taxation is an unpopular measure that governments must employ and enforce in order to do the public's business. the debate over the patient protection and affordable care act 2010 (ppaca or "obamacare"), discussed elsewhere in this and other chapters, shows how bitter the arguments can become, yet the goal of equality of access to health care cannot be denied as a public good, demonstrably contributing to the health of the nation. primary prevention refers to those activities that are undertaken to prevent disease or injury from occurring at all. primary prevention works with both the individual and the community. it may be directed at the host to increase resistance to the agent (such as in immunization or cessation of smoking), or at environmental activities to reduce conditions favorable to the vector for a biological agent, such as mosquito vectors of malaria or dengue fever. landmark examples include the treatment and prevention of scurvy among sailors based on james lind's findings in a classic clinical epidemiological study in 1747, and john snow's removal of the handle from the broad street pump to stop a cholera epidemic in london in 1854 (see chapter 1). primary prevention includes elements of health protection such as ensuring water, food and drug, and workplace safety; chlorination of drinking water to prevent transmission of waterborne enteric diseases; pasteurization of milk to prevent gastrointestinal diseases; mandating wearing seat belts in motor vehicles to prevent serious injury and death in road crashes; and reducing the availability of firearms to reduce injury and death from intentional, accidental, or random violence. it also includes direct measures to prevent diseases, such as immunization to prevent polio, tetanus, pertussis, and diphtheria. health promotion and health protection blend together as a group of activities that reduce risk factors and diseases through many forms of intervention such as changing smoking legislation or preventing birth defects by fortification of flour with folic acid. prevention of hiv transmission by needle exchange for intravenous drug users, promoting condom usage, and promoting male circumcision in africa, and the distribution of condoms and clean needles for hivpositive drug users are recent examples of primary prevention associated with health promotion programs. primary prevention also includes activities within the health system that can lead to better health. this may mean, for example, setting standards and to reduce hospital infections, and ensuring that doctors not only are informed of appropriate immunization practices and modern prenatal care or screening programs for cancer of the cervix, colon, and breast, but also are aware of their vital role in preventing cardiovascular and other non-communicable diseases. in this role, the health care provider serves as a teacher and guide, as well as a diagnostician and therapist. like health promotion, primary prevention does not depend on health care providers alone; health promotion works to increase individual and community consciousness of self-care, mainly by raising awareness and information levels and empowering the individual and the community to improve self-care, to reduce risk factors, and to live healthier lifestyles. secondary prevention is early diagnosis and management to prevent complications from a disease. public health interventions to prevent the spread of disease include the identification of sources of the disease and the implementation of steps to stop it, as shown in snow's closure of the broad street pump. secondary prevention includes steps to isolate cases and treat or immunize contacts so as to prevent further cases of meningitis or measles, for example, in outbreaks. for current epidemics such as hiv/aids, primary prevention is largely based on education, abstinence from any and certainly risky sexual behavior, circumcision, and treatment of patients in order to improve their health and to reduce the risk of spread of hiv. for high-risk groups such as intravenous drug users, needleexchange programs reduce the risk of spread of hiv, and hepatitis b and c. distribution of condoms to teenagers, military personnel, truck drivers, and commercial sex workers helps to prevent the spread of stis and aids in schools and colleges, as well as among the military. the promotion of circumcision is shown to be effective in reducing the transmission of hiv and of hpv (the causative organism for cancer of the cervix). all health care providers have a role in secondary prevention; for example, in preventing strokes by early identification and adequate care of hypertension. the child who has an untreated streptococcal infection of the throat may develop complications which are serious and potentially life-threatening, including rheumatic fever, rheumatic valvular heart disease, and glomerulonephritis. a patient found to have elevated blood pressure should be advised about continuing management by appropriate diet and weight loss if obese, regular physical exercise, and long-term medication with regular follow-up by a health provider in order to reduce the risk of stroke and other complications. in the case of injury, competent emergency care, safe transportation, and good trauma care may reduce the chance of death and/or permanent handicap. screening and high-quality care in the community prevent complications of diabetes, including heart, kidney, eye, and peripheral vascular disease. they can also prevent hospitalizations, amputations, and strokes, thus lengthening and improving the quality of life. health care systems need to be actively engaged in secondary prevention, not only as individual doctors' services, but also as organized systems of care. public health also has a strong interest in promoting highquality care in secondary and tertiary care hospital centers in such areas of treatment as acute myocardial infarction, stroke, and injury in order to prevent irreversible damage. measures include quality of care reviews to promote adequate longterm postmyocardial infarction care with aspirin and betablockers or other medication to prevent or delay recurrence and second or third myocardial infarctions. the role of highquality transportation and care in emergency facilities of hospitals in public health is vital to prevent long-term damage and disability; thus, cardiac care systems including publicly available defibrillators, catheterization, the use of stents, and bypass procedures are important elements of health care policy and resource allocation, which should be accessible not only in capital cities but also to regional populations. tertiary prevention involves activities directed at the host or patient, but also at the social and physical environment in order to promote rehabilitation, restoration, and maintenance of maximum function after the disease and its complications have stabilized. the person who has undergone a cerebrovascular accident or trauma will reach a stage where active rehabilitation can help to restore lost functions and prevent recurrence or further complications. the public health system has a direct role in the promotion of disability-friendly legislation and standards of building, housing, and support services for chronically ill, handicapped, and elderly people. this role also involves working with many governmental social and educational departments, but also with advocacy groups, ngos, and families. it may also include the promotion of disability-friendly workplaces and social service centers. treatment for conditions such as myocardial infarction or a fractured hip now includes early rehabilitation in order to promote early and maximum recovery with restoration to optimal function. the provision of a wheelchair, walkers, modifications to the home such as special toilet facilities, doors, and ramps, along with transportation services for paraplegics are often the most vital factors in rehabilitation. public health agencies work with groups in the community concerned with promoting help for specific categories of risk group, disease, or disability to reduce discrimination. community action is often needed to eliminate financial, physical, or social barriers, promote community awareness, and finance special equipment or other needs of these groups. close follow-up and management of chronic disease, physical and mental, require home care and ensuring an appropriate medical regimen including drugs, diet, exercise, and support services. the follow-up of chronically ill people to supervise the taking of medications, monitor changes, and support them in maximizing their independent capacity in activities of daily living is an essential element of the new public health. public health uses a population approach to achieve many of its objectives. this requires defining the population, including trends of change in the age and gender distribution of the population, fertility and birth rates, spread of disease and disability, mortality, marriage and migration, and socioeconomic factors. the reduction of infectious disease as the major cause of mortality, increased longevity coupled with declining fertility rates, resulted in changes in the age composition, or a demographic transition. demographic changes, such as fertility and mortality patterns, are important factors in changing the age distribution of the population, resulting in a greater proportion of people surviving to older ages. declining infant mortality, increasing educational levels of women, the availability of birth control, and other social and economic factors lead to changes in fertility patterns and the demographic transition -an aging of the population -with important effects on health service needs. the age and gender distribution of a population affects and is affected by patterns of disease. change in epidemiological patterns, or an epidemiological shift, is a change in predominant patterns of morbidity and mortality. the transition of infectious diseases becoming less prominent as causes of morbidity and mortality and being replaced by chronic and non-infectious diseases has occurred in both developed and developing countries. the decline in mortality from chronic diseases, such as cardiovascular disease, represents a new stage of epidemiological transition, creating an aging population with higher standards of health but also long-term community support and care needs. monitoring and responding to these changes are fundamental responsibilities of public health, and a readiness to react to new, local, or generalized changes in epidemiological patterns is vital to the new public health. societies are not totally homogeneous in ethnic composition, levels of affluence, or other social markers. on one hand, a society classified as developing may have substantial numbers of people with incomes that promote overnutrition and obesity, so that disease patterns may include increasing prevalence of diseases of excesses, such as diabetes. on the other hand, affluent societies include population groups with disease patterns of poverty, including poor nutrition and low birth-weight babies. a further stage of epidemiological transition has been occurring in the industrialized countries since the 1960s, with dramatic reductions in mortality from chd, stroke and, to a lesser extent, trauma. the interpretation of this epidemiological transition is still not perfectly clear. how it occurred in the industrialized western countries but not in those of the former soviet union is a question whose answer is vital to the future of health in russia and some countries of eastern europe. developing countries must also prepare to cope with increasing epidemics of non-infectious diseases, and all countries face renewed challenges from infectious diseases with antibiotic resistance or newly appearing infectious agents posing major public health threats. demographic change in a country may reflect social and political decisions and health system priorities from decades before. russia's rapid population decline since the 1990s, china's gender imbalance with a shortage of millions of young women, egypt's rapid population growth outstripping economic capacity, and many other examples indicate the severity and societal importance of capacity to analyze and formulate public health and social policies to address such fundamental sociopolitical issues. aging of the population is now the norm in most developed countries as a result of low birth and declining mortality rates. this change in the age distribution of a population has many associated social and economic issues as to the future of social welfare with a declining age cohort to provide the workforce. the aging population requires pension and health care support which make demands of social security systems that will depend on economic growth with a declining workforce. in times of economic stress, as in europe, this situation is made more difficult by longstanding short working weeks, early pension ages, and high social benefits. however, this results in unemployment among young people in particular and social conflict. the interaction of increasing life expectancy and a declining workforce is a fundamental problem in the high-income countries. this imbalance may be resolved in part through productivity gains and switching of primary production to countries with large still underutilized workforces, while employment in the developed countries will depend on service industries including health and the economic growth generated by higher technology and intellectual property and service industries. the challenge of keeping populations and individuals healthy is reflected in modern health services. each component of a health service may have developed with different historical emphases, operating independently as a separate service under different administrative auspices and funding systems, competing for limited health care resources. in this situation, preventive community care receives less attention and resources than more costly treatment services. figure 2 .5 suggests a set of health services in an interactive relationship to serve a community or defined population, but the emphasis should be on the interdependence of these services with one other and with the comprehensive network in order to achieve effective use of resources and a balanced set of services for the patient, the client or patient population, and the community. clinical medicine and public health each play major roles in primary, secondary, and tertiary prevention. each may function separately in their roles in the community, but optimal success lies in their integrated efforts. allocation of resources should promote management and planning practices to assist this integration. there is a functional interdependence of all elements of health care serving a definable population. the patient should be the central figure in the continuum or complex of services available. effectiveness in use of resources means that providing the service most appropriate for meeting the individual's or group's needs at a point in time are those that should be applied. this is the central concept in currently developing innovations in health care delivery in the usa with organizations using terms such as patient centered medical home, accountable care organizations (acos), and population health management systems, which are being promoted in the obamacare health reforms now in process (see chapter 10) (shortell et al., 2010) . separate organization and financing of services place barriers to appropriate provision of services for both the community and the individual patient. the interdependence of services is a challenge in health care organizations for the future. where there is competition for limited resources, pressures for tertiary services often receive priority over programs to prevent children from dying of preventable diseases. public health must be seen in the context of all health care and must play an influential role in promoting prevention at all levels. clinical services need public health in order to provide prevention and community health services that reduce the burden of disease, disability, and dependence on the institutional setting. health was traditionally thought of as a state of absence of disease, pain, or disability, but has gradually been expanded to include physical, mental, and societal well-being. in 1920, c. e. a. winslow, professor of public health at yale university, defined public health as follows: "public health is the science and art of (1) preventing disease, (2) prolonging life, and (3) winslow's far-reaching definition remains a valid framework but is unfulfilled when clinical medicine and public health have financing and management barriers between them. in many countries, isolation from the financing and provision of medical and nursing care services left public health with the task of meeting the health needs of the indigent and underserved population groups with inadequate resources and recognition. health insurance organizations for medical and hospital care have in recent years been more open to incorporating evidence-based preventive care, but the organization of public health has lacked the same level of attention. in some countries, the limitations have been conceptual in that public health was defined primarily in terms of control of infectious, environmental, and occupational diseases. a more recent and widely used definition is: "public health is the science and art of preventing disease, prolonging life, and promoting health through the organized efforts of society." this definition, coined in 1988 in the public health in england report by sir donald acheson, reflects the broad focus of modern public health. terms such as social hygiene, preventive medicine, community medicine, and social medicine have been used to denote public health practice over the past century. preventive medicine is the application of preventive measures by clinical practitioners combining some elements of public health with clinical practice relating to individual patients. preventive medicine defines medical or clinical personal preventive care, with stress on risk groups in the community and national efforts for health promotion. the focus is on the health of defined populations to promote health and well-being using evidence-based guidelines for cost-effective preventive measures. measures emphasized include screening and follow-up of chronic illnesses, and immunization programs; for example, influenza and pneumococcal pneumonia vaccines are used by people who are vulnerable because of their age, chronic diseases, or risk of exposure, such as medical and nursing personnel and those providing other personal clinical services. clinical medicine also deals in the area of prevention in the management of patients with hypertension or diabetes, and in doing so prevents the serious complications of these diseases. social medicine is also primarily a medical specialty which looks at illness in an individual in the family and social context, but lacks the environmental and regulatory and organized health promotion functions of public health. community health implies a local form of health intervention, whereas public health more clearly implies a global approach, which includes action at the international, national, state, and local levels. some issues in health can be dealt with at the individual, family, or community level; others require global strategies and intervention programs with regional, national, or international collaboration and leadership. the social medicine movement originated to address the harsh conditions of the working population during the industrial revolution in mid-nineteenth-century europe. an eminent pioneer in cellular pathology, rudolph virchow provided leadership in social medicine powered by the revolutionary movements of 1848, and subsequent social democrat political movements. their concern focused on harsh living and health conditions among the urban poor working class and neglectful political norms of the time. social medicine also developed as an academic discipline and advocacy orientation by providing statistical evidence showing, as in various governmental reports in the mid-nineteenth century, that poverty among the working class was associated with short life expectancy and that social conditions were key factors in the health of populations and individuals. this movement provided the basis for departments in medical faculties and public health education throughout the world stressing the close relationship between political priorities and health status. this continued in the twentieth century and in the usa found expression in pioneering work since the 1940s at montefiore hospital in new york and with victor sidel, founding leader of the community health center movement the usa from the 1960s. in the twenty-first century this movement continues to emphasize relationships between politics, society, disease, and medicine, and forms of medical practice derived from it, as enunciated by prominent advocates such as harvardbased paul farmer in haiti, russia and rwanda, and in the uk by martin mckee and others (nolte and mckee, 2008) . similar concepts are current in the usa under headings such as family medicine, preventive medicine, and social medicine. this movement has also influenced sir michael marmot and others in the world health commission of health inequalities of 2008, with a strong influence on the un initiative to promote mdgs, whose first objective is poverty reduction (commission on inequalities report 2010). application of the idea of poverty reduction as a method of reducing health inequalities has been successful recently in a large field trial in brazil showing greater reduction in child mortality where cash bonuses were awarded by municipalities for the poor families than that observed in other similar communities (rasella, 2013). in the usa, this movement is supported by increased health insurance coverage for the working poor, with funding for preventive care and incentives for community health centers in the obamacare plan of 2010 for implementation in the coming years to provide care for uninsured and underserved populations, particularly in urban and rural poverty areas. the political aspect of social medicine is the formulation of and support for national initiatives to widen health care coverage to the 16 percent of the us population who are still uninsured, and to protect those who are arbitrarily excluded owing to previous illnesses, caps on coverage allowed, and other exploitative measures taken by private insurance that frequently deny americans access to the high levels of health care available in the country. the ethical base of public health in europe evolved in the context of its successes in the nineteenth and early twentieth centuries along with ideas of social progress. but the twentieth century was also replete with extremism and wide-scale abuse of human rights, with mass executions, deportations, and starvation as official policy in fascist and stalinist regimes. eugenics, a pseudoscience popularized in the early decades of the twentieth century, promoted social policies meant to improve the hereditary qualities of a race by methods such as sterilization of mentally handicapped people. the "social and racial hygiene" of the eugenics movements led to the medicalization of sterilization in the usa and other countries. this was adopted and extended in nazi germany to a policy of murder, first of the mentally and physically handicapped and then of "racial inferiors". these eugenics theories were widely accepted in the medical community in germany, then used by the nazi regime to justify medically supervised killing of hundreds of thousands of helpless, incapacitated individuals. this practice was linked to wider genocide and the holocaust, with the brutalization and industrialized murder of over 6 million jews and 6 million other people, and corrupt medical experimentation on prisoners. following world war ii, the ethics of medical experimentation (and public health) were codified in the nuremberg code and universal declaration of human rights based on lessons learned from these and other atrocities inflicted on civilian populations (see chapter 15). threats of genocide, ethnic cleansing, and terrorism are still present on the world stage, often justified by current warped versions of racial hygienic theories. genocidal incitement and actual genocide and terrorism have recurred in the last decades of the twentieth century and into the twenty-first century in the former yugoslav republics, africa (rwanda and darfur), south asia, and elsewhere. terrorism against civilians has become a worldwide phenomenon with threats of biological and chemical agents, and potentially with nuclear capacity. asymmetrical warfare of insurgencies which use innocent civilians for cover, as with other forms of warfare, carries with it grave dangers to public health, human rights, and international stability, as seen in the twenty-first century in south sudan, darfur, dr congo, chechnya, iraq, afghanistan, and pakistan. in 1961, kerr white and colleagues defined medical ecology as population-based research providing the foundation for management of health care quality. this concept stresses a population approach, including those not attending and those using health services. this concept was based on previous work on quality of care, randomized clinical trials, medical audit, and structure-process-outcome research. it also addressed health care quality and management. these themes influenced medical research by stressing the population from which clinical cases emerge as well as public health research with clinical outcome measures, themes that recur in the development of health services research and, later, evidence-based medicine. this led to the development of the agency for health care policy and research and development in the us department of health and human services and evidence-based practice centers to synthesize fundamental knowledge for the development of information for decision-making tools such as clinical guidelines, algorithms, or pathways. clinical guidelines and recommended best practices have become part of the new public health to promote quality of patient care and public health programming. these can include recommended standards; for example, follow-up care of the postmyocardial infarction patient, an internationally recommended immunization schedule, recommended dietary intake or food fortification standards, and mandatory vitamin k and eye care for all newborns and many others (see chapter 15). community-oriented primary care (copc) is an approach to primary health care that links community epidemiology and appropriate primary care, using proactive responses to the priority needs identified. copc, originally pioneered in south africa and israel by sidney and emily kark and colleagues in the 1950s and 1960s, stresses medical services in the community which need to be adapted to the needs of the population as defined by epidemiological analysis. copc involves community outreach and education, as well as clinical preventive and treatment services. copc focuses on community epidemiology and an active problem-solving approach. this differs from national or larger scale planning that sometimes loses sight of the local nature of health problems or risk factors. copc combines clinical and epidemiological skills, defines needed interventions, and promotes community involvement and access to health care. it is based on linkages between the different elements of a comprehensive basket of services along with attention to the social and physical environment. a multidisciplinary team and outreach services are important for the program, and community development is part of the process. in the usa, the copc concept has influenced health care planning for poor areas, especially provision of federally funded community health centers in attempts to provide health care for the underserved since the 1960s. in more recent years, copc has gained wider acceptance in the usa, where it is associated with family physician training and community health planning based on the risk approach and "managed care" systems. indeed, the three approaches are mutually complementary (box 2.9). as the emphasis on health care reform in the late 1990s moved towards managed care, the principles of copc were and will continue to be important in promoting health and primary prevention in all its modalities, as well as tertiary prevention with followup and maintenance of the health of the chronically ill. copc stresses that all aspects of health care have moved towards prevention based on measurable health issues in the community. through either formal or informal linkages between health services, the elements of copc are part of the daily work of health care providers and community services systems. the us institute of medicine issued the report on primary care in 1995, defining primary care as "the provision of integrated, accessible health care services by clinicians who are accountable for addressing the majority of personal health care needs, developing a sustained partnership with patients and practicing in the context of the family and the community". this formulation was criticized by the american public health association (apha) as lacking a public health perspective and failing to take into account both the individual and the community health approaches. copc tries to bridge this gap between the perspectives of primary care and public health. the community, whether local, regional, or national, is the site of action for many public health interventions. moreover, understanding the characteristics of the community is vital to a successful community-oriented approach. by the 1980s, new patterns of public health began to emerge, including all measures used to improve the health of the community, and at the same time working to protect and promote the health of the individual. the range of activities to achieve these general goals is very wide, including individual patient care systems and the community-wide activities that affect the health and well-being of the individual. these include the financing and management of health systems, evaluation of the health status of the population, and measures to improve the quality of health care. they place reliance on health promotion activities to change environmental risk factors for disease and death. they promote integrative and multisectoral approaches and the international health teamwork required for global progress in health. the definition of health in the charter of the who as a complete state of physical, mental, and social well-being had a ring of utopianism and irrelevance to states struggling to provide even minimal care in severely adverse political, economic, social, and environmental conditions (box 2.10). in 1977, a more modest goal was set for attainment of a level of health compatible with maximum feasible social and economic productivity. one needs to recognize that health and disease are on a dynamic continuum that affects everyone. the mission for public health is to use a wide range of methods to prevent disease and premature death, and improve quality of life for the benefit of individuals and the community. the world health organization defines health as "a state of complete physical, mental and social well-being, not merely the absence of disease or infirmity" (who constitution, 1948) . in 1978 at the alma-ata conference on primary health care, the who related health to "social and economic productivity in setting as a target the attainment by all the people of the world of a level of health that will permit them to lead a socially and economically productive life". three general programs of work for the periods 1984-1989, 1990-1995, and 1996-2001 were formulated as the basis of national and international activity to promote health. in 1995, the who, recognizing changing world conditions of demography, epidemiology, environment, and political and economic status, addressed the unmet needs of developing countries and health management needs in the industrialized countries, calling for international commitment to "attain targets that will make significant progress towards improving equity and ensuring sustainable health development". the 1999 object of the who is restated as "the attainment by all peoples of the highest possible level of health" as defined in the who constitution, by a wide range of functions in promoting technical cooperation, assisting governments, and providing technical assistance, international cooperation, and standards. in the 1960s, most industrialized countries were concentrating energies and financing in health care on providing access to medical and hospital services through national insurance schemes. developing countries were often spending scarce resources trying to emulate this trend. the who was concentrating on categorical programs, such as eradication of smallpox and malaria, as well as the expanded program of immunization and similar specific efforts. at the same time, there was a growing concern that developing countries were placing too much emphasis and expenditure on curative services and not enough on prevention and primary care. the world health assembly (wha) in 1977 endorsed the primary care approach under the banner of "health for all by the year 2000" (hfa 2000) . this was a landmark decision and has had important practical results. the who and the united nations children's fund (unicef) sponsored a seminal conference held in alma-ata, in the ussr ( kazakhstan), in 1978, which was convened to refocus health policy on primary care. the alma-ata declaration stated that health is a basic human right, and that governments are responsible to assure that right for their citizens and to develop appropriate strategies to fulfill this promise. this proposition has come to be increasingly accepted in the international community. the conference stressed the right and duty of people to participate in the planning and implementation of their health care. it advocated the use of scientifically, socially, and economically sound technology. joint action through intersectoral cooperation was also emphasized. the alma-ata declaration focused on primary health care as the appropriate method of assuming adequate access to health care for all (box 2.11). many countries have gradually come to accept the notion of placing priority on primary care, resisting the temptation to spend high percentages of health care resources on high-tech and costly medicine. spreading these same resources into highly costeffective primary care, such as immunization and nutrition programs, provides greater benefit to individuals and to society as a whole. alma-ata provided a new sense of direction for health policy, applicable to developing countries and in a different way than the approaches of the developed countries. during the 1980s, the health for all concept influenced national health policies in the developing countries with signs of progress in immunization coverage, for example, but the initiative was diluted as an unintended consequence by more categorical programs such as eradication of poliomyelitis. for example, developing countries have accepted immunization and diarrheal disease control as high-priority issues and achieved remarkable success in raising immunization coverage from some 10 percent to over 75 percent in just a decade. developed countries addressed these principles in different ways. in these countries, the concept of primary health care led directly to important conceptual developments in health. national health targets and guidelines are now common in many countries and are integral parts of box 2.11 declaration of alma-ata, 1978: a summary of primary health care (phc) 1. reaffirms that health is a state of complete physical, mental, and social well-being, and not merely the absence of disease or infirmity, and is a fundamental human right. existing gross inequalities in the health status of the people, particularly between developed and developing countries as well as within countries, are of common concern to all countries. 3. governments have a responsibility for the health of their people. the people have the right and duty to participate in planning and implementation of their health care. 4. a main social target is the attainment, by all peoples of the world by the year 2000, of a level of health that will permit them to lead a socially and economically productive life. 5. phc is essential health care based on practical, scientifically sound, and socially acceptable methods and technology. 6. it is the first level of contact of individuals, the family, and the national health system bringing health care as close as possible to where people live and work, as the first element of a continuing health care process. 7. phc evolves from the conditions and characteristics of the country and its communities, based on the application of social, biomedical, and health services research and public health experience. 8. phc addresses the main health problems in the community, providing promotive, preventive, curative, and rehabilitative services accordingly. 9. phc includes the following: (a) education concerning prevailing health problems and methods of preventing and controlling them (b) promotion of food supply and proper nutrition (c) adequate supply of safe water and basic sanitation (d) maternal and child health care, including family planning (e) immunization against the major infectious diseases (f) prevention of locally endemic diseases (g) appropriate treatment of common diseases and injuries (h) the provision of essential drugs (i) relies on all health workers … to work as a health team. 10. all governments should formulate national health policies, strategies and plans, mobilize political will and resources, used rationally, to ensure phc for all people. national health planning. reforms of the nhs -for example, as discussed in chapter 13, remuneration increases for family physicians and encouraging group practice with public health nursing support -have become widespread in the uk. leading health maintenance organizations, such as kaiser permanente in the usa and district health systems in canada, have emphasized integrated approaches to health care for registered or geographically defined populations (see chapters 11-13). this approach is becoming common in the usa in acos, which will be fostered by the 2010 obamacare legislation (ppaca). this systematic approach to individual and community health is an integral part of the new public health. the interactions among community public health, personal health services, and health-related behavior, including their management, are the essence of the new public health. how the health system is organized and managed affects the health of the individual and the population, as does the quality of providers. health information systems with epidemiological, economic, and sociodemographic analysis are vital to monitor health status and allow for changing priorities and management. well-qualified personnel are essential to provide services, manage the system, and carry out relevant research and health policy analysis. diffusion of data, health information, and responsibility helps to provide a responsive and comprehensive approach to meet the health needs of the individual and community. the physical, social, economic, and political environments are all important determinants of the health status of the population and the individual. joint action (intersectoral cooperation) between public and non-governmental or community-based organizations is needed to achieve the well-being of the individual in a healthy society. in the 1980s and 1990s, these ideas contributed to an evolving new public health, spurred on by epidemiological changes, health economics, the development of managed care linking health systems, and prepayment. knowledge and self-care skills, as well as community action to reduce health risks, are no less important in this than the roles of medical practitioners and institutional care. all are parts of a coherent holistic approach to health. the concept of selective primary care, articulated in 1979 by walsh and warren, addresses the needs of developing countries to select those interventions on a broad scale that would have the greatest positive impact on health, taking into account limited resources such as money, facilities, and human resources. the term selective primary care is meant to define national priorities that are based not on the greatest causes of morbidity or mortality, but on common conditions of epidemiological importance for which there are effective and simple preventive measures. throughout health planning, there is an implicit or explicit selection of priorities for allocation of resources. even in primary care, selection of targets is a part of the process of resource allocation. in modern public health, this process is more explicit. a country with limited resources and a high birth rate will emphasize maternal and child health before investing in geriatric care. this concept has become part of the microeconomics of health care and technology assessment, discussed in chapters 11 and 15, respectively, and is used widely in setting priorities and resource allocation. in developing countries, cost-effective primary care interventions have been articulated by many international organizations, including iodization of salt, use of oral rehydration therapy (ort) for diarrheal diseases, vitamin a supplementation for all children, expanded programs of immunization, and others that have the potential for saving hundreds of thousands of lives yearly at low cost. in developed countries, health promotions targeted to reduce accidents and risk factors such as smoking, high-fat diets, and lack of exercise for cardiovascular diseases are low-cost public health interventions that save lives and reduce the use of hospital care. targeting specific diseases is essential for efforts to control tb or eradicate polio, but at the same time, development of a comprehensive primary care infrastructure is equally or even more important than the single-disease approach. some disease entities such as hiv/aids attract donor funding more readily than basic infrastructure services such as immunization, and this can sometimes be detrimental to addressing the overall health needs of the population and other neglected but also important diseases. the risk approach selects population groups on the basis of risk and helps to determine interventional priorities to reduce morbidity and mortality. the measure of health risk is taken as a proxy for need, so that the risk approach provides something for all, but more for those in need, in proportion to that need. in epidemiological terms, these are people with higher relative risk or attributed risk. some groups in the general population are at higher risk than others for specific conditions. the expanded programme on immunization (epi), control of diarrhoeal diseases (cdd), and acute respiratory disease (ard) programs of the who are risk approaches to tackling fundamental public health problems of children in developing countries. public health places considerable emphasis on maternal and child health because these are vulnerable periods in life for specific health problems. pregnancy care is based on a basic level of care for all, with continuous assessment of risk factors that require a higher intensity of follow-up. prenatal care helps to identify factors that increase the risk for the pregnant woman or her fetus/newborn. efforts directed towards these special risk groups have the potential to reduce morbidity and mortality. high-risk case identification, assessment, and management are vital to a successful maternal care program. similarly, routine infant care is designed not only to promote the health of infants, but also to find the earliest possible indications of deviation and the need for further assessment and intervention to prevent a worsening of the condition. low birth-weight babies are at greater risk for many short-and long-term hazards and should be given special treatment. all babies are routinely screened for birth defects or congenital conditions such as hypothyroidism, phenylketonuria, and other metabolic and hematological diseases. screening must be followed by investigating and treating those found to have a clinical deficiency. this is an important element of infant care because infancy itself is a risk factor. as will be discussed in chapters 6 and 7 and others, epidemiology has come to focus on the risk approach with screening based on known genetic, social, nutritional, environmental, occupational, behavioral, or other factors contributing to the risk for disease. the risk approach has the advantage of specificity and is often used to initiate new programs directed at special categories of need. this approach can lead to narrow and somewhat rigid programs that may be difficult to integrate into a more general or comprehensive approach, but until universal programs can be achieved, selective targeted approaches are justifiable. indeed, even with universal health coverage, it is still important to address the health needs or issues of groups at special risk. working to achieve defined targets means making difficult choices. the supply and utilization of some services will limit availability for other services. there is an interaction, sometimes positive, sometimes negative, between competing needs and the health status of a population. public health identifies needs by measuring and comparing the incidence or prevalence of the condition in a defined population with that in other comparable population groups and defines targets to reduce or eliminate the risk of disease. it determines ways of intervening in the natural epidemiology of the disease, and develops a program to reduce or even eliminate the disease. it also assesses the outcomes in terms of reduced morbidity and mortality, as well as the economic justification in cost-effectiveness analysis to establish its value in health priorities. because of the interdependence of health services, as well as the total financial burden of health care, it is essential to look at the costs of providing health care, and how resources should be allocated to achieve the best results possible. health economics has become a fundamental methodology in policy determination. the costs of health care, the supply of services, the needs for health care or other health-promoting interventions, and effective means of using resources to meet goals are fundamental in the new public health. it is possible to err widely in health planning if one set of factors is overemphasized or underemphasized. excessive supply of one service diminishes the availability of resources for other needed investments in health. if diseases are not prevented or their sequelae not well managed, patients must use costly health care services and are unable to perform their normal social functions such as learning at school or performing at work. lack of investment in health promotion and primary prevention creates a larger reliance on institutional care, driving health costs upwards, and restricting flexibility in meeting patients' needs. the interaction of supply and demand for health services is an important determinant of the political economy of health care. health and its place in national priorities are determined by the social-political philosophy and resource allocation of a government. the case for action, or the justification for a public health intervention, is a complex of epidemiological, economic, and public policy factors (table 2 .1). each disease or group of diseases requires its own case for action. the justification for public health intervention requires sufficient evidence of the incidence and prevalence of the disease (see chapter 3). evidence-based public health takes into account the effectiveness and safety of an intervention; risk factors; safe means at hand to intervene; the human, social, and economic cost of the disease; political factors; and a policy decision as to the priority of the problem. this often depends on subjective factors, such as the guiding philosophy of the health system and the way it allocates resources. some interventions are so well established that no new justification is required to make the case, and the only question is how to do it most effectively. for example, infant vaccination is a cost-effective and cost-beneficial program for the protection of the individual child and the population as a whole. whether provided as a public service or as a clinical preventive measure by a private medical practitioner, it is in the interest of public health that all children be immunized. an outbreak of diarrheal disease in a kindergarten presents an obvious case for action, and a public health system must respond on an emergency basis, with selection of the most suitable mode of intervention. the considerations in developing a case for action are outlined above. need is based on clinical and epidemiological evidence, but also on the importance of an intervention in the eyes of the public. the technology available, its effectiveness and safety, and accumulated experience are important in the equation, as are the acceptability and affordability of appropriate interventions. the precedents for use of an intervention are also important. on epidemiological evidence, if the preventive practice has been seen to provide reduction in risk for the individual and for the population, then there is good reason to implement it. the costs, risks and benefits must be examined as part of the justification to help in the selection of health priorities. health systems research examines the efficiency of health care and promotes improved efficiency and effective use of resources. this is a vital function in determining how best to use resources and meet current health needs. past emphasis on hospital care at the expense of less development of primary care and prevention is still a common issue, particularly in former soviet and developing countries, where a high percentage of total health expenditure goes to acute hospital care with long length of stay, with smaller allocation to preventive and community health care. the result of this imbalance is high mortality from preventable diseases. new drugs, vaccines, and medical equipment are continually becoming available, and each new addition needs to be examined among the national health priorities. sometimes, owing to cost, a country cannot afford to add a new vaccine to the routine. however, when there is good evidence for efficacy and safety of new vaccines, drugs, diagnostic methods or other innovations, it could be applied for those at greatest risk. although there are ethical issues involved, it may be necessary to advise parents or family members to purchase the vaccine independently. clearly, recommending individual purchase of a vaccine is counter to the principle of equity and solidarity, benefiting middleclass families, and providing a poor basis of data for evaluation of the vaccine and its target disease. on the other hand, failure to advise parents of potential benefits to their children creates other ethical problems, but may increase public pressure and insurance system acceptance of new methods, e.g., varicella and hpv vaccines. mass screening programs involving complete physical examinations have not been found to be cost-effective or to significantly reduce disease. in the 1950s and 1960s, routine general health examinations were promoted as an effective method of finding disease early. since the late 1970s, a selective and specific approach to screening has become widely accepted. this involves defining risk categories for specific diseases and bearing in mind the potential for remedial action. early case finding of colon cancer by routine fecal blood testing and colonoscopy has been found to be effective, and pap smear testing to discover cancer of the cervix is timed according to risk category. screening for colorectal cancer is essential for modern health programs and has been adopted by most industrialized countries. outreach programs by visits, telephones, emails or other modern methods of communication are important to contact non-attenders to promote utilization, and have been shown to increase compliance with proven effective measures. these programs are important for screening, follow-up, and maintenance of treatment for hypertension, diabetes, and other conditions requiring long-term management. screening technology is changing and often the subject of intense debate as such programs are costly and their cost-effectiveness is an important matter for policy making: screening for lung cancer is becoming a feasible and effective matter for high-risk groups, whereas breast cancer screening frequency is now in dispute; while nanotechnology and bioengineering promises new methods for cancer screening. the factor of contribution to quality of life should be considered. a vaccine for varicella is justified partly for the prevention of deaths or illness from chickenpox. a stronger the right to health public expectation and social norms argument is often based on the fact that this is a disease that causes moderate illness in children for up to 2 weeks and may require parents to stay home with the child, resulting in economic loss to the parent and society. the fact that this vaccination prevents the occurrence of herpes zoster or shingles later in life may also be a justification. widespread adoption of hepatitis b vaccine is justified on the grounds that it prevents cancer of the liver, liver cirrhosis, and hepatic failure in a high percentage of the population affected. how many cases of a disease are enough to justify an intervention? one or several cases of some diseases, such as poliomyelitis, may be considered an epidemic in that each case constitutes or is an indicator of a wider threat. a single case of polio suggests that another 1000 persons are infected but have not developed a recognized clinical condition. such a case constitutes a public health emergency, and forceful organization to meet a crisis is needed. current standards are such that even one case of measles imported into a population free of the disease may cause a large outbreak, as occurred in the uk, france, and israel during 2007 through 2013, by contacts on an aircraft, at family gatherings, or even in medical settings. a measles epidemic indicates a failure of public health policy and practice. screening for some cancers, such as cervix and colon, is cost effective. screening of all newborns for congenital disorders is important because each case discovered early and treated effectively saves a lifetime of care for serious disability. assessing a public health intervention to prevent the disease or reduce its impact requires measurement of the disease in the population and its economic impact. there is no simple formula to justify a particular intervention, but the cost-benefit approach is now commonly required to make such a case for action. sometimes public opinion and political leadership may oppose the views of the professional community, or may impose limitations of policy or funds that prevent its implementation. conversely, professional groups may press for additional resources that compete for limited resources available to provide other needed health activities. both the professionals of the health system and the general public need full access to health-related information to take part in such debates in a constructive way. to maintain progress, a system must examine new technologies and justify their adoption or rejection (see chapter 15). the association between health and political issues was emphasized by european innovators such as rudolf virchow (and in great britain by edwin chadwick; see chapter 1) in the mid-nineteenth century, when the conditions of the working population were such that epidemic diseases were rife and mortality was high, especially in the crowded slums of the industrial revolution. the same observations led bismarck in germany to introduce early forms of social insurance for the health of workers and their families in the 1880s, and to britain's 1911 national health insurance, also for workers and families. the role of government in providing universal access to health care was a struggle in individual countries during the twentieth century and lasting into the second decade of the twenty-first century (e.g. president obama's affordable health care act of 2010). as the concept of public health has evolved, and the cost effectiveness of medical care has improved through scientific and technological advances, societies have identified health as a legitimate area of activity for collective bargaining and government. with this process, the need to manage health care resources has become more clearly defined as a public responsibility. in industrialized countries, each with very different political make-up, national responsibility for universal access to health has become part of the social ethos. with that, the financing and managing of health services have developed into part of a broad concept of public health, and economics, planning, and management have come to be part of the new public health (discussed in chapters 10-13). social, ethical, and political philosophies have profound effects on policy decisions including allocation of public monies and resources. investment in public health is now recognized as an integral part of socioeconomic development. governments are major suppliers of funds and leadership in health infrastructure development, provision of health services, and health payment systems. they also play a central role in the development of health promotion and regulation of the environment, food, and drugs essential for community health. in liberal social democracies, the individual is deemed to have a right to health care. the state accepts responsibility to ensure availability, accessibility, and quality of care. in many developed countries, government has also taken responsibility to arrange funding and services that are equitably accessible and of high quality. health care financing may involve taxation, allocation, or special mandatory requirements on employers to pay for health insurance. services may be provided by a state-financed and -regulated service or through ngos and/or private service mechanisms. these systems allocate between 6 percent and 14 percent of gross national product (gnp) to health services, with some governments funding over 80 percent of health expenditure; for example, canada and the uk. in communist states, the state organizes all aspects of health care with the philosophy that every citizen is entitled to equity in access to health services. the state health system manages research, staff training, and service delivery, even if operational aspects are decentralized to local health authorities. this model applied primarily to the soviet model of health services. these systems, except for cuba, placed financing of health low on the national priority, with funding less than 4 percent of gnp. in the shift to market economies in the 1990s, some former socialist countries, such as russia, are struggling with poor health status and a difficult shift from a strongly centralized health system to a decentralized system with diffusion of powers and responsibilities. promotion of market concepts in former soviet countries has reduced access to care and created a serious dilemma for their governments. former colonial countries, independent since the 1950s and 1960s, largely carried on the governmental health structures established in the colonial times. most developing countries have given health a relatively low place in budgetary allotment, with expenditures under 3 percent of gnp. since the 1980s, there has been a trend in developing countries towards decentralization of health services and greater roles for ngos, and the development of health insurance. some countries, influenced by medical concepts of their former colonial master countries, fostered the development of specialty medicine in the major centers with little emphasis on the rural majority population. soviet influence in many ex-colonial countries promoted state-operated systems. the who promoted primary care, but the allocations favored city-based specialty care. israel, as an ex-colony, adapted british ideas of public health together with central european sick funds and maternal and child health as major streams of development until the mid-1990s. a growing new conservatism in the 1980s and 1990s in the industrialized countries is a restatement of old values in which market economics and individualistic social values are placed above concepts of the "common good" of liberalism and socialism in its various forms. in the more extreme forms of this concept, the individual is responsible for his or her own health, including payment, and has a choice of health care providers that will respond with high-quality personalized care. market forces, meaning competition in financing and provision of health services with rationing of services, based on fees or private insurance and willingness and ability to pay, have become part of the ideology of the new conservatism. it is assumed that the patient (i.e., the consumer) will select the best service for his or her need, while the provider best able to meet consumer expectations will thrive. in its purest form, the state has no role in providing or financing of health services except those directly related to community protection and promotion of a healthful environment without interfering with individual choices. the state ensures that there are sufficient health care providers and allows market forces to determine the prices and distribution of services with minimal regulation. the usa retains this orientation in a highly modified form, with 86 percent of the population covered by some form of private or public insurance systems (see chapters 10 and 13). modified market forces in health care are part of health reforms in many countries as they seek not only to ensure quality health care for all but also to constrain costs. a free market in health care is costly and ultimately inefficient because it encourages inflation of provider incomes or budgets and increasing utilization of highly technical services. further, even in the most free market societies, the economy of health care is highly influenced by many factors outside the control of the consumer and provider. the total national health expenditure in the usa rose rapidly until reaching over 17.7 percent of gross domestic product (gdp) in 2011, the highest of any country, despite serious deficiencies for those without any or with very inadequate health insurance (in total more than 30 percent of the population). this figure compares to some 11.2 percent of gdp in canada, which has universal health insurance under public administration. following the 1994 defeat of president clinton's national health program, the conservative congress and the business community took steps to expand managed care in order to control costs, resulting in a revolution in health care in the usa (see chapters 11 and 13). in the 2011-2019 decade health expenditure in the usa is expected to rise to 19.6 percent of gdp, partly owing to increased population coverage with implementation of the ppaca (obamacare). reforms are being implemented in many "socialized" health systems. these may be through incentives to promote achievement of performance indicators, such as full immunization coverage. others are using control of supply, such as hospital beds or licensed physicians, as methods of reducing overutilization of services that generates increasing costs. market mechanisms in health are aimed not only at the individual but also at the provider. incentive payment systems must work to protect the patient's legitimate needs, and conversely incentives that might reduce quality of care should be avoided. fee-for-service promotes high rates of services such as surgery. increasing private practice and user fees can adversely affect middle-and low-income groups, as well as employers, by raising the costs of health insurance. managed care systems, with restraints on fee-for-service medical practice, have emerged as a positive response to the market approach. incentive systems in payments for services may be altered by government or insurance agencies in order to promote rational use of services, such as reduction of hospital stays. the free market approach is affecting planning of health insurance systems in previously highly centralized health systems in developing countries as well as the redevelopment of health systems in former soviet countries. despite political differences, reform of health systems has become a common factor in virtually all health systems since the 1990s, as each government searches for costeffectiveness, quality of care, and universality of coverage. the new paradigm of health care reform sees the convergence of different systems to common principles. national responsibility for health goals and health promotion leads to national financing of health care with regional and managed care systems. most developed countries have long since adopted national health insurance or service systems. some governments may, as in the usa, insure only the highest risk groups such as the elderly and the poor, leaving the working and middle classes to seek private insurers. the nature and direction of health care reform affecting coverage of the population are of central importance in the new public health because of its effects on allocation of resources and on the health of the population. the effects of the economic crisis in the usa are being felt worldwide. while the downturn has largely occurred in wealthier nations, the poor in low-income countries will be among those affected. past economic downturns have been followed by substantial drops in foreign aid to developing countries. as public health gained from sanitary and other control measures for infectious diseases, along with mother and child care, nutrition, and environmental and occupational health, it also gained strength and applicability from advances in the social and behavioral sciences. social darwinism, a political philosophy that assumed "survival of the fittest" and no intervention of the sate to alleviate this assumption, was popular in the early nineteenth century but became unacceptable in industrialized countries, which adopted social policies to alleviate the worst conditions of poverty, unemployment, poor education, and other societal ills. the political approach to focusing on health and poverty is associated with jeremy bentham in britain in the late eighteenth century, who promoted social and political reform and "the greatest good for the greatest number", or utilitarianism. rudolf virchow, an eminent pathologist and a leader in recognizing ill-health and poverty as cause and effect, called for political action to create better conditions for the poor and working-class population. the struggle for a social contract was promoted by pioneer reformists such as edwin chadwick (general report on the sanitary condition of the labouring population of great britain, 1842), who later became the first head of the board of health in britain, and lemuel shattuck (report of a general plan for the promotion of public and personal health, 1850) . shattuck was the organizer and first president of the american statistical association. the social sciences have become fundamental to public health, with a range of disciplines including vital statistics and demography (seventeenth century), economics and politics (nineteenth century), sociology (twentieth century), history, anthropology, and others, which provide collectively important elements of epidemiology of crucial significance for survey methods and qualitative research (see chapter 3). these advances contributed greatly to the development of methods of studying diseases and risk factors in a population and are still highly relevant to addressing inequalities in health. individuals in good health are better able to study and learn, and be more productive in their work. improvements in the standard of living have long been known to contribute to improved public health; however, the converse has not always been recognized. investment in health care was not considered a high priority in many countries where economic considerations directed investment to the "productive" sectors such as manufacturing and large-scale infrastructure projects, such as hydroelectric dams. whether health is a contributor to economic development or a drain on societies' resources has been a fundamental debate between socially and market-oriented advocates. classic economic theory, both free enterprise and communist, has tended to regard health as a drain on economies, distracting investment needed for economic growth. as a result, in many countries health has been given low priority in budgetary allocation, even when the major source of financing is governmental. this belief among economists and banking institutions prevented loans for health development on the grounds that such funds should focus on creating jobs and better incomes, before investing in health infrastructure. consequently, the development of health care has been hampered. a socially oriented approach sees investment in health as necessary for the protection and development of "human capital", just as investment in education is needed for the long-term benefit of the economy of a country. in 1993, the world bank's world development report: investing in health articulated a new approach to economics in which health, along with education and social development, is seen as an essential precondition for and contributor to economic development. while many in the health field have long recognized the importance of health for social and economic improvement, its adoption by leading international development banking may mark a turning point for investment in developing nations, so that health may be a contender for increased development loans. the concept of an essential package of services discussed in that report establishes priorities in low-and middle-income countries for efficient use of resources based on the burden of disease and cost-effectiveness analysis of services. it includes both preventive and curative services targeted to specific health problems. it also recommends support for comprehensive primary care, such as for children, and infrastructure development including maternity and hospital care, medical and nursing outreach services, and community action to improve sanitation and safe water supplies. reorientation of government spending on health is increasingly being adopted, as in the uk, to improve equity in access for the poor and other neglected sectors or regions of society with added funding for relatively deprived areas to improve primary care services. differential capitation funding as a form of affirmative action to provide for highneeds populations is a useful concept in public health terms to address the inequities still prevalent in many countries. as medical care has gradually become more involved in prevention, and as it has moved into the era of managed care, the gap between public health and clinical medicine has narrowed. as noted above, many countries are engaged in reforms in their health care systems. the motivation is largely derived from the need for cost containment, but also to extend health care coverage to underserved parts of the population. countries without universal health care still have serious inequities in distribution of or access to services, and may seek reform to reduce those inequities, perhaps under political pressures to improve the provision of services. incentives for reform are needed to address regional inequities, and preserving or developing universal access and quality of care, but also on inequities in health between the rich and the poor countries and within even the wealthy countries. in some settings, a health system may fail to keep pace with developments in prevention and in clinical medicine. some countries have overdeveloped medical and hospital care, neglecting important initiatives to reduce the risk of disease. the process of reform requires setting standards to measure health status and the balance of services to optimize health. a health service can set a target of immunizing 95 percent of infants with a national immunization schedule, but requires a system to monitor performance and incentives for changes. a health system may also have failed to adapt to changing needs of the population through lack, or misuse, of health information and monitoring systems. as a result, the system may err seriously in its allocation of resources, with excessive emphasis on hospital care and insufficient attention to primary and preventive care. all health services should have mechanisms for correctly gathering and analyzing needed data for monitoring the incidence of disease and other health indicators, such as hospital utilization, ambulatory care, and preventive care patterns. for example, the uk's nhs periodically undertakes a restructuring process of parts of the system to improve the efficiency of service. this involves organizational changes and decentralization with regional allocation of resources (see chapter 13). health systems are under pressures of changing demographic and epidemiological patterns as well as public expectations, rising costs of new technology, financing, and organizational change. new problems must be continually addressed with selection of priority issues and the most effective methods chosen. reforms may create unanticipated problems, such as professional or public dissatisfaction, which must be evaluated, monitored, and addressed as part of the evolution of public health. literacy, freedom of the press, and increasing public concern for social and health issues have contributed to the development of public health. the british medical community lobbied for restrictions on the sale of gin in the 1780s in order to reduce the damage that it caused to the working class. in the late eighteenth and the nineteenth centuries, reforms in society and sanitation were largely the result of strongly organized advocacy groups influencing public opinion through the press. such pressure stimulated governments to act in regulating the working conditions of mines and factories. abolition of the slave trade and its suppression by the british navy in the early nineteenth century resulted from successful advocacy groups and their effects on public opinion through the press. vaccination against smallpox was promoted by privately organized citizen groups, until later taken up by local and national government authorities. advocacy consists of activities of individuals or groups publicly pleading for, supporting, espousing, or recommending a cause or course of action. the advocacy role of reform movements in the nineteenth century was the basis of the development of modern organized public health. campaigns ranged from the reform of mental hospitals, nutrition for sailors to prevent scurvy and beriberi, and labor laws to improve working conditions for women and children in particular, to the promotion of universal education and improved living conditions for the working population. reforms on these and other issues resulted from the stirring of the public consciousness by advocacy groups and the public media, all of which generated political decisions in parliaments (box 2.12). such reforms were in large part motivated by fear of revolution throughout europe in the mid-nineteenth century and the early part of the twentieth century. trade unions, and before them medieval guilds, fought to improve hours, safety, and conditions of work, as well as social and health benefits for their members. in the usa, collective bargaining through trade unions achieved wage increases and widespread coverage of the working population under voluntary health insurance. unions and some industries pioneered prepaid group practice, the predecessor of health maintenance organizations and managed care or the more recent acos (see chapters 10 and 13). through raising public consciousness on many issues, advocacy groups pressure governments to enact legislation to restrict smoking in public places, prohibit tobacco advertising, and mandate the use of bicycle helmets. advocacy groups play an important role in advancing health based on disease groups, such as cancer, multiple sclerosis, and thalassemia, or advancing health issues, such as the organizations promoting breastfeeding, environmental improvement, or smoking reduction. some organizations finance services or facilities not usually provided within insured health programs. such organizations, which can number in the hundreds in a country, advocate the importance of their special concern and play an important role in innovation and meeting community health needs. advocacy groups, including trade unions, professional groups, women's groups, self-help groups, and many others, focus on specific issues and have made major contributions to advancing the new public health. the history of public health is replete with pioneers whose discoveries led to strong opposition and sometimes violent rejection by conservative elements and vested interests in medical, public, or political circles. opposition to jennerian vaccination, the rejection of semmelweiss by colleagues in vienna, and the contemporary opposition to the work of great pioneers in public health such as pasteur, florence nightingale, and many others may deter or delay implementation of other innovators and new breakthroughs in preventing disease. although opposition to jenner's vaccination lasted well into the late nineteenth century in some areas, its supporters gradually gained ascendancy, ultimately leading to the global eradication of smallpox. these and other pioneers led the way to improved health, often after bitter controversy on topics later accepted and which, in retrospect, seem to be obvious. advocacy has sometimes had the support of the medical profession but elicited a slow response from public authorities. david marine of the cleveland clinic and david cowie, professor of pediatrics at the university of michigan, proposed the prevention of goiter by iodization of salt. marine carried out a series of studies in fish, and then in a controlled clinical trial among schoolgirls in 1917-1919, with startlingly positive results in reducing the prevalence of goiter. cowie campaigned for the iodization of salt, with support from the medical profession. in 1924, he convinced a private manufacturer to produce morton's iodized salt, which rapidly became popular throughout north america. similarly, iodized salt came to be used in many parts of europe, mostly without governmental support or legislation. iodine-deficiency disorders (idds) remain a widespread condition, estimated to have affected 2 billion people worldwide in 2013. the target of international eradication of idds by 2000 was set at the world summit for children in 1990, and the who called for universal iodization of salt in 1994. by 2008, nearly 70 percent of households in developing countries consumed adequately iodized salt. china and nigeria, have had great success in recent years with mandatory salt fortification in increasing iodization rates, in china from 39 percent to 95 percent in 10 years. but the problem is not yet gone and even in europe there is inadequate standardization of iodine levels and population follow-up despite decades of work on the problem. professional organizations have contributed to promoting causes such as children's and women's health, and environmental and occupational health. the american academy of pediatrics has contributed to establishing and promoting high standards of care for infants and children in the usa, and to child health internationally. hospital accreditation has been used for decades in the usa, canada, and more recently in australia and the uk. it has helped to raise standards of health facilities and care by carrying out systematic peer review of hospitals, nursing homes, primary care facilities, and mental hospitals, as well as ambulatory care centers and public health agencies (see chapter 15). public health needs to be aware of negative advocacy, sometimes based on professional conservatism or economic self-interest. professional organizations can also serve as advocates of the status quo in the face of change. opposition by the american medical association (ama) and the health insurance industry to national health insurance in the usa has been strong and successful for many decades. the passage of the ppaca has been achieved despite widespread political and public opposition, yet was sustained in the us supreme court and is gaining widening popular support as the added value to millions of formerly uninsured americans becomes clear. in some cases, the vested interest of one profession may block the legitimate development of others, such as when ophthalmologists lobbied successfully against the development of optometry, now widely accepted as a legitimate profession. political activism for reform in nineteenth-century britain led to banning and suppressing the slave trade, improvements in working conditions for miners and factory workers, and other major political reforms. in keeping with this tradition, samuel plimsoll (1824-1898), british member of parliament elected for derby in 1868, conducted a solo campaign for the safety of seamen. his book, our seamen, described ships sent to sea so heavily laden with coal and iron that their decks were awash. seriously overloaded ships, deliberately sent to sea by unscrupulous owners, frequently capsized, drowning many crew members, with the owners collecting inflated insurance fees. overloading was the major cause of wrecks and thousands of deaths in the british shipping industry. plimsoll pleaded for mandatory load-line certificate markers to be issued to each ship to prevent any ships putting to sea when the marker was not clearly visible. powerful shipping interests fought him every inch of the way, but he succeeded in having a royal commission established, leading to an act of parliament mandating the "plimsoll line", the safe carrying capacity of cargo ships. this regulation was adopted by the us bureau of shipping as the load line act in 1929 and is now standard practice worldwide. jenner's discovery of vaccination with cowpox to prevent smallpox was adopted rapidly and widely. however, intense opposition by organized groups of antivaccinationists, often led by those opposed to government intervention in health issues and supported by doctors with lucrative variolation practices, delayed the implementation of smallpox vaccination for many decades. ultimately, smallpox was eradicated in 1972, owing to a global campaign initiated by the who. opposition to legislated restrictions on private ownership of assault weapons and handguns is intense in the usa, led by well-organized, well-funded, and politically powerful lobby groups, despite the amount of morbidity and mortality due to gun-associated violent acts (see chapter 5). fluoridation of drinking water is the most effective public health measure for preventing dental caries, but it is still widely opposed, and in some places the legislation has been rescinded even after implementation, by wellorganized antifluoridation campaigns. opposition to fluoridation of community water supplies is widespread, and effective lobbying internationally has slowed but has not stopped progress (see chapter 6). despite the life-saving value of immunization, opposition still exists in 2013 and harms public health protection. opposition has slowed progress in poliomyelitis eradication; for example, radical islamists killed polio workers in northern nigeria in 2012, one of the last three countries with endemic poliomyelitis. resistance to immunization in the 1980s has resulted in the recurrence of pertussis and diphtheria and a very large epidemic of measles across western europe, including the uk, with further spread to the western hemisphere in 2010-2013 (see chapter 4). progress may be blocked where all decisions are made in closed discussions, not subject to open scrutiny and debate. public health personnel working in the civil service of organized systems of government may not be at liberty to promote public health causes. however, professional organizations may then serve as forums for the essential professional and public debate needed for progress in the field. professional organizations such as the apha provide effective lobbying for the interests of public health programs and can have an important impact on public policy. in mid-1996, efforts by the secretary of health and human services in the usa brought together leaders of public health with representatives of the ama and academic medical centers to try to find areas of common interest and willingness to promote the health of the population. in europe too, increasing cooperation between public health organizations is stimulating debate on issues of transnational importance across the region, which, for example, has a wide diversity of standards on immunization practices and food policies. public advocacy has played an especially important role in focusing attention on ecological issues (box 2.13). in 1995, greenpeace, an international environmental activist group, fought to prevent the dumping of an oil rig in the north sea and forced a major oil company to find another solution that would be less damaging to the environment. an explosion on an oil rig in the gulf of mexico in 2010 led to enormous ecological and economic damage as well as loss of life. damages levied on the responsible company (british petroleum) amount to some $4.5 billion dollars and several criminal negligence charges are pending. greenpeace also continued its efforts to stop the renewal of testing of atomic bombs by france in the south pacific. international protests led to the cessation of almost all testing of nuclear weapons. international concern over global warming has led to growing efforts to stem the tide of air pollution from fossil fuels, coal-burning electrical production, and other manifestations of carbon dioxide and toxic contamination of the environment. progress is far from certain as newly enriched countries such as china and india follow the rising consumption patterns of western countries. public advocacy and rejection of wanton destruction of the global ecology may be the only way to prod consumers, governments, and corporate entities such as the energy and transportation industries to change direction. the pace of change from fossil fuels is slow but has captured public attention, and private companies are seeking more fuel efficiency in vehicles and electrical power production, mainly though the use of natural gas instead of fuel oil and coal for electricity production or better still by wind and solar energy. the search for "green solutions" to the global warming crisis has become increasingly dynamic, with governments, the private sector, and the general public keenly aware of the importance of the effort and the dangers of failure. in the latter part of the twentieth century and the early twenty-first century, prominent international personalities and entertainers have taken up causes such as the removal of land mines in war-torn countries, illiteracy in disadvantaged advocacy is a function in public health that has been important in promoting advances in the field, and one that sometimes places the advocate in conflict with established patterns and organizations. one of the classic descriptions of this function is in henrik ibsen's play an enemy of the people, in which the hero, a young doctor, thomas stockmann, discovers that the water in his community is contaminated. this knowledge is suppressed by the town's leadership, led by his brother the mayor, because it would adversely affect plans to develop a tourist industry of baths in their small norwegian town in the late nineteenth century. the young doctor is taunted and abused by the townspeople and driven from the town, having been declared an "enemy of the people" and a potential risk. the allegory is a tribute to the man of principle who stands against the hysteria of the crowd. the term also took on a far more sinister and dangerous meaning in george orwell's novel 1984 and in totalitarian regimes of the 1930s to the present time. populations, and funding for antiretroviral drugs for african countries to reduce maternal-fetal transmission of hiv and to provide care for the large numbers of cases of aids devastating many countries of sub-saharan africa. rotary international has played a key role in polio eradication efforts globally. the public-private consortium global alliance for vaccines and immunization (gavi) has been instrumental in promoting immunization in recent years, with participation by the who, unicef, the world bank, the gates foundation, vaccine manufacturers, and others. this has had an important impact on extending immunization to protect and save the lives of millions of children in deprived countries not yet able to provide fundamental prevention programs such as immunization at adequate levels. gavi has brought vaccines to low-income countries around the world, such as rotavirus vaccine, pentavalent vaccine in myanmar, and pneumococcal vaccine for children in 15 countries in sub-saharan africa, including dr congo. the bill & melinda gates foundation pledged us $750 million in 1999 to establish gavi, with us $75 million per year and us $1 billion in 2010 to promote the decade of vaccines. international conferences help to create a worldwide climate of advocacy for health issues. international sanitary conferences in the nineteenth century were convened in response to the cholera epidemics. international conferences continue in the twenty-first century to serve as venues for advocacy on a global scale, bringing forward issues in public health that are beyond the scope of individual nations. the who, unicef, and other international organizations perform this role on a continuing basis (see chapter 16). criticisms of this approach have focused on the lack of similar effort or donors to address ncds, weak public health infrastructure, and that this frees national governments from responsibility to care for their own children. no one can question, however, that this kind of endeavor has saved countless lives and needs the backing of other aid donors and national government participation. consumerism is a movement that promotes the interests of the purchaser of goods or services. in the 1960s, a new form of consumer advocacy emerged from the civil rights and antiwar movement in the usa. concern was focused on the environment, occupational health, and the rights of the consumer. rachel carson stimulated concern by dramatizing the effects of ddt on wildlife and the environment but inadvertently jeopardized anti-malarial efforts in many countries. this period gave rise to environmental advocacy efforts worldwide, and a political movement, the greens, in western europe. ralph nader showed the power of the advocate or "whistle-blower" who publicizes health hazards to stimulate active public debate on a host of issues related to the public well-being. nader, a consumer advocate lawyer, developed a strategy for fighting against business and government activities and products which endangered public health and safety. his 1965 book unsafe at any speed took issue with the us automobile industry for emphasizing profit and style over safety, and led to the enactment of the national traffic and motor safety act of 1966, establishing safety standards for new cars. this was followed by a series of enactments including design and emission standards and seat-belt regulations. nader's work continues to promote consumer interests in a wide variety of fields, including the meat and poultry industries, and coal mining, and promotes greater government regulatory powers regarding pesticide usage, food additives, consumer protection laws, rights to knowledge of contents, and safety standards. consumerism has become an integral part of free market economies, and the educated consumer does influence the quality, content, and price of products. greater awareness of nutrition in health has influenced food manufacturers to improve packaging, content labeling, enrichment with vitamins and minerals, and advertisement to promote those values. low-fat dietary products are available because of an increasingly sophisticated public concerned over dietary factors in cardiovascular diseases. the same process occurred in safe toys and clothing for children, automobile safety features such as mandatory use of car seats for infants, and other innovations that quickly became industry standards in the industrialized world. dangerous practices such as the use of lead paint in toys and melamine contamination of milk products from china capture the public attention quickly and remind public health authorities of the importance of continuous alertness to potential hazards. consumerism can also be exploited by pharmaceutical companies with negative impacts on the health system, especially in the advertising of health products which leads to unnecessary visits to health providers and pressure for approval to obtain the product. the internet has provided people with access to a vast array of information and opinion, and to current literature otherwise unavailable because of the often inadequate library resources of medical and other health professionals. the very freedom of information the internet allows, however, also provides a vehicle for extremist and fringe groups to promote disinformation such as "vaccination causes autism, fluoridation causes cancer", which can cause considerable difficulties for basic public health programs or lead to self-diagnosis of conditions, with often disastrous consequences. advocacy and voluntarism go hand in hand. voluntarism takes many forms, including raising funds for the development of services or operating services needed in the community. it may take the form of fund-raising to build clinics or hospitals in the community, or to provide medical equipment for elderly or handicapped people; or retirees and teenagers working as hospital volunteers to provide services that are not available through paid staff, and to provide a sense of community caring for the sick in the best traditions of religious or municipal concerns. this can also be extended to prevention, as in support for immunization programs, assistance for the handicapped and elderly in transportation, meals-on-wheels, and many other services that may not be included in the "basket of services" provided by the state, health insurance, or public health services. community involvement can take many forms, and so can voluntarism. the pioneering role of women's organizations in promoting literacy, health services, and nutrition in north america during the latter part of the nineteenth and the early twentieth centuries profoundly affected the health of the population. the advocacy function is enhanced when an organization mobilizes voluntary activity and funds to promote changes or needed services, sometimes forcing official health agencies or insurance systems to revise their attitudes and programs to meet these needs. by the early 1970s, canada's system of federally supported provincial health insurance plans covered all of the country. the federal minister of health, marc lalonde, initiated a review of the national health situation, in view of concern over the rapidly increasing costs of health care. this led to articulation of the "health field concept" in 1974, which defined health as a result of four major factors: human biology, environment, behavior, and health care organization (box 2.14). lifestyle and environmental factors were seen as important contributors to the morbidity and mortality in modern societies. this concept gained wide acceptance, promoting new initiatives that emphasized health promotion in response to environmental and lifestyle factors. conversely, reliance primarily on medical care to solve all health problems could be counterproductive. this concept was a fundamental contributor to the idea of health promotion later articulated in the ottawa declaration, discussed below. the health field concept came at a time when many epidemiological studies were identifying risk factors for cardiovascular diseases and cancers that related to personal habits, such as diet, exercise, and smoking. the concept advocated that public policy needed to address individual lifestyle as part of the overall effort to improve health status. as a result, the canadian federal government established health promotion as a new activity. this quickly spread to many other jurisdictions and gained wide acceptance in many industrialized countries. concern was expressed that this concept could become a justification for a "blame the victim" approach, in which those ill with a disease related to personal lifestyles, such as smokers or aids patients, are seen as having chosen to contract the disease. such a patient might then be considered not to be entitled to all benefits of insurance or care that others may receive. the result may be a restrictive approach to care and treatment that would be unethical in the public health tradition and probably illegal in western jurisprudence. this concept was also used to justify withdrawal from federal commitments in cost sharing and escape from facing controversial health reform in the national health insurance program. during the 1960s and 1970s, outspoken critics of health care systems, such as ivan illytch, questioned the value of medical care for the health of the public. this became a widely discussed, somewhat nihilistic, view towards medical care, and was influential in promoting skepticism regarding the value of the biomedical mode of health care, and antagonism towards the medical profession. in 1976, thomas mckeown presented a historicalepidemiological analysis showing that up to the 1950s, medical care had only a limited impact on mortality rates, although improvements in surgery and obstetrics were notable. he showed that crude death rates in england averaged about 30 per 1000 population from 1541 to 1750, declining steeply to 22 per 1000 in 1851, 15 per 1000 in 1901, and 12 per 1000 in 1951, when medical care became truly effective. mckeown concluded that much of the improvement in health status over the past several centuries was due to reduced mortality from infectious diseases. this he related to limitation of family size, increased food supplies, improved nutrition and sanitation, specific preventive and therapeutic measures, and overall gains in quality of life for growing elements of the population. he cautioned against placing excessive reliance for health on medical care, much of which was of unproved effectiveness. this skepticism of the biomedical model of health care was part of wider antiestablishment feelings of the 1960s and 1970s in north america. in 1984, milton roemer pointed out that the advent of vaccines, antibiotics, antihypertensives, and other medications contributed to great improvements in infant and child care, and in the management of infectious diseases, hypertension, diabetes, and other conditions. therapeutic gains continue to arrive from teaching centers around the world. vaccine, pharmaceutical, and diagnostic equipment manufacturers continue to provide important innovations that have major benefits, but also raise the cost of health care. the latter issue is one which has stimulated the search for reforms, and search for lower cost technologies such as in treatment of hepatitis c patients, a huge international public health issue. the value of medical care to public health and vice versa has not always been clear, either to public health personnel or to clinicians. the achievements of modern public health in controlling infectious diseases, and even more so in reducing the mortality and morbidity associated with chronic diseases such as stroke and chd, were in reality a shared achievement between clinical medicine and public health (see chapter 5). preventive medicine has become part of all medical practice, with disease prevention through early diagnosis and health promotion through individual and community-focused activities. risk factor evaluation determines appropriate screening and individual and community-based interventions. medical care is crucial in controlling hypertension and in reducing the complications and mortality from chd. new modalities of treatment are reducing death rates from first time acute myocardial infarctions. better management of diabetes prevents the early onset of complications. at the same time, the contribution of public health to improving outcomes of medical care is equally important. control of the vaccine-preventable diseases, improved nutrition, and preparation for motherhood contribute to improved maternal and infant outcomes. promotions of reduced exposure to risk factors for chronic disease are a task shared by public health and clinical medical services. both clinical medicine and public health contribute to improved health status. they are interdependent and rely on funding systems for recognition as part of the new public health. during the 1950s, many new management concepts emerged in the business community, such as "management by objective", a concept developed by peter drucker at general motors, with variants such as "zero-based budgeting" developed in the us department of defense (see chapter 12). they focused the activities of an organization and its budget on targets, rather than on previous allocation of resources. these concepts were applied in other spheres, but they influenced thinking in health, whose professionals were seeking new ways to approach health planning. the logical application was to define health targets and to promote the efficient use of resources to achieve those targets. this occurred in the usa and soon afterwards in the who european region. in both cases, a wide-scale process of discussion and consensus building was used before reaching definitive targets. this process contributed to the adoption of the targets by many countries in europe as well as by states and many professional and consumer organizations. the usa developed national health objectives in 1979 for the year 1990 and subsequently for the year 2000, with monitoring of progress in their achievement and development of further targets for 2010 and now for 2020. beginning in 1987, state health profiles are prepared by the epidemiology program office of the centers for disease control and prevention based on 18 health indicators recommended by a consensus panel representing public health associations and organizations. the eight mdgs adopted by the un in 2000 include halving extreme poverty, reducing child mortality by twothirds, improving maternal health, halting the spread of hiv/aids, malaria, and other diseases, and providing universal primary education, all by the target date of 2015. the mdgs form a common blueprint agreed to by all countries and the world's leading development institutions. the process has galvanized unprecedented efforts to meet the needs of the world's poorest, yet 2008 reviews of progress indicate that most developing nations will not meet the targets at current rates of progress. the united nations development programme (undp) global partnership for development 2012 report on the mdgs states that if the national development strategies and initiatives are supported by international development partners, the goals can be achieved by 2015. the mdgs were adopted by over 120 nations and provided guidance for national policies and for international aid agencies. the focus was on middle-and low-income countries and their achievements have been considerable but variable (see box 2.15 and chapter 16) . as of july 2012, extreme poverty was falling in every region, the poverty reduction target had been met, the world had met the target of halving the proportion of people without access to improved sources of water, and the world had achieved parity in primary education between girls and boys. further progress will require sustained political commitment to develop the primary care infrastructure: improved reporting and epidemiological monitoring, consultative mechanisms, and consensus by international agencies, national governments, and non-governmental agencies. the achievement of the targets will also require sustained international support and national commitment with all the difficulties of a time of economic recession. nevertheless, defining a target is crucial to the process. there are encouraging signs that national governments are influenced by the general movement to place greater emphasis on resource allocation and planning on primary care to achieve internationally recognized goals and targets. the successful elimination of smallpox, rising immunization coverage in the developing countries, and increasing implementation of salt iodization have shown that such goals are achievable. while the usa has not succeeded in developing universal health care access, it has a strong tradition of public health and health advocacy. federal, state, and local health authorities have worked out cooperative arrangements for financing and supervising public health and other services. with growing recognition in the 1970s that medical services alone would not achieve better health results, health policy leadership in the federal government formulated a new approach, in the form of developing specific health targets for the nation. in 1979, the surgeon general of the usa published the report on health promotion and disease prevention (healthy people). this document set five overall health goals for each of the major age groups for the year 1990, accompanied by 226 specific health objectives. new targets for the year 2000 were developed in three broad areas: to increase healthy lifespans, to reduce health disparities, and to achieve access to preventive health care for all americans. these broad goals are supported by 297 specific targets in 22 health priority areas, each one divided into four major categories: health promotion, health protection, preventive services, and surveillance systems. this set the public health agenda on the basis of measurable indicators that can be assessed year by year. reduce child mortality -progress on child mortality is gaining momentum. the target is to reduce by two-thirds, between 1990 and 2015, the under-5-year-old mortality rate, from 93 children of every 1000 dying to 31 of every 1000. child deaths are falling, but much more needs to be done in order to reach the development goal. revitalizing efforts against pneumonia and diarrhea, while bolstering nutrition, could save millions of children. l mdg5. improve maternal health -maternal mortality has nearly halved since 1990, but levels are far removed from the 2015 target. the targets for improving maternal health include reducing by three-quarters the maternal mortality ratio and achieve universal access to reproductive health. poverty and lack of education perpetuate high adolescent birth rates. inadequate funding for family planning is a major failure in fulfilling commitments to improving women's reproductive health. l mdg6. combat hiv/aids, malaria, tuberculosis, and other diseases -more people than ever are living with hiv owing to fewer aids-related deaths and the continued large number of new infections. in 2011, an estimated 34.2 million were living with hiv, up 17 percent from 2001. this persistent increase reflects the continued large number of new infections along with a significant expansion of access to lifesaving antiretroviral therapy, especially in more recent years. l mdg7. ensure environmental sustainability -the unparalleled success of the montreal protocol shows that action on climate change is within grasp. the 25th anniversary of the montreal protocol on substances that deplete the ozone layer, in 2012, had many achievements to celebrate. most notably, there has been a reduction of over 98 percent in the consumption of ozone-depleting substances. further, because most of these substances are also potent greenhouse gases, the montreal protocol has contributed significantly to the protection of the global climate system. the reductions achieved to date leave hydrochlorofluorocarbons (hcfcs) as the largest group of substances remaining to be phased out. l mdg8. a global partnership for development -core development aid fell in real terms for the first time in more than a decade, as donor countries faced fiscal constraints. in 2011, net aid disbursements amounted to $133.5 billion, representing 0.31 percent of developed countries' combined national income. while constituting an increase in absolute dollars, this was a 2.7 percent drop in real terms over 2010. if debt relief and humanitarian aid are excluded, bilateral aid for development programmes and projects fell by 4.5 percent in real terms. equitable and sustainable funding of health services. 18. developing human resources (educational programs for providers and managers based on the principles of the health for all policy). 19. research and knowledge: health programs based on scientific evidence. 20. mobilizing partners for health (engaging the media/ television/internet). 21. policies and strategies for health for all -national, targeted policies based on health for all. a 2010-2012 review has been commissioned by the european office of the who to assess inequalities in the social determinants of health. while health has improved there are still significant inequalities. factors include variance in local, regional, national, and global economic forces. the european union and the european region of who are both working on health targets for the year 2020. there are competing demands in society for expenditure by the government, and therefore making the best use of resources -money and people -is an important objective. the uk has devolved many of the responsibilities to the constituent countries (england, wales, scotland, and northern ireland) within an overall national framework (box 2.17). of the health consequences of their decisions and to accept responsibility for health. health promotion policy combines diverse but complementary approaches, including legislation, fiscal measures, taxation, and organizational change. it is a coordinated action that leads to health, income, and social policies that foster greater equity. joint action contributes to ensuring safer and healthier goods and services, healthier public services, and cleaner, more enjoyable environments. health promotion policies require the identification of obstacles to the adoption of healthy public policies in non-health sectors, and ways of removing them. built on progress made from the declaration on primary health care at alma-ata, the aim was to make the healthier choice the easier choice for policy makers as well. the logo of the ottawa charter has been maintained by the who as the symbol and logo of health promotion. health promotion represents activities to enhance and embed the concept of building healthy public policy through: l building healthy public policy in all sectors and levels of government and society l enhancing both self help and social support l developing personal skills through information and education for health l enabling, mediating, and advocating healthy public policy in all spheres l creating supportive environments of mutual help and conservation of the natural environment l reorienting health services beyond providing clinical curative services with linkage to broader social, political, economic, and physical environmental components. (adapted from ottawa charter; health and welfare canada and world health organization, 1986) an effective approach to health promotion was developed in australia where, in the state of victoria, revenue from a cigarette tax has been set aside for health promotion purposes. this has the effect of discouraging smoking, and at the same time finances health promotion activities and provides a focus for health advocacy in terms of promoting cessation of cigarette advertising at sports events or on television. it also allows for assistance to community groups and local authorities to develop health promotion activities at the workplace, in schools, and at places of recreation. health activity in the workplace involves reduction of work hazards as well as promotion of a healthy diet and physical fitness, and avoidance of risk factors such as smoking and alcohol abuse. in the australian model, health promotion is not only the persuasion of people to change their life habits; it also involves legislation and enforcement towards environmental changes that promote health. for example, this involves mandatory filtration, chlorination, and fluoridation for community water supplies, vitamin and mineral enrichment of basic foods. primary care alliances of service providers are organized including hospitals, community health services serving a sub-district population for more efficient and comprehensive care. these are at the level of national or state policy, and are vital to a health promotion program and local community action. community-based programs to reduce chronic disease using the concept of community-wide health promotion have developed in a wide variety of settings. such a program to reduce risk factors for cardiovascular disease was pioneered in the north karelia project in finland. this project was initiated as a result of pressures from the affected population of the province, which was aware of the high incidence of mortality from heart disease. finland had the highest rates of chd in the world and in the rural area of north karelia the rate was even higher than the national average. the project was a regional effort involving all levels of society, including official and voluntary organizations, to try to reduce risk factors for chd. after 15 years of follow-up, there was a substantial decline in mortality with a similar decline in a neighboring province taken for comparison, although the decline began earlier in north karelia. in many areas where health promotion has been attempted as a strategy, community-wide activity has developed with participation of ngos or any valid community group as initiators or participants. healthy heart programs have developed widely with health fairs, sponsored by charitable or fraternal societies, schools, or church groups, to provide a focus for leadership in program development. a wider approach to addressing health problems in the community has developed into an international movement of "healthy cities". following deliberations of the health of towns commission chaired by edwin chadwick, the health of towns association was founded in 1844 by southwood smith, a prominent reform leader of the sanitary movement, to advocate change to reduce the terrible living conditions of much of the population of cities in the uk. the association established branches in many cities and promoted sanitary legislation and public awareness of the "sanitary idea" that overcrowding, inadequate sanitation, and absence of safe water and food created the conditions under which epidemic disease could thrive. in the 1980s, iona kickbush, trevor hancock, and others promoted renewal of the idea that local authorities have a responsibility to build health issues into their planning and development processes. this "healthy cities" approach promotes urban community action on a broad front of health promotion issues (table 2. 2). activities include environmental projects (such as recycling of waste products), improved recreational facilities for young people to reduce violence and drug abuse, health fairs to promote health awareness, and screening programs for hypertension, breast cancer, and other diseases. it combines health promotion with consumerism and returns to the tradition of local public health action and advocacy. the municipality, in conjunction with many ngos, develops a consultative process and program development approach to improving the physical and social life of the urban environment and the health of the population. in 1995, the healthy cities movement involved 18 countries with 375 cities in europe, canada, the usa, the uk, south america, israel, and australia, an increase from 18 cities in 1986. the model now extends to small municipalities, often with populations of fewer than 10,000. networks of healthy cities are the backbone of the movement, with more than 1400 member towns and cities across europe. the choice of core themes offers the opportunity to work on priority urban health issues that are relevant to all european cities. topics that are of particular concern to individual cities and/or are challenging and cutting edge for innovative public health action are especially emphasized. healthy cities encourages and supports experimentation with new ideas by developing concepts and implementing them in diverse organizational contexts. a healthy city is a city for all its citizens: inclusive, supportive, sensitive and responsive to their diverse needs and expectations. a healthy city provides conditions and opportunities that encourage, enable and support healthy lifestyles for people of all social groups and ages. a healthy city offers a physical and built environment that encourages, enables and supports health, recreation and well-being, safety, social interaction, accessibility and mobility, a sense of pride and cultural identity and is responsive to the needs of all its citizens. the apha's formulation of the public health role in 1995, entitled the future of public health in america, was presented at the annual meeting in 1996. the apha periodically revises standards and guidelines for organized public health services provided by federal, state, and local governments ( table 2 .4). these reflect the profession of public health as envisioned in the usa where access to medical care is limited for large numbers of the population because of a lack of universal health insurance. public health in the usa has been very innovative in determining risk groups in need of special care and finding direct and indirect methods of meeting those needs. european countries such as finland have called for setting public health into all public policy, which reflects the vital role that local and county governments can play in developing health-oriented policies. these include policies in housing, recreation, regulation of industrial pollution, road safety, promotion of smoke-free environments, bicycle paths, health impact assessment, and many other applications of health principles in public policy. public health involves both direct and indirect approaches. direct measures in public health include immunization of children, modern birth control, and chronic disease case finding -hypertension, diabetes, and cancer. indirect methods used in public health protect the individual by community-wide means, such as raising standards of environmental safety, ensuring a safe water supply, sewage disposal, and improved nutrition (box 2.18). in public health practice, the direct and indirect pproaches are both relevant. to reduce morbidity and mortality from diarrheal diseases requires an adequate supply of safe water and waste disposal, and also education of the individual in hygiene and the mother in use of ort, and rotavirus vaccination of all children. the targets of public health action therefore include the individual, family, community, region, or nation, as well as a functioning and health system adopting current best practices for health care and health protection. the targets for protection in infectious disease control are both the individual and the total group at risk. for vaccine-preventable diseases, immunization protects the individual but also has an indirect effect by reducing the risk even for non-immunized persons. in control of some diseases, individual case finding and management reduce risk of the disease in others and the community. for example, tb requires case finding and adequate care among high-risk groups as a key to community control. in malaria control, case finding and treatment are essential together with environmental action to reduce the vector population, to prevent transmission of the organism by the mosquito to a new host. control of ncds, where there is no vaccine for mass application, depends on the knowledge, attitudes, beliefs, and practices of individuals at risk. in this case, the social context is of importance, as is the quality of care to which the individual has access. control and prevention of noninfectious diseases involve strategies using individual and population-based methods. individual or clinical measures include professional advice on how best to reduce the risk of the disease by early diagnosis and implementation of appropriate therapy. population-based measures involve indirect measures with government action banning cigarette advertising, or direct taxation on cigarettes. mandating food quality standards, such as limiting the fat content of meat, and requiring food labeling laws are part of the control of cardiovascular diseases. the way individuals act is central to the objective of reducing disease, because many non-infectious diseases are dependent on behavioral risk factors of the individual's choosing. changing the behavior of the individual means addressing the way a person sees his or her own needs. this can be influenced by the provision of information, but how someone sees his or her own needs is more complex than that. an individual may define needs differently from the society or the health system. reducing smoking among women may be difficult to achieve if smoking is thought to reduce appetite and food intake, given the social message that "slim is beautiful". reducing smoking among young people is similarly difficult if smoking is seen as fashionable and diseases such as lung cancer seem very remote. recognizing how individuals define needs helps the health system to design programs that influence behavior that is associated with disease. public health has become linked to wider issues as health care systems are reformed to take on both individual and population-based approaches. public health and mainstream medicine have found increasingly common ground in addressing the issues of chronic disease, growing attention to health promotion, and economics-driven health care reform. at the same time, the social ecology approaches have shown success in slowing major causes of disease, including heart disease and aids, and the biomedical sciences have provided major new technology for preventing major health problems, including cancer, heart disease, genetic disorders, and infectious diseases. technological innovations unheard of just a few years ago are now commonplace, in some cases driving up costs of care and in others replacing older and less effective care. at the same time, resistance of important pathogenic microorganisms to antibiotics and pesticides is producing new challenges from diseases once thought to be under control, and newly emerging infectious diseases challenge the entire health community. new generations of antibiotics, antidepressants, antihypertensive medications, and other treatment methods are changing the way many conditions are treated. research and development in the biomedical to improve the quality of public health practice and performance of public health systems sciences are providing means of prevention and treatment that profoundly affect disease patterns where they are effectively applied. the technological and organizational revolutions in health care are accompanied by many ethical, economic, and legal dilemmas. the choices in health care include heart transplantation, an expensive life-saving procedure, which may compete with provision of funds and labor resources for immunizations for poor children or for health promotion to reduce smoking and other risk factors for chronic disease. new means of detecting and treating acute conditions such as myocardial infarction and peptic ulcers are reducing hospital stays, and improving long-term survival and quality of life. imaging technology has been an important development in medicine since the advent of x-rays in the early twentieth century. technology has forged ahead with high-technology instruments and procedures, new medication, genetic engineering, and important low-technology gains such as impregnated bed nets, simplified tests for hiv and tb, and many other "game changers". new technologies that can enable lower cost diagnostic devices, electronic transmission, and distant reading of transmitted imaging all open up possibilities for advanced diagnostic capacities in rural and less developed countries and communities. molecular biology has provided methods of identifying and tracking movement of viruses such as polio and measles from place to place, greatly expanding the potential for appropriate intervention. the choices in resource allocation can be difficult. in part, these add political commitment to improve health, competent professionally trained public health personnel, the public's level of health information, and legal protection, whether through individuals, advocacy, or regulatory approaches for patients' rights. these are factors in a widening methodology of public health. the centers for disease control and prevention (morbidity and mortality weekly report) in 1999 summarized 10 great achievements of public health in the usa, with an extension of the lifespan by over 30 years and improvements in many measures of quality of life. they were updated in a similar summary report in 2011, showing continuous progress, and a global version which was also encouraging in its scope of progress (table 2 .5). these achievements were also seen in all developed countries over the past century and are beginning to be seen in developing countries as well. they reflect a successful application of a broad approach to prevention and health promotion along with improved medical care and growing access to its benefits. in the past several decades alone, major new innovations are leading to greater control of cardiovascular disease, cancer prevention, and many other improvements to health affecting hundreds of millions of people. a similar 2011 report by the cdc shows global progress in the first decade of the twenty-first century, while mdg reports show progress on all eight target topics, although not at uniformly satisfactory rates. these achievements are discussed throughout this text. this successful track record is very much at the center of a new public health involving a wide range of programs and activities, shown to be feasible and benefiting from continuing advances in science and understanding of social and management issues affecting health care systems worldwide. public health issues have received new recognition in recent years because of a number of factors, including a growing understanding among the populace at different levels in different countries that health behavior is a factor in health status and that public health is vital for protection against natural or human-made disasters. the challenges are also increasingly understood: preparation for bioterrorism, avian influenza, rising rates of diabetes and obesity, high mortality rates from cancer, and a wish for prevention to be effective. health systems offer general population benefits that go beyond preventing and treating illness. appropriately designed and managed, they: l provide a vehicle to improve people's lives, protecting them from the vulnerability of sickness, generating a sense of life security, and building common purpose within society l ensure that all population groups are included in the processes and benefits of socioeconomic development l generate the political support needed to sustain them over time. health systems promote health equity when their design and management specifically consider the circumstances and needs of socially disadvantaged and marginalized populations, including women, the poor, and groups who experience stigma and discrimination, enabling social action by these groups and the civil society organizations supporting them. health systems can, when appropriately designed and managed, contribute to achieving the millennium development goals. the mdgs selected by the un in 2000 have eight global targets for the year 2015, including four directly related to public health (discussed above, box 2.15). these are a recognition and a challenge to the international community and public health as a profession and as organized systems. formal education in newly developing schools of public health is increasing in europe, including many countries of eastern europe, and beginning to develop in india and sub-saharan africa. but there is delay in establishing centers of postgraduate education and research in many developing countries which are concentrating their educational resources on training physicians. many physicians from developing nations are moving to the developed countries, which have become dependent on these countries for a significant part of their supply of medical doctors. progress in implementation of the mdgs is mixed in sub-saharan africa, making some progress in immunization, but falling back on other goals. proposals to renew global health targets following the 2015 end-stage of the mdg health goals will need to add a focus on ncds, which account for 60 percent of global deaths, including 8.1 million premature deaths below the age of 60 (undp). economic growth has been hampered by the global recession since 2008, which will affect continued progress with many other factors of changing population dynamics, the economics of prevention versus expensive treatment costs, and the high costs of health care. environmental degradation with high levels of carbon dioxide contamination is a growing concern, with disastrous global warming and consequent effects of drought, flooding, hurricane, and elevated particulate matter-induced asthma and effects on cardiovascular disease. the potential for the development of basic and medical sciences in genetics, nanotechnology, and molecular biology shows enormous promise for health benefits as yet unimagined. at the same time, the effectiveness of health promotion has shown dramatic successes in reducing the toll of aids, reducing smoking, and increasing consciousness of nutrition and physical fitness in the population, and of the tragic effects of poverty and poor education on health status. the ethics of public health issues are complex and changing with awareness that failure to act on strong evidence-based policies is itself ethically problematic. the future of public health is not as a solo professional sector; it is at the heart of health systems, without which societies are open to chronic and infectious diseases that are preventable, affecting the society as a whole in economic and development matters. there is an expanding role of private donors in global health efforts, such as the rotary club and the polio eradication program, gavi with immunization and bed-nets in sub-saharan africa, and bilateral donor countries' help in reducing the toll of aids in sub-saharan africa. the new public health has emerged as a concept to meet a whole new set of conditions, associated with increasing longevity and aging of the population, with the post-world war ii baby-boom generation reaching the over-65 age group facing the growing importance of chronic diseases. inequalities in health exist in and between affluent and developing societies, as well as within countries, even those having advanced health care systems. regional inequalities are seen across the european region in an east-west gradient and globally a north-south divide of extremes of inequality. the global environmental and ecological degradation and pollution of air and water present grave challenges for developed and developing countries worldwide. yet optimism can be derived from proven track records of success in public health measures that have already been implemented. many of the underlying factors are amenable to prevention through social, environmental, or behavioral change and effective use of medical care. the new public health idea has evolved since alma-ata, which articulated the concept of health for all, followed by a trend in the late 1970s to health in all policies and establishing health targets as a basis for health planning. during the late 1980s and early 1990s, the debate on the future of public health in the americas intensified as health professionals looked for new models and approaches to public health research, training, and practice. this debate helped to redefine traditional approaches of social, community, and preventive medicine. the search for the "new" in public health continued with a return to the health for all concept of alma-ata (renewed in 2008) and a growing realization that the health of both the individual and the society involves the management of personal care services and community prevention, with a comprehensive approach taking advantage of advancing technology and experience of best practices globally. the new public health is an extension of the traditional public health. it describes organized efforts of society to develop healthy public policies: to promote health, to prevent disease, and to foster social equity within a framework of sustainable development. a new, revitalized public health must continue to fulfill the traditional functions of sanitation, protection, and related regulatory activities, but in addition to its expanded functions. it is a widened philosophy and practical application of many different methods of addressing health, and preventing disease and avoidable death. it necessarily addresses inequities so that programs need to meet special needs of different groups in the population according to best standards, limited resources, and population needs. it is proactive and advocates interventions within legal and ethical limits to promote health as a value in and of itself and as an economic gain for society as well for its individual members. the new public health is a comprehensive approach to protecting and promoting the health status of the individual and the society, based on a balance of sanitary, environmental, health promotion, personal, and community-oriented preventive services, coordinated with a wide range of curative, rehabilitative, and long-term care services. it evolves with new science, technology, and knowledge of human and systems behavior to maximize health gains for the individual and the population. the new public health requires an organized context of national, regional, and local governmental and non-governmental programs with the object of creating healthful social, nutritional, and physical environmental conditions. the content, quality, organization, and management of component services and programs are all vital to its successful implementation. whether managed in a diffused or centralized structure, the new public health requires a systems approach acting towards achievement of defined objectives and specified targets. the new public health works through many channels to promote better health. these include all levels of government and parallel ministries; groups promoting advocacy, academic, professional, and consumer interests; private and public enterprises; insurance, pharmaceutical, and medical products industries; the farming and food industries; media, entertainment, and sports industries; legislative and law enforcement agencies; and others. the new public health is based on responsibility and accountability for defined populations in which financial systems promote achievement of these targets through effective and efficient management, and cost-effective use of financial, human, and other resources. it requires continuous monitoring of epidemiological, economic, and social aspects of health status as an integral part of the process of management, evaluation, and planning for improved health. the new public health provides a framework for industrialized and developing countries, as well as countries in political-economic transition such as those of the former soviet system. they are at different stages of economic, epidemiological, and sociopolitical development, each attempting to ensure adequate health for its population with limited resources. the challenges are many, and affect all countries with differing balances, but there is a common need to seek better survival and quality of life for their citizens (table 2 .6). the object of public health, like that of clinical medicine, is better health for the individual and for society. public health works to achieve this through indirect methods, such as by improving the environment, or through direct means such as preventive care for mothers and infants or other atrisk groups. clinical care focuses directly on the individual patient, mostly at the time of illness. but the health of the individual depends on the health promotion and social programs of the society, just as the well-being of a society depends on the health of its citizens. the new public health consists of a wide range of programs and activities that link individual and societal health. the "old" public health was concerned largely with the consequences of unhealthy settlements and with safety of food, air, and water. it also targeted the infectious, toxic, and traumatic causes of death, which predominated among young people and were associated with poverty. a summary of the great achievements of public health in the twentieth and in the early twenty-first century in the industrialized world is included in chapter 1 and throughout this text. these achievements are reflective of public health gains throughout the industrialized world and are encourage and leverage national, state, and local partnerships to build a stronger foundation for public health preparedness and investigate health problems and health hazards in the community 3. inform, educate, and empower people about health issues 4. mobilize community partnerships to identify and solve health problems 5. develop policies and plans that support individual and community health efforts 6. enforce laws and regulations that protect health and ensure safety 7 evaluate effectiveness, accessibility, and quality of personal and population-based health services vision, mission and goals guidelines on food fortification with micronutrients. who, geneva. alliance for health policy and systems research 10 essential public health services healthy communities, 2000. model standards for community attainment of the year 2000 national health objectives determinants of adult mortality in russia: estimates from sibling data commission on social determinants and health. closing the gap in a generation: health equity through action on the social determinants of health compression of morbidity in the elderly institute of medicine. who will keep the public healthy? educating public health professionals for the 21st century global alliance for vaccine and immunization (gavi) chronic disease prevention and the new public health the evolution, impact and significance of healthy cities/healthy communities world health organization. ottawa charter for health promotion: an international conference on health promotion behavioral and social sciences and public health at cdc. mmwr health in all policies: seizing opportunities, implementing policies. ministry of social affairs and health new perspectives on the health of canadians: a working document new perspective on the health of canadians: 28 years later the us healthy people initiative: its genesis and its sustainability mortality from cardiovascular and cerebrovascular diseases in europe and other areas of the world: an update strategic review of health inequalities in england post. department of health primary care (extended version): ten key actions could globally ensure a basic human right at almost unnoticeable cost public health in europe: power, politics, and where next health: a vital investment for economic development in eastern europe and central asia. european observatory on health systems and policies. who, european regional office it is not just the broad street pump addressing the epidemiologic transition in the former soviet union: strategies for health systems and public health reform in russia what is the "new public health"? millenium development goals: 2013 progress chart united nations development programme, millennium development goals. eight goals for healthy people 2020 healthy people. the surgeon general's report on health promotion and disease prevention the millennium development goals: a cross-sectoral analysis and principles for goal-setting after selective primary health care: an interim strategy for disease control in developing countries declaration of alma-ata. international conference on primary health care healthy cities networks across the who, european region preamble to the constitution of the world health organization as adopted by the international health conference regional office for europe. health 21 -health for all in the 21st century. who regional office for europe, copenhagen. world health organization, 2012. regional office for europe. who european healthy cities network. available at:. who regional office for europe leading health indicators selected for 2010 incorporate the original 467 objectives in healthy people 2010, which served as a basis for planning public health activities for many state and community health initiatives. for each of the leading health indicators, specific objectives and subobjectives derived from healthy people 2010 are used to monitor progress. the specific objectives set for healthy people 2020 are listed in box 2.16. thirteen new topic areas are listed for 2020, such as older adults, genomics, dementias, and social determinants of health. these provide guidelines for national, state, and local public health agencies as well as insurance providers, primary care services, and health promotion advocates. a key issue will be in reducing regional, ethnic, and socioeconomic health disparities.the process of working towards health targets in the usa has moved down from the federal level of government to the state and local levels. professional organizations, ngos, as well as community and fraternal organizations are also involved. the states are encouraged to prepare their own targets and implementation plans as a condition for federal grants, and many states require county health departments to prepare local profiles and targets.diffusion of this approach encourages state and local initiatives to meet measurable program targets. it also sets a different agenda for local prestige in competitive terms, with less emphasis on the size of the local hospital or other agencies than on having the lowest infant mortality or the least infectious disease among neighboring local authorities. the who european region document "health 21 -health for all in the 21st century" addresses health in the twentyfirst century, with 21 principles and objectives for improving the health of europeans, within and between countries of europe. the health 21 targets include:1. closing the health gap between countries. 2. closing the health gap within countries. 3. a healthy start in life (supportive family policies). 4. health of young people (policies to reduce child abuse, accidents, drug use, and unwanted pregnancies). 5. healthy aging (policies to improve health, self-esteem, and independence before dependence emerges). 6. improving mental health. 7. reducing communicable diseases. 8. reducing non-communicable diseases. 9. reducing injury from violence and accidents. 10. a healthy and safe physical environment. 11. healthier living (fiscal, agricultural, and retail policies that increase the availability of and access to and consumption of vegetables and fruits). 12. reducing harm from alcohol, drugs, and tobacco. 13. a settings approach to health action (homes should be designed and built in a manner conducive to sustainable health and the environment).14. multisectoral responsibility for health.15. an integrated health sector and much stronger emphasis on primary care. 16. managing for quality of care using the european health for all indicators to focus on outcomes and compare the effectiveness of different inputs. the uk national health service (nhs) has semi-autonomous units in england, scotland, wales, and northern ireland. they are funded from the central uk nhs but with autonomy within national guidelines. the nhs has defined national health outcomes for improvements grouped around five domains, each comprised of key indicators aimed at improving health with reducing inequalities. l preventing people from dying prematurely from causes amenable to health care for all ages: l the target diseases include cardiovascular, respiratory, and liver diseases, and cancer (with focus on cancer of breast, lung, and colorectal cancer) l reducing premature death in people with serious mental illnesses l reducing infant mortality, neonatal mortality, still births, and deaths in young children l increasing 5-year survival for children with cancer. health improvement; help people to live healthy lifestyles, healthy choices, reduce health inequalities, protection from major incidents and other threats, while reducing health inequalities. l health care, public health and preventing premature mortality; reduce the numbers of people living with preventable ill-health and people dying prematurely, while reducing the gap between communities.source: uk department of health. available at: https://www.gov.uk/government/organisations/department-of-health/about#our-priorities, https:// www.gov.uk/government/uploads/system/uploads/attachment_data/ file/193619/improving-outcomes-and -supporting-transparency-part-1a.pdf. pdf, and https://www.gov.uk/government/uploads/system/uploads/attach-ment_data/file/127106/121109-nhs-outcomes-framework-2013-14.pdf. pdf [accessed 24 june 2013] . national policy in health ultimately relates to health of the individual. the various concepts outlined in the health field concept, community-oriented primary health care, health targets, and effective management of health systems, can only be effective if the individual and his or her community are knowledgeable participants in seeking solutions. involving the individual in his or her own health status requires raising levels of awareness, knowledge, and action. the methods used to achieve these goals include health counseling, health education, and health promotion (figure 2 .6).health counseling has always been a part of health care between the doctor or nurse and the patient. it raises levels of awareness of health issues of the individual patient. health education has long been part of public health, dealing with promoting consciousness of health issues in selected target population groups. health promotion incorporates the work of health education but takes health issues to the policy level of government and involves all levels of government and ngos in a more comprehensive approach to a healthier environment and personal lifestyles.health counseling, health education, and health promotion are among the most cost-effective interventions for improving the health of the public. while costs of health care are rising rapidly, demands to control cost increases should lead to greater emphasis on prevention, and adoption of health education and promotion as an integral part of modern life. this should be carried out in schools, the workplace, the community, commercial locations (e.g., shopping centers), and recreation centers, and in the political agenda.psychologist abraham maslow described a hierarchy of needs of human beings. every human has basic requirements including physiological needs of safety, water, food, warmth, and shelter. higher levels of needs include recognition, community, and self-fulfillment. these insights supported observations of efficiency studies such as those of elton mayo in the famous hawthorne effect in the 1920s, showing that workers increased productivity when acknowledged by management in the objectives of the organization (see chapter 12). in health terms, these translate into factors that motivate people to positive health activities when all barriers to health care are reduced.modern public health faces the problem of motivating people to change behavior; sometimes this requires legislation, enforcement, and penalties for failure to comply, such as in mandating car seat-belt use. in other circumstances it requires sustained performance by the individual, such as the use of condoms to reduce the risk of sti and/or hiv transmission. over time, this has been developed into a concept known as knowledge, attitudes, beliefs, and practices (kabp), a measurable complex that cumulatively affects health behavior (see chapter 3). there is often a divergence between knowledge and practice; for example, the knowledge of the importance of safe driving, yet not putting this into practice. this concept is sometimes referred to as the "kabp gap". the health belief model has been a basis for health education programs, whereby a person's readiness to take action for health stems from a perceived threat of disease, a recognition of susceptibility to disease and its potential severity, and the value of health. action by an individual may be triggered by concern and by knowledge. barriers to appropriate action may be psychological, financial, or physical, including fear, time loss, and inconvenience. spurring action to avoid risk to health is one of the fundamental goals in modern health care. the health belief model is important in defining any health intervention in that it addresses the emotional, intellectual, and other barriers to taking steps to prevent or treat disease.health awareness at the community and individual levels depends on basic education levels. mothers in developing countries with primary or secondary school education are more successful in infant and child care than less educated women. agricultural and health extension services reaching out to poor and uneducated farm families in north america in the 1920s were able to raise consciousness of safe self-health practices and good nutrition, and when this was supplemented by basic health education in schools, generational differences could be seen in levels of awareness of the importance of balanced nutrition. secondary prevention with diabetics and patients with chd hinges on education and awareness of nutritional and physical activity patterns needed to prevent or delay a subsequent myocardial infarction. the who sponsored the first international conference on health promotion held in ottawa, canada, in 1986 ( figure 2.7) . the resulting ottawa charter defined health promotion and set out five key areas of action: building healthy public policy, creating supportive environments, strengthening community action, developing personal skills, and reorienting health services. the ottawa charter called on all countries to put health on the agenda of policy makers in all sectors and at all levels, directing them to be aware a typical healthy city has a population in the multiple thousands, often multilingual, with an average middleclass income. a healthy cities project builds a coalition of municipal and voluntary groups working together in a continuing effort to improve quality of service, facilities, and living environment. the city is divided into neighborhoods, engaged in a wide range of activities fostered by the project. municipalities have traditionally had a leading role in sanitation, safe water supply, building and zoning laws and regulation, and many other responsibilities in public health (see chapter 10). the healthy cities or communities movement has elevated this to a higher level with policies to promote health in all actions. some examples are listed of municipal, advocacy group, and higher governmental activities for healthier city environments: working with senior levels of government, other departments in the municipalities, religious organizations, private donors, and the ngo sector to innovate and especially to improve conditions in poverty-afflicted areas of cities is a vital role for health-oriented local political leadership. human ecology, a term introduced in the 1920s and revived in the 1970s, attempted to apply theory from plant and animal life to human communities. it evolved as a branch of demography, sociology, and anthropology, addressing the social and cultural contexts of disease, health risks, and human behavior. human ecology addresses the interaction of humans with and adaptation to their social and physical environment.parallel subdisciplines of social, community, and environmental psychology, medical sociology, anthropology, and other social sciences contributed to the development of this academic field with wide applications in health-related issues. this led to the incorporation of qualitative research methods alongside the quantitative research methods traditionally emphasized in public health, providing crucial insights into many public health issues where human behavior is a key risk factor.health education developed as a discipline and function within public health systems in school health, rural nutrition, military medicine, occupational health, and many other aspects of preventive-oriented health care, and is discussed in later chapters of this text. directed at behavior modification through information and raising awareness of consequences of risk behavior, this has become a longstanding and major element of public health practice in recent times, being almost the only effective tool to fight the epidemic of hiv and the rising epidemic of obesity and diabetes.health promotion as an idea evolved, in part, from marc lalonde's health field concepts and from growing realization in the 1970s that access to medical care was necessary but not sufficient to improve the health of a population. the integration of the health behavior model, social ecological approach, environmental enhancement, or social engineering formed the basis of the social ecology approach to defining and addressing health issues (table 2 .3).individual behavior depends on many surrounding factors, while community health also relies on the individual; the two cannot be isolated from one another. the ecological perspective in health promotion works towards changing people's behavior to enhance health. it takes into account factors not related to individual behavior, which are determined by the political, social, and economic environment. it applies broad community, regional, or national approaches that are needed to address severe public health problems, such as controlling hiv infection, tb, malnutrition, stis, cardiovascular disorders, violence and trauma, and cancer. beginning to affect the health situation in countries in transition from the socialist period. countries emerging from developing status are also showing signs of mixed progress in the dual burden of infectious and maternal/child health issues, along with growing exposure to the chronic diseases of developed nations such as cardiovascular diseases, obesity, and diabetes. the new public health synthesizes traditional pub lic health with management of personal services and community action for a holistic approach. evaluation of costeffective public health and medical interventions to reduce the burden of disease also contributes to the need to seek and apply new approaches to health. the new public health will continue to evolve as a framework drawing on new ideas, science, technology, and experiences in public health throughout the world. it must address the growing recognition of social inequality in health, even in developed countries with universal health programs with improved education and social support systems. for a complete bibliography and guidance for student reviews and expected competencies please see companion web site at http://booksite.elsevier.com/9780124157668 bibliography key: cord-263452-y2ral8nx authors: watanabe, yasunori; allen, joel d.; wrapp, daniel; mclellan, jason s.; crispin, max title: site-specific glycan analysis of the sars-cov-2 spike date: 2020-05-04 journal: science doi: 10.1126/science.abb9983 sha: doc_id: 263452 cord_uid: y2ral8nx the emergence of the betacoronavirus, sars-cov-2, the causative agent of covid-19, represents a significant threat to global human health. vaccine development is focused on the principal target of the humoral immune response, the spike (s) glycoprotein, which mediates cell entry and membrane fusion. sars-cov-2 s gene encodes 22 n-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant sars-cov-2 s immunogen. this analysis enables mapping of the glycan-processing states across the trimeric viral spike. we show how sars-cov-2 s glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design. impaired glycan maturation resulting in the presence of oligomannose-type glycans can be a sensitive reporter of nativelike protein architecture (8), and site-specific glycan analysis can be used to compare different immunogens and monitor manufacturing processes (18) . additionally, glycosylation can influence the trafficking of recombinant immunogen to germinal centers (19) . to resolve the site-specific glycosylation of sars-cov-2 s protein and visualize the distribution of glycoforms across the protein surface, we expressed and purified three biological replicates of recombinant soluble material in an identical manner to that which was used to obtain the high-resolution cryo-electron microscopy (cryo-em) structure, albeit without glycan processing blockade using kifunensine (4). this variant of the s protein contains all 22 glycans on the sars-cov-2 s protein (fig. 1a) . stabilization of the trimeric prefusion structure was achieved using the "2p" stabilizing mutations (20) at residues 986 and 987, a "gsas" substitution at the furin cleavage site (residues 682-685), and a c-terminal trimerization motif. this helps to maintain quaternary architecture during glycan processing. prior to analysis, supernatant containing the recombinant sars-cov-2 s was purified by sizeexclusion chromatography ensure only native-like trimeric protein was analyzed ( fig. 1b and fig. s1 ). the trimeric conformation of the purified material was validated using negative-stain electron microscopy (fig. 1c) . to determine the site-specific glycosylation of sars-cov-2 s, we employed trypsin, chymotrypsin, and alpha-lytic protease to generate three glycopeptide samples. these proteases were selected to generate glycopeptides that contain a single nlinked glycan sequon. the glycopeptides were analyzed by liquid-chromatography-mass spectrometry (lc-ms), and the glycan compositions were determined for all 22 n-linked glycan sites (fig. 2) . to convey the main processing features at site-specific glycan analysis of the sars-cov-2 spike yasunori watanabe 1,2,3 *, joel d. allen 1 *, daniel wrapp 4 , jason s. the emergence of the betacoronavirus, sars-cov-2, the causative agent of covid-19, represents a significant threat to global human health. vaccine development is focused on the principal target of the humoral immune response, the spike (s) glycoprotein, which mediates cell entry and membrane fusion. sars-cov-2 s gene encodes 22 n-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant sars-cov-2 s immunogen. this analysis enables mapping of the glycanprocessing states across the trimeric viral spike. we show how sars-cov-2 s glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design. there are two sites on sars-cov-2 s that are principally oligomannose-type: n234 and n709. the predominant oligomannose-type glycan structure observed across the protein, with the exception of n234, is man5glcnac2, which demonstrates that these sites are largely accessible to α1,2-mannosidases but are poor substrates for glcnact-i, which is the gateway enzyme in the formation of hybrid-and complextype glycans in the golgi apparatus. the stage at which processing is impeded is a signature related to the density and presentation of glycans on the viral spike. for example, the more densely glycosylated spikes of hiv-1 env and lassa virus gpc exhibit numerous sites dominated by man9glcnac2 (21) (22) (23) (24) . a mixture of oligomannose-and complex-type glycans can be found at sites n61, n122, n603, n717, n801 and n1074 (fig. 2) . of the 22 sites on the s protein, 8 contain significant populations of oligomannose-type glycans, highlighting how the processing of the sars-cov-2 s glycans is divergent from host glycoproteins (25). the remaining 14 sites are dominated by processed, complex-type glycans. although unoccupied glycosylation sites were detected on sars-cov-2 s, when quantified they were revealed to form a very minor component of the total peptide pool (table s2). in hiv-1 immunogen research, the holes generated by unoccupied glycan sites have been shown to be immunogenic and potentially give rise to distracting epitopes (26) . the high occupancy of n-linked glycan sequons of sars-cov-2 s indicates that recombinant immunogens will not require further optimization to enhance site occupancy. using the cryo-em structure of the trimeric sars-cov-2 s protein (pdb id 6vsb) (4), we mapped the glycosylation status of the coronavirus spike mimetic onto the experimentally determined 3d structure (fig. 3) . this combined mass spectrometric and cryo-em analysis reveals how the n-linked glycans occlude distinct regions across the surface of the sars-cov-2 spike. shielding of the receptor binding sites on the sars-cov-2 spike by proximal glycosylation sites (n165, n234, n343) can be observed, especially when the receptor binding domain is in the "down" conformation. the shielding of receptor binding sites by glycans is a common feature of viral glycoproteins, as observed on sars-cov-1 s (10, 13), hiv-1 env (27) , influenza ha (28, 29) , and lasv gpc (24). given the functional constraints of receptor binding sites and the resulting low mutation rates of these residues, it is likely that there is selective pressure to utilize n-linked glycans to camouflage one of the most conserved and potentially vulnerable areas of their respective glycoproteins (30, 31) . it is interesting to note the dispersion of oligomannosetype glycans across both the s1 and s2 subunits. this is in contrast to other viral glycoproteins, for example the dense glycan clusters in several strains of hiv-1 env induce oligomannose-type glycans that are recognized by antibodies (32, 33) . in sars-cov-2 s the oligomannose-type structures are likely protected by the protein component, as exemplified by the n234 glycan which is partially sandwiched between the n-terminal and receptor-binding domains (fig. 3) . we characterized the n-linked glycans on extended flexible loop structures (n74 and n149) and at the membraneproximal c terminus (n1158, n1173, n1194) that were not resolved in the cryo-em maps (4) these were determined to be complex-type glycans, consistent with steric accessibility of these residues. while the oligomannose-type glycan content (28%) (table s2) is above that observed on typical host glycoproteins, it is lower than other viral glycoproteins. for example, one of the most densely glycosylated viral spike proteins is hiv-1 env, which exhibits ~60% oligomannose-type glycans (21, 34) . this suggests that sars-cov-2 s protein is less densely glycosylated and that the glycans form less of a shield compared with other viral glycoproteins including hiv-1 env and lasv gpc, which may be beneficial for the elicitation of neutralizing antibodies. additionally, the processing of complex-type glycans is an important consideration in immunogen engineering, especially considering that epitopes of neutralizing antibodies against sars-cov-2 s can contain fucosylated glycans at n343 (35) . across the 22 n-linked glycosylation sites, 52% are fucosylated and 15% of the glycans contain at least one sialic acid residue (table s2 and fig. s3 ). our analysis reveals that n343 is highly fucosylated with 98% of detected glycans bearing fucose residues. glycan modifications can be heavily influenced by the cellular expression system utilized. we have previously demonstrated for hiv-1 env glycosylation that the processing of complex-type glycans is driven by the producer cell but that the levels of oligomannose-type glycans were largely independent of the expression system and is much more closely related to the protein structure and glycan density (36) . highly dense glycan shields, such as those observed on lasv gpc and hiv-1 env, feature so-called mannose clusters (22, 24) on the protein surface (fig. 4) . while small mannosetype clusters have been characterized on the s1 subunit of middle east respiratory syndrome (mers) cov s (10), no such phenomenon has been observed for sars-cov-1 or sars-cov-2 s proteins. the site-specific glycosylation analysis reported here suggests that the glycan shield of sars-cov-2 s is consistent with other coronaviruses and similarly exhibits numerous vulnerabilities throughout the glycan shield (10) . finally, we detected trace levels of o-linked glycosylation at t323/s325 with over 99% of these sites unmodified ( fig. s4 ) suggesting that o-linked glycosylation of this region is minimal when the structure is native-like. our glycosylation analysis of sars-cov-2 offers a detailed benchmark of site-specific glycan signatures characteristic of a natively folded trimeric spike. as an increasing number of glycoprotein-based vaccine candidates are being developed, their detailed glycan analysis offers a route for comparing immunogen integrity and will also be important to monitor as manufacturing processes are scaled for clinical use. glycan profiling will therefore also be an important measure of antigen quality in the manufacture of serological testing kits. finally, with the advent of nucleotide-based vaccines, it will be important to understand how those delivery mechanisms impact immunogen processing and presentation. material transfer agreement with the university of texas at austin. this work is licensed under a creative commons attribution 4.0 international (cc by 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. to view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. this license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material. 4), with one rbd in the "up" conformation and the other two rbds in the "down" conformation. the glycans are colored according to oligomannose content as defined by the key. ace2 receptor binding sites are highlighted in light blue. the s1 and s2 subunits are rendered with translucent surface representation, colored light and dark grey, respectively. note that the flexible loops on which n74 and n149 glycan sites reside are represented as dashed lines with glycan sites on the loops mapped at their approximate regions. (8, 21) . site-specific n-linked glycan oligomannose quantifications are colored according to the key. all glycoproteins were expressed as soluble trimers in hek 293f cells apart from lasv gpc, which was derived from virus-like particles from madin-darby canine kidney ii cells. exploiting the defensive sugars of hiv-1 for drug and vaccine design unexpected receptor functional mimicry elucidates activation of coronavirus fusion cryo-em analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans the intracellular sites of early replication and budding of sarscoronavirus identification of n-linked carbohydrates from severe acute respiratory syndrome (sars) spike glycoprotein innate immune recognition of glycans targets hiv nanoparticle immunogens to germinal centers immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen site-specific glycosylation of virion-derived hiv-1 env is mimicked by a soluble trimeric immunogen composition and antigenic effects of individual glycan sites of a trimeric hiv-1 envelope glycoprotein mapping the complete glycoproteome of virionderived hiv-1 gp120 provides insights into broadly neutralizing antibody binding electron-microscopy-based epitope mapping defines specificities of polyclonal antibodies elicited during hiv-1 bg505 envelope trimer immunization rational hiv immunogen design to target specific germline b cell receptors structural basis of preexisting immunity to the 2009 h1n1 pandemic influenza virus antibody neutralization and escape by hiv-1 tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin promiscuous glycan site recognition by antibodies to the high-mannose patch of gp120 broadens neutralization of hiv structural and functional analysis of a potent sarbecovirus neutralizing antibody cell-and protein-directed glycosylation of native cleaved hiv-1 envelope sars-cov-2 spike site-specific n-linked glycan analysis grigorieff, cistem, user-friendly software for single-particle image processing key: cord-278831-gwnfcfvk authors: taniwaki, sueli akemi; figueiredo, andreza soriano; araujo jr., joão pessoa title: virus–host interaction in feline immunodeficiency virus (fiv) infection date: 2013-08-02 journal: comp immunol microbiol infect dis doi: 10.1016/j.cimid.2013.07.001 sha: doc_id: 278831 cord_uid: gwnfcfvk feline immunodeficiency virus (fiv) infection has been the focus of several studies because this virus exhibits genetic and pathogenic characteristics that are similar to those of the human immunodeficiency virus (hiv). fiv causes acquired immunodeficiency syndrome (aids) in cats, nevertheless, a large fraction of infected cats remain asymptomatic throughout life despite of persistent chronic infection. this slow disease progression may be due to the presence of factors that are involved in the natural resistance to infection and the immune response that is mounted by the animals, as well as due to the adaptation of the virus to the host. therefore, the study of virus–host interaction is essential to the understanding of the different patterns of disease course and the virus persistence in the host, and to help with the development of effective vaccines and perhaps the cure of fiv and hiv infections. the feline immunodeficiency virus (fiv) was initially isolated in 1986 from domestic cats that exhibited signs of immunodeficiency but were seronegative for the feline leukaemia virus (felv) [1] . fiv belongs to the family retroviridae, the subfamily orthoretrovirinae, and the genus lentivirus [2] . it exhibits a complex genomic organisation, which includes the structural genes gag, pol, and env, as well as several other genes that encode non-structural proteins. the 5 and 3 ends of the proviral dna that is integrated into the host cell chromosomes comprise long terminal repeats (ltrs), which are repeated regions that contain information on the start and termination of transcription [3, 4] . because the genomic organisation and pathogenesis of fiv are similar to those of human immunodeficiency virus (hiv), fiv has been widely used as an experimental model in studies of immunopathogenesis and virus-host interactions and in the development of vaccines and antiretroviral therapies [4] [5] [6] [7] [8] . fiv is present worldwide and can be classified into five well-characterised subtypes (a-e) [9] [10] [11] [12] [13] . the prevalence of fiv varies from 1% to 44%, depending on the health status of the investigated cats [3, [14] [15] [16] . the prevalence of fiv is influenced by the animals' behaviour. free-ranging male cats in areas of high population density constitute the risk group mainly because of their greater exposure to bites, which are the main path of disease transmission, during territorial demarcation fights [3] . infected cats do not exhibit specific clinical signs, and some animals can remain asymptomatic throughout life. fiv infection causes progressive immunosuppression that results in the development of the acquired immunodeficiency syndrome (aids) in cats, which, similar to human hiv infection, increases susceptibility to secondary and opportunistic infections. concomitantly, fiv also causes immunostimulation, which results in immune-mediated diseases [17, 18] . fiv infection is one of the most important infectious diseases in cats because it causes persistent infection. the aim of the present review is to discuss the virus-host interaction in fiv infection to better understand the immunopathogenesis of this disease, particularly the immune system dysfunction. in addition, the present review seeks to establish the presence of factors that are involved in the natural resistance to fiv infection, similar to those that have been discovered in hiv-exposed seronegative humans. following infection, viral replication occurs in dendritic cells, macrophages, and cd4 + t lymphocytes. a marked viraemia is usually detected in the second week postinfection (pi), with a peak between 8 and 12 weeks, when the virus spreads across the entire organism [3, 4, 18, 19] . during this initial stage, or the acute phase, the animals may exhibit mild to moderate clinical signs, such as fever, transient generalised lymphadenopathy, anorexia, and lethargy [4, 18, 20] . gag-specific cytotoxic t lymphocytes (cd8 + ) may be detected as early as the second week pi, which is prior to seroconversion [21] two to eight weeks after infection, specific antibodies against the surface (su) and transmembrane (tm) proteins of env gene, and the capsid (ca) and matrix (ma) proteins of gag gene might be identified [4, 18] . despite the development of a cellular and humoral immune response, the animals are unable to eliminate the virus, which leads to the establishment of persistent fiv infection [4] . nevertheless, the immune response reduces the circulating viral load, which defines the onset of the asymptomatic stage. the infected cats remain in this stage for many years, or even throughout life, with few clinical signs; however, progressive immunodeficiency with reduction in the number of cd4 + t lymphocytes and an inversion of the cd4 + /cd8 + ratio [3, 18, 19] . finally, a proportion of fiv-positive cats reaches the second stage, or terminal phase, which correlates with the generalised lymphadenopathy, pre-aids, and aids stages of hiv infection [4, 22] . this phase is characterised by severe immunosuppression and a consequent reduction in circulating antibodies, which allows the viral load increase and the development of disease s that are caused by opportunistic or secondary chronic infections, neoplasia, and neurological disorders [4, 19] . despite the progressive immunodeficiency, the course of the disease depends on the virus-host interaction, which explains why only a fraction of the animals develops the terminal phase. the cell tropism and the pathogenesis of the viral disease are determined by the specificity of the interaction between the viral surface proteins and the host cell receptors [4, 23] . similar to hiv infection, infection with fiv requires an initial interaction with a primary receptor and subsequent binding to a co-receptor [24] . in the case of hiv infection, the surface glycoprotein (gp120) interacts with the cd4 receptor expressed on the surface of cd4 + t lymphocytes [25] , which results in a conformational change in gp120 that exposes the co-receptor binding site, thereby allowing binding to the chemokine receptors cxcr4 or ccr5 [23, 25] . the interaction between the coreceptor and gp120 induces a conformational change in the hiv transmembrane glycoprotein (gp41) that exposes its fusion peptide and thus allows the fusion of the viral envelope with the cell membrane [23, 26] . this conformational change in the envelope proteins is an important mechanism for viral escape because the conserved regions of the surface protein, which are the target of neutralising antibodies, are not exposed prior to binding to the receptor and co-receptor [25] . in contrast to hiv, fiv uses cd134 instead of cd4 as its primary receptor. cd134, which is also known as ox40, is a member of the tumour necrosis factor and nerve growth factor receptor superfamily [24] . a large fraction of the fiv primary strains that have been isolated from infected animals, which are most likely the transmissible viruses, require initial binding to the cd134 receptor and subsequent binding to the cxcr4 chemokine receptor for productive infection [24, 27] . however, some cultureadapted fiv strains do not use the cd134 receptor and are able to interact directly with the cxcr4 co-receptor [23] . the interaction between cd134 and the fiv surface protein gp95 leads to structural changes that expose the binding site of the cxcr4 co-receptor [28] . binding to the cxcr4 co-receptor induces the exposure of a serpentine region of the fiv transmembrane protein (gp36) that binds to the cell membrane, thereby originating a hairpin structure that allows for viral fusion and entry [29] . these interactions also induce the exposure of the epitopes that are the target of neutralising antibodies [28] . although the v3 region of gp95 is the principal immunodominant domain of fiv, the antibodies produced exhibit low neutralising activity. however, upon the binding of gp95 to soluble cd134, de perseval et al. [28] observed that monoclonal antibodies against the surface protein inhibited fiv infections in vitro, which denotes a viral escape mechanism during infection. in addition to a mechanism of viral protection against the host's immune response, the binding of the virus to cell surface receptors and co-receptors also accounts for the differentiated cell tropism that occurs throughout the course of the disease. in the case of hiv infection, binding via the cd4 receptor allows for entry into all cd4 + t lymphocytes [24] . however, the main viral strains found during the early stages of infection are those that use the co-receptor ccr5, which is only expressed by activated memory t lymphocytes, whereas the strains isolated at later stages tend to use the cxcr4 co-receptor [30] . in the case of fiv infection, all of the viral strains tested to date interact with the cxcr4 co-receptor, which, in cats, is highly expressed in activated t cells, b cells, and monocytes [23, 31] . nevertheless, the need to interact with the primary receptor cd134, which is more abundantly expressed in activated cd4 + t lymphocytes, constrains the initial infection and consequently results in a depletion of the cd4 + t-cell subgroup that plays a vital role in the development of the antigen-specific cellular immune response [23, 24] . therefore, although these viruses use different entry receptors, the initial target of both fiv and hiv is the activated t lymphocytes [24] . however, with the progression of infection, the host's immune response exerts a positive selective pressure on the virus [32] . the high error rate of the reverse transcriptase enzyme, which is involved in retroviral replication, leads to mutations that result in the generation of a wide variety of different viral genomes within a single individual (quasispecies) and allows for a small changes of the surface proteins that alters their recognition by the immune system (antigenic drift) [33] . kraase et al. [32] found a greater accumulation of mutations in the fiv env gene amongst cats diagnosed with late infection (322 weeks pi) compared with animals with acute infection (12 weeks pi). these mutations were mainly found in the glycosylated regions, which is an additional mechanism that is used by viruses to evade the immune system. in addition, a single mutation involving the loss of a glycosylation site (t271i, n269g, or n269d) might give rise to mutants that are less dependent on the receptor cd134 [32, 34] . similar to the cultureadapted fiv prototypes, the viral variants that bind directly to the co-receptor cxcr4 are likely more susceptible to neutralising antibodies [32] . thus, the presence of these viruses in the circulation during the asymptomatic phase must be transient; however, when the animals reach the terminal phase, which is characterised by severe immunosuppression, the viruses begin to circulate again [32] and increase the number of infected cells, including cd4 + and cd8 + t lymphocytes, b lymphocytes, and astrocytes [3, 23, 35 ]. as mentioned above, a cellular and humoral antiviral immune response occurs during the acute phase of the infection, which reduces the viral load. however, the infection persists as a result of the viral evasion mechanisms (e.g., glycosylation of the viral envelope proteins, camouflage of the immunodominant epitopes through structural changes of the envelope glycoproteins after binding to the host cell, and antigenic drift) and the immune dysfunction that occurs after infection. with the progression of the fiv infection, two opposing events occur that result in immune dysfunction: immunosuppression and hyperactivation of the immune system [7] . a summary of the events that promote the immune dysfunction are shown in fig. 1 . the immunosuppression caused by fiv infection manifests as a loss of response to viral antigens, a loss of response to mitogens, and the inability to launch a primary immune response against secondary pathogens [7] . torten et al. [36] observed that, together with the progression of the disease (25-44 months pi), the infected cats exhibited a reduction in their response to t-dependent immunogens but an adequate response to t-independent immunogens. in addition, these researchers observed a reduction in lymphocyte proliferation, which is induced by mitogens in the infected cats. an alteration in cytokine production, immune anergy, and apoptosis, and a hyperactivation of regulatory t cells are the mechanisms that might explain the immune system dysfunction. although several studies have quantified the cytokines in fiv-infected cats, the profile of cytokine alteration is not yet well established (table 1 ). this cytokine alteration is likely related to the infection stage, especially in relation to the reduction of cd4 + t lymphocytes and the inversion of the cd4 + /cd8 + ratio, as well as the different methods that have been used for the quantification of the proteins or transcripts. lawrence et al. [37] observed a significant increase in the production of interleukin-1 (il-1), il-6, and tumour necrosis factor alpha (tnf␣) in peripheral blood mononuclear cells (pbmcs) in response to mitogens (concanavalin a (cona) and pokeweed mitogen (pwm)) in fiv-positive cats with or without signs of immunosuppression. in addition, a significant reduction of il-2 was observed in symptomatic animals, which also exhibited reduced lymphocyte proliferation in response to mitogens [37] . an increased bioactivity of tnf␣ and il-6 was observed in alveolar macrophages before (four weeks pi) and after (10 weeks pi) the inversion of the cd4 + /cd8 + ratio [38] . the same study also observed an increased expression of tnf␣, il-6, il-10, and interferon-gamma (ifn␥). an increased expression of ifn␥ and a reduced expression of il-2 and il-12 were observed in infected cats after the inversion of the cd4 + /cd8 + ratio 24 weeks pi [39] . fiv-infected cats do not exhibit a primary immune response against a secondary infection by either toxoplasma gondii [39] or listeria monocytogenes [40] . fiv-and t. gondii-co-infected animals exhibit increased expression of il-10 before and after the infection by the secondary pathogen. the high production of il-10 suppresses the production of th1 (il-2, il-12, and ifn␥) and th2 (il-6) cytokines, which explains the inability to contain the infection by t. gondii [39] . dean et al. [41] observed that fiv-positive cats, especially animals with chronic infections (after the inversion of the cd4 + /cd8 + ratio), exhibited higher numbers of colony-forming units of l. monocytogenes in the lymph nodes. in addition, the innate immune response of these cats with chronic infection was unable to contain the secondary infection four days after inoculation, unlike animals with acute infection or fiv-negative cats [41] . this reduction in the response to infection by l. monocytogenes might be due to increased il-10 expression, which inhibits the production of tnf␣ in macrophages [40] . in contrast to levy et al. [39] , dean et al. [40] did not observe a reduction in the levels of il-2 and ifn␥, most likely because the animals from the latter study did not exhibit an alteration of the cd4 + /cd8 + ratio. nevertheless, both studies observed an increase in the il-10/il-12 ratio, which explains the deficient response to the secondary pathogen because il-12 stimulates ifn␥ and tnf␣ production and il-10 suppresses the production of il-12 by dendritic cells [7, 42] . therefore, there is an important change in cytokine production that results in a deficient response to secondary pathogens. both fiv and hiv infections are associated with a progressive loss of t-cell function, which is caused by the suppression of il-2 production, and an increased programmed cell death (apoptosis) of t cells in the lymph nodes [7] . immune anergy is the functional inactivation of t or b lymphocytes following antigenic stimulation due to inadequate amounts of co-stimulatory molecules (e.g., b7.1 and b7.2) in antigen-presenting cells or to the presence of inhibitory ctla-4 (cytotoxic t lymphocyte-associated antigen 4) receptors [43] . the binding of b7 molecules to the cd28 receptor, which is expressed in t lymphocytes, stimulates the release of il-2 and consequently the clonal expansion that is needed to mount an effective immune response, whereas the binding of b7 to the ctla4 receptor suppresses the release of il-2 and thus terminates the immune response [43] . in addition, antigen recognition induces the production of pro-apoptotic proteins, but the activity of these proteins is neutralised by anti-apoptotic proteins in the normal response to microorganisms [43] . guiot et al. [44] observed that fiv-infected cats exhibited increased apoptosis that affected all lymphocyte types (cd4 + and cd8 + t lymphocytes, b lymphocytes, and natural killer (nk) cells). this increased apoptosis was directly correlated with an increase in the animal's age and the duration of the infection and with a reduction in the numbers of cd4 + t cells [44] . the increase in the apoptosis of cd4 + and cd8 + t cells in both the lymph nodes and the blood of fiv-positive cats might be related to an increase in the levels of cd4 + and cd8 + t cells with a b7 + ctla4 + phenotype [45] . an increase in b7 + t cells was observed in animals with acute infection (<6 months), asymptomatic animals (1-5 years), and mainly animals with terminal infection (8-10 years) that exhibit a chronic secondary oral or respiratory infection and, in most cases, the presence of b-cell lymphoma at necropsy [45] . the chronic activation of t cells co-expressing b7 and ctla-4 and the possibility of a t-t interaction mediated by the binding of b7.1/b7.2 to the ctla-4 receptor induce a bidirectional suppression of il-2 that results in anergy and apoptosis [7, 45, 46] . therefore, this mechanism causes immunosuppression with a progressive loss of t cells and premature termination of the immune response against pathogens [45, 46] . the regulatory t (treg) cells are mostly cd4 + cells that express high levels of cd25 (alpha chain of the il-2 receptor), and their function is to regulate the activation of other t cells [43] . the treg cells depend on the transcription factor foxp3 and il-2 to maintain their function and produce cytokines, such as il-10 and tumour growth factor beta (tgf␤), that block the activation of lymphocytes and macrophages [43] . there are two different types of treg cells: the thymus-generated natural treg cells, which are related to the suppression of auto-immune t cells and the maintenance of tolerance to self-antigens, and the adaptive or induced treg cells, which regulate the response of cd4 + and cd8 + t cells to pathogens (e.g., bacteria, fungi, intracellular parasites, and viruses) and are activated in the peripheral lymphoid tissues [47] [48] [49] . the function of the induced treg cells is to avoid the tissue damage that is caused by excessive inflammation in response to pathogens [49, 50] . however, certain pathogens modulate the activity of these treg cells and thus cause persistent chronic infection [48] . mexas et al. [49] showed that treg cells (cd4 + cd25 + ) are the target of acute fiv infection since these cells exhibit high viral mrna levels two weeks after infection, which correlates with the peak of viraemia. the establishment of persistent chronic infection may be related to the infection of cd4 + cd25 + cells because these cells are activated during acute infection in the presence of the foxp3 transcription factor and tgf␤ and suppress the production of il-2, thus inhibiting the antiviral response of the helper t cells [48] . in cats, the cd4 + cd25 + cells are unable to proliferate in response to antigenic or mitotic stimulation due to the lack of il-2 production, and exhibit the ability to suppress the proliferation of the cd4 + cd25 − cells when activated [51] . in addition, during the disease progression, treg cells act as a reservoir of infection, where the virus might replicate at low rates without becoming a target of the immune response because these cells exhibit anergic characteristics [49] . although cats with chronic fiv infection exhibit the same proportion of cd4 + cd25 + cells as fiv-negative animals, the cd4 + cd25 + cells of fiv-positive cats exhibit a greater expression of b7.1 and b7.2 molecules in both the lymph nodes and the blood, as well as a greater ctla-4 expression in the lymph nodes [51] . the expression of these molecules explain the immunosuppression mechanism associated with the treg cells (cd4 + cd25 + b7 + ctla4 + ) that are activated by fiv during the progression of the disease. in addition to progressive immunosuppression, fivpositive cats also exhibit marked hyperactivation of b and t cells [7] . the total protein levels are increased in fiv-infected cats due to an increase in the gamma globulins [52] . in one study, two fiv-positive cats with b-cell lymphoma exhibited an increase in b lymphocytes, increased igg (from the beginning to the end of the follow-up period, which corresponded to 50-300 weeks pi), and a progressive increase in iga [53] . takano et al. [54] also observed higher globulin levels in fiv-positive cats at the terminal phase compared with animals in the asymptomatic phase or those that were fiv-negative. an increase in the levels of b cells with surface immunoglobulins (sig + ) and negative for cd21, which represent cells that are differentiated for antibody production, was observed in the asymptomatic cats and more evident in animals in the terminal phase, which suggests that this finding might be related to the emergence of immunosuppression signs [54] . the activation of b lymphocytes corresponds to a polyclonal and non-virus-specific activation because antibodies against heterologous non-viral antigens were detected six to eight weeks after infection, remained until the end of the study (90 weeks pi), and did not exhibit cross-reactivity with fiv viral recombinant antigens [55] . furthermore, neither the stimulation of b lymphocytes nor hypergammaglobulinemia were caused by co-infection with other pathogens because the study used specific pathogen free (spf) animals that were experimentally infected with fiv [55] . therefore, the hyperactivation caused by fiv is responsible for the appearance of immune-mediated diseases, such as chronic gingivitisstomatitis and glomerulonephritis, myeloproliferative neoplasms, and lymphomas [18] . the stimulation of cd4 + and cd8 + t cells starts during the acute phase of fiv infection and continues throughout the disease progression [7] . the increase in the number of activated cd8 + t cells (cd8␤ low cd62l − ) suggests that the naïve cd8 + t cells are replaced by the activated cd8 + t cells during fiv infection. the same phenomenon occurs with the cd4 + t cells, which reduce the expression of cd62l during the course of infection (reviewed in [7] ). in addition, and as mentioned above, the increased expression of b7 molecules and ctla-4 by activated cd4 + and cd8 + t cells might alter the host's immune response [45] . tompkins and tompkins [7] suggested that fiv infection activates cd4 + , cd8 + and treg (cd4 + cd25 + ) t cells. the activation of treg cells inhibits the production of il-2 and ifn␥ through the production of tgf␤ and thus reduces the immune response against fiv through the induction of anergy and apoptosis, which allows for continuous viral replication and chronic antigenaemia. in turn, chronic antigenaemia causes hyperactivation of the cd4 + /cd8 + t cells and treg cells, which results in anergy and apoptosis due to the activity of the treg cells and an inappropriate t-t cell interaction due to the binding of b7/ctla-4, which are expressed in larger amounts in activated t cells [7] . this uncontrolled hyperactivation of immune system causes progressive immunosuppression and a reduction in the immune response to secondary infections. major efforts have been devoted in recent years to the discovery of novel alternatives to the treatment, and perhaps even cure, of hiv infection. for that purpose, many studies seek to understand the factors that are involved in the natural resistance to hiv-1 infection that occurs in some individuals who, despite a history of frequent (sexual, parenteral, or vertical) exposure to the virus, do not exhibit any serologic evidence of infection or any sign of immunodeficiency [56] . these high-risk exposed but seronegative (esn) individuals seem to have genetic and immunological factors that reduce their susceptibility to infection, although a single factor individually may not account for this characteristic [57] . amongst the genetic factors involved in the slow progression of aids and resistance to hiv infection, the following have been identified: alleles of human leucocyte antigen (hla), the presence of activators or the absence of inhibitors of the genes encoding kir (killer immunoglobulin-like receptor) molecules, a genetic polymorphism of the chemokines rantes (regulated on activation normal t-cell expressed and secreted), macrophage inflammatory protein (mip)-1␣, and mip-1␤ and their receptors [56, 57] . amongst these alterations, the deletion of 32 base pairs of the gene encoding the ccr5 receptor (ccr5 32) is strongly correlated with resistance to hiv infection [58] . dean et al. [59] observed that approximately 10% of the studied population (n > 600), which included healthy controls, esn individuals, and hivpositive individuals with and without signs of aids, had the ccr5 32 allele. this mutation causes a change in the reading frame of amino acid 185 that generates a non-functional protein with a premature termination codon [58, 59] . individuals who are homozygous for the ccr5 32 allele are strongly protected against hiv infection because these individuals lack a functional ccr5 co-receptor on their cell surface [59, 60] . all of the ccr5 32/ 32 homozygous individuals were esn individuals, whereas none amongst the 1343 hiv-positive individuals exhibited this genotype [59] . in addition, ccr5 + / 32 heterozygous individuals exhibited some degree of resistance to infection; and when infected, their viral load is lower, and their progression of aids is slower compared with that observed in ccr5 + / + homozygous individuals [59] . there is still no evidence of any mutations in the fiv receptors that confer a natural resistance to infection in cats. currently, the following are known: the in vitro infectivity of fiv decreases in a large fraction of strains with the use of a feline/human hybrid cd134 receptor, the human cd134 receptor is not functional for fiv entry [61] , and mutations in the human cxcr4 co-receptor (d187a) prevent the in vitro entry of fiv [62] . nevertheless, although a deletion in the ccr5 coreceptor gene grants a high natural resistance to hiv infection, this mutation is not found in all esn individuals. therefore, other factors might also be involved. the host's innate immunity contributes to the protection against hiv infection mainly through the activity of nk cells and a large production of ifn␥. in addition, an increased production of ␤-chemokines (e.g., cc chemokine ligand (ccl)2, ccl3 (mip-1␣), ccl4 (mip-1␤), ccl5 (rantes), and ccl11), ␣-chemokines (stromal cell-derived factor-1 (sdf-1)), peroxiredoxin ii, cd8 + cell antiviral factor (caf), nk cell stimulatory factor b, il-22 (cytokine involved in the production of acute-phase proteins), defensins, and ribonucleases have been associated with protective effects [revised in 56, 57] . the innate immune response mediated by cellular restriction factors, such as apobec3 (apolipoprotein b mrna-editing catalytic polypeptide 3), trim5␣ (tripartite motif protein), and tetherin, inhibits the viral replication of retroviruses and thus blocks the establishment of infection after the virus has entered the host cell [57, 58] . apobec3 deaminates cytidine residues in rna and single-stranded dna, thereby converting cytosine (c) into uracil (u) or thymine (t), which alters the encoding of the viral proteins [8, 63] . according to recent observations in humans, exposure to hiv increases the expression of apobec3g, and esn individuals exhibit a higher expression of this protein compared with non-exposed individuals. in addition, apobec3g reduces the susceptibility to hiv infection in vitro [56] [57] [58] . in cats, the action of apobec3g was investigated with three exogenous feline retroviruses: fiv, felv, and feline syncytium-forming virus (fesfv), which is different from the first two viruses because it is considered non-pathogenic in nature [8] . felv exhibited reduced infectivity in vitro when apobec3 proteins were present, which might explain the occurrence of abortive and regressive infection in some of the cats that had contact with the virus [8, 64] . felv belongs to the genus gammaretrovirus and does not have the accessory genes vif and bet, which are found in species that belong to the genera lentivirus and spumavirus, respectively [33] , and are associated with the inactivation of apobec3 [65] . an exception is the equine infectious anaemia virus (eiav), which is the only lentivirus that lacks the vif gene, but it exhibits resistance to feline apobec3 [8] . several studies have shown that retroviruses are inhibited by the action of apobec3 in non-susceptible species, which explains the host specificity and enables its therapeutic use [63] . although apobec3 exhibits an antiviral effect, viruses have mechanisms that prevent the action of this protein in the natural host, and cause a persistent infection. the same is true with tetherins, which exert an antiviral action by preventing the release of viral particles. dietrich et al. [66] showed that the release of fiv and hiv-1 particles was inhibited by the transient expression of the feline tetherin. however, fiv replication did not decrease when the expression of tetherin was stable, which probably occurs in natural infection. furthermore, the cell-to-cell spread of the virus is facilitated by the increase in syncytium formation [66, 67] . according to some studies, the feline trim5␣ is truncated and does not exhibit any antiretroviral effect [66, 67] . therefore, further investigation of restriction factors, especially their importance in the prevention and treatment of infection, is warranted because orthologous restriction factors were shown to inhibit infection by retroviruses. although acquired immunity, both cellular and humoral, plays a crucial role in the resistance to hiv infection, there is not yet a consensus as to which mechanisms are truly protective. with regard to the cellular immune response in esn individuals, some reports indicate a strong anti-hiv response by non-cytotoxic cd8 + t cells, the recognition of a different epitope compared with seropositive individuals, and a greater th1 response that is characterised by an increased production of il-2 and ifn␥ and a reduced il-10 production [56, 57] . nevertheless, a low lymphocyte activation was also shown to reduce the susceptibility to hiv infection because lymphocyte activation is needed for viral replication and is associated with the pathogenesis of the disease [7, 56] . the resistance to infection might be related to the humoral immune response mainly through the production of anti-hiv immunoglobulin a (iga), which is present in cervicovaginal secretions, saliva, colostrum, and serum, and by the presence of autoantibodies against the cd4 receptor in esn individuals [56, 57] . in cats, anti-cd134 antibodies were observed in 63% (143/226) of fiv-positive animals, whereas these antibodies were not present in any of the spf animals (n = 107) and only in 0.44% (1/225) of the diseased cats, some of which were infected by feline herpesvirus (fhv), feline coronavirus (fcov), or felv but were fiv-negative [68] . in addition, the cats with anti-cd134 antibodies exhibited a lower viral load and a better state of health, which indicates the importance of these antibodies in the progression of disease [68] . the ability of animals to develop an immune response that is sufficiently robust to reduce fiv viral load, although unable to extinguish the fiv infection, may reflect an important factor for reducing the speed of disease progression. however, this factor might also increase the selective pressure and induce the accumulation of a greater number of mutations throughout the course of the infection because the virus has a greater need to adapt to survive in the host. in addition, the early reduction in the number of cd4 + t lymphocytes and the consequent decrease in the cd4 + /cd8 + ratio, which correlates with the emergence of immunosuppression signs, might be related to the presence of virus with mutations that favour its escape or occurs in cats unable to develop a response that restricts viral replication. few studies have examined the presence of factors that are involved in the natural resistance to fiv infection in cats. the identification of these factors may contribute to a better understanding of the various patterns of disease progression and help with the development of a fiv/hiv cure. the complexity of the virus-host interaction, particularly the mechanisms of viral persistence, justifies the difficulty in the development of effective vaccines and treatments. therefore, much of this interaction must still be investigated before a full understanding of fiv infection in cats and hiv infection in humans. 553 3. factors of natural resistance to lentivirus infection isolation of a tlymphotropic virus from domestic cats with an immunodeficiencylike syndrome lentivirus. in: ictvdb -the universal virus database feline immunodeficiency virus infection: an overview vaccination against the feline immunodeficiency virus: the road not taken nucleotide sequence and genomic organization of feline immunodeficiency virus transmission and immunopathogenesis of fiv in cats as a model for hiv lentivirus-induced immune dysregulation restriction of feline retroviruses: lessons from cat apobec3 cytidine deaminases and trim5alpha proteins nucleotide sequence of feline immunodeficiency virus: classification of japanese isolates into two subtypes which are distinct from non-japanese subtypes comparative study of the cell tropism of feline immunodeficiency virus isolates of subtype a, b and d classified on the basis of the env gene v3-v5 sequence genetic diversity of argentine isolates of feline immunodeficiency virus phylogenetic analysis of five portuguese strains of fiv phylogenetic analysis of vietnamese isolates of feline immunodeficiency virus: genetic diversity of subtype c occurrence of feline immunodeficiency virus infection in cats seroprevalence of feline leukemia virus and feline immunodeficiency virus infection among cats in north america and risk factors for seropositivity quality of different in-clinic test systems for feline immunodeficiency virus and feline leukemia virus infection retroviral infections of small animals feline immunodeficiency, abcd guidelines on prevention and management lessons from the cat: development of vaccines against lentiviruses clinical and pathological findings in feline immunodeficiency virus experimental infection a longitudinal study of feline immunodeficiency virus-specific cytotoxic t lymphocytes in experimentally infected cats, using antigen-specific induction vaccine protection against feline immunodeficiency virus: setting the challenge chemokine receptors and co-stimulatory molecules: unravelling feline immunodeficiency virus infection. veterinary use of cd134 as a primary receptor by the feline immunodeficiency virus structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody hiv-1 membrane fusion: targets of opportunity feline immunodeficiency virus targets activated cd4+ t cells by using cd134 as a binding receptor sequential cd134-cxcr4 interactions in feline immunodeficiency virus (fiv): soluble cd134 activates fiv env for cxcr4-dependent entry and reveals a cryptic neutralization epitope mechanism of feline immunodeficiency virus envelope glycoprotein-mediated fusion the hiv coreceptors cxcr4 and ccr5 are differentially expressed and regulated on human t lymphocytes expression of cxcr4 on feline peripheral blood mononuclear cells: effect of feline immunodeficiency virus infection feline immunodeficiency virus env gene evolution in experimentally infected cats virologia veterinária. 1st ed. santa maria: editora da ufsm a single site for n-linked glycosylation in the envelope glycoprotein of feline immunodeficiency virus modulates the virus-receptor interaction adaptation of feline immunodeficiency virus subtype b strain tm2 to a feline astrocyte cell line (g355-5 cells) progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus cytokine production by cats infected with feline immunodeficiency virus: a longitudinal study constitutive expression of types 1 and 2 cytokines by alveolar macrophages from feline immunodeficiency virus-infected cats failure of fiv-infected cats to control toxoplasma gondii correlates with reduced il2, il6, and il12 and elevated il10 expression by lymph node t cells effect of feline immunodeficiency virus on cytokine response to listeria monocytogenes in vivo. veterinary cytokine modulation of the innate immune response in feline immunodeficiency virus-infected cats multivariate statistical analyses demonstrate unique host immune responses to single and dual lentiviral infection imunologia básica: funç ões e distúrbios do sistema imunológico spontaneous programmed cell death (pcd) process of lymphocytes of fiv-infected cats: cellular targets and modulation feline immunodeficiency virus infection is characterized by b7+ctla4+t cell apoptosis a4+t cells engage in t-t cell interactions that mediate apoptosis: a model for lentivirus-induced t cell depletion natural versus adaptive regulatory t cells the role of cd4+cd25+ regulatory t cells in viral infections cd4+cd25+ regulatory t cells are infected and activated during acute fiv infection regulatory cells and infectious agents: détentes cordiale and contraire feline immunodeficiency virus infection phenotypically and functionally activates immunosuppressive cd4+cd25+t regulatory cells hematology and serum biochemistry of feline immunodeficiency virus-infected and feline leukemia virusinfected cats feline immunodeficiency virus (fiv)-associated lymphoma: a potential role for immune dysfunction in tumourigenesis comparative study of the plasma globulin level, cd21(−) b-cell counts and foxp3 mrna expression level in cd4(+) t-cells for different clinical stages of feline immunodeficiency virus infected cats polyclonal jarrett o. b-cell activation in cats infected with feline immunodeficiency virus immunological factors involved in natural resistance to hiv-1 infection host factors involved in low susceptibility to hiv infection determinants of protection among righ risk hiv seronegative persons: an overview genetic restriction of hiv-1 infection and progression to aids by a deletion allele of the ckr5 structural gene, hemophilia growth and development study the evolutionary history of the ccr5-delta32 hiv-resistance mutation mapping the domains of cd134 as a functional receptor for feline immunodeficiency virus effect of mutations in the second extracellular loop of cxcr4 on its utilization by human and feline immunodeficiency viruses restrictions to cross-species transmission of lentiviral infection gleaned from studies of fiv functions, structure, and read-through alternative molecular and functional interactions of cat apobec3 and feline foamy and immunodeficiency virus proteins: different ways to counteract host-encoded restriction feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection cellular restriction factors of feline immunodeficiency virus improved health and survival of fiv-infected cats is associated with the presence of autoantibodies to the primary receptor this work was supported by the são paulo research foundation (fapesp) grant 2009/52979-5. key: cord-287018-g4y5kjju authors: konstantinova, p; de vries, w; haasnoot, j; ter brake, o; de haan, p; berkhout, b title: inhibition of human immunodeficiency virus type 1 by rna interference using long-hairpin rna date: 2006-05-18 journal: gene ther doi: 10.1038/sj.gt.3302786 sha: doc_id: 287018 cord_uid: g4y5kjju inhibition of virus replication by means of rna interference has been reported for several important human pathogens, including human immunodeficiency virus type 1 (hiv-1). rna interference against these pathogens has been accomplished by introduction of virus-specific synthetic small interfering rnas (sirnas) or dna constructs encoding short-hairpin rnas (shrnas). their use as therapeutic antiviral against hiv-1 is limited, because of the emergence of viral escape mutants. in order to solve this durability problem, we tested dna constructs encoding virus-specific long-hairpin rnas (lhrnas) for their ability to inhibit hiv-1 production. expression of lhrnas in mammalian cells may result in the synthesis of many sirnas targeting different viral sequences, thus providing more potent inhibition and reducing the chance of viral escape. the lhrna constructs were compared with in vitro diced double-stranded rna and a dna construct encoding an effective nef-specific shrna for their ability to inhibit hiv-1 production in cells. our results show that dna constructs encoding virus-specific lhrnas are capable of inhibiting hiv-1 production in a sequence-specific manner, without inducing the class i interferon genes. supplementary information: the online version of this article (doi:10.1038/sj.gt.3302786) contains supplementary material, which is available to authorized users. rna silencing or rna interference (rnai) is an evolutionary conserved sequence-specific post-transcriptional gene regulation mechanism that plays an important role in cell differentiation and development. [1] [2] [3] in addition, rnai serves as a defence mechanism against invading viruses and transposons. [4] [5] [6] rna interference is triggered by double-stranded rna (dsrna) molecules, which are processed in the cytoplasm by the dsrnaspecific endonuclease dicer into 19-24 nucleotides (nt) small interfering rnas (sirnas) or micro-rnas (mirnas). 7 these si/mirnas are incorporated into the multiprotein rna-induced silencing complex (risc) that guides the recognition and ultimately the cleavage or translational repression of complementary singlestranded rna, such as messenger rna or viral genomic rna. [8] [9] [10] rna interference has been employed to inhibit the replication of a wide range of viruses, including the human immunodeficiency virus type 1 (hiv-1), hepatitis c virus (hcv), hepatitis b virus (hbv), dengue virus, poliovirus, influenza virus a, coronaviruses, herpesviruses and picornaviruses. 11 human immunodeficiency virus type 1 virions contain a single-stranded rna genome that is a putative rnai target. after entry into a host cell the genomic rna is reverse transcribed into dsdna, which is integrated into the host chromosomal dna. newly synthesized unspliced genome-length and spliced subgenomic viral rnas are possible targets for rnai in the cytoplasm. it has recently been reported that hiv-1 encodes a suppressor of rnai, the tat protein, indicating that hiv-1 replication is controlled by rnai in human cells. 12 due to its sequence specificity, rnai is a potentially powerful and selective method for intracellular immunization against hiv-1 infection. rna interference-mediated suppression of hiv-1 replication has been accomplished by synthetic sirnas in a transient manner [13] [14] [15] [16] and by short-hairpin rna (shrna)-expression vectors in stably transfected cells. [17] [18] [19] despite potent inhibition, the use of sirna/shrna as a therapeutic antiviral is limited, because of the rapid emergence of hiv-1 escape mutants. [19] [20] [21] minor sequence changes in the target sequence, sometimes even a single point mutation, are sufficient to abrogate the inhibition of virus replication. strategies to reduce the chance of viral escape include the simultaneous use of multiple sirnas 22, 23 or the use of long-hairpin rna (lhrna, a single-hairpin molecule) or long dsrna (two complementary molecules that form a duplex). 16, 24 another possibility is the use of mirna-based approaches, which do not require perfect sequence complementarity. 25, 26 several reports describe efficient rnai induction by lhrna and long dsrna as in vitro generated transcripts that are transfected into cells or as gene constructs that produce the transcripts intracellularly. transfection of pre-implantation mouse embryo cells, undifferentiated embryonic stem cells and embryonic carcinoma cells with in vitro synthesized long dsrna confers specific gene silencing. 27, 28 however, exposure of non-embryonic mammalian cells to dsrnas longer than 30 basepairs (bp) leads to rapid induction of a specific set of cytokines, including the class i interferons (ifns). 29 during natural virus infections, the ifn response is activated by virusproduced dsrnas, and acts as an innate defense mechanism. viruses counter this response by encoding ifn antagonists, which are also responsible for the fact that antiviral ifn therapy is often not successful. 30, 31 so far, virus-encoded rnai suppressor factors, like the hiv-1 tat protein, do not appear to be able to suppress induced antiviral rnai. strong induction of rnai by intracellular expression of virus-specific dsrnas is likely to outcompete the inhibiting effects of rnai suppressors. efficient rnai-mediated gene silencing has been shown in mammalian cells by endogenously expressed long dsrnas. 28, 32, 33 in chinese hamster ovary (cho) cells, a dna construct encoding a 700 bp long dsrna specifically inhibits luciferase expression in a sequencespecific manner. 34 complete and specific gene silencing was achieved in different mammalian cell types by expression of 500, 800, or even 1000 bp long dsrnas. 32, [35] [36] [37] interestingly, intact dsrna could not be detected in these cells, suggesting that it is rapidly processed by dicer in the cytoplasm. recently, ski knockdown mice have been produced using a dna construct encoding long dsrna-specific for the murine ski gene. 38 these results suggest that dsrna is tolerated in mammalian cells, most likely because it is rapidly processed by the rnai machinery. several antiviral approaches using extended dsrna have been reported in plant and insect cells lacking the innate antiviral ifn response. although plants and insects lack the ifn response, they also have potent innate antiviral responses, comparable to those in mammals. 39 transient expression of dna constructs encoding virus-specific dsrna in plant protoplasts or insect cells partially protects the cells from infection by the homologous virus. 40, 41 stable expression of such constructs in plant or insect cells renders the cells completely resistant or immune to infection. 42, 43 in vitro made long dsrnas have been used to inhibit hiv-1 production under certain conditions without induction of the ifn response. 16, 24 we have previously demonstrated potent inhibition of hiv-1 replication in t cells that stably express an shrna targeted to viral nef gene sequences. 19 to test whether endogenously expressed lhrna and long dsrna can inhibit hiv-1 at least as potently as sh-nef, we constructed and tested a series of dna constructs. it has previously been demonstrated that hiv-1 replication can be inhibited by sirnas and shrnas directed against viral targets. 11, 14, 15, 18, 19, 44 most of the active sirnas against hiv-1 are targeted to the early regulatory tat, rev and nef genes. 19, 20, 45, 46 interference with an early stage of the hiv-1 replication cycle may be beneficial. for this reason, the dna constructs encoding lhrnas (a single-hairpin molecule) and long dsrnas (two complementary molecules that form a duplex) were designed to target tat, rev and nef sequences as indicated in figure 1 . inhibition of human immunodeficiency virus type 1 by in vitro transcribed ds-nef2 rna and its diced product we initially tested whether in vitro transcribed and annealed nef2 dsrna and its in vitro diced product si-nef2, a mixture of nef-specific sirnas, can inhibit hiv-1. nef2 dsrna of 300 bp was diced in vitro to create si-nef2 rnas of approximately 21 bp (figure 2a) . we cotransfected 500 ng of the hiv-1 molecular clone plai with and without 10 ng inhibitory rna in human embryonic kidney (hek) 293t cells. dna of prl expressing renilla luciferase was included in the transfection mixtures to monitor cell viability and possible non-specific effects, for example, due to ifn induction by dsrna. virus production was measured by ca-p24 enzyme-linked immunosorbent assay (elisa) in the culture supernatant 3 days after transfection. the amount of virus production without an inhibitory rna, generally in the 50-250 ng/ ml ca-p24 range, was set at 100%. nef2 dsrna induced a significant decrease in ca-p24 production, but even more pronounced level of inhibition was obtained with diced si-nef2 ( figure 2b ). this can be explained by the fact that si-nef2 bypasses the intracellular dicing step, which may be a limiting factor in the rnai pathway. one of the hallmarks of the rnai is its sequence specificity. therefore, we tested if nef2 dsrna and its in vitro diced product si-nef2 would inhibit pgl3-nef reporter, in which 250 nt from the nef2 target sequence was placed downstream of the luciferase reporter gene. 21 nef2 dsrna induced a decrease in luciferase expression, but an even more pronounced level of inhibition was figure 1 scheme of the human immunodeficiency virus type 1 (hiv-1) plai proviral genome and target sequences used for the design of long-hairpin rnas (lhrnas). the target sequences are indicated as bars below the hiv-1 coding regions. lhrna (300 basepairs (bp)) tat fuses tat exon 1 (gray bar, 5422-5626) and tat exon 2 (black bar, 7972-8017) sequences, rev fuses rev exon 1 (gray bar, 5562-5626) and rev exon 2 (black bar, 7972-8206) and nef1 contains nef-ltr sequences (8519-8818). double-stranded rna nef2 is a duplex of two separate, complementary sense and antisense nef sequences (8416-8695). the positive control sh-nef is a 21-bp hairpin consisting of nef sequences (8552-8571). 19 inhibition of hiv-1 by lhrna p konstantinova et al obtained with diced si-nef2 ( figure 2c ). the prl expression was not influenced (results not shown). in fact, in vitro diced si-nef2 is a much more effective inhibitor than the in vitro synthesized short-hairpin inhibitor sh-nef rna, which was included as a positive control. we next tested the inhibitory potential of nef2 dsrna in the 1-1000 ng range. at amounts above 10 ng, non-specific decrease of renilla reniformis luciferase (rl) expression was observed, which is most likely due to ifn induction (results not shown, see also figure 8 ). the high level of inhibition obtained with low amounts of dsrna convinced us to design and test a series of dna expression plasmids encoding long hiv-1-specific dsrnas. low-level inhibition of human immunodeficiency virus type 1 by long-hairpin rna expression plasmids to make lhrna constructs, we cloned the hiv-1 tat, rev and nef gene sequences as inverted repeats under the transcriptional control of the constitutive ef1a promoter ( figure 3 ). these vectors should produce lhrna, a longhairpin structure consisting of an approximately 300 bp stem and a 46 nt loop. during transcription of the inverted sequences, an rna molecule is made, which folds back on itself to form a hairpin structure with a stem of approximately 300 bp. in silico rna analysis with the mfold program 47 confirms the folding of these extended hairpins (data not shown). we tested inhibition of hiv-1 in several mammalian cell lines (c33a, hek 293t and vero). cotransfection of plai with the pef1atat, pef1a-rev or pef1a-nef1 vectors resulted in marginal inhibition of hiv-1 production in c33a and hek 293t cells, and only the pef1a-tat vector was inhibitory in vero cells ( figure 4a ). we also designed a control pef1a-green fluorescent protein (gfp) plasmid that expresses a similar extended lhrna against gfp mrna. no inhibition of virus production was observed with the control pef1a-gfp vector, thus providing additional evidence for the specificity of lhrna-mediated inhibition of hiv-1 production. we previously demonstrated potent inhibition of hiv-1 replication in t cells that stably express an shrna targeted to viral nef gene sequences. 19 therefore, if lhrna inhibits hiv-1 potently, it should be at least as active as the sh-nef control. unlike the results with in vitro synthesized nef2 dsrna, either diced or not, all lhrna constructs (tat, rev and nef1) were much less potent inhibitors than the control sh-nef construct, which produces the short hairpin from a polymerase iii promoter. because pef1a-tat and pef1a-nef1 were slightly more effective than pef1a-rev in c33a and 293t cells, we focused on these lhrnas in the subsequent experiments. in order to avoid innate viral responses or possible other side effects and to obtain controllable expression of lhrna, we placed the expression of the lhrnas tat and nef1 under control of inducible promoters: (i) the doxycycline (dox) inducible tet system 48 and (ii) the tat-inducible long-terminal repeat (ltr) promoter/enhancer of hiv-1 49 (figure 3 ). the latter system seems ideally suited to restrict lhrna expression to cells that are infected by hiv-1, thus providing a unique safety feature. furthermore, we replaced the 46 bp spacer between the repeats by the 1000 bp ef-1a intron, since it has been shown that this improves the inhibitory potential of lhrna-encoding dna constructs. 50 the tet-on system is based on the specific, highaffinity binding of the rtta trans-activator to the tet operator (teto) in the presence of dox, triggering transcription of downstream genes. this system has recently been used to express synthetic mirna precursors in mouse or human genomes, following transinhibition of hiv-1 by lhrna p konstantinova et al duction with retroviral or lentiviral vectors. 51 vector plai was cotransfected with p7teto-tat or p7teto-nef1 ( figure 3 ). we also included pcmv-rtta and the control prl, and dox was added 4 h later. p7teto-tat or p7teto-nef1 conferred no or very poor inhibition of hiv-1 as compared to the sh-nef control (figure 4b ). the potency of the lhrnas was not increased by varying the ratio of lhrna-expression vector to pcmv-rtta or the dox concentration. moreover, a non-specific decrease of rl expression was observed at higher pcmv-rtta concentrations, which is probably due to promoter squelching (results not shown). in order to restrict lhrna expression to cells that are infected with hiv-1, the inverted tat and nef1 repeats were cloned downstream of the tat-inducible ltr promoter/enhancer of hiv-1, resulting in plasmids pltr-tat and pltr-nef1 ( figure 3) . a tat-inducible pol ii promoter expressing anti-hiv shrna has been described for inhibition of hiv-1 gene expression in mammalian cells. 52 in this setting, lhrna or shrna expression is activated in trans by the tat protein encoded by hiv-1. to avoid self-targeting, we deleted a large part of the u3 region (up to position à179) in the ltr promoter of both ltr expression plasmids that overlaps with the nef coding domain (323 nt). human embryonic kidney 293t cells were cotransfected with plai and pltr-tat or pltr-nef1. both expression plasmids inhibit virus production by approximately 60% (figure 5a ). the poor inhibitory potency of the different lhrna constructs could be due to expression problems, but the lhrna may also encounter difficulties in entering or proceeding along the rnai pathway. in order to increase the efficacy of the lhrna molecules, we cloned the hiv-1 leader sequence (c) between the ltr and nef1 in pltrc-nef1 ( figure 3 ). control plasmid pltrasc-nef1 was made with the c element inserted in antisense orientation. previously, we reported that sequences from the 5 0 untranslated leader of the hiv-1 genome, such as the rna dimerization signal, can be used to inhibit hiv expression in trans. 53 we presumed that c should bring the antiviral rna along viral pathways, conferring a stronger inhibitory effect. indeed, in hek 293t cells transfected with plai and pltrc-nef1, hiv-1 production was almost completely inhibited ( figure 5a ). the level of inhibition conferred by pltrc-nef1 is comparable to that of the positive control sh-nef. inhibition is specific and not due to a more general cell toxicity problem because no significant decrease in rl expression was observed (results not shown). control plasmid pltrasc-nef1 was much less effective in inhibiting hiv-1 production, with a potency comparable to that of the original pltr-nef1 construct. plasmid pltrc, in which the nef1 sequence has been deleted, failed to inhibit hiv-1 production (figure 5b ). this result indicates that the presence of the c element enhances lh-nef1-mediated inhibition of hiv-1, but the presence of the nef1 sequence is essential for the potent effect of pltrc-nef1. transcript c-nef1 expression from the ltr promoter is induced by plai-encoded tat protein. because plai gene expression is strongly inhibited by pltrc-nef1, a negative feedback loop may have been established, which leads to an underestimation of the inhibitory potential of pltrc-nef1. we therefore added a tatexpression plasmid (pcdna3-tat) in trans to secure pltrc-nef1 expression. human embryonic kidney 293t cells were transfected with 100 ng plai and 10-100 ng pltrc-nef1 with or without pcdna3-tat. as shown in figure 5c , pronounced hiv-1 inhibition was obtained with as little as 10 ng pltrc-nef1. the presence of figure 3 expression vectors for long-hairpin rna (lhrna), double-stranded rna (dsrna) and short-hairpin rna (shrna). longhairpin rnas were created by cloning the 300-nucleotide (nt) inverted repeats from tat, rev and nef1 (see figure 1 ) downstream of the ef1a, 7teto or long-terminal repeat (ltr) promoters. the 1 kb ef1a intron is positioned downstream of the ef1a promoter. a schematic representation of the final hairpin structures is shown on the right. in pef1a constructs, the two complementary rna strands are separated by a 46 nt loop. in the p7teto and pltr constructs, the complementary sequences are separated by a 1 kb spacer that contains splice donor and acceptor sites. vector pltrc-nef1 is a derivative of pltr-nef1 in which the human immunodeficiency virus type 1 (hiv-1) leader sequence (c, 76-630, marked as a gray box) was inserted. the predicted transcript will have the c domain upstream of the rna hairpin. all transcripts contain a polyadenylation signal (pa) downstream of the hairpin sequences. vector pt7-nef2 has 300 basepairs (bp) long doublestranded nef sequences flanked by t7 promoters (t7) and terminators (f) at both 5 0 and 3 0 ends. two separate complementary rna chains, potentially capable of forming dsrna, are transcribed from the convergent promoters by t7 rna polymerase (encoded by expression plasmid pt7-pol). vectors pt7sh-nef and ph1sh-nef express sh-nef from the t7 and h1 promoters, respectively. inhibition of hiv-1 by lhrna p konstantinova et al additional tat did not significantly improve the inhibition conferred by pltrc-nef1, indicating that the background ltr promoter activity produces sufficient amounts of the inhibitory transcript. we next wanted to test if the potent pltrc-nef1 construct was able to inhibit hiv-1 variants that escaped from the sh-nef inhibitor. we described previously a series of viral escape variants with mutations or deletions in the targeted nef sequence. 19 we selected mutant r1 with a 106 nt deletion that includes the complete sh-nef target sequence and mutant r3 0 with two point mutafigure 4 marginal inhibition of human immunodeficiency virus type 1 (hiv-1) production by pef1a-and p7teto-driven longhairpin rna (lhrna) constructs. (a) cells (c33a, human embryonic kidney (hek) 239t and vero) were lipofectamine-transfected with 500 ng plai, 500 ng inhibitory construct, 3 ng pcmv-rtta and 2.5 ng prl. vector pef1a-green fluorescent protein (gfp) was used as a control expressing an irrelevant lhrna against gfp. vectors ph1sh-nef and the empty vector were used as negative and positive controls, respectively. virus production was determined as described in the legend to figure 2 . standard error bars represent the means of three independent experiments. the sh-nef control construct was not tested (n.t.) in c33a cells. (b) hek 239t cells were cotransfected with 100 ng plai, 100 ng p7teto-tat, p7teto-nef1 or ph1sh-nef, 3 ng pcmv-rtta and 2.5 ng prl as an internal control. culture medium was refreshed after 16 h and 1 mg/ml doxycycline was added. virus production was determined as described in the legend to (table 1) . human embryonic kidney 293t cells were cotransfected with 200 ng hiv-1 molecular clone plai (wild-type, r1 or r3 0 ) and 40 ng pltrc-nef1 or ph1sh-nef plasmid. only the wild-type plai construct was equally inhibited by the pltrc-nef1 and sh-nef constructs. the deletion mutant r1 is completely resistant to sh-nef, but was potently inhibited by pltrc-nef1. mutant r3 0 partially escapes from sh-nef, but is potently inhibited by pltrc-nef1. rna interference is mainly a cytoplasmic process, 54 and putative problems in nuclear export of lhrnas may thus hamper their inhibitory activity. in order to test whether there is a difference between the rnai-inducing capacity of dsrnas produced in the nucleus or cytoplasm of cells, we expressed dsrna targeted to the nef-coding domain (nef2 in figure 1 ) using the cytoplasmic t7 rna polymerase system. to generate pt7-nef2, we cloned a 300 bp nef-specific dna fragment between convergent t7 promoters and terminators ( figure 3 ). in addition, the control expression plasmid pt7sh-nef was constructed. cytoplasmic transcription of pt7-nef2 and pt7sh-nef is performed by t7-polymerase encoded by pt7-pol. expression of the firefly luciferase (fl) reporter from the t7 promoter (pt7-luc) was measured as a positive control in all transfection experiments with pt7-pol ( figure 6a ). human embryonic kidney 293t cells were transfected with plai and pt7-nef2 or pt7sh-nef, with and without pt7-pol. virus production was measured in the culture supernatant 3 days after transfection by ca-p24 elisa. in the absence of t7-polymerase, pt7-nef2 and pt7sh-nef confer a low level of inhibition on virus production probably due to leaky rna expression from the plasmids (figure 6b ). with t7-polymerase, pt7-nef2 and pt7sh-nef confer up to 90% inhibition of hiv-1 production (figure 6b ). this level of inhibition is comparable to that obtained with the sh-nef control expression plasmid. renilla luciferase expression was not affected (figure 6b, gray bars) . to demonstrate that the effect of pt7-nef2 is sequence-specific, we cotransfected it with the pgl3-nef reporter (figure 6c ). in the presence of t7-polymerase, pt7-nef2 potently inhibited luciferase expression. we next tested the inhibitory potential of both pt7-nef2 and pt7sh-nef plasmids in the presence of an increasing amount (5-100 ng) of pt7-polymerase. the inhibition was consistently strong even at low amounts (5 ng) of the pt7-pol plasmid (figure 6d ). when more than 100 ng pt7-pol was used in the transfection experiments, a non-specific decrease in rl reporter expression was observed. a high amount of t7-polymerase accumulating in the cytoplasm of cells may be toxic or high amounts of dsrna accumulating in the cytoplasm of cells may induce the innate antiviral ifn response. to measure the rna expression levels of selected lhrna-expression constructs, we performed a reverse transcriptase-polymerase chain reaction (rt-pcr) assay with nef-specific primers. all constructs expressed nef rna (figure 7) . notably, pt7-nef2 produced low amounts of rna even in the absence of the pt7pol inducer. this can explain the slight inhibition of gene expression obtained with pt7-nef2 in the absence of pt7pol (figure 6b and c) . no nef-specific fragment was detected in mock-transfected cells or in cells transfected with poly(i:c). endogenously expressed long-hairpin rna and long double-stranded rna do not induce the interferon response it has been reported that introduction of dsrnas longer than 30 bp in mammalian cells induces the innate antiviral ifn response. we determined ifn-b induction by rt-pcr in hek 293t cells upon transfection with in vitro made nef2 dsrna and dna constructs encoding pef1a-nef1, pltr-nef1, pltrc-nef1, pt7-nef2 or ph1shnef. transfection of 1 mg poly(i:c), a known inducer of ifn and other cytokines, was used as a positive control. the lhrna-and sh-nef-expression plasmids did not induce detectable levels of ifn-b mrna (figure 8 ). both nef2 dsrna and poly(i:c) controls induced high amounts of the ifn-b mrna. the use of synthetic sirnas or shrna-expression plasmids as inducers of rnai-based therapy against hiv-1 faces the major obstacle of the emergence of virus escape variants. similar to the combined use of antivirals in highly active antiretroviral therapy (haart), one could design an rnai therapy with extended lhrna/ dsrna. endogenous expression of two long complementary rnas, with the potential to form an extended dsrna duplex, leads to specific suppression of gene expression in mammalian cells. 32, 34, 35, 37 interestingly, fulllength dsrnas could not be detected, suggesting that inhibition of hiv-1 by lhrna p konstantinova et al processing by dicer precludes their accumulation in the cytoplasm. long dsrnas are indeed processed in vitro by the rnai machinery into multiple active sirnas. 55, 56 several lines of evidence show that lhrnas induce gene silencing by the rnai mechanism. inhibition of dicer abrogated the gene silencing induced by lhrna against gfp, indicating that silencing was mediated by rnai. 32 these results suggest that long dsrna is well tolerated in mammalian cells, most likely because it is processed rapidly by the rnai machinery. it is also relevant to mention that mammalian cells may naturally produce dsrna derived from repetitive and transposable elements. 57, 58 a recently performed bioinformatics study revealed the presence of at least 4520 full-length transcripts, which form sense-antisense gene pairs in the human genome. 59 we designed a set of anti-hiv-1 lhrna/dsrnaexpression constructs and compared their ability to inhibit virus production with a very potent shrnabased inhibitor that we described previously. 19 ideally, a single lhrna should provide more potent inhibition of hiv than an shrna. an additional advantage of lhrna is that it does not require predetermination of optimal shrnas and hiv-1 target sequences because multiple effective sirnas will be produced. a potential disadvantage of the use of lhrna as therapeutic is that the multiple sirnas are more likely to cause off-target effects. (a) pt7-luc (100 ng) construct was cotransfected in human embryonic kidney (hek) 293t cells with or without 30 ng pt7-pol. at 2 days after transfection, cells were lysed and the expression of firefly luciferase was measured. (b) pt7-nef2 (100 ng) and pt7sh-nef vectors were linearized 3 0 of the t7 termination signal with xbai and bamhi, respectively, and cotransfected with 100 ng plai, 30 ng pt7-pol and 2.5 ng prl in hek 293t cells. two separate complementary rna chains, potentially forming dsrna, are transcribed in the cell from convergent t7 promoters. equal amounts of a ph1sh-nef expression vector and the empty vector were added as positive and negative controls, respectively. cotransfections were also performed without pt7-pol to check for non-specific effects of the t7 plasmids. virus production was determined as described in the legend to figure 2 . standard error bars represent the means of four independent experiments. cells were lysed 2 days after transfection to measure renilla luciferase. (c) sequence-specific inhibition of the pgl3-nef reporter, containing the 250-nucleotide (nt) nef2 target sequence downstream of the luciferase coding domain, by pt7-nef2. hek 239t cells were cotransfected with 100 ng of pgl3-nef, 100 ng pt7-nef2, 30 ng pt7-pol and 2.5 ng prl. ph1sh-nef (10 ng) expression vector and the empty vector were added as positive and negative controls, respectively. (d) titration of t7 polymerase. plai (100 ng) was cotransfected with increasing amounts (0-3-10-30-100 ng) of pt7-pol and 100 ng pt7-nef2 or pt7sh-nef. inhibition of hiv-1 by lhrna p konstantinova et al long-hairpin rnas expressed from constitutive (ef1a) and inducible (7teto, hiv-1 ltr) promoters inhibited hiv-1 production marginally, but we demonstrated potent and specific hiv-1 silencing with modified dna constructs. the most active constructs either link the lhrna to viral rna sequences (c) or express the long dsrna directly in the cytoplasm, suggesting that translocation of dsrna from the nucleus to the cytoplasm is a crucial step for these molecules to enter the cytoplasmic rnai pathway. it is widely accepted that rnai is a cytoplasmic process, as most protein components of the rnai pathway, including argonaute 2, trbp and dicer, assemble and function in the cytoplasm. 54, [60] [61] [62] [63] [64] putative problems in nuclear export of lhrna may thus hamper its inhibitory activity. modification of the lhrna by addition of the hiv-1 leader sequence (c) or direct cytoplasmic expression of long dsrna by the t7 polymerase created potent inhibitors. the viral c sequence may provide the transcript with a non-self signature and thereby boost rnai. it has previously been suggested that the inhibitory effect of c-containing transcripts is sequence-specific, possibly due to premature formation of rna dimers, 53 but the exact mechanism of inhibition remains to be elucidated. a major advantage of an lhrna inhibitor over shrna is the reduced chance of viral escape because a larger segment of the viral genome is targeted, as has been shown recently. 65 as an initial test of this idea, we used two hiv-1 mutants that escaped from sh-nef by two point mutations or complete deletion of the target sequence. 19 these escape mutants could be inhibited by the lhrna construct pltrc-nef1, confirming the hypothesis that lhrna may delay or prevent the evolution of hiv-1 escape variants. we showed that endogenously expressed lhrna and dsrna do not activate the innate antiviral response. a similar result has recently been described in literature. 33, 66 exposure of cells to a 50 bp in vitro synthesized dsrna induces the production of class i ifns, but not when such molecules are expressed in the cell from a dna construct with the u6 promoter. modification of lhrnas expressed from a pol iii promoter by inclusion of multiple g:u wobbles induced rnai without any non-specific effects. 66 in fact, most reports on ifn induction by long dsrnas in mammalian cells are based on transfection of cells with in vitro synthesized dsrnas. 40, 67 apparently, endogenously produced dsrna is less active than exogenous dsrna in inducing the ifn response. this finding may have a major impact on the further development of rnai-based antiviral strategies. rna interference-based gene therapy against hiv-1 seems to be a viable option, either as mono-therapy or combined with traditional drug therapy. 22, 68 the outgrowth of rnai-resistant virus mutants presents a major obstacle for all sequence-specific inhibitory strategies. the simultaneous targeting of multiple conserved targets by lhrna may confer increased robustness to future rnai therapies. we will focus on optimization of the dna constructs encoding hiv-1-specific lhrnas, for example, the type of promoter used and the structure of the lhrna. we will also test stable expression of these constructs in hiv-susceptible cells. these cell lines will be extensively tested for the emergence of rnai-resistant virus variants. figure 7 in vivo rna expression from long-hairpin rna (lhrna) constructs. hek 293t cells were transfected with 500 ng of the indicated lhrna/double-stranded rna (dsrna)-expression constructs using lipofectamine 2000. constructs pltr-nef1 and pltrc-nef1 were cotransfected with 30 ng ptat. construct pt7-nef2 was cotransfected with 100 ng pt7-pol. the puc19 plasmid and poly (i:c) were used as negative controls and pt7-nef2 plasmid dna was used as a positive control. total rna was isolated from the cells 48 h after transfection. the nef expression level was determined by reverse transcriptase-polymerase chain reaction (rt-pcr) with primers nef-b/x and antiu3-att, which create a 110 basepairs (bp) amplification product. b-actin mrna expression was analyzed as an internal control. pt7-nef2 rtà is a control reaction without rt step. 293t cells (1.5 â 10 5 ) were cotransfected with plai and the indicated lhrna/double-stranded rna (dsrna)-expression constructs using lipofectamine 2000. construct pt7-nef2 was cotransfected with pt7-pol. renilla luciferase (prl) was used as an internal control. the puc19 plasmid was used as a negative control. in vitro transcribed ds-nef2 rna and poly (i:c) act as positive controls for ifn-b induction. two separate transfections were performed, which were processed for either ifn-b mrna expression or renilla measurement. no significant differences in renilla expression were measured, except for the toxic treatment with ds-nef2 rna and poly(i:c) (results not shown). in addition, we measured ca-p24 in the supernatant, which showed the inhibition characteristics described earlier. total rna was isolated from the cells 24 h after transfection. the ifn-b expression level was determined by reverse transcriptase-polymerase chain reaction (rt-pcr). b-actin mrna expression was analyzed as an internal control. puc19 rt-and poly (i:c) rt-are control reactions without rt step. inhibition of hiv-1 by lhrna p konstantinova et al the full-length hiv-1 molecular clone plai 69 was used to produce wild-type virus and to study inhibition by lhrnas directed against the tat, rev or nef sequences. a detailed description of the construction of all lhrnaexpression constructs is available as supplementary information. plasmid pcdna3-t7pol, expressing bacteriophage t7 polymerase (pt7-pol, a kind gift of dr jean-marc jacque, university of massachusetts medical school, worcester, ma, usa), pgl3-nef and ph1sh-nef have been described previously. 14, 19 the mfold program 47 (http://www.bioinfo.rpi.edu/ applications/mfold) was used to check the correct folding of extended hairpins. in vitro transcription and dicing of ds-nef2 rna the ds-nef2 rna was in vitro transcribed with the megashortscript t7 transcription kit (ambion, austin, tx, usa) from the nef2 pcr template that contains convergent t7 promoters and terminators. the sh-nef rnai inducer 19 was transcribed in vitro from the bamhilinearized pt7sh-nef vector. ds-nef2 rna (1 mg) was diced in vitro at 371c for 18-20 h using recombinant dicer enzyme (stratagene, cedar creek, tx, usa). the fulllength ds-nef2 rna, the cleaved si-nef2 (a mix of sirnas derived from ds-nef2) and sh-nef were purified through microspin g-25 column (amersham biosciences, piscataway, nj, usa). to remove undigested dsrna from the dicing reaction, the si-nef2 was purified further on a microcon ym-100 column (millipore, billerica, ma, usa). si-nef2 rna was analyzed on 2% metaphor (bma, sanver tech, the netherlands) alongside a 20 bp dna marker (gensura, san diego, ca, usa). human embryonic kidney 293t, c33a cervix carcinoma and african green monkey kidney vero cells were grown as a monolayer in dulbecco's modified eagle's medium (dmem) (gibco brl, carlsbad, ca, usa) supplemented with 10% fetal calf serum (fcs) (hybond, escondido, ca, usa), minimal essential medium with non-essential amino acids, penicillin (100 u/ml) and streptomycin (100 mg/ml) at 371c and 5% co 2 . at 1 day before transfection, cells were trypsinized, resuspended in dmem and seeded in 24-well plates at a density of 1.5 â 10 5 cells per well. cells were cotransfected with 100-500 ng plai and 1-1000 ng in vitro transcribed ds-nef2 rna and in vitro diced si-nef2 or 10-500 ng lhrna (tat, rev, nef1) expression constructs using lipofectamine 2000 (invitrogen, carlsbad, ca, usa). virus production was determined by measuring the ca-p24 levels in the culture supernatant by elisa (abbott, abbott park, il, usa) as described previously. 70 for firefly luciferase measurements, cells were cotransfected with 100 ng pgl3-nef and 10 ng in vitro transcribed ds-nef2 rna and in vitro diced si-nef2 or 100 ng pt7-nef2 and 100 ng pt7-pol. in all experiments, vector prl (2.5 ng) (promega, madison, wi, usa), expressing rl under the control of the cmv promoter, was added to the transfection mix as a control for variation in transfection efficiency and cell viability. equal amounts of the ph1sh-nef expression vector or the empty vector were added as positive and negative controls, respectively. vectors pt7-luc and pt7-pol were cotransfected in equimolar amounts (100 ng) and fl reporter expression was measured. briefly, cells were lysed 48-72 h after transfection in 150 ml 1â passive lysis buffer (promega) by shaking for 30 min at room temperature. the cell lysate was centrifuged for 5 min at 4000 r.p.m. and firefly and renilla luciferase expression was measured in 10 ml supernatant with the dual-luciferase reporter assay system or renilla luciferase assay system (promega). induction of the ifn system was measured by a sensitive rt-pcr on the ifn-b mrna. 71 rna was isolated from hek 293t cells with the mirvana mirna isolation kit (ambion) 24 h after transfection with the long dsrna constructs. first-strand cdna was reverse transcribed from approximately 1 mg rna with random hexamer primers (invitrogen) using the mmlv-rt enzyme (invitrogen) according to the manufacturer's instructions. approximately 200 ng cdna was pcr amplified with primers ifn-bf and ifn-br using standard conditions. amplification of the b-actin gene was used as an internal control. transfection of 1 mg poly(i:c) (amersham pharmacia biotech, piscataway, nj, usa), a synthetic inosine/cytosine polymer that mimics viral dsrna, was used as a positive control for ifn-b induction. rna of transfected cells was subjected to pcr with nef-specific primers nef-b/x and antiu3-att, yielding a 110 bp fragment (supplementary table 1 ). induction of stable rna interference in mammalian cells ribo-gnome: the big world of small rnas rnai: the nuts and bolts of the risc machine rna silencing: the genome's immune system sequence-specific inhibition of microrna-and sirna-induced rna silencing a potential role for rna interference in controlling the activity of the human line-1 retrotransposon dicer functions in rna interference and in synthesis of small rna involved in developmental timing in c. elegans potent and specific genetic interference by double-stranded rna in caenorhabditis elegans an rnadirected nuclease mediates post-transcriptional gene silencing in drosophila cells atp requirements and small interfering rna structure in the rna interference pathway inhibition of virus replication by rna interference evidence that hiv-1 encodes an sirna and a suppressor of rna silencing inhibition of hiv-1 infection by small interfering rna-mediated rna interference modulation of hiv-1 replication by rna interference sirna-directed inhibition of hiv-1 infection prevention of hiv-1 infection in human peripheral blood mononuclear cells by specific rna interference bispecific short hairpin sirna constructs targeted to cd4, cxcr4, and ccr5 confer hiv-1 resistance enhanced gene silencing of hiv-1 specific sirna using microrna designed hairpins human immunodeficiency virus type 1 escapes from rna interference-mediated inhibition human immunodeficiency virus type 1 escape from rna interference hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome rna interference as an antiviral approach: targeting hiv-1 a novel approach for inhibition of hiv-1 by rna interference: counteracting viral escape with a second generation of sirnas double-stranded nef rna interferes with human immunodeficiency virus type 1 replication changes in microrna expression profiles in hiv-1-transfected human cells targets for human encoded micrornas in hiv genes specific interference with gene function by double-stranded rna in early mouse development specific interference with gene expression induced by long, doublestranded rna in mouse embryonal teratocarcinoma cell lines specific inhibition of gene expression by small double-stranded rnas in invertebrate and vertebrate systems hiv-1 tat directly interacts with the interferoninduced, double-stranded rna-dependent kinase hiv-i tat inhibits pkr activity by both rna-dependent and rnaindependent mechanisms stable suppression of gene expression by rnai in mammalian cells analysis of double-stranded rna-induced apoptosis pathways using interferon-response noninducible small interfering rna expression vector library sensitive assay of rna interference in drosophila and chinese hamster cultured cells using firefly luciferase gene as target long endogenous dsrnas can induce complete gene silencing in mammalian cells and primary cultures specific and potent rna interference in terminally differentiated myotubes control of specific gene expression in mammalian cells by co-expression of long complementary rnas generation of ski-knockdown mice by expressing a long double-strand rna from an rna polymerase ii promoter plant pathogens and integrated defence responses to infection inhibition of viral gene expression and replication in mosquito cells by dsrnatriggered rna interference dissecting rna silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense rna rna silencing of dengue virus type 2 replication in transformed c6/36 mosquito cells transcribing an inverted-repeat rna derived from the virus genome rna interference of hiv replication expression of small interfering rnas targeted against hiv-1 rev transcripts in human cells choosing ccr5 or rev sirna in hiv-1 mfold web server for nucleic acid folding and hybridization prediction tight control of gene expression in mammalian cells by tetracycline-responsive promoters tat trans-activates the human immunodeficiency virus through a nascent rna target total silencing by intron-spliced hairpin rnas a lentiviral microrna-based system for single-copy polymerase ii-regulated rna interference in mammalian cells negative feedback inhibition of hiv-1 by tat-inducible expression of sirna inhibition of human immunodeficiency virus expression by sense transcripts encoding the retroviral leader rna rna interference in human cells is restricted to the cytoplasm rnai: doublestranded rna directs the atp-dependent cleavage of mrna at 21 to 23 nucleotide intervals role for a bidentate ribonuclease in the initiation step of rna interference antisense transcripts in the human genome effects of length and location on the cellular response to double-stranded rna antisense transcription in the mammalian transcriptome ribonuclease activity and rna binding of recombinant human dicer argonaute2 is the catalytic engine of mammalian rnai assembly and function of rna silencing complexes trbp recruits the dicer complex to ago2 for microrna processing and gene silencing trbp, a regulator of cellular pkr and hiv-1 virus expression, interacts with dicer and functions in rna silencing intracellular-diced dsrna has enhanced efficacy for silencing hcv rna and overcomes variation in the viral genotype escape from the interferon response associated with rna interference using vectors that encode long modified hairpin-rna rna interference is mediated by 21-and 22-nucleotide rnas gene therapy progress and prospects: novel gene therapy approaches for aids changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of hiv-1 lai , hiv-1 mal , and hiv-1 eli functional differences between the long terminal repeat transcriptional promoters of hiv-1 subtypes a through g double-stranded rna-exposed human keratinocytes promote th1 responses by inducing a type-1 polarized phenotype in dendritic cells: role of keratinocyte-derived tumor necrosis factor alpha, type i interferons, and interleukin-18 gt) inhibition of hiv-1 by lhrna p konstantinova et al rnai research in the berkhout lab is sponsored by nwo-cw (top grant) and senter-novem (ts grant with viruvation). we thank stephan heynen for performing ca-p24 elisa; dr p midoux (centre de biophysique moleculaire, orleans, france) for providing us with the pt7-luc plasmid; dr jean-marc jacque (university of massachusetts medical school, usa) for providing the pcdna3-t7pol expressing vector and dr e de jong (university of amsterdam, the netherlands) for the kind gift of ifn-b primers. key: cord-286194-2emwfx89 authors: mirzaei, hossein; mcfarland, willi; karamouzian, mohammad; sharifi, hamid title: covid-19 among people living with hiv: a systematic review date: 2020-07-30 journal: aids behav doi: 10.1007/s10461-020-02983-2 sha: doc_id: 286194 cord_uid: 2emwfx89 this systematic review summarizes the evidence on the earliest patients with covid-19-hiv co-infection. we searched pubmed, scopus, web of science, embase, preprint databases, and google scholar from december 01, 2019, to june 1, 2020. from an initial 547 publications and 75 reports, 25 studies provided specific information on covid-19 patients living with hiv. studies described 252 patients, 80.9% were male, the mean age was 52.7 years, and 98% were on antiretroviral treatment (art). co-morbidities in addition to hiv and covid-19 (multimorbidity) included hypertension (39.3%), obesity or hyperlipidemia (19.3%), chronic obstructive pulmonary disease (18.0%), and diabetes (17.2%). two-thirds (66.5%) had mild to moderate symptoms, the most common being fever (74.0%) and cough (58.3%). among patients who died, the majority (90.5%) were over 50 years old, male (85.7%), and had multimorbidity (64.3%). our findings highlight the importance of identifying co-infections, addressing co-morbidities, and ensuring a secure supply of art for plhiv during the covid-19 pandemic. electronic supplementary material: the online version of this article (10.1007/s10461-020-02983-2) contains supplementary material, which is available to authorized users. coronavirus disease 2019 (covid19) was declared a pandemic on march 11th, 2020 [1] . as of july 10th, 2020, 12,102,328 covid-19 patients and 551,046 covid-19-related deaths have been reported worldwide [2] . the world health organization (who) and center for disease control and prevention (cdc) have issued health alerts and prevention guidelines for people at increased risk for severe health outcomes and death due to covid-19 [3, 4] . these guidelines are generally based on the outcomes and characteristics of patients affected early in the course of the covid-19 pandemic. emerging patterns point to elevated risk for older persons, people living in long-term care facilities, men, and racial/ethnic minorities who have long experienced disparities in health outcomes for many chronic diseases [5] . chronic disease co-morbidities, especially multimorbidity, appear to be driving factors for covid-19 mortality. warnings to take extra precautions include persons with asthma, chronic lung disease, diabetes, serious cardiovascular conditions, chronic kidney disease, obesity, chronic liver disease, and persons who are immunocompromised, such as people living with hiv (plhiv) [3] [4] [5] . the concern over increased risk for severe covid-19 disease for plhiv may be based on the assumption that plhiv are more likely to be immunosuppressed. hiv infection is associated with abnormal humoral and t-cell-mediated immune responses, resulting in increased susceptibility to numerous opportunistic infections [6] . under this rationale, particular caution is warranted for plhiv with low cd4 cell count, advanced disease, high viral load, and those not taking antiretroviral treatment (art). as plhiv are living longer with art, many will also have chronic conditions associated with severe covid-19 disease [7] . however, there have not been large observational studies specifically measuring symptoms, disease severity, complications, multimorbidity, and the proportion of death in reported covid-19-hiv co-infected patients. it is also not known if people with hiv who are clinically and virologically stable will experience any greater risk for covid-19 complications than the population without hiv infection. at present, the available data mainly appear in case reports and case series of covid-19-hiv co-infected patients. given the urgency of the covid-19 pandemic and the rapidly changing information about the disease, a high degree of vigilance is needed on the course of infection among plhiv. as there are 37.9 million plhiv and 1.7 million new infections each year [8] , patients of covid-19-hiv co-infection are likely to increase. this systematic review was therefore undertaken to bring together the existing evidence on the earliest known case reports to provide a baseline for what is known and to alert providers around the world of any emerging patterns. details of inclusion criteria and our analytical approach were conceptualized a priori and are documented in open science framework (https ://osf.io/zj2hu /). following the systematic reviews and meta-analyses (prisma) checklist (see the supplementary file s1) and the peer review of electronic search strategies [9] guideline [10, 11] , we searched pubmed, scopus, web of science, embase, preprint databases (medrxiv, biorxiv, preprints), the references of publications found, and the "cited by" feature of google scholar from december 1, 2019, to june 1, 2020, for studies published in english. search terms were combined using appropriate boolean operators and included subject heading terms/keywords relevant to covid-19 (e.g., sars-cov-2 or coronavirus disease 2019 or covid-19 or severe acute respiratory syndrome coronavirus 2 or coronavirus infection) and hiv (e.g., hiv or human immunodeficiency virus or aids or acquired immunodeficiency syndrome). please see the supplementary file s2 for a sample search strategy. empirical studies including any study design (i.e., case report, case series, cross-sectional, case-control, cohort, and clinical trial) that reported individual-or aggregatelevel data on covid-19 among plhiv were considered for this review. studies that included a mixed sample of hivpositive and hiv-negative covid-19 patients were only considered if subgroup analyses for plhiv were reported or could be extracted. studies were excluded if they did not present original empirical data (e.g., editorials, commentaries, letters to editors, and reviews) or did not report any clinical data on patients with hiv and covid-19 co-infection. the title, abstract, and full-text screening were completed in duplicate and independently by two reviewers. duplicate records were excluded and disagreements over the inclusion of studies for data extraction were resolved through discussion or feedback from the senior author. data extraction was completed in duplicate, and discrepancies were resolved through discussion or feedback from the senior author. data were extracted on publication date, study type, location, sample size, participants' age, and sex, as well as hiv-related characteristics such as cd4 count (cells/mm3), viral load (copies/ml), and antiretroviral therapy before covid-19 diagnosis. data were also extracted for covid-19-related clinical symptoms (e.g., fever, cough, myalgia, dyspnea, headache, sore throat, fatigue, and gastrointestinal symptoms). comorbidities other than hiv included atrial fibrillation, chronic kidney disease, congestive heart failure, chronic liver disease, cerebrovascular accident, cardiovascular disease, dyslipidemia, diabetes mellitus, hyperlipidemia, hypertension, obstructive sleep apnea, pulmonary embolism, obesity, and smoking. the severity of covid-19 disease was classified as mild (i.e., non-pneumonia and mild pneumonia), severe (i.e., dyspnea, respiratory frequency ≥ 30/min, blood oxygen saturation ≤ 93%, and/ or lung infiltrates > 50% within 24-48 h), and critical (i.e., respiratory failure, septic shock, and/or multiple organ dysfunction or failure) [12] . admission to the intensive care unit (icu) and recovery status (cured, died, still in hospital) were also assessed and reported. the joanna briggs institute's critical appraisal tools were used to assess the methodological quality of the included papers [13] . selected studies were examined for inclusion criteria, sample size, description of study participants, setting, and the appropriateness of the statistical analysis. methodological quality was independently assessed by two reviewers, and disagreements were resolved through discussion. the tool was modified to provide a numeric score [14, 15] . quality assessments were done with different tools based on different study designs. tools had nine items for case reports, ten items for case series, nine items for crosssectional studies, and eleven items for cohort studies. quality assessment tools and scores are presented in the supplementary file s3. descriptive analyses were used for reporting results. continuous variables were summarized using mean and standard deviation (sd) with differences compared using a two-tailed student's t test. categorical variables were summarized by frequency and percentage and differences were measured using the fisher's exact test. p-values less than 0.05 were considered as statistically significant. for combining data from studies that reported aggregated data with those reported individual data, aggregated data were weighted by the number of patients. the proportion of death among reported patients in studies included in the review was also measured. the denominator and nominator for this measure are based on people living with hiv whose covid-19 was diagnosed and reported. we also conducted a subgroup analysis by sex. the combined search strategy identified 622 potential publications on covid-19 infection among plhiv (fig. 1) . after screening and removing duplicate studies (n = 222) and those not relevant to covid-19-hiv co-infection (n = 343), the full-texts of 57 reports were sought to assess for eligibility. of these, eight did not report information about patients with co-infection; 23 were editorials, commentaries, or reviews; and the full-text for one abstract was unavailable. the search found 25 studies that met our inclusion criteria and were included in the systematic review (table 1) . of the 25 studies on covid-19-hiv co-infection, 19 reported information on an individual-level, and six reported on an aggregate-level. eleven studies were case reports (i.e., describing one patient), ten studies were case series including 2-33 patients. two studies were cross-sectional investigating covid-19 status among plhiv. two cohort studies were found. eight studies were from the us, seven from china, three from italy, two from spain, and the remaining five studies were from turkey, germany, the uk, republic of cyprus, and uganda. publication dates ranged from march 12, to june 1, 2020. the joanna briggs institute critical appraisal tools assessment scores ranged from 4 to 7 for case reports (out of 8 possible points), and 4-8 for the case series (out of 10 possible points), 6-7 for cross-sectional (out of 9 possible points), and 8-9 for cohort studies (out of 11 possible points). as quality assessments were done with different tools based on different study designs, scores cannot be directly compared. summed across all studies, 252 patients with covid-19-hiv co-infection were described with varying completeness of demographic and clinical data ( based on the available data, no significant differences between male and female patients were observed with regards to age, clinical characteristics, the severity of covid-19, and health outcomes of covid-19-hiv coinfected patients (table 3 ). in this systematic review, we summarized available data from 252 patients co-infected with covid-19 and hiv. the majority of patients with co-infection of hiv and covid-19 were male. also, in line with hiv-negative persons [16] [17] [18] [19] , data point to higher morbidity and mortality among male patients and higher mortality when multimorbidity is present. the main clinical symptoms of covid-19 in plhiv were cough and fever, and comparable to hiv-negative people, the majority had mild covid-19 disease [16] [17] [18] [19] . nonetheless, the proportion of reported plhiv with covid-19 appears to have higher multimorbidity, severity of disease, and a potentially higher proportion of death. multimorbidity was reported in nearly two-thirds of coinfected patients. the most common co-morbidities among patients with hiv and covid-19 were hypertension, obesity or hyperlipidemia, chronic obstructive pulmonary disease, and diabetes. results of a cohort study showed that multimorbidity (mostly hypertension and diabetes) was more prevalent in covid-19-hiv co-infected patients than plhiv without covid-19 [20] . when data were available, the proportion of death among reported covid-19-hiv co-infected patients appears high (14.3%) in our pooled estimate. the result might be confounded by other factors. for example, plhiv may be at increased risk of mortality or severe illness with covid-19 based on their age and other medical conditions. among those who died for whom individual data were recorded, multimorbidity and full-text articles assessed for eligibility (n = 57) full-text articles excluded, with reasons (n = 32) reports were editorial, commentaries or reviews (n = 23) full-text not found (n = 1) study did not report any information about patients with covid-19-hiv coinfection (n = 8) studies included in qualitative synthesis (n = 25) fig. 1 flowchart of studies included in the systematic review of covid-19-hiv co-infection older age were common. current clinical data suggest the main mortality risk factors are linked to older age and multimorbidity, not particular to hiv, including cardiovascular disease, diabetes, chronic respiratory disease, and hypertension [21] . another possible explanation is bias due to the preponderance of publications on hospitalized patients with more severe disease. the observational designs of the available studies and lack of appropriate control do not permit concluding whether art can prevent the acquisition of covid-19 or reduce morbidity and mortality from covid-19. the search for antiviral agents with activity to treat and prevent covid-19 is an area of active research. for example, a clinical trial in 199 patients showed that ritonavir-boosted lopinavir did not have a benefit over standard care for covid-19 [15] . a survey in plhiv in china showed that nucleoside reverse transcriptase inhibitors (nrti) plus non-nucleoside reverse transfer inhibitors (nnrti) did not prevent covid-19 infection [16] . a cohort study showed that there are no differences in previous use of nnrti, insti, or protease inhibitors in individuals with and without covid-19 diagnosis [17] . findings of lower morbidity or mortality among persons on art compared to those not on art would also need to consider higher cd4 count and better immune status. we acknowledge additional limitations of our study. first, while a large proportion of covid-19 patients are asymptomatic or have mild symptoms, all of the patients in this review were symptomatic, and most were admitted to a hospital. this study, therefore, refers only to patients with a confirmed diagnosis of covid-19 and may overrepresent those with severe covid-19 disease. second, the lack of appropriate comparison groups, particularly for the case reports and case series, is a limitation to identifying factors associated with covid-19-hiv co-infection. third, many of the studies included in our review had a small sample size. fourth, without a population-or probability-based survey of plhiv, the true prevalence of covid-19 and disease manifestations among plhiv remain unknown. to the best of our knowledge, no study has yet measured covid-19-hiv co-infection in a wider be divulged, asked for, or recorded and therefore, underassessed among covid-19 patients. the data available to date indicate that plhiv can be infected with covid-19 and are largely affected by similar features of disease risk and progression as hiv-uninfected patients. the presence of multimorbidity and older age appear to be the important factors for severe morbidity and mortality with covid-19-hiv co-infection. results suggest that healthcare providers need to address multimorbidity among plhiv, ensure their art supply is secure, and continue to consider plhiv as a population for whom precautions are needed to prevent the covid-19. given the number of plhiv worldwide, there are likely many more patients with covid-19-hiv co-infection than suggested by the scant literature to date. clinicians and researchers could help fill the data gap by being vigilant to patients who may be co-infected with sars-cov-2 and hiv. a novel coronavirus from patients with pneumonia in china who. who coronavirus disease (covid-19) dashboard cdc. coronavirus disease 2019 covid-19) pandemic epidemiological characteristics of covid-19: a systematic review and meta-analysis hiv and co-infections stigmas, symptom severity and perceived social support predict quality of life for plhiv in urban indian context hiv estimates through 2018: data for decision-making a review of methanol poisoning: a crisis beyond ocular toxicology preferred reporting items for systematic reviews and meta-analyses: the prisma statement press peer review of electronic search strategies: 2015 guideline statement characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention the joanna briggs institute critical appraisal tools for use in jbi systematic reviews checklist for prevalence studies hiv and other sexually transmitted infections among female sex workers in iran: a systematic review and meta-analysis global prevalence of hypertension among people living with hiv: a systematic review and metaanalysis coronavirus disease 2019 (covid-19) in italy epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of coronavirus disease 2019 in china presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance co-infection of sars-cov-2 and hiv in a patient in wuhan city a survey for covid-19 among hiv/aids patients in two districts of wuhan, china. aids patients in two districts of wuhan early virus clearance and delayed antibody response in a case of covid-19 with a history of co-infection with hiv-1 and hcv computed tomography imaging of an hiv-infected patient with coronavirus disease infection of severe acute respiratory syndrome coronavirus 2 in a patient with acquired immunodeficiency syndrome implications for forensic death investigations from first swiss post-mortem ct in a case of non-hospital treatment with covid-19 covid-19 in patients with hiv: clinical case series darunavir does not prevent sars-cov-2 infection in hiv patients case report: one case of coronavirus disease 2019 (covid-19) in patient co-nfected by hiv with a low cd4+ t cell count hiv/sars-cov-2 co-infected patients in istanbul, turkey covid-19 in people living with human immunodeficiency virus: a case series of 33 patients outcomes among hiv-positive patients hospitalized with covid-19 recovery from covid-19 in two patients with coexisted hiv infection clinical features and outcomes of hiv patients with coronavirus disease 2019 4 cases: hiv and sars-cov-2 coinfection in patients from long island. n y encephalopathy and seizure activity in a covid-19 well controlled hiv patient hiv and sars-cov-2 co-infection: a case report from uganda covid-19 in a patient with hiv infection severe sars-cov-2 pneumonia in a 58-year-old patient with hiv: a clinical case report from the republic of cyprus covid-19 in an hiv-positive kidney transplant recipient hospitalized patients with covid-19 and hiv: a case series clinical features and outcome of hiv/sars-cov-2 co-infected patients in the bronx a case series of five people living with hiv hospitalized with covid-19 in chicago, illinois. aids patient care stds clinical characteristics and outcomes in people living with hiv hospitalized for covid-19 the authors did not receive any funding for this key: cord-305085-bv7udg9k authors: lawrence, robert m. title: chapter 13 transmission of infectious diseases through breast milk and breastfeeding date: 2011-12-31 journal: breastfeeding doi: 10.1016/b978-1-4377-0788-5.10013-6 sha: doc_id: 305085 cord_uid: bv7udg9k nan a large body of evidence clearly demonstrates the protective effects of breastfeeding and documents the transmission of specific infections to infants through breast milk. the fear and anxiety that arise with the occurrence of any infectious disease are even greater in the situation of the breastfeeding mother-infant dyad. uncertainty and lack of knowledge often lead to proscribing against breastfeeding out of fear, which then deprives the infant of the potential protective, nutritional, and emotional benefits of breastfeeding exactly at the time when they are most needed (see the discussion of immunologic benefits of human milk in chapter 5). decisions concerning breastfeeding in a mother with an infectious illness should balance the potential benefits of breastfeeding versus the known or estimated risk for the infant acquiring a clinically significant infection via breastfeeding and the potential severity of the infection. documenting transmission of infection from mother to infant by breastfeeding requires not only the exclusion of other possible mechanisms of transmission but also the demonstration of the infectious agent in the breast milk and a subsequent clinically significant infection in an infant that was caused by a plausible infectious process. the first step is to establish the occurrence of a specific infection (clinically or immunologically evident) in a mother and demonstrate the persistence of the infectious agent such that it could be transmitted to the infant. isolation or identification of the infectious agent from the colostrum, breast milk, or an infectious lesion of the breast is important but not necessarily proof of transmission to an infant. epidemiologic evidence of transmission must be considered, including identifying characteristics of the organism that relate an isolate from an infant to the maternal isolate. infectious organisms can reach the breast milk either by secretion in the fluid or cellular components of breast milk or by contamination of the milk at the time of or after expression. a reasonable mechanism of infection via breast milk should be evident and proved through either animal or human studies. demonstration of a subclinical or clinically evident infection in an infant should follow these outlined steps. exclusion of other possible mechanisms of transmission (exposure to mother or other persons/ animals via airborne, droplet, arthropod, or vector modes of transmission or through direct contact with other infectious fluids) would complete the confirmation of transmission of infection via breastfeeding. it is essential to exclude prenatal or perinatal transmission of infection to a fetus/infant, but doing this can often be difficult. clinical case reports or studies confirming the isolation of an infectious agent from the milk are important. to determine a reasonable estimate of the risk for infection via breast milk, larger epidemiologic studies are needed that compare infection rates in breastfed infants versus formula-fed infants, robert m. lawrence addressing the issues just identified. timing of breastfeeding is important relative to the timing of maternal infection and to the presence of a pathogen in colostrum or breast milk. the duration of breastfeeding is another important variable to consider in the estimate of risk because shedding of a pathogen in breast milk may be intermittent. these considerations are only some of the variables to be taken into account, in general, to assess the risk for transmission of an infectious agent from mother to infant via breast milk or breastfeeding. efforts to prove transmission of infection in a particular maternal-infant dyad can be just as difficult and must consider many of the same factors. this chapter focuses on a discussion of specific, clinically relevant, infectious agents and diseases, with reasonable estimates of the risk for infection to infants from breastfeeding. the basic tenet concerning breastfeeding and infection is that breastfeeding is rarely contraindicated in maternal infection. 243 the few exceptions relate to specific infectious agents with strong evidence of transmission and to the association of an infant' s illness with significant morbidity and mortality. the risk or benefit of breastfeeding relative to immunization of a mother or infant is discussed for certain microorganisms. appendix d addresses drugs in breast milk and includes table d-1, on antiinfective agents, and chapter 5 reviews how breastfeeding may protect against infection. chapter 21 addresses specific concerns relating to banked breast milk and includes standards developed by the human milk banking association of north america to guide the appropriate handling of banked human milk relative to possible infectious agents. isolation precautions have undergone some revisions in terminology and conceptualization. 143 understanding that the transmission of microorganisms can occur with a known infection and with unrecognized sources of infection, recommendations have been made for standard precautions to be applied to all patients to protect health care workers from potentially infectious body fluids. additionally, precautions based on the predominant modes of transmission have been recommended to protect against infection through the airborne route, direct contact, or contact with droplets. although these precautions are intended to be used in clinical situations to protect health care workers, they may be applied in certain situations to the mother-infant dyad to prevent transmission of infectious agents from one to the other or to other hospitalized mothers and infants. these precautions are useful most often when a mother and infant are still hospitalized. the use of such precautions within the home is not meant to limit breastfeeding. they are intended to allow breastfeeding in the majority of cases and to facilitate the continuation of breastfeeding with some additional safeguards in certain situations, after short temporary periods of stopping breastfeeding, and when to safely use expressed breast milk (see appendix f). standard precautions include preventing contact with blood, all body fluids, secretions and excretions, nonintact skin, and mucous membranes by (1) careful handwashing before and after every patient contact; (2) use of gloves when touching body fluids, nonintact skin, or mucous membranes or any items contaminated with body fluids (linens, equipment, devices, etc.); (3) use of nonsterile gowns to prevent contact of clothing with body fluids; (4) use of masks, eye protection, or face shields when splashing with body fluids is possible; and (5) appropriate disposal of these materials. standard precautions should be applied to all patients regardless of actual or perceived risks. the centers for disease control and prevention (cdc) does not consider breast milk a body fluid with infectious risks and thus these policies do not apply to breast milk. (see section on misadministration of breast milk later in this chapter as a possible exception to this concept.) in considering breastfeeding infant-mother dyads and standard precautions, body fluids other than breast milk should be avoided, and only in specified situations should breast milk also be avoided. in general, clothing or a gown for the mother and bandages, if necessary, should prevent direct contact with nonintact skin or secretions. avoiding infant contact with maternal mucous membranes requires mothers to be aware of and understand the risks and to make a conscious effort to avoid this type of contact. the use of gloves, gowns, and masks on infants for protection is neither practical nor appropriate. the recommendations concerning the appropriateness of breastfeeding and breast milk are addressed for specific infectious agents throughout this chapter. human immunodeficiency virus (hiv) infection is an example of one infection that can be prevented by the use of standard precautions, including avoiding breast milk and breastfeeding. the recommendations concerning breastfeeding and hiv and the various variables and considerations involved are discussed later. airborne precautions are intended to prevent transmission via droplet nuclei (dried respiratory particles smaller than 5 mcm that contain microorganisms and can remain suspended in the air for long periods) or dust particles containing microorganisms. airborne precautions include the use of a private room with negative-air-pressure ventilation and masks at all times. in the case of pulmonary tuberculosis (tb), respiratory protective devices (requiring personal fitting and seal testing before use) should be worn. airborne precautions are recommended with measles, varicella or disseminated zoster, and tb. breastfeeding in the presence of these maternal infections is prohibited for the infectious period. this is to protect against airborne transmission of the infection from the mother and to allow the infant to be fed the mother' s expressed breast milk by another individual. the exception to allowing breast milk would be local involvement of the breast by varicella-zoster lesions or mycobacterium tuberculosis, such that the milk becomes contaminated by the infectious agent. transmission via droplets occurs when an individual produces droplets that travel only a short distance in the air and then contact a new host's eyes, nose, mouth, or skin. the common mechanisms for producing droplets include coughing, sneezing, talking (singing or yelling), suctioning, intubation, nasogastric tube placement, and bronchoscopy. in addition to standard precautions applied to all patients, droplet precautions include the use of a private room (preferred) and a mask if within 3 feet (0.9 m) of the patient. droplet precautions are recommended for adenovirus, diphtheria, respiratory infections, haemophilus influenzae, neisseria meningitidis or invasive infection, influenza, mumps, mycoplasma, parvovirus, pertussis, plague (pneumonic), rubella, and streptococcal pharyngitis, pneumonia, or scarlet fever. the institution of droplet precautions with a breastfeeding mother who has these infections should be specified for each particular infection. this may require some period of separation for the infant and mother (for duration of the illness, for short-term or complete treatment of the mother, for the infectious period) with use of expressed breast milk for nutrition in the interim. prophylactic treatment of the infant, maternal use of a mask during breastfeeding or close contact combined with meticulous handwashing, and the mother's avoidance of touching her mucous membranes may be adequate and reasonable for certain infections. contact precautions are meant to prevent transmission of infection via direct contact (contact between the body surfaces of one individual with another) and indirect contact (contact of a susceptible host with an object contaminated with microorganisms from another individual). contact precautions include cohorting or a private room, gloves and gowns at all times, and handwashing after removal of gown and gloves. contact precautions are recommended for a long list of infections, such as diarrhea in diapered or incontinent patients with clostridium difficile infection, escherichia coli o157:h7, shigella, rotavirus, hepatitis a, respiratory illness with parainfluenza virus or respiratory syncytial virus (rsv), multidrug-resistant (mdr) bacteria (e.g., enterococci, staphylococci, gramnegative organisms), enteroviral infections, cutaneous diphtheria, impetigo, herpes simplex virus (hsv) infection, herpes zoster (disseminated or in immunocompromised individuals), pediculosis, scabies, staphylococcus aureus skin infection, viral hemorrhagic fevers (e.g., ebola, lassa), conjunctivitis and abscesses, cellulitis, or decubitus that cannot be contained by dressings. 94 for a breastfeeding infant-mother dyad, implementation of precautions for each of these infections in a mother requires meticulous attention to gowning and handwashing by the mother and a specialized plan for each situation. each of these transmission-based precautions can be used together for organisms or illnesses that can be transmitted by more than one route. they should always be used in conjunction with standard precautions, which are recommended for all patients. the red book: report of the committee on infectious diseases by the american academy of pediatrics (aap) 96 remains an excellent resource for infection control guidelines and recommendations to prevent transmission in specific situations and infections. routine culturing of breast milk or culturing breast milk to screen for infectious agents is not recommended except when the milk is intended as donor milk to another mother' s child directly or through human milk banks. see chapter 21 for specific bacterial count standards for raw donor milk and for pasteurization of donor milk. breastfeeding and the expression of or pumping of breast milk (referred to as expressed breast milk) for later use are not sterile activities. in general expressed breast milk should not contain large numbers of microorganisms (less than 10 4 for raw milk and less than 10 6 for milk to be pasteurized), nor should it contain potential pathogens such as s. aureus, β-hemolytic streptococci, pseudomonas species, proteus species, or streptococcus faecalis or faecium. few studies have examined "routine" culturing of milk and the significance of specific bacterial colony counts relative to illness in infants. the studies have been primarily concerned with premature or low-birth-weight (lbw) infants who remain hospitalized and are commonly fed via enteral tubes. a study from canada tested 7610 samples of milk for use in 98 preterm infants. 242 the study did not identify any adverse events in the infants attributed to organisms growing in the milk samples, and routine bacteriological testing of expressed breast milk was not recommended. a study from chicago examined gram-negative bacilli in the milk used in premature infants. 48 samples were tested before feeding and from the nasogastric tubes during feeding. milk samples from before feeding were less likely to contain gram-negative bacilli (36%) than milk samples from the nasogastric tubing (60%). feeding intolerance was observed when there were more than 10 3 colony-forming units per milliliter (cfu/ml), and episodes of sepsis were identified when the bacterial counts in the milk were greater than or equal to 10 6 cfu/ml. this study recommended the routine bacteriologic testing of expressed breast milk. another study from arkansas focused on contamination of feeding tubes during administration of expressed breast milk or formula. 277 ten infants in the neonatal intensive care unit (nicu) were exposed to greater than 10 5 gram-negative bacteria in their feeding tubes. the three infants who were fed expressed breast milk with contamination at greater than 10 5 organisms remained well, but the seven formula-fed infants with high levels of bacterial contamination in the feeding tubes developed necrotizing enterocolitis. the gram-negative bacteria with high level contamination in the feeding tubes were either enterobacter or klebsiella in all cases. many nicus consider 10 5 to 10 6 cfu/ml as the significant bacterial count for gram-negative bacilli in breast milk that places premature and lbw infants at greater risk for infection. even less data are available concerning specific bacterial colony counts for gram-positive organisms and the risk to the infant. generally less than 10 3 gram-positive organisms per ml of milk is considered acceptable, with only case reports and no controlled trials to support this cutoff. when the presence of an infectious illness in an infant and/or the breastfeeding mother' s breast when breast milk is seriously considered as a possible mechanism of transmission to the infant, culturing breast milk to identify the organism may be warranted and useful. more important than hurrying to culture breast milk is the careful instruction of mothers on the proper technique for collecting expressed breast milk, storing it, and cleaning the collection unit. the reinforcement of proper technique from time to time, especially when a question of contamination arises, is equally important. many small reports comment on the contamination of breast milk with different collection methods. relative comparisons suggest decreasing contamination of expressed breast milk when collected by the following methods; drip milk, hand pumped milk, manual expression, modern electric pumped milk. one group from malaysia published results showing no difference in contamination between milk collected by electric pump versus manual expression when collected in the hospital. expressed breast milk collected at home by breast pump had higher rates of contamination with staphylococci and gram-negative bacteria. 46 discussion continues about the need to discard the first few milliliters of milk to lower bacteria numbers in expressed breast milk without any evidence to suggest if this is truly necessary. 62, 337 no evidence shows that cleansing the breast with anything other than tap water decreases the bacterial counts in cultured expressed breast milk. 414 if an infant is directly breastfeeding, collecting milk for culture by manual expression and trying to obtain a "midstream" sample (as is done with "midstream" urine collection for culture) is appropriate. if an infant is being fed expressed breast milk, collecting and culturing the milk at different points during collection (utilizing the same technique the mother uses [manual expression, hand pump, or electric pump]) and administration is appropriate. this might include a sample from immediately after collection, another of stored expressed breast milk, and a sample of milk from the most recent infant feeding at the time the decision to culture is made. please see box 13-1 for the basic steps in culturing expressed breast milk. interpretation of such culture results can be difficult and should involve a pediatric infectious disease expert, a microbiologist, and hospital epidemiologist. additional organism identification is often required, utilizing antibiogram patterns or molecular fingerprinting by various techniques to correlate a bacterial isolate from breast milk with an isolate causing disease in infant or mother. misadministration of breast milk, also known as misappropriation, breast milk exposure, and accidental ingestion of breast milk, and other terms, is a medical-legal issue when it occurs in a hospital. this scenario occurs when one infant receives breast milk from another mother by mistake. this occurrence can be very distressing to the families (recipient patient, recipient parent, and donor mother) and medical staff involved. the actual risk for transmission of an infectious agent to an infant via a single ingestion of expressed breast milk (the most common occurrence) from another mother is exceedingly low. in this scenario, the cdc recommends treating this as an accidental exposure to a body fluid, which could be infectious. 84 bacterial, fungal, or parasitic infection from the one exposure is highly unlikely. the concern is about viral pathogens, known to be blood-borne pathogens, which have been identified in breast milk and include but are not limited to hepatitis b virus (hbv), hepatitis c virus (hcv), cytomegalovirus (cmv), west nile virus, human t-cell lymphotropic virus (htlv), and hiv. most hospitals have protocols for managing the situation from both the infection control/prevention and the medical-legal perspectives. these protocols advise informing both families about what occurred, discussing the theoretical risks of harm from the exposure, and reviewing test results and/ or recommending testing to determine the infectious status of each mother relative to the above mentioned viruses. hcv is not a contraindication to breastfeeding and west nile virus infection in lactating women is rare. 74, 177 neither infection has a documented effective form of prevention or acute treatment. testing either mother (donor or of recipient infant) for these agents is not warranted. prenatal testing for hiv is more commonplace throughout the world. the incidence of hiv among women of childbearing age is low, although it varies significantly by geographic location, and the hospital or locale-specific incidence would be important to know to estimate risk. most women and medical staff are aware that hiv can be transmitted by breastfeeding; therefore breast milk from hiv-positive women is rarely if ever stored in hospitals. the risk for transmission of hiv via breastfeeding is due to the volume of feedings over months (estimated at 400 to 500 feedings in the first 2 months of life) compared with the small "dose of exposure" from one or two "accidental feedings." transmission of hiv from a single breast milk exposure has never been documented. immunologic components in breast milk, along with time and cold of storage, inactivate the hiv in expressed breast milk. for these reasons, the risk for transmission of hiv via expressed breast milk consumed by another child is thought to be extremely low. htlv-i/ii infection in childbearing women is uncommon except in certain geographic regions (japan, africa, the caribbean, and south america). transmission of htlv via breast milk does occur and, like hiv, appears to be related to the volume and duration of breastfeeding. limiting the duration of breastfeeding is effective in decreasing transmission. 407, 409, 446 freezing and thawing expressed breast milk decreases the infectivity of htlv-i. 11 in areas of low prevalence, a positive test in a mother should be suspected to be a false positive test, and retesting with both antibody and polymerase chain reaction (pcr) testing should be performed. for these reasons the transmission of htlv-i/ii via accidental expressed breast milk exposure is thought to be extremely low. although the majority of women are cmv positive by childbearing age and cmv transmission occurs via breastfeeding, the risk for cmv in a full-term infant is low. premature or lbw infants are at greater risk for developing disease with cmv infection. freezing expressed breast milk (at −20° c) for 3 to 5 days significantly decreases the infectivity of cmv. here again the risk for cmv transmission from a single accidental exposure to cmv-positive expressed breast milk is extremely low. with a discussion of theoretical risk should be a discussion of possible preventive interventions, such as vaccination or antimicrobial postexposure prophylaxis. if donor mothers are positive for hbv, it is appropriate to give recipient infants hepatitis b virus immunoglobulin (hbig) and hbv vaccines if they have not already received them. if a box 13-1. culturing breast milk 1. wash hands as per routine. 2. wash breast with warm tap water and a clean washcloth. 3. manually express breast milk ("midstream" collection is not required) or attach breast pump flange (previously cleaned as per routine) for collection and collect milk. 4. place a 3 to 5 ml sample of expressed breast milk in a sterile container with a nonleakable top. 5. deliver to the labatory in less than 1 hour or refrigerate at 4° c until delivery. before sending samples to the viral lab or for nucleic acid/ po lymerase chain reaction (pcr) testing, confirm that the laboratory will accept and process the sample as requested and that the appropriate collection container and prelaboratory management of the specimen are utilized. 6 324 it may also be appropriate to consult a pediatric infectious disease specialist. additional important components of the hospital-based protocols for managing accidental expressed breast milk exposure include ongoing psychosocial support for the families and staff, documentation of medical discussions with the families, investigative steps, consents and interventions, and the demonstration of ongoing infection control efforts to prevent additional events of misadministration of breast milk. microorganisms produce a whole spectrum of clinical illnesses affecting mothers and infants. many situations carry the risk for transmission of the involved organism from a mother to the infant, or vice versa; in general, however, infants are at greater risk because of such factors as inoculum size and immature immune response. as always, an infection must be accurately diagnosed in a timely manner. empiric therapy and initial infection control precautions should begin promptly based on the clinical symptoms and the most likely etiologic agents. when dealing with a maternal infection, clarifying the possible modes of transmission and estimating the relative risk for transmission to the infant are essential first steps to decision-making about isolating a mother from her infant and the appropriateness of continuing breastfeeding or providing expressed breast milk. breastfeeding infrequently is contraindicated in specific maternal infections. 243 often the question of isolation and interruption of breastfeeding arises when symptoms of fever, pain, inflammation, or other manifestations of illness first develop in a mother and the diagnosis is still in doubt. a clinical judgment must be made based on the site of infection, probable organisms involved, possible or actual mechanisms of transmission of these organisms to the infant, estimated virulence of the organism, and likely susceptibility of the infant. additionally, by the time the illness is clearly recognized or diagnosed in a mother, the infant has already been exposed. given the dynamic nature of the immunologic benefits of breast milk, continuation of breastfeeding at the time of diagnosis or illness in a mother can provide the infant protection rather than continued exposure in most illnesses. stopping breastfeeding is rarely necessary. many situations associated with maternal fever do not require separation of mother and infant, such as engorgement of the breasts, atelectasis, localized nonsuppurative phlebitis, or urinary tract infections. appendix f lists a number of clinical syndromes, conditions, and organisms that require infection control precautions in hospitals. this appendix also includes short lists of possible etiologic agents for these conditions and appropriate precautions and recommendations concerning breastfeeding for different scenarios or organisms. this chapter considers specific infectious agents that are common, clinically significant, or of particular interest. bacillus anthracis, a gram-positive, spore-forming rod, causes zoonotic disease worldwide. human infection typically occurs due to contact with animals or their products. three forms of human disease occur: cutaneous anthrax (the most common), inhalation anthrax, and gastrointestinal (gi) disease (rare). person-to-person transmission can occur as a result of discharge from cutaneous lesions, but no evidence of human-to-human transmission of inhalational anthrax is available. no evidence of transmission of anthrax via breast milk exists. standard contact isolation is appropriate for hospitalized patients or patients with draining skin lesions. the issue of anthrax as a biologic weapon has exaggerated its importance as a cause of human disease. the primary concerns regarding anthrax and breastfeeding are antimicrobial therapy or prophylaxis in breastfeeding mothers and the possibility that infant and mother were exposed by intentional aerosolization of anthrax spores. the cdc published recommendations for treatment and prophylaxis in infants, children, and breastfeeding mothers. 72 the recommendations include the use of ciprofloxacin, doxycycline, amoxicillin, and several other agents without discontinuing breastfeeding. little available is information on ciprofloxacin and doxycycline in breast milk for prolonged periods of therapy or prophylaxis (60 days) and possible effects on infants' teeth and bone/cartilage growth during that time period. depending on the clinical situation and sensitivity testing of the identified anthrax strain, other agents can be substituted to complete the 60-day course. the cdc has approved the use of ciprofloxacin and doxycycline for breastfeeding women for short courses of therapy (less than several weeks). simultaneous exposure of infant and mother could occur from primary aerosolization or from spores "contaminating" the local environment. in either case decontamination of the mother-infant dyad' s environment should be considered. breastfeeding can continue during a mother' s therapy for anthrax as long as she is physically well. open cutaneous lesions should be carefully covered and, depending on the situation, simultaneous prophylaxis for the infant may be appropriate. considerable justifiable concern has been expressed because of the reports of sudden infant death from botulism. infant botulism is distinguished from food-borne botulism from improperly preserved food containing the toxin and from wound botulism from spores entering the wound. infant botulism occurs when the spores of clostridium botulinum germinate and multiply in the gut and produce the botulinal toxin in the gi tract. 17 the toxin binds presynaptically at the neuromuscular junction, preventing acetylcholine release. the clinical picture is a descending, symmetric flaccid paralysis. not every individual who has c. botulinum identified in the stool experiences a clinical illness. the age of infants seems to relate to their susceptibility to illness. the illness is mainly in children younger than 12 months of age; the youngest patient described in the literature was 6 days old. 17 most children become ill between 6 weeks and 6 months of age. the onset of illness seems to occur earlier in formula-fed infants compared with breastfed infants. when a previously healthy infant younger than 6 months of age develops constipation, then weakness and difficulty sucking, swallowing, crying, or breathing, botulism is a likely diagnosis. the organisms should be looked for in the stools, and electromyography may or may not be helpful. in a group reviewed by arnon et al, 19 33 of 50 patients hospitalized in california were still being nursed at onset of the illness. a beneficial effect of human milk was observed in the difference in the mean age at onset, with breastfed infants being twice as old as formula-fed infants with the disease. the breastfed infants' symptoms were milder. breastfed infants receiving iron supplements developed the disease earlier than those who were breastfed but unsupplemented. of the cases of sudden infant death from botulism, no infants were breastfed within 10 weeks of death. all were receiving iron-fortified formulas. in most cases, no specific food source of c. botulinum can be identified, but honey is the food most often implicated, and corn syrup has been implicated in infants older than 2 months of age. honey may contain botulism spores, which can germinate in the infant gut. however, botulin toxin has not been identified in honey. it has been recommended that honey not be given to infants younger than 12 months of age. this includes putting honey on a mother' s nipples to initiate an infant' s interest in suckling. arnon 18 reviewed the first 10 years of infant botulism monitoring worldwide. the disease has been reported from 41 of the 50 states in the united states and from eight countries on four continents. the relationship to breastfeeding and human milk is unclear. in general the acid stools (ph 5.1 to 5.4) of human milk fed infants encourage bifidobacterium species. few facultative anaerobic bacteria, or clostridia, existing as spores, are present in breastfed infants. in contrast, formula-fed infants have stool phs ranging from 5.9 to 8.0, with few bifidobacteria, primarily gram-negative bacteria, especially coliforms and bacteroides species. c. botulinum growth and toxin production decrease with declining ph and usually stops below ph 4.6. breast milk also contains additional protective immunologic components, which purportedly have activity against botulinum toxin. 269 the relationship between the introduction of solid foods or weaning in both formula-fed and breastfed infants and the onset of botulism remains unclear. for a breastfed infant, the introduction of solid food may cause a major change in the gut with a rapid rise in the growth of enterobacteria and enterococci followed by progressive colonization by bacteroides species, clostridia, and anaerobic streptococci. feeding solids to formula-fed infants minimally changes the gut flora as these organisms already predominate. although more hospitalized infants have been breastfed, sudden-death victims are younger and have been formula fed, which supports the concept of immunologic protection in the gut of a breastfed infant. much work remains to understand this disease. clinically, constipation, weakness, and hypotonicity in a previously healthy child constitute botulism until ruled out, especially with recent dietary changes. at this time, no reason exists to suspect breastfeeding as a risk for infant botulism, and some evidence suggests a possible protective effect from breastfeeding. breastfeeding should continue if botulism is suspected in mother or infant. brucella melitensis has been isolated in the milk of animals. foods and animals represent the primary sources of infection in humans. brucellosis demonstrates a broad spectrum of illness in humans, from subclinical to subacute to chronic illness with nonspecific signs of weakness, fever, malaise, body aches, fatigue, sweats, arthralgia, and lymphadenitis. in areas where the disease is enzootic, childhood illness has been described more frequently. the clinical manifestations in children are similar to those in adults. 259 infection can occur during pregnancy, leading to abortion (infrequently), and can produce transplacental spread, causing neonatal infection (rarely). the transmission of b. melitensis through breast milk has been implicated in neonatal infection. 259, 260 there have been eight cases of brucellosis in infants that were possibly associated with breastfeeding, but brucella was not isolated from the breast milk in any of those cases.* one case of brucellosis in an infant caused by breast milk transmission, with b. melitensis isolated from the breast milk, before antibiotic treatment was given to the mother has been documented. 415 additionally, brucella melitensis has been cultured from women with breast lumps and abscesses. 295 only one of six women described in this report was lactating at the time of diagnosis, and no information about the infant was given. brucellosis mastitis or abscess should be considered in women presenting with appropriate symptoms and occupational exposure to animals, contact with domestic animals in their environment, or exposure to animal milk or milk products (especially unpasteurized products). the breast inflammation tends to be granulomatous in nature (without caseation) and is often associated with axillary adenopathy; occasionally systemic illness in the woman is evident. treatment of brucellosis mastitis or abscess should be treated with surgery or fine needle aspiration as indicated and 4 to 6 weeks of combination antibiotic therapy with two or three medications. temporary interruption of breastfeeding with breast pumping and discarding the milk to continue stimulation of milk production is appropriate. breastfeeding should then continue after an initial period of 48 to 96 hours of therapy in the mother. acceptable medications for treating the mother while continuing breastfeeding include gentamicin, streptomycin, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, and rifampin (see appendix d). chlamydial infection is the most frequent sexually transmitted disease (std) in the united states and is a frequent cause of conjunctivitis and pneumonitis in an infant from perinatal infection. the major determinant of whether chlamydial infection occurs in a newborn is the prevalence rate of chlamydial infection of the cervix. 364 chlamydial immunoglobulin a (iga) has been found in colostrum and breast milk in a small number of postpartum women who were seropositive for chlamydia. no information is available on the role of milk antibodies in protection against infection in infants. 389 it is not believed that chlamydia is transmitted via breast milk. use of erythromycin or tetracycline to treat mothers and oral erythromycin and ophthalmic preparations of tetracyclines, erythromycin, or sulfonamides to treat suspected infection in infants are appropriate during continued breastfeeding. separating infants from mothers with chlamydial infections or stopping breastfeeding is not indicated. simultaneous treatment of mothers and infants may be appropriate in some situations. corynebacterium diphtheriae causes several forms of clinical disease, including membranous nasopharyngitis, obstructive laryngotracheitis, and cutaneous infection. complications can include airway obstruction from membrane formation and toxinmediated central nervous system (cns) disease or myocarditis. the overall incidence of diphtheria has declined even though immunization does not prevent infection but does prevent severe disease from toxin production. fewer than five cases are reported annually in the united states. transmission occurs via droplets or direct contact with contaminated secretions from the nose, throat, eye, or skin. infection occurs in individuals whether they have been immunized or not, but infection in those not immunized is more severe and prolonged. as long as the skin of the breast is not involved, no risk for transmission exists via breast milk. no toxin-mediated disease from toxin transmitted through breast milk has been reported in an infant. breastfeeding, along with chemoprophylaxis and immunization of affected infants, is appropriate in the absence of cutaneous breast involvement (see appendix f). maternal infection with neisseria gonorrhoeae can produce a large spectrum of illness ranging from uncomplicated vulvovaginitis, proctitis, pharyngitis, conjunctivitis, or more severe and invasive disease, including pelvic inflammatory disease, meningitis, endocarditis, or disseminated gonococcal infection. the risk for transmission from mother to infant occurs mainly during delivery in the passage through the infected birth canal and occasionally from postpartum contact with the mother (or her partner). risk for transmission from breast milk is negligible, and n. gonorrhoeae does not seem to cause local infection of the breasts. infection in neonates is most often ophthalmia neonatorum and less often a scalp abscess or disseminated infection. mothers with presumed or documented gonorrhea should be reevaluated for other stds, especially chlamydia trachomatis and syphilis, because some therapies for gonorrhea are not adequate for either of these infections. with the definitive identification of gonorrhea in a mother, empiric therapy should begin immediately, and the mother should be separated from the infant until completion of 24 hours of adequate therapy. treatment of the mother with ceftriaxone, cefixime, penicillin, or erythromycin is without significant risk to the infant. single-dose treatment with spectinomycin, ciprofloxacin, ofloxacin, or azithromycin has not been adequately studied but presumably would be safe for the infant given the 24-hour separation and a delay in breastfeeding without giving the infant the expressed breast milk (pump and discard). doxycycline use in a nursing mother is not routinely recommended. careful preventive therapy for ophthalmia neonatorum should be provided, and close observation of the infant should continue for 2 to 7 days, the usual incubation period. empiric or definitive therapy against n. gonorrhoeae may be necessary depending on an infant' s clinical status and should be chosen on the basis of the maternal isolate' s sensitivity pattern. the mother should not handle other infants until after 24 hours of adequate therapy, and the infant should be separated from the rest of the nursery population, with or without breastfeeding. haemophilus influenzae type b can cause severe invasive disease such as meningitis, sinusitis, pneumonia, epiglottitis, septic arthritis, pericarditis, and bacteremia. shock can also occur. because the increased utilization of the h. influenzae type b conjugate vaccines, invasive disease caused by haemophilus has decreased dramatically, more than 95%, in the united states. most invasive disease occurs in children 3 months to 3 years of age. older children and adults rarely experience severe disease but do serve as sources of infection for young children. children younger than 3 months of age seem to be protected because of passively acquired antibodies from the mothers, and some additional benefits may be received from breast milk. transmission occurs through contact with respiratory secretions, and droplet precautions are protective. no evidence suggests transmission through breast milk or breastfeeding. evidence supports that breast milk limits the colonization of h. influenzae in the throat. 185 in the rare case of maternal infection, an inadequately immunized infant in a household is an indication to provide rifampin prophylaxis and close observation for all household contacts, including the breastfeeding infant. expressed breast milk can be given to an infant during the 24-hour separation after the mother' s initiation of antimicrobial therapy, or if the mother' s illness prevents breastfeeding, it can be reinitiated when the mother is able (see appendix f). although uncommon in the united states, leprosy occurs throughout the world. this chronic disease presents with a spectrum of symptoms depending on the tissues involved (typically the skin, peripheral nerves, and mucous membranes of the upper respiratory tract) and the cellular immune response to the causative organism, mycobacterium leprae. transmission occurs through long-term contact with individuals with untreated or multibacillary (large numbers of organisms in the tissues) disease. leprosy is not a contraindication to breastfeeding, according to jeliffe and jeliffe. 202 the importance of breastfeeding and urgency of treatment are recognized by experts who treat infants and mothers early and simultaneously. no mother-infant contact is permitted except to breastfeed. dapsone, rifampin, and clofazimine are typically and safely used for infant and mother regardless of the method of feeding (see appendix d). listeriosis is a relatively uncommon infection that can have a broad range of manifestations. in immunocompetent individuals, including pregnant women, the infection can vary from being asymptomatic to presenting as an influenza-like illness, occasionally with gi symptoms or back pain. severe disease occurs more frequently in immunodeficient individuals or infants infected in the perinatal period (pneumonia, sepsis, meningitis, granulomatosis infantisepticum). although listeriosis during pregnancy may manifest as mild disease in a mother and is often difficult to recognize and diagnose, it is typically associated with stillbirth, abortion, and premature delivery. it is thought that transmission occurs through the transplacental hematogenous route, infecting the amniotic fluid, although ascending infection from the genital tract may occur. 122 early and effective treatment of a woman can prevent fetal infection and sequelae. 206, 257 neonatal infection occurs as either early-or late-onset infection from transplacental spread late in pregnancy, ascending infection during labor and delivery, infection during passage through the birth canal, or, rarely, during postnatal exposure. no evidence in the literature suggests that listeria is transmitted through breast milk. treatment of the mother with ampicillin, penicillin, or trimethoprim-sulfamethoxazole is not a contraindication to breastfeeding as long as the mother is well enough. expressed colostrum or breast milk also can be given if the infant is able to feed orally. the management of lactation and feeding in neonatal listeriosis is conducted supportively, as it is in any situation in which an infant is extremely ill, beginning feeding with expressed breast milk or directly breastfeeding as soon as reasonable. n. meningitidis most often causes severe invasive infections, including meningococcemia or meningitis often associated with fever and a rash and progressing to purpura, disseminated intravascular coagulation, shock, coma, and death. transmission occurs via respiratory droplets. spread can occur from an infected, ill individual or from an asymptomatic carrier. droplet precautions are recommended until 24 hours after initiation of effective therapy. despite the frequent occurrence of bacteremia, no evidence indicates breast involvement or transmission through breast milk. the risk for maternal infection to an infant after birth is from droplet exposure and exists whether the infant is breastfeeding or bottle feeding. in either case the exposed infant should receive chemoprophylaxis with rifampin, 10 mg/kg/dose every 12 hours for 2 days (5 mg/kg/dose for infants younger than 1 month of age), or ceftriaxone, 125 mg intramuscularly (im) once, for children younger than 15 years of age. close observation of the infant should continue for 7 days, and breastfeeding during and after prophylaxis is appropriate. the severity of maternal illness may prevent breastfeeding, but it can continue if the mother is able, after the mother and infant have been receiving antibiotics for 24 hours. a period of separation from the index case for the first 24 hours of effective therapy is recommended; expressed breast milk can be given during this period. respiratory illness caused by bordetella pertussis evolves in three stages: catarrhal (nasal discharge, congestion, increasing cough), paroxysmal (severe paroxysms of cough sometimes ending in an inspiratory whoop, i.e., whooping cough), and convalescent (gradual improvement in symptoms). transmission is via respiratory droplets. the greatest risk for transmission occurs in the catarrhal phase, often before the diagnosis of pertussis. the nasopharyngeal culture usually becomes negative after 5 days of antibiotic therapy. chemoprophylaxis for all household contacts is routinely recommended. no evidence indicates transmission through breast milk, with similar risk to breastfed and bottle-fed infants. in the case of maternal infection with pertussis, chemoprophylaxis for all household contacts, regardless of age or immunization status, is indicated. in addition to chemoprophylaxis of the infant, close observation and subsequent immunization (in infants older than 6 weeks of age) are appropriate. despite chemoprophylaxis, droplet precautions and separation of mother and infant during the first 5 days of effective maternal antibiotic therapy are recommended. expressed breast milk can be provided to the infant during this period. staphylococcal infection in neonates can be caused by either s. aureus or coagulase-negative staphylococci (most often s. epidermidis) and can manifest in a wide range of illnesses. localized infection can be impetigo, pustulosis in neonates, cellulitis, or wound infection, and invasive or suppurative disease includes sepsis, pneumonia, osteomyelitis, arthritis, and endocarditis. s. aureus requires only a small inoculum (10 to 250 organisms) to produce colonization in newborns, most often of the nasal mucosa and umbilicus. 193 by the fifth day of life, 40% to 90% of the infants in the nursery will be colonized with s. aureus. 126 the organism is easily transmitted to others from mother, infant, family, or health care personnel through direct contact. outbreaks in nurseries were common in the past. mothers, infants, health care workers, and even contaminated, unpasteurized, banked breast milk were sources of infection. 298, 326 careful use of antibiotics, changes in nursery layout and procedures, standard precautions, and cohorting as needed decreased the spread of s. aureus in nurseries. now the occurrence of methicillin-resistant s. aureus (mrsa) is again a common problem, requiring cohorting, occasionally epidemiologic investigation, and careful infection control intervention. there are numerous reports of mrsa outbreaks in nicus.* the significance of colonization with staphylococcus and the factors leading to development of disease in individual patients are not clear. the morbidity and mortality related to s. aureus infection in neonates is well described. 192, 195, 219 management of such outbreaks has been reviewed. 147, 250 little has been written about the role of breastfeeding in colonization with s. aureus in nicus, wellbaby nurseries, or at home. mrsa is an important pathogen worldwide. community-acquired mrsa is different from hospital-acquired mrsa. community-acquired mrsa is usually defined as occurring in an individual without the common predisposing variables associated with hospital-acquired mrsa, lacking a mdr phenotype (common with hospital-acquired mrsa), frequently carrying multiple exotoxin virulence factors (such as panton-valentine leukocidin toxin), as well as carrying the smaller type iv staphylococcal cassette cartridge for the meca gene on a chromosome (hospital-acquired mrsa carries types i-iii staphylococcal cassette cartridge) and as being molecularly distinct from the common nosocomial strains of hospital-acquired mrsa. community-acquired mrsa is most commonly associated with skin and soft tissue infections and necrotizing pneumonia and less frequently associated with endocarditis, bacteremia, necrotizing fasciitis, myositis, osteomyelitis, or parapneumonic effusions. community-acquired mrsa is so common, it is now being observed in hospital outbreaks. 24, 144, 164, 358 community-acquired mrsa transmission to infants via breast milk has been reported. 34, 144, 210, 253, 286 premature or small-forgestational-age infants are more susceptible to and at increased risk for significant morbidity and mortality due to mrsa due in part to prolonged hospitalization, multiple courses of antibiotics, invasive procedures, and intravenous (iv) lines, their relative immune deficiency due to prematurity and illness, and altered gi tract due to different flora and decreased gastric acidity. therefore colonization with mrsa may pose a greater risk to infants in nicus in the long run. full-term infants develop pustulosis, cellulitis, and soft tissue infections, but rarely has invasive disease been reported. 82, 132, 298 fortunov et al 132 from texas reported 126 infections in term or late-preterm previously well infants including 43 with pustulosis, 68 with celluliltis or abscesses, and 15 invasive infections. family history of soft tissue skin infections and male sex were the only variables associated with risk for infection; cesarean delivery, breastfeeding, and circumcision were not. 132 nguyen et al 298 reported mrsa infections in a well-infant nursery from california. the eleven cases were all in full-term boys with pustularvesicular lesions in the groin. the infections were associated with longer length of stay, lidocaine injection use in infants, maternal age older than 30 years, and circumcision. breastfeeding was not an associated risk factor for mrsa infection. 298 the question of the role of circumcision in mrsa outbreaks was addressed by van howe and robson. 426 they reported that circumcised boys are at greater risk for staphylococcal colonization and infection. 426 others report that s. aureus carriage in infants (and subsequent infection) is most likely affected by multiple variables including infant factors (antibiotics, surgical procedures [circumcision being the most common], duration of hospital stay as a newborn), maternal factors (previous colonization, previous antibiotic usage, mode of delivery, length of stay), and environmental factors (mrsa in the family or hospital, nursery stay versus rooming-in, hand hygiene).* gerber et al 147 from the chicago area published a consensus statement for the management of mrsa outbreaks in the nicu. the recommendations, which were strongly supported by experimental, clinical, and epidemiologic data, included using a waterless, alcohol-based hand hygiene product, monitoring and enforcing hand hygiene, placing mrsa-positive infants in contact precautions with cohorting if possible, using gloves and gowns for direct contact and masks for aerosolgenerating procedures, cohorting nurses for care of mrsa-positive infants when possible, periodic screening of infants for mrsa using nares or nasopharyngeal cultures, clarifying the mrsa status of infants being transferred into the nicu, limiting overcrowding, and maintaining ongoing instruction and monitoring of health care workers in their compliance with infection control and hand hygiene procedures. evaluation of the outbreak could include screening of health care workers and environmental surfaces to corroborate epidemiologic data and laboratory molecular analysis of the mrsa strains if indicated epidemiologically. the use of mupirocin or other decolonizing procedures should be determined on an individual basis for each nicu. s. aureus is the most common cause of mastitis in lactating women. 317, 394, 395, 436 recurrence or persistence of symptoms of mastitis is a well described occurrence and an important issue in the management of mastitis. communityacquired mrsa has been associated with mastitis as well. 342 pasteurization, s. aureus was not detected in any of the 6820 samples of expressed breast milk. colonization of one infant with mrsa was identified, but no mrsa infections were identified in any of the hospitalized infants in the nicu during the 18 months of the study. 26 novak et al 300 identified mrsa in 57 of 500 samples (11%) of expressed fresh-frozen milk from 500 different donors from five brazilian milk banks. only 3 of the 57 samples were positive with high-level bacterial counts of mrsa: greater than 10,000 cfu/ml. these were the only samples that would not have been acceptable by bacteriological criteria according to brazilian or american criteria for raw milk use. they did not investigate other epidemiologic data to identify possible variables associated with low or high level contamination of expressed breast milk with mrsa. 300 management of an infant and/or mother with mrsa infection relative to breastfeeding or use of breast milk should be based on the severity of disease and whether the infant is premature, lbw, very-lowbirth-weight (vlbw), previously ill, or full term. full-term infants who themselves or their mothers develop mild to moderate infections (impetigo, pustulosis, cellulitis/abscess, mastitis/breast abscess, or soft tissue infection) can continue breast feeding after a short period of interruption (24 to 48 hours). during this time, pumping to maintain the milk supply should be supported, an initial evaluation for other evidence of infection should be done in the maternalinfant dyad, the infected child and/or mother should be placed on "commonly" effective therapy for the mrsa infection, and ongoing observation for clinical disease should continue. the mother and infant can "room-in" together in the hospital, if necessary, with standard and contact precautions. culturing the breast milk is not necessary. empiric therapy for the infant may be chosen based on medical concerns for the infant and the known sensitivity testing of the mrsa isolate. appropriate antibiotic choices include short-term use of azithromycin (erythromycin use during infancy [less than 6 weeks of age], or breastfeeding associated with an increased risk for hypertrophic pyloric stenosis), sulfamethoxazoletrimethoprim (in the absence of g6pd deficiency and older than 30 days of age), clindamycin, and perhaps linezolid for mild to moderate infections. infants in nicus (premature, lbw, vlbw, and/ or previously ill), who themselves or their mothers have a mrsa infection, should have the breast milk cultured and suspend breastfeeding or receiving breast milk from their mother until the breast milk is shown to be culture negative for mrsa. the infant should be treated as indicated for their infection or empirically treated if symptomatic (with pending culture results) and closely observed for development of new signs or symptoms of infection. pumping to maintain the milk supply and the use of banked breast milk are appropriate. the infant should be placed on contact precautions, in addition to the routine standard precautions. the infant can be cohorted with other mrsa-positive infants with nursing care cohorted as well. for the mother with mrsa infection, she should be instructed concerning hand hygiene, the careful collection, handling, and storage of breast milk, contact precautions to be used with her infant, and the avoidance of contact with any other infants. the mother can receive several possible antibiotics for mrsa that are compatible with breastfeeding when used for a short period. if the mother remains clinically well, including without evidence of mastitis, but her breast milk is positive for mrsa greater than 10 4 cfu/ml, empiric therapy to diminish or eradicate colonization would be appropriate. various regimens have been proposed to "eradicate" mrsa colonization, but none have been proven to be highly efficacious. these regimens usually include systemic antibiotics with one or two medications (rifampin added as the second medication), nasal mupirocin to the nares twice daily for 1 to 2 weeks with routine hygiene, with or without the usage of hexachlorophene (or similar topical agent or cleanser) for bathing during the 1 to 2 week treatment period. there is no clear information concerning the efficacy of using similar colonization eradication regimens for other household members or pets in preventing recolonization of the mother or infant. before reintroducing the use of the mother' s breast milk to the infant at least two to three negative breast milk cultures should be obtained after completion of therapy. routine screening of breast milk provided by mothers for their infants in nicus for the presence of mrsa is not indicated in the absence of mrsa illness in the maternal-infant dyad, an mrsa outbreak in nicus, or a high frequency of mrsa infection in a specific nicu. one case of staphylococcal scalded skin syndrome was reported by katzman and wald 208 in an infant breastfed by a mother with a lesion on her areola that did not respond to ampicillin therapy for 14 days. subsequently the infant developed conjunctivitis with s. aureus, which produced an exfoliative toxin, and a confluent erythematous rash without mucous membrane involvement or nikolsky sign. no attempt to identify the exfoliative toxin in the breast milk was made, and the breast milk was not cultured for s. aureus. the child responded to iv therapy with nafcillin. this emphasizes the importance of evaluating mother and infant at the time of a suspected infection and the need for continued observation of the infant for evidence of a pyogenic infection or toxin-mediated disease, especially with maternal mastitis or breast lesions. this case also raises the issue of when and how infants and their mothers become colonized with s. aureus and what factors lead to infection and illness in each. the concern is that staphylococcus can be easily transmitted through skin to skin contact, colonization readily occurs, and potentially serious illness can occur later, long after colonization. in the case of staphylococcal scalded skin syndrome or toxic shock syndrome (tss), the primary site of infection can be insignificant (e.g., conjunctivitis, infection of a circumcision, or simple pustulosis), but a clinically significant amount of toxin can be produced and lead to serious disease. toxic shock syndrome can result from s. aureus or streptococcus pyogenes infection and probably from a variety of antigens produced by other organisms. tss-1 has been identified as a "superantigen" that affects the t lymphocytes and other components of the immune response, producing an unregulated and excessive immune response and resulting in an overwhelming systemic clinical response. tss has been reported in association with vaginal delivery, cesarean delivery, mastitis, and other local infections in mothers. mortality rate in the mother may be as high as 5%. the case definition of staphylococcal tss includes meeting all four major criteria: fever greater than 38.9° c, rash (diffuse macular erythroderma), hypotension, and desquamation (associated with subepidermal separation seen on skin biopsy). the definition also includes involvement of three or more organ systems (gi, muscular, mucous membrane, renal, hepatic, hematologic, or central nervous system); negative titers for rocky mountain spotted fever, leptospirosis, and rubeola; and lack of isolation of s. pyogenes from any source or s. aureus from the cerebrospinal fluid (csf). 368 a similar case definition has been proposed for streptococcal tss. 451 aggressive empiric antibiotic therapy against staphylococci and streptococci and careful supportive therapy are essential to decreasing illness and death. oxacillin, nafcillin, first-generation cephalosporins, clindamycin, erythromycin, and vancomycin are acceptable antibiotics, even for a breastfeeding mother. the severity of illness in the mother may preclude breastfeeding, but it can be reinitiated when the mother is improving and wants to restart. standard precautions, but allowing breastfeeding, are recommended. staphylococcal enterotoxin f has been identified in breast milk specimens collected on days 5, 8, and 11 from a mother who developed tss at 22 hours postpartum. 428 s. aureus that produced staphylococcal enterotoxin f was isolated from the mother' s vagina but not from breast milk. infant and mother lacked significant antibody against staphylococcal enterotoxin f in their sera. the infant remained healthy after 60 days of follow-up. staphylococcal enterotoxin f is pepsin inactivated at ph 4.5 and therefore is probably destroyed in the stomach environment, presenting little or no risk to the breastfeeding infant. 35 breastfeeding can continue if the mother is able. coagulase-negative staphylococcal infection (the predominant isolate is staphylococcus epidermidis) produces minimal disease in healthy, full-term infants but is a significant problem in hospitalized or premature infants. factors associated with increased risk for this infection include prematurity, high colonization rates in specific nurseries, invasive therapies (e.g., iv lines, chest tubes, intubation), and antibiotic use. illness produced by coagulasenegative staphylococci can be invasive and severe in high-risk neonates, but rarely in mothers. there are reports of necrotizing enterocolitis associated with coagulase-negative staphylococcus. at 2 weeks of age, for infants still in the nursery, s. epidermidis is a frequent colonizing organism at multiple sites, with colonization rates as high as 75% to 100%. serious infections with coagulase-negative staphylococci (e.g., abscesses, iv line infection, bacteremia/sepsis, endocarditis, osteomyelitis) require effective iv therapy. many strains are resistant to penicillin and the semisynthetic penicillins, so sensitivity testing is essential. empiric or definitive therapy may require treatment with vancomycin, gentamicin, rifampin, teicoplanin, linezolid, or combinations of these for synergistic activity. transmission of infection in association with breastfeeding appears to be no more common than with bottle feeding. as with s. aureus infection control includes contact and standard precautions. occasionally, during presumed outbreaks, careful epidemiologic surveillance may be required, including cohorting, limiting overcrowding and understaffing, surveillance cultures of infants and nursery personnel, reemphasis of meticulous infection control techniques for all individuals entering the nursery, and, rarely, removal of colonized personnel from direct infant contact. s. epidermidis has been identified as part of fecal microbiota of breastfed infants. 203 s. epidermidis has also been identified in the breast milk of women with clinical evidence of mastitis. 107 nevertheless, s. epidermidis is rarely associated with infection in full-term infants. conceivably breast milk for premature infants could be a source of s. epidermidis colonization in the nicus. the other factors associated with hospitalization in a nicu noted previously presumably play a significant role in both colonization and infection in premature infants. the benefits of early full human milk feeding potentially outweigh the risk for colonization with s. epidermidis via breast milk. 348 ongoing education and assistance should be provided to mothers about the careful collection, storage, and delivery of human breast milk for their premature infants. 353 s. pyogenes (β-hemolytic group a streptococcus [gas]) is a common cause of skin and throat infections in children, producing pharyngitis, cellulitis, and impetigo. illnesses produced by gas can be classified in three categories: (1) impetigo, cellulitis, or pharyngitis without invasion or complication; (2) severe invasive infection with bacteremia, necrotizing fasciitis, myositis, or systemic illness (e.g., streptococcal tss); and (3) autoimmune-mediated phenomena, including acute rheumatic fever and acute glomerulonephritis. gas can also cause puerperal sepsis, endometritis, and neonatal omphalitis. significant morbidity and mortality rates are associated with invasive gas infection; mortality rate is 20% to 50%, with almost half the survivors requiring extensive tissue débridement or amputation. 347 infants are not at risk for the autoimmune sequelae of gas (rheumatic fever or poststreptococcal glomerulonephritis). transmission is through direct contact (rarely indirect contact) and droplet spread. outbreaks of gas in the nursery are rare, unlike with staphylococcal infections. either mother or infant can be initially colonized with gas and transmit it to the other. in the situation of maternal illness (extensive cellulitis, necrotizing fasciitis, myositis, pneumonia, tss, mastitis), it is appropriate to separate mother and infant until effective therapy (penicillin, ampicillin, cephalosporins, erythromycin) has been given for at least 24 hours. breastfeeding should also be suspended and may resume after 24 hours of therapy for the mother. group b streptococcus (gbs, streptococcus agalactiae) is a significant cause of perinatal bacterial infection. in parturient women, infection can lead to asymptomatic bacteriuria, urinary tract infection (often associated with premature birth), endometritis, or amnionitis. in infants, infection usually occurs between birth and 3 months of age (1 to 4 cases per 1000 live births). it is routinely classified by the time of onset of illness in the infant: early onset (0 to 7 days, majority less than 24 hours) and late onset (7 to 90 days, generally less than 4 weeks). infants may develop sepsis, pneumonia, meningitis, osteomyelitis, arthritis, or cellulitis. early-onset gbs disease is often fulminant, presenting as sepsis or pneumonia with respiratory failure; three quarters of neonatal disease is early onset. type iii is the most common serotype causing disease. transmission is believed to occur in utero and during delivery. colonization rates of mothers and infants vary between 5% and 35%. postpartum transmission is thought to be uncommon, although it has been documented. risk factors for early-onset gbs disease include delivery before 37 weeks' gestation, rupture of membranes for longer than 18 hours before delivery, intrapartum fever, heavy maternal colonization with gbs, or low concentrations of anti-gbs capsular antibody in maternal sera. 95 the common occurrence of severe gbs disease before 24 hours of age in neonates has lead to prevention strategies. revised guidelines developed by the aap committees on infectious diseases and on the fetus and newborn 95 have tried to combine various variables for increased risk for gbs infection (prenatal colonization with gbs, obstetric and neonatal risk factors for early-onset disease) and provide intrapartum prophylaxis to those at high risk ( figure 13 -1) the utilization of these guidelines and intrapartum prophylaxis across the united states has decreased the incidence of early-onset disease by approximately 80%. in 2005, the incidence of early-onset disease was 0.35 cases per 1000 live births. 95 late-onset gbs disease is thought to be the result of transmission during delivery or in the postnatal period from maternal, hospital, or community sources. dillon et al 112 demonstrated that 10 of 21 infants with late-onset disease were colonized at birth, but the source of colonization was unidentified in the others. gardner et al 141 showed that only 4.3% of 46 children who were culture negative for gbs at discharge from the hospital had acquired gbs by 2 months of age. anthony et al 15 noted that many infants are colonized with gbs, but the actual attack rate for gbs disease is low and difficult to predict. acquisition of gbs through breast milk or breastfeeding is uncommon. cases of late-onset gbs disease associated with gbs in the maternal milk have been reported. 58, 214, 313, 366, 438 some of the mothers had bilateral mastitis, at least one had delayed evidence of unilateral mastitis, and the others were asymptomatic. it was not clear when colonization of the infants occurred or when infection or disease began in the infants. the authors discussed the possibility that the infants were originally colonized during delivery, subsequently colonized the mothers' breasts during breastfeeding, and then became reinfected at a later time. butter and demoor 56 showed that infants initially colonized on their heads at birth had gbs cultured from their throat, nose, or umbilicus 8 days later. whenever they cultured gbs from the nipples of mothers, the authors also found it in the nose or throat of the infants. byrne et al 58 presented a review of gbs disease associated with breastfeeding and made recommendations to decrease the risk for transmission of gbs to infants via breastfeeding or breast milk. some of their recommendations included confirming appropriate collection and processing procedures for gbs cultures 370 in medical facilities to decrease false-negative cultures, reviewing proper hygiene for pumping, collection, and storage of expressed breast milk with mothers, reviewing the signs and symptoms of mastitis with mothers, and utilizing banked human milk as needed instead of mother' s milk. when a breastfed infant develops late-onset gbs disease, it is appropriate to culture the milk. (see discussion of culturing breast milk earlier in this chapter.) consider treatment of the mother to prevent reinfection if the milk is culture positive for gbs (greater than 10 4 cfu/ml), with or without clinical evidence of mastitis in the mother. withholding the mother' s milk until it is confirmed to be culture negative for a pathogen is appropriate and should be accompanied by providing ongoing support and instruction to the mother concerning pumping and maintaining her milk supply. serial culturing of expressed breast milk after treatment of the mother for gbs disease or colonization would be appropriate to insure the ongoing absence of a pathogen in the expressed breast milk. there are reports of reinfection of the infant from breast milk. 23, 225 eradication of gbs mucosal colonization in the infant or the mother may be difficult. some authors have recommended using rifampin prophylactically in both the mother and infant at the end of treatment to eradicate mucosal colonization. 23 (see chapter 16 for management of mastitis in the mother.) a mother or infant colonized or infected with gbs should be managed with standard precautions 94 while in the hospital. ongoing close evaluation of the infant for infection or illness and empiric therapy for gbs in the infant are appropriate until the child has remained well and cultures are subsequently negative at 72 hours. occasionally, epidemiologic investigation in the hospital will utilize culturing medical staff and family members to detect a source of late-onset gbs disease in the nursery. this can be useful when more than one case of late-onset disease is detected with the same serotype. cohorting in such a situation may be appropriate. selective prophylactic therapy for colonized infants to eradicate colonization may be considered, but unlike gas or staphylococcus infection, gbs infection in nurseries has not been reported to cause outbreaks. no data support screening all breastfeeding mothers and their expressed breast 4 cbc including wbc count with differential and blood culture. 5 applies only to penicillin, ampicillin, or cefazolin and assumes recommended dosing regimens. 6 a healthy-appearing infant who was ≥ 38 weeks' gestation at delivery and whose mother received ≥ 4 hours of iap before delivery may be discharged home after 24 hours if other discharge criteria have been met and a person able to comply fully with instructions for home observation will be present. if any one of these conditions is not met, the infant should be observed in the hospital for at least 48 hours and until criteria for discharge are achieved. milk for gbs as a reasonable method for protecting against spread of gbs infection via expressed breast milk. selective culturing of expressed breast milk may be appropriate in certain situations. the face of tuberculosis (tb) is changing throughout the world. in the united states the incidence of tb rose during 1986 through 1993 and has been declining since then. 60 increased rates of tb were noted in adults between 25 and 45 years of age, and because these are the primary childbearing years, the risk for transmission to children increased. tb during pregnancy has always been a significant concern for patients and physicians alike. 340 it is now clear that the course and prognosis of tb in pregnancy are less affected by the pregnancy and more determined by the location and extent of disease, as defined primarily by chest radiograph, and by the susceptibility of the individual patient. untreated tb in pregnancy is associated with maternal and infant mortality rates of 30% to 40%. 365 effective therapy is crucial to the clinical outcome in both pregnant and nonpregnant women. tb during pregnancy rarely results in congenital tb. any individual in a high-risk group for tb should be screened with a tuberculin skin test (tst). no contraindication or altered responsiveness to the tst exists during pregnancy or breastfeeding. interpretation of the tst should follow the most recent guidelines, using different sizes of induration in different-risk populations as cutoffs for a positive test, as proposed by the cdc. 68 figure 13 -2 outlines the evaluation and treatment of a pregnant woman with a positive tst. 398 treatment of active tb should begin as soon as the diagnosis is made, regardless of the fetus' gestational age, because the risk for disease to mother and fetus clearly outweighs the risks of treatment. isoniazid, rifampin, and ethambutol have been used safely in all three trimesters. isoniazid and pyridoxine therapy during breastfeeding is safe, although the risk for hepatotoxicity in the mother may be a concern during the first 2 months postpartum. 391 congenital tb is extremely rare if one considers that 7 to 8 million cases of tb occur each year worldwide and that less than 300 cases of congenital tb have been reported in the literature. as with other infectious diseases presenting in the perinatal period, distinguishing congenital infection from perinatal or postnatal tb in infants can be difficult. postnatal tb infection in infancy typically presents with severe disease and extrapulmonary extension (meningitis, lymphadenopathy, and bone, liver, spleen involvement). airborne transmission of tb to infants is the major mode of postnatal infection because of close and prolonged exposure in enclosed spaces, especially in their own household, to any adult with infectious pulmonary tb. potential infectious sources could be the mother or any adult caregiver, such as babysitters, day care workers, relatives, friends, neighbors, and even health care workers. the suspicion of tb infection or disease in a household with possible exposure of an infant is a highly anxiety-provoking situation ( figure 13 -3). although protection of an infant from infection is foremost in everyone' s mind, separation of the infant from the mother should be avoided when reasonable. every situation is unique, and the best approach will vary according to the specifics of the case and accepted principles of tb management. the first step in caring for the potentially exposed infant is to determine accurately the true tb status of the suspected case (mother or household contact). this prompt evaluation should include a complete history (previous tb infection or disease, previous or ongoing tb treatment, tst status, symptoms suggestive of active tb, results of most recent chest radiograph, sputum smears, or cultures), physical examination, a tst if indicated, a new chest radiograph, and mycobacterial cultures and smears of any suspected sites of infection. all household contacts should be evaluated promptly, including history and tst with further evaluation as indicated. 68 continued risk to the infant can occur from infectious household contacts who have not been effectively evaluated and treated. an infant should be separated temporarily from the suspected source if symptoms suggest active disease or a recent tst documents conversion, and separation should continue until the results of the chest radiograph are seen. because of considerable variability in the course of illness and the concomitant infectious period, debate continues without adequate data about the appropriate period of separation. 278 this should be individualized given the specific situation. hiv testing and assessment of the risk for mdr tb should be done in every case of active tb. sensitivity testing should be done on every mycobacterium tuberculosis isolate. table 13 -1 summarizes the management of the newborn infant whose mother (or other household contact) has tb. initiation of prophylactic isoniazid therapy in the infant has been demonstrated to be effective in preventing tb infection and disease in the infant. therefore continued separation of infant and mother is unnecessary after therapy in both mother and child has begun. 114 the real risk to an infant requiring separation is from airborne transmission. separation of the infant from a mother with active pulmonary tb is appropriate, regardless of the method of feeding. however, in many parts of the world, after therapy in the mother and prophylaxis with isoniazid in the infant has begun, the infant and mother are not separated. with or without separation, the mother and infant should continue to be closely observed throughout the course of maternal therapy to ensure good compliance with medication by both mother and infant and to identify, early on, any symptoms in the infant suggestive of tb. tuberculous mastitis occurs rarely in the united states but does occur in other parts of the world* and can lead to infection in infants, frequently involving the tonsils. a mother usually has a single breast mass and associated axillary lymph node swelling and infrequently develops a draining sinus. tb of the breast can also present as a painless mass or edema. involvement of the breast can occur with or without evidence of disease at other sites. evaluation of extent of disease is appropriate, including lesion cultures by needle aspiration, biopsy, or wedge resection and milk cultures. therapy should be with multiple anti-tb medications, but surgery should supplement this, as needed, to remove extensive necrotic tissue or a persistently draining sinus. 16 neither breastfeeding nor breast milk feeding should be done until the lesion is healed, usually 2 weeks or more. continued anti-tb therapy for 6 months in the mother and isoniazid for the infant for 3 to 6 months is indicated. in the absence of tuberculous breast infection in the mother, transmission of tb through breast milk has not been documented. thus even though temporary separation of infant and mother may occur pending complete evaluation and initiation of adequate therapy in the mother and prophylactic isoniazid therapy (10 mg/kg/day as a single daily dose) in the infant, breast milk can be expressed and given to the infant during the short separation. breastfeeding can safely continue whether the mother, infant, or both are receiving anti-tb therapy. anti-tb medications (isoniazid, rifampin, pyrazinamide, aminoglycosides, ethambutol, ethionamide, p-aminosalicylic acid) have been safely used in infancy, and therefore the presence of these medications in smaller amounts in breast milk is not a contraindication to breastfeeding. although conflicting, reports indicate that breastfeeding by tst-positive mothers does influence infants' responses to bacille calmette-guérin notes: 1 further workup should always include evaluation of tb status of all other household (or close) contacts by tuberculin skin testing (tst), review of symptoms, physical examination, and chest x-ray (cxr). sputum smears and cultures should be done as indicated. 2 separation should occur until interpretation of cxr film confirms absence of active disease, or, with active disease, separation should continue until individual is no longer considered infectious: three negative consecutive sputum smears, adequate ongoing empiric therapy, and decreased fever, cough, and sputum production. separation means in a different house or location, not simply separate rooms in a household. duration of separation should be individualized for each case in consultation with tb specialist. 3 this assumes no evidence of breast involvement, suspected tb mastitis, or lesion (except in status 5, when breast involvement is considered). risk to infant is via aerosolized bacteria in sputum from the lung. expressed breast milk can be given even if separation of mother and infant is advised. 4 tst positive, no symptoms or physical findings suggestive of tb, negative cxr film. 5 prophylactic therapy: isoniazid 10 mg/kg/day, maximum 300 mg for 6 months; pyridoxine 25 to 50 mg/day for 6 months. empiric therapy: standard three-or four-drug regimens for 2 months, and treatment should continue for total of 6 months with isoniazid and rifampin when organism is shown to be sensitive. suspected multidrug-resistant (mdr) tb requires consultation with tb specialist to select optimum empiric regimen and for ongoing monitoring of therapy and clinical response. vaccine, the tst, and perhaps the m. tuberculosis bacillus. despite efforts to identify either a soluble substance or specific cell fractions (gamma/delta t cells) in colostrum and breast milk that affect infants' immune responsiveness, no unified theory explains the various reported changes and no evidence has identified a consistent, clinically significant effect. 39, 213, 319, 367 viral infections arboviruses arboviruses were originally a large collection of viruses grouped together because of the common mode of transmission through arthropods. they have now been reclassified into several different families: bunyaviridae, togaviridae, flaviviridae, reoviridae, and others. they include more than 30 human pathogens. these organisms primarily produce either cns infections (encephalitis, meningoencephalitis) or undifferentiated illnesses associated with fever and rash, severe hemorrhagic manifestations, and involvement of other organs (hepatitis, myalgia, polyarthritis). infection with this array of viruses may also be asymptomatic and subclinical, although how often this occurs is uncertain. some of the notable human pathogens include bunyaviridae (california serogroup viruses), hantavirus, hantaan virus, phlebovirus (rift valley fever), nairovirus (crimean-congo hemorrhagic fever), alphavirus (western, eastern, and venezuelan equine encephalomyelitis viruses, chikungunya virus), flavivirus (st. louis encephalitis virus, japanese encephalitis virus, dengue viruses, yellow fever virus, tick-borne encephalitis viruses), and orbivirus (colorado tick fever). other than for crimean-congo hemorrhagic fever and for reported cases of colorado tick fever associated with transfusion, direct person-to-person spread has rarely been described. recent outbreaks of chikungunya virus infection in reunion island and in india described infection in young infants probably secondary to vertical spread from mother to infant transplacentally. 146, 339, 422 a few cases of early fetal deaths were associated with infection in pregnant women. the cases of vertical transmission occurred with near-term infection in the mothers, and the infants developed illness within 3 to 7 days of delivery. 146, 339 no evidence for transmission via breast milk or breastfeeding is available. little evidence indicates that these organisms can be transmitted through breast milk. the exceptions to this include evidence of transmission of two flaviviruses via breast milk, west nile virus, and yellow fever vaccine virus. standard precautions are generally sufficient. with any of these infections in a breastfeeding mother, the severity of the illness may determine the mother' s ability to continue breastfeeding. providing the infant with expressed breast milk is acceptable. (see the discussion of west nile virus and yellow fever vaccine virus later in this chapter.) in general, treatment for these illnesses is supportive. however, ribavirin appears to decrease the severity of and mortality from hantavirus pulmonary syndrome, hemorrhagic fever with renal failure, and crimean-congo hemorrhagic fever. ribavirin has been described as teratogenic in various animal species and is contraindicated in pregnant women. no information is available concerning ribavirin in breast milk, with little information available on the use of iv or oral ribavirin in infants. arenaviruses are single-stranded ribonucleic acid (rna) viruses that infect rodents and are acquired by humans through the rodents. the six major human pathogens in this group are (1) lymphocytic 6 sensitivity testing should be done on any positive culture. 7 isoniazid 10 mg/kg/day for 3 to 9 months depending on mother's or contact's status; repeat tst at 3 months and obtain normal cxr in infant before stopping isoniazid. before beginning therapy, workup of infant for congenital or active tb may be appropriate. this workup should be determined by clinical status of infant and suspected potential risk, and may include tst after 4 weeks of age, with cxr, complete blood count, and erythrocyte sedimentation rate, liver function tests, cerebrospinal fluid analysis, gastric aspirates, sonography/computed tomography of liver/spleen, and chest if congenital tb is suspected. 8 breastfeeding is proscribed when separation of mother and infant is indicated because of risk for aerosolized transmission of bacteria. expressed breast milk given to infant via bottle is acceptable in absence of mastitis or breast lesions. 9 consult with tb specialist about mdr tb. empiric therapy will be chosen based on the most recent culture sensitivities of index patient or perhaps suspected source case, if known, as well as medication toxicities and other factors. 10 tb mastitis usually involves a single breast with associated axillary lymph node swelling and, infrequently, a draining sinus tract. it can also present as a painless mass or edema of breast. 11 with suspected mastitis or breast lesion caused by tb, even breast milk is contraindicated until lesion or mastitis heals, usually 2 weeks or more. 12 patient has a documented, recent tst conversion but has not been completely evaluated. evaluation should begin and cxr done and evaluated in less than 24 hours to minimize separation of this person from infant. further workup should proceed as indicated by symptoms, physical findings, and cxr results. choriomeningitis virus, (2) lassa fever virus, (3) junin virus (argentine hemorrhagic fever), (4) machupo virus (bolivian hemorrhagic fever), (5) guanarito virus (venezuelan hemorrhagic fever), and (6) sabia virus. the geographic distribution of these viruses and the illness they cause are determined by the living range of the host rodent (reservoir). the exact mechanism of transmission to humans is unknown and hotly debated. 25, 69, 131 direct contact and aerosolization of rodent excretions and secretions are probable mechanisms. lymphocytic choriomeningitis virus is well recognized in europe, the americas, and other areas. perinatal maternal infection can lead to severe disease in the newborn, but no evidence suggests transmission through breast milk. 28, 224 standard precautions with breastfeeding are appropriate. lassa fever (west africa) and argentine hemorrhagic fever (argentine pampas) are usually more severe illnesses with dramatic bleeding and involvement of other organs, including the brain. these fevers more frequently lead to shock and death than do the forms of hemorrhagic fever caused by the other viruses in this group. person-to-person spread of lassa fever is believed to be common, and transmission within households does occur. 212 this may relate to prolonged viremia and excretion of the virus in the urine of humans for up to 30 days. 330 the possibility of persistent virus in human urine, semen, and blood after infection exists for each of the arenaviruses. the possibility of airborne transmission is undecided. current recommendations by the cdc 69 are to use contact precautions for the duration of the illness in situations of suspected viral hemorrhagic fever. no substantial information describes the infectivity of various body fluids, including breast milk, for these different viral hemorrhagic fevers. considering the severity of the illness in mothers and the risk to the infants, it is reasonable to avoid breastfeeding in these situations if alternative forms of infant nutrition can be provided. as more information becomes available, reassessment of these recommendations is advisable. a vaccine is in clinical trials in endemic areas for junin virus and argentine hemorrhagic fever. preliminary studies suggest it is effective, but data are still being accumulated concerning the vaccine' s use in children and pregnant or breastfeeding women. cytomegalovirus (cmv) is one of the human herpesviruses. congenital infection of infants, postnatal infection of premature infants, and infection of immunodeficient individuals represent the most serious forms of this infection in children. the time at which the virus infects the fetus or infant and the presence or absence of antibodies against cmv from the mother are important determinants of the severity of infection and the likelihood of significant sequelae (congenital infection syndrome, deafness, chorioretinitis, abnormal neurodevelopment, learning disabilities). 234 about 1% of all infants are born excreting cmv at birth, and approximately 5% of these congenitally infected infants will demonstrate evidence of infection at birth (approximately five symptomatic cases per 10,000 live births). approximately 15% of infants born after primary infection in a pregnant woman will manifest at least one sequela of prenatal infection. 96 various studies have detected that 3% to 28% of pregnant women have cmv in cervical cultures and that 4% to 5% of pregnant women have cmv in their urine. 120, 172 perinatal infection certainly occurs through contact with virus in these fluids but usually is not associated with clinical illness in fullterm infants. the lack of illness is thought to result from transplacental passive transfer of protective antibodies from the mother. postnatal infection later in infancy occurs via breastfeeding or contact with infected fluids (e.g., saliva, urine) but, again, rarely causes clinical illness in full-term infants. seroepidemiologic studies have documented transmission of infection in infancy, with higher rates of transmission occurring in daycare centers, especially when the prevalence of cmv in the urine and saliva is high. cmv has been identified in the milk of cmv-seropositive women at varying rates (10% to 85%) using viral cultures or cmv deoxyribonucleic acid (dna) pcr. 172, 301, 397, 430 cmv is more often identified in the breast milk of seropositive mothers than in vaginal fluids, urine, and saliva. the cmv isolation rate from colostrum is lower than that from mature milk. 172, 396 the reason for the large degree of variability in identification of cmv in breast milk in these studies probably relates to the intermittent nature of reactivation and excretion of the virus in addition to the variability, frequency, and duration of sampling of breast milk in the different studies. some authors have hypothesized that the difference in isolation rates between breast milk and other fluids is caused by viral reactivation in cells (leukocytes or monocytes) in the breast leading to "selective" excretion in breast milk. 301 vochem et al 430 reported that the rate of virolactia was greatest at 3 to 4 weeks postpartum, and yeager et al 455 reported significant virolactia between 2 and 12 weeks postpartum. antibodies (e.g., secretory iga) to cmv are present in breast milk, along with various cytokines and other proteins (e.g., lactoferrin). these may influence virus binding to cells, but they do not prevent transmission of infection.* several studies have documented increased rates of postnatal cmv infection in breastfed infants (50% to 69%) compared with bottle-fed infants (12% to 27%) observed through the first year of life 120, 281, 397, 430 in these same studies, full-term infants who acquired cmv infection postnatally were only rarely mildly symptomatic at the time of seroconversion or documented viral excretion. also, no evidence of late sequelae from cmv was found in these infants. postnatal exposure of susceptible infants to cmv, including premature infants without passively acquired maternal antibodies against cmv, infants born to cmv-seronegative mothers, and immunodeficient infants, can cause significant clinical illness (pneumonitis, hepatitis, thrombocytopenia).* in one study of premature infants followed up to 12 months, vochem et al 430 found cmv transmission in 17 of 29 infants (59%) exposed to cmv virolactia and breastfed compared with no infants infected of 27 exposed to breast milk without cmv. no infant was given cmv-seropositive donor milk or blood. five of the 12 infants who developed cmv infection after 2 months of age had mild signs of illness, including transient neutropenia, and only one infant had a short increase in episodes of apnea and a period of thrombocytopenia. five other premature infants with cmv infection before 2 months of age had acute illness, including sepsis-like symptoms, apnea with bradycardia, hepatitis, leukopenia, and prolonged thrombocytopenia. 430 vollmer et al 431 followed premature infants with early postnatal cmv infection acquired through breast milk for 2 to 4.5 years to assess neurodevelopment and hearing function. none of the children had sensorineural hearing loss. there was no difference between the 22 cmv-infected children and 22 matched premature control cmv-negative infants in terms of neurologic, speech and language, or motor development. 431 neuberger et al 296 examined the symptoms and neonatal outcome of cmv infection transmitted via human milk in premature infants in a case-control fashion; 40 cmv-infected premature infants were compared with 40 cmv-negative matched premature infants. neutropenia, thrombocytopenia, and cholestasis were associated with cmv infection in these infants. no other serious effects or illnesses were found directly associated with the infection including intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity, necrotizing enterocolitis, bronchopulmonary dysplasia, duration of mechanical ventilation or oxygen therapy, duration of hospital stay or weight, gestational age, or head circumference at the time of discharge. exposure of cmv-seronegative or premature infants to cmv-positive milk (donor or natural mother' s) should be avoided. 379 various methods of inactivating cmv in breast milk have been reported, including holder pasteurization, freezing (−20° c for 3 days), and brief high temperature (72° c for 10 seconds). 120, 135, 155, 393, 455 one small, prospective study suggests that freezing breast milk at −20°c for 72 hours protects premature infants from cmv infection via breast milk. sharland et al 379 reported on 18 premature infants (less than 32 weeks) who were uninfected at birth and exposed to breast milk from their cmv seropositive mothers. only one of 18 (5%) infants became positive for cmv at 62 days of life, and this infant was clinically asymptomatic. this transmission rate is considerably lower than others reported in the literature. cmvseronegative and leukocyte-depleted blood products were used routinely. banked breast milk was pasteurized and stored at −20° c for various time periods and maternal expressed breast milk was frozen at −20° c before use whenever possible. the infants received breast milk for a median of 34 days (range 11 to 74 days) and they were observed for a median of 67 days (range 30 to 192 days). breast milk samples pre-or postfreezing were not analyzed by pcr or culture for the presence of cytomegalovirus. 379 buxmann et al 57 demonstrated no transmission of cmv in 23 premature infants receiving thawed frozen breast milk until 33 weeks (gestational age + postnatal age) (less than or equal to 31 weeks gestational age) born to 19 mothers who were cmv-igg negative. cmv infection was found in five premature infants of 35 infants born to 29 mothers who were cmv-igg positive and who provided breast milk for their infants. three of the five children remained asymptomatic. one child development a respirator-dependent pneumonia and the second developed an upper respiratory tract infection and thrombocytopenia in association with their cmv infections. 57 yasuda et al 454 reported on 43 preterm infants (median gestational age 31 weeks) demonstrating a peak in cmv dna copies, detected by a real-time pcr assay, in breast milk at 4 to 6 weeks postpartum. thirty of the 43 infants received cmv dna-positive breast milk. three of the 30 had cmv dna detected in their sera, but none of the three had symptoms suggestive of cmv infection. much of the breast milk had been stored at −20° c before feeding, which the authors propose is the probable reason for less transmission in this cohort. 454 lee et al 248 reported on the use of maternal milk frozen at −20°c for a minimum of 24 hours before feeding to premature infants in a nicu; 23 infants had cmv-seropositive mothers and 39 infants had cmv-seronegative mothers. two infants developed cmv infection, which was symptomatic. they were both fed frozen thawed milk from cmv-seropositive mothers. 248 others have reported individual cases of cmv infection in premature infants despite freezing and thawing breast milk. 268, 314 simple freezing and thawing of breast milk does not completely prevent transmission of cmv to premature infants. the efficacy of freezing and thawing breast milk for varying lengths of time to prevent cmv infection in premature infants has not been studied prospectively in a randomized controlled trial. eleven of 36 neonatal units in sweden (27 of which have their own milk banks) freeze maternal milk to reduce the risk for cmv transmission to premature infants. 314 a prominent group of neonatologists and pediatric infectious disease experts in california who recognize the significant benefits of providing human milk to premature and lbw infants recommend screening mothers of premature infants for cmv igg at delivery and, when an infant' s mother is cmv igg positive at delivery, using either pasteurized banked human milk or frozen then thawed maternal breast milk for premature infants until they reach the age of 32 weeks. 445 in consideration of the low rates of cmv virolactia in colostrum 169, 397 and the predominant occurrence of virolactia between 2 and 12 weeks (peak at 3 to 4 weeks) postpartum, 430,455 they reasonably propose beginning colostrum and breast milk feedings for all infants until the maternal cmv serologic screening is complete. they appropriately recommend close observation and follow-up of premature infants older than 3 weeks of age for signs, symptoms, and laboratory changes of cmv infection until discharge from the hospital. 445 cmv-seropositive mothers can safely breastfeed their full-term infants because, despite a higher rate of cmv infection than in formula-fed infants observed through the first year of life, infection in this situation is not associated with significant clinical illness or sequelae. dengue viruses (serotypes dengue 1 to 4) are flaviviruses associated primarily with febrile illnesses and rash; dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. the mosquito aedes aegypti is the main vector of transmission of dengue virus in countries lying between latitudes 35 degrees north and 35 degrees south. more than 2.5 billion people live in areas where transmission occurs; dengue virus infects over 100 million individuals a year and casuses approximately 24,000 deaths a year. 159, 163 although dengue hemorrhagic fever and dengue shock syndrome occur frequently in children younger than 1 year of age, they are infrequently described in infants younger than 3 months of age. 167 there are also differences in the clinical and laboratory findings of dengue virus infection in children compared with adults. 222 boussemart et al 49 reported on two cases of perinatal/prenatal transmission of dengue and discussed eight additional cases in neonates from the literature. prenatal or intrapartum transmission of the same type of dengue as the mother was confirmed by serology, culture, or pcr. phongsamart et al 333 described three additional cases of dengue virus infection late in pregnancy and apparent transmission to two of the three infants with passive acquisition of antibody in the third infant. sirinavin et al 386 reported on 17 cases in the literature of vertical dengue infection, all presenting at less than 2 weeks of age, but no observations or discussion of breast milk or breastfeeding as a potential source of infection were published. watanaveeradej et al 439 presented an additional three cases of dengue infection in infants documenting normal growth and development at follow-up at 12 months of age. it has been postulated that more severe disease associated with dengue disease occurs when an individual has specific igg against the same serotype as the infecting strain in a set concentration, leading to antibody-dependent enhancement of infection. the presence of preexisting dengue serotype specific igg in an infant implies either previous primary infection with the same serotype, passive acquisition of igg from the mother (who had a previous primary infection with the same serotype), or perhaps acquisition of specific igg from breast milk. watanaveeradej et al 439 documented transplacentally transferred antibodies against all four serotypes of dengue virus in 97% of 2000 cord sera at delivery. follow-up of 100 infants documented the loss of antibodies to dengue virus over time with losses of 3%, 19%, 72%, 99%, and 100% at 2, 4, 6, 9, and 12 months of age, respectively. no evidence is available in the literature about more severe disease in breastfed infants compared with formula-fed infants. no interhuman transmission of dengue virus in the absence of the mosquito vector and no evidence of transmission via breast milk are known. only one report of a factor in the lipid portion of breast milk, which inhibits the dengue virus, is available, and no evidence for antibody activity against dengue virus in human breast milk is known. 127 breastfeeding during maternal or infant dengue disease should continue as determined by the mother' s or infant' s severity of illness. epstein-barr virus (ebv) is a common infection in children, adolescents, and young adults. it is usually asymptomatic but most notably causes infectious mononucleosis and has been associated with chronic fatigue syndrome, burkitt lymphoma, and nasopharyngeal carcinoma. because ebv is one of the human herpesviruses, concern has been raised about lifelong latent infection and the potential risk for infection to a fetus and neonate from the mother. primary ebv infection during pregnancy is unusual because few pregnant women are susceptible. 149, 189 although abortion, premature birth, and congenital infection from ebv are suspected, no distinct group of anomalies is linked to ebv infection in fetus or neonate. also, no virologic evidence of ebv as the cause of abnormalities was found in association with suspected ebv infection. culturing of ebv from various fluids or sites is difficult. the virus is detected by its capacity to transform b lymphocytes into persistent lymphoblastoid cell lines. pcr and dna hybridization studies have detected ebv in the cervix and in breast milk. one study, which identified ebv dna in breast milk cells in more than 40% of women donating milk to a breast milk bank, demonstrated that only 17% had antibody to ebv (only igg, no igm). 204 another study examining serologic specimens from breastfed and bottle-fed infants showed similar seroprevalence of ebv at 12 to 23 months of age (36/66 [54.5%] and 24/43 [55.8%]) in the breastfed and bottle-fed children, respectively. 236 the question of the timing of ebv infection and the subsequent immune response and clinical disease produced requires continued study. differences exist among the clinical syndromes that manifest at different ages. infants and young children are asymptomatic, have illness not recognized as related to ebv, or have mild episodes of illness, including fever, lymphadenopathy, rhinitis and cough, hepatosplenomegaly, or rash. adolescents or young adults who experience primary ebv infection more often demonstrate infectious mononucleosis syndrome or are asymptomatic. chronic fatigue syndrome is more common in adolescents and young adults. burkitt lymphoma, observed primarily in africa, and nasopharyngeal carcinoma, seen in southeast asia, where primary ebv infection usually occurs in young children, are tumors associated with early ebv infection. 246 these tumors are related to "chronic" ebv infection and tend to occur in individuals with persistently high antibody titers to ebv viral capsid antigen and early antigen. the questions of why these tumors occur with much greater frequency in these geographic areas and what cofactors (including altered immune response to infection associated with coinfections, immune escape by ebv leading to malignancy, or increased resistance to apoptosis secondary to ebv gene mutations) may contribute to their development remain unanswered. 21, 289 it also remains unknown to what degree breast milk could be a source of early ebv infection compared with other sources of ebv infection in an infant' s environment. similar to the situation of postnatal transmission of cmv in immunocompetent infants, clinically significant illness rarely is associated with primary ebv infection in infants. more data concerning the pathogenesis of ebv-associated tumors should be obtained before proscribing against breastfeeding is warranted, especially in areas where these tumors are common but the protective benefits of breastfeeding are high. in areas where burkitt lymphoma and nasopharyngeal carcinoma are uncommon, ebv infection in mother or infant is certainly not a contraindication to breastfeeding. marburg and ebola viruses cause severe and highly fatal hemorrhagic fevers. the illness often presents with nonspecific symptoms (conjunctivitis, frontal headache, malaise, myalgia, bradycardia) and progresses with worsening hemorrhage to shock and subsequent death in 50% to 90% of patients. personto-person transmission through direct contact, droplet spread, or airborne spread is the common mode of transmission. however, the animal reservoir or source of these viruses in nature for human infection has not been identified. attack rates in families are 5% to 16%. 330 no postexposure interventions have proved useful in preventing spread, and no treatment other than supportive is currently available. a recent report documented the presence of ebola virus in numerous body fluids including in breast milk. one acute breast milk sample on day 7 after the onset of illness and a "convalescent" breast milk sample on day 15 from the same woman were positive for ebola virus by both culture and pcr testing. 30 in the same study, saliva remained virus positive for a mean of 16 days after disease onset, urine was positive for a mean of 28 days, and semen for a mean of 43 days after the onset of disease. no information is available concerning the risk for transmission of these viruses in breast milk or additional risks or benefits from breastfeeding. contact precautions are recommended for marburg virus infections and contact and airborne precautions for ebola virus infection. given the high attack and mortality rates, these precautions should be carefully instituted and breastfeeding not allowed. if any other suitable source of nutrition can be found for an infant, expressed breast milk should also be proscribed for the infant of a mother with either of these infections for at least 3 weeks postrecovery. the diagnosis of hepatitis in a pregnant woman or nursing mother causes significant anxiety. the first issue is determining the etiology of the hepatitis, which then allows for an informed discussion of risk to the fetus/infant. the differential diagnosis of acute hepatitis includes (1) common causes of hepatitis, such as hepatitis a, b, c, and d; (2) , igm anti-hbcag, anti-hcv) as the initial diagnostic tests. simultaneous consideration of other etiologies of acute liver dysfunction is appropriate depending on a patient' s history. if the initial diagnostic tests are all negative, subsequent additional testing for anti-hepatitis d virus (hdv), hcv rna, hepatis g virus (hgv) rna, anti-hepatis e virus (hev), or hev rna may be necessary. if initial testing reveals positive hbsag, testing for anti-hdv, hbeag, and hbv dna is appropriate. these additional tests are useful in defining the prognosis for a mother and the risk for infection to an infant. during the diagnostic evaluation, it is appropriate to discuss with the mother or parents the theoretic risk for transmitting infectious agents that cause hepatitis via breastfeeding. the discussion should include an evaluation of the positive and negative effects of suspending or continuing breastfeeding until the exact etiologic diagnosis is determined. the relative risk for transmission of infection to an infant can be estimated and specific preventive measures provided for the infant (table 13-2) . hepatitis a virus (hav) is usually an acute selflimited infection. the illness is typically mild, and generally subclinical in infants. occasionally, hav infection is prolonged or relapsing, extending 3 to 6 months, and rarely it is fulminant, but hav infection does not lead to chronic infection. the incidence of prematurity after maternal hav infection is increased, but no evidence to date indicates obvious birth defects or a congenital syndrome. 372, 464 hav infection in premature infants may lead to prolonged viral shedding. 349 transmission is most often person to person (fecal-oral), and transmission in food-borne or water-borne epidemics has been described. transmission via blood products and vertical transmission (mother to infant) are rare. 440 transmission in daycare settings has been clearly described. infection with hav in newborns is uncommon and does not seem to be a significant problem. the usual period of viral shedding and presumed contagiousness lasts 1 to 3 weeks. acute maternal hav infection in the last trimester or in the postpartum period could lead to infection in an infant. symptomatic infection can be prevented by immunoglobulin (ig) administration, and 80% to 90% of disease can be prevented by ig administration immune serum globulin within 2 weeks of exposure. hav vaccine can be administered simultaneously with ig without affecting the seroconversion rate to produce rapid and prolonged hav serum antibody levels. transmission of hav via breast milk has been implicated in one case report, but no data exist on the frequency of isolating hav from breast milk. 440 because hav infection in infancy is rare and usually subclinical without chronic disease and because exposure has already occurred by the time the etiologic diagnosis of hepatitis in a mother is made, no reason exists to interrupt breastfeeding with maternal hav infection. the infant should receive ig and hav vaccine, administered simultaneously. hepatitis b virus (hbv) infection leads to a broad spectrum of illness, including asymptomatic seroconversion, nonspecific symptoms (fever, malaise, fatigue), clinical hepatitis with or without jaundice, extrahepatic manifestations (arthritis, rash, renal involvement), fulminant hepatitis, and chronic hbv infection. chronic hbv infection occurs in up to 90% of infants infected via perinatal and vertical transmission and in 30% of children infected between 1 to 5 years of age. given the increased risk for significant sequelae from chronic infection (chronic active hepatitis, chronic persistent hepatitis, cirrhosis, primary hepatocellular carcinoma), prevention of hbv infection in infancy is crucial. transmission of hbv is usually through blood or body fluids (stool, semen, saliva, urine, cervical secretions). 94 vertical transmission either transplacentally or perinatally during delivery has been well described throughout the world. vertical transmission rates in areas where hbv is endemic (taiwan and japan) are high, whereas transmission to infants from hbv carrier mothers in other areas where hbv carrier rates are low is uncommon. 399 transmission of hbv to infants occurs in up to 50% of infants when the mothers are acutely infected immediately before, during, or soon after pregnancy. 462 hbsag is found in breast milk, but transmission by this route is not well documented. beasley 31 and beasley et al 32 demonstrated that although breast milk transmission is possible, seroconversion rates are no different between breastfed and nonbreastfed infants in a long-term follow-up study of 147 hbsag-positive mothers. hill et al 176 followed 101 breastfed infants and 268 formula-fed infants born to women who were chronically hbsag positive. all infants received hepatitis b immunoglobulin at birth and a full series of hepatitis b vaccine. none of the breastfed infants and nine of the formulafed infants were positive for hbsag after completion of the hbv vaccine series. breastfeeding had occurred for a mean of 4.9 months (range 2 weeks to 1 year). transmission, when it does happen, probably occurs during labor and delivery. another report from china followed 230 infants born to hbsag-positive women. the infants received appropriate dosing and timing of hbig and hbv vaccine. at 1 year of age, anti-hbs antibody was present in 90.9% of the breastfed infants and 90.3% of the bottle-fed infants. 437 risk factors associated with immunoprophylaxis failure against vertical transmission of hbv include hbeag-seropositive mothers and elevated hbv dna "viral loads" in the mothers. 392 in 2009 the aap committee on infectious diseases stated that "that breastfeeding of the infant by a hbsag-positive mother poses no additional risk for acquisition of hbv infection by the infant with appropriate administration of hepatitis b vaccine and hbig." 96 screening of all pregnant women for hbv infection is an essential first step to preventing vertical transmission. universal hbv vaccination at birth and during infancy, with administration of hepatitis b immunoglobulin (hbig) immediately after birth to infants of hbsag-positive mothers, prevents hbv transmission in more than 95% of cases. breastfeeding by hbsag-positive women is not contraindicated, but immediate administration of hbig and hbv vaccine should occur. two subsequent doses of vaccine should be given at appropriate intervals and dosages for the specific hbv vaccine product. this decreases the small theoretic risk for hbv transmission from breastfeeding to almost zero. when acute peripartum or postpartum hepatitis occurs in a mother and hbv infection is a possibility, with its associated increased risk for transmission to the infant, a discussion with the mother or parents should identify the potential risks and benefits of continuing breastfeeding until the etiology of the hepatitis can be determined. if an appropriate alternative source of nutrition is available for the infant, breast milk should be withheld until the etiology of the hepatitis is identified. hbig and hbv vaccine can be administered to the infant who has not already been immunized or has no documented immunity against hbv. 400 if acute hbv infection is documented in a mother, breastfeeding can continue after immunization has begun. acute infection with hcv can be indistinguishable from hepatitis a or b infection; however, it is typically asymptomatic or mild. hcv infection is the major cause of blood-borne non-a, non-b hepatitis (nanbh). chronic hcv infection is reported to occur 70% to 85% of the time regardless of age at time of infection. sequelae of chronic hcv infection are similar to those associated with chronic hbv infection. bortolotti et al 47 the two commonly identified mechanisms of transmission of hcv are transfusions of blood or blood products and iv drug use. however, other routes of transmission exist because hcv infection occurs even in the absence of obvious direct contact with significant amounts of blood. other body fluids contaminated with blood probably serve as sources of infection. transmission through sexual contact occurs infrequently and probably requires additional contributing factors, such as coinfection with other sexually transmitted agents or high viral loads in serum and other body fluids. studies of transmission in households without other risk factors have demonstrated either low rates of transmission or no transmission. the reported rates of vertical transmission vary widely. in mothers with unknown hiv status or known hiv infection, the rates of vertical transmission were 4% to 100%, whereas the rates varied between 0% and 42% in known hiv-negative mothers. 113 these same studies suggest that maternal coinfection with hiv, hcv genotype, active maternal liver disease, and the serum titer of maternal hcv rna may be associated with increased rates of vertical transmission. 263, 307, 461 the correlation between hcv viremia, the hcv viral load in a mother, and vertical transmission of hcv is well documented. 288, 355, 406, 456 the clinical significance and risk for liver disease after vertical transmission of hcv are still unknown. the timing of hcv infection in vertical transmission is also unknown. in utero transmission has been suggested by some studies, 125 whereas intrapartum or postpartum transmission was proposed by ohto et al 308 when they documented the absence of hcv rna in the cord blood of neonates who later became hcv rna positive at 1 to 2 months of age. more recently, gibb et al 150 reported two pieces of data supporting the likelihood of intrapartum transmission as the predominant time of vertical transmission: (a) low sensitivity of pcr for hcv rna testing in the first month of life with a marked increase in sensitivity after that for diagnosing hcv infection in infants and (b) a lower transmission risk for elective cesarean delivery (without prolonged rupture of membranes) compared with vaginal or emergency cesarean delivery. 150 another group, mcmenamin et al, 275 analyzed vertical transmission in 559 mother-infant pairs. the overall vertical transmission rate was 4.1% (18/441), with another 118 infants not tested or lost to follow-up. comparison of the vertical transmission rate was no different for vaginal delivery or emergency cesarean in labor versus planned cesarean (4.2% vs. 3.0%). this held true even when mothers had hepatitis c rna detected antenatally (7.2% vs. 5.3%). the authors did not support planned cesarean delivery to decrease vertical transmission of hepatitis c infection. no prospective, controlled trials of cesarean versus vaginal delivery and the occurrence of vertical hepatitis c transmission are available. the risk for hcv transmission via breast milk is uncertain. anti-hcv antibody and hcv rna has been demonstrated in colostrum and breast milk, although the levels of hcv rna in milk did not correlate with the titers of hcv rna in serum. 36, 162, 256, 355 nevertheless, transmission of hcv via breastfeeding (and not in utero, intrapartum, or from other postpartum sources) has not been proven in the small number infants studied. transmission rates in breastfed and nonbreastfed infants appear to be similar, but various important factors have not been controlled, such as hcv rna titers in mothers, examination of the milk for hcv rna, exclusive breastfeeding versus exclusive formula feeding versus partial breastfeeding, and duration of breastfeeding.* zanetti et al 461 including 23 infants whose mothers were seropositive for hcv rna. eight infants in that study were infected with hcv, their mothers had both hiv and hcv, and three of these eight infants were infected with both hiv and hcv. the hcv rna levels were significantly higher in the mothers coinfected with hiv compared with those mothers with hcv alone. overall, the risk for hcv infection via breastfeeding is low, the risk for hcv infection appears to be more frequent in association with hiv infection and higher levels of hcv rna in maternal serum, no effective preventive therapies (ig or vaccine) exist, and the risk for chronic hcv infection and subsequent sequelae with any infection is high. it is therefore appropriate to discuss the theoretic risk for breastfeeding in hcv-positive mothers with the mother or parents and to consider proscribing breast milk when appropriate alternative sources of nutrition are available for the infants. hiv infection is a separate contraindication to breastfeeding. additional study is necessary to determine the exact role of breastfeeding in the transmission of hcv, including the quantitative measurement of hcv rna in colostrum and breast milk, the relative risk for hcv transmission in exclusively or partially breastfed infants versus the risk in formulafed infants, and the effect of duration of breastfeeding on transmission. the current position of the cdc is that no data indicate that hcv virus is transmitted through breast milk. 83 therefore breastfeeding by a hcvpositive, hiv-negative mother is not contraindicated. infants born to hcv rna-positive mothers require follow-up through 18 to 24 months of age to determine infants' hcv status, regardless of the mode of infant feeding. infants should be tested for alanine aminotransferase and hcv rna at 3 months and 12 to 15 months of age. alanine aminotransferase and anti-hcv antibody should be tested at 18 to 24 months of age to confirm an infant' s status: uninfected, ongoing hepatitis c infection, or past hcv infection. hepatitis d virus (hdv) is a defective rna virus that causes hepatitis only in persons also infected with hbv. the infection occurs as either an acute coinfection of hbv and hdv or a superinfection of hbv carriers. this "double" infection results in more frequent fulminant hepatitis and chronic hepatitis, which can progress to cirrhosis. the virus uses its own hbv rna (circular, negative-strand rna) with an antigen, hdag, surrounded by the surface antigen of hbv, hbsag. hdv is transmitted in the same way as hbv, especially through the exchange of blood and body fluids. hdv infection is uncommon where the prevalence of hbv is low. in areas where hbv is endemic, the prevalence of hdv is highly variable. hdv is common in tropical africa and south america as well as in greece and italy but is uncommon in the far east and in alaskan inuit despite the endemic occurrence of hbv in these areas. 390 transmission of hdv has been reported to occur from household contacts and, rarely, through vertical transmission. no data are available on transmission of hdv by breastfeeding. hdv infection can be prevented by blocking infection with hbv; therefore hbig and hbv vaccine are the best protection. in addition to hbig and hbv vaccine administration to the infant of a mother infected with both hbv and hdv, discussion with the mother or parents should include the theoretic risk for hbv and hdv transmission through breastfeeding. as with hbv, once hbig and hbv vaccine have been given to the infant, the risk for hbv or hdv infection from breastfeeding is negligible. therefore breastfeeding after an informed discussion with the parents is acceptable. hepatitis e virus (hev) is a cause of sporadic and epidemic, enterically transmitted nanbh, which is typically self-limited and without chronic sequelae. hev is notable for causing high mortality rate in pregnant women. transmission is primarily via the fecal-oral route, commonly via contaminated water or food. high infection rates have been reported in adolescents and young adults (ages 15 to 40 years). tomar 416 reported that 70% of cases of hev infections in the pediatric population in india manifest as acute hepatitis. maternal-neonatal transmission was documented when the mother developed hepatitis e infection in the third trimester. although hev was demonstrated in breast milk, no transmission via breast milk was confirmed in the report. five cases of transfusion-associated hepatitis e were reported. 416 epidemics are usually related to contamination of water. person-to-person spread is minimal, even in households and day care settings. although ig may be protective, no controlled trials have been done. animal studies suggest that a recombinant subunit vaccine may be feasible. 344 hev infection in infancy is rare, and no data exist on transmission of hev by breastfeeding. no evidence of clinically significant postnatal hev infection in infants or of chronic sequelae in association with hev infection and no documented hev transmission through breast milk is available. currently no contraindication exists to breastfeeding with maternal hev infection. ig has not been shown to be effective in preventing infection, and no vaccine is available for hev. hepatitis g virus (hgv) has recently been confirmed as a cause of nanbh distinct from hepatitis viruses a through e. several closely related genomes of hgv, currently named gbv-a, -b, and -c, appear to be related to hcv, the pestiviruses, and the flaviviruses. epidemiologically, hgv is most often associated with transfusion of blood, although studies have identified nontransfusion-related cases. hgv genomic rna has been detected in some patients with acute and chronic hepatitis and a small number of patients with fulminant hepatitis. gbv-c/hgv has also been found in some patients with inflammatory bile duct lesions, but the pathogenicity of this virus is unconfirmed. hgv rna has been detected in 1% to 3% of healthy blood donors in the united states. 8 feucht et al 128 described maternal-to-infant transmission of hgv in three of nine children. two of the three mothers were coinfected with hiv and the third with hcv. none of these infants developed signs of liver disease. neither the timing nor the mode of transmission was clarified. lin et al 255 reported no hgv transmission in three mother-infant pairs after cesarean delivery and discussed transplacental spread via blood as the most likely mode of hgv infection in vertical transmission. wejstal et al 442 reported on perinatal transmission of hgv to 12 of 16 infants born to hgv viremic mothers, identified by pcr. hgv did not appear to cause hepatitis in the children. 442 fischler et al 130 followed eight children born to hgv-positive mothers and found only one to be infected with hgv. that child remained clinically well, while his twin, also born by cesarean delivery and breastfed, remained hgv negative for 3 years of observation. five of the other six children were breastfed for variable periods without evidence of hgv infection. ohto et al 309 examined hgv mother-to-infant transmission. of 2979 pregnant japanese women who were screened, 32 were identified as positive for gbv-c/hgv rna by pcr; 26 of 34 infants born to the 32 hgv positive women were shown to be hgv rna positive. reportedly, none of the infants demonstrated a clinical picture of hepatitis, although two infants had persistent mild elevations (less than two times normal) of alanine aminotransferase. the viral load in mothers, who transmitted hgv to their infants, was significantly higher than in nontransmitting mothers. infants born by elective cesarean delivery had a lower rate of infection (3 in 7) compared with infants born by emergency cesarean delivery (2 of 2) or born vaginally (21 of 25) . in this study, hgv infection in breastfed infants was four times more common than in formula-fed infants, but this difference was not statistically significant because only four infants were formula fed. the authors report no correlation between infection rate and duration of breastfeeding was seen. testing of the infants was not done frequently and early enough routinely through the first year of life to determine the timing of infection in these infants. 309 schröter et al 371 reported transmission of hgv to 3 of 15 infants born to hgv rna positive mothers at 1 week of age. none of 15 breast milk samples were positive for gbv-c/hgv rna, and all of the children who were initially negative for hgv rna in serum remained negative at follow-up between 1 to 28 months of age. 371 the foregoing data suggest that transmission is more likely to be vertical, before, or at delivery rather than via breastfeeding. the pathogenicity and the possibility of chronic disease due to hgv infection remain uncertain at this time. insufficient data are available to make a recommendation concerning breastfeeding by hgv-infected mothers. herpes simplex virus types 1 and 2 (hsv-1, hsv-2) can cause prenatal, perinatal, and postnatal infections in fetuses and infants. prenatal infection can lead to abortion, prematurity, or a recognized congenital syndrome. perinatal infection is the most common form of infection (1 in 2000 to 5000 live births, 700 to 1500 cases per year in the united states) and is often fatal or severely debilitating. the factors that facilitate intrapartum infection and predict the severity of disease have been extensively investigated. postnatal infection is uncommon but can occur from a variety of sources, including oral or genital lesions and secretions in mothers or fathers, hospital workers and home caregivers, and breast lesions in breastfeeding mothers. a number of case reports have documented severe hsv-1 or hsv-2 infections in infants associated with hsvpositive breast lesions in the mothers. 116, 161, 338, 403 cases of infants with hsv gingivostomatitis inoculating the mothers' breasts have also been reported. in the absence of breast lesions breastfeeding in hsv-seropositive or culture-positive women is reasonable when accompanied by careful handwashing, covering the lesions, and avoiding fondling or kissing with oral lesions until all lesions are crusted. breastfeeding during maternal therapy with oral or iv acyclovir can continue safely as well. inadequate information exists concerning valacyclovir, famciclovir, ganciclovir, and foscarnet in breast milk to make a recommendation at this time. breastfeeding by women with active herpetic lesions on their breasts should be proscribed until the lesions are dried. treatment of the mothers' breast lesions with topical, oral, and/or iv antiviral preparations may hasten recovery and decrease the length of viral shedding. human herpesvirus 6 (hhv-6) is a cause of exanthema subitum (roseola, roseola infantum) and is associated with febrile seizures. hhv-6 appears to be most similar to cmv based on genetic analysis. no obvious congenital syndrome of hhv-6 infection has been identified, although prenatal infection has been reported. 118 seroepidemiologic studies show that most adults have already been infected by hhv-6. therefore primary infection during pregnancy is unlikely, but reactivation of latent hhv-6 infection may be more common. no case of symptomatic hhv-6 prenatal infection has been reported. the significance of reactivation of hhv-6 in a pregnant woman and the production of infection and disease in the fetus and infant remains to be determined. primary infection in children occurs most often between 6 and 12 months of age, when maternally acquired passive antibodies against hhv-6 are waning. febrile illnesses in infants younger than 3 months of age have been described with hhv-6 infection, but infection before 3 months or after 3 years is uncommon. various studies involving serology and restriction enzyme analysis of hhv-6 isolates from mother/infant pairs support the idea that postnatal transmission and perhaps perinatal transmission from the mothers are common sources of infection. one study was unable to detect hhv-6 in breast milk by pcr analysis in 120 samples, although positive control samples seeded with hhv-6-infected cells did test positive. 119 given the limited occurrence of clinically significant disease and the absence of sequelae of hhv-6 infection in infants and children, the almost universal acquisition of infection in early childhood (with or without breastfeeding) and the absence of evidence that breast milk is a source of hhv-6 infection, breastfeeding can continue in women known to be seropositive for hhv-6. human herpesvirus 7 (hhv-7) is closely related to hhv-6 biologically. primary infection with hhv-7 occurs primarily in childhood, usually later in life than hhv-6 infection. the median age of infection is 26 months, with 75% of children becoming hhv-7 positive by 5 years of age. 63 seroprevalence of hhv-7 antibody has been reported to be 80% to 98% in adults, and passive antibody is present in almost all newborns. 306, 408 like hhv-6, hhv-7 infection can be associated with acute febrile illness, febrile seizures, and irritability, but in general it is a milder illness than with hhv-6 with fewer hospitalizations. virus excretion of hhv-7 occurs in saliva, and pcr testing of blood cells and saliva are frequently positive in individuals with past infection. 463 congenital infection of hhv-6 was detected via dna pcr testing in 57 of 5638 of cord blood samples (1%), but hhv-7 was not detected in any of 2129 cord blood specimens. 165 hhv-7 dna was detected by pcr in 3 of 29 breast milk mononuclear cell samples from 24 women who were serum positive for hhv-7 antibody. 137 in the same study, small differences were seen in the hhv-7 seropositive rates between breastfed infants and bottle-fed infants at 12 months of age (21.7% versus 20%), at 18 months of age (60% versus 48.1%), and at 24 months of age (77.3% versus 58.3%, respectively,). none of these differences were statistically significant. given that, in general, hhv-6 infection occurs earlier than hhv-7 infection in most infants and that hhv-6 is rarely found in breast milk, it seems unlikely that hhv-7 in breast milk is a common source of infection in infants and children. the infrequent occurrence of significant illness with hhv-7 infection, with the absence of sequelae except in patients who had transplantation surgery at older ages and the common occurrence of infection in childhood argue, that no reason to proscribe against breastfeeding for hhv-7 positive women exists. human papillomavirus (hpv) is a dna virus with at least 100 different types. these viruses cause warts, genital dysplasia, cervical carcinoma (types 6 and 11), and laryngeal papillomatosis. transmission occurs through direct contact and sexual contact. laryngeal papillomas are thought to result from acquiring the virus in passage through the birth canal. infection in pregnant women or during pregnancy does not lead to an increase in abortions or the risk for prematurity, and no evidence indicates intrauterine infection. hpv is one of the most common viruses in adults and one of the most commonly sexually transmitted infections. diagnosis is usually by histologic examination or dna detection. spontaneous resolution does occur, but therapy for persistent lesions or growths in anatomically problematic locations is appropriate. therapy can be with podophyllum preparations, trichloroacetic acid, cryotherapy, electrocautery, and laser surgery. interferon is being tested in the treatment of laryngeal papillomas, with mixed results. 109 prevention against transmission means limiting direct or sexual contact, but this may not be sufficient because lesions may not be evident and transmission may still occur. rintala et al 346 examined the occurrence of hpv dna in the oral and genital mucosa of infants during the first 3 years of life. hpv dna was identified in 12% to 21% of the oral scrape samples and in 4% to 15% of the genital scrape samples by pcr. oral hpv infection was acquired by 42% of children, cleared by 11%, and persisted in 10% of children; 37% of the children were never infected. they did not report on breast milk or breastfeeding in that study. the question of the source of the infection remains undetermined. the breast is a rare site of involvement. 110 hpv types 16 and 18 can immortalize normal breast epithelium in vitro. 441 hpv dna has been detected in breast milk in 10 of 223 (4.5%) of milk samples from 223 mothers, collected 3 days postpartum. 361 no attempt was made to correlate the presence of hpv dna in breast milk with the hpv status of an infant or to assess the "viral load" of hpv in breast milk or its presence over the course of lactation. a second study found dna of cutaneous and mucosal hpv types in 2 of 25 human milk samples and 1 of 10 colostrum samples. 64 no reports of hpv lesions of the breast or nipple and documented transmission to an infant secondary to breastfeeding are available. no increased risk for acquiring hpv from breast milk is apparent, and breastfeeding is acceptable. even in the rare occurrence of an hpv lesion of the nipple or breast, no data suggest that breastfeeding or the use of expressed breast milk is contraindicated. measles is another highly communicable childhood illness that can be more severe in neonates and adults. measles is an exanthematous febrile illness following a prodrome of malaise, coryza, conjunctivitis, cough, and often koplik spots in the mouth. the rash usually appears 10 to 14 days after exposure. complications can include pneumonitis, encephalitis, and bacterial superinfection. with the availability of vaccination, measles in pregnancy is rare (0.4 in 10,000 pregnancies), 148 although respiratory complications (primary viral pneumonitis, secondary bacterial pneumonia), hepatitis, or other secondary bacterial infections often lead to more severe disease in these situations. prenatal infection with measles may cause premature delivery without disrupting normal uterine development. no specific group of congenital malformations have been described in association with in utero measles infection, although teratogenic effects of measles infection in pregnant women may rarely manifest in the infants. perinatal measles includes transplacental infection when measles occurs in an infant in the first 10 days of life. infection from extrauterine exposure usually develops after 14 days of life. the severity of illness after suspected transplacental spread of virus to an infant varies from mild to severe and does not seem to vary with the antepartum or postpartum onset of rash in the mother. it is uncertain what role maternal antibodies play in the severity of an infant's disease. more severe disease seems to be associated with severe respiratory illness and bacterial infection. postnatal exposure leading to measles after 14 days of life is generally mild, probably because of passively acquired antibodies from the mother. severe measles in children younger than 1 year of age may occur because of declining passively acquired antibodies and complications of respiratory illness and rare cases of encephalitis. measles virus has not been identified in breast milk, whereas measles-specific antibodies have been documented. 1 infants exposed to mothers with documented measles while breastfeeding should be given immunoglobulin (ig) and isolated from the mother until 72 hours after the onset of rash, which is often only a short period after diagnosis of measles in the mother. the breast milk can be pumped and given to the infant because secretory iga begins to be secreted in breast milk within 48 hours of onset of the exanthem in the mother. table 13 -3 summarizes management of the hospitalized mother and infant with measles exposure or infection. 148 mumps is an acute transient benign illness with inflammation of the parotid gland and other salivary glands and often involves the pancreas, testicles, and meninges. mumps occurs infrequently in pregnant women (1 to 10 cases in 10,000 pregnancies) and is generally benign. mumps virus has been isolated from saliva, respiratory secretions, blood, testicular tissue, urine, csf in cases of meningeal involvement, and breast milk. the period of infectivity is believed to be between 7 days before and 9 days after the onset of parotitis, with the usual incubation period being 14 to 18 days. prenatal infection with the mumps virus causes an increase in the number of abortions when infection occurs in the first trimester. a small increase in the number of premature births was noted in one prospective study of maternal mumps infection. 383 no conclusive evidence suggests congenital malformations are associated with prenatal infection, not even with endocardial fibroelastosis, as originally reported in the 1960s. perinatal mumps (transplacentally or postnatally acquired) has rarely if ever been documented. natural mumps virus has been demonstrated to infect the placenta and infect the fetus, and live attenuated vaccine virus has been isolated from the placenta but not from fetal tissue in women vaccinated 10 days before induced abortion. antibodies to mumps do cross the placenta. postnatal mumps in the first year of life is typically benign. no epidemiologic data suggest that mumps infection is more or less common or severe in breastfed infants compared with formula-fed infants. although mumps virus has been identified in breast milk and mastitis is a rare complication of mumps in mature women, no evidence indicates that breast involvement occurs more frequently in lactating women. if mumps occurs in the mother breastfeeding can continue because exposure has already occurred throughout the 7 days before the development of symptoms in the mother and secretory iga in the milk may help to mitigate the symptoms in the infant. human parvovirus b19 causes a broad range of clinical manifestations, including asymptomatic infection (most frequent manifestation in all ages), erythema infectiosum (fifth disease), arthralgia and arthritis, red blood cell (rbc) aplasia (less often decreased white blood cells or platelets), chronic infection in immunodeficient individuals, and rarely myocarditis, vasculitis, or hemophagocytic syndrome. vertical transmission can lead to severe anemia and immune-mediated hydrops fetalis, which can be treated, if accurately diagnosed, by intrauterine transfusion. inflammation of the liver or cns can be seen in the infant, along with vasculitis. if the child is clinically well at birth, hidden or persistent abnormalities are rarely identified. no evidence indicates that parvovirus b19 causes an identified pattern of birth defects. postnatal transmission usually occurs person to person via contact with respiratory secretions, saliva, and rarely blood or urine. seroprevalence in children at 5 years of age is less than 5%, with the peak age of infection occurring during the schoolage years (5% to 40% of children infected). the majority of infections are asymptomatic or undiagnosed seroconversions. 417 severe disease, such as prolonged aplastic anemia, occurs in individuals with hemoglobinopathies or abnormal rbc maturation. attack rates have been estimated to be 17% to 30% in casual contacts but up to 50% among household contacts. in one study of 235 susceptible pregnant women, the annual seroconversion rate was 1.4%. 223 no reports of transmission to an infant through breastfeeding are available. excretion in breast milk has not been studied because of limitations in culturing techniques. rat parvovirus has been demonstrated in rat milk. the very low seroconversion rate in young children and the absence of chronic or frequent severe disease suggest that the risk for parvovirus infection via breast milk is not significant. the possibility of antibodies against parvovirus or other protective constituents in breast milk has not been studied. breastfeeding by a mother with parvovirus infection is acceptable. poliovirus infections (types 1, 2, and 3) cause a range of illness, with 90% to 95% subclinical, 4% to 8% abortive, and 1% to 2% manifest as paralytic poliomyelitis. a review by bates 29 from 1955 of 58 cases of poliomyelitis in infants younger than 1 month of age demonstrated paralysis or death in more than 70% and only one child without evidence of even transient paralysis. more than half the cases were ascribed to transmission from the mothers, although no mention was made of breastfeeding. breastfeeding rates at the time were approximately 25%. prenatal infection with polioviruses does cause an increased incidence of abortion. prematurity and stillbirth apparently occur more frequently in mothers who developed paralytic disease versus inapparent infection. 188 although individual reports of congenital malformations in association with maternal poliomyelitis exist, no epidemiologic data suggest that polioviruses are teratogenic. also, no evidence indicates that live attenuated vaccine poliovirus given during pregnancy is associated with congenital malformations. 89, 170 perinatal infection has been noted in several case reports of infants infected in utero several days before birth who had severe disease manifesting with neurologic manifestations (paralysis) but without fever, irritability, or vomiting. additional case reports of infection acquired postnatally demonstrate illness more consistent with poliomyelitis of childhood. these cases were more severe and involved paralysis, which may represent reporting bias. 89 no data are available concerning the presence of poliovirus in breast milk, although antibodies to poliovirus types 1, 2, and 3 have been documented. 270 in this era of increasing worldwide poliovirus vaccination, the likelihood of prenatal or perinatal poliovirus infection is decreasing. maternal susceptibility to poliovirus should be determined before conception and poliovirus vaccine offered to susceptible women. an analysis of the last great epidemic in italy in 1958 was done using a population-based case-control study. 336 in 114,000 births, 942 infants were reported with paralytic poliomyelitis. a group of matched control subjects was selected from infants admitted to the hospital at the same time. using the dichotomous variable of never breastfed and partially breastfed, 75 never-breastfed infants were among the cases and 88 among the control group. the authors determined an odds ratio of 4.2, with 95% confidence interval of 1.4 to 14, demonstrating that the risk for paralytic poliomyelitis was higher in infants never breastfed and lowest among those exclusively breastfed. because by the time the diagnosis of poliomyelitis is made in a breastfeeding mother, the exposure of the infant to poliovirus from maternal secretions has already occurred, and because the breast milk already contains antibodies that may be protective, no reason exists to interrupt breastfeeding. breastfeeding also does not interfere with successful immunization against poliomyelitis with oral or inactivated poliovirus vaccine. 71 the occurrence of human t-cell leukemia virus type i (htlv-i) is endemic in parts of southwestern japan, 66, 105, 207, 450 the caribbean, south america, 156 and sub-saharan africa. htlv-i is associated with adult t-cell leukemia/lymphoma and a chronic condition with progressive neuropathy. the progressive neuropathy is called htlv-i associated myelopathy or tropical spastic paraparesis. 136 other illnesses have been reported in association with htlv-i infection including dermatitis, uveitis, arthritis, sjögren syndrome in adults, and infective dermatitis and persistent lymphadenitis in children. transmission of htlv-i occurs most often through sexual contact, via blood or blood products, and via breast milk. infrequent transmission does occur in utero or at delivery and with casual or household contact. 291 seroprevalence generally increases with age and varies widely in different regions and in populations of different backgrounds. in some areas of japan, seropositivity can be as high as 12% to 16%, but in south america, africa, and some caribbean countries the rates are 2% to 6%. in latin america seropositive rates can be as high as 10% to 25% among female sex workers or attendees to std clinics. 156 in blood donors in europe, the seroprevalence of htlv-i has been reported at 0.001% to 0.03%. the seroprevalence in pregnant women in endemic areas of japan is as high as 4% to 5% and in nonendemic areas as low as 0.1% to 1.0%. htlv-1 is not a major disease in the united states. in studies from europe the seroprevalence in pregnant women has been noted to be up to 0.6%. these pregnant women were primarily of african or caribbean descent. 138 htlv-i antigen has been identified in breast milk of htlv-i positive mothers. 220 another report shows that basal mammary epithelial cells can be infected with htlv-i and can transfer infection to peripheral blood monocytes. 254 human milk from htlv-i positive mothers caused infection in marmosets. 221, 453 htlv-i infection clearly occurs via breastfeeding and a number of reports document an increased rate of transmission of htlv-i to breastfed infants compared with formula-fed infants.* ando et al 12, 13 in two separate reports demonstrated a parallel decline in antibodies against htlv-i in both formula-fed and breastfed infants to a nadir at approximately 1 year of age and a subsequent increase in antibodies from 1 to 2 years of age. the percentage of children seropositive at 1 year of age in the breastfed and formula-fed groups, respectively, was 3.0% and 0.6%, at 1.5 years of age it was 15.2% and 3.9%, and at 2 years of age it was 41.9% and 4.6%. a smaller group of children followed through 11 to 12 years of age demonstrated no newly infected children after 2 years of age and *references 9, 10, 12, 13, 178-180, 407. no loss of antibody in any child who was seropositive at 2 years of age. 12, 13 transmission of htlv-i infection via breastfeeding is also clearly associated with the duration of breastfeeding. 407, 409, 446, 447 it has been postulated that the persistence of passively acquired antibodies against htlv-i offers some protection through 6 months of life (table 13-4) . other factors relating to htlv-i transmission via breast milk have been proposed. yoshinaga et al 460 presented data on the htlv-i antigen producing capacity of peripheral blood and breast milk cells and showed an increased mother-to-child transmission rate when the mother' s blood and breast milk produced large numbers of antigen-producing cells in culture. 460 hisada et al 183 reported on 150 mothers and infants in jamaica, demonstrating that a higher maternal provirus level and a higher htlv-i antibody titer were independently associated with htlv-i transmission to the infant. ureta-vidal et al 421 reported an increased seropositivity rate in children of mothers with a high proviral load and elevated maternal htlv-i antibody titers. various interventions have been proposed to decrease htlv-i transmission via breastfeeding. complete avoidance of breastfeeding was shown to be an effective intervention by hino et al 180, 181 in large population of japanese in nagasaki. avoiding breastfeeding led to an 80% decrease in transmission. breastfeeding for a shorter duration is another effective alternative. ando et al 11 showed that freezing and thawing breast milk decreased the infectivity of htlv-i. sawada et al 363 demonstrated in a rabbit model that htlv-i immunoglobulin protected against htlv-i transmission via milk. it is reasonable to postulate that any measure that would decrease the maternal provirus load or increase the anti-htlv-i antibodies available to infants might decrease the risk for transmission. the overall prevalence of htlv-i infection during childhood is unknown because the majority of individuals do not manifest illness until much later in life. the timing of htlv-i infection in a breastfeeding population has been difficult to assess because of passively acquired antibodies from the mother and issues related to testing. furnia et al 139 in areas where the prevalence of htlv-i infection (in the united states, canada, or europe) is rare, the likelihood that a single test for antibody against htlv-i would be a false positive test is high compared with the number of true positive tests. repeat testing is warranted in many situations. 66 quantification of the antibody titer and the proviral load is appropriate in a situation when mother-to-child transmission is a concern. a greater risk for progression to disease in later life has not been shown for htlv-i infection through breast milk, but early-life infections are associated with the greatest risk for adult t-cell leukemia. 402 the mother and family should be informed about all these issues. if the risk for lack of breast milk is not too great and formula is readily available and culturally acceptable, then the proscription of breastfeeding, or at least a recommendation to limit the duration of breastfeeding to 6 months or less, is appropriate to limit the risk for htlv-i transmission to the infant. freezing and thawing breast milk before giving it to an infant might be another reasonable intervention to decrease the risk for transmission, although no controlled trials document the efficacy of such an intervention. neither ig nor antiviral agents against htlv-i are available at this time. human t-cell leukemia virus type ii (htlv-ii) is endemic in specific geographic locations, including africa, the americas, the caribbean, and japan. transmission is primarily through intravenous drug use, contaminated blood products, and breastfeeding. sexual transmission occurs but its overall contribution to the prevalence of htlv-ii in different populations remains uncertain. many studies have examined the presence of htlv-i and ii in blood products. pcr testing and selective antibody tests suggest that about half of the htlv seropositivity in blood donors is caused by htlv-ii. htlv-ii has been associated with two chronic neurologic disorders similar to those caused by htlv-i, tropical or spastic ataxia. 258 a connection between htlv-ii and glomerulonephritis, myelopathy, arthritis, t-hairy cell leukemia, and large granulocytic leukemia has been reported. mother-to-child transmission has been demonstrated in both breastfed and formula-fed infants. it appears that the rate of transmission is greater in breastfed infants.* htlv-ii has been detected in breast milk. 174 nyambi et al 304 reported that htlv-ii transmission did correlate with the duration of breastfeeding. the estimated rate of transmission was 20%. the time to seroconversion (after the initial loss of passively acquired maternal antibodies) for infected infants seemed to range between 1 and 3 years of age. 304 at this time avoidance of breastfeeding and limiting the duration of breastfeeding are the only two possible interventions with evidence of effectiveness for preventing htlv-ii mother-to-child transmission. 207 with the current understanding of retroviruses, it is appropriate in cases of documented htlv-ii maternal infection to recommend avoiding or limiting the duration of breastfeeding and provide alternative nutrition when financially practical and culturally acceptable. mothers should have confirmatory testing for htlv-ii and measurement of the proviral load. infants should be serially tested for antibodies to htlv-ii and have confirmatory testing if seropositive after 12 to 18 months of age. further investigation into the mechanisms of transmission via breast milk and possible interventions to prevent transmission should occur as they have for hiv-1 and htlv-i. human immunodeficiency virus type 1 (hiv-1) is transmitted through human milk. refraining from breastfeeding is a crucial aspect of preventing perinatal hiv infection in the united states and many other countries. the dilemma is the use of replacement feeding versus breastfeeding in countries where breastfeeding provides infants with significant protection from illness and death due to malnutrition or other infections. the question of the contribution of breastfeeding in mother-to-child hiv-1 transmission is not a trivial one when one considers the following: 3. the who estimates that 2.7 million people were newly infected with hiv-1 in 2007, with children younger than 14 years old making up 370,000 of that 2.7 million. (this number has declined due to increasing access to interventions to prevent mother-to-infant transmission. availability of antiretroviral therapy for prevention of mother-to-child hiv transmission in developing countries in 2007 was estimated to reach 33% of the mothers who needed it.) 419 4. breastfeeding contributes an estimated 10% to 20% increase in the overall mother-to-child transmission rates, over and above intrauterine and intrapartum transmission, when no specific interventions to prevent transmission via breastfeeding are utilized. 5. despite a dramatic increase in the number of people receiving antiretroviral therapy in developing countries (3 million), this represented only 31% of the individuals who needed treatment. 419 the evidence of hiv transmission via breastfeeding is irrefutable. multiple publications summarize the current evidence for hiv transmission via breastfeeding in the literature. 232, 341, 420 since 1985, case reports have documented hiv transmission via breast milk to children around the world. 182, 198, 249, 465 primary hiv infection in breastfeeding mothers, with the concomitant high viral load, is associated with a particularly high rate of hiv transmission via breast milk. palasanthiran et al 322 estimated that risk at 27%.large observational studies have demonstrated higher rates of hiv transmission in breastfed infants of mothers with chronic hiv infection compared with formula-fed infants. 43, 108, 124 a systematic analysis of published reports estimated the additional risk for perinatal hiv transmission due to breastfeeding to be 14% (95% confidence interval 7% to 22%). 117 more recently published cohort studies similarly attributed additional risk for hiv transmission due to breastfeeding at 4% to 22% over and above the risk from prenatal and intrapartum transmission. 38, 104, 121 laboratory reports demonstrate the presence of cell-free virus and cell-associated virus in breast milk as well as various immunologic factors that could block or limit infection.* a dose-response relationship has been observed, correlating the hiv viral load in human milk as well as a mother' s plasma viral load with an increased transmission risk for the breastfed infant. 335, 345, 351, 373 many of the potential risk factors associated with human milk transmission of hiv is higher the longer the duration of breastfeeding. 108, 251, 282, 290, 424 maternal characteristics related to transmission of hiv via human milk include younger maternal age, higher parity, lower cd4+ counts, higher plasma viral loads, and breast abnormalities (mastitis, abscess, or nipple lesions). characteristics of human milk that relate to a higher risk for transmission include higher viral load in the milk, lower concentrations of antiviral substances (lactoferrin, lysozyme), and lower concentrations of virus-specific cytotoxic t-lymphocytes, levels of various interleukins (il-7, il-15), 434, 435 secretory iga, and igm. mixed breastfeeding is also associated with a higher risk for hiv transmission compared with exclusive breastfeeding. 99, 100, 410 the measurable benefits of breast milk versus the relative risk for hiv transmission to the infant due to exclusive breastfeeding (with optimization of other factors to decrease hiv transmission) have been reported in a couple of studies. 97, 229 the measurable benefits of receiving breast milk versus the relative increased risk for hiv transmission will need to be determined in a prospective fashion in different locales. 247 a number of potential interventions to prevent breastfeeding transmission of hiv-1 can be utilized (box 13-3) . the simplest and most effective is the compete avoidance of human milk. this is a practical solution in places like the united states and other countries where replacement feeding as well as other strictly medical interventions are feasible and reasonable, and the risk for not providing breast milk to the infant is negligible. in resourcepoor situations, where the risk for other infections is high without the benefits of breast milk, exclusive breastfeeding is appropriate, with any other reasonable and culturally acceptable interventions to decrease hiv transmission via breast milk. potentially effective interventions include exclusive breastfeeding, early weaning versus breastfeeding for longer durations, education, and support to decrease the likelihood of mastitis or nipple lesions. 191 other possible interventions include treating a mother with antiretroviral therapy for her own health (cd4 counts less than 350) or prophylactically to decrease the human milk viral load, treating an infant prophylactically for a prolonged period of time (6 weeks to 6 months) to protect against transmission via breastfeeding, treating the milk itself to decrease the viral load (by pasteurization or other methods), 316, 318 treating acute conditions in mothers and infants (e.g., mastitis, breast lesions, infant candidiasis), and enhancing an infant' s own defenses via vitamins, immunization, or antiretroviral therapy. some of these may not be feasible in certain settings such as pasteurization or maternal antiretroviral therapy. others may not be culturally acceptable, such as treating expressed breast milk before giving it to an infant or even exclusive breastfeeding. significant data demonstrate the advantage of breastfeeding, even for hiv-infected or hivexposed infants. the complete avoidance of breastfeeding in certain situations may lead to increased risk for illness and death due to other reasons besides hiv transmission. 106 a study from kenya showed improved hiv-1-free survival rates in a formula-fed group of children born to hiv-positive mothers, but the breastfed and formula groups had similar mortality rates (24.4% versus 20.0%, respectively) and similar incidences of diarrhea and pneumonia in the first 2 years of life. 272 no difference in the two groups was seen in the prevalence of malnutrition, but the breastfed infants had better nutritional status in the first 6 months of life. arpadi et al 20 recommend additional nutritional interventions to complement breastfeeding in this population after 6 months of age. two reports from zambia document the benefit of exclusive breastfeeding for decreasing late hiv transmission and the lower mortality at 12 months in infants who had continued breastfeeding rather than had discontinued breastfeeding at 4 months of age. 229, 385 in malawi, hiv-infected and hiv-exposed infants who were breastfed (exclusive breastfeeding for 2 months and mixed feeding after that) had lower mortality at 24 months than those who were not breastfed. 405 a report from botswana examined breastfeeding plus infant zidovudine prophylaxis for 6 months versus formula feeding plus infant zidovudine for 1 month; this study showed a decreased risk for vertical transmission with formula feeding, but also increased cumulative mortality for the hiv-infected infants at 7 months of age who were in the formula-fed group. 411 a study from south africa examining the use of vitamin a also demonstrated less morbidity in hiv-infected children who were breastfed than not breastfed. 102 other abstract reports have shown increased morbidity in hiv-infected children due to diarrhea, gastroenteritis, and hospitalization after weaning from breastfeeding. 205, 226, 315, 413 exclusive breastfeeding in most areas of the world is essential to infant health and survival, even in the situation of maternal hiv infection. 97, 99, 100, 229 the duration of exclusive breastfeeding is crucial to decreasing the risk for hiv infection in infants versus the risk for malnutrition and other infections with early weaning. in the mashi study in botswana, thior et al 411 evaluated infants randomized to breastfeeding plus infant zidovudine for 6 months or formula feeding plus 1 month of infant zidovudine. the cumulative infant mortality was significantly higher at 7 months for the formula-fed group but at 18 months it was similar between the two groups. the breastfed infants were more likely to become hiv infected despite the 6 months of zidovudine prophylaxis. 411 becquet et al 33 analyzed data from cote d'ivoire for 2001 to 2005; 47% of the hivexposed infants were breastfed for a median of 4 months, and 53% were formula fed and observed for 2 years. no significant difference in the rate of hiv infection was seen in the two groups, and no significant difference between the two groups was seen for morbid events (diarrhea, acute respiratory infections or malnutrition) or hospitalization or death. the authors attributed these good outcomes to effective nutritional counseling and care, access to clean water, and the provision of a safe and continuous supply of breast milk substitute. 33 coovadia et al 97 studied exclusive breastfeeding in the first 6 months of life as an intervention in south africa. of the exclusively breastfed infants, 14.1% at 6 weeks of age and 19.5% at 6 months of age were hiv infected. breastfed infants who also were fed solids or formula milk were more likely to acquire infection than exclusively breastfed infants. the cumulative mortality at 3 months of age was markedly lower for exclusively breastfed infants (6.1%) versus 15.1% in the infants receiving mixed feedings. kuhn et al 230 examined the effects of early, abrupt weaning on hiv-free survival of 958 children in zambia. infants were randomly assigned to two different counseling programs that advised either abrupt weaning at 4 months or prolonged breastfeeding. in the weaning intervention group, 69% of mothers stopped breastfeeding by 5 months compared with a median duration of breastfeeding of 16 months in the control group. the study found no significant difference in hiv-free survival at • women and their health care providers need to be aware of the potential risk for transmission of hiv infection to infants during pregnancy and in the peripartum period and through breast milk. • documented, routine hiv education and routine testing with the consent of women seeking prenatal care are strongly recommended so that each woman knows her hiv status and the methods available both to prevent the acquisition and transmission of hiv and to determine whether breastfeeding is appropriate. • at delivery, education about hiv and testing with the consent of women whose hiv status during pregnancy is unknown are strongly recommended. knowledge of a woman' s hiv status assists in counseling on breastfeeding and helps each woman understand the benefits to herself and her infant of knowing her serostatus and the behaviors that would decrease the likelihood of acquisition and transmission of hiv. • women who are known to have hiv infections must be counseled not to breastfeed or provide their milk for the nutrition of their own or other' s infants. • in general, women who are known to be hiv seronegative should be encouraged to breastfeed. however, women who are hiv seronegative but at particularly high risk for seroconversion (e.g., injection drug users and sexual partners of known hiv-positive persons or active drug users) should be educated about hiv with an individualized recommendation concerning the appropriateness of breastfeeding. in addition, during the perinatal period, information should be provided on the potential risk for transmitting hiv through breast milk and about methods to reduce the risk for acquiring hiv infection. • each woman whose hiv status is unknown should be informed of the potential for hiv-infected women to transmit hiv during the peripartum period and through breast milk and the potential benefits to her and her infant of knowing her hiv status and how hiv is acquired and transmitted. the health care provider needs to make an individualized recommendation to assist the woman in deciding whether to breastfeed. 24 months in the two groups (83.9% versus 80.7%). children already infected by 4 months of age had a higher mortality if they were assigned to the early weaning group (73.6% versus 54.8%). additional analysis showed that in mothers with less severe hiv disease early weaning was clearly harmful to the infant. 231 arpadi et al 20 studied the growth of hiv-exposed, uninfected children who were exclusively breastfed for 4 months with rapid weaning to replacement foods or exclusively breastfed until 6 months and then continued breastfeeding with complementary foods. weight-for-age z scores dropped markedly in both groups from 4 to 15 months of age but less so in the continued breastfeeding group. length-for-age z score also dropped dramatically, but was not influenced by continued breastfeeding. even in this hiv-exposed, uninfected group of children, additional nutritional interventions are essential to complement breastfeeding beyond 6 months of age. 20 in recent years the discussion around preventing hiv transmission via breastfeeding and in the number of studies examining the important issues have increased. 98, 233, 283 the fact that intrapartum and perinatal transmission of hiv from mothers to infants has decreased markedly due to the increased utilization of antiretroviral therapy during pregnancy, delivery, and postnatally for prevention emphasizes the importance of now working harder to decrease breast milk transmission of hiv. in considering different possible interventions to decrease mother-infant hiv transmission, it is crucial to reemphasize the goals of optimizing maternal health and survival and optimizing infant health and survival at the same time. a laboratory report shows that mothers receiving highly active antiretroviral therapy (haart) while breastfeeding do have decreased whole breast milk hiv-1 viral loads (23/26 mothers had less than 50 copies/ml) compared with mothers who did not receive haart (9/25 with less than 50 copies/ml). however, the whole milk hiv-1 dna load (measured as "undetectable" at less than 10 copies/10 6 cells) was not significantly different in the haart (13 of 26 mothers)] and non-haart (15 of 23) groups. 378 hiv-1 dna is incorporated into cells found in breast milk. another group showed significantly lower hiv rna levels in the breast milk of women treated with nevirapine, zidovudine, and lamivudine compared with women not receiving antiretroviral therapy. 152 the use of maternal haart seems to decrease hiv-1 transmission via breastfeeding. one group working in mozambique, malawi, and tanzania working with mother-infants pairs receiving haart as prevention during pregnancy compared one cohort (809 mother-infant pairs) who received supplementary formula and water filters for the first 6 months of life with a second cohort (251 motherinfant pairs) breastfeeding exclusively and the mothers receiving haart for the first 6 months. the cumulative incidence rate of hiv infection at 6 months of age was 2.7% for the formula-fed infants and 2.2% for breastfed infants. through 6 months of age no apparent additional risk for late postnatal transmission of hiv was observed. 323 the petra study team working in tanzania, south africa, and uganda examined the efficacy of three shortcourse regimens of zidovudine and lamivudine in preventing early and late hiv transmission in this predominantly breastfeeding population. 332 there were four regimens: a, zidovudine and lamivudine starting at 36 weeks' gestation plus intrapartum medication and 7-days' postpartum treatment; b, same as a without the prepartum component; c, intrapartum zidovudine and lamivudine only; d, placebo. at week 6 the hiv transmission rates were 5.7% in group a, 8.9% in group b, 14.2% in group c, and 15.3% in group d. at 18 months the hiv infection rates were 15% in group a, 18% in group b, 20% in group c, and 22% in group d. although a measurable decrease in transmission at 6 weeks of age was observed, limited protection was seen at 18 months of age. an observational study from tanzania compared maternal haart for 6 months with exclusive breastfeeding and abrupt weaning at 5 to 6 months of age with a historical control of the same feeding schedule without the postnatal maternal haart. in the treatment group the cumulative hiv transmission was 4.1% at 6 weeks, 5.0% at 6 months, and 6.0% at 18 months of age. the cumulative hiv infection or death rate was 8.6% at 6 months and 13.6% at 18 months of age. the cumulative risk for hiv transmission was 1.1% between 6 and 18 months. the hiv transmission in this treatment group was half the transmission rate in the historical control group. 218 another study in sub-saharan africa with 6 months of maternal haart and exclusive breastfeeding for 6 months demonstrated 94% hiv-free survival at 12 months of age; the maternal and infant mortality rates for the treated mother-infant pairs were significantly lower than the country' s maternal and infant mortality rates. 264 antiretroviral therapy prophylaxis for infants is another investigated intervention to decrease hiv transmission via breastfeeding. in a study from cote d'ivoire comparing different groups over time, infants received zidovudine (zdv) alone as zdv prophylaxis, a single dose of nevirapine (nvp), and 7 days of zidovudine (zdv) as nvp/ zdv prophylaxis, or a single dose of nevirapine plus zidovudine and lamivudine (3tc) for 7 days as nvp/zdv/3tc prophylaxis. formula feeding (ff) was compared with exclusive shortened breastfeeding (esb) upto 4 months of age and prolonged breastfeeding (pb). the cumulative transmission rates at 18 months were 22.3% in 238 infants in the zdv + pb group, 15.9% in 169 infants in the nvp/zdv + esb group, 9.4%, in the 195 infants in the nvp/zdv +ff group, 6.8% in the 198 infants in the nvp/zdv/3tc + esb group, and 5.6% in the 126 infants in the nvp/zdv/3tc + ff group. 252 kumwenda et al 235 working in malawi demonstrated decreased hiv transmission with breastfeeding and two different infant prophylaxis regimens. at 9 months of age, they observed a 10.6% occurrence of hiv transmission for infants receiving a single dose of nevirapine plus 1 week of zidovudine compared with 5.2% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of daily nevirapine, and 6.4% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of nevirapine and zidovudine. 235 in the mitra study in tanzania in which the median time of breastfeeding was 18 weeks, the hiv transmission rate at 6 months in the infants who received zidovudine plus 3tc for 1 week plus 3tc alone for breastfeeding through 6 months of age was less than 50% of the transmission rate for those infants receiving only 1 week of zidovudine plus 3tc. 217 a summary of three trials in ethiopa, india, and uganda compared a single dose of nevirapine at birth for infants with 6 weeks of daily nevirapine in predominantly breastfed infants whose mothers were counselled regarding feeding per the who/ unicef guidelines. at 6 months 87 of 986 infants in the single-dose group and 62 of 901 in the extended-dose group were hiv infected, which was not statistically significant. the authors suggested that a longer course of infant antiretroviral prophylaxis might be more effective. 388 the potential effect of breastfeeding on the hivpositive mother needs to be adequately assessed in relation to the mother's health status. from uganda and zimbabwe mbizvo et al 271 reported no difference in the number of hospital admissions or mortality between hiv-positive and hiv-negative women during pregnancy. in the 2 years after delivery the hiv-positive women had higher hospital admission (approximately two times increased risk) and death rates (relative risk greater than 10) than hiv-negative women. 271 chilongozi et al 90 reported on 2292 hiv-positive mothers from four sub-saharan sites followed for 112 months. serious adverse events occurred in 166 women (7.2%); 42 deaths occurred in the hiv-positive women, and no deaths occurred in 331 hiv-negative women. 90 several studies have examined breastfeeding relative to mothers' health and reported conflicting results. the first study from kenya demonstrated a significantly higher mortality rate in breastfeeding mothers compared with a formula-feeding group in the 2 years after delivery. the hypothesized explanation offered by the authors for this difference was increased metabolic demands, greater weight loss, and nutritional depletion. 294 a second study from south africa showed an overall lower mortality rate in the two groups with no significant difference in mortality rate in the 10 months of observation. 101 kuhn et al 227 reported no difference in mortality at 12 months after delivery between 653 women randomly assigned to a short breastfeeding group (326 women, median breastfeeding duration 4 months, 21% still breastfeeding at 12 months) and a long breastfeeding group (327 women, 90% breastfeeding at 5 months, 72% breastfeeding at 12 months, median 15 months). the hiv-related mortality rates were high (4.9%), but not associated with prolonged lactation. 227 walson et al 433 followed 535 hiv-positive women for 1 to 2 years in kenya. the mortality risk was 1.9% at 1 year and 4.8% at 2 years of follow-up. although less than 10% of women reported a hospitalization during the 2 years, they experienced various common infections (pneumonia, diarrhea, tb, malaria, stds, urinary tract infections, mastitis). breastfeeding was a significant cofactor for diarrhea and mastitis but not for pneumonia, tb, or hospitalization. 433 in summary, breastfeeding of infants by hiv-positive mothers does lead to an increased risk for hiv infection in the infants. much remains to be understood about the mechanisms of hiv transmission via breast milk and the action and efficacy of different interventions to prevent such transmission. the complete avoidance of breastfeeding is a crucial component for the prevention of perinatal hiv infection in the united states and many other countries. in resource-poor settings, where breastfeeding is the norm and where it provides vital nutritional and infection protective benefits, the who, unicef, and the joint united nations programme on hiv/ aids (unaids) recommend education, counseling, and support for hiv-infected mothers so they can make an informed choice concerning infant feeding. mothers choosing to breastfeed should receive additional education, support, and medical care to minimize the risk for hiv transmission and to optimize their own health status during and after breastfeeding. mothers choosing to use replacement feedings should receive parallel education, support, and medical care for themselves and their infants to minimize the effect of the lack of breastfeeding. good evidence now shows that antiretroviral prophylactic regimens for mothers or infants while continuing breastfeeding does decrease postnatal hiv transmission. early weaning is associated with increased morbidity and mortality. further carefully controlled research is indicated to adequately assess the risks and benefits to infants and mothers of prolonged breastfeeding with antiretroviral prophylaxis for either or both mothers and infants. along with this, hiv testing rates must be improved at the same time as increased availability and access to antenatal care, hiv prevention services, and hiv medical care for everyone must be increased. the availability and free access to antiretroviral medications must also improve. the decision about infant feeding for hivpositive mothers remains a difficult one, but this is slowly changing with increasing options. the goals remain 100% hiv transmission prevention, optimal maternal health and survival, and long-term infant health and survival. human immunodeficiency virus type 2 (hiv-2) is an rna virus in the nononcogenic, cytopathic lentivirus genus of retroviruses. it is genetically closer to simian immunodeficiency virus than to hiv-1. the clinical disease associated with hiv-2 has similar symptoms to hiv-1 infection but progresses at a slower rate to severe immunosuppression. hiv-2 is endemic in western africa and parts of the caribbean and found infrequently in europe and north and south america. 190, 305 it is transmitted via sexual contact, blood, or blood products and from mother to child. routine testing for hiv-2 is recommended in blood banks. antibody tests used for hiv-1 are only 50% to 90% sensitive for detecting hiv-2. 65 specific testing for hiv-2 is appropriate whenever clinically or epidemiologically indicated. vertical transmission occurs infrequently. ekpini et al 121 followed a large cohort of west african mothers and infants: 138 hiv-1 positive women, 132 hiv-2 positive women, 69 women seropositive for both hiv-1 and 2, and 274 hiv seronegative women. a few cases of perinatal hiv-2 transmission occurred, but no case of late postnatal transmission was observed. 121 it is probable that hiv-2 transmission via breast milk is less common than with hiv-1, but insufficient data support that the risk for transmission is zero. mothers who test positive for hiv-2 should be tested for hiv-1, and guidelines for breastfeeding should follow those for hiv-1 until additional information is available. rabies virus produces a severe infection with progressive cns symptoms (anxiety, seizures, altered mental status) that ultimately proceeds to death; few reports of survival exist. rabies occurs worldwide except in australia, antarctica, and several island groups. in 1992 more than 36,000 cases of rabies were reported to the who, a number that is probably a marked underestimate of the actual cases. 67 between 1990 and 2003, 37 cases of human rabies were reported in the united states. 70, 78 postexposure prophylaxis is given to thousands of patients each year. rabies virus is endemic in various animal populations, including raccoons, skunks, foxes, and bats. because of aggressive immunization programs, rabies in domesticated dogs and cats in the united states is uncommon. the virus is found in the saliva and tears and nervous tissue of infected animals. transmission occurs by bites, licking, or simply contact of oral secretions with mucous membranes or nonintact skin. many cases of rabies in humans now lack a history of some obvious contact with a rabid animal. this may be a result of the long incubation period (generally 4 to 6 weeks, but can be up to 1 year, with reports of incubation periods of several years), a lack of symptoms early in an infectious animal, or airborne transmission from bats in enclosed environments (caves, laboratories, houses). person-to-person transmission via bites has not been documented, although it has occurred in corneal transplants. 44 rabies viremia has not been observed in the spread of the virus. no evidence exists indicating transmission through breast milk. in the case of maternal infection with rabies, many scenarios can occur before the onset of progressive, severe cns symptoms. the progression and severity of maternal illness can preclude breastfeeding, but separation of an infant from the mother is appropriate regardless of the mother' s status and method of infant feeding (especially to avoid contact with saliva and tears). breastfeeding should not continue when the mother has symptoms of rabies, and the infant should receive postexposure immunization and close observation. an infant may received expressed breast milk, but the expression must occur without possible contamination with saliva or tears from the mother. depending on the scenario, the nature of a mother' s illness, the possible exposure of an infant to the same source as the mother, and the exposure of a child to the mother, postexposure immunization of an infant may be appropriate. a more common scenario is a mother' s apparent exposure to rabies (without exposure for the infant), necessitating postexposure immunization of the mother with rabies vaccine. in the majority of cases, in the absence of maternal illness, breastfeeding can reasonably continue during the mother' s five-dose immunization series in 28 days. in a rare situation in which apparent exposure of mother and infant to rabies occurs together, postexposure treatment of both mother and infant should be instituted, and breastfeeding can continue. respiratory syncytial virus (rsv) is a common cause of respiratory illness in children and is relatively common in adults, usually producing milder upper respiratory tract infection in adults. no evidence indicates that rsv causes intrauterine infection, adversely affects the fetus, or causes abortion or prematurity. rsv does produce infection in neonates, causing asymptomatic infection, afebrile upper respiratory tract infection, bronchiolitis, pneumonia, and apnea. mortality rate can be high in neonates, especially in premature infants and ill full-term infants, particularly those with preexisting respiratory disease (hyaline membrane disease, bronchopulmonary dysplasia) or cardiac disease associated with pulmonary hypertension. rsv is believed to be transmitted via droplets or direct contact of the conjunctiva, nasal mucosa, or oropharynx with infected respiratory secretions. documentation of rsv infection is rarely made in adults, and spread from a mother or other household contacts probably occurs before a diagnosis can be made. therefore risk for rsv transmission from breast milk is probably insignificant compared with transmission via direct or droplet contact in families. in nurseries, however, it is appropriate to make a timely diagnosis of rsv infection in neonates to isolate infants from the others and prevent spread in the nursery. ribavirin is not recommended for routine use. it is infrequently used in patients with potentially life-threatening rsv infection. rsv infection should be suspected in any infant with rhinorrhea, nasal congestion, or unexplained apnea, especially in october through march in temperate climates. prophylaxis against rsv with rsv-specific immunoglobulin iv (rsv-igiv) during this season for infants at highest risk for severe disease is appropriate. debate surrounds the topic of the effect of passively acquired antibodies (in infants from mothers before birth) against rsv on the occurrence and severity of illness in neonates and infants. it appears that a higher level of neutralizing antibody against rsv in neonates decreases the risk for severe rsv disease. 153, 239 some controversy remains concerning the measurable benefit of breastfeeding for preventing serious rsv disease. 3, 54, 115 some studies have shown benefit and others no effect. controlling for possible confounding factors (e.g., smoking, crowded living conditions) in these studies has been difficult. at this point, no reason exists to stop breastfeeding with maternal rsv infection; a potential exists for benefit from nonspecific factors in breast milk against the rsv. infants with rsv infection should breastfeed unless their respiratory status precludes it. rotavirus infections usually result in diarrhea, accompanied by emesis and low-grade fever. in severe infections the clinical course can include dehydration, electrolyte abnormalities, and acidosis and can contribute to malnutrition in developing countries. generally, every child will have at least one episode of rotavirus infection by 5 years of age. 157 in developed countries, rotavirus is often associated with diarrhea requiring hospitalization in children younger than 2 years of age, but rarely associated with death. worldwide rotavirus is the leading cause of diarrhea-related deaths in children younger than 5 years old. estimates suggest that in children younger than 5 years old rotavirus infection leads to more than 100 million occurrences of diarrhea, 2 million hospital admissions, and 500,000 deaths each year. 157 fecal-oral transmission is the most common route, but fomites and respiratory spread may also occur. spread of infection occurs most often in homes with young children or in daycare centers and institutions. in hospitalized infants or mothers with rotavirus infection, contact precautions are indicated for the duration of the illness. no evidence indicates prenatal infection from rotavirus, but perinatal or postnatal infection from contact with the mother or others can occur. no case of transmission of rotavirus via breast milk has been documented. breast milk does contain antibodies to rotavirus for up to 2 years. human milk mucin has been demonstrated to inhibit rotavirus replication and prevent experimental gastroenteritis. 457 the mechanisms of rotavirus immunity are not well understood. they are thought to be multifactorial with cell-mediated immunity limiting severity and the course of infection, while humoral immunity protects against subsequent infections. innate and adaptive responses at the level of the mucosa are probably the most important. 134 exclusive breastfeeding may decrease the likelihood of severe rotavirus-related diarrhea by as much as 90%. 93, 377 although breastfeeding does not prevent infection with rotavirus, it seems to decrease the severity of rotavirus-induced illness in children younger than 2 years old. 93, 123, 184 at least one study suggested that this may represent simply the postponement of severe rotavirus infection until an older age. 93 one study suggested that protection against rotavirus rapidly declines upon discontinuation of breastfeeding. 356 this delay in rotavirus infection until the child is older may be beneficial in that the older child may be able to tolerate the infection or illness with a lower likelihood of becoming dehydrated or malnourished. continuing breastfeeding during an episode of rotavirus illness with or without vomiting is appropriate and often helpful to the infant. no reason to suspend breastfeeding by a mother infected with rotavirus is apparent. two rotavirus vaccines (rotateq and rotarix) have been licensed for use in more than 90 countries, but less than 20 countries have routine immunization programs. additional types of rotavirus vaccines are undergoing study in various countries, specifically examining the efficacy of the vaccines in low and medium income countries. 444 some of the explanations for the slow implementation of an effective vaccine globally include differences in protection with specific vaccines in high income countries compared with low or medium income countries, the unfortunate association with intussusception in the united states, the delayed recognition of the significant rotavirus-related morbidity and mortality, and the cost of the new vaccines. the question of variable efficacy of the specific rotavirus vaccines in developed and developing countries remains an important one. several trials are examining this issue and attempting to address factors such as maternal transplacentally transferred antibodies, breastfeeding practices (especially immediately before immunization with a live oral rotavirus vaccine), stomach acid, micronutrient malnutrition, interfering gut flora, and differences in the epidemiology of rotavirus in different locations. 327 evidence indicates that maternal immunization with rotavirus vaccine can increase both transplacental acquisition of antibodies and secretory iga in breast milk. 334 additionally, oral rotavirus vaccines have been able to stimulate a good serologic response in both formula-fed and breastfed infants, although the antigen titers may need to be modified to create an optimal response in all infants. 86 the actual protective effect of these vaccines in different situations and strategies will require measurement in ongoing prospective studies. congenital rubella infection has been well described, and the contributing variables to infection and severe disease have been elucidated. the primary intervention to prevent congenital rubella has been to establish the existence of maternal immunity to rubella before conception, including immunization with rubella vaccine and reimmunization if indicated. perinatal infection is not clinically significant. postnatal infection occurs infrequently in children younger than 1 year of age because of passively acquired maternal antibodies. the predominant age of infection is 5 to 14 years old, and more than half of those with infections are asymptomatic. postnatal rubella is a self-limited, mild viral infection associated with an evanescent rash, shotty adenopathy, and low-grade transient fever. it most often occurs in the late winter and spring. infants with congenital infection shed the virus for prolonged periods from various sites and may serve as a source of infection throughout the year. contact isolation is appropriate for suspected and proved congenital infection for at least 1 year, including exclusion from day care and avoidance of pregnant women, whereas postnatal rubella infection requires droplet precautions for 7 days after the onset of rash. rubella virus has been isolated from breast milk after natural infection (congenital or postnatal) and after immunization with live attenuated vaccine virus. both iga antibodies and immunoreactive cells against rubella have been identified in breast milk. breastfed infants can acquire vaccine virus infection via milk but are asymptomatic. because postpartum infection with this virus (natural or vaccine) is not associated with clinically significant illness, no reason exists to prevent breastfeeding after congenital infection, postpartum infection with this virus, or maternal immunization with rubella vaccine. severe acute respiratory syndrome (sars) is a term that could be applied to any acute serious respiratory illness caused by or associated with a variety of infections agents; since 2003, however, it has been linked with sars-associated coronavirus (sars-cov). in the global outbreak of 2002 to 2003, more than 8400 probable cases of sars and more than 800 deaths occurred. more than the actual number of affected individuals or its associated mortality rate (approximately 10% overall, and closer to 50% in persons older than 65 years of age), it was what we did not know about this new unusual illness, and the tremendous publicity surrounding it, that made sars such a sensation. we now know the cause of this illness, known as the sars-cov. sars-cov was shown not to be closely related to the previously characterized coronavirus groups. 265, 350 despite intense international collaboration to study the illness and the virus, many things are not known, such as the degree of infectiousness, the actual period of transmissibility, all the modes of transmission, how many people have an asymptomatic infection compared with those with symptoms or severe illnesses, how to make a rapid diagnosis of confirmed cases, and where it originated. at least 21 cases of probable sars in children have been described in the literature. 42, 187, 380, 387 in general, the illness in children is a mild, nonspecific respiratory illness, but in adolescents and adults it is more likely to progress to severe respiratory distress. it has been reported that children are less likely to transmit sars than adults. 187 the overall clinical course, the radiologic evolution, and the histologic findings of these illness are consistent with the host' s immune response playing a significant role in disease production. five infants were born to mothers with confirmed sars. the infants were born prematurely (26 to 37 weeks), presumably due to maternal illness. although two of the five infants had serious abdominal illnesses (other coronaviruses have been associated with reported outbreaks of necrotizing enterocolitis), the presence of sars-cov could not be demonstrated in any of these infants. 380 no evidence of vertical transmission of sars is available. the mode of feeding for any of the reported cases of young children with sars or the infants born to mothers with sars was not mentioned. as with other respiratory viruses predominantly transmitted by droplets, transmission via breast milk is an insignificant mode of transmission, if it occurs at all. the benefits of breastfeeding being what they are, mothers with sars should continue breastfeeding if they are able, or expressed breast milk can be given to an infant until the mother is able to breastfeed. in this era of worry about biologic terrorism, smallpox is an important concern. the concern for infants (breastfed or formula-fed) is direct contact with mothers or household members with smallpox. smallpox is highly contagious in the household setting due to person-to-person spread via droplet nuclei or aerosolization from the oropharynx and direct contact with the rash. additional potential exposures for infants include the release of a smallpox aerosol into the environment by terrorists, contact with a smallpox-contaminated space or the clothes of household members exposed to an aerosol, and infection via contact with a mother' s or a household member' s smallpox vaccination site. these risks are the same for breastfed and formulafed infants. no evidence for transmission of the smallpox virus via breast milk exists. a contact is defined as a person who has been in the same household or had face-to-face contact with a patient with smallpox after the onset of fever. patients do not transmit infection until after progression from the fever stage to the development of the rash. an exposed contact does not need to be isolated from others during the postcontact observation period (usually 17 days) until the person develops fever. temperature should be monitored daily in the exposed contact. personal contact and breastfeeding between mother and infant can continue until the onset of fever, when immediate isolation (at home) should begin. providing expressed breast milk for the infant of a mother with smallpox should be avoided because of the extensive nature of the smallpox rash and the possibility of contamination (from the rash) of the milk during the expression process. no literature documents transmission of the smallpox virus via expressed breast milk. the other issue for breastfeeding infants is the question of maternal vaccination with smallpox in a preexposure event vaccination program. children older than 1 year of age can be safely and reasonably vaccinated with smallpox in the face of a probable smallpox exposure. smallpox vaccination of infants younger than 1 year of age is contraindicated. breastfeeding is listed as a contraindication to vaccination in the preevent vaccination program. it is unknown whether vaccine virus or antibodies are present in breast milk. the risk for infection due to contact or aerosolization of virus from a mother' s smallpox vaccination site is the same for breastfed and formula-fed infants. the advisory committee on immunization practices also does not recommend preevent smallpox vaccination of children younger than 18 years old. 443 a report documents tertiary contact vaccinia in a breastfeeding infant. 140 a united states military person received a primary smallpox vaccination and developed a local reaction at the inoculation site. despite reportedly observing appropriate precautions, the individual' s wife developed vesicles on both areolae (secondary contact vaccinia). subsequently, the breastfeeding infant developed lesions on her philtrum, cheek, and tongue. both the mother and infant remained well and the infections resolved without therapy. culture and pcr testing confirmed vaccinia in both the mother' s and the infant' s lesions. the breast milk was not tested. 140 in a review from 1931 to 1981, sepkowitz 375 reported on 27 cases of secondary vaccinia in households. the cdc reported 30 suspected cases of secondary/tertiary vaccinia with 18 of those cases confirmed by culture or pcr. the 30 cases were related to 578,286 vaccinated military personnel. this is an incidence of 5.2 cases per 100,000 vac cinees and 7.4 cases per 100,000 primary vaccinees. 79 in a separate report on the civilian preevent smallpox vaccination program, 37,802 individuals were vaccinated between january and june 2003, and no cases of contact vaccinia were reported. 77 the risk for contact vaccinia is low. the risk is from close or intimate contact. in the above-mentioned case, the risk for the infant was contact with the mother' s breasts, the inadvertent site of her contact vaccinia. breastfed and formulafed infants are equally at risk from close contact in the household of a smallpox vaccinee or a case of secondary vaccinia, and separation from the individual is appropriate in both situations. if the breast of the nursing mother is not involved, expressed breast milk can be given to the infant. tt virus (ttv) is a recently identified virus found in a patient (tt) with posttransfusion hepatitis not associated with the other hepatitis-related viruses a through g. ttv has been described as an unenveloped, circular, single-stranded dna virus. 311 this virus is prevalent in healthy individuals, including healthy blood donors, and has been identified in patients with hepatitis. ttv dna has been detected in infants of ttv-positive and ttvnegative mothers. ohto et al 310 reported no ttv dna was detected in cord blood from 38 infants, and it was detected in only 1 of 14 samples taken at 1 month of age. they noted an increasing prevalence from 6 months (22%) to 2 years (33%), which they ascribed to acquisition via nonparenteral routes. in comparisons of the ttv dna in ttvpositive mothers and their ttv-positive infants, 6 of 13 showed high level nucleotide sequence similarity, and 7 of 13 differed by greater than 10%. 310 schröter et al 371 reported on ttv dna in breast milk examined retrospectively. notably, ttv dna was detected in 22 of 23 serum samples of infants at 1 week of age, who were born to 22 women viremic for ttv dna. twenty-four women who were negative for ttv dna gave birth to 24 children who were initially negative for ttv dna and remained negative throughout the observation period (mean 7.5 months, range 1 to 28 months). ttv dna was detected in 77% of breast milk samples from ttv viremic women and in none of the breast milk samples from ttv-negative women. no clinical or laboratory evidence of hepatitis was found in the 22 children who were observed to be ttv dna positive during the period of the study. 371 other authors have reported ttv in breast milk detected by pcr. they describe the absence of ttv dna in infants at 5 days and 3 months of age, and 4 of 10 infants were positive for ttv dna at 6 months of age, suggesting the late acquisition of infection via breastfeeding. 197 tt virus is transmitted in utero and is found in breast milk. no evidence of clinical hepatitis in infants related to ttv infection and no evidence for a late chronic hepatitis exist. given the current available information, no reason to proscribe breastfeeding by ttv-positive mothers is compelling. certainly more needs to be understood concerning the chronic nature of this infection and the possible pathogenesis of liver disease. no documented evidence indicates that women with breast cancer have rna of tumor virus in their milk. no correlation between rna-directed dna polymerase activity has been found in women with a family history of breast cancer. rna-directed dna polymerase activity, a reserve transcriptase, is a normal feature of the lactating breast. 91, 129, 352 epidemiologic data conflict with the suggestion that the tumor agent is transmitted through the breast milk. the incidence of breast cancer is low among groups who had nursed their infants, including lower economic groups, foreign-born groups, and those in sparsely populated areas. 262 the frequency of breast cancer in mothers and sisters of a woman with breast cancer is two to three times that expected by chance. this could be genetic or environmental. cancer actually is equally common on both sides of the family of an affected woman. if breast milk were the cause, it should be transmitted from mother to daughter. when mother-daughter incidence of cancer was studied, no relationship was found to breastfeeding. sarkar et al 360 reported that human milk, when incubated with mouse mammary tumor virus, caused degradation of the particular morphology and decreased infectivity and reverse transcriptase activity of the virions. they suggest that the significance of this destructive effect of human milk on mouse mammary tumor virus may account for the difficulty in isolating the putative human mammary tumor agent. sanner 359 showed that the inhibitory enzymes in milk can be removed by special sedimentation technique. he ascribes the discrepancies in isolating virus particles in human milk to these factors, which inhibit rna-directed dna po lymerase. the fear of cancer in breastfed female offspring of a woman with breast cancer does not justify avoiding breastfeeding. breastfed women have the same breast cancer experience as nonbreastfed women, and no increase is seen in benign tumors. daughters of breast cancer patients have an increased risk for developing benign and malignant tumors because of their heredity, not because of their breastfeeding history. 280, 287 unilateral breastfeeding (limited to the right breast) is a custom of tanka women of the fishing villages of hong kong. ing et al 194 investigated the question, "does the unsuckled breast have an altered risk for cancer?" they studied breast cancer data from 1958 to 1975. breast cancer occurred equally in the left and the right breasts. comparison of patients who had nursed unilaterally with nulliparous patients and with patients who had borne children but not breastfed indicated a highly significantly increased risk for cancer in the unsuckled breast. the authors conclude that in postmenopausal women who have breastfed unilaterally, the risk for cancer is significantly higher in the unsuckled breast. they thought that breastfeeding may help protect the suckled breast against cancer. 194 others 274 have suggested that tanka women are ethnically a separate people and that left-sided breast cancer may be related to their genetic pool and not to their breastfeeding habits. no mention has been made of other possible influences, such as the impact of their role as "fishermen" or any inherent trauma to the left breast. 274 in 1926, lane-claypon 240 stated that a breast that had never lactated was more liable to become cancerous. nulliparity and absence of breastfeeding had been considered important risk factors for breast cancer. macmahon et al 262 reported in 1970 that age at first full-term pregnancy was the compelling factor, and the younger the mother, the less the risk. in a collective review of the etiologic factors in cancer of the breast in humans, papaioannou 325 concludes, "genetic factors, viruses, hormones, psychogenic stress, diet, and other possible factors, probably in that order of importance, contribute to some extent to the development of cancer of the breast." 325 wing 449 concluded in her 1977 review on human milk and health that "in view of the complete absence of any studies showing a relationship between breastfeeding and increased risk of breast cancer, the presence of virus-like particles in breast milk should not be a contraindication to breastfeeding." henderson et al 173 gradually, studies have appeared challenging the dogma. brinton et al, 52 mctiernan and thomas, 276 and layde et al 245 showed the clearly protective effects of breastfeeding. another example is a study conducted to clarify whether lactation has a protective role against breast cancer in an asian people, regardless of confounding effects of age at first pregnancy, parity, and closely related factors. 458 in a hospital-based case-control study of 521 women without breast cancer, statistical adjustment for potential confounders and a likelihood ratio test for linear trend were done by unconditional logistic regression. total months of lactation regardless of parity was the discriminator. regardless of age of first pregnancy and parity, lactation had an independent protective effect against breast cancer in japanese women. 458 although breast cancer incidence is influenced by genetics, stress, hormones, and pregnancy, breastfeeding clearly has a protective effect. "there is a reduction in the risk of breast cancer among premenopausal women who have lactated. no reduction in the risk of breast cancer occurred among postmenopausal women with a history of lactation," according to newcombe et al, 299 reporting a multicenter study in 1993. varicella-zoster virus infection (varicella/chickenpox, zoster/shingles) is one of the most communicable diseases of humans, in a class with measles and smallpox. transmission is thought to occur via respiratory droplets and virus from vesicles. varicella in pregnancy is a rare event, although disease can be more severe with varicella pneumonia, and can be fatal. congenital varicella-zoster virus infection occurs infrequently, causing abortion, prematurity, and congenital malformations. a syndrome of malformations has been carefully described with congenital varicella-zoster virus infection, typically involving limb deformity, skin scarring, and nerve damage, including to the eye and brain. 148 perinatal infection can lead to severe infection in infants if maternal rash develops 5 days or less before delivery and within 2 days after delivery. illness in infants usually develops before 10 days of age and is believed to be more severe because of the lack of adequate transfer of antibody from the mother during this period and transplacental spread of virus to the fetus and infant during viremia in the mother. varicella in a mother occurring before 5 days before delivery allows sufficient formation and transplacental transfer of antibodies to the infant to ameliorate disease even if the infant is infected with varicella-zoster virus. mothers who develop varicella rash more than 2 days after delivery are less likely to transfer virus to the infant transplacentally; they pose a risk to the infants from postnatal exposure, which can be diminished by the administration of varicellazoster ig to the infant. postnatal transmission is believed to occur through aerosolized virus from skin lesions or the respiratory tract entering the susceptible infant's respiratory tract. airborne precautions are therefore appropriate in the hospital setting. infants infected with varicella-zoster virus in utero or in the perinatal period (younger than 1 month of age) are more likely to develop zoster (reactivation of latent varicella-zoster virus) during childhood or as young adults. postnatal varicella from nonmaternal exposure can occur but is generally mild when it develops after 3 weeks of age or when a mother has passed on antibodies against varicella-zoster virus via the placenta. severe postnatal varicella does occur in premature infants or infants of varicella-susceptible mothers. when a mother' s immune status relative to varicella-zoster virus is uncertain and measurement of antibodies to varicella-zoster virus in mother or infant cannot be performed promptly (less than 72 hours), administration of vzig 81 or ivig to the infant exposed to varicella or zoster in the postnatal period is indicated. ideally a mother' s varicella status should be known before pregnancy, when varicella virus vaccine could be given if indicated. varicella-zoster virus virus has not been cultured from milk, but varicella-zoster virus dna has been identified in breast milk. 459 antibody against varicella-zoster virus has also been found in breast milk. 270 breast milk from mothers who had received the varicella vaccine in the postpartum period was tested for varicella-zoster virus dna. varicella dna was not detected in any of the 217 breast milk samples from the 12 women, all of whom seroconverted after vaccination. 45 one case of suspected transfer of varicella-zoster virus to an infant via breastfeeding has been reported, but virus may have been transmitted by respiratory droplet or exposure to rash before the mother began antiviral therapy. 459 isolation of an infant from the mother with varicella and interruption of breastfeeding should occur only while the mother remains clinically infectious, regardless of the method of feeding. as soon as the infant has received varicella-zoster ig, expressed breast milk can be given to an infant if no skin lesions involve the breasts. persons with varicella rash are considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted, usually in 6 to 10 days. immunocompetent mothers who develop zoster can continue to breastfeed if the lesions do not involve the breast and can be covered because antibodies against varicella-zoster virus are provided to the infant via the placenta and breast milk and will diminish the severity of disease, even if not preventing it. conservative management in this scenario would include giving an infant varicella-zoster ig as well (see table 13 -5). 331 it is estimated that 150 to 300 asymptomatic cases of west nile infection occur for every 20 febrile illnesses and for every one case of meningoencephalitis associated with west nile virus. west nile fever is usually a mild illness of 3 to 6 days' duration. the symptoms are relatively nonspecific, including malaise, nausea, vomiting, headache, myalgia, lymphadenopathy, and rash. west nile disease is characterized by severe neurologic symptoms (e.g., meningitis, encephalitis, or acute flaccid paralysis, and occasionally optic neuritis, cranial nerve abnormalities, and seizures). children are infrequently sick with west nile virus infection and infants younger than 1 year of age have rarely been reported. 331 the case-fatality rate for 2003 in the united states was approximately 2.5%, but has been reported as high as 4% to 18% in hospitalized patients. the case-fatality rate for persons older than 70 years of age is considered to be higher, 15% to 29% among hospitalized patients in outbreaks in romania and israel. 331 the primary mechanism of transmission is via a mosquito bite. mosquitoes from the genus culex are primary vectors. the bird-mosquito-bird cycle serves to maintain and amplify the virus in the environment. humans and horses are incidental hosts. the pathogenesis of the infection is believed to occur via replication of the virus in the skin and lymph nodes, leading to a primary viremia that seeds secondary sites before a second viremia causes the infection of the cns and other affected organs. 59, 111 transmission has been reported in rare instances during pregnancy 7,73 via organ transplant 199 and percutaneously in laboratory workers. 75 a study of west nile virus infection in pregnancy documented four miscarriages, two elective abortions, and 72 live births. cord blood samples were tested in 55 infants and 54 of 55 were negative for anti-west nile virus igm. three infants had west nile virus infection, which could have been acquired congenitally. three of 7 infants who had congenital malformations might have been caused by maternal west nile virus infection based on timing in pregnancy, but no evidence of west nile virus etiology is conclusive. 312 west nile virus transmission occurs via blood and blood product transfusion, 186 and the incidence has been estimated to be as high as 21 per 10,000 donations during epidemics in specific cities. 40 no evidence of direct person-to-person transmission without the mosquito vector has been found. one case of possible west nile virus transmission via breastfeeding has been documented. 74 the mother acquired the virus via packed rbc transfusions after delivery. the second unit of blood she received was associated with other blood products from the same donation causing west nile infection in another transfusion recipient. eight days later the mother had a severe headache and was hospitalized with fever and a csf pleocytosis on day 12 after delivery. the mother' s csf was positive for west nile virus-specific igm antibody. the infant had been breastfed from birth through the second day of hospitalization of the mother. samples of breast milk were west nile virus-specific igg and igm positive on day 16 after delivery and west nile virus-specific igm positive on day 24. the same milk was west nile virus rna positive by pcr testing on day 16, but not on day 24 after delivery. the infant tested positive for west nile virus-specific igm in serum at day 25 of age, but remained well without fever. no clear-cut exposure to mosquitoes for the infant were reported. the cord blood and placenta were not available to be tested. igm antibodies can be found in low concentrations in breast milk, but this is not common or as efficient as the transfer of iga, secretory iga, or igg into breast milk. a review of west nile virus illness during the breastfeeding identified six occurrences of breastfeeding during maternal west nile virus illness. 177 five of the six infants had no illness or detectable antibodies to west nile virus in their blood. one infant developed a rash and was otherwise well after maternal west nile virus illness, but was not tested for west nile virus infection. two infants were identified who developed west nile virus illness while breastfeeding, but no preceding west nile virus infection was demonstrated in their mothers. two other breastfeeding infants developed west nile virus-specific antibodies after their mothers acquired west nile virus illness in the last week of pregnancy, but congenital infection could not be ruled out. live virus was not cultured from 45 samples of breast milk from mothers infected with west nile virus during pregnancy, but west nile virus rna was detected in two samples and 14 samples had igm antibodies to west nile virus. 177 the above data suggest that west nile virus infection through breastfeeding is rare. to date evidence of significant disease due to west nile virus infection in young breastfeeding children is lacking. at this time, no reason exists to proscribe breastfeeding in the case of maternal west nile virus infection if a mother is well enough to breastfeed. as with many other maternal viral illnesses, by the time the diagnosis is made in a mother, the infant may have already been exposed during maternal viremia and possible virolactia. the infant can and should continue to receive breast milk for the potential specific and nonspecific antiviral immunologic benefits. yellow fever virus is a flavivirus which is transmitted to humans by infected aedes and haemogogus mosquitos in tropical areas of south america and africa. large outbreaks occur when mosquitos in a populated area become infected from biting viremic humans infected with yellow fever virus. transmission from the mosquitos to other humans occurs after an incubation period in the mosquito of 8 days. direct person-to-person spread has not been reported. illness due to yellow fever virus usually begins after an incubation period of 3 to 6 days, with acute onset of headache, fever, chills, and myalgia. photophobia, back pain, anorexia, vomiting, and restlessness are other common symptoms. the individual is usually viremic for the first 4 days of illness until the fever and other symptoms diminish. liver dysfunction and even failure can develop as can myocardial dysfunction. cns infection is uncommon but symptoms can include seizures and coma. medical care should include intensive supportive care and fluid management. one case of congenital infection after immunization of a pregnant woman with the attenuated vaccine strain has been reported. one of 41 infants whose mothers had inadvertently received the yellow fever virus vaccine during pregnancy developed igm and elevated neutralizing antibodies against the yellow fever virus without any evidence of illness or abnormalities. 418 a more recent study 404 from brazil examined inadvertent yellow fever virus immunization during pregnancy during a mass vaccination campaign in 2000; 480 pregnant women received the yellow fever virus at a mean of 5.7 weeks' gestation, the majority of whom did not know their pregnancy status at the time. seroconversion occurred in 98.2% of the women after at least 6 weeks after vaccination. mild postvaccination illness (headache, fever, or myalgia) was reported by 19.6% of the 480 women. the frequency of malformations, miscarriages, stillbirths, and premature deliveries was similar to that found in the general population. at the 12-month follow-up point, 7% of the infants still demonstrated neutralizing antibodies against yellow fever virus, but after 12 months only one child was still seropositive. 404 transmission of the yellow fever vaccine virus through breastfeeding was recently reported from brazil. 85 the mother was immunized during a yellow fever epidemic in a nonendemic area in brazil; 15 days after delivering a healthy female infant (39 weeks' gestational age) the mother received the 17dd yellow fever vaccine, and 5 days later the mother reported headache, malaise, and low-grade fever that persisted for 2 days. the mother continued breastfeeding and did not seek medical care for herself. at 23 days of age the infant became irritable, developed fever, and refused to nurse. the infant developed seizures and subsequent evaluation of the infant demonstrated an abnormal csf and ct of the brain showed bilateral areas of diffuse low density suggestive of inflammation and consistent with encephalitis. yellow fever-specific igm antibodies were identified in the infant' s serum and csf. reverse-transcriptase polymerase chain reaction (rt-pcr) testing of the csf also demonstrated yellow fever virus rna identical to the 17dd yellow fever vaccine virus. breast milk and maternal serum were not tested for yellow fever virus. 85 yellow fever virus, wild or vaccine type, has not been identified in human breast milk, although another flavivirus, west nile virus, has been detected in milk from a few lactating women with west nile virus infection. 177 (see the section on west nile virus.) yellow fever vaccine-associated neurologic disease occurs at different rates in different age-groups, including 0.5 to 4.0 cases per 1000 infants younger than 6 months of age. 285 the 17d-derived yellow fever vaccines are contraindicated in infants younger than 6 months of age. since 2002, the advisory committee on immunization practices has recommended, based on theoretical risk, that yellow fever vaccine be avoided in nursing mothers, except when exposure in highrisk yellow fever endemic areas is likely to occur. 76 no case of transmission of yellow fever virus from an infected mother to her infant via breastfeeding or breast milk has been reported. published information on the severity of yellow fever virus infection in infants younger than 1 year of age, potential protection from passively acquired antibodies, or protection from breast milk is limited. no information on a differential risk in breastfed versus formula-fed infants is available. given the well documented method of transmission of yellow fever virus via mosquitos, and the lack of evidence of transmission via breast milk, it makes more sense to protect all infants against mosquito bites than to proscribe breastfeeding, even in the mother infected with yellow fever virus. continued breastfeeding or use of expressed breast milk will depend on a mother's health status and ability to maintain the milk supply while acutely ill. if another source of feeding is readily available then temporarily discarding expressed breast milk for at least 4 days of acute illness in the mother is a reasonable precaution. lyme disease, as with other human illnesses caused by spirochetes, especially syphilis, is characterized by a protean course and distinct phases (stages) of disease. lyme borreliosis was described in europe in the early twentieth century. since the 1970s, tremendous recognition, description, and investigation of lyme disease have occurred in the united states and europe. public concern surrounding this illness is dramatic. lyme disease is a multisystem disease characterized by involvement of the skin, heart, joints, and nervous system (peripheral and central). stages of disease are identified as early localized (erythema migrans, often accompanied by arthralgia, neck stiffness, fever, malaise, and headache), early disseminated (multiple erythema migrans lesions, cranial nerve palsies, meningitis, conjunctivitis, arthralgia, myalgia, headache, fatigue, and, rarely, myocarditis), and late disease (recurrent arthritis, encephalopathy, and neuropathy). the varied manifestations of disease may relate to the degree of spirochetemia, the extent of dissemination to specific tissues, and the host' s immunologic response. the diagnosis of lyme disease is often difficult in part because of the broad spectrum of presentations, inapparent exposure to the tick, and the lack of adequately standardized serologic tests. culture of the spirochete, borrelia burgdorferi, is not readily available. enzyme-linked immunosorbent assay (elisa), immunofluorescent assay, and immunoblot assay are the usual tests. pcr detection of spirochetal dna requires additional testing in clinical situations to clarify and standardize its utility. gardner 142 reviewed infection during pregnancy, summarizing a total of 46 adverse outcomes from 161 cases reported in the literature. the adverse outcomes included miscarriage and stillbirth (11% of cases), perinatal death (3%), congenital anomalies (15%), and both early-and late-onset progressive infection in the infants. silver 384 reviewed 11 published reports and concluded that lyme disease during pregnancy is uncommon, even in endemic areas. although the spirochete can be transmitted transplacentally, a significant immune response in the fetus is often lacking, and the association of lyme infection with congenital abnormalities is weak. 401, 448 little published information exists on whether b. burgdorferi can be transmitted via breast milk. one report showed the detection of b. burgdorferi dna by pcr in the breast milk of two lactating women with untreated erythema migrans, but no evidence of lyme disease or transmission of the spirochete in the one infant followed for 1 year. 369 no attempt to culture the spirochete was made, so it is not possible to determine if the detectable dna was from viable spirochetes or noninfectious fragments. in that same study of 56 women with untreated erythema migrans who had detectable b. burgdorferi dna in the urine, 32 still had detectable dna in the urine 15 to 30 days after starting treatment, but none had it 6 months after initiating therapy. ziska et al 466 reported on the management of nine cases of lyme disease in women associated with pregnancy; seven of the nine women were symptomatic at conception and six received antibiotics throughout pregnancy. follow-up of the infants showed no transmission of lyme disease, even in the seven infants who had been breastfed. 466 the lack of adequate information on transmission of b. burgdorferi via breast milk cannot be taken as proof that it is not occurring. if one extrapolates from data on syphilis and the treponema pallidum spirochete, it would be prudent to discuss the lack of information on the transmission of b. burgdorferi via breast milk with the mother or parents and to consider withholding breast milk at least until therapy for lyme disease has begun or been completed. if the infection occurred during pregnancy and treatment has already been completed, an infant can breastfeed. if infection occurs postpartum or the diagnosis is made postpartum, infant exposure may have already occurred. again, discussion with the mother or parents about withholding versus continuing breastfeeding is appropriate. after prenatal or postnatal exposure, an infant should be closely observed and empiric therapy considered if the infant develops a rash or symptoms suggestive of lyme borreliosis. treatment of mother and infant with ceftriaxone, penicillin, or amoxicillin is acceptable during breastfeeding relative to the infant' s exposure to these medications. doxycycline should not be administered for more than 14 days while continuing breastfeeding because of possible dental staining in the neonate. continued surveillance for viable organisms in breast milk and evidence of transmission through breastfeeding is recommended. a large body of information is available on various "lyme vaccines" used in dogs, but these vaccines are only partially protective and must be repeated yearly. preliminary information suggests that a vaccine for use in humans safely produces good serologic responses, but protective efficacy has not been demonstrated, and no information exists on its use during pregnancy or breastfeeding. syphilis is the classic example of a spirochetal infection that causes multisystem disease in various stages. both acquired syphilis and congenital syphilis are well-described entities. acquired syphilis is almost always transmitted through direct sexual contact with open lesions of the skin or mucous membranes of individuals infected with the spirochete, treponema pallidum. congenital syphilis occurs by infection across the placenta (placentitis) at any time during the pregnancy or by contact with the spirochete during passage through the birth canal. any stage of disease (primary, secondary, tertiary) in a mother can lead to infection of the fetus, but transmission in association with secondary syphilis approaches 100%. infection with primary syphilis during pregnancy, without treatment, leads to spontaneous abortion, stillbirth, or perinatal death in 40% of cases. similar to acquired syphilis, congenital syphilis manifests with moist lesions or secretions from rhinitis (snuffles), condylomata lata, or bullous lesions. these lesions and secretions contain numerous spirochetes and are therefore highly infectious. postnatal infection of an infant can occur through contact with open, moist lesions of the skin or mucous membranes of the mother or other infected individuals. if the mother or infant has potentially infectious lesions, isolation from each other and from other infants and mothers is recommended. if lesions are on the breasts or nipples, breastfeeding or using expressed milk is contraindicated until treatment is complete and the lesions have cleared. spirochetes are rarely identified in open lesions after more than 24 hours of appropriate treatment. penicillin remains the best therapy. evaluation of an infant with suspected syphilis should be based on the mother' s clinical and serologic status, history of adequate therapy in the mother, and the infant' s clinical status. histologic examination of the placenta and umbilical cord, serologic testing of the infant' s blood and csf, complete analysis of the csf, long bone and chest radiographs, liver function tests, and a complete blood cell count are all appropriate given the specific clinical situation. treatment of the infant should follow recommended protocols for suspected, probable, or proven syphilitic infection. 96 no evidence indicates transmission of syphilis via breast milk in the absence of a breast or nipple lesion. when a mother has no suspicious breast lesions, breastfeeding is acceptable as long as appropriate therapy for suspected or proven syphilis is begun in the mother and infant. giardiasis is a localized infection limited to the intestinal tract, causing diarrhea and malabsorption. immunocompetent individuals show no evidence of invasive infection, and no evidence exists that documents fetal infection from maternal infection during pregnancy. giardiasis is rare in children younger than 6 months of age, although neonatal infection from fecal contamination at birth has been described. 22 human milk has an in vivo protective effect against giardia lamblia infection, as documented by work from central africa, where the end of breastfeeding heralds the onset of giardia infection. 145 this has been reaffirmed in undeveloped countries around the world. the protective effect of breast milk has been identified in the milk of noninfected donors. 151 the antiparasitic effect does not result from specific antibodies but rather from lipase enzymatic activity. the lipase acts in the presence of bile salts to destroy the trophozoites as they emerge from their cysts in the gi tract. hernell et al 175 demonstrated that free fatty acids have a marked giardiacidal effect, which supports the conclusion that lipase activity releasing fatty acids is responsible for killing g. lamblia. g. lamblia have also been reported to appear in the mother' s milk, and the parasite has been transmitted to newborns via that route. the exact relationship of breastfeeding to transmission of g. lamblia and the effect on infants continue to be studied, even though symptomatic infection in breastfed infants is rare. 151 one report from the middle east suggests that even partial breastfeeding is protective against infection with intestinal parasites, including cryptosporidium and giardia lamblia. 41 breastfeeding by mothers with giardiasis is problematic mainly because of the medications used for therapy. metronidazole' s safety in infants has not been established, and little information is available on quinacrine hydrochloride and furazolidone in breast milk. paromomycin, a nonabsorbable aminoglycoside, is a reasonable alternative recommended for treatment of pregnant women. breastfeeding by a mother with symptomatic giardiasis is acceptable when consideration is given to the presence of the therapeutic agents in the breast milk. hookworm infection, most often caused by ancylostoma duodenale and necator americanus, is common in children younger than the age of 4 years, and there is at least one report on infantile hookworm disease from china. 374 this publication from the chinese literature reports hundreds of cases of infantile hookworm disease that include the common symptoms of bloody stools, melena, anorexia, listlessness, and edema. anemia, eosinophilia and even leukemoid reactions occur as part of the clinical pictures in young children. they also note at least 20 cases of hookworm diseases in newborn infants younger than 1 month of age. in the discussion of infantile hookworm infection, they note four routes of infection: direct contact with contaminated soil, "sand-stuffed" diapers, contaminated "washed/wet" diapers, and vertical equal to transmammary transmission or transplacental transmission. they postulated that infection of infants before 40 to 50 days of age would most likely be due to transplacental transmission and infection before environmental contact would most likely be due to transmammary transmission. ample evidence is available in veterinary medicine of transmammary spread of helminths. 302, 382 at least two reports suggest the possibility of transmammary transmission of hookworms in humans. setasuban et al 376 described the prevalence of necator americanus in 128 nursing mothers as 61% and identified n. americanus in breast milk in one case. nwosu 303 documented positive stool samples for hookworms in 33 of 316 neonates (10%) at 4 to 5 weeks of age in southern nigeria. the majority of neonatal infections were due to ancylostoma duodenale although necator americanus is more prevalent in that area of nigeria. examination of colostral milk did not demonstrate any hookworm larvae. 303 additional epidemiologic work is necessary to determine the potential significance of transmammary spread of helminths in humans, and more careful examination of breast milk as a source of hookworm infection is required before reasonable recommendation are possible. malaria is recognized as a major health problem in many countries. the effect of malaria infection on pregnant and lactating women and thus on the developing fetus, neonate, and growing infant can be significant. the four species of malaria, plasmodium vivax, p. ovale, p. malariae, and p. falciparum, vary in the specific aspects of the disease they produce. p. vivax exists throughout the world, but p. falciparum predominates in the tropics and is most problematic in its chloroquine-resistant form. malaria in the united states is most often seen in individuals traveling from areas where malaria is endemic. the parasite can exist in the blood for weeks, and infection with p. vivax and p. malariae can lead to relapses years later. transmission occurs through the bite of the anopheline mosquito and can occur via transfusion of blood products and transplacentally. congenital malaria is rare but seems to occur more often with p. vivax and p. falciparum. it usually presents in the first 7 days of life (range 1 day to 2 months). it may resemble neonatal sepsis, with fever, anemia, and splenomegaly occurring in the most neonates and hyperbilirubinemia and hepatomegaly in less than half. malaria in infants younger than 3 months of age generally manifests with less severe disease and death than in older children. possible explanations include the effect of less exposure to mosquitoes, passive antibody acquired from the mother, and the high level of fetal hemoglobin in infants at this age. 22 the variations in the infection rates in children younger 3 months of age during the wet and dry seasons support the idea that postnatal infection is more common than congenital infection. no evidence indicates that malaria is transmitted through breast milk. the greatest risk to infants is exposure to the anopheline mosquito infected with malaria. the main issues relative to malaria and breastfeeding are how to protect both mothers and infants effectively from mosquitoes and what drugs for treating malaria in mothers are appropriate during lactation. protection from mosquito bites includes screened-in living areas, mosquito nets while sleeping, protective clothing with or without repellents on the clothes, and community efforts to eradicate the mosquitoes. chloroquine, quinine, and tetracycline are acceptable during breastfeeding. sulfonamides should be avoided in the first month of an infant' s life, but pyrimethamine-sulfadoxine (fansidar) can be used later. mefloquine is not approved for infants or pregnant women. however, the milk/plasma ratio for mefloquine is less than 0.25, there is a large volume of distribution of the drug, high protein binding of the drug limits its presence in breast milk, and the relative importance of breastfeeding in areas where malaria is prevalent shifts the risk/benefit ratio in favor of treatment with mefloquine. the single dose recommended for treatment or the onceweekly dose for prevention allows for continued breastfeeding with discarding of the milk for short periods after a dose (1 to 6 hours). maternal plasma levels of primaquine range from 53 to 107 ng/ml, but no information is available on levels in human milk. primaquine is used in children, and once daily dosing in the mother would allow discarding milk with peak levels of drug. therefore breastfeeding during maternal malaria even with treatment is appropriate with specific medications. strongyloides stercoralis is a nematode (roundworm). most infections are asymptomatic, but clinically significant infection in humans can include larval skin invasion, tissue migration, intestinal invasion with abdominal pain and gi symptoms, and a loeffler-like syndrome due to migration to the lungs. immune-compromised individuals can develop dissemination of larvae systemically, causing various clinical symptoms. humans are the principal hosts, but other mammals can serve as reservoirs. infection via the skin by filariform larvae is the most common form of transmission; ingestion is an uncommon occurrence. transmammary transmission of strongyloides species has been described in dogs, ewes, and rats. 211, 302, 382 only one report of transmammary passage of strongyloides larvae in humans is available. in 76 infants younger than 200 days of age, 34% demonstrated the presence of strongyloides fuelleborni on stool examination. the clinical significance of this was not elucidated. strongyloides larvae was identified in only one sample of milk from 25 nursing mothers. 53 in the absence of an understanding of the clinical significance of strongyloides in the stools of young infants, given the lack of exclusion of the most common mechanism of transmission (through the skin) in the single report and the apparent infrequent evidence of these larvae in human milk, it is difficult to make any recommendations concerning breastfeeding and strongyloides. toxoplasmosis is one of the most common infections of humans throughout the world. the infective organism, toxoplasma gondii, is ubiquitous in nature. the prevalence of positive serologic test titers increases with age, indicating past exposure and infection. the cat is the definitive host, although infection occurs in most species of warmblooded animals. postnatal infection with toxoplasmosis is usually asymptomatic. symptomatic infection typically manifests with nonspecific symptoms, including fever, malaise, myalgia, sore throat, lymphadenopathy, rash, hepatosplenomegaly, and occasionally a mononucleosis-like illness. the illness usually resolves without treatment or significant complications. congenital infection or infection in an immunodeficient individual can be persistent and severe, causing significant morbidity and even death. although most infants with congenital infection are asymptomatic at birth, visual abnormalities, learning disabilities, and mental retardation can occur months or years later. the syndrome of congenital toxoplasmosis is clearly defined, with the most severe manifestations involving the cns, including hydrocephalus, cerebral calcifications, microcephaly, chorioretinitis, seizures, or simply isolated ocular involvement. the risk for fetal infection is related to the timing of primary maternal infection, although transmission can occur with preexisting maternal toxoplasmosis. 241 in the last months of pregnancy the protozoan is more readily transmitted to the fetus, but the infection is more likely to be subclinical. early in pregnancy the transmission to a fetus occurs less frequently but does result in severe disease. treatment of documented congenital infection is currently recommended, although duration and optimal regimen have not been determined, and reversal of preexisting sequelae generally does not occur. 343 prevention of infection in susceptible pregnant women is possible by avoiding exposure to cat feces or the organism in the soil. pregnant or lactating women should not change cat litter boxes, but if they must, it should be done daily and while wearing gloves. the oocyst is not infective for the first 24 to 48 hours after passage. mothers can avoid ingestion of the organism by fully cooking meats and carefully washing fruits, vegetables, and food preparation surfaces. 94 in various animal models, t. gondii has been transmitted through the milk to the suckling young. the organism has been isolated from colostrum as well. the newborn animals became asymptomatically infected when nursed by an infected mother whose colostrum contained t. gondii. only one report has identified t. gondii in human milk, and some question surrounds the reliability of that report. 241 transmission during breastfeeding in humans has not been demonstrated. breast milk may contain appropriate antibodies against t. gondii. given the benign nature of postnatal infection, the absence of documented transmission in human breast milk, and the potential antibodies in breast milk, no reason exists to proscribe breastfeeding by a mother known to be infected with toxoplasmosis. trichomonas vaginalis is a flagellated protozoan that can produce vaginitis (see chapter 16 for a discussion of vaginitis) but frequently causes asymptomatic infection in both men and women. the parasite is found in 10% to 25% of women in the childbearing years. it is transmitted predominantly by sexual intercourse, but it can be transmitted to the neonate by passage through the birth canal. this parasite often coexists with other stds, especially gonorrhea. infection during pregnancy or while taking oral contraceptives is more difficult to treat. some evidence suggests that infection with and growth of the parasite are enhanced by estrogens or their effect on the vaginal epithelium. no evidence indicates adverse effects on the fetus in association with maternal infection during pregnancy. occasionally female newborns have vaginal discharge during the first weeks of life caused by t. vaginalis. this is thought to be influenced by the effect of maternal estrogen on the infant' s vaginal epithelium and acquisition of the organism during passage through the birth canal. the organism does not seem to cause significant disease in a healthy infant. no documentation exists on transmission of t. vaginalis via breast milk. the difficulty encountered with maternal infection during lactation stems from metronidazole (flagyl), the drug of choice, being contraindicated for infants. case reports describe treatment of neonates with metronidazole without adverse effect. although topical agents containing povidone-iodine (betadine) or sodium lauryl sulfate (trichotine) can be effective when given as douches, creams, or suppositories, metronidazole remains the treatment of choice. the aap advises using metronidazole only with a physician' s discretion and considers its effect on a nursing infant unknown but possibly a concern. the potential concerns are metronidazole' s disulfiram-like effect in association with alcohol, tumorigenicity in animal studies, and leukopenia and neurologic side effects described in adults. on the other hand, metronidazole is given to children beyond the neonatal period to treat serious infections with various other parasites, such as entamoeba histolytica. the current recommendation for lactating women is to try local treatment first, and if these fail, then to try metronidazole. a 2-g single-dose treatment produces peak levels after 1 hour, and discarding expressed breast milk for the next 12 to 24 hours is recommended. if this treatment also fails, a 1-g twice-daily regimen for 7 days or a 2-g single daily dose for 3 to 5 days is recommended, with discarding of breast milk close to the dose and timing of feedings distant from the dose. infants who exclusively breastfeed are presumed at greater risk from exposure to metronidazole than those who are only partially breastfed. candida consists of multiple species. the most common species affecting humans include c. albicans as the dominant agent and c. tropicalis, c. krusei, and c. parapsilosis, as well as many other uncommon species. in general, candida exists as a commensal organism colonizing the oropharynx, gi tract, vagina, and skin without causing disease until some change disrupts the balance between the organism and the host. mild mucocutaneous infection is the most common illness, which can lead to vulvovaginitis, mastitis, or, uncommonly, oral mucositis in a mother, and thrush (oral candidiasis) and candidal diaper rash in an infant. invasive candidal infection occurs infrequently, usually when a person has other illness, impaired resistance to infection (hiv, diabetes mellitus, neutropenia; decreased cell-mediated immunity in premature infants or lbw or vlbw infants), or disrupted normal mucosal and skin barriers and has received antibiotics or corticosteroids. invasive disease can occur through local spread, and may occur more often in the genitourinary tract (urethra, bladder, ureters, kidneys), but usually develops in association with candidemia. the bladder and kidney are more frequently involved, but when dissemination occurs via candidemia, a careful search for other sites of infection should be made (e.g., retina, liver, spleen, lung, meninges). 279 transmission usually occurs from healthy individuals colonized with candida through direct contact with them or through contact with their oral or vaginal secretions. intrauterine infection can occur through ascending infection through the birth canal but is rare. no distinct syndrome of congenital candidal infection exists. most often an infant is infected in passing through the birth canal and remains colonized. postnatal transmission can occur through direct contact with caregivers. the mother and infant serve as an immediate source of recolonization for each other, especially during the direct contact of breastfeeding. for this reason, an infant and breastfeeding mother should be treated simultaneously when treating thrush, vulvovaginitis, diaper candidiasis, or mastitis. colonization with this organism usually occurs in the absence of any clinical evidence of infection. simultaneous treatment should occur even in the absence of any clinical evidence of candida infection or colonization in the apparently uninvolved individual of the breastfeeding dyad. no well-controlled clinical trials define the most appropriate or most effective method(s) of treatment for candidal infection in breastfeeding mother-infant pairs. the list of possible treatment products is extensive and includes many anecdotal and empirical regimens. in the face of this absence of data, brent 51 conducted a survey of members of the academy of breastfeeding medicine concerning the respondents' approach to diagnosis and treatment of thrush in the breastfeeding dyad. most of the respondents relied on the history and physical examination of the infant, but only a third rated the examination of the mother as very important in making a diagnosis. only 7% reported using laboratory testing to make the diagnosis. twentyone percent of the respondents reported using only oral nystatin for the infant when the mother was asymptomatic. almost half treated the infant and the mother with topical nystatin, and 13% used oral nystatin for the infant and oral fluconazole for the mother when the mother had breast pain. less than 5% used oral fluconazole for both infant and mother, and other therapies were used by about 15% of the respondents. for recurrence of persistence of the thrush, more respondents reported treating the mother or both the infant and mother with fluconazole, and almost a quarter reported using other therapies. considerable discussion of mammary candidosis/candidiasis, the clinical diagnosis of candidal involvement of the breast, the significance of pain with breastfeeding, and the presence or absence of candida albicans in milk samples is ongoing. 14, 133, 166 this topic will continue to be debated because additional prospective studies are necessary to clarify specific issues. data are inadequate to make specific recommendations about various clinical situations regarding candida and breastfeeding. clinical practice will vary with experience, especially for the more problematic clinical situations. some general guidelines follow. (see chapter 16 for a discussion of mastitis.) the treatment of mucocutaneous candidiasis should probably begin with a topical agent, such as nystatin, clotrimazole, miconazole, econazole, butaconazole, terconazole, or ciclopirox. treatment should continue for at least 2 weeks, even with obvious improvement in 1 or 2 days. failures most often result from inadequate therapy involving the frequency of application, careful washing and drying before application, or, in the case of diaper candidiasis, decreasing the contact of the skin with moisture. nystatin oral suspension is less effective for the treatment of oral candidiasis in infants, now compared with the past, supposedly due to increasing resistance. 154 gentian violet (diluted to 0.25% to 1.0%) applied to the breast or painted onto an infant' s mouth is being recommended more frequently. other topical preparations have been recommended for the mother' s breast including mupirocin, grapefruit seed extract, or mixtures of mupirocin, betamethasone ointments, and miconazole powder. controlled clinical trials for efficacy and toxicity are not available. when good adherence to the proposed regimen with topical agents fails, or when infant or mother are severely affected by pain and decreased breastfeeding, systemic therapy is appropriate. fluconazole and ketoconazole are the most commonly used systemic agents for oral or diaper candidiasis and vulvovaginitis or mastitis. fluconazole has a better side effect profile than ketoconazole, and more data are available concerning its safe use in children younger than 6 months of age and even neonates and premature infants. 87, 154, 209 fluconazole is not currently approved for use in infants younger than 6 months of age. for severe invasive infections in infants, amphotericin b with or without oral flucytosine, iv fluconazole, voriconazole or caspofungin are reasonable choices in different situations. use of itraconazole in infants has not been adequately studied to date. maternal use of fluconazole during breastfeeding is not contraindicated because only a small amount of medicine compared with the usual infant dose reaches the infant through breast milk. amphotericin or caspofungin therapy in mothers is also not contraindicated because these are both poorly absorbed from the gi tract. whenever a mother is treated for candidal mastitis or vulvovaginitis, the infant should be treated simultaneously, at least with nystatin oral suspension as the first choice of medication. any predisposing risk factors for candidal infection in mothers and infants should be reduced or eliminated to improve the chance of rapid, successful treatment and to decrease the likelihood of chronic or recurrent disease. for mothers, such interventions might include decreasing sugar consumption, stopping antibiotic use as soon as possible, and consuming some form of probiotic bacteria, such as acidophilus (in yogurt, milk, or pill form), to reestablish a normal colonizing bacterial flora. for infants, breastfeeding can enhance the growth of specific colonizing bacterial flora such as lactobacillus, which can successfully limit fungal growth. breastfeeding should continue with appropriate support and problem-solving with a professional who is knowledgeable about breastfeeding. hiv-1, hiv-2, htlv-i, and htlv-ii are the only infectious diseases that are considered absolute contraindications to breastfeeding in developed countries. when the primary route of transmission is via direct contact or respiratory droplets/particles, temporary separation of mother and infant may be appropriate (whether the infant is breastfed or formula fed), but expressed breast milk should be given to the infant for the organism-specific immunologic benefits in the mother' s milk. in most instances, by the time a specific diagnosis of infection is made for a mother, the infant has already been exposed to the organism and providing expressed breast milk to the infant should continue. (refer to appendix f for specific exceptions, such as lassa fever.) regarding antimicrobial therapy for mothers and continued breastfeeding, the majority of the medications commonly used in adults can be used to treat the same infection in infants. the additional amount of medication received by infants via breast milk is usually insignificant. in almost all instances, an appropriate antimicrobial agent for treating mothers that is also compatible with breastfeeding can be chosen. unless the risk to infants for transmission of an infectious agent via breast milk that leads to a clinically significant illness in the infants is documented, breastfeeding should continue. measles antibodies in the breast milk of nursing mothers spectrum of breast tuberculosis respiratory syncytial virus infection among young children with acute respiratory tract infection in iraq probable breast milk borne brucellosis in a young infant breast milk transmission of cytomegalovirus (cmv) infection congenital and perinatal cytomegalovirus infections intrauterine west nile virus: ocular and systemic findings the cloning and clinical implications of hgv and hgbv-c bottle feeding can prevent transmission of htlv-i from mothers to their babies transmission of adult t-cell leukemia retrovirus (htlv-i) from mother to child: comparison of bottle-with breastfed babies effect of freezethawing breast milk on vertical htlv-i transmission from seropositive mothers to children long-term follow up study of vertical htlv-i infection in children breastfed by seropositive mothers long-term followup study of htlv-i infection in bottle-fed children born to seropositive mothers the yeast connection: is candida linked to breastfeeding associated pain? epidemiology of group b streptococcus: maternal and nosocomial sources for acquisition tuberculosis and pregnancy and tuberculous mastitis infant botulism infant botulism: anticipating the second decade protective role of human milk against sudden death from infant botulism growth faltering due to breastfeeding cessation in uninfected children born to hiv-infected mothers in zambia other viral infections of the fetus and newborn protozoan and helminth infections (including pneumocystis carinii) recurrent group b streptococcal disease in infants: who should receive rifampin? methicillin-resistant staphylococcus aureus sccmec type iv: nosocomial transmission and colonisation of healthcare workers in a neonatal intensive care unit stringent precautions are advisable when caring for patients with viral hemorrhagic fevers prevalence of methicillin-resistant staphylococcus aureus in expressed breast milk in a neonatal intensive care unit transmision de brucelosis por lactancia materna: presentacion de dos casos congenital lymphocytic choriomeningitis virus infection in twins poliomyelitis in pregnancy, fetus and newborn assessment of the risk of ebola virus transmission from bodily fluids and fomites transmission of hepatitis by breastfeeding evidence against breast feeding as a mechanism for vertical transmission of hepatitis b two-year morbidity-mortality and alternatives to prolonged breast feeding among children born to hiv infected mothers in cote d'ivorie transmission of methicillin-resistant staphylococcus aureus to preterm infants through breast milk a new staphylococcal enterotoxin, enterotoxin f, associated with tss staphylococcus aureus isolate mother-to-infant transmission of hepatitis c outbreak of methicillin-resistant staphylococcus aureus colonization and infection in a neonatal intensive care unit epidemiologically linked to a healthcare worker with chronic otitis estimating the timing of mother-to-child transmission of human immunodeficiency virus in a breast-feeding population in kinshasa mycobacteriareactive t cells are present in human colostrum from tuberculin-positive, but not tuberculin-negative nursing mothers estimated risk of transmission of the west nile virus through blood transfusion in the us partial breastfeeding protects bedouin infants from infection morbidity: prospective cohort study children hospitalized with severe acute respiratory syndrome-related illness in toronto a prospective study of infants born to women seropositive for human immunodeficiency virus type 1. hiv infection in newborns french collaborative study group clinical virology postpartum varicella vaccination: is the vaccine virus excreted in breast milk? contamination of breast milk obtained by manual expression and breast pumps in mothers of very low birth weight hepatitis c virus infection and related liver disease in children of mothers with antibodies to the virus clinical and laboratory observations, gram-negative bacilli in human milk feedings: quantitation and clinical consequences for premature infants prenatal transmission of dengue: two new cases community associated methicillin-resistant staphylococcus aureus in hospital nursery and maternity units thrush in the breastfeeding dyad: results of a survey on diagnosis and treatment reproductive factors in the aetiology of breast cancer transmammary passage of strongyloides sp. larvae in the human host alaska rsv study group: risk factors for severe respiratory syncytial virus infection among alaska native children detection of human immunodeficiency virus type 1 (hiv-1) proviral dna in breast milk and colostrum of seropositive mothers streptococcus agalactiae as a cause of meningitis in the newborn and bacteraemia in adults incidence and clinical outcome of cytomegalovirus transmission via breast milk in preterm infants </=31 weeks neonatal group b streptococcal infection related to breast milk epidemiology of tuberculosis in the united states probable transmission of brucellosis by breast milk does discarding the first few milliliters of breast milk improve the bacteriological quality of bank breast milk? primary human herpes virus 7 infection: a comparison of human herpes virus 7 and human herpes virus 6 infections in children analysis of the presence of cutaneous and mucosal papillomavirus types in ductal lavage fluid, milk and colostrum to evaluate its role in breast carcinogenesis centers for disease control and prevention: testing for antibodies to hiv-2 in the united states public health service working group: recommendations for counseling persons infected with human t-lymphotropic virus, types i and ii centers for disease control and prevention: screening for tuberculosis and tuberculosis infection in high-risk populations update: management of patients with suspected viral hemorrhagic fever-united states poliomyelitis prevention in the united states: introduction of a sequential vaccination schedule of inactivated poliovirus vaccine followed by oral poliovirus vaccine-united states prevention: recommendations for antimicrobial prophylaxis for children and breastfeeding mothers and treatment of children with anthrax prevention: possible west nile virus transmission to an infant through breastfeeding-michigan prevention: yellow fever vaccine; recommendations of the advisory committee on immunization practices (acip) update: cardiac and other adverse events following civilian smallpox vaccination-united states centers for disease control and prevention: secondary and tertiary transfer of vaccinia virus among u.s. military personnel-united states and worldwide centers for disease control and prevention: a new product (varizig) for post exposure prophylaxis of vericells available under an investigational new drug application expanded access protocol centers for disease control and prevention: communityassociated methicillin-resistant staphylococcus aureus infection among healthy newborns: chicago and los angeles county centers for disease control and prevention: what to do if an infant or child is mistakenly fed another woman' s expressed breast milk prevention: transmission of yellow fever vaccine through breast feeding: brazil take of rhesushuman reassortment tetravalent rotavirus vaccine in breastfed infants candida infections in the neonate prevalence of methicillin-sensitive and methicillin-resistant staphylococcus aureus in pregnant women coxsackieviruses, and enteroviruses morbidity and mortality among a cohort of human immunodeficiency virus type 1 infected and uninfected pregnant women and their infants from malawi, zambia, and tanzania electron microscopic detection of simian-type virus particles in human milk staphylococcus aureus outbreaks among newborns: new frontiers in an old dilemma breastfeeding and the risk of life-threatening rotavirus diarrhea: prevention or postponement? committee on infectious diseases: american academy of pediatrics: red book report of the committee on infectious disease committee on infectious diseases: american academy of pediatrics: red book report of the committee on infectious disease committee on infectious disease: american academy of pediatrics: red book report of the committee on infectious disease mother-tochild transmission of hiv-1 infection during exclusive breastfeeding in the first 6 months of life: an intervention cohort breastfeeding, hiv transmission and infant survival: balancing pros and cons influence of infantfeeding patterns on early mother-to-child transmission of hiv-1 in durban, south africa: a prospective cohort study. south african vitamin a study group method of feeding and transmission of hiv-1 from mothers to children by 15 months of age: prospective cohort study from durban are hivinfected women who breastfeed at increased risk of mortality? morbidity in children born to women infected with human immunodeficiency virus in south africa: does mode of feeding matter? pilot epidemiologic study of transmission of cytomegalovirus from mother to preterm infant by breastfeeding mother-to-child transmission of human immunodeficiency virus type: report from the nairobi study human milk and hiv infection: epidemiologic and laboratory data hiv, breastfeeding and under 5 mortality: modelling the impact of policy decisions for or against breastfeeding staphylococcus epidermidis strains isolated from breast milk of women suffering infectious mastitis: potential virulence traits and resistance to antibiotics hiv-1 transmission through breast-milk: appraisal of risk according to duration of feeding task force on recurrent respiratory papillomatosis presence of papillomavirus in condylomatous lesions of the mamillae and in invasive carcinoma of the breast variations in biological features of west nile viruses group b streptococcal carriage and disease: a 6 year prospective study hepatitis in the obstetric patient prophylactic isoniazid protection of infants in a tuberculosis hospital breast-feeding protects against respiratory syncytial virus infections neonatal herpes simplex infection possibly acquired via maternal breast milk risk of human immunodeficiency virus type 1 transmission through breastfeeding serologic evidence for congenital transmission of human herpesvirus 6 examination of human breast milk for evidence of human herpesvirus 6 by pcr cytomegalovirus infection of breast milk and transmission in infancy late postnatal mother-to-child transmission of hiv-1 in abidjan listeriosis during pregnancy and in neonates rotavirus infections in young nicaraguan children european collaborative study: children born to women with hiv-1 infection: natural history and risk of transmission european paediatric hepatitis c virus network: effects of mode of delivery and infant feeding on the risk for motherto-child transmission of hepatitis c virus flora of the umbilical stump: 2479 cultures a lipid inhibitor of dengue virus in human colostrum and milk, with a note on the absence of anti-dengue secretory antibody vertical transmission of hepatitis g nonspecific antiviral substances in human milk against arbovirus and murine leukemia virus genetic evidence for mother-to-infant transmission of hepatitis g virus stringent precautions are not advisable when caring for patients with viral hemorrhagic fevers evalution and treatment of community acquired staphylococcus aureus infections in term and late-preterm previously healthy neonates diagnostic value of signs and symptoms of mammary candidosis among lactating women immunity and correlates of protection for rotavirus vaccines rate of inactivation of cytomegalovirus in raw banked milk during storage at 20 degrees c and pasteurisation htlv-i transmission from mother to child detection of human herpesvirus 7 (hhv-7) dna in breast milk by polym erase chain reaction and prevalence of hhv-7 antibody in breast-fed and bottle-fed children a new endemic focus of human t-lymphotropic virus type ii carriers among orinoco natives in colombia estimating the time of htlv-i infection following mother-to-child transmission in a breast-feeding population in jamaica tertiary contact vaccinia in a breastfeeding infant community acquisition of group b streptococcus by infants of colonized mothers infectious diseases of the fetus and newborn infant hospital infection control practices advisory committee: guidelines for isolation precautions in hospitals transmission of community-associated methicillian-resistant staphylococcus aureus from breast milk in the neonatal intensive care unit giardiasis and breastfeeding in urban africa multidisciplinary prospective study of mother-to-child chikungunya virus infections on the island of la reunion management of outbreaks of methicillin-resistant staphylococcus aureus infection in the neonatal intensive care unit: a consensus statement chickenpox, measles and mumps seroepidemiology in various population groups of the greater montreal area mother-to-child transmission of hepatitis c virus: evidence for preventable peripartum transmission cholate-dependent killing of giardia lamblia by human milk triple antiretroviral prophylaxis administered during pregnancy and after delivery significantly reduces breast milk viral load: a study within the drug resource enhancement against aids and malnutrition program risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level comparison of fluconazole and nystatin oral suspensions for treatment of oral candidiasis in infants rapid high temperature treatment of human milk htlv-i: a new problem for latin america rotavirus vaccines opportunities and challenges detection of human immunodeficiency virus type 1 (hiv-1) dna and p24 antigen in breast milk of hiv-1-infected ugandan women and vertical transmission denuge and denuge hemorrhagic fever tubercular mastitis child to mother transmission of herpes simples virus-1 infection at an unusal site vertical transmission of hepatitis c virus dengue: an update epidemiology and diagnosis of hospital acquired conjunctivitis among neonatal intensive care unit patients congenital infections with human herpes virus 6 (hhv6) and human herpes virus 7 (hhv7) the absence of candida albicans in milk samples of women with clinical symptoms of ductal candidiasis dengue hemorrhagic fever in infants: research opportunities ignored epidemiology of transmission of cytomegalovirus from mother to preterm infant by breastfeeding cytomegalovirus transmission to preterm infants during lactation congenital malformations and oral poliovirus vaccination during pregnancy mammary tuberculosis: analysis of thirty-eight patient cytomegalovirus in human milk an epidemiologic study of breast cancer detection of htlv-ii in breast milk of htlv-ii infected mothers killing of giardia lamblia by human milk lipases: an effect mediated by lipolysis of milk lipids risk of hepatitis b transmission in breast-fed infants of chronic hepatitis b carriers transmission of west nile virus through human breast milk seems to be rare milk-borne transmission of htlv-i as a major route in the endemic cycle primary prevention of htlv-i in japan primary prevention of htlv-1 in japan apparent vertical transmission of human immunodeficiency virus type 1 by breast-feeding in zambia virus markers associated with vertical transmission of human t lymphotropic virus type 1 in jamaica rotavirus antibodies in the mother and her breastfed infant incidence of haemophilus influenzae in the throats of healthy infants with different feeding methods transfusion transmission of west nile virus: a merging of historical and contemporary perspectives clinical presentations and outcomes of severe acute respiratory syndrome in children poliomyelitis in pregnancy: a twenty-year report from long-term serologic follow-up of patients for epstein-barr virus after recovery from infectious mononucleosis the global distribution of human immunodeficiency virus type 2 (hiv-2) infection interventions for preventing late postnatal mother-to-child transmission of hiv methicillin-resistant staphylococcus aureus colonization and its association with infection among infants hospitalized in neonatal intensive car units staphylococcus aureus in the infant upper respiratory tract. i. observations on hospital-born babies unilateral breast feeding and breast cancer staphylococcus aureus in australasian neonatal nurseries identification of human t-cell lymphotropic virus type iia infection in the kayapo, an indigenous population of brazil mother-to-infant transmission of tt virus in japan italian register for hiv infection in children: hiv-1 infection and breast milk transmission of west nile virus from an organ donor to four transplant recipients tuberculosis mastitis methicillin-resistant staphylococcus aureus infections among healthy full-term newborns human milk in the modern world staphylococcus epidermidis: a differential trait of the fecal microbiota of breastfed infants epstein-barr virus shedding in breast milk post weaning gastroenteritis and mortality in hiv-1 uninfected african infants receiving antiretroviral prophylaxis to prevent mtct of hiv-1 successful antepartum treatment of listeriosis low risk for motherto-child transmission of human t lymphotropic virus type ii in non-breastfed infants staphylococcal scalded skin syndrome in a breastfed infant fluconazole prophylaxis against fungal colonization and infection in preterm infants transmission of staphylococus aureus between healthy lactating mothers and their infants by breastfeeding transmammary transmission of strongyloides ratti case-control study of mastomys natalensis and humans in lassa virusinfected households in sierra leone transfer of tuberculin immunity from mother to infant recurrent group b streptococcal disease in an infant associated with the ingestion of infected mother' s milk mammary tuberculosis: report on 52 cases eradication of methicillin-resistant staphylococcus aureus from a neonatal intensive care unit by active surveillance and aggressive infection control measures prevention of mother-to-child transmission of hiv-1 through breast feeding by treating infants prophylactically with lamivudine in dar es salaam prevention of mother-to-child transmission of hiv through breastfeeding by treating mothers with triple antiretroviral therapy in dar es salaam, tanzania: the mitra plus study clinical outcomes in methicillin-resistant staphylococcus aureus colonized neonates in the neonatal intensive care unit demonstration of adult t-cell leukemia virus antigen in milk from three seropositive mothers oral infection of a common marmoset with human t-cell leukemia virus type i (htlv-i by fresh human milk of htlv-i carrier mothers the differences of clinical manifestations and laboratory findings in children and adults with dengue virus infection human parvovirus b19 infection in women of childbearing age and within families lymphocytic choriomeningitis in the newborn late-onset and recurrent neonatal group b streptococcal disease associated with breast-milk transmission diarrhoea in uninfected infants of hiv-infected mothers who stop breastfeeding at 6 months: the ban study experience accessed 12/19 prolonged breastfeeding and mortality up to two years postpartum among hiv positive women in zambia hiv specific secretory iga in breast milk of hiv-positive mothers is not associated with protection against hiv transmission among breast-fed infants high uptake of exclusive breastfeeding and reduced postnatal hiv transmission; prospective results from the zambia exclusive breastfeeding study accessed 12/19 effect of early, abrupt weaning on hiv-free survival of children in zambia differential effects of early weaning for hiv-free survival of children born to hiv-infected mothers by severity of maternal disease breastfeeding and aids in the developing world hiv prevention is not enough: child survival in the context of prevention of mother to child hi transmission congenital and postnatally acquired cytomegalovirus infections: long-term follow-up extended antiretroviral prophylaxis to reduce breast milk hiv-1 transmission breast milk is not a significant source for early epstein-barr virus or human herpes virus 6 infection in infants: a seroepidemiologic study in 2 endemic areas of human t-cell lymph tropic virus type i in japan evidence for mother-to-child transmission of human t-lymphotropic virus type ii mother-to-child transmission of human t-lymphotropic virus type ii (htlv-ii) role of maternal antibody in pneumonia and bronchiolitis due to respiratory syncytial virus a further report on cancer of the breast repeated congenital infection with toxoplasma gondii is ingestion of milk associated bacteria by premature infants fed raw human milk controlled by routine bacteriologic screening? maternal and child health technical information bulletin cytomegalovirus in human breast milk: risk to the premature infant the independent associations of parity, age at first full term pregnancy, and duration of breastfeeding with risk for breast cancer epstein-barr virus estimating infant mortality from hiv and other causes in breastfeeding and bottle-feeding populations postnatal cytomegalovirus infection from frozen breast milk in preterm low birth weight infants postntal transmission of hiv from mother to child eradication of methicillin-resistant staphylococcus aureus in a neonatal intensive care unit: which measures for which success? international multicentre pooled analysis of late postnatal mother-to-child transmission of hiv-1 infection. ghent international working group on mother-to-child transmission of hiv 18 month effectiveness of short-course antiretroviral regimens combined with alternatives to breastfeeding to prevent hiv mother-tochild transmission breast milk transmission of a panton-valentine leukocidin-producing staphylococcus aureus strain causing infantile pneumonia mammary epithelial cells support and transfer productive human t cell lymphotropic virus infections mechanism of vertical transmission of hepatitis g absence of infection in breast fed infants born to hepatitis c virus-infected mothers intrauterine listeria infection: prenatal diagnosis by biophysical assessment and amniocentesis ejpidemiologic features of htlv-ii: serologic and molecular evidence neonatal brucellosis probable transmission of brucellosis from breast milk to a newborn a multicenter therapeutic study of 1100 children with brucellosis lactation and cancer of the breast: a summary of an international study human immunodeficiency virus infection as risk factor for mother-to-child hepatitis c virus transmission: persistence of anti-hepatitis c virus in children is associated with the mother' s antihepatitis c virus immunoblotting pattern increased infant human immunodeficiency virus type one free survival at one year of age in sub-saharan africa with maternal use of highly active antiretroviral therapy during breast feeding the genome sequence of the sars-associated coronavirus diagnostic approach cytomegalovirus infection of extremely low-birth weight infants via breast milk freezethawing of breast milk does not prevent cytomegalovirus transmission to a preterm infant human milk siga binds to botulinum type b 16 s toxin and limits toxin adherence on t84 cells antimicrobial factors and microbial contaminants in human milk: recent studies morbidity and mortality patterns in hiv-1 seropositive/seronegative women in kampal and harare during pregnancy and in the subsequent two years morbidity and mortality in breastfed and formula-fed infants of hiv-1-infected women: a randomized clinical trial predominance of left-sided breast tumors obstetric management of hepatitis c-positive mothers: analysis of vertical transmission in 559 mother-infant pairs evidence for a protective effect of lactation on risk of breast cancer in young women: results from a case-control study prospective study of the incidence and complications of bacterial contamination of enternal feeding in neonates effect of treatment on the contagiousness of patients with active pulmonary tuberculosis infectious diseases of the fetus and newborn infant does breast feeding increase the child' s risk of breast cancer? role of breast milk in acquisition of cytomegalovirus infection hiv transmission through breastfeeding: a study in malawi prevention of breast milk tranmission of hiv: the time is now mother-to-infant transmission of hepatitis c virus: rate of infection and assessment of viral load and igm anti-hcv as risk factors yellow fever vaccine nosocomial transmission of methicillin-resistant staphylococus aureus from a mother to her preterm quadruplet infants breastfeeding family history and breast disease transmission of hepatitis c virus from mothers to infants: its frequency and risk factors revisited immune escape by epstein-barr virus associated malignancies the duration of breastfeeding by hiv-1 infected mothers in developing countries: balancing benefits and risks search for possible routes of vertical and horizontal transmission of adult t-cell leukemia virus outbreak of invasive disease caused by methicillin-resistant staphylococcus aureus in neonates and prevalence in the neonatal intensive care unit human immunodeficiency virus type 1-infected cells in breast milk: association with immunosuppression and and vitamin a deficiency effect of breastfeeding on mortality among hiv-1 infected women: a randomised trial breast brucellosis in taif, saudi arabia: cluster of six cases with emphasis on fna evaluation case control study of symptoms and neonatal outcome of human milktransmitted cytomegalovirus infection in premature infants human milk glycosaminoglycans inhibit hiv glycoprotein gp 120 binding to its host cell cd4 receptor risk factors for neonatal methicillin-resistant staphylococcus aureus infection in a well-infant nursery lactation and a reduced risk of premenopausal breast cancer contamination of expressed human breast milk with an epidemic multiresistant staphylococus aureus clone human cytomegalovirus infection of breast milk strongyloides papillosus: prenatal and transmammary infection in ewes human neonatal infections with hookworms in an endemic area of southern nigeria. a possible transmammary route mother-to-child transmission of human t-cell lymphotoropic virus types i and ii (htlv-i/ii) in gabon: a prospective follow-up of 4 years transfer of human herpes virus 6 and 7 antibodies from mother to their offspring vertical transmission of hepatitis c virus transmission of hepatitis c virus from mothers to infants for the vertical transmission of hepatitis viruses collaborative study group, motherto-infant transmission of gb virus type c/hgv for the vertical transmission of hepatitis viruses collaborative study group: tt virus infection during childhood tt virus: virological and genomic characteristics and disease associations birth outcomes following west nile virus infection of pregnant women in the united states neonatal group b streptococcal disease associated with infected breast milk transmission of cytomegalovirus to extremely preterm infants through breast milk early breastfeeding cessation among hiv-exposed negative infants and risk of serious gastroenteritis: findings from a perinatal prevention trial in kampala, uganda inactivation of human immunodeficiency virus type i in human milk: effects of intrinsic factors in human milk and of pasteurization lactation mastitis: bacterial cultivation of breast milk, symptoms, treatment, and outcome human immunodeficiency virus and other viruses in human milk: placing the issues in broader prospective effect of breast-feeding on immune response to bcg vaccination brucellosis in a mother and her young infant: probable transmission by breast milk brucellar arthritis of knee in a child breast-feeding during primary maternal human immunodeficiency virus infection and risk of transmission from mother to infant treatment acceleration program and the experience of the dream program in prevention of mother-to-child transmission of hiv updated u.s. public health service guidelines for the management of occupational exposures to hiv and recommendations for post exposure prophylaxis etiologic factors in cancer of the breast in humans: collective review methicillin-resistant staphylococcus aureus in milk oral rotavirus vaccines: how well will they work were they are needed most? determinants of acquisition and carriage of staphylococcus aureus in infancy early acquisition of cytomegalovirus infection clinical virology west nile virus: a primer for the clinician petra study team: efficancy of three short course regimens of zidovudine and lamivudine in preventing early and late transmission of hiv-1 from mother to child in tanzania, south africa, and uganda (petra study): a randomized, double-blind, placebo-controlled trial dengue virus infection in late pregnancy and transmission to the infants effect of maternal rotavirus immunization on milk and serum antibody titers cell-free virus in breast milk of hiv-1-seropositive women role of breastfeeding in paralytic poliomyelitis bacterial contamination of human milk: container type and method of expression maternal herpetic breast infection: another hazard of neonatal herpes simplex mother-to-child transmission of chikungunya virus infection care of the pregnant woman with tuberculosis and her newborn infant: a pediatrician' s perspective read jsand the committee on pediatric aids: human milk, breastfeeding, and transmission of human immunodeficiency virus type 1 in the united states postpartum mastitis and community-acquired methicillin-resistant staphylococcus aureus infectious diseases of the fetus and newborn infant hepatitis e virus breastmilk infectivity in human immunodeficiency virus type 1-infected mothers high risk types of human papillomavirus (hpv) dna in oral and genital mucosa of infants during their first 3 years of life: experience from the finnish hpv family study streptococcal toxic shock syndrome, including necrotizing fasciitis and myositis late-onset septicemia in a norwegian national cohort of extremely premature infants receiving very early full human milk feeding hepatitis a outbreak in a neonatal intensive care unit: risk factors for transmission and evidence of prolonged viral excretion among preterm infants characterization of a novel corona virus associated with severe acute respiratory syndrome longitudinal analysis of human immunodeficiency virus type 1 rna in breast milk and of its relationship to infant infection and maternal disease attempts to detect rna tumour virus in human milk is breast milk collected at home suitable for raw consumption by neonates in brizilian public neonatal intensive care units? prevalence of hiv-1 dna and p24 antigen in breast milk and correlation with maternal factors follow-up of transmission of hepatitis c to babies of human immunodeficiency virus-negative women: the role of breastfeeding in transmission occurrence of acute diarrhea in atopic and nonatopic infants: role of prolonged breast-feeding an outbreak of methicillin-resistant staphylococcus aureus in a neonatal intensive care unit hospital transmission of community acquired methicillin-resistant staphylococcus aureus among postpartum women removal of inhibitors against rna-directed dna polymerase activity in human milk effect of human milk on mouse mammary tumor virus human papillomavirus dna detected in breast milk control of a cluster of a community-associated methicillin-resistant stahpylococcus aureus in neonatology immunoglobulin prophylaxis against milkborne transmission of human t cell leukemia virus type 1 in rabbits chlamydial infections pregnancy and pulmonary tuberculosis possible breast milk transmission of group b streptococcal infection evidence for transmission of lymphocyte responses to tuberculin by breast-feeding, lancet i:529 toxic shock syndrome detection of borrelia burgdorferi dna by pcr in the urine and breast milk of patients with lyme borreliosis prevention of perinatal group b streptococcal disease. revised guidelines from cdc detection of tt virus dna and gb virus type c/hepatitis g virus rna in serum and breast milk: determination of mother-to-child transmission effects of hepatitis a and b in pregnancy on the mother and fetus human immunodeficiency virus load in breast milk, mastitis and mother-to-child transmission of human immunodeficiency virus type 1 how contagious is vaccinia? transmammary transmission of necator americanus larva in the human host a study of infectious intestinal disease in england: risk factors associated with group a rotavirus in children highly active antiretroviral therapy started during pregnancy or postpartum suppresses hiv-1 rna, but not dna, in breast milk prevention of postnatal cytomegalovirus infection in preterm infants infants born to mothers with severe acute respiratory syndrome tuberculosis of the breast masquerading as carcinoma: a study of 100 patients transmammary transmission of strongyloides stercolis in dogs low birth weight and maternal virus diseases: a prospective study of rubella, measles, mumps, chickenpox, and hepatitis lyme disease during pregnancy no benefit of early cessation of breastfeeding at 4 months on hiv-free survival of infants born to hiv-infected mothers in zambia: the zambia exclusive breastfeeding study vertical dengue infection: case reports and reviews a young infant with severe acute respiratory syndrome six week extended-dose neviapine (swen) study team: extended-dose nevirapine to 6 weeks of age for infants to prevent hiv transmission via breastfeeding in ethiopia, india, and uganda: an analysis of three randomized controlled trials chlamydial secretory iga antibodies in human milk hepatitis d virus factors associated with immunoprophylaxis failure against vertical transmission of hepatitis b virus phagocytosis-associated oxidative metabolism in human milk macrophages management of mastitis in breastfeeding women communityacquired methicillin-resistant staphylococcus aureus among patients with puerperal mastitis requiring hospitalization working parents: the impact of day care and breast-feeding on cytomegalovirus infection in offspring breast milk and the risk of cytomegalovirus infection tuberculosis, an old disease but a new threat to mother, fetus, and neonate vertical transmission of hepatitis b antigen in taiwan yeast-recombinant hepatitis b vaccine: efficacy with hepatitis b immune globulin in prevention of perinatal hepatitis b virus transmission maternal lyme disease and congenital heart disease: a case-control study in an endemic area significance of postnatal mother-to-child transmission of htlv-i on the development of adult t-cell leukemia/lymphoma disseminated neonatal herpes simplex virus type i from a maternal breast lesion the effects of yellow fever immunization (17dd) inadvertently used in early pregnancy during a mass campaign in brazil the impact of breastfeeding on the health of hiv positive mothers and their children in sub-saharan africa prospective study of mother-to-infant transmission of hepatitis c virus inhibitory effect of maternal antibody on mother-to-child transmission of human t-lymphotropic virus type i seroepidemiological study of human herpes virus 6 and 7 in children of different ages and detection of these two viruses throat swabs by polymerase chain reaction short-term breast-feeding may reduce the risk of vertical transmission of htlv-i. the tsushima atl study group infant feeding and risk of mother-to-child transmission of hiv-1 in sao paulo state, brazil. sao paulo collaborative study for vertical transmission of hiv-1 breast feeding plus infant zidovudine prophylaxis for 6 months vs. formula feeding plus infant zidovudine for 1 month to reduce mother-to-child hiv transmission in botswana: a randomized trial: the mashi study isolation of the aids virus from cell-free breast milk of three healthy virus carriers rates of diarrhoea associated with early weaning among infants in kisumu, kenya. in program and abstracts of the 14th conference on retroviruses and opportunistic infections contamination in expressed breast milk following breast cleansing brucellar meningitis in an infant-evidence for human breast milk transmission hepatitis e in india human parvovirus b19 congential yellow fever virus infection after immunization in pregnancy unaids/who: report on the global hiv/aids epidemic a review of hiv transmission through breastfeeding mother-to-child transmission of human t-cell leukemia/ lymphoma virus type i: implication of high antiviral antibody titer and high proviral load in carrier mothers clinical profile of chikungunya in infants infective and antiinfective properties of breast milk from hiv-1 infected women postnatal transmission of the human immunodeficiency virus type 1 from mother to infant: a prospective cohort study in kigali, rwanda mother-tochild transmission of human t-lymphotropic virus type ii the possible role of circumcision in newborn outbreaks of community associated methicillin-resistant staphylococcus aureus brucellosis chez un nourrisson de 3 mois recovery of staphylococcal enterotoxin f from the breast milk of a woman with toxic-shock syndrome evidence for sexual and mother-to-child transmission of human t lymphotrophic virus type ii among guaymi indians transmission of cytomegalovirus to preterm infants through breast milk postnatally acquired cytomegalovirus infection via breast milk: effects on hearing and development in preterm infants pregnancy and lactation in relation to breast cancer risk morbidity among hiv-1-infected mothers in kenya: prevalence and correlates of illness during 2-year postpartum follow-up high concentrations of interleukin 15 in breast milk are associated with protection against postnatal hiv transmission low and undectable breast milk interleukin-7 concentrations are associated with reduced risk of postnatal hiv transmission lactation mastitis: a descriptive study of the experience breastfeeding does not pose any additional risk of immunoprophylaxis failure on infants of hbv carrier mothers recurrent neonatal group b streptococcal disease associated with infected breast milk transplacentally transferred maternal-infant antibodies to dengue virus vertical transmission of hepatitis a resulting in an outbreak in a neonatal intensive care unit immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 e6 or e7 perinatal transmission of hepatitis g virus (gb virus type c) and hepatitis c virus infections: a comparison advisory committee on immunization practices; healthcare infection control practices advisory committee: recommendations for using smallpox vaccine in a pre-event vaccination program global rotavirus surveillance: determining the need and measuring the impact of rotavirus vaccines recommendations for minimizing cmv exposure in breast milk fed very low birth weight (vlbw) preterm infants. available at www.breastfeeding. org/articals/cmvpremi.pdf maternal-infant transmission of htlv-i: frequency and time course of seroconversion. abstract w-53 mother-to-child transmission of human t-cell lymphotropic virus type i associated with prolonged breast-feeding maternal lyme disease and congenital malformations: a cord blood serosurvey in endemic and control areas human versus cow' s milk in infant nutrition and health human t-lymphocyte retroviruses working group on severe streptococcal infections: defining gas streptococcal toxic shock syndrome: rationale and consensus definition acquired cytomegalovirus infection of breast milk in infancy oral transmission of human t-cell leukemia virus type 1 into a common marmoset as an experimental model for milk-borne transmission evaluation of cytomegalovirus infections transmitted via breast milk in preterm infants with a real-time polymerase chain reaction assay sequelae of maternally derived cytomegalovirus infections in premature infants mother-to-infant transmission of hepatitis c virus human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis independent protective effect of lactation against breast cancer: a case-control study in japan a maternal factor for mother-to-child transmission: viral antigen-producing capacities in culture of peripheral blood and breast milk cells mother-to-infant transmission of hepatitis c virus infectious diseases of the fetus and newborn infant sensitive method for detection of human herpes viruses 6 and 7 in saliva collected in field studies survey of 34 pregnant women with hepatitis a and their neonates postnatal transmission of aids-associated retrovirus from mother to infant disseminated lyme disease and pregnancy. 9th annual international scientific conference on lyme disease and other tick-borne disorders a. siblings at home have active chickenpox when neonate and mother are ready for discharge from hospital.no no 1. mother: if she has a history of chickenpox, she may return home. without a history, she should be tested for varicellazoster virus antibody titer.* if test is positive, she may return home. if test is positive, she may return home. if test is negative, varicella-zoster ig † is administered and she is discharged home. 2. neonate: may be discharged home with mother if mother has history of varicella or is varicella-zoster virus-antibody positive. if mother is susceptible, administer varicella-zoster ig to infant and discharge home or place in protective isolation. b. mother has no history of chickenpox; exposed during period 6-20 days antepartum. ‡ ‡if exposure occurred less than 6 days antepartum, mother would not be potentially infectious until at least 72 hours postpartum. §considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted. ¶dosage of acyclovir for pregnant woman is 30 mg/kg/day; for seriously ill infant with varicella, 750 to 1500 mg/m 2 /day. elisa, enzyme-linked immunosorbent assay; fama, fluorescent antibody to membrane antigen; la, latex agglutination. key: cord-286574-t9z2ynt5 authors: nan title: speaker presentations date: 2017-09-30 journal: international journal of antimicrobial agents doi: 10.1016/s0924-8579(17)30340-0 sha: doc_id: 286574 cord_uid: t9z2ynt5 nan increased mortality rates in patients infected with major resistant pathogens are as follows; • mrsa: mrsa can cause almost 2-fold higher mortality (or 1.93) compared with mssa strains according to a meta-analysis (cosgrove et al. 2003) . mrsa kills more american people every year (∼19,000) than emphysema, hiv/aids, parkinson's disease and homicide combined (klevens et al. 2007 ). • esbl-producing gram-negative bacilli: a meta-analysis showed that crude rrs were significantly higher mortality in esbl-associated bacteremia ( pooled rr 1.85) (schwaber et al. 2007 ). • carbapenem-resistant k.pneumoniae: in patients with carbapenem-resistant k.pneumoniae bacteremia, the crude mortality rate was 71.9%, while it was 21.9% in control subjects (borer et al. 2009 ). a mortality risk ratio was 3.3 for patients with carbapenem-resistant k. pneumoniae bacteremia. increased mortality and morbidity in resistant infections is due to treatment failure of antibiotic therapy which is associated with bacterial fitness, greater severity of underlying illness, delays in initiating effective therapy and lack of effective therapy (friedman et al. 2016) . due to mismatch between choice of empirical antibiotics and in vitro susceptibility test results, administration of effective antibiotics was delayed by 6-times in the case of antibiotic-resistant e.coli and k.pneumoniae infections compared with susceptible strains (72 hours vs 11 hours) (lautenbach et al. 2001) . also, patients infected with resistant strains are more frequently associated with more severe underlying illness requiring longer hospitalization. for example, patients with cre infections are more likely to be a transplant recipient, require mechanical ventilation, a prolonged hospitalization, icu stays, or use of central venous catheters (borer et al. 2009 ). patients infected with resistant strains are more likely to develop complications and long-term sequelae. for example, mrsa infection caused higher incidence of complications by 69% compared with mssa infection. the most frequent complications are a progression of the local infection (rr 3.25) and pneumonia (rr 2.28) (cecchini et al. 2015) . once amr emerges in the hospital, resistant pathogens can cause additional cases of bacterial infections that affect more patients. emergence of amr in major human pathogens also affects safety and efficacy of surgical procedures, cancer chemotherapy, organ transplantation, and intensive care. therefore, amr is not just an infectious disease issue, but rather an issue in surgery, cancer care, organ transplantation and whole health system. amr can also negatively affect the daily hospital activities such as total closure of an affected ward or unit or cancellation of elective surgery. economic impact of amr is difficult to quantify, which consists of direct costs associated with use of more expensive antibiotics, special equipments, longer hospital stay, and isolation procedures etc., and indirect costs mainly due to loss of productivity of the patients. according to the world bank report to estimate the amr impact on global gdp in -2050 (world bank, 2016 , in the optimistic "low-amr" scenario, global economic output is projected to be 1% lower by 2030 ($1 trillion) and 1.1% lower by 2050 than in the base case. in the pessimistic "high-amr impact" scenario, global economic output would be 3.2% lower in 2030 ($3.4 trillion) and 3.8% lower by 2050 than in the base case. it means that in the "high-amr" scenario, economic damage will be greater than that in 2008-2009 global financial crisis which caused 3.6% drop in the global gdp. economic impact of amr differs by countries with different economic level. low-income and lower middle-income countries would have greater impact of economic loss (5.6% loss of gdp) due to amr compared with high-income countries (3.1% loss of gdp), particularly in the "high-amr" scenario. global economy is negatively affected by amr in various aspects such as international trade, livestock production, and healthcare expenditures. by 2050, the volume of global real exports may decrease by 1.1% in the "low-amr" scenario and by 3.8% in the "high-amr" scenario. in the "high-amr" scenario, healthcare expenditures in 2050 would be as 25% higher than the baseline values for low-income countries, 15% higher for middle-income countries, and 6% higher for high-income countries. the additional expenditures in 2050 would be $1.2 trillion annually in the "high-amr" scenario. in the us, cdc estimated the cost of amr as a total of $55 billion per year : $20 billion in excess for direct healthcare costs and $35 billion for indirect societal costs due to loss of productivity (cdc, 2013) . in europe, the overall economic burden of amr was estimated to be at least 1.5 billion euros; 60 % for direct costs and 40% for indirect costs (ecdc/emea, 2009 ). data on the economic impact of amr in major pathogens showed that healthcare costs significantly increase in the treatment of infections caused by antibiotic-resistant strains (maragakis et al. 2008) ; mrsa bacteremia (us$ 6,916), mrsa surgical site infection (us$ 13, 901) , vre infection (us$ 12, 766) , and esbl or kpc-producing e.coli or klebsiella infection (1.7fold increase). amr is obviously one the most serious and urgent issues with devastating impact on clinical medicine, economic growth and societal system and function. g20 leaders' declaration which was announced in july 2017 also underlined the importance of combating amr through international collaboration. for more effective and robust implementation of global action plan to combat amr, appropriate evaluation of impact of amr should be performed, particularly in developing world which would have greater damage due to amr. school of public health, the university of hong kong, china the development, large-scale production and widespread use of antimicrobial drugs in the twentieth century was a turning point in human history, resulting in dramatic improvements in medical care and reduction of deaths. over time, however, rising levels of antimicrobial resistance (amr) among a wide range of pathogens has placed such gains at risk of being lost. reducing amr is now a top-level global public health priority. in principle, the major high-level goals consist of optimizing the use of such drugs in health and agriculture and minimizing environmental contamination; sustaining the development of new classes of antimicrobials drugs and other medicines and making them affordable and accessible to all who need them; and much more effective application of infection control and prevention principles. for decades, technical solutions have been the primary approach used for addressing amr. more recently, fao, oie and who in combination with like-mined champions have embarked upon a more political and broader "one health" approach to increase awareness and engagement beyond scientific and medical groups. this change is the basis for the 2015 global action for amr, the 2016 high level meeting on amr held at the un general assembly and attention to amr by groups such as the g20. while such results have been instrumental in broadening the awareness and attention paid to amr, it is now critical to adopt concrete and focused activities to consolidate and build upon these gains. the fundamental building blocks will be proposed and discussed. murdoch university, australia methicillin-resistant staphylococcus aureus (mrsa) was initially a healthcare associated pathogen limited to distinct lineages often associated with multi-drug resistance. however, in the late 1980s mrsa began to emerge in the community. in the beginning, this was mostly confined to closed communities, i.e. australian aboriginals, but around the late 1990s community-associated mrsa (ca-mrsa) emerged worldwide in the general population. the ca-mrsa clones evolved independently of the ha-mrsa clones, and typically, possessed type iv or v sccmec elements, were non multi-drug resistant, and were positive for the panton valentine leucocidin (pvl) toxin. whilst regionally many unique ca-mrsa clones have emerged, only a few clones predominate within a region, and even fewer clones have a global distribution. the reasons for the variation in the prevalence and dominance of ca-mrsa clones between different geographical niches remains unclear. although the epidemiology of ca-mrsa varies considerably, four major global ca-mrsa clones have been described: st8-mrsa-iv (usa300) and st30-mrsa-iv (south west pacific clone) which and are considered pandemic; st80-mrsa-iv (european clone) which predominates in europe, africa and the middle east; and st59-mrsa-iv/v t (taiwan/asia pacific clone) in asia. however, using whole genome sequencing analysis, at least two of the clones have different clades that have evolved independently of each other. in clonal complex 8 nine phylogenetic clades with at least eight independent events of methicillin-resistance acquisition have been identified. subsequently, it has been hypothesised usa300 did not evolve from the healthcare-associated st8-iv usa500 clone, the historic ca-mrsa from western australia, or the pvl-positive mssa clone from trinidad and tobago or western africa, but most likely from pvl-positive mssa circulating within the usa. the ancestor of the usa300 clade emerged in central europe in the mid-19 th century and exported to north america in the early 20 th century. once in north america the clone progressively acquired the usa300 characteristic genetic specifications, diversified and spread globally including africa. within the usa300 clade two closely related st8-iv clones are recognised: usa300, primarily isolated in north america (st8-mrsa-iva, spa t008 luks-pv/lukf-pv, acme positive, msra-mediated macrolide resistance genes) and the usa300 latin american variant, primarily isolated in south america (st8-mrsa-ivc, spa t008, luks-pv/lukf-pv, acme negative, tetk, copper and mercury resistance genes). similarly, st59 mrsa is also not a single clone but consists of two major clades, one originating in the usa and the other in east asia. furthermore, two distinct subclades within the east asia clade have been identified: pvlnegative st59-mrsa-iv (asia pacific clone) and the multi-resistant pvl positive st59-mrsa-v t (taiwan clone) which possesses two distinct ccrc genes. recently three newly described pvl-positive ca-mrsa clones have been reported in multiple countries: st93-mrsa-iv (queensland ca-mrsa) from australia, and st772-mrsa-v t (bengal bay) and st22-mrsa-iv from the indian subcontinent. st22-mrsa-iv, first reported in india in 2010, is genetically distinct from the healthcare associated st22-mrsa-iv (emrsa-15) having acquired different sccmec types and sub types. of concern, st772-mrsa-v t and st22-mrsa-iv are multi-resistant hyper-virulent ca-mrsa that have recently been associated with nosocomial transmission. the co-emergence of multiple ca-mrsa lineages that have risen independently in most parts of the world has been striking. to date no single genetic or epidemiological factor has been identified that accounts for the extraordinary success of some genetically distinct ca-mrsa clones. over time, some ca-mrsa clones will replace ha-mrsa clones, which will have significant clinical and public health implications. along the way they will become increasing antibiotic resistant making the distinctions between ha-mrsa and ca-mrsa blurred and antimicrobial treatment difficult. methicillin-resistant staphylococcus aureus (mrsa) has been a major cause of healthcare-associated infections with significant morbidity and mortality, and it has become one of the most important nosocomial pathogens in many countries worldwide since 1980s. in particular, several mrsa clones have been successful in spreading and causing infections in the hospitalized patients. the prevalence rates of mrsa in s. aureus isolates from the hospitalized patients have become high enough to spread into the community. interestingly, during the past two decades, the emergence of community-associated (ca-) mrsa clones has greatly affected the global epidemiology of s. aureus infection. circulating ca-mrsa clone varies according to the region, and it includes st8 (usa300), st80, st30, st59, st72, st772 and st22 which mostly belong to sccmec type iv or v. as a result, the proportion of mrsa in community-acquired s. aureus infections has been significantly increasing in many countries. furthermore, many reports have shown that ca-mrsa clones have replaced traditional hospital-endemic mrsa clones. such an influx of dominant ca-mrsa clones into the hospitals has been making the 'search and destroy' policy which have contributed to maintain low mrsa rates in some countries difficult to maintain. in addition, there has been concern that healthcareassociated infections such as surgical site infection caused by ca-mrsa may increase. in this talk, an update on ca-mrsa epidemiology in healthcare-associated infection and concerns on infection prevention and control will be discussed. the newly emerging middle east respiratory syndrome coronavirus (mers-cov) causes a severe respiratory infection with a high mortality rate (∼35%), and has been a global threat due to continuous outbreaks in the arabian peninsula and international spread by infected travelers since 2012. from may to july 2015, a large outbreak initiated by an infected traveler from the arabian peninsula swept south korea, and resulted in 186 confirmed cases with 38 deaths (case-fatality rate: 20.4%). here, we show the rapid emergence and spreading of a mutant mers-cov with reduced affinity to the human receptor, cd26, during the korean outbreak. we isolated thirteen new viral genomes from fourteen infected patients treated at a hospital and found that twelve of them possess a point mutation in the receptor-binding domain (rbd) of viral spike (s) protein. we also analyzed clinical data and specimens from fourteen mers patients treated in a hospital who collectively represent a wide spectrum of disease severity, ranging from mild febrile illness to fatal pneumonia. comparative and kinetic analyses revealed that high viral loads, weak antibody responses, and lymphopenia accompanying thrombocytopenia were associated with disease mortality, whereas persistent and gradual increases in lymphocyte responses might be required for effective immunity against mers-cov infection. the correlation of the virological and immunological responses with disease severity and mortality, as well as their responses to current antiviral therapy, may have prognostic significance during the early phase of mers. severe fever with thrombocytopenia syndrome (sfts) was discovered as an infectious disease caused by a novel bunyavirus in china in 2011 (n engl j med, 2011 . the causative agent for sfts is named sfts virus (sftsv), which is a tick-borne virus and belongs to the family buniyaviridae, genus phlebovirus. the virus infection causes generalized infections with a high case fatality rate. in late 2012, it was discovered that sfts was also endemic to japan (takahashi t, et al., jid, 2014) . sfts is endemic to china, south korea, and japan. since the discovery of sfts endemic to japan in january 2013, approximately 240 patients with sfts have been reported to the national institute of infectious diseases. the case fatality is about 25%. most of the patients were aged over 40's. the pathophysiology of sfts has been studied through the pathological examination of sfts patients, who died. through the pathological studies on sfts, sftsv is present in the lymph node tissues. there are two pathological types, sftsv-positive lymph node-localized type and sftsv-positive lymph node-generalized types. all the patients, in whom bone marrow aspiration test was performed, showed hemophagocytic syndrome, indicating that cytokine storm plays an important role in pathogenesis of sfts. most fatal sfts patients showed symptoms of hemorrhage and deterioration in consciousness. one of the patients showed a symptom of bloody vomit. real time imaging of the stomach by endoscopic examination revealed the presence of ulcerative lesions with whoozing hemorrhage (kaneyuki s, et al., jjid, 2017) . the pathophysiological features behind the high case fatality rate in sfts are hemophagocytosis, hemorrhagic tendencies due to thrombocytopenia, disseminated intravascular coagulation, and ulcerative lesions appeared in the gastrointestinal tracts, and multi-organ failure. the efficacy of antiviral agent, favipiravir, which was developed by dr. furuta y and his colleagues (toyama chemical co., ltd, japan), in the treatment of sfts was evaluated using mice lacking the type i interferon alpha receptor (ifnar −/− ) (tani h, et al., msphere, 2016) . favipiravir inhibited replication of sftsv in vero cells by 5 log units, with a 50% inhibitory concentration (ic 50 ) and ic 90 of 6.0 and 22 µm, respectively. intraperitoneal or oral administration of favipiravir for 5 days to ifnar−/− mice infected with lethal sftsv significantly improved survival rates (100% survival) without causing body weight loss and reduced the viral load in the serum. although ribavirin also inhibited sftsv replication, it was quite less effective than favipiravir both in vitro and in vivo. a time-of-drug-addition study revealed that therapeutic favipiravir treatment of sftsv infection in ifnar−/− mice was effective. these results suggest that favipiravir might be a promising candidate as antiviral drug against sfts. the study suggested that favipiravir is a candidate drug for the treatment of sfts. in the presentation, i will present the overview of the epidemiology and pathophysiology evaluated through pathological autopsy, and the development strategy of antiviral drug therapy for sfts. this disease, sfts, continues to be endemic to china, korea, and japan for the future. we can not escape from the risk being infected with sftsv. on the other hand, there is a potential that favipiravir has a therapeutic effect in the treatment of sfts. safe and efficacious vaccine against sfts should be developed. furthermore, i will discuss the issues to be addressed to reduce the sfts disease burden. the challenges posed by infectious diseases in the 21 st century are forever growing more complex and numerous. there are no magic bullets or simple solutions that we can employ in our battle against the array of pathogens that every day attack humans, animals and plants. but there is much that we can do to mitigate the changing landscape of infectious diseases threats. this presentation will expand upon dr. osterholm's previous plenary session lecture. it will go into detail describing the major steps we can and must take to tip the balance between the growing threat of infectious diseases and our ability to anticipate these threats and prevent and control them before they can result in increased morbidity and mortality. the polymyxins, colistin ( polymyxin e) and polymyxin b, are effective antibiotics against most multidrug-resistant (mdr) gram-negatives and are currently considered as the last-line drugs for treating severe bacterial infections. polymyxin resistance among gram-negatives has increased gradually for the last few years, and knowledge of its multifaceted resistance mechanisms is expanding. typically, colistin resistance is due to chromosomally mediated modulation of two-component regulatory systems leading to modification of lipid a that resulted in reduced affinity to polymyxins. clones of polymyxin-resistant gram-negatives have spread in some hospitals, but have not significantly affected the use of polymyxins. these resistance genes are generally not transmissible between bacteria and so have not disseminated widely. however, a newly discovered plasmid-mediated polymyxin resistance gene mcr-1 has been detected in many countries in different geographical regions of the world since its first report in 2016. therefore, it seems inevitable that plasmid-mediated transfer of polymyxin resistance will seriously reduce the lifespan of the polymyxins as the backbone of regimens against infections due to mdr gram-negatives. this review provides an update on epidemiology and mechanisms of polymyxin resistance among different commonly encountered mdr gram-negative bacilli. some unresolved questions with respect to polymyxin resistance are also discussed. one of the few remaining options for the infectious disease caused by multiple-drug resistant gram-negative bacilli is colistin. accordingly, emerging mobile colistin resistance mcr-1 for phosphoethanolamine transferase became a death threat to public health. various enterobacteriaceae carrying mcr-1-plasmids from human beings, animals, and environments were reported in asia, europe, africa, and north and south america. in south korea, by the retrospective screening for the animal-oriented escherichia coli strains, the mcr-1 positive isolates have been identified. to evaluate mcr-1-positive clinical strains, a total of 9396 enterobacteriaceae isolates collected between 2010 and 2015 were screened and, finally, three strains, two escherichia coli belonging to either st1011 or st101 and one enterobacter aerogenes, harboring the gene were identified. all possessed inci2 mcr-1-plasmids and colistin mics were 4 g/ml in e. coli strains and 32 g/ml in e. aerogenes. the bla ctx-m-55 gene for extended-spectrum beta-lactamase was co-carried in the mcr-1-plasmid of e. aerogenes and extra plasmids carrying bla ctx-m-55 , bla ctx-m-27 , and bla ndm-9 were co-harbored by e. coli strains. one of the e. coli strains produced complete conjugal machinery presenting the best efficiency of plasmid transfer while e. aerogenes having a truncated prepilin peptidase pilu resulting in complete loss of conjugal activity. the plasmid in e. coli transferred efficiently to e. coli recipients compared to k. pneumoniae and to enterobacter cloacae probably due to the species-specificity allowed by rearranged shufflon. previously identified 11 mcr-1-positive e. coli strains of animal-origin from south korea also possessed inci2 mcr-1-plasmid and the non-self-transferable mcr-1-plasmid in e. aerogenes was closely associated to those, while the self-transferable mcr-1-plasmid in e. coli strains were alike to each other. the plasmid carrying the gene, or the bacterial host harboring the plasmid, has come from animals as the previous reports illustrated. better stewardship for the proper usage of antimicrobials as well as a collaborating surveillance study as a concept of one-health is needed. what to use in the clinical practice? yohei doi md, phd university of pittsburgh, usa while colistin resistance in enterobacteriaceae due to the plasmid-mediated mcr genes has drawn much attention, the threat of colistin resistance that impacts patient care most currently exists in extensively drug-resistant (xdr) bacteria which have acquired colistin resistance through chromosomal mutations in species such as klebsiella pneumoniae, pseudomonas aeruginosa and acinetobacter baumannii. unlike the mcr mechanisms that readily spread horizontally via plasmids, colistin resistance in these xdr species usually arises through selective pressure in patients upon treatment with colistin, though outbreaks have also been reported on occasions. development of colistin resistance in these strains means few or even no remaining active agents. however, patients who are affected by these bacteria are medically complex, heterogenous, and difficult to enroll into clinical trials for many reasons. consequently, clinical data associating colistin resistance, antimicrobial therapy given and patient outcome are scarce and retrospective in nature for the most part. for k. pneumoniae, limited data suggest that mortality of infection from colistin-resistant strains may be lower when treated with gentamicin-containing regimens than those without gentamicin, but this approach is only applicable when the infecting strain is susceptible to this agent. fortunately, the approval of ceftazidime-avibactam has ameliorated concerns over colistin resistance in k. pneumoniae at least for the time being, as therapy with ceftazidime-avibactam appears to improve clinical outcome of infected patients over those treated with colistin-based regimens. interestingly, gastrointestinal decolonization with gentamicin may reduce infections and mortality in those known to be colonized with colistin-resistant k. pneumoniae, but the recent spread of mcr genes in humans brings into question the long-term viability of this approach. there are even less clinical data regarding the treatment of infections caused by colistin-resistant p. aeruginosa and a. baumannii. in vitro data variably support colistin-based combinations with partner agents including carbapenems, rifampicin, fosfomycin, and even gram-positive agents like vancomycin in the case of a. baumannii, but without accompanying robust clinical data. there are several novel agents with activity against colistin-resistant strains in late-stage clinical development. they include novel beta-lactam-beta-lactamase inhibitor combinations, a siderophore cephalosporin, an aminoglycoside and tetracyclines. most of them target carbapenem-resistant enterobacteriaceae, while some are also active against p. aeruginosa and a. baumannii. these new agents are bound to change the paradigm for the treatment of infections caused by colistin-resistant gram-negatives, but uncertainties are still likely to remain, including which agent to use, how to optimize dosing in to maximize efficacy and minimize toxicity as well as potential for development of resistance, and whether use of more than one agent would still be needed. analyte health, usa in the united states, telehealth represents a rapid, convenient, and cost-effective solution for non-life threatening conditions, including acute respiratory infections, urinary tract infection (uti), and sexually transmitted infections (stis). over 1.25 billion outpatient "bricks and mortar" visits occur annually in the us, and it is estimated that one third of these visits (417 million) can be handled through telehealth. furthermore, one third of this subgroup of consults (140 million) are related to infectious disease causes. until recently, the provision of diagnostic testing, in conjunction with telemedicine physician consults, was difficult due to diverse geo-locations of patients, physicians and testing laboratories. for example, in the virtual world of telehealth, a patient can be located in california, with the physician licensed to practice in california located three time zones away in new york. linkage of the physician and patient is now easily accomplished by electronic means (e.g., video conferencing, phone calls, text messaging). however, obtaining specimens from the patient and then transporting the specimen to the appropriate testing laboratory has been a challenge, such that most telemedicine consults are performed without diagnostic testing and empiric treatment is provided. this approach, especially with acute respiratory infection, could result in overuse or underuse of antibiotics. this lecture will describe new approaches to diagnostic testing for infectious diseases in the telehealth ecosystem. included is a description of the infrastructure requirements required and/or recently developed to accommodate specimen acquisition and testing through aggregation ("uber"-ization), of both physicians and laboratory patient service centers, provision of "round the clock" mobile collection (in-home collection) services and self-collection and self-testing. also, the cost-savings of these approaches, in contrast to traditional bricks and mortar approaches will be discussed. rapid molecular, point of care testing instruments are now available for the accurate and complete (no confirmation testing required) testing for group a streptococcus for throat swab specimens, and influenza and rsv for nasopharyngeal swab specimens; future testing capabilities include sexually transmitted diseases like neisseria gonorrhoeae, chlamydia trachomatis and human papilloma virus (hpv). these new devices will accommodate the next inflection point in virtual medicine: point of patient testing, including testing the patients in their homes. the world bank estimates that epidemics will cost $6 trillion dollars in the 21 st centuryroughly $60 billion per year. the $6 billion cost of the ebola epidemic was its economic impact (in countries that can least afford it); however the impact of ebola on families, social customs, and health infrastructure will have long term consequences. in addition, funding for epidemic diseases waxes and wanes with the passage of the epidemic, and as a consequence longer term preventive solutions, such as vaccines, have little means for progressing. in 2016 several nations and charitable foundations launched a new enterprise known as cepi, the coalition for epidemic preparedness innovations. cepi will fund the development of vaccine candidates for diseases of epidemic potential, the initial candidates are mers, nipah and lassa fever. we will review product development activities in these diseases and also detail attempts by international organizations to try to develop a framework around responses to public health emergencies of international concern. carbapenemases, from kpc to ndm to oxa-48: diverse enzymes in diverse clones david m. livermore norwich medical school, university of east anglia, norwich nr4 7tj, uk carbapenemase-producing enterobacteriaceae present a growing problem. the predominant enzymes are members of the kpc, ndm, imp, vim and oxa-48 families. rarer types include imi, sme and fri enzymes, also some members of the ges family. kpc, imi, sme, fri and ges enzymes belong to class a; ndm, imp and vim are class b metallo carbapenemases, whilst oxa-48 and its relatives, e.g. oxa-181, belong to class d. this diversity of enzymes is reflected in a diversity of properties. kpc carbapenemases hydrolyse all widely available β-lactams and are inhibited by avibactam and vaborbactam. by contrast the metallo types evade avibactam and vaborbactam and attack all β-lactams except monobactams, which are often compromised by co-produced esbl and ampc enzymes. oxa-48, which is inhibited by avibactam but not vaborbactam, has little activity against oxyimino-cephalosporins but, again, is often accompanied by cephalosporin-hydrolysing esbls. the origins of most clinically-important carbapenemases are obscure; the exception is oxa-48, which is a chromosomal escape from shewenella spp. it is assumed that other carbapenemase genes similarly escaped from unknown environmental organisms. escape often entails gene mobilisation by insertion sequences, followed by capture by transposons and plasmids, which then facilitate dissemination. bla kpc , regardless of geographic origin, is generally carried by tn4401, often within pkpqil plasmids, whilst bla oxa-48 is often encoded by tn1999-related elements, carried on a small range of plasmids. bla oxa-181 differs only slightly from bla oxa-48 but appears to be a separate escape from shewenella, being associated with an isecp1 element on tn2013, a quite different transposon; moreover bla oxa-181 is epidemiologically linked to india, whereas classical bla oxa-48 links to turkey and the middle east. bla imp and bla vim metallo-carbapenemases occur as cassettes within class i integrons. bla ndm is generally linked to isaba125, carried by a wide diversity of different plasmids and may have arisen as a chimera between apha6, encoding its first six amino acids but otherwise deleted, and a pre-existing metallocarbapenemase gene of unknown origin. the plasmids encoding oxa-48-like enzymes and all the major metallo types (i.e. imp, ndm and vim) spread among strains and species. these enzymes all have a predilection for klebsiella pneumoniae, but frequently occur also in escherichia coli, enterobacter and other enterobacteriaceae species. individual producer strains cause local outbreaks but no single strain has become nationally or globally widespread. rather, when producers became prevalent, the common pattern is one of small clusters within a wider epidemiology of plasmid transfer among strains. by contrast, kpc carbapenemases (carried by tn4401 transposons within pkpqil plasmids) are strongly associated with k. pneumoniae st258. this lineage, along with its variants (e.g. st512), has become internationally widespread and is responsible for the major expansion of 'carbapenemase-producing enterobacteriaceae' in e.g. greece, italy, brazil and israel. only in the last of these countries has it been brought under control, achieved by a determined and centrally mandated infection control effort. not all kpc problems are linked to st258. in manchester, england, the problem is more akin to that with other carbapenemase types, entailing plasmid transfer among strains and species. the plasmids responsible are pkpqil variants with large substitutions resulting in the replacement of the partitioning and replication functions, potentially explaining their spread. the diversity of carbapenemase types and hosts exacerbates the problem of resistance. self-evidently, it harder to devise new pharmaceuticals that inhibit or evade multiple carbapenemase families than to develop those that inhibit or evade single or closely related enzyme types. what is more, experience suggests that it is harder to mobilise infection control staff and efforts against 'plasmid epidemics,' as with most carbapenemases, than against single strains epidemics. carbapenem-resistant enterobacteriaceae (cre) has been increasingly reported worldwide in the past 10 years, which represents a serious threat to public health. invasive cre infections are associated with high mortality, and cre has the potential to spread widely. carbapenem resistance in enterobacteriaceae can mainly result from two different mechanisms. some cre, which possess either ampc or extended-spectrum β-lactamase (esbl) with concomitant porin mutations, can render the organism non-susceptible to carbapenems. more importantly, some cre may result from production of carbapenemases that break down carbapenems. carbapenemases belong to heterogenous group of β-lactamases: molecular class a ( penicillinases), class b (metalloenzymes), and class d (oxacillinases). in 2011, clsi and eucast updated the carbapenem clinical breakpoints for enterobacteriaceae, in order to better predict treatment outcomes and to provide therapeutic alternatives for carbapenem-resistant bacteria. these breakpoints were based on patient response, pharmacokinetic/pharmacodynamic information, and in vitro minimal inhibitory concentration data. with the new lower breakpoints, routine testing for carbapenemase in clinical laboratory became not required for patient care. however, by these updated breakpoints, not all carbapenemase producing cre were non-susceptible to carbapenems. using the updated breakpoints, a study identified that occasional isolates of klebsiella pneumoniae with kpc or vim carbapenemases were susceptible to one or more carbapenem. as of now, few clinical data are available to support that mic is more important than presence of carbapenemase when predicting carbapenem treatment outcome. the different breakpoints for imipenem and meropenem in clsi and eucast may need evaluation for optimal patient care as well as epidemiologic study. carbapenemase detection and characterization are recommended for public health and infection control. currently, there are a variety of the phenotypic assays such as modified hodge test, the carba np test, modified carbapenemase inactivation method, and molecular assays. none of the currently described phenotypic test can detect all carbapenemases. molecular tests are often used as the golden standard, but they have inherent limitations such as missing novel variants and/or previously undescribed enzymes. cre continue to present challenges related to both treatment decisions by physicians and detection methods applied at laboratories. continued efforts to improve detection methods, by making them rapid, sensitive, and unbiased for specific carbapenemase, will hopefully allow for better patient care and prevention of further cre dissemination. ami neuberger, m.d. infections caused by carbapenem-resistant enterobacteriacea (cre) are more difficult to treat both because our current arsenal of antibiotics is limited and because some of the available antibiotics are less efficacious or have not been rigorously studied. the presentation will provide a brief overview of the future of treatment of cre infections. there are a number of controversies regarding the modes of administration of antibotics for cre infections: high versus lower dose, continuous or prolonged versus intermittent administration, duration of antibiotic treatment, and mono versus dual therapy. current evidence supports prolonged or continuous administration of β-lactam antibiotics. new randomized controlled trials of mono versus dual antimicrobial therapy, and short versus long duration of treatment for gram-negative bacteremia are ongoing, with results expected to be available in 2018. older antibiotics, such as colistin, aminoglycosides, fosfomycin, and tigecycline are increasingly used together with newly approved drugs such as ceftazidime-avibactam. some new antibiotics in advanced stages of development ( phase 2 or 3 trials) will be reviewed. these drugs will include combinations of carbapenems or aztreonam with β-lactamses inhibitors, new combinations of cephalosporins with β-lactamses inhibitors, cefiderecola novel cephalosporin, plazomicinan aminoglycoside, and eravacyclinea new fluorocycline. the advantages and disadvantages of these drugs will be discussed. novel approaches to antibiotic development include the use of antibacterials produced from previously "non-culturable" bacteria, and development of new entry mechanisms of antibiotics into gram-negative bacilli. treatment of cre infections is likely to undergo rapid changes in the upcoming years, but any such change will have to be accompanied by comprehensive infection-control programs, antibiotic stewardship programs in hospitals and in the community, and judicious use of new antibiotics. one health approaches, 'one hdealth, one medicine', have been globally recognized to control zoonotic diseases. world organization of animal health (oie) has reported 60% of human pathogens are animal origin and more than 75% of emerging animal diseases are zoonoses. this means collaboration and cooperation between animal and human medicine together can only solve the problem. recent huge outbreaks of highly pathogenic avian influenza (hpai) and middle east respiratory syndrome (mers) in korea have been more pay attention to implement one health approaches in practice. we experienced several hpai epidemics past ten years and the mers in 2015 and had to bear huge damages. one health becomes a key approach to control zoonotic diseases systemically and effectively. a 'one health' approach to minimize the antimicrobial resistance in humans and animals need collaboration among the responsibility of all three parts; human health, animal health and environmental health-communities. surveillance of antimicrobial usage and resistance provides important data for the identification of resistance problems and contributing factors for the development and spread of resistance at a national and local level. through the painful korean experience of these zoonotic diseases and global challenge to amr brings us to establish the effective preventive method and early diagnosis as critical control strategies. prevention and control of infections is essential in fighting antimicrobial resistance. thus, to minimize infections in animal and human and to decrease the volume of antimicrobials used, collaborative efforts should be implemented to improve animal and human health. one health activities on nipah in bangladesh: a high risk country for zoonotic disease spillover to man epidemiological investigations in bangladesh implicated consumption of date palm sap as the major route of transmission of the virus from bats to humans. in bangladesh date palm sap is collected from date palm trees for consumption either as fresh or as fermented beverage. the sap is collected in clay pots attached to the top portion of the tree, where the tree has been denuded of bar, so that the sap can ooze overnight into the collection pots. outbreaks of disease typically occur in the winter months, the interval in which date palm sap is collected. as well, case-control studies have identified consumption of date palm sap beverage as a risk factor at the individual level. videos using infrared cameras have documented that fruit bats visit date palm trees at night and contaminate the collection pots by licking the oozing sap and by urinating in the collection pots. ethnographic studies of affected populations has enabled development of a behavior change intervention to reduce consumption of date palm sap. in addition, bamboo skirts placed around the denuded bark area of the date palm trees and collection pots have been developed to obstruct contamination of collected date palm sap by bat saliva and urine. of great concern, epidemiological studies in bangladesh have also identified person-to-person transmission of the virus between patients and persons who are in close contact with patient secretions, often in hospital settings. a study of nipah outbreaks between 2001 and 2007 attributed 51% of all cases to person-to-person transmission, though only 7% of patients were assessed as having transmitted their infection onward via this route. importantly, handwashing seemed to be effective in reducing person-to-person transmission. in aggregate, these studies illustrate the power of multidisciplinary collaborations under the rubric of one health, and, in view of the documented person-to-person transmission in bangladesh and the potential for emergence of new genetic variants of nipah that are capable of sustained human-to-human transmission, underscore the need for continued surveillance and control efforts, including development of effective vaccines. mycoplasma pneumoniae (mp) is one of the most common causes of community-acquired pneumonia in children and young adults. emerging resistance to macrolides among mp is of great concern since a macrolide-resistant mp strain was first reported in 1997, most notably in japan, china, and korea. although macrolides are recommended for the first-line treatment for mp pneumonia, the efficacy of macrolides in the treatment of m. pneumoniae infection remains unclear. in addition, with the increase in macrolide resistance, concerns about the efficacy of macrolides for the treatment of mp pneumonia in children have been raised. given the versatile features of mp pneumonia, which are determined by the patient's age, the immunologic response of the host, and extrapulmonary manifestations, a more comprehensive approach must be established to analyze the clinical outcome of mp pneumonia according to the presence of macrolide resistance. initially, treatment of mp pneumonia with antimicrobials was supported by a randomized trial of 290 marine recruits that showed a shortening of fever duration, alleviation of cough, and improvement of chest x-rays. a recent systematic review that evaluated the effect of treating mp pneumonia demonstrated that there was no significant clinical benefit of antimicrobial therapy in children with mp pneumonia. although much is not known about clinical impact of macrolide resistance on the severity of mp pneumonia, macrolide resistance alone does not seem to explain the severity of mp pneumonia. some studies have reported that patients infected with macrolide-resistant strains had more febrile days and a longer duration of persistent cough than those infected with macrolide-susceptible strains, suggesting poorer response to macrolide treatment in macrolide-resistant strains. the results highlight the need for well-designed prospective studies to assess a therapeutic benefit from macrolides and an adjunctive therapeutic strategy in the treatment of children with macrolide-resistant mp pneumonia. mayo clinic, usa mycoplasma pneumoniae (mp) can cause upper and lower respiratory tract infections in children and adolescents, with communityacquired pneumonia (cap) comprising the major burden of the disease. mp infections are generally mild and self-limiting. some patients, of any age, may develop severe and fulminant infection with pulmonary complications and/or extrapulmonary manifestations that may affect almost every organ. this presentation will focus on pulmonary infections due to mp. it is estimated that 3-10% of children with mp respiratory infection develop cap and less than 5% are severe enough to require hospitalization, although this may change with increasing prevalence of mrmp. children with mp cap present with a longer duration of fever compared with children with cap due to other organisms. complicating the diagnosis of mp has been the insensitivity of testing as the organism does not grow well in culture, the inability to perform reliable and sequential serologic testing or pcr testing; the co-existence of mp with other pathogens; and asymptomatic carriage of mp in 21-56% of patients. pathogenic effects on the respiratory tract may be direct by active infection, indirect by infection-induced immune mechanisms, or both. the immunopathology is poorly understood, in particular the role of cell-mediated immunity (cmi). studies have demonstrated increased concentrations in il-8 and il-18 in acute phase serum and pleural fluid samples and inf gamma, il-6 and ip-10 in patients with mrmp and that severity of cap correlated positively with the size of cutaneous induration following intradermal injection of mp antigens. it's been postulated that cytoadherence of mp to the respiratory epithelium initiates an immune response with progression to an excessive inflammatory response and a vigorous cmi response leading to pulmonary injury and severe clinical illness. macrolides have been and remain the drugs of choice for treatment of mp infections in children. alternative drugs such as tetracyclines and fluoroquinolones are not first line due to age-related adverse effects. macrolide resistant m. pneumoniae (mrmp) have been reported as early as the 1970s in japan with rates now greater than 90% in areas of japan and china. korea reported mrmp in 2010. mrmp in north america was reported in 2008 with a prevalence now of approximately 13%. europe has reported rates of 2-26% and israel as high as 30%. many countries have unreported rates likely due to the lack of susceptibility testing. the mechanism of resistance is genetic with point mutations in a few positions of the domain v of the peptides transferase loop of the 23s rrna where macrolides bind the 50s rrna subunit. mp pneumonia was more prevalent throughout korea in 2011 and caused more severe clinical features than at any other time, which led to an unpublished observation that it may be associated with mrmp. despite the increasing prevalence of resistance, macrolides remain the drugs of choice for treatment of mp infections in children, but require antimicrobial stewardship. with increasing clinical severity of mp pneumonia and growing evidence for the role of an overreactive host defense, the role of corticosteroids has been investigated. in a study of 12 healthy patients followed retrospectively all had either severe pneumonia (high fever, respiratory distress or initial lobar pneumonic consolidation with or without pleural effusion) or refractory pneumonia ( prolonged fever of >7 days or persistent consolidation of more than one lobe of the lung despite appropriate antimicrobial therapy), were diagnosed serologically, had no viral co-infection, received appropriate antimicrobials (macrolide or beta-lactam) and methylprednisolone at 30 mg/kg/day × 3 days. all improved. although mrmp may cause severe refractory mp, susceptibility testing was not performed in the above study. most often, mp causes a benign respiratory illness that may involve both upper and lower respiratory tracts. but mp has the potential to cause severe disease. the emergence of mrmp has led to severe mp pneumonia. but macrolide resistance is only one potential cause of severe disease. an exuberant host inflammatory response with release of cytokines and, perhaps mediated largely by cmi, appears to play a role in some children and adolescents. in the latter situation, corticosteroids may prove beneficial but more research needs to be done. over the last three decades, many studies have investigated the best approaches to promote hand hygiene practices and improve hand hygiene indicators, in particular healthcare workers' (hcws) compliance. early studies on hand hygiene improvement in healthcare were focused on single interventions promoting the importance of handwashing and introducing the use of antimicrobial soaps. in 1994, our group in geneva conducted the first large-scale epidemiological research on hand hygiene, identifying major risk factors for noncompliance and demonstrating the critical role of alcohol-based handrubs (abhrs) as major system change to replace handwashing with soap and water, while included in a multimodal strategy to change hcw behavior. the strategy combines easy access to abhr ( proven to bypass the time constraint on hcws), hcw education, performance monitoring and feedback, reminders in the workplace and institutional safety climate. the strategy (referenced as the "geneva model of hand hygiene promotion) was proven successful, and hand hygiene improvement was associated with significant reduction in healthcare-associated infections (hais) and spread of multi-resistant organisms. between 2000 and 2006, the "geneva model of hand hygiene promotion" was replicated in single, as well as multiple healthcare institutions, at local, regional and national level, with success. since 2005, the world health organization (who) mandated our group to lead the first global patient safety challenge (clean care is safer care) with the main objectives to raise awareness about hai worldwide, mobilize nations toward ipc activities and promote best ipc practices, in particular hand hygiene. the recent meta-analysis by luangasanatip et al. demonstrates the critical role of the who multimodal approach in successful hand hygiene promotion. nevertheless, several knowledge gaps in hand hygiene monitoring and efficacy remain. in my lecture i will focus on the following topic areas related to key questions in the hand hygiene research agenda: • studies on direct and indirect monitoring of hand hygiene compliance, including new devices for observation and feedback; • studies of the influence of i) handrubbing duration; ii) volume of abhr used, and iii) the optimal sequence of the handrubbing steps within the "how to handrub" 6-step technique, in the reduction of bacterial counts on hcws hands; • studies on the burden of disease and implementation of infection prevention and control strategies worldwide; • studies of the possible role of patient participation and empowerment in hand hygiene promotion. hepatitis c virus (hcv) infection is one of the most common chronic liver disease. globally, it was estimated that in 2005, more than 185 million people had hcv antibodies ( prevalence of 2.8%). most patients infected with hcv acquired the disease through intravenous drug use or blood transfusion, the latter of which has become rare since routine testing of the blood supply for hcv began in 1990. nosocomial transmission of hcv has been documented in several health care settings. where there are stringent infection control protocols to prevent transmission, particularly through unsafe medication injection practices, nosocomial transmission has still been reported, generally because of breaches in protocol. rare sources of transmission of hcv include contaminated equipment used during the performance of procedures and other breakdowns of infection control procedures or aseptic techniques leading to person-to-person transmission. screening for hepatitis c virus (hcv) infection is an important component of successful control of hcv for the infected individual and for public health purposes. diagnostic tests for hepatitis c virus (hcv) are serologic assays that detect antibodies to hepatitis c, and molecular assays that detect or quantify hcv rna, other investigations such as genotype testing, serum fibrosis panels and liver biopsy may help to predict the response to treatment and prognosis. most cases of acute hcv infection are anicteric and asymptomatic, with fewer than 25% being clinically apparent. fulminant hepatitis c is rare. of those who go on to have chronic infection, a substantial proportion will develop cirrhosis, and a subset of those develop hepatocellular carcinoma. active surveillance based on mdr gram-negative pathogens? anucha apisarnthanarak md active surveillance is one of the common strategies employed to control mdr-gram negative pathogens. although listed in the guideline, the true value of active surveillance for mdr gram-negative pathogens has never been demonstrated. several considerations should be made when infection preventionists implement active surveillance as part of control measures. these include the specific reservoir of each specific mdr gram-negative pathogens, resource availability for isolation precaution, the turn-around time of active surveillance culture, whether institution is able to implement other infection prevention strategies together with active surveillance culture. in this session, i will address the pro-and con-for active surveillance culture for mdr gram-negative pathogens. application of active surveillance culture in resource-limited settings will also be discussed. also, rates of catheter associated urinary tract infection (cauti) and c-line associated blood stream infection (clbsi) significantly decreased from 1.85 to 0.88 ( per 1000 catheter-days, f = 10.14, p < 0.0001) and from 3.40 to 2.20 ( per 1,000 catheter-days, f = 14.17, p < 0.0001). in subgroup analysis, rates of vap, cauti and clbsi were significantly decreased regardless of organizational and institutional characteristics of icus. in summary, all of the da-hais have shown a significant reduction in the last 10 years, however v-ur has year-wise significantly increased trend for past 10-years, also uc-ur and cl-ur have not decreased trend significantly. we need effort to make reduction of device utilization ratios and associated infections. invasive fungal infections (ifi) are primary causes of mortality, and rapid and accurate diagnostic laboratory tests are required to improve patient outcomes. although histopathologic examinations of tissues can detect ifis, tissue morphology alone is insufficient to distinguish between aspergillus and other fungi, including fusarium, scedosporium, or mucorales. therefore, species identification using culture and non-culture methods is necessary. recently, assays targeting ribosomal dna have been used to identify infectious fungi in tissues. notably, analyses using frozen tissues were superior to those using formalin-fixed paraffin-embedded tissues. because pan-fungal primers hybridized dna from other eukaryotes, the pan-fungal approach was limited in tissues that primarily contained human dna and little fungal dna. diagnostic tests that rely on fungal cultures are common for ifis. recently, however, dna sequencing and matrix-assisted laser desorption/ ionization time-of-fight mass spectrometry (maldi-tof ms) have been used to rapidly and accurately identify fungal pathogens recovered from cultures. sequence-based identifications have been particularly useful for identifying cryptic species that were misidentified by microscopic analyses or were identified only to the complex level. some cryptic species are resistant to azole antifungal agents, and molecular methods have been developed to detect azole-and echinocandin-resistant candida species as well as azole-resistant aspergillus species. blood cultures are the gold standard for diagnosing candidemia; however, cultures require 1-3 days of growth followed by an additional 1-2 days for identification and antifungal susceptibility testing. such timeframes lead to delays in treatment initiation. additionally, up to onethird of patients with invasive candidiasis fail to test positive in blood cultures. pcr and the 1,3-β-d-glucan (bdg) assay are more sensitive than blood cultures for patients with deep-seated candidiasis. bdg is a cell wall component in candida and other fungal species, except mucorales and cryptococcus, and is included in the eortic/msg revised diagnostic criteria for invasive fungal diseases. however, bdg assays are expensive and labor-intensive, making them unavailable in most developing countries. the bdg assay is highly sensitive but lacks specificity; thus, it is typically suitable only for ruling out the causes of candidiasis. serum galactomannan (gm) is a cell wall component of aspergillus, and can be detected in serum using a commercial test (platelia™ aspergillus eia; biorad, usa). the serum gm test is considered aspergillus-specific, and multiple studies demonstrated high sensitivities (∼70%) in sera from patients with hematological malignancies or allogeneic hematopoietic stem cell transplantations. however, gm sensitivity is low in non-neutropenic patients and recipients of solid organ transplants. additionally, the gm test is associated with poor predictive values in patients receiving mold-active antifungal prophylaxis. however, the detection of gm in bal fluid is high (>70%) even among patients receiving mold-active antifungal therapies. multiple studies have shown that pcr was more sensitive than culture methods at detecting aspergillus in blood and respiratory fluids. multiple pcr assays are commercially available for the detection of aspergillus spp.; however, none is fda-approved. moreover, aspergillusspecific pcr is not recommended for clinical use as only few assays are standardized and validated. the t2candida assay (t2 biosystems, usa) was recently introduced for the detection of five common candida species from whole-blood samples. the t2candida assay is the only fda-approved diagnostic test that offers rapid diagnosis (3-5 hours) and specific organism identification with detection limits of 1 cfu/ml. the development of additional diagnostic tests will facilitate the early diagnosis and management of ifis in high-risk patients. the newest treatment strategies for candidemia thomas f. patterson professor of medicine, ut health san antonio, san antonio, texas usa candidemia is an important cause of morbidity and mortality especially in hospitalized and immunocompromised patients. despite advances in antifungal therapy, the number antifungal drugs and drug classes for treating these serious infectious remains limited. management is further complicated by the fact that it remains difficult to establish an accurate diagnosis which is compounded by the need for early therapy. fluconazole remains a useful antifungal agent especially for follow-on therapy after stabilization of infection but resistance is high for some species including c. glabrata. thus, the echinocandins have become recommended as primary therapy in most patients. however, development of echinocandin drug resistant strains has been reported worldwide. the development of drug resistance has often been tied to antifungal drug use, particularly for some species like candida glabrata. while multidrug resistance is uncommon, increasing reports of multidrug resistance to the azoles, echinocandins and polyenes have occurred in several candida species, most notably candida glabrata and more recently candida auris. new agents are under development for candidemia, including echinocandins improved pharmacokinetic profiles and those available for oral use. in addition, other agents with new targets of action are also undergoing preclinical development. diagnosis of infection and detection of antifungal resistance is critical to the successful management of patients with these infections. new therapies are aimed at improving outcomes in candidemia. epidemiology and management of mucormycosis and invasive aspergillosis: are there lessons to be learned? centre for infectious diseases and microbiology laboratory services, icpmr, westmead hospital, university of sydney, new south wales, australia the epidemiology of aspergillus and mucorales infections may be changing. although invasive aspergillosis (ia) remains a substantive cause of morbidity in patients with neutropenia, hematologic malignancy and organ transplants, there is an expansion in the spectrum of at risk patients. patients in the intensive care unit (icu), with chronic lung disease, hiv/aids and those on immunomodulating drugs are amongst relatively understudied populations. diagnostic difficulties including the interpretation of aspergillus cultures contribute to this limitation. other than the expansion in host risk groups, is the shift in etiology of ia, with cryptic or uncommon species emergent. in addition, azoleresistant a. fumigatus infections pose an increasing dilemma in regions of europe and asia (<5-30% resistance rates). in general, voriconazole is recommended for the primary treatment of ia regardless of site of infection. other azoles, the echinocandins or amphotericin b compounds may be used as second line or salvage therapy. primary combination therapy is not routinely advised. surgical derbridement or resection is an important adjunct where feasible. in the presence of azole resistance, either liposomal amphotericin b (l-amb) or a voriconazole-echinocandin combination is preferred. in vitro susceptibility testing is recommended for all ia cases. isavuconazole has good activity against aspergillus and new antifungal drugs with anti-aspergillus activity are in development. many cases of mucormycosis affect hosts with malignancy and stem cell transplantation, but organ transplantation, diabetes mellitus and iron overload are also important underlying conditions although the use of certain calcineurin inhibitors may be associated with lower risk. emerging risks include underlying rheumatological/autoimmune conditions. trauma-related cases may also be increasing and outbreaks of infection following natural disasters and iatrogenic exposure described. pathogen epidemiology varies with region with emergence of uncommon genera such as apophysomyces. despite best practice management and reversal of risks, mortality is up to 80%. treatment is based on case series and expert opinion. surgical debridement/resection combined with antifungals improves outcomes. initial treatment with l-amb is preferred agent although optimal dosage is uncertain. isavuconazole appears to be as efficacious as l-amb. combination polyene-echinocandin can be considered for extensive disease or salvage therapy, with posaconazole typically employed as step-down therapy. treatment algorithms may change with wider availability of iv posaconazole; other new drugs are in the pipeline. endemic fungal infection in the asia-pacific region (in the context of altered immunity) national university health system, singapore less is known about the incidence and characterization of deep mycoses in the asia pacific region. geoclimatic conditions, population demographics and health resource accessibility vary within the respective asian countries and also differ from west. invasive fungal diseases (ifd) encountered in the region will be discussed with specific highlights on the unique challenges faced pertaining to host immune susceptibility and in the management of these diseases. candidemia and candidiasis constitute the highest proportion of the ifds. in particular, the contribution by candida tropicalis in tropical asia pacific is not to be overlooked. candida tropicalis infection, linked to more severe disease, is seen in patients with weakened immunity such as hematological malignancies and in neonates. the host immune factors recently identified as predisposing to oro-esophageal candidiasis will also be discussed. chronic (cavitatory) pulmonary aspergillosis (cpa) in asia is often a sequelae to old tuberculosis as well as aspergillosis in the critically ill is under-recognized. the immune interplay between the ubiquitous mold and the host defense during severe illness will be explored. cryptococcosis and penicilliosis have traditionally been seen in patients infected with the human immunodeficiency virus (hiv). while cryptococcus infections have been recognized to occur beyond the context of hiv and even in apparently immunocompetent subjects, it is only recently that the immune susceptibility of such patients to cryptococcus are being dissected. similarly the notable predilection of talaormyces marneffei (and other rare fungi) in the asia pacific region is being linked to other non-hiv-attributed host immune defects including a novel disease trait seemingly distinct to asians. pre-exposure prophylaxis: from science to implementation rossana a. ditangco md prep for hiv refers to the preventive strategy of taking a medical agent prior to hiv exposure. this method specifically involves the oral intake of an antiretroviral drug (arv) by hiv-negative individuals who are at high risk of acquiring the virus in order to prevent hiv infection. recent studies have demonstrated safety and efficacy of dual antiretroviral (arv) oral pre-exposure prophylaxis (prep) in preventing sexual and parenteral transmission of hiv infection. the iprex study, a randomized controlled efficacy trial in men who have sex with men (msm) and a small contingent of male-to-female transgender women (tgw) is particularly relevant to asia and the pacific. in this study, emticitrabine/tenofovir disoproxil fumarate (ftc/tdf or truvada ® ) safely achieved a 44% per-protocol reduction in new hiv infections. however, risk reduction was 92% in ftc/tdf recipients having detectable study-drug blood levels, indicative of much greater efficacy associated with increased adherence. subsequent pharmacologic modeling and follow-up studies demonstrated daily use of these agents not being required for achieving optimal protection, declining on a gradual scale associated with decreased frequency of use . this research showed a reduction of hiv infection risk of 99% for 7 doses, 96% for 4 doses and 76% for 2 doses of ftc/tdf per week. the protective efficacy of non-daily dosing (89% risk reduction) was subsequently confirmed in a placebo-controlled trial of intermittent (on-demand) ftc/tdf prep among msm in france and canada. mathematical modeling using iprex findings demonstrated substantial reductions in new hiv infection associated with modest prep program coverage in msm, while increased prep adherence was shown to have the largest population level preventive impact with greatest cost-effectiveness . prep implementation has been shown feasible in open-label extensions and project sites with research and program capacity. despite its excellent safety and efficacy profile, the proof of implementation of prep in resource-limited settings outside of these situations still needs to be delivered. applications of prep in resource limited situations must be grounded in the reality of existing health systems and the interface between community-level primary care clinic-and hospital-based services. a number of unknowns remain with respect to the delivery of hiv prep for msm/tgw in these environments. among others, access, uptake and adherence, effectiveness and behavioral and social impact effects are not described. taking part in a prep project or using arv drugs may disclose same-sex behavior and hiv risk. these in turn may provoke negative reactions or undue pressures in the social, professional and family environment. participants may see certain services or privileges being revoked or denied. in a worst case scenario, disclosure may lead to denial or termination of health or life-insurance, rental agreements, employment or promotion. these negative repercussions constitute violations of individual rights and may adversely impact project participation and prep access, uptake and adherence. of particular concern are poor adherence and increases in sexual risk behavior, the latter potentially accelerating sexually transmitted infections (sti) followed by increased hiv acquisition and transmission in the non-prep serviced portion of at risk population. at present countries are at varying stages of introducing daily oral prep using tdf/ftc, which is now recommended by who as an option for people at substantial risk of hiv. some countries have national programs; others have smaller-scale programs; others aren't offering prep at all. the benefit of prep as a prevention strategy goes beyond its clinical efficacy. prep programs could increase hiv testing uptake and contact with health care provider for hiv counseling and education services and early diagnosis and treatment of sexually transmitted infection. scientific evidence of efficacy and safety of prep, mathematical models for cost effectiveness and potential elimination of new hiv infection and projected benefit beyond clinical effectiveness provide rationale for making prep available as additional layer of hiv prevention strategy. center for aids research, kumamoto university, kumamoto, japan despite the significant reduction in morbidity and mortality following combination antiretroviral therapy (art) there is emerging evidence that people with successfully treated hiv infection by art age prematurely, leading to progressive multi-organ disease referred as comorbidities. one of the major factors involved in this pathogenic process is residual viral replication by persistently infected cells surviving in vivo and subsequent chronic inflammation. in contrast to the current art that only targets viral replication neutralizing or nonneutralizing antibodies against the envelope proteins of hiv-1 have been reported to have an adcc (antibody-mediated cellular cytotoxicity) activity that eliminate hiv-1 infected cells in vitro. the discovery of potent and broadly neutralizing antibodies (bnabs) against hiv has made passive immunization a potential strategy for the prevention and treatment of hiv infection. the bnabs 3bnc117 and vrc01 targeting the hiv cd4-binding site has been tested in treatment naïve patients and on treatment patients for the delay in plasma viral rebound after the discontinuation of art. monoclonal antibody 10-1074 targets the v3 glycan supersite was used at the highest dose of 30 mg/kg and showed a rapid decline in viremia. however, virologic analyses revealed the emergence of multiple independent neutralization resistant mutants in every case. we conducted a phase 1b clinical trial of passive transfer of neutralizing monoclonal antibody kd-247, which is reactive against the tip of the v3 region. eligible subjects were randomized to receive one of the 3 doses of kd-247 and the treatment was found safe and well tolerated. we observed significant decrease in hiv-rna in the 16 mg/kg cohort of kd-247 with two in six cases who achieved >1 log reduction of hiv-rna. long-term suppression of viral load was observed for one patient despite significant decrease in plasma concentration of kd-247, suggesting effects of antibody other than neutralization. we previously reported that the neutralization escape mutants to kd-247 became sensitive to chemokine cc receptor (ccr)5 inhibitors such as maraviroc or cenicriviroc. conversely, resistance mutants to ccr5 inhibitors became sensitive to several neutralizing antibodies including kd-247. furthermore, a series of in vitro experiments suggested synergistic effects of the combination of kd-247 and ccr5 antagonists including maraviroc (figure) . these results taken together, suggest that the combination of neutralizing antibodies with ccr5-inhibitors would be a promising candidate of intensification therapy added onto the current suppressive art aiming toward functional cure of the disease. programmable nucleasesincluding zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens) and rna-guided engineered nucleases (rgens) derived from the bacterial clustered regularly interspaced short palindromic repeat (crispr)-cas (crisprassociated) systemenable targeted genetic modifications in cultured cells, as well as in whole animals and plants. the value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific dna cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. however, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. knowledge of nucleasespecific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications. group cpf1 is a recently reported effector endonuclease protein of the class 2 crispr-cas system. cpf1 has several differences from cas9: cleavage with 5' overhangs, shorter guide rna, and a longer distance between the seed sequence and cleavage site, which could provide potential advantages for some cases of genome editing such as nonhomologous end joining-based gene insertion and efficient genome editing using homology-directed repair. however, limited information is available about cpf1 activity profiles in mammalian cells, precluding its wide use for genome editing. furthermore, both selection of highly efficient guide rnas and collection of big data for determination of parameters of rna-programmable nucleases are currently laborious and costly due to lack of a reliable high-throughput approach to determine rnaprogrammable nuclease activity in mammalian cells. here, we performed en masse evaluation of guide rna and cpf1 activity using synthetic target sequences and deep sequencing. using this in vivo high-throughput approach, we determined on-and off-target activity profiles and protospacer adjacent motif (pam) sequences of cpf1. we found that sequence features of high activity ascpf1 guide rnas are distinct from those of spcas9 and that the pam of as and lbcpf1 in mammalian cells is tttv, rather than tttn, which was previously determined using an in vitro system. evaluation of off-target activity showed that cpf1 target sequences can be divided into three regions: a 6 base pair (bp) seed, a 13 bp trunk, and a 6 bp promiscuous region. these results should serve as a useful guide to select cpf1 as a genome editing tool. more importantly, our in vivo high-throughput evaluation system will greatly facilitate both the selection of efficient and precise guide rnas and the generation of big data for the development of advanced prediction programs for on-and off-target activities. tasp works if an infected person knows his/her hiv status. a diagnosis should have been made as early as possible, and treatment offered promptly, so that the time gap between infection and viral suppression can be narrowed. around the world, late diagnosis is still common. early diagnosis often hinges not just on easy access to voluntary testing, freedom from stigma, but also the availability of screening. selftesting and the submission of specimens (urine, saliva, dried blood spots) for laboratory testing are some solutions, albeit the existence of technical problems, and the need for establishing linkage to care. periodic testing campaigns could impact hiv epidemiology, as illustrated in our modelling study parametrized by hong kong's surveillance data. for patients enrolled in treatment programs, optimization of regimen is crucial to ensure lifelong maintenance of haart without adverse reactions. in the developed world, integrase inhibitor is now used in first-line regimens, which has the capacity of reducing viral load to undetectable range within weeks. their access and that of single tablet regimens, are however limited in places where most hiv patients are. for some antiretrovirals, for example abacavir and efavirenz, knowledge of the host genotype (hla-b5701 and cyp2b6-516gt respectively) could minimize the occurrence of adverse reactions which might otherwise compromise adherence. notably adherence is the key to lifelong maintenance of virus suppression, and there is no simple solution to guarantee adherence over years, or tens of years. whereas all efforts could be made to promote testing and improve treatment coverage, the underdiagnosed interval is often where the achilles heel is. except for a small number of cases presenting as acute infection, the seroconversion time is often unknown. using modified back calculation methods to estimate the seroconversion year of over 3,000 patients in hong kong, we found that the interquartile range of their underdiagnosed intervals was 1-4 years with a range between 0 and 10 years. the lengths of undiagnosed intervals were associated with age and the routes of hiv transmission. obviously people with low perceived risk of hiv infection had longer undiagnosed interval and were more likely to be late for diagnosis. from mathematical modelling, increase of undiagnosed intervals would offset infections averted by tasp even if good coverage and high adherence were assumed. apparently, tasp is a necessary yet insufficient intervention to achieve hiv elimination. tasp targets people infected with hiv, whereas interventions for non-infected individuals would contribute to the reduction of the size of populations requiring tasp. one good example of the latter is pre-exposure prophylaxis, but it's only accessible to handfuls of people at risk around the world. effective protocols are needed, and risk compensation is a new concern. let's not forget promotion of protected sex, provision of methadone maintenance (and other harm reduction measures) for injection drug users, and community-level behavioral interventions. they continue to be powerful components of the armamentarium of measures to combat hiv, complementing tasp to achieve hiv elimination. stephan harbarth health care-associated infection (hcai) is a major global issue in patient safety. it affects hundreds of millions of people worldwide, complicates the delivery of patient care, contributes to patient deaths and disability, promotes resistance to antibiotics, and generates additional expenditure to that already incurred by the patients' underlying disease. indeed, hcai is a growing international problem. patients are becoming more susceptible to infections because of more serious underlying illnesses. poor compliance with hand hygiene by health care staff, increased recourse to invasive medical devices, and care of the critically ill as well as lack of access to safe water and unclean instruments and environmental surfaces all play a role. the environment of patient care is also important. factors such as understaffing, high bed occupancy, and increased patient transfers all create new risks of infection. the first global patient safety challenge created a worldwide focus on reducing hcai as a vital element of the safety of patient care. the last two years provided important and clinically relevant research data for prevention of hcai in different patient populations. my presentation will summarise the results of clinical trials and systematic reviews for the reduction of various types of hcai, and will discuss them in the context of the current relevant scientific and clinical background. in particular, i will discuss recent data on the epidemiology and prevention of nosocomial infections in intensive care units, present new approaches to prevention of surgical site infections as well as catheter-related bloodstream infections, describe recent advances in hand hygiene research and attempt to briefly summarise specific challenges related to the management of infections caused by multidrugresistant microorganisms. overall, hcais remain one of the key challenges of hospital care and significantly contributes to morbidity and mortality. papers published in the last 2 years remind us that further reductions of hcai rates are possibleoften with the help of simple and rather inexpensive interventions. rapid and accurate diagnosis is critical for the effective treatment of life threatening infections, such as bloodstream, respiratory tract and complicated urinary tract infections (utis). these clinical syndromes are difficult to diagnose due to complex aetiology and challenging clinical sample types (e.g. blood, sputum). current culture based diagnosis often has sub-optimal specificity and sensitivity and is too slow to impact on patient management. shotgun metagenomics sequencing has the potential to change the way we diagnose infection, combining rapidity with comprehensiveness beyond that of current methods. real-time nanopore sequencing technology provides the rapid turnaround necessary for infectious diseases diagnostics applications at point-of-care. there are challenges in applying sequencing to infection diagnosis, however, including high human:pathogen nucleic acid ratios, low pathogen numbers and low quality nucleic acid, depending on the disease and the clinical sample type. it is, therefore, vital to carefully design, develop, and optimise diagnostics pipelines before attempting to apply them to clinical samples. i will describe how we develop our minion based infectious diseases diagnostics pipelines with examples from our ongoing research on pneumonia, sepsis and utis. protein synthesis enzymes as primary defense system against infection aminoacyl-trna synthetases (arss) are essential protein synthesis enzymes, making a covalent linkage of their cognate amino acids to trnas. since the catalytic activities of arss are always required for the viability of organisms, they are constitutively expressed and ubiquitously present. due to these features, arss are the first to be exposed to broad spectrum of stresses and challenges. in fact, they have shown diverse roles as rapid responding signal mediators beyond protein synthesis. here we show our recent findings on arss as primary defense system against bacterial and viral infections. for instance, tryptophanyl-trna synthetase (wrs), is rapidly secreted out from monocytes upon bacterial infection and primes innate immune responses. the secretion was far more rapid than the induction of innate immune system. in another case, glutamyl-prolyl-trna synthetase (eprs) plays a unique role in viral clearance. eprs blocks pcbp2mediated ubiquitination of mavs (mitochondrial antiviral signaling protein), leading to the inhibition of viral replication. based on these findings, other arss are expected to play unique roles against infection, thereby collectively serve as a primary shielding system. victor lim international medical university, kuala lumpur, malaysia the world is facing a crisis in antimicrobial resistance (amr). it has been projected that if nothing is done 10 million people will die from antimicrobial-resistant infections annually by 2050 and the economic cost will also be correspondingly high at one hundred trillion us dollars. the overuse of antimicrobial agents is a major driver for the emergence of resistance. improving antibiotic stewardship is therefore crucial in meeting the challenges posed by amr. to do so would require recognition of the problem by all stakeholders at all levels and the political will to solve. adequate planning and provision of resources (including legislation) are essential. most importantly however, it would require a behaviour change among all stakeholders. there is increasing recognition that providing evidence from health research while necessary is insufficient for the delivery of optimal health care. there is a need to translate knowledge into action. the field of knowledge translation (kt) is the scientific study of methods for closing the knowledge-to-practice gap, and of the barriers and facilitators inherent in this process and such methods must be employed if we are to enjoy any success in antibiotic stewardship. the esrc working group on amr report 2014 stated that although amr involves biological processes, the context which determines the operation of these biological mechanisms is shaped by social, cultural, political, and economic processes. 1 behavioural science is a multidisciplinary study of human (and animal) behavior and encompasses the disciplines of psychology, anthropology, sociology and economics. the contribution of behavioural science in improving antibiotic stewardship is crucial but has only been recognized lately. 2 antibiotic prescribing is a complex behaviour, carried out by an informed individual often making a subjectively rational choice. a recent report by the department of health and public health england summarised the evidence on behavioural change and antibiotic prescribing in healthcare settings. it pointed out the lack of underpinning psychological theory and behavioural science in most of the studies which have been done so far. 3 as a result interventions may not be effective in changing behavior. to change behavior we need to understand the drivers of behavior. drivers of prescriber behavior may be intrinsic or extrinsic. intrinsic factors include an altruistic motivation to do one's best for the patient, ignorance, duration of practice, the tendency to yield to patient demands, fear (both of progression of disease and losing the patient), and a lack of understanding of the resistance problem. extrinsic factors would include patient demands, the influence of the pharmaceutical industry, diagnostic uncertainty, patient comorbidities and social class, workload and time pressures, role modelling by senior colleagues and financial incentives. interventions to improve antibiotic stewardship had included prescriber education and training, the issuance of antibiotic guidelines, the use of electronic decision support systems, audit and feedback, rapid and near patient testing to reduce diagnostic uncertainty, restriction strategies (pharmacy and laboratory) as well as financial strategies. to increase awareness of the problem of amr among the public social marketing strategies have been employed including the use of the mass media. although a variety of interventions have been employed over the last 5 decades; their overall effectiveness is subject to question. results have been variable. what works in one setting may not work in another as behavior change is a complex process and influenced by social, economic, ethnic and cultural beliefs. many studies are cross-sectional in nature and the sustainability of the intervention is not measured. to design any strategy or intervention there is a need to understand both prescriber and patient/public behavior and the factors that drive it using methodologies that are established in the behavioural sciences. any intervention has to be specific to the local context and the target population. a combination of strategies may be necessary to modify behavior for desired outcomes. department of infectious diseases, institute of infectious diseases and epidemiology, tan tock seng hospital, singapore antimicrobial resistance in hospitals is characterized by widespread dissemination of multidrug-resistant and extensively drug resistant bacteria, including methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum beta-lactamase and carbapenemase producing enterobacteriaceae, and pandrug-resistant acinetobacter baumannii. a combination of enhanced infection control and antimicrobial stewardship is often recommended to combat antimicrobial resistance in hospitals. multiple strategies are recommended by professional societies for antimicrobial stewardship, with differing levels of evidence and effectiveness. several recent well conducted systematic reviews and meta-analyses have shown that antimicrobial stewardship is effective in reducing broadspectrum antibiotic use and antimicrobial resistance without increasing adverse clinical outcomes. restrictive strategies produce more immediate effects but persuasive strategies are associated with more sustained impact. in addition to commonly adopted strategies of pre-authorization and prospective review and feedback, a greater variety of antimicrobial stewardship interventions are now recommended. in particular, evidence-based clinical care paths for common infections are associated with improved mortality. however antimicrobial use in hospitals is part of the greater context of antimicrobial use and resistance within the one health continuum. increasingly national and regional antimicrobial stewardship efforts must move outside of hospitals. damps: neutrophil dysfunction in experimental sepsis and post-septic complications related to immunosuppression jaroslaw zmijewski university of alabama at birmingham, usa damps: neutrophil dysfunction in experimental sepsis and post-septic complications related to immunosuppression bone using a murine model of polymicrobial septic peritonitis, we demonstrated that treatment with anti-hmgb1 ab significantly diminished sepsis-induced dysfunction of neutrophil nadph oxidase activity. importantly, confirmatory experiments revealed that blocking hmgb1 prevents neutrophils dysfunction. in summary, these results suggest that hmgb1 accumulation in the late phase of sepsis plays a specific role in the development of immunosuppression and specifically affects neutrophil-dependent antibacterial function in sepsis survivors. epidemiology of extensively-resistant gram-negative in mainland china hui wang peking university people's hospital, pr china multidrug resistance in gram-negative bacteria, especially carbapenem-resistant enterobacteriaceae (cre), is a critical public health threat in china advances in next-generation sequencing (ngs) platforms and microbial bioinformatics have positioned ngs to play an increasing role in clinical microbiology laboratories. next-generation sequencing is being applied to microbial isolates as well as directly to clinical specimens benchtop sequencers suitable for use in clinical microbiology laboratories are now available. sequences may be generated with various approaches (e.g., paired-end, mate-pair) and either aligned against a reference strain or assembled de novo with subsequent analytic strategies including single nucleotide polymorphism (snp) analysis and core genome multilocus sequence typing (cgmlst), among others. these approaches impact turnaround time, cost, technical difficulty, and accuracy. how results compare to those of historical methods not only does it allow for pathogen identification, but gene content information can also be used for resistance prediction, typing, and assessment for other relevant genes, such as those encoding virulence factors. our experience applying this approach to the diagnosis of prosthetic joint infection will be presented we found that loss of parkin, ubiquitin ligase implicated in autophagy and mitophagy occurs several hours after pro-inflammatory engagement in macrophages and lungs of mice subjected to intratracheal instillation of endotoxin. parkin dissipation was also accompanied with diminished activity in ampactivated protein kinase (ampk), a major sensor and metabolic regulator of immune homeostasis. we hypothesize that ampk activation will overcome loss in parkin-mediated autophagy and thus, diminish severity of ali. we found that ampk activators metformin or aicar did not recover the amounts of parkin. however, ampk activation promoted autophagy and improved bacterial clearance. in summary, our results show that parkin deficiency increased macrophage pro-inflammatory activation and the severity of ali mayo clinic, usa infectious diseases diagnostic testing is currently in a revolution vis-à-vis delivering new technologies. a myriad of new technologies are in use or under development, including matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (maldi-tof ms), rapid multiplex (i.e., panel) nucleic acid amplification tests (naats), point-of-care microbiology naats, and rapid phenotypic bacterial susceptibility testing, to name a few. these technologies provide new tools to combat antimicrobial resistance, which is especially important in an era of rising resistance. today, there is increased use of unneeded broad spectrum therapy because of the need to address the possibility of resistance until such a time as results of conventional diagnostic tests are available. this situation can potentially be ameliorated by more rapid diagnostics. in addition, over-prescription of antibacterial agents in general can potentially be addressed by rapid tests which exclude the need to prescribe antibacterial agents in the first place. an overview of new rapid diagnostics for infectious diseases that can potentially help combat antimicrobial resistance will be presented.ideal new diagnostics are more rapid, less expensive, and more accurate than existing diagnostics. unfortunately, not all new diagnostics meet all these criteria, with cost often being high. to address this situation, outcomes studies evaluating new diagnostics have assumed an important role. outcomes that should be addressed include antibiotic and further test avoidance, patient outcomes (e.g., length of stay, morbidity, mortality), patient and provider satisfaction, and infection transmission. results of outcomes studies then need to be used to inform the development of practice guidelines for use of these tests (which may vary from practice-to-practice) and also to inform ideal reconfiguration/development of tests by diagnostics companies. our experience with a recent randomized controlled clinical trial evaluating a rapid multiplex naat for testing positive blood culture bottles will be presented.it is an exciting time for diagnostics, with new technologies and opportunities for studies to determine how best to use these technologies in patient care. div. infectious diseases, dept. internal medicine, the catholic university of korea, koreasepsis is a significant syndrome in medicine and one of a major contributing cause of death world-widely.prompt recognition and aggressive management is the key element of improving survival from this brutal disease. unfortunately, sepsis is very complex immunologic event according to etiology and individuals' characteristics. as a result, a degree of organ dysfunction result from sepsis, an effect of protocolized treatment, and outcome could not be same.resuscitation with fluid and vasopressors is an initial and essential treatment of severe sepsis and septic shock. surviving sepsis campaign (ssc) continues to revise the treatment guideline and the initial steps for treatment are converged to use crystalloid fluid and use norepinephrine to whom do not respond to fluid therapy. in 2001, rivers et al suggested protocolized quantitative resuscitation method, otherwise known as early goal-directed therapy (egdt). this method was attractive in hospital settings because of clear notification of parameters, target, and drugs. the uncertainty of the effect of the individual component of bundle approach in treating severe sepsis/septic shock has been debated and egdt has been challenged following the failure to show a mortality reduction in subsequent large multicenter randomized controlled trials. effective fluid resuscitation means an effective restoration of tissue perfusion. although mean arterial pressure (map) can be a representative marker of tissue perfusion, central venous pressure (cvp) and serum lactic acid do not. as you already know, we do not have precise parameters of individual patient's hemodynamic status and also evaluating an effect of therapy we do. what is the proper volume of resuscitating fluid and how to monitor are also important issues we should have interest? although the crystalloid fluid is recommended as an initial therapy of resuscitation, the benefit, and harm of colloidal fluid, and alternative fluid is still evaluating.vasopressor therapy is needed to who do not respond fluid resuscitation. but there are many unresolved questions such as target map, optimal timing, and duration. norepinephrine has been recommended since the first ssc guideline, because more potent and effective at reversing hypotension, and less adverse events compared to dopamine. epinephrine and vasopressin are regarded as next-line or partner drug with norepinephrine. the effect of other vasopressors such as angiotensin ii and endothelin-1 are evaluating.medical experience clearly says that fluid resuscitation and vasopressor are very essential and initial element to improve the survival of the sepsis and septic shock patients. but we do not exactly know how to do accurately and properly. i will briefly present the current consensus and controversies about fluid resuscitation, monitoring of patient's hemodynamics, and use of vasopressors in septic patients in this lecture. the antimicrobial stewardship program (asp) is recognized as the most important tool for proper use of antibiotics. however, in order to properly implement asp, systematic training and tools are needed for medical staff prescribing antibiotics.basic education consists of face-to-face training for medical staffs. however, face-to-face education alone is difficult to maximize its effectiveness. when medical staffs prescribe, a variety of tools are being used to prescribe antibiotics properly.the most basic program is the antibiotic prescription restriction program. this program is a system that antibiotics which can only be used for certain resistant strains in hospitals are used with the permission of an antibiotic manager (usually an infectious diseases specialist).most hospitals in korea with infectious diseases specialists have this system. the infectious disease division of hallym universuty kangnam sacred heart hospital have a list of restricted antibiotics that contain vancomycin, linezolid, carbapenem, piperacillin/tazobactam, tigecycline, liposomal amphotericin b, echinocandin, voriconazole as restricted antibiotics.since the 2000s, antibiotic prescription programs have been introduced in korea and used in various hospitals. the purpose of this program is to recommend appropriate antibiotics according to the impression at the time of admission to the patient. when the causative bacteria are cultured, they help to prescribe appropriate antimicrobial agents according to the cultures. advanced programs automatically adjust the antibiotic dose according to the renal function. the number of antibiotic doses is also automatically recommended. when antibiotics need to be mixed with other solutions, it is automatically recommended that the solutions be mixed.currently, these antibiotic prescription programs have begun to apply the learning function of artificial intelligence and recommend antibiotics that frequently used depending on the name of diagnosis. if you are using antibiotics which could be expected to be drug interactions, you can also show alarms via pop-ups.in 2016, the korean society for chemotherapy developed a mobile app to help prescribe antibiotics. from 2017 to 2018, the korean society of chemotherapy and korea cdc (center for disease control and prevention) are conducting this project together. the antibiotic program is a collaborative project with hospital and clinic antibiotic prescription programs.this lecture will discuss antibiotic prescription programs used in korea and discuss the developmental direction of this program. key: cord-278456-gsv6dh36 authors: qureshi, abid; kaur, gazaldeep; kumar, manoj title: avcpred: an integrated web server for prediction and design of antiviral compounds date: 2016-09-09 journal: chem biol drug des doi: 10.1111/cbdd.12834 sha: doc_id: 278456 cord_uid: gsv6dh36 viral infections constantly jeopardize the global public health due to lack of effective antiviral therapeutics. therefore, there is an imperative need to speed up the drug discovery process to identify novel and efficient drug candidates. in this study, we have developed quantitative structure–activity relationship (qsar)‐based models for predicting antiviral compounds (avcs) against deadly viruses like human immunodeficiency virus (hiv), hepatitis c virus (hcv), hepatitis b virus (hbv), human herpesvirus (hhv) and 26 others using publicly available experimental data from the chembl bioactivity database. support vector machine (svm) models achieved a maximum pearson correlation coefficient of 0.72, 0.74, 0.66, 0.68, and 0.71 in regression mode and a maximum matthew's correlation coefficient 0.91, 0.93, 0.70, 0.89, and 0.71, respectively, in classification mode during 10‐fold cross‐validation. furthermore, similar performance was observed on the independent validation sets. we have integrated these models in the avcpred web server, freely available at http://crdd.osdd.net/servers/avcpred. in addition, the datasets are provided in a searchable format. we hope this web server will assist researchers in the identification of potential antiviral agents. it would also save time and cost by prioritizing new drugs against viruses before their synthesis and experimental testing. validation. furthermore, similar performance was observed on the independent validation sets. we have integrated these models in the avcpred web server, freely available at http://crdd.osdd.net/servers/avcpred. in addition, the datasets are provided in a searchable format. we hope this web server will assist researchers in the identification of potential antiviral agents. it would also save time and cost by prioritizing new drugs against viruses before their synthesis and experimental testing. antiviral compounds (avcs) inhibit the development of viruses in the host cell and are relatively harmless to the host. [1] they can be natural, for example, antivirals found in turmeric [2] and eucalyptus oil, [3] or synthetic, for example, zidovudine (a nucleoside analog) [4] and tamiflu (neuraminidase inhibitor). [5] many compounds and drugs have also been tested and found to be useful in restricting the growth of certain viruses. [6, 7] scientists are endeavoring to broaden the range of antivirals to other families of viruses. [8] however, designing safe and effective antiviral drugs is a difficult task due to the high genetic diversity and consequent drug resistance in viruses. [9] initially, antivirals were discovered using traditional trial-and-error methods. [10] however, it was a very lengthy process for discovering effective antivirals. [10, 11] later, research on virology helped to identify many target pathways to block viral multiplication. [12, 13] scientists are now using rational drug design strategies for developing antivirals that target the viruses at different stages of their life cycles. [14] during the past decade, many new drugs have been successfully identified in controlling the viral replication in host cells, for example, maraviroc (inhibits human immunodeficiency virus or hiv entry), pleconaril (inhibits picornavirus uncoating), acyclovir (inhibits herpesvirus replication), and oseltamivir (inhibits influenza release). [9, 15] to save time and money for discovering a new drug, researchers have widely used various computational methods to screen virtual libraries of compounds before the synthesis and animal testing of chemicals. among the different approaches, quantitative structure-activity relationship (qsar) is mostly used. [16] [17] [18] in this approach, relationships connecting molecular descriptors and activity are used to predict the property of other molecules. [19] molecular descriptors transform the chemical information (structure and linking of groups) of a molecule into simple numbers. [20] qsar-based virtual screening is an effective computational technique leading toward identification and design of novel antiviral agents. [21] lately, many dedicated bioinformatic resources have been developed for antivirals. for example, in the area of rna interference resources published are virsirnadbantiviral sirnas resource for about 42 disease causing viruses, [22] hivsirdb-anti hiv sirnas database, [23] virsirnapred-antiviral sirna inhibition efficacy predictor, [24] and virmirna-database of virus encoded mirnas including antiviral mirnas. [25] similarly, for peptide-based antivirals, a few web servers have also been created like avpdb-collection of antiviral peptides targeting more than 60 medically important viruses, [26] hipdb-hiv inhibiting peptide repository, [27] and avppred-predictor of antiviral activity of peptides. [28] . many general depositories provide information of antiviral molecules. for example, chembl, [29] pubchem-a database of molecules and their activities, [30] zinc-database of commercial compounds for virtual screening, [31] and drugbank-a knowledgebase for drugs and drug targets. [32] in addition, there are a few qsar studies targeting specific viral proteins. [33] [34] [35] [36] [37] [38] [39] [40] [41] however, till date there is no web server/software, which can regressively predict the percentage inhibition value of a compound against different human viruses under a single platform. to cater this need, we developed avcpred, a web server for prediction and design of antiviral compounds. in this method, we used previously known avcs against hiv, hepatitis c virus (hcv), hepatitis b virus (hbv), human herpesvirus (hhv) and 26 other viruses with experimentally validated percentage inhibition from chembl, a large-scale bioactivity database for drug discovery. [29] this was followed by descriptor calculation and selection of best performing molecular descriptors. the latter were then used as input for support vector machine (in regression mode) to develop qsar models for different viruses as well as a general model for other viruses. we have integrated these models in the avcpred web server, which will be helpful for virtual screening of avcs and designing new compounds to target the viruses. in this study, we have used different datasets of avcs having experimentally verified percent inhibition values against hiv, hcv, hhv, hbv and a general dataset having avcs against 26 human viruses. the data were obtained from the chembl resource (https://www.ebi.ac.uk/chembl/). the desired data were fetched using target browser (taxonomy tree) as well as target search using keywords such as hiv, hcv, hbv, hhv, virus, viral, viruses. initially, among the avcs, the majority of data belonged to hiv (1383 compounds), hcv (803 compounds), hhv (473 compounds), hbv (416 compounds), and other viruses (1635 compounds). after filtering entries with desired information and removing redundant entries, we were left with 389 compounds for hiv, 467 in case of hcv, 124 for hhv, 112 against hbv, and 1391 avcs targeting the 26 viruses (table 1 and table s1 ). these datasets were used for descriptor selection and model development. the datasets are available along with references on the web server and can be downloaded from this url: http://crdd.osdd.net/servers/avcpred/datasets.php. to develop virus specific as well as general qsar models, we computed about 18000 chemical descriptors (1d, 2d, and 3d), including geometric, constitutional, electrostatic, topological, hydrophobic, binary fingerprints, using padel, an open-source software to calculate molecular descriptors and fingerprints. [42] t a b l e 1 creation of datasets for the development of prediction models s. no. data filter a percent inhibition [1] reference [2] non-redundant [ the general dataset is comprised of below viruses with unique number of avcs in brackets: dengue virus 1, [1] dengue virus 2, [16] enterovirus, [30] human adenovirus 5, [41] human cox b1, [4] human cox b5, [21] human echovirus 13, [3] human echovirus 9, [2] human enterovirus 71, [19] human enterovirus c, [1] human polio virus 1, [4] human rhinovirus, [1] human rhinovirus 14, [29] human rhinovirus 1b, [18] human rhinovirus 2, [2] human t lymphotropic virus, [42] influenza a, [36] influenza a (h1n1), [16] influenza b, [1] monkeypox virus, [1] respiratory syncytial virus, [4] rift valley fever virus (cercopithecidae), [1] sandfly fever sicilian virus, [2] sars coronavirus, [23] simian virus 40, [45] sindbis virus, [4] vaccinia virus, [12] vaccinia virus wr, [22] variola virus, [1] vesicular stomatitis virus, [63] west nile virus, [17] yellow fever virus. [51] to improve the speed of calculation, we selected the most essential descriptors using 'removeuseless' filter followed by classifiersubseteval (attribute evaluator) with bestfirst (search method) module available in weka package. [43] classifiersubseteval evaluates attribute subsets on training/ testing data using a classifier to estimate the merit of a set of attributes. [44, 45] the selected descriptors were then used to develop the qsar models (table s3 ). we developed individual qsar models for each of the 4 viruses (hiv, hcv, hhv, and hbv) as well as a general model comprising 26 different viruses using smoreg algorithm [46] in weka machine learning software [43] freely available at http://www.cs.waikato.ac.nz/ml/weka. smoreg implements the support vector machine in regression mode. in smoreg, pearson vii function-based universal kernel (puk) and regsmoimproved optimizer were used along with parameters such as (i) the regularization constant/complexity value (c) that allows trade-off between training error and margin, (ii) the omega exponent value (ω) that controls peak half-width, and (iii) the sigma bandwidth value (σ) that controls peak tailing factor. [47, 48] simultaneously, software svm light (freely available at http://svmlight.joachims.org) was employed for machine learning in classification mode. in svm light , radial basis function (rbf) kernel was used with parameters (i) gamma (g) that defines how far the influence of a single training example reaches and (ii) complexity constant (c) that allows trade-off amid training error and margin. [49] selected molecular descriptors and fingerprints were used as input features for the development of qsar models. in order to evaluate performance of our models, we employed a number of statistical parameters including pearson's correlation coefficient, coefficient of determination, mean absolute error root-mean-square error, sensitivity, specificity, accuracy, and mathew's correlation coefficient as briefly described below. the pearson's correlation coefficient (r) is a measure of correlation between two variables. where n is the size of test set, and e i pred and e i act is the predicted and actual efficacy of avcs respectively. a value of 1 denotes total positive correlation, 0 is no correlation, and −1 is total negative correlation. the coefficient of determination (r 2 ) indicates how well data fit a statistical model. an r 2 of 1 indicates that the model perfectly fits the data, while an r 2 of 0 means that the model does not fit the data at all. the mean absolute error (mae) measure indicates how close the predictions are to the eventual outcomes. where e i pred is the prediction, e i act the true value, and maes are negatively oriented scores; that is, lower values are better. the root-mean-square error (rmse) measures the average magnitude of the error. rmses are also negatively oriented scores; that is, lower values are better. sensitivity (sn) or the true positive rate measures the percentage of correctly identified positives. an ideal predictor would be expressed as 100% sensitive. specificity (sp) or the true negative rate measures the percentage of correctly identified negatives an ideal predictor would be expressed as 100% specific. accuracy (ac) is the percentage of correct results (i.e. both true positives and true negatives) among the total number of cases. an ideal predictor would be expressed as 100% accurate. the matthew's correlation coefficient (mcc) is used in machine learning to evaluate the performance of binary classifications. qureshi et al. in the above eqs. (4-7) , tp, fp, tn, and fn represent the true positives, false positives, true negatives, and false negatives respectively. its value ranges from −1 to 1 and a value close to 1 means a better prediction. in order to identify the most effective features or descriptors of antiviral drugs, we computed the correlation between selected chemical features of antiviral drugs and their percent inhibition using comprehensive pharmacological screening datasets from chembl [29] (figure 1) . after attribute selection, the relevant descriptors were 45 for hiv, 52 for hcv, 15 for hbv, 20 for hhv, and 65 for rest of the viruses. a combination of selected chemical descriptors like partial charge, atom-type electrotopological state, extended topochemical atom, chi cluster, weighted path, and fingerprints based on substructure, graph, path, and extended features including pubchem and klekota-roth were found to be useful in prediction. the selected descriptors were then used to develop the qsar models (table s3) (table 2 ). other statistical parameters used in the development of qsar models are depicted in table s2 . a scatter plot between actual and predicted efficacy in each case is shown in figure 2 . in addition, we also checked the performance of our models developed using classification mode of machine learning. the general dataset is comprised of below viruses with unique number of avcs in brackets: dengue virus 1, [1] dengue virus 2, [16] enterovirus, [30] human adenovirus 5, [41] human cox b1, [4] human cox b5, [21] human echovirus 13, [3] human echovirus 9, [2] human enterovirus 71, [19] human enterovirus c, [1] human polio virus 1, [4] human rhinovirus, [1] human rhinovirus 14, [29] human rhinovirus 1b, [18] human rhinovirus 2, [2] human t lymphotropic virus, [42] influenza a, [36] influenza a (h1n1), [16] influenza b, [1] monkeypox virus, [1] respiratory syncytial virus, [4] rift valley fever virus (cercopithecidae), [1] sandfly fever sicilian virus, [2] sars coronavirus, [23] simian virus 40, [45] sindbis virus, [4] vaccinia virus, [12] vaccinia virus wr, [22] variola virus, [1] vesicular stomatitis virus, [63] west nile virus, [17] yellow fever virus. [51] (roc) plots illustrating the performance of the qsar models are shown in figure 3 . the qsar models have been integrated into a freely available and easy to use web server, 'avcpred', where users can predict the antiviral potential of their query molecules against the different viruses in terms of percent inhibition value. avcpred web server includes the following modules: this allows users to submit on or more molecules at a time. users have to choose the viruses on which they want to test their query chemical compounds. on submission, it returns with percent inhibition values against the selected viruses. also users can view the different properties of the query molecule such as structure, charge, molecular weight, logp value, hydrogen and lipinski bond donors/acceptors, rigid and rotatable bonds to identify drug-like molecular structures ( figure 4) . abbreviations: puk: pearson vii function-based universal kernel. regsmoimproved: optimizer for algorithm speed improvement. c: regularization constant/complexity parameter allows trade-off between training error and margin. ω: omega exponent value (controls half-width of the peak) σ: sigma bandwidth value (controls tailing factor of the peak). rbf: radial basis function g: parameter gamma in rbf kernel. it has been found that analogs of known chemical compounds are sometimes more effective than the parent molecule. [50] in order to identify potent analogs of an existing avc, we have included the 'design analogs' tool, where user can design analogs based on given building blocks and predict their inhibition on the viruses. using the 'draw tool', one can sketch the structure of the query molecule using marvin editor ( figure 5 ). this tool also gives the predicted percent inhibition values against the different viruses. in addition, one can view the various properties of the query structure. avcpred also provides the users a search tool to browse the compounds used in our datasets. in this module, different compounds targeting the viruses are stored in a database. the records can be readily searched, filtered/sorted, and downloaded via the web interface. avcpred has been developed using the open-source lamp (linux-apache-mysql-php) system. the prediction software runs on red hat enterprise linux 5 environment using apache httpd server. to inhibit viral growth, the antiviral molecules or drugs target different phases of viral life cycle such as fusion, integration, replication, maturation and should be relatively non-toxic to the host organism. [51, 52] each stage can be targeted using avcs that can, for example, inhibit entry receptors (cd4, ccr5) or viral enzymes (protease, neuraminidase). [53] [54] [55] f i g u r e 4 avcpred submission form with output qureshi et al. various avcs are currently in medical use, and new ones are in clinical trials. [56, 57] finding new and improved viral inhibitors is a major concern in the treatment of deadly human viruses. [58, 59] however, discovery of novel avcs is a tedious process. [60] to speed up the identification of new avcs, a computational approach using qsar method is a rational strategy to decrease cost and time efforts in the wet laboratory. [20] qsar techniques have been widely used in drug designing and further identification of lead molecules. [17] although there are many qsar studies pertaining to different types of viral protein inhibitors, they are very specific in their approach and deal with a particular class of inhibitors such as endonuclease inhibitors [33] in which 40 compounds were used and reached a correlation of 0.76, thiourea derivatives [34] where 85 compounds had a correlation of 0.92, protease inhibitors [39] in which 170 compounds had a correlation of 0.60-0.83, and flavonoid inhibitors [38] where 20 compounds had a correlation of 0.75-0.97 etc. (table 5 ). in most of the cases, the studies are carried out on a limited number of inhibitors. due to this reason, they predict the inhibitors that are similar to the compound type with a high correlation, but do not work on other dissimilar inhibitors for the same target virus. to address these limitations, avcpred models have been developed using diverse and large number of inhibitors. in the current algorithm, we have employed antiviral compound datasets from different studies due to which the overall correlation is less than above studies, yet the models are comparatively more robust to predict different classes of inhibitors. however, as new high-throughput screening data tested under homogeneous conditions on antiviral drugs becomes available, performance of the qsar method can be improved. in this study, we developed virus specific as well as general prediction models to identify the likelihood of a compound being antiviral using selected chemical attributes of experimentally validated avcs. padel, an open-source software, was used to calculate molecular descriptors and fingerprints. however, the software calculates a large number of descriptors, and hence, we used attribute selection approach to reduce their number by eliminating unrelated and extraneous descriptors to get a highly correlated descriptor set. our analysis revealed that several chemical descriptors are important in predicting the compound inhibition activity, for example, partial charge, atom-type electrotopological state, extended topochemical atom, chi cluster, weighted path, and fingerprints. we employed machine learning to train the qsar models on different sets of experimentally validated data. these models were validated on independent datasets, not used during training, and were found to have satisfactory performance. we used the pharmacological data from the chembl resource for training/testing the models developed for general as well as specific viruses. these models were integrated in an open-source web server for evaluation and screening of antiviral compounds. the applicability domain of the qsar models was demonstrated using williams plot ( figure 6 ) in which [40] 9 thymidine kinase n2-phenylguanine inhibitors 20 0.85-0.98 hsv no 2000 [41] f i g u r e 5 web interface of 'avcpred draw' tool standardized residuals are plotted against leverages. [61] if the standardized residual of a compound is greater than three times standard deviation units (±3σ), the compound is treated as an outlier. the warning value of leverage (h*) is considered as 3p/n, where p is the number of model descriptors plus one and n is the number of training compounds. [62, 63] if the leverage of a compound exceeds h*, it is regarded as dissident. the plots demonstrate that the leverages of majority of the compounds do not surpass the critical value (h*) in the regression models, and hence, the compounds are within the chemical domain, implying that the predictivity of the models is reliable. the web server also provides useful services like designing analogs based on given building blocks and drawing structure to sketch novel compounds and predict their inhibition potential against multiple viruses. the avcpred algorithm is hoped to assist the researchers in discovering novel antiviral compounds as well as virtually check the effect of modifications on existing drugs. avcpred is the first web-based algorithm for prediction of avcs based on experimentally validated datasets. five prediction models pertaining to hiv, hcv, hhv, hbv, and a general one were implemented in the web server to make comprehensive predictions. in addition, tools for drug design, virtual screening, and collection of existing avcs have also been integrated. this web server would be helpful for researchers working for the development of antiviral therapeutics. authors are thankful to council of scientific and industrial research (csir) (genesis-bsc0121), department of biotechnology (gap001), and csir-institute of microbial technology for providing infrastructure and financial support. using locally weighted learning to improve smoreg for regression learning to classify text using support vector machines: methods, theory and algorithms cold spring harb the authors declare that they have no competing interests. key: cord-262892-n38r8n70 authors: sheikh, jamila; wynn, bridget a.; chakraborty, rana title: nutritional care of the child with human immunodeficiency virus infection in the united states: a historical and contemporary perspective date: 2015-05-08 journal: health of hiv infected people doi: 10.1016/b978-0-12-800769-3.00009-3 sha: doc_id: 262892 cord_uid: n38r8n70 in well-resourced settings, early infant diagnosis and administration of life-saving antiretrovirals (arvs) have significantly improved clinical outcomes in pediatric human immunodeficiency virus (hiv) infection. the dramatic increase in survival rates is associated with enhancements in overall quality of life, which reflect a multidisciplinary, holistic approach to care. current optimism starkly contrasts with the outlook and prognosis two decades ago, when failure to thrive and wasting syndrome from uncontrolled pediatric hiv infection resulted from poor oral intake, malabsorption, chronic diarrhea, and a persistently catabolic state. the tenets of care developed from that era still hold true in that all infants, children, and adolescents with hiv require comprehensive nutritional services in addition to effective combination antiretroviral therapy (cart). this chapter will review the principles of nutrition in the preand post-cart eras and discuss the etiologic factors associated with malnutrition, with an emphasis on interventions that have favorably impacted the growth and body composition of infants, children and adolescents with hiv. the global pandemic of human immunodeficiency virus (hiv) infection has had grave consequences in the lives of affected infants, children, and adolescents, with more than 33% of infant and child mortality attributed to hiv infection in endemic locations [1] . in settings where voluntary and public resources are insufficient to provide long-term care, millions of children initially cared for by relatives have now been orphaned. however, many guardians themselves get sick or become overwhelmed by the number of dependents for whom they have to provide care. the growing number of street children and child-headed households are often the outcomes of a chain of events that begin with the hiv infection of a mother, her partner, or both. in the united states, perinatal transmission has decreased to such a significant extent that current estimates indicate less than 200 infants born with hiv annually [2, 3] . with the implementation of recommendations for universal prenatal hiv counseling and testing, antiretroviral (arv) prophylaxis, scheduled cesarean section delivery, and avoidance of breastfeeding, the rate of transmission events has decreased to less than 1% in the united states and europe [4] [5] [6] [7] [8] . however, there remains an unacceptable annual rate of newly diagnosed hiv-1 infections among infants in the united states, with the persistence of marked racial and economic disparities [9] . most pediatric hiv infections (>90%) are caused by vertical transmission, with events more common to areas where antenatal hiv seroprevalence is high [10] ; 21 countries in sub-saharan africa and in south, east, and southeast asia account for more than 90% of the pregnant women needing arvs to prevent vertical transmission. however, global rates of new hiv infections and prevalence among young people have fallen in many countries, likely due to reductions in vertical transmission rates and improvement in access to effective cart, which has decreased secondary transmission events. a clinical overview perinatal infection occurs at a time of relative immunologic immaturity. the inability to control viremia exposes the thymus and other lymphoid tissue to hiv-1-mediated destruction at a time of active thymopoiesis and lymphopoiesis [11] . given that the virus is transmitted from the mother and that the degree of human leukocyte antigen class i sharing between mother and infant is high, the virus could evade the protective immune response of the newborn, which results in accelerated disease progression [12] . in contrast to adults, hiv-1-related symptoms, cd4+ t cell depletion, or both develop in most untreated vertically infected children within the first few years of life [13] . in addition, plasma hiv-1 ribonucleic acid (rna) levels remained elevated over the first 2 years among infants [14] and do not decrease to less than 10 5 copies/ml through at least the third year of life [15] . the prolonged elevation of plasma hiv-1 rna levels may be related to the kinetics of viral replication, the size of the pool of host cells that are permissive to viral replication, and immature virus-specific immune responses. ii . nutrition and lifestyle the infection in perinatally infected infants and children progresses more rapidly than in adults. although 4% of the world's population with hiv-1 infection comprises children, 20% of all aids deaths were previously in this group. early studies before the era of cart indicated that a subset of children (~25%) progressed very rapidly to aids within 1 year. the median time to aids for the remaining 75% was 7 years [13] . in adults, opportunistic infections (ois) are often secondary to the reactivation of pathogens acquired before hiv infection. in contrast, in infants and children with vertical infection, ois often reflect primary acquisition of host pathogens during ongoing hiv replication and advancing immunosuppression. for example, young children with active tuberculosis more often present with miliary disease. without effective cart, the most common ois in children include serious bacterial infections such as pneumonia and bacteremia. common copathogens and ois that are difficult to eradicate without successful immune reconstitution include chronic mucosal or disseminated infections with herpesviruses, namely, cytomegalovirus (cmv), herpes simplex virus (hsv), human herpes virus 8 (hhv8) and varicella zoster virus (vzv). primary disseminated and reactivated tuberculosis is a major cause of morbidity and mortality among children with hiv in communities where infection with the pathogen is endemic. disseminated disease with mycobacterium avium complex may occur in children with hiv and advanced immunologic deterioration. pneumocystis jiroveci (formerly carinii) pneumonia (pcp) is a common and serious oi associated with a high mortality rate. pneumonia most often manifests between 3 and 6 months of age in infants with vertically acquired hiv infection. candidiasis (topical, oral, esophageal, and tracheobronchial) is the most common fungal infection in these children. causes of acute and chronic central nervous system (cns) infections include those caused by cryptococcus neoformans, and toxoplasma gondii. less commonly observed ois include cryptosporidiosis and systemic fungal infections. clinical presentations include hepatosplenomegaly, failure to thrive, oral candidiasis, recurrent diarrhea, parotitis, cardiomyopathy, hepatitis, nephropathy, developmental delay, encephalopathy, lymphoid interstitial pneumonitis, recurrent bacterial infections, and specific malignancies. malignancies include non-hodgkin b-cell burkitt-type lymphomas, leiomyosarcomas, and kaposi sarcoma, which are commonly described in children with hiv who are of sub-saharan african ethnicity. in the united states, in clinical practice, the number of ois seen in children with hiv has decreased, reflecting the widespread use and administration of effective cart regimens. however, ois continue to be the presenting symptom of hiv infection in infants due to lack of antenatal testing in mothers or in adolescents and young adults who are increasingly infected through horizontal transmission. the intestine is a primary target organ for hiv. hiv infection causes a depletion of cd4+ t lymphocytes in gut-associated lymphoid tissue, including selective loss of a subset of t helper cells called th17 lymphocytes, which are important in gut mucosal containment of extracellular pathogens such as salmonella typhimurium. th17 cells are lost early in retroviral infection and are not replenished over time. this depletion impairs long-term gastrointestinal (gi) mucosal integrity and permeability, causing increased bacterial translocation and immune activation. the intestinal mucosa is also the main reservoir of hiv in the body despite effective virologic suppression with cart. among untreated children with hiv, as many as 80% will have one or more intestinal disorders at a given time, with iron malabsorption present nearly 50% of the time [16] . hiv enteropathy is secondary to direct hiv-mediated injury and indirect immune-mediated injury to the gi tract mucosa in the absence of specific opportunistic enteropathogens, perhaps reflecting selective loss of th17 lymphocytes. hiv enteropathy can occur in children and adolescents at all stages of hiv infection. clinical manifestations include chronic diarrhea, increased intestinal permeability, malabsorption, and malnutrition. histologic changes include lymphocytic infiltration of the gi tract mucosa, villous atrophy and blunting, and crypt hyperplasia [17, 18] . a direct cytopathic effect of hiv on the intestinal mucosa is supported by the observation that clinical signs and symptoms improve after initiation of effective cart in association with virologic suppression and immune reconstitution of cd4+ t cells [19] . acute, recurrent, and chronic diarrhea associated with malabsorption and growth impairment frequently occur in children with untreated hiv infection and advancing immunosuppression. commonly identified infective enteropathogens include bacteria (salmonella, shigella sp.), viruses (including rotavirus, adenovirus, cmv), parasites (entamoeba, giardia, cryptosporidia, microsporidia, isospora), and opportunistic fungi [20] . in children with hiv, frequent and persistent watery diarrhea is the most common presentation of cryptosporidial, microsporidial, and isosporidial infections, associated with abdominal cramps, fever, vomiting, anorexia, weight loss, and poor weight gain [7] . untreated chronic severe diarrhea may cause malnutrition, failure to thrive, severe dehydration, or a combination of all these problems. gi tract disease caused by cmv may include esophagitis, gastritis, pyloric obstruction, hepatitis, pancreatitis, colitis, ascending cholangitis and cholecystitis. signs and symptoms may include nausea, vomiting, dysphagia, epigastric pain, icterus, and watery diarrhea. stools may be bloody. sigmoidoscopy in cmv colitis provides nonspecific results, showing diffuse erythema, submucosal hemorrhage, and diffuse mucosal ulcerations. specific causes of diarrhea in representative adult subjects with aids are presented in table 9 .1 [21] . data reflect prospective follow-up of 1,933 participants in the swiss hiv cohort study; 560 diarrheal episodes were evaluated by standardized stool examination, with intestinal infections diagnosed in less than 50% of chronic diarrheal episodes [22] . the site and severity of infection vary according to the infecting organism. oral mucosal ulcerations secondary to infectious agents such as candida albicans, cmv, or hsv cause inflammation and pain during swallowing or after eating, which may lead to reduced oral intake. opportunistic enteropathogens such as cryptosporidium, cmv, and microsporidia [23] may affect the hepatobiliary system and pancreas in addition to the gi tract, resulting in vomiting, abdominal pain, and malabsorption. in resource-limited settings, disease with mycobacterium tuberculosis is the most common cause of death in subjects with hiv. hiv and tuberculosis (tb) accelerate disease progression and mortality and are associated with marked clinical wasting; the extent of wasting is related to the severity of tb [24] . the largest proportion of newly diagnosed children with hiv in many us centers are foreign born and at higher risk of prior and potentially ongoing exposure to tb. tb is almost always transmitted to children by an adult, most commonly a household contact, and the infection in children is primary infection rather than reactivated disease as in adults. there should be an increased index of suspicion of tb infection and disease in children with hiv, particularly in the context of clinical wasting and a low threshold for empiric antituberculosis therapy, even when diagnostic investigations fail to identify a tb-causing organism. the combination of underlying hiv infection, nutritional status (particularly protein-energy malnutrition), and host immunity are inextricably interdependent. in the united states, prior to the widespread administration of effective cart, the predominant effect of advancing immunosuppression on nutritional status in children with hiv was wasting and negative energy balance, which predicted both morbidity and mortality [25] . in the pre-cart era, ois were major precipitants of weight loss, necessitating prevention or prompt diagnosis and treatment to prevent wasting and to promote weight recovery [26] . growth in children with hiv was persistently below normal standards, with reduced height and weight velocities, compared with hiv-exposed but uninfected children. in 1994, the centers for disease control and prevention (cdc) defined wasting in children younger than age 13 years as (1) persistent weight loss of more than 10% of baseline; (2) downward crossing of at least two percentile lines on the weight-for-age chart in a child aged 1 year or older; or (3) less than the 5th percentile on the weight-for-height chart on two consecutive measurements at least 30 days apart, plus chronic diarrhea or documented fever for at least 30 days, whether intermittent or constant [27] . in addition to ois, other etiologies that contribute to abnormal growth in untreated hiv infection in children include a synergistic combination of inadequate dietary intake, gi malabsorption, increased energy utilization, and socioeconomic adversity. the prevalence of malnutrition in children with hiv varied among centers in the united states with up to 30-50% of children followed up in pediatric hiv programs having demonstrable evidence of protein-energy malnutrition, which, in turn, exacerbated the immunosuppressive effects of hiv [28] . common patterns of wasting included an early decline in weight and height in the first 3 months of life or early linear stunting with a normal weight-to-height ratio. progressive wasting with low weights and heights were also well recognized and more commonly associated with infectious enteropathogens. sequential follow-up demonstrated that growth in children with untreated hiv infection remained below growth in age-matched and gender-matched uninfected controls. malabsorption also results in macronutrient and micronutrient deficiencies. micronutrient deficiencies are widespread and compound the effects of hiv infection in children. deficiency can manifest in conditions such as fatigue, reduced learning ability due to anemia (iron deficiency), and impaired immunity [29] . such deficiencies reflect inadequate nutrient intake and the consequences of excessive losses due to ois, diarrhea, and malabsorption, as previously described. other micronutrients that can also be malabsorbed resulting in deficiency include vitamin b12, folic acid, thiamine, zinc, selenium, calcium, and magnesium, and fat-soluble vitamins a and d [21] . the evidence base for the specific effect of micronutrient supplementation in children with hiv is limited, but a recent cochrane review of 11 studies with 2,412 participants made the following key recommendations for practice. benefits of periodic vitamin a supplementation in children over 6 months of age with hiv infection in resource-limited settings were supported by data from three african trials and were consistent with evidence of benefits of supplementation in uninfected children. zinc supplements reduced diarrheal morbidity and had no adverse effects on disease ii. nutrition and lifestyle progression in a single safety trial in south african children. children with hiv should therefore receive zinc supplements in the management of diarrhea and severe acute malnutrition in the same way as uninfected children with the same conditions. the review emphasized that micronutrient deficiencies and immune dysfunction in children with hiv would only be restored with effective suppression of hiv replication [30] . cart consists of drugs that target the life cycle of hiv at specific enzymes or receptors to inhibit replication thereby preserving or restoring immune function. specific goals of administration of cart include maximally reduction of the plasma viral load below the limit of detection (<50 copies/ml), prevention of a selection of drug-resistant strains and maintenance of good immunologic status (repopulation with cd4 + -naã¯ve t cells), and prevention of clinical disease progression and ois. clinical trials of cart in infants and children with hiv have demonstrated dramatic reductions in morbidity and mortality (>80-90%) in the united states since widespread implementation from 1996 onward, so the vast majority of infants and children with hiv-1 can now be expected to survive to adulthood [31, 32] . five classes of arv drugs are commonly available for hiv therapy. two classes target the enzyme reverse transcriptase-non-nucleoside reverse transcriptase inhibitors (nrtis) and the non-nrtis. a third class-protease inhibitors (pis)-target viral protease, whereas integrase inhibitors target that corresponding enzyme. in addition, ccr5 inhibitors target the viral co-receptor ccr5 on permissive target cells. hiv-1 mutability is largely the result of errors introduced into the viral genome during replication. the hiv genome is approximately 10,000 nucleotides long, and each new virion has an average of one mutation. this results in a large pool of quasi-species of viral variants that are incapable of productive infection but some of which may provide an adaptive benefit, for example, the development of art resistance, to the virion. drug-resistance of the virus can develop during cart administration because of poor adherence, a regimen that is not potent, or a combination ii. nutrition and lifestyle of these factors resulting in incomplete virologic suppression. in addition, primary drug resistance may occur in arv-naive infants and children who can become infected with the resistant virus. aggressive, multidrug cart as early in infection as possible, with daily adherence for an indefinite period, is advocated to fully suppress viral replication and to preclude the selection or emergence of resistant viral variants. resistance testing has enhanced the ability to choose effective initial regimens as well as second-or third-line regimens. therapeutic strategies continue to focus on timely initiation of arv regimens that are capable of maximally suppressing viral replication in order to prevent disease progression, preserve or restore quantitative and qualitative immunologic function, and reduce the development of drug resistance [33] . difficulties with long-term adherence to cart-particularly in infants and children because of variable drug administration, absorption, and metabolism; pretreatment with maternal cart and vertical transmission of drug-resistant virus; acceptability and palatability of medications; and refrigeration of syrup formulations in warm climates-are all well documented. long-term follow-up of infected infants and children involves longitudinal determinations of prognostic markers, including number and percentage of cd4 t cells, and viral load [34] . such parameters provide a useful framework for the time to initiate and change therapy but involve frequent venipuncture in minors. long-term toxicities include lipodystrophy syndrome [35] and lipid abnormalities, cardiomyopathy, mitochondrial toxicity and lactic acidosis, renal tubular acidosis [36] , hypersensitivity reactions, and cns toxicity. fortunately, the availability of new drugs and drug formulations has led to the use of more potent regimens with reduced short-term toxicity, lower pill burden, and less frequent medication administration, all factors that are associated with better adherence and outcomes. enteral [37, 38] or parenteral supplementation and appetite stimulants [39] can improve the nutritional status and weight in children with untreated hiv infection but have little effect on the growth velocity of height. however, effective virologic suppression with cart was shown to improve mean weight, weight for height, and muscle mass in 67 children with hiv, in whom pi-based therapy was initiated and maintained for a median of 5 months. these effects were independent of virologic suppression and improved cd4 t-lymphocyte counts [40] . these findings were also noted in the pediatric aids clinical trial group 219 study, which ii. nutrition and lifestyle found that pi therapy improved both weight and height z-score annually, after adjusting for cd4 cell count, age, gender, and race [41] . in the era of cart, following the introduction of pi-containing regimens, hiv-associated mortality decreased by greater than 80-90%, with significant declines in opportunistic and related infections [31, 32] . these encouraging outcomes have been tempered by the side effects associated with arvs. altered body composition, lipid abnormalities, and abnormal regulation of glucose metabolism are consequences that result in an increased risk of cardiovascular disease, reflecting complications of inflammation with uncontrolled hiv infection and the specific arv drugs as outlined. in children, adolescents, and adults, a clear syndrome of abnormal fat redistribution or lipodystrophy and metabolic changes associated with administration of cart is well described. patterns of lipodystrophy vary from peripheral fat wasting, or lipoatrophy, in the face, extremities, and buttocks to central fat accumulation, or lipohypertrophy, in the abdomen, dorsocervical spine regions (buffalo hump), and breasts. both conditions may occur alone or in combination [42, 43] and can be difficult to assess in a growing child or adolescent, since changes in body fat occur normally during childhood and puberty [44] . lipodystrophy in children with hiv is clinically evaluated by examination or self-report and has been documented to be as high as 32% [45] . dual-energy x-ray absorptiometry (dexa) quantifies total, trunk, and limb fat. observational studies in children with lipodystrophy show decreased total and extremity fat and a greater trunk-to-extremity fat ratio in children with hiv compared with uninfected children [46, 47] these changes are drug specific and associated with duration of therapy, with prolonged treatment and older age more likely to result in lipodystrophy. treatment with nrtis, including stavudine (d4t), zidovudine (azt), and didanosine (ddi), is associated with a lower percentage of extremity fat and higher percentage of trunk fat and trunk-to-extremity fat ratio even after adjustment for wasting and stunting [21, 43] . these changes in body fat distribution often cannot be reversed even after switching to less lipodystrophic arv regimens. in cohorts of children receiving a pi regimen, higher rates of dyslipidemia have been documented, with higher fasting lipids, cholesterol, and triglycerides. lipodystrophy in patients results in much higher waist-tohip ratios and elevated fasting insulin levels and blood pressure, which ii. nutrition and lifestyle are all significant risk factors for cardiovascular disease [21] . for children without lipodystrophy, up to one fifth show symptoms of dyslipidemia. in summary, when selecting arv regimens, care must be taken to consider the above life-long side effects and their consequences. at a time when newer less lipodystrophic first-line regimens, including tenofovir, abacavir, ritonavir-boosted pis (atazanavir and darunavir), and the integrase inhibitors, most with the added advantage of once daily administration, are available in the united states, regimens that include zidovudine, didanosine, and stavudine should be prescribed less often to children with hiv to reduce these potential long-term toxicities. metabolic syndrome reflects a series of clinical conditions, including elevated triglyceride, low levels of high-density lipoprotein (hdl) cholesterol, hyperglycemia and insulin resistance, increased body fat distribution around the waist, and high blood pressure, all of which collectively increase the risk of cardiovascular disease. in individuals with hiv, the prevalence of metabolic syndrome is higher than in the general population and estimated to be 7-45% [21, 48] . although uncontrolled hiv in the absence of cart can cause low hdl cholesterol and high triglycerides, as discussed previously, arvs also induce body fat redistribution in conjunction with metabolic changes. earlier pis, including treatment doses of ritonavir (without boosting other pis), nelfinavir, and ritonavir-boosted lopinavir (kaletra) were documented to increase lipid plasma concentrations, including serum triglycerides, cholesterol, low-density lipoprotein (ldl) cholesterol, and apolipoprotein e and to lower hdl. virologic control with the newer pis, integrase inhibitors, tenofovir, and abacavir may be associated with increases in serum hdl, in the absence of these metabolic complications. when compared with population norms, children with hiv were noted to have lower-than-expected bone mineral density (bmd) for their age and gender that may have been associated with delays in growth, sexual maturity, duration of hiv infection, ethnicity, and disease severity [49] . a more recent large study of 236 american children and adolescents with hiv, aged 7-24 years, showed that males with hiv had significantly lower bmd at tanner stage 5 compared with uninfected males [50] . reduced bmd secondary to cart administration was first described in 2001 from dexa scans in vertically infected children, with the severity of osteopenia directly related to lipodystrophy [51] . however, a longitudinal study from 2013 in 66 dutch children showed an association between longer cart duration and increases in spinal bmd z-scores [52] . lopinavir-ritonavir ii. nutrition and lifestyle [50] , full-dose ritonavir [53] , and tenofovir [54] are associated with lower bmd in children. the principles of maintaining good bone health in youth with perinatal hiv infection is the same as those recommended for all youth in general. adolescents should therefore receive at least 1,300 mg calcium per day and at least 600 iu vitamin d per day through their diet, by supplementation, or both [55] . immune reconstitution inflammatory syndrome (iris) is a diseasespecific inflammatory response that can occur after treatment with arvs is initiated, reinitiated, or changed, resulting in effective virologic suppression and immune reconstitution of naã¯ve and memory cd4+ t cells. iris has been noted to occur in children who begin art while they have severe malnutrition, are severely immunosuppressed [56] , or both. risk factors therefore include a low cd4 nadir and high viral load levels prior to the initiation of cart. these children and adolescents often have numerous documented ois before, during, and after cart initiation [56] . further research is needed to reduce complications and to optimize clinical management when they do occur. the interaction between hiv infection and nutrition is of great importance, and these two factors are interdependent, since strategies to improve nutritional status both quantitatively and qualitatively have been demonstrated to have a beneficial effect on clinical outcome and the immunologic course of the hiv infection. through the course of their disease, infants and children with hiv have numerous nutritional needs, which reflects, as previously described, impaired absorption, decreased oral intake, and increased nutrient requirements. specific adverse outcomes secondary to specific nutritional deficiencies include the inability to achieve normal weight for height; malnutrition and wasting; growth failure and stunting; and neurocognitive, neurodevelopmental, and oral motor delay often from hiv encephalopathy. early nutrition intervention is, therefore, essential and must be addressed simultaneously with the administration of cart, antimicrobial prophylaxis, and neurodevelopmental interventions. collectively, a ii. nutrition and lifestyle multidisciplinary approach is most effective in improving health outcomes and overall quality of life. in the pre-cart era, the nutritional causes of malnutrition reflected (1) decreased oral intake caused by anorexia and by oral and esophageal lesions often from opportunistic pathogens, (2) gastroesophageal reflux and aspiration, (3) regression or nonattainment of key developmental milestones associated with oromotor dysfunction and impaired mastication, (4) malabsorption, (5) increased energy requirements and metabolism from ois with associated negative energy balance, (6) vomiting and diarrhea from gastrointestinal (opportunistic) enteropathogens, and (7) indirect immune-mediated enteropathy. at a time when effective cart was unavailable and faced with a debilitating catabolic disease and rapid disease progression in infants and children with hiv, nutritional interventions that were developed in the early 1990s by pediatric providers targeted four key areas: 1. prompt management of diarrhea. in addition to isolation of opportunistic enteropathogens and prescribing appropriate antimicrobials for infectious etiologies, management of diarrhea mandates assessment of hydration status and rehydration by the oral or intravenous route. modification of diet in the setting of underlying food intolerance such as lactose or fat malabsorption, including pancreatic enzyme supplementation; and vitamin and mineral supplementation. other recommendations included introduction of a mechanical soft diet and nutritional supplementation. 4. management of nausea and vomiting. in addition to appropriate antiemetic agents, treatment also included recommendation of small frequent meals, liquid intakes between meals, and nutritional supplementation. 5. management of anorexia included small nutrient dense foods, nutritional supplementation, and appetite stimulants such as megestrol acetate. these early nutrition needs related to the unique physiologic demands for growth and development, so even today, interventions should be individualized according to the child's specific needs and relate to disease stage, gastrointestinal function, and growth [57] . as a corollary, the energy and protein requirements for infants and children with hiv have not yet been established because individual needs vary, depending on age, growth, and the clinical and immunologic status that may increase energy and protein needs. infants and children with hiv who have slow weight gain are often prescribed high-protein, high-calorie diets. if nutritional needs are not met through a typical high-calorie, high-protein diet, then additional support may include oral nutritional supplements and overnight feeding through nasogastric or gastrostomy-tube feedings. a commercial formula with intact protein may be appropriate for children without underlying gastrointestinal pathology. infants and children with hiv who have gastrointestinal malabsorption should receive a semi-elemental formula to maximize absorption. elemental formulas are typically prescribed when semi-elemental formulas are not tolerated. infants and children with hiv who are unable to consume adequate calories orally often benefit from supplemental tube feeding. enteral tube feeding supplementation improves weight gain in children with hiv who have growth failure [37, 38] . nasogastric tube feedings should be initially attempted and include night-time feedings, which allow the child to eat normally throughout the day. complications relating to nasogastric tube feedings include sinusitis and the technical inability of the caregiver to place the tube or administer the feedings [21] . if delivery of feedings through a nasogastric tube improves growth, then placement of a more permanent device such as a gastrostomy tube should be considered. enteral supplementation with gastrostomy feeding has improved nutrition in a number of chronic childhood illnesses by providing adequate energy intake to promote weight gain when oral intake is poor. miller et al. [37] first investigated the effects of gastrostomy tube feeding on weight gain, height, body composition, immune parameters, morbidity, and mortality in 1995 on 26 children with hiv. weight z-scores before therapy were -1.6 and had decreased to -2.2 on initiation of nasogastric feedings. gastrostomy tube feedings significantly improved weight z-scores to get back to baseline approximately 5 months after initiation of feeding. significant predictors of response to gastrostomy tube feedings included higher cd4 counts at initiation and lower weight-for-height z-scores at baseline. these findings suggested that early intervention during acute weight loss offers the best chances of improving weight in children with hiv. children with the greatest improvement in weight after gastrostomy tube placement spent less time in hospital and had a greater likelihood of survival compared with children who did not gain weight [37] . this small but important study demonstrated that early nutritional intervention improved quality of life and reduced morbidity in children with hiv at a time when effective cart was unavailable. in the cart era, compliance with medical therapy is often improved with more reliable delivery of arvs through the gastrostomy tube and is associated with improved cd4 t-lymphocyte counts, virologic suppression, and improved longitudinal growth. guarino et al. tested the hypothesis that nutritional support improves intestinal and immune functions in 62 italian children with hiv; 16 received enteral nutrition through continuous feeding, and 46 received total parenteral nutrition. the authors documented a significant increase in cd4 cell count, xylose levels, and body weight in those receiving enteral nutrition, suggesting that nutritional intervention may restore intestinal absorption and increase cd4 cell numbers if initiated early in the course of pediatric hiv infection [58] . enteral feeding is preferred over parenteral nutrition to preserve the gut structure. parenteral nutrition should be used only in those children unable to tolerate or gain weight on enteral supplementation, those who have recurrent or chronic biliary tract or pancreatic disease, and those who have intractable diarrhea with weight loss [21] . megestrol acetate is an oral synthetic progesterone used since the early 1990s as an appetite stimulant. weight gain tends to be associated with increase in body fat rather than muscle. clarick et al. investigated the effects of megestrol acetate treatment on weight gain and linear growth in 19 children with hiv who had growth failure. the average duration of the study was 7 months. the study concluded that megestrol acetate was associated with weight gain but not linear growth during the treatment period. after the megestrol acetate treatment was discontinued, poor weight gain and weight loss were again noted [39] . given the dramatic reductions in morbidity and mortality and the improved longitudinal growth in children with hiv in the united states since the widespread implementation of effective cart, megestrol acetate and other therapeutic agents (including growth hormone and the anabolic steroid oxandrolone) are prescribed very rarely, if at all, to subjects with hiv. in the 1980s and early 1990s, the devastating effects of hiv infection on the health of infants, children, and adolescents became apparent and required a rapid and effective response globally. over time, in the united states, with the introduction of arvs, the clinical manifestations associated with hiv infection as well as its treatment were seen to increase, driven by the short-and long-term toxicities of these new formulations in combination. in children with hiv, these manifestations reflected metabolic changes; wasting and stunting from gastrointestinal dysfunction were most often described in the 1980s, but new clinical concerns in the early 2000s were related to altered body composition, lipid abnormalities, and abnormal regulation of glucose metabolism. these complications were often attributed to the first-generation nrtis and pis. the longterm cardiovascular risks of these arvs on subjects with hiv are still unknown. after 2007, newer pis and integrase inhibitors became more widely available and appear to have fewer metabolic adverse effects, although ongoing surveillance of these arvs and tenofovir will be important to evaluate incidences of renal tubular dysfunction and bmd. in the course of the changes in art over the previous two decades, optimal nutritional support has continued to be a cornerstone of pediatric and adolescent hiv care, applying the same principles developed from the early 1990s to effectively support infants, children, and adolescents with hiv. these principles include ongoing comprehensive nutritional assessments and follow-up. when cart providing effective viral suppression was unavailable, enteral and parenteral support was associated with improved weight and body composition and overall survival and is still a key part of care for children and adolescents who present with advanced hiv disease. in addition, periodic vitamin a supplementation in children with hiv who are older than 6 months of age is supported by clinical trials in africa. children with hiv should also receive zinc supplements in the management of diarrhea and severe acute malnutrition in the same way as uninfected children with the same conditions. investigators should continue to study the effects of oral hypoglycemic agents, lipidlowering medications, and lifestyle changes on cardiovascular risk factors in patients with lipodystrophy and hyperlipidemia at this time when obesity has become endemic in many communities in the united states. this unfortunate development on long-term health also has implications for children and adolescents with hiv across the united states. nevertheless, the overall outlook for children with hiv has improved significantly since the 1990s, as reflected in the reduced rates of morbidity and mortality and improved quality of life. perhaps a measure of the latter is the overall medication burden. figure 9. 1 is a child's medications, as shown by oleske et al. [57] . figure 9 .2 shows the pill burden for a number of adolescent patients in the united states in 2014. the last paragraph of dr. oleske's article still relevant for 2014. to quote directly, "compassionate, comprehensive, and coordinated clinical care services are required for all hiv-infected infants and children through adolescence. we must not underestimate their needs. as we improve their longevity with advances in primary hiv therapies, we must not let quality of life suffer due to a lack of nutritional intervention." global, regional, and national causes of child mortality in 2008: a systematic analysis achievements in public health reduction in perinatal transmission of hiv infection-united states recent trends in the incidence and morbidity that are associated with perinatal human immunodeficiency virus infection in the united states combination antiretroviral strategies for the treatment of pregnant hiv-1-infected women and prevention of perinatal hiv-1 transmission european collaborative study mother-to-child transmission of hiv infection in the era of highly active antiretroviral therapy 2 the high number of medications for an adolescent with hiv in 1 the slew of daily medications for a 13-year-old long-term surviving patient with perinatally acquired hiv in 1996 included: zidovudine (azt), didanosine (ddi), trimethoprim/sulfamethoxazole (tmp/smx), fluconzol, megase, prednisone, acyclovir, dapsone, biaxin, zalcitabine (ddc), albuterol, isonicotinylhydrazine (inh), rifampin, ranitidine (zantac) two-dose intrapartum/newborn nevirapine and standard antiretroviral therapy to reduce perinatal hiv transmission: a randomized trial low rates of mother-to-child transmission of hiv following effective pregnancy interventions in the united kingdom and ireland earlier initiation of art and further decline in mother-to-child hiv transmission rates racial/ethnic disparities among children with diagnoses of perinatal hiv infection -34 states towards universal access: scaling-up priority hiv/ aids interventions in the health sector hiv-1 infection in children: a clinical and immunologic overview evolution and transmission of stable ctl escape mutations in hiv infection the european collaborative study association of human immunodeficiency virus (hiv) load early in life with disease progression among hiv-infected infants the relationship between serum human immunodeficiency virus type 1 (hiv-1) rna level, cd4 lymphocyte percent, and long-term mortality risk in hiv-1-infected children management of gastrointestinal disorders in children with hiv infection hiv enteropathy: crypt stem and transit cell hyperproliferation induces villous atrophy in hiv/microsporidia-infected jejunal mucosa enteropathies in the developing world: neglected effects on global health ritonavir combination therapy restores intestinal function in children with advanced hiv disease aetiology and management of malnutrition in hiv-positive children nutritional aspects of hiv-infected children receiving highly active antiretroviral therapy enteric infections and diarrhea in human immunodeficiency virus-infected persons microsporidia infection in patients with human immunodeficiency virus and unexplained cholangitis nutritional status in malawian patients with pulmonary tuberculosis and response to chemotherapy prospective analysis of patterns of weight change in stage iv human immunodeficiency virus infection nutrition in paediatric hiv infection magnitude of body-cell-mass depletion and the timing of death from wasting in aids centers for disease control and prevention 1994 revised classification system for human immunodeficiency virus infection in children less than 13 years of age unicef tracking progress on child and maternal nutrition: a survival and development priority micronutrient supplementation for children with hiv infection incidence of opportunistic and other infections in hiv-infected children in the haart era declines in mortality rates and changes in causes of death in hiv-1-infected children during the haart era hiv-1 drug resistance in hiv-1-infected children in the united kingdom from national study of hiv in pregnancy and childhood collaborative hiv paediatric study increased lipodystrophy is associated with increased exposure to highly active antiretroviral therapy in hiv-infected children persistent non-gastrointestinal metabolic acidosis in pediatric hiv-1 infection gastrostomy tube supplementation for hiv-infected children effect of enteral tube feeding on growth of children with symptomatic human immunodefi-� ciency virus infection megestrol acetate treatment of growth failure in children infected with human immunodeficiency virus the effect of protease inhibitors on growth and body composition in hiv-infected children impact of protease inhibitor-containing combination antiretroviral therapies on height and weight growth in hiv-1-infected children prevalence, evolution and risk factors for fat atrophy and fat deposition in a cohort of hiv-infected men and women pediatric hiv/aids cohort study. body fat distribution in perinatally hiv infected and hiv-exposed but uninfected children in the era of highly active antiretroviral therapy: outcomes from the pediatric hiv/aids cohort study regional body fat distribution in relation to pubertal stage: a dual-energy x-ray absorptiometry study of new zealand girls and young women european paediatric lipodystrophy group antiretroviral therapy, fat redistribution and hyperlipidaemia in hiv-infected children in europe morphologic and metabolic abnormalities in vertically hiv-infected children and youth longitudinal changes in regional fat content in hiv-infected children and adolescents the metabolic syndrome in hiv predictors of bone mineral density in human immunodeficiency virus-1 infected children total body and spinal bone mineral density across tanner stage in perinatally hiv-infected and uninfected children and youth in pactg 1045 bone mineral loss through increased bone turnover in hiv-infected children treated with highly active antiretroviral therapy bone mineral density increases in hiv-infected children treated with longterm combination antiretroviral therapy antiviral therapy and bone mineral measurements in hiv-infected youths comparison of changes in bone density and turnover with abacavir-lamivudine versus tenofovir-emtricitabine in hiv-infected adults: 48-week results from the assert study vitamin d status in children and young adults with perinatally acquired hiv infection severe malnutrition and metabolic complications of hivinfected children in the antiretroviral era: clinical care and management in resourcelimited settings historical perspectives on the evolution in understanding the importance of nutritional care in pediatric hiv infection effects of nutritional rehabilitation on intestinal function and on cd4 cell number in children with hiv key: cord-273324-xhpv783y authors: land, kevin j.; boeras, debrah i.; chen, xiang-sheng; ramsay, andrew r.; peeling, rosanna w. title: reassured diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes date: 2018-12-13 journal: nat microbiol doi: 10.1038/s41564-018-0295-3 sha: doc_id: 273324 cord_uid: xhpv783y lack of access to quality diagnostics remains a major contributor to health burden in resource-limited settings. it has been more than 10 years since assured (affordable, sensitive, specific, user-friendly, rapid, equipment-free, delivered) was coined to describe the ideal test to meet the needs of the developing world. since its initial publication, technological innovations have led to the development of diagnostics that address the assured criteria, but challenges remain. from this perspective, we assess factors contributing to the success and failure of assured diagnostics, lessons learnt in the implementation of assured tests over the past decade, and highlight additional conditions that should be considered in addressing point-of-care needs. with rapid advances in digital technology and mobile health (m-health), future diagnostics should incorporate these elements to give us reassured diagnostic systems that can inform disease control strategies in real-time, strengthen the efficiency of health care systems and improve patient outcomes. or in sequence. in case of discrepancies, a third test is used as a tiebreaker. an example of this approach is for hiv case detection. specificity. diagnostics should have low false positive rates. the ideal scenario is where the sensitivity and specificity achieved from the diagnostics used at the poc approach those of laboratory-based assays wherever possible. however, a lower specificity can be tolerated if the harm of overtreatment is much less critical than missing the diagnosis of an infection. screening syphilis during pregnancy is an example of this approach, as missing maternal syphilis can lead to stillbirths, preterm birth and congenital syphilis in a third of pregnant women compared to the harm of overtreatment with a single dose of penicillin 5 . user friendliness. tests should be easy to perform in 2-3 steps and require minimal user training with no prior knowledge of diagnostic testing. typically, results should be available in 15-60 minutes after sample collection and enable patient management and treatment during the same visit. the benefits of a rapid test versus a more accurate test that requires patients to return for results have been previously demonstrated 6 . robustness refers to the ability of the test to withstand the supply chain (temperature, humidity, time delays, mechanical stresses) without requiring additional (and often costly) transport and storage conditions (for example, refrigeration). ideally the test does not require any special equipment or can be operated in small portable devices that use solar or battery power. deliverable to end-users. delivery refers to the organizational structures and relationships established with the purpose of coordinating and steering the logistics of selecting, procuring, shipping, storing, distributing and delivering a new health technology to ensure it reaches the end-users in resource-constrained settings 7 . compared to when assured was proposed, access to laboratories in resource-limited settings has improved dramatically both in numbers and in quality 8, 9 , especially in areas such as hiv and malaria testing, cd4 counting and tb. however, there remains a critical need for a wider range of diagnostics that can be performed at the poc. from this perspective, we undertake a review of the assured benchmark over the last 14 years, assess the factors contributing to the success and failure of the assured diagnostics over the past decade, identify areas that have remained difficult to address as well as lessons learned from implementation in resourcelimited settings, and highlight additional conditions that should be considered in addressing poc needs. since 2003, several diagnostic tests that satisfy (or nearly satisfy) the assured criteria have been developed to identify major human pathogens. this includes tests for hiv, malaria, syphilis and tb ( table 1) . the hiv rapid test is the first test to fulfil the assured criteria, followed closely by the malaria and syphilis rapid tests and, recently, a near-poc test for tb. much of this development was the result of advocacy by donors such as the bill & melinda gates hiv rdts. early detection of hiv infection has been a critical component of hiv control programmes worldwide since the early days of the epidemic. since most infected individuals do not show specific symptoms and the period of viraemia is short, screening for hiv antibodies in blood as a marker of exposure has been the most cost-effective means of identifying infected individuals. the who developed a process and criteria for validating the performance of hiv antibody detection assays more than a decade ago and showed that hiv rdts using finger-pricked whole-blood specimens has acceptable performance compared to laboratory-based immunoassays 12, 13 . many donors -for example, global fund and implementation partners, including the who, unaids and pepfar -have helped affected countries with the procurement and introduction of rapid hiv tests and enable early detection and prevent onward transmission. as a result of funding from donors, increased accessibility of rdts, and the 'architecture' provided by national hiv/ aids control programmes, hiv tests successfully reached as many as 150 million children and adults in 129 low-and middle-income countries in 2014 (ref. 14 ) . two major challenges remain: training and quality assurance. with thousands of poc testing sites across a country, even the most well-organized control programmes would find it difficult to provide adequate training on an ongoing basis. this problem is most acute in health facilities where there is high turnover of staff. the us centres for disease control and prevention (cdc) has developed a convenient means of external quality assurance by working with countries to generate thermally stable proficiency panels that can be shipped to every poc testing site to ensure competence of testing personnel. however, a study in south africa found that only 3% of hiv rdts were performed correctly 15 . although not every error will result in an incorrect diagnosis, the alarming reality is that with 150 million tests being performed annually worldwide, assuming a 99% accuracy rate, as many as 1.5 million incorrect results per year could potentially be generated. ongoing quality assurance efforts include developing key policy and quality documents for the implementation of hiv-related poc testing 16 . for example, as poc technologies for hiv viral load and early infant diagnosis were being developed, there was tremendous emphasis on quality, given the complexity of the test and lessons learned from hiv rdts. malaria is estimated to be the cause of at least a million deaths a year worldwide, most of which are in sub-saharan africa. while microscopic identification of parasites in blood smears has been the traditional means of diagnosing malaria in patients presenting with fever, microscopy requires equipment, a source of electricity and trained laboratory technicians. malaria rdts were developed as many rural communities lack these resources, and to date there are over 120 brands of malaria rdts made by approximately 60 companies 17 , which vary widely in performance, manufacturing quality and price. the who has set up a pre-qualification programme with the cdc and the foundation for innovative new diagnostics to evaluate these tests to inform test selection and procurement for national malaria control programmes 17 . in countries that permit the sale of malaria rdts and medicines over the counter, the quality of these commodities cannot be guaranteed. the price of most rdt brands is between us$0.65 and us$2.50 per test 7, 18 , and as with all diagnostics, it is important to caution that price pressure will ultimately affect quality. as with hiv, with the support of donors, selection of high-quality rdts and the architecture provided by national malaria control programmes, malaria rdts have reached millions of patients every year. this promising trend, coupled with the effectiveness of bednets for preventing transmission, has led to the call for malaria elimination in many parts of the world 19 . however, challenges remain for the future of malaria poc testing and the global elimination of malaria. first, highly accurate tests are required for all malaria species affecting humans, but these panspecific tests are usually about 40% more expensive than rdts that only detect plasmodium falciparum. most p. falciparum rdts detect a malarial antigen, histidine-rich protein (hrp) 20 , and the discovery of parasites that have a deletion in this gene has raised concerns about false negative results and ongoing transmission. also, the problems of providing adequate training and quality assurance of malaria tests and testing at remote poc sites are similar to those described for hiv rdts 21, 22 . in the near future, as countries progress towards malaria elimination, funding for rdts may become an issue. as the intensity of transmission decreases due to lower parasite density in infected individuals, more sensitive tests will be needed, which can only be achieved with costlier amplification steps or with ultrasensitive platforms for antigen detection. also, decreasing numbers of cases mean that malaria tests are no longer cost-effective, and thus it is more difficult to justify funding in light of multiple competing priorities for limited health budgets. syphilis. syphilis, caused by the spirochete treponema pallidum, has a long latent period during which patients have no symptoms, but can remain infectious. syphilis in pregnancy can lead to adverse outcomes of stillbirths and miscarriage, and babies born with congenital syphilis in the developing world only have a 50% chance of survival during the first 2 years of life 23, 24 . despite the availability of simple diagnostic tests for antenatal screening and the effectiveness of treatment with a single dose of long-acting penicillin, syphilis is re-emerging as a global public health problem 23 . it is estimated that 500,000 babies die each year as a result of syphilis-associated stillbirths and congenital syphilis, largely because of lack of access to antenatal screening 25, 26 . rdts exist for detecting syphilis and are reported to have acceptable performance 27, 28 and operational characteristics. the introduction of rdts was also acceptable to patients and health care providers and was shown to contribute to the improvement of antenatal care in low-resource settings 29 . despite all this, syphilis rdts have not had the same success as hiv and malaria rdts. the main reasons for this are the lack of advocacy and political will to translate the evidence to national policy, lack of funding to make the tests affordable to those in need and the lack of a national control programmes to provide the architecture needed to coordinate all the different aspects of testing. ensuring adequate training for health care workers and supplies of commodities were cited as key implementation barriers in africa 30, 31 . dual hiv and syphilis rdt testing in countries was prioritized by the who for the elimination of mother-to-child transmission (mtct) of hiv and syphilis by 2020 32,33 , but a recent review showed that while prevention of mother to child transmission (pmtct) programmes for hiv have resulted in a dramatic decrease in the number of hiv-positive infants in sub-saharan africa, the rate of syphilis screening of pregnant women in the same countries has remained at unacceptably low levels of approximately 30% (ref. 34 ). this is due largely to disparity in funding and lack of political will despite the global fund allowing countries to purchase syphilis rdts with hiv rdts since 2007 (ref. 35 ). countries need to harness the architecture provided by hiv pmtct programmes to screen women for both infections using a single drop of blood in a single visit to a health care facility. dual rapid hiv-syphilis rdts with acceptable performance are now available 36 and the who, as well as many countries, has adopted these hiv and/or syphilis rdts into their national guidelines 31, 37 . tuberculosis. tb causes 1.7 million deaths a year, 95% of which occur in low-and middle-income countries. ending the tb epidemic by 2030 is among the health targets of the sustainable development goals 38 . the tb lipoarabinomannan (lam) antigen test provides poc screening for active tb in hiv positive patients. this novel rapid test detects lam in urine samples, providing results in just minutes, enabling earlier treatment for patients 39 . the lam test does not assess tb drug susceptibility, but multidrug-resistant tb is a threat to global health security with an estimated 600,000 new cases a year showing resistance to rifampicin (rif) -the most effective first-line drug 40 . nucleic acid tests (nats) for tb are available and provide highly sensitive and specific means of diagnosis. the recent development of a sample-in-answerout automated testing device that allows for simultaneous detection of mycobacterium tuberculosis (mtb) and rif resistance in 1 h 45 min has improved case detection and could decrease transmission, though nats still require patients to make a return visit for test results and treatment. this test system is available in 1-80 modules, allowing for flexibility in throughput. the mtb/rif near-poc assay can improve time to diagnosis and treatment and increase the efficiency of the health system if introduced appropriately 41-44 , and as of june 2012, two-thirds of high-burden mtb countries and half of countries with a high multidrug-resistant mtb had adopted this assay into their national tb programme guidelines. a wider adoption of such high-performing assays would allow countries to increase their case detection rates and potentially reach the 2020 milestones of the end tb strategy 45 . although national tb programmes provide a robust architecture for the implementation of new technologies, challenges associated with the near-poc nat assay remain as barriers -affordability (molecular assays are device-based and costly, even with subsidy), expertise (more technically demanding than lateral flow rdts) and sustainability 46 , in addition to power and per-test time. we expect that addressing these barriers would improve patient outcomes, but a true poc test is still needed to overcome remaining challenges such as sample preparation and demands on human resources 47 . while culture has been a long-standing microbiological gold-standard and is highly specific, it is also the most technically demanding, costly and slowest of diagnostic options, requiring patients to return for another clinic visit to obtain their test results and treatment if necessary. thus, it does not conform well to the development of assured diagnostics. successful poc tests achieve high sensitivity, which is needed as screening tools to ensure that all true and suspect cases are brought to the attention of control programmes and appropriately managed. in particular, antibody-based detection tests, such as those for hiv and hepatitis c virus, are highly sensitive as antibodies are present in large quantities in blood, and blood samples can be collected with a finger prick and put directly into the test without any prior processing. also, antibody detection can be rapid and easy to perform with minimal training required (table 2) . however, the choice of the antibody target affects a test's effectiveness -unless the diagnostic target is specific to the intended infection, there is the potential for false positive results due to cross-reactivity, which is the case with dengue and zika immunoassays 48, 49 . therefore, antibody detection assays should be interpreted within the appropriate epidemiological context and findings from physical examination. the other major disadvantage of antibody-based tests is that antibodies are usually a marker of exposure to a pathogen and cannot be used to distinguish between those with active and past infection, which is the case of the rk39 tests for visceral leishmaniasis (vl) 50 . a presumptive diagnosis of active vl can only be made using a combination of a positive rk39 test and more than 2 weeks of fever and splenomegaly. the same is true of most syphilis rdts, which detect long-lived antibodies to treponemal antigens and thus do not diagnose active syphilis. antibodies to a non-treponemal antigen, which appears with infection and declines in the months after successful treatment, are used in a flocculation assay called the rapid plasma reagin assay, which can yield a result in 8 minutes but requires electricity to operate, a centrifuge for processing serum from whole blood, a shaker and cardiolipin as an antigen. a combination treponemal and non-treponemal rapid test has been developed in an immunochromatographic test in a lateral flow format with acceptable performance characteristics 51 . antigen detection poc tests, such as those for chlamydia and gonorrhoea, suffer from three major drawbacks (table 2) . first, the specimen used for sexually transmitted infections are usually from urethral, cervical or vaginal swabs that require multiple steps to solubilize the bacteria and free antigen before reaction with the capture antibody. this sometimes requires a heating step and adds to the complexity and cost of the poc test. urine is the preferred specimen in men with such infections, and current poc tests require a urine centrifugation step to concentrate the bacteria before processing and reaction. another common drawback of antigen detection tests is the potential for false positive results due to cross-reactivity with antigens from closely related bacterial or viral species. this is especially true if polyclonal antibodies are used as capture antibodies. finally, a common disadvantage of current antigen detection poc assays is low sensitivity, often requiring 10 4 to 10 5 bacteria for the rdt to become positive. malaria rdts are an exception to these drawbacks in that the parasites are present in large quantities in blood and no pre-processing or concentration steps are required. for most antigen detection poc tests, the low sensitivity may be due to low concentration of target antigens, inefficiency of extraction or limited optimization of reagents, which has hampered chlamydia and gonorrhoea tests 52, 53 . however, gift et al. showed that rapid chlamydia tests with a sensitivity of 65% can lead to more infected patients being treated compared to nats, which require patients to return for their test results 54 . they called this the rapid test paradox, which was also recently seen with a p24 assay for early infant diagnosis of hiv, where a sensitivity of 72% can still lead to a higher 'diagnostic yield' than nats performed on dried blood spots sent from remote antenatal clinics 55 . nats offer a more sensitive and specific alternative to antigen detection assays as nucleic acid targets can be selected from a genome sequence with high specificity, and the amplification process to increase sensitivity can be fairly rapid (table 2) . for example, the near-poc tb diagnostic involves primer-based amplification of mtb dna specifically, including regions associated with rif drug resistance. thus, nat tests are generally highly sensitive and more specific than antigen-or antibody-based tests, and may help inform on drug susceptibilities. while instruments are available for highthroughput screening of patients and subsidies can bring test costs down to us$10, the equipment (capable of four tests at a time) costs more than us$10,000, which may be prohibitive in high-incidence regions. thus, a truly poc diagnostic based on nats without needing any equipment is still a promise and not a reality as yet 56, 57 . of all the criteria originally accepted, the requirement of equipment-free is perhaps the only one which is not as critical as originally defined. a wide range of near-poc nats have been developed for use outside of laboratory settings 58 and most of these assays are automated, requiring only 2-3 minutes of hands-on time and minimal training. these near-poc devices come in sealed units with internal self-calibration making it easier to ensure quality of testing and most are also equipped with data transmission capabilities, which make these platforms very attractive in the context of disease surveillance and epidemic preparedness. most of these near-poc devices have a broad menu of diagnostic targets which makes the capital cost of purchasing the device less of an issue. reagents are pre-measured and dried ready for hydration with the introduction of the specimen. however, near-poc nat cartridges are expensive to manufacture and not affordable to most developing countries without subsidies. rapid advances in miniaturization, material science, electronics and data transmission in recent years have made minimal instrumentation a reality for new diagnostics, including the use of smart phones, which are widely available, custom developed and low cost. the use of a dongle to connect a smart phone to a microfluidic disc to detect hiv and syphilis antibodies in finger-pricked blood allows the phone to power the reaction, interpret the results and transmit the data to a central database. this smart phone-based poc assay is currently undergoing clinical trials 59 . use of a smart phone to power nats is in development and will play an important role in future poc applications 60,61 . in particular, there has been considerable interest shown in utilizing mobile phones as readers and connectivity for rdts using rfid (radio frequency identification) to prevent errors in subjective interpretation and transcription [62] [63] [64] [65] . while phone-based diagnostics are attractive as an option, regulatory approval and rapid updates of phone software that can affect test performance are important challenges. defining and quantifying risks and benefits. while successes need to be celebrated, significant challenges remain. first, as no diagnostic is perfect, countries continue to struggle with defining acceptable risks of false positive or negative results when a novel diagnostic technology may provide incremental clinical benefits. in the developing world where over 60% of the population reside in rural areas, and the sustainable development goals urge countries to provide universal health coverage and leave no one behind, the trade-off between accuracy (sensitive and specific) and accessibility (user friendly, rapid and deliverable) becomes paramount 66 . while it impossible to give absolute values across different tests, both sensitivity and specificity remain critical. in selecting an ideal test, the positive and negative predictive values, which are dependent on the prevalence of the disease as well as the characteristics of the diagnostic test, should be taken into account, but few studies have been performed to determine quantitatively what tradeoffs are acceptable. an analysis for syphilis screening compared the use of a laboratory-based immunoassay with treponemal rdts in tanzania 67 . it showed that a test that has 100% sensitivity but can only be used in sophisticated laboratories can realistically only be accessed by 30-40% of the population, while a rapid lateral flowbased antibody test that has only 90% sensitivity, but can be used at all levels of the health care system effectively gives a correct diagnosis to as many as 81% of the population, highlighting that tests should not be evaluated purely on technical performance, but rather on diagnostic performance and clinical impact. training and quality assurance. assuring the quality of tests and testing is the biggest challenge when testing is decentralized at poc. there are many rapid tests from various manufacturers available, and test quality may impact accurate and inconsistent diagnosis across different sites, with studies showing that high rates of errors were observed even in performance of simple rdts for hiv and malaria 15, 68 . quality of testing requires proficiency panels be sent to all the poc testing sites by a national reference laboratory. including positive and negative controls with each box of tests will allow all tests to be approved once they arrive at their destination and before first use. recently, nine african countries developed a national system for assuring the quality of poc testing for hiv 69, 70 . with data connectivity from each testing site, quality assurance results can be linked to test results at each poc testing site at a centralized database to trigger alerts for corrective action. supply chain software can also be linked through these connectivity solutions, avoiding stock-outs. demonstrating the value of a novel diagnostic technology. the value of a diagnostic goes beyond the technology, as each test needs to be matched to its 'testing environment' , which includes population characteristics, prevalence of target diseases, comorbidities/coinfections and health system characteristics. national programmes should take advantage of the rapid turnaround time of assured tests to streamline patient pathways and increase the efficiency of the health care system. the roll-out of poc diagnostics requires the use of implementation science to ensure success 71 , taking into account the cultural, behavioural, socioeconomic and health systems contexts. this includes a careful assessment of acceptability and feasibility linked to possible increased stresses on the health system/provider when testing is introduced into settings where thus far no or limited testing was performed. studies using the genexpert mtb/rif tb test showed that new diagnostic tests would not have the expected impact on outcomes if test introduction is not accompanied by changes in patient pathways or practices 72, 73 . these studies highlight the importance of programmatic monitoring of the impact of novel technologies beyond studies that are usually conducted under controlled conditions. likewise, same-day testing and treatment using a syphilis rdt strengthened health systems in the amazon forest and rural china when policy makers were involved and testing was introduced within the appropriate social and cultural contexts 71, 74 . for example, in peru, women normally need to present six times to the largest maternal hospital in lima for their prenatal syphilis screening to be completed and treatment provided, but the introduction of the rapid syphilis test streamlined testing and treatment to one visit, reducing patient out-of-pocket expenses and increasing the efficiency of a busy health care facility 75 . there are few detailed analyses which have been conducted to accurately determine the economic impact of many of the available tests, including the diagnostic cost versus the overall economic impact. while these benefits are intuitively important, they are often difficult to quantify 76 . it would, therefore, be extremely useful to have economic analysis tools for each of the major diseases or conditions, so that developers have an understanding of cost implications and what cost structures would be acceptable to health service implementers. as an example, it may be necessary to accept cost trade-offs when addressing global health threats (for example, zika, ebola, and so on), where speed of intervention is critical. this having been stated, low-cost diagnostic tests remain critical in resource-limited settings. in the past two decades, the biggest drivers of diagnostic development were well-resourced diseases such as hiv, malaria and tb. in recent years, the increasing frequency and severity of global health emergencies caused by infectious diseases of epidemic potential, such as severe acute respiratory syndrome (sars) coronavirus, middle east respiratory syndrome coronavirus (mers) co-v, ebola and zika virus, have made the global community realize the need to accelerate assured test development, validation, manufacturing and deployment. there is a need for innovation in rapid detection technologies that are assured but allow multiplex detection of a panel of pathogens such as major causes of respiratory illness, haemorrhagic fevers or enteric infections in a single specimen. these tests should not only identify the cause of outbreak but also be used to process multiple specimens with high throughput throughout the outbreak. another major driver of test development is the need for cheaper, better and faster tests that can be used at poc to reduce inappropriate antimicrobial use. an estimated 700,000 people die from untreatable conditions due to antimicrobial resistance (amr) every year worldwide 77 , and if amr continues to spread, by 2050, 10 million people may die from resistant infections annually at an economic cost estimated at $100 trillion. at the primary care level, a simple rapid test that can be used to distinguish bacterial from nonbacterial causes of fever, respiratory and enteric infections would allow health care providers to reduce antibiotic use and preserve them for future generations. the o'neill report 77 also pointed out that a test that can identify a pathogen and its antibiotic susceptibility at the poc would allow the safe use of first-line drugs or drug combinations with savings to the health care system. to stimulate interest in innovation in diagnostic tests that can be used at poc, several countries have set up challenge prizes. in 2015, the united kingdom announced the longitude prize of â£10 million, which seeks an affordable, accurate, fast and easy-to-use test for bacterial infections that will allow health professionals worldwide to administer the right antibiotics at the right time. the challenge is currently ongoing, with final submission by 2020. more information available at: https://longitudeprize.org. in september 2016, the us department of health and human services announced a challenge prize competition in which up to $20 million will be awarded for one or more novel and innovative poc diagnostics that would have clinical and public health value in combating the development and spread of antibiotic resistant bacteria. more information is available at: https://dpcpsi.nih.gov/amrchallenge. these prizes may catalyse new technologies, but there are still no guarantees to the sustainability of these tests in resource-limited countries given that cost is a critical factor woven into every step needed to bring about and sustain testing. donors and funders of diagnostics, such as the the bill & melinda gates foundation, global fund, unitaid and aid agencies from national governments such as pepfar and dfid, play a critical role in incentivizing diagnostic innovation and addressing market dynamics. technology advances to support innovation. technology 78 , markets and medical devices have matured to enable connected diagnostics to become a useful tool for epidemiology, patient care and tracking, research, and amr and outbreak surveillance. the ability to digitize data from laboratory and poc platforms, including lateral flow rdt results, can standardize the interpretation of results and allows data to be linked to proficiency testing to ensure testing quality, reducing interpretation and transcription errors. remote monitoring of poc instrument functionality and utilization through connectivity allows programmes to optimize instrument placement, algorithm adoption and supply management. alerts can be built into the system to raise alarm at unusual trends such as outbreaks. the application of mobile devices and related technologies to health care is improving patients' access to health information and treatment, offering possibilities to diagnose, track and assess the impact of infectious disease interventions across the world 79, 80 . smart devices can also be used to automatically add data to a central database and allow systems to assess likelihood of a diagnosis or predict disease outbreaks through machine learning, providing a route to smart diagnostics that incorporates historical or epidemiologic data to make diagnoses more accurate. finally, new engineering fields such as the internet of things (iot), industry 4.0 and printed electronics [81] [82] [83] [84] promise to add significantly to the technologies that can be chosen and incorporated into new diagnostic devices. many of these technologies are aimed at high-volume and low-cost distributable manufacturing, thus fitting in well with the goal of poc diagnostics. new detectors and light sources also suggest that incorporating these into tests could allow for more accurate result reading and the possibility of multiplexing. while it is clear that the original assured criteria remain relevant, opportunities exist to improve future diagnostics by incorporating new technological elements to provide real-time quality control for testing and treatment and overcoming the difficulties in specimen collection and/or processing 85 , which currently limits scaling-up of diagnostics in resource-limited areas. we therefore propose two additional criteria of r (real-time connectivity) and e (ease of specimen collection and environmental friendliness) into the original assured, to create a new acronym of reassured (table 3) . to use testing to effectively survey or treat patients, it is critical to obtain and analyse results at the poc 47 . however, one of the main challenges to poc testing is ensuring that results are rapidly provided to the patient once testing has been completed 86 ; this is formidable challenge when testing is decentralized at hundreds or thousands of different sites by health care workers who are not consistently skilled in reading test results, leading to risk that incorrect data is being recorded and/or transmitted. one solution would be to allow the analysis of test results at a centralized level for consistent diagnosis and epidemiological surveillance. many manufacturers are now embedding connectivity into poc and near-poc instruments (for example, the alere pima poc cd4 device 87 ). other manufacturers have developed innovative connectivity solutions that can use mobile phones to read the results from lateral flow assays and provide electronic result exchange, while a third option would be to include connectivity directly onto the test 88 . ideally, connectivity should not only include collection and transmission of test data, but also analysis to provide feedback for immediate patient treatment or for surveillance. with the addition of barcodes, two-dimensional codes or electronic storage into tests, a number of other data points can be collected, including manufacturing information (for example, lot numbers), dates, expiry dates, stock availability and possibly even environmental conditions such as temperature and humidity under which the tests have been manufactured, transported, stored and used. for instrumented tests, maintenance and other machine data can be collected. thus, connectivity solutions can increase quality assurance for poc tests and would allow for centralized and real-time decision-making, even across tiered laboratory systems and during outbreak investigations and global health emergencies. moving forward, it is critically important to make provisions for poor or non-existent internet availability to support sustainability standards and that systems are developed with compatibility so that they can be linked into central databases. ease of specimen collection. specimen collection and processing may need further in-depth development. first, it is ideal to have noninvasive specimen collection given that more tests become available as self or home tests. second, samples come in many different formats (blood, urine, sputum, stool, swab, breath, and so on) and require different preparation depending on the test to be performed. this may include concentrating the sample (or possibly titration), purification, lysing of cells, target amplification, and so on. while most of these processes can be easily performed in the laboratory, there remain few solutions for collecting and performing these tasks at the poc 89 . oral tests for hiv and hepatitis c virus are good examples of advances in non-invasive sampling, although the collection device is more expensive than blood collection via disposable capillary tubes. related, given that rapid lateral flow antigen detection tests often have lower sensitivity, extra processing steps such as heating or other forms of extraction are required when using specimens other than blood. environmental friendliness. advances in technology have made the assured requirement for equipment somewhat less daunting. however, in light of more diagnostic tests now being performed in both urban and rural areas, there could be more future effort devoted to the environmental impact of these tests. the cumulative effect of many tens of thousands of tests performed at remote sites away from laboratory infrastructure may pose health and environmental risks and put a strain on limited resources. some examples include the plastics used in current rapid tests that cannot be recycled and give out toxic fumes when burned, and testing cartridges that may even contain harmful chemicals and need to be disposed of properly. recyclable materials should be used, when possible, for the housing, substrate matrix materials, and reagents used in tests. paper, an obvious choice of substrate material that has many inherent advantages over standard plastic materials, will see increased usage, not only in lateral flow formats but other paper based formats as well such as in micro paperbased analytical devices (î¼ pads) [90] [91] [92] . and recently, cell-free synthetic gene networks for in vitro applications at poc, have been achieved by freeze-drying cell-free systems onto paper, which increases stability at room temperature and can be easily transported and stored, and simply re-activated by adding water 93 . other factors also need to be considered, such as disposal of test reagents, clinical samples and materials used for active components such as electronic tracks and electrodes. the assured criteria have been a valuable framework for developing devices and methods to detect major human diseases in challenging poc contexts. however, over the last decade, new technologies, not envisaged or available at the time of the first assured criteria definition, have given rise to the possibility of including new considerations into the next generation of devices and tests. we propose the acronym reassured for the design of future diagnostic tests to address important priorities such as global health emergencies and amr. an ideal test would be one that combines the best of both existing diagnostic worlds (instrumented laboratory based tests with lateral flow tests) and technologies currently being developed (fig. 2) , which would provide an important extension to diagnostic laboratory systems and fulfil the sustainable development goals of 'no one left behind' in terms of effective health care service delivery. such tests can only be created by forming strong collaborative partnerships across many disciplinary boundaries, and we look toward a future when data connectivity linking cost-effective assured diagnostics to laboratory systems will form the backbone of health care systems and provide real-time data for evidence-based disease control and prevention strategies, more efficient health systems and improved patient outcomes. diagnostics for the developing world mapping the landscape of diagnostics for sexually transmitted infections: key findings and recommandations the costs of accessible quality assured syphilis diagnostics: informing quality systems for rapid syphilis tests in a tanzanian setting reducing the burden of sexually transmitted infections in resource-limited settings: the role of improved diagnostics the rapid test paradox: when fewer cases detected lead to more cases treated: a decision analysis of tests for chlamydia trachomatis access: how do good health technologies get to poor people in poor countries laboratory medicine in low-income and middle-income countries: progress and challenges strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress global health diagnostics requirements for high impact diagnostics in the developing world rapid point-of-care hiv testing in pregnant women: a systematic review and meta-analysis expanding the role of diagnostic and prognostic tools for infectious diseases in resource-poor settings ensuring quality who-find malaria rdt evaluation programme availability and price of malaria rapid diagnostic tests in the public and private health sectors in 2011: results from 10 nationally representative cross-sectional retail surveys plasmodium falciparum parasites lacking histidine-rich protein 2 and 3: a review and recommendations for accurate reporting minimising human error in malaria rapid diagnosis: clarity of written instructions and health worker performance improving community health worker use of malaria rapid diagnostic tests in zambia: package instructions, job aid and job aid-plustraining the global elimination of congenital syphilis: rationale and strategy for action (who, department of reproductive health and research untreated maternal syphilis and adverse outcomes of pregnancy: a systematic review and meta-analysis global estimates of syphilis in pregnancy and associated adverse outcomes: analysis of multinational antenatal surveillance data accelerating worldwide syphilis screening through rapid testing: a systematic review are treponema pallidum specific rapid and point-of-care tests for syphilis accurate enough for screening in resource limited settings? evidence from a meta-analysis introduction of rapid syphilis testing in antenatal care: a systematic review of the impact on hiv and syphilis testing uptake and coverage scaling down to scale up: a health economic analysis of integrating point-of-care syphilis testing into antenatal care in zambia during pilot and national rollout implementation introduction of syphilis point-of-care tests, from pilot study to national programme implementation in zambia: a qualitative study of healthcare workers' perspectives on testing, training and quality assurance avoiding hiv and dying of syphilis elimination of mother-to-child transmission of hiv and syphilis (emtct): process, progress, and program integration global burden of maternal and congenital syphilis in 2008 and 2012: a health systems modelling study celebrating the decline in syphilis in pregnancy: a sobering reminder of what's left to do a systematic review and meta-analysis of studies evaluating the performance and operational characteristics of dual point-of-care tests for hiv and syphilis who guideline on syphilis screening and treatment for pregnant women diagnostic accuracy of a low-cost, urine antigen, point-of-care screening assay for hiv-associated pulmonary tuberculosis before antiretroviral therapy: a descriptive study rolling out xpert mtb/rifâ® for tuberculosis detection in hiv-positive populations: an opportunity for systems strengthening advances in tuberculosis diagnostics: the xpert mtb/rif assay and future prospects for a point-of-care test perspectives on advances in tuberculosis diagnostics, drugs, and vaccines the impact of the roll-out of rapid molecular diagnostic testing for tuberculosis on empirical treatment in cape town drug-resistant tuberculosis: time for visionary political leadership implementation of xpert mtb/rif for routine point-of-care diagnosis of tuberculosis at the primary care level evaluation of diagnostic tests: dengue innovative and new approaches to laboratory diagnosis of zika and dengue: a meeting report visceral leishmaniasis: what are the needs for diagnosis, treatment and control? metaanalysis of the performance of a combined treponemal and nontreponemal rapid diagnostic test for syphilis and yaws systematic reviews of point-of-care tests for the diagnosis of urogenital chlamydia trachomatis infections performance and operational characteristics of point-of-care tests for the diagnosis of urogenital gonococcal infections the rapid test paradox: when fewer cases detected lead to more cases treated: a decision analysis of tests for chlamydia trachomatis sex point-of-care p24 infant testing for hiv may increase patient identification despite low sensitivity emerging technologies in point-of-care molecular diagnostics for resource-limited settings a simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings expert review of molecular diagnostics portable devices and mobile instruments for infectious diseases point-of-care testing a smartphone dongle for diagnosis of infectious diseases at the point of care point-of-care diagnostics in low resource settings: present status and future role of microfluidics the present and future role of microfluidics in biomedical research novel developments in mobile sensing based on the integration of microfluidic devices and smartphones integrating biochemiluminescence detection on smartphones: mobile chemistry platform for point-of-need analysis lateral flow technology for field-based applications-basics and advanced developments using electronic readers to monitor progress toward elimination of mother-to-child transmission of hiv and syphilis: an opinion piece leaving no one behind: a neglected tropical disease indicator and tracers for the sustainable development goals the trade-off between accuracy and accessibility of syphilis screening assays ensuring quality and access for malaria diagnosis: how can it be achieved? assuring the quality of diagnostic testing: the future is now external quality assurance for hiv point-ofcare testing in africa: a collaborative country-partner approach to strengthen diagnostic services implementation science: the laboratory as a command centre cost-effectiveness of xpert mtb/rif for tuberculosis diagnosis in south africa: a real-world cost analysis and economic evaluation xpert mtb/rif versus sputum microscopy as the initial diagnostic test for tuberculosis: a cluster-randomised trial embedded in south african roll-out of xpert mtb/rif point-of-care tests to strengthen health systems and save newborn lives: the case of syphilis rapid syphilis tests as catalysts for health systems strengthening: a case study from peru existing and emerging technologies for point-ofcare testing tackling drug-resistant infections globally: final report and recommendations expert review of molecular diagnostics: the impact of digital technologies on point-of-care diagnostics in resource-limited settings mobile health to improve tuberculosis care and control: a call worth making bringing mhealth connected infectious disease diagnostics to the field integrating electronics and microfluidics on paper from smartphones to diagnostics : low cost electronics for programmable digital microfluidics and sensing a flexible future for paper-based electronics biosensors: sense and sensibility rapid diagnostic tests for neurosyphilis compounding diagnostic delays: a qualitative study of point-of-care testing in south africa data connectivity: a critical tool for external quality assessment functional screen printed radio frequency identification tags on flexible substrates, facilitating low-cost and integrated point-of-care diagnostics a low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami sensing approaches on paper-based devices: a review diagnostics for the developing world: microfluidic paper-based analytical devices recent developments in paper-based microfluidic devices paper-based synthetic gene networks printed microfluidic channels the authors declare no competing interests. reprints and permissions information is available at www.nature.com/reprints.correspondence should be addressed to x.-s.c. or r.w.p.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-294366-swwz4kzd authors: bramwell, vincent w.; perrie, yvonne title: the rational design of vaccines date: 2005-11-15 journal: drug discov today doi: 10.1016/s1359-6446(05)03600-7 sha: doc_id: 294366 cord_uid: swwz4kzd this review provides an insight into the various opportunities for vaccine intervention, analysis of strategies for vaccine development, vaccine ability to modulate immune responses and resultant rational vaccine design. in addition, wider aspects are considered, such as biotechnological advances, advances in immunological understanding and host–pathogen interactions. the key question addressed here is, with all our research and understanding, have we reached a new echelon in vaccine development, that of rational design? the goal of any vaccination is the induction of an appropriate and effective immune response, but precisely what constitutes an effective immune response for many diseases is still unclear. specific levels of antibody are considered protective in vaccine efficacy for vaccines against hepatitis, diphtheria and tetanus; for example, anti-hepatitis surface antigen antibody levels >10 miu/ml, anti-diphtheria antibody levels >100 miu/ml and anti-tetanus antibody levels >100 miu/ml are quoted as protective levels of antibody in humans [1, 2] . however, for many other diseases, correlations of efficacy are less obvious or yet to be agreed. by definition of perceived need, we are most acutely aware of the requirement of effective vaccines against infectious agents, pathogens ancient, re-emergent and new, yet the opportunities for manipulation of immune responses offer potential in the prevention and treatment of a far larger diversity of diseases. from these, immunomodulation in cancer, allergy and autoimmune disease are also considered here. recent developments have brought vaccines against infectious diseases to the forefront of the scientific community. the 'wake up call' that came from sars (severe acute respiratory syndrome) has undoubtedly fuelled present fears of a flu pandemic. the 1918 spanish flu pandemic provides an extreme historical precedent, causing an estimated 50 million deaths after infecting one billion people worldwide [3] . as to the impact of such an outbreak, today there is serious concern over the capacity of the existing infrastructure to limit fatalities by providing suitable healthcare [4] . although development of a suitable vaccine can take a minimum of six months, in an organized and prepared scenario this could provide the major weapon that will ultimately contain such a catastrophe. the latest polio outbreaks look set to delay the long hope for eradication of this disease [5] . in the uk, the substitution of the live oral polio vaccine with an inactive vaccine should provide a step towards safe cessation of polio vaccination when the time is right. the live vaccine has the capacity to generate infectious polio virus through mutation, and altered immunological properties of mutants present concern about virus spread in immunized as well as non-immunized populations [6, 7] . taken as a whole, vaccine research and development encompasses a wide diversity of potential applications, developmental strategies and opportunities. the ongoing elucidation of immunological mechanisms of pathogen recognition and adjuvant action, as well as the knowledge of pathogen mediated mechanisms of immune evasion and gene expression, have transformed our understanding of immunology and disease in recent years. in this complex but increasingly focussed environment of vaccine development, are there opportunities for new solutions for vaccine design? and can new and refined approaches succeed where more conventional approaches appear to have failed? there are several reasons as to why the bacillus calmette-guérin (bcg) vaccine against tuberculosis (tb) is perceived as ineffective, including manipulation of immune responses (as for virulent tb [8, 9] ) and, interestingly, variation between propagated strains of bcg [10] . in the case of tb, recent literature has correlated the production of interleukin 4 (il-4) and il-4δ2 (a splice variant of il-4 and an il-4 antagonist) with the propensity of a latently infected host to succumb to active tuberculosis. the production of il-4δ2 is correlated with the control of the disease [11] . focus on the type of immune response needed to engender protection against tb seems to be orientated (convincingly) towards suppression of il-4 and the long standing dogma advocating the importance of th1 type cytokines (such as interferon gamma, ifn-γ) in tb resistance [12, 13] . in the case of hiv, only one hiv candidate vaccine has completed clinical trials phases i, ii and iii in more than 20 years of the epidemic. the phase iii trial was based on the use of recombinant envelope proteins, with the aim of evoking virus neutralizing antibodies. however, results from this were disappointing. currently available vaccines are mostly effective against viral diseases that are normally cleared during natural infection [influenza, smallpox, polio and all three viruses in the measles, mumps and rubella (mmr) immunisation]. hiv, in common with epstein-barr virus, cytomegalovirus, hepatitis (b and c) and herpes simplex virus, normally establishes chronic infection. opinion is divided as to what type of immune response is required in the control of hiv. neutralizing antibodies might be efficient in blocking virus particles but poorly effective against cellassociated virus, whereas some cytotoxic t lymphocytes (ctls) are effective against virally infected cells but not against free virus particles. latent infection facilitates immune evasion and the rate of mutation inherent in hiv also facilitates evasion (by escape from responding t cells [14] ). promising vaccine strategies will likely depend upon highly conserved epitopes and upon vaccine strategies that induce potent immune responses capable of driving cytotoxicity and producing broadly effective neutralizing antibodies [15] . allergic and autoimmune diseases represent undesired hypersensitive immune responses to normally harmless environmental antigens, in the case of allergy, or normally tolerated self-proteins, in the case of autoimmunity. the understanding of the pathogenesis of allergic and autoimmune diseases has led to several proposed associations and causes for these diseases. it has been shown that highaffinity t cells can out-compete lower-affinity t cells during responses to antigen in vivo [16] . competition between t cells provides the basis for the argument that maintaining a balanced peripheral immune system might also be a side effect of normal competition for shared resources within an intact immune system [17] , a mechanism proposed to be responsible for the increased occurrence of these diseases, where hypersensitivity is normally passively controlled by expansion of immune cells specific for prevalent infectious agents. autoimmunity can have strong associations with exposure to crossreactive proteins, and genes encoding major histocompatibility complex (mhc) are strongly associated with some autoimmune diseases, but this is markedly less so for allergy. allergy and autoimmunity are thought to differ by virtue of their regulation through central and peripheral tolerance. genetic predisposition and environmental exposure are believed to play key roles in the development of asthma and atopy [18] , with the significant increase in the incidence of these disorders thought to provide evidence for the involvement of various environmental factors, especially in developed countries [19] . the rationale for immunological therapeutic intervention is strongly supported by the present lack of curative treatments for autoimmune and allergic disorders, a growing unmet need, where the largely palliative treatment is not without problems [20] . evidence that pathology associated with allergy and autoimmunity is reversible is provided by transient and long-term resolution of disease as well as by the success of specific allergen immunotherapy, where subcutaneous or sublingual administration of the sensitizing protein(s) modifies immunity and reduces allergen sensitivity [20] . serological identification of antigens by recombinant expression cloning has led to the identification of a multitude of new tumour antigens [21] . it is thought that most or all paraneoplastic neurological disorders (neurological disorders remote from the site of a malignant neoplasm or its metastases) are immune mediated [22] and this has been cited as evidence for the involvement of specific immune responses in cancer suppression [23] . indeed, failure to find the relevant antigen in the cancer of a patient should prompt a search for a second cancer www.drugdiscoverytoday.com [24] . immunotherapy has recently achieved much interest as a possible addition to chemotherapeutic treatment [25] and this could open up a new and innovative avenue for clinical trials. effective chemotherapy against schistosoma mansoni with praziquantel is dependent on the presence of antibodies recognizing schistosome glycoprotein epitopes [26] . another interesting approach is the therapeutic vaccination against cancer by targeting anti-apoptotic molecules [27] . infectious agents are implicated as causes of cancer and contribute to a variety of malignancies worldwide. some of the major players in this role are shown in table 1 and they account for several of the most common malignancies and up to 20% of malignancies around the globe [28] . as such, these agents offer increased potential for prevention of cancer by prophylactic vaccination. administration of antisera as a treatment against snake venom dates back to the 1890s and the work of albert calmette. although this article largely focuses on active immunisation and the generation of host antibodies by the host immune system itself, the strategy of passive immunisation is worth consideration, as epitopes identified using modern molecular biology techniques might be targeted by the use of passively administered monoclonal antibodies. examples of immunotherapy using monoclonal antibodies are campath ® (alemtuzumab), manufactured by genzyme for the treatment of b-cell chronic lymphocytic leukaemia, and rituxan ® (rituximab), a chimeric murine-human monoclonal antibody that targets the cd20 antigen found on the surface of normal and malignant b lymphocytes and used in the treatment of non-hodgkin's lymphoma. this strategy is not restricted to cancers; for example remicade ® , a chimeric monoclonal antibody specific for tumour necrosis factor α (tnf-α, is used in the treatment of autoimmune disease. the implementation of rational design was first evident when pasteur reproducibly manufactured attenuated cultures of chicken cholera vaccines, thereby routinely preventing cholera in vaccinated chickens. extrapolation of this strategy for anthrax vaccines in livestock in the 1880s was a success with significant economic benefits. despite the elucidation of an attenuated vaccination strategy against rabies and other inactivated whole-organism vaccines towards the end of the nineteenth century, it wasn't until the cell-culture revolution in 1950-1980 that attenuated viral vaccines really took off, together with the isolation and use of extracts and subunits of infectious agents. pasteur's approaches of attenuation and inactivation still today provide the two poles of vaccine technology [29] . a chronology of important developments and achievements in vaccine research and implementation is shown in figure 1 . recent developments in vaccine technology and application include combination vaccines, new adjuvants and delivery systems, reverse vaccinology and vaccines against noninfectious diseases. the history of vaccine development is very much related to technological advances [30] . subunit vaccines can be produced in bulk, safely and reproducibly, using recombinant dna technology. much research has already been accomplished in the development of suitable carrier systems able to engender enhanced immune responses to entrapped peptide and protein epitopes. the identification of potentially useful antigens is greatly facilitated by improved molecular biology techniques, such as microarray technology (discussed further below), but the elucidation of their full potential might rely on the development of effective delivery systems and adjuvants. many adjuvants are based on microbial components but others, traditionally aluminium salts and more recently emulsions and surfactant based formulations, exploit different mechanisms of action. particulate delivery can be mediated by polymer [often poli(lactideco-glycolide)] microparticles, immunostimulatory complexes, liposomes and virosomes [31] . possible advantages of particulate delivery are that antigen and coadjuvant can be delivered to the same cell and particulates have excellent potential for targeting cells of the immune system [32] . the extensive diversity of adjuvants and delivery systems is more comprehensively reviewed elsewhere [31] [32] [33] [34] and this review will, where appropriate, focus on the application and other aspects of this technology. the 300 million doses of influenza vaccine needed annually worldwide require more than 350 million chicken eggs and six or more months. the development of cell-culture technology that could replace the egg-based manufacturing process might facilitate faster production of much-higher antigen yields [4] . live vaccines generally possess a natural adjuvant capability that is built on the evolved ability of our immune system to recognize many facets of potentially dangerous microbes and they have contributed immeasurably to the control of disease. although some live vaccines might have undesirable characteristics, such as the mutation of polio virus and persistence of viruses such as varicella zoster virus, their use has been, and indeed remains, able to significantly reduce the impact of associated diseases [35, 36] . viruses and bacteria as carriers of heterologous antigens and genes have received considerable attention over previous decades, and generation of auxotrophic mutants, as well as the use of other replication incompetent vectors and of reassortant viruses generated by the addition of rna segments to viruses with segmented genomes, such as influenza and rotavirus, indicates the potential inherent in these efficient types of vaccine. in an interesting utilization of viral replication, a self-replicating rna vector encoding a model antigen (β-gal), which was shown to protect mice against subsequent tumour challenge with colon carcinoma cells engineered to express β-gal, was also shown to effectively induce apoptosis in transduced cells [37] . the observed apoptosis also facilitated enhanced uptake by dendritic cells and was likely mediated by the presence of double-stranded rna in replication of the virally derived genome. double-stranded rna is recognized by toll-like receptor (tlr) binding. different tlrs recognize different surface and intracellular components of microorganisms. the interaction between a tlr and a microbial component triggers the activation of the innate immune system and www.drugdiscoverytoday.com the initiation of acquired immunity [38] . the elucidation of these pathways sheds light on mechanisms of adjuvant action and provides a basis for the inclusion of such potent agents and their analogues in vaccine design. it is important to consider how we classify or view rational design in the context of vaccines. a picture springs to mind of the rapid analysis of pathogens, the identification and allocation of immunological interactions for specific genes, as well as the generation of a superimposed choreography of the infection or disease process, resulting in the identification of credible candidate target antigens. following this, appropriate formulation with adjuvants and delivery systems might elicit immune responses of the type and magnitude predictive of providing protection or therapeutic action. the truth is that we are still very far from this idyll. recent articles have intimated the minor contribution of our knowledge of immunology to the development of vaccines [39, 29] . however, the development of improved and large-scale cellular immunity techniques for the analysis of immune responses, such as elispot for cytokine induction, tetramer staining for peptide specific ctls, along with analysis of the immunological basis for the efficacy of successful vaccines, facilitates the role of immunology in predicting the appropriate context for antigen delivery. extensive characterisation of adjuvant action and the potential to target desired immune responses through the exploitation of this knowledge is key for rational vaccine design [29, 31, 32, 39, 40] . the interaction between different elements involved in rational vaccine design is outlined in figure 2 . the use of microarray technology can allow identification of virulence genes and vaccine targets [41] and is one of the most powerful tools for the study of the transcriptome, the complete set of transcripts of an organism. indeed, in conjunction with proteomics and comparative genome analysis, interpretation of whole-genome sequences through bioinformatics (or genomic mining) can be used to assign putative gene functions to each open reading frame on the basis of homology to known proteins [42] . systematic identification of potential antigens of a pathogen using this information, without the need for cultivation of the pathogen, is termed 'reverse vaccinology' [43] and represents a significant departure towards the idyll of rational vaccine design described above, at least in terms of dissection of potential pathogen-related antigens. knowledge of pathogen interactions during infection can provide an invaluable insight into potentially successful targets for vaccines. for example, knowledge of gene expression in mycobacterium tuberculosis enables us to see why vaccines based on early secreted proteins, such as ag85 and esat-6, can generate effective pre-exposure vaccines and helps us to predict that an effective post-exposure vaccine will utilize dormancy induced proteins, such as α-crystallin, heat shock protein 'hspx' [44] . in the case of hiv, the implication of lipid rafts in viral entry and budding processes might have provided a rationale for the evaluation of peptides, based on highly conserved caveolin-1 binding domains of hiv-1 glycoprotein gp41 in candidate vaccine formulations [45] . encouragingly, this has resulted in peptides capable of the elicitation of neutralizing antibody responses able to inhibit different clades of hiv-1. it is thought that the poor ability of some specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes [46] . important factors in the rational design of vaccines and the cyclical generation of knowledge. the level of involvement of any of these factors depends on the aetiological agent of disease. for example, the level of protection required in a population (herd immunity) will be different and this could allow theoretical flexibility in vaccine efficacy.the application of molecular biology techniques can be crucial in the identification of new candidate antigens and subsequent determination of vaccine efficacy using adjuvants can feed knowledge back to correlates of protection in terms of immunological markers.this knowledge can then be used in choice of appropriate adjuvants and formulation.the key implication projected by this schematic is that for the greatest challenges in vaccine development the cyclical generation of knowledge provides a strong role for rational design. * can be protective or therapeutic. ** including reverse vaccinology and associated technologies. correlates of protection* immunological markers defining protective* antigens antibodies against the caveolin-1 binding domains are rare in many hiv infected individuals, possibly because of the presence of hypervariable immunodominant epitopes or the location and lack of exposure of conserved regions. the proposed opportunities for vaccines against hiv and tb are summarized in figure 3 ; the rationale for elicitation of successfully neutralizing antibodies against hiv is centred upon increasing the immunogenicity of poorly immunogenic peptides, modification and/or removal of hypervariable regions and mimicking viral conformational changes. one of the hottest areas of interest regarding new vaccine development surrounds the phase iii efficacy trials and prospective licensure of two vaccines against human papillomavirus (hpv) [47] . the vaccines, one from merck and one from gsk, are effectively vaccines against cervical cancer; the (now) unequivocal link between this common virus, which rarely causes serious disease in itself, and cervical cancer found credence as long ago as the 1970s. the production of both vaccines (the merck vaccine produced in yeast administered with an aluminium adjuvant and the gsk vaccine in baculovirus with the adjuvant as04; aluminium and bacterial lipid already approved for use in europe) has its basis in work that elucidated the production and self-assembly of virus-like particles of the hpv outer l1 and l2 coat proteins [48, 49] in various cell types. vaccines against most neisseria meningitidis serogroups have been developed using traditional approaches. however, group b meningococcus has represented a particular challenge because of the sequence variation of surface proteins and crossreactivity of the capsular polysaccharide proposed opportunities for vaccination against tb and hiv. in both cases, the utilization of adjuvants capable of driving the required type of immune response can be used.this can be based on our current understanding of tlr interaction, and studies that manipulate this understanding might also serve to elucidate complex interactions during infection and disease. (a) according to recent models, antigen specific cd4+ t cells, cd8+ t cells, γδ t cells and cd1 restricted t cells, all participate in protection against m. tuberculosis infection in response to antigen presentation by macrophages (mφ) and dendritic cells (dc), probably also involving crosspriming, where mycobacterial antigens are transferred from infected mφ to dc. it is unlikely that the induction of ifn-γ or the production of inducible nitric oxide synthase alone is enough to control tb infection, and evidence indicates that there might be another role for cd4+ t cells.the development of pre-and post-exposure vaccines will likely require antigens expressed by m. tuberculosis under pre-and post-exposure conditions, respectively. interestingly, nonpolymorphic cd1 restricted t cells are thought to have evolved as a result of prolonged interaction with mycobacteria, indicating the extent of the impact of mycobacteria on its mammalian host. (b) for vaccines against hiv, it seems increasingly likely that strategies will depend upon highly conserved epitopes and on vaccine strategies that induce potent immune responses capable of driving cytotoxicity, as well as broadly reactive, highly effective neutralizing antibodies.target antigens might very probably be different for each of these strategies. figure adapted, with permission, from ref. [32] . adsorption of antigens; maintaining conformational integrity with host polysialic acid [50] . in this case, reverse vaccinology was convincingly applied to overcome these problems by alternative means [51] . screening of dna fragments, discarding likely cytoplasmic protein sequences and known neisseria antigens, extensive cloning and expression of identified candidate genes allowed selection of antigens that were found not only to offer significant potential for protection against group b meningococcus but also to facilitate crossprotection against heterologous strains. also worth mentioning is the technique of molecular breeding or gene shuffling, where reassembled chimeric genes are created from a selection of homologous gene sequences. the related sequences are denatured, annealed and subsequently extended in what is in effect a self-priming polymerase chain reaction (pcr) that takes advantage of crossovers, deletions, insertions, inversions and point mutations, as occurs in natural evolution (hence the term 'molecular breeding'). proteins and genes with enhanced activity are then selected for inclusion in further rounds of molecular breeding. this has a proven application for the generation of enzymes with markedly enhanced activity [52] and the enhancement of the functions of a diverse range of proteins, such as green fluorescent protein [53] and ifn-α [54] . this technique is thought to be superior to other methods that employ random mutagenesis, such as error-prone pcr (hence the phrase 'the rational basis for irrational design' [55] ), but the screening of candidate antigen genes is necessarily expensive in terms of resources and time if it is compared for example to the screening of an enzyme. their potential has been noted with relevance to allergy vaccines [56] , as pre-existing immunity is the therapeutic target and screening of allergen variants might be somewhat more practicable. bringing together developmental strategies with the knowledge gleaned from host pathogen interactions represents a highly evolved design strategy. although each individual molecular biology technique appears to have something to contribute to vaccine development, it is the holistic application of a combined approach that is closest to a premeditated and planned rational vaccine design. pathogens such as streptococcus pneumoniae, porphyromonas gingivalis, staphylococcus aureus, chlamydia pneumoniae and bacillus anthracis have all been investigated using bioinformatics techniques in the context of reverse vaccinology [43] , with continued interesting results. in terms of antigen identification, the national institute of allergy and infectious diseases (niaid) recently announced initiation of the large-scale antibody and t cell epitope discovery program; utilizing complementary methods for epitope discovery, it is designed to identify immune epitopes from selected infectious agents and will make this information freely available to scientists worldwide through the immune epitope database and analysis resource (iedb), currently under development [57] . recent discoveries have resulted in a wealth of options, when considering what we can do in terms of vaccine design and production. mechanisms of adjuvant action have been elucidated, pattern recognition receptors including toll-like receptors, nod1, nod2, scavenger receptors, mannose and other receptors are becoming increasingly well defined [38] and intracellular events related to these, such as activation of transcription factors and expression of inflammatory cytokines, shed further light on how these ligands and adjuvants that bind to pattern recognition receptors mediate their immunological actions. the ability of infectious agents to induce rapid, innate immune responses to molecular patterns provides the basis for adjuvant immunotherapy of cancers. studies, such as the recent work examining the adjuvant activity of bcg cell-wall skeleton, peptidoglycan and lipopolysaccharide by the induction of genes in dendritic cells [58] , will very possibly help to achieve definition of efficient effector output with minimal toxicity. the easier and safer production of vaccines and vaccine components facilitated by modern biological techniques underpins a rational approach that is an integral part of the rational design of vaccines. the extensive knowledge of immunological mechanisms and host pathogen interactions is able to contribute to the design of effective vaccines in addition to the elucidation of the mechanisms of action of many candidate vaccine agents. in fact, rational design of vaccines represents a driving force born in the first revelations that components from or relating to a microbial pathogen can be used to protect against that disease and present throughout development of vaccines in the modern era, honing our understanding and preparing the way for the next steps in the battle to combat today's significant pathogens and diseases, such as cancer, hiv, tuberculosis and malaria. in this era, we begin to explore the very limits of immunotherapeutic and prophylactic intervention and the tools that have been developed to elucidate gene expression and function might have at last begun to find their application in our most significant of challenges. anamnestic response to administration of purified non-adsorbed hepatitis b surface antigen in healthy responders to hepatitis b vaccine with longterm non-protective antibody titres the safety, reactogenicity and immunogenicity of a 7-valent pneumococcal conjugate vaccine (7vpnc) concurrently administered with a combination dtap-ipv-hib vaccine vaccines in the public eye preparing for the next pandemic who's attempts to eradicate polio are thwarted in africa and asia spread of vaccinederived poliovirus from a paralytic case in an immunodeficient child: an insight into the natural evolution of oral polio vaccine long-term excretion of vaccine-derived poliovirus by a healthy child bacillus calmette-guerin shares with virulent mycobacterium tuberculosis the capacity to subvert monocyte differentiation into dendritic cell: implication for its efficacy as a vaccine preventing tuberculosis autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages bcg-different strains, different vaccines? healthy individuals that control a latent infection with mycobacterium tuberculosis express high levels of th1 cytokines and the il-4 antagonist il-4δ2 disseminated tuberculosis in interferon gamma genedisrupted mice an essential role for interferon gamma in resistance to mycobacterium tuberculosis infection positive selection of hiv-1 cytotoxic t lymphocyte escape variants during primary infection requirement of diverse t-helper responses elicited by hiv vaccines: induction of highly targeted humoral and ctl responses t cells down-modulate peptide-mhc complexes on apcs in vivo t cell regulation as a side effect of homeostasis and competition recent findings on genes associated with inflammatory disease peptidebased therapeutic vaccines for allergic and autoimmune diseases analysis of the b-cell repertoire against antigens expressed by human neoplasms paraneoplastic syndromes involving the nervous system dna vaccines to attack cancer anti-hu-associated paraneoplastic encephalomyelitis: analysis of 200 patients immunotherapy and chemotherapy-a practical partnership role of host antibody in the chemotherapeutic action of praziquantel against schistosoma mansoni: identification of target antigens regulators of apoptosis: suitable targets for immune therapy of cancer infectious agents and cancer: criteria for a causal relation vaccines: past, present and future six revolutions in vaccinology targeting the innate immune response with improved vaccine adjuvants particulate delivery systems for vaccines particulate delivery systems for biodefense subunit vaccines meeting on novel adjuvants currently in/close to human clinical testing: world health organization-organisation mondiale de la sante fondation merieux eradication and cessation of programmes: vaccination and public health care a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults cancer therapy using a self-replicating rna vaccine toll-like receptor signalling can successful vaccines teach us how to induce efficient protective immune responses? current status of dna vaccines and their route of administration dna content analysis on microarrays biotechnology and vaccines: application of functional genomics to neisseria meningitidis and other bacterial pathogens reverse vaccinology annulling a dangerous liaison: vaccination strategies against aids and tuberculosis the caveolin-1 binding domain of hiv-1 glycoprotein gp41 is an efficient b cell epitope vaccine candidate against virus infection access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1 public health. high hopes and dilemmas for a cervical cancer vaccine expression of human papillomavirus type 11 l1 protein in insect cells: in vivo and in vitro assembly of viruslike particles expression of vaccinia recombinant hpv 16 l1 and l2 orf proteins in epithelial cells is sufficient for assembly of hpv virion-like particles development of vaccines against meningococcal disease identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing rapid evolution of a protein in vitro by dna shuffling improved green fluorescent protein by molecular evolution using dna shuffling evolution of a cytokine using dna family shuffling directed evolution: the 'rational' basis for 'irrational' design molecular breeding of allergy vaccines and antiallergic cytokines a roadmap for the immunomics of category a-c pathogens gene-inducing program of human dendritic cells in response to bcg cellwall skeleton (cws), which reflects adjuvancy required for tumor immunotherapy the authors thank graham smith at aston university, birmingham b4 7et, for his rendering of figure 3 . key: cord-272925-xag1yaie authors: carter, gemma c.; bernstone, laura; sangani, dhaval; bee, jessica wynter; harder, thomas; james, william title: hiv entry in macrophages is dependent on intact lipid rafts date: 2009-03-30 journal: virology doi: 10.1016/j.virol.2008.12.031 sha: doc_id: 272925 cord_uid: xag1yaie macrophages are an important natural target cell for hiv-1, but previous studies of virus entry into these cells are limited, and the involvement of membrane cholesterol and lipid rafts is unknown. cholesterol disruption of macrophage membranes using four pharmacological agents acting by different mechanisms: methyl-β cyclodextrin, nystatin, filipin complex and lovastatin, all significantly inhibited productive hiv entry and reverse transcription. the inhibitory effects of these drugs resulted in decreased virus release from infected cells, and could be substantially reversed by the addition of water-soluble cholesterol. the virus bound equally to cholesterol-disrupted cells even though hiv receptor expression levels were significantly reduced. macrophage cd4 and ccr5 were found to partition with the detergent-resistant membranes with a typical raft-associating protein flotillin-1. hiv particles were observed co-localising with a marker of lipid rafts (ctb-fitc) early post infection. these data suggest that macrophage membrane cholesterol is essential for hiv entry, and implicate lipid raft involvement. virus entry into target cells is a complex process involving many cellular and viral proteins with the primary goal of overcoming the barrier of the cellular membrane. some viruses achieve this by acquiring a lipid envelope during exit from the host cell; these enveloped viruses enter cells by fusion of their envelope with the cellular membrane at either the cell surface or within an endosome. viruses without a lipid envelope have acquired mechanisms to penetrate the membrane barrier, entering cells mainly via an endocytic pathway using either clathrin-coated vesicles or caveolae, or by the formation of a pore at the cell surface as has been proposed for poliovirus (hogle, 2002; marsh and helenius, 2006; smith and helenius, 2004) . there is a growing amount of evidence that cholesterol, an important structural component that modulates the fluidity of biological membranes, is essential for the uptake of many viruses. successful virus entry may require cholesterol in the host cell membrane or in the viral envelope. many enveloped viruses, such as vaccinia virus (chung et al., 2005) , herpes simplex virus (bender et al., 2003) and severe acute respiratory syndrome-coronavirus (li et al., 2007) require cholesterol in the target cell membrane. the same is true for many non-enveloped viruses; sv40 (anderson et al., 1996) , foot-and-mouse disease virus and echovirus (marjomaki et al., 2002; pietiainen et al., 2004) require cholesterol to enter cells by the lipid raft-dependent caveolae endocytosis pathway. in the case of influenza virus, the presence of cholesterol in its viral envelope is critical, but it is not essential in the target cell (sun and whittaker, 2003) , whereas the converse has been found for murine leukaemia virus (lu and silver, 2000) and duck hepatitis b virus (funk et al., 2008) . in contrast, various strains of dengue virus and yellow fever virus enter and infect cells independently of cholesterol (umashankar et al., 2008) . cholesterol is concentrated within the fluid mosaic bilayer of the plasma membrane, along with sphingolipids and glycerophospholipids, in specialised dynamic microdomains known as lipid rafts. these lipid assemblies are tightly packaged and ordered, allowing them to float within the rest of the fluid sea of disordered lipid bilayer (simons and ikonen, 1997) . the presence of cholesterol in the lipid rafts confers some resistance to detergents at low temperatures, allowing their separation from detergent-sensitive membranes by ultracentrifugation (wilflingseder and stoiber, 2007) . due to this property, they are often referred to as detergent-resistant membranes (drms). membrane rafts act as mobile platforms within the plasma membrane, and are involved in many important diverse cellular processes. many proteins associate with lipid rafts, and these include gpi-anchored proteins, gtpases and kinases. mounting evidence for the cholesterol dependency of productive virus entry suggests that many viruses may hijack these dynamic lipid raft platforms to utilise them as an entry portal to the cell. cholesterol is already known to be important at multiple stages of the hiv lifecycle. the virus assembles and buds out of lipid raft domains, and in doing so acquires a cholesterol and sphingomyelinrich envelope (aloia et al., 1988 (aloia et al., , 1993 campbell et al., 2004 ) that is resistant to detergents but has a strikingly different composition to host cell membranes chan et al., 2008) . the hiv accessory protein, nef, functions on many levels to manipulate cellular cholesterol, including binding to cholesterol to enhance its trafficking virology 386 (2009) to rafts (zheng et al., 2003) , increasing cellular cholesterol biosynthesis (van 't wout et al., 2005) and inhibiting cholesterol efflux by redistributing a major component of the cholesterol efflux pathway castrillo et al., 2003) . although in contrast, other studies have found that nef specifically enhances the incorporation of sphingomyelin but not cholesterol into budding viral particles (brugger et al., 2007) . the importance of hiv envelope lipid composition is highlighted by the finding that depletion of viral envelope cholesterol causes a dramatic reduction in viral infectivity campbell et al., 2004 campbell et al., , 2002 guyader et al., 2002; liao et al., 2003) . the early events in hiv entry, in which the viral envelope protein, gp120-gp 41, engages with the primary receptor cd4 on target cells, are well characterised. this interaction permits conformational changes within gp120 that allow additional binding to a co-receptor, usually cxcr4 for t-lymphocyte tropic strains, or ccr5 for macrophage tropic isolates. co-receptor binding is the trigger for virus entry, during which the fusion peptide gp41, inserts into the cellular membrane to drive the fusion event. the lipid composition of the host cell is important for hiv entry. cholesterol depletion of target cells using pharmacological agents prevents hiv entry, and render cells resistant to infection (kozak et al., 2002; liao et al., 2001; nguyen and taub, 2002b; popik et al., 2002) . the association of cd4 with lipid raft domains is well accepted (kozak et al., 2002; nguyen et al., 2005; percherancier et al., 2003; popik et al., 2002) , but whether this association is required to support productive virus entry is disputed. one study found that mutant cd4 targeted to non-raft membranes did not permit efficient hiv entry (del real et al., 2002) , whereas alternative cd4 mutants that also prevent cd4 association with lipid rafts were found to support hiv entry (percherancier et al., 2003; popik and alce, 2004) . the membrane localisation of the co-receptor molecules is somewhat controversial. ccr5 has been shown to associate with lipid rafts microdomains in cell lines by its presence in drm fractions and its co-localisation with the raft associated lipid gm1 (manes et al., 1999; popik et al., 2002) . in contrast, studies in primary t cells identified ccr5 in the detergent-soluble membrane (dsm) fractions representing the non-raft membranes (percherancier et al., 2003) . cxcr4 has been reported to be lipid raft-associated in 293 cells (manes et al., 2000) but non-raft-associated in t cell lines (kozak et al., 2002; popik et al., 2002) . hiv-1 gp120 binding to cd4 molecules in lipid rafts has been proposed to cause recruitment of the hiv coreceptor into the lipid rafts or its interface, thus bringing all molecules required for hiv entry together (kozak et al., 2002; nguyen et al., 2005; popik et al., 2002) . a different productive entry pathway for hiv into macrophages has been described, in which virus is taken up by an endocytic route involving macropinocytosis, a constitutive process whereby the cell takes up large quantities of the extracellular fluid (marechal et al., 2001) . numerous intracellular pathogens are taken up into macrophages by macropinocytosis, including brucella (watarai et al., 2002) and afipia felis (schneider et al., 2007) , and in these cases a requirement for lipid rafts has been established. cholesterol is already known to play an important role in hiv replication of macrophages, with specific studies showing that nef affects the normal function of atp-binding cassette transporter a1 to impair cholesterol efflux from infected macrophages (mujawar et al., 2006) . modulation of intracellular lipid metabolism in hiv-infected macrophages may facilitate virus budding from lipid raft associated membranes, but it is tempting to infer that cholesterol might also play an important role in the entry of hiv into macrophages. all of the studies regarding the involvement of cholesterol and lipid rafts in hiv entry have been performed using immortalised cell lines, t cell lines and primary cd4+ lymphocytes. given their critical position in hiv pathogenesis, and their highly specialized cellular architecture, we thought it necessary to investigate the requirement of cholesterol and role of lipid rafts in hiv-1 entry into macrophages. we have assessed the effect of depleting cholesterol in the macrophage plasma membrane, using four different pharmacological agents, on virus binding, entry and hiv receptor levels. here we show that membrane cholesterol is essential for productive hiv entry into macrophages. virus binding to cholesterol-depleted macrophages is not reduced but there are alterations to the surface expression of the receptor molecules. we observed virus co-localising with a marker of lipid rafts early after infection, and isolated macrophage cd4 and ccr5 in the membrane fractions representing raft microdomains. this, together with the sensitivity of virus entry to membrane cholesterol depletion, implies a role for macrophage lipid rafts in hiv-1 entry. to investigate the involvement of lipid rafts in hiv uptake into macrophages, we first sought to visualise virus entry and lipid rafts using fluorescent cholera toxin b subunit (ctb-fitc) the binding of which to gm1 clusters these membrane domains together into larger patches that can be observed microscopically. hiv nl4.3 pseudotyped with jrfl envelope was bound to macrophages on ice, and warmed to 37°c in the presence of ctb-fitc and analysed at various times post entry. a representative experiment taken at 20 min post entry is shown in figs. 1a-d, and shows virus co-localising with ctb-positive areas of the macrophage plasma membrane. the number of particles directly associated with gm1-positive membranes on the cell surface or in endocytic vesicles was quantified by taking 0.8 μm z-slices through the cell and scoring the percentage of the total particles colocalising with ctb-fitc. three consecutive z-slice focal planes are shown in panels a-c. these slices start towards the bottom of the cell (a) and proceed upwards, and show many cell-associated virus particles clearly co-localising with ctb-fitc. the distribution of all virus particles and ctb-fitc is shown in a projection of z-stack slices in panel d. analysis of multiple cells (n = 10) at 20 min post entry shows approximately 34% (±3.72 sem) of total particles are colocalising with ctb-fitc. at later times post entry (40 and 60 min), there is a reduction in association of virus with ctb-fitc perhaps due to productive entry of most virus particles into the cytoplasm or degradation of the virus preventing its detection (fig. 1e ). membrane cholesterol can be disrupted by a number of pharmacological agents; the manipulation of this key component of lipid microdomains is frequently used to implicate these assemblies in virus entry. the effects of methyl-β cyclodextrin (mβcd) are well characterised. this cholesterol-binding agent is not incorporated into the membrane but selectively extracts membrane cholesterol by binding it in a central non-polar cavity (ilangumaran and hoessli, 1998) . we incubated macrophages with various concentrations of mβcd in serum-free media, and measured the depletion of lipid rafts by detecting fluorescent ctb-fitc binding to the raft glycolipid gm1 by flow cytometry. ctb-fitc binding to drug-treated cells on ice was reduced in a dose-dependent manner compared to untreated cells confirming that mβcd effectively disrupts macrophage lipid rafts ( fig. 2a) . the effect of this drug on cell viability was measured using the mts cytotoxicity assay (fig. 2b ) and showed that 1 h treatment with mβcd had no toxic effect on the macrophages. quantification of the amount of free cholesterol and cholesteryl esters in 10 mm mβcdtreated macrophages using amplex red cholesterol quantification assay revealed a significant decrease (p = 0.01 by paired t-test) in all 6 donors tested with an average of 28.7% (±20.4%) reduction compared to untreated control cells. the potential anti-viral properties of mβcd on hiv entry into macrophages was tested by measuring virus reverse transcription in drug-treated macrophages. the hiv envelope acquires cholesterol during budding, and mβcd can deplete this virion cholesterol, resulting in reduced virus infectivity (guyader et al., 2002; liao et al., 2003) . therefore, to avoid this potentially confounding effect, cells were pre-treated with mβcd for 1 h, and washed extensively before the addition of virus, to ensure that any anti-viral effects observed were not due to decreased particle infectivity. mβcd treatment of macrophages was found to significantly reduce the generation of reverse transcription products following challenge with hiv bal, at both concentrations tested, in macrophages derived from six different donors (fig. 2c) . the inhibitory effect of mβcd could be partially reversed by the replenishment of cellular cholesterol with watersoluble cholesterol added for 1 h directly after mβcd treatment. cholesterol replenishment restored virus reverse transcription to 43.5% of the untreated control (fig. 2d ). the inhibitory effects of mβcd were also observed when p24 release from infected cells was measured by elisa (fig. 2e ). reduced p24 release kinetics were observed in the early days post infection for all donors treated with mβcd, but the effects were less pronounced due to the longer length of this experiment and the measurement of multiple rounds of infection. restoration of p24 release kinetics was observed upon cholesterol replenishment. therefore, depletion of macrophage membrane cholesterol with mβcd severely inhibits the early steps of productive hiv infection of macrophages. to further pinpoint the stage at which cholesterol-containing lipid rafts are required in hiv infection, we determined the effect of cholesterol depletion on virus binding to macrophages. equal amounts of hiv bal were added to untreated and mβcd-treated macrophages for 2 h on ice. unbound virions were removed by extensive washing before cells were lysed and the amount of cellassociated virus determined by p24 elisa (fig. 2f) . a slight decrease in virus adsorption to mβcd-treated macrophages compared to untreated control cells was observed. however, this decrease was not significant and would not account for the 8-fold decrease observed in reverse transcription in mβcd-treated cells. therefore, cholesterol and lipid rafts might play a minor role in virus binding but they are more likely to be required for entry or post-entry events. decreased entry of hiv might be attributed to alterations in receptor or co-receptor levels; therefore we sought to determine if mβcd treatment of macrophages alters the levels of cd4, ccr5 and cxcr4. detection of these cell surface molecules using antibody labelling followed by flow cytometry shows that control macrophages express cd4, ccr5 and cxcr4 at detectable levels ( fig. 3a) . treatment of macrophages with mβcd significantly decreases the cell surface expression of all receptors; cd4 by 35%, ccr5 by 100% and cxcr4 by 59%. both ccr5 and cxcr4 expression levels are restored upon cholesterol replenishment, implying that the decrease is due solely to cholesterol depletion. interestingly, replenishment with cholesterol does not restore the cd4 expression levels, but reduces them further to almost undetectable levels. this further reduction in cd4 expression may explain why cholesterol replenishment only partially restores hiv reverse transcription (see above). mβcd treatment of macrophages does not reduce the expression of all membrane proteins because the expression of cd71 (transferrin receptor), a known non-raft associated protein, was not reduced. therefore, mβcd selectively alters the exposure of proteins found in the cholesterolrich lipid raft membranes without effecting non-raft associated proteins. the combined decrease in cd4 and ccr5 receptor levels may account for the decrease in productive hiv entry. the reduction in hiv receptor levels in cholesterol-depleted macrophages and the co-localisation of virus with a marker of lipid rafts may imply that the receptors are localised in these microdomains. traditional methods to isolate lipid rafts rely on cholesterol's property of favouring the formation of membranes that are resistant to detergent at low temperature, and can be separated from soluble membranes by ultracentrifugation. we adapted a procedure for isolating macrophage drms by disrupting cells by a combination of nitrogen cavitation and homogenisation followed by fractionation using an optiprep density gradient. western blot analysis of equal volumes of each fraction shows the expected localisation of the control proteins: flotillin-1 is located in fractions 4 and 5 representing the drms, and transferrin receptor is in fractions 8 and 9 indicative of the dsms (fig. 3b) . the receptor cd4 is present in the detergent-resistant fractions 4 and 5, and has a similar distribution to that of flotillin-1, although it is also present at low levels in fractions 6 and 7. ccr5 is present in detergent-resistant fractions 4-5 only. therefore, the receptors required for macrophage-tropic hiv entry reside in the drm fractions of macrophage membranes, which have properties of lipid rafts and contain raft-associated protein flotillin-1. macrophages for 2 h on ice was measured by determining the amount of cell-associated virus using p24 elisa. data represent mean ± sd of results obtained with multiple independent experiments using several donors (4 donors for ctb, viability and hiv binding assays, 4-6 donors for qpcr and p24 data is representative of 3 donors). ⁎⁎ very significant difference, p b 0.01 and ⁎⁎⁎ extremely significant difference p b 0.001 (paired t-test). to exclude any drug-specific effects of mβcd, we used two other compounds that can bind to cholesterol and disrupt lipid rafts by directly inserting into membranes and sequestering cholesterol into complexes. both nystatin and filipin complex, at concentrations significantly inhibiting ctb-fitc binding (fig. 4a) and not compromising cell viability (fig. 4b) , inhibited hiv infection in a dosedependent manner. nystatin at concentrations of 66 and 50 μg/ml significantly reduced reverse transcription in infected cells by 8.4-and 4.1-fold respectively compared to those treated with the same concentration of dmso vehicle (fig. 4c ). filipin complex significantly inhibited the number of hiv reverse transcripts by 3.2-fold compared to the untreated control (fig. 4d) . similarly, these inhibitory effects can be observed as reduced p24 release kinetics at early time points post infection (fig. 4e) . both nystatin and filipin complex do not reduce virus binding to macrophages (fig. 4f) . this supports the findings obtained by mβcd treatment, implying that cholesterol and lipid rafts are likely to be required for entry and/or post-entry events and not binding. studies with mβcd indicate that the hiv receptors are sensitive to cholesterol depletion so we sought to determine if receptor levels would also be altered when cholesterol organisation is disrupted by another mechanism (fig. 4g) . nystatin significantly decreased cd4 and ccr5 expression by 59% and 91%, respectively. the expression level of cxcr4 was only slightly decreased. however, nystatin did significantly reduce levels of cd71 by 43% indicating that this drug might reduce the cell surface expression of many proteins including those outside lipid rafts. conversely, filipin complex marginally decreased cd4 and ccr5 but had no effect on the levels of cxcr4 or cd71. the anti-viral effects of nystatin might be explained by the lower levels of cd4 and ccr5 but it is likely that filipin complex might inhibit infection by a different mechanism than decreased receptor levels. the cholesterol-binding drugs, mβcd, nystatin and filipin, mechanically disrupt lipid rafts either by actively removing or by sequestering cholesterol. lipid rafts can also be disrupted using statins, such as lovastatin. these drugs inhibit the 3-hydroxy-3methylglutaryl coenzyme a (hmg-coa) reductase enzyme responsible for the production of mevalonate, a precursor in cholesterol biosynthesis. cells were treated with lovastatin for 4 days in the presence of serum-free medium supplemented with lipoproteindeficient serum after which cell viability was unaffected (fig. 5a) . the binding of ctb-fitc to lovastatin-treated cells was reduced by 33% (± 11.1%) compared to the untreated cells (fig. 5b) . lovastatin treatment of macrophages very significantly inhibited hiv reverse transcription in a dose-dependent manner by 16.6-and 5-fold with 12.5 μm and 5 μm lovastatin, respectively (fig. 5c) . a marked decrease the surface expression of hiv receptors after nystatin or filipin complex treatment was determined by staining macrophages with fluorescent antibodies to cd4, ccr5, cxcr4, cd71 or an appropriate isotype control. cell surface binding was measured by flow cytometry, and mean fluorescence intensity values were calculated by subtracting the isotype control fluorescence from the specific antibody fluorescence. data represent mean ± sd of results obtained with multiple independent experiments (3 donors for qpcr, ctb and p24 binding assays, 4 donors for receptor expression, viability and p24 data is representative of at least 2 donors). ⁎ significant difference p b 0.05, ⁎⁎ very significant difference, p b 0.01 (paired t-test). in p24 release kinetics compared to untreated control was also observed (fig. 5d) . the effects of lovastatin on hiv reverse transcription were partially reversed by the addition of mevalonate, the product of hmg-coa reductase, to the culture medium (fig. 5e ). the surface expression levels of all hiv receptors (but not cd71) were decreased after lovastatin treatment but not to significant levels (fig. 5f ). although these effects consistently implicate normal cholesterol synthesis for productive virus entry and receptor expression levels, we were unable to detect significant changes in cholesterol abundance per se in lovastatin-treated cells using the standard assay. here we have demonstrated the critical importance of cholesterol for entry of hiv-1 into macrophages. firstly, we showed that productive virus entry is significantly reduced when the macrophage target cells are treated with mβcd, a cholesterol-depleting drug. by incubating cells with this drug before the addition of virus, only cholesterol from the target cells would be removed allowing virus particle cholesterol and infectivity to remain intact. the addition of exogenous cholesterol to macrophage membranes directly after macrophages were pre-treated with 12.5 μm lovastatin and/or 800 μg/ml mevalonate (whose production is blocked by lovastatin) for 4 days. productive hiv entry was measured by qpcr detection of hiv bal reverse transcription after 30 h of infection. (f) the surface expression of hiv receptors after lovastatin treatment was determined by staining macrophages with fluorescent antibodies to cd4, ccr5, cxcr4, cd71 or an appropriate isotype control. data represent mean ± sd of results obtained with multiple independent experiments (2 donors for ctb and viability assays, 2-5 donors for qpcr, 5-6 donors for receptor expression and p24 data are representative for 2 donors). ⁎⁎⁎ extremely significant difference p b 0.001 (paired t-test). mβcd treatment substantially restored hiv infection, indicating that the decreased infectivity was at least in part due to the depletion of cholesterol from the cell membrane. secondly, we modified the properties of cholesterol-rich macrophage membranes using nystatin and filipin complex to sequester cholesterol into large aggregates. treatment of macrophages with these drugs significantly inhibited productive hiv entry in a concentration-dependent manner. thirdly, depletion of macrophage cholesterol using lovastatin, a compound that inhibits hmg-coa reductase, the rate limiting enzyme in cholesterol biosynthesis, significantly inhibited hiv productive entry after a prolonged 4 day treatment. incubation of macrophages with mevalonate, whose production is prevented by lovastatin, partially restored productive hiv entry into macrophages. therefore, modification of macrophage membrane cholesterol content using 4 different pharmacological inhibitors acting on cholesterol by different mechanisms all significantly inhibited hiv entry. cholesterol is a major component of lipid raft microdomains and manipulation of its localisation or presence within membranes is frequently used to implicate these lipid assemblies in virus entry. here we provide additional evidence for the involvement of macrophage lipid raft microdomains in hiv-1 entry by observing virus colocalisation with a marker (ctb-fitc) of these microdomains at an early time post-infection. furthermore, we have isolated the receptor molecules cd4 and ccr5, the receptors typically utilised by macrophage-tropic viruses, in drm fractions partitioning with a known-raft associated protein flotillin-1. these results confirm observations regarding the importance of target cell plasma membrane cholesterol and lipid raft microdomains in virus entry into t cell lines and primary cd4+ t cells (liao et al., 2001; manes et al., 2000; percherancier et al., 2003; popik et al., 2002) and extend them to macrophages, a critical natural target cell for hiv-1. for viruses to infect target cells, they have to firstly bind to attachment factors or receptor molecules on the cell surface. a decrease in productive virus entry may reflect a reduction in virus binding to cells. we confirmed which stage of infection was being affected by cholesterol depletion by investigating virus binding to the macrophage membranes. no significant decrease in the amount of bound virus after a 2 h incubation on ice was observed between drugtreated and control untreated macrophages. this indicates that cholesterol depletion of macrophages does not perturb the attachment of virus, perhaps to alternative molecules such as heparan sulphate glycosaminoglycans (saphire et al., 2001) , but must alter virus entry events after attachment. the surface expression levels of the hiv receptors were found to be altered to differing extents by the 4 pharmacological agents. mβcd treatment had the most pronounced effect on the receptors, with cd4 and cxcr4 being significantly reduced and ccr5 surface levels disappearing to almost undetectable levels. cholesterol replenishment restored the levels of ccr5 and cxcr4 to similar levels as the untreated macrophages. interestingly, cd4 expression was decreased further by cholesterol replenishment to almost undetectable levels. this complete knockdown in cd4 surface expression may explain why only a partial restoration of hiv reverse transcription was observed upon cholesterol replenishment of mβcd treatment macrophages. the addition of cholesterol may reduce cd4 expression to a level that is not favourable for hiv infection. the mechanism by which cholesterol further decreases cd4 surface expression is unknown but as these cholesterol-treated macrophages have a more granular appearance by flow cytometry, we can speculate that these macrophages actively take up cholesterol via an endocytic mechanism that may simultaneously internalise cd4. it is not uncommon for cholesterol replenishment to give rise to only partial restoration of infection; it has been reported for other viruses with the suggested explanation being that only one form of cholesterol may have been restored (tang et al., 2008) . the reduction in surface expression of hiv receptors following depletion of membrane cholesterol contrasts with findings using different cells. treatment with similar concentrations of mβcd caused no reduction in plasma membrane expression of cd4 on primary tcells (percherancier et al., 2003) , pbls (viard et al., 2002) or pm1 t cell line . the reduction of cd4 surface expression upon mβcd and cholesterol treatment, to the best of our knowledge, seems to be unique to macrophages. this observation may result from the differences in cd4 expression and internalisation on macrophages compared to t lymphocytes. in macrophages, cd4 expression levels are 10 to 20-fold lower than they are in cd4+ t cells (collman et al., 1990; kazazi et al., 1989; lee et al., 1999) , and cd4 internalisation rates are enhanced, likely due to the absence of lck (pelchen-matthews et al., 1998) . reduction in cxcr4 and ccr5 surface expression has been reported after treatment with a cyclodextrin derivative bcd in pm1 and primary t cells (liao et al., 2001) . more importantly, cholesterol has been shown to be essential for the conformational integrity of ccr5 and cxcr4, and in these studies bcd treatment prevented the cell surface binding of antibodies specific for distinct ccr5 epitopes taub, 2002a, 2002b) . molecular simulations, and work with model membranes of defined composition show that the integrity of lipid raft microdomains is very sensitive to minor changes in overall cholesterol concentrations (risselada and marrink, 2008) . most likely, cholesterol depletion in macrophages disrupts lipid rafts, thereby redistributing raft-associated proteins such as ccr5 to other membrane domains. it is possible that, in this new context, their conformation is altered to one that is no longer recognised by antibodies, and unable to sustain hiv entry. nystatin and filipin complex modify cholesterol organisation within cells, but do not affect overall cholesterol concentration, and have a differing effect on hiv receptor levels. nystatin significantly decreases cd4 and ccr5 surface expression but also significantly decreased non-raft associated cd71, implying that effect of nystatin may not be restricted to proteins located in cholesterol rich domains. conversely, filipin complex treatment significantly reduced cd4 expression levels and slightly decreased ccr5 levels but did not appear to have such a dramatic effect on the expression of other membrane proteins. the effects of cholesterol sequesteration on the surface expression of the hiv receptors, after nystatin and filipin treatment, have not been investigated before. disruption of cholesterol biosynthesis with lovastatin also reduced the surface expression of the hiv receptors on macrophages but not by significant levels. this is in contrast to observations with cd4 t lymphocytes, in which lovastatin down-modulated the mrna and cell surface protein expression of ccr5 (but not cd4 or cxcr4), resulting in reduced hiv-1 infection (nabatov et al., 2007) . the sensitivity of virus entry to depletion of cellular cholesterol could be explained by the association of the viral receptors, cd4 and ccr5, with lipid rafts. the tightly structured cholesterol-rich lipid rafts, known as drms, can be isolated in the presence of ice cold 1% triton x-100 and separated by ultracentrifugation. macrophage cd4 and ccr5 were found in the drm fractions along with flotillin-1 whose location in these membranes is well known. in contrast, cd71 was found in the soluble fractions reflecting its association with the non-raft membranes. decreased hiv receptor, but not cd71, expression after mβcd treatment supports the notion that the hiv receptors are located in cholesterol-rich domains in macrophages. we provide additional evidence for the involvement of lipid rafts in virus entry by visualising virus uptake in parallel with a fluorescent marker of lipid rafts. at 20 min post infection, approximately a third of all cellassociated virions were co-localised with lipid raft membranes in punctate structures that appear to have been internalised and may resemble an endosomal compartment. cd4 association with drms has been established in many different cell types and we can show this to be true for macrophage cd4 (kozak et al., 2002; liao et al., 2001; nguyen et al., 2005; percherancier et al., 2003; popik et al., 2002) . ccr5 has been reported to be present in lipid rafts in cell lines artificially over-expressing this receptor (manes et al., 1999; popik et al., 2002) , but not in primary cd4 t lymphocytes, where this receptor was found in non-raft membrane domains (percherancier et al., 2003) . here, we show that in macrophages, ccr5 is also present in or associated with lipid rafts, and both receptors required for macrophage-tropic virus entry are localised in similar membrane domains. this is in agreement with electron micrographs showing cd4 and ccr5 (but not cxcr4) localised on the outer membranes of microvilli and blebs in macrophages, often in closely apposed microdomains (singer et al., 2001) . however, despite existing together in drms it is possible that further aggregation of receptors is still required and may occur upon gp120 binding. the most likely explanation for the inhibition of productive hiv entry into cholesterol-depleted macrophages is that redistribution of (or conformational changes to) the hiv receptors render the macrophages unsusceptible to hiv infection. it is plausible that manipulation of membrane cholesterol may inhibit entry by other mechanisms, of which we suggest five possibilities. 1) depletion of macrophage cholesterol may alter the lipid composition of the cellular plasma membrane so that it is no longer competent for fusion with the hiv lipid envelope. 2) disruption of membrane rafts may prevent receptor migration and co-localisation. both cd4 and ccr5 are mobile within cellular membranes (shown in cho cells) with the chemokine receptor being significantly more mobile than cd4 and requiring membrane cholesterol for this mobility (steffens and hope, 2004) . 3) signal transduction pathways induced upon ccr5 and envelope engagement may be required for virus entry (harmon and ratner, 2008) . cholesterol has been shown to be essential for ccr5 signalling and its depletion may prevent these important signalling events in macrophages (cardaba et al., 2008) . 4) hiv entry into macrophages may proceed by macropinocytosis, and cholesterol-depletion may block this uptake pathway in macrophages, as has been reported in brain microvascular endothelia (liu et al., 2002) . 5) lovastatin may inhibit hiv entry by another mechanism, as it has been shown that blockade of hmg coa reductase impedes prenylation of small rho gtpases that may be involved in post-entry signalling events (del real et al., 2004) , or by diminishing hiv-1 attachment to target cells by suppressing icam1 lfa1 interactions (giguere and tremblay, 2004) . however, addressing these possibilities to determine the exact role of cholesterol will be challenging, especially using infectious macrophage-tropic virus and primary macrophages. our study highlights the importance of cholesterol and lipid rafts in macrophage hiv infection. this provides further support for the use of cholesterol lowering pharmacological agents as anti-retroviral therapy by confirming their anti-viral effect in another pathogenically significant hiv target cell, the macrophage. peripheral blood mononuclear cells (pbmcs) were isolated using ficoll-paque plus (ge healthcare) density gradient centrifugation from the blood of healthy donors. monocytes were isolated by cd14 positive selection using anti-cd14 magnetic beads (miltenyi biotec) according to the manufacturer's instructions. monocytes were seeded at a density of 1.5 × 10 5 cells/cm 2 in 6-well plates, 12-well plates, 24-well plates or t75 flasks. culture media referred to as rpmi fcs m-csf: consisted of rpmi 1640 (paa) with 10% fetal calf serum (fcs; paa), 2 mm l-glutamine (paa), 100 u/ml penicillin and 100 μg/ml streptomycin (paa), supplemented with 100 ng/ml (approximately 1.7 × 10 4 u/ml) recombinant human m-csf (r&d systems). monocytes were differentiated for 7-9 days prior to use. hiv-1 bal was obtained through the aids research and reference reagent program, division of aids, niaid, nih, from s. gartner (gartner et al., 1986) and viral stocks were generated in differentiated, unstimulated pbmcs. infected cell supernatants were harvested 14-21 days after infection and frozen for use in infectivity assays. virus stocks with proviral dna removed were generated for qpcr experiments by treatment with 100 μg/ml dnase i (sigma). to generate single round replication competent virus, 293t cells were regularly passaged in dmem (paa) with 10% fcs, 2 mm l-glutamine, 100 u/ml penicillin and 100 μg/ml streptomycin. cells seeded into t75 flasks were transfected with pnl4.3lucr-e-(2 μg) and pjrfl env (1.5 μg) using fugene 6 according to manufacturers instructions. the nl4.3 jrfl virus was harvested 48 h later, passed through a 0.44 μm filter and frozen. macrophages were washed once with serum-free rpmi, and treated for 1 h at 37°c with varying concentrations of mβcd, filipin complex, or nystatin (all from sigma) diluted in serum-free rpmi. as nystatin is dissolved in dmso (mβcd and filipin in water), to exclude for any effects of this solvent the control cells were incubated in the same dilution of dmso as used for nystatin. after incubation, cells were washed once with serum-free rpmi prior to infection or flow cytometry analysis. following infection, cells were cultured in rpmi fcs m-csf. water soluble cholesterol (400 μg/ml, sigma) was added for a further 1 h where stated. for lovastatin treatment, macrophages were washed once with serum-free rpmi, before the addition of varying concentrations of lovastatin (sigma) with or without mevalonate (sigma), diluted in rpmi with 10% lipoprotein-deficient serum (sigma). cells were left for 4 days at 37°c before infection or flow cytometry analysis. infected cells were cultured in rpmi fcs m-csf following the infection procedure. to determine cell viability following treatment with cholesterol disrupting agents, macrophages were incubated with celltiter 96® aqueous one solution cell proliferation assay reagent (promega) diluted 1/6 in serum-free rpmi or rpmi with lipoprotein-deficient serum containing lovastatin or mevalonate, for 1 h at 37°c. in triplicate, 100 μl samples of the supernatant were then transferred to a 96 well plate and the absorbance read at 490 nm. background absorbance readings from reagent and media alone were deducted and the values expressed as a percentage of untreated cells. to quantify cellular cholesterol levels, drug treated or control macrophages were detached by pipetting with pbs containing 5 mm edta and 12 mm lidocaine (sigma), on ice. cells were lysed and the cholesterol was extracted as previously described (chung et al., 2005) . the amount of cholesterol was assayed with amplex red cholesterol assay kit (molecular probes) according to the manufacturer's instructions and the concentrations were determined from a standard curve and normalised to the number of cells. macrophages were infected in 12-well plates with 500 μl of dnase i treated hiv-1 bal by spinoculation in a sealed plate spinning centrifuge at 2000 ×g for 90 min at 37°c. the virus inoculum was removed, cells were overlaid with rpmi fcs m-csf and infections were left to proceed for 28-30 h. cells were harvested by scraping and dna was extracted using dneasy blood and tissue kit (qiagen) according to the manufacturer's instructions. qpcr reactions to detect late stage reverse transcription products were carried out as previously described (butler et al., 2001; cohen et al., 2008) . hiv-1 standards consisting of pnl4.3lucr -e -hiv-1 backbone plasmid diluted with 30 ng/μl hela dna were used in duplicate at dilutions ranging from 10 to 1 × 10 7 copies, and actin standards consisting of human xsomal dna (eurogentec) were used at 10-fold dilutions from 80 ng. to measure multiple rounds of hiv infection, macrophages were infected in 12-well plates with hiv-1 bal for 2 h at 37°c. the inoculum was removed and replaced with rpmi fcs m-csf. over a 14-16 day period, supernatant samples were taken at intervals and kept at −80°c. for analysis of hiv-1 binding, macrophages were incubated with hiv bal for 2 h on ice. the cells were then washed 5 times in ice-cold pbs to remove unbound virus, and lysed in 200 μl tes (1 × tris buffered saline [tbs], 1% empigen [fluka], 10% fcs, 0.05% tween 20) before heat inactivation. for analysis of hiv-1 production, samples from infected cells were diluted in tes and heat inactivated. to create a standard curve, recombinant p24 protein (aalto) was diluted in doubling dilutions in tes. costar eia/ria flat bottom high binding 96-well plates (corning) were pre-coated with 10 μg/ml sheep anti-hiv-1-p24 gag antibody (clone d7320, aalto) diluted in 0.05 m carbonate-bicarbonate buffer ph 9.6 (sigma) and blocked with 2% bsa (sigma) in tes. diluted samples or standards were added to the plates and after overnight incubation at room temperature, bound p24 was detected using a biotinylated mouse anti-hiv-1-p24 gag antibody (clone eh12ei, centre for aids reagents, nibsc) at 0.23 μg/ml in tt/ss (tbs, 20% fcs, 0.05% tween 20) followed by 0.625 μg/ml streptavidin-hrp (pierce). plates were developed using tmb substrate (pierce), the reaction was stopped with 1 m h 2 so 4 (sigma) and plates were then read using a molecular devices e max precision microplate reader and softmax pro software version 4.0. graphpad prism software version 4 was used to create standard curves (2-site binding equation) and to calculate unknown values. detached macrophages were washed once with facs buffer (pbs, 0.01% nan 3 , 1% fcs, 10 μg/ml human igg), and 1 × 10 5 cells were incubated with 5 μg/ml directly conjugated mouse anti-human antibodies to cd4 (clone 1180), ccr5 (clone 45531), cxcr4 (clone 44717.111) or appropriate isotype control (all r & d systems). as a control, the expression of the non-raft associated protein transferrin receptor was also measured using mouse anti-human cd71 (clone rvs10) with isotype control mouse igg1 (clone 203) (both diluted 1/ 10 and from immunotools). cells were fixed in 4% formaldehyde. flow cytometry was carried out using a becton-dickinson facscalibur flow cytometer with 10000 events collected and data analysed using flowjo software, version 7.1.3. macrophages were washed once in serum-free rpmi, and incubated with ctb-fitc (sigma) at 10 μg/ml in serum-free rpmi, for 30 min on ice or at 37°c. detached cells were then analysed by flow cytometry as described. to visualise virus and ctb, hiv-1 capable of a single round of infection nl4.3 jrfl was spinoculated (1 h at 4°c at 700 ×g) onto day 7 macrophage monolayers seeded onto glass coverslips in 24 well plates. cells were washed with ice-cold pbs and incubated in rpmi containing 10 μg/ml ctb-fitc for 20 min at 37°c. cells were fixed in 4% formaldehyde, quenched in 80 mm glycine in pbs and permeabilised in buffer containing 1% fcs, 0.1% saponin and human igg 10 μg/ml in pbs. to detect virus, cells were stained with mouse anti-hiv p17 diluted 1/500 (clone 4c9, centre for aids reagents, nibsc) followed by alexa fluor 647 conjugated goat antimouse igg2a antibody diluted 1/400 (clone a21131, invitrogen). coverslips were mounted on slides using mowiol and images collected using a zeiss pascal microscope. macrophages (1 × 10 7 ) were harvested by scraping, pelleted and suspended in 1 ml h buffer (10 mm sodium hepes, ph 7.2, 250 mm sucrose, 2 mm mgcl 2 , 10 mm naf, and 1 mm vanadate) containing protease inhibitors (roche). the cells were nitrogen-cavitated using a nitrogen cavitation bomb (model 4639; parr instrument company) equilibrated at 4°c, 50 bar for 10 min (harder and kuhn, 2000) . cells were further disrupted by adding 1 ml hne buffer (10 mm hepes, ph 7.0, 150 mm nacl, and 5 mm edta), passaged 15 times through a 23g syringe and 1% triton x-100 was added before cells were incubated for 1 h at 4°c. a 200 μl extract containing approximately 1 × 10 6 cells was adjusted to 45% optiprep™ (nycomed pharma) and transferred to an sw55 centrifuge tube (beckman coulter) and overlaid with another 200 μl 45% optiprep to make a 1 ml layer. this was overlaid with 2 ml of 35% and 0% optiprep in hne and gradients were spun at 40,000 rpm for 3 h at 4°c. nine fractions of 550 μl were collected and precipitated in an equal vol of 20% trichloroacetic acid, washed twice in ice-cold acetone and resuspended in 8 m urea and sds sample buffer. equal volumes of each fraction were run on a 10% sds-page protein gel with biorad precision plus protein standards. protein was transferred onto 0.2 μm pvdf membranes, and membranes were incubated with primary mouse anti-human antibodies to cd4 (clone 34915, 2.5 μg/ml r & d systems) ccr5 (clone ctc5, 2.5 μg/ml r & d systems), flotillin-1 (clone 18, 1.25 μg/ml, bd biosciences) and transferrin receptor (clone 2, 1.25 μg/ml, bd biosciences). to detect primary antibody binding, membranes were incubated with goat antimouse igg (fab specific) antibody conjugated to alkaline phosphatase (sigma) diluted 1/30,000. protein was detected using ecf substrate (pierce), and membranes were imaged using storm imager. statistical analysis was performed by two-tailed paired t-test (unpaired with 2 replicates) using graphpad prism version 4. stars indicate the p value: ⁎ = significant p = 0.05-0.01, ⁎⁎ = very significant p = 0.01-0.001, ⁎⁎⁎ = extremely significant p b 0.001. lipid composition and fluidity of the human immunodeficiency virus lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae specific association of glycoprotein b with lipid rafts during herpes simplex virus entry the hiv lipidome: a raft with an unusual composition human immunodeficiency virus type 1 nef protein modulates the lipid composition of virions and host cell membrane microdomains a quantitative assay for hiv dna integration in vivo virion-associated cholesterol is critical for the maintenance of hiv-1 structure and infectivity the raftpromoting property of virion-associated cholesterol, but not the presence of virionassociated brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity ccr5 internalisation and signalling have different dependence on membrane lipid raft integrity crosstalk between lxr and toll-like receptor signaling mediates bacterial and viral antagonism of cholesterol metabolism retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts an aptamer that neutralizes r5 strains of hiv-1 binds to core residues of gp120 in the ccr5 binding site macrophage-tropic strains of human immunodeficiency virus type 1 utilize the cd4 receptor blocking of hiv-1 infection by targeting cd4 to nonraft membrane domains statins inhibit hiv-1 infection by down-regulating rho activity duck hepatitis b virus requires cholesterol for endosomal escape during virus entry virus isolation from and identification of htlv-iii/lav-producing cells in brain tissue from a patient with aids statin compounds reduce human immunodeficiency virus type 1 replication by preventing the interaction between virion-associated host intercellular adhesion molecule 1 and its natural cell surface ligand lfa-1 role for human immunodeficiency virus type 1 membrane cholesterol in viral internalization selective accumulation of raft-associated membrane protein lat in t cell receptor signaling assemblies induction of the g{alpha}q signaling cascade by the human immunodeficiency virus envelope is required for virus entry poliovirus cell entry: common structural themes in viral cell entry pathways effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane variations in cd4 expression by human monocytes and macrophages and their relationships to infection with the human immunodeficiency virus segregation of cd4 and cxcr4 into distinct lipid microdomains in t lymphocytes suggests a mechanism for membrane destabilization by human immunodeficiency virus quantification of cd4, ccr5, and cxcr4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle lipid rafts and hiv pathogenesis: host membrane cholesterol is required for infection by hiv type 1 lipid rafts and hiv pathogenesis: virionassociated cholesterol is required for fusion and infection of susceptible cells human immunodeficiency virus type 1 enters brain microvascular endothelia by macropinocytosis dependent on lipid rafts and the mitogen-activated protein kinase signaling pathway ecotropic murine leukemia virus receptor is physically associated with caveolin and membrane rafts membrane raft microdomains mediate front-rear polarity in migrating cells membrane raft microdomains mediate lateral assemblies required for hiv-1 infection human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis internalization of echovirus 1 in caveolae virus entry: open sesame human immunodeficiency virus impairs reverse cholesterol transport from macrophages statins disrupt ccr5 and rantes expression levels in cd4+ t lymphocytes in vitro and preferentially decrease infection of r5 versus x4 hiv-1 cxcr4 function requires membrane cholesterol: implications for hiv infection dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of cd4 engagement lack of p56lck expression correlates with cd4 endocytosis in primary lymphoid and myeloid cells hiv-1 entry into t-cells is not dependent on cd4 and ccr5 localization to sphingolipid-enriched, detergentresistant, raft membrane domains echovirus 1 endocytosis into caveosomes requires lipid rafts, dynamin ii, and signaling events cd4 receptor localized to non-raft membrane microdomains supports hiv-1 entry. identification of a novel raft localization marker in cd4 human immunodeficiency virus type 1 uses lipid raft-colocalized cd4 and chemokine receptors for productive entry into cd4(+) t cells the molecular face of lipid rafts in model membranes syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages lipid microdomaindependent macropinocytosis determines compartmentation of afipia felis functional rafts in cell membranes ccr5, cxcr4, and cd4 are clustered and closely apposed on microvilli of human macrophages and t cells how viruses enter animal cells mobility of the human immunodeficiency virus (hiv) receptor cd4 and coreceptor ccr5 in living cells: implications for hiv fusion and entry events role for influenza virus envelope cholesterol in virus entry and infection human herpesvirus-6 infection induces the reorganization of membrane microdomains in target cells, which are required for virus entry differential cholesterol binding by class ii fusion proteins determines membrane fusion properties nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected t cells role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells modulation of brucella-induced macropinocytosis by lipid rafts mediates intracellular replication float on: lipid rafts in the lifecycle of hiv nef increases the synthesis of and transports cholesterol to lipid rafts and hiv-1 progeny virions this research was supported by grant number 9746 and a phd studentship from the uk medical research council. key: cord-268901-7cm6m1ol authors: ku, therese; lopresti, natalie; shirley, matthew; mori, mattia; marchant, jan; heng, xiao; botta, maurizio; summers, michael f.; seley-radtke, katherine l. title: synthesis of distal and proximal fleximer base analogues and evaluation in the nucleocapsid protein of hiv-1 date: 2019-07-01 journal: bioorganic & medicinal chemistry doi: 10.1016/j.bmc.2019.05.019 sha: doc_id: 268901 cord_uid: 7cm6m1ol abstract anti-hiv-1 drug design has been notably challenging due to the virus’ ability to mutate and develop immunity against commercially available drugs. the aims of this project were to develop a series of fleximer base analogues that not only possess inherent flexibility that can remain active when faced with binding site mutations, but also target a non-canonical, highly conserved target: the nucleocapsid protein of hiv (nc). the compounds were predicted by computational studies not to function via zinc ejection, which would endow them with significant advantages over non-specific and thus toxic zinc-ejectors. the target fleximer bases were synthesized using palladium-catalyzed cross-coupling techniques and subsequently tested against nc and hiv-1. the results of those studies are described herein. for some time, the seley-radtke group has designed and synthesized various classes of flexible purine nucleos(t)ides, or "fleximers". [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] these novel nucleosides were designed to investigate how flexibility in the nucleobase could potentially affect receptor-ligand recognition and function. in addition, their flexible design allows them to overcome issues with binding site mutations thus retaining their activity. to date, fleximers have shown key advantages over the corresponding purine-base nucleosides. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] for example, the distal guanosine fleximer (flex-g, fig. 1 ) proved to be an inhibitor of s-adenosyl-lhomocysteine hydrolase by adapting a syn conformation, thereby placing the exocyclic amino group such that it mimicked the amino group from an adenosine nucleobase. 3, 4 moreover, the guanosine fleximer triphosphate (flex-gtp) was shown to be a superior substrate of human gdp-l-fucose pyrophosphorylase compared to the natural substrate gtp, 5 likely due to the fleximer's ability to interact with amino acids in the active site not accessible by gtp. 6 this also allowed flex-gtp to retain all activity when essential catalytic residues needed for gtp binding were mutated. 5, 6 recently, a series of fleximers possessing acyclic sugars exhibited broad spectrum antiviral activities against coronaviruses, flaviviruses and filoviruses, further supporting their significance. 10, 11 in an effort to explore the fleximer approach to other viruses such as hiv-1, a virus with the ability to readily mutate and develop antiviral resistance, a series of fleximers were designed and studied in silico for their ability to inhibit the virus' nucleocapsid protein, nc. [14] [15] [16] [17] nc plays several key roles in hiv-1 replication. through non-specific binding, it acts as a chaperone protein, partially protecting the viral nucleic acids (nas). 18 during reverse transcription, nc directs the annealing of cellular trna (lys, 3) primer to the hiv-1 primer binding site, thus initiating the synthesis of the (−)-strong stop dna. 19, 20 nc then facilitates the two strand transfers required for (−) and (+) strand synthesis. it is also implicated to be a vital element in vdna integration. 21, 22 prior to encapsidation, nc discriminates viral from host na by selectively binding to the hiv-1 ψ-encapsidation signal sequence. [23] [24] [25] in addition, in vitro studies have shown that nc may chaperone the dimerization of the two copies of hiv-1 viral genomic rna by rearranging the kissing complex into an extended duplex through a series of stabilizing and destabilizing events, an important step prior to encapsidation. 26, 27 because of nc's interaction with multiple highly conserved sequences of the hiv-1 genome, and being essential in all hiv-1 subtypes nc represents a powerful drug target for developing novel antivirals. [28] [29] [30] [31] more importantly, it is thought to be highly resistant to mutation due to its multifunctional role, thus providing a significant advantage over other protein targets. 26, 32 thus, inhibitors of the interaction between nc and the viral nucleic acids could provide a new approach to antiretroviral therapy. 14, 33 for this purpose, a series of fleximers were computationally designed with that goal in mind. herein we report the synthesis and biological results for a series of compounds that were predicted to interact with nc. in many structural studies done to date on nc, 16, 17, [34] [35] [36] a guanosine residue was shown to consistently stack with the w37 residue, whether bound to dna (pbs-dna) or rna (ψ-rna). as such, the inherent flexibility of the fleximer guanine analogue was predicted to affect binding and potentially result in inhibition. based on this hypothesis, a number of fleximer nucleosides were initially designed, however the early results showed that the sugar moiety on the fleximer nucleoside provided no benefit over the fleximer base itself. as a result, the corresponding fleximer base analogues were therefore pursued, since this would signfiicantly shorten the synthetic route. thus, the fleximer bases were then tested computationally against the nmr structure of the nc in complex with a small molecule inhibitor (figs. 2 and 3). 37 to this end, a computational protocol was established and refined in the group of botta. 14, 33, 38 several nc binding small molecules have already been discovered through this protocol, supporting its validity. 14, 15, 33, 38 the docking results of the fleximer bases on nc revealed several key advantages for the proposed target compounds. the docking conformation of fleximer bases 1-4 ( fig. 3 ) within the hydrophobic pocket that is located in correspondence of w37 showed an excellent structural overlay with respect to the guanine base. moreover, all of the fleximer bases were able to establish a network of h-bond interactions with the backbone atoms of key residues in the hydrophobic pocket (i.e. k33, g35, w37, and m46, fig. 3a -d) that is highly comparable to that established by the guanine base (fig. 3e) . additionally, 1-4 adopted a similar stacking conformation to w37 as is observed with the natural guanine. however the additional rotatable bond allowed for the pyrimidine moiety to extend and interact with the neighboring residues (i.e. k47 in the binding mode of 4, fig. 3d ) into the solvent area. the distal guanine fleximer base (1) was predicted to bind stronger than the proximal base (2) and, notably, slightly stronger than the guanine base (table 1) , although scoring values calculated by the fred docking program with the chemgauss4 function were highly comparable to each other. 39, 40 thus, the observed differences between guanine, 1 and 2 may not be significant. in addition, the docking study surprisingly showed that the bipyrimidine scaffold (3 and 4, fig. 2 ), would also be highly advantageous, although to a slightly lesser extent than the parent imidazole-pyrimidine scaffold (table 1) , thus those targets were pursued as well. as a result of this molecular modeling analysis, compounds 1-4 ( fig. 2) were selected as the best starting candidates as proof of concept. the intended route to achieve both sets of flexible purine and bipyrimidine bases utilized palladium-catalyzed cross-coupling, with the pyrimidine as the organometallic coupling partner and the imidazole as the halogenated coupling partner. the goal was to achieve two products from one reaction as the organometallic moiety has been shown to undergo homocoupling during cross-coupling reactions. [41] [42] [43] [44] [45] as the distal compounds were predicted to be better binders to nc, the first goal was to install the organometal on the c-6 of 5 to avoid protecting the exocyclic amine. to obtain the iodinated intermediate, commercially available 2-amino-6-chloro-4-methoxypyrimidine was iodinated using hydroiodic acid (scheme 1). the reaction was then neutralized and filtered, and the precipitate recrystallized in ethanol to obtain 5. since the proximal intermediate 2-amino-4-methoxy-5-tributylstannylpyrimidine was easily achievable starting with 2-amino-5-iodo-4-methoxypyrimidine, [9] [10] [11] similar methodologies were applied to obtain the 2-amino-4-methoxy-6-tributylstannylpyrimidine intermediate 6, using various palladium catalysts as well as a range of temperatures, but none of the conditions yielded the desired organostannane 6 (scheme 2). the organoborane 7 was then considered for subsequent suzuki coupling (scheme 3), however, the boronic ester could not be generated under any conditions. because the cross-coupling intermediates could not be obtained through palladium catalysis, harsher conditions were used to achieve the desired coupling partners. the exocyclic amine was protected (scheme 4) with tms as it is immune to grignard and lithium reagents but can be cleaved easily in mildly acidic conditions. 46 each tms group was added sequentially as opposed to simultaneously to form 8 in situ. lithium halogen exchange using n-buli, followed by metalation with tributyltin chloride finally produced deprotected organostannane 6. characterization through 1 h and 13 c nmr in addition to ms confirmed the presence of the product however 6 proved highly unstable and decomposed rapidly. as installation of the metal on the pyrimidine proved to be difficult, and literature showed that an organozinc intermediate could be generated in situ with the imidazole moiety for subsequent negishi coupling, the previous methodology was abandoned. 47 a benzyl (bn) protecting group was initially chosen as bn's are robust against many conditions. 46 the organozinc 10 was generated in situ and subsequent negishi coupling with 5 (scheme 5) yielded the protected distal fleximer 11, albeit in poor yield (24%). moreover, the bn and methyl groups were extremely difficult to deprotect (scheme 6). hydrogenation using pd/c in the presence of h 2 at room temperature was thought to be sufficient to remove the bn group, however no reaction occurred. this was ultimately achieved by heating the reaction mixture of 11 with ammonium formate and pd/c in etoh to 120°c. boron tribromide (bbr 3 ) was employed to deprotected the methyl, however, a solubility problem arose when attempting to demethylate 13 in dcm, and therefore deprotection of the methyl was more efficient when performed prior to deprotection of the bn (scheme 6). unfortunately, these molecules proved difficult to purify via column chromatography, likely due to either their polar nature or their ability to stack efficiently from the presence of two heteroaromatic moieties and multiple hydrogen bonding elements. to bypass these challenges, a trityl protecting group was employed to protect the imidazole, and the exocyclic amine of the pyrimidine was protected with tert-butyloxycarbonyl (boc, schemes 7 and 8). negishi coupling using these two heterocycles proved more facile and the cross-coupling reaction proceeded at room temperature (scheme 8). 47 trityl deprotection was accomplished using acetic acid while boc deprotection required trifluoroacetic acid. as previously mentioned, the methyl protected fleximer guanine 12 was insoluble in dcm, however, it was soluble in etoac, and final deprotection using bbr 3 in etoac was successful. since 1 was ultimately obtained through negishi coupling, a similar strategy was employed to obtain the analogous bipyrimidine (scheme 9). unexpectedly, the amine-linked compound 23 was produced instead. the compound was then subjected to aminolysis to convert the chloro group to an exocyclic amine, however, no starting material or product was recovered (scheme 9). in hindsight, the presence of a palladium catalyst likely induced a buchwald-hartwig amination, 48 leading to the synthesis of 22 and the absence of compound 21. because the correct cross-couplings procedures could not be easily performed on the c-6 of 5, 6-bromo-2,4-dimethoxypyrimidine (25, scheme 10) was pursued instead, which which was envisioned by first brominating barbituric acid with pobr 3 followed by nucleophilic substitution using sodium methoxide to afford compound 25. to achieve the targeted bipyrimidine 3 through stille coupling, 25 was first converted to stannane 26. interestingly, 26 was never synthesized, however the bipyrimidine 27 was recovered at good yields (80%, scheme 11). from there, two strategies were attempted to attain the desired bipyrimidine 3. first, removal of the methyl protecting groups followed by chlorination via pocl 3 was tried, and intermediate 28 was used crude as it was insoluble in various purification solvents. the tetrachlorinated intermediate 29 could not be isolated as the crude reaction mixture was difficult to purify. the next approach was to directly convert the methoxy groups to amines (30) and enzymatically convert the c4-nh 2 group to an eoh using adenosine deaminase. neither the starting material nor product were recovered, likely due to the harsh conditions used. since homocoupling of the pyrimidines had occurred with the dimethoxypyrimidines, it was speculated that the same conditions would likely produce a homocoupled product for the 2-amino-4-methoxypyrimidines as well, which fortuitously proved true (scheme 12). unfortunately, the product 21 proved to be highly insoluble, and impossible to purify, and was only observed via ms. as achieving the distal analogues proved challenging, the focus then turned to synthesizing the proximal analogues. considering the synthesis of compound 31 was facile, a straightforward stille with the halogenated pyrimidine produced the bipyrimidine 33 (scheme 13), however, similar purification and solubility issues as 21 occurred. to complete this series, the proximal fleximer guanine 2 was also pursued. instead of using stille cross-coupling techniques, the negishi method used for achieving the distal fleximer guanine was employed (scheme 14). 47 surprisingly, no product was observed when the reaction was allowed to stir at room temperature, and poor yields were found even after reflux. the failure of the reaction was speculated to be due to the placement of the halogen on the electron-rich carbon of the c-5 position of the pyrimidine. as palladium prefers to react with electron-deficient carbons, the oxidative addition reaction between the electron-rich halogenated coupling partner and palladium likely did not occur, which led to the absence of the desired coupled product. 49 hence, the organozinc was placed on the pyrimidine in subsequent reactions. as predicted, the organozinc on the pyrimidine was successfully synthesized in situ, and negishi cross-coupling followed by deprotection of the trityl group was accomplished to yield 37 (scheme 15). 47 regrettably, methyl deprotection to produce the proximal fleximer guanine 2 was unsuccessful using bbr 3 in either ch 2 cl 2 or etoac as the intermediate was insoluble in both. deprotection using catalytic sulfuric acid and tmscl in acetic anhydride also did not produce 2 (confirmed through ms). the hypothesis to explain this phenomenon ties into the in silico results by bardon et al. that showed that the most thermodynamically stable conformation of the proximal fleximer bases is in a planar form. 50 this conformation could be promoting the stacking of the bases that consequently does not allow 37 to solubilize in usual solvents. the dimethoxypyrimidine series was also pursued and proved more facile to obtain as no additional protection steps were required. following the approach to realize the 2-amino-4-methoxy series, the dimethoxy proximal compounds were synthesized using the pyrimidine as the organometallic moiety (scheme 16). deprotection of the methyl groups were attempted, however, the resulting crude mixture was insoluble and thus purification was not possible. similar conditions were used to synthesize the dimethyl protected distal fleximer xanthosine as scheme 8 (scheme 17). the yield of 42 (68%) was higher than that of 18 (43%), with the major structural difference being the c-2 substitution (ome versus nhboc). with compound 1 in hand, a 1 h nmr experiment was performed with nc. if the fleximer did bind to the na binding site of nc where w37 resides, a shift should be observed in the aromatic region of the protein, similar to that shown in goudreau et al. 37 after recording the 1 h nmr spectra of nc in d 2 o without 1, a one equivalent molar ratio of 1 was added to the sample (dissolved in dmso-d 6 ). both the aliphatic region and aromatic region (fig. 4) were recorded but no significant changes in the nc signals were detected. this study does not necessarily exclude 1 as an nc binder, but does prove that the interaction is at most very weak, thus undetectable via nmr experiments. unfortunately, none of the other products that were successfully purified would dissolve in the nmr solvent or in the presence of the protein, therefore the nmr studies were abandoned. finally, all of the target compounds were sent to the national cancer institute (nci) to be tested against hiv-1 by dr. eric freed. disappointingly, none of the analogues exhibited any meaningful antiviral activity. a number of fleximer bases were successfully synthesized via palladium-catalysed cross-couplings in this study, albeit with more difficulty than anticipated, especially during purification. the synthetic routes developed for the distal fleximer base bypassed the classic tricyclic route that has been used in the seley-radtke group for over a decade. if yields could be improved, this would potentially be useful for synthesis of distal fleximer nucleosides in the future. biologically however, these molecules were not recognized by nc based on the nmr experiment performed, and were inactive against hiv-1. while the biological results were disappointing, the project did provide a better understanding of palladium-catalysed cross-coupling strategies for fleximer bases in terms of choosing the optimum crosscoupling partners. this which has proven advantageous for other projects ongoing in our group and others, the results of which will be published as they become available. all chemicals were obtained from commercial sources and used without further purification unless otherwise noted. anhydrous dmf, ch 3 oh, dmso and etoh were purchased from fisher scientific. anhydrous thf, acetone, ch 2 cl 2 , ch 3 cn, and ether were obtained using a solvent purification system (mbraun labmaster 130). nmr solvents were purchased from cambridge isotope laboratories (andover, ma). all 1 h and 13 c nmr spectra were obtained either on a jeol ecx 400 mhz nmr, operated at 400 and 100 mhz, respectively, or a bruker avance iii hd 500 mhz nmr, operated at 500 and 125 mhz, respectively, and referenced to internal tetramethylsilane (tms) at 0.0 ppm. the spin multiplicities are indicated by the symbols s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and br (broad). reactions were monitored by thin-layer chromatography (tlc) using 0.25 mm whatman diamond silica gel 60-f254 pre-coated plates. purification was performed on a teledyne isco combiflash rf 200, and eluted with the indicated solvent system. yields refer to chromatographically and spectroscopically ( 1 h and 13 c nmr) homogeneous materials. mass spectra were recorded at the umbc mcac for nominal using bruker apollo™ ii esi/apci -maldi dual source for apex(r)-qe ftms or johns hopkins mass spectrometry facility for high resolution using vg analytical vg-70se magnetic sector mass spectrometer. commercially available 2-amino-4-chloro-6-methoxypyrimidine (5.0 g, 31.3 mmol) was suspended in 20 ml of 57 wt% hi in h 2 o at 0°c. the mixture was stirred at room temperature for 72 h. the resulting sludge was diluted in 20 ml h2o and neutralized to ph 7-8 using sat. na 2 co 3 . the precipitate was filtered and recrystallized in etoh to yield a white solid (4.1 g, 16.3 mmol, 52%). 1 (6) 5 (139 mg, 0.55 mmol) was dissolved under n 2 in 20 ml of anhydrous thf and cooled to −78°c. etmgbr (3.0 m, 0.20 ml, 0.61 mmol) was added dropwise and allowed to stir for 2 min. tmscl (0.08 ml, 0.61 mmol) was added and allowed to stir for 5 min. again, etmgbr (3. 0 m, 0.20 ml, 0.61 mmol) was added dropwise and allowed to stir for 2 min, then tmscl (0.08 ml, 0.61 mmol) was added and allowed to stir for 5 min. n-butyllithium (1.6 m, 0.4 ml, 0.61 mmol) was added dropwise and allowed warm to room temperature and stirred for 3.5 h. tributyltin chloride (0.3 ml, 1.11 mmol) was added and the mixture was stirred for 18 min. the reaction was quenched using 10 ml nh 4 cl and the solvent was removed in vacuo. the crude material was extracted into ch 2 cl 2 (20 ml × 3), washed with brine (10 ml × 2) and the organic layer was dried over mgso 4 . the crude material was purified using silica gel column chromatography (hexanes/ etoac = 9:1-3:1) to yield a yellow oil (126 mg, 0. 30 1-benzyl-4-iodo-1h-imidazole 9 (118 mg, 0.42 mmol) 51 was dissolved under n 2 in 25 ml of anhydrous thf and cooled to −78°c. etmgbr (3.0 m, 0.15 ml, 0.44 mmol) was added dropwise and allowed to stir for 10 min. zncl 2 (0.7 m in thf, 1.2 ml, 0.84 mmol) was subsequently added dropwise, stirred at −78°c for 10 min, warmed to room temperature and stirred for 2 h. the organozinc was added dropwise to a mixture of 5 (105 mg, 0.42 mmol), pd(pph 3 ) 4 (48 mg, 0.04 mmol), cui (19 mg, 0.1 mmol) in 40 ml of anhydrous thf and allowed to stir at room temperature for 24 h. the reaction was quenched using 10 ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch 2 cl 2 (50 ml × 3), washed with brine (10 ml × 2) and the organic layer was dried over mgso 4 . the crude material was purified using silica gel column chromatography (hexanes/etoac = 1:1-0:1) to yield a white solid (25 mg, 0.10 mmol, 24% yield). r f = 0.35, (etoac). 1 synthetic procedure related to of scheme 8 reported. 19 (100 mg, 0.34 mmol) was dissolved in 20 ml trifluoroacetic acid and stirred for 48 h. the solvent was removed and the crude material was purified using silica gel column chromatography (ch 2 cl 2 /ch 3 oh = 19:1-4:1) to yield a white solid (59 mg, 0.31 mmol, 91% yield). r f = 0.50, (ch 2 cl 2 /ch 3 oh = 4:1). 1 11 (25 mg, 0.10 mmol) was dissolved in 15 ml anhydrous ch 2 cl 2 under n 2 and cooled to −78°c. bbr 3 (3 m, 0.4 ml, 1.2 mmol) was added dropwise and the reaction was allowed to warm to room temperature and stirred for 72 h. the mixture was dripped slowly into 20 ml iced water and stirred for 30 min. the solvent was removed in vacuo and the crude material was purified using silica gel column chromatography (ch 2 cl 2 /ch 3 oh = 9:1-4:1) to yield a white solid (16 mg, 0.06 mmol, 60% yield). r f = 0.70, (ch 2 cl 2 /ch 3 oh = 9:1). 1 2-amino-4-iodo-6-methoxypyrimidine 5 (4.1 g, 16.3 mmol) was suspended in 50 ml ch 2 cl 2 under n 2 . di-tert-butyl decarbonate (8.9 g, 40.8 mmol) and 4-dimethylaminopyridine (5.0 g, 40.9 mmol) were added. the reaction was allowed to stir at room temperature for 18 h. tlc showed absence of starting material and two products. the solvent commercially available 4-iodo-1-trityl-1h-imidazole (500 mg, 1.15 mmol) was dissolved under n 2 in 25 ml of anhydrous thf and cooled to −78°c. etmgbr (3.0 m, 0.4 ml, 1.20 mmol) was added dropwise and allowed to stir for 10 min. zncl 2 (0.7 m in thf, 3.3 ml, 2.3 mmol) was subsequently added dropwise, stirred at −78°c for 10 min, warmed to room temperature, and stirred for 2 h. the organozinc was added dropwise to a mixture of 15 (451 mg, 1.0 mmol), pd (pph 3 ) 4 (115 mg, 0.1 mmol) and cui (10 mg, 0.05 mmol) in 40 ml of anhydrous thf and allowed to stir at room temperature for 24 h. the reaction was quenched using 10 ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch 2 cl 2 (50 ml × 3), washed with brine (10 ml × 2) and the organic layer was dried over mgso 4 . the crude material was purified using silica gel column chromatography (hexanes/etoac = 1:1-0:1) to yield a yellow oil (267 mg, 0.5 mmol, 43% yield). r f = 0.60, (hexanes/etoac = 1:2). 1 (19) 18 (267 mg, 0.50 mmol) was dissolved in 20 ml acetic acid and stirred for 48 h. the solvent was removed and the crude material was purified using silica gel column chromatography (ch 2 cl 2 / ch 3 oh = 19:1-4:1) to yield an off-white solid (140 mg, 0.48 mmol, 96% yield). r f = 0.85, (ch 2 cl 2 /ch 3 oh = 9:1). 1 12 (41 mg, 0.21 mmol) was dissolved in 20 ml anhydrous etoac under n 2 and cooled to −78°c. boron tribromide (1.0 m, 0.6 ml, 0.6 mmol) was added dropwise. the reaction was allowed to warm to room temperature and stirred for 36 h. the mixture was dripped slowly into 20 ml iced water and stirred for 30 min. the solvent was removed in vacuo and the crude material was purified using silica gel column chromatography (ch 2 cl 2 /ch 3 oh = 9:1-2:1) to yield a white solid (32 mg, 0.18 mmol, 86% yield). r f = 0.25, (ch 2 cl 2 /ch 3 oh = 2:1). 1 6.10. synthesis of 6,6′-dimethoxy-4,4′-bipyrimidine-2,2′-diamine (21) 5 (101.3 mg, 0.40 mmol) was dissolved in 30 ml degassed 1,4-dioxane in a glass tube. bis(tributyltin) (0.2 ml, 0.4 mmol) was added, followed by pd(pph 3 ) 2 cl 2 (28.3 mg, 0.04 mmol). the glass tube was sealed and heated to 130°c for 48 h. the tube was cooled to 0°c, opened and warmed to room temperature. the crude content was filtered over a pad of celite and the solvent was removed. the crude material was purified using silica gel column chromatography (ch 2 cl 2 / ch 3 oh = 9:1-2:1) to yield an impure mixture. another attempt at purification with silica gel column chromatography using the pharmasset conditions (etoac/ch 3 5 (212 mg, 0.84 mmol) was dissolved under n 2 in 20 ml of anhydrous thf and cooled to −78°c. etmgbr (3.0 m, 0.3 ml, 0.93 mmol) was added dropwise and allowed to stir for 2 min. tmscl (0.1 ml, 0.93 mmol) was added and allowed to stir for 5 min. again, etmgbr (3.0 m, 0.3 ml, 0.93 mmol) was added dropwise and allowed to stir for 2 min, then tmscl (0.1 ml, 0.93 mmol) was added and allowed to stir for 5 min. etmgbr (3.0 m, 0.3 ml, 0.93 mmol) was added dropwise and allowed to stir for 10 min followed by addition of zncl 2 (1 m in thf, 1.7 ml, 1.7 mmol) dropwise, stirred at −78°c for 10 min, warmed to room temperature and stirred for 2 h. the organozinc was added dropwise to a mixture of 2-amino-6-chloro-4-methoxypyrimidine (80 mg, 0.50 mmol), pdcl 2 (pph 3 ) 2 (59 mg, 0.08 mmol) and cui (16 mg, 0.08 mmol) in 30 ml of anhydrous thf and allowed to stir at reflux for 24 h. the reaction was quenched using 20 ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch 2 cl 2 (30 ml × 3), washed with brine (10 ml × 2) and the organic layer was dried over mgso 4 . the crude material was purified using silica gel column chromatography (hexanes/etoac = 1:3) to yield a white solid (107 mg, 0.38 mmol, 76% yield). r f = 0.50, (hexanes/ etoac = 1:3). 1 h nmr (400 mhz, dmso-d 6 ) δ 3.76 (s, 3h), 3.91 (s, 3h), 6.30 (br, 2h), 6.56 (s, 1h), 6.85 (s, 1h), 9.80 (br, 1h). 13 commercially available barbituric acid (2.0 g, 15.6 mmol) was suspended in 30 ml of anhydrous toluene under n 2 and cooled to 0°c. phosphorus (v) oxybromide (17.9 g, 62.4 mmol) was added and n,ndimethylaniline (3.6 ml, 28.4 mmol) was added dropwise. the mixture was heated to 110°c and stirred vigorously for 3 h. the reaction was cooled to room temperature and quenched with 30 ml iced water. the mixture was transferred to a separatory funnel and the remaining insoluble gum was washed with etoac (10 ml × 3). all organic layers were combined and washed with sat. nahco 3 (10 ml × 3), then brine (10 ml × 2), and the organic layer was dried over mgso 4 . the crude material was purified using silica gel column chromatography (hexanes/etoac = 99:1-9:1) to yield 25 as a while solid (3.6 g, 11.4 mmol, 73% yield). r f = 0.80, (hexanes/et 2 o = 4:1). 1 (0.5 m, 13.2 ml, 6.60 mmol) was added dropwise and the mixture was warmed to room temperature and stirred for 18 h. the reaction was quenched using 20 ml nh 4 cl and the solvent was removed. the crude material was extracted into ch 2 cl 2 (50 ml × 3), washed with brine (10 ml × 2) and the organic layer was dried over mgso 4 . the crude material was purified using silica gel column chromatography (hexanes/etoac 6.14. synthesis of 2,2′,6,6′-tetramethoxy-4,4′-bipyrimidine (27) 25 (157 mg, 0.72 mmol) was dissolved in 20 ml degassed 1,4-dioxane in a glass tube. bis(tributyltin) (0.4 ml, 0.72 mmol) was added, followed by pd(pph 3 ) 4 (83 mg, 0.07 mmol). the glass tube was sealed and heated to 120°c for 18 h. the tube was cooled to 0°c, opened and warmed to room temperature. the crude content was filtered over a pad of celite and the solvent was removed. the crude material was purified using silica gel column chromatography (hexanes/ etoac = 1:0-9:1) to yield a white fluffy solid (81 mg, 0.29 mmol, 80% yield). r f = 0.80, (hexanes/etoac = 9:1). 1 31 (206 mg, 0.5 mmol) 9 and 32 (124 mg, 0.5 mmol) were dissolved in 50 ml degassed dmf. pd(pph 3 ) 4 (57 mg, 0.05 mmol), cui (19 mg, 0.1 mmol) and csf (150 mg, 0.99 mmol) were added. the reaction was allowed to stir at 90°c for 18 h. the contents were cooled and filtered over celite. the solvent was removed, and the crude material was purified using silica gel column chromatography (ch 2 cl 2 / ch 3 oh = 4:1-2:1) to yield contaminated 33. the contaminated sample was recrystallized in etoac, followed by ethanol, followed by methanol and finally dmso to obtain a white solid (15 mg, 0.06 mmol, 12 % yield). 1 h nmr (400 mhz, dmso-d 6 ) δ 3.73 (s, 6h), 6.54 (br, 4h), 7.78 (s, 1h). 13 32 (500 mg, 1.99 mmol) was dissolved under n 2 in 50 ml of thf and cooled to −78°c. etmgbr (3.0 m, 0.7 ml, 2.10 mmol) was added dropwise and allowed to stir for 2 min. tmscl (0.3 ml, 2.19 mmol) was added and allowed to stir for 5 min. again, etmgbr (3. 0 m, 0.7 ml, 2.10 mmol) was added dropwise and allowed to stir for 2 min, then tmscl (0.3 ml, 2.19 mmol) was added and allowed to stir for 5 min. etmgbr (3.0 m, 0.7 ml, 2.10 mmol) was added dropwise and allowed to stir for 10 min followed by addition of zncl 2 (0.7 m in thf, 5.7 ml, 3.98 mmol) dropwise, stirred at −78°c for 10 min, warmed to room temperature and stirred for 2 h. the organozinc was added dropwise to a mixture of 16 (850 mg, 1.95 mmol), pd(pph 3 ) 4 (230 mg, 0.2 mmol), cui (20 mg, 0.1 mmol) in 80 ml of thf and allowed to stir at room temperature for 24 h. the reaction was quenched using 10 ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch 2 cl 2 (50 ml × 3), washed with brine (10 ml × 2) and the organic layer was dried over mgso 4 . the crude material was purified using silica gel column chromatography (hexanes/ etoac = 1:1-0:1) to yield a yellow solid (252 mg, 0.58 mmol, 29% yield). r f = 0.45, (etoac). 1 (37) 34 (252 mg, 0.58 mmol) was dissolved in 20 ml acetic acid and stirred for 48 h. the solvent was removed and the crude material was purified using silica gel column chromatography (ch 2 cl 2 / ch 3 oh = 19:1-4:1) to yield an off-white solid (108 mg, 0.56 mmol, 97% yield). r f = 0.60, (ch 2 cl 2 /ch 3 oh = 4:1). 1 commercially available 5-bromo-2,4-dimethoxypyrimidine (200 mg, 0.91 mmol) was dissolved under n 2 in 20 ml of anhydrous thf and cooled to −78°c. etmgbr (3.0 m, 0.3 ml, 0.96 mmol) was added dropwise and allowed to stir for 10 min. zncl 2 (0.7 m in thf, 2.6 ml, 1.83 mmol) was subsequently added dropwise, stirred at −78°c for 10 min, warmed to room temperature and stirred for 2 h. the organozinc was added dropwise to a mixture of 4(5)-iodo-1himidazole (155 mg, 0.8 mmol), pd(pph 3 ) 4 (92 mg, 0.08 mmol) and cui (8 mg, 0.04 mmol) in 30 ml of thf and allowed to stir at reflux for 18 h. the reaction was quenched using 10 ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch 2 cl 2 (50 ml × 3), washed with brine (10 ml × 2) and the organic layer was dried over mgso 4 . the crude material was purified using silica gel column chromatography (ch 2 cl 2 /ch 3 oh = 1:0-9:1) to yield a white solid (72 mg, 0.35 mmol, 44% yield). r f = 0.75, (ch 2 cl 2 / ch 3 oh = 9:1). 1 (2,4-dimethoxy-5-pyrimidinyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (40) commercially available 5-bromo-2,4-dimethoxypyrimidine (578 mg, 2.64 mmol) was suspended in 30 ml degassed dmf under n 2 , followed by addition of bis(pinacolato)diboron (804 mg, 3.17 mmol), potassium acetate (777 mg, 7.92 mmol) and pdcl 2 (dppf) 2 ·chcl 3 (108 mg, 0.13 mmol). the mixture was heated to 100°c and stirred for 1 h. the reaction was cooled and transferred to 50 ml dh 2 o. the mixture was extracted in etoac/toluene (1:1, 20 ml × 3), washed with brine (10 ml × 2) and the organic layer was dried over mgso 4 . the solvent was removed in vacuo and the crude product was used without further purification. 6.20. synthesis of 2,2′,4,4′-tetramethoxy-5,5′-bipyrimidine (41) commercially available 5-bromo-2,4-dimethoxypyrimidine (250 mg, 1.14 mmol) and crude 40 (2.64 mmol) were suspended in 25 ml degassed 1,4-dioxane/dh 2 o (4:1) under n 2 in a sealed glass flask. cs 2 co 3 (1.12 g, 3.44 mmol) and pdcl 2 (dppf) 2 ·chcl 3 (46 mg, 0.06 mmol). the glass flask was sealed, heated to 105°c and stirred for 1 h. the flask was chilled to 0°c, opened, and warmed to room temperature. the crude content was filtered over a pad of celite and the solvent was evaporated in vacuo. the crude material was purified using silica gel column chromatography (hexanes/etoac = 2:1-3:7) to yield a white solid (212 mg, 0.76 mmol, 67% yield). r f = 0.40, (hexanes/ etoac = 2:1). 1 4-iodo-1-trityl-1h-imidazole (500 mg, 1.15 mmol) was dissolved under n 2 in 25 ml of thf and cooled to −78°c. etmgbr (3.0 m, 0.4 ml, 1.20 mmol) was added dropwise and allowed to stir for 10 min. zncl 2 (0.7 m in thf, 3.3 ml, 2.3 mmol) was subsequently added dropwise, stirred at −78°c for 10 min, warmed to room temperature and stirred for 2 h. the organozinc was added dropwise to a mixture of 26 (219 mg, 1.0 mmol), pd(pph 3 ) 4 (115 mg, 0.1 mmol), cui (10 mg, 0.05 mmol) in 40 ml of thf and allowed to stir at room temperature for 6 h. the reaction was quenched using 10 ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch 2 cl 2 (50 ml × 3), washed with brine (10 ml × 2) and the organic layer was dried over mgso 4 . the crude material was purified using silica gel column chromatography (hexanes/etoac = 2:1-1:4) to yield a white solid (307 mg, 0.68 mmol, 68% yield). r f = 0.50, (hexanes/ etoac = 1:1). 1 (43) 42 (307 mg, 0.68 mmol) was dissolved in 30 ml acetic acid and stirred for 48 h. the solvent was removed and the crude material was purified using silica gel column chromatography (ch 2 cl 2 / ch 3 oh = 1:0-9:1) to yield a white solid (133 mg, 0.64 mmol, 95% yield). r f = 0.80, (ch 2 cl 2 /ch 3 oh = 9:1). 1 molecular modeling study was performed as described in previous studies. 14, 38 briefly, the nmr structure of the nc in complex with a small molecule inhibitor was used as rigid receptor in molecular docking simulations, 37 which were carried out by the fred docking program from openeye, version 3.0.1. 39, 40 ligand conformational analysis was performed with omega from openeye. 52,53 the hiv-1 nc coding region in pnl4-3 55 was pcr amplified using the 5′-primer ccagctaccatacatatgcagaaaggc (ndei site underlined) and the 3′-primer ggccggatcctccctaactaattagcct gtc-tctc (bamhi and stop codon underlined). the expression vector pet-3a (novagen, madison, wi) was doubly digested with ndei and bamhi and treated with calf intestinal alkaline phosphate. the pcr product was purified by phenol-extraction and ethanol-precipitation and doubly digested with ndei and bamhi. the insert and vector were ligated using phage t4 dna ligase at 16°c for five hours and transformed into competent hms174. dna from transformants were sequenced and found to be identical with the hiv-1 nc coding sequence in pnl4-3. a clone, designated as prd2, overexpressed the 55-residue nc protein with the sequence m q k g n f r n q r k t v k c f n c g k e g h i a k n c r a p r k k g c w k c g k e g h q m k d c t e r q a n (the two zinc knuckles are underlined). ion-spray mass spectrometry confirmed the mass of the apoprotein to be 6369( ± 2) da (calculated 6369 da) and 6501( ± 2) da (calculated 6500 da) for the zn-bound protein. for protein expression of hiv-1 nc in escherichia coli, prd2 was transformed into bl21(de3) plyse. the purification scheme for the recombinant hiv-1 nc was adapted from ji et al. 56 and you & mchenry. 57 culture media were supplemented with 100 μg/l ampicillin and 34 μg/l chloramphenicol. a starter culture of 20 ml of zb 58 inoculated from a single colony was grown at 37°c overnight. the starter culture was added to 2 l of m9zb 58 supplemented with 0.1 mm zncl 2 and grown at 37°c to an absorbance at 600 nm of 0.5 to 0.6 before induction with 1 mm iptg (isopropyl-β-d-thiogalactopyranoside). after three hours, the cells were harvested by centrifugation, resuspended in 30 ml of lysis buffer (50 mm tris-hcl (ph 8.0), 10% (v/v) glycerol, 0.1 m nacl, 0.1 mm zncl 2 , 5 mm dithiothreitol, 2 mm edta), and stored at 70°c. to lyse the cells, the cells were thawed in ice-water, and 172 ml of 10 mm pmsf (phenylmethylsulfonyl fluoride), 30 ml of 1 mg/ ml pepstatin a, and 2.1 ml of 1% (w/v) sodium deoxycholate were added. the cells were sonicated by five bursts of 20 seconds to reduce the viscosity. the nucleic acids were precipitated by adding 4% (w/v) polyethyleneimine (ph 7.9) dropwise to a final concentration of 0.4% and stirred for 15 minutes before centrifugation at 23,000 g for 30 minutes at 4°c. the supernatant was collected (42 ml), filtered (0.45 μm pore size), and loaded at 1 ml/minute onto a 20 ml q-sepharose and a 20 ml sp-sepharose column (pharmacia) connected in series and previously equilibrated with 200 ml of buffer a (50 mm tris-hcl (ph 8.0), 10% glycerol, 0.1 m nacl, 0.1 mm zncl 2 , 10 mm bme (βmercaptoethanol)). after washing with 60 ml of buffer a, the q-sepharose column was detached, and the sp-sepharose column was washed with 1.5 column volumes of buffer a. a ten column volume linear gradient from 40% to 50% buffer b (50 mm tris-hcl (ph 8.0), 10% glycerol, 1.0 m nacl, 0.1 mm zncl 2 , 10 mm bme) was applied to elute the hiv-1 nc protein. the protein fractions were pooled (15 ml) and loaded at 0.5 ml/minute onto a 320 ml sephadex g-50 column (pharmacia) pre-equilibrated with two volumes of buffer c (50 mm tris-hcl (ph 7.0), 10% glycerol, 0.1 m nacl, 0.1 mm zncl2, 10 mm bme). the nc protein eluted at 175 ml and fractions were pooled (35 ml) for concentration and dialysis into nmr buffer (see below). nmr buffer (10 mm tris-hcl, ph 7.0, 140 mm kcl, 10 mm nacl, 1 mm mgcl 2 ) was deoxygenated by sparging with argon for 15 minutes and filter-sterilized (0.2 μm pore size). the protein sample was dialyzed using centricon-3 by adding nmr buffer five or six times (total volume 40-50 ml). the buffered protein sample was lyophilized for ease of storage. 25 μm protein samples were made in 500 μl of d 2 o and loaded into a 5 mm nmr tube. after taking the blank 1 h spectrum, the test compound was titrated into the protein sample such that 1:1 ratio of compound/protein could be established. data for 1 h nmr signal assignments were collected at a sample temperature of 10°c with a bruker dmx 600 mhz ( 1 h) nmr and bruker avance iii hd 500 mhz nmr spectrometers. the authors have no conflicts to declare. fleximers" design and synthesis of two novel split nucleosides fleximers" design and synthesis of a new class of novel shape-modified nucleosides unexpected inhibition of s-adenosyl-lhomocysteine hydrolase by a guanosine nucleoside conformational properties of shape modified nucleosides -fleximers substrate discrimination by the human gtp fucose pyrophosphorylase identification of catalytic amino acids in the human gtp fucose pyrophosphorylase active site molecular chameleons". design and synthesis of c-4-substituted imidazole fleximers design and synthesis of a second series of flexible nucleosides synthetic routes to a series of proximal and distal 2 '-deoxy fleximers design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity flex-nucleoside analogues -novel therapeutics against filoviruses carbocyclic 5 '-nor "reverse" fleximers. design, synthesis, and preliminary biological activity reverse" carbocyclic fleximers: synthesis of a new class of adenosine deaminase inhibitors structure-based identification of hiv-1 nucleocapsid protein inhibitors active against wild-type and drug-resistant hiv-1 strains use of virtual screening for discovering antiretroviral compounds interacting with the hiv-1 nucleocapsid protein molecular dynamics and dft study on hiv-1 nucleocapsid protein-7 in complex with viral genome predicting the binding mode of known ncp7 inhibitors to facilitate the design of novel modulators dna condensation by the nucleocapsid protein of hiv-1: a mechanism ensuring dna protection hiv-1 nucleocapsid protein zinc finger structures induce trna(lys,3) structural changes but are not critical for primer/template annealing genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1 nucleocapsid protein function in early infection processes analysis of ncp7-dependent activation of hiv-1 cdna integration and its conservation among retroviral nucleocapsid proteins structure of the hiv-1 nucleocapsid protein bound to the sl3 psi-rna recognition element affinities of packaging domain loops in hiv-1 rna for the nucleocapsid protein effects of the nature and concentration of salt on the interaction of the hiv-1 nucleocapsid protein with sl3 rna flexible nature and specific functions of the hiv-1 nucleocapsid protein retroviral rna dimerization and packaging: the what, how, when, where, and why properties, functions, and drug targeting of the multifunctional nucleocapsid protein of the human immunodeficiency virus identification of hiv-1 inhibitors targeting the nucleocapsid protein targeting the viral nucleocapsid protein in anti-hiv-1 therapy nucleocapsid protein: a desirable target for future therapies against hiv-1 the nucleocapsid protein of hiv-1 as a promising therapeutic target for antiviral drugs synthesis and evaluation of bifunctional aminothiazoles as antiretrovirals targeting the hiv-1 nucleocapsid protein time-resolved fluorescence investigation of the human immunodeficiency virus type 1 nucleocapsid protein: influence of the binding of nucleic acids probing the hiv-1 ncp7 nucleocapsid protein with site-specific gold(i)-phosphine complexes structure of the complex between the hiv-1 nucleocapsid protein ncp7 and the single-stranded pentanucleotide d(acgcc) discovery and structural characterization of a new inhibitor series of hiv-1 nucleocapsid function: nmr solution structure determination of a ternary complex involving a 2:1 inhibitor/nc stoichiometry identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the hiv-1 nucleocapsid protein fred pose prediction and virtual screening accuracy fred 3.0.1 openeye scientific software suppression of a palladium-mediated homocoupling in a suzuki cross-coupling reaction. development of an impurity control strategy supporting synthesis of ly451395 preparation of 1,3-diarylpropenes by phosphine-free palladium(o)-catalyzed suzuki-type coupling of allyl bromides with arylboronic acids highly efficient and accelerated suzuki aryl couplings mediated by phosphine-free palladium sources aryl couplings with heterogeneous palladium catalysts high yields of unsymmetrical biaryls via cross-coupling of arylboronic acids with haloarenes using a modified suzuki-beletskaya procedure greene's protective groups in organic synthesis synthesis and significant cytostatic activity of 7-hetaryl-7-deazaadenosines applications of palladium-catalyzed c-n cross-coupling reactions metal-catalyzed cross-coupling reactions. 2nd, completely rev. and enl ed how flexible are fleximer nucleobases? a computational study a convenient synthesis of 1,4-disubstituted imidazoles conformer generation with omega: algorithm and validation using high quality structures from the protein databank and cambridge structural database openeye omega 3.0.0.1: openeye scientific software dynamical behavior of the hiv-1 nucleocapsid protein production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone effect of human immunodeficiency virus type 1 (hiv-1) nucleocapsid protein on hiv-1 reverse transcriptase activity in vitro hiv nucleocapsid protein. expression in escherichia coli, purification, and characterization use of t7 rna polymerase to direct expression of cloned genes this work was supported by the national institutes of health t32 gm066706, r21 ai118470 (ksr), and r01 gm42561, r25 gm055036 (mfs). the authors also wish to thank the openeye free academic licensing program for providing a free academic license for molecular modeling and chemoinformatics software. supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmc.2019.05.019. key: cord-273777-qb0vp9gr authors: happel, anna-ursula; varsani, arvind; balle, christina; passmore, jo-ann; jaspan, heather title: the vaginal virome—balancing female genital tract bacteriome, mucosal immunity, and sexual and reproductive health outcomes? date: 2020-07-30 journal: viruses doi: 10.3390/v12080832 sha: doc_id: 273777 cord_uid: qb0vp9gr besides bacteria, fungi, protists and archaea, the vaginal ecosystem also contains a range of prokaryoteand eukaryote-infecting viruses, which are collectively referred to as the “virome”. despite its well-described role in the gut and other environmental niches, the vaginal virome remains understudied. with a focus on sexual and reproductive health, we summarize the currently known components of the vaginal virome, its relationship with other constituents of the vaginal microbiota and its association with adverse health outcomes. while a range of eukaryote-infecting viruses has been described to be present in the female genital tract (fgt), few prokaryote-infecting viruses have been described. literature suggests that various vaginal viruses interact with vaginal bacterial microbiota and host immunity and that any imbalance thereof may contribute to the risk of adverse reproductive health outcomes, including infertility and adverse birth outcomes. current limitations of vaginal virome research include experimental and analytical constraints. considering the vaginal virome may represent the missing link in our understanding of the relationship between fgt bacteria, mucosal immunity, and adverse sexual and reproductive health outcomes, future studies evaluating the vaginal microbiome and its population dynamics holistically will be important for understanding the role of the vaginal virome in balancing health and disease. despite the considerable literature published on bacterial communities, only a few human microbiota studies have focused on viruses, fungi, protists and archaea communities [1] [2] [3] , yet shifts in one community likely modulate community structure of others and vice versa. it is estimated that only about 1% of the human virome has been described at the sequence level [4] , and even less has been characterized functionally and hardly any studies have evaluated the virome in relation to reproductive health and the female genital tract (fgt). from various biological niches, such as the human gut, we know that viruses interact with other components of the microbiota and the human host, consequently influencing human health [5] [6] [7] [8] . however, little is known about the interactions of prokaryote-and eukaryote-infecting dna and rna viruses present in the fgt microbiota, collectively making up the vaginal virome, with other components of the vaginal microbiota or the human host, and its consequent impact on health outcomes. the primary outcome of this review was to summarize our current understanding of the pro-and eukaryote-infecting viruses making up the vaginal virome. secondary outcomes included describing interactions between the vaginal virome and other constituents of the vaginal microbiota, and possible associated adverse health outcomes. the literature search for this non-systematic review was conducted on 31 march 2020, using two databases (pubmed and googlescholar), with the search terms ("vagina" or "female genital tract" or "female reproductive tract") and ("virome" or "virus" or "viral" or "microbiome") and was limited to studies that were in published in english. a range of vaginal dna viruses infecting eukaryote cells have been identified by shotgun metagenomics of vaginal samples from generally healthy, asymptomatic women of reproductive age participating in the human microbiome project (hmp) [9] , including double-stranded (ds) dna (families adenoviridae, herpesviridae, papillomaviridae and polyomaviridae) and single-stranded (ss) dna viruses (families anelloviridae) ( table 1 and figure 1 ). the most common viruses detected in the lower reproductive tract were alphapapillomaviruses, with 38% of the participants being infected with at least one alphapapillomavirus [9] . to date, more than 220 human papillomavirus (hpv) types have been identified, including at least 50 that preferentially infect the genital mucosa [10, 11] (table 1 and figure 1 ). longitudinal sampling in the hmp suggests that up to 50% of these papillomaviruses establish productive infections and were replication competent, as many of the viruses were detected over multiple time points in the same women. additional dsdna viruses have been identified in vaginal samples from pregnant women and women with reproductive disorders [12] [13] [14] that share sequence similarities to those in the families alloherpesviridae, iridoviridae, marseilleviridae, mimiviridae, phycodnaviridae and poxviridae (table 1 and figure 1 ). in a cohort of women undergoing in vitro fertilisation, herpesviridae, polyomaviridae, papillomaviridae and anelloviridae were present, with papillomaviridae being the most common virus family detected [15] , while in a cohort of sixty pregnant women, anelloviruses were the most commonly detected viruses in vaginal samples, being present in 42% of women screened [12] . in a cohort of women coinfected with human immunodeficiency virus (hiv) and hpv, four viral families were identified in the fgt: papillomaviridae, anelloviridae, genomoviridae and herpesviridae [16] . papillomavirus reads were more abundant in women with premalignant cervical lesions, which were also strongly associated with carrying multiple high-risk hpvs, while anellovirus read abundance was negatively correlated with host cd4+ t-cell counts [16] . similarly, 46 known hpv types were detected in the fgt of women living with hiv, in addition to viruses belonging to the polyomaand anelloviridae families [17] . whether a core vaginal virome exists or whether differences in vaginal viruses identified in these studies are due to variable demographic or clinical characteristics of the cohorts, to differences in laboratory or sequencing methods, or to the viral databases and viral annotation tools applied remains unclear. to our knowledge, only one sequencing study [13] reported rna viruses in the reproductive tract, belonging to the partitiviridae family (dsrna), of which fungi is the natural host. this is likely to be due to the poor stability of stored rna and more complex laboratory assays being required to sequence rna viruses. hiv-1, an ssrna reverse transcribing virus (ssrna-rt) in the family of retroviridae, is also detectable in fgt secretions of women living with hiv during viral shedding [18] [19] [20] . viruses 2020, 12, x for peer review 3 of 18 stored rna and more complex laboratory assays being required to sequence rna viruses. hiv-1, an ssrna reverse transcribing virus (ssrna-rt) in the family of retroviridae, is also detectable in fgt secretions of women living with hiv during viral shedding [18] [19] [20] . while none of the women participating in the hmp had any genital symptoms, the high prevalence of eukaryote-infecting vaginal viruses raises the question of whether these vaginal viruses play a role in reproductive health. as the majority of humans remain asymptomatic to some viral infections, it has been proposed that viruses have become part of the metagenome of "healthy" individuals, rarely causing disease and remaining dormant within the host [21] . however, some welldescribed disease-causing viruses, such as high-risk hpv types, herpes simplex virus (hsv)-2 and polyomaviruses were also described to be part of the vaginal virome of some individuals despite being asymptomatic (table 1 and figure 1 ). furthermore, viruses that do not overtly cause disease yet establish chronic infections have been shown to influence immunity at other mucosal surfaces, such as the gut [22, 23] , and this is also likely to occur in the lower reproductive tract and is thus discussed in more detail below. while none of the women participating in the hmp had any genital symptoms, the high prevalence of eukaryote-infecting vaginal viruses raises the question of whether these vaginal viruses play a role in reproductive health. as the majority of humans remain asymptomatic to some viral infections, it has been proposed that viruses have become part of the metagenome of "healthy" individuals, rarely causing disease and remaining dormant within the host [21] . however, some well-described disease-causing viruses, such as high-risk hpv types, herpes simplex virus (hsv)-2 and polyomaviruses were also described to be part of the vaginal virome of some individuals despite being asymptomatic (table 1 and figure 1 ). furthermore, viruses that do not overtly cause disease yet establish chronic infections have been shown to influence immunity at other mucosal surfaces, such as the gut [22, 23] , and this is also likely to occur in the lower reproductive tract and is thus discussed in more detail below. * including only subfamilies, genera, species and types that have been described to infect the genital mucosa. risk of hpv types is indicated using different font types: high-risk, possibly high-risk and low risk. few shotgun metagenomic studies have investigated the presence or function of prokaryotic viruses in the lower reproductive tract of women. although prokaryotic-infecting viruses, from now on referred to as bacteriophages, are estimated to be amongst the most abundant living entities on earth [24] and are thought to play an important role in shaping the bacterial microbiota and associated health outcomes in the human gut [5, [25] [26] [27] , oral cavity [28, 29] , skin [30, 31] and lungs [32] , their role in the lower reproductive tract is understudied. functionally, bacteriophages are divided into lytic (virulent) and temperate types based on their differential ability to either lyse host cells (to release progeny bacteriophages) or to incorporate their genomes into the host cell genome as prophages and remain dormant, respectively [33] . several groups have identified functional and nonfunctional prophages in the genomes of vaginal bacterial species [34] [35] [36] [37] [38] . strong bioinformatic and in vitro evidence indicates that vaginal lactobacillus strains (including l. crispatus, l. gasseri, l. jensenii and l. plantarum isolates) carry inducible prophages [34] [35] [36] 38] . one third of all vaginal lactobacillus strains from south african women that have been examined to date were found to harbour at least one prophage, with l. crispatus more commonly harbouring prophages than l. jensenii [36] . for most of these lactobacillus prophages, however, factors determining their induction and permissive bacterial hosts are yet to be determined. prophages have also been described in the genomes of vaginal and urinary bacterial species that have been associated with adverse reproductive health outcomes, including gardnerella vaginalis [37] , group b streptococcus (gbs) [39] and enterococcus spp. [40] . among 39 gardnerella strains, a species associated with bacterial vaginosis (bv), more than 400 annotated prophage sequences have been identified and evidence of ongoing prophage acquisition within these gardnerella populations was present [37] . another study found that almost 90% of the examined genomes of vaginal and urinary gardnerella strains contained at least one prophage sequence [40] . similarly, almost 80% of gbs isolates had at least one prophage, which carried genes encoding factors previously associated with host adaptation and virulence [39] . the high abundance of prophage sequences within the genomes of vaginal bacterial species suggests that bacteriophages might play a role in shaping the bacterial microbiota of the fgt and associated health outcomes, similar to other biological niches. recent metagenomic sequencing of vaginal samples revealed that the majority of identified vaginal dna viruses are dsdna bacteriophages, similar to those in the families myo-, podo-and siphoviridae [14] . in addition, various unclassified viruses within the order caudovirales were found to be present. notably, only 4% of vaginal viruses identified by metagenomic sequencing by jakobsen et al. (2019) targeted eukaryotes [14] , confirming the high abundance of prokaryote-infecting viruses within the fgt microbiota (table 1 and figure 1 ). considering that a shift from lysogeny to a lytic lifecycle has been correlated with disease within the gut microbiota [41] , the role of bacteriophages for fgt health remains a crucially important yet underexplored area. limited studies have examined the interactions between all components of the vaginal microbiota and the human host. observational studies have shown that the acquisition and transmission of viral sexually transmitted infections (stis), including hsv-2, hpv and hiv, are more common in women with high diversity, nonoptimal vaginal bacterial microbiota [42] [43] [44] . however, many studies did not adjust for confounders to this relationship, such as the presence of other stis, sexual risk behaviour (condomless sex) and circumcision status of sexual partners. recent evidence from longitudinal studies suggests that changes in the bacterial microbiota and associated immunomodulatory metabolites precede incidental stis [45] and have been associated with a higher rate of hpv persistence and genital hsv-2 shedding [43] . data from the hmp further indicated that alpha-papillomaviruses were more common in women with a high vaginal bacterial diversity than in those who had a lactobacillus-dominant microbiota [9] . strong co-abundances between bacteriophages and predicted bacterial hosts were observed in a metagenomic study [14] , further suggesting that viral and bacterial communities interact within the lower reproductive tract. as such, links between viral community composition and the presence of l. crispatus, l. iners, g, vaginalis and a. vaginae in the fgt were found [14] . diversity changes in the vaginal eukaryotic dna virome over the course of pregnancy appeared similar to concomitant changes in the vaginal bacteriome, and pregnant women with highly diverse vaginal viromes tended to also have highly diverse vaginal bacteriomes [12] . while a relationship between bacterial and viral communities within the human lower reproductive tract appears to be supported by these studies, the directionality of this relationship is not clear and causality is yet to be established. it remains to be determined if changes in the bacterial microbiota are driven by changes in the viral community structure, or if susceptibility, persistence and clearance of viral infections is influenced by the vaginal bacterial microbiota. it is also important to acknowledge that underlying indirect mechanisms may regulate both, bacterial and viral communities, in addition to a more direct microbial mechanism implied by available studies. there is clinical and in vitro evidence that the vaginal bacterial microbiota influences colonisation with fungal strains [46, 47] , albeit studies investigating yeast-viral interactions in the fgt are limited. the majority of available research of yeast-viral interactions focuses on the relationship between viral stis and candida coinfections, in which vaginal candida infection was investigated as a risk factor for hiv transmission [48, 49] . however, this is likely to be the results of mucosal barrier disruption and/or inflammation associated with vulvovaginal candidiasis. seventy-five percent of women experience at least one episode of candidiasis during their life, and the risk for frequent, more invasive and resistant infections in persons living with hiv is high, likely due to t cell immune defects [50] . other in vitro studies have shown that hsv-2 significantly enhances binding of candida albicans to hela cells [51] , which the authors conclude suggests that hsv-2 increases candida persistence in the fgt. interestingly, in vitro studies have also shown that hsv-1 and coxsackievirus-b5 were retained in and released by c. albicans biofilms, in which the viruses remained viable and protected from antiviral agents as well host factors [52] . pseudomonas phages have been described to inhibit c. albicans biofilms as well as their planktonic growth [53] . whether yeast-infecting bacteriophages are commonly present in the vagina is unknown, although as mentioned previously, partitiviridae, typically a fungal phage, have been identified in the female reproductive tract [13] . in turn, hsv-1 has been shown to protect c. albicans by downregulating monocyte-mediated anti-candida immune system responses [54] , suggesting bilateral interactions between fungal and viral communities in the fgt. hiv-1 envelope and transactivating proteins have also been shown to bind to c. albicans, which may promote fungal virulence by inducing hyphae formation [55] [56] [57] . although the clinical relevance of these in vitro results remains to be elucidated and research on a broader range of vaginal viruses and fungi is needed, these studies suggest that there may be clinically relevant interactions between viral and fungal communities in the fgt and that an imbalance between these may influence reproductive health. microbial communities also interact with the host and influence innate immunity, immune cell activation and cytokine secretion. a balanced interplay between the vaginal microbiome and host immunity is crucial to prevent infections on the one hand but to maintain an immunotolerant environment, particularly during pregnancy, on the other hand. while the interaction of the vaginal bacteriome with the host has been reviewed by others [43, 58, 59] , there are less data available on the effect of eukaryote-infecting viruses other than viral stis, prokaryote-infecting viruses and the collective virome on host immunity. the innate immune system, including epithelial cells and mucus, toll-like receptors, antimicrobial peptides and defensins, cytokines and innate immune cells, is an important host immune defence mechanism in the fgt, collectively minimizing the risk of viral infection upon exposure [60, 61] . similarly, cellular and humoral adaptive immune responses to viral stis have been described in detail. while hiv-1 infection leads to induction of cd8+ t-cell responses in the cervical mucosa [61] , hsv-2 infection has been associated with cervical cd4+ t cell numbers and a distinct cytokine profile including various cytokines without significant alterations in local proinflammatory cytokines [42] , and hpv infection has been associated with a t helper (th) 1 cytokine immune response and elevated proinflammatory and regulatory cytokines [62] [63] [64] . in germ-free mice, expansion of enteric bacteriophages has been linked to immune cell expansion and increased inflammation in the gut, and phage dna isolated from human faeces stimulated interferon (ifn)-γ production by dendritic cells in mice [8] . further, certain viruses may be important for conditioning of immune responses. in the gut, infection by a persistent strain of murine norovirus compensated for the absence of bacteria in germ-free mice by restoring intestinal morphology and by promoting lymphocyte differentiation [22] . the same virus protected mice from lung injury following infection with pseudomonas aeruginosa and restored serum immunoglobulins in germ-free mice to levels observed in conventional mice [65] , yet the relevance of these findings for humans and specifically the fgt remains to be confirmed. these initial studies point towards an important role of the vaginal virome in innate and adaptive immunity of the fgt. while many of these initial findings are based primarily on disease-associated viruses, they may suggest that changes in the collective virome may similarly lead to altered mucosal immunity in the fgt and thus may impact sexual and reproductive health outcomes. as reviewed elsewhere, sexually transmitted viruses, like hsv-2, hpv, cytomegalovirus (cmv), hepatitis b or hiv, have been associated with a range of adverse health outcomes [66] [67] [68] [69] [70] [71] , including cervical cancer (hpv) [66] , genital ulcers (hsv-2), aseptic meningitis as well as vertical infections in infants, such as neonatal herpes (hsv-2) [67] or cirrhosis and liver cancer (hepatitis b) [69, 70] . bv is a risk factor for severe reproductive complications [72] [73] [74] and stis [49, 75, 76] , and it has been suggested by several authors [34, 77, 78] that bacteriophages are a contributing underlying biological cause for the rapid change in composition of vaginal bacterial communities associated with onset of bv and its difficulty to treat. with advances in shotgun metagenomics, further evidence is emerging that eukaryote-and prokaryote-infecting dna viruses, including bacteriophage communities, may differ between women with and without bv [14] , although others previously did not find any differences by vaginal bacterial community state type [44] , possibly due to different laboratory and analysis methods used. jakobsen et al. (2019) found that, while no significant difference in overall viral alpha diversity was present between groups, bacteriophage operational taxonomic units (otus) and predicted bacterial host otus strongly correlated in women with bv. miller-ensminger et al. (2018) observed variations between the prophage populations of women with and without overreactive bladder symptoms, suggesting that bacteriophages may contribute to genitourinary health [40] . infertility is defined by the failure to establish pregnancy after 12 months of regular sexual intercourse. while the most predictive factor for female infertility is a woman's age, biological and environmental factors are believed to contribute as well [79] . changes in the vaginal bacterial microbiota have been described as a risk factor for infertility [80] [81] [82] , and it is likely that viruses, such as human herpesviruses (hhv), also play a role. in up to 20% of infertile couples, the male or female partner had urogenital bacterial infections [83] , and in agreement with older women being more likely to be infertile, it has been described that seroprevalence of hhv-8, which similarly to hhv-6 can be transmitted via saliva, is increasing with increasing age [84] . hhv-6 dna was found in 43% of endometrial epithelial cells from infertile women in contrast to being completely absent from the endometrium of fertile women [85, 86] . hhv-6a-specific endometrial natural killer (nk) cell numbers and cytokine responses were elevated in women who had hhv-6a present [85] , suggesting that hhv-6 infections may modify endometrial immune cell and inflammatory profiles, resulting in the inability to sustain a successful pregnancy. further, endometrial nk cells had increased expression of several chemokine receptors and endometrial epithelial cells upmodulated the corresponding ligands, including monocyte chemotactic protein 1 (mcp1 and ccl2), interferon gamma-induced protein 10 (ip-10 and cxcl10) and eotaxin-3 (ccl26) [86] . others assessed whether hhv-6 played a role in preventing embryo implantation and found that 50% of women with two or more failed embryo transfers had detectable hhv-6 late viral proteins in endometrial epithelial cells and that women who were hhv-6 positive had undergone significantly more failed transfers than those who were hhv-6 negative [87] , further providing evidence that hhv-6 infection might influence fertility and pregnancy outcomes. in subfertile women undergoing in vitro fertilisation with a fresh embryo transfer, an association of the eukaryotic vaginal virome with prophylactic antibiotic exposure and reproductive outcomes has been described [15] . while there was no association between viral diversity and clinical pregnancy overall, a higher diversity of herpesviruses and alphapapillomaviruses was present in women who received prophylactic azithromycin treatment compared to women not receiving azithromycin. further, in women receiving azithromycin, viral diversity was higher in women whose embryo transfer did not result in clinical pregnancy compared with those who achieved clinical pregnancy [15] , suggesting that both bacterial and viral components of the vaginal microbiota may influence the ability to achieve clinical pregnancy. a number of clinically relevant viruses can be vertically transmitted from mothers to their foetus, including zika virus, hiv, hepatitis b virus, hepatitis c virus, hsv-1 and -2, varicella zoster virus, rubella virus, parvovirus b19 and cmv, and can cause stillbirth or severe morbidity in infants [88] [89] [90] [91] [92] [93] [94] . in the context of preterm birth (ptb) and small-for-gestational age (sga) births, biological mechanisms are still poorly understood. sequence-based studies of the vaginal bacterial microbiota have revealed that high-diversity bacterial communities [95] [96] [97] and possibly increased concentrations of vaginal inflammatory markers [95, 98] might contribute to increased risk for these adverse birth outcomes (abos). recently, a higher richness of the vaginal eukaryotic dna virome, including viral sequences similar to those in the families adenoviridae, anelloviridae, herpesviridae, papillomaviridae, polyomaviridae and poxviridae, has been linked to spontaneous and medically indicated ptb in north american women, but not one specific virus or group of viruses could be linked to ptb [12] . having both high bacterial and viral diversity in the first trimester of pregnancy was linked to the highest risk for ptb, indicating that the interplay of bacterial and viral communities or an imbalance thereof may be a mechanism by which ptb is triggered. neither whether higher viral diversity would also be associated with ptb in other populations nor how demographic factors influence the vaginal virome during pregnancy, are clear. focusing on specific viral groups rather than the collective virome, vaginal hpv infections have been linked to sga and low birth weight in observational studies, independently of other risk factors [99, 100] . in silico analyses have revealed significant associations between invasive neonatal-infecting gbs isolates and harbouring of a specific group of prophages within their genomes [39] . various viruses were also identified in amniotic fluid samples obtained from pregnancies with adverse outcomes, with adenovirus, cmv and enterovirus being the most common, while few healthy pregnancy controls had any virus detected [101] . more than half of women delivering infants with intrauterine growth restriction had viruses in their amniotic fluid during pregnancy, and adenovirus was detected in the amniotic fluid collected by amniocentesis in 60% of these women [101] . culture-and sequencing-based studies have found evidence of bacterial colonisation in amniotic fluid from pregnancies with abos [102] [103] [104] , suggesting that similarly viruses might be present in amniotic fluid in pregnancies with adverse outcomes, where anatomical, physiological or immunological barriers are compromised. it remains to be determined whether bacterial and viral communities are present in amniotic fluid from healthy pregnancies, as recent sequencing-based studies have drawn conflicting conclusions [105] [106] [107] [108] . it also has been shown in vitro that adenoviral infection of extravillous trophoblast cells in the presence of maternal decidual lymphocytes induces trophoblast apoptosis [109] , indicating that the maternal inflammatory response to adenovirus might induce placental cell death and subsequent abo. in mouse models of pregnancy, however, a combination of bacteria and eukaryotic-infecting viruses induced ptb, but neither alone was capable of causing ptb [110, 111] . similarly, viral infection of the murine cervix and placenta has been shown to alter the inflammatory responses to subsequent bacterial infection of the fgt [110] [111] [112] , indicating that viral infection of the fgt during pregnancy may alter the capacity to control ascending bacterial infections, which subsequently may lead to abo. data relating to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection of the fgt and the consequent relationship with pregnancy outcomes is sparse but continually accruing. a recent systematic review and meta-analysis of 79 pregnant women with coronavirus-related symptoms, of which half had confirmed sars-cov-2 infection, reported a significant prevalence of placenta-mediated disorders with high rates of miscarriage, ptb, preeclampsia and foetal growth restrictions [113] . while there is currently still conflicting yet continuously accumulating evidence for the presence of sars-cov-2 in vaginal secretions, amniotic fluid, cord blood or breastmilk in infected women [114] [115] [116] [117] , infection and visualization of sars-cov-2 in placental tissue has been demonstrated thoroughly [116, 118] . the first case of transplacental transmission of sars-cov-2 from a pregnant woman affected by covid-19 during late pregnancy to her neonate has been described [116] , suggesting potential perinatal sars-cov-2 transmission. further investigations are warranted to confirm vertical transmission of sars-cov-2 and long-term consequences thereof. the growing interest in the field of vaginal virome requires standardisation of laboratory protocols and analysis pipelines, including identification of rna viruses, adequate use of negative controls to account for contamination with environmental viruses, continued development of high-throughput sequencing accessibility, and advancement in viral annotation databases and tools. adequate use of viral nucleic acid extraction methods or kits that have not been associated with contamination as well as removal of bacterial and human nucleic acids either during the extraction process or after sequencing are crucial. it is concerning that various sequencing-based studies have described the presence of viruses in the fgt that share similarities to nonhuman, nonbacterial and nonfungal virus families. for example, vaginal iridoviridae have been described, which were previously only thought to infect ectothermic vertebrates, insects and crustaceans [119] . similarly, phycodnaviridae were also described to be present in vaginal samples, which were previously only thought to infect algae [120] . while this suggests that these viral sequences might have been present due to contamination or might have been incorrectly taxonomically classified or that they share similarities to sequences of viruses in these families (since there are clearly protein homologues found across viral families, especially the rna-dependent rna polymerases, helicase domains of replication proteins and, in some cases, capsid proteins [121] ), it might be possible that these findings are real due to vaginal insertional or hygiene practices. further, analysis of low biomass samples, such as breastmilk, placenta or amniotic fluid, requires rigorous use of controls, including nucleic acid extraction and pcr controls, as well as environmental swabs collected and processed as samples as additional negative controls. there is a paucity of research on the vaginal virome and the interplay of vaginal bacterial and viral communities and host immunity and its likely effect on sexual and reproductive health outcomes. once systematic laboratory and analysis pipelines have been established for both dna and particularly rna viruses, the vaginal virome should be characterized in broader populations, such as pre-and postmenopausal women and women from different demographics, and in connection with other components of the vaginal microbiota and the human host. functional characterization of viruses present in the vaginal virome and evaluation of their effect on reproductive and sexual health outcomes are crucial, and highly detailed longitudinal clinical cohorts with frequent sampling or animal models will be required to assess causality. funding: this review was funded in part by r01 hd083040-02s1. the authors declare no conflict of interest. a comprehensive non-redundant gene catalog reveals extensive within-community intraspecies diversity in the human vagina archaea as emerging organisms in complex human microbiomes methanogenic bacteria in human vaginal samples the virome in mammalian physiology and disease bacteriophages in gut samples from pediatric crohn's disease patients: metagenomic analysis using 454 pyrosequencing disease-specific alterations in the enteric virome in inflammatory bowel disease altered virome and bacterial microbiome in human immunodeficiency virus-associated acquired immunodeficiency syndrome expansion of bacteriophages is linked to aggravated intestinal inflammation and colitis metagenomic analysis of double-stranded dna viruses in healthy adults prevalence and viral load of 51 genital human papillomavirus types and three subtypes international human papillomavirus reference center the vaginal eukaryotic dna virome and preterm birth the metagenome of the female upper reproductive tract characterization of the vaginal dna virome in health and dysbiosis: an opening study in patients with non-female factor infertility association of the eukaryotic vaginal virome with prophylactic antibiotic exposure and reproductive outcomes in a subfertile population undergoing in vitro fertilisation: a prospective exploratory study composite analysis of the virome and bacteriome of hiv/hpv co-infected women reveals proxies for immunodeficiency comprehensive profiling of the vaginal microbiome in hiv positive women using massive parallel semiconductor sequencing vaginal hiv-1 shedding among hiv-1 infected women in the current era of combined antiretroviral therapy: a cross sectional study determinants of hiv-1 shedding in the genital tract of women hiv-1 shedding from the female genital tract is associated with increased th1 cytokines/chemokines that maintain tissue homeostasis and proportions of cd8+foxp3+ t cells viral immunity. transkingdom control of viral infection and immunity in the mammalian intestine an enteric virus can replace the beneficial function of commensal bacteria enteric virome sensing-its role in intestinal homeostasis and immunity bacteriophage resistance mechanisms healthy human gut phageome dysbiosis in inflammatory bowel disease: a role for bacteriophages ? smoking in inflammatory bowel disease: impact on disease course and insights into the aetiology of its effect evidence of a robust resident bacteriophage population revealed through analysis of the human salivary virome metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity propionibacterium acnes bacteriophages display limited genetic diversity and broad killing activity against bacterial skin isolates the diversity and host interactions of propionibacterium acnes bacteriophages on human skin puchhammer-stockl, e. temporal dynamics of the lung and plasma viromes in lung transplant recipients diversity of phage infection types and associated terminology: the problem with 'lytic or lysogenic induction of vaginal lactobacillus phages by the cigarette smoke chemical benzo[a]pyrene diol epoxide comparative study of vaginal lactobacillus phages isolated from women in the united states and turkey: prevalence, morphology, host range, and dna homology identification of predominant culturable vaginal lactobacillus species and associated bacteriophages from women with and without vaginal discharge syndrome in south africa genomes of gardnerella strains reveal an abundance of prophages within the bladder microbiome bacteriophage induction versus vaginal homeostasis: role of h 2 o 2 in the selection of lactobacillus defective prophages analysis of the prophages carried by human infecting isolates provides new insight into the evolution of group b streptococcus species bacteriophages of the urinary microbiome the human gut phage community and its implications for health and disease distinct effects of the cervicovaginal microbiota and herpes simplex type 2 infection on female genital tract immunology interplay among vaginal microbiome, immune response and sexually transmitted viral infections lactobacillus-deficient cervicovaginal bacterial communities are associated with increased hiv acquisition in young south african women 3 the vaginal microenvironment prior to incident sti vaginal lactobacilli inhibit growth and hyphae formation of candida albicans associations between the vaginal microbiome and candida colonization in women of reproductive age candida infection as a risk factor for hiv transmission bacterial vaginosis and vaginal yeast, but not vaginal cleansing, increase hiv-1 acquisition in african women determinants of symptomatic vulvovaginal candidiasis among human immunodeficiency virus type 1 infected women in rural herpes simplex virus (hsv) modulation of staphylococcus aureus and candida albicans initiation of hela 299 cell-associated biofilm human pathogenic viruses are retained in and released by candida albicans biofilm in vitro pseudomonas phage inhibition of candida albicans herpes simplex virus type 1 dysregulates anti-fungal defenses preventing monocyte activation and downregulating toll-like receptor-2 human immunodeficiency virus type 1 tat binds to candida albicans, inducing hyphae but augmenting phagocytosis in vitro human immunodeficiency virus type 1 gp41 binds to candida albicans via complement c3-like region human immunodeficiency virus type 1 gp160 and gp41 binding to candida albicans selectively enhances candidal virulence in vitro the interplay of host immunity and environment on risk of bacterial vaginosisand associated reproductive health outcomes host-vaginal microbiota interactions in the pathogenesis of bacterial vaginosis role of the innate immunity in female reproductive tract innate and adaptive immune responses in male and female reproductive tracts in homeostasis and following hiv infection evaluation of cytokines in endocervical secretion and vaginal ph from women with bacterial vaginosis or human papillomavirus cervical cytokines and clearance of incident human papillomavirus infection: hawaii hpv cohort study hpv infection and the genital cytokine milieu in women at high risk of hiv acquisition protective role of murine norovirus against pseudomonas aeruginosa acute pneumonia human papillomavirus vaccination: the population impact review of mathematical models of hsv-2 vaccination: implications for vaccine development effect of hsv-2 infection on subsequent hiv acquisition: an updated systematic review and meta-analysis chronic hepatitis b virus infection and total and cause-specific mortality: a prospective cohort study of 0.5 million people hepatitis b vaccination during pregnancy for preventing infant infection systematic review of the birth prevalence of congenital cytomegalovirus infection in developing countries role of bacterial vaginosis in peripartum infections early pregnancy changes in bacterial vaginosis-associated bacteria and preterm delivery evaluation, p.i.d.; study investigators, c.h. bacterial vaginosis and anaerobic bacteria are associated with endometritis bacterial vaginosis associated with increased risk of female-to-male hiv-1 transmission: a prospective cohort analysis among african couples vaginal bacterial phaginosis ? phage infection in vaginal lactobacilli: an in vitro study fertility and infertility: definition and epidemiology anaerobes and bacterial vaginosis in pregnancy: virulence factors contributing to vaginal colonisation risks associated with bacterial vaginosis in infertility patients: a systematic review and meta-analysis bacterial vaginosis and infertility: cause or association? bacterial agents as a cause of infertility in humans age-specific seroprevalence of human herpesvirus 8 in mediterranean regions presence of hhv-6a in endometrial epithelial cells from women with primary unexplained infertility hhv-6a infection of prospective evaluation of human herpesvirus 6 (hhv-6) in endometrial tissues of women with repeat implantation failure maternal-fetal interplay in zika virus infection and adverse perinatal outcomes. front hepatitis e virus infection among pregnant women in africa: systematic review and meta-analysis a systematic review and meta-analysis of vertical transmission route of hiv in ethiopia vertical transmission of herpes simplex virus: an update cytomegalovirus infection in pregnancy outcome of fetuses with congenital parvovirus b19 infection: systematic review and meta-analysis fetal varicella-diagnosis, management, and outcome the vaginal microbiome and preterm birth vaginal microbiome signature is associated with spontaneous preterm delivery establishment of vaginal microbiota composition in early pregnancy and its association with subsequent preterm prelabor rupture of the fetal membranes mid-gestational changes in cervicovaginal fluid cytokine levels in asymptomatic pregnant women are predictive markers of inflammation-associated spontaneous preterm birth human papillomavirus infection and intrauterine growth restriction: a data-linkage study preconception risk factors and sga babies: papilloma virus, omega 3 and fat soluble vitamin deficiencies detection of intrauterine viral infection using the polymerase chain reaction prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre-labor rupture of membranes microbial invasion of the amniotic cavity in pregnancies with small-for-gestational-age fetuses microbial invasion of the amniotic cavity in preeclampsia as assessed by cultivation and sequence-based methods human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community differential expression of the coxsackievirus and adenovirus receptor regulates adenovirus infection of the placenta1 viral infection of the placenta leads to fetal inflammation and sensitization to bacterial products predisposing to preterm labor viral infection of the pregnant cervix predisposes to ascending bacterial infection type i interferon regulates the placental inflammatory response to bacteria and is targeted by virus: mechanism of polymicrobial infection-induced preterm birth outcome of coronavirus spectrum infections (sars, mers, covid 1-19) during pregnancy: a systematic review and meta-analysis sars-cov-2 is not detectable in the vaginal fluid of women with severe covid-19 infection clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records transplacental transmission of sars-cov-2 infection detection of sars-cov-2 in human breastmilk visualization of sars-cov-2 virus invading the human placenta using electron microscopy unveiling crucivirus diversity by mining metagenomic data key: cord-254279-7u6ap4g4 authors: zwick, michael b; saphire, erica o; burton, dennis r title: gp41: hiv's shy protein date: 2004 journal: nat med doi: 10.1038/nm0204-133 sha: doc_id: 254279 cord_uid: 7u6ap4g4 the first x-ray crystal structures of gp41, the protein that mediates fusion of hiv-1 to target cells, were solved in the mid-1990s. the structures provide a foundation for understanding viral entry and the mechanism of action of compounds that block fusion. the first fusion inhibitor has recently entered the clinic, and the hope is that more potent and broadly active compounds, based on molecular design, will follow. the structure of gp41 was a long-sought and elusive goal for hiv biologists. the difficulties of working with a membrane protein, the strong tendency of gp41 to aggregate, and other hurdles stood in the way. when chan et al. 1 and weissenhorn et al. 2 finally produced the first structures in 1997, followed shortly by tan et al. 3 , they began to give molecular form to the apparatus of viral fusion, whose outline had emerged earlier in the decade. the structures are the focus of much work to design new inhibitors of the fusion process, and serve as an outstanding example of how findings in basic science can stimulate exploration of new therapeutics. this is what fusion looks like: envelope spikes composed of gp41 and gp120 decorate the surface of hiv-1. the surface glycoprotein of the spike, gp120, engages cd4 and ccr5 or cxcr4 coreceptors, primarily on macrophages and cd4 + t cells. gp120 undergoes conformational changes to activate gp41, a transmembrane glycoprotein that uses conserved structural elements to mediate the fusion of the host and viral membranes. genetic information can then flow from the virus to the target cell. the structure of the spikes is dictated by the virus' need to bind and enter target cells while avoiding a host neutralizing antibody response. in such a response, host antibodies bind to the virus and prevent it from entering the target cell to manage this problem, hiv-1 has evolved to keep the vulnerable conserved regions of the envelope spike-such as those on gp41 that comprise the fusion machinery-hidden as much as possible. this serves two purposes: it restricts the ability of conserved epitopes to elicit antibodies (low immunogenicity), and it helps prevent antibodies that are elicited from binding to conserved epitopes (low antigenicity). neutralizing antibodies against conserved epitopes are a particular threat to hiv-1, as escape via mutation is more difficult than for variable regions of the envelope spikes. in the mature, 'untriggered' spike, much of the surface of gp41 is masked by gp120. after receptor engagement, during which gp41 is perhaps most vulnerable, access to the fusionintermediate state of gp41 is restricted by the closely opposing membranes. the fusion process itself takes only minutes, which significantly limits access to the transiently exposed epitopes on gp41. in contrast, the postfusion conformation of gp41 is a stable repeating unit that is highly immunogenic. this postfusion conformation appears to be so radically different from the fusion-intermediate conformations of gp41 that elicited antibodies are unable to bind to the intermediate conformations and therefore are unable to inhibit fusion. this description was given a molecular underpinning in 1997, with the x-ray crystal structures of the putative postfusion form of the first x-ray crystal structures of gp41, the protein that mediates fusion of hiv-1 to target cells, were solved in the mid-1990s. the structures provide a foundation for understanding viral entry and the mechanism of action of compounds that block fusion. the first fusion inhibitor has recently entered the clinic, and the hope is that more potent and broadly active compounds, based on molecular design, will follow. and coreceptor, gp120 undergoes conformational changes that expose gp41 and activate the fusion machinery, notably the n-hr regions and the fusion peptide (fp). gp41 is probably in a somewhat extended arrangement in which the n-hr region of three gp41 molecules form an α-helical bundle, positioning the fusion peptides for insertion into the host-cell membrane. the c-hr region of gp41 is drawn here without defined secondary structure, but some models show it in an α-helical conformation. gp41-targeting fusion inhibitors such as t20 act on the fusion-intermediate conformation(s) of gp41 to inhibit six-helix bundle formation. (c) after fusion. fusion seems to coincide with chain reversal, or 'jackknifing', such that the antiparallel n-hr and c-hr regions form a six-helix bundle, represented here by the structure of weissenhorn et al. 2 . hiv-1 gp41 (refs. 1-3). before then, it was known that the n-and c-terminal portions of the external domain of gp41 contain heptad repeat sequences. peptides corresponding to the n-terminal (n-hr) and c-terminal (c-hr) heptad repeat regions of gp41, notably t21 (dp107) and t20 (dp178), respectively, could inhibit hiv-1 entry into cells [4] [5] [6] . t20, rebranded as enfuvirtide, was approved by the us food and drug administration for patients last fall. to overcome the technical hurdles of crystallizing gp41, it was necessary to truncate the protein to make it stable and soluble. each of the three laboratories crystallized a proteolytically stable core of the extraviral domain of gp41; (devoid of the n-terminal hydrophobic fusion peptide, a disulfide-loop region linking the n-hr and c-hr regions, and the membrane-proximal region). although each of the groups crystallized fragments of different sizes, the resulting structures were nearly identical. gp41 was found to have a trimeric core consisting of parallel α-helices corresponding to the n-hr region of gp41. packed around this trimeric core were three, slightly oblique c-hr α-helices, arranged antiparallel to the inner core to create a trimer of heterodimers, or a six-helix bundle (fig. 1) . notably, this arrangement had also been observed in the ph-induced conformation of the influenza hemagglutinin molecule 7 and the transmembrane subunit of moloney murine leukemia virus 8 . the structural motif has since been found in ebola virus, newcastle disease virus, respiratory syncytial virus and the coronavirus linked to severe acute respiratory syndrome 9,10 . the motif is a striking example of convergent evolution, and has revealed a vulnerability to be exploited in drug design. the gp41 structures provide an understanding of how t20 and other gp41-targeted fusion inhibitors work, and shed light on the difficulties of designing vaccines that elicit neutralizing antibodies to gp41. the t20 peptide corresponds to the c-hr region of gp41 and inhibits hiv-1 entry by packing against the outer grooves on the trimer of α-helices, thereby preventing the formation of six-helix bundles 11 . resistance to t20 has been documented, typically involving one or two substitutions along the outer grooves in the n-hr region of gp41. the relatively new drug t1249, a peptide analog of t20, is an effective inhibitor of t20resistant isolates of hiv-1 and is in clinical trials (http://www.trimeris.com/pipeline/clinical.html). resistance to t1249 is anticipated but has yet to be documented. peptides corresponding to the n-hr region of gp41 inhibit six-helix bundle formation by targeting the c-hr region. monomeric n-hr peptides such as t21 are prone to aggregation, making them much less potent than t20; potency is increased substantially with soluble, trimeric n-hr constructs 12, 13 . in principle, antibodies that could bind to the n-hr inner helical core after receptor activation should also block entry of hiv-1, similar to the fusion inhibitors. in terms of vaccine design, however, eliciting neutralizing antibodies to this target may be difficult because of spatial and temporal limitations during the fusion process. the broadly neutralizing antibodies defined to date seem to recognize gp41 epitopes that are close to the viral membrane but accessible during fusion 14, 15 . vaccinologists await structures for the fusion intermediates. the gp41 structures have stimulated progress in the design and selection of fusion inhibitors. in one study, an engineered trimer of the inner n-hr helical peptides was used to select peptides from a random phage display library. these peptides, selected as proteolytically stable mirror-image or d-peptides, potently inhibit hiv-1 entry into cells 13 . in another study, investigators constructed a fivehelix bundle that lacks one of the outer c-hr peptides. the five-helix bundle potently inhibited hiv-1 entry, probably by binding to a single c-hr region on hiv-1 gp41 and preventing six-helix bundle formation 16 . in a particularly inspired demonstration of structure-based design, the solvent-exposed residues on one face of the α-helix of a gp41 c-hr peptide were substituted onto the solvent-exposed face of another helical scaffold, gcn-4. indeed, the resulting hybrid peptide blocked hiv-1 entry into cells 17 . there are major problems with peptide therapeutics, including the cost and difficulty of manufacturing them. t20 currently costs $20,000 per year per patient, and the supply has yet to satisfy the demand. in an initial effort to develop nonpeptide inhibitors of hiv-1 entry using the crystal structures of gp41, molecular docking algorithms have been used to search small molecule libraries in silico for compounds that might bind in a deep hydrophobic cavity in the inner n-hr peptide core. some of the bestranking compounds inhibited hiv-1 infectivity with modest potency in vivo 18 . another group used a short c-hr peptide as a scaffold for attaching a nonpeptide combinatorial library onto its n terminus. the library was screened against an engineered trimer of n-hr peptides, and a hybrid ligand, composed of a synthetic moiety linked to the c-hr peptide, was selected that was able to inhibit hiv-1 envelope-mediated cell-cell fusion 19 . further optimization of the nonpeptide moiety is envisioned as a route to a stand-alone, small-molecule fusion inhibitor. in another approach, a small molecule was designed to mimic a hydrophobic surface along one face of a heptad repeat α-helix; the proteomimetic was able to partially disrupt the sixhelix bundle and inhibit hiv-1 mediated fusion 20 . as the hidden secrets of gp41 continue to be revealed, new leads for hiv-1 therapies and vaccines will emerge. interesting new targets for intervention may be uncovered by obtaining detailed structural information on the native and fusion-intermediate conformations of gp41. it will also be useful to elucidate the influence on gp41 structure and function of the segments that were omitted in the gp41 crystal structures. small-molecule drug design has been extremely effective in inhibiting the hiv-1 protease and reverse transcriptase, but it remains to be seen how well it will work against the envelope complex. since drug resistance is so prevalent for hiv-1 and has already been documented for t20, drug cocktails may be the best solution. indeed, the range of potential targets on gp41 and a growing arsenal of other entry inhibitors 21 make synergy with existing and future drug combinations an exciting possibility. this material is part of nature medicine's 10 year anniversary series. for more content related to these special focus issues, please see http://www.nature.com/ nm/special_focus/anniversary/index.html proc. natl. acad. sci. usa 91 key: cord-300522-okbupw61 authors: sansone, clementina; brunet, christophe; noonan, douglas m.; albini, adriana title: marine algal antioxidants as potential vectors for controlling viral diseases date: 2020-05-07 journal: antioxidants (basel) doi: 10.3390/antiox9050392 sha: doc_id: 300522 cord_uid: okbupw61 as the covid-19 epidemic expands in the world, and with the previous sars epidemic, avian flu, ebola and aids serving as a warning, biomedical and biotechnological research has the task to find solutions to counteract viral entry and pathogenesis. a novel approach can come from marine chemodiversity, recognized as a relevant source for developing a future natural “antiviral pharmacy”. activities of antioxidants against viruses can be exploited to cope with human viral infection, from single individual infections to protection of populations. there is a potentially rich and fruitful reservoir of such compounds thanks to the plethora of bioactive molecules and families present in marine microorganisms. the aim of this communication is to present the state-of-play of what is known on the antiviral activities recognized in (micro)algae, highlighting the different molecules from various algae and their mechanisms of actions, when known. given the ability of various algal molecules—mainly sulfated polysaccharides—to inhibit viral infection at stage i (adsorption and invasion of cells), we envisage a need to further investigate the antiviral ability of algae, and their mechanisms of action. given the advantages of microalgal production compared to other organisms, the opportunity might become reality in a short period of time. the ongoing global emergency linked to the covid-19 pandemic [1] teaches us that viral diseases can be dramatic all over the world in the present period of climate, political, social and globalization change. scientific solutions were not available and society was unprepared, claiming that research activities for the discovery of compounds for the prevention and treatment of severe and acute viral infections are nowadays a priority. oxidative stress-a loss in the balance between the production of free radicals including reactive oxygen species (ros) and antioxidant cell signaling pathways [2] -can be a key factor of the pathogenesis in many acute or chronical human diseases [3] . dietary intake of exogenous antioxidants in humans has a well-known role in preventing and protecting cellular oxidative stress. they operate diverse mechanisms of action and possess different therapeutic effects. as a result, modern medicine tends to use them in the prevention and treatment of various oxidative-stress-associated diseases [4] . viral infections are promoted by oxidative processes, the latter acting on replication in infected cells, cell proliferation inhibition and cell apoptosis induction [5] . in patients affected by herpes simplex [6] , it was observed that an increase of the ros-induced membrane phospholipids peroxidation caused dysfunction of vital cellular processes such as membrane transport and mitochondrial respiration [7] . epstein-barr virus (ebv) infections cause an increase in dna damage and significant ros accumulation, and, interestingly, treatment with ros scavengers was able to lower dna damage in both mitogen-stimulated and ebv-infected cells [8] . in this framework, discovery and screening of antioxidant compounds with antiviral properties is promising since the treatment of viral diseases requires the suppression of viral replication and cell survival promotion. the most known ros scavenger compounds belong to different chemical families, such as polyphenols (phenolic acids, flavonoids, anthocyanins, lignans and stilbenes), carotenoids (xanthophylls and carotenes), sterols, or vitamins (vitamins b, d, e, and c) [9] . for instance, there is a lot of evidence of the capability of natural antioxidants such as vitamins c and e (ascorbic acid and tocopherol, respectively) or polyphenols (e.g., curcumin) scavenging ros levels in infected cells, inhibiting proapoptotic factors and thus reestablishing the intracellular equilibrium between the stress-related proteins (such as c-jun n-terminal kinases-jnk) and promitotic (mapk) and transcription factors (nf-kb) [10] [11] [12] [13] . the most effective antioxidants are mainly synthetized by photosynthetic organisms, sharing these precious compounds with herbivorous animals through diet, and bioaccumulating along the trophic web [14] , as occurs in marine systems [15] . indeed, marine organisms represent a rich source of antioxidants, in terms of quantity and/or diversity. vitamins b 12 , c, d, e, peptides, amino acids, chitooligosaccharide derivatives, carotenoids, sulphated polysaccharides, sterols, phlorotannins, phenolic compounds and flavones are examples of marine antioxidant richness [16] . it is not surprising, therefore, that marine pharmacology is increasingly growing. recently, mayer et al. [17] reported that 21 studies were published in 2014/2015, focusing on marine antiviral drugs acting against human enterovirus 71, human cytomegalovirus, human immunodeficiency virus type-1 (hiv-1), human herpes simplex virus (hsv), influenza virus, hepatitis b virus, murine norovirus, respiratory syncytial virus (rsv) or sindbis virus. the mechanism of action of these compounds is known for five of them, although for the others it is still undetermined [17] . the aim of the present study is to show the state of the art, exploring the potential antiviral activity of known marine antioxidant compounds. for that, we explore the relationship between oxidative stress and viral infections, looking for solutions through the deciphering of cell signaling pathways that can inhibit virus replication and infections, and the mechanisms of action of potential antiviral molecules. the global public health emergency of international concern by 30 january 2020 regards the novel coronavirus [18] , which is the direct cause of the severe pneumonia (covid-19), with a high rate of transmission and infectivity. the 85% of critically ill patients with covid-19 showed leukocytosis, high levels of monocytes and neutrophils, and lymphopenia [19] , which is a lack of lymphocytes, with patients dying with comorbidity and high levels of plasma cytokines [20] . lymphopenia and hypercytokinemia are directly correlated with increased severity, mortality and a dysregulated immunological response [21] . first epidemiological indications reveal that the covid-19 patients requiring intensive care are older and are more likely to have hypertension, diabetes, cardiovascular or cerebrovascular pathologies [22] . aging and chronic diseases induce chronic endothelial dysfunction that provokes disassembly of intercellular junctions, endothelial cell death and blood-tissue barrier disruption, along with enhanced leukocyte adhesion and extravasation, which could contribute to explaining the lymphopenia observed in severe covid-19 patients [23] . endothelial dysfunction increases oxidative stress and systemic inflammation, glycocalyx degradation and inducing a procoagulant and antifibrinolytic state [24] . in some, especially old patients, with covid19 infection together with other persistent comorbidity chronic states, these pathways could induce severe respiratory failure [25] . covid-19 infection enters into the elective targets for viral diseases, which are, more generally, epithelial tissues, lung (influenza virus, coronavirus [26] ), liver (hcv and hcv [27] ) and cervix (hpv [28] ), all of which are strongly sensitive to oxidative stress and damages [29] . ros play a fundamental role in the normal functioning of the immune system and in the induction of proliferation of t cells and immunological defense [30] . oxidative stress represents a key factor in inflammation cell signaling for the regulation of cytokines and growth factors, as well as for immunomodulation and apoptosis [31] . recent observations have drawn attention to the possibility that interactions between ros and the human immunodeficiency virus (hiv) may also play a role in the pathogenesis of many other viruses as well [32] . indeed, the role of oxidative stress has been demonstrated in a number of viral infection diseases [33] , where cell pro-oxidative signaling co-occurred with viral infections [34] . viral infection is generally divided into three stages: • stage i, which consists of virus adhesion, adsorption, entry and invasion of cells; • stage ii, or eclipse phase, during which the cell is forced to replicate multiple copies of virus genome; • stage iii, or maturity and release of virus particles (virions). viruses enter the cell through specific receptors and coreceptors using the phagocytosis mechanism. the virus must then break out of the vesicle by which it was taken up in order to gain access to the cytoplasm [35] . the activation of phagocytes induces the release of pro-oxidant cytokines, such as tumor necrosis factor alpha (tnfa), or interleukin-1 and 6, paralleled with a significant iron uptake by the reticuloendothelial system [36] . in turn, the release of tnfa from activated phagocytes induces different intracellular responses. first, it acts on cell mitochondria inducing a pro-oxidant effect through the inhibition of the site of superoxide production (site ii). second, tnfa induces the release of nuclear transcription factor kappa b(nf-k-b) from the cytoplasmic inhibitor protein ikb, activating at a genomic level the transcription of cellular and/or viral genes. many viruses induce host cell death, by apoptosis or pyroptosis among others [37] , probably to enhance replication and expansion into the entire organism [38] . for example, hiv-1 activates pathways causing cd4 t-cell death in infected hosts due to caspase-1-mediated pyroptosis triggered by abortive viral infection. pyroptosis corresponds to an intensely inflammatory form of programmed cell death in which cytoplasmic contents and proinflammatory cytokines, including il-1β, are released. this death pathway relies on the two signature events in hiv infection-cd4 t-cell depletion and chronic inflammation-and creates a pathogenic vicious cycle in which dying cd4 t cells release inflammatory signals inducing enlarged cell death [39] . considering this evidence, antioxidant compounds are able to inhibit immune system cells (lymphocytes) through apoptosis and then release proinflammatory cytokines (il-1, il-6 and tnfa) that are likely to prevent lymphopenia, hypoferremia and, thus lower the viral replication in patients suffering severe viral diseases [40] . antioxidants such as vitamin e [41] are able to prevent the inhibition of the site of superoxide production (site ii), while the nf-kb-induced gene transcription can be inhibited by thiol groups [42] belonging to molecules that strongly interact with the antioxidant system [43, 44] . antioxidant therapy might further be related to the inhibition of virally induced cell death by antioxidants that scavenge peroxides, such as n-acetylcysteine and glutathione peroxidase [45] . it was observed that the antioxidant n-acetylcysteine blocks viral production in monocyte cell lines, which are the major reservoir for hiv in infected patients [46] . the epigallocatechin-3-gallate from green tea blocks hiv entry [47] due to its antioxidant properties. therapeutic treatments to inhibit or reduce virus replication in human cells are unfortunately restricted. known examples are mostly anti-hiv drugs, such as ritonavir, sequinavir, or antiflu, such as lopinavir, abifìgavan, or anti-ebola reagents. they all can have short-term or long-term adverse effects [48] , which makes it necessary to explore new molecular moieties in the perspective of producing new pharmaceutical tools [49] . many antiviral drugs are synthetic organic chemicals or natural derived products, for example, from secondary metabolites of plants [50] . in the emergent "blue" technology era, there is a growing interest in marine-derived antiviral compounds. marine organisms represent a rich source of many antioxidants that are promising for the development of drugs for the prevention and treatment of various chronic and acute human diseases [51] . thus, thousands of compounds from various marine organisms such as algae, bacteria, fungi, marine invertebrates or sponges have been screened and 21 of them have demonstrated antiviral activities [17] . in 2018, fedoreyev et al. [2] reported that an antioxidant "soup", containing echinochrome a (pigment of sea urchins), ascorbic acid (vitamin c) and α-tocopherol (vitamin e) possess in vitro antiviral activity against rna-containing tick-borne encephalitis virus and dna-containing herpes simplex virus type 1 [2] . this study also demonstrated that a synergistic effect of antioxidant molecules can be effective against virus infections. while the research and development of antiviral compounds are mainly focused on macroalgae [52] , the interest in microalgae and cyanobacteria has increased since they are known to produce many antioxidant molecules, which also possess antimicrobial, anticancer and antiviral activities [53] . moreover, cyanobacteria and microalgae in general are promising since the relatively low-compared to higher plants-requirements for growth make them good candidates for the production of antiviral agents at an industrial scale [54] . the compounds mainly capable of antiviral activities comprise sulfated polysaccharides, phenolic compounds, and organic acids [55] . in recent years, it has been shown that marine sulfated polysaccharides possess a wide range of bioactivities, such as anticoagulant, antioxidant, anti-inflammatory, antiviral, antibacterial, antiproliferative, antitumor, anticomplementary and antiadhesive activities [56] . interestingly, the antiviral activity exhibited by these compounds is often related to their immunomodulation and anticancer activities [57] . the double effect, anticancer and antiviral, of these natural compounds is mainly related to their capability to inhibit cell proliferation and to activate ifn signaling pathways that inhibit kidney and liver cancer progression [58] . in general, antiviral compounds extracted from (micro)algae are mainly polysaccharides that have been screened against pathogenic human viruses ( table 1 ). the mechanisms of action of these compounds against viruses are not fully clarified, but the activity seems to be related to their antioxidant power inhibiting the different stages of the viral infection, and interfering with its adhesion, penetration or replication [59] . for example, the sulfated polysaccharide isolated from the cyanobacteria spirulina platensis, named spirulan, has exhibited potent antiviral activity against both the herpes simplex virus type 1 (hsv-1) and the human immunodeficiency virus type 1 (hiv-1) [87] . the sulfated polysaccharides (sps) carrageenans from red algae (rhodophyta) display antiviral effects on several viral agents [60] , and are significantly active against hpv [61] . they predominantly act by inhibiting the binding or the internalization of virus into the host cells [62] . the carrageenan isolated from gigartina skottsbergii exert promising antiviral activities towards diverse strains of hsv-1 and hsv-2 during virus attachment stage [63] and against human rhinovirus (hrv) proliferation by preventing the primary phases of virus replication [64] . a recent in vivo study in mice indicated that low molecular weight carrageenans (3, 5, and 10 kda), as well as acetylated and sulfated derivatives, were active against influenza virus and reduced the hiv activity by depolymerization and sulfation processes [65] . another class of sulfated polysaccharides is represented by galactans, present in the external membranes of red algae [66] . the various structural types of this class of polysaccharides display relevant antiviral activity against several enveloped viruses, such as hsv-1 and hsv-2, denv, hiv-1 and hiv-2, and hepatitis a virus [67] . the galactan sulfate (gs), isolated from the red alga agardhiella tenera, displays an effective control against hiv-1 and hiv-2, blocking the virus adhesion to the cell as well as the attachment of gp120 on cd4+ t cell receptor to hiv-1 gp120 [68] . another sulfated galactan isolated from the red seaweed, schizymenia binderi, presented highly selective antiviral effects against hsv types 1 and 2 by the inhibition of the attachment of virus to host cells [69] . the d,l-galactan hybrid c2s-3, extracted from the marine red alga cryptonemia crenulata, blocks the multiplication of denv-2 in vero cell line [70] . alginates are polysaccharides widely distributed in brown algae (phaeophyceae), which have also been particularly attractive for their antiviral activities [71] . in particular, an alginate named 911 exhibited promising activity against hiv-1 decrementing the activity of reverse transcriptase (rtase), discontinuing the virus adsorption, and immunostimulating the host cells [72] . alternative inhibitory results were also reported against the hepatitis b virus (hbv), where 911 alginate could inhibit the virus replication by suppressing the activity of dna polymerase [73] . furthermore, the sulfated polymannuroguluronate (spmg) is a promising anti-aids drug candidate, inhibiting the robust attachment of hiv-1 gp120 protein with cd4 molecules on the surface of t cells [74] . fucans are high-molecular-weight sulfated polysaccharides, present in the intercellular tissues or mucilaginous matrix of brown algae [75] . beside many bioactivities, such as antiproliferative, antiadhesive properties can protect the cells from viral infections [76] . for instance, the sulfated fucans from the brown seaweeds, dictyota mertensii, lobophora variegata, fucus vesiculosus, and spatoglossum schroederi, could prevent hiv infection via the blocking effect of the activity of reverse transcriptase [77] . this study highlighted the necessity of sulfate and carboxyl groups in the inhibitory viral activity of the polysaccharides. the compound named mc26, a new type of fucose polysaccharide isolated from the marine brown alga, sargassum piluliferum exhibited a strong anti-influenza virus activity [78] . furthermore, the sulfated fucans extracted from the brown seaweed cystoseira indica showed a promising activity against hsv-1 and hsv-2 [79] . it was suggested that these compounds might act against viral infection through the inhibition of virus adsorption [80] . the polysaccharides fucoidan, based on the sulfated l-fucose, possesses various biological activities, among them activities against many rna and dna viruses such as hiv, hsv1-2, dengue virus, and cytomegalovirus [81] . fucoidans exert their antiviral activities by blocking the interaction of viruses with the cells, thus inhibiting viral-induced syncytium formation [82] . this property led to attachment of fucoidan to the f0 protein resulting in a great antiviral potency of fucoidan, e.g., higher than the antiviral drug ribavirin [83] . last but not least, fucoidans also display bioactivity on the immune system at cellular and humoral level by increasing macrophage phagocytosis [84] . the glucan laminaran, one of the most common polysaccharides in brown algae, exhibits a great antiviral activity and low toxicity in vivo [78] . laminaran polysaccharides extracted from brown algae are proficient to prevent the activity of hiv by preventing its adsorption on human-derived lymphocytes and by blocking the ability of hiv reverse transcriptase, thus the virus' proliferation [72] . three polysaccharides extracted from marine microalgae, naviculan from the diatom navicula directa, and two others (named a1 and a2) from the dinoflagellate cochlodinium polykrikoides also displayed antiviral activities against several enveloped viruses, such as hiv-1, hsv-1 or influenza virus type a (ifv-a) [71] . the sulfated polysaccharide p-kg03 extracted from another dinoflagellate gyrodinium impudicum has been also reported active against the encephalomyocarditis rnavirus (emcv) [85] , and against several strains of influenza viruses, with efficiency comparable to some existing drugs [86] . the common characteristic between the above-described examples of antiviral activity from algal polysaccharide is the presence of sulfated groups in their chemical structure [96] . although these compounds might act on stage iii of viral infection, selectively inhibiting reverse transcriptase in the case of hiv, thus hampering production of new viral particles after infection [97] , the inhibitory effect generally might refer to stage i of viral infection. this is a crucial aspect to investigate since the antiviral property starts with the interaction between the molecule and the positive charges of the virus or on the cell surface, preventing penetration of the former into the host cells [98] . indeed, sulfated exopolysaccharides from some marine microalgae have been hypothesized to interfere with stage i of viral infection [99] . therefore, small molecules, such as sulfated polysaccharides, represent a good challenge in antiviral drug discovery studies, since the actual antiviral pharmaceutics are proteins and act at the stage ii of the infection. considering the covid19 disease [100] , this virus is very infective due to the high adhesion capacity on the oral cell surface [101] and for the easy entrance ability through the ace2 receptor on the lung cell surface (stage i of the viral infection) [102] . less investigated, but with a high antioxidant activity, and therefore as potential antiviral, are the polyphenolic compounds: antioxidants produced by marine algae, such as flavonoids, cinnamic acid, benzoic acid, gallic acid, quercetin [91] and phlorotannins, the latter being found in brown macroalgae [92] , or diatoms [93] . phlorotannins are tannin derivatives exhibiting numerous bioactivities, such as antioxidant, anti-inflammatory [94] antibacterial, and antimatrix metallopeptidases (mmp) activities [95] . ahn et al. [93] reported that phloroglucinol derivative 8,8 -bieckol inhibited the activity of recombinant rt and protease of hiv-1 in vitro. another peculiar polyphenol discovered in some diatoms, (haslea sp.) marennine, presented relevant bioactivity and antiviral properties [95] . viral infections are often promoted by oxidative processes, favoring replication in infected cells, induction and inhibition of cell proliferation. antiviral activities of antioxidants acting in the antiviral infection task can be exploited as the sea is a fruitful reservoir of such compounds. many algal sulfated polysaccharides-several of them being exclusively marine-present strong antiviral activities. furthermore, tannins, e.g., phlorotannin-exclusive in brown algae-as well as some phycobiliproteins-exclusive in marine algae-exert antiviral activities. to implement natural antiviral tools, there is a priority of: (i) investigating the antiviral activities and mechanisms of action, and (ii) focusing on microalgae, as fast dividing and easily exploitable organisms. the antiviral activity of various microalgae has been demonstrated, although there is a lack of information on many classes. since microalgae are known to be rich in bioactive molecules-the so-called biofactory cells-and present a high diversity and complementarity of antioxidant compounds, they are the ideal reservoir of a sea-derived antiviral pharmacy. this is a fundamental aspect since synergy between molecules, and combinations of diverse backbones, can be further effective against virus infections, as demonstrated in the development and success of the haart combination therapy for hiv. the promising results reviewed in this communication suggest and urge for investment and advanced research into the development of knowledge on marine antiviral drugs and to develop a pipeline reaching into the biodiversity and chemodiversity of (micro)algae screening to screen them with an antiviral focus. author contributions: all authors have equally contributed for the realization of this paper. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases antiviral and antioxidant properties of echinochrome a oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases mitochondria-targeted antioxidants for treatment of parkinson's disease: preclinical and clinical outcomes oxidative stress influences positive strand rna virus genome oxidative stress favors herpes virus infection in vertebrates: a meta-analysis herpes simplex virus type 1 infection induces oxidative stress and the release of bioactive lipid peroxidation by-products in mouse p19n neural cell cultures oxidative stress enables epstein-barr virus-induced b-cell transformation by posttranscriptional regulation of viral and cellular growth-promoting factors natural antioxidants in foods and medicinal plants: extraction, assessment and resources effects of vitamin e and c supplementation on oxidative stress and viral load in hiv-infected subjects antiviral potential of curcumin antiviral effect of polyphenol rich plant extracts on herpes simplex virus type 1 the role of food antioxidants, benefits of functional foods, and influence of feeding habits on the health of the older person: an overview aquatic primary producers as a driving force for ecosystem responses to large-scale environmental changes promises and challenges of microalgal antioxidant production marine compounds with antibacterial, antidiabetic, antifungal, anti-inflammatory, antiprotozoal, antituberculosis, antiviral, and anthelmintic activities; affecting the immune and nervous systems, and other miscellaneous mechanisms of action. mar. drugs emergence of a novel coronavirus causing respiratory illness from wuhan lymphopenic community acquired pneumonia as signature of severe covid-19 infection clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study pathological alteration and therapeutic implications of sepsis-induced immune cell apoptosis comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis shared features of endothelial dysfunction between sepsis and its preceding risk factors (aging and chronic disease) degradation of the endothelial glycocalyx in clinical settings: searching for the sheddases respiratory syncytial virus-induced oxidative stress in lung pathogenesis the pathology of influenza virus infections how hepatitis c virus invades hepatocytes: the mystery of viral entry human papilloma virus' life cycle and carcinogenesis oxidative stress and hpv carcinogenesis redox regulation of t-cell function: from molecular mechanisms to significance in human health and disease immune mechanisms linked to depression via oxidative stress and neuroprogression oxidative stress during viral infection: a review. free radic the role of oxidative stress in viral infections oxidative stress, prooxidants, and antioxidants: the interplay endocytosis of viruses and bacteria tumor necrosis factor-α signaling in macrophages staying alive: cell death in antiviral immunity how viral and intracellular bacterial pathogens reprogram the metabolism of host cells to allow their intracellular replication cell death by pyroptosis drives cd4 t-cell depletion in hiv-1 infection disabling of lymphocyte immune response by ebola virus the importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state inhibiting nf-κb activation by small molecules as a therapeutic strategy the role of thiols in antioxidant systems. free radic first evidence of the biosynthesis of the marine antioxidant ovothiol b in diatoms. free radic free radicals and antioxidants in normal physiological functions and human disease oxidative stress during hiv infection: mechanisms and consequences polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-hiv agent the treatment of influenza with antiviral drugs new perspectives on how to discover drugs from herbal medicines: cam's outstanding contribution to modern therapeutics a historical overview of natural products in drug discovery exploring the ocean for new drug developments: marine pharmacology antimicrobial action of compounds from marine seaweed evaluation of antibacterial, antifungal and antiviral activities of ten marine macroalgae from red sea microalgae as a source of bioactive molecules-experience from cyanophyte research the promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products sulfated polysaccharides as bioactive agents from marine algae partial characterization, the immune modulation and anticancer activities of sulfated polysaccharides from filamentous microalgae tribonema sp immunomodulatory and antitumor effects of type i interferons and their application in cancer therapy flavonoids as antiviral agents for enterovirus a71 (ev-a71) antiviral activity of sulfated polysaccharides carrageenan from some marine seaweeds in vitro inhibition of human papillomavirus following use of a carrageenan-containing vaginal gel iota-carrageenan is a potent inhibitor of influenza a virus infection antiviral activity of a carrageenan from gigartina skottsbergii against intraperitoneal murine herpes simplex virus infection iota-carrageenan is a potent inhibitor of rhinovirus infection preparation and potential in vivo anti-influenza virus activity of low molecular-weight κ-carrageenans and their derivatives galactans: an overview of their most important sourcing and applications as natural polysaccharides antiviral potential of algae polysaccharides isolated from marine sources: a review marine compounds and their antiviral activities antiviral activities of sulfated polysaccharides isolated from sphaerococcus coronopifolius (rhodophytha, gigartinales) and boergeseniella thuyoides (rhodophyta, ceramiales) an algal-derived dl -galactan hybrid is an efficient preventing agent for in vitro dengue virus infection current trends in marine algae polysaccharides: the digestive tract, microbial catabolism, and prebiotic potential marine algae metabolites as promising therapeutics for the prevention and treatment of hiv/aids. metabolites inhibition of hepatitis b virus replication by n-hydroxyisoquinolinediones and related polyoxygenated heterocycles the potential molecular targets of marine sulfated polymannuroguluronate interfering with hiv-1 entry. interaction between spmg and hiv-1 rgp120 and cd4 molecule fucoidans of brown algae: biosynthesis, localization, and physiological role in thallus marine polysaccharides from algae with potential biomedical applications current trends on seaweeds: looking at chemical composition, phytopharmacology, and cosmetic applications the antiviral activities and mechanisms of marine polysaccharides: an overview structural features and antiviral activity of sulphated fucans from the brown seaweed cystoseira indica the signaling pathways, and therapeutic targets of antiviral agents: focusing on the antiviral approaches and clinical perspectives of anthocyanins in the management of viral diseases defensive effects of a fucoidan from brown alga undaria pinnatifida against herpes simplex virus infection in vitro characterization of the antiviral activity of fucoidan from cladosiphon okamuranus against newcastle disease virus sulphated polysaccharides from ulva clathrata and cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion, in ndv infection biological activities of fucoidan and the factors mediating its therapeutic effects: a review of recent studies antiviral effects of sulfated exopolysaccharide from the marine microalga gyrodinium impudicum strain kg03 in vitro inhibition of influenza a virus infection by marine microalga-derived sulfated polysaccharide p-kg03 antiviral activity of arthrospira-derived spirulan-like substances anti-herpes simplex virus target of an acidic polysaccharide, nostoflan, from the edible blue-green alga nostoc flagelliforme inhibition of enterovirus 71-induced apoptosis by allophycocyanin isolated from a blue-green alga spirulina platensis anti-herpes simplex virus substances produced by the marine green alga, dunaliella primolecta antioxidant activity of marine algal polyphenolic compounds: a mechanistic approach phlorotannins as bioactive agents from brown algae phlorotannins from ecklonia cava (phaeophyceae): biological activities and potential health benefits antibacterial activity of crude extracts and phlorotannin isolated from the diatom cymbella spp haslea karadagensis (bacillariophyta): a second blue diatom, recorded from the black sea and producing a novel blue pigment sulfated seaweed polysaccharides as multifunctional materials in drug delivery applications molecular strategies to inhibit hiv-1 replication cell entry mechanisms of hsv: what we have learned in recent years antimicrobial activities of microalgae: an invited review. in science against microbial pathogens: communicating current research and technological advances; mendez-vilas, a., ed.; formatex microbiology book series coronavirus disease 2019 (covid-19) in italy mechanisms of coronavirus cell entry mediated by the viral spike protein ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia acknowledgments: the authors acknowledge the two reviewers for their comments on the previous version of the manuscript. the authors declare no conflict of interest. key: cord-266226-gxbrgy6g authors: lee, choongho title: griffithsin, a highly potent broad-spectrum antiviral lectin from red algae: from discovery to clinical application date: 2019-10-06 journal: mar drugs doi: 10.3390/md17100567 sha: doc_id: 266226 cord_uid: gxbrgy6g virus entry into a susceptible host cell is the first step in the formation of all viral diseases. controlling viral infections by disrupting viral entry is advantageous for antibody-mediated neutralization by the host’s immune system and as a preventive and therapeutic antiviral strategy. recently, several plant-derived carbohydrate-binding proteins (lectins) have emerged as a new class of antiviral biologics by taking advantage of a unique glycosylation pattern only found on the surface of viruses. in particular, a red algae-derived griffithsin (grft) protein has demonstrated superior in vitro and in vivo antiviral activity with minimum host toxicity against a variety of clinically relevant, enveloped viruses. this review examines the structural characteristics of grft, focusing on its carbohydrate-binding capability. its in vitro antiviral profiles against human immunodeficiency virus (hiv) are also discussed followed by a description of the results from a combination study using anti-hiv drugs. the results of several studies regarding its novel antiviral mechanism of action are provided in conjunction with an explanation of viral resistance profiles to grft. in addition, its in vitro and in vivo host toxicity profiles are summarized with its pharmacokinetic behavior using in vivo efficacy study results. also, a large-scale production and formulation strategy, as well as a drug delivery strategy, for grft as a new class of broad-spectrum microbicides is discussed. finally, results from two ongoing clinical studies examining grft’s effects on viruses are presented. every virus starts its life cycle by entering a susceptible host cell. the host cell-targeting ability of a virus is mainly determined by the presence of appropriate host cell receptors which are engaged by a virus. for successful host cell entry, enveloped viruses have evolved a diverse array of envelope glycoproteins on their membrane with different receptor association capabilities. the best-characterized example of this viral glycoprotein-host cell receptor relationship might be the interaction between the human immunodeficiency virus (hiv) glycoprotein gp120 and the human t lymphocyte cd4 molecule. due to its essential role in the overall virus life cycle, viral entry has been an attractive target for both vaccine and antiviral drug development with the goal of disrupting the binding of viral glycoproteins to host cell receptors. in addition, heavy and unique glycosylation patterns found only on viral glycoproteins provide another level of virus specificity, which could increase the selectivity of virus-targeting therapeutics and prophylactics. in this regard, the use of carbohydrate-binding proteins, collectively called "lectins", has been explored as a new therapeutic antiviral strategy. recently discovered virus-targeting lectins include banana lectin [1] , cyanovirin [2] , microvirin [3] , scytovirin [4] , and griffithsin (grft) [5] . among them, grft has been studied extensively for the development of corresponding secondary -sheet structures are marked with yellow arrows based on the crystal structure of a grft dimer with mannose at a resolution of 1.78 å [19] . tyrosine and aspartate residues in three carbohydrate-binding domains, which are essential for mannose-binding, are noted with blue and orange arrows, respectively. wildtype grft has a non-standard amino acid at position 31, but it is replaced by alanine (colored in red) in recombinant grft. the sequence display was generated by the rcsb protein data bank website (www.rcsb.org). the amino acid sequence of griffithsin (grft). corresponding secondary β-sheet structures are marked with yellow arrows based on the crystal structure of a grft dimer with mannose at a resolution of 1.78 å [19] . tyrosine and aspartate residues in three carbohydrate-binding domains, which are essential for mannose-binding, are noted with blue and orange arrows, respectively. wild-type grft has a non-standard amino acid at position 31, but it is replaced by alanine (colored in red) in recombinant grft. the sequence display was generated by the rcsb protein data bank website (www.rcsb.org). marine drug 2019, x, x; doi: www.mdpi.com/journal/marinedrugs resolution of 1.78 å [19] . tyrosine and aspartate residues in three carbohydrate-binding domains, which are essential for mannose-binding, are noted with blue and orange arrows, respectively. wildtype grft has a non-standard amino acid at position 31, but it is replaced by alanine (colored in red) in recombinant grft. the sequence display was generated by the rcsb protein data bank website (www.rcsb.org). grft is a carbohydrate-binding protein made of 121 amino acids and is 12.7 kda in size [9] . it has a unique amino acid residue at position 31 which does not match any of the 20 standard amino acids [5] (figure 1 ). it was initially isolated from an aqueous extract of the red algae griffithsia sp. collected from waters off the shores of new zealand. researchers at the u.s. national cancer institute first reported its potent cytoprotective activity against hiv-1 in t-lymphoblastoid cells [5] . five research papers reported structural results on grft by using either x-ray crystallography or nuclear magnetic resonance (nmr) techniques (table 1) [19] [20] [21] [22] [23] . in terms of structural classification, grft is a jacalin-related lectin harboring three repeats of an antiparallel four-stranded -sheet with a triangular prism shape ( figure 2 ) [19] . it is called a domain-swapped dimer because two -strands from one protein forming the dimer display domain-exchanging properties with the same two strands of its counterpart [19] . it has three identical carbohydrate-binding sites (located within residues 20-34, 58-76, and 96-120) on each monomer with three conserved glycine-rich repeats (ggsgg) [19] . it is estimated that as many as 11 high-mannose oligosaccharides are present on one hiv-1 gp120 protein. therefore, grft's multivalent interactions with gp120 via these three carbohydrate-binding domains seem to be essential for its high-affinity and anti-hiv-1 potency at figure 2 . the crystal structure of a grft dimer with six mannoses at a resolution of 1.78 å [19] . each grft monomer is presented in either red or blue. six bound mannose molecules are shown with ball and stick models. this image was created with pdb id and associated publication, ngl viewer (as rose et al. (2018) ngl viewer: web-based molecular graphics for large complexes. bioinformatics doi:10.1093/bioinformatics/bty419), and rcsb pdb. grft is a carbohydrate-binding protein made of 121 amino acids and is 12.7 kda in size [9] . it has a unique amino acid residue at position 31 which does not match any of the 20 standard amino acids [5] ( figure 1 ). it was initially isolated from an aqueous extract of the red algae griffithsia sp. collected from waters off the shores of new zealand. researchers at the u.s. national cancer institute first reported its potent cytoprotective activity against hiv-1 in t-lymphoblastoid cells [5] . five research papers reported structural results on grft by using either x-ray crystallography or nuclear magnetic resonance (nmr) techniques (table 1) [19] [20] [21] [22] [23] . in terms of structural classification, grft is a jacalin-related lectin harboring three repeats of an antiparallel four-stranded β-sheet with a triangular prism shape ( figure 2 ) [19] . it is called a domain-swapped dimer because two β-strands from one protein forming the dimer display domain-exchanging properties with the same two β-strands of its counterpart [19] . it has three identical carbohydrate-binding sites (located within residues 20-34, 58-76, and 96-120) on each monomer with three conserved glycine-rich repeats (ggsgg) [19] . it is estimated that as many as 11 high-mannose oligosaccharides are present on one hiv-1 gp120 protein. therefore, grft's multivalent interactions with gp120 via these three carbohydrate-binding domains seem to be essential for its high-affinity and anti-hiv-1 potency at low concentrations (picomolar range) [22] . in particular, three aspartate residues in these carbohydrate-binding sites (asp30, asp70, and asp112) play a critical role in the interaction of grft with high-mannose type oligosaccharides such as man9glcnac2, which is composed of nine mannose molecules and two n-acetyl glucosamines [22] . this was supported by a study demonstrating that grft point mutations (d30a, d70a, or d112a) partially inhibit its ability to bind to gp120, and a grft mutant with all three mutations loses almost all of its binding capacity [21] . in addition, tyrosine residues such as tyr28, tyr68, and tyr110 are necessary for grft to bind to carbohydrates [24] . however, the apparent disparity between the gp120-binding ability and hiv-1 inhibitory potency for these grft variants indicates the existence of another antiviral mechanism beyond simple gp120-grft binding [21] . with regard to carbohydrate-binding specificity, all six grft dimer carbohydrate-binding sites can be occupied by mannose, glucose, n-acetyl glucosamine, and 1-6 α-mannobiose with similar affinities [23] . to determine whether grft must form a dimer to exert its antiviral activity, a monomeric form of grft (mgrft) was created by inserting either two or four amino acids at its dimerization interface. mgrft possesses greatly reduced antiviral activity against hiv-1 in spite of its comparable association with high-mannose oligosaccharides, since mgrft possesses all three carbohydrate-binding sites [20] . this suggests that the intact dimeric form of grft is required to efficiently disrupt hiv-1 infectivity [20] . table 1 . five structural studies involving grft. the structure resolution of grft and its major characteristics are presented. grft alone or with mannose 1.3 and 0.94 a domain-swapped dimer, a jacalin-related lectin with a β-prism motif, and three identical mannose-binding sites on each monomer [19] grft with glucose or n-acetylglucosamine 30-1.50 and 30-1.56 all six monosaccharide binding sites of grft are occupied by both glucose and n-acetylglucosamine with a mode of binding similar to that of mannose [23] grft with 1-6α-mannobiose or maltose 2.0 and 1.5 the binding of 1-6α-mannobiose is similar to that of mannose and the binding of maltose is weaker than that of mannose [22] monomeric grft 30-0.97 reduced activity against hiv-1 due to a loss of multivalent interaction and the binding of a monomeric grft to two different nona-mannosides [20] grft with a disrupted carbohydrate-binding site nmr study reduced binding to mannose and a weaker correlation between anti-hiv-1 activity and gp120 binding [21] several studies have described the in vitro anti-hiv activities and cytotoxicity of grft (table 2) . in their publication, they observed that grft inhibited soluble gp120 from binding to cd4 receptor-expressing cells [5] . cell-to-cell fusion and transmission of hiv-1 infection were also blocked by grft at similar concentrations [5] . in addition, the coadministration of monosaccharides such as glucose, mannose, and n-acetyl glucosamine hindered glycosylation-dependent binding of grft to soluble gp120 [5]. in parallel with this observation, emau et al. also demonstrated that grft could block the infectivity of cxcr4-and ccr5-tropic hiv viruses at picomolar concentrations. they also confirmed the long-term stability of grft in a cervical/vaginal lavage [27] . in order to harness the hiv-inactivating power of grft, a new peptide called grifonin-1 was designed based on the three carbohydrate-binding amino acid sequences of grft. its sub-micromolar anti-hiv activity was confirmed using an in vitro tzm-bl cell line and p24 gag antigen release assays [24] . in addition to its ability to suppress hiv-1 transmission in cd4+ t-lymphocytes, grft also subverted dc-sign (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-mediated hiv capture [30] . mechanistically, a binding competition between grft and gp120 for dc-sign was proposed as a potential mode of action for grft-mediated inhibition of hiv-1 capture by dc-sign [21] . corresponding to this dc-sign-dependent antiviral activity, grft also potently inhibited giant cell formation between persistently hiv-infected t cells and noninfected cd4+ target t cells. this led to the suppression of hiv transmission, cd4+ t-cell destruction, and ultimately viral replication through the dc-sign mediated pathway [33] . this efficient blockage of the binding of dc-sign to immobilized gp120 by grft was further confirmed [21] . this observation was in harmony with the grft-mediated expulsion of gp120 from the gp120/dc-sign complex [21] . interestingly, this highly potent inhibition of dc-sign-mediated capture and transmission by grft was markedly impaired when grft was mutated in one of its three carbohydrate-binding sites (d30a, d70a, or d112a) [21] , further implicating its carbohydrate-binding sites as critical determinants for anti-hiv-1 activity. regarding its cytotoxicity, two studies demonstrated that the cc 50 concentration for grft was several hundred nanomolar [5,27], thousands of times higher than its reported antiviral concentrations. four studies have described the combination effects of grft with either current anti-hiv drugs or potential therapeutics under development (table 3) [26, 28, 29, 32] . grft showed synergistic antiviral activity with tenofovir, maraviroc (a ccr5 antagonist), and enfuvirtide (a gp41 fusion peptide inhibitor) [29] . the different glycosylation patterns on the viral envelope of clade b and clade c gp120 had no observable effect on their synergistic antiviral action [29] . covalently linking grft to the gp41-binding peptide c37 also exhibited a potency several-fold greater than that of grft alone in inhibiting hiv env-mediated fusion in a ccr5-tropic cell-cell fusion assay [32] . in line with this observation, all grft/antiretroviral drug (entry inhibitors, reverse transcriptase inhibitors, integrase inhibitors, and protease inhibitors) combinations displayed either synergistic or additive effects in inhibiting cell-cell fusion and protected against target cd4+ t cell destruction [33] . grft/antiretroviral combinations also potently inhibited short-term viral replication in t-cells via dc-sign-mediated transmission [33] . combinations of grft and other carbohydrate-binding agents (cbas) including hippeastrum hybrid agglutinin, galanthus nivalis agglutinin, a mannose-specific monoclonal antibody (mab) (2g12), microvirin, and banana lectin also showed synergistic activity against hiv-1, hiv-2, and even against certain cba-resistant hiv-1 strains [28] . none of the cbas competed with each other's glycan-binding sites on gp120 since they have distinct binding patterns on the gp120 envelope [28] . in addition to antiviral synergy, gp120-grft complexes showed higher immunogenicity than the individual proteins per se. this suggests that removing the mannose moieties on monomeric gp120 improves the humoral immune response to this protein [34] . six different antiviral mechanisms of action against hiv-1 by grft have been proposed (table 4) [26, 30, [34] [35] [36] [37] . a carbohydrate binding-dependent antiviral mechanism of grft was explored by using hiv-specific neutralizing monoclonal antibodies (mabs) such as 2g12, 48d, b12, and b6 [25, 26] . grft preferentially inhibited gp120 from binding to the 2g12 mab, which targets n-linked glycans at positions 332, 339, and 392 on gp120. this suggests an overlapping binding specificity with the 2g12 mab [25] . in addition, grft increased the interaction between gp120 and the 48d mab, which recognizes a cd4-induced epitope [25] . grft also enhanced the binding of hiv-1 to plates coated with b12 and b6 mabs [26] . this indicates that the binding of grft to gp120 triggers the exposure of the cd4-binding site on gp120. in particular, the glycan at position 386, which shields the cd4 binding domain of gp120, is also involved in the grft-mediated binding enhancements and the neutralization synergy between grft and b12 [26] . in addition, a synergy between grft and b12 was also exhibited when hiv-1 isolates became more sensitive to neutralization upon increasing hiv-1 binding of grft to b12 and b6 mabs [26] . together, these data suggest that grft-mediated blockage of a post-cd4 receptor binding event, such as the coreceptor binding with gp120, might be another plausible mechanism through which grft inhibits hiv-1 infection [26] . since the glycans on hiv-1 gp120 play an important role in shielding neutralization-sensitive epitopes from antibody recognition [26] , disruption of the mannose molecules on gp120 by grft may also increase antibody-dependent neutralization of hiv-1 particles. although the multivalent interaction of grft with high-mannose oligosaccharides is believed to account for most of its picomolar antiviral potency, the looser correlation between gp120-binding ability and hiv inhibitory potency for the binding site mutants of grft suggests the possibility of another unknown antiviral mechanism of grft that is not based on simple gp120 binding [21] . according to an isothermal titration calorimetry binding study, grft bound to glucose and n-acetyl glucosamine in a similar fashion to that of mannose, demonstrating its flexible specificity in binding carbohydrates [23] . this binding flexibility might have an implication for its broad antiviral spectrum. to study the potential role of the grft dimer in the suppression of hiv-1 infectivity, either two or four amino acids were inserted at the dimerization interface of grft. this resulted in a monomeric form of grft (mgrft) with greatly reduced antiviral activity against hiv-1. these results further emphasize the importance of multivalent interactions between dimeric grft and oligosaccharides present on hiv envelope glycoproteins for the successful cross-linking and aggregation of viral particles [20] . interestingly, an obligate dimer of grft with a peptide linker between the two subunits altered the structure of gp120 by exposing the cd4 binding site. however, grft-linker-grft with mutated carbohydrate-binding sites largely lost this ability [37] . on the other hand, the glycan-specific dc-sign receptor binds the virus and mediates its transfer to cd4+ cells [38] . in this regard, grft's ability to partially block gp120 from binding to human dc-sign [34] and its potent inhibition of dc-sign-dependent transfer of hiv-1 [38] could synergize with its antiviral action by blocking viral entry. to maximize the antiviral synergy caused by grft multimerization, tandem repeats of mgrft (mgrft tandemers) were engineered. they displayed picomolar-level antiviral activity in whole-cell anti-hiv assays [37] . however, since mgrft tandemers could not aggregate hiv virions, moulaei et al. suggested the intra-virion crosslinking of hiv envelope glycoproteins may be more integral to their antiviral activity [36] . inter-virion aggregation or clustering of hiv-1 gp120 on the viral membrane was found to be related to neutralization potency [35] . table 4 . grft anti-hiv-1 mechanisms of action. ref. exposure of the cd4 binding site of gp120 through the glycan at position 386 and blockage of coreceptor binding step [26] inhibition of mannose-binding to gp120 and improvement of the humoral immune response to gp120 [34] inhibition of gp120 binding to dc-sign and expulsion of gp120 from the gp120/dc-sign complex [30] alteration of gp120 structure through the exposure of the cd4 binding site [37] intra-virion crosslinking of gp120 [36] inter-virion aggregation or clustering of gp120 [35] five studies have characterized viral resistance profiles caused by chronic grft treatment (table 5) [25, 26, 31, 39, 40] . since the antiviral activity of grft mainly depends on disrupting the biological functions of multiple mannose molecules on viral glycoproteins, a reduction in the glycosylation levels of a target protein can lead to grft resistance. for example, the 234 and 295 glycosylation sites are involved in grft-induced hiv-1 neutralization since a concomitant lack of glycans at both positions resulted in natural grft resistance [25] . conversely, introducing glycosylation sites at n234 and n295 in hiv-1 clade c virus increased grft antiviral potency [25] . in line with these observations, deglycosylation at position 295 or 448 decreased the sensitivity of a single transmitter/founder hiv env to grft [31] . since n295 and n448 are grft-specific, high-mannose, n-linked glycosylation sites on gp120, a single deglycosylation at n295 or n448 in primary or t-cell-line-adapted hiv-1 isolates also resulted in marked resistance to grft [40] . furthermore, glycosylation sites at positions 230, 234, 241, and 289 located in the c2 region and 339, 392, and 448 in the c3-c4 region were also implicated in grft resistance [39] . a loss of glycosylation sites on gp120 as well as a rearrangement of glycans in v4 also led to hiv-1 subtype c resistance against grft [39] . in the case of dc-sign-dependent antiviral activity of grft, the effects of extra glycosylation seem to be dependent on the location of the glycosylation. for example, the introduction of the 234 glycosylation site abolished hiv-1 sensitivity to lectin's ability to inhibit binding to dc-sign and virus transfer [38] . however, the addition of the 295 glycosylation site enhanced the inhibition of dc-sign-dependent hiv-1 transfer by grft [38] . given the overlapping nature of the binding specificity displayed by grft and neutralizing antibodies against hiv-1, grft resistance could also affect hiv-1 sensitivity to antibody-dependent neutralization. the ubiquitous nature of glycosylated proteins in the body raises concerns that grft may cause toxicity by interacting with glycosylated host proteins. for this reason, the effects of grft on host cells and animals have been studied extensively. according to five previous reports, grft does not exhibit any toxicity at its active antiviral concentrations (table 6 ) [28, [41] [42] [43] [44] [45] . grft induced only minimal changes in the secretion of inflammatory cytokines and chemokines by epithelial cells and human peripheral blood mononuclear cells (pbmcs) [43] . in addition, it had no measurable effect on cell viability or the levels of t-cell activation markers [43] . grft treatment induced only minimal alterations in the gene expression profile of human ectocervical cells [43] . it also caused no significant cell death, mitogenicity, activation, or cytokine release in mouse pbmcs [44] . no obvious changes were observed in animal fitness, blood chemistry, or complete blood count parameters in grft-treated mice [44] . interestingly, the pbmc-bound form of grft was still able to maintain its antiviral activity, raising the potential of its versatile in vivo antiviral activity [43] . chronic intravaginal or systemic administration of 2 mg/kg of grft was also non-toxic in mice [44] . grft, when administered in gel form, was not associated with any changes in the rectal proteome [42] . an increased abundance of two common and beneficial microbial taxa after grft treatment was due to placebo formulations and not to grft, itself [42] . this association between the placebo gel and changes in the rectal proteome and microbiota indicates the need to alter the components of the placebo gel in future studies [42] . grft was also well tolerated after subcutaneous administration in guinea pig and mouse models [41] . in addition, grft was found to be non-irritating and non-inflammatory in human cervical explants and in an in vivo rabbit vaginal irritation model [45] . in this study, no mitogenic activity was reported in cultured human lymphocytes treated with grft [45] . however, following grft treatment, reversible splenomegaly was observed with the activation of certain spleen b and t cells [44] . this grft-associated increase in spleen and liver mass was also noted in another study [41] . therefore, an immune response elicited by grft treatment should be controlled in order to avoid potential grft immune-related toxicity [41] . table 6 . toxicity studies of grft in various cell and animal models. dose tested effects of grft ref. no effect on the production of proinflammatory cytokines and chemokines. no vaginal irritation in rabbits. [45] human cervical epithelial cells, cervicovaginal cells, and pbmcs up to 84 µm minimal changes in secretion of inflammatory cytokines and chemokines no measurable effect on cell viability and t-cell activation markers. [43] guinea pig balb/c mice single 50 mg/kg and 10 daily subcutaneous injections of 10 mg/kg minimal overall toxicity. well tolerated. increase in spleen and liver mass. [44] rhesus macaques intravaginal 0.1% gels no change in rental proteome or microbiome [42] three studies were conducted to examine the in vivo antiviral efficacy and pharmacokinetic behavior of grft using various small animal models (table 7) [41, 46, 47] . subcutaneous injections of grft into guinea pigs and mice were very well tolerated, resulting in the accumulation of grft up to relevant therapeutic concentrations [41] . the serum from grft-treated animals was found to retain antiviral activity against hiv-1-enveloped pseudoviruses in a cell-based neutralization assay [41] . in addition, active grft, which is capable of neutralizing hiv-env pseudoviruses, was also detected in rat fecal extracts after chronic oral dosing [46] . the in vivo efficacy of grft was also demonstrated in the humanized bone marrow-liver-thymus mice which were protected from vaginal infection with hiv-1 after being treated with recombinant c. crescentus expressing grft [47] . however, grft was not orally bioavailable even after chronic treatment [46] . table 7 . in vivo anti-hiv-1 activity of grft in animal models. dose tested effects of grft ref. guinea pig balb/c mice single 50 mg/kg and 10 daily subcutaneous injections of 10 mg/kg. retention of antiviral activity in serum [41] sprague dawley (sd) rats a single dose of 10 mg/ml intravenously or subcutaneously. ten 40 mg/kg doses for 10 days. neutralization activity found in fecal extracts [46] humanized bone marrow-liver-thymus mice 10 8 grft-expressing recombinant c. crescentus intravaginally. protection against hiv-1 infection [47] the clinical application of protein drugs requires a cost-effective large-scale production procedure to meet the high volume needed in a clinical setting. for efficient grft production, seven different production methods have been developed and optimized (table 8 ) [45, [48] [49] [50] [51] [52] [53] . giomarelli et al. used a fermenter and a rich, auto-inducing medium, which led to an approximately 45-fold increase in the total amount of grft per liter with approximately 70% of the protein expressed in the soluble fraction [49] . o'keefe et al. were able to produce grft in multigram quantities by using nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing grft [45] . hahn et al. employed a simple spraying method to introduce agrobacterium vectors into nicotiana plants in the presence of a surfactant as a substitute for vacuum inoculation [50] . they found that recombinant grft is stable during the storage of plant biomass as silage, which is suitable for mass production of cost-sensitive products [50] . fuqua et al., however, developed a simplified grft purification method [48] . they achieved >99% pure grft by generating the initial green juice extract in ph 4 buffer, heating the extract to 55 • c, incubating it overnight with a bentonite mgcl 2 mixture, and purifying it via chromatography [48] . vamvaka et al. successfully produced grft by using the endosperm of transgenic rice plants (oryza sativa) [52] . they found that both crude and pure grft had potent anti-hiv activity, and the crude extracts were not toxic in human cell lines [52] . this suggests that crude grft with minimal processing could be administered to reduce costs associated with purification [52] . petrova et al. expressed grft in the probiotic strains lactobacillus rhamnosus gg and l. rhamnosus gr-1 for gastrointestinal and vaginal mucosal delivery, respectively [51] . hoelscher et al. used stably transformed tobacco chloroplasts to produce grft, which accounted for up to 5% of the total soluble protein of the plant [53] . they also found that the tobacco, when dried, provides a storable source of grft that can be purified at a later date [53] . however, when produced in nicotiana benthamiana, kim et al. found that grft had pathologic effects on plants because it was directed to the apoplast during production, resulting in necrotic symptoms associated with hypersensitive response (hr)-like cell death [54] . they found that a specific interaction between grft and an apoplast-located endogenous glycoprotein, xyl1, initiated the hr response. to suppress grft-induced cell death in nicotiana benthamiana, exogenous expression of naphthalene hydroxylase was suggested by the authors [54] . to avoid the emergence of hiv-1 strains resistant to single microbicides, vamvaka et al. expressed components of multiple anti-hiv proteins including grft, 2g12 mab, and cyanovirin-n in rice endosperm [55] . they found that extracts from plants expressing all three proteins showed enhanced in vitro binding to gp120 and synergistic hiv-1 neutralization [55] . unexpectedly, they also found that synergistic hiv neutralization caused by the triple microbicide was enhanced by production in rice endosperm because rice globulin protein enhances gp120 binding in the expressed proteins [55] . table 8 . large-scale grft production methods. n/a indicates "not applicable." ref. transformation and the use of an autoinduction fermentor escherichia coli 45-fold increase [49] transduction with tobacco mosaic virus nicotiana benthamiana multigram quantity [45] agrobacterium vectors nicotiana benthamiana 90% of the leaf cells and 50% of the total soluble protein [50] transduction with tobacco mosaic virus in ph 4 buffer, heating the extract to 55 • c, a bentonite mgcl 2 mixture, and chromatography. nicotiana benthamiana 88% ± 5% of griffithsin from the initial extract [48] particle bombardment rice endosperm 223 µg/g dry seed weight [52] use of probiotic microorganisms lactobacillus rhamnosus gg and l. rhamnosus gr-1 n/a [51] chloroplast transformation nicotiana tabacum 360 µg of pure griffithsin per gram [53] protein stability is critical for long-term maintenance of pharmacological activity. for this reason, the susceptibility of grft to proteolytic digestion should be considered. grft was resistant to digestion by eight different proteases including pepsin, papain, leucine aminopeptidase, pronase, α-chymotrypsin, proteinase k, endoproteinase, and trypsin [56] . a number of different nanoparticle drug delivery systems have been tested to provide sustained and controlled delivery of grft, improve its solubility, protect its payloads, and enhance its mucosal permeability (table 9 ) [57] [58] [59] [60] . plga (poly lactic-co-glycolic acid) nanoparticles with a diameter of approximately 180-200 nm were successfully used in the co-delivery of grft and dapivirine in vitro [60] . both drugs showed a biphasic release with an initial burst phase followed by a sustained release phase [60] . grooms et al. successfully generated grft-modified electrospun fibers to inactivate hiv prior to cellular entry [57] . furthermore, lal et al. developed a self-administered, vaginal fast-dissolving insert (fdi) produced by freeze-drying that delivered safe and effective amounts of grft and carrageenan (gc), a sulfated polysaccharide extracted from seaweed [58] . fibers comprised of methoxy polyethylene glycol-plga: poly n-butyl acrylate-co-acrylic acid (mpeg-plga: pba-co-paa) were able to achieve high grft loading. these grft-loaded fibers were well maintained within a simulated vaginal fluid (svf) and showed ph-dependent release upon exposure to buffered svf and simulated semen solutions [59] . table 9 . pharmaceutical formulations for the efficient delivery of grft. delivery route effects on delivery ref. a biphasic release with an initial burst phase followed by a sustained release phase [60] electrospun fibers in vitro maintenance of antiviral efficacy [57] fdi comprised of vaginal good friability, hardness, and stability [58] mpeg-plga:pba-co-paa vaginal high grft loading and ph-dependent release [59] to date, two phase-one clinical studies have been initiated to investigate the potential toxicity of grft in healthy populations [61, 62] . the first study aimed to evaluate the safety of grft in a cg gel used vaginally for a single dose and then for 14 consecutive days in healthy women [61] . this was a two-part study with the first part consisting of a single-dose and an open-label design, and the second part consisted of a multiple-dose, randomized, placebo-controlled study design. during the second part, 30 subjects were administered a placebo and 30 subjects were given the grft gel. in this study, the pharmacokinetic behavior of grft was also analyzed by forming a time-concentration curve of grft in blood samples. however, rising dose tolerance was not studied because of the minimal systemic absorbance of grft, which was reported in preclinical studies. although the official study results are not available yet, the population council website reported grft to be safe for vaginal use for up to 14 days with potent anti-hiv activity in cell-based assays and cervical explants up to 8 h after receiving the dose [63] . in 2014, another phase-one clinical study for grft started as an integrated preclinical/clinical program. this program, which was named prevent (pre-exposure prevention of viral entry), aimed to provide a comprehensive set of data to facilitate an informed decision on whether grft should progress within the topical microbicides pipeline [62] . this was a randomized, double-blind phase-one safety and pharmacokinetic study of grft enema administered rectally to hiv-1 seronegative adults who practice urai. the number and frequency of adverse events, the blood concentration of grft, and changes in humoral antibody response were analyzed. this on-going clinical study will be completed in 2021. grft not only displays antiviral activity against hiv but also for other enveloped viruses such as sars-cov [10] , mers-cov [11] , hcv [12, 13] , hsv [14, 15, 64] , jev [16, 17] , and pedv [18] . even human papillomavirus (hpv), which is a non-enveloped virus, was inactivated by grft via a glycosylation-independent mechanism [15] . grft specifically bound to the sars-cov spike glycoprotein and inhibited viral entry [10] . grft also inhibited particles pseudotyped with the mers-cov spike protein from entering host cells [11] . preincubation of hcv particles with grft prevented infection of huh-7 hepatoma cells [13] . furthermore, grft was able to interfere with the direct cell-to-cell transmission of hcv [13] . this anti-hcv activity was further demonstrated in vivo when hcv infection was mitigated in chimeric mice [12] . in this report, grft was readily bioavailable after subcutaneous injection, and it showed significant in vivo efficacy by reducing hcv viral titers in a mouse model system with engrafted human hepatocytes [12] . in contrast to hiv, hcv resistance to grft was not directly conferred by mutations in the envelope protein genes, but it could occur through an indirect mechanism involving mutations in other viral proteins [65] . grft displayed modest inhibitory activity against hsv-2 if it was present during viral entry, but if it was present post-entry, it completely blocked plaque formation, reduced plaque size, and prevented cell-to-cell propagation [14] . these in vitro findings translated into significant protection against genital herpes in mice treated with a 0.1% griffithsin gel [14] . the in vivo anti-hsv activity of grft was further demonstrated when murine model test subjects were protected by c. crescentus expressing grft after intravaginal infection with hsv-2 [64] . levendosky et al. explored the antiviral properties of a combination product composed of grft and cg against hsv-2 and hpv. they found that grft was able to block the entry of hsv-2 and hpv into target cells but not the adsorption of hsv-2 and hpv onto target cells [15] . this grft/cg combination was also tested as a freeze-dried, fdi formulation. this product protected rhesus macaques against a high-dose vaginal simian hiv challenge 4 h after fdi insertion [66] . furthermore, this grft/cg fdi also protected mice, vaginally, against hsv-2 and hpv pseudovirus [66] . in vitro experiments showed that the treatment of jev with grft before inoculation into bhk-21 cells inhibited infection in a dose-dependent manner [17] . in vivo experiments showed that grft (5 mg/kg) administered intraperitoneally before virus infection prevented mortality in mice challenged intraperitoneally with a lethal dose of jev [17] . with regard to its antiviral mechanism, grft was shown to bind to jev glycosylated viral proteins, specifically the enveloped and pre-mature glycoproteins [16] . in addition, grft was able to reduce pedv infection in vero cells [18] . due to its novel carbohydrate-targeting antiviral mechanism of action, superior anti-hiv-1 efficacy, excellent host toxicity profile, synergistic interaction with other antiretroviral drugs, optimized large-scale production methodology, and various formulation methods for its efficient delivery, grft holds great promise as the first topical protein-based anti-hiv pre-exposure prophylactic. even though a potentially adverse immunotoxicity issue observed during preclinical animal studies needs to be resolved, its broad antiviral spectrum, which is applicable to other enveloped viruses, could make grft the first universal antiviral therapeutic that can specifically target virus carbohydrates. favorable outcomes from two ongoing phase-one clinical trials will accelerate the drug development process. if approved, grft will provide a superior way to prevent many transmissible viral infections. a lectin isolated from bananas is a potent inhibitor of hiv replication potent anti-influenza activity of cyanovirin-n and interactions with viral hemagglutinin microvirin, a novel alpha(1,2)-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv-1 activity comparable with that of cyanovirin-n but a much higher safety profile bioengineered intravaginal isolate of lactobacillus plantarum expresses algal lectin scytovirin demonstrating anti-hiv-1 activity isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp toxicity of antiretroviral therapy and implications for drug development effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of hiv infection in women global epidemiology of hiv infection in men who have sex with men griffithsin: an antiviral lectin with outstanding therapeutic potential broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae middle east respiratory syndrome coronavirus infection is inhibited by griffithsin antiviral lectins from red and blue-green algae show potent in vitro and in vivo activity against hepatitis c virus griffithsin has antiviral activity against hepatitis c virus griffithsin protects mice from genital herpes by preventing cell-to-cell spread griffithsin and carrageenan combination to target herpes simplex virus 2 and human papillomavirus griffithsin binds to the glycosylated proteins (e and prm) of japanese encephalitis virus and inhibit its infection griffithsin inhibits japanese encephalitis virus infection in vitro and in vivo in vitro antiviral activity of griffithsin against porcine epidemic diarrhea virus domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-hiv activity the role of individual carbohydrate-binding sites in the function of the potent anti-hiv lectin griffithsin crystallographic, thermodynamic, and molecular modeling studies of the mode of binding of oligosaccharides to the potent antiviral protein griffithsin crystallographic studies of the complexes of antiviral protein griffithsin with glucose and n-acetylglucosamine grifonin-1: a small hiv-1 entry inhibitor derived from the algal lectin, griffithsin mannose-rich glycosylation patterns on hiv-1 subtype c gp120 and sensitivity to the lectins, griffithsin, cyanovirin-n and scytovirin binding of the mannose-specific lectin, griffithsin, to hiv-1 gp120 exposes the cd4-binding site griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide combinations of griffithsin with other carbohydrate-binding agents demonstrate superior activity against hiv type 1, hiv type 2, and selected carbohydrate-binding agent-resistant hiv type 1 strains synergistic activity profile of griffithsin in combination with tenofovir, maraviroc and enfuvirtide against hiv-1 clade c role of the carbohydrate-binding sites of griffithsin in the prevention of dc-sign-mediated capture and transmission of hiv-1 sensitivity of transmitted and founder human immunodeficiency virus type 1 envelopes to carbohydrate-binding agents griffithsin, cyanovirin-n and galanthus nivalis agglutinin potent strategy to inhibit hiv-1 by binding both gp120 and gp41 griffithsin, alone and combined with all classes of antiretroviral drugs, potently inhibits hiv cell-cell transmission and destruction of cd4+ t cells occluding the mannose moieties on human immunodeficiency virus type 1 gp120 with griffithsin improves the antibody responses to both proteins in mice binding site geometry and subdomain valency control effects of neutralizing lectins on hiv-1 viral particles griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus the griffithsin dimer is required for high-potency inhibition of hiv-1: evidence for manipulation of the structure of gp120 as part of the griffithsin dimer mechanism the lectins griffithsin, cyanovirin-n and scytovirin inhibit hiv-1 binding to the dc-sign receptor and transfer to cd4(+) cells mechanisms of hiv-1 subtype c resistance to grft, cv-n and svn removal of two high-mannose n-linked glycans on gp120 renders human immunodeficiency virus 1 largely resistant to the carbohydrate-binding agent griffithsin activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models impact of the griffithsin anti-hiv microbicide and placebo gels on the rectal mucosal proteome and microbiome in non-human primates investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate studies in a murine model confirm the safety of griffithsin and advocate its further development as a microbicide targeting hiv-1 and other enveloped viruses scaleable manufacture of hiv-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component pharmacokinetics of the antiviral lectin griffithsin administered by different routes indicates multiple potential uses a caulobacter crescentus microbicide protects from vaginal infection with hiv-1jr-csf in humanized bone marrow-liver-thymus mice improving the large scale purification of the hiv microbicide, griffithsin recombinant production of anti-hiv protein, griffithsin, by auto-induction in a fermentor culture a novel and fully scalable agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants engineering lactobacillus rhamnosus gg and gr-1 to express hiv-inhibiting griffithsin rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against hiv high-level expression of the hiv entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves characterization of the hypersensitive response-like cell death phenomenon induced by targeting antiviral lectin griffithsin to the secretory pathway unexpected synergistic hiv neutralization by a triple microbicide produced in rice endosperm degradation of naturally occurring and engineered antimicrobial peptides by proteases griffithsin-modified electrospun fibers as a delivery scaffold to prevent hiv infection development of a vaginal fast-dissolving insert combining griffithsin and carrageenan for potential use against sexually transmitted infections ph-responsive delivery of griffithsin from electrospun fibers design of poly(lactic-co-glycolic acid) (plga) nanoparticles for vaginal co-delivery of griffithsin and dapivirine and their synergistic effect for hiv prophylaxis study to evaluate the safety of griffithsin in a carrageenan gel in healthy women griffithsin-based rectal microbicides for prevention of viral entry (prevent) developing and testing a griffithsin (non-arv) microbicide generation of a dual-target, safe, inexpensive microbicide that protects against hiv-1 and hsv-2 disease hepatitis c virus resistance to carbohydrate-binding agents griffithsin carrageenan fast dissolving inserts prevent shiv hsv-2 and hpv infections in vivo this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-269759-1n1oo6wc authors: villamil-gómez, wilmer e.; sánchez, álvaro; gelis, libardo; silvera, luz alba; barbosa, juliana; otero-nader, octavio; bonilla-salgado, carlos david; rodríguez-morales, alfonso j. title: fatal human coronavirus 229e (hcov-229e) and rsv–related pneumonia in an aids patient from colombia date: 2020-02-06 journal: travel med infect dis doi: 10.1016/j.tmaid.2020.101573 sha: doc_id: 269759 cord_uid: 1n1oo6wc nan we have read the article of yavarian et al. [1] , showing the prevalence of influenza and not of middle east respiratory syndrome coronavirus (mers-cov) in pilgrims and the general population. we would like to discuss the relevance of other respiratory viruses, including other cov different to mers-cov, the severe acute respiratory syndrome cov (sars-cov) and the 2019 novel cov (2019ncov) [2] in relation to a case of coinfection between hcov-229e, respiratory syncytial virus (rsv) and hiv we had in colombia. the three highly pathogenic viruses, sars-cov, mers-cov, 2019n-cov, cause severe respiratory syndrome in humans, and the other four human coronaviruses (hcov-nl63, hcov-229e, hcov-oc43 and hcov-hku1) induce only mild upper respiratory diseases in immunocompetent hosts, although some of them can cause severe infections in infants, young children and elderly individuals [3] . two years ago, a 32-year-old man admitted to the intensive care unit (icu) for acute respiratory failure, five days after hospitalization, due to continuous productive cough (yellow secretion), nasal flaring, respiratory distress, intercostal retractions, sweating, chills and mucocutaneous paleness. during this admission, the patient tested hiv positive by elisa and western-blot. his initial cd4 cell count and viral load were 20 cells/μl and 759,780 copies/ml (pcr), respectively. patient was started on combination antiretroviral treatment (art) with a regimen of abacavir, lamivudine and efavirenz. on icu admission, he had fever of 37.6°c (99.68°f) and was mildly tachypnoeic. his blood pressure was 110/60 mmhg, with a pulse rate of 128/min. a chest radiograph showed bilateral micronodular infiltrates (fig. 1) . the patient's condition deteriorated rapidly, requiring endotracheal intubation and mechanical ventilation. arterial blood gas analysis showed severe hypoxemia with pao 2 of 70 mmhg and oxygen saturation of 60%, respiratory acidosis with paco 2 of 52. fourteen days after admission to icu, and despite treatment with oseltamivir and aggressive supportive care with mechanical ventilation, fluid resuscitation, and high dose norepinephrine infusion, refractory hypoxia rapidly led to a fatal multiorgan failure. no autopsy was performed. cultures of bal fluid, blood and urine specimens remained negative for mycobacteria, and fungi. only escherichia coli from urine and proteus mirabilis from bal (both were susceptible to meropenem) were detected. there is a lack of reported cases of a human coronavirus infection in hiv infected patients from colombia and south america, confirmed by rt-pcr. hcov-229e causes common cold but occasionally it can be associated with more severe respiratory infections in children [3, 4] , elderly and persons with underlying illness [3, 5] , which would be the case of hiv infection, as seen in this report [4, 6] . the identification of coronavirus in high-risk immunocompromised patients may lead to early adoption of a specific therapeutic strategy, but, in the absence of proof of the efficacy of antiviral drugs, the treatment remains only supportive [5, 7] . evidence for a zoonotic origin of hcov (eg. involving bats, camels), have been documented extensively over the past decade, including sars-cov, mers-cov, and now the 2019n-cov, which is causing epidemic and led to the world health organization to declare it as a public health emergency of international concern (pheic) [5] . in colombia, the unique previous reference to coronaviruses was the identification of avian infectious bronchitis virus strains (an avian coronavirus, genus gammacoronavirus) in antioquia [8] , a department close to sucre, where our patient was diagnosed. the patient denied travelling recently to other regions of the country as well as internationally. this case, similar to other non-mers hcov infection cases reported [7] , is a reminder that although most infections with human coronaviruses are mild and associated with common colds, certain animal and human coronaviruses may cause severe and sometimes fatal infections in humans. influenza virus but not mers coronavirus circulation in iran, 2013-2016: comparison between pilgrims and general population the next big threat to global health? 2019 novel coronavirus (2019-ncov): what advice can we give to travellers? -interim recommendations origin and evolution of pathogenic coronaviruses clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in hiv-infected and hiv-uninfected south african children coronavirus 229e-related pneumonia in immunocompromised patients coronavirus infection in an aids patient isolation of a novel coronavirus from a man with pneumonia in saudi arabia molecular characterization of avian infectious bronchitis virus strains isolated in colombia during sincelejo, colombia doctoral program of tropical medicine to dr. ziad memish, chair, working group on zoonoses, international society for chemotherapy, for his critical review and advice for improve of the manuscript. this manuscript is dedicated to the memory of luz alba silvera, in memoriam. the authors have no reported conflicts of interest. key: cord-256477-dftt5m6i authors: feller, john m. title: potential ebola prophylaxis date: 2015-07-01 journal: j paediatr child health doi: 10.1111/jpc.12955 sha: doc_id: 256477 cord_uid: dftt5m6i nan your editorial provides insights into ebola and the impact of such terrible infectious diseases. 1 in addition to providing international emergency health services and developing vaccines and treatments, it would be wise to consider prevention measures. health workers in africa need effective ebola prophylaxis. while waiting for effective vaccines, an epidemiological survey could show if a non-infected cohort had been prescribed chloroquine (cq) for malaria. cq-resistant malaria predominates, but cq is sometimes given with proguanil. the hypothesis that cq might afford ebola prophylaxis comes from our own work showing hydroxychloroquine (hcq) induces apoptosis (programmed cell death) in peripheral blood mononuclear cells. 2 hcq also inhibits the replication of hiv in vitro. 3 there is a possible clinical analogy. prior to anti-retroviral therapy (art), hiv-positive pregnant women in uganda taking cq for concurrent malaria had reduced transmission of hiv to their newborns. also, viral loads were far lower if hiv subjects were taking cq, and the hiv infected children on concurrent cq were well and lived longer. 4, 5 a randomised controlled trial (rct) in 13 art-naïve patients found cq was associated with decreased memory cd8 t-cell activation, cd4 and cd8 t-cell proliferation and lipopolysaccharide levels compared with baseline, but there were no changes in plasma hiv rna. 6 however, another rct of hcq in art-naïve patients demonstrated no change in t-cell activation and proliferation, an increase in hiv rna and a decrease in cd4 t-cell counts. 7 in a small non-randomised study of 20 patients receiving suppressive art, hcq administration was associated with a reduction in multiple markers of immune activation but no significant increase in cd4 t-cell recovery. 8 hcq or cq could possibly stop the spread of ebola, similar to hiv. ebola infects dendritic cells, which display signals of infection on their surfaces to activate t lymphocytes that destroy other infected cells before the virus replicates further. defective dendritic cells fail to give the right signal to t-cells to limit infection. ebola works in part by inhibiting interferon: an ebola protein, vp24, binds to and blocks a transport protein on the surface of immune cells that plays an important role in the interferon pathway. 6 finally, the recent emergence of the coronavirus mers, as a serious threat to life in asia and the middle east, has led to investigation of possible therapeutic agents against this virus. the team of dr. e.j. snijder, in holland, has identified benefit with four agents -chloroquine, chlorpromazine, loperamide, and lopinavir -out of 348 food and drug administrationapproved compounds. 9 cq could be a useful first-line treatment for ebola and warrants further investigation. pandemic paranoia: a heretical view of ebola and other infectious threats induction of apoptosis in peripheral blood lymphocytes following treatment in vitro with hydroxychloroquine inhibition of human immunodeficiency virus type 1 replication by hydroxychloroquine in t cells and monocytes hiv status and delayed type hypersensitivity skin testing cord blood chloroquine levels and hiv transmission in ugandan infants ebola virus vp24 targets a unique nls binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated stat1 reduction of immune activation with chloroquine therapy during chronic hiv infection effects of hydroxychloroquine on immune activation and disease progression among hiv-infected patients not receiving antiretroviral therapy: a randomized controlled trial screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture key: cord-261830-5o5epn6v authors: rheinemann, lara; sundquist, wesley i. title: virus budding date: 2020-08-07 journal: reference module in life sciences doi: 10.1016/b978-0-12-814515-9.00023-0 sha: doc_id: 261830 cord_uid: 5o5epn6v enveloped viruses exit producer cells and acquire their external lipid envelopes by budding through limiting cellular membranes. most viruses encode multifunctional structural proteins that coordinate the processes of virion assembly, membrane envelopment, budding, and maturation. in many cases, the cellular escrt pathway is recruited to facilitate the membrane fission step of budding, but alternative strategies are also employed. recently, many viruses previously considered to be non-enveloped have been shown to exit cells non-lytically within vesicles, adding further complexity to the intricacies of virus budding and egress. lara rheinemann and wesley i sundquist, university of utah, salt lake city, ut, united states r 2020 elsevier inc. all rights reserved. to spread infection, viruses must exit producer cells and transmit their genetic material into target cells. viruses have evolved two general strategies for cellular egress: (1) enveloped viruses acquire a host-derived lipid membrane as they breach the limiting membranes of the cell. during this process, the viral membrane typically also acquires one or more viral glycoproteins that bind target cell receptors and facilitate the membrane fusion step required for viral entry. (2) non-enveloped viruses, by contrast, exit cells when the plasma membrane is disrupted, typically by cell lysis. non-enveloped viruses then infect target cells either by disrupting limiting membranes to gain access to the cytoplasm or by pumping their nucleic acids directly into the cell. in recent years, the distinction between enveloped and non-enveloped viruses has been blurred by the discovery that some viruses, traditionally thought of as non-enveloped, can exit cells within vesicles. these are termed "quasi-enveloped" viruses. the process of enveloped virus release comprises a series of coordinated steps, which are illustrated for human immunodeficiency virus type 1 (hiv-1) in fig. 1 : (1) assembly: viral proteins and other essential components co-assemble to form virions. many viruses assemble at the plasma membrane, but others assemble at internal membranes or in the cytoplasm before trafficking to the plasma membrane or exiting via the secretory system. (2) envelopment: the host membrane is bent and wrapped around the nascent virion. (3) budding: the membrane stalk connecting the virion to the host membrane is constricted and severed to release the enveloped particle. (4) maturation: most enveloped viruses undergo further proteolytic and conformational maturation steps during or after budding. maturation converts the assembly-competent virion into an infectious virus that can enter, uncoat, and replicate in the new target cell. the complex series of events that accompany enveloped viral egress must be coordinated spatially and temporally, and these events are typically orchestrated by virally-encoded, multifunctional structural proteins. these proteins bind and remodel the membrane, self-assemble into virions, package other essential components such as nucleic acids into the nascent virion, and contain or recruit all of the activities necessary for budding and maturation. this article will describe general principles of enveloped virus assembly and release using the well-characterized hiv-1 gag protein as a paradigm for a viral structural protein. important principles employed by other viral families will also be discussed. all retroviruses, including hiv-1, express a gag polyprotein that coordinates assembly of the immature virion (sundquist and krausslich, 2012; meng and lever, 2013; lingappa et al., 2014) . when expressed alone, gag is released within virus-like particles (vlps), indicating that it contains or recruits all activities necessary for assembly, envelopment, and budding. an exciting new fig. 1 stages of hiv-1 virion formation. hiv-1 virion formation is coordinated by the multifunctional structural polyprotein gag. complexes of gag with the viral rna genome (blue) traffic to the plasma membrane where gag assembles and binds other virion components, including the longer gag-pol polyprotein, which contains the viral protease, reverse transcriptase and rnase h enzymes. gag also recruits three different host factors to facilitate membrane constriction and fission: amot-nedd4l (orange and light purple, respectively), and the early-acting escrtassociated factors, escrt-i/ii (pink) and alix (blue), which in turn recruit escrt-iii proteins (green). escrt-iii proteins form polymeric filaments that constrict the bud neck with the help of the vps4 aaa þ atpase and its cofactor lip5. protease activation during budding leads to gag and gag-pol processing and formation of the mature, infectious virion. variation on this same theme is the discovery of gag-related cellular proteins that can self-assemble, exit producer cells, and transfer rna molecules between cells (pastuzyn et al., 2018; ashley et al., 2018) . as illustrated in fig. 2 , hiv-1 gag comprises four functional elements: ma, ca, nc and p6, connected by two spacer peptides: sp1 and sp2. the ma domain in gag (ma gag ) targets assembly to the plasma membrane, nc gag binds the dimeric viral rna genome, the ca gag and sp1 gag elements mediate spherical particle assembly, and p6 gag recruits cellular escrt (endosomal sorting complexes required for transport) pathway factors required for virus budding. gag also packages several other components required for full infectivity, including the viral enzymes, which are packaged as co-assembling gag-pol fusion proteins, the viral env protein, whose intracellular domain interacts with ma gag , and viral protein r (vpr), which binds p6 gag . (1) rna packaging: following synthesis in the cytoplasm, gag monomers or low-order multimers bind two associated copies of the full-length viral genomic rna through a highly structured packaging site located near the 5 0 end of the genome (jouvenet et al., 2009; kutluay and bieniasz, 2010; keane et al., 2015) . this interaction is mediated by nc gag and enables specific selection of the hiv-1 genome over cellular rnas and viral rnas undergoing translation (kaddis maldonado and parent, 2016; dubois et al., 2018; kuzembayeva et al., 2014 , brown et al., 2020 . several host proteins, including abce1, staufen1, and ddx6, can associate with gag-rna complexes and have been proposed to promote gag trafficking, multimerization, and/or genome encapsidation, although these activities are not yet fully defined (reed et al., 2012; zimmerman et al., 2002; chatel-chaix et al., 2007) . (2) membrane trafficking: gag and gag-rna complexes move to the plasma membrane and associate with cholesterol-rich microdomains, commonly termed "lipid rafts" (ono and freed, 2001) . binding of gag to the plasma membrane is mediated by a bipartite signal in the ma gag domain that comprises a post-translational myristic acid modification at the amino-terminus and a cluster of basic residues, the highly basic region (hbr). prior to membrane binding, the myristate is sequestered in a hydrophobic cavity within the globular ma gag domain. the hbr interacts with acidic head groups of the plasma membranespecific phospholipid phosphatidylinositol-(4,5)-bisphosphate (pi(4,5)p 2 ) and this interaction, perhaps in concert with gag multimerization, exposes the myristate moiety. pi(4,5)p 2 and the conformational "myristoyl switch" enable targeting and stable gag association with the plasma membrane (chukkapalli and ono, 2011; tang et al., 2004) . in addition to pi(4,5)p 2 , ma gag can also bind cellular rna, especially trnas. the ma-trna interactions likely prevent nonspecific binding of gag to cellular membranes other than the plasma membrane (chukkapalli et al., 2010; alfadhli et al., 2011; kutluay et al., 2014; gaines et al., 2018) . (3) virion assembly: at the plasma membrane, gag molecules multimerize and assemble into a hexagonal lattice through lateral protein-protein interactions that are mediated by the ca-sp1 region wagner et al., 2016) . host-derived inositol hexakisphosphate (ip 6 ) binds within the ca-sp1 hexamers and facilitates formation of the immature gag lattice (dick et al., 2018; mallery et al., 2018) . in addition to the genomic rna, gag interacts directly with other viral proteins that are incorporated into the nascent virion, including the multifunctional accessory protein vpr, which binds a leucine-rich element located within p6 gag (kondo et al., 1995) , and the cytoplasmic tail of the gp41 subunit of the heterotrimeric transmembrane env protein, which is incorporated within holes in the associating gag lattice created by ma trimerization (tedbury and freed, 2014; tedbury et al., 2016; hill et al., 1996; pezeshkian et al., 2019) . in addition to the gag polyprotein, the full-length viral rna also encodes the gag-pol polyprotein. the longer gag-pol protein is translated by a ribosomal frameshifting mechanism, contains the viral enzymes, and is incorporated into the nascent virion through interactions with gag (smith et al., 1993) . fig. 2 the hiv-1 gag polyprotein. hiv-1 gag comprises four functional elements connected by two spacer peptides sp1 and sp2 (gray). ma (yellow) facilitates membrane binding and env incorporation. ca (orange) mediates assembly of the immature capsid and, after proteolytic processing, forms the mature conical capsid. nc (red) binds the viral rna genome through two zinc finger motifs. p6 (brown) binds vpr and recruits early-acting escrt proteins tsg101 (a subunit of the escrt-i complex) and alix to facilitate membrane fission. pink arrowheads denote proteolytic cleavage sites during maturation. the multifunctional structural proteins that mediate assembly and membrane targeting also appear to facilitate virion envelopment by inducing membrane curvature. the hexagonal hiv-1 gag lattice contains small discontinuities that accommodate declination and allow the immature lattice to bend the membrane and create a spherical virion . the host factor angiomotin (amot) has also been implicated in hiv-1 virion envelopment because fully enveloped spherical particles are not formed efficiently in the absence of amot (mercenne et al., 2015) . the bar domain of amot likely contributes to this activity as this domain has been shown to bend and tubulate membranes in other contexts (nishimura et al., 2018) . unlike hiv-1, betaretroviruses and spumaviruses assemble in the cytoplasm before being trafficked to the plasma membrane. thus, assembly and budding are spatially and temporally separated in these viruses. for example, gag proteins from prototypical foamy virus, mason-pfizer monkey virus, and mouse mammary tumor virus preassemble into immature virions near the pericentriolar region and are then trafficked on microtubules to associate with membranes and the viral env protein (müllers, 2013; hütter et al., 2013; swanstrom and wills, 1997) . hepatitis b virus (hbv), a hepadnavirus, similarly forms capsids in the cytoplasm, which then associate with envelope proteins in cellular membranes to mediate particle envelopment and budding (prange, 2012; blondot et al., 2016) . more broadly, most enveloped viruses have multifunctional structural proteins that mediate assembly and envelopment. often, this role is fulfilled by matrix proteins that bind to the viral membrane, assemble into higher-order structures, and link internal ribonucleoprotein complexes to external envelope glycoproteins. in viruses that lack classical matrix proteins, such as hantaviruses (hepojoki et al., 2012; cifuentes-munoz et al., 2014; muyangwa et al., 2015) and alphaviruses (brown et al., 2018) , cytoplasmic tails of envelope glycoproteins act as matrix protein surrogates by directly interacting with nucleoproteins. larger, more complex dna viruses such as pox viruses (roberts and smith, 2008; liu et al., 2014) and herpes viruses (lv et al., 2019) divide these functions between multiple viral proteins (see table 1 ). budding as membrane envelopment proceeds, the membrane is constricted until nascent virions are connected to the plasma membrane by a thin stalk that must be severed to separate the viral and cellular membranes. many enveloped viruses accomplish membrane constriction and fission by recruiting the machinery of the cellular escrt pathway (votteler and sundquist, 2013) . escrt-independent enveloped viruses also exist, however, and these viruses must therefore either recruit other, as yet unidentified, host factors or encode viral proteins that mediate budding. the escrt pathway mediates cellular membrane fission events throughout eukarya and also in some archaeal species (mccullough et al., 2018; christ et al., 2017; scourfield and martin-serrano, 2017; henne et al., 2013) . the pathway was initially identified as the machinery that mediates intraluminal vesicle budding into specialized late endosomes, termed multivesicular bodies (mvb) (hanson and cashikar, 2012) , but is now known to act at many other cellular membranes, including during cytokinetic abscission (carlton and martin-serrano, 2007; morita et al., 2007) , resealing of the post-mitotic nuclear envelope (olmos et al., 2015; vietri et al., 2015) , membrane repair (scheffer et al., 2014; jimenez et al., 2014; skowyra et al., 2018) , closure of autophagosomes (takahashi et al., 2018; zhou et al., 2019) , and neuronal pruning (zhang et al., 2014; loncle et al., 2015; issman-zecharya and schuldiner, 2014) . notably, all of these membrane fission events involve constricting membranes toward a cytoplasm-filled neck and are therefore topologically equivalent to virus budding from the plasma membrane. escrt-mediated membrane fission events are catalyzed by a common core machinery (mccullough et al., 2018; christ et al., 2017; scourfield and martin-serrano, 2017; banjade et al., 2019) , which is recruited to different membranes by adapter proteins. these membrane-specific adapters recruit early-acting escrt proteins, which help to stabilize membrane curvature and also nucleate assembly of late-acting escrt-iii proteins, which form the core fission machinery. escrt-iii proteins can be recruited by three known mechanisms: (1) adapters can recruit bro1 domain-containing proteins such as alix, which serves as a bridge to the escrt-iii proteins, (2) adapters can bind the escrt-i complex, which in turn recruits escrt-iii proteins via intermediate escrt-ii complexes, and (3) the nuclear lem2 adapter binds chmp7, a hybrid escrt-ii/escrt-iii protein, which then binds other escrt-iii proteins. humans express 12 related escrt-iii proteins that are divided into eight subfamilies, chmp1-7 and ist1, with some subfamilies comprising several homologs. escrt-iii proteins can adopt "closed" and "open" conformations. in the autoinhibited closed state, escrt-iii proteins are soluble and monomeric. when autoinhibition is relieved, escrt-iii subunits open and can then bind membranes and form curved helical filaments. these filaments constrict membranes and recruit vps4 aaa þ atpases. vps4 enzymes dynamically remodel and disassemble escrt-iii filaments, using the energy of atp hydrolysis to extract individual escrt-iii subunits and release them back into the cytoplasm. these enzymes thereby power the virus budding cycle, although the precise mechanism by which escrt-iii filaments and vps4 enzymes collaborate to mediate fission is not yet fully understood. enveloped viral structural proteins recruit the escrt pathway using motifs that mimic cellular escrt adapters (votteler and sundquist, 2013; hurley and cada, 2018; lippincott-schwartz et al., 2017) . these motifs were initially discovered in retroviral gag proteins (gottlinger et al., 1991; huang et al., 1995; parent et al., 1995; xiang et al., 1996) and were termed "late domains" because they exerted their effects at a late stage of assembly. analogous late domains were subsequently identified in many other enveloped viruses. late domains can frequently function from different positions within viral structural proteins and can be swapped between viruses (parent et al., 1995; yuan et al., 2000) , consistent with the idea that although they have different primary binding partners, they all ultimately converge on common downstream escrt-iii proteins. several different classes of late domains are now well understood, and others have been identified but remain to be linked to escrt binding partners. p(s/t)ap: the p(s/t)ap late domain (where the second residue can be either a serine or a threonine) was first identified in the p6 polypeptide of hiv-1 gag (gottlinger et al., 1991; huang et al., 1995) , and subsequently identified in structural proteins of other retroviruses, filoviruses, arenaviruses and reoviruses (votteler and sundquist, 2013) . the p(s/t)ap motif recruits the four-protein escrt-i complex by binding the uev domain of the tsg101 subunit (demirov et al., 2002; garrus et al., 2001; verplank et al., 2001; martin-serrano et al., 2001) . p(s/t)ap motifs are found in several cellular escrt adapter proteins, such as the early endosomal protein hrs (bache et al., 2003) . ypx n l: ypx n l late domains (where x n can vary in sequence and length) recruit alix by binding the central v domain (strack et al., 2003; martin-serrano et al., 2003; von schwedler et al., 2003) . hiv-1 contains a ypx n l late domain, although this motif is less critical for budding than the ptap motif in most cell types. other viruses rely exclusively or primarily on ypx n l domains for budding, including other retroviruses, paramyxoviruses, flaviviruses, and possibly herpesviruses (votteler and sundquist, 2013) . divergent structural proteins that lack a readily detectable ypx n l motif, yet still bind to alix, have also been described (boonyaratanakornkit et al., 2013; lee et al., 2012) , suggesting that alix-recruiting sequence motifs may accommodate more variability than has been documented to date. cellular ypx n l-containing escrt adapters recruit alix during exosome biogenesis and lysosomal sorting (baietti et al., 2012; dores et al., 2012) . ppxy: the ppxy late domain (where x can be any residue but is often a proline) binds ww domains in nedd4-like hect ubiquitin e3 ligases. ppxy late domains are found in retroviruses, filoviruses, arenaviruses, rhabdoviruses, and hepadnaviruses (votteler and sundquist, 2013) . hiv-1 gag does not contain a recognized ppxy motif. nevertheless, overexpression of the ubiquitin e3 ligase nedd4l induces budding of hiv-1 gag constructs that lack ptap and ypx n l late domains (usami et al., 2008; chung et al., 2008) . in this context, nedd4l is recruited by a cellular ppxy-containing protein amot, which also binds hiv-1 gag (mercenne et al., 2015) . the related angiomotin-like 1 (amotl-1) recruits nedd4 family members to paramyxovirus m proteins, which also lack discernible ppxy domains (pei et al., 2010; ray et al., 2019) . nedd4 e3 enzymes transfer k63-linked ubiquitin chains onto ppxy-containing cellular proteins, and they regulate endocytosis, escrt-dependent mvb cargo selection and protein trafficking through ubiquitination (rotin and kumar, 2009 ). it is not yet fully understood how nedd4 family members promote budding, but it has been suggested that ubiquitination of viral structural proteins, or other proteins within the budding site, recruits the escrt pathway because alix and tsg101 both contain ubiquitin-binding domains that can recognize ubiquitinated cargos for mvb incorporation and lysosomal degradation (shields and piper, 2011) . consistent with this idea, retroviral gag proteins are typically ubiquitinated (although this requirement is not absolute (zhadina et al., 2007) ), and ubiquitin depletion or mutations that prevent ubiquitin ligase recruitment inhibit retrovirus budding (patnaik et al., 2000; strack et al., 2000) . fpiv: the paramyxovirus sv5 employs an fpiv motif to facilitate budding. sv5 release is escrt-dependent and is augmented by amotl1, but the binding partner for the fpiv motif remains to be defined (el najjar et al., 2014) . viral structural proteins typically encode multiple late domains that function in synergy. for example, hiv-1 p6 gag contains both a p(s/ t)ap and a ypx n l motif, the htlv-i gag and ebola virus (ebov) structural protein vp40 proteins contain adjacent pppy and ptap late domains that bind both tsg101 and nedd4, and murine leukemia virus gag contains all three canonical late domains (votteler and sundquist, 2013) . nevertheless, some viruses contain a single (or at least dominant), late domain. for example, the retroviral eiav gag protein appears to recruit alix exclusively through a single ypx n l motif (votteler and sundquist, 2013) . all modes of viral escrt pathway recruitment ultimately converge on escrt-iii, the machinery that catalyzes membrane fission. in the best-studied cases such as hiv-1, multiple different mammalian escrt-iii proteins have been shown to localize to the bud neck (jouvenet et al., 2011) , but only chmp2 and chmp4 family members seem to perform indispensable functional roles (sandrin and sundquist, 2013; morita et al., 2011) . escrt-iii proteins form helical filaments in the bud neck and progressively constrict it with the help of vps4 enzymes, as described above. ultimately, a membrane fission reaction severs the neck, releasing the virion from the cell. some enveloped viruses bud independently of the escrt pathway, including alphaviruses (brown et al., 2018), some paramyxoviruses (salditt et al., 2010; utley et al., 2008) , and influenza a virus (rossman and lamb, 2011) . escrt-independent membrane scission mechanisms are generally not well understood, but appear to involve as yet unidentified cellular factors, virally encoded proteins, or a combination. some rna viruses, such as alphaviruses, contain an outer glycoprotein shell that completely covers the exterior of the viral membrane. the formation of this external protein coat has been suggested to play a role in virus budding, both in escrtindependent and escrt-dependent viruses. the alphaviral transmembrane glycoproteins e1 and e2 are embedded in the viral lipid envelope and form heterodimers that further trimerize into a continuous icosahedral lattice. these interactions are required to complete the budding step, and completion of the e1-e2 protein lattice, together with the nucleocapsid interactions across the viral membrane may be sufficient to drive both membrane curvature and fission (brown et al., 2018; weissenhorn et al., 2013) . in contrast, flaviviruses and hepaciviruses also contain an outer protein coat, but still depend on the escrt pathway to complete their replication cycle (see below). thus, external protein coats can apparently either replace or act in concert with the escrt pathway. influenza a is another escrt-independent virus (bruce et al., 2009; watanabe and lamb, 2010; chen et al., 2007) , but in this case, the viral transmembrane protein m2 appears to mediate membrane fission (rossman et al., 2010) . during particle assembly, the hemagglutinin (ha) and neuraminidase (na) glycoproteins are targeted to the plasma membrane and cluster in lipid rafts. the matrix protein m1 interacts with the cytoplasmic tails of ha and na, polymerizes against the membrane, and apparently acts in concert with ha to induce membrane curvature. m1 also recruits m2 to the bud neck, and an amphipathic helix in the cytoplasmic m2 tail inserts, deforms and promotes plasma membrane fission (martyna and rossman, 2014; chlanda and zimmerberg, 2016) . m2 also functions as a ph-regulated ion channel that facilitates the release of viral ribonucleoprotein complexes from the endosome into the cytoplasm, but ion channel and membrane fission appear to be independent activities (rossman et al., 2010) . some viral families envelop and bud into internal cellular membranes rather than at the plasma membrane. budding into the lumen of a cellular organelle is topologically equivalent to budding from the plasma membrane, but in these cases the viral particle is temporarily surrounded by two membranes, the viral envelope and the organelle membrane. virion release into the extracellular space therefore requires transport to and fusion of the virion-containing organelle with the plasma membrane. one such case is the hepatitis b virus (hbv), which co-opts the mvb pathway for egress (prange, 2012; blondot et al., 2016; patient et al., 2009) . the three viral envelope proteins s, m, and l form budding sites at the mvb membrane that recruit mature cytoplasmic nucleocapsids. the assembling hbv particles bud into the mvb lumen in an escrt-dependent reaction that creates intraluminal virions. mvbs then fuse with the plasma membrane to release the enveloped viral particles from the cell, a process that resembles exosome release. herpesviruses are released via the secretory pathway in a complex series of events that requires several viral and cellular proteins (lv et al., 2019; fradkin and budnik, 2016; owen et al., 2015) . herpesviral genome replication, capsid assembly, and genome packaging all take place in the nucleus. the fully assembled nucleocapsids are too large to escape into the cytoplasm through nuclear pores and instead exit the nucleus and cell by undergoing several steps of envelopment and de-envelopment at multiple cellular membranes. during primary envelopment, the nucleocapsids bud through the inner nuclear membrane into the perinuclear space, thereby acquiring a lipid envelope. this process requires the virus to remodel the nuclear lamina. cellular and viral kinases phosphorylate components of the nuclear lamina, leading to its disassembly. two viral proteins, pul31 and pul34, then assemble into a cage-like nuclear egress complex, that carries the virion across the inner nuclear membrane (bigalke and heldwein, 2015) . there is some evidence that the escrt machinery may also be involved in facilitating the membrane fission step required to release the enveloped virion into the intermembrane space (arii et al., 2018) . the primary envelope then fuses with the outer nuclear membrane in a process termed de-envelopment. the viral glycoproteins are necessary for de-envelopment, likely because they mediate fusion with the outer nuclear membrane. once in the cytoplasm, nucleocapsids associate with tegument proteins, which in the mature virion occupy the space between the nucleocapsid and the envelope. the nucleocapsids then bud into vesicles, whose origins have been variously described as the trans-golgi network, endosomes, or autophagic membranes. recruitment to sites of secondary envelopment is promoted by tegument proteins through interactions with vesicle membranes and viral glycoproteins. early-acting escrt proteins are not required for this process, but there are reports that escrt-iii and vps4 activity are required for virion release (crump et al., 2007; calistri et al., 2007; pawliczek and crump, 2009) , and an exciting new structure of the herpes simplex virus pul7: pul51 complex, which is required for efficient virion assembly, reveals that the n-terminal region of pul51 adopts a chmp4b-like fold that may function as a viral escrt-iii-like protein (butt et al., 2020). following membrane fission, the enveloped virions end up inside intracellular vesicles and are released into the extracellular space when the vesicles fuse with the plasma membrane. in a related process, flaviviruses and hepaciviruses are released through the secretory pathway (chatel-chaix and bartenschlager, 2014; falcon et al., 2017; gerold et al., 2017) . both genera of viruses induce extensive remodeling of endoplasmic reticulum (er) membranes to form replication compartments. these vesicle-like structures remain connected to the er, enclose viral proteins and the viral genome, and serve as protected compartments where almost all steps of the life cycle are carried out. assembled nucleocapsids then bud into the er lumen and are released through the secretory pathway. historically, viruses have been divided into enveloped and non-enveloped classes based on the presence or absence of a hostderived membrane envelope, and it was thought that non-enveloped viruses were released exclusively by host cell lysis. this simple paradigm has now been overturned by studies showing that many different classes of non-enveloped viruses can acquire hostderived lipid envelopes and exit cells within vesicles. these extracellular vesicles resemble exosomes, and viruses that use this egress method are termed "quasi-enveloped" (feng et al., 2014) . the picornavirus hepatitis a virus (hav) was the first virus definitively shown to be quasi-enveloped (feng et al., 2013) , and hav still serves as a paradigm for the process. quasi-enveloped hav particles (ehav) are released in exosome-like vesicles that typically contain 1-4 particles per vesicle. these vesicles are formed when hav capsids bud into endosomes in an escrt-dependent manner. to promote budding, the vp2 capsid protein recruits alix, apparently using tandem ypx 3 l domains that become buried in the fully assembled virion (gonzalez-lopez et al., 2018; mcknight et al., 2017) . virion-containing multivesicular bodies then fuse with the plasma membrane and release ehav particles into the extracellular space. there is now good evidence that this is the primary mode of hav release from hepatocytes in vivo, and that hav circulates in the blood exclusively within small vesicles (feng et al., 2013) . hav can then shed its envelope in the biliary tract, which produces a non-enveloped particle that may be more stable in harsher environmental conditions (feng et al., 2014) . importantly, hav is highly infectious in both its enveloped and non-enveloped states. the capacity for quasi-envelopment has since been described for several other viruses that were traditionally considered to be non-enveloped, including many other picornaviruses (chen et al., 2015; mutsafi and altan-bonnet, 2018) , hepatitis e virus (qi et al., 2015) , rotaviruses and noroviruses (santiana et al., 2018) . furthermore, some picornaviruses, including poliovirus and coxsackievirus, differ from the hav paradigm in that they form quasi-enveloped virions by subverting the autophagy pathway. in these cases, double-membraned autophagosomes engulf multiple naked viral particles, which then release quasi-enveloped viruses when the outer autophagosomal membrane fuses with the plasma membrane (mutsafi and altan-bonnet, 2018; bird et al., 2014) . a final variation on this theme is the exosomal transfer of viral nucleic acids between cells, which apparently can, in some viruses like hepatitis c, spread productive infections without requiring full viral assembly (ramakrishnaiah et al., 2013; bukong et al., 2014) . the membrane appears to perform several important functions for quasi-enveloped viruses, including protecting the capsid from antibody-mediated neutralization (feng et al., 2013) , and clustering together of multiple virions so that they can enter target cells as a swarm or "quasi-species" that can cooperate genetically through cross-complementation. the later activity may be most important for enteroviruses, whose larger vesicles can each contain tens or even hundreds of viral particles (chen et al., 2015; santiana et al., 2018) . the outer membranes of quasi-enveloped viruses lack viral glycoproteins, and therefore cannot fuse with target cell membranes. instead, ehav particles are taken up into the host cells by endocytosis and trafficked toward the lysosome, where the membrane is degraded, and the released naked virions can cross into the cytoplasm by disrupting the endolysosomal membrane (rivera-serrano et al., 2019) . most enveloped viruses undergo additional maturation steps during and after budding. before maturation, the virion functions as an assembly machinery that can package components and leave the producer cell. conformational changes, typically triggered by proteolytic cleavage or ph changes, then convert the virion into a particle that is capable of entering and replicating in a new target cell (veesler and johnson, 2012; steven et al., 2005) . in the case of hiv-1, the viral protease (pr) is activated by autoproteolysis as the virus assembles and buds, and it cleaves the gag polyprotein at five different sites, producing three new proteins (ma, ca and nc) and three smaller peptides (sp1, sp2, and p6). gag processing drives a series of major rearrangements in which the ca protein forms a conical internal capsid that surrounds viral rna in complex with nc protein and viral enzymes. gag cleavage is a sequential, ordered process, and each processing event appears to perform a different function. cleavage at the sp1-nc junction releases the nc-rna complex to condense to the center of the virion, cleavage at the ma-ca junction promotes folding of the ca n-terminus into a b-hairpin that will ultimately form an ntp-permeable pore in the assembled capsid, and cleavage at the ca-sp1 junction destabilizes the immature lattice and promotes formation of the mature capsid lattice. the nc-sp2 and sp2-p6 cleavages are also required for infectivity, as is cleavage of the longer gag-pol polyprotein, which liberates the viral enzymes. the mature conical capsid is a fullerene cone, with a curved hexagonal lattice comprising ca hexamers, and the cone ends closed through the incorporation of 12 ca pentamers. ca hexamers are stabilized by binding ip 6 (dick et al., 2018; mallery et al., 2018) , and differential placement of the hexamers and pentamers produces a variety of related capsid structures that each differ slightly in length and shape (sundquist and krausslich, 2012; mattei et al., 2016; freed, 2015) . viral glycoproteins and their fusion peptides that enable entry into target cells must also typically be proteolytically processed to be functional. for example, the hiv-1 env glycoprotein is synthesized as a polyprotein precursor (gp160), which is inserted into the endoplasmic reticulum membrane co-translationally. env is glycosylated and then proteolytically cleaved by the host golgiassociated protease furin as it traffics through the secretory pathway, producing the mature surface gp120 and transmembrane gp41 glycoprotein subunits, which remain non-covalently associated as heterotrimeric spikes. proteolytic processing exposes the fusion peptide at the gp41 n-terminus and is required for fusogenic activity (checkley et al., 2011) . in other viruses, proteolytic activation of viral fusion proteins can occur following entry into the target cell. activation of the ebov glycoprotein is a particularly well-understood case. ebov particles associate with the host cell surface by interactions with host receptors that bind to glycans on the viral glycoprotein gp and phosphatidylserine in the viral envelope. after internalization through macropinocytosis, endosomal cysteine proteases such as cathepsins l and b proteolytically process gp to remove a mucinlike subdomain and the glycan cap and expose the receptor-binding site (rbs). the rbs binds the late endosomal/lysosomal protein npc1, which induces a conformational change in gp, insertion of a fusion loop into the endosomal membrane, fusion of viral and endosomal membranes and release of the nucleocapsid into the cytoplasm (lee and saphire, 2009; carette et al., 2011; cote et al., 2011; gong et al., 2016; wang et al., 2016) . after budding, viruses can spread in two different ways; through cell-free transmission and cell-to-cell transmission. cell-free virions diffuse freely through the extracellular space, and even between organisms, before entering target cells. this process can promote dissemination over long distances, to new tissues, and between hosts. however, untargeted diffusion through aqueous media is relatively inefficient, and free viruses are susceptible to immune recognition. in contrast, viral spread through direct sites of cell-to-cell contact increases transmission efficiency and can help evade antibody recognition. to promote cell-to-cell transmission, viruses often subvert cellular structures that are normally used for cell-cell communication or cargo transfer. retroviruses such as hiv-1 and mlv actively promote the formation of adhesive structures between donor and target cells. these stable contact sites are termed virological synapses because they resemble the immunological synapses that mediate antigen presentation, and even employ some of the same molecular components. virological synapse formation requires interactions between the viral glycoprotein on the donor cell and its cognate target cell receptor. coreceptors and adhesion molecules are then recruited to stabilize and further organize these contact sites, and the producer cell cytoskeleton is repolarized towards the synapse to promote directional viral assembly and release. virions bud directly into the intersynaptic space and are transferred efficiently to the closely opposed target cell (agosto et al., 2015; bracq et al., 2018; dufloo et al., 2018; nejmeddine and bangham, 2010) . other viruses hijack existing cell-cell contacts for transmission. for example, neurotropic viruses such as herpesviruses and rabies viruses are transported along axons and spread across synaptic contacts (koyuncu et al., 2013) . viruses can also achieve targeted release by exploiting membrane protrusions such as nanotubes and filopodia, which normally transmit information and cargoes between cells (agosto et al., 2015; bracq et al., 2018; dufloo et al., 2018) . the principles of enveloped virus budding are remarkably conserved between different virus families, presumably owing to evolutionary history and common functional requirements. viral egress is typically orchestrated by multifunctional structural proteins that recruit components, assemble the virion, bend host membranes, and facilitate membrane fission. in many, but not all cases, the cellular escrt pathway is recruited to mediate the final membrane fission step. recent studies have also revealed that many traditional "non-enveloped" viruses can be released within vesicles as quasi-enveloped viruses and that viruses frequently alter cellular pathways to promote directional release and synapse formation. although the general strategies for enveloped virus egress are increasingly well understood, important challenges remain, including characterizing the release mechanisms of escrt-independent viruses, the biology and entry mechanisms of quasienveloped viruses, and the molecular mechanisms and pathogenesis associated with cell-to-cell viral spread. these and other advances will help reveal the best approaches for inhibiting virus release for therapeutic benefit and harnessing release activities in new systems that can be used to deliver biomolecular cargoes into target cells in vivo. ultrastructural and biochemical basis for hepatitis c virus morphogenesis a pathogenic picornavirus acquires an envelope by hijacking cellular membranes naked viruses that aren't always naked: quasi-enveloped agents of acute hepatitis this bud's for you: mechanisms of cellular nucleocytoplasmic trafficking via nuclear envelope budding hiv-1 assembly, release and maturation hiv-1 matrix protein interactions with trna: implications for membrane targeting tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding structural insights into the niemann-pick c1 (npc1)-mediated cholesterol transfer and ebola infection redundant late domain functions of tandem vp2 ypx 3 l motifs in nonlytic cellular egress of quasi-enveloped hepatitis a virus effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release multivesicular body morphogenesis hantavirus structure -molecular interactions behind the scene crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly p6gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease inside job: how the escrts release hiv-1 from infected cells foamy virus budding and release the pi3k class iii complex promotes axon pruning by downregulating a ptc-derived signal via endosome-lysosomal degradation escrt machinery is required for plasma membrane repair imaging the interaction of hiv-1 genomes and gag during assembly of individual viral particles dynamics of escrt protein recruitment during retroviral assembly orchestrating the selection and packaging of genomic rna by retroviruses: an ensemble of viral and host factors the p6gag domain of human immunodeficiency virus type 1 is sufficient for the incorporation of vpr into heterologous viral particles virus infections in the nervous system global changes in the rna binding specificity of hiv-1 gag regulate virion genesis life of psi: how full-length hiv-1 rnas become packaged genomes in the viral particles the escrt machinery is recruited by the viral bfrf1 protein to the nucleus-associated membrane for the maturation of epstein-barr virus ebolavirus glycoprotein structure and mechanism of entry how hiv-1 gag assembles in cells: putting together pieces of the puzzle a consensus view of escrt-mediated human immunodeficiency virus type 1 abscission from crescent to mature virion: vaccinia virus assembly and maturation remodeling of host membranes during herpesvirus assembly and egress hiv-1 and ebola virus encode small peptide motifs that recruit tsg101 to sites of particle assembly to facilitate egress divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins alterations of membrane curvature during influenza virus budding retrovirus maturation-an extraordinary structural transformation structures, functions, and dynamics of escrt-iii/vps4 membrane remodeling and fission complexes protein composition of the hepatitis a virus quasi-envelope wrapping up the bad news: hiv assembly and release angiomotin functions in hiv-1 assembly and budding human escrt and alix proteins interact with proteins of the midbody and function in cytokinesis escrt-iii protein requirements for hiv-1 budding the foamy virus gag proteins: what makes them different? enterovirus transmission by secretory autophagy hantaviral proteins: structure, functions, and role in hantavirus infection paramyxovirus glycoprotein incorporation, assembly and budding: a three way dance for infectious particle production the htlv-1 virological synapse membrane re-modelling by bar domain superfamily proteins via molecular and non-molecular factors escrt-iii controls nuclear envelope reformation plasma membrane rafts play a critical role in hiv-1 assembly and release tegument assembly and secondary envelopment of alphaherpesviruses positionally independent and exchangeable late budding functions of the rous sarcoma virus and human immunodeficiency virus gag proteins the neuronal gene arc encodes a repurposed retrotransposon gag protein that mediates intercellular rna transfer ubiquitin is part of the retrovirus budding machinery herpes simplex virus type 1 production requires a functional escrt-iii complex but is independent of tsg101 and alix expression piv5 m protein interaction with host protein angiomotin-like 1 single-molecule imaging of hiv-1 envelope glycoprotein dynamics and gag lattice association exposes determinants responsible for virus incorporation host factors involved in hepatitis b virus maturation, assembly, and egress hepatitis e virus produced from cell culture has a lipid envelope exosome-mediated transmission of hepatitis c virus between human hepatoma huh7.5 cells angiomotin-like 1 links paramyxovirus m proteins to nedd4 family ubiquitin ligases hiv-1 gag co-opts a cellular complex containing ddx6, a helicase that facilitates capsid assembly influenza virus assembly and budding influenza virus m2 protein mediates escrt-independent membrane scission physiological functions of the hect family of ubiquitin ligases measles virus m protein-driven particle production does not involve the endosomal sorting complex required for transport (escrt) system escrt requirements for eiav budding vesicle-cloaked virus clusters are optimal units for inter-organismal viral transmission an atomic model of hiv-1 capsid-sp1 reveals structures regulating assembly and maturation growing functions of the escrt machinery in cell biology and viral replication requirements for incorporation of pr160gag-pol from human immunodeficiency virus type 1 into virus-like particles virus maturation: dynamics and mechanism of a stabilizing structural transition that leads to infectivity a role for ubiquitin ligase recruitment in retrovirus release aip1/alix is a binding partner for hiv-1 p6 and eiav p9 functioning in virus budding hiv-1 assembly, budding, and maturation. cold spring harbor perspectives synthesis, assembly, and processing of viral proteins an autophagy assay reveals the escrt-iii component chmp2a as a regulator of phagophore closure the role of matrix in hiv-1 envelope glycoprotein incorporation biochemical evidence of a role for matrix trimerization in hiv-1 envelope glycoprotein incorporation efficient and specific rescue of human immunodeficiency virus type 1 budding defects by a nedd4-like ubiquitin ligase respiratory syncytial virus uses a vps4-independent budding mechanism controlled by rab11-fip2 virus maturation tsg101, a homologue of ubiquitin-conjugating (e2) enzymes, binds the l domain in hiv type 1 pr55(gag) spastin and escrt-iii coordinate mitotic spindle disassembly and nuclear envelope sealing virus budding and the escrt pathway crystal structure of an hiv assembly and maturation switch. elife 5, e17063 influenza virus budding does not require a functional aaa þ atpase, vps4 how to get out: ssrna enveloped viruses and membrane fission fine mapping and characterization of the rous sarcoma virus pr76gag late assembly domain infectivity of moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses ubiquitin-dependent virus particle budding without viral protein ubiquitination endocytic pathways downregulate the l1-type cell adhesion molecule neuroglian to promote dendrite pruning in drosophila rab5-dependent autophagosome closure by escrt identification of a host protein essential for assembly of immature hiv-1 capsids we thank moona huttunen and jörg votteler for helpful comments on the manuscript, and janet iwasa for creating the figures. this work was funded by nih r37 ai051174. we apologize to our many colleagues whose primary literature references we were unable to cite owing to space limitations. key: cord-259503-dkfrk71a authors: smith, sarah e.; wash, rachael s. title: sherlock genomes — viral investigator date: 2013-02-15 journal: nat rev microbiol doi: 10.1038/nrmicro2979 sha: doc_id: 259503 cord_uid: dkfrk71a this month's genome watch highlights how deep sequencing technologies have vastly reduced the time and prior knowledge needed to generate viral genomes. deep sequencing technologies and stateof-the-art bioinformatics techniques have revolutionized the way that rna viruses, a notoriously variable group of pathogens, can be identified and characterized. traditionally, specific information about the viral genome was required to design primers for the reverse transcription and amplification of viral rna prior to sequencing. in addition, a reference genome was often used to assemble short read sequences into a complete genome. however, recent methodological advances have negated some of these prerequisites. gall et al. 1 developed a method to generate full genomes of hiv-1. they designed a 'pan-hiv-1' reverse transcription pcr primer set, based on sequences available in the los alamos hiv sequence database, that could give rise to four overlapping amplicons from any hiv-1 virus. the addition of multiplex identifier (mid) adaptors (tags that enable the source of each read to be identified) meant that many samples could be sequenced simultaneously on deep sequencing platforms. by using de novo assembly of short reads, the authors showed that this method could generate full genomes for viruses within all four major hiv-1 genetic groups. this shows the potential of deep sequencing for high-throughput studies involving hiv-1 genomes with a broad range of sequence diversity. the high mutation rate of hiv-1 means that an infected individual can be host to many viral genome variants. some of these can confer drug resistance, which in hiv-1 can involve mutations in different genes along the whole genome. the protocol described above offers a read depth that would allow the detection of lowfrequency genotypes, facilitating the analysis of rare mutations associated with drug resistance, unlike capillary sequencing, which offers low read coverage. even when almost nothing is known about the agent of a viral infection, deep sequencing technologies can identify a pathogen and produce the full genome, fast. in 2012, a saudi arabian patient was admitted to hospital suffering from acute severe pneumonia of unknown cause. preliminary diagnostics indicated that he was infected by a coron avirus (cov), a single-stranded rna virus that can infect many species, with bats being an important zoonotic reservoir. six cov species have been detected in humans, although only one was known to cause severe disease: severe acute respiratory syndrome (sars)-cov. when sars-cov began infecting people in 2002, it took a large team to generate a full genome by capillary sequencing. first, primers were designed for conserved regions of known cov species, followed by further primer design as the sequence of sars-cov was generated. by contrast, in 2012 van boheemen et al. 2 used random priming to amplify viral rna isolated from the saudi arabian patient, and then deep-sequenced the genome. using a massively parallel approach, whereby all the randomly amplified template dna was sequenced simultaneously on one plate, the team pieced together 90% of the genome in a single run, with an average read depth of 1,006-fold. primers could then easily be designed to fill in small gaps by capillary sequencing; the whole process took just a few days. knowing the sequence of the whole genome enabled the authors to further characterize this new virus, named human cov (hcov)-emc2012. phylogenetic analysis was carried out on sequence alignments of genome portions, including those encoding the replicase and other structural proteins, and this revealed that the virus was a novel cov. the authors showed that hcov-emc2012 is most closely related to two members of the c lineage of betacoronaviruses: bat cov hku4 and bat cov hku5. however, the conserved synteny between hcov-emc2012 and these two bat cov isolates is well below the 90% threshold for members of the same species. more recently, annan et al. 3 screened faecal samples from >4,000 bats across africa and europe, with the hope of finding viruses related to hcov-emc2012. sequencing facilitated the detection of betacoronaviruses in 25% and 15% of tested nycteris and pipistrellus bats, respectively. phylogenetic analysis of these viruses indicated that hcov-emc2012 originated from bats. fast, whole-genome sequencing of viruses is important for improved diagnostics, accurate geographical mapping of viral spread, and drug treatments. as sequencing technologies continue to improve, so will the value of whole-genome sequencing to both clinical and basic virology research. universal amplification, next-generation sequencing, and assembly of hiv-1 genomes genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe. emerg. infect dis the authors declare no competing financial interests. 150 | march 2013 | volume 11 www.nature.com/reviews/micro key: cord-298033-kzdp9edn authors: domingo, esteban title: quasispecies dynamics in disease prevention and control date: 2019-11-08 journal: virus as populations doi: 10.1016/b978-0-12-816331-3.00008-8 sha: doc_id: 298033 cord_uid: kzdp9edn medical interventions to prevent and treat viral disease constitute evolutionary forces that may modify the genetic composition of viral populations that replicate in an infected host and influence the genomic composition of those viruses that are transmitted and progress at the epidemiological level. given the adaptive potential of viruses in general and the rna viruses in particular, the selection of viral mutants that display some degree of resistance to inhibitors or vaccines is a tangible challenge. mutant selection may jeopardize control of the viral disease. strategies intended to minimize vaccination and treatment failures are proposed and justified based on fundamental features of viral dynamics explained in the preceding chapters. the recommended use of complex, multiepitopic vaccines, and combination therapies as early as possible after initiation of infection falls under the general concept that complexity cannot be combated with simplicity. it also follows that sociopolitical action to interrupt virus replication and spread as soon as possible is as important as scientifically sound treatment designs to control viral disease on a global scale. medical interventions have dramatically increased over the last century, and in the case of infectious diseases, the discovery and development of antibiotics and antiviral agents have represented a very powerful external selective constraint imposed upon replicating microbes. hundreds of antiviral agents have been developed since the second half of the 20th century, and viruses can generally find evolutionary pathways to continue replication in their presence. the same is true of antibiotics and replication of bacteria. the belief that bacterial diseases were on their way toward extinction was quite widespread in the middle of the 20th century. sir m. burnet wrote in his 1966 textbook: "and since bacterial infections are, with unimportant exceptions, amenable to treatment with one or other of the new drugs, our real problems are likely to be concerned with virus diseases" (burnet, 1966) . in 1932, a prominent spanish medical doctor, g. marañ on, declared: "in the year 2000 cancer will be a historical disease. infections will be almost entirely absent as a cause of mortality." (on a personal note, when i joined the university of california, irvine in 1969, to work as postdoctoral student with r.c. warner, i attended some biology courses in which the teachers expressed to students that infectious diseases would disappear in a few decades as a consequence of the use of antibiotics and antiviral agents). predictions in science tend to fail. the optimistic view was not unanimous. a. fleming, the discoverer of penicillin, recognized the adaptive capacity of bacteria and suggested virus as populations https://doi.org/10.1016/b978-0-12-816331-3.00008-8 that bacteria would inevitably find ways of resisting the damage to them caused by antimicrobial drugs [quoted from the document "new antimicrobial drugs" from the european academies science advisory council, november 2014 (www.easac.eu); see also chapter 10]. furthermore, there was early evidence of selection of mycobacterium tuberculosis mutants resistant to streptomycin (mitchison, 1950) . antibiotic resistance in bacteria has similarities and differences with antiviral resistance in viruses, and they are compared in chapter 10. we are now very aware that one of the major problems in antiviral therapy is the nearly systematic selection of drug-resistant virus mutants, which is often associated with treatment failure. other external influences, such as vaccination or immunotherapy, particularly using monoclonal antibodies, can also evoke the selection of viral subpopulations capable of replicating in the presence of those components inherent to an immune response. thus, selective constraints intended to limit rna virus replication meet with the broad and dynamic repertoire of variants ingrained in quasispecies dynamics. two space-time levels of the effects of drugs or vaccines are distinguished in coming sections: (i) short-term consequences for the individual in the form of treatment or vaccination failure and (ii) long-term consequences at the population level in the field, or vaccine-driven evolution of the antigenic properties of viruses. there are other medical interventions that may alter virus survival. individuals who are immunocompromised as a consequence of treatment after organ transplantation or those subjected to anticancer chemotherapy become particularly vulnerable to viral infections. enhanced viral replication can favor pathological manifestations in the affected individual as well as the spread of a large number of viruses into the environment, with consequences for the emergence and reemergence of viral disease (section 7.7 in chapter 7). viral diseases are an important burden for human health and agriculture (bloom and lambert, 2003) . virus evolution, through the basic mechanisms exposed in previous chapters, can influence the two major strategies to combat viral infections: prevention by vaccination and treatment by antiviral inhibitors. for the design of new antiviral vaccines, a critical issue is the diversity displayed in the field by the virus to be controlled. the natural evolution of the virus may result in the circulation of one major antigenic type or the cocirculation of multiple antigenic forms. the vaccine composition (independently of the type of vaccine; see section 8.3.1) must match the antigenic composition of the virus to be controlled. hepatitis a virus (hav) circulates as a single serotype, while foot-and-mouth disease virus (fmdv) circulates as seven serotypes and diverse subtypes, and the antigenic types are unevenly distributed in different geographical locations. a monovalent vaccine made of the prevailing antigenic type of hav should be sufficient to confer protection, while a multivalent vaccine composed of several types or subtypes is required to confer protection against fmdv, and the antigenic composition of the vaccine should match the circulating viruses in each geographical region. this is why antifmd vaccines of different compositions are used in different world areas at a given time, and vaccine composition must be periodically updated to maintain its efficacy. thus, one effect of virus evolution relevant to vaccine design derives from the necessity to prepare a vaccine that mirrors the antigenic composition of the virus to be controlled. in the case of live-attenuated antiviral vaccines, the evolution of the vaccine virus while it replicates in the vaccinee is a risk factor to produce virulent derivatives. the invasion of a susceptible host by a virus and the ensuing viral replication can be regarded as a step-wise process during which the virus must adapt to a series of selective pressures presented by the host, notably the immune response. the outcome can be either viral clearance (elimination of the infection) or virus survival and progression toward an acute or a persistent infection. administration of antiviral agents is an additional selective constraint that limits viral replication. evolutionary mechanisms may either succeed in the selection of mutants resistant to the antiviral agent that will permit the infection to continue or fail in sustaining the infection, resulting in the clearing of the virus from the organism. treatment planning, one of the aims of the new antiviral pharmacological interventions, based on information of viral genomic sequences present in each infected patient, has parallels with vaccine composition design. for vaccines, the information comes from the analyses of antigenic composition of circulating viruses, and for antiviral agents, the information comes from the mutant spectrum composition of the virus to be controlled in the infected patient. world-wide vaccination campaigns made possible the eradication of human smallpox [with the official declaration by the world health organization (who) in 1980] and animal rinderpest [with the official declaration by the world organization for animal health, office international des epizooties (oie) in 2011]. the number of new cases has decreased as a result of vaccination programs against several viral diseases, including measles or hepatitis b (bloom and lambert, 2003) , and substantial progress has been made toward the eradication of poliomyelitis (chumakov and kew, 2010; himman, 2017) . only the deliberate decision not to vaccinate (for religious reasons or misinformation campaigns) or lack of vaccine accessibility (for socioeconomic circumstances) jeopardizes vaccine efficacy. these facts demonstrate that at least some viral diseases can be controlled on a global basis by vaccination, an unprecedented achievement of human and animal health. despite the huge economic investment, however, there are important viral diseases such as acquired immunodeficiency syndrome (aids), hepatitis c, or viral hemorrhagic fevers for which no effective vaccines are available. for some diseases such as human influenza or animal fmd, vaccines are accessible, but they require periodic updating to approximate the antigenic composition of the vaccine to that of the circulating virus (section 8.2). in the case of influenza virus (iv), a major change in antigenic composition can occur through antigenic shift, in which the virus acquires new hemagglutinin and neuraminidase genes by genome segment reassortment (section 7.4 in chapter 7), with the first evidence obtained by g. laver as early as 1971 (for the early history of influenza, its causative virus, and vaccine designs, see beveridge, 1977; kilbourne, 1987) . antigenic variation of viruses, whatever the mechanism might be, can affect vaccine efficacy and in some cases, the extreme rapid intra and interhost evolution of a virus may render an effective vaccine unfeasible at least with the current tools of vaccinology. cd8 þ t cell responses may act soon after infection and promote the selection of escape mutants (bull et al., 2015) . the difficulties for the control of virus disease derived from the adaptive potential of viruses (domingo, 1989; domingo and holland, 1992; bailey et al., 2004; hamelaar et al., 2019) require the judicious application of existing tools and innovative approaches that are still in their infancy. a first basic requisite for the preparation of a vaccine against a viral agent is the understanding 8.3 antiviral vaccines and the adaptive potential of viruses of the immune response evoked by the virus when it infects the organism to be protected (activation of b and t lymphocytes for antibody production, cellular responses, and generation of memory cells) and correlates of protection (bloom and lambert, 2003; van regenmortel, 2012; hagan et al., 2015; cunningham et al., 2016; rolland, 2019) . despite social pressure to rapidly obtain a vaccine, for each virus-host system, well-designed experiments are necessary to try to establish the determinants of protection, which is not a simple issue. the discussions in coming paragraphs are focused on the relevance of virus evolution in vaccine efficacy, irrespective of the type of protection afforded by the vaccine. what we term "protection" may mean the total absence of replication of the infecting virus (termed "sterilizing" immunity) or absence of disease manifestations despite infection and virus replication. as a general initial statement, which is widely accepted by vaccinologists, a vaccine is likely to be effective when it evokes an immune response that is similar to the response elicited by the authentic viral pathogen when it produces disease successfully overcome by the infected organism (evans and kaslow, 1997; bloom and lambert, 2003) . we refer to this as the basic principle of vaccinology. when infection by an antigenically constant virus produces lifelong immunity (i.e., measles virus infection), a vaccine is likely to evoke longlasting protection. in contrast, if a patient cured of a virus can be reinfected by the same (or a closely related) virus (i.e., hepatitis c virus infection) a vaccinedat least one prepared by standard methodologydis unlikely to evoke protection. some points to be considered in the design of antiviral vaccines are listed in box 8.1. they are intended to minimize the selection of vaccineescape mutants and favor the success of vaccination campaigns. some of the recommendations deserve further comment. first, a basic knowledge of virus evolutionary dynamics and how it affects virus antigenic stability (or lack of) is essential. the fact that a methodology is available (i.e., vectors that can express large amounts of based on domingo and holland (1992) . 8 . quasispecies dynamics in disease prevention and control antigens displaying good immunogenicity) does not guarantee vaccine efficacy, and even less if correlates of protection are not understood. the order of efficacy of different vaccine designs proposed in box 8.1 is justified both by the basic principle of vaccinology and by the mechanisms of selection of antibody (ab)-and cytotoxic tcell (ctl)-escape mutants by viruses. single amino acid substitutions at b-and t-cell epitopes in viral proteins are often sufficient to elude neutralization by the corresponding cognatespecific antibody or to escape recognition by a clonal ctl population. for many viruses, the frequency of monoclonal antibody-escape mutants has been measured in 10 à4 to 10 à6 , even in clonal populations obtained under controlled laboratory conditions and that have undergone a limited number of replication rounds (section 7.4.2 in chapter 7). generation of immune-escape variants can result in lack of vaccine efficacy, contribute to viral persistence (pircher et al., 1990; weidt et al., 1995; ciurea et al., 2000 ciurea et al., , 2001 richman et al., 2003; pawlotsky, 2006) , and provoke vaccination-induced virus evolution (section 8.3.2). in human immunodeficiency virus type 1 (hiv-1), antibody-escape variants are incessantly being produced in vivo to the point that virus replication continues despite the antibody response (richman et al., 2003; bailey et al., 2004) . the frequency of selection of mutants that can escape a number (n) of components in which we could hypothetically separate a global immune response is far lower than the frequency of escape to a single (a, b, c, etc.) of the i components of the response. making a simple mathematical abstraction that is applicable also to antiviral-escape mutants (section 8.4), the frequency of mutants that escape n components of an immune response is the product of frequencies of escape to each individual component [10 àa â 10 àb â 10 àc â . . this is an oversimplification because it is not realistic to dissect the selective impact of a complex immune response into discrete components. a virus generally includes multiple antigenic sites and each of them is often composed of several overlapping or nonoverlapping epitopes; in addition, a virus has several t-cell epitopes in different structural and nonstructural proteins, and each epitope displays a different degree of relative dominance. however, the above abstraction reflects the advantage of stimulating the host immune system with a sufficiently broad array of b-and t-cell epitopes to prevent selection of vaccineescape mutants due to a high genetic and phenotypic barrier (compare with the barrier to drug resistance described in section 8.4.2). therefore, selection of vaccine-escape viral mutants is more likely with synthetic peptidic vaccines, than with whole virus-attenuated or inactivated vaccines because the latter present a broad epitope repertoire to the immune system. selection of escape-mutants by peptidic vaccines that evoked partial protection of cattle was documented with fmdv (taboga et al., 1997; tami et al., 2003) . the arguments in favor of multiepitopic presentation are also endorsed by a notorious scarcity of licensed peptidic vaccines for viral diseases despite horrendous economic investments (orders of magnitude greater than investments in quasispecies research!). use of a complex, multiepitopic vaccine, however, need not prevent long-term selection of antigenic virus variants as a result of vaccine usage, an important still largely underexplored topic discussed in section 8.3.2. experimental evolution with fmdv has opened the way to a new generation of antiviral vaccines that share features of attenuated and inactivated vaccines. this new design is based on the conversion of the monopartite fmdv genome into a segmented genome version that dominated the population after extensive high moi passages (section 2.12 in chapter 2 and section 6.6 in chapter 6). for the segmented virus version to be able to produce progeny, the two genome classes (which are encapsidated into separate particles) must reach the same cell. therefore, administration of the segmented virus preparation should lead to an 8.3 antiviral vaccines and the adaptive potential of viruses immune response akin to that evoked by inactivated viral particles, followed by a self-limiting infection. the vaccine was tested successfully in mice and swine, the authentic host of the parental virus (rodriguez-calvo et al., 2010) . potential concerns with this type of vaccine are that the standard (monopartite) genome can be reconstructed by recombination in the early stages of replication in the animal. a safety level is included in that particular fmd vaccine because of multiple mutations that accumulated during the transition toward genome segmentation, which deviated the genome sequence from the one present in the original swine isolate . exploration of evolutionary mechanisms that result in altered forms of viruses may open new possibilities for vaccine design. new prospects for attenuated vaccines have been opened with the engineering of viruses with suboptimal replication fidelity or deoptimized codon or codon pair usage (coleman et al., 2008; vignuzzi et al., 2008; cheng et al., 2015) . altered polymerase copying fidelity often leads to virus attenuation (gnadig et al., 2012; graham et al., 2012; korbouk et al., 2014; rozen-gagnon et al., 2014; van slyke et al., 2015) . a critical issue with live-attenuated vaccines based on deviation from the standard mutation rate is the stability of the attenuation trait. not only true revertants or other site revertants of the polymerase may arise and displace the vaccine virus, but other viral proteins may affect nucleotide incorporation (smith et al., 2013 stapleford et al., 2015; agudo et al., 2016 ). when a virus circulates in a population where vaccinated and unvaccinated host individuals coexist, and the vaccine does not induce sterilizing immunity, viruses with an altered antigenic profile might be selected. the larger the overall effective population size of the circulating virus, and the longer the virus is allowed to replicate in such a scenario, the higher the probability of incorporation of compensatory mutations that yield high-fitness antigenic variants. these events in the case of vaccines used in veterinary medicine are particularly significant because they may alter the cell tropism and host range of viruses, thus increasing the possibilities of their zoonotic transmission into humans (schat and baranowski, 2007) . evidence of vaccination-induced dna and rna virus evolution is increasing, and it has been documented with bovine respiratory syncytial virus, bovine herpesvirus-1, marek's disease virus, porcine circovirus 2, and classical swine fever virus, among others [ (valarcher et al., 2000; muylkens et al., 2006; ji et al., 2014; kekarainen et al., 2014; constans et al., 2015; yoo et al., 2018) ; reviews in gandon et al., 2003; schat and baranowski, 2007) ]. the timing of dominance of ctl-escape mutants of the simian immunodeficiency virus (siv) was influenced by vaccination, and the process could be analyzed by penetration into the mutant spectra of the relevant viral populations (loh et al., 2008) . for human viruses, evidence of vaccineescape mutants has been obtained for hepatitis a and b viruses. vaccination-associated escape mutants of hav with substitutions around the immunodominant site of the virus were identified in a cohort of hiv-1, hav doubly infected individuals (perez-sautu et al., 2011) . the study suggested that an incomplete vaccination schedule, combined with the hiv-1-produced immunosuppression might have contributed to high-hav loads, thus facilitating the generation and dominance of antigenic variants. in taiwan, the prevalence of mutants at a major antigenic determinant of the surface antigen of hepatitis b virus (hbv) tripled in 1 decade, and it has been suggested that this increase of prevalence might be due to the ample vaccination coverage in the region (hsu et al., 1999) . other studies also suggest the circulation of hbv mutants 8. quasispecies dynamics in disease prevention and control associated with vaccine escape and diagnosis failures (di lello et al., 2019) . vaccines can rarely afford protection to all vaccinated individuals due to many factors that include variations in vaccine receptivity factors due to polymorphisms in genes involved in the adaptive immune response, immunosuppression of the vaccine recipient, the insufficient time between vaccination and exposure to the viral pathogen, and antigenic differences between the vaccine strain and circulating viruses. in addition, for massive vaccinations in veterinary medicine, damage to the vaccine (during transport, storage, etc.) and improper administration are additional problems. vaccination may occasionally promote the selection not only of antigenic variants but also host cell tropism, host range, or virulent variants (swayne and kapczynski, 2008; kirkwood, 2010; read et al., 2015; rolland, 2019) . it is not known to what extent the widespread use of vaccination can contribute to antigenic variation relative to other factors (persistence of antibodies from previous infections, genetic drift due to genetic bottlenecks, etc.). however, our current understanding of virus dynamics should encourage investigations on the genetic and antigenic modifications of breakthrough viruses that arise from vaccinated individuals as compared with changes in viruses from unvaccinated host populations. reversion of live-attenuated vaccine viruses into virulent forms is a cause of disease derived from the evolutionary potential of viruses. in the case of attenuated sabin poliovirus vaccine, the rate of vaccine-associated poliomyelitis among those vaccinated for the first time was one per 500,000 to 750,000 vaccinees, and the rate of those receiving the second vaccine dose was about one in 12 million (reviewed in rowlands and minor, 2010) . attenuated antifmd vaccines were used in some countries during the second half of the 20th century, but a reversion to virulence forced the halting of the vaccination programs. vaccine-escape mutants may arise due to ineffective vaccines, and concomitant factors, such as immunosuppression. the escape mutants may remain confined to the unsuccessfully vaccinated host or may spread to other susceptible individuals, and attain different degrees of epidemiological relevance. escape mutants may be direct mutants of the infecting virus or may originate by recombination between the infecting virus and other coinfecting related viruses, as observed with poliovirus and bovine herpesvirus-1 [kew et al., 2002; thiry et al., 2006 ; among other studies]. reiteration of vaccine selection and fitness increase processes over many generations of vaccinees (be it humans or animals) may result in accelerated virus evolution. since systematic use of vaccines for humans and animals in intensive production units is relatively recent in terms of evolutionary time (less than 100 years, and in some cases even only a few decades), it is still premature to evaluate whether vaccination is a significant factor in promoting long-term virus evolution. the first description of virus resistant to an antiviral inhibitor was by j. barrera-oro, h.j. eggers, i. tamm, and colleagues working with enteroviruses and guanidine hydrochloride and 2-(alpha-hydroxybenzyl)-benzimidazole as inhibitors (eggers and tamm, 1961; melnick et al., 1961) . these early results that suggested that antiviral-resistant mutants could be readily selected have been amply confirmed with many viruses and inhibitors in cell culture and in vivo. indeed, the selection of viral mutants resistant to antiviral agents is an extremely frequent occurrence that has been known for decades, although it became widely recognized in the course of development and clinical use of antiretroviral agents to treat hiv-1 infections and aids. the description of drug-escape mutants has been based on three main groups of observations: • detection of antiviral-resistant mutants in patients during treatment. when a reverse genetics system is available, the suspected mutation should be introduced in an infectious clone and resistance ascertained and quantified in cell culture or in vitro enzyme assays. • selection of resistant mutants in cell culture, by subjecting the viruses to passages in the presence of inhibitors. the viral population size is an important variable in this type of experiment (section 8.4.1). • calculation of the frequency of resistant mutants by plating a virus in the absence and presence of the antiviral agent similar to the assays to calculate the frequency of monoclonal antibody (mab)-resistant mutants (described in chapter 7, section 7.4.2). in the three groups of observations, the frequency at which a specific escape mutant is found depends on a number of barriers to resistance (section 8.4.2). traditionally, the fact that a drug can select virus-resistant mutants is regarded as a proof of the selectivity of the drug, as opposed to unspecific or toxic effects on the host cell that indirectly impair virus replication (herrmann and herrmann, 1977; golan and tashjian, 2011) . selection of viral mutants resistant to antiviral inhibitors is a major problem for the control of viral disease for two main reasons: (i) because it often results in virus breakthrough (increase of viral load) resulting in treatment failure and (ii) because resistant virus variants may become epidemiologically relevant, with the consequent decrease of inhibitor efficacy at the population level (domingo and holland, 1992; huang et al., 2019) . increasing numbers of antiviral agents have been developed based on the three-dimensional structure of viral proteins and their complexes with natural and synthetic ligands, in efforts that have engaged academic institutions and pharmaceutical companies. others have been incorporated as a result of drug repositioning, that is, the discovery of antiviral activity of compounds licensed for other medical purposes, often with the help of computational tools (ab ghani et al., 2019) . antiviral agents may target viral or cellular proteins involved in any step of the virus life cycle. they may interact with virions and inhibit an early step of infection, such as the attachment to the host cell, penetration into the cell, or uncoating to liberate the genetic material of the virus inside the cell. other agents interfere with the synthesis of viral nucleic acids or viral protein processing, particle assembly, or virus release from cells. selection of resistant mutants has been described for virtually any chemical type of antiviral agent directed to any step of the infectious cycle of dna or rna viruses, including important pathogens, such as herpesviruses, picornaviruses, iv, hbv, and hepatitis c virus (hcv). several reviews and articles have covered the theoretical basis of drug resistance, and consequences for treatment management [as examples see (domingo et al., 2001b) and previous versions in progress in drug research (richman, 1994 (richman, , 1996 ribeiro and bonhoeffer, 2000; domingo et al., 2001a domingo et al., , 2012 menendez-arias, 2013; perales, 2018; mokaya et al., 2018; nitta et al., 2019; pawlotsky, 2019) , and the articles in the current opinion of virology volume edited by l. menendez-arias and d. richman (menendez-arias and richman, 2014) ]. therefore, the general mechanisms that confer adaptability to viruses are very effective in finding drug-escape pathways through molecular mechanisms that are summarized in section 8.5. considering the implications of quasispecies dynamics explained in previous chapters, the 8 . quasispecies dynamics in disease prevention and control following statement will be obvious to the reader: "if a single mutation is able to confer resistance to an antiviral agent, and the mutation does not cause a significant selective disadvantage to the virus (fitness decrease) in the considered environment, a drug-resistant virus mutant will be present in most, if not all, virus populations" (domingo, 1989) . if a virus replicates in such a way that a population size of 10 4 can never be achieved in a single population, it is extremely unlikely that any drug-resistance mutation (or any mutation associated with a phenotypic change) that is generated at a frequency of 10 à4 or lower will be propagated from that viral population (perales et al., 2011) . selection of escape mutants depends on the replicative load, and the concentration of inhibitor attained at the sites of virus replication. consider different cell or tissue compartments in which an antiviral inhibitor reaches different concentrations (exerts different intensity of selection) ( fig. 8.1 ). in each compartment, there are multiple replication complexes. a mutation conferring resistance to the inhibitor will occur at the same rate in each of them, assuming that the mutation rate is independent of the presence of the inhibitor. however, after its occurrence, the proportion of viral rnas harboring the mutation will decrease depending on the inhibitor concentration. the time at which the effect of the inhibitor will be manifested depends on the inhibitor target. in the example of fig. 8 .1 we assume that the concentration of inhibitorresistant mutants will decrease in the replication complexes, reaching resistant mutant frequencies of 10 à3 , 10 à4 , and 10 à5 in compartments 1, 2, and 3, respectively. the frequency of inhibitor-resistant mutants in the entire cell, tissue, or organism at that time will be given by the weighted average of mutation frequencies at the individual compartments. in the case of a virus-producing viremia, assuming no bottleneck effects or differential selection for other figure 8 .1 frequency of a drug-resistant mutant in different compartments (subcellular site, cell, tissue, or organ) of the same organism. three compartments labeled 1, 2, and three are drawn. replication complexes are depicted as ellipses and replicating genomes as lines. a replicative unit is defined here as a set of replication complexes. the inhibitor-resistant mutant is represented by a red circle in a genome. compartments 1, 2, and 3 reach increasing concentration of the inhibitor, rendering inhibitor-resistant mutant frequencies of 10 à3 , 10 à4 , and 10 à5 , respectively. see text for the difference between occurrence and presence of the resistant mutant, and implications of compartmentalization. 8.4 resistance to antiviral inhibitors traits, the frequency of resistant mutants calculated for the virus in blood should reflect the average frequency in all compartments that supply virus to blood. low inhibitor concentration in a compartment will favor the selection of the resistant mutant that can either be archived as an adaptive reservoir or penetrate other compartments, depending on the sequence of events of virus spread. if two or more independent mutations can confer resistance to an inhibitor, the probability of occurrence of an inhibitor-resistant mutant is equal to the sum of probabilities of occurrence of each mutation. for multiple mutations, the probability will be the sum of probabilities of the different mutations, a frequent case in viruses since they often display several evolutionary pathways to drug resistance. the probability of finding a viral genome resistant to two or more inhibitors directed to different targets is given by the product of probabilities of resistance to each of the individual inhibitors. the basic probability considerations regarding the frequency of occurrence of inhibitor-resistant viral mutants are summarized in box 8.2. when two or more mutations occur in the same genome, they may be subjected to epistatic effects, meaning either increase (positive epistasis) or decrease (negative epistasis) of viral fitness (see section 2.3 of chapter 2 for the concept of epistasis). the diversity of chemical structures of the antiviral compounds that can select for escape mutants is illustrated in figs. 8.2 and 8.3 with the formulae of some antiviral agents in current or historical use. they include relatively simple organic molecules, nucleoside analogs, and complex heterocyclic compounds with a variety of residues (ch 3 -, c═ o, nh, nh 2 , f, and cl) that may contribute to interactions with viral proteins or alter the electronic structure of neighbor bonds thus modifying the interaction behavior of some atoms. for all of them, resistant viral mutants have been identified, despite barriers imposed upon the virus to reach a drug-resistance phenotype. • if there are two or more different mutations that produce the same inhibitor-resistance phenotype, and once one of the mutations is present additional mutations are no longer necessary to produce the phenotype, the probability of achieving the phenotypic change is equal to the sum of probabilities of finding each mutation individually. • if two or more independent mutations must happen to produce resistance to an inhibitor, the probability of occurrence of the necessary mutations is equal to the product of probabilities of occurrence of each mutation individually. • if a virus is inhibited by an inhibitor combination, and the mutations that confer resistance to each inhibitor are independent (no cross-resistance is involved), the probability of a combination-resistant mutant to arise is equal to the product of probabilities of resistance to the individual mutations. these probability calculations are applicable to other mutation-dependent virus variations. the impediments for a virus to attain resistance to an inhibitor are divided into genetic, phenotypic, and mutant swarm (population) barriers to resistance (box 8.3). the genetic barrier to resistance to a specific inhibitor is not a universal value for a virus group, since it may be affected by genetic differences among natural viral isolates. the diversification of hcv into genotypes 1a and 1b influenced the genetic barrier to resistance to the ns3/4a protease inhibitor telaprevir [formula (20) in fig. 8 .3]. one of the amino acid substitutions that confer resistance to telaprevir is r155k in ns3. in genotype 1a, the triplet encoding r155 is aga; therefore, a single nucleotide transition g / a can yield the triplet aaa, which encodes k. in genotype 2b, the triplet encoding r-155 is cga; therefore, two nucleotide changes (transversion c / a and transition g / a) are required to reach aaa, the triplet encoding k. reaching the alternative aag codon for k would require the same or a larger number of mutations (see section 4.3.1 in chapter 4 for another example of how the synonymous codon usage can influence an 7) rimantadine, (a-methyl-1-adamantane methylamine). (8) oseltamivir (trade name tamiflu), ethyl (3r, 4r, 5s)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-1-ene-1-carboxylate. (9) zanamivir (trade name relenza), (2r, 3r, 4s)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1r, 2r)-1,2,3-trihydroxypropyl)-3,4-dihydro-2h-pyran-6-carboxylic acid. 8.4 resistance to antiviral inhibitors figure 8.3 some inhibitors of human immunodeficiency virus type 1 (antiretroviral agents) and hepatitis c virus. the inhibitors are (10) zidovudine (azt), 1-[(2r, 4s, 5s)-4-azido-5(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione. (11) zalcitabine (ddc), 4-amino-1-((2r, 5s)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1h)-one. (12) 2r,3r ,4r,5r)-5-(2,4-dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4methyl-tetrahydrofuran-2-yl]methoxy-phenoxy-phosphoryl]amino]propanoate. additional drugs currently used in antiviral therapy can be found in the references quoted in the text and in chapter 9. 8 . quasispecies dynamics in disease prevention and control evolutionary outcome). the requirement of transitions versus transversions to reach the resistant phenotype will affect the genetic barrier to resistance. most viral polymerases tend to produce transition mutations more readily than transversions presumably because in the course of rna elongation it is easier to misincorporate a purine by another purine than by a pyrimidine, and the same for pyrimidine misincorporations (chapter 2). mutation preference is one of several factors that determine the frequency of drug-escape mutants. thus, evolution may diversify viruses to display different genetic barriers to the same drugs. since in many cases, several independent mutations may confer resistance to the same drug to complicate matters even more, it also has to be considered that the genetic barrier to one inhibitor may be affected by the presence of other inhibitors (beerenwinkel et al., 2005) . the phenotypic barrier to drug resistance is equivalent to the fitness cost inflicted upon the virus by the mutations and corresponding amino acid substitution(s) required for resistance [fitness cost is treated in chapter 4 (section 4.6) and in chapter 7 (section 7.4.2) in connection with the frequency of monoclonal antibody-or cytotoxic t-cell-escape mutants in viral populations]. when a drug-resistance mutation inflicts a high fitness cost, a likely result is a reversion of the mutation when the virus replicates in the absence of the drug. an alternative outcome is that compensatory mutations are introduced in the genome so that viral fitness increases while maintaining the inhibitor-resistance mutation. the two outcomes are not mutually exclusive and may contribute to the multiple, transient selection pathways observed by the application of deep sequencing to monitor the response of a viral population to specific selective force (tsibris et al., 2009; fischer et al., 2010; cale et al., 2011; kortenhoeven et al., 2015) (see section 6.3 in chapter 6). a high fitness cost may prevent or delay the selection of escape mutants. sofosbuvir [formula (21) in fig. 8 .3] is a very effective ns5b (viral polymerase) inhibitor of hcv. amino acid substitution s282t in ns5b has been associated with sofosbuvir resistance, and the substitution has been detected in patients and in some natural isolates of hcv. in one of several clinical studies on sofosbuvir efficacy, the mutant spectrum composition of hcv genotype 2b in an infected patient treated with the drug was followed by deep sequencing of the virus at baseline (prior to initiation of treatment), in the course of treatment, and posttreatment. the frequency of s282t was 0.05% at baseline, indicating preexistence of resistance mutations despite no exposure of the virus to the drug (section 8.6). two days after initiation of sofosbuvir treatment, the level of s282t decreased to 0.03%, and viral breakthrough was detected 4 weeks later when 99.8% of the viral population included s282t. during the posttreatment period, genomes with the wild-type s282 amino acid regained dominance that was attributed to the true reversion of mutant genomes rather than the outgrowth of baseline wild-type genomes (hedskog et al., 2015) . this result suggests a high phenotypic barrier for sofosbuvir, and that hcv has mechanisms to overcome the barrier. the complexities of virus-host interactions render the elucidation of the pathways exploited by a virus to overcome the phenotypic barrier to a drug a highly empirical endeavor. the hope is that a combination of inhibitors that display a high barrier to resistance may impede escape and drive the virus to extinction (chapter 9). the mutant swarm barrier to resistance is a consequence of the interfering interactions that operate within quasispecies, and that are described in chapter 3 (section 3.8). it is a particular case of interference that can delay or impede the increase of frequency of a resistance mutation (crowder and kirkegaard, 2005; kirkegaard et al., 2016) . the possible contribution of mutant swarms to facilitate or prevent the dominance of drug-resistant mutants in infected patients is still largely unexplored. it is difficult to anticipate how the three types of barrier listed in box 8.3 may result in a level of drug resistance for a particular virus, in a particular host individual, in a specific target organ, at a given time. additional influences are drug pharmacokinetics, drug penetration into different cells, tissues, and organs where the virus replicates (see fig. 8 .1), and prior history of virus replication in the infected host. it is not surprising that the study of drug resistance in viruses remains fundamentally descriptive. when independent amino acid substitutions can lead to resistance to the same drug, alternative evolutionary pathways may be followed depending on trna abundances, mutational preferences, and relative nucleotide substrate concentrations at the virus replication sites. if resistance requires two or more amino acid substitutions, the genetic barrier will be correspondingly increased (section 8.4.1 and box 8.2). quantification of barriers to resistance in experiments in cell culture requires a prior characterization of the drug to be tested when acting on the cell culture-adapted virus as it infects a specific cell line. the two basic parameters to be determined are the toxicity of the drug for the host cell, and its capacity to inhibit the production of infectious virus. toxicity is quantified by the concentration of drug that kills a given percentage (generally 50%, but sometimes another value) of cells under the conditions used in the infection. it is expressed as the cytotoxic concentration 50 (cc 50 ), as depicted in fig. 8 .4. toxicity may depend on the cell concentration, the extent of confluence in a cell monolayer, and the metabolic state of the cell (resting vs. actively dividing). the capacity of inhibition is quantified by the concentration of inhibitor that reduces the infectious progeny production by a given percentage (generally 50%, but sometimes another value) under the defined conditions of the infection, including a multiplicity of infection (moi). it is expressed as the inhibitory concentration 50 (ic 50 ), as depicted in fig. 8 .4. the therapeutic index (ti) is given by the quotient cc 50 /ic 50 , and although generally used for in vivo experiments of drug efficacy testing, it can also be applied to cell culture measurements. the three parameters, cc 50 , ic 50, and ti, are not universal for a virus and a drug since they may be influenced by the composition of the viral population and environmental factors, as repeatedly expressed for other features of viruses in the present book. as a guide, ti values of 100 or more suggest the excellent performance of an antiviral agent; values higher than ten are acceptable, but values lower than ten predict limited efficacy. the quantitative effects of a drug may vary when analyzing a single round of infection versus multiple rounds in serial passages, or when comparing in vivo versus cell culture experiments. cc 50 and ic 50 values serve as a guide to decide the range of the drug concentration to be used in serial passage experiments to evaluate the possible selection of inhibitor-resistant mutants and to estimate the genetic barrier. the possibility to overcome a genetic barrier depends on the virus population size. for viruses that replicate in cell culture, it is possible to estimate the minimal viral population size needed to select a drug-resistant mutant which is generally positively correlated with the genetic barrier ( fig. 8.5 ). in the hypothetical example of the figure, a viral population is composed of inhibitor-sensitive viruses (blue spheres), and a low level of inhibitor-resistant viruses (red spheres). the proportion of inhibitorresistant viruses is given by the mutational pressure (e.g., at a frequency of 10 à4 , which is increased in the picture for clarity). passage of a small amount of virus (e.g., 10 2 infectious virus in the small circle at the upper part of the figure) will exclude the mutant virus (red spheres) that will be maintained at the basal level dictated by mutational pressure in the course of passages (limited to two in the figure for simplicity). selection of escape mutants is precluded by the limited population size at each transfer. in contrast, if the population size used for the successive infections is sufficiently large (>10 4 , larger circles at the bottom that surround both sensitive and resistant viruses), the resistant mutant can become figure 8.4 schematic representation of two experiments to determine the concentration of an inhibitor needed to kill 50% of cells in culture (cc 50 value, left) and the concentration of inhibitor that reduces the viral production to 50% (ic 50 value, right). the therapeutic index is the quotient between cc 50 and ic 50 (box at the bottom). similar tests can be performed with tissue explants or animals, under controlled environmental conditions. see text for pharmacological implications. 8.4 resistance to antiviral inhibitors gradually dominant and can be isolated for further studies. to give another example, a single amino acid replacement that requires two mutations (the change from cag to aug to attain substitution q151m in hiv-1 reverse transcriptase, associated with resistance to multiple antiretroviral nucleosides) will occur at a lower frequency than replacements that require a single mutation. if each of the two mutations reaches a frequency of 2 â 10 à4 , the expected frequency of the drug-resistant genomes (ignoring fitness effects) will be (2 â 10 à4 ) â (2 â 10 à4 ) ¼ 4 â 10 à8 . thus, at least 4 â 10 8 viral genomes must undergo one round of copying (or a lower number of genomes a proportionally higher number of rounds of copying) to approach a good probability to obtain a drug-resistant genome in that viral population. population size limitation of a drug selection event is a specific example of how random events may intervene in the process of positive selection (compare with section 3.2 and fig. 3 .2 in chapter 3; in that figure, the random event that excludes the positively selected population is conceptually equivalent to the insufficient population size depicted in the upper infection series of fig. 8 .5). when two or more mutations are needed to confer the resistance phenotype, drug resistance will be less likely not only due to the lower probability of generating the two required mutations but also because of the increased chances of two mutations in the same genome entailing a fitness cost. a virus that requires three or more mutations to overcome a selective constraint may occur at a frequency in the range of 10 à 12 or lower which will often be insufficient for the mutant to be present in the mutant spectrum of the infected host ( fig. 8.6 ). failure to select for a drug-resistant mutant in cell culture does not necessarily mean that the resistant mutant is not present in the population. it may mean that due to a high genetic barrier, the selection experiment was designed to infect with an insufficient amount of virus. similar and even more accentuated problems are encountered in selection experiments in vivo, since not only the phenotypic barrier or fitness cost inflicted by a drug-resistance mutation (box 8.3) is often estimated empirically from the frequency of the relevant substitution in patients treated with the drug or in cell culture assays. an adequate procedure to quantify the phenotypic barrier to resistance is to determine the fitness of the virus expressing the protein with the wild-type amino acid (the one that confers drug sensitivity) relative to the virus expressing the protein with the substituted amino acid that confers resistance; fitness is measured in the absence and presence of the drug (double assay). this is an extension of the determination of fitness vectors described in section 5.1.1 of chapter 5, as depicted in fig. 8.7 ; the assays are best performed in cell culture, although the use of explants or in vivo assays is also feasible. two parameters can be calculated: the fitness cost inflicted by the amino acid substitution associated with resistance, in the absence of the drug (reflected in a fitness value f à drug 1 relative to the wild type), and the selective advantage conferred by the substitution in the presence of the drug (reflected in a fitness value f þ drug > 1 relative to the wild type). the lower the value of f à drug , the higher the fitness cost. we define the selective strength of the resistance mutation as f þ drug /f à drug . for example, if we put arbitrary numbers (unrelated to values shown in ordinate) to the fitness values in the first graph of fig. 8 .7, figure 8.6 decreased frequency and fitness of mutant genomes resistant to one, two, or three inhibitors. the genome frequency level decreases by several orders of magnitude when resistance to one inhibitor (red asterisk in the upper mutant spectrum), or two inhibitors (red and green asterisks in the middle-mutant spectrum), or three inhibitors (red, green, and blue asterisk in the bottom mutant spectrum) must occur in the same genome (left part of the figure and numerical values at the center). increased number of mutations generally implies fitness decrease (right part of the figure) . see text for implications. 8.4 resistance to antiviral inhibitors f þ drug ¼ 1.4 and f à drug ¼ 0.8, we obtain a selective strength of 1.7. for the vectors in the second graph, if f þ drug ¼ 3.6 and f à drug ¼ 0.1, the selective strength is 36. high selective strength means an important selective advantage conferred by the amino acid substitution for virus replication in the presence of the drug despite a high fitness cost inflicted by the substitution (compare with section 4.2 in chapter 4 for the trade-off and "no free lunch" concepts). if the substitution does not entail any fitness cost (f à drug ¼ 1), the fitness value in the presence of the drug equals the selective strength. selective strength can be calculated for a mutation or group of mutations that confer resistance to a drug used at a given concentration in a defined environment [example in (de la higuera et al., 2017) ]. the limitations of fitness measurements (environment dependence, etc.) described in section 5.1.2 of chapter 5 apply here. since viral genomic sequences may vary in the course of fitness assays, a limited number of passages and triplicate parallel assays are recommended. if a substitution entails a high fitness cost, direct reversion of the substitution or incorporation of compensatory mutations may occur. nucleotide sequence monitoring in the course of the assay should reinforce the conclusions. most drug-resistance mutations inflict a fitness cost upon the virus and yet very rarely drug resistance represents an unsurmountable barrier to maintain viral infectivity. several possibilities can account for the pertinacious occurrence and selection of drug-resistant, viable viral mutants. one possibility, supported by some experimental and clinical observations, is that a drug-resistant phenotype may be achieved through a number of alternative genetic modifications. even if a specific amino acid substitutiondthat would serve as the most direct and effective determinant of drug resistancedwere highly detrimental or lethal for the virus, alternative mutations can often be found that lead to a similar resistance phenotype, or at least a sufficient resistance to permit virus replication and exploration of sequence space to find compensatory mutations. the connectivity among points of sequence space and the fact that several space positions map into the same (or similar) drug-resistance phenotype contribute to the extended occurrence of drug resistance. this phenotypic redundancy applies to both standard nonmutagenic inhibitors and to mutagenic inhibitors. the cascade of mutations that confer resistance of picornaviruses to the mutagenic purine analog ribavirin illustrates how alternative amino acid substitutions in the viral polymerase (some being genuine resistance mutations and others acting as compensatory substitutions to maintain polymerase function) can lead to the ribavirin-resistance phenotype (discussed in chapter 9). a speculative interpretation of the systematic occurrence of drug-resistant viral mutants is that the majority of the chemicals used in antiviral therapy (figs. 8.2 and 8.3) have a structure which may be related to natural compounds that viruses and their ancestral replicative machineries encountered in their continuous struggle to survive. in this view, drug resistance would have been gradually built as a consequence of coevolution (section 4.5 of chapter 4) between virus replicative and gene expression machineries and the "space" of chemical compounds that interacted with them. mechanisms of drug resistance might have had their roots in molecular events repeatedly experienced as viruses evolved in an interactive manner with protocellular and cellular metabolites in our biosphere. discrimination in favor of small molecule substrates compatible with a flow of genome replication and gene expression and avoidance of perturbing intruders that could alter catalytic activities should have been positively selected. unfortunately, this is a possibility we will never be able to test. whatever the reasons behind, the unfortunate reality is that drug resistance is an extremely frequent event that complicates enormously the control of the viral disease. multiple drug evasion mechanisms have been identified or proposed for rna and dna viruses, and they can operate depending on basic replicative parameters in connection with drug levels and their variation with time. delayed lysis of bacteriophage vx174 that reduced the replication rounds in the presence of 5-fluorouracil (fu) produced resistance to this analog (pereira-g omez and sanju an, 2014). in this line, synchronization of a virus life cycle so that replication occurs when drug levels are minimal has been proposed as a potential resistance mechanism (neagu et al., 2018) . virus plasticity favors modification of life cycle parameters for virus survival in the presence of drugs, provided time for the relevant selection events is allowed once the treatment has been implemented. the great majority of inhibitor-resistance mechanisms involve amino acid substitutions in viral proteins that directly or indirectly diminish the binding of the drug to the viral target. the following major mechanisms have been documented: • substitutions in the protein targeted by the drug that decreases the affinity of the protein for the drug. mutations that modify nucleotide selectivity in viral polymerases belong to this group. substitutions may affect the neighborhood of the polymerase catalytic site, other polymerase domains, or even nonstructural proteins that interact with the polymerase. • if the inhibitor acts on a viral protein that itself has some other viral protein or genomic structure as a target, amino acid substitutions, or mutations that affect that target may also contribute to resistance. correlated mutations 8.5 molecular mechanisms of antiviral resistance in the first and second target may also yield the resistance phenotype. this is the case of some protease inhibitor-resistance mutations in hiv-1. • in the case of viral polymerases, some mutations permit the excision of a chainterminating nucleotide at the 3 0 -end of the primer. it is achieved through phosphorolysis mediated by a pyrophosphate donor, probably atp. resistance to nucleoside/nucleotide reverse transcriptase (rt) inhibitors (nrtis) is achieved by one of at least two mechanisms: (i) discrimination against the incorporation of the triphosphate form of the nrti and (ii) excision of the chain-terminating nucleotide once incorporated at the 3 0 -end of the growing dna chain. this occurs with thymidine analog resistance mutation (or tams) that are typically selected under treatment with azt or d4t [formulae (10) and (13) in fig. 8.3 ]. groups of amino acid substitutions may yield a multidrugresistance phenotype. a well-studied example in hiv-1 is the q151m complex in the reverse transcriptase, which includes substitutions a62v, v75i, f77l, f116y, and q151m. the phenotype consists of the limitation of incorporation of several nucleotide analogs. these and other mutants are characterized by a decrease in the catalytic rate constant (k pol ) of incorporation of the analog or analogs relative to standard nucleotides (see section 2.6 in chapter 2 for the basic kinetic parameters for polymerase activity). the combination of enzymological and structural studies has provided a molecular interpretation of the mechanism of inhibition of hiv-1 by nrtis (reviews in men endez-arias, 2013; men endez-arias and alvarez, 2014; clutter et al., 2016; g€ unthard et al., 2019) ; for a predictive model that includes nucleotide levels, see von kleist et al. (2012) . nonnucleoside rt inhibitors (nnrtis) bind to an rt pocket 10å away (a considerable distance) from the catalytic site composed of residues of the p66 and p51 subunits of the enzyme. the hydrophobic nature of nnrtis is illustrated in fig. 8.3 with the structures of nevirapine, efavirenz, and delavirdine [formulae (15), (16), and (17), respectively]. several mechanisms have been proposed to explain their inhibitory activity, including alteration of the catalytic amino acids ymdd at the rt active site, distortion of the nucleotidebinding site, or modification of the position of the primer that receives the incoming nucleotides (men endez-arias, 2013). amino acid substitutions that confer resistance to nnrtis block the access of the inhibitors to their binding sites or alter the conformation and volume of the binding pocket. the essential viral proteases are a target for the development of specific antiviral inhibitors. examples are saquinavir and ritonavir for the hiv-1 [formulae (18) and (19), respectively, in fig. 8.3 ]. multiple resistance mutations have been described for protease inhibitors. they can affect the substrate-binding site or neighbor positions, often accompanied of compensatory substitutions at distant positions including sites of the target viral protein [e.g., the gag cleavage sites in hiv-1 (fun et al., 2012; flynn et al., 2015) ]. some hiv-1 protease inhibitor combinations display a high genetic barrier to resistance but, significantly for the capacity of viruses to explore sequence space, resistant mutants with 20e30 amino acid substitutions in the proteasecoding region have been isolated (rhee et al., 2010) . the reviews by men endez-arias (2013) and men endez-arias and alvarez (2014) provide excellent and detailed accounts of mechanisms of resistance of hiv-1 and hiv-2 to inhibitors that include virus entry and integrase inhibitors, in addition to the rt and protease inhibitor briefly described here. 8 . quasispecies dynamics in disease prevention and control because of the distance effects that can be exerted among amino acids on the structure of proteins, substitutions that confer resistance to inhibitors of viral enzymes may lie far from the catalytic site. the effect of drug-resistance mutations on the general catalytic efficiency of a viral enzyme is one of the determinants of the functional barrier to resistance, since it may diminish replicative fitness. when an enzyme activity assay in vitro is available, the effects of specific drug-resistance amino acid substitutions on enzyme activity can be tested, although the observed alteration may not be the only influence on the fitness modification of the corresponding mutant virus. the reason is that most viral proteins (including viral enzymes) are multifunctional, and an enzyme activity assay may not capture the range of influences exerted by the enzyme. regarding direct-acting antiviral (daa) agents for hcv, the inhibitors that target the hcv polymerase (ns5b) generally display a higher functional barrier to resistance than the protease inhibitors and manifest a broader genotype coverage. fitness decreases entail reductions in viral load and, consequently, lower probability of viral breakthrough (treatment failure). nucleotide analogs that bind to conserved residues at or near the active site of the viral polymerase tend to show subtypeindependent antiviral activity. because amino acid substitutions at or near the active site of viral enzymes are likely to inflict a fitness cost, such substitutions may not preexist in treatment-naive patients (margeridon-thermet and shafer, 2010; sarrazin and zeuzem, 2010) . interestingly, in the case of hiv-1, mutations conferring resistance to rt inhibitors inflict a lower fitness cost than mutations that confer resistance to protease inhibitors (reviewed in martinez-picado and martinez, 2008) . thus, enzymes that perform similar functions for different viruses may have evolved to display different tolerance to amino acid substitutions. it is not possible to generalize which types of resistance mutations will display high or low functional barriers. there is a broad range of frequencies of antibody-and drug-escape mutants in viral populations, although values of 10 à4 to 10 à6 mutants per infectious unit are frequent for many rna and dna viruses (see table 7 .2 in chapter 7 for antibody-escape mutants). a few estimates for drug-escape mutants are listed in table 8 .1; several observations and characterization of escape mutants have not been accompanied by frequency measurements. a point worth emphasizing is that quantification of viruses harboring biologically relevant mutations has been possible because an adequate biological assay is available. an antiviral agent or a neutralizing antibody measures the proportion of infectious viral particles that differ from the majority of the population in the relevant resistance trait. there is no reason to suspect that the viral amino acid residues (that are the target of an inhibitor or an antibody) that are substituted to confer the resistance phenotype are more prone to accept variations than many other amino acids in viral proteins. if we had additional selective agents to probe other viral sites, we expect a similar range of variant amino acids than using inhibitors or antibodies. this quite straightforward prediction is another way to state that there is general agreement in the mutation rates and frequencies for viruses being in the range of 10 à3 to 10 à5 substitutions per nucleotide (s/nt), calculated using a variety of biochemical and genetic methods (chapter 2). a quite general observation is that in antibody neutralization experiments, a fraction of the virus population remains infectious despite the 8.5 molecular mechanisms of antiviral resistance addition of high antibody concentrations. although the resistance mechanism is unclear, a possibility is that the heterogeneous viral population includes a small proportion of antigenic variants with decreased affinity for antibodies. incomplete neutralization with the nonsigmoidal slope in the neutralization curves has been characterized for broadly neutralizing antibodies directed to hiv-1 (mccoy et al., 2015) . this general observation is an added complication to preventive designs that consider administration of neutralizing antibodies either as vaccine additives or in combination with antiviral inhibitors (section 9.2 in chapter 9). the calculated frequencies of antibody-or drug-resistant mutants in viral populations may be lower than the rate at which they originate by mutation due to the fitness cost of the mutation (section 8.4.4) . the argument is parallel to that used to justify why mutation rates and frequencies differ due to the fitness effects of mutations (chapter 2). another prediction derived from the above considerations is that mutations conferring resistance to antiviral agents are expected to be detected in viral populations never exposed to the relevant drugs. all forms of genetic variation of viruses can contribute to dominance and spread of drugresistant mutants, including the combined effect of mutation, recombination, and genome segment reassortment (richman, 1996; neher and leitner, 2010; rogers et al., 2015) . the basal level of mutational pressure may be sufficient to provide a detectable proportion of escape mutants without the need for selection by the selective agent. this is an important aspect of antiviral therapy that is addressed next. the first demonstration that the baseline mutation level in viral quasispecies can include a detectable level of mutations that confer resistance to inhibitors in the absence of selection by the inhibitors, was obtained by d.d. ho, i. n ajera, c. l opez-galíndez, and their colleagues working with hiv-1 (mohri et al., 1993; n ajera et al., 1994 , 1995 . one of the studies examined the pol gene of 60 hiv-1 genomes obtained directly from lymphocytes of infected patients. mutation frequencies for independent viral isolates were in eggers and tamm (1965) amantadine and rimantadine iv 1 â 10 à3 to 4 â 10 à4 measurements in cell culture appleyard (1977) , lubeck et al. (1978) rimantadine a iv 27% percentage of children treated with rimantadine that shed resistant iv belshe et al. (1988) disoxaril a hrv14 1 â 10 à3 to 4 â 10 à4 low-level resistance in cell culture heinz et al. (1989) 4 â 10 à5 high-level resistance in cell culture guanidine d pv 1.8 â 10 à5 to 4 â 10 à8 measurements in cell culture pincus and wimmer (1986) a the formula of these drugs is included in figure 8 .2 with the following number in parenthesis: amantadine (6); rimantadine (7); disoxaril (2) the range of 1.6.10 à2 to 3.4.10 à2 s/nt, while for mutant spectrum components of individual isolates the values were 3.6.10 à3 to 1.1.10 à2 s/nt. in the virus from these patients, mutation frequencies at the codons for amino acids involved in antiretroviral resistance were very similar to the average mutation frequency for the entire pol gene. consistently with the mutation frequency values, several mutations that led to amino acid substitutions that conferred resistance to reverse transcriptase inhibitors were identified in patients not subjected to therapy. at the time of the study, the number of antiretroviral agents was still limited, and a considerable number of patients were not treated. the authors gave convincing epidemiological arguments that the background of mutations related to antiretroviral resistance was a consequence of high mutation rates and quasispecies dynamics, and not due to the transmission of resistant virus from individuals that had been subjected to therapy (primary resistance) (n ajera et al., 1995) . the presence of inhibitor-resistance mutations in viral populations never exposed to the corresponding inhibitor has been confirmed for hiv-1 and for several other viruses, including hcv, and it is supported by the calculated mutant frequencies in viral quasispecies (havlir et al., 1996; lech et al., 1996; ribeiro et al., 1998; ribeiro and bonhoeffer, 2000; cubero et al., 2008; johnson et al., 2008; toni et al., 2009; tsibris et al., 2009; peres-da-silva et al., 2017) . ample support has also come from deep sequencing analyses of mutant spectra, opening a point of debate on the basal frequency of inhibitor-resistance mutations that constitutes an indication to avoid the use of the corresponding inhibitors in therapy. at least in the case of hcv (and probably applicable to other viruses), there is no basis to suggest that the presence of a drug resistance mutation below a certain level in a patient will not have relevance for treatment failure when the inhibitor is administered. the understanding of quasispecies dynamics cautions against such reductionist arguments as evidenced by clinical cases (perales et al., 2018) . the data underline the relevance of mutant spectra as phenotypic reservoirs to confront selective constraints before constraints are in operation. for treatments, including a drug that has already been administered to a patient in the past, the influence of quasispecies memory should also be considered (section 5.5.1 in chapter 5). mutant spectra can be viewed as an anticipatory reservoir of phenotypes. in addition to the presence of drug resistance mutations in viral populations due to mutant frequency levels, the transmission of drug-resistant mutants from treated to na € ive patients may contribute to epidemiological relevance of resistance mutations. such primary resistance has been amply documented with hiv-1, and it appears to increase in the case of hcv (franco et al., 2014; echeverría et al., 2016; huang et al., 2019) . higher levels of resistance mutations as a function of time in untreated patients is an indication that the mutations are not due to basal mutant frequencies but to the epidemiological expansion of virus mutants that originated in treated patients. from the clinical data available, the rate of expansion of virus harboring resistance mutations may vary depending on transmission and epidemiological features of each pathogen, but it seems unavoidable in the face of extended treatments for genetically variable pathogenic viruses. we confront a situation with parallels with antibiotic resistance in bacteria (chapter 10). the major mechanism of drug resistance in viruses is based on amino acid substitutions that render the drug ineffective through the several molecular mechanisms summarized in section 8.5. application of the cell culture system of hcv replication in human hepatoma cells (lindenbach et al., 2005; wakita et al., 2005; zhong et al., 2005) to examine the effects of 8.7 fitness or a fitness-associated trait as a multidrug-resistance mechanism long-term evolution has indicated that viral fitness can be an additional mechanism of drug resistance. the evidence was obtained when addressing the important issue of hcv resistance to interferon-alpha (ifn-a). ifn-a and ribavirin were the two components of the standard of care treatment against hcv infections until the advent of new therapies based on daa agents in 2014. natural hcv isolates differ in ifn-a sensitivity, and the molecular basis of the difference is largely unknown. the study in cell culture consisted in subjecting a clonal population of hcv (termed hcvp0, prepared by electroporation of hepatoma cells with rna encoding the viral genome, transcribed from a plasmid) to 100 serial passages (of the type described in section 6.1 of chapter 6) in the absence or presence of increasing concentrations of ifn-a added to the culture medium. several mutations scattered throughout the hcv genome were associated with ifn-a resistance (perales et al., 2013) . the selection of multiple alternative mutations is most likely influenced by the fact that ifn-a evokes a multicomponent antiviral response, which is not focused toward a single viral protein (perales et al., 2014) . unexpectedly, even the control hcv populations (those passaged 100 times in the absence of ifn-a) displayed a partial (but statistically significant) resistance to ifn-a that could not be attributed to endogenous ifn production by the hepatoma cells (perales et al., 2013) . in view of this intriguing result, the initial hcvp0 population and the hcv population passaged 45 and 100 times in the absence of ifn-a (termed hcvp45 and hcvp100, respectively) were tested for their resistance to other inhibitors of hcv replication: the protease inhibitor telaprevir [formula (20) in fig. 8 .3], the ns5a inhibitor daclatasvir, the cellular protein cyclophilin a inhibitor cyclosporin a, the mutagenic purine nucleoside ribavirin, and the high barrier inhibitor sofosbuvir [formula (21) in fig. 8.3 ]. hcvp45 and hcvp100 displayed significantly increased resistance to all inhibitors tested, as compared with the parental population hcvp0 gallego et al., 2016) (fig. 8.8) . passage of hcv entailed a 2-to 3-fold increase of viral fitness and a broadening of the mutant spectrum that might have increased the frequency of mutations associated with drug resistance, thus explaining the behavior of the multiply passaged hcv populations. the search for the resistant mutations was easier for telaprevir, daclatasvir, and cyclosporin a than for the other drugs because amino acid substitutions in the target protein had been previously identified as responsible for drug resistance. (in the case of cyclosporin a resistance, substitutions map in ns5a and ns5b, because the drug binds to cyclophilin a, which in turn interacts with ns5a). analysis of the mutant spectra of hcvp45 and hcvp100 by molecular cloning and sanger sequencing and by deep sequencing failed to identify specific drugresistance mutations. since it could not be excluded that the broadening of the mutant spectrum might have increased the frequency of resistance mutations still to be characterized, two additional tests were performed. one was to determine the kinetics of viral production over a 1000-fold range of moi in the absence and presence of telaprevir. both the unpassaged and multiply passaged hcv displayed parallel kinetics at the different mois, which excludes that drug resistance was due to the presence of resistance mutations in minority components of the mutant spectrum ( fig. 8.8 ). to further substantiate the findings, biological clones obtained by end-point dilution of the corresponding hcvp0 and hcvp100 populations were tested regarding drug resistance. a biological clone should have eliminated the minority genomes that harbored drug-resistance mutations since biological cloning is the most severe form of bottleneck event (sections 6.2 and 6.5 in chapter 6). the biological clones did not display any decrease in drug resistance as compared 8. quasispecies dynamics in disease prevention and control moreno, e., gallego, i., pineiro, d., et al., 2014 . increased replicative fitness can lead to decreased drug sensitivity of hepatitis c virus. j. virol. 88, 12098e12111, with permission from the american society for microbiology, washington dc, usa. 8.7 fitness or a fitness-associated trait as a multidrug-resistance mechanism with their corresponding parental, uncloned populations . the above observations have established viral fitness as a multidrug-resistance determinant in hcv that may also apply to other viruses. one possible molecular mechanism may consist of competition between replicative complexes and inhibitory molecules inside the infected cells. this model implies that fitness increase is reflected either in more replicating molecules per each replicative unit or in an increase in the number of replicative units per cell, without any influence on the number of inhibitor molecules that reach the replication sites. exploration of this competition model and alternative models and the extension to other viral-host systems are important challenges in the field of antiviral research. to sum up, mutant spectra and quasispecies dynamics can mediate antiviral resistance by at least two mechanisms: (i) by increases in the proportion of resistance mutations in the mutant spectra and (ii) by a fitness increase promoted by continued viral replication in the same environment. both mechanisms may act conjointly during viral infections in vivo. some studies with hcv have documented drugresistance phenotypes in infected patients, in the absence of specific drug-resistance mutations (sullivan et al., 2013; svarovskaia et al., 2014; sato et al., 2015; stross et al., 2016; di maio et al., 2017; dietz et al., 2018) . in fact, prolonged chronic hcv infections represent an adequate scenario for fitness increase due to extended rounds of infections in the same host liver. as a consequence, chronic infections may be prone to display fitness-associated multidrug-resistance phenotypes in the absence of drug-resistance mutations. the multiple mechanisms of drug resistance related to quasispecies dynamics justify even further the need for new antiviral strategies, as presented in chapter 9. high viral loads are predictors of disease progression. for hiv-1 and other lentiviruses, efficient early control of virus replication by the host immune response is generally associated with limited disease severity. the viral load that follows after the initial immune response to hiv-1 is referred to as the "viral set point." in the absence of early therapy, low set points in hiv-1 are generally attributed to a strong cellular immune response, likely influenced by additional host and viral factors. [this and other aspects of hiv-1 replication and pathogenesis have been reviewed in excellent monographs by levy (2007 levy ( , 2015 ]. a low set point predicts an asymptomatic outcome, and this is generally the case for viruses that establish persistent rather than acute infections. high viral fitness during the early stages of viral replication can promote disease manifestations. this was suggested by the progression toward the disease of a cohort of individuals that were infected during blood transfusion with an hiv-1 containing a large deletion in nef, an adaptor protein that mediates replication and pathogenesis (reviewed in arien and verhasselt, 2008) . after more than 15 years, some of the infected individuals showed clinical signs, probably as a result of the accumulation of mutations in the hiv-1 genome that compensated for the lack of nef. more generally, fitness-decreasing (but not lethal) genetic lesions in a viral genome may be compensated by additional genomic mutations that become increasingly dominant in the course of further viral replication. the kinetics of fitness gain will depend on the nature of the lesion and the functional implications of the altered protein or genomic regulatory region (chapter 5). fitness, replicative capacity, and viral load are directly interconnected parameters, and they 8. quasispecies dynamics in disease prevention and control affect disease progression (domingo et al., 2012) ( fig. 8.9 ). fitness gain will be more effective with a high load of actively replicating virus in the infected organism. elevated replicative capacity and fitness sustain high viral loads. the reason for this basic feature of viral population dynamics is that given a basal mutation rate, a large number of replicating genomes entails a correspondingly higher probability that a required mutation for fitness gain can be produced. the events involved are a specific case of search for adaptive mutations in terms of exploration of sequence space, as discussed in section 3.7 of chapter 3. while active viral replication, high load, and high fitness favor progression of the infection and disease manifestations, the fourth parameter included in the large arrow of fig. 8 .9, mutant spectrum diversity, has an optimal range. too low or too high intrapopulation diversity is detrimental to virus adaptability. insufficient diversity limits adaptability to complex environments (pfeiffer and kirkegaard, 2005; vignuzzi et al., 2006) , while excess diversity may lead the virus to cross an extinction threshold, and this is the basis of lethal mutagenesis as an antiviral therapy (chapter 9). an additional implication of the parameters shown in fig. 8 .9 for antiviral interventions is that fitness decrease is recognized as an alternative to inhibition of viral replication to control viral infections[ (clementi, 2008; clementi and lazzarin, 2010) ; reviewed in (domingo et al. 2012) ]. thus, key features of quasispecies dynamics have a direct implication on the management of viral infections. external interventions that have been applied or have been envisaged to limit or suppress virus infection include not only vaccination and administration of antiviral agents as described in previous sections, but also passive immunotherapy, antisense rnas, or oligonucleotides with various chemical modifications, interfering rnas, ribozymes, or their combinations. biotechnological developments have favored the design of chemically defined vaccines (consisting of expressed immunogenic proteins, synthetic peptides, or peptide arrays), without the need to handle or administer live virus. one of the most na € ive manifestations of trust in biotechnology in the middle of the 20th century was the belief that a catalog of plasmids encoding the antigenic proteins of the circulating types of pathogenic viruses would suffice to prepare the required vaccine as needed. concerning influenza vaccines, w.i. beveridge wrote the following: "the first objective would be to capture the full range of influenza a subtypes. their antigens would be studied by specialists at central laboratories and made available for the preparation of particular vaccines if and when required. it might be feasible to stockpile some vaccine against all the principal hemagglutinin antigens to be used in a figure 8.9 a schematic representation of interconnected parameters of viral replication that often relate to disease progression. an understanding of quasispecies dynamics has made it evident that the aim of antiviral therapy need be not only to directly diminish the viral load but also to affect other parameters that can then reduce the viral load. see text for justification and references and chapter 9 for new antiviral strategies that follow the concept expressed in this figure. 8.9 limitations of simplified reagents and small molecules as antiviral agents fire brigade type of action as soon as an incipient pandemic is spotted" (beveridge, 1977) . naivety is also perceived in current designs of universal vaccines based on conserved antigens, considering the different escape mechanisms that operate in viruses to elude neutralization by antibodies (chai et al., 2016) . it is remarkable how our present understanding of viral populations renders obsolete the views expressed in the w.i. beveridge book, and in other writings at the boom of implementation of dna recombinant techniques. yet, attempts to produce vaccines against highly variable viruses, based on antigenic structures or some viral isolates, are ongoing (christiansen et al., 2018) . a critical issue is if the host immune response against a vaccine engineered with "universal" (conserved) antigenic motifs will be sufficient to prevent disease upon infection by other forms of the same pathogen (freeman and cox, 2016) . from our current knowledge of viral population dynamics, it seems unlikely but time will tell, since efforts toward the manufacturing of universal antiviral vaccines are under way. with a conceptual similarity to vaccines, in medical practice, monotherapy with an antiviral agent was traditionally preferred over drug mixtures. (the change of paradigm was largely a consequence of the aids epidemic, and it was publicly expressed by the pioneer hepatologist s. sherlock in a summary address of an international symposium on viral hepatitis held in madrid in 1998). the change of perspective is clear. some antiviral strategies, such as antisense nucleic acids or virus-directed ribozymes, were intensely investigated decades ago. it is unlikely that when used in isolation, they can be converted into useful antiviral therapies because resistant mutants are likely to be selected. yet, they could be part of combinations with other antiviral inhibitors to provide a larger antiviral barrier (chapter 9). a similar fate is likely for interfering rnas (boden et al., 2003; gitlin et al., 2005; herrera-carrillo and berkhout, 2015; mcdonagh et al., 2015) . to a large extent, the failures of defined chemical entities (oligonucleotides, ribozymes, small molecule inhibitors, etc.) to control virus replication and spread are a consequence of their targeting a very defined viral genomic sequence combined with the adaptability of viral populations. combination of such multiple elements have been envisaged and tested, but off-target effects and the adaptive potential of viruses are likely to limit their efficacy. unfortunately, biotechnological developments that have been so positive for many research areas and practical applications tend to simplify the types of agents to prevent disease or inhibit virus replication, ignoring the inherent complexity of the object to be controlled. success is unlikely when "complexity" is combated with "simplicity." an increased understanding of viral population dynamics over the last decades has changed the picture dramatically by providing an interpretation of "virus escape" as a general and largely unavoidable phenomenon. such awareness has pushed the development of new antiviral designs, which are fundamentally centered in two strategies: a combination of multiple, independently acting elements or fitness decrease through excess mutations (chapter 9). a pronouncement by p. ehrlich at the international congress of medicine held in london in 1913, reflected old traditions on how to treat microbial infections ["here, therefore, the old therapeutic motto is applicable: frapper fort et frapper vitte" and "therefore, it is in my opinion necessary to allow the therapeutic treatment to come into action as early as possible, ..."; sentences taken from (ehrlich, 1913) , with emphases as in the original text]. this pronouncement fits our current understanding of viral populations. following ehrlich, the full implications of quasispecies-mediated adaptation of viruses for 8. quasispecies dynamics in disease prevention and control antiviral therapy were expressed by d.d. ho in an influential article entitled "time to hit hiv, early and hard" (ho, 1995) . the article title captures what is needed to prevent adaptation of a virus in the infected host. any opportunity to replicate is exploited by the virus to increase its fitness and to become less vulnerable to internal (intrahost) or external interventions such as antiviral therapy. treatment interruptions during chronic infections, such as "drug holidays" that in the case of hiv-1-infected patients were justified to alleviate side effects associated with administration of antiretroviral agents, provided an opportunity for the virus to gain fitness. in principle, given our current understanding of viral quasispecies dynamics, the proposal of p. ehrlich and d.d. ho is applicable to other viral pathogens. one argument that tones down the strength of the "hit early and hit hard" proposal is that some infected patients may not progress to disease, but maintain an asymptomatic lifelong persistent infection. this is the case with elite controllers in the case of hiv-1 infection, and individuals infected with hcv who will not progress toward liver disease. in cases in which such nonprogression can be anticipated by viral and host parameters, it may be justified to exclude some patients from aggressive interventions (suthar and harries, 2015; casado et al., 2018) . as a general rule, however, the potential benefits of early treatment are obvious not only to avoid disease on an individual basis, but also to diminish the chances of virus transmission (reviewed in suthar and harries, 2015; see also sections 7.1 and 7.7 in chapter 7 regarding the relevance of viral population numbers in transmission). restricting the number of treated patients for economic reasons will result in more expensive public health interventions when infected individuals develop the disease. box 8.4 includes recommendations for the use of antiviral agents and recapitulates concepts explained in this and preceding sections. despite the emphasis on evolutionary aspects, prevention and treatment of viral disease have many other angles some of which were box 8.4 • avoid monotherapy. ideally, use two or more antiviral agents which do not share a mechanism of action ( fig. 8.6 ). • treat as soon as possible after virus diagnosis, to avoid virus adaptability associated with high virus population size and to minimize transmission of inhibitor-resistant mutants. • individual patients should be treated only during the time at which the drug proves effective. when viral load rebounds, treatment should be discontinued. • use deep sequencing methodology to determine mutant spectrum composition for an adequate choice of inhibitors. the aim is to design personalized treatments that consider the probability of drug combination efficacy with minimal side effects. • consider temporary shelving of effective drugs when resistant mutants acquire epidemiological relevance. considered in chapter 7 in connection with factors of disease emergence (smolinski et al., 2003) . three of them should be mentioned here because they are as important as the adequate treatment designs described in this chapter: (i) adequacy of public health measures, (ii) public information about virus sources and means of contagion and (iii) need of global political action. information to the public should aim at limiting the spread of disease that is, undertaking personal and collective actions to reduce the r 0 value for a given virus (chapter 7, section 7.2). as an illustration of this key point, there was a quite extensive information campaign on hiv-1 and aids in developed countries during the early decades of hiv-1 spread, while the information about other potentially threatening viruses such as ebola or the severe acute or the middle east respiratory syndrome (sars and mers, respectively) coronaviruses was more limited. the need of a global response to limit the extension of disease episodes at the sites where they are initiated has been recognized for a long time, but it became obvious with the 2014e15 west african ebola epidemic (see siedner et al., 2015) . there is a need for international organizations and governments of developed countries to provide the health-care workforce to assist low-and middle-income countries to control viral episodes at an early stage. "help early, help effectively" is the recognized need at a global scale, which is the parallel to "hit early, hit hard" for the treatment of infected patients. global early action and adequate information can be as important as an adequate treatment design to control viral disease. it can restrict viral replication rounds and consequent adaptability. information is thought to have been critical for the control of ebola epidemic in nigeria (siedner et al., 2015) . however, information must also be planned to reach the target population in a convincing manner, as learned from the poliovirus vaccination and eradication campaign (renne, 2010) . the uncertainties regarding whether an initial, limited episode of viral disease will expand or die out do not help in decision making. however, the best choice in the case of emerging and reemerging infections is to act assuming the worst scenario. medical interventions represent a totally new set of selective constraints that viruses are facing only since decades ago, an infinitesimal time of their existence as biological entities. however, the evolutionary mechanisms available to viruses have successfully coped with many selective pressures, notably the effect of vaccination when a broad immune response is not evoked, or treatment with antiviral agents. a common way to proceed is to test a new vaccine with an animal host, be it the authentic host or an animal model, and obtain full protection when the animal is challenged with a virus that matches the antigenic composition of the vaccine. following the initial excitement, very often the vaccine displays only partial protection when tested in the natural environment. somewhat parallel arguments can be made about clinical trials for antiviral agents, usually performed initially with selected groups of patients. clinicians have coined the term "reallife" treatment studies when patients are not selected for optimal results (often pushed by commercial and political interests). this chapter has emphasized how the complexity of viral populations is a serious (often underestimated) difficulty to prevent and treat viral disease. examination of the molecular mechanisms exploited by viruses to survive despite antiviral interventions suggests two major lines of action: first, more judicious use of existing tools that should consider the complexity of viral populations and their dynamics; complexity 8. quasispecies dynamics in disease prevention and control cannot be combated with simplicity. second, the need to design new antiviral strategies, a topic addressed in chapter 9. several interconnected parameters determine the probability of success of an antiviral intervention. most of them follow from the general concepts of darwinian evolution explained in preceding chapters. it is important, however, to quantify as much as possible the evolutionary events that determine therapy success or failure. for this reason, the importance of viral population size, basic probability calculations of developing resistance, and the selective strength of mutations, have been explained with numerical examples. hopefully, these simple quantifications will permit a higher awareness of when and why treatment may succeed or fail. we live in a very unequal society. the chapter closes with the recognition that there are many social economic issues that are as important as scientific planning to combat the pathogenic viruses around us (see summary box). • medical interventions represent a new class of selective constraints acting on viral populations. • viral evolution affects antiviral preventive and treatment strategies in two different ways: (i) through the molecular mechanisms of short-term response in treated individuals that select escape mutants, and (ii) through the epidemiological impact of viruses that have acquired escape mutations. • ineffective vaccines can contribute to the selection of antigenic viral variants. • selection of viral mutants resistant to antiviral agents is a general phenomenon. selection is favored by suboptimal treatments and is delayed by the combined administration of multiple inhibitors. resistance may also occur in the absence of specific resistance mutations, and it is associated with viral fitness. • the aim of therapy should be to increase the functional barrier to resistance and to give no opportunity to the virus to pursue replication that increases its replicative fitness. • replication rate, viral load, fitness, and mutant spectrum complexity are interconnected parameters that may tip the balance toward either control of the infection or disease progression. each of these parameters can be targeted in an antiviral design. • health care resources, adequate public information, and firm political action are as important as antiviral designs to control virus infections at a global level. drug reposer: a web server for predicting similar amino acid arrangements to know drug binding interfaces for potential drug repositioning involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism amantadine-resistance as a genetic marker for influenza viruses hiv nef: role in pathogenesis and viral fitness mechanisms of hiv-1 escape from immune responses and antiretroviral drugs estimating hiv evolutionary pathways and the genetic barrier to drug resistance genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection influenza: the last great plague; and unfinished story of discovery the vaccine book transmitted/founder viruses rapidly escape from cd8 þ t cell responses in acute hepatitis c virus infection natural history of infections disease epitope-specific cd8 þ t lymphocytes cross-recognize mutant simian immunodeficiency virus (siv) sequences but fail to contain very early evolution and eventual fixation of epitope escape mutations during siv infection two escape mechanisms of influenza a virus to a broadly neutralizing stalk-binding antibody development of live-attenuated arenavirus vaccines based on codon deoptimization immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent hcv vlp vaccine the poliovirus eradication initiative viral persistence in vivo through selection of neutralizing antibody-escape variants cd4þ t-cell-epitope escape mutant virus selected in vivo perspectives and opportunities for novel antiviral treatments targeting virus fitness human immunodeficiency virus type 1 fitness and tropism: concept, quantification, and clinical relevance hiv-1 drug resistance and resistance testing antigenic determinants of possible vaccine escape by porcine circovirus subtype 2b viruses trans-dominant inhibition of rna viral replication can slow growth of drugresistant viruses naturally occurring ns3-protease-inhibitor resistant mutant a156t in the liver of an untreated chronic hepatitis c patient vaccine development: from concept to early clinical testing molecular and functional bases of selection against a mutation bias in an rna virus molecular epidemiology of hepatitis b virus mutants associated with vaccine escape, drug resistance and diagnosis failure patterns of resistanceassociated substitutions in patients with chronic hcv infection following treatment with direct-acting antivirals multiclass hcv resistance to direct-acting antiviral failure in real-life patients advocates for tailored secondline therapies rna virus evolution and the control of viral disease complications of rna heterogeneity for the engineering of virus vaccines and antiviral agents quasispecies dynamics in disease prevention and control quasispecies and rna virus evolution: principles and consequences virus population dynamics, fitness variations and the control of viral disease: an update viral quasispecies evolution. microbiol naturally occurring ns3 resistance-associated variants in hepatitis c virus genotype 1: their relevance for developing countries. virus res spectrum and characteristics of the virus inhibitory action of 2-(alpha-hydroxybenzyl)-benzimidazole coxsackie a9 virus: mutation from drug dependence to drug independence address in pathology on chemotherapy viral infections of humans. epidemiology and control. plenum medical book company transmission of single hiv-1 genomes and dynamics of early immune escape revealed by ultra-deep sequencing deep sequencing of protease inhibitor resistant hiv patient isolates reveals patterns of correlated mutations in gag and protease detection of a sexually transmitted hepatitis c virus protease inhibitor-resistance variant in a human immunodeficiency virus-infected homosexual man lessons from nature: understanding immunity to hcv to guide vaccine design human immunodeficiency virus gag and protease: partners in resistance barrier-independent, fitness-associated differences in sofosbuvir efficacy against hepatitis c virus imperfect vaccination: some epidemiological and evolutionary consequences poliovirus escape from rna interference: short interfering rna-target recognition and implications for therapeutic approaches coxsackievirus b3 mutator strains are attenuated in vivo principles of pharmacology: the pathophysiologic basis of drug therapy a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease systems vaccinology: enabling rational vaccine design with systems biological approaches global and regional molecular epidemiology of hiv-1, 1990-2015: a systematic review, global survey, and trend analysis nevirapine-resistant human immunodeficiency virus: kinetics of replication and estimated prevalence in untreated patients evolution of the hcv viral population from a patient with s282t detected at relapse after sofosbuvir monotherapy genetic and molecular analyses of spontaneous mutants of human rhinovirus 14 that are resistant to an antiviral compound the impact of hiv-1 genetic diversity on the efficacy of a combinatorial rnaibased gene therapy a working hypothesisevirus resistance development as an indicator of specific antiviral activity time to hit hiv, early and hard changes of hepatitis b surface antigen variants in carrier children before and after universal vaccination in taiwan comparison of naturally occurring resistanceassociated substitutions between 2008 and 2016 in chinese patients with chronic hepatitis c virus infection vaccination influences the evolution of classical swine fever virus minority hiv-1 drug resistance mutations are present in antiretroviral treatment-naive populations and associate with reduced treatment efficacy genetic variability of porcine circovirus 2 in vaccinating and non-vaccinating commercial farms outbreak of poliomyelitis in hispaniola associated with circulating type 1 vaccinederived poliovirus influenza. plenum medical book company my cousin, my enemy: quasispecies suppression of drug resistance genetic and antigenic diversity of human rotaviruses: potential impact on vaccination programs rna virus population diversity, an optimum for maximal fitness and virulence in vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects hiv and the pathogenesis of aids dispelling myths and focusing on notable concepts in hiv pathogenesis vaccination and timing influence siv immune escape viral dynamics in vivo susceptibility of influenza a viruses to amantadine is influenced by the gene coding for m protein comparison of the mechanisms of drug resistance among hiv, hepatitis b, and hepatitis c hiv-1 reverse transcriptase inhibitor resistance mutations and fitness: a view from the clinic and ex vivo incomplete neutralization and deviation from sigmoidal neutralization curves for hiv broadly neutralizing monoclonal antibodies combination sirna therapy against feline coronavirus can delay the emergence of antiviral resistance in vitro rapid development of drug-resistant mutants of poliovirus molecular basis of human immunodeficiency virus type 1 drug resistance: overview and recent developments antiretroviral therapy and drug resistance in human immunodeficiency virus type 2 infection development of streptomycin resistant strains of tubercle bacilli in pulmonary tuberculosis; results of simultaneous sensitivity tests in liquid and on solid media quantitation of zidovudine-resistant human immunodeficiency virus type 1 in the blood of treated and untreated patients a systematic review of hepatitis b virus (hbv) drug and vaccine escape mutations in africa: a call for urgent action quasispecies dynamics in disease prevention and control exploration of sequence space as the basis of viral rna genome segmentation pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy impact of novel ns5a resistance-associated substitutions of hepatitis c virus detected in treatment-experienced patients hepatitis c virus population dynamics during infection retreatment of hepatitis c virus infected patients with direct-acting antiviral failures influence of mutagenesis and viral load on the sustained low-level replication of an rna virus response of hepatitis c virus to long-term passage in the presence of alpha interferon: multiple mutations and a common phenotype molecular basis of interferon resistance in hepatitis c virus baseline hepatitis c virus resistance-associated substitutions present at frequencies lower than 15% may be clinically significant delayed lysis confers resistance to the nucleoside analogue 5-fluorouracil and alleviates mutation accumulation in the single-stranded dna bacteriophage vx174 hepatitis a virus vaccine escape variants and potential new serotype emergence increased fidelity reduces poliovirus fitness under selective pressure in mice production of guanidineresistant and -dependent poliovirus mutants from cloned cdna: mutations in polypeptide 2c are directly responsible for altered guanidine sensitivity imperfect vaccination can enhance the transmission of highly virulent pathogens the politics of polio in northern nigeria hiv-1 protease mutations and protease inhibitor cross-resistance production of resistant hiv mutants during antiretroviral therapy resistance, drug failure, and disease progression antiviral drug resistance rapid evolution of the neutralizing antibody response to hiv type 1 infection new vaccine design based on defective genomes that combines features of attenuated and inactivated vaccines intrahost dynamics of antiviral resistance in influenza a virus reflect complex patterns of segment linkage, reassortment and natural selection hiv-1 phylogenetics and vaccines vaccine strategies alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models resistance to direct antiviral agents in patients with hepatitis c virus infection deep sequencing and phylogenetic analysis of variants resistant to interferon-based protease inhibitor therapy in chronic hepatitis induced by genotype-1b hepatitis c virus animal vaccination and the evolution of viral pathogens increased replicative fitness can lead to decreased drug sensitivity of hepatitis c virus coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics mutations in coronavirus nonstructural protein 10 decrease virus replication fidelity microbial threats to health. emergence, detection and response natural hcv variants with increased replicative fitness due to ns3 helicase mutations in the c-terminal helix a18 evolution of treatmentemergent resistant variants in telaprevir phase 3 clinical trials a public health approach to hepatitis c control in low-and middle-income countries infrequent development of resistance to genotype 1-6 hepatitis c virus-infected subjects treated with sofosbuvir in phase 2 and 3 clinical trials strategies and challenges for eliciting immunity against avian influenza virus in birds a large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants evidence of the coevolution of antigenicity and host cell tropism of foot-and-mouth disease virus in vivo recombination in the alphaherpesvirus bovine herpesvirus 1 detection of human immunodeficiency virus (hiv) type 1 m184v and k103n minority variants in patients with primary hiv infection quantitative deep sequencing reveals dynamic hiv-1 escape and large population shifts during ccr5 antagonist therapy in vivo basic research in hiv vaccinology is hampered by reductionist thinking sequence-specific fidelity alterations associated with west nile virus attenuation in mosquitoes quasispecies diversity determines pathogenesis through cooperative interactions in a viral population hiv-1 polymerase inhibition by nucleoside analogs: cellular-and kinetic parameters of efficacy, susceptibility and resistance selection production of infectious hepatitis c virus in tissue culture from a cloned viral genome emergence of virus escape mutants after immunization with epitope vaccine genetic evolution of classical swine fever virus under immune environments conditioned by genotype 1-based modified live virus vaccine robust hepatitis c virus infection in vitro key: cord-259237-aty0vrat authors: frabutt, dylan a.; zheng, yong-hui title: arms race between enveloped viruses and the host erad machinery date: 2016-09-19 journal: viruses doi: 10.3390/v8090255 sha: doc_id: 259237 cord_uid: aty0vrat enveloped viruses represent a significant category of pathogens that cause serious diseases in animals. these viruses express envelope glycoproteins that are singularly important during the infection of host cells by mediating fusion between the viral envelope and host cell membranes. despite low homology at protein levels, three classes of viral fusion proteins have, as of yet, been identified based on structural similarities. their incorporation into viral particles is dependent upon their proper sub-cellular localization after being expressed and folded properly in the endoplasmic reticulum (er). however, viral protein expression can cause stress in the er, and host cells respond to alleviate the er stress in the form of the unfolded protein response (upr); the effects of which have been observed to potentiate or inhibit viral infection. one important arm of upr is to elevate the capacity of the er-associated protein degradation (erad) pathway, which is comprised of host quality control machinery that ensures proper protein folding. in this review, we provide relevant details regarding viral envelope glycoproteins, upr, erad, and their interactions in host cells. despite their vast diversity, animal viruses can be simply divided into two categories: non-enveloped viruses and enveloped viruses [1] . while non-enveloped viruses are wrapped with naked shells made of viral capsid proteins, enveloped viruses are covered with a lipid-bilayer, which is called a viral envelope. the viral envelope is obtained from progenitor host cells during the budding process, which can be a portion of plasma membrane or intracellular membrane. on the surface of the enveloped viruses, there are peplomers that project from the viral envelope, and play a critical role in viral infection. these peplomers are also described as spikes, which are made of viral envelope glycoproteins. envelope spikes serve to identify and bind to viral receptors on the host cell surface, allowing viral entry into cells and the initiation of infection by mediating virus-cell fusion. thus, the infectivity of enveloped viruses is absolutely dependent on the integrity of the viral envelope, and the functionality of the viral glycoproteins found therein. enveloped viruses are more stable than non-enveloped viruses under physiological conditions, at the expense of their sensitivity to high-temperature, low-ph, desiccation, or detergent-treatment, which limits their ability to withstand severe environments [2] . the entry of enveloped viruses requires the formation of a fusion pore between the viral envelope and the cell membrane, through which the viral genome is released into the cell. this fusion process is triggered by interactions between viral glycoproteins on the viral envelope and viral receptors on the cell surface, which can occur directly at the plasma membrane at neutral ph or in endocytic compartments at either low or neutral ph [3] . in addition, enveloped viruses can also enter cells through direct cell-to-cell contacts via virological glycosylation, which is one of the most common post-translational modifications in eukaryotic cells, is required for protein folding and maintaining protein structure. viruses have taken advantage of this benefit at nearly every step of the viral life cycle [12] . n-glycosylation significantly promotes their folding and solubility, enhances subsequent trafficking of these viral proteins to their destinations, and ensures that they are properly processed and incorporated into virions. nevertheless, glycosylation can have distinct effects that are both advantageous and detrimental to viral fitness. for example, if glycosylation occurs close to the glycoprotein processing sites, it may block the precursor cleavage by proteases and inhibit viral infection [13] ; if glycosylation occurs adjacent to the receptor-binding site, it may enhance the binding affinity and promote viral infection [14, 15] . in addition, the high density of glycans on virions may form a shield to impede antibody attack and promote immune evasion. however, these glycans can also become epitopes for stimulating neutralizing antibodies and the innate immune response, making viruses more vulnerable to immune clearance [16] . thus, there are multiple selective pressures on viral envelope glycosylation that can influence the pattern of glycosylation in order to achieve the optimal fitness in their hosts [17] . viruses are obligate intracellular parasites, and their glycoprotein biosynthesis and modification rely entirely on host cell machinery in the secretory pathway. therefore, viral and host proteins are glycosylated in a similar manner by the same mechanism. although glycans can be attached to polypeptide structures via several different mechanisms, asparagine n-linked glycosylation represents a fundamental and well characterized post-translational modification in eukaryotic organisms [18] . n-linked glycosylation starts from the membrane of the endoplasmic reticulum (er), where the tetradecasaccharide precursor is assembled. this precursor consists of two n-acetylglucosamine (glcnac), nine mannose (man, 4 are α1,2-linked), and three terminal glucose (glc) residues distributed on three extended man branches: a, b, and c (glc 3 man 9 glcnac 2 ) ( figure 1a ) [19, 20] . when nascent polypeptides enter the er lumen, the precursor is en bloc attached to asn residues of a nascent polypeptide in a consensus asn-x-(ser/thr) motif. after the attachment, these precursors are processed by a series of enzymes in both the er and the golgi apparatus to remold the core oligosaccharide into diverse n-linked glycan structures ( figure 1b ). the first step in this process is the sequential removal of the two outermost glc residues on branch a. the first glc residue is removed by glucosidase i (gi), resulting in the di-glycosylated oligosaccharide glc 2 man 9 glcnac 2 , which is recognized by an er transmembrane lectin malectin [21] . the second glc residue is then removed by glucosidase ii (gii), resulting in the mono-glucosylated oligosaccharide glc 1 man 9 glcnac 2 , which is recognized by two other er lectins, the membrane-bound calnexin (cnx) and/or soluble calreticulin (crt). interaction with these two chaperones segregates the newly formed glycoprotein and provides access to protein disulfide isomerases (pdis) such as erp57, which promotes disulfide bond formation, resulting in protein folding into a native conformation. once a protein is properly folded, gii cleaves the last glc residue on branch a, which releases the protein from the cnx/crt cycle. the er class i α-mannosidase (ermani) then cleaves the outermost man residue on branch b on native proteins, resulting in the oligosaccharide man 8 glcnac 2 . these high-man glycans are then recognized by lectins including er-golgi intermediate compartment-53 (ergic-53), vesicular integral membrane protein of 36kda (vip36), and vip36-like (vipl), which promote trafficking from the er to the golgi [22] . the remaining man residues are cleaved by the golgi mannosidases, and the glycan remolding process is continued through the remainder of the n-glycosylation pathway, which generates functional glycoproteins that are delivered to the cell surface ( figure 1b ). in addition to these chaperones and enzymes that promote protein folding, the er is also equipped with a unique quality control mechanism that extracts and degrades proteins that are not correctly folded or assembled into their native conformation, which is called er-associated protein degradation (erad) [23] . in fact, the folding efficiency of glycoproteins in the er is very low, which requires cycles of association and dissociation from cnx/crt to ensure proper glycoprotein maturation. if glycoproteins with the man 9 glcnac 2 oligosaccharide display non-native conformations, they are reglucosylated by the udp-glc:unfolded glycoprotein glucosyltransferase (ugt1 or uggt), and are subject to additional rounds of re-engagement with the cnx/crt machinery until folding is achieved. however, if a certain time frame for the folding is exceeded, proteins may never fold properly. misfolded proteins are sequestered into coat protein complex ii (cop-ii) -dependent, highly mobile er-derived quality control vesicles (qcvs), where ermani is enriched ( figure 1b ) [24] . because ermani is able to excise all α1,2-man residues when it is expressed at much higher levels in vitro [25] , the enzyme may catalyze extensive demannosylation, resulting in the production of low-man oligosaccharide man 5 glcnac 2 -containing glycoprotein precursors. the removal of the a branch terminal man residue, which is the acceptor for glc transferred by uggt, disables these proteins from reengagement with cnx/crt and re-entering into the folding cycle. importantly, the low-man n-glycans represent a tag for defective glycoproteins, targeting them to erad [26] . figure 1b ) [24] . because ermani is able to excise all α1,2-man residues when it is expressed at much higher levels in vitro [25] , the enzyme may catalyze extensive demannosylation, resulting in the production of low-man oligosaccharide man5glcnac2-containing glycoprotein precursors. the removal of the a branch terminal man residue, which is the acceptor for glc transferred by uggt, disables these proteins from reengagement with cnx/crt and re-entering into the folding cycle. importantly, the low-man n-glycans represent a tag for defective glycoproteins, targeting them to erad [26] . , and endoplasmic reticulum (er) stress pathways. nascent polypeptides are translocated through sec61 into the rough er, where the core oligosaccharide is transferred from a dolichol phosphate onto asparagine residues in asparagine-x-serine/threonine (nxs/t) motifs (i). the two terminal glucose residues on the core oligosaccharide are trimmed by glucosidase i, (gi) (ii), and gii (iii), respectively, allowing for the association with the chaperones, membrane-bound calnexin (cnx) and and/or soluble calreticulin (crt), which promote folding to a native conformation. eventually, the last terminal glucose residue will be trimmed by gii, and the glycoprotein will attain a native conformation (iv), or misfold (vii). glycoproteins that reach a native conformation will have the terminal α1,2-man residue on the b branch removed by er class i α-mannosidase (ermani) (v), as a signal to allow it to traverse the canonical secretory pathway for surface presentation or secretion (vi). polypeptides unable to reach a native conformation (vii) will engage in multiple rounds of the cnx/crt cycle, facilitated by reglucosylation of the terminal glucose by udp-glc:unfolded glycoprotein glucosyltransferase (uggt) (viii), and trafficking between quality control vesicles (qcv) (ix) and the the er-derived quality compartments (erqc) (x) under er stress. terminally misfolded glycoproteins will be demannosylated to remove all α1,2-man residues (xi), followed by association with lectins osteosarcoma amplified 9 (os9) and xtp3-transactivated gene b protein (xtp3-b) for erad (xii). figure 1 . (a) schematic presentation of the n-linked core oligosaccharide structure. the core is composed of two n-acetylglucosamine (glcnac, blue), nine mannose (man, red), and three glucose (glc, yellow) residues. a, b, and c are three oligosaccharide branches. (b) schematic description of n-glycosylation, endoplasmic reticulum-associated protein degradation (erad), and endoplasmic reticulum (er) stress pathways. nascent polypeptides are translocated through sec61 into the rough er, where the core oligosaccharide is transferred from a dolichol phosphate onto asparagine residues in asparagine-x-serine/threonine (nxs/t) motifs (i). the two terminal glucose residues on the core oligosaccharide are trimmed by glucosidase i, (gi) (ii), and gii (iii), respectively, allowing for the association with the chaperones, membrane-bound calnexin (cnx) and and/or soluble calreticulin (crt), which promote folding to a native conformation. eventually, the last terminal glucose residue will be trimmed by gii, and the glycoprotein will attain a native conformation (iv), or misfold (vii). glycoproteins that reach a native conformation will have the terminal α1,2-man residue on the b branch removed by er class i α-mannosidase (ermani) (v), as a signal to allow it to traverse the canonical secretory pathway for surface presentation or secretion (vi). polypeptides unable to reach a native conformation (vii) will engage in multiple rounds of the cnx/crt cycle, facilitated by reglucosylation of the terminal glucose by udp-glc:unfolded glycoprotein glucosyltransferase (uggt) (viii), and trafficking between quality control vesicles (qcv) (ix) and the the er-derived quality compartments (erqc) (x) under er stress. terminally misfolded glycoproteins will be demannosylated to remove all α1,2-man residues (xi), followed by association with lectins osteosarcoma amplified 9 (os9) and xtp3-transactivated gene b protein (xtp3-b) for erad (xii). ermani containing qcv are rapidly recycled through autophagy/lysosome pathways (xiii). without interactions with client glycoproteins, edemosome components are degraded through an autophagy-like mechanism (xiv). viruses can hijack edemosomes to form double membrane vesicles (dmvs) that serve as platforms for their replication (xv). with only one-tenth of the total cell volume, the er is responsible for the synthesis of the vast majority of the secreted or membrane proteins, which account for one-third of total cellular proteins. therefore, the er has extremely high protein concentrations (100 mg/ml), which renders this organelle very susceptible to protein aggregation [27] . in addition, the protein folding is error prone, and this process can be further compromised by physiological and pathological perturbations. moreover, genetic mutations may prohibit proteins from being folded properly. all these factors may cause the accumulation of unfolded or misfolded proteins. when the level of these aberrant proteins exceeds the folding and clearance capacity of the er, known as er homeostasis, it leads to a cellular stress response termed "er stress", which in turn activates the unfolded protein response (upr) to restore the er homeostasis [28] . er stress is sensed by three er transmembrane receptors: double-stranded rna (dsrna)-activated protein kinase (pkr)-like er kinase (perk), inositol-requiring enzyme 1 (ire1), and activating transcription factor 6 (atf6). perk and atf6 are in association with another er chaperone, the binding immunoglobulin protein (bip, or grp78), when the cell is not under stress. bip preferentially binds to misfolded proteins and dissociates from perk and atf6 under er stress, resulting in their activation and upr to mitigate this stress [29, 30] . ire1 is activated by the direct binding of unfolded proteins [31] . ire1 then activates the transcription factor x-box binding protein 1 (xbp-1), which in turn up-regulates er chaperones to assist in the folding capacity of the er as well as erad components to boost protein degradation. perk phosphorylates the eukaryotic initiation factor (eif)-2α and halts protein translation, and atf6 up-regulates protein expression to boost the er protein folding capacity and erad. however, if these objectives are not achieved within a certain time span or if the disruption is prolonged, upr also activates pathways leading to cell death. although perk activation causes global inhibition of protein translation by blocking eif-2α activity, it paradoxically enhances translation of the transcription factor atf4. atf4 then trans-activates the ccaat/enhancer-binding protein-homologous protein (chop), which is a pro-apoptotic transcription factor, resulting in cell death by apoptosis [32] . erad is a protein quality control mechanism conserved in all eukaryotic cells, which is an important arm of upr, necessary to alleviate er stress [33] . erad results in the selective dislocation of unfolded and misfolded proteins from the er to the cytosol via specific membrane machinery. erad targets are subsequently degraded by the cytosolic ubiquitin proteasome system (ups) [34] . quality control of functional proteins produced from the er is also critical for maintenance of the er homeostasis by eliminating unfolded and misfolded proteins. thus, erad is a central element of both the secretory pathway and upr, which targets a number of physiological and pathological substrates such as the t cell antigen receptor (tcr) [35] , 3-hydroxy-3-methylglutaryl coenzyme-a (hmg-coa) reductase (hmgcr) [35] , squalene monooxygenase (sqle) [36] , inositol 1,4,5-trisphosphate (ip 3 ) receptor [37] , diacylglycerol acyltransferase 2 (dgat2) [38] , heme oxygenase-1 (ho-1) [39] , alpha-1 antitrypsin [35] , and cystic fibrosis transmembrane regulator (cftr) [40] . so far, more than 60 human diseases have been attributed to this pathway [41] . although the vast majority of secreted proteins are glycosylated, the er is responsible for the folding and assembly of both glycosylated and non-glycosylated proteins into functional complexes, which are subjected to erad quality control if they are misfolded. the process of erad can be divided into three steps: substrate recognition, retrotranslocation, and ubiquitylation/proteasomal degradation. in fact, extensive excision of α1,2-man residues from n-glycans sends an important signal to trigger misfolded glycoprotein degradation, which is dependent on class i mannosidases [42] . class i mannosidases belong to the glycoside hydrolase family 47 (gh47), which are exo-acting α1,2-mannosidases that are divided into three subfamilies [43] . the first subfamily consists of ermani, which is supposed to cleave the outmost α1,2-man residue on the b branch from n-linked glycans in the er. the second subfamily consists of three golgi α-mannosidase i, including golgimania, golgimanib, and golgimanic, which cleave the remaining three α1,2-man residues in the golgi complex for n-glycan maturation. the third subfamily consists of the er degradation-enhancing α-mannosidase-like proteins (edem) 1, 2, and 3. although some edem orthologs in lower eukaryotes have detectable α1,2-mannosidase activity, such activity has not been reported for any mammalian edem proteins in vitro. nevertheless, there is evidence suggesting that these edem proteins should have enzymatic activity in vivo [44, 45] . indeed, the extent of man excision determines the fate of a glycoprotein, which could be either targeted to erad for degradation or sent to the golgi for normal trafficking. ermani exhibits a slow rate of enzymatic activity, which allows nascent proteins to perform multiple rounds of reglucosylation and achieve proper folding [46] . properly folded glycoproteins should have one man residue trimmed off from n-glycans by ermani. these glycoproteins then interact with the high-man binding lectin, er-golgi intermediate compartment 53 kda protein (ergic-53) [47] , for trafficking from the er to the golgi ( figure 1b) . however, if glycoproteins are misfolded terminally, the remaining three α1,2-man residues are excised from these molecules, which targets misfolded proteins for degradation [48] . it is still not completely understood how misfolded glycoproteins are subjected to such extensive demannosylation in the er and then targeted for erad. although ermani alone may be able to complete this task, there is evidence suggesting that additional gh47 enzymes are involved. elevation of the golgi mannosidases has been shown to accelerate erad, so these enzymes may possibly be responsible for such extensive excision, likely by trafficking back to the er via an unknown mechanism [49] . in fact, the localization of the golgimania has recently been observed in qcv with other canonical erad machinery such as ermani, and overexpression and knockdown can, respectively, increase or retard trimming of misfolded glycoproteins from man 9 glcnac 2 to man 5 glcnac 2 in vitro [50] . in addition, upon er stress, qcv converge to form the er-derived quality compartments (erqc), where edem proteins are also sequestered ( figure 1b ) [48] . edem1 and edem3 boost mannose trimming when overexpressed [45, 51, 52] . in addition, using a genomic knockout approach, it has been recently proposed that edem2 plays a central role in the trimming of the outmost man residue on the b branch, whereas edem1 and edem3 should be responsible for trimming of the remaining α1,2-man residues. accordingly, a "double check" model for misfolded glycoproteins has been proposed, which suggests that edem2 catalyzes the first step of man trimming, and edem1 and edem3 contribute the second step [44] . under these joint actions, all four α1,2-man residues are removed from the oligosaccharide, which is then recognized by the lectins osteosarcoma amplified 9 (os9) and xtp3-transactivated gene b protein (xtp3-b) via the mannose 6-phosphate receptor homology (mrh) domain ( figure 1b) [53, 54] . misfolded proteins are targeted to specific translocation channels (retrotranslocons) for retrotranslocation in an energy-dependent manner. this process is facilitated by p97, a member of the atpases associated with the diverse cellular activities (aaa) family, by catalyzing atp hydrolysis [55] . the p97 atpase is recruited by the ubiquitin-like (ubx)-domain-containing protein ubxd8, an er-membrane protein that plays a role in erad [56] . it is still mysterious how these retrotranslocons are formed and how integral membrane and lumenal erad substrates are exported across the er membrane through these retrotranslocons. the first candidate channel is composed of the sec61 complex, which is comprised of α, β, and γ subunits. the α subunit crosses the membrane 10 times, and forms a channel with the other subunits. the sec61 heterotrimeric channel is the main translocon involved in co-translational protein transport into the er [57] . however, there is evidence suggesting that the sec61 translocon is also involved in retrotranslocation of erad substrates, implying the non-specificity and bi-directional property of this channel [58] . the second candidate is a member of the derlin family (derlin-1, -2, and -3) [59, 60] . derlins are integral membrane proteins that likely span the er membrane four times and contain a rhomboid-like domain [61] . the third candidate includes the erad-specific e3 ligases. they have a large number of transmembrane domains, which are not only responsible for polyubiquitylation, but could act as potential exit channels for erad substrates [62] . in saccharomyces cerevisiae, there are two major types of really interesting new gene (ring)-finger e3 ligase complexes, hrd1 and doa10, which mediate erad by targeting discrete substrates [63] . hrd1 was the first e3 enzyme identified in the erad pathway during the study of hmg-coa reductase degradation (hrd) [64] . hrd1 has 6 transmembrane helices in its n-terminal transmembrane domain and a catalytic ring domain in the soluble c-terminal region extended to the cytosol. using an elegant erad assay reconstituted in vitro, the hrd1-mediated formation of ubiquitin-gated protein-conducting membrane channels has been demonstrated [65, 66] . hrd1 has two mammalian orthologs named hrd1 and gp78, and its functioning e2 enzyme is known as ubc7, which also has two mammalian orthologs, ube2g2 and ube2g1 [67] . hrd1 is unstable, and must be stabilized by its co-factor hrd3 in an equimolar ratio [68] . the mammalian ortholog of hrd3 is sel1l, which is required for erad substrate retrotranslocation [69] . hrd1 interacts with der1, and the der1-hrd1 interaction is bridged by another integral membrane protein usa1, which is also required for hrd1 oligomerization [70] . the usa1 mammalian ortholog is called herp, which also interacts with hrd1 and derlin-1 and plays an important role in erad [71] . doa10 was identified in degradation studies of mating type (mat)-α2-10 (doa), which is a yeast transcription factor. doa10 is a~150 kda protein that has 14 transmembrane domains, which requires both ubc6 and ubc7 as e2 enzymes. the doa10 mammalian ortholog is teb4 (march6), which functions in the erad pathway with similar subcellular distribution and topology [72] . the ubc6 e2 enzyme has two mammalian orthologs, ube2j1 and ube2j2; both are involved in erad [73, 74] . in saccharomyces cerevisiae, erad is executed synergistically by hrd1 and doa10 with minimal redundancy because they exhibit different substrate specificity. doa10 mainly triggers ubiquitylation of specific soluble proteins and membrane proteins with degrons exposed to the cytosol; a process referred to as erad-c [75, 76] . hrd1 interacts with two other types of substrates, whose degradation is termed erad-l and erad-m. erad-l includes soluble lumenal proteins in the er and transmembrane proteins with degrons exposed to the er lumen; erad-m includes transmembrane proteins with degrons embedded into the er membrane [63] . simultaneous inactivation of both genes has been shown to increase the sensitivity to heavy metal-induced cellular stress and exhibit an elevated upr. the regulation of protein folding and the functional relation between erad and the upr are much more complex in mammalian cells. in saccharomyces cerevisiae, the cnx cycle does not exist due to the lack of uggt. in addition, protein synthesis is tightly controlled at the translational level by determination of the stoichiometry to avoid surplus production, resulting in minimal dependence on the post-translational regulation of protein expression. moreover, although the yeast ermani ortholog mns1p and edem ortholog htm1p are indispensable for erad, only one edem ortholog is present in yeast [77, 78] . because overly active erad may interfere with the regular protein folding process in the er, mammalian cells have evolved mechanisms to tightly regulate this quality control device by a combination of compartmentalization and tuning. edem1 is segregated into er-derived, lc3-i-associated vesicles, which are called edemosomes, where edem1, os-9, and sel1l are concentrated when they lack client glycoproteins to dislocate ( figure 1b) [79] . notably, unlike chaperones and the other enzymes, many erad regulators including ermani, edem1, os-9, herp, and sel1l are short-lived proteins, and ermani, edem1, sel1l, and os-9 are targeted to the lysosomal pathway for degradation [79, 80] . thus, edemosomes are called erad tuning vesicles, which deliver their content to lysosomes for disposal via an autophagy-like pathway to reduce the erad capacity under natural conditions [81] . additionally, lysosomal inhibitors are able to cause the accumulation of an aggregating mutant of dysferlin in the er when compared to the wild-type, which was used as evidence to propose that large protein aggregates are disposed of via an autophagy/lysosomal pathway, dubbed erad ii [82] . however, under er stress, most of these factors are highly induced, including the edem proteins, but not ermani [45, 83, 84] . under stress, qcv are recruited to the erqc, resulting in the accumulation of ermani and its glycoprotein substrates [85] . moreover, many other erad components, including edem1, hrd1, derlin-1, sec61β, and herp, are also concentrated in erqc. importantly, it has been found that edem1 stabilizes ermani and increases its protein expression at steady-state levels [86] . such enrichment of these critical components accelerates efficient assembly of the erad machinery, potentiating the degradation of misfolded glycoproteins and alleviating er stress. during infection, viruses are able to hijack the host translational machinery and saturate the er with viral proteins. not only do viruses use the er to generate their glycoproteins, but some even utilize the er as their site to assemble progeny particles [87] . such accumulation of viral proteins in the er places a heavy demand on the protein folding machinery, which may cause er stress, and in turn, activate the upr, resulting in restoration of the er homeostasis or apoptosis. so far, at least 36 viruses have been found to be able to induce er stress, and activate the three upr stress signaling pathways [88] . enveloped viruses may bud through the plasma membrane or an intracellular compartment. in addition, their envelope glycoproteins are targeted to the er for post-translational modifications and folding. not surprisingly, many viral envelope glycoproteins are significant inducers of the upr, which includes hcv [89] , hepatitis b virus (hbv) [90] , coronaviruses [91] , chikungunya virus (chikv) [92] , and retroviruses [93] . as introduced earlier, the upr utilizes three different mechanisms to alleviate er stress: reducing global protein translation, increasing the er folding capacity, and enhancing erad by activating the perk, atf6, or ire1-xbp1 pathways, respectively. viral infections may activate these pathways, resulting in the inhibition or enhancement of viral replication (table 1) . for example, the perk-mediated global translation shutdown is a very effective antiviral mechanism, and a similar shutdown by pkr has been used in the interferon pathway to defend against viral infection [94] . conceivably, viruses have evolved a number of strategies to circumvent the detrimental effect of upr to establish productive infection. hcv is still able to produce viral proteins even when the cellular translational machinery is shut down, because these viruses have their own internal ribosome entry site (ires) to recruit and assemble the ribosomal initiation complex for protein expression [95] . epstein-barr virus (ebv), herpes simplex virus (hsv), and african swine fever virus (asfv) can counteract the perk-mediated eif2α phosphorylation by activating an eif2α phosphatase pp1 [96] [97] [98] . in another example, the hcv e2 protein directly interacts with perk to prevent er stress sensing by acting as a pseudo-substrate to block perk activity [99] . in addition to combating the upr, viruses also take advantage of the upr pathways to benefit their replication. for example, influenza a virus (iav) replication is promoted by activation of the ire1-xbp1 pathway [100] ; atf6 activation promotes asfv, lymphocytic choriomeningitis virus (lcmv), denv, human cytomegalovirus (hcmv), and japan encephalitis virus (jev) replication [101, 102] , and atf4 activation enhances hiv-1 replication [103] . thus, despite the detrimental effects, viruses have evolved to manipulate host upr signaling pathways to promote viral infections. below, we will focus on the roles of erad played in virus replication, which is the main target of this review. as introduced earlier, erad transports unfolded/misfolded proteins from the er into the cytosol for proteasomal degradation. conceivably, viruses can manipulate and exploit this cellular machinery to degrade several important host factors to promote their propagation. herpesviruses have evolved multiple mechanisms to suppress the host immune response via erad. major histocompatibility complex (mhc) molecules play an indispensable role in triggering an immediate immune response to inhibit virus infections. herpesviruses inhibit mhc class i (mhc-i) expression by targeting these molecules to erad for degradation. for example, hcmv produces two transmembrane proteins, us2 and us11, and each is sufficient to bind to mhc-i heavy chains, causing their dislocation from the er to the cytosol for degradation [108] . notably, us2 and us11 use different mechanisms to degrade mhc-i. us2-dependent mhc-i degradation is mediated through an interaction with the e3 ligase, trc8. this us2/trc8 complex has been implicated in the degradation of other membrane proteins including multiple alpha-integrins, the interleukin 12 receptor (il-12r), thrombomodulin (thbd), protein tyrosine phosphatase receptor type j (ptprj), and cd112 [109] . although the signal peptide peptidase (spp) has been shown to bind to trc8, the us2/trc8 complex maintains its mhc-1 degradation activity in spp−/− knockout cells, suggesting that spp binding is not related to mhc-1 degradation [110, 111] . recent reports now regard the us2/trc8 complex as a multifunctional hub that is able to degrade a multitude of targets in order to further hcmv immune evasion [109, 112] . a complex formed between us11, derlin-1, and the e3 ligase, tmem129, mediates mhc-i degradation via us11 [113] . initial reports concerning us11 found an association with sel1l and assumed that us11 mediated mhc-1 degradation could be sel1l/hrd1 dependent. recent literature has confirmed that while the us11/tmem129 complex degrades mhc-1, us11 itself is degraded through a sel1l/hrd1 axis in the absence of the client mhc-1 [113, 114] . recruitment of p97 by ubxd8 is also crucial for us11-mediated mhc-i degradation [115] . with regard to us11, hcmv utilizes erad to dispose of mhc-i and its own effector protein using discrete axes for ubiquitination. mouse gammaherpesvirus 68 (mhv68) uses another mechanism to inhibit mhc-i. mhv68 produces a protein termed mk3, which is a ring-finger e3 ligase anchored on the er membrane. mk3 interacts with mhc-i heavy chain molecules, and it also associates with the transporter associated with antigen processing (tap), p97, derlin-1, and the e2 ube2j2. association with ube2j2 results in an interesting pattern of ubiquitination of non-lysine residues (the mk3/ube2j2 complex can ubiquitinate serines as well as lysines) that leads to rapid degradation of the mhc-i by proteasomes [73, 116] . thus, herpesviruses have evolved numerous strategies to block the mhc antigen presentation and evade the host immune response to establish a persistent infection. primate lentiviruses also harness the erad pathway to promote their replication via downregulation of their receptor cd4. cd4 downregulation prevents superinfection and promotes viral release by interrupting viral receptor-envelope interactions on the plasma membrane, leading to a controlled and productive viral infection and immunodeficiency [117] . these viruses produce two accessory proteins, nef and vpu, to trigger cd4 degradation via two distinctive mechanisms [118] . nef uses the endocytic pathway to redirect cd4 from the cell surface, or to interfere with the transport of newly synthesized cd4 from the trans-golgi network (tgn) to the cell surface, resulting in cd4 dislocation to endosomes and degradation by lysosomes [119] . however, vpu interacts with cd4 in the er and induces cd4 proteasomal degradation via erad [120] . vpu is a small transmembrane protein encoded by hiv-1 and some simian immunodeficiency virus (siv) isolates. vpu forms ion conductive membrane pores; it also interacts with β-transducin repeat-containing proteins (βtrcp), which are f-box/wd repeat-containing proteins that are part of the skp1-cul1-f-box (scf) e3 ubiquitin ligase complex [121] . the vpu-induced cd4 degradation is strictly dependent on the scf-β-trcp complex [122] . notably, this e3 ligase complex is not associated with the er membrane, and therefore does not normally function in erad. however, the degradation also requires the cytosolic atpase p97 and its cofactors ufd1l and npl4, which are key components of the erad machinery, suggesting that cd4 is degraded via erad [122] . nevertheless, the degradation is not dependent on hrd1, sel1l, and ubc7. in addition to degradation, viruses may harness erad components to benefit their replication. first, erad can promote viral protein expression. mouse mammary tumor virus (mmtv) is a betaretrovirus, which expresses the rem protein in the er. rem has a n-terminal 98-amino acid signal peptide (sp), which is cleaved off by signal peptidase and retrotranslocated in a p97-dependent manner [123] . rem sp then promotes the nuclear export of viral unspliced rnas to the cytosol for protein expression. similarly, hepatitis e virus (hev) orf2 is an n-linked glycoprotein, but functions as the major capsid protein. although orf2 is expressed in the er, it depends on erad components to exit from the er to the cytoplasm without being polyubiquitylated [124] . second, erad can promote virus entry. polyomaviruses (pyv) enter cells through the er and then replicate in the nuclei [125] . to get from the er to the nucleus, these viruses can cross the er membrane into the cytosol via the erad retrotranslocons [126] . an example of this is mouse pyv, which uses derlin-2, whereas simian virus 40 (sv40) uses derlin-1 and the sel1l complex for dislocation [126, 127] . in addition, the proteasome machinery is also required for the human bk pyv exit from the er [127] . third, erad can promote virus replication. the replication of positive-strand rna viruses normally involves the formation of double-membrane vesicles (dmvs) and convoluted membranes (cms) by rearrangement of cellular membranes, which segregates and protects viral proteins and genomes from the host's innate immune response. as introduced earlier, the erad activity can be adjusted by erad tuning vesicles termed edemosomes ( figure 1b) , which display non-lipidated lc3 and segregate the erad factors edem1, os-9, and sel1l from the er lumen [81] . by comparing the similarity between dmvs and edemosomes, it has been discovered that mouse hepatitis virus (mhv), equine arteritis virus (eav), and jev indeed replicate in these erad tuning vesicles [128] . thus, these viruses can subvert edemosomes as their replication vesicles to promote infection [129] . although erad has been frequently manipulated by a number of viruses to promote infection or attenuate immune responses, it may also function directly as an antiviral device to protect host cells from infection. because viral envelope glycoprotein production and folding take place in the er, these viral proteins may become the primary targets for erad, resulting in the inhibition of viral infection. primate lentiviruses, including hiv and siv, have low levels of envelope glycoproteins on their surface, and the average copy number is~14 env trimers per virion [130, 131] . in contrast, ifa, sendai virus, hsv, and moloney murine leukemia virus (momulv) have much more envelope glycoproteins on their surfaces [132] [133] [134] [135] . the exceptionally low number of env spikes may protect hiv-1 from host immune responses [136] since almost 85% of env proteins are retained in the er and are degraded [137] [138] [139] . this degradation mechanism was not clear until we recently reported that hiv-1 env glycoproteins are targeted for erad. from completely unrelated studies, we isolated hiv-1 non-permissive (np) and permissive (p) t cell clones n2-np and n5-p from the original cem.nkr human t cell line [140] . our initial analysis uncovered that hiv-1 replication is restricted from the second round of the viral life cycle in n2-np cells, resulting in~1000-fold inhibition when compared to n5-p. further transcriptome analysis by microarrays revealed that n2-np cells overexpress the mitochondrial translocator protein (tspo), which strongly inhibits hiv-1 env expression [141] . tspo interacts with the mitochondrial permeability transition pore (mptp) complex, which includes the outer membrane protein voltage-dependent anion channel (vdac) protein, the inner membrane protein adenine nucleotide translocase (ant), and the mitochondrial matrix protein cyclophilin d (cypd) [142] . tspo binds to vdac and contributes to the regulation of the mitochondrial membrane permeability by the mptp complex [143] . our results suggested that tspo overexpression could reduce the oxidative redox status in the er, which interferes with the env oxidative folding process, resulting in env degradation. consistently, the rapid env degradation in n2-np cells was rescued by kifunesine, an effective inhibitor of glycoside hydrolase family 47 (gh47) enzymes [144] , suggesting that hiv-1 is degraded via erad in n2-np cells. to further explore the env degradation mechanism, we investigated which of those four er-associated gh47 enzymes was responsible for the env degradation. notably, when ermani, edem1, edem2, and edem3 were ectopically expressed in 293t cells, only ermani strongly inhibited env expression in a dose-dependent manner. in addition, when the endogenous ermani was knocked out by crispr/cas9, tspo was no longer able to suppress the env expression [145] . these results demonstrated that ermani should be responsible for the initiation of hiv-1 env degradation via erad. human ermani is a 699-amino-acid, 79.5-kda, type ii membrane protein, which is divided into an n-terminal cytoplasmic domain (cd), transmembrane (tm) helix, lumenal 'stem' region, and a catalytic domain [146, 147] . using an immunoprecipitation assay, we found that hiv-1 env interacts with the catalytic domain of ermani [145] . the structure of this catalytic domain shows an (αα) 7 -barrel composed of 14 consecutive helices [148] . in the catalytic domain, there are seven residues that are critical for ermani function. c527 and c556 form a highly conserved disulfide bond and were reportedly critical for protein folding [149] , whereas e330, d463, and e599 were proposed as catalytic residues [148] . r334c and e397k mutations are found in nonsyndromic autosomal-recessive intellectual disability (ns-arid) disease [150] , and the r334c mutation is also found in the congenital disorders of glycosylation [151] . all these residues are required for hiv-1 env degradation, suggesting that the mannosidase activity is important for the ermani activity. ermani also targets the terminally misfolded human alpha1-antitrypsin variant null (hong kong) (nhk) for degradation via erad, but neither its catalytic activity nor its catalytic domain is required for this degradation, suggesting that different mechanisms are involved in hiv-1 env and nhk degradation [152] . we have also found that the viral protein r (vpr) of hiv-1 enhances viral replication in monocyte-derived macrophages (mdms) and dendritic cells (mddcs) by rescuing env from erad degradation through the erad (ii) autophagy pathway. compounds known to facilitate glycoprotein folding (pk11195 and as 2 o 3 ) and inhibit er α-mannosidases crucial for erad (kifunensine), and those that block lysosomal proteases (bafilomycin) rescued envelope expression and infectivity in a ∆vpr background to that of wild-type virus [153] . as aforementioned, unlike ermani, whose expression is not responsive to upr, the expression of the edems is induced upon upr via the ire1/xbp activation pathway, which boosts erad and alleviates er stress. although ectopic expression of edems did not inhibit hiv-1 env expression [145] , these proteins inhibit the expression of some other envelope glycoproteins. hbv expresses three surface glycoproteins, the large (l), middle (m), and small (s), which are translated from different initiation codons within the same open reading frame (orf) and share the tetra-spanning transmembrane domains in the s protein. the n-terminus of the m and l protein contain additional pres2 and pres1-pres2 domains, respectively. the common s domain has an n-glycosylation site, and the m pres2 domain has another site. overexpression of the surface proteins is sufficient to activate the ire1/xbp1 pathway and elevate edem1, edem2, and edem3 expression. importantly, edem1 overexpression destabilizes s, m, and l, and edem1 silencing stabilizes their expression [154] . in addition, the autophagy/lysosomal pathway, but not the proteasomal pathway, is involved in the degradation of hbv surface glycoproteins, further complicating our understanding of the viral protein degradation process via erad [154] . hcv has two n-glycosylated envelope proteins e1 and e2 on the surface of virions, which are type i transmembrane proteins expressed from a common viral polyprotein precursor. hcv infection strongly induces the activation of the ire1 stress sensor, resulting in elevation of edem1, edem2, and edem3, but not the ermani expression. both edem1 and edem3, but not edem2, interact with e2, and overexpression of these two proteins induces e2 polyubiquitylation and degradation. conversely, knockdown of edem1 expression or treatment with kifunesine increases e2 expression, and also reduces the interaction of edem1 and edem3 with sel1l [155] . taken together, these results strongly suggest that edem proteins are able to extract viral polypeptides from the er quality control cycle, and degrade them via erad. however, since none of these proteins can target the jev e protein to erad for degradation, not every viral glycoprotein is recognizable by these proteins [155] . in vivo experiments on patients with chronic liver injury were unable to identify up-regulation of upr and erad elements in diseased versus control patients [156] . erad has also been implicated in the degradation of hcmv glycoproteins, gh and gl, via the 26s proteasome. hcmv produces at least 65 unique glycoproteins, with four homologues to the hsv glycoproteins, gh, gb, gl, and gm [157] . the glycoproteins, gh and gl, are constituents of the gcii type complexes found on the surface of hcmv virions. the gcii trimeric complex between gh, gl, and go can initiate ph independent fusion [158] . in addition, a pentameric complex between gh, gl, and the gene products u128, u130, and u131 is able to mediate entry into different cell types via ph-dependent receptor-mediated endocytosis; a process that requires the trimeric gh/gl/go complex [159] . although previous studies have shown that the glycoprotein gl stabilizes the expression of gh and potentiates its surface localization [160] , recent work revealed that gh is degraded via erad in the absence of gl [161] . replacement of the cytoplasmic tail of gh with that of the human cd4 protein subverted gh degradation via erad, potentiating surface expression. current studies describe two paradigms for erad to target viral glycoproteins for degradation: ermani-mediated, which targets hiv-1 env, and edem-mediated, which can target hcv and hbv surface glycoproteins. gh47 family members share a common catalytic mannosidase homology domain of~440-residues [52] , and the three catalytic residues e330, d463, and e599 found in ermani are all conserved in these proteins [43] . nevertheless, there is little protein sequence homology beyond this domain among these proteins. unlike ermani, all three edems are er-lumenal proteins, although the signal sequence of edem1 is resistant to cleavage [162] . edem3 has two novel features including an additional protease-associated domain of unknown function and a kdel signal for er retention [45] . whether or how the coordination between the edems and ermani facilitates erad is still a convoluted issue. due to lysosomal degradation mediated by the n-terminal cytoplasmic tail, ermani is expressed at very low basal levels in cells, and its expression is not induced by upr [86] . such proteolytically driven checkpoint control of ermani expression may contribute to establish glycoprotein quality control at a baseline level, which maintains er homeostasis without activation of ire1/xbp1. however, if this basic mechanism fails to restore er homeostasis, ire1/xbp1 is induced to elevate expression of the edems, which will increase erad. unlike hcv and hbv, hiv-1 induces upr, but barely activates the ire1/xbp1 pathway, which may explain why hiv-1 env is not directly targeted by edem proteins [93] . nevertheless, these two different arms of erad do not exclude the role of the edems in ermani-mediated degradation. edems may accelerate the release of terminally misfolded glycoproteins from the cnx/crt cycle, and thereby help ermani to conduct more extensive demannosylation [163] ; and the association of edem with sel1l may further accelerate the cytosolic delivery of misfolded proteins [164] . moreover, edem1 may form a complex with ermani, which stabilizes ermani by the suppression of its proteolytic degradation [86] . discrepancies concerning the localization of ermani with various labs determining colocalization with the er, golgi, or er-golgi intermediate compartments and quality control vesicles, lends credence to both current theories that ermani is either a golgi checkpoint in quality control that will return misfolded proteins back to the er for further processing, or that it resides in quality control vesicles with glycoprotein substrates as part of the cnx/crt cycle [24, 165] . it is well established that viruses have evolved to manipulate host upr and erad to optimize their replication, whether they are 'tuning' host quality control to ensure the proper folding of their envelope glycoproteins, circumventing erad in order to prevent degradation of their viral envelope glycoproteins, or hijacking erad to dispose of host proteins. there are still many questions left to be answered, including the identities of the dislocons that each envelope glycoprotein is targeted to, the motifs or patterns that allow α1,2-mannosidases to differentiate between native and misfolded glycoproteins, why some viral proteins are disproportionately targeted (hcmv gh), and the roles that the upr and erad play in vivo during viral infections. these exciting areas merit more extensive studies. virus entry: molecular mechanisms and biomedical applications the cell biology of receptor-mediated virus entry recruitment of hiv and its receptors to dendritic cell-t cell junctions penetration of nonenveloped viruses into the cytoplasm virus and cell fusion mechanisms both e protein glycans adversely affect dengue virus infectivity but are beneficial for virion release a systematic study of the n-glycosylation sites of hiv-1 envelope protein on infectivity and antibody-mediated neutralization playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection the hepatitis c virus glycan shield and evasion of the humoral immune response comprehensive functional analysis of n-linked glycans on ebola virus gp1 tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin glycosylation affects cleavage of an h5n2 influenza virus hemagglutinin and regulates virulence importance of hemagglutinin glycosylation for the biological functions of influenza virus interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics the hiv glycan shield as a target for broadly neutralizing antibodies virus glycosylation: role in virulence and immune interactions vertebrate protein glycosylation: diversity, synthesis and function roles of n-linked glycans in the endoplasmic reticulum n-glycan-based er molecular chaperone and protein quality control system: the calnexin binding cycle malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein n-glycosylation the role of lectin-carbohydrate interactions in the regulation of er-associated protein degradation the long road to destruction mammalian er mannosidase i resides in quality control vesicles, where it encounters its glycoprotein substrates the specificity of the yeast and human class i er alpha 1,2-mannosidases involved in er quality control is not as strict previously reported endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to man6-5glcnac2 the unfolded protein response: integrating stress signals through the stress sensor ire1alpha endoplasmic reticulum stress sensing in the unfolded protein response dynamic interaction of bip and er stress transducers in the unfolded-protein response the unfolded protein response: from stress pathway to homeostatic regulation unfolded proteins are ire1-activating ligands that directly induce the unfolded protein response er-stress-induced transcriptional regulation increases protein synthesis leading to cell death endoplasmic reticulum-associated degradation regulation of endoplasmic reticulum-associated protein degradation (erad) by ubiquitin. cells how early studies on secreted and membrane protein quality control gave rise to the er associated degradation (erad) pathway: the early history of erad sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase doa10/teb4. elife 2013, 2, e00953 when worlds collide: ip(3) receptors and the erad pathway regulation of diacylglycerol acyltransferase 2 protein stability by gp78-associated endoplasmicreticulum-associated degradation ubiquitin-proteasome system mediates heme oxygenase-1 degradation through endoplasmic reticulum-associated degradation pathway selective inhibition of endoplasmic reticulum-associated degradation rescues deltaf508-cystic fibrosis transmembrane regulator and suppresses interleukin-8 levels: therapeutic implications the delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology flagging and docking: dual roles for n-glycans in protein quality control and cellular proteostasis family 47 alpha-mannosidases in n-glycan processing edem2 initiates mammalian glycoprotein erad by catalyzing the first mannose trimming step edem3, a soluble edem homolog, enhances glycoprotein endoplasmic reticulumassociated degradation and mannose trimming in vitro mannose trimming property of human er alpha-1,2 mannosidase i ergic-53 and traffic in the secretory pathway endoplasmic reticulum (er) mannosidase i is compartmentalized and required for n-glycan trimming to man5-6glcnac2 in glycoprotein er-associated degradation stimulation of erad of misfolded null hong kong alpha1-antitrypsin by golgi alpha1,2-mannosidases mannosidase ia is in quality control vesicles and participates in glycoprotein targeting to erad edem1 regulates er-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation glycoprotein folding and the role of edem1, edem2 and edem3 in degradation of folding-defective glycoproteins os-9 and grp94 deliver mutant alpha1-antitrypsin to the hrd1-sel1l ubiquitin ligase complex for erad the mrh domain suggests a shared ancestry for the mannose 6-phosphate receptors and other n-glycan-recognising proteins in vitro analysis of hrd1p-mediated retrotranslocation of its multispanning membrane substrate 3-hydroxy-3-methylglutaryl (hmg)-coa reductase derlin-1 and ubxd8 are engaged in dislocation and degradation of lipidated apob-100 at lipid droplets structure of the native sec61 protein-conducting channel proteasome 19s rp binding to the sec61 channel plays a key role in erad a membrane protein required for dislocation of misfolded proteins from the er a membrane protein complex mediates retro-translocation from the er lumen into the cytosol derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant alpha-1 antitrypsin from the endoplasmic reticulum protein quality control in the er: balancing the ubiquitin checkbook the ubiquitylation machinery of the endoplasmic reticulum role of 26s proteasome and hrd genes in the degradation of 3-hydroxy-3-methylglutaryl-coa reductase, an integral endoplasmic reticulum membrane protein autoubiquitination of the hrd1 ligase triggers protein retrotranslocation in erad key steps in erad of luminal er proteins reconstituted with purified components selective ubiquitylation of p21 and cdt1 by ubch8 and ube2g ubiquitin-conjugating enzymes via the crl4cdt2 ubiquitin ligase complex endoplasmic reticulum degradation requires lumen to cytosol signaling. transmembrane control of hrd1p by hrd3p association of the sel1l protein transmembrane domain with hrd1 ubiquitin ligase regulates erad-l usa1 functions as a scaffold of the hrd-ubiquitin ligase the ubiquitin-domain protein herp forms a complex with components of the endoplasmic reticulum associated degradation pathway membrane topology of the yeast endoplasmic reticulum-localized ubiquitin ligase doa10 and comparison with its human ortholog teb4 (march-iv) ube2j2 ubiquitinates hydroxylated amino acids on er-associated degradation substrates hrd1 and ube2j1 target misfolded mhc class i heavy chains for endoplasmic reticulum-associated degradation distinct ubiquitin-ligase complexes define convergent pathways for the degradation of er proteins the yeast erad-c ubiquitin ligase doa10 recognizes an intramembrane degron htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast the saccharomyces cerevisiae processing alpha 1,2-mannosidase is localized in the endoplasmic reticulum, independently of known retrieval motifs segregation and rapid turnover of edem1 by an autophagy-like mechanism modulates standard erad and folding activities human endoplasmic reticulum mannosidase i is subject to regulated proteolysis disposal of cargo and of erad regulators from the mammalian er two endoplasmic reticulum-associated degradation (erad) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome erad(i) and autophagy/lysosome erad(ii) a novel er alpha-mannosidase-like protein accelerates er-associated degradation a novel stress-induced edem variant regulating endoplasmic reticulum-associated glycoprotein degradation glycan regulation of er-associated degradation through compartmentalization the mammalian upr boosts glycoprotein erad by suppressing the proteolytic downregulation of er mannosidase i how viruses use the endoplasmic reticulum for entry, replication, and assembly the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis unfolded protein response in hepatitis c virus infection modulation of the unfolded protein response by the human hepatitis b virus coronavirus infection, er stress, apoptosis and innate immunity differential unfolded protein response during chikungunya and sindbis virus infection: chikv nsp4 suppresses eif2alpha phosphorylation hiv infection and antiretroviral therapy lead to unfolded protein response activation signal integration via pkr the pathway of hcv ires-mediated translation initiation the lmp1 oncogene of ebv activates perk and the unfolded protein response to drive its own synthesis herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase perl and mediates eif-2alpha dephosphorylation by the gamma(1)34.5 protein the african swine fever virus dp71l protein recruits the protein phosphatase 1 catalytic subunit to dephosphorylate eif2alpha and inhibits chop induction but is dispensable for these activities during virus infection protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e2 through the eukaryotic initiation factor 2alpha kinase perk influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (ier1) stress pathway the atf6 branch of unfolded protein response and apoptosis are activated to promote african swine fever virus infection er stress, autophagy, and rna viruses short communication: activating transcription factor 4 (atf4) promotes hiv type 1 activation pb1-f2 attenuates virulence of highly pathogenic avian h5n1 influenza virus in chickens dengue virus modulates the unfolded protein response in a time-dependent manner cytomegalovirus downregulates ire1 to repress the unfolded protein response the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor the hcmn gene products us2 and us11 target mhc class i molecules for degradation in the cytosol plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by hcmv us2 in cooperation with ul141 cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins signal peptide peptidase is required for dislocation from the endoplasmic reticulum the trc8 e3 ligase ubiquitinates mhc class i molecules before dislocation from the er tmem129 is a derlin-1 associated erad e3 ligase essential for virus-induced degradation of mhc-i identifying the erad ubiquitin e3 ligases for viral and cellular targeting of mhc class i sel1l nucleates a protein complex required for dislocation of misfolded glycoproteins mhc class i ubiquitination by a viral phd/lap finger protein role of cd4 receptor down-regulation during hiv-1 infection mechanisms of cd4 downregulation by the nef and vpu proteins of primate immunodeficiency viruses a novel human wd protein, h-beta trcp, that interacts with hiv-1 vpu connects cd4 to the er degradation pathway through an f-box motif hiv-1 vpu -an ion channel in search of a job multilayered mechanism of cd4 downregulation by hiv-1 vpu involving distinct er retention and erad targeting steps retroviral rem protein requires processing by signal peptidase and retrotranslocation for nuclear function cytoplasmic localization of the orf2 protein of hepatitis e virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum the polyomaviridae: contributions of virus structure to our understanding of virus receptors and infectious entry murine polyomavirus requires the endoplasmic reticulum protein derlin-2 to initiate infection simian virus 40 depends on er protein folding and quality control factors for entry into host cells coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication how viruses hijack the erad tuning machinery electron tomography analysis of envelope glycoprotein trimers on hiv and simian immunodeficiency virus virions distribution and three-dimensional structure of aids virus envelope spikes retrovirus envelope protein complex structure in situ studied by cryo-electron tomography three-dimensional structure of herpes simplex virus from cryo-electron tomography paramyxovirus ultrastructure and genome packaging: cryo-electron tomography of sendai virus zernike phase contrast electron microscopy of ice-embedded influenza a virus why hiv virions have low numbers of envelope spikes: implications for vaccine development model for intracellular folding of the human immunodeficiency virus type 1 gp120 secretion of a truncated form of the human immunodeficiency virus type 1 envelope glycoprotein biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160 a novel hiv-1 restriction factor that is biologically distinct from apobec3 cytidine deaminases in a human t cell line cem the mitochondrial translocator protein, tspo, inhibits hiv-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway the mitochondrion in apoptosis: how pandora's box opens tspo interacts with vdac1 and triggers a ros-mediated inhibition of mitochondrial quality control elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation ermani (endoplasmic reticulum class i alphamannosidase) is required for hiv-1 envelope glycoprotein degradation via endoplasmic reticulum-associated protein degradation pathway identification, expression, and characterization of a cdna encoding human endoplasmic reticulum mannosidase i, the enzyme that catalyzes the first mannose trimming step in mammalian asn-linked oligosaccharide biosynthesis cloning and expression of a specific human alpha 1,2-mannosidase that trims man(9)glcnac(2) to man(8)glcnac(2) isomer b during n-glycan biosynthesis structural basis for catalysis and inhibition of n-glycan processing class i alpha 1,2-mannosidases role of the cysteine residues in the alpha1,2-mannosidase involved in n-glycan biosynthesis in saccharomyces cerevisiae. the conserved cys340 and cys385 residues form an essential disulfide bond mutations in the alpha 1,2-mannosidase gene, man1b1, cause autosomal-recessive intellectual disability somatic overgrowth associated with homozygous mutations in both man1b! and sec23a. cold spring harb. mol. case stud a golgi-localized mannosidase (man1b1) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control hiv-1 vpr increases env expression by preventing env from endoplasmic reticulum-associated protein degradation (erad) activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles no evidence of the unfolded protein response in patients with chronic hepatitis c virus infection human cytomegalovirus glycoproteins human cytomegalovirus penetrates host cells by ph-independent fusion at the cell surface human cytomegalovirus gh/gl/go promotes the fusion step of entry into all cell types, whereas gh/gl/ul128-131 broadens virus tropism through a distinct mechanism a novel herpes simplex virus glycoprotein, gl, forms a complex with glycoprotein h (gh) and affects normal folding and surface expression of gh human cytomegalovirus gh stability and trafficking are regulated by er-associated degradation and transmembrane architecture characterization of early edem1 protein maturation events and their functional implications role of edem in the release of misfolded glycoproteins from the calnexin cycle edem1 recognition and delivery of misfolded proteins to the sel1l-containing erad complex golgi localization of ermani defines spatial separation of the mammalian glycoprotein quality control system the authors declare no conflict of interest. key: cord-253997-imwjoecx authors: lotter-stark, hester c.t.; rybicki, edward p.; chikwamba, rachel k. title: plant made anti-hiv microbicides—a field of opportunity date: 2012-12-31 journal: biotechnology advances doi: 10.1016/j.biotechadv.2012.06.002 sha: doc_id: 253997 cord_uid: imwjoecx abstract hiv remains a significant global burden and without an effective vaccine, it is crucial to develop microbicides to halt the initial transmission of the virus. several microbicides have been researched with various levels of success. amongst these, the broadly neutralising antibodies and peptide lectins are promising in that they can immediately act on the virus and have proven efficacious in in vitro and in vivo protection studies. for the purpose of development and access by the relevant population groups, it is crucial that these microbicides be produced at low cost. for the promising protein and peptide candidate molecules, it appears that current production systems are overburdened and expensive to establish and maintain. with recent developments in vector systems for protein expression coupled with downstream protein purification technologies, plants are rapidly gaining credibility as alternative production systems. here we evaluate the advances made in host and vector system development for plant expression as well as the progress made in expressing hiv neutralising antibodies and peptide lectins using plant-based platforms. recent reports show that the number of new hiv infections has declined by 21% since the peak of the disease almost 15 years ago (unaids, 2011) . however, worldwide more than 34 million people are still living with the disease (unaids, 2011) . furthermore, in sub-saharan africa, the most heavily hiv affected region, it is estimated that only 6.6% of the population have been tested for hiv in 2009 (unaids, 2010 . thus, globally there still exists a huge reservoir of hiv-infected people with the potential to infect millions more. despite commendable research efforts over nearly 30 years, a protective hiv vaccine is still not available. thus, it has become crucial to develop other strategies for disease prevention, such as microbicides that would effectively block the initial transmission of the virus. women comprise 50% of the hiv infected population and are high risk candidates who are in many cases unable to protect themselves due to domestic violence, cultural and social habits, lack of education and financial security (unaids, 2010) . due to these difficult socioeconomic conditions a successful microbicide should further lend itself to formulations that can be applied topically or orally in order for women to self-manage the use of it (moscicki, 2008) . the microbicide development field received a boost with the progress made in caprisa004 studies where it was demonstrated that a microbicide gel containing 1% tenofovir, a reverse transcriptase inhibitor, could prevent the risk of hiv infection by 38% (karim et al., 2010) . anti-hiv microbicide candidates include surfactants, vaginal milieu protectors, viral entry inhibitors, reverse transcriptase inhibitors and other agents with an unknown mode of action (cutler and justman, 2008) . surfactants and vaginal milieu protectors were the first generation candidate microbicides (cutler and justman, 2008) . these were broad acting and failed to produce effective hiv inhibition, even enhancing infection in some instances (van damme et al., 2002) . of these, n-9 was the first surfactant that was tested in a clinical trial (garg et al., 2009) . although no adverse effects were reported for n-9 in preclinical and phase i clinical trials, genital ulcers, irritation, inflammation and subsequent higher hiv risk were reported from phase iii trials (garg et al., 2009; moscicki, 2008) . further surfactant development as microbicides faded under the risk of vaginal damage and inconclusive clinical trials. vaginal milieu protectors stabilise the low mucosal ph. in this class of microbicides acidform (amphora, instead inc., dallas, tx, usa) and buffergel (carbopol 974p, reprotect, baltimore, md, usa) have been evaluated extensively, displayed good contraceptive properties and were shown to be well tolerated in human subjects . whilst in vitro anti-hiv activity has been reported for acidform, it has only been subjected to safety and acceptability pre-clinical studies (reviewed by cutler and justman, 2008 ; http:// www.insteadsciences.com/amphora.htm#results). buffergel failed to show reduction of hiv infectivity when compared to the placebo gel in a study that evaluated its effectiveness in reduction of hiv incidence in a high risk study group (karim et al., 2011) . it is thus likely that these vaginal milieu protectors will not be effective in preventing hiv transmission in single formulations and will probably be used in combination with other antiviral entities. in fact, carbopol 974p is being used as the polymer base to formulate gels for the application of reverse transcriptase inhibitors such as tenofovir and uc781 (garg et al., 2010) . other strategies to maintain a healthy mucosal environment include the restoration of the microflora population by products such as lactin v and mucocept from osel, santa clara, ca, usa (moscicki, 2008) . entry inhibitors are a group of microbicides that interact either with viral or host cell structures to prevent attachment, fusion and entry. the first type of entry inhibitors was chemical molecules such as anionic polymers that establish an interaction with the virus based on surface charges (reviewed by cutler and justman, 2008) . most of these compounds failed to show significant protection in clinical trials, were associated with unwanted side effects and in some instances associated with an enhanced hiv infection risk (pironne et al., 2011) . subsequent microbicide development focused on more potent specialised molecules such as reverse transcriptase inhibitors, ccr5 antagonists and viral entry inhibitors. reverse transcriptase inhibitors target viral enzymes (campiani et al., 2002; cihlar, 2006) , ccr5 antagonists compete with the virus for host cell co-receptors (baba, 2006; schols, 2006 schols, , 2011 , whilst entry inhibitors bind to viral envelope components to prevent entry of the virus into the cell (balzarini, 2006; botos and wlodawer, 2005) . in the later group, antibody and peptide lectins represent a class of molecules that are in advanced stages of development as microbicides. to avoid repeating past failures, newly researched microbicide candidate molecules are currently undergoing strict evaluation in several preclinical test studies using specialised models and formulations (buckheit et al.; , 2010; doncel and clark, 2010; mcgowan, 2009) . rigour is necessary in preclinical testing because clinical trials are complex and expensive (minces and mcgowan, 2010) and the largest market segment for hiv prophylaxis resides in resource limited countries that can ill afford the development costs. because of these cost hurdles, it is crucial that microbicides be produced with minimum upfront capital outlay so as to facilitate development, testing and ultimate availability of the final product. plants are emerging as cost friendly alternative production systems for a variety of pharmaceuticals. numerous therapeutic proteins have been produced in plant systems (giddings et al., 2000; ma et al., 2003) . protein based microbicides, namely, neutralising antibodies and peptide lectins-lend themselves to production in plants (de muynck et al., 2010; matoba et al., 2010; o'keefe et al., 2009; sexton et al., 2006) . although these microbicides have been extensively studied in terms of their structure and mode of action, their production in plant host expression systems has not been audited to date. in this study, we evaluate the progress made in the expression and development of peptide and antibody candidate microbicides in plants. over the past two decades plants have been extensively investigated as alternative production systems for pharmaceutical proteins. even with careful consideration of existing production systems, plants provide several attractive features that are equivalent or more beneficial (mett et al., 2008; twyman et al., 2003 twyman et al., , 2005 . like mammalian and yeast cells, plants possess the cellular machinery which enables them to perform the post translational modifications essential for maturation and sometimes function of proteins. unlike mammalian fermentation systems, plants are not at risk of being contaminated with human pathogens. furthermore compared to mammalian and yeast systems, plant production systems are more easily scaled up; plants can either be propagated in large numbers in designated land plots or in contained greenhouses. maintenance of plants in soil, hydroponic or cell culture is simple and cheap compared to the complex growth media and requirements of yeast and mammalian cell systems (knäblein, 2005) . furthermore, plants provide a huge biomass in the form of green leafy tissue or as the numerous seeds of certain crops. the latter provides a further advantage of stable storage over longer time periods and high protein content that can be exploited for recombinant protein production (cunha et al., 2011a, b; lau and sun, 2009) . whilst costs of downstream processing remain as high as that required for purifying proteins made from conventional systems, the burden can be alleviated by maximising production yields and utilising innovative purification strategies to improve product recovery (paul and ma, 2011) . thus, for plant production systems the upfront investment required for infrastructure is lower, which potentially lowers the barriers to entry by more players or especially players in developing countries. considering the time and effort invested over twenty-years, relatively few plant made pharmaceuticals (pmps) are currently marketed (faye and gomord, 2010) . the main reasons for this are that the production levels in plants were often too low to be commercially viable, and regulatory approval of pmps was perceived as being difficult to obtain. recombinant proteins were initially expressed in transgenic plants through stable nuclear transformation using agrobacterium-mediated delivery of binary vectors or alternative methods such as biolistic introduction of dna into plant cells (banta and montenegro, 2008; vianna et al., 2011) . these are lengthy, labour intensive processes which mostly generated progeny with variable and, for the most part, low target protein accumulation levels. expression of multiple component proteins such as antibodies required numerous crossings and screening of plants in breeding programmes. transient expression with binary vectors was mainly used as a rapid screen to validate the expression potential of a gene and did not generally result in high protein accumulation (gleba et al., 2005) . whilst viral vectors were useful to produce proteins in plants, these were limited by the insert size or the fidelity of the transcript (pogue et al., 2002) . furthermore, systemic spread of the virus sometimes resulted in loss of the foreign gene insert and raised concerns of containment. however developments in this arena have resulted in significantly improved state-of-the-art technologies for plant based protein expression. another perceived limitation for pmps in clinical applications is the variation in plant glycan structure compared to that of humans (gomord et al., 2005) . this shortcoming is also typical of other systems such as yeast and insect cells (mett et al., 2008) . thus far there has been no clinically significant evidence that plant-specific glycans are inappropriately immunogenic in humans (van der veen et al., 1997) . another development that has done a lot to allay such fears is the recent fda approval of a plant made therapeutic for gaucher disease; glucocerebrosidase (also known as taliglucerase alfa or elelyso), which is produced in carrot cells, has proven to be not only well tolerated but perhaps even more efficacious and stable-in other words, to be a "biobetter" (aviezer et al., 2009 ). however, the pressure remains on plant production systems to deliver therapeutic proteins with a humanised glycan profile. several advances in expression vector systems and plant hosts have addressed some of these limitations to a large extent. to increase protein yield, second generation agrobacterium binary vectors have incorporated various elements to enhance transcription and translation (veluthambi et al., 2003) . these improved vectors, used in conjunction with transient infiltration, have improved protein expression levels in plants. for instance, transient expression of a human optimised hpv-16 l1 capsid protein gene using a specialised binary ptra vector resulted in a yield of more than 0.5 g/kg of fresh leaf weight (17% total soluble protein, maclean et al., 2007) . significant lower levels were obtained when the same protein was transgenically produced in tobacco and potato resulting in the l1 protein accumulating to 0.5 and 0.2% of total soluble protein respectively (biemelt et al., 2003) . further development in vector systems has seen the merging of viral and binary vector technology to increase yields and address insert size restrictions, retention of target genes and containment issues. icon genetics gmbh (halle, germany) developed a deconstructed viral vector system initially based on tobacco mosaic virus (tmv) in which target genes and different viral vector components are carried on several pro-module vectors (marillonnet et al., 2004) . in this system the viral coat protein has been removed to eliminate systemic spread. instead agroinfiltration provides the delivery of the modules to the plant cell and limits replication to the infiltrated area. in the cell, high-level expression is facilitated by a rna dependent rna polymerase. a site-specific recombinase facilitates the assembly of the modules into a dna molecule which is transcribed and spliced into a functional transcript. the transcript moves to the cytosol where it is translated into the specified protein. alternatively target signals can be incorporated to direct proteins to specified subcellular compartments (marillonnet et al., 2004) . this system has been used to accumulate various proteins at levels over 4 g/kg plant material (bendandi et al., 2010) . one shortcoming of the initial icon system vectors was the inability to co-express more than one protein in the same spatial location (giritch et al., 2006) . this was problematic with the production of multi-component proteins such as immunoglobulins. a solution to this came by co-expression from two non-competing monopartite viral genomes such as tmv and pvx (giritch et al., 2006) . alternatively, viral vectors derived from bi-or tri-partite viral rna genomes do not seem to be competing and are able to co-function in the same area. the cowpea mosaic virus (cpmv) is a bipartite viral rna genome from which two types of vector systems were developed. in the full-length system (wild type, wt) the coding sequence for the protein of interest is fused to the c-terminus of the rna-2 polypeptide which is co-translationally released via 2a-peptide mediated cleavage . replication is facilitated by the co-expression of rna-1. the full-length version allows for local co-expression of two different proteins; however segregation of the co-expressed proteins occurs with systemic movement . a deleted cpmv version of rna-1 (hypertranslatable, ht) was developed which lacked the ability of systemic spread and was thus able to co-express more than one protein without the occurrence of segregation. moreover the deleted cpmv system obtained higher expression levels than the full-length version (sainsbury et al., , 2010 . using this system, protein expression levels exceeded 0.3 g/kg protein . several pharmaceutically important molecules have been expressed in plants using vectors based on the ssdna geminivirus bean yellow dwarf virus (beydv) (chen et al., 2011 ): huang et al. (2010 and regnard et al. (2010) have independently developed vector systems based on the dna genome of the virus. the beydv system requires only two viral components for co-expressing heteromeric proteins: these are the long intergenic region (lir) and the short intergenic region (sir) control sequences, and the single alternatively spliced gene for the replication-associated proteins rep/repa. in the system non-competing co-expression can be achieved either from two replicons encoding different proteins, or from a single replicon containing the different proteins. in nicotiana benthamiana, transient expression levels from the beydv vector were 3-to 7-fold more for egfp and hiv-1 p24 compared to levels obtained using a binary ptra agrobacterium tumefaciens vector (regnard et al., 2010) . furthermore, expression with beydv resulted in accumulation levels of 0.5 g/kg monoclonal antibody against ebola virus gp1 (mab 6d8) (huang et al., 2010) . it is anticipated that the system will be able to simultaneously produce as many as four different protein subunits. several other geminiviruses and also the multicomponent ssdna nanoviruses have been investigated for their potential as expression vectors; their further development could greatly expand the range of plant species that can be used for transient production of recombinant proteins (rybicki and martin, 2011) . in eukaryotes the n-glycan biosynthesis pathway is conserved for the endoplasmic reticulum (er) (kukuruzinska and lennon, 1998) . variations between species occur in modifications to glycan structures in processing steps after the protein exits from the er. in plants these variations depend on the protein itself, plant species and plant organ used for expression . unless the plant glycosylated form of the therapeutic protein is more attractive as is the case with the carrot cell produced glucocerebrosidase (shaaltiel et al., 2007) , it is considered more ideal if plants are able to produce therapeutic proteins that have mammal-like glycans. where glycan structure is not critical to protein function, recombinant proteins without glycan structures are desirable (rodriguez et al., 2004) . therapeutic proteins that are produced as aglycosylated forms are only feasible if the proteins need to stimulate an inflammatory response or in the case of a recombinantly produced antibody that does not require an effector function since glycan structures are often crucial for this biological function of the protein (jefferis, 2009 ). the current gel formulation of hiv neutralising antibodies and peptide lectins (tsai et al., 2003) suggests that these microbicides will most likely be applied topically. when these are exposed to the mucosal surfaces it will only have a limited interaction with the immune system and thus not cause inflammation. thus for the production of these microbicides in plants the nature of the glycan structures on these microbicides might not be as important as the yield obtained. another means to produce a protein that more closely resembles a humanised glycan profile is to restrict the recombinant protein to the er by using kdel, hdel or sekdel er retention signals (ko et al., 2003; triguero et al., 2005) . for regulatory purposes it is generally regarded as "safer" to produce a native version of the protein . however in the case of er retention signals regulatory approval might be less stringent seeing that these sequences are also found in mammalian proteins. of note is that protalix's carrot cell produced glucocerebrosidase, which has just received fda approval, was produced with a storage vacuole targeting signal (shaaltiel et al., 2007) . another argument for er retention is that for the production of some proteins, er retention is needed to increase the accumulation levels (bortesi et al., 2009; yang et al., 2005) . on the other hand er retention can result in the degradation of the protein or low stability and reduced half life of the therapeutic in vivo (ko et al., 2003; loos et al., 2010) . studies have also shown that er retention of recombinantly produced proteins is not always successfully achieved, leading to some proteins leaking from the er that are then further processed to contain complex immunogenic plant glycans (floss et al., 2008; loos et al., 2010; rademacher et al., 2008) . subsequently, improved plant hosts have been developed with the aim of humanising the glycan patterns of recombinant proteins. plants such as arabidopsis, tobacco and moss have been generated in which the plant-specific glycosylation genes have been knocked out (koprivova et al., 2003; schähs et al., 2007; strasser et al., 2009) . in these mutants, plant specific α1,3-fucose and β1,2-xylose residues are replaced by complex n-acetylglucosamine (gngn) structures. further glycan improvements are made by co-expressing mammal like glycosylation and sialylation enzymes such as β1,4galactosyltransferase (galt), n-acetylglucosaminyltransferase iii (gntiii), core α1,6-fucosyltransferase, udp-n-acetylglucosamine 2epimerase/n-acetylmannosamine kinase (gne), n-acetylneuraminic acid phosphatase synthase (nans), cmp-n-acetylneuraminic acid synthetase (cmas), cmp-n-acetylneuraminic acid transporter (cmp-neu5ac) and α2,6-sialyltransferase (st) in these plant glycosylation knock-out mutants (castilho et al., 2010 (castilho et al., , 2011 strasser et al., 2009 ). resulting proteins not only lack plant-specific glycans but also contain human glycan structures. neutralising antibodies play a very important role in the development of vaccines for passive immunisation and as viral entry inhibitors (reina et al., 2010) . they are directed against the viral envelope protein and interfere with viral docking and fusion. they thus inhibit the infectivity of the virus and also potentially facilitate viral clearance via their fc related effector functions . several hiv-1 neutralising antibodies have been isolated from hiv infected individuals (simek et al., 2009; walker et al., 2009; wu et al., 2010) . novel broad acting neutralising antibodies such as vrc01, vrc02, pg16 and pg9 were isolated which displayed a larger breadth and potency than some of the well-known neutralising antibodies (wu et al., 2010) . however the four well-known hiv-1 neutralising antibodies, namely, 2g12, 4e10, 2f5 and b12 have been well researched in terms of structure, interaction with the virus, protection in animal models and safety in clinical trials and were produced in various plant platforms. these antibodies have fared well in protecting macaques from systemic or vaginal simian/human immunodeficiency virus (shiv) challenges and are well tolerated in human subjects (armbruster et al., 2002 (armbruster et al., , 2004 hessell et al., 2010; mascola et al., 1999 mascola et al., , 2000 parren et al., 2001) . furthermore, passive administration of neutralising monoclonal antibodies (mabs) 4e10, 2f5 and 2g12 reduced viral rebound in established hiv infections (trkola et al., 2005) . three of these neutralising monoclonal antibodies (mabs)-2g12, 4e10, 2f5 are being assessed as a gel formulated microbicide in a phase i clinical trial for their safety and pharmacokinetic effects . so far vaccination attempts with these antibody epitopes have failed to produce equivalent neutralising antibodies in humans (coëffier et al., 2001; lenz et al., 2005; mcgaughey et al., 2003) . it is thus most likely therefore, that these antibodies will have to be administered passively and that a large production quantity will be required. mammalian cells are currently used for fda-approved therapeutic antibody production . given that the capacity of these traditional fermentor systems will not meet the demand, plants can be employed as alternative manufacturing platforms (knäblein, 2005) . several antibodies have been successfully produced in plant platforms (de muynck et al., 2010) . apart from yield, the glycan composition and efficacy will be important criteria for plant made manufacturing of these antibodies. therefore we evaluated the progress of plant production of these neutralising antibodies in light of these criteria. table 1 summarises the expression of these four antibodies in plant systems. it has been reported that 2g12 neutralises a and b clade hiv-1 virus entry by recognition of a manα1→2man rich epitope on the exterior face of the gp120 protein (binley et al., 2004; scanlan et al., 2002) . monoclonal antibody (mab) 2g12 can activate the complement system and display antibody dependent cellular cytotoxicity (adcc) against virus infected cells (trkola et al., 1996) . passive infusion of 2g12 combined with other neutralising antibodies including 2f5, protected macaques from a vaginal and intravenous challenge with shiv (baba et al., 2000; mascola et al., 2000) . passively infused 2g12 and 2f5 were well tolerated in human subjects in a phase-i clinical trial (armbruster et al., 2002) . clinical trials with 2g12 produced in transgenic tobacco have commenced in 2009 (paul and ma, 2011) . mab 2g12 is unique in its structure in that it naturally forms a single fab region via domain swapping between the variable regions of the light chain (v l ) and heavy chain (v h ) and between the constant region of the light chain (c l ) and heavy chain (c h 1) respectively (calarese et al., 2003; west et al., 2009 ). the 2g12 dimer has shown to be more than 50 times more efficient as the monomer in neutralising several hiv-1 strains in both in vitro (west et al., 2009) and in vivo assays (luo et al., 2010) . the production of 2g12 has been actively pursued in maize, arabidopsis and tobacco. mab 2g12 was produced in the seed endosperm of two different maize lines under control of the rice glutelin (gt-1) promoter. the antibody was produced in hi-ii maize with endoplasmic reticulum (er) retention signals and in the elite maize line m37w as a secreted form (ramessar et al., 2008) . er retained antibody accumulated to 30 μg/g not mentioned both b12 and b12-cv-n fusion were able to bind gp120. in a virus neutralisation assay the sexton et al. (2009) in the t1 generation and 60 μg/g in the t3 generation whilst the secreted form reached 100 μg/g. identification of the glycan structures showed that the majority of the er retained antibodies contained oligo-mannose type glycans (omt). however a few immunoglobulins contained glycans of the vacuolar type, indicating that er retention was not completely successful. different glycoforms were detected for the secreted 2g12 with the majority being single n-acetylglucosamine (glcnac) residues and the rest containing complex type fucose and xylose glycans with a small number also containing omt type glycans. the efficacy of the maize produced antibody was compared with the chinese hamster ovary cells (cho)-produced 2g12 derivative. both the er retained and secreted 2g12 had a similar antigen binding ability as the cho produced 2g12. however in an hiv neutralisation assay, the er retained and secreted forms were four and threefold more effective than the cho produced equivalent respectively. the increased potency was attributed to the dimerisation and aggregation of the antibody. in arabidopsis, 2g12 was expressed in both the leaves (schähs et al., 2007) and seeds (loos et al., 2010 ) of a knockout line (δxt/ft), that lacked the ability to generate immunogenic plant-specific β 1,2-xylose and α1,3-fucose glycans (strasser et al., 2004) . in leaves, expression was driven by the 35s camv promoter without any er retention signals. the antibody levels varied between 0.05 and 0.2% tsp in both the wildtype (wt) and δxt/ft line (schähs et al., 2007) . the glycans on the antibody that was produced in the δxt/ft line were mainly terminal n-acetylglucosamine (gn) residues that lacked plant-specific β 1,2-xylose and α1,3-fucose residues. a small population of the antibody molecules produced contained omt residues indicating that the processing of all antibodies was incomplete. notably, the binding capacity of the δxt/ft produced 2g12 antibody was similar to the cho produced 2g12. for seed expression in arabidopsis δxt/ft, expression of 2g12 was driven by the β-phaseolin promoter (loos et al., 2010) . the antibody was expressed with and without er retention signals. there was no significant difference in accumulation levels between secreted and er retained antibody. expression levels peaked around 3.6 μg/mg. the n-glycan profile of the purified antibodies revealed that the secreted antibody of the wild type line contained complex n-glycans containing n-acetylglucosamine-xylose-fucose (gngnxf) residues whilst the 2g12 produced in the mutant line contained a homogenous n-glycan structure consisting of n-acetylglucosamine (gngn). the majority of the er retrieved antibody of the wild type line contained oligo-mannosidic n-glycans, with a small amount of antibody carrying gngnxf. the efficacy of the seed produced 2g12 in an hiv neutralisation assay was slightly inferior to the cho derivative. in n. benthamiana leaves, 2g12 was transiently expressed using the full length and the deleted rna-2 (ht) version of the cpmv vector sainsbury et al., 2010) . the antibody was expressed in both systems with and without er retention signals. overall higher antibody accumulation was obtained by using the deleted cpmv vector and er retention signals. levels of 325 mg/kg were reported. the glycan analysis of the antibodies showed that er retained forms consisted mainly of oligo-mannose type structures (omt) with a few containing more complex glycans. secreted antibodies contained complex gngnxf, n-acetylglucosamine-mannose-xylose-fucose (gnmxf) with a few omt also present. in vitro evaluation of the binding ability and neutralisation efficacy of the n. benthamiana produced antibody showed that it was equal to the mammalian cell derived 2g12. to further humanise the glycan structures on 2g12, strasser et al. (2009) produced 2g12 in a n. benthamiana δxt/xt galt + mutant line. this n. benthamiana line does not produce plant-specific xylose and fucose glycans but produces partially humanised glycans via the activity of a highly active human derived β1,4-galactosyltransferase. although no mention was made of the accumulation levels, the 2g12 antibody produced in this system was fully galactosylated and was more effective in neutralising hiv-1 than the cho produced version. mab 2f5 displays a broader neutralisation activity than 2g12, inhibiting hiv isolates from clades a, b, d and e (binley et al., 2004) . it docks onto to the core epitope eldkwa on the lipid embedded membrane proximal exterior region (mper) of gp41 and potentially interferes with the fusion step of the virus (binley et al., 2004; de rosny et al., 2004; franquelim et al., 2011; muster et al., 1993) . on its own and in combination with other antibodies including 2g12 and 4e10, 2f5 displayed the ability to protect against an intravenous, vaginal and oral challenge of shiv in macaques (baba et al., 2000; hessell et al., 2010; mascola et al., 1999 mascola et al., , 2000 . furthermore passive administration of this antibody did not seem to cause immune responses or other adverse effects in hiv infected human participants (armbruster et al., 2004) . production of 2f5 was explored in nicotiana species. the heavy chain (hc) and light chain (lc) were expressed with sekdel retention sequences in tandem under control of the 35s camv promoter in nicotiana tabacum l. cv bright yellow (by-2) cell cultures (sack et al., 2007) . accumulation of 2f5 reached a maximum of 2.9 mg/kg fresh weight and was further enriched by protein-a purification to 6.44 mg/kg wet cell weight. no degradation products were observed following purification, however minor impurities were detected. n-glycans were expected to be of the omt, but this was not confirmed by analyses. fc region binding between the by-2 and cho produced 2f5 was equivalent. however, the antigen binding capacity of cho produced 2f5 (97%) was slightly superior to the plant derived 2f5 (89%). in hiv neutralisation studies the by-2 produced antibody was threefold less efficient than the cho produced counterpart. this lower potency was attributed either to the presence of impurities, the added sekdel motif or different glycan structures that could have interfered with the antibody access to the epitope. to further enhance the accumulation of 2f5 in tobacco, the antibody was expressed as er retained elastin-like polypeptide (elp) fusions (floss et al., 2008) . the elp peptide has been used to facilitate accumulation of proteins in green leaf tissue (patel et al., 2007) . four transgenes; hc unfused, hc-elp fused, lc unfused and lc-elp fused were introduced in n. tabacum cv. samsun nn. plants were subsequently crossed resulting in combinations with neither gene carrying the fusion or both hc and lc carrying elp fusions or either the hc or lc fused to elp. prior to crossing the transgenic lines, it was observed that the presence of elp increased the accumulation of the chains with the lc accumulating to higher levels than the hc. in the crossed lines, the lc-elp fusion facilitated a higher accumulation of the unfused hc as well. accumulated total soluble protein (tsp) levels reached 0.3% for the lcelp-hc, 0.2% hcelp-lc, 0.6% for hcelp-lcelp and 0.1% for hclc. the elp fusion eased the purification process of the plant-produced antibodies and did not interfere with the assembly of the antibody. the glycans of the plant-produced 2f5 were mainly oligo-mannose type (omt) with lesser amounts of complex glycans consisting of n-acetylglucosamine (gngn), nacetylglucosamine-xylose (gngnx), galactosyl-n-acetylglucosamine-xylose (agnx), n-acetylglucosamine-mannose-xylose (gnmx) and n-acetylglucosamine-xylose-fucose (gngnxf) moieties. 2f5 variants were all similar to the cho produced 2f5 in their antigen binding capacity. mab 4e10 is one of the most broadly neutralising antibodies that are active against several viral isolates of different clades including clade c, which is the most prevalent clade in the heavily affected sub-saharan africa region (binley et al., 2004; walker et al., 2009) . both 4e10 and vrc01 were able to neutralise over 90% of the key hiv subtypes (walker et al., 2009; wu et al., 2010) . although vrc01 is more potent, it uses a different mode of action with the virus than 4e10. vrc01 interacts with the envelope in a way that resembles the cd41-gp120 interaction (li et al., 2011) . the 4e10 epitope interaction is also somewhat complex; the antibody recognises a linear epitope adjacent to the 2f5 epitope on the membrane proximal exterior region (mper) and interacts with the lipids on the cell membrane (franquelim et al., 2011; zwick et al., 2001) . whether lipid binding is involved in the broadly neutralising ability of 4e10 is still debatable (scherer et al., 2010; xu et al., 2010) . thus both antibodies can be used in combination against several hiv isolates. in a phase i clinical trial, it was demonstrated that 4e10 can be safely administered to hiv infected participants alone or combination with 2f5 and 2g12 (armbruster et al., 2004) . when 4e10 was administered intravenously, rhesus macaques were protected from a mucosal challenge with shiv (hessell et al., 2010) . mab 4e10 has been expressed via nuclear transformation in n. benthamiana (strasser et al., 2009) . it was produced in a wild type (wt), a glycoengineered δxt/fx mutant line and in a δxt/fx galt + line that produced an altered version of the human β1,4-galactosyltransferase. the glycans of the wt produced 4e10 contained n-acetylglucosamine-xylose-fucose (gngnxf), n-acetylglucosamine (gngn) for the xt/fx mutant and galactosylated (aa) glycans for the xt/fx galt + line. the later mab form was more potent than the other plant made forms and more efficient than the cho produced derivative in a neutralisation assay, possibly because of the galactosylated glycans that enhance the stability, half-life and functionality of the antibody. mab b12 can effect hiv neutralisation across different clades from different geographic locations (binley et al., 2004) . unlike other neutralising antibodies that are restricted to certain conformations of the virus, in vitro studies show that b12 can bind different conformations of the envelope (eggink et al., 2007; zhou et al., 2007) . this antibody has been shown to protect macaques in a vaginal challenge with shiv when administered systemically or topically (parren et al., 2001; veazy et al., 2003) . mab b12 was expressed in the milk of female mice and displayed the same hiv neutralisation ability as the cho cell derived antibody (yu et al., 2010) . in combination with cd4-igg2 (pro542), b12 potently inhibited hiv infection of cervical tissue (hu et al., 2004) . more importantly, in this combination or administered alone, mab b12 is able to stay associated with the virus that leaves the mucosal environment on migrating cells and prevents subsequent infection of target lymphocytes (hu et al., 2004; van montfort et al., 2011) . other neutralising antibodies in the study did not display this property. sexton et al. (2009) produced b12 and a b12-cv-n (cyanovirin) fusion in n. tabacum. cv-n is a cyanobacterium lectin that displays potent anti-hiv activity . plants were generated that expressed b12 hc, lc or a fusion where cv-n was fused to the b12 hc. subsequent crosses were performed to generate progeny that expressed both an unfused b12 (7.55 μg/ml) as well as b12-cv-n fusion (2.45 μg/ml). the authors demonstrated that both modules of the fusion molecule were functional and the fusion molecule to be more potent than cv-n or b12 alone in an hiv neutralisation assay. the glycan profile of the plant made proteins was not presented. lectins are proteins of non-immune origin that selectively bind to carbohydrate moieties (goldstein and hayes, 1978) . these proteins have been isolated from all life forms including bacteria, viruses, algae, mushrooms, nematodes and plants. based on plant lectin information, 12 distinct families have already been described (van damme et al., 1998) . lectins have been useful for several applications including pest resistance in crop plants (peumans and van damme, 1995) , therapeutic agents for cancer treatment (liu et al., 2009 (liu et al., , 2010 and as antiviral microbicide candidates (francois and balzarini, 2010) . the hiv envelope is heavily populated with mainly high mannose type glycans (doores et al., 2010; geyer et al., 1988) . it comes as no surprise that the majority of these anti-hiv lectins show an affinity for mannose moieties (botos and wlodawer, 2005) . by interacting with the glycan residues on the viral envelope they prevent attachment and fusion. many of these lectins have a broad range of activity against different viral clades of various serotypes and co-receptor dependability. furthermore, some have displayed the potential to inhibit viral capture and dissemination by dc-sign bearing host cells (balzarini et al., 2007; nabatov et al., 2008) . the anti-hiv peptide lectins fall into different families with different modes of interaction with mannose glycans. furthermore variations occur in their quaternary structures, efficacy level towards hiv and immune stimulatory effect of human cells (barre et al., 1996; ziółkowska and wlodawer, 2006) . anti-hiv lectins have been reviewed extensively with regards to structure and mode of binding (balzarini, 2006; francois and balzarini, 2010; ziółkowska and wlodawer, 2006) . the majority of these lectins are remarkably stable across broad ph ranges and high temperatures. this allows their manipulation in expression, purification, formulation and applications as microbicides. they represent a rich source of proteins that can be developed as anti-hiv microbicides. the first group of anti-hiv lectins was originally isolated from plants (van damme et al., 1987) . thereafter numerous others have been isolated from other organisms including prokaryotic algae, bacteria, fungi and nematodes bulgheresi et al., 2006; chiba et al., 2004; inokoshi et al., 2001; mori et al., 2005; zhao et al., 2010) . recombinant expression of lectins in plants has been applied to some extent to introduce pest resistance in valuable crops or promote rhizosphere symbiotic associations (rovenská and zemek, 2006; sreevidya et al., 2005; wang et al., 2005) . plant lectins which show anti-hiv activity have been isolated directly from their natural source such as the case of galathus nivalus agglutinin (gna) where the bulbs of snowdrop (g. nivalus) contain reasonably high levels of the lectin (van damme et al., 1987) . on the other hand, hiv inhibiting lectins such as those from prokaryotes and some plants are produced in low quantities and it is thus not feasible for direct isolation from the source (koshte et al., 1990; . lectins isolated from cyanobacteria, red algae and fungae displayed generally higher potency than most plant lectins and have been extensively researched as topical anti-hiv microbicides. since the proteins seem to occur in low quantities in their native host, recombinant production in alternative systems, such as plants has been explored. as lectins occur naturally in plants, it seems that production of recombinant lectins from other sources will not be problematic. however, it has come to light that plants produce two types of lectins, classical and nucleocytoplasmic (lannoo and van damme, 2010; van damme et al., 2004) . classical lectins reside in storage organelles whilst the nucleocytoplasmic lectins occur mainly in the cytoplasm. generally classical lectins are synthesised with signal peptides, are produced in abundance and serve a defence and storage purpose for the plant. nucleocytoplasmic lectins are produced without any signal peptides in small quantities and are thought to play a role in regulatory processes in the plant cell (lannoo and van damme, 2010) . it is thus evident that in a plant cell there is a clear distinction in signalling and abundance of different lectins with different roles. thus, heterologous production of lectins in plants could have an effect on the viability of the plant cell. the native roles of many of these hiv neutralising lectins have not been resolved and although the majority mainly bind to mannose residues on the viral envelope, one cannot rule out the possibility that other ligands may exist in the plant cell environment which may affect their expression, accumulation or recovery from the plant matrix. thus subcellular targeting may play an important role in the resolution of the optimal compartment for high yield lectin accumulation that is not detrimental for plant cells during heterologous expression. here the progress that has been made with heterologous expression of anti-hiv lectins in plants is briefly reviewed. table 2 highlights the major findings. cyanovirin (cv-n) is an 11 kda protein that was isolated from the blue-green algae nostoc ellipsosporum with an ec 50 value of 0.1 nm . cv-n inhibited in vitro fusion of hiv-1 with target cells as well as subsequent viral spread between virus infected and uninfected cells. it displayed antiviral activity against primary and laboratory modified hiv strains of several clades including m, t and dual tropic viruses . furthermore cv-n inhibits gp120 binding to ccr5 or cxcr4 co-receptor dependent strains (dey et al., 2000; mori and boyd, 2001) . antiviral activity of cv-n against hepatitis c (helle et al., 2006) , ebola (barrientos et al., 2003; smee et al., 2008) , shiv , measles and herpes virus 6 (dey et al., 2000) has also been reported. cv-n has very low homology to other known protein sequences, but contains a sequence motif that is typical to the cv-n type lectin family gustafson et al., 1997; percudani et al., 2005; van damme et al., 1998) . in solution, cv-n exists as a monomer or dimer depending on ph and temperature conditions (barrientos and gronenborn, 2002; barrientos et al., 2004) . cv-n interacts with terminal mannose residues of the oligomannose glycan structures of gp120 (bewley and otero-quintero, 2001; shenoy et al., 2001) . the monomer contains two carbohydrate binding domains with different affinities to di-and trimannose respectively (bewley and otero-quintero, 2001) . although anti-hiv activity has been reported for both monomeric and dimeric forms, it appears that the potency of cv-n depends more on the formation of multisite interactions with glycan residues rather than the affinity and presence of each binding domain (barrientos et al., 2004; fromme et al., 2007; kelley et al., 2002) . in vitro test with host cells and cv-n displayed no or little loss of cell viability as a result of host cell exposure to cv-n. furthermore, in vivo studies with gel-formulated cv-n caused no adverse effects in the test animals (tsai et al., 2003) . however, more extensive tests showed that cv-n induced the production of chemokines and cytokines and stimulated cell proliferation (huskens et al., 2008) . this cytotoxicity was however not linked to its carbohydrate-binding property. thus with further development such as mutations or pegylations (zappe et al., 2008) that could potentially reduce the cytotoxicity of cv-n, the lectin might still be considered as a potential microbicide. cv-n has been used in ground-breaking microbicide development work to pave the way for future development of lectins as viable microbicide molecules. it displayed the potential for lectins to be used as a gel formulated microbicide to protect against vaginal and rectal challenged with hiv (tsai et al., 2003) . cv-n has been recombinantly produced in commercial bacteria such as streptococcis gordonni (giomarelli et al., 2002; pozzi et al., 2001) and lactobacillus jensinii (liu et al., 2006) . cv-n displayed on the surface of s. gordonni was able to capture hiv virions, whilst if secreted from the bacteria it could bind to gp120. recombinant l. jensinii were able to colonise the vagina in mice and secrete full length cv-n. l. jensinii produced cv-n was able to inhibit ccr5 hiv in vitro in nanomolar concentrations. several fusions of cv-n have been explored with different applications in mind: for example, to form high potency chimaeras cv-n has been fused to the broadly neutralising b12 antibody (sexton et al., 2009) and to the linear 12pi peptide (mcfadden et al., 2007) . both partners in the fusions were active and the new chimaeras displayed similar stability and higher antiviral activity. cv-n was also fused to a pseudomonas exotoxin a . the chimeric protein potently eliminated hiv infected cells that expressed gp120 on their surface. the recombinant expression of cv-n in alternative systems has recently been reviewed by xiong et al. (2010) . in brief, initial production and purification in escherichia coli was not optimal, resulting in low levels of cv-n accumulation. further optimisations resulted in high accumulation levels but consisted of a heterogeneous cv-n population of intact, truncated and signal peptide containing cv-n forms. chaperone fusions of cv-n resulted in homogenous cv-n that accumulated to 100 mg/l. expression of cv-n was also pursued in yeast, which only resulted in low yields of non-functional protein. sexton et al. (2006) showed that it is feasible to produce cv-n in tobacco plants as well as hydroponic cultures. the cv-n gene was transformed into n. tabacum and expressed under the 35s camv constitutive promoter and er targeting signal peptide. cv-n accumulated to 130 ng/mg fresh leaf weight (or 0.85% tsp). hydroponic cultures derived from the transgenic plants secreted cv-n at 0.4 μg/ml. crude cv-n extracts from tobacco were able to inhibit hiv infection of tzm-bl cells comparable to that of purified e. coli derived cv-n. cv-n was also produced in tobacco as a fusion with the monoclonal antibody b12 (sexton et al., 2009 ). the fusion accumulated at 2.45 μg/ml and was more active than cv-n or b12 alone. griffithsin (grft) was isolated from the red alga griffithsia (mori et al., 2005) . its 121 amino acid sequence contains an unknown amino acid at position 31 and codes for a 12.7 kda protein that is sequence unrelated to any known protein. in its folded state, the monomer displays the β-prism-i motif found in other lectins such as jacalin, whilst the dimer is formed by a unique domain swopping between two grft molecules that are not typically found in this lectin family . grft displayed broad activity against corona viruses and hiv (o'keefe et al., 2010) . grft has shown antiviral activity against hiv clades a, b and c that are prevalent in sub-saharan africa, india and the west. it is active against both clinical and laboratory adapted t and m tropic hiv-isolates and inhibits both ccr5 and crcx4 orientated strains. of all the prokaryotic anti-hiv lectins, grft is thus far one of the most potent and promising lectins for microbicide development with an ec 50 value as low as 0.04 nm (mori et al., 2005) . the potency of this lectin is attributed to its remarkable dimeric structure that contains six mannose binding sites that are most likely spaced for optimal interaction and subsequent cross linking of the glycans of the gp120 coat protein (moulaei et al., 2010; . any successful topically applied microbicide must ultimately be able to function in the mucosal environment. given that the infection rate of hiv is quite rapid, the microbicide should remain stable and efficacious to neutralise hiv immediately on contact. its presence furthermore should not compromise tissue viability or initiate an inflammatory response. in the light of these criteria, preclinical test shows that grft is a good microbicide candidate. grft is virucidal upon contact with the virus and remains stable over several hours prior to or after application (emau et al., 2007) . grft is stable and functional in cervical lavage fluid over a wide ph range. furthermore grft was not cytotoxic to human and primate cell lines, does not initiate an inflammatory response and did not cause adverse effects in a rabbit vaginal irritation model (emau et al., 2007; o'keefe et al., 2009) . it is likely that an effective anti-hiv therapeutic will consist of more than one microbicide to limit the risk of viral resistance. it is thus important that candidate microbicides should be compatible with other microbicides without compromising efficacy and safety of the component molecules. grft was tested in combination with tenofovir (nucleotide reverse transcriptase inhibitor), maraviroc (ccr5 hiv co-receptor inhibitor) and enfuvirtide (a gp41 fusion inhibitor) against calde b and c virus isolates to evaluate possible synergistic effects of the lectin in combination with other microbicides (férir et al., 2011) . when grft was combined with other microbicides the potency of the combination was higher than that of the individual molecules. recombinant production of grft was initially pursued in e. coli. although the lectin accumulated to 819 mg/l, 33% was irreversibly lost to inclusion bodies . whilst the expression of grft in e. coli illustrated the feasibility of an alternative production system of functional grft, it remains an expensive production platform with high optimisation and maintenance demands. o'keefe et al. (2009) used a tmv based vector system for the transient production of grft in the cytosol of n. benthamiana leaves. grft accumulated to more than 1 g/kg fresh weight and allowed the purification of 60 g grft from 226.5 kg processed leaf material. the gp120 binding potential and efficacy of the plant made grft were similar to e. coli produced and native grft respectively, demonstrating the potential of plant expression approaches as viable alternatives for the production of the lectin for use as a candidate microbicide. actinohivin (ah) is a lectin isolated from the actinomycete longispora abida with a reported ic 50 value of 2 nm (chiba et al., 2004) . ah harbours a lot of potential to be developed as an anti-hiv microbicide; the lectin inhibits both t and m tropic hiv strains and is particularly potent against c clade viruses (chiba et al., 2004; matoba et al., 2010) . furthermore ah exhibits an impressive safety profile; the lectin did not cause proliferation or mitogenic stimulation on host cells (hoorelbeke et al., 2010) . unlike other prokaryotic lectins, ah binds to clustered high mannose type glycans instead of single moieties (chiba et al., 2004; tanaka et al., 2009) . it is thus possible that the cluster binding of ah confers its specificity towards glycan types that are typical of the hiv envelope and is linked to its low mitogenic effects. matoba et al. (2010) investigated a plant based production system for ah. the native gene (chiba et al., 2004) was expressed using the icon vector system. expression levels between 20 and 120 mg/kg were obtained. when the small ubiquitin-like modifier (sumo) was fused to the n-terminus of ah, the protein levels accumulated to over 200 mg/kg in the apoplast (davis, 2010) . the plant-produced ah was able to inhibit hiv mediated syncytium formation. the burden of the continuing hiv pandemic together with the lack of an effective vaccine, has spurred the development of several microbicidal candidate molecules to curb hiv transmission. of these, neutralising antibodies and peptide lectins have shown encouraging potency and protection against the virus in in vivo and in vitro studies. plants present a viable option for the cost effective production of protein based candidate microbicides. we reviewed here the progress made with the production of hiv neutralising antibodies and peptide lectins in plant systems. so far four hiv neutralising antibodies-2g12, b12, 2f5 and 4e10have been successfully made in plants. expression levels in transgenic plant leaves and seeds and in plant cell cultures were relatively low compared to that obtained with transient expression technologies. for instance, transient infiltration using new generation viral vector technologies such as the deleted cpmv vector resulted in 2g12 accumulating to 325 mg/kg. expression and targeting of product to various subcellular locations are generally used to optimise recombinant protein yield. in the case of antibodies, this aspect of expression also influences the glycan structure of the molecule. also, depending on plant organ, subcellular targeting can influence the overall yield and efficacy of the plant made antibody. where er retention of 2g12 in arabidopsis seed showed no significant difference in expression levels, it resulted in almost twofold lower levels of 2g12 in maize seed. in contrast to maize seed, er retention of 2g12 in tobacco leaves improved yields. furthermore er retention does not seem to be entirely optimal, leaving a small fraction of immunoglobulins that will contain immunogenic plant glycans. separating these during downstream processing will only add to production costs. a promising solution lies in the use of a modified host system that is incapable of producing plant glycans but instead adds human type glycans to secreted proteins. both 2g12 and 4e10 were produced in such a modified n. benthamiana plant. the resulting antibodies contained only human like galactosylated glycan structures. furthermore these galactosylated antibodies were more efficacious than the cho produced equivalent. no mention was made of the accumulation levels of the antibodies in the mutant plant, but the ability to express mab products with a human glycan profile represents a significant step towards the production in plants of fully functional proteins that are likely to be well tolerated. it is difficult to say which factors have the most influence on the efficacy of plant made immunoglobulins. whilst some plant made antibodies were as active as their cho counterparts, others were less effective and others yet up to fourfold more active. impurities in the recovered products and glycan structure might play a role in decreasing activity, whilst aggregation and human-like glycan structures appear to improve activity as was the case for mab 2g12 antibody produced in maize and a modified tobacco. furthermore, fusion with other anti-hiv agents also showed an increase in the potency of the antibody against the virus. a few lectins with highly potent anti-hiv activity have been isolated from different biological organisms. in general, these lectins do not occur in large amounts in their natural sources, or these sources are difficult to propagate. thus recombinant expression of the lectins was pursued in other production systems such as bacteria and yeast. with the later systems, lectin production was not optimal resulting in heterogeneous products, non-functional products or products that formed insoluble inclusion bodies. subsequently these lectins were produced in plants with generally improved outcomes with respect to these challenges. three potent anti-hiv lectins-namely, cv-n, grft and ah-have been expressed in tobacco. stable transgenic expression resulted in significantly lower levels of accumulated product relative to transient production of lectins. the highest accumulation level reported thus far for a lectin produced in a plant is for grft. by using a tmv-based viral vector the lectin accumulated in tobacco leaves to more than 1 g/kg protein. the ah lectin also accumulated to a good level of 120 mg/kg using the icon viral vector system. furthermore all the plant-produced lectins reviewed here seem to retain their native efficacy against the virus. subcellular targeting is an important aspect to consider when producing these lectins in plants, since lectin expression may affect plant processes, yields and viability of the plant cell. the lectins reviewed here have either been produced as secreted or cytosolic proteins. it is not clear which compartment suits which lectin since high levels for both a cytosolic (grft) and a secreted lectin (ah) have been reported. thus each lectin that is expressed in a plant system might have to be produced in different cell compartments to evaluate the optimal expression conditions for that lectin. apart from expressing solitary candidate microbicides, plants were also able to produce fusions of lectins and antibodies, either with each other (antibody-lectin fusions) or with entirely different molecules. any administered hiv microbicide will most likely consist of several compounds with a different mode of action to ensure broad maximum activity without the risk of developing resistance. by producing fusion microbicides one combines the neutralisation potential of two molecules in a single production run, with positive implications for lowering cost and increasing efficacy. additionally fusions can stabilise the target protein to ensure higher yields, as in the example of elp fused to anti-hiv antibodies (floss et al., 2008) . clearly, production of microbicidal candidate molecules offers advantages beyond simple challenges of expression of efficacious molecules. after twenty years of research, plants are on the brink of entering the playing field of protein production platforms for human therapeutics. their progress from potential to actual production platform has been facilitated largely by technical developments in vector systems and plant hosts. for a disease such as hiv, where there is a desperate demand for an effective microbicide, these advances could potentially enable plants to meet the supply gap. although several anti-hiv neutralising antibodies and peptide lectins have been produced in plants, only two have entered clinical trials . mapp66 is a cocktail of several antibodies produced by the magnicon system in n. benthamiana whilst plant-made 2g12 entered clinical trials in 2011 . in addition, plant-produced grft has passed pre-clinical studies and is considered safe to be evaluated in clinical trials. advancements in plant-made therapeutics in clinical development and regulatory approval such as the use in advanced broad access trials of carrot cell glucocerebrosidase, provide a new perspective on the potency and utility of pmps. the hope is that as production and purification technology become more standardised in the field, and as more plant-made candidates progress along the preclinical and clinical developmental pipeline, plants will become a source of routinely used, effective therapeutic and preventative biologics. a phase i trial with two human monoclonal antibodies (hmab 2f5, 2g12) against hiv-1 passive immunization with the anti-hiv-1 human monoclonal antibody (hmab) 4e10 and the hmab combination 4e10/2f5/2g12 a plant-derived recombinant human glucocerebrosidase enzyme-a preclinical and phase i investigation recent advances of ccr5 antagonists human neutralizing monoclonal antibodies of the igg1 subtype protect against mucosal simian-human immunodeficiency virus infection large-molecular-weight carbohydrate-binding agents as hiv entry inhibitors targeting glycoprotein gp120 carbohydrate-binding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign)-directed hiv-1 transmission to t lymphocytes agrobacterium and plant biotechnology structure-function relationship of monocot mannose-binding lectins the domain-swapped dimer of cyanovirin-n contains two sets of oligosaccharide binding sites in solution cyanovirin-n binds to the viral surface glycoprotein, gp1, 2 and inhibits infectivity of ebola virus 1 flipping the switch from monomeric to dimeric cv-n has little effect on antiviral activity rapid, high-yield production in plants of individualized idiotype vaccines for non-hodgkin's lymphoma the potent anti-hiv protein cyanovirin-n contains two novel carbohydrate binding sites that selectively bind to man8 d1d3 and man9 with nanomolar affinity: implications for binding to the hiv envelope protein gp120 production of human papillomavirus type 16 virus-like particles in transgenic plants comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco proteins that bind high-mannose sugars of the hiv envelope discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development development of topical microbicides to prevent the sexual transmission of hiv a new c-type lectin similar to the human immunoreceptor dc-sign mediates symbiont acquisition by a marine nematode antibody domain exchange is an immunological solution to carbohydrate cluster recognition non-nucleoside hiv-1 reverse transcriptase (rt) inhibitors: past, present, and future perspectives in planta protein sialylation through over-expression of the respective mammalian pathway rapid high yield production of different glycoforms of ebola virus monoclonal antibody geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp120 nucleotide hiv reverse transcriptase inhibitors: tenofovir and beyond antigenicity and immunogenicity of the hiv-1 gp41 epitope eldkwa inserted into permissive sites of the male protein accumulation of functional recombinant human coagulation factor ix in transgenic soybean seeds expression of functional recombinant human growth hormone in transgenic soybean seeds vaginal microbicides and the prevention of hiv transmission development of novel vaccines and therapeutics using plant-based expression systems production of antibodies in plants: status after twenty years binding of the 2f5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes multiple antiviral activities of cyanovirin-n: blocking of human immunodeficiency virus type 1 gp120 interaction with cd4 and coreceptor and inhibition of diverse enveloped viruses preclinical evaluation of anti-hiv microbicide products: new models and biomarkers envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens antibodies to hiv-1: aiming at the right target griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide success stories in molecular farming-a brief overview synergistic activity profile of griffithsin in combination with tenofovir, maraviroc and enfuvirtide against hiv-1 clade c gmp issues for recombinant plant-derived pharmaceutical proteins biochemical and functional characterization of anti-hiv antibody-elp fusion proteins from transgenic plants potential of carbohydrate-binding agents as therapeutics against enveloped viruses anti-hiv-1 antibodies 2f5 and 4e10 interact differently with lipids to bind their epitopes a monovalent mutant of cyanovirin-n provides insight into the role of multiple interactions with gp120 for antiviral activity the future of hiv microbicides: challenges and opportunities advances in development, scale-up and manufacturing of microbicide gels, films, and tablets carbohydrates of human immunodeficiency virus. structures of oligosaccharides linked to the envelope glycoprotein 120 transgenic plants as factories for biopharmaceuticals the microbicide cyanovirin-n expressed on the surface of commensal bacterium streptococcus gordonii captures hiv-1 recombinant production of anti-hiv protein, griffithsin, by auto-induction in a fermentor culture rapid high-yield expression of full-size igg antibodies in plants co-infected with noncompeting viral vectors magnifection-a new platform for expressing recombinant vaccines in plants the lectins: carbohydrate-binding proteins of plants and animals biopharmaceutical production in plants: problems, solutions and opportunities plant-specific glycosylation patterns in the context of therapeutic protein production isolation, primary sequence determination, and disulfide bond structure of cyanovirin-n, an anti-hiv (human immunodeficiency virus) protein from the cyanobacterium nostoc ellipsosporum cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans fc receptor but not complement binding is important in antibody protection against hiv broadly neutralizing monoclonal antibodies 2f5 and 4e10 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus shiv ba-l actinohivin, a broadly neutralizing prokaryotic lectin, inhibits hiv-1 infection by specifically targeting high-mannose-type glycans on the gp120 envelope blockade of attachment and fusion receptors inhibits hiv-1 infection of human cervical tissue high-level rapid production of full-size monoclonal antibodies in plants by a single-vector dna replicon system safety concerns for the potential use of cyanovirin-n as a microbicidal anti-hiv agent molecular cloning of actinohivin, a novel anti-hiv protein from an actinomycete, and its expression in escherichia coli recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of hiv infection in women safety and effectiveness of buffergel and 0.5% pro2000 gel for the prevention of hiv infection in women engineering an obligate domain-swapped dimer of cyanovirin-n with enhanced anti-hiv activity encyclopedia of molecular cell biology and molecular medicine function and glycosylation of plant-derived antiviral monoclonal antibody n-glycosylation in the moss physcomitrella patens is organized similarly to that in higher plants isolation and characterization of banlec-i, a mannoside-binding lectin from musa paradisiac (banana) protein n-glycosylation: molecular genetics and functional significance nucleocytoplasmic plant lectins plant seeds as bioreactors for recombinant protein production trimeric membrane-anchored gp41 inhibits hiv membrane fusion cell culture processes for monoclonal antibody production mechanism of neutralization by the broadly neutralizing hiv-1 monoclonal antibody vrc01 engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-n induction of apoptosis by polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma l929 cells plant lectins: potential antineoplastic drugs from bench to clinic production of monoclonal antibodies with a controlled n-glycosylation pattern in seeds of arabidopsis thaliana dimeric 2g12 as a potent protection against hiv-1 the production of recombinant pharmaceutical proteins in plants optimization of human papillomavirus type 16 (hpv-16) l1 expression in plants: comparison of the suitability of different hpv-16 l1 gene variants and different cell-compartment localization in planta engineering of viral rna replicons: efficient assembly by recombination of dna modules delivered by agrobacterium protection of macaques against pathogenic simian/human immunodeficiency virus 89.6 pd by passive transfer of neutralizing antibodies protection of macaques against vaginal transmission of a pathogenic hiv-1/siv chimeric virus by passive infusion of neutralizing antibodies hiv-1 neutralization profile and plant-based recombinant expression of actinohivin, an env glycan-specific lectin devoid of t-cell mitogenic activity safety and tolerability of buffergel, a novel vaginal microbicide, in women in the united states a recombinant allosteric lectin antagonist of hiv-1 envelope gp120 interactions hiv-1 vaccine development: constrained peptide immunogens show improved binding to the anti-hiv-1 gp41 mab public health aspects of hiv/aids in low and middle income countries plants as biofactories advances in the development of microbicides for the prevention of hiv infection cyanovirin-n, a potent human immunodeficiency virus-inactivating protein, blocks both cd4-dependent and cd4-independent binding of soluble gp120 (sgp120) to target cells, inhibits scd4-induced binding of sgp120 to cell-associated cxcr4, and dissociates bound sgp120 from target cells construction and enhanced cytotoxicity of a cyanovirin-n-pseudomonas exotoxin conjugate against human immunodeficiency virus-infected cells isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp microbicides and hiv prevention: lessons from the past, looking to the future a prospective randomized double blind placebo-controlled phase 1 pharmacokinetic and safety study of a vaginal microbicide gel containing three potent broadly neutralizing antibodies (2fs, 2g12, 4e10) (mabgel) vaginal microbicides: where are we and where are we going? monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-hiv activity a conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1 c-type lectin mermaid inhibits dendritic cell mediated hiv-1 transmission to cd4+ t cells biologically active proteins from natural product extracts scaleable manufacture of hiv-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae antibody protects macaques against vaginal challenge with a pathogenic r5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves plant-made pharmaceuticals: leading products and production platforms the anti-hiv cyanovirin-n domain is evolutionarily conserved and occurs as a protein module in eukaryotes lectins as plant defense proteins the rise and fall of polyanionic inhibitors of the human immunodeficiency virus type 1 making an ally from an enemy: plant virology and the new agriculture mucosal delivery of microbicides by recombinant commensal bacteria: expression of the hiv-inactivating protein cyanovirin-n in gram-positive bacteria recombinant antibody 2g12 produced in maize endosperm efficiently neutralizes hiv-1 and contains predominantly single-glcnac n-glycans cost-effective production of a vaginal protein microbicide to prevent hiv transmission high level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector hiv microbicides: state-of-the-art and new perspectives on the development of entry inhibitors transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor host plant preference of aphids, thrips and spider mites on gna-expressing and control potatoes virus-derived ssdna vectors for the expression of foreign proteins in plants functional analysis of the broadly neutralizing human anti-hiv-1 antibody 2f5 produced in transgenic by-2 suspension cultures extremely high-level and rapid transient protein production in plants without the use of viral replication expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus rna-2 rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-hiv antibody the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2g12 recognizes a cluster of 132 mannose residues on the outer face of gp120 production of a monoclonal antibody in plants with a humanized n-glycosylation pattern aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions hiv coreceptor cxcr4 antagonists hiv co-receptor inhibitors as novel class of anti-hiv drugs transgenic plant production of cyanovirin-n, an hiv microbicide design, expression, and characterization of a multivalent, combination hiv microbicide production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of gaucher's disease using a plant cell system selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-n with high-mannose oligosaccharides on gp120 and other glycoproteins human immunodeficiency virus type 1 elite neutralizers: individuals with broad and potent neutralizing activity identified by using a high-throughput neutralization assay together with an analytical selection algorithm treatment of influenza a (h1n1) virus infections in mice and ferrets with cyanovirin-n expression of the legume symbiotic lectin genes psl and gs52 promotes rhizobial colonization of roots in rice generation of arabidopsis thaliana plants with complex n-glycans lacking β1,2-linked xylose and core α1,3-linked fucose improved virus neutralization by plant-produced anti-hiv antibodies with a homogeneous 1,4-galactosylated n-glycan profile mechanism by which the lectin actinohivin blocks hiv infection of target cells plant-derived mouse igg monoclonal antibody fused to kdel endoplasmic reticulum-retention signal is n-glycosylated homogeneously throughout the plant with mostly high-mannose-type n-glycans human monoclonal antibody 2g12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1 delay of hiv-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies cyanovirin-n gel as a topical microbicide prevents rectal transmission of shiv89. 6p in macaques molecular farming in plants: host systems and expression technology transgenic plants in the biopharmaceutical market the production of vaccines and therapeutic antibodies in plants report on the global hiv-1/aids epidemic isolation and characterization of a lectin with exclusive specificity towards mannose from snowdrop (galanthus nivalis) bulbs plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles effectiveness of col-1492, a nonoxynol-9 vaginal gel, on hiv-1 transmission in female sex workers: a randomised controlled trial cytoplasmic/nuclear plant lectins: a new story poor biologic activity of cross-reactive ige directed to carbohydrate determinants of glycoproteins efficient capture of antibody neutralized hiv-1 by cells expressing dc-sign and transfer to cd4+ t lymphocytes prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to hiv-1 gp120 the current status of plant transformation technologies a minimal dna cassette as a vector for genetic transformation of soybean (glycine max) broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target enhancement of resistance to aphids by introducing the snowdrop lectin gene gna into maize plants design and expression of a dimeric form of human immunodeficiency virus type 1 antibody 2g12 with increased neutralization potency rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1 the antiviral protein cyanovirin-n: the current state of its production and applications interactions between lipids and human anti-hiv antibody 4e10 can be reduced without ablating neutralizing activity high yield recombinant silk-like protein production in transgenic plants through protein targeting neutralization of hiv by milk expressed antibody. poster presented at federation of pegylation of cyanovirin-n, an entry inhibitor of hiv a novel lectin with highly potent antiproliferative and hiv-1 reverse transcriptase inhibitory activities from the edible wild mushroom russula delica structural definition of a conserved neutralization epitope on hiv-1 gp120 structural studies of algal lectins with anti-hiv activity domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41 this research forms part of a phd study that is funded by the council for scientific and industrial research (csir), biosciences, pretoria, south africa. key: cord-260476-whfyczcj authors: seissler, tanja; marquet, roland; paillart, jean-christophe title: hijacking of the ubiquitin/proteasome pathway by the hiv auxiliary proteins date: 2017-10-31 journal: viruses doi: 10.3390/v9110322 sha: doc_id: 260476 cord_uid: whfyczcj the ubiquitin-proteasome system (ups) ensures regulation of the protein pool in the cell by ubiquitination of proteins followed by their degradation by the proteasome. it plays a central role in the cell under normal physiological conditions as well as during viral infections. on the one hand, the ups can be used by the cell to degrade viral proteins, thereby restricting the viral infection. on the other hand, it can also be subverted by the virus to its own advantage, notably to induce degradation of cellular restriction factors. this makes the ups a central player in viral restriction and counter-restriction. in this respect, the human immunodeficiency viruses (hiv-1 and 2) represent excellent examples. indeed, many steps of the hiv life cycle are restricted by cellular proteins, some of which are themselves components of the ups. however, hiv itself hijacks the ups to mediate defense against several cellular restriction factors. for example, the hiv auxiliary proteins vif, vpx and vpu counteract specific restriction factors by the recruitment of cellular ups components. in this review, we describe the interplay between hiv and the ups to illustrate its role in the restriction of viral infections and its hijacking by viral proteins for counter-restriction. the human cell is in a continuous arms race with various viruses. this has led to the coevolution of cellular restriction factors on the one hand and viral proteins for counter-defense on the other hand. restriction factors are generally induced as a result of an interferon response-they use unique mechanisms to impair specific steps of the replication cycle and they exhibit a dominant and autonomous effect. in this continuous fight, the ubiquitin-proteasome system (ups) plays a central role on the cellular as well as on the viral side. the cell expresses restriction factors, some of which are themselves components of the ups, targeting viral proteins for degradation and thereby inhibiting some crucial steps of the viral life cycle. however, viruses have evolved to use the ups to their own benefits, subverting components of the ups to degrade restriction factors, thereby protecting themselves from the cellular defense machinery to allow their dissemination. in this review, we will describe the mechanisms by which the human immunodeficiency viruses (hiv-1 and 2) use and subvert the ups in the continuous battle between cellular defense and viral counter-defense. the ubiquitin-proteasome system (ups) is an important pathway in the cell, ensuring regulation of the protein pool in the cytoplasm as well as in the nucleus. the ups is constituted by three main components: the proteasome holoenzymes, several ubiquitin ligases and a large variety of deubiquitinating enzymes (dubs) [1] . ubiquitin is a ubiquitously expressed and well-conserved the ups plays a central role in many viral infections (reviewed in [18] [19] [20] ), with five main modes of action on the viral life cycle: (1) some cellular e3 ubiquitin ligases recognize viral proteins and induce their ubiquitination, which can have a positive effect on viral replication. for instance, ubiquitination of the p6 domain of the hiv-1 gag polyprotein is important for the interaction of p6 with the escrt machinery. however, the mono-ubiquitination of lysine residues within the p6 domain (k27 and k33) does not seem to be sufficient to facilitate budding of new virions, the latter being also dependent on the cumulative ubiquitination of nc-p2 (nucleocapsid-peptide 2) domain [21] [22] [23] [24] . ubiquitination of the hiv-1 accessory protein tat by cellular e3 ligases stimulates transcription of viral rna [25, 26] . (2) ubiquitination of viral proteins can also induce their degradation, thereby blocking the viral life cycle. this is a strategy used by certain restriction factors. the polymerase pb1 (protein binding 1) of the influenza a virus (iav) for example is ubiquitinated (k48-linked ubiquitin) by the cellular e3 ubiquitin ligase trim32 (tripartite motif-containing protein 32), followed by its degradation by the proteasome [27] . this seems to be a general mechanism as pb1 proteins derived from various iav serotypes (h1n1 (hemagglutinin 1 neuraminidase 1), h3n2, h5n1 or h7n9) associate with trim32 in multiple cell types and this suggests that pb1 has not yet adapted to avoid trim32 targeting [28] . the human herpesvirus type i (hsv-1) capsid protein vp5 has also been shown to be degraded by the ubiquitin proteasome system, leaving the viral genome exposed to innate immune sensors [29] . interestingly, trim5α was reported to inhibit hsv-1 and -2 replication at an early stage of the infection cycle [30] , suggesting a role for this or related protein in cytosolic sensing of herpesvirus capsids. (3) certain viruses have evolved to recruit the cellular e3 ligases to induce the degradation of cellular proteins that might have harmful effects on the viral life cycle. for instance, the protein e6 of human papillomavirus (hpv) recruits the cellular e3 ubiquitin ligase e6-ap to induce ubiquitination and degradation of p53, thereby allowing viral replication [31, 32] . the nsp1 (non-structural rna binding protein 1) protein of rotaviruses subverts the skp1-cul1-fbox (scf) e3 ligase to induce the ubiquitination and degradation of β-trcp (β-transducin repeat containing protein). β-trcp is by itself a substrate adaptor of an e3 ligase and its degradation leads to accumulation of the nf-κb inhibitor iκb, resulting in inhibition of the nf-κb induced antiviral responses [33, 34] . these mechanisms are important for hiv replication and will be detailed in section 5. (4) other viruses directly encode their own e3 ligases. kaposi sarcoma herpesvirus (kshv) protein k3 and k5 (ring-ch family of ligases) ubiquitinate mhc-i (major histocompatibility complex i), resulting in its down-regulation from the cell surface through a clathrin-dependent sorting pathway to an endolysosomal compartment [35, 36] . this endolysosomal sorting requires k63-linked instead of k48-linked polyubiquitin chains [19] . another well-known example is the icp0 protein (infected cell protein 0) of hsv-1, an e3 ubiquitin ligase which induces the degradation of the nd10 (nuclear domain 10) nuclear body components pml (promyelocytic leukemia protein) and sp100 through the ups, thereby avoiding antiviral sensing [37, 38] . icp0 has also been shown to have a ring-independent e3 ligase activity that polyubiquitinates the e2 enzyme cdc34. icp0 influences many cellular pathways and is required for the activation of most viral and many cellular genes, for reactivation from latency and suppression of innate immunity [19] . (5) finally, ubiquitin modifications can be reversed by the isopeptide-bond specific proteolytic activity of dubs. in addition to cellular dubs, it has been reported that various virus families code their own dubs (coronavirus, herpesvirus etc.) to evade host antiviral immune response and promote virus replication (for a recent review see [1] ). for instance, in the herpesviridae family, a variety of dubs play an important role in the virus life cycle (e.g., ul36usp (ubiquitin ligase 36 ubiquitin specific protease) of hsv-1, tegument protein pul48 of human cytomegalovirus (hcmv)). regarding hiv-1, a recent study reported that several cellular dubs (usp7 and usp47, ubiquitin specific protease family) play an important role in its replication by regulating gag processing and thus the infectivity of released virions and simultaneously the entry of gag into the ups and mhc-i pathway [39] . moreover, this study showed that treatment with dub inhibitors targeting usp47 causes a general gag processing defect, indicating that usp47 interacts with gag and prevents its entry into the ups. similarly, proteasome inhibitors have been shown to impact hiv-1 replication by reducing the release and maturation of infectious particles [40, 41] or by suppressing its transcription [42] . taken together, these studies suggest a potential antiretroviral activity of dub and proteasome inhibitors. the importance of the ups in antiviral restriction will be discussed here using hiv as an example. hiv-1 and 2 are retroviruses of the genus lentivirus. their genome is composed of two (+) single stranded rnas encoding the gag, pol and env polyproteins, which correspond to the structural (matrix, capsid, nucleocapsid and p6), enzymatic (protease, reverse-transcriptase and integrase) and envelope (transmembrane and surface) viral proteins. in addition, the genome of these two viruses express two regulatory (tat and rev) and four auxiliary (nef, vpu/vpx, vpr and vif) proteins, which regulate several steps in the viral life cycle [43, 44] . the main difference between hiv-2 and hiv-1 is the lack of the vpu protein in the former, which is replaced by vpx [45] . following viral attachment and entry into the target cell, the dimeric viral genomic rna is partially uncoated and transported to the cell nucleus. concomitantly, reverse transcription of the viral genomic rna takes place to form the pre-proviral dna, which is then integrated into the cellular genome. the integrated provirus mediates the synthesis of new full-length viral rna (or unspliced rna), which will be used as genomic rna encapsidated into viral particles and as mrna for structural and enzymatic proteins and mono-and multi-spliced viral mrnas, which encode the viral envelope and the regulatory and auxiliary proteins in the infected cell. finally, the components of the viral particle assemble at the plasma membrane, where new viral particles bud, maturate and disseminate to other host cells in the infected organism ( figure 2 ) [43, 44, [46] [47] [48] [49] . during its life cycle, hiv is subjected to different cellular restriction factors (figure 2 ), the first line of defense of cellular immunity. the newly discovered serinc3 (serine incorporator 3) and serinc5 proteins target the very beginning of the viral life cycle by inhibiting correct fusion of the viral envelope with the plasma membrane, thereby preventing the virus from entering into a new host cell [50, 51] . ifitm (interferon-induced transmembrane) proteins 1, 2 and 3 also target the viral entry into the cell by inhibiting viral fusion with target cells. the exact mechanism of restriction is yet a matter of debate, as well as whether ifitm incorporation in virions or its expression in target cells is responsible for the antiviral effect. ifitm proteins might act on env to inhibit its functions in viral fusion and it has been shown that some mutations in the env protein can indeed confer resistance to ifitm restriction [52] [53] [54] [55] [56] [57] . once the virus has entered the cell, trim5α (tripartite motif-containing protein 5α) can inhibit the early steps of the viral life cycle in a species-specific manner by accelerating viral uncoating [58] [59] [60] . the viral capsid protein also seems to be the target of myxovirus resistance 2 (mx2/b), a restriction factor that inhibits nuclear import and subsequent integration of the provirus through an unknown mechanism. some mutations in the capsid protein have been shown to confer resistance to mx2 and particularly some mutations located at the site of interaction with cyclophilin a, an important host factor for hiv-1 infectivity [61] [62] [63] [64] [65] [66] . samhd1 (sterile alpha motif and histidine aspartate domain-containing protein 1) also targets the early phase of viral infection: this deoxynucleotide-triphosphohydrolase inhibits reverse transcription by depleting the pool of cellular dntps (deoxy nucleotide triphosphates) [67, 68] . during reverse transcription of the viral rna, the restriction factor apobec3g (apolipoprotein b mrna editing enzyme, catalytic polypeptide-like 3g, or a3g) and other factors from the apobec3 family, can induce g to a hypermutations, which prevent production of functional viral proteins [69] [70] [71] . the amount of viral proteins that are produced in an infected cell can be limited by schlafen11 (slfn11). due to the bias of hiv-1 towards a/u rich codons, the virus stimulates production of corresponding trnas by the cell to increase viral translation, a mechanism that seems to be partly counteracted by slfn11, which binds trnas in a codon-specific manner [72] [73] [74] . the final steps in the viral life cycle can be targeted by tetherin/bst2 (bone marrow stromal antigen 2), which inhibits release of new viral particles from the host cell [75] [76] [77] and march8 (membrane-associated ring-ch 8 protein), which decreases incorporation of envelope proteins into newly produced virions, thereby decreasing their infectivity [78] . two of these restriction factors, trim5α and march8, use the ups to exert their restricting activity. hiv is able to counteract restriction factors using its accessory proteins ( figure 2 ): nef prevents serinc5 incorporation into virions by mediating its relocalization to late endosomes through interaction with the clathrin adaptor ap-2 [50, 79] . vif counteracts a3g by inducing its proteasomal degradation as well as by reducing its transcription and translation [69, [80] [81] [82] . vpx (and vpr of certain simian immunodeficiency virus (siv) strains) counteracts samhd1 by inducing its proteasomal degradation [67, 83, 84] . vpu (env for hiv-2 and nef or vpu for siv) counteracts bst2/tetherin by sequestering it away from sites of viral budding [76, 77, 85] . amongst these accessory proteins, vif, vpx and vpu hijack the ups to exert their counter-defense. in the following section, we will discuss in detail the restriction factors as well as the viral proteins which use the ups for their respective mechanisms. infected cell can be limited by schlafen11 (slfn11). due to the bias of hiv-1 towards a/u rich codons, the virus stimulates production of corresponding trnas by the cell to increase viral translation, a mechanism that seems to be partly counteracted by slfn11, which binds trnas in a codon-specific manner [72] [73] [74] . the final steps in the viral life cycle can be targeted by tetherin/bst2 (bone marrow stromal antigen 2), which inhibits release of new viral particles from the host cell [75] [76] [77] and march8 (membrane-associated ring-ch 8 protein), which decreases incorporation of envelope proteins into newly produced virions, thereby decreasing their infectivity [78] . two of these restriction factors, trim5α and march8, use the ups to exert their restricting activity. hiv is able to counteract restriction factors using its accessory proteins ( figure 2 ): nef prevents serinc5 incorporation into virions by mediating its relocalization to late endosomes through interaction with the clathrin adaptor ap-2 [50, 79] . vif counteracts a3g by inducing its proteasomal degradation as well as by reducing its transcription and translation [69, [80] [81] [82] . vpx (and vpr of certain simian immunodeficiency virus (siv) strains) counteracts samhd1 by inducing its proteasomal degradation [67, 83, 84] . vpu (env for hiv-2 and nef or vpu for siv) counteracts bst2/tetherin by sequestering it away from sites of viral budding [76, 77, 85] . amongst these accessory proteins, vif, vpx and vpu hijack the ups to exert their counter-defense. in the following section, we will discuss in detail the restriction factors as well as the viral proteins which use the ups for their respective mechanisms. one example of the cell using the ups to restrict hiv is trim5α, an e3-ubiquitin ligase that interacts with the viral capsid after its entry into the cell. trim5α mediates a species-specific block: hiv-1 is restricted by the trim5α proteins of old world monkeys like rhesus or cynomolgous one example of the cell using the ups to restrict hiv is trim5α, an e3-ubiquitin ligase that interacts with the viral capsid after its entry into the cell. trim5α mediates a species-specific block: hiv-1 is restricted by the trim5α proteins of old world monkeys like rhesus or cynomolgous monkeys, while the trim5α of human or new world monkeys have no or only a very weak effect on hiv-1 [59, 60, 86, 87] . trim5α thereby constitutes one of the factors responsible for the interspecies barrier. the restriction of hiv-1 by trim5α is mediated by the interaction of the trim5α spry (spia and ryanodine receptor) domain ( figure 3a ) with the viral capsid in the cytoplasm of newly infected cells [59] . this interaction leads to premature decapsidation of the viral core. moreover, viral capsid and integrase proteins are degraded ( figure 3c 1 ) and the reverse transcription of the viral genome is inhibited in the presence of a restricting trim5α. these effects seem to be mediated by the ups, since treatment with proteasome inhibitors restores a normal decapsidation rate and reverse transcription. it has also been shown that the proteasome co-localizes with trim5α and viral cores in the cytoplasm [88, 89] . trim5α is also degraded by the proteasome but only in the presence of susceptible viral cores [90] , suggesting that trim5α recruits the proteasome to the viral cores and induces their degradation. this mechanism seems to be mediated by the e3-ubiquitin ligase activity of trim5α, through its ring domain ( figure 3a ) [58, 91] . nevertheless, trim5α inhibits integration of the proviral dna independently of the proteasome, suggesting that trim5α uses an additional, yet uncharacterized, strategy to block viral infection ( figure 3c 2 ) [92, 93] . finally, the association of trim5α with the viral capsid enhances its e3-ubiquitin ligase activity, which, in conjunction with the e2 enzyme ubc13/uev1a (ubiquitin-conjugating enzyme 13/ubiquitin-conjugating enzyme variant 1a), leads to the synthesis of free k63-linked ubiquitin chains, thus stimulating tak1 (transforming growth factor β-activated kinase 1) and finally activating ap1 and nf-κb signaling ( figure 3c 3 ) [94, 95] . this indicates that trim5α, in addition to its direct antiviral activity, also functions as a sensor that induces a general antiviral state of the cell. figure 3a ) with the viral capsid in the cytoplasm of newly infected cells [59] . this interaction leads to premature decapsidation of the viral core. moreover, viral capsid and integrase proteins are degraded ( figure 3c ) and the reverse transcription of the viral genome is inhibited in the presence of a restricting trim5α. these effects seem to be mediated by the ups, since treatment with proteasome inhibitors restores a normal decapsidation rate and reverse transcription. it has also been shown that the proteasome co-localizes with trim5α and viral cores in the cytoplasm [88, 89] . trim5α is also degraded by the proteasome but only in the presence of susceptible viral cores [90] , suggesting that trim5α recruits the proteasome to the viral cores and induces their degradation. this mechanism seems to be mediated by the e3-ubiquitin ligase activity of trim5α, through its ring domain ( figure 3a ) [58, 91] . nevertheless, trim5α inhibits integration of the proviral dna independently of the proteasome, suggesting that trim5α uses an additional, yet uncharacterized, strategy to block viral infection ( figure 3c ) [92, 93] . finally, the association of trim5α with the viral capsid enhances its e3-ubiquitin ligase activity, which, in conjunction with the e2 enzyme ubc13/uev1a (ubiquitin-conjugating enzyme 13/ubiquitin-conjugating enzyme variant 1a), leads to the synthesis of free k63-linked ubiquitin chains, thus stimulating tak1 (transforming growth factor β-activated kinase 1) and finally activating ap1 and nf-ϰb signaling ( figure 3c ) [94, 95] . this indicates that trim5α, in addition to its direct antiviral activity, also functions as a sensor that induces a general antiviral state of the cell. (d) march8 (red) mediates intracellular retention of envelope proteins (env, brown), leading to reduced env incorporation into virions, thereby decreasing infectivity. march8 has recently been identified as a restriction factor of hiv-1, expressed by differentiated myeloid cells like monocyte derived macrophages and dendritic cells [78] . march8 significantly reduces infectivity of virions produced from march8-expressing cells by decreasing the number of env-proteins incorporated into budding virions. march8 is a transmembrane e3-ubiquitin ligase, possessing an n-terminal, cytoplasmic ring domain ( figure 3b ), known to downregulate multiple cellular proteins from the plasma membrane by ubiquitination followed by degradation in the endo-lysosomal pathway [96] [97] [98] [99] . in the case of hiv-1 restriction, it has been shown that march8 interacts with env and causes its downregulation from the cell surface. the ring-domain of march8 is necessary for this mechanism, suggesting that ubiquitination plays a role. however, env does not seem to be degraded in the endo-lysosomal pathway like cellular proteins targeted by march8 but seems rather to be retained in intracellular compartments. march8 thus sequesters env away from hiv-1 budding sites, thereby reducing env incorporation into newly formed virions, making them less competent for infection of new target cells ( figure 3d ) [78] . the family of apolipoprotein b mrna-editing enzyme, catalytic polypeptide-like 3 (apobec3/a3) proteins, is a family of 7 cytosine deaminases (a3a to a3h) which induce transition of cytosine to uracil on single-stranded dna, with a preferential recognition of cc sequence motifs by a3g and tc motifs by the others [100] [101] [102] . a3g ( figure 4a ) has been the first member of this family to be identified as a potent antiviral factor. it is incorporated into budding hiv virions and is thereby carried over into the next infected cell [69] . during reverse transcription of the viral genomic rna, the single stranded negative sense dna is sensitive to the cytosine-deaminase activity of a3g, leading to c to u transitions [70, 71] . these mutations can either be recognized by uracil dna glycosylases, like the virion-associated ung2 (uracyl n-glycosylase 2), leading to the degradation of the provirus by abasic site endonucleases [103] , or they can be conserved in the provirus. due to the sequence preference of a3g, these mutations very frequently introduce new stop codons in the viral genome, thus leading to the expression of non-functional mutated or/and truncated viral proteins ( figure 4c ). hiv-1 counteracts a3g with its vif protein, which prevents a3g incorporation into virions by inducing its degradation through the proteasome [80] . to do so, vif recruits an scf-like e3-ubiquitin ligase, composed of cullin5, rbx2, elongin b and c. in this complex, vif possesses the role of a substrate adaptor, directly interacting with a3g through its n-terminal domain ( figure 4b ), thereby recruiting it for ubiquitination ( figure 4c 3 ) [104] . the recruitment of cullin5 is mediated by the zinc-binding domain of vif [105] and cullin5 in turn recruits the e2-ubiquitin-conjugating enzyme rbx2. the recruitment of elongin b and c is mediated by the bc-box domain of vif ( figure 4b ), which can be negatively regulated by phosphorylation. in this complex, not only a3g but also vif is ubiquitinated, which might contribute to the transport of a3g to the proteasome [106] . the cellular protein hdac6 (histone deacetylase 6) has been shown to play a role in this process, by inducing vif degradation through autophagosomes as well as by protecting a3g from ubiquitination and degradation [107] . the expression level of vif is also regulated by mdm2 (mouse double minute 2 homolog), an e3-ubiquitin ligase that can induce the ubiquitination of vif and its proteasomal degradation [108] . cbf-β (core binding factor β), a co-factor of the runx transcription factor family, is recruited by vif and ensures its stability by inhibition of mdm2 binding [109] . cbf-β is also necessary to allow assembly of the scf-like e3-ubiquitin ligase mediated by vif, resulting in the inability of vif to induce ubiquitination and degradation of a3g in the absence of cbf-β [110, 111] . moreover, by sequestering cbf-β in the e3-ubiquitin ligase complex, vif indirectly causes a decrease in a3g transcription as the a3g gene is regulated by the runx transcription factor family, which requires cbf-β as cofactor ( figure 4c 1 ) [81] . degradation of a3g through the ups has been known for a long time as the main mechanism for hiv-1 to counteract cellular restriction; however it has been shown that vif can also inhibit a3g translation [82, 112, 113] and this inhibition significantly contributes to the counteraction mechanism ( figure 4c 2 ) [82, 112, 113] . while a3g is the main member of the a3-family that efficiently restricts hiv, a3d, f and h also showed a restricting activity towards hiv-1 in the absence of vif, even though to a lesser extent than a3g [114] . vif is also able to recruit these a3 proteins by different motifs of its n-terminal domain ( figure 4b ), thus inducing their degradation by the proteasome similarly to a3g [115] [116] [117] . translation [82, 112, 113] and this inhibition significantly contributes to the counteraction mechanism ( figure 4c ) [82, 112, 113] . while a3g is the main member of the a3-family that efficiently restricts hiv, a3d, f and h also showed a restricting activity towards hiv-1 in the absence of vif, even though to a lesser extent than a3g [114] . vif is also able to recruit these a3 proteins by different motifs of its n-terminal domain ( figure 4b ), thus inducing their degradation by the proteasome similarly to a3g [115] [116] [117] . sterile alpha motif and histidine-aspartate domain-containing protein 1 (samhd1, figure 5a ) is a dgtp-regulated deoxynucleoside-triphosphohydrolase that catalyzes the hydrolysis of dntps to deoxynucleosides and inorganic triphosphate [118, 119] . in non-cycling myeloid cells as well as in resting cd4 + t cells, this restriction factor causes a block in the early steps of the hiv-1 life cycle [67] by depleting the intracellular pool of dntps [68] , which leads to abortion of the viral genomic rna reverse transcription and accumulation of defective viral cdna ( figure 5c ) [120] . this block strongly (c) the mechanism of apobec3g restriction and vif counteraction. apobec3g (red) is incorporated into virions and induces hypermutations of the provirus leading either to its degradation or production of truncated viral proteins. vif (blue) decreases a3g transcription 1 , inhibits its translation 2 (red t bar) and induces its degradation by the proteasome 3 . sterile alpha motif and histidine-aspartate domain-containing protein 1 (samhd1, figure 5a ) is a dgtp-regulated deoxynucleoside-triphosphohydrolase that catalyzes the hydrolysis of dntps to deoxynucleosides and inorganic triphosphate [118, 119] . in non-cycling myeloid cells as well as in resting cd4 + t cells, this restriction factor causes a block in the early steps of the hiv-1 life cycle [67] by depleting the intracellular pool of dntps [68] , which leads to abortion of the viral genomic rna reverse transcription and accumulation of defective viral cdna ( figure 5c ) [120] . this block strongly affects infectivity of hiv-1 in these cell types but has no effect on hiv-2 infectivity [121] . indeed, hiv-2 possesses the viral protein x (vpx, figure 5b ) which alleviates the post-entry block mediated by samhd1 by inducing its degradation by the proteasome. vpx has been found to recruit the cul4a-ddb1-dcaf1(ddb1 and cul4 associated factor 1) e3 ubiquitin ligase through a direct interaction with its substrate recognition protein dcaf1 (ddb1 and cul4 associated factor 1) [122] while also interacting with the c-terminal domain of samhd1, thereby loading samhd1 onto the e3 complex and inducing its ubiquitination followed by its proteasomal degradation ( figure 5c ). the nuclear localization of samhd1 is required for its vpx-induced proteasomal degradation, suggesting the nuclear ups is important in this mechanism [123, 124] . degradation of samhd1 leads to an increase in cellular dntp levels and the efficiency of proviral dna synthesis [120] . in this manner, the vpx protein allows hiv-2 to efficiently infect human dendritic and myeloid cells and it significantly increases the infection by hiv-1 [83] . vpx therefore seems to be an important protein for viral replication, however it is present exclusively in hiv-2 and some siv strains. in these lineages, the vpx gene has evolved from vpr which is present in all hiv and siv strains and whose main function is the induction of cell cycle arrest [125] [126] [127] [128] [129] . vpx and vpr share many similarities, like for example their interaction with the same cul4a e3 ubiquitin ligase [122, 125] . interestingly, the vpr protein of some siv strains has been shown to induce proteasomal degradation of samhd1, thereby compensating for the lack of vpx. indeed it seems that the ability to degrade samhd1 has first been acquired by the vpr protein in certain lentiviral strains before the evolution of a separate vpx gene which has subsequently conserved the function of samhd1 antagonism [84, 130] . nevertheless, many lineages, like hiv-1 for example, lack an anti-samhd1 activity. hiv interestingly, samhd1 seems to be regulated in cells by phosphorylation mediated by cdk6-(cyclin-dependent kinase 6) dependent cdk2, which links its activity to cell cycle control. indeed, samhd1 is phosphorylated in cycling cells which blocks its activity as a dntp hydrolase [131] . this correlates with the permissiveness of cycling cells for hiv-1 infection as opposed to non-cycling cells. moreover, hiv infection is made possible despite the lack of a viral factor counteracting samhd1 by different cellular proteins: cd81 for example has recently been shown to favor hiv-1 infection by interacting with samhd1 and stimulation of its proteasome-dependent degradation [132] . cyclin l2 also induces samhd1 proteasomal degradation through interaction with samhd1 and dcaf1, a mechanism interestingly similar to the one used by vpx [133] . affects infectivity of hiv-1 in these cell types but has no effect on hiv-2 infectivity [121] . indeed, hiv-2 possesses the viral protein x (vpx, figure 5b ) which alleviates the post-entry block mediated by samhd1 by inducing its degradation by the proteasome. vpx has been found to recruit the cul4a-ddb1-dcaf1(ddb1 and cul4 associated factor 1) e3 ubiquitin ligase through a direct interaction with its substrate recognition protein dcaf1 (ddb1 and cul4 associated factor 1) [122] while also interacting with the c-terminal domain of samhd1, thereby loading samhd1 onto the e3 complex and inducing its ubiquitination followed by its proteasomal degradation ( figure 5c ). the nuclear localization of samhd1 is required for its vpx-induced proteasomal degradation, suggesting the nuclear ups is important in this mechanism [123, 124] . degradation of samhd1 leads to an increase in cellular dntp levels and the efficiency of proviral dna synthesis [120] . in this manner, the vpx protein allows hiv-2 to efficiently infect human dendritic and myeloid cells and it significantly increases the infection by hiv-1 [83] . vpx therefore seems to be an important protein for viral replication, however it is present exclusively in hiv-2 and some siv strains. in these lineages, the vpx gene has evolved from vpr which is present in all hiv and siv strains and whose main function is the induction of cell cycle arrest [125] [126] [127] [128] [129] . vpx and vpr share many similarities, like for example their interaction with the same cul4a e3 ubiquitin ligase [122, 125] . interestingly, the vpr protein of some siv strains has been shown to induce proteasomal degradation of samhd1, thereby compensating for the lack of vpx. indeed it seems that the ability to degrade samhd1 has first been acquired by the vpr protein in certain lentiviral strains before the evolution of a separate vpx gene which has subsequently conserved the function of samhd1 antagonism [84, 130] . nevertheless, many lineages, like hiv-1 for example, lack an anti-samhd1 activity. hiv interestingly, samhd1 seems to be regulated in cells by phosphorylation mediated by cdk6-(cyclin-dependent kinase 6) dependent cdk2, which links its activity to cell cycle control. indeed, samhd1 is phosphorylated in cycling cells which blocks its activity as a dntp hydrolase [131] . this correlates with the permissiveness of cycling cells for hiv-1 infection as opposed to non-cycling cells. moreover, hiv infection is made possible despite the lack of a viral factor counteracting samhd1 by different cellular proteins: cd81 for example has recently been shown to favor hiv-1 infection by interacting with samhd1 and stimulation of its proteasome-dependent degradation [132] . cyclin l2 also induces samhd1 proteasomal degradation through interaction with samhd1 and dcaf1, a mechanism interestingly similar to the one used by vpx [133] . in the absence of vpu, newly formed virions remain tethered to the plasma membrane of their host cell after budding and are eventually endocytosed and degraded [75] . the cellular restriction factor responsible for the block of virion release is tetherin/bst-2. bst-2 is found as a disulfide-bond-linked dimer which is anchored into the plasma membrane by two domains: a transmembrane domain close to its n-terminus and an extracellular c-terminal glycosyl-phosphatidylinositol (gpi)-anchor ( figure 6a ) [134] . these two domains mediate virion-tethering to the host cell, one remaining in the plasma membrane and the other one being inserted into the viral envelope ( figure 6c ). it has been shown that this tethering involves approximately a dozen of bst-2 dimers and that among the two membrane-associated domains, the gpi-anchor is preferentially incorporated into budding virions [135] . the extracellular domain of bst-2 thereby acts like a molecular ruler, maintaining the virus at a constant distance of the plasma membrane, preventing it from disseminating to other target cells [134] . the viral protein vpu counteracts bst-2 by direct interaction of their transmembrane domains embedded in the plasma membrane [136] . the exact mode of action of vpu is still a matter of debate, but it seems clear now that vpu sequesters bst-2 away from virion budding sites, thereby preventing it from incorporation into the viral envelope ( figure 6c 1 ) [77, 85, 137, 138] . several studies have shown that in the presence of vpu, newly synthesized bst-2 is sequestered in intracellular compartments, particularly the trans-golgi-network ( figure 6c 2 ). this finally results in the downregulation of surface levels of bst-2, thereby allowing normal rates of virion release in the presence of vpu [77, 85, 137, 138] . bst-2 is constitutively regulated by ubiquitination and lysosomal degradation mediated by the cellular e3 ubiquitin ligases march8 and nedd4 (neural precursor cell expressed developmentally down-regulated protein 4) [139] . it is still a matter of debate however, whether vpu also uses the endo-lysosomal system for bst-2 counteraction. the e3-ubiquitin ligase adaptor β-trcp is known to be recruited by the cytoplasmic dsgxxs motif of vpu ( figure 6b ) [140] , which might lead to ubiquitination of bst-2 followed by its degradation in the endo-lysosomal system ( figure 6c 3 ) [137, 141] . even though the interaction of vpu with β-trcp, as well as the capacity of β-trcp to recruit an e3-ubiquitin ligase seem to be required for bst-2 counteraction by vpu [137, 142, 143] , conflicting data have also been reported [144] [145] [146] . certain components of the autophagy pathway, as well as clathrin adaptors ap-1 and 2 and components of the escrt system might also be involved in the downregulation of bst-2 by vpu, which would corroborate transport of bst-2 in the endosomal system [137, [147] [148] [149] . however, degradation of bst-2 might not be absolutely required for viral counteraction of bst-2, since vpu is capable of intracellular sequestration of bst-2 independently of its degradation [85, 138] . the guanylate binding protein 5 (gbp5) has very recently been discovered as a new restriction factor of hiv-1 infection, that interferes with viral env proteins, thereby decreasing infectivity of produced virions [150, 151] . as vpu and env are expressed from the same transcript by leaky scanning, the loss of vpu expression can in this case lead to an increase of env expression, as observed in the macrophage tropic ad8 isolate [152] , allowing the virus to partly overcome gbp5 restriction. surprisingly, such vpu mutants seem to occur frequently despite the presence of bst-2. hiv-2 and siv are also counteracted by bst-2 proteins expressed by their respective host species in a species-dependent manner, but some of them lack vpu to counteract this mechanism. it has been shown that the hiv-2 env protein can enhance virion release in the presence of bst-2 thereby substituting for vpu [153, 154] . certain siv strains, like sivagm, sivblu and sivmac also lack the vpu gene and rely on the accessory protein nef to counteract bst-2. other siv strains like sivmon, sivmus, sivgsn and sivden express vpu and use it to counteract bst-2. even though sivgor and sivcpz express vpu, nef seems to take over the role of bst-2 counteraction. this gives interesting clues about the evolution of hiv and siv strains [155] [156] [157] . vpu can also induce bst-2 degradation in the endo-lysosomal pathway . the ups is hijacked by hiv and plays an important role for the viral defense against multiple cellular restriction mechanisms. apart from restriction factors, several other cellular proteins can also be targeted by hiv through the ups. the viral auxiliary protein vpu for example possesses the ability to associate with the cul1-skp1 e3 ubiquitin ligase through interaction with its substrate receptor β-trcp. this association not only seems to play a role in the counteraction of bst-2 but has also been shown to induce degradation of the hiv receptor cd4. indeed, vpu induces cd4 ubiquitination followed by its extraction from the endoplasmic reticulum (er) [140, [158] [159] [160] [161] [162] . the mechanism used by vpu to induce cd4 depletion involves the cellular er-associated degradation (erad) pathway, which operates as a quality control mechanism to dispose of unwanted er membrane proteins into the cytosol for subsequent proteasomal degradation. the dislocation of protein from the membrane is achieved by the recruitment of the vcp-ufd1l-npl4 (valosin-containing protein -ubiquitin fusion degradation protein 1-nuclear protein localization protein 4) complex through recognition by ufd1l of k48-linked poly-ubiquitin chains on the cd4 cytosolic tail. interestingly, the degradation of cd4 depends also on ubiquitination of serine/threonine residues [140, [158] [159] [160] [161] . the atpase activity of vcp then drives dislocation of cd4 from the er membrane into the cytosol and eventually its degradation in proteasomes. the multiple levels at which vpu acts to prevent export the ups is hijacked by hiv and plays an important role for the viral defense against multiple cellular restriction mechanisms. apart from restriction factors, several other cellular proteins can also be targeted by hiv through the ups. the viral auxiliary protein vpu for example possesses the ability to associate with the cul1-skp1 e3 ubiquitin ligase through interaction with its substrate receptor β-trcp. this association not only seems to play a role in the counteraction of bst-2 but has also been shown to induce degradation of the hiv receptor cd4. indeed, vpu induces cd4 ubiquitination followed by its extraction from the endoplasmic reticulum (er) [140, [158] [159] [160] [161] [162] . the mechanism used by vpu to induce cd4 depletion involves the cellular er-associated degradation (erad) pathway, which operates as a quality control mechanism to dispose of unwanted er membrane proteins into the cytosol for subsequent proteasomal degradation. the dislocation of protein from the membrane is achieved by the recruitment of the vcp-ufd1l-npl4 (valosin-containing protein-ubiquitin fusion degradation protein 1-nuclear protein localization protein 4) complex through recognition by ufd1l of k48-linked poly-ubiquitin chains on the cd4 cytosolic tail. interestingly, the degradation of cd4 depends also on ubiquitination of serine/threonine residues [140, [158] [159] [160] [161] . the atpase activity of vcp then drives dislocation of cd4 from the er membrane into the cytosol and eventually its degradation in proteasomes. the multiple levels at which vpu acts to prevent export of cd4 from the er underscore the importance of ensuring complete depletion of cd4 from the plasma membrane for progression of the infection [143, 160, 161, 163] . other targets of vpu-induced ubiquitination and degradation include the cell surface glycoprotein icam-1 and the amino acid transporter snat-1, both involved in immune signaling [164, 165] . it is well established that the viral auxiliary protein vpr associates with the cul4a-ring e3 ligase through interaction with its substrate recognition subunit dcaf1. this complex has been shown to induce ubiquitination followed by proteasomal degradation of the dna glycosylase ung2. thereby vpr reduces encapsidation of ung2, ultimately contributing to the protection against the restriction factor a3g. ung2 recognizes c to u mutations induced by a3g and generates abasic sites, leading to degradation of viral dna. indeed a virus lacking vif can be partially rescued by vpr-mediated reduction of ung2 compared to viruses lacking both vif and vpr [166] [167] [168] . moreover, it has recently been shown that vpr can also induce the degradation of a3g itself through the ups [169] . vpr seems to also enhance hiv-1 production in macrophages by ups-mediated degradation of the cellular protein dicer, which is involved in rna silencing [170] . the main function of vpr known to date is the induction of a cell cycle arrest at the g2 phase. the association of vpr with the cul4a e3 ubiquitin ligase has been shown to be important for this process, although the exact mechanism is still unknown [125] [126] [127] [128] . cell cycle arrest seems to involve vpr association with the slx4-slx1-mus81-eme1 complex, leading to slx4 (structure-specific endonuclease subunit) activation and ultimately proteasomal degradation of mus81 (crossover junction endonuclease) and eme1 (essential meiotic structure-specific endonuclease 1) [127, 128] . vpr also induces the degradation of multiple other cellular proteins such as the dna translocase hltf (helicase-like transcription factor) [171] , the dna replication factor mcm10 (mini chromosome maintenance 10) [172] , as well as the chromatin associated proteins zip (leucine zipper), szip and class i hdacs (histone deacetylase 6) [173, 174] . the ups plays an important role in viral infections in general and especially in the process of viral restriction and counter-restriction. in this continuous battle between the virus and the cell, the ups constitutes an efficient tool for both sides. several hiv auxiliary proteins have evolved the ability to interact with components of the ups, subverting it for its own means. this allows the targeting of a multitude of different cellular proteins through a single platform. this strategy is not limited to hiv, but is used by a plethora of different viruses to ensure various aspects of their life cycles. overall, the specific degradation of certain cellular proteins in the ups allows viruses to generate a favorable environment for their own replication. the almost universal role of the ups in counteraction of cellular restriction factors by hiv makes the ups an interesting target for antiviral therapy. one of the main difficulties in therapy-design against hiv is the rapid evolution of the virus, which easily escapes therapeutic molecules by mutation of the targeted viral proteins. targeting the human ups represents a promising antiviral strategy because it would allow to avoid the escape through mutations [175, 176] . a better knowledge on how the virus hijacks the ups and which components are involved in viral replication is crucial in this attempt. structure and function of viral deubiquitinating enzymes the ubiquitin-proteasome pathway ubiquitin: same molecule, different degradation pathways ubiquitination in the antiviral immune response function and regulation of cullin-ring ubiquitin ligases components of ubiquitin-protein ligase system. resolution, affinity purification, and role in protein breakdown protein ubiquitination involving an e1-e2-e3 enzyme ubiquitin thioester cascade a uniform isopeptide-linked multiubiquitin chain is sufficient to target substrate for degradation in ubiquitin-mediated proteolysis a multiubiquitin chain is confined to specific lysine in a targeted short-lived protein the logic of the 26s proteasome involvement of the proteasome in various degradative processes in mammalian cells ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26s proteasome redundant roles of rpn10 and rpn13 in recognition of ubiquitinated proteins and cellular homeostasis crystal structure of the human 20s proteasome in complex with carfilzomib the escrt complexes: structure and mechanism of a membrane-trafficking network k63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, escrt-i the ubiquitin-conjugating system: multiple roles in viral replication and infection viral avoidance and exploitation of the ubiquitin system hiv-1, ubiquitin and ubiquitin-like proteins: the dialectic interactions of a virus with a sophisticated network of post-translational modifications ubiquitin is covalently attached to the p6gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12gag protein of moloney murine leukemia virus cumulative mutations of ubiquitin acceptor sites in human immunodeficiency virus type 1 gag cause a late budding defect tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding ubiquitination of hiv-1 and mulv gag a non-proteolytic role for ubiquitin in tat-mediated transactivation of the hiv-1 promoter pja2 ubiquitinates the hiv-1 tat protein with atypical chain linkages to activate viral transcription trim32 senses and restricts influenza a virus by ubiquitination of pb1 polymerase the trimendous role of trims in virus-host interactions proteasomal degradation of herpes simplex virus capsids in macrophages releases dna to the cytosol for recognition by dna sensors simian trim5α proteins reduce replication of herpes simplex virus the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 the hpv-16 e6 and e6-ap complex functions as a ubiquitin-protein ligase in the ubiquitination of p53 rotavirus nsp1 inhibits nfκb activation by inducing proteasome-dependent degradation of β-trcp: a novel mechanism of ifn antagonism putative e3 ubiquitin ligase of human rotavirus inhibits nf-κb activation by using molecular mimicry to target β-trcp downregulation of major histocompatibility complex class i molecules by kaposi's sarcoma-associated herpesvirus k3 and k5 proteins kaposi's sarcoma-associated herpesvirus k3 utilizes the ubiquitin-proteasome system in routing class major histocompatibility complexes to late endocytic compartments herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated pml and sp100 proteins pml contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by icp0 inhibitors of deubiquitinating enzymes block hiv-1 replication and augment the presentation of gag-derived mhc-i epitopes proteasome inhibition interferes with gag polyprotein processing, release, and maturation of hiv-1 and hiv-2 retroviruses have differing requirements for proteasome function in the budding process proteasome inhibitors block hiv-1 replication by affecting both cellular and viral targets hiv-1: fifteen proteins and an rna hiv-1 replication hiv interaction with human host: hiv-2 as a model of a less virulent infection on the whereabouts of hiv-1 cellular entry and its fusion ports hiv-1 reverse transcription hiv dna integration. cold spring harb hiv-1 assembly, budding, and maturation. cold spring harb serinc3 and serinc5 restrict hiv-1 infectivity and are counteracted by nef hiv-1 nef promotes infection by excluding serinc5 from virion incorporation ifitm proteins incorporated into hiv-1 virions impair viral fusion and spread the ifitm proteins inhibit hiv-1 infection the v3 loop of hiv-1 env determines viral susceptibility to ifitm3 impairment of viral infectivity ifitm proteins are incorporated onto hiv-1 virion particles and negatively imprint their infectivity ifitm proteins restrict hiv-1 infection by antagonizing the envelope glycoprotein resistance of transmitted founder hiv-1 to ifitm-mediated restriction ring domain mutations uncouple trim5α restriction of hiv-1 from inhibition of reverse transcription and acceleration of uncoating specific recognition and accelerated uncoating of retroviral capsids by the trim5α restriction factor the cytoplasmic body component trim5α restricts hiv-1 infection in old world monkeys mx2 is an interferon-induced inhibitor of hiv-1 infection human mx2 is an interferon-induced post-entry inhibitor of hiv-1 infection restriction of hiv-1 requires the n-terminal region of mxb as a capsid-binding motif but not as a nuclear localization signal the highly polymorphic cyclophilin a-binding loop in hiv-1 capsid modulates viral resistance to mxb host and viral determinants of mx2 antiretroviral activity host and viral determinants for mxb restriction of hiv-1 infection samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx samhd1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein the cytidine deaminase cem15 induces hypermutation in newly synthesized hiv-1 dna broad antiretroviral defence by human apobec3g through lethal editing of nascent reverse transcripts codon-usage-based inhibition of hiv protein synthesis by human schlafen 11 non-human primate schlafen11 inhibits production of both host and viral proteins hiv-1 modulates the trna pool to improve translation efficiency hiv-1 vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu the interferon-induced protein bst-2 restricts hiv-1 release and is downregulated from the cell surface by the viral vpu protein march8 inhibits hiv-1 infection by reducing virion incorporation of envelope glycoproteins the antagonism of hiv-1 nef to serinc5 particle infectivity restriction involves the counteraction of virion-associated pools of the restriction factor the antiretroviral enzyme apobec3g is degraded by the proteasome in response to hiv-1 vif transcriptional regulation of apobec3 antiviral immunity through the cbf-β/runx axis hiv-1 vif blocks the antiviral activity of apobec3g by impairing both its translation and intracellular stability vpx relieves inhibition of hiv-1 infection of macrophages mediated by the samhd1 protein the ability of primate lentiviruses to degrade the monocyte restriction factor samhd1 preceded the birth of the viral accessory protein vpx vpu binds directly to tetherin and displaces it from nascent virions early replication block of human immunodeficiency virus type 1 in monkey cells restriction of hiv-1 (subtype b) replication at the entry step in rhesus macaque cells recruitment and dynamics of proteasome association with rhtrim5α cytoplasmic complexes during hiv-1 infection trim5α associates with proteasomal subunits in cells while in complex with hiv-1 virions proteasomal degradation of trim5α during retrovirus restriction trim5α-mediated ubiquitin chain conjugation is required for inhibition of hiv-1 reverse transcription and capsid destabilization fates of retroviral core components during unrestricted and trim5-restricted infection proteasome inhibition reveals that a functional preintegration complex intermediate can be generated during restriction by diverse trim5 proteins an innate immune sensor for the retrovirus capsid lattice ring dimerization links higher-order assembly of trim5α to synthesis of k63-linked polyubiquitin inhibition of mhc class ii expression and immune responses by c-mir ubiquitination by the membrane-associated ring-ch-8 (march-8) ligase controls steady-state cell surface expression of tumor necrosis factor-related apoptosis inducing ligand (trail) receptor 1 the e3 ubiquitin ligase march8 negatively regulates il-1β-induced nf-κb activation by targeting the il1rap coreceptor for ubiquitination and degradation march ubiquitin ligases alter the itinerary of clathrin-independent cargo from recycling to degradation family-wide comparative analysis of cytidine and methylcytidine deamination by eleven human apobec proteins apobec3f properties and hypermutation preferences indicate activity against hiv-1 in vivo dna deamination mediates innate immunity to retroviral infection virion-associated uracil dna glycosylase-2 and apurinic/apyrimidinic endonuclease are involved in the degradation of apobec3g-edited nascent hiv-1 dna induction of apobec3g ubiquitination and degradation by an hiv-1 vif-cul5-scf complex a zinc-binding region in vif binds cul5 and determines cullin selection phosphorylation of a novel socs-box regulates assembly of the hiv-1 vif-cul5 complex that promotes apobec3g degradation the hdac6/apobec3g complex regulates hiv-1 infectiveness by inducing vif autophagic degradation mdm2 is a novel e3 ligase for hiv-1 vif core binding factor β protects hiv, type 1 accessory protein viral infectivity factor from mdm2-mediated degradation vif hijacks cbf-β to degrade apobec3g and promote hiv-1 infection t-cell differentiation factor cbf-β regulates hiv-1 vif-mediated evasion of host restriction hiv-1 vif binds to apobec3g mrna and inhibits its translation translational regulation of apobec3g mrna by vif requires its 5'utr and contributes to restoring hiv-1 infectivity human and rhesus apobec3d, apobec3f, apobec3g, and apobec3h demonstrate a conserved capacity to restrict vif-deficient hiv-1 the activity spectrum of vif from multiple hiv-1 subtypes against apobec3g, apobec3f, and apobec3h suppression of apobec3-mediated restriction of hiv-1 by vif regulation of apobec3f and human immunodeficiency virus type 1 vif by vif-cul5-elonb/c e3 ubiquitin ligase hiv-1 restriction factor samhd1 is a deoxynucleoside triphosphate triphosphohydrolase aicardi-goutieres syndrome gene and hiv-1 restriction factor samhd1 is a dgtp-regulated deoxynucleotide triphosphohydrolase tight interplay among samhd1 protein level, cellular dntp levels, and hiv-1 proviral dna synthesis kinetics in human primary monocyte-derived macrophages samhd1 restricts hiv-1 infection in resting cd4(+) t cells the human immunodeficiency virus type 2 vpx protein usurps the cul4a-ddb1 dcaf1 ubiquitin ligase to overcome a postentry block in macrophage infection role of samhd1 nuclear localization in restriction of hiv-1 and sivmac the vpx lentiviral accessory protein targets samhd1 for degradation in the nucleus ddb1 and cul4a are required for human immunodeficiency virus type 1 vpr-induced g2 arrest hiv-1 vpr-mediated g2 arrest involves the ddb1-cul4avprbp e3 ubiquitin ligase premature activation of the slx4 complex by vpr promotes g2/m arrest and escape from innate immune sensing slx4-slx1 protein-independent down-regulation of mus81-eme1 protein by hiv-1 viral protein r (vpr) complex evolutionary history of primate lentiviralvprgenes evolutionary toggling of vpx/vpr specificity results in divergent recognition of the restriction factor samhd1 cell cycle control and hiv-1 susceptibility are linked by cdk6-dependent cdk2 phosphorylation of samhd1 in myeloid and lymphoid cells cd81 association with samhd1 enhances hiv-1 reverse transcription by increasing dntp levels cyclin l2 is a critical hiv dependency factor in macrophages that controls samhd1 abundance structural basis of hiv-1 tethering to membranes by the bst-2/tetherin ectodomain mechanism of hiv-1 virion entrapment by tetherin hiv-1 vpu protein antagonizes innate restriction factor bst-2 via lipid-embedded helix-helix interactions vpu antagonizes bst-2-mediated restriction of hiv-1 release via β-trcp and endo-lysosomal trafficking antagonism of tetherin restriction of hiv-1 release by vpu involves binding and sequestration of the restriction factor in a perinuclear compartment characterization of e3 ligases involved in lysosomal sorting of the hiv-1 restriction factor bst2 a novel human wd protein, h-β trcp, that interacts with hiv-1 vpu connects cd4 to the er degradation pathway through an f-box motif hiv-1 accessory protein vpu internalizes cell-surface bst-2/tetherin through transmembrane interactions leading to lysosomes vpu directs the degradation of the human immunodeficiency virus restriction factor bst-2/tetherin via a {β}trcp-dependent mechanism hiv-1 vpu neutralizes the antiviral factor tetherin/bst-2 by binding it and directing its β-trcp2-dependent degradation hiv-1 vpu utilizes both cullin-ring ligase (crl) dependent and independent mechanisms to downmodulate host proteins serine phosphorylation of hiv-1 vpu and its binding to tetherin regulates interaction with clathrin adaptors β-trcp is dispensable for vpu's ability to overcome the cd317/tetherin-imposed restriction to hiv-1 release berlioz-torrent, c. lc3c contributes to vpu-mediated antagonism of bst2/tetherin restriction on hiv-1 release through a non-canonical autophagy pathway hiv-1 vpu antagonizes cd317/tetherin by adaptor protein-1-mediated exclusion from virus assembly sites berlioz-torrent, c. the escrt-0 component hrs is required for hiv-1 vpu-mediated bst-2/tetherin down-regulation guanylate binding protein (gbp) 5 is an interferon-inducible inhibitor of hiv-1 infectivity identification of potential hiv restriction factors by combining evolutionary genomic signatures with functional analyses regulation of virus release by the macrophage-tropic human immunodeficiency virus type 1 ad8 isolate is redundant and can be controlled by either vpu or env the envelope glycoprotein of human immunodeficiency virus type 2 enhances viral particle release: a vpu-like factor? the human immunodeficiency virus (hiv) type 2 envelope protein is a functional complement to hiv type 1 vpu that enhances particle release of heterologous retroviruses nef proteins from simian immunodeficiency viruses are tetherin antagonists species-specific activity of siv nef and hiv-1 vpu in overcoming restriction by tetherin/bst2 the evolution of pandemic and non-pandemic hiv-1 strains has been driven by tetherin antagonism human immunodeficiency virus type 1 vpu protein induces rapid degradation of cd4 cd4 glycoprotein degradation induced by human immunodeficiency virus type 1 vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway requirements for the selective degradation of cd4 receptor molecules by the human immunodeficiency virus type 1 vpu protein in the endoplasmic reticulum multilayered mechanism of cd4 downregulation by hiv-1 vpu involving distinct er retention and erad targeting steps transmembrane domain determinants of cd4 downregulation by hiv-1 vpu cd4 and bst-2/tetherin proteins retro-translocate from endoplasmic reticulum to cytosol as partially folded and multimeric molecules hiv-1 vpu downmodulates icam-1 expression, resulting in decreased killing of infected cd4(+) t cells by nk cells cell surface proteomic map of hiv infection reveals antagonism of amino acid metabolism by vpu and nef hiv-1 vpr loads uracil dna glycosylase-2 onto dcaf1, a substrate recognition subunit of a cullin 4a-ring e3 ubiquitin ligase for proteasome-dependent degradation the ddb1-dcaf1-vpr-ung2 crystal structure reveals how hiv-1 vpr steers human ung2 toward destruction human immunodeficiency virus type 1 vpr induces the degradation of the ung and smug uracil-dna glycosylases the hiv-1 accessory protein vpr induces the degradation of the anti-hiv-1 agent apobec3g through a vprbp-mediated proteasomal pathway the hiv-1 protein vpr targets the endoribonuclease dicer for proteasomal degradation to boost macrophage infection hiv-1 vpr degrades the hltf dna translocase in t cells and macrophages hiv-1 vpr protein enhances proteasomal degradation of mcm10 dna replication factor through the cul4-ddb1[vprbp] e3 ubiquitin ligase to induce g2/m cell cycle arrest hiv-1 vpr induces the degradation of zip and szip, adaptors of the nurd chromatin remodeling complex, by hijacking dcaf1/vprbp hiv-1 vpr protein induces proteasomal degradation of chromatin-associated class i hdacs to overcome latent infection of macrophages the ubiquitin-proteasome system in hiv replication: potential targets for antiretroviral therapy inhibition of vpx-mediated samhd1 and vpr-mediated host helicase transcription factor degradation by selective disruption of viral crl4 (dcaf1) e3 ubiquitin ligase assembly key: cord-103662-a4ok5wqc authors: tarek, m.; elhefnawi, m.; maricato, j. t.; diaz, r. s.; shytaj, i. l.; savarino, a. title: custommune: a web tool to design personalized and population-targeted vaccine epitopes date: 2020-04-29 journal: nan doi: 10.1101/2020.04.25.20079426 sha: doc_id: 103662 cord_uid: a4ok5wqc computational prediction of immunogenic epitopes is a promising platform for therapeutic and preventive vaccine design. a potential target for this strategy is human immunodeficiency virus (hiv-1), for which, despite decades of efforts, no vaccine is available. in particular, a therapeutic vaccine devised to eliminate infected cells would represent a key component of cure strategies. hiv peptides designed based on individual viro-immunological data from people living with hiv/aids have recently shown able to induce post-therapy viral set point abatement. however, the reproducibility and scalability of this method is curtailed by the errors and arbitrariness associated with manual peptide design as well as by the time-consuming process. we herein introduce custommune, a user-friendly web tool to design personalized and population-targeted vaccines. when applied to hiv-1, custommune predicted personalized epitopes using patient specific human leukocyte antigen (hla) alleles and viral sequences, as well as the expected hla-peptide binding strength and potential immune escape mutations. of note, custommune predictions compared favorably with manually designed peptides administered in a recent phase ii clinical trial (nct02961829). furthermore, we utilized custommune to design preventive vaccines targeted for populations highly affected by covid-19. the results allowed the identification of peptides tailored for each population and predicted to elicit both cd8+ t-cell immunity and neutralizing antibodies against structurally conserved epitopes of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). overall, our data describe a new tool for rapid development of personalized or population-based immunotherapy against chronic and acute viral infections. the rapid development of automated platforms for data generation and analysis are increasingly making precision medicine a concrete option for several diseases. due to its potential for high selectivity and efficacy, immunotherapy is an optimal choice for the design of personalized therapeutic interventions 1 . while most efforts in this direction have focused on cancer 1-3 , viral infections can be a relevant application as well, particularly chronic infections characterized by extensive genetic diversity, in part due to in-host viral evolution. human immunodeficiency virus (hiv-1) is case in point, as the large number of circulating strains and its high replicative mutation rates have hampered the development of effective vaccines, both preventive and therapeutic 4, 5 . several lines of evidence highlight the relevance of immune control in hiv-1 infection. spontaneous long-term control of hiv-1 replication can be accompanied by the presence of broadly neutralizing antibodies 6, 7 or, more frequently, effective cell-mediated immune responses 8 . moreover, protective class i hla alleles have been identified both in people living with hiv/aids (plwha) and macaques infected with the hiv homolog simian immunodeficiency virus (siv) [9] [10] [11] [12] [13] [14] . in line with this, temporary depletion of cd8 + t-cells is associated with a rapid viral load increase, while their replenishment can revert this effect [15] [16] [17] [18] . a therapeutic vaccine based on cell-mediated immunity might offer the advantage of decreasing the number of infected cells. on the one hand, hiv-1 latently infected cells, which constitute the main barrier to a cure [19] [20] [21] , are not targeted by antiretroviral drugs or cd8 + t-cells 22 . on the other hand, effective cell-mediated immune responses could preserve drug-free control of the infection by keeping viral load low/undetectable and by eliminating the infected cells undergoing spontaneous hiv-1 reactivation from latency. such therapeutic vaccines could also be combined with strategies aimed at purging the hiv-1 latent reservoirs by inducing pharmacologic reactivation of latently infected cells 23 . the strong correlation between the host's genetic background and immune-mediated control of the infection suggests that effective immunity is mainly directed against a subset of hiv-1 epitopes. consistently, several studies have shown that cell-mediated immune responses against the hiv-1 gag protein correlate with lower viral loads in plwha and with post-therapy control of the infection in macaques 18, [24] [25] [26] [27] . the peculiar efficacy of anti-gag immunity might be partially explained by the higher fitness cost associated with mutations in this viral protein 28 . in particular, specific regions of gag, which are essential for hiv-1 packaging and assembly, are structurally and evolutionarily conserved, displaying low shannon entropy both in humans and primate lentiviruses 29 . however, it is noteworthy that low diversity is not sufficient per se to induce viral load control, as vaccine approaches designed exclusively by selecting epitopes based on their evolutionary conservation have shown only modest effects 30, 31 . a recent phase ii clinical trial (nct02961829) has attempted to induce anti-gag immunity against conserved epitopes using a personalized approach based on patient hla sequences 32 . although the study enrolled only a small number of plwha and tested multiple interventions, preliminary results suggest that therapeutic vaccination with autologous dendritic cells pulsed with individually designed peptides decreased the viral set point in some patients during analytical treatment interruption (ati) 32 . in the present work we describe and test a new automated, user-friendly web-based tool to design personalized peptides for vaccination. the tool, named custommune, was principally interrogated to develop therapeutic vaccine candidates for hiv-1. to this aim, by intersecting input data from patient-specific viral sequences and hla alleles, custommune provides an output of epitopes of desired length filtered for their predicted specificity, immunogenicity and mutation potential. of note, in our simulations, custommune performance was superior to that of manual vaccine design (applied in clinical trial nct02961829) in terms of prediction of clinical response. one advantage of custommune over traditional vaccine design techniques is the ability to quickly adapt the tool for different targets and strategies. in this regard, we applied custommune to the novel pandemic covid19, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) 33 . due to the acute manifestations of the disease, the design of a population-targeted preventive vaccine was chosen as a more practical approach as compared to a personalized therapeutic vaccine. moreover, to broaden the expected coverage and increase the likelihood of achieving herd immunity 34 , a strategy able to potentially evoke both neutralizing antibodies and cell-mediated immunity was preferred. using input regions of sars-cov-2 identified as viable targets, custommune was able to design vaccine candidates specific for regionally prevalent hla genotypes. in addition, custommune selected those hla class ii restricted epitopes that could induce neutralizing antibodies and thus provide a two-layered protection against the infection. taken as a whole, our results show the potential of the custommune algorithm to quickly design personalized or population-specific peptides for preventive and therapeutic vaccination. due to its intuitive and scalable approach, custommune might provide an effective tool for rapid vaccine development against chronic and acute conditions. the custommune web tool (available at: http://www.custommune.com) was written in python (http://www.python.org) using the django framework (https://www.djangoproject.com) and provides the user with an easy online interface for accessing and downloading prediction datasets without any coding knowledge requirements. the tool utilizes a pipeline ( figure 1 ) to design epitopes for preventive and therapeutic vaccines. for hiv-1 therapeutic vaccine design, custommune crosses input data from patient-specific viral sequences (dna in fasta format or raw dna sequencing inputs) and patient's hla-i and/or hla-ii alleles, providing an output of epitopes of desired k-mer length. to facilitate the allele input step, the tool provides two links directing the user to a list of supported class i and class ii hla alleles, respectively. these lists mirror those of the netmhcpan 4.0 algorithm 35 , for either hla class. although the approach could potentially be extended to encompass entire hiv-1 sequences, we decided to limit the search for viable epitopes to the gag gene only, because of the previously described distinctive efficacy of anti-gag cell-mediated immunity 18, [24] [25] [26] [27] . the tool pipeline ( figure 1 ) starts by translating input gag genomic sequences to protein sequences. custommune then performs multiple sequence alignment using the clustal omega (rest) web service python client 36 and builds a consensus translated sequence. the consensus sequence is then used to predict epitopes restricted to patient-specific hla-alleles for both classes. the hla-specific epitopes provided as final output by custommune are pre-filtered by the algorithm. this pre-filtering follows a set of parameters that compute epitope affinity in terms of sequence variation and conservation degree, allele-restricted affinities, and previous clinical evidence of immune response ( figure 1 ). for calculating evolutionary conservation, each epitope is compared, in terms of similarity, to an internal database of gag amino acid sequences (supplementary file 1) collected mainly from curated alignments retrieved from the los alamos hiv sequence database (http://www.hiv.lanl.gov/). moreover, to verify whether antigenicity has already been reported for the candidate epitopes, the tool compares potential epitopes to those already described in the los alamos hiv immunology site (http://www.hiv.lanl.gov/content/immunology). to further refine the structural assessment of epitope binding to hla-alleles, custommune performs structural epitope modelling followed by epitope-hla docking to determine the structural stability of the hla-predicted epitope binding ( figure 1 ). the custommune pipeline also computes some related physicochemical parameters of the personalized epitope sequence to aid in the assessment of the structural stability of candidate peptides. overall, the tool is optimized to identify immunogenic peptides characterized by the lowest variability (mutation potential). in line with this, the tool specifically highlights potential epitopes that are contained in regions which were previously described as essential for viral fitness 29, 37 . this is a novel and fundamental feature of this approach, as rna viruses are characterized by a high ability to mutate 38 . the custommune pipeline can be applied to other vaccine strategies by following a parallel workflow ( figure 1 ). an example of these applications are acute infections, such as covid-19. in this case, an approach combining neutralizing antibody responses and recognition by hla haplotypes most represented in a given population might provide a reasonable compromise between specificity and scalability. to this aim, using bepipred-2.0 39 , custommune can identify potential neutralizing epitopes which overlap with epitopes consistent with recognition by population-specific class ii hla haplotypes. at the same time, custommune can predict another set of epitopes optimized for recognition by hla class i haplotypes of the same population, thus providing two levels of potential immune recognition. overall, the custommune pipeline provides a flexible and fast tool to generate epitope predictions according to the genetic diversity of the virus and the genetic hla profile/s of the host or susceptible populations. we tested custommune predictions against manual epitope selection using results from an ongoing multi-interventional phase ii clinical trial enrolling plwha (nct02961829) 32 . in this trial, autologous dendritic cells were pulsed with a personalized vaccine designed manually from gag sequences generated from each patient´s circulating virus. in the study groups (g5 and g6) that had received this vaccine (along with other interventions), the patients showed variable responses including two individuals who displayed significant control of viral load during ati 32 . when viral and hla sequences of patients from g5 and g6 were used as input for custommune, the epitopes predicted by the tool generally displayed some overlap with those administered in the study ( figure 2a) . therefore, to investigate the potential therapeutic efficacy of custommune predictions, we stratified patients based on the virologic response during ati, which was defined as > 1 log10 ∆ viral load set point (i.e. the difference between median pre-and post-therapy copies of hiv-1 rna/ml of plasma). of note, non-responders were the only patients for whom there was no overlapping prediction between epitopes calculated by custommune and those administered in vivo ( figure 2b ). conversely, patients who had been administered vaccine epitopes highly overlapping (>50%) with those predicted by custommune, were characterized by higher viral load abatement ( figure 2c ). these data suggest that custommune can predict epitopes with therapeutic potential and could improve both efficacy and speed of personalized vaccine design. as the ongoing covid-19 outbreak is an urgent challenge for vaccine development 40 , we decided to test the potential of custommune for rapid identification of vaccine targets. in order to utilize custommune for sars-cov-2 predictions we first decided to identify the viral regions that could act as optimal input for the tool. due to the recent evolution of sars-cov-2, there is no equivalent of hiv-1 gag, i.e. a validated viral target for effective immunity. however, sars-cov-2 shares approximately 80% sequence identity with sars-cov 41 , the causative agent of an epidemic burst of acute respiratory distress syndrome (ards) in 2003. therefore, we decided to use previously described strategies successfully targeting sars-cov replication as a template to restrict custommune predictions. in particular, our efforts were directed at two validated sub-targets within the s-glycoprotein necessary for viral attachment to host cells 42 : 1) the portion of the s-glycoprotein that mediates the main protein-protein interaction with the cellular entry receptor, i.e. angiotensin converting enzyme 2 (ace2), as this was described as an optimal target for neutralizing antibodies 43 ; 2) the viral s-glycoprotein region binding the glycosylated portion of ace2, an interaction inhibited by pretreatment with chloroquine 44, 45 , a drug recently shown to effectively hamper sars-cov-2 replication in vitro and in patients 46, 47 . in order to translate these approaches into vaccine design: 1) we performed a thorough analysis for molecular complexes of the viral s-glycoprotein with the entry receptor ace2. considering the configuration of ace2, we superimposed complexes of s-glycoprotein/ace2 in both states of the receptor, i.e. free or bound (in this case with the competitive inhibitor mln-4760) 48 . our analyses indicated that the receptor-binding domain (rbd) surface of s-glycoprotein interacting with the bound configuration of ace2 is relatively smaller than (though 100% overlapping with) that interacting with the unbound configuration of ace2 ( figure 3a ,b). in light of this, we decided to restrict the custommune input to the rbd sequence interacting with bound ace2 and the linker amino acids (henceforth rbdp) ( figure 3a ,b). it is expected that this approach will be able to evoke antibodies against the rbdp irrespective of the ace2 bound/unbound configuration. 2) we inspected the possible contribution of oligosaccharide moieties of ace2 to the sglycoprotein/ace2 binding interface. the oligosaccharide moiety of ace2 was described as fundamental for optimal binding of the s-glycoprotein of sars-covs 44 . so far, in published structures, only partial ace2-bound oligosaccharide data is available. therefore, we decided to study this phenomenon by analyzing a published structure of inhibitor-bound ace2 (1r4l), which presents an n-acetylglucosamine (nag) covalently bound to residue asn90 and remaining from the oligosaccharide originally attached to this protein 49 . this evidence suggests that the nag present in the 1r4l structure is a marker of the position of the oligosaccharide originally attached to ace2 before being altered by the crystallization process. by superimposing this structure to the structure of the s-glycoprotein with ace2 and measuring the atomic distances at the binding interface between nag and the s-glycoprotein, we were able to determine the specific segment of the s-glycoprotein rbd that could be responsible for the interaction with the ace2-bound oligosaccharide. two specific residues of the s-glycoprotein (gly416 -lys417) were found to interact with nag, being within a 10 å radius from nag, i.e. a distance associated with significant intermolecular interactions ( figure 3c ,d). using s-glycoprotein gly416 as a starting point, we selected a core peptide spanning 20 amino acids in both directions of the translation frame. this led to the identification of a segment of the s-glycoprotein rbd, which we henceforth name rbdg, as a bona fide target for vaccine epitope design ( figure 4a ). of note, a structure of the sars-cov-2 s-glycoprotein and ace2 interaction (pdb: 6m17) was recently published while the present report was in preparation 50 . the authors concluded that the binding interface to ace2 is similar for sars-cov and sars-cov2, and their conclusions are largely overlapping with the results of the present analyses. overall, this evidence shows that the rbdp and rbdg dna sequences of sars-cov-2 can be used as optimal inputs for custommune. to mimic the approach described for hiv-1, we first analyzed the variability of rbdp and rbdg by multiple alignment of all sars-cov-2 s-glycoprotein sequences available at ncbi and gisaid (including isolates from humans, bats and pangolins) (supplementary file 2, 3 and figure 4a ). in line with the predicted key structural role of rbdp and rbdg, both sequences displayed very limited variability, mostly deriving from non-human isolates (supplementary file 2 and 3) . moreover, every amino acid variant (except one in rbdg) fully preserved the main physicochemical characteristics of the consensus residue (according to the scoring system of 51 ). these results suggest that both rbdp and rbdg represent bona fide equivalents of the conserved gag sequences used as privileged targets for custommune hiv-1 predictions. to adapt custommune predictions to some of the populations most affected by the sars-cov-2 pandemic (at the time at which these analyses were performed), we retrieved the relative hla allele frequencies in individuals from northern italy and south korea (supplementary file 4) (allele frequency net database; http://www.allelefrequencies.net) 52 . moreover, we applied the same approach to hla alleles of individuals from southern china and from the city of wuhan, where the outbreak had initially spread (supplementary file 4). when the rbdp and rbdg sequences were used as inputs along with population-specific hlas, custommune returned a set of epitopes (supplementary file 5) for either class i or class ii hlas. the hla class ii specific epitopes were further filtered to highlight those predicted as targets for . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . neutralizing antibodies using bepipred-2.0 39 . this was done to ensure that a unique peptide may provide the double stimulus necessary for optimal b-cell activation and antibody production (supplementary file 5). in line with the custommune pipeline, and in order to improve the likelihood of immune recognition, the binding stability and affinity of the most promising epitopes was validated by molecular docking. in particular, epitopes were selected for docking if they had been predicted to bind with an ic50 < 600 nm 53 to hla alleles described at four digit resolution for the population of interest in the allele frequency net database (supplementary file 5 and figure 4b ,c). interestingly, the identified epitopes included key residues involved in hydrogen bond formation between the s-glycoprotein of sars-cov-2 and ace2 (e.g. gln 474 in epitope steiyqagstpcngveg, gln498 in epitope lqsygfqp and lys417 in epitope irgdevrqiapgqtgkiadynyklpd of s-glycoprotein, engaged, respectively, in hydrogen bonds with residues gln24, tyr41, asp30 of ace2). since hydrogen bonds were recently described as crucial for the stability of the virus-receptor interaction 50 , epitopes containing the hydrogen-bonding residues might be particularly suitable targets to evoke immunity against structural determinants of sars-cov-2 infection. moreover, in order to ensure the best coverage likelihood of the target population, we also included the predicted epitopes for the most prevalent class i hla antigens. our results show that a peptide set specific for both neutralizing antibody/hla class ii and for hla class i could provide a good population coverage upon simultaneous delivery, potentially achieving herd immunity (fig 4c) . of note, one of the most promising epitope candidates designed by custommune for two of the populations examined (i.e. epitope klpddftgc for southern china/wuhan and northern italy) (supplementary file 5 and figure 4c ) is equivalent to a highly immunogenic peptide previously identified by stimulating cells of patients who had successfully recovered from sars infection 54 . taken as a whole, these results show the application of custommune to predict epitopes for specific populations and highlight a set of vaccine candidates to curb the spread of sars-cov-2 in highly affected areas. the precision medicine era, albeit still in its early stages, is expected to supersede traditional, onesize-fits-all therapeutic approaches. the development of personalized, yet scalable, treatments would allow accounting for the genetic variability of individuals, pathogens, or cancer profiles, and pave the way for more accurate efficacy predictions while reducing side effects. the implementation of our custommune pipeline in the context of hiv/aids shows that the tool algorithm may be used to predict novel immune-based treatments with in-vivo potential. even though the pipeline was applied to the hiv-1 gag protein in the present work, it can potentially be extended to other hiv genomic regions or other chronic viral infections. crucial prerequirements of the personalized custommune approach are the identification of a key structural component of the target pathogen and the obtainment of sequencing data from both the host hla . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . alleles and the infecting virus. while cost considerations might represent a limiting factor in some settings, the quick advances in sequencing technology, coupled to the steep reduction in price 55 , make the approach already feasible in developed countries. moreover, a personalized intervention aimed at a cure could make the cost-benefit analysis attractive also in developing countries, which often bear the main burden of chronic viral infections 56 . in terms of potential efficacy, the custommune approach relies on minimizing epitope diversity while maximizing predicted binding strength and immunogenicity of said epitopes. it is noteworthy that, when compared to a real clinical scenario, epitopes predicted by custommune correlated with treatment response. this was likely aided by the large amount of immunologic data available on hiv-1 (e.g. los alamos hiv immunology site). therefore, due to its low cost and scalability, custommune could be immediately applied to the design of therapeutic hiv peptide vaccines 57 or autologous dendritic cell vaccines pulsed with tailored gag peptides. compared to previous attempts at streamlining vaccine design in the context of cancer 58 , the custommune pipeline includes multiple layers of epitope ranking with scoring parameters accounting for: mutational potential, structural conservation, hla docking, escape mutations, location of the neomutation and previous evidence of antigenicity. these partially redundant filtration stages are envisaged to maximize the chances for durable and potent epitope recognition. moreover, other filters such as predicted epitope processing and cleavage have been included to the pipeline when this manuscript was in preparation, confirming the versatility of the custommune approach. our implementation of custommune was here extended to include vaccine design for sars-cov-2. current predictions suggest that traditional vaccine strategies might be too slow to address the spread of the pandemic and mitigate the death toll 59 . furthermore, immune responses developed during natural infection might be insufficient to provide long-term protection against reinfection 60 . the approach herein proposed is aimed at a flexible response customized for the populations most affected at a given time. as a novel pathogen will necessarily lack the wealth of immunologic data available for heavily studied viruses like hiv-1, our vaccine strategy attempts both induction of cell-mediated immunity and neutralizing antibodies. indeed, early evidence indicates that a broad immune response might correlate with successful clearance of the infection 61 . custommune predicted epitopes would further combine this broad immune stimulation with a design based on the most common hla alleles in the population of interest, potentially providing enough immune coverage for the induction of herd immunity. moreover, the choice of a highly conserved viral target as a source of vaccine epitopes should ensure a broadly effective response in those individuals for whom the vaccine should prove immunogenic. by utilizing custommune, the whole vaccine design process should last less than a working day. therefore, this approach, if successful, could be quickly adopted to blunt the pandemics during its spread or, ideally to preempt it. the binding affinities predicted by custommune for epitopes derived from rbdp and rbdg were generally higher for hla class i alleles in the populations here considered. while this is not sufficient to predict that cell mediated immunity would be preferentially induced by the proposed vaccine, previous evidence in mice suggests that memory cd8 + t-cells might alone be sufficient . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . to provide effective protection against sars-cov 62 . corroborating this hypothesis, one of the peptides designed by custommune was equivalent to an epitope associated with clearance of sars-cov infection during the previous epidemics and with immunogenicity in mice when used as a vaccine 54 . our estimate of the probability to reach herd immunity in the populations considered is based on the assumption that the development of immune responses against each of the vaccine peptides would be per se sufficient to guarantee some level of protection. while this prerequisite might prove optimistic, it is noteworthy that the viral targets selected for vaccine design (i.e. rbdp and rbdg of the s-glycoprotein) display exceptional evolutionary conservation and that no polymorphism in these regions was detected in the viral isolates from either italy, south korea or china. this conservation, coupled with the generally moderate mutation rate of sars-cov 63 and sars-cov-2 64 as compared to other rna viruses, yields credibility to the idea of achieving protection by targeting single immunodominant epitopes 62 . moreover, the expected population coverage of each of the vaccines designed in the present study is theoretically sufficient to achieve herd immunity based on the estimated reproductive number of sars-cov-2 34,65 . in the current work, to simplify administration schedule and increase scalability, we envisage, among other possibilities, a strategy synthesizing one multi-epitope peptide for each target population. this peptide would link class ii hla-restricted and neutralizing antibody epitopes as well as class i hla-restricted cd8 + t-cell epitopes. however, this approach will require empirical validation and could be modified, e.g. by administering hla class i and class ii restricted epitopes in separate formulations. while reduced immunogenicity is a well-known caveat of epitope-based vaccines, recent advances in adjuvant and delivery technology might allow overcoming this limitation 66 . apart from classical adjuvants, the use of an "adjuvant" drug such as chloroquine, is of particular interest for sars-cov-2. this treatment option could enhance vaccine immunogenicity 67,68 while possibly providing per se some protection against the virus 69 . in terms of delivery, carriers such as liposomes and nanoparticles, or strategies employing chemical conjugation or cell-penetrating peptides could increase epitope presentation by antigen presenting cells 66, 70 . finally, in our current model, we envisaged the use of linker sequences with protease cleavage sites between different epitopes 71 . this strategy might increase the chances of presenting peptides of optimal size to both hla alleles of class i and class ii. however, covalent linkage of epitopes has also been described to increase immunogenicity 66 . in-vivo studies will be required to optimize these strategies for inducing immunity against sars-cov-2. due to the ongoing rapid expansion of the epidemics and the relatively good safety profile of peptide vaccines 66 , pilot clinical testing in significantly affected areas might be envisaged. overall, our study describes a novel tool to improve multi-epitope vaccine design specificity while drastically reducing the associated time and cost. the pipeline herein described can be directly applied for testing personalized therapeutic vaccines for hiv-1 and to identify the core epitopes of preventive vaccines aimed at populations heavily affected by sars-cov-2. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04. 25.20079426 doi: medrxiv preprint the web application of custommune is available at http://www.custommune.com. written in python (v3.7) using django (v2.2.6) custommune is a tool that provides an integrated pipeline (figure 1 ) for prediction and filtration of personalized epitopes. the biopython package 72 is used for translating input sequences. alignment of translated sequences is then performed using the python client of clustal omega (rest) web service 36 . a consensus of the aligned sequences is generated using the biopython module with a 50% similarity cutoff. the biopython "proteinanalysis" function is used to estimate physicochemical parameters and secondary structure of the consensus sequence, including: molecular weight, gravity, specific count of amino acids, isoelectric point and fractions of secondary structures. custommune is connected with restful interface (iedb-api) 73 which serves as a platform for using netmhcpan v4.0 35 for class i and ii hla predictions as well as bepipred v2.0 39 for antibody epitope predictions. the pandas package (mckinney et al. 2010) is then used to structure epitope sorting tables and allow for comparative filtration. the primary filtration is based on ic50 values, a cutoff of 1000 nm is used to prevent loss of potentially false negatives. the los alamos hiv database (http://www.hiv.lanl.gov/content/immunology) was used to create internal hla class-specific datasets of previously reported immunogenic epitopes against hiv gag. using pandas 74 , high-affinity epitopes are compared to these datasets to highlight epitopes with previously described immunogenicity. moreover, another filtration layer is designed to report escape variants by comparing each epitope to an internal database collected from various literature sources including: dataset of hla-associated polymorphisms in hiv-1 gag as reported in ref. 75 , as well as the datasets reported in ref. 76 and the datasets of ctl/cd8 + and t helper/cd4 + epitope variants and escape mutations reported in the los alamos hiv database (http://www.hiv.lanl.gov/content/immunology/). additional filtration is obtained by comparing the epitope location within the gag sequence, to gag regions essential for viral assembly and packaging, which tend to be structurally and evolutionarily conserved, as reported in ref. 29 . to further refine this filtration, custommune computes the degree of conservation for each epitope by comparing the epitope sequence to the hiv sequence compendium database 77 which includes 680 alignments of hiv-1/sivcpz gag protein sequences. the degree of conservation (cscore) of each epitope is calculated as a fraction represented by the subset of sequences{ }in which the epitope scored a local alignment of more than 80% using clustal omega 36 over the total sequences in the internal database. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. the next layer of filtration selects only epitopes that rank high for multiple alleles in case a multiple-allele input was selected by the user for both hla classes. for further assessment of the impact of predictable mutations, custommune computes the effect of these mutations (retrieved from the internal gag sequence database; supplementary file 1) on the binding affinity of epitopes to the patient hlas. this refined analysis is performed only on the top three ranking epitopes initially predicted by the tool. by computing affinities to the same allele the user can estimate the impact of mutations in this specific segment on the affinity to the restricted allele. the degree of deviation of the mutated version is estimated based on , which are calculated as a standard deviation (sd) of the set of ic50 values for the candidate epitope and its mutant versions. the deviation value is therefore considered to negatively reflect the binding stability of this peptide segment to a restricted allele, in respect to a set of predicted mutant versions of the same segment. the python package peptidebuilder 78 is used for generation of 3d models of top epitopes, while the package lightdock 79,80 is implemented to perform epitope-hla docking based on the glowworm swarm optimization (gso) algorithm 81 . solved structures of hla alleles were collected from the phla3d database 82 and the protein data bank (pdb) 83 . homology modelling of structurally unsolved hla alleles was generated using swissmodel 84 . distance-scaled, finite ideal-gas reference (dfire) function 85 is used to calculate mean force potential of all atoms in a residue-specific manner within a resolution of less than 2 å, which has been found to accurately predict stabilities of structural (hla-epitope) complexes. dfire was implemented as a scoring function for lightdock simulations and docking scores were added in the final filtration layer for the highest ranking epitope candidates. for highly ranking epitope candidates, a scoring function is designed to account for each filtration layer. in this function each continuous parameter ( 50, , ) is represented by a quantitative value, according to the following rules: 1) the ic50 value is rescaled by calculating its reciprocal multiplied by a weighting factor of 10 4 ; 2) docking scores are preceded by a negative sign to weight the negative binding energies of the dfire scoring function of lightdock; 3) cscore is considered as a percentile of the cscore fraction weighted by a factor of 10 3 ; 4) sdaffinities are preceded by a negative sign to weight the positive values of deviation values. categorical parameters ( and ) are represented by binary values weighted by a factor of 500 for favorable states while non favorable states are given null values. overall the formula to calculate the final ranking (s) can be calculated as follows: s = 10000 * (ic50) -1 -dfire + escapem *500 + cscore *1000 + locationscore * 500 -sdaffinities + doverlap *500 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04. 25.20079426 doi: medrxiv preprint the top three epitopes ranked by s score are further analyzed based on their possible overlap with epitope data sets previously associated with: post-art control, efficacy in vaccine studies and the lack of reported escape mutations. finally, predicted antibody epitopes estimated by bepipred 2.0 39 are reported if they overlap with the top candidate epitopes ranked by s score. to allow manual inspection of results, sequence processing data and unfiltered predictions are provided in a separate section of the results page with a downloading link for a text file. s-glycoprotein sequences were retrieved from ncbi (https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/) and gisaid 86 . multiple alignments were performed using clustal omega web service 36 . consensus sequences and sequence conservation scores and histograms were generated with jalview (v. 2.11) 87 according to the amino acid conservation scoring criteria described in 51 . class i and ii hla allele frequencies were retrieved from the allele frequency net database (http://www.allelefrequencies.net/hla.asp) 52 using the "hla classical allele freq search" option. population-specific datasets (shown in supplementary file 4) were employed to identify the most represented hla alleles in areas heavily affected by sars-cov-2 spread, namely northern italy, south korea and china (wuhan and southern china). for all alleles analyzed, each data set provided values of frequency, which were determined as the number of copies of a given allele (x) divided by the total number of alleles in the population (of size n) assayed (i.e. frequency = x/2n). for each population of interest, only hla alleles with frequency ≥ 0.1 in at least one dataset of the same population were considered for further analysis. when a given hla allele was represented in more than one dataset of the same population, a weighted frequency was calculated. specifically, given an allele of interest represented in n datasets with population sizes n1, n2...nn, with a frequency of f1, f2...fn, the weighted frequency (fw) of the allele was calculated as: as the datasets employed included hlas characterized at different resolutions, allele frequencies were considered separately in case a 2 or ≥4 digit resolution 88 was available (supplementary file 4). alleles at 4 digit resolution and ≥ 0.1 (weighted) frequency were used as direct input for custommune. alleles at 2 digit resolution and ≥ 0.1 (weighted) frequency were instead analyzed with custommune by including all potential second field 88 options currently supported by custommune. class i and class ii hla alleles which were predicted by custommune to bind rbdp and rbdg epitopes of sars-cov-2 were used to estimate potential vaccine coverage in the populations of interest. to this aim, only (weighted) frequencies of hla alleles available at four digit resolution in the population of interest were included (supplementary file 4) . moreover, among these alleles, . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04. 25.20079426 doi: medrxiv preprint only those with high predicted binding affinity (ic50 < 600 nm) for an rbdp or rbdg epitope were included in the vaccine design (supplementary file 5) . to estimate the percentage of individuals (p) of a given population expected to carry an hla allele, the (weighted) frequency of that allele (f) in the same population (supplementary file 4) was used, according to the formula: for heterodimers (e.g. hla-dqa1 and dqb1) an overall frequency of the heterodimer was first calculated as: frequency of heterodimer 1 * frequency of heterodimer 2. this overall heterodimer frequency was then used to calculate p as described above. hla alleles of the population that were predicted to recognize more than one epitope of the vaccine were considered only once in the calculation of m. hiv-1 viral loads of individuals enrolled in trial nct02961829 were measured by q-pcr as described in 89 . clinical data were analyzed by unpaired t-test using graphpad prism (v. 6 graphpad software, la jolla california usa). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020 following these filtration layers, custommune identifies whether any predicted epitope displays highaffinity to multiple hla alleles and (final epitope filtration) discards any epitopes that have reported escape mutations and/or are not located in an evolutionary conserved region. (affinity robustness) among remaining candidates, custommune restricts further analyses on the three top scoring epitopes for both hla classes. for these, custommune computes the hla binding affinities of potential mutant versions, though not classified as escape mutations, to estimate the impact of these mutations on epitope recognition (sdaffinities). (hla-epitope docking) on the same three top ranking epitopes, custommune computes epitope-hla allele docking scores, calculated using the lightdock 79 python package and scored using the dfire 85 scoring function. (final output and annotation) in a parallel process, the bepipred 2.0 39 algorithm is implemented to predict neutralizing antibody epitopes from the initial consensus sequence, that can be further intersected with class ii restricted epitopes to increase immunogenicity. as a final output, for both class i and ii hlas, custommune ranks the top 3 epitopes according to a score (custoscore) which accounts for all aforementioned filtration parameters. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . by two-tailed student t-test. panel c) ∆ viral load set point in trial participants who received peptides with high or low overlap to custommune predictions (≥ 50% or < 50% overlap, respectively). the ∆ viral load set point was calculated as the difference between pre-and post-therapy viral load set points, with post-therapy viral load set point calculated as the median of all available measurements (up to 9 weeks post-treatment interruption). each data point in panels b and c indicates a trial participant. consensus sequence and evolutionary conservation were calculated based on the multiple sequence alignments in supplementary files 2 and 3 using jalview 87 . the conservation score is based on 51 . (b) example of epitope-hla docking pose generated using lightdock 79 . the custommune-predicted epitope "kiadynykl" (magenta) is shown restricted by the hla class i histocompatibility antigen a-2 α-chain (hla-a*02:01, green), which is highly expressed in northern italy (supplementary file 4) . also shown is the invariant β2-microglobulin (cyan). the docking pose was scored using the dfire function 85 as listed in supplementary file 5. (c) custommune vaccine predictions and expected coverage for each target population. predicted epitopes were selected from those on which docking was performed (supplementary file 5). maximum expected population coverage was calculated based on allele frequencies in each population (listed in supplementary file 4) according to the formula described in the "materials and methods" section. linker regions between vaccine peptides are an example of a vaccine strategy based on a single, multi-epitope, formulation. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 29, 2020. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04. 25.20079426 doi: medrxiv preprint r + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + * + + + + + + + + rbdp * * * + * 9 + * * + * + * * * * * * * * + * * * * * * * * * * * * + * * * + * * * personalized vaccines for cancer immunotherapy cancer precision medicine: from cancer screening to drug selection and personalized immunotherapy cancer stem cells: at the forefront of personalized medicine and immunotherapy advancing an hiv vaccine; advancing vaccinology therapeutic vaccination for hiv coexistence of potent hiv-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller neutralizing antibodies are positively associated with cd4+ t-cell counts and t-cell function in longterm aids-free infection the international hiv controllers study. the major genetic determinants of hiv-1 control affect hla class i peptide presentation influence of combinations of human major histocompatibility complex genes on the course of hiv-1 infection hla b*5701 is highly associated with restriction of virus replication in a subgroup of hiv-infected long term nonprogressors dominant influence of hla-b in mediating the potential co-evolution of hiv and hla two mhc class i molecules associated with elite control of immunodeficiency virus replication, mamu-b*08 and hla-b*2705, bind peptides with sequence similarity mamu-b*08-positive macaques control simian immunodeficiency virus replication characterization of the peptide binding motif of a rhesus mhc class i molecule (mamu-a*01) that binds an immunodominant ctl epitope from simian immunodeficiency virus cd8(+) lymphocytes are required for maintaining viral suppression in siv-infected macaques treated with short-term antiretroviral therapy dramatic rise in plasma viremia after cd8(+) t cell depletion in simian immunodeficiency virus-infected macaques cd8+-cell-mediated suppression of virulent simian immunodeficiency virus during tenofovir treatment two-year follow-up of macaques developing intermittent control of the human immunodeficiency virus homolog simian immunodeficiency virus sivmac251 in the chronic phase of infection presence of an inducible hiv-1 latent reservoir during highly active antiretroviral therapy identification of a reservoir for hiv-1 in patients on highly active antiretroviral therapy replication-competent noninduced proviruses in the latent reservoir increase barrier to hiv-1 cure cold spring harb shock and kill cd8+ t-cell responses to different hiv proteins have discordant associations with viral load gag-specific cytotoxic responses to hiv type 1 are associated with a decreased risk of progression to aids-related complex or aids relative dominance of gag p24-specific cytotoxic t lymphocytes is associated with human immunodeficiency virus control preferential ctl targeting of gag is associated with relative viral control in long-term surviving hiv-1 infected former plasma donors from china fitness cost of escape mutations in p24 gag in association with control of human immunodeficiency virus type 1 cell-mediated anti-gag immunity in pharmacologically induced functional cure of simian aids: a 'bottleneck effect'? therapeutic conserved elements (ce) dna vaccine induces strong t-cell responses against highly conserved viral sequences during simian-human immunodeficiency virus infection hiv-1 conserved-element vaccines: relationship between sequence conservation and replicative capacity post-therapy viral set-point abatement following combined antiproliferative and immune-boosting interventions: results from a randomised clinical trial the covid-19 epidemic evaluation of the establishment of herd immunity in the population by means of serological surveys and vaccination coverage netmhcpan-4.0: improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega mature hiv-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics mechanisms of viral mutation bepipred-2.0: improving sequencebased b-cell epitope prediction using conformational epitopes chloroquine is a potent inhibitor of sars coronavirus infection and spread new insights into the antiviral effects of chloroquine remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro hydroxychloroquine and azithromycin as a treatment of covid-19: preliminary results of an open-label non-randomized clinical trial substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ace2) inhibitors ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis structural basis for the recognition of the sars-cov-2 by full-length human ace2 protein sequence alignments: a strategy for the hierarchical analysis of residue conservation allele frequency net 2015 update: new features for hla epitopes, kir and disease and hla adverse drug reaction associations the relationship between class i binding affinity and immunogenicity of potential cytotoxic t cell epitopes screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes the $1,000 genome: the revolution in dna sequencing and the new era of personalized medicine the epidemiological transition: the current status of infectious diseases in the developed world versus the developing world therapeutic hiv peptide vaccine openvax: an open-source computational pipeline for cancer neoantigen prediction how will country-based mitigation measures influence the course of the covid-19 epidemic? duration of antibody responses after severe acute respiratory syndrome breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection moderate mutation rate in the sars coronavirus genome and its implications the establishment of reference sequence for sars-cov-2 and variation analysis the sars-cov-2 outbreak: what we know peptide-based synthetic vaccines enhancement of t cell-mediated immune responses to whole inactivated influenza virus by chloroquine treatment in vivo chloroquine enhances human cd8+ t cell responses against soluble antigens in vivo hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro cell-penetrating peptides: from basic research to clinics fusion protein linkers: property, design and functionality iedb-ar: immune epitope database-analysis resource in 2019 python for data analysis: data wrangling with pandas, numpy, and ipython. ('o'reilly media human leukocyte antigen-specific polymorphisms in hiv-1 gag and their association with viral load in chronic untreated infection frequent and variable cytotoxic-t-lymphocyte escape-associated fitness costs in the human immunodeficiency virus type 1 subtype b gag proteins peptidebuilder: a simple python library to generate model peptides lightdock: a new multi-scale approach to protein-protein docking lightdock goes informationdriven glowworm swarm optimization for simultaneous capture of multiple local optima of multimodal functions phla3d: an online database of predicted threedimensional structures of hla molecules the protein data bank and the challenge of structural genomics swiss-model: homology modelling of protein structures and complexes distance-scaled, finite ideal-gas reference state improves structurederived potentials of mean force for structure selection and stability prediction data, disease and diplomacy: gisaid's innovative contribution to global health jalview version 2--a multiple sequence alignment editor and analysis workbench key: cord-286219-qcx5ehnh authors: calistri, arianna; munegato, denis; carli, ilaria; parolin, cristina; palù, giorgio title: the ubiquitin-conjugating system: multiple roles in viral replication and infection date: 2014-05-06 journal: cells doi: 10.3390/cells3020386 sha: doc_id: 286219 cord_uid: qcx5ehnh through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to by-pass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. in this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells. ubiquitin (ub) is a highly conserved protein of 76 aminoacids that can be covalently linked to target proteins through a multistep process known as ubiquitination. protein ubiquitination represents open access one of the best characterized post-translational modifications that controls the fate/function of proteins [1] . protein ubiquitination involves a series of cellular enzymes in an enzymatic cascade, starting with the ub-activating enzyme e1, followed by the ubiquitin-conjugating enzyme e2 and by the ub ligase e3, which form an isopeptide bond between the carboxyl terminus of ub and the ε-amino group of a lysine residue on the target protein [2] . the e3 ligase usually determines the substrate specificity, although the e2-conjugating enzyme can also play a role in the substrate selection. accordingly, while there are few known e1 enzymes in mammals and roughly thirty five e2s in humans, hundreds of e3/ub ligases have been identified so far. e3 enzymes are currently classified into three main classes with different structural and functional characteristics: the hect domain family of ub ligases, the cullin-ring family of ub ligases, and the u-box containing ub ligases [3] [4] [5] . the final outcome of the first round of the ubiquitination cascade is the mono-ubiquitination of the target protein. after mono-ubiquitination, a specific lysine of the first ub can be used by the same set of proteins to mediate the consecutive attachment of additional ubs, resulting in the formation of poly-ub chains ( figure 1 ). in the most studied event, the ubiquitinated misfolded or damaged cytoplasmic and nuclear proteins are delivered to the proteasome for degradation as final event of the well-known ub-proteasome system (ups) [6] . on the other hand, ubiquitination of the cytoplasmic domains of transmembrane proteins results in their sorting to lysosomes via the multivesicular body (mvb) pathway [7] . ub-mediated degradation is important not only for the regulation of protein turn-over, but it also plays a role in dna damage repair, cell-cycle regulation, cellular growth, as well as in the immune system functions [8] . moreover, it has been demonstrated that ub can also function act independently from its proteolytic activity, by regulating protein function and protein/protein interaction [8, 9] . an important role in this context is played by specific ub hydrolases (deubiquitinating enzymes or dubs) that catalyze the removal of ub from the target proteins. similar to the function of kinases and phosphatases during the phosphorylation process, ub ligases and dubs can affect substrate function by transient ubiquitination. thus, protein ubiquitination represents a highly versatile and reversible event that may influence different features of a protein and not only its stability. part of this versatility is clearly linked to the fact that ub contains at least seven lysines (k) and additional residues that can be employed by the ub ligases to generate different types of ub chains on the target proteins, which, in turn, will interact with different downstream factors ( figure 1 ) [1] . for instance, it is well established that k-48-based linkages lead mainly to the proteasome-mediated degradation of the ubiquitinated protein, while k-63-based ub chains control primarily protein endocytosis, as well as trafficking and enzyme activity ( figure 1 ) [10, 11] . in addition to ub, a number of ub-like (ubl) proteins can also be conjugated to target substrates by specific e1, e2, and e3 enzyme-like proteins. among them are sumo (small ub modifier), isg15 (interferon-stimulated gene 15), nedd8 (neural precursor cell expressed, developmentally downregulated 8), fat10 (hla-f adjacent transcript 10), atg12 (autophagy-related protein 12) and lc3 (microtubule-associated protein 1 light chain 3) which display different functions and roles in the cellular physiology [12] . schematic representation of the ub molecule and of the enzymatic cascade leading to protein ubiquitination. the seven lysines (k) involved in the process, the ubiquitin-activating enzyme e1, the ubiquitin-conjugating enzyme e2 and the ubiquitin ligase enzyme e3 are highlighted, along with the main fate of the target proteins. as obligate intracellular parasites with limited genome size, viruses must co-opt the host cellular machineries in almost every step of their life cycle, from entry into the host cell, replication and transcription of their genome (either rna or dna), synthesis of proteins, assembly of new particles, till the egress from the infected cell. in addition, in order to establish an infection, viruses must overcome different host immune defenses. thus, there is a constant interaction between the virus and its host, with the virus trying to either counteract or exploit different complex cellular mechanisms and pathways to efficiently produce infectious progenies, or to establish a long-term persistence in the host, depending on the virus taken into consideration. under this respect, given the role played in many cellular fundamental processes, it is to be expected that ub and ubl proteins are involved in almost every aspect of the viral life cycle and pathogenesis [13] . in this review, we will present different examples of how viruses interact with ub/ubl-mediated cellular processes in order to optimize their chance of survival, with a special focus on the mechanisms evolved, in this context, by one of the most important human pathogens, the human immunodeficiency virus type 1 (hiv-1). the first evidence supporting the ability of viruses to utilize the ups to their own advantage came from the study of small dna tumor viruses and of their capability to interfere with the regulation of the cell cycle [14] . since then, it has become clear that members of almost all viral families subvert or exploit both the cellular ub-conjugating and -deconjugating machineries in different phases of their replication cycle [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . under this respect, studies based on the treatment of infected cells with proteasome inhibitors have been instrumental, as such a treatment not only blocks the ups, but also depletes the cellular pool of free ub, affecting de facto all the cellular pathways involving this protein. proteasome inhibitors have been shown to interfere with the replication of major human pathogens such as herpesviruses [15, 16] , poxviruses [17, 18] , hepadnaviruses [19] , adenoviruses [20] , influenzaviruses [21] , retroviruses [22] [23] [24] , coronaviruses [25] , paramyxoviruses [26] , picornaviruses [27] and rotaviruses [28] . ub already plays a role in the first steps of viral replication. as an example, proteasome inhibitors have been reported to inhibit herpes simplex virus (hsv) entry at an early step, immediately after the penetration of the viral capsid into the target cell [15, 29] . the kaposi sarcoma associated herpesvirus (kshv) [30] , the influenza virus [31] and adenoviruses [32] are additional examples of viruses which rely upon the ups for their entry into target cells. for instance, it has been demonstrated that the ups is linked to the ability of the human pathogen kshv to penetrate into endothelial cells and to traffic to the nuclei. not only entry and early post-entry events, but also other steps of the viral life cycle can be affected by impairment of the proteasome activity. indeed, ub-mediated mechanisms regulate gene expression in epstein-barr virus (ebv) [33] , hiv-1 [34] , and human t lymphotropic virus (htlv) [35] . in all these cases, specific viral proteins which function as transcriptional transactivators are able to interact with the ub/ubl-conjugating machinery, leading to an increase in viral protein activity. ups is also involved in the ability of herpesviruses to establish lifelong infections in their hosts, a phenomenon known as latency, as reported for kshv [36, 37] and ebv [38, 39] . specifically, in the case of ebv, the viral latent membrane protein 2a (lamp2a) [38] and lmp1 [39] regulate viral lytic/latent replication through the interaction with specific ubligases and dubs. finally, ub plays a role in viral release from infected cells [40] , as it will be discussed in more details later on. innate immunity represents a first line of defense employed by the cells against microorganisms. microorganisms are recognized by specific molecules named pattern recognition receptors (prr) that bind to pathogen-associated molecular patterns (pamps). this binding leads to the activation of different signaling cascades, with the involvement of the pro-inflammatory transcription factors ap-1, nf-kb and/or one or more members of the interferon-regulatory factor (irf) family, with the final production of pro-inflammatory cytokines and interferon (ifn). not only regulation of innate immune signaling relies on post-translational modifications such as conjugation of ub/ubls to keys cellular proteins [41] , but the ub/ubl conjugating system itself is adopted by many viruses to counteract this host defense response [42] . first of all, viruses prevent the induction of nf-kb and/or ifn. to this end, the use of the cellular ub conjugating machinery represents one of the most exploited strategy. nf-kb is a complex of dimeric transcription factors, which in mammals comprises rela (p65), relb, c-rel, nf-kb1 (p50) and nf-kb2 (p52) [43] . in normal conditions, nf-kb dimers are bound to nf-kb inhibitory proteins (ikbs) and retained in the cytoplasm. upon activativation, a multiprotein complex constituted by the enzyme transforming growth factor beta activated kinase-1 (tak1), the nf-kb essential modifier (nemo), and the ikb kinase (ikk) is formed. this complex phosphorylates the nfkb inhibitor (ikb), leading to its ubiquitination and degradation by the proteasome. once released by ikb, nf-kb can translocate into the nucleus where, along with specific irfs and other co-factors, is able to stimulate ifn transcription. among other mechanisms, it has been described that viruses can directly influence nf-kb stability. for instance, the case of the murid herpesvirus-4 (muhv-4) latency associated protein orf73 leads to p65/rela degradation. [44] . viruses can also use ups to impair nf-kb translocation to the nucleus by blocking ikb degradation. for instance, the rotavirus nsp1 protein mediates the ubiquitination and degradation of the β-transducin repeat containing protein (β-trcp) that, otherwise, would bind to and degrade ikb [45] . viruses can also affect the stability of irfs and in particular of irf3. one example is given by the varicella zoster virus (vzv) orf61, a protein displaying a ring finger e3 ubiquitin ligase activity. orf61 specifically interacts with the phosphorylated/activated form of irf3 leading to its ubiquitination and proteasome mediated degradation [46] . in addition, viruses can act downstream the ifn induction, by inhibiting the signal cascade activated by ifn binding to its receptor [47] . as an example of ups-mediated viral interference with proteins belonging to this cascade, viruses in the rubulavirus genus of the paramyxoviridae family lead stat proteins to proteasome-mediated degradation, by assembling stat-specific ubiquitin ligase complexes from cellular components [48] . finally, viruses can directly target the antiviral ifn-induced proteins (isgs) [49] . some of these proteins, such as the protein kinase r (pkr), the 2',5'-oligoadenylate-synthetase (oas), the ribonuclease l(rnasel), and the mx gtp-ase, along with the respective viral counteracting mechanisms have been extensively studied [49] [50] [51] . recently, different reports have been focused on the isgs belonging to the tripartite motif (trim) containing family, especially after the discovery of the role played by trim5α in the establishment of a cross-species barrier against hiv-1 infection [52] . interestingly, trim5α and many other members of this family of isgs function by ubiquitinating, sumoylating or isgylating host/viral proteins with different outcomes on the antiviral response [53, 54] . not only, the isg15 ub-like protein is itself an isg, and one of the most potently induced upon viral infection. it has been demonstrated that isg15 displays a broad antiviral activity [55] , even though the mechanism/s accounting for this effect is/are still under investigation. what is known is that isgylation can specifically inhibit the functions of an ub ligase, nedd4, which plays a role in the replication cycle of different rna viruses, such as ebola virus and oncoretroviruses [56] . moreover, it has been reported that viruses have evolved the ability to remove isg15 from target proteins. for instance, the coronavirus papain-like protease (plp) protein acts as a de-ubiquitinating and de-isgylating enzyme [57] . another interesting isg is represented by a peculiar cellular protein, tetherin, that being one of the most recently characterized targets of the hiv-1 accessory protein vpu, will be described in more details later. this rapid overview on the involvement of ups in crucial steps of the viral life cycle suggests immediately that viruses are connected to ub in different ways, either by usurping the host's ub-conjugating system or by evolving their own one. first of all, viral proteins have been described that can modify the substrate specificity of cellular ub ligases. as a consequence, specific cellular proteins are targeted for degradation. such a strategy, is exploited by viruses in several of the examples described in the previous paragraphs. for instance, the muhv-4 orf73 represents one of the viral proteins that can subvert the substrate recognition of a cellular e3 enzyme. indeed, by binding to orf73, the elonginc/cullin5/socs ub-ligase complex is able to poly-ubiquitinate p65/rela with its subsequent proteasomal degradation [44] . the rotavirus nsp1 protein leads to β-trcp ubiquitination and degradation through the recruitment of the ubiquitin-ligase complex skp-1/cul1/f-box (scf) [45] . furthermore, small dna viruses with known oncogenic activity, such as the human papillomavirus (hpv), adenoviruses and polyomaviruses, take control of the cell cycle by usurping specific cellular ub ligase complexes to target crucial cell cycle regulators such as p53 and the protein of the retinoblastoma (prb) for degradation [58] . in this way, two of the best studied tumor suppressor cellular pathways are inactivated. indeed, different types of hpv (high-risk) are strongly linked to the onset of cervical cancers, as well as to other type of cancer such as those affecting vagina, vulva, penis, and the head and neck [59] , while many others, classified as low-risk, are only very rarely associated with cancer. although the high-risk and low-risk hpvs share several biological features, the two groups display significant structural/functional differences at the level of the two main viral encoded oncogenes: e6 and e7. indeed, high-and low-risk e6 and e7 proteins have a different ability in affecting p53/prb stability and in modulating the activity of additional cellular proteins with an effect on cell cycle control and cellular proliferation [59] . interestingly, the studies of viral interactions with the host cell cycle have been instrumental in the identification and characterization of p53 and prb [60] . in addition to p53 and prb, another protein complex involved in cell cycle control, the anaphase-promoting complex (apc) is emerging as a key target for viral proteins. apc is a cullin-ring e3 ub ligase that leads to the proteasome degradation of multiple cell cycle regulators and, as a consequence, to the correct progression of the cell cycle itself [61] . different viruses have been reported to affect the apc function. in particular, the human cytomegalovirus (hcmv), a known human pathogen, encodes a protein, pul21a, that is able to induce proteasome-dependent degradation of apc subunits during viral infection [62] . additional viruses, and among them important human pathogens with known oncogenic activity, such as the htlv-1, hpv, hepatitis b virus (hbv) have been reported to interfere with apc [61] . viral proteins themselves can be directly modified by ub or ub-like proteins and, as a consequence, they can be recognized by cellular pathways that can be then exploited by the virus to perform specific tasks. examples of these processes are found in the mechanisms evolved by several enveloped viruses to egress from infected cells, as it will be explained later. viruses can also alter the activity of cellular de-ubiquitinating enzymes, by encoding, for instance, proteins which are able to interact with cellular dubs. under this respect, the ebv ebna1 protein, which is involved in several crucial aspects of viral replication and pathogenesis such as maintenance of the viral genome, transcription and translation of the viral dna, viral persistence, and cellular transformation, interacts with usp7 or herpesvirus-associated ub-specific protease (hausp) [63] [64] [65] . this cellular dub is able to remove ub from p53 and from ebna1 itself, thus preventing their degradation [66] . during ebv infection, not only usp7 but several additional cellular dubs are known to increase their activity and one of the effects appears to be the stabilization of β-catenin, a key factor of the wnt signaling pathway. the disregulation of this signaling pathway has been implicated in tumor development [67] . since ebv is associated, as well, with different types of cancers, such as burkitt's and hodgkin's and the nasopharyngeal carcinoma, the study of ebv interference with the wnt pathway and the role played by the cellular dubs in this context are relevant. some viruses, especially the large dna viruses such as herpeviruses and poxviruses, encode their own ubiquitinating enzymes (table 1) . vzv orf61 mentioned above is an example of such viral encoded ub ligases. additional examples are found in the processes evolved by different viruses to overcome the host immune defenses. under this respect, kshv encodes two e3 ub ligases, k3 and k5, able to ubiquitinate the class i major histocompatibility complex (mhc), leading to its downregulation from the cell surface or from the er and thus interfering with cell antigen presentation [68] . k5 also affects other surface proteins important for the stimulation of t cells, such as icam-1 and b7-2 [69] . interestingly, several other members of the herpeviridae family have been described to down-regulate mhci and t-cell activation markers from the cell surface by employing different mechanisms [70] , some relying again on ub, as in the case of hcmv [71] . another interesting example is given by the hsv-1 icp0 protein, a multifunctional factor which displays, among other features, a ring domain e3 ub ligase activity [72] . thanks to this activity, icp0 is able to disassemble cellular protein aggregates, known as promyelocytic leukemia nuclear bodies (pml nbs), that are present in the nucleus of infected cells where they are involved in different processes such as the interferon (ifn) response to viral infection [73] . both herpesviruses and adenoviruses have been described to interfere with pml nbs [73] and ups is at the basis of some of the mechanisms employed by these viruses to this end. hsv-1 icp0, in particular, mediates the proteasomal degradation of one of the protein complexes that constitute these bodies [74] . moreover, viral dubs have been described (table 2 ). among them, the large tegument protein of herpesviruses belonging to all the three known families of these pathogens (α, β and γ) not only is an essential component of the viral particle, but displays a conserved and unique deubiquitinating activity [96] . taking into account the functions played by the large tegument proteins in the herpesviral replication cycle, a role for these viral dubs during both the entry and the egress of the virus from infected cells is very likely. for instance, the ebv dub, bplf1, is known to deubiquitinate and downregulate the viral ribonucleotide reductase [97] , the cellular processivity factor pcna [98] and the e3 ubiquitin ligase rad18 with a positive effect on the production of infectious particles [99] . it is interesting to note that the herpes simplex virus 1 ub-specific protease, ul36, has been recently reported to inhibit β-interferon production by deubiquitinating the tnf receptor associated factor 3 (traf3) [100] . this finding would indicate a role for viral dubs in overcoming the cellular immune response, as in the case of several viral ub ligases. the impact played by the ub/ubl system on viral replication and on the establishment of a successful infection is particularly clear when the life cycle and the pathogenetic mechanisms evolved by one of the most studied human pathogens, the hiv-1, is analyzed. hiv-1 is a lentivirus and, like the other members of the retroviridae family, is characterized by a non-icosahedral particle enwrapped in a lipidic envelope. its genome comprises two copies of single-stranded rna with a short dimerized region. hiv-1 is the etiological agent of the acquired immunodeficiency syndrome, aids, a condition that after 30 years from its discovery and the introduction of the highly active antiretroviral therapy (haart), still represents one of the major public health problem world-wide [112] . the hiv-1 life cycle is a typical retroviral replication cycle starting with viral entry into specific target cells, reverse transcription of the viral rna into double stranded proviral dna, integration of this proviral genome into the host chromosomal dna, transcription and translation of viral proteins, assembly of viral particles, followed by their budding from the cell surface with the acquisition of an envelope. all retroviral genomes consist of at least 3 genes, gag, pol and env. in addition to these three main genes, complex retroviruses such as hiv-1 encode accessory proteins that enhance their replication and infectivity ( figure 2 ). in particular, hiv-1 is characterized by six auxiliary genes (tat, rev, nef, vpr, vpu and vif), of which only two, tat and rev, are essential for viral replication in vivo and in vitro [113] . on the other hand, nef, vpu, vif, vpr genes encode factors that are known as accessory proteins, as they appear to be dispensable for viral replication in several in vitro experimental settings. however, the high degree of conservation of these proteins suggest crucial functions in vivo. the relevance of hiv-1 as human pathogen and the consequent development and availability of different tools and techniques to manipulate its genome, has allowed to deeply dissect several aspects of hiv biology. even though different questions still need to be answered, the reliance of hiv-1 on numerous cellular pathways and factors for nearly every step of its replication is well appreciated [114] [115] [116] . moreover, it is becoming clear that hiv-1 proteins, and especially the accessory proteins, have the ability to antagonize host molecules that represent first lines of defense against retroviral infections. these cellular proteins are known as intrinsic immunity factors or restriction factors [117] . recent studies have highlighted how the hiv-1 accessory proteins nef, vif, vpu, and vpr have evolved in order to enable the virus to evade the host immune system [118] . interestingly, a common mechanism of action of these proteins is the use of the ups to interfere with cellular proteins that would affect hiv-1 replication. in particular, vif, vpu, and vpr exploit and subvert the physiological activity of specific cullin-ring finger ub ligases (crls) to induce the polyubiquitination and proteasomal degradation of specific cellular targets [119, 120] (table 3) . crls are the largest family of ub ligases and are responsible for ubiquitination of almost 20% of cellular proteins degraded through the ups. the choice as target of members of this particular family of e3 enzymes operated by these hiv-1 proteins is not accidental. indeed, crls are multisubunit complexes composed of a cullin (at least seven cullins are known in vertebrates), an rbx/roc ring finger protein, a variable substrate-recognition subunit (srs), and in most cases, an adaptor that links the srs to the complex [4, 121, 122] . thus, crls are extremely versatile comprising hundreds of distinct complexes with the potential to recruit several protein. this feature has been exploited by vif, vpu and vpr to optimize the chances of hiv-1 to overcome different restriction factors evolved by the cells to inhibit viral replication and spreading. vif is a 23kda protein that is required for production of infectious virus in a cell type-specific manner [134] . experimental evidence indicated that cells non permissive to vif-defective hiv-1 express a host factor inhibiting viral replication [135] , next identified in the cytidine deaminase apobec3g [136] . apobec3g is clearly expressed in vif-defective hiv-1 nonpermissive cells where it acts as an intrinsic restriction factor. in the absence of vif, apobec3g is incorporated into budding virions, and, in the newly infected cells, it determines cytidine to uracil mutations in the single stranded dna of hiv-1, during the process of reverse transcription. this hypermutation results in viral replication impairment. when expressed, vif recruits a multi-subunit e3 ub ligase complex composed of a scaffold protein, cullin 5, ring-box protein, a socs box binding protein complex, elongins b/c, as well as the core binding factor beta (cbf-β) [137] . vif directly binds cullin 5 [137] . the vif-cbf-β-elonginb-elonginc-cullin5-rbx e3 complex is then able to polyubiquitinate apobec3g leading to its protesomal degradation [138] . as a consequence, apobec3g is not incorporated into viral particle and hiv-1 can replicate in de novo infected cells. it has to be mentioned that additional apobec molecules (i.e. apobec3de/3f) have been characterized for their ability to restrict vif-defective hiv-1. in vif expressing cells, all these restriction factors are substrates of the same e3 complex described in the case of abobec3g and are as well subjected to proteasomal degradation [139] . interestingly the crystal structure of the vif/crl complex has been recently resolved [123] . this finding will help to further clarify the molecular basis of vif function. vpu is an 81 amino acid dimeric integral membrane protein. one of the first characterized functions of this hiv-1 accessory protein was the ability of recruiting a crl (skp1-cullin1 e3 ligase complex) to the cytoplasmic tail of cd4, inducing its downregulation at the level of the er [124] . in particular, vpu acts as an adaptor protein, directly interacting with cd4 in the er and with β-trcp, a component of the skp1-cullin-f box (scf) ubiquitin ligase complex, leading to the cd4 polyubiquitination, dislocation from the er to the cytosol and proteasomal degradation [124] . in addition to this important function, which is likely required for proper trafficking and maturation of the viral envelope glycoproteins, vpu has been more recently characterized for yet another crucial role, connected with the ability of the virus to evade a specific ifn-1 induced antiviral factor: the b cell stromal factor 2 (bst-2) or tetherin. the latter name perfectly reflects the activity of bst-2 that, in the absence of vpu, physically retains (tethers) fully assembled hiv-1 particles to the surface of the infected cells [140] . indeed, bst-2 is a type ii transmembrane protein with an unusual topology consisting of an n-terminal cytoplasmic tail, a transmembrane domain, a coiled-coil extracellular domain, and a glycosylphosphatidylinositol anchor at the carboxy-terminus. it has been demonstrated that this peculiar conformation, rather than the primary sequence, confers to tetherin the ability to physically retain budding virions on the cellular membrane [141] . hiv-1 is not the only virus sensitive to bst-2 [142] and human cells are not the only one encoding for such a restriction factor [142] [143] [144] [145] . indeed, tetherin represents also one of the major cross-species barrier factor especially in the context of lentiviral infection [143, 144, [146] [147] [148] . there is still some uncertainty as to how vpu antagonism is accomplished, as different mechanisms have been reported. indeed, it has been described that vpu directly interacts with tetherin and can mediate its down-regulation from the cell surface with an increase in viral release [125] . while some studies have indicated a vpu mediated β-trcp-dependent proteasomal degradation of tetherin [125, 126] , there are also data supporting a role for the β-trcp-dependent endo-lysosomal pathway in bst-2 degradation [127, 149] . in agreement with these latter studies rab7a, a small gtpase essential for the maturation of late endosomes and lysosomal fusion, appears to be required for tetherin degradation [150] , as along with the endosomal sorting complex required for transport (escrt)-0 [151] . interestingly, β-trcp2-dependent ubiquitination and subsequent degradation of tetherin do not seem to require lysine residues in the cytoplasmic domain of tetherin [152] . it has to be mentioned that other studies have revealed a β-trcp2-independent mechanism of tetherin antagonism by vpu, with the delocalization and retention of tetherin in a perinuclear compartment in the absence of degradation [153, 154] . even though degradation does not appear to be the primary system by which vpu counteracts bst-2, sequestration and degradation might be parallel existing and not mutually exclusive mechanisms by which vpu optimizes the chances to counteract the antiviral effect of tetherin. in agreement with the concept of multiple mechanisms, other viral proteins are able to counteract tetherin function, both by inducing its ub-dependent endosomal degradation, as in the case of the khsv k5 protein [82] , or by sequestration in a perinuclear compartment, as in the case of the hiv-2 env [155] . vpr is a 96 amino acid protein whose function has been difficult to elucidate. one of the clearest activities of vpr is its ability to delay or arrest cells in the g2 phase of the cell cycle. in addition, in this case, the recruitment of a specific crl appears to be essential. indeed, vpr interacts with the cullin4a-ddb1-dcaf1 ub ligase complex [130, [156] [157] [158] [159] [160] [161] [162] , a binding that is required for the induction of the g2 arrest [163] . however, the exact target/s of vpr-cullin4a-ddb1-dcaf1 activity that are linked to the g2 cell-cycle arrest is/are still unknown [164] . in particular, the two substrates of vpr-mediated degradation, that have been better characterized so far, the uracil-dna glycosylase (ung) 2 and the single-strand selective monofunctional uracil-dna glycosylase (smug1) [131] do not seem to account for the above described vpr effect. recently, it has been demonstrated that also the endoribonuclease dicer is subjected to proteosomal degradation via vpr-recruited cullin4a-ddb1-dcaf1 ub ligase, with enhanced hiv-2 replication in monocyte-derived macrophages [132] . it is well known that dicer is involved in the generation of mirna, a major component of the rna silencing machinery interfering with viral replication. thus, vpr would also act as a suppressor of silencing (srs) and vpr-mediated degradation of dicer would represent another example of a cellular restriction mechanism to hiv-1 infection bypassed by a viral "usurped" crl complex. while the vpr accessory protein is encoded by all primate lentiviruses, including hiv-1 and hiv-2, its paralog vpx is expressed only by hiv-2 and by certain simian lentiviruses. myeloid cell types are known to be less permissive to hiv-1 infection with respect to cd4-positive t lymphocytes [133, 165] . this difference in susceptibility to hiv-1 infection has been linked to the expression in myeloid cell types of the vpx-interacting protein samhd1 [133] . samhd1 is a nucleotide triphosphohydrolase that can inhibit lentiviral reverse transcription by depleting the intracellular pool of available dntps [133, 166] . the hiv-2 and siv vpx proteins counteract this restriction by inducing samhd1 degradation following its ubiquitination. once again, as just described in the case of vpr, the crl involved in such a process is the vpx recruited cullin4a-ddb1-dcaf1 [118, 167] . it has been recently reported that hiv vpr and vpx exploit not only cullin4a but also cullin4b to mediate ubiquitination of target proteins. interestingly, in primary macrophages vpx appears to need both cullin4a and cullin4b to obtain maximal samhd1 degradation [168] . since hiv-1 does not have a vpx protein and hiv-1 vpr is not capable of interacting with samhd1, myeloid cell types display resistance to hiv-1 infection. thus, samhd1 represents an additional restriction factor that interferes with hiv-1 infection, at least in a specific cell type, and that can be overcome by the virally recruited cullin4a-ddb1-dcaf1 ub ligase. viruses have developed sophisticated mechanisms for exiting from infected cells. enveloped viruses leave the cells through a complex process, known as budding, that requires two main steps, (i) the cell membrane deformation around the assembling virions; and (ii) a fission, resulting in the detachment of the viral particles from the cellular surface [169, 170] . studies on retroviruses, and in particular on hiv-1, have been instrumental in dissecting the complex viral/cellular interplay which ensures a successful egress of enveloped viruses. indeed, the gag polyprotein is the only retroviral protein necessary at this level. this feature allowed the development of viral mutants and tools to identify the molecular mechanisms involved in budding. in 1991, göttlinger and co-workers, along with other groups, identified in the c-terminal p6 domain of hiv-1 gag a highly conserved motif (pt/sap) as crucial player in the detachment of budded virions from the cell surface [171, 172] . starting from these initial findings, short proline-rich sequences, named late assembly or l-domains, functionally equivalent to the hiv-1 pt/sap motif, have been identified in the gag of different retroviruses [173] . to date, in addition to the pt/sap motif, typical of most lentiviruses, two further l-domains have been well characterized: the ppxy-type l-domain present in the gag proteins of oncoretroviruses and the ypx n l-type motif, identified in the gag protein of the equine infectious anemia virus (eiav) [56] . besides retroviruses, l-domains have been also found in the structural proteins of most rna enveloped viruses such as rhabdoviruses, filoviruses, arenaviruses, and paramyxoviruses [56] , and in some dna enveloped viruses [174] [175] [176] [177] [178] . several data have indicated a connection between ub, l-domains and retroviral egress from infected cells. firstly, a functional l-domain leads to gag ubiquitination [23] and when directly fused to different retroviral gags, ub can functionally replace the l-domains [179, 180] . furthermore, ub can be found in retroviral mature particles [181, 182] and its depletion inhibits virus budding [22, 23] , while the recruitment of hect ub ligases, belonging to the nedd4-like family, is clearly involved in retroviral particle release [23, [183] [184] [185] [186] [187] . interestingly, the ppxy type of l-domain interacts with the members of the nedd4-like family of ubiquitin ligases by directly binding the ww domain characteristic of these cellular proteins [183] . finally, the l-domains act independently from their position in the viral protein, frequently occur in combination and can be exchanged between unrelated viruses without losing their ability to mediate budding [188] [189] [190] [191] [192] . overall, these features are suggestive of a role for the l-domains as docking sites for cellular factors belonging to a specific pathway involving ub and exploited by retroviruses to efficiently execute budding. the role played by ub in the endocytosis of transmembrane proteins suggested initially that the endocytic pathway could represent this cellular pathway [6] . furthermore, some transmembrane proteins, such as the epithelial na+ channel, were known to contain sequences perfectly overlapping retroviral l-domains which are involved in the process of endocytosis [193] [194] [195] . moreover, it was demonstrated that the ub residues involved in retroviral budding were indeed the residues involved in protein endocytosis [196] . however, the vesiculation process taking place during endocytosis is topologically opposite to the one occurring during viral budding. indeed, while the budding of a virus happens from the cytosol toward the extracellular space and the factors that catalyze membrane fission must work from within the bud neck, during endocytosis the vesicles bud into the cytoplasm and membrane fission is driven by dynamin from outside of the bud neck. thus, the cellular machinery involved in the formation of endocytic vesicles could not be the one exploited by viruses for their egress. this apparent discrepancy between experimental data started to find a solution thanks to the seminal discoveries of the carter's laboratory [197] and of the sundquist's group [198] , that identified in the cellular protein tumor suppressor gene 101 (tsg101) the binding partner of the hiv1-1 pt/sap motif, an interaction which is crucial for hiv budding. these initial findings, along with the work done beforehand and in parallel by cellular biologists, allowed to establish the connection between a cellular pathway, to which tsg101 belongs, and the hiv-1 egress from infected cells: the biogenesis pathway of an organelle of the endocytic pathway, the multivesicular bodies (mvb) [7] . since then, more than 10 years of research have clarified that retroviruses, and in general most enveloped rna and some dna enveloped viruses, exploit mvbs during the latest steps of their replication cycle [173] . these organelles give reason of the connection between viral budding, ubiquitin, l-domains and the endocytosis of transmembrane proteins. indeed, mvbs represent the organelles that eukaryotic cells have evolved to make the degradation of transmembrane proteins possible [7] . when such a protein needs to be removed from the plasma membrane, it is ubiquitinated and endocytosed on the surface of endosome. then, a budding of vesicles from the membrane into the lumen of the endosome takes place, which allows the delivery of the trans-membrane protein into the lumen of the endosomes. this vesiculation event leads to the biogenesis of the mvb, which will eventually fuse with a lysosome resulting in the degradation of its cargo ( figure 3 ) [199, 200] . it is clear that, if the transmembrane protein would not get access to the lumen of the endosome, thanks to the formation of the mvb, its degradation through the lysosome could not occur. thus, the budding of vesicles from the endosomal membrane into the interior of the organelle (the biogenesis of the mvb) is the crucial step in this process. this vesiculation process, which takes place from the cytosol towards an environment that is equivalent to the extracellular space (the endosomal lumen), is now an event topologically identical to the budding of viruses from the plasma membrane. as a consequence, the connection between viral budding and mvbs biogenesis is functionally and physiologically sustainable. this vesicle budding step requires the sequential recruitment from the cytosol to the endosomal membrane of a highly conserved set of proteins, the escrt machinery [201] and different members of such a machinery, as tsg101 and aip1/alix are recruited by the viral l-domains to allow viral budding [56, 173] . what is still apparently missing is the link between ub, mvb and viral budding. instead, several links do exist, with the most interesting one represented by the evidence that ub plays a central role in the regulation of the mvb biogenesis pathway. indeed, ubiquitination is necessary and sufficient to trigger the escrt-dependent endosomal sorting of membrane proteins and their degradation through the mvb/lysosomal pathway [202] . moreover, precise and finely controlled cycles of ubiquitination and deubiquitination of target proteins and of specific components of the escrt machinery are essential for the function of the entire pathway of mvb biogenesis, till the vesicle budding [203] [204] [205] . indeed, different upstream components of the escrt machinery with an essential role in viral budding, such as tsg101 and aip1/alix, are ubiquitinated and are able to bind ub [206] [207] [208] [209] . the sequential recruitment of the escrt complexes to the mvb membrane is described along with the additional factors involved in the cargo protein delivery into the organelle lumen. the extracellular environment and its equivalents are colored in light blue, while the cytoplasmic environment is colored in yellow. details on the escrt proteins and on the other mvb key factors can be found in several comprehensive reviews [7, 169, 173, 201, 202] . overall, the aforementioned crucial role played by ub in the control of the mvb biogenesis pathway by itself supports the strong evidence of an involvement of this cellular protein in viral budding. however, in addition or in parallel to this regulatory function, there is evidence that also direct ubiquitination of specific viral/cellular proteins might be important for the escrt-dependent viral budding. in particular, the importance of a direct gag ubiquitination is supported by several studies, as reviewed by votteler and sundquist [173] . the working model foresees that ubiquitination of gag would facilitate its interaction with the escrt machinery. in other words, ubiquitination of gag would mimic ubiquitination of transmembrane proteins along the endocytic/mvb pathway, thus mediating the engagement of the escrt components. under this respect, it has been shown that the binding of tsg101 to gag has an increased affinity when gag is linked to ub [208, 209] . furthermore, the bouamr's group, by fusing the dub domain of the hsv-1 ul36 to gag and to specific escrt proteins, has recently reported evidence supporting a crucial role for gag ubiquitination in hiv budding [210] . however, along with these data, other studies indicate a more complex scenario. for instance, it has been demonstrated that ubiquitination of gag proteins can increase also in the absence of a functional l-domain and when budding is inhibited [211] . moreover, the artificial targeting of ub ligases to gag leads to its ubiquitination, but not always enhances budding [187] . finally, zhadina and coworkers have nicely shown that ub-dependent viral budding can take place without viral protein ubiquitination [212] . thus, the question whether gag ubiquitination is the result of a bystander effect or has a functional relevance is still open. interestingly, it has been reported that the identity of the protein to which ub is bound might not be the key element for viral budding [192] . what appears to be crucial is, instead, the presence of ub at the site of budding, thus emphasizing its crucial role in the process. as very simple intracellular parasites, viruses rely on host cell factors and pathways to perform their life cycle and to establish a successful infection. the extensive use of ub and ubl-mediated pathways by a number of unrelated viruses in different steps of their life cycle emphasizes the central importance of these cellular machinery in the cell physiology and, as a consequence, for viral replication and spreading. in this review, we have tried to give an overview of the complexity of the interplay between viruses and ub/ubl-conjugating systems. while it appears evident that different aspects have been deeply analyzed and better understood, such as the role played by ups in the context of the mechanisms evolved by viruses to control the cell cycle and to counteract innate immunity responses, more work needs to be done in order to clarify the functional relevance of ub involvement in specific steps of viral replication, such as viral budding. moreover, different emerging findings require further clarification both from the cellular and from the viral point of view, such as the role and the substrates of the newly characterized viral and cellular ubl proteins. under this respect, the study of the viral connection with the ub/ubl-conjugating machinery still represents one of the better examples of how viruses can serve as potent tools to dissect complex cellular processes and pathways. ubiquitin linkages make a difference mechanisms underlying ubiquitination the hect family of e3 ubiquitin ligases: multiple players in cancer development cullin-ring ubiquitin ligases: global regulation and activation cycles u-box proteins as a new family of ubiquitin ligases ubiquitin enters the new millennium multivesicular body morphogenesis ubiquitin and ubiquitin-like proteins as multifunctional signals back to the future with ubiquitin ubiquitin in chains polyubiquitin chains: polymeric protein signals ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets viral avoidance and exploitation of the ubiquitin system the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 cellular proteasome activity facilitates herpes simplex virus entry at a postpenetration step proteasome subunits relocalize during human cytomegalovirus infection, and proteasome activity is necessary for efficient viral gene transcription inhibition of the ubiquitin-proteasome system prevents vaccinia virus dna replication and expression of intermediate and late genes orthopoxviruses require a functional ubiquitin-proteasome system for productive replication bortezomib inhibits hepatitis b virus replication in transgenic mice tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator eia inhibition of the ubiquitin-proteasome system affects influenza a virus infection at a postfusion step proteasome inhibition interferes with gag polyprotein processing, release, and maturation of hiv-1 and hiv-2 a role for ubiquitin ligase recruitment in retrovirus release retroviruses have differing requirements for proteasome function in the budding process the ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry decreased replication of human respiratory syncytial virus treated with the proteasome inhibitor mg-132 ubiquitination is required for effective replication of coxsackievirus b3 replication of the rotavirus genome requires an active ubiquitin-proteasome system a pre-immediate-early role for tegument icp0 in the proteasomedependent entry of herpes simplex virus the ubiquitin/proteasome system mediates entry and endosomal trafficking of kaposi's sarcoma-associated herpesvirus in endothelial cells epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus a capsid-encoded ppxy-motif facilitates adenovirus entry ebna1-mediated recruitment of a histone h2b deubiquitylating complex to the epstein-barr virus latent origin of dna replication a non-proteolytic role for ubiquitin in tat-mediated transactivation of the hiv-1 promoter ubiquitination of human t-cell leukemia virus type 1 tax modulates its activity kaposi's sarcoma-associated herpesvirus transactivator rta promotes degradation of the repressors to regulate viral lytic replication kaposi's sarcoma-associated herpesvirus rta promotes degradation of the hey1 repressor protein through the ubiquitin proteasome pathway the epstein-barr virus latent membrane protein 2a py motif recruits ww domain-containing ubiquitin-protein ligases the a20 deubiquitinase activity negatively regulates lmp1 activation of irf7 wrapping up the bad news: hiv assembly and release regulation of the innate immune system by ubiquitin and ubiquitin-like modifiers ubiquitylation in innate and adaptive immunity shared principles in nf-kappab signaling termination of nf-kappab activity through a gammaherpesvirus protein that assembles an ec5s ubiquitin-ligase rotavirus nsp1 inhibits nfkappab activation by inducing proteasome-dependent degradation of beta-trcp: a novel mechanism of ifn antagonism varicella-zoster virus immediate-early protein orf61 abrogates the irf3-mediated innate immune response through degradation of activated irf3 viral evasion of the interferon system paramyxoviruses sv5 and hpiv2 assemble stat protein ubiquitin ligase complexes from cellular components interferon-inducible antiviral effectors the tiers and dimensions of evasion of the type i interferon response by human cytomegalovirus interferon-stimulated genes: a complex web of host defenses innate immune interferon responses to human immunodeficiency virus-1 infection trim family proteins and their emerging roles in innate immunity the roles of the trim e3-ubiquitin ligase family in innate antiviral immunity ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses role of multivesicular bodies and their components in the egress of enveloped rna viruses deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases e3 ubiquitin ligases and human papillomavirus-induced carcinogenesis review of the current knowledge on the epidemiology, pathogenesis, and prevention of human papillomavirus infection how the rb tumor suppressor structure and function was revealed by the study of adenovirus and sv40 control the host cell cycle: viral regulation of the anaphase-promoting complex proteasome-dependent disruption of the e3 ubiquitin ligase anaphase-promoting complex by hcmv protein pul21a protein interaction domains of the ubiquitin-specific protease, usp7/huasp protein profiling with epstein-barr nuclear antigen-1 reveals an interaction with the herpesvirus-associated ubiquitin-specific protease huasp/usp7 huasp/usp7 as an epstein-barr virus target contributions of epstein-barr nuclear antigen 1 (ebna1) to cell immortalization and survival wnt signaling in stem cells and cancer what has the study of the k3 and k5 viral ubiquitin e3 ligases taught us about ubiquitin-mediated receptor regulation? viruses a viral protein that selectively downregulates icam-1 and b7-2 and modulates t cell costimulation herpesviruses and immunity: the art of evasion human cytomegalovirus immunity and immune evasion herpes simplex virus type 1 immediate-early protein icp0 and is isolated ring finger domain act as ubiquitin e3 ligases in vitro pml and pml nuclear bodies: implications in antiviral defence the two functions of herpes simplex virus 1 icp0, inhibition of silencing by the corest/rest/hdac complex and degradation of pml, are executed in tandem a proteomic perspective of inbuilt viral protein regulation: pul46 tegument protein is targeted for degradation by icp0 during herpes simplex virus type 1 infection herpes simplex virus 1 e3 ubiquitin ligase icp0 protein inhibits tumor necrosis factor alpha-induced nf-κb activation by interacting with p65/rela and p50/nf-κb1 centromere architecture breakdown induced by the viral e3 ubiquitin ligase icp0 protein of herpes simplex virus type 1 icp0 dismantles microtubule networks in herpes simplex virus-infected cells a viral e3 ligase targets rnf8 and rnf168 to control histone ubiquitination and dna damage responses ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection haploid genetic screens identify an essential role for plp2 in the downregulation of novel plasma membrane targets by viral e3 ubiquitin ligases the ring-ch ligase k5 antagonizes restriction of kshv and hiv-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin kaposi's sarcoma-associated herpesvirus k3 and k5 proteins down regulate both dc-sign and dc-signr the march family e3 ubiquitin ligase k5 alters monocyte metabolism and proliferation through receptor tyrosine kinase modulation the ring finger domain of varicella-zoster virus orf61p has e3 ubiquitin ligase activity that is essential for efficient autoubiquitination and dispersion of sp100-containing nuclear bodies adenovirus ubiquitin-protein ligase stimulates viral late mrna nuclear export murine gammaherpesvirus 68 orf75c contains ubiquitin e3 ligase activity and requires pml sumoylation but not other known cellular pml regulators, ck2 and e6ap, to mediate pml degradation newly discovered viral e3 ligase pk3 induces endoplasmic reticulum-associated degradation of class i major histocompatibility proteins and their membrane-bound chaperones the poxvirus p28 virulence factor is an e3 ubiquitin ligase viral ubiquitin ligase wssv222 is required for efficient white spot syndrome virus replication in shrimp arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling plp2 of mouse hepatitis virus a59 (mhv-a59) targets tbk1 to negatively regulate cellular type i interferon signaling pathway plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase mechanism of inhibiting type i interferon induction by hepatitis b virus x protein structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes the epstein-barr virus (ebv) deubiquitinating enzyme bplf1 reduces ebv ribonucleotide reductase activity epstein-barr virus bplf1 deubiquitinates pcna and attenuates polymerase η recruitment to dna damage sites the rad6/18 ubiquitin complex interacts with the epstein-barr virus deubiquitinating enzyme, bplf1, and contributes to virus infectivity herpes simplex virus 1 ubiquitin-specific protease ul36 inhibits beta interferon production by deubiquitinating traf3 autocatalytic activity of the ubiquitinspecific protease domain of herpes simplex virus 1 vp1-2 cleavage specificity of the ul48 deubiquitinating protease activity of human cytomegalovirus and the growth of an active-site mutant virus in cultured cells mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pul36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf64 molecular basis for ubiquitin and isg15 cross-reactivity in viral ovarian tumor domains mers-cov papain-like protease has deisgylating and deubiquitinating activities a compact viral processing proteinase/ubiquitin hydrolase from the otu family a viral deubiquitylating enzyme targets viral rna-dependent rna polymerase and affects viral infectivity the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions deubiquitinating function of adenovirus proteinase past, present and future: 30 years of hiv research regulation of hiv-1 gene expression identification of host proteins required for hiv infection through a functional genomic screen host cell factors in hiv replication: meta-analysis of genome-wide studies host factors mediating hiv-1 replication host restriction factors in retroviral infection: promises in virus-host interaction hiv accessory proteins versus host restriction factors viral modulators of cullin ring ubiquitin ligases: culling the host defense hiv: ringside views selective protein degradation: a rheostat to modulate cell-cycle phase transitions the emerging family of cullin3-ring ubiquitin ligases (crl3s): cellular functions and disease implications structural basis for hijacking cbf-β and cul5 e3 ligase complex by hiv-1 vif role of hiv-1 vpu protein for virus spread and pathogenesis hiv-1 antagonism of cd317 is species specific and involves vpu-mediated proteasomal degradation of the restriction factor hiv-1 vpu neutralizes the antiviral factor tetherin/bst-2 by binding it and directing its beta-trcp2-dependent degradation vpu directs the degradation of the human immunodeficiency virus restriction factor bst-2/tetherin via a {beta}trcp-dependent mechanism inhibition of β-trcp-dependent ubiquitination of p53 by hiv-1 vpu promotes p53-mediated apoptosis in human t cells besnard-gué rin, c. involvement of the betatrcp in the ubiquitination and stability of the hiv-1 vpu protein hiv1 vpr arrests the cell cycle by recruiting dcaf1/vprbp, a receptor of the cul4-ddb1 ubiquitin ligase vpr expression abolishes the capacity of hiv-1 infected cells to repair uracilated dna the hiv-1 protein vpr targets the endoribonuclease dicer for proteasomal degradation to boost macrophage infection samhd1 host restriction factor: a link with innate immune sensing of retrovirus infection new insights into the role of vif in hiv-1 replication evidence for a newly discovered cellular anti-hiv-1 phenotype isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein hiv-1 vif n-terminal motif is required for recruitment of cul5 to suppress apobec3 hiv-1 vif, apobec, and intrinsic immunity interactions between hiv-1 vif and human elonginb-elonginc are important for cbf-β binding to vif tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu tetherin inhibits hiv-1 release by directly tethering virions to cells the antiviral activities of tetherin tetherin-driven adaptation of vpu and nef function and the evolution of pandemic and nonpandemic hiv-1 strains tetherin antagonism by primate lentiviral nef proteins feline tetherin is characterized by a short n-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein ancient origin of a deletion in human bst2/tetherin that confers protection against viral zoonoses reacquisition of nef-mediated tetherin antagonism in a single in vivo passage of hiv-1 through its original chimpanzee host vpu antagonizes bst-2-mediated restriction of hiv-1 release via beta-trcp and endo-lysosomal trafficking rab7a is required for efficient production of infectious hiv-1 berlioz-torrent, c. the escrt-0 component hrs is required for hiv-1 vpu-mediated bst-2/tetherin downregulation serine-threonine ubiquitination mediates downregulation of bst-2/tetherin and relief of restricted virion release by hiv-1 vpu antagonism of tetherin restriction of hiv-1 release by vpu involves binding and sequestration of the restriction factor in a perinuclear compartment β-trcp is dispensable for vpu's ability to overcome the cd317/tetherin-imposed restriction to hiv-1 release hiv-1 vpu and hiv-2 env counteract bst-2/tetherin by sequestration in a perinuclear compartment hiv-1 vpr-mediated g2 arrest involves the ddb1-cul4avprbp e3 ubiquitin ligase hiv-1 vpr activates the g2 checkpoint through manipulation of the ubiquitin proteasome system lentiviral vpr usurps cul4-ddb1[vprbp] e3 ubiquitin ligase to modulate cell cycle hiv-1 vpr function is mediated by interaction with the damage-specific dna-binding protein ddb1 ddb1 and cul4a are required for human immunodeficiency virus type 1 vpr-induced g2 arrest the hiv1 protein vpr acts to promote g2 cell cycle arrest by engaging a ddb1 and cullin4a-containing ubiquitin ligase complex using vprbp/dcaf1 as an adaptor assembly with the cul4a-ddb1dcaf1 ubiquitin ligase protects hiv-1 vpr from proteasomal degradation human immunodeficiency virus type 1 vif induces cell cycle delay via recruitment of the same e3 ubiquitin ligase complex that targets apobec3 proteins for degradation defining the interactions and role of dcaf1/vprbp in the ddb1-cullin4a e3 ubiquitin ligase complex engaged by hiv-1 vpr to induce a g2 cell cycle arrest molecular mechanisms of hiv immune evasion of the innate immune response in myeloid cells intracellular nucleotide levels and the control of retroviral infections structural basis of lentiviral subversion of a cellular protein degradation pathway cullin 4a and cullin4b are interchangeable for hiv vpr and vpx action through the crl4 ubiquitin ligase complex membrane budding and scission by the escrt machinery: it's all in the neck viral membrane scission effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release p6gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease virus budding and the escrt pathway herpes simplex virus type 1 cytoplasmic envelopment requires functional vps4 intracellular trafficking and maturation of herpes simplex virus type 1 gb and virus egress require functional biogenesis of multivesicular bodies human cytomegalovirus exploits escrt machinery in the process of virion maturation cellular vps4 is required for efficient entry and egress of budded virions of autographa californica multiple nucleopolyhedrovirus functional characterization of the putative hepatitis b virus core protein late domain using retrovirus chimeras ubiquitin is part of the retrovirus budding machinery functional replacement of a retroviral late domain by ubiquitin fusion ubiquitin in avian leukosis virus particles ubiquitin is covalently attached to the p6gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12gag protein of moloney murine leukemia virus hect ubiquitin ligases link viral and cellular ppxy motifs to the vacuolar protein-sorting pathway efficient and specific rescue of human immunodeficiency virus type 1 budding defects by a nedd4-like ubiquitin ligase sundquist, w.i. nedd4l overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking ptap and ypxl late domains role of the feline immunodeficiency virus l-domain in the presence or absence of gag processing: involvement of ubiquitin and nedd4-2s ligase in viral egress rescue of hiv-1 release by targeting widely divergent nedd4-type ubiquitin ligases and isolated catalytic hect domains to gag positionally independent and exchangeable late budding functions of the rous sarcoma virus and human immunodeficiency virus gag proteins infectivity of moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses functional replacement and positional dependence of homologous and heterologous l domains in equine infectious anemia virus replication aip1/alix is a binding partner for hiv-1 p6 and eiav p9 functioning in virus budding functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding ww domains of nedd4 bind to the proline-rich py motifs in the epithelial na+ channel deleted in liddle's syndrome regulation of the epithelial na+ channel by nedd4 and ubiquitination late assembly domain function can exhibit context dependence and involves ubiquitin residues implicated in endocytosis pr55(gag) tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding the role of ubiquitination in lysosomal trafficking of δ-opioid receptors ubiquitination in the first cytoplasmic loop of μ-opioid receptors reveals a hierarchical mechanism of lysosomal down-regulation dynamics of escrt proteins ubiquitin: same molecule, different degradation pathways how ubiquitin functions with escrts escrt ubiquitin-binding domains function cooperatively during mvb cargo sorting the circuitry of cargo flux in the escrt pathway tsg101-specific e3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding the yeast alix homolog bro1 functions as a ubiquitin receptor for protein sorting into multivesicular endosomes structure and functional interactions of the tsg101 uev domain structure of the tsg101 uev domain in complex with the ptap motif of the hiv-1 p6 protein ubiquitin conjugation to gag is essential for escrt-mediated hiv-1 budding analysis of human immunodeficiency virus type 1 gag ubiquitination ubiquitin-dependent virus particle budding without viral protein ubiquitination owing to space limitations and to the complexity of the topics we have predominantly cited recent publications and reviews, therefore we apologize to those whose original contributions have not been specifically referenced. the studies carried on in the authors' laboratory have been supported by the miur-prin-2005 (#2005069559_004 to cp), miur-prin-2007 (#20072j9rwm_004 and 2007m52htt_004 to cp and gp, respectively), aids grants from the istituto superiore di sanità (rome-aids projects #30g.24 and 40h98 to cp, #30g.55 and 40f.57 to gp), grants from the university of padova (ex-60% to cp, gp, ac and cpda061582 to ac) and from regione veneto to gp. dm is a phd student in biomedicine, at the department of molecular medicine, university of padova. the authors declare no conflict of interest. key: cord-307817-2vy28i4m authors: lou, zhiyong; sun, yuna; rao, zihe title: current progress in antiviral strategies date: 2014-01-14 journal: trends pharmacol sci doi: 10.1016/j.tips.2013.11.006 sha: doc_id: 307817 cord_uid: 2vy28i4m the prevalence of chronic viral infectious diseases, such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), and influenza virus; the emergence and re-emergence of new viral infections, such as picornaviruses and coronaviruses; and, particularly, resistance to currently used antiviral drugs have led to increased demand for new antiviral strategies and reagents. increased understanding of the molecular mechanisms of viral infection has provided great potential for the discovery of new antiviral agents that target viral proteins or host factors. virus-targeting antivirals can function directly or indirectly to inhibit the biological functions of viral proteins, mostly enzymatic activities, or to block viral replication machinery. host-targeting antivirals target the host proteins that are involved in the viral life cycle, regulating the function of the immune system or other cellular processes in host cells. here we review key targets and considerations for the development of both antiviral strategies. the prevalence of chronic viral infectious diseases, such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), and influenza virus; the emergence and re-emergence of new viral infections, such as picornaviruses and coronaviruses; and, particularly, resistance to currently used antiviral drugs have led to increased demand for new antiviral strategies and reagents. increased understanding of the molecular mechanisms of viral infection has provided great potential for the discovery of new antiviral agents that target viral proteins or host factors. virus-targeting antivirals can function directly or indirectly to inhibit the biological functions of viral proteins, mostly enzymatic activities, or to block viral replication machinery. host-targeting antivirals target the host proteins that are involved in the viral life cycle, regulating the function of the immune system or other cellular processes in host cells. here we review key targets and considerations for the development of both antiviral strategies. viruses comprise a large group of pathogens that are responsible for causing severe infectious diseases. over the past 30 years, antiviral agents that target viral proteins or host factors have been successfully developed. based on their inhibitory mechanisms, antiviral reagents can be divided into two groups: (i) inhibitors that target the viruses themselves or (ii) inhibitors that target host cell factors. virus-targeting antivirals (vtas) can function directly (dvtas) or indirectly (indvtas) to inhibit biological functions of viral proteins, mostly enzymatic activities, or they block the correct formation of the viral replication machinery (table 1) . host-targeting antivirals (htas) include reagents that target the host proteins that are involved in the viral life cycle (figure 1 ), regulating the function of the immune system or other cellular processes in host cells. with increased knowledge of viral protein and host factors, the scientific community has achieved great progress in mechanism-based antiviral discovery against chronic viral infectious diseases, and in understanding of the emergence of new viral diseases and of the resistance to traditional antivirals. this review will highlight recent achievements in antiviral development and discuss various strategies for preventing virus attachment and entry into the host cell, as well as strategies for preventing virus replication and transcription within the host cell. the first step in viral invasion is the attachment to host cells via an interaction with functional receptor(s). for enveloped viruses, the viral proteins located on the outer envelope of the virion are responsible for the recognition of receptors and the attachment to host cells. hiv (a member of the retroviridae family) is a typical enveloped virus, and its invasion is mediated by the envelope proteins gp120 and gp41, which are arranged on the viral membrane as a trimer of three trans-membrane gp41 and three noncovalently attached gp120 surface subunits [1] (figure 2 ). gp120 recognizes the cd4 receptor and launches the conformational changes that expose the binding sites for the binding of a co-receptor, (i.e., ccr5 and cxcr4 [2] ). antagonists that block the interactions between hiv and its receptor and co-receptors have therefore been developed as anti-hiv therapeutics. attempts to find specific inhibitors that block the interaction between hiv-1 and cd4 were initiated using a soluble extracellular domain of cd4 protein that retained the ability to bind gp120. although the preliminary results revealed that either soluble cd4 protein or a cd4-immunoglobulin fusion protein showed good in vitro anti-hiv activity, all failed in clinical trials due to poor pharmacokinetic features (e.g., the half-life of cd4-immunoglobulin fusion protein in mice is only 2.4 h) [3] [4] [5] . small molecule inhibitors that occupy a specific region within the cd4-binding pocket of gp120 were subsequently developed to block the gp120-cd4 interaction (figure 2a ). for example, bms488043 [6] and bms663068 [7] were found to significantly reduce hiv-1 proliferation and have good pharmaceutical characteristics. another success is influenza neuraminidase (na) inhibitor (nai). influenza na is a surface glycoprotein and functions at two steps of the viral life cycle: (i) cleaves the cell receptor sialic acid residues, which bind to influenza hemagglutinin (ha), and allows the release of the progeny virus; and (ii) cleaves the sialic acid moieties on the mucin that bathes the airway epithelial cells or cobinds the receptor with ha [8] . in line with the structure of na [9, 10] , several nais have been successfully developed to competitively occupy the sialic acid-binding pocket of na. among these nais, oseltamivir and zanamivir were first used clinically as an anti-flu therapy [11] . oseltamivir is a prodrug that is readily absorbed by the gastrointestinal tract and is converted by hepatic esterases to the active compound (oseltamivir carboxylate). zanamivir has poor oral bioavailability and is currently available as a dry powder mixed with lactose. moreover, laninamivir and peramivir were also approved in north asia recently. laninamivir has excellent in vitro activity against wild type, as well as oseltamivir-resistant, influenza viruses currently circulating [12] . additionally, peramivir is another nai that differs structurally from other inhibitors through novel substitutions that result in multiple binding interactions with the active site and allows the antiviral to be active against nai-resistant viruses [13] . non-enveloped viruses, such as the picornavirus (picornaviridae family) and human papillomavirus (hpv) (papillomaviridae family), interact with their functional receptors through viral capsid proteins. picornaviruses are typical non-enveloped viruses, and some members, including enterovirus 71 (ev71) and human rhinoviruses (hrvs), are responsible for causing severe human infection diseases. the non-enveloped capsids of picornaviruses are icosahedral structures comprising 60 copies of viral structural proteins vp1-4 [14, 15] . vp1-3 each adopt a b-barrel configuration and are arranged with icosahedral symmetry such that vp1 surrounds the 5-fold axes and vp2 and vp3 alternate around the 2-and 3-fold axes [16] . although the receptor-binding sites on the surface of picornavirus capsids are not conserved [17] , these sites have been used to discover inhibitors that block virus-receptor interactions. for example, the canyon structure on the surface of the hrv capsid serves to bind to the hrv receptor, and the soluble portion of the intercellular adhesion molecule-1 (icam-1) [18] and numerous compounds that compete with the putative hrv receptor binding site have been shown to bind in a nearby hydrophobic pocket to inhibit virus attachment to the receptor [19] . however, none of these compounds have been clinical successes to date. after attaching to host cells, a virus will release its genome into the cytoplasm through endocytosis or direct membrane fusion. because this viral entry is one of the key early steps in the viral life cycle (figure 1 ), entry inhibitors have been successfully developed for antiviral therapies. as an enveloped virus, hiv-1 uses gp41 to facilitate its entry process after gp41 is activated by the binding of gp120 to the receptor. the extracellular portion of gp41 contains two heptad repeat domains (hr1 and hr2) separated by a loop region and a hydrophobic fusion peptide (fp) at the n terminus. during the fusion process, the fp of gp41 is inserted into the host cell membrane, and hr1 adopts a triple-stranded coiled-coil structure, forming a meta-stable prefusion intermediate. hr2 subsequently folds into the hydrophobic grooves of the hr1 coiled-coil to form a stable six-helix bundle that juxtaposes the viral and cellular membranes for fusion ( figure 2c ). fusion inhibitors are designed to block the conformational changes that are required for membrane fusion. the t20 peptide (enfuvirtide), which is a peptidic mimic of hr2 and acts by competitively binding to hr1, is the first and the only clinically approved fusion inhibitor [20, 21] . t20 can inhibit a broad range of hiv strains at the nanomolar level, but the poor bioavailability of the drug (it has a plasma half-life of 4 h) make the clinical application of this drug difficult [22, 23] . to overcome this pharmacokinetic disadvantage, a series of modified peptides including cp32m [24] , sifuvirtide [25] , and t2635 [26] have been generated to stabilize the helical structure of the hr2-like peptide by incorporating intrahelical salt bridges between the helix turns. in particular, the plasma half-life of sifuvirtide is 5-fold greater than that of t20 [27] . moreover, chemical modifications including the pegylation [28] , glycosylation [29] , and more have also been introduced to improve the pharmacokinetics of hr2-based fusion inhibitors. alternatively, a subset of bioavailable small molecule fusion inhibitors have been developed targeting the hr2binding pocket on the hr1 trimer [30] [31] [32] or other undefined regions [33] . a similar strategy has been successfully used against many viruses that use a class i fusion mechanism, including influenza virus, ebola virus (filoviridae family), and severe acute respiratory syndrome coronavirus (sars-cov) (coronaviridae family) [34] , but this approach is rare in antiviral development targeting viruses with a class ii/iii fusion mechanism. however, the difficulty in production and delivery means that none of these small molecule fusion inhibitors can be approved for clinical use. non-enveloped viruses use a different strategy for virus entry. after attaching to their receptors, a non-enveloped virus releases its genome into the host cell through a conformational shift of its capsid protein(s). for example, the capsid of ev71 harbors 60 copies of a hydrophobic 'pocket factor', a natural lipid (sphingosine), at the base of the canyon in the capsid protein vp1 [35] (figure 3 ). the expulsion of sphingosine following the binding of the virus to its receptor triggers the opening of the capsid to release the viral genome. pleconaril [36] and bta798 are two examples of hydrophobic compounds that can replace the natural pocket factor and inhibit picornaviral uncoating by generating resistance to the expulsion of pocket factor [37] [38] [39] . using the skeletons of pleconaril and related molecules, a novel class of imidazolidinones has been further synthesized with significant anti-ev71 activity (ic 50 in the range of 0.001-25 mm) [40] . most viruses encode one or several proteases that play crucial roles in the viral life cycle. the viral proteases carry out the proteolysis of a polyprotein precursor and release functional viral proteins, allowing them to function correctly and individually in replication/transcription and maturation [41, 42] . viral proteases also effectively protect viral proteins by modulating host cell pathways, including ubiquitination and isgylation [43] [44] [45] [46] [47] [48] . in contrast to the diversity of viral protease functions and structures, the catalytic active site of viral proteases generates stringent substrate specificity in protein cleavage. synthetic substrate peptides, which can be designed according to the natural substrates of individual viral proteases, usually generate high-affinity binding and thus provide potent candidates for further drug discovery. one of the great successes is the hiv-1 protease inhibitors (pis). there are ten pis currently approved to treat hiv-1 infection: amprenavir (apv), atazanavir (atz), darunavir (tmc114), fosamprenavir (lexiva), indinavir (idv), lopinavir (lpv), nelfinavir (nfv), ritonavir (rtv), saquinavir (sqv), and tipranavir (tpv) [49] . all hiv-1 pis share relatively similar chemical structures derived from its natural peptidic substrate and, therefore, the cross-resistance to pis occurs at the active site of hiv-1 protease [50] . as a result, pis are commonly used as the combination with other anti-hiv drugs to avoid drug resistance. because most of the pharmaceutical disadvantages have been overcome, pis have become the most potent types of antiviral drugs. current progress in generating antiviral pis has been systematically reviewed elsewhere [51, 52] . almost all viruses encode polymerases in the central steps of replication and transcription. thus, polymerases are becoming the most attractive and suitable targets for antiviral development. based on the function and structure of viral polymerases, there are two major types of polymerase inhibitors: (i) nucleoside and nucleotide substrate analogs and (ii) allosteric inhibitors. nucleoside/ nucleotide analogs play a dominant role in antiviral drugs targeting viral polymerases. nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids. the disadvantage of nucleoside analogs is that the initial phosphorylation step, that is, production of the monophosphorylated form, required for activation to a triphosphate may not correctly occur in the host cell [53] . therefore, monophosphate nucleotide analogs were developed as polymerase inhibitors to avoid this problem. to date, most of the approved antiviral drugs for anti-hiv therapy utilize this mechanism, including zidovudine (azt, , and others. the same strategy was also successfully used in the development of antivirals against a wide range of viruses, including cytomegalovirus (cmv) [54] and herpes simplex virus (hsv) [55] (herpesviridae family), hepatitis b virus (hbv) (hepadnaviridae family) [56] , and hcv [57] . during the course of a polymerase cycle, the relative orientation of the polymerase domains undergoes a slight shift and this shift causes the conformational change of a specific site, the allosteric site, in the viral polymerase. therefore, compounds that bind to the allosteric site could conceivably block the structural movement of polymerase domains and thus inhibit the function of viral polymerases. antiviral inhibitors that work by this mechanism are known as 'allosteric inhibitors'. the allosteric inhibitors of hiv reverse transcriptase (rt) are also known as nonnucleoside reverse transcriptase inhibitors (nnrtis). nnrtis and the hydrophobic binding site on hiv rt were first identified by screening compound libraries against hiv-1 rt combined with structural biological analysis [58] [59] [60] [61] . the binding of nnrti to hiv-1 rt prevents the flexibility and movement of rt required for the elongation of the nucleic acid. to date, there are five nnrtis, that is, nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine, that have been approved by the fda for clinical use to treat hiv-1 infection. furthermore, the development of nnrtis against other viral polymerases, such as that of hcv, is ongoing, and other polymerases are likely to have suitable sites for allosteric inhibitors [62] . integrase is an enzyme that helps the retrovirus to facilitate the incorporation of proviral dna into the host cell genome and catalyzes a vital function in viral replication. inhibitors of integrase represent the newest class of figure 4) . raltegravir, an integrase strand transfer inhibitor (insti), was the first generation drug of this class to be approved and is a potent and welltolerated antiviral agent [63] . dolutegravir is the most advanced second generation insti, and it possess good tolerability, once-daily dosing with no need for a pharmacological enhancer, and relatively little cross-resistance with raltegravir [64] . another insti, elvitegravir, was recently approved for the treatment of hiv infection as part of a fixed dose combination known as stribild [65] . furthermore, inhibitors of the lens epithelium-derived growth factor (ledgf)/p75 binding site of integrase (led-gins) have also been developed, but these are still in a very early stage. each of these drugs contributes a new benefit to the class and will extend the treatment options for patients with hiv-1 infection. the 5 0 terminus of the genomic rna in a subset of rna viruses requires a cap structure, which can be directly taken from the mrna of host cells, as in influenza virus, or synthesized by viral enzymes, as in flavivirus and coronavirus. in the latter cases, a cap structure is generated by a series of enzymes and attached to the first nucleotide of the genome rna via a 5 0 -5 0 triphosphate linker. for example, the genome of flavivirus is capped by a type 1 cap structure (m 7 gpppam), which is methylated at the n-7 position of the guanine (m 7 g) and on the 2 0 oh of the ribose of the first nucleotide (am) of the genome rna [66] . three enzymatic steps are required to form the cap structure, including an rna triphosphatase (ns3), a guanylyl transferase (gtase), and a methyltransferase (mtase), provided by the n terminus of ns5 protein. based on the structure of mtase and its complex with different substrates, three key ligand-binding sites were identified ( figure 5 ). the binding site for s-adenosyl methionine (sam), which acts as the methyl donor, is located at the core domain of mtase, whereas the binding site for the receiver gtp molecule and the guanine moiety of the rna cap is formed by the core domain and the n-terminal extension. moreover, there is another site that binds rna between the sam and gtp pockets; this site is known to be a second, lower affinity binding site that is formed by the core domain and by a channel between two helices of the nterminal extension. because these sites are crucial for mtase activity and virus replication, all are valid sites for the design of dvtas (figure 4) . ribavirin was first shown to inhibit the binding of the rna cap to dengue virus (denv) mtase by competitively binding to the gtp/rna cap pocket [67] . through structure-based drug design, aurintricarboxylic acid was found to inhibit the 2 0 -o activity of denv mtase with an ic 50 of 2 mm by binding to the lower affinity site [68] . another inhibitor, sinefungin, which is an analog of sam with the methylated sulfur of sam replaced by a carbon and amine, can inhibit flavivirus activity with an ic 50 as low as 0.7 mm [69] . recently, the compound bg323 has been discovered using high-throughput screening (hts) methods as an in vitro inhibitor of the guanylyltransferase activity of denv mtase (and thus denv replication) [70] . however, these inhibitors must be used at a high concentration to outcompete the high-affinity endogenous ligand, and it is necessary to solve this problem before this mechanism can be clinically exploited. some viruses utilize a viral helicase to separate one strand of dna or rna from the complementary strand in a process driven by adenosine triphosphate (atp) hydrolysis. the most widely studied viral helicase is the flaviviral helicase, which is the helicase domain of nonstructural protein 3 (ns3) encoded by hcv. sars-cov (nsp13 protein), simian virus 40 (sv40) (polyomaviridae family) (tag protein), and hpv (e1 protein) are also known to encode helicases for their replication [71] . the inhibitors of helicase-catalyzed atp hydrolysis are the most straightforward class, and several nucleotide and nucleobase analogs have been discovered targeting this mechanism. biphenyls and triphenylmethanes have been studied as inhibitors of sv40 tag [72] , hpv e1 [73] , and hcv ns3 helicase [74] . biphenyls and biphenysulfonacetic acid were found to inhibit hpv growth, an inhibitor of hpv e1-catalyzed atp hydrolysis with an ic 50 value >2 mm [75] . compounds similar to triclocarban (cid7547) and bisphenol a (bpa; cid6623) were found to inhibit the helicase activity of sv40 tag. based on the complex structure of the hcv ns3 helicase domain and soluble blue ht (pdb code 2zjo), triphenylmethanes, a more potent triphenylmethane [76] , and aurintricarboxylic acid (ata) [77] were developed to inhibit the helicase activity of hcv ns3. the nucleic acid binding site of helicase is also a promising site for the discovery of antiviral compounds that directly inhibit helicase-catalyzed unwinding [78] . for example, many dna-binding pharmacophores, such as anthracyclines, acridones, tropolones, and amidinoanthracyclines, have been optimized as hcv helicase inhibitors [78] . although there are numerous efforts that have been launched to find antivirals targeting viral helicase, little progress had been made to push them into clinical usage, and these projects have met great challenges on cytotoxicity, bioavailability, and pharmacokinetic properties [79] . replication and transcription complex blockers following viral entry, viral proteins, together with a number of host cell factors, assemble a viral replication and transcription complex (rtc) that is responsible for the production of the viral genome or other nucleic acids [80] . therefore, reagents that can efficiently block the formation of the viral rtc could conceivably inhibit viral proliferation. for example, once hcv enters the host cell, the genome of hcv will work as mrnas to produce viral proteins and form an rtc in which the host factors account for viral replication and transcription. ns5a is a membrane-associated nonstructural phosphoprotein, and it is believed that ns5a has no enzymatic activity but plays a critical role in regulating the formation of the hcv rtc. gao et al. identified a potent hcv inhibitor, bms790052, that targets ns5a and produced few side effects in a phase i clinical study [81] . although the exact mechanism by which bms790052 exerts its effects is yet to be defined, the resistance profile reveals that inhibitor sensitivity maps to the n terminus of domain 1 of ns5a. this inhibitor is further shown to block the hyperphosphorylation of ns5a, which is believed to play an essential role in the viral life cycle. this work, for the first time, proved the concept that small molecules targeting a non-traditional viral protein without any known enzymatic activity can also have profound antiviral effects with considerable promise for the treatment of hcv infection. during the replication of ev71 and other picornaviruses, polymerase (also named 3d pol ), 3b (also named vpg), and protease (also named 3c pro ) participate in the formation of viral rtc, together with host polya-binding protein 1 (pabp-1) and polyc-binding protein 2 (pcbp-2) [15] . unlike many other viruses, the replication of the picornavirus genome is initiated by the 5 0 end of genome covalently linked to vpg through a so-called vpg uridylylation process [82] . although vpg-binding sites vary in picornaviruses [14, 15, 83, 84] , the binding of vpg to 3d pol is known to be critical for vpg uridylylation and virus replication. in our recent study, we demonstrated that a ten amino acid peptide of vpg can effectively inhibit the vpg uridylylation process with an ec 50 as low as 50 nm (z. lou et al., unpublished). these results shed light on discovering future indvtas. throughout the life cycle of a negative sense single-stranded rna (ssrna) virus, the genome length rna is encapsidated by a virally encoded nucleoprotein (np), instead of a naked rna, and associated with rdrp (polymerase complex) to form a stable ribonucleoprotein (rnp) complex, which is responsible for virus replication, transcription, and assembly [85] . during this process, np can protect the rna against exogenous nucleases or the innate immune system in the host cell. based on the crystallographic achievements regarding the ssrna virus rnp complex [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] over the past few years, great progress has been made in this new putative antiviral strategy. kao et al. first identified a compound, nucleozin, that triggers the aggregation of and inhibits the nuclear accumulation of np; this compound can inhibit the replication of influenza virus with a nanomolar median effective concentration (ec 50 ) [97] . in a parallel effort, gerritz et al. discovered a series of influenza replication inhibitors and showed that they interfere with np-dependent processes via the formation of higher order np trends in pharmacological sciences february 2014, vol. 35, no. 2 oligomers with an ec 50 of 60 nm [98] . notably, the structure of the np in complex with a representative compound from this class of inhibitors revealed that two molecules of an inhibitor in an antiparallel orientation lock two adjacent np protomers. this unexpected quaternary complex explained the viral inhibition via ligand-induced formation of stable np oligomers [98] . in addition to inhibiting np, the disruption of the polymerase complex of influenza virus has been proposed for antiviral strategies. based on the complex structure of the pa c-terminal domain (pa c ) and the first 25 amino acids of pb1 [99] , a subset of modifications on n-terminal peptide of pb1 was shown to diminish the binding affinity of pa and pb1, inhibit polymerase activity, and attenuate the replication of influenza virus [100] [101] [102] . moreover, the structure of sftsv np in complex with suramin, an antiviral inhibitor, revealed that the blockers bind to the rna-binding cavity and can attenuate sftsv replication; this indicated that targeting rnp formation may be a new therapeutic antiviral approach [103, 104] . because both the polymerase complex and np show significant conservation between different influenza viruses, these results demonstrated that targeting the formation of viral rnp is a valid approach to the development of small molecule therapies against serious antiviral resistance to currently available drugs, such as adamantanes or neuraminidase inhibitors. other key interactions between viral proteins and/or host factors play essential roles in virus entry, replication, and maturation. a very interesting example is the interaction between hpv e1 and e2 protein, helping e1 helicase to tether to the hpv origin of replication. this interaction can therefore be used to guide the development of antivirals treating hpv infection. the disruption of the hpv e1-e2 interaction was first facilitated by a very potent inhibitor, chembl1207308. this inhibitor can effectively diminish the interaction between hpv e1 and e2 and thus inhibit hpv proliferation with an ec 50 of 6 nm [105] . although a few indvtas have been discovered for antiviral therapy, their mechanisms of working are still required to be further investigated. we cannot conclude the possibility that indvtas may have additional functions (or may be the major function) and the category of antivirals will be refined according to further knowledge. viruses utilize many host factors for their efficient proliferation. the correct functions of these host factors are crucial for virus replication and, therefore, compounds that regulate the function of host factors can be introduced as antiviral agents. next we discuss two host factors that are involved in broad spectrum virus infection. apart from the acquired immune response, host cells mount a number of defenses, such as the innate immune response, to virus infection. interferon (ifn) is one of the most crucial molecules in the innate immune response and acts as the primary switch for initiating antiviral immunity in vertebrates. upon being infected by a virus, host cells produce and secrete type i (mainly ifn-a and ifn-b) and type iii (ifn-l) ifns. these secreted ifns interact with the membrane-anchored ifn receptors (ifnars), mainly ifnar1 and infar2, and subsequently stimulate and upregulate the expression of hundreds of ifn-stimulated genes (isgs) to inhibit the replication of viruses [106] . among these isgs, ifn-inducible transmembrane (ifitm) proteins restrict the entry of influenza virus, west nile virus (wnv), and denv. additionally, mx (myxovirus resistance) gtpases can inhibit the correct function of viral nucleocapsids and polymerases, such as influenza virus [107] . due to the significance of ifn in the host cells to restrict viral infections, ifn is used in certain instances as a primary antiviral therapy, particularly in the absence of an effective antiviral or vaccination strategy. the use of ifn for antiviral therapy was first developed for treating hbv infection [108] and subsequently applied to treat hcv in combination with ribavirin [109] . ifn-b [110] and ifn-g [111] have also been evaluated for use in anti-hcv treatment. to further improve the therapeutic efficacy of ifn-a therapy, several options are being investigated. some clinical benefits were observed in pilot studies with ofloxacin [112, 113] and an immunomodulatory peptide, a1-thymosin [114] . in particular, peg-modified ifn-a [115] combined with ribavirin is now standard treatment for hcv infection. cyclophilins (cyps) are key cellular factors that function in numerous cellular processes, including transcriptional regulation, the immune response, protein secretion, and mitochondrial function [116, 117] . cyclophilin a (cypa) is a key member of the cyp family and was first identified as a mediator of the immunosuppressive function of cyclosporin a (csa) through the formation of a csa-cypa complex. this complex binds to and inhibits the function of the protein phosphatase calcineurin [118] , which normally functions to dephosphorylate nf-at, a transcription factor important for t cell activation. cypa is also known to play critical roles in the proliferation of viruses, including hiv-1, influenza virus, hcv, vesicular stomatitis virus (vsv), vaccinia virus, sars-cov, rotavirus (rv), and hpv [117, 119] , by interacting with viral proteins or facilitating ifn-b production [14, [120] [121] [122] [123] . cypa was first revealed to be incorporated into hiv-1 virions by interacting with the capsid protein (ca), and an interaction between newly synthesized hiv-1 ca and cypa is required for hiv-1 to induce dendritic cell maturation [114, 124] . cypa also interacts with other hiv-1 proteins, such as vpr and p6, to regulate hiv infection [88, 91, [125] [126] [127] [128] . several lines of evidence indicate that cypa and cyclophilin b (cypb) [129] function in the replication of hcv by either increasing the affinity of hcv polymerase ns5b for viral rna to enhance hcv replication [130] or binding to the hcv ns5a protein to aid viral replication [131] . the discovery of cypa inhibitors as antiviral agents started with the immunosuppressive drug csa that inhibits hcv [129] and hiv [132] . on the basis of these results, cypa antagonists, which are often derived from review trends in pharmacological sciences february 2014, vol. 35, no. 2 csa, have been developed, including alisporivir (debio-025), nim811, and scy635; these compounds lack immunosuppressive effects but retain high-affinity cypa binding and show very good antiviral effects against hiv or hcv infection [133] [134] [135] . hiv-1 co-receptors antagonists hiv-1 co-receptor antagonists that block the interactions between hiv-1 and ccr5 and/or cxcr4 have also been introduced to the anti-hiv efforts, and a few of these have been successful. for instance, ccr5 antagonists that potently inhibit hiv-1 replication and have good pharmaceutical properties, including aplaviroc [136] , maraviroc (the only clinically used anti-hiv drug as a co-receptor antagonist) [137] , vicriviroc [138] , and cenicriviroc [139, 140] , have been successfully advanced to phase ii/iii clinical trials or approved for clinical usage in the past 5 years. however, the precise mechanisms of these co-receptor antagonists are still not clear. recently, the crystal structures of ccr5 [141] and cxcr4 [142] have been reported. these two structures provided the atomic information of the ligand-binding pocket and the sites for co-receptor oligomerization. further structural study on the complex of ccr5 and cxcr4 with their antagonists will promote the investigation into the inhibitory mechanisms of these inhibitors and help us to increase their anti-hiv efficacy. at present, diverse antiviral drugs are clinically approved or in the later stages of clinical trials. most are based on the conserved mechanisms described in this review, in particular through targeting of viral polymerases and proteases. however, the majority of drug-resistant infection cases have been reported with the usage of antivirals based on this strategy. the requirement for new antiviral drugs to treat chronic infectious diseases and the emergence of more efficient new viruses serve as catalysts for research to find additional targets and mechanisms for antiviral development. a few novel strategies have been introduced for antiviral research, including inhibitors of viral mtase and helicase, blockages of viral rtc formation (e.g., nucleozin to influenza), and host factor antagonists or agonists. however, protease and polymerase inhibitors still occupy a dominant place among antivirals. this is because we still do not have very precise knowledge on the mechanisms underlying alternative strategies, and this requires further investigations on their mechanism before these strategies can be widely used for antiviral development, in particular those targeting drug-resistant viral infections. in our opinion, protease and polymerase inhibitors will still be the first, and probably the major, choice in the development of therapies against emerging novel viruses. in addition, we must consider another important aspect for antiviral development. for some viruses, currently available drugs can effectively eliminate the virus in the host, but genetic components are left behind. thus, the host still suffers from the infection because the virus can integrate its genetics into the host cell. for example, treatment with the combination of inf and adefovir (or other antivirals) can effectively, if not completely, reduce hbv titer in human liver. however, the covalently closed circular dna (cccdna) cannot be removed and still exists in the nuclei of infected liver cells, where it continuously coordinates the expression of hbv antigens. therefore, new mechanisms and strategies to completely remove viral components integrated in host cells, as well as to kill the virus itself, are required in future antiviral development. molecular architecture of the uncleaved hiv-1 envelope glycoprotein trimer elicitation of hiv-1-neutralizing antibodies against the cd4-binding site biological and pharmacokinetic properties of a novel immunoglobulin-cd4 fusion protein recombinant soluble cd4 therapy in patients with the acquired immunodeficiency syndrome (aids) and aids-related complex. a phase i-ii escalating dosage trial treatment of advanced human immunodeficiency virus type 1 disease with the viral entry inhibitor pro 542 antiviral activity, pharmacokinetics, and safety of bms-488043, a novel oral small-molecule hiv-1 attachment inhibitor, in hiv-1-infected subjects in vitro antiviral characteristics of hiv-1 attachment inhibitor bms-626529, the active component of the prodrug bms-663068 a mutant influenza virus that uses an n1 neuraminidase as the receptor-binding protein structure of the catalytic and antigenic sites in influenza virus neuraminidase structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 a resolution antivirals and resistance: influenza virus laninamivir and its prodrug, cs-8958: longacting neuraminidase inhibitors for the treatment of influenza clinical experience in adults and children treated with intravenous peramivir for 2009 influenza a (h1n1) under an emergency ind program in the united states crystal structure of enterovirus 71 rnadependent rna polymerase complexed with its protein primer vpg: implication for a trans mechanism of vpg uridylylation enterovirus 71 vpg uridylation uses a twomolecular mechanism of 3d polymerase rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen crystal structure of equine rhinitis a virus in complex with its sialic acid receptor a soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection inhibitory effect of dibenzofuran and dibenzosuberol derivatives on rhinovirus replication in vitro; effective prevention of viral entry by dibenzosuberenone peptides corresponding to a predictive a-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry the safety, plasma pharmacokinetics, and antiviral activity of subcutaneous enfuvirtide (t-20), a peptide inhibitor of gp41-mediated virus fusion, in hiv-infected adults a phase ii clinical study of the long-term safety and antiviral activity of enfuvirtide-based antiretroviral therapy potent hiv fusion inhibitors against enfuvirtideresistant hiv-1 strains synthesized peptide inhibitors of hiv-1 gp41-dependent membrane fusion design of helical, oligomeric hiv-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus design and evaluation of sifuvirtide, a novel hiv-1 fusion inhibitor pegylation, successful approach to drug delivery glycoengineering: the effect of glycosylation on the properties of therapeutic proteins ads-j1 inhibits human immunodeficiency virus type 1 entry by interacting with the gp41 pocket region and blocking fusion-active gp41 core formation design, synthesis, and biological activity of novel 5-((arylfuran/1h-pyrrol-2-yl)methylene)-2-thioxo-3-(3-(trifluoromethyl)phenyl)thi azolidin-4-ones as hiv-1 fusion inhibitors targeting gp41 design, synthesis, and structure-activity relationship of a novel series of 2-aryl 5-(4-oxo-3-phenethyl-2-thioxothiazolidinylidenemethyl)furans as hiv-1 entry inhibitors a low-molecular-weight entry inhibitor of both ccr5-and cxcr4-tropic strains of human immunodeficiency virus type 1 targets a novel site on gp41 viral membrane fusion picornavirus uncoating intermediate captured in atomic detail in vitro and in vivo evaluation of ribavirin and pleconaril antiviral activity against enterovirus 71 infection treatment of picornavirus infections a novel basis of capsid stabilization by antiviral compounds stabilization of poliovirus by capsidbinding antiviral drugs is due to entropic effects design, synthesis, and structure-activity relationship of pyridyl imidazolidinones: a novel class of potent and selective human enterovirus 71 inhibitors jr (1968) evidence for large precursor proteins in poliovirus synthesis translation of encephalomyocarditis virus rna in vitro yields an active proteolytic processing enzyme mechanisms and enzymes involved in sars coronavirus genome expression identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes deubiquitinating function of adenovirus proteinase the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism function from tmc114 to darunavir: five years of data on efficacy implications of antiretroviral resistance on viral fitness protease inhibitors for the treatment of chronic hepatitis c genotype-1 infection: the new standard of care hiv protease inhibitors in pregnancy: pharmacology and clinical use rational design of polymerase inhibitors as antiviral drugs drug targets in cytomegalovirus infection entecavir (bristol-myers squibb) new drugs for chronic hepatitis b: a review antiviral drug advances in the treatment of human immunodeficiency virus (hiv) and chronic hepatitis c virus (hcv) a novel lead for specific anti-hiv-1 agents: 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine a new class of hiv-1-specific 6-substituted acyclouridine derivatives: synthesis and anti-hiv-1 activity of 5-or 6-substituted analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (hept) pyridinone derivatives: specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity structure-activity relationship studies on clinically relevant hiv-1 nnrtis non-nucleoside inhibitors of the hcv ns5b polymerase: progress in the discovery and development of novel agents for the treatment of hcv infections next-generation integrase inhibitors: where to after raltegravir? clinical use of hiv integrase inhibitors: a systematic review and meta-analysis co-formulated elvitegravir, cobicistat, emtricitabine, and tenofovir versus co-formulated efavirenz, emtricitabine, and tenofovir for initial treatment of hiv-1 infection: a randomised, double-blind, phase 3 trial, analysis of results after 48 weeks structural biology of dengue virus enzymes: towards rational design of therapeutics a structural basis for the inhibition of the ns5 dengue virus mrna 2 0 -o-methyltransferase domain by ribavirin 5 0 -triphosphate flaviviral methyltransferase/rna interaction: structural basis for enzyme inhibition structural and functional analyses of a conserved hydrophobic pocket of flavivirus methyltransferase identification of a novel antiviral inhibitor of the flavivirus guanylyltransferase enzyme development of chemical inhibitors of the sars coronavirus: viral helicase as a potential target a screen for modulators of large t antigen's atpase activity uncovers novel inhibitors of simian virus 40 and bk virus replication biphenylsulfonacetic acid inhibitors of the human papillomavirus type 6 e1 helicase inhibit atp hydrolysis by an allosteric mechanism involving tyrosine 486 discovering new medicines targeting helicases: challenges and recent progress discovery of small-molecule inhibitors of the atpase activity of human papillomavirus e1 helicase structure-based discovery of triphenylmethane derivatives as inhibitors of hepatitis c virus helicase characterization of aurintricarboxylic acid as a potent hepatitis c virus replicase inhibitor ns3 helicase inhibitors viral and cellular rna helicases as antiviral targets the dependence of viral rna replication on co-opted host factors chemical genetics strategy identifies an hcv ns5a inhibitor with a potent clinical effect protein is linked to the 5 0 end of poliovirus rna by a phosphodiester linkage to tyrosine the structure of a protein primerpolymerase complex in the initiation of genome replication the crystal structure of coxsackievirus b3 rnadependent rna polymerase in complex with its protein primer vpg confirms the existence of a second vpg binding site on picornaviridae polymerases architecture and regulation of negative-strand viral enzymatic machinery nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and rna polymerization the structure of native influenza virion ribonucleoproteins crimean-congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses structure of the lassa virus nucleoprotein reveals a dsrna-specific 3 0 to 5 0 exonuclease activity essential for immune suppression crystal structure of the lassa virus nucleoprotein-rna complex reveals a gating mechanism for rna binding bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation organization of the influenza virus replication machinery cap binding and immune evasion revealed by lassa nucleoprotein structure phleboviruses encapsidate their genomes by sequestering rna bases structure of the rift valley fever virus nucleocapsid protein reveals another architecture for rna encapsidation structural basis for encapsidation of genomic rna by la crosse orthobunyavirus nucleoprotein identification of influenza a nucleoprotein as an antiviral target inhibition of influenza virus replication via small molecules that induce the formation of higherorder nucleoprotein oligomers crystal structure of the polymerase pa(c)-pb1(n) complex from an avian influenza h5n1 virus identification of high-affinity pb1-derived peptides with enhanced affinity to the pa protein of influenza a virus polymerase identification of a pa-binding peptide with inhibitory activity against influenza a and b virus replication disruption of the viral polymerase complex assembly as a novel approach to attenuate influenza a virus the nucleoprotein of severe fever with thrombocytopenia syndrome virus processes a stable hexameric ring to facilitate rna encapsidation structure of crimean-congo haemorraghic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase-3 cleavage optimization and determination of the absolute configuration of a series of potent inhibitors of human papillomavirus type-11 e1-e2 protein-protein interaction: a combined medicinal chemistry, nmr and computational chemistry approach structural linkage between ligand discrimination and receptor activation by type i interferons intrinsic antiviral immunity natural killer cell activity and function in chronic hcv-infected patients during peg interferon and ribavirin: early effects of active substance use interferon-a-2b plus ribavirin: a review of its use in the management of chronic hepatitis c dynamics of hepatitis c virus monitored by real-time quantitative polymerase chain reaction during first 2 weeks of ifn-b treatment are predictive of long-term therapeutic response interferon-g brings additive anti-viral environment when combined with interferon-a in patients with chronic hepatitis c treatment of chronic hepatitis c with ainterferon plus ofloxacin in patients not responding to a-interferon alone effects of combination therapy with interferon and ofloxacin on chronic type c hepatitis: a pilot study interferon and thymosin combination therapy in naive patients with chronic hepatitis c: preliminary results development of pegylated interferons for the treatment of chronic hepatitis c cyclophilin: a specific cytosolic binding protein for cyclosporin a insights into the roles of cyclophilin a during influenza virus infection calcineurin is a common target of cyclophilincyclosporin a and fkbp-fk506 complexes target cell cyclophilins facilitate human papillomavirus type 16 infection hepatitis b virus (hbv) surface antigen interacts with and promotes cyclophilin a secretion: possible link to pathogenesis of hbv infection requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a cyclophilin a inhibits rotavirus replication by facilitating host ifn-i production binding of the human immunodeficiency virus type 1 gag polyprotein to cyclophilin a is mediated by the central region of capsid and requires gag dimerization cyclophilin a interacts with hiv-1 vpr and is required for its functional expression structural characterization of the hiv-1 vpr n terminus: evidence of cis/trans-proline isomerism hiv-1 p6-another viral interaction partner to the host cellular protein cyclophilin a the host-pathogen interaction of human cyclophilin a and hiv-1 vpr requires specific n-terminal and novel c-terminal domains cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes critical role of cyclophilin a and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis c virus replication complex identification of residues required for rna replication in domains ii and iii of the hepatitis c virus ns5a protein the effect of cyclosporine a on infection of susceptible cells by human immunodeficiency virus type 1 debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis c virus (hcv) repliconcontaining cells when used alone or in combination with specifically targeted antiviral therapy for hcv (stat-c) inhibitor scy-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis c virus rna replication in vitro inhibition of human immunodeficiency virus type 1 replication in human cells by debio-025, a novel cyclophilin binding agent antiviral activity and safety of 873140, a novel ccr5 antagonist, during short-term monotherapy in hiv-infected adults maraviroc versus efavirenz, both in combination with zidovudine-lamivudine, for the treatment of antiretroviral-naive subjects with ccr5-tropic hiv-1 infection vicriviroc plus optimized background therapy for treatment-experienced subjects with ccr5 hiv-1 infection: final results of two randomized phase iii trials pharmacokinetics and pharmacodynamics of tbr-652, a novel ccr5 antagonist, in hiv-1-infected, antiretroviral treatment-experienced, ccr5 antagonist-naive patients safety, efficacy, and pharmacokinetics of tbr-652, a ccr5/ccr2 antagonist, in hiv-1-infected, treatmentexperienced, ccr5 antagonist-naive subjects structure of the ccr5 chemokine receptor-hiv entry inhibitor maraviroc complex structures of the cxcr4 chemokine gpcr with small-molecule and cyclic peptide antagonists parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses structure of a v3-containing hiv-1 gp120 core a sensor-adaptor mechanism for enterovirus uncoating from structures of ev71 this work was supported by the ministry of science and technology of china '973' project (grant numbers 2013cb911103 and 2014cb542800) and the national natural science foundation of china (grant numbers 81322023, 31000332, 31100208, 31170678, and 31370733). key: cord-275677-hbv49e01 authors: ramana, lakshmi narashimhan; anand, appakkudal r.; sethuraman, swaminathan; krishnan, uma maheswari title: targeting strategies for delivery of anti-hiv drugs date: 2014-10-28 journal: j control release doi: 10.1016/j.jconrel.2014.08.003 sha: doc_id: 275677 cord_uid: hbv49e01 human immunodeficiency virus (hiv) infection remains a significant cause of mortality globally. though antiretroviral therapy has significantly reduced aids-related morbidity and mortality, there are several drawbacks in the current therapy, including toxicity, drug–drug interactions, development of drug resistance, necessity for long-term drug therapy, poor bio-availability and lack of access to tissues and reservoirs. to circumvent these problems, recent anti-hiv therapeutic research has focused on improving drug delivery systems through drug delivery targeted specifically to host cells infected with hiv or could potentially get infected with hiv. in this regard, several surface molecules of both viral and host cell origin have been described in recent years, that would enable targeted drug delivery in hiv infection. in the present review, we provide a comprehensive overview of the need for novel drug delivery systems, and the successes and challenges in the identification of novel viral and host-cell molecules for the targeted drug delivery of anti-hiv drugs. such targeted anti-retroviral drug delivery approaches could pave the way for effective treatment and eradication of hiv from the body. cells i.e. cd4 + t cells, monocytes, macrophages, and dendritic cells. the decline in cd4 + t-cells in the more advanced stages of infection is responsible for the profound immune suppression that characterizes the advanced stage of aids. however, the advent of highly active anti-retroviral therapy (haart), a combination of drugs that inhibit hiv-1 replication, has led to reduced viremia and the onset of opportunistic infections in most patients and prolonged survival. chemotherapy has been the main mode of treatment of aids. the common classes of drugs that have been employed in the treatment of hiv infections are designed to inhibit a particular stage in the infectious cycle of hiv. fig. 1 depicts the different targets for the various antiretroviral drugs. entry inhibitors comprising fusion inhibitors and coreceptor inhibitors prevent the entry of hiv into the host cell. while fusion inhibitors interact with the glycoprotein-41 (gp-41) on the viral envelope thereby blocking its fusion with the cell membrane, the coreceptor inhibitors interact with the ccr5 receptor in the host cell and prevent their interactions with the glycoprotein-120 (gp-120) of the virus [2] . protease inhibitors (pis) interfere with the proteolytic processing of viral proteins through binding interactions with the active site of the hiv protease [3] . the reverse transcriptase inhibitors form a major class of anti-retroviral drugs. these molecules disrupt the transformation of the single stranded rna of the viral genome into a double stranded dna by the referred to as nucleoside reverse transcriptase inhibitors (nrtis) and nucleotide reverse transcriptase inhibitors (ntrtis) respectively. these inhibitors binds directly to the reverse transcriptase enzyme and thus block the hiv life cycle. a related class of drugs known as non-nucleoside reverse transcriptase inhibitors (nnrtis) binds to an allosteric site of reverse transcriptase resulting in inhibition of the enzyme activity. a recent addition to the repertoire of anti-retroviral drugs is the integrase inhibitors that interfere with the integration of the viral genome with the host cell. list of anti-hiv drugs are listed in table 1 . anti-retroviral therapy (arv) has significantly reduced aids-related morbidity and mortality. however there are several drawbacks in the current arv therapy. a) drug resistance: one other major problem with anti-hiv drug therapy is that of resistance. the process of hiv replication is rapid and error-prone (~10 billion viral particles are produced on a daily basis), while generating at least one mutation per genome. these genetic mutations enable the virus to develop resistance to anti-hiv drug therapy, especially when monotherapy is employed. an example is the emergence and selection of hiv subtype c virus in india, which contains multiple nf-kb (sites?) and strengthens the promoters of the virus. b) long-term drug therapy: the nature of hiv infection, including viral persistence in reservoirs necessitates long-term uninterrupted multi-drug arv therapy. treatment compliance is critical regardless of whether a patient is treatment-naïve or treatment-experienced since poor patient compliance is often a factor in treatment failure and viral rebound. the lack of patient adherence to complicated drug administration regimens is further exacerbated by the cumulative costs of combined arv therapy. c) toxicity and drug-drug interactions: prolonged treatment with arv drugs has resulted in several side-effects, including constipation, fever, liver disorders, muscular dystrophy, metabolic disorders, and peripheral neuropathy. the use of a combination of drugs in the conventional therapy can also lead to undesirable drug-drug interactions thereby reducing the efficacy of the drugs. for instance, when a combination of the reverse transcriptase inhibitor nevirapine and protease inhibitor saquinavir was used to treat hiv infections, saquinavir levels in the plasma were found to drop rapidly. this is due to the induction of the liver cytochrome p450 by nevirapine leading to the metabolization of saquinavir and its subsequent elimination therefore reducing its availability and efficacy [4] . d) poor bio-availability: arv drugs are burdened by poor pharmacokinetics. most of the arv drugs are formulated as solid dosage forms for oral route of administration. the oral dosage forms offer convenience, but the combined dose of compounds that make up a therapeutic regimen is usually high. high doses are preferred because the treatment objective is to completely inhibit viral proliferation, an effect which is proportional to drug concentrations. the delivery of drugs via oral route suffers from significant first-pass effect, variation of absorption and degradation in the gastrointestinal tract due to enzymes and extreme ph conditions leading to low and erratic bioavailability. for example, the expression of multidrug resistant efflux proteins (mrps) such as p-glycoprotein (p-gp) on the gastrointestinal tract further decreases their oral bioavailability. the metabolism/elimination and transport barriers will substantially reduce the effective amount of anti-hiv drug reaching the target action site. the half-life for several arv drugs is short, which then requires frequent administration of doses leading to poor patient compliance. the short residence time and reduced half-life of the drug in the plasma necessitate frequent administration of booster dosages as well as increased drug dosages contributing to the development of drug resistance. some anti-retroviral drugs such as saquinavir possess poor bioavailability due to their metabolization by liver enzymes [5] . e) lack of access to tissues and reservoirs: most of the anti-retroviral drugs cannot cross the blood-brain barrier (bbb) as a result of which they are ineffective in entering microglial cells such as astrocytes that hoards hiv particles [6] . a major factor is the high protein binding of most anti-hiv drugs that prevent their diffusion across the bbb. also, it has been observed that the anti-retroviral drugs are unable to reach the lymphatic system that harbors hiv. thus the conventional therapy is ineffective in annihilating viral reservoirs as less than 2% of the lymphatic cells are present in systemic circulation [7] . the cells of the lymphatic system like macrophages and dendritic cells are involved in the transmission of the virus to cd4 + t h cells (helper t lymphocytes) [8] and failure to target such reservoirs of hiv increases the risk of a viral relapse post-treatment. 3. emergence of newer approaches to counter problems in conventional arv therapy to circumvent the problems mentioned above and effectively treat the hiv infection, recent anti-aids therapeutic development efforts have been focusing on improving drug delivery and not just on discovering new chemical entities. efforts have been made to design novel drug delivery systems for anti-hiv agents to reduce the dosing frequency, to enhance the bioavailability, to improve the central nervous system (cns) penetration, to inhibit the cns efflux and to deliver them to the target cells selectively with minimal side effects. among the recent approaches of novel drug delivery system for anti-hiv drugs, targeted/intracellular drug delivery only in host cells capable of getting infected with hiv or more specifically hiv-infected cells and reservoirs holds great promise. the main aim of drug targeting is to optimize a drug's therapeutic index by strictly localizing its pharmacological activity to the site or organ of action. the result of the targeting would be a significant reduction in drug toxicity, reduction of the drug dose, and increased treatment efficacy. during hiv infection, the virus infects only specific cells of the immune systems including cd4 + t-cells, macrophages and dendritic cells, which forms a strong rationale for targeting delivery of anti-hiv drugs specifically to these cells for increasing the efficacy of therapy. drug delivery to hiv-infected tissues/ cells with selectivity can be achieved by targeting surface markers on cd4 + t-cells, macrophages, dendritic cells and towards the cns. targeting these receptors can have two effects: i) prophylactic protection of the cell by occupying the receptor necessary for virus-cell interaction, and/or ii) improving the specific uptake of a nanocarrier loaded with anti-viral drugs. in this regard, several molecules of both viral and host cell origin have been described in recent years, that would enable targeted drug delivery in hiv infection. in the following review, we provide a brief overview of the recent developments in the identification of novel viral and host-cell molecules for the targeted drug delivery of anti-hiv drugs. a schematic representation of the different types of viral and host targets investigated for specifically targeting hivinfected cells, thus far is shown in fig. 2 . the conserved regions in hiv envelope glycoproteins gp41 and gp120 are involved in binding of the virus to the host cells [9] . many targeting strategies have focused on binding to these protein sequences to achieve specificity, though the high rates of mutation exhibited by the retrovirus make it challenging to identify a suitable targeting sequence on the envelope [10] . the glycoprotein-120 (gp120) present in the outer envelope of hiv enables the entry of the virus particles into cd4 + t-cells. it mainly binds to the cd4 receptor of the t-lymphocytes (t h cells) and macrophages and aids in the viral entry with the help of the fusion protein gp41 [11] . upon infection, the gp120 is mainly exposed on the surface of the hiv infected cells and hence can be an attractive target for cellspecific delivery of anti-retroviral drugs. an immunoliposomal carrier encapsulating the protease inhibitor p11 and surface modified with anti-gp120 was reported to exhibit excellent specificity towards hiv infected cells due to binding of the anti-gp120 to the exposed domains of gp120 present on the surface of hiv infected cells [12] . actinohivin, a prokaryotic lectin found in actinomycetes was identified to possess anti-hiv activity and was found to specifically target hiv infected cells. this lectin belongs to the family of carbohydrate binding agents (cbas) and displays high affinity binding to the mannose residues of the d1 chains of high mannose-type glycans associated with gp120 of hiv [13] . in an earlier work, a glycosylphosphatidylinositol (gpi) moiety conjugated to gp120 was incorporated in liposomes and was found to selectively bind to cd4 + cells [14] . comparison of the gpi-gp120 liposomes with liposomal vesicles formed from components of the viral envelope revealed differences in the intracellular localization in the two systems. as both carriers were internalized through cd4 mediated endocytosis, it was inferred that differences in the lipid composition in the two systems influenced their intracellular compartmentalization. this facet, however, remains largely unexplored in the context of targeted nanocarrier delivery of anti-retrovirals. mesoporous silica nanoparticles with highly oriented and accessible pores were functionalized with an 18-mer fragment of cd4 designated as scd4 either through amide conjugation or glycosyl linkage [15] . the functionalized nanoparticles exhibited excellent binding to gp120 protein. recently, v3 loop of gp120 has been identified as the region involved in the initial entry of virus into the cell [16] . antibodies against the v3 loop have been found to inhibit the entry of the virions [17] . antibodies against the v3 loop can also be used to target hiv infected cells thereby conferring both target specificity and anti-viral activity. another strategy involving the use of a tri-functional igg-like bi-specific antibody with anti-gp120 and anti-c3d domains has been reported [18] . a tri-functional antibody contains two binding sites for different molecules and has been reported to display nearly 1000-fold enhancement in the targeting specificity when compared with the conventional antibodies that seek only a single target [18] . the bi-specific antibody reported against hiv infected cells was found to bind specifically with the gp120 fragment exposed in the infected cells as well as to the c3d, a component of the complement resulting in the activation of the complement-mediated lysis of the infected cells. a broad activity antibody against gp120, f105 has also been used to target the hiv infected cells [19] . f105 displays higher affinity to gp120 even at nanomolar concentrations. crystal structure analysis of the binding fragment of f105 reveals a h-loop containing a unique sequence of serine and tyrosine residues and a phenyl alanine residue at the apex [19] . it is proposed that this phenyl alanine residue in f105 associates with the binding pocket of gp120 similar to the phenyl alanine residue (phe43) from cd4 of the host cells. the serine and tyrosine residues in the h-loop stabilize the f105-gp120 binding through formation of hydrogen bonds [20] . in a novel approach, a cd4 mimetic peptide covalently linked with heparan sulfate was synthesized to inhibit hiv-1 entry into the host cell [21] . the cd4 mimetic peptide specifically binds to gp120 resulting in a conformation change that exposes the glycoprotein-binding domain of gp120, which interacts with the heparan sulfate moiety thereby blocking further interactions of the viral gp120 with the host cell receptors. a recombinant peptide expressing the cd4 binding region for gp120 designated as cd4(178)-pe40 and pseudomonas toxin was developed to specifically target and destroy hiv infected cells. the peptide was found to specifically bind to the exposed regions of gp120 found on the surface of hiv infected cells [22] . a surfactant protein extracted from the lung surfactant, sp-d, which belongs to the lectin family has been found to display high affinity calcium-dependent binding to the glycosylated residues of gp120 [23] . the use of soluble cd4 and the immunoadhesin molecule cd4-igg for targeting hiv infected cells has also been reported [24] . these molecules were found to bind to the hiv infected cells through the gp120 domain exposed on the surface of the infected cells. as conjugation of large antibodies to the surface of the nanoparticles is tedious and the risk of denaturation or loss of proper orientation conducive to ligand binding is high, smaller molecules that display similar specificity and affinity to the target antigen have been explored. aptamers, a class of small molecules that can be either an oligonucleotide or a peptide, that exhibit high binding affinities to the target molecules can be a suitable alternative to antibodies [25] . a 2-fluoropyrimidine substituted rna binding aptamer that specifically binds to the surface glycoprotein gp120, has been reported to display excellent target specificity [26] . further studies on the use of such aptamer conjugated nanoparticle systems are warranted for improved therapy. fig. 3 depicts a schematic representation of the concept of targeting hiv infected cells through gp120. glycoprotein-41 (gp41) is a trans-membrane protein present in the hiv envelope that facilitates the fusion of the virus with cd4 + cells [27] . the gp41 moiety is attached non-covalently to the larger glycopro-tein120 (gp120), which is involved in the binding of the viral particles to cd4 + cells. the binding of gp120 to the cd4 receptors of the host cells causes conformational changes leading to the release of the stable gp41 [28] . the ectodomain of gp41 aids the fusion between the virus and host cell. the gp41 protein contains various subunits such as fusion peptides, n-terminal heptad repeats, c-terminal heptad repeats and membrane proximal extracellular region [29] . similar to gp120, gp41 is also expressed on the surface of hiv infected cells and hence is an attractive target for therapeutic strategies involving selective delivery of the therapeutic agent to hiv infected cells. antibodies against gp41 have been employed for targeting hiv infected cells [30] . in few cases, radio immunotherapy (rit) using gp41 antibody labeled with 188 re (rhenium) has been employed for targeting hiv infected cells containing viral particles budding on the cell [31] . the radioactive element causes the death of the hiv infected cells. this strategy was successfully demonstrated in vivo with about 99% reduction in the viral load reported. recently, a new strategy has been reported for targeting both gp41 and gp120 using two antigenic determinants in a single antibody known as double variable domain immunoglobulins (dvds). the use of dvds promotes greater interactions with the two glycoprotein domains in the hiv infected cells resulting in higher uptake of the immunoconjugates and thereby leading to superior viral load reduction [32] . in a similar strategy, lu et al. have reported a recombinant bivalent protein 2dlt, which contains the did2 fragment of the cd4 and a peptide t1144 [33] . the did2 domain of the 2dlt binds to the gp120 and then interacts with the nhr trimer repeat of the gp41 through t1144 resulting in the decay of the gp41 thus preventing further fusion of the hiv virions with other uninfected cells. such recombinant proteins may also be used in conjugation with nanoparticles for targeting hiv infected cells, though such approaches have not been explored thus far. many engineered peptides have been developed for targeting gp41. for instance, it has been identified that the peptide t20 binds specifically to the n-terminal heptad region of gp41 and inhibits infection by the virions due to prevention of conformational changes in gp41 responsible for viral entry [34] . the sequence of t20 is derived from the gp41. similar peptides such as c34 and c37 that are also derived from gp41 have been designed to bind and inactivate the interactions between gp120 and the c-heptad repeat (c-hr) domain of gp41 leading to the inactivation of the env gene. these engineered peptides interact with specific exposed regions of the gp41 of hiv and prevent the entry of the virus into the cells [34] . the membrane proximal external region (mper) in gp41 is also a highly conserved region containing 23 amino acids and antibodies against this domain have proved to be successful in targeting the hiv infected cells [34] . surprisingly, though several molecules targeting conserved regions on the virus have been identified, integrating these targeting molecules with anti-retroviral drug containing nanoparticles has not been extensively investigated and remains in its infancy. yet another interesting approach to target hiv-infected cells is to identify unique markers on the host cells that are also sought by the virus. compared with virus-based targets, a relatively greater number of host cell-based targets have been identified. these include cd4 (receptor for hiv), chemokine receptors, cxcr4 and ccr5 (co-receptors for hiv), carbohydrate-binding antigens (cbas), tuftsin, hla-dr, dc-sign and lfa-1. however, recent studies indicate that the most promising targets include hla-dr, dc-sign and lfa-1. in addition to infection with cell-free virions, the importance of cellcell spread across connecting membrane bridges and close cell-cell contacts referred to as virological synapse (vs) for hiv-1 propagation is increasingly being recognized as the predominant mechanism of hiv-1 propagation between t-cells. assembly of the hiv-1 t-cell vs requires engagement of the hiv-1 env surface subunit gp120, expressed on the effector cell, with its cellular receptors cd4 and cxcr4 on the target cell. further recruitment of receptors and hiv-1 proteins to the conjugate interface is a cytoskeleton-dependent process in both target and effector t cells. specifically, the vs is characterized by clustering of leukocyte function-associated antigen 1 (lfa-1, also known as α l β 2 or cd11a/cd18) at the effector-target cell interface. the lymphocyte function associated antigen 1 (lfa-1) belongs to the integrin family of adhesion molecules and is hypothesized to contribute to the formation of a stable adhesive junction between the effector t-cell and the target t-cell. the major cognate ligand of lfa-1 on t cells is icam-1 (cd54). furthermore, integrins have been implicated in cell-cell transmission of hiv-1 from dendritic cells (dcs) to t cells via lfa-1 and dc-sign (dc-specific intercellular adhesion molecule 3 [icam-3]-grabbing nonintegrin), and their probable role in this setting is to maintain robust dc-t-cell contacts. since lfa-1 is a key molecule involved in transmission of hiv during the formation of the vs between t-cells and in dc-tcell interaction, recent studies have targeted lfa-1 as a potential anti-hiv target. lfa-1 is present in t lymphocytes, b lymphocytes, neutrophils and macrophages and its expression is high during hiv infections. in addition, it has also been recognized that the hiv envelope glycoprotein gp120 induces high levels of lfa-1 expression [35] . immunoliposomes modified with antibody targeting the lfa-1 integrin receptor were developed to deliver si-rna against ccr5 gene and were evaluated for its efficacy in vitro and in vivo [36] . the antibody linked liposomes were able to deliver the si-rna successfully into the infected cells. cyclic peptides cibr, cibl and cibc derived from icam-1 were able to bind and internalize into cells that express lfa-1 through the i-domain of lfa-1 and thus can be used to target hiv infected cells [37] . a monoclonal antibody al-57 has also been demonstrated to exhibit high affinity to cells that express lfa-1 [38] . it was observed that the binding of al-57 occurred with lfa-1 present only in lymphocytes activated by the hiv infection while the antibody binding did not occur in inactive cells thus displaying a high degree of selectivity towards hiv infected cells [39, 40] . an emerging paradigm in the use of monoclonal antibodies directed against lfa-1 is their ability to inhibit viral replication. in a recent study focused to understand the mechanism of viral replication inhibition by anti-lfa-1, it was found that the binding of the antibody to free virus did not influence its replication efficiency. however, binding of the antibody with the lfa-1 present in hiv infected cells resulted in the production of a soluble factor that interfered with the viral replication [40] . hence, use of anti-lfa-1 could be doubly beneficial by ensuring specific targeting to hiv infected cells as well as inhibiting viral inhibition. this facet, however, needs to be explored in-depth with additional experiments in vivo. fig. 3 represents the concept of targeting lfa-1 using anti-lfa-1 modified drug loaded nanoparticles. though the above literature reports that studies in targeting lfa-1 with nanoparticles are few, the studies are promising and will surely open up avenues for more detailed studies on targeting lfa-1 as an anti-hiv target. the human leukocyte antigen (hla) receptor belongs to the major histocompatibility class (mhc) of proteins and is involved in presenting of the antigens to the lymphocytes leading to the activation of the immune response [41] . among the various mhc class ii types, hla-dr, hla-dq and hla-dp are heterodimeric cell surface receptors found in macrophages, dendritic cells and b cells and present the antigen to the cd4 + t helper cells. due to their localization on the surface of cells that serve as hiv reservoirs and their ability to attract cd4 + t helper cells, the hla cell surface receptors are an attractive targeting moiety for hiv infected cells. interestingly, hla-dr molecule expression is increased during hiv infection along with cd25, a trans-membrane receptor of interleukin 2 [42] . recently, anti-hla-dr molecule has been used to target the hla-dr expressed on the hiv infected cells [43] . immunoliposomes loaded with the protease inhibitor indinavir and surface modified with anti-hla-dr were found to effectively bind to hiv infected cells and deliver the antiretroviral drug leading to a significant reduction in the viral load [44] . in another study, the biodistribution of anti-hla-dr conjugated immunoliposomes was evaluated using mice models and it was found that the immunoliposomes were concentrated in the secondary lymphoid organs like spleen and lymph nodes which are the main reservoirs of the hiv virions [101] . a unique advantage acquired through use of anti-hla-dr modified liposomes was that it was able to bind both hiv infected cells and also the free hiv virions found entrapped in the follicular dendritic cell network [45] . it has also been reported that the other viral reservoirs such as monocytes, macrophages, and dendritic cells that are difficult to target due to the dormant nature of the viruses, can be easily targeted using the anti-hla-dr due to the high levels of hla-dr expression on these cells and can therefore reduce undesirable toxicity of the anti-retroviral drugs to normal cells. it was also observed that the liposomes with anti-hla-dr preferentially accumulated in the lymph nodes rich in the hiv infected cells [46] . immunoliposomes conjugated with anti-hla-dr loaded with amphotericin b, an inhibitor of hiv replication, were also reported to target hiv infected cells and reduce the viral load [45] . immunoliposomal systems encapsulating si-rna against rev and tat genes of hiv that are involved in the replication process were successfully delivered to the target cells through conjugation of antibodies against hla-b and hla-c that belong to the mhc class i molecules. it is evident from the scan of literature that the anti-hla antibodies conjugated to liposomes alone have been reported while other nanocarriers modified with hla-dr are yet to be explored. one of the major challenges in hla targeting lies in the design of an antibody with high specificity due to the presence of large number of serotypes for hla. furthermore, hla-dr cd38 + cd4 + t cells show not only increased susceptibility to hiv, but once infected also produce support higher viral replication levels compared to other cells [47] . the hiv peptides show high affinity to the hla-dr moieties. a study has also demonstrated the high affinity of certain hiv-derived peptides to the hla-dr on the cell surface [48] . studies indicate that the hla-dr is incorporated into the viral envelope at the budding stage of the hiv cycle and facilitates further infection of the other uninfected cells using the cd4 receptor as the receptor. taken together, these studies indicate that targeting of the hla-dr will not only deliver the anti-retroviral drugs to the possible sites of hiv infection, but may also attenuate the hla-dr-mediated spread of viral infection in the body [49] . the c-type lectin dc-sign (dc specific icam-3-grabbing nonintegrin) has been identified as a cell surface receptor on immature dcs that binds hiv and mediates transfer of virus to cd4 + permissive t cells. contact of hiv-1 with dc-sign is thus the first event in the pathogenic cascade and, therefore, it is the primary target point for therapies aimed at hiv infection prevention. dc-sign binding to hiv results in internalization of virus to a non-endolysosomal compartment. from this compartment, internalized virus moves rapidly to synapses formed by infected dcs and cd4 + t cells. the transmission of virions is mainly due to the interaction of the cd4-dc-sign and followed by tetramer formation resulting in the higher affinity of the dc-sign to the gp120 residues thus resulting in the fusion of the membrane and capture and transfer of the hiv virions by the dendritic cells to the cd4 positive cell [50] . the silencing of the dc-sign expression in hiv infected cells has been shown to reduce the viral load, thus confirming the potential for dc-sign to function as a potential target [50] . some of the molecules which bind dc-sign are important in targeting the hiv infected cells. these include polyman 19 which binds specifically to the dc-sign expressed on the hiv infected t cells. thus dc-sign could be a critical target to combat early hiv infection. 4.2.4.1. cell surface glycoprotein cd4. the glycoprotein cd4 (cluster of differentiation 4) belongs to the immunoglobulin family and is expressed on the surface of several immune cells including t lymphocytes, monocytes, macrophages and dendritic cells [51] . cd4 elicits an immune response through interactions with the antigen presented by the major histocompatibility complex (mhc) molecule in the antigen presenting cells [52] . hiv hijacks cd4 as its primary receptor to invade its target cells. therefore, targeting the cd4 moiety is an attractive stratagem to selectively deliver anti-hiv drugs to its target cells. the targeting of the cd4 + cells can be achieved through antibodies designed to bind specifically to any of the four domains d1 to d2 present in cd4. a liposomal carrier conjugated with antibodies against the d2 or d3 domains of cd4 was developed that specifically bound to the hiv infected cells [53] . similarly, anti-cd4 conjugated liposomes were employed for the delivery of si-rna to silence the rev gene that is implicated in the regulation and expression of virion proteins. a significant amount of viral inhibition was observed due to the effective targeting to cd4 + cells. in a recent work, lipid nanoparticles conjugated with two peptide sequences that were derived from the binding domains of cd4 designated as cd4-bp2 and cd4-bp4 were used for targeted delivery of the protease inhibitor indinavir to hiv infected cd4 + cells with high specificity [54] . one of the major challenges in targeting cd4 + cells is its possible interference with the normal functions of cd4 expressing cells. in normal conditions, cd4 is used by the t lymphocytes to bind to mhc (major histocompatibility complex) class ii proteins for antigen recognition [55] . the binding domain of cd4 involved in its interactions with mhc class ii overlaps with the binding domain involved in its interactions with gp120 of hiv, the differences being the higher contact area and stronger affinity of the gp120 with cd4 [56] . therefore, design of peptidomimetics with greater affinity to cd4 has been explored that may also serve as competitive inhibitors of the gp120 binding to cd4 [57] . the use of such anti-cd4 peptides for targeting drug-loaded nanoparticles however has not been explored thus far. hiv-infected cell targeting has also been achieved using an engineered cytotoxic t lymphocyte (ctl) expressing a chimera cd4 receptor that employs the extracellular portion of cd4 as a targeting moiety. the specific binding of the engineered cell through the cd4 receptor with the cd4 + cells results in the activation of signaling pathways in ctl that causes the lysis of the hiv infected cells [58] . a novel concept that had been experimented during the late 90s was the development of 'viraceuticals' that involved engineered viruses of the rhabdoviridae family devoid of their envelope proteins but expressing the co-receptors cd4 and cxcr4 that enables it to selectively seek gp120 bound cells and destroy them [59] . however, their successful demonstration in a clinical set-up is yet to be realized. in a departure from the conventional concept of targeting cd4 + cells for delivery of anti-retrovirals or inhibit viral entry, a recent report has employed anti-cd4 conjugated gold nanoparticles for visualizing the lymph nodes through x-ray computed tomography [60] . such applications can be employed in the future for development of 'theranostic' systems that enable simultaneous visualization of the infected cells and their treatment using a co-administered therapeutic agent. the viral entry into the cell is facilitated by the presence of co-receptors cxcr4 and ccr5 that have also been employed for targeting hiv infected cells. the ccr5 or c-c chemokine receptor type 5 belongs to the beta chemokine receptor family and acts as the receptor for many chemokines such as ccl5 and macrophage inflammatory proteins (mips) [61] . it is expressed by t cells, macrophages, microglial cells and dendritic cells [62] . it is also an important co-receptor recruited by the hiv virus to gain entry into the host cell. while cxcr4 is believed to be involved in the later stages of the viral infection, ccr5 is involved in the entry of virus during the early phases of the hiv infection [63] . as a result, ccr5 remains among the most extensively investigated chemokine receptor target for controlling the spread of hiv infections. a liposomal system covalently linked with ccr5 and encapsulating the model drug ethylene diaminetetraacetic acid (edta) was targeted to the hiv infected cells expressing gp120 along with soluble cd4 [63] . as ccr5 possesses affinity towards the hiv gp120, the ccr5-modified liposomes bind to the infected cells causing their selective death in the presence of soluble cd4 [64] . monoclonal antibodies developed against ccr5 have also been employed for homing into hiv infected cells. the anti-ccr5 monoclonal antibody pro140 can bind to multiple extracellular domains of ccr5 and therefore can selectively deliver anti-retroviral drugs efficiently to hiv infected cells on conjugation with nanoparticles [65] . the natural ligand for ccr5 is chemokine c-c ligand 5 (ccl5) also referred to as rantes (regulated on activation, normal t cell expressed and secreted) can also be used to deliver the therapeutic moieties to hiv infected cells [65] . synthetic peptides derived from the sequences in the extracellular loop of ccr5 that is involved in interactions with the viral proteins have also been employed for targeting hiv infected cells. these synthetic peptides have been demonstrated to bind to gp120 present in the surface of the virus infected cells [66] . a classic example of this category of peptides is the e51-derived peptide referred to as ccr5mim, which resembles the extracellular loop of ccr5 that has high affinity to gp120 and can therefore be used to target hiv infected cells [67] . in a novel approach, peptide nucleic acids (pnas) with specificity towards the ccr5 gene were encapsulated in biodegradable nanoparticles of plga (poly(l-lactide-co-glycolide)) and delivered to hiv infected cells [68] . the pnas were associated with the ccr5 gene through hoogsteen bonding and resulted in the suppression of the gene expression leading to a condition akin to those found in cells carrying a ccr5-δ32 mutation. these cells turn hiv resistant due to the absence of ccr5 expression on the cell surface thereby preventing the entry of hiv. the cxcr4 (chemokine receptor type 4) also known as fusin, is a trans-membrane protein that belongs to the g protein coupled receptor family and is ubiquitously expressed by several cell types including t lymphocytes, endothelial cells and tumor cells [69] . the natural ligands for this receptor are the stromal derived factor-1alpha (sdf-1α) (cxcl12) that serves as a chemoattractant for lymphocytes [70] and ubiquitin that is implicated in mitigating pro-inflammatory molecules [71] . hiv-1 strains designated as t-tropic gain entry into the host cell through binding interactions with cd4 and cxcr4. it is believed that the primary binding with cd4 facilitates a conformation change in the viral protein that then binds to cxcr4. it is also found that several strains of hiv-2 are able to gain entry even in cd4 − cells through the cxcr4 receptors suggesting that these viruses possess an envelope protein that binds specifically to the chemokine receptor [72] . a small basic bicyclam molecule amd3100 has been synthesized that exhibits high affinity binding to cxcr4 [73] . this molecule has been found to be a useful target in cancer therapy and wound healing apart from potential anti-hiv properties [74] . however, its therapeutic use against hiv infections is limited as the viral load reduction post-administration of this drug was not significant. this is because in most cases the virus was able to gain entry through other chemokine receptors. antibodies and peptide sequences directed against cxcr4 have been used to target the hiv infected cells. peptide sequences derived from the viral macrophage protein vmip-ii designated as dv1, dv3 and dv1-k-(dv3) were investigated for their targeting affinity towards cxcr4 [75] . it was found that dv1 and dv3 exhibited moderate affinity while dv1-k-(dv3) displayed high affinity towards cxcr4 and hence can be explored further for targeting cxcr4 positive cells. in a recent study, a peptide n15p derived from the amino acid sequence 1-15 of the n-terminus of the viral macrophage inflammatory protein (vmip-ii) was encapsulated in a nanoliposomal carrier [76] . this liposomal system was found to inhibit the sdf-1 induced chemotactic activity of peripheral blood mononuclear cells through competitive binding to cxcr4. the antibody 12g5 against cxcr4 has also been used for targeting the hiv infected cells possibly through recognition of a specific sequence in the extracellular loop 2 (ecl2) of cxcr4 [77] . currently, a humanized anti-cxcr4 antibody designated as 515h7 is under preclinical trials to evaluate its targeting efficacy [78] . stromal cell derived factor-1 (sdf-1) which is secreted by the bone marrow stromal cells has also been used for targeting hiv infected cells. two types of sdf namely sdf-1α and sdf-1β have been investigated due to their high affinity and easy binding to the cxcr4 receptors thereby serving as promising targeting moieties towards cd4 + t h cells infected with hiv [79] . conversely, cxcr4 can also be linked to the nanocarrier to target hiv infected cells due to its affinity for the viral gp120 that is present on the surface of hiv infected cells [80] . a trans-membrane protein, tm4 derived from cxcr4 has also been demonstrated to bind to cd4 + cells and can be used for targeting the hiv infected cells [81] . the synthetic pentapeptides 15k and 15d have been employed as inhibitory peptides for hiv infection and have been found to exhibit superior binding affinity towards cxcr4 co-receptor than the anti-cxcr4 monoclonal antibody 12g5 [82] . the selective binding of two synthetic antagonistic polypeptides tn140 and tn14003 to cxcr4 has been demonstrated in different cancer cell types and hence can be a promising moiety to target hiv infected cells [83, 84] . however, these peptides have not been evaluated in hiv infected cells yet. the recent emergence of 'nanobodies', single domain antibodies that contains a single chain of the variable domain binding to the antigen, has resulted in the development of alx-0651 that displayed high affinity binding to cxcr4 comparable to amd3100 [85] . this nanobody that binds to the ecl2 loop of cxcr4 is currently in phase ii clinical trials where its potential as a targeting ligand towards hiv infected cells is being evaluated. . the viral envelope contains many glycan residues that aid in specific binding to the host cells and also serve to evade immune recognition by masking the immune epitopes. mannose residues are important binding moieties on the surface of the hiv glycoprotein gp120 [86] . targeting the sugar moieties can be an effective strategy for specific homing onto hiv infected cells because of the numerous glycan residues that are exposed on the surface of the hiv infected cells. this concept has led to the emergence of carbohydrate binding agents (cbas) that can be broadly classified either as lectins that belong to the protein family or as non-peptidic molecules with specificity towards a particular type of sugar moiety [87] . apart from imparting the ability to selectively bind to the viral envelope thereby blocking its ability to interact with the host cell surface receptors, the repeated use of cbas also induces the virus to delete the glycan residues thereby leading to unmasking of the hidden immune epitopes resulting in immune activation [88] . most of the glycan residues are also involved in the folding of a protein to its native conformation. deletion of the glycan residues by the virus in response to the treatment with cbas can therefore indirectly hamper the function of the glycans in protein folding thereby reducing the virulence of the virus [89] . a host of peptidic cbas, namely, lectins has been identified from different plant, microbial and mammalian sources. apart from their origin, these lectins differ in their size and specificity towards the sugar residues that have an impact on their binding affinity as well as viral inhibition activity [90] . in general, it has been identified that lectins that display specificity towards α-1,2, α-1,3 and α-1,6-mannose oligomers exhibit better viral binding and inhibition when compared with lectins that exhibit specificity to other carbohydrate moieties [91] . an exception to this generalization is the lectin isolated from the stinging nettle urtica dioica referred as uda (u. dioica agglutinin) that displays specificity towards n-acetyl glucosamine moieties and has also displayed significant affinity towards the hiv glycan envelope [92] . the lectin cyanovirin-n (cv-n) extracted from the cyanobacterium nostoc ellipsosporum [93] was found to bind to the α-1,2-mannose residues of gp120 with high affinity. similarly, scyctovirin (svn), a lectin extracted from the cyanobacterium scytonema varium exhibited selectivity to α-1,2-α-1,6-mannose residues and was found to bind to hiv glycoproteins gp41 and gp120 present on the surface of the hiv infected cells [94] . the lectins from sources of eukaryotic origin gsa (gerardia savaglia agglutinin) from the sea coral g. savaglia displays calcium dependent binding to the dmannose residues in the hiv-1 glycoprotein envelope resulting in complete viral inhibition in in vitro conditions [95] . the lectin actinohivin isolated from the actinomycete longisporum albida has also been identified as an important targeting moiety that binds to the mannose residues present in the gp120 at lower concentrations with an affinity greater than anti-gp120 antibodies thereby preventing the interaction of the viral glycoprotein with cxcr4 and ccr5 chemokine receptors [13] . griffithsin, a lectin isolated from the red algae griffithsia species has been identified to possess specific binding to mannose, glucose, fucose and n-acetyl glucosamine moieties thus conferring a broad spectrum of activity to this lectin towards different viral strains [96] . griffithsin has been found to be effective in interfering with the interactions of the viral glycoproteins with cxcr4 as well as ccr5 [97] . it has also shown promise in inhibiting the sars coronavirus by binding to the outer glycoprotein coat of sars (severely compromised acute respiratory distress syndrome) virus. the lectin bca (boodlea coacta agglutinin) extracted from the green algae b. coacta shows structural similarities with the galanthus nivalis agglutinin (gna) isolated from the snowdrop plant and acts as a potent targeting moiety for the hiv infected cells [97, 98] . it has been shown to bind mainly to the α-1,2-mannose type n-glycans present in the hiv envelope glycoprotein gp120 with high affinity of about 3.7 × 10 8 m −1 making it a promising targeting agent against hiv infected cells [97] . the binding affinity of bca was found to increase with increasing number of mannose residues in the cluster. other lectin molecules that have been identified to possess specific affinity towards the mannose residues of the hiv glycoproteins are cvl (chaetopterus variapedatus lectin) isolated from the annelid worm c. variapedatus, npa (narcissus pseudonarcissus agglutinin) from the daffodil plant n. pseudonarcissus, scl (scilla campanulata lectin) from the bluebell plant s. campanulata, cona (concanavalin a) from the jack bean canavalia ensiformis, jacalin from the jack fruit artocarpus integrifolia, mbl (mannose binding lectin) that is found in the serum of mammalian systems and displays calcium-dependent binding to mannose residues, dc-sign (dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin) expressed in dendritic cells of mammalian systems with specificity towards α-1,3 and α-1,6 mannotriose residues, etc. [98, 99] . a unique lectin referred to as mermaid, which is a c-type lectin expressed on the surface of the marine nematode laxus oneistus was identified to possess a structure similar to dc-sign [100] . this lectin binds to gp120 with high affinity and has been found to inhibit the binding of the virus to the dendritic cells as well as to inhibit the transmission of the virus from dendritic cells to cd4 + t lymphocytes [100] . yet another class of cationic cysteine-rich peptides expressed in the leukocytes is the defensins that possess anti-viral properties. different isoforms of defensins namely α, β, and θ have been identified [101] . the defensins promote chemotaxis of the t lymphocytes and have also displayed the ability to inhibit viral entry and replication [102] . the anti-viral activities of α and β defensins have been mainly attributed to their ability to interfere with the pkc (protein kinase c) signaling in hiv infected cd4 + cells as well as enhancement in the c-c chemokine levels in the cells leading to competitive inhibition of the viral binding to the host cells [102] . the θ defensins also known as retrocyclins display similarity with lectins in binding specifically to the glycan residues of the hiv envelope and hence could serve as a glycan-targeting moiety. a recent attempt to encapsulate defensin peptides along with gp41 residue in biodegradable poly(l-lactide-co-glycolide) microparticles was reported [103] . the presence of defensin resulted in an elevated immunogenic response against hiv and hence it was proposed that such strategies could be effective as vaccines against hiv infections. early work on glycan targets have employed mannose as the targeting moiety towards the mannose receptors present in hiv infected cells like macrophages, dendritic cells and astrocytes resulting in increased uptake of the mannose-linked drug-loaded nanoparticles through receptor-mediated endocytosis leading to a decrease in the viral load [104] [105] [106] . antibodies against the glycan residues of hiv have also been used as a targeting moiety. monoclonal antibodies against the oligosaccharides in the glycoproteins gp120/gp41 were reported to bind to the exposed glycan residues on the surface of the hiv infected cells with high affinity [107] . one of the common monoclonal antibody employed to target the glycan residues is 2g12 that binds specifically to hiv gp120 through terminal mannose oligomers [107] . however, any mutation in this region resulting in the deletion of this residue might render the antibody ineffective. such issues are not commonly encountered with the use of cbas because they are capable of binding to multiple residues in the same glycoprotein sequence. as a result, numerous deletions and mutations have to be effected by the virus to achieve resistance against the cbas [108] . this high genetic barrier achieved by the cbas makes them attractive therapeutic moieties that can also be used to achieve target specificity. among the non-peptidic cbas, the antibiotics pradimicin (prm-a) and nanomicin a (bnm-a) from actinomycete species have been found to exhibit calcium dependent binding to the terminal d-mannose of the viral glycoprotein forming a ternary complex [109, 110] . concerted efforts to develop low molecular weight non-peptidic molecules with carbohydrate-specific binding are underway in many research laboratories. a liposomal carrier containing a c-type lectin with high binding specificity towards dc-sign was developed and was found to exhibit excellent target-specific delivery of its cargo, the fluorescent calcein suggesting that such carriers may be useful in the context of site-directed delivery of therapeutic agents [111] . the use of glycan residues for immuno-stimulatory purposes has also been attempted as a strategy to reduce viral infections. in order to achieve highly effective multivalent display of carbohydrate moieties to stimulate the immune system, novel structures such as glycoclusters and glycodendrimers have also been reported in literature [112] . the concept of using cbas as part of the anti-hiv arsenal is however hampered by many factors. the high expense involved in the isolation and purification of the cbas, storage and stability issues, mitogenic nature of the lectins, poor bioavailability and their inability to distinguish between pathogenic glycan targets from native host glycan residues remains to be addressed before these could be used in a clinical set-up. tuftsin is a tetrapeptide derived from the immunoglobin igg in the spleen and consists of the amino acid sequence thr-lys-pro-arg [113] . since its discovery in 1970, tuftsin has found applications in immunotherapy due to its ability to bind to and activate macrophages and dendritic cells [113] . binding of tuftsin to the tuftsin receptors expressed on the surface of the cells of the immune system activates their chemotaxis and phagocytosis. as these cells also serve as viral reservoirs during hiv infections, employing tuftsin to target the infected cells has been explored as a potential treatment strategy against aids. a drug-tuftsin conjugate was developed by covalently linking tuftsin to the reverse transcriptase inhibitor 3′-azido-3′-deoxythymidine (azt) [114] . the conjugate was effectively used for targeting of macrophages, which hoards the hiv virions. a significant decrease in the viral load compared with the free drug was achieved thus illustrating the specific targeting potential of tuftsin. the tuftsin moiety also stimulated the release of the cytokine interleukin 1 (il-1) from macrophages thereby increasing the immune response [114] . ag5poly(propylene imine) (ppi) dendrimer loaded with the reverse transcriptase inhibitor efavirenz was conjugated with tuftsin and evaluated for its anti-viral efficacy in vitro using macrophages [115] . the tuftsin-conjugated dendrimer significantly decreased the viral load when compared with the unconjugated ppi dendrimer suggesting that tuftsin conjugation enhances uptake in infected cells [115] . tuftsin conjugated liposomes have also been extensively investigated for treatment of tuberculosis [116] , malaria [116] , leishmaniasis [117] and fungal infections [118] . however, apart from a few reports of tuftsin conjugated dendrimer systems, studies involving tuftsin-conjugated nanocarriers remain largely unexplored in the context of hiv therapy. transferrin is an important iron-binding glycoprotein present in blood that is primarily involved in the maintenance of iron levels in the biological system [119] . the affinity of iron to transferrin is extremely high at physiological ph but decreases with decrease in ph. transferrin receptors are abundantly present in liver and brain in physiological conditions [120] . as the brain is an important sanctuary for hiv, it is essential to deliver anti-retroviral drugs across the bloodbrain barrier. as the transferrin receptor expression is high in the brain, it is possible to deliver therapeutic agents into the brain through transferrin-conjugated nanocarriers that will be internalized using receptor-mediated endocytosis [121] . transferrin-conjugated plga nanoparticles encapsulating the reverse transcriptase inhibitor nevirapine were successfully transported across the blood-brain barrier into human brain microvascular endothelial cells [122] . a similar strategy was reported to enable delivery of a hiv model antigen cn54gp140 through the epithelial barrier in the mucosal layer [123] . the transferrin-conjugated system was able to elicit higher immune response as evident from the high titers obtained for the antibodies igg and iga in the genital tract of female mice when compared with the unconjugated system. a specific antibody against the transferrin receptor namely ox26 that binds to the cells expressing the transferrin receptor was conjugated to a streptavidin moiety to deliver antisense oligonucleotides against the rev gene of hiv that was modified covalently with a biotin molecule. the biotinylation protected the oligonucleotides from exonuclease degradation and enabled its binding to the transferrin conjugated streptavidin. this system was found to increase the inactivation of the rev mrna in the cerebral region when compared with the free oligonucleotides highlighting the ability of transferrin conjugated systems to cross the blood-brain barrier [124] . albumin nanoparticles encapsulating the nucleoside reverse transcriptase inhibitor azidothymidine (azt) were surface modified with transferrin and were found to cross the blood-brain barrier efficiently and decrease the viral load [125] . the biodistribution studies of the nanoparticulate system in albino rats revealed selective enhancement in the uptake of the nanoparticles in the brain illustrating the targeting efficacy of transferrin. investigations on the relation between iron levels and hiv infections have revealed that hiv infections lead to an increase in the oxidative stress in the cells and also cause the release of intracellular iron stores. as a result of the iron overload, it has been observed that the transferrin receptor expression in the infected cells is reduced. hence, in the advanced stages of infection, it remains to be seen if transferrin receptor could still be an effective target. in a departure from conventional carriers, use of a modified transferrin as a drug carrier was reported as a proof of principle work. the authors had inserted a peptide sequence in transferrin that was recognized by the hiv protease enzyme. this system was successfully demonstrated to enter hiv infected cells and was lysed by the hiv protease enzyme and can be explored for drug delivery to hiv infected cells. quantum rods comprising of a cadmium selenide core and a graded cadmium sulfide-zinc sulfide shell were conjugated with transferrin and complexed with saquinavir for treatment of neuro-aids [126] . these complexes were found to be extremely effective in transporting saquinavir across the blood-brain barrier. 4.2.4.6. aptamers. the challenges in conjugation of antibodies to carriers primarily due to changes in conformation induced by the chemical treatment steps result in a loss of target specificity. this has necessitated the development of novel small molecule ligands that retain the target specificity of antibodies but with greater structural stability. aptamers are small molecules of single stranded oligonucleotides (dna or rna) or peptides that have excellent target specificity. aptamers are developed using selex (systematic evolution of ligands by exponential enrichment) after a series of sequential steps that involve binding, separation, purification and amplification [127] . aptamers also possess the ability to hybridize through watson-crick pairing and hence can be used to form chimeric structures with other molecules such as si-rna and ribozymes that can be used for delivery into desired locations [128] . a chimeric aptamer-si-rna system exhibiting specificity towards gp120 has been reported to enter hiv infected cells through gp120mediated endocytosis [129] . the aptamer-si-rna chimera is cleaved by dicer inside the cell, leading to the release of si-rna, which binds to the mrna of the hiv tat and rev genes and inhibits the hiv life cycle. aptamers that bind specifically to hiv infected cells have been developed. a 2′f-rna aptamer that binds to the gp120 of infected cd4 + cells has been reported and could be employed for targeting hiv infected cells [130] . several aptamers with specificities towards the hiv reverse transcriptase, integrase, nucleoprotein, gp120, gag protein, tat protein, rev protein and transactivation response element (tar) have been reported [131] . novel aptamers with g-rich repeats that spontaneously form g-quadruplexes in the presence of divalent ions through hoogsteen pairing have been developed for hiv integrase and gp120 through terminal modifications. these quadruplex aptamers display extraordinary stability and binding specificity to their target [132] . these aptamers have immense potential in the field of targeted delivery of anti-retrovirals that remain to be explored in-depth. low-density lipoprotein (ldl). low-density lipoprotein (ldl) is a type of lipoprotein that is involved in the transport of lipids into the cells [133] . ldl has the ability to attract macrophages and hence has been explored as a possible targeting moiety against hiv infected macrophages. ldl conjugated azt (azidothymidine) was demonstrated to internalize into macrophages through ldl receptor-mediated endocytosis and deliver the drug [134] . similar studies were carried out using ldl conjugated fluorothymidine, which binds to the macrophages infected with hiv and delivered the drug effectively causing a decrease in the viral load when compared with the free drug [135] . another study had employed acetylated ldl loaded azt for site-specific delivery to macrophages. the uptake of acetylated ldl modified carrier was achieved through scavenger receptors and resulted in a decrease in the viral load [136] . however, it was observed that hiv infections result in a decreased expression of the ldl receptor and hence targeting this receptor during advanced stages of the disease might not be an effective strategy. passive targeting does not involve any chemical modification of the carrier and its internalization into the desired location is mainly driven by specific property of the targeted cells. the concept of passive targeting has been well exploited in cancer therapeutics by utilizing the enhanced permeation and retention (epr) property of cancer cells [137] . in case of hiv infections, the dendritic cells and macrophages having high amount of virions were found to transfer them to the cd4 + t h cells through virological synapses. these macrophages and dendritic cells have been observed to be highly active during hiv infections and exhibit higher phagocytic properties than uninfected cells [138] . this enhanced phagocytic activity of the macrophages can be harnessed to entrap anti-retroviral loaded carriers in the cells that are infected with hiv. this strategy of passive targeting was successfully demonstrated using indinavir encapsulated liposomal nanoparticles prepared using the synthetic phospholipids phosphatidyl choline, phosphatidyl ethanolamine and a stabilizing agent [139] . these nanoparticles exhibited good uptake in hiv infected macrophages and reduced the viral load significantly when compared with soluble indinavir. this difference between the encapsulated and free form of the drug was mainly attributed to the phagocytosis of the nanoparticles by macrophages. in another approach, a self-assembled dual drug conjugate of azt (zidovudine) and didanosine (ddi) separated by adeoxycholyl spacer was synthesized [140] . this zidovudine-phosphoryl-deoxycholyl didanosine (zpdd) conjugate was easily internalized by the phagocytic macrophages and resulted in the destruction of the virions. a schematic representation of nanoparticles internalized in infected cells through passive targeting is shown in fig. 4 . other targeting moieties for hiv positive cells that have been explored are thiamine [141] , glutathione and albumin [142] . these moieties can easily pass through the blood-brain barrier and are efficient in delivering the drugs on conjugating with the nanocarriers. yet another strategy that is still in the 'proof of principle' stage is the use of pluronics® (tri-block copolymer of poly(ethylene oxide) and poly(propylene oxide)) micelle carriers to disable the efflux pumps in the hiv infected cells that are triggered by the infection to achieve multidrug resistance [143] . the pluronics polymer and unimer alter the mitochondrial membrane fluidity thereby disrupting the electron transport chain. this impairs atp production leading to the shutdown of the efflux pumps [144] . this strategy may enable sensitization of hiv infected cells to anti-retrovirals thereby enhancing their therapeutic efficiency. investigations using knockdown experiments and next generation sequencing to unravel complex interactions between hiv and human proteins have helped in identification of potential targets that could be explored in future for therapeutic applications. in a recent report, a novel viral accessory factor, vif has been identified to recruit a human transcription factor cbf-β (core binding factor subunit beta) and uses the ubiquitin-ligase complex to degrade the restriction factors such as retroviral complementary dna deaminase in the human system that can inactivate the viral replication [145] . targeting cbf-β or vif might offer better chances of curbing the viral replication. another novel target could be slit2n, a glycoprotein that has been found to inhibit the replication of both t tropic and m tropic hivs probably through modification of the cytoskeleton dynamics [146] . this could also be explored indepth as a therapeutic target for viral infections. in a novel approach, melittin, an active component of the bee toxin was encapsulated in liposome nanoparticles and evaluated for their anti-hiv activity [147] . melittin is an activator of phospholipase enzyme and can destroy cells through disruption of the lipid coat. this cytolytic agent was found to selectively internalize to a greater extent in hiv infected cells where it destroyed the viral lipid coat. though free melittin also was found to destroy the virus, its cytotoxicity towards normal cells was significantly high when compared to its nanoparticle counterpart, which displayed selective toxicity only to the virus. this system displayed toxicity towards both cxcr4 and ccr5 tropic viruses. as it attacked the lipid coat, the probability of the virus developing a resistance towards this molecule is remote. this system needs to be further evaluated and validated for clinical significance. rna silencing has emerged as an important strategy to combat aids infections. many new viral targets are being identified to be silenced using rnai technology. some of the targets that have been targeted through rnai technology include the structural, regulatory, and accessory genes such as gag, pol, env, rev, tat, tar, vif, nef, vpx, vpr, etc. attempts to silence factors triggered in the host cell as a result of hiv infection such as the golgi transport proteins rab6 and vps53, karyopherin tnpo3, mediator complex med28, akt1, prkaa1, cd97, neil3, bmp2k and serpinb6 have also been made to control hiv infection [148] . however, the success of rnai depends on its complexation with a suitable nanocarrier as well as identification of a targeting moiety to selectively deliver these into the infected cells. also, a new emerging paradigm is that it may be necessary to silence more than one gene target simultaneously to ensure total disruption of the hiv virions. but such studies are yet to be explored in-depth thereby leaving the field wide open for further research. table 2 lists the numerous targeted nanocarriers that have been explored for hiv therapy, indicating the tremendous scope for further investigations employing a combination of therapeutic agents, nanocarriers and targeting agents. there continues to be a vital need for newer agents to confront the emergence of drug resistance and various adverse effects with longterm use of arv therapy. also the half-life for several arv drugs is short, requiring frequent administration of doses, which in turn leads to poor patient compliance. therefore, the usage of novel drug delivery systems is a logical approach to overcome these problems and effectively treat hiv infection. among the recent therapeutic approaches, a fig. 4 . schematic representation of a passive targeting strategy employing nanoparticles. major thrust area is in developing effective drug delivery systems for the existing drugs, which have been tested and proven effective in reducing the viral load. in this review, the need for novel drug delivery, advantages, and recent developments in identification of viral and host surface molecules as markers for targeted drug delivery of antiretroviral drugs were discussed. these studies open new avenues for more indepth studies on the effective use of these targeting strategies for hiv therapy. such a comprehensive approach could ultimately prove effective not only in reducing viral load, but also in eradication of virus from the reservoirs. we envisage that future directions in the field will involve a multipronged strategy to target hiv at various stages of infection. these include the transmission of the virions from dendritic cells and macrophages to the cd4 + t cells with potential targets being hla-dr and dc-sign. furthermore, cell-to-cell transmission of hiv between cd4 + t cells through the virological synapses is also a critical stage of viral transmission in the body. in case of cell-to-cell transmission, lfa-1 is likely to emerge as a potential target for targeted anti-hiv therapy. another key area should involve countering the challenges in targeting latent hiv in multiple reservoirs. in this regard, strategies that combine the release the hiv virions from latently infected cells such as activation of the protein kinase pathway along with anti-hiv drug delivery hold tremendous potential. in summary, recent scientific advances in the development of targeted drug delivery of anti-hiv drugs hold great promise for the development of improved treatment strategies and should certainly pave the way towards global eradication of hiv/aids. chimeric anti-gp120 aptamer rnai in vitro [165] the global impact of hiv/ aids a binding pocket for a small molecule inhibitor of hiv-1 entry within the transmembrane helices of ccr5 pathogenesis of hiv-1-protease inhibitor-associated peripheral lipodystrophy, hyperlipidaemia, and insulin resistance using pharmacokinetics to optimize antiretroviral drug-drug interactions in the treatment of human immunodeficiency virus infection cyp3a4-mediated hepatic metabolism of the hiv-1 protease inhibitor saquinavir in vitro hiv type 1 infection of human astrocytes is restricted by inefficient viral entry surface modifications of nanocarriers for effective intracellular delivery of anti-hiv drugs 3d visualization of hiv transfer at the virological synapse between dendritic cells and t cells atomic structure of the ectodomain from hiv-1 gp41 adaptive mutations in a human immunodeficiency virus type 1 envelope protein with a truncated v3 loop restore function by improving interactions with cd4 energetics of the hiv gp120-cd4 binding reaction, proc. natl. acad. sci. 97 sustained and specific in vitro inhibition of hiv-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes actinohivin, a broadly neutralizing prokaryotic lectin, inhibits hiv-1 infection by specifically targeting high-mannose-type glycans on the gp120 envelope targeting of liposomes to cells expressing cd4 using glycosylphosphatidylinositol-anchored gp120. influence of liposome composition on intracellular trafficking binding of hiv-1 gp120 glycoprotein to silica nanoparticles modified with cd4 glycoprotein and cd4 peptide fragments a conserved hiv gp120 glycoprotein structure involved in chemokine receptor binding cd4-induced interaction of primary hiv-1 gp120 glycoproteins with the chemokine receptor ccr-5 hypothesis a novel trifunctional igg-like bispecific antibody to inhibit hiv-1 infection and enhance lysis of hiv by targeting activation of complement binding kinetics, uptake and intracellular accumulation of f105, an anti-gp120 human igg1κ monoclonal antibody, in hiv-1 infected cells structure of the fab fragment of f105, a broadly reactive anti-human immunodeficiency virus (hiv) antibody that recognizes the cd4 binding site of hiv type 1 gp120 a synthetic heparan sulfate-mimetic peptide conjugated to a mini cd4 displays very high anti-hiv-1 activity independently of coreceptor usage cd4-pseudomonas exotoxin hybrid protein blocks the spread of human immunodeficiency virus infection in vitro and is active against cells expressing the envelope glycoproteins from diverse primate immunodeficiency retroviruses surfactant protein d modulates hiv infection of both t-cells and dendritic cells liposome targeting to human immunodeficiency virus type 1-infected cells via recombinant soluble cd4 and cd4 immunoadhesin (cd4-igg) aptamers as therapeutic and diagnostic agents structural characterization of an anti-gp120 rna aptamer that neutralizes r5 strains of hiv-1 characterization of gp41 as the transmembrane protein coded by the htlv-iii/lav envelope gene structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody biochemistry and biophysics of hiv-1 gp41 -membrane interactions and implications for hiv-1 envelope protein mediated viral-cell fusion and fusion inhibitor design a broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1 targeted killing of virally infected cells by radiolabeled antibodies to viral proteins anti-hiv double variable domain immunoglobulins binding both gp41 and gp120 for targeted delivery of immunoconjugates a bivalent recombinant protein inactivates hiv-1 by targeting the gp41 prehairpin fusion intermediate induced by cd4 d1d2 domains asymmetric deactivation of hiv-1 gp41 following fusion inhibitor binding hiv envelope gp120 activates lfa-1 on cd4 t-lymphocytes and increases cell susceptibility to lfa-1-targeting leukotoxin (ltxa) rnai-mediated ccr5 silencing by lfa-1-targeted nanoparticles prevents hiv infection in blt mice biodegradable nanoparticles surface modification techniques with cibr peptide targeting to lfa-1 expressing leukemic cells al-57, a ligand-mimetic antibody to integrin lfa-1, reveals chemokine-induced affinity up-regulation in lymphocytes selective gene silencing in activated leukocytes by targeting sirnas to the integrin lymphocyte functionassociated antigen-1 a cytotoxic t lymphocyte inhibits acquired immunodeficiency syndrome virus replication in peripheral blood lymphocytes hla, immune response and disease hiv-1-induced activation of cd4 + t cells creates new targets for hiv-1 infection in human lymphoid tissue ex vivo frequency of class i hla-restricted anti-hiv cd8+ t cells in individuals receiving highly active antiretroviral therapy (haart) targeted nanomedicines: effective treatment modalities for cancer, aids and brain disorders targeting cell-free hiv and virally-infected cells with anti-hla-dr immunoliposomes containing amphotericin b targeting lymph nodes with liposomes bearing anti-hla-dr fab′ fragments expression of activation antigens, hla-dr and cd38, on cd8 lymphocytes during hiv-1 infection role of polymorphic residues of human leucocyte antigen-dr molecules on the binding of human immunodeficiency virus peptides incorporation of hla-dr into the envelope of human immunodeficiency virus type 1 in vivo: correlation with stage of disease and presence of opportunistic infection dc-sign plays a stronger role than dcir in mediating hiv-1 capture and transfer cd4: its structure, role in immune function and aids pathogenesis, and potential as a pharmacological target mhc class ii-dependent basophil-cd4 + t cell interactions promote th2 cytokine-dependent immunity targeting of liposomes to hiv-1-infected cells by peptides derived from the cd4 receptor enhanced anti-hiv efficacy of indinavir after inclusion in cd4-targeted lipid nanoparticles recognition of cluster of differentiation 1 antigens by human cd4-cd8-cytolytic t lymphocyte truncated variants of gp120 bind cd4 with high affinity and suggest a minimum cd4 binding region rational optimization of the binding affinity of cd4 targeting peptidomimetics with potential anti hiv activity targeted cytolysis of hiv-infected cells by chimeric cd4 receptor-bearing cells, in, google patents drug development in the 21st century: medicines, man and receptors anti-cd4-targeted gold nanoparticles induce specific contrast enhancement of peripheral lymph nodes in x-ray computed tomography of live mice host genes and hiv: the role of the chemokine receptor gene ccr5 and its allele quantification of cd4, ccr5, and cxcr4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages ccr3 and ccr5 are co-receptors for hiv-1 infection of microglia cell derived liposomes expressing ccr5 as a new targeted drug-delivery system for hiv infected cells potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the ccr5 monoclonal antibody pro 140 specific inhibition of hiv-1 coreceptor activity by synthetic peptides corresponding to the predicted extracellular loops of ccr5 a tyrosine-sulfated ccr5-mimetic peptide promotes conformational transitions in the hiv-1 envelope glycoprotein site-specific genome editing in pbmcs with plga nanoparticle-delivered pnas confers hiv-1 resistance in humanized mice apoptosis of cd8 + ; t cells is mediated by macrophages through interaction of hiv gp120 with chemokine receptor cxcr4 cxcr4-sdf-1 signalling, locomotion, chemotaxis and adhesion initial assessment of the role of cxc chemokine receptor 4 after polytrauma cd4-independent infection by hiv-2 is mediated by fusin/cxcr4 amd3100, a small molecule inhibitor of hiv-1 entry via the cxcr4 co-receptor molecular biology of cancer-associated fibroblasts: can these cells be targeted in anti-cancer therapy? semin a synthetic bivalent ligand of cxcr4 inhibits hiv infection anti-hiv effect of liposomes bearing cxcr4 receptor antagonist n15p inhibition of hiv-1 infection by down-regulation of the cxcr4 co-receptor using an intracellular single chain variable fragment against cxcr4 novel antibodies for the treatment of hiv, in, google patents identification and characterization of the cxcr4 chemokine receptor in human t cell lines: ligand binding, biological activity, and hiv-1 infectivity anti-cxcr4 monoclonal antibodies recognizing overlapping epitopes differ significantly in their ability to inhibit entry of human immunodeficiency virus type 1 dimerization of cxcr4 in living malignant cells: control of cell migration by a synthetic peptide that reduces homologous cxcr4 interactions novel peptides based on hiv-1 gp120 sequence with homology to chemokines inhibit hiv infection in cell culture inhibition of breast cancer metastasis by selective synthetic polypeptide against cxcr4 development of specific cxcr4 inhibitors possessing high selectivity indexes as well as complete stability in serum based on an anti-hiv peptide t140 halting metastasis through cxcr4 inhibition mannose binding lectin (mbl) and hiv carbohydrate-binding agents (cbas) inhibit hiv-1 infection in human primary monocyte-derived macrophages (mdms) and efficiently prevent mdmdirected viral capture and subsequent transmission to cd4 + t lymphocytes exposure of hiv-1 to a combination of two carbohydratebinding agents markedly delays drug resistance development and selects for virus strains with compromised fitness carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp120: a new therapeutic concept to hit the achilles heel of hiv inhibition of hiv entry by carbohydrate-binding proteins differences in the mannose oligomer specificities of the closely related lectins from galanthus nivalis and zea mays strongly determine their eventual anti-hiv activity the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborine and the (n-acetylglucosamine)n-specific plant lectin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro high-mannose-specific deglycosylation of hiv-1 gp120 induced by resistance to cyanovirin-n and the impact on antibody neutralization scytovirin engineering improves carbohydrate affinity and hiv-1 entry inhibition a d-mannose-specific lectin from gerardia savaglia that inhibits nucleocytoplasmic transport of mrna isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp high mannose-binding lectin with preference for the cluster of α1-2-mannose from the green alga boodlea coacta is a potent entry inhibitor of hiv-1 and influenza viruses targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy proteins that bind high-mannose sugars of the hiv envelope a new c-type lectin similar to the human immunoreceptor dc-sign mediates symbiont acquisition by a marine nematode defensins: natural anti-hiv peptides defensins in innate antiviral immunity modulation of hiv peptide antigen specific cellular immune response by synthetic α-and β-defensin peptides targeting potential and anti-hiv activity of lamivudine loaded mannosylated poly (propyleneimine) dendrimer mannosylated gelatin nanoparticles bearing an anti-hiv drug didanosine for site-specific delivery biodegradable polymeric nanoparticles based drug delivery systems antibody domain exchange is an immunological solution to carbohydrate cluster recognition identification of two n-linked glycosylation sites within the core of the simian immunodeficiency virus glycoprotein whose removal enhances sensitivity to soluble cd4 pradimicin, a novel class of potent antifungal antibiotics new antifungal antibiotics, benanomicins a and b from an actinomycete dc-sign-specific liposomal targeting and selective intracellular compound delivery to human myeloid dendritic cells: implications for hiv disease targeting the carbohydrates on hiv-1: interaction of oligomannose dendrons with human monoclonal antibody 2g12 and dc-sign, proc. natl. acad. sci. 105 tuftsin (an igassociated tetrapeptide) triggers the immunogenic function of macrophages: implications for activation of programmed cells dendrimer toxicity: let's meet the challenge targeting of efavirenz loaded tuftsin conjugated poly (propyleneimine) dendrimers to hiv infected macrophages in vitro tuftsin-bearing liposomes as rifampin vehicles in treatment of tuberculosis in mice drug targeting in leishmania donovani infections using tuftsin-bearing liposomes as drug vehicles tuftsin-bearing liposomes as drug vehicles in the treatment of experimental aspergillosis iron status in hiv-1 infection: implications in disease pathology transferrin trojan horses as a rational approach for the biological delivery of therapeutic peptide domains targeted drug delivery via the transferrin receptormediated endocytosis pathway targeting nevirapine delivery across human brain microvascular endothelial cells using transferrin-grafted poly (lactide-coglycolide) nanoparticles transferrin conjugation confers mucosal molecular targeting to a model hiv-1 trimeric gp140 vaccine antigen drug delivery of antisense molecules to the brain for treatment of alzheimer's disease and cerebral aids targeted brain delivery of azt via transferrin anchored pegylated albumin nanoparticles enhancing the delivery of anti retroviral drug "saquinavir" across the blood brain barrier using nanoparticles development of dna aptamers using cell-selex rna-based therapeutics: current progress and future prospects selection, characterization and application of new rna hiv gp 120 aptamers for facile delivery of dicer substrate sirnas into hiv infected cells novel dual inhibitory function aptamer-sirna delivery system for hiv-1 therapy molecular strategies to inhibit hiv-1 replication investigation of formation, recognition, stabilization, and conversion of dimeric g-quadruplexes of hiv-1 integrase inhibitors by electrospray ionization mass spectrometry transport of lipids from high and low density lipoproteins via scavenger receptor-bi cell specific uptake of antiretroviral drugs: azt coupled to ldl inhibits hiv replication in human macrophages selective endocytosis of fluorothymidine and azidothymidine coupled to ldl into hiv infected mononuclear cells enhanced delivery of azt to macrophages via acetylated ldl enhanced permeability and retention effect for selective targeting of anticancer nanomedicine: are we there yet? macrophage targeting of azidothymidine: a promising strategy for aids therapy* laboratory investigations for the morphologic, pharmacokinetic, and anti-retroviral properties of indinavir nanoparticles in human monocyte-derived macrophages combination anti-hiv therapy with the selfassemblies of an asymmetric bolaamphiphilic zidovudine/didanosine prodrug drug delivery to the central nervous system: a review inhibition of hiv in vitro by antiviral drug-targeting using nanoparticles influence of pluronic® f68 on ceftazidime biological activity in parenteral solutions pluronic® block copolymers for overcoming drug resistance in cancer viral infectivity factor: a novel therapeutic strategy to block hiv-1 replication n-terminal slit2 inhibits hiv-1 replication by regulating the actin cytoskeleton melittin-loaded immunoliposomes against viral surface proteins, a new approach to antiviral therapy antiviral stratagems against hiv-1 using rna interference (rnai) technology, evol mannan-coated gelatin nanoparticles for sustained and targeted delivery of didanosine: in vitro and in vivo evaluation tat-conjugated nanoparticles for the cns delivery of anti-hiv drugs targeted delivery of indinavir to hiv-1 primary reservoirs with immunoliposomes optimizing size and copy number for peg-fmlf (n-formyl-methionyl-leucylphenylalanine) nanocarrier uptake by macrophages optimization of lipid-indinavir complexes for localization in lymphoid tissues of hiv-infected macaques reduced hematopoietic toxicity, enhanced cellular uptake and altered pharmacokinetics of azidothymidine loaded galactosylated liposomes lymphatic targeting of zidovudine using surfaceengineered liposomes stavudine-loaded mannosylated liposomes: in-vitro anti-hiv-i activity, tissue distribution and pharmacokinetics reduced hepatic toxicity, enhanced cellular uptake and altered pharmacokinetics of stavudine loaded galactosylated liposomes efficient sirna delivery into primary cells by a peptide transduction domain-dsrna binding domain fusion protein antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors dendritic cells loaded with hiv-1 p24 proteins adsorbed on surfactant-free anionic pla nanoparticles induce enhanced cellular immune responses against hiv-1 after vaccination nanochemistry-based immunotherapy for hiv-1 targeting of efavirenz loaded tuftsin conjugated poly(propyleneimine) dendrimers to hiv infected macrophages in vitro tuftsin-azt conjugate: potential macrophage targeting for aids therapy sustained and specific in vitro inhibition of hiv-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes dual functional rna nanoparticles containing phi29 motor prna and anti-gp120 aptamer for cell-type specific delivery and hiv-1 inhibition the authors gratefully acknowledge the financial support from the indian council for medical research (icmr, grant no. 35/9/2009-bms) and infrastructural support from sastra university. key: cord-254098-4imkkptg authors: chutiwitoonchai, nopporn; hiyoshi, masateru; hiyoshi-yoshidomi, yuka; hashimoto, michihiro; tokunaga, kenzo; suzu, shinya title: characteristics of ifitm, the newly identified ifn-inducible anti-hiv-1 family proteins date: 2013-01-30 journal: microbes infect doi: 10.1016/j.micinf.2012.12.003 sha: doc_id: 254098 cord_uid: 4imkkptg ifn-inducible ifitm proteins (ifitm1, 2, and 3) inhibit the replication of various viruses including hiv-1 through poorly understood mechanisms. here, we further analyzed characteristics of these newly identified hiv-1 restriction factors. firstly, in contrast to other anti-hiv-1 proteins, such as tetherin and apobec3g, ifitms were resistant to a down-regulation of surface expression or degradation by hiv-1 proteins. secondly, the enforced expression of ifitms reduced the production of hiv-1 viruses from cells transfected with proviral plasmids containing whole viral sequences. although their inhibitory activities were modest when compared to that of tetherin, ifitms, but not tetherin, directly reduced the expression of hiv-1 proteins including gag, vif and nef. of importance, however, ifitms had no inhibitory effect when these viral proteins were expressed by codon-optimized cdnas that bypassed the viral-specific expression machinery. indeed, our results supported the idea that ifitms interfere with viral protein expression mediated by double-stranded viral rnas, such as rre and tar. finally, the s-palmitoylation of ifitms, which is crucial for their anti-influenza virus activity, was not required for their anti-hiv-1 activity, indicating that ifitms restrict these viruses at different steps. these characteristics lead to a better understanding of the mechanism by which ifitms restrict hiv-1 and other viruses. type i interferons (ifn) are cytokines of the innate immune system that induce the expression of antiviral proteins upon viral infection. viral recognition induces the activation of cellular signaling pathways that trigger the production of ifn, which leads to the expression of a set of ifn-stimulated genes that inhibit viral replication through diverse mechanisms [1, 2] . the ifn-induced transmembrane (ifitm) genes were identified as ifn-stimulated genes [3] . among this family of proteins, ifitm1, 2, and 3 are ubiquitously expressed. although it has been reported that ifitm1 and ifitm3 play distinct roles in mouse primordial germ cell homing and repulsion [4] , their precise physiological functions remain largely unknown. intriguingly, recent studies have revealed that ifitm family proteins are potent inhibitors of influenza virus, west nile virus, dengue virus [5e8], marburg and ebola filoviruses, sars coronavirus [9] , vesicular stomatitis virus [10] , hcv [11] , and hiv-1 [12, 13] . however, it remains unclear how these small proteins, which are composed of approximately 130 amino acids, exert antiviral activity against a broad range of viruses. recently, lu et al. showed that the knockdown of all three ifitm genes increased the susceptibility of tzm-bl hela cells to hiv-1 infection [12] . consistent with this result, the enforced expression of ifitm1, 2, or 3 markedly suppressed hiv-1 replication in supt1 t cells without affecting cell proliferation, the cell cycle, or the cell surface expression of the hiv-1 entry receptor cd4 [12] . schoggins et al. also reported that the enforced expression of ifitm2 or 3 had a similar inhibitory effect on hiv-1 replication using another t cell line, mt-4 [13] . although hiv-1 entry step was clearly inhibited by ifitm2 and 3, the entry step might not be the primary target of ifitm1 during its restriction of hiv-1 replication, since ifitm1 did not affect hiv-1 entry but efficiently suppressed hiv-1 replication in supt1 cells [12] . indeed, it was shown that the intracellular region, rather than the n-or c-terminal extracellular domains, of ifitm1 is required for its inhibitory effect on hiv-1 [12] . importantly, lu et al. also showed that the enforced expression of ifitms led to a reduction in the expression of the structural hiv-1 protein gag, which might be simply due to the reduced viral replication. nevertheless, it is also possible that ifitms directly affect gag expression. in addition to the mechanism by which they suppress gag expression, there are several unanswered questions regarding ifitms. hiv-1 encodes proteins that antagonize the activities of ifn-inducible antiviral proteins. for instance, tetherin (also known as bst-2 or cd317) blocks the release of nascent virions from infected cells, but the hiv-1 accessory protein vpu acts as a viral antagonist by inducing the down-regulation of tetherin expression at the cell surface [14e19]. similarly, the cytidine deaminase apobec3g, whose expression is elevated by ifn [20] , causes the hyper-mutation of hiv-1 cdna, but another hiv-1 accessory protein, vif, antagonizes its anti-hiv-1 activity by inducing the degradation of apobec3g [21e27]. however, it has not been examined whether hiv-1 antagonizes the anti-hiv-1 activity of ifitms. curiously, in contrast to the findings of two independent studies [12, 13] , neil et al. failed to observe an inhibitory effect of the enforced expression of ifitms on hiv-1 production in a study in which they identified tetherin as an hiv-1 restriction factor [15] . in this study, we therefore attempted to investigate whether hiv-1 proteins affect the expression and localization of ifitms, how anti-hiv-1 activity of ifitms and tetherin are different, and how ifitms affect the expression of hiv-1 proteins in order to further understand the characteristics of these newly identified hiv-1 restriction factors. the n-terminal flag-tagged human ifitm1, 2, and 3 cdnas were provided by liang [12] , and subcloned into the pcdna3.1 vector (invitrogen). it was shown that ifitm3 was s-palmitoylated at three membrane-proximal cysteine residues (c71, c72, and c105) [7] . we therefore prepared three different mutants of flag-tagged ifitm3, in which these cysteines had been mutated (singly or in combination) to alanine (c1/2a, c3a, and c1/2/3a; see fig. 6a for details). as these cysteines are conserved in ifitm2 (c70, c71, and c104), similar mutants of ifitm2 were also prepared. the mutants were prepared using the quikchange ii site-directed mutagenesis kit (stratagene) and appropriate mutagenic primers and cloned into the pcdna3.1 vector. the nucleotide sequences of the mutants were verified using the bigdye terminator v3.1 cycle sequencing kit and an abi prism 3100 genetic analyzer (applied biosystems). extracellular flagtagged human tetherin (bst-2-exflag) cloned into the pcaggs vector was prepared as described previously [28] . a proviral nl43 plasmid and a vpu-deleted mutant version of the plasmid (pnl-ue65) were provided by adachi [29] . a proviral jrfl plasmid in which the nef gene had been replaced with that of the sf2 strain was prepared as described previously [30] . a codon-optimized gagegfp fusion expression plasmid (syngagegfp) cloned into the pcdna3.1 vector (ohmine et al., manuscript under review) was kindly provided by y. ikeda (mayo clinic, rochester, mn). codon-optimized vpu (vphu) and vif (hvif) expression plasmids cloned into the pcdna3.1 vector were obtained through the nih aids research and reference reagent program (division of aids, national institute of allergy and infectious diseases, national institutes of health, bethesda, md), both of which were derived from the nl43 strain [31] . two different nef (the sf2 strain) expression plasmids were prepared as described previously [32, 33] : nef fused to the c-terminus of the extracellular/ transmembrane regions of cd8 (cd8-nef) was cloned into the prc/cmv vector and nef fused to the n-terminus of gfp (nefegfp) was cloned into the pacgfp-n1 vector (clontech). the nl43 strain rev expression plasmid cloned into the pcaggs vector was prepared as described previously [28] . in this study, we also created gag and gag/pol expression plasmids (pca-gag-rre and pca-gagpol-rre, respectively) by inserting pcr-amplified nl43-derived gag and gag/pol genes (nucleotides 790e2292 and 790e5096, respectively) together with a rev-response element (rre; nucleotides 7759e7992) into the pcaggs vector. hek293 cells (invitrogen) were maintained in dme medium (wako, osaka, japan) supplemented with 10% fcs (nichirei biosciences, tokyo, japan). the cells were seeded onto 12-well tissue culture plates at a density of 1.8 â 10 5 cells/well and transfected with various plasmids using 4 ml/ well lipofectamine 2000 reagent (invitrogen), as described previously [30, 32] . the total amount (1.6 mg/well) of the plasmid was normalized using appropriate control (empty) vectors. after 6 h of transfection, the culture medium was replaced with fresh medium, and the cells were cultured for an additional 42 h and then subjected to p24 gag protein elisa, western blotting, flow cytometric analysis, or immunofluorescence analysis. in another experiment (see fig. 5 ), the double-stranded rna-dependent protein kinase (pkr) inhibitor c16 (imidazolo-oxindole; sigma) was added to the culture (0.1% v/v) during the changing of the medium. the inhibitor was dissolved in dmso (wako), and the same volume of dmso was used as a vehicle control. viral production was assessed by measuring the concentration of p24 gag protein in the culture supernatant [30] . the supernatants of the transfected 293 cells were clarified by brief centrifugation, and their p24 concentrations were analyzed by elisa (zeptometrix, buffalo, ny). the absorbance of each well was measured at 450 nm with a microplate reader (bio-rad laboratories). the preparation of the total cell lysates and western blotting was performed essentially as described previously [34] . briefly, the cells were lysed on ice for 30 min with nonidet p-40 lysis buffer (1% nonidet p-40, 50 mm tris, and 150 mm nacl) containing protease inhibitors (1 mm edta, 1 mm pmsf, 1 mg/ ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin). the cell lysates were centrifuged and the resultant supernatants were resolved by sds-polyacrylamide gel electrophoresis (sds-page) under reducing conditions. the proteins were transferred to a nylon membrane (hybond-p; ge healthcare). the antibodies used were as follows: anti-gag (#65-004; bio-academia, osaka, japan), anti-nef (#2949; nih aids research and reference reagent program), anti-vif (#319; nih aids research and reference reagent program), anti-gfp (#fl; santa cruz biotechnology), anti-flag (clone m2; sigma), and anti-actin (#c-2; santa cruz biotechnology). the detection was performed with hrp-labeled secondary antibodies (anti-rabbit or anti-mouse igg; ge healthcare), the immunostar ld western blotting detection reagent (wako), and an image analyzer (imagequant las 4000; ge healthcare). the cell surface expression of the flag-tagged ifitm or flag-tagged tetherin was assessed by the flow cytometric analysis, essentially as described previously [35] . the transfected 293 cells were detached using the enzyme-free cell dissociation buffer (gibco), stained on ice for 30 min with pelabeled anti-flag antibody (60 mg/ml; columbia biosciences, columbia, md), and analyzed using a facscalibur (becton dickinson) and cell quest software (becton dickinson). in a selected experiment (see fig. 2c ), 293 cells stably expressing human cd4 [32] were transfected and analyzed for the cell surface expression of cd4 using pe-labeled anti-cd4 antibody (clone rpa-t4, ebioscience). for immunostaining, the transfected 293 cells were directly fixed in 2% paraformaldehyde, permeabilized with 0.2% triton x-100, and stained with the primary antibodies for 12 h followed by labeled secondary antibodies [32, 33] . the following primary antibodies were used: anti-flag (clone m2; sigma, to detect flag-tagged ifitm proteins) and anti-gag (#65-004; bioacademia). anti-mouse igg-alexafluor488 and anti-rabbit igg-alexafluor568 (both from molecular probes) were used as the labeled secondary antibodies. nuclei were stained with dapi (molecular probes), and fluorescent signals were visualized with a bz-8000 fluorescent microscope (keyence, osaka, japan) equipped with plan-fluor elwd 20â/0.45 objective lenses (nikon). image processing was performed using a bz-analyzer (keyence) and the adobe photoshop software (adobe systems). the statistical significance of differences between samples was determined using the student's t-test. p values less than 0.05 were considered significant. it has been shown that the knockdown of ifitms increases the susceptibility of tzm-bl hela cells to hiv-1 infection and that their enforced expression suppresses hiv-1 replication and gag expression in supt1 cells [12] . however, in a study in which the enforced expression of tetherin was found to strongly inhibit viral release from 293 cells, ifitms failed to display a similar inhibitory effect [15] . therefore, we initially compared their anti-hiv-1 activities in the same system, i.e., the co-transfection into 293 cells. viral production was monitored by assessing the concentration of p24 gag protein in the culture supernatants. in this study, we found that ifitm3 significantly reduced viral production when it was cotransfected with the proviral nl43 plasmid containing the whole viral sequence (fig. 1a, left graph) . a similar inhibitory effect was also observed when ifitm3 was co-transfected with the vpu-deleted nl43 mutant (nl43-dvpu; right graph). when analyzed using the tzm-bl reporter cells, the infectivity of the wt viruses produced in the presence of ifitm3 was comparable to that of the control viruses (data not shown). tetherin also reduced viral production, but its inhibitory effect was more marked when it was co-transfected with nl43-dvpu (fig. 1a) . this was due to the ability of vpu to down-regulate the cell surface expression of tetherin [14e19]. in the experiment shown in fig. 1a , we used 0.6 mg ifitm3 expression plasmid. when the same amount of plasmid was used, tetherin displayed a lower expression level (fig. 1b) but stronger inhibitory activity (fig. 1a ) than ifitm3. these results indicated that the inhibitory effect of ifitm3 on viral production is modest when compared with that of tetherin, which explains why neil et al. [15] failed to observe an inhibitory effect of ifitms in their study. the importance was that ifitm3 and tetherin restricted hiv-1 at different steps because ifitm3 but not tetherin significantly reduced the expression levels of the p55 and p24 gag proteins in the cells (fig. 1c) . it therefore appears that tetherin inhibits the release of viruses without affecting intracellular gag expression whereas ifitm3 directly reduces intracellular gag expression, and thereby suppresses viral production. tetherin and apobec3g are well-characterized hiv-1 restriction factors, but it is also known that hiv-1 proteins counteract their activities. vpu and vif induce the downregulation of the cell surface expression of tetherin [14e19] and the degradation of apobec3g [21e27], respectively. indeed, tetherin inhibited the release of the vpu (à) viruses more strongly than it inhibited the release of the wild-type viruses (fig. 1a) . on the other hand, ifitm3 exhibited comparable inhibitory activity to these two viruses (fig. 1a) . therefore, we next examined whether hiv-1 proteins affect the localization or expression of ifitm family proteins. the flagtagged ifitm1, 2, or 3 expression plasmid was co-transfected with the proviral plasmid (jrfl or nl43), but we did not detect any obvious changes in the cell surface expression of ifitms ( fig. 2a) . although ifitm3 has been found to localize not only to the plasma membrane but also to the perinuclear region [7] , we did not detect any obvious changes in the intracellular distribution of ifitms after their co-transfection with the proviral jrfl plasmid (fig. 2b) . we also tested the effect of the overexpression of individual hiv-1 proteins. the flag-tagged ifitm1, 2, or 3 expression plasmid was co-transfected with the codon-optimized version of the vpu (vphu) or vif (hvif) plasmid, which bypassed the complicated viral-specific expression machinery and allowed efficient expression [31] , and the cell surface expression of flag-ifitms was analyzed by flow cytometry using anti-flag antibody. nef was also added to the analysis, as it down-regulates the expression of multiple cell surface proteins including cd4 [36] . however, none of the hiv-1 proteins down-regulated the cell surface expression of ifitms (data not shown). the expression levels of vpu and nef under our experimental conditions were sufficient to downregulate tetherin and cd4, respectively (fig. 2c ). the expression level of vif under our experimental conditions was sufficient to induce the degradation of apobec3g (western blotting, data not shown). these results indicate that ifitms are resistant to the down-regulation of cell surface expression or the degradation by hiv-1 proteins. these findings are important because they imply that any hiv-1 proteins, unlike those of tetherin and apobec3g, do not counteract the anti-hiv-1 activities of ifitms. we next attempted to understand how ifitms suppress the production of hiv-1 viruses. there was no obvious difference in the inhibitory effect on viral production among ifitms (fig. 3a) . all the ifitms reduced the production of both the nl43 and jrfl strain viruses (fig. 3b, bar graph) , and this effect was strongly associated with reduced gag expression (fig. 3b, gag blot) . importantly, we found that ifitms also reduced the expression of nef and vif (nef and vif blots). however, more important finding was that ifitm proteins did not induce any reduction in the expression of these viral proteins when they were co-transfected with the codonoptimized gag expression plasmid (fig. 4a , syngagegfp blot), the nef expression plasmid (fig. 4a, nefegfp blot) , or the codon-optimized vif expression plasmid (fig. 4b, hvif blot) . it is well characterized that gag and vif rnas contain a double-stranded region (rev response element; rre), and the incompletely spliced rnas encoding these viral proteins require the binding of hiv-1 rev protein to the rre sequences for their nuclear export and subsequent expression [37, 38] . codon-optimization bypasses this complicated mechanism and allows these proteins to be expressed in a revindependent manner [31, 37, 38] . thus, our results raised the possibility that ifitms selectively interfere with the rev/ rre-mediated expression of gag and vif. indeed, ifitms displayed an inhibitory effect on gag expression in an artificial rev-dependent expression system (fig. 4c ). in this system, the codon-unoptimized and rre sequence-containing gagepol and gag expression plasmids (gagepolerre and gag-rre) were expressed at normal levels only in the presence of the rev plasmid (fig. 5b, first 2 lanes) . as a result, we found that ifitm1, 2, and 3 significantly reduced rev/rremediated gag expression (lanes 3e5), as they did in the proviral plasmid-mediated expression system (see fig. 3b ). however, the finding that ifitms interfere with rev/rremediated expression does not explain the fact that they also reduced the proviral plasmid-mediated expression of nef (see fig. 3b ) because nef rna does not contain rre, and therefore its expression is independent of the function of rev [37, 38] . thus, we next examined whether another viral double-stranded rna, i.e., the trans-activation response (tar) element, was involved in the inhibitory activity of ifitms. all of the hiv-1 rnas including that encoding nef contain the tar element at their 5 0 end, which binds and activates the double-stranded rna-dependent protein kinase (pkr) [39] . once activated, pkr phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eif2a), which reduces the efficiency and rate of the translation initiation of proteins including viral proteins [39] . on the other hand, the hiv-1 tat protein counteracts pkr activation via various complicated mechanisms, and the combination of the inhibitory pathways that prevent pkr activation determines the level of viral expression [39] . in this study, we found that the small molecule pkr inhibitor c16 restored the expression of p55gag, vif, and nef, when it was added at effective but noncytotoxic concentrations (0.2e0.8 mm) [40, 41] to the cells that had been co-transfected with the proviral nl43 plasmid and ifitm3 (fig. 5a) . meanwhile, c16 did not restore gag expression in the tat/tar-independent but rev/rredependent expression system (fig. 5b) , which was consistent with the fact that tar, but not rre, induces strong pkr activation [39] . therefore, our results (figs. 3e5) suggested that ifitms reduced the expression of gag, and possibly vif, by interfering with both rev/rre-and tat/tarmediated expression, and reduced the expression of nef by interfering with tat/tar-mediated expression. again, the inhibitory effect was not non-specific because ifitms did not affect the levels of these viral proteins when they were expressed via the system that bypassed the viral-specific machinery (see fig. 4a and b). a previous study demonstrated that ifitm3 was posttranslationally modified by s-palmitoylation, which is crucial for its activity against influenza virus infection [7] . therefore, we finally examined whether the s-palmitoylation of ifitms is important for their anti-hiv-1 activity. to this end, we prepared three different mutants of flag-tagged ifitm3 and ifitm2, in which the s-palmitoylated cysteine residues were mutated singly or in combination to alanine (fig. 6a) . consistent with the finding that the first two cysteines were more heavily s-palmitoylated than the third cysteine [7] , the mutants in which the first two cysteines were substituted (c1/2a and c1/2/3a) displayed more marked changes in their intracellular localization (fig. 6b, arrows) . however, unlike in the case of the anti-influenza virus activity of ifitm3, it was found that the s-palmitoylation was not necessary for the anti-hiv-1 activity of ifitms because all of the mutants of ifitm3 and ifitm2 reduced the concentrations of p24 gag in the supernatants (fig. 6c , bar graph) and the expression levels of p55 and p24 gag proteins in the cells (gag blot). these results clearly indicated that ifitms restrict hiv-1 and influenza viruses at distinct steps. both knockdown and enforced expression experiments demonstrated that ifitms restrict hiv-1 replication [12, 13] . in this study, we extended the findings of lu et al. [12] and revealed that the enforced expression of ifitms interfered with the production of hiv-1 proteins such as gag, vif, and nef only when viral double-stranded rnas (rre and/or tar) mediated their expression. these findings suggested that ifitm bind directly to viral double-stranded rna. indeed, a previous report raised the possibility that ifitm1 is an rrebinding protein [p. constantoulakis et al., inhibition of revmediated hiv-1 expression by an rna binding protein encoded by the interferon-inducible 9e27 gene, science 259 (1993) 1314e1318.]. however, as the report was retracted [m. campbell et al., science 264 (1994) 492.], careful studies will be necessary in order to clarify the exact mechanisms by which ifitms interfere with the viral protein expression mediated by the double-stranded viral rnas such as rre and tar. studies will be also necessary to explain why there was no obvious difference in the anti-hiv-1 activity among three ifitm proteins in our transfection assay, in contrast to the study by lu et al. [12] . despite these unanswered questions, the present study demonstrated that ifitms possess different characteristics from other anti-hiv-1 proteins such as tetherin and apobec3g and supported the idea that ifitms restrict hiv-1 and influenza viruses at distinct steps. new developments in the induction and antiviral effectors of type i interferon interferon-inducible antiviral effectors transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells ifitm/mil/ fragilis family proteins ifitm1 and ifitm3 play distinct roles in mouse primordial germ cell homing and repulsion the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus a.l. brass, ifitm3 inhibits influenza a virus infection by preventing cytosolic entry palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms isg56 and ifitm1 proteins inhibit hepatitis c virus replication the ifitm proteins inhibit hiv-1 infection a diverse range of gene products are effectors of the type i interferon antiviral response an interferon-alphainduced tethering mechanism inhibits hiv-1 and ebola virus particle release but is counteracted by the hiv-1 vpu protein tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu the interferon-induced protein bst-2 restricts hiv-1 release and is downregulated from the cell surface by the viral vpu protein suppression of tetherin-restricting activity upon human immunodeficiency virus type 1 particle release correlates with localization of vpu in the trans-golgi network hiv-1 antagonism of cd317 is species specific and involves vpu-mediated proteasomal degradation of the restriction factor vpu enhances hiv-1 virus release in the absence of bst-2 cell surface down-modulation and intracellular depletion alpha interferon potently enhances the anti-human immunodeficiency virus type 1 activity of apobec3g in resting primary cd4 t cells isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein speciesspecific exclusion of apobec3g from hiv-1 virions by vif hiv-1 vif blocks the antiviral activity of apobec3g by impairing both its translation and intracellular stability hiv-1 vif protein binds the editing enzyme apobec3g and induces its degradation induction of apobec3g ubiquitination and degradation by an hiv-1 vif-cul5-scf complex a single amino acid determinant governs the species-specific sensitivity of apobec3g to vif action vif overcomes the innate antiviral activity of apobec3g by promoting its degradation in the ubiquitin-proteasome pathway hiv-1 accessory protein vpu internalizes cellsurface bst-2/tetherin through transmembrane interactions leading to lysosomes site-directed mutagenesis of hiv-1 vpu gene demonstrates two clusters of replicationdefective mutants with distinct ability to down-modulate cell surface cd4 and tetherin the identification of a small molecule compound that reduces hiv-1 nef-mediated viral infectivity enhancement codon optimization of the hiv-1 vpu and vif genes stabilizes their mrna and allows for highly efficient rev-independent expression interaction between hck and hiv-1 nef negatively regulates cell surface expression of m-csf receptor dys-regulated activation of a src tyroine kinase hck at the golgi disturbs n-glycosylation of a cytokine receptor fms il-34 and m-csf share the receptor fms but are not identical in biological activity and signal activation hiv-1 nef interferes with m-csf receptor signaling through hck activation and inhibits m-csf bioactivities serine phosphorylation-independent downregulation of cell-surface cd4 by nef cellular proteins and hiv-1 rev function cessation of hiv-1 transcription by inhibiting regulatory protein rev-mediated rna transport multiple levels of pkr inhibition during hiv-1 replication small molecule inhibitors of the rna-dependent protein kinase prevention of the b-amyloid peptide-induced inflammatory process by inhibition of double-stranded rna-dependent protein kinase in primary murine mixed co-cultures we thank h. motoyama for her secretarial assistance. this work was supported by the global coe program "global education and research center aiming at the control of aids", which was commissioned by the ministry of education, culture, sports, science, and technology of japan (to n.c. and s.s.). key: cord-029416-738t6rk1 authors: mandal, sandip; bhatia, vineet; sharma, mukta; mandal, partha pratim; arinaminpathy, nimalan title: the potential impact of preventive therapy against tuberculosis in the who south-east asian region: a modelling approach date: 2020-07-20 journal: bmc med doi: 10.1186/s12916-020-01651-5 sha: doc_id: 29416 cord_uid: 738t6rk1 background: the prevention of tuberculosis (tb) is key for accelerating current, slow declines in tb burden. the 2018 world health organization (who) guidelines on eligibility for preventive therapy to treat latent tb infection (ltbi) include people living with human immunodeficiency virus (plhiv), household contacts of tb patients including children, and those with clinical conditions including silicosis, dialysis, transplantation, etc. and other country-specific groups. we aimed to estimate the potential impact of full implementation of these guidelines in the who south-east asian (sea) region, which bears the largest burden of tb and ltbi amongst the who regions. methods: we developed mathematical models of tb transmission dynamics, calibrated individually to each of the 11 countries in the region. we modelled preventive therapy in the absence of other tb interventions. as an alternative comparator, reflecting ongoing developments in tb control in the region, we also simulated improvements in the treatment cascade for active tb, including private sector engagement and intensified case-finding. relative to both scenarios, for each country in the region, we projected tb cases and deaths averted between 2020 and 2030, by full uptake of preventive therapy, defined as comprehensive coverage amongst eligible populations as per who guidelines, and assuming outcomes consistent with clinical trials. we also performed sensitivity analysis to illustrate impact under less-than-optimal conditions. results: at the regional level, full uptake of preventive therapy amongst identified risk groups would reduce annual incidence rates in 2030 by 8.30% (95% cri 6.48–10.83) relative to 2015, in the absence of any additional interventions. if implemented against a backdrop of improved tb treatment cascades, preventive therapy would achieve an incremental 6.93 percentage points (95% cri 5.81–8.51) of reduction in annual incidence rates, compared to 2015. at the regional level, the numbers of individuals with latent tb infection that need to be treated to avert 1 tb case is 64 (95% cri 55–74). sensitivity analysis illustrates that results for impact are roughly proportional to ‘effective coverage’ (the product of actual coverage and effectiveness of the regimen). conclusions: full implementation of who guidelines is important for ending tb in the sea region. although future strategies will need to be expanded to the population level, to achieve large declines in tb incidence, the uptake of current tools can offer a valuable step in this direction. despite large-scale efforts to improve tb services over the last two decades, global tb burden today is decreasing at a rate of only 1-2% per year. there remain critical challenges in the tb treatment cascade, for example, limited outreach of service, and missed opportunities for diagnosis in many high-burden settings [1, 2] . however, it is widely recognised that prevention, as well as optimising tb diagnosis and treatment, will be critical in any strategy aimed towards ending tb [3] [4] [5] . despite ongoing vaccine development [6, 7] and increasing attention on the social determinants of tb [8, 9] , preventive therapy arguably remains the primary option immediately available for tb prevention. in the absence of a widely deployable test to identify who would benefit most from preventive therapy, world health organization (who) guidelines identify high-risk groups for eligibility: for example, those with human immunodeficiency virus (hiv) coinfection [10] and, in the most recently updated guidelines, all household contacts of diagnosed tb cases and those with clinical conditions including silicosis, those on anti-tnf treatment, and other country-specific groups [11] . however, global uptake has been slow, partly owing to challenges in initiating and administering preventive therapy regimens, lasting several months, amongst otherwise healthy individuals [12, 13] . in the who south-east asian region, only 15% of plhiv and 26% of eligible children < 5 years of age were reported to have received preventive treatment in 2018 [14] . nonetheless, options for preventive therapy have seen important developments in recent years. isoniazid preventive therapy (ipt) involves 6 months daily treatment with isoniazid, and could lower the risk of active tb by 60%, amongst those with latent tb infection [15] . more recent developments include 3hr (isoniazid and rifampicin daily for 3 months), 3hp (isoniazid and rifapentine once weekly for 3 months) and 1hp (isoniazid and rifapentine once weekly for 1 month), all regimen options achieving non-inferior effectiveness to ipt, with shortened and simplified treatment [16] [17] [18] . these and future regimens may offer new opportunities to accelerate uptake of tb prevention, in line with who recommendations. in this context, strategic planning could benefit from estimation of the potential epidemiological impact of preventive therapy. in the present work, we aimed to address this need, using dynamical mathematical models. we concentrated on the who south-east asian (sea) region [19] , estimated to account for > 30% of global prevalence of latent tb infection and 44% of global tb incidence, the highest of any of the who regions [4, 14] . we developed mathematical models of tb transmission, calibrated to the tb epidemic in each of the 11 countries in the region. using this framework, we estimated the potential reductions in tb incidence and mortality that could be achieved by 2030, with implementation of who guidelines in each of the 11 countries in the region. the who sea region consists of 11 countries: bangladesh, bhutan, dpr korea, india, indonesia, maldives, myanmar, nepal, sri lanka, thailand and timor-leste. we built on a deterministic, compartmental framework developed in earlier work, in support of strategic planning in the region [20] . here we outline the model structure and approach, with further technical details given in the supplementary document (see additional file 1 [1, 11, 14, [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] ). the model structure is illustrated schematically in fig. 1 . a characteristic feature of many countries in the sea region is the presence of a large private healthcare sector, particularly in bangladesh, india, indonesia, myanmar, nepal and thailand. in these countries, the private sector plays a major role in tb care, often not reporting tb cases to public health authorities [33] [34] [35] [36] [37] . there is evidence to indicate that tb care in the private sector does not uniformly meet national standards [36, 38] , leading to delays in diagnosis and suboptimal treatment outcomes. accordingly, in the model, we distinguished between public and private sectors, allowing for a lower standard of diagnosis and less favourable treatment outcomes in the latter than the former (see tables s3 and s4 for parameters in additional file 1 [1, 14, 15, 17, 26, 27, 31, 32, [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] ). we calibrated the model independently for each of the 11 sea regional countries, to who estimates of tb burden from 2014 to 2018, the estimated proportion of tb cases having hiv coinfection in 2018, and who data for public sector notifications in 2018 (see table s2 in additional file 1 [14] ). we combined this data with uncertainty ranges for model parameters (see table s3 for parameter ranges in additional file 1 [1, 14, 15, 17, 26, 27, 31, 32, [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] ), ultimately using bayesian markov-chain monte-carlo (mcmc) [21] methods, a technique widely used in modelling studies [22] [23] [24] to sample from the posterior distribution of model parameters. as described below, we then used these samples to create model projections for the future impact of preventive therapy. this method allows a systematic approach for propagating uncertainty from model inputs to model outputs. for each country, we conducted the mcmc and recorded 10,000 iterations following initial 'burn-in'. for any given model projection, we estimated 95% credible intervals as the 2.5th and 97.5th percentiles arising from these samples. who guidelines for preventive therapy have adapted over time, incorporating steadily expanding eligibility criteria. the most recent guidelines identify as priorities for preventive therapy: those living with hiv, hivnegative contacts of tb patients (prioritising < 5 years old but also including adult and adolescent contacts), in whom active tb disease has been ruled out; and those with other clinical risk factors, including silicosis, transplant patients, and those receiving dialysis [11] . in the present analysis, we defined 'full uptake' as a scenario where (i) all eligible individuals receive preventive therapy, and (ii) where the outcomes of preventive therapy are consistent with those observed in clinical trials, assuming that both of these conditions are achieved over a 3-year scale-up period. we also performed sensitivity analysis to examine how the modelled impact would vary under less optimal conditions for coverage and effectiveness of preventive therapy. hiv coinfection is a prominent risk factor for developing tb, elevating a 10% lifetime risk of developing tb (amongst hiv-negative individuals) to a 10% annual risk [10] . preventive therapy has been shown to mitigate this risk by up to 60% [25] . in the sea region, hiv accounted for an estimated 3.2% of tb incidence in 2018, on the country level ranging from 0% in maldives to 8.5% in thailand. accordingly, we modelled the impact of preventive therapy amongst people living with hiv by a reduced rate of progression to active disease, reflecting both the coverage and efficacy of preventive therapy (see additional file 1 [11, 26, 27, [29] [30] [31] [32] ). hiv-negative household contacts are an important risk group because (i) the prevalence of latent tb infection in households of tb patients can be twice as high as in the general community, and (ii) those with latent infection are likely to have been recently infected with tb, the interval in which they are at highest risk of developing active disease [11] . as a result of both factors, the incidence rate of tb diseases amongst a cohort of household contacts with ltbi is substantially greater than in the general population. to quantify this relative risk, we drew from a recent case-finding study in vietnam [26] that conducted longitudinal follow-up amongst household contacts of tb patients, finding an incidence of 894 per 100,000 contacts in the first year of follow-up, 7 times greater than who estimates of country-level incidence in vietnam. this rate ratio is consistent with findings from ongoing household cohort studies in india [27] . it is not feasible to model households explicitly within a compartmental modelling framework. rather than aiming to capture this type of population structure, we asked the question: 'what are the population-level implications of reducing tb incidence amongst a defined cohort (of household contacts) in the population?'. we first estimated the direct effect on incidence: that is, the reduction in incidence that would arise from a given coverage of preventive therapy amongst household contacts, in the absence of secondary transmission effects. subsequently, and to capture indirect (transmission-mediated) effects, we incorporated this direct effect into the transmission model, in order to simulate the full incidence reductions at the population level. approaches for estimating direct and indirect effects have been described previously [28] and, in brief, involve controlling for the force-of-infection under different scenarios. further technical details are given in the supplementary document (see additional file 1). we modelled other clinical risk factors (e.g., silicosis and receiving dialysis) as an aggregate group and using a similar approach as that described above for household contacts. we simulated all scenarios from 2018 to 2030, assuming preventive therapy coverage to be scaled up in a linear way over 3 years from 2020 to 2023. we captured current levels of preventive therapy coverage in each country, amongst both household contacts and plhiv (at the regional level, current coverages of 26% and 15%, respectively); we assumed that the intervention would gradually replace existing ipt with new, rifamycin-based shorter regimens [17] as per eligibility criteria when scaling up to cover all household contacts of notified tb cases, as well as to cover all plhiv. to model the impact of preventive therapy, it is also important to model what may have occurred in future, in the absence of this intervention (the 'comparator' scenario, against which the incremental impact of preventive therapy is assessed). we adopted two comparators: (i) a 'status quo' comparator, with current tb services continuing without change indefinitely, and (ii) an 'improved tb cascade' comparator to capture ongoing efforts across the region to improve tb control, independent of preventive therapy. the latter includes comprehensive scale-up of services including engagement of the private/outside the national tb programmes (non-ntp sector), along with improvement in tb diagnostics and treatment outcomes, and additional efforts to accelerate the diagnosis of active tb, including measures such as intensified case-finding and generation of demand for tb services [50, 51] . modelled in previous work [20] , these scenarios are described in more detail in the supporting information. in the context of each of these comparators, we measured the impact of preventive therapy by projecting annual incidence and mortality rates in 2030, and comparing with rates in 2015, in line with the who end tb strategy [52] . preventive therapy is complicated by drug-resistant infection, as these infections may be resistant to standard preventive therapy regimens, and it is currently not possible to test the drug sensitivity status of ltbi. as a conservative approach, we modelled only the use of a 3hplike regimen amongst household contacts and amongst plhiv, assuming that all those with drug-resistant ltbi would either be ruled out for preventive therapy or would not respond to it. this assumption tends to underestimate overall impact, as in practice, contacts of drug-resistant tb patients may be offered fluoroquinolone-based preventive regimens [11] . finally, to identify the most influential model parameters, we selected a focal model output: the percent reduction in incidence rates as a result of full uptake of preventive therapy, in 2030 relative to 2015. we computed the partial rank correlation of this outcome against all model parameters (e.g. as described in ref. [53] ), aiming to identify the most influential parameter as that showing the greatest correlation. figures s1 and s2 in the supplementary document (see additional file 1) show results of model calibrations for each of the 11 countries in the region, and tables s3, s4 shows the bayesian posterior estimates for model parameters. figure 2 shows model projections for incidence and mortality impact in the region, as an aggregate of projections over all 11 countries in the region, under the two comparator scenarios described above. in the absence of other additional tb interventions ('status quo' comparator), full adoption of who guidelines would result in a 8.30% (95% cri 6.48-10.83) reduction of the annual incidence rate in 2030 relative to 2015, and a reduction of tb deaths in 2030 by 6.75% (95% cri 5.19-8.54) relative to 2015. in the context of background interventions to improve tb services (the 'improved tb cascade' comparator), the incremental impact from preventive therapy would be to reduce incidence rates in 2030 by a further 6.93% (95% 5.81-8.51) and tb deaths in 2030 by a further 3.52% (95% 2.72-5.08) (both relative to 2015). table 1 shows these results by country, while tables s5 and s6 (additional file 1) show impact in terms of cumulative cases and tb deaths averted. while these results focus on the incremental impact attributable to preventive therapy alone, table s7 in the supporting information also shows the combined impact of improving the tb cascade and implementing preventive therapy. table 2 additionally stratifies impact amongst those living with hiv and amongst household contacts of tb cases, taking the example of the improved tb cascade comparator. at the regional level, preventive therapy limited to people living with hiv would reduce annual incidence rates in 2030 by 1.81% (95% 1.30-2.38), relative to 2015. a larger impact arises from limiting preventive therapy to household contacts, yielding an incidence rate reduction of 6.93% (95% 5.81-8.51); this latter group accounts for the bulk of epidemiological impact in the sea region. table 1 for impact by country, as well as in terms of incidence and mortality. shaded intervals show 95% bayesian credible intervals. as described in the main text, the 'status quo comparator represents current tb services continuing indefinitely without change, while the 'improved cascade' comparator incorporates background improvements in tb care, including comprehensive engagement with the private healthcare sector, and intensified case-finding, throughout the region table 1 impact on tb incidence and mortality, by country and for the whole region, by 2030 relative to 2015. estimates show the incremental impact attributable to preventive therapy alone, in the context of each comparator. thus estimates under the 'status quo' comparator reflect the difference between the blue and orange curves in the left-hand panel of fig. 2 and estimates under the 'improved tb cascade' comparator reflect the difference between the green and yellow curves in the right-hand panel of fig. 2 incremental impact of preventive therapy relative to 'status quo comparator' as a simple proxy for resource needs, we sought to estimate the numbers of individuals that would need to receive preventive therapy, in order to avert 1 tb case and to avert 1 tb death. figure 3 shows model projections, illustrating these to vary widely by country setting. numbers-needed-to-treat to avert 1 tb case are generally higher for low-burden countries (e.g. 208 (95% cri 165-283) for sri lanka) than for higher-burden settings (e.g. 73 (95% cri 59-91) for india), potentially because higher levels of transmission in higher-burden settings also amount to higher levels of indirect protection. corresponding numbers to avert tb deaths are typically 10-20 times higher, for example with india needing 848 (95% cri 552-1375) individuals to receive preventive therapy in order to avert 1 tb death. finally, figs.s3, s4 in the supplementary document (see additional file 1) shows results of sensitivity analysis. we first examined the model sensitivity to our assumption of 'full uptake' of preventive therapy, meaning that all eligible groups receive preventive therapy, with efficacy similar to those observed in clinical trials. figure s3 relaxes these assumptions, showing that incidence impact is roughly proportional to 'effective coverage', defined as the product of the coverage and effectiveness of preventive therapy. figure s4 shows additional sensitivity analysis, using partial rank correlation coefficients to identify the model inputs that are most influential for model outcomes. results illustrate that the key uncertainty is the incidence rate amongst household contacts, relative to the general population (k hh ). notably less sensitivity is attached to the incidence rate amongst those with silicosis, dialysis patients, etc. (k rg ), despite the wide uncertainty intervals adopted for this population: despite the clear value of preventive therapy amongst these patients, their relatively small size, as a proportion of the overall population, limits their potential contribution to overall declines in tb burden. with preventive therapy forming an important part of the end tb strategy worldwide, it is important to assess the impact that could be achieved with currently available tools. our results suggest that, amongst the 11 countries of the sea region, full adoption of who guidelines could have a meaningful impact in reducing tb burden between now and 2030. in particular, our results suggest that such measures could reduce tb incidence and deaths in the region by 8.30% (95% cri 6.48-10.83) and 6.75% (95% cri 5.19-8.54), respectively. the relatively modest contribution of prevention amongst plhiv to a reduction in incidence is because hiv coinfection does not play the strong role in driving tb transmission in sear as it does, for example, in south africa: at the regional level in sear, an estimated 3.4% of incident tb cases are hiv coinfected [14] . nonetheless, given the sheer mortality toll caused by tb amongst those living with hiv and the proven effectiveness of preventive therapy in this group, tb prevention amongst plhiv justifiably continues to be a public health priority in the region. moreover, there is substantial variation between countries within the region, with an estimated 9.8% of incident tb cases in thailand being hiv-coinfected. in such settings, prevention amongst plhiv plays a notably stronger role than on the regional level (table 2) . broadly, the relative reductions in tb burden that are achievable by preventive therapy appear greater in those countries with higher tb incidence rates (table 1 ). if the impact of preventive therapy is amplified by its indirect effects (i.e. those mediated by transmission), then it might be expected that this impact is greatest in settings where transmission is strongest. it will be important for any future scale-up of preventive therapy to be accompanied by monitoring for drug resistance. in the context of isoniazid preventive therapy (ipt), despite concerns that widespread use could drive the emergence of drug resistance, the available evidence suggests otherwise [54, 55] . the risks of newer regimens driving drug resistance may be still smaller than the risk for ipt, given that they involve combinations of drugs, such as 3hp. nonetheless, continued surveillance for drug resistance will remain essential. household contacts account for the bulk of projected impact of preventive therapy in the region, but more is required in order to meet the ambitious goals of the end tb strategy by 2030. as argued in previous work, there is a need for future preventive strategies to move beyond specific risk groups, to achieve tb prevention on a population level [20] . such levels of coverage are infeasible with currently available tools, for example with the unsuitability of current preventive regimens for widescale deployment [13, 56] . nonetheless, with the advent of new diagnostics and therapies [17, 18] , the full implementation of current guidelines would form a valuable transitional step to the deployment of future preventive strategies. intersectoral approaches, such as addressing comorbidities including undernutrition and diabetes, are also likely to play an important role in the populationlevel prevention of tb [8, 57, 58] . as with any modelling analysis, our work has entailed several simplifications. we have ignored age structure, as well as averaging over pulmonary and extrapulmonary forms of tb. we have also not addressed subnational fig. 3 numbers-needed-to-treat with preventive therapy, to prevent 1 tb case. figure shows estimates stratified by the 11 countries in the region, as a simple proxy for the effort required to achieve the incidence declines shown in fig. 2 . error bars show 95% bayesian credible intervals. in the second panel, numbers-needed-to-treat are disproportionately high for the maldives because of a low incidence (33 per 100 k population), as well as a low reported tb mortality rate (0.15 per 100 k population) variation within countries with, for example, different states within india having widely varying levels of tb burden [59] . as noted above, compartmental models are not readily suited for capturing household structure in the population: we have sought to address this challenge, while maintaining the overall tractability of the modelling approach, by abstracting from household structure. future work could aim to test these assumptions by (i) building the evidence basis for tb incidence amongst household contacts in different settings, particularly though longitudinal designs such as in ref. [26] ; (ii) seeking to measure the community benefits of preventive therapy; and (iii) comparing against alternative modelling approaches, including individual-based modelling. we have additionally not addressed the very real implementation challenges of preventive therapy [25] , assuming scenarios where these challenges are sufficiently overcome to realise the full potential of preventive therapy. to some extent, these assumptions may be justified with the emergence of new, simplified preventive therapy regimens [17, 18] , but this remains to be demonstrated. while a full costing analysis is outside the scope of the current study, we have nonetheless estimated a simple proxy, the number of individuals with latent tb infection who would need to be treated with preventive therapy, in order to avert 1 tb case (fig. 3 ). an important area for future analysis is to perform a more systematic costing analysis, including not only the costs for testing and treating ltbi, but also the cost savings that would arise from prevention of future tb episodes. important developments, including the recently announced price reductions for the 3-month regimen [60] , will impact any assessment of cost-effectiveness of preventive therapy. future costing analysis will also be invaluable in informing investment strategies in the implementation of preventive therapy strategies, as well as in the development of new diagnostics and regimens. in conclusion, there is a need to expand current tb efforts beyond the tb treatment cascade, to a comprehensive strategy incorporating tb prevention as well. although current strategies are necessarily limited to specific risk groups, in the who sea region, they could nonetheless achieve as much incidence and mortality impact as parallel measures to optimise the tb treatment cascade. looking to the future, full implementation of these current strategies will form an important stepping stone to more wide-ranging preventive measures. potentially facilitated by new technologies for targeted prevention, such measures will form a critical component of collective efforts to accelerate reductions in global tb incidence. supplementary information accompanies this paper at https://doi.org/10. 1186/s12916-020-01651-5. additional file 1 : figure s1 . calibration results to incidence and mortality. figure s2 . calibration results to additional indicators. figure s3 . sensitivity analysis to assumptions for coverage and effectiveness of preventive therapy. figure s4 . sensitivity analysis to model parameters. table s1 . list of state variables used in the model. table s2 . epidemiological indicators for model calibration. table s3 . list of regional-level parameters used in the model. table s4 . summary of country-wise parameter estimates. table s5 . impact on cumulative tb incidence by country, and for the whole region, from 2019-2030. table s6 . impact on cumulative tb mortality by country, and for the whole region, from 2019-2030. table s7 . total impact of improved tb cascade and preventive therapy. the tuberculosis cascade of care in india's public sector: a systematic review and metaanalysis the south african tuberculosis care cascade: estimated losses and methodological challenges tuberculosis preventive treatment: the next chapter of tuberculosis elimination in india the global burden of latent tuberculosis infection: a re-estimation using mathematical modelling eliminating human tuberculosis in the twenty-first century progress and challenges in tb vaccine development phase 2b controlled trial of m72/as01 e vaccine to prevent tuberculosis the social determinants of tuberculosis: from evidence to action the impact of social protection and poverty elimination on global tuberculosis incidence: a statistical modelling analysis of sustainable development goal 1 hiv and tuberculosis: a deadly human syndemic world health organization, who. latent tb infection: updated and consolidated guidelines for programmatic management contact screening and chemoprophylaxis in india's revised tuberculosis control programme: a situational analysis the cascade of care in diagnosis and treatment of latent tuberculosis infection: a systematic review and meta-analysis isoniazid for preventing tuberculosis in non-hiv infected persons. cochrane database syst rev short-course therapy with rifampin plus isoniazid, compared with standard therapy with isoniazid, for latent tuberculosis infection: a meta-analysis three months of rifapentine and isoniazid for latent tuberculosis infection one month of rifapentine plus isoniazid to prevent hiv-related tuberculosis tuberculosis in the who south-east asian region. world health organization, searo strategies for ending tuberculosis in the south-east asian region: a modelling approach an adaptive metropolis algorithm bayesian melding for estimating uncertainty in national hiv prevalence estimates population health impact and cost-effectiveness of tuberculosis diagnosis with xpert mtb/rif: a dynamic simulation and economic evaluation assessing tuberculosis control priorities in high-burden settings: a modelling approach tuberculosis preventive therapy in the era of hiv infection: overview and research priorities household-contact investigation for detection of tuberculosis in vietnam report international: advancing tuberculosis biomarker research through global collaboration counting the lives saved by dots in india: a model-based approach use of tuberculosis interferon-gamma release assays (igras) in low-and middle-income countries. policy statement. geneva: world health organization incorporating household structure and demography into models of endemic disease the epidemiology of tuberculosis in gold miners with silicosis guidelines for the prevention and management of mycobacterium tuberculosis infection and disease in adult patients with chronic kidney disease effectiveness of involving the private medical sector in the national tb control programme in bangladesh: evidence from mixed methods how do private general practitioners manage tuberculosis cases? a survey in eight cities in indonesia missed opportunity for standardized diagnosis and treatment among adult tuberculosis patients in hospitals involved in public-private mix for directly observed treatment short-course strategy in indonesia: a cross-sectional study tuberculosis management by private practitioners in mumbai, india: has anything changed in two decades? use of standardised patients to assess quality of tuberculosis care: a pilot, cross-sectional study delays in diagnosis and treatment of pulmonary tuberculosis in india: a systematic review the natural history of tuberculosis: the implications of age-dependent risks of disease and the role of reinfection modeling the dynamic relationship between hiv and the risk of drug-resistant tuberculosis revisiting rates of reactivation tuberculosis: a population-based approach toman's tuberculosis: case detection, treatment, and monitoring relapse in persons treated for drug-susceptible tuberculosis in a population with high coinfection with human immunodeficiency virus in new york city use of xpert mtb/rif in decentralized public health settings and its effect on pulmonary tb and dr-tb case finding in india world health organization. guidelines for treatment of tuberculosis and patient care durations and delays in care seeking, diagnosis and treatment initiation in uncomplicated pulmonary tuberculosis patients in mumbai, india. plos one risk of progression to active tuberculosis following reinfection with mycobacterium tuberculosis efficacy and completion rates of rifapentine and isoniazid (3hp) compared to other treatment regimens for latent tuberculosis infection: a systematic review with network meta-analyses united nations, department of economic and social affairs pd. household size and composition around the world 2017 achieving systemic and scalable private sector engagement in tuberculosis care and prevention in asia joint effort for elimination of tuberculosis world health organisation. the end tb strategy a methodology for performing global uncertainty and sensitivity analysis in systems biology isoniazid preventive therapy and risk for resistant tuberculosis. emerg infect dis tuberculosis outcomes and drug susceptibility in individuals exposed to isoniazid preventive therapy in a high hiv prevalence setting biomarkers and diagnostics for tuberculosis: progress, needs, and translation into practice the relationship between malnutrition and tuberculosis: evidence from studies in humans and experimental animals tuberculosis control and elimination 2010-50: cure, care, and social development sub-national tb prevalence surveys in india, 2006-2012: results of uniformly conducted data analysis a powerful tuberculosis drug gets a deep price cut publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions vb, ms and na conceived the study, sm performed the modelling analysis, and vb, ms and ppm contributed to the interpretation of the results. sm and na wrote a first draft of the manuscript. all authors read and approved the final manuscript. key: cord-263438-9ra94uda authors: snowden, frank m. title: emerging and reemerging diseases: a historical perspective date: 2008-09-19 journal: immunol rev doi: 10.1111/j.1600-065x.2008.00677.x sha: doc_id: 263438 cord_uid: 9ra94uda summary: between mid‐century and 1992, there was a consensus that the battle against infectious diseases had been won, and the surgeon general announced that it was time to close the book. experience with human immunodeficiency virus/acquired immunodeficiency syndrome, the return of cholera to the americas in 1991, the plague outbreak in india in 1994, and the emergence of ebola in zaire in 1995 created awareness of a new vulnerability to epidemics due to population growth, unplanned urbanization, antimicrobial resistance, poverty, societal change, and rapid mass movement of people. the increasing virulence of dengue fever with dengue hemorrhagic fever and dengue shock syndrome disproved the theory of the evolution toward commensalism, and the discovery of the microbial origins of peptic ulcer demonstrated the reach of infectious diseases. the institute of medicine coined the term ‘emerging and reemerging diseases’ to explain that the world had entered an era in which the vulnerability to epidemics in the united states and globally was greater than ever. the united states and the world health organization took devised rapid response systems to monitor and contain disease outbreaks and to develop new weapons against microbes. these mechanisms were tested by severe acute respiratory syndrome in 2003, and a series of practical and conceptual blind spots in preparedness were revealed. in the long contest between humans and microbes, the years from mid-century until 1992 marked a distinctive era. in those euphoric decades, there was a consensus that the decisive battle had been joined and that the moment was at hand to announce the final victory. almost as if introducing the new period, the us secretary of state george marshall declared in 1948 that the world now had the means to eradicate infectious diseases from the earth. marshall's view was by no means exceptional. for some, in the early postwar years, the triumphant vision applied primarily to a single disease. the heady goal arose first of all within the field of malariology, where the rockefeller foundation scientists fred soper and paul russell thought that they had discovered in ddt (dichlorodiphenyltrichloroethane) a weapon of such unparalleled power that it would enable the world to eliminate the ancient scourge forever. with premature confidence in 1955, russell published man's mastery of malaria (1), frank m. snowden in which he envisaged a global spraying campaign that would free mankind from malaria -cheaply, rapidly, and without great difficulty. rallying to russell's optimism, the world health organization (who) adopted a global campaign of malaria eradication with ddt as its weapon of choice. the director of the campaign, emilio pampana, elaborated a one-size-fits-all program of eradication through four textbook steps -'preparation, attack, consolidation, and maintenance' (2). russell's followers alberto missiroli, the director of the postwar campaign in italy, and george macdonald, the founder of quantitative epidemiology, reasoned that so signal a victory over mosquitoes could be readily expanded to include the elimination of all other vector-borne tropical diseases, ushering in what missiroli called a contagion-free eden, where medicine would make man not only healthy but also happy (3) (4) (5) . if malariologists, who dominated the international public health community, launched the idea of the final conquest of infectious diseases, it rapidly developed into the prevailing orthodoxy. e. harold hinman, chief malariologist to the tennessee valley authority and member of the who expert committee on malaria, extrapolated from the conquest of malaria to the conquest of all contagion in his influential work world eradication of infectious diseases (6) . aidan cockburn, a distinguished epidemiologist at johns hopkins and advisor to the who, gave expression to this new creed in his revealingly titled work the evolution and eradication of infectious diseases (7) . as cockburn noted, '''eradication'' of infectious disease as a concept in public health has been advanced only within the past two decades, yet it is replacing ''control'' as an objective' (7) . although not a single disease had yet been destroyed by his time of writing in 1962, cockburn believed that the objective of eradication was 'entirely practical,' not just for individual illnesses but for the whole category of communicable diseases. indeed, he argued, 'it seems reasonable to anticipate that within some measurable time, such as 100 years, all the major infections will have disappeared' (7) . by that time, he explained, 'the major infections of today should have disappeared, and only remaining should be their memories in textbooks, and some specimens in museums. . . . with science progressing so rapidly, such an end-point is almost inevitable, the main matter of interest at the moment is how and when the necessary actions should be taken' (8) . cockburn's timetable of total eradication by 2060 was, in fact, too slow for some. just a decade later, in 1973, the australian virologist and nobel laureate frank macfarlane burnet went so far as to proclaim, together with his colleague david white, that 'at least in the affluent west,' the grand objective had already been reached. 'one of the immemorial hazards of human existence has gone,' he reported, because there is a 'virtual absence of serious infectious disease today' (9) . the who also saw the entire planet as ready to enter the new era by the end of the century. meeting at alma ata in 1979, the world health assembly adopted the goal of 'health for all, 2000' (10) . what could possibly have led to such overweening confidence in the power of science, technology, and civilization to vanquish communicable disease? one factor was historical. in the industrialized west, rates of mortality and morbidity from infectious diseases began to plummet in the second half of the 19th century, in large part as a result of 'social uplift' -dramatic improvements in wages, housing, diet, and education. at the same time, developed nations erected the solid fortifications of sanitation and public health: sewers, drains, sand filtration, and chlorination of water as defenses against cholera and typhoid; sanitary cordons, quarantine, and isolation against bubonic plague; vaccination against smallpox; and the first effective 'magic bullet' -quinine -against malaria. meanwhile, improvements in the handling of food, pasteurization, retort canning, and the sanitation of seafood beds, yielded major advances against bovine tuberculosis (tb), botulism, and a variety of food-borne maladies. already by the early 20th century, therefore, many of the most feared epidemic diseases of the past were in headlong retreat for reasons that were initially more empirical and spontaneous than the result of the application of science. science, however, soon added new and powerful weapons. the foundational work of louis pasteur and robert koch had established the biomedical model of disease that promoted unprecedented understanding and yielded a cascade of scientific discoveries and new sub-specialties (microbiology, immunology, parasitology, and tropical medicine). the dawn of the antibiotic era with penicillin and streptomycin provided means to treat syphilis, staph infections, and tb. the development of a series of vaccines dramatically lowered the incidence of smallpox, pertussis, diphtheria, tetanus, rubella, measles, mumps, and polio. ddt seemed to furnish a means to abolish malaria and other insect-borne pathogens. by the 1950s, therefore, scientific discoveries had provided effective weapons against many of the most prevalent infectious diseases. extrapolating from such dramatic developments, many concluded that it was reasonable to expect that communicable diseases could be eliminated one at a time until the vanishing point was reached. indeed, the worldwide campaign against smallpox provided just such an example when the who announced in 1979 that the disease had become the first ever to be eradicated by intentional human action. those who asserted the doctrine of the conquest of infection viewed the microbial world as largely static or only very slowly evolving. for that reason, there was little concern that the victory over existing infections would be challenged by the appearance of new diseases for which humanity was unprepared and immunologically naive. falling victim to historical amnesia, they ignored the fact that the last 500 years even in the west had been punctuated by the appearance of a series of catastrophic new diseases: bubonic plague in 1347, syphilis in the 1490s, cholera in 1830, spanish influenza in 1918-1919. macfarlane burnet in this regard was typical. burnet was a founding figure in evolutionary medicine who acknowledged, in theory, the possibility of the emergence of new diseases as a result of mutation. but, in practice, he believed that such appearances are infrequent and that they occur only at such distant intervals as to occasion little concern. 'there may,' he wrote, 'be some wholly unexpected emergence of a new and dangerous infectious disease, but nothing of the sort has marked the last fifty years' (9) . the notion of microbial fixity, that the diseases that we have are the ones that we will face, even underpinned the international health regulations adopted in 1969 (ihr 1969) , which specified that the three great epidemic killers of the 19th century were the only diseases requiring notification: plague, yellow fever, and cholera. the regulations gave no thought to what action would be required if an unknown but deadly and transmissible new microbe should appear (11, 12) . if belief in the stability of the microbial world was one of the major articles of faith underpinning the eradicationists' vision, a second misplaced evolutionary idea also played a crucial role. this was the doctrine that nature was fundamentally benign. over time, eradicationists believed, the pressure of natural selection would drive all communicable diseases toward a decline in virulence. the principle was that excessively lethal infectious diseases would prevent their own transmission by prematurely destroying their hosts. the long-term tendency, the proponents of victory asserted, is toward commensalism and equilibrium. new epidemic diseases are virulent almost by accident as a temporary maladaptation, and they therefore evolve toward mildness, ultimately becoming readily treatable diseases of childhood. examples were the evolution of smallpox from variola major to variola minor; the transformation of syphilis from the fulminant 'great pox' of the 16th century into the slow-acting disease of today; and the transformation of classic cholera into the far milder el tor biotype. similarly, the doctrine held a priori that, in the family of four diseases of human malaria, the most virulent, i.e. falciparum malaria, was an evolutionary newcomer relative to the less lethal vivax, ovale, and malariae malaria, which were believed to be older and to have evolved toward commensalism. against this background, the standard textbook of internal medicine in the eradicationist era, the 7th edn of harrison's principles of internal medicine of 1974, claimed that a feature of infectious diseases is that they 'as a class are more easily prevented and more easily cured than any other major group of diseases' (13, 14) . the most fully elaborated and most cited theory of the new era was the 'epidemiologic transition' or 'health transition' theory represented by abdel omran, professor of epidemiology at johns hopkins, in 1971 and refined by him in 1977 and 1983. omran's theory of the transition was an account of the encounter of human societies with disease in the modern period. according to omran and his followers in such journals as the health transition review, humanity has passed through three eras of modernity in health and disease. although omran is ambiguous about the precise chronology of the first era, the 'age of pestilence and famine,' it is clear that it lasted until the 18th century in the west and was marked by malthusian positive checks on demography: epidemics, famines, and wars. there followed the 'age of receding pandemics' that extended from the mid-18th century until the early 20th in the developed west and until later in non-western countries. during this period there was a declining mortality from infectious diseases in general and from tb in particular. finally, after world war i in the west and after world war ii in the rest of the globe, humanity entered the 'age of degenerative and man-made diseases.' whereas in the earlier stages of disease evolution, social and economic conditions played the dominant role in determining health and the risk of infection, in the final phase medical technology and science played a major part. in this period, mortality and morbidity from infectious diseases have been progressively replaced by the rise of degenerative diseases such as cardiovascular disease, cancer, diabetes, and metabolic disorders, by man-made diseases such as occupational and environmental illnesses, and by accidents (15) (16) (17) . adopting the perspective of 'health transition' theory, us surgeon general julius b. richmond announced in 1979 that infectious diseases were simply the 'predecessors' of the degenerative diseases that succeed and replace them. the course of nature, in his view, was simple, unidirectional, and benign (18) . if memory of the power of public health and science provided a major impetus to overconfidence, forgetfulness also played a vital role. us surgeon general william steward reported in 1969 that the time had come to 'close the book on infectious diseases.' this view was profoundly eurocentric. even as medical experts in europe and north america snowden á emerging and reemerging diseases r 2008 the authors journal compilation r 2008 blackwell munksgaard immunological reviews 225/2008 declared final victory, infectious diseases remained the leading cause of death worldwide, and nowhere more disastrously than in the poorest and most vulnerable countries of africa, asia, and latin america. while the tb sanatoria were closing their doors in the developed north, the disease continued its ravages in the south. indeed, the disease continued to ravage the marginalized underclasses of the north itself: the homeless, prisoners, intravenous drug users, immigrants, and racial minorities. as paul farmer has argued, tb was emphatically not disappearing; it was just that the bodies it affected were either distant or hidden from sight (19, 20) . indeed, in 2008 the best estimates suggest that there are more people ill with tb today than at any time in human history and that nearly two million will die of it during the course of the year (21, 22) . ultimately, by the early 1990s, the eradicationist position became untenable. rather than witnessing the rapid fulfillment of the prediction that science and technology would eliminate all infectious diseases from the globe, the industrial west discovered that it remained painfully vulnerable and to a degree that had seemed unimaginable. the decisive event, of course, was the arrival and upsurge of human immunodeficiency virus (hiv)/acquired immunodeficiency syndrome (aids). aids was first recognized as a new disease entity in 1981, and its etiologic pathogen was identified in 1983. by the end of the decade, it was clear that hiv/aids embodied everything that the eradicationists had considered unthinkable. aids was a new infectious disease for which there was no cure, it reached the industrial world as well as developing countries, and it unleashed in its train a series of exotic additional opportunistic infections. furthermore, it had the potential to become the worst pandemic in history as measured not only by mortality and suffering but also by its profound social, economic, and security consequences. from the front lines of the battle against aids, a series of voices sounded the alarm in the 1980s about the severity of the new threat. most famous of all was the case of the us surgeon general c. everett koop, who became the chief federal spokesman on the disease. in 1988 he produced the brochure understanding aids and took the pioneering step of having it mailed to all 107 million households in the nation (23) . working in greater obscurity in sub-saharan africa, peter piot, who later directed unaids, warned in 1983 that aids in africa was not a 'gay plague' but an epidemic of the general population. he warned that it was transmitted by heterosexual as well as homosexual intercourse and that in fact it affected women more readily than men. the warnings of the 1980s, however, were confined to the issues of aids: they did not directly confront the larger issue of eradicationism or announce a new era in medicine and public health. that task fell first to the national academy of science's institute of medicine (iom) and its landmark publications on emerging diseases that began in 1992 with emerging infections: microbial threats to health in the united states (24) . once raised by the iom, the cry of alarm was taken up widely and almost immediately: by the centers for disease control and prevention (cdc), which devised its own response to the crisis in 1994 and founded a new journal emerging infectious diseases devoted to the issue; by the national science and technology council (nstc) in 1995; and by 36 of the world's leading medical journals that agreed to take the unprecedented step by which each devoted a theme issue to emerging diseases in january 1996, which they proclaimed 'emergent diseases month' (25) (26) (27) . in 1996, in addition, president bill clinton (28) issued a fact sheet entitled 'addressing the threat of emerging infectious diseases' in which he declared them 'one of the most significant health and security challenges facing the global community.' there were also highly visible hearings on emerging infections in the us congress (29) . in opening those hearings before the senate committee on labor and human resources, senator nancy kassebaum, the committee chairperson, noted, new strategies for the future begin with increasing the awareness that we must re-arm the nation and the world to vanquish enemies that we thought we had already conquered. these battles, as we have learned from the 15year experience with aids, will not be easy, inexpensive, nor quickly resolved. (29) finally, to attract attention at the international level, the who, which had designated april 7 of each year world health day, declared that the theme for 1997 was 'emerging infectious diseases -global alert, global response' with the lesson that in a global village, no nation is immune (30) . in addition to the voices of scientists, elected officials, and the public health community, the popular press gave extensive coverage to the new and unexpected danger, especially when the lesson was driven home by three events of the 1990s that captured attention worldwide. the first was the onset of a large-scale epidemic of asiatic cholera in south and central america, beginning in peru in 1991 and rapidly spreading across the continent until 400 000 cases and 4000 deaths were reported in 16 countries (31) . since the americas had been free of the disease for a century, the arrival of the unwelcome visitor reminded the world of the fragility of painfully won advances in public health. because cholera is transmitted by the contamination of food and water by fecal matter, it is a 'misery thermometer' -an infallible indicator of societal neglect and substandard living conditions (32) . its outbreak in the west late in the 20th century, therefore, caused shock and a sudden awareness of unexpected danger. indeed, the press informed its readers of the 'dickensian slums of latin america,' where the residents of lima and other cities drew their drinking water directly from the 'sewage-choked river rimac' and similarly polluted sources (33, 34) . who director-general hiroshi nakajima proclaimed the south american epidemic an 'emergency situation. ' the second news-catching event in the matter of epidemic diseases was the outbreak of plague in the indian states of gujarat and maharashtra in september and october 1994. the final toll for the epidemic was limited -700 cases and 56 deaths were reported (31) . nevertheless, the news that plague had broken out in both bubonic and pneumonic forms unleashed an almost biblical exodus of hundreds of thousands of people from the industrial city of surat. it cost india an estimated $1.8 billion in lost trade and tourism, and it sent waves of panic around the world. the disproportionate fear, as the new york times explained, was due to the fact that the very word plague was explosively charged. it evoked cultural memories of the black death that killed a quarter of the population of europe in the 14th century. india's plague, the paper continued, 'is a vivid reminder that old disease, once thought to have been conquered, can strike unexpectedly anytime, anywhere' (35) . the third major epidemic shock of the 1990s was an outbreak of the frightening disease of ebola hemorrhagic fever at the city of kikwit, zaire (now democratic republic of the congo), in 1995. cholera claimed international attention because of the numbers of those it afflicted, even though it had a low case fatality rate if treated early. plague demanded attention because of its all too familiar potential. ebola, by contrast, did not inspire terror by giving rise to a major epidemic: it infected only 315 people between january and july 1995. nor did it create fear because of historical memories of disaster since it was a new disease first recognized in 1976. nevertheless, ebola set off a tidal wave of fear -a 'modern nightmare' in the words of le monde -across the globe. the reasons were that it dramatically revealed the lack of preparedness of both industrial and developing nations to deal with a public health emergency. it ignited primordial western fears of the jungle and of untamed nature, and it fed on racial anxieties about 'darkest' africa. as a result, a prominent aspect of the kikwit outbreak was its capacity to generate what the journal of infectious diseases termed 'extraordinary' and 'unprecedented' press coverage that amounted at times to the commercial 'exploitation' of human misery and a 'national obsession' (36) . descending onto the banks of the kwilu river, the world's tabloids stressed in vivid hyperbole that ebola was a zoonotic disease that had sprung directly from the jungles of africa as a result of the encounter between native charcoal burners and monkeys and now threatened the west. in the revealing headline of the daily telegraph of sydney, 'out of the jungle a monster comes' (37) . even the most legitimate investigators, however, were disturbed to discover that ebola had eluded public health attention for 12 weeks between the death of the index case on january 6 and the notification of the international community on april 10, despite the fact that the disease had left clusters of severely ill and dying patients in its train. with such a porous surveillance network in place, ebola aroused the fear that it might spread unnoticed 500 km from kikwit to kinshasa, and then throughout the world by means of the zairian capital's intercontinental airport. there the virus could be loaded on board as 'a ticking, airborne time bomb' (38) . most of all, however, the kikwit outbreak commanded attention because ebola is almost invariably fatal and because its course in the human body is excruciating, dehumanizing, and dramatic. commenting on the scenes that he had observed in zaire, the author richard preston explained on television at the height of the outbreak that the mortality rate among sufferers was 90% and that there was no known remedy or prophylactic. he continued: the victims suffer what amounts to a full-blown biological meltdown. . . . when you die of ebola, there's this enormous production of blood, and that can often be accompanied by thrashing or epileptic seizures. and at the end you go into catastrophic shock and then you die with blood pouring out of any or all of the orifices of the body. and in africa where this outbreak is going on now, medical facilities are not all that great. i've had reliable reports that doctors . . . were literally struggling up to their elbows in blood -in blood and black vomit and in bloody diarrhea that looks like tomato soup, and they know they're going to die. (39) in combination with the announcement by scientists that the world was highly susceptible to new pandemics of just such infections, these events on three continents generated hordes, and of nature exacting its revenge for human presumption. as forrest sawyer reported on abc news, 'once the western world thought it was safe from these invisible killers. not anymore. we are now biologically connected in a web or a net.' in addition, there was an outpouring of films devoted to the possibility of pandemic disaster such as wolfgang petersen's thriller outbreak and of widely read books on the same theme, including richard preston's best-seller, the hot zone; laurie garrett, the coming plague: newly emerging diseases in a world out of balance; and william close, ebola. in the words of david satcher, director of the cdc, the result was the 'cnn effect' -the perception by the public that it was at immediate risk even at times when the actual danger was small (29) . in this climate of anxiety, the term 'emerging and reemerging diseases' was coined for the iom by joshua lederberg, winner of the nobel prize for medicine, to mark a new era. lederberg defined these disease entities as follows: 'emerging infectious diseases are diseases of infectious origin whose incidence in humans has increased within the past two decades or threatens to increase in the near future' (40) . emerging diseases were those that, like aids and ebola, were previously unknown to have afflicted humans; reemerging diseases, such as cholera and plague, were familiar scourges whose incidence was rising, or whose geographical range was expanding. lederberg's purpose in devising a new category of diseases was to give notice that the age of euphoria was over. instead of receding to a vanishing point, he declared, communicable diseases 'remain the major cause of death worldwide and will not be conquered during our lifetimes . . . we can also be confident that new diseases will emerge, although it is impossible to predict their individual emergence in time and place' (24) . indeed, the contest between humans and microbes was a darwinian contest with the advantage tilted toward the microbes. the stark message of the iom was that, far from being secure from danger, the united states and the west were at greater risk from contagious and epidemic diseases than at any time in history. an important reason for this new vulnerability was the legacy of eradicationism itself. the belief that the time had come to close the books on infectious diseases had produced a pervasive climate that critics labeled variously as 'complacency,' 'optimism,' 'overconfidence,' and 'arrogance.' the conviction that victory was imminent had led the industrial world to premature and unilateral disarmament. assured by a consensus of the leading medical authorities for 50 years that the danger was past, federal and state governments in the united states dismantled their public health programs dealing with communicable diseases and slashed their spending. at the same time, investment by private industry on the development of new vaccines and classes of antibiotics dried up, the training of health care workers failed to keep abreast of new knowledge, vaccine development and manufacture were concentrated in fewer laboratories, and the discipline of infectious diseases struggled to attract its aliquot share of research funds and of the best minds. at the nadir in 1992, the united states spent only $74 million for infectious disease surveillance as public health officials prioritized other concerns -chronic diseases, substance abuse, tobacco use, geriatrics, and environmental issues. for these reasons, the assessment of american preparedness to face the challenges of the new era was disheartening. in the words of the cdc in 1994: the public health infrastructure of this country is poorly prepared for the emerging disease problems of a rapidly changing world. current systems that monitor infectious diseases domestically and internationally are inadequate to confront the present and future challenges of emerging infections. many foodborne and waterborne disease outbreaks go unrecognized or are detected late; the magnitude of the problem of antimicrobial drug resistance is unknown; and global surveillance is fragmentary. (25) more bluntly, michael osterholm, the minnesota state epidemiologist, informed congress in 1996 that, 'i am here to bring you the sobering and unfortunate news that our ability to detect and monitor infectious disease threats to health in this country is in serious jeopardy. . . . for twelve of the states or territories, there is no one who is responsible for food or water-borne disease surveillance. you could sink the titanic in their back yard and they would not know they had water' (29) . a striking example of the effects of complacency on infectious disease is the case of tb in new york city. tb had once been the leading cause of death in the city, but improvements in hygiene and education, followed by the discovery of streptomycin, led to the conviction by the middle of the 20th century that the disease was on the verge of being entirely conquered. as a result, funding was diverted, and demonstrably effective tb programs were dismantled although the social determinants of the disease worsened dramaticallyimmigration, crowding, homelessness, and rates of incarceration. meanwhile, hiv/aids continued to provide large numbers of patients with compromised immunity. as a result, the risk of infection increased, while access to health care became increasingly difficult, and the city experienced a remarkable and entirely preventable resurgence of the 'white plague,' primarily among african american and hispanic residents. between 1978 and 1992, the numbers of cases tripled, while drug resistance developed as a significant additional problem. new york city led the way in a national resurgence of tb as cases increased by 20% between 1985 and 1992. overweening confidence led directly and rapidly to a local epidemic and a partial reversal nationally of decades of tireless campaigning (29) . if the experience of the united states with tb suggests how fragile advances in health remained even in the industrial world, the situation in developing countries was still more disquieting. there, progress toward the germ-free eden during the eradicationist era was nil. in david satcher's uncompromising observation, 'persons living in tropical climates are still as vulnerable to infectious disease as their early ancestors were' (41) . the critique of 50 years of hubris went deeper than just a protest against a decline in vigilance. in addition, the theorists of emerging diseases argued that, unnoticed by the eradicationists, society since world war ii had changed in ways that actively promoted the emergence and reemergence of epidemic diseases. one of the leading features most commonly cited was the impact of globalization in the form of the rapid mass movement of goods and populations. as william mcneill noted in plagues and peoples (14) , the migration of people throughout history has been one of the most dynamic factors affecting the balance between microbes and man. humans are permanently engaged in a kind of war in which the social and ecological conditions that they create exert powerful evolutionary pressure on micro-parasites. by mixing gene pools and by providing access for microbes to populations of non-immunes living in conditions in which the microbes thrive, globalization gave microorganisms a powerful advantage. in the closing decades of the 20th century and the early years of the 21st, the speed and scale of this phenomenon amounted to a quantum leap, as 2.1 billion passengers boarded airplanes in 2006 (31, 42) . in the words of the popular press, the daily movement of people around the globe by airplane means that a disease breaking out today in kikwit can arrive in new york, mumbai, and mexico city tomorrow. the numbers of voluntary travelers, moreover, are massively supplemented by millions of involuntary refugees and displaced persons in flight from warfare, famine, and religious, ethnic, or political persecution. for lederberg and the iom, these rapid mass movements have tilted the advantage in favor of microbes, 'defining us as a very different species from what we were 100 years ago. we are enabled by a different set of technologies. but despite many potential defenses -vaccines, antibiotics, diagnostic tools -we are intrinsically more vulnerable than before, at least in terms of pandemic and communicable diseases' (43) . after globalization, the second factor most frequently underlined was demographic growth, especially because this growth occurred in circumstances that were the delight of microorganisms and of the insects that often transmit them. in the postwar era, population has soared above all in the poorest and most vulnerable regions of the world, with the global urban population growing at four times the rate of the rural. its hallmark has been wholesale, chaotic, and unplanned urbanization, led by the resource-poor nations of sub-saharan africa, which is the most rapidly urbanizing region on the planet (44) . the results have been escalating poverty, widening social inequality, the birth of 'megacities' exceeding 10 million inhabitants, and the spawning of teeming peri-urban slums without sanitary, educational, or other infrastructures. such places were ready-made for ancient diseases to expand, as cholera demonstrated in the shantytowns and barrios of cities like lima, mexico city, and rio de janeiro, where millions lived without sewers, drains, secure supplies of drinking water, or appropriate waste management. already in the 19th century, cholera had flourished in the conditions created in european cities by rapid and unplanned urbanization. in the final decades of the 20th century and the start of the 21st, a much larger process on a global scale reproduced in the cities of africa, asia, and latin america the anomalous sanitary conditions propitious for cholera (45) . another clear indication of socio-economic conditions in these new urban ecosystems is the appearance of trench fever (bartonella quintana) among the inhabitants of homeless shelters in north american cities. trench fever first emerged in the filth and crowding of soldiers in the trenches of the western front in the first world war, when millions of combatants were infected by the lice that covered their bodies. bartonella quintana, however, had never been documented apart from the vermin and the grime of wartime. the reemergence of the disease in urban america is therefore a clear measure of the insalubrious conditions of marginalized populations among the urban poor (46, 47) . here too in urban poverty were the social determinants that made possible the global pandemic of dengue fever that began in 1950 and has continued unabated until today, when 2.5 billion people are at risk every year and 50-100 million people are infected. dengue is the ideal type of an emerging disease. an arborovirus transmitted primarily by the highly urban, daybiting, and domestic aedes aegypti mosquito, dengue thrives in crowded tropical and semi-tropical slums whenever there is standing and unregulated water. it breeds abundantly in gutters, uncovered cisterns, unmounted tires, stagnant puddles, and plastic containers, and it takes full advantage of societal neglect and the absence or cessation of vector control programs. particularly important for the theorists of 'emerging diseases' was the manner in which dengue demonstrated the hollowness of the reassuring dogma that infectious diseases evolve inexorably toward commensalism and reduced virulence. the dengue virus is a complex of four closely related serotypes (den-1, den-2, den-3, and den-4) that have been known to infect humans since the 18th century. until 1950, however, dengue infections in any geographical area were caused by a single virus that gave rise to a painful illness marked by fever, rash, headache behind the eyes, vomiting, diarrhea, prostration, and joint pains so severe that the infection earned its nickname 'break-bone fever.' but 'classical' dengue was a self-limiting disease that was followed by lifelong immunity. the movement and mobility of populations, however, have allowed all four serotypes to spread indiscriminately around the globe, so that for the first time individuals who have already experienced infection with one dengue virus can subsequently be infected with one or more of the others, as there is no crossover immunity from one serotype to another. through mechanisms that are still imperfectly understood, the disease is much more severe in patients suffering re-infections with different serotypes. instead of becoming milder, therefore, dengue has become a growing threat, giving rise to far more frequent outbursts and to sudden, devastating epidemics in which large numbers of patients suffer the severe and often lethal complications of dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) that were once unknown. in the americas, the first modern epidemic of dengue fever broke out in 1983 in cuba, producing 344 000 cases, of whom 24 000 suffered dhf and 10 000 dss (48) . moreover, since the dengue vectors a. aegypti and aedes albopictus are present in the united states, scientists at the national institute of allergy and infectious diseases (niaid), such as its director anthony fauci, have noted that dengue fever has broken out in both hawaii and puerto rico, and that they see no inherent reason it could not include the continental united states in its ongoing global expansion (49) . dengue therefore demonstrates the following important evolutionary lessons: (i) infectious diseases that do not depend on the mobility of their host for transmission (because they are vector-borne, waterborne, or foodborne) are not under selective pressure to become less virulent; (ii) overpopulated and unplanned urban or peri-urban slums provide ideal habitats for microbes and their arthropod vectors; and (iii) modern transportation and the movements of tourists, migrants, refugees, and pilgrims facilitate the process by which microbes and vectors gain access to these ecological niches. paradoxically, the very successes of modern medical science also prepared the way for the emergence of new infections. by prolonging life, medicine gives rise to ever larger numbers of elderly people with compromised immune systems. as part of this process, significant numbers of immunocompromised populations have appeared at earlier ages as well-diabetics, cancer and transplant patients undergoing chemotherapy, and aids patients whose lives have been radically extended by antiretroviral treatment. furthermore, such people are frequently concentrated in settings where the transmission of microbes from body to body is amplified, such as hospitals, facilities for the elderly, and prisons. the proliferation of invasive procedures has also increased the opportunities for such diseases. modern nosocomial infections emerged in these conditions, and have become a major problem of public health as well as an ever growing economic burden. of these infections, the so-called 'superbug' staphylococcus aureus -the leading cause of nosocomial pneumonia, of surgical site infections, and of nosocomial bloodstream infections -is the most important and widespread. a recent study notes that in the united states by 2008: each year approximately two million hospitalizations result in nosocomial infections. in a study of critically ill patients in a large teaching hospital, illness attributable to nosocomial bacteria increased intensive care unit stay by 8 days, hospital stay by 14 days, and the death rate by 35%. an earlier study found that postoperative wound infections increased hospital stay an average of 7.4 days. (50) a further threatening byproduct of the advance of medical science is the development of ever increasing antimicrobial resistance. already in his 1945 nobel prize acceptance speech, alexander fleming, who discovered penicillin, the first antibiotic, issued a prophetic warning. penicillin, he advised, needed to be administered with care, because the bacteria susceptible to it were likely to develop resistance. the selective pressure of so powerful a medicine would make it inevitable. echoing fleming's warning, the emerging diseases theorists argue that antibiotics are a 'non-renewable resource' whose duration of benefit is biologically limited. by the late 20th century, this prediction was reaching fulfillment. on the one hand, the discovery of new classes of antimicrobials had slowed to a trickle, especially in a market in which profit margins are compressed by competition, by regulations requiring large and expensive clinical trials before approval, and by the low tolerance for risk on the part of regulatory agencies charged with the safety of the public. on the other hand, while antiinfective development stagnates, many microorganisms have evolved extensive resistance. as a result, in one telling metaphor, physicians are rapidly emptying their quiver, and the world stands poised to enter the postantibiotic era (51) . some of the most troubling examples of the emergence of resistant microbial strains are the emergence of plasmodia that are resistant to all synthetic antimalarials, of s. aureus that is resistant both to penicillin and to methycillin (mrsa), and of strains of mycobacterium tuberculosis that are resistant not only to first-line medications (mdr-tb) but to second-line medications as well (xdr-tb) (52) . antimicrobial resistance has become a global crisis, and many anticipate the early appearance of strains of hiv, tb, staph a, and malaria that are not susceptible to any available therapy. in part the problem of antimicrobial resistance is a simple result of darwinian evolution. as a rand corporation study (53) notes, there are tens of thousands of viruses and 300 000 species of bacteria that are capable of infecting human beings, and many of them replicate and evolve billions of times in the course of a single human generation. evolutionary pressures, in this context, work to the long-term disadvantage of human beings. but unwise human actions have dramatically hastened the process. farmers spray crops with pesticides and fruit trees with antibiotics, and they add subtherapeutic doses of antibiotics such as virginiamycin and avoparcin wholesale to animal feed to prevent disease, promote growth, and increase the productivity of chickens, pigs, and feedlot cattle. indeed, half the world output of antimicrobials by tonnage is used in agriculture (54) . at the same time, the popular confidence that microorganisms will succumb to a chemical barrage has led to a profusion of antimicrobials in domestic settings where they serve no purpose (55) . physicians, pressured to give priority in clinical settings to the immediate risk of individual patients over the long-term interest of the species and to meet patients' expectations, have succumbed to profligate prescribing fashions, administering antibiotics even for non-bacterial conditions for which they are unnecessary or entirely useless. the classic case in this regard is the pediatric treatment of otitis media (or middle ear infection), for which the overwhelming majority of practitioners in the 1990s prescribed antibiotics, even though two-thirds of the children derived no benefit from the medication. widespread possibilities of self-medication in countries with few regulations or through opportunities created by the internet amplify the difficulties. in the case of diseases such as malaria and tb that require a long and complicated therapeutic regimen, there is also the issue of patients who interrupt their treatment after the alleviation of their symptoms instead of persevering until their condition is cured. here the problem is not the overuse but the underuse of antibiotics. sometimes described as simple non-compliance by patients, the issue in fact raises complex questions of education, poverty, and lack of access to health care. here the who strategies of dots (directly observed treatment short course) and dots-plus are helpful but cannot solve the underlying problems. a further issue raised by the new era was the overly rigid conceptualization of disease by the eradicationists, who drew too sharp a distinction between chronic and contagious diseases. infectious diseases, it became clear during the 1990s, are a more expansive category than scientists previously realized because many diseases long considered noninfectious in fact have infectious origins. in demonstrating these causal connections, the decisive work was that of the australian nobel laureates barry j. marshall and robin warren with regard to peptic ulcers in the 1980s. peptic ulcers are a significant cause of suffering, cost, and even death, as one american in 10 develops one during the course of a life time, over one million people are hospitalized by them every year, and 6000 die. marshall noted in his acceptance speech for the nobel prize in 2005, however, that the chronic etiology of peptic ulcer in the 1980s was universally accepted as scientific truth. in his words, 'i realized that the medical understanding of ulcer disease was akin to a religion. no amount of logical reasoning could budge what people knew in their hearts to be true. ulcers were caused by stress, bad diet, smoking, alcohol and susceptible genes. a bacterial cause was preposterous.' what marshall and warren were able to demonstrate, therefore, was a medical watershed. they proved, in part by means of an auto-experiment, that the bacterium helicobacter pylori was the infectious cause of the disease and that antibiotics rather than diet, lifestyle change, and surgery were the appropriate therapy (56) . this insight led to the realization that many other non-acute diseases, such as certain forms of cancer, chronic liver disease, and neurological disorders, are due to infections. human papillomavirus, for instance, is thought to give rise to cervical cancer, hepatitis b and c viruses to chronic liver disease, campylobacter jejuni to guillain-barré syndrome, and certain strains of escherichia coli to renal disease (57, 58) . there are indications as well that infections serve as an important trigger to atherosclerosis and arthritis, and there is a growing recognition that epidemics and the fear that accompanies them leave psychological sequelae in their wake, including posttraumatic stress (59, 60) . this understanding of these processes is what some have termed finally, and most emphatically, the concept of emerging and reemerging diseases was intended to raise the most important threat of all -that the spectrum of diseases that humans confront is broadening with unprecedented and unpredictable rapidity. the number of previously unknown conditions that have emerged to afflict humanity since 1970 exceeds 40, with a new disease discovered on average more than once a year. the list includes such frightening names as hiv, hantavirus, lassa fever, marburg fever, legionnaires' disease, hepatitis c, lyme disease, rift valley fever, ebola hemorrhagic fever, nipah virus, west nile virus, sars (severe acute respiratory syndrome), bovine spongiform encephalopathy, avian flu h5n1, chikungunya virus, and group a streptococcus -the so-called 'flesh-eating bacterium.' skeptics argue that simply to list the diseases that have emerged since 1970s gives the misleading impression that diseases are emerging at an accelerating rate. this impression, they suggest, is largely an artifact of heightened surveillance and improved diagnostic techniques rather than a new development. the who has countered that not only have diseases emerged at record rapidity as one would expect from the transformed social and economic conditions of the postwar world, but also that they gave rise between the years 2002 and 2007 to a record 1100 worldwide epidemic events (31) . the most recent and comprehensive examination of the question (62), published in february 2008 in nature, involved the study of 335 emerging infectious disease (eid) 'events' between 1940 and 2004, controlling for reporting effort through more efficient diagnostic methods and more thorough surveillance. the conclusion was that, 'the incidence of eid events has increased since 1940, reaching a maximum in the 1980s. . . . controlling for reporting effort, the number of eid events still shows a highly significant relationship with time. this provides the first analytical support for previous suggestions that the threat of eids to global health is increasing' (62) . there are no rational grounds, the public health community concluded, to fail to expect that as diseases emerge in the future, some of them will be as virulent and as transmissible as hiv or the spanish influenza of 1918/1919. discussion has therefore shifted dramatically from the question of whether new diseases will emerge and old ones resurge to the issue of how the international community can best prepare to face them. in the stark words of the us department of defense, 'historians in the next millennium may find that the twentieth century's greatest fallacy was the belief that infectious diseases were nearing elimination. the resultant complacency has actually increased the threat' (63) . a major aspect of the official response to the challenge of emerging and reemerging diseases is that microbes now are regarded as threats to the security of states and to the stability of the international order. for the first time, therefore, not only public health authorities but also intelligence agencies and conservative think tanks have classified infectious diseases as a 'non-traditional threat' to national and global security. they assumed therefore the task of envisaging the future and the challenge that communicable diseases would play. here a turning point was the central intelligence agency (cia)'s national intelligence estimate (nie) for 2000 (64) , which was devoted to the danger posed by disease and presented defense against epidemic diseases as a major security goal for the united states. as a document, nie 99-17d (64) was divided into four major sections: alternative scenarios, impact, implications, and discussion. in the first section, the cia attempted to outline three possible scenarios for the course of infectious diseases over the next 20 years: (i) the optimistic contemplation of steady progress in combating communicable disease; to (ii) the forecast of a stalemate with no decisive gains either by microbes or by humans in their long war of attrition; and (iii) the consideration of the most pessimistic prospect of deterioration in the position of humans, especially if the world population continues, as seems probable, to expand and if megacities continue to spring up with their attendant problems of crowding, sanitation, and unprotected drinking water. unfortunately, the cia regarded the optimistic first case as extremely unlikely. the probable course of events, in its view, is that 170 000 americans will die from infectious diseases every year or considerably more if a pandemic of influenza or of a still unknown disease occurs, if there is a dramatic decline in the effectiveness of antiretroviral treatments for hiv/aids. only toward the end of the 20 years did the report foresee possible advances due to enhanced public health initiatives, the development of new drugs and vaccines, and economic development (64) . against this background, the succeeding sections on 'impact' and 'implications' outlined a series of likely economic, social, and political results that would occur in the new age of increasing disease burdens. in the most afflicted regions of the world, such as sub-saharan africa, the report anticipated 'economic decay, social fragmentation, and political destabilization.' the international consequences of these developments would be growing struggles to control increasingly scarce resources, accompanied by crime, displacement, and the degradation of familial ties. disease, therefore, would heighten international tensions while it weakened forces, such as international peacekeepers, who might otherwise have played a larger role in controlling regional tensions. us or european military forces deployed abroad in support of humanitarian or other operations would be at high risk. because the economic and social consequences of increasing burdens of communicable diseases in the developing world are certain to impede economic development, the nie also predicted that democracy would be imperiled, that civil conflicts and emergencies would multiply, and that the tensions between north and south would deepen. three years later, motivated by the cia's report, an influential national security think tank, the rand corporation, turned to the intersection of disease and security when it attempted to provide 'a more comprehensive analysis than has been done to date, encompassing both disease and security' (53) . in so doing, it envisaged even more somber probabilities than the cia in the new global environment. the rand corporation intelligence report the global threat of new and reemerging infectious diseases: reconciling u.s. national security and public health policy (53) had two leading themes. the first was that in the postwar era there was a sharp decline in the importance of direct military threats to security. the second was that there is a corresponding rise in the impact of 'non-traditional challenges,' of which diseases are the major but inadequately recognized component. it has always been accepted, the report stressed, that diseases kill and undermine the quality of individual lives. in addition, it was essential to recognize that the transition to the era of emerging and reemerging diseases marked the opening of a period in which infectious diseases would profoundly affect the ability of states to function and to preserve social order. the most striking portion of the global threat of new and reemerging infectious diseases (53) was its imagining of a probable scenario in which south africa could become the first modern state to fail specifically because of infectious diseases in general and the hiv/aids pandemic in particular. as the report explained, 'the contemporary hiv/aids crisis in south africa represents an acute example of how infectious diseases can undermine national resilience and regional stability.' in absolute numbers, south africa has the highest number of hiv-positive inhabitants in africa -4.7 million people in 2000, or 25% of the country's adult population. already, such extreme prevalence of the disease has pervasive impacts, affecting all aspects of south african security. but south africa is just emerging from the first phase of the aids pandemic and is therefore far from experiencing the full effects of the crisis, which even in the absence of resistance to antiretroviral therapy, is expected to produce 6 200 000 patients with hiv and 800 000 with full-blown aids by 2010. in these circumstances, over a quarter of the economically active population will have the disease, causing severe skill shortages, creating poverty, destroying economic development, undermining participation in political life, and giving rise to more than two million orphans who will be impoverished, uneducated, and easily drawn into crime and prostitution. the effects will also be deeply felt in the military, the police, and the legal system, which will be severely deprived of manpower and unable to function just as social tensions deepened. 'the net effect,' it concluded, 'will be entirely negative for south africa's civil stability, possibly reducing the country to widespread social anarchy within the next five to twenty years.' this disturbing outcome, moreover, could be hastened by the public health policies of president thabo mbeki, who espoused the theories of the aids denier peter duesberg and rejected the link between the hiv virus and the disease. the point the rand corporation stressed most about south africa, however, was that it was simply a dramatic illustrative example. what was occurring there as a result of hiv/aids could happen without warning elsewhere. 'a crisis of similar proportions,' it explained, 'could therefore break out in any country at any time.' indeed, in the context of a growing danger of bioterrorist attack, such an outbreak could be launched intentionally. it was precisely this point -the growing vulnerability of all in the age of globalization -that led the world community, the european union, and individual nations to rearm in preparation for the inevitable threats to come. in the new climate of preparedness, the united states took a prominent role, beginning almost immediately in the aftermath of the 1992 iom report. in 1994 the cdc -the chief monitoring agency -drafted a strategic plan that it then updated in 1998, while niaid -the principal basic research center -established a research agenda. both agencies' plans were endorsed by the white house, where the nstc under the chairmanship of vice president al gore issued a 'fact sheet: addressing the threat of emerging infectious diseases,' which in turn was backed by a presidential decision directive of june 12, 1966 . the result, as gore explained, was the first national policy by the united states to confront the international problem of infectious diseases (65) . the essential starting point of the plan envisaged by the cdc, niaid, and the white house was the iom's description of the darwinian struggle under way between humans and microbes. in the iom's analysis of that struggle, microbes possess formidable advantages. they outnumber human beings a billionfold, they enjoy enormous mutability, and they replicate, in lederberg's estimate, a billion times more quickly than man, with generations measured in minutes rather decades. in terms of natural evolutionary adaptation, therefore, microbes are genetically favored to win the contest. in lederberg's observation, 'pitted against microbial genes, we have mainly our wits' (66) . taking this iom analysis as its starting point, the american response to the new challenge is best seen as the attempt to organize and deploy human wit, backed by newly found financial resources, to counter the microbial genetic challenge (25) . the white house 'fact sheet' declared in clear alarm that, 'the national and international system of infectious disease surveillance, prevention, and response is inadequate to protect the health of u.s. citizens.' to remedy the situation, the white house established six policy goals, as follows: 1. strengthen the domestic infectious disease surveillance and response system, both at the federal, state, and local levels and at ports of entry into the united states, in cooperation with the private sector and with public health and medical communities. 2. establish a global infectious disease surveillance and response system, based on regional hubs and linked by modern communications. 3. strengthen research activities to improve diagnostics, treatment, and prevention, and to improve the understanding of the biology of infectious disease agents. 4. ensure the availability of the drugs, vaccines, and diagnostic tests needed to combat infectious diseases and infectious disease emergencies through public and private sector cooperation. 5. expand missions and establish the authority of relevant us government agencies to contribute to a worldwide infectious disease surveillance, prevention, and response network. 6. promote public awareness of eids through cooperation with non-governmental organizations and the private sector (65) . in pursuit of goals 2, 3, and 4, nih funding was doubled between 1998 and 2003. niaid established a research agenda to develop new weapons to combat epidemic diseases, giving rise to an explosion in knowledge while publications on infectious diseases burgeoned. indeed, the agency director, anthony s. fauci, claimed in 2008 that hiv/aids in particular has become the most extensively studied disease in human history. niaid's priority is the development of safe and effective vaccines and medications to combat hiv/aids, malaria, tb, and influenza. to that end, it has evaluated over 50 hiv vaccine candidates, funded 70 clinical trials, and developed 20 antiretroviral medications. in the field of malariology, it has completed the genomic sequencing of plasmodium falciparum and of the feared malaria vector anopheles gambiae with the expectation that this genetic knowledge is the first step toward the capacity to design anti-malarial drugs, vaccines, and pesticides. the work of the federal agency, moreover, has been complemented by the work of private organizations such as the bill and melinda gates foundation, and university laboratories (67) . at the same time that niaid stressed basic research, the cdc developed a defensive strategy against emerging pathogens in compliance with goal 1 of the president's directive. the cdc articulated its plan in two seminal works published in 1994 and 1998. there it articulated its objectives in four principal areas: surveillance; applied research; prevention and control; and the enhancement of the infrastructure and trained personnel needed for diagnostic laboratories at the federal, state, local, and international levels. in addition, the atlanta-based agency strengthened its links with the international public health community and with other surveillance agencies such as the fda and the department of defense. it enhanced its capacity to respond to outbreaks, and it launched the journal emerging infectious diseases as a forum to pool information on communicable diseases. it sponsored a series of major international conferences on the topic of emerging and reemerging diseases, beginning in 1998 with the participation of representatives from all 50 states and 70 countries. the cdc initiatives were widely regarded as a model for the establishment of surveillance and response capabilities in other countries as well (25, 68) . at the global level, the un and its agency who also took major steps to strengthen international preparedness for the ongoing siege by microbial pathogens. a first step was the creation in 1996 of the disease-specific organization unaids with the function of raising awareness, mobilizing resources, and monitoring the pandemic. funding levels in the fight against the disease increased from $300 million in 1996 to nearly $9 billion a decade later (69). a further step was that like the united states, the united nations announced that it regarded infectious diseases as threats to international security. in acknowledgement of this new development, the security council took the unprecedented step in june 2001 of devoting a special session to the hiv/aids crisis. the session adopted a 'declaration of commitment on hiv/aids: global crisis -global action.' the declaration declared the global epidemic a 'global emergency and one of the most formidable challenges to human life and dignity' (70) . five years later, in june 2006, the general assembly reaffirmed its commitment to the campaign, and adopted the '2006 political declaration on hiv/aids,' whose chief goal was the establishment of national campaigns to improve access to care and treatment (69). a third step was the establishment of a new set of international sanitary regulations -ihr (2005) -to replace the outdated ihr (1969). whereas the old framework was disease-specific and required notification only in the event of plague, yellow fever, and cholera, the new rules required notification for any 'public health emergency of international concern,' thereby including unknown pathogens and emerging infections. the regulations specified the nature of the 'events' that should trigger international concern. they also committed all of the 193 who member states to improve their capacity for surveillance and response and to designate 'national ihr focal points' as the units responsible for providing notification while requiring, in exchange, that the who provide assistance to member states in fulfilling their obligations (71, 72) . in addition, recognizing that microbes do not acknowledge political frontiers, ihr (2005) called for effective responses wherever necessary to contain an outbreak on the basis of realtime epidemiological evidence instead of concentrating on taking defensive measures at international borders. finally, the who organized a rapid response capacity with the necessary supporting infrastructure. this was the global outbreak alert and response network (goarn), which was established in 2000 with the goal of ensuring that even most resource-poor countries would have access to the experts and resources needed to respond to an epidemic emergency. to that end, goarn pooled the resources of 60 countries and organized 500 experts in the field. in addition, it stockpiles vaccines and drugs, and supervises their distribution during epidemic events. between its founding and 2005, goarn responded to 70 outbreaks and attempted to learn from experience by establishing protocols to standardize such matters as field logistics, security, communication, and the deployment of field teams (73). in addition to goarn, the who set up surveillance systems specifically designed to deal with pandemic influenza, which the un agency determined as its most feared security threat. these disease-specific networks are (i) the global influenza surveillance network, which provides recommendations twice a year on the appropriate vaccine for the subsequent influenza season by collecting samples from patients in 94 countries and forwarding them to who collaborating laboratories for analysis, and (ii) flunet, which compiles the surveillance data thus collected to establish a global real-time early-alert system for the disease (74, 75) . in practice, the first test of the effectiveness of the new structures was the sars pandemic of 2002/2003 -the first major emerging disease threat of the 21st century. after first appearing in the chinese province of guangdong in november 2002, it erupted as an international health threat in march 2003, when the who received notification and declared a global travel alert. between march and the declaration on july 5 that the disease had been contained, sars affected 8098 people, caused 774 deaths, brought international travel to a halt in entire regions, and cost $60 billion in gross expenditure and business losses to asian countries alone. as retrospective studies have demonstrated, sars presented many of the features that most severely expose the vulnerabilities of the global system: sars is a respiratory disease capable of spreading from person to person without a vector; it has an asymptomatic incubation period of more than a week; it generates symptoms that closely resemble those of other diseases; it takes a heavy toll on caregivers and hospital staff; it readily spreads unobserved aboard aircraft; and it has a case fatality rate of 10%. at the time this new disease appeared, moreover, its causative pathogen (sars-associated coronavirus) was unknown, and there was neither a diagnostic test nor a specific treatment. for all of these reasons, it dramatically confirmed the iom's 1992 prediction that all countries were more vulnerable than ever to eids. sars demonstrated no predilection for any region of the globe and was no respecter of prosperity, education, technology, or access to health care. indeed, after its outbreak in china, sars spread by airplane primarily to affluent cities such as singapore, hong kong, and toronto, where it struck relatively prosperous travelers and their contacts, hospital workers, patients, and hospital visitors, rather than targeting the poor and the marginalized. more than half of the recognized cases occurred in well-equipped and technologically advanced hospital settings such as the prince of wales hospital in hong kong, the scarborough hospital in toronto, and the tan tock seng hospital in singapore (31, 76, 77) . in terms of response to the crisis, the sars outbreak demonstrated and vindicated the reforms taken on both the national and international levels. after the debacle of chinese obfuscation at the start of the epidemic, national governments cooperated fully with ihr (2005). the world's most equipped laboratories and foremost epidemiologists, working in realtime collaboration via the internet, succeeded, with unprecedented speed, in identifying sars-cov in just 2 weeks. at the same time the newly created goarn, together with such national partners as the canadian public health intelligence network, the cdc, and the who global influenza network, took rapid action to issue global alerts, monitor the progress of the disease, and supervise containment strategies before the disease could establish itself endemically. ironically, given the high-tech quality of the diagnostic and monitoring effort, the containment policies were based on traditional methods dating from the public health strategies against bubonic plague by the 17th century and the foundation of epidemiology as a discipline in the 19th. these measures were case tracking, isolation, quarantine, the cancellation of mass gatherings, the surveillance of travelers, recommendations to increase personal hygiene, and barrier protection by means of masks, gowns, gloves, and eye protection (78) . although sars affected 27 countries and every continent, the containment operation coordinated by goarn successfully limited the outbreak overwhelmingly to hospital settings with only sporadic community involvement, so that by july 5 the who could announce that the pandemic was over. although sars tested the newly established global defenses against emerging diseases and the protective ramparts withstood the challenge, doubts relentlessly surfaced. the chinese policy of concealment between november 2002 and march 2003 had placed international health in jeopardy and revealed that even a single weak link in the response network could undermine the ihr (2005) system. indeed, resourcepoor countries that were compliant with the new framework of obligations nonetheless found it difficult or impossible to maintain the surveillance effort for the full 4-month duration of the emergency. still more tellingly, it was also clear that a major factor in the containment of sars was simple good fortune. the world was lucky that sars is spread by droplets and therefore requires extended contact for transmission, unlike classic airborne diseases such as influenza and smallpox. it was, relatively, much easier to contain, because except in the infrequent and still poorly understood case of so-called 'super shedders,' it is not readily communicable from person to person. as poorly transmissible as it was, however, sars exposed the absence of 'surge capacity' in the hospitals and health care systems of the prosperous and well-resourced countries it affected. the events of 2003 thereby raised the specter of what might have happened had sars been pandemic influenza, and if it had traveled to resource-poor nations at the outset instead of mercifully visiting cities with well-equipped and well-staffed modern hospitals and public health care systems. furthermore, sars arrived in peacetime rather than in the midst of the devastation and the dislocations of war. in that respect, too, it did not repeat the challenge of the spanish lady of 1918-1919. the physician paul caulford, who fought the sars epidemic in the front lines at scarborough hospital in toronto, raised these matters. in december 2003, after the passing of the emergency, he reflected: sars must change us, the way we treat our planet, and how we deliver health care, forever. will we be ready when it returns? sars brought one of the finest publiclyfunded health systems in the world to its knees in a matter of weeks. it has unnerved me to contemplate what the disease might do to a community without our resources and technologies. without substantive changes to the way we manage the delivery of health care, both locally and on a worldwide scale, we risk the otherwise preventable annihilation of millions of people, either by this virus, or the next. (79) at the end of the victory over sars, the nagging question therefore remains: even after the impressive efforts at rearmament since 1992, how prepared is the international community for upcoming emerging diseases? have we been forever changed? the reforms introduced since the iom report in 1992 have been profound and important. indeed, the manner in which the international community responded to sars was innovative and, in the circumstances, highly successful. there is, however, a disconcerting sense of a systematic blindness in the responses -at all levels -to the crisis described by the iom, the cia, the rand corporation, the who, and the white house. what has been done has been necessary but probably far from sufficient. some of the issues raised by those who sounded the alarm have been forcefully addressed, but others have been largely ignored. the responses to date have fit into two chief categories, both of which are essential and both of which were evident during the sars pandemic. the first is reactive: the ability to respond rapidly and effectively to the outbreak of new epidemic threats. through a series of initiatives, the years since 1992 have witnessed the establishment of organized networks for gathering public health intelligence, of an international legal framework to structure emergency interventions, and of well equipped response teams of experts to contain and monitor outbreaks. if one were to compare outbreaks of infectious diseases to forest fires, the world has provided itself with surveillance satellites, advanced communications infrastructures, and a well-equipped fire department. one could question details of the response to sars, such as implementation lapses that risked the spread of the disease from the hospital environment into the community, but overall the world's 'dress rehearsal' demonstrated far-sighted planning and coordination beyond anything ever attempted before on an international scale. the second category of initiatives is proactive and scientific: the attempt to discover new weapons to attack microbial threats. after half a century of dwindling resources for the fight against infectious diseases, the scientific and public health communities have successfully aroused worldwide awareness of the threat to health and security. they have, at least initially, attracted new levels of funding for basic research from both public and private sources, and they have set research agendas. the result has been an explosion of knowledge, grants, and publications with priority given to genomic approaches to microbes and vectors, to the development of vaccines, and to the search for new medications and diagnostic tools. naturally there are grounds for criticism of various aspects of these initiatives. there is, for example, general agreement that overall levels of funding remain inadequate to the extent of the crisis and that after initial enthusiasm, governments have not continued to increase their support. there are also reasonable grounds for disagreement as to the relative distribution of research efforts, with discussion, for example, about the balance struck between research against hiv/aids and that against such other major diseases as malaria, tb, and pandemic influenza. some have also questioned whether developing vaccines is the right paradigm on all fronts. for example, should priority be given to those diseases for which the human immune response gives grounds for optimism thaton the basis of historical experience -a safe and effective vaccine can be developed (e.g. influenza and dengue)? or should other strategies be followed with respect to diseases for which the human immune response makes the development of a vaccine a far more arduous and unpredictable endeavor (e.g. cholera and malaria)? nevertheless, although there is no basis for false confidence, global research efforts have been galvanized, and major advances have been made in the field of infectious diseases in comparison with the early decades after world war ii. there is also a consensus that the effort to find vaccines and medicines is vital and that it must be enhanced in order to replenish the quivers of clinicians and public health officials. what is more troubling in principle is that there are also systematic blind spots -areas of danger raised by those who first sounded the tocsin regarding emerging diseases that have not been addressed at all or only marginally and sporadically. broadly speaking, the global community has chosen to address those issues for which scientific and technological responses are appropriate, while giving little sustained priority to what might be termed the social, economic, and environmental determinants of infectious disease. here there is a considerable irony. the founding figures of the modern concept of emerging and reemerging diseases such as joshua lederberg and robert shope stressed that epidemics do not strike societies randomly or in accord with the caprices of angry gods. diseases instead reflect the relationships that human beings establish with one another and with the natural and built environments. they then spread by taking advantages of the fault lines created by demography, poverty, environmental degradation, warfare, mass transportation, and societal neglect. the very beginning of the iom's discussion of the new dangers was the recognition that our new vulnerability is not accidental but is the logical result of the type of society that we have become. in defining this vulnerability in a keynote speech in 1998, for instance, lederberg stated: to our disadvantage, we have crowding; we have social, political, economic, and hygienic stratification. we have crowded together a hotbed of opportunity for infectious agents to spread over a significant part of the population. this condensation, stratification, and mobility is unique, defining us as a very different species from what we were 100 years ago. (80) if our problem results from 'condensation, stratification, and mobility,' there is a disturbing silence in the government response. ironically, the various agencies -niaid, the cia, the department of defense -tasked by the presidential directive with augmenting american preparedness in the fight against infectious diseases neither mention socioeconomic factors nor elaborate a long-term strategy to address them. the call to action aroused the will to find new means to attack microbes and their vectors, and to contain disease outbreaks in human populations, but not to ameliorate the underlying conditions that have made modern societies vulnerable in the first place. three crucial examples illustrate the problem. the first is condensation or the press of overpopulation. clearly unrestrained demographic growth as the world population approaches seven billion strains all resources, degrades the environment, gives rise to the megacities and peri-urban slums where dengue, tb, and cholera thrive, drives populations to intrude into forests where they are exposed to new zoonotic infections, and overwhelms educational, housing, and hygienic infrastructures. here, the medical and public health communities agree, is a driving factor in the new human vulnerability to emerging diseases. the remedies, moreover, are already known, involving voluntary universal access for women to family planning education and technologies. one of the few forums even to raise the issue was the 'first international conference on women and infectious diseases' held in atlanta, february 27-28, 2004 , where it was noted that, 'women's health, in and of itself, rarely has been at the forefront of international development programs or national health planning and policies' (81) . in the field of infectious diseases, this lacuna is especially glaring because women are, as the conference stressed, more susceptible to infections than men, both for biological reasons and due to their caregiving roles and their relative burden of unemployment and poverty. women, moreover, suffer more serious complications from infectious diseases, above all during pregnancy. a second illustration is stratification, the burden of poverty and inequality. nearly all of the leading studies on emerging diseases regard poverty and its sequelae of poor diet, substandard housing, lack of education, and inadequate access to health care as one of the chief determinants of epidemic disease. poverty prevents people from taking measures to protect their own health, it undermines the immune system, it complicates access to safe water supplies, it leads to overcrowding in unhygienic housing, and it creates patterns of labor mobility and migration that compromise health. health care workers and clinicians recognize the link between inadequate resources and disease, with the result that many of the leading epidemic infections are widely termed 'diseases of poverty' (82) . the issue therefore surfaces in who campaigns to combat the three most important contemporary epidemics: hiv/aids, malaria, and tb. as the 2005 report addressing poverty in tb control stated: poverty is the greatest impediment to human and socioeconomic development. the united nations and its specialized agencies are focusing on poverty reduction as a leading priority. in the health sector, poverty represents a principal barrier to health and health care and, consequently, the world health organization has committed to integrate the promotion of pro-poor policies throughout its work. (83) the reduction of extreme poverty and hunger also form part of the un 'millennium development goals' to be achieved by 2015. except for exhortation and moral suasion, however, it is not clear that the who has developed specific plans to tackle the problem of poverty as a primary determinant of public health, and the promotion of greater equality is entirely ignored. more strikingly, neither issue forms part of the strategic public health thinking of the united states. american analyses recognize poverty as a factor creating an environment favorable for infectious diseases, but they avoid both poverty and inequality as matters of practical health policy. here is the antithesis of the strategic recommendation of the south african pediatrician nulda beyers, who commented: the western cape is in some ways a model of tb epidemiology . . .. tb is almost non-existent in the white population, but in the black and coloured populations, where unemployment is running at 60%, and malnutrition and crowded slum housing are the norm, tb deaths can reach 3000 per 100 000. if i had to put my money on only one option -science or social upliftthere is no doubt that social uplift would have the bigger impact. (84) poverty, moreover, reinforces both condensation and mobility. poverty creates a vicious downward spiral by interacting with population pressure because impoverished women are unable to practice effective family planning. the population explosion of the 21st century is based in the poorest regions of the planet. given a free and informed choice, privileged families in the industrial world limit their fertility. at the same time, however, poverty also augments vulnerability to infectious disease by setting in motion great streams of mobile people -the poor who become migrants, refugees, and displaced persons, and who then crowd into slums, mining compounds, refugee camps, and homeless shelters. these are people who are at disproportionately high risk of falling ill and of transporting their microbial burden with them. finally, there is the question of access to care. here the position of the leading figures in the campaign to recognize the importance of emerging and reemerging diseases is strangely contradictory. the iom examined the managed care revolution in the united states and the implications of for-profit medicine for the preparedness of the nation to face infectious diseases (85) . by 2000, managed care already enrolled 150 million americans and therefore dominated health care delivery. the performance of the managed care revolution, however, did not inspire the iom. on the contrary, it produced a list of the major problems that, in its view, managed care created for public health. this list was lengthy and devastating. according to the iom, managed care creates severe public health difficulties because it does the following: (i) it places such strict controls on reimbursements that it becomes an impediment to effective collaboration with the public health community; (ii) it lowers costs by fostering management of infectious diseases by nonspecialists; (iii) it promotes the shift from inpatient to outpatient treatment, where there are neither the specialists nor the infrastructure to diagnose or contain infectious diseases; (iv) it proliferates bureaucratic complexities that complicate prompt response to disease outbreaks; (v) it reduces the commitment to training and research; and (vi) it encourages excessive antibiotic use (85) . by leaving tens of million of people in the united states without insurance coverage and therefore without effective access to care, for-profit medicine effectively removes them from the disease surveillance network. to the extent that uninsured people avoid care entirely or seek it only at a late stage of their illness, the prompt information on which effective public health depends is undermined. in addition, excluding people from coverage drives them further into poverty and creates an underclass of the marginalized. finally, managed care relentlessly cuts costs by squeezing out of the system the surge capacity on which populations depend in the event of a disease outbreak. nevertheless, despite these observations, the iom reached perfectly anodyne conclusions. it did not conclude that only a system that guaranteed universal access is compatible with defense against infectious disease threats. instead, it lamely urged a deeper partnership between the managed care industry and public health officials. for these reasons, one can only conclude that we are not, in fact, forever changed. on the contrary, on both the national and international levels the response to the challenge of emerging disease threats remains partial with major gaps that are potentially costly in terms of human life and suffering. the united states and the world health community have established a sophisticated and necessary rapid response system. they have also proclaimed -and partially funded -a new commitment to basic research aimed at finding new antimicrobial weapons. they have not, however, systematically addressed the underlying causes for the new vulnerability. man's mastery of malaria textbook of malaria eradication dynamics of tropical disease: a selection of papers with biographical introduction and bibliography by the late george macdonald: chapters 16-19 the malaria program -from euphoria to anarchy the conquest of malaria: italy, 1900-1962 world eradication of infectious diseases the evolution and eradication of infectious diseases infectious diseases: their evolution and eradication natural history of infectious disease world health assembly resolution 32.30 of 1979, ''health for all international health regulations international health regulations harrison's principles of internal medicine, 7th edn plagues and peoples epidemiologic transition: changes of fertility and mortality with modernization a century of epidemiologic transition in the united states the epidemiologic transition theory. a preliminary update healthy people: the surgeon general's report on health promotion and disease prevention health, human rights, and the new war on the poor infections and inequalities: the modern plagues could a tuberculosis epidemic occur in london as it did in new york? the continued threat of tuberculosis emerging infections: microbial threats to health in the united states addressing emerging infectious disease threats: a prevention strategy for the united states. atlanta: us department of health and human services infectious disease -a global threat: report of the national science and technology council infectious diseases: a global approach to a global problem fact sheet: addressing the threat of emerging infectious diseases emerging infections: a significant threat to the nation's health how the cholera scare is waking latin america feeding on 19th century conditions, cholera spreads in latin america a 30-year respite ends: cases of plague reported in india's largest cities an introduction to ebola: the virus and the disease out of the jungle a monster comes. the daily telegraph quarantine at 30,000 feet: ebola virus as a ticking, airborne time bomb arthur richard preston discusses the deadly outbreak of the ebola virus in zaire public health systems and emerging infections: assessing the capabilities of the public and private sectors emerging infections: getting ahead of the curve transmission of infectious diseases during commercial air travel infectious diseases as an evolutionary paradigm malaria on the move: human population movement and malaria transmission naples in the time of cholera emergence of bartonella quintana infection among homeless persons trench fever identified in seattle's homeless. npr transcripts: ''all things considered dengue cases on the rise niaid experts see dengue as potential threat to u.s. public health. news release of the economic impact of staphylococcus aureus infection in new york city hospitals an emptying quiver: antimicrobial drugs and resistance world wide emergence of extensively drug-resistant tuberculosis the global threat of new and reemerging infectious diseases: reconciling u.s. national security and public health policy who. the medical impact of antimicrobial use in food animals antibacterial household products: cause for concern helicobacter connections chronic sequelae of foodborne disease emerging infectious determinants of chronic diseases potential infectious etiologies of atherosclerosis: a multifactorial perspective the global threat of new and reemerging infectious diseases: reconciling u.s. national security and public health policy the global infectious disease threat and its implications for the united states. nie 99-17d global trends in emerging infectious diseases addressing emerging infectious disease threats: a strategic plan for the department of defense the global infectious disease threat and its implications for the united states. nie 99-17d presidential decision directive ntsc-7 addressing the threat of emerging infectious diseases infectious disease -a threat to global health and security emerging infectious diseases: a 10-year perspective from the national institute of allergy and infectious diseases preventing emerging infectious diseases: a strategy for the 21st century. atlanta: us department of health and human services declaration of commitment on hiv/aids global public health security who. international health regulations flunet as a tool for global monitoring of influenza on the web global surveillance, national surveillance, and sars bell dmworld health organization working group on international and community transmission of sars. public health interventions and sars spread sars: aftermath of an outbreak infectious disease as an evolutionary paradigm steps for preventing infectious diseases in women targets now set by g8 countries to reduce ''diseases of poverty addressing poverty in tb control: options for national tb control programmes tuberculosis experts back social reform managed care systems and emerging infections: challenges and opportunities for strengthening surveillance, research and prevention key: cord-262076-b5u5hp2r authors: liu, ying poi; haasnoot, joost; ter brake, olivier; berkhout, ben; konstantinova, pavlina title: inhibition of hiv-1 by multiple sirnas expressed from a single microrna polycistron date: 2008-03-16 journal: nucleic acids res doi: 10.1093/nar/gkn109 sha: doc_id: 262076 cord_uid: b5u5hp2r rna interference (rnai) is a powerful approach to inhibit human immunodeficiency virus type 1 (hiv-1) replication. however, hiv-1 can escape from rnai-mediated antiviral therapy by selection of mutations in the targeted sequence. to prevent viral escape, multiple small interfering rnas (sirnas) against conserved viral sequences should be combined. ideally, these rna inhibitors should be expressed simultaneously from a single transgene transcript. in this study, we tested a multiplex microrna (mirna) expression strategy by inserting multiple effective anti-hiv sirna sequences in the mirna polycistron mir-17-92. individual anti-hiv mirnas that resemble the natural mirna structures were optimized by varying the sirna position in the hairpin stem to obtain maximal effectiveness against luciferase reporters and hiv-1. we show that an antiviral mirna construct can have a greater intrinsic inhibitory activity than a conventional short hairpin (shrna) construct. when combined in a polycistron setting, the silencing activity of an individual mirna is strongly boosted. we demonstrate that hiv-1 replication can be efficiently inhibited by simultaneous expression of four antiviral sirnas from the polycistronic mirna transcript. these combined results indicate that a multiplex mirna strategy may be a promising therapeutic approach to attack escape-prone viral pathogens. rnai is an evolutionary conserved and sequence-specific gene silencing mechanism in eukaryotes (1, 2) . rnai can be induced by double-stranded rna that is processed by the rnase iii-like enzyme dicer into 21-25 bp sirnas (3) (4) (5) . the sirna is incorporated into the rna-induced silencing complex (risc) in the cytoplasm and directs risc to degrade an mrna that is perfectly complementary to one (guide) strand of the sirna (5) . cellular mirnas are the natural inducers of rnai. most mirnas are synthesized as part of longer primary rna transcripts (pri-mirnas) (6) (7) (8) . the pri-mirnas are cleaved by the nuclear drosha-dgcr8 complex to produce mirna precursors (pre-mirnas) of $70 nt (9) (10) (11) (12) . pre-mirnas are transported by exportin-5 to the cytoplasm, where they are cleaved by dicer to produce the mirna duplex of $22 bp. the single-stranded mature mirna programs risc for mrna cleavage (perfect complementarity) or translational repression (incomplete complementarity) (13, 14) . several mirnas are encoded in genomic clusters that are transcribed as polycistronic pri-mirnas, allowing the production of multiple mirnas from a single transcription unit (15, 16) . rnai can be induced using rna polymerase iii promoterdriven shrna expression vectors that direct sirna expression. another sirna expression strategy uses a pre-mirna backbone that is transcribed by polymerase ii or iii (17) (18) (19) (20) . an optimized pre-mirna design includes the single-stranded flanks of the pri-mirna (21) (22) (23) . currently, rnai has been employed to inhibit the replication of a wide range of viruses including hiv-1, hepatitis c virus (hcv), hepatitis b virus (hbv), dengue virus, poliovirus, influenza virus a, coronavirus, herpesvirus and picornavirus (24, 25) . for hiv-1, potent inhibition has been reported with single shrna and mirna expression constructs (17, (26) (27) (28) . however, the therapeutic use of a single inhibitor is limited because of the rapid emergence of hiv-1 escape mutants (27, 29, 30) . minor sequence changes in the target sequence, sometimes even a single-point mutation, are sufficient to overcome rnaimediated inhibition (30) , thus demonstrating the exquisite sequence-specificity of rnai. strategies to reduce the chance of viral escape include the simultaneous use of multiple shrnas in a combinatorial rnai approach, which increases the genetic barrier for viral escape (31, 32) . similar strategies have previously been validated for antisense dna and ribozymes (33) (34) (35) . however, expression of these shrnas necessitates multiple expression cassettes and the construction of rather complex vectors that will not easily provide equimolar shrna expression levels. furthermore, when the same promoter is reiterated in a lentiviral vector, recombination occurs with high frequency on the repeated sequences (36) . alternative antiescape strategies include the use of a second-generation of sirnas that target-specific escape variants (37) , the use of tandem sirna transcripts (38) , long hairpin rnas (39) or the targeting of cellular co-factors that are critically involved in viral replication (40) (41) (42) (43) (44) . another attractive approach is to express multiple antiviral sirnas from a single polycistronic mirna transcript, such as a natural genomic mirna cluster that can be expressed from an rna polymerase ii promoter. this strategy is of particular interest for antiviral purposes because mirnalike transcripts were shown to be more effective antivirals than regular shrnas (17, 45) . furthermore, using an rna polymerase ii promoter will allow lower and regulated expression, thereby reducing the risk of toxicity due to oversaturation of the rnai machinery (46) . in this study, we designed a polycistronic transcript based on the mir-17-92 backbone to simultaneously express four anti-hiv sirnas. to generate this transcript, we first constructed individual anti-hiv mirnas that resemble the natural pri-mirna structures. these hairpins were optimized for viral inhibition by varying the sirna position in the hairpin stem. we show that the expression of individual mirnas is greatly enhanced in multiplex hairpin transcripts that are properly processed into functional mirnas. hiv-1 replication can be potently inhibited by simultaneous expression of four antiviral mirnas. these combined results indicate that the multiplex mirna strategy is a promising therapeutic approach against escape-prone viral pathogens. the wild-type mir-17-19b polycistron was amplified from genomic dna of 293t cells with primers oncof1 and oncor1 and ta-cloned in topo2.1 (invitrogen). the mir-17-19b polycistron sequence was verified and is identical to the sequence in the ncbi database (nt_009952.14). the topo2.1/mir-17-19b construct was used as a template to generate the antiviral mirna constructs. the construction consists of a four-step fusion pcr as shown in the supplementary figure 1 . briefly, the 5 0 -flank of the pri-mirna was amplified with a forward primer encoding a bamhi site and a reverse primer encoding the hiv-1 sequence at its 3 0 -end (step 1). similarly, the 3 0 -flank of the pri-mirna was amplified with a forward primer containing hiv-1 sequences and a reverse primer encoding bglii and xhoi sites (step 2). two complementary oligonucleotides, creating the stem-loop structure of the antiviral mirna, were annealed as described previously (step 3) (32) . the partial sequence similarity between the fragments generated in steps 1, 2 and 3 allowed their fusion by pcr with the outer forward f1 and reverse r2 primers (step 4), resulting in the antiviral mirna. supplementary table 1 lists all oligonucleotides used in this study. pcr amplification was performed in a 50 ml reaction containing 1 â pcr amplification buffer (invitrogen), 1.5-2 mm mgcl 2 (optimized for each reaction), 25 pmol of each primer, 0.2 mm dntps and 2.5 units of amplitaq dna polymerase (perkin elmer applied biosystem). the pcr program was as follows: 958c for 5 min, 35 cycles of 2 min at 948c, 1.5 min at 608c, 2.5 min at 728c and a final extension for 8 min at 728c. the pcr products were separated on a 1% agarose gel stained with ethidium bromide and compared to a standard dna size marker (eurogentec). mirna pcr products were excised from gel, purified with the qiaquick gel extraction kit (qiagen), digested with bamhi and xhoi and cloned in the corresponding sites of pcdna6.2-gw/emgfp-mir (invitrogen). all mirna constructs were sequenced with primers gfpseqf and mirr using the bigdye terminator v1.1 cycle sequencing kit (perkin elmer applied biosystem). multimerization of the individual pri-mirna units was performed by digestion of a single mirna hairpin construct with bamhi and xhoi and religation into the bglii/xhoi sites of pcdna6-mirna. by repeating this procedure we obtained constructs expressing different combinations of 1, 2, 3, 4 and 6 pri-mirnas. the rna structures formed by the transcripts were predicted with the mfold program (47) at http://frontend.bioinfo.rpi.edu/ applications/mfold/ and found to be similar to the predicted conformation of the wild-type pri-mirnas. the firefly luciferase (fl) reporters containing hiv-1 target sequences pol47 (luc-a pol47 ), pol1 (luc-b pol1 ), gag5 (luc-c gag5 ), r/t5 (luc-d r/t5 ), ldr9 (luc-e ldr9 ) and the anti-hiv shrnas have been described previously (32) . the full-length hiv-1 molecular clone lai (accession number af33819.3) (48) was used to produce wildtype virus and to study its inhibition by the antiviral mirnas and shrnas. human embryonic kidney (hek) 293t cells were grown as a monolayer in dmem (invitrogen) supplemented with 10% fetal calf serum (fcs) (hybond), minimal essential medium nonessential amino acids, penicillin (100 u/ml) and streptomycin (100 mg/ml) at 378c and 5% co 2 . cells were trypsinized one day before transfection, resuspended in dmem without antibiotics and seeded in 24-well plates at a density of 1.2 â 10 5 cells per well. cells were co-transfected with the indicated dna constructs using lipofectamine 2000 reagent (invitrogen) according to the manufacturer's instructions. one nanogram of prl plasmid (promega) expressing renilla luciferase (rl) from the cmv promoter was added as an internal control for cell viability and transfection efficiency. all transfection experiments were controlled for equal dna input by adding pbluescript sk-(promega). luciferase and renilla expression was measured with the dual-luciferase reporter assay system (promega) according to the manufacturer's instructions. virus production was determined by measuring the ca-p24 levels in the culture supernatant by elisa (abbott) (49) . subsequently, the cells were lysed to measure the renilla luciferase activities using the renilla luciferase assay system (promega) according to the manufacturer's protocol. transfection experiments were corrected for between session variations as described previously (50) . the human t-cell line supt1 was cultured in 25 cm 2 flasks in rpmi 1640 medium supplemented with 10% fcs, penicillin (100 u/ml) and streptomycin (100 mg/ml) at 378c and 5% co 2 . 200.000 supt1 cells were infected with equal amounts virus (0.5 ng ca-p24) produced in 293t cells. when hiv-induced cytopathic effects were observed, cell and supernatant samples were stored at à808c. virus spread was followed by measuring the ca-p24 levels in the culture supernatant by elisa. one day before transfection 7.5 â 10 5 293t cells were plated in 6-well plates. cells were transfected with 39-5000 ng of the shrna construct and 2500 or 5000 ng of the mirna-expression construct using lipofectamine 2000 reagent (invitrogen) according to the manufacturer's instructions. in all transfection experiments we added pbluescript sk-(promega) to obtain identical dna concentrations. total cellular rna was extracted 2 days post-transfection with the mirvana mirna isolation kit (ambion) according to the manufacturer's protocol. for northern blot analysis, 15 mg total rna was heated for 5 min at 958c before electrophoresis on a 15% denaturing polyacrylamide gel (precast novex tbu gel, invitrogen). to check for equal sample loading, the gel was stained with 2 mg/ml ethidium bromide in milliq water for 20 min. destaining was performed by rinsing the gel three times with milliq water for 10 min. the ribosomal rna (rrna) bands were visualized under uv light. the rna samples were electrotransferred to a positively charged nylon membrane (boehringer mannheim, gmbh, mannheim, germany) and crosslinked to the membrane using uv light at a wavelength of 254 nm (1200 mj â 100). lna oligonucleotide probes were 5 0 -end labeled with the kinasemax kit (ambion) in the presence of 1 ml [g-32 p]atp (0.37 mbq/ml amersham biosciences). to remove unincorporated nucleotides, the probes were purified on sephadex g-25 spin columns (amersham biosciences) according to the manufacturer's protocol. hybridizations were performed at 428c with labeled lna oligonucleotides in 10 ml ultrahyb hybridization buffer (ambion) according to the manufacturer's instructions. we used the oligonucleotide probes (lna-positions underlined): 5 0 -gtgaaggggcagtagtaat-3 0 (pol47 probe), 5 0 -acaggagcagatgatacag-3 0 (pol1 probe), 5 0 -gaagaaatgatgacagcat-3 0 (gag5 probe), 5 0 -atggcaggaagaagcggag-3 0 (r/ t5 probe) and 5 0 -agatgggtgcgagagcgtc-3 0 (ldr9 probe). the membranes were washed twice for 5 min at 428c in 2 â ssc/0.1% sds and twice for 15 min at 428c in 0.1 â ssc/0.1% sds. signals were detected and quantified using a phosphorimager (amersham biosciences). lentiviral vector plasmids are derived from the construct plenti6/v5-dest (invitrogen), which we renamed plv. the mirna cassettes, containing a gfp marker, were inserted into plv using the gateway-adapted block-it lentiviral pol ii mir rnai expression system (invitrogen) according to the manufacturer's instructions. the sequences of all mirna constructs were verified using the primers cmvf and v5r. the mirna inhibitory potential was determined by co-transfection with appropriate luciferase reporters (results not shown). lentiviral vector was produced in 293t cells (2.2 â 10 6 ) seeded in a 25 cm 2 flask. the next day, medium was replaced with 2.2-ml medium without antibiotics. subsequently, plv vectors expressing a mirna (2.4 mg) were co-transfected with packaging plasmids psyngp (1.5 mg) (51), rsv-rev (0.6 mg) and pvsvg (0.8 mg) (52) with 16 ml of lipofectamine 2000 reagent and 1.5 ml optimem (gibco brl). the second day, medium was replaced with fresh medium. on the third and fourth day, medium containing lentiviral vector was harvested and pooled. cellular debris was removed by filtration through a fp30/0.45 ca-s filter (schleicher and schuell microscience) and 1-ml aliquots were stored at à808c. lentiviral stocks were titrated on supt1 and 293t cells to determine the vector titer. supt1 and 293t cells (1 â 10 5 ) were transduced with the plv vector expressing an unrelated mirna n (invitrogen) or the antiviral mirnas a pol47 , b pol1 , c gag5 , d r/t5 , e ldr9 and acde at a multiplicity of infection (moi) of 0.15 as described previously (32) . the plv vector contains the blasticidin resistance gene, which allows the selection of transduced cells using 3.5 mg/ml blasticidin. the gfp marker encoded by the mirna cassette was used to select gfp+ cells by facs sorting approximately 10 days post-transduction. the human mir-17-92 cluster on chromosome 13 encodes a 1 kb pri-mirna polycistronic transcript with six pre-mirnas that produces seven mature mirnas ( figure 1a , upper panel) (15) . the pre-mir-17 hairpin encodes two mirnas, one on the 5 0 side of the hairpin (mir-17-5p) and one on the 3 0 side (mir-17-3p). we amplified the sequences encoding the first five pri-mirnas from the mir-17-92 polycistron and cloned it under the control of the cytomegalovirus (cmv) immediate early promoter ( figure 1a , lower panel). we subcloned each individual pre-mirna with at least 40-nt flanks on each side of the hairpin and systematically replaced the mature mirna sequences with antiviral sequences as explained in detail in the supplementary figure 1 . the original mirna names were replaced with letters a-e and we inserted 19-to 24-nt antiviral sequences against five hiv-1 genes ( figure 1b, upper panel) . the hiv-1 targets represent highly conserved sequences to which we successfully raised potent shrna inhibitors (32) . we set out to combine 2, 3, 4 or 5 antiviral mirnas, which will eventually result in an antiviral pri-mirna polycistron ( figure 1b , lower panel). we first determined if we could produce an optimal antiviral mirna construct by varying the position and length of the anti-hiv sequence. for the initial experiments we chose mirnas mir-20 and mir-19b (d wt and e wt ) because they contain the smallest number of bulges and thus most resemble the original shrna structure ( figure 2 ). the original mature mirna and the antiviral mirna strand are boxed. we modified the passenger strand of the basepaired stem to mimic structural features (mismatches, bulges and thermodynamic stability) of the natural pre-mirna. we constructed a series of antiviral mirnas with the effective r/t5 and ldr9 sirnas in the d wt backbone, which produces the antiviral guide strand from the 5 0 side of the hairpin duplex. the 19-nt antiviral sirnas were positioned either at the 5 0 (d r/t5-19 0 and d ldr9-19 0 ) or 3 0 -end (d r/t5-19 0 and d ldr9-19 0 ) of the original mirna sequence (figure 2 , upper panel). additional constructs were made in which we extended the antiviral sirnas at the 3 0 -end to 23-nt, which is the actual size of the wild-type mature mirna (d r/t5-23 and d ldr9-23 ) ( figure 2 , table 1 ). we repeated this strategy for the r/t5 inhibitor in the e wt hairpin, which produces the antiviral guide strand from the 3 0 side of the hairpin stem ( figure 2 , lower panel). we designed a similar set of constructs in which the 19-nt r/t5 sirna was positioned at the 5 0 (e r/t5-19 0 ) or 3 0 -end (e r/t5-19 0 ) of the wild-type mirna and an extended 23-nt version (e r/t5-23 ). to determine the inhibitory activity of the mirna constructs, we co-transfected 293t cells with the inhibitors and luciferase reporter constructs containing either the 50-nt r/t5 target (luc-d r/t5 and luc-e r/t5 ) or the 50-nt ldr9 target (luc-d ldr9 ). a plasmid encoding renilla luciferase (prl) was included to correct for transfection variation and to monitor for cell viability that may be affected by off-target effects of the modified mirnas and/or the antiviral sirnas. firefly and renilla luciferase expression was measured 48 h post-transfection. firefly luciferase expression was normalized to the control renilla luciferase expression. firefly luciferase expression in the presence of pbluescript (pbs) was set at 100% ( figure 3a) . a common pattern was observed in that most efficient inhibition was scored for the 19-and 23-nt sirna positioned at the 5 0 -end of the original mirna sequence, which are the 19 and 23 variants of the d hairpin and the 19 0 and 23 variants of the e hairpin. a similar trend was observed in hiv-1 inhibition studies ( figure 3b ). we co-transfected the hiv-1 molecular clone lai and the mirna constructs into 293t cells and virus production was measured as the ca-p24 level in the culture supernatant at 2 days post-transfection. we observed the strongest inhibition when the sirna inhibitor was situated at the 5 0 -end of the mirna sequence. the optimized hairpins have a similar efficiency of inhibiting hiv-1 production as the shrna constructs that were used as positive controls. based on these initial findings, we designed additional mirna constructs with 19-24 nt antiviral sequences at the 5 0 -end of the mirna: a pol47-24 , a pol47-19 , b pol1-22 , b pol1-19 , c gag5-23 , c gag5-19' , e ldr9-23 and e ldr9-19' (table 1) . of the original d r/t5 , d ldr9 and e r/t5 constructs, we selected d r/t5-19 because it is the best inhibitor. as a consequence, the ldr9 inhibitor was introduced in the e hairpin. we tested the effectiveness of all new constructs against luciferase reporters and hiv-1 ( table 1 ). the observed inhibition is sequence-specific because non-matching mirnas did not have any effect. furthermore, 19 19 a . figure 2 . structure of the antiviral mirnas based on pre-mirnas d wt and e wt . upper panel: the r/t5 and ldr9 sirnas were incorporated into the d wt backbone, which produces the guide strand from the 5 0 -side of the hairpin. the guide strand is marked in grey. we modified the passenger strand to mimic structural features (mismatches, bulges and thermodynamic stability) of the natural pre-mirna. the 19-nt antiviral sirnas were positioned either at the 5 0 (d r/t5-19 and d ldr9-19 ) or 3 0 -end (d r/t5-19' and d ldr9-19' ) of the original mirna and the length of the sirnas was extended at the 3 0 -end to 23-nt (d r/t5-23 and d ldr9-23 ). lower panel: the r/t5 sirna was similarly incorporated into the e wt pre-mirna, which produces the guide strand from the 3 0 -side of the hairpin. the structure of the original shrnas sh r/t5 and sh ldr9 are presented. hiv-1 sequences are blue, mature wild-type mirna sequences are red, pre-mirna sequences are black, watson-crick base pairs are shown with dashes and gu wobbles with dots. the expression of the control renilla reporter was stable in all experiments. the mirna constructs with 19-nt anti-hiv sequences showed a somewhat higher activity than the extended versions. we therefore selected the 19-nt inhibitors for the construction of antiviral mirna polycistrons. for simplicity, we removed the 19 indications from the mirna names (e.g. a pol47-19 becomes a pol47 ). the original shrna inhibitor sh r/t5 seems slightly more active than the optimized d r/t5 mirna molecule ( figure 3 ). this could be due to the use of different promoters (polymerase iii u6 versus polymerase ii cmv, respectively), but may also be due to differential rna processing efficiencies. we therefore wanted to compare the sirna level expressed from each construct. we titrated 39-2500 ng sh r/t5 construct in 293t cells and compared that with 2500 ng d r/t5 construct. two days post-transfection, total cellular rna was isolated from the transfected cells and analyzed on a northern blot with an r/t5 probe that detects the guide (antisense) strand ( figure 4 ). the original d wt hairpin was used as a negative control. quantification of the rna bands showed that expression of the antiviral 19-nt sirna by construct d r/t5 is about 15-30-fold lower than the expression by construct sh r/t5 (figure 4 , see numbers below the blot). although the sirna expression level of d r/t5 is significantly lower than that of sh r/t5 , the inhibitory effect measured in the luciferase and hiv-1 inhibition assays is comparable. this result suggests that the intrinsic inhibitory capacity of d r/t5 is in fact much greater than that of sh r/t5 . to address the effect of chaining of different hairpins for the silencing activity of the individual hairpins, we coupled the a pol47 inhibitor to wild-type pri-mirnas. we constructed two variants of a pol47 with two or six hairpins in a single transcript: a pol47 b wt and a pol47 a wt -e wt (a wt -e wt represents the complete wild-type mir-17-19b polycistron). we tested the ability of these hairpins to inhibit the luc-a pol47 reporter ( figure 5a , left). firefly luciferase expression was normalized to the renilla luciferase expression from the co-transfected prl plasmid. we set the luciferase expression in the presence of pbs at 100%. the knockdown efficiency of the single a pol47 hairpin was $40%, but chaining it with the b wt hairpin or the a wt -e wt cluster enhanced the silencing activity. as a negative control, we included the a wt -e wt construct. as a positive control, we included the original shrna. a similar pattern was observed for inhibition of hiv-1 production ( figure 5a, right) . thus, expression of a mirna inhibitor as part of a mirna polycistronic transcript enhances the silencing activity. next, we constructed polycistronic hairpin constructs with different combinations of two, three or four hairpins of the mirna inhibitors a pol47 , b pol1 , c gag5 , d r/t5 and e ldr9 : ab, ac, ad, acc, acd, accd, acde and acdb wt . we first determined the knockdown efficiency of each individual hairpin within the multiplex transcripts by co-transfection with the corresponding luciferase reporter into 293t cells ( figure 5b, supplementary table 2 ). constructs encoding the a wt -e wt cistron and individual wild-type or non-matching mirnas were used as negative controls. for inhibitors a pol47 , c gag5 and e ldr9 , we observed a remarkable enhancement of silencing activity hiv-1 sequences are blue; pre-mirna sequences are black; mature mirna sequences are red; guide strand is bold; à, no; +, 10-30%; ++, 30-70%; +++, 70-100%; nd, not determined. when chained to other hairpins. for instance, construct c gag5 is poorly active compared to construct ac or acde ( figure 5b ). construct b pol1 does not exhibit any inhibitory activity, either alone or when combined with another hairpin as in ab. since the b pol1 hairpin structure is rather unstable as predicted by the mfold algorithm, this could be due to misfolding of the hairpin rna. we therefore will not use hairpin b pol1 in the final polycistron construct. hairpin d r/t5 does not benefit from linkage to other hairpins, but this is likely due to the high inhibitory activity of the individual d r/t5 inhibitor. strong inhibition of luc-d r/t5 was observed with d r/t5 , ad, acd, accd and acde, indicating that generation of effective sirnas from the multiplex hairpin transcripts does not depend on the mirna position in the polycistron. we further tested the ability of the antiviral mirnas to inhibit hiv-1 by co-transfection with the hiv-1 molecular clone lai ( figure 5c, supplementary table 2) . consistent with the luciferase results, we observed a moderate hiv-1 inhibition by the single mirna constructs, except for construct b pol1 (inactive) and d r/t5 (highly active) ( figure 5c ). multimerization of the hairpins strongly enhanced the inhibition of hiv-1 production, which is due both to enhancement of the silencing activity of the individual hairpins and to the presence of multiple antiviral sirnas. the a wt -e wt construct was used as a negative control and the shrna constructs were used as positive controls. we next performed northern blot analysis of the antiviral sirnas made by the different polycistron constructs ( figure 6 ). the results are remarkably similar to the activity data in figure 5 . for instance, sirna expression of a pol47 is greatly increased by linkage to another hairpin as in ad ( figure 6a ). the northern blot analysis of the b pol1 and the ab inhibitors provides an explanation for its inactivity as only the $60-nt b pol1 precursor is observed ( figure 6b , indicated by an arrow). to study whether the passenger sirna strand of b pol1 is made, we performed a northern blot analysis with the corresponding probe, but failed to detect any passenger sirnas (results not shown). in contrast, properly processed sirnas are produced from hairpin a pol47 in construct ab, indicating that the inactive b unit in the two-hairpin transcript does not negatively influence the active a unit ( figure 6a and b) . in addition, sirna production from the a hairpin is boosted for the polycistronic transcripts compared to the single a pol47 hairpin transcript. for inhibitor c gag5 , we also observed a strong increase in sirna production when the hairpin is combined with other hairpins in a polycistronic transcript ( figure 6c ). the d r/t5 inhibitor is expressed individually and does not benefit from chaining to other hairpins ( figure 6d ), which correlates nicely with the luciferase results ( figure 5b ). for inhibitor e ldr9 , we observed increased sirna levels for acde compared to the single hairpin ( figure 6e ). the combined luciferase inhibition results and the northern blot analyses demonstrate that the silencing activity of an individual hairpin rna can be significantly enhanced when expressed in a polycistronic transcript. we next created stably transduced supt1 cells with a lentiviral vector (plv) expressing the individual mirna constructs a pol47 , b pol1 , c gag5 , d r/t5 , e ldr9 and the polycistronic acde construct. plv expressing the control mirna n (invitrogen) was used as negative control. to study the impact of the antiviral mirnas on supt1 cell viability, we set up a sensitive toxicity screen for cells transduced with n, a pol47 and acde. we cultured the cells for 36 days after lentiviral transduction and followed the percentage of gfp+ (transduced) and gfp-(untransduced) cells by facs (table 2) . we did not observe a decrease in the fraction of transduced cells, indicating a similar growth rate as untransduced cells. the stably transduced supt1 cells were selected by facs sorting and subsequently infected with hiv-1 (0.5 ng of ca-p24). virus replication was followed for 7 days by measuring the ca-p24 level in the culture supernatant. fast virus replication and virus-induced cytopathic effects were observed in cells expressing mirnas n, b pol1 , c gag5 and untransduced supt1 cells (figure 7) . hiv-1 replication was inhibited by the individual hairpins a pol47 , d r/t5 and e ldr9 . virus replication was profoundly inhibited in supt1 cells expressing the polycistronic mirna construct acde and control cells that express an extended triple shrna construct (e-shrna 3) (manuscript in preparation). in this study, we designed a combinatorial rnai approach against hiv-1 using the human mir-17-92 polycistron. we first constructed individual pri-mirna transcripts against five conserved regions of hiv-1 under the control of a cmv promoter. we maintained the secondary structure of the original pre-mirnas and included the single-stranded flanks because these are important for proper mirna processing and subsequent risc loading (12) . we used the cmv promoter to express the transcripts because most primary mirnas are transcribed by rna polymerase ii (7) , which also allows inducible or tissue-specific mirna expression (22, 53, 54) . previously, several reports have demonstrated effective gene knockdown in mammalian cells with sirnas derived from mirna precursors (17, 18, 22, 53) . however, none of these studies addressed the issue that a mature mirna is typically 22-24 nt, whereas an sirna is only 19-nt in length. here, we demonstrate that positioning of 19-24 nt antiviral sirna sequences at the 5 0 -end of the pre-mirna hairpin stem results in optimal hiv-1 inhibition. despite the optimization of the mirna-like inhibitors, their activity is less than that of the original shrna antivirals that were used to design the mirnas. we therefore addressed whether the mirna-like inhibitor is correctly processed and compared the amount of sirna produced from the mirna versus shrna constructs. the sirna level produced by the shrna construct sh r/t5 is 15-30-fold higher than the d r/t5 mirna, which is likely due to the use of different promoters (rna polymerase iii versus rna polymerase ii). interestingly, the shrna inhibitor did only show marginally higher inhibitory activity, suggesting that the intrinsic inhibitory activity of the mirna-like inhibitor is in fact much greater than the shrna variant. consistent with these results, vectors encoding hairpin structures that closely resemble a natural pre-mirna produced $12-fold more mature sirnas than vectors encoding simple hairpin structures (21) . the superior activity of natural mirna-like inhibitors is likely attributed by the intrinsic properties of a mirna, which is processed in the nucleus by drosha, exported to the cytoplasm, processed further by dicer and loaded into risc. in the case of an shrna, the drosha step is bypassed, which could provide a less-efficient entry into the rnai pathway. moderate hiv-1 inhibitory activity was observed with constructs expressing a single mirna hairpin. interestingly, we demonstrate that co-expression of two or more hairpins in a single transcript greatly enhanced the silencing activity of each individual hairpin in the transcript. northern blot analyses showed that the increased inhibitory activity correlates with higher sirna expression levels. multimerization of different mirna hairpins is of particular interest for targeting of rna viruses such as hiv-1 and hcv because of their extreme genetic diversity and potential for mutational escape. two recent papers presented the potential of combinatorial rnai using two mirna-30 hairpins (55, 56) . in agreement with these studies, we showed an increase in rnai activity upon multimerization of 2, 3, 4 or 6 hairpins in a single transcript. another study used the mir-30 backbone to multiplex artificial mirnas and reported decreased rnai activity for the tandem plasmid, which may be due to mirna processing problems (57 studied extensively. our study focuses on the natural mir-17-92 polycistron and we demonstrate that insertion of four antiviral sirnas creates a transcript that is properly processed into functional antiviral mirnas that effectively inhibit hiv-1 production and replication. a major concern of the mirna approach is the offtarget effect on cellular transcripts with partial sequence complementarity. such an off-target effect may require only a complementarity of 6-8 nt between the seed region of the mirna and the target (58, 59) . such a weak restraint results in numerous potential off-target genes for any mirna. when multiple mirnas are used, the number of potential off-targets will increase, increasing the chance of a negative effect on the treated cells. however, we observed no obvious cellular changes in a sensitive toxicity screen. furthermore, the observed inhibition of firefly luciferase reporters clearly showed sequence-specificity as non-matching mirnas did not have any effect and the expression of the control renilla reporter was not affected. another study, in which artificial mirnas were expressed in arabidopsis thaliana, conferred viral resistance without cellular alterations, suggesting that off-target effects are not significant (60) . furthermore, recently evidence emerges that sirna sequences inserted in a mirna backbone do not compete for transport and incorporation into risc, while competition was observed when the same sirna sequences were presented as synthetic sirnas or shrnas (61) . nonetheless, off-targeting is a genuine concern for the development of any rnai-based gene therapy against hiv-1 and the potential risk should be assessed properly in relevant in vivo models prior to an eventual clinical application (62) . we have shown in stably transduced t-cell lines that multiple effective mirnas inhibit hiv-1 replication much stronger than a single mirna. these data, together with our previous shrna studies (32, 63) , indicate that a combinatorial rnai approach against hiv-1 results in an increased magnitude of inhibition and consequently a restriction of viral escape. current strategies combine multiple polymerase iii shrna expression cassettes (63) , which results in high expression levels. this may not always be desired in a gene therapy setting because of increased toxicity due to saturation of the rnai machinery with sirnas (46) . the use of polymerase ii promoters to express the mirna polycistron will reduce this risk because of lower expression levels. in addition, polymerase ii cassettes allow expression in a tissue-specific manner and inducible gene expression, which increases the flexibity for gene therapy and functional genomic applications (22, 54) . for hiv-1 gene therapy applications the use of a hematopoietic or t-cell-specific promoter can increase the target cell specificity. an interesting candidate is the was promoter that is active in human hematopoietic precursor cells (cd34+) and t lymphocytes, b cells and dendritic cells (64) . another intriguing possibility is to use the hiv-1 ltr promoter to express the mirna polycistron. transcriptional activation of the hiv-1 ltr requires the viral tat protein, which is produced only in hiv-1-infected cells, thereby allowing exquisite target cell specificity. this approach has previously been employed for shrna expression (65) . thus, several approaches can be tested experimentally in order to optimize the antiviral mirna polycistron strategy for hiv-1 inhibition. in summary, one can effectively combat hiv-1 with multiple mirna effector molecules transcribed from a single polycistronic transcript. we showed that expression of the mirna polycistron results in the production of functional mature mirnas that can efficiently and selectively inhibit hiv-1. further optimization of this construct by increasing the target cell specificity and inducibility will be a further step towards a gene therapeutic approach against hiv-1. with lentiviral vectors expressing antiviral mirnas a pol47 , b pol1 , c gag5 , d r/t5 , e ldr9 and acde. untransduced supt1 cells and cells transduced with the unrelated mirna n were used as negative controls. cells expressing an extended triple shrna construct (e-shrna 3) serve as positive control. supt1 cells stably expressing mirnas a pol47 , b pol1 , c gag5 , d r/t5 , e ldr9 and acde were infected with hiv-1 and virus replication was monitored for 7 days by measuring ca-p24 in the culture supernatant. one representative experiment is shown, similar results were obtained in three independent experiments. potent and specific genetic interference by double-stranded rna in caenorhabditis elegans mechanisms of gene silencing by double-stranded rna rnai: double-stranded rna directs the atp-dependent cleavage of mrna at 21 to 23 nucleotide intervals a species of small antisense rna in posttranscriptional gene silencing in plants posttranscriptional gene silencing by double-stranded rna microrna maturation: stepwise processing and subcellular localization microrna genes are transcribed by rna polymerase ii human micrornas are processed from capped, polyadenylated transcripts that can also function as mrnas the nuclear rnase iii drosha initiates microrna processing processing of primary micrornas by the microprocessor complex the drosha-dgcr8 complex in primary microrna processing recognition and cleavage of primary microrna precursors by the nuclear processing enzyme drosha transcription and processing of human microrna precursors microrna biogenesis: coordinated cropping and dicing a microrna polycistron as a potential human oncogene human embryonic stem cells express a unique set of micrornas enhanced gene silencing of hiv-1 specific sirna using microrna designed hairpins both natural and designed micro rnas can inhibit the expression of cognate mrnas when expressed in human cells micrornas and small interfering rnas can inhibit mrna expression by similar mechanisms use of rna polymerase ii to transcribe artificial micrornas second-generation shrna libraries covering the mouse and human genomes a lentiviral microrna-based system for single-copy polymerase ii-regulated rna interference in mammalian cells probing tumor phenotypes using stable and regulated synthetic microrna precursors inhibition of virus replication by rna interference rna interference: its use as antiviral therapy bispecific short hairpin sirna constructs targeted to cd4, cxcr4, and ccr5 confer hiv-1 resistance human immunodeficiency virus type 1 escapes from rna interference-mediated inhibition inhibition of hiv-1 replication with designed mirnas expressed from rna polymerase ii promoters human immunodeficiency virus type 1 escape from rna interference hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome rna interference as an antiviral approach: targeting hiv-1 silencing of hiv-1 with rna interference: a multiple shrna approach specific inhibition of human immunodeficiency virus type 1 replication by antisense oligonucleotides: an in vitro model for treatment can hammerhead ribozymes be efficient tools to inactivate gene function? importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus lentiviral vectors that carry anti-hiv shrnas: problems and solutions a novel approach for inhibition of hiv-1 by rna interference: counteracting viral escape with a second generation of sirnas design of extended short hairpin rnas for hiv-1 inhibition inhibition of human immunodeficiency virus type 1 by rna interference using long-hairpin rna ) sirna-directed inhibition of hiv-1 infection suppression of chemokine receptor expression by rna interference allows for inhibition of hiv-1 replication inhibition of hiv-1 fusion with small interfering rnas targeting the chemokine coreceptor cxcr4 the inner-nuclear-envelope protein emerin regulates hiv-1 infectivity unpaking'' human immunodeficiency virus (hiv) replication: using small interfering rna screening to identify novel cofactors and elucidate the role of group i paks in hiv infection artificial microrna-mediated virus resistance in plants fatality in mice due to oversaturation of cellular microrna/short hairpin rna pathways mfold web server for nucleic acid folding and hybridization prediction changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of hiv-1 lai , hiv-1 mal , and hiv-1 eli functional differences between the long terminal repeat transcriptional promoters of hiv-1 subtypes a through g factor correction as a tool to eliminate between-session variation in replicate experiments: application to molecular biology and retrovirology a rev-independent human immunodeficiency virus type 1 (hiv-1)-based vector that exploits a codonoptimized hiv-1 gag-pol gene self-inactivating lentivirus vector for safe and efficient in vivo gene delivery polycistronic rna polymerase ii expression vectors for rna interference based on bic/mir-155 a single lentiviral vector platform for microrna-based conditional rna interference and coordinated transgene expression multi-mirna hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown multiple shrnas expressed by an inducible pol ii promoter can knock down the expression of multiple target genes an rna polymerase ii construct synthesizes short-hairpin rna with a quantitative indicator and mediates highly efficient rnai conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microrna targets utr seed matches, but not overall identity, are associated with rnai off-targets expression of artificial micrornas in transgenic arabidopsis thaliana confers virus resistance combinatorial delivery of small interfering rnas reduces rnai efficacy by selective incorporation into risc rna interference against viruses: strike and counterstrike lentiviral vector design for multiple shrna expression and durable hiv-1 inhibition lentiviral vectors targeting wasp expression to hematopoietic cells, efficiently transduce and correct cells from was patients negative feedback inhibition of hiv-1 by tatinducible expression of sirna hiv-1 rna research in the berkhoutlab is sponsored by zonmw (vici grant) and nwo-cw (top grant). we thank stephan heynen for performing ca-p24 elisa and jens gruber for useful discussions. funding to pay the open access publication charges for this article was provided by zonmw and nwo-cw. supplementary data are available at nar online.conflict of interest statement. none declared. key: cord-262752-bwofzbwa authors: li, qianqian; liu, qiang; huang, weijin; li, xuguang; wang, youchun title: current status on the development of pseudoviruses for enveloped viruses date: 2017-12-07 journal: rev med virol doi: 10.1002/rmv.1963 sha: doc_id: 262752 cord_uid: bwofzbwa emerging and reemerging infectious diseases have a strong negative impact on public health. however, because many of these pathogens must be handled in biosafety level, 3 or 4 containment laboratories, research and development of antivirals or vaccines against these diseases are often impeded. alternative approaches to address this issue have been vigorously pursued, particularly the use of pseudoviruses in place of wild‐type viruses. as pseudoviruses have been deprived of certain gene sequences of the virulent virus, they can be handled in biosafety level 2 laboratories. importantly, the envelopes of these viral particles may have similar conformational structures to those of the wild‐type viruses, making it feasible to conduct mechanistic investigation on viral entry and to evaluate potential neutralizing antibodies. however, a variety of challenging issues remain, including the production of a sufficient pseudovirus yield and the inability to produce an appropriate pseudotype of certain viruses. this review discusses current progress in the development of pseudoviruses and dissects the factors that contribute to low viral yields. a pseudovirus is a recombinant viral particle with its core/backbone and envelope proteins derived from different viruses 1 ; moreover, the genes inside the pseudovirus are usually altered or modified so that they are unable to produce the surface protein on their own. as such, an additional plasmid or stable cell line expressing the surface proteins is needed to make the pseudovirus. 2 pseudoviruses are capable of infecting susceptible cells, but they only replicate for 1 round in the infected host cells. 3 compared with wild-type (wt) viruses, pseudoviruses can be safely handled in biosafety level (bsl)-2 laboratories 4 and are usually easier to manipulate experimentally. 5 nevertheless, the conformational structure of pseudoviral surface proteins bears high similarity to that of the native viral proteins, and these surface proteins can effectively mediate viral entry into host cells. therefore, pseudoviruses are widely used for the study of cellular tropism, 6 receptor recognition, 7 and virus inhibition, 8 as well as for developing and evaluating antibodies 9 and vaccines. 10 in addition, data from in vitro pseudovirus-based antiviral assays and in vivo biodistribution analyses have been found to correlate very well with the results generated by using live wt viruses. 11, 12 as pseudoviruses have usually been engineered to carry reporter genes, it is much easier to perform quantitative analyses on these viruses than on wt viruses, 13 and the number of pseudovirus-infected cells has been shown to be directly proportional to reporter gene expression. the reporter genes usually encode either an enzyme or a fluorescent protein, with each option having its particular strengths and weaknesses. specifically, chemiluminescence assays usually have lower background and are more sensitive, but the data acquisition and analyses for these assays are time-consuming and more expensive. in contrast, assays using a florescence protein, such as green fluorescent protein, are cheaper and easier to operate in both in vitro and in vivo systems; however, they are less sensitive and may have higher background. [14] [15] [16] in this review, we provide an update on the development and application of pseudoviral systems and discuss some challenging technical issues. the hiv-1 packaging system is the most widely used pseudovirus packaging system. to make this packaging system, hiv genes are selectively cloned into dna vectors. specifically, 2 to 4 plasmids are used as the vectors, a strategy that aims to minimize viral gene recombination and thereby reduce the possibility of reversion to the wt virus. table 1 lists the currently used hiv-1-based systems. the original 2-plasmid system comprises 1 envelope plasmid and 1 hiv-1 backbone plasmid, ie, psg3δenv and pnl4-3 (the env gene sequence in psg3δenv is destroyed). 51 however, this system is not ideal, as its viral yield is usually very low. improvements have been made through the addition of other sequences for better reporter gene expression. specifically, our research group inserted the reporting gene, firefly luciferase (fluc), into psg3δenv between env and nef to produce psg3δenv.fluc.δnef. in addition, we also generated psg3δenv.cmvfluc, which carries a functional nef and cmv promoter to drive the reporter gene. 10 by using these optimized backbone and envelope protein expression plasmids, our group succeeded in producing several hiv pseudoviruses carrying the envelope proteins of ebov, marv, lasv, middle east respiratory syndrome-coronavirus, rabies virus (rv), chikungunya virus, and nipah virus (niv). the yields of pseudoviruses constructed with this optimized system were improved by 100 to 1000-folds as compared with those of pseudoviruses constructed with pnl4-3.luc.r-e. 52 the hiv 3-plasmid system is usually comprised of a packaging plasmid, a transfer plasmid containing the reporter gene, and an envelope-expressing plasmid. specifically, this system is made by splitting the hiv-1 backbone into separate packing and transfer plasmids. the packaging plasmid expresses the gag and pol proteins, while the transfer plasmid contains the cis-regulatory elements needed for hiv reverse transcription, integration, and packaging as well as multiple cloning sites and a reporter gene under the control of a heterogeneous promoter. 48-50 the envelope-expressing plasmid is made of a vector carrying the envelope gene driven by a cmv promoter. the hiv 4-plasmid system is based on the 3-plasmid system, with the rev protein being expressed by an additional, separate plasmid. specifically, this system comprises 1 packaging plasmid expressing the gag and pol proteins, a second packaging plasmid encoding rev, 1 plasmid producing the wt envelope protein, and a transfer plasmid with cis-regulatory elements. 53 these 3 hiv-based systems were reported by different groups, and, as yet, no comparison has been made among the different systems in safety and efficiency. our group is able to drastically improve the viral yield with 2-plasmid system, while no safety issue of this hiv pseudoviral systems was observed in animals. 52 2.1.2 | the simian immunodeficiency virus packaging system hiv env cellular tropism, neutralization antibody assay, impact of env amino acid mutation, and glycosylation on the neutralization epitope, drug screening and validation, and receptor recognition psg3δenv; pnl4-3.luc.r-e the vsv packaging system is a versatile tool for making pseudotyped viruses; this system is advantageous in that it has no stringent selectivity for the envelope proteins, and the resulting virus may be manipulated in a bsl-2 laboratory. early studies jointly employed vsv and a second virus to coinfect cells, resulting in the pseudotyped virus carrying the core of vsv with envelope proteins derived from the other virus. 60 stillman et al were the first to clone the vsv genome into a plasmid to make stable vsv, 61 which was subsequently used to generate pseudoviruses carrying heterogeneous glycoprotein. 62, 63 various reporter genes were successively inserted into this plasmid to facilitate its easy detection. 64, 65 some examples of vsv-based pseudovirus system are listed in table 2 . notably, when the vsv packaging system is used to make a pseudovirus, there may be residual vsv virus mixed with the pseudovirus, thereby complicating the neutralization assay in which it is used or producing false-positive results. preferably, the amount of vsv should be minimized; however, if excess vsv is believed to be interfering with a pseudovirus-based assay, treatment of the pseudovirus preparation with a vsv neutralizing antibody could be considered before its use in future assays. the mlv packaging system, also called the retroviral system, is commonly used to make pseudoviruses. table 3 lists the pseudoviruses packaged by the mlv system that have been reported in the literature. early work by witte and colleagues showed that when they used vsv to infect the cells in which mlv is packaged, they were able to harvest pseudovirus for use in neutralization antibody assays. 98 since then, the genome of mlv had been split into 2 parts: one encoding gag-pol and the other containing the reporter gene. the 2 gene sets were further cloned into plasmids to generate highly efficient mlv packaging systems. 99 to improve the stability of this system, investigators established several cell lines that were confirmed to be stable in transfection and expression. murine leukemia virus may actually be a better choice than hiv as a packaging system in some cases. for example, in studying lasv-mediated entry into cells, cosset et al compared the mlv and hiv systems and found that the former is 5-fold more efficient than the latter. 100 the aforementioned pseudovirus packaging systems have not always been successful in generating certain types of pseudoviruses. in those cases, other alternatives such as reverse genetics have been reported. for example, hu et al prepared a pseudotyped dengue virus (denv) types 1 to 4 by using the hiv system, but its titer was insufficiently high. 101 however, by using reverse genetics, reporter genes were a high yield is needed for practical applications of the pseudoviruses. there are several factors that can critically influence their yield/titer. in general, the subcellular localization for viral packaging and maturasuccessfully used the mlv packaging system to generate a pseudotyped influenza virus. 108 cosset et al reached the same conclusion for pseudotyped lasv preparations. 100 moreover, in our experiences, pseudotyped lasv packaged by the vsv system had a higher titer than that produced by using the hiv system. furthermore, the vsv system was able to incorporate hantavirus glycoprotein that had failed to be packaged by hiv. nonetheless, our group developed a modification of the hiv packaging system that could improve the pseudoviral yield by 100-fold; specifically, the backbone plasmid psg3δenv.cmvfluc that was developed in our lab was superior to pnl4-3.luc.r-e-. 10 the packaging conditions can also drastically influence the pseudovirus yield. in the hiv packaging system, the pseudovirus titer could be pseudoviruses have been widely used for conducting in vitro studies on the interaction between the virus and the host cells. 112 they have also proven to be very useful for in vivo studies, particularly studies on the mechanism of viral infection as well as on the biodistribution. 113 our lab used a pseudotyped rv carrying reporter genes to establish an in vivo imaging model in mice. this mouse model was used to study viral tissue tropism and its dynamic change over time. 10 we also established a pseudotyped ebov mouse model; the ebov pseudoviruses were mainly detected in the thymus and spleen following viral infection, revealing that the pseudotyped ebov and wt ebov have the same tissue tropism. 52 other groups have also used pseudotyped hsv-1 and marv in small animal models to investigate viral infections. 114 4.2 | application of pseudoviral systems to neutralization antibody and antibody-dependent cellmediated cytotoxicity assay antibody neutralization assay based on pseudoviruses has been widely used, particularly for the analyses of some virulent viruses that would otherwise need to be handled in bsl-3 or bsl-4 laboratories. compared with the traditional assays, the reported pseudovirus-based assays have demonstrated a good correlation with the wt virus-based assay; the pseudovirus-based assays are usually high-throughput procedures with fewer amounts of serum samples needed. 1, 104, 115, 116 wilkinson et al compared 22 platform technologies for assaying antibody against ebov with neutralization assays by using the wt virus and found that the 5 best assays included methods based on wt and vsv pseudotype neutralization and elisa, but the lentiviral and other platforms were problematic. 117 no false start for novel pseudotyped vectors vesicular stomatitis virus pseudotypes of retroviruses a bioluminescent imaging mouse model for marburg virus based on a pseudovirus system lassa and ebola virus inhibitors identified using minigenome and recombinant virus reporter systems high-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses infectious hepatitis c virus pseudo-particles containing functional e1-e2 envelope protein complexes transferrin receptor 1 is a cellular receptor for new world haemorrhagic fever arenaviruses a comparative high-throughput screening protocol to identify entry inhibitors of enveloped viruses most neutralizing human monoclonal antibodies target novel epitopes requiring both lassa virus glycoprotein subunits development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison a safe and sensitive enterovirus a71 infection model based on human scarb2 knock-in mice comparison of two high-throughput assays for quantification of adenovirus type 5 neutralizing antibodies in a population of donors in china development of a moloney murine leukemia virus-based pseudotype anti-hiv assay suitable for accurate and rapid evaluation of hiv entry inhibitors characterization of retroviral and lentiviral vectors pseudotyped with xenotropic murine leukemia virus-related virus envelope glycoprotein development of a triple-color pseudovirion-based assay to detect neutralizing antibodies against human papillomavirus temperature-dependent production of pseudoinfectious dengue reporter virus particles by complementation dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes characterization of chikungunya pseudotyped viruses: identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in e1 glycoprotein development of a pseudotyped-lentiviral-vector-based neutralization assay for chikungunya virus infection chikungunya virus glycoproteins pseudotype with lentiviral vectors and reveal a broad spectrum of cellular tropism filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo toremifene interacts with and destabilizes the ebola virus glycoprotein distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses in vitro evaluation of cyanovirin-n antiviral activity, by use of lentiviral vectors pseudotyped with filovirus envelope glycoproteins packaging hiv-or fiv-based lentivector expression constructs and transduction of vsv-g pseudotyped viral particles pseudotype formation between enveloped rna and dna viruses replication and amplification of novel vesicular stomatitis virus minigenomes encoding viral structural proteins foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles generation of vsv pseudotypes using recombinant deltag-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines quantification of lyssavirus-neutralizing antibodies using vesicular stomatitis virus pseudotype particles second generation of pseudotype-based serum neutralization assay for nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase a system for functional analysis of ebola virus glycoprotein rho gtpases modulate entry of ebola virus and vesicular stomatitis virus pseudotyped vectors characterization of pseudotype vsv possessing hcv envelope proteins use of vesicular stomatitis virus pseudotypes bearing hantaan or seoul virus envelope proteins in a rapid and safe neutralization test a pseudotype vesicular stomatitis virus containing hantaan virus envelope glycoproteins g1 and g2 as an alternative to hantavirus vaccine in mice study of andes virus entry and neutralization using a pseudovirion system efficient production of hantaan and puumala pseudovirions for viral tropism and neutralization studies analyses of entry mechanisms of novel emerging viruses using pseudotype vsv system characterization of the interaction of lassa fever virus with its cellular receptor alpha-dystroglycan analysis of lujo virus cell entry using pseudotype vesicular stomatitis virus development of a neutralization assay for nipah virus using pseudotype particles ephrinb2 is the entry receptor for nipah virus, an emergent deadly paramyxovirus a neutralization test for specific detection of nipah virus antibodies using pseudotyped vesicular stomatitis virus expressing green fluorescent protein involvement of ceramide in the propagation of japanese encephalitis virus preparation of vesicular stomatitis virus pseudotype with chikungunya virus envelope protein efficient generation of vesicular stomatitis virus (vsv)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses a vesicular stomatitis pseudovirus expressing the surface glycoproteins of influenza a virus analysis of the entry mechanism of crimean-congo hemorrhagic fever virus, using a vesicular stomatitis virus pseudotyping system characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines murine leukemia virus pseudotypes of la crosse and hantaan bunyaviruses: a system for analysis of cell tropism truncation of the human immunodeficiency virus-type-2 envelope glycoprotein allows efficient pseudotyping of murine leukemia virus retroviral vector particles assessment of hiv-1 entry inhibitors by mlv/hiv-1 pseudotyped vectors functional murine leukemia virus vectors pseudotyped with the visna virus envelope show expanded visna virus cell tropism ross river virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production coreceptor switch of [mlv(sivagm)] pseudotype vectors by v3-loop exchange development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein murine leukemia virus (mlv)-based coronavirus spike-pseudotyped particle production and infection transduction of schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped moloney murine leukemia retrovirus transduction of schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes establishment of retroviral pseudotypes with influenza hemagglutinins from h1, h3, and h5 subtypes for sensitive and specific detection of neutralizing antibodies detection of antibodies against h5 and h7 strains in birds: evaluation of influenza pseudovirus particle neutralization tests mechanism of formation of pseudotypes between vesicular stomatitis virus and murine leukemia virus a transient three-plasmid expression system for the production of high titer retroviral vectors characterization of lassa virus cell entry and neutralization with lassa virus pseudoparticles characterization of retrovirusbased reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses a rapid and quantitative assay for measuring antibody-mediated neutralization of west nile virus infection a high-throughput assay using dengue-1 virus-like particles for drug discovery pseudotypes: your flexible friends a plasma membrane localization signal in the hiv-1 envelope cytoplasmic domain prevents localization at sites of vesicular stomatitis virus budding and incorporation into vsv virions truncation of the enzootic nasal tumor virus envelope protein cytoplasmic tail increases env-mediated fusion and infectivity polybasic kkr motif in the cytoplasmic tail of nipah virus fusion protein modulates membrane fusion by inside-out signaling a sensitive retroviral pseudotype assay for influenza h5n1-neutralizing antibodies optimized large-scale production of high titer lentivirus vector pseudotypes a high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes the optimisation of pseudotyped viruses for the characterisation of immune responses to equine influenza virus pseudotyping viral vectors with emerging virus envelope proteins applications of bioluminescence imaging to the study of infectious diseases transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice the use of pseudotypes to study viruses, virus sero-epidemiology and vaccination optimization and proficiency testing of a pseudovirus-based assay for detection of hiv-1 neutralizing antibody in china comparison of platform technologies for assaying antibody to ebola virus novel cross-reactive monoclonal antibodies against ebola virus glycoproteins show protection in a murine challenge model high-throughput screening of viral entry inhibitors using pseudotyped virus teicoplanin inhibits ebola pseudovirus infection in cell culture identification of a broadspectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay a systematic screen of fdaapproved drugs for inhibitors of biological threat agents characterization of the inhibitory effect of an extract of prunella vulgaris on ebola virus glycoprotein (gp)-mediated virus entry and infection properties of wild-type, c-terminally truncated, and chimeric maedi-visna virus glycoprotein and putative pseudotyping of retroviral vector particles cholesterol supplementation during production increases the infectivity of retroviral and lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (vsv-g) short communication: potential risk of replication-competent virus in hiv-1 env-pseudotyped virus preparations current status on the development of pseudoviruses for enveloped viruses the authors have no competing interest. key: cord-301449-5okb7wf2 authors: nixon, douglas f. title: comments on “coinfection of sars‐cov‐2 and hiv in a patient in wuhan city, china” date: 2020-04-08 journal: j med virol doi: 10.1002/jmv.25821 sha: doc_id: 301449 cord_uid: 5okb7wf2 zhu et al. report in their letter, co-infection of sars-cov-2 and hiv in a patient in wuhan city, china, a case of covid19 in an hiv infected patient1 . however, from the details given in the report, there are doubts that this patient is living with hiv, and additional information would be needed to confirm it. in addition, the antiretroviral drug treatment mentioned is not within the standard guidelines for someone infected with hiv. this article is protected by copyright. all rights reserved. comments on "coinfection of sars-cov-2 and hiv in a patient in wuhan city, china" zhu et al 1 report in their letter, coinfection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and human immunodeficiency virus (hiv) in a patient in wuhan city, china, a case of coronavirus disease 2019 in an hiv-infected patient. however, from the details given in the report, there are doubts that this patient is living with hiv, and additional information would be needed to confirm it. in addition, the antiretroviral drug treatment mentioned is not within the standard guidelines for someone infected with hiv. information on how sars-cov-2 infection impacts immunocompromised people, such as those people living with hiv (plwh), off therapy or on therapy, is of urgent concern to those managing plwh. in this case report, the authors describe a 61-yearold male, who was a heavy smoker with type ii diabetes. he presented with recurrent fever and dry cough and was diagnosed with sars-cov-2 pneumonia on a chest computed tomography scan. there is no other demographic information given in the case report, but he does not appear to fall into traditional hiv infection risk groups. there is no information on sexual history, drug use, or blood transfusion, which would assist in evaluating his hiv risk. the authors then mention "an antigen/antibody combination test on blood gave the hiv-positive result," without discussion of why the test was given, nor what the test was, or if it was repeated more than once. there is no mention of an hiv viral load measurement, or any subsequent follow up for confirmation of his hiv diagnosis. they then say the patient was put on "oral therapy with an anti-hiv drug, lopinavir/ritonavir" for 12 days. this is not an adequate regimen for someone diagnosed with hiv. while they end on an encouraging note about the patient, with a discussion about his improvement and subsequent isolation at home to recover, they do not discuss any additional follow up for his purported hiv infection with no mention of an adequate treatment regimen. in this rapidly changing time, it is essential to monitor and discuss reports on covid-19, and its' impact on the hiv-infected community. it is also important to point out the limitations of studies so that further studies are not compromised. co-infection of sars-cov-2 and hiv in a patient in wuhan city key: cord-274080-884x48on authors: rumlová, michaela; ruml, tomáš title: in vitro methods for testing antiviral drugs date: 2018-06-30 journal: biotechnology advances doi: 10.1016/j.biotechadv.2017.12.016 sha: doc_id: 274080 cord_uid: 884x48on abstract despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. a combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. we provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses. viral pandemics remain serious threats to humankind. every year, known viruses such as hiv-1 and hepatitis b virus newly infect millions of people across the globe. in addition, recent outbreaks of ebola virus, influenza virus, severe acute respiratory syndrome (sars) coronavirus and middle east respiratory syndrome-coronavirus (mers-cov) serve as a reminder of the silent danger. the world health organization reported in 2015 that over 1.34 million causalities per year are connected with hepatitis b and almost 500,000 with hepatitis c. occasional epidemic outbreaks of other viruses are also striking. these include outbreaks of noroviruses, flaviviruses (zika and dengue viruses), new strains of influenza viruses, the re-emergence of west nile virus in italy and the united states. despite relatively long pauses, viruses such as influenza virus can re-emerge and cause global pandemic health problems. unlike cellular genomes, which consist of double-stranded (ds) dna, viral genomes can be formed by a broad variety of nucleic acids (nas), including ds or single-stranded (ss) circular or linear dna or positive-, negative-or sometimes ambi-sense rna. as viral life cycles are dependent on cellular factors and cellular metabolic and signaling pathways, the number of possible antiviral drug targets is limited. however, almost all viruses encode unique proteins and enzymes that may serve as specific targets for antiviral therapy. the overarching goal of modern drug development efforts is to design compounds that specifically inhibit viral targets or cellular targets essential for virus replication. the purpose of this review is to provide insight into the broad variety of cell-based and biochemical assays used for identification and evaluation of antivirotics, including high-throughput screening (hts) methods. an overview of available inhibitors and vaccines, which has been reviewed elsewhere [e.g. in (de clercq and li, 2016) ], is beyond the scope of this paper. to package their genomes into particles of very limited size, viruses encode few genes. virions consist of an rna or dna genome that is protected by an outer shell called a capsid (also nucleocapsid) formed by a lattice of capsid proteins. in enveloped viruses, the capsid is additionally surrounded by a lipid bilayer spiked with viral proteins. the size of animal viruses ranges from approximately 25 nm to over 300 nm (cohen et al., 2011) . the capsids and virions of rna viruses adopt various shapes. viral capsids may be icosahedral (e.g. picornaviridae, astroviridae, reoviridae, togaviridae), bullet-shaped (rhabdoviridae), helical (coronaviridae), helical filamentous (filoviridae, orthomyxoviridae, paramyxoviridae), or filamentous (arenaviridae, bunyaviridae). the retroviral capsid core may be conical, spherical, or rod-shaped. the morphology of dna viruses is similarly diverse, ranging from icosahedral (enveloped -hepadnaviridae, herpesvridae; nonenveloped -adenoviridae, parvovirdae, polyomaviridae and papillomaviridae) to rodshaped (baculoviridae) or pleomorphic (poxviridae). the viral life cycle is the process of viral replication in a host cell. first, viruses enter the host cell and replicate their genomes. following translation of viral proteins by the host cell machinery, viruses package their genomic material into protective proteinaceous capsids and exit the cell to infect another host cell. nonenveloped viruses consist only of a protein capsid shell enclosing the viral genome and enzymes, while the capsid shell of enveloped viruses is enclosed in a lipid envelope derived from the host cell membrane. regardless of whether the virus is enveloped, its surface must display (glyco)proteins suitable for specific interactions with host cell receptors. in contrast to the surface proteins of nonenveloped viruses, those of enveloped viruses usually serve another function in addition to host cell recognition and attachment. for example, they may enable fusion of the viral and cellular membranes, usually through an interaction with a secondary receptor(s) or co-receptor(s). the fusion domains of viral surface glycoproteins thus can lower the kinetic barrier for the energetically demanding membrane fusion. the viral particle must be sufficiently stable to protect the genome until its delivery into the host cell. simultaneously, it must be metastable, to allow its disassembly and release of the genome for replication in the infected cell at the appropriate time. the energetic barrier that prevents viral disassembly outside the cell is lowered upon infection by structural changes in the viral components. these changes may be induced by binding of a cellular ligand or by changes in the environment, such as ph change upon entering a specific cellular compartment. numerous viruses preassemble immature particles that undergo irreversible (usually proteolytic) transitions into mature structures of fully infectious virions. mutual interactions of viral capsid proteins are typically different from those of cellular proteins, which predominantly create binary interactions. viral structural proteins interact with multiple neighboring partners to form multicomponent macromolecular structures [reviewed in (cheng and brooks, 2015; jayaraman et al., 2016) ]. the economy of the packaged viral genomes due to the limited capsid size implies formation of only a few types of structural proteins, which are usually symmetrically organized [reviewed in (prasad and schmid, 2012; raguram et al., 2017) ]. in the initial stage of infection, viruses must overcome several obstacles. the first is the cellular plasma membrane with an actin cortex. then, they must traffic through dense cytoplasm to reach their final destination for replication and assembly. these pathways are specific for different viruses and often are dictated by the size of the particle and its structure. the viral life cycle can be divided into several common stages, including entry, uncoating, genome replication, genome packaging and assembly, release, and maturation ( fig. 1) . in general, the first phase of viral infection is specific recognition of the target host cell and binding to a surface receptor displayed on the cell membrane. this process is common to both enveloped and nonenveloped viruses. in enveloped viruses, the binding is mediated by viral surface components, typically oligomers of integral glycoproteins. nonenveloped viruses bind receptors through sites or projections on the capsid surface. viruses can use either a single receptor (e.g. tim-1 for hepatitis a virus, gm1 for sv40, cd155 for poliovirus, low-density lipoprotein receptor for human rhinovirus) or multiple receptors with equivalent roles (e.g., nectin-1/2 or hvem for herpes simplex virus 1/ 2, ace or l-sign for sars coronavirus). for other viruses (e.g. hiv, hcv, adenoviruses, rotaviruses, picornaviruses, and some herpesviruses), the presence of at least two cytoplasmic membrane components is required. for example, hiv-1 binds to the primary receptor cd4 and one of two co-receptors (ccr5 or cxcr4) [reviewed in (cossart and helenius, 2014; grove and marsh, 2011) ]. to infect a cell, viruses must overcome the plasma membrane to deliver their genetic material into the cytosol. viruses enter cells by two main mechanisms. a majority of animal viruses, both enveloped and nonenveloped, enter cells by one or two types of endocytosis, such as clathrin-mediated endocytosis (e.g. vsv, influenza a virus, rhinovirus), caveolin-mediated uptake (echovirus, polyoma virus), clathrin/caveolin-independent endocytosis such as caveolar or lipid raft-mediated (e.g. sv40, polyomavirus mouse), or macropinocytosis (e.g. vaccinia virus, respiratory syncytial virus, ebola virus, hiv-1) (blaas, 2016; cossart and helenius, 2014; fields and knipe, 2013; kirkham and parton, 2005; marechal et al., 2001; mayor and pagano, 2007; mercer and helenius, 2009; parton and simons, 2007; rasmussen and vilhardt, 2015; saeed et al., 2010) . these endocytic mechanisms enable the virus to be transported by the cell's machinery through the plasma membrane and to pass through the dense actin cortex. cellular entry of some viruses is coupled with receptor-mediated signaling, resulting in activation of molecules that facilitate virus entry by cytoskeleton reorganization, induction of long-distance transport of the virus-containing vesicles, or actin cortex disassembly. the second type of entry is used by some enveloped viruses (paramyxo-, herpes-, and retroviruses), which upon cell surface receptor binding, infect the cell by direct fusion of viral and plasma membranes at neutral ph. upon internalization, most viruses become trapped and enclosed in vesicles that pinch off the inner side of the plasma membrane and transport their cargo into the cytoplasm. on their way through the cell, these transport vesicles undergo maturation by fusing with other vesicles. to fulfill their task of genome replication, viruses have to escape from these endosomes. the "great escape" is triggered by activation of a fusion or penetration mechanism, such as changes in conditions in the endosomal interior [e.g. ph, ionic environment (e.g. calcium ion concentration), oxido-reducing conditions], changes in membrane composition, and other physico-chemical cues (blaas, 2016; cossart and helenius, 2014; inoue and tsai, 2013) . depending on the requirements for a particular virus, these events can occur in early endosomes (ph 6.5-6.0; e.g. hepatitis c virus, vesicular stomatitis virus), late endosomes (ph 6.0-5.0; e.g. influenza a virus, dengue virus, sars coronavirus), recycling endosomes, macropinosomes, the endoplasmic reticulum, the golgi apparatus, or lysosomes (ph 5.0-4.5) (blaas, 2016; cossart and helenius, 2014; grove and marsh, 2011; inoue and tsai, 2013) . for the majority of animal viruses, the activation of these fusion or penetration mechanisms occurs through conformational changes and structural rearrangements in viral surface proteins and/or the whole virion shell that may destabilize the capsid core. structural rearrangements in enveloped viruses usually mediate fusion of viral and endosomal membranes. in nonenveloped viruses, the structural changes uncover amphipathic or hydrophobic domains that may induce pore formation or disruption of endosomal membranes. to deliver genetic material to the replication site, these mechanisms ultimately release viral capsid structures from endosomal vesicles into the cytosol either by fusing with the endosomal membrane (enveloped viruses) or by penetrating the endosomal membrane (nonenveloped viruses). uncoating is the partial or complete disassembly of the protective capsid shell and/or lipid envelope to liberate the viral genome. for many viruses, this process is closely connected with conformational changes induced by the virus binding to the cell surface receptor; a low ph environment; or changes in oxido-reducing conditions, ion concentrations, or other factors. for enveloped viruses, uncoating involves a loss of the viral membrane by fusion either with the plasma membrane or with intracellular vesicles, followed by stepwise uncoating of the protective capsid shell. in nonenveloped viruses, the uncoating process typically involves conformational changes that result in the weakening of intermolecular interactions, loss of structural proteins, proteolytic cleavage, and so on (fields and knipe, 2013) . depending on the virus, uncoating can take place at the plasma membrane, in the cytoplasm, during endocytosis in early or late endosomes, in lysosomes, in the nucleus, or at the nuclear pore complex (npc). the ssrna genome of retroviruses, which is fully enclosed inside a protective capsid shell, must be reverse transcribed into dsdna and released from the capsid. although it is accepted that hiv-1 uncoating is linked to reverse transcription and nuclear import (ambrose and aiken, 2014) and is controlled by host factors (e.g. cyclophilin a, trim5α), the precise molecular mechanisms that trigger the uncoating remain unknown. several hypotheses have been proposed, including breakage of the capsid shell due to increased inner pressure caused by accumulation of the reverse transcription product (rankovic et al., 2017) , the requirement of intact microtubules and dynein and kinesin motors (lukic et al., 2014) , and phosphorylation of capsid shell protein by the host cell kinase melk (takeuchi et al., 2017) . in contrast to the majority of rna viruses, which replicate in the cytoplasm, most dna viruses (with the exception of large dna viruses including poxviridae, asfarviridae, and mimiviridae) and several negative stranded rna viruses enter the nucleus to replicate their genomes (kobiler et al., 2012; koonin and yutin, 2010) . passive diffusion into the nucleus is suitable for molecules smaller than 9 nm in diameter, but larger structures must enter through the nuclear pore complex (npc), which can accommodate molecules of up to 39 nm (pante and kann, 2002) . translocation through the npc is tightly regulated and requires the presence of a nuclear localization signal (nls) on the passing molecule and nuclear import receptors (importins or karyopherins) (cautain et al., 2015) . due to the diversity of viral particle sizes (from 25 nm to over 300 nm) and structures, viruses have evolved several strategies to export their dna or rna genome into the nucleoplasm. with regard to the npc, these pathways can be divided into npc-dependent and npcindependent mechanisms. due to size limitations, only a few, very small simplified scheme of common stages of viral life cycle targeted by antiviral drugs. these stages including: 1) attachment and entry, 2) uncoating, 3) genome replication, 4) genome packaging and assembly of viral particle and 5) virus release and maturation. m. rumlová, t. ruml biotechnology advances 36 (2018) 557-576 viral capsid particles, such as hepatitis b virus (hbv; 32-36 nm diameter), can pass through the npc (cohen et al., 2011; rabe et al., 2003) in an importin-dependent manner. hbv then disassembles at the nuclear side of the npc (the nuclear basket), releasing its genome into the nucleoplasm (fay and panté, 2015) . the capsid shells or ribonucleoprotein complexes (rnps) of some larger viruses, such as influenza a virus and hiv-1, disassemble within the cytoplasm. the viral genome, released from the shell and complexed with nls-containing components, is then translocated through the npc (ambrose and aiken, 2014; campbell and hope, 2015; cohen et al., 2011; fay and panté, 2015; hutchinson and fodor, 2013) . another mechanism, used by herpes simplex virus (hsv) and adenoviruses, involves cellular (importins, nup) or viral protein-mediated attachment (docking) of the capsid shell at the cytoplasmic side of the npc. this facilitates the passage of genomic dna into the npc either by ejection from an almost intact particle (through the capsid portal in hsv-1) or upon complete disassembly of the capsid shell (in adenoviruses) (kremer and nemerow, 2015; ojala et al., 2000; pasdeloup et al., 2009) . npc-independent mechanisms are used by some retroviruses (e.g. mlv) that enter the nucleus during the mitotic phase of the cell division cycle when the nuclear envelope is dissolved (matreyek and engelman, 2013; roe et al., 1993) . another mechanism, described for parvoviruses, involves partial disruption of the nuclear envelope (cohen and pante, 2005; fay and panté, 2015) . viral genomes can be encoded by various types of nas, as summarized in table 1 for dna viruses and table 2 for rna viruses. by convention, ssdna of equivalent polarity to mrna is designated as the positive (+) strand. the complementary ssnas are of minus polarity (−). the majority of dna viruses replicate in the nucleus, where cellular dna replication and transcription also occur. in contrast, rna viruses usually replicate in the cytoplasm. rna viruses are the only "organisms" that store their genetic information in the form of rna. replication of their genomes is accomplished either by rna-dependent rna synthesis ( fig. 2a , b) [reviewed in (ferron et al., 2017; lu and gong, 2017; menéndez-arias and andino, 2017; pietilä et al., 2017; tao and ye, 2010) ] or through rnadependent dna synthesis (reverse transcription) followed by dna integration, replication, and transcription ( fig. 2c ). as some of these enzymatic activities are not commonly found in uninfected host cells, rna viruses must encode enzymes to aid replication (table 2 ). rna viruses are divided according to the baltimore classification into dsrna viruses (birna-and reoviridae); positive-sense ssrna viruses (corona-, flavi-, and picornaviriade); negative-sense ssrna viruses (filo-, rhabdo-, paramyxo-, and orthomyxoviriade), and ambisense rna viruses (arena-and some members of bunyaviridae) with both positive-and negativesense rnas (nguyen and haenni, 2003 ) (see table 2 ). the nature of the genome not only dictates the mechanism of replication, but also has other important consequences. the genome of (+)rna viruses may serve directly as mrna for production of viral proteins. therefore, the mere introduction of genetic material (e.g. in exocytic vesicles) may result in productive infection. the rna polymerases that copy the genetic material of rna viruses are error-prone, which provides considerable genetic flexibility and the propensity to evolve drug-resistant mutants. these features are amplified in viruses with segmented genomes that undergo reassortment. in viruses with segmented genomes (orthomyxoviridae, arenaviridae and bunyaviridae), each segment is transcribed in an autonomous transcription-replication unit by viral rna polymerase that binds to the 5′ end cap structure. of note, the genomic rna (grna) is capped by a unique mechanism called cap-snatching (ferron et al., 2017) , in which the cap is cleaved from cellular mrna and transferred onto the viral grna by a subunit of viral rna polymerase (pflug et al., 2017) . some dsrna viruses (birnaviridae and reoviridae) also contain segmented genomes. upon replication, these viruses must ensure stoichiometric incorporation of single copies of each grna segment into new particles. this is guided by specific packaging signals on each segment of grna that interact with positively charged domains in the capsid proteins [reviewed in (isel et al., 2016; pohl et al., 2016) ]. although different models of packaging have been proposed for various segmented genome viruses, a common feature is co-assembly of the capsid proteins with the grna and rna polymerase. the genomes of dna viruses come in a considerable variety of sizes and shapes, from small ss to large ds molecules that may be linear or circular. the size range of these genomes (from 1.8 kb to 1200 kb) reflects the necessity for some viruses to encode specific proteins required for viral replication. small-genome dna viruses (polyoma-, papilloma-, and parvoviruses) use only host cell enzymes for replication and transcription. the only exceptions are some hepadnaviruses (e.g. hepatitis b virus) that despite having small genomes (approximately 3 kb), encode their own specific dna polymerase/reverse transcriptase that reverse transcribes pregenomic rna (pgrna) into genomic viral dna (fig. 2d , ii) (beck and nassal, 2007) . viruses with intermediate-size genomes (up to 35 kb) (e.g. adenoviruses) encode their own dna polymerase for genome replication, but they usually utilize cellular rna polymerases ii and iii for transcription (fields and knipe, 2013) . viruses with large genomes (larger than 100 kb) (e.g. herpesviruses) encode proteins for replication, including dna polymerase and dna helicase-primase, as well as some enzymes necessary for biosynthesis of deoxyribonucleotide triphosphates (dntps) and several transcription factors (boehmer and nimonkar, 2003) . poxviruses (e.g. vaccinia virus), are another type of virus with large dsdna genomes, and their replication, transcription, and translation take place entirely in the cytoplasm within discrete juxtanuclear sites called virus factories (moss, 2013) , (fig. 2d , iii). these viruses encode all enzymes and specific factors necessary for genomic replication and transcription. with their own replicative machinery, large-genome dna viruses can replicate in nondividing cells. in contrast, the replication of small-genome dna viruses, which depends on cellular dna polymerases, must occur simultaneously with the s-phase of the cell cycle (e.g. parvoviruses), or must express some viral protein/ oncogene to re-program the host cell-cycle regulatory proteins p53 or retinoblastoma protein (prb), triggering entry into the s-phase (e.g. polyomaviruses and papillomaviruses). by affecting the g1/s checkpoint (controlled by p53 and prb), the viruses ensure the production of host enzymes required for viral replication (fields and knipe, 2013) . production of viral proteins of dna viruses with intermediate and large size genomes is divided into early and late phases. in the early (prereplicative) phase, nonstructural proteins required for dna replication are translated. the late phase, during which the late structural proteins needed for assembly are translated, begins after viral dna replication ( fig. 2d , i). depending on the species, viruses assemble either in contact with the cellular membrane or independent of the membrane in either the nucleus or cytoplasm. the membrane-independent route is used by nonenveloped viruses and a few enveloped viruses (e.g. orthomyxoviruses, herpesviruses, and some retroviruses) that acquire the membrane envelope after intracellular assembly during budding from the cell. the limiting step for nuclear assembly is the size of npcs, which are large enough to transport rna and import proteins required for assembly into the nucleus; however, npc size limits the transport of larger assemblies. therefore, some viruses form assembly intermediates in the nucleus. these structures are then exported to the cytoplasm, where they come together to form viral particles. nuclear export is specific and depends on the presence of nuclear export signals (nes) in the transported proteins. one example of a nonenveloped icosahedral virus that assembles in the nucleus is adenovirus, the assembly of which has been studied intensely due to its potential use in gene therapies [reviewed in (ahi and mittal, 2016) ]. recent data suggest that upon accumulation of multiple copies of adenoviral dsdna genomes, coordinated assembly and packaging occur by two interlinked mechanisms that involve both capsid proteins and core components (condezo and san martín, 2017) . the assembly occurs in the so-called peripheral replicative zone with the assistance of scaffolding proteins that facilitate formation of adenoviral particles but are excluded from mature viruses. adenoviruses are finally released upon lysis of the infected host cell. orthomyxoviruses and herpesviruses are enveloped viruses that assemble their nucleoproteins in the nucleus. herpesviruses package their dsdna genome as head-to-tail concatemers and assemble icosahedral procapsids on scaffold proteins in the nucleus [reviewed in (heming et al., 2017) ]. however, subsequent steps of herpesvirus assembly proceed in the cytoplasm. the preassembled procapsid is too large to pass through the npc, but it exits the nucleus by viral proteindriven vesicular transport across the nuclear inner membrane leaflet. thus, the herpesvirus acquires an initial envelope from the inner nuclear membrane [for review see (fields and knipe, 2013; hellberg et al., 2016) ]. next, the herpesviral membrane fuses with the outer nuclear membrane, and the naked particle is released from the nucleus into the cytosol. here, the virus acquires tegument (a protein layer between the capsid and the envelope) and other proteins. final herpesvirus envelopment occurs at the golgi membrane containing the viral glycoproteins (fields and knipe, 2013) . preassembled orthomyxoviral ribonucleoproteins, upon their export from the nucleus, are driven to the plasma membrane to which they attach through electrostatic interactions of the matrix protein m1 with membrane phosphatidylserine. the virions then assemble simultaneously with budding during which they also acquire ha, na and m2 membrane proteins. poxviruses undergo an even more complicated pathway. they are enveloped with multiple membranes acquired from er/intermediate compartments and golgi or early endosomes (moss, 2015 (moss, , 2016 . these membranes also provide the poxviruses with their membrane proteins. most viruses assemble upon interaction of their structural proteins with cellular membranes. the target membrane for assembly differs according to the virus type. flaviviruses assemble at the surface of the er and then upon their budding into the er lumen, the immature particles are then transported into the tgn. some viruses, such as coronaviruses, assemble at the er-golgi intermediate compartment [reviewed in (ujike and taguchi, 2015) ]. the assembly of bunyaviruses occurs concurrently with replication of grna segments in virus factories located along the golgi (guu et al., 2012; strandin et al., 2013) . the presence of membrane glycoproteins at the golgi membrane determines the sites where assembling bunyavirus particles bud into the golgi lumen, similar to other enveloped viruses. numerous viruses, including paramyxoviruses (cox and plemper, 2017) , orthomyxoviruses (pohl et al., 2016) , alphaviruses (jose et al., 2009) , rhabdoviruses (okumura and harty, 2011) , and most retroviruses (freed, 2015) , assemble underneath the cytoplasmic membrane, which facilitates assembly by providing a scaffolding function. the virus then acquires a lipid envelope through budding across the plasma membrane. during this process, it also gains the envelope glycoproteins (env) that are anchored in the plasma membrane by hydrophobic transmembrane domains. env reaches the plasma membrane by a cellular secretory pathway upon synthesis in the er and subsequent glycosylation in the golgi. usually, a specific interaction between viral structural proteins and glycoproteins is required. this may be either direct or mediated through the interaction with matrix protein (e.g. in (-)rna viruses such as ortho-and paramyxoviruses or rhabdoviruses). most retroviruses, including hiv (so-called morphogenetic c-type), also assemble at the plasma membrane of the host cell. the interaction of the structural polyprotein precursor (gag) with the plasma membrane is usually facilitated by a bipartite signal in the n-terminal domain of gag (i.e. matrix protein) comprising both the basic patch and n-terminally linked myristoyl residue (added co-translationally to gag). in contrast to c-type assembly at the membrane, morphogenetic b/d-type retroviruses assemble at pericentriolar sites where gag polyproteins are transported along microtubules by dynein molecular motors (sfakianos et al., 2003; vlach et al., 2008) . for both morphogenetic pathways, the packaging of grna facilitates assembly. (+)rna viruses have adopted a mechanism of extensive rearrangement of intracellular membranes to provide a milieu for virus replication and assembly. this mechanism effectively protects dsrna intermediates from degradation by the host cell machinery (delgui and colombo, 2017; jackson, 2014) . this process has been well-documented for poliovirus, in which the newly formed membranous structures exclusively serve for virus production. various types of vesicles that are formed upon viral infection have different roles in viral replication (rossignol et al., 2015) . some nonenveloped mammalian viruses, such as reoviruses, assemble in the cytosol in so-called viroplasms or viral factories into icosahedral particles consisting of three concentric layers encircling segments of genomic dsdna (benavente and martinez-costas, 2006; jones, 2000; shah et al., 2017) . rotavirus seems to be the only exemption in the reovirus family, as it enters the endoplasmic reticulum to gain its outer protein shell (trask et al., 2012) . numerous viruses assemble from polyprotein precursors that must be specifically cleaved by a viral protease to generate infectious particles. this mechanism, which is an irreversible step in the virus life cycle, ensures equimolar packaging of structural proteins and proportional co-assembly of viral enzymes in the form of precursors. upon proteolytic release, the liberated proteins may undergo different trafficking pathways or fulfill various functions in the virus. maturation changes the energy of interaction forces among those interfaces required for intracellular assembly of immature particles to those suitable for viral stability in the environment and for disassembly and uncoating of genetic material for replication [reviewed in (veesler and johnson, 2012) ]. in poliovirus, the autocatalytic and subsequent viral proteasemediated cleavage of p1 precursor protein allows formation of pentamers that interact with grna. additional cleavage of vp0, yielding vp2 and vp4, is required to form infectious poliovirus particles. in retroviruses, proteolytic processing is initiated by the autocatalytic liberation of viral protease, which subsequently cleaves the polyproteins to trigger major structural rearrangements in the virus and release of other viral enzymes (reverse transcriptase and integrase) and structural proteins. in herpesviruses, maturation involves proteolytic processing of the scaffolding protein and recruitment of tegument proteins that stabilize the particle and mediate interactions with the membrane during envelopment (gibson, 2008; tandon and mocarski, 2012; tandon et al., 2015) . adenoviruses undergo a maturation process that involves processing of six structural components of the core and one non-structural precursor that initiates replication of gdna (gaba et al., 2017) . one interesting feature of adenoviral maturation is that dna is required as a co-factor for protease activity. this means that maturation occurs only in particles that have packaged gdna (mangel and san martín, 2014) . flaviviruses form icosahedral particles upon budding into the neutral milieu of the er. the particles then translocate to the more acidic environment of the trans-golgi network. this ph shift results in disassembly of the immature lattice and extensive rearrangement of the flaviviral particle. this is connected with the exposure of a viral structural glycoprotein (prm) that is specifically processed by the , retroviruses (c) and dna viruses (d) (the schemes a-c were modified from: (ahlquist, 2006) . a: following endocytosis, the genome of (+)rna viruses may directly serve as mrna for translation of virus encoded proteins. among them, there are proteins of rna-replication machinery that recruit (+)rna into a membrane-associated replication complex. the genomic rna is replicated by using (-)rna template, which is transcribed in a low copy number amplified (+)rna is then packaged into newly assembled virions that exit the host cell either through secretion or cell lysis. b: upon the virus attachment, a core containing both grna and virus encoded rna polymerase is delivered into the cytoplasm by endocytosis. cytoplasmic transcription of the (-)rna template provides (+)rna that serves as mrna for translation of viral proteins. in dsrna viruses, the (+)rna is packaged into new cores which undergo maturation by synthesizing (-) rna (dotted strand) and acquiring surface proteins. in the (-)rna viruses, the (+)rna strand is transcribed into genomic (-)rna in the cytoplasm and then packaged. the new virions egress the cell either through secretion or cell lysis. c: following fusion of viral and cell plasma membrane, the retroviral core is released into cytosol, rna genome is transcribed into dsdna by viral reverse transcriptase concomitantly with uncoating of the viral core, ds dna is transferred into nucleus where it is integrated into host cell genome by viral integrase, following translation of retroviral structural and enzymatic polyproteins, the unspliced grna is packaged into the immature particles that usually assemble at the plasma membrane and the viral particles bud from the cell. d: three mechanisms (i.-iii.) of dna viruses replication are shown: (i): following entry and uncoating, the dna genome is transported to the nucleus; products of early genes (regulatory proteins, transcription factors) regulate the synthesis of viral enzymes (e.g. dna polymerase) required for genome replication; expression of late genes encoding structural capsid proteins in the cytosol, they are then transported into nucleus where packaging and pre-assembly take place; preassembled procapsids exit the nucleus and leave the cell (e.g. herpesviruses). (ii) unique replication cycle of hepatitis b virus (hbv): following entry, the viral particle is internalized by endocytosis and the nucleocapsid is released into the cytoplasm; the genome (relaxed circular rcdna) is transported into the nucleus, where it is converted to a covalently closed circular form (cccdna); which serves as a template for transcription of pregenomic rna (pgrna) for translation of the core protein and the viral polymerase and three subgenomic mrnas used for translation of regulatory and envelope proteins; following viral transcription and translation, the hbv core proteins self-assemble in the cytoplasm into viral nucleocapsid with concurrent incorporation of pgrna and hbv polymerase, pgrna is reversely transcribed into a rcdna within the nucleocapsid; nucleocapsid containing rcdna can either re-enter the nucleus to amplify cccdna, or can be enveloped by hbv envelope proteins in the endoplasmic reticulum. the particles are then secreted from the infected hepatocyte by exocytosis. (iii) upon entering the cell, the replication, transcription and translation take place entirely in the cytoplasm, within discrete juxtanuclear sites called virus factories (e.g. poxviruses) adapted from: ahlquist, p., 2006. parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses. nat rev microbiol 4(5), 371-382. cellular protease furin in the trans-golgi network. the liberated globular heads of prm remain attached at the low ph, but are released when the virus enters neutral body fluids (rey et al., 2017) . one frequently used mechanism of release of nonenveloped and some enveloped viruses is lysis of the infected cell. this is the simplest release mechanism, although the transition to lysis from latent infection is delicately regulated (aneja and yuan, 2017; levings and roth, 2013; schmiedel et al., 2016) . however, viruses that are usually lysogenic may also use alternative release pathways (bird and kirkegaard, 2015a) . these include non-lytic spread of viruses mediated by vesicles, which has been observed for poliovirus (bird and kirkegaard, 2015b; jackson et al., 2005) , coxsackievirus (alirezaei et al., 2012) , and hepatitis a virus . another possibility is that vesicles released from a cell infected with (+)rna viruses contain naked viral grna. this (+)grna-containing vesicle functions itself as an infectious agent as it is transferred to another cell (bird and kirkegaard, 2015a) . the standard way for enveloped viruses to leave the host cell is budding, which includes membrane extrusion and separation of the viral and cellular membranes (so-called pinching off). the lipid envelope layer acquired during viral particle budding through the plasma membrane protects the virus particle. there are three basic mechanisms of budding: i) mediated by envelope glycoproteins, such as the e protein of coronaviruses; ii) independent of glycoproteins, in which the viral structural proteins trigger the extrusion of the plasma membrane, such as retroviral budding in which strong interactions between the gag n-terminal domain (matrix protein) and the plasma membrane induce bulging of the membrane; and iii) requiring interaction between the viral glycoproteins and the capsid for membrane extrusion, as in alphaviruses. the final step that results in the separation of virus from the cell surface is pinching off the particle. this is a controlled process that involves both viral protein domains and cellular factors. during this process, viruses apparently use cellular machinery similar to that used during the last step of cell division (the release of the daughter cell) called endosomal sorting complex required for transport (escrt) [reviewed in (campsteijn et al., 2016; hurley, 2015; olmos and carlton, 2016; scourfield and martin-serrano, 2017) ]. direct interactions of numerous viral domains with escrt complex subunits have been identified (bieniasz, 2006) . in retroviruses, short specific amino acid sequences (ptap or psap) interact with the escrt components, and the interaction of hiv with the escrt proteins nedd4 and alix is wellknown (freed, 2002; fujii et al., 2007; gomez and hope, 2005) . however, this mechanism has also been adopted by other viruses that interact with the same escrt components, such as filoviruses (han et al., 2015; jasenosky and kawaoka, 2004; liu and harty, 2010) . interactions with escrt proteins have also been reported for vesicular stomatitis virus luyet et al., 2008 ), rhabdovirus (taylor et al., 2007 , arenaviruses (ziegler et al., 2016) , picornaviruses , paramyxoviruses (park et al., 2016) , and hepatitis c virus, a representative of the flavivirus family (falcon et al., 2017) . the typical release pathways used by viruses may vary under some conditions. for example, during chronic infection, numerous viruses use alternative cell-to-cell transmission that may help the virus avoid host neutralization (hulo et al., 2017) . syncytium formation, an hivinduced cell fusion, was recognized in the early 1990s (callahan, 1994) . another type of cell-to-cell transmission through tight junctions was shown for hiv (hübner et al., 2009; wang et al., 2017) and murine leukemia virus (sherer et al., 2010) . receptors on tight junctions that specifically recognize hepatitis c virus (carloni et al., 2012; ploss et al., 2009 ) and reovirus (barton et al., 2001) have been identified. some viruses are able to modify cellular pathways to reprogram both the synthesis and metabolism of lipids and membrane compartmentalization for their transmission and to evade cellular defense mechanisms (mazzon and mercer, 2014) . despite a general understanding that poliovirus spreads through cellular lysis, it was recently found that it may also be transferred between cells by an autophagy-dependent mechanism, called autophagosome-mediated exit without lysis (bird and kirkegaard, 2015b; bird et al., 2014; lai et al., 2016; richards and jackson, 2012) . similar mechanisms have been described for varicellazoster virus and human cytomegalovirus (grose et al., 2016; meier and grose, 2017) . poxviruses encode several proteins that block the apoptotic cellular response to the presence of their dsdna in the cytoplasm. this allows cell-to-cell passage of poxviruses by a mechanism known as apoptotic mimicry (amara and mercer, 2015; nichols et al., 2017) . in this process, enveloped viruses expose surface phosphatidylserine to mimic apoptotic bodies. these cells are then macropinocytosed by either dendritic cells or macrophages (mercer and helenius, 2010) . among the plethora of compounds designed to inhibit infectious viruses, only a few (< 100) have been approved for clinical use (de clercq, 2004; de clercq and li, 2016) . nevertheless, some effective antiviral drugs have been on the market for several decades, such as the anti-influenza a virus drug amantadine, marketed under the trade name symmetrel (by dupont), which was approved in 1966. in 1982, burroughs-wellcome introduced acyclovir as an inhibitor of herpesviruses. its remarkable specificity is connected with its activation by viral thymidine kinase-catalyzed phosphorylation. however, due to development of drug resistance in a number of viruses, especially rna viruses, there is a continuous need to design and test new inhibitors, preferably targeting different steps of viral life cycles. here, we provide insights into the world of biochemical and cell-based assays that were developed to test antivirotics targeting various steps in the viral life cycle. different types of assays, including cell-cell fusion assays, cell-virus fusion assays with pseudotyped viral particles, and in vitro biochemical assays have been developed to screen inhibitors of viral entry. in enveloped viruses, entry is initiated by fusion of the viral envelope with the target cellular membrane. the entry mechanism has been well-described for hiv-1, in which it is mediated by env glycoprotein, consisting of transmembrane gp41 and surface gp120 subunits. binding of gp120 to its cellular receptor, cd4, and to one of two co-receptors, cxcr4 or ccr5, triggers a refolding of gp41 that promotes fusion of the viral and cellular membranes. the refolding involves oligomerization of the extracellular n-and c-terminal heptad repeat (hr) domains of gp41, which leads to the formation of a 6-helical bundle [reviewed in (melikyan, 2008) ]. jiang et al. established an in vitro system to quantify formation of the hiv-1 gp41 6-helical bundle (jiang et al., 1999 ). in their system, the bundle is formed by mixing equimolar concentrations of peptides derived from the n-and c-hr regions of gp41. elisa using the monoclonal antibody nc-1, which specifically recognizes and binds an epitope formed on the gp41 6-helix bundle but not the individual peptides, enables screening for compounds that prevent formation of fusion-active gp41. for hts of hiv-1 fusion inhibitors, this method was modified to use a fluorescence-linked immunosorbent assay (flisa), in which the c-hr peptide was replaced with fitc-conjugated c-hr peptide (boyer-chatenet et al., 2003) . cell-based assays are routinely used to screen viral entry inhibitors. high throughput formats have been developed for screening of hiv-1 fusion inhibitors. cell-cell fusion assays involve two types of cells: effector cells that stably (bradley et al., 2004) or inducibly (herschhorn et al., 2011; ji et al., 2006) express hiv-env glycoprotein and target cells that express cd4 and either cxcr4 or ccr5. co-cultivation of these cells leads to hiv-1 env-mediated cell membrane fusion, resulting in formation of multinucleated syncytia. membrane fusion induces expression of a reporter protein such as luciferase (herschhorn et al., 2011; ji et al., 2006) or β-galactosidase (bradley et al., 2004) . one such assay enables determination of both the efficiency and specificity of fusion inhibitors (herschhorn et al., 2011) . this approach uses effector cells that express both hiv-1 env and the renilla luciferase (r-luc) reporter protein using inducible tetracycline-controlled transactivator (tta) and target cells that express the hiv-1 receptor (cd4) and coreceptor (ccr5) and contain the firefly luciferase (f-luc) reporter gene under the control of a tta-responsive promoter. upon fusion of the effector and target cells, tta enters the target cells and activates the expression of the f-luc reporter. the inhibition of fusion of cellular membranes is determined as a decrease in f-luc luminescence, and the inhibitor specificity is measured as the r-luc activity (herschhorn et al., 2011) . based on an hiv-1 cell-cell fusion method that uses a computercontrolled digital image analysis system for automatic quantitation , a modified method was developed to screen inhibitors targeting mers-cov s protein (lu et al., 2014) . to test potential fusion inhibitors, effector cells stably expressing the mers-cov spike protein s2s and egfp were used to mediate fusion with target cells expressing the dpp4 receptor (lu et al., 2014) . virion-based fusion assays are another category of cell-based fusion assays. one such approach is based on production of chimeric hiv-1 virions carrying β-lactamase-vpr chimeric protein (blam-vpr). chimeric hiv released into the cell culture media is isolated by ultracentrifugation and used to infect target cells. entry of the virions into the cytoplasm is detected by cleavage of a fluorescent substrate by βlactamase. the fluorescence shift corresponds to the fusion efficacy and is measurable by fluorescence microscopy, flow cytometry, or fluorometric plate reader (cavrois et al., 2002; cavrois et al., 2004 cavrois et al., , 2014 . modification of this assay by constructing pseudotyped hiv-1 virions in which hiv-1 env was replaced with ebola virus glycoprotein (gp) has also been described (yonezawa et al., 2005) . tscherne et al. developed an approach to monitor viral entry using the blam reporter (tscherne et al., 2010) . their assay uses influenza virus-like particles (vlps) bearing the influenza membrane proteins hemagglutinin and neuraminidase. blam tagged with influenza matrix protein (m1) is incorporated into the vlps and delivered into target cells. upon release, blam can be detected by flow cytometry, microscopically, or fluorometrically. a rapid cell-based hts method was developed to assess sars-cov entry inhibitors (zhou et al., 2011) . this dual envelope pseudovirion (dep) assay employs two hiv pseudoviruses: one encodes an envelope protein from the target virus and firefly luciferase and the second encodes a control, unrelated viral envelope protein and renilla luciferase. reporter expression is determined with the dual-glo luciferase assay system (promega). inclusion of the unrelated envelope protein greatly reduced false positive hits (zhou et al., 2011) . the method was further used to screen compounds that inhibit entry of filoviruses, including ebola virus . a similar approach employing four pseudotyped hiv viruses, carrying marburg virus glycoprotein, hemagglutinin and neuraminidase isolated from a/goose/qinghai/59/05 (h5n1) influenza virus, ebolavirus zaire envelope glycoprotein, and lassa virus envelope glycoprotein, has been used for entry inhibitor screening . this screening identified multiple compounds with potent inhibitory activity against entry of both marburg and ebola viruses in human cancer cell lines, and confirmed their anti-ebola activity in human primary cells . other pseudotyped viral assays have been used to screen entry inhibitors of sars-cov, ebola, hendra, and nipah viruses. to establish infection, the glycoproteins of these viruses must be processed by the host intracellular lysosomal protease cathepsin l (catl). hts resulted in identification of several inhibitors that block the cleavage of viral glycoprotein but not catl itself (elshabrawy et al., 2014) . until recently, the development of anti-hbv therapeutics had been limited by the lack of an in vitro infection system. several aspects of the hbv life cycle have been elucidated using in vitro production of hbv particles after transfection of human hepatoma cell lines (hepg2) with recombinant hbv dna and by establishment of hepatoma cell lines with the entire hbv genome integrated, such as the hepg2.2.15 cell line (ladner et al., 1997; sells et al., 1987; sureau et al., 1986) . in addition to differentiated human (phhs) (gripon et al., 1988) and tupaia belangeri (glebe et al., 2003) hepatocytes, the heparg cell line, which exhibits hepatocyte-like characteristics, also supports hbv replication (gripon et al., 2002) . the identification of sodium taurocholate cotransporting polypeptide (ntcp) as an hbv receptor by yan et al. opened possibilities to use ntcp-complemented hepg2 cells not only for studies of hbv replication mechanisms but also for hts of inhibitors (yan et al., 2012) . in an infection competition assay, hbv particles were isolated and used to infect heparg and phhs cells that had been pre-incubated with hbv envelope protein-derived peptides to test their potential activity to block hbv infection. twelve days after infection, viral rnas were quantified by northern blot (gripon et al., 2002 (gripon et al., , 2005 . using this assay, researchers identified a peptide that specifically prevents hbv and hdv entry into heparg and phhs cell lines (gripon et al., 2005) , primary cultures of tupaia hepatocytes (glebe et al., 2005) , and cells in vivo (lütgehetmann et al., 2012; petersen et al., 2008; volz et al., 2013) . recently, a phase iia clinical trial of a first-in-class entry inhibitor (myrcludex-b) that functions as an ntcp inhibitor was promisingly completed (bogomolov et al., 2016) . development of cell culture systems producing hcv pseudoparticles (hcvpp) and infectious hcv particles (hcvcc) has shed light on hcv interactions and enabled discovery of antiviral drugs and vaccines (colpitts et al., 2016) . hcvpp consist of hcv e1 and e2 glycoproteins enveloping a retroviral particle that packages gfp mrna during assembly (bartosch et al., 2003) . the entry efficiency of hcvpp can be quantified by facs analysis as the number of gfp-positive cells. use of this screening system led to discovery of a triazine derivative that blocks the entry of hcv (baldick et al., 2010) . production of hcvcc is a robust system to generate infectious hcv in naïve cells (kato et al., 2006; zhong et al., 2005) . the anti-hcv activity of hundreds of compounds approved for a wide variety of indications was determined immunochemically with anti-hcv e2 antibody in 96-well plate format. over thirty compounds displayed anti-hcv activities, most of which were directed against hcv entry (gastaminza et al., 2010) . the majority of viruses enter cells by endocytosis. unfortunately, there are no suitable experimental techniques for endosome handling, making it difficult to study early steps in the viral life cycle such as uncoating and capsid disassembly. viruses that enter cells by direct membrane fusion, such as hiv-1, are an exception. there are several methods available to monitor and quantify hiv uncoating. as some of these were reviewed recently by campbell and hope (campbell and hope, 2015) , we will discuss them very briefly. two main techniques are used in these assays: ultracentrifugation or utilization of hiv-1 specific cellular restriction factors. the "in vitro core-stability assay" is based on ultracentrifugation of released hiv-1 virions through a detergent layer, where the viral membrane dissolves, into a sucrose gradient, where the viral cores are concentrated (shah and aiken, 2011) . the "fate of capsid" assay uses ultracentrifugation through a sucrose cushion to separate the hiv-1 core from a whole cell lysate prepared shortly after infection (stremlau et al., 2006) . the "csa-washout assay" involves specific cellular factors (trim-cyclophilin) that restrict hiv-1 infection by binding to the capsid core and cyclosporine a (csa), which can effectively turn off restriction (hulme et al., 2011) . recently, a novel entry/uncoating assay (eurt), an alternative to blam-vpr (described in section 3.1), was reported (da silva santos et al., 2016). it quantifies the protein product of a virion-packaged mrna reporter upon uncoating. a method to monitor the uncoating/disassembly of the capsid of influenza a virus, which enters the cell by endocytosis, also is based on ultracentrifugation (stauffer et al., 2016) . purified virions are separated using velocity gradient centrifugation through a two-layer glycerol gradient. similar to the "in vitro core stability" assay for hiv, the sedimenting viruses migrate through the detergent-containing layer of the gradient, which dissolves the membrane to release the viral core. moreover, modification of the detergent-containing glycerol layer-for example, by lowering ph, changing ionic strength, or adding putative viral uncoating factors-enables study of the factors or compounds that affect viral uncoating in vitro. depending on the virus type, either dna or rna polymerases replicate the viral genome. thus, these enzymes play a key role in viral life cycles. reverse transcriptase, an rna-dependent dna polymerase of retro-and hepadnaviruses, is unique among nucleic acid polymerases. despite the different mechanisms of viral replication, polymerases, which are essential for all viruses, are excellent targets for antiviral therapies. polymerase inhibitors represent the vast majority of clinically approved antiviral drugs, followed by protease inhibitors, immunostimulators, entry inhibitors, and integrase inhibitors [reviewed in (de clercq and li, 2016) ]. polymerases continue to be a preferred target for newly designed inhibitors (clercq, 2008; de clercq and li, 2016) . polymerase inhibitors may be nucleoside and nucleotide analogs, pyrophosphate analogs, or nonnucleosides, such as allosteric inhibitors (de clercq and li, 2016; öberg, 2006) . non-specific approaches such as plaque assays were initially used to monitor the effectiveness of polymerase inhibitors (tino et al., 1993; tisdale et al., 1993) . the activity of these inhibitors also can be evaluated based on their ability to prevent the cytopathic effects of the virus (zhang et al., 2017) . more straightforward assays involve measurement of incorporated radio-labeled nucleotides, which directly reflects the polymerase activity (coates et al., 1992; hirashima et al., 2006; joyce, 2010) ; these include hts methods using homopolymeric polycytidylic acid and [ 3 h]-gtp (amraiz et al., 2016) . alternatively, the pyrophosphate released during the polymerase reaction can be measured luminometrically by combining the primer extension with the commercial piper assay (malvezzi et al., 2015) . the pyrophosphate anion also can be detected with dna-attached magnetic nanoparticles (tong et al., 2015) or quantum dots (chai et al., 2015) . frequently used fluorescence methods avoid the use of radiochemicals. these methods exploit a fluorescent label, such as dsdna binding dyes like sybr green or pi-cogreen (driscoll et al., 2014; holden et al., 2009; zipper et al., 2004) , or fret between two nucleotides (schwartz and quake, 2009; weiss, 2000) . numerous kits for quantification of products of both dna and rna polymerases, including reverse transcriptase, are commercially available (bustin, 2000) . quantitative real-time pcr is a preferred method to monitor the activity of dna polymerases (cellular as well as viral) in the presence of inhibitors (beadle et al., 2016; zweitzig et al., 2012) , and quantitative real-time reverse-transcription pcr has become standard for screening inhibitors of viral rna polymerases okon et al., 2017; pelliccia et al., 2017; zhang et al., 2017) . white et al. described a hts method using a microfluidic apparatus combined with digital pcr for single-cell analysis (white et al., 2013) . in their method, a fluorescently labeled template also serves as a primer, due to stem formation. it emits a measurable polarization signal both upon binding of the polymerase and extension (mestas et al., 2007) . the assay has been used for hts of inhibitors of poliovirus rna polymerase (campagnola et al., 2011) . when available, viral genomes modified with reporter genes can be used for cell-based luminescent or fluorescent screening of viral inhibitors (beadle et al., 2016; edwards et al., 2015; fenaux et al., 2016; feng et al., 2014; lo et al., 2017; madhvi et al., 2017; wang et al., 2015c) . this approach can be extended to screen inhibitors of other enzymes. in the aptamer-based approach, a dna template encodes rna of interest joined to a fluorescence module and ribozyme sequence. when transcribed, the fluorescence module is released and detected (höfer et al., 2013) . hcv uses an rna-dependent rna polymerase (rdrp). a unique cell-based assay for monitoring hcv rna polymerase (ns5b) activity, based on the innate immune signaling molecule retionic acid-inducible gene i product (rig-i), has been developed (ranjith-kumar et al., 2011) . the rig-i-like receptors are cytosolic proteins that recognize viral rna and induce production of proinflammatory cytokines and interferon-activated genes (jensen and thomsen, 2012) . rig-i triggers cytokine production stimulated by various viral rnas from different families, including flaviviridae, paramyxoviridae, rhabdoviridae, orthomyxoviridae, and arenaviridae, as well as ebola virus rna (jensen and thomsen, 2012) . the assay is based on recognition of hcv rna produced by active ns5b by rig-i, followed by rig-i-mediated activation of firefly luciferase expression controlled by the interferon b promoter. renilla luciferase expression is used for normalization of transfection efficacy. the viral grnas of some rna viruses are modified at the 5′ ends with cap structures, which may be either acquired from the host cell mrna (e.g. in influenza virus) or newly synthesized (e.g. flaviviruses). the methylation of viral grna mimics that of cellular mrna cap structures to enhance the chances of the virus to escape from cellular defense mechanisms and also to increase the efficiency of translation of viral proteins. virus-specific methyltransferases are thus a promising therapeutic target. virus-encoded methyltransferases have been identified and characterized in flaviviruses such as zika virus coutard et al., 2017; duan et al., 2017; munjal et al., 2017; zhao et al., 2017) , west nile virus, and dengue virus (dong et al., 2012) ; rhabdoviruses such as vesicular stomatitis virus (vsv) (rahmeh et al., 2009 ); coronaviruses such as sars (wang et al., 2015b) ; and roniviruses (zeng et al., 2016) . in flaviviruses, the n-terminal part of ns5 methyltransferase catalyzes cap formation via both guanine n-7 and ribose 2′-oh methylations at the 5′ end of grna, and the c-terminus of the enzyme is responsible for the rna polymerase activity (ray et al., 2006; zhao et al., 2015) . the c-terminal part of the sars nsp14 protein exhibits guanine n7-methyltransferase activity, forming the grna cap, while the 3′-5′ exoribonuclease activity of the n-terminus enhances the fidelity of viral replication (case et al., 2016; minskaia et al., 2006) . in vsv, the methyltransferase activity resides in the conserved region vi of the multifunctional large polymerase protein (li et al., 2005; ma et al., 2014) . some cellular methyltransferases regulate viral infections, as shown for herpes simplex virus, for which epigenetic control is involved in viral latency (cliffe and wilson, 2017) . inhibition of human histone methyltransferases such as histone-lysine n-methyltransferase (ezh2/ 1) (arbuckle et al., 2017) or lysine-specific demethylase-1 (lsd1) induces antiviral signaling pathways (liang et al., 2009) . the inhibitory effect of histone demethylase activity has been demonstrated for human cytomegalovirus (gan et al., 2017) and herpes simplex virus (hill et al., 2014; liang et al., 2013) . the activity of purified recombinant methyltransferases can be determined by measuring the methylation by-product s-adenosyl homocysteine (sah) using commercially available kits. in one such assay, conversion of s-adenosyl methionine (sam) to sah is monitored luminometrically via luciferase reaction, in which measurable atp is generated through a sequence of reactions with mtase-glo™ reagent (promega). the epigeneous™ methyltransferase kit (cisbio bioassays) is based on competition of produced sah with fluorescently labeled sah (sah-d2 tracer) for binding to a terbium cryptate-labeled anti-sah antibody. the decrease in fret between the tracer and antibody is then evaluated. elisa using anti-5-methylcytosine antibody can be used to quantify methylation of immobilized cytosine-rich dna substrate (e.g. epiquik™ dna methyltransferase activity/inhibition assay kit; epigentek). this method was modified with homogenous time resolved fret (degorce et al., 2009) to screen a library of inhibitors against sars-cov nsp14 . flaviviral and human cap n7-mtases have been screened with radioactive assays using 3 h-labeled sam and gpppac4 or 7m gpppac4 synthetic rnas. the 3 h methyl transferred onto deae filter-bound rna can be measured by scintillation after multiple washings to remove unincorporated 3 h-sam . alternatively, in vitro transcription can be carried out using 3 h-sam. the radioactively labeled products are then resolved by thin-layer chromatography and developed using a phosphorimager (li et al., 2007) . a yeast cell-based method was established based on the finding that coronavirus methyltransferase can functionally replace a methyltransferase essential for yeast viability sun et al., 2014) . in this method, sun et al. constructed recombinant yeasts producing the viral methyltransferase instead of the yeast one (sun et al., 2014) . this strain was used for a hts of methyltransferase inhibitor activity that negatively correlated with the cell density after 20 h incubation. virus-encoded atpase-driven helicases have been identified in numerous human pathogens. helicases from several (+)rna viruses have been characterized, including ns3 helicases from flaviviruses such as dengue virus, hcv, west nile virus, yellow fever virus, and japanese encephalitis virus (cao et al., 2016; gu and rice, 2016; jain et al., 2016; lin et al., 2017; nedjadi et al., 2015; wu et al., 2005) . sars and mers coronaviruses encode nsp13 with helicase activity (adedeji and lazarus, 2016; hao et al., 2017; seybert et al., 2000) . in semliki forest virus, a representative member of the togavirus family, helicase activity is encoded by the n-terminal domain of nsp2 protein. helicases are also common in some dna viruses, including poxviruses. these include the vaccinia virus helicase-primase d5 (bayliss and smith, 1996; hutin et al., 2016) , e1 protein of bovine papilloma virus 1 (yang et al., 1993) , and the large tumor antigen of sv40 (stahl et al., 1986) . helicases appear to be attractive targets for antiviral drugs (briguglio et al., 2011; frick, 2003; reynolds et al., 2015) , but development of such compounds is challenged by cytotoxicity and bioavailability issues (kwong et al., 2005) . traditional methods for monitoring the activity of rna helicases use radioactively labeled dsrna substrates and follow the unwinding reaction by electrophoretic separation (nondenaturing page) of the ss reaction products, which are detected autoradiographically (adedeji et al., 2012; utama et al., 2000) . to determine whether the inhibitor affects binding of helicase to nucleic acid, a standard gel mobility shift assay is usually used (adedeji et al., 2012) . helicase activity is fueled by atp hydrolysis; thus, inhibition of atpase activity became another possible antiviral strategy. atpase activity can be determined either by the decrease in atp or formation of adp (using commercially available fluorescent anti-adp antibodies) or inorganic pyrophosphate. phosphates released by atp hydrolysis can form a molybdophosphate complex that can be measured colorimetrically using malachite green, quinaldine red, or rhodamine b (baykov et al., 1988; debruyne, 1983; miyata et al., 2010) or by lightscattering (oshima et al., 1996) . the colorimetric methods can be miniaturized for hts (zuck et al., 2005) . the absorbance-based assay was also converted into a hts method based on fluorescence quenching by a colored quinaldine red complex ). an alternative method employs a europium-tetracycline complex for luminescent determination of inorganic phosphate (schäferling and wolfbeis, 2007) . a more complex coupled enzyme colorimetric assay (with maltose phosphorylase, glucose oxidase, and horseradish peroxidase) was used for hts of atpase inhibitors (avila et al., 2006) . assays for luminescent and fluorescent screening of atpase activity, including an immunochemical method using fluorescently labeled anti-adp antibodies, have been reviewed by shadrick and colleagues (shadrick et al., 2013) . in a radioactive method using [γ-32 p]atp, the amp product was separated from unreacted atp by thin-layer chromatography on polyethyleneimine-cellulose and visualized autoradiographically (adedeji et al., 2012) . several research groups have described fluorescence assays to identify inhibitors targeting sars-cov helicase (nsp13) (adedeji et al., 2012; cho et al., 2015; özeş et al., 2014) . the substrate is usually a dsdna oligonucleotide consisting of a fluorescently labeled strand annealed to the complementary strand carrying a quencher. this approach was also adapted for real-time determination of the rna helicase activity of hcv (tani et al., 2010) . in this assay, an ssdna capture strand complementary to the strand carrying the quencher was used to prevent reannealing of the unwound duplex. recently, a colorimetric assay for monitoring helicase activity using dna-conjugated gold nanoparticles was developed (deka et al., 2017) . this method is based on shifts in the optical properties of nanoparticles due to dna unwinding and allows simple screening of inhibitor activity. the dsdna melting curves can be determined spectrophotometrically (at 524 nm and 260 nm) or even by the naked eye. another fluorescence hts of dengue virus ns3 helicase inhibitors measures the unwinding of a double-labeled molecular beacon (basavannacharya and vasudevan, 2014) . other approaches involve graphene oxide-based fluorescence monitoring of viral helicase activity [reviewed in (jang et al., 2013) ]. a gquadruplex-based method for label-free determination of hcv helicase ns3 activity measures changes in the luminescence of transition metal complexes with dna upon helicase-mediated quadruplex melting (leung et al., 2015) . both protein tyrosine kinases and protein serine/threonine (s/t) kinases have been found in viruses. tyrosine kinase function is wellunderstood in connection with oncogenic retroviruses, [for review on retroviral oncogenes, see (vogt, 2012) ]. in contrast to tyrosine kinases from oncogenic viruses, such as rous sarcoma virus src tyrosine kinase, viral s/t kinases share little homology with cellular enzymes. they are exclusively encoded by large dna viruses (e.g. herpesviruses), in which they play important roles in viral virulence, helping the virus to escape defense mechanisms such as those regulated by cytokine signaling pathways (jacob et al., 2011; sato et al., 2017) . their autophosphorylation and transphosphorylation activities mimic those of cellular cyclin-dependent kinases such as cdc2. for example, viral kinases can phosphorylate translation elongation factor 1 delta (ef-1δ) (jacob et al., 2011; kawaguchi and kato, 2003) . all herpesviruses encode the s/t protein kinase ul13, and us3 s/t kinases have been described in the alphaherpesvirus subfamily (kato, 2016; kawaguchi and kato, 2003) . in addition to protein kinases, hsv encodes a thymidine kinase. unlike cellular thymidine kinase, hsv thymidine kinase has a wide substrate specificity that includes pyrimidine and purine phosphonate analogs (e.g. acyclovir, ganciclovir, penciclovir) (de clercq and li, 2016) . in the body, ganciclovir is phosphorylated by cellular kinases and penciclovir and acyclovir by virus-encoded thymidine kinases to the active nucleoside triphosphate forms of the drugs that inhibit viral dna synthesis (de clercq and li, 2016; kokoris and black, 2002) . some cellular protein kinases appear to support viral replication. for example, polo-like kinases induce early stages in the influenza virus life cycle (pohl et al., 2017) , and human protein kinase c regulates the assembly of the ribonucleoprotein complexes in influenza virus (mondal et al., 2017) . inhibitors of abelson tyrosine-protein kinase 2 are active against sars and mers coronaviruses (coleman et al., 2016) . several in vitro approaches can be used to determine kinase activity as well as the activity of kinase inhibitors. analyses of the cellular phosphoproteome, sometimes accompanied by phosphopeptide enrichment, have become standard to determine kinase activity (lea and simeonov, 2011; meyer et al., 2017; vyse et al., 2017) . these assays can be used to assess the impact of inhibitors on the overall phosphoproteome in mammalian cells. although these methods provide complex information about the overall array of kinases and phosphatases in the cell (olsen et al., 2006) , they are not applicable to screening inhibitors of a single viral kinase. for these purposes, in vitro assays with recombinant kinases have been developed [for review see ], including some hts fluorescence methods (zegzouti et al., 2009 ) that can replace radioactive techniques using [γ 32 p]atp (sanghera et al., 2009) . mass spectrometric analysis also can be used to identify in vitro kinase inhibitors. for these analyses, synthetic peptides, proteins, or phosphatase-and heat-treated tissue samples (to dephosphorylate the proteins and inactivate all enzymes, respectively) are subjected to kinase treatment in the presence of inhibitors (huang et al., 2007; meyer et al., 2017; xue et al., 2012) . recombinant kinase activity in the presence of inhibitors can be quantified as atp consumption or adp production in the phosphorylation reaction by numerous commercial kits. other methods monitor binding of inhibitors to phage-displayed kinases in ligand competition assays (fabian et al., 2005) . fluorescence methods including fret, fluorescence polarization or intensity endpoint measurement, and lifetime imaging of fluorescence including fluorescence biosensors (zhang and allen, 2007) have been reviewed elsewhere. sulfonamido-oxine labeled peptides can be used as chromophores that bind mg 2 + upon phosphorylation and emit chelation-enhanced fluorescence (devkota et al., 2013; luković et al., 2008) . kinase-catalyzed phosphorylation of fluorescent peptides promotes their binding to metal-coated nanoparticles, which decreases their mobility and enhances measurable fluorescence polarization (lea and simeonov, 2011; sportsman et al., 2004) . tbiii complexes, in which phosphotyrosine induces fluorescence emission (wang et al., 2015a) , may be used to evaluate protein tyrosine kinase activity (akiba et al., 2015; sumaoka et al., 2016) . fluorescence polarization methods also can be useful for drug screening [reviewed in (hall et al., 2016) ]. other alternatives are immunochemical methods that use antibodies specific to the phosphorylated amino acids, such as phosphotyrosine (li et al., 2001; youngren et al., 1997) or phosphoserine/ phosphothreonine. these antibodies can be used to detect protein/ peptide phosphorylation by western blotting, elisa, or immunoprecipitation of phosphorylated proteins for further mass spectrometry-based analysis (grønborg et al., 2002; zhang et al., 2002) . an elegant approach that limits the false-positive hits in screening of specific kinase inhibitors is based on an in situ proximity ligation assay using both an antibody against the target protein and an anti-phosphotyrosine antibody (leuchowius et al., 2010) . both antibodies are coupled with oligonucleotides, which when brought together due to antibody binding, can be enzymatically ligated and replicated through rolling circle amplification to form a long linear tandem repeat of sequences detectable by a complementary fluorescent oligonucleotide. in addition to protein kinases, some lipid kinases have been targeted by antivirals. one example is sphingosine kinase 1, which affects replication of dengue virus (aloia et al., 2017) . its activity can be determined by measuring the production of 32 p-labeled sphingosine-1phosphate from sphingosine and [γ 32 p]atp (clarke et al., 2016; pitman et al., 2012) . retroviral integrase inhibitors are a new type approved new type of inhibitors imposed by the emergence of drug-resistant mutants. hiv integrase activities, integrase inhibitors, and drug resistance have been discussed in detail elsewhere (andrake and skalka, 2015; anstett et al., 2017; hajimahdi and zarghi, 2016; liao et al., 2010; podany et al., 2017; thierry et al., 2016) . methods to assess the two major activities of integrase-end processing of the reverse transcription product and its joining to target chromosomal dna-have been reviewed in detail by several groups (engelman and cherepanov, 2014; marchand et al., 2001; merkel et al., 2009) . initial methods used radioactively labeled dna oligonucleotides comprising the terminal cis-acting sequences of linear viral dna required for integration. the joining of the processed strand to the other strand (self-integration) or to supplemented target dnas can be analyzed by page (katz et al., 1990; katzman et al., 1989) . a less time-consuming, non-radioactive method involves timeresolved fluorescence anisotropy measurement using a 21-meric oligonucleotide fluorescently labeled on the terminal gt dinucleotide. this assay monitors the binding of integrase to the substrate as well as the subsequent 3′-processing reaction, which both change the anisotropy (guiot et al., 2006) . alternatively, the yields of both the processing and joining reactions can be measured upon separation of the radioactively labeled product from the rest of the dna molecule using adsorption to pei-cellulose (muller et al., 1993) . a real-time hts method measures fluorescence emission resulting from removal of the 3′-terminal dinucleotide, labeled with a quencher, by integrase (he et al., 2007) . han et al. described a fluorescence method to screen molecules that inhibit binding of integrase to viral dna . methods evaluating the integrase strand transfer reaction have been modified to a high-throughput format using magnetic beads (he et al., 2008) or streptavidin-coated microplates (john et al., 2005) . a method to assess strand transfer by time-resolved fret with a europium-streptavidin-labeled substrate has been optimized for 384-and 1536-well plate formats (wang et al., 2005) . the hbv capsid protein is the building block of the viral core, surrounding the viral nucleic acid (pre-genomic rna, pgrna) and reverse transcriptase. the hbv core is icosahedral, formed by 240 copies of capsid protein dimers. in vitro hts of hbv core assembly inhibitors using a modified hbv capsid protein has been described (stray et al., 2006) . the capsid protein was modified by deleting the nucleic acid binding domain, which is dispensable for capsid assembly, and the nterminal assembly domain alone was used in the assay. to fluorescently label the hbv capsid protein, all cysteine residues dispensable for assembly were replaced with alanines. a unique cysteine residue (c150) was c-terminally joined to the assembly domain and labeled with fluorescent bodipy-fl dye (c150bo). during assembly, capsid proteins dimerize, bringing c150bo residues close together and resulting in c150bo fluorescence self-quenching. following incubation of the labeled hbv protein with inhibitors, fluorescence was measured in black 96-well microtiter plates. development of in vitro assembly systems has contributed greatly to current understanding of the structure of retroviral particles and mechanisms of virion formation. these systems also became the base for several high throughput assays for screening assembly inhibitors. during the last 20 years, a number of in vitro assembly assays have been established, mainly for hiv-1 (campbell et al., 2001; campbell and rein, 1999; ehrlich et al., 1992; gross et al., 2000; lanman et al., 2002) , mason-pfizer monkey virus (bohmova et al., 2010; klikova et al., 1995; rumlova-klikova et al., 2000; rumlova-klikova et al., 1999; ulbrich et al., 2006) , rous sarcoma virus vogt, 1995, 1997; purdy et al., 2008 purdy et al., , 2009 , and murine leukemia virus (dolezal et al., 2016; hadravova et al., 2012; cheslock et al., 2003) . hts assays for inhibitors of hiv-1 assembly include several methods using purified hiv-1 ca or ca-nc proteins. one of them, the turbidimetric assay, is based on the observation that direct dilution of the hiv-1 capsid protein (ca) into a high-salt solution (1.6-2.2 m nacl) leads to the formation of tubular structures. as the tube formation is accompanied by an increase in light scattering, assembly can be detected as an increase in turbidity, and the rate of turbidity change is proportional to the rate of the assembly (lanman et al., 2002) . other method published by lemke et al., exploits the affinity of nucleocapsid (nc) to a short tg-rich deoxyribooligonucleotide, d(tg25), which is used as a scaffold (lemke et al., 2012) . this arrangement enables ca to assemble at much lower protein and salt concentrations than in the turbidimetric assay (lanman et al., 2002) . biotin-labeled d(tg25) bound on the surface of neutravidin-coated microtiter well plates nucleates assembly of complexes of ca-nc and soluble fluorescein-labeled d(tg25). fluorescence is measured after washing to remove the unbound and unassembled material from the captured assembly products (lemke et al., 2012) . similarly, the faith assay uses a dually labeled oligonucleotide (tqon). however, in this case, the ssdna oligonucleotide tqon is labeled with the reporter dye fluorescein (fam) as well as black hole quencher (bhq); thus, it does not emit any fluorescence. the assembly reaction is triggered by mixing hiv-1 ca-nc or a gagtruncated assembly-competent version with tqon. following incubation, during which tqon is incorporated into the particles, exonuclease is added to degrade unbound tqon, while co-assembled tqon is protected from cleavage. degradation of free unbound tqon with exoi results in separation of fam from its quencher, and the emitted fluorescence is measured (hadravova et al., 2015) . phage display has been employed to screen peptide inhibitors of hiv-1 assembly (sticht et al., 2005) . a commercial library of m13-derived phages presenting random 12-amino-acid peptides was analyzed for specific binding to purified ca or ca-nc proteins. the specifically bound phages were sequenced, and corresponding peptides were chemically synthesized and re-tested in an in vitro assembly assay (gross et al., 2000) . late in the retroviral life cycle, grna is incorporated into the nascent particle during assembly at the plasma membrane. nc contains two zinc-finger domains that are responsible for specific binding of grna. to screen for compounds that would prevent nc-rna/dna interactions, a hts system consisting of two sequential screens was developed (breuer et al., 2012) . the primary screen uses fluorescence polarization (fp), while the secondary one uses differential scanning fluorimetry (dsf). the combination and order of these two techniques were selected to first identify compounds that disrupt interactions between dna and nc, and then identify the compounds binding to nc during the secondary screen. a rapid and simple turbidimetric method was developed to screen inhibitors of assembly of hcv core protein (fromentin et al., 2007) . for the in vitro assembly reaction, two components, an n-terminal part of the hcv core protein corresponding to the minimal assembly competent domain and rna corresponding to the full-length 5′utr of hcv, were used. assembly of hcv nucleocapsid-like particles was initiated by mixing the purified protein and nucleic acid, and the assembly process was monitored by measuring turbidity at 350 nm. numerous viruses, including picornaviruses, retroviruses, alphaviruses, and flaviviruses, encode proteases that are essential for their virulence. the majority of viral proteases specifically cleave viral polyprotein precursors to liberate the functional proteins of the virion. some viral proteases, such as the papain-like proteases of coronaviruses, also reprogram cellular signaling pathways, including ubiquitination mechanisms and interferon controlled responses, to prevent degradation of viral components (clementz et al., 2010; frieman et al., 2009; randow and lehner, 2009; xing et al., 2013) . numerous viruses, including papillomaviruses (bronnimann et al., 2016; buck et al., 2005) and retroviruses (hallenberger et al., 1992) , use also host cell proteases, mainly furin, to trim their envelope and surface proteins. this triggers conformational changes required for interaction with cellular receptors and membrane fusion in enveloped viruses. some viruses use proteases other than furin to modulate their infectivity, as shown for viruses entering airway epithelial cells [reviewed in (laporte and naesens, 2017) ] such as influenza virus (böttcher-friebertshäuser et al., 2010; kühn et al., 2016) , newcastle disease virus (gotoh et al., 1992) , and respiratory syncytial virus (sugrue et al., 2001) . extensive research efforts have yielded detailed information about hiv-1 protease and its inhibitors [reviewed in (de clercq, 2004; konvalinka et al., 2015; midde et al., 2016) ]. large amounts of data also are available for hcv (foote et al., 2011; pawlotsky et al., 2007; razonable, 2011) and sars-cov 3cl protease inhibitors (pillaiyar et al., 2016) , although there is not yet an inhibitor of the latter target approved for clinical use. numerous approved drugs are synthetic peptides derived from the natural proteolytic substrates of target viruses modified to improve the in vivo effects related to bioavailability, stability, and so on. numerous in vitro assays to monitor the activity of proteases and their inhibitors, including commercial kits, have been developed. classical methods use synthetic peptides that mimic the target sites of the protease. although some of the methods described here were not originally designed to screen the activity of viral proteases in the presence of inhibitors, they can be adapted for this purpose by simply changing the peptide sequence to the target site of the protease of interest. the cleavage yield is usually monitored either colorimetrically (ding and yang, 2015; zhou et al., 2014) or as a change in fluorescence triggered by the release of fluorescent labels such as 7-amido-4-methylcoumarin (amc) or rhodamine (grant et al., 2002) . fluorogenic substrates have been used to determine the activity of coronavirus proteases and screen inhibitors (kuo et al., 2004; lee et al., 2014; park et al., 2017; song et al., 2014; tomar et al., 2015; wang et al., 2016; yang et al., 2005; zhao et al., 2008) . some recently described arrangements employ nanoparticles (feltrup and singh, 2012; khalilzadeh et al., 2016; udukala et al., 2016; wang et al., 2014; zeng et al., 2015) or quantum dot bioconjugates (lee and kim, 2015; li et al., 2014; medintz et al., 2006) with immobilized fluorescently or luminescently labeled peptide substrates. alternatively, cleavage products may be monitored by analysis of proteolytic products by mass spectrometric methods (hu et al., 2015; joshi et al., 2017; lathia et al., 2011; rumlová et al., 2003) , analytical hplc (teruya et al., 2016) , or electrochemical methods based on the difference in penetration of substrate and cleavage products through the membrane of a polyionselective sensor (gemene and meyerhoff, 2011; han et al., 1996) . to study the specificity of inhibitor binding and to extend the research to rational design of inhibitors, x-ray or nmr structures of proteases in complex with the inhibitor may be determined, as reported in numerous cases for the proteases of hiv-1 [reviewed in (ghosh et al., 2016) ], hcv (yilmaz et al., 2016) , and mers . cell-based assays can provide additional information, including the capability of the inhibitor to pass through the cell membrane and its stability in the cytoplasm. a general determination of infectivity, such as a plaque cytotoxicity assay in the presence of protease inhibitor, may be used for confirmation. one elegant example exploits the cytotoxicity of hiv protease. cells are transfected with a protease precursor fused to gfp. in the absence of inhibitor, hiv-1 protease is autocatalytically activated and cleaves a broad variety of cellular proteins, resulting in activation of apoptosis and cell death (cummins and badley, 2010; rumlova et al., 2014) . this toxic effect, when suppressed by active inhibitors, results in production of a gfp signal in surviving cells (lindsten et al., 2001) . another elegant approach for hiv and coxsackievirus b3 proteases, which both undergo autocatalytic cleavage, employs constructs in which the protease gene is inserted between sequences encoding the dna-binding domain and the domain that activates transcription of the gal1-lacz reporter gene (dasmahapatra et al., 1992; murray et al., 1993) . the protease-mediated cleavage separates the dna-binding domain from the trans-activating domain and results in failure of reporter gene transcription. this approach has also been modified with gfp as a reporter gene (hilton and wolkowicz, 2010) . co-expression of cleavage-activated luciferase substrate and mers-cov protease permits both live-cell imaging and quantification of the enzyme activity (kilianski et al., 2013) . the need to develop new antiviral compounds will likely persist over the long term, although there has been enormous progress in molecular biology methods, especially rna silencing, bioinformatics, imaging, and structural biology techniques. viruses present challenging targets for drug development due their flexibility and adaptability caused by the error-prone copying of their genomes, which can result in emergence of drug-resistant mutants. viral integration into the host genome and inhibitor toxicity are other obstacles. here, we provide an overview of in vitro methods, including cell-based assays, that may be suitable for screening of antivirotics that interfere with the key steps of viral life cycles and target either virus or cell-encoded proteins required for the infectivity. biochemical characterization of middle east respiratory syndrome coronavirus helicase severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase components of adenovirus genome packaging click conjugation of a binuclear terbium(iii) complex for real-time detection of tyrosine phosphorylation pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo investigation of sphingosine kinase 1 in interferon responses during dengue virus infection viral apoptotic mimicry hiv-1 uncoating: connection to nuclear entry and regulation by host proteins development of robust in vitro rnadependent rna polymerase assay as a possible platform for antiviral drug testing against dengue retroviral integrase: then and now reactivation and lytic replication of kaposi's sarcoma-associated herpesvirus: an update hiv drug resistance against strand transfer integrase inhibitors toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay inhibitors of the histone methyltransferases ezh2/1 induce a potent antiviral state and suppress infection by diverse viral pathogens highthroughput screening for hsp90 atpase inhibitors a novel small molecule inhibitor of hepatitis c virus entry junction adhesion molecule is a receptor for reovirus infectious hepatitis c virus pseudoparticles containing functional e1-e2 envelope protein complexes suramin inhibits helicase activity of ns3 protein of dengue virus in a fluorescence-based high throughput assay format a malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay vaccinia virion protein i8r has both dna and rna helicase activities: implications for vaccinia virus transcription -phosphonomethoxy)ethyl]guanine (ode-bn-pmeg), a potent inhibitor of transient hpv dna amplification hepatitis b virus replication early steps in avian reovirus morphogenesis late budding domains and host proteins in enveloped virus release escape of non-enveloped virus from intact cells nonlytic spread of naked viruses nonlytic viral spread enhanced by autophagy components viral entry pathways: the example of common cold viruses herpes virus replication treatment of chronic hepatitis d with the entry inhibitor myrcludex b: first results of a phase ib/iia study effect of dimerizing domains and basic residues on in vitro and in vivo assembly of mason-pfizer monkey virus and human immunodeficiency virus cleavage of influenza virus hemagglutinin by airway proteases tmprss2 and hat differs in subcellular localization and susceptibility to protease inhibitors development and automation of a 384-well cell fusion assay to identify inhibitors of ccr5/cd4-mediated hiv virus entry identification of hiv-1 inhibitors targeting the nucleocapsid protein inhibition of rna helicases of ssrna(+) virus belonging to flaviviridae, coronaviridae and picornaviridae families furin cleavage of l2 during papillomavirus infection: minimal dependence on cyclophilins maturation of papillomavirus capsids absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays hiv-1 virion-cell interactions: an electrostatic model of pathogenicity and syncytium formation high-throughput screening identification of poliovirus rna-dependent rna polymerase inhibitors hiv-1 capsid: the multifaceted key player in hiv-1 infection in vitro assembly properties of human immunodeficiency virus type 1 gag protein lacking the p6 domain self-assembly in-vitro of purified ca-nc proteins from rous-sarcoma virus and human-immunodeficiency-virus type-1 in vitro assembly of virus-like particles with rous sarcoma virus gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles modulation of hiv-like particle assembly in vitro by inositol phosphates novel escrt functions in cell biology: spiraling out of control? molecular mechanism of divalentmetal-induced activation of ns3 helicase and insights into zika virus inhibitor design hcv infection by cell-to-cell transmission: choice or necessity? mutagenesis of s-adenosyl-l-methionine-binding residues in coronavirus nsp14 n7-methyltransferase m demonstrates differing requirements for genome translation and resistance to innate immunity components and regulation of nuclear transport processes a sensitive and specific enzyme-based assay detecting hiv-1 virion fusion in primary t lymphocytes fluorescence resonance energy transfer-based hiv-1 virion fusion assay hiv-1 fusion assay a reversible fluorescence nanoswitch based on carbon quantum dots nanoassembly for detection of pyrophosphate ion functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase tsg101: a novel anti-hiv-1 drug target protein-protein interfaces in viral capsids are structurally unique identification of a coumarin-based antihistamine as an anti-filoviral entry inhibitor charged assembly helix motif in murine leukemia virus capsid: an important region for virus assembly and particle size determination identification of a novel small molecule inhibitor against sars coronavirus helicase reduction in sphingosine kinase 1 influences the susceptibility to dengue virus infection by altering antiviral responses deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases emerging antiviral drugs restarting lytic gene transcription at the onset of herpes simplex virus reactivation (-)-2'-deoxy-3'-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type 1 and type 2 replication in vitro pushing the envelope: microinjection of minute virus of mice into xenopus oocytes causes damage to the nuclear envelope how viruses access the nucleus abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion structures of ns5 methyltransferase from zika virus highthroughput approaches to unravel hepatitis c virus-host interactions localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center endocytosis of viruses and bacteria zika virus methyltransferase: structure and functions for drug design perspectives structure and organization of paramyxovirus particles mechanisms of hiv-associated lymphocyte apoptosis: 2010 a novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized hiv-1 infection a genetic system for studying the activity of a proteolytic enzyme antiviral drugs in current clinical use approved antiviral drugs over the past 50 years inorganic phosphate determination: colorimetric assay based on the formation of a rhodamine b-phosphomolybdate complex htrf: a technology tailored for drug discovery -a review of theoretical aspects and recent applications dna-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors a novel mechanism underlying the innate immune response induction upon viral-dependent replication of host cell mrna: a mistake of + srna viruses' replicases. front high-throughput screens for eef-2 kinase quantitative serine protease assays based on formation of copper(ii)-oligopeptide complexes functional and structural characterization of novel type of linker connecting capsid and nucleocapsid protein domains in murine leukemia virus 2′-o methylation of internal adenosine by flavivirus ns5 methyltransferase a quantitative fluorescence-based steadystate assay of dna polymerase the crystal structure of zika virus ns5 reveals conserved drug targets high-throughput minigenome system for identifying small-molecule inhibitors of ebola virus replication assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay retroviral integrase structure and dna recombination mechanism a small molecule-kinase interaction map for clinical kinase inhibitors ultrastructural and biochemical basis for hepatitis c virus morphogenesis nuclear entry of dna viruses development of a fluorescence internal quenching correction factor to correct bont/a endopeptidase kinetics using snaptide antiviral nucleotide incorporation by recombinant human mitochondrial rna polymerase is predictive of increased in vivo mitochondrial toxicity risk a pathogenic picornavirus acquires an envelope by hijacking cellular membranes inhibition of hepatitis c virus replication by gs-6620, a potent c-nucleoside monophosphate prodrug transcription and replication mechanisms of bunyaviridae and arenaviridae l proteins fields virology boceprevir: a protease inhibitor for the treatment of chronic hepatitis c viral late domains hiv-1 assembly, release and maturation helicases as antiviral drug targets severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-κb signaling a method for in vitro assembly of hepatitis c virus core protein and for screening of inhibitors beyond tsg101: the role of alix in 'escrting' hiv-1 proteolytic cleavage of bovine m adenovirus 3-encoded pviii epigenetically repressing human cytomegalovirus lytic infection and reactivation from latency in thp-1 model by targeting h3k9 and h3k27 histone demethylases unbiased probing of the entire hepatitis c virus life cycle identifies clinical compounds that target multiple aspects of the infection detection of protease activities by flash chronopotentiometry using a reversible polycation-sensitive polymeric membrane electrode recent progress in the development of hiv-1 protease inhibitors for the treatment of hiv/aids structure and formation of the cytomegalovirus virion pre-s1 antigen-dependent infection of tupaia hepatocyte cultures with human hepatitis b virus mapping of the hepatitis b virus attachment site by use of infection-inhibiting pres1 lipopeptides and tupaia hepatocytes the ins and outs of hiv replication mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/pace to pc2 or pc1/ pc3 development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates hepatitis b virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide infection of a human hepatoma cell line by hepatitis b virus efficient inhibition of hepatitis b virus infection by acylated peptides derived from the large viral surface protein a mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, frigg, as a protein kinase a substrate varicella-zoster virus infectious cycle: er stress, autophagic flux, and amphisome-mediated trafficking a conformational switch controlling hiv-1 morphogenesis the cell biology of receptor-mediated virus entry the spring α-helix coordinates multiple modes of hcv (hepatitis c virus) ns3 helicase action relationship between the oligomeric status of hiv-1 integrase on dna and enzymatic activity bunyavirus: structure and replication in vitro assembly of virus-like particles of a gammaretrovirus, the murine leukemia virus xmrv faith -fast assembly inhibitor test for hiv progress in hiv-1 integrase inhibitors: a review of their chemical structure diversity fluorescence polarization assays in high-throughput screening and drug discovery: a review inhibition of furin-mediated cleavage activation of hiv-1 glycoprotein gpl60 selective monitoring of peptidase activities with synthetic polypeptide substrates and polyion-sensitive membrane electrode detection development of a fluorescence-based hiv-1 integrase dna binding assay for identification of novel hiv-1 integrase inhibitors alix rescues budding of a double ptap/ppey l-domain deletion mutant of ebola vp40: a role for alix in ebola virus egress crystal structure of middle east respiratory syndrome coronavirus helicase highthroughput real-time assay based on molecular beacons for hiv-1 integrase 3'-processing reaction a novel highthroughput format assay for hiv-1 integrase strand transfer reaction using magnetic beads nuclear egress of herpesviruses: the prototypic vesicular nucleocytoplasmic transport herpesvirus capsid assembly and dna packaging an inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of hiv-1 entry inhibitors inhibition of lsd1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes an assay to monitor hiv-1 protease activity for the identification of novel inhibitors in t-cells benzimidazole derivatives bearing substituted biphenyls as hepatitis c virus ns5b rna-dependent rna polymerase inhibitors: structure − -activity relationship studies and identification of a potent and highly selective inhibitor jtk-109 universal aptamer-based real-time monitoring of enzymatic rna synthesis factors affecting quantification of total dna by uv spectroscopy and picogreen fluorescence peptide code-on-a-microplate for protease activity analysis via maldi-tof mass spectrometric quantitation a systematic ms-based approach for identifying in vitro substrates of pka and pkg in rat uteri quantitative 3d video microscopy of hiv transfer across t cell virological synapses complementary assays reveal a relationship between hiv-1 uncoating and reverse transcription the ins and outs of eukaryotic viruses: knowledge base and ontology of a viral infection escrts are everywhere transport of the influenza virus genome from nucleus to nucleus domain organization of vaccinia virus helicase-primase d5 how viruses use the endoplasmic reticulum for entry, replication, and assembly experimental approaches to study genome packaging of influenza a viruses poliovirus-induced changes in cellular membranes throughout infection subversion of cellular autophagosomal machinery by rna viruses viral serine/threonine protein kinases structure of the ns3 helicase from zika virus a new helicase assay based on graphene oxide for anti-viral drug development filovirus budding oligomeric viral proteins: small in size, large in presence sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion development of a novel dual ccr5-dependent and cxcr4-dependent cell-cell fusion assay system with inducible gp160 expression a screening assay for antiviral compounds targeted to the hiv-1 gp41 core structure using a conformation-specific monoclonal antibody development and application of a highthroughput screening assay for hiv-1 integrase enzyme activities avian reovirus infections. revue scientifique et technique a structural and functional perspective of m. rumlová alphavirus replication and assembly the rational design of therapeutic peptides for aminopeptidase n using a substrate-based approach techniques used to study the dna polymerase reaction pathway molecular mechanism by which us3 protein kinase regulates the pathogenicity of herpes simplex virus type-1 cell culture and infection system for hepatitis c virus the avian retroviral in protein is both necessary and sufficient for integrative recombination in vitro the avian retroviral integration protein cleaves the terminal sequences of linear viral dna at the in vivo sites of integration protein kinases conserved in herpesviruses potentially share a function mimicking the cellular protein kinase cdc2 reduced graphene oxide decorated with gold nanoparticle as signal amplification element on ultra-sensitive electrochemiluminescence determination of caspase-3 activity and apoptosis using peptide based biosensor assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers efficient in-vivo and in-vitro assembly of retroviral capsids from gag precursor proteins expressed in bacteria virus strategies for passing the nuclear envelope barrier characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity retroviral proteases and their roles in virion maturation origin and evolution of eukaryotic large nucleo-cytoplasmic dna viruses adenovirus tales: from the cell surface to the nuclear pore complex the proteolytic activation of (h3n2) influenza a virus hemagglutinin is facilitated by different type ii transmembrane serine proteases characterization of sars main protease and inhibitor assay using a fluorogenic substrate viral and cellular rna helicases as antiviral targets inducible expression of human hepatitis b virus (hbv) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of hbv replication the autophagic machinery in enterovirus infection kinetic analysis of the role of intersubunit interactions in human immunodeficiency virus type 1 capsid protein assembly in vitro airway proteases: an emerging drug target for influenza and other respiratory virus infections multiplexed protease assays using element-tagged substrates fluorescence polarization assays in small molecule screening fluorescent and bioluminescent nanoprobes for in vitro and in vivo detection of matrix metalloproteinase activity identification of novel drug scaffolds for inhibition of sars-cov 3-chymotrypsin-like protease using virtual and high-throughput screenings characterization of the activity of 2′-c-methylcytidine against dengue virus replication distinct effects of two hiv-1 capsid assembly inhibitor families that bind the same site within the n-terminal domain of the viral ca protein high content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation label-free luminescence switch-on detection of hepatitis c virus ns3 helicase activity using a g-quadruplex-selective probe †electronic supplementary information (esi) available: compound characterisation and supplementary data immunity to bovine herpesvirus 1: i. viral lifecycle and innate immunity small molecule insulin receptor activators potentiate insulin action in insulin-resistant cells amino acid residues within conserved domain vi of the vesicular stomatitis virus large polymerase protein essential for mrna cap methyltransferase activity vesicular stomatitis viruses resistant to the methylase inhibitor sinefungin upregulate rna synthesis and reveal mutations that affect mrna cap methylation fluorescence detection techniques for protein kinase assay quantum dots based molecular beacons for in vitro and in vivo detection of mmp-2 on tumor inhibition of the histone demethylase lsd1 blocks α-herpesvirus lytic replication and reactivation from latency a novel selective lsd1/kdm1a inhibitor epigenetically blocks herpes simplex virus lytic replication and reactivation from latency authentic hiv-1 integrase inhibitors single-molecule imaging reveals the translocation and dna looping dynamics of hepatitis c virus ns3 helicase cellbased fluorescence assay for human immunodeficiency virus type 1 protease activity viral and host proteins that modulate filovirus budding rapid and automated fluorescencelinked immunosorbent assay for high-throughput screening of hiv-1 fusion inhibitors targeting gp41 gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses. sci. rep. 7 a structural view of the rna-dependent rna polymerases from the flavivirus genus automatic quantitation of hiv-1 mediated cell-tocell fusion with a digital image analysis system (dias): application for rapid screening of hiv-1 fusion inhibitors structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor hiv-1 uncoating is facilitated by dynein and kinesin 1 recognition-domain focused (rdf) chemosensors: versatile and efficient reporters of protein kinase activity humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation the escrt-i subunit tsg101 controls endosome-to-cytosol release of viral rna the challenge of selecting protein kinase assays for lead discovery optimization mrna cap methylation influences pathogenesis of vesicular stomatitis virus in vivo a screen for novel hepatitis c virus rdrp inhibitor identifies a broad-spectrum antiviral compound quantification of pyrophosphate as a universal approach to determine polymerase activity and assay polymerase inhibitors structure, function and dynamics in adenovirus maturation in vitro human immunodeficiency virus type 1 integrase assays human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis viral and cellular requirements for the nuclear entry of retroviral preintegration nucleoprotein complexes pathways of clathrin-independent endocytosis lipid interactions during virus entry and infection proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates variable effects of autophagy induction by trehalose on herpesviruses depending on conditions of infection common principles and intermediates of viral protein-mediated fusion: the hiv-1 paradigm viral polymerases virus entry by macropinocytosis apoptotic mimicry: phosphatidylserine-mediated macropinocytosis of vaccinia virus oligonucleotide-based assays for integrase activity a fluorescence polarization based screening assay for nucleic acid polymerase elongation activity multiplex substrate profiling by mass spectrometry for kinases as a method for revealing quantitative substrate motifs investigational protease inhibitors as antiretroviral therapies discovery of an rna virus 3′ → 5′ exoribonuclease that is critically involved in coronavirus rna synthesis high-throughput screen for escherichia coli heat shock protein 70 (hsp70/dnak): atpase assay in low volume by exploiting energy transfer influenza virus recruits host protein kinase c to control assembly and activity of its replication machinery poxvirus dna replication poxvirus membrane biogenesis. virology 479-480 membrane fusion during poxvirus entry rapid solution assays for retroviral integration reactions and their use in kinetic analyses of wild-type and mutant rous sarcoma virus integrases advances in developing therapies to combat zika virus: current knowledge and future perspectives inactivation of a yeast transactivator by the fused hiv-1 proteinase: a simple assay for inhibitors of the viral enzyme activity tackling dengue fever: current status and challenges expression strategies of ambisense viruses poxviruses utilize multiple strategies to inhibit apoptosis rational design of polymerase inhibitors as antiviral drugs herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro anchimerically activatable antiviral protides rabies virus assembly and budding the escrt machinery: new roles at new holes global, in vivo, and site-specific phosphorylation dynamics in signaling networks determination of phosphate as aggregates of ion associates by light-scattering detection and application to flow injection real-time fluorescence assays to monitor duplex unwinding and atpase activities of helicases nuclear pore complex is able to transport macromolecules with diameters of about 39 nm nipah virus c protein recruits tsg101 to promote the efficient release of virus in an escrt-dependent pathway evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors the multiple faces of caveolae herpesvirus capsid association with the nuclear pore complex and viral dna release involve the nucleoporin can/nup214 and the capsid protein pul25 the hepatitis c virus life cycle as a target for new antiviral therapies inhibition of dengue virus replication by novel inhibitors of rna-dependent rna polymerase and protease activities prevention of hepatitis b virus infection in vivo by entry inhibitors derived from the large envelope protein structural insights into rna synthesis by the influenza virus transcription-replication machine alphavirus polymerase and rna replication an overview of severe acute respiratory syndrome-coronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small molecule chemotherapy isoform-selective assays for sphingosine kinase activity human occludin is a hepatitis c virus entry factor required for infection of mouse cells comparative clinical pharmacokinetics and pharmacodynamics of hiv-1 integrase strand transfer inhibitors late stages of the influenza a virus replication cyclea tight interplay between virus and host identification of pololike kinases as potential novel drug targets for influenza a virus principles of virus structural organization critical role of conserved hydrophobic residues within the major homology region in mature retroviral capsid assembly retroviral capsid assembly: a role for the ca dimer in initiation nuclear import of hepatitis b virus capsids and release of the viral genome a chiral pentagonal polyhedral framework for characterizing virus capsid structures ribose 2'-o methylation of the vesicular stomatitis virus mrna cap precedes and facilitates subsequent guanine-n-7 methylation by the large polymerase protein viral avoidance and exploitation of the ubiquitin system a cell-based assay for rna synthesis by the hcv polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by ns5a reverse transcription mechanically initiates hiv-1 capsid disassembly macropinocytosis is the entry mechanism of amphotropic murine leukemia virus west nile virus 5′-cap structure is formed by sequential guanine n-7 and ribose 2′-o methylations by nonstructural protein 5 antiviral drugs for viruses other than human immunodeficiency virus flavivirus structural heterogeneity: implications for cell entry melting of duplex dna in the absence of atp by ns3 helicase domain through specific interaction with a single-strand/ double-strand junction intracellular vesicle acidification promotes maturation of infectious poliovirus particles integration of murine leukemia virus dna depends on mitosis the role of electron microscopy in studying the continuum of changes in membranous structures during poliovirus infection specific in vitro cleavage of mason-pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection hiv-1 protease-induced apoptosis conditions resulting in formation of properly assembled retroviral capsids within inclusion bodies of escherichia coli analysis of mason-pfizer monkey virus gag domains required for capsid assembly in bacteria: role of the n-terminal proline residue of ca in directing particle shape cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes comparison of the luminescent adp-glo assay to a standard radiometric assay for measurement of protein kinase activity involvement of herpes simplex virus type 1 ul13 protein kinase in induction of socs genes, the negative regulators of cytokine signaling europium tetracycline as a luminescent probe for nucleoside phosphates and its application to the determination of kinase activity human herpesvirus 6b downregulates expression of activating ligands during lytic infection to escape elimination by natural killer cells single molecule measurement of the "speed limit" of dna polymerase growing functions of the escrt machinery in cell biology and viral replication production of hepatitis b virus particles in hep g2 cells transfected with cloned hepatitis b virus dna the human coronavirus 229e superfamily 1 helicase has rna and dna duplex-unwinding activities with 5′-to-3′ polarity the m-pmv cytoplasmic targeting-retention signal directs nascent gag polypeptides to a pericentriolar region of the cell discovering new medicines targeting helicases: challenges and recent progress in vitro uncoating of hiv-1 cores genome packaging of reovirus is mediated by the scaffolding property of the microtubule network directional spread of surface-associated retroviruses regulated by differential virus-cell interactions papain-like protease (plpro) inhibitory effects of cinnamic amides from < i > tribulus terrestris < /i > fruits immobilized metal ion affinity-based fluorescence polarization (imap): advances in kinase screening targeting zoonotic viruses: structure-based inhibition of the 3c-like protease from bat coronavirus hku4 -the likely reservoir host to the human coronavirus that causes middle east respiratory syndrome (mers) dna helicase activity of sv40 large tumor antigen in vitro disassembly of influenza a virus capsids by gradient centrifugation a peptide inhibitor of hiv-1 assembly in vitro cytoplasmic tails of bunyavirus gn glycoproteins-could they act as matrix protein surrogates an in vitro fluorescence screen to identify antivirals that disrupt hepatitis b virus capsid assembly specific recognition and accelerated uncoating of retroviral capsids by the trim5alpha restriction factor furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells selective sensing of tyrosine phosphorylation in peptides using terbium(iii) complexes yeast-based assays for the high-throughput screening of inhibitors of coronavirus rna cap guanine-n7-methyltransferase production of hepatitis b virus by a differentiated human hepatoma cell line after transfection with cloned circular hbv dna phosphorylation of the hiv-1 capsid by melk triggers uncoating to promote viral cdna synthesis viral and host control of cytomegalovirus maturation the a, b, cs of herpesvirus capsids real-time monitoring of rna helicase activity using fluorescence resonance energy transfer in vitro ubiquitin depletion and dominant-negative vps4 inhibit rhabdovirus budding without affecting alphavirus budding structural basis for the development of sars 3cl protease inhibitors from a peptide mimic to an aza-decaline scaffold different pathways leading to integrase inhibitors resistance synthesis and antiviral activity of novel isonucleoside analogs rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the ymdd region of reverse transcriptase ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3cl(pro)): implications for nsp5 regulation and the development of antivirals fluorescent sensing of pyrophosphate anion in synovial fluid based on dna-attached magnetic nanoparticles structural insights into the coupling of virion assembly and rotavirus replication an enzymatic virus-like particle assay for sensitive detection of virus entry early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection incorporation of spike and membrane glycoproteins into coronavirus virions distinct roles for nucleic acid in in vitro assembly of purified mason-pfizer monkey virus canc proteins identification and characterization of the rna helicase activity of japanese encephalitis virus ns3 protein virus maturation d-retrovirus morphogenetic switch driven by the targeting signal accessibility to tctex-1 of dynein retroviral oncogenes: a historical primer the entry inhibitor myrcludex-b efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis b virus advances in mass spectrometry based strategies to study receptor tyrosine kinases homogeneous highthroughput screening assays for hiv-1 integrase 3β-processing and strand transfer activities nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases a terbium (iii)-complex-based on-off fluorescent chemosensor for phosphate anions in aqueous solution and its application in molecular logic gates coronavirus nsp10/nsp16 methyltransferase can be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis establishment of a high-throughput assay to monitor influenza a virus rna transcription and replication structure of main protease from human coronavirus nl63: insights for wide spectrum anti-coronavirus drug design mathematical analysis of an hiv latent infection model including both virus-to-cell infection and cell-to-cell transmission measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy highthroughput microfluidic single-cell digital polymerase chain reaction structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus the e1 protein of bovine papilloma virus 1 is an atp-dependent dna helicase design of wide-spectrum inhibitors targeting coronavirus main proteases improving viral protease inhibitors to counter drug resistance studies of ebola virus glycoproteinmediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha decreased muscle insulin receptor kinase correlates with insulin resistance in normoglycemic pima indians adp-glo: a bioluminescent and homogeneous adp monitoring assay for kinases compact, programmable, and stable biofunctionalized upconversion nanoparticles prepared through peptide-mediated phase transfer for high-sensitive protease sensing and in vivo apoptosis imaging identification and characterization of a ribose 2′-o-methyltransferase encoded by the ronivirus branch of nidovirales fret-based biosensors for protein kinases: illuminating the kinome phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs cell-based high-throughput screening assay identifies 2′,2′-difluoro-2′-deoxycytidine gemcitabine as a potential antipoliovirus agent structure of the main protease from a global infectious human coronavirus, hcov-hku1 molecular basis for specific viral rna recognition and 2′-oribose methylation by the dengue virus nonstructural protein 5 (ns5) structure and function of the zika virus full-length ns5 protein robust hepatitis c virus infection in vitro inhibitors of sars-cov entryidentification using an internally-controlled dual envelope pseudovirion assay a new colorimetric strategy for monitoring caspase 3 activity by hrp-mimicking dnazyme-peptide conjugates protease inhibitors targeting coronavirus and filovirus entry the lymphocytic choriomeningitis virus matrix protein ppxy late domain drives the production of defective interfering particles investigations on dna intercalation and surface binding by sybr green i, its structure determination and methodological implications miniaturization of absorbance assays using the fluorescent properties of white microplates characterization of a novel dna polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes this work was supported by ga čr (cz) ga17-25602s to mr, ga17-24281s to tr and the ministry of education, youth and sport of the czech republic(oppc cz.2.16/3.1.00/24503), through its "national program of sustainability i" npu lo 1601. key: cord-255075-6azu6k3h authors: zhuang, jianjian; yin, juxin; lv, shaowu; wang, ben; mu, ying title: advanced “lab-on-a-chip” to detect viruses – current challenges and future perspectives date: 2020-05-12 journal: biosens bioelectron doi: 10.1016/j.bios.2020.112291 sha: doc_id: 255075 cord_uid: 6azu6k3h massive viral outbreaks draw attention to viruses that have not been thoroughly studied or understood. in recent decades, microfluidic chips, known as “lab-on-a-chip”, appears as a promising tool for the detection of viruses. here, we review the development of microfluidic chips that could be used in response to viral detection, specifically for viruses involved in more recent outbreaks. the advantages as well as the disadvantages of microfluidic systems are discussed and analyzed. we also propose ideas for future development of these microfluidic chips and we expect this advanced technology to be used in the future for viral outbreaks. viruses infect millions of individuals each year resulting in serious morbidity (hutchinson 2018 the lod of this system is 1 copy/μl for sudv, 100 copies/μl for ebov, 1000 copies/μl for bdbv 128 and 10 copies/μl for tafv detected in 50 minutes. 4 minutes) and accurate (as low as 1 copy) detection for ebov at poc. however, some chips lack 131 sample preparation or require additional instrumentation, which is not suitable in resource-limited 132 settings for poc. moreover, due to the high contagious rate and characteristics of the multiple ebov 133 subtypes, low-cost multiplex detection chips should be developed. hiv is a single-stranded rna virus that results in acquired immunodeficiency syndrome (aids) 136 (watts et al. 2009 ). hiv attacks t lymphocytes and integrates into the chromosomes of its host, which 137 in turn leads to defects in the human immune system causing irreparable damage to the body (druce et simultaneously. this chip meets serological requirements and detects hiv rna as low as 10 3 viral 166 particles/ml from saliva or blood. paper-based microfluidics is also an effective tool for hiv detection. zhao with the goal of detecting hiv, these studies were mainly performed analyzing cd4 + cells, currently, the influenza virus is a health concern for "lab on a chip" ( this method utilizes electromagnetically-driven magnetic beads and enzyme-linked immunosorbent 208 (elisa)-like assays on the platform to detect h1n1 viruses. this system can also reach a lod of moreover, rapid and ongoing evolution of influenza viruses will make this even more challenging. in recent years, microfluidics has also shown to be an effective tool for the early diagnosis of (figure 7 a ) . this strategy used bead beating and heating procedures to 303 obtain stable rna directly from whole blood, which could be used to detect viruses. in this system, 304 direct buffer was used to extract rna from whole blood and obtain a lod of 10 2 pfu/200 μl in less 305 than 1 hour. yin et al (yin et al. 2020 ) reported a microfluidic system that integrated rna extraction 306 and multiplex pcr to detect four denv serotypes (figure 7 b ). in this system, chitosan-modified 307 paper chip to extra rna and on-chip pcr product was detected using a membrane sensor. this system detection reagents were prestored into the chip and the freeze-dried reagents were kept in 2−8 °c for 6 377 months in the disc chip. moreover, the separation step of plasma or serum is avoided since it is directly 378 detected from whole blood. this system can detect 10 2 copies/ml hbv dna in ~48 minutes from 500 379 μl of whole blood. our group also proposed a digital isothermal chip for the quantitative detection of will give more precise results. in addition, the integration of sample preparation with detection is important especially for 420 nucleic acid-based detection methods (yin et al. 2019 ). however, very few studies have been able to 421 integrate virus sample preparation into chips. integrated sample preparation will reduce testing time, improve accuracy and minimize labor. moreover, sample-in-answer-out is the ideal detection process. therefore, sample preparation should be considered in a single chip. whether lab-on-a-chip is used in 424 the clinic or the home, sample preparation integration is necessary. in addition, affordable and 425 user-friendly qualities should be considered especially for poc in resource-limited settings. automated and high-throughput microfluidics should also be considered. therefore, additional sample preparation 427 methods should be tested and integrated into chips that will be used for virus outbreaks. chip is further combined with the "biological mobile phone", "mobile detection station", or "artificial intelligence"，its potential for virus detection will be extended even further. in the future, microfluidic products that meet the criteria for poc proposed by who including (1) 478 being affordable to those at risk of infection, containing (2) high sensitivity, (3) high specificity, (4) 479 user-friendly capabilities, being (5) rapid and robust, (6) equipment free, and (7) comparison of herpes 603 simplex virus pcr with culture for virus detection in multisource surface swab specimens from 604 neonates nanoparticle-enhanced electrical detection of zika virus on paper 607 microchips improving hiv proteome annotation: new features of bioafrica hiv proteomics resource multiplexed efficient on-chip sample preparation 613 and sensitive amplification-free detection of ebola virus microfluidic 615 system for detection of viral rna in blood using a barcode fluorescence reporter photocleavable capture probe dengue virus: a review on 619 advances in detection and trends -from conventional methods to novel biosensors differential serum cytokine profiles in patients with chronic hepatitis b, 623 c, and hepatocellular carcinoma the propagation of a christmas carol produced by adolescent cancer patients at the istituto 626 hands-free smartphone-based diagnostics for simultaneous detection of zika dengue at point-of-care wearable microfluidic diaphragm pressure sensor for health and tactile touch 632 monitoring microfluidic chips for point-of-care immunodiagnostics genomic surveillance elucidates ebola virus origin and 643 transmission during the 2014 outbreak rapid, low-cost and instrument-free cd4+ cell counting 645 16 for hiv diagnostics in resource-poor settings prevalence and seasonal distribution of respiratory viruses 647 during the 2014-2015 season in istanbul the ebola epidemic a global health emergency on-chip 651 multiplex electrochemical immunosensor based on disposable 24-site fluidic micro-array screen 652 printing analytical device for multi-component quantitative analysis point of care diagnostics: status 655 and future zika virus is a global public health emergency, declares who new fronts emerge in the influenza cytokine storm dengue virus -mosquito interactions biology of zika virus infection in human skin cells a multi-virus detectable microfluidic 666 electrochemical immunosensor for simultaneous detection of h1n1, h5n1, and h7n9 virus using zno 667 nanorods for sensitivity enhancement deaths from norovirus 669 among the elderly, england and wales a comparison study of zika virus outbreaks 671 in french polynesia, colombia and the state of bahia in brazil high-throughput and all-solution phase african swine fever virus (asfv) 674 detection using crispr-cas12a and fluorescence based point-of-care system traditional and modern cell culture in virus diagnosis. osong public 678 health and microsphere integrated microfluidic disk: synergy 681 of two techniques for rapid and ultrasensitive dengue detection efficient 683 inhibition of african swine fever virus replication by crispr/cas9 targeting of the viral p30 gene 684 (cp204l) what's the point? how point-of-care sti tests can 686 impact infected patients influenza virus a bead-based 689 immunofluorescence-assay on a microfluidic dielectrophoresis platform for rapid dengue virus 690 detection in silico cd4+, cd8+ t-cell and b-cell immunity 693 associated immunogenic epitope prediction and hla distribution analysis of zika virus a point-of-care pcr test for hiv-1 detection 696 in resource-limited settings evaluation of a manual 698 dna extraction protocol and an isothermal amplification assay for detecting hiv-1 dna from dried 699 blood spots for use in resource-limited settings simpler, faster, and sensitive zika virus assay using 701 smartphone detection of loop-mediated isothermal amplification on paper microfluidic chips a one-size-fits-all flu vaccine? towards detection and diagnosis of 705 ebola virus disease at point-of-care estimating the basic reproductive ratio for 707 the ebola outbreak in liberia and sierra leone the architecture of 709 sars-cov-2 transcriptome microfluidic sample preparation: cell lysis and nucleic 711 acid purification virus concentration and purification by a microfluidic filtering 714 system with an integrated pegylated antifouling membrane development of a set of community-informed ebola messages for 717 a soft, wearable microfluidic device for the capture, storage, and colorimetric sensing 720 of sweat enabling miniaturised personalised diagnostics: 722 from lab-on-a-chip to lab-in-a-drop micro total analysis systems: fundamental advances and applications in the 725 laboratory, clinic, and field a historical perspective on paper 727 microfluidic based point-of-care diagnostics comparative performance evaluation of carbon dot-based paper immunoassay on whatman filter 730 paper and nitrocellulose paper in the detection of hiv infection development of an enzyme-linked immunosorbent assay 737 for rapid detection of dengue virus (denv) ns1 and differentiation of denv serotypes during 738 early infection modeling dengue virus-hepatic cell interactions 740 using human pluripotent stem cell-derived hepatocyte-like cells hepatitis b virus epidemiology, disease burden, treatment, and current and 742 emerging prevention and control measures high-speed fabrication of patterned colloidal 744 photonic structures in centrifugal microfluidic chips role of cell culture for virus detection in the age of 746 technology prevalence and 748 impact of cardiovascular metabolic diseases on covid-19 in china smartphone assisted 750 immunodetection of hiv p24 antigen using reusable, centrifugal microchannel array chip therapeutic options for the 2019 novel coronavirus (2019-ncov) sample-to-answer hepatitis b virus dna detection 755 from whole blood on a centrifugal microfluidic platform with double rotation axes a microfluidic paper-based origami nanobiosensor for label-free research progress on detection and traceability 760 technology of stacked transgenic plants and their products comparison of viral isolation and 763 multiplex real-time reverse transcription-pcr for confirmation of respiratory syncytial virus and 764 influenza virus detection by antigen immunoassays cross-subtype detection of hiv-1 using reverse transcription and 767 recombinase polymerase amplification fast and parallel detection of four ebola virus species on a microfluidic-chip-based portable 770 a 773 sample-to-answer labdisc platform integrated novel membrane-resistance valves for detection of highly 774 pathogenic avian influenza viruses novel coronavirus (sars-cov-2) 776 epidemic: a veterinary perspective a structure-free digital microfluidic platform for 778 detection of influenza a virus by using magnetic beads and electromagnetic forces an integrated self-driven microfluidic device for rapid 781 detection of the influenza a (h1n1) virus by reverse transcription loop-mediated isothermal 782 amplification paper-based rna detection and 785 multiplexed analysis for ebola virus diagnostics ebola virus disease tissue and cellular tropism, 789 pathology and pathogenesis of ebola and marburg viruses miniaturized 791 devices for point of care molecular detection of hiv lower respiratory tract infection caused by respiratory syncytial virus: 795 current management and new therapeutics molecular techniques for the detection of organisms in aquatic 797 integration of sample preparation and analysis into an optofluidic chip for multi-target disease 800 detection oligomeric state of the zikv e protein defines protective immune responses multiplex microfluidic paper-based 805 immunoassay for the diagnosis of hepatitis c virus infection an information value based analysis of physical and climatic 807 factors affecting dengue fever and dengue haemorrhagic fever incidence knowledge and perceptions 809 of zika virus transmission in the community of puerto plata future developments in biosensors for field-ready 812 biological characterization of african swine fever virus genotype ii strains from 815 north-eastern estonia in european wild boar guillain-barre syndrome: pathogenesis, diagnosis, treatment and prognosis current approaches for 958 diagnosis of influenza virus infections in humans good laboratory practices guarantee biosafety in the sierra leone-china friendship 962 biosafety laboratory simultaneous and automated detection of influenza a virus hemagglutinin h7 and h9 based on 965 magnetism and size mediated microfluidic chip architecture and secondary structure of an entire hiv-1 rna genome unaids: global report: unaids report on the global aids epidemic 10 nervous system injury and neuroimaging of 971 a picoliter microfluidic chip driven by negative pressure for 973 quantifying nucleic acid accurately with isothermal amplification. chin immunoassay for ultrasensitive multiplex avian influenza virus detection based on fluorescent 976 magnetic multifunctional nanospheres smartphone-based point-of-care microfluidic platform fabricated with a zno nanorod template for 979 a case series of atypical presentation of zika 981 virus infection in singapore a 983 self-contained all-in-one cartridge for sample preparation and real-time pcr in rapid influenza 984 diagnosis multiplex 986 detection of bacteria on an integrated centrifugal disk using bead-beating lysis and loop-mediated 987 amplification hiv-seronegative aids patient with pneumocystis jirovecii pneumonia bandage-like wearable flexible microfluidic recombinase 991 polymerase amplification sensor for the rapid visual detection of nucleic acids microfluidic-cfpa chip for the point-of-care 993 detection of african swine fever virus with a median time to threshold in about 10 min a rapid and label-free platform for virus capture and 997 identification from clinical samples multicolored silver nanoparticles for multiplexed disease diagnostics: distinguishing 1000 dengue, yellow fever, and ebola viruses based microfluidic point-of-care diagnostic 1002 devices integrated microfluidic systems with sample preparation and nucleic acid amplification a non-optical multiplexed pcr diagnostic platform for serotype-specific detection of dengue 1008 integrated microsystems for in situ genetic detection of 1010 dengue virus in whole blood using direct sample preparation and isothermal amplification. the 1011 analyst viral metagenomics reveal blooms of 1014 anelloviruses in the respiratory tract of lung transplant recipients mosquitocidal and oviposition repellent activities 1016 of the extracts of seaweed bryopsis pennata on aedes aegypti and aedes albopictus a novel paper-plastic microfluidic hybrid chip 1019 integrated with a lateral flow immunoassay for dengue nonstructural protein 1 antigen detection isothermal amplification methods for the detection of nucleic acids in 1022 microfluidic devices immunosensor-based label-free and multiplex detection of influenza 1024 viruses: state of the art a 1026 point of care platform based on microfluidic chip for nucleic acid extraction in less than 1 minute multiplex snp genotyping in whole blood using an integrated 1030 microfluidic lab-on-a-chip a portable paper-based microfluidic platform for multiplexed electrochemical 1032 detection of human immunodeficiency virus and hepatitis c virus antibodies in serum covid-19 and the cardiovascular system recent advances in lab-on-a-chip 1037 technologies for viral diagnosis digital pcr on an integrated self-priming compartmentalization chip molecular aspects of the dengue virus schematic representation of the structure of representative viruses. a: ebola virus de (wit et 1085 al. 2011)；b：human immunodeficiency virus (druce et al. 2016)；c：influenza virus ；e：dengue virus (zonetti et al. 2018)；f：sars-cov figure 3：microfluidic system to detect ebov. a: microfluidic chips proposed by qin et al adapted from ref. 60 with permission from nature publications. c: paper chip 1114 proposed by brangel et al. serum forms complexes between the labeled gold nanoparticles (aunps) 1115 and the target analytes. targeted igg serum antibodies against single or multiple recombinant ebola 1116 viral proteins bind to preprinted test lines, forming a visual red-purple line. a control line is used to 1117 validate assay function for the detection of antihuman antibody-gold nanoparticle conjugates. results 1118 can be obtained in 15 minutes. adapted from ref. 66 with permission from nature publications. d: 1119 schematic of the microfluidic chip for detection of four ebov species figure 4 ：microfluidic systems for the detection of hiv. a: schematic and photograph of the manual 1136 magnetophoretic cd4+ isolation chip proposed by glynn et al. prior to the test, the chip was perfused 1137 through a degassing process b: schematic and photograph of the rt-lamp substrate and smartphone apparatus for hiv 1140 (i) heating stage. the microfluidic chip was put on the stage (ii) copper 1141 base containing mineral oil, (iii) wavelength filters placed in front of the led and smartphone camera, 1142 (iv) smartphone ,(v) blue led light source, and (vi) apparatus. adapted from ref. 79 with permission 1143 from the elsevier. c：schematic and photograph of microraad for hiv testing when using this system, 1) the paper-based chip should be assembled into plastic housing with a 1145 temperature control circuit, 2) buffer should be added into inlets and sealed with adhesive tape to 1146 minimize evaporation, 3) the chip should be connected with a phone to heat, 4) one should wait 90 1147 minutes for automated fluid delivery and sample incubation in μpad d: schematic of a 1149 paper-based chip for the detection of hiv developed by li et al. image shows the components (left) 1150 and the assembly (right) of origami chip this is a magnetism and size mediated platform. different 1172 influenza subtypes could be simultaneously separated and detected depending on the different-sizes 1173 magnetic beads. adapted from ref. 106 with permission from the elsevier. d: photograph microfluidic 1174 chip for the detection of 12 influenza subtypes (shen et al 2019). arrayed reaction chambers contain 1175 primer sets for amplifying specific regions of the ha and na genes such that the rt-pcr-derived 1176 signal output could be used for viral subtyping figure 6： microfluidic systems for the detection of zikv. a: workflow of the portable cup device 1188 based on the microfluidic system developed by song et al. (a) schematic of saliva sample preparation saliva samples are collected in a saliva collection tube and then lysed in qiagen binding/lysis (avl) the lysed sample is filtered through the isolation membrane of a microfluidic cassette for 1191 nucleic acid extraction. (c) enlarged view of the chemically-heated cup. the cup consists of a thermos 1192 cup body, a 3dprinted cup lid, a chip holder, pcm material, heat sink and single-use mg-fe alloy pack 1193 heat source. (d) a photograph of the chemically-heated cup for point of care molecular diagnostics of 1194 zikv b: schematic of the wearable microfluidic sensor for detection of zikv nucleic acids the detection method was based on the rpa. the reaction can start on this wearable microfluidic 1197 system effectively by human epidermal heat that was close to the optimal temperature of rpa. adapted 1198 from ref. 123 with permission from the elsevier the performance of microfluidic systems in virus detection are discussed and analyzed the challenges of microfluidic systems with regard to sample preparation, throughput, and multiplexing are highlighted ideas for future development of microfluidic systems were proposed. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord-269862-krcu3hfa authors: wang, shui-mei; wang, chin-tien title: apobec3g cytidine deaminase association with coronavirus nucleocapsid protein date: 2009-05-25 journal: virology doi: 10.1016/j.virol.2009.03.010 sha: doc_id: 269862 cord_uid: krcu3hfa we previously reported that replacing hiv-1 nucleocapsid (nc) domain with sars-cov nucleocapsid (n) residues 2–213, 215–421, or 234–421 results in efficient virus-like particle (vlp) production at a level comparable to that of wild-type hiv-1. in this study we demonstrate that these chimeras are capable of packaging large amounts of human apobec3g (ha3g), and that an hiv-1 gag chimera containing the carboxyl-terminal half of human coronavirus 229e (hcov-229e) n as a substitute for nc is capable of directing vlp assembly and efficiently packaging ha3g. when co-expressed with sars-cov n and m (membrane) proteins, ha3g was efficiently incorporated into sars-cov vlps. data from gst pull-down assays suggest that the n sequence involved in n–ha3g interactions is located between residues 86 and 302. like hiv-1 nc, the sars-cov or hcov-229e n-associated with ha3g depends on the presence of rna, with the first linker region essential for ha3g packaging into both hiv-1 and sars-cov vlps. the results raise the possibility that ha3g is capable of associating with different species of viral structural proteins through a potentially common, rna-mediated mechanism. apolipoprotein b mrna-editing enzyme-catalytic polypeptide-like 3g (apobec3g) is a member of the apobec3 family of cytidine deaminases (cullen, 2006) . human apobec3g (ha3g) can be packaged into hiv-1 virions to mediate c-to-u editing on nascent proviral minus strands during reverse transcription, resulting in the inhibition of viral replication mangeat et al., 2003; yu et al., 2004; zhang et al., 2003) . accordingly, ha3g as an anti-viral factor confers innate immunity to hiv-1. however, hiv-1 encodes vif, which counteracts ha3g by inducing ha3g degradation via the ubiquitin-proteasome pathway, thereby blocking its incorporation into budding virions (mehle et al., 2004; yu et al., 2003) . the ability of vif to negate ha3g function is both dependent on physical association and partly species-specific-in other words, vif cannot counteract a form of a3g from a different species. this inefficient vif-ha3g interaction means that hiv-1 vif is capable of counteracting human and chimpanzee a3g, but not mouse a3g (ma3g) or african green monkey a3g (mariani et al., 2003) . other researchers have shown that ha3g or other apobec family members can confer innate immunity to a wide range of retroviruses as well as hbv (which is similar to retroviruses in that it also goes through a reverse transcription step during genomic replication) (cullen, 2006) . the incorporation of a3g into hiv-1 virions is mediated by the gag nucleocapsid (nc) domain (alce and popik, 2004; cen et al., 2004; khan et al., 2005; luo et al., 2004; schafer et al., 2004; zennou et al., 2004) . in addition to playing a key role in viral rna packaging (berkowitz et al., 1993 (berkowitz et al., , 1995 poon et al., 1996; zhang and barklis, 1997) , nc contains an i domain that is responsible for gag-gag interactions (bennett et al., 1993; bowzard et al., 1998) . heterologous polypeptides capable of self-association have been shown to confer the ability to efficiently produce chimeric vlps when substituted for hiv-1 nc (accola et al., 2000; burniston et al., 1999; johnson et al., 2002; zhang et al., 1998) . however, replacing nc with a leucine-zipper motif that does not encapsidate rna abolishes ha3g packaging without significantly affecting hiv-1 virion production (zennou et al., 2004) , suggesting rna involvement in ha3g incorporation. this is consistent with the proposal that rna is required for either ha3g viral incorporation or gag-ha3g interaction (khan et al., 2005; schafer et al., 2004; svarovskaia et al., 2004; zennou et al., 2004) . we previously demonstrated that hiv-1 gag mutants containing severe acute respiratory syndrome coronavirus nucleocapsid (sars-cov n) coding sequences as nc substitutes can effectively assemble vlps . while completely unrelated, the sars-cov n protein is similar to hiv-1 nc in that it contains putative protein-protein interaction domains (he et al., 2004; narayanan et al., 2003; surjit et al., 2004; yu et al., 2005) and plays a role in viral rna packaging (hsieh et al., 2005; huang et al., 2004a) . given that sars-cov n possesses a rna-binding property, it is likely that assembly-competent chimeras containing a replacement of hiv-1 nc by an sars-cov n sequence may support the incorporation of ha3g into vlps. here we demonstrate that the carboxyl-terminal half of the sars-cov or human coronavirus 229e (hcov-229e) n protein not only enables efficient vlp production, but also confers the ability to efficiently package human apobec3g (ha3g) when substituted for hiv-1 nc. direct evidence comes in the form of ha3g being efficiently packaged into sars-cov vlps. the interaction between coronavirus n and ha3g is also rnadependent. this implies that in addition to retroviruses, sars-cov and other rna viruses may be capable of packaging ha3g via nucleocapsid domains. we previously reported that the hiv-1 gag chimeras containing the sars-cov n carboxyl-terminal 215-421 [nc(n2)] or 234-421 [nc (n4)] codons in the deleted nc region (fig. 1) are capable of assembling vlps as efficiently as wt gag . this raises the question of whether the inserted sars-cov n sequence confers the chimeric ability to package ha3g. we therefore coexpressed assembly-competent nc(n2) and nc(n4) with ha3g in 293t cells and used western immunoblotting to detect vlpassociated ha3g. our controls were the hiv-1 nc-deleted mutants δnc, δpc, and delnc, all with different numbers of deleted nc codons ( fig. 1) . our results show that nc(n2) and nc(n4) were capable of packaging ha3g at the same efficiency level as the wild-type ( fig. 2a , lanes 6 and 7 vs. lane 2). low but detectable vlp-associated ha3g was observed in the nc-deletion mutants (lanes 4 and 5). since hiv-1 vif inhibits ha3g virion incorporation by triggering ha3g degradation (conticello et al., 2003; kao et al., 2003; liu et al., 2004; mariani et al., 2003; marin et al., 2003; mehle et al., 2004; sheehy et al., 2003; stopak et al., 2003; yu et al., 2003) , the ha3g expression level or the presence of vif may affect the level of virus-associated ha3g. to test whether vif or ha3g expression level affects ha3g packaging into chimeric vlps, we introduced mutation-blocking vif expression into wt and nc(n2) and co-transfected each vif-deficient (δvif) result with different amounts of the ha3g-coding plasmid. in the absence of vif, we observed a relatively higher steady-state ha3g expression level accompanied by an increased level of vlp-associated ha3g (fig. 2b, right top panel) . members of the δvif group occasionally expressed higher vlp levels than their vif-intact counterparts (fig. 2b, . however, we also repeatedly observed δvif group members producing higher levels of vlp-associated ha3g. in the presence or absence of vif, nc(n2) displayed a level of vlpassociated ha3g comparable to that displayed by wt. these results suggest that the sars-cov n carboxyl-terminal region can be substituted for hiv-1 nc without affecting vlp assembly or ha3g packaging functions. to determine the required sars-cov n sequences for conferring ha3g packaging ability, we co-expressed ha3g with wt or with chimeras containing a variety of n coding regions as hiv-1 nc substitutes. as illustrated in fig. 1 , full-length sars-cov n sequences were inserted into the δnc (δnc [con] ) and delnc (nc[con]) constructs. nc(n1), nc(n3), nc(n5), nc(n6), and nc(n7) respectively contain n codons 2-213, 215-359, 302-421, 2-168, and 2-86 in the deleted nc region. we again found that both nc(n2) and nc(n4) were capable of efficiently packaging ha3g at vlp-associated ha3g levels comparable to or higher than those of wt (fig. 2c, lanes 5 and 7) . even though δnc(con) and nc(con) were found to be defective in vlp production, they incorporated ha3g at an efficiency level comparable to that of wt (lanes 2 and 3). further, despite producing substantial amounts of vlps, the nc(n1) was incapable of packaging ha3g as efficiently as wt (i.e., it produced a vlp-associated ha3g level approximately 60% that of wt) (lane 4). the barely detectable level of vlp-associated ha3g found in nc(n6) may be due to lower levels of vlps in the blot and/or lower levels of ha3g expression (fig. 2c) . however, in a separate blot containing higher vlp and ha3g expression levels, vlp-associated ha3g in nc(n6) was approximately 50% that measured in wt (data not shown). both ha3g viral incorporation and gag-ha3g interaction require the presence of rna (khan et al., 2005; schafer et al., 2004; svarovskaia et al., 2004; zennou et al., 2004) . we tested whether the association between sars-cov n and ha3g has the same requirement by fusing the sars-cov n amino-terminal (n1, n6, and n7) or carboxyl-terminal sequence (n2, and n5) to the carboxyl terminus of gst and co-expressing each gst fusion construct with ha3g. cell lysates containing ha3g and gst fusions were treated with rnase prior to performing a gst pull-down assay. results indicate that gst-con, gst-n1, gst-n2, and gst-n6 were capable of pulling down ha3g, whereas the amount of ha3g associated with gst fusion was markedly reduced following rnase treatment (fig. 3) . neither gst-n5 nor gst-n7 was capable of pulling down ha3g, regardless of whether or not they were treated with rnase (fig. 3, lanes 7, 9, 16 and 18 ). these results suggest that (a) the ha3g-binding domain of sars-cov n is largely contained within the sequence between residues 86 and 302, and (b) efficient n-ha3g interactions occur in a rna-dependent manner. , nucleocapsid (nc), p6, and amino acid residues in sp1-nc-sp2 junction. δnc has ten hiv-1 nc residues remaining in the deleted region; δpc has nc almost deleted, with sp1 partially removed. delnc has the two methionine residues (bracketed) in the sp1-nc junction removed. pcr-amplified fragments containing various portions of sars-cov n coding sequences were inserted into the deleted nc regions. numbers denote codon positions at inserted sars-cov n protein sequence boundaries. arrows indicate sp1-nc and nc-sp2 junction sites. remaining hiv-1 nc residues in deleted regions are underlined. altered or foreign amino acid residues inserted in juncture area are italicized. all constructs were expressed in hivgptd25 (a hiv-1 pr-defective expression vector). to test whether ha3g can be efficiently packaged into sars-cov, we transfected 293t cells containing the ha3g-coding plasmid with sars-cov m and n expression vectors. sars-cov vlps were generated by co-expressing m and n proteins (huang et al., 2004b) . results indicate that the amount of sars-cov vlp-associated ha3g was comparable to that of hiv-1 (fig. 4a , lane 4 vs. lane 5). medium ha3g was not detected when co-expressed with m or n alone. extracellular n was also undetectable without m co-expression (data not shown). results from co-immunoprecipitation experiments indicate that n was capable of associating with ha3g but m was not (data not shown). the data suggest that ha3g packaging into sars-cov vlps largely depends on ha3g-n association. to further confirm an association between released ha3g and sars-cov vlps formed by m and n, supernatant pellets derived from incorporation of human apobec3g into vlps. 293t cells were co-transfected with a human apobec3g (ha3g) expression vector and indicated plasmid. at 48-72 h posttransfection, cells and supernatant were collected and subjected to western immunoblotting. ha3g plasmid dna (2 or 8 μg) was used for co-transfection as indicated. δvif is a vifdeficient mutation introduced into wt and nc(n2) (panel b, lanes 6-9). wild-type gag and chimeric proteins were probed with a monoclonal antibody directed against hiv-1 ca; an anti-ha monoclonal antibody was used to detect ha3g. vif antiserum was used to detect vif. relative levels of vlp-associated ha3g are indicated along the bottom (panel c). ha3g, wt gag, and chimeric proteins were quantified by scanning ha3g and p24ca-associated band densities from immunoblots. ratios of ha3g to p24 gag were determined and normalized to that of wt. cells co-transfected with ha3g, m, and n expression vectors were subjected to sucrose density gradient fractionation experiments. the results shown in fig. 4b indicate that both m and n had fraction 6 peaks at a density of 1.18 g/ml, which is consistent with a previous report on sars-cov virus-like particle density (huang et al., 2004b) . a ha3g peak was also found at fraction 6, suggesting an association between ha3g and sars-cov vlps generated via m and n coexpression. members of the apobec3 protein family share a highly conserved zinc-coordinating motif: his-x-glu-x 23-28 -pro-cys-x 2-4 -cys (chen et al., 2007b) . similar to apobec1, apobec3g contains two zinccoordinating motifs, with the carboxyl-terminal zinc-coordinating motif possessing catalytic cytidine deaminase activity. to identify ha3g regions responsible for ha3g incorporation into sars-cov vlps, we co-transfected 293t cells with m and n expression vectors and a plasmid encoding wt or a ha3g mutant containing various carboxyl-or amino-terminus deletions (fig. 5a) . the results indicate that ha3g mutants lacking residues 1-104, 157-384, or 246-384 were capable of being incorporated into vlps, and that ha3g mutants lacking residues 1-156 or 309-384 were undetectable in the medium samples (fig. 5b, lanes 4 and 7) . in the cases of δ246-384 and δ309-384, poor particle incorporation may be due in part to low expression levels. in particular, δ309-384 was barely detectable in repeat experiments. low steady-state expression levels of the carboxylterminal truncated ha3g mutant have been reported previously (cen et al., 2004) . in comparison, the δ157-384 mutant expressed greater steady-state stability and a higher level of vlp-associated ha3g (lanes 5 and 12). to test whether the mutations had similar effects on ha3g packaging into chimeric vlps, wt or each ha3g mutant was individually co-expressed with nc(nc2) (which contains the carboxyl-terminal half of the n coding sequence and is known to efficiently produce vlps and package ha3g) (figs. 1 and 2). our results indicate that the ha3g mutations exerted similar effects on ha3g packaging into nc(n2) vlps with no detectable vlp-associated δ1-156 or δ309-384 (fig. 5c) . combined, these results suggest that the essential region for ha3g packaging into sars-cov vlps is located between residues 104 and 156, corresponding to the first linker region. to test whether the deletion mutations exerted similar effects on ha3g incorporation into hiv-1 vlps, we co-expressed wt or each ha3g deletion mutant . equal amounts of cell lysates were treated with (lanes 10-18) or without (lanes 1-9) 0.2 mg/ml dnase-free rnase a for 30 min at 25°c, followed by mixing with glutathione-agarose beads for 2 h at 4°c. complexes bound to the beads were pelleted, washed, and subjected to western immunoblotting with anti-ha and anti-gst antibodies. individually with hiv-1 gag. in agreement with previous reports (cen et al., 2004; luo et al., 2004) , the δ1-156 mutation (with a deletion involving the first linker region) blocked ha3g incorporation (fig. 5d, lane 4) . overall, the deletion mutations had similar effects on ha3g packaging into hiv-1, nc(n2), and sars-cov vlps. specifically, the essential domain for incorporating ha3g into hiv-1 and sars-cov vlps was located in the same (first) linker region. to test whether the sars-cov n ability to confer ha3g packaging is dependent on n-ha3g interaction and therefore distinct from its selfinteraction capacity, we co-expressed ha3g with an assemblycompetent gag containing a leucine-zipper domain as a nc substitute. inserting a wild-type leucine-zipper (wtzip) domain into the deleted nc region restored vlp production to wt level (fig. 6a, lane 4) . a mutant leucine-zipper motif (kzip) replacement was incapable of rescuing the δnc assembly defect (fig. 6a, lane 5) , but in spite of producing a wt-comparable level of vlps, δnc(wtzip) did not package ha3g as efficiently as wt (lane 4 vs. lane 2). we also examined whether human coronavirus 229e (hcov-229e) n protein (having approximately 30% homology with sars-cov n at the amino acid level) (tswen-kei tang, 2005) is capable of conferring the ability to package ha3g when substituted for hiv-1 nc. to perform this test, we constructed nc(229en2) (an nc[n2] counterpart) by inserting the hcov-229e carboxyl-terminal half nucleocapsid coding sequence into the deleted hiv-1 nc region. the resulting construct was transiently co-expressed with ha3g in 293t cells. the results indicate that nc(229en2) can also package ha3g to a degree comparable to that of wt when released vlps are at a similar level (data not shown). to mitigate the impact of overexpressed ha3g on this conclusion, we repeated the experiment by co-expressing ha3g with wt or the chimeric construct in 293 cells. as the results in fig. 6b indicate, the carboxyl-terminal half of hcov-229e n (which also contains a putative self-association domain) (tswen-kei tang, 2005) was capable of replacing the hiv-1 nc function with respect to vlp assembly and ha3g packaging-that is, substantial amounts of vlps and ha3g were detected in nc(229en2) transfectant supernatant (lane 4). to determine whether the interaction between ha3g and hcov-229e n also requires rna, we performed a gst pull-down assay in the presence or absence of rnase. as expected, the gst fusions containing the full-length (gst-229en), amino-terminal half (gst-229en1), or carboxyl-terminal half (gst-229en2) of the hcov-229e n sequence were capable of efficient ha3g pull-down, which is also dependent on the presence of rna (fig. 7) . these data suggest that hcov-229e n and sars-cov n may share a propensity for ha3gassociation. open boxes indicate the zinccoordinating motif, hxe-x 23-28 -cx 2-4 c. the asterisk denotes the cytidine deaminase catalytic site. domains that previously referred to the "linker" peptides are depicted by arrowheads. numbers denote amino acid positions in ha3g. indicated positions of deleted ha3g amino acid residue boundaries were used to designate mutant ha3g constructs. note that each ha3g deletion mutant was tagged with a single copy of ha at the carboxyl terminus and that the wt ha3g was triple-tagged with ha. this may have caused a higher signal for the detected wt ha3g, thus leading to underestimates of the vlp-associated ha3g deletion mutants. (b-d) incorporation of wt and mutant ha3g into vlps. 293t cells were cotransfected with m and n, nc(n2) or an hiv-1 gag expression vector (hivgptd25) plus each of the ha3g expression vectors. to avoid effects from vif on ha3g expression level, nc (n2) and hivgptd25 were expressed in vif-deficient (δvif) contexts. at 48-72 h post-transfection, supernatant and cells were harvested, prepared, and subjected to western immunoblotting. m and n were probed with m antiserum and an anti-n monoclonal antibody. ha3g was probed with an anti-ha antibody and wt gag and chimeric proteins were detected with an anti-p24ca antibody. levels of p24ca-and n-associated proteins and virus-associated ha3g in each sample were quantified by scanning immunoblot band densities. ratios of total ha3g versus p24ca-associated or n-associated protein levels were calculated for each sample and normalized to that of wt ha3g in parallel experiments. dashes (-) denote ratios below 0.01. similar results were observed in repeat and independent experiments. the immunoblots presented here were intentionally overexposed to allow for visualization of the individual ha3g mutants. any conclusion that sars-cov n enables efficient ha3g packaging into vlps when substituted for hiv-1 nc must be tempered when over-expression systems are considered. still, the evidence presented in this paper suggests an association between sars-cov n and ha3gthat is, the leucine-zipper domain is incapable of conferring the ability to package ha3g when substituted for hiv-1 nc even though it enables efficient vlp production. moreover, assembly-competent chimeras with various sars-cov n protein sequences inserted into the deleted hiv-1 nc did not have equal ha3g packaging capabilities: nc(n2) and nc(n4) exhibited a higher level of vlp-associated ha3g than nc(n1). results from a gst pull-down assay suggest that the sars-cov n domain associated with ha3g is largely contained within the central n region between residues 86 and 302 (fig. 3) . although gst-n1 and gst-n6 can pull down ha3g, we found that nc(n1) and nc(n6) are incapable of packaging ha3g as efficiently as wt or nc (n2). therefore, insufficient ha3g incorporation into nc(n1) and nc (n6) may be due in part to steric hindrance in a chimeric protein context. evidence that a carboxyl-terminal self-association domain of sars-cov n or hcov-229e n (as opposed to the amino-terminal rna-binding domain) confers the ability to efficiently package ha3g suggests that for this specific purpose, hiv-1 nc can be replaced by a protein sequence that does not encapsidate hiv-1 genomic rna. given the number of studies demonstrating that viral genomic rna is dispensable for efficient a3g packaging (alce and popik, 2004; cen et al., 2004; khan et al., 2005; luo et al., 2004; navarro et al., 2005; schafer et al., 2004; svarovskaia et al., 2004; zennou et al., 2004) , any genomic rna packaging defect that does exist should not exert a significant impact on the ha3g package. the finding that efficient sars-cov n association with ha3g is rna-dependent (fig. 3) agrees with the proposal that rna (viral or nonspecific) is required for mediating ha3g-gag association and ha3g incorporation into virions (svarovskaia et al., 2004; zennou et al., 2004) . we assumed that the chimeras considered competent in ha3g packaging are capable of packaging considerable amounts of rna, since the sars-cov n (hsieh et al., 2005; luo et al., 2006) and hiv-1 ma domains (burniston et al., 1999; ott et al., 2005) both possess nucleic acid binding properties that may confer rna packaging ability. using a rna quantification assay as described by chang et al. (2008) , we found that nc(n2) vlps contain rna at approximately 80% of the level measured in hiv-1 gag vlps (data not shown). recent studies have suggested that ha3g preferentially binds with cellular 7sl rna, which in turn facilitates an association between rna-bound ha3g and nc for viral incorporation (bogerd and cullen, 2008; wang et al., 2007) . accordingly, 7sl rna apparently plays a key role in determining ha3g packaging into hiv-1 virions. additional experiments are required to determine whether sars-cov or nc(n2) are as capable as hiv-1 in terms of packaging 7sl rna. to date, one in vitro study has demonstrated that a3g binds to rna prior to hiv-1 nc association (bogerd and cullen, 2008) . accordingly, it may be that the interactions required for a3g/rna/nc ternary complex formation involve specific contacts between nc and both a3g and a3g-bound rna. sars-cov n association with ha3g may in large part depend on n binding to ha3g-bound rna, since the sars-cov n contains clusters of basic residues in the amino-and carboxyl-terminal regions that may confer rna-binding ability (hsieh et al., 2005) . extensive genetic analyses indicate that sars-cov n rna-binding domains are largely located between amino acid residues 45-181 (huang et al., 2004a) , 248-280 (chen et al., 2007a; luo et al., 2006) , and 363-382 equal amounts of cell lysates were treated with (lanes 6-10) or without (lanes 1-5) 0.2 mg/ml dnasefree rnase a, followed by gst pull-down assay as described in the legend of fig. 3 . (luo et al., 2006) , all of which contain large quantities of basic residues. it is conceivable that basic residue clusters within sars-cov n make a significant contribution to ha3g-n association by enhancing n binding to ha3g-associated rna. compatible with this hypothesis, we found that the gst fusions gst-con, gst-n1, gst-n2 and gst-n6, all of which contain most of the residues involved in rna-binding, are competent in their associations with ha3g (fig. 3) . the exception to this rule is gst-n5, which is unable to pull down ha3g despite bearing a putative rna-binding domain (residues 363-382). our finding that the essential region for ha3g packaging into hiv-1 or sars-cov vlps resides between codons 104 and 156 is compatible with a genetic analysis demonstrating that residues 124 to 127 are responsible for ha3g viral incorporation (huthoff and malim, 2007) . a very recent study suggests that ha3g contains a necessary and sufficient cytoplasmic retention signal located between residues 113 and 128 (bennett et al., 2008) . consistent with this report, our immunofluorescence experiment results indicate that cells expressing the ha3g deletion mutant δ1-156 show fluorescence staining in both the nucleus and cytoplasm; in contrast, fluorescence in cells expressing wt or ha3g mutants δ1-104, δ157-384, or δ246-384 is predominantly present in cell cytoplasm (data not shown). however, the mislocalization of δ1-156 does not account for its defective incorporation into hiv-1 or sars-cov vlps, since gst pull-down assay results suggest that cytoplasmic ha3g(δ1-156) is still incapable of associating with sars-cov n (data not shown). hcov-229e n and sars-cov n share two conserved sequences: one from residues 50 to 170 and the other from 250 to 360 (tswen-kei tang, 2005) . combined, the ability of nc(229en2) to package ha3g and the rna-dependent association of hcov-229e n with ha3g ( fig. 7) imply that in addition to sars-cov n, coronaviral n products may associate with ha3g via binding with rna. however, human tcell leukemia virus type 1 (htlv-1) has been found to prevent ha3g packaging through gag nc domain action (derse et al., 2007) , suggesting that some rna viruses are incapable of packaging a3g. our results indicate that the presence of the ha3g first linker region is required for ha3g to be packaged into either hiv-1 gag or sars-cov vlps. it is possible that an rna/ha3g complex, either alone or in combination with other cellular factors, is an acceptable structure for an association between hiv-1 nc and sars-cov n. here we demonstrated for the first time that sars-cov is capable of packaging ha3g via the sars-cov n protein. it remains to be determined whether chimeric nc(n2) or sars-cov can package endogenous a3g as efficiently as hiv-1. the biological significance of a sars-cov n association with ha3g also requires further exploration. according to one report, ha3g is capable of editing virus-associated hiv-1 rna (bishop et al., 2004) ; the ha3g-mediated inhibition of viral replication may be independent of its cytidine deaminase activity (rösler et al., 2005; turelli et al., 2004) . since humans are not natural hosts for sars-cov , human susceptibility implies that cells infected by sars-cov may not express a3g, or sars-cov may have evolved to counteract a3g anti-viral activity. further studies are required to address this issue. the parental hiv-1 proviral plasmid dna used in this study was hxb 2 (ratner et al., 1985) . the cdna clone of the sars-cov n (sars coronavirus strain twc, genbank accession number ay321118) was provided by the centers for disease control of the department of health, taiwan. hiv-1 nc-deletion mutants δnc, delnc, and δpc and chimeras δnc(con), nc(con), nc(n1), nc(n2), nc(n3), nc(n4), nc (n5), nc(n6), nc(n7), δnc(wtzip) and δnc(kzip) were all as previously described . primers used for making nc(229en2) were 5′-cgcaatcgattcatgaaggcagttgct-3′ (for-ward) and 5′-cttcggatcccgtttacttcatcaat-3′ (reverse) using a coronavirus 229e (hcov-229e) nucleocapsid expression vector ptre-hn (schelle et al., 2005) as template (kindly provided by volker thiel). to construct a vif-deficient (δvif) mutation, vif-encoding plasmid dna was digested with ndei, filled in, and relegated, resulting in a shift in the vif open reading frame. all of the engineered constructs were cloned into the pr-defective hiv-1 proviral expression vector hivgptd25 (wang et al., 2000) . human apobec3g (ha3g) expression vector pcdna3.1-apobec3g-ha (sheehy et al., 2002; stopak et al., 2003) was obtained through the nih aids research and reference reagent program. ha3g deletion mutants were gifts from s. cen (cen et al., 2004) . mammalian expression vectors encoding sars-cov m and n were provided by g.j. nabel (huang et al., 2004b) . to construct gst fusions, amplicons containing sars-cov n or hcov-229e n coding sequences were digested with bamhi and clai and fused to the c-terminus of gst, which is directed by a mammalian elongation factor 1a promoter (cortes et al., 1996) . primers for cloning gst fusions were gst-con, 5′-taaaggatcctctgataatggaccc-3′ (forward), 5′-tcatatcgatttatgcctgagttgaatc-3′ (reverse); gst-n1, 5′-taaaggatcctctgataatggaccc-3′ (forward), 5′-gctcat-cgattagctagccattcgagc-3′ (reverse); gst-n2, 5′-tggctggat-ccggtggtgaaactgcc-3′ (forward), 5′-tcatatcgatttatgcct-gagttgaatc-3′ (reverse); gst-n5, 5′-attacggatcctggccg-caaattgca-3′ (forward), 5′-tcatatcgatttatgcctgagttgaatc-3′ (reverse); gst-n6, 5′-taaaggatcctctgataatggaccc-3′ (forward), 5′-gctcatcgattatgttgttccttgagg-3′(reverse); gst-n7, 5′-taaaggatcctctgataatggaccc-3′ (forward), 5′-gctcatcgat-tagccaatttggtcat-3′ (reverse); gst-229en, 5′-cgggatccgcta-cagtcaaatgg-3′ (forward), 5′-ccatcgattagtttacttcatcaat-3′ (reverse); gst-229en1, 5′-cgggatccgctacagtcaaatgg-3′ (forward), 5′-ccatcgattattcctgaggcttgtc-3′ (reverse); and gst-229en2, 5′-cgggatccatgaaggcagttgct-3′ (forward), 5′-ccatc-gattagtttacttcatcaat-3′ (reverse). mutations were confirmed by restriction enzyme digestion or dna sequencing. 293t and 293 cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal calf serum (gibco). confluent 293t or 293 cells were trypsinized and split 1:10 onto 10cm dishes 24 h prior to transfection. for each construct, cells were transfected with 20 μg of plasmid dna using the calcium phosphate precipitation method; 50 μm chloroquine was added to enhance transfection efficiency. unless otherwise indicated, 10 μg of each plasmid was used for co-transfection. culture supernatant and cells were harvested for protein analysis 2-3 d post-transfection. at 48-72 h post-transfection, supernatant from transfected 293t or 293 cells was collected, filtered, and centrifuged through 2 ml of 20% sucrose in tse (10 mm tris-hcl [ph 7.5], 100 mm nacl, 1 mm edta plus 0.1 mm phenylmethylsulfonyl fluoride [pmsf]) at 4°c for 40 min at 274,000 ×g. pellets were suspended in ipb (20 mm tris-hcl [ph 7.5], 150 mm nacl, 1 mm edta, 0.1% sds, 0.5% sodium deoxycholate, 1% triton x-100, 0.02% sodium azide) plus 0.1 mm pmsf. next, cells were rinsed with ice-cold phosphate-buffered saline (pbs), collected in ipb plus 0.1 mm pmsf, and microcentrifuged at 4°c for 15 min at 13,700 ×g to remove cell debris. supernatant and cell samples were mixed with equal volumes of 2x sample buffer (12.5 mm tris-hcl [ph 6.8], 2% sds, 20% glycerol, 0.25% bromophenol blue) and 5% β-mercaptoethanol and boiled for 5 min. samples were resolved by electrophoresis on sds-polyacrylamide gels and electroblotted onto nitrocellulose membranes. membrane-bound gag or chimeric proteins were immunodetected using a mouse monoclonal antibody directed against hiv-1 p24ca (wang et al., 1998) or the sars-cov nucleocapsid . sars-cov m was detected with a rabbit anti-m polyclonal antibody (rockland). hatagged ha3g was probed with a mouse anti-ha (sigma) monoclonal antibody at a dilution of 1:5000. for hiv-1 vif detection, a rabbit anti-vif polyclonal antibody (goncalves et al., 1994) was used at a 1:2000 dilution. the secondary antibody was a sheep anti-mouse or donkey anti-rabbit horseradish peroxidase-(hrp) conjugated antibody (invitrogen), both at 1:5000 dilutions. to detect gst and gst fusions, anti-gst hrp conjugate (amersham) was used at a 1:5000 dilution. horseradish peroxidase activity was detected according to the manufacturer's protocol. gst pull-down assay 293t cells either mock transfected or transfected with gst fusion expression vectors were collected, lysed in ripa buffer (140 mm nacl, 8 mm na 2 hpo 4 , 2 mm nah 2 po 4 , 1% np-40, 0.5% sodium deoxycholate, 0.05% sds) containing complete protease inhibitor cocktail (roche), and microcentrifuged at 4°c for 15 min at 13,700 ×g (14,000 rpm) to remove cell debris. aliquots of post-nuclear supernatant (pns) were mixed with equal amounts of 2× sample buffer and 5% β-mercaptoethanol and held for western blot analysis. ripa buffer was added to the remaining pns samples to final volumes of 500 μl. each sample was mixed with glutathione-agarose beads (30 μl) (sigma) and rocked for 2 h at 4°c. bead-bound complexes were pelleted, washed tree times with ripa buffer, twice with pbs, eluted at 1× sample buffer with 5% β-mercaptoethanol, boiled for 5 min, and subjected to sds-10% page as described above. supernatant cultures of transfected 293t cells were collected, filtered, and centrifuged through 2 ml 20% sucrose cushions as described above. viral pellets were suspended in pbs buffer and laid on top of a pre-made 20-60% sucrose gradient consisting of 1 ml layers of 20, 30, 40, 50 and 60% sucrose in tse that had been allowed to sit for 2 h. gradients were centrifuged in an sw50.1 rotor at 40,000 rpm (274,000 ×g) for 16 h at 4°c; 500 μl fractions were collected from top to bottom. sucrose density was measured for each fraction. proteins in each fraction were precipitated with 10% trichloroacetic acid (tca) and subjected to western immunoblotting. confluent 293 cells were split 1:80 onto coverslips 24 h before transfection. two days post-transfection, cells were fixed at 4°c for 20 min with ice-cold pbs containing 3.7% formaldehyde, washed once with pbs and once with dmem plus 10% heat-inactivated calf serum (dmem/calf serum), and permeabilized at room temperature for 10 min in pbs plus 0.2% triton x-100. samples were incubated with the primary antibody for 1 h and secondary antibody for 30 min. following each incubation, samples were subjected to three washes (5 to 10 min each) with dmem/calf serum. the primary antibody was an anti-ha (sigma) at a 1:200 dilution. a rabbit antimouse rhodamine-conjugated antibody at a 1:100 dilution served as the secondary antibody (cappel, icn pharmaceuticals, aurora, ohio, usa). after the final dmem/calf serum wash, the coverslips were washed three times with pbs and mounted in 50% glycerol in pbs for viewing. images were taken using an epifluorescence microscope (olympus ax-80) or laser scanning confocal microscope (olympus fv300). efficient particle production by minimal gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain apobec3g is incorporated into virus-like particles by a direct interaction with hiv-1 gag nucleocapsid protein functional chimeras of the rous sarcoma virus and human immunodeficiency virus gag proteins nuclear exclusion of the hiv-1 host defense factor apobec3g requires a novel cytoplasmic retention signal and is not dependent on rna binding specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral rnas detected by rna mobility shift assays retroviral nucleocapsid domains mediate the specific recognition of genomic viral rnas by chimeric gag polyproteins during rna packaging in vivo apobec-mediated editing of viral rna single-stranded rna facilitates nucleocapsid: apobec3g complex formation importance of basic residues in the nucleocapsid sequence for retrovirus gag assembly and complementation rescue human immunodeficiency virus type 1 gag polyprotein multimerization requires the nucleocapsid domain and rna and is promoted by the capsid-dimer interface and the basic region of matrix protein the interaction between hiv-1 gag and apobec3g hiv-1 matrix protein repositioning in nucleocapsid region fails to confer virus-like particle assembly structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna extensive mutagenesis experiments corroborate a structural model for the dna deaminase domain of apobec3g the vif protein of hiv triggers degradation of the human antiretroviral dna deaminase apobec3g in vitro v(d)j recombination: signal joint formation role and mechanism of action of the apobec3 family of antiretroviral resistance factors resistance of human t cell leukemia virus type 1 to apobec3g restriction is mediated by elements in nucleocapsid subcellular localization of the vif protein of human immunodeficiency virus type 1 dna deamination mediates innate immunity to retroviral infection analysis of multimerization of the sars coronavirus nucleocapsid protein assembly of severe acute respiratory syndrome coronavirus rna packaging signal into virus-like particles is nucleocapsid dependent structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production identification of amino acid residues in apobec3g required for regulation by human immunodeficiency virus type 1 vif and virion encapsidation nucleic acid-independent retrovirus assembly can be driven by dimerization the human immunodeficiency virus type 1 vif protein reduces intracellular expression and inhibits packaging of apobec3g (cem15), a cellular inhibitor of virus infectivity viral rna is required for the association of apobec3g with human immunodeficiency virus type 1 nucleoprotein complexes bats are natural reservoirs of sars-like coronaviruses influence of primate lentiviral vif and proteasome inhibitors on human immunodeficiency virus type 1 virion packaging of apobec3g amino-terminal region of the human immunodeficiency virus type 1 nucleocapsid is required for human apobec3g packaging carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization broad antiretroviral defence by human apobec3g through lethal editing of nascent reverse transcripts species-specific exclusion of apobec3g from hiv-1 virions by vif hiv-1 vif protein binds the editing enzyme apobec3g and induces its degradation vif overcomes the innate antiviral activity of apobec3g by promoting its degradation in the ubiquitin-proteasome pathway characterization of n protein self-association in coronavirus ribonucleoprotein complexes complementary function of the two catalytic domains of apobec3g redundant roles for nucleocapsid and matrix rna-binding sequences in human immunodeficiency virus type 1 assembly charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in rna packaging and infectivity apobec-mediated interference with hepadnavirus production complete nucleotide sequence of the aids virus, htlv-iii specific packaging of apobec3g into hiv-1 virions is mediated by the nucleocapsid domain of the gag polyprotein precursor selective replication of coronavirus genomes that express nucleocapsid protein isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein the antiretroviral enzyme apobec3g is degraded by the proteasome in response to hiv-1 vif hiv-1 vif blocks the antiviral activity of apobec3g by impairing both its translation and intracellular stability the nucleocapsid protein of the sars coronavirus is capable of self-association through a c-terminal 209 amino acid interaction domain human apolipoprotein b mrna-editing enzymecatalytic polypeptide-like 3g (apobec3g) is incorporated into hiv-1 virions through interactions with viral and nonviral rnas biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229e human coronaviruses inhibition of hepatitis b virus replication by apobec3g analysis of minimal human immunodeficiency virus type 1 gag coding sequences capable of virus-like particle assembly and release assembly and processing of human immunodeficiency virus gag mutants containing a partial replacement of the matrix domain by the viral protease domain 7sl rna mediates virion packaging of the antiviral cytidine deaminase apobec3g severe acute respiratory syndrome coronavirus nucleocapsid protein confers ability to efficiently produce virus-like particles when substituted for the human immunodeficiency virus nucleocapsid domain induction of apobec3g ubiquitination and degradation by an hiv-1 vif-cul5-scf complex single-strand specificity of apobec3g accounts for minusstrand deamination of the hiv genome recombinant severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein forms a dimer through its c-terminal domain apobec3g incorporation into human immunodeficiency virus type 1 particles effects of nucleocapsid mutations on human immunodeficiency virus assembly and rna encapsidation analysis of the assembly function of the human immunodeficiency virus type 1 gag protein nucleocapsid domain the cytidine deaminase cem15 induces hypermutation in newly synthesized hiv-1 dna we thank y.-p. li for the reagents and technical assistance, cen shan for providing the ha3g deletion mutant plasmids, volker thiel for the hcov-229e n cdna, and g. nabel for the sars-cov m and n expression vectors. the following reagents were obtained through the nih aids research and reference reagent program: pcdna3.1-apobec3g-ha from warner c. greene and hiv-1 vif antiserum from d. gabuzda. this work was supported by grants vgh94-314 from the taipei veterans general hospital and nsc94-2320-b-010-035 from the national science council, taiwan, republic of china. key: cord-271970-i35pic5o authors: boris, bonaventure; antoine, rebendenne; de gracia francisco, garcia; marine, tauziet; joe, mckellar; valadão ana luiza, chaves; valérie, courgnaud; eric, bernard; laurence, briant; nathalie, gros; wassila, djilli; mary, arnaud-arnould; hugues, parrinello; stéphanie, rialle; olivier, moncorgé; caroline, goujon title: a genome-wide crispr/cas9 knock-out screen identifies the dead box rna helicase ddx42 as a broad antiviral inhibitor date: 2020-10-28 journal: biorxiv doi: 10.1101/2020.10.28.359356 sha: doc_id: 271970 cord_uid: i35pic5o genome-wide crispr/cas9 knock-out genetic screens are powerful approaches to unravel new regulators of viral infections. with the aim of identifying new cellular inhibitors of hiv-1, we have developed a strategy in which we took advantage of the ability of type 1 interferon (ifn) to potently inhibit hiv-1 infection, in order to create a cellular environment hostile to viral replication. this approach led to the identification of the dead-box rna helicase ddx42 as an intrinsic inhibitor of hiv-1. depletion of endogenous ddx42 using sirna or crispr/cas9 knock-out increased hiv-1 infection, both in model cell lines and in physiological targets of hiv-1, primary cd4+ t cells and monocyte-derived macrophages (mdms), and irrespectively of the ifn treatment. similarly, the overexpression of a dominant-negative mutant of ddx42 positively impacted hiv-1 infection, whereas wild-type ddx42 overexpression potently inhibited hiv-1 infection. the positive impact of endogenous ddx42 depletion on hiv-1 infection was directly correlated to an increase in viral dna accumulation. interestingly, proximity ligation assays showed that ddx42, which can be mainly found in the nucleus but is also present in the cytoplasm, was in the close vicinity of hiv-1 capsid during infection of primary monocyte-derived macrophages. moreover, we show that ddx42 is also able to substantially decrease infection with other retroviruses and retrotransposition of long interspersed elements-1 (line-1). finally, we reveal that ddx42 potently inhibits other pathogenic viruses, including chikungunya virus and severe acute respiratory syndrome coronavirus 2 (sars-cov-2). over the past 20 years, a growing list of cellular proteins with various functions have been identified as capable of limiting different steps of hiv-1 life cycle (doyle et al., 2015; ghimire et al., 2018) . lentiviruses have generally evolved to counteract the action of these so-called restriction factors. however, type 1 interferons (ifns) induce, through the expression of interferonstimulated genes, an antiviral state particularly efficient at inhibiting hiv-1 when cells are preexposed to ifn (ho et al., 1985; bednarik et al., 1989; coccia et al., 1994; baca-regen et al., 1994; goujon and malim, 2010; cheney and mcknight, 2010) . the dynamin-like gtpase mx2, and, very recently, the restriction factor trim5a, have both been shown to participate in this ifninduced inhibition (goujon et al., 2013a; kane et al., 2013; ohainle et al., 2018; jimenez-guardeño et al., 2019) . with the hypothesis that additional hiv-1 inhibitors remained to be identified, we took advantage of the hostile environment induced by ifn to develop a wholegenome screen strategy in order to reveal such inhibitors. the development of crispr/cas9 as a genome editing tool in mammalian cells has been a major breakthrough, notably with the generation of pooled single guide (sg)rna libraries delivered with lentiviral vectors (lvs), allowing high-throughput screens at the whole-genome scale (shalem et al., , 2015 doench, 2018) . we used the genome-scale crispr knock-out (gecko) sgrna library developed by feng zhang's laboratory shalem et al., 2014 shalem et al., , 2015 to generate cell populations knocked-out for almost every human gene in the t98g glioblastoma cell line. this model cell line is both highly permissive to lentiviral infection and potently able to suppress hiv-1 infection following ifn treatment (supplementary information, si figure 1 ). the screen strategy is depicted in figure 1a . t98g cells were first modified to stably express cas9 and a high number of t98g-cas9 cells were then transduced with lvs coding sublibraries a or b, the two halves of the gecko library. a low multiplicity of infection (moi) was used to avoid multiple integration events and increase the probability to express only 1 sgrna per cell. deep sequencing analysis of the gecko cell populations showed more than 94% coverage for both libraries (≥ 10 reads for 61,598 and 54,609 sgrna-coding sequences out of gecko populations were subjected to type 1 ifn treatment in order to induce the antiviral state and, 24h later, incubated with vsv-g-pseudotyped, hiv-1 based lvs coding for an antibiotic resistance cassette. two days later, the cells successfully infected despite the ifn treatment were selected by cell survival in the presence of the corresponding antibiotic. in order to enrich the population with mutants of interest and to limit the presence of false-positives, two additional rounds of ifn treatment, infection and selection (using different antibiotics) were performed ( figure 1a ). as expected, the cells enriched after each round of the screen became less refractory to hiv-1 infection following ifn treatment (si figure 2) . . the gecko populations were then exposed to ifn for 24h and challenged with hiv-1-based lvs coding for an antibiotic resistance gene. after selection by antibiotic addition, the surviving cells (i.e. efficiently infected despite the ifn treatment) were amplified. in total, the gecko population underwent three successive rounds of ifn treatment, infection and selection using lvs coding for different resistance cassettes. the genomic dnas of the initial gecko populations and the three-time selected populations were extracted, the sgrnacoding sequences were amplified by pcr and sequenced by next generation sequencing (ngs). b. the candidate genes were identified using the mageck computational statistical tool (li et al., 2014) . mageck establishes a robust rank aggregation (rra) score for each gene based on the sgrna enrichment and the ctrl ifnar1 mx2 wars2 ddx42 cldn12 reep1 ikbip topbp1 copg1 fgf4 spryd7 rdh8 dctpp1 prlr sun3 cxorf27 tmem161a kcn6 lsm5 loc339862 pkd2 ndpc1 kiaa1549 mir-4737 vstm2a calu smarca2 dnajb2 fam212a etnppl rhoc mir-105-1 cxorf56 ccser1 slc25a2 or6n2 uba1 hspa1b siae krt222 adra1a lin54 tectb cdrt1 tceal7 trerf1 cd14 hsp90aa1 ctnna2 plac1l relb bsnd 0 gecko population versus selected population number of sgrnas targeting the same gene. here, genes belonging to the ifn-response pathway (indicated in blue) and ddx42 (in red) are represented (together with their respective rank into brackets) for the 2 independent screens (the results of which were merged in the analysis). c. t98g/cd4/cxcr4/cas9/firefly ko populations were generated for the 25 best candidate genes of each screen. the control (ctrl) condition represents the mean of four negative control cell populations (i.e. expressing 4 different non-targeting sgrnas) and ifnar1 and mx2 ko cell populations were used as positive controls. ko cell populations were pre-treated with ifn and infected with hiv-1 renilla. the cells were lysed 24 h post-infection and the two luciferase signals were measured (renilla signals were normalized to internal firefly control). the ifn inhibition (corresponding to the ratio of the untreated / ifn-treated conditions) was calculated and sets at 100% inhibition for the average of the 4 negative ctrl populations. a representative experiment is shown (mean and standard deviation from technical duplicates). to identify the genes of interest, the differential sgrna abundance between the starting gecko populations and the enriched (3-times selected) populations was analysed by ngs. 2,332 and 3,900 different sgrnas were identified (≥ 10 reads) for screens a and b, which represented 3,6% and 7% of the sgrnas present in the initial gecko population a and b, respectively. the mageck algorithm, which assigns a robust ranking aggregation (rra) score, was used to rank the gene candidates from each screen ( figure 1b ). for both screens, we observed a positive enrichment for 200 genes (rra score > 0,01), with the best hits being ifnar1, jak1 and stat2 ( figure 1b ). all the crucial mediators of the type 1 ifn signalling cascade were present among the top hits in both screens (with the notable exception of stat1), validating our approach and confirming the identification of relevant genes. interestingly, most of the other 195 positively selected genes displayed unknown functions or functions that were a priori unrelated to the ifn response pathway or to innate immunity. of note, very little overlap was observed between the two independent screens, performed with two different sub-libraries. however, a poor overlap between independent screens has been observed before and does not preclude obtaining valid data (doench, 2018) . therefore, the top 25 candidate genes from each independent screen were selected for further validation. as a first validation step, the sequences of the most enriched sgrna for each gene were chosen and cloned into the lentiguide-puro vector . t98g-cas9 cells expressing hiv-1 cd4 and cxcr4 receptors, as well as the firefly luciferase as internal control (t98g cas9/cd4/cxcr4/firefly cells), were transduced with the sgrna-expressing lvs to generate individual ko populations. four irrelevant, non-targeting sgrnas, as well as sgrnas targeting ifnar1 and mx2, were used to generate negative and positive control populations, respectively. the ko cell populations were pre-treated with ifn and infected with an hiv-1 reporter virus expressing the renilla luciferase reporter and bearing hiv-1 envelope (nl4-3/nef-ires-renilla, hereafter called hiv-1 renilla). infection efficiency was analysed 30h later ( figure 1c ). as expected, ifnar1 and mx2 ko fully and partially rescued hiv-1 infection from the protective effect of ifn, respectively (goujon et al., 2013a; bulli et al., 2016; xu et al., 2018) . the ko of two candidate genes, namely wars2 and ddx42, allowed a partial rescue of hiv-1 infection from the ifn-induced inhibition, suggesting a potential role of these candidate genes. ddx42 is a member of the dexd/h box family of rna helicases with rna chaperone activities (uhlmann-schiffler et al., 2006) and, as such, retained our attention. indeed, various dead box helicases, such as ddx3, ddx6 and ddx17, are well-known to regulate hiv-1 life cycle (gringhuis et al., 2017; sithole et al., 2018 sithole et al., , 2020 soto-rifo et al., 2013; williams et al., 2015; yedavalli et al., 2004) . however, to our knowledge, the impact of ddx42 on hiv-1 replication had never been studied. in order to validate the effect of ddx42 ko on hiv-1 infection in another model cell line, two additional sgrnas were designed (sgrna-2 and -3) and used in parallel to the one identified in the gecko screen (sgddx42-1) (figure 2a ). u87-mg/cd4/cxcr4 cells were used here, as we previously extensively characterized the ifn phenotype in these cells (goujon et al., 2013a) . control and ddx42 ko cell populations were treated or not with ifn for 24h prior to infection with increasing amounts of hiv-1 renilla. ddx42 depletion improved hiv-1 infection with all three sgrnas used, confirming that endogenous ddx42 had a negative impact on hiv-1 infection. interestingly, the increase in infection efficiency induced by ddx42 ko was observed irrespectively of the ifn treatment. ddx42 is not known to be an isg (interferome database and our previous study (goujon et al., 2013a) , geo accession number: gse46599), which we confirmed in a number of cell types (si figure 3 ). the fact that the ifn-induced state is at least partially saturable (si figure 1 ) explains why an intrinsic inhibitor of hiv-1, which is not regulated by ifn, could be identified by our approach: removing one barrier to infection presumably rendered the cells generally more permissive and, in this context, ifn had less of an impact. sgddx42-2, and sgddx42-3) and 4 different non-targeting sgrnas, respectively (for the ctrl condition, the average of the data obtained with the four cell populations is shown). cells were pre-treated or not with ifn 24 h prior to infection with hiv-1 renilla (50 ng p24 gag ) and the ratio of renilla/firefly activity was analysed, as in 3) and the infection efficiency was measured by p24 gag intracellular staining followed by flow cytometry analysis. when indicated, the cells were treated with 10 µm zidovudine (azidothymidine, azt) and lamivudine (3tc) reverse transcription inhibitors for 2h prior to infection. d. blood monocytes from healthy donors were isolated, differentiated into mdms, and transfected with non-targeting sirnas (sictrl1 and sictrl2) or sirnas targeting ddx42 (siddx42-1 and siddx42-2). two days after transfection, mdms were infected with a ccr5tropic version of nl4-3-renilla (100 ng p24 gag ). infection efficiencies were monitored 24h later by measuring renilla activity. the relative luminescence results from experiments performed with cells from 3 different donors are shown. e. cd4+ t cells were isolated from peripheral blood mononuclear cells, activated with il-2 and phytohemagglutinin, and electroporated with cas9-sgrna rnp complexes using two non-targeting sgrnas (sgctrl1 and sgctrl2) and five sgrnas targeting ddx42 (sgddx42-1, -2, -3, -4, -5) two days later. four days after electroporation, the activated cd4+ t cells were infected with nl4-3 renilla for 24h. relative infection efficiencies obtained with cells from three independent donors are shown. ddx42 protein levels were determined by immunoblot and actin served as a loading control (a representative experiment is shown in order to confirm ddx42's effect on hiv-1 infection with an independent approach, we used 3 different sirnas to knockdown ddx42 expression. we observed that depleting ddx42 with sirnas (with >90% efficiency both at the mrna and protein levels, figure 2b ) improved hiv-1 infection efficiency by 3 to 8-fold when using an hiv-1 renilla reporter in u87-mg/cd4/cxcr4 cells, irrespectively of the presence of ifn ( figure 2b , right panel). of note, wild-type hiv-1 infection was also impacted by ddx42 silencing, as shown by capsid (p24 gag ) intracellular staining 30h post-infection ( figure 2c ). we then investigated whether ddx42 had an impact in hiv-1 primary target cells. in mdms, we observed that hiv-1 infection was increased by about 2fold following ddx42 silencing ( figure 2d ), whereas ddx42 mrna abundance was decreased by only 40% in these cells using sirnas (si figure 4 ). as the sirna approach did not work in our hands in primary t cells, we used electroporation of pre-assembled cas9-sgrna ribonucleoprotein complexes (rnps) to deplete ddx42 in primary cd4+ t cells ( figure 2e ). highly efficient depletion of ddx42 was obtained with all 5 sgrnas as compared to the 2 sgctrls ( figure 2e , bottom panel) and this depletion increased hiv-1 infection by 2-to 3-fold, showing a role of ddx42 as an intrinsic inhibitor of hiv-1 in primary cd4+ t cells. having established that endogenous ddx42 had an impact on hiv-1 infection, we then analysed the consequences of ddx42 overexpression. an irrelevant control (firefly) or ddx42 were ectopically expressed in u87-mg/cd4/cxcr4 and the cells were challenged with hiv-1 renilla ( figure 2f ). ddx42 ectopic expression induced a substantial inhibition of hiv-1 infection (about 5-fold decrease in infection efficiency in comparison to the control) ( figure 2f ). we then tested a mutant version of ddx42 that is unable to hydrolyse atp and may supposedly act as a dominant negative, ddx42 k303e (granneman et al., 2006; rocak, 2005 ) (si figure 5) . interestingly, the expression of ddx42 k303e mutant increased hiv-1 infection by 3-fold, reminiscent of what we observed with ddx42 depletion. altogether, these data showed for the first time that endogenous ddx42 is able to intrinsically inhibit hiv-1 infection. in order to determine the step of hiv-1 life cycle affected by ddx42, we first analysed viral entry with a blam-vpr assay (cavrois et al., 2002) . consistent with the observation that vsv-gpseudotyping did not bypass ddx42-mediated inhibition of infection (si figure 6 ), we observed that ddx42 silencing did not impact hiv-1 entry (si figure 7) . we then quantified hiv-1 dna accumulation over time in ddx42-silenced and control cells. ddx42 depletion increased by 2-to 5-fold the accumulation of early and late reverse transcript products ( figure 3a , b and c), as well as integrated provirus and 2-ltr circles at 48h post-infection ( figure 3d and e). more than 85% knockdown was achieved with both sirnas targeting ddx42 ( figure 3f ). these data suggested that ddx42 rna helicase could inhibit the reverse transcription process and/or impact the stability of hiv-1 genome, leading to a decrease in viral dna accumulation. we hypothesized that if that was the case, ddx42 should be found in close proximity to hiv-1 reverse transcription complexes during infection. in agreement with this, proximity ligation assay (pla) performed on mdms infected with hiv-1 showed that ddx42 could indeed be found in close vicinity of capsid ( figure 3f and g). we next examined the ability of ddx42 to inhibit infection by a range of primate lentiviruses including laboratory-adapted strains of hiv-1, hiv-1-transmitted founder strains, hiv-2 and simian immunodeficiency virus derived from the rhesus macaque (sivmac). tzm-bl cells were transfected with ddx42-targeting or scramble sirnas and infected with vsv-g-pseudotyped lentiviruses. infection efficiencies were monitored after 24h by measuring β-galactosidase activity ( figure 3h ). ddx42 depletion increased infection levels with all the tested hiv-1 strains to the same extent than what was observed with hiv-1 nl4-3 (i.e. 3-to 5-fold). hiv-2rod10 and sivmac infection efficiencies were also slightly improved in the absence of ddx42 (by about 2-fold). the analysis was then extended to two non-primate lentiviruses, the equine infectious anaemia virus (eiav) and feline immunodeficiency virus (fiv), using gfp-coding lvs derived from these viruses in comparison to hiv-1 and hiv-2 lvs (si figure 8 ). ddx42 antiviral activity appeared less potent on hiv-1 lvs compared to replication-competent, full-length hiv-1, which might suggest that viral components, absent in lvs, could be playing a role in ddx42-mediated hiv-1 inhibition. nevertheless, ddx42 depletion appeared to increase hiv-1, hiv-2 and fiv lv infection to the same extent, i.e. by about 2-fold, whereas eiav infection was less impacted by ddx42 (si figure 8 ). we extended this study to the gammaretrovirus murine leukaemia virus (mlv) and observed that ddx42 depletion led to an increase in infection with gfp-coding mlv vectors ( figure 3i ). these results strongly support a general antiviral activity of ddx42 against retroviruses. ddx42 can be found in the cytoplasm but is predominantly located in the nucleus (si figure 9 ; uhlmann-schiffler et al., 2009; zyner et al., 2019) . considering that ddx42 showed a broad activity against retroviruses and seemed to act at the level of reverse transcription, we sought to investigate whether ddx42 could inhibit retrotransposons. long interspersed nuclear elements (line)-1 are non-ltr retrotransposons, which have been found to be active in the germ line (branciforte and martin, 1994; ergün et al., 2004; trelogan and martin, 1995) and in some somatic cells (belancio et al., 2010; muotri et al., 2005; rangwala et al., 2009) . interestingly, ddx42 was identified among the suppressors of line-1 retrotransposition through a genome-wide screen in k562 cells, although not further characterized (liu et al., 2018) . to confirm that ddx42 could inhibit line-1 retrotransposition, hek293t cells were co-transfected with two different, gfpexpressing line-1 plasmids (rps or lre3) or an inactive line-1 (jm111) together with a ddx42-or a control (firefly)-expressing plasmid ( figure 3j ). gfp-line-1 retrotransposition was quantified by flow-cytometry 7 days post-transfection (moran et al., 1996) . because the gfp cassette is cloned in antisense and disrupted by an intron in this reporter system, gfp is only expressed after line-1 transcription, splicing and orf2p-mediated reverse-transcription and integration into the host genome (moran et al., 1996) . considering that most line-1 replication cycles lead to truncations and defective integrations (gilbert et al., 2005) , gfp expression derived from a new integration is a relatively rare event and, as expected, the percentage of gfp+ cells observed was very low ( figure 3j ) (figure 4) . strikingly, ddx42 depletion did not have an impact on iav or vsv replication ( figure 4a and b), thereby confirming that manipulating ddx42 expression did not have a broad and unspecific impact on target cells. however, depletion of endogenous ddx42 increased infection with zikv, chikv and sars-cov-2, and had a particularly high impact on the latter two (up to 1 log and 3 log increase in infection efficiency in ddx42-depleted cells in comparison to control cells, for chikv and sars-cov-2, respectively, figure 4d and f). of note, silencing efficiency was similar in the two types of target cells used here ( figure 4e and g). interestingly, ddx42 was recently identified as a potential inhibitor of sars-cov-2 replication in a whole-genome crispr/cas9 screen in simian vero e6 cells (wei et al., 2020) , supporting our observations that endogenous ddx42 potently inhibits the replication of this highly pathogenic coronavirus. taken together, our data showed that ddx42 is a broad inhibitor of viral infections, albeit presenting some specificity. further work is now warranted to explore in depth the breadth of ddx42 antiviral activity. . in contrast, our study revealed broad activity of endogenous ddx42 among retroviruses and retroelements, which was observed in various cell types, including primary cd4+ t cells. interestingly, our pla assays showed a close proximity between ddx42 and hiv-1 capsid, which is a viral protein recently shown to remain associated with reverse transcription complexes until proviral dna integration in the nucleus (burdick et al., 2020; dharan et al., 2020; peng et al., 2014) . this observation could suggest a direct mode of action on viral ribonucleoprotein (rnp) complexes. interestingly, we observed that ddx42 was able to inhibit viruses from other families, which possess different replication strategies, including sars-cov-2 and chikv. however, ddx42 did not have an impact on all the viruses we tested, reminiscent of broad-spectrum antiviral inhibitors such as mxa, which show some specificity (haller et al., 2015) . ddx42 is known to be a non-processive helicase, which also possesses rna annealing activities and the ability to displace rna-binding proteins from single-stranded rnas (uhlmann-schiffler et al., 2006) . moreover, ddx42 binds g-quadruplexes (zyner et al., 2019) , which are secondary structures found in cellular and viral nucleic acids and involved in various processes, such as transcription, translation and replication (fay et al., 2017; ruggiero and richter, 2018) . all these known activities of ddx42 would be consistent with a potential role in rnp remodeling (uhlmann-schiffler et al., 2006; will et al., 2002) . nonetheless, further investigation will be needed to determine whether ddx42 acts directly by altering viral rnps, and, if that's the case, what are the determinants for viral rnp recognition. in conclusion, this work highlights the importance of understanding the mechanism of action of ddx42 rna helicase and its contribution to the control of rna virus replication, an understanding which may contribute to the development of future antiviral interventional strategies. plasmids were a gift from prof. f. zhang (addgene #52962, #52963, and #1000000048, respectively ). lvs coding for sgrnas targeting the candidate genes and bearing hiv-1 nl4-3, iiib and hiv-2 proviral clones have been described (adachi et al., 1986; simon et al., 1995; schaller et al., 2011) , as well as the transmitted founder hiv-1 molecular clones ch077.t, ch106.c, rejo.c (gifts from prof. b. hahn, (ochsenbauer et al., 2012) ) and hiv-2rod10 and sivmac239 (ryan-graham and peden, 1995; gaddis et al., 2004) . pblam-vpr and padvantage have been described (cavrois et al., 2002) . gfp-coding hiv-1 based lv system (i.e. p8.91 hiv-1 gag-pol, pmd.g, and gfp-coding minigenome), and hiv-2, fiv, and eiavderived, gfp coding lvs, as well as mlv-derived, gfp coding retroviral vectors have all been described (naldini et al., 1996; bainbridge et al., 2001) , (o'rourke et al., 2002; saenz et al., 2005) . the line-1 plasmid 99 rps-gfp pur (prps-gfp), 99 rps-gfp jm111 pur (pjm111) and plre3-gfp were developed by prof. kazazian's lab (moran et al., 1996; ostertag et al., 2000; goodier et al., 2012) . was added at 1000 u/ml for 16-24h prior to virus infection or rna extraction, and azt and 3tc (aids reagent program) at 10 µm for 2 h prior to infection. sang, under agreement n°21pler2019-0106. peripheral blood mononuclear cells (pbmcs) were isolated by centrifugation through a ficoll® paque plus cushion (sigma-aldrich). primary human cd4+ t cells and monocytes were purified by positive selection using cd3 and cd14 microbeads, respectively (miltenyi biotec), as previously described (goujon et al., 2013a) . hiv-1 renilla and nl4-3 hiv-1 were produced by standard pei transfection of hek293t. when indicated, pmd.g was cotransfected with the provirus at a 3:1 ratio. the culture medium was changed 6h later, and virus-containing supernatants were harvested 42h later. viral particles were filtered, purified by ultracentrifugation through a sucrose cushion (25% weight/volume in tris-nacl-edta buffer) for 75 min at 4°c and 28,000 rpm using a sw 32 ti rotor (beckman coulter), resuspended in serum-free rpmi 1640 or dmem medium and stored in small aliquots at -80°c. β-lactamase-vpr (blam-vpr)-carrying viruses, bearing the wild-type env, were produced by cotransfection of hek293t cells with the nl4-3/nef-ires-renilla provirus expression vector, pblam-vpr and padvantage at a ratio of 4:1:0.5, as previously described (cavrois et al., 2002) . viral particles were titrated using an hiv-1 p24 gag alpha-lisa kit and an envision plate reader (perkin elmer) and/or by determining their infection titers on target cells. wild-type and/or vsv-g pseudotyped-hiv-1, target cells were plated at 2.5 x 10 4 cells per well in 96-well plates or at 2 x 10 5 cells per well in 12-well plates and infected for 24-48 h before lysis and renilla (and firefly) luciferase activity measure (dual-luciferase® reporter assay system promega) or fixation with 2% paraformaldehyde (pfa)-pbs, permeabilization (perm/wash buffer, bdbiosciences) and intracellular staining with the anti-p24 gag kc57-fitc antibody (beckman coulter), as described previously (goujon and malim, 2010) . for tzm-bl assays, the bgalactosidase activity was measured using the galacto-star™ system (thermofisher scientific). (corman et al., 2020) . blam-vpr assay for hiv-1 entry. these assays were performed as described previously (goujon and malim, 2010) . briefly, 2 pnl4-3 or ptopo-2ltr (generated by ptopo cloning of a 2-ltr circle junction amplified from nl4-3 infected cells, using ohc64 and u3-reverse primers into pcr™2.1-topo™) were diluted in 20 ng/ml of salmon sperm dna to create dilution standards used to quantify relative cdna copy numbers and confirm the linearity of all assays. proximity ligation assay. the proximity ligation assays were performed using the duolink® in situ detection reagents (sigma-aldrich, duo92014). for this, mdms were plated in 24-well plates with coverslips pre-treated with poly-l-lysin (sigma-aldrich) and infected with 1 µg p24 gag of hiv-1 nl4-3 (ba-l env) or mock-infected. 24 h later, the cells were fixed with 4% paraformaldehyde in pbs1x for 10 min, washed in pbs1x and permeabilized with 0.2% triton x-100 for 10 min. after a couple of washes in pbs1x, either ngb buffer (50 mm nh4cl, 2% goat serum and 2% bovine serum albumin in pbs) or duolink® blocking solution was added for 1h. cells were incubated with ag3.0 mouse anti-capsid antibody obtained from the national institutes of health (nih) aids reagent program (#4121) and anti-ddx42 rabbit antibody (hpa023571, sigma-aldrich) diluted in ngb buffer or in duolink® blocking solution for 1h. after 2 washes in pbs1x, the cells were incubated with the duolink® in situ pla® probe anti-rabbit minus (duo92006) and duolink® in situ pla® probe anti-mouse plus (duo92001) for 1h at 37°c. after 2 washes in pbs1x, the ligation mix was added for 30 min at 37°c. after 2 washes in pbs1x, the cells were incubated with the amplification mix for 100 min at 37°c. finally, the cells were washed twice with pbs1x and stained with hoechst at 1 µg/ml for 5 min, washed again and the coverslips mounted on slides in prolong mounting media (thermofisher scientific). zstack images were acquired using an lsm 880 confocal microscope (zeiss) using a 63x lens. pla punctae quantification was performed using the fiji software (schindelin et al., 2012) . briefly, maximum z-projections were performed on each z-stack and the number of nuclei per field were quantified. then, by using a median filter and thresholding, pla punctae were isolated and quantified automatically using the analyse particles function. to obtain a mean number of dots per cell, the number of pla dots per field were averaged by the number of nuclei. for representative images, single cells were imaged using a lsm880 confocal microscope coupled with an airyscan module. processing of the raw airyscan images was performed on the zen black software. immunoblot analysis. cell pellets were lysed in sample buffer (200 mm tris-hcl, ph 6.8, 5.2% sds, 20% glycerol, 0.1% bromophenol blue, 5% β-mercaptoethanol), resolved by sds-page and analysed by immunoblotting using primary antibodies specific for human ddx42 (hpa023571, sigma-aldrich) and actin (mouse monoclonal a1978, sigma-aldrich), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence (bio-rad). images were acquired on a chemidoc™ gel imaging system (bio-rad). we have described previously iav nanoluciferase reporter virus generation (doyle et al., 2018) . stocks were titrated by plaque assays on mdck cells. iav challenges were performed in serumzikv production and infection. the nanoluciferase expressing zikv construct has been described (mutso et al., 2017) . the corresponding linearized plasmid was transcribed in vitro using the sp6 mmessage mmachine™ (thermofischer scientific) and hek293t cells were transfected with the transcribed rna. after 7 days, supernatants were harvested, filtered and stock titers were determined by plaque assays on vero cells. for infections, 2.5 x 10 4 cells per well in 96-well plates were infected, at the indicated mois. 24h after infection, cells were lysed and nanoluciferase activity was measured using the kit nano glo luciferase assay (promega). chikv production and infection. the gaussi luciferase coding chikv construct has been described (pohjala et al., 2011) . the linearized plasmid coding chikv genome was transcribed with the t7 mmessage mmachine kit (thermofischer scientific) and 5 x 10 5 hek293t were transfected with 1-4 µg of transcribed rna, using lipofectamine 2000 (thermofischer scientific). after 24h, supernatants were harvested, filtered and viruses were then amplified on baby hamster kidney (bhk21) cells. stock titers were determined by plaque assays on vero cells. for infections, 2.5 x 10 4 cells per well in 96-well plates were infected at the indicated mois. 24h after infection, cells were lysed and gaussia luciferase activity was measured using the pierce™ gaussia luciferase flash assay kit (thermofischer scientific). the sars-cov-2 betacov/france/idf0372/2020 isolate was supplied by pr. sylvie van der werf and the national reference centre for respiratory viruses hosted by institut pasteur (paris, france). the virus was amplified in vero e6 cells (moi 0,005) in serum-free media supplemented with 0,1 µg/ml l-1-p-tosylamino-2-phenylethyl chloromethylketone (tpck)-treated trypsin (sigma-aldrich). the supernatant was harvested at 72 h post infection when cytopathic effects were observed (with around 50% cell death), cell debris were removed by centrifugation, and aliquots stored at -80c. viral supernatants were titrated by plaque assays in vero e6 cells. typical titers were 5.10 6 plaque forming units (pfu)/ml. infections of a549-ace2 cells were performed at the indicated multiplicity of infection (moi; as calculated from titers obtained in vero e6 cells) in serum-free dmem and 5% serum-containing dmem, respectively. the viral input was left for the duration of the experiment and cells lysed at 48 h post-infection for rt-qpcr analysis. the datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request. requests for material should be addressed to caroline goujon or olivier moncorgé at the corresponding address above, or to addgene for the plasmids with an addgene number. b.b. and cg designed the study, analysed the data and wrote the manuscript. b.b. and c.g. and mageck analyses, respectively. all authors have read and approved the manuscript. the authors have no conflicts of interest to declare in relation to this manuscript. supplementary production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone alpha interferoninduced antiretroviral activities: restriction of viral nucleic acid synthesis and progeny virion production in human immunodeficiency virus type 1-infected monocytes in vivo gene transfer to the mouse eye using an hiv-based lentiviral vector; efficient long-term transduction of corneal endothelium and retinal pigment epithelium inhibition of human immunodeficiency virus (hiv) replication by hiv-trans-activated alpha 2-interferon somatic expression of line-1 elements in human tissues developmental and cell type specificity of line-1 expression in mouse testis: implications for transposition complex interplay between hiv-1 capsid and mx2-independent ifnα-induced antiviral factors hiv-1 uncoats in the nucleus near sites of integration a sensitive and specific enzyme-based assay detecting hiv-1 virion fusion in primary t lymphocytes specific inhibition of viral protein synthesis in hiv-infected cells in response to interferon treatment detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr nuclear pore blockade reveals that hiv-1 completes reverse transcription and uncoating in the nucleus am i ready for crispr? a user's guide to genetic screens hiv-1 and interferons: who's interfering with whom? the interferon-inducible isoform of ncoa7 inhibits endosome-mediated viral entry cell type-specific expression of line open reading frames 1 and 2 in fetal and adult human tissues rna g-quadruplexes in biology: principles and molecular mechanisms further investigation of simian immunodeficiency virus vif function in human cells novel host restriction factors implicated in hiv-1 replication multiple fates of l1 retrotransposition intermediates in cultured human cells mov10 rna helicase is a potent inhibitor of retrotransposition in cells characterization of the alpha interferon-induced postentry block to hiv-1 infection in primary human macrophages and t cells characterization of simian immunodeficiency virus sivsm/human immunodeficiency virus type 2 vpx function in human myeloid cells human mx2 is an interferon-induced post-entry inhibitor of hiv-1 infection evidence for ifnα-induced, samhd1-independent inhibitors of early hiv-1 infection comprehensive mutational analysis of yeast dexd/h box rna helicases required for small ribosomal subunit synthesis hiv-1 blocks the signaling adaptor mavs to evade antiviral host defense after sensing of abortive hiv-1 rna by the host helicase ddx3 mx gtpases: dynamin-like antiviral machines of innate immunity recombinant human interferon alfa-a suppresses htlv-iii replication in vitro immunoproteasome activation enables human trim5α restriction of hiv-1 mx2 is an interferon-induced inhibitor of hiv-1 infection ultrafast and memory-efficient alignment of short dna sequences to the human genome the sequence alignment/map format and samtools mageck enables robust identification of essential genes from genome-scale crispr/cas9 knockout screens selective silencing of euchromatic l1s revealed by genome-wide screens for l1 regulators the highly polymorphic cyclophilin a-binding loop in hiv-1 capsid modulates viral resistance to mxb cutadapt removes adapter sequences from high-throughput sequencing reads investigation of influenza virus polymerase activity in pig cells high frequency retrotransposition in cultured mammalian cells somatic mosaicism in neuronal precursor cells mediated by l1 retrotransposition reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a brazilian zika virus isolate in vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector generation of transmitted/founder hiv-1 infectious molecular clones and characterization of their replication capacity in cd4 t lymphocytes and monocyte-derived macrophages a virus-packageable crispr screen identifies host factors mediating interferon inhibition of hiv comparison of gene transfer efficiencies and gene expression levels achieved with equine infectious anemia virus-and human immunodeficiency virus type 1-derived lentivirus vectors determination of l1 retrotransposition kinetics in cultured cells quantitative microscopy of functional hiv post-entry complexes reveals association of replication with the viral capsid inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays many line1 elements contribute to the transcriptome of human somatic cells a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon characterization of the atpase and unwinding activities of the yeast deadbox protein has1p and the analysis of the roles of the conserved motifs g-quadruplexes and g-quadruplex ligands: targets and tools in antiviral therapy both virus and host components are important for the manifestation of a nef-phenotype in hiv-1 and hiv-2 restriction of feline immunodeficiency virus by ref1, lv1, and primate trim5alpha proteins improved vectors and genome-wide libraries for crispr screening hiv-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency fiji: an open-source platform for biologicalimage analysis genome-scale crispr-cas9 knockout screening in human cells high-throughput functional genomics using crispr-cas9 complementation of vif-defective human immunodeficiency virus type 1 by primate, but not nonprimate, lentivirus vif genes controls use of the hiv a4/5 splice acceptor cluster and is essential for efficient replication of hiv ddx5 potentiates hiv-1 transcription as a co-factor of tat the dead-box helicase ddx3 substitutes for the cap-binding protein eif4e to promote compartmentalized translation initiation of the hiv-1 genomic rna tightly regulated, developmentally specific expression of the first open reading frame from line-1 during mouse embryogenesis ddx42p--a human dead box protein with rna chaperone activities the dead box protein ddx42p modulates the function of aspp2, a stimulator of apoptosis genome-wide crispr screens reveal host factors critical for sars-cov-2 infection characterization of novel sf3b and 17s u2 snrnp proteins, including a human prp5p homologue and an sf3b dead-box protein identification of rna helicases in human immunodeficiency virus 1 (hiv-1) replication -a targeted small interfering rna library screen using pseudotyped and wt hiv-1 fastq screen: a tool for multi-genome mapping and quality control role of mxb in alpha interferon-mediated inhibition of hiv requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function genetic interactions of g-quadruplexes in humans indirect immunofluorescence analysis of endogenous ddx42 in primary mdms. mdms were fixed, endogenous ddx42 and the nuclei were visualized using ddx42-specific antibodies and hoechst staining, respectively, and confocal microscopy we wish to thank tom doyle and chad swanson for their useful comments on the manuscript, key: cord-268776-yfq9oky5 authors: mattson, mark p. title: infectious agents and age-related neurodegenerative disorders date: 2003-11-05 journal: ageing res rev doi: 10.1016/j.arr.2003.08.005 sha: doc_id: 268776 cord_uid: yfq9oky5 as with other organ systems, the vulnerability of the nervous system to infectious agents increases with aging. several different infectious agents can cause neurodegenerative conditions, with prominent examples being human immunodeficiency virus (hiv-1) dementia and prion disorders. such infections of the central nervous system (cns) typically have a relatively long incubation period and a chronic progressive course, and are therefore increasing in frequency as more people live longer. infectious agents may enter the central nervous system in infected migratory macrophages, by transcytosis across blood–brain barrier cells or by intraneuronal transfer from peripheral nerves. synapses and lipid rafts are important sites at which infectious agents may enter neurons and/or exert their cytotoxic effects. recent findings suggest the possibility that infectious agents may increase the risk of common age-related neurodegenerative disorders such as alzheimer’s disease (ad) and parkinson’s disease (pd), amyotrophic lateral sclerosis (als) and stroke. while scenarios can be envisioned whereby viruses such as chlamydia pneumoniae, herpes simplex and influenza promote damage to neurons during aging, there is no conclusive evidence for a major role of these pathogens in neurodegenerative disorders. in the case of stroke, blood vessels may be adversely affected by bacteria or viruses resulting in atherosclerosis. other articles in this special issue of aging research reviews describe the alterations in immune function that occur during normal aging that may increase the vulnerability of the elderly to many different types of infectious agents. bacteria, viruses and unconventional pathogens such as prion proteins can cause inflammatory processes and neuronal degeneration in the central nervous system (cns). the cns may be particularly vulnerable to infectious agents during aging ( fig. 1) because: (1) the bloodbrain barrier and cellular immune mechanisms are compromised during aging such that infectious agents can "hide" in the cns without being detected by the immune system; (2) some infectious agents (e.g. herpes virus) can be transferred transcellularly from infected peripheral nerves into the cns; (3) age-related increases in oxidative stress and impaired energy production can render neurons vulnerable to the toxicity of viral or prion proteins; (4) signaling pathways that promote the survival and plasticity of neurons, such as those activated by neurotrophic factors, may be impaired during aging. fig. 1 . mechanisms whereby viruses infect the cns and cause neuronal dysfunction and death. two routes of entry of viruses into the cns are by infection of cells that cross the blood-brain barrier (e.g. cells of the monocyte-macrophage/microglia lineage) and intra-and transneuronal transfer from peripheral neurons. microglia and neural stem cells appear to be cell types in which viruses may replicate at a high rate; viruses may also infect neurons and astrocytes, but may not replicate in such postmitotic cells. viruses can produce toxic viral proteins (tvps) with the hiv-1 proteins gp120 and tat being exemplary. due in part to the low level of immune surveillance the cns is particularly vulnerable to chronic infection and long-term damage as the result of systemic infection with many relatively common pathogens. because of the decline in immune function that occurs during aging, and because of an age-related increase in vulnerability of neurons to many different insults, the brain and spinal cord may be hot spots of smoldering infections in the elderly. for example, it is well-established that the elderly are vulnerable to central nervous system damage caused by west nile virus (nedry and mahon, 2003) , lyme disease (hayes, 2002) and tuberculosis (hosoglu et al., 2002) . acquired immune deficiency syndrome (aids) is caused by the human immunodeficiency virus (hiv-1) virus. hiv-1 infects lymphocytes and impairs their immune function, thereby increasing the risk of several different opportunistic infections and certain types of cancers. although effective treatments for aids, such as protease inhibitor cocktails have been developed, the resulting increase in survival of infected patients has resulted in a dramatic increase in the number of individuals in which serious neurological consequences of hiv-1 infection arise (gartner, 2000; mcarthur et al., 2003) . dementia is now common in treated aids patients and results from degeneration of synapses and neurons in brain regions such as the hippocampus and related limbic and cortical structures. hiv encephalitis also takes a toll on neurons in the basal ganglia-dysfunction and degeneration of neurons in the basal ganglia result in motor dysfunction and may also contribute to cognitive impairment, particularly in older patients infected with hiv (berger and arendt, 2000; nath et al., 2000) . as is the case with alzheimer's disease (ad), dementia in hiv-infected patients is more severe in those with an e4 allele of apolipoprotein e (corder et al., 1998) . the mechanism by which hiv-1 causes dysfunction and degeneration of neurons in the brain is not fully understood. the cells in which hiv-1 replicates in the cns may include microglia/brain macrophages and neural stem cells (haughey and mattson, unpublished data) . hiv-1 does not infect neurons and it is therefore believed that neurons are damaged by hiv-1 proteins released from infected cells. two such neurotoxic hiv-1 proteins have been identified, namely, the coat protein gp120 and the transcriptional regulator tat, both of which have been shown to be present in soluble forms in the brains of hiv-1 dementia patients. both gp120 and tat can kill cultured neurons and can increase the vulnerability of neurons to excitotoxicity and apoptosis (haughey and mattson, 2002) . patients with hiv dementia exhibit evidence of oxidative damage in their brains including increased amounts of peroxynitrite, 4-hydroxynonenal and protein carbonyls . gp120 and tat have been shown to increase oxidative stress, and antioxidants can protect cells from the damaging effects of these proteins, suggesting an important role for oxidative damage in the pathogenesis of hiv dementia (kruman et al., 1998) . inflammatory cytokines that are known to be increased in the brains of hiv dementia patients (tnf␣, il1, ifn␥ and fas/fasl) can induce ceramide production in non-neural cells by increasing the g rtk 7tmr gpi virus ca2+ gp120 tat cholesterol sphingomyelin ceramide dendrite fig. 2 . roles of membrane lipid rafts in infections of the central nervous system. lipid rafts are microdomains of the plasma membrane in which the outer leaflet of the phospholipid bilayer contains relatively high levels of cholesterol and sphingomyelin. a variety of receptors and associated signal transduction proteins (receptor tyrosine kinases, rtk; seven transmembrane receptors, 7tmr coupled to gtp-binding proteins), and ion channels are concentrated in lipid rafts. viruses may gain entry to cells by binding to proteins (e.g. gpi-anchored receptors) or lipids in lipid rafts. lipid rafts may also be regions of membranes that are damaged by cytotoxic viral proteins such as the hiv-1 proteins gp120 and tat. activity of sphingomylinases (shi et al., 1998; wiegmann et al., 1994) . prominent increases in the pro-inflammatory cytokines tnf␣, il1, il2, il6 and fas/fasl have been reported in brain and csf of hiv dementia patients (wesselingh et al., 1993) . antiretroviral treatment can dramatically slow cognitive decline and restore the cytokine balance, suggesting that inflammatory products play important roles in hiv associated cns dysfunction . emerging findings suggest that membrane microdomains called lipid rafts play important roles in the pathogenesis of hiv dementia. lipid rafts are regions of the plasma membrane that have high levels of cholesterol and sphingomyelin, and receptors for many different cytokines and growth factors are concentrated in these membrane microdomains (fig. 2) . lipid rafts are believed to be portals through which many different types of viruses, including hiv-1 enter cells (campbell et al., 2001) . in addition, lipid rafts may be regions of the cell at which gp120 and tat exert their neurotoxic actions. activation of cytokine receptors and oxidative stress can induce the production of ceramide from membrane sphingomyelin and ceramide, in turn can trigger a form of programmed cell death called apoptosis. we discovered that levels of ceramide and sphingomyelin are significantly increased in brain tissues and cerebrospinal fluid of patients with hiv dementia . when cultured neurons were exposed to gp120 and tat, levels of ceramide increased greatly. the ceramide precursor palmitoyl-coa sensitized neurons to tat and gp120 toxicities, and an inhibitor of ceramide production protected the neurons, demonstrating a critical role for ceramide in the neurotoxic actions of hiv-1. a better understanding of the roles of lipid rafts in the pathogenesis of hiv-1 dementia may lead to novel approaches for preventing and treating this disorder. there has been much interest in prion disorders because of their transmissibility among humans and the potential for their transmission from animals, and animal products such as beef, to humans. prion disorders affect primarily the cns. examples include scrapie in sheep, bovine spongiform encephalopathy in cattle, and kuru, creutzfeldt-jakob disease, gerstmann-straussler-scheinker disease and fatal familial insomnia in humans (mastrianni and roos, 2000; giese and kretzschmar, 2001; mckintosh et al., 2003) . these disorders are characterized by the intracellular accumulation of insoluble aggregates of prion protein, abnormal (scrapie) forms (prpsc) of a normal protein called the cellular prion protein (prpc). because prion disorders can be transmitted from one individual to another it was initially assumed that these diseases were caused by a virus. however, intensive investigations failed to identify a virus and the emerging evidence led to the remarkable conclusion that the disease is transmitted by an abnormal form of the prion protein itself. prpsc adopts a structure that fosters an interaction with prpc that results in prpc being converted to the pathogenic prpsc form (fig. 3) . in some cases the abnormal prion protein may result from a mutation in the prpc gene, while in other cases the abnormal conformation of prpsc may result from posttranslational modifications (wiessmann, 2002) . prion disorders may have a long incubation time and acquired cases of prion disease are more common in older individuals. prion proteins may damage and kill neurons by inducing oxidative stress, disrupting calcium homeostasis and triggering apoptosis (haughey and mattson, 2002) . inflammatory processes involving microglial activation and production of pro-inflammatory cytokines may also contribute to the pathogenesis of prion disorders (eikelenboom et al., 2002) . changes that occur in brain cells during normal aging, including increased oxidative stress and metabolic impairment, may render neurons vulnerable to the toxicity of prpsc. it is known that inflammation-like processes occur in the brains of patients with alzheimer's, parkinson's (pd) and huntington's diseases, but whether such inflammatory processes are pathogenic or simply represent responses to neuronal degeneration is unclear. it has been suggested that certain childhood infections may increase the risk of age-related neurodegenerative disorders, although the evidence is as yet meager (martyn, 1997) . there are certainly a variety of viruses that infect cells in the nervous system and some of these viruses can be retained in neurons for long periods and even for the lifetime of an individual examples include herpes simplex-1, sindbis virus, measles and rabies (kristensson et al., 1996; griffin, 1998; schneider-schaulies et al., 2001; mettenleiter, 2003 normal forms of a␤ (soluble a␤) and prion proteins do not self aggregate, whereas in prion disorders and alzheimer's disease the proteins generate reactive oxygen species (ros) and acquire abnormal conformations. genetic factors (e.g. mutations in the genes encoding the prion protein or a␤) and environmental factors (e.g. high caloric intake or folate deficiency) and the aging process may cause or promote formation of pathogenic forms of a␤ and prpsc. during the process of peptide oligomer and fibril formation, a␤ and prpsc induce membrane-associated oxidative stress and disrupt membrane ion transporter and channel functions resulting in synaptic and mitochondrial dysfunction and apoptosis. it has recently become evident that autoantibodies against abnormal conformations of a␤ and prpsc can be generated by the immune systems in patients and in subjects immunized with a␤ or prion peptides. the autoantibodies may promote clearance of the endogenous pathogenic proteins from the brain and/or they may enhance the neurotoxicity of a␤ or prpsc. transient infections might trigger neurodegenerative cascades when the infection occurs in aged individuals who may be at risk of the disease. ad is characterized by the accumulation of amyloid ␤-peptide (a␤), synaptic dysfunction and degeneration, and neuronal death in brain regions involved in learning and memory processes (mattson, 1997a (mattson, ,b, 2002 . mutations in three different genes (the amyloid precursor protein, presenilin-1 and presenlin-2), and studies of cultured cells and transgenic mice expressing ad-linked app and presenilin mutations have provided evidence that a critical event in ad is altered proteolytic processing of app resulting in increased production of a␤. a␤ may damage neurons and render them vulnerable to excitotoxicity and apoptosis by inducing membrane lipid peroxidation and impairing the function of ion-motive atpases, glucose and gluatmate transporters, and ion channels (mattson et al., 1992; mark et al., 1997; guo et al., 1998 guo et al., , 1999a (fig. 3) . in addition to the degeneration of synapses and neurons that occurs in ad, damage to the oligodendrocytes that myelinate axons in the brain have been documented in mouse models of ad (pak et al., 2003) . environmental factors that may increase the risk of ad include a high caloric intake and dietary folate deficiency (zhu et al., 1999; kruman et al., 2002a,b) . altered immune responses have been documented in studies of ad patients and in animal models and may result, in part, from perturbed calcium signaling in lymphocytes and microglia lee et al., 2002) . a␤ may play an important role in triggering activation of microglia in ad by a mechanism involving the activation of cd40 (tan et al., 1999) , and a␤ can also perturb astrocyte function in ways that may impair their ability to communicate with neurons . peripheral or central stimulation with lipopolysaccharide induces a transient increase in expression of pro-inflammatory cytokines such as tnf, interleukin 1 beta and interleukin 6, and these cytokine changes are associated with microglial activation and altered processing of app (brugg et al., 1995; lee et al., 2002) , suggesting roles of immune responses in neurodegenerative cascades in ad. while it has been proposed that the immune system can be stimulated by immunization with a␤, the specific antibodies produced may determine whether such immunization is beneficial or promotes neuronal degeneration instead . systemic infection can result in inflammatory processes in the brain including microglial activation. infections in the elderly can result in cognitive impairment that outlasts the infection; in patients with ad who encounter a systemic infection, cognitive function can be impaired for several months after the resolution of a systemic infection and the cognitive impairment is preceded by raised serum levels of interleukin 1 beta (holmes et al., 2003) . soininen et al. (1993) analyzed circulating immune complexes in the blood of patients with ad and individuals with age-associated memory impairment. ad patients with severe dementia had significantly elevated levels of circulating immune complexes compared with ad patients with mild or moderate disease and to control subjects and individuals with age-associated memory impairment. it is unclear whether the increased immune complexes in severe ad patients contribute to the pathogenic process. nucleic acids prepared from postmortem brain tissue samples from ad patients and control subjects were screened by polymerase chain reaction assay for dna sequences from chlamydia pneumoniae; brain areas with typical ad-related neuropathology were positive for the organism in 17/19 ad patients, whereas 18/19 control patients were negative (balin et al., 1998) . the latter study also showed that cultures from affected ad brain tissues were strongly positive for c. pneumoniae, while identically performed analyses of non-ad brain tissues were negative. in a subsequent study by this the same investigators it was reported the c. pneumoniae is present in glial cells in areas of neuropathology in the brains of ad patients (balin and appelt, 2001) . the cells in the brains of ad patients that might be infected by c. pneumoniae include vascular endothelial cells and monocytes; these cells may play roles in the inflammatory processes and neuronal degeneration in ad (macintyre et al., 2003) . however, a role for c. pneumoniae in ad pathogenesis remains to be established and negative data have been obtained. for example, in a study of centenarians there was no association between c. pneumoniae infection and dementia (bruunsgaard et al., 2002) . dobson and itzhaki reported that herpes simplex type 1 virus (hsv1) is present in the brain of many elderly people, and that it may be a risk factor for ad, particularly in individuals with the apolipoprotein e4 allele (dobson and itzhaki, 1999) . however, in other studies there was no evidence of herpes virus rna in the hippocampus of demented individuals with extensive neuropathological changes of ad (deatly et al., 1990) . finally, in an interesting study it was shown that caregivers of ad patients have significantly poorer immune responses to influenza virus vaccine when compared to age-matched control subjects (kiecolt-glaser et al., 1996) , which may result from the chronic stress associated with caregiving leading to increased vulnerability to infection and perhaps to increased vulnerability to age-related neurodegenerative disorders as well. pd is characterized by degeneration of dopamine-producing neuron in the substantia nigra resulting in progressive impairment of the patient's ability to control their body movements. most cases of pd are sporadic and the causes are unknown, although rare cases are caused by inherited mutations in ␣-synuclein, parkin or dj-1 (moore et al., 2003) . it is believed that mitochondrial dysfunction and associated atp depletion and oxidative stress are pivotal and relatively early events in the neurodegenerative process in pd (duan et al., 1999a) (fig. 4) . dopaminergic neurons may die in pd by apoptosis mediated by par-4 (duan et al., 1999b) and the tumor suppressor protein p53 (duan et al., 2002a; gilman et al., 2003) . epidemiological and experimental findings suggest a potential role for environmental toxins such as the pesticide rotenone in the pathogenesis of pd, although the data are not yet conclusive (di monte et al., 2002) . dietary factors such as high calorie intake and folate deficiency (duan and mattson, 1999; duan et al., 2002b) and exposure to pesticides and excitotoxins (lockwood, 2000; ludolph et al., 2000) may also increase the risk of pd. some epidemiological data suggest that influenza a viral infections may increase the risk of pd and may be responsible for the formation of lewy bodies and the later death of nigral neurons (takahashi and yamada, 1999) . based upon data suggesting that peptic ulcer is more frequent in pd patients and that helicobacter pylori can cause ulcers, a study was performed to determine whether h. pylori seropositivity was associated with pd (charlett et al., 1999) . it was shown that siblings of pd patients had an increased probability of h. pylori seropositivity compared to control subjects. in one study there was an increased percentage of teachers and healthcare workers with pd in a large tertiary care movement disorders clinic suggesting the possibility that a high level of exposure to viral or other respiratory infections in these occupations might be a risk factor for pd (tsui et al., 1999) . the possibility that viruses can induce pd is suggested by studies showing that certain viruses can induce pd-like pathology in rodents. for example, rats infected with japanese encephalitis virus exhibited neuronal loss with gliosis which was confined mainly to the zona compacta of the substantia nigra (ogata et al., 1997) . amyotrophic lateral sclerosis (als) is characterized by degeneration of lower and upper motor neurons resulting in progressive paralysis and death. the cause(s) of most fig. 4 . cellular and molecular mechanisms responsible for motor dysfunction in parkinson's disease (pd) and amyotrophic lateral sclerosis (als). some rare cases of pd and als are caused by mutations. three genes have been identified in which mutations cause early onset inherited pd (parkin, ␣-synuclein and dj-1) and mutations in two genes (cu/zn-sod and alsin) are known to cause als. insight into the molecular and cellular abnormalities that lead to the dysfunction and death of midbrain dopaminergic neurons in pd and of motor neurons in als has come from studies of cultured cells and transgenic mice expressing the disease-causing human genes. in the case of pd, each of the mutations has been linked to abnormal ubiquitin/proteasome-mediated proteolysis, cellular oxidative and metabolic stress and triggering of apoptosis. in the case of als, cu/zn-sod mutations cause oxidative stress and disrupt cellular calcium homeostasis. the aging process and environmental factors may perturb the same or similar regulatory systems that are adversely affected by genetic mutations. synapses are particularly vulnerable to genetic, aging and environmental factors, and are sites where excitotoxic and apoptotic cascades that cause the death of the neurons are initiated. cases of als is unknown, but a few cases result from mutations in the gene encoding cu/zn-superoxide dismutase. the pathogenic process in als involves increased oxidative stress and disruption of cellular calcium homeostasis which may trigger apoptosis (pedersen et al., 1998 (pedersen et al., , 2000 kruman et al., 1999; guo et al., 2000) (fig. 4) . recent findings suggest that abnormalities in lipid metabolism, involving increased levels of ceramides and cholesterol esters, also plays a role in the pathogenesis of als (cutler et al., 2002) . the possibility that retroviral infection can cause als has recently been suggested by studies of hiv-infected patients who developed a rapid progressive als-like disorder as the first manifestation of their hiv-infection (portegies and cohen, 2002) . the patients stabilized or improved with antiretroviral therapy. enterovirus rna has also been detected in the spinal cords of als patients, although it remains to be established if such viruses cause the disease (portegies and cohen, 2002) . in a study of gulf war veterans there was a significant association between systemic mycoplasmal infections and als (nicolson et al., 2002) , although it was not established if mycoplasma plays a role in the pathogenesis of als or if als patients are more susceptible to als. the results of another study suggested an association between the risk of als and infection with certain enteroviruses and herpes virus (cermelli et al., 2003) . ischemic stroke, a major cause of death and disability in the elderly, occurs when a blood vessel becomes occluded or ruptures. risk factors for stroke include high calorie/high fat diets, hypertension and physical inactivity (gorelick, 1995; yu and mattson, 1999; kurth et al., 2002) . neurons in the brain region normally perfused by the affected blood vessel degenerate after a stroke as the result of decreased glucose and oxygen availability. ischemic neuronal death involves metabolic compromise, dysregulation of cellular calcium homeostasis and increased oxidative stress liu et al., 2002) . activation of microglia and the production of pro-inflammatory cytokines also play an important role in ischemic neuronal death (bruce et al., 1996; yu et al., 2000) . in addition to the cascades that result in the death of neurons, several important neuroprotective signaling pathways are activated after a stroke including intercellular signaling involving neurotrophic factors and cytokines, and intracellular pathways involving calcium and transcription factors such as nf-b and creb (mattson, 1997b; mattson et al., 2000; . because atherosclerosis is a major antecedent process in stroke, there is evidence that infectious agents that may promote atherosclerosis increase the risk of stroke. there is a growing body of evidence that c. pneumoniae can promote cerebrovascular atherosclerosis and may thereby increase the risk of stroke. johnston et al. (2001) documented increased c-reactive protein levels and viable c. pneumoniae in atherosclerotic carotid arteries. however, it remains to be established whether c. pneumoniae induces/enhances atherosclerosis or if atherosclerotic plaques provide an environment in which c. pneumoniae accumulates. similarly, it was reported that c. pneumoniae is present in symptomatic atherosclerotic carotids and that this is associated with increased serum antibodies, inflammation and apoptosis of t cells (neureiter et al., 2003) . other systemic bacterial and viral infections may promote stroke by enhancing atherosclerosis and by weakening cerebral blood vessels (emsley and tyrrell, 2002) . acute infectious episodes may increase the risk of acute ischemic stroke in the elderly independently of other predisposing factors (nencini et al., 2003) . another infectious agent that may increase the risk of stroke is influenza which is suggested by studies showing that elderly individuals receiving the influenza vaccine have a significantly lower risk of stroke than do those not receiving the vaccine (meyers, 2003) . while atherosclerosis is an inflammatory process and is the major pathogenic process that predisposes to stroke, inflammatory mediators also play roles in modifying the neurodegenerative process that occurs as the result of an ischemic stroke. activated lymphocytes, macrophages and microglia accumulate in ischemic brain tissue after a stroke, and these cells may produce neurotoxic cytokines and excitotoxins. accordingly, it has been shown that anti-inflammatory drugs can reduce the extent of brain injury in animal models of stroke (antezana et al., 2003) . not only is aging a risk factor for infection with agents that may promote atherosclerosis, but older persons who suffer a stroke have a significantly worse outcome-more brain tissue is damaged and recovery of function is limited (duverger and mackenzie, 1988) . the myelinating cells of the central (oligodendrocytes) and peripheral (schwann cells) nervous systems are vulnerable to being damaged and killed by several viruses (fazakerley and walker, 2003) . progressive multifocal leukoencephalopathy is caused by the infection of oligodendrocytes by jc papovirus and is characterized by slowly progressing dementia, visual problems and ataxia. subacute sclerosing panencephalitis is caused by the measles virus and is characterized by behavioral changes mental and visual disturbances and ataxia; it is almost always fatal. the damage to oligodendrocytes may result from infection by the virus, exposure to viral proteins and/or an autoimmune response. certain coronaviruses can also cause demyelination. for example, the mouse hepatitis virus can cause demyelination in the brains of non-human primates and human coronaviruses related to mouse hepatitis virus have been detected in brain tissue samples from patients with multiple sclerosis. multiple sclerosis is the most common demyelinating disorder in humans (cluskey and ramsden, 2001) . although not yet conclusive, accumulating data suggest that infectious agent could be involved in the pathogenesis of multiple sclerosis. associations between infections with measles virus, parainfluenza virus, canine distemper, human herpes virus-6 and c. pneumoniae have been reported (for review see steiner et al., 2001) . it is clear that there is an autoimmune component to multiple sclerosis and it is possible that infectious agents may initiate damage to oligodendrocytes or otherwise elicit targeting of oligodendrocytes by the immune system. because it is well-established that many different viruses can cause demyelination, it will be important to establish which specific viruses play a role in the pathogenesis of multiple sclerosis. as a result of the large and repetitive ion fluxes associated with membrane depolarization, and neurotransmitter release and receptor activation, synapses are sites at which neurons are subjected to very high levels of oxidative and metabolic stress. accordingly, synapses have been shown to vulnerable to dysfunction and degeneration in each of the infectious and age-related neurodegenerative disorders described above. synapses are where excitotoxicity is initiated, a process in which glutamate receptors are overactivated under conditions of oxidative and metabolic stress, resulting in excessive calcium influx (gilman and mattson, 2002; mattson, 2003) . apoptosis may also be triggered at synapses and, indeed, biochemical cascades can be locally engaged in synapses in experimental models relevant to the pathogenesis of neurodegenerative disorders (mattson et al., 1998a,b; duan et al., 1999b; glazner et al., 2000; gilman et al., 2003) . thus, synapses are vulnerable to the kinds of factors that are promoted by infectious agents. for example, the hiv-1 proteins gp120 and tat induce oxidative stress and promote calcium influx through glutamate receptor channels and voltage-dependent channels which are concentrated in synapses (haughey and mattson, 2002) . in addition to their vulnerability to aging and infectious agents, synapses may play important roles in the propagation of infectious agents in the cns. synapses may portals of entry of viruses because cell surface receptors to which the viruses bind are located in lipid rafts which are concentrated in synaptic membranes. for example, putative receptors for botulinum and tetanus toxins are located in synapses (herreros et al., 1997 (herreros et al., , 2000 . some viruses, such as herpes simplex, can be transferred between neurons; this transcellular transfer occurs at synapses and likely involves specific exocytotic and endocytotic mechanisms (labetoulle et al., 2000) . high-dose ibuprofen for reduction of striatal infarcts during middle cerebral artery occlusion in rats role of infection in alzheimer's disease identification and localization of chlamydia pneumoniae in the alzheimer's brain hiv dementia: the role of the basal ganglia and dopaminergic systems altered neuronal and microglial responses to brain injury in mice lacking tnf receptors inflammatory processes induce beta-amyloid precursor protein changes in mouse brain proinflammatory cytokines, antibodies to chlamydia pneumoniae and age-associated diseases in danish centenarians: is there a link? scand lipid rafts and hiv-1: from viral entry to assembly of progeny virions risk of sporadic amyotrophic lateral sclerosis associated with seropositivity for herpesviruses and echovirus-7 parkinsonism: siblings share helicobacter pylori seropositivity and facets of syndrome mechanisms of neurodegeneration in amyotrophic lateral sclerosis hiv-infected subjects with the e4 allele for apoe have excess dementia and peripheral neuropathy evidence that accumulation of ceramides and cholesterol esters mediates oxidative stress-induced death of motor neurons in als human herpes virus infections and alzheimer's disease environmental factors in parkinson's disease herpes simplex virus type 1 and alzheimer's disease dietary restriction and 2-deoxyglucose administration improve behavioral outcome and reduce degeneration of dopaminergic neurons in models of parkinson's disease participation of prostate apoptosis response-4 in degeneration of dopaminergic neurons in models of parkinson's disease prostate apoptosis response-4 production in synaptic compartments following apoptotic and excitotoxic insults: evidence for a pivotal role in mitochondrial dysfunction and neuronal degeneration p53 inhibitors preserve dopamine neurons and motor function in experimental parkinsonism dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of parkinson's disease the quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration, and age neuroinflammation in alzheimer's disease and prion disease inflammation and infection in clinical stroke virus demyelination hiv infection and dementia prion-induced neuronal damage-the mechanisms of neuronal destruction in the subacute spongiform encephalopathies do apoptotic mechanisms regulate synaptic plasticity and growth-cone motility p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to dna damage, and oxidative and excitotoxic insults caspase-mediated degradation of ampa receptor subunits: a mechanism for preventing excitotoxic necrosis and ensuring apoptosis stroke prevention a review of alphavirus replication in neurons par-4 is a mediator of neuronal degeneration associated with the pathogenesis of alzheimer disease neurotrophic factors [activity-dependent neurotrophic factor (adnf) and basic fibroblast growth factor (bfgf)] interrupt excitotoxic neurodegenerative cascades promoted by a presenilin-1 mutation increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock-in mice als-linked cu/zn-sod mutation impairs cerebral synaptic glucose and glutamate transport and exacerbates ischemic brain injury calcium dysregulation and neuronal apoptosis by the hiv-1 proteins tat and gp120 alzheimer's amyloid beta-peptide enhances atp/gap junction-mediated calcium-wave propagation in astrocytes involvement of perturbed sphingolipid metabolism and ceramide production in the pathogenesis of hiv dementia lyme disease localization of putative receptors for tetanus toxin and botulinum neurotoxin type a in rat central nervous system c-terminal half of tetanus toxin fragment c is sufficient for neuronal binding and interaction with a putative protein receptor systemic infection predictors of outcome in patients with tuberculous meningitis c-reactive protein levels and viable chlamydia pneumoniae in carotid artery atherosclerosis mitochondrial mnsod prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation and mitochondrial dysfunction chronic stress alters the immune response to influenza virus vaccine in older adults rabies: interactions between neurons and viruses. a review of the history of negri inclusion bodies als-linked cu/zn-sod mutation increases vulnerability of motor neurons to excitotoxicity by a mechanism involving increased oxidative stress and perturbed calcium homeostasis hiv-1 protein tat induces apoptosis of hippocampal neurons by a mechanism involving caspase activation, calcium overload, and oxidative stress folic acid deficiency and homocysteine impair dna repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of alzheimer's disease body mass index and the risk of stroke in men neuronal pathways for the propagation of herpes simplex virus type 1 from one retina to the other in a murine model adverse effect of a presenilin-1 mutation in microglia results in enhanced nitric oxide and inflammatory cytokine responses to immune challenge in the brain activation of mitochondrial atp-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of bax translocation and cytochrome c release pesticides and parkinsonism: is there an etiological link? the role of excitotoxicity in als-what is the evidence? chlamydia pneumoniae infection promotes the transmigration of monocytes through human brain endothelial cells amyloid beta-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation infection in childhood and neurological diseases in adult life the prion diseases cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives neuroprotective signal transduction: relevance to stroke oxidative stress, perturbed calcium homeostasis, and immune dysfunction in alzheimer's disease excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders nf-b in neurodegenerative disorders ␤-amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity evidence for synaptic apoptosis amyloid ␤-peptide induces apoptosis-related events in synapses and dendrites apoptotic and antiapoptotic mechanisms in stroke presenilin mutations and calcium signaling defects in the nervous and immune systems human immunodeficiency virus-associated dementia: an evolving disease prion diseases pathogenesis of neurotropic herpesviruses: role of viral glycoproteins in neuroinvasion and transneuronal spread myocardial infarction, stroke, and sudden cardiac death may be prevented by influenza vaccination a role for the ubiquitin-proteasome system in parkinson's disease and other neurodegenerative brain amyloidoses neurotoxicity and dysfunction of dopaminergic systems associated with aids dementia autoantibodies to amyloid beta-peptide (abeta) are increased in alzheimer's disease patients and abeta antibodies can enhance abeta neurotoxicity: implications for disease pathogenesis and vaccine development west nile virus: an emerging virus in north america acute inflammatory events and ischemic stroke subtypes detection of chlamydia pneumoniae but not of helicobacter pylori in symptomatic atherosclerotic carotids associated with enhanced serum antibodies, inflammation and apoptosis rate high frequency of systemic mycoplasmal infections in gulf war veterans and civilians with amyotrophic lateral sclerosis (als) a rat model of parkinson's disease induced by japanese encephalitis virus presenilin-1 mutation sensitizes oligodendrocytes to glutamate and amyloid toxicities, and exacerbates white matter damage and memory impairment in mice protein modification by the lipid peroxidation product 4-hydroxynonenal in the spinal cords of amyotrophic lateral sclerosis patients the prostate apoptosis response-4 protein participates in motor neuron degeneration in amyotrophic lateral sclerosis possible etiological role retroviruses and enteroviruses in the development of amyotrophic lateral sclerosis measles virus interactions with cellular receptors: consequences for viral pathogenesis neuronal apoptosis induced by hiv-1 tat protein and tnf-alpha: potentiation of neurotoxicity mediated by oxidative stress and implications for hiv-1 dementia circulating immune complexes in sera from patients with alzheimer's disease and subjects with age-associated memory impairment infection and the etiology and pathogenesis of multiple sclerosis viral etiology for parkinson's disease-a possible role of influenza a virus infection microglial activation resulting from cd40-cd40l interaction after beta-amyloid stimulation occupational risk factors in parkinson's disease oxidative stress in hiv demented patients and protection ex vivo with novel antioxidants intracerebral cytokine messenger rna expression in acquired immunodeficiency syndrome dementia functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling molecular genetics of transmissible spongiform encephalopathies: an introduction dietary restriction and 2-deoxyglucose administration reduce focal ischemic brain damage and improve behavioral outcome: evidence for a preconditioning mechanism pivotal role for acidic sphingomyelinase in cerebral ischemia-induced ceramide and cytokine production, and neuronal apoptosis dietary restriction protects hippocampal neurons against the death-promoting action of a presenilin-1 mutation key: cord-253768-y35m3vh1 authors: springer, sandra a; barocas, joshua a; wurcel, alysse; nijhawan, ank; thakarar, kinna; lynfield, ruth; hurley, hermione; snowden, jessica; thornton, alice; del rio, carlos title: federal and state action needed to end the infectious complications of illicit drug use in the united states: idsa and hivma’s advocacy agenda date: 2020-10-01 journal: j infect dis doi: 10.1093/infdis/jiz673 sha: doc_id: 253768 cord_uid: y35m3vh1 in response to the opioid crisis, idsa and hivma established a working group to drive an evidenceand human rights-based response to illicit drug use and associated infectious diseases. infectious diseases and hiv physicians have an opportunity to intervene, addressing both conditions. idsa and hivma have developed a policy agenda highlighting evidence-based practices that need further dissemination. this paper reviews (1) programs most relevant to infectious diseases in the 2018 support act; (2) opportunities offered by the “end the hiv epidemic” initiative; and (3) policy changes necessary to affect the trajectory of the opioid epidemic and associated infections. issues addressed include leveraging harm reduction tools and improving integrated prevention and treatment services for the infectious diseases and substance use disorder care continuum. by strengthening collaborations between infectious diseases and addiction specialists, including increasing training in substance use disorder treatment among infectious diseases and addiction specialists, we can decrease morbidity and mortality associated with these overlapping epidemics. the epidemiology of the us opioid epidemic continues to evolve and presents new challenges. in recent years, the epidemic has shifted from prescription opioid pills to injection of illicitly produced opioids, including heroin and fentanyl, with concomitant increasing injection of stimulants including cocaine and methamphetamine [1] [2] [3] . as a result, the incidence of injection drug use (idu)-related infections such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), hepatitis b virus (hbv), and invasive bacterial and fungal infections, including staphylococcus aureus bacteremia, endocarditis, and skin and soft tissue infections, is rising [2, [4] [5] [6] [7] [8] [9] [10] [11] . injection of fentanyl or heroin alone and in combination with stimulants have led to new hiv outbreaks among people who use drugs throughout the country [4, [10] [11] [12] . in addition to hiv, both acute hcv and hbv infection incidence has mirrored the rise in injection opioids [5, 13] and hospitalizations for injection opioid-related endocarditis have increased more than 12-fold in recent years [6, 8] . at the state of the union address in february 2019, president trump called for a plan to end hiv as an epidemic in the united states. this plan seeks to reduce new infections by 75% in the next 5 years and by 90% in the next 10 years. even amid the opioid epidemic, such ambitious goals can be achieved if policy changes occur and adequate resources are provided. thus, more than ever, addressing the hiv epidemic as well as hcv and other idu-related infections also requires a focus on the opioid and co-occurring stimulant epidemics. doing so will improve patients' outcomes and reduce the public health risk of infectious disease transmission. nevertheless, a number of barriers to care in people who use drugs need to be addressed to end the opioid and hiv epidemics in the united states as well as reduce the other infectious disease health outcomes. to address these barriers we recommend expanding medicaid, expanding access to harm reduction services, improving treatment and surveillance to enhance the continuum of care, and treating opioid and other substance use disorders (sud), including through lowbarrier hospital and community-based treatment, as well as in the criminal justice setting. the authors of this paper are members of a working group created by the infectious diseases society of america (idsa) and the hiv medicine association (hivma) in 2017 to enhance their efforts to educate and advocate on the urgent need to better prevent and treat serious infections linked to the opioid and stimulant epidemics and underlying sud. the working group developed a policy agenda reflecting issues raised by infectious diseases and hiv physicians and health care professionals working at the intersection of infectious diseases and opioid use disorder (oud) and other sud epidemics more broadly. in this paper, we outline practice and policy suggestions that are likely to positively impact the oud, stimulant epidemics, and infectious diseases epidemics, and that have been reviewed and approved by the idsa and hivma board of directors as a call to action for infectious diseases and hiv practitioners. medications for treatment of opioid use disorder (mouds, which is now the preferred term to medication-assisted therapy) are recognized as the most effective treatments for oud [14] . there are 3 food and drug administrationapproved mouds-methadone, buprenorphine, and extended-release naltrexone (xr-ntx). methadone is a full opioid agonist and buprenorphine is a partial opioid agonist, while xr-ntx is an opioid antagonist. all are successful in treating oud and in decreasing mortality. all reduce illicit opioid use, opioid craving, overdose, and hiv and hcv transmission [14, 15] ; and buprenorphine and xr-ntx also improve hiv viral suppression in people living with hiv, the gold standard of care in treatment of hiv that is associated with reduced mortality and reduced transmission [16, 17] . of the 3 mouds, access to methadone and buprenorphine are limited by regulations. prescribing requires special training outside postgraduate programs and either a waiver from the drug enforcement agency in the case of buprenorphine or treatment in a federally certified opioid treatment program in the case of methadone. unfortunately, many clinical settings lack physicians trained in oud treatment. only about 5% of the nation's physicians have waivers to prescribe buprenorphine and most substance use treatment programs do not have opioid treatment programs, which makes methadone treatment challenging to obtain [18] . therefore, the prevailing care for these patients typically consists of withdrawal management or detoxification and referral to outpatient resources for follow-up treatment. this asks patients with severe oud to tolerate withdrawal symptoms, risking premature exit from hospital, and relapse to opioid use after failure to connect with oud treatment referrals. such inadequate care results in prolonged hospitalizations due to concern about relapse and nonadherence if patients leave the hospital, readmissions after oud relapse, and, if concomitant infection is present, lack of antibiotic adherence and reinfection. ultimately, this cycle leads to poor clinical outcomes, high health care costs, and excess deaths. infectious disease specialists are at the frontlines in many hospitals treating infectious diseases in people who use drugs and have an opportunity to screen and treat co-occurring suds. in 2018, congress passed legislation offering opportunities to heighten the response to the opioid epidemic and its infectious diseases complications. on 24 october 2018, president trump signed into law the substance use disorder prevention that promotes opioid recovery and treatment (support act). this bill includes a range of prevention, care, workforce, and public health provisions to strengthen the response to the opioid epidemic (table 1 ) [19] . the bill was passed with strong bipartisan support from congressional members recognizing that the status quo was woefully inadequate to respond to the opioid epidemic. idsa and hivma supported the support act, including provisions that improved medicaid and medicare coverage of sud treatment and services, and that increased the patient cap for which physicians could prescribe moud. priority issues for idsa and hivma were provisions authorizing funding for the centers for disease control and prevention (cdc) to eliminate opioid related infections through improved surveillance and prevention for infections linked to idu and funding for the health resources and services administration to build workforce capacity through a new substance use treatment provider loan forgiveness program, offering up to $250 000 in loan repayment over 6 years for providers working in substance use treatment facilities [20] . both programs depend on congress to appropriate funding. five million dollars was appropriated for fiscal year 2019 for the cdc eliminate opioid related infections funding provision. the fiscal year 2020 appropriations bills were signed into law on december 20, 2019 and included $10 million for the cdc's eliminate opioid related infections program and $12 million for the new substance use disorder loan forgiveness program [21] . other legislative proposals supported by idsa and hivma that have been introduced in the 116th congress include: the medicaid re-entry act that would allow medicaid coverage for inmates during the 30-day period preceding release from a public institution [22] ; the comprehensive addiction resources emergency act modeled after the highly successful ryan white hiv/aids program and that would provide funding to states to support comprehensive prevention, care, and treatment programs [23] ; and the mainstreaming addiction treatment act that would eliminate the requirement for clinicians to obtain a waiver to prescribe buprenorphine [24] . as outlined in this paper, urgent policy action is needed to reduce illness and death due to our nation's substance use epidemics. in addition to state and jurisdictional bans or restrictions on syringe services programs (ssp), funding remains a significant barrier to expanding access to ssp services [25] . increased state and federal funding are needed to expand ssp and other harm reduction services, including access to moud and infectious diseases treatment services in order to decrease hcv, hiv, idu-related infections, and vaccine-preventable diseases, and improve oud-related outcomes [26, 27] . studies have demonstrated that incorporation of ssps combined with moud is associated with a decrease in hcv and hiv acquisition risk by 76% and 34%, respectively [28] . ssps also can facilitate vaccine uptake for hepatitis a virus (hav), hbv, influenza, and invasive pneumococcal disease, which disproportionately impact people who inject drugs or experience unstable housing or homelessness [29, 30] . ssps can offer a safe space without stigma for individuals with suds to also receive counseling regarding safe sexual practice, safe injection practice, and provision of hiv preexposure prophylaxis (prep) and contraception. ssps that also provide treatment for oud can facilitate linkage to care for effective evidence-based treatments [31] . furthermore, persons with suds may be reluctant to seek nonemergent care for skin and soft tissue infections and postpone medical evaluation until the need is more urgent. providing care for skin and soft tissue infections in a supported setting may reduce progression to serious infections and reduce complications like wound botulism or partial drainage of abscesses. given the increased incidence of idu-associated infections and overdose deaths [1, 2] , there is a need to provide ongoing support for and increased access to ssps. the hiv outbreak in scott county, indiana, in addition to other emerging hbv and hcv epidemics, has highlighted the need to expand ssps, particularly in nonurban areas [32] . although federal funds to support ssps was an important step, additional federal funding and flexibility are needed to fully cover services and costs associated with these programs, including purchasing of sterile syringes and to support delivery of moud at ssps [33] . in jurisdictions where ssps are prohibited or sparse, cities and states should be incentivized to modify their laws and to encourage uptake by local jurisdictions. in some states, there is a limit on the number or location of such programs, or ssps may only be allowed during certain circumstances (ie, public health emergencies) [34] . such limitations should be eliminated given the documented need for these programs and their potential to reduce infectious diseases [34, 35] . additionally, drug paraphernalia laws, which prohibit possession of syringes, pose barriers to ssp expansion and effectiveness [36] . state and local governments should be encouraged to employ innovative programming, including mobile delivery and contracting with communitybased organizations. additionally, states should be incentivized to eliminate 1-for-1 syringe exchange (ie, exchanging 1 used syringe for 1 sterile syringe) because they create barriers to individuals who inject drugs having an adequate supply of sterile 2. incentivize states to give more authority to local governments to establish ssps and to eliminate barriers to sterile syringes, such as one-for-one needle exchange requirements. 3. allow jurisdictions that have approved overdose prevention sites or supervised injection facilities to implement and evaluate the intervention in the united states. 4. urge all states to expand medicaid. 5. fund demonstration projects and pilot studies to identify effective care models for comanagement of infectious diseases and sud. 6. increase funding for national and regional warmlines and peer-to-peer mentoring, programs for prescribers of mouds, and for cotreatment of related infections. 7. eliminate the buprenorphine waiver, remove patient caps, and offer grant funding for case management and other support services to clinics and practices that prescribe mouds. 8. increase funding and reimbursement for telehealth and other low-barrier access care delivery models. 9. support implementation of universal hcv testing. 10 . develop a national surveillance system to report and track idu-related infections to predict and respond to emerging epidemics. 11. integrate moud and counseling services during incarceration. 12. integrate screening for oud and treatment with moud into jails and prisons. 13. expand access to harm reduction during and after incarceration. 14. allow states to initiate medicaid coverage 30 days prior to release from criminal justice settings to facilitate care initiation and coordination during the transition to the community. syringes. secondary exchange, or the distribution of sterile syringes from 1 person to a social network, is often necessary due to distance and transportation barriers. in addition to ssps, other harm reduction services are needed to address the expanding epidemics. overdose prevention sites (also known as supervised injection facilities or safe injection sites) are facilities in which persons can inject drugs in a safe, clean environment under medical supervision. overdose prevention sites enable rapid, life-saving intervention in the case of drug overdose and can also provide injection equipment and referrals to care for sud and other health care services. overdose prevention sites have existed for many years in europe, australia, and canada. studies of overdose prevention sites in vancouver and sydney have found an increase in withdrawal management or detoxification service referrals and a decrease in drug overdose rates, syringe sharing, public injections, and publicly discarded syringes [37] [38] [39] . several us municipalities have advocated for overdose prevention sites, but political opposition has so far impeded implementation. a recent modeling study in seattle estimated that an overdose prevention site would yield cost savings through prevention of overdose deaths, enrollment in mouds, prevention of emergency medical services deployments, and emergency department visits and hospitalizations [40] . although concerns have been raised about violation of federal and state drug laws, overdose prevention sites have been legally established successfully in areas outside of the united states. review of the processes and experience could facilitate implementation in us jurisdictions that have approved overdose prevention sites. jurisdictions that have approved overdose prevention sites should be allowed to implement and evaluate the intervention in the united states. significant work needs to be done to improve the care continuum for people with infectious diseases and co-occurring sud. the first step needs to be ensuring that everyone has access to health care. federal support for the medicaid expansion must continue and the 14 states that have not expanded medicaid should be incentivized to do so [19] . recent studies have shown that expansion of health care services, mostly via medicaid expansion, increased utilization of moud [3, 41, 42] . expanding access to health coverage is necessary to prevent and treat the infection, underlying sud, and improve overall mortality and quality of life as evidenced by studies finding an association between enrollment in an affordable care act qualified health plan and improved outcomes for people with hiv [43] . as a next step, treatment programs that integrate substance use care and treatment for infectious complications in order to improve outcomes are needed. treatment of both the sud and associated infections (eg, hiv, hcv) can be cost-effective and is associated with improved infection and sud outcomes [44] . previous studies have shown that patients with either hiv or hcv who receive mouds have improved viral suppression (hiv) [16, 17] , achieve sustained virologic response/ cure (hcv) [45, 46] , and have increased retention in care [47] . however, significant gaps remain in understanding the role substance use treatment plays in caring for people with other idurelated infections, such as endocarditis, deep tissue abscesses, skin and soft tissue infections, and bone and joint infections. one innovative care model combined outpatient parenteral therapy with buprenorphine treatment and showed similar clinical and drug use outcomes to completing inpatient therapy and resulted in reduced hospital length of stay by 24 days [48] . studies are needed to evaluate novel approaches to antimicrobial treatment for idu-associated infections such as the role of long-acting glycopeptides. increased funding is necessary for other demonstration projects and pilot studies to identify effective care models for comanagement of infectious diseases and oud as well as other suds. additionally, we need to expand the network of providers prescribing moud. most infectious diseases and hiv physicians receive little to no formal training in the management of oud and other suds. training to identify and treat oud and other suds should be increased in medical schools, nursing schools, physician assistant schools, residency programs, and within hospitals. while all infectious diseases and hiv physicians should become familiar with harm reduction principles and be able to counsel patients regarding safe injection practices, we need broader national support for physicians table 2 to comanage oud, suds, and co-occurring infectious diseases. lack of confidence has been identified as a major barrier preventing some physicians from integrating buprenorphine into their practice for the treatment of oud [49] . warmlines, such as the one run by the clinical consultation center at the university of california san francisco, and videoconferencingbased learning communities such as project echo, are excellent resources to provide support on a number of clinical aspects of disease management (table 2 ) [44] . increasing funding for national and regional warmlines, telehealth-based learning communities, peer-to-peer mentoring programs, and other technical assistance programs such as the opioid response network will help decrease barriers to providing substance use treatment. the opioid response network is a network of experienced clinicians that is funded by the substance abuse mental health and services administration to provide technical assistance to improve access to substance use treatment. in addition, a reorganization of the buprenorphine prescribing system is needed. in order to increase the number of providers who prescribe moud and improve patient access, we recommend eliminating the buprenorphine waiver requirement, removing the patient caps, and dedicating grant funding for case management and other support services to clinics that prescribe mouds. increased funding and reimbursement are also needed for low-barrier care delivery models such as telehealth. these innovative programs, which have already begun to be tested in infectious diseases/oud comanagement [50] , have the potential to increase medication uptake and improve outcomes by increasing access to treatment where people reside. in addition, multidisciplinary team meetings, including surgeons, sud specialists, inpatient internal medicine clinicians, nurses, social work, and case management, are being piloted in several hospitals across the country in order to make informed and collaborative decisions on complex patients, such as those with recurrent endocarditis following valve repair. evaluation of the impact of these types of collaborative efforts, both on patient outcomes and workplace satisfaction, can help inform best practice for all hospitals. we also need to address the requirements of particularly highrisk patients, including pregnant women and infants born to mothers with oud, and persons experiencing homelessness who may be unable to access traditional care. oud among pregnant women has increased significantly and there is an urgent need to build capacity to manage oud among pregnant women [51] . infants born to mothers with oud during pregnancy are at increased risk for hiv, hbv, and hcv. screening for hiv, hbv, and hcv is recommended for all pregnant women [52] and has been successfully integrated into most prenatal screening paradigms, allowing for perinatal management that decreases the risk of infant infection. in september 2006, the cdc recommended screening all sexually active persons 13-65 years old for hiv at least once, but this has not occurred and needs emphasis in order to end the epidemic. in august 2019, the us preventive services task force issued a draft recommendation for universal hcv screening [53] . given that overall incidence of hcv is increasing alongside the opioid epidemic [54] , strategies including provider education and increased resources are needed to ensure universal hcv testing is performed, particularly among women of child-bearing age and in prenatal care to prevent infant infection [55] [56] [57] . persons experiencing homelessness and unstable housing are similarly at increased risk for infections associated with sud. this is, in part, due to the high prevalence of concomitant untreated mental illness and sud among these individuals and sanitation issues [58, 59] . it is also due to our inability to implement effective management strategies for sud and infections in this vulnerable population. in addition to ensuring persons who experience homelessness receive treatment of their infectious diseases and sud through low-barrier and street-based medicine programs, expanding access to stable housing would also improve short-and long-term outcomes and should be part of a comprehensive strategy [60, 61] . finally, to monitor progress of these interventions, we need a standardized mechanism for reporting idu-related infections. other than for hiv and, in some states, for hcv infection, there is no national database of idu-related infections for surveillance, prevention activities, and program evaluation. this makes it difficult-if not impossible-to identify, predict, and prevent new infectious disease epidemics related to substance use in the united states. in addition, the majority of federal funding has been directed towards opioid overdose treatment and hiv resultant from idu, but not toward the bacterial and fungal infection complications, partly due to lack of integrated surveillance systems for serious idurelated infections, such as endocarditis. developing national surveillance systems to track and predict new epidemics before they happen and increasing national institutes of health funding for research into other infectious diseases related to the worsening sud epidemics in this country are urgently needed. over half of the criminal justice-involved population (cjip) have oud or sud, with a 10-fold higher prevalence than found in the general adult population [62] . as such, employing and enforcing evidence-based treatment guidelines that address the overlap of sud and infectious diseases in the criminal justice system has the potential to improve morbidity and mortality substantially. key components include evaluating new entrants for sud and idu-related infections, integrating moud and counseling services during incarceration, providing both appropriate medical care during incarceration and harm reduction during and after incarceration, care coordination, seamless referral to outpatient care for sud and chronic infections, and uninterrupted insurance coverage for cjip. intertwined with national increases in sud and incarceration rates, there have been substantial increases in hiv and hcv in cjip, as well as outbreaks of hav and hbv [63, 64] . inequities that exist in health care access in the community are amplified by criminal justice involvement, leading to premature deaths [65] . mortality rates are high following release, primarily driven by untreated oud leading to fatal overdose, progression of hiv, and hbv/hcv-induced liver disease [66] [67] [68] . additionally, overall infectious disease testing rates and rates of vaccination against hav and hbv are low [69, 70] . integration of infectious disease management with treatment for oud in cjip is an endorsed strategy for reducing these health inequities that will likely lead to improved infectious disease outcomes and facilitates linkage to care postrelease [16, 17, 71] . despite the evidence, however, few incarcerated settings offer moud. sud screening in jails and prisons with linkage to substance use treatment also decreases postrelease mortality [72, 73] and increases postrelease hiv viral suppression [16, 17] . clearly, prevention and treatment for oud and associated infections in this population can improve both individual outcomes and public health, especially when initiated during incarceration. time spent in prison or jail provides a reachable moment-an opportunity to engage a vulnerable population. screening for oud and treatment with moud need to be integrated into jails and prisons to improve substance use and infectious diseases outcomes. in addition to testing and treatment, access to harm reduction tools to prevent infection needs to be prioritized in the cjip. harm reduction tools like condoms and clean needles are not routinely available in prisons or jails despite several research studies demonstrating the need for such tools, and the consequences of not providing them [74, 75] . increasing awareness and availability of prep in jails and prisons-continued from the community, initiating while detained, or initiated before release-need to be urgently deployed, especially in communities deemed to be at high risk of hiv outbreak [76] . as evidenced by previous successful implementation of intensified harm reduction, expansion can be implemented in jails, effectively containing outbreaks [77] . substance use treatment coupled with uninterrupted health insurance is needed to improve outcomes among persons who are released from jail and prison. as a case study, expansion of hcv treatment during incarceration is feasible, cost-effective, and the best option to move closer to national hcv elimination [78, 79] . hcv diagnosis in jails with linkage postrelease is a feasible alternative if hcv treatment costs are deemed prohibitive [80] . a major barrier in hcv linkage to care postrelease is that 90% of states have policies that withdraw enrollment in insurance programs when people are incarcerated, outsourcing health care to medical corporations hired by criminal justice administrators [81] . prior to release, there are often attempts to reestablish health insurance, but this is complex because of uncertainty around the date of release and place the person will live. the process of re-entry is a vulnerable time for people who are incarcerated, with high mortality related to drug use but also associated with suboptimal postrelease management of chronic conditions like liver disease [82] . increased flexibility of medicaid, allowing initiation of insurance before release and sustained coverage prior to conviction, would improve health care transitions into and out of correction settings. finally, additional research funding is needed to develop and evaluate strategies to manage non-hiv/hcv-related infections secondary to idu, such as skin and soft tissue infections or infective endocarditis. despite increasing frequency of endocarditis in people with oud/sud [7] , and high rates of history of incarceration in people with skin and soft-tissue infections [7] , there are limited data on the epidemiology of disseminated bacterial and fungal infections in cjip. since the time this manuscript was accepted in december of 2019 the covid-19 pandemic has changed the world and has made the implementation of many of these recommendations even more urgent. we are at a pivotal moment in the opioid epidemic in the united states. as we desperately attempt to decrease the staggering number of overdose deaths, we must also grapple more broadly with idu in general, which is causing increases in hiv, hcv, and other idu-related infections. as a result, we as infectious disease specialists need a paradigm shift in our clinical approach, and we need broad and aggressive policy changes to support that shift. throughout history, infectious diseases clinicians have risen to the challenge. in 1998, dr jonathan mann said in an address, "when the history of aids and the global response is written, our most precious contribution may well be that, at a time of plague, we did not flee, we did not hide, we did not separate ourselves. " this time is no different-it is our epidemic too. disclaimer. the funders were not involved in the research design, analysis, or interpretation of the data or the decision to publish the manuscript. financial support. this work was supported by the national institute on drug abuse, national institutes of health (grant number k02 da032322 for career development to s. a. s.). supplement sponsorship. this supplement is sponsored by the centers for disease control and prevention. potential conflict of interests. s. a. s. has provided scientific consultation to alkermes inc. j. a. b. reports providing waiver training courses for providers to become buprenorphine prescribers through the providers clinical support system. a. w. is a consultant to gensler architectural firm. a. n. reports grants from gilead focus program outside the submitted work. r. l. reports royalties for a book on infectious disease surveillance donated to minnesota department of health. all other authors report no potential conflicts. increases in drug and opioid-involved overdose deaths-united states hospitalizations related to opioid abuse/dependence and associated serious infections increased sharply nonopioid overdose death rates rose almost as fast as those involving opioids community outbreak of hiv infection linked to injection drug use of oxymorphone-indiana increases in acute hepatitis c virus infection related to a growing opioid epidemic and associated injection drug use, united states trends in drug use-associated infective endocarditis and heart valve surgery increasing infectious endocarditis admissions among young people who inject drugs hospitalizations for endocarditis and associated health care costs among persons with diagnosed drug dependence-north carolina invasive methicillinresistant staphylococcus aureus infections among persons who inject drugs-six sites notes from the field: hiv diagnoses among persons who inject drugs-northeastern massachusetts outbreak of human immunodeficiency virus infection among heterosexual persons who are living homeless and inject drugs notes from the field: hiv infection investigation in a rural area-west virginia increases in acute hepatitis b virus infections-kentucky tip 63: medications for opioid use disorder-executive summary oral substitution treatment of injecting opioid users for prevention of hiv infection retention on buprenorphine is associated with high levels of maximal viral suppression among hiv-infected opioid dependent released prisoners extended-release naltrexone improves viral suppression among incarcerated persons living with hiv with opioid use disorders transitioning to the community: results of a double-blind, placebo-controlled randomized trial opioid treatment programs and related federal regulations education, defense, state, foreign operations, and energy and water development appropriations act, 2020. passed by the us house of representatives hr 1865 -further consolidated appropriations act hr 2569 comprehensive addiction resources emergency act of 2019 hr 2482 mainstreaming addiction treatment act of 2019 national academies of sciences, engineering, and medicine. integrating responses at the intersection of opioid use disorder and infectious disease epidemics: proceedings of a workshop integrating treatment at the intersection of opioid use disorder and infectious disease epidemics in medical settings: a call for action after a national academies of sciences, engineering, and medicine workshop needle and syringe programmes and opioid substitution therapy for preventing hcv transmission among people who inject drugs: findings from a cochrane review and meta-analysis homelessness and hepatitis a opioid analgesic use and risk for invasive pneumococcal diseases optimising health and safety of people who inject drugs during transition from acute to outpatient care: narrative review with clinical checklist reduction of injectionrelated risk behaviors after emergency implementation of a syringe services program during an hiv outbreak the policy surveillance program. a lawatlas project. syringe service program laws interventions to prevent hiv and hepatitis c in people who inject drugs: a review of reviews to assess evidence of effectiveness state syringe and drug possession laws potentially influencing safe syringe disposal by injection drug users impact of a medically supervised safer injection facility on community drug use patterns: a before and after study attendance at supervised injecting facilities and use of detoxification services medically supervised injecting centres the projected costs and benefits of a supervised injection the role of health insurance on treatment for opioid use disorders: evidence from the affordable care act medicaid expansion the affordable care act in the heart of the opioid crisis: evidence from west virginia evidence from a multistate cohort: enrollment in affordable care act qualified health plans' association with viral suppression addiction medicine consultations reduce readmission rates for patients with serious infections from opioid use disorder intensive models of hepatitis c care for people who inject drugs receiving opioid agonist therapy: a randomized controlled trial low hepatitis reinfection following direct acting antiviral therapy among people who inject drugs on opioid agonist therapy treatment of medical, psychiatric, and substance-use comorbidities in people infected with hiv who use drugs outpatient parenteral antimicrobial therapy plus buprenorphine for opioid use disorder and severe injection-related infections prescribing practices of rural physicians waivered to prescribe buprenorphine integrated, co-located, telemedicine-based treatment approaches for hepatitis c virus management in opioid use disorder patients on methadone state strategies to address opioid use disorder among pregnant and postpartum women and infants prenatally exposed to substances, including infants with neonatal abstinence syndrome american college of obstetricians and gynecologists. routine tests during pregnancy us preventive services task force. draft recommendation statement hepatitis c virus infection in adolescents and adults: screening increases in acute hepatitis c virus infection related to a growing opioid epidemic and associated injection drug use, united states hepatitis c virus infection among reproductive-aged women and children in the united states reducing risk for mother-to-infant transmission of hepatitis c virus: a systematic review for the u.s. preventive services task force hepatitis c screening in mothers and infants exposed to opioids the opioid epidemic in veterans who were homeless or unstably housed experience and outcomes of hepatitis c treatment in a cohort of homeless and marginally housed adults hepatitis c treatment outcomes among homelessexperienced individuals at a community health centre in boston adherence to protease inhibitors, hiv-1 viral load, and development of drug resistance in an indigent population drug use, dependence, and abuse among state prisoners and jail inmates prevalence of infection with hepatitis b and c viruses and co-infection with hiv in three jails: a case for viral hepatitis prevention in jails in the united states incarceration, drug use, and infectious diseases: a syndemic still not addressed hivpositive and in jail: race, risk factors, and prior access to care risks of drug-related death, suicide, and homicide during the immediate postrelease period among people released from new york city jails mortality after prison release: opioid overdose and other causes of death, risk factors, and time trends from substance use disorders, psychiatric disorders, and mortality after release from prison: a nationwide longitudinal cohort study preventive healthcare for underserved women: results of a prison survey hepatitis b vaccination practices in state and federal prisons linkage to hepatitis c care after incarceration in jail: a prospective, single arm clinical trial postincarceration fatal overdoses after implementing medications for addiction treatment in a statewide correctional system the impact of opioid substitution therapy on mortality post-release from prison: retrospective data linkage study the furthest left behind: the urgent need to scale up harm reduction in prisons a spray bottle and a lollipop stick": an examination of policy prohibiting sterile injecting equipment in prison and effects on young men with injecting drug use histories the path to implementation of hiv pre-exposure prophylaxis for people involved in criminal justice systems the global state of harm reduction in prisons cost-effectiveness and budgetary impact of hcv testing, treatment and linkage to care in u.s. prisons prevention of hepatitis c by screening and treatment in u.s. prisons a budget impact analysis of newly available hepatitis c therapeutics and the financial burden on a state correctional system filling the gap: the importance of medicaid continuity for former inmates public health implications for adequate transitional care for hivinfected prisoners: five essential components key: cord-261287-l4649du3 authors: puoti, massimo; zanini, barbara; quinzan, gian paolo; ravasio, laura; paraninfo, giuseppe; santantonio, teresa; rollo, adriano; artioli, stefania; maggiolo, franco; zaltron, serena; master hiv/hcv co-infection study group; raise, enzo; mignani, ermenegildo; resta, francesco; verucchi, gabriella; pastore, giuseppe; suter, fredy; carosi, giampiero title: a randomized, controlled trial of triple antiviral therapy as initial treatment of chronic hepatitis c in hiv-infected patients() date: 2004-05-06 journal: j hepatol doi: 10.1016/j.jhep.2004.04.016 sha: doc_id: 261287 cord_uid: l4649du3 background/aims: interferon and ribavirin combination therapy for chronic hepatitis c induces a low response rate in human immunodeficiency virus (hiv) infected patients. to assess the impact of intensification of interferon administration and of the addition of amantadine on the efficacy and safety of standard anti-hepatitis c virus (hcv) treatment in hiv-infected patients. methods: multicentre, prospective, open-label, randomized, phase iii clinical trial. eighty co-infected patients were randomized to receive ribavirin 800–1000 mg/day in combination with, group a: interferon alpha2a 3 miu thrice weekly; group b: ifnα2a 3 miu daily, plus amantadine 200 mg/day; treatment duration was 24–48 weeks according to hcv genotype. results: forty-one patients were randomized in group a and 39 in group b. intention-to-treat analysis showed a sustained virological response, defined as hcv-rna negativization, 6 months after stopping treatment in 22% of patients from group a and 13% from group b (p>0.05). the lack of a 2-log drop in hcv-rna levels after 12 weeks of treatment showed a 100% predictive value of lack of sustained response. conclusions: amantadine addition and interferon intensification do not improve the low efficacy of combination of interferon alfa plus ribavirin in hiv/hcv co-infected patients. patients with no early virologic response did not have any probability of sustained response. prevalence of hepatitis c virus (hcv) infection among anti-human immunodeficiency virus (hiv) seropositive patients with a history of intravenous drug use (idu) or transfusion is greater than 80%. the extensive use of highly active antiretroviral therapy (haart) has dramatically changed the prognosis of hiv infection, prolonging and improving life of anti-hiv seropositives [1] . on the other hand, mortality and morbidity for liver disease have increased significantly [2] . consequently, treatment of chronic hepatitis c in co-infected patients has become mandatory. interferon in combination with ribavirin was the gold standard for treatment of chronic hepatitis c in hiv-uninfected patients, inducing a 40% rate of sustained response [3] . however, a cumulative sustained virological response (svr) was observed in only 22% (95% confidence interval (ci), 14 -30%) of 111 patients enrolled in four pilot uncontrolled studies aiming to assess the efficacy and tolerability of ribavirin plus interferon alfa1 administered thrice weekly in hiv/hcv co-infected patients [4 -7] . so there is an urgent need for new and more effective treatment schedules for hepatitis c in hiv co-infected patients. among patients treated with interferon alfa three times weekly an intermittent increase of hcv viral load is observed on treatmentfree days [8] . daily administration of interferon could maintain a sustained antiviral effect on hcv; this schedule is expected to increase the efficacy of interferon alfa 2. amantadine (1-aminoamantadan) is a tricyclic amine with antiviral activity against toga-, myxo-, arena-, flavi-and coronavirus [9] . a comparative study recently demonstrated a statistically significant advantage of the addition of amantadine to interferon and ribavirin combination in hiv-uninfected patients with chronic hepatitis c and nonresponders to a previous cycle of interferon monotherapy [10] . thus addition of amantadine and intensification of interferon schedule with daily administration could be expected to increase the efficacy of standard combination treatment with ribavirin and interferon administered thrice weekly. in order to test this hypothesis we designed a multicentre, randomized controlled trial to compare the efficacy and safety of a new treatment schedule for chronic hepatitis c including ribavirin, amantadine and daily interferon alfa administration with the standard combination treatment. from april 2000 to october 2001, 80 hiv/hcv co-infected patients were consecutively enrolled in a multicentre, prospective, open-label, randomized, phase iii clinical trial. the study was conducted by the master hiv/hcv co-infection study group. eligibility criteria included: age between 18 and 60 years; alanine aminotransferase (alt) levels above the upper limit of normal (uln) 6 months before enrolment in the study; detectable plasma hcvrna by qualitative test (polymerase chain reaction (pcr) with amplicor w roche diagnostic system, hoffman-la roche, basel, switzerland); proven hivab seropositivity by elisa confirmed by western blot; stable hiv disease with cd4 cell count persistently over 300/ml during the last 8 months; anti-retroviral treatment (art) started at least 3 months before enrolment and demonstrated to be effective or no need for art; exclusion of hepatocellular carcinoma by imaging and alphafetoprotein level lower than 100 ng/ml; willingness not to consume alcohol during the treatment period. exclusion criteria were: reactivity for hepatitis b surface antigen (hbsag), neutropenia (fewer than 1500 neutrophils per ml), anaemia (less than 12 g/dl of haemoglobin in women and less than 13 g/dl in men), thrombocytopenia (fewer than 90,000 platelets per ml), decompensated liver disease, serum creatinine level more than 1.5 times the uln, poorly controlled psychiatric disease, alcohol or drug dependence in the year prior to enrolment; substantial coexisting medical conditions except hiv co-infection, previous treatment with ifn alfa or ribavirin or amantadine; systemic anti-neoplastic or immunomodulatory treatment in the preceding 6 months; current/present hiv-related opportunistic infection or malignancy classified as aids defining events (according to 1993 cdc aids surveillance case definitions); concomitant medication with rifampicin and/or rifabutin and/or isoniazid and/or pyrazinamide and/or gancyclovir; evidence of excessive alcohol consumption (.40 g in males and 20 g in females) and/or illegal substance abuse within the last 6 months; coexisting causes of liver disease; any additional contraindication to any of the drugs used in the study. additional exclusion criteria were pregnancy or lactation, and refusal to practise effective contraception during treatment and follow-up. this randomized controlled clinical trial was conducted at 15 italian centres from april 2000 to december 2002. patients were randomly assigned at a 1:1 ratio (with a block size of five) to receive: group a: interferon alpha 2a (ifna) 3 million units (miu) subcutaneously (sc) three times per week plus daily per oral3 (po) ribavirin (copegus w , roche, 200 mg tablets) or group b: ifna 2a 3 miu sc daily plus ribavirin (copegus w , roche, 200 mg tablets) and amantadine (mantadan w , boheringer ingelheim, 100 mg tablets) po 100 mg every 12 h. ribavirin was given orally with food at a dose of 800 mg/day (two tablets bid every 12 h) for patients ,75 kg of body weight and 1000 mg/day (two tablets in the morning and three in the evening after 12 h) for patients .75 kg of body weight. treatment duration differed according to hcv genotype: 24 weeks for hcv genotype 2 or 3, and 48 weeks for hcv genotype 1 or 4, only if hcvrna was negative at week 24 according to international guidelines [11] . randomization was centralized in the coordinating centre and stratified according to hcv genotype (genotype 1 or 4 vs. 2 or 3). genotype was performed by reverse hybridization assay (inno lipa hcv ii; innogenetics, ghent, belgium). patients were followed up for a treatment-free period of 24 weeks after cessation of therapy. the institutional review boards of the participating centres approved the protocol and all patients provided written informed consent. the study was conducted according to the declaration of helsinki, the applicable regulatory requirements and the ich/cpmp guidelines 'good clinical practice'. clinical examination, laboratory testing (including lactic acid and bicarbonate) and haematological count, including cd4 cell count, were performed monthly; plasma hivrna was monitored every 3 months using commercially available tests (quantiplex hiv-1 rna v.3.0 assay chiron corporation emeryville california, usa); hcvrna was measured at time 0 and after 1, 2, 4 and 12 weeks by a commercially available secondgeneration rt-pcr test (amplicor monitor version 2.0, roche diagnostic system, pleasanton, ca) according to the manufacturer's instructions. presence of hcvrna in plasma was determined at weeks 4, 12 and 24, at the end of treatment and 24 weeks after cessation of therapy using a commercially available second-generation rt-pcr test (amplicor hcv 2.0; roche diagnostic systems, pleasanton, ca) with a low end detection limit of 50 iu/ml according to the manufacturer's instructions. primary measures of efficacy were end-of-treatment response (eotr) and svr, respectively, defined as hcvrna levels below 50 iu/ml at the end of treatment, and 24 weeks after treatment. measures of safety were: any change in cd4 cell count, hivrna level, rate of withdrawal for adverse events (ae) or drop-out (do), rate of withdrawal from art or switch of anti-retrovirals. in difficult-to-treat patients (such as 'non-responders' to interferon monotherapy), triple therapy has been proven to increase by at least three times the sustained viral response rate obtained with standard interferon and ribavirin combination [10] . in order to establish that the svr in the triple therapy arm is at least three times higher than the 18% sustained response rate observed in hiv-co-infected patients treated with interferon and ribavirin in pilot studies [4] [5] [6] [7] , it was calculated that at least 64 patients should have been enrolled. a confidence probability of 80% and a significance level of 0.05 were used. intention to treat (itt) and per protocol (pp) analyses were performed. categorical variables were compared using fisher's exact test; distribution of continuous variables was compared using the t-test, mann-whitney two-sample statistic test or wilcoxon ranksum test. relationship between patients' baseline characteristics and svr was examined by univariate logistic regression analysis. to assess the independence of these factors, a multivariate logistic regression analysis was performed with backward selection ðp . 0:2þ: all p values reported are two-sided, and p was considered significant when ,0.05. statistical analysis was performed using stata software, version 7.0 (stata corporation, college station, tx, usa). eighty patients were enrolled in the study; 41 were randomly assigned to group a and 39 to group b; their baseline characteristics are shown in table 1 . no difference was found in demographics or clinical and immunovirological characteristics between the two groups. sixty patients (75%) were taking art, all for more than 3 months according to inclusion criteria, when they started anti-hcv treatment. the rates of hcvrna clearance in both groups are shown in table 2 . itt analysis showed 32.5% eotr, 31.7% in group a and 33.3% in group b. at the end of 6 months, follow-up response rates had decreased by about half: svr was observed in 17.5, 22% in group a and 12.9% in group b. differences in absolute hcvrna levels or hcvrna change-over baseline between the two groups at any time were not statistically significant. table 3 shows the distribution of some baseline characteristics in patients with and without svr; viroimmunologic and art characteristics were also analyzed, but the tests did not reveal a relationship with svr; genotype 2 or 3 and gamma glutamyl transferase (ggt) baseline level less than 1.5 times the upper limit of the normal range were more frequent in patients with svr (p , 0:05 fisher's exact test); univariate logistic regression showed their role as predictive factors of svr, with an odds ratio of 6 (1.2 -28.9 ci 95%) and 13.5 (1.6 -111.8 ci 95%), respectively. aspartate aminotransferase (ast) and neutrophil baseline levels were, respectively, lower and higher in patients who obtained an svr (p , 0:05 with mann -whitney test). by performing a multivariate logistic regression we found that genotype 2 or 3 and ggt baseline value were independent predictive factors for svr (table 4 ). by week 12, 32 of the 68 patients (47%) still on active treatment had a virologic response defined as a 2-log decrease from baseline hcvrna levels or no detectable serum hcvrna (fig. 1) . of those with a 12-week virologic response 14 (44%) subsequently had an svr, 5 dropped out (2 because of an adverse event and 3 spontaneously stopped treatment), 5 did have sustained hcvrna negativization, 2 showed a breakthrough after reducing the dose of ribavirin, and 6 patients relapsed after stopping treatment. eighteen out of 32 tested hcvrna negative at 12 weeks. the sustained response rate in this group (9/18, 50%) did not differ significantly from that observed in subjects with detectable hcvrna at 12 weeks (5/14; 36%). by contrast, of the 36 patients who did not have a 12-week virologic response 20 dropped out (8 because of ae and 12 prematurely stopped treatment), and 14 did not show hcvrna negativization; only two patients showed an etr, but none showed an svr. twenty-five out of 80 patients (31.3%) stopped treatment prematurely: 10 patients (18.8%) withdrew due to ae and 15 independently stopped therapy: distribution of ae and do was similar in the two treatment groups. pp analysis showed 32.1% of svr in group a and 14.8% in group b: there was not a significant difference between the two treatment arms. the treatment schedule was modified for more than showed an increase in hivrna level at any time during treatment and only 4 of them needed a switch of art due to loss of efficacy. no statistically significant difference was found in cd4 and hivrna levels between the two treatment groups at any time. eleven patients were undergoing treatment with didanosine, 39 with stavudine, and six with both of them; lactic acidosis was not observed in any patients. the main finding of this study was the very poor rate of svr to combination of standard interferon and ribavirin observed in both treatment arms, with and without amantadine and interferon dose intensification. cumulatively 17.5% of treated patients and 23.6% of those who completed the treatment course cleared hcvrna. this response rate is significantly lower than the 40% svr rate reported in trials performed on hiv seronegatives [12] the high withdrawal rate (31%), the large number of subjects requiring adjustment of the treatment schedule (31%) and the low response rate observed in those who completed the treatment schedule suggest that the tolerability and efficacy of interferon alfa and ribavirin in combination are reduced in hiv-co-infected subjects. the proportion of patients who needed interferon dose reduction was significantly higher in group b. we did not find a statistically significant difference in the rates of eotr and svr between the two treatment arms. therefore, the combination of interferon alfa schedule intensification and amantadine addition neither increase the efficacy nor improve the tolerability of combination therapy in co-infected hiv/hcv patients. clinical trials with pegylated interferon formulations [13, 14] in combination with ribavirin are ongoing in hiv/hcv-co-infected patients; preliminary results suggest that increased interferon levels induced by pegylated interferons could improve response rates in hiv/hcv-co-infected patients [15, 16] . amantadine addition did not improve the efficacy or tolerability of interferon alpha and ribavirin in chronic hcv infection. however, given the low power of this study, additional studies are needed before this drug is discarded from the therapeutic armamentarium for hiv/hcv co-infection. univariate logistic regression analysis of factors predictive of svr did not confirm that of age, sex, degree of fibrosis or baseline hcvrna levels, reported in hiv seronegative patients, but this was probably due to the small size of the sample under study [17] . the results of our study show that half of the patients with svr had in the past reached a cd4 cell count of less than 250/ml; such a low nadir of cd4 count in the years preceding treatment did not preclude achievement of a svr. these findings, together with the absence of an association between fibrosis score and cd4 counts and svr, suggest that progression of both hcv and hiv infections does not decrease the response rate to interferon and ribavirin combination in patients without severe immune depletion. we can therefore hypothesize that in patients without a high probability of response and early stage of liver fibrosis a watchful waiting strategy would not reduce the potential efficacy of anti-hcv treatment. this hypothesis needs to be confirmed by larger studies or by a meta-analysis of multiple pilot studies. hcv genotype, early hcvrna clearance and ggt levels were significant predictors of svr in multivariate analysis. the association between hcv genotypes 2 and 3 and a higher rate of svr is well known and has been confirmed in anti-hiv seropositives by pilot studies [4 -7] . all but two responders were infected by hcv genotype 2 or 3 and the svr rate in patients infected by genotype 1 or 4 was 5%. so, taking into account the side effects of treatment, these data suggest a very low cost effectiveness of treatment with standard interferon and ribavirin in hiv seropositives infected by hcv genotype 1 or 4. the association between earlier hcv clearance and svr suggests that suppression of hcv replication in the early phase of treatment is necessary but not sufficient to induce an svr. high serum ggt in chronic hepatitis c patients was frequently associated with more severe hepatic fibrosis or cirrhosis, or with steatosis, and may in part account for poor response to interferon therapy [18 -21] . ggt alteration in hiv seropositive patients is frequent and possibly correlated with one or more of the following factors: hepatic steatosis and/or lipidic dismetabolism, drug-related hepatic toxicity (especially in patients undergoing art), excessive alcoholic consumption, hcv-related hepatic damage (especially genotype 3) and immuno-mediated biliary duct damage. although all the included patients denied alcohol abuse, we cannot rule out that high ggt levels were due to undeclared excessive alcohol consumption. body mass index was not significantly associated with treatment response. only one of the svrs showed high ggt at baseline; in this patient ggt normalized the third month after hcvrna negativization. evaluation of art toxicity, careful assessment of alcohol intake and correction of alcohol abuse, diagnosis and treatment of altered lipid metabolism are factors to evaluate before starting treatment for chronic hepatitis c in patients co-infected with hiv. in this study the lack of a 2-log drop in hcvrna levels after 12 weeks of treatment showed a 100% negative predictive value of svr. although this data should be interpreted with caution, given the high proportion of dos, this result reinforces/supports the observation of a nearly absolute negative predictive value of the lack of a 12-week virologic response in hcv non-infected patients treated with pegylated interferons [13, 14] . as we enter an era in which hcv treatment is going to be pursued aggressively in hiv/hcv-co-infected persons, more and more emphasis must be placed on identifying at an early stage subjects who will not benefit from an expensive and poorly tolerated therapy. if these results are confirmed by larger studies, anti-hcv treatment could be stopped after 12 weeks, without decreasing the expected rate of svr, in hiv-infected patients who did not show at least a 2-log drop in hcvrna level. in conclusion, intensification of interferon alpha schedule and amantadine addition do not appear to improve the limited efficacy of standard combination therapy including interferon thrice weekly plus ribavirin for the treatment of chronic hepatitis c in hiv-co-infected patients. the best candidates for anti-hcv treatment are patients infected by hcv genotype 2 or 3 with normal ggt levels. the lack of a 2-log drop in hcvrna level after 12 weeks of treatment seems to be highly predictive of the poor efficacy of anti-hcv treatment. paolo and ospedale umberto i venezia-mestre) a.poggio, v. mondino (divisione di malattie infettive, ospedale di verbania), m. tinelli, mc. cerri (divisione di malattie infettive ospedale di lodi). mortality for liver disease in patients with hiv infection: a cohort study mortality due to chronic viral liver disease among patients with human immunodeficiency virus randomised trial of interferon a2b plu ribavirin for 48 weeks or for 24 weeks vs. interferon a2b plus placebo for 48 weeks for treatment of chronic hepatitis c virus chronic hepatitis c in hiv infection: feasibility and sustained efficacy of therapy with interferon alfa-2b and ribavirin long term efficacy of combination therapy with interferon alfa 2b and ribavirin for severe chronic hepatitis c in hiv infected patients interferon and ribavirin combination therapy in chronic hepatitis c in human immunodeficiency virus co-infected patients with congenital coagulation disorders pilot study of interferon alpha high-dose induction therapy in combination with ribavirin for chronic hepatitis c in hivco-infected patients the effects of a high dose, short course of interferon on hepatitis c a controlled trial of amantadine and rimantadine in the prophylaxis of influenza a infection triple antiviral therapy as a new option for patients with interferon non-responsive chronic hepatitis c international consensus conference on hepatitis c. consensus statement introduction to therapy of hepatitis c peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis c: a randomised trial multicenter, randomized trial comparing pegylated interferon alpha-2b (peg-ifn) plus ribavirin (rbv) vs. peg-ifn for treatment of hiv/hcv co-infected patients anrs hc02-ribavic: a randomised controlled trial of pegylatedinterferona-2b plus ribavirin vs interferona-2b plus ribavirin as primary treatment of chronic hepatitis c in hiv co-infected patients is an 'à la carte' combination interferon alfa-2b plus ribavirin regimen possible for the first line treatment in patients with chronic hepatitis c? retreatment with interferon plus ribavirin of chronic hepatitis c non-responders to interferon monotherapy: a metaanalysis of individual patient data gamma-glutamyl transpeptidase as a response predictor when using alpha-interferon to treat hepatitis c treatment of chronic sporadic-type non-a, non-b hepatitis with lymphoblastoid interferon: gamma gt levels predictive for response steatosis accelerates the progression of liver damage of chronic hepatitis c patients and correlates with specific hcv genotype and visceral obesity the authors wish to thank monica bertoletti and angela braga for their invaluable help and technical assistance in preparing the manuscript. key: cord-264159-e9071tyv authors: lin, weikang nicholas; tay, matthew zirui; lu, ri; liu, yi; chen, chia-hung; cheow, lih feng title: the role of single-cell technology in the study and control of infectious diseases date: 2020-06-10 journal: cells doi: 10.3390/cells9061440 sha: doc_id: 264159 cord_uid: e9071tyv the advent of single-cell research in the recent decade has allowed biological studies at an unprecedented resolution and scale. in particular, single-cell analysis techniques such as next-generation sequencing (ngs) and fluorescence-activated cell sorting (facs) have helped show substantial links between cellular heterogeneity and infectious disease progression. the extensive characterization of genomic and phenotypic biomarkers, in addition to host–pathogen interactions at the single-cell level, has resulted in the discovery of previously unknown infection mechanisms as well as potential treatment options. in this article, we review the various single-cell technologies and their applications in the ongoing fight against infectious diseases, as well as discuss the potential opportunities for future development. five months since the first reported infection cluster, covid-19 has turned into a vicious worldwide pandemic that infected more than 3.6 million people and caused over 250,000 deaths [1] . the pandemic will also have large spillover effects in terms of economic damage both in the form of healthcare costs and in monetary losses from the disruption of global supply chains, with world trade expected to fall between 13% and 32% in 2020 [2] . the covid-19 pandemic serves as a grim reminder that infectious disease is, and will always be, a major threat to the continued existence of mankind. to date, there are about 1400 microorganisms known to be pathogenic to humans. these pathogens can be broadly classified as viral, bacterial, fungal, and parasitic pathogens [3] . in particular, there have been 3 infectious diseases that have been persistently difficult to eradicate, namely, human immunodeficiency virus and acquired immune deficiency syndrome (hiv/aids), tuberculosis, and malaria. aids, due to hiv, is responsible for nearly 1 million deaths per year [4] . the death toll from tuberculosis, caused by mycobacterium tuberculosis (mtb) bacteria, is the highest amongst all infectious diseases, which is a problem that is exacerbated by the rise of antimicrobial resistance variants of the disease [4] . malaria, a parasitic infection, has afflicted humans for thousands of years and continues to do so today [5] . in light of the above-mentioned examples, among other infectious diseases, further efforts have to be directed for the continued management of the global burden of these diseases. the covid-19 pandemic has highlighted many questions that are relevant in the context of infectious disease as a whole. why are certain people more susceptible to infections? why are some infected individuals asymptomatic or display only mild symptoms? why are there differences in terms of disease progression and outcomes among patients? this diverse response to infection could be explained by the interactions of inherently heterogeneous populations of pathogens, host cells, and immune cells. however, discerning this heterogeneity is difficult in conventional bulk analyses, as they fail to recognize the following: (1) the genomic variability of pathogens, (2) the coexistence and interactions of infected host cells and bystanders, and (3) the diverse functional roles of immune surveillance participants. aside from the limited resolving power in pathophysiological studies, bulk analyses often fall short in terms of the level of precision and the amount of derived information needed for early diagnostics and high-efficacy vaccine development against infectious diseases. just as how microscopy revolutionized our understanding of biology, the enhanced resolution, precision, and breadth of information offered by single-cell technologies has brought an exciting overhaul to our perception of infectious diseases in recent years. the use of single-cell genomics, transcriptomics, proteomics, and epigenetics (referred to as omics altogether in this article) has flourished in many areas of the battlefield against infectious diseases. table 1 presents commonly used single-cell technologies in infectious disease studies alongside several of other non-single-cell systems. a scoring heatmap is used to represent the complexity of information in various aspects that they can provide (e.g., genetic, epigenetic, proteomic, spatial). the heatmap also provides an overall ranking of throughput, cost, and downstream assay compatibility amongst all the listed techniques. hence, it serves as a general guide for future users to select the methods that match their desired outputs. for instance, if the primary target for the study is to collect genetic information (e.g., analysis of invading virus gene heterogeneity in single host cell), single-cell sequencing might be the best candidate. likewise, if the focus is on proteomics orchestrating the immune responses, mass cytometry can furnish the most detailed insight. for infectious disease models that involve the interplay of genomics and proteomics, both cite-seq and reap-seq can be the suitable candidates. while flow cytometry has the highest throughput and lowest cost per experiment amongst the listed single-cell methods, it yields very limited aspects of information. other single-cell assays capable of providing more complex data typically come at the expense of a decreased throughput and increased cost. it is also worth mentioning that microfluidics has made great success in boosting the throughput and cost efficiency of existing single-cell assays. one prominent example would be the transition of well plate systems into microchambers or microdroplets, which ultimately reduces the required amount of reagent required per experiment and in turn reduces costs. in this review, we identified infection pathophysiology, therapeutic discovery, and disease diagnostics as three major areas in which single-cell omics has contributed substantially in the past decade. in pathophysiological studies of infectious disease, single-cell omics offer excellent spatial-temporal resolution that help to not only reconstruct the uneven subcellular distribution of pathogen across the entire host cell population, but also reveal the sequence of immune events accompanied by the change of immune cell profiles. single-cell omics also extrapolates meaningful molecular details that describe the dynamic host-pathogen interplay and immune activation. furthermore, single-cell omics identifies the rare molecules and cell subtypes that exhibit significant functionality in the pathogen-host immune interactions. insights in fundamental pathophysiology naturally have spillover benefits for translational science, such as in vaccine development where single-cell omics has the capability to enhance the discovery of mechanistic correlates of protection through multi-parameter measurements of the immune state with respect to disease, and it enables precision quality control checkpoints to aid the evaluation of vaccine efficacy. in the field of antibody discovery, single-cell omics can simultaneously interrogate antigen specificity and recover the b cell receptor gene sequences, which in turn shortens the previously prolonged and labor-intensive research cycle in the search for effective therapeutic or diagnostic antibodies. in the application of infectious disease diagnostics, single-cell omics are on the verge of practical clinical deployment, as demonstrated by some examples of automated and miniaturized devices. the diagnostic power of single-cell omics can be further enhanced by incorporating digital assays or integrating with other label free single-cell technologies. while there are many merits of single-cell analysis, we also discuss the new sets of challenges that need to be addressed in these systems. finally, we will conclude with our insight on future prospects of single-cell research in infectious disease and highlight several emerging single-cell technologies that may further enrich our arsenal against infections. understanding the pathophysiology of infection is critical to the rational design of prophylactic and therapeutic strategies to tackle infectious diseases. the course of infection, determined by the encounter of pathogens and host cells, is often measured as population-averaged results, leaving the important cell-to-cell heterogeneity out of the picture. the heterogeneity arises from both the pathogens and the infected cells. for example, pathogen heterogeneity can be reflected in the case of viruses, as a mixture of mutated viral particles displaying different infection ability [6] , or in the cases of bacteria, as a population of cells having different resistance to the same antibiotics [7] . host cellular heterogeneity is a combined result of variances in metabolism, composition, activation status, cell cycle, or infection history [6] . recent advances in single-cell analysis provide an attractive approach to probe the cellular population diversity and characterize infection pathophysiology at single-cell resolution. in this section, we will review how the recent advancement of single-cell technologies has helped deepen the understanding of pathogen and host cell heterogeneity and how the complex immune system reacts against infectious pathogens, with a focus on the contributions of single-cell sequencing. pathogen heterogeneity can be inherent or as a result of heterogeneous host-pathogen interactions. it is a favorable feature for pathogens because varied genomic sequences or functional properties enable immune evasion, colonization in novel hosts, and drug resistance acquisition; therefore, they increase the possibility of survival. besides, stochastic fluctuation in biochemical reactions may also contribute to cell-to-cell variability. single-cell technologies provide high-resolution insights into different aspects of intracellular pathogen replication. one area of virology that has benefited from the enhanced resolution of single-cell technologies is the study of variation in infection across single cells and the reasons for such variation. in the study by heldt et al., cells were infected in a population, isolated into microwells, and incubated. the supernatant was subjected to viral plaques measurement, and viral rna was quantified from lysed single infected cells [8] . it was shown that cells infected by influenza a virus (iav) under the same conditions produced largely heterogenous progeny virus titers, ranging from 1 to 970 plaque-forming units (pfu) and intracellular viral rna (vrna) levels varied three orders of magnitude. similarly, using scrna-seq, another study determined the percentage of viral transcripts in the total mrna generated from iav-infected cells, and it revealed that while most cells contained less than 1% of viral transcripts, some cells generated more than 50%, demonstrating infection heterogeneity from the angle of viral load [9] . reasons for this variation can be further explored through the use of high-throughput imaging technology. for instance, akpninar et al. used virus expressing red fluorescence protein (rfp) to study the effect of defective interfering particles (dip) on viral infection kinetics. dip are noninfectious progeny particles lacking genes essential for replication, and they are commonly produced during infection due to the high mutation rate. when participating in infection along with viable viral particles, they compete for host cellular machinery and result in viral replication inhibition. in this study, cells in a bulk population were infected with a mixture of vesicular stomatitis virus (vsv) expressing rfp and vsv-dips, and they were either untreated or isolated by serial dilution. rfp expression was observed during incubation as a surrogate for viral replication levels. the results showed that dip inhibited viral replication 10 times more on single cells, suggesting that the inhibition of viral replication is mitigated by cell-cell interactions when infection happens in a population [10] . table 1 . overview of commonly used single-cell technologies and their respective characteristics: a higher color intensity corresponds to a higher score (e.g., higher throughput, ease of moving cells of interest onto subsequent assays, higher information content, higher cost). the genomic mutation of pathogens during infection can be also detected directly. the sequencing of transcriptome and viral genes in single infected cells showed that iav is highly prone to mutation during infection [11] . detected mutations can cause consequences include viral polymerase malfunction and failure to express the interferon (ifn) antagonist protein, which is correlated to heterogeneous immune activation among infected cells [11] . the sequencing of 881 plaques from 90 vsv-infected cells detected 36 parental single nucleotide polymorphism (snp) and 496 snp generated during infection ( figure 1a -e) [12] . although extremely low multiplicity of infection (moi) was adopted, resulting in 85% of the cells statistically infected with only one pfu, 56% contained more than one parental variant, indicating that pre-existing differences in viral genomes can be spread within the same infectious unit, in this case, the host cell population. moreover, by measuring the viral titers produced by each infected cell, a significant correlation was found between the number of mutations in the viral progeny and the log yield of the initially infected cell. (e) correlation between the abundance of each type of substitution in single-cell-derived plaques and natural isolates. all panels adapted with permission from [12] . copyright 2015, elsevier. genomic variability also widely exists among bacteria populations. fluorescence labeling enables the quantification of bacterial growth in single host cells [13] [14] [15] , and by correlating the heterogenous growth with host response, it was found that the salmonella population exhibits different induction levels of the phop/q two-component system, which modulates lipopolysaccharides (lps) on the surface of individual bacteria [14] . to understand the pathophysiology of infectious diseases, it is important to study the identities of targeted cells. mounting evidence has shown that even under identical conditions, individual host cells manifest differential susceptibility and responses to infection in a population. how does this preference arise? do they share similar features that might be reasons for their susceptibility of infection? how do the states of infected cells affect pathogen replication and infection outcome? furthermore, how are host cells' phenotypes influenced by infection individually and temporally? answers to these questions are critical for the identification of target cells and individuals of novel pathogens, as well as for the understanding of infection pathophysiology. analysis of cells exposed to pathogens at single-cell resolution requires, first and foremost, strategies to distinguish infected cells from uninfected ones. pathogen-specific proteins, such as viral glycoproteins embedded in the cell membrane, or intracellular proteins such as viral capsid or polymerases, as well as pathogen nucleic acids, including genomic dna/rna and transcripts, can serve this purpose. these microbial elements can be labeled with specific antibodies or oligonucleotide probes for detection and quantification. alternatively, pathogen nucleic acids can be directly captured in deep sequencing. by combining tools for pathogen identification with host cell phenotyping assays, infected cells can be profiled at the single-cell level. xin et al. investigated the effects of host cell heterogeneity on both acute and persistent infection by foot-and-mouth disease virus (fmdv) [16] . by sorting single infected cells with facs based on cellular parameters, and quantifying viral genome replication with rt-pcr, they showed that the host cell size and inclusion numbers affected fmdv infection. cells with larger size and more inclusions contained more viral rna copies and viral protein and yielded a higher proportion of infectious virions, which is likely due to favorable virus absorption. additionally, the viral titer was 10-to 100-fold higher in cells in g2/m than those in other cell cycles, suggesting that cells in the g2/m phase were more favorable to viral infection or for viral replication. such findings have also been reported for other viruses [9, 17, 18] , revealing a general effect of heterogeneous cell cycle status in a population on virus infection. golumbeanu et al. demonstrated host cell heterogeneity using scrna-seq: they showed that latently hiv-infected primary cd4 + t cells are transcriptionally heterogeneous and can be separated in two main cell clusters [19] . their distinct transcriptional profiles correlate with the susceptibility to act upon stimulation and reactivate hiv expression. in particular, 134 genes were identified as differentially expressed, involving processes related to the metabolism of rna and protein, electron transport, rna splicing, and translational regulation. the findings based on in vitro infected cells were further confirmed on cd4 + t cells isolated from hiv-infected individuals. similarly, enabled by scrna-seq and immunohistochemistry, several candidate zika virus (zikv) entry receptors were examined in the human developing cerebral cortex and developing retina, and axl was identified to show particularly high transcript and expression levels [20, 21] . scrna-seq can also be used to identify potential target cells of novel pathogens and facilitate the understanding of disease pathogenesis and treatment. the spike protein of the virus sars-cov-2, the pathogen responsible for the covid-19 pandemic, binds with the human angiotensin-converting enzyme 2 (ace2) [22, 23] . this binding, together with a host protease type ii transmembrane serine protease tmprss2, facilitates viral entry [22, 23] . by analyzing the existing human scrna-seq data, it was identified that lung type ii pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells co-express ace2 and tmprss2, which suggests that they might be the putative targets of sars-cov-2 [24] . in the preparation of scrna-seq library, standard poly-t oligonucleotide (oligo-dt) is commonly used to capture mrna from single cells, which can also capture polyadenylated viral transcripts from dna virus or negative-sense single stranded rna virus. a simultaneous analysis of host transcriptome profiles and viral dna/rna offers information on the presence of the studied pathogen and its activities and allows a more accurate characterization on the dynamics of host-pathogen interactions. wyler et al. profiled the transcriptome of single human primary fibroblasts before and at several time points post-infection with herpes simplex virus-1 (hsv-1), and they described a temporal order of viral gene expression at the early infection stage [25] . more importantly, by simultaneously profiling the host and viral mrna, they identified that transcription factor nrf2 is related to the resistance to hsv infection. the finding was verified with the evidence that nrf2 agonists impaired virus production. steuerman et al. performed scrna-seq of cells from mice lung tissues obtained 2 days after influenza infection [26] . facs was applied to sort immune and non-immune cells based on cd45 expression. nine cell types were clustered (figure 2a) , and viral load was determined by the proportion of reads aligned to influenza virus gene segments, with higher than 0.05% considered infected. the authors found that viral infection can be detected in all cell types, and the percentage ranges from 62% in epithelial cells to 22% in t cells. however, the high variability of viral load was only observed among epithelial cells, while the majority of infected cells of other cell types showed to have low viral load (less than 0.5%) ( figure 2b ). for positive sense rna virus whose transcripts lack polyadenylation and cannot be captured by oligo-dt, a reverse complementary dna oligo probe to the positive-strand viral rna was employed. zanini et al. described this method and correlated gene expression with virus level in the same cell to study the infection of dengue virus (denv) and zika virus (zikv). they identified several cellular functions involved in denv and zikv replication, including er translocation, n-linked glycosylation, and intracellular membrane trafficking [27] . interestingly, by contrasting the transcriptional dynamics in denv versus zikv-infected cells, differences were spotted in the specificity of these cellular factors, with a few genes playing opposite roles in the two infections. genes in favor of denv (such as rpl31, tram1, and tmed2) and against denv infection (such as id2 and ctnnb1) was also validated with gain/loss-of-function experiments. analysis methods have been advancing for the detection of genetic variant-based scrna-seq data [28] [29] [30] . they could contribute, in the study of infectious diseases, to the characterization of temporal changes in viral mutational prevalence [31] . moreover, viral mutation can be correlated with host gene expression status at the single-cell level to further investigate their potential mutual effect on one another throughout the course of infection and reveal the dynamic host responses and pathogen adaptations in the progression of infection [32] . in spite of the above-mentioned examples characterizing virus presence with scrna-seq, it is worth noticing that viral mrna or genome occurrence is not necessarily equivalent to viral progeny, due to reasons such as missing essential genes caused by mutations. experimental techniques enabling the joint analysis of host transcriptional responses and viral titers will be needed to reveal the underlying mechanisms of virus production levels and host cell heterogeneity. another challenge of analyzing viral rna data is distinguishing infected cells with intracellular viral transcription from uninfected cells acquiring exogenous viral rna. combining single-cell transcriptomics data with flow cytometry or mass cytometry by time-of-flight (cytof) to measure the intracellular viral protein may help overcome this issue. immune and non-immune single cells were isolated from the whole lung of control and influenza-treated mice for massively parallel single-cell rna sequencing. host and the viral mrna were simultaneously measured, allowing the identification of infected as opposed to bystander cells, the quantification of intracellular viral load, and the profiling of transcriptomes. nine cell types were distinguished based on their transcriptional identities (b) the single-cell heterogeneity of intracellular viral load during influenza infection. percentages of low (yellow), medium (light brown), and high (dark brown) viral-load states (y axis) within the population of infected cells are shown for each of the nine cell types (x axis; total numbers of infected cells are indicated). (c) host genetic responses across all cell types. differential expression in influenza-treated and control mice (color bar) of nuclear-encoded genes (rows) across the nine major cell types (columns). right column indicates membership in four type i interferon (ifn)-related categories. all panels adapted with permission from [26] . copyright 2018, elsevier. immune responses activated by infection, since it is the innate immune responses that are primarily initiated in infected cells, or adaptive immune responses by lymphocytes carrying specific roles, are dynamic and complex, and they often happen in specific tissue microenvironments. heterogeneity in immune responses is also a long-recognized phenomenon. for instance, the activation of antiviral responses in dendritic cells (dcs) by bacterial lps starts with a small fraction of cells initiating the reaction, followed by the response by the rest of the population via paracrine responses [33] . technologies that enable the simultaneous measurement of multiple parameters facilitate high-resolution characterization of transcripts and protein at the single-cell level and boost our understanding of how host immune responses are initiated and orchestrated against infection. although pathogens usually dominate the war with host immune responses, hence the prevalence of infectious diseases, in-depth understanding of the interplay provides valuable information for the design of strategies to fight against infectious diseases. in this section, we cover the single-cell characterization of both innate immune responses from infected cells and adaptive immune responses activated in infected units. type i interferon (ifn), a key cytokine in innate immunity, orchestrates the first line of host defense against infection. its production is initiated upon host cells sensing pathogen-specific molecules, and it turns on the antiviral state of host cells by activating the transcription of hundreds of ifn-stimulated genes (isgs), some of which are crucial for coordinating adaptive immune responses. many studies have shown a large variability of ifn expression among infected cells. in the case of influenza virus infection, this can be partially explained by the high mutation rate during replication, revealed by sequencing viral genes in single infected ifn reporter cells [11] . however, such viability was also found to exist in infected cells expressing unmutated copies of all viral genes, which might be a result of the stochastic nature of immune activation irrelevant to viral genotypes [11] . in another study, pbmcs from patients with latent tuberculosis infection (ltbi) or active tuberculosis (tb), and from healthy individuals were analyzed with scrna-seq [34] . t cells, b cells, and myeloid cells were distinguished, and 29 subsets were clustered. the novel finding in this work is the consistent depletion of one natural killer (nk) cell subset from healthy individual samples to samples from ltbi and tb, which was also validated by flow cytometry. the discovered nk cell subset could potentially serve as a biomarker for distinguishing tb from ltbi patients, which is valuable for predicting disease outcome and developing treatment strategies. by analyzing scrna-seq data of pbmcs derived from individuals before and at multiple time points after virus detection, kazer et al. investigated the dynamics of immune responses during acute hiv infection [35] . after identifying well-established cell types and subsets in pbmcs, the authors examined how each cell type varies in phenotype during the course of infection. genes involved in cell-type specific activities, including monocyte antiviral activity, dendritic cell activation, naïve cd4 + t cell differentiation, and nk trafficking manifested similar changes with plasma virus levels: peaking closer to detection and gradually descending with time. phenotypic variations in bacteria populations were shown to influence host cell responses. avraham et al. investigated macrophage responses against salmonella infection with fluorescent reporter-expressing bacteria and scrna-seq on host cells [14] . transcriptional profiling revealed the bimodal activation of type i ifn responses in infected cells, and this was correlated with the level of induction of the bacterial phop/q two-component system. macrophages that engulfed the bacterium with a high level of induction of phop/q displayed high levels of the type i ifn response, which was presumably due to the surface lps level related to phop/q induction. with a similar setup, saliba et al. studied the salmonella proliferation rate heterogeneity in infected macrophages [13] . the varied growth rate of bacteria, indicated by fluorescent expression by engineered salmonella in single host cells, influenced the polarization of macrophages. those bearing nongrowing salmonella manifested proinflammatory m1 macrophages markers, similar with bystander cells, which were exposed to pathogens but not infected. in comparison, cells containing fast-growing salmonella turned to anti-inflammatory, m2-like state, showing that bacteria can reprogram host cell activities for the benefit of their survival. the above-mentioned strategy to simultaneously profile host cell transcriptome and viral rna also plays an important role in characterizing immune responses against infection by identifying infected immune cells and analyzing the transcriptomes simultaneously. for instance, it was applied to study the heterogeneous innate immune activation during infection by west nile virus (wnv) [17] . high variability was revealed for both viral rna abundance and ifn and isgs expression. interestingly, the expression of some isgs, with tnfsf10, ifi44l, and mx1 being the most prominent examples, was found to be negatively correlated with viral rna abundance, which could be a direction for future studies on wnv-mediated immune suppression in infected cells. similarly, zanini et al. studied the molecular signatures indicating the development of severe dengue (sd) infection by analyzing single pbmcs derived from patients [36] . facs was employed to sort pbmcs into different cell types (t cells, b cells, nk cells, dcs, monocytes), and then scrna-seq was performed. the majority of viral rna-containing cells in the blood of patients who progressed to sd were naïve immunoglobulin m (igm) b cells expressing cd69 and cxcr4 receptors, as well as monocytes. transcriptomic profiling data indicated that various ifn regulated genes, especially mx2 in naive b cells and cd163 in cd14 + cd16 + monocytes, were upregulated prior to progression to sd. comparison of the single-cell transcriptomes of lung tissue from health and influenza-infected mice revealed that 101 genes, among which the majority are isgs and targets of antiviral transcription factors, were consistently upregulated among all nine identified infected cell types, including both immune and non-immune cells [26] . this finding suggested that antiviral innate responses against influenza infection generically exist ( figure 2c ). moreover, by contrasting the expression profiles among infected, bystander, and unexposed cells, it was shown that the non-specific ifn gene module is a result of extracellular exposure and responses of environmental signals. while single-cell transcriptomics analysis provides an unbiased determination on host cell states, proteomics analysis offers direct characterizations of proteins expressed upon pathogen activation. going beyond traditional flow cytometry, mass spectrometry, or cytometry by time-of-flight (cytof) offers vastly increased numbers of parameters that can be investigated simultaneously, exponentially increasing the depth of the dataset collected. for instance, to investigate the effect of a precedent dengue virus infection on the outcome of subsequent zika infections, pbmcs derived from patients with either acute dengue infection or health individuals were incubated with dengue virus or zika virus, and the treated pbmcs were assessed by multiparameter cytof [37] . cytof in this study allowed the simultaneous detection of changes in the frequency of immune cell subpopulations and quantification of functional activation markers and cytokines in distinct cell subsets. while secondary infection with dengue virus led to increases of cd4+ t cells and t cell subsets, which are involved in adaptive immunity, secondary infection with zika virus induced the upregulation of several functional markers including ifnγ and macrophage inflammatory protein-1β (mip-1β) in nk cells, dcs, and monocytes, indicating an intact innate immunity against zika virus in the cases of possible concurrent dengue infection. hamlin et al. compared two denv serotypes (denv-2 and denv-4) in their infection in human dcs using cytof, which allowed simultaneous analysis on denv replication, dc activation, cytokine production, and apoptosis [38] . the tracking of intracellular denv proteins and extracellular viral particles showed different replication kinetics yet similar peak viral titers by these two serotypes, as well as the percentage of infected dcs. moreover, denv-4 infection was found to induce a higher expression of cd80, cd40, and greater production of tumor necrosis factor-α (tnfα) and interleukin-1β (il-1β), compared to denv-2 infection. additionally, bystander cells, which were identified by the absence of intracellular viral proteins, were identified to produce less tnfα and il-1β, but show more activation of interferon-inducible protein-1 (ip-1), which is a member of isgs. besides cytof, host cell secretomes can also be measured with customized miniatured systems, and the level of multiplexing and flexibility of sample handling is often improved. for instance, lu et al. showed the co-detection of 42 secreted proteins from immune effector cells stimulated with lps [39] . in a similar setup, chen et al. performed a longitudinal tracking of secreted proteins from single macrophages in response to lps treatment [40] . these studies provide valuable insights into the dynamic and comprehensive responses to pathogen over time. notably, such methods require microfabrication tools and skills, which is not always available and thus hinder their accessibility, compared with flow cytometery and cytof. epigenetic profiling at the single-cell level is also important, especially for elucidating the influence of host immune responses in chronic infection. the assay for transposase-accessible chromatin with high throughput sequencing (atac-seq) utilizes tn5 transposase to insert sequencing adapters into regions of open chromatin, in order to study genome-wide chromatin accessibility. buggert et al. applied atac-seq and established the epigenetic signatures of hiv-specific memory c8 + t cells resident in lymphoid tissue [41] . yao et al. used chromatin immunoprecipitation followed by high-throughput sequencing (chip-seq) to examine the histone modification of progenitor-like cd8 + t cells from mice chronically infected with lymphocytic choriomeningitis virus (lcmv) [42] . they found that progenitor-like cd8 + t cells showed distinct epigenomic features compared with memory precursor cells, exhibiting more abundant active histone markers (h3k37ac modification) at genes co-expressed with tox, which encodes the thymocyte selection-associated high mobility group box protein tox. this might promote the long-term persistence of virus-specific cd8 + t cells during chronic infection. in some cases, deep sequencing can be implemented together with other single-cell technologies for a comprehensive and systematic profiling of immune responses against infection. for instance, michlmayr et al. performed 37-plex cytof on peripheral blood mononuclear cells (pmbcs), rna seq on whole blood, and serum cytokine measurement of blood samples from patients with chikungunya virus (chikv) infection [43] . moreover, samples collected at acute and convalescent phases were compared to study the disease progression. such multidimensional analysis allows the large-scale, unbiased characterization of gene expression, cytokine/chemokine secretion, and cell subpopulation changes in response to infection. one important result of this study is revealing monocyte-centric immune response against chikv, with the frequency of two subsets both related to antibody titers and antiviral cytokine secretion. in addition, significant viral protein expression was found in two b cell subpopulations. while multiple assays can be done on the same bulk sample to obtain different data parameters (e.g., transcriptomic, proteomic), such datasets are not able to correlate the data parameters at the resolution of a single cell. newer advances allow the simultaneous collection of multiple types of parameters for the same cell. for instance, cellular indexing of transcriptomes and epitopes by sequencing (cite-seq) and rna expression and protein sequencing (reap-seq) are techniques for the simultaneous collection of transcriptomic and high-dimensional information on specified proteomic targets. by using antibodies tagged with unique nucleotide sequences, the subsequent transcriptomic sequencing simultaneously sequences these tags to allow the quantification of the antibody targets. corresponding transcriptomic and proteomic data at the single-cell level allows the opportunity to study the role of post-translational gene regulation in the immune response. the increased dimensionality of the information obtained may also allow more accurate machine learning to identify signatures of healthy or dysfunctional immune responses. for instance, using cite-seq, kotliarov et al. were able to identify a common signature of activation in a plasmacytoid dendritic cell-type i interferon/b lymphocyte network that was associated both with flares of systemic lupus erythematosus (sle) and influenza vaccination response level [44] . as noted above, the ability to study biological processes at the single-cell level gives an unprecedented to attribute bulk phenotypes in immunology and host-pathogen interaction to specific cell subpopulations, including rare cell populations, in a relatively unbiased fashion. apart from basic science discovery, how do these insights affect clinical practice in infectious disease? biomarker discovery is one obvious area of impact-the molecular differences found to underpin broader disease phenotypes can be used to diagnose or even predict disease. in particular, diagnosis is a notable problem in infectious disease, where identification of the causative pathogen can take days to weeks for culture-based systems, which may delay appropriate, targeted treatment [45] . apart from biomarker discovery, single-cell technology is also revolutionizing the discovery of vaccines and therapeutics, which will be elaborated upon in the sections below. other clinical uses of single-cell technology may require an increased uptake of such technologies within the hospital setting. for instance, one potential area of impact is antimicrobial resistance. the bulk genotype or phenotype of a pathogen population may not accurately identify its ability to become resistant to antimicrobials, since antimicrobial resistance can involve the selection of a previously rare, resistant population. should single-cell technology become routinely used in hospitals, the increased resolution could enables the identification of such rare populations, which can inform the choice of antimicrobials prescribed. to generalize, this similarly applies to any disease phenotype that can be triggered by a rare host or pathogen cell population. the complexity of current single-cell technologies hinders their implementation in the clinic, and in the section titled diagnostics, we highlight various steps that have been taken toward simplifying single-cell technology platforms to allow their clinical use. the first step of the vaccine development pipeline would be to identify a promising disease antigen, which could be in the form of a recombinant protein or inactivated/attenuated virus. unlike traditional vaccinology where vaccines were generated via pathogen growth and inactivation, the reverse vaccinology approach relies on predicting antigen features that are likely to trigger protective functions and engineering the antigen accordingly [46] . to predict these antigen features, two main approaches have been used: via whole genome sequencing and more recently, identifying and mapping the structural epitopes of neutralizing antibodies using the methods discussed later in this review [47] . after identifying a vaccine candidate, the next step would be to verify its efficacy. this efficacy is quantified based on its ability to bring about a set of specific immune responses which are specifically linked with protective functions, which are known as correlates of protection (cops) [48] . it is important to identify the cops for each vaccine for multiple reasons, including the following: (1) to understand the mechanisms of vaccine protection for improvement of vaccines, (2) to understand the mechanisms of vaccine protection for improvement of vaccines, (3) to determine the consistency of the vaccines produced, (4) to evaluate the levels of protection to patients before and after treatment, and (5) for the licensure of said vaccine [49] . historically, most of the cops in commercial vaccines typically involve quantifying the titer of neutralizing antibody produced by antigen-specific memory b cells. in the past decade, better understanding of the in vivo vaccine response has led researchers to identify several relevant memory t-cell responses as cops, and these t-cell responses are usually quantified by measuring the expressed cytokines via techniques such as elispot, flow cytometry, and elisa [50] . however, it remains difficult to define vaccine cops for a number of diseases. these include those diseases that cannot yet be eliminated by vaccine or infection-elicited immune responses (e.g., hiv-1 infection, tuberculosis), since a suitable end point of protection is not attainable. they also include those diseases for which vaccines do not yet exist but vaccine cops may be expected to differ from infection-related cops, including diseases for which natural clearance occurs via the innate immune response or early adaptive immune response (e.g., . even when immune parameters that correlate with disease risk are found, the causative mechanism of immune protection, or mechanistic cop, may remain elusive if multiple immune parameters are elicited in parallel by a protective response. as seen in the excellent review by plotkin [51] , the cops may not always be as obvious or limited to humoral immunity, and since vaccines typically elicit multiple immune responses. this is especially true for the case of vaccines against complex pathogens such as hiv and malaria, where the resultant network of immune responses may not always be easily identifiable. single-cell approaches may define a greater space of immune parameters to be explored as cops. furthermore, the increased breadth of data that can be obtained from a single sample is useful in increasing the number of hypotheses that can be probed, especially in longitudinal analyses, which are most useful for mechanistic immune studies but where the sample volume is often limited. furthermore, using a systems vaccinology approach via omics technology, researchers have begun to uncover these potential cops early in the vaccine development process [52] . in one of the earliest proof-of-concepts, querec et al. successfully identified a cop for vaccine efficacy on humans vaccinated against yellow fever. a gene marker present in cd8+ t cells which could predict for protection was discovered by using a multivariate analysis of the immune response via a combination of flow cytometry and microarray techniques [53] . with the rapid developments in single-cell omics technology, a deeper understanding of vaccine response can be obtained through an even more detailed mapping of the interactions between the various immune cell populations at the single-cell level, as well as identify the causes of heterogeneous vaccine response in individual immune cells [54] . this could be seen from the recent work by waickman et al. [55] where a dengue vaccine elicited a highly polyclonal repertoire of cd8+ t cells that was identified using scrna-seq. combined with transcriptional analysis of the cd8+ t cells, the authors established a set of metabolic markers that could be potential cops for vaccine efficacy evaluation. combining the simultaneous analysis of single-cell transcriptomic and tcr sequence data, tu et al. identified preferential transcriptional phenotypes among subsets of expanded tcr clonotypes. this is a strategy that may be highly valuable in assessing the functionality of t cells and their correlation to protection in vaccine responses [56] . antibodies are widely used in therapeutics and diagnostics due to their high specificity and generally low toxicity. antibodies are capable of mediating protective functions against infectious diseases, including pathogen neutralization, antibody-mediated phagocytosis, antibody-mediated cellular cytotoxicity, and complement-dependent cytotoxicity. antibody-containing sera remains in use for diseases where there are no other therapeutic options, including for viruses such as hepatitis a or b, rabies, vaccinia, sars-cov-2 at the point of writing, and for toxins (e.g., snake venom). however, there are limitations to this approach: serum therapy from animal sources causes a risk of serum sickness due to immune reaction against animal protein, while pooled hyperimmune sera from humans is difficult to collect and standardize. instead, the appropriate b cell clone that secretes antibody with protective activity can be isolated, and its antibody sequence can be obtained and expressed in culture to obtain monoclonal antibodies as therapeutics. similarly, in diagnostics, monoclonal antibodies provide the specific recognition of pathogen antigens that allow the rapid diagnosis of infection. in order to identify the correct b cell clone from thousands or millions of b cells, its antigen specificity and/or protective activity must be interrogated. this is classically done by cell immortalization (such as by hybridoma production or epstein-barr virus infection to generate b lymphoblastoid cell lines), followed by single-cell plating and expansion to obtain sufficient antibody from a single clone, and then the well-based screening of the antibody-containing cell supernatants. however, these techniques are low in throughput and efficiency, losing more than 99% of potential cells [57, 58] for hybridomas, and 70-99% of potential cells for b lymphoblastoid cell lines [59, 60] . moreover, there remains a bottleneck in throughput at the subsequent stage of subcloning and screening the resulting clones to determine which clones are antigen-specific and functional for the desired purpose-even large experiments are limited to screening several thousand cells [61, 62] , or up to 100,000 cells for robot-assisted operations [63] , whereas a single 30 ml human blood draw contains an order of magnitude more (approximately 900,000) candidate cd27+ igd-class-switched memory b cells [64] . more recently, techniques that avoid the need for cell expansion have been developed-these speeds up the life cycle for monoclonal antibody discovery. primary b cells expressing antigen-specific b cell receptors (bcrs) are labeled using fluorescent antigens, allowing flow cytometry-based single-cell sorting to isolate these antigen-specific b cells [65] . this technique is useful especially for the interrogation of memory b cells, which express the bcr on their surface. the interrogation of plasmablasts and plasma cells, which secrete antibodies but have low or no surface expression of the bcr, require other procedures such as the formation of an ig capture matrix on the b cells [66] , or alternative methods of screening that allow the physical separation of single cells such as droplets [67] , nanowells [68] [69] [70] [71] , or microcapillaries [72] . following the isolation of the desired b cells, they are lysed and their rna is interrogated to recover the antibody heavy and light chain genes. with these techniques, both antigen interrogation and antibody gene recovery do not require large clonal cell populations, removing the need for inefficient and time-consuming cell expansion processes. for the recovery of antibody genes, rt-pcr is commonly used, but recovery rates are typically low (<70% success rate for each pair of heavy and light chains) due to the large variability across the v gene families. single-cell rna-seq (smart-seq2) is an alternative to rt-pcr, which results in improved recovery rates (>90%) [73] . bcr recovery can also be done in the same step as antigen-specific sorting via the use of dna-barcoded antigens, such that both the antigen barcodes and bcr sequence are recovered simultaneously during single-cell ngs [74] . this has been used to successfully isolate broadly neutralizing hiv-1-specific antibodies and influenza-specific antibodies simultaneously from a single sample, although the resulting antibody candidates had variable neutralization functions, which required subsequent in vitro confirmation. another method for monoclonal antibody discovery is the use of phage display libraries, where phages expressing antibody genes are selected for using an antigen-coated surface in an iterative process of biopanning [75] . this has been a fast and effective method for monoclonal antibody discovery. the main limitation of phage library display is the random, largely non-native pairing of vh and vl genes, which may cause problems in subsequent antibody expression and production, and it may also have a higher likelihood of triggering anti-idiotypic allergic responses. more recently, a single-cell emulsion technique has been used for the interrogation of antigen specificity and high-throughput sequencing, allowing the interrogation of a yeast library utilizing natively paired human antibody repertoires [76] . using it, rare broadly neutralizing antibodies against hiv-1 could be identified, albeit with the correct antigen required for identifying the desired b cell clones. antigen binding is the most common form of screening for monoclonal antibodies due to its compatibility with high-throughput methods including flow cytometry, biopanning, and nanowell-based elisa. however, antigen binding may not correlate with functional activity against the intended target. for example, this may occur if the protein antigen used does not accurately mimic the native form of the antigen; monoclonal antibodies generated against the protein antigen may not be active against the native target [77] [78] [79] [80] . another example would be if functional activity requires binding in a specific orientation, such as virus neutralization requiring the monoclonal antibody to disrupt the receptor binding site [81, 82] . assays for monoclonal antibody function include assays for virus neutralization, opsonophagocytosis, antibody-dependent cellular cytotoxicity, and receptor agonism/antagonism [83] . currently, these assays are typically done in bulk with relatively low throughput, creating a bottleneck in monoclonal antibody screening. microfluidic technologies, such as water-in-oil emulsions or nanowells, are being developed to increase the throughput of such assays. for instance, a high-throughput screen for enzyme antagonism using a droplet-based assay has been reported [84] . using water-in-oil microdroplets, el debs et al. co-encapsulated single hybridoma cells with an enzyme (ace-1) and an enzyme substrate that emits a fluorescence signal upon enzyme hydrolysis, and they were able to sort out hybridomas secreting ace-1-inhibiting antibodies through fluorescence-activated droplet sorting. another group has recently also reported assays that are capable of assaying cellular internalization, opsonization, and the functional modulation of cellular signaling pathways [67] , and several companies have also reported proprietary platforms that may be able to carry out some other functional assays [85] . however, the specificity and sensitivity of these assays have not been reported. the ability to immobilize single cells in nanowells allows repeated longitudinal profiling, which is a property that was utilized by story et al. to obtain antibody-antigen binding curves that can classify related populations of b cells [71] . the characterization of diverse bacterial populations, including microbiome studies, has traditionally been done at the bulk level. for instance, the selection of particular organisms out of a diverse population has been done by the plating and amplification of single colonies. however, this method is limited in throughput. the enhancement of throughput can be done via miniaturization-for instance, one study isolated antibiotic-resistant e. coli mutants by encapsulating and culturing single bacteria in nanoliter-scale droplets containing the antibiotic [86] . this approach can be applied to accelerate the identification of targets acted upon by antibiotics of unknown mechanisms. apart from being limited in throughput, traditional microbial selection systems also require the ability to culture the microorganism of interest in vitro. however, it is estimated that the bulk of microorganisms cannot be cultured and expanded in typical cell culture media [87] . one potential solution is to use microfluidic devices to physically separate and phenotype individual bacteria while immersing them in media derived from their natural environment. this method was adopted to identify a new antibiotic, teixobactin, from a previously unculturable β-proteobacteria belonging to a group of gram-negative organisms not previously known to produce antibiotics [88] . in the clinical setting, single-cell analysis techniques are currently rarely routinely used in infectious disease diagnostics and monitoring. it is still impractical to apply most of the other conventional single-cell analysis techniques for diagnostic applications due to the associated high costs, long workflow durations, and high degree of technical expertise required. one notable exception would be flow cytometry, where aside from its high initial equipment cost, its fast turnaround times, high sensitivity, and ease of operation make it a staple tool in clinical institutions worldwide [89] . flow cytometry is mainly used to perform the immunophenotyping of blood cells against various disease-specific biomarkers [90] [91] [92] . the most prominent example would be in the routine monitoring of human immunodeficiency virus (hiv) progression by counting the number of cd4 + t cells in a patient's blood sample [91] . the other single-cell technique that has seen some use in diagnostics against pathogens would be fluorescence in situ hybridization (fish). as a diagnostic tool, fish has numerous advantages that include low cost and complexity; its rapid turnaround time allows the diagnosis of fastidious bacteria and the ability to distinguish between mixed populations of pathogens at a single-cell resolution [93] . while fish has been successfully used for the direct identification of panels of pathogens from blood samples [94, 95] , its reliance on image analysis as the readout limits the throughput of this technique, and the results are subject to user-to-user variation and bias [96] . to resolve these issues, a variant of the technique, fish-flow, was developed. fish-flow combines fish with flow cytometry to achieve higher throughputs as well as automates the signal readout through the cytometric system [97] , and it has been used to detect hiv reservoirs in t cells [98] as well as bacteria from blood [99] . while the ability to identify biomarkers at a single-cell resolution is certainly invaluable in the fight against infectious diseases, current flow cytometer systems are typically bulky and expensive, thereby limiting their use in a laboratory setting [100] . fortunately, advancements in microfluidics and low-cost electronics have given rise to the development of portable platforms that can perform single-cell analysis in a point-of-care (poc) setting. recent examples of portable cytometric systems that are relevant to infectious disease diagnosis include a miniaturized modular coulter counter capable of label-free detection and the differentiation of particles of varying sizes [101] , a low-cost and portable image-based cytometer for the quantification of malaria-infected erythrocytes [102] (figure 3a) , and a portable miniaturized flow cytometer that is capable of multi-channel fluorescence interrogation of whole blood samples [103] . the portability of such cytometers could mean faster turnaround test timings through on-site diagnostics and disease monitoring, hence expediting clinical decisions and improving healthcare outcomes in general [104] . in addition, the portability of such microfluidic systems lends to other practical applications of flow cytometry, especially in pathogen detection in water and food sources. particularly, diarrheal diseases (a leading cause of death for children under the ages of 5) are closely linked to the consumption of contaminated water sources and could be mitigated via regular, on-demand pathogenic testing of drinking water [105] . however, adapting current single-cell technologies into a portable format holds its own set of unique challenges. most of the existing literature surrounding such technologies still report separate sample enrichment or staining steps prior to cell analysis [106] [107] [108] ; such additional preparatory steps increase assay complexity, which may not be desirable in a poc setting [109] . while a gamut of existing microfluidic technology has already been established for sample purification as well as for reagent addition and mixing, integrating the various modules into a single platform is typically not a trivial process [110] . for single-cell technology to make the successful transition from the lab to the bedside, such practicalities must be considered and successfully implemented. digital assays are a relatively recent assay format comprised of the following steps: (1) the discretization of a single initial larger sample volume into multiple smaller volumes (typically via microwell, microvalve, or droplet emulsion partitioning techniques [111] ), and (2) performing the chemical or biological assay on each individual volume to obtain a quantifiable signal [112] . due to the ability to individually assay a large number of cells at the single-cell level, the digital assay format has been widely employed in single-cell omics studies [113] . in the field of infectious disease research, while most of the applications of digital assays have been centered on answering fundamental questions relating to pathophysiology, there are other single-cell diagnostic applications that can benefit tremendously from such an assay format. an example mentioned earlier in the review would be rapid antimicrobial-susceptibility testing (ast) to address the surge of antimicrobial-resistant infections worldwide as a result of the misuse of antimicrobials. phenotypic ast, which involves the culture of the pathogen in the presence or absence of antibiotics, may help guide treatment options, but existing conventional assays have low sensitivity and require a long time of 12-48 h for cell regrowth to achieve measureable assay outcomes [114, 115] . higher-sensitivity single-cell digital assays that have been recently reported can obtain measurable signals without requiring cell regrowth and could be the answer to reducing ast turnaround times ( figure 3b ) [116] [117] [118] . another application of digital assays could be in quantifying viral reservoirs in patients at a single-cell resolution. in hiv eradication studies, latent reservoirs are reactivated using latency-reversing agents (lras) for subsequent inhibition via antiretroviral therapy [119] . the ability to isolate and individually assay the patients' blood to obtain the distribution of reactivation states in the heterogenous cell population can give clinicians an idea of antiretroviral treatment efficacy in the future [120, 121] . pump-free droplet emulsion generation system that is capable of performing antimicrobial-susceptibility testing (ast) of different species of bacteria with a turnaround time of ≈5 h. image reproduced with permission from reference [117] . copyright 2020 royal society of chemistry. (c) microfluidic impedance cytometry is able to differentiate between healthy and malaria-infected red blood cells at a single-cell resolution based on the difference in electrical impedance measured across two electrodes. image reproduced from [122] under a creative commons license. the development of label-free single-cell analysis techniques has been gaining considerable attention over the last decade with the advent of microfluidics because of their numerous advantages over their counterparts that require cell labeling. some of the advantages include: (1) lower technical complexity and turnaround times in assay workflow because the preparatory step is omitted, (2) not requiring knowledge of cell biomarkers beforehand, making them suitable for assaying novel cell populations, and (3) by avoiding the use of labels that might affect the natural state of the cells, results might be more representative of actual in vivo cellular conditions [123] . coupled with the precise fluid handling capabilities afforded by microfluidics systems, there is a burgeoning number of label-free single-cell analysis platforms that have been reported in recent years that are able to measure infection based on the inherent properties of the cells. one prime example would be the identification of cells via their electrical properties, specifically electrical impedance. this impedance is derived from the change of voltage or current signal when single cells flow across a pair of miniaturized electrodes, and it has been shown to be able to differentiate between healthy and malaria-infected erythrocytes ( figure 3c ) [122] , as well as the viability and species of parasitic protozoa [124] . another promising direction for label-free single-cell analysis is via measuring the inherent optical properties of cells. this has been shown in recent work such as the single-cell identification of parasites through their raman spectra [125] , the quantification of single-cell viral infection titer through laser force cytology [126] , and single bacteria detection via refractive index measurements [127] . lastly, the mechanical and size properties of cells have also been exploited for identifying infected single cells. for example, using inertial microfluidics, white blood cells could be hydrodynamically isolated from lysed blood containing ring-stage malaria parasites as a result of the white blood cells' larger sizes [128] . other recent works that demonstrate potential applications for single-cell label-free infectious disease analysis includes cell identification via their acoustophoretic responses [129] , as well as their deformability and hydrodynamic resistance [130] . evidently, there is a host of promising label-free single-cell analysis technology that could be translated to clinical diagnostic applications. in the near future, these technologies would be useful complement poc applications where labeling steps in the assay workflow would increase the technical complexity and hinder the transition from the lab to bedside. among the methods discussed, scrna-seq is the primary tool for single-cell studies. in the following section, we briefly cover some important points that needs to be considered when designing and conducting such experiments. for a more in-depth coverage of this subject matter, the reader is invited to read other excellent reviews from luecken [131] , see [132] , and lähnemann [133] . as covered earlier in this review, the main applications of scrna-seq in infectious disease study comprise of the following: (1) studying effect of host cell heterogeneity on infection, (2) identifying host immune responses, and (3) antibody discovery. however, the number of sequenced cells and depth of sequencing ultimately depend on the end goal of each experiment as well as the amount of financial resources at hand. the availability of a variety of commercial platforms for single-cell analysis with different throughput and sensitivity can provide users with different options to best suit the purpose of their studies [134] . for studies that involve identifying the cell types of a heterogeneous sample, a minimum of 50,000 reads per cell would be sufficient [135] , while testing on a significantly large number of cells would ensure that rare subpopulations do not get missed out. one such application in infectious disease studies would be the systemic characterization of immune cell populations in response to an infection, wherein a large number of cells has to be screened in order to encompass the extensive diversity of b and t cells [136] . on the other hand, for studies which the main goal is to obtain a high resolution readout of the transcriptome for a small number of cells, 1,000,000 reads per cell would be a reasonable estimate [33] . in typical bulk analysis, multiple biological and technical replicates can be performed in order to ensure the reproducibility of data. however, for single-cell experiments, particularly for scrna-seq, there are two main issues to contend with. firstly, measurements typically have high technical variability as replicate measurements cannot be performed on the same cell, which is lysed as part of the rna extraction process. secondly, the resulting single-cell data are typically noisy due to technical variations from the multitude of steps in scrna-seq, as well as biological variation stemming from cell heterogeneity [137] . as such, great care has to be taken at each step of the scrna-seq workflow (i.e., sample preparation, library preparation and sequencing, data analysis) to minimize such technical variability and batch effects. one of the major sources of such variability arises from the initial sample preparation process. regardless of how the cells are dissociated, purified, or enriched, cell expression is likely to change in response to the stress induced from these processes. to minimize such undesired changes which might affect downstream data analysis, the sample preparation protocol should be optimized iteratively for each cell type [138] . to reduce technical variability, one common method would be to spike = 0 in known quantities of synthetic rna into the samples as controls to normalize read counts prior to data analysis [139] . a recent advancement in such rna spike-in normalization methodology would be the bearscc (bayesian ercc assessment of robustness of single-cell clusters), which generates simulated technical replicates based on the readout signal variation from spike-in measurements [140] . an alternative to rna spike-ins would be the use of unique molecular identifiers (umis) incorporated into the primers during reverse transcription, which essentially act as unique barcodes that allows the identification and subsequent tracking of transcribed mrna. then, the resulting data can be normalized against the umi levels to account for amplification bias during the library generation step [141] . however, both rna spike-in and umi have their own set of limitations to consider; rna spike-ins are unsuitable for protocols that utilize poly-t priming and template switching, and since they are typically used in large amounts relative to the endogenous rna, they could potentially occupy a lot of reads. protocols utilizing umi need to ensure that library sequencing is sufficiently deep to cover all umi transcripts; otherwise, there will be a risk of incorrectly quantifying the initial sample rna [142] . another source of error for scrna-seq comes from batch effects, which are brought about by unavoidable variations between batches of experimental runs due to changes in environmental conditions, temperature, reagent lot, etc. in response, several computational methods have been developed to mitigate said batch effects from the scrna-seq data. for example, one of the more commonly used batch-effect correction methods, combat, utilizes an empirical bayesian framework that removes batch effects via a linear model, which factors in both the mean and variance of the scrna-seq data [143] . for a more in-depth study on the comparative performance between the various batch-effect correction methods, we urge readers to consult a recent study by tran et al. [144] . while single-cell platforms have indeed come a long way in the past two decades, the plethora of existing techniques still face a few general concerns that could present themselves as opportunities for development in the near future. one of the inherent challenges in single-cell studies stems from the simple fact that the total amount of biological material present in a single cell is pretty limited and as a result, the resulting data are typically noisy from multiple biological and technical sources. making sense of the data requires downstream data pre-processing and analysis, which are non-trivial components of the workflow that limits the accessibility of such studies to groups with the essential background. additionally, with the increasing number and complexity of parameters at which single-cell assays are being performed, the curse of dimensionality is a pertinent problem that still requires further examination [133] . another concern for single-cell platforms would be inter-experiment variability, as mentioned in the previous section. single-cell technologies innately have high measurement sensitivity and thus are more susceptible to variations in results obtained from technical replicates, and methods to bioinformatically correct for such differences are required. coupled with the fact that single-cell studies are typically expensive and therefore sample sizes are small, ensuring that results are comparable between each sample becomes an even more important issue. finally, the inability to maintain viable cells after analysis, particularly for high-throughput methods such as flow cytometry or scrna-seq, gives rise to a couple of problems. firstly, a majority of the conventional single-cell studies are limited to a single time point of study, following which the cells are discarded. secondly, the irrecoverability of the cells makes it difficult to integrate back-to-back assays, which required measuring different parameters. as such, improvements in cell handling to improve cell viability would be invaluable in obtain multi-parametric datasets required for a more holistic understanding of cellular behavior. to date, the applications of single-cell technology have revolutionized our understanding of host-pathogen interactions. while many studies have focused on immune cells from the blood, the study of immune responses in the context of solid tissues or foci of infection (in both acute and chronic disease phases) is important to understand the local context of host-pathogen interaction. for this, techniques allowing the integration of spatial information with other single-cell technologies will be useful. for instance, imaging mass cytometry has been used to obtain quantitative information on 32 proteins at a spatial resolution of 1 µm [145] . this is done by systematically ablating a formalin-fixed tissue sample spatially line by line. the increased number of markers allows the fine distinction of cell subsets and activation states, providing valuable information on cellular roles in immune effector function or immunopathogenesis. it may even be possible to simultaneously obtain information on specific dna and rna targets via in situ hybridization. similarly, several techniques have been recently developed to obtain simultaneous spatial and transcriptomic data, including multiplexed error-robust fish (merfish) [146] , laser capture microdissection sequencing (lcm-sequencing) [147] , tomo-seq [148] , slide-seq [149] , and spatial transcriptomics [150] . these techniques may similarly be helpful in infectious disease to better define the interplay of immune cells, susceptible cells, stromal cells, and pathogens. another important gap that remains to be bridged is the ability to comprehensively access the state of a single cell across time. both immune and infection processes are highly dynamic, but because most of the single-cell technologies listed above are destructive, changes over time must be assessed either by careful time-point studies, or by assuming the presence of a range of cells in a population that represent early and late stages of the process (e.g., cellular activation or infection stage). with the advent of microfluidic devices that can immobilize single cells for continued study, the same cell can be assessed at multiple points for longitudinal study. in addition to typical proteomic marker analyses and rna or dna in situ hybridization techniques with live cell imaging, it is already possible even to measure more complex phenotypes such as bioenergy metabolism [151] . since microfluidic devices are also used for 3d organoid growth to simulate in vivo conditions, it is conceivable that future developments in technology will allow similar types of information to be collected in the context of organoids. this will represent one approximation toward high-dimensional in vivo data, which remains impossible with current methods. most of the single-cell studies reviewed in this article are based on end-point assays that are destructive and can therefore only measure a single time point of these single-cell targets. however, several recent studies outside the sphere of infectious disease have highlighted time as a prominent variable that influences the level of heterogeneity in host cells, immune cells, and pathogens. to that end, microfluidic platforms that enable the automated and precise control of media and reagents are ideal for performing such dynamic studies. for example, wu et al. [152] , using a customized microwell-microvalve system, performed a continuous measurement of a disintegrin and metalloproteinases (adams) and matrix metalloproteinase (mmps) secretions by single hepg2 cells upon a phorbol 12-myristate 13-acetate (pma) challenge. using their microfluidic platform, heterogenous changes in the secretion rates of adam and mmps were observed in response to pma stimulation, which may be used to predict hepg2 cell fates. in another recent study, a microfluidic platform that combined mutation visualization (mv) and microfluidic mutation accumulation (µma) enabled real-time tracing of mutations of single bacteria [153] . evidently, the utilization of microfluidic technology could enable high temporal resolution single-cell studies suited for uncovering the pathophysiology of infectious diseases. the advantages offered by microfluidics technologies include the efficient capture and compartmentalization of single cells, the precise control of fluid exchange, and ensuring a viable microenvironment for cell survival. these characteristics enable the dynamic study of large populations of single cells in parallel, which may eventually provide us with a more comprehensive understanding of the causes and effects of single-cell and single-pathogen heterogeneity. through the various applications of single-cell technology, we have gained a more thorough understanding of infectious disease pathophysiology at an unprecedented resolution. revealing the heterogeneity within populations of pathogens has allowed a finer dissection of virulence factors, and similarly, heterogeneity within populations of infected cells has given us a deeper understanding of host immune defenses. in addition, the high-dimensional single-cell information that can be collected even from primary cells has allowed us to identify rare but important cell subtypes, and it has shed light on the complex interplay between the different cells of the immune system. with the advent of antibody and t cell-based therapeutics, and antibody-based diagnostics, the contributions of single-cell technology to the high-throughput identification of candidate b and t cell receptor sequences that are target-specific have also accelerated the development of new therapeutics and diagnostics for both newly emerging and existing diseases. the adoption of single-cell technologies is likely also to revolutionize clinical studies for both drugs and vaccines, given its immense potential for biomarker discovery. with the field of single-cell technology only just taking off in the last decade, there remain vast prospects in both the increased adoption of existing technologies and the development of new technologies. the authors declare no conflict of interest. covid-19 coronavirus pandemic trade set to plunge as covid-19 pandemic upends global economy microbiology by numbers history of the discovery of the malaria parasites and their vectors the use of single-cell rna-seq to understand virus-host interactions breaking the population barrier by single cell analysis: one host against one pathogen single-cell analysis and stochastic modelling unveil large cell-to-cell variability in influenza a virus infection extreme heterogeneity of influenza virus infection in single cells high-throughput single-cell kinetics of virus infections in the presence of defective interfering particles single-cell virus sequencing of influenza infections that trigger innate immunity single-cell analysis of rna virus infection identifies multiple genetically diverse viral genomes within single infectious units single-cell rna-seq ties macrophage polarization to growth rate of intracellular salmonella pathogen cell-to-cell variability drives heterogeneity in host immune responses phenotypic variation of salmonella in host tissues delays eradication by antimicrobial chemotherapy single-cell analysis of the impact of host cell heterogeneity on infection with foot-and-mouth disease virus nile virus-inclusive single-cell rna sequencing reveals heterogeneity in the type i interferon response within single cells single-cell virology: on-chip investigation of viral infection dynamics single-cell rna-seq reveals transcriptional heterogeneity in latent and reactivated hiv-infected cells expression analysis highlights axl as a candidate zika virus entry receptor in neural stem cells zika virus disrupts phospho-tbk1 localization and mitosis in human neuroepithelial stem cells and radial glia sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structure, function, and antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues single-cell rna-sequencing of herpes simplex virus 1-infected cells connects nrf2 activation to an antiviral program dissection of influenza infection in vivo by single-cell rna sequencing single-cell transcriptional dynamics of flavivirus infection fast and accurate calling of germline and somatic variants systematic comparative analysis of single-nucleotide variant detection methods from single-cell rna sequencing data estimating the allele-specific expression of snvs from 10× genomics single-cell rna-sequencing data trajectory-based differential expression analysis for single-cell sequencing data identifying correlations between expressed snvs and gene expression using rna-sequencing data single-cell rna-seq reveals dynamic paracrine control of cellular variation single-cell transcriptomics of blood reveals a natural killer cell subset depletion in tuberculosis integrated single-cell analysis of multicellular immune dynamics during hyperacute hiv-1 infection virus-inclusive single-cell rna sequencing reveals the molecular signature of progression to severe dengue single cell immune profiling of dengue virus patients reveals intact immune responses to zika virus with enrichment of innate immune signatures high-dimensional cytof analysis of dengue virus-infected human dcs reveals distinct viral signatures highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands sequential secretion analysis of the same single cells reveals distinct effector response dynamics dependent on the initial basal state identification and characterization of hiv-specific resident memory cd8+ t cells in human lymphoid tissue single-cell rna-seq reveals tox as a key regulator of cd8+ t cell persistence in chronic infection comprehensive innate immune profiling of chikungunya virus infection in pediatric cases broad immune activation underlies shared set point signatures for vaccine responsiveness in healthy individuals and disease activity in patients with lupus host-based peripheral blood gene expression analysis for diagnosis of infectious diseases exploiting genomes for vaccine design reverse vaccinology 2.0: human immunology instructs vaccine antigen design 3-correlates of protection correlates of protection induced by vaccination methods for measuring t-cell memory to vaccination: from mouse to man updates on immunologic correlates of vaccine-induced protection systems vaccinology: probing humanity's diverse immune systems with vaccines systems biology approach predicts immunogenicity of the yellow fever vaccine in humans tracking the immune response with single-cell genomics dissecting the heterogeneity of denv vaccine-elicited cellular immunity using single-cell rna sequencing and metabolic profiling tcr sequencing paired with massively parallel 3' rna-seq reveals clonotypic t cell signatures antibodies: a laboratory manual an optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus efficient methods to isolate human monoclonal antibodies from memory b cells and plasma cells analysis of a clonal lineage of hiv-1 envelope v2/v3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors staged induction of hiv-1 glycan-dependent broadly neutralizing antibodies a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins reference values for b cell subpopulations from infancy to adulthood a method for identification of hiv gp140 binding memory b cells in human blood isolation and characterization of antigen-specific plasmablasts using a novel flow cytometry-based ig capture assay high-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array a microengraving method for rapid selection of single cells producing antigen-specific antibodies screening individual hybridomas by microengraving to discover monoclonal antibodies profiling antibody responses by multiparametric analysis of primary b cells exploiting highly ordered subnanoliter volume microcapillaries as microtools for the analysis of antibody producing cells smart-seq2 for sensitive full-length transcriptome profiling in single cells high-throughput mapping of b cell receptor sequences to antigen specificity phage antibodies: filamentous phage displaying antibody variable domains functional interrogation and mining of natively paired human v h: v l antibody repertoires therapeutic antibodies directed at g protein-coupled receptors identification of human neutralizing antibodies that bind to complex epitopes on dengue virions nature of nonfunctional envelope proteins on the surface of human immunodeficiency virus type 1 stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1 human antibody responses to hiv type 1 glycoprotein 41 cloned in phage display libraries suggest three major epitopes are recognized and give evidence for conserved antibody motifs in antigen binding initial b-cell responses to transmitted human immunodeficiency virus type 1: virion-binding immunoglobulin m (igm) and igg antibodies followed by plasma anti-gp41 antibodies with ineffective control of initial viremia systems serology: profiling vaccine induced humoral immunity against hiv functional single-cell hybridoma screening using droplet-based microfluidics single-cell analysis deepens antibody discovery high-throughput screening of antibiotic-resistant bacteria in picodroplets platforms for antibiotic discovery a new antibiotic kills pathogens without detectable resistance simplified cytometry for routine monitoring of infectious diseases methodology and application of flow cytometry for investigation of human malaria parasites cd4 immunophenotyping in hiv infection analysis of the phenotype of mycobacterium tuberculosis-specific cd4+ t cells to discriminate latent from active tuberculosis in hiv-uninfected and hiv-infected individuals fluorescence in situ hybridization (fish) in the microbiological diagnostic routine laboratory: a review a novel fluorescence in situ hybridization test for rapid pathogen identification in positive blood cultures situ hybridization (fish) assays for diagnosing malaria in endemic areas fluorescence in situ hybridization, a complementary molecular tool for the clinical diagnosis of infectious diseases by intracellular and fastidious bacteria fish-flow, a protocol for the concurrent detection of mrna and protein in single cells using fluorescence in situ hybridization and flow cytometry a novel single-cell fish-flow assay identifies effector memory cd4 + t cells as a major niche for hiv-1 transcription in hiv-infected patients accelerated bacterial detection in blood culture by enhanced acoustic flow cytometry (afc) following peptide nucleic acid fluorescence from chip-in-a-lab to lab-on-a-chip: a portable coulter counter using a modular platform a portable image-based cytometer for rapid malaria detection and quantification cellular immunity monitoring in long-duration spaceflights based on an automatic miniature flow cytometer improving the accessibility and efficiency of point-of-care diagnostics services in low-and middle-income countries: lean and agile supply chain management estimates of the global, regional, and national morbidity, mortality, and aetiologies of diarrhoea in 195 countries: a systematic analysis for the global burden of disease study field-portable microfluidics-based imaging flow cytometer a flow cytometry-based submicron-sized bacterial detection system using a movable virtual wall rapid quantification of pathogenic salmonella typhimurium and total bacteria in eggs by nano-flow cytometry point-of-care testing for infectious diseases: diversity, complexity, and barriers in low-and middle-income countries microfluidic single-cell analysis-toward integration and total on-chip analysis scaling by shrinking: empowering single-cell 'omics' with microfluidic devices digital assays part i: partitioning statistics and digital pcr integrative single-cell analysis advances in rapid identification and susceptibility testing of bacteria in the clinical microbiology laboratory: implications for patient care and antimicrobial stewardship programs developmental roadmap for antimicrobial susceptibility testing systems rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital lamp quantification in clinical samples gravity-driven microfluidic assay for digital enumeration of bacteria and for antibiotic susceptibility testing digital antimicrobial susceptibility testing using the millidrop technology the alphabet soup of hiv reservoir markers high-throughput characterization of hiv-1 reservoir reactivation using a single-cell-in-droplet pcr assay single-cell characterization of viral translation-competent reservoirs in hiv-infected individuals dielectric characterization of plasmodium falciparum-infected red blood cells using microfluidic impedance cytometry developments in label-free microfluidic methods for single-cell analysis and sorting analysis of parasitic protozoa at the single-cell level using microfluidic impedance cytometry notingher, i. induction and measurement of the early stage of a host-parasite interaction using a combined optical trapping and raman microspectroscopy system rapid quantification of vesicular stomatitis virus in vero cells using laser force cytology an optofluidic imaging system to measure the biophysical signature of single waterborne bacteria. lab. a chip malaria detection using inertial microfluidics. lab. a chip a continuous-flow acoustofluidic cytometer for single-cell mechanotyping characterization and sorting of cells based on stiffness contrast in a microfluidic channel current best practices in single-cell rna-seq analysis: a tutorial low-coverage single-cell mrna sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex immune cell profiling of covid-19 patients in the recovery stageby single-cell sequencing accounting for technical noise in single-cell rna-seq experiments experimental considerations for single-cell rna sequencing approaches. front a test metric for assessing single-cell rna-seq batch correction bearscc determines robustness of single-cell clusters using simulated technical replicates quantitative single-cell rna-seq with unique molecular identifiers how to design a single-cell rna-sequencing experiment: pitfalls, challenges and perspectives adjusting batch effects in microarray expression data using empirical bayes methods a benchmark of batch-effect correction methods for single-cell rna sequencing data highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry rna imaging. spatially resolved, highly multiplexed rna profiling in single cells laser capture microscopy coupled with smart-seq2 for precise spatial transcriptomic profiling genome-wide rna tomography in the zebrafish embryo slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution visualization and analysis of gene expression in tissue sections by spatial transcriptomics a platform for high-throughput bioenergy production phenotype characterization in single cells high-throughput protease activity cytometry reveals dose-dependent heterogeneity in pma-mediated adam17 activation real-time visualization of mutations and their fitness effects in single bacteria this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-295494-wal0gtrs authors: limeres posse, jacobo; diz dios, pedro; scully, crispian title: infection transmission by saliva and the paradoxical protective role of saliva date: 2017-07-31 journal: saliva protection and transmissible diseases doi: 10.1016/b978-0-12-813681-2.00001-9 sha: doc_id: 295494 cord_uid: wal0gtrs saliva is produced by both major (parotid and submandibular and sublingual) and minor (located in the mouth) glands, with different constituents and properties between the two groups. in the mouth saliva is a colorless, odorless, tasteless, watery liquid containing 99% water and 1% organic and inorganic substances and dissolved gases, mainly oxygen and carbon dioxide. salivary constituents can be grouped into proteins (e.g., amylase and lysozyme), organic molecules (e.g., urea, lipids, and glucose mainly), and electrolytes (e.g., sodium, calcium, chlorine, and phosphates). cellular elements such as epithelial cells, leukocytes and various hormones, and vitamins have also been detected. the composition of saliva is modified, depending on factors such as secreted amount, circadian rhythm, duration and nature of stimuli, diet, and medication intake, among others. infection transmission by saliva and the paradoxical protective role of saliva saliva is produced by both major (parotid and submandibular and sublingual) and minor (located in the mouth) glands, with different constituents and properties between the two groups. in the mouth saliva is a colorless, odorless, tasteless, watery liquid containing 99% water and 1% organic and inorganic substances and dissolved gases, mainly oxygen and carbon dioxide. salivary constituents can be grouped into proteins (e.g., amylase and lysozyme), organic molecules (e.g., urea, lipids, and glucose mainly), and electrolytes (e.g., sodium, calcium, chlorine, and phosphates). 1 cellular elements such as epithelial cells, leucocytes and various hormones, and vitamins have also been detected. the composition of saliva is modified, depending on factors such as secreted amount, circadian rhythm, duration and nature of stimuli, diet, and medication intake, among others. despite this heterogeneous composition, from the functional point of view saliva has to be considered as a unique biological fluid, and not as the sum of its biochemical components. 2, 3 salivary secretion and maintenance of a film of saliva on oral surfaces is dependent upon nerve-mediated, reflex salivary gland secretion mainly stimulated by taste. the afferent arm is mainly activated by stimulation of chemoreceptors (located in the taste buds) and mechanoreceptors (located in the periodontal ligament). 4 olfaction, mental processes, and stretch of the stomach are weak stimuli. impulses affecting secretion depending on the emotional state are carried by afferent cranial nerves v, vii, ix, and x to the cns salivary nuclei (salivation center) in the medulla oblongata. the efferent part of the reflex is mainly parasympathetic. the cranial nerve vii provides control of the submandibular, sublingual, and minor glands, whereas the cranial nerve ix controls the parotid glands. the flow of saliva is enhanced by sympathetic innervation, which promotes contraction of muscle fibers around the salivary ducts. 5 autonomic nerves also have an important role in both gland development and function. 6 a dry mouth is a common experience where there is fear. saliva may be secreted in the absence of exogenous stimuli, then referred to as the resting or unstimulated salivary flow. in the resting state 70% of saliva is secreted by the submandibular and sublingual glands. when stimulated, the parotids provide most of the saliva and flow can increase by up to fivefold. on average, in healthy nonmedicated adults, the unstimulated and chewing-stimulated salivary flow rates are about 0.3 and 1.5 ml/min respectively, 1 but the range is wide and the limits of normality in all age groups and both genders are considerable. the normal daily production of saliva varies from 700 ml to 1.5 l. a decrease in the daily production of saliva below 500 ml/ day is termed hyposecretion or hyposialia. 7 sialorrhea, hypersialia, hypersalivation, and ptyalism are terms used to describe salivary flow above the limits of the normal. 8 saliva plays a central role in oral health monitoring, regulating and maintaining the integrity of the oral hard and soft tissues. 1 it lubricates and cleans the oral cavity, possesses antibacterial, antiviral, and antifungal properties, buffers the ph, helps in chewing, speech, swallowing, and digestion, promotes taste, and contributes to the maintenance and remineralization of teeth. 9 moreover, it may be useful in the diagnosis of various diseases. 10 the characterization by proteomic approachesof more than 1000 salivary proteins and peptides-has allowed the identification of new salivary markers in oncology, salivary gland dysfunction, sjögren's syndrome, systemic sclerosis, psychiatric and neurological diseases, and dental and periodontal pathology. 11à13 the infectivity of microorganisms can depend on the infective load, virulence, with some, such as the notorious norovirus, being extremely contagious and able to survive weeks on surfaces and fomites. 14 the detection and continuous shedding of infectious agents in saliva does not necessarily mean transmission by this route. factors including the microorganism load, the existence of specific receptors on oral epithelial cells, and host defenses may play an important defensive role. 15 moreover, blood contamination in saliva-often invisible to the eye-is not uncommon mainly among active smokers 16 and individuals with poor oral health status, those with gingivitis or periodontitis, 17 and those with certain infectious diseases including human immunodeficiency virus (hiv) infection. 18 saliva contact can cause overt concern when using utensils such as cutlery or oral health devices, or if kissing a person with an infectious disease. however, the apparent absence of obvious disease does not guarantee the absence of infection or infective agents in saliva (or other body fluids): many diseases (especially viral) can be incubating or be subclinical (causing no or nonspecific symptoms or signs). intimate mucosal contacts, particularly where there are epithelial breaches or substances that may impede salivary defenses (e.g., other body fluids), predispose to infection transmission. kissing is not exclusive to humans or primates, though it may have different connotations in different species. 19 theories to explain kissing behavior consider it to have an origin in social and sexual interactions, premastication of foods for newborns or even the intentional transfer of microorganisms to promote immunity. 20 kissing is seen in most human cultures, 20 and often is part of daily behavior, playing important roles in building and maintaining interpersonal relationships 21,22 and in partner selection. 23 there are, however, huge intercultural differences related to kissing; this being considered an acceptable behavior in some cultures but totally offensive in others. for example, social kissing is an accepted form of salutation in the mediterranean and latin cultures, in muslim-majority societies governed by religious law there are strict taboos about whom one can kiss, or people from some areas in sudan refuse to kiss because they fear having their soul stolen through kissing. 24 in general, kissing is considered by many of the public to have few or no serious health implications. different types of kissing are evident and the type of kissing may well be relevant with respect to the transmission of microorganisms, as it not only determines the capacity of the kiss to spread infectious diseases, 25 but it can also have a bearing on the chemoprophylaxis strategy to be used in "kissing contacts" in certain situations (e.g., during an outbreak of meningococcal disease). 26 "air kissing" is a cheek-to-cheek approximation; "osculum" is when the lips make contact with the body, usually the cheeks; "basium kiss" consists of mutual approximation of the lips with the mouth closed, exercising light pressure; and finally, there is the "saviolum kiss" in which, in addition to lip contact, the tongue is inserted into the opposite person's mouth ("french kissing," "passionate kissing," deep kissing," "active kissing," or "intimate kissing"). 27 finally "kiss of life" refers to direct, intense, and recurrent lip contact during mouth-to-mouth resuscitation-the therapy of choice for cardiorespiratory arrest in the community. couples may exchange an average of 5 ml of saliva during active kissing, 28 making this an activity that could favor the transmission of infectious diseases. evidence for person-to-person transmission by kissing is limited to a few microorganisms and even this evidence can be often based on only weak scientific evidence. published studies are heterogeneous, from isolated case reports of kissing as a "possible" cause of transmission of diseases (e.g., hiv infection) 29 to studies analyzing the inhibitory activity of the saliva on specific microorganisms (e.g., herpes simplex). 30 few studies have been designed specifically to demonstrate the degree of infectivity if any of kissing, but one study showed it does not efficiently spread common cold infection by rhinoviruses. 31 studies on the risks from mouth-to-mouth ventilation without barrier devices 32 demonstrated isolated cases of transmission of tuberculosis, 33 herpes simplex infection, 34 shigellosis, 35 salmonellosis, 36 and meningococcal infection. 37 despite this, evidence for infection transmission by kissing is not strong, so this does not justify philemaphobia (morbid fear of kissing). paradoxically, it has even been suggested that kissing could be an evolutionary adaptation to protect against some neonatal infections (e.g., cytomegalovirus). 38 in reality, saliva may also have a protective role, and many animals, even humans, instinctively lick wounds-an act that may be defensive-possibly via histatins mainly. 39, 40 saliva also contains an array of factors which facilitate protection ( adequate salivary flow has a cleansing action and saliva also contains potentially protective constituents (table 1 .1). 40à42 antimicrobial proteins can arise from epithelial cells, innate immune, and other cells and can modulate the microbial flora in the mouth. for example, viruses such as noroviruses are affected by host genetic factors 43 including histoblood group antigens (hbgas) (i.e., the abo blood group, the lewis phenotype, and the secretor status). salivary proteins which can be protective at least against certain agents, include scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), mucins, histatins, and human neutrophil defensins. the protein gp340-formerly salivary agglutinin-aggregates a variety of bacteria and can function as a specific inhibitor of hiv-1 and influenza a. 41 salivary mucins muc5b and muc7 reduce the attachment and biofilm formation of streptococcus mutans by keeping bacteria in the planktonic state. 44 several studies have shown that salivary mucins induce phenotypic changes in candida albicans at the level of mrna transcription, which downregulate genes necessary for hyphal development and some virulence factors. 45 saliva also contains an array of other protective proteins including tissue factor, growth factorsit has been reported that saliva inhibits oral transmission of hiv through kissing, dental treatment, biting, and aerosolization; both crude saliva and mucins muc5b and muc7 inhibit hiv-1 activity, probably because they trap or aggregate the virus and prevent its entry into host cells. 46 slpi is also important. hantaviruses are also sensitive to the antiviral actions of mucins, 47 and sialic acid type molecules have high activity against human influenza viruses. 48 histatins provide the first line of defense against c. albicans 49 and other fungi. 50 cystatin may inhibit coronaviruses. 51 moreover, saliva mediates antibody-dependent cell-mediated cytotoxicity as in hiv-1infected individuals 52 and can regulate specific humoral defense mechanisms against microorganisms including cryptococcus neoformans 53 or paracoccidioides. 54 oral carriage of microorganisms and infections are more likely where there is hyposalivation and/or immunoincompetence-and so infections may be more prevalent in neonates who lack acquired immunity, or where immunity wanes such as in older or patients with immunocompromising conditions (e.g., malignant disease and its treatment, hiv/aids, or corticosteroid therapy)-particularly where the load of infecting agents is high or the microbe is virulent. • specific saliva protection against oral bacteria saliva has a mechanical flushing action and there are innate immune defenses and complex interactions with microorganisms. 55, 56 for example, the gene dmbt1 (deleted in malignant brain tumor 1) encodes antimicrobial proteins involved in mucosal innate immunity, and salivary dmbt1 glycoprotein (gp340) and salivary agglutinin (dmbt1(sag)) glycoproteins which are identical, agglutinate s. mutans and some other gram-positive bacteria, as well as several gram-negative bacteria. 57 some of the salivary components can change with disease; e.g., higher interleukin (il)-6/ il-1β, secretory iga, and lower lysozyme, and histatins 1 and 5 have been found in hepatic cirrhosis. 58 the innate and acquired immune defenses in saliva persist even after removal of lymphoid tissue in tonsillectomy: serum-derived antimicrobial proteins (myeloperoxidase, lactoferrin, igg) remain in high concentrations in whole saliva with no effect on the numbers of oral cariogenic s. mutans or on the total aerobic flora. 59 • specific saliva protection against bacteria such as pseudomonas aeruginosa pseudomonas aeruginosa often colonizes the airways in cystic fibrosis. p. aeruginosa binds to oral and bronchial epithelial cells, 60,61 by pili and fimbriae which promote adherence to glycosphingolipid adhesins asialo-gm1 on surfaces of host epithelia and phagocytes such as polymorphonuclear leukocytes. 62à64 failure to isolate pathogenic organisms consistently from the upper airways in all patients with positive sputum argues against a local epithelial factor predisposing to bacterial colonization 65 and also suggests that defensive processes are in play. p. aeruginosa are aggregated by saliva. the sero-mucous products of the submandibular gland have a greater role than the serous secretions of the parotids and are possibly responsible for the differences in oral colonization by p. aeruginosa in different subjects. 66 the low-molecular-weight mucin (mg2) of human submandibularàsublingual saliva, and neutral cystatin, bind to pili. 67 p. aeruginosa interactions with s. aureus may be predicated on the formation of mg2àsecretory iga antibody complex, which may facilitate clearance from the oral cavity. 68 interbacterial adherence between strains of p. aeruginosa with oral actinomyces viscosus indigenous to the human mouth and with strains of streptococcus pyogenes, and streptococcus agalactiae, appear to involve galactosyl-binding adhesins. 69 most oral viridans streptococci have potentially bacteriocin-like activity against p. aeruginosa. 70 • specific saliva protection against viruses such as hiv saliva may also be inhibitory to hiv. though complete inactivation may require 30 minutes of exposure, saliva may inhibit hiv-1. 71à76 a main protective mechanism of saliva may be the inactivation of hiv-transmitting leukocytes by the hypotonicity of saliva 77 and the oral transmission of hiv by seminal and other fluids introduced into the mouth may be due to their isotonicity overcoming the inactivation of hiv by isotonic saliva. 78 hiv transmission across mucosae involves complex mechanisms and the oral mucosal epithelia mucosa is less permissive for hiv replication than other sites (e.g., vagina/cervix and anal/rectal). 79 innate immunity plays a role in protection. 80 viral reception appears to involve both cd4 (cluster of differentiation 4) and a co-receptor-particularly ccr5 (chemokine receptor type 5). 81 the mhc appears to have a role in hiv-1 control, particularly the hla complex p5 (hcp5) and human leukocyte antigen-c (hla-c) and this may explain the occurrence of "elite supressor" patients. 82 the scavenger receptor protein gp340-encoded by the dmbt1 gene-interacts with surfactant proteins (sp-d), and both gp340 and sp-d can individually and together interact and agglutinate some viruses and dmbt1(gp340) binds to a variety of other host proteins, including serum and secretory iga, c1q, lactoferrin, muc5b, and trefoil factor 2 (tff2), all molecules involved in innate immunity and/or wound healing. 57 the protein gp340 appears to facilitate hiv transmission across genital but not oral mucosa. 83à86 acquired immunity might confer some protection in re-exposures: immunization with an hiv peptide may produce hiv-inhibitory antibodies in saliva. 87 various glycoproteins may also be inhibitory to hiv. crude saliva and salivary mucins muc5b and muc7 (both from hiv-positive people and uninfected controls) can inhibit hiv-1. 46, 88 other glycoproteins may also be implicated. 89, 90 slpi may have an important hiv-inhibitory role, 91,92 as might human β-defensins (hbds) from the epithelium. 93, 94 saliva may also mediate antibody-dependent cytotoxicity against hiv. 52 other viruses as. e.g., h5n1 influenza virus are particularly susceptible to human saliva, which may play a role in its infectivity and transmissibility. 48 many salivary antibacterial proteins have antiviral activity, typically against specific pathogens. 41 antiviral activities of saliva against influenza a virus (iav) and hiv differ both in terms of specific glandular secretions and the inhibitory proteins. whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibits iav, whereas only submandibular/sublingual secretions are inhibitory to hiv. among salivary proteins, scavenger receptor cysteine-rich glycoprotein 340 (gp340), muc5b, histatins, and human neutrophil defensins at concentrations present in whole saliva inhibit iav, while acidic proline-rich proteins and amylase have no activity nor do several less abundant salivary proteins (e.g., thrombospondin or serum slpi). 95 gp340 interacts with surfactant proteins a and d (sp-d) and can interact and agglutinate iva virus and also binds to proteins involved in innate immunity and/or wound healing, including serum and secretory iga, c1q, lactoferrin, muc5b and tff2. 57 salivary gp340 can antagonize sp-d antiviral activities-which may be relevant to the effects of aspiration of oral contents on sp-d-mediated lung functions. 96 other components responsible for antiviral activity on influenza virus, in particular swine origin influenza a virus (s-oiv), include an α-2-macroglobulin (a2m) and an a2m-like protein. 97 salivary glycoproteins which have significant roles against iva also include lectins (e.g., mal-ii and sna). 98 muc5b inhibits iav by presenting a sialic acid ligand for the viral hemagglutinin. 95 other sialic acidàcontaining molecules may be effective against human influenza viruses more so than against h5n1. 48 • specific saliva protection against fungi such as candida spp. both innate immunity and cell-mediated immune response are involved in defenses against fungal infections. saliva has a mechanical defense action and components including secretory immunoglobulin a, lactoferrin, and polymorphonuclear leukocyte (pmnl) superoxide are protective. 99 a low, stimulated salivary flow rate-not a low, unstimulated flow rate-is associated with candida spp. carriage. 100 salivary components mediate microbial attachment to oral surfaces and interact with planktonic microbial surfaces to facilitate agglutination often mediated by lectin-like proteins that bind to glycan motifs on salivary glycoproteins and help eliminate pathogens. antimicrobial peptides in saliva appear to play a crucial role in the regulation of oral candida growth. oral candidiasis may be associated with salivary gland hypofunction and decreases of salivary lactoferrin, secretory immunoglobulin a, β-defensin 1, and β-defensin 2 antibacterial proteins. 101 histatins are basic histidine-rich cationic proteins present in saliva that provide the first line of defense against oral candidiasis-an important antimicrobial is histatin 5 (hst 5), 102,103 which shows potent and selective antifungal activity and with the carrier molecule spermidine which, by binding to fungal cell wall proteins (ssa1/2) and glycans, significantly enhances c. albicans killing. 49 histatins effectively kill c. albicans, c. glabrata, and c. krusei, and histatin 3 acts against c. dubliniensis. 104 other antimicrobial proteins include calprotectin, 105 cystatin sa1, 106 and β-defensin 2, 107 deficiencies of which predispose to chronic candidiasis. salivary lysozyme can also be protective. 108 the development of candidiasis in hiv-infected patients could be a consequence of inefficient lysozyme and lactoferrin concentrations and of decreased cooperation between innate and adaptive immune systems. 109 the vast majority of candida isolates appear to succumb to nonspecific host immune mediators 110 but innate immunity alone is unable to stop yeast expansion in hiv-infected patients. 111 c. albicans-secreted aspartyl proteinase (sap1-sap8) and phospholipase b (plb1 and plb2) genes are expressed during both infection and carriage of candida spp. the differential expression of these hydrolytic enzyme genes correlates the expression of specific candida spp. virulence genes with active candidiasis and anatomical location. 112 salivary anti-somatic, anti-sap2, and anti-sap6 antibodies are not efficient in limiting candidal infection 113 and although hiv-infected patients have a high mucosal response against c. albicans virulence antigens, such as somatic antigen, sap1, and sap6, 114 this is not totally protective. defensins such as α-defensin (human neutrophil peptides, hnps) and β-defensin-2 (hbd-2) peptides can have antifungal and cytotoxic activities. 115 defensins that exhibit antibacterial, antifungal, and antiviral properties are a component of the innate immune response. β-defensins (hbd-1) are cationic antimicrobial peptides encoded by the defb1 gene expressed in oral epithelia that may have a major role in mediating and/or contributing to susceptibility to candidiasis. 116 nitric oxide (no) is involved in host resistance to infection with c. albicans at least in animal models. il-4 is associated with resistance to oral candidiasis and suggests that no is involved in controlling colonization of the oral mucosal surface with c. albicans. 117 oral epithelial cells may play a role in innate resistance against candidiasis. 118 host defenses against c. albicans include epithelial cell defenses and innate and specific immune mechanisms. cell-mediated immunity by th1-type cd4 1 t-cells is important for protection against mucosal infections, and pmnls are important for protection against systemic infections. 119 when cd8(1) t-cell migration is inhibited by reduced tissue e-cadherin, there is susceptibility to infection which supports a role for cd8(1) t cells in host defense against oropharyngeal candidiasis. 120 fungal pattern recognition receptors such as c-type lectin receptors trigger protective t-helper (th)17 responses in the oral mucosa. the th17/il-17 axis is vital for immunity to fungi, especially c. albicans. the inflammatory cytokine il-17 induces tumor necrosis factor (tnf)-α, and interleukins il-1β and il-6. 121 a systemic immune response involving t-helper 1 (th1) cells with the production of tnfα and ifn-γ is seen in patients with oral candidiasis. 122 th17 cells may act through il-17, to confer defenses via neutrophils and antimicrobial factors. 123 oral epithelial cells also are involved in local host defenses against c. albicans infections via ifn-γ induced il-18. 124 biofilms, some 15% of which may be due to dual candida spp., contribute to the pathogenesis of oral candidiasis, 125 biofilm formation of c. albicans appearing to be modulated by salivary and dietary factors. 126 c. albicans hyphal wall protein 1 (hwp1) mrna is present in candidiasis regardless of symptoms, implicating hyphal and possibly pseudohyphal forms in mucosal carriage as well as disease. 127 overall, hwp1 and hyphal growth forms appear to be important factors in both benign and invasive interactions of c. albicans with human hosts. transmission of infection by saliva may be prevented or minimized by avoidance of exposure, by good oral hygiene (plaque removal), and by the use of the various substances such as some mouthwashes, and probiotics that may inhibit salivary microorganisms. 128,129 bacterial pathogens have been identified in salivary samples by specific antibody reactivity, antigen detection, or via pcr, including escherichia coli, mycobacterium tuberculosis, treponema pallidum, and a wide range of streptococcus spp. more than 20 viruses have also been detected; these include a number of herpes viruses, hepatitis viruses, human immunodeficiency viruses, papillomavirus, influenza virus, or poliovirus. nonviral and nonbacterial infectious agents including fungi and protozoa are also detectable, usually by antibodies to these infectious agents. recognition of the components of the oral microbiota can help in the prediction of the onset, progression, and prognosis of oral and systemic diseases. tests for these pathogens are currently under development. omics methods, such as 16s rrna sequencing, metagenomics, and metabolomics, can play an essential role to explore microbial community and its metabolite production, without the biases of microbial culture. saliva contains many antibacterial, antiviral, and antifungal agents which modulate the oral microbial flora. consequently, detection and shedding of infectious agents in saliva does not necessarily mean transmission by this route. anyway, the presence of these pathogens in saliva is particularly important in immunosuppressed patients in whom infections can result fatal. moreover, the defensive ability of saliva against emerging infectious diseases caused by new or previously unrecognized microorganisms remains unknown. saliva in health and disease: an appraisal and update salivary glands and saliva saliva: its secretion, composition and functions saliva and gastrointestinal functions of taste, mastication, swallowing and digestion sialorrhea: a management challenge salivary secretion: mechanism and neural regulation the physiology and biochemistry of the mouth physiological factors affecting salivary flow rate, oral sugar clearance, and the sensation of dry mouth in man saliva as a diagnostic fluid potential applications of human saliva as diagnostic fluid clinical and diagnostic utility of saliva as a noninvasive diagnostic fluid: a systematic review transmission of salmonella via mouth-tomouth resuscitation some recollections of the meningococcal diseases. the first harry f. dowling lecture kissing as an evolutionary adaptation to protect against human cytomegalovirus-like teratogenesis histatins are the major wound-closure stimulating factors in human saliva as identified in a cell culture assay saliva and wound healing antiviral activities in human saliva the functions of human saliva: a review sponsored by the world workshop on oral medicine vi host-pathogen co-evolution and glycan interactions salivary mucin as related to oral streptococcus mutans in elderly people mucins suppress virulence traits of candida albicans the role of crude human saliva and purified salivary muc5b and muc7 mucins in the inhibition of human immunodeficiency virus type 1 in an inhibition assay antiviral effect of human saliva against hantavirus sialic acid content in human saliva and anti-influenza activity against human and avian influenza viruses how does it kill? understanding the candidacidal mechanism of salivary histatin 5 the antifungal activity of human parotid secretion is species-specific cystatin d, a natural salivary cysteine protease inhibitor, inhibits coronavirus replication at its physiologic concentration oral herpes simplex virus type 2 reactivation in hivpositive and -negative men humoral defense mechanisms in cryptococcosis: substances in normal human serum, saliva, and cerebrospinal fluid affecting the growth of cryptococcus neoformans levels of specific antigen (gp43), specific antibodies, and antigen-antibody complexes in saliva and serum of paracoccidioidomycosis patients saliva-bacterium interactions in oral microbial ecology influence of saliva on the oral microbiota review: gp-340/dmbt1 in mucosal innate immunity salivary microbiota reflects changes in gut microbiota in cirrhosis with hepatic encephalopathy salivary antimicrobial proteins and mutans streptococci in tonsillectomized children glycosphingolipid receptors for pseudomonas aeruginosa identification of enterococcus faecalis and pseudomonas aeruginosa on and in implants in individuals with peri-implant disease: a crosssectional study fimbriae (pili): molecular basis of pseudomonas aeruginosa adherence human buccal epithelial cell receptors of pseudomonas aeruginosa: identification of glycoproteins with pilus binding activity inhibition of pilusmediated adhesion of pseudomonas aeruginosa to human buccal epithelial cells by monoclonal antibodies directed against pili bacterial reservoirs in cystic fibrosis whole, submandibular, and parotid saliva-mediated aggregation of pseudomonas aeruginosa in cystic fibrosis binding of the pili of pseudomonas aeruginosa to a low-molecular-weight mucin and neutral cystatin of human submandibular-sublingual saliva interaction of a salivary mucin-secretory immunoglobulin a complex with mucosal pathogens interbacterial adherence between actinomyces viscosus and strains of streptococcus pyogenes, streptococcus agalactiae, and pseudomonas aeruginosa )o(2) produced by viridans group streptococci may contribute to inhibition of methicillin-resistant staphylococcus aureus colonization of oral cavities in newborns components of saliva inactivate human immunodeficiency virus saliva inhibits hiv-1 infectivity salivary inhibition of hiv-1 infectivity: functional properties and distribution in men, women, and children in vitro inhibition of hiv-1 infectivity by human salivas hiv recovery from saliva before and after dental treatment: inhibitors may have critical role in viral inactivation anti-infectivity activity of human salivary secretions toward human immunodeficiency virus why is hiv rarely transmitted by oral secretions? saliva can disrupt orally shed, infected leukocytes oral transmission of human immunodeficiency virus by infected seminal fluid and milk: a novel mechanism mucosal transmission of human immunodeficiency virus innate immunity in hiv-1 infection: epithelial and non-specific host factors of mucosal immunity-a workshop report hiv-1 receptors and cell tropism the role of protective hcp5 and hla-c associated polymorphisms in the control of hiv-1 replication in a subset of elite suppressors the n-terminal srcr-sid domain of gp-340 interacts with hiv type 1 gp120 sequences and inhibits viral infection hiv envelope binding by macrophage-expressed gp340 promotes hiv-1 infection gp340 promotes transcytosis of human immunodeficiency virus type 1 in genital tract-derived cell lines and primary endocervical tissue periluminal distribution of hiv-binding target cells and gp340 in the oral, cervical and sigmoid/rectal mucosae: a mapping study neutralization of hiv-1 by secretory iga induced by oral immunization with a new macromolecular multicomponent peptide vaccine candidate the role of crude saliva and purified salivary mucins in the inhibition of the human immunodeficiency virus type 1 identification of a cd36-related thrombospondin 1-binding domain in hiv-1 envelope glycoprotein gp120: relationship to hiv-1-specific inhibitory factors in human saliva human submandibular saliva inhibits human immunodeficiency virus type 1 infection by displacing envelope glycoprotein gp120 from the virus secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro inhibition of human immunodeficiency virus type 1 infectivity by secretory leukocyte protease inhibitor occurs prior to viral reverse transcription human beta-defensins suppress human immunodeficiency virus infection: potential role in mucosal protection role of human beta-defensins in hiv infection multiple components contribute to ability of saliva to inhibit influenza viruses salivary agglutinin and lung scavenger receptor cysteine-rich glycoprotein 340 have broad anti-influenza activities and interactions with surfactant protein d that vary according to donor source and sialylation the essentiality of alpha-2-macroglobulin in human salivary innate immunity against new h1n1 swine origin influenza a virus age-and sex-associated differences in the glycopatterns of human salivary glycoproteins and their roles against influenza a virus regulation of candida albicans growth and adhesion by saliva fungal load and candidiasis in sjogren's syndrome. oral surg oral med oral pathol oral radiol endod decreased excretion of antimicrobial proteins and peptides in saliva of patients with oral candidiasis susceptibility of candida albicans isolates from the oral cavities of hiv-positive patients to histatin-5 salivary histatin-5 and oral fungal colonisation in hiv 1 individuals susceptibility of candida dubliniensis to salivary histatin 3 salivary calprotectin levels are raised in patients with oral candidiasis or sjogren's syndrome but decreased by hiv infection autoimmunity and cystatin sa1 deficiency behind chronic mucocutaneous candidiasis in autoimmune polyendocrine syndrome type 1 new mechanism of oral immunity to mucosal candidiasis in hyper-ige syndrome salivary anticandidal activity and saliva composition in an hiv-infected cohort new sensitive method for the measurement of lysozyme and lactoferrin to explore mucosal innate immunity. part ii: time-resolved immunofluorometric assay used in hiv patients with oral candidiasis antifungal effects of lysozyme and lactoferrin against genetically similar, sequential candida albicans isolates from a human immunodeficiency virus-infected southern chinese cohort nonspecific secretory immunity in hiv-infected patients with oral candidiasis differential expression of candida albicans secreted aspartyl proteinase and phospholipase b genes in humans correlates with active oral and vaginal infections longitudinal study of anti-candida albicans mucosal immunity against aspartic proteinases in hiv-infected patients increased serum and salivary immunoglobulins against candida albicans in hiv-infected patients with oral candidiasis immunohistochemical study on expression of alpha-defensin and beta-defensin-2 in human buccal epithelia with candidiasis single-nucleotide polymorphisms (snps) in human beta-defensin 1: high-throughput snp assays and association with candida carriage in type i diabetics and nondiabetic controls nitric oxide-enhanced resistance to oral candidiasis growth inhibition of candida by human oral epithelial cells immunity to candida cd8 t cells and e-cadherin in host responses against oropharyngeal candidiasis rheumatoid arthritis patients exhibit impaired candida albicans-specific th17 responses microbiological and immunological features of oral candidiasis th17 cells and il-17 receptor signaling are essential for mucosal host defense against oral candidiasis involvement of interleukin-18 in the inflammatory response against oropharyngeal candidiasis characteristics of dual species candida biofilms on denture acrylic surfaces biofilm formation of candida albicans is variably affected by saliva and dietary sugars candida albicans hwp1 gene expression and host antibody responses in colonization and disease effect of xylitol and xylitol-fluoride lozenges on approximal caries development in high-caries-risk children salivary mutans streptococci and lactobacilli modulations in young children on consumption of probiotic ice-cream containing bifidobacterium lactis bb12 and lactobacillus acidophilus la5 key: cord-027859-citynr6c authors: p. shetty, nandini; s. shetty, prakash title: epidemiology of disease in the tropics date: 2020-06-22 journal: manson's tropical diseases doi: 10.1016/b978-1-4160-4470-3.50007-0 sha: doc_id: 27859 cord_uid: citynr6c nan the study of epidemiology in the tropics has undergone major changes since its infancy when it was largely a documentation of epidemics. it has now evolved into a dynamic phenomenon involving the ecology of the infectious agent, the host, reservoirs and vectors as well as the complex mechanisms concerned in the spread of infection and the extent to which this spread occurs. 1 similar concepts in the study of epidemiology apply to communicable as well as non-communicable diseases. the understanding of epidemiological principles has its origins in the study of the great epidemics. arguably, the most powerful example of this is the study of that ancient scourge of mankind, the so-called black death or plague. a study of any of the plague epidemics throughout history has all the factors that govern current epidemiological analysis: infectious agent, host, vector, reservoir, complex population dynamics including migration, famine, fi re and war; resulting in spread followed by quarantine and control. the world health report 1996: 'fighting disease, fostering development', states that infectious diseases are the world's leading cause of premature death. 2 infectious diseases account for 45% of deaths in low-income countries (figure 3 .1) and up to 63% of deaths in children under 4 years of age worldwide. africa and south-east asia carry the highest mortality due to infectious diseases (figure 3 .2). in addition, new and emerging infections pose a rising global threat (table 3 .1). no more than six deadly infectious diseases: pneumonia, tuberculosis, diarrhoeal diseases, malaria, measles and more recently, hiv/aids, account for half of all premature deaths, killing mostly children and young adults (figure 3 .3). acute respiratory infections (aris) are the leading cause of death of infectious aetiology, killing more than 4 million people a year, 1.9 million of which constitute children under the age of fi ve. 3 among the 42 countries of the world that carry 90% of the child mortality burden, 14-24% of the under-5 mortality is due to pneumonia and nearly 70% of this pneumonia mortality occurs in the africa and south and south-east asia regions. the majority of this burden is borne during early childhood, with the greatest risk from mortality occurring during the neonatal period. the global incidence of ari in children is estimated to be 154 million cases per year. 3 this range of infections, which includes pneumonia in its most serious form, accounts for more than 8% of the global burden of disease. pneumonia often affects children with low birth weight or those whose immune systems are weakened by malnutrition or other diseases. caused by different viruses or bacteria, ari is closely associated with poverty, overcrowding and unsanitary household conditions. several other factors seem to exacerbate the disease. exposure to tobacco smoke increases the risk of contracting these infections, and many studies implicate both indoor and outdoor air pollution. indoor air pollution has been the focus of particular concern: specifi cally, the soot and smoke associated with the burning of biomass fuels such as wood, coal, or dung. many people in the developing world, mostly in rural areas, rely on biomass fuels for heating or cooking. a cause-and-effect relationship between indoor air pollution and ari has been diffi cult to prove. even so, the world bank estimated in 1992 that switching to better fuels could halve the number of pneumonia deaths. 4 approaches to the management of childhood pneumonia in the tropics are hampered by lack of diagnostic facilities to identify the aetiological agent. the who has devised a simple algorithm for use in fi eld situations, by primary healthcare workers, using clinical criteria such as respiratory rate and indrawing of ribs to decide whether a child needs hospitalization. proper implementation of this strategy has been shown to reduce the mortality from childhood pneumonias by 25-50%. 5 however, implementation of community ari treatment programmes remains patchy and current rates of children with ari being taken to a health provider are ~40% in africa and south asia. in nearly half of the 81 countries with available data, less than 50% of the children with ari were taken to an appropriate healthcare provider. 5 the aids pandemic has emerged as the single most defi ning occurrence in the history of infectious diseases of the late twentieth and early twenty-fi rst centuries. according to the aids epidemic update of december 2005 (unaids and who), 6 the epidemiology of hiv in the tropics varies enormously from place to place (figure 3 latest estimates show some 8.3 million people (2 million adult women) were living with hiv in 2005, including the 1.1 million people who became newly infected in the past year. aids claimed some 520 000 lives in 2005. these estimates are in line with known risk behaviour in this region, where men account for the majority of injecting drug users, and are responsible for sexual transmission of hiv, largely through commercial sex. commercial sex accounts for a large part of the estimated 20% of hiv infections in china that are due to unprotected heterosexual contact. it also features in the transmission of the virus among men who have sex with men: a recent survey among male sex workers in the southern city of shenzhen found that 5% of them were hivpositive. however, it is the potential overlap between commercial sex and injecting drug use that is likely to become the main driver of china's epidemic. diverse epidemics are underway in india, where, in 2003, an estimated 5.1 million indians were living with hiv. although levels of hiv infection prevalence appear to have stabilized in some states (such as tamil nadu, andhra pradesh, karnataka and maharashtra), it is still increasing in at-risk population groups in several other states. as a result, overall hiv prevalence has continued to rise. a signifi cant proportion of new infections is occurring in women who are married and who have been infected by husbands who (either currently or in the past) frequented sex workers. commercial sex (along with injecting drug use, in the states of nagaland and tamil nadu) serves as a major driver of the epidemics in most parts of india. hiv surveillance in 2003 found 14% of commercial sex workers in karnataka (26% in the city of mysore) and 19% in andhra pradesh were infected with hiv. the wellknown achievements among sex workers of kolkata's sonagachi red-light area (in west bengal, india) have shown that safe sex programmes that empower sex workers can curb the spread of hiv. condom use in sonagachi has risen as high as 85% and hiv prevalence among commercial sex workers declined to fewer than the combination of high levels of risk behaviour and limited knowledge about aids among drug injectors and sex workers in pakistan favours the rapid spread of hiv, and new data suggest that the country could be on the verge of serious hiv epidemics. most countries in asia still have the opportunity to prevent major epidemics. bangladesh, where national adult hiv prevalence is well below 1%, began initiating hiv prevention programmes early in its epidemic. indonesia is on the brink of a rapidly worsening aids epidemic. with risk behaviour among injecting drug users common, a mainly drug-injection epidemic is already spreading into remote parts of this archipelago. in malaysia, approximately 52 000 people were living with hiv in 2004, the vast majority of them young men (aged 20-29 years), of whom approximately 75% were injecting drug users. after peaking at 3% in 1997, national adult hiv prevalence in cambodia fell by one-third, to 1.9% in 2003. the reasons for this are two-fold: increasing mortality and a decline in hiv incidence due to changes in risk behaviour. thailand has been widely hailed as one of the success stories in the response to aids. by 2003, estimated national adult hiv prevalence had dropped to its lowest level ever, approximately 1.5%. however, thailand's epidemic is far from over; infection levels in the most at-risk populations are much higher: just over 10% of brothel-based female sex workers were hiv-infected in 2003, as were 45% of injecting drug users who attended treatment clinics. while cambodia and thailand in the 1990s were planning and introducing strategies to reverse the spread of hiv, another serious epidemic was gaining ground in neighbouring myanmar. there, limited prevention efforts led to hiv spreading freely. consequently, myanmar has one of the most serious aids epidemics in the region, with hiv prevalence among pregnant women estimated at 1.8% in 2004. the main hiv-related risk for many of the women now living with the virus was to have had unprotected sex with husbands or boyfriends who had been infected while injecting drugs or buying sex. in japan, the number of reported annual hiv cases has more than doubled since 1994-1995, and reached 780 in 2004; the highest number to date. much of this trend is due to increasing infections among men who have sex with men. prevalence of hiv remains low in the philippines and lao pdr. the advance of aids in the middle east and north africa has continued, with latest estimates showing that 67 000 people became infected with hiv in 2005. approximately 510 000 people are living with hiv in this region. an estimated 58 000 adults and children died of aids-related conditions in 2005. although hiv surveillance remains weak in this region, more comprehensive information is available in some countries (including algeria, libya, morocco, somalia and sudan). available evidence reveals trends of increasing hiv infections (especially in younger age groups) in such countries as algeria, libya, morocco and somalia. the main mode of hiv transmission in this region is unprotected sexual contact, although injecting drug use is becoming an increasingly important factor (and is the predominant mode of infection in at least two countries: iran and libya). infections as a result of contaminated blood products, blood transfusions or a lack of infection control measures in healthcare settings are generally on the decline. by far the worst-affected country in this region is sudan. in a country with a long history of civil confl ict and forced displacement, internally displaced persons face higher rates of hiv infection. for instance, among displaced pregnant women seeking antenatal care in khartoum in 2004, hiv prevalence of 1.6% was found compared with under 0.3% for other pregnant women. the epidemic in latin america is a complex mosaic of transmission patterns in which hiv continues to spread through male-tomale sex, sex between men and women, and injecting drug use. sub-saharan africa has just over 10% of the world's population, but is home to more than 60% of all people living with hiv -25. the rights and status of women and young girls deserve special attention. around the world -from south of the sahara in africa and asia to europe, latin america and the pacifi c -an increasing number of women are being infected with hiv. it is often women with little or no income who are most at risk. widespread inequalities including political, social, cultural and human security factors also exacerbate the situation for women and girls. in several southern african countries, more than three quarters of all young people living with hiv are women, while in sub-saharan africa overall, young women between 15 and 24 years old are at least three times more likely to be hiv-positive than young men (figure 3 .5). 6 in many countries, marriage and women's own fi delity are not enough to protect them against hiv infection. among women surveyed in harare (zimbabwe), durban and soweto (south africa), 66% reported having one lifetime partner, 79% had abstained from sex at least until the age of 17 (roughly the average age of fi rst sexual encounter in most countries in the world). yet, 40% of the young women were hiv-positive. many had been infected despite staying faithful to one partner. diarrhoea remains one of the most common diseases affl icting children under 5 years of age and accounts for considerable mortality in childhood. estimates from studies published between 1992 and 2000 show that there was a median of 3.2 episodes of diarrhoea per child-year in developing countries. this indicates little change from previously described incidences. estimates of mortality revealed that 4.9 children per 1000/year in these countries died as a result of diarrhoeal illness in the fi rst 5 years of life, a decline from the previous estimates of 5.6-3.6 per 1000/year. the decrease was most pronounced in children aged under one year. despite improving trends in mortality rates, diarrhoea accounted for a median of 21% of all deaths of children aged under 5 years in developing countries, being responsible for 2.5 million deaths per year. there has not been a concurrent decrease in morbidity rates attributable to diarrhoea. as population growth is focused in the poorest areas, the total morbidity component of the disease burden is greater than previously. 7 diarrhoea remains a disease of poverty affl icting malnourished children in crowded and contaminated environments. efforts to immunize children against measles, provide safe water and adequate sanitation facilities, and to encourage mothers to exclusively breast-feed infants through to 6 months of age can blunt an increase in diarrhoea morbidity and mortality. preventive strategies to limit the transmission of diarrhoeal disease need to go hand in hand with national diarrhoea disease control programmes that concentrate on effective diarrhoea case management and the prevention of dehydration. 8 the factors contributing to childhood mortality and morbidity due to diarrhoea are described in table 3 .2. 8 studies in asia and africa have clearly shown that establishment of an oral rehydration therapy (ort) unit with training of hospital staff can signifi cantly reduce diarrhoea case fatality rates. for instance, at mama yemo hospital in kinshasa, zaire, there was a 69% decline in diarrhoea deaths after creation of an ort unit. 9 in may 2002, the world health organization and the united nations children's fund recommended that the formulation of oral rehydration solution (ors) for treatment of patients with diarrhoea be changed to one with a reduced osmolarity and that safety of the new formulation, particularly development of symptomatic hyponatremia, be monitored. 10 a total of 53 280 patients, including 22 536 children younger than 60 months, were monitored at the dhaka and matlab hospitals, bangladesh. the risk of symptoms associated with hyponatraemia in patients diarrhoeal disease treated with the reduced osmolarity ors was found to be minimal and did not increase with the change in formulation. 10 changing patterns in the epidemiology of diarrhoea have been noted in many studies. in matlab, bangladesh, acute watery diarrhoea accounted for 34% of diarrhoea deaths in under-fi ves, while the remaining 66% were related to dysentery or persistent diarrhoea and malnutrition. this pattern was age dependent, with acute watery deaths being more important in infancy, being associated with 40% of deaths, and less important in later childhood, being associated with 10% of deaths. 11 rotavirus is the most common cause of severe diarrhoeal disease in infants and young children all over the world, and an important public health problem, particularly in developing countries where 600 000 deaths each year are associated with this infection. more than 125 million cases of diarrhoea each year are attributed to rotavirus. in tropical developing countries, rotavirus disease occurs either throughout the year or in the cold dry season. almost all children are already infected by the age of 3-5 years. although the infection is usually mild, severe disease may rapidly result in life-threatening dehydration if not appropriately treated. natural infection protects children against subsequent severe disease. globally, four serotypes are responsible for the majority of rotaviral disease, but additional serotypes are prevalent in some countries. the only control measure likely to have a signifi cant impact on the incidence of severe disease is vaccination. since the withdrawal from the market of the tetravalent rhesus-human reassortant vaccine (rotashield, wyeth laboratories) because of an association with intussusception, ruling out such a risk has become critical for the licensure and universal use of any new rotavirus vaccine. recent studies have shown that two oral doses of the live attenuated g1p [8] human rotavirus vaccine were highly effi cacious in protecting infants against severe rotavirus gastroenteritis, signifi cantly reduced the rate of severe gastroenteritis from any cause, and were not associated with the increased risk of intussusception linked with the previous vaccine. 12 man is both the reservoir and natural host of shigella, the commonest cause of dysentery in the tropics. the most severe infections are caused by the s. dysenteriae type 1 (also known as shiga's bacillus); it is also the only serotype implicated in epidemics. infection is by the faecal-oral route and is usually spread by personto-person transmission. it takes only 10-100 shigella organisms to produce dysentery, a low infectious dose, whereas 1 million to 10 million organisms may need to be swallowed to cause cholera. during the late 1960s, shiga's bacillus was responsible for a series of devastating epidemics of dysentery in latin america, asia and africa. in 1967, it was detected in the mexican-guatemalan border area and spread into much of central america. an estimated half a million cases, with 20 000 deaths, were reported in the region between 1967 and 1971. in some villages the case fatality rate was as high as 15%; delayed diagnosis and incorrect treatment may have been responsible for this high death rate. one particularly disturbing feature was the resistance of the bacteria to the most commonly used antibacterial drugs: sulfonamides, tetracycline and chloramphenicol. 13 serious epidemics due to the multiple-drug resistant s. dysenteriae type 1 have occurred recently in bangladesh, somalia, south india, burma, sri lanka, nepal, bhutan, rwanda and zaire. west bengal in india has always been an endemic area for bacillary dysentery. preventive measures include boiling or chlorination of drinking water, covering faeces with soil, protecting food from fl ies, avoiding eating exposed raw vegetables and cut fruits, and washing hands with soap and water before eating and after using the latrine. however, such measures are not easy to implement in most areas. consequently epidemics take their own course and subside only gradually. 13 tuberculosis tuberculosis (tb) is the leading cause of death associated with infectious diseases globally. the incidence of tb will continue to increase substantially worldwide because of the interaction between the tb and hiv epidemics. 14 in many developing countries, tb is mainly a disease of young adults affecting carers and wage-earners in a household, thus placing a huge economic burden on society as a whole. chemotherapy, if properly used, can reduce the burden of tb in the community, but because of the fragile structure of treatment programmes in many countries tb cases are not completely cured and patients remain infectious for a much longer time. another important consequence of poor treatment compliance is development of drug resistance in many developing countries. resistance to tuberculosis drugs is probably present everywhere in the world. 15 worldwide attention was focused on south africa, when in october 2006 a research project publicized a deadly outbreak of xdr-tb in the small town of tugela ferry in kwazulu-natal. xdr-tb is the abbreviation for extensively drug-resistant tuberculosis (tb). this strain of mycobacterium tuberculosis is resistant to fi rstand second-line drugs, and treatment options are seriously limited. of 536 tb patients at the church of scotland hospital, which serves a rural area with high hiv rates, some 221 were found to have multi-drug resistance and of these, 53 were diagnosed with xdr-tb. some 52 of these patients died, most within 25 days of diagnosis. of the 53 patients, 44 had been tested for hiv and all 44 were found to be hiv-positive. the patients were receiving antiretrovirals and responding well to hiv-related treatment, but they died of xdr-tb. since the study, 10 more patients have been diagnosed with xdr-tb in kwazulu-natal. only three of them are still alive (see: http://www.who.int/tb/xdr/xdr_jan.pdf). directly observed treatment, short course (dots), is the most effective strategy available for controlling the tb epidemic today. dots uses sound technology and packages it with good management practices for widespread use through the existing primary healthcare network. it has proven to be a successful, innovative approach to tb control in countries such as china, bangladesh, vietnam, peru and countries of west africa. however, new challenges to the implementation of dots include health sector reforms, the worsening hiv epidemic, and the emergence of drugresistant strains of tb. the technical, logistical, operational and political aspects of dots work together to ensure its success and applicability in a wide variety of contexts. 14 million africans who die from malaria each year, most are children under 5 years of age. in addition to acute disease episodes and deaths in africa, malaria also contributes signifi cantly to anaemia in children and pregnant women, adverse birth outcomes such as spontaneous abortion, stillbirth, premature delivery and low birth weight, and overall child mortality. the disease is estimated to be responsible for an estimated average annual reduction of 1.3% in economic growth for those countries with the highest burden. 16 of the four species of plasmodium that infect humans: p. falciparum, p. vivax, p. malariae and p. ovale, p. falciparum causes most of the severe disease and deaths attributable to malaria and is most prevalent in africa south of the sahara and in certain areas of south-east asia and the western pacifi c (figure 3.7) . the second most common malaria species, p. vivax, is rarely fatal and is commonly found in most of asia, and in parts of the americas, europe and north africa. there are over 40 species of anopheline mosquitoes that transmit human malaria, which differ in their transmission potential. the most competent and effi cient malaria vector, anopheles gambiae, occurs exclusively in africa and is also one of the most diffi cult to control. climatic conditions determine the presence or absence of anopheline vectors. tropical areas of the world have the best combination of adequate rainfall, temperature and humidity allowing for breeding and survival of anophelines. in areas of malaria transmission where sustained vector control is required, insecticide treated nets are the principal strategy for malaria prevention. all countries in africa south of the sahara, the majority of asian malaria-endemic countries and some american countries have adopted insecticide treated nets as a key malaria control strategy. 16 one of the greatest challenges facing malaria control worldwide is the spread and intensifi cation of parasite resistance to antimalarial drugs. the limited number of such drugs has led to increasing diffi culties in the development of antimalarial drug policies and adequate disease management. 16 resistance of p. falciparum to chloroquine is now common in practically all malariaendemic countries of africa (figure 3.7) , especially in east africa. resistance to sulfadoxine/pyrimethamine, the main alternative to chloroquine, is widespread in south-east asia and south america. mefl oquine resistance is now common in the border areas of thailand with cambodia and myanmar. parasite sensitivity to quinine is declining in several other countries of south-east asia and in the amazon region, where it has been used in combination with tetracycline for the treatment of uncomplicated malaria. 16 in response to widespread resistance of p. falciparum to monotherapy with conventional antimalarial drugs such as chloroquine and sulfadoxine-pyrimethamine, who now recommends combination therapies as the treatment policy for falciparum malaria in all countries experiencing such resistance. the preferred combinations contain a derivative of the plant artemisia annua, which is presently cultivated mainly in china and vietnam. artemisininbased combination therapies (acts) are the most highly effi cacious treatment regimens now available. resistance of p. vivax to chloroquine has now been reported from indonesia (irian jaya), myanmar, papua new guinea and vanuatu. 17 urban and periurban malaria are on the increase in south asia and in many areas of africa. military confl icts and civil unrest, along with unfavourable ecological changes, have greatly contributed to malaria epidemics, as large numbers of unprotected, nonimmune and physically weakened refugees move into malarious areas. such population movements contribute to new malaria outbreaks and make epidemic-prone situations more explosive. 16 another disquieting factor is the re-emergence of malaria in areas where it had been eradicated (e.g. democratic people's republic of korea, republic of korea and tadjikistan), or its increase in countries where it was nearly eradicated (e.g. azerbaijan, northern iraq and turkey). current malaria epidemics in a majority of these countries are the result of a rapid deterioration of malaria prevention and control operations. climatic changes have also been implicated in the re-emergence of malaria. in the past 5 years, the worldwide incidence of malaria has quadrupled, infl uenced by changes in both land development and regional climate. in brazil, satellite images depict a 'fi sh bone' pattern where roads have opened the tropical forest to localized development. in these 'edge' areas malaria has resurged. temperature changes have encouraged a redistribution of the disease; malaria is now found at higher elevations in central africa and could threaten cities such as nairobi, kenya. this threat has been hypothesized to extend to temperate regions of the world that are now experiencing hotter summers year on year. 18 although substantial progress has been made in reducing measles deaths globally, in 2000 measles was estimated to be the fi fth leading cause of mortality worldwide for children aged <5 years. measles deaths occur disproportionately in africa and south-east asia. in 2000, the african region of who, with 10% of the world's population, accounted for 41% of estimated measles cases and 58% of measles deaths; the south-east asia region, with 25% of the world's population and 28% of measles cases, accounted for 26% of measles deaths. the burden of mortality in africa refl ects low routine vaccination coverage and high case-fatality ratios. in south-east asia, where vaccination coverage is slightly below average worldwide levels, the large population amplifi es the number of cases and deaths resulting from ongoing measles transmission. the overwhelming majority of measles deaths in 2000 occurred in countries eligible to receive fi nancial support from the global alliance for vaccines and immunization's vaccine fund (who, unpublished data 2003). the majority of measles deaths occur among young children living in poor countries with inadequate vaccination services. like human immunodefi ciency virus, malaria, and tuberculosis, measles can be considered a disease of poverty. however, unlike these diseases, measles can be prevented through vaccination. 19, 20 in much of the world, particularly sub-saharan africa, south-east asia, china and the pacifi c basin, infection with hepatitis b virus (hbv) is very widespread. the carrier rate in some of these populations may be as high as 10-20%. in developing countries most hepatitis b transmission occurs during the perinatal period. infection between children is another common route of infection; it is not uncommon to fi nd up to 90% of 15-year-olds have serological evidence of infection with hbv. intermediate levels of infection (2-7%) are seen in parts of the former soviet union, south asia, central america and the northern zones of south america. these high rates of infection lead to a high burden of disease, mainly from the clinical consequences of long-term carriage of the virus, which may include chronic hepatitis, cirrhosis and liver cancer. it has been estimated that hbv infection is the second most common cause of cancer deaths in the world (after tobacco consumption). in india hepatitis b is linked to 60% of cases of hepatocellular carcinoma and 80% of cases of cirrhosis of the liver. 21 on the basis of disease burden and the availability of safe and effective vaccines, the who recommended that by the end of the twentieth century, hepatitis b vaccine be incorporated into routine infant and childhood immunization programmes for all countries. the effi cacy of universal immunization has been shown in different countries, with striking reductions of the prevalence of hbv carriage in children. most important, hepatitis b vaccination can protect children against hepatocellular carcinoma and fulminant hepatitis, as has been shown in taiwan. nevertheless, the implementation of worldwide vaccination against hbv requires greater effort to overcome the social and economic hurdles. safe and effective antiviral treatments are available but are still far from ideal, a situation that, hopefully, will be improved soon. with hepatitis b immunization, the global control of hbv infection is possible by the end of the fi rst half of twenty-fi rst century. 22 tetanus is a vaccine-preventable disease that causes a total of 309 000 deaths annually. of particular concern is maternal and neonatal tetanus (mnt), which can be prevented through immunization of the mother in pregnancy. in 2000, neonatal tetanus alone was responsible for an estimated 200 000 deaths. in addition, an estimated 15 000-30 000 non-immunized women worldwide die each year from maternal tetanus that results from postpartum, postabortal or postsurgical wound infection with clostridium tetani. while the focus is on 57 priority countries, 90% of the neonatal tetanus deaths occur in 27 countries. unicef spearheaded the effort to eliminate mnt by the year 2005, with the support of numerous partners. mnt elimination is defi ned as less than one case of neonatal tetanus per 1000 live births at district level. the main strategies consist of promotion of clean delivery practices, immunization of women with a tetanus toxoid (tt) containing vaccine, and surveillance. maternal tetanus immunization is, in most developing countries, implemented as part of the routine immunization programme. however, large areas remain underserved, due to logistical, cultural, economical or other reasons. in order to achieve the target of mnt elimination by 2005, and to offer protection to women and children otherwise deprived from regular immunization services, countries are encouraged to adopt the high risk approach. this approach implies that, in addition to routine immunization of pregnant women, all women of child-bearing age living in high risk areas are targeted for immunization with three doses of a tetanus toxoid containing vaccine (tt or td). 23 by the end of 2007 vaccination against a range of bacterial and viral diseases is an integral part of communicable disease control worldwide. vaccination against a specifi c disease not only reduces the incidence of that disease, but it also reduces the social and economic burden of the disease on communities. very high immunization coverage can lead to complete blocking of transmission for many vaccinepreventable diseases. the worldwide eradication of smallpox and the near-eradication of polio from many countries provide excellent examples of the role of immunization in disease control. despite these advances many of the world's poorest countries do not have access to vaccines and these infections remain among the leading global causes of death. the special programme for research and training in tropical diseases (tdr) of the world health organization has designated several infectious diseases as 'neglected tropical diseases' (ntds) that disproportionately affl ict the poor and marginalized populations in the developing regions of sub-saharan africa, asia and the americas. 24 infectious diseases are considered as 'neglected' or 'orphan' diseases when there is a lack of effective, affordable, or easy to use drug treatments. as most patients with such diseases live in developing countries and are too poor to pay for drugs, the pharmaceutical industry has traditionally ignored these diseases. ntds cause an estimated 500 000 to 1 million deaths annually and cause a global disease burden equivalent to that of hiv-aids. who estimates that at least 1 billion people, i.e. onesixth of the world's population suffers from one or more neglected tropical diseases, while other estimates suggest the number to be much higher. some diseases affect individuals throughout their lives, causing a high degree of morbidity and physical disability and, in certain cases, gross disfi gurement. others are acute infections, with transient, severe and sometimes fatal outcomes. patients can face social stigmatization and abuse, which only add to the already heavy health burden. neglected tropical diseases are contrasted with the 'big three' diseases (hiv/aids, tuberculosis and malaria) which receive much more attention and funding. the current neglected diseases portfolio includes parasitic diseases of protozoan origin like kala-azar (leishmaniasis), african sleeping sickness (african trypanosomiasis) and chagas' disease (american trypanosomiasis) as well as those caused by helminths such as schistosomiasis, lymphatic fi lariasis, onchocerciasis (river blindness) and dracunculiasis (guinea worm). infestations due to soil transmitted helminths such as ascariasis, trichuriais and hookworm also belong to the latter category. other neglected diseases include those of bacterial origin such as leprosy, buruli ulcer and trachoma as well as those of viral origin like dengue fever which are vector-borne. even cholera and yellow fever are considered by some as ntds, while some include cysticercosis, hydatidosis and food-borne trematode infections. it is now believed that ramped up efforts against the 'big three', will yield far bigger dividends if they are coupled with concerted attack on ntds 25 . evidence now points to substantial geographical overlap between the neglected tropical diseases and the 'big three', suggesting that control of the neglected tropical diseases could become a powerful tool for effectively combating hiv/aids, tuberculosis, and malaria. 25 since 1991, resurgent and emerging infectious disease outbreaks have occurred worldwide. in addition, many diseases widely believed to be under control, such as cholera, dengue and diphtheria, have re-emerged in many areas or spread to new regions or populations throughout the world (figure 3.8) . 26 a growing population and increasing urbanization contribute to emerging infectious disease problems. in many parts of the world, urban population growth has been accompanied by overcrowding, poor hygiene, inadequate sanitation and unclean drinking water. urban development has also caused ecological damage. in these circumstances, certain disease-causing organisms and some of the vectors that transmit them have thrived, making it more likely that people will be infected with new or re-emerging pathogens. the existing public health infrastructure is already overtaxed and ill prepared to deal with new health threats. breakdown of public health measures due to civil unrest, war and the movement of refugees has also contributed to the re-emergence of infectious diseases (table 3. 3). 26 international travel and commerce have made it possible for pathogens to be quickly transported from one side of the globe to the other (figure 3.9) . 26 examples of new and resurgent infections include ebola, dengue fever, rift valley fever, diphtheria, cholera, nipah virus infection, west nile virus infection, severe acute respiratory syndrome (sars) and avian infl uenza. in 1976 ebola (named after the ebola river in zaire) fi rst emerged in sudan and the democratic republic of the congo (formerly zaire). ebola virus occurs as four distinct subtypes: zaã¯re, sudan, cã´te d'ivoire and reston. three subtypes, occurring in the democratic republic of the congo, sudan and cã´te d'ivoire, have been identifi ed as causing illness in humans. ebola haemorrhagic fever (ehf) is a febrile haemorrhagic illness which causes death in 50-90% of all clinically ill cases. the natural reservoir of the ebola virus is unknown despite extensive studies, but seems to reside in the rain forests on the african continent and in the western pacifi c. through the global prevalence of dengue and dengue haemorrhagic fever (dhf) has grown dramatically in recent decades. the disease is now endemic in more than 100 countries in africa, the americas, the eastern mediterranean, south-east asia and the western pacifi c. south-east asia and the western pacifi c are most seriously affected. some 2500 million people -two-fi fths of the world's population -are now at risk from dengue. who currently estimates there may be 50 million cases of dengue infection worldwide every year. in 2001 alone, there were more than 609 000 reported cases of dengue in the americas, of which 15 000 cases were dhf. this is greater than double the number of dengue cases which were recorded in the same region in 1995. not only is the number of cases increasing as the disease is spreading to new areas, but explosive outbreaks are occurring. in 2001, brazil reported over 390 000 cases including more than 670 cases of dhf. during epidemics of dengue, attack rates among the susceptible are often 40-50%, but may reach 80-90%. an estimated 500 000 cases of dhf require hospitalization each year, microbial adaptation changes in virulence and toxin production; development and change of drug resistance; microbes as co-factors in chronic diseases of whom a very large proportion are children. without proper treatment, dhf case fatality rates can exceed 20%. with modern intensive supportive therapy, such rates can be reduced to less than 1%. the spread of dengue is attributed to expanding geographical distribution of the four dengue viruses and of their mosquito vectors, the most important of which is the predominantly urban species aedes aegypti. a rapid rise in urban populations is bringing ever greater numbers of people into contact with this vector, especially in areas that are favourable for mosquito breeding, e.g. where household water storage is common and where solid waste disposal services are inadequate. 28 rift valley fever (rvf) is a zoonotic disease typically affecting sheep and cattle in africa. mosquitoes are the principal means by which rvf virus is transmitted among animals and to humans. following abnormally heavy rainfall in kenya and somalia in late 1997 and early 1998, rvf occurred over vast areas, producing disease in livestock and causing haemorrhagic fever and death among the human population. as of december 2006, who fi gures indicate that the outbreak continues to affect the north western provinces of kenya. in september 2000 who documented the fi rst ever rvf outbreak outside africa, in yemen and the kingdom of saudi arabia (ksa). rna sequencing of the virus from ksa indicated that it was similar to the rvf viruses isolated from east africa in 1998. a total of 1087 suspected cases were identifi ed, of which 121 (11%) persons died. of the 1087, 815 (75%) cases reported exposure to sick animals, handling an abortus or slaughtering animals in the week before onset of illness. 29 the vibrio responsible for the seventh pandemic, now in progress, is known as v. cholerae o1, biotype el tor. according to the who, it continues to spread in angola and sudan; more than 40 000 cases have been documented with over 1500 deaths: a case fatality rate of 3.5-4%. cholera (biotype el tor) broke out explosively in peru in 1991, after an absence of 100 years, and spread rapidly in central and south america, with recurrent epidemics in 1992 and 1993. from the onset of the epidemic in january 1991 to 1 september 1994, a total of 1 041 422 cases and 9642 deaths (overall case fatality rate 0.9%) were reported from countries in the western hemisphere to the pan american health organization. in december 1992, a large epidemic of a new strain of cholera v. cholerae 0139 began in south india, and spread rapidly through the subcontinent (figure 3.10) . this strain has changed its antigenic structure such that there is no existing immunity and all ages, even in endemic areas, are susceptible. the epidemic has continued to spread and v. cholerae o139 has been reported from 11 countries in south asia. because humans are the only reservoirs, survival of the cholera vibrios during interepidemic periods probably depends on low-level undiagnosed cases and transiently infected, asymptomatic individuals. recent studies have suggested that cholera vibrios can persist for some time in shellfi sh, algae or plankton in coastal regions of emerging and resurgent infectious diseases infected areas and it has been claimed that they can exist in a viable but non-culturable state. 30 in early 1999, health offi cials in malaysia and singapore investigated reports of febrile encephalitis and respiratory illnesses among workers who had been exposed to pigs. a previously unrecognized paramyxovirus (formerly known as hendra-like virus), now called nipah virus, was implicated by laboratory testing in many of these cases. as of april 1999, 257 cases of febrile encephalitis were reported to the malaysian ministry of health, including 100 deaths. laboratory results from 65 patients who died suggested recent nipah virus infection. the apparent source of infection among most human cases continues to be exposure to pigs. human-tohuman transmission of nipah virus has not been documented. outbreak control in malaysia has focused on culling pigs; approximately 890 000 pigs have been killed. other measures include a ban on transporting pigs within the country, education about contact with pigs, use of personal protective equipment among persons exposed to pigs, and a national surveillance and control system to detect and cull additional infected herds. 31 nipah virus cases and deaths have also been reported from bangladesh. since then, no more human cases have been reported. sars is due to infection with a newly identifi ed coronavirus named as sars-associated coronavirus (sars-cov). 32 the source of infection is likely to be a direct cross-species transmission from an animal reservoir. this is supported by the fact that the early sars cases in guangdong province had some history of exposure to live wild animals in markets serving the restaurant trade. animal traders working with animals in these markets had higher seroprevalence for sars coronavirus, though they did not report any illness compatible with sars. more importantly, sars-cov-like virus detected from some animal species had more than a 99% homology with human sars-cov. 32 the clinical course of sars varies from a mild upper respiratory tract illness, usually seen in young children, to respiratory failure which occurred in around 20-25% of mainly adult patients. as the disease progresses, patients start to develop shortness of breath. from the second week onwards, patients progress to respiratory failure and acute respiratory distress syndrome, often requiring intensive care. 32 in may 1997, a 3-year-old boy in hong kong contracted an infl uenza-like illness, was treated with salicylates, and died 12 days later with complications consistent with reye's syndrome. laboratory diagnosis included the isolation in cell culture of a virus that was identifi ed locally as infl uenza type a but could not be further characterized with reagents distributed for diagnosis of human infl uenza viruses. by august, further investigation with serological and molecular techniques in the netherlands and in the usa had confi rmed that the isolate was a/hong kong/156/97 (h5n1), which was very closely related to isolate a/chicken/hong kong/258/97 (h5n1). the latter virus was considered representative of those responsible for severe outbreaks of disease on three rural chicken farms in hong kong during march 1997, during which several thousand chickens had died. molecular analysis of the viral haemagglutinins showed a proteolytic cleavage site of the type found in highly pathogenic avian infl uenza viruses. by late december, the total number of confi rmed new human cases had climbed to 17, of which fi ve were fatal; the case fatality rates were 18% in children and 57% in adults older than 17 years. almost all laboratory evidence of infection was in patients who had been near live chickens (e.g. in marketplaces) in the days before onset of illness, which suggested direct transmission of virus from chicken to human rather than person-to-person spread. in december 1997, veterinary authorities began to slaughter all (1.6 million) chickens present in wholesale facilities or with vendors within hong kong, and importation of chickens from neighbouring areas was stopped. knowledge of how humans are infected, the real level of humanto-human transmission, the spectrum of disease presentation and the effectiveness of treatment remains scanty. human-to human transmission is known to have occurred, but there is no evidence that transmission has become more effi cient. all the human-tohuman infections with h5n1 to date seem not to have transmitted on further. therefore, although the case fatality rate for human infection remains high (around 57% for cases reported to who), it seems that h5n1 avian viruses remain poorly adapted to humans. 33 global prevalence studies (figure 3 .11) indicate that indonesia is currently the most active site of bird to human h5n1 transmission in the asia pacifi c region, and a large number of human cases have been detected here in 2005-06. china and cambodia have also reported human cases in 2006. in south asia (india and pakistan), there have only been sporadic reports of infection in poultry to date. in vietnam and thailand there have been offi cial reports of poultry outbreaks; these show a decline since 2006. surveillance in africa is especially weak, and there is evidence of widespread infection in domestic poultry in parts of north, west and central africa. prospects of control are bleak here because of weaknesses in veterinary services, and a number of competing animal and human health problems. the outbreaks in egypt have been well described. these involved both commercial and backyard fl ocks, with considerable impact on economic life and food security. it is probable that large numbers of people in african countries are at risk of h5n1 infection. if that virus had pandemic potential then a pandemic arising from africa must be considered a possibility. 33 non-infectious diseases take an enormous toll on lives and health worldwide. non-communicable diseases (ncds) account for nearly 60% of deaths globally, mostly due to heart disease, stroke, cancer, diabetes and lung diseases. the rapid rise of ncds represents one of the major health challenges to global development in the twenty-fi rst century and threatens the economic and social development of nations as well as the lives and health of millions of their subjects. in 1998 alone, ncds were estimated to have contributed to 31.7 million deaths globally and 43% of the global burden of disease. 34 until recently, it was believed that ncds were a minor or even non-existent problem in developing countries in the tropics. a recent analysis of mortality trends from ncds suggests that large increases in ncds have occurred in developing countries, 35 particularly those in rapid transition like china and india (table 3 .4). according to these estimates at least 40% of all deaths in the tropical developing countries are attributable to ncds, while in industrialized countries ncds account for 75% of all deaths. low-and middle-income countries suffer the greatest impact of ncds. the rapid increase in these diseases is seen disproportionately in poor and disadvantaged populations and is contributing to widening health gaps between and within countries. in 1998, of the total number of deaths attributable to ncds 77% occurred in developing countries, and of the disease burden they represent 85% was borne by low-and middle-income countries. 34 it has now been projected that, by 2020, ncds will account for almost three-quarters of all deaths worldwide, and that 71% of deaths due to ischaemic heart disease (ihd), 75% of deaths due to stroke, and 70% of deaths due to diabetes will occur in developing countries 36 and the number of people in the developing world with diabetes is expected to increase by more than 2.5-fold, from 84 million in 1995 to 228 million in 2025. 37 on a global basis, 60% of the burden of ncds will occur in developing countries and the rate at which it is increasing annually is unprecedented. the public health and economic implications of this phenomenon are staggering, and are already becoming apparent. it is important to recognize that these trends, indicative of an increase in ncds, may be partly confounded by factors such as an increase in life expectancy, a progressive reduction in deaths due to communicable diseases in adulthood, and improvements in case detection and reporting in the tropics. however, increase in the incidence of these chronic degenerative diseases is real. the complex range of determinants (below) that interact to determine the nature and course of this epidemic 38 needs to be understood in order to adopt preventive strategies to help developing societies in the tropics to deal with this burgeoning problem. the determinants of non-communicable diseases in developing societies are as follows: 1 â�¢ demographic changes in population â�¢ epidemiological transition â�¢ urbanization and internal migration â�¢ changes in dietary and food consumption patterns â�¢ lifestyle changes (changes in physical activity patterns, sociocultural milieu and stress as well as increased tobacco consumption) â�¢ adult-onset effects of low birth weight and the effects of early life programming â�¢ infections and their associations with chronic disease risk â�¢ effect of malnutrition and nutrient defi ciencies â�¢ poverty, inequalities and social exclusion â�¢ deleterious effects of environmental degradation â�¢ impacts of globalization. four of the most prominent ncds: cardiovascular disease, cancer, chronic obstructive pulmonary disease and diabetes, are linked to common preventable risk factors related to diet and lifestyle. these factors are tobacco use, unhealthy diet and lack of physical activity. interventions to prevent these diseases should focus on controlling these risk factors in an integrated manner and at the family and community level since the causal risk factors are deeply entrenched in the social and cultural framework of society. developing countries in the tropics have to recognize that the emerging accelerated epidemic of ncds is a cause for concern and that it needs to be dealt with as a national priority. they have to learn from the experience of industrialized and affl uent countries to tackle the emerging crisis of chronic diseases that they are likely to face in the near future. the emerging health burden of chronic disease affecting mainly the economically productive adult population will consume scarce resources. it is important, however, to realize that the poorer countries will be burdened even more in the long run, if attempts are not made to evolve and implement interventions to address these emerging health issues on an urgent basis. ensuring that health policies are aimed at tackling the 'double burden' of the continued existence of the huge burden of infectious/communicable diseases alongside the emerging epidemic of non-communicable diseases in developing countries of the tropics becomes a priority. 39 the world we live in is constantly changing. in the past 25 years, we have witnessed signifi cant progress in sustainable and technological development. however, increases in mass population movements, continuing civil unrest and deforestation have helped carry diseases into areas where they have never been seen before. this has been aided by the massive growth in international travel. effective medicines and control strategies are available to dra-matically reduce the deaths and suffering caused by communicable and non-communicable diseases. despite reduced global military spending many governments are failing to ensure that these strategies receive enough funding to succeed. who priorities for the control of infectious diseases in developing countries include childhood immunization, integrated management of childhood illnesses, use of the dots strategy to control tb, a package of interventions to control malaria, a package of interventions to prevent hiv/aids, access to essential drugs, and the overall strengthening of surveillance and health service delivery systems. over 10% of all preventable ill-health today is due to poor environmental quality-conditions such as bad housing, overcrowding, indoor air pollution, poor sanitation and unsafe water. the challenge of disease in the tropics has continued into the new millennium -never before have we been so well equipped to deal with disease threats. it remains for humankind to summon the collective will to pursue these challenges and break the chain of infection and disease. national and international surveillance of communicable diseases health report: fighting disease fostering development. geneva: world health organization acute respiratory infections. geneva: world health organization indoor air pollution energy and health for the poor estimate of global incidence of clinical pneumonia in children under fi ve years the global burden of diarrhoeal disease number evl-97-002. a global review of diarrhoeal disease control new parameters for evaluating oral rehydration therapy: one year's experience in a major urban hospital in zaire symptomatic hyponatremia during treatment of dehydrating diarrheal disease with reduced osmolarity oral rehydration solution diarrhoea mortality in rural bangladeshi children for the human rotavirus vaccine study group. safety and effi cacy of an attenuated vaccine against severe rotavirus gastroenteritis guidelines for the control of epidemics due to shigella dysenteriae 1. publication no. who/cdr/95.4. epidemiology of dysentery caused by shigella. geneva: world health organization global tuberculosis control-surveillance, planning financing, geneva: world health organization who/international union against tuberculosis and lung disease global project on anti-tuberculosis drug resistance surveillance. epidemiology of antituberculosis drug resistance (the global project on anti-tuberculosis drug resistance surveillance): an updated analysis geneva: world health organization climate, ecology and human health global burden of disease and risk factors. geneva: world health organization update: global measles control and mortality reduction -worldwide towards the elimination of hepatitis b: a guide to the implementation of national immunization programs in the developing world. the international task force on hepatitis b immunization. geneva: world health organization global control of hepatitis b virus infection tetanus in developing countries: an update on the maternal and neonatal tetanus elimination control of neglected tropical diseases (ntd) incorporating a rapid-impact package for neglected tropical diseases with programs for hiv/aids, tuberculosis, and malaria emerging infectious diseases review of state and federal diseases surveillance fact sheet: ebola haemorrhagic fever. fact sheet no. 103. geneva: world health organization report of the public health laboratories division. who collaborating centre for research and training in viral diagnostics national institute of health update: vibrio cholerae o1-western hemisphere, 1991-1994, and v. cholerae o139-asia update: outbreak of nipah virus: malaysia and singapore sars and emerging infectious diseases: a challenge to place global solidarity above national sovereignty world avian infl uenza update: h5n1 could become endemic in africa global strategy for the prevention and control of non-communicable diseases. geneva: world health organization global comparative assessments in the health sector. geneva: world health organization life in the 21st century: a vision for all. geneva: world health organization life course perspectives on coronary heart disease, stroke and diabetes: key issues and implications for policy and research diet and life-style and chronic non-communicable diseases: what determines the epidemic in developing societies? in: krishnaswami k, ed. nutrition research: current scenario and future trends the double burden of communicable and non-communicable diseases in developing countries key: cord-256459-6h358si5 authors: sharpstone, d; gazzard, b title: gastrointestinal manifestations of hiv infection date: 1996-08-10 journal: lancet doi: 10.1016/s0140-6736(96)01034-3 sha: doc_id: 256459 cord_uid: 6h358si5 the harrowing picture of emaciated terminally ill aids patients is a reminder of our lack of understanding of immunological mechanisms that normally control opportunistic infections. many gastrointestinal pathogens in patients with aids are resistant to treatment and lead inexorably to weight loss and death. although knowledge of the pathogenesis and clinical significance of weight loss has improved considerably, this has not yet led to a sustained effort to improve nutritional status during early stages of disease. most of the morbidity and mortality of late aids is associated with gastrointestinal disease. whilst oesophageal candidosis responds readily to treatment and cytomegalovirus enteritis may be cleared by antiviral agents, the two commonest protozoal pathogens-microsporidia and cryptosporidia-cannot be eradicated. these protozoa cause disruption of small-bowel villus architecture by unknown mechanisms and severe malabsorption, maldigestion, and diarrhoea. weight loss is a major contributor to death in most patients with aids and is commonly caused by protozoal gut infection. the host metabolic response to these infections is typical of starvation and patients are likely to respond to the provision of additional calories, ideally by the enteral route. by contrast, weight loss associated with other opportunistic infections-mycobacterium avium intracellulare and cytomegalovirus-is characterised by cachexia and is unlikely to respond to simple refeeding. the commonest cause of oesophageal symptoms is candidosis; odynophagia or dysphagia with oral candidosis is an aids-defining diagnosis and can be treated empirically with a systemic azole. the second most frequent cause is cytomegalovirus (cmv), which produces either diffuse oesophagitis or discrete ulceration. the aetiology of these lesions can be confirmed by biopsy of the centre of ulcerated areas. detection of cmv inclusion bodies is enhanced by immunoperoxidase staining. oesophagoscopy occasionally reveals vesicles typical of herpes simplex oesophagitis, diagnosed either by biopsy or smears from brushings showing typical giant cells. as many as 10% of oesophageal ulcers leading to dysphagia are idiopathic, possible causes being unknown opportunistic viruses or hiv itself. 1 these ulcers may respond to thalidomide. pathogenesis hiv was initially believed to cause intestinal symptoms since hiv-seropositive individuals free from intestinal pathogens commonly have diarrhoea. however, the frequency of "pathogen-negative diarrhoea" depends on the extent of the diagnostic investigations and the definition of diarrhoea. in a prospective study 2 of pathogen-negative diarrhoea, follow-up revealed an unsuspected pathogen in only a minority. most patients had low-volume diarrhoea that either resolved spontaneously or was controlled with antimotility agents, a response that accords with the features of irritable bowel syndrome. hiv-seropositive persons free from intestinal pathogens do have minor abnormalities of villus architecture, characteristically a mild villus atrophy associated with either crypt hypoplasia or hyperplasia. 3 these minor abnormalities are unlikely to cause diarrhoea since they occur in symptom-free individuals and are associated either with no or with very mild malabsorption of carbohydrates. 4 however, there is a consistent increase in small-bowel permeability in hiv-seropositive individuals; 5 this functional abnormality and the minor structural abnormalities of the small bowel mucosa may be due to the immunological changes produced by hiv infection of lamina propria lymphocytes. activated t cells cause villus atrophy in fetal gut explants, 5 and hiv causes activation of these cells. hiv can be identified in the lamina propria of both the large and small intestine but its detection or quantification is not related to diarrhoea. 6 whether hiv also infects small-bowel mucosal cells, as was initially shown by in-situ hybridisation, is unknown. 7 other possible mechanisms for villus damage include ingress of foreign antigens because of the increased permeability that sets in motion a vicious circle of release of cytokines and other inflammatory mediators. alternatively, bacterial overgrowth in the small intestine of hiv-seropositive patients, as reported in some small studies, might produce an inflammatory response and villus atrophy. 8 bacterial overgrowth might occur because of the hypochlorhydria reported in aids patients. 9 hypochlorhydria is common in those with starvation and systemic infection 10 and may therefore occur in advanced aids without any hiv-induced defects in gastric secretion. neither hypochlorhydria nor bacterial overgrowth is a universal finding in patients with advanced hiv infection, so these mechanisms are unlikely to have an important role in villus atrophy. the harrowing picture of emaciated terminally ill aids patients is a reminder of our lack of understanding of immunological mechanisms that normally control opportunistic infections. many gastrointestinal pathogens in patients with aids are resistant to treatment and lead inexorably to weight loss and death. although knowledge of the pathogenesis and clinical significance of weight loss has improved considerably, this has not yet led to a sustained effort to improve nutritional status during early stages of disease. fluoresce with agents such as calcofluor, 29 thereby providing an excellent screening test. results can be confirmed with giemsa staining. cryptosporidia in the stool is diagnosed by a modified ziehl-neelsen or auramine stain. enteric bacteria are usually detected by routine stool cultures but are occasionally found only in the blood. 30 stool culture of mycobacterium avium intracellulare is tedious and of uncertain pathogenic significance since colonisation can occur without diarrhoea. the value of stool electron microscopy for enteroviruses is debatable since in some studies these organisms have a similar prevalence in individuals with solid stools and in those with diarrhoea. 31 detection is of limited value because enteroviruses produce a self-limiting infection, even in most hiv-seropositive persons, and are untreatable. mucosal biopsy: although diagnosis by stool analysis alone has been suggested by johanson and sonnenberg, 32 this study may have overestimated the value of symptomatic treatment and ignored the possibility that cytomegalovirus infection sometimes responds to therapy. gut biopsy is the only way to diagnose infection with cmv or adenovirus. infections are commonly found in the large intestine, and sigmoidoscopy with rectal biopsies is probably the best initial invasive gastroenterological investigation in patients with diarrhoea. 10-30% of cases of cmv colitis affect only the right side of the colon or small intestine, so colonoscopy or small-intestinal biopsy may be indicated in individuals with persistent diarrhoea and negative rectal biopsy. 33 starvation, and diarrhoea is often out of proportion to the mucosal abnormalities, so a secretory component is likely. a secretory response was shown in isolated segments of human jejunum exposed to faeces from calves infected with cryptosporidia 19 but not in patients with moderately increased stool volumes due to cryptosporidiosis. 20 decreased intestinal transit time may exacerbate diarrhoea in protozoal infection, 21 but whether this is a primary motility defect or a consequence of malabsorption or enteric nerve damage is unclear. 22 another possibility is that motility changes or nerve damage are mediated by nitric oxide release as a result of stimulation of nitric oxide synthase in macrophages infected by hiv or opportunistic organisms; this possibility remains unexplored. changes in the immunological responses in the gut mucosa allow the persistence of protozoal infection in hiv-seropositive individuals, and abnormal immune responses themselves may be important in the pathogenesis of diarrhoea. the cd4 lymphocyte population of the lamina propria in hiv-seropositive patients shows a disproportionate reduction compared with the circulating cd4 count. 23 experiments in immunocompromised mice have shown that cd4-positive cells are essential for the eradication of cryptosporidia. 24 cd8 responses may be important in controlling infection in cd4-count-depleted mice, and interferon-gamma released from macrophages may also have a role. 25 altered cd8 or macrophage responses in hiv-seropositive individuals who are unable to mount a cd4 response to cryptosporidial infection may lead to aberrant cytokine release and consquent intestinal damage. increased concentrations of tumour necrosis factor-alpha have been reported in the faeces 26 but not in the mucosa of patients with protozoal diarrhoea. 27 studies of the complex balance between various cytokines and their tissue receptors are awaited. stool analysis is the initial investigation for hivseropositive subjects with diarrhoea and three samples will identify about 80% of pathogens. 28 cryptosporidia may not always take up appropriate stains in the stool and some infections are diagnosed only by histological examination of small or large bowel biopsy specimens. histological examination of duodenal biopsy specimens may also reveal previously unsuspected microsporidia, giardia, m avium intracellulare; or isospora. ultrastructural examination of enteric biopsy specimens by electromicroscopy is not done routinely but enables speciation of organisms such as microsporidia and maybe a useful addition to light microscopy ( figure) . analysis of six stool samples and histological examination of small and large bowel biopsy speicmens detect more than 90% of infectious causes of diarrhoea in hiv-seropositive individuals. 28 most individuals in whom no pathogens are found by these initial investigations have minor symptoms that usually resolve spontaneously. a few have continued large-volume diarrhoea, and in some of these widespread kaposi's sarcoma of the gut or lymphoma is subsequently diagnosed. 2 after stool analysis, acutely ill individuals with diarrhoea should be treated with ciprofloxacin, to which most enteric bacteria in hiv-seropositive individuals are sensitive. in those with chronic diarrhoea, antimotility agents reduce symptoms. oral rehydration therapy may also be important to maintain electrolyte balance. the somatostatin analogues octreotide and vapreotide are used in hiv-seropositive individuals with diarrhoea because of a motility effect and possibly because hiv has aminoacid sequences similar to vasoactive intestinal peptide (vip) and may induce diarrhoea by upregulating vip receptors. 34 increased concentrations of vip have been shown in some patients with pathogen-negative diarrhoea, in whom octreotide has likewise been successful. 35 somatostatin analogues are most effective for pathogen-negative diarrhoea and less so in protozoal diarrhoea, with one placebo-controlled study showing no effect. 36 these expensive agents are unlikely to have a wide role in the treatment of hiv-related diarrhoea. microsporidiosis: two genera of microsporidia are associated with diarrhoea. enterocytozoon bieneusi is found only in human beings. there is no known effective treatment, although uncontrolled studies suggest that some patients may respond to albendazole, 37 thalidomide, 38 or atovaquone. 39 encephalitozoon spp are much less commonly associated with diarrhoea, but infection with these organisms can also cause disseminated disease; organisms are eradicated by albendazole, with resolution of symptoms. cryptosporidiosis: there are many anecdotal reports of treatment for cryptosporidiosis that are difficult to interpret in view of the variable natural history of this infection. in at least a third of patients, diarrhoea resolves spontaneously, especially in those with an initial cd4 count above 200/l. investigation of suitable therapeutic agents is hampered by inability to culture the organism in vitro and the major differences between animal models and human infection. although many agents have been used, the only one with proven efficacy is paromomycin, which reduces stool volumes by about 50%. 40 quantitative stool analyses have shown some reduction in oocyst excretion with therapy but the organism is seldom eradicated. 41 because therapy of this infection is so difficult, primary prophylaxis would be a highly desirable goal. human cryptosporidiosis is a waterborne infection. since the minimum infectious dose is low-between one and five oocysts in gnotobiotic animals 42 and 20-50 oocysts in healthy human beings 43 -standard screening tests of metropolitan water supplies may not be sensitive enough for detection. boiling water eliminates infective oocysts, so this strategy is sensible for hiv-seropositive patients with cd4 counts of less than 200/l. enteritis: the treatment of cytomegalovirus enteritis remains controversial. both ganciclovir and foscarnet reduce symptoms and improve macroscopic and microscopic appearances of the colon with 3 weeks' therapy. 44 however, relapse with a median time of 12 weeks is very common. both initial treatment and the decision to give maintenance therapy will therefore depend on the initial severity of symptoms. since diagnosis of cytomegalovirus enteritis is improving, patients with milder symptoms are being detected and the quality of life with treatment-anti-cmv agents have to be given intravenously and have considerable toxicitymay not be enhanced compared with no therapy. whether such patients left untreated will develop complications of cmv enteritis such as toxic dilation and perforation, which were common in the early years of the epidemic, remains to be determined. oral ganciclovir in a high dose to overcome problems of bioavailability may have a limited role as primary prophylaxis in preventing the development of cmv disease. nevertheless, such therapy has major toxicity, only limited benefit, and is extremely expensive. widespread use of primary prophylaxis must await the development of more effective agents or of other markers that allow better prediction of patients who are most likely to develop cmv disease. mycobacterium avium-intracellulare (mai): about 5% of severely immunosuppressed hiv-seropositive patients have mai-induced diarrhoea. 45 primary prophylaxis with rifabutin of clarithromycin may reduce the incidence of mai but established small-bowel infection necessitates treatment with conventional quadruple therapy. such treatment must include the most effective agentsclarithromycin or azithromycin. resistance to these drugs will probably be limited by the addition of rifabutin and their efficacy improved by ethambutol. weight loss is a common feature of late hiv infection. initially wasting was thought to be a feature of hiv infection per se. 46 however, although there is a slight increase in resting energy expenditure (ree) in individuals at most levels of immunosuppression, weight and lean body mass are normal unless an opportunistic infection supervenes. 47 the pattern of weight loss characteristic of starvation, with decreased ree and relative preservation of lean body mass, is seen with enteric protozoal infection. 48 the starvation response is probably caused by a combination of voluntary reduction of intake to reduce diarrhoea and anorexia related to malabsorption. certain opportunistic infections, especially m avium intracellulare and cmv, produce a cachectic response in which the ree is further raised and there is a more pronounced loss of lean body mass, likely to be related to inappropriate cytokine release. 48 although increase in fat mass may improve body image, repletion of lean body mass is probably required to improve locomotor function and many aspects of quality of life. hiv-seropositive individuals losing weight because of starvation are likely to respond to food supplements given orally, parenterally, or enterally via a nasogastric tube or percutaneously by gastroenterostomy tube. such patients may also benefit from appetite stimulants. however, refeeding is likely to be ineffective in those with cachexia, who may improve lean body mass in response to anabolic agents (eg, recombinant human growth hormone). 49 abdominal pain cmv infection is associated with an arteritis and the consequent ischaemia may be associated with acalculous cholecystitis or appendicitis. cmv colitis commonly gives rise to abdominal pain with bloody diarrhoea and rebound tenderness. the other origin of abdominal pain unique to hiv-seropositive patients is an aids-related sclerosing cholangitis caused by various opportunists including microsporidia, cmv, and cryptosporidia. 50 this condition is associated with sclerosis of both the intrinsic and the extrinsic hepatic system and with changes in the pancreatic duct. sclerosing cholangitis may also be common in those without pain, but it is usually the discomfort that leads to investigation. since the natural history of this pain is variable, how frequently sphincterotomy improves symptoms is unclear. background low adherence of patients to prescribed, selfadministered medical interventions is ubiquitous. low adherence limits the benefits of current medical care. efforts to assist patients to follow treatments might improve the efficiency of care and substantially enhance benefits. our objective was to summarise the results of randomised controlled trials (rcts) of interventions to help patients follow prescriptions for medications. methods a previous systematic review was updated through computerised searches in medline, international pharmaceutical abstracts, psychinfo, and hstar online databases; bibliographies in articles on patient adherence; articles in the reviewers' personal collections; and contact with authors. articles were judged of interest if they reported original data concerning an unconfounded rct of an intervention to improve adherence with prescribed medications, with one or more measure of medication adherence, one or more measure of treatment outcome, at least 80% follow-up of each group studied, and, for longterm treatments, at least 6 months of follow-up for studies with positive initial findings. information on study design features, interventions and controls, and findings were ex tracted by one reviewer (rk) and checked by the other two reviewers. findings 1553 relevant citations and abstracts were screened, 252 full text articles were reviewed in detail, and 13 rcts met all criteria. the studies were too disparate in clinical problems, adherence interventions, measures and reporting of adherence, and the clinical outcomes studied to warrant meta-analysis. seven of 15 interventions were associated with improvements in adherence and six interventions led to improvements in treatment outcomes. for short-term treatments, one study showed an effect on adherence and outcome of counselling and w ritten information. the interventions that were effective for longterm care were complex , including various combinations of more convenient care, information, counselling, reminders, self-monitoring, reinforcement, family therapy, and other forms of additional supervision or attention. even the most effective interventions did not lead to substantial improvements in adherence. interpretation although adherence and treatment outcomes can be improved by certain-usually complexinterventions, full benefits of medications cannot be realised at currently achievable levels of adherence. it is time that additional efforts be directed towards developing and testing innovative approaches to assist patients to follow treatment prescriptions. lancet 1996; 348: 383-86 introduction non-adherence with prescribed treatments is very common. typical adherence rates for prescribed medications are about 50% with a range from 0% to over 100%. 1 to the extent that treatment response is related to dose, non-adherence reduces treatment benefits 2 and can bias assessment of the efficacy of treatments. 3 with increasing numbers of efficacious self-administered treatments, the need is apparent for better understanding and management of non-adherence. in previous reviews, we have examned the accuracy of clinical measures of non-adherence, 4 interventions to improve attendance at appointments for medical services, 5 and interventions to enhance medication adherence. 6 in the last review some of the included trials were acute hiv infection presenting with painful swallowing and esophageal ulcers natural history and prognosis of diarrhoea of unknown cause in patients with acquired immunodeficiency syndrome (aids) effects of zidovudine treatment on the small intestinal mucosa in patients infected with the human immunodeficiency virus intestinal absorptive capacity, intestinal permeability and jejunal histology in hiv and their relation to diarrhoea t-cell activation can induce either mucosal destruction or adaptation in cultured human fetal small intestine intestinal mucosal inflammation associated with human immunodeficiency virus infection human immunodeficiency virus detected in bowel epithelium from patients with gastrointestinal symptoms duodenal mucosal t cell subpopulation and bacterial cultures in acquired immunodeficiency syndrome association of gastric hypoacidity with opportunistic enteric infections in patients with aids decreased gastric acid secretion and bacterial colonisation of the stomach in severely malnourished bangladeshi children prevalance of enteric pathogens in homosexual men with and without acquired immunodeficiency syndrome infectious diarrhea in patients with aids gastrointestinal symptoms in patients infected with human immunodeficiency virus: relevance of infective agents isolated from gastrointestinal tract prevalence of intestinal protozoans in french patients infected with aids prevalence of intestinal microsporidiosis in hiv-infected individuals referred for gastroenterological evaluation faecal tumour necrosis factor-alpha in hiv-related diarrhoea the diagnosis of aids-related chronic diarrhoea: a prospective study in 155 patients light and electron microscopic appearances of pathological changes in hiv gut infection enterotoxic effect of stool supernatant of cryptosporidium-infected calves on human jejunum jejunal water and electrolyte transport in aids-related cryptosporidiosis small bowel transit in hivseropositive individuals autonomic denervation in jejunal mucosa of homosexual men infected with hiv loss of mucosal cd4 lymphocytes is an early feature of hiv infection mechanisms of innate and acquired resistance to cryptosporidium parvum infection in scid mice cryptosporidium infection in an adult mouse model: independent roles for ifn-â�¥ and cd4+ t lymphocytes in protective immunity faecal tumour necrosis factor-alpha and faecal alpha-one-antitrypsin in hiv infection diarrhoea in hiv-infected patients: no evidence of cytokine-mediated inflammation in jejunal mucosa an algorithm for the investigation of diarrhoea in hiv infection use of the fluorochrome calcofluor white in the screening of stool specimens for spores of microsporidia salmonella, campylobacter and shigella in hiv positive patients gastrointestinal viral infections in homosexual men who were symptomatic and seropositive for human immunodeficiency virus efficient management of diarrhea in the acquired immunodeficiency syndrome (aids): a medical decision analysis cytomegalovirus in aids: presentation in 44 patients and a review of the literature spectral and sequence similarity between vasoactive intestinal peptide and the second conserved region of human immunodeficiency virus type 1 envelope glycoprotein (gp 120): possible consequences on prevention and therapy of aids elevated plasma levels of vasoactive intestinal peptide in aids patients with refractory idiopathic diarrhea: effects of treatment with octreotide octreotide therapy of large volume refractory aids-associated diarrhea: a randomized controlled trial treatment with albendazole for intestinal disease due to enterocytozoon bieneusi in patients with aids thalidomide for microsporidiosis atrovaquone is effective treatment for the symptoms of gastrointestinal microsporidiosis in hiv-1 infected patients paromomycin for cryptosporidiosis in aids: a health information research unit intestinal function and injury acquired immunodeficiency syndrome-related cryptosporidiosis infective dose size studies on cryptosporidium parvum using gnotobiotic lambs the infectivity of cryptosporidium parvum in healthy volunteers treatment of aids-associated gastrointestinal cytomegalovirus infection with foscarnet and ganciclovir: a randomized comparison the microbial causes of diarrhea in patients infected with the human immunodeficiency virus body composition studies in patients with the acquired immunodeficiency syndrome prospective analysis of patterns of weight change in stage iv human immunodeficiency virus infcction the metabolic sequelae of specific opportunistic infections in aids anabolic effects of recombinant human growth hormone in patients with wasting associated with human immunodeficiency virus infection natural history of aids related sclerosing cholangitis: a study of 20 cases key: cord-260496-s2ba7uy3 authors: moncany, maurice l.j.; dalet, karine; courtois, pascal r.r. title: identification of conserved lentiviral sequences as landmarks of genomic flexibility date: 2006-08-08 journal: c r biol doi: 10.1016/j.crvi.2006.07.001 sha: doc_id: 260496 cord_uid: s2ba7uy3 considering that recombinations produce quasispecies in lentivirus spreading, we identified and localized highly conserved sequences that may play an important role in viral ontology. comparison of entire genomes, including 237 human, simian and non-primate mammal lentiviruses and 103 negative control viruses, led to identify 28 conserved lentiviral sequences (clss). they were located mainly in the structural genes forming hot spots particularly in the gag and pol genes and to a lesser extent in ltrs and regulatory genes. the cls pattern was the same throughout the different hiv-1 subtypes, except for some hiv-1-o strains. only cls 3 and 4 were detected in both negative control htlv-1 oncornaviruses and d-particle-forming simian viruses, which are not immunodeficiency inducers and display a genetic stability. clss divided the virus genomes into domains allowing us to distinguish sequence families leading to the notion of ‘species self’ besides that of ‘lentiviral self’. most of acutely localized clss in hiv-1s (82%) corresponded to wide recombination segments being currently reported. to cite this article: m.l.j. moncany et al., c. r. biologies 329 (2006). hiv genome flexibility is characterized by a high frequency of spontaneous mutations and a variety of rearrangements. the error-prone replication by hiv reverse transcriptase is generally held responsible for the impressive fraction of defective viruses observed in productively infected lymphocytes. a variety of other mechanisms can contribute to generate modifications in the hiv genomes by restabilizing the viral information under new forms, among them recombination phenomena now thought to be mainly responsible for hiv genomic flexibility throughout an infection process [1] [2] [3] [4] . this raised the notion of 'mosaic viruses' composed of parts inherited from divergent entire or partial viral genomes that could be present in the cells when the replication steps occur [5] [6] [7] . as lentiviral genomes are composed of an alternation of long variable and short conserved sequences, this appeared to be a characteristic of each part of the genomes organized as a succession of segments that can evolve differently and independently. the sequence conservation can be considered as the maintenance of either the general function of a protein or a potential precise function associated with a nucleotide segment (e.g., restriction or binding site). the extended nucleotide variability allowed by natural selection could lead to the evolution or to the disappearance of a function. in view of this analysis, the short conserved sequences may play crucial roles both in viral ontology and viral divergence independently of gene products functions (e.g., regulatory or enzymatic processes) as some of these sequences overlap genes. they could correspond to recombinogenic sequences important to understand lentiviral genomic flexibility. studies of the hiv genetic variability required computerized methods to investigate genomic divergences [8] [9] [10] [11] . a recombinant identification program was applied to the hiv-1 gag and env coding regions and allowed to determine putative large recombination segments -thus delimiting 'recombination cassettes' -and to create phylogenetic trees [12] . however, theses trees highly differed when the currently used computation methods were applied to either the gag, pol, or env genes, and when reference genome in the same population was changed [6, 7, [12] [13] [14] . the situation was made more complex when fragment analysis of a single gene showed divergent phylogenetic trees for each studied fragment [5] [6] [7] [14] [15] [16] , this being due to the independent evolution of each gene. in lentiviral genomes, the programs have rarely revealed precise recombinogenic segments, but rather computerized plots or large domains possibly implied in the recombination process [10, 11, 17] . comparison of whole genomes of related 'species' (with the meaning of taxons) is currently considered to identify the genomic organization and functionality without prior biological characterization [8, 18] . in our global approach concerning complete genomes in all situations, we carried out the detection of conserved lentiviral sequences (clss), their precise location being harmonized thanks to the use of a single mmy 1 ® sequence starting reference (see section 2). the analysis was made on genomes belonging to mammal lentiviruses, negative control viruses and randomgenerated genome-like sequences. this scan of the dna lentiviral sequences for conserved stretches allowed to identify 28 clss mainly situated in gag, pol and, in a minor proportion, env structural genes. a few of them were also noticed in the ltrs and the regulatory genes. a large part (82%) of clss located in hiv-1s is situated in currently described recombination segments where they might form recombinogenic hot spots [6, 7, [13] [14] [15] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . the similar position of each sequence in the different viral families led to establish the notion of 'lentiviral' and 'retroviral self' that was defined by the specificity of the sequences for restricted viral families. the viral genomes were retrieved from the genbank database and their loci are listed in appendix a. immunodeficiency lentiviral genomes correspond to 171 human viruses (155 hiv-1s and 16 hiv-2s), 33 simian viruses (3 cpz, 9 agm, 8 macaque, 2 mandrill, 10 sooty mangabey, 1 sykes' monkey viruses) and 33 non-primate mammal viruses (2 bovine, 2 caprine, 11 equine, 9 feline, 3 ovine and 6 ovine/caprine viruses). to test cls specificity, three kinds of negative controls were examined. first, non lentiviral yet retroviral genomes were screened (5 spumaviruses, 25 oncornaviruses and 4 d-particle-forming simian viruses). then, a set of human, animal and vegetal viruses was tested: 4 herpes viruses, 47 human and animal viruses (1 adenovirus, 6 coronaviruses, 2 filoviruses, 12 flaviviruses, 13 parvoviruses, 10 picornaviruses and 3 rhabdoviruses) and 18 vegetal viruses (5 geminiviruses, 2 potyviruses, 5 tobamoviruses, 1 tombusvirus, 3 tymoviruses and 2 necroviruses). finally, 50 10-kilobase-dna-like structures were randomly generated with a computer in order to eliminate any bias due to the possible subsequent biological role. using a previously published program [8, 37] , a general analysis was carried out to identify the highly conserved sequences common to all or a maximum of lentiviral genomes. we first established the length of sub-sequences with the following trial/error method selecting parts of genomes. when the length was inferior to 10 nucleotides, numerous sub-sequences were found in every genome, including the control ones. when it was superior to 50 nucleotides, very few sub-sequences were found. the correct length was determined when it corresponded to that of sub-sequences common to most of the immunodeficiency viral genomes in either all or certain viral families. a similar approach was used for the determination of the number of accepted transitions. the relationship between both parameters led to an optimized choice of a 15-nucleotide-long sequence with a maximum of three admitted transitions. all the sequences showing such a determined length were tested and positioned in genomes thanks to the expasy program available on internet. some of them overlapped and created longer conserved domains. sequences present in most lentiviral genomes and checked not to be found in negative controls were selected. the number of accepted transitions was retained according to their lentiviral specificity to allow a variability of 15 to 20%. in some cases the limit of 25% was retained, which is thought to delimit the generally admitted jump from one family to another and might fit with a possible biological significance. the genomes supplied by the databases show variable lengths because ltrs are reported with different lengths. thus, as the first considered nucleotides at the 5 extremity vary from one genome to another, the rough detection of the sub-sequences gave heterogeneous localizations. another approach could use the real starting atg (as given in the databases) of the coding sequences as the initial nucleotide, but this was too complex because of the frequent presence of several other atgs. to sharpen and to harmonize the location of clss, we calculated their relative positions as compared to that of one of them chosen as the reference sequence (position +1). this sequence -mmy 1 ® -was previously described for pcr purpose [38, 39] and corresponds to the beginning of the pbs (reverse transcription primer binding site) at the 5 extremity of the genomes. it is situated, for example, between nucleotides 182 and 199 on the hiv-1 bru/lai genome reported in genbank. mmy 1 ® was found in hiv-1, hiv-2, agm, macaque, cpz, sooty mangabey, mandrill, equine and feline lentiviruses. when two ltrs were present in the genomic banks (hiv-1, hiv-2 and equine viruses), a second mmy 1 ® sequence detected at the 3 extremity of the genomes was not considered in the analysis. in the sykes' monkey, bovine, caprine, ovine and ovine/caprine viruses lacking mmy 1 ® , the indicated numbers corresponding to cls locations were determined as crude positions directly detected on the genomes. when investigated on 237 complete viral genomes, 28 clss were detected, six of them being the mmy ® ones that have been previously used as pcr primers for the early detection of possible hiv infection in highly exposed population [38, 39] . these sequences together with some new determined clss classified from cls1 to cls 22 are shown in table 1 . the relationship between precise positions of clss and genomic organization of the viral families are depicted in figs. 1 to 3. the cls characteristics together with their specific gene locations shown in tables 1-3 were divided into categories according to their lentiviral specificity. these sequences included all the mmy ones, cls 1 to 6 and cls 16. almost all of them were also present in simian viruses. it is worth mentioning the following particularities for some clss: cls 2 was detected twice in most hiv-1s, all cpz and mandrill (pol and nef genes) and most agm virus genomes (nef gene). it was observed only once (nef gene) for most hiv-2s, macaque and sooty mangabey viruses. besides, a detection degree of the tested cls in the genomes: (") at least 90%; (2) comprised between 10% and 89%; (!) less than 10%. b gene location of the clss: genes separated by ( / ) when cls was detected on each of the two genes; genes separated by (-) when cls overlapped the 2 genes; gene indicated in italics when cls was present at an occasional position. c a gradual detection of cls 14 was observed when the admitted transitions in this sequence varied from 1 (66%) to 7 (85%). the particular sykes' monkey virus presented the single cls 2 in the vif gene. when viral genomes did not possess the second cls 2 (nef gene), cls 16 (from which cls 2 derived) was detected in the pol gene. for cls 4 (gag gene), a maximum of three transitions (16.7%) and sometimes four transitions (22.2%) were introduced in the computation. in the control viruses, this sequence was only detected in one herpes virus after three permitted transitions. when reaching four transitions, it were detected in almost all lentiviruses and in some negative genomes. while cls 3 (pol gene) as well as cls 4 was found in many genomes in most viral families, they were the only ones present in the four simian d-particle-forming viruses whose presence does not develop aids-like syndrome. cls 6 (pol gene) was present in all the primate viruses, except the sykes' monkey's ones. these sequences corresponded to cls 7 up to cls 15, in addition to those common to a maximum of hiv-1s and hiv-2s. the pattern of detection of hiv-1s was also found for cpzs except that cls 13 and 14 were missing. cls 7, 8 and 9 were only detected in hiv-1s and in cpz viruses. in particular, cls 10 was detected in most hiv-1s (pol gene), sometimes twice for 34/141 positive strains (gag and pol genes). it was not found in 5/6 hiv-1-o viruses (af407418, him302646, him302647, hivant70c and hivmvp5180) but was in af407419. when cls 10 was present, its position highly varied in all cpz viruses (pol gene), the two caprine viruses (vif gene), hiv-2s and sooty mangabey (env gene), and mandrill viruses (pol gene). cls 13 was present in most hiv-1 genomes (env gene), except for 12 out of 155 hiv-1s including the six hiv-1-o viruses, and was found twice in 40 out of 155 ones (env and pol gene). cls 14 only found in most hiv-1s was located mainly in gag gene and gradually detected (66% to 85%) when the admitted transitions varied from 1 to 7. cls 15 (pol gene) was specific to most hiv-1s, but was absent from all the hiv-1-o group, and was present in one cpz virus. a detection degree of the tested cls in the genomes: (") at least 90%; (2) comprised between 10% and 89%; (!) less than 10%. b gene location of the clss: genes separated by ( / ) when cls was detected on each of the two genes; genes separated by (-) when cls overlapped the 2 genes; gene indicated in italics when cls was present at an occasional position. c n.a.: not attributed. these sequences correspond to cls 17 up to 21, in addition to those common to a maximum of hiv-1s and hiv-2s. the five clss characteristic of hiv-2s were also conserved in most simian viruses except for cpz, mandrill and sykes' monkey viruses. cls 17 was present in most hiv-2s and macaque viruses (env gene) and sometimes in sooty mangabey and agm viruses (rev gene). cls 18 was found in gag gene in all hiv-2, agm and macaque genomes and in most sooty fig. 3 . clss localization and genomic organization of cpz, d-particle-forming viruses, feline and equine viruses. the numbers represent the relative positions of the detected cls calculated versus that of the mmy 1 ® , except for d-particle-forming viruses that lack mmy 1 ® , whose numbers correspond to the crude positions of the clss directly referenced from the genomes (see section 2). the reference organization of cpz, d-particle-forming viruses, feline and equine viruses was represented using: af103818, af033815, fivz1 and af247394 viruses, respectively. occp.: cls found at an occasional position. mangabey and mandrill ones. cls 19 (gag gene) and 21 (vpx gene) were detected in most hiv-2s and cls 20 (env gene) in all of them. cls 19, 20 and 21 were also present in all macaque and sooty mangabey viruses in the gag, env and vpx genes, respectively. in addition to the sequences common to a maximum of hiv-2s, cls 22 was characteristic of all agm and sykes' monkey lentiviruses in the env and tat overlapping genes, respectively. when present in one of the two mandrill viruses (sivmndgb1), cls 22 was located at an unusual position in the gag gene. non-primate lentiviruses showed less clss than primate ones, with 13/28 not present at all (table 3) , bovine lentiviruses showing the minimal number of two clss. the missing clss concerned particularly those detected in hiv-1s. it is worth mentioning that cls 2 was uniquely observed in all equine viruses where it was detected once at the unusual position in gag gene. cls 10 was only present in all caprine viruses (unusually in vif gene) as well as cls 16 in most feline ones (pol gene). mmy 1 ® was restricted to all equine and feline viruses. a few clss were displayed in some negative control viral genomes, while the random-generated tenkilobase-dna-like structures did not present any of them. cls 3 was found in 8/9 htlv-1s together with cls 4 in 5/9 of them while htlv-2s did not present any sequence. cls 3 and 4 were the only ones detected in d-particle-forming simian viruses in the pol and gag genes, respectively. mmy 1 ® was in 1/6 murine retroviral genomes. one should note that all these different oncornaviruses belong to families that are genetically stable and do not induce immunodeficiency. cls 1 and 22 were in retroviral spumaviruses (3/5 and 2/5, respectively). cls 3, 4, 10, 17, 20 and 21 were found in 1/4 herpes viruses. remarkably, hiv-1s present a crosstransactivation activity on herpes viruses [40, 41] as well as htlv-1s [42] . cls 18 was in 1/10 picornaviruses while cls 16 was in 5/13 parvoviruses. the observed sequences were mapped on all the genomes of the different viral families. from the particular organization of the hiv-1 and hiv-2 ( fig. 1) , agm and macaque (fig. 2) , cpz, feline, equine and d-particle-forming viruses (fig. 3) and that of sooty mangabey, mandrill and other non-primate lentiviruses (supplementary data), it appears that a given cls occupied on the viral genome a specific position that was roughly conserved in the different viral families. at first sight, the clss were detected mainly in the gag and pol structural genes and, to a lesser extent, in the ltrs, the env structural gene and the nef, vpr, vpu, vpx, vif, rev and tat regulatory genes in a decreasing order. when analyzing the data among families, the hiv-1 genome displayed the highest number of clss that were mainly found in the first half of the genome, covering 5 ltr, gag and pol genes and a part of vif gene. particularly, the sequence detection evidenced the p17 and p24 proteins for gag gene and the p31 and p51 proteins for pol gene, while cls 11 and 13 were found in the gp120 protein of env gene (see tables 2 and 3) . moreover, it was noticeable that cls 7 was at the hinge of the two ltrs. in hiv-2 genome, clss were also mainly related to gag, pol and env genes. hiv-2 and siv genomes presented similar organizations, but some differences led us to differentiate the sivs in several categories (figs. 2 and 3; supplementary data) . for example, cpz viruses exhibited a striking analogy with hiv-1s, while agm, macaque and sooty mangabey genomes showed a cls organization rather similar to that found in hiv-2s, confirming molecular data. cpz viruses also presented such a similarity with hiv-1s at the molecular level, yet they belonged to the simian viruses concerning the immunological characterization (e.g., [1, 8, 43] ). besides, the sykes' monkey virus appeared to be particular since it presented only six clss, four of them being at the hinge of the pol/vif genes. the d-particleforming viruses showed the simplest organization with only cls 3 and 4 that framed the beginning of pol gene. for the non-primate mammal viruses, the data must be cautiously interpreted, because the low number of studied genomes was not representative for some of these families. however, it is noteworthy that their detected sequences were mainly situated in the gag-pol region. the clss of these viruses whose number increased from bovine, equine, ovine/caprine, ovine, feline and finally up to caprine genomes showed a similar pattern ( fig. 3; supplementary data) . lentiviral genomes contained 28 clss allowing them to divide into regions corresponding to 'evolutive cassettes' that defined viral specific subtypes. about one third of clss were common to almost all primate lentiviral genomes. as to clss specific to hivs, their detection fitted with the known immunological families. maps of the viruses that can be reconstructed from building blocks delimited by clss were specific to each viral type, though they presented a similar high density in the gag-pol region. the revealed homology in gene location of clss between hivs and simian viruses confirmed the separation into two categories (hiv-1/cpz viruses, hiv-2s/other sivs). a clear barrier between primate and other mammal viruses appeared, due to shifting locations for some clss in the non-primate ones. many studies describe lentiviruses as mosaic viruses and numerous examples of recombination between different hiv-1s strains have been reported [6, 7, [13] [14] [15] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] . the correlation between wide recombination segments (whose list is not exhaustive) and clss acutely localized in the same regions is shown in table 4 . most of clss located in hiv-1s (82%) corresponded to such reported sequences, an underestimated value since multiple recombinogenic segments lacking of precise location (e.g., [35, 36] ) are not mentioned. cls 2 and 4 as well as mmy 28 were situated in the hiv-1 recombination sequence shown in the gag-pol region [21] . other domains contained cls 2 and cls 7 in the nef -ltr region [19] , mmy 1 (ltr), mmy 3 (gag gene) and mmy 31, as well as cls 9 (pol gene) in a study using hiv-1 strains deleted in the env gene [22] . in complete hiv-1 genomes, recombinogenic segments have been reported in the pol gene at rt level (p51 protein), to which corresponded cls 8, 9, 10 and 12, and in the env/nef common part, containing cls 2 [20] . they concerned also the gag (p17 protein), env (gp 41 and 120 proteins) and nef genes that carried mmy 3 as well as cls 10 and 14, table 4 correlation between recombinogenic segments described in hiv-1s and clss gene location of reported recombinogenic segments clss detected in corresponding segments [22, 25] 5 ltr mmy 1 [6, 15, 21, 22, [26] [27] [28] gag mmy 3, 28, cls 4, 10, 14 [6, 7, 14, 20, [22] [23] [24] [29] [30] [31] pol mmy 31, 32, cls 1-3, 8-10, 12, 16 [13, 26, 27, 30, 32] vpr/tat/rev cls 14, 17 [6, 20, 26, 27, 33, 34] env cls 11, 13 [7, 19] env/nef cls 2, 7 [19] nef/ 3 ltr cls 7 cls 11 and 13, and cls 2 and 7, respectively [6] . recombinogenic segments corresponded to clss 11 and 13 in env gene (gp120 protein) and cls 14 in the tat/rev overlapping region [34] . among the 22 clss present in hiv-1s, 82% belonged to the large recombination domains cited above. mmy 4 and cls 5 that corresponded to gag p24 protein (core protein) and clss 6 and 15 that corresponded to pol p31 protein (integrase) have not been correlated until now to recombinogenic segments. clss were characterized by their nucleotide composition and exhibited at first look a clear gap in detection between primate and other mammal viruses. in fact, well-conserved clss can represent a signal specific of the strain, the sub-group, the group or the family of virus to which they belong. these sequences can be classified as a function of the possible recombination events they could induce: 'hiv-1 type' between the different hiv-1 and cpz strains; 'hiv-2 type' between the different hiv-2s, macaque and sooty mangabey viruses and the feline ones; 'simian type' between agm, mandrill and sykes' monkey viruses; 'lentiviral type' between approximately all mammal lentiviruses; 'retroviral type' between almost all retroviruses. thus clss can be considered as the 'identifying self' of the viruses and their presence permitted the denomination of 'viral self', which could be a 'species self' (an hiv-1-type, for example), or an 'inter-species self' -'lentiviral' or 'retroviral' self. our global study of the entire lentiviral genomes suggested the involvement of recombinations in genome flexibility. it allowed us to postulate that if one recombination induced the formation of one variant, the association of series of related variants formed one subtype. the latter was produced by recombinations between distinct variants that could cause or not the emergence of a new subtype. the genomic flexibility is associated with the viral-derived dna sequences that can recombine [1, 19, 30] and/or produce complementing and/or recombining rnas [4, 44, 45] . once several elements of the viral genome have penetrated into a cell -and sometimes together with hiv dna pieces carried by virions [46] -they may be rearranged to possibly elicit productive infection in a new target tissue. for example, cls 11 and cls 13 (hiv-1 env gene) were located at the level of the v3 and v4 variable loops of the gp120 cellbinding protein, respectively corresponding to the recombinogenic segments previously described [6, 20, 26, 27, 33, 34] . this cls 11/cls 13 tandem seems to correspond to an important building block for the creation of a new cellular tropism. the v3 domain is critical for chemokine-mediated blockade of infection [47] and the v3 and v4 regions that are separated by the c3 constant domain represent the targets of many trials for the establishment of a candidate vaccine. a productive viral dissemination has been revealed that evidences recombinations implying independent gene evolutions [1, 14, 30] , and possibly leads to a new gene acquisition [48, 49] . such genomic divergences both maintain viral specificity and allow the emergence of new families raising the question: are these divergences selected by the specificity or are they specificityselectors while maintaining the viral integrity? so a new viral cell tropism can be created that correlates with the use of the ccr5 or cxcr4 classical co-receptors [50, 51] or a postulated new one [52] . another essential step was to link the determined landmarks of genomic flexibility with precise viral functions. it is worth mentioning that clss 16 and 2 have a very close function. cls 16 was a part of the cppt involved in the initiation of lentivirus reverse transcription [53] where dna synthesis enhances dna/dna recombination [54] . cls 2 represented the well-conserved part of the distal ppt, and perhaps the action site, while the neighbouring sequences which varied highly from a virus to another one might constitute a specific recognition site for the reverse transcrip-tase associated with a given virus. hiv-1s displayed two cls 2 at positions shown to be the cppt (pol gene) [44] and the distal ppt (nef gene) [53] . also, hiv-2s revealed a slightly different organization, since cls 16 was part of the cppt (pol gene) and cls 2 part of the distal ppt when the nef gene overlaps the 3 ltr. the role in recombination of clss situated in the conserved ppt was emphasized by the determination in the pol gene (protein p31, integrase) of a recombinogenic segment that could affect the viral productive cycle [14] . another interesting point concerns the sequence structure which could also indicate a specific additional function for some clss. for example, the presence of the repeated aatt motif inside cls 15 (3 motives) and 13 (2 motives) is similar to a folding-like inducer of the dna [55] . cls 15 presented the noticeable triplicate aatt structure (aaactaaagaatt acaaaaacaaattacaaaaatt) neighbouring cls 6 to form a termination structure. in view of the structure of the cts (pol gene, p31 protein) [56] , cls 15 together with cls 6 showed the aaaaatt and aaatttt motives corresponding respectively to 1 and 2, strong and weak, stop signals [57, 58] . the cts approximately represents the succession of these two sequences. the raison d'être of a cls -simply recombining, participating in the genetic expression or both -can imply important differences in the detected sequence function and/or viral ontology such as generation of a new subtype (e.g., [26, 44] ). clss specificity revealed that two kinds of recombinations seemed to coexist. one involved sequences mostly common to all the lentiviral genomes separated by large domains, which could allow interspecific recombinations. the other type of recombination involved sequences that within a same family were separated by short distances or even overlapped as shown in hiv-1s for cls 10 and 14 (gag gene). these findings led us to discriminate between restricted or expanded specificity. in a gene-to-gene study on gag and env genes, wide segments have been described where recombinations could be present, which implied that one recombinant virus characterizes one subtype [12] . the multiplicity of viral subspecies present in the same infected host may be a cause and/or a consequence of the recombination phenomena [1, 3, 4, 30] , the generation of recombining strains increasing this process. the presence of clss is not directly associated with the role of a gene since a recombination can be either intra-or intergenic. such a process involves two adjacent or distant clss that can be situated in the same gene or in two different genes. considering all the possibilities of recombination that happen during an infection, the viability of the newly recombinant genome is the single criterion of selection. the cascade of slow genomic divergences is an integral part of the viral ontogeny to ensure long-term survival. such genetic approaches could benefit from the defined clss, whose characteristic is to be well-conserved and to keep functional the viral genome. thus the mechanism that maintains the genomic flexibility would be an excellent tool to impede viral growth, particularly when sequences implied in recombination and genetic expression are modified or blocked. as an ad absurdum argument, d-particle-forming viruses that do not cause immunodeficiency presented a single sequence cls 3 (pol gene), and occasionally cls 4 (gag gene), a situation leading to the suppression of some necessary specificity steps. these two clss were the only sequences detected in htlv-1s showing a genetic stability like that in htlv-2s yet without cls, which suggests that they could play a role in gene exchange between oncorna-and/or retroviruses. during lentiviruses adaptation to changes caused by drug administration [2, 45, 59, 60] or by environmental conditions to ensure continued reproduction, the viral propagation beyond a critical level is constantly in a situation of flux. thus, clss can allow the spontaneous replacement of defective variants with newly 'recruited' recombined (or complementing) hiv genomes. the high degree of divergence is a vital part of the viral ontogeny, and recombinations induce sustained viral multiplication allowing the environment to select the most efficient genomic alternative. this emphasizes the importance of the number, the position and the specificity of clss on the viral genome, especially since most of them fitted with recombinogenic segments already described. the biological role validity of some clss not associated until now with a known function had to be checked in vitro, for example by reverse genetic experiments that could reveal the importance of the different sequences. clss, which represent essential landmarks of genomic flexibility, may become key targets for the establishment of new drug and/or gene therapies that can escape the resistances encountered with treatments presently in use. chimpanzee (3): af103818, af115393, sivcpzgab. african green monkeys af074965, af139382, af326583, af326584, af412314, hl2g12gnom, hl2v2cg, htlvcge, nc_001488. murine viruses a.3. negative control viruses herpes viruses recombinant hiv sequences: their role in the global epidemic different evolutionary patterns are found within human immunodeficiency virus type 1-infected patients recombination in hiv: an important viral evolutionary strategy mechanisms of retroviral recombination mosaic structure of the human immunodeficiency virus type 1 genome infecting lymphoid cells and the brain: evidence for frequent in vivo recombination events in the evolution of regional populations high frequency of recombinant genomes in hiv type 1 samples from brazilian southeastern and southern regions morgado, molecular epidemiology of hiv-1 in venezuela: high prevalence of hiv-1 subtype b and identification of a b/f recombinant infection fast analysis of genomic homologies: primate immunodeficiency virus a likelihood method for the detection of selection and recombination using nucleotide sequences in vivo characteristics of human immunodeficiency virus type 1 intersubtype recombination: determination of hot spots and correlation with sequence similarity a novel exploratory method for visual recombination detection scanning the database for recombinant hiv-1 genomes characterization of a highly replicative intergroup m/o human immunodeficiency virus type 1 recombinant isolated from a cameroonian patient sequence variability of the integrase protein from a diverse collection of hiv type 1 isolates representing several subtypes high prevalence of diverse forms of hiv-1 intersubtype recombinants in central myanmar: geographical hot spot of extensive recombination development and application of a highthroughput hiv type-1 genotyping assay to identify crf02_ag in west/west central africa stepwise detection of recombination breakpoints in sequence alignments sequencing and comparison of yeast species to identify genes and regulatory elements genetic characterization of the nef gene from human immunodeficiency virus type 1 group m strains representing genetic subtypes a precise mapping of recombination breakpoints suggests a common parent of two bc recombinant hiv type 1 strains circulating in china genotypic and phenotypic analysis of hiv type 1 primary isolates from western cameroon human immunodeficiency virus type 1 recombination: rate, fidelity, and putative hot spots v118i substitution in the reverse transcriptase gene of hiv type 1 crf02_ag strains infecting drug-naive individuals in cameroon hiv type-1 circulating recombinant form crf09_cpx from west africa combines subtypes a, f, g, and may share ancestors with crf02_ag and z321 isolation and characterization of a fulllength molecular dna clone of ghanaian hiv type 1 intersubtype a/g recombinant crf02_ag, which is replication competent in a restricted host range emergence of new forms of human immunodeficiency virus type 1 intersubtype recombinants in central myanmar independent introduction of transmissible f/d recombinant hiv-1 from africa into belgium and the netherlands mother-to-child hiv type-1 transmission in argentina: bf recombinants have predominated in infected children since the mid-1980s identification of ugandan hiv type-1 variants with unique patterns of recombination in pol involving subtypes a and d evolution and diversity of hiv-1 in africa -a review prevalence and origin of hiv-1 group m subtypes among patients attending a belgian hospital in 1999 dual human immunodeficiency virus type 1 infection and recombination in a dually exposed transfusion recipient. the transfusion safety study group an ab recombinant and its parental hiv type 1 strains in the area of the former soviet union: low requirements for sequence identity in recombination, unaids virus isolation network new hiv type-1 crf01_ae/b recombinants displaying unique distribution of breakpoints from incident infections among injecting drug users in thailand hiv type-1 bf recombinant strains exhibit different pol gene mosaic patterns: descriptive analysis from 284 patients under treatment failure the structure of hiv-1 genomic rna in the gp120 gene determines a recombination hot spot in vivo a probabilistic algorithm for interactive huge genome comparison late seroconversion in three multitransfused young haemophiliacs confirmed by hiv pcr analysis in vitro non-productive infection of purified natural killer cells by the bru isolate of the human immunodeficiency virus type 1 post-transcriptional transactivation of human retroviral envelope glycoprotein expression by herpes simplex virus us11 protein cross-talk between human herpesvirus 8 and the transactivator protein in the pathogenesis of kaposi's sarcoma in hiv-infected patients functional replacement of the hiv-1 rev protein by the htlv-1 rex protein wain-hobson, genetic organization of a chimpanzee lentivirus related to hiv-1 mechanisms associated with the generation of biologically active human immunodeficiency virus type-1 particles from defective proviruses how rna viruses exchange their genetic material viral dna carried by human immunodeficiency virus type 1 virions the v3 domain of the hiv-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection gene acquisition in hiv and siv evolution of the primate lentiviruses: evidence from vpx and vpr biological and molecular aspects of hiv-1 coreceptor usage impact of antiretroviral treatment on the tropism of hiv-1 plasma virus populations identification and characterization of hiv-2 strains obtained from asymptomatic patients that do not use ccr5 or cxcr4 coreceptors a single-stranded gap in human immunodeficiency virus unintegrated linear dna defined by a central copy of the polypurine tract strand displacement synthesis in the central polypurine tract region of hiv-1 promotes dna to dna strand transfer recombination the structure of an oligo(da).oligo(dt) tract and its biological implications hiv-1 reverse transcription. a termination step at the center of the genome synthesis of dna by human immunodeficiency virus reverse transcriptase is preferentially blocked at template oligo(deoxyadenosine) tracts template-directed pausing of dna synthesis by hiv-1 reverse transcriptase during polymerization of hiv-1 sequences in vitro mutations in retroviral genes associated with drug resistance characterization of resistant hiv variants generated by in vitro passage with lopinavir/ritonavir we thank dr christiane plas for critical reading of this manuscript and for helpful discussions. appendix a af408631, af408632, a04321, a07867, him237565, him245481, him271445, him291719, him302646, him302647, hivant70c, hivbcsg3x, hivbrucg, hivcam1, hivelicg, hivf12cg, hivhxb2cg, hivibng, hivjrcsf, hivmalcg, hivmncg, hivmvp5180, hivndk, hivnl43, hivny5cg, hivoyi, hivpv22, hivp896c, hivrf, hivsf2cg, hivth475a, hivu455a, hivu43096, hivu43141, hivu46016, hivu51188, hivu51189, hivu54771, hivu69584 key: cord-260444-ooi5x9p3 authors: gadelha farias, luís arthur brasil; gomes moreira, ana livia; austregésilo corrêa, eduardo; landim de oliveira lima, cicero allan; lopes, isadora maria praciano; de holanda, pablo eliack linhares; nunes, fernanda remígio; pires neto, roberto da justa title: case report: coronavirus disease and pulmonary tuberculosis in patients with human immunodeficiency virus: report of two cases date: 2020-08-18 journal: am j trop med hyg doi: 10.4269/ajtmh.20-0737 sha: doc_id: 260444 cord_uid: ooi5x9p3 coinfection of sars-cov-2/mycobacterium tuberculosis (mtb) in patients with hiv/aids has not been previously reported. here, we present two cases of coinfection of sars-cov-2 and mtb in patients with hiv. the first case is a 39-year-old patient who was admitted with a 7-day history of fever, myalgia, headache, and cough. the second patient is a 43-year-old man who had a 1-month history of cough with hemoptoic sputum, evolving to mild respiratory distress in the last 7 days. both patients already had pulmonary tuberculosis and subsequently developed sars-cov-2 infection during the 2020 pandemic. nonadherence to antiretroviral treatment may have been a factor in the clinical worsening of the patients. covid-19, caused by sars-cov-2, represents a new fatal disease mainly characterized by a respiratory illness. 1 on march 11, 2020, the who officially declared the infection to have risen to a pandemic state. 2, 3 in brazil, the first case of covid-19 was confirmed on february 26, 2020, and community transmission was recognized on march 20, 2020. 4, 5 on may 24, 2020, there were almost 350,000 confirmed cases and approximately 22,000 deaths, with a mortality rate of 6.9%. the most affected states were são paulo (82,161), rio de janeiro (37,912), and ceará (35,595). 6, 7 the coinfection of sars-cov-2 and mycobacterium tuberculosis (mtb) in patients with hiv infection is a matter of concern and has not been well studied. here, we present two cases of triple coinfections (hiv/sars-cov-2/mtb) in patients admitted to sao josé hospital of infectious diseases, fortaleza, ceará, brazil. both patients provided informed consent to publish their clinical data. nasopharyngeal swab samples were used for covid-19 diagnoses based on the amplification of the betacoronavirus e gene and the specific sars-cov-2 rdrp gene using pcr. sputum samples were used for mtb diagnosis. case report case 1. a 39-year-old italian man who had been living in brazil for more than 20 years arrived at the emergency department (ed) in april 2020 with a known diagnosis of hiv/ aids (hiv viral load 293,313 copies/mm 3 and cd4 cell count 145/mm 3 ) and a 7-day history of sudden fever (temperature 38.0°c), myalgia, headache, and cough. he had poor adherence to antiretroviral therapy and previous use of marijuana and crack. he denied recent travel. he also tested positive for the surface antigen of the hepatitis b virus. he reported previous tuberculosis (tb) treatment for 3 months, although he did not complete the scheme. on physical examination, he was cachectic (weight 38 kg), with mild respiratory distress, a heart rate of 108 bpm, a respiratory rate of 18 rpm, and blood oxygen saturation (spo 2 ) levels of 93% without supplementary oxygen. no lymphadenopathy was observed. a chest examination revealed bilateral crepitations, rhonchi, and wheezes. chest computed tomography (ct) revealed a large cavitation located in the left lung associated with bilateral glass-ground opacities ( figure 1a -d). blood cell count presented low hemoglobin (5.4 g/dl) and hematocrit levels (16.5%), lymphopenia (408/mm 3 ), and normal platelet and white cell count ranges. the c-reactive protein level was markedly elevated (118.8 mg/l). hepatic and renal functions were normal. d-dimer, troponin, aspartate transaminase (ast), alanine transminase (alt), and ferritin levels were at normal range. acid-fast bacilli smears were positive, and mtb dna was detected using pcr (genexpert mtb/rifampicin [rif] assay) without rif resistance. during hospitalization, the patient experienced respiratory distress and required continuous oxygen via nasal cannula 2 l/minute during the first 3 days. admission to the intensive care unit and invasive mechanical ventilation was not required. treatment with isoniazid, ethambutol, pyrazinamide, and rif was initiated. he was also treated with azithromycin (500 mg/day), hydroxychloroquine (hcq) (400 mg/day) for 5 days, and ceftriaxone (2 g/day). antiretroviral therapy was not started to avoid complications with anti-tb treatment. after being hospitalized for 3 weeks, the patient was clinically stable and discharged to home with outpatient follow-up. antiretroviral therapy was planned to be initiated after the first 8 weeks of anti-tb treatment. 8 case 2. a 43-year-old brazilian man arrived at the ed in may 2020 with a history of cough with hemoptoic sputum for more than 1 month, evolving to mild respiratory distress in the last 7 days. he did not show signs of a fever and other symptoms. he had a previous history of hiv/aids with no adherence to antiretroviral therapy, illicit drug abuse, and had generalized anxiety disorder. he had lost clinical and laboratory follow-up in the last 5 years. in 2015, the hiv viral load was 9,054 copies/ mm 3 , and the t-cd4 cell count was 407/mm 3 . on physical examination, his weight was 55 kg, heart rate was 79 bpm, respiratory rate was 18 rpm, and spo 2 was 96%. no lymphadenopathy was observed. blood cell count presented low hemoglobin (9.9 g/dl) and hematocrit levels (29.7%), and normal platelet and white cell count ranges. lactate dehydrogenase and c-reactive protein levels were 312 u/l, above reference limits (140-271 u/l), and 52.3 mg/dl, respectively. the d-dimer level (0.6 mcg/ml) exceeded the normal range. troponin, ast, alt, and ferritin levels were in normal range. chest ct revealed bilateral glass-ground opacities occupying approximately 25% of both lungs (figure 2a-d) . respiratory secretion was collected with a nasopharyngeal swab and tested positive for sars-cov-2. mycobacterium tuberculosis dna was detected using pcr (genexpert mtb/ rif assay) without rif resistance. treatment was initiated with azithromycin (500 mg/day), hcq (400 mg/day) for 5 days, and ceftriaxone (2 g/day). he was treated with isoniazid, ethambutol, pyrazinamide, and rif. antiretroviral therapy was not started to avoid complications with anti-tb treatment. the patient was clinically stable and discharged to home after 1 week. currently, the patient is under follow-up and remains asymptomatic without developing relapses. to the best of our knowledge, this is the first reported case of coinfection of sars-cov-2/mtb in patients with hiv/aids. hiv/mtb coinfection is prevalent worldwide-especially in low-income and developing countries such as brazil-and its clinical consequences are well known. hiv patients with mtb latent infection have a higher risk of reactivation. tuberculosis is the most common opportunistic disease and tends to be more lethal in cases of hiv. extrapulmonary forms and disseminated disease occur more frequently in hiv, especially in those with high viral load and low t-cd4 cell count. however, as the covid-19 pandemic develops, there is a concern regarding its clinical and epidemiological relevance in hiv/ mtb-coinfected patients. covid-19 is an acute viral infection with frequent and severe pulmonary involvement, which can lead to hospitalizations and deaths. the lungs can be directly affected by the virus, leading to viral pneumonia. the immune system is also markedly affected and challenged by sars-cov-2. leukopenia, lymphopenia, and an inflammatory cytokine storm are some of these immunological changes. treatment with immunosuppressive drugs, such as corticosteroids, has been adopted in some cases. in this context, the impact of covid-19 on hiv viral replication in hiv patients with mtb latent infection and in patients with hiv and active tb must be subjects of research. this includes the spectrum of clinical manifestations of the triple coinfections. there are some reports that show that sars/mtb and middle east respiratory syndrome-cov/mtb coinfections can augment other infections. 9 one observational study on sars-cov-2/mtb coinfection concluded that mtb infection possibly increases covid-19 susceptibility and severity. 10 blanco et al. 11 recently published a case series of five hivinfected patients with covid-19, of whom none of them died, although two needed intensive care supportive therapy. gervasoni et al. 12 described 47 hiv-positive patients hospitalized by covid-19 and concluded that hiv patients were not at greater risk of severe disease or death than hiv-negative patients. motta et al. 13 compared 69 patients with sars-cov-2/ mtb coinfections, although they could not show tb as a major predictor of mortality in these patients. this appeared to be true in patients reported in the present study because even with hiv infection association, the patients did not evolve to a greater gravity. however, according to he et al., 14 patients with previous lung disease such as treated or untreated tb could affect the prognosis of patients with covid-19, making greater vigilance necessary during outpatient follow-up. the present article reports two cases of covid-19 in patients with hiv/mtb coinfections. both patients already had active tb before being infected with sars-cov-2. they both had a history of irregular antiretroviral treatment, detectable hiv viral loads, and low t-cd4 lymphocyte counts (< 500). molecular tests (pcr and reverse transcription pcr) were positive for mtb and sars-cov-2 simultaneously. there was an overlap of symptoms commonly seen in active tb and covid-19, such as cough and fever. one of the patients had hemoptoic sputum. chest ct findings showed patterns that could be related to both diseases, although they cannot be differentiated from other pulmonary diseases such as bacterial pneumonia and chronic obstructive pulmonary disease. although the risk factors for covid-19 still need to be fully understood, the two cases presented here may indicate that hiv/mtb coinfection could be another risk factor to be considered when evaluating sars-cov-2-infected patients. nonadherence to antiretroviral treatment may have been a factor in the worsening of mtb/sars-cov-2 coinfection. 15 the possibility of associated bacterial pneumonia could not be ruled out in both cases, and antibiotic coverage with ceftriaxone and azithromycin was chosen. both patients were treated with hcq. thus, the usefulness of this drug is controversial. on june 17, 2020, based on evidence from the solidarity trial, u.k. recovery trial and a cochrane review, who announced that the hcq arm of the solidarity trial to find an effective covid-19 treatment was being stopped. both showed that hcq does not result in the reduction of mortality of hospitalized covid-19 patients. 16 this study has some limitations. herein, we studied only two cases of sars-cov-2 and mtb coinfection in hiv-infected patients. interleukin-6 was not available at our center. it is important to carefully evaluate suspected sars-cov-2 patients in the presence of other infectious diseases, such as tb, especially if cohorting is performed for suspected sars-cov-2 to avoid nosocomial transmission. a pneumonia outbreak associated with a new coronavirus of probable bat origin discurso de abertura do diretor-geral da oms na conferência de imprensa sobre covid-19 how brazil can hold back covid-19 ministério da saúde declara transmissão comunitária nacional.brasília, brazil: ministério da saúde covid-19 and pulmonary tuberculosis in patients with hiv da-saude-declara-transmissao-comunitaria-nacional painel de casos de doença pelo coronavírus 2019 (covid-19) no brasil pelo ministério da saúde. brasília, brazil: ministério da saúde decreto no33.519, de 19 de março de 2020. intensifica as medidas para enfrentamento da infecção humana pelo novo coronavírus. ceará, brazil: editoração casa civil painel de casos de doença pelo coronavírus 2019 (covid-19) no brasil pelo ministério da saúde consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection: recommendations for a public health approach middle east respiratory syndrome coronavirus and pulmonary tuberculosis coinfection: implications for infection control active or latent tuberculosis increases susceptibility to covid-19 and disease severity. medrxiv covid-19 in patients with hiv: clinical case series clinical features and outcomes of hiv patients with coronavirus disease 2019. clin infect dis (epub ahead of print tuberculosis, covid-19 and migrants: preliminary analysis of deaths occurring in 69 patients from two cohorts epub ahead of print the burden of covid-19 in people living with hiv: a syndemic perspective hydroxychloroquine and covid-19 this is an open-access article distributed under the terms of the creative commons attribution (cc-by) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord-252433-0e9lonq4 authors: cullen, bryan r. title: viral rnas: lessons from the enemy date: 2009-02-20 journal: cell doi: 10.1016/j.cell.2009.01.048 sha: doc_id: 252433 cord_uid: 0e9lonq4 viruses are adept at evolving or co-opting genomic elements that allow them to maximize their replication potential in the infected host. this evolutionary plasticity makes viruses an invaluable system to identify new mechanisms used not only by viruses but also by vertebrate cells to modulate gene expression. here, i discuss the identification and characterization of viral mrna structures and noncoding rnas that have led to important insights into the molecular mechanisms of eukaryotic cells. viruses are adept at evolving or co-opting genomic elements that allow them to maximize their replication potential in the infected host. this evolutionary plasticity makes viruses an invaluable system to identify new mechanisms used not only by viruses but also by vertebrate cells to modulate gene expression. here, i discuss the identification and characterization of viral mrna structures and noncoding rnas that have led to important insights into the molecular mechanisms of eukaryotic cells. the hiv-1 tat protein activates transcription of the hiv-1 provirus by recruiting the cellular p-tefb complex to the viral tar rna hairpin. nuclear export of incompletely spliced hiv-1 transcripts is facilitated by the rre rna structure, which recruits a complex consisting of the viral rev protein and cellular crm1. a similar function is performed by the cte rna structure present in mpmv, which recruits the cellular tap nuclear export factor. the cytoplasmic translation of picornaviral mrnas is facilitated by ires elements, and the translation of retroviral and coronaviral mrnas is modulated by sequences that induce ribosomal frameshifting. finally, the translation of both viral and cellular mrnas can be specifically downregulated by virally encoded micrornas. to tar, p-tefb mediates the phosphorylation of negative regulators of transcription elongation and of the carboxy-terminal domain of rnap ii molecules that have initiated transcription of hiv-1 proviral dna. these phosphorylation events render rnap ii elongation competent and allow it to transcribe the entire viral genome (barboric and peterlin, 2005; kao et al., 1987) . in contrast, in the absence of tat or tar, transcription initiation at the ltr promoter still occurs but almost all of these initiating rnap ii molecules fall off the dna template within ~200 bp of the transcription start site. analysis of tat function led to the realization that not only transcription initiation but also transcription elongation can regulate gene expression levels in animal cells (barboric and peterlin, 2005) . almost all retroviruses contain a single rnap ii-dependent promoter element in the viral ltr that drives transcription of an initial genome-length rna that also acts as an mrna for translation of the viral gag and pol proteins (cullen, 2003) . in the case of hiv-1, this initial transcript can also be processed into fully spliced transcripts encoding the tat and rev proteins of hiv-1 as well as the auxiliary protein nef. alternatively, this transcript can be processed into partially spliced mrnas encoding the three other viral auxiliary proteins vif, vpu, and vpr. the hiv-1 replication cycle, therefore, requires that the single initial viral transcript is exported out of the nucleus in several differentially spliced forms. these include an unspliced form that programs gag and pol expression and that is packaged into virion progeny; partially spliced forms that program expression of env, vif, vpr, and vpu; and fully spliced forms that program expression of tat, rev, and nef (cullen, 2003) . the difficulty with this scenario is that eukaryotic cells do not normally permit the nuclear export of intron-containing mrnas. almost all cellular mrnas are transcribed as intron-containing pre-mrnas, and these introns are recognized in the nascent transcript by splicing factors, including commitment factors. commitment factors both commit the pre-mrna to the splicing pathway and retain the pre-mrna in the nucleus until all introns are removed (legrain and rosbash, 1989) . hiv-1 mrnas rely entirely on cellular factors for appropriate splicing, and intron-containing hiv-1 transcripts are therefore also retained in the infected cell nucleus by splicing commitment factors. the strategy that hiv-1 has evolved to circumvent this nuclear retention is dependent on the viral rev protein, which is translated from a fully spliced viral mrna that is constitutively exported from the nucleus. as a result, hiv-1 mutants lacking a functional rev gene are able to express the proteins encoded by fully spliced viral mrnas, that is, tat, nef, and the defective rev protein itself, but cannot express any of the proteins encoded by incompletely spliced viral mrnas, including gag, pol, and env. the transcripts encoding these viral structural proteins, however, can be detected in the nucleus of cells infected by rev-deficient viruses, where they are either degraded or eventually fully spliced and then exported (cullen, 2003) . rev function requires a highly structured rna target, located in the hiv-1 env gene, called the rev response element or rre (malim et al., 1989) . the rre contains a single, high-affinity rev-binding site and also functions as a scaffold for the multimerization of rev on viral mrnas. rev in turn interacts with a cellular factor called crm1 that belongs to the karyopherin family of nucleocytoplasmic transport proteins (fischer et al., 1995) . this interaction is mediated by a leucine-rich motif located toward the carboxyl terminus of rev that was the first nuclear export signal (nes) to be identified and is the prototype of the leucine-rich class of ness. karyopherin function is regulated by the action of a g protein called ran, which, like all g proteins, is active when bound by gtp and inactive when bound by gdp (kohler and hurt, 2007) . cells contain high levels of a ran-specific g nucleotide exchange factor (gef) in the nucleus and of a ran-specific gtpase activating protein (gap) in the cytoplasm. as a result, ran:gtp is largely nuclear and ran:gdp is mainly cytoplasmic. ran:gtp binds to crm1 in the nucleus and activates the binding of crm1 to leucine-rich ness. the ribonucleoprotein complex, consisting of ran:gtp, crm1, and rev, that forms on the hiv-1 rre directs incompletely spliced hiv-1 transcripts to the nuclear pore complex and then into the cytoplasm, where hydrolysis of the gtp moiety by cytoplasmic gap disassembles this complex. although rev was the first nuclear mrna export factor to be identified, it soon became clear that crm1 is not required for the nuclear export of most cellular mrnas. in fact, crm1 is involved largely in the nuclear export of small nuclear rnas (snrnas) and preribosomal subunits, as well as in protein nuclear export (kohler and hurt, 2007) . so which factors are required for the export of cellular mrnas? an important part of the answer emerged from analysis of a second retrovirus called mason-pfizer monkey virus (mpmv). mpmv has a simpler genomic organization than hiv-1 and only encodes the three structural proteins picornaviruses and some flaviviruses recruits cellular translation factors and ribosomal subunits to viral translation initiation codons in the absence of an mrna cap gag, pol, and env. nevertheless, mpmv expresses both a genome-length gag/ pol mrna and a spliced env mrna. as mpmv does not encode a rev homolog, how do the incompletely spliced genomic mpmv mrnas reach the cytoplasm? this question led to the discovery of an rna stem-loop structure within the mpmv genome, the constitutive transport element (cte), that mediates the nuclear export of incompletely spliced mrnas in the absence of any viral proteins (bray et al., 1994) . further analysis revealed that the cte recruits a heterodimer of two cellular proteins, tap and p15, that also plays a critical role in the nuclear export of the majority of cellular mrnas (grüter et al., 1988; kohler and hurt, 2007) . normally, the tap/p15 heterodimer is only recruited to mature, fully spliced mrnas. however, the cte is able to prematurely recruit tap/p15 to partially spliced mrnas and thereby circumvents the nuclear retention of incompletely spliced mpmv mrnas. although ctes have now been defined in several other exogenous and endogenous retroviruses, not all ctes act by directly recruiting tap/p15. in particular, the avian leukemia virus cte does not appear to bind to tap or p15 directly, although tap may be required for its function (leblanc et al., 2007) . further analysis may reveal new insights into how the export of retroviral nuclear mrnas is regulated. after an mrna is exported to the cytoplasm, it must recruit cellular ribosomes in order for the translation of the encoded open reading frame to occur (figure 1 ). picornaviruses presented two mysteries in terms of how these pathogenic viruses are able to translate the single large polyprotein encoded by their positive-sense rna genome. first, the single genome-length picornavirus mrna is uncapped. second, infection by picornaviruses such as poliovirus results in the efficient translation of viral mrnas, yet cellular mrna translation is largely blocked. so, why is this uncapped viral mrna translated more efficiently than capped cellular mrnas? the key discovery that led to the resolution of this conundrum was the identification of the poliovirus internal ribosome entry site (ires), a ~450 nucleotide (nt) highly structured rna element found in the 5′ untranslated region (5′utr) of poliovirus mrnas (jang et al., 1988; pelletier and sonenberg, 1988) . the ires directly recruits several eifs and the 40s ribosomal subunit to an internal viral translation initiation codon without the requirement for either cap binding or 5′utr scanning. as a result, poliovirus translation is independent of the host cell cap recognition factor eif4e. moreover, although poliovirus translation initiation does require eif4g, it functions perfectly well with the carboxy-terminal fragment of eif4g that is generated by the proteolytic cleavage of eif4g by a virus-encoded protease. because capdependent translation requires fulllength eif4g, this cleavage blocks host cell translation, whereas viral mrna translation is not only unimpeded but in fact is enhanced by the access of viral mrnas to the entire pool of available eifs and ribosomal subunits (martinez-salas et al., 2008) . subsequent work has demonstrated that all picornaviruses as well as some flaviviruses, including hepatitis c virus (hcv), contain ires elements. surprisingly, these exist in several functionally distinct classes. for example, the hcv ires, unlike the poliovirus ires, can directly recruit 40s ribosomal subunits to the viral internal translation initiation codon in the absence of eifs, although eifs do participate in the process of translation initiation (martinez-salas et al., 2008 ). an even more unusual ires is found in cricket paralysis virus (crpv), a picornavirus-like insect virus (jan et al., 2003; pestova and hellen, 2003) . the crpv ires not only is able to recruit both the 40s and 60s ribosomal subunits to assemble elongation-competent 80s ribosomes on viral mrnas but also acts as a mimic of met-trnai to permit initiation of the translation of viral capsid proteins in the absence of met-trnai. although ires elements were first discovered in rna viruses, a subset of cellular mrnas are now known to also contain iress. interestingly, iress seem to be especially prevalent in mrnas whose expression is activated by stress, when cap-dependent translation may be inefficient. many ires-containing host mrnas encode proteins that protect cells from stress, whereas the proteins encoded by other ires-containing cellular mrnas seem to be important during apoptosis (bushell et al., 2006; komar and hatzoglou, 2005) . another interesting translational phenomenon observed in several virus families, including many species of retroviruses and all coronaviruses, is programmed ribosomal frameshifting (brierley and dos ramos, 2006) . in retroviruses such as hiv-1, frameshifting prevents some ribosomes from terminating translation at the end of the open reading frame (orf) encoding the gag structural protein and instead induces ribosomes to shift into the overlapping pol orf, resulting in the production of the large gag-pol polyprotein. similarly, in coronaviruses, ribosomal frameshifting is used to produce the 1a/1b replicase polyprotein rather than the shorter 1a variant. frameshifting is induced by a bipartite element consisting of a 5′ frameshifting site and an adjacent 3′ rna structure (brierley and dos ramos, 2006; jacks et al., 1988) . the frameshift site has the consensus sequence x_xxy_yyz (where the translational phase is indicated), which then slips into the −1 frame, that is, xxx_yyy_z. the actual shift sites found in hiv-1 and the sars coronavirus are u_uuu_uua and u_uua_aac, respectively. the 3′ rna structure found in hiv-1 is thought to be a simple rna hairpin but other −1 frameshifting signals instead contain a pseudoknot 3′ to the frameshift signal (brierley and dos ramos, 2006) . it has been proposed that the function of the 3′ rna structure is to induce transient ribosomal pausing at the frameshift site to facilitate ribosomal slippage in the −1 direction. although frameshifting in hiv-1 occurs with an efficiency of ~5%, frameshifting efficiency in other viruses can be as high as ~25% and may be facilitated by a direct interaction between the paused ribosome and the downstream pseudoknot structure. programmed frameshifting is not unique to viruses but is found also in a small number of cellular genes in both eukaryotes and bacteria (shigemoto et al., 2001; tsuchihashi and kornberg, 1990) . viruses, rna interference, and micrornas rna interference (rnai) was first discovered by genetic analysis in nematodes (fire et al., 1998) ; however, it is likely that rnai first evolved as an innate immune response to viral infection. indeed, rnai continues to represent a key component of the antiviral response in plants and invertebrates (cullen, 2006) . the triggers for rnai in these species are the long double-stranded rnas (dsrnas) that form critical intermediates in the replication of all rna viruses except retroviruses. these dsrnas are bound by the rnase iii-related enzyme dicer, which progressively cleaves these dsrnas into ~22 bp rna duplexes containing terminal 2 nt 3′ overhangs (see review by r.w. carthew and e.j. sontheimer in this issue of cell). one strand of this duplex, called a small-interfering rna (sirna), is then incorporated into the rna-induced silencing complex (risc), where it acts as a guide for rna to target risc to complementary regions of viral genomic, anti-genomic, or mrna species. risc then cleaves these viral rnas, leading to their degradation. the first sirnas to be identified were in fact antiviral sirnas produced in tobacco cells infected by a pathogenic rna virus, potato virus x (hamilton and baulcombe, 1999) . rnai is a critical component of the antiviral immune response in plants and invertebrates, but emerging evidence indicates that rnai responses to viral infection are not induced in mammalian somatic cells (cullen, 2006) . instead, mammalian cells have evolved other innate responses that are induced by viral dsrnas, including the interferon response. because of the importance of rnai as an antiviral defense in plants and insects, many rna viruses that infect these species have evolved gene products that inhibit rnai and, hence, enhance virus replication. conversely, the absence of antiviral rnai responses in mammalian cells means that the rnai machinery in these cells generally remains active during viral infection (figure 1) . although the role of rnai as an antiviral response appears to have been lost in mammalian somatic cells, the residual rnai machinery still plays a very important role by mediating the function of cellular micrornas (mirnas). unlike sirnas, which are derived from long dsrnas (frequently of exogenous origin), mirnas are encoded within the cell's genome as part of one arm of an ~80 nt rna hairpin located in a larger rnap ii transcript called a primary mirna (bartel, 2004) . after excision, by the sequential action of the host cell rnase iii-related enzymes drosha and dicer, mirnas are loaded into risc and downregulate the expression of cellular mrnas. unlike viral mrna targets of viral sirnas, cellular mrnas are rarely fully complementary to cellular mirnas. as full complementarity is a prerequisite for efficient cleavage by risc, cellular mrnas are generally not subject to degradation by cellular mirnas. instead, cellular mirnas can induce the translational inhibition of cellular mrnas by binding to partially complementary target sites (bartel, 2004) . as most mammalian viruses do not seem to interfere with the loading or function of risc, mirnas remain active in infected cells, thus offering the possibility for viruses to use the cellular rnai machinery to regulate cellular or viral gene expression by programming risc with viral mirnas. analysis of a range of virally infected cells has revealed that several dna viruses, including herpesviruses, encode multiple distinct mirnas. of note, most viral mirnas appear to be processed by the same drosha and dicer dependent pathway used by the majority of cellular mirnas, although there are a few examples of viral mirnas that are transcribed by rna polymerase iii, not rnap ii, and then excised directly by dicer (gottwein and cullen, 2008) . similarly, riscs programmed by viral mirnas appear to function in the same way as riscs programmed by cellular mirnas. although beyond the scope of this article, it is interesting to note that several cellular and viral mrna targets of viral mirnas have now been defined (gottwein and cullen, 2008) . in general, it appears that viral mirnas either downregulate cellular or viral genes that increase the sensitivity of virally infected cells to host innate or adaptive immune responses or, in the case of herpesvirus mirnas, stabilize viral latency by downregulating the expression of the viral immediate early proteins, which favor entry into the lytic replication cycle. in addition to mirnas, a number of dna viruses also encode long noncoding rnas that play a role in regulating viral replication and pathogenesis (table 2 ; reviewed in sullivan and cullen, 2009; see review by c.p. ponting, p.l. oliver, and w. reik in this issue of cell). but how do viruses use noncoding rnas to promote their replication? one interesting noncoding rna is the latency associated the instability of the 6.3 kb lat rna appears to be due to the fact that it is processed into several viral mirnas that may play a key role in regulating hsv-1 latency (umbach et al., 2008) . the role of the stable 2 kb lat intron is less clear, but evidence has been presented arguing that the lat intron is exported out of the nucleus by crm1 and associates with cellular ribosomes, thus suggesting a role in modulating mrna translation in neurons latently infected with hsv-1 (atanasiu and fraser, 2007) . another interesting viral noncoding rna is the polyadenylated nuclear (pan) rna encoded by kaposi's sarcomaassociated herpesvirus (kshv). pan is an unspliced rnap ii transcript that is the most highly expressed viral rna during lytic kshv infection, comprising up to 80% of all viral rnas. remarkably, the function of this rna in the viral life cycle is still unclear. however, recent data demonstrate that pan contains a novel ~80 nt-long rna element that stabilizes pan rna in the infected cell nucleus. insertion of this viral rna element in cis also increases the nuclear abundance of cellular mrnas, such as β-globin mrnas, that are normally unstable when expressed in an intronless form (conrad et al., 2006) . it is unclear whether this element simply stabilizes pan rnas or whether it also acts in trans to stabilize other kshv-coding mrnas, which are also largely intronless. finally, several viral noncoding rnas seem to be inhibitors of cellular innate antiviral immune responses. for example, the β2.7 noncoding rna encoded by human cytomegalovirus (hcmv) binds to mitochondrial enzyme complex i of the host cell. this interaction stabilizes the production of atp in infected cells and also inhibits virally induced apoptosis (reeves et al., 2007) . another noncoding rna, the va1 rna expressed by adenovirus, also binds to a cellular factor to inhibit an antiviral response. in this case, the target is protein kinase r (pkr), a cellular protein that binds to the long dsrnas produced by adenoviruses and many other pathogenic viruses. binding of dsrna by pkr induces pkr dimerization and autophosphorylation as well as phosphorylation of cellular eif2α, which results in a global inhibition of translation in the infected host cell. va1, a highly structured ~160 nt-long noncoding rna, binds to pkr with high affinity and blocks pkr dimerization and activation. this prevents the inhibition of translation induced by adenovirus-derived dsrnas and allows virus replication to proceed unimpeded (mathews and shenk, 1991) . interestingly, both hcmv β2.7 and adenovirus va1, like kshv pan and hsv-1 lat, are also expressed at high levels in infected cells. β2.7 comprises up to 20% of all viral transcripts in hcmv-infected cells, and adenovirus va1 is expressed at an extraordinarily high number of copies (~10 8 ) per infected cell. presumably, these high expression levels facilitate the saturation of cellular binding sites for these rnas. efforts to understand the replication cycles of viruses are often motivated by the pathogenic potential of these intracellular parasites. however, such analyses have also led to several key insights into how not only infected cells but also uninfected cells regulate the expression of their genome. moreover, as noted in the brief discussion of viral noncoding rnas, our knowledge of how virally encoded transcripts work in the host cell remains far from complete. clearly, future research into virus replication will provide unexpected and exciting insights into the complex molecular machinery that makes human cells tick. proc. natl. acad. sci. usa 91 proc. natl. acad. sci. usa proc. natl. acad. sci. usa non-coding regulatory rnas of the dna tumor viruses proc. natl. acad. sci. usa proc. natl. acad. sci. usa key: cord-264050-6zpw6itb authors: pirofski, liise-anne; casadevall, arturo title: immune-mediated damage completes the parabola: cryptococcus neoformans pathogenesis can reflect the outcome of a weak or strong immune response date: 2017-12-12 journal: mbio doi: 10.1128/mbio.02063-17 sha: doc_id: 264050 cord_uid: 6zpw6itb cryptococcosis occurs most frequently in immunocompromised individuals. this has led to the prevailing view that this disease is the result of weak immune responses that cannot control the fungus. however, increasingly, clinical and experimental studies have revealed that the host immune response can contribute to cryptococcal pathogenesis, including the recent study of l. m. neal et al. (mbio 8:e01415-17, 2017, https://doi.org/10.1128/mbio.01415-17) that reports that cd4(+) t cells mediate tissue damage in experimental murine cryptococcosis. this finding has fundamental implications for our understanding of the pathogenesis of cryptococcal disease; it helps explain why immunotherapy has been largely unsuccessful in treatment and provides insight into the paradoxical observation that hiv-associated cryptococcosis may have a better prognosis than cryptococcosis in those with no known immune impairment. the demonstration that host-mediated damage can drive cryptococcal disease provides proof of concept that the parabola put forth in the damage-response framework has the flexibility to depict complex and changing outcomes of host-microbe interaction. c ryptococcosis is usually observed in hosts with impaired immunity. consequently, the agent causing cryptococcosis, cryptococcus neoformans, is often considered pathogenic only when the immune system is unable to control its growth. this has led to its characterization as an "opportunistic pathogen" (1) . in their article in mbio, l. m. neal et al. (2) report that the cd4 ϩ t cell-mediated response to c. neoformans is a major contributor to tissue damage in cryptococcal meningitis in mice even though it also mediates fungal clearance. this observation adds to increasing evidence that the host response can drive cryptococcal disease and highlights the ability of the damageresponse framework (drf) to guide our understanding of microbial pathogenesis. the drf was first put forth in 1999 (3) to provide a theory of microbial pathogenesis that could incorporate the contributions of both host and microbe to host damage that stems from host-microbe interaction. prior to the drf, microbial pathogenesis was largely viewed as a singular outcome of either microbial factors or host factors. while such microbe-or host-centered views were able to explain the pathogenesis of certain infectious diseases, they could not explain others, especially those caused by microbes only rarely associated with disease. this shortcoming became glaring in the late 1970s and early 1980s as the hiv/aids pandemic led to the emergence of previously rare and unusual diseases, including cryptococcosis (4). in the original formulation of the drf, the outcome of host-microbe interaction with different microbes was depicted by six curves that plotted host damage as a function of the strength of the immune response. these curves, referred to as pathogen classes, were based on what was known at the time about the outcome of infection with given microbes. the rationale for the pathogen classes was underpinned by the tenet that host damage can stem from microbial factors, host factors, or both. central to this tenet was the idea that host damage stemming from the immune response to a microbe can drive disease pathogenesis. at the time the drf was proposed, the host inflammatory response was not generally viewed as a causal factor in the pathogenesis of infectious diseases. however, this has changed. the occurrence of severe acute respiratory syndrome (sars) in young, previously well persons who presented with excessive pulmonary inflammation (5), reminiscent of influenza epidemics that struck young, robust persons (6), highlighted the role that host-mediated damage can play in viral disease pathogenesis. the ability of the drf to account for host damage due to inflammation stemming from the immune response to certain microbes (3) highlighted its flexibility and capacity to incorporate new diseases and information. despite the ability of the drf to classify most microbes into one of the original six pathogen classes, new knowledge from clinical and experimental studies led to the conclusion that some of the original classifications were incorrect. again, lessons learned from the hiv/aids pandemic provided new insights into microbial pathogenesis, perhaps best exemplified by cryptococcosis. c. neoformans was first classified as a class 2 pathogen, the definition of which was "pathogens that cause damage either in hosts with weak immune responses or in the setting of normal immune responses" (3). this characterization was consistent with available knowledge in 1999. however, the emergence of cryptococcus gattii in apparently healthy persons in the pacific northwest (7) and the unexpected appearance of immune reconstitution inflammatory syndrome (iris)-associated cryptococcosis in patients with hiv/aids after initiation of antiretroviral therapy (art) (8, 9) revealed that the host immune response itself can contribute to the pathogenesis of cryptococcosis. thus, classification of c. neoformans as a class 2 pathogen needed to be revisited. another example of a pathogen needing reclassification is pneumocystis jirovecii, which was originally classified as a class 1 pathogen, defined as "pathogens that cause damage only in the setting of weak immune responses" (3). however, pneumocystis pneumonia is associated with intense inflammation in children and after initiation of therapy in patients with hiv/aids (10, 11) . in fact, a major advance in treating pneumocystis pneumonia was the realization that corticosteroid therapy reduced morbidity and mortality, a finding that was counterintuitive, since this disease arose in the setting of profound hiv-associated immunodeficiency. thus, inflammation may drive pneumocystis-and cryptococcus-mediated damage, even in patients with severely impaired immunity, defying their initial classifications as class 1 and 2 pathogens, respectively. although one might propose that these microbes be reclassified as class 3 pathogens, "pathogens that cause damage in the setting of appropriate immune responses and cause damage at both ends of the continuum of immune response" (3), most persons with normal immune responses never exhibit clinically detected damage as an outcome of interaction with these microbes. these examples highlight limitations of the original drf pathogen classes and illustrate that each of the six classes is actually a derivative of a single parabola that can shift to the left, right, up, or down to depict the impact of the immune response on disease pathogenesis (fig. 1) . the emergence of iris-associated cryptococcosis in the setting of art initiation provided clear evidence that host damage in patients with cryptococcal disease may be driven by inflammation (12) . iris often occurred in patients with very low initial cerebrospinal fluid (csf) lymphocyte counts (8) , and has been associated with chemokine dysregulation (13) and activated cd4 ϩ t cells and macrophages (12, 14, 15) . similar findings have also been reported in some non-hiv-infected patients with cryptococcal meningitis (16, 17) . a role for cd4 ϩ t cells in the pathogenesis of cryptococcusassociated iris has also been demonstrated in experimental mouse models. eschke and colleagues induced iris in rag1 ϫ/ϫ mice by reconstituting them with naive (wild-type) cd4 ϩ t cells 1 month after pulmonary infection with 500 cfu of c. neoformans (18) . in this model, mice developed wasting and systemic inflammation evidenced by high levels of th1-type cytokines (interleukin 6 [il-6], tumor necrosis factor alpha [tnf-␣], and gamma interferon [ifn-␥]), activated cd4 ϩ t cells, and granulomatous inflammation in the liver compared to control mice that did not receive cd4 ϩ t cells (and mice that received cd4 ϩ t cells and were not infected). reconstitution did not affect fungal clearance. this model demonstrated that cd4 ϩ t cell reconstitution induced systemic inflammation in c. neoformans-infected mice that closely resembled art-induced iris in hiv-infected patients with cryptococcosis. neal and colleagues (2) describe another mouse model in which cd4 ϩ t cells mediate inflammation and host damage in the setting of c. neoformans infection. their study was undertaken to gain insight into the role that cd4 ϩ t cells may play in the pathogenesis of iris-and postinfectious inflammatory syndromes (piirs) in hivinfected and hiv-uninfected patients with cryptococcosis. mice infected intravenously with 10 6 cfu of c. neoformans were monitored clinically or analyzed to determine their central nervous system (cns) fungal burden (cfu), inflammatory pathology, and cellular and cytokine responses. the number of c. neoformans cfu in the brains of mice increased from 1 to 3 weeks after infection and then decreased from 3 to 5 weeks thereafter. interestingly, the mice developed symptomatic disease and neurological deterioration 3 to 4 weeks after infection, corresponding to fungal clearance and surprisingly, mortality. cns pathology revealed cellular inflammation marked by leukocytes beginning 3 weeks after infection that subsequently increased due to infiltration of cd4 ϩ and cd8 ϩ t cells, with cd4 ϩ t cells being the predominant population and both populations being antigen experienced. infiltrating cd4 ϩ and cd8 ϩ t cells exhibited a th1-type bias, producing ifn-␥. depletion of cd4 ϩ t cells resulted in reduced mortality and inflammatory pathology, providing proof of concept that cd4 ϩ outcomes of host-cryptococcus neoformans interaction depicted by the basic parabola of the damage-response framework. the left side of the parabola, shaded in green and labeled weak, depicts the original circa 1999 concept that c. neoformans was a class 2 pathogen that caused damage in the setting of a weak or normal immune response. here, the immune response fails to limit fungal growth, which results in host damage. the right side of the parabola, shaded in red and labeled strong, depicts information that has emerged since 1999, which has revealed that c. neoformans can elicit strong immune responses that produce host damage stemming from inflammation. the portion of the parabola that rises above the black dotted line represents the threshold for clinical disease. the portion of the parabola that lies below the black dotted line represents the state of c. neoformans latency, which is not associated with clinically evident host damage. proposed therapeutic interventions, based on counteracting the main source of host damage are depicted by red (to enhance weak response) or green (to reduce strong response) arrows. some factors that contribute to weak and strong immune responses are shown along the y axis for each type of response, though immunity is likely multifactorial, and additional factors are also likely to contribute (16, 29 -35) . commentary ® t cells were the principal mediators of inflammation and damage in this model. notably, survival of infected, cd4 ϩ t cell-depleted mice was improved despite an inability to induce fungal clearance and markedly elevated fungal burdens compared to mice with sufficient cd4 ϩ t cells. the data generated by neal et al. (2) demonstrate that cd4 ϩ t cells can play dichotomous roles in c. neoformans infection. on one hand, they can mediate fungal clearance in the cns, but on the other hand, they can induce inflammation that leads to neurological deterioration and death. this suggests a possible explanation for why inflammation is often a prominent feature of cryptococcal meningitis in hiv-uninfected, but not hiv-infected patients, and raises the intriguing possibility that the profound cd4 ϩ t cell deficiency, which portends risk for cryptococcosis, may actually limit inflammatory pathology in hiv-infected persons not on art. this dichotomy may also provide insight into the failure of adjunctive steroid therapy to reduce mortality or improve morbidity in hiv-associated cryptococcosis (19) and the fact that cryptococcosis may have a better prognosis in immunosuppressed patients than in nonimmunosuppressed patients (20) (21) (22) . the detrimental effect of activated cd4 ϩ t cells in mice also suggests the possibility that antifungal therapy-induced fungal clearance and reduced levels of glucuronoxylomannan (gxm) (an immune suppressing molecule [23] [24] [25] ), could enhance cd4 ϩ t cell activation and damage. this may help explain paradoxical worsening of cryptococcosis in certain patients after initiation of antifungal therapy (26) . the aforementioned experimental models provide possible mechanistic explanations for the dichotomous role that the immune response may play in the pathogenesis of iris-and piirs-associated cryptococcosis while contributing to fungal clearance. thus, microbe and host both contribute to host damage and where an individual patient's immune response lies on the continuum of the drf parabola determines the nature of the disease process. at one end, on the left, a weak immune response can result in tissue damage due to fungal proliferation that results in compression of brain tissue with little or no inflammation in what is primarily microbe-mediated damage. at the other end, on the right, a strong cellular immune response can also damage brain tissue due to excessive inflammation in what is primarily host-mediated damage. the occurrence of host damage in the setting of weak and strong immune responses provides important clues as to why it has been so difficult to consistently use agents that modulate inflammation, such as corticosteroids and interferon, to improve the outcome of cryptococcosis. although adjunctive ifn-␥ therapy in hiv-associated cryptococcosis (27) and corticosteroids can ameliorate iris (28) in some patients, such immune-modulating agents may not be effective in other patients, because physicians do not usually know whether the preponderance of damage is due to host factors, microbial factors, or both. in this regard, the failure and detrimental effects of steroid therapy in patients with hiv-associated cryptococcosis (19) probably represent additional suppression of weak immunity resulting in enhancement of fungus-mediated damage. hence, the effective use of immunotherapy is likely to require a rational approach that incorporates knowledge of where the immune response of an individual lies along the immune response co ntinuum depicted by the drf parabola. host-pathogen interactions: the attributes of virulence cd4 ϩ t cells orchestrate lethal immune pathology despite fungal clearance during cryptococcus neoformans meningoencephalitis host-pathogen interactions: redefining the basic concepts of virulence and pathogenicity what is a host? incorporating the microbiota into the damage-response framework a clinicopathological study of three cases of severe acute respiratory syndrome (sars) the pathogenesis of influenza virus infections: the contributions of virus and host factors spread of cryptococcus gattii into pacific northwest region of the united states paucity of initial cerebrospinal fluid inflammation in cryptococcal meningitis is associated with subsequent immune reconstitution inflammatory syndrome international network for the study of hiv-associated iris (inshi). 2010. cryptococcal immune reconstitution inflammatory syndrome in hiv-1-infected individuals: proposed clinical case definitions the expanding role of co-trimoxazole in developing countries anti-inflammatory treatment of acute and chronic pneumonia immune reconstitution inflammatory syndrome in hiv-associated cryptococcal meningitis: a prospective study chemokine levels and chemokine receptor expression in the blood and the cerebrospinal fluid of hiv-infected patients with cryptococcal meningitis and cryptococcosis-associated immune reconstitution inflammatory syndrome cellular immune activation in cerebrospinal fluid from ugandans with cryptococcal meningitis and immune reconstitution inflammatory syndrome the immunopathogenesis of cryptococcal immune reconstitution inflammatory syndrome: understanding a conundrum paradoxical immune responses in non-hiv cryptococcal meningitis fighting the monster: applying the host damage framework to human central nervous system infections a novel experimental model of cryptococcus neoformans-related immune reconstitution inflammatory syndrome (iris) provides insights into pathogenesis adjunctive dexamethasone in hiv-associated cryptococcal meningitis predictors of mortality and differences in clinical features among patients with cryptococcosis according to immune status different presentations and outcomes between hivinfected and hiv-uninfected patients with cryptococcal meningitis outcomes of central nervous system cryptococcosis vary with host immune function: results from a multi-center, prospective study effects of the capsular polysaccharides of cryptococcus neoformans on phagocyte migration and inflammatory mediators downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes purified capsular polysaccharide of cryptococcus neoformans induces interleukin-10 secretion by human monocytes an immune reconstitution syndrome-like illness associated with cryptococcus neoformans infection in organ transplant recipients adjunctive interferon-gamma immunotherapy for the treatment of hiv-associated cryptococcal meningitis: a randomized controlled trial central nervous system immune reconstitution inflammatory syndrome naive b cells reduce fungal dissemination in cryptococcus neoformans infected rag1ϫ/ϫ mice igm ϩ memory b cell expression predicts hiv-associated cryptococcosis status susceptibility to cryptococcal meningoencephalitis associated with idiopathic cd4 ϩ lymphopenia and secondary germline or acquired defects fc gamma receptor 3a polymorphism and risk for hiv-associated cryptococcal disease study of common functional genetic polymorphisms of fcgr2a, 3a and 3b genes and the risk for cryptococcosis in hiv-uninfected patients anti-gm-csf autoantibodies in patients with cryptococcal meningitis genotypes coding for mannose-binding lectin deficiency correlated with cryptococcal meningitis in hiv-uninfected chinese patients l.p. is supported in part by nih grant r01ai97096. a.c. is supported in part by nih grants 5r01hl059842, 5r01ai033774, 5r37ai033142, and 5r01ai052733. key: cord-269222-g2ibmo75 authors: valenti, piera; rosa, luigi; capobianco, daniela; lepanto, maria stefania; schiavi, elisa; cutone, antimo; paesano, rosalba; mastromarino, paola title: role of lactobacilli and lactoferrin in the mucosal cervicovaginal defense date: 2018-03-01 journal: front immunol doi: 10.3389/fimmu.2018.00376 sha: doc_id: 269222 cord_uid: g2ibmo75 the innate defense system of the female mucosal genital tract involves a close and complex interaction among the healthy vaginal microbiota, different cells, and various proteins that protect the host from pathogens. vaginal lactobacilli and lactoferrin represent two essential actors in the vaginal environment. lactobacilli represent the dominant bacterial species able to prevent facultative and obligate anaerobes outnumber in vaginal microbiota maintaining healthy microbial homeostasis. several mechanisms underlie the protection exerted by lactobacilli: competition for nutrients and tissue adherence, reduction of the vaginal ph, modulation of immunity, and production of bioactive compounds. among bioactive factors of cervicovaginal mucosa, lactoferrin, an iron-binding cationic glycoprotein, is a multifunctional glycoprotein with antibacterial, antifungal, antiviral, and antiparasitic activities, recently emerging as an important modulator of inflammation. lactobacilli and lactoferrin are largely under the influence of female hormones and of paracrine production of various cytokines. lactoferrin is strongly increased in lower genital tract mucosal fluid of women affected by neisseria gonorrheae, chlamydia trachomatis, and trichomonas vaginalis infections promoting both innate and adaptive immune responses. in vaginal dysbiosis characterized by low amounts of vaginal lactobacilli and increased levels of endogenous anaerobic bacteria, the increase in lactoferrin could act as an immune modulator assuming the role normally played by the healthy microbiota in vaginal mucosa. then lactoferrin and lactobacilli may be considered as biomarkers of altered microbial homeostasis at vaginal level. considering the shortage of effective treatments to counteract recurrent and/or antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lactoferrin could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis. the innate defense system of the female mucosal genital tract involves a close and complex interaction among the healthy vaginal microbiota, different cells, and various proteins that protect the host from pathogens. vaginal lactobacilli and lactoferrin represent two essential actors in the vaginal environment. lactobacilli represent the dominant bacterial species able to prevent facultative and obligate anaerobes outnumber in vaginal microbiota maintaining healthy microbial homeostasis. several mechanisms underlie the protection exerted by lactobacilli: competition for nutrients and tissue adherence, reduction of the vaginal ph, modulation of immunity, and production of bioactive compounds. among bioactive factors of cervicovaginal mucosa, lactoferrin, an iron-binding cationic glycoprotein, is a multifunctional glycoprotein with antibacterial, antifungal, antiviral, and antiparasitic activities, recently emerging as an important modulator of inflammation. lactobacilli and lactoferrin are largely under the influence of female hormones and of paracrine production of various cytokines. lactoferrin is strongly increased in lower genital tract mucosal fluid of women affected by neisseria gonorrheae, chlamydia trachomatis, and trichomonas vaginalis infections promoting both innate and adaptive immune responses. in vaginal dysbiosis characterized by low amounts of vaginal lactobacilli and increased levels of endogenous anaerobic bacteria, the increase in lactoferrin could act as an immune modulator assuming the role normally played by the healthy microbiota in vaginal mucosa. then lactoferrin and lactobacilli may be considered as biomarkers of altered microbial homeostasis at vaginal level. considering the shortage of effective treatments to counteract recurrent and/or antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lactoferrin could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis. keywords: lactobacilli, lactoferrin, cervicovaginal defense, vaginal homeostasis, inflammation introduction the basic structures of the female reproductive system are ovaries, fallopian tubes, uterus, cervix, and vagina. ovaries are responsible for the production of the ovum and secrete both estrogen and progesterone. when an ovum is developing in an ovary, it is encapsulated in a sac known as an ovarian follicle. on maturity of the ovum, the follicle and the ovary's wall rupture, allowing the ovum to escape and enter the fallopian tube to reach uterus. the uterus is a muscular organ useful to accept a fertilized ovum, which becomes implanted into the endometrium. the cervix is the neck of the uterus, which protrudes through the upper anterior vaginal wall. the vagina is a fibromuscular canal that connects the upper part of female genital tract to the outside of the body and represents the portal of entry of pathogenic microorganisms. the epithelial mucosa of the lower genital tract is extensively colonized by commensal microorganisms, while the tissues of the upper genital tract are generally considered to be sterile (1) . however, bacterial colonization of the upper genital tract of healthy asymptomatic women remains a somehow controversial issue (1) . the vaginal tract is colonized by microorganisms, recognized as the vaginal microbiota (vm). these microorganisms, in addition to a complex synergism among secretion's proteins and peptides, epithelial, and immune cells, perform a pivotal role in the defense of female genital tract against infectious and inflammatory processes. in the state of mucosal health, the various components are in balance. the rupture of mucosal homeostasis determined by the alteration of one of the various actors often results in an increased host susceptibility to infections. healthy vm is dominated by lactobacillus spp., but other microorganisms can be present at lesser extent (gardnerella, prevotella, streptococcus, ureaplasma, peptostreptococcus, staphylococcus, corynebacterium, clostridium, mycoplasma, enterococcus, bacteroides, escherichia, bifidobac terium, veillonella, and candida) (2) (3) (4) (5) (6) . over 20 species of lactobacillus have been detected in the vagina. however, in the majority of women, the healthy vaginal microflora contains one or two lactobacillus species among lactobacillus crispatus, lactobacillus gasseri, lactobacillus jense nii, and lactobacillus iners (7, 8) . currently, the role of l. iners in vaginal health is still unclear (9) . indeed, l. iners has been recently detected in both dysbiotic and healthy women, and its presence and amount are inversely correlated with l. crispatus (8, 10, 11) . lactobacilli are involved in maintaining the healthy vaginal environment by counteracting overgrowth of other resident microorganisms (12) . lactobacilli can also colonize the human cervix. in different studies, a range of 29-52% of women resulted colonized by lactobacilli in the cervix, and within a single subject, usually the same lactobacillus strains colonized both the cervix and the vaginal tract (13) (14) (15) . lactobacilli exert their protective effects by several mechanisms: (i) microbial competition for the nutrients and for adherence to the vaginal epithelium; (ii) reduction of the vaginal ph by the production of organic acids, especially lactic acid, through the degradation of glycogen released by vaginal cells thus exerting selective antimicrobial activity against non-resident microbiota; (iii) production of antimicrobial substances, such as bacteriocins and hydrogen peroxide (h2o2) able to suppress the growth of several microorganisms; and (iv) modulation of the local immune system (16) . homeostasis of vaginal environment results from complex interactions and synergies among the host and different microorganisms that colonize the vaginal mucosa, and the maintenance of high numbers of resident lactobacilli is an effective hallmark of woman's health and a wellorganized protection against pathogens causing sexually tranmitted infections (stis). abnormal vm involving a strong reduction or disappearance of lactobacilli characterizes a pathologic condition known as bacterial vaginosis (bv) that afflicts fertile, premenopausal, and pregnant women with an incidence rate ranging from 20 to 50% (17) . bv is a polymicrobial clinical syndrome resulting from the replacement of the normal lactobacillus spp. with high number of anaerobic bacteria such as gardnerella vaginalis, prevotella spp., mobiluncus spp., ureaplasma, mycoplasma, and other fastidious or not culturable anaerobes (5, 18) . in bv, the overgrowing anaerobes produce compounds such as polyamines and other molecules capable of inducing the release of proinflammatory cytokines such as il-1β, il-6, and il-8 (19, 20) . bv represents an independent risk factor for severe reproductive tract sequelae associated with pelvic inflammatory disease and tubal factor infertility (21, 22) . changes in the vm have been also associated with obstetrical complications such as late miscarriage and premature birth (23), thus exerting a profound impact also on the health of newborns. moreover, women with lactobacillus poor flora show an increased susceptibility to sexually transmitted pathogens. several studies indicate that abnormal vm lacking lactobacilli is associated with the acquisition of infections by neisseria gonorrhoeae, chlamydia trachomatis, and trichomonas vaginalis (24) (25) (26) (27) (28) . furthermore, the alterations in vm are associated with increased risk of acquiring viral sexually transmitted diseases (stds). indeed, longitudinal and cross-sectional studies demonstrated the association between altered vm and the increased prevalence/incidence of many viral stis such as human immunodeficiency virus (hiv), herpes simplex virus (hsv), human papillomavirus, and cytomegalovirus infection (29) . in addition to lactobacilli, the cervicovaginal fluid (cvf) exerts a significant microbicidal activity against gram-positive and gram-negative bacteria, fungi, and certain viruses as well as an anti-inflammatory activity through several peptides and proteins, all characterized by common cationic features (30) . the main antimicrobial peptides and proteins present in the cvf are shown in table 1 (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) . these peptides and proteins act through different mechanisms: (i) microbial lysis; (ii) depletion of environmental nutrients essential for microbial growth; (iii) competitive binding to host cells; (iv) degradation of negatively charged microbial surface components; (v) interference with host cell signaling pathways; and (vi) modulation of inflammation and other functions involved in host defense (34, 47) . the bactericidal activity of many of these compounds is strictly associated with their cationic features. the concentration of some of these molecules in cvf is lower than that required for in vitro inhibition of pathogens; however, it is known that several antimicrobials display synergistic effects. indeed, human β defensin 2 and cathelicidin antimicrobial peptide ll-37 (48) , secretory leukocyte protease inhibitor (slpi) and lysozyme (49) , and lactoferrin (lf) and lysozyme (49) display synergistic effects that potentially increase innate immune protection in the female reproductive tract (50) . many of these antimicrobial/immunomodulatory compounds appear to be under hormonal control (31) . in cvf α and β defensins, slpi and lysozyme levels are high during the proliferative phase, greatly decrease at mid-cycle/ovulation, and increase again during the late secretory phase. lactoferrin, belonging to transferrin family, is a multifunctional glycoprotein of about 690 amino acids and a mw of (44, 51, 52) . lf, abundantly found in most biological fluids of mammals, is synthesized by exocrine glands, many mucosal epithelial cells and released by neutrophils during inflammation. the highest level of the human lf (hlf) is found in colostrum (7 mg/ml), while decreases in mature milk (1.5-4.0 mg/ml). in the tears, hlf is detected at low concentration (about 2.0 mg/ml), while in saliva, small intestine, earwax, vaginal fluid, amniotic fluid, upper airway fluid, seminal plasma, and the cervical mucus at very low levels (<0.1 mg/ml) (53) . in particular, the concentration of hlf in human vaginal fluid corresponds to 1-3 µg/ml, while it is extremely high (100 µg/ml) in the cervical mucus plug (30, 54, 55) . of note, a total number of 10 6 neutrophils release 15 µg of hlf in sites of inflammation and infection (53) . in human body fluids, the concentration of free available iron must not overcome 10 -18 m to avoid microbial multiplication and to hinder the precipitation of insoluble ferric hydroxides as well as the formation of reactive oxygen species. hlf, by its iron-binding ability, guarantees that free available iron does not exceed 10 -18 m. hlf and bovine milk derivative lf (blf) possess high homology of sequence. from three-dimensional structure, lf is folded into two homologous lobes, each structured in two domains (n1 and n2, c1 and c2). one fe (iii) ion is chelated by each lobe. when lf is completely iron saturated, its conformation appears in a closed state, more resistant to proteolytic enzymes than the unsaturated open form (51) . the low iron availability (10 -18 m), hindering microbial growth, is a signal of health and wellness, while iron concentration higher than 10 -18 m favors not only microbial replication but also the biofilm formation (56, 57) and persistence (44) . interestingly, wiesner and vilcinskas have reported that proteins and peptides of mucosal secretions possess several functions (58) . accordingly, hlf and blf are multifunctional glycoproteins effective against bacteria, mycetes, viruses, and parasites, possessing also anti-inflammatory and immunomodulatory properties (44) . in particular, blf, available in large quantities and recognized by food and drug administration (fda, usa) as a safe substance, is the main lf used in in vitro studies (44) as well as in clinical trials (52, (59) (60) (61) (62) and in mice (63) . the level of vaginal hlf and others antimicrobial peptides change in response to microbial infections. it has been demonstrated that hlf and defensin levels increase in genital secretion of women with c. trachomatis, n. gonorrhoeae, t. vaginalis, or candida spp. infection and bv in comparison to healthy condition (64, 65) . lactobacilli the innate defense system of the female mucosal genital tract involves a complex interaction among the healthy vaginal flora, immune cells, and several proteins that defend the host from pathogens. it is broadly suggested that the crucial role of vaginal lactobacilli is to protect female genital tract through the production of lactic acid responsible for low vaginal ph that inhibits sexually transmitted pathogens. lactic acid is in equilibrium with lactate anion. the former is the predominant form in healthy vaginal conditions and low ph (<4.5), thus exerting antimicrobial activity against pathogens. lactate anion predominates at higher ph (>4.5) conditions in women with dysbiosis (66) . in vitro experiments demonstrated that at physiological concentrations (55-111 mm) of lactic acid and at ph 4.5, bv-associated bacteria such as g. vaginalis and atopobium vaginae were inactivated without effects on typical vaginal species of lactobacilli (67) . furthermore, inactivation of bv-associated species was dependent on lactic acid itself rather than ph, since a ph value of 4.5 determined by other acids was significantly less microbicidal. recent in vitro studies, however, demonstrated that c. trachomatis multiplication is inhibited by different strains of vaginal lactobacilli, independently from ph alterations (68) . c. trachomatis, an obligate intracellular pathogen, responsible for the most common bacterial std worldwide, causes acute and chronic infections. unlike acute infections, which can be cured with oral or topical administration of antibiotics, chronic infections are difficult to eradicate and need prolonged therapies, thus increasing the risk of developing antibiotic resistance (69) . therefore, novel alternative therapies are needed. the difficulty in finding new agents against c. trachomatis infection resides in the complex life cycle of this peculiar pathogen. in fact, c. trachomatis has a unique biphasic developmental cycle, alternating between the extracellular infectious elementary bodies (ebs), metabolically inactive, and the intracellular non-infectious reticular bodies (rbs), which are metabolically active. it has been recently demonstrated that vaginal lactobacilli inhibit ebs adhesion to epithelial cells as well as the intracellular rbs replication (68) . the effect on the early phases of infection was related both to co-aggregation between lactobacilli and c. trachomatis and to competition for epithelial cell adhesion. the inhibition of chlamydial infection by lactobacilli was strain and dose dependent, suggesting that the strains and the amount of lactobacilli in the vagina are responsible for the protection from chlamydial infection. lactobacilli have been demonstrated to protect lower female genital tract also from n. gonorrhoeae infection, the second most common bacterial sti. the interaction between bacteria and host cells determines the success of the pathogen mucosal colonization or its elimination through the continuous fluid flow. in this respect, vielfort et al. (15) showed that lactobacilli compete with n. gonorrhoeae for adhesion to human cervical cells. it has been also demonstrated that l. jensenii atcc 25258 could reduce both adhesion and invasion of n. gonorrhoeae, whereas l. gasseri atcc 33323 could displace adherent gonococci from the cell surface (70) . the protection exerted by healthy vm toward viral infections can be ascribed to a direct virucidal effect or to the maintenance of natural defense factors present in the vaginal milieu. some mechanisms have been suggested by results obtained from both in vitro experiments and clinical observations in infected women (29) . lactobacillus metabolites possessing antimicrobial activity may be directly protective against viral infections. hydrogen peroxide (h2o2) produced by lactobacilli plays an important role as a natural microbicide within the vaginal ecosystem due to its toxic activity against a number of microorganims and viruses, including hiv-1 (71) and hsv-2 (72) . it has been observed that a range of 70-95% of lactobacilli present in the vaginal flora of healthy women produce h2o2. this percentage drops to 5% in women affected by vaginal infections (73) . the physiological acid vaginal ph value (≤4.5) determined by lactobacilli inactivates hiv (74) and hsv-2 (75) . in addition, hsv-2 is inactivated by lactic acid concentrations leading to ph values similar to the ones detected in the healthy human vagina (72) . several compounds released from lactobacilli can impair the efficiency of target cells in supporting viral replication. a nonprotein cell wall component extracted from a vaginal strain of lactobacillus brevis strongly reduces hsv-2 replication in cell culture (76) , whereas acid lactobacillus metabolic products decrease activation of t lymphocytes, with a consequent lower lymphocyte susceptibility to hiv-1 infection (77) . a healthy vm contributes to the maintenance of the natural defense mechanisms from invading pathogens. the gel layer coat of the vaginal and cervical mucosa represents a physical barrier that hinders viral binding to cell membrane receptors, thus protecting women from viral infections. indeed, in vitro studies demonstrated that hsv could be trapped into the viscous cervical mucus (78) . bv-related microorganisms are able to produce higher levels of mucin-degrading enzymes, such as mucinase and sialidase, in comparison to lactobacilli-dominated healthy vaginal flora (79) (80) (81) . therefore, an increased degradation of the protective mucus layer may promote binding of hsv-2 and other viruses to the underlying epithelial cell receptors. further studies have demonstrated that vaginal lactobacilli are able to inhibit the first steps of hsv-2 infection in cell culture (72, 76) . the antiviral activity exerted by the presence of lactobacilli during hsv-2 binding to the cell membrane is strain dependent and appears directly related to the adhesion capacity of lactobacillus strains (82) . in conclusion, several mechanisms may be involved in the antimicrobial effect of vaginal lactobacilli: interference with microorganisms in the process of adhesion or entry into host cells, production of metabolites with a direct antimicrobial effect, production of compounds able to inhibit obligate intracellular pathogen replication, and contribution to the maintenance of natural defense factors present in the vaginal milieu. as already discussed, hlf is one of the most important defense proteins of cvf. in fact, endogenous hlf has been found increased in the genital fluid of women affected by neisseria gonorrheae, c. trachomatis, and t. vaginalis infections and/or vaginal dysbiosis (64) . in this respect, hlf released by neutrophils recruited in situ could represent a marker of non-healthy conditions and one of the mediators involved in counteracting the inflammatory mileu. the first function of hlf, recognized in vitro, was the bacteriostatic activity depending on its ability to sequester iron necessary for bacterial survival and growth (83) . hlf and blf establish a battle for iron acquisition with pathogens, capable to counteract these iron-binding proteins by synthesizing siderophores, small high affinity iron-chelating molecules, or through iron acquisition from other sources (65) . as a matter of fact, g. vaginalis, lacking of siderophores, acquires iron by the lysis of erythrocytes, using hemoglobin as iron source. this is consistent with the observation that g. vaginalis level increases during menses (6) . moreover, independently from iron-binding ability, blf exerts several antibacterial activities: (i) bacterial lysis through its binding to lipopolysaccharide (lps); (ii) inhibition of bacterial adhesion to the epithelial cells; and (iii) inhibition of the entry into host cells by facultative or obligate intracellular bacteria through competitive binding to host cells and/or to microbial surface components [(44) and references therein (46, 84) ]. of note, facultative intracellular pathogens require intracellular nutrients, including iron, for replication in mammalian cells, and obligate intracellular c. trachomatis is no exception (85) . a preparation of blf, iron saturated at 20% to consent further iron chelation, was utilized in in vitro model to check its antichlamydial activity (84) . similar to that observed using vaginal lactobacilli, the incubation of cell monolayers with blf before the infection or at the moment of the infection significantly inhibited the adhesion and entry of c. trachomatis into epithelial cells. therefore, the inhibition of c. trachomatis infectivity by blf was dependent on its interaction with the cell surface and especially with glycosaminoglycans and heparan sulfate proteoglycans (86) , which are potential receptors for c. trachomatis adhesion (87) . conversely, the preincubation of blf with c. trachomatis ebs did not influence its infectivity, supporting the idea that the specific interaction between blf and epithelial host cells could be the sole mechanism responsible for the inhibition of c. trachomatis invasion (84) . recently, the inhibition of il-6 and il-8 synthesis by blf has been demonstrated in in vitro model, mimicking the in vivo chlamydial infection. blf, added to infected cells 3 h postinfection, produced a significant decrease of il-6 and il-8 without any effect on the number of intracellular chlamydia. similarly, il-6 levels were reduced when blf was added 3 h postinfection to epithelial monolayers infected with other facultative intracellular bacteria or to lps-stimulated macrophages (88) (89) (90) . the anti-chlamydial activity of blf related to its anti-inflammatory function has been shown in vivo. in a pilot study, 7 of 176 pregnant women showing cervical specimens positive for c. trachomatis were treated with the intravaginal administration of blf (100 mg) every 8 h for 30 days. interestingly, after 1 month, six women resulted negative for c. trachomatis and showed significant decreased il-6 levels in their cvf (84) . similar to what observed in in vitro model, intravaginal administration of blf seems to act by protecting mucosal host cells against the adhesion and entry of chlamydial ebs, which are released extracellularly after redifferentiation of rbs to ebs. the decrease of il-6 levels could be a marker for the inhibition of c. trachomatis ebs infection of host cells due to the presence of blf. in other words, blf protects host cells and prevents the early phase of infection by ebs. unlike lactobacilli (68), blf does not affect the replication of rbs (84) . the potential influence of exogenous blf on microbial communities populating vagina has been recently investigated. vaginal blf administration to 60 women with bv has been shown to be able to modify vm composition. in fact, the treatment induced a reduction of bv-associated gardnerella, prevotella, and lachnospira genera as well as an increase of lactobacillus species (91) . these data suggest the therapeutic potential of blf in counteracting female genital tract diseases. indeed, it would be relevant to unveil the molecular mechanisms as well as the immunological changes accompanying blf effects on microbiota. furthermore, blf antiviral activity, verified against both enveloped and naked viruses, is exerted in the early stage of infection, thus inhibiting viral binding and entry into the host cell. this activity is mainly due to blf binding to heparan sulfate glycosaminoglycan cell receptors or viral particles or both (45) . similar to viral particles, the inhibition of plasmodium endocytosis is attributed to the interaction between blf and both cell surface heparan sulfate and lipoprotein receptor-related protein (92) (93) (94) (95) . in this respect, blf represents the most relevant protein symbolizing a brick in the wall of natural non-immune defenses of human mucosal fluids against microbial infections (44) . it is well known that lactobacilli are endowed with healthpromoting and immunomodulatory properties. along with bifidobacteria, they have been proposed as candidates for prevention and/or treatment of allergy, colitis, infections, and other inflammatory conditions (96) . some lactobacillus strains have been also proposed as vaginal microbicide candidates against sti (e.g., n. gonorrhoeae, candida albicans, and hiv). besides mechanisms related to the bacterium itself (e.g., enhancement of epithelial barrier function and competition with pathogens), the capability to redirect the immune response underlies many of the beneficial effects of lactobacilli. in vitro data demonstrate that l. crispatus (97) (98) (99) and l. jensenii (100) act not only through colonization of epithelial cells but also influencing the cytokine secretion pattern. in particular, upon recognition through tolllike receptor (tlr) 2/6 and 2/4, nf-κb signaling is activated without induction of pro-inflammatory mediators (il-1β, il-1α, and tnf-α). furthermore, secretion of cytokines as il-8 is inhibited, while production of il-10, il-6, and defensins can be induced ensuring homeostasis of immune responses. although innate immunity is the first level to be influenced by the probiotic interaction with mucosal epithelium, other cells (e.g., dendritic cells) can be shaped by lactobacilli to skew adaptive responses. an example is represented by l. crispatus sj-3c-us strain, which was shown to confer anti-inflammatory properties to dendritic cells by inducing upregulation of il-10 production and induction of regulatory t cells (101). as mentioned above, a key metabolite produced by lactobacilli is lactic acid. besides its antimicrobial properties, many of the immune modulation mechanisms exerted by lactobacilli can be ascribed to this compound. in particular, lactic acid has been shown to induce an anti-inflammatory response from vaginal and cervical epithelial cells by inhibiting il-6, il-8, rantes, and tnf-α secretion stimulated by tlr agonists used to mimic pathogen-associated molecular patterns (pamps) from microbes (102) . given that il-6, il-8, and tnf-α are known to promote replication of hiv through activation of nf-κb transcription in hiv target cells, lactic acid produced by lactobacilli in the vaginal environment could be relevant in the context of viral infection acquisition. furthermore, the anti-inflammatory cytokine il-1ra was induced by lactic acid treatment of cervicovaginal epithelial cells. all these anti-inflammatory effects were mediated by both l-and d-lactic acid isomers and by the protonated form which predominates in healthy conditions with low values of vaginal ph (102, 103) . very few data about the immunological changes associated with benefits induced by lactobacilli administration are available in the vaginal tract in vivo in both physiological and pathological conditions. lactobacillus salivarius crl 1328 and l. gasseri crl 1263 have been proposed as good candidates to keep a balanced microbiota and immune surveillance. indeed, in a murine model set up to evaluate the benefits of lactobacilli and their effects on the mouse vaginal mucosa and innate immune cells, lactobacilli inoculation did not modify the amounts of granulocytes and macrophages in vaginal washings (104) . in humans, it has been shown that administration of probiotic lactobacillus vaginal tablets produces a significant reduction in the levels of vaginal il-1β and il-6 cytokines demonstrating the capacity of lactobacilli to modulate the production of inflammatory cytokines in both women with bv and women with healthy vaginal flora (105, 106) . a link between oral probiotic administration and vm/immune markers has been recently demonstrated by vitali et al. on pregnant women (107) . the authors investigated the effects of dietary supplementation with vsl#3 probiotic mixture containing eight species of lactobacillus, bifidobacterium, and streptococcus on the vm during late pregnancy. interestingly, no changes in the bacterial counts of the most represented populations were revealed upon probiotic administration. however, the probiotic mixture was able to change the composition of less abundant vaginal microorganisms by avoiding the reduction of bifidobacterium and the increase of atopobium recorded in the last trimester of pregnancy in control healthy women. significant modifications of the local immune system were also associated with the consumption of the probiotic showing anti-inflammatory effects. in particular, vaginal levels of il-4 and il-10 were maintained in balance compared to the reduction observed in control group. furthermore, vaginal levels of eotaxin, a pro-inflammatory chemokine, were reduced upon probiotic dietary supplementation. similar to lactobacilli, also blf exhibits effects on the host immune system, ranging from inhibition of inflammation to promotion of both innate and adaptive immune responses (108) . innate immunity is shaped by endogenous hlf through its interaction with pamps and/or pattern recognition receptors (prrs) expressed by host cells. in vitro studies demonstrated that carbohydrate chains of hlf make it able to interact directly with tlr4 resulting in moderate activation of tlr4 associated pathways (109) . lps is a typical pamp, which is bound by hlf, resulting in the inhibition of cell activation and inflammatory responses (110, 111) . it has been proposed that in vivo hlf inhibits lps-stimulated tlr4 signaling and depresses endotoxemia (112) . in particular, upon lps binding, hlf acts in reducing tnf-α, il-1, and il-6 production by immune cells (macrophages, neutrophils, and lymphocytes), as well as il-8 release by endothelial cells (113) . it has been demonstrated that levels of hlf are increased in inflammatory diseases such as rheumatoid arthritis (114) , severe acute respiratory syndrome (115) , inflammatory bowel disease (116) , and as mentioned above, some std and bv (64) . these observations suggest that hlf could be used as a clinical marker of inflammatory conditions. besides prrs and pamps binding, hlf displaces proteases from heparin in mast cells thus playing antiallergic effects (117) . furthermore, hlf competes with il-8 through the binding to proteoglycans on endothelium, thus interfering with neutrophils recruitment to the site of inflammation (118) . hlf has been shown to bind also dna in the neutrophil extracellular traps (nets), and this capability plays a key role in the context of netosis, which is the nets production by neutrophils. hlf adheres to the dna structures released due to chromatin decondensation and spreading exerting its antimicrobial properties (119) . in vitro experiments showed that recombinant hlf is able to induce maturation of antigen-presenting cells such as dendritic cells, thus suggesting that it can represent a link in shaping adaptive immunity (120) . depending on the external stimulus (pathogens, allergen, tumor antigens, etc.) and host immune status, hlf can modulate il-4, il-2, il-12, or ifn-γ levels, thus providing different outcomes: strong th1 polarization (infections, tumor), reduction of excessive th1 responses, and correction of th1/th2 balance (allergy, autoimmunity). furthermore, lf is able to support proliferation of t cell precursors and their differentiation. besides its role in cellular-mediated immunity, hlf influences activation of b cells, thus playing a role also in humoral responses (113) . the effects of exogenous lf on immune responses have been evaluated in different in vitro systems. different epithelial cell monolayers infected with various facultative or obligate intracellular pathogens produce pro-inflammatory cytokines and the addition of blf significantly decreased il-1β, il-6, il-8, and nf-kb levels (84, 88, 121, 122) . the results obtained in different in vitro models and in various clinical trials confirm the blf ability in downregulating pro-inflammatory cytokine synthesis. it has been demonstrated that exogenous blf localizes into cell nucleus, thus acting as transcriptional factor and inhibiting pro-inflammatory cytokines (52, (122) (123) (124) . although the mechanisms by which blf exerts its anti-inflammatory activity are still under debate, recently, the blf ability to decrease the high levels of il-6 in cvf seems strictly related to its capacity to restore iron homeostasis disorders (52, 84) . therefore, lf is not only a key element in the host defense system (44, 125, 126) but also a pivotal component that is able to regulate the inflammatory response and iron homeostasis (52, (60) (61) (62) . recently, blf is emerging as an attractive molecule for treating inflammation by ranging pro-inflammatory macrophagic phenotypes m1 to regulatory/ anti-inflammatory m2 phenotypes (90). women life: estrogens, lactobacilli, and lf lactobacilli defenses of female mucosal genital tract are largely under the influence of hormones and paracrine production of various cytokines. vaginal environment undergoes overtime shifts in the representation and abundance of microbial key species that are influenced by factors that may include age, hormonal fluctuations, sexual activity, use of medication, and hygiene (12) . the vagina is lined by stratified not keratinizing squamous epithelium, which is variable in thickness and structure depending on life stages. the vaginal epithelium consists of three cell layers: superficial, intermediate, and basal capable of storing glycogen under the influence of estrogen. in the pre-pubertal and the postmenopausal women, the epithelium is thin and characterized by a basal layer of cells and several layers of parabasal cells. this thin atrophic epithelium is susceptible to infection and frequently shows degenerative and inflammatory changes. vaginal epithelium reflects the hormonal changes of the menstrual cycle with increased mitosis of the basal layers. under the influence of estrogen in the proliferative phase of the cycle, the whole epithelium thickens and is multilayered. during the secretory phase of the cycle, the intermediate layers become thick and the cells stuffed with glycogen. therefore, the glycogen content of the vaginal epithelium co-variates with estrogen levels. breakdown of glycogen by resident healthy vm produces an acid ph in the vagina, which deters infections. indeed, reproductive-age women carry lactobacillus species (predominant lactic acid bacteria) and genera of streptococcus and atopobium, which conserved the ecological function of lactate production in the vaginal microbiome (127) . it is well demonstrated that fluctuations in the vm occur not only based on intercourse and infections but also during menstrual cycle. in general, high levels of estradiol may favor a lactobacillidominant environment, especially l. crispatus, l. gasseri, and/or l. jensenii (128) , which can be underrepresented in low estrogen conditions such as the beginning of a menstrual cycle or in postmenopausal women. in one study, l. crispatus appears to decline during menses, while g. vaginalis increases along with l. iners and subsequently the concentration of both species decreases after menses (6) . in other studies, l. crispatus has also been reported to decline 100-fold during menses, while the numbers of l. iners strongly increase (10, 11, 129) . recently, cultivation-independent methods have highlighted the complexity and temporal variability of the vm (8, 130) . in particular, gajer et al. described temporal changes in the composition of vm in reproductive-age women within a 16-week period (129) . in general, the highest variability of microbiota community was associated with menses. for example, a subject can show a community dominated by l. crispatus, which could be replaced by l. iners during menses or by streptococcus spp. in a different subject. the same community could then revert to a community dominated by l. crispatus at the end of menses. moreover, the results also showed how some bacterial communities changed greatly over short time periods, while others were more stable. hormone levels were combined with diversity data showing that an increase in estradiol and progesterone corresponded to decreased microbial variability. vaginal metabolome data added information about community function, which was maintained despite changes in its composition. indeed, shifts in community composition involved only changes in the relative dominance of a little number of different bacteria that are able to produce lactic acid (129) . interestingly, a recent study performed to investigate timing and sequence of changes that occur in the vaginal and vulvar microbiota during puberty showed that vm of perimenarcheal girls resembles those of reproductive-age women (131) . in fact, l. crispatus, l. iners, l. gasseri, l. jensenii, and, in some subjects, streptococcus spp. were dominant in the microbiota of girls before the onset of menarche in the early to middle stages of puberty. further studies should be performed to increase knowledge about the link between estrogen, vaginal glycogen levels, lactic acid bacteria abundance, and vaginal ph. other important fluctuations in the vaginal microbiome are recorded during pregnancy. aagaard et al. showed that microbiome was enriched in l. iners, l. crispatus, l. jensenii, and l. johnsonii (132) . the increase in lactobacilli may be due to the increase in estrogen levels that occurs during pregnancy although further investigations are needed to better understand the relationship between specific species of lactobacillus and estrogen levels. however, another study shows that l. crispatus and l. iners dominate the vaginal flora as pregnancy progresses and maternal age seems to be important for the dominance of l. crispatus or l. iners, with l. iners being dominant in older gravidae (133) . as mentioned above, menopause usually represents a phase in which lactobacilli levels are low, but it seems that their implication is even more pronounced involving other features. actually, inverse correlation has been found between lactobacillus levels and vaginal dryness, a common condition of postmenopausal period, which was shown to be associated with changes in vaginal epithelial cell integrity and inflammation (134) . besides the endogenous hormonal fluctuations, clinical evidences demonstrate that the use of hormonal contraceptives is also able to induce changes in vm, thus influencing the susceptibility to sti and bv. therefore, sti and bv (which in turn predisposes to sti) depend, in part, on modification of vaginal bacterial communities induced by some contraception methods. clinical trials based on nugent score endpoint and questionnaires have revealed a reduced bv rate in women who use estrogen-containing contraceptives (135, 136) . the effects of progestin-containing contraceptives such as depot medroxyprogesterone acetate (dmpa) and levonorgestrel are less clear. in a systematic review of 36 eligible studies, authors have shown that combined oral contraceptives (cocs; combination of an estrogen and a progestin) and dmpa reduce bv by a range of 10-20 and 18-30%, respectively (137) . accordingly, in a recent retrospective study on 682 fertile women, based on 16s rrna sequencing, authors found that women using coc or dmpa showed a reduced colonization by bv-associated bacteria compared to women using condoms. in the same study, women using progestinbased therapies have a significantly higher abundance of taxa associated with a dysbiotic vm. on the other hand, coc users have a lactobacilli-dominated vm and a higher proportion of h2o2-producing lactobacillus species (l. crispatus, l. gasseri, and l. jensenii) correlating with vaginal health compared to progestin-containing contraceptives (138) differences in the results reported in other studies, which do not reveal any changes in vaginal bacterial communities associated with progestin use, could be due to the different study population characteristics (e.g., ethnicity of subjects) and/or the design of the study itself such as the specific bv status examination (139, 140) . in women with bv, vm is not the only level that hormonal contraception acts on. in fact, changes in genital tract immunity by effect of hormonal contraceptives have been shown in terms of both suppression and activation of responses. in a study on 81 women, cervical secretions contained lower levels of pro-inflammatory molecules (tnf, ifn-γ, and gm-csf) in subjects with bv using hormonal contraceptives compared to those not using them (141) . on the other hand, an increase of inflammatory cytokines (mip-1α, mip-1β, il-6, il-8, ip-10, and rantes) has been associated with hormonal contraception in a different cross-sectional analysis including 376 african women (142) . conflicting data existing in these studies on immunomodulatory potential of hormonal contraception in female genital tract are complicated by their variability in terms of in vitro versus in vivo models used and sample tested (plasma or blood versus cervical fluid). however, the investigation on these features may represent a key point in understanding the association between hormonal contraception and hiv acquisition. in fact, women using progestin dmpa, but not those who use coc have been found to be at significantly increased risk of hiv infection compared to women not using hormonal contraception (143) . in a more recent case-control selection of specimens from a large, prospective, clinical study, the same authors have investigated on innate immunity mediators in cervical samples collected from 199 women at their visit before hiv seroconversion and matched visits from 633 women remaining hiv uninfected. higher levels of pro-inflammatory markers such as rantes have been found in hiv seroconversion and in dmpa users, suggesting a possible role of this cytokine in the association between the contraceptive and hiv risk acquisition (144) . the possible explanation could be that the upregulation of rantes, observed in women using dmpa and women with bv, may simultaneously block the ccr5 cellular hiv receptor but also facilitate transmission through recruiment of target cells. also other sti can be influenced by hormonal contraception, such as candidiasis, which has been found increased in women using coc but not dmpa (137) . taken together, these observations suggest that altered immune responses by effect of hormonal contraception may predispose to infections from pathogens. in cases of a pre-existing condition of dysbiosis or specific cervicovaginal infection, suppression or activation of immunity by pathogens could increase the risk of infections by cumulative action with exogenous hormones. similar to the estrogen-induced changes in the species and number of lactobacilli, the hlf concentration fluctuates accordingly to circulating estrogen levels (145) (146) (147) . in addition to the hlf levels produced by uterine epithelium and released in cvf, the synthesis of iga and igg is also modulated by estrogen and progesterone, thus exerting an immune protection against sexually transmitted pathogens (148) . figure 1 shows a comparison among estrogen, progesterone, and lf levels during the proliferative, ovulatory, and secretory phase. the hlf levels increase in line with estrogen production, reaching the highest levels during ovulatory phase, while they are inversely correlated with progesterone levels. as a matter of fact, secretory phase accordingly to the increase of progesterone shows the lowest levels of hlf. at the end of the secretory phase, hlf returns to increase in parallel to the progesterone decrease (145) (146) (147) (148) (149) . obviously, the use of oral contraceptives by decreasing estrogen synthesis suppresses the production of hlf as well as immunoglobulins for the duration of hormone exposure, thus possibly increasing the susceptibility of women to infections (148) . in rats, pretreatment with progesterone prior to exposure to chlamydia thracomatis infection induced a persistent infection (150) . furthermore, the rats treated with progesterone were also found to be more vulnerable to chlamydial intrauterine infection, whereas the treatment with estradiol, the major female estrogen sex hormone, reduced the susceptibility to infection (151) . the sex steroid hormones are also important mediators of inflammation (152) and may influence resistance or susceptibility to parasitic infections (153) . sex steroid hormones are also involved in viral infections. it has been proposed that hiv-1 utilizes a window of vulnerability during the menstrual cycle. this crucial period overlaps with the mid-cycle when innate and adaptive immune responses are suppressed by estrogens and/or progesterone to facilitate reproductive processes. hiv-1 presumably exploits this time frame, during which antiviral factors are suppressed, to establish and propagate infection in the female mucosal genital tract (31, 148, 154) . in menopausal women, the low concentration of hlf in the secretions, related to the low levels of sexual hormones (155) , may lead to recurrent infections. it is important to underline that during menses the decrease of this important natural defense glycoprotein is balanced by the presence of neutrophils that, through the synthesis of granules, can restore, at least partially, hlf concentration and its antimicrobial activity when the epithelial barrier is disrupted. the recruitment of neutrophils occurs through high levels of inflammatory biomarkers as pro-inflammatory cytokine as il-8, il-6 or c-reactive protein (119) . as matter of fact, during menstruation, as well as in aging and menopause, the decrease of estrogens is related to the increase of inflammatory processes (156) . interestingly, hlf expression in endometrium suggests that, during the gestational period, hlf produced by uterine epithelium and neutrophils and released in cvf is controlled by sex steroid hormones (149) . it has been reported that high levels of progesterone parallel low levels of estrogens in normal pregnancy, while this ratio is inverted in pregnant women with the preterm delivery threat (157) . determining the existence of a regulatory circuit linking hlf synthesis and sex steroid hormone fluctuations may unravel novel mechanisms leading to preterm birth. figure 2 | lactobacillus spp. and lactoferrin interplay on infection and inflammation in female genital tract. a schematic representation of lactobacillus spp. and lactoferrin balance: a multitasking strategy to protect against pathogen challenge and maintain immune homeostasis. (a) healthy genital tract; (b) vaginosis: high levels of pro-inflammatory cytokines, decrease of lactobacilli and increase of gram-negative anaerobes, and increase of lactoferrin concentration released by neutrophils; (c) decrease of pro-inflammatory cytokines by lactoferrin and restoration of healthy microbiota. lactobacillus spp. and lf are pivotal components of first-line defense in the female mucosal genital tract involved in protection against a multitude of microbial infections and the most effective natural mechanism to dampen inflammatory processes. to inhibit cervicovaginal infections, an ideal drug should inhibit: • microbial growth; • microbial adhesion and entry into host cells; • microbial intracellular replication; and • infection of new host cells by microbes extracellularly released from the infected cells. in the vaginal environment of women of childbearing age, the inhibition of bacterial multiplication through the synthesis of antibacterial substances by lactobacilli or by competition between microbes and lf for iron acquisition represents an effective natural defense mechanism. both lactobacilli and lf can inhibit the adhesion and consequently the microbial entry inside the cells through an interaction with the cell surface components potential receptors for pathogens. lactobacilli and lf appear complementary since lactobacilli inhibit microbial intracellular replication and together with lf hinder the infection of still healthy cells by microbes extracellularly released. this close cooperation is also exerted through their anti-inflammatory function. in this scenario, the mucosal environment represents a good model of mutualism and reciprocity against the injury by microbes. a schematic representation of lactobacillus spp. and lf balance in protecting against pathogens and maintaining immune homeostasis in the vaginal tract is shown in figure 2 . considering the shortage of effective treatments to counteract antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lf could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis. author contributions pm, rp, and pv conceived the topic concept and wrote and revised the final manuscript; lr, ac, ml, dc, and es provided figures and contributed to manuscript preparation and editing. all authors read and approved the final version. acknowledgments this work was granted by sapienza university of rome funds to pv. microbiota of the upper and lower genital tract emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora understanding the bacterial flora of the female genital tract characterization of vaginal flora and bacterial vaginosis in women who have sex with women the human vaginal bacterial biota and bacterial vaginosis temporal variability of human vaginal bacteria and relationship with bacterial vaginosis the vaginal microbiome: new information about genital tract flora using molecular based techniques vaginal microbiome of reproductive-age women lactobacillus iners: friend or foe? longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles longitudinal qpcr study of the dynamics of l. crispatus, l. iners, a. vaginae, (sialidase positive) g. vaginalis, and p. bivia in the vagina lactobacilli in the female genital tract in relation to other genital microbes and vaginal ph cervical bacterial flora in infertile and pregnant women microbial flora of the vagina and cervix adherence of clinically isolated lactobacilli to human cervical cells in competition with neisseria gonorrhoeae defense factors of vaginal lactobacilli prevalence of bacterial vaginosis: 2001-2004 national health and nutrition examination survey data the microbiology of bacterial vaginosis local and systemic cytokine levels in relation to changes in vaginal flora cytokines, pregnancy, and bacterial vaginosis: comparison of levels of cervical cytokines in pregnant and non-pregnant women with bacterial vaginosis bacterial vaginosis as a risk factor for upper genital tract infection rates of bacterial vaginosis in women undergoing in vitro fertilisation for different types of infertility bacterial vaginosis as a risk factor for preterm delivery: a meta-analysis vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition bacterial vaginosis is a strong predictor of neisseria gonorrhoeae and chlamydia trachomatis infection bacterial vaginosis (bv) and the risk of incident gonococcal or chlamydial genital infection in a predominantly black population bacterial vaginosis, race, and sexually transmitted infections: does race modify the association? bacterial vaginosis assessed by gram stain and diminished colonization resistance to incident gonococcal, chlamydial, and trichomonal genital infection vaginal microbiota and viral sexually transmitted diseases antimicrobial components of vaginal fluid innate immunity in the human female reproductive tract: endocrine regulation of endogenous antimicrobial protection against hiv and other sexually transmitted infections antibacterial activity of antileukoprotease antibacterial activity and specificity of the six human α defensins antiviral mechanisms of human defensins gene expression, immunolocalization, and secretion of human defensin-5 in human female reproductive tract human β-defensins the antibacterial and antifungal properties of trappin-2 (pre-elafin) do not depend on its protease inhibitory function antiviral activity of trappin-2 and elafin in vitro and in vivo against genital herpes calprotectin -a pleiotropic molecule in acute and chronic inflammation metal chelation and inhibition of bacterial growth in tissue abscesses cathelicidins: family of antimicrobial peptides. a review protective roles of the skin against infection: implication of naturally occurring human antimicrobial agents β-defensins, cathelicidin ll-37 and lysozyme female genital tract secretions inhibit herpes simplex virus infection: correlation with soluble mucosal immune mediators and impact of hormonal contraception lactoferrin: an important host defence against microbial and viral attack antiviral properties of lactoferrin -a natural immunity molecule lactoferrin: a natural glycoprotein involved in iron and inflammatory homeostasis the role of cationic polypeptides in modulating hiv-1 infection of the cervicovaginal mucosa endogenous antimicrobial peptides and skin infections in atopic dermatitis synergistic and additive killing by antimicrobial factors found in human airway surface liquid host antimicrobial defence peptides in human disease molecular structure, binding properties and dynamics of lactoferrin body iron delocalization: the serious drawback in iron disorders in both developing and developed countries lactoferrin: an alternative view of its role in human biological fluids innate host defense of human vaginal and cervical mucosae antimicrobial factors in the cervical mucus plug a component of innate immunity prevents bacterial biofilm development iron availability influences aggregation, biofilm, adhesion and invasion of pseudomonas aeruginosa and burkholderia cenocepacia antimicrobial peptides: the ancient arm of the human immune system oral administration of lactoferrin increases hemoglobin and total serum iron in pregnant women the influence of lactoferrin, orally administered, on systemic iron homeostasis in pregnant women suffering of iron deficiency and iron deficiency anaemia lactoferrin efficacy versus ferrous sulfate in curing iron disorders in pregnant and non pregnant women safety and efficacy of lactoferrin versus ferrous sulphate in curing iron deficiency and iron deficiency anaemia in hereditary thrombophilia pregnant women: an interventional study aerosolized bovine lactoferrin reduces neutrophils and pro-inflammatory cytokines in mouse models of pseudomonas aeruginosa lung infections multiplex immunoassay of lower genital tract mucosal fluid from women attending an urban std clinic shows broadly increased il1ß and lactoferrin siderophores in iron metabolism: from mechanism to therapy potential antimicrobial and immune modulatory effects of lactic acid and short chain fatty acids produced by vaginal microbiota associated with eubiosis and bacterial vaginosis in vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide effects of vaginal lactobacilli in chlamydia trachomatis infection treatment of chlamydial infections: 2014 update inhibition of neisseria gonorrhoeae epithelial cell interactions by vaginal lactobacillus species viricidal effect of lactobacillus acidophilus on human immunodeficiency virus type 1: possible role in heterosexual transmission inhibition of herpes simplex virus type 2 by vaginal lactobacilli prevalence of hydrogen peroxide-producing lactobacillus species in normal women and women with bacterial vaginosis disinfection and inactivation of the human lymphotropic virus type iii/lymphadenopathy-associated virus acidform inactivates herpes simplex virus and prevents genital herpes in a mouse model: optimal candidate for microbicide combinations antiviral activity of lactobacillus brevis towards herpes simplex virus type 2: role of cell wall associated components human vaginal leukocytes and the effects of vaginal fluid lymphocyte and macrophage defense functions diffusion of macromolecules and virus-like particles in human cervical mucus glycosidase and proteinase activity of anaerobic gram negative bacteria isolated from women with bacterial vaginosis a novel bacterial mucinase, glycosulfatase, is associated with bacterial vaginosis among pregnant women with bacterial vaginosis, the hydrolytic enzymes sialidase and prolidase are positively associated with interleukin-1β technological and biological evaluation of tablets containing different strains of lactobacilli for vaginal administration iron-binding proteins in milk and resistance to escherichia coli infections in infants effect of bovine lactoferrin on chlamydia trachomatis infection and inflammation response of chlamydia trachomatis serovar e to iron restriction vitro and evidence for iron-regulated chlamydial proteins characterization of the glycosaminoglycan-binding region of lactoferrin the chlamydia trachomatis ctad1 invasin exploits the human integrin 1 receptor for host cell entry lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases lactoferrin prevents lps-induced decrease of the iron exporter ferroportin in human monocytes/macrophages lactoferrin efficiently counteracts the inflammation-induced changes of the iron homeostasis system in macrophages bacterial biota of women with bacterial vaginosis treated with lactoferrin: an open prospective randomized trial plasmodium falciparum inhibition in vitro with lactoferrin, desferrithiocin, and desferricrocin iron chelators as therapeutic agents against pneumocystis carinii dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (lrp) and heparan sulfate proteoglycans reduction of the infectivity of toxoplasma gondii and eimeria stiedai sporozoites by treatment with bovine lactoferricin challenges in translational research on probiotic lactobacilli: from in vitro assays to clinical trials lactobacillus crispatus modulates epithelial cell defense against candida albicans through toll-like receptors 2 and 4, interleukin 8 and human β-defensins 2 and 3 probiotic properties of lactobacillus crispatus 2,029: homeostatic interaction with cervicovaginal epithelial cells and antagonistic activity to genitourinary pathogens lactobacillus crispatus mediates anti-inflammatory cytokine interleukin-10 induction in response to chlamydia trachomatis infection in vitro homeostatic properties of lactobacillus jensenii engineered as a live vaginal anti-hiv microbicide lactobacillus crispatus strain sj-3c-us induces human dendritic cells (dcs) maturation and confers an anti-inflammatory phenotype to dcs vaginal lactic acid elicits an anti-inflammatory response from human cervicovaginal epithelial cells and inhibits production of pro-inflammatory mediators associated with hiv acquisition the role of lactic acid production by probiotic lactobacillus species in vaginal health effectiveness of vaginal tablets containing lactobacilli versus ph tablets on vaginal health and inflammatory cytokines: a randomized, double-blind study biological control of vaginosis to improve reproductive health dietary supplementation with probiotics during late pregnancy: outcome on vaginal microbiota and cytokine secretion bovine lactoferrin counteracts toll-like receptor mediated activation signals in antigen presenting cells human lactoferrin activates nf-kappab through the toll-like receptor 4 pathway while it interferes with the lipopolysaccharide stimulated tlr4 signaling lactoferrin is a lipid a-binding protein lactoferrin binds cpg-containing oligonucleotides and inhibits their immunostimulatory effects on human b cells human milk components modulate toll-like receptor-mediated inflammation lactoferrin, a key molecule in immune and inflammatory processes increased levels of lactoferrin in synovial fluid but not in serum from patients with rheumatoid arthritis expression profile of immune response genes in patients with severe acute respiratory syndrome update of fecal markers of inflammation in inflammatory bowel disease the inhibition of mast cell activation by neutrophil lactoferrin: uptake by mast cells and interaction with tryptase, chymase and cathepsin g lactoferrin inhibits the lipopolysaccharide-induced expression and proteoglycan-binding ability of interleukin-8 in human endothelial cells neutrophil extracellular traps and its implications in inflammation: an overview recombinant human lactoferrin induces human and mouse dendritic cell maturation via toll-like receptors 2 and 4 lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive escherichia coli strains lactoferrin decreases inflammatory response by cystic fibrosis bronchial cells invaded with burkholderia cenocepacia iron-modulated biofilm cellular internalization of lactoferrin in intestinal epithelial cells the n1 domain of human lactoferrin is required for internalization by caco-2 cells and targeting to the nucleus lactoferrin: a modulator of immune and inflammatory responses multifunctional roles of lactoferrin: a critical overview differences in the composition of vaginal microbial communities found in healthy caucasian and black women changes in the predominant human lactobacillus flora during in vitro fertilisation temporal dynamics of the human vaginal microbiota daily temporal dynamics of vaginal microbiota before, during and after episodes of bacterial vaginosis vaginal microbiota of adolescent girls prior to the onset of menarche resemble those of reproductive-age women a metagenomic approach to characterization of the vaginal microbiome signature in pregnancy pregnancy's stronghold on the vaginal microbiome vaginal microbiome and epithelial gene array in post-menopausal women with moderate to severe dryness prevalent and incident bacterial vaginosis are associated with sexual and contraceptive behaviours in young australian women recurrence of bacterial vaginosis is significantly associated with posttreatment sexual activities and hormonal contraceptive use hormonal contraception decreases bacterial vaginosis but oral contraception may increase candidiasis: implications for hiv transmission changes in vaginal community state types reflect major shifts in the microbiome cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract association between injectable progestin-only contraceptives and hiv acquisition and hiv target cell frequency in the female genital tract in south african women: a prospective cohort study hormonal contraceptive use modulates the local inflammatory response to bacterial vaginosis injectable progestin-only contraception is associated with increased levels of proinflammatory cytokines in the female genital tract hormonal contraception and hiv acquisition: reanalysis using marginal structural modeling cervical inflammation and immunity associated with hormonal contraception, pregnancy, and hiv-1 seroconversion estrogen regulation of lactoferrin expression in human endometrium lactoferrin gene expression and regulation: an overview lactoferrin: the path from protein to gene regulation of mucosal immunity in the female reproductive tract: the role of sex hormones in immune protection against sexually transmitted pathogens differential expression and estrogen response of lactoferrin gene in the female reproductive tract of mouse, rat, and hamster chlamydia trachomatis infection in the female reproductive tract of the rat: influence of progesterone on infectivity and immune response antigen-presenting cells in the female reproductive tract: influence of estradiol on antigen presentation by vaginal cells the complex role of estrogens in inflammation immunoendocrine mechanisms associated with resistance or susceptibility to parasitic diseases during pregnancy a new strategy to understand how hiv infects women: identification of a window of vulnerability during the menstrual cycle bone health in menopausal women: a role for general practitioners. clin cases miner bone metab molecular mechanisms of aging-associated inflammation dydrogesterone supplementation in women with threatened preterm delivery -the impact on cytokine profile, hormone profile, andprogesterone-induced blocking factor key: cord-286719-1xjmlwqr authors: draz, mohamed shehata; shafiee, hadi title: applications of gold nanoparticles in virus detection date: 2018-02-15 journal: theranostics doi: 10.7150/thno.23856 sha: doc_id: 286719 cord_uid: 1xjmlwqr viruses are the smallest known microbes, yet they cause the most significant losses in human health. most of the time, the best-known cure for viruses is the innate immunological defense system of the host; otherwise, the initial prevention of viral infection is the only alternative. therefore, diagnosis is the primary strategy toward the overarching goal of virus control and elimination. the introduction of a new class of nanoscale materials with multiple unique properties and functions has sparked a series of breakthrough applications. gold nanoparticles (aunps) are widely reported to guide an impressive resurgence in biomedical and diagnostic applications. here, we review the applications of aunps in virus testing and detection. the developed aunp-based detection techniques are reported for various groups of clinically relevant viruses with a special focus on the applied types of bio-aunp hybrid structures, virus detection targets, and assay modalities and formats. we pay particular attention to highlighting the functional role and activity of each core au nanostructure and the resultant detection improvements in terms of sensitivity, detection range, and time. in addition, we provide a general summary of the contributions of aunps to the mainstream methods of virus detection, technical measures, and recommendations required in guidance toward commercial in-field applications. viruses are remarkable pathogens that are causing prominently increasing morbidity and mortality worldwide. their highly contagious nature and the absence of immediate and efficient control systems are the main reasons behind their potential health impacts. currently, viral infections and associated diseases are major causes of death in mankind, and under the present context of industrialization and immigration, they continue to emerge at a rapid pace, causing significant human, social, and financial costs [1] . additionally, gaps in currently applied detection systems potentially contribute to increasing the likelihood of international incidences and outbreak of viral infections [2] [3] [4] [5] . the implementation of highly sensitive and specific diagnostic tools has the potential to rapidly identify viral infections, initiate and guide judicious controls, and subsequently curtail their dissemination. toward this endeavor and beyond the pitfalls of current immunological and molecular techniques commonly applied to virus detection, several new approaches based on nanoparticles (nps) have recently been developed. aunps are widely described to be suitable for numerous biosensing functions and applications. their unique photonic, electric, and catalytic properties, coupled with the molecular interaction specificity of various biomolecules (e.g., antibodies, single-stranded (ss) dna, and rna aptamers, among others), represent the design principles of a wide range of virus detection systems [6] [7] [8] . the advantages of being simple, rapid, and sensitive and facilitating quantitative detection with excellent multiplexing capabilities have greatly promoted these systems to be envisioned as state-of-the-art technologies for virus ivyspring international publisher detection [9, 10] . however, there are no available reviews of the applications of bio-aunp hybrid structures for sensing and detecting viruses. in this article, we provide a current review of aunp-based virus detection at the level of virus type, including the types and structures of the applied aunps. we highlight their role in enhancing sensory and detection performance in comparison with current techniques in terms of analytical sensitivity, detection range, and time. furthermore, this review specifically summarizes detection designs, formats, functions, and contributions, which are of special importance to both scientific and applied research. viruses are particulate in nature and usually exist in different morphological forms that generally range in size from 20 to 900 nm [11] [12] [13] . their intact, mature infectious particles are typically composed of definite units of proteins and nucleic acid that self-assemble to form nanoparticulate structures called virions. the viral proteins are usually arranged in a surface layer called the capsid and sometimes in an outer envelope surrounding an inner core of nucleic acids. this core of viral nucleic acids can be single-or double-stranded (ds), dna or rna, one or several molecules, linear or circular in shape, and a few thousands of nucleotides to one million base pairs in size. the nanoscale size and the relatively simple structure of viruses tend to impose technical difficulties in establishing wide-use and long-term systems for virus detection. the small size of viruses increases the difficulty of their isolation and visualization compared with other microbes, such as bacteria and fungi that can be readily examined using ordinary light microscopes. only electron microscopy (em) with a high-magnification power of ~100,000× can allow the direct visualization of viruses and the study of their structures [14, 15] . therefore, em remains crucial for many purposes in virus research. however, em is certainly inappropriate for routine clinical diagnosis because of the required time and cost, as well as many safety concerns. furthermore, although the simple structure of virus particles allows the features of diagnostic relevance to be easily defined and tested, it presents limitations on the use of their characteristics for practical applications, especially with the increasing number of discovered viruses and recorded viral infections [14, 16, 17] . in addition, this structural simplicity confers viruses with rapid rates of spontaneous adaptation and evolution that may occur through direct genetic mutation, genetic substitution, or recombination. in this way, viruses not only outpace our attempts to develop sustainable control strategies but also raise more questions about the appropriateness and validity of current diagnostic techniques for long-term use [18, 19] . along with these limitations in virus detection and control, the possibility of viral infection emergence or reemergence has become increasingly prevalent, and severe pandemics and epidemics have occurred around the world. in the past, many outbreaks of viruses, such as human immunodeficiency virus (hiv), severe acute respiratory syndrome (sars), influenza virus (h5n1 and h1n1) and zika virus, started as local events and then expanded to have global consequences. hiv was discovered in central africa as a new virus causing acquired immune deficiency syndrome (aids) three decades ago, and it now represents one of the most significant public health threats worldwide [20, 21] . the highly contagious respiratory virus sars has fueled global fears of pandemics since its first appearance in china [22, 23] . in late 2012, the world health organization (who) raised a global alert for a new sars-like respiratory coronavirus, which is now called middle east respiratory syndrome (mers) coronavirus. mers is now classified as one of the most prominent viral threats to the middle east and has caused hundreds of deaths and thousands of infections in a short time. moreover, in the past few years, the world witnessed the worst ebola outbreak ever that started to hit west africa in early 2014 and the pandemic reemergence of zika virus in 2016 [11, 12, 24] . with these evident possibilities of massive outbreaks and implications, the situation is rapidly growing more serious, and the demand for the development of rapid diagnostics and effective control strategies is becoming more urgent. the first studies on virus isolation and detection were started early in the 1950s when the first cell culture system and electron microscope were developed [25, 26] . for decades, these techniques represented the main tools for studying and investigating the biochemical and morphological properties of viruses that remain the main foundation of all known classification and detection systems. however, their practical use in virus detection has remained greatly debated due to several considerations, including laboratory equipment expenses and time and safety concerns [27, 28] . in the early 1980s, the field of diagnostic virology was boosted with two other major developments: 1) the birth of various immunoassays; and 2) the invention of polymerase chain reaction (pcr). this was followed by the development of a very wide range of serological and molecular detection techniques, which rapidly evolved to constitute the mainstream approaches of both laboratory research and the clinical diagnosis of viruses ( fig. 1) [24, 29] . serology remains the standard method for virus detection. it primarily relies on testing for the presence of specific viral antigens or the corresponding antibody responses of the immune system. the most common types of serological tests include the neutralization assay, complement-fixation test, immunoprecipitation assay (ipa), hemagglutination-inhibition (hai) assay, enzyme immunoassay (eia), radioimmunoassay (ria), chemiluminescent immunoassay (cia), particle agglutination, immunostaining, immunofluorescence assay, single radial hemolysis, immunoblotting assay (iba), and immunochromatographic test (ict). the main working principle of these techniques is the use of specific antibodies in conjugation with different signal reporting systems, such as red blood cells, enzymes, and radioactive or fluorescent materials [30, 31] . most of these techniques are relatively easy to perform and flexible in their timing of specimen collection, and most of the needed reagents are usually commercially available. furthermore, they are generally unrestricted by the practical limitations of virus isolation and propagation usually involved with other direct detection methods, such as cell culture and em [9, 32] . therefore, serological methods are widely accepted and recognized techniques for simple, safe, and cheap virus detection [25, 30] . in addition, they are usually described as the first choice for large-scale testing in epidemiological studies and for evaluating antiviral therapies and vaccinations. however, the accuracy and reliability of serological methods are usually challenged by the cross-reactivity of the used antibodies, and the risk of false-positive results is usually very high. in addition, most immune responses can only be detected for a period of time after the initial virus infection; thus, serological detection does not normally benefit patients. molecular techniques are attracting more interest and have found an increasing number of applications in virus detection. the discovery of the genetic enzyme systems involved in the cellular machinery of nucleic acid replication and the stunning invention of an in vitro nucleic acid amplification system, commonly called pcr, by mullis in the early 1980s, opened new frontiers in nucleic acid-based detection [33] . in addition, the known high specificity of nucleic acid hybridization and the absolute availability of its synthesis and modification guided the development of many figure 1 . the onset of nanotechnology in virus detection applications compared with the development of the most common virus detection techniques. cell culture and electron microscopy techniques that are now commonly applied in the direct testing for and detection of viruses were discovered in the mid-20th century [25] . then, different serological and molecular techniques were developed. the serological detection of viruses with immunoassays was first reported in 1970: a radioimmunoassay was applied for the detection of the australia antigen, later called hepatitis b virus surface antigen [218] . pcr was discovered in the 1980s and first reported in virus detection in 1988 for acquired immune deficiency syndrome detection [219] . later, many molecular techniques, including amplification-and nonamplification-based techniques, were reported in virus detection. the concept of nanotechnology was envisioned as early as 1959 by the renowned physicist richard feynman [36] . however, nanotechnology was only applied to virus detection in 1997, when gold nanoparticles were employed for the detection of single-copy human papillomavirus [39] . nanotechnology has recently come to represent one of the most outstanding trends in virus detection and diagnosis via the wide variety of assays described in this review. detection and genotyping techniques known for the rapid and specific detection of viruses. these techniques can be classified as amplification-or nonamplification-based molecular detection techniques. amplification-based molecular techniques usually employ one or more forms of nucleic acid amplification to allow the indirect detection of the target virus. these techniques represent the majority of molecular techniques applied in virus detection and include various types of target amplification techniques (e.g., pcr, loop-mediated isothermal amplification (lamp), transcription-mediated amplification, and nucleic acid sequence-based amplification), signal amplification techniques (e.g., branched dna and hybrid capture), and probe amplification techniques (e.g., ligase chain reaction and strand-displacement amplification). nonamplification-based techniques are mainly applied to the direct testing for the presence of a specific virus in clinical samples (e.g., in situ hybridization, southern blot hybridization, and dot blot hybridization). generally, molecular methods are relatively rapid and more sensitive than immunoassays and can be applied either in a simple form for the manual detection of viruses or as an embedded component of more advanced systems. numerous fully automated and high-throughput detection systems are now available and widely used in clinical settings. in fact, the development of advanced molecular detection systems has revolutionized the way in which diagnostic tests are delivered and greatly enhanced the control of many viral infections, whether in hospitals or communities. in addition, these systems have eliminated the biosafety and time concerns usually associated with the clinical and academic study of viruses. however, despite these wide and promising applications, most molecular techniques still have several potential limitations in repeatability, accuracy, sensitivity, and specificity that are mainly caused by the high genetic variability of some viruses. furthermore, these assays are expensive and time consuming and typically call for specialized laboratory instruments and skilled personnel [34, 35] . the concept of nanotechnology was introduced in early 1959 [36] . subsequently, nanotechnology was realized via various types of new materials that rapidly emerged as promising tools for biological and chemical analyses. nanomaterials are known to possess multiple unique optical, electronic, magnetic, and mechanical properties enabling very attractive applications, especially in the fields of biomedical imaging and clinical diagnosis [37, 38] . the first reported application of nanomaterials in the detection of viruses was attempted in the late 1990s: aunps were coupled with silver staining and applied for the detection of human papillomavirus in cervical carcinoma cells (fig. l) [39] . currently, there is a very wide range of nanomaterials, including metal nps, carbon nanotubes, silica nps, quantum dots (qds), upconversion nps, and polymeric nps, that are being heavily investigated for virus detection [37, 38, 40] . one of the most common approaches for exploiting these nanostructures in virus detection is the development of nanobio hybrid systems that contain one or more biomolecules derived from viruses (e.g., dna, rna, antibody, pentabody, antigen, or peptide) conjugated to the surface of different np forms. these systems leverage the significant labeling properties and signal transduction functions of nps and the specific activity of the conjugated biomolecules to act as multivalent-np probes [37, 38, 41] . such virus-specific np probes have surprisingly been used to build up various optical, fluorometric, electrochemical, and electrical assays that have been extensively reported for single and multiple detection modes ( table 1 ). the results of most of these studies clearly demonstrate the inherent potential of these probes, along with numerous advantages over traditional approaches, in terms of size, performance, specificity, signal sensitivity, and stability. additionally, these studies have extensively described their application to allow simple, rapid, highly sensitive and label-free detection. aunps are a leading class of metal nanostructures that is widely known for its chemical stability, water solubility, and broad size and shape controllability. aunps can range from 1 to 800 nm in size and have different morphological shapes, including spheres, rods, prisms, tetrapods, dog bones, cubes, shells and several hollow structures [42, 43] . the synthesis of aunps can be performed using different methods such as chemical reduction of salts, ultraviolet irradiation, lithography, aerosol technologies, laser ablation, ultrasonic fields, photochemical reduction of au, and biological synthesis [9, 32] . aunps possess a high surface density of free electrons that results in inherent optical, electrical, and catalytic properties. the excitation of aunps with light can cause these free surface electrons, i.e., "plasmons," to oscillate to one side away from the atomic core, which remains as a positive charge on the other side, thereby creating a dipole or plasmon polariton [44, 45] . this dipole plasmon can change its direction in accordance with the frequency of incident light, and the resonance condition is reached when their frequency is approximately the same. this condition has been referred to as surface plasmon resonance (spr). the most widely used aunps exhibit intense spr bands that usually exist between 510-1100 nm and are known to be weakly dependent on the size of the aunps and the refractive index of the surrounding media but very sensitive to both the shape of the nps and the interparticle distance [46] . the spr of aunps has been observed to cause intense enhancing or quenching effects upon interactions with nearby photon emitters. these distance-dependent coupling effects are dipole-dipole interactions that usually include surface plasmon-mediated energy nanotransfer processes similar to fluorescence resonance energy transfer (fret) and may be either destructive (resulting in quenched emission) or constructive (resulting in enhanced emission). compared to other types of nanomaterials, metal nanoparticles, particularly aunps, constitute ideal tools in virus detection for numerous reasons, including the ease of synthesis, characterization, and surface modification, outstanding stability, biocompatibility, and exceptionally high absorption coefficients [47, 48] . furthermore, as labeling agents, aunps are easily visualized due to their intense colors and are known to form stable and highly active bioconjugates with common targeting biomolecules, such as dna and proteins, thereby enabling highly sensitive and specific sensing and detection applications [48] . aunps have been preferentially employed to perform numerous optical signal transduction functions in virus detection, such as resonance light scattering [49] [50] [51] [52] [53] , color amplification [54] [55] [56] [57] [58] [59] , and fluorescence quenching or enhancing [60] [61] [62] [63] [64] [65] (fig. 2) . resonance light-scattering-based detections usually involve measuring the amount of light scattered by different light spectroscopy techniques, including localized surface plasmon resonance (lspr) [52] , raman spectroscopy [49, 50] and dynamic light scattering (dls) [51, 53] . the colorimetric detection of viruses using aunps usually relies on two main techniques: 1) a color amplification technique in which aunps are applied to act as direct coloring labels with their characteristic, intense red color [55, 58, 59, 66] ; and 2) color change technique in which a color change from red to purple occurs in response to particle aggregation. aunp aggregation can be noncrosslinked aggregation caused by the target-triggered removal of stabilizing ligands from the surfaces of aunps [54, 56] or interparticle-crosslinked aggregation caused by the binding of ligands on modified aunps with target analytes [57, 67] . analogous to fret, aunp-based fluorescence quenching is a distance-dependent process in which aunps act to reduce the radiative rate of fluorophores in their proximity. thus, as efficient acceptors, aunps have been paired with dyeand qd-based fluorophores in different analyte-induced donor-acceptor crosslinking and coupling protocols [60-62, 64, 65] . aunps are widely described as electroactive and catalytic tags in various electrochemical assays applied to virus detection (fig. 2 ). based on their redox and electrical properties, aunps can directly act as electrochemical tags detected either by their acid dissolution followed by electrochemical stripping measurements of gold ions [68] or by their direct deposition on the surface of electro transducers, allowing an enhanced electrical conduction and resistance change [69] [70] [71] [72] [73] . aunps can indirectly perform electrochemical transduction functions based on their catalytic activity toward some chemical reduction reactions of metal ions (e.g., silver and copper) [72, 74, 75] or other species, such as h2o2 [76] [77] [78] . this potential to catalyze metal deposition is preferentially called np-metal enhancement amplification, and it represents the basic function of multiple scanometric [50, 70, [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] , photoelectric [89] , light-scattering [90] , and colorimetric schemes [91] for virus detection. additionally, aunp-based catalysis for the oxidation-reduction reaction of hydroquinone has been reported in another simple, colorimetricbased virus detection method [92] . other studies have exploited aunps to enhance the detection sensitivity of some bioanalytical techniques, such as quartz crystal microbalance (qcm) [93] , inductively coupled plasma mass spectrometry (icp-ms) [94] , and atomic force microscopy (afm) [95] in virus detection. through these schemes, aunps have been applied as effective nanoparticulate amplification tags to enhance mass, elemental, and topographic signal transduction, respectively (fig. 2) . interestingly, the enhanced surface area-to-volume ratio of aunps has allowed them to further act as exquisite scaffolds for biorecognition and detection reactions (fig. 2 ); in addition, it has granted these structures unprecedented capabilities for bioimmobilization, which is a basic principle of most aunp-based diagnostic designs. by coupling these bioimmobilization and signal transduction functions, aunps can achieve virus detection with highly improved analytical sensitivity and specificity. furthermore, the direct increase in loading efficiency allows new possibilities of controlled multifunctionalization with different biomolecules. based on this principle, developed aunp tags are characterized by enhanced reactivity and stability. increased loading efficiency also allows the design of new probes modified with multiple structures for biotargeting (e.g., antibodies and dna), together with other signal amplification structures (e.g., enzymes and dna oligonucleotides) [69, 76, 77, [96] [97] [98] , both types of which are stridently included in the development of new diagnostic schemes and designs. for example, the aunpfigure 2 . a generalized scheme for the basic characteristics and functions of aunps applied in virus detection. the size and metal nature of aunps are the key characteristics that enable them to directly transduce multiple types of signals, including optical, catalytic and electrical signals that can be detected by dynamic light scattering (dls), localized surface plasmon resonance (lspr), surface-enhanced raman scattering (sers), ultraviolet-visible (uv-vis) spectroscopy, fluorometry and other electrochemical analysis techniques. in addition, aunps act as nanoparticulate tags to enhance the signal detection of several analytical techniques, such as quartz crystal microbalance (qcm), atomic force microscopy (afm), and inductively coupled plasma mass spectrometry (icp-ms). their large surface area-to-volume ratio is known to permit them to function as an excellent scaffold for bioimmobilization and target-probe interactions with highly enhanced specificity and sensitivity. barcoding assay and its descendent technique of immuno-pcr universally rely on aunp tags modified with antibodies and dna oligonucleotides [81, 96, 97] , and aunp-based enzymatic electrochemical assays are based on using aunps dually modified with dna and antibodies together with an enzyme to induce a chemical reduction reaction allowing electrochemical signal transduction [76, 77] . several recent studies have attempted to harness the unique properties of aunps to develop various advanced schemes for virus detection. the developed assays are greatly variable in design and underlying principle. however, the utilization of aunps conjugated with specific virus-targeting biomolecules is a key component in most of these assays. various aunp bioconjugates have been widely employed in many colorimetric, scanometric, electrochemical, and fluorometric systems for the detection of many groups of well-known human viruses (table 1 and fig. 3 ). each of these groups will be discussed in more detail in the following sections. the bunyaviridae family comprises more than 300 members that are primarily organized into four main genera: hantavirus, orthobunyavirus, phlebovirus and nairovirus [99] . bunyaviruses are spherical, enveloped rna viruses 80-100 nm in size. their genome is composed of three segments of negative-sense, ssrna: a large segment (l, 6.3-12 kb) that encodes rna polymerase, a medium segment (m, 3.5-6 kb) that encodes viral glycoprotein, and a small segment (s, 1-2.2 kb) that encodes nucleocapsid protein [100] . these viruses are diverse in their host range and are frequently involved in a wide range of diseases in plants, animals, and humans. human pathogenic bunyaviruses, such as crimean-congo hemorrhagic fever virus, hantaan virus, la crosse virus, oropouche virus, rift valley fever virus, and toscana virus, continue to present increasingly important health concerns worldwide [101, 102] . the genus hantavirus was recently expanded to include more than 24 antigenically and genetically distinct htnvs [99] . among them, rodent-borne htnvs can cause serious diseases in humans, including hemorrhagic fever with renal syndrome, and hantavirus cardiopulmonary syndrome, and hospitalizes more than 150,000 persons each year with a mortality rate reaching 10%. recently, the health impacts of htnvs are expected to dramatically increase in the near future due to the increasing number of reports on newly discovered htnvs [102] [103] [104] . aunps were utilized to develop a novel immuno-pcr assay for the immunological detection of htnv nucleocapsid protein [96] . this assay mainly relies on the enhanced surface area of aunps to prepare dually functionalized antibody-oligonucleotide conjugates that exceptionally carry specific monoclonal antibodies to label the target htnv antigen; the assay also involves barcoding dna for signal amplification (fig. 4a ). this assay could optimally detect concentrations as low as 200 am of purified or spiked antigen samples, which is ~7 orders of magnitude more sensitive than conventional elisa. the high detection sensitivity together with the targeting of htnv nucleocapsid protein [105] , which is the most abundant viral component synthesized post virus infection, makes this assay a promising candidate method for the early diagnosis and control of htnv. rvfv is an arthropod-borne pathogen primarily known to affect animals and later discovered to infect humans. rvfv infection in humans can progress to serious hemorrhagic fevers that often lead to death, with prominent mortality rates of up to 10-12%. infected persons can also develop other clinical manifestations, including retinitis, encephalitis, and paralysis [106, 107] . rvfv is historically endemic to africa and has had a long history of outbreaks ranging from its reemergence in egypt in 1977 to the most recent outbreak in south africa in 2011 [108, 109] . thus far, rvfv outbreaks are unpredictable and usually associated with very high socio-economic and public health consequences [110, 111] . therefore, diagnostic methods that can rapidly identify the virus have become crucial not only for avoiding and preventing such outbreaks but also for monitoring cross-border dissemination. a novel immunoassay based on aunps coupled with a surface-enhanced raman scattering (sers) technique has been developed for the detection of rvfv capsid antigen [49] . in this assay, aunps are applied as both immobilization scaffolds for the target-capturing antibodies and metal promoters to enhance the raman reporter dyes. the target antigen is first captured by antibody-magnetic particle conjugates and then labeled with dye/antibody-aunp conjugates, forming a three-component immunocomplex that is magnetically concentrated and finally detected by sers (fig. 4b) . this approach has shown an outstanding sensitivity level down to 5 fg/ml of the target antigen. this high sensitivity with the possibility for the direct detection of rvfv in complex media or samples allowed by magnetic particles support the application of this assay for the rapid and sensitive detection of rvfv and the control of any future outbreaks. immuno-pcr assay for htnv detection using aunp probes dually functionalized with antibody (ab) and double-stranded (ds)dna. au-nanoprobes are directly applied to label virus antigens precaptured on a microplate. the aunps are carrying dsdna that includes barcode single-stranded (ss) dna for signal amplification. the barcode dna is separated, amplified and detected by gel electrophoresis [96] . additionally, this assay can be modified in a more complex detection scheme that has been reported for human immunodeficiency virus detection [81] . (b) surface-enhanced raman spectroscopy (sers)-based assay for detection of rvfv using raman reporter dye-coated aunps and magnetic nps (mnps). aunps and mnps are conjugated with a polyclonal ab specific for the target virus antigen forming aunp/virus antigen/mnp complexes; then, a 785 nm laser excites the magnetically concentrated aunp/virus antigen/mnp complexes. the presence of the target antigen yields a reduction in the intensification of raman dye signature spectrum peaks, thereby providing an estimation of its concentration [49] . this assay has been applied for the detection of wnv infection [49] . the coronaviridae family comprises two subfamilies, coronavirinae and torovirinae, that were recently expanded to include many viruses. coronavirinae are classified into four genetically distinct genera (alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus) [112] . coronaviruses are enveloped rna viruses that are pleomorphic in shape (spherical and 120-160 nm or bacilliform and 170-200 nm by 75-88 nm). they have a relatively large, positive-sense, ssrna genome of 27-32 kb that encodes four to five structural proteins (s, the spike glycoprotein; m, the membrane glycoprotein; n, the nucleocapsid interrupt phosphoprotein; e, the envelope protein; and he, the hemagglutinin-esterase glycoprotein) and 2 nonstructural polyprotein precursors (pp1a, polyprotein 1a; and pp1ab, polyprotein 1ab), which are later processed into several other nonstructural proteins and viral polymerase [113, 114] . coronaviruses are clinically significant to humans and usually associated with different respiratory, intestinal, hepatic, or neurological diseases [115] . hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku1 are among the most disseminating coronaviruses that usually infect the upper respiratory tract in humans, causing mild common cold-like diseases [116] [117] [118] [119] . other coronaviruses, such as sars-cov identified in china in 2002 and mers-cov discovered in the middle east in 2012, are notorious pathogens able to cause widespread outbreaks of pneumonia and pneumonialike conditions with very high morbidity and mortality rates of up to 35% [120, 121] . due to their high clinical significance and contagious nature, there has recently been great interest in testing and developing advanced sars detection methods. sars detection using aunps is primarily focused on developing rapid and specific molecular detection through two main assays: 1) a colorimetric assay for pp1ab gene detection; and 2) an electrochemical assay for nucleocapsid protein gene detection ( table 1 ). the colorimetric assay involves the ability of aunps to preferentially adsorb ssdna over dsdna and specifically sense the presence of the target dna [54] . specific, short ssdna probes adsorb to the surface of aunps, resulting in an increased particle colloidal stability and an increased ability to withstand slightly elevated salt concentrations without significant aggregation or color changes. upon addition, the target dna specifically forms dsdna with the adsorbed ssdna probes, which then easily detach, leaving the aunps to aggregate due to the salt. the particle aggregation causes the red color of the solution to change to blue, indicating the presence of target sars nucleic acids; this change can be directly assessed by the naked eye or precisely quantified by ultraviolet-visible (uv-vis) spectroscopy in correlation to the target dna concentration (fig. 5a ). in the electrochemical assay, aunps are applied to enhance the electrode conductivity and increase the surface area available for detection probe immobilization [72] . sars-specific dna-capturing probes are first immobilized on the surface of an electrode structured with aunps; then, they are allowed to hybridize with the biotinylated targets. then, streptavidin-labeled alkaline phosphatase is applied to catalyze the indirect reduction and deposition of silver ions, which are eventually measured by anodic stripping voltammetry in correlation with the target dna concentration (fig. 5b) . the aunp-based assays developed for the molecular detection of sars are relatively rapid and simple; especially the colorimetric assay, which completely eliminates the need for instrumentation or trained personnel and yields results exclusively in the liquid phase that can be rapidly identified as positive/negative by the naked eye within 5 min. this technique can allow the detection of target sars nucleic acids with a sensitivity limit down to 100 fm; thus, it can be very beneficial for the early diagnosis of the sars virus, which is critical for such a contagious pathogen. in addition, the simplicity and very high sensitivity of aunp-based colorimetric assays for nucleic acids have promoted the application of aunps for the detection of other viruses either using slightly modified procedures or in combination with other sensing characteristics of aunps [56] . the family filoviridae can be classified into two main genera, ebolavirus [119, 122] . filoviruses are filamentous, enveloped rna viruses 80 nm in diameter and approximately 800-1000 nm in length. their genome is nonsegmented, negative-sense, ssrna 19 kb in size that encodes for at least 7 proteins (nucleoprotein, viral proteins vp24, vp30, vp35, and vp40, glycoprotein, and polymerase protein) [123] . aunps stabilized by single-stranded (ss)dna probes. in the presence of the target dna sequence, double-stranded (ds)dna is formed and desorbs from the surface of citrate-reduced aunps, leaving them to aggregate. this aggregation results in a color change from red to blue, indicating the presence of target nucleic acids [54, 56] . (b) enzymatic electrochemical detection of sars by anodic stripping voltammetry using a aunp-screen-printed carbon electrode. the aunp-modified electrode is modified with capture dna probes that are allowed to hybridize with biotinylated targets. streptavidin-alkaline phosphatase (s-ap) is then applied for the indirect reduction of silver ions in the solution into a metallic deposit. the formed silver metals are then electrochemically measured to determine the target virus dna concentration [72] . most ebovs primarily originate from africa, where they are frequently associated with large outbreaks of ebola hemorrhagic fever (ehf), which is now known to be one of the most deadly and virulent diseases affecting humans, with a case fatality approaching 90% [124] . the early outbreaks of ehf were confined to sudan and its neighbor, democratic republic of the congo, from 1976 to 1978, followed by several independent outbreaks in different countries: [124, 125] . most of these outbreaks were caused by zebov and sebov, while some were caused by new species later named ciebov and bebov [125] . these heavy series of ehf outbreaks are continuing, and most recently, multiple countries in west africa have struggled against a massive outbreak of ebov beginning in march 2014. ebola is expected to remain a major global public health threat, especially with the current absence of effective treatments; therefore, it is necessary to have flexible rapid-detection schemes in place to diagnose and control ebov outbreaks [126] . a very interesting aunp-based molecular assay has been developed for the bimodal scanometric and sers-based detection of ebov by using aunps to promote silver staining on their large surface area [50] . the target ebov dna sequence is first captured on chip surfaces by specific, short dna probes and labeled with aunp-dna-cy3 probes. then, the formed 3-component hybridization complexes are subjected to a silver enhancement step. the deposited silver metal eventually enables scanometric detection (based on a silver-caused dark color) and sers detection (based on using cy3 as a raman-active tag) (fig. 6) . this assay has additional benefits beyond its high specificity and sensitivity that reach down to 20 fm of the target rna. because a very large number of probes can be designed based on using different raman tags, this assay can be readily adapted for multiplex detection, allowing the simultaneous detection of different diagnostic targets of the same virus or even completely different viruses. furthermore, it does not require dna amplification and eliminates the need for specific thermal cyclic equipment. in addition, the capability for bimodal detection simplifies evaluation of the results, as the output can be positive/negative either by traditional scanner systems or by sers when the probes are labeled with raman dyes. such simplicity based on bimodal detection without the need for complicated thermal amplification equipment, multiplexing, and high sensitivity supports the application of this technique for ebov, which is very contagious and usually necessitates the detection of several targets. it is a chip-based detection scheme in which the target dna is captured by immobilized, specific dna oligonucleotides and then directly labeled by aunp-dna probes, followed by silver enhancement. the aunps enhance the surface area available for silver deposition and dna immobilization to achieve highly sensitive scanometric detection signals. modification of the aunp probes with cy3 allows the additional sers-based detection of target dna. this scheme has been reported for the detection of a wide range of viral nucleic acids, including those of hcv, hbv, and hav, which belong to different virus groups [50] . furthermore, the scanometric detection reported in this scheme has been considered a universal step in other detection schemes described for other viruses, such as hev [82] and hiv [81, 84] . this assay has been reported by the same authors for multiplex detection of hepatitis a virus (hav), hepatitis b virus (hbv), hiv, and variola virus (smallpox, vv), which confirms the broad capability of this assay for virus detection [50] . flaviviridae is a large virus family comprising three main genera: flavivirus (53 species, type species is yellow fever virus), hepacivirus (one species, hepatitis c virus; hcv), and pestivirus (four species, type species is bovine virus diarrhea) [127] . flaviviruses are spherical, enveloped rna viruses 40-65 nm in size. their genome is nonsegmented, positive-sense ssrna approximately 10-11 kb in size and includes one open reading frame (orf) encoding three structural proteins (c capsid; prm, premembrane; and e, envelope), and seven nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5). several well-known human pathogens are included in this family, such as dengue virus (denv), hcv, yellow fever virus, and west nile virus (wnv), japanese encephalitis virus, and tick-borne encephalitis virus. these viruses have become increasingly serious and have been frequently involved in causing many endemics and epidemics around the world [128, 129] . denv is the most rapidly spreading vector-borne virus in the world. denv is now known to be epidemic in more than 100 subtropical and tropical countries, threatening up to 2.5 billion people. denv causes more than 350 million new annual infections, including 90 million with symptoms varying from flu-like, mild, undifferentiated fever to classic dengue fever (df) or df with hemorrhagic manifestations [130, 131] . although many of these infections are self-limiting and resolve without hospitalization, some progress to severe disease that needs to be quickly identified and treated [132, 133] . currently, there are no specific treatments or protective vaccines available against denv, and as a result of its rapid expansion, accurate and sensitive diagnostic techniques have become crucial for effective control and treatment applications [134] . aunps have been combined with the well-known analytical techniques qcm and icp-ms to develop two novel molecular assays for denv detection. for the first time, these assays implemented aunps to increase the mass of target dna to allow its detection by qcm in a highly quantitative and sensitive manner (fig. 7a) or to amplify the detection signal by releasing many gold ions during icp-ms analysis of the target dna (fig. 7b ). in the aunp-based qcm assay, the presence of target dna initiates a layer-by-layer hybridization of the target dna and several specific multivalent aunp-dna probes, resulting in significant mass changes detected by qcm [93] (fig. 8a) . whereas, in the icp-ms-based assay, two types of probes prepared from specific aunp-dna and mnp-dna conjugates are applied to specifically recognize and sandwich the target virus dna sequences. the mnp probes then help to magnetically separate the formed sandwich complexes, and the target dna concentration is indirectly estimated by detection of the au concentration existing in the sample [94] (fig. 8b) . it is worth noting that both techniques are among the most novel applications of aunps in virus detection. the implementation of aunps extends the ability of qcm and icp-ms to detect dna. moreover, aunp-based icp-ms is among the most sensitive molecular dna assays applied for virus detection, with a detection limit down to 80 zmol, which can greatly help to control denv and other viruses [94] . the bio-barcode ssdna is released and employed to establish multilayered aunps over the electrodes to enhance the produced electric current [72] . (b) dry-reagent strip-based dna assay using aunp-oligo (dt) reporters. aunps with poly (dt) are applied to label biotinylated target dna modified with a poly (da)-tail pre-captured by immobilized streptavidin in the test zone of the strip and generate characteristic red bands [55] . (c) single-particle fluorometric detection of hcv using cy3-tagged ssrna that is electrostatically adsorbed or covalently coupled through thiol (sh) chemistry to the surface of aunps. the coupling of aunps and ssrna probes brings cy3 in the vicinity of the aunp surface and eventually results in emission quenching; upon hybridization with the target rna, the quenched emission is released [55] . hcv is one of the leading causes of chronic liver diseases in humans. approximately 3% of the global population has experienced hcv infection. currently, there are 170 million chronic carriers, and more than three million new infections occur globally each year [135, 136] . chronic hcv infection is frequently associated with marked hepatic injuries, including cirrhosis, liver failure or hepatocellular carcinoma. these types of injuries can be very severe and often end in death or liver transplantation [135, 137] . hcv infection is generally resilient and usually requires treatment that combines numerous medications. the most popular treatment regimen for hcv is a combination of pegylated interferon alfa (peg-ifnα) and ribavirin (rbv) [138] . other three-drug regimens based on the addition of the serine protease inhibitor telaprevir (tvr) or boceprevir (bec) to the standard peg-ifnα/rbv treatment have also been described [139] . in addition, several recently developed oral drugs, such as simeprevir and sofosbuvir, have been approved by the fda for more effective care and shorter treatment duration [140] [141] [142] [143] . despite these numerous drugs and due to the lack of efficient vaccines against hcv, reliable and sensitive detection methods are essential for viral infection prevention and therapeutic responses. numerous aunp-based scanometric, fluorometric, electrochemical, and colorimetric assays have been reported for the molecular detection of hcv (table 1 ). in these assays, aunps are utilized for different sensing functions: 1) aunps have been modified with poly(dt)-tailed dna and directly applied to label target dna sequences terminally modified with a poly(da)-tail in a lfa assay to allow the colorimetric detection of hcv (fig. 7b) [55] . 2) aunps electrostatically or covalently modified with cy3-tagged ssrna sequences have been applied for the fluorometric detection of target hcv sequences, in which the fluorophores are quenched in a process analogous to fret. the presence of cy3 in the vicinity of the surface of aunps eventually results in cy3-emission quenching. upon hybridization with target viral rna, the electrostatically adsorbed probes labeled with cy3 are detached from the surface of the particles, releasing the quenched fluorescence. the covalently bound probes form rigid dsrna with a linear configuration, causing cy3 to move away from the particle surface and release the quenched emission [60] (fig. 7c) . 3) aunps have also been applied to enhance electrical conduction and catalyze a reduction reaction on the surface of detection electrodes. a specifically designed hairpin dna probe terminally modified with aunps and bound to the surface of a glassy carbon electrode is utilized to detect the amplified viral rna. the presence of target dna results in the formation of rigid dsdna, and the hairpin conformation consequently changes, moving the aunps away from the electrode surface, which eventually appears as a detectable decline in the current value in proportion to the target dna concentration [78] . 4) aunps decorated with a specific nucleic acid probe were used for colorimetric detection of hcv. the assay is based on using cationic aunps to induce the aggregation of aunps probes. in the absence of hcv, the cationic aunps electrostatically bind to negatively charged aunps-dna probes causing their aggregation and change of the solution color to blue. the presence of hcv nucleic acid protects the probes and prevents their aggregation by cationic aunps and the solution color remains red. this assay was specific and showed a sensitivity of 93.3% and a detection limit of 4.57 iu/μl [13] . in the immunological detection of hcv, aunps dually modified with capturing antibodies and barcoding dna, similar to those described for the immuno-pcr technique, in combination with magnetic np (mnp)-antibody conjugates, have been applied to capture and separate the target antigen. subsequently, the barcode dna is further allowed to act as a bridge, guiding the formation of a multilayer of aunp-dna probes over the surface of a nanogap electrode (fig. 7a) . the formation of such barcode dna-guided aunp multilayers significantly increases the detected electrical current and is utilized to quantitatively measure the target hcv antigen concentration in applied solutions. the results of this immunoassay indicate a detection limit of 1 pg/µl [69] . in a similar protocol, the barcoding dna was further applied to hcv core antigen detection based on an enzyme to release the barcode dnas to be quantified with rt-pcr. this assay showed a detection limit of 1 fg/ml, which is one magnitude greater than the standard elisa (2 ng/ml) [14] . it is worth mentioning that the primary focus of the developed aunp-based assays was the detection of hcv rna rather than hcv antigens or antibodies. this is due to the broader potential of molecular assays for the detection and management of active hcv infections. hepadnaviridae comprises two main genera, orthohepadnavirus and avihepadnavirus. the genus orthohepadnavirus includes 4 species (hbv, woodchuck hepatitis virus, ground squirrel hepatitis virus, and woolly monkey hbv) that infect mammals, while the genus avihepadnavirusincludes2 species (duck hbv and heron hbv) infect birds [144] . hepadnaviruses are spherical, enveloped dna viruses 40-48 nm in size. they possess a unique gapped genome of 3.2-kb circular dsdna, which contains 4 partly overlapping orfs encoding the different viral proteins: the core protein (c), e antigen, polymerase protein (pol), envelope proteins, and transcriptional transactivator protein [144, 145] . the prototype member of the family hepadnaviridae, hbv, is a health threat to ~88% of the global population [145, 146] . hbv is the etiological agent of hepatitis type b in humans. it is estimated that nearly 2 billion people around the world have been exposed to hbv infection, and there are more than 360 million chronic carriers suffering from the serious risk of developing liver cirrhosis and cancer [146, 147] . the availability of an effective protective vaccine against hbv has greatly reduced the number of new infections. in addition, there have been significant advances in the available treatments for chronic hbv infection. viral dna polymerase inhibitors and peg-ifnα are now well known to significantly control hbv infection and prevent its progression to chronic hepatitis b-associated liver failure and hepatocellular carcinoma [148, 149] . however, chronic hbv infections remain highly contagious, and the virus can easily be transmitted to other persons through any close contact allowing the transfer of infected bodily fluids. the highly contagious nature of hbv and its ability to spread among people are the main reasons why this pathogen continues to threaten the public. therefore, diagnostic methods that are highly sensitive to low concentrations of virus are needed to curtail this virus and prevent its dissemination. hbv is by far the most common virus reported in the published literature regarding the applications of aunps in virus detection (fig. 3) . interestingly, the developed assays cover most of the known diagnostic markers of hbv through many different electrochemical, scanometric, fluorometric, lightscattering, colorimetric, and sers detection schemes (table 1 and fig. 6, 14-18) . several aunp-based electrochemical assays have been developed for the detection of hbv antigens and dna. two enzyme-amplified electrochemical assays have been reported for the detection hbv surface antigen (hbsag) using conjugates of aunps and horseradish peroxidase (hrp)-labeled antibodies against hbv surface antigen (hbsabs) [76, 77] . the target hbsag is captured and labeled with aunp-hbsab/hrp as secondary antibody conjugates. then the reduction of h2o2 catalyzed by the bound hrp is measured either by using an aunp/thionin/dna-modified au electrode coupled with a cyclic voltammeter [76] or through using a nanoporous au electrode coupled with differential pulse voltammeter [77] (fig. 9a) . alizadeh et al. reported aunps modified with horseradish peroxidase mimicking dnazyme to label hbsag magnetically captured and concentrated on the surface of a au sheet-like electrode. due to the efficient catalytic activity of hrp-mimicking dnazyme, the proposed immunoassay allowed quantitative detection of hbsag with a linear concentration range of 0.3-1000 pg/ml and detection limit of 0.19 pg/ml [15] . other studies described the electrochemical detection of hbv based on aunp-metal enhancement amplification, including copper and silver metal enhancement [74, 75] . using copper metal enhancement, hbsag is sandwiched by mnp-hbsab conjugates and aunp-hbsab probes to form a 3-component immunocomplex, which is magnetically separated and stained through a copper enhancement step (fig. 9b ). after copper acidic dissolution, the resulting ions are measured by anodic stripping voltammetry (asv) [75] . using silver enhancement, hbv dna is detected using probes prepared of streptavidin-modified aunps. biotinylated hbsag gene sequences are magnetically pre-separated and concentrated using magnetic bead-dna conjugates. the deposited silver ions are then analyzed by an electrochemical stripping technique to detect the target hbv dna quantitatively [74] . hbv detection through aunp-based scanometric assays mainly relies on the well-described aunp-promoted silver-staining protocol, in which aunp probes are applied to facilitate the reduction of silver ions into metallic silver that is deposited as visible black spots, indicating the presence of hbv. following this protocol, aunps modified with staphylococcal protein a (spa) [88] or specific dna probes [50, 80] have been utilized in multiple chip-based assays to permit the detection of different hbv antibodies and dna (fig. 6 ). in addition, wang et al. combined this aunp-promoted silver-staining protocol integrated with a bio-barcode-amplification (bca) technique to allow an enhanced scanometric detection of hbv dna [85] . this bca-based scanometric assay employs two different sets of dna conjugates to capture and detect a specific hbv sequence. the first set is a group of aunps modified with the short dsdna of a signal amplification dna strand (barcode dna) partially complemented with a detection probe strand, while the second set is a group of magnetic microparticles (mmps) modified with ssdna specific to the target hbv dna. the presence of the complementary target sequences of dna guides the assembly of aunp/dna/mmp sandwich hybrids. subsequently, the sandwiched hybrids are magnetically isolated and washed, and the barcode dna is eventually released from the aunps. the barcode dna is then added to the surface of a chip modified with specific dna-capturing probes and directly labeled with aunp-dna conjugates, followed by silver staining to further amplify the detection signal (fig. 10) . the aunps-fluorometric assays for hbv are performed through different schemes that basically depict the quenching efficiency of aunps in a manner remarkably similar to the traditional fret protocol. zheng et al. developed a multiplex detection system based on applying gold nanorods (aunrs) as acceptors and qds of different colors as donors to simultaneously detect hbsag and hbv e antigen [62] . this assay follows the direct sandwich immunoassay format, and the presence of target antigens is manifested by the fret-induced quenching of qd fluorescence in the formed sandwich nanostructure of aunr-ab1/ag/qd-ab2 (fig. 9c ). draz et al. further coupled multiple aunp-peptide conjugateacceptors with single-core qd-antibody fab conjugate-donors, forming a preassembled hybrid nanocluster plasmonic resonator complex for hbsag and hbv particle detection (fig. 9c) . this scheme follows a competitive assay format, and the addition of hbv target surface antigen or particles to the preassembled nanocluster releases the quenched fluorescence signal of the qds in proportion to the in this assay, the target hbsag is captured and labeled with aunp-ab/hrp, and the detection signal is measured by using an ab-modified au electrode to assess the reduction of h2o2 catalyzed by the bound hrp using differential pulse voltammetry (dpv) technique [76] . (b) copper (cu)-enhanced electrochemical immunoassay. aunps serve as a scaffold for a horseradish peroxidase (hrp) enzyme that catalyzes a redox reaction in the first scheme or enhances metal deposition in the second scheme [75] . (c) fluorometric detection by the fret-induced quenching of fluorophores on the surface of aunps. aunps and gold nanorods (aunrs) are applied to quench the fluorescence of fluorophores, such as (i and ii) quantum dots (qds) [62, 65] and (iii) fluorescein amidite (fam) [61] , through a fret-based interaction in the presence of the target virus. a similar aunp fret-based scheme has been reported for the detection of h1n1, which belongs to the family orthomyxoviridae [63] . (d) light-scattering assays using aunp-antibody probes. (i) light-scattering immunoassay of virus antigen based on using aunps with different sizes (10 nm and 50 nm) conjugated with antibodies (abs) to sandwich target antigens and enhance the dynamic light scattering [51] . additionally, this scheme has been reported with the use of aunps of the same size for h1n1 detection [53] . (ii) plasmonic immunoassay based on allowing aunr-ab conjugates to interact with the target antigen, thus changing the aunr localized surface plasmon resonance (lspr) behavior [52] . target concentration [65] . furthermore, lu et al. developed a fluorometric assay for hbv dna detection in which positively charged aunrs are employed to chelate fluorescein amidite (fam)tagged ssdna electrostatically onto their surface, forming fam-ssdna-aunr ternary complexes [61] . the formation of these complex results in the fret-based quenching of fam fluorescence, and when complementary target dna is present in the system, a fam-ssdna/cdna duplex is formed. this dna duplex is comparatively more negative than the fam-ssdna and can mediate stronger electrostatic interactions with aunrs, leading to increased fret efficiency, which manifests as a further decline in the fluorescence intensity of fam. this change in fluorescence intensity is utilized to sense and estimate the exact concentration of the added target dna (fig. 9c) . aunp-based light-scattering assays have been described for the detection of hbsag through two main schemes: the first scheme applies spherical aunps of different diameters (10 nm and 50 nm) modified with hbsab to sandwich hbsag, and the presence of hbsag results in specific immuno-based aggregations that can be estimated by measuring the increases in hydrodynamic diameter using dls (fig. 9d) [47, 51] ; the second scheme relies on the lspr characteristic behavior of aunrs to directly sense the binding of hbsag to the antibodies conjugated to their surface by measuring lspr peak shifts via uv-vis spectroscopy (fig. 9d) [52] . a aunps-based colorimetric lateral flow assay (lfa) assay was developed for the detection of hbsag. this assay relies on the characteristic red color of different sized aunps to label hbsag on an immunochromatographic test strip. a strip composed of a sample pad, conjugation pad, control line, test line and absorbent pad was used to capture hbsag labeled with aunps and the test line becomes red in color. the results showed that the signal visibility of lfa could be improved by increasing the diameter of aunps, and ~43 nm aunp enabled lfa assay with a detection limit of 100 ng/ml [16] . a aunps-based sers assay for hbv dna detection was developed following a sandwich assay format performed on a heat-responsive hybrid silicon substrate modified with aunps. the target hbv dna is captured on the surface of the silicon substrate and forms a sandwich complex with specifically designed aunps modified with dnaindocyanine green proximally to the surface, leading to high sers signals. this heat-responsive hybrid silicon substrate-based sers platform can detect remarkably low hbv dna concentrations of ~0.44 fm at 25 °c and ~0.14 fm at 37 °c [17] . aunp-based fluorometric detection is the most sensitive among the reported methods for hbv detection, with a detection limit of 1 particle/μl. due to the highly infectious nature of hbv, this limit of detection can be very useful for exploiting these assays for efficient control and management of hbv infection. in addition, the simplicity of scanometric and colorimetric detections is very interesting, allowing flexibility in the detection design and target, and was reported for single and multiple target detection for more efficient and accurate screening of hbv. on the other hand, the developed dls assay is very novel to hbv research, simple and sensitive; however, the need for bulky equipment for dls measurement remains the major challenge for its wide use and real application in hbv detection. the family hepeviridae is monogeneric with one exclusive member called hev, which is a spherical, small, nonenveloped rna virus with a ~7.2 kb ssrna genome [150] . the viral rna is a positive-sense molecule organized into 3 partially overlapping orfs flanked by short untranslated regions at both ends. the first orf encodes 4 mature nonstructural proteins (methyltransferase, protease, helicase and replicase), the second orf encodes 2 structural proteins (c and vp1), and the third orf encodes a small protein of unknown function [150] . hev is known for causing human liver inflammation figure 10 . aunp-based nucleic acid assay for hepatitis b virus (hbv) detection. hbv dna was detected using a scanometric detection that integrates magnetic separation and aunps-based bio-barcode-amplification method. aunps provide a large surface area for silver deposition and dna immobilization, thereby achieving highly sensitive scanometric detection signals through a chip-based silver deposition scheme [85] . called hepatitis e, which is widely disseminated in developing countries with poor sanitation conditions and has recently become apparently endemic in some industrialized countries, including the usa [151] . one aunp-based colorimetric assay has been reported for the molecular detection of hev [152] . this assay targets a conservative fragment of hev in orf1 that is pre-amplified by one-step rt-pcr/pcr amplification, and the generated cdna is labeled by aunp-dna conjugates, forming three-component sandwich hybrids in a manner very similar to that previously depicted in fig. 6 . this hybridization is followed by a silver-staining step to allow the scanometric detection of target dna either by the naked eye or using simple scanners, with a detection limit of 100 fm of hev amplicon. one of the major advantages of this hev detection assay is its sensitivity, which can help control hev. however, this assay suffers from the same disadvantages of conventional pcr because of the requirements of the amplification step. herpesviridae is a taxonomically large family of viruses, including 3 major subfamilies (alphaherpesvirinae, betaherpesvirinae and gammaherpesvirinae), which together comprise nearly 9 genera with over 100 viruses [153] . repeats. the rearrangements of these repeated sequences result in different genome isomers. in epstein-barr virus (ebv) and kaposi sarcoma-associated herpes virus (kshv), from the subfamily gammaherpesvirinae, there are repeated sequences at the termini of their genomes that differ from those of other human pathogenic herpesviruses, including herpes simplex virus type 1 (hsv-1) and type 2 (hsv-2), human cytomegalovirus (hcmv), and varicella zoster virus (vzv); these viruses are characterized by the definite presence of us and ul regions in their genomes [153] [154] [155] . herpesviruses can infect humans and many animals. they account for significant and recurrent human cutaneous diseases, including oral herpes, genital herpes, and many cancers. eight herpesviridae family members are commonly involved in human infection: hsv-1 (human herpesvirus1; hhv-1), hsv-2 (hhv-2), vzv (hhv-3), ebv (hhv-4), hcmv (hhv-5), hhv-6, hhv-7, and kshv (hhv-8). hsvs are among the most prevalent viruses in humans. there are two serotypically and genetically different hsvs, types 1 (hsv-1) and 2 (hsv-2) [156] . hsv-1 is estimated to infect up to one-third of the world population, while hsv-2 infects nearly 500 million people around the globe, with more than 20 million new cases occurring every year [157] [158] [159] . both hsv types are renowned for infecting epithelial cells of the skin or mucosal surfaces and then infecting the central nervous system, causing lifelong, incurable infections in humans. these infections are primarily asymptomatic but can progressively result in serious health complications. for example, hsv-1 infection is the main cause of infectious blindness in the world and has emerged as an important cause of genital disease in the developed world, and hsv-2 infection is the leading cause of genital ulcers worldwide [159, 160] . nevertheless, these viruses are involved in many other clinical conditions, such as encephalitis, conjunctivitis, zosteriform skin lesions, pneumonia, and systemic infections that compromise vital organs in the body [161] . therefore, desperate efforts have been directed toward developing an effective vaccine against hsvs. however, there are no protective vaccines against hsvs, and only a few anti-herpes drugs, such as acyclovir, valacyclovir, and famciclovir, are commercially available [162] [163] [164] . these drugs are only helpful to control symptoms and signs of infection and do not eradicate the virus or even decrease the frequency or severity of infection recurrence and due to the drug resistance rapidly developed by hsvs during treatment, the efficacy of anti-herpes drugs is increasingly hampered over time. thus, the definitive diagnosis of hsv virus infection is essential for effectively controlling its severity and applying treatment regimens. with their characteristic red color, aunps have been exploited as colorimetric labels in a lateral-flow immunochromatographic assay (lfia) to allow the immunological detection of hsv antigen. in this assay, the lfia strip is preloaded with the labeling aunp-anti-human igg conjugates onto the conjugate pad, and the capturing igg-1 and igg-2 antigens, along with the anti-igg antibody, are immobilized onto separate test and control lines directly next to the sampling area (fig. 11a) . the sample is loaded and allowed to migrate across the strip with the chase buffer to react with the capturing antigens (igg-1 and igg-2) loaded on the test lines, as well as the anti-igg antibody loaded on the control line. as a result, hsv antibodies are captured on the test lines and labeled with the loaded aunp-dna conjugates, causing characteristic red lines to appear. according to the color of the test lines, the samples can be qualitatively determined to be positive or negative within 20 min [59] . lateral-flow immunochromatographic assay of hsv-2 antibodies using aunp anti-igg probes. sample addition followed by chase buffer addition causes gold nanoparticle (aunp)-anti-igg conjugates and the sample to migrate, contacting the test lines sequentially and resulting in the capture of type-specific antibodies to hsv-2. the concentration of hsv-2 antibody-aunp complexes on the test line causes a corresponding pink color to form [59] . this scheme is similar to that reported for the detection of h5n1 [58] and hiv-1 [91] . (b) electrochemical detection of hcmv by the acidic dissolution of aunps. hcmv dna is directly labeled with specific aunp-dna probes. after the acidic dissolution of aunps, au iii ions are measured by anodic stripping voltammetry (asa) [68] . (c) colorimetric one-pot np-based assay of kshv. this assay has been reported for the differential diagnosis of kshv dna and dna from a frequently confounding bartonella species. upon the addition of sample dna to a mixture of silver np (agnp)-dna and aunp-dna conjugates specific for kshv and bartonella dna, respectively, complementary dna hybridization triggers the aggregation of bare np conjugates and the subsequent decrease or disappearance of its corresponding color [60] . hcmv/hhv-5 is the largest known endemic virus in humans. it persistently infects over 70% of the population worldwide [165] . hcmv can infect a broad range of cells, including endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, neuronal cells, hepatocytes, and many types of immune cells [165, 166] . nearly all hcmv infections are asymptomatic, except in immunocompromised individuals and neonates, and can lead to severe symptoms. hcmv infection is the leading cause of congenital malformations in live births worldwide, while in immunocompromised individuals hcmv is usually opportunistic, causing very serious clinical manifestations, such as pneumonia, hepatitis, encephalitis, colitis, and retinitis [167, 168] . moreover, hcmv has been described to be associated with several human malignancies, especially in the brain, breast, prostate, skin and colon [169] . numerous hcmv vaccine candidates have been developed and tested, but a vaccine has yet to be licensed. antiviral therapy with several anti-hcmv drugs, such as ganciclovir, valganciclovir, foscarnet, and cidofovir, which target the viral dna polymerase, is currently the main strategy for controlling hcmv infection [166, 170, 171] . unfortunately, these drugs result in substantial hematotoxicity and nephrotoxicity in patients, and hcmv remains a considerable public health threat without an efficient treatment or control strategy. in such conditions, the need for new diagnostic methods that can help to control the dissemination of this pathogen is very important. a novel aunp-based electrochemical assay for the molecular detection of hcmv dna has been reported [68] . in this assay, aunps are applied to amplify the detected electrochemical signal by releasing many au ions upon exposure to acid dissolution. typically, the immobilized target hcmv ssdna is initially labeled with oligonucleotidemodified aunp probes followed by a metal dissolution step, releasing many gold ions. the concentration of solubilized gold ions is then indirectly assessed by asv (fig. 11b ). this assay allows the highly sensitive detection of target dna concentrations down to 5 pm [68] . kshv/hhv-8 is a leading cancer-causing virus. it is commonly responsible for inducing a considerable range of neoplastic diseases in immunocompromised patients, such as kaposi sarcoma, primary effusion lymphoma, and some kinds of multicentric castleman disease [172, 173] . owing to the dramatically increasing rates of immunocompromised patients over the past two decades, the clinical significance of kshv is clearly increasing; additionally, kshv has a prevalence of 3-40% in this class of patients worldwide [173, 174] . while kshv is a relatively newly discovered virus, there are no standard diagnostic approaches, and the detection of this virus through traditional serological and molecular methods remains challenging. this difficulty is mainly due to the inconsistency observed among serological assays targeting different antigens, and the low levels of viral dna that are usually present in the peripheral blood of infected individuals [175, 176] . hence, there is a continued interest in developing new reliable and sensitive methods for kshv detection. through using a mixture of aunps and silver nps (agnps), a colorimetric assay has been established for the multiplex, one-pot molecular detection of kshv and its frequently confounding disease bacillary angiomatosis [57] . in this assay, two types of probes are prepared from aunps and agnps separately functionalized with kshv and bartonellaspecific dna-capturing dna probes, respectively, and mixed with the target dna. then, the color of the reaction mixture changes in a manner dependent on the type of target dna added to the reaction solution: the addition of kshv dna causes aunp conjugates to aggregate and the solution to appear murky yellow-orange, i.e., the specific color of the nonaggregated agnps, while the addition of bartonella dna causes agnp conjugates to aggregate and the solution to appear pink, i.e., the color of nonaggregated aunps (fig. 11c ). in addition to being simple, this multicolor-change system successfully allows the multiplex detection of targets present in nanomolar concentrations. the family orthomyxoviridae includes five genera: influenzavirusa, influenzavirus b, influenzavirus c, isavirus, and thogotovirus [177] . the first three genera are widely known to infect birds, humans, and other mammals, causing influenza. the influenza-virusa genus contains many strains and can be further divided into several antigenic subtypes according to the antigenic properties of their envelope glycoproteins, i.e., hemagglutinin (ha) and neuraminidase (na). in contrast, the influenzavirusb genus has no distinct antigenic subtypes of hemagglutinin or neuraminidase. influenzavirusc is similar to types a and b but less common and lacks the na protein. the genus isavirus is known to infect salmon, while thogotoviruses usually cause infection in some vertebrates and invertebrates [177, 178] . orthomyxoviruses can be spherical (80-120 nm) or filamentous (20 nm in diameter and 200-300 nm long) in shape, and their genome contains 7 or 8 segments of linear negative-sense ssrna with a total length of 12-15 kb encoding different structural and nonstructural proteins, including nucleoprotein, three large proteins (pb1, pb2, and pa), matrix proteins (m1 and m2), the glycoproteins ha and na, and two nonstructural proteins (ns1 and ns2) [177, 179, 180] . several strains of the genus influenzavirusa cause seasonal and occasional acute respiratory diseases called influenza, which is profoundly and rapidly spread in humans. influenza is reported to affect up to 5% of adults and 20% of children of the global population each year [181, 182] . over the past decade, the world witnessed two major influenza outbreaks caused by newly emerging influenzavirusa strains: h5n1 in 2003 (avian influenza), and h1n1 in 2009 (swine influenza). both were severe enough to incite the who to issue a pandemic warning alert, which reached the highest level, i.e., "phase 6," for h1n1 in 2009 [21, 183] . the severity of influenza infection depends on both the virus strain and the age of the infected person. in elderly persons, influenza can result in hospitalization or death, while in younger adults, influenza-associated morbidity usually results in restricted activity, impaired work performance, and increased health care use [184, 185] . most people with the flu recover within one to two weeks without treatment. however, serious complications usually require medications to reduce the severity and duration of infection. some antiviral drugs, such as oseltamivir and zanamivir, can effectively treat or prevent influenza [186] . on the other hand, immunization with vaccine preparations of the most-circulating virus strains (trivalent or quadrivalent vaccines) remains the key strategy for preventing both influenza and its serious complications [187, 188] . in fact, both the treatment and management of influenza infection, either by antivirals or vaccination, depend on the influenza virus strains and the timing and severity of the infection. therefore, the rapid and accurate testing and typing of influenza is critical for treating infection and plays an important role in public health measures. the application of aunps in iav detection has primarily been focused on developing assays that enable the direct detection of whole iav particles [53, 58, 92] or viral rna [64, 66, 67, 70, 71, 83, 86] through numerous aunp-based fluorometric, sers, scanometric, light-scattering, colorimetric, and electrochemical detection schemes ( table 1) . the aunp-based fluorometric assays developed for h5n1 detection have simply exploited the intense plasmonic-based quenching properties of aunps through different detection formats. one of these assays targets the detection of the whole virus particle through a lateral-flow immunoassay format that is similar to that previously described for the detection of hsv-2, as shown in fig. 11a . in this assay, the virus is first captured on a lateral-flow test strip by pre-immobilized antibodies; then, aunp-antibody conjugates are applied to label the captured virus particles. after washing, the aunps are collected and dissolved to release gold ions that are eventually utilized to quench the fluorescence of qds in an additional, separate step [58] . in addition, ganbold et al. have developed a bimodal assay for the fluorometric and sers detection of viral rna using aunps modified with ssdna-dye probes [64] . the surface ssdna-dye probes are weakly adsorbed to the surface of aunps by electrostatic interactions, and with the addition of the complementary target dna, perfectly matched dsdna forms and desorbs from the surface of aunps, resulting in measurable changes in the conjugated dye fluorescence and raman fingerprint that are more substantial than those resulting from either mismatched dsdna or ssdna (fig. 12a) . aunp-based colorimetric assays have been developed for iav detection through four main detection schemes: 1) the first scheme involves the application of aunps conjugated with anti-digoxigenin as colorimetric labels to detect the target viral dna premodified with digoxigenin and electrophoresed through a membrane-based lateral-flow technique [66] . 2) the second scheme is simply based on the electrostatic interactions between the negatively charged phosphate groups of the target dna amplified with lamp and the positively charged ctab bilayer on aunrs, resulting in the aggregation of aunrs and a color change from red to purple [67] . 3) the third scheme relies on a magnetic-based hydroquinone oxidation reaction involving two metal np bioconjugates: iav-specific pentabody-mnps as capture probes and anti-aiv monoclonal antibody-aunps as detection probes. in the presence of virus particles, an immunocomplex forms and is separated by a magnetic field; after washing, aunps in the separated complex act to catalyze the oxidation of hydroquinone, which is detected optically in correlation with the aiv sample concentration [92] . 4) the fourth scheme relies on the peroxidase-like catalytic activity of au np films and colloidal aunps toward tmb-h2o2 mixtures. the virus is captured on a 96-well plate modified with aunp film biofunctionalized with ha antibody. then it is labeled with aunps modified with na antibody for signal generation. tmb-h 2 o 2 is added, and rapid color changes are observed because of the oxidation of peroxidase substrate tmb. using h1n1, the response of the developed assay was linear in the range from 10 pg/ml to 10 μg/ml and the limit of detection was 50.5 pg/ml, while with h3n2, the optical density of the developed color was dependent on the virus concentration in the range of 10-50,000 pfu/ml and the limit of detection was 24.3 pfu/ml [22] . aunp-based electrochemical assays rely on using aunps to directly modify the target dna or electron transducer surfaces, allowing enhanced electrical conduction and increased surface area for probe immobilization and target interaction. following this protocol, liu et al. [71] successfully detected aiv ha gene sequences using a dna aptamer immobilized onto an electrode; its surface was modified with a composite of carbon nanotubes, polypyrrole nanowires and aunps (fig. 12b) . furthermore, bonanni et al. [70] developed an impedimetric electrochemical assay for the detection of aiv m gene sequences based on measuring changes in the impedimetric behavior of the electrode when the target dna hybridizes with the capture dna probes immobilized onto its surface and is subsequently labeled by aunps via streptavidin/ biotin interaction (fig. 12c) . aunp-based scanometric assays for detecting aiv are mainly performed using the well-described aunp-promoted metal staining protocol shown in fig. 12d . in this protocol, the target nucleic acid is recaptured on the surface of glass chips modified with specific capture dna probes [86] . then, the captured probes are labeled with aunps either modified with specific dna probes or with the renowned dsdna intercalator daunorubicin [83] to mediate the metal staining. following this labeling step with aunp probes, the presence of the target nucleic acids is visually determined with an additional silver-or gold-staining step. specific dna aptamers immobilized on a gold electrode (ge) modified with a layer of multiwall carbon nanotubes (mwcnts), polypyrrole nanowires (ppnws) and aunps are collectively applied as an electrochemical platform for the detection of dna hybridization using dpv [71] . (c) impedimetric electrochemical assay using streptavidin-aunps probes. the direct/sandwich hybridization of biotinylated target dna with capture probes immobilized on the surface of a screen-printed electrode (spe) and additional conjugation with streptavidin-aunps results in impedimetric signal enhancement [70] . (d) label-free scanometric detection using positively charged aunps. the electrostatic interaction between positively charged aunps and negatively charged dna is utilized to visualize the double-stranded (ds)dna formed by the hybridization of single-stranded probe dna and complementary target dna on the array of chips through aunp-mediated metal staining [83, 86, 87] . the light-scattering detection of h1n1 virus using aunps relies on dls to measure the aggregation of aunp-antibody conjugates induced by the addition of target virus particles (similar to fig. 9d ). the magnitude of aunp aggregation and the corresponding increase in their hydrodynamic size is correlated with the concentration of virus particles [53] . finally, the immunological detection of h1n1 hemagglutinin (ha) antigen has only been reported through a novel lspr-based immunoassay. in this assay, the presence of target antigen induces a sandwich immunoreaction between the fluorophorelabeled detection antibodies and the aunp-capturing antibody conjugates, directly resulting in enhanced fluorescence caused by the coupling of fluorophore emission with the localized surface plasmons of aunps in a manner similar to that shown in fig. 9c . the sensitivity of this method has been described to be 10 3 -fold, improved over that of the conventional elisa technique, with a reported limit of detection of 13.9 pg/ml [63] . papillomaviridae is a widely diverse virus family that includes hundreds of recognized viruses classified into 16 taxonomically distinct genera designated with specific greek alphabet letters, from alpha to pi. the first five genera (alphapapillomavirus, betapapillomavirus, gammapapillomavirus, mupapillomavirus and nupapillomavirus) include the human papillomaviruses, while the remaining genera specifically include other mammalian and avian papillomaviruses [189, 190] . papillomaviruses are spherical, nonenveloped dna viruses 40-55 nm in size. their genome consists of a single molecule of circular, supercoiled, dsdna 5.3-8 kbp in size and encodes 6 early expressed (e) and 2 late (l) expressed proteins, in addition to a noncoding long control region [189, 191] . hpv infections are the most epidemic sexually transmitted viral infections worldwide [192, 193] . hpvs are highly epitheliotropic pathogens that usually infect the deepest layer of the skin or the genital surfaces. most hpv infections cause no symptoms and usually self-clear without causing clinical problems, but persistent infections lead to serious clinical complications [194] . this persistence typically depends on the type of hpv causing the infection. among more than 100 types of hpvs, approximately 40 types infect the mucous membranes and can be either high-or low-risk hpvs. high-risk types, such as hpvs 16, 18, 31, 51 and 52, are usually associated with many cancers of the cervix, vagina, vulva, penis, anus, neck, and head, as well as oropharyngeal cancer, while low-risk types, such as hpvs 6 and 11, are mainly related to anogenital warts and recurrent respiratory papillomatosis [195, 196] . currently, hpv is considered the most important cancer-causing virus, as it is associated with ~4.8% of all human cancers [197] . despite this high clinical significance of hpv, there are no available treatments, and vaccination using the bivalent vaccine against hpvs 16 and 18 or the quadrivalent vaccine against hpvs 6, 11, 16, and 18 remains the first and only choice for controlling the global hpv disease burden [198] [199] [200] . in fact, these vaccines are relatively expensive, and their efficacy is very restricted to specific hpv types. therefore, the development of accurate diagnostic techniques for hpv detection and typing is essential for acquiring surveillance data and initiating appropriate vaccination programs. aunp-based assays have been developed for the molecular detection of hpv through different photoelectric, fluorometric, electric and colorimetric schemes [89, 98] . the photoelectric assay essentially leverages aunps to enhance the deposition of silver metal on their surfaces, as previously described for several other assays. however, the enhanced precipitation of silver metal in this assay is used to block the light generated from a photodiode array (pda), and the target dna concentration is measured in proportion to the decrease in light intensity as electrically detected using a bipolar integrated circuit pda chip [89] . specifically, pcr-amplified biotinylated target dna is captured on the surface of the pda chip, and then labeled with aunp-biotin antibody conjugates; subsequently, a silver enhancement step decreases the light intensity, which is quantitatively translated into an electrical signal depending on the amount of target dna (fig. 13a) . on the other hand, a fluorometric assay integrating aunps and a microfluidic bead-based array has been developed as a rapid and controlled hpv dna detection system [98] . in this assay, aunps dually functionalized with hrp enzyme and capture dna strands are applied to label the target dna captured on the surface of microbeads (fig. 4a) , causing hrp to be in proximity to biotin-tyramine loaded on the surface of the microbeads and catalyze their oxidation by hydrogen peroxide. this process eventually increases the deposition of biotin moieties on the surface of the beads, subsequently increasing the labeling efficiency with qd-streptavidin conjugates and yielding an enhanced fluorescence signal that varies significantly based on the target dna concentration (fig. 13b) . in the electrical detection of hpv, aunps were used as an immobilization scaffold for dna immobilization on a silicon substrate carrying nanogapped interdigitated electrodes. this aunps deposition technique was shown to reduce the size of the nanogaps, allowing enhanced electric detection of the target dna captured on the silicon substrate by specific hpv dna probes. the results demonstrated that this assay was able to specifically detect hpv dna within 30 min and at concentrations down to 1 pm [201] . based on the color change of aunps upon aggregation, aunps carrying hpv dna probe were coupled with the lamp technique for colorimetric detection of hpv. lamp reaction was performed at a temperature of 65°c for 20 min and the generated products were hybridized with aunp-dna probes for 5 min. then the presence of the target dna was detected by the addition of magnesium salt. the addition of the salt causes aunps aggregation and the color changes from red to blue. the presence of lamp amplicons can protect aunps from aggregation and prevents aunps aggregation. the sensitivity of the developed lamp-aunp assay was greater than the lamp turbidity assay by up to 10-fold with detection limits of 10 2 and 10 0 copies for hpv16 and hpv18, respectively [27] . the family picornaviridae is taxonomically very diverse and has recently been expanded to involve 12 genera: eight are originally identified genera (aphthovirus, cardiovirus, enterovirus, erbovirus, hepatovirus, kobuvirus, parechovirus, and teschovirus), and four newly proposed genera (avihepatovirus, sapelovirus, senecavirus, and tremovirus) [202] . picornaviruses are spherical, relatively small (22-30 nm) , nonenveloped rna viruses. their genome is positive-sense ssrna that is 7.0-9.5 kb in size and terminally bonded to a noncapsid viral protein (vpg) at its 5' end and to a polyadenylated tail at its 3' end. the expression of this genome produces a single large polyprotein that undergoes a cascade of cleavage and processing reactions to form 10-12 final structural (vp1-4) and nonstructural (2a-c and 3a-d) proteins [203] . picornaviruses can infect a broad spectrum of hosts, including birds, humans, and other mammals [202, 204] . the genera enterovirus, hepatovirus, parechovirus, kobuvirus, and cardiovirus include several important species that affect humans. particularly, the genus hepatovirus encompasses hav, which is the most likely causal agent of acute viral hepatitis in humans. hav infection is usually asymptomatic and self-limiting, and the related symptoms can be mild or sporadically progress to fulminant hepatic failure [205, 206] . the availability of an effective vaccine against hav has substantially reduced the number of people who become infected with hav [207, 208] . however, the ability of this virus to survive harsh environmental conditions and remain in seawater, fresh water, wastewater, and soil for extended periods increases the possibility of outbreaks, especially in developing countries where sanitation is not typically available [205] . the transmission of the virus usually occurs through the consumption of contaminated materials. as such, the virus does not replicate in the environment, water, or foods, and traces of viral contamination are difficult to identify, which remains a challenge for controlling hav. the need for sensitive assays for hav remains an urgent public health demand. hav detection using aunps was accomplished through scanometric and sers-based assays. both assays rely on the extensively described virtue of aunps to enhance silver staining, thereby allowing the molecular detection of hav dna in a microarray chip format (table 1 and fig. 6 ). in the scanometric aunps modified with anti-biotin igg are applied to label biotinylated target dna captured on the pda chip. after a silver enhancement step, the precipitated silver metal at the surfaces of the aunps blocks the light irradiated from above by light-emitting diodes (leds) and decreases the resulting photocurrent [89] . (b) microfluidic bead-based enzyme-aunp amplification method for the fluorometric detection of hpv. streptavidin-coated beads are dually functionalized with biotin-electron-rich protein and biotin-dna capture probes. the captured target dna first reacts with horseradish peroxidase (hrp)-functionalized aunp-dna labels, bringing hrp close to the surface of the microbeads. the oxidation of tyramine-biotin by hydrogen peroxide results in the deposition of multiple biotin moieties onto the surface of the beads. this deposition is markedly increased in the presence of immobilized electron-rich proteins. streptavidin-labeled quantum dots (qds) are then allowed to bind to the deposited biotin moieties and display amplified fluorescence signals in relation to the target dna concentration [98] . detection scheme, aunp-dna conjugates are applied as specific detection probes to label target hav rna captured by specific vp1 probes on the surface of a microarray chip. a silver metal enhancement step is subsequently used for signal amplification, followed by the visual detection of nucleic acids [79] . in the sers detection, the captured hav dna is labeled by aunp-dna probes loaded with the raman reporter dye cy3; this approach has been applied as previously described for ebov and depicted in fig. 6 of this review [50] . these assays are very simple and can detect target concentrations ranging from 3.37×10 -7 -3.37×10 -8 m, with 100 fm as a limit of detection, which can be superior to the relatively expensive pcr-based approaches currently used for hav detection and management. the family retroviridae is broadly classified into 7 genera (alpharetrovirus, betaretrovirus, deltaretrovirus, epsilonretrovirus, gammaretrovirus, lentivirus, and spumavirus), which include nearly 51 viruses [209] . retroviruses are spherical, small (80-100 nm), enveloped rna viruses. their viral genome is typically bipartite and composed of two positive-sense ssrna molecules (10 kb in total length). they comprise 4 major genes that encode the different structural and nonstructural proteins of the virus: gag encodes the large protein form matrix, capsid, and nucleocapsid; pro encodes the protease enzyme; pol encodes the reverse transcriptase and integrase enzymes; and env encodes the envelope glycoproteins and transmembrane proteins [209] [210] [211] . retroviruses infect a wide variety of vertebrates, including humans. their infection in humans is usually very serious and can potentially cause oncogenesis and variable hematopoietic and neurological conditions. among retroviruses, hiv is a well-known, formidable human pathogen responsible for the most devastating viral pandemic that has ever occurred in human history [212] . hiv infection is usually correlated with the progression of what is clinically called aids, in which the immune system is harshly destroyed, and the entire body becomes vulnerable to terrible, life-threatening opportunistic infections and cancers. people with aids also suffer from other systemic symptoms, such as fevers, sweats, swollen glands, chills, weakness, and weight loss [213] . currently, there is no effective vaccine against hiv, and most of the treatment strategies rely on using antiretroviral drugs, which in most cases maintain the viral load at low levels to prevent the formation of acute hiv infection rather than cure the virus [214] . therefore, the accurate detection of hiv is a key determinant for monitoring hiv treatment and reducing its transmission and dissemination. currently, aunps have been extensively tested for the immunological and molecular detection of hiv through various scanometric, colorimetric, electrochemical, ipcr, and afm assays ( table 1) . the scanometric assays target the detection of the hiv p24 antigen and hiv nucleic acid using the silver metal enhancement protocol. for hiv p24, the target antigen is precaptured by magnetic particles [81] or isolated in a microplate [84] , followed by a silver enhancement step for the quantitative visual immunological detection of hiv (fig. 4a, b and fig. 6 ). for hiv nucleic acid detection, aunp-dna probes specific to the hiv pol gene are applied to detect hiv nucleic acids precaptured and isolated by magnetic particles, followed by a silver metal enhancement step (fig. 14a) . the deposition of silver on the aunp surface increases the np size and scattering efficiency and additionally allows the target detection via dls [90] . in aunp-based colorimetric detection of hiv, the large surface area of aunps was exploited to amplify the detection signal by enhancing the gold metal staining on their surfaces, allowing naked-eye detection of hiv nucleic acids (hiv gag gene) in a lateral-flow format, similar to the scheme presented in fig. 11a [91] . on the other hand, the ability of aunps to enhance electron transfer has been applied to develop an electrochemical assay for hiv detection (fig. 14b) . in this electrochemical assay, the target hiv genome is detected in correlation with the change in electrical impedance of graphene sheets decorated with spherical aunp-dna probes specific to the hiv pol gene [73] . the developed aunp-based rna detection of hiv using the aunp-lfa technique has a sensitivity limit of ~11 log10 copies/ml, while the best dna detection can be achieved through the light-scattering scheme with a sensitivity limit of 10 fm. furthermore, afm coupled with aunps as nanoparticulate labels has been applied for the detection of hiv p24 as follows: the target antigen is precaptured on the surface of a nanoarray specifically coated with anti-p24 and labeled with aunp-antibody conjugates that significantly changes the surface topography of the array, allowing the detection of p24 (fig. 14c) [95] . so far the developed aunps-based assays for hiv are among the most innovative in virus detection and offer a significantly improved detection limit down to attomolar range, which is hundreds of times lower than that of conventional techniques used in hiv detection, including pcr and elisa. therefore, aunps assays can be very beneficial in detecting hiv early, preventing its dissemination, and treatment monitoring. furthermore, simple detection methods such as lateral flow and scanometric assays can be easily incorporated into the point-of-care detection of hiv, which is currently of great interest and more suitable for developing countries. aunps have made a tremendous impact in the detection of viruses, as demonstrated by the numerous assays for most of the clinically relevant groups of human viruses (fig. 3) . coupled with different biomolecules, aunps have enabled the development of a completely new class of probes that are specifically composed of a aunp core (spherical or rod-like in shape) for signal readout and a surface layer of one or multiple types of biomolecules for target recognition and interaction. these probes have mostly been incorporated as a new set of functional and highly interactive components in different traditional techniques (e.g., eias and pcr) and have derived substantial improvements in design, detection performance, sensitivity, specificity and stability. this created a new trend in applying aunps to overcome the limitations of traditional techniques. aunp-based immunological and serological detection methods are the most common and have been accomplished through multiple scanometric, colorimetric, dls, immuno-pcr, electrochemical, electrical, sers, lspr, fluorometric, and afm-based approaches (table 1) . aunp-based dls and lspr assays are especially novel, simple and among the most sensitive reported assays for the immunodetection of viruses with a detection limit that is hundreds of times lower than that of the standard elisa technique [51, 52] . in contrast, the developed aunp-based molecular assays are not as sensitive as the conventional pcr technique or other pcr-based molecular techniques. however, the addition of aunps greatly simplifies the detection procedures of viral nucleic acids, and largely eliminates the need for complex procedures and expensive thermal cycling machines, which greatly reduces the detection time and total cost. in particular, colorimetric assays based on the intense red color of aunps represent an interesting qualitative shift in nucleic acid detection due to the complete elimination magnetic np (mnp)-dna probes scavenge target hiv sequences, which are then magnetically collected and labeled with aunp-dna probes. after repetitive washing, aunps are released into solution by a dehybridization step and subjected to silver metal enhancement, resulting in particle growth and increased scattering [90] . (b) electrochemical impedance assay of hiv rna. graphene oxide sheets decorated with aunps are applied for the immobilization of dna detection probes. hybridization with specific dna sequence results in increased impedance [73] . (c) atomic force microscopy (afm)-based immunoassay of hiv. aunp-antibody probes form a three-component sandwich immunocomplex with the target virus antigen captured on the surface of a specific antibody nanoarray; eventually, the increase in the surface height of the array is detected by afm [95] . of the pcr amplification step and the possibility of direct and rapid detection of the target virus within a few minutes. other assays rely on well-known analytical techniques, such as icp-ms and qcm, which have been introduced to virus nucleic acid detection for the first time through the application of aunps and have achieved detection sensitivity down to femtomolar concentrations [93, 94] . aside from these immunological and molecular assays, aunps have also been described for viral load testing by detecting intact virus particles, which are of special interest as a substitution to traditional cell culture technique [58, 65, 92] . in this context, the small size of aunps probes that is comparable to virus particles, allows rapid, and highly dynamic interaction with viruses for gaining information about infectivity. the application of aunps, especially in biology and medicine, require specific structural and physicochemical characteristics, and any subtle alterations to the intended characteristics can compromise the function and performance of the whole system. for bioanalytic and diagnostic applications, aunps exhibiting homogeneous morphological and fine structural characteristics are important specifically for effective and accurate response to target detection. therefore, many pre-analytical factors related to np synthesis and surface modification are critical to the development of an efficient virus detection assay. the synthesis of aunps commonly relies on methods that basically employ wet chemistry reduction techniques, as presented in other reviews [9, 32] . although these techniques are generally simple and fast and can support the large-scale production of aunps, the synthesis of uniform aunps with a discrete size and shape remains challenging [215] . recently, some approaches have been reported for the controlled synthesis and modification of aunps. however, these methods remain unsuitable either due to the protocol complexity or difficulty of particle separation, which might require additional steps or specific surface modifications. therefore, the development of more stringent and advanced methods for the facile and controlled synthesis of uniform aunps with the option of performing multiple surface modifications is strongly recommended. because of the direct relationship between aunp size and shape and their function as labels, such characteristics are also of inherent importance to both the performance of au nanoprobes and their efficiency for diagnostic applications. for instance, small aunps are preferred in colorimetric and light scattering-based assays due to their spr band sensitivity. in contrast, large aunps are preferentially employed in assays such as bca, immuno-pcr, and enzymatic-based electrochemical detection techniques, which rely on the surface functionalization of aunps with targeting or signaling biomolecules. additionally, a large np size is highly desirable in assays that depend on aunps as metal labels, such as electrochemical stripping, qcm, and icp-ms detection methods. in addition, spherical aunps are more commonly applied than rod-shaped nps. however, the latter exclusively enable a very interesting class of lspr-based detection methods that have shown an unprecedented detection sensitivity range. since aunps by themselves are "blind" with respect to sensing the target, the addition of specific biomolecules to their surfaces is a key step for detecting viruses [37, 38, 40] . unfortunately, most biomolecules have been reported to be very sensitive to harsh chemical modifications that can adversely affect their reactivity and specificity. thus, the type of targeting biomolecule and the surface conjugation reaction method applied are crucial and require optimization. short oligonucleotides and other dna sequences are physically rigid and more stable than protein biotargeting structures, including antibodies, which usually have several secondary and tertiary conformations that are important for their full, specific interaction with a target molecule. furthermore, the full reactivity of surface biomolecules via a suitable conjugation reaction is critical and should be retained and confirmed with additional assessment steps before use in virus detection. while the application of traditional conjugation techniques, such as electrostatic adsorption, is still widely reported, it is strongly recommended to be avoided due to the incident loss of conjugated biomolecule activity and large inequality in loading efficiency, which directly result in decreased assay sensitivity and reproducibility [216] . in this regard, aunps have an enhanced surface area-to-volume ratio that greatly improves the efficacy of current conjugation techniques, and several directional and controlled conjugation methods based on covalent and noncovalent interactions have been thoroughly described in the literature [9, 32, 35] . although aunps are widely employed in various sensing functions and formats, their use for monitoring of virus infection is challenged by long-term stability and reusability of the developed aunp-based sensors. the reusability of sensing platforms has a crucial role in developing applications that are reliable, economic and sustainable. nonetheless, the reusability of aunps in virus detection is limited to electrical and electrochemical assays, in which the main function of aunps is limited to enhancing the performance of the modified electrodes [68, [70] [71] [72] [73] [75] [76] [77] [78] . the long-term stability of aunps is reported and well investigated in numerous studies. however, their conjugates to different biomolecules (i.e., dna, rna, antibody, enzymes) are sensitive to storage time and conditions and the reusability of these conjugates is usually hampered by the loss of activity of surface molecules and irreversible aggregations. it was demonstrated that exposure to elevated temperatures, high salt concentrations, and reducing agents such as dithiothreitol (dtt), mercaptoethanol, and glutathione (commonly existing in biological fluids) results in the loss of stability and activity of aunp-conjugates [9, 41, 43] . thus, the development of new chemistries and novel conjugation strategies for the synthesis of stable aunp-conjugates becomes very crucial for practical use of aunps in virus detection. considering these limitations and challenges, important analytical parameters, such as the use of positive and negative controls, follow-up patient specimens, and internal reference tests, are recommended to confirm the overall accuracy and practicality of developed schemes. a restricted set of measures related to the assay detection sensitivity, specificity, and performance is required for the quality assurance and assessment of these applications [217] . due to the rapid development rate of aunp-based assays, international data analysis measures and standards are also critical to reflect the true assay sensitivity and specificity and to allow the evaluation of their ability and potential to substitute current detection techniques. furthermore, more efforts are needed to test the practicality and generalizability of these assays through clinical sample testing and in-field evaluation of the newly developed aunp-based methods along with the conventional methods using standard evaluation systems and protocols. aunps are a powerful addition to the range of techniques available for virus testing and control. when used as probes, aunps enable simple, rapid, and sensitive detection with an unprecedented multiplexing capability. importantly, aunp-based probes have matured from tools being added to improve traditional techniques, such as pcr, eliza, and many others, to the basis of completely novel techniques developed and introduced for the first time for virus detection. with aunps, virologists can create an unlimited number of detection assays that match the diagnostic needs of each virus in terms of simplicity, speed and cost with sensitivity levels that reach zeptomolar concentrations. aunps can be further developed to become components of more routine techniques, and they will likely engage the interest of scientists from all the disciplines, whether fundamental, environmental, clinical or industrial, in the future. the challenge of emerging and re-emerging infectious diseases globalization and infectious diseases in women globalization, international law, and emerging infectious diseases globalization and infectious diseases globalization of infectious diseases: the impact of migration gold nanoparticles in biomedical applications: recent advances and perspectives gold nanoparticles: opportunities and challenges in nanomedicine biomedical applications of plasmon resonant metal nanoparticles a review on functionalized gold nanoparticles for biosensing applications optical resonator biosensors: molecular diagnostic and nanoparticle detection on an integrated platform ebola virus disease (the killer virus): another threat to humans and bioterrorism: brief review and recent updates insights from clinical research completed during the west africa ebola virus disease epidemic gold aggregating gold: a novel nanoparticle biosensor approach for the direct quantification of hepatitis c virus rna in clinical samples an improved gold nanoparticle probe-based assay for hcv core antigen ultrasensitive detection a highly sensitive electrochemical immunosensor for hepatitis b virus surface antigen detection based on hemin/g-quadruplex horseradish peroxidase-mimicking dnazyme-signal amplification development of lateral flow assay based on size-controlled gold nanoparticles for detection of hepatitis b surface antigen sers detection of hepatitis b virus dna in a temperature-responsive sandwich-hybridization assay cross-species virus transmission and the emergence of new epidemic diseases genetic variability: the key problem in the prevention and therapy of rna-based virus infections factors in the emergence of infectious-diseases updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic in situ self-assembly of gold nanoparticles on hydrophilic and hydrophobic substrates for influenza virus-sensing platform avian influenza virus (h5n1): a threat to human health re-emergence of zika virus: a review on pathogenesis, clinical manifestations, diagnosis, treatment, and prevention diagnostic virology protocols clinical and diagnostic virology high sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18 the brazilian zika virus strain causes birth defects in experimental models molecular diagnosis of influenza diagnostic virology viral serology and detection functionalized gold nanoparticles: synthesis, properties and applications-a review molecular techniques for clinical diagnostic virology molecular diagnosis of medical viruses nanoparticle-mediated systemic delivery of sirna for treatment of cancers and viral infections there's plenty of room at the bottom engineering nanomaterial surfaces for biomedical applications nanostructures in biodiagnostics sensitive in situ hybridization with catalyzed reporter deposition, streptavidin-nanogold, and silver acetate autometallography: detection of single-copy human papillomavirus the use of nanocrystals in biological detection enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity various methods of gold nanoparticles (gnps) conjugation to antibodies chemical synthesis of novel plasmonic nanoparticles a molecular ruler based on plasmon coupling of single gold and silver nanoparticles gold and silver nanoparticles in sensing and imaging: sensitivity of plasmon response to size, shape, and metal composition review: synthesis of fluorescent metallic nanoclusters toward biomedical application: recent progress and present challenges gold nanoparticles for the development of clinical diagnosis methods surface-enhanced raman scattering (sers) detection of multiple viral antigens using magnetic capture of sers-active nanoparticles nanoparticles with raman spectroscopic fingerprints for dna and rna detection detection of hepatitis b surface antigen by target-induced aggregation monitored by dynamic light scattering gold nanorod-based localized surface plasmon resonance biosensor for sensitive detection of hepatitis b virus in buffer, blood serum and plasma one-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles colorimetric detection of dna sequences based on electrostatic interactions with unmodified gold nanoparticles oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for dna analysis by hybridization direct detection of unamplified hepatitis c virus rna using unmodified gold nanoparticles multiplexed colorimetric detection of kaposi's sarcoma associated herpesvirus and bartonella dna using gold and silver nanoparticles a fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of herpes simplex virus type 2-specific immunoglobulin g antibodies in serum and whole blood sizeand distance-dependent nanoparticle surface-energy transfer (nset) method for selective sensing of hepatitis c virus rna a gold nanorods-based fluorescent biosensor for the detection of hepatitis b virus dna based on fluorescence resonance energy transfer multiple homogeneous immunoassays based on a quantum dots-gold nanorods fret nanoplatform detection of swine-origin influenza a (h1n1) viruses using a localized surface plasmon coupled fluorescence fiber-optic biosensor aggregation effects of gold nanoparticles for single-base mismatch detection in influenza a (h1n1) dna sequences using fluorescence and raman measurements hybrid nanocluster plasmonic resonator for immunological detection of hepatitis b virus electrophoresis-enhanced detection of deoxyribonucleic acids on a membrane-based lateral flow strip using avian influenza h5 genetic sequence as the model nanomolecular detection of human influenza virus type a using reverse transcription loop-mediated isothermal amplification assisted with rod-shaped gold nanoparticles gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus dna using disposable microband electrodes ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based dna amplification impedimetric detection of influenza a (h1n1) dna sequence using carbon nanotubes platform and gold nanoparticles amplification electrochemical detection of avian influenza virus h5n1 gene sequence using a dna aptamer immobilized onto a hybrid nanomaterial-modified electrode genosensor for sars virus detection based on gold nanostructured screen-printed carbon electrodes green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance dna hybridization biosensing nanoparticle-based electrochemical detection of hepatitis b virus using stripping chronopotentiometry highly sensitive electrochemical stripping detection of hepatitis b surface antigen based on copper-enhanced gold nanoparticle tags and magnetic nanoparticles gold nanoparticle-labeled detection antibodies for use in an enhanced electrochemical immunoassay of hepatitis b surface antigen in human serum electrochemical immunoassay of hepatitis b surface antigen by the amplification of gold nanoparticles based on the nanoporous gold electrode catalytic signal amplification of gold nanoparticles combining with conformation-switched hairpin dna probe for hepatitis c virus quantification development of array-based technology for detection of hav using gold-dna probes visual gene diagnosis of hbv and hcv based on nanoparticle probe amplification and silver staining enhancement detection of hiv-1 p24 gag in plasma by a nanoparticle-based bio-barcode-amplification method array-based nano-amplification technique was applied in detection of hepatitis e virus scanometric analysis of dna microarrays using dna intercalator-conjugated gold nanoparticles nanoparticle-based immunoassays for sensitive and early detection of hiv-1 capsid (p24) antigen detection of hepatitis b virus deoxyribonucleic acid based on gold nanoparticle probe chip multiplexed, rapid detection of h5n1 using a pcr-free nanoparticle-based genomic microarray assay label-free and naked eye detection of pna/dna hybridization using enhancement of gold nanoparticles rapid and simultaneous detection of human hepatitis b virus and hepatitis c virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus homogeneous detection of nucleic acids based upon the light scattering properties of silver-coated nanoparticle probes a lateral flow assay for quantitative detection of amplified hiv-1 rna influenza virus detection with pentabody-activated nanoparticles a method of layer-by-layer gold nanoparticle hybridization in a quartz crystal microbalance dna sensing system used to detect dengue virus gold nanoparticle-based inductively coupled plasma mass spectrometry amplification and magnetic separation for the sensitive detection of a virus-specific rna sequence the use of nanoarrays for highly sensitive and selective detection of human immunodeficiency virus type 1 in plasma gold nanoparticle enhanced immuno-pcr for ultrasensitive detection of hantaan virus nucleocapsid protein two types of nanoparticle-based bio-barcode amplification assays to detect hiv-1 p24 antigen multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using au nanoparticle probes and quantum dots as labels virus taxonomy: classification and nomenclature of viruses fields virology bunyaviruses and the type i interferon system hantaviruses: a global disease problem hantavirus infections: epidemiology and pathogenesis hantavirus infection: a review and global update isolation of the causative agent of hantavirus pulmonary syndrome rift valley fever virus rift valley fever virus(bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention epidemiologic investigations into outbreaks of rift valley fever in humans outbreak of rift valley fever affecting veterinarians and farmers in south africa an assessment of the regional and national socio-economic impacts of the 2007 rift valley fever outbreak in kenya rift valley fever and a new paradigm of research and development for zoonotic disease control supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron ciyomicroscopy coronaviruses post-sars: update on replication and pathogenesis coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus a previously undescribed coronavirus associated with respiratory disease in humans identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia no novel coronaviruses identified in a large collection of human nasopharyngeal specimens using family-wide codehop-based primers isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease filoviruses in bats: current knowledge and future directions marburg and ebola viruses evolutionary history of ebola virus ebola virus outbreaks in africa: past and present efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial. the lancet virus taxonomy: ninth report of the international committee on taxonomy of viruses opinion -architects of assembly: roles of flaviviridae non-structural proteins in virion morphogenesis phylogeny of the genus flavivirus perspectives for the treatment of infections with flaviviridae a brief review on dengue molecular virology, diagnosis, treatment and prevalence in pakistan world health organization. dengue guidelines for diagnosis, treatment, prevention and control: new edition the global distribution and burden of dengue the burden of hepatitis c in the united states estimating the future health burden of chronic hepatitis c and human immunodeficiency virus infections in the united states histological and clinical outcome after liver transplantation for hepatitis c peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection telaprevir for previously untreated chronic hepatitis c virus infection sofosbuvir for previously untreated chronic hepatitis c infection sofosbuvir and ribavirin in hcv genotypes 2 and 3 simeprevir with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients with chronic hepatitis c virus genotype 1 infection (quest-1): a phase 3, randomised, double-blind, placebo-controlled trial quest for a cure for hepatitis c virus: the end is in sight virus taxonomy: classification and nomenclature of viruses (ninth report of the international committee on taxonomy of viruses) hepatitis b virus infection--natural history and clinical consequences natural history and clinical consequences of hepatitis b virus infection world health organization best practice in the treatment of chronic hepatitis b: a summary of the european viral hepatitis educational initiative (evhei) diagnosis and management of chronic hepatitis b in children, young people, and adults: summary of nice guidance virus taxonomy: ninth report of the international committee on taxonomy of viruses hepatitis e virus --an update array-based nano-amplification technique was applied in detection of hepatitis e virus virus taxonomy: ninth report of the international committee on taxonomy of viruses herpesvirus entry: an update herpesviruses remodel host membranes for virus egress genital herpes. the lancet the nine ages of herpes simplex virus an estimate of the global prevalence and incidence of herpes simplex virus type 2 infection epidemiology, clinical presentation, and antibody response to primary infection with herpes simplex virus type 1 and type 2 in young women herpesviruses: latency and reactivation-viral strategies and host response herpes simplex virus 2 infection: molecular association with hiv and novel microbicides to prevent disease hsv-2 vaccine: current status and insight into factors for developing an efficient vaccine immune evasion by herpes simplex virus evasion of early antiviral responses by herpes simplex viruses review of cytomegalovirus seroprevalence and demographic characteristics associated with infection cytomegalovirus: pathogen, paradigm, and puzzle international consensus guidelines on the management of cytomegalovirus in solid organ transplantation neonatal cytomegalovirus blood load and risk of sequelae in symptomatic and asymptomatic congenitally infected newborns is hcmv a tumor promoter? vaccine prevention of maternal cytomegalovirus infection identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma kshv infection and the pathogenesis of kaposi's sarcoma kaposi's sarcoma-associated herpesvirus kaposi's sarcoma-associated herpesvirus infection prior to onset of kaposi's sarcoma detection of kaposi sarcoma associated herpesvirus in peripheral blood of hiv-infected individuals and progression to kaposi's sarcoma. the lancet virus taxonomy: ninth report of the international committee on taxonomy of viruses avian influenza viruses and their implication for human health influenza: lessons from past pandemics, warnings from current incidents the viruses and their replication seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america influenza and rsv: know the diagnostic options world health organization. pandemic h1n1 2009 update 112 cost-benefit analysis of a strategy to vaccinate healthy working adults against influenza burden of influenza-like illness and effectiveness of influenza vaccination among working adults aged 50-64 years antiviral drugs for seasonal influenza efficacy of high-dose versus standard-dose influenza vaccine in older adults acip releases updated recommendations on influenza vaccination to include the 2014-2015 season virus taxonomy: ninth report of the international committee on taxonomy of viruses cross-roads in the classification of papillomaviruses the biology and life-cycle of human papillomaviruses chapter 1: hpv in the etiology of human cancer screening to prevent cervical cancer: guidelines for the management of asymptomatic women with screen detected abnormalities: commonwealth of australia epidemiology of acquisition and clearance of cervical human papillomavirus infection in women from a high-risk area for cervical cancer human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update multi-site study of hpv type-specific prevalence in women with cervical cancer, intraepithelial neoplasia and normal cytology, in england global burden of human papillomavirus and related diseases efficacy and safety of prophylactic vaccines against cervical hpv infection and diseases among women: a systematic review & meta-analysis safety and persistent immunogenicity of a quadrivalent human papillomavirus types 6, 11, 16, 18 l1 virus-like particle vaccine in preadolescents and adolescents: a randomized controlled trial prophylactic hpv vaccines: prospects for eliminating ano-genital cancer gold nanoparticle mediated method for spatially resolved deposition of dna on nano-gapped interdigitated electrodes, and its application to the detection of the human papillomavirus natural occurrence and characterization of two internal ribosome entry site elements in a novel virus, canine picodicistrovirus, in the picornavirus-like superfamily clinical significance, diagnosis, and treatment of picornavirus infections hepatitis a virus. mandell, douglas, and bennett's principles and practice of infectious diseases hepatitis a acute liver failure: follow-up of paediatric patients in southern brazil centers for disease c, prevention. surveillance for acute viral hepatitis -united states hepatitis a in the era of vaccination virus taxonomy, ninth report of the international committee on taxonomy of viruses historical introduction to the general properties of retroviruses translational control of retroviruses global report: unaids report on the global aids epidemic abc of aids: natural history and management of early hiv infection new paradigms for hiv/aids vaccine development synthetic approaches to metallic nanomaterials aspects of recent development of immunosensors towards complete and accurate reporting of studies of diagnostic accuracy: the stard initiative radioimmunossay of australia antigen detecting the aids virus directly with pcr nanoparticle-based electrochemical detection of hepatitis b virus using stripping chronopotentiometry development of array-based technology for detection of hav using gold-dna probes we would like to thank frank fanqing chen the authors have declared that no competing interest exists. key: cord-285898-rtqkvf63 authors: padberg, stephanie title: anti-infective agents date: 2014-09-29 journal: drugs during pregnancy and lactation doi: 10.1016/b978-0-12-408078-2.00007-x sha: doc_id: 285898 cord_uid: rtqkvf63 infections may be hazardous to the health of the mother, the course of pregnancy, and the unborn child. they can lead to premature labor or premature rupture of membranes and thereby increase the risk for spontaneous abortion and prematurity. furthermore, certain germs can pass to the unborn child and harm it directly. therefore, an anti-infective treatment which should be both effective and safe for the mother and the unborn child is often required. the use of penicillines and older cephalosporines is well documented and considered to be safe. consequently, they are the drug of choice during pregnancy. in selected cases of bacterial resistance or intolerance to first-line antibiotics, other anti-infective agents might be recommended. especially for life-threatening infections, a therapy with not so well-tried agents might be needed. the potential benefit of treatment in such cases most often outbalances the potential risk for the unborn child. infections may be hazardous to the health of the mother, the course of pregnancy, and the unborn child. they can lead to premature labor or premature rupture of membranes and thereby increase the risk for spontaneous abortion and prematurity. furthermore, certain germs can pass to the unborn child and harm it directly. therefore, an anti-infective treatment which should be both effective and safe for the mother and the unborn child is often required. the use of penicillins and older cephalosporins is well documented and considered to be safe. consequently, they are the drug of choice during pregnancy. in selected cases of bacterial resistance or intolerance to first-line antibiotics, other anti-infective agents might be recommended. especially for life-threatening infections, a therapy with not so well-tried agents might be needed. the potential benefit of treatment in such cases most often outbalances the potential risk for the unborn child. stephanie padberg 2.6 2.6.4 erythromycin and other macrolides pharmacology erythromycin and other macrolides inhibit bacterial protein synthesis and are bacteriostatic. macrolides are primarily applied in the treatment of infections with gram-positive germs, but are also effective against haemophilus influenzae and intracellular pathogens such as chlamydia. macrolides offer an alternative for patients with penicillin allergy. erythromycin is the oldest medication of this group. its resorption can be delayed in the third trimester. gastrointestinal side effects can lead to lower than therapeutic plasma concentrations, resulting in treatment failure (larsen 1998) . only 5-20% of the maternal erythromycin concentration is obtained in the fetus. therefore, erythromycin is not a sufficiently reliable drug for fetal or amniotic infections. the newer macrolide antibiotics azithromycin, clarithromycin, dirithromycin, josamycin, midecamycin, roxithromycin and troleandomycin have a similar antibacterial spectrum as erythromycin, but to some degree less gastrointestinal side effects. spiramycin is used for toxoplasmosis in the first trimester. telithromycin is the first ketolide antibiotic for clinical use. it is structurally related to erythromycin. erythromycin has always been considered a safe and effective antibiotic during pregnancy. data on several thousand first trimester exposures do not support an association between erythromycin and congenital malformations (e.g. czeizel 1999a ). however, an analysis of the data from the swedish birth registry showed a weakly significant increase in malformations in 1,844 children whose mothers took erythromycin in early pregnancy compared to offspring whose mothers used phenoxymethylpenicillin (källén 2005a ). this was based on an increased rate of cardiovascular malformations, especially ventricular and atrial septal defects. an update of the swedish data verified an association between the use of erythromycin during early pregnancy and cardiovascular defects (källén 2014 ). an increased incidence of pyloric stenosis was discussed by the same author (källén 2005a) . this observation after intrauterine exposure in the first trimester is biologically not plausible; but it should be mentioned that a link has been suggested between a neonatal treatment with erythromycin during the first two weeks and the development of pylorus stenosis (e.g. mahon 2001) . other studies have failed to find a higher rate of septum defects, pyloric stenosis or other malformations (bahat dinur 2013 , lin 2013 , romøren 2012 , malm 2008 , cooper 2002 , louik 2002 . in summary, the experiences argue against an increased embryo-and fetotoxic risk for erythromycin. there are several reports of maternal hepatotoxic changes when erythromycin estolate was administered in the second half of pregnancy. these women developed a cholestatic icterus during the second week of treatment that abated within weeks when the treatment was discontinued, without evidence of permanent damage or signs of fetal compromise (e.g. mccormack 1977) . azithromycin, clarithromycin and roxithromycin have also been studied in several publications without any indication of embryo-or fetotoxic effects (bar-oz 2012, bar-oz 2008 , chun 2006 , sarkar 2006 , drinkard 2000 , einarson 1998 ). in the case of clarithromycin, there was some 2.6 anti-infective agents 2 pregnancy initial concern as animal experiments demonstrated teratogenic effects, and for instance, in some studies cardiovascular defects were induced in rats. recently a danish cohort study based on a prescription register observed an increased risk of miscarriage after clarithromycin in early pregnancy, but no increased risk for major malformations (andersen 2013a) . experience with dirithromycin, josamycin, midecamycin, spiramycin, and troleandomycin is very limited (czeizel 2000b) . spiramycin has been used in many first trimesters for the treatment of toxoplamosis. although these reports did not focus on a possible teratogenic effect, numerous normal births after spiramycin exposure are reassuring. there is no published experience with the use of the ketolide telithromycin in the first trimester. the animal experiments did not show that this agent is teratogenic. a local treatment with macrolides is quite safe for the fetus. yet, because resistance develops quickly and allergies are frequent, macrolides should be used with some reservation. clindamycin and lincomycin belong to the lincosamide group. they inhibit bacterial protein synthesis and can be bactericidal or bacteriostatic depending on concentration and sensitivity. after an oral dose the resorption is almost complete. about half of the maternal concentration can be attained in the umbilical veins. there were no signs of embryo-or fetotoxic effects in several hundred pregnant women treated with lincomycin at different points in pregnancy (czeizel 2000c , mickal 1975 . there were also no problems found for clindamycin. pseudomembranous enterocolitis is a dangerous maternal complication of clindamycin treatment that may also happen after vaginal application. pregnancy complications due to bacterial vaginosis are not sufficiently preventable by vaginal clindamycin therapy (joesoef 1999) . it should be noted though, that other investigators found a reduction in late abortions and prematurity when treating several hundred patients with oral clindamycin for an abnormal vaginal flora (ugwumadu 2003) . recommendation. erythromycin, clarithromycin, azithromycin, and roxithromycin may be used in pregnancy when the resistance spectrum requires them, or in cases of an allergy to penicillin. because of hepatotoxicity, erythomycin estolate should not be given during the second and third trimester. spiramycin is the treatment of choice for toxoplasmosis in the first trimester. telithromycin and other makrolides should only be given during pregnancy when no alternatives are available. recommendation. clindamycin and lincomycin should only be used when penicillins, cephalosporins and macrolides have failed. clindamycin should not be routinely used after dental procedures. the bacteriostatic effect of tetracyclines is based on an inhibition of the bacterial protein synthesis. these broad-spectrum antibiotics, especially tetracycline itself, form stable chelates with calcium ions. the standard agent today is doxycycline. minocycline is especially lipophilic and displays a somewhat wider antibacterial spectrum than doxycycline. the older derivatives such as oxytetracycline and tetracycline are now rarely used as they are poorly resorbed. chlortetracycline, demeclocycline, and meclocycline are only used as local agents. tigecycline is a minocycline derivative that belongs to the glycylcyclines; it has a very broad-spectrum and is especially effective against multi-resistant pathogens such as mrsa. tetracyclines cross the placenta. according to current knowledge an increased risk of malformation is not expected when tetracyclines are used (cooper 2009 , czeizel 1997 . the results of a population-based case-control study suggested that oxytetracycline was associated with an increased incidence of congenital malformations (czeizel 2000d) . however, the number of cases in this study was small, and there are no other studies confirming this suspicion. a danish cohort study found an association between oral clefts and maternal tetracycline exposure in the second month, but this result was based on only two exposed cases (mølgaard-nielsen 2012) . from the sixteenth week of pregnancy when fetal mineralization takes place, tetracyclines can bind to calcium ions in developing teeth and bones. in the 1950s numerous publications described the brown/yellow discoloration of teeth in children who were prenatally exposed to tetracyclines. such dental discoloration is the only proven prenatal side effect of tetracyclines in humans. under discussion were also enamel defects leading to an increased risk of caries, inhibition of the growth of the long bones, specifically the fibula and further, cataracts due to depositions into the lens. as doxycycline has a weaker affinity to calcium ions than the older tetracyclines, the risk appears to be lower for doxycycline exposures. a discoloration of milk teeth is not to be expected prior to the sixteenth week of gestation. even thereafter, at worst, only the first molars of the permanent teeth would be affected when the usual therapeutic regimens if current dosings are adhered to. a bigger risk for the described development abnormalities can possibly expected with higher tetracycline doses during the second and third trimester that are necessary, for example, in malaria treatment. in the past, the use of tetracyclines, especially in high doses or via intravenous administration in the second half of pregnancy, has been associated with severe maternal hepatic toxicity (e.g. lewis 1991) . in most cases these were patients with kidney problems whose serum concentrations were markedly above the therapeutic range. no untoward effects have been described in pregnant women who applied tetracyclines locally during pregnancy. there is a lack of experience with tigecycline; no statement can be made about its tolerance in pregnancy. for local application silver sulfadiazine is used for burn injuries and sulfacetamide for eye infections. sulfonamides attain 50-90% of the maternal concentration in the fetus and compete with bilirubin for binding sites on albumin. today, sulfonamides are seldom used as monotherapy because their spectrum is limited and resistance develops rapidly. combined with a folate antagonist such as trimethoprim or pyrimethamine (section 2.6.16), sulfonamides are indicated among others in the treatment of toxoplasmosis and malaria. the fixed combination of the sulfonamide sulfamethoxazole and trimethoprim is available as co-trimoxazole. both agents in this combination are not subject to pregnancy-induced variation in clearance that would require dose modifications. trimethoprim is effective as a monotherapy in uncomplicated urinary tract infections with sensitive pathogens. to date, there are no indications that sulfonamides, trimethoprim, and their combinations have a teratogenic potential in humans (nørgård 2001 , czeizel 1990 ). an embryotoxic potential has been discussed from time to time, because antagonists to folic acid can lead to malformations in animal experiments, and in humans the spontaneous incidence of neural tube defects (spina bifida) can be decreased by the administration of folic acid during early pregnancy (chapter 2.18). the fact that human folic acid reductase is much less sensitive to trimethoprim than the bacterial enzyme, could explain that teratogenic problems have so far not been documented in humans when antibiotics with folic acid antagonists were used. trimethoprim has been used for many decades in pregnant women. at present, there is an ongoing discussion concerning the association between the use of folic acid antagonists and an increased risk of congenital malformations. a retrospective case-control study discusses the causal relationship between treatment with trimethoprim and other folic acid antagonists, and the development of neural tube defects, cardiovascular abnormalities, cleft lip and palate, and urinary tract anomalies (hernandez-diaz 2000) . authors' views on a preventative dose of multivitamin and folic acid preparations vary. additional case-control studies, some of them with notable methodological problems, found weakly recommendation. all tetracyclines are contraindicated after the fifteenth gestational week. prior to this, they are antibiotics of second choice. doxycycline should be preferred in such cases. inadvertent use of tetracyclines, even after the fifteenth week, is not an indication for termination of pregnancy (chapter 1.15). if really necessary, a local application to a small area may be conducted throughout pregnancy. tigecyclin is reserved for special situations when sufficiently tested antibiotic are not effective. significant evidence for the development of cardiovascular defects, urinary tract anomalies, anencephaly, limb defects, and orofacial clefts (e.g. mølgaard-nielsen 2012 , crider 2009 , czeizel 2001c ). an increased risk for preterm birth and low birth weight has also been observed after exposure to trimethoprim/sulfamethoxazole (santos 2011 , yang 2011 . a danish cohort study based on a prescription register found a doubling of the hazard of miscarriage after trimethoprim exposure in the first trimester (andersen 2013b ). based on the same prescription register, an increased risk of heart and limb defects was observed after preconceptional exposure (during the 12 weeks before conception) to trimethoprim (andersen 2013c) . beside methodological problems, such an association seems unlikely because a short-term therapy with trimethoprim does not usually lead to a relevant folic acid deficiency as a possible cause for birth defects. trimethoprim and sulfonamides are not drugs of first choice, but they exhibit no established teratogens. according to current knowledge the teratogenic risk of a trimethoprim and sulfonamide therapy is negligible. actually, there are no sufficiently convincing arguments to support the recommendation of an additional folic acid administration during an antibiotic therapy with the discussed medications, see chapter 2.18.8 for additional discussion concerning folic acid usage. extensive, generally reassuring experiences in the use of co-trimoxazole for common urinary tract infections during pregnancy, do not include the conclusion that this medication is safe when used at a much higher dose for opportunistic infections such as a pneumocystis pneumonia in the context of an hiv infection. so far, there have been no reports of malformations when such therapy was used in pregnant women. there are no systematic studies about the local application of sulfonamides during pregnancy. as sulfonamides compete with bilirubin for binding sites with plasma proteins, it has been argued that the risk of neonatal kernicterus is increased when sulfonamides are given at the end of gestation. with current surveillance, the danger of kernicterus is not tangible. however, a rise in bilirubin, especially in premature infants, cannot be excluded when sulfonamides have been used until birth. a danish population-based study could not find an association between sulfamethoxzole exposure near term and an increased risk of neonatal jaundice (klarskov 2013 ). quinolones inhibit the bacterial enzymes topoisomerase ii and iv that are important for the nucleic acid metabolism of bacteria. quinolones recommendation. sulfonamides, trimethoprim, and co-trimoxazole are antibiotics of second choice throughout pregnancy. if high dose co-trimoxazole is used for a pneumocystis pneumonia during the first trimester, based on theoretical grounds, folic acid should be supplemented and a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. if a premature birth is threatening, sulfonamides should be avoided in view of the bilirubin levels of the newborn. a short-term local treatment is acceptable, especially if the site is small. have a high affinity for cartilage and bone tissue which is highest in immature cartilage. pipemidic acid and nalidixid acid belong to the group of older quinolones. they have been displaced by the newer fluoroquinolones. the most important fluoroquinolones include ciprofloxacin, enoxacin, levofloxacin, moxifloxacin, norfloxacin, and ofloxacin. several substances have been removed from the market because of severe side effects. garenoxacin, lomefloxacin, pefloxacin, rosoxacin, and sparfloxacin are still available in some countries. gatifloxacin and nadifloxacin are only used as local agent. quinolones cross the placenta and are found in the amniotic fluid at low concentrations.when moxifloxacin is used about 8% of the maternal serum concentration can be measured in the amniotic fluid, and with lovofloxacin about 16% (ozyüncü 2010) . quinolones have not been found to be teratogenic in animals but severe, irreversible damage to joint cartilages was noted in young dogs treated after birth with quinolones (e.g. gough 1992 ). such alterations have not been described in prenatally exposed children. many publications failed to show indications of joint cartilage damage or an increased risk of malformations (bar-oz 2009 , cooper 2009 , larsen 2001 , loebstein 1998 , schaefer 1996 , berkovitch 1994 . one study expressed concern that the prenatal use of fluoroquinolones may be associated with an increased risk of bone malformations (wogelius 2005) . although not resembling each other, in three out of four birth defects the skeleton was affected. however, in this study of 130 women who redeemed a prescription for fluoroquinolones during the first trimester, or 30 days before conception, the total malformation rate was not increased (wogelius 2005) . in a prospective cohort study with 949 women who were exposed to a fluorquinolone during the first trimester, neither the rate of major birth defects, nor the risk of spontaneous abortion were increased compared to a control group (padberg 2014) . altogether, most data are available for norfloxacin and ciprofloxacin and, to a lesser extent, for levofloxacin, moxifloxacin, ofloxacin and pefloxacin. there are few or no data for the other fluoroquinolones. there have been no reports of undesirable side effects after topical use of quinolones during pregnancy. nitrofurantoin is a chemotherapeutic agent for drug-resistant urinary tract infections (utis) and for the prevention of recurrent utis. it acts as a bacteriostatic, but is also bactericidal at higher concentrations. details of its mechanism of action remain to be clarified. after an oral dose, therapeutic effective levels are attained only in the urinary tract. several publications do not support an association between nitrofurantoin and congenital malformations (nordeng 2013 , goldberg 2013 quinolones are antibiotics of second choice during pregnancy. in well-founded situations, when better studied antibiotics are ineffective, those quinolones that are well documented may be preferred such as norfloxacin or ciprofloxacin. a detailed ultrasound examination may be offered after exposure with the other fluoroquinolones during the first trimester. local treatment with quinolones is acceptable throughout pregnancy. czeizel 2001d , ben david 1995 , although in a number of studies, some of them with methodological faults, weakly significant findings were noted for craniosysnostosis, ophthalmic malformations, oral clefts, and cardiovascular defects (crider 2009 , källén 2005b , källén 2003 . a case-control study observed an increased risk of craniosynostosis after intrauterine exposure to nitrosatable drugs (gardner 1998) . as nitrofurantoin lowers the activity of glutathione reductase, discussions arise periodically as to whether an intrauterine exposure could trigger a fetal hemolysis. bruel (2000) reported a mature newborn with hemolytic anemia whose mother took nitrofurantoin during the last gestational month. nitrofurantoin is often used during pregnancy, and fetal hemolysis has not been commonly observed; therefore, a relevant risk is not likely. however, nordeng (2013) observed an increased risk of neonatal jaundice after maternal nitofurantoin treatment in the last 30 days before delivery. there is a case report of a pregnant woman who developed a toxic hepatitis after having been exposed to nitrofurantoin in her thirty-sixth week (aksamija 2009 ). in another case a woman took nitrofurantoin in her thirty-third week and was interpreted to present a gestational nitrofurantoin-induced pneumonia (mohamed 2007) . the nitrofurantoin derivative nifuroxazide is used for the treatment of diarrhea. there are no documented reports of its tolerance in pregnancy nor evidence of effectiveness. nifurtimox is a nitrofuran used for treatment of chagas disease. experience for pregnancy is very limited and the world health organization recommends that nifurtimox should not be taken by pregnant women (who 2013a). one study about safety included 14 pregnant women, but did not give information about the pregnancy outcome (schmid 2012) . for local treatment the nitrofurans furazolidone, nitrofural, and nifuratel are available. there has been no evidence of embryo-or fetotoxic risk in local applications. the use of local nitrofurans, especially as vaginal therapy, remains controversial and needs to be critically assessed not only during pregnancy. methenamine is a uti medication that releases the antiseptic formaldehyde into the urine. methenamine mandelate had been used for chronic utis due to e. coli and unproblematic germs. effectiveness and tolerance of the agent remain controversial. embryo-or fetotoxic problems have not been reported. there are no reports about the use of the hydroxy-quinolone derivative nitroxoline in pregnancy. fosfomycin is a broad-spectrum antibiotic that is bactericidal by inhibiting the synthesis of the bacterial cell wall. it is used as an intravenous injectable and as a reserve antibiotic in severe infections such as osteomyelitis. fosfomycin tromethamine is an orally taken salt of fosfomycin used for the treatment of uncomplicated utis. some authors also recommend the oral use during pregnancy (e.g. falagas 2010 , bayrak 2007 . these studies, however, are primarily focused on the effectiveness of fosfomycin tromethamine, not on the risk for the newborn. overall, the experience argues against a teratogenic and fetotoxic potential in humans. nitroimidazoles are effective bactericidal agents against anaerobes and protozoa. they are converted into metabolites that impede intracellular bacterial dna synthesis. the main representative of the nitroimidazoles is metronidazole. metronidazole is now being recommended by some investigators for the treatment of bacterial vaginosis in pregnancies at high risk for preterm delivery, as a strategy to decrease this risk (review by joesoef 1999) . others, however, failed to notice an improvement in the incidence of prematurity ( shennan 2006 , andrews 2003 , klebanoff 2001 . after oral and intravenous administration, concentrations as high as those in the mother are reached in the embryo/fetus. significant systemic absorption occurs after vaginal application, exposing the fetus as well. the pharmacokinetic profile of metronidazole did not change at the different time points assessed during pregnancy, and did not differ from nonpregnant patients (wang 2011) . like all nitroimidazoles, metronidazole displays an experimentally mutagenic and cancerogenic potential (review by dobias 1994) that has not been confirmed in humans. an investigation that ranged over 20 years did not show any indication of an increased risk of cancer when metronidazole was used (beard 1988) . on the basis of over 3,000 analyzed pregnancies, it can be stated that metronidazole has no teratogenic potential in humans (e.g. koss 2012 , diav-citrin 2001 , czeizel 1998 . suggestions from the hungarian malformation registry of a link between vaginal therapy with metronidazole and miconazole during the second and third month, and an increased appearance of syndactylies and hexadactylies have not been confirmed by other investigators (kazy 2005a) . nimorazole and tinidazole, both registered for the treatment of trichomonas infections, amebiasis, and bacterial vaginosis, cannot be evaluated sufficiently because of the lack of human data -the same applies to ornidazole. so far, there are no reports of human teratogenicity. the aminoglycoside antibiotics amikacin, framycetin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, streptomycin, recommendation. nitrofurantoin can be given during pregnancy to treat urinary tract infections when the antibiotics of choice have been ineffective. if possible, it should be avoided towards the end of pregnancy. the use of nifuroxazide, nifurtimox, local nitrofurans, methenamine, and nitroxoline should be avoided during pregnancy. when the antibiotics of choice in pregnancy cannot be used, fosfomycin tromethamine may be used to treat urinary tract infections in pregnancy. the intravenous application of fosfomycin should be restricted to severe bacterial infections with problematic germs. recommendation. metronidazol may be used in pregnancy when indicated. a single oral dose of 2 g is preferable to vaginal administration spread over several days, particularly as there are doubts about the effectiveness of the vaginal application. a parenteral administration is only indicated for a serious anaerobic infection. metronidazole is to be preferred to the less examined nitroimidazoles. and tobramycin inhibit protein synthesis and are bactericidal primarily for gram-negative germs. after oral administration only a minimal portion of aminoglycosides is resorbed. after parenteral administration of about 20-40% of the maternal plasma concentration is detectable in the fetus. spectinomycin is an aminocyclitol antibiotic closely related to the aminoglycosides. oto-and nephrotoxic side effects are also known to occur in nonpregnant patients when aminolgycosides are used parenterally. there are case reports about the parenteral use of kanamycin and streptomycin during pregnancy describing auditory problems, even deafness, in children exposed in utero (e.g. jones 1973 , conway 1965 , robinson 1964 . a similar case was reported in connection with gentamicin (sánchez sainz-trápaga 1998). an investigation of the hearing ability of 39 children whose mothers had received gentamicin intravenously during pregnancy found no deficiencies. this argues against a major ototoxic risk of gentamicin when used in pregnancy (kirkwood 2007) . theoretically, a fetal nephrotoxic risk exists because aminoglycosides concentrate in the fetal kidneys. a case report about a connatal kidney dysplasia after maternal gentamicin therapy (hulton 1995) does not prove a clinically relevant human risk, nor does a case of a hydronephrosis and suspected stenosis at the uteropelvic junction with lethal outcome, where the mother had been treated for uti first with ciprofloxacin and then with gentamicin at weeks 4-5 (yaris 2004) . except for these case reports, studies argue against a high oto-or nephrotoxic risk of gentamicin in the fetus and newborn. there has been no increase in the observation of malformations (czeizel 2000e) . no untoward effects have been described with aminoglycosides as local treatment during pregnancy. experience with spectinomycin is insufficient to analyze a risk in pregnancy. glycopeptide and polypeptide antibiotics  glycopeptide antibiotics the glycopeptides vancomycin and teicoplanin are bactericidal only for gram-positive pathogens by inhibiting their cell wall synthesis. they are considered reserve antibiotics to be used against msra and multiresistant enterococci. to avoid the development of resistance, their application should be critically appraised, and possibly limited only to fighting problematic pathogens. oral glycopeptides are hardly resorbed. this is useful when treating pseudomembranous enterocolitis with vancomycin. however, in this situation metronidazole (section 2.6.10) should be considered as an alternative, as vancomycin therapy is more expensive, and to prevent the selection for vancomycin-resistant enterococci. vancomycin crosses the placenta reaching the fetus in relevant quantities (laiprasert 2007) . it has not shown teratogenic effects in animal studies. experience with treatment in human pregnancy is limited to a recommendation. aminoglycosides should only be used parenterally in life-threatening infections with difficult gram-negative pathogens and when firstchoice antibiotics fail. the serum levels need to be monitored regularly during the treatment. a risk-based termination of pregnancy or invasive diagnostic are not required (chapter 1.15). if the parenteral therapy had been extensive, renal function should be monitored in the neonate and an auditory test should be performed. if local or oral application of aminoglycosides is indicated, they can be given because systemic absorption is minimal by these routes. pregnancy few case reports. there were no observations of malformations, kidney damage, or hearing deficits (reyes 1989) . experience with teicoplanin and the new lipoglycopeptides dalbavancin, oritavancin and telavancin is insufficient to analyze a risk in pregnancy. in vitro telavancin crosses the human placenta, with fetal concentrations reaching less than 3% of maternal concentrations (nanovskaya 2012) . daptomycin belongs to a new class of cyclic lipopeptides and is effective exclusively against gram-positive bacteria. it works by interfering with the bacterial cell membrane and protein synthesis, and is indicated to treat complicated infections with difficult pathogens. in animal experiments, daptomycin crossed the placenta and was not teratogenic. two children whose mothers took daptomycin in the fourteenth and twentyseventh weeks were unremarkable (stroup 2010 , shea 2008 ). polymyxins belong to the polypeptide antibiotics that are bactericidal by interfering with the transport mechanism of the cell wall. while the polymyxin colistin is today mostly used locally, it can also be applied parenterally where there is an infection with multi-resistant gram-negative germs. in patients with mucoviscidosis it is used as an inhalative. enterally colistin is not resorbed; therefore its oral administration is used to selectively decontaminate the intestinal tract. the polypeptide antibiotics bacitracin, polymyxin b, and tyrothricin are used locally. only limited experience is available in the application of polypeptide antibiotics during pregnancy and do not indicate a substantial risk (kazy 2005b ). other antibiotics chloramphenicol and tiamphenicol inhibit bacterial protein synthesis and have bacteriostatic activity. chloramphenicol is relatively toxic, and can cause severe agranulocytosis. it crosses the placenta well and can reach therapeutic concentrations in the fetus. in premature and term births it may lead to the grey baby syndrome. chloramphenicol can reach toxic levels in the neonate even when only the mother has been treated. there have been no suggestions of malformations (czeizel 2000f ). recommendation. glycopeptides should only be used in cases of life-threatening bacterial infections; vancomycin should then be preferred. recommendation. the use of daptomycin is limited to cases of life-threatening bacterial infections. recommendation. the parental use of colistin is limited to cases of lifethreatening bacterial infections. the local and oral application of polypeptide antibiotics need to be critically assessed. experience with thiamphenicol is insufficient to analyze a risk in pregnancy. dapsone, used among other indications against leprosis, apparently has no teratogenic potential (e.g. lush 2000 , bhargava 1996 . however, cases of hemolytic anemia have been reported in mothers and newborns. as dapsone bears a structural similarity to the sulfonamides, it has been argued that it might compete with bilirubin for protein binding, and thus could lead to hyperbilirubinemia in the newborn. fidaxomicin is a macrocyclic antibiotic which is approved for the treatment of infections with clostridium difficile. enterally fidaxomicin is very poorly resorbed. no experiences have been reported about its use during pregnancy. linezolid is a member of the oxazolidinone class, a new group of antibiotics. it acts bactericidally by inhibiting bacterial protein synthesis and is indicated in the treatment of multi-resistant pathogens. there is just one case report about the use of linezolid during pregnancy. after intrauterine exposure from gestational weeks 14 to 18 a healthy infant was delivered at term (mercieri 2010 ). the antiprotozoal agent pentamidine, among others effective in pneumocystis pneumonia, has not been evaluated sufficiently in pregnancy to recommendation. the systemic use of chloramphenicol and thiamphenicol is contraindicated throughout pregnancy. exceptions are life-threatening maternal infections that do not respond to less toxic antibiotics. when systemic treatment is absolutely necessary before birth, it is important to observe the newborn for toxic symptoms. a local application is also to be avoided during pregnancy. recommendation. during pregnancy, dapsone should be reserved for specific indications. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. fidaxomicin should be avoided in pregnancy. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. with the lack of experience, linezolid should only be used for severe infections with problematic germs. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. pregnancy estimate its embryotoxic potential for humans. usually it can be replaced by other antibiotics, e.g. co-trimoxazole (section 2.6.7). rifaximin is an antibiotic to treat travelers' diarrhea. there is not enough experience regarding its use in pregnancy. minimal enteral resorption and negative animal testing suggest that a high embryotoxic risk is unlikely. streptogramins are a group of cyclic peptide antibiotics that inhibit, like macrolides and lincosamides, the synthesis of bacterial proteins. they are derivatives of the naturally occurring pristinamycin. the later developed derivatives quinupristin and dalfopristin are used in a fixed combination. streptogramins should only be applied as reserve antibiotics for infections with highly resistant gram-positive germs. reports about use in pregnancy have not been available. active tuberculosis (tb) requires treatment in pregnancy, as the disease endangers not only the mother, but also the fetus. pregnancy does not seem to affect the course of tb. the prevalence of congenital tb is less than 1% where no treatment is initiated. lin (2010) investigated 761 newborns of mothers who had received treatment for tb during the gestation. their children were smaller and had lower birth weights than the control group of children of healthy mothers. there are slight differences in the recommendations of the different organizations in the world, such as the who (2010a), the international union against tuberculosis and lung disease (iuatld), and several national organizations (e.g. blumberg 2003) . treatment considerations depend on disease status and drug resistance. first-line drugs for the treatment of tb during pregnancy are isoniazid (+pyridoxine), rifampicin, ethambutol and pyrazinamide. these standard medications have not shown teratogenic or fetotoxic effects in humans (e.g. bothamley 2001) . as far as we know today, tb drugs reach the fetus in relevant quantities. an increasing development of resistance makes it harder to choose the right medication in pregnancy. pregnant women with multidrugresistant tb (mdr-tb) may also require second-line antituberculous recommendation. pentamidine is to be reserved in pregnancy for special situations when better tested antibiotics are not effective. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. if possible, rifaximin should be avoided during pregnancy. recommendation. streptogramins are to be avoided during pregnancy. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. drugs; the necessity for treatment should be weighed against the risk for the fetus on an individual base. current experiences in the management of mdr-tb argue against a high risk of the reserve drugs for the newborn (drobac 2005 , shin 2003 . streptomycin, however, should be avoided because of its ototoxic potential. ethambutol is a bacteriostatic drug used against tuberculosis. it can cross the placenta, but the risk of congenital malformations when used during pregnancy appears to be low. there are no reports indicating that ethambutol can cause ocular toxicity in the fetus, as it does in adults, when given in higher doses. isoniazid (inh) has proven to be a highly effective drug against many strains of mycobacterium, and can be used for tuberculous prophylaxis and for treatment of an active disease during pregnancy. although inh can cross the placenta, it does not appear to be teratogenic, even when given during the first trimester. the older literature contains case reports of different malformations and neurological damages in prenatally exposed children. inh intake, lack of pyridoxine, co-medication, and even the tb disease itself was blamed. newer publications did not confirm a teratogenic risk (e.g. taylor 2013 . in summary, experiences speak against a major risk. inh increases pyridoxine metabolism, which may be responsible for cns toxicity. to prevent a possible vitamin b6 deficiency, inh should be given during pregnancy in combination with pyridoxine. pyrazinamide (pza) is an antibiotic with specific effectiveness against mycobacterium tuberculosis. as its structure resembles nicotinamide, it is assumed that it intervenes with the nucleic acid metabolism of the bacterial cell. pza has effective bactericidal properties. systematic studies of its tolerance in pregnancy are lacking. so far, there has been no evidence of embryo-or fetotoxic effects in humans. the use of pza during pregnancy is recommended in several guidelines (e.g. who 2010a). the american thoracic society recommends in its guidelines to hold pza as a reserve drug during pregnancy, as there are currently insufficient data about its teratogenicity (blumberg 2003) . if pza is not used, treatment may be prolonged. recommendation. ethambutol is a first-line drug for treatment of tuberculosis during pregnancy. recommendation. isoniazid is a first-line drug for treatment of tuberculosis during pregnancy. it needs to be given together with pyridoxine. rifampicin also called rifampin, inhibits bacterial rna polymerase and is effective as a bactericidal agent against different pathogens, particularly mycobacteria. rifampicin can cross the placenta. in animal experiments, teratogenic effects were seen with doses 5-10 times higher than in human treatment. because rifampicin inhibits dna-dependent rna polymerase, there has been concern that it might interfere with fetal development. until now, no reports in the literature have confirmed this fear. there is apparently no increased risk of malformations. a long-term therapy of the mother could result in inhibition of vitamin k synthesis, and result in a higher bleeding tendency in neonates. streptomycin is an aminoglycoside that is used parenterally in the treatment of tb. it is bactericidal, particularly affecting germs that proliferate extracellularly. its ototoxicity can also hurt the fetus (section 2.6.11). aside from the above discussed first-line drugs for tb, reserve medications are available and used in cases of resistance or intolerance. no systematic studies exist on the tolerance of 4-aminosalicylic acid (p-aminosalicylic acid; pas). so far, no evidence for embryo-or fetotoxic effects has been found in humans (e.g. lowe 1964) . capreomycin, ethionamide, protionamide, rifabutin, rifapentine, thioacetazone, and terizidone, a prodrug of cycloserine, are all second-line agents used internationally for mdr-tb. the extent of documented experiences in pregnancy is limited, and insufficient for a differentiated risk assessment. single case reports argue against a high teratogenic risk of these drugs (e.g. lessnau 2003 , drobac 2005 . for additional reserve drugs for multi-resistant tb such as amikacin, see section 2.6.11, and diverse quinolones, see section 2.6.8; for other anti-infective agents, view the relevant sections of this chapter. recommendation. pyrazinamide may be used during pregnancy to treat active tb. recommendation. rifampicin is a first-line drug for treatment of tuberculosis during pregnancy. when used near term the newborn should receive an extended vitamin k prophylaxis (chapter 2.9). regarding other infections such as mrsa, rifampicin should only be administered when the drugs of first choice for pregnancy cannot be used. recommendation. streptomycin is contraindicated during pregnancy because of its ototoxic properties. inadvertent exposure does not require risk-based termination of pregnancy or invasive diagnostic procedures, but hearing tests should be performed after birth (chapter 1.15). generally, each external antibiotic treatment needs to be examined carefully to see whether or not the bacterial infection is more effectively treated with systemic medication. the potential of local treatment is often overestimated. further, with topical therapy, sensitization and resistance development need to be considered. fusafungine has bacteriostatic and anti-inflammatory effects and is used as a spray for the treatment of infections of the nose and throat area. there is insufficient experience about its application in pregnancy. fusidic acid is an antibiotic that is almost exclusively used externally; its prenatal tolerance has not been examined systematically, although the medication has been available for a long time. it has a narrow spectrum of effectiveness against gram-positive bacteria (staphylococci) and is not recommended for an untargeted treatment. mupirocin is primarily bacteriostatic, affecting staphylococci and streptococci by inhibiting bacterial protein synthesis. it is especially used as a nasal ointment to eliminate mrsa. mupirocin has not been examined systematically, but there is no evidence of undesirable effects in pregnancy. retapamulin is the first representative of the pleuromutilins that is approved for human treatment. it is applied as an ointment for short-term treatment of superficial skin infections. retapamulin inhibits bacterial protein synthesis and is bacteriostatic, primarily for gram-positive germs. systemic resorption is minimal with topical use, but nevertheless, as experience in pregnancy has been limited, its application needs to be critically examined. taurolidine is an antimicrobial solution that can be used for lavage in peritonitis and for the prevention of infections with catheters. as a bactericidal agent, its mechanism of action is only partially clarified. there are no reported experiences in pregnancy. see the corresponding sections for the local application of aminoglycosides (section 2.6.11), chloramphenicol (section 2.6.13), quinolones (section 2.6.8), macrolides (section 2.6.4), nitrofurans (section 2.6.9), nitroimidazoles (section 2.6.10), polypeptide antibiotics (section 2.6.12), sulfonamides (section 2.6.7), and tetracyclines (section 2.6.6). apart from pregnant women living in malaria areas, pregnant women are increasingly traveling to tropical countries and need a suitable malaria prophylaxis. increased resistance of malaria pathogens make it more difficult to suggest a general recommendation. the guidelines of tropical medicine recommendation. the reserve drugs discussed here should only be used for multi-resistant tuberculosis when standard therapy is not indicated. an inadvertent exposure during pregnancy does not require a risk-based termination or invasive diagnostic, but a detailed ultrasound examination should be carried out (chapter 1.15). recommendation. externally used antibiotics are not suspected to be teratogenic. nevertheless, the application of local antibiotics needs to be critically assessed. antibiotics that are safe when used systemically may also be used locally. if another local antibiotic is absolutely necessary, it may be used in pregnancy. should be followed, also in pregnancy, according to the travel destination. especially difficult is the management of malaria tropica caused by plasmodium falciparum. pregnancy enhances the clinical severity of falciparum malaria, especially in the primiparous and non-immune woman. pregnancy alters a woman's immunity to malaria, making her more susceptible to malaria infection and increasing the risk of illness, severe anaemia, and death. maternal malaria increases the risk of spontaneous abortion, stillbirth, prematurity, and low birth weight, and thus results in excess infant mortality (e.g. bardaji 2011 , shulman 2003 . therefore, mosquito-bite prevention, prophylaxis, and treatment of malaria should not be shortened or omitted in an ongoing pregnancy. traveling to areas with multidrug-resistant malaria should be avoided if possible. the choice of drug for malaria prophylaxis and treatment during pregnancy depends on the local pattern of antimalarial drug resistance, the severity of the malaria, and the degree of pre-existing immunity. it is important to be well informed about the current recommendations for prophylaxis and treatment of malaria in the area to be visited. for travelers to malaria-endemic areas, a general recommendation is difficult because of increasing resistances. depending on the drug, the chemoprophylaxis must be continued for up to 4 weeks after leaving the malarial region. for women living in falciparum-endemic areas with stable transmission, the world health organization recommends the use of insecticide-treated nets (itns) and intermittent preventive treatment (ipt) with sulfadoxine-pyrimethamine during pregnancy (who 2013b , nyunt 2010 . ipt reduces maternal malaria episodes, maternal anaemia, placental parasitaemia, low birth weight, and neonatal mortality (review by mcclure 2013). a prompt diagnosis and effective treatment of malaria infections is vital. although data from prospective studies are limited quinine, chloroquine, proguanil, and clindamycin (section 2.6.5) are considered safe during early pregnancy. pregnant women in the first trimester with uncomplicated malaria tropica should be treated with quinine plus clindamycin (if available) (who 2010b). for the second and third trimester the world health organization recommends artemisinin derivatives. the choice of combination partner is difficult because of limited information. reserve medications include the following: amodiaquine, atovaquone, dapsone (section 2.6.13), lumefantrine, mefloquine, piperaquine, and pyrimethamine plus sulfadoxine. doxycycline is contraindicated after the sixteenth gestational week (section 2.6.6). halofantrine and primaquine should be avoided. see the relevant sections of this chapter about the specific active substrates. during gestation plasma concentrations of many antimalaria agents are lower and their elimination is enhanced. this can result in treatment failure. thus, in each patient dose and dose interval need to be assessed individually. recommendation. generally, the physician should discuss with a patient if the trip to a tropical region could be postponed (section 2.6.36). the risk of exposure can be reduced by long clothes, mosquito netting, and repellents. in no case should medications be denied for prophylaxis or treatment on behalf of a pregnancy, as the potential risk for the unborn child predominates. if medications with inadequate pregnancy experience are used in the first trimester, a detailed ultrasound examination should be offered. a risk-based termination is not justified when the above-described medications have been used in pregnancy (chapter 1.15). amodiaquine, like chloroquine, belongs to the group of 4-aminoquinolines. it can cause severe side effects such as liver damage and agranulocytosis, and for this reason, is unsuitable for prophylaxis. its use is limited as a reserve medication for malaria. there has been no evidence of tetatogenicity (review by thomas 2004) , but experiences are limited. with regard to early pregnancy, only single case reports have been published. one study found only mild maternal side effects in 450 pregnant women who had been treated in the second or third trimester. an increase in miscarriages, prematurity, stillbirth, or malformations was not observed (tagbor 2006 ). artemisinin and its derivatives artemether, artemotil, artesunate, and dihydroartemisinin, are increasingly used against malaria as plasmodium falciparum has developed resistance to other drugs. these compounds combine rapid blood schizonticide activity with a wide therapeutic index. artemisinins should be given as combination therapy to protect them from resistance. typical combinations of such artemisinin-based combination therapy (act) are artemether plus lumefantrine, artesunate plus amodiaquine, artesunate plus mefloquine, artesunate plus sulfadoxine-pyrimethamine, and dihydroartemisinin plus piperaquine first trimester experiences with the use of artemisinin derivatives are limited. a number of studies contain data of more than 250 pregnant women treated with an artemisinin derivative during the first trimester, without showing evidence of a teratogenic risk (mosha 2014 , adam 2009 , clark 2009 , who 2006 . manyando (2010) more commonly found umbilical hernias in an additional 140 children whose mothers had been treated with artemether and lumefantrine. after 12 months most of these hernias were not detectable anymore. there are experiences with more than 1,500 pregnant women who used artemisinin derivatives in the second and third trimester (e.g. piola 2010 , bounyasong 2001 , deen 2001 , mcgready 2001 , phillips-howard 1996 . in summary, these studies did not find an increased risk in miscarriages, stillbirths, and malformations. to some degree the artemisisin derivatives were better tolerated by pregnant women, and were more effective than treatments of the control group. as plasma levels of artemether are decreased during pregnancy, it has been suggested that the dose and the dose interval may have to be adjusted (e.g. tarning 2013, morris 2011). these reassuring data led the who (2010b) to recommend using artemisinin derivatives as medications of choice for malaria tropica in the second and third trimester. it does not specify what combination is recommended in the context of act. during the first trimester, based on a lack of experiences, the who views artemisinin derivatives as reserve medications that should not be withheld in an individual case where needed. recommendation. amodiaquine may be used as a reserve medication for the treatment of malaria. atovaquone is a broad-spectrum anti-protozoal drug that is also used in pneumocystis pneumonia. monotherapy quickly leads to resistance, thus it is combined with proguanil when used for malaria prophylaxis and treatment. experience with atovaquone is limited in pregnancy. a danish cohort study based on a prescription register with 149 women exposed during their first trimester to atovaquone, 93 of them exposed at any time in weeks 3 through 8 after conception, found no increased risk for birth defects (pasternak 2011) . when used in the second and third trimester, small studies observed no adverse effects (mcgready 2005 , na-bangchang 2005 . available data are insufficient for a differentiated risk assessment, but do not suggest a teratogenic risk. mcgready (2003) discusses the need of a dose adjustment as clearance increases and levels decrease during pregnancy. chloroquine, an antimalaria drug of the group of 4-aminoquinolines, works well and effectively as a schizonticidal drug against the erythrocytic forms of all types of plasmodia. today though, almost all pathogens of the potentially lethal malaria tropica have become resistant to this rather well tolerated, and for many decades, useful medication. resistance has also been noted for plasmodium vivax, the pathogen of the less severe malaria tertiana. plasmodium ovale and plasmodium malariae still remain mainly sensitive to chloroquine. chloroquine is not embryo-and fetotoxic when used at the usual dose for malaria prophylaxis or for a three-day treatment of a typical malaria attack (mcgready 2002 , phillips-howard 1996 . current evidence does not suggest fetal ocular toxicity when chloroquine was used as antimalarian medication during pregnancy (review by osadchy 2011). lee (2008) examined 12 pregnant women and nonpregnant controls, and did not find any changes in the pharmacokinetics or the serum level of chloroquine. the anti-inflammatory properties of chloroquine are used also for antirheumatic therapy (section 2.12.8). antirheumatic doses of chloroquine are higher than those used for malaria prevention. recommendation. artemisinin derivatives may be used in the second and third trimester. in the first trimester they are reserve medications for the treatment of malaria. recommendation. atovaquone may be used as a reserve medication for the treatment of malaria. recommendation. chloroquine may be used throughout pregnancy for the prophylaxis and treatment of malaria. if chloroquine resistance of the parasite is likely or has been demonstrated, other drugs must be used. halofantrine has a rapid schizonticidal effect upon the erythrocytic forms of those plasmodia that are resistant to chloroquine and other antimalarials. halofantrine prolongs the qt interval in the ekg. becauses it can provoke life-threatening cardiac arrhythmias in patients with heart disease, or in conjunction with other arrhythmogenic medications, halofantrine is no longer recommended. the limited experiences in pregnancy allow no differentiated risk analysis. lumefantrine belongs to the group of arylamine alcohols like quinine, mefloquine, and halofantrine. artemether plus lumefantrine is currently a popular artemisinin-based combination therapy. few experiences are available regarding its application in the first trimester without showing evidence of a teratogenic risk (e.g. mosha 2014) . for the second and third trimester, studies with several hundred patients have been reported and do not indicate a major risk (piola 2010 . manyando (2010) found only a mild increase in umbilical hernias in 140 children whose mothers took artemether and lumefantrine during the first trimester. most of these had disappeared when follow-up examination was conducted 12 months later. in summary, current experiences do not suggest a major embryo-or fetotoxic risk of lumefantrine. during pregnancy, the plasma concentration is lower and the elimination enhanced, thus increasing the risk of treatment failure (e.g. tarning 2009 ). mefloquine displays an effective and rapid activity against the erythrocytic forms of all plasmodia. current experiences with more than 2,000 treated pregnant women, several hundred of them in the first trimester, do not suggest a teratogenic or fetotoxic potential in humans (e.g. schlagenhauf 2012 , bounyasong 2001 , mcgready 2000 . one single study of the use of mefloquine has been debated as finding an increased rate of stillbirths. this study compared 200 pregnant women who received mefloquine for malaria, and found them to have a significantly higher rate of stillbirth than those who had been treated with quinine and other antimalarials (nosten 1999) . other studies, however, have not confirmed this risk, and mefloquine has been an established medication in pregnancy for some time. recommendation. halofantrine is only to be used in cases of acutely threatening malaria that cannot be managed with better tested and less toxic drugs. when cardiac problems are an issue, other antimalaria medications must be used. recommendation. lumefantrine may be used as a reserve medication for the treatment of malaria. recommendation. mefloquine may be used throughout pregnancy for the prophylaxis and treatment of malaria if there is no resistance. a fixed oral combination of the bisquinolone piperaquine and dihydroartemisinin (dhp) is a new and promising artemisinin-based combination therapy. the mechanism of action of piperaquine is unknown. an indonesian observational study detected a higher rate of abortion after first trimester exposure to dihydroartemisinin-piperaquine (poespoprodjo 2014) . this observation was based on five abortions among eight pregnancies (63%). the same study found a lower risk of perinatal mortality after dihydroartemisinin-piperaquine in the second and third trimester compared to quinine-based regimens. the limited experiences in pregnancy allow no differentiated risk analysis. no significant pharmacokinetic differences between pregnant and nonpregnant women were reported in two small studies , hoglund 2012 ). primaquine, an 8-aminoquinoline derivative, is effective against the intrahepatic permanent forms of plasmodium vivax and plasmodium ovale. it is used for the complete elimination of pathogens in combination with a blood schizontocide for the erythrocytic parasites. primaquine should not be used in pregnancy because of the potential risk of hemolytic effects in the fetus. as yet, there are no studies that permit a well-grounded risk assessment. however, there is no substantial evidence for a teratogenic potential in humans (phillips-howard 1996). proguanil, an older medication for malaria prophylaxis belonging to the folic acid antagonists, is experiencing a renaissance as it has become useful in the face of increasing chloroquine resistance. most often it is applied in combination with the synergistic atovaquone. there is no evidence of an embryotoxic potential in humans (e.g. pasternak 2011 , mcgready 2005 . mcgready (2003) discuss the need to adjust the dose as clearance is increased and blood levels decreased during pregnancy. pyrimethamine is an inhibitor of folic acid synthesis that is also used in the treatment of toxoplasmosis and pneumocystis pneumonia. in malaria recommendation. piperaquine may be used as a reserve medication for the treatment of malaria. recommendation. primaquine is not a therapeutic option during pregnancy. a prophylactic elimination of hepatic spores should usually be postponed for a time after birth. recommendation. proguanil may be used throughout pregnancy for prophylaxis and treatment of malaria provided there is no resistance. treatment it is only applied in combination with another folic acid antagonist such as sulfadoxine (section 2.6.7). this particular combination is used for intermittent preventive treatment (ipt) during pregnancy. however, increasing resistance has started to limit the effectiveness of this popular combination (newman 2003) . as animal experiments indicated embryotoxic effects, concerns had been raised about the use of these folic acid antagonists in early pregnancy. numerous investigations, however, have not demonstrated an increased malformation risk in humans (e.g. manyando 2010 , phillips-howard 1996 . some studies suggest that pregnancy adversely alters the pharmacokinetics of pyrimethamine and sulfadoxine (e.g. karunajeewa 2009 , green 2007 . as data are inconsistent, a general recommendation about dose adjustments in pregnancy is difficult. when sulfadoxine-pyrimethamine is given in early pregnancy, it should be supplemented by folic acid until the tenth week. the who recommends 0.4-0.5 mg per day, as a coadministration of high dose (5 mg daily) compromises the efficacy of sulfadoxine-pyrimethamine in pregnancy (who 2010b). quinine is the oldest antimalarial agent. it works well and effectively as a schizonticidal drug against the erythrocytic forms of all plasmodium species. despite a relatively high toxicity and a narrow therapeutic range, it is used again increasingly in the treatment of chloroquine-resistant malaria. in combination with clindamycin (section 2.6.5) its effectiveness is increased. concentrations in the fetus are just as high as in the mother, and are potentially toxic. in some case reports it was observed that children had auditory or visual defects after the use of quinine in pregnancy. however, in those cases considerably higher doses had been administered than currently in use. there is no evidence of an increased risk of abortion or preterm delivery with the use of a standard dosage of quinine for treatment of acute malaria (phillips-howard 1996) . these findings were confirmed by other studies with several hundred pregnant women exposed during the first trimester, where no increased rates of spontaneous abortion, congenital malformations, stillbirth or low birth weight were found (e.g. adam 2004b , mcgready 2002 . quinine increases the secretion of insulin (elbadawi 2011) . especially in the last part of pregnancy, severe maternal hypoglycaemia has been induced by quinine therapy. due to the risk of hypoglycemia, the who (2010b) guidelines prefer to use an artemisinin combination for the management of malaria tropica from the second trimester on. a study of the metabolism of quinine in pregnant and nonpregnant women failed to show significant pharmacokinetic differences. the authors concluded that dose adjustment is not necessary during pregnancy (abdelrahim 2007) . induction of contractions with high doses of quinine cannot be excluded. recommendation. pyrimethamine in combination with sulfadoxine may be administered for the treatment of malaria. for toxoplasmosis it is the drug of choice when combined with a long-term sulfonamide, especially after the first trimester. when pyrimethamine is given in early pregnancy, it should be supplemented with folic acid, see also chapter 2.18.8. quinine is a component of some analgesic compounds and of certain beverages, although in lower and apparently nonembryotoxic doses. azole antifungals azole derivatives inhibit the ergosterol biosynthesis, thereby causing disturbances in the permeability and functions of the fungal cell membrane. azole antifungals include two broad classes, imidazoles and triazoles. in animal experiments azole antifungals cross the placenta and are teratogenic at high doses. with regard to the use of the triazole derivative fluconazole in pregnancy, there was a report of three children (two of them siblings) with craniofacial, skeletal, and cardiac malformations, similar to those seen in animal studies (pursley 1996) . because of meningitis, their mother had used high doses of fluconazole (400-800 mg daily) through or beyond the first trimester on a long-term basis. additional case reports have described two births involving craniofacial, limb, and cardiac defects in two mothers who used fluconazole (lopez-rangel 2005 , aleck 1997 ). those cases shared some characteristics with the antley-bixler syndrome. however, there was no evidence of an increased risk of malformation in a prospective cohort study with 226 women exposed during the first trimester (mastroiacovo 1996) . in several other studies, first trimester exposure to low-dosage regimens of fluconazole for vaginal candidiasis did not appear to cause an increased risk of malformations (e.g. jick 1999 , campomori 1997 , inman 1994 . danish cohort studies based on a prescription register also could not find an increased risk of birth defects after first trimester exposure in several thousand pregnant women (nørgaard 2008 , sørensen 1999 ). an extended analysis of the danish data observed an increased risk for tetralogy of fallot based on seven cases (prevalence 0.1%) compared to unexposed pregnancies (or 3.16; 95% ci 1.49-6.71). the rate of major birth defects was not increased (mølgaard-nielsen 2013). in most cases the low and single dose consisted of 150 mg fluconazole usually used for a vaginal yeast infection. itraconazole is a triazole derivative with wide-spectrum activity. there has been no evidence of teratogenicity in prospective studies examining several hundred women with first trimester exposure (e.g. de santis 2009 , bar-oz 2000 ; most of the exposures were short-term. a danish register analysis did not find an increased risk of birth defects among 687 women with a first trimester prescription of itraconazole (mølgaard-nielsen 2013). recommendation. despite its toxicity, quinine belongs to the drugs of choice when dealing with chloroquine-resistant malaria tropica in pregnancy. in this situation the potential risk of treatment is much smaller for the fetus than the danger of a severe maternal disease. attention needs to be paid to possible maternal hypoglycemia. even though embryotoxic effects due to quinine in analgesic compounds are not to be expected, these agents should be avoided because they do not conform to good therapeutical practice. the same holds for the regular or excessive consumption of quinine drinks. the imidazole derivative ketoconazole is usually avoided in systemic use because it is poorly tolerated and has many suitable alternatives. ketoconazole is administered on occasion for the treatment of cushing syndrome as it inhibits steroid synthesis. theoretically, by decreasing testosterone synthesis, it might impede the sexual development of male foetuses; however, this has not been described. ketoconazole has been used in several cases in pregnancy with good maternal and fetal outcome (e.g. boronat 2011 , berwaerts 1999 , amado 1990 . a retrospective study from data of the hungarian malformation registry based on 18 exposed subjects shows no evidence of an increased risk of malformations after systemic use of ketoconazole (kazy 2005c ). an analysis of a danish register did not observe a significantly increased risk of birth defects among 72 pregnant women with a prescription for this agent during first trimester (mølgaard-nielsen 2013). for posaconazole and voriconazole which are used for aspergillosis and other invasive mycoses, information is lacking about use during pregnancy. there is only one published case report of a normal child, born after voriconazole treatment of the mother in the second and third trimester (shoai tehrani 2013). a multitude of poorly resorbed topical azole derivatives are available for the treatment of superficial fungal infections. the drugs of this group that had been introduced first, namely clotrimazole and miconazole, are most thoroughly investigated for use in pregnancy. regarding clotrimazole, there are extensive studies about the treatment of vaginal yeast infections that do not indicate an embryotoxic potential (e.g. czeizel 1999b , king 1998 . also, there is no suggestion that there is an increase in miscarriages. czeizel (2004a) noted a decrease in prematurity when vaginosis was treated locally with clotrimazole. experiences with several thousand pregnant women are available for miconazole (e.g. czeizel 2004b , mcnellis 1977 . a suggestion by the hungarian malformation registry about a link between vaginal therapy with miconazole plus metronidazole during the second and third gestational month, and an increase in syndactyly and hexadactyly, has not been substantiated by other studies (kazy 2005a ). an israeli report describes two cases of severe skeletonal anomalies after the use of bifonazole that are reminiscent of anomalies seen after systemic use of fluconazole. in the first case, bifonazole was taken orally from week 6 to 16, in the second case 500 mg/d vaginally throughout pregnancy and clearly at a higher dose than recommended (linder 2010) . at dose levels which are reached with nomally recommended topical application no teratogenic risks have been noted, yet systematic studies are lacking. for ketoconazole see above (azole antifungals for systemic use). recommendation. if a systemic treatment with an azole derivative becomes absolutely necessary, fluconazole and itraconazole are to be preferred as the better-tested medications. if possible, treatment should start after the first trimester. an inadvertent exposure during pregnancy does not require a risk-based termination or invasive diagnostic, but a detailed ultrasound examination should be carried out (chapter 1.15). clearly less experience has been collected about the local applications of butoconazole, croconazole, econazole, fenticonazole, isoconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, terconazole, and tioconazole. teratogenic effects have not been observed (king 1998) . this was confirmed for vaginal econazole treatment in a study of 68 pregnant patients (czeizel 2003a ). amphotericin b is a broad-spectrum antifungal agent of the polyene group. it binds to ergosterol at the cell membrane of fungi and disturbs cell wall permeability. it can be used intravenously, orally, and locally. with oral application, it is poorly resorbed and thus has only a local effect in the intestinal tract. conventional amphotericin b given parenterally has a number of side effects, primarily nephrotoxicity. newer lipid formulations of amphotericin b such as liposomal amphotericin b are characterized by a markedly better tolerance and less nephrotoxicity. amphotericin passes the placenta. relevant plasma concentrations could be measured in a newborn, although the mother had taken her last dose four months prior (dean 1994 ). this could be due to placental accumulation or delayed elimination by the fetal kidneys. several case reports do not indicate an increased risk of malformations with amphotericin b (e.g. costa 2009 , ely 1998 , king 1998 ). more than 10 pregnancy courses with liposomal amphotericin b also argue against an embryo-or fetotoxic risk (e.g. mueller 2006 , pagliano 2005 , pipitone 2005 ). these experiences are insufficient for a differentiated risk assessment. as resorption is minimal with oral or local use, a risk appears unlikely. echinocandins are a new antifungal medication group. these parenteral synthetic lipopeptides inhibit the synthesis of 1,3-β-d-glucan, a key ingredient of the fungal cell wall. anidulafungin, caspofungin, and micafungin are currently approved. in animal experiments echinocandins cross the placenta. there have been no reports of their use in pregnancy. yalaz (2006) described the successful postnatal application of caspofungin in a dystrophic premature newborn of the twenth-seventh gestational week. recommendation. clotrimazole and miconazole belong to the local antifungal medications of choice in pregnancy. the other azole derivates are antimycotic drugs of second choice. recommendation. amphotericin b should only be used parenterally in cases of serious disseminated fungal infections. the liposomal formulation may be preferred. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. oral and local use of amphotericin b is acceptable in pregnancy. flucytosine is effective against cryptococcus neoformans and many candida species. it inhibits the dna synthesis. within the mycotic cell flucytosine is partially converted into the cytostatic 5-fluorouracil. to a smaller degree this reaction has to be expected in humans as well. due to a high incidence of resistance, flucytosine should only be administered in combination with another antifungal drug such as amphotericin b. in animal experiment, fluycytosine has a teratogenic effect at doses below those used in humans. as yet, no malformations have been reported in humans; however, there is, as yet, no published experience with the use of flucytosine in the first trimester. case reports about application in the second and third trimester for dangerous disseminated cryptococcosis have not shown evidence of fetal damage (e.g. ely 1998). griseofulvin is an organically derived antifungal agent that is used orally for several weeks against fungal infections of the skin, hair and nails. as it is deposited within the keratin, it is especially suited for the management of fungal infections of nail mykoses. in animal experiments griseofulvin is teratogenic and, at high doses, cancerogenic. it crosses the placenta at term (rubin 1965) . one publication, based on birth defects data, reported two pairs of conjoined twins after the use of griseofulvin in early pregnancy (rosa 1987) . this observation could not be confirmed in other publications (knudsen 1987 , metneki 1987 . a population based case-control study with some 31 exposed pregnant women did not demonstrate an increased risk of malformations (czeizel 2004c ). these experiences are insufficient for a differentiated risk assessment. recommendation. as there is insufficient data regarding the use of echinocandins in pregnancy, they should only be used where no alternatives are available and the mycosis is life-threatening. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. flucytosine should only be used for life-threatening disseminated fungal infections during pregnancy. as it is not indicated as a monotherapy, it needs to be assessed critically if its use as a second mycotic drug is really necessary. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. as griseofulvin is not used to treat life-threatening fungal infections, its application in pregnancy should be avoided. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. terbinafine is used for both oral and topical treatment of fungal infections of the nails and other dermatophytoses. a prospective study reported on 54 pregnant women exposed to terbinafine which showed no evidence of a teratogenic potential (sarkar 2003) . of these women 24 were exposed during the first trimester and 26 had an oral exposure. these data are insufficient for a differentiated risk analysis. when used topically, less than 5% is resorbed making a risk unlikely. regarding the topical use of azole derivatives such as clotrimazole and miconazole, see section 2.6.17; for amphotericin b, section 2.6.18; and for terbinafine, section 2.6.22. nystatin is an antifungal drug from the polyene group. like the closely related amphotericin b it binds with ergosterol of the mycotic cell wall and interferes with its function. nystatin is an effective local antifungal drug for candidiasis of the skin or mucosa. when taken orally, it is poorly resorbed and only works locally in the intestinal tract. the indication for intestinal cleansing needs to be critically assessed in immunocompromized patients. nystatin is used frequently, and there is no evidence of embryo-or fetotoxic effects (e.g. king 1998) . a population-based case control study did not show an increased risk of malformation after first trimester exposure. when treatment was performed in the second and third trimester, slightly more cases of hypospadia were noted (czeizel 2003b ). however, a low resorption rate, methodological weaknesses of the study, and the low number of only 106 pregnant women place the result in question. a retrospective study of the hungarian malformation register, with 160 exposed subjects, did not reveal signs of an increased risk of malformation when natamycin was applied vaginally (czeizel 2003c) . based on the same register, a case-control study discussed a possible association between cardiovascular malformations and maternal use of tolnaftate in pregnancy (czeizel 2004d) . this observation was based on 26 exposed cases, of which four cases had varying types of cardiac defects (or 3.1, 95%; ci 1.0-9.7). these data are insufficient for a differentiated risk analysis. amorolfine, butenafine, ciclopirox, haloprogin, naftifin, and tolciclate are insufficiently investigated with regard to prenatal human toxicity. as yet, there is no substantial indication for an increased risk of malformations after local use. recommendation. terbinafine should be avoided during pregnancy as safety data are lacking and fungal nail infections do not require urgent treatment. if treatment took place in the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. a topical application is likely to be harmless. recommendation. nystatin, like clotrimazole and miconazole is an antifungal drug of choice during pregnancy. where possible, these drugs should be preferred. external treatment with amorolfine, butenafine, ciclopirox, haloprogin, natamycin, naftifin, tolciclate, and tolnaftate should be avoided during pregnancy. more than 2 billion people are infected with helminths worldwide. soil-transmitted helminths have been recognized as an important public health problem in many developing countries. severe hookworm and other helminth infections during pregnancy may cause anemia, reduced birth weight and increased perinatal mortality. a routine application of anthelmintics during the second and third trimester for women in areas endemic for hookworm infection has been suggested, with the argument that this may improve maternal anemia, birth weight, and neonatal mortility (e.g. who 2013c, christian 2004 ). however, a randomized placebo-controlled study showed no advantage for newborns whose pregnant mothers had received albendazole or praziquantel . recently, it has been discussed if routine anthelmintic treatment during pregnancy might lead to an increased risk for allergies in infancy (mpairwe 2011 ). the benzimidazole derivatives albendazole, flubendazole, mebendazole, thiabendazole, and triclabendazole inhibit the uptake of glucose and thereby kill the parasites. in animal experiments benzimidazole derivative with anthelmintic activity showed teratogenic effects. albendazole and mebendazole are poorly resorbed from the gastrointestinal tract, except when there is an inflammation. however, enteral absorption may be increased due to high-fat diet. mebendazole is a highly effective and well tolerated anthelmintic drug used against nematodes (such as pinworms, roundworms, whipworms, and hookworms). there have been reports describing children with various malformations after in utero exposure to mebendazole, but a distinct pattern of malformations could not be discerned (review by schardein 2000) . an increased risk of congenital malformations was not observed in a study of over 400 pregnant women exposed to mebendazole in the first trimester (de silva 1999) . this was confirmed in a controlled prospective study covering 192 first trimester exposed pregnant women (diav-citrin 2003) . another study with 48 first trimester exposures also found no increased risk for malformations or miscarriages ( mcelhatton 2007) . although numbers are too small for any definite conclusion, mebendazole does not appear to represent a major teratogenic risk. significantly more experience has been collected with exposure during the second and third trimester showing no evidence of a fetal risk (e.g. gyorkos 2006) . albendazole is a newer, highly effective broad-spectrum anthelmintic which combined with operative interventions has become the treatment of choice for alveolar and cystic echinococcosis. limited experience in the first trimester has not shown evidence of a major risk (gyapong 2003 , cowden 2000 . there are several thousand pregnancies with the use of albendazole in the second or third trimester without any obvious adverse reactions reported (e.g. , ndyomugyenyi 2008 . two abstracts from korea which reported the outcome of 16 pregnant women after the first trimester exposure to flubendazole showed no evidence of a teratogenic potential (choi 2008 (choi , 2005 . however, the data is insufficient for a differentiated risk assessment. there are no reports of thiabendazole and triclabendazole use during human pregnancies. ivermectin is a broad-spectrum anthelmintic agent which is mainly used in humans in the treatment of onchocerciasis (river blindness), lymphatic filiriasis and strongyloidiasis. it is also effective against other worm infections and some epidermal parasitic skin diseases, such as scabies. ivermectin is well resorbed after oral administration. animal experiments do not suggest a teratogenic potential, although at maternally toxic exposures malformations were noted in rodents. a number of case reports describing accidental treatments during the first trimester have not shown malformations in the children (gyapong 2003 , chippaux 1993 , pacque 1990 ). however, data are insufficient for a differentiated risk assessment. a study encompassing more than 100 women who took ivermectin during the second trimester found no significant anomalies in the newborns (ndyomugyenyi 2008 ). niclosamide is an anthelmintic that is effective against tapeworms (cestodes). it affects the energy metabolism of the parasites and is practically not resorbed by the intestinal tract. this agent had been used extensively in the past and is not suspected to cause malformations, but has not been systematically studied in humans. praziquantel is a highly effective broad-spectrum anthelmintic agent against many trematodes and cestodes. it is mainly used for the treatment of schistosomiasis (bilharziosis). no teratogenicity has been reported in animal studies. over the last decades millions of pregnant women have been inadvertently treated with praziquatel during routine anthelmintic programs without an obvious adverse reactions reported. a few publications recommendation. mebendazole may be used during pregnancy to treat relevant worm diseases. albendazole may be used in cases of echinococcosis. the other benzimidazole anthelmintics should only be used with a compelling indication, and when more established anthelmintics are ineffective. after first trimester exposure a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. with a compelling indication ivermectin may be used in pregnancy. after first trimester exposure a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. niclosamide may be given during pregnancy to treat relevant tapeworm infections. application in the first trimester needs to be critically assessed as tapeworm infections are generally not a great hazard to the mother or unborn child. after first trimester exposure, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. also found no evidence of a teratogenic potential after mothers had been treated in the first trimester (adam 2004a , paparone 1996 . in a study from uganda encompassing more than 1,000 pregnant women, treatment with praziquantel in the second and third trimester was not associated with an increase in adverse outcomes (ndibazza 2010) . the who (2002) recommends the use of praziquantel for schistosomiasis during pregnancy. pyrantel is a broad-spectrum anthelmintic that acts by inhibition of cholinesterase, causing spastic paralysis and subsequent death of the parasite. no teratogenicity has been reported in animal studies. pyrantel is poorly absorbed from the gastrointestinal tract. published experience on its use during pregnancy is not sufficient to determine risk. pyrvinium is effective against pinworms (enterobius). after oral administration it is hardly absorbed. therefore, it is unlikely to reach the fetus in relevant amounts. there are no reports of embryo-or fetotoxic effects. however, there has been no published experience with the use of pyrvinium during pregnancy. a danish cohort study based on prescription registers identified 1606 women redeeming a prescription for pyrvinium (449 during first trimester). the pregnancy outcome was not considered in this article (torp-pedersen 2012). diethylcarbamazine is used for the treatment of filiriasis and onchocercosis. no teratogenicity was reported in animal studies. no publications regarding its use during human pregnancies have been located. levamisole is used as anthelmintic and as an immunomodulator. a retrospective study with data from the hungarian malformation registry based on 14 subjects (four first trimester exposures), shows no evidence of an increased risk of malformations after use of levamisole (kazy 2004) . oxamniquine is used for the treatment of schistosomiasis. no experiences have been reported about its use during pregnancy. recommendation. praziquantel should be reserved for specific severe indications like schistosomiasis. usually for other indications better-established anthelmintics are available. after first trimester exposure a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. pyrantel should be avoided in pregnancy because better tested alternatives are available for all indications. after first trimester exposure a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. pyrvinium may be used during pregnancy. the standard agent of this group is acyclovir which is used against the varicella-zoster virus (vzv) and herpes simplex virus (hsv) type 1 and 2. the manufacturer's case collection contains over 1,000 women treated systemically with acyclovir during pregnancy, 756 of them during the first trimester; with no evidence of embryo-or fetotoxic risk (stone 2004) . a study of a danish registry with 1,561 women with prescriptions in the first trimester, showed no increased risk after acyclovir (pasternak 2010) . although these studies had some methodological weaknesses, the experiences argue against the risk of acyclovir in pregnancy. valacyclovir, the prodrug of acyclovir, is converted quickly and completely to acyclovir in the body. orally it is distinctly better resorbed than acyclovir of which only about 20% is resorbed. the manufacturer did not find an increased risk of malformation in 56 women who had received valacyclovir during pregnancy, 14 of these during the first trimester (glaxo wellcome 1997) . also, the above cited study of the danish registry did not show evidence of embryo-or fetotoxic risk in 299 pregnancies, in which the mother filled a prescription for valacyclovir during the first trimester (pasternak 2010) . ganciclovir and its prodrug valganciclovir are effective in cytomegalus virus infections (cmv). in animal experiment, teratogenic effects were only seen with plasma levels that were twice as high as those recommended in human therapy. there are a few case reports describing normal pregnancy outcome after the first trimester treatment during early pregnancy (pescovitz 1999) . puliyanda (2005) describes a successful oral treatment with ganciclovir for an intrauterine cmv infection after the 22nd week. these experiences are insufficient to evaluate the safety of ganciclovir in pregnancy. famciclovir is quickly converted after enteral resorption into the virostatic penciclovir. neou (2004) reported a newborn whose mother took 250 mg famciclovir daily in her fifth week. the boy who succumbed to a severe neonatal infection had a hypoplastic thymus, a mild stenosis of the pulmonary valve, an ostium secundum defect, and an enlarged liver with stenotic extrahepatic biliary ducts. a retrospective study of data from the danish birth registry contained 26 women who took oral famciclovir during the first trimester, and showed no increase in the malformation rate (pasternak 2010) . there is insufficient data about the use in pregnancy for brivudine, cidofovir, foscarnet, and fomivirsen. in animal experiments, small doses of foscarnet sodium trigger skeletal anomalies in rats and rabbits. no experience is reported for the combination therapy of dimepranol and inosine that is used to stimulate the immune system against viruses of the herpes group. recommendation. diethylcarbamazine, levamizole and oxamniquine should be avoided during pregnancy as better tested alternatives are available for most indications. after first trimester exposure a detailed ultrasound examination should be offered to ascertain the normal development of the fetus.  herpes medication for local use acyclovir, foscarnet, ganciclovir, idoxuridine, penciclovir, trifluridine, and tromantadine are locally applied in hsv infections. none of these agents has been suspected to give rise to teratogenic effects. acyclovir may be used in pregnancy systemically and is harmless in local application. in the above cited danish registry study 2,850 women had used acyclovir and 118 women penciclovir locally during the first trimester, and no increased malformation risk was noted (pasternak 2010) . the other agents lack studies about local application. docosanol is a newly approved agent for topical application in herpetic cold sores. the mechanism of action is unknown. there has been no experience about its use in pregnancy; however, a risk is unlikely with its minimal resoption. the local application of zinc sulfate and of patches containing hydrocolloid particles is harmless in pregnancy. antiviral drugs for hepatitis  antiviral drugs for hepatitis b nucleoside/nucleotide analogs and α-interferon (chapter 2.12) are used for the management of chronic hepatitis b. a general therapeutic recommendation cannot be made for pregnancy as data are inadequate. experience so far did not reveal serious signs of teratogenic or fetotoxic damage in humans. if there is a very active hepatitis b or cirrhosis, antiviral treatment might be considered. passive-active immunoprophylaxis of infants have reduced mother-to-child-transmissions. however, in high viremic mothers immunoprophylaxis might fail. no consensus has been reached if pregnant women who are hbsag positive, and highly viremic should be treated in the third trimester to prevent a perinatal transmission to the infant (e.g. pan 2012). for lamivudine and tenofovir see section 2.6.30. adefovir dipivoxil, the prodrug of adefovir, is an orally-administered nucleotide analog. no teratogenicity has been reported in animal studies. the antiretroviral pregnancy registry (2013) received reports of 48 births after a maternal adefovir dipivoxil regimen in the first trimester. no birth defects were observed in the infants. entecavir has shown teratogenic effects in animal studies where, in high doses, more vertebral and tail malformations occurred. of 55 recommendation. if an antiviral therapy is indicated for a severe maternal disease, or to protect the fetus from an intrauterine infection, acyclovir or valacyclovir should be used as the best evaluated medication whenever possible. the other antiviral agents are only indicated in infections where they have a therapeutic advantage over acyclovir. after the application of one of the less well examined drugs during first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. recommendation. where indicated, local remedies for herpes may be used during pregnancy. drying agents and patches for herpes are harmless. where possible, acyclovir should be preferred as the best evaluated antiviral drug. infants whose mothers were exposed to entecavir during first trimester, two babies were born with birth defects (no details available) (antiretroviral pregnancy registry 2013). one case report describes a healthy baby born after entecavir exposure for 32 days in the second trimester (kakogawa 2011) . telbivudine raised no suspicions for teratogenicity in animal experiments. among 86 pregnancies of women who received telbivudine before or in early pregnancy the abortion rate was 7.9%. fifty mothers delivered 52 infants. one pregnancy was terminated because of cleft lip and palate and one infant showed right ear accessories, no other birth defects were reported (liu 2013) . in the antiretroviral pregnancy registry (2013) no birth defects were observed in 10 infants after first trimester exposure to telbivudine. in a prospective study, 136 infants were born after maternal treatment with telbivudine in late pregnancy to prevent perinatal transmission. exposure took place from the twentieth to thirty-second gestational week until at least 1 month after delivery. there were no significant differences in infant outcomes compared to a control group. no serious adverse events were noted in the infants . there is an ongoing discussion as to whether telbivudine should be given to women with a high virus load during late pregnancy to prevent intrauterine transmission (review by deng 2012). the nucleoside analog ribavirin inhibits both dna-and rna-viruses, displaying a relatively broad antiviral spectrum experimentally. among other applications, it is used to treat respiratory syncytial virus (rsv) infections in infants, and, combined with α-interferon (chapter 2.12), against hepatitis c. ribavirin has teratogenic and mutagenic effects in animal experiments. nine women who were treated during the second half of pregnancy for severe measles delivered healthy infants (atmar 1992) . a woman treated for sars (severe acute respiratory syndrome) in the first trimester with ribavirin by injection for 3 days gave birth to a normal child (rezvani 2006) . in its pregnancy registry, the manufacturer noted eight women with ribavirin exposure in the first trimester, and 77 women with exposure within 6 months of the last menstrual period (roberts 2010) . the authors found no evidence of a teratogenic risk for humans. in summary, current data is insufficient for a risk assessment for ribavirin. an embryo-or fetotoxic risk is not apparent with the available case reports. the level of ribavirin is twice as high in seminal fluid as in sperm. there has been no increased risk of malformations after paternal ribavirin treatment and interferon in 20 pregnancies reported as case reports (review by hofer 2010), and 110 pregnancies of the ribavirin pregnancy registry (roberts 2010) . these numbers are inadequate to assess a possible risk after paternal exposure. the protease inhibitors boceprevir, simeprevir and telaprevir have been approved for the treatment of chronic hepatitis c. there are no experiences with their use in pregnancy. the same applies to sofosbuvira recently approved polymerase inhibitor for the treatment of chronic hepatitis c. amantadine enhances dopamine activity at the receptor and thus is also used as an antiparkinson drug. as an antiviral medication, it inhibits the membrane protein hampering the ability of the virus to enter the cell nucleus. because of rapid resistance and frequent neurologic side effects, it is not recommended any more as an antiviral agent. for amantadin in parkinson disease, see chapter 2.11. the neuraminidase inhibitors oseltamivir, peramivir and zanamivir are used to treat patients whose influenza requires therapy. oseltamivir has not shown teratogenic effects in animal studies. a prospective investigation at two japanese centers did not see an increase in malformations where 90 women had been treated in the first trimester (review by tanaka 2009 ). another study involving 137 exposed offspring, 18 of them in the first trimester, also did not find a higher risk (greer 2010) . the manufacturer, too, noticed no increased risk in 115 women who had used oseltamivir during pregnancy, 44 of these during the first 3 months (donner 2010) . one study with 81 pregnant women exposed to oseltamivir, 24 in the first trimester, found an increased risk of late transient hypoglycaemia compared to an unexposed control group. no other increased risks of adverse birth outcomes among the infants have been observed. one child had a ventricular septal defect. this was the only major malformation after exposure in the first trimester (svensson 2011) . another publication included 619 pregnant women exposed to oseltamivir, 159 of them in first trimester. the overall rate of major malformation after first trimester exposure was 1.3% (saito 2013) . in a french publication, a total of 337 mothers received at least one prescription of oseltamivir during pregnancy. one congenital heart defect was observed among 49 infants who were exposed during first trimester. no significant association between adverse fetal outcomes and exposure to oseltamivir during pregnancy could be found (beau 2014) . dunstan (2014) could also find no signs of embryo-or fetotoxic effects in 27 exposed pregnant women. no birth defects were observed in eight first trimester exposures. a population-based retrospective cohort study analyzed data from 1,237 women who received oseltamivir during pregnancy. compared to a control group, there were no associations between maternal use of oseltamivir with preterm birth and low apgar score. women who recommendation. ribavirin and the other antiviral agents discussed here should only be used during pregnancy when compellingly indicated. treatment during the first trimester is not a justification for a risk-based termination of pregnancy (chapter 1.15 ). in such a situation a detailed ultrasound examination should be offered to ascertain normal fetal development. pregnancy took oseltamivir during pregnancy were less likely to have a small for gestational age infant. however, birth defects and time of exposure were not mentioned (xie 2013) . two studies looked into the pharmacokinetics of oseltamivir and its active metabolite oseltamivir carboxylate during gestation. greer (2011) compared the pharmacokinetics of 10 pregnant women in each group during the last trimester and found no significant differences. beigi (2011) examined the pharmacokinetcs in 16 pregnant women (average gestational age 24.6 weeks) in comparison to 23 nonpregnant women, and found the pregnant group to have lower oseltamivir carboxylate level. however, it remains unclear if the dose needs to be adjusted during pregnancy. zanamivir is applied by inhalation and very little is resorbed. no teratogenicity was found in animal experiments. a case series study from japan reported 50 infants born after intrauterine zanamivir exposure, 15 of them were exposed in the first trimester. no malformations have been observed (saito 2013) . a prospective surveillance study did not provide a case that use of zanamivir in pregnancy is associated with an increased risk of adverse pregnancy outcomes among 180 women exposed to zanamivir during pregnancy. no major malformations were reported in 37 zanamivir first trimester exposures (dunstan 2014) . experience and the presence of low systemic concentrations, make it unlikely that there is an increased embryo-or fetotoxic risk. experience during pregnancy with peramivir is insufficient for a risk assessment. the aim of antiretroviral therapy (art) during pregnancy is the prevention of a vertical transmission of the human immunodeficiency virus (hiv) from mother to child, and also the optimal management of the hiv-infected mother, whereby unwanted side effects are to be kept at a minimum for her and the child. art in pregnancy has become an integral part in the prophylaxis of hiv transmission after data revealed the protective effect of perinatal prophylaxis, with the nucleoside analog reverse transcriptase inhibitor (nrti) zidovudine that could prevent a possible vertical transmission during the last trimester and labor (connor 1994) . national and international guidelines recommend a standard therapy for both nonpregnant and pregnant hiv-infected women take a combination of at least three antiretroviral medications (eacs 2013 , oarac 2012 , who 2010c . this highly active antiretroviral therapy (haart) typically consists of two nrtis and either a protease inhibitor (pi), or a non-nucleoside analog reverse transcriptase inhibitor (nnrti). the intention is that the suppression of the plasma hiv load (hiv-rna) should be as close to <50 copies/ml at least by the end of the pregnancy. when an effective haart is applied during pregnancy and lactation, the hiv rate of transmission can be decreased from its former levels of recommendation. if indicated, neuraminidase inhibitors oseltamivir and tanamivir may be used in pregnancy. peramivir should be avoided. amantadine is no longer recommended for the treatment of influenza. when used during the first trimester, a detailed ultrasound examination should be offered to ascertain normal fetal development. 20-30% to <1% (townsend 2008 , warszawski 2008 . the decision of what regimen to use is already complicated in nonpregnant patients, but more so in pregnancy. how to balance individual needs and risks should be considered, especially in view of the timing of the start of treatment, a possible interruption of therapy during the first trimester in women already under treatment, and the selection of appropriate antiretroviral medications. the risks from intrauterine exposure to combinations of antiretroviral agents are difficult to assess, as data are limited concerning the pharmakinetics and the developmental toxicity for most of the drugs. there is no data about the long-term toxicity of the exposure to intrauterine retroviral substances. information about the safety of retroviral drugs in pregnancy are limited to experiments in animals, single case reports, a few clinical studies, and analyses of registries such as the antiretroviral pregnancy registry (2013) in the usa that contains most of the information about the safety of antiviral substances in pregnancy. overview of the antiretroviral medications , darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir. 4 . entry inhibitors: enfuvirtide and maraviroc. 5. integrase inhibitors: raltegravir, dolutegravir and elvitegravir. data currently available do not allow for a summarizing differentiated risk analysis for antiretroviral medications in pregnancy. with the exception of efavirenz, there have been no serious signs of teratogenic or fetotoxic damages in humans (e.g. watts 2011 , ecs 2003 . prospectively documented pregnancies do not demonstrate a higher risk of malformations and, like retrospective case reports, fail to reveal any distinct pattern of anomalies. when antiretroviral agents are used in the first trimester, the embryotoxic risk appears to be generally small (phiri 2014 , floridia 2013 , antiretroviral pregnancy registry 2010 , joao 2010 . nevertheless, substances that might be embryotoxic should be eschewed in early pregnancy. common side effects in children treated in utero or after birth with zidovudine or antiretroviral combinations consist of hematologic problems, especially anemias and neutropenias (dryden-peterson 2011 , feiterna-sperling 2007 , le chenadec 2003 . it is being debated if antiretroviral treatment with or without protease inhibitors favors prematurity (chen 2012 , patel 2010 , kourtis 2007 , cotter 2006 , tuomala 2005 . the maternal risks of therapy are discussed with the specific medications. the medical treatment of hiv infection during pregnancy is a prime example for the need to sometimes utilize insufficiently tested medications -because of the acute danger for mother and child. in individual cases it needs to be critically assessed if an ongoing or maternally indicated treatment is absolutely necessary during the time of embryogenesis, or if it can be temporarily suspended. data from clinical studies during pregnancy in women are available for abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, and zidovudine. with the exception of didanosine, the nrtis showed comparable levels in the maternal serum, and the umbilical cord blood suggested an easy placental passage (pacifici 2005) . having an affinity to mitochondrial γ-dna polymerases, nrtis can induce mitochondrial dysfunction. the greatest risk for mitochondrial toxicity is exhibited in vitro for didanosine, stavudine, and zidovudine. the question if a perinatal nrti exposure could lead to mitochondrial problems in children is currently under discussion; a final consensus has not been reached (benhammou 2007 , blanche 1999 . lamivudine and zidovudine are the nrtis that should be preferred during pregnancy because of extensive experience. abacavir, emtricitabine and tenofovir are alternative nrtis which also might be used. didanosine and stavudine should only be used in special circumstances (oarac 2012). abacavir can lead to skeletal anomalies when given to rats at a high dosage. there is no evidence of teratogenicity in humans. abacavir readily crosses the placenta (chappuy 2004) . data from the antiretroviral pregnancy registry (2013) with 27 birth defects in 905 cases, indicate a malformation rate of 3.0% after exposure during the first trimester, similarly as seen in the general population of the usa. in animal experiments didanosine given at high doses did not show teratogenic effects. didanosine crosses the placenta only in limited recommendation. antiretroviral medications may be used in pregnancy. specific risks for the prophylaxis of transmission and the therapy of maternal hiv infection need to be observed. the choice of medication and the timing of treatment have to be decided on an individual basis. when choosing medications it should be noted that some of the retroviral substances should be avoided during pregnancy, if possible. this concerns efavirenz (teratogenic effects) and the combination stavudine/didanosine (lactic acidosis). for newer medications such as maraviroc, raltegravir and etravirine, few or no data are available concerning their use in pregnancy. caution is called for when nevirapine is used in women with cd4 cell counts of <250/mm mm 3 (hepatotoxicity). if nevirapine is used during pregnancy, transaminases need to be checked regularly, especially during the first 18 weeks of treatment; also, clinical symptoms are to be watched. the short-term use of nevirapine for transmission prophylaxis does not seem to carry a similar risk. when exposure occurs during the first trimester, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. it is recommended that the pregnant patient is cared for in a specialized center. physicians should report pregnancies involving the use of hiv medications shortly after diagnosis to the antiviral pregnancy registry (www.apregistry.com). amounts (wang 1999) . the data of the antiretroviral pregnancy registry (2013) show a slightly increased malformation rate after first trimester exposure at 4.8% (20 of 416 births), in comparison to 2.7% in the general us population. however, no distinct pattern of birth defects has been discovered. in a study where 14 hiv infected women were treated at 26-36 weeks with didanosine, neither maternal nor neonatal side effects were noted (wang 1999) . cases of lethal lactic acidosis have been described in pregnant women treated with a combination of stavudine and didanosine (mandelbrot 2003 , sarner 2002 . due to the risk of fatal lactic acidosis, combination treatment with didanosine and stavudine should only be used in cases where no alternatives are available (bristol-myers squibb 2001) . emtricitabine has not shown evidence of teratogenicity in animal experiments or in humans. it crosses the placenta readily (stek 2012 , hirt 2009b ). among cases of first trimester exposures reported to the antiretroviral pregnancy registry (2013), the prevalence of birth defects was 2.4% (34 of 1,400 births), similar to the rate in the general us population. lamivudine, one of the best evaluated nrtis, is also approved for the treatment of chronic hepatitis b. levels measured in the umbilical cord blood correspond to those of the mother. data from the antiretroviral pregnancy registry (2013) indicate an unsuspicious malformation rate of 3.1% (136 of 4,360 births). a larger study to prevent perinatal transmission was conducted in france where 445 pregnant women received zidovudine and lamivudine after gestational week 31, and their newborns were also given the combination for 6 weeks ( mandelbrot 2001) . in this study newborns displayed significant side effects that included lethal mitochondriopathies. however, lamivudine and zidovudine are medications that are preferred in pregnancy because of extensive experience. there is no evidence that stavudine leads to teratogenic effects in animal experiments or humans. stavudine crosses the placenta easily ( chappuy 2004) . the malformation rate after exposure in the first trimester is 2.6% (21 of 805 births) according to data from the antiretroviral pregnancy registry (2013), thus similar as in the general us population (2.7%). good tolerance of a staduvine-lamivudine combination has been described in a small phase i/ii study with 14 mother-child pairs (wade 2004) . cases of lethal lactic acidosis have been described in pregnant women treated with a combination of stavudine and didanosine (mandelbrot 2003 , sarner 2002 . due to the risk of a fatal lactic acidosis, combination treatment with didanosine and stavudine should only be used in cases where no alternatives are available (bristol-myers squibb 2001). in animal experiments, offspring of monkeys that received high doses of tenofovir have a decreased fetal growth rate and diminished fetal bone density (tarantal 2002) . during pregnancy tenofovir crosses the placenta easily (flynn 2011 , hirt 2009a . there is no evidence that tenofovir is teratogenic in humans. according to the data of the antiretroviral pregnancy registry (2013) the malformation rate after exposure during the first trimester is 2.3% (46 of 1,982 births), similar to the 2.7 % rate in the general us population. in clinical studies hiv patients, primarily children, displayed decreased bone density when treated with tenofovir. the clinical significance of these findings is still unclear. one study did not reveal any risk for adverse effects of in utero tenofovir exposure in 141 pregnant women (gibb 2012 ). however, tenofovir should be used with caution during pregnancy, because of the risk of fetal bone changes and the paucity of other data about its pregnancy-related risks. zidovudine, also known as azidothymidine (azt), is the oldest antiviral drug used for antiretroviral therapy. it readily crosses the placenta. in rats, maternal toxic doses lead to an increased malformation rate during organogenesis, an effect not seen with lower doses. there are no signs of teratogenicity in humans. according to data from the antiretroviral pregnancy registry (2013) the malformation rate of 3.2% (129 of 4,000 births) was not significant higher than that of the general us population. the application of zidovudine has been well studied in pregnancy and is considered to be safe in regard to shortterm and medium-term toxicities. a common side effect, when zidovudine is used in the perinatal period, is a transient anemia in newborns (sperling 1998 , connor 1994 . a follow-up study of 234 children who had been exposed to zidovudine in utero did not display any physical, immunological, or cognitive anomalies. the median age of children at the time of last follow-up was 4.2 years (range, 3.2-5.6 years) (culnane 1999) . also, there was no evidence of an increased risk for neoplasia in more than 700 children after pre-and perinatal exposure (culnane 1999 , hanson 1999 . there are no data regarding long-term toxicity, especially for cancerogenicity. data from clinical studies about the safety in human pregnancy for nnrt is are limited. nevirapine is the agent that should be preferred if a nnrti is required during pregnancy. efavirenz might be used in special circumstances. for etravirine and rilpivirine the data are insufficient to recommend use during pregnancy (oarac 2012). delavirdine is not recommended as part of an initial therapy. delavirdine caused an increased incidence of ventricular septal defects in rats. experience in humans is limited to 11 births after first trimester exposure reported to the antiretroviral pregnancy registry (2013). although no birth defects have been observed, these data allow no differentiated risk analysis. most guidelines do not recommend delavirdine as a part of antiretroviral regimens for initial treatment of hiv infection because of inferior efficacy. in animal experiments efavirenz showed evidence of teratogenicity. three of 20 prenatally exposed cynomolgus monkeys showed malformations when plasma levels were similar to the therapeutic levels in humans. anencephaly with unilateral anophthalmia was observed in one fetus, microphthalmia in another, and cleft palate in a third. there are case reports in humans about neural tube defects in children whose mothers had received efavirenz during the first trimester (de santis 2002 , fundaro 2002 . according to the data of the antiretroviral pregnancy registry (2013) the malformation rate of 2.3% (18 of 766 births) after first trimester exposure is comparable to the background rate of 2.7% in the general us population. the 18 birth defects included one infant with myelomeningocele. another child was born with anophthalmia, severe facial cleft and amniotic banding. in total, the antiretrorviral pregnancy register received six retrospective reports of neural tube defects; four of them were exposed to efavirenz. a meta-analysis, including nine prospective studies together with 1,132 live births, did not detect an increased risk of overall birth defects after exposure to an efavirenz-containing regimen during the first trimester. including retrospective studies, one neural tube defect was reported in 1,256 live births (ford 2010 ). an update of this meta-analysis which included 181 additional subjects had similar results (ford 2011) . in contrast to these reassuring findings, another study analyzes data of 1,112 infants born between 2002 and 2007. a significantly increased risk of congenital anomalies after exposure to efavirenz during first trimester was observed. six of 47 infants with first trimester exposure to efavirenz had congenital anomalies (adj.or 2.84, 95%; ci: 1.13-7.16) (knapp 2012) . however, the six observed major and minor defects (patent foramen ovale, gastroschisis, postaxial polydactyly, arnold-chiari malformation, talipes equinovarus, plagiocephaly), do not present a distinct pattern. with the available published experience, the british hiv association guidelines panels concluded that there are insufficient data to support the former position and furthermore recommend that efavirenz can be both continued and commenced in pregnancy (taylor 2012) . however, the united state guidelines are more restrictive. they recommend that an efavirenz-based regimen may be continued in women who present for antenatal care in the first trimester, provided the regimen produces virologic suppression (oarac 2012). animal experiments have not shown that etravirine is teratogenic. experience in pregnancy is limited to case reports (jaworsky 2010 , furco 2009 ). according to the data of the antiretroviral pregnancy registry (2013) no birth defects were reported in 39 infants born after first trimester pregnancy exposure to etravirine. experiences are insufficient to analyze a possible risk in pregnancy. there is no evidence in animal experiments or human experience that nevirapine is teratogenic. nevirapine crosses the placenta easily and attains levels in the neonate that correspond to those of the mother ( benaboud 2011 , mirochnick 1998 . according to the data of the antiretroviral pregnancy registry (2013) the malformation rate after first trimester exposure is 2.9% (31 of 1,061 births), which is no higher than that of the general us population. studies indicate that viral transmission is blocked when 200 mg p.o. nevirapine is given to the mother at the beginning of labor, and the newborn receives a single dose of 2 mg/kg 48 to 72 hours after delivery (guay 1999) . there is a high risk of developing viral resistance even after a single dose (low resistance barrier and long half-life of nevirapine), thus nevirapine should only be administered in a combination regimen. reports have been published describing single cases of liver toxicity in pregnant women who took nevirapine (e.g. knudtson 2003 ). this event is often rash-associated and potentially fatal. liver toxicity is primarily observed in patients with higher cd4 cell counts (>250/mm 3 ); in these patients the risk of symptomatic hepatic events is twelve times greater than in women with lower cd4 cell counts (<250/mm 3 ). studies indicate that pregnancy per se is a risk factor for liver toxicity. pregnant patients using haart that includes nevirapin have no higher risk of hepatotoxicity than those who use haart without nevirapine (ouyang 2010 , ouyang 2009 ). these data suggest that the risk of liver toxicity of nevirapine is similar in pregnant and nonpregnant patients. however, if nevirapine is used in pregnancy, physicians should be aware of hepatotoxicity. animal experiments failed to show that rilpivirine is teratogenic. in the antiretroviral pregnancy registry (2013) no birth defects were reported in 31 infants born after first trimester exposure to rilpivirine. one publication describes two healthy infants after rilpivirine exposure during pregnancy (colbers 2014) . experiences are insufficient to analyze a possible risk in pregnancy. pis are being used increasingly in pregnancy. they are recommended in regimens combined with two nrti drugs. pi therapy can lead to the disturbance of glucose tolerance and even to the manifestation or exacerbation of diabetes mellitus. it remains unclear if pregnancy itself increases the risk even further. generally, pis pass the placenta poorly ( gingelmaier 2006 , marzolini 2002 , mirochnick 2002 . therefore, fetal toxicity would seem to be unlikely. lopinavir/ritonavir and atazanavir with low-dose ritonavir boosting are the preferred pis during pregnancy. alternative pis include ritonavir-boosted saquinavir and darunavir. indinavir and nelfinavir should only be used in special circumstances. data is too limited to recommend the routine use of fosamprenavir and tipranavir in pregnant women (oarac 2012). atazanavir has not shown evidence of teratogenicity in animal experiments or human experience. according to the data of the antiretroviral pregnancy registry (2013), the malformation rate of 2.2% (19 of 878 births) after first trimester exposure is comparable to the rate of 2.7% in the general us population. a number of studies are available, including pharmacokinetic evaluations in pregnant women using haart with atazanavir , ripamonti 2007 . some experts recommend an increased dose in late pregnancy. the umbilical cord blood of neonates shows atazanavir levels of 13-16% of those seen in the maternal serum. atazanavir inhibits the uridin glucuronosyl transferase that metabolizes indirect bilirubin. thus, as a common side effect, atazanavir treatment may lead to higher indirect bilirubin levels. while case numbers are relatively small, investigations showed that neonates of atazanavir-treated mothers did not show pathological elevations of indirect bilirubin. , ripamonti 2007 ). darunavir did not demonstrate evidence of teratogenicity in animal experiments. some case reports demonstrated a limited placental transfer. like with other pis a reduction in plasma levels has been observed in late pregnancy (pinnetti 2010) . in the antiretroviral pregnancy registry (2013) five birth defects were reported in 212 infants born after first trimester exposure to rilpivirine (prevalence 2.4%). few experiences about its use in pregnancy are available (e.g. jaworsky 2010, ivanovic 2010). these data are insufficient for a differentiated risk assessment. in animal experiments no evidence was found that fosamprenavir leads to teratogenicity. human data about its use in pregnancy are very limited. transplacental passage analyzed in seven cases was relatively high compared to other pis. the authors detected a median ratio of 0.27 of cord blood to maternal amprenavir level (the active metabolite of fosamprenavir) (cespedes 2013) . one publication did not report adverse effects in nine infants after intrauterine exposure to fosamprenavir (martorell 2010) . two birth defects among 102 births were reported to the antiretroviral pregnancy registry (2013) after first trimester exposure to fosamprenavir. these data are insufficient for a differentiated risk assessment. evidence for teratogenicity is not evident for indinavir in animal experiments or human reports. little of indinavir crosses the placenta ( mirochnick 2002) . according to the data of the antiretroviral pregnancy pregnancy registry (2013) the malformation rate of 2.4% (7 of 289 births) after first trimester exposure is comparable to that in the general us population. these data are insufficient for a differentiated risk assessment. there is a theoretical concern that physiologic hyperbilirubinemia might be exacerbated due to indinavir. lopinavir is used in conjunction with its pharmacological booster ritonavir. in animal experiments with high doses of lopinavir, rats displayed evidence of embryotoxicity with an increased rate of miscarriages, less fetal viability, lower fetal weight, and skeletal changes. these problems were not apparent in rabbits. there is no evidence of teratogenicity in humans. like most pis, lopinavir/ritonavir crosses the placenta poorly (gingelmaier 2006) . according to the data of the antiretroviral pregnancy registry (2013) the malformation rate is 2.3% (26 of 1,125 births) after first trimester exposure, and thus not increased in comparison to the general us population. studies with hiv-infected pregnant women indicate that the treatment with lopinavir/ritonavir is well tolerated. pharmacokinetic investigations show lower plasma levels, primarilty in the last trimester (best 2010) . it is unclear if pregnant women require a higher dose or just a continuation of the pi standard therapy. a report of 50 infants who received lopinavir/ ritonavir after birth observed an association with transient adrenal dysfunction in the infants (simon 2011) . a systematic review about the safety and efficacy of lopinavir/ritonavir during pregnancy included nine studies involving 2,675 pregnant women. no concerns with the use of these agents were suggested (pasley 2013 ). nelfinavir did not display evidence of teratogenicity in animal experiments. according to the data of the antiretroviral pregnancy registry (2013), the malformation rate is 3.9% (47 of 1,211 births) after first trimester exposure which is a modest evaluation compared to the general population (2.7%). no distinct pattern of birth defects defects has been discovered. in studies with hiv-infected pregnant women it was noted that a small amount crosses the placenta (bryson 2008 , mirochnick 2002 . when nelfinavir is used as an unboosted pi in pregnant women who need treatment for hiv, it is inferior to newer, low-dose ritonavir boosted pis, but is useful as an alternative pi in combination with 2 nrtis for the prophylaxis of hiv transmission. however, nevirapine should only be used under special circumstances during pregnancy. ritonavir should be used in combination with other pis as a low-dose booster to increase levels of a second pi. only a small amount crosses the placenta (mirochnick 2002) . there is no evidence that ritonavir is teratogenic in animal experiments or humans. according to the data of the antiretroviral pregnancy registry (2013) the malformation rate is 2.3% (52 of 2,260 births) after first trimester exposure, thus similar to the general us population. saquinavir has not demonstrated evidence of teratogenicity in animal experiments or human experience. like with other pis only small amounts of the drug cross the placenta (mirochnick 2002) . pharmacokinetic studies indicate that the newer tablet formulation that has replaced the former capsule formulation, leads to plasma concentrations similar to nonpregnant patients (van der lugt 2009). thus, it is not necessary to adjust the doses in pregnancy. seven birth defects among 182 first trimester exposures were reported to the antiretroviral pregnancy registry (2013). these data are insufficient for a differentiated risk assessment. tipranavir shows no teratogenicity in animal experiments. there are no data about its ability to cross the placenta. aside from single case reports of pregnant patients with multiple resistances (weizsaecker 2011 , wensing 2006 , there are no other data about the use of tipranavir in pregnancy. no birth defects were reported to the antiretroviral pregnancy registry (2013) among four first trimester exposures to tipranavir. experiences are insufficient to analyze a possible risk in pregnancy. entry inhibitors are antiretroviral agents that inhibit viral binding or fusion of hiv to the cell, either by inhibition of the fusion of the viral capsule with the cell membrane or by blocking cd4-or co-receptors. data about the use of enfuvirtide or maravorioc during pregnancy are insufficient to recommend their use during pregnancy (oarac 2012). in animal experiments no evidence was observed that enfuvirtide is teratogenic. a number of single case reports suggest that enfuvirtide apparently does not cross the placenta (weizsaecker 2011 , brennan-benson 2006 . according to the data of the antiretroviral pregnancy registry (2013) no birth defects have been reported among 20 first trimester exposure to enfuvirtide. thus, it can be assumed that the risk of fetal toxicity is likely to be small. enfuvirtide may be used in pregnant women with multi-resistent hiv in combination with other potent agents as a therapeutic option, but current experience in pregnancy is very limited. maraviroc is a ccr5 inhibitor that is used to treat pretreated hivinfected adults in combination with other antiretroviral medications, when exclusively ccr5-tropic hiv type-1 have been proven to be present. animal experiments using rats and rabbits did not show evidence of teratogenicity for maraviroc. there are no data indicating to what degree pregnancy maraviroc crosses the placenta. while there has been no indication that the use of maraviroc leads to a higher rate of malignancy, a theoretical concern remains based on the method of its action. maraviroc should only be used when the benefit justifies the potential fetal risk. there is a lack of data about its application in pregnancy. among 13 cases with first trimester exposure reported to the antiretroviral pregnancy registry (2013) no birth defects have been observed. integrase inhibitors block integrase, a hiv-coded enzyme, and thereby hiv replication. the use of raltegravir during pregnancy can be considered in special circumstances when preferred and alternative agents cannot be used (oarac 2012) . there is insufficient data for the new integrase inhibitors dolutegravir and elvitegravir. in animal experiments no evidence was seen that dolutegravir is teratogenic. placental transfer has been described in animals. no experiences have been reported about its use during human pregnancy. there are also no reports about the use of dolutegravir to the antiretroviral pregnancy registry (2013). elvitegravir is combined with colbicistat which has no known antiretroviral activity. colbicistat is a pharmacokinetic enhancer which inhibits enzymes that metabolize elvitegravir. animal studies of elvitegravir have shown no evidence of teratogenicity. only one report about the use of elvitagravir during the first trimester has been reported to the antiretroviral pregnancy registry (2013). no birth defects were observed in this case. development studies in rats and rabbits did not show raltegravir to be teratogenic. however, there was a slightly increased incidence of supernumerary ribs in the offspring of rats that had received raltegravir at doses about 4.4 times higher than those recommended in human treatment. potential human risks are not known at this time. according to the few data about its use during pregnancy, raltegravir crosses the placenta well (mckeown 2010). in a case series of five women raltegravir was well tolerated (taylor 2011) . three birth defects were observed among 141 pregnant women with first trimester exposures reported to the antiretroviral pregnancy registry (2013). because experience is increasing, the united states guidelines recommend allowing a regimen including raltegravir in special circumstances, when preferred and alternative agents cannot be used (oarac 2012) . however, the data on the use of raltegravir during pregnancy allow no differentiated risk analysis. more than 30 years ago animal experiments demonstrated that an increase in the body temperature can cause malformations (review by graham 2005 , edwards 1995 , miller 2013 . this problem has also been discussed for humans. neural tube defects, in particular (suarez 2004 , shaw 1998 ), but also kidney, heart and abdominal wall defects (abe 2003 , chambers 1998 , have been reported in association with febrile infections in early pregnancy, even though the overall malformation risk is absent or only mildly increased. moretti (2005) performed a meta-analysis about the risk of neural tube defects and hyperthermia. they included 15 studies with 1,719 cases and found a significant correlation (or 1.9; 95%; ci 1.61-2.29), both in the nine case-control studies and the six cohort studies. lowering fever in pregnant women seems to reduce the risk (suarez 2004) . it has been debated if the use of sauna, electric blankets, or other factors that bring about a short-term increase in body temperature could lead to similar effects as high fever (suarez 2004) . in finland, where this issue had been investigated repeatedly, visits to saunas occur frequently during pregnancy and is considered safe. the use of electric blankets and heated water beds has not shown, in other investigations, that they are linked to an increased malformation risk. one study observed that children between the ages of 5 and 12 had more frequent emotional and cognitive deficits where there were reports about high fever during the second and third trimester (dombrowski 2003) . in summary, it appears that there is a slightly higher risk of malformations when high fever (>39°c and >24 hours) occurs, especially during the first 4 weeks after conception. during long-distance travel and flights during pregnancy, a number of potential risks need to be considered: □ prevention of infections (malaria prophylaxis, see section 2.6.16.; vaccinations, see chapter 2.7). □ the risk of other infections (fever, fluid loss), and required therapy. □ during long-distance flights: -risks of thrombosis -ionizing cosmic radiation recommendation. if there is an infection with high fever, especially during early pregnancy, the fever should be controlled with acetaminophen (paracetamol) or ibuprofen (chapter 2.1). ibuprofen should not be taken after 28 gestational weeks. non pharmacological measures of fever control such as cool wrappings, and sufficient fluid intake should also be considered. in cases of high fever episodes in early pregnancy, a detailed ultrasound examination should be offered to ascertain the normal development of the fetus. a fever episode does not justify a risk-based termination of pregnancy (chapter 1.15). visits to a sauna should be limited to less than 10 minutes, and hot or long baths need to be avoided as well as other sources that can overheat the body. -decrease of the partial oxygen pressure equivalent to an altitude of 2,500 m -dry air. □ physical and psychological stress. specific developmental anomalies have not been found in pregnant women undergoing vaccinations or recommended malaria prophylaxis, nor were such problems seen as a result of long-distance flights. however, it needs to be noted that the stress of a long-distance trip, especially in predisposed women, might increase the risk of miscarriage. also, aside from typical infectious diseases, "common" infections may be more prevalent due to altered hygienic standards in the destination country. the accompanying dehydration, fever, or other complications may also endanger the fetus. the dose of cosmic radiation on a long-distance flight varies markedlydepending on solar activity. yet, according to current knowledge, no doses are reached that are high enough to lead to an increased risk of malformations. pharmacokinetics of quinine and its metabolites in pregnant sudanese women with uncomplicated plasmodium falciparum malaria maternal febrile illnesses, medication use, and the risk of congenital renal anomalies is praziquantel therapy safe during pregnancy? safety of artemisinins during early pregnancy, assessed in 62 sudanese women quinine for chloroquine-resistant falciparum malaria in pregnant sudanese women in the first trimester pharmacokinetics of piperaquine in pregnant women in sudan with uncomplicated plasmodium falciparum malaria nitrofurantoin-induced acute liver damage in pregnancy multiple malformation syndrome following fluconazole use in pregnancy: report of an additional patient successful treatment with ketoconazole of cushing's syndrome in pregnancy clarithromycin in early pregnancy and the risk of miscarriage and malformation: a register based nationwide cohort study trimethoprim use in early pregnancy and the risk of miscarriage: a register-based nationwide cohort study trimethoprim use prior to pregnancy and the risk of congenital malformation: a register-based nationwide cohort study randomized clinical trial of metronidazole plus erythromycin to prevent spontaneous preterm delivery in fetal fibronectin-positive women antiretroviral pregnancy registry international interim report for 1 complications of measles during pregnancy fetal safety of macrolides pregnancy outcome after gestational exposure to the new macrolides: a prospective multi-center observational study pregnancy outcome after in utero exposure to itraconazole: a prospective cohort study the safety of quinolones -a meta-analysis of pregnancy outcomes the outcomes of pregnancy in women exposed to the new macrolides in the first trimester: a prospective, multicentre, observational study impact of malaria at the end of pregnancy on infant mortality and morbidity is single-dose fosfomycin trometamol a good alternative for asymptomatic bacteriuria in the second trimesterof pregnancy? cancer after exposure to metronidazole safety of oseltamivir during pregnancy: a comparative study using the efemeris database pharmacokinetics of oseltamivir among pregnant and nonpregnant women the safety of nitrofurantoin during the first trimester of pregnancy: meta-analysis population pharmacokinetics of nevirapine in hiv-1-infected pregnant women and their neonates clinical mitochondrial dysfunction in uninfected children born to hiv-infected mothers following perinatal exposure to nucleoside analogues first-trimester exposure to amoxycillin/ clavulanic acid: a prospective, controlled study safety of the new quinolones in pregnancy first trimester exposure to cefuroxime: a prospective cohort study cushing's syndrome in pregnancy treated by ketoconazole: case report and review of the literature lopinavir tablet pharmacokinetics with an increased dose during pregnancy antileprosy drugs, pregnancy and fetal outcome persistent mitochondrial dysfunction and perinatal exposure to antiretroviral nucleoside analogues american thoracic society/centers for disease control and prevention/infectious diseases society of america: treatment of tuberculosis successful outcome of pregnancy in a patient with cushing's disease under treatment with ketoconazole during the first trimester of gestation drug treatment for tuberculosis during pregnancy: safety considerations randomized trial of artesunate and mefloquine in comparison with quinine sulfate to treat p. falciparum malaria pregnant women enfurvitide prevents vertical transmission of multidrug-resistant hiv-1 in pregnancy but does not cross the placenta healthcare provider important drug warning letter hemolytic anemia in a newborn after maternal treatment with nitrofurantoin at the end of pregnancy pharmacokinetics and safety of nelfinavir when used in combination with zidovudine and lamivudine in hiv-infected pregnant women: pediatric fluconazole treatment for vulvovaginal candidiasis during pregnancy steady-state pharmacokinetics, cord blood concentrations, and safety of ritonavir-boosted fosamprenavir in pregnancy maternal fever and birth outcome: a prospective study maternal-fetal transfer and amniotic fluid accumulation of nucleoside analogue reverse transcriptase inhibitors in human immunodeficiency virus-infected pregnant women highly active antiretroviral therapy and adverse birth outcomes among hiv-infected women in botswana absence of any adverse effect of inadvertent ivermectin treatment during pregnancy fetal outcome after exposure to antihelminthics albendazole and flubendazole during early pregnancy {abstract} fetal outcome after exposure to flubendazole during pregnancy {abstract} antenatal anthelmintic treatment, birthweight, and infant survival in rural nepal fetal outcome following roxithromycin exposure in early pregnancy embryotoxicity of the artemisinin antimalarials and potential consequences for use in women in the first trimester pharmacokinetics, safety and transplacental passage of rilpivirine in pregnancy: two cases reduction of maternal-infant transmission of human immunodeficiency virus type 1 with zidovudine treatment. pediatric aids clinical trials group protocol 076 study group streptomycin in pregnancy: effect on the foetal ear prenatal prescription of macrolide antibiotics and infantile hypertrophic pyloric stenosis antibiotics potentially used in response to bioterrorism and the risk of major congenital malformations cryptococcal meningitis in hiv negative pregnant women: case report and review of literature is antiretroviral therapy during pregnancy associated with an increased risk of preterm delivery, low birth weight, or stillbirth? mebendazole and albendazole treatment of geohelminth infections in children and pregnant women antibacterial medication use during pregnancy and risk of birth defects: national birth defects prevention study lack of long-term effects of in utero exposure to zidovudine among uninfected children born to hiv-infected women. pediatric aids clinical trials group protocol 219/076 teams a case-control analysis of the teratogenic effects of co-trimoxazole teratogenic study of doxycycline a population based case-control teratologic study of oral metronidazole treatment during pregnancy a population-based case-control teratologic study of oral erythromycin treatment during pregnancy no teratogenic effect after clotrimazole therapy during pregnancy oral phenoxymethylpenicillin treatment during pregnancy. results of a population-based hungarian case-control study a case-control teratological study of spiramycin, roxithromycin, oleandomycin and josamycin a teratological study of lincosamides a population-based case-control teratologic study of oral oxytetracycline treatment during pregnancy a teratological study of aminoglycoside antibiotic treatment during pregnancy a population-based case-control teratologic study of oral chloramphenicol treatment during pregnancy augmentin treatment during pregnancy and the prevalence of congenital abnormalities: a population-based case-control teratologic study use of cephalosporins during pregnancy and in the presence of congenital abnormalities: a population-based, case-control study the teratogenic risk of trimethoprim-sulfonamides: a population based case-control study nitrofurantoin and congenital abnormalities a population-based case-control study of the safety of oral anti-tuberculosis drug treatment during pregnancy a population-based case-control teratological study of vaginal econazole treatment during pregnancy a population-based case-control teratological study of oral nystatin treatment during pregnancy a case-control teratological study of vaginal natamycin treatment during pregnancy preterm birth reduction after clotrimazole treatment during pregnancy population-based case-control teratologic study of topical miconazole a population-based case-control study of oral griseofulvin treatment during pregnancy tolnaftate spray treatment during pregnancy effect of mebendazole therapy during pregnancy on birth outcome periconceptional exposure to efavirenz and neural tube defects first-trimester itraconazole exposure and pregnancy outcome: a prospective cohort study of women contacting teratology information services in italy use of amphotericin b during pregnancy: case report and review the safety of the combination artesunate and pyrimethamine-sulfadoxine given during pregnancy birth outcome of 1886 pregnancies after exposure to phenoxymethylpenicillin in utero the effects of telbivudine in late pregnancy to prevent intrauterine transmission of the hepatitis b virus: a systematic review and meta-analysis pregnancy outcome after gestational exposure to mebendazole: a prospective controlled cohort study pregnancy outcome after gestational exposure to metronidazole: a prospective controlled cohort study genotoxicity and carcinogenicity of metronidazole association between maternal fever and psychological/behavior outcomes: a hypothesis safety of oseltamivir in pregnancy: a review of preclinical and clinical data postmarketing surveillance of medications and pregnancy outcomes: clarithromycin and birth malformations treatment of multidrug-resistant tuberculosis during pregnancy: long-term follow-up of 6 children with intrauterine exposure to second-line agents increased risk of severe infant anemia after exposure to maternal haart pregnancy outcome following maternal use of zanamivir or oseltamivir during the 2009 influenza a/h1n1 pandemic: a national prospective surveillance study europeans guidelines for treatment of hivinfected adults in europe hyperthermia and birth defects a retrospective review of ampicillin-sulbactam and amoxicillin + clavulanate vs cefazolin/cephalexin and erythromycin in the setting of preterm premature rupture of membranes: maternal and neonatal outcomes a prospective controlled multicentre study of clarithromycin in pregnancy effect of quinine therapy on plasma glucose and plasma insulin levels in pregnant women infected with plasmodium falciparum malaria in gezira state cryptococcal pneumonia complicating pregnancy exposure to antiretroviral therapy in utero or early life: the health of uninfected children born to hiv-infected women fosfomycin versus other antibiotics for the treatment of cystitis: a meta-analysis of randomized controlled trials hematologic effects of maternal antiretroviral therapy and transmission prophylaxis in hiv-1-exposed uninfected newborn infants birth defects in a national cohort of pregnant women with hiv infection in italy pharmacokinetics and safety of single-dose tenofovir disoproxil fumarate and emtricitabine in hiv-1-infected pregnant women and their infants safety of efavirenz in the first trimester of pregnancy: an updated systematic review and meta-analysis safety of efavirenz in first-trimester of pregnancy: a systematic review and meta-analysis of outcomes from observational cohorts myelomeningocele in a child with intrauterine exposure to efavirenz successful use of darunavir, etravirine, enfuvirtide and tenofovir/emtricitabine in pregnant woman with multiclass hiv resistance maternal exposure to prescription and non-prescription pharmaceuticals or drugs of abuse and risk of craniosynostosis pregnancy and infant outcomes among hiv-infected women taking long-term art with and without tenofovir in the dart trial placental transfer and pharmacokinetics of lopinavir and other protease inhibitors in combination with nevirapine at delivery acyclovir pregnancy registry and valacyclovir pregnancy registry: interim report for 1 exposure to nitrofurantoin during the first trimester of pregnancy and the risk for major malformations quinolone arthropathy -acute toxicity to immature articular cartilage edwards: discoverer of maternal hyperthermia as a human teratogen pharmacokinetics of sulfadoxinepyrimethamine in hiv-infected and uninfected pregnant women in western kenya pharmacokinetics of oseltamivir according to trimester of pregnancy maternal and neonatal outcomes after antepartum treatment of influenza with antiviral medications intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of hiv-1 in kampala, uganda: hivnet 012 randomised trial inadvertent exposure of pregnant women to ivermectin and albendazole during mass drug administration for lymphatic filariasis lack of risk of adverse birth outcomes after deworming in pregnant women a prospective and open-label study for the efficacy and safety of telbivudine in pregnancy for the prevention of perinatal transmission of hepatitis b virus infection lack of tumors in infants with perinatal hiv-1 exposure and fetal/neonatal exposure to zidovudine review of beta-lactam antibiotics in pregnancy. the need for adjustment of dosage schedules pharmacokinetics and transplacental passage of imipenem during pregnancy folic acid antagonists during pregnancy and the risk of birth defects population pharmacokinetics of tenofovir in hiv-1-infected pregnant women and their neonates (anrs 12109) population pharmacokinetics of emtricitabine in human immunodeficiency virus type 1-infected pregnant women and their neonates seminal fluid ribavirin level and functional semen parameters in patients with chronic hepatitis c on antiviral combination therapy a population pharmacokinetic model of piperaquine in pregnant and non-pregnant women with uncomplicated plasmodium falciparum malaria in sudan renal dysplasia associated with in utero exposure to gentamicin and corticosteroids safety of fluconazole in the treatment of vaginal candidiasis. a prescription-event monitoring study, with special reference to the outcome of pregnancy use of darunavir/ritonavir once daily in treatment-naive pregnant woman: pharmacokinetics, compartmental exposure, efficacy and safety use of newer antiretroviral agents, darunavir and etravirine with or without raltegravir, in pregnancy: a report of two cases a population-based study of maternal use of amoxicillin and pregnancy outcome in denmark pregnancy outcomes after maternal exposure to fluconazole maternal antiretroviral use during pregnancy and infant congenital anomalies: the nisdi perinatal study bacterial vaginosis: review of treatment options and potential clinical indications for therapy intrauterine ototoxicity. a case report and review of literature chronic hepatitis b infection in pregnancy illustrated by a case of successful treatment with entecavir maternal drug use in early pregnancy and infant cardiovascular defect is erythromycin therapy teratogenic in humans? maternal drug use, fertility problems, and infant craniostenosis fetal safety of erythromycin. an update of swedish data pharmacokinetic properties of sulfadoxine-pyrimethamine in pregnant women levamisole (decaris) treatment during pregnancy the possible association between the combination of vaginal metronidazole and miconazole treatment and poly-syndactyly population-based case-control teratologic study parenteral polymyxin b treatment during pregnancy population-based case-control study of oral ketoconazole treatment for birth outcomes broad-spectrum antibiotics for preterm, prelabour rupture of fetal membranes: the oracle i randomised trial. oracle collaborative group antifungal therapy during pregnancy is gentamicin ototoxic to the fetus? short-acting sulfonamides near term and neonatal jaundice failure of metronidazole to prevent preterm delivery among pregnant women with asymptomatic trichomonas vaginalis infection prevalence of congenital anomalies in infants with in utero exposure to antiretrovirals no association between griseofulvin and conjoined twinning drug rash with eosinophilia and systemic symptoms syndrome and renal toxicity with a nevirapine-containing regimen in a pregnant patient with human immunodeficiency virus investigation of metronidazole use during pregnancy and adverse birth outcomes use of antiretroviral therapy in pregnant hivinfected women and the risk of premature delivery: a meta-analysis transplacental passage of vancomycin in noninfected term pregnant women serum erythromycin levels in pregnancy birth outcome following maternal use of fluoroquinolones perinatal antiretroviral treatment and hematopoiesis in hiv-uninfected infants chloroquine pharmacokinetics in pregnant and nonpregnant women with vivax malaria multidrug-resistant tuberculosis in pregnancy: case report and review of the literature drug hepatotoxicity in pregnancy increased risk of low birthweight and small for gestational age infants among women with tuberculosis maternal exposure to amoxicillin and the risk of oral clefts safety of macrolides during pregnancy association of high-dose bifonazole administration during early pregnancy and severe limb reduction defects in the newborn safety of telbivudine treatment for chronic hepatitis b for the entire pregnancy pregnancy outcome following gestational exposure to fluoroquinolones: a multicenter prospective controlled study prenatal exposure to fluconazole: an identifiable dysmorphic phenotype erythromycin use during pregnancy in relation to pyloric stenosis congenital defects among children born to women under supervision or treatment for pulmonary tuberculosis successful use of dapsone in refractory pregnancy-associated idiopathic thrombocytopenic purpura maternal and infant use of erythromycin and other macrolide antibiotics as risk factors for infantile hypertrophic pyloric stenosis first trimester use of macrolides and risk of major malformations {otis abstract} case report: nucleoside analogue-induced lactic acidosis in the third trimester of pregnancy lamivudine-zidovudine combination for prevention of maternal-infant transmission of hiv-1 assessment of infant development during an 18-month follow-up after treatment of infections in pregnant women with cefuroxime axetil safety of artemether-lumefantrine in pregnant women with malaria: results of a prospective cohort study in zambia safety and efficacy of fosamprenavir in human immunodeficiency virus-infected pregnant women transplacental passage of protease inhibitors at delivery prospective assessment of pregnancy outcomes after first-trimester exposure to fluconazole a systematic review of the impact of malaria prevention in pregnancy on low birth weight and maternal anemia hepatotoxicity of erythromycin estolate during pregnancy preliminary data on exposure to mebendazole during pregnancy {abstract} a randomized comparison of artesunate-atovaquoneproguanil versus quinine in treatment for uncomplicated falciparum malaria during pregnancy randomized comparison of mefloquine-artesunate versus quinine in the treatment of multidrug-resistant falciparum malaria in pregnancy artemisinin antimalarials in pregnancy: a prospective treatment study of 539 episodes of multidrug-resistant plasmodium falciparum the pharmacokinetics of atovaquone and proguanil in pregnant women with acute falciparum malaria a randomised controlled trial of artemetherlumefantrine versus artesunate for uncomplicated plasmodium falciparum treatment in pregnancy the effects of quinine and chloroquine antimalarial treatments in the first trimester of pregnancy high neonatal concentrations of raltegravir following transplacental transfer in hiv-1 positive pregnant women treatment of vulvovaginal candidiasis in pregnancy. a comparative study critical pneumonia complicating early-stage pregnancy griseofulvin teratology the safety of lincomycin in pregnancy arrhenius thermodynamics and birth defects: chemical teratogen synergy. untested, testable, and projected relevance pharmacokinetics of nevirapine in human immunodeficiency virus type 1-infected pregnant women and their neonates. pediatric aids clinical trials group protocol 250 team atazanavir pharmacokinetics with and without tenofovir during pregnancy concentrations of protease inhibitors in cord blood after in utero exposure acute respiratory failure during pregnancy: a case of nitrofurantoin-induced pneumonitis maternal use of antibiotics and the risk of orofacial clefts: a nationwide cohort study use of oral fluconazole during pregnancy and the risk of birth defects maternal hyperthermia and the risk for neural tube defects in offspring: systematic review and meta-analysis population pharmacokinetics of artesunate and dihydroartemisinin in pregnant and non-pregnant women with malaria safety of artemether-lumefantrine exposue in first trimester of pregnancy: an observational cohort anthelminthic treatment during pregnancy is associated with increased risk of infantile eczema: randomized-controlled trial results a comparison of liposomal amphotericin b with sodium stibogluconate for the treatment of visceral leishmaniasis in pregnancy in sudan the influence of labour on the pharmacokinetics of intravenously administered amoxicillin in pregnant women the jarisch-herxheimer reaction and fetal monitoring changes in pregnant women treated for syphilis the pharmacokinetics and pharmacodynamics of atovaquone and proguanil for the treatment of uncomplicated falciparum malaria in third-trimester pregnant women transplacental transfer of vancomycin and telavancin effects of deworming during pregnancy on maternal and perinatal outcomes in entebbe, uganda: a randomized controlled trial efficacy of ivermectin and albendazole alone and in combination for treatment of soil-transmitted helminths in pregnancy and adverse events: a randomized open label controlled intervention trial in masindi district, western uganda famciclovir exposure during organogenesis {abstract} safety, efficacy and determinants of effectiveness of antimalarial drugs during pregnancy: implications for prevention programmes in plasmodium falciparum-endemic sub-saharan africa neonatal outcomes after gestational exposure to nitrofurantoin maternal use of fluconazole and risk of congenital malformations: a danish population-based cohort study population-based case control study of the safety of sulfasalazine use during pregnancy the effects of mefloquine treatment in pregnancy pharmacokinetics of sulfadoxine and pyrimethamine in intermittent preventive treatment of malaria in pregnancy panel on treatment of hiv-infected pregnant women and prevention of perinatal transmission. recommendations for use of antiretroviral drugs in pregnant hiv-1-infected women for maternal health and interventions to reduce perinatal hiv transmission in the united states ocular toxicity in children exposed in utero to antimalarial drugs: review of the literature lack of increased hepatotoxicity in hiv-infected pregnant women receiving nevirapine compared with other antiretrovirals increased risk of hepatotoxicity in hiv-infected pregnant women receiving antiretroviral therapy independent of nevirapine exposure maternal blood and amniotic fluid levels of moxifloxacin, levofloxacin and cefixime transfer of antivirals across the human placenta pregnancy outcome after inadvertent ivermectin treatment during community-based distribution observational cohort study of pregnancy outcome after first-trimester exposure to fluoroquinolones visceral leishmaniasis in pregnancy: a case series and a systematic review of the literature an algorithm for risk assessment and intervention of mother to child transmission of hepatitis b virus case report: neurocysticercosis in pregnancy safety and efficacy of lopinavir/ritonavir during pregnancy: a systematic review use of acyclovir, valacyclovir, and famciclovir in the first trimester of pregnancy and the risk of birth defects atovaquone-proguanil use in early pregnancy and the risk of birth defects prenatal protease inhibitor use and risk of preterm birth among hiv-infected women initiating antiretroviral drugs during pregnancy absence of teratogenicity of oral ganciclovir used during early pregnancy in a liver transplant recipient the safety of antimalarial drugs in pregnancy first trimester exposure to antiretroviral therapy and risk of birth defects decreased plasma levels of darunavir/ritonavir in a vertically infected pregnant woman carrying multiclass-resistant hiv type-1 efficacy and safety of artemether-lumefantrine compared with quinine in pregnant women with uncomplicated plasmodium falciparum malaria: an open-label, randomised, non-inferiority trial a case of blastomycosis in pregnancy dihydroartemisinin-piperaquine treatment of multidrug resistant falciparum and vivax malaria in pregnancy drug treatment during pregnancy and isolated orofacial clefts in hungary successful use of oral ganciclovir for the treatment of intrauterine cytomegalovirus infection in a renal allograft recipient fluconazole-induced congenital anomalies in three infants vancomycin during pregnancy: does it cause hearing loss or nephrotoxicity in the infant? pregnancy outcome after exposure to injectable ribavirin during embryogenesis atazanavir plus low-dose ritonavir in pregnancy: pharmacokinetics and placental transfer the ribavirin pregnancy registry: findings after 5 years of enrollment hearing loss in infants of tuberculous mothers treated with streptomycin during pregnancy pregnancy outcome after gestational exposure to erythromycin -a population-based register study from norway griseofulvin teratology, including two thoracopagus conjoined twins placental transfer of griseofulvin outcomes of infants exposed to oseltamivir or zanamivir in utero during pandemic (h1n1) 2009 relationship between a case of severe hearing loss and use of gentamycin in the pregnant mother exposure to anti-infective drugs during pregnancy and the risk of small-for-gestational-age newborns: a case-control study pregnancy outcome following gestational exposure to terbinafine: a prospective comparative study pregnancy outcome following gestational exposure to azithromycin acute onset lactic acidosis and pancreatitis in the third trimester of pregnancy in hiv-1 positive women taking antiretroviral medication evaluation of a case registry of the european network of teratology information services (entis) chemically induced birth defects pregnancy and fetal outcomes after exposure to mefloquine in the pre-and periconception period and during pregnancy in-hospital safety in field conditions of nifurtimox eflornithine combination therapy (nect) for t.b. gambiense sleeping sickness maternal illness, including fever and medication use as risk factors for neural tube defects successful treatment of vancomycin-resistant enterococcus faecium pyelonephritis with daptomycin during pregnancy a randomised controlled trial of metronidazole for the prevention of preterm birth in women positive for cervicovaginal fetal fibronectin: the premet study treatment of multidrug-resistant tuberculosis during pregnancy: a report of 7 cases case report of exposure to voriconazole in the second and third trimesters of pregnancy importance and prevention of malaria in pregnancy association of prenatal and postnatal exposure to lopinavir-ritonavir and adrenal dysfunction among uninfected infants of hiv-infected mothers risk of malformations and other outcomes in children exposed to fluconazole in utero safety of the maternal-infant zidovudine regimen utilized in the pediatric aids clinical trial group 076 study effect of pregnancy on emtricitabine pharmacokinetics pregnancy outcomes following systemic prenatal acyclovir exposure: conclusions from the international acyclovir pregnancy registry, 1984-1999 use of daptomycin in a pregnant patient with staphylococcus aureus endocarditis the effect of fever, febrile illnesses, and heat exposures on the risk of neural tube defects in a texas-mexico border population birth outcomes among women exposed to neuraminidase inhibitors during pregnancy efficacy, safety, and tolerability of amodiaquine plus sulphadoxine-pyrimethamine used alone or in combination for malaria treatment in pregnancy: a randomised trial safety of neuraminidase inhibitors against novel influenza a (h1n1) in pregnant and breastfeeding women fetal and maternal outcome after administration of tenofovir to gravid rhesus monkeys (macaca mulatta) pharmacokinetic properties of artemether, dihydroartemisinin, lumefantrine, and quinine in pregnant women with uncomplicated plasmodium falciparum malaria in uganda population pharmacokinetics of lumefantrine in pregnant women treated with artemether-lumefantrine for uncomplicated plasmodium falciparum malaria pregnancy outcomes in hiv-infected women receiving long-term isoniazid prophylaxis for tuberculosis and antiretroviral therapy british hiv association guidelines for the management of hiv infection in pregnant women 2012 raltegravir in pregnancy: a case series presentation can amodiaquine be used safely during pregnancy? exposure to mebendazole and pyrvinium during pregnancy: a danish nationwide cohort study low rates of mother-to-child transmission of hiv following effective pregnancy interventions in the united kingdom and ireland improved obstetric outcomes and few maternal toxicities are associated with antiretroviral therapy, including highly active antiretroviral therapy during pregnancy effect of early oral clindamycin on late miscarriage and preterm delivery in asymptomatic women with abnormal vaginal flora and bacterial vaginosis: a randomised controlled trial the pharmacokinetics, safety and efficacy of boosted saquinavir tablets in hiv type-1-infected pregnant women pivmecillinam and adverse birth and neonatal outcomes: a population-based cohort study pharmacokinetics and safety of stavudine in hivinfected pregnant women and their infants: pediatric aids clinical trials group protocol 332 pharmacokinetics of didanosine in antepartum and postpartum human immunodeficiency virus-infected pregnant women and their neonates: an aids clinical trials group study pharmacokinetics of metronidazole in pregnant patients with bacterial vaginosis mother-to-child hiv transmission despite antiretroviral therapy in the anrs french perinatal cohort birth defects among a cohort of infants born to hiv-infected women on antiretroviral medication effect of single-dose anthelmintic treatment during pregnancy on an infant's response to immunisation and on susceptibility to infectious diseases in infancy: a randomised, double-blind, placebo-controlled trial pharmacokinetic profile in late pregnancy and cord blood concentration of tipranavir and enfuvirtide prevention of mother-to-child transmission of multi-drug resistant hiv-1 using maternal therapy with both enfuvirtide and tipranavir report of the who informal consultation on the use of praziquantel during pregnancy/ lactation and albendazole/mebendazole in children under 24 months assessment of the safety of artemisinin compounds in pregnancy guidelines for treatment of tuberculosis guidelines for the treatment of malaria antiretroviral drugs for treating pregnant women and preventing hiv infection in infants: recommendations for a public health approach who. chagas disease (american trypanosomiasis) malaria in pregnant women soil-transmitted helminth infections further analysis of the risk of adverse birth outcome after maternal use of fluoroquinolones infant outcomes among pregnant women who used oseltamivir for treatment of influenza during the h1n1 epidemic successful caspofungin treatment of multidrug resistant candida parapsilosis septicaemia in an extremely low birth weight neonate exposure to trimethoprim/sulfamethoxazole but not other fda category c and d anti-infectives is associated with increased risks of preterm birth and low birth weight gentamicin use in pregnancy. a renal anomaly key: cord-103350-jj9pc4a6 authors: tang, pingtao; das, jharna r.; li, jinliang; yu, jing; ray, patricio e. title: an hiv-tat inducible mouse model system of childhood hiv-associated nephropathy date: 2020-05-08 journal: biorxiv doi: 10.1101/2020.05.06.081851 sha: doc_id: 103350 cord_uid: jj9pc4a6 background modern antiretroviral therapies (art) have decreased the prevalence of hiv-associated nephropathy (hivan). nonetheless, we continue to see children and adolescents with hivan all over the world. furthermore, once hivan is established in children, it is difficult to revert its long-term progression, and we need better animal models of childhood hivan to test new treatments. objectives to define whether the hiv-1 trans-activator (tat) gene precipitates hivan in young mice, and to develop an inducible mouse model of childhood hivan. design/methods an hiv-tat gene cloned from a child with hivan was used to generate recombinant adenoviral vectors (rad-tat). rad-tat and lacz control vectors (2 × 109) were expressed in the kidney of newborn wild type and hiv-transgenic (tg26) fvb/n mice without significant proteinuria (n = 5 8 per group). mice were sacrificed 7 and 35 days later to assess their renal outcome, the expression of hiv-genes and growth factors, and markers of cell growth and differentiation by rt-qpcr, immunohistochemistry, and/or western blots. results hiv-tat induced the expression of hiv-1 genes (env) and heparin binding growth factors in the kidney of hiv-tg26 mice, and precipitated hivan in the first month of life. no significant renal changes were detected in wild type mice infected with rad-tat vectors, suggesting that hiv-tat alone does not induce renal disease. conclusion this new mouse model of childhood hivan highlights the critical role that hiv-tat plays in the pathogenesis of hivan, and could be used to study the pathogenesis and treatment of hivan in children and adolescents. summary statement we developed a new inducible mouse model system of childhood hiv-associated nephropathy, and demonstrated that hiv-tat plays a critical role in this renal disease acting in synergy with other hiv-1 genes and heparin binding cytokines. modern combined antiretroviral therapies (cart) have improved the clinical outcome of children and adolescents living with hiv and decreased the prevalence of hiv-associated nephropathy (hivan) in a significant manner. however, physicians have had less success providing chronic cart to children and adolescents living with hiv, and we continue to see hivan in this group of people all over the world. over 80% of the estimated 2.1 million hiv-infected children are living in the sub saharan africa [1], and it is anticipated that approximately 10% of these children will develop hivan if they do not receive appropriate art [1] . furthermore, we have noticed that once the typical renal histological features of hivan are established in children, it is difficult to prevent its long-term progression to eskd with current treatments available. in addition, previous reports in adults [2, 3] and children [4] suggest that hivan can occur in people with suppressed viral load. these studies suggest that inflammatory cytokines released by hivinfected cells can play a role in the pathogenesis of hivan independently of the viral load. taken together, all these findings underscore the importance of acquiring a better understanding of the pathogenesis and treatment of childhood hivan during the modern cart era. childhood hivan is a renal disease seen predominantly in black children and adolescents who acquired hiv-1 through vertical transmission and do not receive appropriate antiretroviral therapy [5, 6] . from the clinical point of view it is characterized by persistent proteinuria, often in the nephrotic range, and in the late stages is associated with edema, reduced gfr, hypertension, and rapid progression to end stage kidney disease (eskd) [1, 5, 6] . the renal histological lesions of childhood hivan reveal mesangial hyperplasia, focal segmental or collapsing glomerulosclerosis, and multicystic tubular dilatation leading to renal enlargement [1, 5, 6] . several hiv-transgenic (hiv-tg) animal models are available to study the pathogenesis and treatment of hivan [7] [8] [9] [10] [11] . however, these animals develop hivan at different time points, usually after they reach adulthood, and we lack a reliable mouse model system to study the pathogenesis of childhood hivan. therefore, we carried out this study to determine whether the hiv-1 trans-activator (tat) gene precipitates hivan in young mice, and define whether this approach could be used to generate an inducible mouse model system of childhood hivan. to accomplish this goal, we infected newborn wild type and heterozygous hiv-tg 26 the protein sequence of the hiv-tat derived from a child with hivan (tat-hivan) was aligned with tat protein sequences derived from the lymphotropic virus hiv-1 iiib and the monocytetropic hiv-1 virus ada (nih aids research and reference reagent program). as shown in figure 1a , tat-hivan contains the basic domain that is essential for hiv-1 activation, but is missing the rgd motif that interacts with cytokines and integrin receptors [12, 13] . using an adenovirus gene transferring technique developed in our laboratory [14] , we were able to express hiv-tat in the kidney and liver of wild type and hiv-tg 26 mice ( figure 1b ). as expected, by western blots, higher tat protein expression levels where detected in the liver compared to kidneys [14] ( figure 1b ). hiv-tg 26 newborn mice infected with rad-tat showed higher tat mrna expression levels when compared to transgenic mice injected with rad-lac-z vectors ( figure 1b) . the tat mrna detected in hiv-tg 26 mice injected with rad-lac-z vectors was transcribed from the hiv pro-viral dna d1443 transgenic construct. figure 4a -b). furthermore, the bun levels of these mice were elevated, when compared to hiv-tg 26 mice injected with rad-lac-z vectors (35 ± 1 mg/dl* vs. 24.5 ± 2.6 mean± sem; *p<0.05). overall, these findings suggest that tat plays an important role precipitating hivan in hiv-tg 26 mice. figures 1-2) . taken together, these findings suggest that hiv-tat interacts with other hiv-1 genes and/or cytokines to induce the proliferation of renal epithelial cells [15, 16, 18] . alternatively, using an in situ apoptosis detection kit and western blots to to develop the mouse model of childhood hivan we took advantage of the hiv-tg 26 mouse line [7, 15, 19] . these mice carry a 7.4-kb hiv-1 construct lacking 3-kb sequence overlapping the gag/pol region of the provirus pnl4-3 [7, 15, 19] and express hiv-1 transcripts in many tissues, including kidney glomerular and tubular epithelial cells. homozygous hiv-tg 26 mice are born sick and usually died with multiple systemic lesions during the first days or weeks of life [7, 19] . in contrast, heterozygous mice can be followed until they reach adulthood, and have been used by several investigators to explore the pathogenesis of hivan [7, 15, 16, 19] . because the majority of heterozygous hiv-tg 26 mice develop hivan at different time points after they reach adulthood, currently we do not have a reliable mouse model system to study the pathogenesis of childhood hivan. therefore, we carried out this study to test the hypothesis that the induction of hiv-genes in the kidney of newborn mice precipitates hivan during the first month of life. to accomplish this goal, we used an adenovirus gene transferring technique developed in our laboratory, which is based on the principle that the retention of adenoviral vectors in the circulation improves the transduction of renal glomerular cells in rodents [20, 21] in previous studies, we showed that newborn mice have delayed clearance of rad vectors from the circulation, and therefore, more efficient transduction of glomerular cells after a systemic injection of adenoviruses via the retro-orbital plexus [14] . following this approach, we expressed the coding sequences of a tat gene derived from a child with hivan (tat-hivan) in the kidney of newborn hiv-tg 26 mice, and precipitated the development of hivan during the first month of life. our findings support the results of previous studies showing that hiv-1 genes expressed in the kidney play a critical role in this process, although we do not yet understand the exact mechanisms involved. further studies are warranted to explore this issue. the hiv-tat protein is a powerful transcriptional factor encoded by two exons. the first exon encodes the hiv activation and basic binding domains, which are required for hiv-transcription and nuclear localization of tat [22] . the second exon encodes the rgd motif (c-terminal amino acids 73-86), which enhances the angiogenic activity of tat acting through cytokines and integrin receptors [23] . tat plays an essential role in hiv-replication by recruiting a cellular human protein called cyclin t1, which efficiently increases the transcription of the hiv-ltr via nf-κb [24] . however, cloning and characterization of the murine cyct1 protein revealed that mouse cyclin 1 lacks a critical cysteine residue that is needed to form a complex with tat and induce its full transcriptional activity [25, 26] . for this reason, tat has limited direct transcriptional activity in mice, but it can induce the expression of tnf-α [27] and other cytokines that increase the transcription of hiv-1 via nf-κb dependent mechanisms [28] . our study showed that the activation and basic binding domains of tat are sufficient to induce the renal expression of hiv-genes and precipitate hivan in young mice. in contrast, we found tat's rgd motif is not essential in this process. in addition to being a powerful activator of hiv-1 transcription, tat is released into the circulation by infected cells and can be taken up by uninfected cells [13, 18] . in this manner, tat mimics the action of several cytokines involved in the pathogenesis of aids, including sdf-1α, rantes, and mif1-β [13, 18, 29] . furthermore, acting in synergy with fgf-2, tat can induce the de-differentiation and proliferation of cultured human podocytes [30] [31] [32] . for these reasons, we explored the effects of tat in wild type mice, but were unable to detect significant renal lesions in these mice. our findings suggest that tat alone cannot induce renal disease in wild type mice. however, we should mention that tat-hivan has an incomplete rgd sequence, and its ability to interact with cytokines and integrin receptors in vivo might be impaired [12, 13] . thus, further studies are needed to determine whether tat proteins containing rdg sequences can cause kidney damage per se in wild type mice. we speculate that an additional mechanism through which tat could precipitate hivan in hiv-tg 26 mice is by increasing the production and/or activity of tnf-α [27] , since high levels of tnf-α are detected in the circulation of hiv-tg 26 mice [33] , and tnf-α worsens the outcome of hivan in adult mice [34] . alternatively, tat could act in synergy with fgf-2 and vegf-a [15, 16, 28, 31] , considering that both heparin binding growth factors were up-regulated by tat in 7 day old hiv-tg 26 overall, our mouse model reproduces all the renal histological features characteristic of childhood hivan [1, 36] . interestingly, the expression levels of the podocyte specific proteins, nephrin, wt-1, and synaptopodin, did not change in correlation with the induction of hiv-1 genes during the first week of life. these findings suggest that the podocyte de-differentiation changes characteristic of hivan, might be a secondary event associated with the regeneration of these cells. it is possible that podocytes that express high levels of hiv-1 genes died, and were replaced by parietal epithelial or renal progenitor cells [37, 38] , which do not express podocytes markers, and are sensitive to the growth promoting effects of several growth factors [39] . in addition, we noted a reduced number of renal epithelial cells undergoing apoptosis in 7 days old hiv-tg 26 mice infected with rad-tat vectors, when compared to the controls. it is tempting to speculate that tat, in combination with bfgf-2 and vegf-a, may have an antiapoptotic effect [40] , since both heparin binding growth factors were up-regulated at this time point. however, more studies are needed to test this hypothesis. finally, we found that tat induced direct renal epithelial proliferative changes in 7 and 35 days old hiv-tg 26 mice. these changes appear to be driven by the mapk signaling pathway, which can be activated directly by hiv-nef [41] [42] [43] [44] , as well as fgf-2 [15] or vegf-a [16] in humans, the risk variants of the apolipoprotein-1 (apol1) increase the lifetime risk of untreated hiv+ people to develop hivan by ~ 50% [45] [46] [47] . therefore, one limitation of our animal model is that hiv-tg 26 mice do not express the apol-1 gene. this limitation could be overcome by generating dual transgenic hiv-tg 26 / apol1 mice [48] , and infecting newborn mice with rad-tat vectors. in addition, a significant number of black children living with hiv develop hivan independently of the apol1 risk variants [49, 50] , and previous studies suggest that the apol1 risk variants may play more relevant role in adults, when compared to young children [50, 51] . thus, young kidneys might be more sensitive to the cytotoxic effects of hiv-1 genes, tnf-α, and heparin binding growth factors, and less dependent on the apol1 risk variants to develop hivan. alternatively, it is possible that other unknown genetic factors may play an additional role precipitating hivan in black children, as reported in hiv-tg 26 mice [52] . taken together, these studies show that a strong genetic influence modulates the outcome of hivan both in mice and humans, and more work needs to be done to define these factors in children. in conclusion, we have developed an inducible mouse model system of childhood hivan that reproduces the full hivan phenotype during their first month of life. in addition, we showed that tat plays a relevant role in this process by inducing the renal expression of hiv-1 genes, fgf-2, and vegf-a, leading to the activation of the mapk pathway. hopefully, this animal model will facilitate the discovery of new therapeutic targets to prevent the progression of hivan in children and adolescents. care and use committee. heterozygous newborn hiv-tg 26 fvb/n mice [7, 19] and their respective wild type (wt) littermates were injected through the retro-orbital plexus with 2 x 10 9 pfu/mouse of recombinant adenoviral (rad) vectors carrying either hiv-tat derived from a child with hivan (rad-tat vector) or the e. coli lacz gene (rad-lac-z). to express the hiv-tat rad vector in the kidney of newborn mice, we used a gene transferring technique developed in our laboratory [14] . wild type and hiv-tg 26 mice expressing the pro-viral dna construct d1443 [15, 19] , were divided in groups (n = 8 mice each) and sacrificed at 7 days (peak of rad-tat expression) and 35 days (renal clearance of the viral vectors) after the adenoviral injections. all mice had free access to water and standard food and were treated in accordance with the national institutes of health (nih) guidelines for care and use of research animals. the generation of the rad-tat vector derived from a child with hivan was described in detail before [31] . briefly, a cdna fragment encoding the full-length tat protein was cloned into the pcxn2-flag vector and used to generate e1-deleted recombinant adenoviruses carrying tat-hivan-flag [31] . the protein sequence of the tat-hivan gene was aligned and compared to other tat genes using the clustal omega multiple sequence alignment program (http:// www.ebi.ac.uk/tools/msa/clustalo/). both tat-flag and lac-z control adenoviruses were amplified, purified, desalted, and titrated as previously described [14, 53, 54] . the particle-to-plaque-forming unit (pfu) ratio of the virus stock used in these experiments was 100. blood, urine and kidney sample collection. mice were sacrificed 7 and 35 days after the rad injections. urine, blood and kidney samples were harvested and kept frozen at -80 0 c. blood urea nitrogen was assessed using the quantichrom urea assay kit from bioassay systems (catalog no. diur-500) as described before [55] . the urinary creatinine levels were measured using colorimetric assay from r&d systems (catalog no. kge005). urinary protein was measured using the bayer multistix 10 sg reagent strips for urinalysis. in addition, 5 microliters of urine were run on 4-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and stained with coomassie blue stain solution (bio-rad) to detect proteinuria. the protein band corresponding to albumin was quantified by densitometry analysis using adobe photoshop 6.0 (adobe systems, san jose, ca). results were expressed in arbitrary optical density units adjusted to the urinary creatinine values as described before [32] . amplification reaction, we used 5µl and 2µl cdna respectively. the densitometry analysis was conducted using adobe photoshop 6.0 as described before [31, 56] . western blot analysis. the kidneys were lysed using ripa lysis buffer containing protease inhibitors and phosphatase inhibitor cocktail 2 (sigma-aldrich), and processed by western blots as described before [3] . the following primary antibodies were used: phospo-p44/42 mitogen(progen biotechnik gmbh). all primary antibodies were diluted 1:1000 except wt-1, which was diluted 1:500 dilution and incubated overnight at 4 o c. protein bands were detected using supersignal west pico chemiluminescent substrate (thermo scientific) according to the manufacturer's instruction. all membranes were exposed to a kodak film (x-omat) and developed using an automated developer. densitometry analysis of the data expressed as a β actin ratio was conducted using adobe photoshop 6.0 as described before immunohistochemistry. paraffin embedded sections were cut at 5µm, deparaffinized, rehydrated, and stained as previously described [54] . immunostaining was performed with a commercial streptavidin-biotin-peroxidase complex (histostain sp kit, zymed, san francisco, ca) according to the manufacturer's instructions as described before [57] . the peroxidase activity was monitored after the addition of substrate using a dab kit (vector laboratories, catalog no sk-4100) or aec substrate kit (catalog no. 002007,invitrogen, frederick, md). sections were counterstained with hematoxylin. the pcna staining kit from invitrogen was used to detect pcna. ki-67 and wilms' tumor 1 (wt-1) staining were assessed using a 1:50 dilution of a monoclonal rat anti-mouse ki-67 antibody (clone tec-3) and a mouse monoclonal anti-human wt-1 antibody (clone 6f-h2) respectively, both from dako north american inc). synaptopodin was detected with a ready-to-use a mouse monoclonal antibody (clone g1d4, if not specified otherwise, the data were expressed as mean + sem. differences between two groups were compared using an unpaired t test. multiple sets of data were compared by the expression of pcna, wt-1 and nephrin was quantified as a ratio of beta actin. the graphs show the results of the densitometry analysis and quantification of the results in optical density units (mean ± sem), as described in the methods sections. all changes between groups were not statistically significant (p > 0.05). kidney disease in hiv-positive children hiv-associated kidney glomerular diseases: changes with time and haart viral load and hiv-associated nephropathy hiv-associated nephropathy in the setting of maximal virologic suppression renal disease in children with the acquired immunodeficiency syndrome human immunodeficiency virus (hiv)-associated nephropathy in children from the hivassociated nephropathy in transgenic mice expressing hiv-1 genes nephropathy in hiv-transgenic mice a novel hiv-1 transgenic rat model of childhood hiv-1-associated nephropathy expression of hiv-1 genes in podocytes alone can lead to the full spectrum of hiv-1-associated nephropathy transgenic and infectious animal models of hiv-associated nephropathy the tat protein of human immunodeficiency virus type-1 promotes vascular cell growth and locomotion by engaging the alpha5beta1 and alphavbeta3 integrins and by mobilizing sequestered basic fibroblast growth factor inflammatory cytokines synergize with the hiv-1 tat protein to promote angiogenesis and kaposi's sarcoma via induction of basic fibroblast growth factor and the alpha v beta 3 integrin adenovirus-mediated gene transfer to glomerular cells in newborn mice bfgf and its low affinity receptors in the pathogenesis of hivassociated nephropathy in transgenic mice hiv-1 upregulates vegf in podocytes a urinary biomarker profile for children with hiv-associated renal diseases synergy between basic fibroblast growth factor and hiv-1 tat protein in induction of kaposi's sarcoma progressive glomerulosclerosis and enhanced renal accumulation of basement membrane components in mice transgenic for human immunodeficiency virus type 1 genes liver bypass significantly increases the transduction efficiency of recombinant adenoviral vectors in the lung, intestine, and kidney efficient gene transfer to rat renal glomeruli with recombinant adenoviral vectors mutational analysis of the conserved basic domain of human immunodeficiency virus tat protein cutting edge: a short polypeptide domain of hiv-1-tat protein mediates pathogenesis tat trans-activates the human immunodeficiency virus through a nascent rna target the interaction between hiv-1 tat and human cyclin t1 requires zinc and a critical cysteine residue that is not conserved in the murine cyct1 protein recruitment of a protein complex containing tat and cyclin t1 to tar governs the species specificity of hiv-1 tat the tat protein of hiv-1 induces tumor necrosis factor-alpha production. implications for hiv-1-associated neurological diseases hiv-1 tat protein induces production of proinflammatory cytokines by human dendritic cells and monocytes/macrophages through engagement of tlr4-md2-cd14 complex and activation of nf-kappab pathway effects of the human immunodeficiency virus type 1 tat protein on the expression of inflammatory cytokines human immunodeficiency virus-1 tat induces hyperproliferation and dysregulation of renal glomerular epithelial cells the basic domain of hiv-tat transactivating protein is essential for its targeting to lipid rafts and regulating fibroblast growth factor-2 signaling in podocytes isolated from children with hiv-1-associated nephropathy circulating fibroblast growth factor-2, hiv-tat, and vascular endothelial cell growth factor-a in hiv-infected children with renal disease activate rho-a and src in cultured renal endothelial cells elevated levels of tumor necrosis factor alpha (tnf-alpha) in human immunodeficiency virus type 1-transgenic mice: prevention of death by antibody to tnf-alpha interposes the proliferative and nf-kappab-mediated inflammatory response by podocytes to tnf-alpha role of fibroblast growth factor-binding protein in the pathogenesis of hiv-associated hemolytic uremic syndrome hiv-associated nephropathy proliferating cells in hiv and pamidronateassociated collapsing focal segmental glomerulosclerosis are parietal epithelial cells regeneration of glomerular podocytes by human renal progenitors ureteric bud cells secrete multiple factors, including bfgf, which rescue renal progenitors from apoptosis fibroblast growth factor-2 and the hiv-1 tat protein synergize in promoting bcl-2 expression and preventing endothelial cell apoptosis: implications for the pathogenesis of aids-associated kaposi's sarcoma hiv-1 nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein critical role for nef in hiv-1-induced podocyte dedifferentiation nef stimulates proliferation of glomerular podocytes through activation of src-dependent stat3 and mapk1,2 pathways hiv-1 viral protein r induces erk and caspase-8-dependent apoptosis in renal tubular epithelial cells association of trypanolytic apol1 variants with kidney disease in african americans hiv-associated nephropathy in african americans apol1 risk variants are strongly associated with hiv-associated nephropathy in black south africans apol1-g0 or apol1-g2 transgenic models develop preeclampsia but not kidney disease apol1-associated glomerular disease among african-american children: a collaboration of the chronic kidney disease in children (ckid) and nephrotic syndrome study network (neptune) cohorts brief report: apol1 renal risk variants are associated with chronic kidney disease in children and youth with perinatal hiv infection renal and cardiovascular morbidities associated with apol1 status among african-american and non-african mapping a locus for susceptibility to hiv-1-associated nephropathy to mouse chromosome 3 adenovirus-mediated correction of the genetic defect in hepatocytes from patients with familial hypercholesterolemia a novel role of fibroblast growth factor-2 and pentosan polysulfate in the pathogenesis of intestinal bleeding in mice role of circulating fibroblast growth factor-2 in lipopolysaccharide-induced acute kidney injury in mice transmembrane tnf-alpha facilitates hiv-1 infection of podocytes cultured from children with hiv-associated nephropathy fibroblast growth factor-2 increases the renal recruitment and attachment of hiv-infected mononuclear cells to renal tubular epithelial cells acknowledgements. we thank dr. marina jerebtsova phd. for her advice expanding adenoviral vectors and perfoming injections in mice, and dr. xuefang xie phd. for her contribution performing the sequencing and analysis of the tat-hivan gene. competing interests. all authors declare no conflicts or competing interest related to this manuscript.funding sources. this study was supported by national institutes of health awards: r01 dk-108368; r01 dk-115968; and r01 dk-04941. key: cord-025172-qg3jxgch authors: covarrubias, jose; grigorian, areg; kuza, catherine m.; dolich, matthew; dosch, austin; kojayan, greg g.; delaplain, patrick; lekawa, michael; nahmias, jeffry title: trauma patients with human immunodeficiency virus (hiv): a propensity matched analysis date: 2020-05-24 journal: eur j trauma emerg surg doi: 10.1007/s00068-020-01402-4 sha: doc_id: 25172 cord_uid: qg3jxgch background: given the growing number of people worldwide living with huma immunodeficiency virus (hiv), a larger subset of these patients are now susceptible to sustaining a traumatic injury. however, the impact of hiv on outcomes in trauma with modern antiretroviral treatment remains unclear. we hypothesized mortality and rates of infectious and inflammatory complications would be higher in hiv positive (hiv+) trauma patients. methods: the trauma quality improvement program was queried to identify trauma patients ≥ 18 years of age with hiv. due to the imbalance between hiv+ and hiv negative (hiv−) trauma patients, a 1:2 propensity-matched model was utilized. matched variables included age, injury severity score, mechanism of injury, systolic blood pressure, pulse rate, glasgow coma scale score, and patient comorbidities. results: 84 hiv+ patients were matched to 168 hiv− patients. compared to hiv− patients, hiv+ patients had no significant differences in mortality rate (9.5% vs. 4.8%, p = 0.144) or infectious complications, including pneumonia (6.0% vs. 4.2%, p = 0.530), urinary tract infection (1.2% vs. 1.2%, p = 1.000), or severe sepsis (1.2% vs. 0.0%, p = 0.156). however, higher rates of acute respiratory distress syndrome (ards) (9.5% vs. 0.6%, p < 0.001) and acute kidney injury (aki) (4.8% vs. 0.0%, p = 0.004) were observed. conclusion: hiv+ trauma patients are not at higher risk of mortality or infectious complications, likely due to the advent and prevalence of combination antiretroviral therapy. however, hiv positivity appears to increase the risk of aki and ards in trauma patients. further research is needed to confirm this finding to elucidate the etiology underlying this association. as per the joint united nations program on human immunodeficiency virus/acquired immunodeficiency syndrome (hiv/aids), there were an estimated 36.9 million people worldwide living with hiv/aids in 2017 [1] . within the united states, the center for disease control and prevention (cdc) estimates there were approximately 1.1 million people aged ≥ 13 years living with hiv in 2015 [2] . interestingly, most people with hiv are between the ages of 45-54, despite a much higher infection rate in younger individuals, indicating that people are surviving much longer with the disease [2] . in fact, since 2010, there has been a 34% decrease in aids-related deaths [3] . with fewer hiv positive (hiv+) patients succumbing to the progression of their disease, there is now a larger subset of these patients at risk for sustaining a traumatic injury. to optimize care for these patients, a keen understanding of the impact hiv has on outcomes in trauma is needed. to date, the effects of hiv on trauma has been mixed in the literature. martin et al. showed increased 30-day mortality in trauma patients [4] while other studies demonstrated no association with mortality [6] [7] [8] [9] [10] . similarly, some studies have shown higher renal and pulmonary complications [5, 6] as well as higher infectious complications [5, 7, 8] , whereas others have demonstrated no difference in renal [7, 8] , pulmonary [7, 8] , infectious [6] , or overall complication rates [4, 9] in hiv+ trauma patients. furthermore, much of the existing literature evaluating the impact of hiv on clinical outcomes in trauma is nearing a decade old [5] [6] [7] [8] . since publication of these studies, the life expectancy in hiv+ patients has improved dramatically with the use of combination antiretroviral therapy (cart) such that the life expectancy gap relative to hiv negative (hiv−) patients is now as low as 8 years [10] [11] [12] . in addition, many of the available studies either analyze a small population and/or are conducted at a single center [4] [5] [6] 9] . therefore, we sought to provide a large contemporary descriptive analysis of clinical outcomes in hiv+ trauma patients. we hypothesized that a hiv+ trauma cohort would have increased mortality rates, infectious complications (i.e., pneumonia, urinary tract infection (uti), and sepsis), and inflammatory complications [i.e., acute respiratory distress syndrome (ards) and acute kidney injury (aki)], compared to a propensity-matched hiv− group. this study was approved by the institutional review board at our institution. furthermore, the study utilized a large national database with de-identified patients, thus no consent was required. the database used was the trauma quality improvement program (tqip), a database comprised of a retrospective cohort of trauma patients from participating level i or level ii trauma centers in the united states and canada. tqip patient inclusion criteria consists of age ≥ 16, presence of at least one valid trauma international statistical classification of diseases and related health problems (icd) diagnosis code, blunt or penetrating mechanisms of injury, abbreviated injury scale (ais) score ≥ 3, and having data for hospital or emergency department disposition [13] . tqip patient exclusion criteria consists of patients with advanced directives preventing life-sustaining care or age ≥ 65 with isolated hip fractures [13] . a retrospective analysis of the tqip was performed from january 2010 to december 2016 to identify trauma patients who were ≥ 18 years of age with hiv. patients considered hiv+ were those with an icd-9 diagnosis code for hiv: 72 and 79.53. patients considered hiv− were those without an icd-9 diagnosis code for hiv. due to the observed imbalance between hiv+ and hiv− trauma patients, we elected to perform a propensitymatched analysis using a 1:2 model. this was derived from a logistic regression model in which the dependent variable was hiv. the variables utilized in our model included age, injury severity score (iss), mechanism of injury, systolic blood pressure (sbp), pulse rate, glasgow coma scale (gcs) score on admission, and comorbidities such as history of hypertension, chronic obstructive pulmonary disease (copd), cirrhosis, congestive heart failure (chf), steroid use, end-stage renal disease (esrd), and disseminated cancer. patients with similar propensity scores were matched to compare outcomes among patients with and without hiv. only cases that were within 0.001 of the estimated logit were selected. this technique of defining the closeness of a matched case is termed caliper matching and is a validated method of emulating randomization in observational studies [14] . once propensity scores were calculated for each case, one hiv+ and two hiv− matched trauma patients were included for further analysis. if a close match was not available for a hiv+ trauma patient, they were excluded from analysis. we performed bivariate analyses for all variables to confirm a successfully matched cohort. this was done with either a chi-square or mann-whitney-u test for categorial and continuous variables, respectively. the primary outcome was in-hospital mortality. secondary outcomes included infectious and inflammatory complications such as aki, ards, pneumonia, and uti as well as other in-hospital complications. other measured outcomes included total hospital length of stay (los), ventilator days and intensive care unit (icu) los. all p-values were two-sided, with a statistical significance level of < 0.05. all analyses were performed with ibm spss statistics for windows (version 24, ibm corp., armonk, ny). from 1,403,466 trauma patients, 94 (0.007%) patients were hiv+ . after propensity-score matching, 84 hiv+ trauma patients were compared to a cohort of 168 hiv− trauma patients. there were 10 patients that did not fit into our propensity-matched model and were thus excluded from analysis ( fig. 1 ). compared to a matched cohort of hiv− patients, the hiv+ patients had no differences in demographics, injury patterns, or vital signs upon hospital arrival. this confirmed successful matching between the two groups. hypertension was the most common comorbid condition in both groups, followed by copd, and diabetes. traumatic brain injury (tbi) was the most common injury in both groups followed by injuries to the lower extremity (table 1) . there was no difference observed in mortality rate (4.8% vs 9.5%, p = 0.144) or in the rate of infectious complications including uti (1.2% vs 1.2%, p = 1.000), pneumonia (6.0% vs 4.2%, p = 0.530), and sepsis (1.2% vs 0.0%, p = 0.156) between the hiv+ and hiv− group. however, higher rates of ards (0.6% vs. 9.5%, p < 0.001) and aki (0.0% vs. 4.8%, p = 0.004) were observed in the hiv+ group relative to the hiv− group. the los, icu los, and median ventilator days did not differ between the two groups ( table 2 ). though once considered a fatal disease, hiv/aids now more closely resembles a chronic disease with the advent and rise of cart medications [3, 15, 16] . in this study, we evaluated the clinical outcomes in this modern-day hiv+ trauma patient population and compared our findings to those reported in earlier studies. using seven years of data derived from the tqip database, we demonstrated no differences in mortality or rates of infectious complications between hiv+ and hiv− trauma patients. however, the hiv+ group was found to have higher rates of aki and ards. prior literature generally has shown no association between hiv and mortality in trauma patients [5] [6] [7] [8] . however, a recent study by martin et al. evaluating trauma patients in rwanda found hiv to be associated with increased 30-day mortality although no in-hospital mortality was reported [4] . our study supports the findings of earlier domestic studies but appears to contrast the findings of the international study by martin et al. it should be noted that direct comparisons between this study and the one by martin et al. are difficult given the geographic disparities as well as the differing primary outcome of interest. however, their finding of no in-hospital mortality appears to support our results. the association between hiv and development of infectious complications in trauma patients has been mixed, with some studies demonstrating higher [5, 7, 8] rates and others demonstrating no difference [6] in the rate of infectious complications like pneumonia, uti, bacteremia, and sepsis. however, these studies are relatively old with data ranging from 1989 to 2006, a time when treatment for hiv was not widely available. in fact, the cdc estimates only 28% of all hiv+ patients in the u.s. had achieved viral suppression by 2010 [17] . the only research addressing infectious complications in modern-day hiv+ trauma patients has been through relatively small single center or multi-center studies conducted outside of the u.s. both, martin [4, 9] . similarly, we did not find a difference in infectious complications when comparing u.s. hiv+ to hiv− trauma patients. this is likely reflective of the growing prevalence of cart and should thus be considered as physicians counsel patients and/or make decisions regarding management of hiv+ trauma patients. the association between hiv and ards/aki in trauma has varied in previous reports [5] [6] [7] [8] . here we reported a higher rate of ards and aki in hiv+ trauma patients, a finding that supports the work by stawicki et al. and duane et al. [5, 6] . the mechanism for this might be related to the chronic inflammatory state that remains in hiv+ patients despite adequate viral suppression from cart [18] . as a known inducer of systemic inflammation [19] , traumatic injury in hiv+ patients may serve to further augment the inflammatory state already present. this can amplify the activity of neutrophils known to be a component of the chronic inflammatory state observed in hiv [20] . neutrophil activation is known to be key in the pathogenesis of ards [21] so additional neutrophil involvement may make hiv+ trauma patients susceptible for the development of ards. this augmented inflammatory state may also amplify pre-existing renal tissue injury and contribute to the development of aki. however, the mechanism for higher rates of aki is likely multifactorial since aki in hiv+ patients has typically been shown to have two or more contributing etiological factors such as infection, drug nephrotoxicity, and/ or decreased renal perfusion [22] . the nephrotoxic effects of antiretroviral agents such as atazanavir, lopinavir, indinavir, and tenofovir are well documented [23] [24] [25] and may occur insidiously [26] . it is possible that traumatic injury in hiv+ patients led to decreased renal perfusion from hypovolemia, exacerbating pre-existing renal injury and increasing the likelihood for aki. more research is needed to evaluate the pathophysiology behind the increased rates of ards and aki among hiv+ trauma patients. clinicians should be aware of this finding to attempt to mitigate this through vigilant monitoring and/or targeted interventions. as a large retrospective database study, there are inherent limitations to this work such as reporting bias and coding errors. the estimated prevalence of hiv in the us population in 2015 was 0.3%, which is much higher than the observed prevalence in this study. this could indicate a significant population of missed hiv+ patients in the cohort. another limitation is in how hiv positivity was determined. to be considered hiv+ for this study, an icd-9 diagnosis code for hiv must have been present. however, hiv is not a mandated comorbid condition. therefore, it is possible hiv+ patients went unaccounted for, potentially further explaining the much lower prevalence of hiv in our study relative to the united states general population. additionally, the tqip database does not provide information on a patient's cd4 count, viral load, or treatment, which creates a non-homogenous population of hiv+ patients. furthermore, we do not have any information regarding baseline renal function or prior history of infectious complications in our patient population, which may be serving as potential confounders. an additional important point to consider is the finding of no statistically significant difference in mortality rate between the two study groups despite the rate being nearly double in the hiv+ group. this may represent a type ii error and should be investigated further with a larger multicenter study. finally, as a retrospective study, our findings cannot be interpreted as causation. our findings can only serve as markers of a correlation that merits future exploration. hiv+ trauma patients had a similar rate of mortality and infectious complications compared to a matched population of hiv− trauma patients. this may be due to the advent of cart. however, hiv positivity is associated with an increased incidence of aki and ards in trauma patients. future clinical studies and basic science research investigating biochemical and/or physiological pathways that may predispose hiv+ patients to these inflammatory complications are warranted. author contributions jc, jn, ag were involved in the development of the study design as well as completion of the manuscript. ag compiled the necessary study statistics. all authors were involved in the analysis of data and critical revisions to the manuscript. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors report no conflict of interest, financial or otherwise. informed consent this retrospective study involved humans but since a national database was used with de-identified patients, risk was minimal to participants. no consent was required. joint united nations programme on hiv/ aids (unaids) cdc. estimated hiv incidence and prevalence in the united states world health organization (who) assessing the impact of hiv status on injury outcomes: a multicenter study of trauma patients in rwanda human immunodeficiency virus infection in trauma patients: where do we stand? does hiv influence outcomes after trauma? effects of human immunodeficiency virus status on trauma outcome: a review of the national trauma database the impact of select chronic diseases on outcomes after trauma: a study from the national trauma data bank the effect of human-immunodeficiency virus status on outcomes in penetrating abdominal trauma: an interim analysis narrowing the gap in life expectancy between hiv-infected and hiv-uninfected individuals with access to care trends in life expectancy of hiv-positive adults on antiretroviral therapy across the globe: comparisons with general population life expectancy of hivpositive people after starting combination antiretroviral therapy: a meta-analysis methodology and analytic rationale for the american college of surgeons trauma quality improvement program optimal caliper widths for propensity-score matching when estimating differences in means and differences in proportions in observational studies past, present, and future: 30 years of hiv research monitoring selected national hiv prevention and care objectives by using hiv surveillance data-united states and 6 dependent areas vital signs: hiv prevention through care and treatment-united states hiv-associated nephropathies: epidemiology, pathology, mechanisms and treatment systemic inflammation after trauma the role of polymorphonuclear neutrophils during hiv-1 infection acute respiratory distress syndrome: pathophysiology and therapeutic options hiv care and the incidence of acute renal failure antiretrovirals and the kidney in current clinical practice: renal pharmacokinetics, alterations of renal function and renal toxicity estimated glomerular filtration rate, chronic kidney disease and antiretroviral drug use in hiv-positive patients association between antiretroviral exposure and renal impairment among hiv-positive persons with normal baseline renal function: the d:a:d study hiv and kidney diseases: 35 years of history and consequences key: cord-350571-6tapkjb6 authors: nan title: 45th escp-nsf international symposium on clinical pharmacy: clinical pharmacy tackling inequalities and access to health care. oslo, norway, 5–7 october 2016 date: 2017-01-10 journal: int j clin pharm doi: 10.1007/s11096-016-0404-4 sha: doc_id: 350571 cord_uid: 6tapkjb6 nan pharmacy, sint maartenskliniek, ubbergen, 2 pharmacy, radboud university medical centre, nijmegen, 3 clinical pharmacy and toxicology, maastricht university medical centre, maastricht, netherlands please specify your abstract type: research abstract background and objective: according to literature adherence to statins ranges from 32 to 71%. medication adherence is affected by both practical barriers and patient's beliefs about medication. however, physicians also have their beliefs about medication. several studies have shown that these beliefs also impact the decision of patients to agree with a particular treatment or not. as current published interventions on medication adherence (which focus predominantly on patients) are not or just partly effective, physicians' beliefs might be a promising target for interventions to improve adherence. however, there is currently no information available on physician's beliefs about statins and whether these beliefs affect patient's beliefs and adherence. therefore, the objective of this study is to examine whether physicians' beliefs about statins influence the beliefs and adherence of patients using a statin. setting and method: this cross-sectional study was conducted in gp practices and community pharmacies, between september 3, 2014 and march 20, 2015. physicians' and patients' beliefs about statins were assessed with the beliefs about medicine questionnaire (bmq) specific. patients' adherence on statins was assessed with both the mars-5 and the morisky-8 questionnaires. please specify your abstract type: research abstract background and objective: nhs highland and nhs western isles are the most remote and rural health boards in the united kingdom, with high numbers of dispensing medical practices. a pilot is underway in dispensing practices with clinical pharmacists undertaking targeted medication reviews. a previous quantitative service evaluation demonstrated its value, with pharmaceutical care issues identified in almost all patients, the vast majority of which (86.7%) were managed by the pharmacist without any need for general practitioner (gp) referral. the objective was to undertake a qualitative exploration of the service. setting and method: all patients and staff involved in the service were invited to participate. a semi-structured interview schedule was developed and piloted. telephone interviews were conducted with all consenting staff and a purposive sample of consenting patients recruited to the point of data saturation. interviews were audiorecorded, transcribed verbatim and analysed thematically. nhs ethics and research and development approvals were obtained. were the most confident with doacs (range from 73.4 to 75.7%) please specify your abstract type: research abstract background and objective: patients are at risk of drug-related problems (drps) at transition points during hospitalization. the community pharmacist (cp) is often the first healthcare professional patients visit after discharge. cps lack sufficient information about the patient and so they may be unable to identify problems in medications, which may lead to dispensing the wrong drugs or dosage, and/or giving wrong information. we aim to assess the impact of a complex intervention comprising of medication reconciliation performed at discharge by a hospital pharmacist (hp) with communication between the hp and cp on drps during the 7 days following discharge. setting and method: cluster randomized crossover trial involving medical and surgery care units (each unit corresponding to a cluster) in french hospitals during two consecutive 14-day periods, randomly assigned as 'experimental'(e) or 'control' c (usual care) periods. during the experimental period, the hp performed a medication reconciliation that was communicated to the patient's cp. main outcome measures: the primary outcome was a composite outcome of any kind of drp (prescription/dispensation, gap or patient) during the 7 days following discharge assessed at day seven post-discharge by phone from patient and cp. the secondary outcomes were 1/unplanned hospitalizations assessed by phone contact at day 35 after discharge and 2/the iatrogenic potential exposure scale from 0 to 3 for each patient established by a clinical team. analysis was conducted in intention to treat. results: 22 hospitals corresponding to 48 clusters enrolled 1092 patients (536 e group v/s 548 c group). no difference was observed on age, sex, autonomy, and number of drugs in home medication at admission and discharge. at day 7; 236 (45.6%) patients in e group had at least one drp v/s 280 (52.6%) in c group (or 0.77; ic 95% [0.61; 0.98] p = 0.034). intervention was especially efficient for patient discharged from surgery unit (or 0.64 ic 95% [0.43; 0.94]) and aged less than 75 years (or 0.71 ic 95% [0.53; 0.94] . although intervention decreased patient exposure to drp with high iatrogenic potential (from 8.7 to 5.2% p \ 0.0006), un-planned hospitalizations at day 35 weren't different between groups (5.8 vs. 4.5% p = 0.50). conclusion: medication reconciliation associated to communication between hospital and community pharmacists is efficient to decrease patient exposure to drp but not sufficient to decrease un-planed hospitalization. hp-pc003: clinical pharmacists bridging health care levels by medication reviews in primary care katherine wendelbo *,1 , kristine lundereng 2 1 namsos hospital pharmacy, central norway hospital pharmacy trust, namsos, 2 levanger hospital pharmacy, central norway hospital pharmacy trust, levanger, norway please specify your abstract type: descriptive abstract (for projects) background and objective: nord-trøndelag county is sparsely populated and many inhabitants live far from the hospital. additionally, only half (12 of 23) of the municipalities have a local pharmacy. traditionally, namsos and levanger hospital pharmacies have performed quality audits of the implementation of drug administration procedures in primary care units. since 2013, a service where clinical pharmacists participate in multidisciplinary medication reviews in 19 municipalities throughout the county has been established. the objective of this poster is to describe the practical approach and design of the service. design: a descriptive report of an implemented clinical pharmacy service in primary care where clinical pharmacists, as part of multidisciplinary teams, perform medication reviews. results: medication reviews are performed on patients admitted to nursing homes and patients in home care, receiving help with handling of their drugs. primary care nurses prioritise patients (by selecting frail elderly with multiple co-morbidities and polypharmacy), usually five patients in each meeting. prior to the review, nurses collect medical information using a checklist including; diagnosis, drug-related symptoms, standard laboratory tests and an updated medication list. the clinical pharmacist receives de-identified medical information by postal mail or e-mail before the meeting. based on this information the pharmacist identifies possible drugrelated problems (drps) and provides recommendations on how to solve them. this is performed in a structured approach according to the integrated medicine management (imm) model. subsequently, the pharmacist visits the municipality and discusses the medication reviews in a multidisciplinary team meeting with nurses and physicians. in addition, the pharmacist gives lectures in a medication related topic (e.g. treatment of insomnia and anxiety, oral anticoagulants and cognitive side effects). following the meeting, the pharmacist reports the drps and suggested interventions to the multidisciplinary team, for further follow-up. during 2015, totally 220 medication reviews were performed in 19 municipalities. in the same period, 25 lectures were given by the clinical pharmacists. conclusion: this clinical pharmacy service enables multidisciplinary medication reviews even in municipalities with limited health professionals and resources. as a part of multidisciplinary teams, the clinical pharmacists contribute with medical competence. camille castel 1 , arnaud de la blanchardière 2 , vincent cattoir 3 , guillaume saint-lorant *,1 1 pharmacy, 2 infectious and tropical diseases, 3 microbiology, chu caen, caen, france please specify your abstract type: research abstract background and objective: antimicrobial stewardship have clearly demonstrated their efficiency towards a more adequate use of antibiotics. since 2014, the use of daptomycin, a ''critical last resort antibiotic'' has intensified in our hospital, occasionally outside the scope of its approved indications. this situation has led to the implementation of an antimicrobial stewardship and the drafting of local guidelines. the aim of this study is to analyse the evolution and pertinence of daptomycin prescriptions, after distribution of these guidelines within our institution. setting and method: a monocentric prospective study was conducted between july 2015 and november 2015 in a 1500-bed university hospital. each daptomycin prescription recorded by pharmacy department was analysed by an infectious diseases specialist in the presence of the prescriber and considering local guidelines and the patient's clinical conditions. main outcome measures: the indicators chosen to determine prescription pertinence were: treatment indication, prescribed dose and other antibiotics associated with the daptomycin prescription. results: 20 daptomycin prescriptions were analysed. observed indications were: sepsis (35%), infective endocarditis (25%), bone and joint infections (25%) and vascular prosthetic infections (10%). identified pathogens were: mrsa (35%), methicillin-resistant coagulase-negative staphylococci (25%), methicillin-sensitive staphylococcus aureus (10%), enterococci (10%) and methicillinsensitive coagulase-negative staphylococci (5%). daptomycin was prescribed as first-line treatment in 50% of cases. the mean dose was 7 mg/kg/day [3-10 mg/kg/day] for a mean duration of 17 days [2; 55 days] . local guidelines were followed in 20% of cases. daptomycin use was relevant for 70% of prescriptions. the irrelevant prescriptions triggered the modification or stoppage of antibiotic therapy in 50% of cases, respectively, generating an 18% decrease in consumption and an economy of over €6700 for our institution. conclusion: this study shows the efficiency of antimicrobial stewardship in adequately using antibiotics, limitating ecological impacts, improving patient care and decreasing healthcare costs. it also shows that guidelines alone are insufficient to ensure a proper use of antibiotics. without a close prescription follow-up, constant reminders and sustainable evaluations, guidelines only affect a few prescribers. within the context of an ''antimicrobial crisis'', further development of guidelines and antimicrobial stewardship is essential to fight increasing bacterial resistances and requires a close collaboration between all healthcare professionals including pharmacists. interviews were transcribed verbatim and data were analysed using systematic text condensation. results: three major themes were identified: benefits, unrealised potential and criteria and barriers for success. (1) benefits described by physicians included increased patient safety, increased awareness on drugs, and an ease of workload. drug interaction management was emphasized as one of the clinical pharmacists' most important work tasks, as well as being a resource for collaborating healthcare professions and to the patient himself. (2) the clinical pharmacists expressed that they had an unrealised potential and could contribute to a greater extent in the multidisciplinary team than they did already. they mentioned education towards physicians and nurses, contribution in treatment decision-making and patient counselling as examples for possible extended work tasks. (3) as criteria to succeed as a clinical pharmacist, physicians highlighted the importance of oral communication and physical presence on the wards. as barriers for integration in the team, the clinical pharmacists identified the physicians' lack of knowledge about the clinical pharmacists' skills as well as unclear expectations regarding their responsibilities. conclusion: physicians agreed that the clinical pharmacist represent a valuable contribution to the multidisciplinary team, where patient safety and drug interaction management are highlighted as main benefits. clinical pharmacists should to a greater extent educate healthcare professions in drug related topics and provide patient counselling. continuous effort on making the clinical pharmacist a natural part of the multidisciplinary team is crucial for the development of clinical pharmacy. by gathering perceptions from the collaborating professions as well as educating them on what clinical pharmacists can provide, we can develop a multidisciplinary team that enhances patient safety. hp-pc006: assessment of dual antiplatelet therapy following acute coronary syndrome using grace and crusade sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: mortality and morbidity benefits of dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) have been unequivocally demonstrated in a large body of evidence. with the availability of more potent antiplatelet agents, balancing ischemic and bleeding risks to prevent adverse outcomes is an on-going challenge, in particular, recognising that patients with high bleeding risk were excluded from clinical trials. grace and crusade scores stratify risk of mortality and in-hospital major bleeding post acs respectively. these tools should be used to support antiplatelet choice in light of newer more potent agents that equally pose a greater risk of bleeding. design: grace and crusade scores were calculated for patients presenting with acs. clopidogrel was recommended for patients with a high or very high crusade score (greater bleeding risk). ticagrelor was recommended for patients presenting with st-elevation myocardial infarction (stemi) or those with nsteacs with a grace score of intermediate or above (greater ischemic risk) and a crusade score of moderate or less (low bleeding risk). in either case, treatment was at the discretion of the clinician and patients received concomitant aspirin. a registry was collated of risk scores, diagnosis and choice of antiplatelet therapy. results: 1030 patients were included in the registry, of which 587 (57%) presented with stemi and 443 (43%) presented with nsteacs. 558 of 752 (74%) patients with a greater ischemic risk received ticagrelor as part of their dapt regime. advanced age, concomitant anticoagulation and those awaiting surgery were the most common reasons for patients with a greater ischemic risk to receive clopidogrel. 145 (14%) had a high or very high crusade score. of these, 130 (90%) received clopidogrel as part of their dapt regime. conclusion: risk stratification was streamlined using the data collection tool and useful to support choice of dapt. european society of cardiology (esc) guidance recommends use of established risk scores for prognosis and bleeding; however evidence to correlate to choice of dapt is lacking. outcome data is currently being reviewed and will provide further evidence to correlate choice of dapt to grace and crusade scores. please specify your abstract type: descriptive abstract (for projects) background and objective: in europe, approximately 25% of the patients with the human immunodeficiency virus (hiv) infection are co-infected with the hepatitis c virus (hcv). treatment recommendations in hiv/hcv co-infected patients are identical to those in patients with hcv mono-infection. however, potential drug-drug interactions (ddis) between antiretroviral agents and new direct-antiviral agents (daas) imply the need of a careful selection of the hcv treatment regimen. the aim of the present study was to evaluate the need of a change in the antiretroviral therapy (art) due to potential ddis in patients with hiv/hcv co-infection who started treatment for hcv with new daas. we also assessed the effectiveness of hcv treatment 12 weeks after hcv treatment completion. design: we retrospectively registered clinical data about hcv and hiv management: hcv genotype, fibrosis metavir score, initial hcv viral load, hcv treatment and previous art regimen. we recorded the changes in art prior to starting hcv treatment and the reason of this switch (ddi, simplification or duplication of the therapy). results: between february 2015 and january 2016, 50 hiv/hcv coinfected patients started hcv treatment with a daas regimen. of them, 39 had advanced liver disease (fibrosis score: f3/f4) and 27 were infected with hcv genotype 1. prior to starting hcv treatment, 29 patients needed a switch in art regimen due to potential ddis with daas. simeprevir and the co-formulation ombitasvir/paritaprevir/ritonavir were the daas most frequently implicated in ddi with protease inhibitors or non-nucleoside reverse transcriptase inhibitors: 19/29 and 5/29, respectively. also, we observed some changes of art due to other causes. five switches occurred to adequate the regimen (discontinuation of ritonavir in candidates to take the co-formulation ombitasvir/paritaprevir/ ritonavir or art improvement to decrease pill burden). as for hcv treatment effectiveness, 42/50 (84%) patients achieved sustained viral response 12 weeks after therapy completion. conclusion: a large proportion of patients with hiv/hcv co-infection who initiate treatment with daas for hcv need to switch art due to potential interactions that may impact on effectiveness and safety of both treatments. additionally, some changes in art treatment are made to facilitate therapeutic adherence. these results highlight the need of a multidisciplinary approach in which interactions between art and hcv treatments should be carefully assessed. please specify your abstract type: descriptive abstract (for projects) background and objective: the potential impact of polymedication, iatrogenic events and medication error is a serious concern in hospitalized patients. clinical pharmacists can limit these risks by identify high risk. the aim of this study are to identify in six medical units high risk patients by using three predictive scores of rehospitalisation (8 ps) 1 , early mortality (charlson) 2 and drug related problems (drp) 3 . design: 49 clinical and therapeutic variables in 216 patients were collected through medical records and prescriptions by clinical pharmacists. scores were calculated during 2 months in six units (internal medicine, n = 30; nephrology, n = 35; geriatrics, n = 35; rheumatology, n = 45; cardiology, n = 54 and endocrinology, n = 29). the data were analysed by mann and whitney test for the continuous variables and chi square test for the qualitative variables. the coefficient of correlation between the three scores were calculated by a pearson test for normal distribution and by a spearman test for non normal distribution. patients were considered at a high risk for re-hospitalization (8ps [ 2) , early mortality (charlson [ 5) and iatrogenic events (drp c 8) . results: in the general population, the average age was 67.4 ± 18.1 years old and the sex ratio was 0.94. the average treatment used was 7.8 ± 4. charlson scores were higher in geriatric unit (6.3 ± 1.8) follow by medical interne unit (5.7 ± 2.7). the 8ps and drp scores were higher in nephrology unit respectively 2.5 ± 0.9 and 9.1 ± 2.6 follow by internal medecine unit 2.2 ± 1.3 and 7.6 ± 2.7. on contrary the rheumatology unit presented the lower level for the three scores. 51 patients were considered at high risk for three scores, 33% (n = 17) in nephrology unit (almost 49% of unit), 20% (n = 10) in geriatric unit, 18% (n = 9) in internal medicine unit, 18% (n = 9) in cardiology unit, 6% (n = 3) in endocrinology unit and 6% (n = 3) in rheumatology unit. conclusion: knowledge of the variables associated with these predictor scores could help clinical pharmacists to prioritise various medicine units and target those at risk. we identified especially three units at risk: nephrology, geriatric and internal medicine. thanks to these results, clinical pharmacists can rapidly and efficiently target patients who present iatrogenic and/or re-hospitalization risks. design: a retrospective observational analysis was conducted in our hospital, based on medical records of patients presenting atrial fibrillation (af) and treated by doacs from january 2011 to may 2016. to identify patients hospitalized due to severe bleeding, we analysed prothrombin complex concentrates (pccs) and activated pccs prescriptions, as well as pharmacovigilance declarations. results: 1328 patients were treated with doacs: 763 with rivaroxaban (57.5%), 286 with dabigatran (21.5%) and 279 with apixaban (21%). fifty-nine (4.4%) patients experienced at least one bleeding leading to hospitalization: 35 with rivaroxaban (4.6%), 16 with dagibatran (5.6%) and 8 with apixaban (2.9%). thirty-eight severe bleeding were identified (2.9%): 24 occurred with rivaroxaban (3.1%), 10 with dabigatran (3.5%) and 4 with apixaban (1.4%). they included 10 intracranial bleeding (26%) and 21 gastro-intestinal bleeding (55%). seven haemorrhages resulted in hypovolemic shock (dabigatran:4, rivaroxaban:2, apixaban:1) and 2 of them were fatal (dabigatran:2). rates of bleeding (p = 0.86, v 2 test) and of severe bleeding (p = 0.85, v 2 test) were not statistically different for the three molecules. in case of major haemorrhage, the recommended factor concentrate in our protocols differs between the anticoagulant. with dabigatran, the antidote idarucizumab (5 g, intravenously) should be administered, without waiting for plasma concentration results. with rivaroxaban, apixaban or unknown doacs, pcc (30-50 units/kg) is indicated. in case of pcc failure, activated pcc (30-50 units/kg) is suggested. pcc, activated pcc or idarucizumab (2) were used in 15/38 patients (40%). in rivaroxaban and apixaban-related haemorrhages, 10 patients received activated pcc: two had a 20 ui/kg dose and one had a 60 ui/kg dose. regarding dabigatran-related bleeding, one patient received pcc instead of idarucizumab. compliance with local recommendations was 92% (35, p [ 0.003, v 2 test) 0.9 pharmacovigilance reports were issued. conclusion: management of doacs-associated severe bleeding in our hospital respects local protocols. it should also be pointed out that patients with life threatening bleeding may benefit from pcc. however, the risk of thrombosis associated with pcc must be weighed against the risk of haemorrhage. since specific antidotes are emerging, like idarucizumab or andexanet alpha, new guidelines for doacs-related haemorrhage are expected. please specify your abstract type: descriptive abstract (for projects) background and objective: this project is part of a prospective quasi experimental proof-of-concept investigation of a clinical pharmacist intervention to reduce drug-related problems among people admitted to a ward in a rural hospital in northern sweden. the aim of this particular study is to explore doctors' and nurses' expectations of having a ward-based pharmacist providing clinical pharmacy services in a rural hospital. design: eighteen face to face semi-structured interviews were conducted with a purposive sample of doctors and nurses working on the ward were the clinical pharmacy service was going to be implemented. semi-structured interviews were digitally recorded, transcribed and analysed using thematic analysis. results: the majority of participants had limited experience or a vague idea of what pharmacists are able to do in a ward. most participants described traditional roles such as inventory, drug distribution and dispensing. most respondents were unaware of the pharmacists' knowledge, skills and competences. for some it was unclear how having a clinical pharmacist in the ward was going to impact on their workload this was particularly important for the nurses. some doctors (mainly experienced) were concerned that having a pharmacist may mean losing or not gaining competence on drugs. for others it was unclear how the pharmacists' will work with patients or what clinical skills they have. however most participants were positive about the implementation of the new service. conclusion: this study provided a rare opportunity to explore the doctors' and nurses expectations of the role of clinical pharmacists before a clinical pharmacy service was implemented. the results showed that the participants' expectations of the clinical pharmacist role were unclear. to successfully implement clinical pharmacy services in a clinical pharmacy ''naïve'' setting; roles, clinical competence and responsibilities should be clearly described. furthermore, it is important to focus on inter professional collaborations between doctors, nurses and pharmacists. practical for the local hospital setting. seven out of 9 experts agreed with pharmacist prescribing for the conditions identified. pharmacists (n = 31) were more willing to prescribe antihypertensive and antidiabetic medication (87.1%) when compared to oral anticoagulants (64.5%). these values are higher than those obtained by vella in 2014. the majority of pharmacists (87.1%) recommended that pharmacists should take up further studies to a master or doctorate level degree in a clinical aspect in order to be authorised to prescribe. conclusion: the developed framework for pharmacist prescribing and the guidelines developed for pharmacist prescribing of oral anticoagulants and pharmacotherapy of hypertension and diabetes mellitus were shown to be reliable and were accepted by pharmacists and physicians. please specify your abstract type: research abstract background and objective: valproic acid (vpa) and its derivates and mycophenolate mofetil (mmf) and mycophenolic acid used during pregnancy increase risk of congenital malformation and cognitive impairment. thus, the french national agency for medicines and health products safety (ansm) decided to establish new conditions of prescription and dispensation of drugs containing vpa (may 2015) and mmf (april 2016). a signed care agreement and a co-prescription of contraceptives are now mandatory in the drug dispensation for reproductive-age adolescent girls and adult women. this study will describe the impact of these new guidelines on our practice. our objective is to compare the vpa and mmf media coverage and the impact on the prescriptions. setting and method: we compared the mass communication between vpa and mmf on social media, webpages and journal article (public and professional journal) on google and googletrends in the first months around these new rules. we combined different keywords such as ''accord de soins'' and the drug name. in the same time, we collected and analysed vpa and mmf prescribing and dispensing data and compared it to the 2015 data for the first 6 months. main outcome measures: results: just before the vpa rule, the vpa was presented in the general press as the new health scandal after benfluorex mediator°w ith 100 google searches in march 2016 compared to 10 searches usually per month. simple research combining keywords reveal always more than twice more webpages concerning vpa than mmf. at the same time, a patients association (renaloo for renal failure) wrote to the ansm to contest the new rule with the double contraception and without any consultation of patients association. in our daily practice we also faced some physician reluctant to sign this prescription agreement with patient (too many agreements already asked, decision of ansm without any consultation of learned societies). the care agreements are kept in the patient records, a statement ''care agreement signed'' is reported in the electronic prescription of vpa and mmf. the overall consumption of mmf and vpa increase for respectively the first 2 and 3 months after rule implementation (from +122 to +326%) except for the micropakin 500 mg. the 6 months mmf data will be presented for the final communication. conclusion: the media pressure and the new regulation have an impact on prescription trends. these new prescription and dispensing rules concerned two different contexts: pathology, media coverage, possible drug alternatives. we were faced to some difficulty in implementing the new guidelines, which reveals a certain reluctance of the prescribers or the patients represented by associations. tdmp001: vancomycin trough serum concentrations are frequently subtherapeutic in a population of critically ill patients: a prospective observational study please specify your abstract type: descriptive abstract (for projects) background and objective: to design and characterise a framework of international pharmacy standards for pharmaceutical care application on oral anticoagulation for prevention of atrial fibrillation (af) related strokes. design: literature review (including existing international guidelines and quality measures) was conducted to characterise the standards and design an international framework for pharmaceutical practice application on oral anticoagulation for prevention of af-related strokes. expert opinions were sought through a delphi method to reach consensus on the framework domains and standards. results: the framework consisted of twelve overarching standards, which were defined and grouped into four domains as follows; ([personal care package]:-communication with patients, support decision making process, education and counselling, adherence. [medicines optimisation]:-clinical review and therapy optimisation, initiation and control, maintenance, supply and transfer between care settings. [workforce]:-workforce planning, training and development, analysing information; and [governance]:-assurance of service provision) specific to oral anticoagulation in prevention of af-related stroke. each standard was also categorised within dimensions and supporting statements to describe what a quality pharmacy service should deliver. a total of forty-five dimensions and twelve statements were incorporated into the framework. conclusion: a clearly defined framework of international standards was developed as a clinical tool and quality assurance to optimise the delivery of care for oral anticoagulation in prevention of af-related strokes. it will support pharmacists and their teams to develop their professional practice, improve services, and deliver safe and high quality patient care across all pharmacy settings. poster discussion forum i: community pharmacy and public health cp-pc004: nurses' and pharmacists' learning experiences from participating in inter professional medication reviews in primary health care: a qualitative study hege t. bell *,1 , anne gerd granås 2 , ragnhild omli 3 , ingela enmarker 4 , aslak steinsbekk 5 1 nord university/ntnu, trondheim, 2 hioa, oslo, 3 nord university, namsos, norway, 4 department of nursing, østersund, sweden, 5 ntnu, trondheim, norway please specify your abstract type: research abstract background and objective: traditionally, drug prescription and follow up have been the sole responsibility of physicians. however, interprofessional medication reviews (imrs) have been developed to prevent drug discrepancies and patient harm. what participating nurses and pharmacists learn from each other during imr is poorly studied. the aim of this study was to investigate nurses' and pharmacists' perceived learning experience after participating in imrs in primary health care for up to 2 years. setting and method: a qualitative study with semi-structured focus group interviews and telephone interviews with nurses and pharmacists with experience from imrs in nursing homes and home based services. the data was analysed thematically by using systematic text condensation. main outcome measures: a qualitative method is useful when looking at objects from the perspective of how they are experienced. results: sixteen nurses and four pharmacists were interviewed. the nurses' perception of the pharmacist changed from being a controller of drug management routines towards being a source of pharmacotherapy knowledge and a discussant partner of appropriate drug therapy in the elderly. the pharmacists became more aware of the nurses' crucial role of providing clinical information about the patient to enable individual advice. increasingly the nurses learned to link the patient's symptoms of effect and side effect to the drugs prescribed. with time both professions jointly spoke of an increased awareness of the benefit of working as a team and the perception of contributing to better and more individual care. conclusion: imrs in primary health care meet some challenges especially concerning how to ensure participation of all three professions and how to get thorough information about the patient. possible solutions might be to use shared communication tools like internet based communication programs and to introduce the patient as a participant at the imrs. please specify your abstract type: research abstract background and objective: international good pharmacy practice guidelines describe how pharmacists should counsel the patients about their medicines, offer additional services where needed, and intervene at drug related problems. daily practice often differs from theory. this study aimed at illustrating the whole process of prescribed medicines dispensing in daily community pharmacy practice. part b of the project focuses on pharmacists' opinions. setting and method: community pharmacies in basel, switzerland, were invited in random order for study participation. one master student in pharmacy performed non-participant observations during 1 day at each included community pharmacy. at dispensing of prescribed medicines, patient data, content of counselling, communication style, and provision of further services (e.g. follow-up offer) were documented on a checklist with predefined themes. interventions were documented systematically. a semi-structured interview on the pharmacists' opinions about the counselling, triggers, facilitators and barriers, and the documentation of interventions was conducted at each community pharmacy. main outcome measures: counselling content at prescription dispensing by numbers and by pharmacists' opinions; barriers, facilitators, and triggers for counselling at prescription dispensing. results: in march and april 2016, 18 of 49 invited community pharmacies participated in the study. out of 561 documented observation periods, 556 encounters were analysed (first prescription: 269/refill prescription: 287). counselling was provided to 367 (66.0%) clients with an average of 2.9 (±3.1) themes per encounter. a total of 148 clients refused counselling. themes most counselled at first and refill prescription dispensing were: drug intake (476/80), dosage (191/50) , and administration (163/38). for the pharmacists (n = 18), most important themes to be discussed at first prescription dispensing were indication (11), administration (9), and anamnesis (8); for refill prescription dispensing they were adherence (9), therapy benefits (9), and adverse effects (7). the majority of pharmacists (13) felt that it was their obligation to ask questions about patients' health during the dispensing of prescription medicines and named trigger (e.g. patient knowledge gap, patient motivation, interactions), but one-third reported difficulties with it. barriers were refusal by patients (13), communication problems (language, 7), lack of medical data (3) , and lack of time (3) . conclusion: a discrepancy in counselling content by observation compared to pharmacists' opinions was revealed. this might indicate that pharmacists are aware but hindered by barriers to practice according to good pharmacy practice guidelines. please specify your abstract type: research abstract background and objective: the 2020 workforce vision in scotland envisages 'making more and better use of technology …to increase access to services and improve efficiency' across the healthcare interface. services offered by community pharmacy remain limited by lack of shared access to patients' clinical information. in scotland, every patient has a unique identifier, their chi (community health index), which facilitates identification of/searching for patient records. the aim of this research was to explore the experiences of community pharmacists granted clinical portal access to patients' records. setting and method: from april 2015, community pharmacists across nhs tayside (n = 21) who had completed technical and information governance training were invited to maintain a portal log of their experiences of using the clinical portal to access patient records. each was asked to record when/why they considered accessing a patient's record and whether their information needs were met. portal logs were subject to independent summative content analysis by two researchers. this study gained ethical approval from robert gordon university. main outcome measures: not applicable. results: clinical portal logs were received from most participating pharmacists (n = 18/21). two were unavailable (moved to hospital setting; maternity leave). a third had not had occasion to access the clinical portal which he speculated was due to not working at weekends but also raised concerns about gaining patient consent. frequency of seven identified themes provided a partial indication of balance of reasons for usage. firstly (#1), to confirm a patient's prescription (n = 48), secondly (#2), for additional information (n = 46). less frequently (#3), portal access was to check repeat medications (n = 23). other reasons for access were (#4) to check hospital discharge (n = 21) followed by (#5) check on multi-compartment appliance aid status (n = 14) or (#6) check the emergency care summary (n = 11). there were also instances (#7) when portal access was not found to be helpful (n = 16) so traditional offline routes were followed. conclusion: preliminary findings indicate mainly positive experiences with no technical issues raised and community pharmacists' information needs largely met. although limited to a small number of pharmacists in only one health board, findings support scottish policy aims, including 'prescription for excellence.' further work is underway around patients' perspectives of community pharmacist access. please specify your abstract type: research abstract background and objective: multicompartment compliance aids (mcas) such as the multidrug punch cards pharmis ò are used to support patients in the daily management of their medication. solid oral medicines are unpacked from their original packaging and repacked in mcas although controversy exists about the stability of repackaged medicines. different countries published contradictory lists of medicines not recommended for repackaging. we aimed to define and apply criteria able to assess visual alteration of medicines repackaged in mcas. setting and method: eight criteria describing physical alteration of tablets/capsules were retrieved from the who international pharmacopoeia 2015: chipping, swelling, capping, rough surface, cracking, crushing under pressure, mottling, and discoloration. absence of one criteria gives 1 point. a maximum score of 8 points can be obtained. twenty-two critical medicines and three half tablets were repackaged in the multidrug punch cards pharmis ò and stored at accelerated conditions (40°c/75% rh) for 4 weeks. original blisters of medicines were stored at room temperature as control. each tablet/capsule was visually inspected after 7, 14, 21 and 28 days. main outcome measures: score according to eight criteria. results: after 4 weeks, 17 tablets/capsules including 2 of the half tablets showed no visual alteration and were identical to controls. a reduced score (3-7 points) was given to seven repackaged medicines and one half tablet within 4 weeks: madopar ò (3), pravastatin sandoz ò (4), carvedilol mepha ò (6), plavix ò (6), pantoprazol nycomed ò (7), adalat ò cr (7). swelling and rough surface were the most frequent. chipping and capping were not observed. conclusion: our eight visual criteria are able to detect physical alteration of repackaged solid oral medicines under stress conditions. in absence of reliable data we suggest to apply this simple quality control for repackaged medicines in pharmacy practice. because chemical stability testing is not feasible in practice, pragmatic solutions are sought. further studies are needed for storage at room temperature. cp-pc008: drug-related problems and symptom burden in nursing home residents kerstin bitter *,1 , ulrich jaehde 1 , christina pehe 2 , gabriela heuer 3 , manfred krü ger 3 1 clinical pharmacy, university of bonn, bonn, 2 aok rheinland/ hamburg, 3 pharmacists' association north rhine, düsseldorf, germany please specify your abstract type: research abstract background and objective: drug-related problems (drp) are common in the elderly due to polymedication. community pharmacies supplying drugs to nursing homes may play an important role in detecting and solving drp in nursing home residents. this project aims to evaluate whether community pharmacists can enhance the medication safety of nursing home residents by solving drp by means of a simple medication review (mr). furthermore, the applicability of a new tool to detect symptoms as potential adverse drug reactions in elderly patients should be tested. setting and method: nursing home residents at the minimum age of 65 years insured by aok rheinland/hamburg and regularly taking at least five drugs per day were invited to participate. pharmacists performed a mr based solely on the patients' medication data, including dosage regimens of the nursing home and self-medication data. the detected and solved drp were counted. additionally, a simple questionnaire (sympel) was distributed to the patients periodically in order to assess their symptom burden. main outcome measures: frequency and type of the patients' drp as well as their symptom burden before and after the mr. results: after testing the feasibility of this intervention in a pilot study, 54 patients were included in the main study so far. in average, the pharmacists identified two drp per patient and reported to the responsible general practitioner (gp) . as in the pilot study, most frequent drp documented by pharmacists were drugdrug-interactions (34%). 29% of the pharmacists' recommendations were accepted by the gp. 46% of the patients took at least one drug considered as potentially inadequate in the elderly. out of six different symptoms, patients reported dizziness and bruises most frequently. conclusion: pharmacists can detect many drp in nursing home residents by means of a simple mr. however, the full potential of this service to solve drp can only be exploited if the cooperation with the gps is improved. please specify your abstract type: research abstract background and objective: medicines for rare diseases (rd) are costly and have limited efficacy evidence but they represent around 20% of all innovative medicines. therefore, countries are facing challenges in providing patient access to them. the purpose of the study was to assess the patient access for slovenia and compare it with 23 other european countries in the last decade. setting and method: the medicines for rd that obtained marketing approval between 2005 and 2014 via centralised procedure were included in the study based on the orphanet list from january 2016. using the quarterly ims health database, sales data were analysed for slovenia and 21 other european union countries, norway and switzerland. patient access was assessed for each country and comparisons between the countries were made using the three main outcome measures. main outcome measures: the number of medicines for rd available; time to first continuous use after marketing approval; total pharmaceutical expenditure for medicines for rd in euros. results: altogether, 125 medicines for rd were approved between 2005 and 2014. complete sales data were available for 120 medicines which were included in the comparison. for germany and the united kingdom the continuous use of 102 (85%) and 94 (78%) was observed, respectively. the following italy, france, denmark and sweden use 60-70% of all the medicines. in slovenia, 66 (55%) medicines for rd were introduced. germany and the united kingdom times to first continuous use were the shortest (median time 1-3 months after marketing approval). median times below 12 months were observed for norway, sweden, austria, the netherlands, france and switzerland. other countries were slower in enabling first continuous use (median time from 12 to 34 months). germany, france, switzerland had the largest pharmaceutical expenditure per inhabitant in 2014 (29.2, 25.0 and 24.0 euros/inhabitant) while slovenia amounted to 18.5 euros/inhabitant. in slovenia more than a half of the medicines for rd approved in europe are used which ranks it in the middle of all the countries in comparison. comparing to the important european pharmaceutical markets like germany, united kingdom, france and italy, slovenia's median time to first continuous use is longer and pharmaceutical expenditure per inhabitant for these medicines is lower. pec002: mapping of the dlqi scores to eq-5d utility values using ordinal logistic regression and monte carlo simulations: is it plausible? please specify your abstract type: research abstract background and objective: converting dermatology life quality index (dlqi) scores to generic measure data would allow utility calculations and enable cost-effective analysis. this would meet the needs of health technology assessment agencies (htas) such as nice, who preferentially use the general health measure eq-5d. the dlqi is a specialty-specific measure unlike the eq-5d, a generic measure from which utility values can be derived. often several measures are implemented in studies with increased cost and patient burden. ordinal logistic regression (olr) was used to develop a model to convert dlqi scores to eq-5d based utility values for use in economic appraisal of medicines. setting and method: data from 4010 patients were randomly divided into estimation and validation sets to fit and test the model. a series of ordinal logistic regressions were fitted in spss v22 for the five eq-5d dimensions based on age, sex and all 10 individual items of the dlqi as predictors. the model produced three estimated probabilities per subject per eq-5d domain. using these estimated probabilities, a series of 10 monte carlo (mc) simulations were run for each subject resulting in predicted domain responses. from these, utility values were calculated and compared to actual patient values. main outcome measures: conversion of dlqi scores to eq-5d domain data results: there are conceptual overlaps between items of the dlqi and eq-5d. the validation data set (which was not included in the creation of the model), demonstrated that the models were highly predictive compared to actual responses, except for minor differences for the pain/discomfort domain. for example, for the eq-5d ' mobility' domain, 1389 patients answered 'no' (predicted 1392 and 440 patients answered 'some or extreme' (predicted 437) . we examined the model's latent variables, which also demonstrated high predictability at individual level. for example for the 'usual activities' domain the mean latent variable scores were -2.49, -1.62 and -0.86 for those responding 'no', 'some' and 'extreme' respectively, showing a clear increase in the scores with response. after excluding subjects with missing variable data there were 1769 patients in the estimation set and 1773 in the validation set. the model was shown to be highly predictive and repeated simulations demonstrated a stable model. the average predicted utility value for the entire validation set ranged from 0.742 to 0.753 across the 10 mc simulations compared to the actual average utility value of 0.754. conclusion: using olr, we have developed a method of mapping the disease-specific dlqi onto the eq-5d: utility values may then be derived for population data sets, and possibly for individuals. the olr technique could be used to convert data from other disease-specific quality-of-life measures to generic utility data for incorporation into cost-effective analyses, greatly enhancing the potential value of such information. tomi laptoš *,1 , tanja kersnik levart 2 1 hospital pharmacy, 2 the division of paediatrics, department of nephrology, university medical centre ljubljana, ljubljana, slovenia please specify your abstract type: descriptive abstract (for projects) background and objective: having to take medication during time in school may present certain discomfort for some children. suboptimal dosing regimens (i.e. dosing more frequent than determined by drug's trough:peak ratio) can be prevented by a clinical pharmacist's overview and therapy optimization. the aim of the study was to review and evaluate dosage regiments in paediatric patients on antihypertensive therapy. dosage regiments are defined as schedule of doses of a therapeutic agent per unit of time and the amount of a medicine to be given at specific time. design: electronic health records (ehr) review, evaluation of actual dosage regiments against regiments recommended in smpcs and clinical database (uptodate ò ), a consecutive case series study. results: ehrs of 254 patients, admitted to or discharged from the department of nephrology of the division of paediatrics, umcl in 2015 with suspected icd-10 diagnoses from i10 to i15 were reviewed. 107 patients were excluded (diagnosis not confirmed or lifestyle-change disease management only). the remaining 147 patients (average age 15.7 years (range 3-20)) received daily on average 1.59 medications (range 1-4) in 2.06 individual doses (range [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . most frequently used drugs were perindopril (n = 57), ramipril (n = 47), amlodipine (n = 34), bisoprolol (n = 32) and doxazosin (n = 13). 10 patients received ex tempore oral suspensions. dosage regimen was not optimized in 17% (n = 25) of the patients, among those 4% (n = 6) receiving one medication only and 13% (n = 19) receiving more than one medication where at least one was not optimized. furthermore, 9% (n = 13) patients on stabledosage therapy (no dosage change of either medication in last 6 months with satisfactory clinical outcomes) were eligible for fixeddose combination medication. with optimized dosage regimens patients would receive daily on average 1.51 medication (range 1-4) (-5%) in 1.76 individual doses (range 1-9) (-15%). the difference was statistically significant (95% ci, p \ 0.05) in both cases. conclusion: our data show that the majority of the paediatric patients on antihypertensive therapy (83%) received their medication in optimal dosage regimens. however, with an estimated every fifth patient not being on optimal dosage regimen, a multidisciplinary approach is crucial to assure that the individual patient achieves the best clinical, humanistic and economic therapy outcomes. ph005: polypharmacy management programmes in the elderly: a case study in greece dimitra gennimata *,1 , christos kampolis 1 , aggelos vontetsianos 1 , jennifer mcintosh 2 , alpana mair 3 , on behalf of simpathy consortium please specify your abstract type: descriptive abstract (for projects) background and objective: polypharmacy management and medication adherence in the elderly are significant public health issues throughout the european union (eu). simpathy (stimulating innovation management of polypharmacy and adherence in the elderly) is a consortium of 10 organizations representing eight european countries, aiming at stimulating innovation around management of appropriate polypharmacy and adherence, ultimately providing tools for eu policy makers to develop and/or improve, implement and evaluate programs addressing these issues. design: a mixed-methods case study was carried out in greece, to identify policies on the management of polypharmacy and adherence issues in the elderly. a desk review of the polypharmacy and adherence policies at the government, regional and institutional level has been completed. key informant interviews were conducted with policymakers and health professionals responsible for developing and implementing strategies. focus groups consisting of policymakers, clinicians and patients validated the research findings. results: although e-prescription implementation is widespread (&98% coverage nationwide) and disease-specific guidelines have been developed, polypharmacy management is only associated with direct economic indicators. no formal policies or programmes are identified. significant contributions are coming from different health professional organizations that have chosen to provide expanded services to their patients, aiming at optimising drug therapy and thus polypharmacy management and medication adherence in the elderly. community pharmacists offer pharmaceutical care to patients, which includes management of prescribed medication, otc remedies, vitamins and supplements and food-drug interactions. hospital pharmacists in some state (public) hospitals review medication for inpatients and out-patients, communicate with prescribers and confirm the ''benefit-no harm'' principle in the prescribed medication (e.g. incompatibilities, side effects, 7-rights of medication). medical doctors, mostly general practitioners and some specialized ones, usually in primary healthcare settings, keep health records of their patients and have an overview of all administered medication. however, key barriers still remain the lack of coordination of institutions and authorities and overlap of their responsibilities, healthcare workforce and infrastructure shortages and several cultural issues. conclusion: all initiatives to medication management and medicines optimisation are provided without directive from national policies or guidelines. therefore, these activities rely on the goodwill of the health professionals to address pharmacotherapy and therefore polypharmacy management but they are not necessarily representative of what is happening nationwide. a national policy to implement the management of polypharmacy nationwide could mobilise the willingness of health professionals and ensure consistency of care. development and implementation of this policy should build on the grassroots efforts currently underway in this area. ph006: clinical profile and treatment discontinuation in a tuberculosis control state programme in brazil: preliminary results from sinan database simone s. bezerra 1 , mara guerreiro *,2,3 , nathany pessoa 4 , maria paula athayde 5 , rodrigo auad 6 , joão josé gomes 7 , josé lamartine soares sobrinho 1 1 post graduation program in therapeutic innovation, federal university of pernambuco, recife, pernambuco, brazil, 2 centro de investigação interdisciplinar (ciiem), instituto superior de ciências da saúde egas moniz, monte de caparica, 3 please specify your abstract type: research abstract background and objective: challenges remain in tuberculosis (tb) control. discontinuing treatment can leave patients infectious and contributes to the emergence of resistance. this study aimed to describe the clinical profile and cure and discontinuation rates of tb patients enrolled in the pernambuco tuberculosis control programme (pect). setting and method: the study was conducted in three sites in recife, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were extracted from the notifiable diseases information system (sinan) for all pect outpatients, from 01/2012 to 12/2014 (n = 440). analysis was performed with the aid of action for excel; there is on-going analysis to further explore differences across sites. ethical approval was granted. main outcome measures: clinical form of the disease, hiv testing, new cases, cure and treatment discontinuation. results: sociodemographic data were available for sites a and b only. most patients were male (70%, n = 291), with age raging from 20 to 49 years old (60.5%, n = 252); most had a low education level (46%, n = 192) and low socioeconomic status (92%, n = 382). the most common clinical presentation was pulmonary tb. most cases were new (78.4%, n = 345); recurrence and enrolment after discontinuation were respectively 5.9 and 10.9% (n = 26 and n = 48). with respect to hiv, 37.27% of patients were seronegative (n = 164); about a third (35%; n = 155) had not performed hiv test. rates for cure were respectively 59.1% (n = 260), 28.95% (n = 128) and 16.6% (n = 73) in the sites a, b and c. correspondent rates for treatment discontinuation were 21.2%(n = 93), 24.9% (n = 110), 4.3% (n = 19), respectively. tb-related mortality ranged from 0 in site c to 5.4% in site b. conclusion: missed hiv tests may represent undetected tuberculosis/ hiv coinfection. site b presented the highest rates of intrapulmonary plus mixed tb forms, discontinuation of treatment and tb-related mortality; additionally, it had the lowest rate of enrolment after treatment discontinuation. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings may help managers allocating resources and assist clinical pharmacists in planning their interventions. findings also suggest the need of more intensive interventions in tb patients, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: pharmacists working in primary health clinics have various roles. pharmaceutical care is one of them. how to provide this service varies across countries and settings. the most optimal way to provide pharmaceutical care is important to define when developing clinical pharmacy services in a new setting such as primary care practices. general practitioners are key stakeholders in this endeavour. the aim of this study was to find the most optimal approach to providing pharmacist-led pharmaceutical care in primary health care clinics in iceland in collaboration with general practitioners. setting and method: action research provided the framework for this research. data was collected from pharmaceutical care interventions with patients, field observations, field notes, and interviews with general practitioners over the period of the study. the study ran from september 2012 to june 2015. three separate semi-structured in-depth interviews were conducted with five general practitioners from one primary health care clinic in iceland at different time points throughout the study. pharmacist-led pharmaceutical care was provided to patients (n = 125) before and between general practitioners' interviews. the study settings was a primary health care clinic in reykjavik area and the patients' homes. main outcome measures: how to provide pharmaceutical care in collaboration with general practitioners in the icelandic health care environment. results: direct contact between pharmacists and general practitioners over short distances are essential to providing optimal pharmaceutical care services. pharmacist's access to medical records is necessary even though face-to-face communication between pharmacist and patients are most effective in providing pharmaceutical care. pharmacist-led clinical service was deemed most needed in dose dispensing polypharmacy patients. patients require more information about drugs prescribed to them coupled with an accurate drug list with greater detail. conclusion: the most efficient collaboration when pharmacist and general practitioner is obtained when they work side by side at the primary health care clinic. when new services are developed it is vital to identify different requirements of the primary health care clinics to optimize the running of a clinical pharmacist service. ph008: accompaniment of patients treated with oral chemotherapy: a survey on patients' experience laure napoly *,1 , pascal paubel 2,3 , sylvie burnel 1 1 oncorif -regional cancer network, 2 health law and health economics deparment, 3 health law institute, inserm, umr s 1145, paris descartes university, sorbonne paris cité, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: antineoplastic agents taken orally are more and more used in cancer care. these medicines confer autonomy to patients. although, they may cause adverse effects that can lead to treatment adherence issue, unjustified hospitalizations, or premature treatment interruption. proper accompaniment of patient can prevent these issues. in order to define how to organize this accompaniment, we conducted a survey on patients' experience. the objective is to describe patients care pathway and identify their needs. design: a descriptive, qualitative, prospective, survey was conducted using self-administered questionnaires, in hospitals of ile-de-france region. inclusion criteria were: having being treated with oral neoplastic agents during minimum 2 months, age [18, solid tumor, no concomitant iv chemotherapy. collected data were: patients' sociodemographic and clinical profile, their insight about different steps of care pathway (information, treatment delivery, follow up), their behaviours and interactions with healthcare professionals, and their overall opinion. results: 44 patients were recruited in six hospitals. their sociodemographic and clinical characteristics were variable. 72% were treated with targeted therapy, 12% with cytotoxic agent, and 16% with endocrine therapy. patients showed high satisfaction for given information at the beginning of the treatment. principal source of information identified was the oncologist (100%). while the delivery of treatment, 40% of patients beneficiated of advices from the pharmacist. accompaniment of patients during treatment seemed unequal, with: a frequency of visits with the oncologist ranging from less than 1-3 months; 36% of patients not knowing any telephone number they can call in case of worries or questions about their condition or treatment; 39% not remembering any particular accompaniment treatment (visits or calls from nurses or doctors for example). for adverse effects management, three principals actors were identified: oncologist (95%), general practitioner (52%) and pharmacist (31%). regarding the use of oral chemotherapy, patient are satisfied with: it's comfort (93%); being more actively involved in their cancer care (90%); and state not having any anxiety taking chemotherapy home (95%) . asked openly about their concerned and needs three major concepts were identified: high concern about adverse effects, positive feedback about maintaining contact with health professionals between cures and difficulties of communication between health professionals. conclusion: there is a high satisfaction regarding oral chemotherapy and health professionals. the oncologist has a primordial place in patient pathway, whereas implication of pharmacist and general practitioner stays variable. adverse effects are a major concern that needs proactive accompaniment. the variability of our results suggests that accompaniment must be flexible and adapted to the needs of each patients. the key to flexibility could be good coordination and communication between healthcare professionals. ph009: general beliefs about medicines among independent elderly adults in sweden: data from an rct lina hellström *,1,2 , victoria throfast 2 1 the pharmaceutical department, kalmar county council, 2 ehealth institute, linnaeus university, kalmar, sweden please specify your abstract type: research abstract background and objective: there is a need to improve prescription and use of medications by the elderly. the objective of a recent rct was to investigate the effects of e-learning about medicines among elderly adults. a positive impact on the primary outcome measure, knowledge about medicines, is reported elsewhere. a secondary outcome measure, general beliefs about medicines, is reported below. setting and method: the study was a randomized controlled trial in elderly people (aged c65 years). participants were recruited from patient associations and pensionerś associations. participants were randomized to either an intervention group that participated in the e-learning, i.e. internet modules with video and audio, or a control group that did not take part in the e-learning. post-intervention data was collected using paper-based questionnaires, completed within two weeks after agreeing to participate in the study. the general beliefs about medicines questionnaire (bmqgeneral), comprising the subscales necessity, harm and overuse, was used to elicit beliefs. higher scores indicate stronger endorsement of scale constructs (range [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . main outcome measures: bmq-general subscale scores. results: a total of 195 elderly people were included in the study, 96 in the intervention and 99 in the control group. the mean age in the total population was 74.5 years and 53% were women. eleven percent did not use any prescribed drugs while 27% used more than five prescribed drugs. the patients' scores were very similar in the two groups for all three bmq subscales. the median ''overuse'' score was 12 in the intervention group versus 13 in the control group, the median ''harm'' score was 10 (iqr 8-12) in both groups and the median ''necessity'' score was 18 in both groups. in the total population the most commonly expressed negative beliefs referred to overuse of drugs. 62.4% of respondents agreed with the statement ''if doctors had more time they would prescribe fewer medicines'', 43.4% stated ''doctors prescribe too many medicines'', and 37.9% stated ''doctors place too much trust in medicines''. a majority of the respondents agreed with the four items on the ''necessity'' scale. for example ''medicines help people to live a better life (96.9% agreed)''. conclusion: the studied e-learning intervention was not shown to have any impact on general beliefs about medicines. beliefs about medicines have been associated with a number of background variables which might explain why increased knowledge about medicines alone cannot change such beliefs. in general, respondents in the study had highly positive beliefs about the necessity of medicines. nevertheless, the results indicate that overuse of medicines is regarded as a problem. ph010: maf-plus: pharmacists' contributions to provision of financial assistance for medications ian wee * , charlene ong, niron naganathar 1 changi general hospital, singapore, singapore please specify your abstract type: descriptive abstract (for projects) background and objective: the medication assistance fund plus (maf-plus) is a government scheme introduced in singapore in 2011 to provide financial assistance to needy patients who meet pre-set criteria based on means testing. unlike previous schemes, maf-plus provides broader discretion to institutions when providing financial assistance. in our institution, pharmacists reviewed patients' case and medication histories, and filed recommendations to a multidisciplinary committee tasked with approving deserving maf-plus applications. the pharmacists' contributions to the committee, and the outcomes of the applications, are presented in this study. design: all maf-plus applications received between 1st october 2011 and 31st december 2015 were reviewed. pharmacists' comments for each application, where provided, were noted, as well as the range of medicines applied for, review approval rates, and cumulative percentage of available funds utilised. the effect of a recent widening of the scheme's scope to include notable high-cost items was also evaluated. results: between 2011 and 2015, 2001 maf-plus applications were reviewed, of which 1737 (86.8%) were approved. of the 264 rejected applications, 145 (54.9%) were channelled to alternative financial assistance schemes on the recommendation of the pharmacists. the medicines most commonly applied for were intended for the treatment of cardiac (24.3%), respiratory (14.7%), and psychiatric (13.5%) conditions. pharmacists' recommendations also led to a gradual expansion of our institution's list of pre-approved medicines-from 5 in 2011-2012 to 47 by end-2015. from 2014 onwards, pharmacists previewed increasing numbers of applications for high-cost medicines, particularly those for treatment of retroviral disease, hepatitis c, and rare diseases. cumulative utilisation of maf-plus funds (inclusive of annual replenishment) rose from 3.3% in 2011-2012 to 38.8% by end-2015, representing an average year-on-year growth of 151.1%. conclusion: using a process of judicious previewing of maf-plus applications, and recommendations to the maf-plus committee, pharmacists contributed to a high percentage of patients receiving financial assistance for medications. despite a steep growth in the number of applications received between 2011 and 2015, this approach helped to prevent over-extension in fund utilisation. pharmacists will likely be increasingly relied upon due to an anticipated rise in the number of applications for high-cost medicines. please specify your abstract type: research abstract background and objective: adolescents often treat themselves and take medications without parental supervision. lack of experience and knowledge of medicines in this age group frequently leads to inappropriate use of medicines and adverse drug reactions. data about the use of medicines among slovak adolescents and their knowledge of medicines have not been studied yet. setting and method: for our study we used the questionnaire method. the questionnaire contained 23 multidimensional items with closed-ended and open-ended questions, which focused on the characteristics of the adolescentś health status, use of medicines, also in relation to parents and adolescentś knowledge and perception of medicineś risk. we distributed 930 validated questionnaires for adolescents aged from 12 to 18 at secondary schools in all regions of slovakia. response rate was 70.6%. 657 questionnaires were finally analysed. the differences in the distribution of categorical variables between groups were evaluated using the chi square test. sas 9.4. was used as statistical software. main outcome measures: to determine adolescentś knowledge of medicines in terms of efficacy, self-medication, safety of therapy and analyse which medicines are the most frequently used by adolescents. to compare adolescents with chronic disease and healthy ones from the perception of pharmacotherapy point of view. results: in the analysed group 40.8% (n = 268) of adolescents are treated for chronic disease. mostly they suffer from allergy (25.0%, n = 67) and skin diseases (6.8%, n = 18). adolescents with chronic disease use regularly prescription medicines (36.9%, n = 99, p \ 0.001) and over the counter medicines (11.6%, n = 31, p \ 0.001). this group of adolescents better accept the pharmacotherapy with parental supervision (33.2%, n = 89, p = 0.0047 vs. healthy adolescents) and they believe in effectiveness of prescription medicines (71.3%, n = 191, p = 0.0226 vs. healthy adolescents). most frequently prescribed medicines were azithromycin, levocetirizine, ofloxacin and over the counter medicines were ibuprofen, paracetamol, ascorbic acid. we found out in all group of adolescents that 81.3% (n = 534) prefer self-medication without check-ups, 73.5% (n = 483) used drugs in the last 6 months without a prescription, 51.8% (n = 340) take over the counter medications independently without the supervision of parents, 14.9% (n = 154) buy medicines themselves in the pharmacy, 44.6% (n = 293) do not take medications as recommended, 56.6% (n = 372) believe that they have enough knowledge of medicines which they take, 51.4% (n = 338) resp. 49.6%, (n = 326) believe that prescription medicines resp. over the counter medicines are safe. conclusion: questionnaire analysis pointed out that slovak adolescents have not enough knowledge of medicines. the study provides new information in the field of risk perception and adolescentś knowledge of medicines in the slovak republic and highlights the areas that need to be studied in the future in terms of adolescentś education. federal university of pernambuco, 6 state technical school prof. agamenon magalhães, recife, pernambuco, brazil please specify your abstract type: research abstract background and objective: the effectiveness of tuberculosis (tb) control programmes depends critically on patients completing appropriate treatment. this study aimed to outline the cure and discontinuation rates of patients enrolled in the pernambuco tuberculosis control program (pect), based on dispensing data. setting and method: the study was carried out in three sites in recife public health system, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were collected between 07-11/2014, through reports from the stock management software for public pharmacies (horus) for pect outpatients. reports corresponded to a total of 948 patients (232, 348 and 368 in sites a, b and c, respectively). horus defines ''cure'' as medicines collection for three, six or nine consecutive months without interruption, depending on the treatment scheme; discontinuation is defined as non-sequential collection of medicines or treatment interruption for two consecutive months or more. patients were assigned an ''undetermined'' status if treatment was ongoing. data were inputted onto an excel spreadsheet and checked for accuracy. quisquared test, fisher's exact test and bootstrap analysis were performed with r statistical computing. ethical approval was granted. main outcome measures: cure and discontinuation rates for pect outpatients. results: demographic data are not available for the sample. rates for cure were respectively 35.9% (83), 23.6% (82) and 31% (114) in the sites a, b and c, while rates for treatment discontinuation were 3.4% (8), 27.8% (97) and 9% (33), respectively. discontinuation rates were significantly different among the sites a, b and c (p \ 0.05). bootstrap analysis showed that overall the proportion of patients with an ''undetermined'' status in each site did not significantly change these differences. conclusion: only site a had an acceptable discontinuation rate, in light of the world health organization recommendations. this deserves attention as default treatment leaves patients infectious for longer, increases the risk of poor outcomes and fosters resistance to antibiotics. pharmacists could use dispensing data to signal tb patients at-risk of discontinuation, and subsequently tailor interventions addressing its causes. site b had the greater number of patients which discontinued treatment. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings suggest the need of more intensive interventions in patients co-infected with tb and hiv, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: many efforts are done to organise good quality and safe pharmaceutical care. in general, the involvement of a hospital pharmacist or hospital pharmacy personnel in the process of medication reconciliation results in a reduction of the number of medication discrepancies. however, in case of emergency admissions this topic is still insufficiently studied. the introduction of good medication reconciliation on the emergency department (er) requires firm logistical and organisational efforts. we investigated the effects of a drug reconciliation intervention by pharmacy personnel during emergency admissions in order to identify discrepancies between medication lists taken by er physicians and by pharmacy personnel. setting and method: this observational, comparative, non-randomised intervention study was performed in 2011. we calculated that a population size of 65 patients was sufficient to perform reliable measurements. inclusion criteria: all patients presented at the er and admitted to a hospital ward \24 after presentation with usage of one or more prescription drugs. exclusion criteria: age \18 years, residency outside the region delftland, inability to undergo an oral interview, absence of a medication list of the public pharmacy (ozislist), decease of the patient during er-stay and patients undergoing surgical procedures. discrepancies between both medication lists taken by an er physician or pharmacy technician were classified in four categories of increasing severity (1 = no discrepancy to 4 = clinical relevant discrepancy) using the index of the national coordinating council for medication error reporting and prevention (www.nccmerp.org). discrepancies were categorised by a panel consisting of a pharmacy technician, a (senior) hospital pharmacist and a 6th year pharmacy student. statistical analysis was carried out with a statistical software package (spss 18) using the mann-whitney u test and chi squares test. main outcome measures: during the intervention measurement we analysed the reconciliated medication by comparing the er's physician's list with the list of the pharmacy technician after a medication verification interview. the number of discrepancies were measured and judged by the panel. discrepancies were given a category 1, 2, 3 or 4 as defined. results: during the intervention measurement 768 patients were admitted to the er. sixty-five (65) patients (8.5%) met the in-and exclusion criteria. the number of medication discrepancies decreased significantly after intervention of the pharmacy technician by 52%, from 161 to 77 discrepancies. the average number of discrepancies per patient after intervention decreased by 68.0%, from average 2.5 to 0.8 discrepancies per patient. conclusion: medication verification by pharmacy personnel in the er reduces the number of medication discrepancies by half. medication lists generated with a standard interview by pharmacy technicians in combination with an ozis-list on admission of patients at the er is more complete and accurate than the current method. hp-pc015: discharged patients: a problem for community pharmacists? information transfer, as well as the role, needs, and objectives of pharmacists when they care for recently discharged patients. setting and method: a focus group was conducted with a sample of six community pharmacists from personal contacts to represent different characteristics. the focus group consisted of different questions and the recording was transcribed, fragmented and categorised. based on these results, a nationwide online-survey was created with the following questions: a) responder's characteristics, b) number and origin of prescriptions, c) role fulfilment of the joint-who/fip-guideline on good pharmacy practice, rated with a 5-point likertscale, d) 30 information items derived from the focus group discussion grouped into four categories and evaluated for their availability and for their usefulness by likert-scales, e) goals for discharge optimisation, f) additional comments. the questionnaire was piloted and translated forward and backward to french and italian by native speakers. it was sent to all managers of pharmacies belonging to the swiss pharmacist's association in summer 2015 (n = 1348). main outcome measures: conclusions from focus group discussion and responses to questions a-f from the nationwide questionnaire. results: the focus group participants (47.3 ± 13.7 years, 50% female, 50% employees) emphasised the importance of an expanded information transfer, especially for medication changes, unclear prescriptions, and information about a patient's medication acquisition. they were concerned about their extensive workload of discharge prescriptions, and mentioned treatment continuity as one of their goals. the questionnaire was answered by 194 pharmacists (response rate 14.4%, 49.7 ± 10.8 years, 50.5% female). there were 56.7% of responders who reported to fulfil their role (to manage a patient's therapy, function b) not satisfyingly. unavailable but essential information were allergies and the specification of off-label use prescription. unavailable although desired information were the reasons for therapy changes, indications, appointments, contact information, or compounding formulations. concerning design and transfer, information should be written in a structured way but no clear preference for a transfer method was found. goals of community pharmacists were: improved treatment continuity, patient safety, and pharmaceutical care. conclusion: swiss community pharmacists rarely receive sufficient information on discharge prescriptions. appropriate pharmaceutical care is therefore impeded. the knowledge and application of the findings enable directed optimisation of discharge. hp-pc016: patients attitude for using antipsychotic medication in the norwegian early intervention in psychosis, tips 2 study rafal yeisen *,1 , stein opjordsmoen 1,2,3 , inge joa 1,4 , jan olav johannessen 1,4 , jone bjørnestad 1 on behalf of centre for clinical research in psychosis, psychiatric division, stavanger university hospital, stavanger, norway background and objective: poor drug adherence in patients with psychosis leads to relapse, re-hospitalization, poor outcome and increased consumption of health services. pharmacoclinical studies have demonstrated that the treatment response decreases with each relapse. it is estimated that 50% of patients suffering from chronic illness are not taking medication as prescribed after 6 months. the purpose of this study is to investigate which experiential factors that potentially might affect adherence with medication in adults with psychotic disorders. setting and method: in a descriptive qualitative sub-study in the ongoing norwegian early intervention in psychosis, tips 2 study, where twenty-first episode patients (7 male, 13 female) participated in semi-structured interviews 2 years after inclusion. they were still using or had used antipsychotics during the last 2 years. data were analysed using interpretative phenomenological analysis. main outcome measures: adherence to antipsychotics. results: the data suggested four main themes, reflecting the patients' subjective experiences and their impact on the desire to adhere to antipsychotics: (1) admission experience as a psychotic's patient; (2) information from healthcare staff; (3) limited involvement in decision-making; (4) attitude to antipsychotics. conclusion: a number of factors had a positive influence on adherence to antipsychotics. pleasant admission/stay experiences, feeling that antipsychotics had therapeutic effects, mild or no side effects, and believing that antipsychotics are necessary and useful, were typical statements. please specify your abstract type: research abstract background and objective: the hospital-to-home transition is a vulnerable stage in a patient's care. patients can experience problems with medication supply, which possibly lead to therapy interruptions. the objectives of this study were to investigate medication supply after discharge, and patients' and physicians' opinions about the current discharge process and possible optimisations. setting and method: a telephone interview was conducted with 100 discharged patients from the surgical and internal medical wards from the cantonal hospital in baden (switzerland). inclusion criteria were: patients c50 years old, discharged home with a discharge prescription. patients were called between the 2nd and 6th day after discharge and a piloted, structured interview was performed, consisting of questions on experiences and optimisations. afterwards, semi-structured interviews were conducted with five physicians from the study hospital. results from patient interviews and the general discharge process were discussed. main outcome measures: proportion of filled prescription, frequency and type of supply problems including therapy interruptions. opinions of physicians and patients on current discharge process and possible optimisations. results: discharged patients were 65.6 ± 17.4 years old, 39% female, 53% from internal medicine, and 97% regularly visit the same pharmacy. of the 100 interviewed patients, 23 have not filled their prescriptions yet and 77 had their prescription filled when they were called. of these, 78% of them visited the pharmacy on the day of discharge, but it took up to the 6th day until all of them received their medication. supply problems were encountered by 14 patients (18%), mainly because of the medication not being in stock in the community pharmacy. only four patients experienced therapy interruptions, which took up to the 3rd day post-discharge. patients discharged from internal medical wards had more supply problems compared to surgical wards (relative risk = 5.56, p = 0.007). patients experiencing supply problems had statistically significant more medicines on a daily basis (8.0 ± 4.32 vs. 4.9 ± 3.04, p = 0.010). physicians were surprised about the late prescription filling and worried about the disease outcomes. however, interruptions were interpreted as unfrequent. when asked if, in future, hospitals should transfer prescription to the community pharmacy prior to discharge, 71% of patients refused and physicians were undecided, mainly because of a questionable benefit. but both groups indicated that giving some bridging supply would be welcome. conclusion: this study showed that patients discharged from a swiss hospital encounter supply problems, but therapy interruptions are seldom. giving some bridging supply was preferred over an early information transfer by patients and physicians. interventions should consider these opinions and focus on internal medicine patients with high number of medication. please specify your abstract type: research abstract background and objective: adherence to secondary prevention evidence-based medical (ebm) therapies for patients with st-segment elevation myocardial infarction (stemi) is essential to reduce long-term rates of major adverse cardiovascular events. current guidelines recommend the long-term use of low-dose aspirin, highintensity statins, angiotensin-converting enzyme inhibitors (acei)/ angiotensin receptor blockers (arb) and beta-blockers (bb), in addition to p2y 12 inhibitors for 1 year. we aimed to assess the adherence to secondary prevention ebm therapies from discharge to one-year follow-up among patients with stemi undergoing primary percutaneous coronary intervention (pci) in contemporary practice. setting and method: observational single-centre study including consecutive patients with stemi undergoing primary pci in a tertiary hospital in switzerland over a one-year period. secondary prevention ebm therapies were assessed at discharge and at one-year follow-up. main outcome measures: prescription of key secondary prevention ebm therapies (aspirin, p2y 12 inhibitors, statins, acei/arb and bb) from discharge to one-year follow-up after stemi. bb was recommended only for patients with heart failure or left ventricular ejection fraction (lvef) \40%. results: a total of 179 patients were included. ebm drug prescription at discharge was 99.4% for aspirin (n = 178), 97.8% for p2y 12 receptor inhibitor (n = 175), 97.2% for statin (n = 174), 93.9% for acei/arb (n = 168) and 87.5% for bb (n = 28, among 32 patients with lvef \ 40%). ticagrelor (84.6%) was the major p2y 12 inhibitor prescribed. overall, 25 ebm drugs were missing at discharge, with 13 of these missing drugs having no justification for no-prescription (contraindications, allergy or intolerance). at one-year follow-up (median 13.4 months, n = 156), aspirin, statins and acei/ arb prescription rates were 92.9% (n = 145), 92.3% (n = 144) and 82.1% (n = 128) respectively. 19 out of 23 patients (82.6%) with lvef \ 40% received a bb. among patients treated with ticagrelor at discharge, 31 (23.5%) were receiving ticagrelor at follow-up, whereas 21 (15.9%) were switched to another p2y 12 inhibitor. among patients who discontinued ticagrelor (n = 80, 60.6%), duration of dual antiplatelet therapy was 12 months for 80% (n = 64) and discontinued prematurely (\1 year) for 15% (n = 12) patients. reasons for ticagrelor early discontinuation or switch were not specified. conclusion: in a real-world cohort of patients with stemi undergoing primary pci, prescription of recommended secondary prevention medications at discharge is excellent. adherence to ebm therapies at 1 year remains high with more than 80% of patients receiving all ebm drugs. early discontinuation of dual antiplatelet therapy was observed in 15% of patients, whereas ticagrelor was switched for another p2y 12 inhibitor in 15.9% of patients. these observations highlight key opportunities to improve longitudinal use of secondary prevention therapies after stemi in routine clinical practice. although side effects are less common than traditional chemotherapies, certain ones such as pain, fatigue, nausea and vomiting can still be bothersome. in oncology outpatient clinics, side effects are monitored by oncology nurses; however due to high patient turnover and limited numbers of nurses, the assessment of side effects might not be performed adequately. therefore, aim of this study was to determine side effects of immunotherapy and targeted therapy and to compare the severity assessment of side effects by clinical pharmacist and nurses. setting and method: the study was conducted in the hacettepe university oncology hospital outpatient clinic. the patients who have been taking ipilimumab, nivolumab, pembrolizumab, bevacizumab, panitimumab or cetuximab during october 2015-march 2016 were included. the assessment of side effects were undertaken by a clinical pharmacist and nurses separately on each visit using the common terminology criteria for adverse events version-2 toxicity assessment scale. an independent clinical pharmacist compared the side effects' assessments by pharmacist and nurses for analysis. ethical approval was obtained from hacettepe university ethics committee. main outcome measures: to compare the severity of side effects of targeted drug therapies which were assessed by a clinical pharmacist and nurses. results: during the study period 204 visits of 43 patients were evaluated. a total of 5508 side effects assessments were recorded. among those assessments 909(16.5%) was assessed in different ranking by nurses and pharmacist. the differences in the number of assessments were mainly seen in criteria related to pain (n = 34; 51), sensory loss (n = 24; 59), fatigue (n = 114; 26), stress (n = 16; 28), insomnia (n = 18; 28) which was performed by nurses and pharmacist respectively. other side effects detected only by clinical pharmacist were oedema, cough, gastrointestinal complaints (heartburn, cramp) and sensitivity of odour which require close monitoring and in-depth counselling by clinical pharmacist. conclusion: this study explores the differences in assessment of side effects by pharmacist and nurses in targeted therapies. routine assessment of side effects between chemotherapy cycles might yield to misinterpretation or inadequate assessment due to workload of outpatient clinic. therefore, inter-professional interactions in outpatient clinics might close the communicational gaps and improve patient care. hp-pc021: implementation of clinical pharmacy in the acute psychiatric wards: improving quality of medical treatment across health care sectors amila zekovic *,1 , signe kristensen 1 , lisbeth lund pedersen 2 1 clinical pharmaceutical services, capital regional pharmacy, 2 head of clinic, mental health services, copenhagen, denmark please specify your abstract type: descriptive abstract (for projects) background and objective: a study from 2011 shows that people with a mental disorder had a two-to threefold mortality compared with the general population in denmark. life style diseases are the major reason for the excess mortality, partly due to undertreatment of physical disease and well known side effects from medicines such as obesity, diabetes, and heart disease. in may 2015, a clinical pharmacy service (cps) was implemented in all acute psychiatric wards (apw) in the capital region as a part of a three-year project funded by the danish health authorities. the objective is to illustrate how the implementation of clinical pharmacy in the apw in copenhagen increases the focus on drug related problems, rational pharmacotherapy and side effects, increasing the quality of medical treatment and patient safety across health care sectors. design: data was collected at the apw in copenhagen which consists of three wards and has a total capacity of 39 beds. inclusion criteria were patients to which two or more of the following apply: • c65 years of age • c6 drugs • high risk drugs (clozapine, sertindole and opioids) • combination of antipsychotics and benzodiazepines • diagnosed with liver/kidney disease the secondary inclusion criteria were all patients receiving c6 drugs as a single criterion. to obtain a valid medication history and secure medication reconciliation, the pharmacist interviewed included patients. the patients were also asked about side effects, compliance, and perceived effects of treatment. a medication review was conducted based on the patient interview, screening for interactions in an interaction database, and consideration of biomedical data in order to evaluate if treatments should be adjusted, initiated or discontinued. the pharmacist's input was discussed with the doctor, as inputs are more likely to be considered if they are communicated orally. finally, all inputs were documented in the patient's journal as a pharmacist note. the model for improvement was used as a tool for implementing the cps and is being used continuously for improving the service. results: between may 26th 2015 and june 24th 2016, 2285 patients were screened at admission to the apw, of which 30.7% met the inclusion criteria (702 patients). in this period the pharmacist conducted 475 notes, indicating that 67.7% of the included patients were seen by the pharmacist. in april 2016, 30 patients were in average admitted with 8.3 drugs and 1.5 inconsistencies between the hospital's medication orders and the medications that the patient had been taking. regarding patients who are discharged to community care shortly after admission, the pharmacist note is sent to their general practitioner for follow up. conclusion: overall, the implementation of a cps in the apw has been successful. medication reconciliation ensures that the patient is provided with correct medicine at admission, transfer or discharge. by performing a thorough medication review based on a consultation with the patient, the service contributes to an increase in quality of medical treatment. please specify your abstract type: descriptive abstract (for projects) background and objective: the importance of the role of a clinical pharmacist resident in the operating room during 6 months, in a private hospital belonging to a group devoted to healthcare for over 70 years. the hospital is recognized as a reference centre of excellence of hospital care in portugal. it has 145 inpatient beds, two surgical blocks with 9 rooms and 12 beds in the intensive care unit. the aim of the clinical pharmacist in the operating room is to ensure compliance with good clinical practice, safety and pharmacotherapeutic effectiveness, as well as optimization of drug costs. design: 1. logistics restructuring of pharmaceutical services and the need of the physical presence of the pharmacist in the operating room. 2. furthermore, the workstation of the pharmacist is moved to the operating room and the in-depth study of all medicines used in the operating room. 3. in compliance with the joint commission, definition, optimization and adjustment of drug stocks to the needs of the service itself. in close collaboration with the nursing staff, consumer kits were created for registration of drugs by type of surgery in order to facilitate registration and ensure billing efficiency. control of the analgesic drug's dispensation circuit in hospitalized surgical patients that stay less than 24 h in the hospital. ensure compliance with the project through which the health regulatory authority evaluates several hospitals in the country, creating a national ranking among hospital specialties. 4. clinical phase: creation of prescription protocols by type of surgical intervention based on national clinical guidelines. validate prescriptions in the intra-surgical block in compliance with antibiotic prophylaxis, antiemetic and thromboembolic, checking deviations in therapy according to good practice. identify pharmacologic hypersensitivities of patients by consulting the clinical process and anaesthesiology records. provide information on drugs, drug efficacy monitoring and adverse drug reactions in risk management platform. check off label use of drugs. results: of a total of 772 interventions, 360 relate to revenue optimization and 412 relate to clinical interventions. there was an increase of approximately 25% in billing. on what regards to clinical interventions, the majority of them showed deviations from good clinical practice. the physical presence of a clinical pharmacist in the surgical block is essential as the prescription and administration of drugs is carried out simultaneously, allowing immediate therapy validation, in order to increase the safety and efficacy. the pharmacist has the ability to interact with the multidisciplinary team, as well as monitoring the patient's clinical process, the pharmacotherapeutic profile and drug allergies, allowing the detection of any adverse drug reaction on-time. all these interventions are possible in the pre, intra and postoperative phases. results: counselling (av.(±sd) duration: 9 ± 4 min) was performed in 773 patients (57.9% female; av.(±sd) age: 51.7 (±21) years; av.(±sd) medicines at discharge: 5.4 (±3.9)). in 30% of patients mrps were intercepted. the five most common mrps (%) were: need for organisational support (30.5, e.g. proper prescriptions' writing), therapy-related discussions (14.6), untreated indications (12.8), errors in documentation (11.1), and medicines without an indication (9.7). 437 patients (56.5%) classified for study inclusion, of whom 157 (35.9%) consented to be followed-up and 113 (72%) provided data. roughly 80% of patients report having received information about medicines at discharge, of which three-fourths remember being informed by the pharmacist. more then every second patient (54.7%) reported having received valuable new information. changes in chronic-use medicines occurred in 40.6, 42.4, and 34.5% of patients at 1-, 3-, and 6-month, respectively. at 6-month, in 27.4% of patients chronic-use medicines were newly prescribed, in 23.9% discontinued. medical specialists initiated these changes in 72.8% of patients. one out of five patients couldn't recall the reasons for changes in medication. nearly 30% of patients showed moderate to little medication adherence at 6-month. it did not significantly change during the follow-up period. conclusion: clinical pharmacists' counselling prevents mrps at the transition from hospital to home. follow-up data show that changes occur in one out of three patients. medication adherence remains stable, but generally needs to be improved. please specify your abstract type: research abstract background and objective: until 2013, prescription analysis was based in our hospital pharmacy. clinical pharmacy has been deployed in care units since 2014. many clinical pharmacy services were developed: medication reconciliation, patient's therapeutic education and counselling, and prescription analysis unit based. the purpose of this study is to assess the impact of the clinical pharmacist as a direct patient-care team member on prescription analysis. setting and method: we collected pharmaceutical interventions (pis) of the first 6 months of 2013 and at the same period of the year 2015 in the neurology unit when the pharmacist was unit based. we studied and compared type of pis (medication, drug related problem-drp), rate of pis acceptance and clinical impact. focus was made on high alert risk medications and potentially inappropriate medications. 4.5% in 2015 versus none in 2013. when prescription analysis was based in the pharmacy unit, 31% of drp detected by the pharmacist had a potential clinical impact versus 59% when the pharmacist performed prescription analysis in the care unit (p \ 0.05). three drp detected in 2015 had serious potential harm. results: ward-based prescription analysis allowed detecting five more times drp with a significant more important clinical impact than pharmacy unit based prescription analysis. the clinical pharmacist as a direct patient-care team member is more efficient in detecting serious potential harm. indeed, the pharmacist has a greater knowledge of the patient's clinical condition. nevertheless the global rate of acceptance of pis was greater when the prescription analysis was based in the pharmacy unit even if the difference is not significant. but prescription analysis is more complex when performed in the care unit, taking account adherence of the patient, and potentially inappropriate medications resulting in much higher risk-taking by the ward-based pharmacist. conclusion: this study showed that unit based prescription analysis is the best way to detect drug related problem. it must be competed by medication reconciliation and medication review to improve medication safety process. hp-pc027: qt-prolongation in an acute psychiatric setting: fact or fiction? eva jacxsens *,1 , hans van den ameele 2 , jü rgen de fruyt 2 , yves vandekerckhove 3 , frank vancoillie 1 , veerle grootaert 1 1 pharmacy, 2 psychiatry, 3 cardiology, az sint-jan brugge-oostende av, bruges, belgium please specify your abstract type: research abstract background and objective: several psychotropic drugs can induce qt-prolongation, which is a well-known risk factor for developing torsade de pointes (tdp) and sudden death. the clinical relevance of this side effect of psychotropic medication remains unclear, especially in patients hospitalized in an acute hospital. to interpret the clinical importance of psychotropic drug induced qt-prolongation, we investigated the prevalence of these electrocardiographic changes. setting and method: a prospective study was conducted on four psychiatric wards in a general hospital: two acute, short-term psychiatric units (asp1 and asp2), one addiction service unit (asu) and one geriatric-psychiatric ward (gpw). all adult patients admitted between october 1st 2015 and march 15th 2016 on a psychiatric ward were eligible for inclusion. at admission, an ecg (ecg0) was performed and creatinine and potassium levels were measured. a second ecg (ecg1) was performed at least 7 days after the start of a psychotropic drug associated with a risk of qt-prolongation. qtcprolongation was defined as 470 ms for males and 480 ms for females. clinically relevant qtc-prolongation was defined as c500 ms. statistical analysis (r software) was done as appropriate. main outcome measures: prevalence of psychotropic drug induced qtc-changes and correlating factors. results: 268 patients (mean age 55 years, 59%female) were enrolled in the analysis. in 85 patients, an ecg1 was performed. qtc 0+1 were prolonged in 2.3%(5/220) of females and 3.7%(5/136) of males. no clinical relevant prolongation (c500 ms) was registered. higher qtc intervals were measured in the geriatric population. 28.5%(36/126) of all measured qtc were situated between [450 c qtc 0+1 b500 ms] in gpw versus 9.4%(22/233) in the other units. significant difference in qtc-changes was associated with sex (p = 0.02246). there was no correlation assessed between qtc-prolongation and age, number of psychotropic drugs or a specific single psychotropic drug (p [ 0.05). conclusion: in this study qtc-prolongation due to psychotropic drugs is less common than previously described. ecg monitoring may be unnecessary in the follow up of patients without risk factors and could reduce hospital and community costs. however, considering the potential harm associated with tdp, qt-prolongation should be avoided. we recommend recording an ecg before the start of a qt-prolonging psychotropic drug in risk patients: patients with a chronic alcohol or drug addiction, a cardiac history, on concomitant therapy with at least two qt-prolonging psychotropic drugs, or geriatric patients ([65 years). hp-pc028: implementation of medication reconciliation aase m. raddum *,1 , anne-lise sagen major 2 1 sykehusapotekene i midt-norge, sjukehusapoteket i å lesund, avd. volda sjukehus, volda, 2 sykehusapotekene i midt-norge, sjukehusapoteket i å lesund, å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: a correct and accurate medication list should accompany patients at transitions in care from one setting to another, including admission to hospital. complete information on drug use is a prerequisite for all hospital treatment, whereas incomplete information represents a potential patient safety risk. medication reconciliation is defined by the world health organization (who) as ''…the formal process in which health care professionals partner with patients to ensure accurate and complete medication information transfer at interfaces of care.'' the objective of this study was to investigate the quality of the medication history obtained for admitted patients. furthermore, measures to improve the quality of medication histories, i.e. implementation of medication reconciliation, were initiated. design: the study included patients admitted to the internal medicine ward. a comprehensive medication history was determined by performing a standardized patient interview and/or by using relevant sources of information. the primary endpoint was discrepancies between the medication history obtained on admission and the one determined prospectively by a clinical pharmacist. the clinical relevance of the discrepancies was not determined, but sorted according to six major categories, such as: medication not in chart, but patient reports using (omission) and medication in chart, but patient reports not using (commission). further on, in order to minimize the risk of discrepancies, it was focused on implementation of medication reconciliation. a campaign was initiated, where a clinical pharmacist held information meetings regarding the medication reconciliation procedure. for the next 9 weeks, the degree of medication reconciliation was recorded. to spur the degree of medication reconciliation, each ward's weekly numbers were published and the ward with the highest degree of medication reconciliation won a prize. results: among the 74 patients included, a total of 120 discrepancies were revealed. in summary, 50 patients had at least one discrepancy in their medication history, resulting in discrepancies in the medication lists of 68% of the included patients. at the start of the study, the level of medication reconciliation varied among the wards (23-54%), while at the end of the study the levels were increased (75-92%). conclusion: all the included wards improved their level of medication reconciliation during the study period. however, these new combinations have potential drug-drug and herb-drug interactions which can affect the safety and effectiveness of the treatment. in our clinical practice, the clinical pharmacist provides patient education about direct acting antiviral drugs (daa) based-regimens, promotes medication adherence and manages potential interactions with hcv treatment. the aim of the present study was to determine the prevalence of use of herbal products in the patients on hcv treatment, and to describe the potential hepatotoxicity of the herbal products and their interactions with hcv treatment. design: we included all adult patients on daa treatment for hcv who were dispensed drugs from 01/09/2014 to 31/12/2015. we retrospectively recorded demographic data (age and gender), clinical data related to hcv infection (hcv genotype, fibrosis stage, daa regimen and treatment outcomes) and type of herbal products consumed. we then assessed the presence of herb-drug interactions and the potential hepatotoxicity of herbal products. results: we obtained data from 359 patients on daa-based treatment for hcv. the prevalence of consumption of herbal products prior to starting the treatment was 20.61% (74/359). the most consumed herbal products were (prevalence [ 10% among herbal products users): milk thistle, green tea, chamomile, valerian, pennyroyal, boldo and artichoke. we detected four herbal products with potential hepatotoxic effects according to the literature: milk thistle, green tea, pennyroyal and aloe vera. the prevalence of consumption of these hepatotoxic plants among herbal products consumers were, respectively: 21.62, 17.57, 13.51 and 5.41%. we detected herb-drug interactions or potential for hepatotoxicity in 47 out of 74 patients who consumed herbal products. the management of these potential interactions consisted of stopping the herbal product before starting the hcv treatment. conclusion: the consumption of herbal products in our hcv patients was frequent. the management of potential interactions was conservative, recommending to stop herbal products. clinical pharmacists have an important role in the counselling, detection and management of potential herb-drug interactions and herbal products-related hepatotoxicity. poster discussion forum iii: hospital pharmacy and pharmaceutical care 2 please specify your abstract type: research abstract background and objective: anaemia is a common comorbidity of chronic kidney disease. intravenous (iv) iron is used when oral iron formulation became insufficient or to reduce the use of erythropoiesis-stimulating agents (esas) in haemodialysis (hd) patients. the lack of generic group for iv iron sucrose (is) preparations leads to a controversial issue about their clinical effectiveness. in this study, we evaluated the effectiveness of original is compared to is similar (iss) in hd patients. setting and method: a retrospective monocentric observational cohort study was conducted from 01/09/2014 to 31/08/2015, in a stable hd population to compare is and iss. the follow-up periods lasted 24 weeks and were separated by a one-month wash-out period. original is and iss were administered respectively during the first (p1) and the second (p2) periods. the comparisons were performed using the paired student's t test or the paired wilcoxon test for continuous data and the fisher's exact test for categorical data. main outcome measures: the main endpoint was the difference in haemoglobin (hb) levels between p1 and p2 per patient. anaemia parameters (serum iron, serum ferritin, transferrin saturation ratio), the number of transfused patients, the doses of iv is and the doses of erythropoiesis stimulating agents (esas) were compared before and after the switch from is to iss, as secondary endpoints. results: a total of 105 patients were included. there was no significant difference in mean hb value between p1 and p2 (6.78 ± 0.63 mmol/l versus 6.79 ± 0.59 mmol/l p = 0.97). anaemia parameters were significantly different between p1 and p2 (mean serum ferritin, serum transferrin and transferrin saturation ratio) with p \ 0.0001, except to the mean serum iron. the mean monthly dose of iv iron per patient and the mean dose of esas were respectively in p1 and in p2: 248.31 ± 159.18 mg versus 259.74 ± 158.92 mg (p = 0.39) and 0.46 ± 0.50ui/kg/week versus 0.59 ± 0.60ui/kg/ week (p = 0.005). transfusions occurred less frequently in p1 than in p2 (p = 0.02). conclusion: this study showed that iss was as effective as original is regarding hb levels. however anaemia parameters appeared to be in favour of is; the mean dose of esas seemed to be higher after switching from is to iss. these outcomes should be further explored using prospective comparative clinical studies. please specify your abstract type: research abstract background and objective: the pharmacy residents are sometimes up to deliver chemotherapy when they are on night or week-end duty at the hospital. a dispensation's error (delivery of metoject ò (methotrexate) for intrathecal (it) injection whereas it doesn't have the indication for this use), led us to test the pharmacy residents' knowledges about the it access in order to underscore the points to be improved. the final aim of this work is to secure the pharmaceutical care of the patient 24 h a day, 7 days a week. setting and method: an online and anonymous survey of 14 questions was sent to the 35 residents of our area. it was composed of three parts: specific general information, questions about the chemotherapy specifically (indication, maximum dose and volumes, molecules used), illustrated questions about real situation for the dispensation on duty. the answers were collected over a two weeks period. main outcome measures: we studied the rate of good answers in global and by respondent. results: twenty-five residents answered the survey, among them 60% never achieved any internship in a centralized unit of reconstitution of chemotherapy (urc). all the levels of internship are represented: 1st year (n = 4), 2nd year (n = 10), 3rd year (n = 7) and 4th year (n = 4). only 32% know where a medicine is injected intrathecally on the spinal column, 64% know on which level of the meninges. three residents think that a nurse can inject intrathecally. they also had to select the molecules which can be injected by this access: 20% answered vincristine, 8% vinblastine, 4% bortezomib; despite these three molecules are mortals if they are injected intrathecally. the majority know the indication of the it chemotherapy: prevention and treatment of cancers' meningeal localizations. sixty percent do not know that several molecules can be injected for the same patient in the same time. the maximum dose of methotrexate is known for half of the respondents, but only 28% for the cytarabine'one. only 3 residents out of 25 know that 10 ml is the maximum volume allowed to be injected for an adult. six residents would have delivered metoject ò 15 mg in pre syringe filled if a doctor had asked for an it during a night. lastly, there are only two people who know that aracytine ò (cytarabine) 100 mg must be reconstituted with sodium chloride for it use and not with the provided solvent containing benzylic alcohol. the score of the residents having already done an internship in an urc is 8.4/14 compared with 6.3/14 for those who never did. the respective scores per year of internship are 6.2 (1st year), 6.4 (2nd year), 7.4 (3rd year) and 7.9 (4th year). conclusion: results and answers have been presented in a meeting and sent to the residents. we initially note many gaps in knowledge. the residents who already worked in an urc and the elders got better results. all the residents could be on duty at the hospital and all must be formed. a second session will be organized in a month to evaluate the formation's impact. it also has been presented to the assistants during an interactive lesson. this formation is essential to guarantee the dispensation of the adequate product and a secured medical care of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: patient adherence to prescribed medications is crucial for reaching metabolic control goal. to better understand the impact of polypharmacy on medication adherence, we undertook a detailed survey of medication use among patients with endocrinologic diseases. the aim of this study was to determine medication adherence in a cohort of patients with endocrinologic diseases and to test the hypothesis that adherence decreases with increased number of medicines prescribed. design: we conducted structured interviews to determine self-reported adherence of patient on a scale of 0 (high) to 4 (low observance) (srap-4) and a measurement using morisky medication adherence scales . demographic and medication information were collected from medical record. for statistical analysis, mann-whiney u-test for continuous variables, with chi square for categorical variables and kendall test for correlation were used. results: our cohort included 149 patients, 52% were women and 76% were diabetic (65% suffering from type 2 diabetes). the mean age was 56 ± 14 years, the average number of medication was 7.3 ± 4.1. 53 (36%) patients were not able to estimate their adherence. patients reported srap-4 scale with an average of 1.1 ± 0.9, this estimation was significantly higher than mmas-4 with an average of 0.9 ± 0.9 (p \ 0.05). the proportion of adherence level were identical between srap-4 and mmas-4 with respectively 51 and 60% of high, 45 and 35% of medium and 4 and 5% of low adherence. a significand correlation between srap-4 and mmas-4 scales (r 2 = 0.58, p \ 0.0001) was found. however no correlation between adherence scale and number of treatment (r 2 = 0.0001 for mmas-4 and 0.01 for srap-4 scale) nor number of daily doses (r 2 = 0.0011 for mmas-4 and 0.016 for srap-4 scale). on the 1080 medications, 11% presented difficulties with observance. cardiovascular (34.5%), diabetes (25.9%) and psychiatric (13.8%) treatment are the three most involved drug classes in nonadherence. conclusion: in this cohort, patients reported high medication adherence. we highlighted a correlation between srap-4 and mmas-4 scales. surprisingly, we didn't find correlation between adherence scales and number of treatment or dose by day. the next step of this work will be the identification of risk factor of nonadherence using logistic regression analysis. hp-pc033: the office of access to healthcare: how to optimize secured access to treatments? claire chatron, adeline flatres, claudine hecquard, guillaume saint-lorant * , alexandra muzard pharmacy, chu caen, caen, france please specify your abstract type: research abstract background and objective: office of access to healthcare (oah) is an organization which offers a medical and social coverage to people who can't access to care and to medication because of the absence of social welfare, living conditions, or financial difficulties. medications are free dispensed thanks to retrocession activity in hospitals pharmacies. the aim of this study is to analyse this activity and to improve communication with patients and access to treatments by an adapted pharmaceutical interview. setting and method: this study includes all dispensations of year 2015. in order to get medications in our hospital, a social worker and the patient come at the hospital's pharmacy. one people of retrocession team (four assistants, two externs, two residents and two pharmacists) dispenses necessary drugs to the patient according to hospital drug formulary and operating protocol. a switch or a special order can be purposed if the drug is not available. then, we give the patient a medication management plan (mmp) to explain him how to take his treatment at home. retrocession team filled a quiz about this activity and ways to improve it. main outcome measures: the main topics included in the quiz and evaluated were: dispensation organization, english talking, feeling during the interview and evaluation of the mmp. results: three hundreds and ten patients were admitted in oah 2015. these patients come mainly from the eastern europe and do not speak french in most of cases. social workers, who can help for communication, are not always present because these patients can come during on-call duty. quiz results showed that weak points occurred during the interview: explanation of the mmp, languages barriers, mention '' if needed '' not understood by the patient. explanation of the order for a particular drug was difficult to operate too. mmp were only drafted in french which was not convenient for foreign people. however, modalities of dispensation were well understood by the retrocession team. following quiz results, mmp was translated into english by the retrocession team. mentions '' if needed '', '' number of maximum tablet a day: … '', ''your medication is in order, thank you for coming to look for this treatment back to the hospital on … '', '' … is the same as …, prescribed by your doctor on your medication list '' have been added and translated. results of the study and new mmp will be presented to the pharmaceutical team and to social workers in staff. an index card for ''communication in english with a patient'' has also been drafted. it contains sentences meadow drafted in english. conclusion: access quality health care service is important to achieve health equity and to increase the quality of everyone's life. these documents improve communication with patients and by the way their understanding about their treatment. the use and the impact of these documents on well understanding will be soon evaluated with social workers and patients. hp-pc034: improve the medication in an associated to general hospital nursing home luisa alonso * , marta vidal iglesias, lucia gómez carrasco, guillermo goda, laura garcia, laura marin, ana hernandez, alvaro moreno please specify your abstract type: descriptive abstract (for projects) background and objective: in order to improve the medication reconciliation and to implement training programs for the medical team in an associated to general hospital nursing (asnh) home we measured the discrepancies between pharmacy registered treatments (prt) and medical prescriptions (mp), and we analysed potentially inappropriate prescriptions according to ''american geriatrics society 2015 beers criteria'' and ''stopp-start 2014 criteria. design: retrospective observational study that included 143 patients admitted in the asnh. the ''consensus document on terminology and classification in medication reconciliation'' was considered for discrepancy classification. data collected: discrepancies between mp and prt. in 84 patients from the original group of 143, we reviewed potentially severe drug interactions, potentially inappropriate mp and drug classes to avoid in older adults and medications to be used with caution in older adults (according to stopp-start2014 and beers 2015) . all data were registered, measured and analysed in excel ò . results: 143 patients and a total of 1487 mp were reviewed. 367 discrepancies (24.7%) were found between the medical order and the prt, those discrepancies included errors of omission in prt (11.2%), absence of discontinuation of medication (8.3%), incorrect dosage (5.2%). potentially moderate to severe interactions: the most frequent drug groups were proton pump inhibitors (ppis) (12.9%), benzodiazepines (bzds) (9.7%), oral hypoglycemiants (9.7%), other groups with frequency over 5%, oral antihistaminic, statines, low molecular weight heparine (lmwh), laxatives, calcium salts and iron salts. 176 stopp criteria were identified that affected to 440 mp and the distribution was as follows: laxative combinations (40.5%), long term ppis (15.5%), cns depressants combinations (11.4%), long half life bzds combinations (10.9%), aspirin incorrect drug strength (9.1%) and other groups with lower frequencies, nsaids and prokinetics. start criteria: 12 being all of them by omission of the drug at the time of admission. beers 2015 criteria: 90 prescriptions in the ''avoid prescription in adults'' group of which corresponded largely to concomitantly cns depressants and long term use of ppis in no risk patients. conclusion: the difficult working conditions, the excessive workload and the high staff turnover, where doctors have a patient ratio over 100/1, make difficult to update treatments according to patient daily needs. a clear communication problem between the hospital pharmacy and the asnh prescribers exists due to lack of infrastructures, and it has been demonstrated with the high percentage of discrepancy, that implies an important logistic problem (not a safety problem) since the nurse team works directly with the original medical orders. the analysis of prescriptions showed the need for updating the medical knowledge. the high volume of stop and beers criteria and lack of doctors time made impossible the individual acting upon each patient, so short summaries of continuous training related to most frequent problems have been designed. please specify your abstract type: descriptive abstract (for projects) background and objective: our french university hospital is one of the most active centre for liver transplant (100 transplants annually). various professionals are involved in the graft patient care and education. much information and education sessions are exempted before and after the transplant. the objective of this work was to realize a short movie for patients (1) to get them ready for transplant (2) to give the key messages to support their transplant (3) to make family understanding the process and to promote the life behaviour changes. design: three members of the pharmaceutical team with nurses-led care coordination and a surgeon wrote the scenario. we requested two directors for 3 days of shooting. we defined the key points for the patient and places to film, and fixed the duration (7-12 min). the scenario was validated by the chief of the liver transplant unit and nurses-led care coordination. after the 3 days of film shooting, we selected sequences. results: the movie was a succession of six parts. (1) the movie has been burned onto cds, put on flash-drives and will be uploaded on the internet. because of the international origin of our patients, the video will be subtitled at least in english. the video will be broadcast to hospitals which do not transplant patients and refer them to our hospital. since the medical team was involved in a collaborative project, the making of the video has permitted to strengthen the cohesion. indeed, this work would not succeed if everybody did not express himself. patients understood the interest to testify about their lived experience with the liver transplant, because they wished to have such information when they were waiting for the graft. conclusion: this movie is very useful for patients and families who are looking for information before and after liver transplant. it is a tool to get them into condition patients. this video presents the advantage of being personalized (local and caregivers that the patient will encounter are filmed). furthermore, it maintains a dynamic involvement of the pharmacy (already well established with clinical pharmacy, patient education and medication reconciliation) in the liver transplant unit. the making of the film has been an opportunity to bind the members of the team together, by valuing the work of everyone. the film could be screened if this abstract is selected for an oral communication. please specify your abstract type: descriptive abstract (for projects) background and objective: numerous procedures on medication management at oslo university hospital aim to minimize the risk of medication-related errors. error reports and observations show great variation in the use of these procedures, primarily due to difficulties in their implementation and maintenance. our aim was to assess the effect of a novel teaching strategy, the impala project, on doctors and nurses compliance with the medication management procedures. design: the project was carried out at 12 general medicine wards at oslo university hospital for a period of 6 weeks at each ward. assessment of medication-related error reports yielded the following areas of focus: (i) correct medication prescription, (ii) specification of doses for medications given on an ''as required''-basis, (iii) double control of medication dosing, (iv) correct and documented generic substitution. weekly presentations by pharmacist(s), lasting for a maximum of 15 min, were given to doctors and nurses as part of daily ward routines. this was repeated over 4 weeks. data on medicationmanagement procedure compliance were recorded before the start of the intervention, during and after each intervention period. the results were presented and made available to both leaders and employees throughout the project period both as an incentive to improvement and as a motivation factor for continued effort. results: there was a marked increase in medication-management procedure compliance among the nurses, especially after the second week of intervention. the most marked increase was shown for double control. increase in medication-management procedure compliance was also present among the doctors, but was less prominent. the data presented gave an extra motivational kick according to the participants. the leaders and the employees stated that the impala strategy was easy to follow and gave results without much organizational effort. conclusion: fifteen minutes presentations given by a pharmacist(s) as part of daily ward routines, combined with presentation of results demonstrated considerable improvement in medication-management procedure compliance. please specify your abstract type: research abstract background and objective: high unexpected serum vancomycin concentrations (svcs) were observed in patients without impaired renal function during the therapeutic drug monitoring (tdm) in our pharmacokinetic service. the aim of this study was to analyse the evolution of the svcs and its relationship with the markers of renal function. setting and method: retrospective study conducted at a university hospital with a follow-up period of 3 months. only adult patients having at least two tdm were selected. trough svcs were measured by cmia (architect i-1000 analyser, abbott ò ) and fitted to a two-compartment model by using bayesian analysis (pks ò , abbott). clinical and demographic data and daily dose, as well as timings of vancomycin administration and of blood sample collection were accurately recorded. spss ò , version 22.0 was used to compare data from both tdm by student t-test (parametric data) and wilcoxon (nonparametric data). main outcome measures: concentration-to-dose ratio (cdr: trough concentration *1000/daily dose); glomerular filtration rates (gfr) estimated by cockroft-gault formula; measured and predicted svcs levels. results: 30 adult patients were included (females: 20%); median age 68 [35-93] years).the first and the second tdm were carried out after 1.5 [0.5-4 .0] and 3 [1.5-6.5 ] days from the beginning of the treatment, respectively. in the first tdm, no difference was found between the measured concentrations (11.93 (5.07) lg/ml) and those predicted (12.22 (5.58) mg/l. however, predictions were less accurate in the second tdm and predicted concentrations were significantly higher svcs (15.96 (4.32) mg/l vs. 12.88 (3.70) mg/ml, p \ 0.05). the median cdr in the second tdm was significantly higher than that calculated in the first one (6.2 [2.2-13 .6] l -1 vs. 9.6 [3.3-20.2] l-1; p \ 0.05), indicating a lower clearance and a drug accumulation. however, no statistically significant differences in the glomerular filtration rates were found (114 [30-230] ml/min vs. 107 ml/min) in the first and second tdm, respectively. conclusion: although the markers of renal function did not change during the treatment, a decrease in vancomycin clearance was observed. the pharmacokinetic model does not accurately predict evolution of the svcs over the treatment. the introduction of covariates such as the length of treatment or the cumulative dose in the pharmacokinetic model could improve its predictive performance. please specify your abstract type: descriptive abstract (for projects) background and objective: genetic polymorphism or major physiological changes have to be considered in patient therapeutic management. clinical pharmacists have a role to evaluate and optimize the appropriateness and effectiveness of patient's medications. we report here the impact of the clinical pharmacist and his collaboration with the clinical pharmacologist in the therapeutic management of a patient suffered from anorexia nervosa, a psychiatric disorder leading to body composition change that may influence drug pharmacokinetics and efficacy. design: case report. results: the patient was a 35-year old woman hospitalized for chronic pulmonary aspergillosis previously treated by voriconazole, posaconazole and itraconazole. her medical history included anorexia nervosa since 1997 with a body mass index of 9.8 kg m -2 , pulmonary tuberculosis in 2011 with relapse in 2013, and chronic pulmonary aspergillosis since 2014. at admission, a treatment by oral voriconazole at 100 mg/12 h was introduced. the trough concentration of voriconazole at steady state was .1 mg/l (therapeutic range 1-4 mg/l) despite taking drug on empty stomach. although the voriconazole dosage increased in 200 mg/12 h, the trough concentration did not increase significantly (0.2 mg/l). we hypothesized anorexia led to a significant mucosal atrophy and accordingly, a significant decrease in intestinal absorption surface which is a major determinant of the level of drug absorbed. thus, a switch from oral to intravenous route was performed (voriconazole 200 mg/12 h). according to subtherapeutic voriconazole concentrations (trough concentration: 0.2 mg/l) despite the use of intravenous route, we decided to perform genotyping to look for mutations of cytochromes p450 3a4*22, 2c19*2 and 2c19*17, particularly implicated in voriconazole metabolism. the presence of an ultrarapid metabolizer genotype (17* allelic variant of the 2c19 isoenzyme) in our patient should lead to increase drug dosage from 50 to 75%. finally, the patient was treated by intravenous voriconazole at 7 mg/kg/12 h (i.e., increase by 75%). the maximum concentration performed 24 h after iv route initiation was at 2.8 mg/l, suggesting a better efficacy. conclusion: this case report highlights the potential complexity of therapeutic management in some patients given anatomical and functional changes or genetic polymorphism, which can affect drug efficacy. clinical pharmacists in collaboration with clinical pharmacologists have to be able to help physicians in this type of situations. please specify your abstract type: research abstract background and objective: posaconazole (pcz) is widely used for invasive fungal infections as prophylactic, pre-emptive or curative therapy in lung transplantation. recently, a new formulation of pcz has been available in enteric-coated tablets. this new formulation improves pcz bioavailability, as compared to the oral suspension, which leads to increase pcz plasma trough concentrations (c min ) in haematological patients. no data related to pcz exposure and its effects on tacrolimus (tac), an immunosuppressant with narrow therapeutic index widely used, exists in lung transplantation. we aimed to assess the consequences of the treatment by pcz entericcoated tablets on pcz and tac exposure in lung transplant patients. setting and method: a single-centre retrospective study was conducted among lung transplant patients receiving tac and either enteric-coated pcz or both galenic forms. main outcome measures: pcz and tac exposure were estimated by the measurement of c min . to overcome the influence of dose (d), c min were adjusted on dose (c min /d) for both pcz and tac. a spearman test (nonparametric distribution) was performed to assess the correlation between pcz c min /d and tac c min /d. results: eighteen lung transplant patients (median age [q1; q3] = 48. 5 [34.7; 57.4] years; 50% female) were included between june 2015 and march 2016. eight patients received only pcz entericcoated tablets. pcz enteric-coated tablets were associated to an increase in pcz c min /d as compared to oral suspension (0.008 ± 0.006 l -1 vs. 0.001 ± 0.001 l -1 , p \ 0.0001). overall, pcz therapy initiation led to an increase in tac c min /d (0.002 ± 0.001 l -1 before initiation vs. 0.008 ± 0.007 l -1 after initiation, p = 0.02). tac c min /d was significantly higher with pcz enteric-coated tablets, as compared to pcz oral suspension (0.004 ± 0.004 l -1 vs. 0.009 ± 0.006 l -1 , p \ 0.0001). a weak correlation was observed between pcz c min /d and tac c min /d, independently to pcz galenic form (r = 0.37, p = 0.0001 with pcz enteric-coated tablets and r = 0.33, p = 0.02 with pcz oral suspension). conclusion: this pilot study in lung transplantation confirms the better bioavailability of pcz enteric-coated tablets as compared to oral suspension. our results show a more important increase in tac exposure with pcz enteric-coated tablets compared to pcz oral suspension, suggesting a concentration-dependent cyp450 3a4 inhibitor effect of pcz. these findings are of interest in clinical practice to monitor transplant patients treated by the new formulation of pcz. further analyses, including the consideration of confounders, will be conducted. please specify your abstract type: descriptive abstract (for projects) background and objective: within 8 months, two patients receiving apixaban developed agranulocytosis. based on temporal and clinical plausibility as well as published literature, the objective was to determine the causal relationship between agranulocytosis and apixaban. design: description of two agranulocytosis cases reported in our hospital. results: first case is an 89 years old male, admitted to the neurology unit (d0) for ischemic stroke. at admission, blood count showed no abnormalities. four days after admission, treatment administered consisted in: dextrose 5% infusion iv, sodic heparin iv, acetaminophen, atorvastatin, metoprolol. neutrophils count was normal (5.6 g/l). heparin was stopped at d9 and replaced with apixaban according to following dose regimen 2.5 mg twice a day. at d15, patient presented with hyperleukocytosis (neutrophils count 15 g/l) and high crp (109 mg/l). thus, a cytobacteriological urine test was performed. at d17, patient presented with hypothermia followed by hyperthermia related to acute sepsis. blood count showed agranulocytosis (neutrophils count 0.45 g/l). broad spectrum antibiotherapy was started (ceftriaxone and gentamycin). despite treatment, death of patient occurred at d17. the suspected cause of death was septic shock added to severe febrile neutropenia. following haemocultures confirmed sepsis (e. coli) possibly originating from urinary tract infection. second patient is a 76 years old male, admitted to the cardiology unit (d0) for bronchopneumopathy associated with tachycardia and atrial fibrillation. a treatment with heparin was immediately started in association with patient usual treatment (bisoprolol, valsartan, rosuvastatin, hydrochlorothiazide and manidipine). in addition, broad spectrum iv antibiotherapy was started with ceftriaxone and spiramycine followed at d7 by an oral treatment with cefixime and spiramycine until d12. heparin was replaced by apixaban at d1 (2.5 mg twice daily). antihypertensive treatment was adapted throughout patient's stay. patient presented neutropenia at d15 (neutrophils count 0.8 g/l), followed by agranulocytosis at d19 (neutrophils count 0.42 g/l) when it was decided to replace treatment with apixaban by fluindione. the following day, neutrophils count was about 0.28 g/l and patient received filgrastim. a myelogram showed a possible peripheral neutropenia. in the absence of other confounding factors (hiv, hbv, hcv, cmv), an iatrogenic agranulocytosis related to apixaban was suspected. conclusion: causal association with heparin is unlikely as neutropenia is not an adverse drug reaction known included in the smpc of this drug having a well-established safety profile. since the two patients were taking their usual treatment for a significant period of time, a causal relationship is deemed unlikely. temporal and clinical plausibility seem to indicate a possible relationship between agranulocytosis and apixaban. as this medicine has been recently approved, this might explain why no case has been reported in the literature and the absence of agranulocytosis as an adverse drug reaction of apixaban. please specify your abstract type: research abstract background and objective: taste is tightly connected to children's acceptability of medicines. two ways to overcome lack of acceptability are to administer solid formulations which are easier to taste mask and change to better tasting medicines. dicloxacillin is an antibiotic known for its unpalatability, and taste studies suggest that this might jeopardize its adherence. the aim of this study was to explore if prescription data can be used to estimate acceptability of antibiotics among children on a population level using dicloxacillin as an example drug. the research questions were: when comparing dicloxacillin with other antibiotics commonly used in children, (1) is there a difference in the age of conversion from liquid to solid formulation and (2) is there a difference in re-prescription rates on day 1 and 2 after the initial prescription? setting and method: we included all initial prescriptions of oral dicloxacillin, phenoxymethylpenicillin, amoxicillin and erythromycin for children 0-12 years registered in the norwegian prescription database (norpd) 2005-2007 due to dicloxacillin mixture being discontinued from the norwegian market in 2007. the age of conversion was defined as the age where half of the children were prescribed liquids and the other half prescribed solid formulations. re-prescription rates were defined as re-prescriptions of a different antibiotic or formulation on day 1 and 2 after the initial prescription, divided by the total number of prescriptions. main outcome measures: age of conversion and re-prescription rates of dicloxacillin compared with other common antibiotics. results: the age of conversion for dicloxacillin was 4.5 years, compared to 7 years for other common antibiotics. the average represcription rate for dicloxacillin was 8.2% for children 0-6 years and 1.8% % for children 7-12 years. the highest re-prescription rate of 13.3% was found in 2-year olds. corresponding numbers were 2.1, 1.8 and 2.3% for common antibiotics. conclusion: the lower age of conversion from liquid to solid formulation and higher re-prescription rate of dicloxacillin mixture compared to common antibiotics indicates that prescription data can be used to identify antibiotics with low acceptability for children 0-12 years. further studies are needed to investigate if this also holds true for other antibiotics. please specify your abstract type: research abstract background and objective: attention deficit/hyperactivity disorder (adhd) or hyperkinetic disorders (hkf) is among the most common mental disorders in children, and may persist through adolescence into adulthood. pharmacotherapy used for treating the disorders also has potential for misuse/abuse. the aim was to describe the prevalence and magnitude of use of stimulant drugs and atomoxetine, and compare consumption in the nordic countries. setting and method: a descriptive pharmacoepidemiological study from the * 26 million inhabitants of the five nordic countries in the period 2004-2014. data were collected from national prescription registers, public drug reports and by correspondence with public health institutions. population data were obtained from official statistical databases or by correspondence with public health institutions. main outcome measures: trend over time, comparison between countries, type of pharmaceutical, gender, age, comparability of data. results: the annual consumption has been increasing from 2004 to 2014, both in volume and prevalence of use. denmark had the largest increase in volume, from 0.6 to 8.2 ddd/1000 inhabitants/day. sweden had the highest increase in prevalence of use over the period, from 1.6 to 8.6 users/1000 inhabitants. iceland had the largest consumption of adhd medications in 2014, 21.9 ddd/1000 inhabitants/day. prevalence data was not available for iceland but sweden was highest in prevalence of use among the other countries in 2014: 8.6 users/1000 inhabitants. males aged 10-14 years had the largest volume and prevalence of use in 2014, but females' consumption had been increasing faster both in terms of numbers of users (* 1.5 9 faster) and in volume (*29 faster) than men's consumption. conclusion: variation in consumption is considerable and cannot be explained by diagnostic and prescription guidelines, as these are similar in the five countries. consumption has been increasing fast in the period in all the countries, and faster for women than for men, although men still consume larger volumes than women, and are more frequent users. please specify your abstract type: research abstract background and objective: in 2009 and 2013, regulatory bodies in usa (fda) and europe (ema), issued warnings on use of metoclopramide due to an increased risk for serious adverse drug reactions (adr), especially neurological adrs. ema recommended that metoclopramide only should be prescribed for up to 5 days while fda concluded that treatment longer than 12 weeks should be avoided. metoclopramide is commonly used to treat nausea and vomiting in pregnancy (nvp) and deficient breast milk production (10 days course). ema did not make any recommendations concerning use during pregnancy and lactation. the objective of this study is to assess the disproportionality of reporting of adr from metoclopramide, with special emphasis on neurological adrs and women in reproductive age. setting and method: data from whos global adr database vigibase ò for the time period november 1967 to may 2016 was used. the measure of disproportionality of reporting calculated was the proportional reporting ratio (prr), and 95% confidence intervals (ci). analyses were performed according to gender and age. time-toonset of adr was calculated. main outcome measures: proportional reporting ratio (prr) results: vigibase contains over 13 million adr reports. metoclopramide is a suspected/interacting drug in 47 407 of the reports, most common (72%) are neurological adrs. the majority (84%) of the metoclopramide adrs occurred within the first 3 days of use. a total of 65% of the reports was received the last 5 years (2011) (2012) (2013) (2014) (2015) . the reporting of neurological adrs was higher for metoclopramide than other medications in vigibase. women in reproductive age (18-44 years) reported higher proportion of neurological adrs (prr = 3.0, 95% ci 2.9-3.0, n = 4533) than women 45 + years (prr = 2.5, 95% ci 2.4-2.5, n = 3057) but a similar proportion as men 18-44 years (prr = 3.1, 95% ci 3.0-3.2, n = 1823). conclusion: there is a 2.5 to three fold higher proportion of all reports regarding neurological adrs for metoclopramide than for other drugs. patients initiating treatment with metoclopramide should be informed about risks of adrs and that most adrs occur within 3 days, and instructed to contact health care personnel and stop treatment if adrs occur. please specify your abstract type: descriptive abstract (for projects) background and objective: self-induced drug intoxications (sidi) are one of the most frequent reasons of hospitalization in emergency service (1%) with around 4-5/1000 inhabitants and represent around 6% of admissions in intensive care unit (icu). it is the most frequently used method of suicide attempts (sa) and the leading cause of hospitalization for young people under 30. the main objective of our study was to analyse, stratify and pharmaceutically map the different sidi identified in our icu. design: this is a prospective study over 20 months, including all icu patients following sidi from june 2014 to january 2016. we have collected psychiatric history and previous sa by sidi, usual treatment, state of consciousness, incriminating drugs, drug classes stratified according to the clinical severity score igsii, evolution, transfer in a specialized centre and average cost of stay. results: ninety-two cases were reported, representing 6% of icu admissions. the average hospital stay was 3 days for an average cost of 3396.5€. this amount is low compared with the average cost of all stays gone through the icu for the period (14,957€). ninety percent of patients had a psychiatric history and 48% a previous sa. the usual treatment was involved in 77% of sidi. half of the patients arrived conscious with an average of severity score igsii of 35/163, 88 being the highest found for a patient who had swallowed simultaneously pregabalin and nitrazepam. clinical severity of these patients is less than that found on average for all patients in the icu in this period (59/163). eighty-seven percent had a favorable evolution. only one death was observed after ingestion of propranolol. fifty-six and a half percent of patients were then hospitalized in a specialized centre. the great family of psychotropic is the most frequent with benzodiazepines 70%, neuroleptics 36%, antidepressants 31.5% and antiepileptic 17.5%. the main drugs involved are oxazepam 16%, alprazolam 15%, cyamemazine 14%, bromazepam 13% and quetiapine 8%. antihypertensives then arrive and represent 14% of sidi. the stratification of severity scores does not appear to show significant differences between drug classes, nor between mono or polydrug ingestions. conclusion: sa by drug ingestion are very common and are often linked to risky behaviours. for these epidemiological and economic findings, it is necessary to continue and develop prevention strategies avoiding the appearance of intoxication (primary), limiting the consequences (secondary), and reducing the risk of recurrence (tertiary). please specify your abstract type: research abstract background and objective: interpretation of quality of life scores to render them meaningful to aid clinical decision-making is an ongoing challenge. interventions often result in statistically significant quality of life (qol) improvement, but may not reach the threshold of clinical importance. the minimal clinically important difference (mcid) is the minimal score change of relevance clinically. the aim of this systematic review was to assess the impact on quality of life of topical, systemic and biologic treatments for psoriasis in randomised controlled trials (rcts). setting and method: prisma guidelines were followed. all available articles describing rcts of therapies for psoriasis that included qol measurements published up to november 2014 were identified. six databases were examined with 388 search terms. abstracts of articles were reviewed independently by two assessors: a third adjudicator resolved any opinion differences. risk of bias was assessed using the jadad scale. main outcome measures: reporting of the use of qol endpoints and impact of interventions in psoriasis. results: of 3597 screened article abstracts, 329 articles were selected for detailed review: 100 trials met the eligibility criteria, describing research on a total of 33,215 patients. reports of psoriasis interventions that fulfilled inclusion criteria have gradually increased over time : 1998-2004 = 12, 2005-2009 = 33, and 2010-2014 = 55 (1) to evaluate the relationship between the use of different therapeutic agents and the severity of osa, and (2) to determine the effects of commonly used medications on continuous positive airway pressure (cpap). setting and method: patient medical records (n = 2688) of 183 patients, that underwent sleep studies between the years 2009 and 2013 were collected over an eight-month period from the sleep laboratory department at mater dei hospital using a random sampling technique. data collected included body mass index, gender, age, epworth sleepiness score (ess), drug history, apnoea hypopnoea index (ahi) and cpap therapy prescription. likelihood ratio chi square test, paired samples t-test and multinomial logistic regression were the statistical tests used for data analysis. main outcome measures: assessment of the drug history in response to osa control using the ess and ahi scores. results: one hundred and seventy (92.9%) patients of the 183 patients (131 males, 52 females) were diagnosed with osa. forty-five (24.6%), 43 (23.5%) and 82 (44.8%) patients suffered from mild, moderate and severe osa respectively. patients had a mean age of 54 years. angiotensin ii receptor antagonists (arbs) (p-value = 0.022), sulphonylureas (p-value = 0.050), insulin therapy (p-value = 0.040) and non-benzodiazepine sedating agents (p-value = 0.037) were found to be associated with the presence of osa. a decline in the use of the arbs (p-value = 0.000), angiotensin converting enzyme inhibitors (p-value = 0.000) and non-benzodiazepine hypnotics (pvalue = 0.001) was observed over the study year period. reduction in the cpap therapy benefit was detected with the use of histamine (h 1 ) antagonists (p-value = 0.015), b-adrenergic blocking agents (pvalue = 0.001) and antiplatelets (p-value = 0.003). conclusion: it is confirmed that hypertension and diabetes mellitus type ii are the main co-morbidities associated with the presence of osa. reduction in the use of certain therapeutic agents is observed secondary to cpap therapy use. patients using specific drugs have been identified as being at risk of a reduced cpap therapy benefit. please specify your abstract type: research abstract background and objective: people are using increasingly more common of social networks such as facebook, twitter and youtube for different purposes. many people are using these networks with the aim of getting information and knowledge sharing. there are many groups that pharmacist is a member in social networks at turkey. the largest of these groups has 14,000 members. pharmacists are shared common problems, information and experiences in these groups. but the accuracy of the information shared on social networks are not always conclusive. the study aim to evaluate the impact of social network information sharing in the knowledge and attitude of pharmacists. setting and method: clinical pharmacy group has been created to share information on facebook. 2400 pharmacist joined this clinical pharmacy group. the group was fed by information which include new drugs, fda alerts, adverse event and case report and also drug related problems during the 6 months. pharmacists were assigned in two major groups, group a active pharmacist who becomes a member of our clinical pharmacy group, share and discuss information through the network and group b who is not a member. a knowledge measurement survey (ams) was given to both of them. main outcome measures: acknowledge measurement survey (ams) was developed and the difference in the score was used to evaluate the difference between the two study groups. results: 142 pharmacists participated in the study, 34.50% of the participants were a member of our facebook group and 65.49% of participants were not. 4.9% of the participants have doctoral degree or student, 15.4% have master degree or student, 32% have bachelor degree from 4 year-pharmacy faculty, 47.1% have bachelor degree from 5 year-pharmacy faculty. the education level distribution between the two groups was not statistically significant. while 63.64% of the ams questions were answered correctly in the member group only 44.09% were answered correctly in the non-member group. conclusion: the study emphasizes the importance of social network in providing the accurate and fastest information for the daily use of the pharmacists, there is a significant difference in knowledge between the pharmacist who join, share and discuss information on the social network and the one who do not join. cp-ce004: impacts of a community pharmacy practice experiences on student professionalism yunn-fang ho 1,2 , hung-wei lin *,1 , fang-ju lin 1,2 , sheng-ping chang 2 , yen-ming huang 1 1 graduate institute of clinical pharmacy, 2 school of pharmacy, college of medicine, national taiwan university, taipei, taiwan, r.o.c please specify your abstract type: research abstract background and objective: professionalism is valued globally and pharmacy schools are expected to nurture competent practitioners to better serve the public with humanity attitudes and behaviours. the study aims: (1) to understand possible differences in professionalism between pharmacy students and potential community pharmacist preceptors, and (2) to evaluate student changes in professionalism upon completing the community pharmacy practice experiences (cppe) at the end of the third (p3) year. setting and method: a modified chisholm's pharmacy professionalism instrument (18-item, 10-point likert scale) was administered to p3 students, pre-cppe and hopefully post-cppe in september, and community pharmacist practitioners who participated in a two-day preceptor training workshop. participants also provided their significance ratings toward ten traits, namely altruism, accountability, excellence, duty, honor and integrity, respect for others, communication, ethics, humanism, and teamwork. main outcome measures: differences or changes in chisholm professionalism scores. results: thirty-two students and fifty pharmacists participated in the survey. honor and integrity (8.9 ± 1.5) and communication (9.2 ± 1.1) were recognized by students (31.3%) and pharmacists (23.5%), respectively, as the most significant trait. humanism was rated the lowest in both groups (students, 8.2 ± 1.9; pharmacists, 8.1 ± 1.9). the 18-item professionalism scores ranged from 6.6 ± 1.6 (''i do not expect anything in return when i help someone.'') to 9.3 ± 0.9 (''i am respectful to individuals who have different backgrounds than mine.'') in the student group; whereas 8.1 ± 1.9 (''i do not expect anything in return when i help someone.'') to 9.5 ± 1.1 (''it is wrong to cheat to achieve higher rewards (i.e., grades, money).'') in the pharmacist group. in general, pharmacists' professionalism scores were higher and, in certain items, statistically significant differences were achieved. conclusion: professionalism might grow with professional competency and practice experiences as demonstrated by potential pharmacist preceptors. upon completion of cppe, students could probably exhibit gains in professionalism. more investigations are still underway. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, a significant consumption of benzodiazepines (bzd) is observed in prisons. they are widely used during incarceration to treat or prevent anxiety and insomnia. furthermore, it is known that, an important traffic exists with these drugs because of the releasing properties of bzd in case of misuse. based on these observations, the pharmacist has set up a plan to improve the use of bzd in prison. the purpose of the study was to evaluate the impact of these measures after 1 year of implementation. design: in january 2015, we shared with physicians in a meeting to explain our plan for a better use of bzd and to set up new rules of prescription in prison: • regularly reducing the dose to limit drug tolerance • promoting the use of long half-life molecules which allow reducing addiction and misuse • advising sedatives anti-histaminics to treat insomnia • providing information to patients about addictives risks of bzd on the tv channel please specify your abstract type: descriptive abstract (for projects) background and objective: some drug combinations (described in thesaurus of national agency of drug) are contraindicated because they appear to increase the risk of torsade de pointes. the aim of this work is to standardize our pharmacists' intervention and to propose guidelines for doctors and pharmacists, depending on the situation and drugs, to limit these combinations and to reduce this risk at our hospital centre (1105 beds). design: a prospective survey was realized over a period of 5 months to identify the drug combinations prescribed in medical prescription software, from the national drug agency thesaurus, that might be inducing torsade de pointes. a multidisciplinary staff was then constituted composed of a cardiologist, a geriatrician, a paediatrician, an anaesthesiologist, a psychiatrist and pharmacists to identify the different situations and to establish guidelines. results: from the survey 18 drug combinations were found to be contraindicated due to increased risk of inducing torsade de pointes on a list 415 interventions realized by pharmacists. the work group identified three drugs with a therapeutic alternative: hydroxyzine, domperidone, escitalopram, the other drugs can't be switched because they are vital or have no alternative. the work group decided to maintain hydroxyzine but only on premedication and child anxiety, to eject domperidone from our therapeutic index and substitute it with metoclopramide or metopimazine, to not initialize escitalopram but to keep it if the patient has no have others risk factors associated or no contraindication. if the patient has a contraindication with a risk factor the doctor could prescribe other ssri. in addition, pharmacists alert doctors about the risk of torsade de pointes on medical prescription software if some contraindications are identified. conclusion: the contraindications identified must not be underestimated. this work allows identification of torsadogenic drugs commonly prescribed and provides guidance for doctors and pharmacists regarding drug combinations. the collective decision will be disseminated to sensitize all the doctors in the establishment. some treatments could not be substituted despite the contraindication; these must be retained but with clinical monitoring. conclusion: a substantial proportion of medication waste in the community pharmacy could have been prevented. unused medicines in the community pharmacy are generally of low economic value, making it unlikely that the costs that pharmacies will make with the redispensing of unused medicines will be covered. therefore, other actions to decrease medication waste in the community pharmacy, such as preventing that too much medicines are dispensed, should be considered. please specify your abstract type: research abstract background and objective: flaws in usage technique for inhalationmedicines is common, as much as half of the users may need some correction measures, to get the active substances down to the lungs and provide the intended effect. inadequate compliance, especially for regular-use preventive medications, is common. good guidance in pharmacies enhances correct use of medicines. the new norwegian pharmaceuticals policy (legemiddelmeldingen) from 2015 opened up for paid cognitive services, leading to the first such service being implemented in march 2016. the service can contribute to a more correct use of the medicines and, as a consequence, lead to better control of the symptoms for patients with asthma or copd. our objective was to map the variation in pharmacies' handling of an inquiry regarding lack of effect of an inhalation-medicine. the study was done prior to the implementation of the standardized service ''inhalation-guidance'' in norwegian pharmacies. setting and method: simulated patient (mystery shopper) visits in 100 pharmacies in oslo, akershus and buskerud in november/december 2015. the mystery shopper expressed just having started to use an inhaler because of her asthma, but not experiencing effect. structured data collection sheets were used to register the handling immediately after the visit. main outcome measures: scoring of the quality and contents of the information based on the products' patient information leaflets. results: the issue of inhalation-technique was mentioned in 74 of the pharmacies, whereof 18 asked the ''patient'' to show their inhalationtechnique, in order to correct and advice and 32 used an inhaler or demo-inhaler as an aid in the guidance. going through the instructions or watching a video-demonstration with the simulated patient also occurred, or referring the patient to read the instructions and/or watch the video-demonstration on his own. half of the pharmacies discussed the difference between use for preventive treatment of asthma and inhaler that is being used for treatment of attacks. sixty-five pharmacies gave no information about the importance of regular use of the preventive treatment. conclusion: there was considerable variation in how the pharmacies guided, which indicates a potential for improvement. the new guidance-service, implemented in norwegian pharmacies in march 2016, will contribute to better guidance. please specify your abstract type: research abstract background and objective: in portugal, tobacco addiction was responsible for over 12,000 deaths in 2013 (11% of the total deaths). the community pharmacist's contribution to control this public health problem is insufficiently documented. the aim of this study is to assess the contribution of the community pharmacist for smoking cessation. setting and method: a retrospective and longitudinal study of a convenience sample of patients integrating quit tobacco consultations, as part of a pharmaceutical care programme implemented by an outsourced pharmacist was performed at several community pharmacies. the smokers, aged 18 or over, were invited to join the programme. patients signed an informed consent and were submitted to a comprehensive approach by face-to-face consultations and telephone contacts. richmond and fagerström tests were used to evaluate motivation and nicotine dependence, respectively. the therapeutic plan (pharmacotherapy and behavioural counselling) was personalised to each smoker. the quit rates were evaluated by patient selfreport and confirmed by carbon monoxide measurements. the continuous variables are expressed as mean ± standard error of the mean. main outcome measures: quit rates at 1, 3, 6 and 12 months. results: between january 2009 and june 2016, 87 smokers joined the programme, 22 dropouts (25.3%). the remaining 65 smokers, 40 (61.5%) were male, with mean age of 48.4 ± 1.91 years. on average, each smoker consumed 21.0 ± 1.53 cigarettes per day. the mean age of initial tobacco use was 15.8 ± 0.51 years with 31.4 ± 1.98 years of consumption. about 70% reported moderate or high motivation and 60% medium or high dependence. a total of 315 consultations were held and, on average, each patient received 7.3 ± 0.82 interventions. all smokers received non-pharmacological interventions (e.g. motivational approach) and 61 (93.8%) also accepted pharmacological interventions, usually nicotine replacement products. the quit day was achieved by 50 patients (57.5%). a month after quit date, 41 patients were abstinent (41.7%). the number reduced to 32 after 3 months (36.8%), to 27 after 6 months (31.0%) and to 19 after 1 year (21.8%). these data upgrade and are consistent with our previously published results (2014). the smoking cessation consultation in the scope of a pharmaceutical care programme in community pharmacy seems to effectively contribute to the reduction of tobacco addiction in portugal. cp-pc013: patient counselling at dispensing of oral anticancer drugs in european countries from the pharmacists' perspective andreja eberl * , on behalf of epic working group pharmacy, institute of oncology ljubljana, ljubljana, slovenia please specify your abstract type: research abstract background and objective: the number of oral anticancer drugs (oads) available on the market grows constantly. consequently the number of patients, which have to manage the complex treatment with oads at home is increasing. the pharmacists present an important member of healthcare team, since they are dispensing oads to the patients, which need a high quality information at that crucial moment. therefore, our aim was to evaluate pharmacists perceived confidence and needs for specific continuing education in connection to oads dispensing in european countries. setting and method: we used an electronic mailing approach and a standardized online survey to ask practicing pharmacists in european countries about their experience with dispensing of oads. main outcome measures: frequency of patient counselling and fields of counselling, assessment of knowledge and skills. results: the frequency of patient counselling varied widely in participating countries between ''never'' and ''more than 80%'' at initial fill of an oad. at following refills the frequency of counselling was generally even lower. counselling mostly encompassed directions of use, the proper use of antiemetics and side effects. however many pharmacists stated, that they do not feel comfortable counselling patients of oads (25%) and even more acknowledged that they were uncomfortable with managing patients' side effects (20%). on the other hand only 45% of pharmacists believed, that they have received adequate knowledge of oads through undergraduate program, continuing education (ce) events and professional practice. many of pharmacists (70%) have not attended any of ce events related to oncology in last 2 years. pharmacists' responses differed little between the countries. conclusion: the proportion of pharmacists who regularly counsel their patients on oads is insufficient in view of importance of the patients' needs to manage their therapy at home. however the pharmacists seems to be aware of their knowledge deficits and educational needs. the field of oads needs better coverage in under-and postgraduate education. the number of ces has to be increased in order to improve the knowledge and skills in the areas of oads counselling. please specify your abstract type: research abstract background and objective: treatment guidelines for diabetes recommend that patients are well-informed about their disease, treatments and treatment goals, e.g. glycosylated haemoglobin (hba1c). the objective was to describe diabetes patients' self-monitoring of blood glucose (smbg) and potential need of guidance. please specify your abstract type: descriptive abstract (for projects) background and objective: in 2015, the international pharmaceutical federation collected data of remuneration models for community and hospital pharmacy and identified large variations between remuneration models and highlighted that the focus is largely on products and not on cognitive services. the aim of the study is to map the remuneration models of different pharmacist-led cognitive services in primary care across europe, with a special interest on medication reviews and to update a prior survey by bulajeva (bulajeva a et al. medication review practices in european countries. res social adm pharm 2014;10:731-40.). the definition of terms is pivotal for such a european survey to avoid results based on pseudoconceptions. hereafter we present the development of the survey and we will present first results from pilot tests. design: pharmacist-led cognitive services were selected based on a previous study by our group and by searching the literature, official government websites, the pcne wiki and arising links. the definitions of the terms of these services were based on searches in the mesh browser, medline and google scholar. additionally, a search in grey literature and in the internet was conducted to find appropriate foundation for the formulation of the definitions. the questionnaire will consist, of a first part about the remuneration of the pharmacist-led cognitive services. the focus is on country-specific differences in remuneration and the different levels of supply across europe. the second part of the survey is about the different types of medication review services with a focus on e.g. the implementation level, addressed issues, eligibility criteria. this survey will have a cross-sectional study design with an online questionnaire specific for invited participants across europe. to achieve the best quality of answers we will send this survey to at least two researchers with references in pharmacy practice, in each european country (purposive sample). the answers from each country will be checked for discrepancies and these potential discrepancies will be solved by a discussion with the responders. results: by the end of the pre-pilot phase, 22 different pharmacist-led cognitive services were identified and the correlating definitions of the terms were developed. conclusion: at the time of submission the pre-pilot phase has been finished and the pilot will start july 2016. please specify your abstract type: research abstract background and objective: medication adherence is one of the key aspects in assuring optimal health outcomes in majority of chronic diseases. the aim of the study was to evaluate copd patients' medication adherence in slovenia and its association with health outcomes. setting and method: patients were recruited by community pharmacists at the time of dispensing medication for copd. medication adherence was evaluated by using morisky medication adherence scale (mmas-8). patients who scored b6 points, 6.25-7.75 points and 8 points were regarded to have poor, moderate and good adherence, respectively. quality of life was evaluated by saint george's respiratory questionnaire (sgrq) and the impact of disease by copd assessment test (cat). the study was conducted in september 2014 and february 2015. the association between potential predictors and copd impact or quality of life was estimated using multiple linear regression in ibm spss statistics version 23. main outcome measures: medication adherence rate (mmas-8), quality of life (sgrq total score) and impact of disease (cat score). results: of 65 patients, majority were men (68%) with mean age 70 years. in average, patients were prescribed 2.5 medicines for copd and 3.8 medicines for other diseases. good, moderate and low adherence to copd medication regimens was found in 53.4, 32.8 and 13.8% of patients, respectively. mean cat scores and sgrq scores were 17.3 (range 3-34) and 41.3 (range 2-79), respectively. thirtyeight percent of patients experienced an exacerbation in the past year. linear regression showed no statistically significant association between medication adherence and quality of life or copd impact on patient. factors that statistically significantly predicted patients' quality of life were exacerbation in the past year, education level and number of concomitant medicines for other diseases. the latter was found to be the only factor associated with copd impact. conclusion: the study showed half of the copd patients to be optimally adherent to their treatment and only a small proportion of patients not taking their medicines regularly. due to the nature of the disease medication adherence does not seem to play the most important role in assuring optimal health outcomes in copd patients. please specify your abstract type: descriptive abstract (for projects) background and objective: intermediate care units (imcu) are designed to serve patients in need of more advanced medical care than the ordinary nursing home units can provide. the aim of this study was to see; (1) how medication information follows patients in and out of icmu and nursing home short-termcare units (stcu) (2) the type and amount of drug related problems (drp), focusing inappropriate drugs, and (3) if there are differences between the icmu and stcu in drug use and drps. design: patients c65 years old admitted and submitted at the imcu or stcu in the study period (10 weeks) were included. transfer of medication information were evaluated and given a score. the clinical pharmacist provided medication reconciliation upon admission, medication review and monitoring, and presented identified drps and a suggestion for solving the problem, to the multidisciplinary team. inappropriate drugs, identified by screening tools (stopp/norgep), and systematic medication reviews, were recorded. results: 14 patients from imcu and five from stcu were included. a hospital discharge summary including medical history followed mostly all patients. the score of the medication history was 7.5 points out of 16. by submission from either imcu or stcu, the score was 8.6. systematic drug review identified 3.9 drp in the imcu and 2.0 in the stcu. imcu patients used 9.5 drugs, stcu patients 10. in the icu, 14% of the identified drps was inappropriate drugs, none in the stcu. the clinical pharmacist in the multidisciplinary team presented 92% of the identified drps. the doctors agreed in 80% of the suggestions for solution, and started immediate changes in 43%. conclusion: a hospital discharge summary followed the patients, but the medical history part needs improvement. although few patients, the results suggest that imcu patients had more complicated medication and more inappropriate drugs than stcu patients did. clinical pharmacist in a multidisciplinary team provides useful contribution to identify, solve and prevent clinical relevant drps, including inappropriate drugs. please specify your abstract type: research abstract background and objective: lack of clinical effects of medication review on health-related quality of life of older people may be due to insufficient focus on health-related complaints. goal attainment scales (gas) are an instrument to formulate specific health-related goals. the objective of this early process-evaluation of the dreamer-study (drug use reconsidered in the elderly using goal attainment scales during medication review) is to investigate if pharmacists are able to formulate gas during a medication review of older people with polypharmacy. setting and method: older patients aged 70 years or older using 7 or more medicines are included in this study. half of the patients were randomized into the intervention group, where they received a medication review. during the patient interview, the pharmacist formulated gas in concordance with the patient. recommendations were made to reach these goals in collaboration with the gp. main outcome measures: number of performed medication reviews, total number of formulated gas and the three most frequent types of gas. results: until now 453 patients have been included in the drea-mer study (60% of the target). half of them (220) were randomized into the intervention group. by now 179 (81%) of these patients have received a patient interview. 143 goal attainment scales were formulated yet. the number of gas ranged from 0 to 3 per patient. the four most frequent gas were: polypharmacy-reducing the number of medicines (28), reducing pain (23), increasing mobility (16), reducing fatigue (15). conclusion: gas seem to be a feasible approach during medication review that increased focus on patient's needs and health-related complaints. cp-pc019: oral transmucosal fentanyl citrate: a regional survey of dispensing practices in community pharmacy please specify your abstract type: descriptive abstract (for projects) background and objective: oral transmucosal fentanyl citrate (otfc) is an opioid analgesic indicated for management of breakthrough cancer pain in patients with malignancies who are already receiving and who are tolerant to opioid therapy for their underlying persistent cancer pain. otfc are usually use off-label prescription, especially in noncancer patients or patients without opioid maintenance treatment. this practice can expose to iatrogenic risks, lack of efficacy, abuse and addiction. the observatory of drugs, medical devices and therapeutic innovation of upper normandy, conducted a study to assess the knowledge of pharmacists on these medications and assess dispensing practices (pharmaceutical analysis and advice to patients). design: between june and september 2015, two quizzes were sent to the 1344 pharmacists and 512 pharmacies in upper normandy: one included questions of knowledge and general practice, the other assess dispensing practices of otfc prescriptions received at the counter, regarding indication, dosage and associated opioid medication. results: of the 93 pharmacists who participate in the survey, 21% know the all of the 7 oftc specialties, 46% of them confuse transdermal and transmucosal fentanyl specialties. indication, dosage, titration methods and the main interest of oftc are known by 52, 43, 18 and 71% of them. only 30% have dispensed oftc more than 10 times over the past 12 months, 28% never have. they already have dispensed oftc in noncancer patients (45%) or without opioid maintenance treatment (36%). they consider not know enough about these drugs to be able to provide the necessary advice to patient (57%) and would like specific training on oftc (89%). of the 21 analyzed prescriptions, only 24% are consistent with the marketing authorization: otfc medicines are prescribing in noncancer patient (52%) and/or dosage is higher than four units per day (24%) and/or there is no prescribed opioid maintenance treatment (24%). only two prescriptions have been discussed with the prescriber, and all were approved and dispensed. conclusion: otfc specialties are occasionally dispensed and often misunderstanding by pharmacists. a good knowledge of otfc is necessary to achieve the pharmaceutical analysis and provided appropriate advice to patients, in order to guarantee the good use of these medicines. support tools for dispensation, recalling indication, . the most frequent interventions were drug substitution (n = 132), dose adjustment (n = 57), and clarification of information (n = 48). common services were reconstitution of suspension (n = 7), provision in advance for continuing supply (n = 26), and follow up offers (n = 3). conclusion: the observation of the dispensing process in community pharmacies revealed a broad range of tasks performed by the pharmacy and identified several variables likely to influence the counselling. in addition, pharmacy activities could be pictured by the documentation of pharmaceutical interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a multidisciplinary process to correct medication errors resulting from miscommunicated information at transitions of care. development of this activity is essential but it is hindered by the time required for its implementation. we must carefully choose which services can develop this activity. as it was recently introduced in cardiac surgery unit, this study aims to evaluate impact of this process to hospital admission (severity of potential harm of medication error intercepted) and to determine the relevance of this activity in this unit. design: prospective study conducted from january 2016 to april 2016. the data is recorded in an excel table, filled after each mr. there are five items: patient's age, best possible medication histories (bpmh), implementation period of the mr, inadvertent discrepancies (ids) and clinical impact. to assess the severity of ids, a scoring method was used (doerper et al. 2015) with the cooperation of surgeon and pharmacist. results: eighty-two patients (mean age 68 ± 8 years old) were included in the study, which represents 52% of the patients hospitalized in this service. the mean number of drugs per patient was 6 ± 3. the bpmh were obtained within 24 h to 72 h of admission to hospital. a total of 34 ids were detected, with a mean of 0.41 ids per patient. the most frequent type of ids was omission (52%, n = 16), error of dose (39%, n = 12). the three most common classes involved in ids were hypolipaemic drug (n = 9), antidiabetic drugs (n = 6) and the drugs for acid related disorders (n = 4). the mean of ids per patient (0.41) as well as the percentage of patients affected by a ids (32%) are less important in cardiac surgery than those observed in other services of the institution and in the literature. about clinical impact, 31% of patients presented with ids considered as minor, 44% significant and 25% major. among the major ids, none was evaluated as critical or catastrophic. in our study, this process remains retroactive. conclusion: one of challenge experienced when implementing mr process in hospitals is demonstrating its clinical impact. in order to address this concern, we found that the little ids with a serious clinical impact in this unit. mr is an interesting process to detect drug errors. to optimize our study we will improve our organization in order to be closer to the patient and to strengthen the doctor-pharmacist collaboration. please specify your abstract type: research abstract background and objective: special packaging like multidose drug dispensing (mdd) may optimize medication use in patients with a decreased ability to manage their own medication. however, it remains unclear how a 'decreased ability to manage medication' is defined. the objective of this study is to assess potential medication problems that contribute to a decreased ability to manage medication in patients starting with mdd compared to patients who use manually-dispensed drugs. setting and method: patients starting with mdd (cases) and patients using manually-dispensed drugs (controls) were interviewed in 45 community pharmacies. questions to assess potential medication problems covered three domains; medication adherence (12), practical management issues (12) and medication knowledge (2) . every potential medication problem was scored with one point. cognition was assessed with the mini-cog and frailty with the groningen frailty index (gfi). main outcome measures: mean scores of potential medication problems on the domains medication adherence, practical management issues and medication knowledge. results: 188 patients starting with mdd and 230 patients using manually-dispensed drugs were interviewed. patients starting with mdd scored more potential medication problems on all domains: adherence 5.5 versus 1.7, practical management issues 3.7 versus 2.1, medication knowledge 1.1 versus 0.3. on the three domains together, patients starting with mdd scored 10.2 [9.6-10.9] potential medication problems compared to 4.1 [3.7-4.6 ] for patients with manuallydispensed drugs. forty-two percent of the patients starting with mdd might be cognitive impaired and 63% was classified as frail compared to 20 and 27% respectively of the patients using manually-dispensed drugs. conclusion: patients starting with mdd reported significantly more potential problems on three domains that may contribute to a decreased ability to manage their medication. cp-pc024: fifteen key questions to assess patient knowledge on new oral anticoagulants corina metaxas * , valerie wentzky, sonja luginbü hl, kurt e. hersberger, isabelle arnet please specify your abstract type: research abstract background and objective: knowledge on new oral anticoagulants (noacs) is crucial for their safe and effective use. validated tools that assess patient knowledge exist for vitamin k antagonists, but not for noacs. we aimed to identify which questions are relevant for patient knowledge on noacs. setting and method: based on a systematic literature search, 45 questions were compiled for the assessment of noacs knowledge. key questions were selected through three rounds of ranking by an expert panel (four physicians, four pharmacists, four nurses). round 1 (online survey; importance): the 45 questions grouped into the nine educational topics of wofford,adapted for noac (disease, mode of action, risk-benefit, adherence, accessing healthcare professionals, diet/life-style, lab-monitoring, medication interactions, self-care) were to be rated as important/not important and educational topics were to be ranked according to decreasing importance. round 2 (online survey; relevance): the questions were to be ranked according to decreasing relevance. round 3 (focus group): number of questions was reduced by voting. main outcome measures: ranking of educational topics and questions (1 = most important/relevant) in march/april 2016. results: experts ranked adherence (2.2 ± 0.9) as the most important topic, followed by risk-benefit (3.0 ± 1.6), disease (3.7 ± 2.7), accessing healthcare professionals (4.1 ± 2.0), self-care (5.3 ± 2.6), lab-monitoring (5.6 ± 1.6), medication interactions (6.5 ± 1.8), diet/life-style (7.3 ± 0.9) and mode of action (7.8 ± 1.5). one question was judged as unimportant by all experts. out of the remaining 44 questions, 10 (22.7%) were selected as relevant for basic knowledge, 7 (15.9%) were combined into four questions and one new question was generated. a total of 15 key questions remained after the focus group discussion. conclusion: a multiprofessional expert panel was able to select key questions retrieved from literature and ensured content validity. the selected questions will be compiled into a tool to assess patient knowledge on noacs. background and objective: medicines use review (mur) was defined by the slovene chamber of pharmacies in december 2014 and an education program was set to assure pharmacists competencies. in june 2015 the first pharmacists were certified and implemented the service in the community pharmacies. additionally, an online database was established to collect mur reports and provide feedback on pharmacists' performance. the aim of the study was to evaluate identified drug related problems (drp) as well as pharmacists' interventions from mur documentation. setting and method: a preliminary retrospective analysis of documentation for mur services provided in the first year after implementation was performed. drps were classified using a slovenian drp classification system, which is based on the pcne classification v 6.2 [1] . data were analysed with descriptive statistics measures. main outcome measures: number and type of identified drp and pharmacists' intervention. results: a preliminary analysis was performed on 129 mur cases, performed by 11 certified pharmacists. in total 258 drps were identified: 116 (44.96%) manifested and 142 (55.04%) potential. patient had on average two drps, however 11 patients had none. main risk factor for potential drps was inappropriate use of medicines. adverse drug events (ades) presented 50.86% of manifested drps; the main risk factor was again inappropriate use. in two cases ades happened due to an allergic reaction. 29 different medicines were the cause of ades; mainly statins resulting in muscles pain and sleeplessness. another frequently manifested drp was insufficient effectiveness of treatment. drug interactions were risk factors in 12 cases of manifested drps, mainly in connection with antidepressants: serotonin syndrome due to escitalopram, bleedings in concurrent use of escitalopram and ginkgo, sleepiness, etc. pharmacist intervened independently in 74.4% of cases; 57 times recommendations were given to physicians. however, in 92.6% of cases the outcome of intervention is unknown. the preliminary results of the first mur cases points to a high number of identified manifested drps. however, the knowledge of intervention outcomes is lacking and therefore more attention has to be put on establishing adequate follow up on this issue. official definition represented harmonisation of several similar activities that have already been performed in slovenian pharmacies and also provided an educational program to assure pharmacists competencies. in may 2015 the first 18 pharmacists were certified and implemented the service in the community pharmacies. therefore, the aim of the research was to get an insight into the implementation of mur in slovenia from the perspective of the first community pharmacists that provide the service in practice. setting and method: a focus group with seven community pharmacists, that provide mur in practice, was run in february 2016. guided discussion included three main themes: the development and assurance of competencies, experience with the provision of service in practice and the future of the service. the discussion was voice recorded and analysed with the nvivo 11. written consent from included participants was obtained. main outcome measures: views, challenges and opportunities for the medicines use review service in slovenia. results: in total 364 themes were identified and organized in three main categories: competencies for quality provision of mur, mur's recognisability and organizational aspects of mur provision. participants emphasized broad knowledge in pharmacotherapy is pharmacists' key competence and advantage in performing mur when compared with other healthcare professions. recognisability of mur among other health care professions as well as participants' work environments is low. hence a comprehensive approach in marketing of the service is needed. positive patient's feedbacks were reported, however persuading patients to attend mur presented a challenge. another barrier was the time to perform mur, which could be overcome by suitable work organization and special time intended for mur. conclusion: participants of the focus panel had positive experience with the development of competencies and implementation of the service in the practice. several challenges were presented connected with the recognition of the service by patients, physicians and health care payer. they strongly believe that continuing professional development forms the base for quality of the service in the future. cp-pc027: evaluation of rational antibiotic dispensing in the community pharmacy setting: a simulated patient study betul okuyan * , mehmet ali savan, fikret vehbi izzettin, mesut sancar please specify your abstract type: research abstract background and objective: in the present study, it is aimed to evaluate rationale antibiotic dispensing without prescription in the community pharmacy setting; this will be done by using a simulated patient methods. setting and method: this study was conducted in malatya, located in the east part of turkey. the simulated patient visited the community pharmacies to meet the pharmacist, posing as the husband of a patient with acute uncomplicated rhinosinusitis. the simulated patient was trained regarding the standard information to be provided by the researchers and informed about the privacy of all information that would be gathered during the present study. the sample size was sixty-seven pharmacies, with a confidence interval of 95% and error of margin of 10%. the study was conducted over a total of 70 pharmacies. all the pharmacies were listed alphabetically and were randomly selected and allocated random numbers by a computerbased program. main outcome measures: after each community pharmacy was visited, the simulated patient filled the check list which had been drawn up for the purpose of the present study. due to ethical concerns, no audio or video records were used during the study. any suggested medications were not purchased from the community pharmacy. results: of the total community pharmacies that were visited 55.7% of them had female pharmacists and 44.3% were run by male pharmacists. the mean number of questions asked by pharmacists to the simulated patient was 3.17 ± 1.65. only eleven pharmacists did not suggest any medication for the simulated patient. however, thirty-two (45.7%) pharmacists recommended various medication regimens, including antibiotics. of them, 67.1% referred the simulated patient to a physician. conclusion: in conclusion, it was observed that dispensing antibiotics without prescription was still high, pharmacists did not take comprehensive medical or medication history from patients, and pharmacists provided insufficient medication information to the patient regarding suggested medications at community pharmacy setting. to avoid irrational antibiotic dispensing, it is essential to educate both health care providers and the general population. although dispensing antibiotics without prescription is illegal in some countries, it is necessary to actualize new regulations to avoid antibiotic dispensing without prescription. please specify your abstract type: research abstract background and objective: the medication adherence is an important part of active (as well as passive) attitude of a patient to the disease treatment. it represents the level of keeping the treating procedure as well as the recommendations of doctors, pharmacists and other healthcare professionals. this study deals with the adherence in patients with hypertension. the hypertensive patients are a substantial part of patients, daily visiting the community pharmacy to pick their prescriptions. these patients represent group of patients with typical asymptomatic disease. this means that they do not take the medicines or use them according to their own will. the result of their non-adherence could lead to later complications. the aim of the study was to evaluate the level of adherence and its relation to the clinical outcome-the blood pressure in hypertensive patients. setting and method: the methodology was based on a single anonymous questionnaire survey combined with the blood pressure measuring in a community pharmacy in slovakia. the modified morisky 4-item medication adherence tool was used in this study. main outcome measures: the results of medication adherence were evaluated as follows: 3-4 points = full adherence, 2 points = partial adherence and 0-1 = non-adherence. each participant should use at least one antihypertensive agent and fulfil the anonymous questionnaire in the community pharmacy. the pharmacist measured the blood pressure in each participant twice, within the interval of 10 min and used the average value in data sheet. results: the research included 150 hypertensive patients (55.33% females and 44.67% males). the results showed that almost 46% of the respondents were non-adherent to the prescribed pharmacotherapy (57.98% of those were males and 42.02% were females). the group of partially adherent patients consisted of 35.33% of the respondents (66.04% of those were female). only 18.67% respondents were fully adherent according to modified morisky score (67.86% of those were women). fully adherent patients reached an average blood pressure 117.393/76.464 mmhg; partially adherent hypertensive patients recorded an average blood pressure 124.162/80.623 mmhg; and in the non-adherent patients has been observed the average blood pressure 154.116/95.319 mmhg. the results showed an alarming situation, and confirm the published data. non-adherent patients could not goal the good clinical outcomes. this leads to adding of another medications, raising the risk of interactions and adverse drug reactions, complications of undertreated disease, and finally, to pharmacotherapy costs increasing. please specify your abstract type: research abstract background and objective: in psychology, depression is a mental state characterized by feelings of sadness, dejection, inner tension and indecision. in psychiatry, the depression is defined as a severe mental affective disorder which paralyzes clarity of thought, psychomotorics, sleep cycle and raises pessimistic and depressing emotions often lead to pathological changes of personality. during treatment of depression is often needed psychotherapy and pharmacotherapy as well. using of antidepressants requires the sufficient level of medication adherence in patients. non-adherence to antidepressant medication significantly contributes to the undertreatment of depression in primary care populations. the aim of this study was to evaluate the level of medication adherence to antidepressants to better understand the socio-behavioural factors associated with non-adherence. setting and method: the anonymous, face-to-face questionnaire survey was set in the community pharmacy in slovakia. questionnaire obtained questions on socio-behavioural factors and adherence tool-modified 8-item morisky score (mmmas-8). main outcome measures: respondents were 150 patients (39 males, 111 females) using at least one antidepressant. the results were evaluated as follows: 8 points = full adherence, 6-7 points = partial adherence and 0-5 = non-adherence. results were evaluated in relation to socio-behavioural factors. results: average level of the medication adherence in our group was 5113, which means the line between partial and full adherence. the results showed non-significant higher medication adherence level in males (5179) compared to females (5090). the highest level of medication adherence (5367) has been shown in patients 45-64 years old, the lowest average adherence level (non-adherence) was observed in patients up to 24 years old (4656). patient living in the city were more adherent to their medication (5127) compared to patients living in countryside (5083). the highest level of the partial medication adherence has been shown in secondary educated patients (5344). partial adherence level was higher in patients with monthly income over 1000 € (5750) compared to non-adherent patients with monthly income up to 300€ (4878). in patients using no other medications, only antidepressant, we have observed the highest partial adherence (5219). conclusion: our survey showed the partial antidepressant medication adherence levels in our study group. poor adherence results in low stabilization of clinical state in patient, in using more types of therapy and in increasing costs. there might be very important role of the community pharmacists and other health care professionals to improve the medication adherence and persistence through counselling and education patients on importance and need of antidepressant medication. (1) and medication regimen complexity was assessed by using the medication regimen complexity index (mrci) (2) . five and more medication usage has been defined as polypharmacy. results: a hundred and two elderly subjects (74.5 ± 6.0; 65 male) were included in this study. of them, 89.2% had two and more chronic diseases. the most common chronic diseases determined in study population were cardiovascular diseases (especially hypertension), diabetes and hyperlipidaemia. the polypharmacy has been defined in 77.5% of them. the mean of mrci per elderly patient was 12.5 ± 7.0. one or more pims use was observed in seventy-four elderly subjects (72.5%). of all elderly subjects, 59.8% were dispensed one and more medicines with a potential for drug-disease/ syndrome interaction. pims use was more frequently determined in patients with polypharmacy (78.5 vs. 52.2%, p \ 0.05). the total score of mrci was significantly increased with elevated number of pims (r = 0.304, p \ 0.01). conclusion: this study highlights a significant association between utilization of pims and both polypharmacy and higher total score of mrci in elderly patients. pharmacists could be evaluated utilization of pims in especially elderly patients with used five or more medications and/or higher total score of mrci. please specify your abstract type: research abstract background and objective: nursing home patients with multimorbidity often use multiple drugs simultaneously, which makes these patients more susceptible to adverse drug events. several studies have pointed to a need to increase the quality of prescribing to this population. to achieve this there is a need for reliable information about patients' diagnosis, and what is recorded as the drug's indication in different electronical and handwritten health records. the aim of this study was to examine the registered diagnoses, and indications for drug use in nursing home patients. we also wanted to study the extent to which diagnoses are untreated with drugs, as well as the extent to which drugs have a registered indication for use and a suitable recorded diagnosis. setting and method: data was collected for 70 long-term patients, on average 83 years old, and 66% females from four nursing homes in tromsø municipality, norway. we retrieved information about patients' diagnoses and indication for drug use from the electronic health record and written drug charts. two pharmacists conducted the linkage between the reported diagnoses and drug use. main outcome measures: percentage of untreated diagnoses and the percentage of drugs with a registered indication for use. results: as considered by the pharmacists, 70% of the registered diagnoses was untreated with drugs. dementia, gout and osteoporosis were the most commonly untreated diagnoses with, 97, 60 and 57%, respectively. in comparison, the indication for use listed on the patients' drug charts was reported for 82% of the drugs. the drugs with the highest percentage of recorded indications were acetylcysteine (n = 15), oxycodone (n = 14) and zopiclone (n = 11), where 100, 93 and 90% had a listed indication, respectively. conclusion: a high percentage of nursing home patients' diagnoses seem to be untreated. however, most drugs that patients received were listed with indication for use in the drug charts. to increase quality of drug prescribing, one should put emphasis on improving the recorded information in electronical health records. cp-pc033: personal changes in drug regimen: dangerous for health system? inga urtane, raivis pastars, dace bandere please specify your abstract type: research abstract background and objective: patient compliance is a key factor for a successful treatment and lack of it is the main reason for predicting treatment failure. in multiple researches patient adherence is determined to be as low as 50%. therefore it is important to identify the reasons of patients not following their drug regimen. objective. to analyse the patient comprehension of their drug regimen depending on the duration of hypertension and received treatment. setting and method: during the period from december 2015 to march 2016 a quantitative survey was conducted to include respondents who have been diagnosed with arterial hypertension and whose regimen includes at least one fixed dose combination drug. main outcome measures: in an anonymous survey data was collected about their demographic information, co-morbidity, other prescribed medication, intake regime, the average blood pressure during treatment, and patient's assessment of the prescribed therapy. collected data was analysed with spss. results: the study included 103 participants, most of whom (64.1%) were women. participants average age was 62.5 ± 12.5 years and the median arterial hypertension duration was 8 (5; 16) years. the study participants, who sometimes consciously adjusted dosing regimen, observed arterial hypertension for a longer period of time compared to the group, which follows the prescribed regimen according to their doctor's recommendation, respectively, 10 (6; 28) vs. 8 (4; 15); p = 0.110. group of respondents (n = 16) receiving c3 prescription drugs, more often deliberately adjusted treatment regimen compared to respondents (n = 8) treated with b2 prescription drugs, respectively 26.7 versus 18.6%; p = 0.310. respondents who deliberately adjusted drug were more often not satisfied with the number of longterm daily use of tablets (n = 13) compared to the group (n = 11), which had to intake fewer tablets every day, respectively, 54.2 versus 45.8%; p = 0.005. conclusion: arterial hypertension duration was associated with more frequent conscious adjustment of therapy without consulting a doctor. more individual prescriptions (c3) and an increase in the number of tablets per day at the same time also increases the risk of patients deliberately changing their dosing regimen. long-term drug users should receive additional attention during pharmaceutical care process to their respective treatment schedule in order to promote proper use of medication. please specify your abstract type: research abstract background and objective: diabetes is a health issue and real burden for 1 in 6 belgians. better adherence to the treatment could potentially reduce complications, decrease morbidity and mortality, and have a beneficial economic impact due to fewer consultations and hospitalizations. setting and method: a one-year program was started in 370 belgian pharmacies to accompany diabetes patients taking dpp-4 inhibitors and encourage them to be compliant with their treatment. this study concerns 270 of these pharmacies, all part of the same cooperative group. all pharmacists received prior training in motivational techniques and reviewed the bases of diabetes therapy with an e-learning program. materials developed for the patients included brochures on diabetes and its treatment, nutritional advice, physical exercise, foot care and tips and tricks for diabetics. main outcome measures: the impact on pharmacological adherence was measured using mmpr and pdc. two control groups were included: a historical control group and a group of patients that were not included in the project. non-pharmacological adherence was assessed using questionnaires. results: in the subgroup of 270 pharmacies, 495 patients were included in the program. by the end of april 2016, only 44 of them had completed the program; 78 patients came only once to the pharmacy. they either stopped their treatment after one prescription, or were occasional clients. adherence rates were found to be high in all groups (5.7-9.1% of patients with mmpr b 80%). only for the pdc, a statistically significant difference was measured between the intervention and control group (135.37 vs. 129.22%; p = 0.015). no other statistically significant impact was measured (neither pharmacological, nor non-pharmacological). conclusion: adherence was very high in all groups. the underlying reasons still need to be investigated (choice of adherence measure, healthy user effect, etc.). however, both patients and pharmacists were very pleased with this type of program. this new role of the pharmacist will definitely be more developed in the future. please specify your abstract type: research abstract background and objective: oral anticoagulants (oac) have a beneficial effect on the long term survival of patients with atrial fibrillation and venous thromboembolism. however oacs have also side effects such as bleeding, especially when used inappropriately. pharmaceutical care interventions aim to optimize medicines use and improve patient health outcomes. the literature lacks a review on the impact of pharmaceutical care interventions in patients using oac. therefore, we systematically assessed the impact of pharmaceutical care interventions on the effective and safe use of oac compared to usual care. setting and method: a systematic review was performed in pubmed and embase with synonyms/detailed specifications of the terms oral int j clin pharm (2017) . it was motivate for the need to sort the instruments for urm, including professional participation, and on the basis of the clinical management unit, and reduce variability in decisions. the p&t or ''multidisciplinary commission rational use of medicines'' is constituted by 13 people: one hospital medical director (president), head of pharmacy (secretary), and three directors of healthcare centre, three directors of department of specialities, one epidemiology, one hospital pharmacy, one primary care pharmacy and one paediatric. because some of these members are far between them, and normally dose not have too much time, we create an online platform to work, discuss and download all the necessary documents. setting and method: we used the facilities of the andalusian agency for healthcare quality (www.acsa.junta-andalucia.es), and as a base the law of the administrative decision. we have organized a session to discuss methodology with the participation of all members. main outcome measures: number of meeting and number of internal discussion emails. drug or protocol decisions. design of the platform. results: the design platform consists of five tabs: (1) has the member information, position, telephone and address, (2) email forum, following a subject line, (3) a place for meeting requests and then hang up the meeting minutes. (4) a tool allows you to upload documents to the portal (5) a search engine. two sessions are schedules and total of 28 mails. we have 4 of 13 members who have never participated online. at this moment we have adopted two decisions. conclusion: it is an online experience of one andalusian p&t committees, the low turnout makes go slower than expected, therefore physical meetings are necessary in this moment. we are working how to get more participation and involve in the project the committee members. please specify your abstract type: research abstract background and objective: liver cirrhosis can have a major impact on drug metabolism, requiring evaluation of drug safety and dosage in individual patients. currently, there are no guidelines on safe prescribing for medications in patients with liver cirrhosis, and these patients have many questions about safety and side effects of medication. the objective of this study is to explore the patient's needs on information about medication. setting and method: qualitative, semi-structured interviews were performed in 28 patients with a (history of) liver cirrhosis. the patients were approached through an item in the newsletter of de dutch association of liver patients. topics in the interview guide were preferences about information about medication, side effects, safety, drug dosage, and how patients preferred to receive this information. interviews were audiotaped and transcribed verbatim. interviews were analysed using thematic content analysis. main outcome measures: the experiences and needs of patients with liver cirrhosis concerning information about medication. results: patients indicated they had received sufficient information about the indication, possible drug-drug interactions and the duration of treatment. they preferred (more) information about how medications work, what adverse drug reactions could be expected and practical aspects concerning intake of medication. informational needs were related to questions 'how to act': patients with more informational needs took a more active role in responsibility for their own medication management. patients needed information to know what to do, e.g. in case of adverse drug reactions or when a dosage was forgotten. the doctor and internet were the preferred sources of information: doctors because of the personal contact and internet because of the accessibility. facilitating factors were 'taking time' in healthcare provider-patient contact and 'everyday language' for texts on the internet and in package leaflets. a combination of verbal information by the healthcare professional and written information was preferred. conclusion: patients with liver cirrhosis need information about medication to take an active role in their drug management. comission for medicines and medical devices, chu de toulouse, toulouse, france please specify your abstract type: descriptive abstract (for projects) background and objective: due to its common use, insulin is often considered as a harmless medication by lots of health professionals while an overdose can lead to dramatic consequences and death. between january 2014 and june 2016, in our university hospital, 6% (7 out of 121) of the declared adverse drug events have involved insulin: 2 were caused by prescription errors and 5 by administration errors. all were discovered after the medicines have been administered but thankfully none had serious consequences. the british national health service (nhs) and the french medication safety national agency (ansm) made a list of ''never events'': avoidable events which should never happen and misadministration of insulin is among them. the objective was to increase patient safety in the hospital by setting different actions to promote and improve the appropriate use of insulin and warn health professionals about the real dangers of this medicine. design: different actors participated in the implementation of these actions: the commission for medicines and medical devices (which is composed by doctors and pharmacists) directed a group made by physicians and clinical pharmacists from the department of cardiological and metabolic diseases'. results: in addition of the usual analysis of any adverse event linked to medication declared in the hospital, several actions were set up: • a didactic document summarizing all the ''sensitive'' steps during the prescription, stocking, dispensation and administration of insulin has made the front page of the hospital's intranet and was also diffused throughout the establishment. • a chart resuming all the different insulins commercialized in france has also been diffused. it contains their types, durations and onsets of action, conditions of storage and pictures of their packaging. • the 49 computerized protocols involving insulin are going to be reviewed in order to lower their numbers and harmonize their content. • a revision of the list of insulins available at the hospital is in progress to reduce their number and avoid any confusion between the different products. • an evaluation of insulin's computerized prescription practices will be made via a data request. : this topic about insulin shows a greater willingness to secure the medication circuit in the hospital. other action plans such as this one will be set up involving other medications among the never-events list. meanwhile, the commission for medicines and medical devices pursues its actions of promoting the appropriate use of medication. please specify your abstract type: descriptive abstract (for projects) background and objective: one of the hospital pharmacist tasks is to suggest substitutions to ensure conformity of medical prescription with the hospital formulary. indeed, when an eye drop isn't available at the hospital, there is a specific supply circuit which has to remain exceptional: it's ordered directly to the pharmaceutical wholesaler. in this context, ophthalmologists and clinical pharmacists created a table proposing therapeutic equivalencies with eye drops available at the hospital. after approval by the commission for medicines and medical devices, this tool has been diffused within the establishment via the intranet website since the beginning of 2013 to the medical and paramedical staff. the purpose of the study is to evaluate professional practices concerning the use of the eye drops' equivalence chart. design: in the study, we compared eye drops' orders made to the pharmaceutical wholesaler before and after the table's diffusion, thus between january 2012 and december 2015. for each order, we used the table available at this time to determine if equivalencies could have been proposed or not. if so, we identified the hospital ward and the pharmaceutical specialty. market changes have also been considered. results: we noticed a decreased frequency of eye-drops ordered despite available equivalencies: 52% in 2012 (before the table's diffusion), 25% in 2013 (after its diffusion), 33% in 2014 and 33% in 2015. prisons units are responsible of 95% of these orders: they have the lowest rates of substitutions. their most ordered pharmaceutical specialties are ophthalmic glaucoma agents: 46% ganfort ò (bimatoprost 0.3 mg/ml/timolol 0.5%), 20% xalacom ò (latanoprost + timolol 0.5%), 23% azopt ò (brinzolamide) for which the authorized substitutions are for the first two specialties: monoprost ò (latanoprost) + ophtim ò (timolol 0.5%) and for the third: dorzolamide ò . conclusion: equivalence table diffusion throughout the hospital has facilitated and improved the prescription and substitution of eyedrops. orders of pharmaceutical specialties despite authorized equivalencies available have declined by half. probably for practical reasons regarding long-term treatments, prison units make less substitutions but an awareness campaign will be carried out to reduce these rates. please specify your abstract type: research abstract background and objective: the patient's education and information is a mean to reduce medicine misuse and it can be performed with support of a leaflet or informative material about medicines. in brazil, there is a lack in regulation about this type of informative material to compounded medicines. the aim of this study was to evaluate the quality and effectiveness of leaflets developed to compounded medicines' users through knowledge's level and medicine treatment adherence. setting and method: analytical and quantitative study; 3 month prospective study through interviews, at time zero (t0) and after 30 days (t1) in a university pharmacy in goiás, brasil; fisher's exact test to measure effectiveness; ethics committee number 008/12. main outcome measures: categorization into high adherence and low adherence by morisky test; categorization into sufficient, regular or insufficient knowledge about medicine prescription; perceptions and suggestions about delivered leaflet in medicine dispensing process. results: of 52 patients (82.7% female, mean = 48.7 years), 93.5% considered as relevant the leaflet's content, as well 88.5% of them kept it and 67.4% of them read it. suggestions of 16.1% included a desire in increase font size, more emphasis on drug interactions and images. there was a predominance of regular knowledge in both analysed times (48.4% e 38.7%), however there was a decrease in high adherence to medicine treatment (19.2-7.7%). among patients who read the leaflet, no statistically significant association was found on these two variables at t0 and t1 (p = 0.24 and p = 0.84, respectively). knowledge about ''administration schedules'' showed a significant improvement after intervention (p = 0.02). 61.3% of patients considered that there was no need to obtain more information. conclusion: this study demonstrates the evaluated leaflets had relevance to patients and demonstrate clinical relevance. however was not observed statistically significance. this highlights the need of using different ways to measure the effectiveness of an informative material to promote rational use of medicines and depth studies and stimulation of greater attention from the health professionals to the topic. di006: chlormethine gel: effectiveness and tolerance to treat mycosis fungoides françois dugre *,1 , anne lefebure 1 , sonia martelli 1 , marion pin 1 , eve maubec 2 , philippe arnaud 1 1 pharmacy, 2 dermatology, bichat-claude bernard hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: to determine the effectiveness and tolerance of chlormethine gel in treating mycosis fungoides. design: mycosis fungoides is the most common form of cutaneous t-cell lymphoma (mf-ctcl). early stages (ia and ib) can be controlled by skin-directed therapies such as chlormethine and carmustine. these drugs which are solutions for injection are usually used for skin application. chlormethine or mechlorethamine gel is an alkylating agent representing an alternative for previously treated patients diagnosed with mf-ctcl, in case of therapeutic failure and intolerance, or in case of chlormethine and carmustine solutions supply disruption. a retrospective observational analysis was conducted based on medical records of all patients treated by chlormethine gel in our hospital from the first of july to the first of september 2015. the following data were collected with an excel table: body surface area or bsa affected by disease, location of the lesions, therapeutic management, effectiveness and treatment tolerance. results: fourteen patients (7 women, 7 men, mean age 55 [min 33; max 84]) were treated with chlormethine gel in our hospital. twelve (86%) were treated three times per week, 2 (14%) once a day. before treatment by chlormethine gel, 4 (29%) patients were treated by dermocorticoids, 4 (29%) by dermocorticoids and phototherapy, and 1 (7%) by bexarotene, all of them stopped their treatment on account of inefficacity. one (7%) patient was treated by carmustine and dermocorticoid, and 3 (21%) by only carmustin, all of them stopped it because of supply disruption. one (7%) patient received it in first line therapy. ten (71%) patients showed a response (partial or complete), one (7%) experienced a stabilization of his disease. before treatment with topical chlormethine, seven patients (50%) had an involved bsa [ 10% and four of them (57.1%) experienced adverse effects. seven patients (50%) had an involved bsa \10% and three of them had (42.9%) side effects. a total of seven patients (50%) presented at least one adverse effect. five patients (36%) stopped the treatment on account of adverse effects; two of them (14%) interrupted it temporarily. reported side effects were: irritant dermatitis and erosive toxicity (5), rash (2) and telangiectasia (2) . conclusion: our results indicate that chlormethine gel can be effective to treat mycosis fungoides. however, it involves side effects that seems to be more frequent than those observed with chlormethine solution (used for skin application). indeed, the french national authority for health reports 28% of adverse effects for chlormethine solution versus 50% in our study for chlormethine gel. moreover, telangiectasia was never documented with chlormethine. this significant number of side effects of chlormethine gel can be explained by the gel formulation which induces patients to apply more product, especially in patients with plaques affecting more than 10% of the bsa. it is important to explain to patients to apply a thin film of chlormethine gel to involved skin areas and allow the skin to dry completely. sophie dumas *,1 , capucine devaux 1 , nathalie le guyader 2 1 diaconesses croix saint-simon hospital, 2 diaconesses croix saint-simon hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: aprepitant, a neurokinin-1 receptor antagonist, prevents nausea and vomiting due to high and moderate emetogenic chemotherapy in combination with other antiemetic agents. it induces cytochrome p450 (cyp) 2c9 and moderately inhibits cyp3a4. drug-drug interaction could occur with intravenous anticancer or antiemetic drugs metabolised by these isoenzymes. it may lead to adverse effects or loss in efficacy. regarding recent international antiemetic guidelines, emergence of new intravenous chemotherapy and lack of bibliographic data, a report on aprepitant interactions is performed in oncology. the aim of this study is to review pharmacokinetic interactions with aprepitant in order to prevent potential toxic effects of intravenous anticancer or antiemetic agents and provide the best patient care. design: anticancer and antiemetic agents metabolised by cyp3a4 and 2c9 were identified. pharmacokinetic literature review was performed using medline ò database and laboratory data. clinical assessment and non-aprepitant pharmacokinetic studies were excluded. a table was established to summarize data. results: ten intravenous anticancer agents used in oncology are identified as cyp3a4 substrates. pharmacokinetic assessments are achieved for docetaxel, cyclophosphamide, vinorelbine, irinotecan and trabectedin. studies dealing with the five other drugs are strictly clinical assessments. among the different pharmacokinetic studies, only trabectedin showed relevant interaction with aprepitant. in this association, aprepitant dose needs to be adjusted. cyp2c9 catalyses the cyclophosphamide activation pathway with minor contribution. however, it would have few repercussions on cyclophosphamide pharmacokinetic. corticosteroids and 5 hydroxytryptamine type 3 (5ht-3) receptor antagonists are also metabolised by cyp3a4. aprepitant significantly increases corticosteroid plasma concentrations. in this case, corticosteroid dose adjustment should be applied. furthermore, no interaction has been found with 5ht-3 receptor antagonist. conclusion: regardless of the emetogenic level of anticancer agents, all drugs have been studied because of theirs potential combinations. two relevant pharmacokinetic interactions have been demonstrated leading to dose adjustment recommendation. corticosteroids doses, in association with aprepitant, should be reduced one-fourth for intravenous form and one half for oral form. aprepitant first dose should be decreased to 80 mg when it is co-administrated with trabectedin. these two results lead us to re-evaluate our prescription practices. please specify your abstract type: research abstract background and objective: nsaids are associated with serious adverse reactions which in turn are responsible for significant risks of morbidity and mortality. the aims of this project is to identify risks involved in nsaid administration including over-usage and significant drug interactions, and to analyse occurrence of side-effects. the trends of nsaid prescribing by physicians and pharmacists are also determined. setting and method: a pharmacy from each electoral district was chosen by stratified sampling. a sample population (n = 100) was obtained from 13 pharmacies in malta. data was collected through the completion of questionnaires carried out by the patients. the trends of nsaid prescribing were determined by another questionnaire directed to 49 pharmacists and physicians that was available online. main outcome measures: use of nsaids by patients and prescribing trends. results: back pain (n = 19), muscular pain (n = 13), headache (n = 12) and arthritic pain (n = 10) accounted for the most frequent use of nsaids. diclofenac accounted for the most commonly administered nsaid, taken by 58 of the patients, of which 48 use the 50 mg dose. chronic disorders of symptoms experienced by the patients included hypertension (n = 24), heartburn (n = 22), dyspepsia (n = 21), asthma (n = 4) and a history of helicobacter pylori infection (n = 2). other disorders suffered by single individuals include epilepsy, crohn's disease and renal dysfunction. more than half of the respondents (n = 55) admitted to self-prescribing regardless the fact that the majority of nsaids are prescription-only medications. epigastric pain (64.6%), stomach ulcers or gi bleeding (30.6%) and elevated blood pressure (29.8%) were the most common sideeffects that pharmacists and physicians come across. nsaids were frequently found to be co-administered with antihypertensives (68.1%) and ssris (37%) regardless of their significant risks of interacting with nsaids. 75.5% of the pharmacists and doctors believe that nsaids are being over-used and 95.9% state that closer monitoring of nsaid adverse effects is necessary. conclusion: the risk involved with nsaid administration due to over-usage and drug interactions is identified, and healthcare professionals are aware of this risk. pierre leduc, antoine lanneluc, christophe gellis * , sylvie poux, dominique plats, regine larnaudie corrèze, ch brive la gaillarde, brive la gaillarde, france please specify your abstract type: research abstract background and objective: proton pump inhibitors (ppi) are widely prescribed in hospital while their long-term use may be responsible of many potentially serious long-term side effects (hypomagnesemia, neutropenia, gastric cancer) and drug interactions (ppi are inhibitors of cyp2c19). the objective of this study was to assess the appropriateness of ppi prescriptions in a geriatric department in order to optimize their conditions of prescriptions. setting and method: this prospective study involved patients hospitalized between january 2016 and april 2016 in a geriatric department. the accordance of the prescriptions with the marketing authorization indications and the french guidelines 1 was analysed. data collection was done using a table excel. main outcome measures: collected information were related to patients (age, sex) and ppi prescriptions (active substance, administration route, dosage, duration of therapy, therapy indication and reassessment of ppi therapy). results: ninety-one patients were included: sr: 0.3, mean age: 87.3 years [78; 102]. ppi therapy prevalence over the period was 38%. the ppi were prescribed in the geriatric department in 33 patients (mostly esomeprazole) whereas 58 patients had ppi therapy (mostly esomeprazole) at the admission, for more than 2 years in 42 patients. oral route was the most frequent one (n = 86). 89 ppi were administered once a day and only three ppi were administered in the morning. 40% of ppi prescriptions were considered unjustified; the indications were prevention of haemorrhage with antiplatelet therapy (n = 19), prevention of haemorrhage with corticoid (n = 4), prevention of haemorrhage with anti-vitamin k (n = 3), dyspeptic disorders (n = 2), gastralgy (n = 4) and others reasons (n = 4). 60% of ppi prescriptions were considered relevant. the reassessment of ipp therapy (n = 25) lead to prescribe another dosage (n = 9), to stop therapy (n = 11) or no change (n = 5). conclusion: the study showed that the majority of ipp prescriptions were not in accordance with french guidelines. limiting the prescription to the indications, reassessing the therapy or respecting the therapy duration should reduce the risk of long term side effects and the economic burden of ppi in a long term use. please specify your abstract type: descriptive abstract (for projects) background and objective: to evaluate the effectiveness and safety of the use of high dose of tigecycline (200 mg followed by 100 mg every 12 h) a tertiary care hospital. design: retrospective observational study. period: january to december 2015. inclusion criteria: episodes use of tigecycline (200 mg followed by 100 mg every 12 h. exclusion criteria: time less than 4 days treatment. data source: corporate program stories electronic health. results: we identified 24 episodes in 23 patients (15 men, mean age: 71 years (23-88)). treatment was directed to multidrug-resistant organism infection in 11 cases (seven klebsiella pneumoniae oxa-48, two enterobacter cloacae, two enterococcus faecium and one methicillin resistant staphylococcus aureus. in one episode they coincided e. cloacae and e. faecium). in 14 cases had severe sepsis or septic shock (seven abdominal focus, six respiratory focus and one unknown focus). the median number of days of treatment was 12 (4-119). tigecycline was administered as monotherapy in three cases, bitherapy 13 and triple combination therapy in 13. the antibiotics were associated were: beta-lactam (11), aminoglycosides (10), quinolones (3) colistin (three, two inhaled cases), cotrimoxazole (1) and vancomycin (1) . in 13 episodes produced clinical and/or microbiological resolution and 7 antibiotics are rotated by progression picture or lack of improvement, death occurred in three cases and 1 was suspended on suspicion of hepatotoxicity. among the seven episodes of klebsiella pneumoniae oxa-48 infection there were four pneumonias, three with favourable evolution and one patient died, two bacteraemia, both with resolution clinical and microbiological, and one urinary tract infection resolved. among the 14 episodes in severe/ septic shock were five cures, six cases of antibiotic rotation progression or lack of improvement and three deaths while patients receiving therapy tigecycline. 2 patients showed an adverse effect possibly related to therapy tigecycline: 1 diarrhoea after 15 days of treatment and 1case of liver toxicity after 4 days of tigecycline and piperacillin-tazobactam which led to their withdrawal. int j clin pharm (2017) 39:208-341 253 conclusion: tigecycline has been used in double dose defined in data sheet especially in situations of severe sepsis or septic shock and infection multiresistant microorganisms. the effectiveness is conditioned by the clinical situation patient, being worse in severe/septic shock sepsis. tigecycline high dose was well tolerated and there was only a case of stopping the medication for suspected damage hepatic. di012: wikipedia and medicines: who edits medicine articles on the english wikipedia? kristian husvik skancke 1 , kristian svendsen *,2 1 department of history, uit -the arctic university of norway, 2 hospital pharmacy of tromsø, tromsø, norway please specify your abstract type: research abstract background and objective: the medical profession and pharmacists are divided on the usability of wikipedia for looking up health information. nevertheless wikipedia is widely used, more than half of us physicians and 94 percent of all medical students use wikipedia as a source of health-related information. there is a potential for incorrect and biased information being added by the pharmaceutical industry. the aim of this project was to examine who edits wikipedia articles on medicines and to investigate whether the pharmaceutical industry edits these articles. setting and method: two different groups of articles has been examined; the top ten bestselling medicines (substances) in the world in 2014 and the ten most recently approved medicines on the european market (until december 2014). the top ten medicines were selected from a consultancy report by evaluatepharma/ep vantage. the ten most recently approved medicines (new substances) were found on the european medicines agency webpage. we queried the english wikipedia on 11 january 2015 and information from the edit history and the editors' user information were extracted. unregistered editors were checked using a whois service. for the new medicines all editors were checked, while for the bestselling medicines large edits and initial edits was checked. main outcome measures: edits suspected of being made by the pharmaceutical industry. results: ten bestselling medicines: there are many users editing these articles and/or watching them, limiting the risk of misinformation from the industry. there was no indication that the pharmaceutical industry had edited any of the articles. ten most recent medicines: no article existed for dasabuvir. for the nine other substances there were relatively few editors and watchers. in four out of the nine articles we found evidence of edits from the pharmaceutical industry. these edits, were done by registered editors with very few edits except for the medicine in question and they had made large additions to the articles sometimes even before the medicine was marketed. conclusion: the pharmaceutical industry seems to edit articles about medicines on english wikipedia however we found no evidence of harmful edits and bestselling medicines have many editors monitoring the quality of articles. please specify your abstract type: research abstract background and objective: the pharmaceutical professional service of the monitored dosage systems (mds) tries to improve the adherence of the patients to the treatment. the aim was to analyse the relevance of the repackaging of the most sold medicines in our country being used by patients included in the mds professional service and to determine the information discrepancies according to the source used by the pharmacist. setting and method: cross-sectional descriptive study. community pharmacy and healthcare institutions. all the patients included in the pharmaceutical professional service of mds on june 1, 2016. data source: patients' records in the professional service, database of medicines ranked by sales in units in our country to december 2015 (400 medicines), information sources on medicines: (1) vademecum of medicines and (2) the centre of drug information of our agency of medicines. main outcome measures: number of institutionalized and ambulatory patients included in the professional service of the mds and demographic characteristics, sum of different repackaged medicines belonging to the studied patients, analysis of the repackaged medicines of major use, number of discrepancies on the repackaging of the medicines according to the information source. results: 88 patients were included in the professional service of the mds. 67 of them were institutionalized (average age: 39.9 years, 65.7% men, 76.1% polymedicated defined as using c4 prescribed chronic medicines) and the remaining 21 were ambulatory (average age: 76.8 years, 57.1% women, 23.7% polymedicated). 130 different medicines prescribed in the institutionalized patients were taken into account, 29 of them included in the sales ranking in our country. according to the first source, 18 of 29 medicines were eligible for repackaging, 6 medicines could be repackaged according to the laboratory manufacturer and the 5 remaining ones could not be repackaged. according to the second source, 21 of 29 medicines could be repackaged, and the 8 remaining ones could not. 128 different medicines prescribed in the ambulatory patients were taken into account, 37 of them included in the sales ranking in our country. according to the first source, 27 of 37 medicines could be repackaged, 8 medicines would depend on the laboratory manufacturer and the 2 remaining ones could not be repackaged. according to the second source, 27 of 37 medicines could be repackaged, and the 10 remaining ones must remain in the original package. discrepancies were observed in the information for 8 (27.6%) and 14 (37.8%) medicines in institutionalized and ambulatory patients, respectively, based on the sources used. conclusion: a considerable number of discrepancies in the information on the relevance of the repackaging of medications in the mds were found between two analysed sources. these findings have already improved the quality of this professional service. it would be necessary to alert the pharmacist of the existence of the above mentioned discrepancies to be able to prevent errors from occurring at the time of repackaging the medicines in the mds and, thus, increasing patient safety. please specify your abstract type: research abstract background and objective: despite the global advances of pharmacy practice and subsequently pharmacy education, students experience insufficient opportunities to practice the activities, tasks and processes essential to deliver pharmaceutical care. objective: to describe the development, implementation, and assessment of a clinical pharmacy practice (cpp) experience course in internal medicine, cardiovascular, respiratory clinics and drug information centre that is newly integrated into pharmacy curriculum at a university in north cyprus. setting and method: a 8 weeks structured pharmacy practice experience was designed for fifth year students. student competence was assessed using formative osces and summative written exams before and after the course, and mapped in eight main cpp competences. the course utilized a wide variety of learning and practical activities including rounds participation, morning case reports, interdisciplinary activities, carrying interventions, role-play, direct patient care, formal case presentations, journal clubs and answering drug queries. competencies tested and strengthened include: taking medication history, response to the symptoms, pharmacotherapy knowledge application, comprehensive patient assessment, data interpretation using evidence-based approach, public health counselling, drug related problems management, patient counselling and communication skills. student perceptions and experience was assessed using semi-structured group interview and a questionnaire. main outcome measures: student scores in osce; student's perceptions. results: student reported that the course met pre-set objectives with substantial learning in different areas of cpp. students scored best in communication skills (83.4 ± 1.74%), public health promotion (76.6 ± 2.32%) and patient counselling (68.0 ± 2.74%) than in resolution of drps (49.0 ± 4.33%) and pharmacotherapy application (49.0 ± 3.37%), while they significantly enhanced in di manipulation (88.1 ± 2.6%) compared to baseline assessment (33.1 ± 2.41%)(p = 0.0038). conclusion: the course provided a rich experiential learning environment rather than just theoretical knowledge of clinical pharmacy. students well perceived the course structure assessment and knowledge attained. this could be implemented in other faculties of pharmacy through turkey. please specify your abstract type: descriptive abstract (for projects) background and objective: clinical pharmacy and clinical pharmacology have many similar aspects. both areas present professionals who have groundings in drug therapy principles and who aim to optimize the efficacy and safety of therapies for patient's benefits. however, there are clear distinctions. clinical pharmacologists are in general doctors with an additional education in clinical pharmacology. many of these are prescribers of drugs in practice but are in usual connected to academic parts responsible for education and research. they belong to a well-recognized but small sub-specialty of medicine. in contrast, clinical pharmacists are part of a much greater group of professionals working in most hospitals in developed countries. while the former one is restricted and subordinate to distributing the drugs requested by the medical prescribers, the role of the pharmacist has increasingly developed to encircle monitoring outcomes of medicine treatment and report management, patient safety and budgetary responsibilities. pharmacists are currently capable to take on prescribing responsibilities in developed countries and have been actively involved in collaboration in practice of prescribing with doctors. they also take on a great part in education related to rational prescribing that was once thought the area of the clinical pharmacologist. given the difference in size of the two areas there is understandably increasing confusion in the minds of managers in health services as to the continuing role and identity of clinical pharmacology. this may illuminate, in part, the diminishing in numbers and visibility of clinical pharmacologists in certain countries. in fact, some might see the continuous development of clinical pharmacy as a direct danger to the viability and future existence of the specialty of clinical pharmacology. however, clinical pharmacy and clinical pharmacology working synergistically would serve for the well-being of the public. design: . results: . conclusion: . maxime apparuit *,1 , lea boissinot 1 , ngauv melodie 1 , stephanie charles weber 2 , isabelle lopez 1 , françois chast 1 1 pharmacy, hopital cochin, 2 pharmacy, hopital hotel-dieu, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: hereditary angioedema (hae) is a rare disease characterized by episodic attacks of swelling which can be life-threatening. treatment for hae involves prophylaxis and management of acute attacks. the objective of this study was to evaluate patients' knowledge of their disease and their treatment. design: a questionnaire about the disease and drug treatment has been implemented. it was distributed to patients through either a pharmacist during patients stay at the hospital, or the french association des malades souffrant d'angioedèmes (amsao). answers were collected by electronic or conventional mail. results: 39 patients completed the questionnaire. the average patients age is 25.8 ± 27.2 years. all of those interviewed could name their disease. for 23% of patients, the crisis happened unpredictably but in most cases a triggering factor was described, such as stress (21%), fatigue (18%) or an emotional shock (17%). oedema were located mainly in extremities (31%), abdomen (19%), ent sphere (12%) or face (11%). 17 patients (43%) reported having more than 12 crisis each year (eligible to prophylaxis), among them, 5 patients (13%) said they had no preventive treatment. all patients knew the difference between prophylactic and curative treatment of crisis. among the 38 patients receiving treatment for crisis, 16 were able to define which treatment to be used depending on the intensity and location of the crisis. the majority of patients used icatibant during a crisis, but the most frequently cited prophylaxis treatments were tranexamic acid (38%) and danazol (35%). for injectable drugs to treat acute episodes, icatibant (subcutaneous) and c1 esterase inhibitor (intravenous) were self-administrated respectively in 79 and 8% of patients. conclusion: this study showed that patients generally knew their disease and its treatment. however, they are insufficiently informed on drugs to be used according to the clinical situation and especially intravenous self-administration. therefore, it seems necessary to increase pharmacist involvement in patient's information about therapeutic strategy and drugs routes of administration. this for a major objective: an optimal self-care in a skilled patient. please specify your abstract type: descriptive abstract (for projects) background and objective: hospital pharmaceutical educations (hpe) on patients with oral anticoagulant (oa) can improve their overall management by providing skills on proper use. an ambulatory monitoring is necessary to ensure good compliance and understanding of the treatment. our study aimed at the establishment of hpe for patients with oa, the establishment of a hospital-city link in burgundy, and an evaluation of the expectations of ambulatory health professionals (ahp). design: the development of hpe has been performed in our centre for patients with oa and assessed between may and september 2015. in order to ensure continuity in their support, patients then received a binding document to the attending physician, pharmacist and nursing home stating the treatment and acquired skills. a satisfaction survey, with anonymous electronic questionnaire circulated by the representative boards evaluating the expectations of ahp, took place in order to improve and make the programme more attractive. results: two hundred and ninety-one patients could benefit from hpe and 252 came out with an oa. one hundred and forty-three answers were collected: 38 officinal pharmacists and 105 nurses. ninety-seven percent of ahp have judged relevant the following stated security goals: the name of the drug, its use, its risks and to be able to inform all ahp. ''associated pathologies and treatments,'' ''the last coagulation test'' and ''potential factors for non-adherence'' seem necessary for the binding document. more than 90% of participants found that this action will facilitate the establishment of pharmaceutical anticoagulant educations in cities, the dialogue around the oa with the doctor, patient's compliance and will secure the treatment. conclusion: hpe certainly help patients. its implementation for patients with oa in our hospital has generated a real interest. the addition of an ambulatory link allows continuing at best their support. the questionnaire has also allowed us to know the opinions of ahp involved and some improvements to the binding document may have been done. participants were asked to associate the task to the profession by determining whether each profession had the main responsibility for undertaking the task, a supportive responsibility, or whether they should not be involved at all. data was analysed using spss ò, version 22. the chi squared test was used to assess any significant association between categorical variables. main outcome measures: perception of the oncology pharmacist's role by healthcare professionals. results: from a total of 84 completed questionnaires, it was found that for tasks listed as ''patient education and counselling'', 18% were considered as the pharmacists' main responsibility, whereas 42% were believed to be supportive roles. main tasks included educating the patient regarding which medication to avoid during their treatment. for tasks listed as ''drug related problems'', 20 and 45% of tasks were found to include pharmacists as having main and supportive roles respectively. supportive tasks included dose calculation of anti-tumour therapy required per patient. in the ''authorisation of medication'' category pharmacists' main roles carried a total of 13% and supportive that of 50% of the total number of tasks. this included ordering anti-tumour medication. further analysis of data revealed that years of experience did not have a significant association with results obtained (p-value = 0.074); however physicians, pharmacists and allied healthcare professionals were found to involve the pharmacist most extensively (pvalue = 0.000). conclusion: tasks associated with the pharmacist were representative of the current role they possess within the oncology setting; however this association was limited to professionals having a close working relationship with pharmacists. this may be due to the lack of an established multidisciplinary team approach within this scenario thus limiting the perception of the oncology pharmacist's contribution. an implemented multidisciplinary team may improve communication between the professionals involved and optimises patient care. the aim of the study is to analyse from a qualitative and quantitative point of view the pharmacy resident's activity in pneumology service. setting and method: the study included all the daily prescriptions of three units of pneumology from january to april 2016. pi and data were extracted from the software pharma ò and collected in a summary excel ò table: nature of potential errors, nature of the proposals offered by residents, way of transmitting pi, and rate of pis' acceptance. main outcome measures: potential errors are collected by following the validated and standardized criterions of french society of clinical pharmacy. results: over 4 months, 7968 lines of prescriptions from 412 patients aged 67 years old (median [28-85]) were evaluated. sex ratio (m/f) was 1.46. one hundred and two medication problems have been found: overdose (21.6%), contraindication (ic) (20.7%), under dosage (7.8%), wrong rhythm of administration (7.8%), forgotten treatment (7.8%), dose unit error (5.9%), antibiotic indication missing (5.9%), drug not listed in the hospital formulary (4.9%), potassemie unchecked (4.9%), dose unadapted to renal function (4.9%) or to inr (2.9%), treatment not indicated (1.9%), wrong administration route (0.98%), antibiotic unreevaluated (0.98%), redundancy (0.98%). the proposals made to the doctors were: stopping treatment (25.5%), posology adaptation (19.6%), substitution (19.6%), dose unit modification (7.8%), adding information about the indication (6.7%), treatment renewal (6.7%), administration modalities changing (4.9%), biological monitoring (3.9%), therapeutical monitoring (2.9%), antibiotic treatment reevaluation (2.94%). all pi were made by informatical way. all medicinal classes were found in this study. hydroxyzine, cyamemazine and escitalopram were often found in contraindication errors. they are involved in cardiac disorders with qt extension. pis' acceptance rate was 82%. conclusion: this study shows the importance of pharmaceutical analysis on the quality of access to healthcare. the statement of pi allows us to identify the most frequent errors, warn and prevent doctors from these potentials errors by proposing solutions. the rate of acceptance is high which means that doctors agree with our proposals. pharmacists' implication in clinical pharmacy activities and their participation to medical rounds will improve this activity and by the way optimization of the management of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: ppis consumption is largely practiced in europe, because of their excellent tolerance in short time, and their misuse with regard to indications, dosage and treatment duration (in 2007, france was the 2nd,ppi consumer in eu). the result is drug iatrogenic disease and unjustified expenses in health insurance. objectives: assess the ppis consumption and appreciate conformity according to the latest recommendations for relevant prescriptions of ppis. design: prospective study via an audit (model created internally), every hospitalized patients with a ppis prescription, in two hospitals, on a given day. data collected through the patient's medical record. prescriptions conformity defined, by taking account of indication level (1: approved by the ma (marketing authorization), 2: non-valid but certified by international publications or learned society, 3: nonvalid without scientific proofs, 4: non-indicated), dosage and treatment duration. analysed situations with no conformity (inappropriate dosage despite conform indication, treatment duration unjustified and ppis prescribed in wrong indications (3 and 4) . results: 172 patients have ppis prescriptions (78 male, 25 £ 99 years] among 325 patients (53%). 45% ppis prescriptions began during the hospitalization. 53 (30%) of the ppis prescriptions are in accordance with the experiment (indication + dosage +treatment duration), as well in community than in hospitals. details: indication level 1 (92.5%), indication level 2-gi bleedings-(7.5%). 119 of the ppis prescription aren't in accordance. details: treatment duration (6%), dosage (3%), indication level 3-prevention of iatrogenic bleeding risk without nsaids prescription-(75%), indication level 4 (16%). regarding level 3 indications, ppis are always taken with anticoagulant and/or platelet aggregation inhibitors and/or corticoid. conclusion: the part of ppis prescriptions in this study is high. the majority of non-conformity is caused by ppis prescribed with an indication level 3. the improvement program will involve feeding back ppis' good use, to educate physicians (junior and senior) about the relevant ppis prescription and give advice in complex situations (indication 3 and 4). in collaboration with prescribers, shutdown protocol of ppis, prescribed in long term, could be implemented in order to avoid the acid rebound effect after brutal treatment discontinuation. hp-ce014: impact of a self-management program on inflammatory bowel disease patient in a university hospital caroline egon * , xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: inflammatory bowel disease (ibd) is a group of chronic inflammatory diseases that affects the colon and the small intestine. crohn's disease and ulcerative colitis are the principal types of ibd and involve severe diarrhoea, pain, fatigue and weight loss. ibd affects young adult with an increasing annual incidence (2.5 million concerned people in europe). patients with ibd are affected by somatic or psychosocial problems and patient education may contribute to their well-being. since september 2010, individual educational sessions have been set up and since september 2015, collective educational sessions. these sessions have been developed to improve patient's understanding of treatment options and medical adherence. the aim of this study was to demonstrate that a therapeutic education program (tep) could have a significant effect on ibd patient's skills with regards to their disease. design: after individual education sessions with a nurse, a group education session was introduced for outpatients with ibd. the collective session include approximately six to ten patients and is organized in a half day workshops (about disease and treatment) conducted by a multidisciplinary team. the workshops were performed by an education nurse, two hospital gastroenterologists, two hospital pharmacists and a community pharmacist. these sessions were wrapped up by a short satisfaction and knowledge questionnaires. results: in total, 141 ibd outpatients participated to the educational program, 112 patients with crohn's disease and 29 patients with ulcerative colitis (52.5% male; median age: 39). for the individual educational sessions, two competence questionnaires were performed about anti-tumour necrosis factor alpha (tnfa) therapy: one about general knowledge, another one about self-administration subcutaneous injection. 43 patients completed these questionnaires. for the collective educational session, the competence questionnaire developed consisting of six questions covering few items: disease, symptoms, treatment and complications. 14 patients completed this questionnaire. after the questionnaire, each participant received a summary document about drugs, side effects, therapeutic and medical advice. conclusion: the patient education program contributes to the improvement of self-management skills when it comes to ibd. pharmacists joining medical specialists and nurses provided pharmaceutical care with a positive impact on compliance, which is a determining factor for the success of the treatment and the quality of life in patients living with an ibd. this program will be continued and a new program for teenagers is to be established as well. hp-ce015: desensitization study of paclitaxel and carboplatin drug in the ovary tumor protocol in cuf descobertas hospital miguel â . freitas * , daniela brites, ana bota pharmacy, hospital cuf descobertas, lisbon, portugal please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacy should be an integral part of the multidisciplinary team and implement strategies that meet the patient's needs. pharmacy, in oncological area, is in constant renewal. josé de mello saúde uses paclitaxel/carboplatin protocol as first line in ovarian tumor. although the antineoplastic agents are essential for the treatment of cancer, they can also cause hypersensitivity reactions, which may carry serious consequences. both immunoallergologist and oncologist create a desensitization protocol, which allows the reintroduction of the drug with greater security. the desensitization protocols involves the gradual administration of small quantities of the drug, resulting in a refractory period of the white blood cells (mastocytes) and a lower production of cytokines until the dose has been totally administrated. objective:to evaluate the efficacy of methods used to prevent and treat hypersensitivity reactions of carboplatin and paclitaxel, in order to carry on the treatment. design: a retrospective review of the patient files was performed in the day hospital between 2014 and 2016. we included only patients with moderate to severe immediate hypersensitivity reactions (b24 h) receiving carboplatin and paclitaxel. the desensitization protocol brigham and women's hospital was applied using three solutions with increasing concentrations (dilution 1: 100, 1:10 and 1:1) in twelve successive steps for about 6 h. results: in the period 2014-2016 were desensitized five patients with platinum group drugs, carboplatin (n = 4) and paclitaxel (n = 2) and the total elapsed six desensitization. almost all patients reached the scheduled daily dose, except a patient, which suspended the desensitization program for disease progression. conclusion: the desensitization protocol allowed the successful reintroduction of antineoplastic drugs in patients with a history of hypersensitivity reactions, in order to treat the disease. please specify your abstract type: research abstract background and objective: in the context of harmonization of clinical pharmacy activities within our region, a common medication reconciliation project was developed between two general hospitals. the objectives of this study were to initiate, a common medication reconciliation activity in the two hospitals, to analyse the results, and to communicate to all professionals in the area. setting and method: a working group composed of pharmacists of each hospital was formed to develop analytical documents. a 3-month prospective study was conducted in two general hospitals: in the first one, in an emergency department, and the other one, in a medicine department. patients included in the study were either elderly and/or had polypharmacy and/or were hospitalized for iatrogenic reason. at int j clin pharm (2017) 39:208-341 259 the point of admission and discharge for each patient, the pharmacist has completed a conciliation record, and has detected potential discrepancy. unintentional discrepancies were reviewed and corrected by doctors. at the discharge, medication changes were sent to general practitioner and community pharmacies. a satisfaction survey about this process was sent to 39 healthcare professionals (gp, pharmacist and nurses). a medication reconciliation's workshop was organized for a hundred healthcare professionals in the area. main outcome measures: at the point of admission, the conciliation record included the list of patient's home medication, admission medical orders, and the types of discrepancies. at the discharge, drugs prescribed were compared to admission medical orders. the satisfaction survey included seven questions to assess the process. results: during the study period, 35 patients were included corresponding to 351 prescription lines. reconciliation process required about 47 min per patient. we identified at admission 33 unintentional discrepancies. the most common unintentional discrepancy was the omission of medication (61%). 25% concerned alimentary tract and metabolism group. at the discharge, no discrepancies were found; the process required 35 min per patient.41% of healthcare professionals answered to our satisfaction survey to date. 100% are satisfied and believe that the process of medication reconciliation secures the patient medicinal treatment.56 healthcare professionals were present at the medication reconciliation's workshop, indicating an interest in the process. conclusion: in this experience of medication reconciliation, due to unintentional discrepancies observed, we had better implement this activity in the two general hospitals. a pharmacist devoted to this activity will be hire in each hospital. this relevant practice is well accepted by clinician. thus, we will improve communication with gp and community pharmacies. please specify your abstract type: descriptive abstract (for projects) background and objective: the sickle cell disease (scd) is a genetic, chronic disease, paroxystic in its unpredictable and polymorphic acute events. this most frequent genetic illness in the world is a major public health concern in french overseas territories. haute autorité de santé (has) recommendations for the care of scd advocate the development of therapeutic patient education (tpe). in martinique (french west indies), we consider the population of patients with scd in 1500 among which 1000 are followed in the adults sickle cell centre (ascc). one of the actions carried out by the ascc of our hospital is the tpe. the objective is to set up an original (because specific in the scd) tpe method, which enables the patient to live better with his disease on a daily basis, by teaching him and his family to recognize prematurely certain complications. design: we analysed needs from the outcomes of a national french survey has which one participated martinique and retained the following themes: the red blood cell, the genetic transmission, the main symptoms, the role of the water, the medicinal treatments and the questions of everyday life. we chose the innovative educational tools called the ''malles des savoirs ò **'', a set of unusual experiments, accessories and models, which, by using a method of active pedagogy ''omca*: observer, manipuler, comprendre, agir ***'', value the learner by offering to him to manipulate and to experiment by himself. results: in 2016, 24 healthcare professionals (doctors, pharmacists, nurses) and a president of patients with scd association followed one week of formation in the omca* method for the animation of six workshops for 10-15 teenagers and adults. every ''malle des savoirs ò **'' contains the necessary material for the animation and a guide of the organizer, including, for every tackled issue, a generic introduction, a presentation of the themes, the index cards of educational animations proposing the activities and one time of synthesis grouping the approaches concepts. the interactive manipulation allows the appropriation of the discoveries become then long-lasting experiences. a final evaluation allows to spot the problems met by learners to understand, to analyse the difficulties and to proceed to the useful adaptations during the next activity. conclusion: this tool, playful and perfectly adapted to the scd, engages, accompanies and helps patients in the construction of their own knowledges to return them actors of their disease. in 2017, we shall estimate the impact of the development of this specific tpe programme of the patient with scd. please specify your abstract type: research abstract background and objective: to investigate the frequencies and clinical relevance of unintentional medication discrepancies, between preadmission medication lists and discharge medication lists, at discharge from hospital. a discrepancy is considered unintentional if there is no documentation explaining the intent of the medication change or if it is unintentional according to the prescribing physician. setting and method: systematic literature review. main outcome measures: frequency of unintentional medication discrepancies per patient and per medication; frequency of clinically relevant medication discrepancies. results: of the patients included 14-88% experienced at least one unintentional medication discrepancy. of the medications used by the patients, 3-50% were involved in unintentional medication discrepancies. of unintentional medication discrepancies found in five studies, 15-58% were clinically relevant. conclusion: the review documented a high frequency of medication discrepancies, of which many were clinically relevant. ensuring sufficient communication of correct and complete medication information in transitions of care is a process which should be better implemented, to enhance patient safety. please specify your abstract type: research abstract background and objective: to investigate the frequency of medication changes not documented in the discharge letter, at discharge from hospital, for both regular, as needed and over-the-counter medications, supplements and herbal remedies (otc). secondary, differences between variables and patients with undocumented medication changes were investigated. setting and method: the patients included were all part of the intervention groups from an intervention study, conducted by one of the authors (tg), from april 2013 to december 2014. the best possible discharge medication list was compared against the medication list in the discharge letter and any discrepancy between the two lists was noted, taking into account the text in the discharge summary. main outcome measures: the proportion of patients affected by at least one undocumented medication change at discharge and proportion of medications with undocumented changes. the proportion of patients was compared using a test according to gender, age, number of preadmission/discharge medications and length of hospital stay. results: two hundred patients were included in the study. the proportion of patients experiencing at least one undocumented medication change for the three subgroups: regular medications; as needed; otc, were 78, 65 and 55% respectively. the proportion of medications involved in undocumented changes for the three subgroups were 34, 71 and 77% respectively. the proportion of patients experiencing undocumented medication changes was significantly higher in patients with more than five regular medications at admission, (p \ 0.001) and at discharge (p \ 0.001). in both regular and as needed medications, the proportion of patients experiencing undocumented medication changes was higher in patients hospitalized longer than 2 days (p \ 0.001 and p: \ 0.05 respectively). for otc, the rate of patients experiencing undocumented medication changes, was higher in females (p: \ 0.05). conclusion: a high proportion of patients are affected by at least one undocumented medication change and many medications are involved in undocumented changes. correct and complete medication information at admission and discharge may resolve many of these errors, ensuring patent safety at transitions of care. hp-ce020: participation in courses at learning and mastery centre and the impact on patients' beliefs about medicines merethe nilsen *,1 , erik oie 2 , kirsten k viktil 1 1 diakonhjemmet hospital pharmacy, 2 department of internal medicine, diakonhjemmet hospital, oslo, norway please specify your abstract type: descriptive abstract (for projects) background and objective: patients with chronic diseases are referred to learning and mastery centre (lmc) where the main objective is to support patients to cope with chronic diseases. education about the disease(s) (by a physician) and the medication treatment (by a clinical pharmacist) are important elements of these courses. little is known about how the participation at lmc influences the patients' beliefs about medicines. design: patients c18 years participating at a 2 days course at lmc regarding acute coronary disease or atrial fibrillation were included in the period september 2014-december 2015. the patients filled out 'beliefs about medicines questionnaire'(bmq) before and immediately after the course, and also 3 months after the course to evaluate their concern (bmq-concern) and necessity (bmq-necessity) of their cardiovascular medications. the bmq scores were dichotomized at scale midpoint (scale 1-5) to evaluate high and low concern and necessity, and these scores were combined to calculate the 'ambivalence'and 'acceptance', 'sceptical', and 'indifferent'rate to medications, and also the mean scores of the bmq were calculated. results: fifty patients were included, mean age 65 years, 14% were women, using a mean of 3.5 cardiovascular drugs taken regularly. fifty-eight percent of the patients had high concern prior to the course, whereas 37 and 60% had high concern immediately after and 3 months after the course, respectively. ninety-nine percent of the patients assessed their medication as highly necessary before the course, 100% immediately after, and 97% 3 months after the course. the mean score for bmq-necessity was 3.82 (sd 0.64) prior to course and 3.88 (0.56) and 3.78 (0.69) immediately after and 3 months after the course, respectively. the corresponding scores for bmq-concern were 2.58 (0.78), 2.39 (0.72), and 2.57 (0.77), respectively. the proportions of patients classified to be 'accepting'were 40, 63, and 43% at the three time points, respectively, and the corresponding numbers for patients classified as 'ambivalent'were 56, 38, and 60%, respectively. conclusion: the lmc course had an immediate positive influence on the patients' concern about their medicines and on 'acceptance'. however, the effect seems not to persist over time. a closer follow-up could be discussed. please specify your abstract type: research abstract background and objective: the narrative-based medicine was intended primarily for health care professionals, and the use of narratives can be applied in any settings to better understand the meaning of own profession, to rediscover/strengthen the motivation to work as a team. the italian society of hospital pharmacist (sifo) promotes a qualitative study aimed at getting the real picture of pharmacist's role within the national health system (nhs), the interaction with other health professionals and patients through the narratives of under specialization pharmacists (ui) and pharmacists already working in the nhs (hp). these data can be further investigated to increase the perceived value/role of the pharmacist. setting and method: sifo hps and uis joining the national pharmacy school specialization network were invited to participate. all pharmacists participating to the study were given a semi-structure interview. the methodology was developed within the conceptual framework of the grounded theory (gt) a research methodology that arises in the context of qualitative research. gt is a systematic methodology involving the construction of theory grounded in data systematically gathered and analysed. main outcome measures: analysis of narratives. narratives were analysed according to the classifications of kleinman, frank and launer and robinson together with transitional analysis (ta). results: a total number of 31 narratives were collected (16 ups and 15 hps). narratives from both group of participants show the need of strengthening the professional identity already in the early years of the pharmacy curriculum and more effectively during the years of specialization as well as the need of being educated to deliver patientcentred care as members of an interdisciplinary team. conclusion: this is the first step of a study that also includes patient's contribution to the definition of pharmacist's professional identity. hp-ce023: impact of pharmaceutical counselling on cancer patients' information desire and treatment satisfaction stephanie wuyts *,1 , jacques de grève 2 , veerle foulon 3 , hilde collier 1 , pieter-jan cortoos 1 1 pharmacy, 2 medical oncology, university hospital brussels, brussels, 3 faculty of pharmaceutical sciences, catholic university of leuven, leuven, belgium please specify your abstract type: research abstract background and objective: appropriately educating onco-/haematological patients is a prerequisite to improve patient empowerment, satisfaction and outcomes. objective: to quantify patients' information need and satisfaction on cancer drug therapy and how this can be improved by clinical pharmacist's counselling. additionally, the pharmacist's impact on therapy quality and costs is assessed. setting and method: setting: prospective, randomised study in the ambulatory (26 beds) and in-hospital onco-/haematology unit (34 beds) in a tertiary hospital. inclusion criteria: adult patients on intravenous or oral cancer therapy, with informed consent. methods: all patients were asked to complete standardised surveys (extent of information desired, eid; patient satisfaction with cancer treatment education, ps-cate and cancer satisfaction of treatment questionnaire, ctsq) on three occasions (at the start of a new therapy, during the second cycle and after 3 months). patients in the intervention group received additional counselling by a clinical pharmacist including medication reconciliation and review. control patients received standard of care (information on drug therapy was provided by the onco-/haematologist, followed by limited administration instructions by nursing staff). main outcome measures: patient information desire and satisfaction on cancer treatment results: 83 patients were included over a period of 6 months (control (n = 43); intervention (n = 40)). no significant differences were found between contact moments or patient groups for eid, ps-cate and ctsq-scores. however, scores for ps-cate on medication side effects were positively correlated with contact moment (r s = 0.198; p = 0.022). multiple linear regression analysis showed a similar trend (b = 0.207; p = 0.101). patients receiving first-line therapy (b = 0.270; p \ 0.001) and ambulatory patients (b = 0.027; p = 0.018) were more satisfied on treatment education. the clinical pharmacist documented more drugs than were recorded in the patient file (8 vs. 4.9 drugs/patient; p \ 0.001). on average, each patient required two pharmacist's interventions per occasion. intervention acceptance rate on drug related problems was high (72%). during the study, interventions shifted from therapy adjustments towards advice on supportive measures (1st contact: 12%; 3rd contact: 41%). improved medication stock control on the ward led to a savings of €36,890. conclusion: the clinical pharmacist can play an important role on the onco-/haematological ward, leading to improved drug reconciliation, patient counselling and cost savings. hospitalised patients and patients receiving salvage therapy appear to have higher educational needs, making them possibly overlooked target groups. finally, pharmaceutical counselling should be repeated and primarily focused on side-effect management to have a meaningful impact on patient satisfaction. please specify your abstract type: research abstract background and objective: europe is ahead of the usa and canada on approval, regulatory and marketing aspects of biosimilars. however, there is still uncertainty about interchangeability and substitution of biosimilars. the aim of the study is to assess pharmacists' perceptions about biosimilar interchangeability. setting and method: a cross-sectional study was carried out in june-july 2016. hospital pharmacists from quebec and france were invited to respond to an online survey of nine questions (surveymonkey ò , palo alto, ca, usa). the survey focuses on pharmacist's exposition to biosimilars (general knowledge, dispensing) and their perceptions about biosimilar interchangeability. a 5-item likert scale was used to answer to 15 statements based on key issues about biosimilar interchangeability. main outcome measures: levels of agreement on biosimilar interchangeability key issues. results: a total of 229 pharmacists responded (62% in quebec vs. 38% in france). the global response rate is: 27% (23% quebec vs. 34% france) (n = 229/880). 64% attended at least to one conference on biosimilars (57 vs. 74%). 36% had already dispensed biosimilars (7 vs. 81%). more than 95% of the pharmacists knew that: biosimilars can cause immunogenicity, clinical studies are requested for their approval, automatic substitution is not permitted. 43% considered that post-marketing surveillance for biosimilars should be reinforced. pharmacists considered that biosimilars are cheaper than the reference product (89 vs. 75%). there was no difference between the level of agreement of french and quebec pharmacists for the 15 statements. pharmacists agree that a list of biosimilar and interchangeable biologic products is necessary (85 vs. 77%), using the international nonproprietary name to prescribe a biological product can create confusion between the reference product and its biosimilar (60 vs. 55%), pharmacists should check if patients already experienced an immunogenic reaction before dispensing a biological product (82 vs. 70%). pharmacists disagree that a biosimilar can be used for all the indications of the reference product (53 vs. 46%). conclusion: perceptions of quebec and french hospital pharmacists about biosimilar interchangeability issues are very similar. this study highlights the need to deal with the lack of clarity of national guidances. clinical studies on biosimilar interchangeability must be conducted in the future to help pharmacists and physicians to take clear-headed decisions. please specify your abstract type: research abstract background and objective: analgesics are essential drugs in hospitals and especially in emergency units. medical and nurse staffs are used to the narcotic status of opioids. for some drugs, a regulatory change to narcotic status can discourage their use. for others, it could limit their access particularly in developing countries; that's why who did not recommend ketamine to be placed under international control (http://www.who.int/medicines/access/controlled-substances/ recommends_against_ick/en/). yet, the french drug agency has recently considered to register drugs containing ketamine as narcotics. the aim of this study was to assess the impact of this possible regulatory change on the pharmaceutical and medical practices in some paediatric french hospitals. setting and method: the survey was conducted in january-february 2016 in four parisian paediatric hospitals: four pharmacies, paediatric neurology and anaesthesia departments, intensive care units and pain management services. main outcome measures: pharmacists, clinicians, health managers and nurses were interviewed, using a standardized questionnaire with closed and opened questions, on the drug circuit including ordering, storage, distribution, prescription, administration and destruction. results: all the 20 health professionals (five pharmacists, ten clinicians, five nurses) indicate that the change to narcotic status would not preclude the use of an analgesic drug. they consider that the pharmaceutical aspects (dispensation, storage and transport, etc.) are not limiting, provided that clinical usefulness is demonstrated: short action onset allowing rapid efficacy, short duration of action allowing the replacement by another drugs if needed, and moderate clinical monitoring. change to narcotic status was rather seen as advantageous since allowing better traceability, use and prescription. half of the pharmacies (n = 2/4) had a computerized register of narcotics and 80% of care units (n = 4/5) had a drug staffing in addition to nominative prescriptions, which was used in all care services. the drugs were kept into secured rooms. none of the emergency units (n = 0/5) had a computerized secured cabinet. conclusion: according to this survey, narcotic status is not a limiting factor for a drug use in paediatric hospitals, when its clinical usefulness is clearly demonstrated. to promote its use, it is important to inform medical and nurse staffs and include it into care protocols. beyond the nominative prescription, implementation staffing is a key step. please specify your abstract type: research abstract background and objective: port-a-cath is an implanted venous access device most commonly used for frequent or continuous chemotherapy administration. however, the procedure and its subsequent maintenance are not free of complications and requires additional intervention by the clinical pharmacist who can provide further patient care to make a positive impact on. to assess the effective provision of appropriate patient counselling offered by a clinical pharmacist on reducing port-a-cath relatedcomplications in cancer patients. setting and method: a controlled prospective observational study carried out on 110 patients newly diagnosed with cancer eligible for chemotherapy administration at the oncology unit. assessment of port-a-cath related-complications were assessed at regular schedule of chemotherapeutic protocols administration. main outcome measures: to assess, reduce and solve port-a-cath related-complications. results: the most significant port-a-cath related complications were skin rash 55.5% (p \ 0.05) with occurrence in males (n = 40) and females (n = 21), skin erythema 5.5% with equal occurrence in both genders, followed by skin discharge 1.8% with also equal occurrence in both genders. a high occurrence of skin rash 88.2% occurred among diabetic cancer patients. a significant improvement in port-a-cath related complications after the provision of patient counselling by the clinical pharmacist was observed as skin rash (3.6%), skin discharge (0.9%), and skin erythema (0.9%). conclusion: results of this study pointed out the essential role of clinical pharmacist in argumenting patient care and improving port-a-cath related-complications in cancer patients. please specify your abstract type: research abstract background and objective: polytherapy, frequently used in the elderly, is associated to an increased risk of potential drug-drug interactions (pddis) and adverse drug reactions (adrs). literature demonstrated that medication reconciliation and medication review performed by hospital pharmacists are correlated to drug related problems (drps). aim: to define a structured and feasible model where hospital pharmacists support clinicians identifying drps and promote the safe use of medicines. setting and method: prospective, feasibility study conducted in four internal medicine wards of a hospital in northern italy. inpatients (c65 years old, treated with c5 drugs) were consecutively included; the recognition/reconciliation process was performed by pharmacists in order to identify changes between prescription profile at home and during the admission (active principles, dose, administration route). these changes were classified as intentional documented discrepancies (id), not documented (ind), not intentional (ni). prescriptions during the first 24-hours of hospitalisation were analysed to retrieve drps (ddis, inappropriate medications for elderly, off-label, over/ under dosage, duplications, adrs) then discussed with clinicians. based on literature, referring almost 1 drp in 70% of patients, a sample size of 50 patients should allow an estimate of drp rate over 50% (need of intervention) with a 80% power and a confidence interval of 95% (software stata version 12.0). main outcome measures: rate and type of: discrepancies, drps at admission and discharge, pharmacists consultations accepted by clinicians. results: ad interim results are presented. between october/2015-february/2016, 30 inpatients (16 male, 85.4 mean age) were included. overall, patients were admitted with 257 drugs used at home and 240 prescribed during the first 24-hours; pharmacists retrieved 99 discrepancies (45%id, 52%ind, 3%ni) and 118 drps, of which 57% ddis, 1% off-label, 3% overdoses, 3% duplications, 30% inappropriate drugs, 6% not notified adrs. the 70% of drps was known to clinicians and 52% considered clinically relevant for the patients. please specify your abstract type: research abstract background and objective: hypertension is a major risk factor for cardiovascular morbidity and mortality worldwide, for which management is based on two principal, complementary approacheslifestyle modification and lifelong treatment with antihypertensive medication. adherence to hypertension therapy is a major public health challenge, despite the availability of multiple classes of antihypertensive agents. factors contributing to non-adherence are multifactorial and include intolerances to drugs at standard doses that result in therapy discontinuation. medication intolerance (mi-htn) refers to patients who experience adverse drug reactions (adrs) to at least one antihypertensive medication, without a known immunological mechanism and the need to discontinue them. we sought to determine factors associated with mi-htn and to identify patients' beliefs and concerns about their antihypertensive treatment and medication in general. setting and method: a cross sectional survey consisting of selfreported questionnaires including beliefs about medicines questionnaire (bmq), perceived sensitivity to medication (psm) and quality of life was undertaken in an unselected patients attending a hypertension centre of excellence out-patient clinic based in london. main outcome measures: to determine factors associated with mi-htn and the impact of health beliefs and self-reported perceived sensitivity to medications on mi-htn and bp control. chi squared tests for comparisons between cases/controls and multiple logistic regression analysis were used for statistical analysis. results: 102 participants were included, of which 46 (45%) participants had mi-htn. two-thirds were female (p = 0.002) with a mean age of 70 ± 10 years (p 0.001), of whom 67.4% had uncontrolled hypertension (p = 0.063). calcium channel blockers were the most commonly reported intolerance by drug class followed by diuretics. being female and age [60 were statistically associated with a greater likelihood of reporting medicines intolerance (p \ 0.05). patients who believed that medicines are harmful were [5-times more likely to report mi-htn (p = 0.009) and 4-times more likely to have uncontrolled bp ([140/90 mmhg) (p = 0.024). patients with high self-perceived sensitivity to medication was 4-times more prone to mi-htn (p = 0.007). conclusion: our findings suggests the need for greater focus on behavioural change interventions to both improve patients' perception of the necessity to persist with lifelong antihypertensive medication and allay concerns regarding harmful effects of drugs may help with long term control of hypertension. please specify your abstract type: research abstract background and objective: today, the number of medical problems in heart transplant recipients has increased due to aging and complications common to immunosuppressive drugs. the co-existence or emergence of other disease states such as renal dysfunction, infection, diabetes, obesity, hypertension, hyperlipidaemia, malignancies, and osteoporosis necessitates the use of other medications. the use of these medications in combination with immunosuppressive agents increases the risk of drug-drug interactions. the aim of this study is to identify the frequency and significance of drug-drug interactions for the patients who received cardiac transplantation. setting and method: this retrospective study was conducted at a cardiovascular specialty hospital. all patients who received cardiac transplantation from the same surgery team between 2009 and 2014 (6 years) were included in the study. all data were collected from the medical records of the patients. only the most recent prescription before discharge was analysed for the presence and significance of drug-drug interactions. drug-drug interactions were checked using micromedex(r) interaction checker. main outcome measures: main outcome measures were the frequency and significance of drug-drug interactions. results: a total of 58 patients met the inclusion criteria and 58 prescriptions were analysed. each prescription contained an average of 11 drugs. a total of 529 drug-drug interactions were identified: 51.8% was classified as moderate; 43.4% as major and 3.7% as contraindicated. almost half of all interactions (n = 271) included immunosuppressive agents (59.4% was classified as moderate; 35.4% as major and 4.8% as contraindicated). conclusion: cardiac transplant recipients were found to have a high number of drug-drug interactions. in order to advise on these interactions which increase with poly-pharmacy, drugs with narrow therapeutic index or drugs that require intensive monitoring, it is recommended to include a transplantation pharmacist in the transplantation team. please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of patient education provided by the pharmacist on gylcemic control, medication knowledge level and medication adherence of patients with type 2 diabetes. patients who were diagnosed with type 2 diabetes for at least one-year time and were receiving at least one antidiabetic medication, attending to the outpatient diabetes clinic for the control visit were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. setting and method: the setting is a diabetes outpatient clinic of a state hospital. the medication knowledge levels, medication adherence scores, fasting blood glucose levels, hba1c levels and blood pressure of the patients were measured before pharmacist's education. after provision of standard information and individualized patient education all these parameters were measured again after 3 monthstime and the impact of the education was assessed. main outcome measures: main outcome measures are change in the clinical parameters (hba1c; fasting blood glucose; blood pressure), as well as improvements in medication knowledge and adherence levels. results: the study was conducted on 54 patients who met the inclusion criteria; none of the patients were lost to follow-up. majority (83%) of the patients was female and the mean age was 53.7 years. pharmacist intervention resulted in positive outcomes at all clinical parameters. systolic blood pressure decreased by 6 mmhg, while diastolic blood pressure decreased by 1.76 mmhg (p \ 0.05). hba1c level decreased by 0.39% (from 6.94 to 6.55%; p \ 0.05) and fasting blood glucose level by 7.1 mg/dl (p [ 0.05). on the other hand, the number of patients reaching the blood pressure goal increased from 36 to 46; and those reaching to hba1c goal increased from 23 to 32 (p \ 0.05 for all). similarly, the medication knowledge level [usual range 0-8] increased from 4.43 to 5.82 (p \ 0.001); and the medication adherence score [usual range 0-4] increased from 3.4 to 3.09 (p \ 0.001). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control and management of co-morbid conditions of type 2 diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of chronic diseases. hp-pc043: impact of a pharmaceutical care program on glycemic control, medication knowledge and medication adherence levels of type 2 diabetic patients residing at a nursing home nimet saglam *,1 , sule apikoglu-rabus 1 , betul okuyan 1 , fikret v. izzettin 1 , nuran yildirim 2 1 clinical pharmacy department, marmara university faculty of pharmacy, 2 darulaceze nursing home, istanbul, turkey please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of pharmaceutical care provided by the pharmacist on glycaemic control, medication knowledge level and medication adherence of patients with type 2 diabetes residing at a nursing home. setting and method: this prospective cohort study was conducted in a state nursing home (darülacaze nursing home) in istanbul, turkey on 39 patients who completed the whole study. all the patients received pharmaceutical care provided by the pharmacist. this pharmaceutical care program was held for 3 months. it consisted of an initial visit, followed by 5 ''care and control'' visits and a final control visit; each visit was held at two-week time intervals. at the initial visit, demographic and general clinical data were collected and medication knowledge and medication adherence levels of the patients were also assessed. pharmaceutical care needs were identified for each patient and recommendations addressing these issues were structured. education regarding the medications of the patients was provided in both verbal and written forms using the standard patient education leaflets prepared by the pharmacist. at each visit pharmaceutical care needs are assessed and pharmaceutical care is tailored accordingly. main outcome measures: main outcome measures are change in the clinical parameters (hba1c; fasting blood glucose), as well as improvements in medication knowledge and adherence levels. results: majority (74%) of the patients was male and the mean age was 68.8 years. pharmacist intervention resulted in positive outcomes regarding hba1c levels. hba1c level decreased by 0.35% (from 7.02 to 6.67%; p \ 0.05) and fasting blood glucose level by 11 mg/dl (p [ 0.05). similarly, the medication knowledge level [usual range 0-8] increased from 2.67 to 5.44 (p \ 0.001); and the medication adherence score [usual range 0-4] increased from 2.9 to 3.28 (p \ 0.01). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control of type 2 diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of type 2 diabetes at the nursing home setting. please specify your abstract type: research abstract background and objective: haemoglobin variability is related to mortality and morbidity in haemodialysis, renal transplantation and pre-dialysis patients. some demographic, haematological and pharmacological variables may affect hb variability. but there are some controversies about the influences of different erythropoiesis stimulating agents (esa).the objective of this study is to determine the influence of different esa on haemoglobin variability in pre-dialysis patients. setting and method: we conducted a prospective observational study with chronic kidney disease patients recruited from outpatients of nephrology department of a tertiary university hospital (from january 2011 to june 2012). exclusion criteria were: stage i and ii, not treated with esa, haemodialysis, peritoneal dialysis, renal transplantation, thalassemia, and deficit of glucose-6-phosphate dehydrogenase . main outcome measures: patients included were treated with esa in maintenance phase (stable 6 months prior).hb variability was calculated by standard deviation (sd) and residual standard deviation (residual sd) of hb levels. statistical analysis was performed with spss 15.0 (spss inc, chicago). observation period was 18 months and data were recorded from the clinical records. (2) 5.7%, sofosbuvir/daclatasvir/ribavirin (2) 5.7%, sofosbuvir/simeprevir (4) 11.4%, sofosbuvir/ledipasvir (6) 17.1%, sofosbuvir/ledipasvir/ribavirin (3) 8.5%, dasabuvir/ombitasvir/paritaprevir/ritonavir (2) 5.7%, dasabuvir/ombitasvir/pari taprevir/ritonavir/ribavirin (14) 40% ombitasvir/paritaprevir/ritonavir/ ribavirin (1) 2.8%, sofosbuvir/ribavirin (1) 2.8%. viral load at week 4 was \15 iu/ml in 32 patients and at the end of treatment 33. conclusion: the results of rapid viral response at end of treatment were similar to those obtained in studies published to date. due to its recent access to these treatments it is necessary to continue monitoring these patients to assess virologic sustained response at 24 weeks after end of treatment. please specify your abstract type: research abstract background and objective: fragile patients are considered those vulnerable patients with a certain degree of complexity in their care (polypharmacy, multi-pathological, palliative and/or residents in social and healthcare institutions). to ensure their continuity of care and safety in the use of drugs we applied a medication reconciliation process at admission, transition of care and/or hospital discharge. objective: to analyse the results of the medication reconciliation process of a fragile patient. setting and method: we developed a list of current medication with the following sources of information: medical history, clinical databases and information provided by the patient (interview). clinical case: 95-year-old woman admitted through emergency department due to severe dyspnoea. no known drug allergy. background: heart failure, chronic hypertension, hypercholesterolemia, hyperthyroidism, hyperuricemia, gouty arthritis, chronic kidney disease and cognitive impairment by alzheimer disease. exploration and complementary tests: echocardiogram and analytical control. clinical judgment: acute decompensated heart failure. acute myocardial infarction. prerenal acute kidney injury. main outcome measures: medication reconciliation made at admission with the detection of discrepancies and deprescribing criteria at hospital discharge. results: fragile patient (high-risk) with 13 medicines as home treatment. patient was hemodynamically stable during the hospital stay. 9 discrepancies were detected between the prescribed medication and the home treatment. discrepancies justified (7): five by omission of medication (two new clinical situation, two therapeutic exchanges to adapt to the pharmacotherapy guide and one wrong drug) and two beginning of medication. discrepancies unjustified (2): by omission of medication. to discharge: one antiplatelet therapy was. after the comprehensive review, we made the following recommendations of deprescription: suspend one non-steroidal anti-inflammatory drug-nsaid (by risk of bleeding in association with concomitant antiplatelet and antidepressant therapy) and one benzodiazepine (central nervous system-cns side effects); modify treatment: reduce doses of diuretics (blood pressure lowering effect). pharmacotherapeutic recommendations were accepted. conclusion: detection of discrepancies in the medication reconciliation and deprescription process are effective and safe strategies that allow optimization of pharmacotherapy in fragile patients. the use of drugs such as nsaids (gastrolesive effect), the combination of drugs with cns side effects and hypotensive action (associated with falls) in elderly patients constitute situations of risk that should be reviewed in fragile patients, as an essential part of the clinical evaluation. please specify your abstract type: descriptive abstract (for projects) background and objective: there are no positions for clinical pharmacists at the hospital, so we are dependent on projects to be able to show how pharmacists can contribute in the clinical team. our aim in this project was to introduce pharmaceutical knowledge by implementing medication reconciliation and medication review in different hospital wards. we wanted to show that many patients have discrepancies in their medication lists during hospital stay and that some of the drugs or doses given can cause drug related problems for the patient. our final goal was to get the physicians to be more aware of these issues when treating their patients. design: the method used was based on the two first parts of the integrated medicines management. the pharmacist conducted a standardized drug interview with patients who prior to admission were responsible for their own drugs. for patients who could not be interviewed or were not responsible for administering their own drugs, a current medication list from relevant care level was obtained. the medication lists obtained were compared to the documentation in the patient's drug chart and discrepancies communicated to the physician. during the hospital stay, a medication review and monitoring was also conducted by the pharmacist. results were presented to the patients physician and discussed. results: a total of 129 patients were included and of these 63% had c1 discrepancy identified by the process. the most frequent type of discrepancy was the use of a drug that was not registered on admission (omission discrepancies). other discrepancies were wrong dose, dosage or formulation and registration of a drug the patient didn't use. drug-related problems were discovered in 61% of the patients and the most frequent were use of anticholinergic drugs in elderly, interactions, lack of treatment and monitoring and too high doses regarding kidney function. many of the detected drug related problems results in change in medication, other times the physician addresses the problem to the gp. the physicians were surprised of the high numbers of discrepancies in medication lists and drug related problems discovered. almost all the physicians considered that the pharmacist could be an important part of the treatment team and they wanted the participation of the pharmacist to be permanent. conclusion: the project led to increased awareness of the importance of medication reconciliation and medication review and showed the importance of pharmaceutical knowledge in the treatment team. unfortunately this was not sufficient to create positions for pharmacists in our hospital. new projects will focus on pharmacists teaching interns to improve the reconciliation at admission. please specify your abstract type: descriptive abstract (for projects) background and objective: we aimed to assess the quality of fluoroquinolones (fq) prescriptions at the toulouse university hospital emergency department as part of significant increase in consumption. design: retrospective mono-centric study of fq prescriptions written to adult patients managed at the emergency department (february 29th, 2016 -march 6th, 2016 . a pair consisting of a biologist pharmacist and a clinical pharmacist has analysed them using tools provided by the centre de coordination de lutte contre les infections nosocomiales (cclin). various criteria (pertinence of prescription, choice of antibiotic, dosage, duration of treatment, method of administration…) were faced with the guidelines issued by the société de pathologie infectieuse de langue française (spilf). results: about 1229 files were examined, 17 contained fq prescriptions for systemic use. the most frequently prescribed antibiotic was ofloxacin (59%) and the most frequent indications were urinary tract infections (47%). among the 17 prescriptions of fq, the establishment of fq was justified in 71% of cases and the antibiotic chosen was always the most suitable. nonconformities of dosage and/ or treatment time were found in a quarter of cases. overall, 47% did comply with guidelines. the prescriptions, due to the particularity of emergency were still permormed probabilistic. however, a reassessment of them was scheduled for two-third of outpatients. conclusion: this study highlights the conformity of less than half of the prescriptions. this demonstrates that there are still actions to ensure the accuracy of fq prescriptions. and it is in this sense that this audit should be registered under the impetus of the committee on anti-infectives. it will raise awareness among doctors in the proper use of this family of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the number of persons suffering from end-stage renal disease (esrd) is growing worldwide, mainly due to the aging of the population. esrd incidence has been increasing by 3-7% per year for 10 years. it is estimated that worldwide, more than 1.5 million patients with established renal failure are being treated with haemodialysis (hd). water for haemodialysis must meet the physicochemical and bacteriological compliance standards defined by the european pharmacopoeia. as a medicine, this water is placed under the responsibility of hospital pharmacists. addressed to hospital pharmacists, this methodology guide will enable them not only to validate controls of haemodialysis water as well as drug prescriptions for dialysis patients, but also to familiarize themselves with the best currently existing dialysis techniques and medical devices. we have tried to simplify and synthesize existing circulars and guidelines so as to render them more readily understandable for the pharmacist in charge of a haemodialysis service, and thereby help to ensure optimally safe treatment of haemodialysis patients. design: the themes developed in this guide are: • a review of the different existing dialysis techniques, • a review of the different sampling points for controls of hd water, • a review of the physicochemical and bacteriological standards of these controls according to the latest recommendations of the european pharmacopeia, and of appropriate conduct for exceeding established thresholds, • a review of the main international recommendations with regard to clinical signs of chronic kidney disease: anaemia, mineral and bone disorders (ckd-mbd), high blood pressure. • a review of the various medical devices used in haemodialysis and haemodiafiltration. results: the recommendations of good practices summarized in this guide are integrated perfectly adapted to the concept of quality assurance and its role in the accreditation process. they are focused on improving patient safety by harmonizing pharmaceutical haemodialysis practices in different dialysis centres. conclusion: these types of recommendations may be transposable to other pharmaceutical fields and/or be used as a training tool for pharmacy students or young pharmacy school graduates. the format of this guide makes it convenient, easy to use every day. it will be revised regularly to ensure the sustainability of quality plans. please specify your abstract type: descriptive abstract (for projects) background and objective: combination antiretroviral therapy (cart) has strongly improved disease control in hiv-infected patients. however, aging and comorbidities are becoming a major problem in this group of patients. most hiv-infected patients are treated with five or more medications, and harms by polypharmacy increase proportionally with number of medications. possible risks include: poor medication adherence and consequently inefficient care, increased risk of drug interactions and adverse events, with prolonged hospitalization. the problem is worsened when patients are of nonnative language and so their comprehension and adherence to drug therapy can be very poor, compromising efficacy. the hivig study is designed to evaluate the impact of the interventions promoted by the clinical pharmacist in the optimization and comprehension to personal drug therapy, favouring compliance, in a cohort of patients, hiv infected with comorbidities like cancer; the cohort includes a high number of non-native italian language individuals. design: hivig is a randomised, parallel groups clinical trial. in april 2016 the study protocol was approved by the local ethical committee, aviano. the project is scheduled to start in autumn 2016. main objective: evaluation of the impact of a series of tools-''drug therapy setting interventions'' (dtsis) applied by the clinical pharmacist on a cohort pf hiv-infected patients with comorbidities, afferent for care at cro aviano. the treatment arm will be submitted to dtsis. dtsis interventions (treatment group) consist in: motivational interview, sharing and delivery of printed, explanatory material in the patient's native language, reconciliation of patients medications at hospital admission and at discharge; identification of potential risks due to drug-drug interactions; monitoring of compliance to drug therapy, and finally detection of adverse drug reactions (adrs) occurring in the course of care. the control group will undergo only to scheduled standard medical visits at cro. results: we expect to recruit a total 350 patients for a 24-months period of follow-up. statistical analysis will be performed by intention-to-treat and by protocol. at cro aviano, the italian cooperative group on aids and tumors (gicat) has studied malignancies in hiv-positive patients since 1986 and has a leading role for studies conducted in italy (vaccher, 2014) conclusion: previous collected data from the previous trial performed at cro aviano (target-vig), showed a positive impact in the optimization of individual drug therapy and in the reporting of adrs. hiv has an enormous impact on life of infected patients and represents a priority issue for the entire community. we consider the method of dtsi, combined with a close monitoring of patients by means of telephonic motivational interviews, the best added value performed by the profile of the clinical pharmacist in optimizing drug therapy and personal awareness about medicines. please specify your abstract type: research abstract background and objective: the world health organization reports that ''one in four people in the world will be affected by mental or neurological disorders at some point in their lives. around 450 million people currently suffer from such conditions, placing mental disorders among the leading causes of ill-health and disability worldwide». to review the evidences published about the roles and the impact of pharmacists in psychiatry. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, mental illness and psychiatry from january 1st 1990 until june 14th 2016. manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in psychiatry. results: a total of 62 articles were included. described pharmaceutical interventions included patient-pharmacist relationship (7), medication reconciliation (26), patient care needs assessment (25), drug therapy assessment (74), patient follow-up (61), interdisciplinary work (26), knowledge transfer (104), competencies maintenance (2). the impact of pharmacists interventions was studied using a total of 369 indicators from which 146 (40%) had outcome measures. of these 146 outcome indicators, 68 (47%) were positive, 77 neutral and 1 negative (knowledge transfer strategy). positive impacts of pharmaceutical interventions were identified in the following areas: morbidity (24), patient adherence (11), patients or clinicians satisfaction (7), side effects management (2), medication errors prevention (2), mortality (2) please specify your abstract type: research abstract background and objective: the world health organization reports that 8.2 million people die each year from cancer, an estimated 13% of all deaths worldwide and that there is a 70% increase in new cases of cancer expected over the next two decades. to review the evidences published about the roles and the impact of pharmacists in cancer. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, neoplasms from january 1st 1990 until june 20th 2016. manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions as well as descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in cancer. results: a total of 42 articles were included. described pharmaceutical interventions included patient-pharmacist relationship (6), patient care needs assessment (76), drug therapy assessment (84), drug compounding/dispensing (1), patient follow-up (93), interdisciplinary work (15), knowledge transfer (39). the impact of pharmacists interventions was studied using a total of 274 indicators from which 112 (41%) had outcome measures. of these 112 outcomes indicators, 98 (88%) were positive, 13 (12%) neutral and 1 (1%) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity (59), patient adherence (2), patients or clinicians' satisfaction (3), side effects management (8), medication errors prevention (7), mortality (0), costs (2) setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, myocardial infarction, acute coronary syndrome from january 1st 1990 until june 14th 2016. manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in myocardial infarction. results: a total of 35 articles were included. described pharmaceutical interventions included patient-pharmacist relationship (11), medication reconciliation (39), patient care needs assessment (2), drug therapy assessment (102), drug compounding/dispensing (8), patient follow-up (51), interdisciplinary work (60), knowledge transfer (52). the impact of pharmacists interventions was studied using a total of 177 indicators from which 107 (61%) had outcome int j clin pharm (2017) 39:208-341 269 measures. of these 107 outcome indicators, 49 (46%) were positive, 56 (52%) neutral and 2 (2%) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity (8), patient adherence (10), side effects management (1), mortality (5) and others (21). conclusion: the role and the impact of pharmacists have been studied in myocardial infarction and 46% of outcome indicators used in these studies show a positive impact of pharmaceutical interventions. pharmacists should pay attention to these evidences to improve their practice, contribute to prevention or insure treatment of patients with potential or found myocardial infarction. hp-pc055: impact of pharmaceutical care in vaccination: a review of literature please specify your abstract type: research abstract background and objective: the world health organization reports that ''immunization is the process whereby a person is made immune or resistant to an infectious disease, typically by the administration of a vaccine and a proven tool for controlling and eliminating lifethreatening infectious diseases and is estimated to avert between 2 and 3 million deaths each year. it is one of the most cost-effective health investments, with proven strategies that make it accessible to even the most hard-to-reach and vulnerable populations». to review the evidences published about the roles and the impact of pharmacists in vaccination. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, vaccination and immunization from january 1st 1990 until july 5th 2016. manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in vaccination. results: a total of 43 articles were included. described pharmaceutical interventions included patient-pharmacist relationship (37), medication reconciliation (19), patient care needs assessment (37), drug therapy assessment (26), patient follow-up (39), interdisciplinary work (11), knowledge transfer (47), competencies maintenance (1). the impact of pharmacists interventions was studied using a total of 212 indicators from which 81 (38%) had outcome measures. of these 81 outcome indicators, 65 (80%) were positive, 15 (19%) neutral and 1 negative. positive impacts of pharmaceutical interventions were identified in the following areas: cost (3), errors (2), morbidity (21), patient adherence (14), patients or clinicians satisfaction (3) please specify your abstract type: descriptive abstract (for projects) background and objective: evaluating the appropriateness and effectiveness of the patient's medications by analysing prescriptions is pharmacist side work. bedside drug administration and computerised drug administration traceability (cdat) in nursing care plan (ncp) are nurse's one. however, in order to check adherence, efficiency and tolerance of a drug, pharmacist has to ensure that the patient takes the medication appropriately. therefor ncp could be a useful tool. the aim of this study is to evaluate the effectiveness of cdat, and if not, define causes of divergences with real life situation. design: • comparison between unused drugs remained in individual patients' seven daily pill dispensers (considered as not taken) which come back from the evaluated service to the pharmacy, and their cdat status completed by nurses (taken, not taken or no status) • two recorded data, each collecting a three-week period, separated by a period of discussion with nurses: first results presentation, analysis of divergences by taking into account their feedbacks, and actions to raise their awareness about the importance of cdat. • pill dispensers' cdat is correct only if all returned drugs' status in ncp is ''not taken''. results: during the first period (n = 112 pill dispensers), 29.5% of pill dispensers had an incorrect cdat status. on average, 1.4 drug per pill dispenser didn't have an appropriate status in ncp (taken or no status). major causes of divergences were the lack of time and insufficient human resources, the fact that they often are interrupted in the middle of this task, a software which isn't ''user-friendly'' and a deficit of information about the issue. corrective actions were implemented, prior to the second recorded data period, targeting human factor of divergences (oral and written reminders about cdat with didactic memorandum on computers). after awareness actions, results (n = 110) were 23.6 and 1.5 respectively. conclusion: efforts about cdat have been done but not enough to observe a significantly improvement in short terms. ncp's level of reliability is not optimal yet and still dependent on nurses' practices. this study allowed us to strengthen the relationship between clinical service and pharmacy, and opens the way for further works particularly through corrective actions targeting material and organizational causes of divergences. please specify your abstract type: descriptive abstract (for projects) background and objective: in 2015, our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. because broad spectrum antibiotics have to be followed with the attention of resistance prevention, we focused our analysis on these antibiotics in our hospital (carbapenems, piperacillin/tazobactam and amoxicillin/clavulanic acid). design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j01, j02, j04a, p01ab, a07, j05ah, p01b from the atc classification) prescribed at 8 a.m. on the day of the survey, were involved. from those who were treated by carbapenems, pip/taz or amx ac, following data were collected: age, gender, weight, doses, indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. results: the survey was carried out from april to june 2015 in 51 wards. among the 1435 patients included, 369 patients (25.7%) were treated with antimicrobial agents.114 patients (30.9%) were treated with broad spectrum antibiotics: 53 (14.1%) with amx-ac, 47 with pip-tz, 6 with imipenem and 8 with meropenem. the mean age of patients was 60.5 ± 17.7 and the weight was 67.9 ± 13.9 kg. their prescriptions were concentrated in three types of wards: 24 (21.1%) in icu, 47 (41.2%) in medicine, 43 (37.7%) in surgery. moreover, we observe that bsa were used to treat 33 (28.9%) community acquired infections, 54 (47.4%) nosocomial infections, 6 (5.2%) used as medical prophylaxis, or surgical prophylaxis (n = 19, 16.7%). in relation to the type of treatment: 66 were empirical treatment (including 25 prophylaxes) and 48 were targeted treatments (3 bacteraemia, 5 joint and bones infections, 1 cardiovascular system infection, 12 urinary tract infections, 10 lower respiratory tract infections, 10 skin and soft tissues infections and 7 others infections). finally, 23 extended spectrum beta-lactamase (esbl) producing enterobacteriaceae and 1 third generation cephalosporin resistant enterobacteriaceae non-esbl producing were targeted by bsa regimen. conclusion: in this survey, use of bsa is globally compliant to french guidelines and we identified no improper prescription: multidrug resistant bacteria infections, several diseases and empirical treatments with limited duration of regimen. this shows that control of the proper use of antibiotics especially those with a broad spectrum is efficient in our hospital and has to be continued. this has been made possible due to a multidisciplinary approach including physicians, bacteriologists and pharmacists. please specify your abstract type: descriptive abstract (for projects) background and objective: in 2015, our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. from these results, we observed that sulfamethoxazole/trimethoprim (tmp/smx) was largely prescribed in our hospital. we focused then our analysis on these results with the attention of check of its proper use. design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j01, j02, j04a, p01ab, a07, j05ah, p01b from the atc classification) prescribed at 8 a.m. on the day of the survey, were involved. from those who were treated by tmp/smx, following data were collected: age, gender, weight, doses, and indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. please specify your abstract type: research abstract background and objective: in france, benzodiazepine (bzd) is frequently prescribed in elderly people (ep). long-term efficacy is often questioned, and treatment has to be regularly re-examined, especially in ep. in our geriatric day-hospital for assessment of frailty, a multidisciplinary team evaluates the patients and gives them preventative measures against the loss of autonomy. medication evaluation is part of these measures. the aim of our study was to evaluate the impact of a standardized intervention on the optimization of bzd treatment. setting and method: after a short interview and the delivery of an information booklet about bzd, patients were proposed an optimization of their bzd treatment (dosage reduction, occasional medication, switch to a short half-life bzd, or total discontinuation). patients were followed up monthly by a phone-interview over a 6-months period. main outcome measures: the main outcome measure was the prevalence of bzd optimized treatments after a 6 months follow-up. results: 18 patients were included. among them, 50% have been taking a bzd for more than 10 years, and 29% were prescribed a long half-life bzd, which can be qualified as inappropriate in ep. 50% of the subjects were frail and 44% pre-frail according to the fried criteria. at the end of the study, 33% of the patients had their bzd treatments optimized, including 17% of total discontinuation. conclusion: in frail or pre-frail elderly population, a standardized intervention can be useful to improve bzd treatment. an extension to this intervention would be the creation of an organisation tasked with routinely monitoring the patients withdrawal over a 6 month period. hba1c and weight were significantly reduced by 1.36 ± 1.79%, p \ 0.000 and 3.21 ± 3.52 kg, p \ 0.000, respectively; systolic bp (12.91 ± 10.57 mmhg, p \ 0.000), diastolic bp (6.39 ± 7.34 mmhg p \ 0.000) and triglycerides (45.58 ± 115.65 mg/dl, p .011).genital-and urinary tract infections were reported by 6.7% patients. any diabetic ketoacidosis case was reported. conclusion: sglt-2 inhibitors added to other oral antidiabetic drugs or insulin in patients with uncontrolled t2dm significantly improved glycaemic control, reduced weight, blood pressure and triglycerides, and was generally well tolerated. in conclusion, sglt-2 inhibitors, appears to be an important addition to the therapeutic options for the management of type 2 diabetes, particularly when used as add-on therapy. (1). treatment safety takes part of the decision to undergo bariatric surgery. during multidisciplinary team meetings, the clinical pharmacist must rely on guidelines to limit drug-induced iatrogenesis. this review aims at assessing influence of bariatric surgery on the clinical impact and pk of cardiotropic drugs so as to document pharmacists' notifications. setting and method: literature review on medline-1946 to may 2016-with terms: cardiovascular drugs and bariatric surgery or malabsorption syndrome. related articles were reviewed. main outcome measures: pharmacokinetic or pharmacodynamic data and clinical impact of cardiotropic drugs. results: a total of 924 titles, and abstracts when necessary, were screened for eligibility. after reviewing process, 15 studies were included: nine concerning digoxin, five beta-blockers (bb) and one amiodarone. published studies varied in methodology: five case report, seven case control and three cohort studies. studies reported variations of digoxin plasmatic concentrations among 22 patients versus 66, suggesting liquid oral form are preferred. no clinical event was notified. more the bb is liposoluble (propranolol), the higher the toxicity is, such as heart rate and blood pressure decreasing, with potential fatal outcomes. a case of amiodarone-induced hyperthyroidism is described after bariatric procedure showing an increase plasma concentration adjusted to weight. conclusion: while the impact on narrow therapeutic range drugs is documented, others cardiotropic drugs may cause serious patient injury justifying their monitoring. therefore, risk must be identified for all patients undergoing bariatric surgery to setting up closely therapeutic monitoring. further studies are still expected to lead to recommendations about posology and treatment withdrawal to improve patient safety. please specify your abstract type: research abstract background and objective: the issue of non-compliance to prescribed medical treatment has been reported to be a crucial problem in psychiatric outpatients. the aims of this study were to assess the extent of non-compliance in a cohort of psychiatric outpatients in malta and to investigate the applicability of using a 7-day multi-dose pill box in terms of practicality, ease of use and impact on compliance within this patient group. setting and method: the study was conducted at mount carmel hospital, a psychiatric hospital in malta. twenty outpatients were recruited by convenience sampling. the study was divided into two phases. during phase 1, patient compliance was assessed using the medication adherence rating scale (mars) survey and patients were administered part a of a questionnaire entitled 'assessment of the 7-day multi dose pill box'. this questionnaire evaluated the patients' opinion regarding the 7-day multi dose pill box before and after its use. in phase 2, the chosen patients were given a demonstration on how to use the 7-day multi dose pill box and the device was given to them to use at home for one week. after one week, part b of the questionnaire was completed and compliance was re-assessed using mars . main outcome measures: evaluation of adherence before and after use of the compliance aid device. results: of the 20 patients recruited, 9 were male and 11 were female. the mean age was 46 years (range 33-70) and the mean number of daily medications 6 (range 3-16). upon initial scoring using mars, 15 patients were adherent and 5 patients were nonadherent. a higher adherence was observed in patients taking 5 or more medications daily. ten patients accepted to move on to phase 2 of the study and took the device home to use for one week. out of these 10 patients, 4 felt that the way they take their medication improved following use of the device and 7 out of 10 patients would consider buying the device since they found it practical and easy to use. statistical analysis of mars score before and after use of the device showed no significant improvement in compliance (p [ 0.05). there was no significant association between level of adherence and type of psychiatric condition (p [ 0.05). furthermore, results did not indicate increased adherence in patients who have a carer in-charge of their medication administration or in patients using a compliance aid device (p [ 0.05). conclusion: the use of a compliance aid device in psychiatric patients is challenging due to difficulty in establishing patient communication and motivation. the pharmacist is in a position to identify patients who would benefit from the compliance aid device. adrien borowik * , anne fratta, fabien hernandez pharmacy, ap-hp, armand trousseau paediatric hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: enoxaparin, a low molecular weight heparin, is the most prescribed anticoagulation treatment in paediatric indications. however, the marketing authorization mentions that due to the lack of data, the use of enoxaparin is not recommended to children nor anyone weighing less than 40 kg. thus, expert recommendations described specific dosage for the paediatric use: we aimed to compare these with our hospital practices. (3), sofosbuvir/ledipasvir (20), ombitasvir/ paritaprevir/ritonavir (4), ombitasvir/paritaprevir/ritonavir + dasabuvir (12), simeprevir + ifn (1) . twenty-six patients (44%) were treated for 24 weeks (12 week pay-back policy). twenty pharmacists' interventions were carried out with an acceptance rate of 80%. the interventions included treatment adjustments due to drug interactions (4), inappropriate treatment according to genotype (2), duration of treatment (5) and switch to a more cost-effective therapy (9). seven pharmacists' interventions concerning treatment switch were applied (78%) resulting in a cost saving of €102,102.7. all assessable patients (28) have a negative serum hcv rna 12 weeks after the end of treatment (svr = 100%) while 1 patient died during follow-up (due to the disease). conclusion: the hospital pharmacist, as an active member of the multidisciplinary team, has an essential role in guaranteeing optimal care for hcv patients at the best cost. monitoring has also shown to be fundamental to evaluate the real world effectiveness of these drugs approved with surrogate endpoints. hp-pc068: are hospital pharmaceutical staff educated on the criticality of thermosensitive drugs? camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: in the use of thermosensitive drugs, the safety of patient care involves compliance with allowed temperatures. having the right information at time of care is essential. the aim of this study is to assess, within a french university hospital, pharmaceutical staff knowledge on the criticality of thermosensitive drugs and to educate them accordingly, including associated patient risks. setting and method: an assessment of knowledge using a questionnaire was led in january 2016 among pharmaceutical staff in a 1500-bed hospital (11 pharmacists, 14 pharmacy residents, 28 pharmacy technicians). evaluation criteria were: storage temperature of refrigerated drugs and frozen drugs, thermosensitive drug retention period after removal from the refrigerator, highest risk situation for a thermosensitive drug (t [ 8°c or t \ 2°c) and action to be taken during a temperature excursion. main outcome measures: to determine shortcomings in the management of thermosensitive drugs in order to adapt appropriate tools. results: 43 completed questionnaires were collected. collected questionnaires included 12% from pharmacists (n = 5), 23% from pharmacy residents (n = 10) and 65% from pharmacy technicians (n = 28). regulatory variations in storage temperatures of refrigerated and frozen drugs are known in respectively 79 and 32% of cases. 3% of pharmaceutical staff are aware of thermosensitive drug retention periods after removal from the refrigerator and 51% of the highest risk situation for a thermosensitive drug (t \ 2°c). the measures to adopt during a temperature excursion are understood in 84% of cases. conclusion: this study highlights the lack of knowledge on the management and criticality of thermosensitive drugs and the lack of information available to pharmaceutical staff. dissemination of data and questionnaire reponses have been beneficial for the pharmacy department and have reduced inequalities in available information among pharmaceutical staff. subsequent to the study, thermosensitive drug management procedures have been revised. the deployment of this questionnaire is continuing via the university hospital intranet in order to train all health professionals in good patient care. please specify your abstract type: research abstract background and objective: temocillin is a beta-lactam antibiotic exclusively active against gram-negative pathogens. its use can avoid that of broad spectrum antibiotics, such as carbapenems, for the treatment of infections due to extended-spectrum beta-lactamase producing enterobacteriaceae. however, the absence of recommendations by learned societies on temocillin use could lead to misuse and the emergence of resistance. the aim of this study is to identify the role of temocillin in a french university hospital arsenal in order to limit ecological risks. setting and method: a retrospective study was conducted in a 1500-bed university hospital. all adult patients having received at least 2 days of treatment between june 2015 and april 2016 were included. data collected for the study were: age, sex, treatment indication (type of infection, identified pathogen, dosage and treatment duration), previous antibiotics and therapeutic outcomes. main outcome measures: the indicators chosen were: treatment indication, prescribed dose and treatment duration. results: two patients were included. in july 2015, temocillin was used in a 47 year old female as first-line treatment of intraperitoneal haematoma infection due to multiresistant klebsiella pneumoniae. prescribed at a dose of 2 g twice daily by an infectious diseases specialist, treatment was continued at the same dose for up 3 weeks with therapeutic success. in august 2015, temocillin was used in a 59 year old male for the treatment of bacteraemia due to multiresistant enterobacter aerogenes. previously treated by imipenem/cilastatin, temocillin was prescribed as second-line treatment at a dose of 2 g twice daily by an infectious diseases specialist. treatment was continued at the same dose for up 6 weeks with therapeutic success. conclusion: the dissemination of antibiotic resistance among gramnegative enterobacteriaceae continues to be an increasing threat for healthcare worldwide. within this context, temocillin could be an interesting alternative. determining the role of temocillin in a therapeutic arsenal is essential. our hospital considers temocillin as a ''critical antibiotic'' although its use is not exclusively limited to the new drug application. therefore, temocillin prescriptions are monitored permanently by infectious diseases specialists, microbiologists and pharmacists in order to improve the good use of this antibiotic and to optimise patient safety. please specify your abstract type: research abstract background and objective: drinkable solutions are more susceptible to deterioration and can lead to a potential risk for patient care. having the right information at time of care is essential. the aim of this study is to assess nursing staff knowledge in a french university hospital on the management of drinkable solutions to elaborate tools to help health professionals and to enhance equality of information in order to optimise patient care. setting and method: an assessment of practice using a questionnaire was conducted in may 2016 among a share of the nursing staff in a int j clin pharm (2017) results: 133 completed questionnaires were collected. 20% of nursing staff replied that the period-after-opening is the same for all of drinkable solutions. this period is estimated at 1 month in 21% of cases, 2 weeks in 10% of cases and 7 days in 8% of cases. 58% of nursing staff do not know how to store drinkable solutions after opening. the date of opening or the date of expiry after opening are specified on the medicine bottle in respectively 77 and 3% of cases. only 11% of nursing staff have tools pertaining to the management of drinkable solutions. these observations led the pharmacy to create and distribute appropriate tools. storage methods for the drinkable solutions available in our hospital were collected directly from pharmaceutical laboratories. this information has been made available to nursing staff via drug control software (pharma ò , computer engineering, paris). conclusion: this study highlights the lack of knowledge on the management of drinkable solutions and the lack of information available to nursing staff. in our hospital, the dissemination of appropriate data reduced inequalities in available information between care units. data will soon be integrated within the drug prescription software (mc kesson usv2 ò , crossway, san francisco) in order to homogeneously train all health professionals in good patient care. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a process which allows prevention of iatrogenic injuries during patient's hospitalisation and transfers. since 2013, a clinical pharmacist has been integrated into the orthopaedic surgery care. he has performed mr at patients' admission. the aim of this study was to evaluate the impact of medication reconciliation performed by a clinical pharmacist. design: a prospective monocentric study was conducted on patients admitted in an orthopaedic surgery care (elective or unplanned surgery), during 3 months. the clinical pharmacist established the best possible medication history (bpmh) from at least three sources of information (including patient interview when possible). then, it was compared to the admission medication order (amo) (from anaesthetists when elective or orthopaedists when unplanned). unintended medication discrepancies (umd) detected were discussed with prescribers in order to be corrected. epidemiological data, number and type of umd, therapeutic classes involved and the percentage of corrected umd were collected and their potential clinical impact was assessed. results: in this study, 325 patients were included during 3 months. elective surgeries were concerned in 72% of the cases. at least one umd was identified in 158 patients (49%) (median age: 77.1 years old; male/female ratio: 0.65). of these, 133 (84%) were older than 65 years old. finally, 406 umd were detected, being 2.6 by patient. main therapeutic classes concerned cardiovascular system (31%), nervous system (20%) and digestive system (18%). of the 406 umd detected by mr, there were 55% of omissions, 27% of inappropriate dosing and 13% of renewal prescriptions stopped by the patient. finally, 87% of umd were corrected. of these 406 umd, 2% were major errors (i.e. causing potential harm), 39% were significant errors (i.e. monitoring or intervention potentially required to preclude harm) and 59% were minor errors (i.e. without potential harm to the patient). conclusion: medication reconciliation process performed by a clinical pharmacist allows detection and correction of umd on half of patients in surgery care particularly on elderly patients. the high proportion of umd can be explained by the multiplicity of actors involved in medication management. health information technology could help to focus mr on patients at high-risk of adverse drug events. please specify your abstract type: research abstract background and objective: darunavir plus ritonavir (drv/r) have shown optimized results in simplification strategies (monotherapy (mt) or dual therapy (dt)) for selected hiv + in randomized clinical trials and real life experience. recent introduction of one pill drv plus cobicistat co-formulation (drv/c) may be particularly suited for both mt/dt allowing once daily administration optimizing dosage and adherence. the objective of our study is to evaluate efficacy and security of drv/c in mt and dt. setting and method: all hiv + adults with antiretroviral change to drv/c in mt/dt at a reference hospital in the northwest of spain were included in this retrospective study. a statistical analysis was performed using the spss v.19.software. main outcome measures: epidemiological, clinical, antiretroviral regimen, serum creatinine, lipids and inmunovirological data (rna-hiv and lymphocytes cd4) were compared previous and after change to drv/c. results: 71 hiv treatment-experienced patients have received drv/c in dt (27) or mt (44). 76.1% were men with a mean age of 48 years. main risk factors were: 45.1% heterosexual, 28.2% msm, 18.3% injection drug users, 2.8% mother-to-child transmission, 1.4% transfusion in haemophiliac patient and 4.2% unknown. cdc category distribution was 64.8% a, 4.2% b, 28.2% c and 2.8% unknown. overall mean nadir cd4 counts were 196.7 ± 115.1cells/mcl. mean time since drv/c prescription to discontinuation or until analysis was 182.5 days [range 61-296]. 86.4% drv/c mt were prescribed to patients with prior drv/r mt in order to simplify treatment and the mean time of the duration of these prior therapies were 3.1 years. in case of dt, 66.7% were prescribed on patients with prior drv/ r + 3tc with a mean duration of 1.3 years. serum creatinine increases (1.03 vs. 1.08; p \ 0.001) and cd4 decrease (714.1 vs. 663.3; p = 0.031) when patients move to drv/c. no significant change in the other analytical parameters and all patients maintained undetectable. 2 patients discontinue drv/c due to intolerance and inability to swallow in each case. conclusion: this preliminary study concludes that drv/c in mt or dt is efficacy (no viral rebound) and safety. although an increase in creatinine was observed, it would not be considered clinically significant. of note, lymphocytes decreased significantly and it will be important closely monitored to check that maintain effectiveness during the follow up. hp-pc073: developing clinical pharmacy in emergency department setting up a medication reconciliation process marion collignon *,1 , antoine gantier 1 , florent lapacherie 1 , hélène dewaele 1 , laura foucault 1 , anne-laure raso 1 , emmanuel cirot 1 , said laribi 2 , xavier pourrat 1 1 pharmacy, 2 emergency department, chru tours, tours, france please specify your abstract type: descriptive abstract (for projects) background and objective: in emergency department (ed), if a drug related problem (drp) happens at the patient admission, the risk is the error remains until discharge. one part of drp may be avoided with using medication reconciliation (mr). the objective of this study was to evaluate the feasibility of setting up a medication history (mh) of patients in ed in an acceptable lap of time before they were transferred in another unit or discharged. design: a 6 months prospective study was conducted in ed in a university hospital in france. two junior pharmacists coached by a senior pharmacist, after a 2 months training for mr, were in charge of the data and mh collection. for all patients, we collected age, mh according to number of sources, discrepancies identified, adherence to treatment (according to the social security questionnaire), type of sources. mh were established according to community pharmacies, patients, previous electronic patient files, prescription sheets, patient's family, packs of pills and to the general practitioner (gp). for patients from long term care facilities (ltcf), the mh was established only by communication with the ltcf. then, the current prescription was compared with the home medication regimen. mh and discrepancies (omitting medication, incorrect dose, ambiguous name) were recorded in the electronic patient files to be available during hospitalization. because ed does not have a pharmaceutical review of prescriptions, only major discrepancies were transmitted to physicians. results: we collected 1426 mh (187 from ltcf), with a sex ratio of 1.0 and a medium age of 74.5 years old. it represented an average of 9.5 mh per day or 19 min per mh. among patients who did not come from ltcf, sources used by pharmacy students were patient's community pharmacy (967, 78% of cases), patient (890, 72%), previous electronic patient file (769, 62%), prescription sheets (634, 51%), call to gp (80, 6.5%), gp mail (78, 6.3%), patient's family (47, 3.8%), packs of pills (29, 2.3%), community nurse (26, 2.1%). finally, 683 patients (48%) had been hospitalized, others were discharged. we analysed mh for 1099 patients: at least one drp occurred for 599 patients (55%). among 386 patients, 150 (39%) had an immediate pharmaceutical intervention because of the risk due to discrepancy. among 203 patients who did not come from ltcf and who could communicate, 162 were good adherent to treatment (80%). conclusion: this study highlights the great interest of the mh by pharmacists at ed, which avoids many drp. the presence of pharmacists in ed contributes to maintain a safe environment for medication and to assist prescribers in the continuity of treatment between home and hospital. spending 20 min by mh, we identify one drp every 11 min. nevertheless, it could be benefit to develop this activity because of the satisfaction of the emergency physicians. currently, mr is the first step to develop clinical pharmacy in the ed. please specify your abstract type: research abstract background and objective: emerging evidence in the literature suggests a high prevalence of suboptimal vitamin d (vitd) and an association between lower serum levels and higher mortality in cancer. the objective of this study was to quantify vitd deficiency in patients after surgery for head and neck cancer, and to determine the effect of one cholecalciferol intramuscular dose. setting and method: intervention study with a follow-up period of 5 months (november 2015-february 2016) performed on patients followed by the nutrition support unit after surgery for head and neck cancer. demographic and physiopatological data, including admission diagnosis, age, gender, calcium, magnesium and phosphate were collected. nutrition screening by conut index was carried out. a single intramuscular dose of 200.000 ui cholecalciferol (vitamine d3 bon ò ) was administered to vitd-deficient patients and serum 25-hidroxy-vitamin d (s25ohd) records after the administration, including primary carés records after discharge, were evaluated (reference range 30-70 ng/ml). main outcome measures: s25ohd (\20 ng/ml: deficiency; 20-31 ng/ml: insufficiency; c32 ng/ml: sufficiency). results: data from 25 patients with a mean (sd) age of 63.8 (14.8) years were collected (males: 92%). the admission diagnosis was laryngeal squamosis cell carcinoma (n = 14), glottis carcinoma (n = 6) and nasopharynx, tongue and skull base cancer (n = 5). at baseline, 1, 17 and 7 patients were considered have high, medium and low risk of malnutrition, respectively. the mean (sd) serum 25ohd was 8.46 (5.68) ng/ml (deficiency: 24 patients; insufficiency: 1 patient). despite the role of vitd in mineral balance, calcium, magnesium and phosphate mean (sd) serum levels were between the normal range 9.15 (0.36) mg/dl, 2.08 (0.22) mg/dl, and 2.49 (0.89) mg/dl, respectively. 17 s25ohd records were available 1 week after the administration (mean (sd) = 21.46 (13.79) ng/ml). 7 and 4 patients still showed deficiency and insufficiency, respectively. primary care's records from 3 patients were available after discharge (30.2, 36.5 and 52.5 ng/ml). conclusion: poor nutritional status and high prevalence of suboptimal vitd in patients with head and neck cancer were found. a single dose of intramuscular cholecalciferol slowly raises s25ohd. follow-up after discharge is essential to evaluate the achievement of the therapeutic objective. setting and method: this is a descriptive retrospective study. it took place in a teaching hospital. antifungal broad spectrum therapies (liposomal amphotericin b, caspofungin, micafungin, posaconazole, voriconazole) used between 1st january 2013 and 31st december 2015 were included. main outcome measures: indications, type of combination and patients specifications were analysed. results: only 19 patients (1.9% over all patients receiving antifungal therapy; n = 19/977) received an antifungal combination therapy during the study period. majority of patients presented risk factors: 31% of patients had an organ transplant (n = 6), 53% suffered from malignant blood disorders (four acute myeloid leukaemia, two chronic lymphoid leukemia, one non-hodgkin's lymphoma, one hodgkin's lymphoma and two refractory anaemia with excessive blast), 11% suffered from solid cancer (one lung cancer and one breast cancer) and 5% suffered from chronic obstructive bronchopneumopathy (n = 1). antifungal combination therapy was used against invasive aspergillosis in 68% of cases (n = 13) among which complications such as brain and cardiac impairment were found in 32% of patients (n = 6). the six remaining patients (32%) were co-infected with candidiasis for three patients and mucomycosis for three patients. voriconazole was logically the most used in combination, and just one patient received oral form. it was in majority prescribed with caspofungin (38%, n = 8) and intravenous liposomal amphotericin b (33%; n = 7). combination including liposomal amphotericin b and caspofungin (n = 3, 14%) or posaconazole with liposomal amphotericin b (n = 1) were found in our study. five patients deceased during the hospitalization of the fungal infection (26%) which shows the gravity of these cases. majority of patients ([50%) was treated less than 10 days with these combinations. conclusion: this retrospective study shows that patients who received antifungal combination therapy were mostly immunocompromised, co-infected or experienced a severe infection with severity factors. the antifungal combination was in majority initiated because monotherapy failed to cure the patient. all prescriptions were discussed with a mycologist who tried to shorter the combination treatment duration. this multidisciplinary approach is a major key in the process of these type of treatments. please specify your abstract type: research abstract background and objective: because of its broad spectrum and the risk of resistance mutation, delivery of posaconazole is nominative and controlled by hospital pharmacists. the aim of this work was to describe the use and pharmaceutical follow-up of posaconazole tablets over a 7-months period. setting and method: this is a descriptive retrospective study over a 7-months period from november 2015 to may 2016 in a teaching hospital. all patients who received posaconazole tablets were included. main outcome measures: indications and dosage were reported. results: 23 patients were included in the study. posaconazole tablets were used for: fungal invasive infection prophylaxis in case of stem cell transplantation (52%; n = 12), fungal invasive infection prophylaxis if a chemotherapy was started to treat a chronic myeloid leukaemia or a myelodysplasic syndrome (26%; n = 6); treatment of invasive aspergillosis (13%; n = 3); mycetoma (5%; n = 1); zygomycosis or mucormycosis while patient had renal impairment (5%; n = 1). all of these indications were approved for posaconazole (marketing authorization and local guidelines). only 10 patients (43%) received a loading dose (300 milligrams twice a day) as recommended in approval authorization. posaconazole blood levels were monitored by pharmacologists: 70% of patients (n = 16) did not need dosage modulation which shows that variability is not so important. but three patients did not have any assay to monitor posaconazole blood concentration. 1 patient received a loading dose and was switched to intravenous voriconazole after icu transfer. 3 patients needed increase and/or reduction dose to obtain optimal posaconazole blood levels. conclusion: this study describes the use and the follow-up of posaconazole tablets during the first months after its approval in europe. all indications are approved for posaconazole but this analysis shows that pharmacist have to remind the necessity of a loading dose. dosage can be adjusted according to assays results. please specify your abstract type: research abstract background and objective: due to the acute, hectic environment in a fast-paced work-flow emergency department (ed) it is a challenge to verify the correct and updated medication list for the admitted patients. when performing medication reconciliation (mr) in this environment, these challenge has to be taken into account and prioritizing patients for mr could be necessary. the objective of this study was to identify risk factors correlated to clinical relevant medication discrepancies (crmds) among patients admitted to ed, and based on these revealed risk factors, develop a model for prioritizing patients for mr in the fast-paced work-flow at the ed. setting and method: 276 patients continuously included at the ed, diakonhjemmet hospital (dh), oslo, norway. trained pharmacists and emergency nurse conducted mr. patient specific factors and revealed crmds, between hospital admission records and information about prehospital medication use, were recorded. binary linear regression was used to identify risk factors correlated to crmds. the prioritizing model was built using statics and clinical experiences. main outcome measures: what risk factors is correlated to crmds and how precisely do the prioritizing model classify the patients as high-and low-risk patients. results: 62% of the patients had c1 crmd. the following were identified as risk factors correlated to crmd and were suitable for inclusion in the prioritizing model; gender (woman), age (c60), c1 admission to hospital last 12 months, admission causes; surgical, malfunction, cancer. the model correctly classified 76.1% of the patients with crmds as high risk. further, 23.9% of the patients with crmds were classified by the model as low-risk patients (false negatives). the model classified 27.1% of the patients who did not have a crmd as high-risk patients (false positives). conclusion: the prioritizing model developed can be helpful in identifying what patients are at increased risk of having crmds in the fast-paced work-flow at the ed. identifying these patients will result in using the resources available in the ed in the most efficient manner and utilizing the full potential of the mr method. as a consequence of this, patient safety would be increased. hp-pc078: intravenous potassium chloride: quick audit of prescribers knowledge and recommendations regarding safe practice and proper usage asmaa damou * , vincent zaugg, martine postaire please specify your abstract type: descriptive abstract (for projects) background and objective: our hospital has established methods that try to ensure the safe use of high alert medications. intravenous potassium chloride (kcl) was the subject of preventive measures: separation of different dosages (kcl 7.46% vials reserved for paediatric services and kcl 10% vials reserved for adult services); creation of an advice record for doctors and nurses; specific labelling of storage areas; double-check the prescription and administration. the objective of this study was to evaluate the knowledge of the safe use of intravenous kcl by prescribers. design: multiple-choice questions were developed for prescribing recommendations established by our hospital with the collaboration of the doctor who is chairman of the central committee of vigilance and risk associated with care (cvris). a link to the online survey was sent by email to 85 physicians practicing in 14 departments (eight paediatric services and six adult services). the results were extracted and interpreted in excel ò . results: 57% of physicians responded to the survey (17 medicine residents, 32 hospital doctors). in paediatric services, 93% of doctors know that only the kcl 7.46% should be used. 87% know the unit of prescription to be used (mmol/kg or meq/kg), and 90% know that the maximum recommended infusion rate is 0.5 mmol/kg/hour (or 1 mmol/kg/h in recovery unit). in adult services, the recommended maximum rate of infusion (1 g/h) is known to all prescribers, but only 56% know that the concentration of kcl must be less than 4 g/l. 74% of paediatric doctors say that their kcl prescriptions are checked by a second doctor, but the answers in the same service area are sometimes contradictory. in adult services, only 6% of physicians say that the prescriptions are double-checked. the information brochure available on the intranet of the hospital is known by 16% of prescribers. the response rate of physicians to the survey was satisfactory. therefore, the recommendations are rather well known by prescribers, except the value of the maximum concentration of infusion for adults. the results of this audit were returned to the doctors, accompanied by a reminder stating the need to double-check the prescription and the existence of advice records on the website of the hospital. conclusion: this audit is an approach to increase the safety of the use of high alert medications. it will be completed a second time, by an evaluation of prescriptions collected and the storage conditions of potassium chloride in the care units. please specify your abstract type: research abstract background and objective: data listed behind each unit dose of a primary packaging of a pharmaceutical product are essential for a safe identification for the patient. however, the last medical services of the lausanne university hospital where nurses remove the solid form drugs (sfd) from their blisters when they prepare in advance the week container were in the vaud's prisons. the aims of the study were: (194), quetiapine (173) and ibuprofen (124) and 978 were psychotropics (39.8%). part 2. the four data identified as essential: brand name, dosage [mg], batch number, expiration date. the sfd unit doses were classified as green when the blister included four data, yellow with two or three and red with less than two. of the 273 sfd in cupboards, 90 were green (33%), 57 yellow (21%) and 126 red (46%); an infovigilance was sent to each manufacturers. part 3. potential barriers identified: trays' sizes and space in drug's cupboards; preparation time to cut versus to remove the blisters; risks of self/hetero-aggression with pre-cut blisters; drugs packaged in bulk; multidose liquid medications. using containers larger than is usual was rarely necessary; space in cupboards was sufficient. the preparation time gradually decreased during the study. ingestion or aggression with pre-cut blisters was considered as limited, based on literature and experiences of two others prisons (geneva; lyon). for bulk sfd and multidose liquid drugs: proposals to the pharmacy to store some alternatives blistered sfd; blistering expensive bulked drugs; availability of the entire package delivered to inmates. the pilot phase was initiated in may 2015. conclusion: a majority of inmates takes a drug treatment. half of sfd unit dose is identifiable (trade name and dosage) but an effort from manufacturers would better secure the drug supply chain. the study of the barriers helped to further implement the pilot phase. since early 2016, none of the five prisons medical wards are removing the blisters and no incident was reported. please specify your abstract type: research abstract background and objective: nefopam is a widely used antalgic in hospital. its use is contraindicated in the epileptic patient as it results in lowering the epileptogen threshold and is likely to trigger epileptic seizures. the clinical pharmacist should systematically warn the prescriber against this contraindication when analysing prescriptions. following the onset in our establishment of an epileptic condition in a patient treated with nefopam, who had not been subject to any pharmaceutical intervention (pi), we set about analysing the validation practices regarding this contraindication and possibly implementing actions designed to improve those practices. setting and method: retrospective collection over a period of 17 months of prescriptions for patients hospitalized in 210 hospital beds with clinical pharmacy service (associating med-reconciliation, checking prescription according to medical file and participation to medical rounds): orthopaedic surgery, hepatic-gastro-enterology, general surgery, liver transplant and chest surgery. records of patients with nefopam prescription associated to medication belonging to the therapeutic class of antiepileptics were consulted with a view to finding cases of epilepsy. the pharmaceutical alerts were extracted from the pharmaceutical software. main outcome measures: number of epileptic patients treated with nefopam, number of pharmaceutical interventions issued when prescribing nefopam in epileptic patients. the study focused on 11,252 patients. 3980 (35.4%) of them were prescribed nefopam, and 143 (1.3%) of them were prescribed nefopam associated to medication belonging to the therapeutic class of antiepileptics. after analysis of the patients' records has shown that 55 of them were really epileptic. only 29 pi's were effected (52.3% of problematic prescriptions), and 25 (86%) of them had an immediate prescription change. 77.8% (14/18) of the patients have a pi in medicine services compared to 40.5 (15/37) in surgery services (p \ 0.01). the results of this study show that 47% of the contraindications related to the use of nefopam in epileptic patients are not reported to the prescriber. these results will be presented to our pharmacists so they can take them into account. subsequently a new study will be conducted to measure the relevance and efficiency of this program. hélène dewaele * , anne-laure raso, emmanuel cirot, marion collignon, laura foucault, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) has been demonstrated to reduce drug-related problems in inpatients. in our university hospital, mr has been performed for 200 beds for 10 years at the same time as prescriptions review. the aim of this study was to assess the impact of mr on pharmaceutical interventions (pi) during prescriptions review. design: a 6-month prospective study in orthopaedic surgery, hepato-gastro-enterology, general surgery, liver transplant and chest surgery was conducted. during medication review all pis were collected and those related to mr (rpi) were identified. thereafter for each patient we collected age, type of hospitalization unit (med or surgery) and for pis the drug associated and its acceptance by the medical team. results: during the study 4756 patients had a daily prescription review. 1576 patients (33%) had at least one drug-related problem. 928 lines of prescriptions were mentioned to have at least one rpi. rpi represent 33% of drug-related problems. 697 (75%) discrepancies were corrected by prescribers. the age of the patient was significantly different between patients with rpi (mean age: 70 years old) and with pi (mean age: 65 years old; p \ 0.05). the type of unit did impact the percentage of prescriptions with drug-related problems (medicine: 46.8; surgery: 66.8; p \ 0.01), the rate of corrected pi (medicine:75%, surgery: 61%, p \ 0.01), but did not impact the rate of corrected rpi (p = 0.49). in surgery units the rate of corrected rpi (967/1571) is significantly higher than corrected pi (601/804; p \ 0.001). medicines belonging to the four classes of: digestive and metabolism system, blood and blood flow, cardiovascular system, neurological system represent more than 75% of all the medication concerned by a resolved pi or rpi. the proportion of medicines from the digestive and metabolism class is the only class among those four that is not significantly different between resolved pi and rpi. conclusion: mr highlights a large number of discrepancies in inpatients. a modification of prescriptions due to mr occurs in 10% of the patients. in surgery units, these rpi are more frequently taken into account than drug-related problem warned by pis. indentifying patients for whom mr has the bigger impact could help us to reinforce our actions. please specify your abstract type: research abstract background and objective: diabetes is very frequently causing cardiovascular complications, thus impairing various systems and organs. therapy for these multiple conditions has to be revised and improved constantly. the aim of this closed retrospective study lead in bucharest emergency clinical hospital was the assessment of some of the diabetes mellitus (dm) complications and the related medication. setting and method: data was collected from cardiology, neurology, gastroenterology, internal medicine wards from bucharest emergency clinical hospital. only patients diagnosed with type 2 dm were included in the study. there were analysed 105 records from patients aged 36-89 of whom 65 were men, following the presence, signalling and monitoring of diabetic nephropathy and arteriopathy. main outcome measures: we investigated the relationship between diagnosis and/or biochemical signs of kidney disease (serum urea, serum creatinine levels), diagnosis of arteriopathy, and the drug therapy administered in the respective cases. we also assessed the sex and age distribution of the patients diagnosed with diabetes mellitus and facing at least one of its complications. results: kidney disease, as a dm complication, was present in 30% of cases, patients aged 55-89, of whom 57% were men. 53 patients received diuretic treatment, 5 of them being given hydrochlorothiazide, contraindicated in dm because of its hyperglycaemia-inducing effect. of the 105 patients, 36 had high serum urea levels ([50 mg/dl), 39 had high levels of serum creatinine ([1.2 mg/dl), and 26 presented risen levels for both, but only 16 were also diagnosed with kidney disease. 9 patients with kidney disease were given furosemide, known for altering the renal function. circulatory failure was found in 10% of the patients, aged 61-80 and 6% of subjects, aged 62-79, had both diabetic complications. conclusion: the present study emphasizes the role of the clinical pharmacist in adapting the medication of the diabetic patient, an inappropriate pharmacotherapy worsening dm complications. this is essential especially for elders, where polypathology and polymedication lead to a significant increase of dm complications risk. hp-pc084: epileptic seizure after treatment with thiocolchicoside: discussion about a case report valérie dobremez *,1 , adeline martin-dupray 2 , jacqueline berlioz 1 , pierric giraud 2 1 pharmacy, 2 neurology, centre hospitalier annecy-genevois, metz-tessy, france please specify your abstract type: descriptive abstract (for projects) background and objective: thiocolchicoside is a semisynthetic derivate of naturally occurring colchicoside, which is largely used in humans as a centrally acting muscle relaxant. this compound also has anti-inflammatory and analgesic effects. the objective of this work is to report a recent case of serious adverse effect of thiocolchicoside occurring in context post traumatic brain damage without sequalae. design: a 28-year-old woman suffered from headaches and neck pain since 5 days, she was treated with thiocolchicoside. she took 8 mg in the evening and 8 mg the next morning. five generalized tonic-clonic seizures, without recovery of normal consciousness between seizures, have occurred suddenly 15-30 min after the second administration. the patient was admitted to intensive care unit in order to control the epileptic seizures. a status epilepticus was diagnosed requiring intravenous drugs with clonazepam, phenobarbital and propofol. the patient was controlled and transferred in neurologic unit in order to complete paraclinical investigations. its main antecedent was a severe head injury at the age of 7 years following a public road accident. the brain scan revealed an old frontal hypodensity. rest of etiological assessment was negative (lumbar puncture, no infectious disease), numbers were normal. the definitive diagnosis was a status epilepticus on post-traumatic sequelae, sensitized by taking a proconvulsant drug. a treatment with levitiracetam was initiated at 750 mg twice a day. outcome was favourable with no recurrence 10 months later, a recommendation was requested to pharmacovigilance. results: the muscle relaxant activity of thiocolchicoside results of an agonist action on glycinergic receptors located primarily in the brain stem and spinal cord. however, thiocolchicoside also acts as an antagonist of the gaba-a receptor (mainly located in the cerebral cortex), this pharmacological action can cause a proconvulsant effect. epilepsy is a very rare adverse effect, only few cases have been reported in literature. the epileptogenic activity of thiocolchicoside occur mainly in patients with a history of epilepsy, acute brain injury or possible blood-brain barrier disruption. the chronology is consistent with the responsibility of the drug as a promoting factor. pharmacovigilance retains after analysing drug causality. conclusion: the case history indicates that thiocolchicoside has a powerful epileptogenic activity. thiocolchicoside can precipitate seizures in predisposed patients, and that its use should be avoided in patients with brain diseases (and therefore lower seizure thresholds) or blood-brain barrier disruption. pharmacists could warn physicians and should verify the absence of notable history before dispensing thiocolchicoside. hp-pc085: acute exacerbation generalized myasthenia after red yeast rice use: a case report valérie dobremez *,1 , amélie serra 2 , déborah grosset-janin 2 , jacqueline berlioz 1 , aymeric dopter 3 , jean-henri ruel 2 1 pharmacy, 2 neurology, centre hospitalier annecy-genevois, metz-tessy, 3 nutrivigilance, french agency for food, environmental and occupational health and safety, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: many drugs can induce acute exacerbations or reveal myasthenia gravis. self-medication or complementary and alternatives medicines expose patients. the objective of this work is to report a recent case of acute exacerbation of myasthenia gravis because of a dietary supplement use. design: intermittent vertical diplopia and ptosis of the left eye settled in a 69-year-old man. its main antecedent is hypertension treated with perindopril. the neurovascular origin was ruled out. the electromyogram (emg) found a significant decrement (11%) of a postsynaptic block in the tongue and right orbicularis muscle. acetylcholine receptor-antibodies were positive. myasthenia gravis was diagnosed (osserman score 90/100) and the patient was treated with pyridostigmine. the identification of carotid atheroma required a treatment with a statin that the patient refused. he preferred a cholesterol lowering dietary supplement, containing red yeast rice. six days later, he was hospitalized for an acute decompensation of myasthenia with bilateral ptosis, oculomotor paresis, drooping head, int j clin pharm (2017) 39:208-341 281 chewing trouble and dysphagia (osserman score 42/100). the patient is treated with high-dose intravenous immunoglobulins then corticosteroids. the dietary supplement is stopped. an opinion was requested to the clinical pharmacist of neurology. the osserman score gradually increases to 78/100. results: red yeast rice contains a range of compounds known as monacolins, of which monacolin k-renamed lovastatin, which was found to be an inhibitor of cholesterol synthesis and the progenitor of the statin family. a literature review has highlighted the responsibility of statins in acute exacerbations or reveal myasthenia gravis occurrences. in this case, the chronology is consistent with the responsibility of red yeast rice. the case was reported to the french system of nutrivigilance, which retained after analysing a probable intrinsic imputability score. conclusion: dietary supplement with red rice yeast are not recommended in case of myasthenia gravis. this is the first case of acute decompensation of myasthenia recorded with red yeast rice in the french system of nutrivigilance. multidisciplinary collaboration (neurologists, clinical pharmacist) has optimized the patient management. fanny durand * , camille lambert, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: development of computerized prescription highlights the need to harmonize pharmaceutical analysis practices. the aim of this study is to analyse the antibiotics prescriptions in the treatment of urinary tract infections, to develop a pharmaceutical validation tool. design: a prospective observational study was conducted for one week, in 20 care units. pharmacists, interns, and pharmacy students were trained on spilf (french society of infectious pathology) recommendations, on pharmacist's role in the management of urinary tract infections, and on the data collection. all patients with antibiotic prescription for urinary tract infection were included. some data were collected: reason for hospitalization, clinical signs, results of susceptibility testing, risk factors for complications (organic or functional abnormality of urinary tract, male, pregnancy, elderly, severe immunodeficiency, severe renal impairment) and signs of severity (severe sepsis, septic shock, interventional surgical drainage). then, the treatments prescribed to the patient, probabilistic on the one hand and documented on the other hand, were compared to spilf recommendations. finally, during a multidisciplinary meeting (pharmacist, expert in infectious diseases), we selected the relevant pharmacist interventions. results: twenty-three patients were included (14 women, 9 men), 42% had a urinary catheter. 52.7% of prescriptions were concordant with spilf recommendations: probabilistic and documented treatment, and duration. among the non-conforming prescriptions, nine pharmacist interventions have been formulated: four prescriptions did not specify the duration of treatment, one antibiotic was prescribed on an insufficient period, two cases of severe acute pyelonephritis without prescription of aminoglycoside, one prescription was not reassessed according to results of susceptibility testing, one pregnant woman with urinary colonization without clinical signs, was treated before obtaining results of susceptibility testing. three cases of poor management are identified: two cases which treatment began only after results of susceptibility testing (a urinary tract infection linked to care, an acute pyelonephritis with complication risk), and a cystitis treated with nitrofurantoin while the germ was resistant. conclusion: a synthetic tool was created. there are three elements for helping pharmaceutical analysis: the questions to ask oneself facing a prescription of antibiotic for urinary tract infection, a flowchart to identify the recommendation adapted to the case, and finally a summary table showing spilf recommendations. this tool will be distributed and evaluated. hp-pc088: off-label use of rituximab in refractory antisynthetase syndrome (as) through a long-time experience in a neuromuscular diesases center lise durand * , carole metz, patrick tilleul, helga junot pharmacy, gh pitié salpêtrière, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: as is an idiopathic autoimmune inflammatory myopathy, characterized by presence of antisynthetase antibodies: anti-jo1, anti-pl7, anti-pl12. patients are usually first treated by corticosteroids (cs) or immunomodulating drugs. rituximab (rtx) has become another option for refractory as, supported by few uncontrolled studies 1 . because of its off-label use, our hospital pharmacy has implemented a controlled drug delivery. this work assesses a 2-years follow-up of patients treated by rtx and the resulted drug costs. design: patients registered in our database since december 2014 who received c1 injections of rtx to treat as, were analysed to describe their eligibility criteria, conditions of management and the clinical and biological effects of the treatment (creatine kinase (cpk) used as biomarker). patient files were consulted to collect all individual data and pharmaceutical software was used to review deliveries. drug costs were also reckoned based on prices from french health insurance. results: for 18 months, 14 patients (median age (min-max): 52 (20-76), 64% women) have been treated with rtx for refractory as, the majority with anti-jo1 antibodies (8). all patients suffer from muscular and lung affections, particularly interstitial pneumonia. many are also living with arthropathies (10) or cutaneous disorders (9). cardiac involvement is seldom (4 symptomatic patients). the mean age of diagnostic is 7.3 years and the mean treatment period is 2.6 years. the common treatment is 1 g at day1 (d1) and d15, then 1 g all 6 months. before rtx treatment, seven patients received c5 other drugs such as cs (93%), azathioprine (71%), methotrexate or mycophenolate mofetil (57%). prednisone and azathioprine are also prescribed with rtx respectively for 79 and 23%. treatment is associated with cures of intravenous immunoglobulins for four patients. to date, median number of administrations per patient is 4 (1-8), d1 and d15 included. all patients have presented positive effects on both clinical and biological markers, mainly during the first 6 months after treatment induction. wilcoxon tests show a significant difference in cpk level between d1 and m6, also between d1 and the last known result. today, three complete remissions are specified in patient file; only one hepatitis b virus reactivation is reported. since 2014, budget impact due to drug cost amounts to 119 000€. conclusion: whereas the use of rtx is controverted for treatment of all types of myopathy, as could have one of the best response 1 . our cohort shows real clinical results and positive effect on usual biomarker. our experience demonstrates the safe and successful use of repeated administrations in refractory as. however, there is a need for further controlled studies to assess the efficacy/safety of rtx and to define its place in the strategy in view of its cost-effectiveness ratio. the pharmaceutical controlled drug delivery has to be continued to supervise, support and document its proper off-label use. please specify your abstract type: descriptive abstract (for projects) background and objective: as a part of the national patient safety program, the northern norway regional health authority are implementing new procedures for medication reconciliation (mr) in hospitals in the region. the procedure defines that mr is the doctor's responsibility and describes how it should be performed. the aim of this study was to investigate whether the implementation of the procedure reduces medication discrepancies (mds) in the charts at bodø hospital. and 53.9% (7/13) of the patients died before discharge. parenteral nutrition was administered an average of 21.5 days (95% ci 2.5-40.5), of which 7.7 (95% ci 3.0-12.4) were with ipn. previous spn had been administered in 84.6% (11/13) of the patients. before beginning ipn, the average triglycerides level was 608.1 mg/ dl (95% ci 388.1-828.0) but at the end of the ipn it was 324.9 mg/ dl (95% ci 245.4-404.5), which lead to a mean reduction of 283.2 mg/dl (95% ci 45.3-520.0; p = 0.02). regarding to the total amount of lipids provided with parenteral nutrition, with ipn there was a mean reduction of 30.4 g (95% ci 12.4-48.5; p = 0.002) comparing to those administered with spn. conclusion: usage of ipn in critically ill patients with htg permits to adjust parenteral nutrition formulations to meet specific nutrition needs, enables to reduce the total amount of lipids administered and, therefore, it allows to significantly decrease triglycerides levels. jennifer a. esteban gonzález * , elisabet nogué pujadas, angels andreu crespo, xavier bonafont pujol, nuria romero pascual please specify your abstract type: descriptive abstract (for projects) background and objective: the incidents involving patient misidentification (pm), or wrong patient medical errors (wpme), are medication errors (me), near-miss or close-call situations which can pose a considerable threat to patient health. pm may be under-reported due to the unawareness of the error or the difficulty of identifying them. the aim of this study is to describe the incidence and categories of wpme in a university hospital. design: observational, retrospective analysis of the voluntary reported wpme in the pharmacy database since march 2010 until june 2016. these were classified in prescription, transcription, dispensing, administration and drug system errors. in addition, the national coordinating council for medication error reporting and prevention (nccmerp) taxonomy was used for classifying me according to the severity of the outcome. results: of 1767 me registered, 50 of them were wpme (2.8%). 40.0% of them were due to prescription errors, which consist on wrong labelled medical orders, intermingled patient prescriptions or patient misidentification in computerized physician order entry (cpoe). the administration errors supposed a 30.0% of the total amount of wpme and dispensing errors were 18.0%. 6% of wpme were transcription errors, which occurred previously to the implementation of cpoe, and the remaining 6.0% were system errors after cpoe. the wpme reported took place in the hospitalization wards (44.0%), pharmacy (20.0%), outpatient services (16.0%), intensive care unit (16.0%) and day-care hospital unit (4.0%). 88.0% occurred at working days and 12.0% at the weekends. wpme were notified by pharmacists (62.0%), nurses (34.0%) and physicians (4.0%). referring to the classification according to nccmerp, 48.0% of wpme didn't reach the patient (category b) whereas 40.0% reached the patient but didn't cause harm (category c) and 8.0% required patient monitoring (category d). the remaining wpme (4.0%) caused harm to patients and required medical intervention (category e). finally, in int j clin pharm (2017) 39:208-341 283 more than half of wpme (62.0%), reporters suggested measures to prevent these errors. conclusion: wpme represents near 3% of total me reported in our hospital. given that more than 50% reached the patient, safety measures must be implemented to reduce the risk of hazardous events. additionally, further encouragement in notification is necessary in order to improve patient safety. results: two men diagnosed with rrms aggressive evolution were included in the study. age: 23 and 31. both of them without any treatment by the time they started being treated with alemtuzumab (previously one of the patients had been treated with fingolimod, suspended by inefficiency). the protocol design for the elaboration and control of alemtuzumab in the pharmacy service ensures greater safety and represents a saving strategy. in addition, the development of the protocol in the electronic prescription system (silicon ò ) facilitates the prescription, proper administration and standardization of treatment among patients. the protocol includes daily alemtuzumab infusion for 5 days and other necessary medications including premedication (metylprednisolone, omeprazole, paracetamol and metoclopramide) and anti-infective prophylaxis (aciclovir). developed adverse effects during infusion were skin erythema, pruritus and fever. it was not necessary to stop the alemtuzumab infusion in any patient. during treatment, one patient developed a severe lymphopenia and upper respiratory tract infection (influenza a). conclusion: the role of the pharmacist is critical at various stages, from the preparation and the administration guidelines, to detection, monitoring and reporting of adverse effects. alemtuzumab is presented as an alternative for those patients who do not respond to standard therapies or who have rapidly evolving severe rrms. because of its mechanism of action it is important to closely monitor patients, with particular emphasis on prophylaxis of possible infections. hp-pc093: descriptive analysis of patients receiving oral anticoagulation following acute coronary syndromes sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: triple therapy with concomitant anticoagulant and dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) increases bleeding risk by 50% compared to patients on dapt. bleeding post acs increases mortality and reinfarction risk; balancing ischemic and bleeding risks is particularly challenging in this population. european society of cardiology (esc) produced a consensus document, providing guidance for patients presenting with acs requiring concomitant anticoagulation; however optimal duration of triple therapy and safety and efficacy of novel oral anticoagulants (noacs) and more potent antiplatelet agents requires further evidence. design: a registry was collated of patients presenting with acs requiring concomitant anticoagulation. baseline characteristics, bleeding and ischemic risk scores, periprocedural treatment and antiplatelet/anticoagulant choice and duration was recorded and analysed for trends in prescribing. results: 71 patients have been included in the registry between oct 2015 and june 2016, of which 40 (56%) were naïve to anticoagulation prior to admission, 24 (34%) were taking warfarin and 7 (10%) were on noacs. atrial fibrillation (af) accounted for 51 (73%) cases, (average chadsvasc score of 5, hasbled score of 2), and 15 (21%) were for lv thrombus. of those naïve to anticoagulation, 25 (63%) were initiated on warfarin and 15 (37%) on a noac (last 10 patients all received noacs). of those on a noac for af, 17 (81%) were dose reduced on triple therapy; apixaban being the most commonly prescribed (59% apixaban, 35% rivaroxaban, 6% dabigatran). background and objective: solid oral formulations are more convenient than liquids to manufacture, store and administer for most adults. given this superiority, one would think that children were prompted to use solid formulations when available in an eligible dose. there are indications, however, that the conversion from liquid to solid formulation in children is influenced by characteristics of the liquid medication, rather than the child's ability to swallow solid medications. the aim of this study was therefore to explore if the proportion of oral liquid formulations differed between antibiotics commonly used for upper respiratory tract infections (urti) in hospitalized children. setting and method: we collected the sales data for 2015 for the children's department of the five university hospitals in norway. the three most common oral antibiotics used for urti in children were included: penicillin v, amoxicillin and erythromycin. the proportion of oral liquids was calculated by dividing the number of defined daily doses (ddd) of liquids by the total oral ddds for each substance. main outcome measures: the proportion of ddds of oral liquid antibiotics. results: a total of 2575 ddds of common oral urti antibiotics were sold in 2015, distributed as 30% erythromycin 31% amoxicillin and 39% penicillin v. amoxicillin had the highest proportion of liquid with 97%, followed closely by erythromycin at 94%. in contrast, only 70% of the ddds sold of oral penicillin v were liquids. conclusion: higher proportions of liquid amoxicillin and erythromycin compared to penicillin v were sold to children's departments in hospitals. there are several limitations regarding the quality of sales data, as we lack information of the administered doses as well as the child's age, gender, infection and specific needs. infections in hospitals often require initial intravenous treatment, and oral switch will often be based on the initial treatment. despite these limitations, the results fit well with earlier findings which indicate that children prefer liquid amoxicillin and erythromycin to penicillin v. hp-pc095: proactive medication reconciliation: a preliminary study to identify barriers before its implementation in surgery departments laura foucault * , marion collignon, hélène dewaele, anne laure raso, emmanuel cirot, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: it's well known that medication reconciliation (mr) decreases drug-related problems at patient admission (pa). in surgery departments, for planned hospitalizations, mr is performed 24-48 h after the pa (pourrat x and al, 2013). during this period, some chronic treatments are unintentionally not prescribed to patients. the aim of proactive mr (pmr) is to anticipate the pa by collecting their medication history before their hospitalization. the objective of this study was to identify the barriers preventing pmr implementation in our hospital. design: one week prospective study in digestive and orthopaedic surgery units in a 600 beds' university hospital. the main outcome is to identify which barriers prevent the collection of mr before pa including the evaluation of time required to collect the relevant information, reconcile any discrepancies after the pa and identify the right sources from which to perform the mr. results: eighteen patients with a median age of 59 years old (14-84) were contacted by phone one week before their scheduled surgery. these calls were conducted by pharmacy residents mainly between 6 and 8 p.m. (a more practical time for patients and at the end of pharmacist's routine tasks). an average of 1.7 (1-3) calls per patient were conducted. one patient was unreachable by phone. the average duration of the calls was 7 min (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) . twelve community pharmacy (cp) were contacted. in all cases, cp have accepted to share information about the patient's prescriptions by phone and sending it by fax during the day. five pharmacists were not contacted because patients had no chronic treatment and consequently no regular cp. on 53 lines of prescriptions, 12 discrepancies between the patient's information and prescriptions were identified and 7 between prescriptions and the anaesthesia records. drug history was reported in the patient's records by pharmacy students on the day of pa in order to be used immediately by prescribers. surgery was cancelled for one patient. conclusion: the first step of an mr is made by a hospital anaesthetist some weeks before hospitalization but we have demonstrated that this step is not able to avert all potential errors. our study highlights that the time necessary to perform an mpr appears to be shorter than for an mr. in fact, it's sometimes difficult to properly interview patients during hospitalization (patient in operating room, drug-induced drowsiness). additionally, a key hurdle is to obtain any necessary modification of the prescriptions by surgeons. pmr can be expected to produce time saving efficiencies given that at pa, prescribers will have their full medication history. this study also allowed us to highlight the good cooperation between patients, cp and the hospital. it is worth noting that efforts were made to accommodate the schedules of a majority of working patients. however, as we would expect pharmacy student to perform the pmr, they will most likely attempt to contact patients during standard working hours which may impact the number of patients they are able to reach. laura foucault * , hélène dewaele, marion collignon, emmanuel cirot, anne laure raso, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: the french legislation has clearly defined and integrated the therapeutic education of patient (tep) for healthcare professionals. the pharmacist is invited to get involved in tep as a caregiver around the patient. in our study, we are investigating how the pharmacist's role is viewed by patients with chronic diseases that are included in a tep program. design: prospective study on 17 patients included in a tep program (chronic inflammatory bowel diseases, rheumatoid arthritis, ankylosing spondylitis) between september 2015 and april 2016. in july 2016, the participants of group sessions (gs) conducted with health professionals, including a pharmacist, were interviewed on the phone. the principal outcome of the interviews was to evaluate how their view of the involved health professional's roles evolved before and after gs; to evaluate if they would consider being followed by their pharmacist for individual sessions (is) in a community pharmacy (cp); and if the information supplied by the pharmacist during gs was understandable. to health care. however, discussions between patients appear to be essential to facilitate their acceptance of a chronic condition. some patients also questioned the cp's skills and knowledge when it comes to their particular disease. nevertheless, 88.2% of patients have found that the vocabulary and documents used by pharmacist during gs was adapted and that the information supplied was very useful. conclusion: this study highlights that although the pharmacist is the drug's specialist, a majority of patients will more likely ask their physician about medication. their participation to the gs hasn't changed their habits even if the pharmacist intervention was relevant and understandable. the fact that the pharmacists took into account the level of health literacy of each participant was an appreciated aspect. cp should be more proactive in their relationship with the patients in order to highlight their skills and the assistance they can provide in a chronic disease. however, it's important to take in consideration that in some cases, patients have lived with their disease since childhood. the role of is is likely to be much more limited than in other situations given their key need is to interact with patients afflicted with the same condition. hp-pc097: use and safety of trastuzumab emtansin in her2 + metastatic breast cancer in a tertiary hospital c. chaguaceda galisteo * , alba manzaneque gordon, héctor josé del río torres, natália creus baró please specify your abstract type: descriptive abstract (for projects) background and objective: novel anti her2 drugs have changed the management of her2 + metastatic breast cancer patients. the aim of this study is to describe the use of trastuzumab emtansin (tdm-1) in clinical practice in a tertiary hospital and to evaluate its safety profile. design: we performed a retrospective study of patients who started tdm-1 between january 2013 and december 2015. we recorded demographic data, clinical and treatment variables, number of doses received, reasons for discontinuation, progression-free survival (pfs) and adverse effects (aes). data were obtained from the chemotherapy prescription program and medical records. aes were classified according to the common terminology criteria for adverse events version 4.0 of the national cancer institute. results: eleven female patients with a median age of 55.6 years [40.7-80.1] and an ecog 1 (9/11) were included. tdm-1 was prescribed as a third or further line treatment in 8/11 patients and as firstline in one patient who develop disease recurrence within 6 months of completing adjuvant therapy. median number of tdm-1 cycles was 8.5 . all treatments discontinuations were due to disease progression (6/11). pfs was 6.0 [1.9-20.7 months] (patients that received less than three cycles were excluded (n = 2)). most frequent aes were plaquetopenia, neutropenia and transaminitis but only grade 3 in three patients (two transaminitis and one neutropenia). conclusion: the lower pfs obtained comparing to the pivotal study (6.0 vs. 9.6 months) could be explained by the later use of tdm-1 in clinical practice (8/11 patients received tdm-1 as third or further line while 61% in the pivotal study were first or second line). tdm-1 safety profile was according to the summary product characteristics. few data are currently available regarding the use of tdm-1 in clinical practice. further data are required to position this drug in clinical practice. please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacist for their specialized training in the area of medicines, possess a greater responsibility in the detection and reporting of adverse drug reactions (adrs), as well as other problems related to treatment, which may be subject to monitoring and reporting to the regulatory authorities and the respective laboratories. thus, the pharmaceutical services of the cuf infante santo hospital has implemented a pharmacovigilance program, with two main objectives: 1. optimization of the detection and reporting of problems related to therapy; 2. implementation of corrective and/or risk minimization measures. the pharmacovigilance program is based on the following methodology: 1. detection of adrs/problems related to therapy/medical device: the detection can be performed by the pharmacist or other health professional that guides the process to the pharmaceutical services. 2. information processing by the pharmaceutical services and realization of spontaneous reporting: the notification is performed both for the portuguese regulator (infarmed) as to the appropriate laboratory (if applicable). after evaluation by both entity, the conclusions are communicated to the pharmaceutical services, which has the responsibility to share it with all the other hospital services. 3. report of the event in the internal risk management platform: when applicable, the pharmaceutical services internally report the adverse event to the hospital's risk management department, leading to an internal evaluation of the current process. 4. completion of the process and implementation of corrective measures: when the regulatory authority and/or the laboratory sends the report/technical advice about the notification, the pharmaceutical service in partnership with the risk management team perform a reassessment of the whole process. if needed, corrective and/or monitoring measures are implemented. 5. monitoring of implemented measures: after the implementation of corrective and/or monitoring measures there is a period of evaluation. results: the implementation of this program for the period of 1 year, has led to a total of fourteen spontaneous reports. from all of these notifications, seven were related to quality defect of medicines, four were of adr, one was due to suspected lack of therapeutic efficacy, and lastly, one of the notifications was medication error derived. conclusion: the obtained results, over a 1 year period, by the pharmacovigilance program were satisfactory but the aim of the pharmaceutical services is to consolidate and optimize the same program with a view to achieving better results. the pharmaceutical services will continue to take responsibility for the pharmacovigilance circuit management in this hospital, by promoting a proactive approach to monitoring the safety, quality and efficacy of medicines, which possess the primary objective to patient safety assurance. please specify your abstract type: descriptive abstract (for projects) background and objective: for prematures, parenteral nutrition (pn) is essential for medical care but is complex (specific needs, daily change of intakes…). now, the software logipren ò , developed by the french society of neonatology, allows the prescription of pn as well as all the childish therapeutics. it is also in link with our production robot (baxa pomp) for individual pn bags. our objective was to integrate this software while optimizing our pharmaceutical validation process. design: the software implementation was lead by a physician/ pharmacist collaboration with several preliminary steps: • identification of pharmaceutical validation settings (pertinence of individual pn vs. industrial bags, parenteral approach, elements…). before the life-sized use of logipren ò , a base test has been experimented to identify possible difficulties and to realize some correctives actions of the software or our process. results: logipren ò leads us to a change in our pharmaceutical validation process, by introducing new elements: • the pharmaceutical validation of pn bags is done in collaboration with the physician, during the prescription step. • all the therapeutics are known, which allow the pharmacist to take into consideration all the intakes (micro-nutrients, vitamins…). • remove the transcription step of pn bags in our production software (abacus ò ) thanks to an interface with our production robot. • less production problems because of the coverage of those pharmaceutical aspects during the prescription. since 2 months, this reorganization helped us to propose 22 pharmaceutical notices for 234 prescriptions: • omissions (remove lipids, levocarnyl ò , micro-nutrients, electrolytes, remove industrial bags…) • modification (reduce proteins according to urea level, micronutrients and electrolytes posology, duration of lipids infusion…) conclusion: the implementation of logipren ò enabled us to reorganize of the pharmaceutical validation process with a consolidation of the role of the pharmacist during the prescription step, in the paediatric ward. it had a beneficial aspect by the reduction of the validation and production time, a decreased risk of error (suppression of job interrupts and better communication) and an improved production by the end of transcription step to abacus ò . furthermore, during our experimentation, we could bring to the software editor new ways to improve it and make it more efficient. 57% (52.9% in 2015) , difficulties in swallowing/psycho-behavioural distress in 60. 71% (41.2% in 2015) , and rejection of oral drug in 10. 71% (5.9% in 2015) . physicians and nurses indicate the reason in the medical record in 77.78% of case versus 50% last year. this year, 287 drug were crushed versus 134 drugs in 2015: 22% concerned nervous system group (vs. 25% in 2015), 18% concerned cardiovascular system group (vs. 19% in 2015) , and 15% concerned alimentary tract and metabolism group (vs. 13% in 2015) . nurses use guideline in 50% of cases versus 2.9% last year. as the previous year, in 100% of cases, washing hands before preparation and after administration are met. last year, none of them was wearing mask and gloves during this operation while this year, 17% was wearing mask and gloves. finally, in the two assessment, for each patient, drugs are systematically crushed together and then mixed with the patient's meal. conclusion: this study shows that crushing drugs is still problematic in our units. however, best practices were observed, such as the indication of the reason of crushing in the medical record, or the consultation of guideline. a new training for nurses will be conducted to create awareness about risks of crushing drug. please specify your abstract type: research abstract background and objective: in invasive candidemia, three echinocandins are indicated: caspofungin, mycafungin and anidulafungin. the aim of this work is to establish which echinocandin to prescribe in a french university hospital, given the scarcity of available clinical data in the literature regarding obese patients. setting and method: in a french uhc with 1500 beds, a multidisciplinary working group composed of a microbiologist, an infectious disease specialist and a pharmacist has been set up to analyse the various therapeutic options. main outcome measures: analysis of the literature, pharmacoeconomic study. results: four medications have been identified as possible therapeutic options. their adverse effects are similar and their administration rhythm is the same. according to recommendations by the esmid (2012) and the idsa (2016), the level of evidence for these three echinocandins in initial treatment of candidemia is equivalent. concerning obese patients, no weight limit is mentioned, int j clin pharm (2017) 39:208-341 287 despite recommended dosage adjustment. caspofungin must be prescribed at a dose of 70 mg/day for patients weighing over 80 kg. micafungin must be administered at a dose of 100 mg/day regardless of patient weight. in the case of persistence of cultures or if clinical condition does not improve, the dose may be increased to 200 mg/day. anidulafungin, which is not referenced in our establishment, must be prescribed at the same dose regardless of patient weight. from an economic point of view, in our hospital, micafungin at a dose 100 mg/day remains the least costly therapy. however, if its posology is doubled as indicated, caspofungin then becomes the most economic therapy. amphothericin b, an optional treatment, is never the most economically advantageous therapy. conclusion: as a result of this study, the chosen prescribed therapy for obese patients is caspofungin at a dose of 70 mg/day. this work has improved access to healthcare for obese patients. pharmacokinetics and survival data must be collected on the basis of various patient weights in order to predict clinical efficacy. kristin f. heier * , liv czynski please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to develop a system to prioritize patients for medication reconciliation by pharmacists in the emergency department. it also proved a useful setting for evaluating how other health care professionals perceived the role of the pharmacist performing medical reconciliations within the emergency department. design: the study was located in the østfold municipal hospital, located in kalnes, norway. pharmacists used a prioritization model to identify ''high-risk patients'' having clinically relevant prehospital medication discrepancies between hospital admission records and the information obtained via medication histories, general physician referrals and nursing homes. pharmacists registered patient information such as age, gender and drug-related problems (drps). seventeen physicians and thirty nurses in the emergency department answered structured questionnaires anonymously. main outcome measures: • number of patients with medication reconciliation performed by a pharmacist. • number of drug-related problems denoted in the electronical journal and presented to the physician. • the overall experience physicians and the nurses had with pharmacists when located in the emergency department. results: pharmacists performed medication reconciliation for 262 patients, identifying 443 drps and 178 potential drps in total. fourteen of the physicians had read the journal notes from pharmacist and found them helpful (n = 4, 29%) or greatly beneficial (n = 10, 71%). most physicians (n = 14, 82%) and nurses (n = 19, 63%) reported a good cooperation with the pharmacist in the care of the patients. some of the physicians (n = 4, 29%) and most nurses (n = 21, 70%) wanted more information about the pharmacists work in the emergency department. the majority of ed staff (100% of physicians and 63% of nurses) found pharmacist as a good academic resource in the emergency department. conclusion: the physicians reported an improvement regarding the quality in the medication reconciliation made by pharmacists in the emergency department and both physicians and nurses expressed a need that pharmacists work in the emergency department on a more permanent basis. more information in general and especially better communication with nurses regarding the care of the patients are important actions need to optimise collaboration with pharmacist in the emergency department. results: a total of 105 patients were included in the study, median age was 53 years and 63% were males. they used in average four drugs regularly (range 1-15). almost three-quarters (70%) of the patients reported high or moderate adherence to all their regularly used drugs (mmas-8 c 6 (max 8)). of the 39 patients using oral spasmolytics, 74% reported high or moderate adherence to these drugs. the majority (97% of the patients) had high perceptions of necessity to their treatment (bmq [ 2.5 (max 5)), and 54% had a high level of concern (bmq [ 2.5 (max 5)). logistic regression analysis showed that there was no association between adherence and pain, nor between adherence and spasticity. younger age was found to be associated with higher risk of nonadherence. conclusion: even though overall adherence was high, the patients were more concerned to take their medicines compared to other patients with other chronic conditions. further studies are required for understanding adherence and attitudes toward medication in this population, and to help the patients feel safe about their medication regime. please specify your abstract type: descriptive abstract (for projects) background and objective: errors in medication lists often emerge in transition between health care levels, and there is need for strategies to communicate medication information. therefor we aimed to describe reasons why medication discrepancies (md) occurs in the transfer of patients between hospital and primary care service. design: in conjunction to a study based on use of structured medication report at transition from hospital to primary care service, we observed different reasons to why mds occurs. our observations and experiences linked to communication between health care levels is outlined. results: we observed that many md's disclosed at discharge could most likely be attributed to lack of medicines reconciliation at admission to hospital. for instance, several medicines were prescribed in primary care service prior to admission, but not at admission to the hospital. in addition, at admission, some medicines were listed as prescribed medications although not found in the medication lists in primary care service. we also observed that newly started and discontinued medicines were documented in the hospital discharge letter, but not implemented in primary care service. according to health care personnel in primary care service, insufficient communication about the patients' medications at discharge from hospital, led to corrections in the medication lists based on their previous knowledge about the patients. in addition, justified medication changes at discharge from hospital were not always implemented in primary care service due to professional disagreement. some stated that lack of trust was one reason for not always taking changes into account, often based on earlier experience. conclusion: these observations indicated that mds occurred both with and without intent when patients from primary care service were admitted to hospital and returned back due to poor communication. medication errors during hospitalisation and unproven intentional changes may be the consequences. due to this, it is important to improve the communication and confidence between professionals in the hospital and primary care service in order to reduce the number of mds and to enhance patient safety. please specify your abstract type: descriptive abstract (for projects) background and objective: intravenous human immunoglobulins (iv igs), plasma protein products, may cause in patient to a range of adverse side effects (headache, skin rash, kidney failure, thromboembolic event). in the framework of securing medicinal care, an assessment of professional practices has been conducted within our university hospital. the overall goal of this study is to evaluate the process of intravenous administration of human immunoglobulins done by the nurse staff. design: this prospective study has been carried out in three departments of neurology. an observation grid was established on the basis of guidelines on good practices. all in all, 53 criterions have been examined resuming: prerequisites before administration, patient setup, iv igs administration, monitoring, traceability of drug delivery and management of adverse side effects. results: during the course of this investigation, 51 administrations were observed. only 26% of nurses deliver information about the treatment to their patients before administration and 46% question patients about previous hypersensitivity reaction. the presence of spontaneous diuresis is verified in 14% of cases. emergency cart is not reachable in 33% of all cases. 78% of nurses ask patients to decline their identity. the use-by date on the bottles is checked in 51% of cases. at the time of preparation of perfusion, labelling does not mention either patient's name (48%) or date and hour of perfusion (93%). int j clin pharm (2017) 39:208-341 289 during perfusion, only 11% of nurses follow diuresis and 70% watch rate of administration. hydration is not always kept 20 min after the end of perfusion (80%). patient monitoring varies between 5 min and 1 h after perfusion's end. in 14% of cases, diuresis is monitored after the end of administration. 85% of nurses explain to patients side effects that may occur remotely. finally, administration traceability is was conform in 100% of all cases and in the event of adverse side effects, statement was made in 96% of cases. conclusion: best compliance scores have been achieved in myology department where patients are fewer than in the two others departments (6 vs. 20 and 25). a presentation of those results will be given in theses three departments in order to improve patient management and securitization of iv igs administration. this audit will be carried out soon in other departments. please specify your abstract type: descriptive abstract (for projects) background and objective: a new human polyvalent immunoglobulins dose (40 g) for intravenous administration is available on our establishment since 2014. in order to secure the administration, this new dosage was initially reserved for the healthcares using administration pumps (being four health-care). the aim of this survey was to evaluate the satisfaction of the nursing staff already user of the new 40 g dose and to estimate the motivation of the nonuser nursing staff by the audit date. design: this satisfaction survey was carried out with the most igiv consumer services (being internal medicine, neurology, cardiology and haematology). the questionnaire was structured in two sections: the first section regarding igiv in general, the second section concerning the new 40 g dose. the survey included multiple choice questions or questions with answers based on a four levels evaluation scale (not satisfied, mildly satisfied, satisfied, and very satisfied). results: the audit was realized on eight health-care, involving 41 nurses. among the 41 interviewed, 17 (42%) have already used the 40 g dosage. in 80% of cases, users were very satisfied and 20% were satisfied. the most positive points noted were: gain of time provided (89.5% of satisfaction), less manipulation needed (99.9% of satisfaction), and reducing of infectious risk (94.7%). moreover, the influence of the injection technique on users' satisfaction was further reported. indeed, according to nurses interviewed, the use of an injection pump is safer and improves the job comfort of nursing staff, unlike the injection by gravity (used in 14% of cases), which seems to slow down the use of this new dosage. in two cases, a positive opinion given by patient was also reported. finally, negatives points noted were related to administration instruments (use of pump or not) and to less flexibility in daily dose regulation. among the 58% not-user of this new dose, the 89% showed a strong interest for the product apart from services making the igiv administration by gravity. conclusion: in light of these results, the use of 40 g dose will be spread to other services. the general diffusion of this dosage will provide a gain of time also at the pharmacy, during the unitary delivery and the computer-based administration of every units. a second survey will be soon effected within patients involved in the switch 20 g/40 g. the capital region pharmacy, 2 clinic of neurology, rigshospitalet, blegdamsvej, copenhagen, denmark please specify your abstract type: research abstract background and objective: the clinic of neurology, rigshospitalet, copenhagen, denmark experience continuous medicine-related patient safety incidents (psi) related to newly admitted patients and patient transfers between wards. in order to prevent drug related problems (drp), the pharmacists increased their focus on these patients and provided systematic medication reconciliation. thus, the objective of this pilot study was to investigate if the intervention would help identify drug discrepancies (dd) and prevent drp. four wards were included in this study; two neurological, one neuro-anaesthetic (icu), and one neurosurgical ward(s). three wards use electronic medication module (epm), whereas the icu uses critical information system (cis). furthermore, all patients' prescriptions are registered on shared medication record (smr), which provides an overview of prescribed medicine. prescriptions cannot be transferred from smr and epm to cis and vice versa. we suspected that psi resulted from these system incompatibilities. setting and method: patients admitted or transferred from may 2nd 2016 to june 3rd 2016 were included. medication reconciliations using smr, epm and cis were conducted by a pharmacist on weekdays. dd were presented to a physician orally and documented. only dd accepted by physicians led to drug prescribing change. main outcome measures: number of identified dd. results: the study included 186 patients, of which 147 (79%) were newly-admitted. 39 patients (21%) were transferred between wards. of the transferred patients, 37 (95%) were transferred from the icu to other wards and 2 (5%) were transferred from other wards to the icu. of the newly-admitted patients, 44 (30%) were admitted to the icu and 103 (70%) were admitted to other wards. the pharmacists identified 16 dd; 3 dd (19%) in the transferred and 13 dd (81%) in the newly-admitted patients. in the transferred, 3 dd were all related to the icu. in the newly-admitted, 11 dd (85%) was related to the icu and 2 dd (15%) to other wards. of the 16 dds, 11 (69%) were accepted by the physician. an example of a severe dd identified was an omission of prednisolone to a patient admitted to the icu. conclusion: most dd were identified in patients admitted to or transferred from icu, which uses the incompatible system cis. pharmacist systematic medication reconciliation helps identify these dd and prevent drp. please specify your abstract type: research abstract background and objective: antibiotic related drug interactions are more likely in intensive care unit patients due to common polypharmacy and antibiotic usage. the aim of this study is to determine the antibiotic related drug interactions with three different online databases (micromedex-paid, medscape-free and drugs.com-free) and to evaluate these interaction information by clinical pharmacist. setting and method: a retrospective, descriptive study was set up in hacettepe university hospital's intensive care units, between november 10 and december 31, 2015. 62 patients who use at least one antibiotic were involved in this study. all drugs were assessed by each three databases and only antibiotic drug interactions were evaluated. clinical significance of identified drug interactions were evaluated by clinical pharmacist. main outcome measures: clinical pharmacist's assessment in significance of drug interactions indicated by three online databases. please specify your abstract type: research abstract background and objective: an implementation of clinical pharmacy practice by postgraduate students in intensive care units is a new way of learning in postgraduate education which creates opportunities in multidisciplinary collaboration in clinical pharmacy research, and also has influence on clinicians' routine patient care process. this system in educational program was ongoing in the department of clinical pharmacy since 2014. as a part of this educational program, drug related problems in intensive care units were described and analysed, an influence of clinical pharmacy postgraduate students on patient treatment process was sought. setting and method: a prospective, cross-sectional study was performed between the march-june 2016 in hacettepe university hospitals, department of internal diseases intensive care units which consists of 17 beds. three postgraduate pharmacy students from the department of clinical pharmacy, faculty of pharmacy conducted medication reconciliation in order to identify any problems in patients' medical orders. drug related problems (drps) were identified by the students and recommendations for management were approved by a supervisor of clinical pharmacy department before they were directed to physicians for approval. the students were not authorized to undertake any action in patient care process, therefore all required interventions for drp were undertaken by physicians and the acceptance ratio of the interventions were recorded. the pharmaceutical care network europe foundation classification system (v.6.2) was used to asses drps. main outcome measures: determination of drps by pharmacists and evaluation of their interventions' acceptance by physicians in intensive care units. results: during the study period, 106 patients were admitted to the intensive care units. each patient's medication orders were evaluated and 80 interventions were recommended by postgraduate students. the number of interventions per patient was 0.75. the acceptability rate of interventions by physicians was 96.3%. in addition, physicians were provided drug information on seven different occasions. recommendations regarding drug therapy were mainly related with treatment effectiveness and adverse reactions. the common causes of drps were requiring dose adjustment due to pharmacokinetic problems (42.5%), no therapeutic drug monitoring (18.8%), inappropriate timing of administration and/or dosing intervals (11.3%), requiring dose adjustment due to deterioration/improvement of diseases (6.3%), inappropriate drug selection (5%) and new indication for drug treatment presented (5%). the most common drugs responsible for drps were ranitidin, levothyroxine, allopurinol, pantoprazol, piperacillintazobactam and vancomycin. the study showed that the most common drps was dose-related, therefore close monitorisation of the intensive care unit patients by students in clinical pharmacy postgraduate program can help physicians in terms of detecting, preventing and minimizing drps in order to improve patients' health outcomes. please specify your abstract type: research abstract background and objective: antibiotic stewardship is the process of salvaging important antibiotic agents from becoming ineffective due to bacterial resistance. this is important because throughout the world antibiotics continue to be one of the most important classes of therapeutic agents due to their vital role in saving patient lives. key goals of antimicrobial stewardship are to improve clinical outcomes, prevent antibiotic resistance, promote patient safety, and reduce health care cost. pharmacist are in the frontlines because they perform antibiotic stewardship activities, such as selecting the most optimal antibiotic agent, adjusting drug-dosage, and stopping use of unnecessary antibiotics. as a result of the continuous rise in antibiotic resistance and decline in development of new antibiotics, antibiotic stewardship programs are proving to be indispensable in a health care settings. setting and method: 100 adult and paediatric inpatients receiving antibiotic therapy in the hospital medipol university has been evaluated. patients were selected randomly in the hospital system. patients were evaluated for antibiotic susceptibility results and compliance with antibiotic management guidelines. main outcome measures: to evaluate the antibiotic therapy in patients with culture results and to determine according the treatment guidelines. results: it was observed 10 different pathogens in blood culture results of 51 inpatients out of 100 patients who were treated with antibiotics in hospital. antibiotic susceptibility results for acinetobacter spp, staphylococcus spp, enterococcus spp, pseudomonas aeruginosa, klebsiella spp, e. coli spp, streptococcus spp, corynebacterium spp, streptococcus pneumonia and enterobacter spp are evaluated in the study. klebsiella spp was the most isolated pathogen at total of 85 culture results. most frequently resistance were int j clin pharm (2017) results: a total of 289 (28%) questionnaires were completed. of these, approximately 80% were answered by hospital nurses, the remaining mainly by physicians (18%) and 2% ''other''. on the question ''what is your general perception of the benefit of the clinical pharmacy service; for collaborating health professionals? for the patient?'' the total benefit was ranked 5.45 and 5.54 respectively (scale from 0 (''no benefit'') to 6 (''beneficial to a very large extent''). the open questions: ''what disadvantages/advantages have you experienced by the introduction of clinical pharmacist into multidisciplinary teams?'' received 153/185 comments respectively. physical obstacles regarding office space, interference with the decision making process, more time consuming processes and the issue of relying too much upon the advices given was reported as possible disadvantages. 120 respondents answered ''none'' to this question. the comments regarding advantages dealt mainly with general increased patient safety and quality assurance. in addition, advantages as work-load relieve, time saved, collegial support, practical help, and learning interchange between professions, were highlighted. conclusion: health-professionals assessed the clinical pharmacy service as highly beneficial. the advantages outlined were higher patient safety and quality regarding medication, in addition to collegial support, practical help and learning interchange. please specify your abstract type: descriptive abstract (for projects) background and objective: in june 2015, the french health authority, the «has», published an index resuming the recommendations of benzodiazépines (bzd) prescriptions and proposing an approach to stop using it. indeed, it has been established that there is a too high and too long consumption of bzd in france. a study of prescriptions' prevalence has been done in our hospital centre. the aim of this study was to know our situation regarding the use of bzd in order to set up some improvements and take part in their proper use. design: a prospective study has been done on a 5 months period in different services: geriatric, post-op and rehabilitation facilities, endocrinology, internal medicine, pneumology and cardiology services. the data were raised on a given day in each services and recovered thanks to the prescriptions software but also through interviews with the patients and their doctors. it was examined whether there was a bzd prescription (hypnotic or anxiolytic), whether the duration was superior or not to the duration of the amm and whether the prescription was done in our hospital centre. if the prescription was already part of the patient treatment, we looked if it was possible for the patient to stop using it, according to the has criteria. on their discharge, the letters and bzd prescriptions were also analysed and some patients' general practitioner were contacted after their discharge. results: 185 patients (median age 83 years old) were included from november 2015 to march 2016. 59.5% (110/185) of the patients had at least one bzd prescription the day we collected the data. we found only one bzd in 81 prescriptions (73.6%) and among them 77.7% (63/81) were anxiolytic bzd. among those prescriptions, 62.7% (69/ 110) already existed before the hospitalization and 37.3% (41/110) were given during the hospitalization (24 were prescribed automatically). 59.4% (60/110) of the prescriptions did not respect the legal duration of the amm (9 pieces of data were not found). 12.2% (5/41) already exceeded this duration limit. among the patients who already had a bzd treatment before going to hospital, 24.6% (17 out of 69) could consider stopping their use of bzd. by the end of this study, 143 patients were discharged from hospital, among them 48.3% (69/ 143) with a prescription of bzd. 55.2% (16/29) of the prescriptions established during the hospitalization had been renewed when the patient came out of the hospital, we managed to contact ten general practitioners (approximately 53.4 days after their discharge), nine patients carried on their bzd treatment, among them one patient had reduced his consumption. conclusion: this study is an example of the high proportion of bzd prescriptions in france which the majority doesn't respect the legal length of the amm. the prescriptions of bzd in the hospital are generally systematically renewed by the general practitioners. the patients must be informed about the risks of using those molecules. in order to ameliorate this practice in the hospital, a proper use leaflet, reminding the prescriptions of bzd, has been created and distributed in each services to make people aware. main causes of admission were infections (34%) (respiratory disease (23%) and other (11%)), hepatic disease (20%) and neoplasias complications (13.5%). 11 patients died during their admission; 5 due to hepatic disorder, 3 due to neoplasia, and 3 due to infections. conclusion: last diagnosis of hiv or no art treatment are causes of admission. immunovirological situation is related with their adherence but isńt with admissions. coinfection with hcv or hbv or others infections are risk factors for admission. center for psychopharmacology, diakonhjemmet hospital, oslo, norway please specify your abstract type: research abstract background and objective: complex medical history and treatment can potentially cause problems. the objective of this study was to investigate the prevalence of drug-related problems (drps) and medication discrepancies in internal medical patients with complex treatment at hospital admission. further, to investigate to which extent drps were identified as a result of medication reconciliation, and to which extent drps could be associated to the hospitalization. setting and method: patients with at least four regular medicines from two different therapeutic groups were consecutively included at admission to an internal medicine ward at a university hospital in norway in the period 01.09.14-15.02.15. pharmacists used the integrated medicines management (imm) model for medication reconciliation and medication reviews at admission. a medication discrepancy was defined as any discrepancy between the recorded medication list at admission and the patient's actual use of medications, as revealed by medication reconciliation. the patients' actual use of medications, medical journal and laboratory results, were used to perform a medication review at admission time and identify drps. the proportion of drps revealed due to medication reconciliation was calculated. moreover, the project group retrospectively assessed possible drp-induced hospitalizations based on clinical history, cause(s) of admission and identified drps. main outcome measures: the main outcome was the median number of drps per patient at admission. the proportion of drps revealed due to medication reconciliation, the proportion of patients with drps possibly associated to the hospitalization, and the median number of medication discrepancies, were included as secondary outcomes. results: 120 patients were included, 50.0% women. median patient age was 79 (range 27-96) and most of the patients were home-living before admittance (89.2%). in total 1359 drps were identified at admission, with a median number of 11 (range 2-27) per patient. 99 drps (7.3%) were identified due to medication reconciliation. for 25 patients (20.8%) a causal relationship between the hospitalization and the drps was assessed as ''possible''. medication discrepancies were revealed in 113 of the 120 included patients (94.2%), with a median number of 4 (range 0-14) per patient. conclusion: internal medical patients with complex drug regime are frequently exposed to drps and medication discrepancies at hospitalization. medication reconciliation could be essential to identify drps, which is likely a common cause of hospitalization in the studied patient population. hp-pc117: assessment of oral anticoagulant prescriptions and pharmaceutical analysis at the hospital by regional audit damien fuss *,1 , clélia monchablon 1 , anaïs breteau 1 , marie lefebvre-caussin 1 , rémi varin 2 , jean doucet 1 , mikael daouphars 3 , doreya monzat 1 1 omedit normandie -chu rouen, 2 chu rouen, 3 please specify your abstract type: descriptive abstract (for projects) background and objective: oral anticoagulants (oa) are the most common drug class associated with preventable adverse drug events in hospitalized patients that require optimizing the pharmaceutical analysis (pa) process. in this context, a regional audit was conducted on pa of prescriptions oral of oa. the aim of this study is to provide an overview of the treatment by oa in the hospital by evaluating the consistency of the oa prescriptions compared with national and european guidelines and evaluate the pharmaceutical interventions. design: this study is based on the collection of pa data (demographics, indication, posology, drug interactions, monitoring) as well as the collection of pharmaceutical interventions and discordance int j clin pharm (2017) 39:208-341 293 between guidelines recommendations and clinical practice. the inclusion criteria were any patient treated with oa (vitamin k antagonists (vka), non-vitamin k antagonist oral anticoagulants (noacs)). included patients were followed minimum 2 months. the primary outcomes include description of baseline characteristics of patients, the number of inappropriate prescriptions compared to the different clinical recommendations, the number of pharmaceutical interventions, the number of adverse drug reactions (adrs) related to oa use and the assessment of patient monitoring. results: during the 6-months study period, 588 patients were included in six health institutions. the average age was 78 years (70% of patients over 75 years old) and 59% of the patients were women. 32% of patients had renal impairment. 73% of patients were treated with vka, and 27% with noacs. it was the first prescription of oa for 27% of patients (56% with vka; 44% with noacs). the most common indication was the non-valvular atrial fibrillation (60%). in this indication, 99% of patients had cha2ds2-vasc score c2, and nearly 30% had a high risk of bleeding (has-bled score c3). 8 drug interactions were observed, and 35 adrs occurred related to oa. 42% of patients with an adrs had a has-bled score c3. 12.5% of prescriptions were considered inappropriate, including 45% noacs (no monitoring renal function in 13% of patients over 75 years initiating treatment, inappropriate posology in 14%, and 3% of contraindications). the rate of pharmaceutical interventions was 4%. nearly 60% of the prescriptions were already adapted when the pharmacist was starting analysis. conclusion: prescribers are sensitized of the risks on the oa prescriptions, which explained the delay upon pa and low rate of pharmaceutical interventions. however, the high number of inappropriate prescriptions shows the necessity to improve the pa process on these drugs, particularly by actions on therapy initiation and patient monitoring, especially for noacs. for this class, the impossibility of assess the level of anticoagulation by laboratory monitoring requires appropriate initiation and monitoring, especially an assessment of baseline renal function. please specify your abstract type: descriptive abstract (for projects) background and objective: the development of bacterial resistance these last 10 years is a public health major problem in the world and needs to implement actions. in france, the national drug safety agency has defined a list of ''critical antibiotics''. this list includes antibiotics particularly generator of bacterial resistance (amoxicillinclavulanate, cephalosporine, fluroquinolone) and antibiotics called ''last resort'' (antibiotics against gram-positive cocci, car-bapenem…). at our regional level, an evaluation of prescription of these critical antibiotics was proposed to all medical centers. the aim was to evaluate the quality of prescription of these critical antibiotics. design: the regional working group (pharmacists, infectious diseases physicians and biologists) had developed a collection grid including data on patients, antibiotics and four criteria: adequate molecule, compliance with medical prescriptions, duration of antibiotic therapy and reassessment at 72 h. this is a prospective study proposed to all health institutions (public and private), which had to be completed on a given day in all care units and had to be conducted by a team of multi-professional evaluators. the study included a quantitative part (number of patients hospitalized in the audited units, number of patients receiving antibiotics and number of critical antibiotic prescriptions) and a qualitative part (adequate to the four criteria). results: response rate was of 84%. the study investigated on 7026 patients hospitalized in the audited units, including 1391 patients (20%) receiving antibiotics. among the 1391 patients, 89% were hospitalized in medical, surgery or obstetrics units we recorded 865 prescriptions of ''critical antibiotics particularly generator of bacterial resistance'' (53% amoxicillin-clavulanate, 30% ceftriaxone, 14% fluoroquinolone and 3% other third-generation cephalosporine) and 42 prescriptions of antibiotics called ''last resort'' (74% carbapenem). the average age of the population was 69.9 years (±20 years). sex ratio was 1.1. 91% corresponded to curative use and 9% to prophylactic use. the expertise of infections diseases physician was requested in only 13% of the cases. the antibiotics were prescribed in majority to treat bronchopulmonary infections (38%), urinary tract infections (16%) and intraabdominal infections (16%). ninety-two percent of the prescriptions had a proper indication. 66% of the prescriptions complied to the guidelines. the duration of antibiotic therapy was adequate in 82% of the cases. only 45% of the prescriptions were correct according to these three criteria. forty-four percent of the prescriptions were reassessed and adapted by the physician. conclusion: this study is original because of its regional dimension and antibiotic analysis. the number of analysed prescriptions was significant with an overall proper prescription in adequate with the guidelines. however, actions must be implemented on duration and reassessment and adjustment of treatment. these results were presented to the participating hospitals. these three points will be reevaluated during a new regional audit. the criterion «no more 2 psychotropic drugs» has been met in 88.95% of assessment. otherwise, 2 or more psychotropic drugs are prescribed in 88.89% of assessment from the point of admission. the criterion «no more a benzodiazepine drug» has been met in 91.72% of assessment. otherwise, more than one benzodiazepine drug is prescribed in 59.26% of assessment from the point of admission. no contra-indication is detected in 93.25% otherwise, a contra-indication between two drugs causing torsade de pointes is detected from the point of admission in this department. no more 1 anticholinergic drug is prescribed in 84.05% of assessment. according to the french criteria, one or more inappropriate drug is prescribe in 46.93% of assessment. the most common inappropriate drug group prescribed was alimentary tract and metabolism drug (60.85%) (the hospital at home team needs these class of drug) followed by nervous system (25.95%) (prescribed at the point of admission) and by cardiovascular drugs (12.34%) (prescribed at the point of admission). finally, the criteria «no more one non-steroidal anti-inflammatory drug» and «no illogical association» have been met in all cases. conclusion: this analysis shows that most of criteria for «assessment of prescription among elderly in a «hospital at home» department have been met. when one has not been met, either the hospital at home team needs the drug prescribed, or this drug have been yet prescribed from the point of admission in this department. this study could be used for the next certification. hp-pc120: access to health care: case of autologous serum eye drops batiste martel, fabien lindenberg, camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: autologous serum eye drops (ase), prepared from patient's serum, are indicated in the treatment of severe dry eye syndrome and defective epithelial healing. its in-hospital preparation within a controlled-atmosphere zone unable it to be dispensed by non-equipped hospital pharmacies. the aim of this study was to implement security measures to allow transport towards distant hospital pharmacies and all patients even those residing far from a regional university hospital (uh). setting and method: this study was conducted in a 1495-bed french university hospital. patient blood samples were taken within the university hospital every 12 weeks. serum was then biologically controlled (negative tests for hiv, hbv, hcv, tpha, vdrl). preparation was conducted 3 days after blood sampling. sterile preparations were then stored at a temperature of -18°c. studies showed that eye drops were stable 14 days after being thawed. transport of eye drops to distant hospital pharmacies requires to be conducted under controlled temperature i.e. below 8°c, to ensure the stability of eye drops. these pharmacies are located close to patient's homes. the entire process was examined by a pharmacy team in order to study and secure each step, transport in particular. main outcome measures: validation of each step of the autologous serum eye drop dispensing process, from sampling to receipt by different hospital pharmacies, transport in particular. results: 4 patients benefitted from the preparation. all patients resided more than 100 kilometres from a uh. a follow-up form was completed to qualify dispatching and to trace each step during transport. a temperature sensor was placed inside the box. the receiving agent was required to stop and control the sensor. a double retrospective control was performed by a pharmaceutical team via the recording of temperature sensors. a second follow-up form was drafted in order to track dispensation reviews, ongoing dispensation and future planning. a patient information booklet was distributed to hospital pharmacies to inform patients about good practice concerning eye drops. conclusion: technological necessities concerning autologous serum eye drop preparation and transport limit access to health care. in this study, the role of the pharmacist consisted in reducing inequalities among patients residing at a distance from the only regional uh. the role of the pharmacist is to ensure absolute quality of preparation between the uh and the patient. hp-pc121: computerized medication reconciliation: overview of pharmaceutical software used and support for development of integrated modules julie mocquard, anaïs berthe, elise rochais * , nicolas prévost, jean-claude maupetit, on behalf of centre de ressources régional en conciliation médicamenteuse omedit pays de la loire, nantes, france please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation aims to improve continuity of care for patients. in 2015, a national survey identified barriers for implementation of this activity in france, among which computerized systems were judged unsuitable for hospital practices. in the absence of appropriate hospital information systems (his), medication reconciliation remains a time consuming process implying manual transcriptions, potentially leading to a lack of traceability and medication errors. the objective of the study was to assess the current his used in a french region including the integration of medication reconciliation into the software and to define courses of action to assist this integration. design: an online survey conducted in may 2016 was addressed to head pharmacists of the 134 health facilities in the region, giving a total of 100 head pharmacists concerned. it included questions on the software used by the health facility, the development of medication reconciliation and its traceability, formulation of operational requirements to the editors of software and availability of a module integrating medication reconciliation provided by the software. results: seventy-eight pharmacists (78%) participated in the study, with all types of health facilities represented: public hospitals, clinics, home health agencies, haemodialysis structures and after care and rehabilitation facilities. thirty different software were identified in the region. 27 (35%) pharmacists planned to develop medication reconciliation in their health facility and 20 (26%) were already carrying out this activity. within these 26%, medication reconciliation was conducted on paper only for 9 (45%) of them, while 11 (55%) were using a computerized system (patient file, pharmaceutical software, other) for traceability. the most widely used software in the region contains a module enable for computerized medication reconciliation, and three other editors are currently developing one. no development is scheduled for another three editors nonetheless commonly used in the region. 15 (19%) pharmacists had contact with the editor of the software, and 10 had given thought to the preparation of requirement specifications to the editor to develop an integrated module of medication reconciliation. conclusion: despite the interest attributed to medication reconciliation and despite the need of a fully integrated module of medication reconciliation to his, only a few health facilities of the region possess an appropriate computerized system to develop this activity. this study underlines the approaches already made by pharmacists to editors in order to integrate medication reconciliation to the his. subsequently, retrieving these approaches and writing specifications common to all health facilities is scheduled, in order to assist them in providing a strong incentive for the editors to integrate medication reconciliation to existing his. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is an interactive and multiprofessional process that ensures the continuity of care by integrating the ongoing treatment to the new hospital prescription. this helps securing the patient's care pathway particularly at transition points. the objective is to initiate the mr process in our medical institution with a pilot study in the department of internal medicineemergency downstream to validate a methodology and adapted tools. design: the mr takes place in three steps performed by a pharmacy student: (1) realization of best possible medication histories (bpmh), combining at least three sources of information and using sources' collection form. this research begins with a patient interview done in pairs with a medical student using an interview guide. (2) comparison bpmh with the initial hospital prescription in the department (after passing through the emergency department) on the treatment reconciliation form. a status is assigned to each line of drugs and then the differences are identified (stopped, changed or added). these two steps are validated by a pharmacist. (3) discussion and characterization of observed differences (intentional/unintentional and documented/undocumented) with the senior physician. results: twenty-six mr were performed over 3 weeks in 2015. the mr is performed within 2 days after admission. on average, 2.3 information sources per patient were used for the bpmh: mainly drug prescription (dispensed in pharmacies community); analysis of emergency medical records and patient interview. for the 26 patients included, 268 drugs were listed. 148 discrepancies were observed and 62 were studied (status stopped or changed only): one documented intentional discrepancy, 43 undocumented intentional discrepancies and 18 unintentional discrepancies (ud). these uds affected 12 patients (1-3 medication errors per patient) and corresponded to a non-prescribed drug in 90% of the cases. vitamins, antihistamines, anti-reflux and proton-pump inhibitors were involved in 59% of cases; cardiovascular drugs in 17% and antiinfectious in 12%. through this pilot study, the methodology was validated: (a) need to have a minimum of three sources to achieve a relevant bpmh and to confirm each information with two sources; (b) need for a dedicated time with trained staffs; (c) development of tools to improve the traceability of information obtained from each source and traceability of medication reconciliation activity. conclusion: the mr establishment in the internal medicine department was helpful in identifying 18 medication errors that have been corrected. it is proposed to archive the treatment reconciliation form in the patient file to contribute to the traceability of information on treatment. this study strengthens the deployment of this method and mr tools to other services of the hospital. alma mulac * please specify your abstract type: research abstract background and objective: clinical pharmacists have an important role in improving healthcare services. there is lack of knowledge of clinical pharmacists' experiences in interprofessional collaboration. our objective was to explore the challenges and barriers experienced by clinical pharmacists in interdisciplinary teams in norway and incorporation of expanded pharmacist roles in hospital settings. setting and method: this qualitative study was conducted using semi-structured interviews. a total of 13 clinical pharmacists from four (government) hospitals were included in the study. the interviews were audio recorded using a digital recorder. the recordings were transcribed verbatim. main outcome measures: challenges and barriers clinical pharmacists experience in interdisciplinary teams in hospital setting. results: the main findings are that the pharmacists' role is little known to other health care professionals, particularly at hospitals with short tradition for clinical pharmacy services. clinical pharmacists have great motivation from being able to influence drug treatment for patients. from the perspective of the participating pharmacists they succeed in interdisciplinary cooperation when their professional knowledge solves the patients' drug-related problems. communicating recommendations to physicians with professional credibility has great importance for the intervention to be implemented. using the theoretical framework of communicating tensions, we argue that the pharmacists in our study use indirect communication to prevent physicians defensiveness to recommendations. lack of education in interprofessional cooperation and communication is apparent in this study. the participants also stated that there should be some form of quality assurance or education requirements before one can work as a clinical pharmacist. conclusion: training in communication for graduates and interprofessional collaboration during the undergraduate pharmacy education, can possibly help pharmacists with integration in interdisciplinary teams. increased attention to teamwork from the hospital leadership is essential for the implementation of interprofessional collaboration in a larger context. please specify your abstract type: descriptive abstract (for projects) background and objective: antifungal therapy in the icu, particularly therapy targeting resistant aspergillosis, mucormycosis and systemic candida, is often of lifesaving importance. posaconazole and voriconazole are the antifungal agents of choice. our aim was to compile a tool that can be used at the icu to address aspergillosis, mucormycosis and systemic candida in an optimal manner. design: female patient, age 50 + , liver transplant, crp [ 300 mg/ l, creatinine [ 150 lmol/l. abdominal x-ray imaging revealed four large abscesses and laboratory analyses confirmed mucormycosis. posaconazole intravenous (300 mg one times daily) and liposomal amphotericin b (1 mg/kg/day) were initiated. the inflammatory markers remained unchanged 5 days following initiation of therapy with no change in size or number of abscesses and the patient developed sepsis. amphotericin b dose was increased to 3 mg/ kg/day. after 1 week the inflammatory parameters and size of abscesses began to fall. the dosage form of posaconazole was switched from intravenous to mixture. the dose remained the same and within 24 h the crp rose to 600 mg/l. results: pharmacist intervention revealed a missing loading dose of intravenous posaconazole as well as incorrect dosage of the per oral form due to bioavailability variation. posaconazole mixture dose was increased to 400 mg two times daily. through serum concentration analysis of posaconazole was suggested prior to the dose increase. the serum concentration was 0.6 mg/l (range [1.0-1.25) . through serum concentration 4 days later was 1.2 mg/l. both crp and abscess size were on the decline. a dosage and tdm pocket card for posaconazole therapy of mucormycosis, aspergillosis and candida was compiled. conclusion: optimal systemic fungal infection therapy is essential, especially in the critically ill. of special importance is tdm and correct dose adjustment when dosage-form changes occur. please specify your abstract type: research abstract background and objective: potentially inappropriate prescriptions and omission of prescription, respectively ip and op, are common issues in the pharmacotherapy, especially in vulnerable population, such as elderly and children. there are many available tools detecting ip and op for geriatrics, however, similar tools are less common in paediatrics. therefore, a first target tool for paediatric population: popi «paediatrics: omission of prescriptions and inappropriate prescriptions» was created and was validated by delphi method in 2014. we aim to evaluate inter-rater reliability between health care professionals, who apply popi. our study also assessed their satisfaction and the accessibility of this tool. setting and method: twenty cases with or without ip or op were selected. these cases were identified in a previous retrospective ip-op prevalence study on 15.973 patients. these patients were admitted to the emergency department of a university mother and child hospital, between october 2014 and march 2015. one doctor and one pharmacist, who participated in the creation of popi tool, identified ip and op in 20 cases and composed ''standard answers''. these cases were then reviewed independently by eleven clinicians (including generalists, paediatricians, pharmacists, residents, general practitioners), who did not experience this tool before. inter-evaluator agreement was calculated by using the agreement kappa test. the satisfaction of users was also evaluated. main outcome measures: inter-evaluator agreement, the median time of use and the satisfaction of users. results: a high level of agreement of ip and op detection was recorded (ip: k median = 0.80; op: k median = 0.71). the easy use of popi was approved by 91% evaluators. the median time of use was 2 min 45 s per case (quartiles : 2.4-3.4) . as a result, there were 82% of clinicians satisfied with the provided popi and they would like to apply this tool in their daily practice. conclusion: popi demonstrated a good interrater reliability and is easy to use. this strong validation by many specialists prove popi is a reliable tool. it can be applied daily at work in paediatric section by doctors and pharmacists. other multicentre and prospective study should be conducted to evaluate economical and clinical impacts of popi. please specify your abstract type: descriptive abstract (for projects) background and objective: drug dosing during cvvh is challenging due to changes in pharmacokinetic parameters brought about by the patients' deterioration in health and factors associated with the physical process of filtration. this is of particular significance in the icu. in addition, there is the issue of the patients' diuresis or lack of such. this will affect the total clearance (cl total ) of the drug. the dose of antibiotics must therefore be calculated individually taking into account all of the above as well as changes of filtration parameters. our aim was to illustrate how such dosage calculations can be undertaken. design: a 50-year-old male patient, weight 75 kg, diagnosed with stenotrophomonas maltophilia infection. the trimethoprim/sulfamethoxazole dose was 7.5 mg trimethoprim/kg/day every 8 h as specified for anuric patients on cvvh. patient was initially anuric for 3 days after which diuresis was started. the dose was recalculated. results: creatinine clearance (crcl) related to cvvh during the anuric period was calculated accounting for ultrafiltration rate, sieving coefficient, blood-flow, haematocrit concentration and pre-dilution. the value was 22 ml/min. following diuresis on day 4, remaining kidney function was assessed by measuring urine and serum creatinine. the value for crcl renal (18 ml/min) was added to the extracorporeal clearance, and gave a total clearance of40 ml/min. this warranted dose adjustment of trimethoprim/sulfamethoxazole since this drug requires normal dosage at crcl [ 30 ml/min. conclusion: during cvvh, the presence or absence of diuresis must be taken into consideration when dosing antibiotics. in anuric patients, the cvvh-machine set up constitutes crcl total , but in patients with diuresis, the remaining crcl renal should be added. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of the study is; to evaluate patients' home (prescribed and non-prescribed) and hospital medication during hospital admission by computing medication regimen complexity index and investigating possible drug-drug interactions. design: patients (aged 18 and older) who applied internal service during 6 months (2 days/a week) were included to the study. patients' medical profile were obtained from patients' file. their home medication and hospital medication were calculated with medication regimen complexity index (1) and checked drug interactions with micromedex drug interaction program. results: a total of 151 from 360 of patients who applied to the internal service (male 46.4%, female 53.6; the mean age of patients was 69.01 ± 16.28.) were included to this study during 6 months. of them, 75.5% had low education level (\8 education years), 53.6% had 2 and more chronic diseases of them, 45% hospitalized last 6 months before this hospital admission. the most prevalent diagnoses documented at admission were kidney disease (19.8%), cardiovascular disease (15.3%) and cancer (13.2%). the mean of patients' home medication number was 5.64 ± 3.50 and the mean of their mrci scores was 16.02 ± 10.89. 45% in patients hospitalized in the last 6 months. at least one possible drug-drug interactions were found in 66.6% of patients at home medication and in 78.8% of patients at hospital medication, respectively. the mean number of possible drug-drug interactions at patients' home medications was 2.88 ± 3.62, while the mean number of possible drug-drug interactions at patients' hospital medications was 4.07 ± 4.06. of them, 53.6% had polypharmacy at home medication. the frequency of possible drug-drug interactions and the score of medication complexity index was found high among patients' hospital medications when compared with their home medications. conclusion: the potential role of pharmacist including medication reconciliation and medication review could improve rationale drug use during hospital admission. coronavirus. experts' local committee has approved to use oral ribavirin for the treatment of these respiratory viral infections. we aimed to assess the effectiveness and safety of oral ribavirin as main treatment in respiratory viral infections. setting and method: from may 2013 to october 2015, we performed a retrospective monocentric study including patients who received oral ribavirin for non-hcv infections. main outcome measures: viremia negativation was used to determine the response rate to oral ribavirin. specific toxicities (anaemia, cytopenia, liver dysfunction) and renal function were assessed biologically. results: thirty-five immunocompromised patients (f/m: 3/4, age: 57) were included. underlying conditions were lung transplant (n = 32), heart transplant (n = 1), pulmonary fibrosis (n = 1) and acute myeloblastic leukaemia (n = 1). the median duration between transplantation and infection was 1.8 years (0.1-10.8). nine patients were exclusively infected by rsv, 19 by hpiv (2 hpiv-1; 2 hpiv-2; 10 hpiv-3; 4 hpiv-4; 1 non-identified hpiv), 4 by hmpv and 1 by coronavirus. there were six co-infections: rsv/ hpiv-1, rsv/coronavirus, hpiv-2/hpiv-4 and hpiv-3 or 4/coronavirus (3 patients). all the patients were admitted in pulmonary division, except for the patient with heart transplant who was in cardiac intensive care unit. the administered dose was 400 mg tid or 200 mg tid if there was renal insufficiency (9 patients). the median duration of the treatment was 8 days . four patients prematurely discontinued the treatment due to severe toxicity or therapeutic change; three didn't respond to the treatment (no data for the last one). four patients were re-treated despite having a virological response to the first cure. one patient treated for a hpiv-3/coronavirus coinfection had an hpiv-3 relapse 64 days after ribavirin discontinuation. concerning the three other patients, they received a second cure to treat a new infection (coronavirus, hpiv-4 and hmpv, in opposition to hpiv-3 twice and hpiv-2 respectively). virological response rate was 82% (7/11 for rsv, 22/25 for hpiv, 4/5 for coronavirus and 4/4 for hmpv). two non-negative viremia patients (rsv and hpiv-4/coronavirus) received intravenous ribavirin after oral ribavirin therapy. no patient died from viral infection. twelve patients presented specific toxicity: one hepatic cytolysis and cholestasis, eight haemoglobin decrease, two pancytopenia and one mucositis. conclusion: despite the poor number of patient, our study shows that oral ribavirin seems to be efficient to treat hpiv, hmpv and coronavirus in immunocompromised adults. we observed known side effects that could generally be managed. oral ribavirin may thus represent a therapeutic strategy in several respiratory viral infections. please specify your abstract type: descriptive abstract (for projects) background and objective: reconciliation of medicine lists is important to ensure correct medical treatment of patients both in hospital and other healthcare levels. while reconciliation upon admission is part of the normal routine at surgical ward b, molde hospital, there has been less focus on reconciliation at discharge. as such, this study aimed to ensure reconciliation and correct transfer of medical information at discharge. design: medicine lists of all patients discharged from surgical ward b, molde hospital between week 39 and 51 in 2015 (n = 240) were investigated. the forms were gathered and counted based on the tasks signed for to ensure completed reconciliation and sufficient information given to the patient. the count was performed every 2-3 weeks, and the forms in each count was pooled together as one point of measure. the quality of medicine lists in discharge lists was evaluated based on the norwegian patient safety program criteria. medicine lists in discharge lists from week 39 to 51 (n = 102) were pooled together and compared to medicine lists in weeks 36-39 (n = 46). results: the results of reconciliation was divided into the subsections of surgical ward b, and represent the number of completed tasks as signed for in the reconciliation form. the surgical subsection showed a significant increase in patients with pre-checked medicine lists and reconciled medicine lists over the measured time period. similar results were not found in the orthopaedic subsection. as for the quality of medicine lists in the discharge lists, significant improvement was seen in all set criteria, with the exception of ''source'' in the surgical subsection. in the orthopaedic subsection however, no significant improvement was seen in any of the criteria other than ''indication for use''. conclusion: the implementation of reconciling medicine lists at discharge was successful. however, both subsections need to work further to ensure continuation and improvement of the process. furthermore we found varying results in the writing of medicine lists depending on subsection. still, regardless of the individual results of the two subsections there is big room for improvement to ensure that sufficient medical information is included in the discharge papers. please specify your abstract type: descriptive abstract (for projects) background and objective: from july 2015 clinical pharmacists began conducting medication histories and reviews (pharmacist notes) at the emergency surgical ward (esw), north zealand hospital (nzh). inclusion criteria are acute patients using c5 drugs or c1 risk drug (antidiabetics, anticoagulants, antipsychotics, benzodiazepines, opioids and digoxin). the aim of the service is to identify drugrelated problems and secure correct medication reconciliation between the medicine the patient is admitted with and the medicine in the electronic medication system (ems) in the hospital. the service ensures that the patients' medication follows across healthcare sectors. the objective is to determine if the discrepancies between the medicine the patient is admitted with and the medicine in ems (documented in the pharmacist notes) are used by the physicians. in addition to determine if the pharmacist interventions increase the physicians' acceptance rate of the discrepancies. design: data were collected at the esw at nzh (capacity of 27 beds). data consist of pharmacist notes conducted from august 2015 to may 2016. pharmacist notes were compared to the patient record and ems to identify if the pharmacist notes were considered by the physicians. in order to increase physicians' acceptance rate of the discrepancies suggested in the pharmacist notes, interventions were made according to the model for improvement. throughout the period, the focus was on oral delivery of the pharmacist notes. in december 2015 the pharmacist optimized the clinical relevance of the discrepancies, by creating and testing a list of products (including vitamins, herbal drugs, glucosamine etc.) which the pharmacist should not intervene on. in december 2015 the pharmacist also started to follow up on the pharmacist notes not considered by a physician the previous day to ensure that the physician considered the discrepancies. results: there were identified 599 discrepancies between the patients' actual medication at admission and ems at the hospital in 306 patient records (1.96 discrepancies per patient). in total 424 discrepancies were accepted by the physicians (1.39 discrepancies per patient). the physicians' acceptance rate was based on the acceptance of one or more of the discrepancies in the pharmacist note. baseline data were collected from august to november 2015, where 54 out of 85 pharmacist notes were accepted by the physicians resulting in an acceptance rate of 63.5%. from december 2015 to may 2016 the interventions made by the pharmacist contributed to an increase in acceptance rate to 81.8% (108 out of 132 notes accepted). if the pharmacist notes were not delivered orally to the treating physician the acceptance rate was 9% (8 out of 89 notes accepted). conclusion: the pharmacist interventions contributed to an increase in the physicians' acceptance rate of discrepancies from 56.5 to 81.7%. a result indicating that the pharmacist notes contributes to an increase the quality of the medication process across sectors. hp-pc134: how the centralization of medicines manufacturing enable to generalize the pharmaceutical validation? samantha oses * , soizic vandierdonck, vincent servant, dominique breilh please specify your abstract type: research abstract background and objective: the centralization of the reconstitution of injectable anti-infective drugs enhance to decrease costs and several risks. this minimization of the risks operates at several levels such as i) reduction of the staff exposure and external contamination of preparations during the reconstitution phase (with controlled atmosphere areas, isolators, etc.), ii) improvement in the quality of the management of infective diseases thanks to a pharmaceutical validation systematically performed after the prescription and before the reconstitution phase. the main objective of the study was to describe and quantify pharmaceutical validation on injectable anti-infective drugs prescriptions restored in a pharmaceutical reconstitution unit. setting and method: an observational descriptive study was carried out on each prescription with at least one injectable anti-infective drug that has to be reconstituted before administration. the process was as follows: 1-prescription by the physician on an electronic prescription software, 2-pharmaceutical validation and if necessary pharmaceutical intervention (pi) made by phone call, 3-reconstitution at the pharmacy, 4-administration to the patient. the pharmaceutical validation methodology followed the french society of clinical pharmacy (sfpc) guidelines ''prescriptions screening and analyses level'' published in the good practices of clinical pharmacy and one resident and one pharmacist were devoted to the activity every day. main outcome measures: the pharmaceutical validation was quantified by the number of pi by patient, which were categorized according to the sfpc guidelines. results: during 4 months, a total of 222 pi were collected. they concerned 93 patients with an average of 2.4 pi per patient. among them, 11.8% (11) concerned paediatric population. antibiotics were involved in 53.2% (118) then followed by 36.9% (82) cases (59.5% (69) biological assessment issues, 37.1% (43) absence of therapeutic drug monitoring (tdm) and 3.4% (4) the drug hasn't been adapted to the weight), dosage adjustments in 25.2% (56), information missing concerning the treatment indication in 15.8% (35) and miscellaneous pi in 6.8% (15) such as wrong clinical service on the prescription, etc. approval rate of physicians was 79.3% (176). conclusion: this study has shown that even if prescriptions were secured by electronic prescription software, the pharmaceutical validation remains essential. in that case, the centralization of the reconstitution of injectable anti-infective drugs enabled to generalize this activity on all prescriptions of the hospital. however, the pharmaceutical validation was focused only on anti-infective drugs, that was not fully efficient and must be extended to the whole prescription. it is a priority to develop a comprehensive and exhaustive validation on every medical prescription; however, this activity is highly time consuming and needs larger and more trained staff. hp-pc135: the start/stopp criteria as a helping tool to the pharmacists' medication review in the acute admissions unit of the regional hospital in horsens hans pedersen * please specify your abstract type: research abstract background and objective: polypharmacy occurs often increasing the need for patients' medications to be reviewed. the start/ stopp criteria help detects potentially inappropriate prescriptions in older people. in this study we aimed to measure and categorize the different start/stopp criteria found in medication reviews in the acute admissions unit of horsens and the acceptance rate. setting and method: patients admitted to the acute admissions unit were selected based on their age and the number of prescriptions in a period of 4 months. patients 65 years or more which received six or more drugs were included in the study. only patients who later were transferred to another medicine ward were included in the study. the pharmaceutical medicine review was performed by a clinical pharmacist using minimum two different sources; the electronic medical record and medication-lists. the guideline of pharmaceutical medicine review in the hospital pharmacy central denmark region was used as the standard-guideline. in addition, thestart/stopp criteria version 2 was used. main outcome measures: the number of start/stopp criteria found in medication reviews. the different start/stopp criteria were scored equally with one point each. results: 33 patients, 17 males and 16 females, out of 55, were included. the mean age was 79 years and the patients received in average 14 prescribed drugs. at admission the average number of stopp criteria were 1.1 ± 0.9 and 0.4 ± 0.6 for the start criteria. in average, 27% of the purposed stopp criteria were accepted by the physicians. the most frequently accepted stopp criteria were in the category of drugs that predictably increases the risk of falls in older people. the benzodiazepines where the most common drugs to be discontinued. in the start category, 25% of the suggested start criteria were accepted, which included: calcium and vitamin d3 supplement, beta-2 agonist and bisphosphonate. conclusion: the present study demonstrates that it was possible to integrate the start/stopp criteria as a helping tool in the medication reviews in the acute admissions unit of horsens. the start/stopp criteria were found within the different categories, however only a minor part of the registered start/stopp criteria were accepted by the physician. please specify your abstract type: research abstract background and objective: the objective of this work is to assess prescribing practices of somatostatin analogues in a surgery department, and to analyse the conformity of switching from immediateacting octreotide to the long-acting release (lar) form, in accordance to laboratories' guidelines. setting and method: retrospective observational study. a focus was realized on patients admitted in a digestive surgery unit between january 1 and december 31, 2015. the patients' medical records were reviewed for clinical features, diagnosis workup and treatment strategies. main outcome measures: medical records for patients with diagnosis of gastro-entero-pancreatic or endocrine tumors who had received injections of lar octreotide during hospitalization were reviewed and the economic impact of prescriptions errors has been evaluated. results: of the evaluated 234 patients, 73 (31%) were hospitalized in surgery digestive unit; mean age at first administration of octreotide was 66 years and 58% were male. the male and female ratio was 1.35:1. reasons for hospitalization were: digestive system neoplasms (75%), fistula (7%), intestinal obstructions (4%) and other pathologies (14%). of the 73 patients treated with octreotide, 41 (56%) received a lar form. only four patients received doses in accordance with guidelines: one at 20 mg/month lar form and three at 30 mg/month lar form, after having respectively been treated by intravenous octreotide at 300 and 600 mcg/day during 7-10 days. medical prescriptions of the 37 remaining patients did not comply: all patients received 30 mg/month after an intravenous treatment of 300 mcg/day, instead of 20 mg/month. from a financial perspective, these misuses have led to an additional cost of 6659.7 euros for the hospital, excluding tax (30 mg: 1389.29€/unit and 20 mg: 1212€/unit). conclusion: despite the publication of octreotide release form proper use recommendation in our hospital, 90% of patients of digestive unit are not right treated. a new guideline will be written added by doses of long-acting release and economic data. this work will be transmitted to specialists by clinical pharmacists. hp-pc137: pharmaceutical process for intrathecal analgesia in clinical oncology practice vivien pigeon * , guillaume binson, claire grignon, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: in some cases, patients with cancer pain remain painful despite the use of high dose of intravenous opioids and intrathecal analgesia becomes the ultima recourse to manage acute pain. until 2015, intrathecal syringes were prepared by nurses in the unit care which involve a risk for patients. therefore, the aim of this work is to describe the set-up of the prescription and preparation process with the potential benefits for the safety. design: multidisciplinary concertation took place between pharmacists, physicians and surgical teams and several points were discussed to secure the process: • identification of patients with high level of infection risk; • identification of critical points of the pharmaceutical process; • validation of quality control and drug stability studies regarding drug compounding involving morphine, ropivacaine, baclofen and clonidine, alone or in admixture. results: multidisciplinary concertation lead us to define the most important points to set up the pharmaceutical process for intrathecal analgesia: • chosen patients are cancer patients; • implementation of a prescription software to secure the prescription step; • production of syringes by the pharmacy department implying several criteria: • preparation in controlled atmosphere area; • training of pharmacy technicians; • implementation of quality control and drug stability studies at 4°c in syringe over 48 h and at 37°c in pumps over 1 month; • microbiological control and bacterial endotoxin level. the implementation of a pharmaceutical process for intrathecal analgesia gave us the opportunity to reorganize the care of cancer patients tolerant to high dose of opioids. in this process, the pharmacy department plays a major role leading to decrease the risk of infections and errors of dosing. ingrid plessala *,1 , xavier deviot 1 , thomas sidibe 1 , zohra mostefaï 2 , michèle minvielle 2 , marta wyrtwal 2 , roselyne gervais 1 1 pharmacy, 2 geriatrics, saint-denis hospital centre, saint-denis, france please specify your abstract type: descriptive abstract (for projects) background and objective: proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. design: prospective study in three geriatric wards. the study included ppis treated patients from these three geriatric wards. dose, indication of the ppi, age, gender and duration of treatment have been recorded for each patient. the relevance of each ppi treatment has been reassessed by a geriatrician, a pharmacist and a junior pharmacist, regarding the indication and the patient's clinical condition. following this re-evaluation, three situations arose: • to maintain ppi at the same dose (30 mg or 15 mg) • to maintain ppi but half dose (from 30 mg to 15 mg) • to stop ppi corrective actions have been recorded in patients' files to allow their traceability. results: 109 patients were included in the study. 61% of ppis prescriptions were off-label, 24% had no indication mentioned in patient's file and 11% were conform to the marketing authorization. 98% of patients have been on ppis medication longer than 2 months, which is the recommended treatment' duration in france, 55% longer than a year and 29% longer than 4 years. in collaboration with the geriatricians, ppi prescriptions were maintained for 52% of patients. we reduced the dose in 14% of cases. finally, we decided to stop a third of the ppis prescriptions. conclusion: ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since 2009 may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in 2017. ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since 2009 may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in 2017. hp-pc139: oral anticoagulants and heparin for children: standardized protocols for prescription, dispensation and administration alexandra liauzu 1 , marie-françoise hurtaud-roux 2 , ronan bonnefoy 3 , caroline farnoux 4 , philippe sachs 5 , theresa kwon 6 , olivier bourdon 7 , sophie ajzenfisz 8 , sonia prot-labarthe *,7 1 pharmacy, 2 hématologie clinique, ap-hp hôpital robert-debré, 3 cardiologie, 4 néonatologie, ap-hp hôpital robert debré, 5 réanimation pédiatrique, ap-hp hôpital robert-debré, 6 néphrologie, 7 pharmacy, ap-hp hôpital robert debré, 8 coordonnateur de la gestion des risques associés aux soins/ responsable du système de management de la qualité de la prise en charge médicamenteuse, ap-hp hôpital robert-debré, paris, france please specify your abstract type: research abstract background and objective: high-alert medications (ham) are medications that are associated with a high risk of serious harm if used improperly. we already identified paediatric ham used in our institution to identify safety measures for their use. anticoagulants and heparin were part of these high-alert medications. we aim to write standard protocol of use for low weight heparin and oral anticoagulant used in our mother-child teaching hospital. our secondary objectives were to decrease medication errors, anti-xa and inr unexplained variability and to help nurses to administer the drugs (standard dilution, oral solution available) setting and method: we carried out a literature search on pubmed ò , on websites of several learned or professional societies and agencies. the results of the literature search were compiled on written protocol and presented to our institute drug safety-steering committee composed of four doctors, two head nurses, two pharmacists, and one risk manager. main outcome measures: not applicable. results: the protocols concerned enoxaparin, tinzaparin, warfarin but we chose to also include protamine. the most difficult issue was to have standardized dilution and protocol for all ages and weight: from premature to adolescents and all units of care (from cardiology to intensive care unit, nephrology and neonatology). we took into account the administration errors we had in our hospital and the preexisting protocol to avoid any drastic error-prone change. the final version of these protocols will be presented on the final communication with web link to upload them. conclusion: for now we did note evaluate the impact of these protocols but a before/after analysis of error reports and users evaluation will be done. however, these protocols can help all health professionals working in paediatric units for benchmarking. hp-pc140: does a hospital formulary system impact timely medication administration and quality of inpatient care? anne-valérie putallaz *,1 , vera jordan-von gunten 1 , pierre-auguste petignat 2 , pierre turini 3 , johnny beney 1 1 division of pharmacy, institut central des hôpitaux, 2 division of internal medicine, 3 medical coordinator for quality of care and patient safety, hôpital du valais, sion, switzerland please specify your abstract type: research abstract background and objective: the prevalence of drug omissions is often underestimated but their impact can be clinically relevant. we hypothesized that delays in the administration of non-formulary/nonstored drugs could impair the quality of care. the aims of this study were: 1°to determine the time between the prescription and the administration of the first prescribed dose and, if applicable, to calculate how many doses were omitted. 2°to analyse the clinical relevance of the identified delays. setting and method: three months retrospective study of electronic records of patients hospitalized on the internal medicine wards of a network of hospitals supplied by a centralized pharmacy. this pharmacy is located in one of the sites; other sites are 15-45 km apart. main outcome measures: 1. for the main hospital site and the three distant sites: • median time between the prescription and the administration of the first prescribed dose • mean number of omitted doses for formulary and non-formulary/ non-stored drugs. 2. categorization of patient's harm caused by the delays of timecritical drugs, according to the ncc-merp taxonomy of medication errors. results: 16'954 prescriptions were analysed. calculated delays for non-stored/non-formulary drugs were longer than for formulary drugs. however, the median time to administration is less than 1 h for both formulary and non-stored/non-formulary drugs; and more than 95% of formulary drugs and around 90% of non-stored/non-formulary drugs were administered within 24 h following their prescription. there was no significant difference in the mean number of omitted doses or in the delays between the site where the centralized pharmacy is located and the other sites, except for one of them. a delay representing 1.5 or more omitted doses was found for 332 (1.96%) prescriptions. among them, only 17 were considered potentially clinically relevant. none of them caused severe harm to the patients involved. conclusion: in our setting, non-stored/non-formulary drugs take more time to be delivered than formulary drugs, but more than 95% of formulary drugs and around 90% of non-stored/non-formulary drugs are administered within 24 h following their prescription. none of the 17 patients who experienced delays underwent severe harm. our study showed that delays also occur for formulary drugs but no systematic cause of omission was identified; further studies should focus on all dose omissions during hospitalization. penelope randuineau *,1 , roger jeremy 1 , lauriane cornuault 1 , anne lecoeur 1 , franck lemercier 1 , isabelle javerliat 2 , thomas tritz 1 1 service de pharmacie à usage intérieur, 2 service de chirurgie vasculaire, hôpital ambroise paré, boulogne-billancourt, france please specify your abstract type: descriptive abstract (for projects) background and objective: a french national survey of inpatient adverse events reveals that nearly half of adverse drug events (ade) are preventable. medication errors behind these ade occur mainly during the transition steps of care pathway. in this context, medication reconciliation process has been implemented in our vascular surgery department. the objective of this study is to identify unintentional discrepancies (uid) and assess their potential clinical impact design: a pharmacy resident or a pharmacy student reconciliated patients: aged older than 65 or with at least five chronic treatments at admission or suffering from chronic diseases. patients were considered reconcilable if at least two reliable sources on usual patient's treatment were available. these many sources of data (patient interview, prescription or interview of general practitioner, reference dispensary, drug box …) were compared to the admission prescription during the first 48 h of hospitalization to detect and correct uid. based on gravity scale promoted by the french high authority of health, two pharmacists (a resident and a senior) and a vascular surgeon reviewed every uid in order to define their potential clinical impact. the uids were considered minor if it leads to no consequence for the patient, clinically significant if it leads to essential monitoring, major if it could cause temporary clinical consequences, and critical if it could result in permanent clinical consequences or the involvement of the prognosis. results: between february 15th and may 31st 2016, a total of 102 patients have been reconciled. 10 patients were excluded due to a lack of reliable sources. mean age was 73.9 years old (±11.4) and sex ratio m/f was 0.7. 85% of the reconciliated patients' admissions were scheduled. the mean number of medication was 10.8 (±2.9). 38 patients (41%) had at least one uid and the mean uids per patient was 2.0 (±1.6). the most common types of uids were omission (73%), incorrect dose (15%) and incorrect administration frequency (11%). more than 60% of these uid presented a potential clinical impact: an adverse effect (high blood pressure, hyperglycaemia) was observed for nine patients and lead to therapeutic optimization and monitoring; 37 uid were considered to have potential clinically significant impact (49%), 11 a potential major impact (15%) and 1 a potential critical impact. conclusion: these results appear consistent with those reported in literature. vascular surgeons have appreciated the approach and would like systematic medication reconciliation before surgery. as a major part of admissions were scheduled, we would like to establish the reconciliation before the patient's hospitalization every time it's possible. this new organization should facilitate the care pathway before surgery and decrease preventable postoperative adverse events. hp-pc142: delirium in elderly patients: successful use of melatonin gaëlle jouin 1 , aurélie reiter-schatz 1 , pierre bentzinger 2 , fatem-zohra laalou 2 , bénédicte gourieux *,1 1 pharmacy-sterilization, 2 orthopedic's intensive care unit, university hospital of strasbourg, strasbourg, france please specify your abstract type: descriptive abstract (for projects) background and objective: postoperative delirium happens to about one-third of elderly patients and is a major cause of morbidity and mortality. it is reported that haloperidol, an antipsychotic, has been the agent of choice for managing delirium. however, it induces cerebrovascular adverse effects and greater mortality. the hyperactive type of delirium is known to be associated with a low melatonin level and the loss of a normal melatonin secretion rhythm. the postoperative administration of melatonin to elderly could decrease the symptoms of delirium. the purpose of this study was to evaluate melatonin effectiveness in a cohort of patients suffering from postoperative delirium. design: a retrospective study of melatonin prescriptions has been conducted over a 12 months period. medical background, type of surgery, symptoms of delirium, use of antipsychotics and benzodiazepines have been studied in all patients who received melatonin in an orthopaedic surgery unit. length of hospital stay, time between delirium and melatonin administration and the effect of melatonin had been evaluated. results: a total of 14 patients were included: average age was 77.6 years (64-87), sex ratio m/f = 1. twelve patients (86%) were hospitalized because of an infection (prosthesis or osteoarticular). in 64% of cases (n = 9), the prescription of melatonin was started when the patients were hospitalized in our intensive care unit. nine patients (64%) were under chronic treatments like benzodiazepines or antipsychotics. the average length of hospital stay was 76 days (11-186). melatonin was started on an average of 14 days after surgery , and administered at the dose of 2 mg xr in the evening, during an average of 35 days (7-113). cognitive impairments requiring a prescription of melatonin were: confusion (86%, n = 12), agitation (64%, n = 9), daytime sleepiness (64%, n = 9), temporal-spatial disorientation (57%, n = 8), nocturnal awakening (14%, n = 2), hallucination (14%, n = 2), difficult falling asleep (7%, n = 1). the average time to recover from confusion was 8 days, agitation 4 days, daytime sleepiness 10 days, temporal-spatial disorientation 7 days, nocturnal awakening 16 days, hallucination 5 days and falling asleep 24 days. melatonin treatment helped stopping benzodiazepines treatment in six patients (66%). conclusion: after administration of melatonin, delirium symptoms were improved for all patients and benzodiazepines treatment stopped for six patients. earlier prescription of melatonin could regulate sleep-wake cycle and reduce the duration and incidence of delirium. please specify your abstract type: research abstract background and objective: denosumab (xgeva ò ), a fully human monoclonal antibody targeting rankl, which inhibits bone resorption, is indicated to prevent skeletal complications in patients with solid tumors and bone metastases. about 10% of patients develop hypocalcaemia, a common adverse event that may induce spasms, muscle cramps, paraesthesia, prolonged qt interval, tetany, convulsions… we report the management of ionic supplementation and physicochemical incompatibilities in a case of hypocalcaemia due to denosumab. setting and method: the clinical case was analysed with the pharmacovigilance regional center. main outcome measures: a 61 year old patient, with nodal and bone metastasis in prostate cancer, was treated with denosumab (stopped with the last injection 2 months before, on the 29th of march). he went to emergency on the 30th of may with asthenia, anorexia, nausea, diarrhoea, qt prolongation. biological results showed hypocalcaemia (corrected calcaemic = 1.94 mmol/l) and hypophosphatemia (phosphorus \ 0.21 mmol/l). concomitant calcium and phosphorus intravenous supplementation started with loading doses (10 g of calcium and 1.8 g of phosphorus) and then a week of following daily intakes: phosphorus (1 g iv and 1.2 g oral); calcium (1 g iv and 4.6 g oral). however, low-serum corrected calcium and phosphorus levels persist at 1.89 mmol/l and 0.24 mmol/l. results: incompatibility between phosphorus and calcium by formation of soluble or not-soluble complexes is described in literature. in our case, calcium and phosphorus were mixed in a same infusion. after a week of supplementation, calcium infusion is continued with increased dose (4 g/day) and phosphorus infusion is stopped. phosphorus oral supplementation remains stable (1.2 g per day); calcium oral supplementation is increased (9.3 g per day). 2 h between intakes is applied to avoid digestive complexation. 48 h later, corrected calcium levels are normalized at 2.19 mmol/l and phosphorus levels are still low. therefore, as hypocalcaemia due to denosumab induced a secondary hyperparathyroidism and thus hypophosphatemia; phosphorus levels are expected to increase subsequently. conclusion: this case report shows that recurrent hypocalcaemia with denosumab is possible few months after administration. supplementation with large amount of calcium is needed and administration methods may impact the effectiveness of supplementation. indeed, it seems that the incompatibility between phosphorus and calcium did not allow an effective supplementation. gunnhild langdal *,1,2 , ida rudberg 1 , lone holst 2 , anne-lise sagen major 1,3 1 central norway hospital pharmacy trust, å lesund, 2 centre for pharmacy, university of bergen, bergen, 3 møre og romsdal health trust, å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: drug interactions (dis) can cause side effects and lack of therapeutic effect. the objective of this study was to describe the prevalence of dis at the medical department of å lesund hospital, and to investigate how dis were managed by clinical pharmacists and physicians. design: at the medical department, å lesund hospital, clinical pharmacists serve seven out of ten wards, from which patients were included during a five weeks period. the clinical pharmacists selected patients for screening for potential dis (www.interaksjoner.no) as int j clin pharm (2017) 39:208-341 303 usual (= pharmacist group). detected dis were classified according to a predetermined classification system, and it was registered whether the physician implemented suggested changes in prescription. for patients not selected by clinical pharmacists (= non-pharmacist group), a pharmacy student performed the search for dis. results: in total 373 patients were admitted. on average, each patient had 1.6 dis, and 56.6% of the admitted patients had at least one di. the prevalence of dis was significantly higher among the 194 patients in the pharmacist group compared to the 179 patients in the non-pharmacist group (median@@@ 1 vs. 0, respectively, p \ 0.001). the groups differed significantly regarding number of drugs used, age, duration of hospital stay and number of warfarin users. 10.5% of the dis detected in the pharmacist group were discussed with the physician. the remaining 89.5% were considered not necessary to discuss for various reasons e.g. because they were considered not clinically relevant (30%) or already adjusted for in clinical practice (20%). for 24 dis the clinical pharmacist suggested a change in prescription, and 20 of these suggestions (83%) were implemented by the physician. conclusion: just over half of the patients were selected by the clinical pharmacist for screening of dis, and the pharmacist seemingly made a reasonable priority of patients with many drugs, old age, a long hospital stay and users of warfarin. only 1 of 10 dis was discussed with physicians. this indicated that pharmacists do a considerable work in assessing the relevance of dis before discussing with the physicians. it also seemed that changes in prescription suggested by the clinical pharmacist were reasonable. hp-pc145: securing the paediatric use of oral chemotherapy: a proactive risk assessment samia mouffak *,1 , linda an 1 , anne fratta 1 , anne auvrignon 2,3 , nadia marquis 3 , karine morand 1 1 pharmacy, 2 risk management committee, 3 hematology, armand trousseau hospital -aphp, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: oral chemotherapy is an important part of the therapeutic strategy in childhood cancer or haematological malignancy. it also represents an emerging risk area in oncology practice. several medications errors involving oral chemotherapy were reported in children of our onco-haematology department, fortunately without clinical consequences. nevertheless, the potential severity of such errors led us to implement a failure analysis of the paediatric oncology care pathway in order to identify and prevent potential risks, and secure the paediatric use of oral chemotherapy. design: we conducted a failure modes, effects and criticality analysis (fmeca) which is a proactive risk assessment approach. first, process maps were detailed for each step of the oncology care pathway. it was performed by a multi-disciplinary group composed of 2 physicians, 1 coordinating nurse, 2 hospital pharmacists and 1 pharmacy resident. then, for each step of the medication-use process, the team identified the failure modes, their main causes and effects. finally, participants rated the expected severity, frequency and detectability for each failure mode, assigning a score on a five-point scale. a risk priority number (rpn) was then calculated by multiplying those three indexes. the risks getting a high rpn were categorized as critical risks and have been the object of safety improvements. results: 69 failure modes were identified, including 15 critical risk failure modes. 9 critical failures were related to hospital discharge prescriptions and 6 were about the dispensation of oral chemotherapy by pharmacy assistants. most failures were due to prescriptions heterogeneity, lack of clinical information reported on prescriptions, and lack of training of pharmacy assistants in reading oral chemotherapy prescriptions and in mistake detection. two improvement strategies were implemented. first, physicians' awareness led to the harmonisation of practices and to the standardisation of discharge prescriptions. then, to enhance pharmacy assistants' abilities, an educational program on oral chemotherapy dispensation was planned. conclusion: the implementation of a fmeca has highlighted the most critical risks of oral chemotherapy medication-use process. the awareness of all caregivers and the targeted changes in our practices allowed us to improve the safety of the paediatric oncology care pathway. please specify your abstract type: descriptive abstract (for projects) background and objective: the purpose of this study was to investigate if medication reconciliation and medication review, by using the integrated medicines management (imm) model, were suitable to assure the quality of patients' medical treatment at a gastrointestinal surgical ward. furthermore, to analyse frequency, type, handling and clinical relevance of medication discrepancies (mds) and other medication related problems (mrps). design: patients, above 18 years of age, from two departments at a gastrointestinal surgical ward at a norwegian university hospital were included consecutively. medication reconciliation was performed at admission by a clinical pharmacist. the resulting medication histories were compared with the medications documented in the medical records. mds were detected and categorized. thereafter the clinical pharmacist identified mrps by reviewing the medical records systematically and categorized the revealed mrps. mds and mrps were presented for the physician with proposed solutions. the physician's actions to manage the mrps were registered. later a multidisciplinary team assessed the clinical significance of mds and mrps in a subset of patients. results: a total of 48 patients were included. overall, 144 mds and 153 mrps were identified. at least one md was revealed in 90% of the included patients, whereas at least one mrp was identified in 88% of the patients. the most frequent type of mds was omission, whereas mrps most often were related to medications that were considered unnecessary. totally, 76% of the mds and mrps were discussed with the treating physician. the physicians followed the pharmacist's input in 85% of the discussed md-cases and 62% of the mrp-issues. longterm consequences of mds and mrps were considered more serious than short-term consequences for the patients. conclusion: medication reconciliation and medication review revealed, solved and prevented a large number of mds and mrps in this study. the results emphasize that pharmacist involvement, by using the multidisciplinary imm-model, contributed to more correct medical records and furthermore to quality assurance of the patients' medical treatment at a gastrointestinal ward. hp-pc147: prevention and management of drug interactions in oncology day-hospital: results from a 6 months study involving drug assessment and pharmaceutical report to oncologist pauline-saraï zeller *,1 , chloé hugard 2 , céline mongaret 1,3 , juliette vella-boucaud 2 , antonin maréchal 1 , olivier bouché 2 , dominique hettler 1 , florian slimano 1,3 1 pharmacy, 2 oncology day hospital, university hospital reims, 3 clinical pharmacy, faculty of pharmacy, reims, france please specify your abstract type: research abstract background and objective: quality during transitions of care is a major concern in drug safety for patients. traditional hospitalization allows to reconciliation medication but there is not possible for dayhospitalization (patient's hospitalization short time and no outpatient medication prescribe by oncologists). however, lack of communication between health professionals may expose patients to drug-drug interactions (ddi). while ddi between oral antineoplastic and other drugs are well known, there is a lack of knowledge about ddi between parenteral antineoplastic (ak) and other drugs. in this pilot study, we aim to investigate prevalence and characteristics of ddi between ak and other drugs in real life and to propose a pharmaceutical report model to enhance patient's drugs safety. setting and method: during 6 months, all new oncologic patients (thoracic and digestive) receiving chemotherapy in day-hospital have been recruited by clinical pharmacist. first it was conducted a patient-clinical pharmacist interview and carried out the best possible medication history (bpmh) by contacting at least three different sources of drug information. then, the bpmh has been confronted with oncologic treatment (including concomitant medications such as like antiemetic) with support at least with two different database of ddi analysis. finally pharmaceutical recommendations in order to manage potential relevant ddi were reviewed with oncologists then reported and inserted in personal health record (phr). main outcome measures: prevalence and description of potential clinically relevant ddi in an ambulatory oncology population. results: from november, 2015 to april, 2016 n = 90 oncologic patients were included with following characteristics: mean age of 63.2, sex ratio 2:1, majority of oncologic thoracic localization (56%). number of oncologic concomitant medications per patient was 3.97 ± 0.94 (mean ± standard deviation). patients present an average of 2.2 ± 1.93 comorbidities (excluding cancer) and 5.97 ± 3.76 linked medications per patient. pharmaceutical analysis revealed 33 potential clinically relevant ddi (0.37 ± 0.97 per patient): 72% of them concern antiemetics (ondansetron and aprepitant): pharmaceutical interventions were formulated (including recommendations to adapt chronic treatment) and 39% of them involved biological monitoring (for renal function, inr, potassemie or magnesemie). conclusion: our pilot study confirms high prevalence of ddi between oncologic and non-oncologic drugs. clinical pharmacy services with bpmh performing and pharmaceutical recommendation appears to be useful to enhance patient' drug safety in oncology dayhospital. we currently are deploying our study in order to convey a pharmaceutical letter to general practitioner and community pharmacist. hp-pc148: loading dose of anti-infectives: elaboration of a tool helping pharmaceutical analysis julie soyer *,1 , cécile sanchez 1 , guillaume beraud 2 , nicolas venisse 3 , pauline lazaro 1 , antoine dupuis 1 1 pharmacy, 2 infectiology, 3 pharmacokinetics, university of poitiers, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: the recent data on vancomycin and ceftazidime confirm that continuous infusion is the best way of administration of these antibiotics. moreover a loading dose before the administration is required for the antibiotics to prevent from the infratherapeutic period at the start of infusion and limiting the risk of resistance emergence. long half-life antibiotics and antifungals also require a loading dose to be effective. the aim of this study is to analyse the prescriptions of anti-infective requiring a loading dose in order to develop a tool to help pharmaceutical analysis. design: a prospective observational study was carried out during 15 days in 16 units. initially, pharmacists, residents and students were trained (role of the loading dose, drugs concerned). then, all patients with anti-infective requiring loading dose were included. some data were collected: weight patient, creatinine clearance, loading dose or not, dose, administration mode, monitoring of steady state concentrations (vancomycin and ceftazidime) and dose adjustment. the results were analysed and compared to bibliographic research before discussion during a multi-disciplinary meeting (pharmacists, infection control specialist and pharmacokinetic specialist). finally, a list of relevant pharmacist interventions was selected. results: out of the 393 patients, 44 were enrolled for prescription of anti-infective requiring loading dose. twenty-six prescriptions including vancomycin, 8 ceftazidime, the others fluconazole, caspofungine, voriconazole and posaconazole. concerning vancomycin, the loading dose was prescribed in 85% of case, monitoring of steady state concentrations was performed in 90% of case and dose adjustment after first dosage was required in 56% of case. selected pharmacist interventions were: • to favour continuous infusion (excepted paediatric) • to keep loading dose at full dose even in patient with renal failure • to monitor steady state concentrations after the first 24 h in patient with renal failure or obesity • to adapt dosage when the target concentration is not reached concerning ceftazidime, the interventions were: • to recommend continuous infusion: 6 g/24 h after loading dose of 25 mg/kg • to monitor steady state concentrations in patient with renal failure a total of 27 interventions (dosage, adaption of posology at the monitoring, patients with renal failure, obese, paediatric patient, administration…) were identified by the group of experts. conclusion: this study allowed creating a recap data sheet for students and hospital pharmacists. the selected interventions will allow the harmonization of practices. these recommendations have been validated by the commission of the anti-infective. finally, this study shows that the pharmacist has a key role in the management of antiinfective requiring loading dose. hp-pc149: assessment of potentially inappropriate medications in orthogeriatric patients using the rasp list the detection of inappropriate prescribing. the objective of this study was to investigate if the rasp list (rationalization of home medication by an adjusted stopp list in older patients), an explicit screening method adapted to the belgian context, can be used to reduce the number of potentially inappropriate medications (pims) in orthogeriatric patients. setting and method: single-centre, interventional study conducted at the orthogeriatric department of the uz brussel, a 721-bed university hospital. the rasp list was first applied by a last year pharmacy student to the admission medication of orthogeriatric patients hospitalised in october 2015. after potential adaptations to the medication by a liaising geriatrician, the rasp list was additionally applied by the same pharmacy student to the discharge medication of these patients. main outcome measures: detection and reduction of the number of pims. results: in total, 59 orthogeriatric patients, from whom an informed consent was obtained, participated in this study. on admission, a total of 136 pims were detected in this population. at discharge, the number of pims decreased to 101. the median number of pims per patient decreased from 2 (on admission) to 1 (at discharge). this difference was statistically significant (p \ 0.001; wilcoxon signed rank test). drugs of atc class n (nervous system) were responsible for the highest number of pims. conclusion: pims can be detected and reduced in the hospital using the rasp list. a structured and collaborative medication review between (student) pharmacists and physicians appears a good approach to reduce the number of potentially inadequate drugs. nevertheless, more research is necessary to substantiate this further as well as to assess the clinical impact of the findings. hp-pc150: impact of implementing ward based dispensaries across a hospital site on both service delivery and patient care michelle sullivan, paul wright, christopher watson, malcolm smith, sotiris antoniou * please specify your abstract type: descriptive abstract (for projects) background and objective: waiting for medication at discharge is often quoted as a key factor for delaying patients leaving hospital. feedback from service users (patients and healthcare professionals) was for a more patient facing pharmacy service. this led to a phased installation of remote dispensaries on wards within the hospital to supply medicines. this new and innovative service enabled the supply function to be fully co-ordinated on the ward. this model was initially implemented on 8 wards, which coupled with one-stop dispensing meant 91% of discharges require nothing to be supplied at the point of validation, 100% of discharge prescriptions meeting key performance indicator of being dispensed and ready within 1 h with average turnaround time of 18 min for a discharge prescription and a reduction in missed doses-3.1% in september 2015 to 2.1% in march 2016. this success prompted further installation of remote dispensaries in all clinical areas on site. design: implementation included; purchasing hardware, pharmacy labellers, locating appropriate computer terminals and stock cupboards. the main pharmacy labelling and stock control system was fully integrated at ward level, enabling the automatic reordering of replacement stock. identification of items and quantities to stock for remote dispensaries was also needed prior to role out. there was a need to scope staffing requirements including the redeploying of roles from a main inpatient pharmacy to patient facing areas. results: over 6000 items are supplied at ward level each month via satellite pharmacies for all wards, equating to more than 80% of the total dispensing workload for the site allowing for pharmacy staff to be redistributed from dispensary to the ward. this offered the benefit of being more patient facing and supporting other initiatives such as patient counselling and medicines reconciliation. the project has impacted the pharmacists as it has enabled them to focus on clinical aspects of service delivery, including attendance of ward rounds as well as supporting a ward team approach with the pharmacy technician. results of missed dose audit from june 2016 shows across the site 41% (7) wards scored below the national 1.4% target and (29%) 5 wards had no unintentional missed doses. conclusion: ward based dispensing has led to pharmacists and pharmacy technicians being 100% ward based. as a constant presence on the ward, the team offer consistency within the pharmacy service for patients, nursing and medical staff. impact of pre-discharge planning has been beneficial to nurses, patients and work flow of the pharmacy teams. ward based dispensing has improved supply at discharge as well as promoting a more patient facing pharmacy service that has seen the pharmacy team instilled as integral to service delivery at ward level. kutay demirkan * , nursel surmelioglu, aygin bayraktar-ekincioglu clinical pharmacy, hacettepe university, ankara, turkey please specify your abstract type: research abstract background and objective: hacettepe university hospitals clinical pharmacy unit was established in april 2014. this unit runs its services by clinical pharmacy postgraduate students under the supervision of two qualified clinical pharmacists as part-time and oncall basis, in adults, paediatrics and oncology hospitals. the aim of this study was to identify drug related problems and describe its management strategies in inpatient and outpatient settings by pharmacists in clinical pharmacy postgraduate education program. setting and method: during a total of 9 months study period (period i: february-july 2015, and period ii: november-february 2016), clinical pharmacy postgraduate students followed patients for 2-3 times in a week in different services in hospitals (internal medicine, internal medicine intensive care, infectious diseases, neurology intensive care, paediatric bone marrow transplant/haematology unit, paediatric intensive care, geriatrics and nutrition units) and drug related problems were identified and pharmacists' recommendations were listed. main outcome measures: determination and evaluation of drug related problems by pharmacist in hospital. results: a total of 114 recommendations was provided for 93 patients. those recommendations were classified as alteration or discontinuation of drug treatment (33.3%), dose adjustment (31.6%), change in drug administration time (14.9%), inadequate treatment (7.9%), healthcare staff training/consulting (6.1%), patient education (5.3%) and error/deficit in therapeutic drug monitoring (1.7%). a majority of recommendations (n = 38) were related with alteration or discontinuation of drug treatment provided mainly in departments of internal medicine (n = 9, geriatrics (n = 7), neurology intensive care (n = 5) and infectious diseases service (n = 5). the following main reason for pharmacist's recommendation was related with dose adjustment (n = 36) which were provided in departments of internal intensive care (n = 16), infectious diseases service (n = 6), neurology (n = 5) and internal medicine (n = 5). conclusion: clinical pharmacy practices are being carried out effectively in many services, particularly in internal medicine services, internal medicine intensive care unit and infectious diseases services. a collaborative and bed-side education in postgraduate programs in clinical pharmacy help to increase the knowledge and skills of students in real life circumstances and also maintain safe and effective drug therapy by an involvement of clinical pharmacists in hospital services. hp-pc152: development of a tool to help pharmaceutical analysis in patients with hepatic failure barbara troussier *,1 , eric gautier 1 , astrid bacle 1 , florian charier 2 , christine silvain 3 , pauline lazaro 1 1 pharmacy, 2 gastroenterology, 3 hepatology and gastroenterology, university hospital of poitiers, france, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: hepatic impairment can cause significant changes in the pharmacokinetics of many medicines. however hospital pharmacists can be helpless in performing pharmaceutical analysis behind the lack of precise guidelines. we need a strategy to first detect accurately patients with hepatic impairment, then lead us in dose adjustments. the objectives of this project were to develop a helping tool for hospital pharmacists in the pharmaceutical analysis of patients with hepatic failure's prescription and to select relevant pharmacist interventions. design: we first planned an investigation of patients with hepatic failure's management, with multidisciplinary experts groups. the study was conducted during one week in post-surgical, gastro-enterology, endocrinology, cardiology, pulmonology, geriatric departments and reanimation care units. a flowchart based on hepatic's biomarkers helped us including patients. criteria used to assess hepatic impairment could be: a stage c child-pugh score, prothrombin score inferior to 70%, bilirubin superior to 50 micrograms per millilitres of blood without haemolysis, aspartate and alanin aminotransferases superior to three times the high normal value, and presence of a vitamin k antagonist interfering with those results. after a review of each included patient's prescription, we checked the major pharmacokinetic elimination pathway of each prescribed molecule (biliary or renal) and if hepatic biotransformation was expected. we also checked if the molecule could cause hepatic side effects. results: out of 474 patients, 15 patients were included for liver failure (3.2%) and 4 for a cholestasis (0.8%) mainly in reanimation care units (17.5%) and gastro-enterology (10%). among the 232 lines of prescribed medicines, the main pharmacological classes encountered were cardiology, (5%) pain (5%), psychiatry (4%), haemostasis (2%) and antibiotics (1%). at the end of the investigation, the expert group decided on the relevant pharmacist interventions. these were based on dose adjustment of anti-infectious, psychotropic drugs, painkillers, oral anti-diabetics, anti-coagulants and corticosteroids. alternatives are proposed for each class. conclusion: to conduct a better pharmaceutical analysis, 3 steps are necessary. first, any liver failure or cirrhosis must be detected thanks to the patient's biological results and medical record. then the patient's prescription can be analysed in order to highlight drugs that need a dose adjustment in a context of hepatic impairment. finally, the physicist and the pharmacist discuss about dose adjustments or alternatives if presence of contraindication with the drugs prescribed. soon the designed tool will be available to all pharmacists to harmonize clinical pharmacy practices. please specify your abstract type: research abstract background and objective: data regarding adherence rates to oral chemotherapy in lymphoma patients is limited. the aim was to assess pharmacist intervention on adherence to oral chemotherapy in patients suffering from hodgkin's (hl) and non-hodgkin's lymphoma (nhl). setting and method: following ethics approval, 5 hl and 41 nhl patients attending chemotherapy sessions at the medical investigations and treatment (mitu) at mater dei hospital accepted to participate. a questionnaire was compiled to evaluate adherence to oral chemotherapy and to assess pharmacist intervention. the questionnaire was divided into 3 sections (a-c). the same questionnaire was used for both the first interview (t = 0) and after 6 weeks (t = 1). an additional section (d) was incorporated at t = 1 to evaluate pharmacist intervention. section a consisted of questions regarding patient management of lymphoma. section b incorporated the morisky 8-item medication adherence scale (mmas-8) 1 to evaluate adherence to oral chemotherapy. a total mmas-8 score of zero indicates high adherence, a score between 1 and 2 indicates medium adherence and a score between 3 and 8 indicates low adherence. section c consisted of additional questions regarding medication adherence. between t = 0 and t = 1, pharmacist intervention involved providing each patient with an information leaflet which was developed in this study, an individualised treatment chart and verbal advice. ibm ò spss version 22 and the wilcoxon signed-rank test were used to assess changes in medication adherence between t = 0 and t = 1. main outcome measures: evaluation of pharmacist intervention on adherence to oral chemotherapy in patients suffering from hl and nhl. results: out of the 5 patients with hl at t = 0, 3 'never' missed a dose, 1 missed a dose 'once in a while' and 1 'sometimes' missed a dose. for the 41 patients with nhl at t = 0, 38 'never' missed a dose, 2 missed a dose 'once in a while' and 1 'sometimes' missed a dose. the reason for missing a dose was forgetfulness. all 41 nhl and 5 hl patients indicated the haematologist as their source of information about the management of lymphoma. of the 41 nhl patients, 3 scored low adherence and 38 scored medium adherence at t = 0 and after 6 weeks (t = 1) all 41 nhl patients who participated scored medium adherence. of the 5 hl patients, 2 scored low adherence and 3 scored medium adherence in the first interview (t = 0) and after 6 weeks (t = 1) all 5 hl patients who participated scored medium adherence. there was a statistically significant increase (p \ 0.05) in the number of patients who scored medium medication adherence between t = 0 and t = 1 for both nhl and hl patients. conclusion: this study shows how pharmacist intervention and extended professional services could be implemented in the clinical setting to impact on the management of hl and nhl patients. please specify your abstract type: descriptive abstract (for projects) background and objective: in may 2015, an activity of medication reconciliation was implemented in the gastroenterology service to carry on the optimization of the medication care of patients due to the recent computerization of their prescriptions. design: this project, worked in collaboration with the gastroenterology service has been introduced in two medical committees. this activity gathers pharmacy students, the pharmacist, senior and junior doctors. reconciled patients are selected according to several criteria (advanced age, poly pathological, poly-medicated and those for whom a drug background is difficult to retrieve for the medical team). a minimum of 3 information sources is used for the collection of the drug background. all information are synthetized on a paper, validated by the pharmacist and discussed again with the prescriber. results: on a 10-month period, 61 patients were reconciled with on average age of 72. the reconciliation is executed on average 2.4 days after the entry in the service. 96.7% of reconciliations are retroactive. the main sources of information used for the collection of the drug background are: in 85.2% of the cases an oral interview with the patient and/or the family; in 82% of the cases the prescriptions, the hometown pharmacist (78.7%) and a medical letter (67.2%). 6.7 drugs are on average on the hospital prescription, and 47.5% (29/61) of the patients are concerned with at least one non intentional divergence (nid). on average there are 1.2 nid/patient and 3.9 intentional divergences (id)/patient. the main types of nid are omissions (44.7%), drug dose errors (19.7%) and errors in administration frequency (11.8%). after the detection of nid, the proposed modifications to the prescribers are accepted in more than 50% of the cases (31/61). the average time of a reconciliation is 49 min. exchanges on the id and nid are made with the junior doctors in 85.2% of the cases. conclusion: some nid are occurring for 47.5% of the reconciled patients. it is therefore necessary to extend this new activity to reconciliation in other services in order to increase the interception of eventual medication mistakes and allow their correction. please specify your abstract type: research abstract background and objective: diuretic therapy is routinely used in the management of congestive heart failure (chf).,compliance with clinical practice guidelines is reported to result in improved outcomes for patients with chf such as reduced exacerbations. the aim was to assess the effect of pharmacist intervention on adherence to diuretic treatment in a hospital and community pharmacy scenario. setting and method: the study was undertaken at karin grech hospital (kgh), a geriatric and rehabilitation hospital, and in one community pharmacy. inclusion criteria for patients recruited from kgh were age over 60 years, suffering from chf and on bumetanide therapy. the validated 8-item morisky medication adherence scale (mmas-8) 1 was administered to patients on admission (t = 0), repeated after two weeks hospital stay (t = 1) and again one-month post-discharge (t = 2). a total mmas-8 score of zero indicates high adherence, a score between 1 and 2 indicates medium adherence and a score between 3 and 8 indicates low adherence. in the community setting patients on diuretic therapy were chosen by convenience sampling. the same adherence scale was administered prior to pharmacist intervention (t = 0) and one-month after pharmacist intervention (t = 1). pharmacist intervention in the community setting involved dissemination of an informative leaflet regarding chf and diuretic therapy developed for the purpose of this study. main outcome measures: impact of pharmacist intervention on adherence to diuretic therapy in chf patients. results: a total of 37 patients were recruited from the hospital setting, of whom 19 were female and 18 were male with a mean age of 80 years (range 67-97 years). on admission (t = 0), 16 patients scored high adherence, 11 scored medium adherence and 10 scored low adherence to bumetanide therapy. following 2 weeks at the hospital (t = 1), the number of patients scoring high adherence increased from 16 to 31 and the number of patients scoring low adherence decreased from 10 to 1. one-month post-discharge (t = 2), patients scoring high adherence decreased from 31 to 19 and patients scoring low adherence increased from 1 to 3 (p \ 0.05). a total of 38 patients were recruited from the community pharmacy, of whom 21 were female and 17 were male, with a mean age of 79 years (range 68-97 years). after pharmacist intervention (t = 1), the number of patients scoring high adherence increased from 21 to 23, while the patients scoring low adherence decreased from 9 to 5 (p [ 0.05). conclusion: pharmacist intervention in the hospital setting improved adherence to bumetanide therapy. in the community pharmacy setting, there was a slight improvement in the compliance. pharmacist monitoring and patient support is important post-discharge to ensure patient compliance to therapy. conclusion: surveillance of aeds may be followed by combination of data from adverse drug reaction databases and drug utilisation data from prescription databases. focus on reporting adverse reactions is important for pharmacists and clinicians, especially for newly approved drugs. awareness of increased exposure of aeds to new groups of patients followed by data regarding safety aspects is important and contributes to improved pharmacovigilance. please specify your abstract type: research abstract background and objective: the medication review of polymedicated patients is a priority shared among all healthcare professionals. a multidisciplinary approach of these patients is necessary to achieve the best results for their treatment (1) . the objective was to analyse the rate of acceptance of the recommendations made by the primary care pharmacist (pcp) to the general practitioner (gp) regarding the treatment of polymedicated patients. setting and method: setting: a primary health care centre (27,521 population). method: a review of the medical records of polymedicated patients (c5 chronic drugs for c6 months). the patients' data were collected from january to june 2015 from their clinical records. statistical descriptive analysis of data was performed. main outcome measures: drug related problems (drp) for each patient: interactions, contraindications, inadequate dosages, nonindicated drugs, omission of a necessary drug, duplications, medication with low therapeutic effect, and inappropriate medication for patients c75 years old. treatment alternatives proposed to gp's by pcp were also measured. results: 34 patients were included in the study (average age: 68.6 ± 11.7, 74% women). out of the 34 patients, 88 interventions were laid out to reduce the risks of drp's and to improve the efficiency of treatments. 97% of patients presented some drp or some intervention to improve the efficiency of their treatment, this mean an average 2.5 interventions for patient. the prevalence of intervention proposals were: non-indicated drugs (28%), interventions for improve the efficiency of treatments (20%), interactions (16%), inappropriate medication for patients c75 years old (10%), contraindicated drugs (9%), duplications (6%), medication with low therapeutic effect (5%), inadequate dosages (5%) and omission of a necessary drug (1%). 97% of these intervention proposals were accepted by the gp: 38% of the accepted proposals were carried out and from the remaining 62, 43.6% led to a prescription from a specialist physician. in 51% of the cases, the patient did not accept the changes. 5.4% were not carried out due to other issues. the main drug related problem was the prescription of non-indicated drugs and the most involved drug was omeprazole. conclusion: acceptance by gp's to changes proposed by primary care pharmacists was high. a significant number of changes was not accomplished due to the negative response by some patients and led prescriptions from a specialist physician. the gp greatly values the multidisciplinary aid in approaching the complexity of polymedicated patients. background and objective: case-reports provided evidence that influenza infections, particularly severe episodes, may exert neuronal damage in the cns and thereby increase the risk of depression. it was the aim of this study to analyse the association between influenza infections and the risk of developing incident depression. setting and method: we conducted a case-control analysis using the large uk-based primary care database clinical practice research datalink (cprd). this database contains anonymous longitudinal data from primary care. at present, it contains over 100 million person-years of data from some 10million active patients. the study encompassed 103,307 patients below the age of 80 years with an incident major depression diagnosis between 2000 and 2013, and we matched each case to one control patient on age, sex, general practice, number of medical encounters, and years of history in the cprd prior to the index date. main outcome measures: major depression diagnosis was identified by read-codes based on icd-10 codes (f32), with a minimum of three prescriptions for antidepressant drugs recorded after the diagnosis. we calculated relative risk estimates of developing depression in association with previous influenza infections, stratified by the number, timing and severity of such events, and we adjusted for a variety of comorbidities, smoking status, alcohol intake, body mass index, use of oral corticosteroids, and benzodiazepines. results: patients with a previous influenza infection had an increased risk of developing depression (or 1.30, 95% ci 1.25-1.34) compared to patients with no history of influenza infections. a recent influenza infection recorded within 30-180 days prior to the index date yielded an adjusted or of 1.57 (95% ci 1.36-1.81), and an increasing number of previous influenza infections was associated with increasing odds ratios (c3 recorded influenza infections, adjusted or 1.48, 95% ci 1.22-1.81). we did not see any differences in the relative depression risk associated with influenza with regard to a previous influenza vaccination. conclusion: this study suggests that influenza infections are associated with a moderately increased risk of developing depression. please specify your abstract type: research abstract background and objective: warfarin is known for its interactions with many drugs. elderly patients are particularly sensitive to warfarin interactions. to evaluate the incidence of potential drug interactions when prescribing new drugs to elderly patients on warfarin, a prospective observational study was conducted. setting and method: patients on warfarin older than 65 years were included and monitored for 6 months in 4 community pharmacies in croatia. data regarding new prescribed drugs was obtained from pharmacy records at the moment of dispensing or by patient selfreporting. the potential interacting drugs were identified using the lexicomp ò lexi-interact online software. only the clinically significant (levels c, d, x of clinical significance as classified by lexicomp ò lexi-interact online) interactions were included in this analysis. main outcome measures: number of new proscribed drugs, level of interaction with warfarin, mechanism of interactions. results: we included 157 elderly patients with an average age of 73 years. in the follow-up period, new drugs were prescribed to 54 patients (34.4%). there were 79 prescriptions of new drugs and 57 (72.2%) of those were drugs with a clinically significant interaction with warfarin. there were 39 prescriptions of drugs with level c of interaction (68.4%), and 18 (31.6%) with level d. there were no drug interactions of level x. in the group with level c the most prescribed drugs were antibiotics with 26 prescriptions: amoxicillin/clavulanate 28%, clindamycin 8%, ciprofloxacin 8%, norfloxacin 8%, azithromycin 5%, cefuroxime 5%, clarithromycin 3%, doxycycline 3%. the remaining 13 prescriptions included tramadol with paracetamol 18%, rosuvastatin 5%, simvastatin 3%, fluvastatin 3%, levothyroxine 3% and torasemide 3%. the dominant mechanism of the potential interactions was pharmacokinetic. in the group with level d the most prescribed drugs were nonsteroidal anti-inflammatory drugs with 12 prescriptions-diclofenac 35%, ibuprofen 23%, indomethacin 12%. among other drugs, 6 prescriptions were antibiotic sulfamethoxazole with trimethoprim 12%, fenofibrate 6%, miconazole 6%, and fluconazole 6%. the dominant mechanism of the potential interactions was pharmacodynamic. conclusion: pharmacists should actively monitor prescribing of new drugs to elderly patients on warfarin in order to reduce the risk of clinically significant drug interactions. please specify your abstract type: research abstract background and objective: explicit criteria of potentially inappropriate medications in the elderly (pims) have been published in the usa, canada, australia and many eu countries. there is a lack of studies describing prevalence of pim use in central and eastern europe. the aim of the eu cost action 1402 initiative wg1b (2015 wg1b ( -2018 is to evaluate the registration rates and use of pims in central and eastern europe compared to other eu countries participating in this initiative. this abstract describes preliminary findings on different registration rates of pims in different eu countries. setting and method: researchers/members of the eu cost action 1402 initiative from the czech republic, serbia, hungary, spain, turkey and portugal were asked to fill in evaluation tables for the list of 484 pims in the period 01-06/2016. items available in these evaluation tables related to: registration of individual pims on the pharmaceutical market, registered doses, drug forms, availability of pims on prescription or as otc drugs, prescription limits and the most frequently used brand names. data were evaluated using comparative descriptive statistics. main outcome measures: overall prevalence of registered pims in different countries, cross-country differences in availability of individual pims. results: of 484 pims 81.8% were registered in at least 1 participating country. for the czech republic (45.2%), turkey (48.6%), spain (52.1%) and hungary (54.1%) overall prevalence rates of registered pims were found to be similar. however, these prevalence rates substantially differed in serbia (low prevalence-33.9%) and portugal (high prevalence-68.5%). substantial differences were found also in the lists of individual pims registered in different countries. these lists were similar in spain and portugal compared to the czech republic, hungary, serbia and to turkey. conclusion: although overall prevalence rates of registered pims were similar in the majority of evaluated countries (except serbia and portugal), availability of individual pims was substantially different. our pilot results confirmed that there are substantial geographical/ regional differences in europe in the lists of pims available (in spain and portugal compared to central and eastern europe and compared to turkey). please specify your abstract type: research abstract background and objective: inappropriate prescribing is a common circumstance found in polymedicated patients. screening tools for identifying potentially inappropriate prescription (pip) and pharmacist interventions for evaluating them have been developed to decrease this (1) . the aim of this study was to evaluate the effectiveness of a pharmacist provided intervention to reduce pips in polymedicated patients. setting and method: the design was a quasi-experimental study focusing on a single group before and after intervention. the study took place from july to december of 2015 at three primary care centres (52,992 population). polymedicated patients were those using c10 chronic drugs for c6 months. main outcome measures: reduction in the rate of pip per polymedicated patient (number of pips found divided by the total number of polymedicated patients) before and after intervention, and the influence of the following variables: type of pip (inappropriate medication for patients c75 years old, medication with low therapeutic effect, duplication of benzodiazepines (bzd) or angiotensinconverting enzyme (ace) inhibitors, combination of anticoagulant and antiplatelet, combination of non-steroidal anti-inflammatory drug (nsaid) with a diuretic and ace inhibitor, nsaid in cardiovascular disease, chronic antipsychotic in dementia, chronic bzd, or chronic nsaid), gender and age of patients with at least one pip, and the main prescribed drugs involved in the pips based on atc classification system of world health organization. results: there were 1093 and 959 polymedicated patients before and after intervention, respectively. 71.36% (n = 780, before) and 68.30% (n = 655, after) of the total patients had at least one pip. the number of pips was reduced from 1373 to 1108, while the rate of pip per polymedicated patient decreased from 1.26 to 1.15, achieving the limit established by the regional health authority. 50.90% (before) and 48.70% (after) of patients had more than one pip at the same time, up to 5 pips per patient. before and after intervention, more than half of patients with at least one pip were c75 years old, and approximately 9 out of 10 were c65 years old. also before and after intervention, 8 out of 10 patients with chronic nsaid and with bzd duplication were women. 6 out of 10 patients with combination of anticoagulant and antiplatelet were men. the main pips before and after intervention were, respectively: chronic prescription of bzd (39.77 vs. 37.99% of the total pip), medications with low therapeutic effect (19.81 vs. 21.30%) and inappropriate medication for patients c75 years old (16.82 vs. 17.42%). the main atc group involved in the total of pips was drugs for the nervous system, and the five most prescribed drugs were all bzd (lorazepam being the first). conclusion: pharmacist provided intervention was able to reduce pip in polymedicated patients. gender, age and atc classification of drugs involved were factors in the pips. please specify your abstract type: research abstract background and objective: up to 10% of women are exposed to selective serotonin reuptake inhibitors (ssris) during pregnancy. information on their effect on birthweight and gestational age remains conflicting. the aim of this sibling-controlled prospective cohort study is to address shared genetic and family-level confounding to investigate the effects of prenatal ssri exposure and maternal depression on birthweight and gestational age. setting and method: we used the norwegian mother and child cohort study (moba) and the medical birth registry of norway (mbrn). our study population consisted of 27 756 siblings; 194 were prenatally exposed to ssris and 27 500 were unexposed to any antidepressant medication. random and fixed effects analysis with propensity score adjustment was used to evaluate the effects on birthweight and gestational age. main outcome measures: birth weight. gestational age. results: ssri exposure during two or more trimesters was associated with a decrease in birthweight of 205 g [95% confidence interval (ci) 372 to38] and a decrease in gestational length of 4.9 days (95% ci9.1 to1.4). neither maternal ssri use in one trimester, lifetime history of major depression nor depressive symptoms during pregnancy were associated with these pregnancy outcomes. conclusion: prenatal exposure to ssris during two or more trimesters may decrease birthweight and gestational length. our results indicate that neither maternal depression nor shared genetics and family environment fully explain this association. please specify your abstract type: research abstract background and objective: the drugs burden index (dbi) is a tool to evaluate the burden of medications with anticholinergic and sedative effects and this exposure has been associated with poorer physical and cognitive function in older people. objectives were; to determine the cumulative burden of anticholinergic and sedative medicines in older adults with intellectual disability (id) using the dbi, to examine the relationship between dbi score with demographics and comorbidity. setting and method: data from wave 2 of the intellectual disability supplement to the irish longitudinal study on ageing (ids-tilda), a nationally representative study of ageing people with id in ireland. dbi scores were calculated for all participants with available medication data (n = 677). bivariate associations between dbi and demographic and clinical characteristics were examined with a significance level of 0.05 main outcome measures: dbi scores of participants categorised into low (0), medium (0-1) and high (c1). dbi score categories were related to demographics, cognitive effects and to a modified functional comorbidity index (fci), which is associated with physical function in older adults. results: of 677 participants, 95.1% (644) had dbi exposure; 51.3% were exposed to any anticholinergic medication, 32.1% to any sedative medication; mean number of dbi medications 2.91 (±1.68), mean dbi score: 1.30 (±1.24). 145 (21.4%) participants had dbi score 0, 165 (24.4%) 0-1, and 367 (54.2%) c1. antiepileptics accounted for the greatest contribution to cumulative score (27.6%), antipsychotics (25%) and antidepressants (14%). there was no significant association between higher dbi score and sleep difficulties (p = 0.135). there was a significant age gradient associated with higher dbi score (p = 0.016) and significant association between higher scores and increased comorbidity scores; mean fci of 3.28 in those with dbi c 1, 2.73 in dbi 0-1 and 2.35 in those with dbi 0. conclusion: cumulative exposure to sedative and anticholinergic medicines was high in older adults with id. higher dbi scores were associated with higher comorbidity and associated poorer physical function. optimising use of medications with anticholinergic and sedative effects through medicines review by pharmacists as part of multidisciplinary teams using a tool such as the drug burden index may reduce functional decline and improve quality of life among older adults with id. please specify your abstract type: research abstract background and objective: poor adherence to pharmacotherapy may have considerable consequences for the patients' health and for healthcare costs to society. there was observed that diabetes patients have higher risk of later health complications development. it is necessary to be adherent to non-and pharmacological recommendations as well, to improve the clinical outcomes and decrease the cardiovascular risk (cvr). the aim of this study was to evaluate the medication adherence and cvr in group of patients with diabetes, and to find an association between them. setting and method: the methods were based on a questionnaire survey using a modified 8-item morisky score and score charts (2012). medication adherence and cvr were evaluated in the whole group (n = 107, 51 males and 56 females, range 22-86 years) as well as in subgroups according to age, gender, (no-/ex-) smoking, level of education, residence, number of used medicines, exercises, compliance to the diabetic diet, and total cholesterol levels. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava -ruzinov. all participants signed an informed consent. main outcome measures: the results of medication adherence were evaluated as follows: 8 points = full adherence, 6-7 points = partial adherence and 0-5 = non-adherence. the cvr (estimating 10-year cardiovascular attack risk) was evaluated according to score charts using data from questionnaire and medical records-gender, age, smoking, total cholesterol levels and blood pressure. the results showed a partial medication adherence in the study group in average (6.84 ± 0.28). the average value of cvr in the study group was 3.7%. the highest average medication adherence has been observed in males b65 years (7.03), with elementary education (7.0), in ex-smokers (7.1), in patients with regular physical activity-at least 3 times a week (7.29), in patients non-adherent to the diabetic diet (7.05), in patients using 2 medications (7.11), and in patients with satisfactory (4.5-5.0 mmol/l) total cholesterol levels (6.97). the lowest cvr has been observed in females b65 years (1.8%), in no-smokers (3.2%), with elementary education (3.0%), in patients with irregular physical activity (2.9%), in patients adherent to the diabetic diet (3.14) , in patients using 4 medications (2.9%) and in patients with satisfactory (4.5-5.0 mmol/l) total cholesterol levels (2.97) . on the other hand, the highest cvr has been observed in males [65 years (7.3%), smokers (5.9%), secondary educated patients (3.8%), without any physical activity (4.7%), in patients partially adherent to diabetic diet (4.7%), using 6 medications (4.1%) and, surprisingly, in patients with satisfactory (\4.5 mmol/l) total cholesterol levels (4.76%). conclusion: our survey has showed that medication adherence in our study group has been decreased and cvr has been increased. cvr and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, cvr and their relationships are specific in every patient. please specify your abstract type: research abstract background and objective: studies show that quality of life (qol) of patients with diabetes mellitus can influence medication adherence, satisfactorily improving clinical outcomes and reducing the morbidity and mortality rates and disease progression. this applies even upside down-medication adherence could significant contribute to improving patient qol. the aim of this study was to evaluate the medication adherence in group of patients with diabetes, to evaluate their qol and find a correlation between them. setting and method: the methodology was based on a questionnaire survey using a modified 8-item morisky score and questionnaire eq-5d-5l, including visual analogue scale (vas). medication adherence and qol were evaluated in the whole group (n = 107) as well as in subgroups according to age, gender, level of education, monthly income, number of used medicines and type antidiabetic treatment. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava-ruzinov. main outcome measures: the results of medication adherence were evaluated as follows: 8 points = full adherence, 6-7 points = partial adherence and 0-5 = non-adherence. the qol in 5 levels of 5 dimensions results were evaluated as follows: the lowest qol in every dimension = 1 point, the highest = 5 points. the highest vas evaluation has been 100 points and every patient should mark number on the scale 0-100 to indicate his/her health on current day. results: the results showed a partial medication adherence in the whole group in average (6.84 ± 0.28). the average value of the qol in the study group was 21.21 and vas 69.29. the highest medication adherence has been observed in males (7.04 ± 1.29), patients \40 years old (7.0 ± 1.05), with primary education (7.07 ± 1.04), with monthly income over 600€ (7.38 ± 0.99) and in patients using 2 medications (7.11 ± 1.6). the highest qol and vas (qol; vas) has been observed in males (22.0; 74.24), patients \40 years old (23.22; 77.22), university educated (23.11; 75.55) , with monthly income over 600€ (22.75; 75.63) . qol has been highest in patients using 3 medications (23.92), vas has been highest in patients using 1 medication (83.33). we have observed the highest level of medication adherence in patients treated with combined therapy-with oral antidiabetic agents and insulin (7.06), the lowest in patients treated with only insulin therapy (6.95). highest qol was recorded in patients treated with oral antidiabetic agents (22.04), and the lowest qol in patients with insulin therapy (19.97). the highest vas has been observed in patients using only oral antidiabetic agents (72.45), the lowest in patients using combined therapy (62.06). conclusion: survey has showed that medication adherence and qol in our study group has been decreased. qol and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, qol and their relationships are specific in every patient. the role of health care professionals should be in education and counselling with patients to improve qol and medication adherence as well. please specify your abstract type: research abstract background and objective: to assess the appropriateness of antibiotic prescriptions used for urinary tract infections (uti) in the elderly. setting and method: we included patients aged 70 years and older, hospitalized in the geriatric department and for whom a urine culture was performed between march and may 2016. a prescription was qualified as inappropriate: when the antibiotic prescribed was not the narrowest compared to the culture result, or when there was a contra-indication, or when the treatment duration was shorter or longer than recommended. prescriptions were consistent with the guidelines when they were identical to those adopted by the french society for infectious diseases in december 2015. main outcome measures: appropriateness of antibiotic prescription (type and duration) results: 47 elderly patients were included (women: 74.5% (n = 35), mean age: 85.9 years). 68% of antibiotic choices were appropriate and 64% of treatment durations were consistent to the guidelines. urinary clinical signs were mentioned in the medical files for 29.8% of the cases (n = 14). 28 patients received an empirical antibiotherapy (59.6%). 70.2% (n = 33) of urine cultures were positive with bacteria, escherichia coli being the most prevalent (n = 18). the urine culture results led to a change in antibiotics for 66.7% of the cases. for cystitis, 53.8% of the antibiotics chosen were appropriate (n = 7). the main reasons of non-conformity were the lack of deescalation (to amoxicillin or pivmecillinam), and the prescription of ciprofloxacin when the bacteria was in vitro resistant to other fluoroquinolones. the average duration of effective antibiotherapy for cystitis was 9.3 days (appropriateness: 53.8% (n = 7)). for pyelonephritis, 88.9% of the antibiotics chosen were appropriate (n = 8). the average duration of effective antibiotic treatment was 10.1 days (appropriateness: 77.8% (n = 7)). 40.4% of the patients had a transurethral catheterization (n = 19). another infection was diagnosed for 48.9% of the patients (n = 23). conclusion: according to these results, it appears important to reemphasize to the prescribers the guidelines around the uti diagnosis and treatment in order to improve the prescriptions appropriateness in elderly patients. it is particularly necessary to promote the de-escalation of antibiotherapy (with pivmecillinam for example which has recently become available in our hospital) and to insist about the recommended durations of treatment. please specify your abstract type: research abstract background and objective: to measure the use of potentially inappropriate medications (pim) in the general elderly population several criteria lists exist, e.g., beers criteria. last year, a set of explicit criteria for assessing pharmacologically inappropriate medication use in nursing homes was developed; the norwegian general practice-nursing home criteria (norgep-nh). the aim of this study was to investigate the prevalence of pims in nursing home patients using this new assessment tool. furthermore, we studied possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. setting and method: cross-sectional study comprising 103 nursing home patients from two geographical different regions in norway; tromsø city (n = 70) and lofoten islands (n = 33). data was collected from november 2015 to january 2016. pims were identified by norgep-nh. we used logistic and poisson regression to examine possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. main outcome measures: number of pims per patient, and odds ratios (or) and marginal effects for associations. results: nursing home patient used a mean (sd) of 10.9 (4.3) drugs; 7.2 (3.6) regularly and 3.7 (1.9) as needed. at least 69% of patients used one pim. concomitant use of three or more psychotropic drugs was the criterion most commonly identified (33%), followed by the use of antidepressant (26%) and hypnotics (23%). an increasing number of regularly used drugs increased the odds of having pims (or: 1.74), as well as it lead to 0.18 more pims per extra drug used. on average, patients c80 years had 0.46 fewer pims than patients \80 years. no statistical significant associations were seen between having pims and gender, nor geographical area and the use of as-needed medication. yet, statistical significant differences were identified in some criteria. conclusion: this is the first study that explicit uses norgep-nh. our results confirm that nursing home patients often use potentially inappropriate medications. this is an area where further work is necessary, not to measure the prevalence of pim, but to develop interventions in order to prevent pims from being used. pe020: use of pharmacy dispensing data to measure adherence and identify nonadherence with oral hypoglycaemic agents please specify your abstract type: research abstract background and objective: a framework for calculation of adherence for oral hypoglycaemic agents (ohas) based on data from health-insurance claims is available. pharmacy dispensing data aid identification of nonadherent patients in pharmacy practices. however, use of these data for calculation of oha adherence requires additional methodological categories. we examined the impact of different methodological choices on estimation of oha adherence using pharmacy dispensing data. setting and method: a framework for adherence calculation for pharmacy dispensing data was developed from health-insurance claims. a basic scenario was developed from 16 methodological categories. consequences of choices for different parameters within these categories on the scores of the three adherence measures were calculated from dispensing data. main outcome measures: for oha use between july 2013 and july 2014, three adherence measures were calculated: (1) average medication availability (ama); (2) mean rate of adherent patients with an ama c80% (mrap80); (3) please specify your abstract type: research abstract background and objective: ulcerative colitis (uc) is a chronic inflammatory disease usually affecting young adults and impacting on patient's quality of life. although many biological agents (bas) have been approved for the treatment of moderate-to-severe uc in patients who have responded inadequately to conventional therapy, the selection of bas is controversial due to the lack of head-to-head trials. indirect economic comparisons of these costly drugs are available from national healthcare perspectives that are not the italian ones. therefore, the objective is to evaluate cost-utility of bas for the treatment of refractory moderate-to-severe uc both in italy and in the lombardy region. setting and method: a markov model (considering 3 transition states: remission, clinical response, relapse) was constructed using the software r 3.3.1 markovchain-package to evaluate incremental cost-utility ratios (icur) of adalimumab, infliximab, infliximab biosimilar, golimumab and vedolizumab treatments of patients over a ten-year time horizon from the perspective of the italian (n) and lombardy region (r) healthcare system. clinical parameters were derived from clinical trials. costs (which have been actualised-1.5%) were obtained from the national database and regional public tender. utility was expressed as qaly (quality adjusted life years). main outcome measures: icur. results: costs per treatment were different from a n and r perspective (adalimumab -55%; infliximab -16.7%; infliximab biosimilar -29.6%; golimumab -9.6%; vedolizumab -10%). direct healthcare costs (treatment cost, visits, lab tests, hospital admissions) were calculated over 10 years of treatment per patient: adalimumab (n: €114,226.70, r: €68,314.12, -40.2%), infliximab (n: €130,594.90, r: €103,081.00, -21%), infliximab biosimilar (n: €110,437.80, r: €78,852.03, -28.6%), golimumab (n: €118,602.10, r: €96,922.20, -18.3%), vedolizumab (n: €113,851.80, r: €102,932.20, -9 .6%) with associated qaly respectively of 6.68, 6.66, 6.66, 6.70, 7.02. from a n perspective, infliximab biosimilar was dominating compared to all other treatments. the icur of vedolizumab/infliximab biosimilar was €9483.33 for 10 years (willingness to pay (wtp) €948.33/qaly). from a r perspective, adalimumab was dominating compared to all other treatments. the icur of vedolizumab/adalimumab was €101,817.88 for 10 years (wtp €10,181.78/qaly). conclusion: national and regional cua produced different results. as regional price discounts can occur, local analyses are needed to estimate the economic impact of therapies to ensure optimal choice. please specify your abstract type: research abstract background and objective: automated dispensing systems (ads) have been implemented to reduce overall medication errors related to picking, preparation and administration of drugs. costs of drug storage between ads and classic dispensing system (cds) had not been yet performed in france. our objective was to assess economic impact of ads compared to cds. setting and method: retrospective quasi experimental study was conducted in 2 university hospitals in 2015, one with ads (800 beds, 43 ads) and one with cds (600 beds, 31 cds (17) for ads and 53 (15) for cds (p \ 0.001). mean number of costly drug per system was 3 for ads and 1 for cds. the global stock value in the wards was 205,915€ in ads and 54,908€ in cds representing respectively 14.5 and 6.1% of total pharmacy stock value. conclusion: our data demonstrate that despite the same storage capacity, ads allow the storage of more expensive drugs such as innovative drugs fully reimbursed up to national reimbursement prices, due to the lower risk of pilferage. this preliminary study was focused mainly on stock value. subsequently, another study is conducted to evaluate cost of these two drug storage systems, satisfaction of pharmaceutical technicians and nurses and time allowed for systems reloading. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, pharmacists are not entitled to substitute an original biological drug with its biosimilar, due to specific issues of efficiency, safety, and patient monitoring. our hospital referenced a biosimilar of infliximab on 6 january 2015. according to the french medication safety national agency's recommendations, it has been decided that naïve patients would be treated with biosimilars, and changes between specialties would be proscribed. the objective is to compare prescribing practices between infliximab and its biosimilar, 1 year after its introduction. design: a database tracking patients treated with infliximab was set up. data comparing prescribing practices of biosimilar and reference treatment were analysed between june 2015 and may 2016. regional and national infliximab consumption between january 2015 and february 2016 were used to compare the practices of our hospital with other hospitals. the past and future savings were estimated from repayments data of the regional health agency. results: infliximab was administered to 633 patients, of which 201 (32%) were naive. 111 patients were treated with biosimilar (i.e. 17.5% of all patients), of which 97 were naive. in the end, nearly 48% of naive patients actually received the biosimilar and 2.5% of patients treated with infliximab switched specialties during treatment. in 80% of cases, biosimilar prescriptions were consistent with the recommendations (vs. 94% for infliximab). in 79% of cases the off-label prescriptions of the biosimilar were explained in the patient record (vs. 75% for infliximab). in february 2016, the share of biosimilars was 14% in france, 12% at regional level and 15% locally. in 1 year, infliximab and its biosimilar's consumption in our hospital have increased by 12% in quantity and only 2% in expenditure (+€ 6 m expenditure). negotiating a lower purchase price and costs has enabled the hospital to save € 137,629 (vs. € 182,961 during the previous year). because of the decline of refund rates, the gains would have been zero without using the biosimilar but € 377,172 if it had been prescribed to every naive patient. conclusion: current data from the literature on security and effectiveness of infliximab biosimilars are very reassuring and the french medication safety national agency doesn't exclude the possibility of changing specialties during treatment. in our hospital, there is room to improve the efficiency of treatment with infliximab. feedback on prescribing practices will be given to prescribers and a campaign to widespread prescriptions of biosimilars will be made. the arrival of biosimilars on the market is a real economic opportunity for hospitals, which are increasingly financially constrained in particular by the arrival of therapeutic innovations which are more and more expensive. setting and method: the study used health claims data on prescription ppis from 1st january 2011 to 31st july 2014 obtained from the health insurance institute of slovenia. to assess medicine use and costs before and after trp implementation data were aggregated into four periods: jan-dec 2011, pre-baseline period; jan-dec 2012, baseline period; jan-sept 2013, transition period between announcement and introduction of trp; oct 2013 to jul 2014, period after trp enforcement. main outcome measures: medicine costs; defined daily doses (ddds) dispensed per 1000 inhabitants per day; market share; herfindahl-hirschaman index (hhi); number of active substance switches; number of exceptions when medicine is fully reimbursed since physicians may choose option ''not to switch medicine'' when adverse consequences are predicted. results: average monthly cost of ppis declined from € 1,350,289 in pre-baseline period to € 800,125 in period after trp introduction although the consumption increased from 52.4 to 55.2 ddds/1000 inhabitants/day. cost of ppis decreased the most in baseline period (26%), however trp induced 9.5% cost reduction compared to the transition period. the reference pantoprazole was market leader already in the transition period, but its use increased significantly after trp introduction and represented 51% of total ppis consumption. manufacturers' market shares were constant before trp, whereas trp caused decrease of the largest market share for 5%. still, this resulted in the minor market concentration change; hhi was on average 0.351 before and 0.307 after trp introduction. further, at least one active substance switch was detected in approx. 15 and 21% of patients before and after trp introduction, respectively. similarly, the proportion of exceptions when medicine was fully reimbursed increased from 6.7% in transition period to 23.7% in period after trp introduction. conclusion: enforcement of trp for ppi contributed to approx. € 1 m annual cost savings. from the payer's perspective the new policy was proven to be effective in reducing pharmaceutical expenditure; however trp also affected physician prescribing pattern and use of ppis. pec007: blood coagulation factor: improvements of the supply chain samantha oses * , serri traore, sonia caroline sorli, lea damery, philippe cestac, sylvie pomies, julien tourel please specify your abstract type: descriptive abstract (for projects) background and objective: most of the antihemophilic factor (ahf) must be held by a teaching hospital to face serious bleeding events. to ensure better availability, offsite-stocks at critical points are required (emergency unit, intensive care unit, etc.). however, this management system increases the risk of economic loss and alteration of the quality due to expired products. in this context, we carried out an optimization of the supply and management system of the ahf. to identify critical points of the supply and management system and to implement improvement solutions. design: a multidisciplinary working group belonging to a regional management centre of haemophilia was set up. two lines of improvement were discussed: i) optimization of stocks ii) optimization of the supply system. results: the optimization of stocks has led to the modification of the threshold of the lowest stock (ls) for 19 ahf out of 38. in 70% of cases, this stock modification has exceeded 15%. the overall cost of ls has been reduced by 15.0% (83,000 €) for the general stock at the central hospital pharmacy (hp) and by 8.5% for offsite-stocks (20,000 €). the ahf mainly involved in this reduction was fvii 5 mg (27,000 €), then followed by the strengths of 2 mg and 1 mg (13,000 € for each). in order to improve the ahf management, several propositions have been implemented: (1) developing an online, easily accessible and monthly updated spreadsheet that displayed several accurate data such as the shortest expiry date and the storage location. this operative tool is shared between all pharmacists involved in ahf management in order to facilitate a stock rotation and decrease economic losses, (2) regular reminders to physicians and health care staff concerning the guidelines for inventory management and the importance of checking the drug expiry date, (3) presentation of the financial results and raising awareness on ahf costs to the medical consultant[ppip1] and (4) optimizing stock distribution based on consumption on the different hospital sites for better patient care management (pcm). conclusion: this optimization of stocks and improvement of the supply chain have led to a direct cost saving of 83,000 €. however, a more accurate assessment has to be performed to quantify the direct and indirect impact on pcm and cost saving. this work has been done in a context of a sharing operative network at a regional level. the aim of such project is to share, to optimize and to improve practices, knowledge, human and medical health resources at a widespread level to enhance the security and quality of health services and to promote cost and time saving. please specify your abstract type: descriptive abstract (for projects) background and objective: the overall pharmaceuticals consumption in hospitals is rising, which has led to an increasing expenditure, challenging health care professionals and threatening patients safety. clinical trials in hospitals have increased over the past few years and currently play an important role, giving access to new investigational medicinal products and also avoiding costs with standard treatments. the objective of this study is to evaluate the savings of centro hospitalar do porto, a central university hospital with 800 beds and currently 80 clinical trials, with patients included in clinical trials between january 2013 and may 2016. design: retrospective observational study over 41 months. all the clinical trials ongoing between january 2013 and may 2016 were analysed and the data was collected based on: pathology and doses established; number of treatments per patient and the medium prices of standard treatments that patients would be receiving if they were not in the clinical trial. results: there were 112 clinical trials ongoing between january 2013 and may 2016, but only 30 were selected to be included in this study. the total number of patients included was 652. the clinical trials selected for this study were conducted in 6 medical specialties: 4 in dermatology, 6 in immunology clinical unit, 11 in hemato oncology, 1 in gastroenterology, 5 in ophtalmology and 3 in neurology. during these 41 months, with all ongoing clinical trials, centro hospitalar do porto was able to save, in medical products, more than 2 million euros. conclusion: during the period of time established, 82 of the clinical trials ongoing, were not selected due to: not including patients or not having an alternative treatment. hospitals and patients can benefit from clinical trials not only financially but also by preserving resources and medication. on centro hospitalar do porto, the pharmacists specialized in clinical trials, as members of the study team, are more and more required to perform specific tasks, their contribution has been increasing over the years and also have become more aware of all the advantages from participating in clinical trials. these savings can be used to provide a better assistance and contribute, in general, to a higher quality health care. please specify your abstract type: descriptive abstract (for projects) background and objective: several studies show a misuse of opioid maintenance treatment (omt) in detention. in fact, buprenorphine (bup) when it's misused, could present the same effects as heroine. in order to reduce misuses, the pharmacist decided to switch all the patients under bup to buprenorphine/naloxone (bup/nlx). bup/ nlx prevents patients from misusing by a withdrawal syndrome when it's issued by another route of administration than sublingual route. in france, bup/nlx is more expensive than bup which may explain why this therapeutic strategy is not often observed. the purpose of this study is to evaluate the extra cost after switching patients from bup to bup/nlx in order to decide if this choice could be maintained. design: to identify our population, we used the administration reports drugs written by nurses. please specify your abstract type: research abstract background and objective: haemophilia b is an x linked genetic disorder characterized by spontaneous or prolonged haemorrhages due to factor ix (fix) deficiency 1 . within the next few years, new treatments are willing to hit the market. among them are recombinant extended half-life products that will reduce by half the number of injections and will potentially improve the patient quality of life. the aim of the study is to describe the development of haemophilia treatments market between 2011 and 2014 and to forecast the potential impact of these new therapies on the haemophilia market. setting and method: national and french hospitals of paris (aphp) consumption data of 4 fix between 2011 and 2014 have been studied. new therapies in development or soon to be marketed have been identified. potential benefits and interest in the therapeutic care of these new products were discussed with haemophilia's medical experts. main outcome measures: quantity (ui) and value (euros) of fix aphp and national consumption. results: in 2014, 1 recombinant (rfix) and 3 plasma-derived factors (pfix) were on the french market. the ap-hp's purchases of these 4 factors represent almost 15 million ui and 10 million euros, which comprise 24% of national fix expenditures. in france and aphp, ambulatory care is a major part of the use of these treatments with nearly 90% of the fix purchases in 2014. french rfix consumptions are higher than pfix consumptions (64% against 36%). in the ap-hp hospitals, rfix even account for 89% of consumptions against 11% for pfix. both national and ap-hp rfix purchases have steadily increased between 2011 and 2014. the added competition arising from new treatments may lead to more competitive market procedures in hospitals and may reduce costs of haemophilia treatments. according to haemophilia doctor, long-acting (la) fix would offer obvious benefits like fewer infusions and presumably fewer bleeds. these treatments will mainly be used in a prophylactic wayin ambulatory care-than in a curative way (such as surgical use). conclusion: the therapeutic extent of these new treatments is still hard to define. the choice of treatment must remain consensual between physicians and patients. please specify your abstract type: descriptive abstract (for projects) background and objective: good practice about medicines imposes to health institutions a close monitoring of prescriptions, especially off-label prescriptions. patient care should take into account clinical profile, respect of guidelines and health expense control. we report here a case highlighting the significant role of the clinical pharmacist in care units to ensure medication good use in a castleman syndrome, a rare disease due to human herpesvirus 8 (hhv-8) and associated with human immunodeficiency virus (hiv) infection. design: case report. results: our patient, a 49 years old man (creatinine clearance rate (crcl): 95 ml/min), was diagnosed with hiv infection in february 2016 (cd4 at 160ui/l), leading to introduce a therapy by emtricitabine-tenofovir, darunavir, and ritonavir. the evolution was hampered by repeated episodes of acute renal failure (arf; crcl: 21 ml/min) and pancytopenia (hemoglobinemia at 8.6 g/dl, leucopoenia at 3.3g/l, and thrombopenia at 55g/l). because of hhv8 blood pcr at 30 000copies/ml, transient crises with pancytopenia, arf, and hiv infection, a diagnostic of kaposi sarcoma herpesvirus (kics), an atypical castleman syndrome, was retained. given the lake of data in literature for this rare disease, a multidisciplinary team (medical specialists and clinical pharmacists) was gathered to choose an appropriate therapeutic strategy. treatment regimen consisted of: day 1, intravenous etoposide at 250 mg; day 4, rituximab at 375 mg/ m 2 ; following one week later by rituximab 1 day and oral etoposide at 250 mg the day after. good communication between medical specialists and pharmacists enables the patient to get an optimal and personal treatment. relaying the information by clinical pharmacists in care units to pharmacists in charge of good practice facilitate the reimbursement. conclusion: clinical pharmacists in care unit help to optimize therapeutic strategies according to their experiences and scientific works. cooperation with physicians is improved, as well as prescriptions follow-up of off-label drugs, and health patients fully respected. quality and relevance of prescriptions are strengthened, with a better control of economic expenses. please specify your abstract type: research abstract background and objective: the maltese government launched the hpv vaccination scheme in 2013 and the national healthcare system (nhs) has since provided the cervarix ò vaccine free of charge to girls aged 12. the aim of this study was to assess the cost of the administration of hpv vaccines in the healthcare system of malta. this study was based on the scheme provided by the nhs. the number of girls born per year was used to estimate the annual cost for vaccinating 12 year old girls, based on the wholesale price and tender price respectively. the estimated yearly cost using the wholesale price was approximately €547,000 while the average estimated cost based on the tender price was approximately €157,000. this signifies that cost savings based on the tender price compared to wholesale costs were of approximately €390,000. the cost for the cohort who completed the three dose schedule using the tender price on average was of €171,000 per year. this result proved to be more than the anticipated cost. a reason for this could be that the number of girls aged 12 increased possibly due to an influx of immigrants. including boys in the vaccination scheme would increase costs by an average of €165,000 per year. conclusion: this study shows that procuring branded vaccines using the tendering process reduces expenditure for the government and the tax payer. wholesale prices were found to be more expensive than tender prices. this proves that the tendering system in malta is a potent system with many advantages for the tax paying public. the impact of the tendering process must therefore, be safeguarded. please specify your abstract type: research abstract background and objective: with the old age, presence of comorbidities, and overcrowding in mass gatherings such as the annual hajj pilgrimage in saudi arabia, there is a high risk of spreading infectious diseases among pilgrims and then within their country of origin. knowledge and application of hygiene principles in such an environment is therefore important to reduce the transmission of infectious diseases. up to date, there have been no studies to evaluate pilgrims' knowledge, attitude and practices toward mers-cov during the annual hajj pilgrimage in order to see whether there is a need for these aspects to be improved. setting and method: a cross-sectional survey study was conducted with a convenience sample of 257 participants. participants were pilgrims, aged over 18, and able to speak arabic or english. a selfadministered structured questionnaire was distributed during hajj season in mecca. descriptive and multiple linear regression analysis were used in data analysis. main outcome measures: assessing pilgrims' knowledge, attitude and practices regarding mers-cov. results: two hundred and fifty-seven participants completed the study, 80% of whom were female, and the median (iqr) age was 35 (24.5-43.5) years. pilgrims had moderately correct knowledge and accurate attitudes towards mers-cov with median scores of 5 (iqr 4-7) and 6 (iqr: 5-7) respectively. they were less educated about management (80%), hallmark symptoms (77%), high-risk individuals (45%) and source of coronavirus (38%). almost 40% of participants showed a negative attitude towards the use of protective measures such as avoiding food prepared under unsanitary conditions and contact with live animals. some participants (30%) were unable to comply with hygiene practices, particularly washing hands with soap and water or disinfectant after sneezing/coughing and wearing a face mask in crowded areas. educational level and employment status were significantly associated with knowledge whereas gender and age were significantly associated with attitude and practices respectively (p \ 0.05). the correlation between knowledge, attitude and practices was significant (correlation coefficient: 0.207; p \ 0.05). better knowledge was found to be a predictor for positive practice. conclusion: these findings aided in the assessment of the adequacy of current pilgrims' educational measures. they will also provide insight when designing future interventions to promote specific messages to improve knowledge, change attitude and improve practice regarding mers-cov. please specify your abstract type: research abstract background and objective: the prevalence of type 2 diabetes significantly increased in the paediatric population, which is affected by obesity worldwide. today, type 2 diabetes accounts for 45% of all cases of new-onset diabetes in adolescents. preventive health care particularly taking place at community pharmacies may involve risk assessment for the children and the adolescents, early referral for seeking relevant medical care and patient education on healthy lifestyle choices. the aim of the study is to conduct a type 2 diabetes risk assessment program for the kids b18 years of age of whose parents visited the community pharmacies involved in the study and also to identify the behavioural parameters that might be associated with this risk. setting and method: the study was conducted in 4 community pharmacies. all patients with kids aged b18 years who visited the study pharmacies during one-week period were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. all data were provided by the parents. demographic data, height and weight of the kid, as well as data regarding the behavioural features (eating habits, exercising, time spent in front of a screen, etc.) of both the children and the parents were collected using standardized forms. type 2 diabetes risk test consisted of 8 questions and identified subjects at risk. the parent of the kid who was identified to have risk for type 2 diabetes was referred to a physician for further examination. also, information regarding type 2 diabetes and the importance of preventive measures such as converting to a healthy life-style was provided. main outcome measures: main outcome measures were the percentage of kids identified to be at risk of developing type 2 diabetes and the behavioural parameters associated with type 2 diabetes risk. results: the study involved 212 subjects. of the subjects 26% were identified to be at risk of type 2 diabetes. more girls than the boys had the risk (36 vs. 9.3%). those with type 2 diabetes risk were older, taller, heavier and had higher body mass index. they were spending more time in front of a screen (tv, pc, tablet, smart phone); 22.6% were spending more than 6 h a day. although the kids' eating habits were similar for those with and without risk, the parents' of the kids with risk ate out more frequently, consumed rice, pasta and pastry more frequently. both the kids with risk and their parents exercised more regularly and frequently. conclusion: this study shows that pharmacist have a vital role in identifying children and adolescents at risk for type 2 diabetes; thus at early management of this condition. identifying and addressing the behavioural parameters associated with the risk will be helpful in lifestyle modification interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: analyse and promote the reporting of adverse drug events (ade), to improve the quality and safety of care to be able to control the risks. design: a software is available on the intranet website of the institution, to enable health professionals to report ade. the drug and medical devices commission (comedims) of the hospital, centralizes these statements and always makes a multidisciplinary and overall analysis of the event, using a collection sheet which is based on the pdca model (plan, do, check, act). it proposes the nursing and medical teams axes of improvement. results: in 2015, only 48 ade were reported and analysed by the comedims, including 20 from the paediatric centre (44%), particularly sensitized to this issue. health professionals are divided as follows: healthcare executives (60%), nurses (23%), pharmacists (11%), residential students (2%), doctors (2%) and others (2%). the main impacted steps of the drug circuit are: administration (62%), prescription (27%) and the use or implementation of a sterile medical device (4%). identified causes include related following factors: operational tasks and procedures (37%), health professionals (31%), work environment (13%), organization and management (7%), drugs or associated medical devices (5%). the number of ade reports, taking into account the size of the institution, remains very low. in january 2016, the comedims decided to broadcast a communication campaign to promote ade reporting, on the hospital website via the intranet. three months after the release, this document was viewed 1059 times, and the number of reports increased by 229% compared to the same period in 2015. conclusion: in front of the low number of returns of adverse drug events, and relying on the charter of non-punishment, the come-dims wants to increase health professionals' awareness. in our hospital, where e-learning about drug-related iatrogenesis is already available, the communication campaign with poster and analysis of adverse events seems to be a useful complementary tool to enhance awareness of medication safety concerns. please specify your abstract type: research abstract background and objective: the migration of modern social networks to the internet has facilitated the transition of traditional pharmacy networks online. the ubiquitous nature of social media (some) combined with merging of personal and professional personas have led to organisations publishing guidance on online behaviour and responsible use of social media. the research to date on the use of social media as a support for professional practice in general is limited. as the pharmacy profession evolves to embrace the technologies which underpin core services and mainstream online daily social activities, it is important that research tracks and evaluates its use and impact within the profession. the objective of this research was to explore and describe how and why pharmacists interact with hosted networks on social media. setting and method: two one-hour online hosted micro-blogging twitter chats were held in december 2015 via the #weph network. topic guides were developed around 'exploring the use of twitter and wepharmacists' in line with the wenetwork guidelines (#wecommunities), informed by existing literature, discussion with the #weph moderator after review by an expert panel. all research was carried out in accordance with university governance processes and association of internet researchers guidelines. themes were inducted from analysing the textual content of the chats using the topic guide as a framework. the research was approved by the school of pharmacy and life sciences ethics committee. main outcome measures: tweets per chat results: each of the chats had over 2 million impressions with participants representing international pharmacy practice. themes of e-professionalism and online privacy emerged as concerns; however, the benefits included using social media for education, networking, support mechanisms and career development. tweets highlighted personal experiences of 'trolling' (angry, offensive behaviour) and the effect on user interaction with social media. twitter was also recognised as a career development tool and, in particular, collaborative outcomes around mentorship networking early career pharmacists with more experienced colleagues. conclusion: results support the responsible use of social media as a force for inclusion, breaking down geographical barriers in support of pharmacy practice. further research is underway including a systematic review of guidance on the use of social media by registered healthcare professionals. please specify your abstract type: research abstract background and objective: it is estimated that half of the 350,000 persons with diabetes in norway have not been diagnosed. with early treatment, life expectancy can be increased and the incidence of longterm complications and health costs reduced. community pharmacies may be able to help uncover undiagnosed diabetes, but being diagnosed with diabetes can lead to strong emotional reactions, and how the diagnosis is given may influence the experience. the aim of this study was to explore how norwegian people living with type 2 diabetes (t2d) experienced being diagnosed, and what led up to the diagnosis. in addition, their attitudes towards a planned community pharmacy service to identify undiagnosed t2d was investigated. setting and method: three focus group interviews with people with t2d were conducted using a semi-structured interview guide. eleven participants were recruited through a course about type 2 diabetes. the interviews were audio-taped and transcribed in modified verbatim form and analysed in accordance with malteruds principles of systematic text condensation. the study was approved by the norwegian data protection authority, and did not require approval from the regional committee for medical and health research ethics. main outcome measures: how people with t2d describe their experiences of being diagnosed with t2d, how the disease was revealed and reactions towards using community pharmacies to perform risk assessment for t2d. results: none of the participants were diagnosed due to their own suspicion of having diabetes. some saw their doctor because of unspecific symptoms such as fatigue and thirst, and were thereafter diagnosed with t2d. others were diagnosed through a routine checkup. negative reactions like shock, discontent and denial were commonly used to describe the experience of being diagnosed with t2d, but some participants also expressed a more relaxed attitude, especially if they were familiar with the disease through family members. participants expressed a strong wish for more and better information following the diagnosis. ''it's a jungle out there'' was used to describe how difficult they felt it was to find trustworthy and understandable information. they described change of lifestyle, side effects from drug use, and stigma as challenges following the diagnosis. while in general the participants were positive to using community pharmacies to uncover undiagnosed diabetes as this could help reduce the number of people who were undiagnosed, some were sceptical. they questioned whether the pharmacy staff had the necessary competence of the for this type of service, and saw it as the doctor's responsibility. conclusion: more information and support when people are diagnosed with diabetes may lead to that the experience being diagnosed will be more adaptable and that the challenges living with diabetes are reduced. community pharmacies are important healthcare providers, and risk assessment of t2d at the pharmacy can be valuable. however, the pharmacies may also be helpful to reduce the information gap. please specify your abstract type: research abstract background and objective: chemotherapy-induced nausea and vomiting (cinv) is a disruptive and unpleasant side effect in chemotherapy patients and is associated with decline in patients' quality of life and decrement in the adherence to effective chemotherapy regimens. setting and method: 100 chemotherapy naive patients were included in this study. consistency with guidelines were assessed according to mascc/esmo 2014. flie questionnaire was administered to patients before chemotherapy, and 5 days after receiving chemotherapy to assess the difference in the quality of life due to chemotherapy administration. main outcome measures: patients were categorized into two groups as consistent with guidelines group (acute (gcga) and delayed (gcgd)) and inconsistent with guidelines group (acute (giga) and delayed (gigd)). flie score differences between the two groups were assessed. results: the median flie score for patients prior to chemotherapy was 126 and a dramatic decline was noticed post chemotherapy (flie score 108; p \ 0.001). the post-chemotherapy score were for nausea and for vomiting (49.5, 63 respectively). although the flie score differed significantly between gcgd and gigd (p \ 0.01), these differences were not significant in gcga and giga. conclusion: the significant drop in flie scores in the study (126 pre-to 108 post-chemotherapy) reflected substantial declination in patients' quality of life. the lower postchemotherapy flie score of nausea emphasized the negative impact of nausea, and to a lesser extent vomiting on the patients ability to complete normal daily activities such as enjoying meals and maintaining social activities. although there were no significant differences in flie scores between giga and gcga groups for acute cinv prevention, significant differences were noted between gigd and gcgd (p \ 0.001). the flie score was lower for gigd patients. this result implied guideline inconsistency associated with high incidence of nausea which negatively affect patient quality of life. as for the degree of compliance with gp, the results are expressed as percentage of compliance compared to the ideal of 100%. prescription criterion was fulfilled to 100%: all requirements of pntb were performed using standardized procedure. in what concerns validation, 94% of pntb prescriptions were validated by a pharmacist. the invalidated prescriptions were made outside opening hours of the pharmacy service, which is open monday to friday from 08:00 to 20:00 and on weekends and holidays from 08:00 to 15:00. 100% of the dispensations were individualized and not pntb stocks were found in hospital wards. as for preparation, 36% were supplemented with micronutrients. pntb of kabiven peripheral administration 1920 ml are not supplemented in our centre. of the remaining 325 prescriptions central administration, 81% were supplemented. in all cases, the addition of micronutrients was performed in laminar flow hood in pharmacy service and the corresponding galenic validation was performed. finally, in the process of administration, 94% of pntb identified with a complete label: name of the patient, medical record number, type of pntb, qualitative and quantitative composition, date of administration and infusion rate. conclusion: use practices of pntb of our centre are far from those recommended by the sefh standards. this initial evaluation will serve for improvement measures that increase the quality of prescribing and safe use of pntb, in order to minimize errors that can occur with the use of this therapeutic modality. please specify your abstract type: research abstract background and objective: methadone maintenance treatment was developed in malta in 1987 and is provided to patients by sedqa, the national agency against drug and alcohol abuse. methadone is the most frequently prescribed opioid in opioid substitution treatment and is dispensed through a centralised service through the substance misuse outpatients unit. in 2013, 1078 patients were in opioid substitution treatment, 976 of who were on methadone. in 2005, the government introduced a take-home methadone program. the prescribing, purchasing and dispensing of methadone are regulated by subsidiary legislation 101.06. the objectives were to determine whether community pharmacists in malta would be willing to dispense and supervise the consumption of methadone and to investigate the involvement of community pharmacies in the development of a regionalised methadone dispensing service. setting and method: the study was set in community pharmacies. a cross-sectional study, through the use of a questionnaire, was performed to quantitatively analyse whether pharmacists in malta would be willing to dispense methadone. the questionnaire consisted of 19 questions divided into 3 sections, with each section assessing a particular aspect of community pharmacists' attitudes towards methadone dispensing. community pharmacies were then chosen via a systematic sampling procedure. a hard copy of the questionnaire, addressed to the managing pharmacist, along with a cover letter, instructions on how the questionnaire was to be returned, and a prepaid self-addressed envelope was distributed via postage to 103 community pharmacies. an online format of the questionnaire was also circulated to 311 community pharmacists through the pharmacy council. data was analysed using spss version 21. main outcome measures: community pharmacist's attitudes towards methadone dispensing. results: a total of 109 responses were obtained and a response rate of 35.04% was achieved. eighteen percent of the pharmacists (n = 109) who responded to the questionnaire worked in a community pharmacy located in the north of malta, 24% in the centre, 17% in the south, 8% in the southeast and 4% in gozo. thirty-two percent of community pharmacists were willing to dispense methadone to drug misusers. the number of community pharmacists who are willing to dispense methadone increased to 41% if they were provided with appropriate education and support. twenty-nine percent of community pharmacists were prepared to handle the duty of supervising the consumption of methadone while 86% had never learnt about methadone and its clinical application within opioid substitution treatment. conclusion: community pharmacists should be provided with education and training regarding methadone substitution treatment before embarking on a new regionalised methadone dispensing service within community pharmacies. this would allow more community pharmacists to become involved in a new dispensing methadone service. pt009: evaluation of regorafenib in patients with colorectal cancer please specify your abstract type: research abstract background and objective: the colorectal cancer is the second more frequent cancer in europe and the third in the world. regorafenib is only approved in adult patients with metastatic colorectal cancer who are previously been treated with available therapies or are not considered suitable candidates to these treatments. regorafenib is an oral anti-tumor drug that blocks the kinases involved in the tumor angiogenesis (vegfr1, -2, -3, tie2), the oncogenesis (kit, ret, raf-1, braf, brafv600e) and the tumor microenvironment (pdgfr, fgfr).in this study, we are reviewed the reports of the patients with colorectal cancer who are been treated with regorafenib in our hospital and analysed the information in order to evaluate the efficacy and safety of regorafenib. setting and method: descriptive and observational study about the use of regorafenib from april 2015 to the present day. the variables studied, obtained from the software applications archinet and diraya, were: sex, age, pathology, location of metastasis, posology and adverse effects of regorafenib, tumor markers (cea y ca 19.9) before and after the treatment with this drug and the mutational state of kras. main outcome measures: the tumor markers cea and ca 19.9 only decreased in the 22.22% of the patients after the regorafenib treatment. results: regorafenib was taken by 9 patients (78%men).the average age of these patients was 64.78 ± 8.48 years old. the patients took regorafenib to treat: metastatic and non-intervened gastrointestinal stromal tumors (gist) e-iv that progressed with the previous treatment of imatinib and sunitinib (11.11% patients), intervened colon adenocarcinoma e-iv (33.33% patients), sigma adenocarcinoma e-iv (33.33% patients) and unresectable and non-intervened rectal adenocarcinoma e-iv (22.22% patients).all patients presented metastasis in different locations on the body: liver (55.55% patients), diaphragm (11.11% patients), intestine (11.11% patients) and lung (44.44% patients).the 77% of the patients started the treatment with 160 mg of regorafenib, administrated once a day for 3 weeks followed by one week without this drug; while the 22.22% of the patients started the treatment with 120 mg. however, the 33.33% had to decrease the initial dose and the 55.56% of the total patients had to get off the treatment because of the development of side effects. the most frequent adverse effects were: hypertension associated with headache, hyperbilirubinemia, elevation of ast and alt, intense asthenia. the 66.67% of the patients presents native kras. the native kras was presented in the 100% of the patients treated with regorafenib who had an appropriate development of the illness (decrease of cea and ca 19.9) conclusion: the decrease of cea in the 22.22% of the patients and the high development of side effects reveal that regorafenib has low effectiveness and security in the control of the progression of colorectal cancer. in addition, it is supposed that this drug has better results in native kras patients. however, more studies are necessaries in order to demonstrate the effectiveness of regorafenib in this pathology. pt010: evaluation of nintedanib in patients with non-small-cell lung carcinoma (nsclc) please specify your abstract type: research abstract background and objective: the nsclc means a high rate of mortality in developed countries. patients diagnosed with nsclc who debut with advanced or metastatic disease have a median survival of 13 months. one of the innovative drugs approved to improve survival in nsclc is nintedanib: an inhibitor of multiple tyrosine kinases, which can be found in some receptors on the surface of cells involves in the growth and spread of cancer cells (''pdgfr'', ''fgfr'' and ''vegfr''). nintedanib is not yet marketed in spain. hospital pharmacists are responsible for applying this treatment as ''expanded drug'', only after the elaboration of an exhaustive report. in this study, we have reviewed all the reports and classified the information in order to present our clinical practice. the objective of this study is to evaluate the effectiveness and safety of nintedanib in patients with nsclc treated in a tertiary hospital. setting and method: descriptive observational study of the use of nintedanib from november 2014 to september 2015. sex, age, body mass index (bmi), pathology, smoking habits, line of treatment, posology and adverse reactions of the treatment with nintedanib and tumor markers (cea an ca 19.9) before and later the treatment with nintedanib were collected from medical history through archinet informatic application. main outcome measures: the tumor marker cea decreased in 57% of the patients and ca 19.9 no decreased in any patient after nintedanib treatment. results: nintedanib was used in 7 patients (57% men and 43% smoker).the average age of these patients was 58 years old. the average bmi was 28 kg/m 2 (18-50).all patients received nintedanib together with docetaxel for metastatic nsclc with adenocarcinoma histology and with non-mutated egfr and alk in third line treatments. posology: all patients started the treatment with nintedanib 200 mg/12 h from day 2 to day 21 every 3 weeks; but 2 patients had to reduce the initial dose to 300 mg/24 h (1 patient) and 150 mg/12 h (1 patient) because of some adverse reactions. the side effects were: asthenia, diarrhoea, alteration of transaminases, muscle pain and cramps, weight loss and mucositis. conclusion: the decrease of cea in 57% of the patients reveals that nintedanib is effective in controlling nsclc progression which involves an increase of the survival and the quality of life of these patients. however, more studies are required to demonstrate the efficacy of nintedanib in this illness. please specify your abstract type: research abstract background and objective: patients with sore throat symptoms often seek fast, meaningful relief when presenting to their local pharmacy. flurbiprofen is a non-steroidal anti-inflammatory drug, which has been developed as a spray and lozenge to provide targeted relief for the main underlying process responsible for the symptoms of sore throats, inflammation. to study the relief provided by flurbiprofen 8.75 mg delivered as a spray or lozenge, we conducted a multicentre, randomised, double-blind, double-dummy, parallel group, activecontrolled, single-dose, non-inferiority study. setting and method: adult patients with acute sore throat were randomly assigned to take one dose of either flurbiprofen 8.75 mg spray plus a placebo lozenge, or flurbiprofen 8.75 mg lozenge plus placebo spray at 16 sites across russia. main outcome measures: patients rated sore throat relief using the sore throat relief rating scale (strrs; a 7-point scale, 0 = no relief, 1 = slight relief, 2 = mild relief, 3 = moderate relief, 4 = considerable relief, 5 = almost complete relief, 6 = complete relief) at timed intervals throughout 2 h starting from 1 min post completion of first dosing (1 min after administration of the spray, and 1 min after the lozenge had fully dissolved). adverse events (aes) were recorded over 2 h post-dose. results: 417 patients were assessed (n = 205 for spray, n = 212 for lozenge). [90% of patients in either treatment group experienced some relief (a score of [1 on the strrs) at 1 min post-dose, which increased to 98% of patients by 2 h. 55-60% of patients reported 'at least moderate relief', which is a well-recognised measure of a clinically meaningful effect at 1 min post-dose, which increased to 74-78% of patients by 2 h. over the 2 h post-dose, a total of 17 drugrelated aes were reported by 13 patients across both treatments and no severe adverse events were reported. conclusion: flurbiprofen 8.75 mg delivered as a lozenge or spray provides fast, clinically meaningful relief from sore throat. pt012: analising antiangiogenics prescription in an ophtalmology service after a protocol implementation silvia cornejo-uixeda * , ivan de la vega-zamorano, celia aparicio-rubio, olga carrascosa-piquer, manuel prieto-castello, agustin sanchez-alcaraz pharmacy, hospital universitario de la ribera, alzira, spain please specify your abstract type: descriptive abstract (for projects) background and objective: after some years using antiangiogenics in our hospital, we observed a large variety of use. considering the high cost of these treatments, we proposed ophthalmology service to develop a protocol of use, attending efficiency criteria. in this paper, we analyse the protocol implementation repercussion. design: a protocol of use was designed with the main of unify criteria and to use the most efficient treatment depending on the specific situation on each patient. once it was implemented, we compared two periods, the period after the implementation (january-may2016) and the period before of it (january-may2015). the protocol designed is the following: the cost for each injection and patient was the following: aflibercept 207€, bevacizumab 10€, ranibizumab 857€. results: in the 2015 period, 303 patients were treated with antiangiogenics.181(60%) with aflibercept, 110(36%) with bevacizumab and 12(4%) with ranibizumab. in the 2016 period, 297 patients were treated, 147(49%) with aflibercept, 134(46%) with bevacizumab and 16(5%) with ranibizumab. the consumption of aflibercept decreased a 19%, bevacizumab consumption increased 22% an ranibizumab increased a 33%.we also observed, some patients had more than one diagnostic at the same time. once the protocol was implemented, the percentage of use was the following: 38% 1. please specify your abstract type: research abstract background and objective: drug prescribing is the most common medical intervention in the elderly. however, elderly patients are more sensitive to the drug's effects due to pharmacokinetic and pharmacodynamic changes associated with aging. chronic diseases and co-morbidities often require the use of a large number of medications. therefore, when prescribing drugs for the elderly, the choice of suitable drugs, dosage and duration of treatment should be carefully considered as well as clinically significant drug interactions. inappropriate prescribing is often associated with an increased risk of adverse drug reactions, increased morbidity and mortality, and health care costs. the aim of this study was to determine the incidence of potentially inappropriate medications (pim) prescriptions in the elderly (c65 years) using the original protocol developed by mimica matanovic and vlahovic-palcevski. setting and method: we enrolled 240 patients hospitalized in clinic of internal medicine. data about patients' medications was collected during patient interview taken by the pharmacists on hospital admission. pharmacotherapy was analysed using the original protocol developed by mimica matanovic and vlahovic-palcevski in order to detect pims. main outcome measures: number and type of potentially inappropriate medications, potential clinically significant interactions. results: the average age of patients was 74 years (range 65-92), and the average number of drugs per respondent was 6.7 (range 1-15). a total of 109 patients (45.4%) were taking at least one pim. the most common pim were long-acting benzodiazepines, central antihypertensive moxonidine and non-steroidal anti-inflammatory drugs (nsaids) in patients with hypertension. in the study population, 110 patients (45.8%) have taken at least one combination of drugs that could result in a clinically significant interaction. the most common combinations included application of nsaids and antihypertensive drugs or diuretics, concomitant use of multiple medications with effects on the central nervous system and drug combinations that can cause hyperkalaemia. conclusion: this study revealed the high prevalence of inappropriate prescribing. clinical application of this protocol could be an effective method for improving and optimizing drug prescription with the aim to reduce the number of side effects and the morbidity and mortality associated with the drug use in the elderly. please specify your abstract type: research abstract background and objective: to reduce adverse effects of conventional amphotericin b formulation (deoxycholate or d-amb) it can be infused in intralipid ò (a fat parenteral nutrition), or lipid-based formulations can be used (i.e. amphotericin b lipid complex (ablc), amphotericin b colloidal dispersion (adcd) and liposomal amphotericin b (l-amb)). studies evaluating safety profiles present conflicting results. the aim of our study was to gather evidence on nephrotoxicity rates of d-amb versus lipid-based formulations in immunosuppressed patients susceptible to invasive fungal infection. setting and method: a systematic review, including randomized controlled trials (rcts) that compared the use of d-amb and amphotericin b lipid-based was performed. a search was conducted in pubmed, scopus, web of science and scielo. results were synthetized and meta-analysis was performed using software review manager 5.3. main outcome measures: nephrotoxicity rates. results: eighteen rcts were identified (n = 2525 participants). the result from the meta-analysis favours the treatment with the lipidbased amphotericin b formulations (or: 0.32 (0.25, 0.41) and presents a low heterogeneity (i 2 = 18%). about 22% of patients from lipid-based treatment group presented an increase in serum creatinine of one to two times, which corresponds to stage one or two of acute renal failure (arf). and 2% presented an increase of tree times in serum creatinine achieving a stage three in arf (severe) which will require dialysis. while in group treated with conventional formulation int j clin pharm (2017) all of these 23 patients, except one whose treatment adherence was inadequate, were cirrhotic (15/23), liver transplanted (5/23) and/ or presented hepatocellular carcinoma (3/23). 6/23 patients were coinfected with hiv. 8/23 patients (35%) were genotype 3. the total genotype 3 patients treated with daas (svr12/relapsed) were 79, which means that 10.1% (8/79) of all genotype 3 patients has had a relapse. 10/23 patients (43%) were treated with ledispavir/sofosbuvir (2.4% of a total of 411 patients (svr12/relapsed) treated with this option). 39% of patients who suffered a relapse were treated with daas sofosbuvir, simeprevir, daclatasvir, previously to the introduction of the newest antivirals (dasabuvir + ombitasvir/ paritaprevir/ritonavir, ledispavir/sofosbuvir), which represents 6.3% of the total of 142 patients treated with the older option. conclusion: relapses rate was 3.1%, slightly lower than reported in other studies. according to the references, these results show that genotype 3 is the one presenting more relapses. all the patients presented a deteriorated performance status, except for one whose treatment adherence was inadequate. patients treated before april 2015, when the newest daas where introduced, showed more relapses. more studies have to be developed in the near future since other daas will appear, the treatment options will be amplified and the number of relapses is expected to decrease. please specify your abstract type: research abstract background and objective: the inappropriate use of antibiotics remains a major issue since it causes bacterial resistance, longer hospital stay and increased mortality. antibiotic prescriptions must be monitored: the clinical pharmacist has a key role in ensuring patient safety and quality of pharmaceutical care. therefore, an antimicrobial stewardship program has been implemented as part of a national project of the italian society of hospital pharmacy (sifo). the objective is to describe the results obtained at the hospital. setting and method: a multidisciplinary antimicrobial management team has been implemented including clinical pharmacists, microbiologists and infectious disease specialists. the pharmacist examines drug charts on a daily basis in the department of medicine and supports clinicians to improve the appropriate use of antibiotics. data from 2 time-points were extracted from medical records and collected in an excel database: t0 (november 2015-january 2016) and t1 (february 2016-april 2016). main outcome measures: type of infection, antibiotic consumption data, type of isolated pathogens, patient allergies, clostridium difficile infection assessment and adverse drug reactions (adr). results: 465 records were analysed (t0-t1), 277 of which contained at least one antibiotic prescription. the most frequent infections were urinary tract (27%), respiratory (20%) and gastro-intestinal (14%). antibiotic therapy was started in 15.9% of cases due to aspecific increase of c-reactive protein (crp). ddds were calculated for each treatment and were grouped by type of infection and setting (empiric vs targeted): ceftriaxone, meropenem and metronidazole were the most widely used antibiotics for empiric therapy. at t1, an increase in the use of piperacillin-tazobactam instead of meropenem was observed. the ddd of ceftriaxone for targeted therapies decreased significantly, while an increase was observed for carbapenems, levofloxacin, glycopeptides and, in case of mdr bacteria, tigecycline. three allergies to antibiotics were reported in medical history. there were 20 clostridium difficile infections (5 relapses), confirmed by antibiogram. a total of 22 adrs were identified: 3 of these were related to antibiotics. conclusion: antimicrobial stewardship is a fundamental step to optimise antibiotic management, ensure patient safety and improve quality of care. the results obtained so far demonstrate the added value of a multidisciplinary team in controlling bacteria resistance and in the improving the use of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to analyse effectiveness and safety of pirfenidone, an anti-inflammatory and antifibrotic agent used for treatment of idiopathic pulmonary fibrosis. design: a retrospective, descriptive, observational study including all patients treated with pirfenidone at the hospital between march 2015 and june 2016 (15 month) was carried out. to identify patients and collect data the outpatient medication dispensation software farhos ò and the electronic medical record software hcis ò were used. statistical analysis was carried out using microsoft excel ò . demographic (age and sex), clinical (forced vital capacity (fvc), diffusing co capacity (dlco) and six-minute walk test (wt6 m)) and therapeutic (dosage and adverse reactions) variables were collected. results: throughout the study period, a total of 22 patients (16 males) started treatment with pirfenidone, with a median age of 74.5 years (46-81). during this period 5 patients were excluded for lack of monitoring. the median fvc, dlco, wt6 m values prior to pirfenidone therapy, were 59% (50 [ 81%), 38.5% (17 [ 65%) and 357 m (200-620 m) respectively. all patients met the inclusion criteria of capacity trial according to fvc and wt6 m; however 8 of them didn't meet the dlco criteria (at least 35%).'' all patients were monitored every 3 months. the median in fvc percentage change at the end of the study was -1% (-13% to +9%). 8 patients (50%) showed an improvement on fvc during treatment with a median change of 7%. in the other eight patients fvc value decreased with a median of -6%. only one patient would be candidate to discontinue treatment due to a lack of efficacy, according to discontinuation criteria established at the hospital (absolute decrease of c10% in fvc during first year of treatment). dlco percentage was measured in 14 patients, with a median change of 2% (-17% to +10%). dlco decreased in 6 patients. wt6 m was monitored in 12 patients, with a median change of -44.5 m (-222 m to +35 m). adverse effects related to pirfenidone were gastrointestinal disorders (9/17), increase of hepatic ggt (5/17), and dermatologic toxicity (2/17). six patients (35%) required a dose reduction because of gastrointestinal adverse effects. five patients (29%) discontinued treatment with pirfenidone due to hepatotoxicity (2), gastrointestinal (1) and dermatologic effects (1). one patient died. conclusion: half of the patients improved fvc during the period of the study. the other half, showed a decrease in fvc value which was similar to the median obtained in capacity trial. gastrointestinal disorders were the most frequent adverse effects and cause of discontinuing treatment. treatment monitoring is important to achieve therapeutic benefit and control the adverse effects. the national centre for epilepsy, oslo university hospital, oslo, norway please specify your abstract type: research abstract background and objective: systematic medication reviews in interdisciplinary teams can help to identify potential and actual drugrelated problems (drp). the centre for development of institutional and home care services in oslo, norway, conducted medication reviews for polypharmacy patients with mental disabilities in 2015-2016, based on a lack of knowledge about drug-related problems in this patient group. the objective was to examine prescribing patterns, frequencies and types of drp in patients with mental disabilities. setting and method: the forms for medication reviews were developed by the national patient safety campaign in norway. the nurse/social educator recruited eligible patients, observed them, and ordered test if needed. the clinical pharmacist (jwa) reviewed the medications to identify drps. the interdisciplinary case conference took place at the different general practitioners' offices being responsible for the individual patients. the general practitioner, the nurse/social educator and the pharmacist were present, and in some cases, also patients took part. main outcome measures: an independent researcher (aqm) collected and analysed the data based on the drp-forms containing information on the prescribed medicines, strength, dose, indication, a description of drp and suggested interventions to resolve them. results: overall, 40 patients with mental disabilities, aged 34-77 years, consented to have a medication review. they used on int j clin pharm (2017) 39:208-341 327 average 12 medicines (range 5-23). the team identified 191 drp in 39 of the 40 patients (average 4.9, range 0-13). overall, 79% of all drp were resolved. for one-third of the medicines, an action was taken to improve the prescribing. the most commonly medicines were analgesics (62%), antiepileptics (58%) and anxiolytics (52%). the most frequent drps were unnecessary drug choice (24%), side effects (11%) and too low dose (11%). drps were most common in antipsychotics (10%), antidepressants (9%) and anxiolytics (7%). conclusion: patients with intellectual disabilities take more medicines and have many drps compared to other patient groups. they are also more prone to taking combinations of cns-active medicines and therefore more at risk of side effects and drug interactions. pt020: protocol feasibility and patient findings when using a dry extract of zingiber officinale roscoe (ginger extract gr10) during pregnancy please specify your abstract type: research abstract background and objective: there is limited information about the use of dry extracts of ginger root. the objectives of this study are (1) to evaluate the feasibility of a pilot study with a food supplement among pregnant women (2) to learn what the patient findings are when using the dry extract of ginger during pregnancy. this abstract deals with the intermediate evaluation of a study conceived to investigate the safety of the ginger extract gr10 during pregnancy. setting and method: a prospective, interventional and real life pilot study with pregnant women between 4 and 14 weeks of gestation and having symptoms of nausea and vomiting or digestive complaints. the included patients can use the ginger extract gr10 for digestive comfort during pregnancy when needed. during the use, the score of digestive discomfort is noted and the researcher reports adverse events. main outcome measures: (1) number of included patients as an indicator of feasibility: including a number of 50 patients was taken as a target (2) analysis (qualitative and quantitative) of the patient diaries, more particularly patient behaviour, wellbeing and impressions. results: within twelve weeks, 51 patients were included with an average age of 29.9 years and a median age of 29 (19-42) years. 45 patients used gr10: 3 patients were dissatisfied, 15 patients had a neutral opinion and 19 patients were satisfied to very satisfied. one miscarriage occurred at a gestational age of almost 17 weeks (only 2 tablets of gr10 were used, with no relevant medical history in preceding pregnancies). two patients were hospitalized, of which 1 with hyperemesis gravidarum. one patient complained about heartburn and one patient experienced a bad taste and heartburn. three patients have indicated that they experienced more nausea after taking the tablets. 29 patients experienced no adverse events. the remaining 8 patients were not yet evaluated. of the 51 included patients, six patients decided not to use the product: 3 because their gastrointestinal complaints were not serious enough, 1 because problems of swallowing (using ginger gums instead). one patient was afraid for the negative consequences for her unborn child. the last of the nonusers indicated that she had no confidence in the product. conclusion: conducting a pilot study with the ginger extract gr10 in case of pregnancy is feasible. the majority of the evaluated patients were satisfied. signing the consent form does not guarantee the intake of the product. pregnant women remain very cautious in the use of unknown products during their pregnancy, even though it concerns a food supplement and not a drug. the severity of symptoms does not give a good indication whether or not and how often the product will be used. please specify your abstract type: descriptive abstract (for projects) background and objective: to analyse effectiveness and safety of ibrutinib, an oral inhibitor of bruton tyrosine kinase, in patients with mantle cell lymphoma (mcl) who have received at least one prior therapy. design: a descriptive observational study was carried out. all patients with relapsed or refractory mcl who started treatment with 560 mg of daily ibrutinib between september 2014 and june 2016 were included. patients were identified and followed through electronic medical record. demographic and baseline clinical characteristics of patients were collected: age, sex, ecog (eastern cooperative oncology group scale), number and type of prior regimens, simplified mipi status (mantle-cell lymphoma international prognostic index), and disease stage (relapsed or refractory). progression free survival (pfs) and response to treatment were recorded to evaluate effectiveness. adverse effects related to ibrutinib and possible interactions with concomitant medication were documented to measure safety. statistical analysis of the data was carried out using microsoft excel 2013 ò and spss ò 18. results: throughout the period of study a total of 5 patients (4 males and 1 female) with a mean age of 62.5 ± 8.2 years started treatment with ibrutinib. the median of previous treatments were 2 (1) (2) (3) (4) (5) including first-line treatment with high dose chemotherapy (100%), steam-cell transplantation (80%), rituximab (100%), bortezomib (60%) and lenalidomide (20%). the median ecog value prior to ibrutinib therapy was 0 (range 0-1). the mipi status was intermediate risk in 4 patients and high risk in 1, the disease stage was relapsed in 80% of the patients. partial response was reported in 3 patients. the mean pfs estimated at the end of the study period was 13 months (95% 4.4-21.5). adverse effects related to ibrutinib were: fatigue (10%), diarrhoea (10%) leucocytosis (30%) and infections (50%), including upper respiratory and urinary tract infections, sinusitis and pneumonia. one possible interaction between ibrutinib and everolimus was found in a liver transplant patient. close monitoring of everolimus plasmatic levels was recommended. conclusion: the mean pfs estimated in our study was similar to the median obtained in the pivotal phase ii trial. infections were the most frequent adverse effects. concomitant medication to ibrutinib should be checked, as ibrutibib is metabolised by cyp3a4 and interactions may be frequently present. 1 pharmacy, 2 hiv unit, germans trias i pujol hospital, badalona, spain please specify your abstract type: descriptive abstract (for projects) background and objective: dolutegravir (dtg) is one of the preferred options for initial antiretroviral therapy (art) due to its high efficacy, good tolerability and low potential for drug-drug interactions. nevertheless, an unexpectedly high rate of dtg discontinuation (up to 16%) due to adverse events in the clinical practice has been recently reported. therefore, we aimed at assessing the dtg discontinuation rate and reasons for discontinuation in our hospital. design: single-centre, retrospective study from september 2014 to june 2016 of 2709 patients cohort with art both naive and pretreated. patients who had started dtg-based art containing regimen were identified and the reasons for the discontinuations were analysed. data were collected using the primary care service program and the electronic prescription program. results: out of 2700 patients attended by pharmacy department in our hospital, 563 patients (494 males, mean age 47 years (range 16-84)) had started a dtg-based art. out of them, 61 patients were art naive and 502 art-experienced. at the moment of starting dtg, mean cd4 cells were 654cell/mm 3 (range 7-2147) and hiv-1 rna load in plasma was detectable in 69 patients. treatment discontinuation was reported in 52/563 patients (9.2%) with a median treatment time of 241 days (range 7-842). 8/52 patients (15.4%) were naïve and 44/52 patients (84.6%) pre-treated. most of the patients (404) were in single tablet regimens (str) containing dtg in combination with abacavir and lamivudine, whereas the rest were in combination with other antiretroviral drugs. the main reason for treatment discontinuation was toxicity in 38/52 patients (73.1%). the rest of the patients discontinued due to other motives (clinical trial inclusion (3/52), treated in another hospital (4/52), exitus (1/52) and others (6/52). reasons for the discontinuation were classified in different side effects: 17/38 (44.7%) related to central nervous system (cns) (insomnia, psychiatric disorders such as anxiety, nightmares and depression), 14/38 (36.8%) gastrointestinal effects, 4/38 (10.5%) headaches, 6/38 (15.8%) musculoskeletal effects, 4/38 (10.5%) fatigue, 1/38 (2.7%) allergy and 6/38 (15.8%) for other reasons. some patients reported various toxicities at once. conclusion: more than 6% of patients treated with dtg discontinued by toxicity reasons. it is important to note that half of these patients had cns adverse effects. please specify your abstract type: research abstract background and objective: hcv therapy has been revolutionised recently by the approval of antiviral agents direct-acting (daa) facilitating the treatment of patients coinfected with hiv/hcv. however, potential drug interactions and overlapping toxicities of both treatments represent the major challenges in adapting therapy. to analyse the prescription profile of direct acting antivirals (aad) in patients coinfected with hiv/hcv. setting and method: retrospective observational study from january 2015 to january 2016 in a specialty hospital. the data were collected from the hospital program of clinical stories, archinet ò , and the outpatient program farmatools ò . the results were analysed using the statistical program r-commander. main outcome measures: inclusion criteria: adult patients coinfected with hiv/hcv with undetectable viral load. the following variables were collected: age, gender, hcv genotype, degree of fibrosis, patient type (naïve or pre-treated), baseline cd4 count, cd4 levels end of treatment, sustained viral response (svr) and hcv treatment. results: 20 patients, of whom 16 were men, mean age 52 years were included. 9 patients received daclatasvir and sofosbuvir for hcv, 3 patients had genotype 1a and 1b respectively, 2 patients genotype 3 and 1 patient genotype 4. 8 patients had fibrosis f3 f4, 1. of the 9 patients 3 they had not received previous treatment (naïve) and 6 had failed to treatment. hiv treatment was modified in 8 patients, 6 patients achieved svr. the cv was undetectable to hiv treatment change for all patients. cd4 levels increased in all patients at the end of treatment for hcv with a median of 304 cells/ul and 398 at the beginning and end respectively. 2 patients received ombitasvir/paritaprevir/ritonavir and dasabuvir, who had a genotype 1a. these two patients had received previous treatment and had a f2 and f4 fibrosis. none of them was modified hiv treatment and only one got svr. cv remained undetectable and cd4 slightly increased after the treatment. 9 patients received ledipasvir and sofosbuvir, 6 patients had genotype 1a, 2 patients genotype 1b and 1 patient genotype 4. 4 patients had f4 fibrosis and 5 had f3. 9 patients had received previous treatment (naïve). the hiv treatment was modified only in one of the patients, 8 patients achieved svr. cv increase in 2 patients after the treatment while cd4 followed the trend of increasing. conclusion: the aad that caused fewer changes in the hiv treatment were ombitasvir/paritaprevir/ritonavir and dasabuvir followed by ledipasvir/sofosbuvir. sofosbuvir and daclatasvir present a greater number of interactions with hiv drugs so they behaved to a major change. more patients are needed to assess more accurately the aad leading to a minor modification. please specify your abstract type: research abstract background and objective: the simplification strategies reduce the amount of tablets and the toxicity in order to facilitate adherence in patients with virological suppression. the strategy more studied is monotherapy with a ritonavir-boosted protease inhibitors (pi/r). to analyse the effectiveness of monotherapy with pi/r in pre-treated patients infected with hiv. setting and method: retrospective observational study. selected hiv patients treated with pi/r monotherapy at any time of pharmacotherapeutic history to 30/12/2015, with at least one clinical and analytical control 6 months before the beginning. data were collected from the medical record archinet ò and outpatient farmatools ò program. variables included were age, sex, duration of monotherapy, virological failure, treatment failure, cd4% during monotherapy. main outcome measures: inclusion criteria: virological suppression for 1 year prior to the start of monotherapy, no previous ip virological failure, high cd4 count ([300 cell/ml) and a high level of drug adherence. the effectiveness is defined as the percentage of patients without virological failure (2 consecutive plasma viral load (vl) [200 copies/ml) and without treatment failure (any event causing retirement monotherapy). results: 141 patients with monotherapy, which represent 22% of patients with antiretroviral therapy (art) at our institution were identified. 29 were excluded (8 co-infected with hepatitis virus, 3 with insufficient data and 18 no had more than 6 months included), including 112 patients in the analysis, with a mean age of 45 years and 60% were men. the median of time monotherapy treatment was 1.75 years (639.5 days), 89(79.4%) patients received darunavir/r and 23 (20.53%) lopinavir/r. the effectiveness of monotherapy treatment during the follow up period was 100% with undetectable pvl at follow-up. the median of cd4% over the treatment time was 794 cell/ml (34%). conclusion: the effectiveness of treatment with ip/r monotherapy in our hospital obtained good results. according with our results treatment adherence plays a very important role. this is a current and valid strategy that brings benefits to the patient and to the healthcare system. please specify your abstract type: research abstract background and objective: the access to investigational drugs for patients who are not included in a clinical trial and without authorized therapeutic alternatives is known as compassionate use. the incorporation of the evidence-based medicine in the area of oncohaematology has implied that an important part of clinic therapy validated by evidence that could not be controlled from an administrative point of view. this is due to the continuous and progressive development of investigation and information on cancer treatment and the delay of the administration regulation. the use of drugs in this way is regulated by royal decree 1015/2009 (19/6). the objective of the study is to describe the use of cancer drugs through compassionate use in the last 5 years in a specialty hospital. setting and method: descriptive retrospective study on a specialty hospital. all the applications for a compassionate use drugs were analysed from january 2011 until october 2015. the data were obtained from medical records programme diraya ò and from an excel database of medicines in compassionate use of the pharmacy service. main outcome measures: the following variables were registered: • number of patient clinic history • authorized medicine • authorization date • applicant service results: we recorded 80 requests of cancer drugs in compassionate use during the 5 years of study. oncology was the service that recorded more authorizations with 95%, followed with gynaecology with 2.5% and finally endocrinology and haematology with 1.25%. 64 drugs of the 80 requests were approved (80%) and 16 unauthorized (20%) in the 5 years of study. the year in which more applications were received was 2013 (31.25%) and the least requests were received in 2012 (6.25%), being the year where all requests were authorized. in 2015 fewer applications were authorized, 75%. in the years 2011, 2013 and 2014 were authorized 88.3, 76 and 88.3%, respectively. a total of 34 different active drugs were received during the study, the most requested bevacizumab (24%) for grade iii oligoastrocytoma, ovarian cancer (monotherapy), metastatic gall bladder cancer and metastatic platinum-resistant ovarian cancer, everolimus (18%) for indications of neuroendocrine carcinoid tumour and metastatic breast cancer, nab-paclitaxel (18%) for invasive lobular carcinoma indications of high-grade and metastatic pancreatic cancer, ipilimumab (12%) for the indication of metastatic melanoma, and regorafenib for indications of colorectal cancer and metastatic gist i pre-treat with imatinib (12%). the solicitude of drugs through compassionate use needs effective commissions of pharmacy and therapeutics, along with the medical management to establish an agile and faster requesting circuit and the consequent use monitoring. please specify your abstract type: research abstract background and objective: to describe the standard procedure for the elaboration and control of a magistral formula (mf) to assess their effectiveness in two patients with cutaneous metastases of malignant melanoma refractory to other treatment. setting and method: medication for compassionate use was requested for two patients of 78 and 49 years with histopathologic diagnosis of cutaneous metastases of malignant melanoma in the left thigh and left heel in which the lack of response to first-line treatments made to be valued to start with adesleukina intralesional therapy. the first week was infiltrated 3 mu (1 ml) in 5 lesions less than 1 cm, 9mu (3 ml) in the larger lesions and repeating each week until complete remission of the lesions. in the 2nd patient we proceed in the same way but the second week was infiltrated 5mu (8 ml). the following week, infiltrated 9 mml, in 15 metastases and we turn to 2 weekly infiltrations. the response was assessed by clinical disappearance of the lesions treated. complete response (cr) is defined as a clinical disappearance of lesions and partial response (pr) greater than 50% reduction of the lesion diameter. main outcome measures: we performed a literature search (pubmed, trissel, spc) for all studies published to determine the standard procedure for preparing and monitoring the mf (processing, preservation, stability, dose and indication). results: the standard procedure of preparation and quality control was carried out following the rules established in rd 175/2001. it was made in a vertical laminar flow cabinet. the aldesleukin vial was reconstituted with 1.2 ml api (18 mu/ml) and then diluted with 4.8 ml of a solution of 0.1% albumin, 5% glucose as stabilizer, to avoid aggregate formation, preparing 1 ml syringes (3 mu/ml). it was obtained a homogeneous and clear solution without precipitate or opalescence appearance. stable 6 days in a refrigerator (2-8°c), protected from light. initially patients had approximately a total of 60 injuries. after 2 months of treatment it was obtained a cr of most lesions in the first patient and rp of the second patient injuries. treatment was well tolerated. the side effects presented were only a flu-like syndrome in the second patient. conclusion: intralesional administration aldeslukina has been effective in treating malignant melanoma skin metastases in our patients, allowing the extension of its use in patients with the same involvement refractory to other primary treatments. the results are similar to those of the publications consulted. please specify your abstract type: research abstract background and objective: chronic infection with hepatitis virus c (hcv) affects about 170 million people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. the new direct acting antivirals against hcv have revolutionized the treatment of this disease. due to the high cost of these drugs it is necessary to assess their use in clinical practice. to evaluate the effectiveness of daclatasvir in combination with sofosbuvir in patients with hcv monoinfected in a specialty hospital. setting and method: retrospective observational study of patients who began treatment with the combination of daclatasvir and sofosbuvir from january 2015 to january 2016 in a specialty hospital. the data were collected from the hospital program of clinical stories archinet ò and the outpatient program farmatools ò . the results were analysed using the statistical program r-commander. main outcome measures: the sustained virologic response (svr) was considered the primary endpoint of the study. as secondary variables were analysed: sex, duration of treatment, naïve patients or pre-treated, degree of fibrosis, hcv genotype, concomitant use with ribavirin, viral load (vl) before treatment and medical service. results: there were included 28 patients of whom 23 were men. baseline characteristics were: 17 patients with genotype 3, 8 genotype 1b, 2 with genotype 1a and 1 genotype 4. the degree of fibrosis in the study was 15 patients with f4, f3 9 and 3 to f2. among the 17 patients infected with hcv genotype 3, 9 had not received prior treatment (naïve) and 8 had failed therapy. the duration of the treatment was 12 weeks to 16 patients and 24 weeks for 12 patients. only 7 patients receiving ribavirin of these 5 had genotype 3 and 2 genotype 1b. from ribavirin patients it was greater the number of patients in whom the treatment duration was 24 weeks (6 patients versus 1 with p-value = 0.008151). the digestive service attended to 17 patients while 11 patients were followed by infectious. the median cv was 3,067,940 iu/ml. svr was achieved in 81.2% of patients with hcv genotype 3 in 87.5% with genotype 1b and 100% with genotype 4 and 1a. after 12 weeks of treatment 61% of patients achieved svr and 39% after 24 weeks. only one patient died during treatment. the results are similar to those obtained in clinical trials. svr has not been influenced by hcv subtype, duration of treatment, degree of fibrosis, pre-treatment or by concomitant use of ribavirin. further studies are needed to evaluate the efficacy of this treatment. please specify your abstract type: research abstract background and objective: the safety and efficacy of medications can vary significantly between patients as a result of genetic variability. as genomic screening technologies become more widely available, pharmacists are ideally suited to utilize this tool to optimize medication management. the objective of this study is to evaluate the feasibility of implementing personalized medication services into community pharmacy practice and to assess the number of drug therapy problems identified as a result of pharmacogenomic screening. setting and method: the study was designed as open-label, nonrandomized, and observational. two community pharmacies in toronto, ontario offered pharmacogenomic screening as part of their professional services program. prior to initiation, participating pharmacists received structured, comprehensive training in pharmacogenetics. pharmacists then facilitated voluntary subject enrolment among patients who they believed would benefit from screening and met inclusion criteria. eligible patients received a simple buccal swab followed by dna analysis using pillcheck ò . pillcheck ò is a genotyping assay that translates genomic data and generates a personalized, evidence-based, report that provides insight into patients' inherited drug metabolic profile. upon receiving the report, pharmacists invited patients back to the clinic for interpretation of the results. clinically significant drug therapy problems were identified and recommendations for medication optimization were forwarded to the primary care physician. main outcome measures: number of clinically significant drug therapy problems identified by pharmacists as a result of pharmacogenomic testing. results: 100 patients were enrolled in the study. average age was 57.4 years and patients were taking a mean of 5.6 chronic medications. pharmacists cited the most common reasons for testing as ineffective therapy (44.6%), to address an adverse reaction (35.5%), and to guide initiation of therapy (11.8%). an average of 1.3 drug therapy problems were identified per patient. pharmacist recommendations included change in therapy (57.1%), dose adjustment (14.3%), discontinuation of a drug (7.1%), and increased monitoring (19.6%). generally, physician feedback was positive but did reveal an opportunity for a broader understanding of the technology. conclusion: these results highlight the readiness of community pharmacists to adopt pharmacogenetic screening into practice and their ability to leverage this novel technology to positively impact medication management. community pharmacists are ideally suited to both offer personalized medication services and interpret genomic results. please specify your abstract type: descriptive abstract (for projects) background and objective: visual impairment is a common geriatric syndrome and glaucoma/miotic eye drops treatment is a frequent therapeutic option. pharmacist's role in medication reconciliation is an effective process for reducing medication errors and supporting safe medication use. we observed that mentioned medication reconciliation was occasionally not performed during hospital stay and could be cause of delirium because of visual impairment. the aim of this study was to evaluate the influence of omission errors of eye drops treatment on incidence of acute confusional state. design: we conducted an observational, descriptive and retrospective study in an orthogeriatric unit with an average of 600 patients with hip fractures per year (95% surgically treated). data collection was performed from june 2015 to march 2016. reconciling medications at admission was performed by implementing the tools and resources of the canadian patient safety institute (cpsi). we extracted from our electronic database (filemaker pro ò ): • demographic patient data (age and gender). • name and posology of the glaucoma/miotic eye drops treatment. • medication reconciliation performed and identification of professional in charge (pharmacist, geriatrician or orthopaedic surgeon) registration during hospital stay. • protocolar management of delirium with tiapride occasional intramuscular administration performed if necessary was also registered to establish the incidence of acute confusional state. results: thirty-two patients (26 women and 6 men) were included, median age 86 year-old . in 21 patients, eye drops reconciliation treatment was performed by the pharmacist in 17 of the 21 patients, the geriatrician in 3 cases and the orthopaedic surgeon in 1. in 11 patients, the mentioned medication reconciliation was not performed (pharmacist absentism). considering the 21 patients on eye drops treatment during hospital stay, 4 (19.0%) of them suffer from acute confusional state. on the other hand, among the 11 patients without medication reconciliation, delirium was registered in 7 cases (63.6%). concerning ocular topic treatment, 2.4 ± 1.1 active principles per patient were observed, being the most frequent timolol (68.8%), brinzolamide (40.6%) and latanoprost (37.5%). conclusion: we consider of paramount importance the pharmacist evaluation availability at an orthogeriatric unit, minimizing the impact of acute confusional state during hospital stay by medication reconciliation. please specify your abstract type: descriptive abstract (for projects) background and objective: to report the therapeutic management of haemorrhagic rectocolitis onset in a lung-transplanted patient with mycophenolate-induced diarrhoea. design: case report. results: a 54-year-old-man lung transplant patient for alpha 1-antitrypsin deficiency in 2003 receiving mycophenolate mofetil, tacrolimus and corticosteroid developed chronic diarrhoea worsened by sigmoid and cecal necrosis in 2011, and treated successfully by sigmoidectomy. severe diarrhoea attributed to mycophenolate mofetil reappeared in april 2015, which motivated a switch to mycophenolate sodium. the absence of clinical improvement in june 2015 led to stop mycophenolate sodium and introduce azathioprine at 100 mg/day (absence of mutation for the thiopurine methyl transferase gene). one month later, the patient presented melena, diarrhoea, bloating, nausea, and knee pain, attributed to azathioprine. this latter was stopped and mycophenolate mofetil was rechallenged associated with symptomatic treatment (i.e., diosmectite and loperamide). in january 2016, a colonoscopy, performed in a context of profuse chronic diarrhoea with mucus during 3 months, highlighted haemorrhagic rectocolitis. therefore, the patient initiated sulfazalasine therapy with no clinical improvement, and then high doses of oral corticosteroids. because high-dose of oral corticosteroids was not recommended as a long-term treatment, mercaptopurine was proposed as a new therapeutic option. mercaptopurine has no indication as an immunosuppressive treatment in solid organ post-transplant supportive care. however, as the active metabolite of azathioprin, an immunosuppressive drug widely used in transplantation, mercaptopurine has immunosuppressive functions towards t-lymphocytes. after multiprofessional collaboration between gastroenterology, pneumology and pharmacy specialists, it was decided to stop mycophenolate mofetil and introduce mecaptopurine at 1.5 mg/kg/day, as immunosuppressant for haemorrhagic rectocolitis as well as lung transplantation. this unusual lung transplant immunosuppressive therapy, associated with tacrolimus, improved digestive disorders and patient's quality of life. currently, mercaptopurine is biologically and clinically well tolerated. the dosage of blood residual concentrations of purinethol metabolites (6-thioguanine and 6-methylmercaptopurine) is going to be performed. conclusion: immunosuppressive therapy in solid organ transplantation is a real challenge for patients who have comorbidity onset. despite a lack of data in the literature, a multidisciplinary collaboration based on comprehensive pharmacology skills is essential to choose the best therapeutic option in this type of patients. please specify your abstract type: descriptive abstract (for projects) background and objective: the use of complementary medicines (cm) in oncology is the subject of broad but still controversial interest. a large part of patients with cancer uses cm, including complementary drugs, during their treatment period. indeed, according to different studies, this proportion ranges from 18 to 83%. importantly, the risk of interaction between cm and anti-cancer drugs is not negligible; hence we need to identify these cm to ensure the security of our patients and the success of their treatment. design: to achieve this purpose, a monocentric retrospective analysis was conducted with collection of data by pharmacy students during medication reconciliation of hospitalized patients from january to june 2016. collected data are patients' characteristics, prevalence of cm use and potential cm-anticancer drug interactions. results: 161 patients were included in the study (91 men-70 women); median age was 65 [34-88 years]. a total of 24.2% (n = 39) were using a least one cm, most frequently homeopathy (62%, n = 24) or phytotherapy (36%, n = 14); some patients were using a combination of two cm (41%, n = 16). cm are mainly used by women in comparison to men (32.9% versus 17.6% and p = 0.025, chi square test). for phytotherapy, at least 36 different herbs were described by patients and among them the most frequently used were mistletoe (viscum album), propolis and fireweed (epilobium angustifolium). data analysis showed that 23% (n = 9) of patients were at risk of potential cm-anticancer drug interaction. moreover this risk was increased to 50% if we considered only patients taking phytotherapy. interactions included pharmacokinetic (15%, n = 3), such as altered hepatic metabolism, and pharmacodynamics ones (85%, n = 17). conclusion: in conclusion, our work clearly demonstrates that the use of cm by patients is associated with high risk of relevant drug interaction with their anti-cancer treatment. even if further investigations are necessary to clarify the clinical impact of these interactions, the use of cm must be considered during prescribing process. please specify your abstract type: research abstract background and objective: since their reimbursement, the direct oral anticoagulants (doacs) are increasingly used for stroke prevention in atrial fibrillation (af). the objective of this study was to identify the proportion of real life patients with af eligible for doac therapy, based on the inclusion and exclusion criteria used in the clinical studies and based on the officially approved indications as mentioned in the summary of product characteristics (smpc). setting and method: data for this retrospective cross-sectional study was extracted from the uz brussel stroke registry, containing anonymized data of 2205 patients with a suspected stroke. characteristics of patients with documented af were compared with the patient characteristics in clinical trials and the approved indications in the smpc. main outcome measures: proportion of real life patients with af eligible for doac therapy. results: data of 468 patients with af was analysed. based on the selection criteria of the clinical trials, significantly less patients were eligible for treatment with rivaroxaban compared to dabigatran etexilate (39.3% versus 47.6%; p = 0.010), but not compared to apixaban (45.5%; p = 0.055). based on the indications and contraindications in the smpc, significantly fewer patients were eligible for apixaban compared to dabigatran etexilate and rivaroxaban (62.0% for apixaban, 72.9% for dabigatran etexilate and 75.6% for rivaroxaban; p \ 0.001 and p \ 0.001, respectively). significantly more patients were eligible for doac therapy based on the indications and contraindications in the smpc compared to the inclusion and exclusion criteria of the clinical trials (72.9% versus 47.6%; p \ 0.001 for dabigatran; 75.6% versus 39.3%; p \ 0.001 for rivaroxaban and 62.0% versus 45.5%; p \ 0.001 for apixaban). conclusion: when taking into account the selection criteria from the pivotal clinical trials with doacs for stroke prevention in af, less than half of real life patients are eligible for therapy with one of the doacs. however, the indications mentioned in the smpcs of these drugs are less strict. please specify your abstract type: research abstract background and objective: idiopathic pulmonary fibrosis (ipf) is a disease in which tissue deep in the lungs becomes thick and stiff, or scarred, over time. the formation of scar tissue is called fibrosis. pirfenidone is an anti-fibrotic and anti-inflammatory agent, thus offers a new hope for treating progressive fibrotic diseases. int j clin pharm (2017) 39:208-341 333 our objective is to set a description of idiopathic pulmonary fibrosis patients treated with pirfenidone, as well as the adverse reactions observed. setting and method: descriptive study in which all patients have received pirfenidone. the data were obtained through the dispensing program of outpatient (farmatools) and review of medical records of the hospital database (archinet) and clinical station (diraya). main outcome measures: we have extracted from each patient baseline data, comorbidities, dose received, reported adverse reactions and data about haematology and biochemistry. results: we have a total amount of 5 patients treated with pirfenidone, all diagnosed with idiopathic pulmonary fibrosis, including 2 women and 3 men. the age of patients is between 66 and 81 years, with an average of 70.8 years. all patients are ex-smokers and one of them is also ex-alcoholic. concerning concomitant pathologies, 3 patients have diabetes mellitus, 2 have arterial hypertension, and one of them has ischemic heart disease. another has upper gastrointestinal bleeding prior, among others chronic pathologies. pirfenidone dose received was the usual dose in 4 of the 5 patients: days 1-7 267 mg every 8 h, days 8-15 534 mg every 8 h and a maintenance dose of 801 mg every 8 h. in one patient due to its low imc the dose received was smaller (1-7 days 267 mg every 12 h, days 8-15 267 mg every 8 h and maintenance dose of 534 mg every 8 h). in relation with the adverse effects, digestive discomfort were observed in 2 of the 5 patients, causing the interruption of the treatment in one of them (with prior gastrointestinal bleeding). in the other patient it was relieved by lowering the dose received. also, one patient has experienced photosensitivity. alterations in transaminase levels were observed in 2 patients but that didn't force to discontinue the treatment. no alterations were observed in the blood count. conclusion: treatment with pirfenidone is being generally well tolerated by patients. it has improved their life-quality and reached the objective data of a slowdown in disease progression. currently, the number of patients is no enough to give conclusive information in relation to the drug effectiveness. please specify your abstract type: research abstract background and objective: to describe the total amount of patients treated with a magistral formula of sodium cromoglycate 200 mg without excipients: indications, concomitant therapy and the response to therapy. setting and method: we run a descriptive study in which we included the totality of patients in treatment with a magistral formula of sodium cromoglycate 200 mg without excipients in a tertiary hospital. the data were obtained through paracelso (development of magistral formulas program), as well as with farmatools (dispensation program of outpatient) and the review of medical records from the hospital database (archinet), and diraya clinical station. main outcome measures: from each patient we extracted data relative to sex, age, diagnosis, time in treatment with the formula, dose received, response to therapy, concomitant antihistamines treatments and adverse effects. results: a total of 9 patients in treatment with a magistral formula of sodium cromoglycate 200 mg without excipients were reviewed: 2 women and 6 men with a mean age of 45.3 years old (range 38-59 years). regarding the indication of the prescription, 3 patients have been diagnosed of indolent systemic mastocytosis and the remaining 5 were diagnosed of mast cell activation syndrome. in all cases, the diagnosis was established by examination of the bone marrow in the mastocytosis studies institute of castilla la mancha (spain). on average, patients took the treatment 12.75 months, with a range between 3 months and 24 months. the dose received was 200 mg every 8 h in 7 patients, having to be increased to 400 mg 3 times daily in a case with poor response to the therapy. in the remaining patients, the treatment response has been optimal. in relation to the concomitant anti-allergic treatment received, 6 patients took fexofenadine daily during the study. no cases of adverse effects related to the therapy received have been reported. conclusion: both indolent systemic mastocytosis and mast cell activation syndrome are considered rare diseases, and we should indicate that in spain there are no commercial medicines available of sodium cromoglycate without excipients for its treatment. the treatment with this magistral formula of sodium cromoglycate 200 mg without excipients has been effective and well tolerated in all patients, improving the symptoms associated with their condition as well as their quality of life, and also, assuming a solution to the lack of marketing of the drug currently in spain. please specify your abstract type: research abstract background and objective: to analyse the prescription profile, safety and effectiveness of new therapies available for the treatment of hcv genotype 1b in a tertiary hospital. setting and method: we run a retrospective observational study in which we included a total amount of 59 patients infected with hcv genotype 1b treated with the new therapies against hcv from february 2015 to december 2015 in a tertiary hospital. the data were obtained through the outpatient dispensing program farmatools and the review of the medical records from the hospital database, archinet and prescription hepatitis c portal of the andalusian health service. main outcome measures: from each patient the following information was collected: sex, age, viral genotype (gen.), naive/nonnaive, hiv coinfection, presence of cirrhosis, degree of hepatic fibrosis measured by fibroscan, treatment prescribed and duration, adverse effects, sustained viral response (svr) and the service that made the prescription. results: a totality of 59 patients with hcv gen. 1b were reviewed which 64.4% of them were men with a mean age of 58.76 years (range 38-78 years). 21 of the patients were naive and only 2 of them were hiv co-infected, there were a 57.63% of cirrhotic patients. regarding the degree of hepatic fibrosis, 36 patients had grade f4, f3 grade 13 patients, 8 patients grade f2 and f1 grade 2 patients. the most commonly therapy prescribed was lepidasvir + sofosbuvir in 28 patients (14 without ribavirin and with ribavirin 14) using a treatment schedule of 12 weeks in 23 of them. the treatment was discontinued in one case because of the adverse effects, achieving svr in the remaining patients. the combo treatment with paritaprevir/ombitasvir/r + dasabuvir was prescribed in 16 times (10 without ribavirin and 6 with ribavirin) choosing only in one of them for a treatment period of 24 weeks. there were no treatment discontinuations and svr was achieved in all patients treated in this way. 8 patients received simeprevir + sofosbuvir for 12 weeks (3 without ribavirin and 5 with ribavirin), one patient of the left the treatment due to adverse effects. svr was found in the remaining patients who completed treatment. sofosbuvir + daclatasvir was prescribed to 5 patients, associating ribavirin in only one case. a treatment duration of 12 weeks was used in 3 patients and 24 weeks in the remaining two. one patient failed rvs without any incidences of adverse effects in any case. interferon + ribavirin sofosbuvir + was prescribed to 2 patients in 12-week regimen which was well tolerated achieving svr. digestivo service treated the 83% of the total amount of patients. conclusion: new therapies for hcv have been used in all the treated patients and the older drugs have been relegated. about the effectiveness, svr was achieved in 98.30% of patients. regarding the safety, only 2 patients have discontinued the treatment due to adverse effects representing less than 5% dropout rate of the therapy. please specify your abstract type: descriptive abstract (for projects) background and objective: thanks to pharmacogenetics we can identify and predict different responses to the same drug among different individuals. during these last years we have noted a big increase of dosing guidelines and advices about the use of several drugs due to the influence of different polymorphisms. the aim of this study is to describe and evaluate the use of pharmacogenetics in our hospital from april 2012, when we started our first research about pharmacogenetics, to the actual time, using these information in our daily clinical practice; and indeed quantify the number of different tests and the number of different clinical advices done because of pharmacogenetic information, by different healthcare specialty areas and drugs. design: we reviewed all the pharmacogenetic test requests in our hospital from april 2012 to april 2016, noting which health specialty and for which drug was asked the test. polymorphisms were genotyped using taqman ò genotyping assays technology by 2 independent laboratories to confirm the results. results: from april 2012 we were asked for 2208 pharmacogenetic tests from 7 different healthcare specialty areas: rheumatology (9.78%), infectious diseases (1.49%), oncology (3.53%), cardiology (71.51%), vascular surgery (5.11%), neurology (4.53%), ophthalmology (4.03%); this information was asked about 5 different drugs: clopidogrel (81.16%), trastuzumab (3.53%), ranibizumab (4.03%), azathioprine (2.99%) and tocilizumab (8.29%). from all the genotypes, 713 (32.29%) were done after using the drug (study phase) and 1495 (67.71%) were done previous to the use of the drug in daily clinical practice to make a ''clinical recommendation''; from these recommendations 1429 affected to the prescription of clopidogrel. conclusion: during the last 4 years we could implement the use of pharmacogenetics in the daily clinical practice in our hospital in 5 different healthcare areas affecting 2 drugs and we started research studies previous to its use on the clinical practice for other three different drugs. please specify your abstract type: descriptive abstract (for projects) background and objective: the drug burden index (dbi) is a tool used to quantify the anticholinergic and sedative burden of medication on an individual. it has been independently associated with poor physical and cognitive performance in community-dwelling older people. objectives were: to create an inventory of medications used in ireland with clinically significant anticholinergic and/or sedative activity and to decide upon the minimum daily dose (mdd) for each medication. design: medications with potential anticholinergic and/or sedative burden were identified by literature review and examination of the summary of product characteristics (smpc) for all medications registered in ireland. each medicine was classified as anticholinergic or sedative. drugs with both anticholinergic and sedative properties were classified as primarily anticholinergic. the mdd, a key component of the dbi score calculation, was selected by reference to the irish smpc. other options which were also considered for this value include the defined daily dose (ddd) of a medication, as available from the world health organisation (who), and the mdd as outlined in the british national formulary (bnf). mdds were decided upon regardless of indication as the lowest effective therapeutic dose as specified in the smpc for the medication. the final list of medicines and mdds to be included in the inventory was then defined by consensus of three pharmacists. results: in total, 383 medicines with potential anticholinergic and/or sedative activity were considered for inclusion. a final list of 258 medications was identified by consensus (117 anticholinergic, 141 sedative). of these, 128 (50%) were agents which act primarily on the nervous system. the three main therapeutic groups contributing to the inventory of dbi medications were antipsychotics (25 medications), antidepressants (21 medications) and antiepileptics (20 medications). conclusion: creation of an inventory of medications with anticholinergic and/or sedative properties, in combination with the individual mdds, was achieved. this is a useful resource for use in analysis of drug burden in an older population. it could help in both identifying patients who would benefit from medication review as well as analysing population medication data. please specify your abstract type: research abstract background and objective: vancomycin is an antibiotic widely used to treat infections such as bacteraemia, infective endocarditis, osteomyelitis, meningitis and pneumonia. nowadays, optimal trough concentration is stablished between 10 and 15 mg/l to avoid development of resistance or 15-20 mg/l to improve penetration in complicated infections. some articles 1 have been published explaining the methodology to calculate an expected trough level in steady state. our aim was to compare the trough serum value estimated by the mathematical method with a two-compartimental bayesian forecasting model. setting and method: observational retrospective study carried out in a tertiary hospital from january to december 2015. non obese adult patients with creatinine clearance (crcl) \ 120 ml/min and who have achieved steady state level were included. vancomycin serum values were measured using a chemiluminescence's immunoassay (cmia) and bayesian analysis was performed with abbottbase pksystem ò (pks ò ). the statistical analysis was made with medcalc software ò . bland-altman plot and passing-bablok regression were used to compare both methods. main outcome measures: sex, age, weight, dose, creatinine, and size were collected from clinical history. serum trough values (cminr) were collected from cmia. trough values were estimated using two methods: mathematical method (cminf) and bayesian calculations (cminb). results: 50 patients were included, with a mean age of 61 (±16.17) years. 52% were male and 48% female. they received a median dose per 24 h of 2000 (1000-3000) mg. the mean of cminr was 17.21 mg/l (95% ci 13.86-20.58), cminb 17.28 mg/l (95% ci 13.87-20.69), cminf 18.21 (95% ci 14. 2399-22.1856) . correlation coefficients (r) comparing both methods were significantly different: r between cminf and cminr was 0.75 (95% ci 0.5866-0.8467), while r between cminb and cminr was higher: 0.99 (95% 0.9900-0.9968). bland-alman plot analysis showed both methods cannot be used interchangeably. the regression equations estimated by passing-bablok regression were y = -3.873368 + 1.309775x and y = -0.110207 + 1.003266x. conclusion: bayesian method has demonstrated better correlation with real measures than mathematical method. most part of our patients could be underestimated or overestimated using mathematical methods which could cause toxicity or lack of efficacy, so this method is unsuitable for clinical use. bayesian estimation remains the best option for optimal dosing of vancomycin. please specify your abstract type: research abstract background and objective: combination therapy with digoxin and acenocoumarol is common in patients with atrial fibrillation (af). getting optimal concentrations of digoxin leads an appropriate response; taking into account its narrow therapeutic range and all the factors which can affect to its pharmacokinetics. interaction between them has been studied, even though its mechanism is not clear yet. patients who are taking both drugs need higher doses of digoxin; because they get lower concentrations by using the same dosage. the objective of this study was to analyse digoxin concentrations in patients treated with this combination compared to expected concentrations according to population parameters. setting and method: retrospective observational study from december 2015 to march 2016 performed by pharmacokinetic unit. patients included had chronic treatment with acenocoumarol and digoxin, which determination were realized in the steady state before the next dosage. patients with toxics concentrations of digoxin, or who were suspected nonadherence, were excluded. the plasma digoxin concentrations were determined through the autoanalyzer architect c-4000 ò (petinia). dosage adjustment was realized by the program abbot pharmacokinetics system (pks). a comparative between the real measured concentrations in patients and estimated concentrations were realized based on population parameters. finally, in order to get optimal concentrations, some dosage changes were proposed based on pharmacokinetic monitoring. data collected: population characteristics (gender, age, weight, and height), analytical data (potassium, urea, creatinine and clearance). main outcome measures: digoxin serum concentrations (optimal range 0.8-1 ng/ml). results: data from 73 patients, 65.8% women with a mean (sd) age of 77.84 (9.07) years were included in the study. at baseline, potassium, urea, creatinine and clearance mean (sd) was 4.43 (0.52) mmol/l; 55.99 (34.05) mg/dl; 0.96 (0.39) mg/dl; 48.76 (22.14) ml/min. 69.86% of the patients had lower concentrations than expected according to population parameters. finally, digoxin dosage was increased in 41.07% of patients, it was maintained in 47.96%, and it was decreased in 10.97%. conclusion: digoxin concentrations in patients with af in combination therapy of digoxin and acenocoumarol are lower than would be expected in most cases. it is important monitoring digoxinaemia to achieve optimal concentrations and a good clinical response. further studies are needed to determine the relevance of this interaction in clinical practice. please specify your abstract type: research abstract background and objective: tocilizumab (tcz) is a humanized monoclonal antibody inhibitor of il-6 receptor, indicated in combination with methotrexate in the treatment of rheumatoid arthritis (ra) in patients with inadequate response or intolerance to prior therapy. interleukin 6 is involved in the pathogenesis of rheumatoid arthritis via its broad effects on immune and inflammatory responses. previous studies have shown that c-allele at the -174g[c (rs1800795) polymorphism is related with a bad response to tocilizumab (according to eular criteria). the aim of our study was to explore the potential role of il-6 genetic polymorphisms as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients and check this association depending on the genotype. setting and method: the il-6 (g[c) (rs1800795) genetic variant was genotyped using predesigned taqman ò genotyping assays technology and analysed on a viia7 ò real-time pcr system. main outcome measures: clinical response was evaluated at 3, 6, 9 and 12 months according to the eular criteria. patients were classified as ''responders'' (good and moderate response according to eular criteria) and ''non-responders''. the statistical analysis was performed using spss v.20. results: we recruited 163 patients with ra treated with tocilizumab, these were aged 54.02 ± 11.11 (mean ± sd), 132 (81%) were women. the mean das28 at baseline was 5.66 ± 1.14. of these 163 patients, the il-6 g[c genetic polymorphism was significantly associated with ''responders'' at 3 months after the baseline (cc vs non-cc p = 0.039, or 0.270, 95% ci 0.072-1.005) but not at 6 (p = 0.666), 9 (p = 0.233) and 12 (p = 0.244) months. conclusion: the il-6 g[c may be useful as a genetic marker of tocilizumab efficacy at 3 months. other polymorphisms, clinical parameters and other pharmacological treatment during the follow-up may be checked about their influence on the response to tocilizumab. tdmp014: daptomycin pk/pd profile in neutropenic cancer patients with beta-lactam-resistant gram-positive infection nancy perrottet *,1 , frederic tissot 2 , laurent decosterd 3 , thierry buclin 3 , guy prod'hom 4 , christina orasch 2 , oscar marchetti 2 , farshid sadeghipour 1,5 , thierry calandra 2 , véronique erard 2 1 pharmacy service, 2 infectious diseases service, 3 laboratory and division of clinical pharmacology, service of biomedicine, 4 institute of microbiology, lausanne university hospital, lausanne, 5 school of pharmaceutical sciences, university of geneva, university of lausanne, geneva, switzerland please specify your abstract type: research abstract background and objective: the pharmacokinetics (pk) and pharmacodynamics (pd) of many antibiotics are modified in neutropenic patients and few data are available on daptomycin in this population. this prospective study aimed to assess the pk/pd profile of daptomycin in the treatment of neutropenic patients with beta-lactamresistant gram-positive cocci infections. setting and method: this substudy was performed in the context of a prospective pilot study on daptomycin versus vancomycin in adult hemato-oncological patients with febrile neutropenia and proven or suspected infection with methicillin-resistant staphylococci or betalactam-resistant enterococci. patients received daptomycin 6 mg/ kg/day (8 mg/kg/day for enterococci) for c7 days as a 2-min infusion. main outcome measures: pk analysis using a published non-linear mixed effect model with nonmem ò , followed by comparison of parameters with values published for healthy subjects. pd analysis based on auc/mic (area under the concentration-time curve/minimal inhibitory concentration). according to eucast, an auc/mic ratio [438 is required for bacteriostatic effect against staphylococci and [800 for a two-log reduction in bacterial count. for e. faecium, an auc/mic ratio of 0.94 has been suggested for bacteriostasis and 4.14 for a 1-log bacterial count reduction. results: model-derived mean auc observed in 13 patients was 539.3 ± 340.1 mg h/l, maximum concentration (cmax) 88 ± 28 mg/ l, minimal concentration (cmin) 7.2 ± 6.7 mg/l. clearance was 0.98 ± 0.36 l/h and volume of distribution at steady sate 11.3 ± 3.3 l, both values found higher than those reported in healthy subjects. all patients (7/7) with a staphylococcal infection achieved auc/mic values predictive of bacteriostatic effect on staphylococci, and 6 out of 7 values associated with two-log bacterial killing. of note, infection relapse occurred in the only patient with suboptimal daptomycin exposure (auc/mic of 580). the pd targets were also reached in the two patients with e. faecium infection. an asymptomatic elevation of creatine phosphokinase was reported in two patients (568 u/l and 218 u/l) with cmin of 25.7 and 13.7 mg/l, respectively. conclusion: daptomycin pk profile in 13 neutropenic cancer patients indicated higher total clearance and volume of distribution, along with lower total exposure, compared to healthy subjects. despite this, standard dosages allowed attainment of pd targets in 6/7 patients with a staphylococcal infection (two-log drop) and 2/2 with e. faecium infection (1-log drop) . please specify your abstract type: research abstract background and objective: individual clinical response to infliximab can be influenced by their pharmacokinetics and immunogenicity, so therapeutic monitoring of drug levels (tdm) can guide these biologic treatments. the objective was to analyse the suitability of serum infliximab trough levels (sitls) in patients with inflammatory bowel diseases (ibd) receiving dose schemes based only on clinical response. setting and method: prospective and descriptive study of patients with ibd treated with infliximab and under tdm. medical records were reviewed. dose schemes were established according to clinical guidelines (5 mg/kg every 8 weeks) and optimized based on an index of clinical response (mayo, pcr…). sitls (therapeutic range 3-9 mcg/ml) and anti-drug antibodies (ada) were measured in all of patients by elisa (promonitor ò ). ada presence was considered as a therapeutic failure indicator. informed voluntary consent was obtained from all patients. main outcome measures: sitls and ada. results: a total of 61 patients, with a median age of 48 years (range ), were included in the analysis. infliximab standard dose according to clinical guidelines were administered to 39 patients: 46.1% showed sitls under the therapeutic range (11.1% with ada). in eight patients with maintained good clinical response, dose decrease or interval elongation had been implemented: 25% of these patients showed sitls below the therapeutic range (100% with ada). it had been necessary to increase the dose or shorten the interval in 14 patients due to inadequate clinical response: 28.6% of these patients with sitls below the therapeutic range (50% with ada). conclusion: optimization based on clinical response of infliximab treatments in patients with ibd is not always an effective strategy, since it leads to a high percentage of patients with sitls below the therapeutic range and adas. tdm together with clinical response should guide the optimization of infliximab treatments. please specify your abstract type: research abstract background and objective: in addition to its anticonvulsive properties, valproate is also used as a mood stabiliser in bipolar disorder and as augmentation treatment of other psychiatric disorders. the unpredictable relationship between dose-plasma valproate concentrations and correlation between concentrations-efficacy suggest therapeutic drug monitoring (tdm) of plasma valproate concentrations might be useful. the aim of our study was to evaluate the rationale of a new protocol for measuring valproate concentrations and the incorporation of a clinical pharmacist in the process of valproate tdm service, compared to pre-existing standard measuring. setting and method: in the retrospective study we analysed the process of measuring plasma valproate concentrations at the department of psychiatry and at the unit for forensic psychiatry of a large teaching hospital in slovenia before the enrolment of a clinical pharmacist. for the prospective study we created a protocol for tdm of valproate in adults based on literature research. the protocol included reference range, sampling time, indications for sampling and schedule of other laboratory tests that have to be monitored during valproate therapy. main outcome measures: percentage of plasma valproate concentrations in reference range (c trough = 50-125 mg/l) before/after the enrolment of a clinical pharmacist, percentage of measured valproate c trough . results: in the retrospective study 30 randomly chosen patients with measured plasma valproate concentrations were included (56% male, age 49 ± 15 years, length of hospital stay 56 ± 40 days). plasma valproate concentrations were measured 5.8 ± 4.4 times per patient, 22% were in the reference range (other 78% subtherapeutic), 3% were drawn at c though , 15.5% were drawn for assessing compliance (nontrough). in the prospective study 19 patients were included (37% male, age 46 ± 16 years, length of hospital stay 36 ± 22 days). plasma valproate concentrations were measured 2.95 ± 1.5 times per patient, 43% were in the reference range (other subtherapeutic), 71% were drawn at c trough , 14.3% were drawn for assessing compliance. conclusion: the inclusion of a clinical pharmacist in valproate tdm service increased the number of valproate plasma concentrations in the reference range by almost 100% and increased the number of concentrations drawn at c trough , when indicated. including a clinical pharmacist in valproate tdm is beneficial and the new protocol is useful for optimising valproate therapy. concurrent and predictive validity of a self-reported measure of medication adherence the effect of pharmacist-led interventions in optimising prescribing in older adults in primary care: a systematic review aflibercept: 1. neovascular membranes with visual acuity higher than 0.1 2. one eye affection severe cardiovascular pathology (severe episodes in the last 6 months) non-responders to other anti-vegf bevacizumab: 1. diabetic macular edema macular edema secondary to vascular pathology setting and method: a longitudinal study was carried out in primary care centres. participants: patients aged c65, under treatment with 5 or more drugs and belonging to 7 primary care areas in 5 different towns. patients should have at least one of the following potential safety problems: (a) concomitant use of a non-steroidal anti-inflammatory drug (nsaid) with an antihypertensive drug, anticoagulant or antithrombotic drug; (b) use of two or more benzodiazepines. two clinical management units (cmu) were randomized per area to be included in the study. thirty patients per cmu were randomized to be enrolled and monitored during 30 months number of adverse effects (0.40; p \ 0.001) and number of clinical problems (0.19; p \ 0.001).with each year increase in age 026) and a significant rise in physician (0.08; p \ 0.001) and nurse (0.06; p \ 0.001) home visits. women compared to men resulted in a significant decrease 043) but a significant increase in visits to nurses (0.43; p \ 0.001), hospital admissions (0.39; p \ 0.053) and hospital visits (0.35; p \ 0.030). age, sex and npsp had no significant effect on falls, fractures or cardiovascular events. conclusion: the npsp in elderly patients contributes to an increase in the use of health services and comorbidity. effective interventions should be addressed to general practitioners to reduce inappropriate prescriptions bpa was found in the dialysate (39 ng/l) and ls (1033 ng/l) wherein the concentration of bpa decreases over time to reach 265 ng/l at the end of a session. finally, bpa was present in all tested dialysis at concentrations of up to 149.0 ng/dialyzer in the compartment mimicking the blood and to 174.0 ng/dialyzer in the dialysate despite prior rinsing with 2l of 0.9% nacl. conclusion: our study is the first one to show the risk of exposure to bpa and bpa-clx hdf-ol. while assessment of the impact of this exposure in a patient under treatment remains to be done, it is now possible to better master contamination by bpa and its four chlorinated derivatives through better practices (choice of medical devices) and improvement of the overall water treatment process san cecilio university hospital, 2 genomic unit san cecilio university hospital, 2 genomic unit, genyo, centre for genomics and oncological research the aim of this study is to compare the apparition of stroke, acs, cardio-vascular death and the need of surgery in patients after percutaneous transluminal angioplasty (pta) or stroke depending on the presence of cyp2c19*2*3 polymorphisms. setting and method: retrospective cohort study. we recruited patients treated with clopidogrel after a pta of the lower limb or stroke (without surgery) from 2010 to 2013 in our hospital. data collected: age, sex, cyp2c19*2 (rs4244285) and cyp2c19*3 (rs4986893) genotypes and the primary end-point: stroke, acs, cv death and surgery of the affected vessel during 12 months after discharge. polymorphisms were genotyped using taqman ò genotyping assays technology. main outcome measures: we recruited 58 patients with stroke (62.07% men; mean age 68.30) and 72 patients after pta (77.7% men; mean age 67.44) treated with clopidogrel after discharge 8%) suffered the primary end-point during 12 months after discharge; 1 of these patients had the cyp2c19*2 allele. among patients with pta of the lower limb: 25% of them had the cyp2c19*2 allele and no one a cyp2c19*3 allele; 25 (34.72%) of these 72 patients suffered the primary end-point during 12 months after the discharge and 11 of these had the *2 allele of the cyp2c19 isoenzyme *2 allele and treated with clopidogrel have a higher risk of the primary end-point than those patients not carrying it spain please specify your abstract type: research abstract background and objective: the engagement of fcgrs by tnf antagonists could affect to macrophage-mediated clearance of immune-complexes. the aim of our study was to evaluate the potential role of fcgr3a (a[c) (rs366911) single nucleotide polymorphism (snp) as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients. setting and method: the fcgr3a (a[c) (rs366911) snp was genotyped using predesigned taqman ò genotyping assays technology and analysed on a viia7 ò real-time pcr system. the statistical analysis was performed using spss v the mean age of the patients was 53.25 ± 12.42 years and 79% were women. the mean das28 at baseline was 5.71 ± 1.13. we found no statistically significant association between our end-point and the genetic polymorphisms studied tdmp011: therapeutic drug monitoring of infliximab biosimilar and anti-infliximab antibodies in inflammatory diseases patients with dermatological conditions and inflammatory bowel disease being treated with ifx-b (5 mg/kg/8 weeks after the induction dose) were included. the concentrations of ifx-b and ati-b were quantified by two sandwich-type elisa immunoassays (triturus ò analyser). main outcome measures: plasma levels of ifx-b and ati-b, clinical response and infusion reactions. the clinical response was assessed according to pathology of each patient (based on specific clinical variables for the pathology into the electronic history) pharmacokinetic results (% assessments): (a) 30.0% no ifx-b detection (c \ 0.035 mcg/ml) and positive ati-b (c [ 2 ua/ml) (3 assessments/1 patient). atis = 114, 282 y 309 ua/ml. no clinical response (nr) in 66.6% assessments. (b) 10.0% ifx-b and ati-b (c b 2 ua/ml) no detection (1 assessments/1 patient). nr 0%. (c) 60.0% ifx-b detection (c [ 0.035 mcg/ml) and negative ati-b (6 1 assessments/4 patients) weight: 63 (21-90) kg. twenty assessments, 2.5 (1-5) assessments per patient, 4 (4-8) ifx-b doses, 80% concomitant treatment (16/16-azathioprine, 8/16-corticosteroid) the incidence of ati-b was low. a correlation was observed between the presence of ati-b and loss of clinical response, as infliximab original. tdmp012: serum concentration of non-vitamin k antagonist oral anticoagulants (noacs) in older hip fracture patients ina linnerud 1 , mette i martinsen 2 estimation of t of noacs by t1/2 = ln2/kel [kel; elimination constant] using two s-concentration measurements. results: we included 167 patients (median age 84 years, 73.1% women). noac use was detected be serum analysis in 11 patients (6.6%; 100% coherent with mr), while 15 patients (9.0%) used warfarin. 7 of the 11 noac users (63.6%) had s-concentrations of noacs above the reference range at admission, and five patients (45.5%) had s-concentrations within the reference range before surgery. patients using noac had significantly longer median waitingtime for surgery than warfarin-users (50 vs 36 h, p = 0.004). blood transfusions were given to 36.4% of noac-users vs 21.4% of warfarin-users (p = 0.651). mean estimated t of noacs were 33, 16.5 and 14.5 h for dabigatran (n = 2), apixaban (n = 4) and rivaroxaban (n = 2), respectively. conclusion: mr is effective in detecting noac use in older hip fracture patients, but importantly s-concentrations are higher than expected in this population. this might reflect the significantly longer waiting-time for surgery this column is supplied with packing material made of totally porous spherical silica coated with a silicone polymer monolayer containing octadecyl (c18) groups. the mobile phase was composed of 0.5% na 2 po 4 h 2 o (ph 2.5), acetonitrile, and methanol (55:25:20, v/v/v), which was degassed in an ultrasonic bath prior to use. the flow rate was 1.0 ml/min at ambient temperature and sample detection was carried out at 250 nm. plasma samples were obtained from 11 patients with cml receiving nilotinib treatment. sampling was performed at the steady state. blood samples were collected by venipuncture 24 h after oral administration of nilotinib. plasma was separated by centrifugation at 1900 9 g for 15 min and stored at -40°c until analysis. plasma samples (100 ll) were then extracted as described above. the same samples were also sent to a commercial laboratory (bml, inc.) for assaying nilotinib concentration by liquid chromatography-tandem mass spectrometry (lc-ms/ms). in addition, we applied this method to tdm of cml patients receiving nilotinib at our hospital. main outcome measures: the calibration curve exhibited linearity over the nilotinib concentration range of 50-2500 ng/ml at 250 nm, with relative standard deviations (n = 5) of 7.1, 2.5, and 2.9% for 250, 1500, and 2500 ng/ml, respectively. the detection limit for nilotinib was 5 ng/ml due to three blank determinations (q = 3). in addition, we compared the results with those measured by lc-ms/ ms at bml, inc. (a commercial laboratory). as a result, a strong correlation was observed between the nilotinib concentrations measured by our hplc method and those obtained by lc/ms-ms (r 2 = 0.988, p \ 0.01). in addition, tdm of nilotinib was performed to six cml patients. there was the case which participated in dosage adjustment of nilotinib in hepatic dysfunction and poor glycaemic control. results: we have developed a simple ultraviolet detection method for the determination of nilotinib, which has high sensitivity and large dynamic range please specify your abstract type: research abstract key: cord-076081-ue9azoyf authors: hardon, anita; desclaux, alice; egrot, marc; simon, emmanuelle; micollier, evelyne; kyakuwa, margaret title: alternative medicines for aids in resource-poor settings: insights from exploratory anthropological studies in asia and africa date: 2008-07-10 journal: j ethnobiol ethnomed doi: 10.1186/1746-4269-4-16 sha: doc_id: 76081 cord_uid: ue9azoyf the emergence of alternative medicines for aids in asia and africa was discussed at a satellite symposium and the parallel session on alternative and traditional treatments of the aidsimpact meeting, held in marseille, in july 2007. these medicines are heterogeneous, both in their presentation and in their geographic and cultural origin. the sessions focused on the role of these medications in selected resource poor settings in africa and asia now that access to anti-retroviral therapy is increasing. the aims of the sessions were to (1) identify the actors involved in the diffusion of these alternative medicines for hiv/aids, (2) explore uses and forms, and the way these medicines are given legitimacy, (3) reflect on underlying processes of globalisation and cultural differentiation, and (4) define priority questions for future research in this area. this article presents the insights generated at the meeting, illustrated with some findings from the case studies (uganda, senegal, benin, burkina faso, china and indonesia) that were presented. these case studies reveal the wide range of actors who are involved in the marketing and supply of alternative medicines. regulatory mechanisms are weak. the efficacy claims of alternative medicines often reinforce a biomedical paradigm for hiv/aids, and fit with a healthy living ideology promoted by aids care programs and support groups. the aidsimpact session concluded that more interdisciplinary research is needed on the experience of people living with hiv/aids with these alternative medicines, and on the ways in which these products interact (or not) with anti-retroviral therapy at pharmacological as well as psychosocial levels. a large number of new treatments offered to people living with hiv/aids (plwa) have appeared over the last fifteen years in the therapeutic domain of aids. these med-icines are particularly heterogeneous, both in their presentation and in their geographic and cultural origin. they constitute a group of products with a therapeutic aim that occupies a space between the customary traditional, popular and biomedical sectors of health care [1] . these products often mix reference to biomedicine and science with notions of traditional health culture and nature in a syncretic way. they consist mainly of herbs and nutritional substances and are packaged as 'modern' pharmaceuticals: capsules, tablets, and solutions. the names of these alternative treatments reflect their reference to biomedicine: immunocomplex, viralgic, virjint, etc. their accompanying leaflets provide detailed information on substance, as well as dosage, indications, and biomedical efficacy claims. their diffusion follows contemporary paths in the global economy and makes use of new information technologies. in this paper, we will use the term "alternative" to consider a generic category including medicines that recently appeared for aids which have not been authorised by drug regulatory authorities, nor recommended by who. other terms, such as neo-traditional or neo-phytotherapeutic, may be discussed for the characterization of some of these treatments, related to their local meanings or their social status. the emergence of alternative medicines for aids in asia and africa was discussed at a satellite symposium and the parallel session on alternative and traditional treatments of the aidsimpact meeting, held in marseille, in july 2007. we were especially interested in the role of these medications since the introduction and rapid scale-up of highly active anti-retroviral therapy (haart) in resource poor settings. twenty anthropologists and health researchers attended the satellite session and presented exploratory findings from asia and africa (uganda, senegal, benin, burkina faso, china and indonesia). the aims of the satellite, the results of which were presented at the parallel session [2] , were to (1) identify the actors involved in the diffusion of these alternative medicines for hiv/aids, (2) explore uses and forms of these medicines, and the way they are given legitimacy, (3) reflect on underlying processes of globalisation and cultural differentiation, and (4) define priority questions for future research in this area. we present here the insights generated at the meeting, illustrated with some findings from the studies that were discussed. there has been an increased professionalisation and commercialisation of traditional medicine in response to the development of biomedicine. this trend is not specific to aids and not necessarily a recent development. social scientists first noted this trend in the late 1980s: charles leslie [3] for example has shown how, in india, in response to an increased authority of biomedicine and the globalisation of health markets, unani and ayurvedic medicine production changed; and afdhal and welsch [4] described the rise of 'modern' jamu in indonesia. jamu is the traditional term for indonesian indigenous medicines usually prepared from herbal medicines such as leaves, bark, roots and flowers. nowadays a multimillion dollar industry is involved in the production of ayurvedic and unani medicines in india, and of jamu in indonesia. a rapidly expanding assortment of powders, creams, pills, capsules and cosmetics has been manufactured both in small cottage industries as in large factories with increasingly sophisticated technologies [3, 4] . the modernization of the manufacturing of these drugs has been accompanied with more modern biomedical modes of presenting their efficacy [5] . under globalization, similar trends occurred in other regions and these products diffused more rapidly. at the seminars in marseille, we discussed the ways in which such alternative remedies operate in the therapeutic domain of aids care. in the first decade of the aids epidemic there was no effective treatment for hiv/aids and patients were faced with nearly certain premature death. at that time, there were regular hypes offering hope for life. but with the introduction of art, alternative treatments are now marketed for many additional purposes too: to prevent aids, to kill viruses, to delay the need for art, to restore and enhance health while on art, to treat opportunistic infections, and to alleviate adverse side effects of other treatments. biomedical practitioners generally discourage the use of alternative medicines, fearing interactions with art and also through the concern that patients may stop using art. at the aidsimpact sessions egrot and colleagues [6] presented findings on the supply of what they label "neo-traditional medicines" to refer to the boundary-crossing nature of these treatments in west africa. the "designers" of the inventoried products are extremely heterogeneous. in some cases these people are nationals of african countries who present themselves as healers. some say they have undertaken "research" on the basis of therapeutic products that were already known locally. others refer to a dream revelation (classic in the universe of healers in africa) of a plant composition that is "efficacious" against aids, while yet others speak of a divine revelation. physicians, scientists and academics are solicitated, brought into involvement or spontaneously engage themselves in the exploitation of neo-traditional products. the case studies in west africa show that other treatments, such as immunicomplex or aloe vera, originate in europe and the usa. alternative medicines from europe and the usa occupy the same shelves in ordinary pharmacies as those originating from africa and china, often along with a few 'immune-boosting' food products (honey, olive oil). specialized "bio", "natural health" and "health food" shops make these products available to the more affluent. the distributors and marketing men of these products also target health workers and clinics directly. the west africa case studies noted that health workers also have started to prescribe alternative products such as immuboost (nhi2t) or viralgic (pharma concept) (see figure 1) . a case study from uganda showed how health workers operating an anti-retroviral treatment program adopted a locally available traditional ointment as an alternative medication for skins problems of people living with hiv and aids. the skin problems result from adverse effects of art or symptoms of opportunistic infections. the health workers obtained the recipe from local traditional healers (patients had told them that the cream works well), and the patients help collect the ingredients. they 'repackage' this traditional remedy into what is now called 'mobile cream' (to make clear it is produced by the so called 'mobile' art program). one of the nurses reports: "the mobile cream, which we ourselves prepare either at our chief nurse's home or here at the office depending on how busy we are at the office, is very efficacious for many kinds of skin related conditions. we are quick to prescribe it to the patients because we know it works and it is popular among patients too because it works for them [7] ." content analysis of drug information leaflets, advertisements, product catalogues, and brochures distributed by medical representatives in the west africa case studies [6, 8] casts light on the range of effects that are attributed to these drugs. most commonly cited (biomedical) properties are immune-stimulation and antioxidant. some manufacturers suggest that the products have antiviral properties as well. the antiviral dimension refers either to the opportunistic infections such as herpes (mentioned for example in the product information for immuboost) or eventually to the immunodeficiency syndrome itself. indeed, some products boldly claim anti-hiv activity as well, and are marketed as natural art (see figure 2 ). however, such efficacy claims are not static. the producer of virusinest (nesto-pharma) recently withdrew the antiviral claim, stating in its information leaflet: "the analyses carried out among patients do not allow the anti-hiv assertion to be upheld". there may be also inconsistencies between various information sources. the brochure for viralgic (pharma concept) says that this is a product which renders the virus undetectable, but the website of the manufacturer presents the drug as immunostimulant (result of trials published on the web site), and present the product as treatment for opportunistic infections: "antiherpes...for healthy persons". a case study on indonesia [9] dealt with the demand for alternative medicines among plwa. as afdhal and welsch noted two decades ago, indonesia has a thriving market for jamu. jamu are sold for a wide range of indications: common colds, influenza, headaches, aches and pains, high blood pressure, beauty, improvements in sexual performance, and recently to treat and prevent hivrelated health problems. aids prevalence is below 1% (i.e. this is a low prevalence area), but the disease is stigmatised, because of its association with intravenous drug use and prostitution. hardon and her colleagues conducted interviews with women and men who live with hiv and use anti-retroviral therapy, mainly intravenous drug users and their partners. all of them had better health since taking these modern drugs. nonetheless, all of them see the need to take jamu as well. they do so in tobacoak's, west africa part out of their intention to live positively (i.e. eating and sleeping well, and keeping a positive outlook on life), as promoted by many of the support groups in which plwa participate. the respondents do not make distinctions between modern medicines and jamu in these health maintenance and restoring practices. rather they distinguish the drugs by their effects. they use popular jamu to treat side effects of haart, such as itchiness. these jamus are not specifically promoted for hiv and aids in indonesia, perhaps partly because the disease is so stigmatised. however one neo-traditional preparation stood out in the narratives of our respondents as a product which can treat hiv/aids: virgin coconut oil. ceri, for example, started using coconut oil shortly after she found out she was hivpositive. she says: actually, the effect is not only for your immune system. so, i feel better, don't feel tired, and have more energy. i think what influences most is self-suggestion. it's self-suggestion that matters... mia (a 28 year old woman from jakarta) was given virgin coconut oil by a friend from yogjakarta: i got 70 boxes. a box contains 60 capsules. it took it every day until i felt sick, but there was no effect. my cd4 level did not increase. three months, three months made me look like a coconut you just needed to squeeze (laughing). i became very oily. the good effect when you take vco is that your skin is silk smooth, your face is fairer and if you take a shower, you don't need any lotion, because your skin is naturally oily. that is the positive effect. your hair is also stronger. but buli, a 29-year-old ex-drug user from karawang, one of the most active members of the support group in karawang says: in indonesia, the drug sellers were not very willing to discuss the effects of vco. they would acknowledge that indeed these drugs are used by plwa, or they would deny knowing anything about the drugs. but their pharmacies are full of advertisements for the products and they have prominent positions on their shelves (see figure 3 ). content analysis of the package information for vco in indonesia revealed that they are marketed as real 'curealls', i.e. to kill viruses and bacteria and/or strengthen the immune system, efficacy claims that we also found in west africa. for example the package leaflet for vicofit (manufactured by sumber dinamis in bogor) states that the drug has "a high content of lauric acid which has antivirus, anti-bacterials and anti-protozoa properties." and that it is "believed to help improve the health condition of those with cholesterol, diabetes, coronary heart disease, hepatitis c, hiv positive, cancer, prostate, uric acid, osteoporosis, influenza and weight problems". the package for virjint (produced by pt vermindo international) states that the medicine is safe for daily use and without side effects. it lists two dosages: one for prevention (2 × 2 capsules per day) and another for treatment (2 × 3 capsules per day). the leaflet stipulates that the indications are: -"to increase energy and body stamina -to increase body resistance (meningkatkan daya tahan tubuh) against bacterial, viral and fungal pathogens -to reduce weight -anti-oxidant, anticancer, and anti-hiv -to overcome uric acid, hypertension, stroke, heart disease, atherosclerosis, osteoporosis, influenza, hepatitis, chickenpox, herpes, tb, diabetes, epilepsy, eczema, liver, haemorrhoids, kidney, peradangan (burning sensation), infection, degenerative disease." the packages cite clinical research conducted elsewhere (philippines, usa) to give legitimacy to the products. for example the leaflet of holistic virgin coconut oil states: "based on research conducted in the philippines, holistic virgin coconut oil is very effective to fight against sars and aids". one of the key characteristics of alternative medicines in asia and africa is that they move from one cultural and geographic space to another, apparently without being constrained by trade-barriers, or regulatory mechanisms. some governments promote the production and diffusion of neo-traditional medicines. they do so for economic reasons: alternative medicines are big business, but they also do so for ideological reasons: neo-traditional medicines reflect an attractive hybrid of modernity and national heritage, providing a sense of national identity in the globalized health economy [10] . the governments of india, china, indonesia, and some african countries support research programs to further advance these neo-traditional products, and facilitate market diffusion. while registered pharmaceuticals are regulated heavily upon market entry (proof of efficacy is assessed by national drug regulatory authorities), this is not the case for alternative medicines. art programs, which are sponsored by the same governments, usually discourage the use of alternative medicines, fearing the toxicity of the drugs, or that these medicines will interact with anti-retroviral medication and lead to discontinuation of art therapy [11] . governmental agencies may have contradictory attitudes towards the use of alternative medicines for aids, discouraging it within art programs and supporting it within divisions of traditional medicine. an exception is the chinese government, which officially supports a complementary medicine program for aids care and research [12] . mass-produced alternative medicines meet an increasing demand for health products, a trend which has been labelled the "commodification of health" [13, 14] : from the slums of djakarta to rural settings in burkina faso, people believe more and more that they need pharmaceutical 'things' to protect their health and to treat illness symptoms. people living with hiv and aids are particularly uncertain about their health and their future: art may be accessible and improve health now, but they wonder if this will be the case in the future. this uncertainty makes them an attractive market for the 'best of both worlds', alternative medicines, which come with assertions of 'natural' safety and 'biomedical' efficacy [15] . however the case studies presented in marseille suggest that people especially want to use alternative medicines to delay onset of art, treat opportunistic infections, restore health and alleviate adverse effects once on art. immuneboosters are popular, though our case studies suggest that plwa are often ambivalent about alternative medicines that claim anti-hiv efficacy. the case studies make clear that the market of alternative medicines for hiv/aids is dynamic. it adapts to progress in biomedicine, which has produced potent anti-retrovi-ral medications. in some cases, the efficacy claims for alternative medicines reinforce a biomedical paradigm for hiv/aids, and fit with a healthy living ideology promoted by aids care programs and support groups. more interdisciplinary research is needed on the experience of people living with hiv/aids with these alternative medicines, the ways in which the products and their representations move from one cultural setting to another, and on the ways in which these products interact (or not) with anti-retroviral therapy at pharmacological as well as psychosocial levels. more research is also needed to assess the economic impact of these therapies, since people seem to be spending much on these 'other' medicines while art is provided for free. a blanket denial of the relevance of these products for the quality of life of plwa does not make sense for patients, who need precise information that make clear which products are likely to have negative interactions with art, and which ones could be beneficial. unfortunately research on the interactions between alternative medicine and antiretroviral drugs is sparse [11] . to be able to inform patients better, more clinical research is needed on the benefits and risks of those alternative medicines that are perceived to be beneficial by people living with hiv and aids. patients and healers in the context of culture: an exploration of the borderland between anthropology, medicine and psychiatry berkeley alternative and traditional treatments for aids in the time of art in resource-poor settings: a comparative analysis of recent anthropological studies abstracts aids impact 8th international conference indigenous pharmaceuticals, the capitalist world system, and civilization the rise of the modern jamu industry in indonesia: a preliminary overview ayurvedic and unani health and beauty products: reworking india's medical traditions an overview on neotraditional medicines for hiv/ aids in west africa. naarps workshop alternative and traditional treatments for hiv/aids staying healthy while on haart: the experiences of providers and patients on haart in uganda's resource limited settings. naarps workshop alternative and traditional treatments for hiv/aids non-conventional hiv/aids treatments in west-africa: based on the case of benin. proceedings naarps workshop alternative and traditional treatments for hiv/aids on coconut oil, buah merah and other treatments used by people living with aids in west-java neo-traditional treatments for aids in china: national aids treatment policy and local/global use of tcm (traditional chinese medicine use of traditional herbal medicine by aids patients in kabarole district, western uganda facettes de la recherche médicale et de la gestion du vih-sida dans le système de santé chinois: un autre exemple d'adaptation locale de la biomédecine' (an outline of aids medical research and management of hiv and aids in the chinese public health system: another example of biomedicine localisation) the anthropology of pharmaceuticals: a biographical approach. annual review of anthropology agenda for an anthropology of pharmaceutical practice use of traditional medicine by hiv-infected individuals in south africa in the era of antiretroviral theraphy the authors thank john kinsman for his editorial suggestions and rosalijn both for her assistance in preparing the manuscript. the authors prepared papers for a joint seminar held in aix-en-provence, see reference list for the titles of the contributing papers. a summary of the insights from the papers was subsequently presented at the marseille impact meeting, based on which a draft of this manuscript was written by ah and ad. all authors have contributed to the final manuscript. key: cord-259748-x7dq1sy4 authors: wan, dongshan; jiang, wei; hao, junwei title: research advances in how the cgas-sting pathway controls the cellular inflammatory response date: 2020-04-28 journal: front immunol doi: 10.3389/fimmu.2020.00615 sha: doc_id: 259748 cord_uid: x7dq1sy4 double-stranded dna (dsdna) sensor cyclic-gmp-amp synthase (cgas) along with the downstream stimulator of interferon genes (sting) acting as essential immune-surveillance mediators have become hot topics of research. the intrinsic function of the cgas-sting pathway facilitates type-i interferon (ifn) inflammatory signaling responses and other cellular processes such as autophagy, cell survival, senescence. cgas-sting pathway interplays with other innate immune pathways, by which it participates in regulating infection, inflammatory disease, and cancer. the therapeutic approaches targeting this pathway show promise for future translation into clinical applications. here, we present a review of the important previous works and recent advances regarding the cgas-sting pathway, and provide a comprehensive understanding of the modulatory pattern of the cgas-sting pathway under multifarious pathologic states. pattern-recognition receptors (prrs) serve as innate cellular sensors of danger signals, such as pathogen-associated molecular patterns (pamps) or danger-associated molecular patterns (damps), and yield cellular-stress response. dna molecules are vital genetic components within cells, which are compartmentalized restrictively into specific regions. the occasionally misplaced dna is degraded rapidly by scavenger cells and extracellular or intracellular ribonucleases. aberrant accumulation of dna is relevant to tissue damage (1) . in 2008, several research teams discovered a new protein on the endoplasmic reticulum (er) which can be activated by immune-stimulatory dna (isd) and initiate type-i interferon (ifn) responses, which was named "stimulator of interferon genes" (sting, also known as mita, eris) (2) (3) (4) . sting does not bind to dna directly, and bacteria-derived cyclic di-guanylate monophosphate (c-dgmp) or cyclic di-adenosine monophosphate (c-damp) were confirmed to be ligands for sting (5, 6) . subsequently, it was found that some dna sensors can facilitate sting activation, such as interferon gamma inducible protein 16 (ifi16) (7) . however, sting activation could not be fully explained by the upstream factors/ligands that had been found. it was postulated that an unknown upstream regulator might be responsible for sting activation. in 2013, wu and sun found that cyclic guanosine monophosphate-adenosine monophosphate (cgamp) was a novel secondary messenger serving as a ligand of sting (8) . beside it, they purified a new protein named "cyclic-gmp-amp synthase" (cgas) that had cytosolic dna-sensing ability and can synthesize cgamp (8) . also, they found that the cgas-cgamp-sting pathway was indispensable for host anti-viral immunity (9) . their work filled in the gaps missing from upstream of sting. stimulator of interferon genes or cyclic-gmp-amp synthase is expressed widely in a broad spectrum of cells including immune, non-immune, cancer cells (10) . mounting evidence has demonstrated that the cgas-sting pathway is important for mediating cellular immune sensing, and shows particular responses pattern to the isd distinguished from other nucleotide-sensing pathways. it is also regulated delicately by several molecules or feedback loops to maintain cellular homeostasis. nevertheless, cgas-cgamp-sting pathway itself has distinctive or even opposing effects under different conditions. in this review, we cover the roles of cgas-sting pathway in cellular type-i ifn immune response, and several cellular processes including autophagy, survival and senescence. we also summarize the literature on intrinsic cellular mechanisms modulating cgas-sting pathway as well as its cross-regulations with other dna-sensing pathways. moreover, the inflammationmodulation capacities of this pathway in infectious disease, inflammation and cancers have been elucidated too, and a pervasive pattern of this pathway has been described, which could provide a plausible explanation of the contradictory findings of studies. finally, current or prospective therapeutic strategies targeting the pathway, and issues that need to be addressed in the future, are discussed. cyclic-gmp-amp synthase belongs to the structurally conserved cgas/dncv-like nucleotidyltransferase (cd-ntases) superfamily. the latter is expressed universally in prokaryotes and eukaryotes, and can use purines or pyrimidines selectively as substrates for the production of linear or cyclic di-or even tri-nucleotide compounds, which act as secondary intracellular messengers (11) . cyclic-gmp-amp synthase is distributed mainly in the cytosol (also nucleus in some specific conditions) (8) . generally speaking, cgas is activated upon the recognition of b-type double-stranded dna (dsdna) without sequence-specificity but not a-type dsdna or rna (12, 13) . hybrid dna:rna or stemlike single-stranded dna (ssdna) are also low-affinity ligands for cgas (14, 15) . after binding with ligands, cgas undergoes an allosteric structural change, and subsequently catalyzes its substrates guanosine triphosphate (gtp) and adenosine triphosphate (atp) to produce a mixed phosphodiester-linked cyclic dinucleotide: g(2 -5 )pa (3 -5 ) p cgamp (abbreviated as 2 ,3 -cgamp or cgamp) (16) . cgas also catalyze the synthesis of linear dinucleotides such as amp-2 -atp, gmp-2 -gtp, and amp-2 -gtp as intermediate products (17) . there are two major dsdna-binding sites on opposite sides of the catalytic pocket: a and b site. site a is the primary contact surface for dsdna, whereas site b is complementary, binding another dsdna. it allows for cgas to the formation of a 2:2 cgas:dsdna complex structure directed into two orientations with dsdna at least 20 bp (18) (19) (20) (figure 1a) . increased numbers of back-to-back dimers of cgas hold the two dsdna molecules together and permit successive recruitment of cgas which, consequently, forms a 2n:2 cgas:dsdna higherordered "ladder-like" oligomerization, with cgas arrayed "head to head/tail to tail" (19, 21) . the dna-binding protein hu, mitochondrial transcription factor a (tfam), or bacterial high mobility group box 1 protein (hmgb1) can bend the dsdna into a u-shaped structure and, thus, facilitate binding of cgas dimers to the same strand as it travels in opposite directions (21) (figure 1b) . human cgas, unlike mouse cgas, is prone to formation of this ladder-like network with long dsdna, because of the human-specific residues k187 and l195. these two dsdna-interfacing residues of site a loosen the interaction of dsdna with cgas, leading to dsdna curving and allowing more convenient binding for the next adjacent cgas (20, 21) ( figure 1c) . finally, accumulated cgas-dsdna complexes can go through a liquid-phase separation and condense into gellike droplets as a reaction unit ( figure 1d) . this conformation requires a sufficiently long dsdna strand to form multivalent interaction positions, also requires the function of the n-terminal tail of cgas and a recently discovered dsdna-binding site in the catalytic domain of cgas (site c) (22, 23) . meanwhile, the n-terminal tail of cgas mediates cgas localization onto the membrane by binding to phosphatidylinositol 4,5bisphosphate (pi (4, 5) p2) and prevents liberation of cgas and oligomerization, but can release cgas during cell stress (24). the structure of cgas determines long strand dsdna (>500-1,000 bp) could potentially stimulate the enzyme activity and cgamp production of cgas (25). the ability of human cgas to discriminate long dsdna strands from shorter dsdna may contribute to the specific sensing and recognition of the "danger dna" of pathogens, necrotic cells or cancer cells rather than irrelevant shorter dsdna, thereby enhancing the immunity against them specifically. double-stranded dna is restricted into the nucleus or mitochondria and is rarely present in the cytoplasm. extrinsic dsdna from pathogens such as viruses, bacteria, transcellular vesicles or rupture of dying cells can be internalized into the cytosol in several diverse ways (26-28). these extrinsic dsdna sources are engulfed by endosome through phagocytosis and digested immediately by dnaseii when fusing with lysosomes (29, 30). however, some escaping mechanisms under certain conditions could help protect them from being degraded. for example, antimicrobial peptide ll37 could efficiently transports self-dna from endosome into cytosol of monocytes (28). cell oxidative stress can lead to phagosomal acidification delay and probably release endosome context including dsdna owing to increased membrane permeability (27, 31). the intrinsic self-dsdna can also be segregated inaccurately and released into the cytosol (32, 33). for example, genomic dna (gdna) injury as a result of genotoxic stress and dna self-instability or replication errors leads to double-strand breaks (dsbs) and can be repaired by several ways (34). impaired mediators of dna-damage repair response mediators, such as ataxia telangiectasia mutated (atm)-rad3, poly adpribose polymerase (parp) and breast cancer1/2 (brca1/2) multiple cgas molecules can bind two double-stranded dnas (dsdna) to form a 2n:2 cgas:dsdna higher-ordered "ladder-like" oligomerization. mitochondrial transcription factor a (tfam) can bend the dsdna into a u-shaped structure and promote polymerization. (c) cgas can recognize b-type dsdna. in humans, the cgas dna-interfacing residue of site a loosens the interaction of dsdna to curve dsdna away for more convenient binding with next adjacent cgas. cgas can catalyze gtp and atp to synthesize cyclic guanosine monophosphate-adenosine monophosphate (cgamp). the n-terminal tail binds to the cell membrane, associating with phosphatidylinositol 4,5-bisphosphate [pi (4,5)p2]. (d) accumulation of cgas-dna complex goes through a liquid-phase separation and condenses into gel-like droplets. are associated with persisting dsbs and accumulation of cytosolic dna (35-37). extra-nuclear micronuclei formation during mitosis is a source of cytosolic dsdna caused by dsbs (32, 38). followed by homologous recombination repair of collapsed replication forks, dna cleavage by methyl methanesulphonate (mms) and ultraviolet-sensitive 81 (mus81) also lead to cytosolic dsdna presenting (39). furthermore, manually cre/loxp recombination technology can induce dsdna damage during dna cleavage, which results in the accumulation of cytoplasmic dsdna (40). in normal cellular mitotic processes, chromosomal dna can be exposed to the cytoplasm, while it is hard to bind and trigger cgas (41). in addition, mitochondrial dna (mtdna) is also a considerable ligand of cgas and can be released into the cytosol under mitochondrial stress or dysfunction of proteins which participates in maintaining mitochondrial operations (33, 42, 43) (figure 2a) . cells have several types of nucleases to restrict cytosolic dna to avoid cgas activation. for example, three-prime repair exonuclease 1 (trex1 also known as dnaseiii) is a cytosolic dna exonuclease which removes unprotected dsdna from the cytosol (44) . rnaseh2 locates to the nucleus and specifically degrades the rna in rna:dna hybrids participating in dna replication (45) . dnaseii is a lysosomal dnase which degrades undigested dna in endosomes or autophagosomes to prevent their entry into the cytoplasm (30). sam domain and hd domain-containing protein 1 (samhd1) is characterized as a dntpase and restricts reverse transcription of the rna virus (46, 47) . samhd1 can also stimulate the exonuclease (but not the endonuclease) activity of mre11 to degrade nascent cdns (including cgamp) might be exported by some ways. (c) inflammatory signaling mediated by the cgas-sting pathway. after sensing dna, cgas produces cgamp and extracellular cdns, promoting stimulator of interferon genes (sting) to undergo dimerization. sting can exit from the endoplasmic reticulum (er), and be translocated from the er to the er-golgi vesicle, and arrives at the golgi. sting and tank binding kinase 1 (tbk1) can be oligomerized and cluster at the golgi. the sting-tbk1/iκb kinase (ikk) signalosome forms a scaffold to phosphorylate interferon regulatory factor 3 (irf3) and inhibitor of nf-κbα (iκbα). then, dimerized irf3 and the activated canonical nf-κb p50/p65 complex can be translocated into the nucleus as transcription factors to promote transcription of type-i ifn. (d) autophagy initiation and degradation. sting activation on er triggers er stress and mechanistic target of rapamycin complex1 (mtorc1) dysfunction. er stress and mtorc1 dysfunction can stimulate the unc-51 like autophagy activating kinase (ulk1) complex and beclin1-phosphatidylinositol 3-kinase catalytic subunit type 3 (pi3kc3) complex. autophagy-related protein 9 (atg9) and light chain 3 (lc3) are associated with genesis and elongation of the autophagosome. after autophagy initiation, cgas-sting is ubiquitinated and binds with p62. then, they are packaged into autophagosomes and terminally sorted to lysosomes (bold arrows represent main signaling pathways, thinner arrows represent regulatory signaling pathways, and dashed arrows represent bypass or suspicious pathways). ssdna, and start dna-repair responses at stalled replication forks (48) . depletion of samhd1 leads to the cleaving of nascent ssdna by the activity of mre11 endonuclease and cytosolic translocation of gdna (48) . deficiency of any of these nucleases can lead to accumulation of self-dna in the cytoplasm, thereby activating the cgas-sting pathway against dna molecules (30) (figure 2a ). the production of asymmetrically linked 2 ,3 -cgamp catalyzed by cgas has the highest affinity for sting to promotes sting dimerization (49, 50) . cgamp as a second messenger can be also transferred among cells in several ways to pass danger signaling of frontiers in immunology | www.frontiersin.org cytosolic dna. intercellular gap junction consists of two docking hexamer channels formed by different connexins, which allows many small molecules, including cgamp, to pass bi-directionally through cells. and intercellular transfer of cgamp through gap junction is largely dependent on connexin 43 (51) (52) (53) . additionally, cgamp can be packaged into virons and pre-notify newly infected cells (54, 55) . cell fusion is a distinct manner for intracellular transmission of the human immunodeficiency virus (hiv); cgamp also enter membrane-fused bystander cells in this way (56) . extracellular vesicles such as exosomes can contain cgamp along with viral dna, host gdna or mtdna, and mediate cells communication (57, 58) . there were no evidences that cgamp could be pumped out to extracellular space by a channel/transporter. however, it was found that slc19a1 can transmit cyclic dinucleotides (cdns) into cell plasma (59, 60) . notably, ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (enpp-1) can degrade extracellular cgamp (61) (figure 2b ). besides triggering sting, these exogenous cgamp can directly bind to cgas and prompt its activation as well (62) . after binding to cgamp, the "lid" region of the sting dimer undergoes a conformational change that converts sting from an inactive "open" formation to an active "closed" formation. following that, the sting dimer translocates from the er to perinuclear er-golgi intermediate compartment (ergic) vesicles, finally arriving at the golgi to form punctuate structures with downstream molecules (2, 63) . er-retention of sting caused by mutations results in reduced ifn signaling (64, 65) . the translocon-associated protein β (trapβ) recruited by inactive rhomboid protein 2 (irhom2) initially forms the trap translocon complex that mediates sting exit from the er (2, 66). they both assist cytoplasmic coat protein complex-ii (copii) to drive er-vesicle formation and carry the sting complex to the golgi (67, 68) . trafficking sting can bind directly to and be phosphorylated by tank binding kinase 1 (tbk1) dimer or iκb kinase (ikk) complex (3, 69, 70) . the c-terminal tail (ctt) of sting is a linear unfolded segment, which determines the optimization of combination specificity. sting ctt in mammals tends to bind tbk1, whereas in fish it tends to activate nuclear factor-kappab (nf-κb) signal (71) . the sting phosphorylation site ser366 in the ctt cannot reach the kinase-domain active site of its directly bound tbk1, instead can reach the kinase-domain active site of the next adjacent tbk1 binding with another sting and be phosphorylated, while tbk1 phosphorylate each other (72, 73) . hence, sting and tbk1 can aggregate on the golgi to form the sting signalosome. clustering sting undergoes palmitoylation and full activation (74) . it is also possible for sting-ikk to cluster and form the sting signalosome in this manner. the sting-tbk1/ikk signalosome produces a scaffold to phosphorylate interferon regulatory factor 3 (irf3) or inhibitor of nf-κbα (iκbα). activated irf3 undergo dimerization (70) . the activation of iκbα leads to its polyubiquitination and degradation by the proteosome, thereby eliminating its inhibition of nf-κb. there is also evidence suggesting that nf-κb activation might not require sting trafficking from the er (75) . then, the dimerized irf3 or activated nf-κb p50/p65 (p65 is also known as rela) complex are translocated into the nucleus as transcription factors and bind to the promoter of type-i ifn to aid the transcription of type-i ifn (2, 3, 70) . meanwhile, activation of nf-κb p52/relb can prevent recruitment of p65 and inhibit the p50/p65 signal (76) (figure 2c ). expressed type-i ifn can propagate among cells in paracrine or autocrine manners. the binding of ifnα/β with its receptor triggers janus kinase (jak) and signal transducer and activator of transcription (stat) pathways, then induce transcription of type-i ifn-stimulated genes (isgs), which have ifn-sensitive response elements (isres) in their 5 -untranslated regions (utrs) (77) . irf3 can also bind partially to several isres alone (78) . herein, the expression of some isgs including interferon-induced protein with tetratricopeptide repeats (ifit) and pro-inflammatory cytokines such as tumor necrosis factor α (tnfα), interleukin (il)-6, c-x-c motif chemokine ligand 10 (cxcl10) and c-c motif chemokine ligand 5 (ccl5) is increased remarkably by the cgas-sting pathway (79) . furthermore, cgas and sting are both isgs, suggesting a positive feedback loop in spreading of the ifn signal (80, 81) . stimulator of interferon genes activation on the er also triggers an er stress response with an "unfolded protein response (upr) motif " on the c-terminus of sting, which leads to and er stress-mediated autophagy (82, 83) . sting-tbk1 activation and er stress also induce mechanistic target of rapamycin complex 1 (mtorc1) dysfunction (84) . er stress or reduced mtorc1 signaling activates unc-51-likeautophagy activating kinase (ulk1) complex and the beclin-1-class iii phosphatylinositol 3-kinase (pi3kc3 also known as vps34) complex, which promotes initiation of the classical autophagy path (85) . cgas can also interact directly with the autophagy protein beclin-1-pi3kc3 complex and trigger autophagy (86) . furthermore, cgas-dsdna polymer can form a liquid-phase condensate (as mentioned above), which could theoretically be an initiator of autophagy (87) . after autophagy initiation, autophagy-related protein 9 (atg9) undertakes the genesis of the autophagosome along with light chain3 (lc3) undergoing lipidation, thereby resulting in elongation of the autophagosome (88) . lc3 can also be recruited directly by ergic-loading sting and bypass the classical autophagy pathway (68, 89) . cgas-sting-mediated autophagy can spread to the whole cell and help the elimination of intracellular microorganisms, subcellular organelles or misfolded proteins, as well as the er itself that loads the sting signalosome (90-92) ( figure 2d ). cgas-sting-mediated autophagy is also indispensable for removing cytosolic dna and inflammatory signaling factors to restrict the inflammatory response raised by the pathway itself (93) . excessive signaling of the autophagy cascade can lead to irreversible apoptosis termed "autophagic cell death" (94) . consequently, oligomerized cgas or sting undergoes ubiquitination and is packaged into autophagosomes with the help of p62, to be terminally sorted into lysosomes (79, 83, 95, 96) . cgas or sting is digested immediately in the autophagolysosome after transient activation of downstream signaling (68, 79, 83, 89) . autophagy functions as a negative feedback loop which ensures transient cgas-sting signaling and avoids consistent over-reaction of the pathway. thus, impairment of autophagy may give rise to destructive inflammatory diseases (31). we cataloged factors in the literature that could potentially up-or down-regulate expression of cgas/cgamp/sting in pre-translational or post-translational stages (tables 1, 2) . the regulatory mechanisms of tbk1, irf, and nf-κb in signaling pathways associated with expression of type-i ifn are outside the scope of this review. pyhin family member absent in melanoma 2 (aim2) is a cytoplasmic dsdna sensor. it can recruit apoptosis-associated speck-like protein containing a card (asc) by its pyhin domain and form the aim2 inflammasome. the inflammasome activates caspase-1, which activates il-1 and trigger pyroptosis (97) . the aim2 pathway could counteract the cgas-sting pathway (98) . first, cgas is a target for caspase-1 cleavage (99) . second, gasdermin d activated by caspase-1 can lead to potassium ion (k + ) efflux which inhibits cgas (100) . conversely, the cgas-sting pathway can trigger the aim2 inflammasome or nlr family pyrin domain containing 3 (nlrp3) by several means, and the process lags behind canonical ifn signaling (96, 101) . in this way, the inhibitory nucleic-acid sensor nlr family card domain containing 3 (nlrc3) can counteract sting by binding and occupying it, but viral dna as a possible nlrc3 ligand can reverse its occupation of sting (102) (figure 3a) . another pyhin family member, ifi16, is a dna sensor located in the nucleus. ifi16 can bind to viral dna sequences or damaged chromatin dna and be translocated to the cytoplasm to recruit sting cooperatively with tnf receptor associated factor 6 (traf6) and p53 (103, 104) . several studies have shown that ifi16 (which can stimulate the phosphorylation and recruitment of sting and tbk1) is required for the full response of sting (105, 106) ( figure 3b) . conversely, cgas can partially enter the nucleus and interact with ifi16 to promote its stability (107) . therefore, it is inferred that during viral infection, ifi16 can facilitate recognition of decapsidated viral dna in the nucleus, while cgas in the cytoplasm engages with viral gene transcription products (104, 108) . however, sting signaling can trigger ifi16 degradation by tripartite motif-containing 21 (trim21) ubiquitination (109) . tlr is also an important prr for multiple pamps (110) . tir domain-containing adaptor-inducing ifnβ (trif) is downstream of several subtypes of tlrs (including tlr3). trif may be responsible for interacting with sting and helping the dimerization of sting (111) . during viral infection, the tlr9-myeloid differentiation primary response 88 (myd88)-irf3/7 pathway is necessary for mouse monocytes recruitment to lymph nodes, whereas the sting pathway is necessary for local production of type-i ifn (112) . however, sting signaling can induce suppressor of cytokine signaling1 (socs1) expression, which can negatively regulate myd88 activity (113) (figure 3c ). oxidized mtdna can be released into the cytoplasm during cell stress elicited by hypoxia, viral infection and mitochondrial damage, etc.; oxidized mtdna is resistant to degradation by the cytosolic nuclease trex1 (114) . in addition, mtdna accompanied with tfam (a mtdna-binding protein that can bend mtdna) is also a reasonable target for recognition by cgas (21, 33) . however, during regulated cell death (as represented by apoptosis), it undergoes mtdna release but has certain mechanisms to ensure a minimal cgas-stingmediated immune response. mitochondrial outer membrane permeabilization (momp) activation, which is executed by bcl-2-associated x protein (bax) and bcl-2 antagonist or killer (bak), is a highly controlled conserved process in regulated cell figure 3 | interaction of the cgas-sting pathway with other dna-sensing pathways and its role in cell survival. (a) absent in melanoma2 (aim2) pathway and pyroptosis and necroptosis. aim2 can be triggered by cgamp and form an inflammasome, consequently triggering interleukin (il)-1 production and pyroptosis. stimulator of interferon genes (sting) trafficking to the lysosome ruptures the lysosome membrane, resulting in k + efflux and activation of the nlrp3 inflammasome, leading to pyroptosis. cyclic-gmp-amp synthase (cgas) and interferon regulatory factor 3 (irf3) can be a target for caspase-1 cleaving. gasdermin d can lead to k + efflux and inhibition of cgas. (b) interferon gamma inducible protein 16 (ifi16). ifi16 can be transported to the cytoplasm to help to recruit sting and tank binding kinase 1 (tbk1). ifi16 as a pyhin family protein may form the inflammasome only in theory. (c) toll-like receptor (tlr) pathway. tir domain-containing adaptor-inducing ifnβ (trif) may be responsible for helping the dimerization of sting. sting signaling can induce suppressor of cytokine signaling 1 (socs1) expression, which negatively regulates myd88 activity. (d) apoptosis. mitochondrial outer membrane permeabilization (momp) formed by bax/bak induced by mitochondrial stress can release oxidized mitochondrial dna (mtdna) and cytochrome c into the cytosol. oxidized mtdna is a suitable ligand for cgas recognition and is resistant to dnaseiii (trex1) degradation. cytochrome c binds to apoptotic protease-activating factor 1 (apaf1) and initiates the formation of an apoptosome cooperatively with caspase-9 to activate caspase-3, which can induce apoptosis. caspase-3 can cleave cgas. death. bak and bax activated by apoptosis signals cooperatively form a pore-like conformation on the mitochondrial outer membrane, leading to a permeability change of outer and also inner membranes (115, 116) . consequently, the mitochondrial matrix, including cytochrome c and oxidized mtdna-tfam, is released into the cytoplasm (115, 117) . cytochrome c binds to apoptotic protease-activating factor 1 (apaf1) and initiates the formation of the apoptosome cooperatively with caspase-9, which further triggers the intrinsic apoptosis program (117) . in vivo and in vitro studies have shown that an absence of caspase-9 is associated with greater release of type-i ifn (43, 117) . this occurs because caspase-9 and its downstream caspase-3 can cleave cgas and irf3 to restrain deleterious inflammation (118) (figure 3d) . the cgas-sting pathway can also initiate programmed cell death. activation of sting enhances phosphorylation and activation of receptor interacting serine/threonine kinase 3 (rip3) and mixed lineage kinase domain-like pseudokinase (mlkl). proapoptotic bcl2 binding component 3 (puma), a member of bh3-only family, is subsequently activated in a rip3/mlkl-dependent manner, which promotes leakage of mtdna by momp (119, 120) . activated irf3 can bind directly to bax to form irf3/bax complex and induce apoptosis (47) . excessive cgas-sting-mediated autophagy signaling can cause "autophagic cell death" and prevent malignant transformation of cells through dna damage (94, 121) . sting trafficking to the lysosome can broaden permeabilization of the lysosome membrane, thereby rupturing the lysosome and releasing its contents, resulting in "lysosomal cell death (lcd)". lcd further triggers k + efflux and nlrp3 activation, ultimately resulting in pyroptosis (96, 101) (figure 3d) . moreover, stimulating sting-dependent type-i ifn and tnfα signals simultaneously can lead to necroptosis of tumor cells (122, 123) . cell senescence is recognized as a permanent arrest of the cell cycle, and is common in aging, immunity, ontogenesis and infectious defense (124) . it lacks a specific biomarker but can be identified by the expression of several antiproliferative molecules (representatively rb-p16 andp53-p21 pathway) (125) . during senescence, changes in the nuclear structure and loss of the nuclear lamina protein disrupt the integrity of the nuclear envelope, leading ultimately to dna damage and cytoplasmic chromatin fragments (126) . cellular senescence can be accelerated by accumulation of cytoplasmic chromatin in turn (127) . these senescent cells produce the senescence-associated secretory phenotype (sasp), which shapes an inflammatory microenvironment (128) . the cgas-sting pathway has been reported to be involved in the recognition of cytoplasmic chromatin fragments from senescence-related dna damage, and mediate the expression of sasp genes (129) (130) (131) (132) . along with these actions, the expression of trex1 and dnaseii is inhibited by dna damage through the inhibition of e2f/dp (a potential transcription factor of trex1 and dnaseii) (130) . for hematopoietic stem cells (hscs), dna damage can promote excessive secretion of type-i ifn in the hsc niche and activate p53 pathway, both of which can lead to long-term senescence and exhaustion of hscs (133, 134) . hscs expressing a circular rna named "cia-cgas" in the nucleus, however, are protected from this exhaustion as a result of cia-cgas having stronger affinity than that of self-dna, which prevents it from being sensed (135) . it implied a novel target to manipulate the immune environment in bone marrow and help for finding treatment approaches for hematopoiesis-based diseases, such as aplastic anemia. utilizing cellular senescence to restrain tumor growth is discussed below. cgas-sting signaling has an essential role in defense against a broad spectrum of intracellular dna and rna viruses (9, 26, 50) . hiv is a typical rna retrovirus: there is neither dsdna in its genome, nor production of nucleic acids (50) . nevertheless, cgas can detect the presence of hiv. rna:dna hybrids synthesized during reverse transcription that can be sensed by cgas explain (at least in part) this phenomenon (14) . cgas may be triggered by endogenous dna broken and released during hiv infection as well theoretically. however, some studies found the new mechanisms. the early reverse-transcription production of hiv-1 can flank short stem loops with paired base, which lead to the production of y-type dna containing unpaired guanosines that can activate cgas well (15) . moreover, nucleolus protein non-pou domain-containing octamer-binding protein (nono), as a sensor of capsid components of hiv, can help cgas to be translocated to the nucleus and assist cgas to sense hiv dna accompanied by polyglutamine-binding protein 1 (pqbp1) (136, 137) . the assistance proffered by nono in assisting cgas to sense dna is also associated with its role in constructing a ribonuclear complex with dna-dependent protein kinase (dna-pk) subunits around hexamethylene bisacetamide-inducible protein1 (hexim1), termed as "hexim1-dna-pk -paraspeckle components-ribonuclear protein complex (hdp-rnp), " which also has a role in repair of dna damage and transduction of genotoxic signals (138) . this complex is also required to accompany cgas-pqbp1 in sensing dna virus, such as kaposi's sarcoma-associated herpes virus (139) . in addition, during virus infection, sting activation can lead to global suppression of translation in cells, which restricts viral replication (140) . compared with hiv-1, hiv-2 is less infective because it can infect dendritic cells (dcs) and elicit an anti-virus immune response. as a result, hiv-2 can cross-protect against hiv-1 (141). this phenomenon has been attributed to the fact that hiv-2 (instead of hiv-1) can encode protein vpx, which overcomes the samhd1 restriction of dntp in dcs (46, 142) . hiv-1 can infect dcs via vpx presentation, nevertheless, hiv-1 still cannot be fully sensed and induce an efficient immune response owing to certain escape mechanisms. whether it is hiv-1 or hiv-2, a completely robust ifn response is required at pre-and post-integration sensing stages (143) . cgas in dcs can detect reverse-transcribed cdna of hiv-2 before and after integration, whereas hiv-1 sensing is after genome integration owing to its capsid protection (144, 145) . it was suggested that during initial infection by hiv-1, nucleotides are recruited into the intact capsid through the hexamer pores on the hiv-1 capsid. therefore, the capsid-coated hiv-1 virus prevents the encapsidated reverse-transcription production from being sensed by the cytosolic nucleic-acid sensors (146) . hiv-1 capsids can be ubiquitinated and then degraded by the host e3 ubiquitin ligase function of trim5, which leads to detection of viral dna, meanwhile hiv-1 could use some host protein like cyclophilins to evade the sensing (147, 148) (figure 2a) . similarly, other viruses also have evasion mechanisms to escape cgas-sting pathway surveillance (table 3) . therefore, identifying and preventing such viral-evasion factors could be a viable means to design novel anti-viral drugs. cgas-sting pathway is responsible to protect against intracellular or extracellular bacterial infection (especially hsv, herpes simplex virus; cmv, cytomegalovirus; irhom2, inactive rhomboid protein 2; trapβ, translocon-associated protein β; kshv, kaposi's sarcoma-associated herpes virus; hdp-rnp, hexim1-dna-pk-paraspecklecomponents-ribonuclear protein complex; lana, latency-associated nuclear antigen; virf1, viral interferon regulatory factor 1; plpro, papain-like protease; lc3, light chain3; mtor, mechanistic target of rapamycin; traf3, tnf receptor associated factor 3; hcv, hepatitis c virus; hpv, human papilloma virus; hbv, hepatitis b virus; hiv, human immunodeficiency virus; nf-κb, nuclear factor-κ b. intracellular infections). cdns (e.g., c-dgmp, c-damp, and cgamp) produced by bacteria are essential for the regulation of bacterial function, such as biofilm formation, colonization, and reproduction (149, 150) . as ligands for sting, cdns can bind directly to and activate sting independently of cgas, which contributes to several immune responses from bacteria (151) . usually, bacteria can enter or be engulfed by the cell through the endophagosome and be sequestered from the cytosolic sense receptor. some bacteria, such as mycobacterium tuberculosis (mtb), can survive in vacuoles, resulting in an insufficient cellular immune response to defend against it (152) . in contrast, the esx-1 secretion system of the mycobacterium can translocate the phagosomal vacuolar matrix including bacterial genome molecules into the cytoplasm and trigger the cgas-sensing pathway (153) . for other bacteria, such as legionella pneumophila or and brucella abortus, the host guanylate binding proteins (gbps) facilitate rupture of phagosome vacuoles and are indispensable for controlling their infection (154, 155) . autophagy signaling mediated by cgas/sting is also involved in microorganism clearance mentioned above (90, 91) . bacteria have evolved strategies to confront this pathway too. bacterial phosphodiesterase cdnp produced by mtb or group-b streptococci can degrade cdns (156, 157) . cpsa (a type of mtb lytr-cpsa-psr domain-containing protein) can prevent autophagy responses for eliminating pathogens (90) . chlamydia trachomatis inclusion membrane proteins can maintain the stability of the inclusion membrane and avoid inclusion lysis (leading to pathogen antigens leaking out and being detected by the host cell) (158, 159) . yersinia outer protein j (yopj) deubiquitinates sting and impedes the formation of the sting signalosome (160) . the cgas-sting pathway activation even impedes the elimination of listeria monocytogenes because bacterial dna can be packaged into evs and transferred into t cells, where it induces apoptosis of t cells (161, 162) . several protozoans, such as toxoplasma gondii and malaria parasites, have an intracellular period in their lifecycle. t. gondii could engage cgas-sting exclusively (163) . however, irf3 activation inducing isg expression promotes t. gondii development independently of ifn expression (163) . p. falciparum can target erythrocytes, lacking a nucleus and unable produce ifn, but infected erythrocytes can secrete evs containing parasitic gdna to monocytes and trigger cgas (164) . the immune system is regulated by a complicated network. disorder of immune signaling can elicit non-infectious inflammatory or autoimmune diseases. excessive, uncontrolled production of type-i ifn can lead to a spectrum of inflammation diseases termed "type-i interferonopathies, " which have some common manifestations (165) . cgas-sting is the one of main sources of type-i ifn, acts as a cellular immune-sensing signaling axis, and is involved in type-i interferonopathies. stimulator of interferon genes -associated vasculopathy with onset in infancy (savi) is a typical sting-related hereditary inflammatory type-i interferonopathy, and is manifested by interstitial lung disease, dermatomyositis and arthritis. its pathology is featured by leukocytoclastic vasculitis and microthrombotic angiopathy of small dermal vessels (166, 167) and patients can also suffer from lymphopenia (166) (167) (168) (169) . the etiology of savi is a gain of function (gof) mutant in sting which leads to constitutive sting activation without cdns stimulation (166) . currently, several mutant amino acids residues have been found in or close to the dimerization domain (v155m, n154s, g166e, v147l, and v147m) (64, 166, 168, 170) , as well as r284g, r284s, r281q, and c206y in the cgamp-binding domain (171) . other types of type-i interferonopathies, such as systemic lupus erythematosus (sle) and aicardi-goutières syndrome (ags), have relationships with defective clearance of cytosolic nucleic acids caused by congenital dysfunction of trex1, rnaseh2, and samhd1. sle is a heterogeneous autoimmune disease which has prominent type-i and also -ii ifn signatures (172) . ags comprises some systemic autoimmune syndromes overlapping with sle, and can be classified as a "lupuslike disease" (173) . additionally, ags also causes severe developmental neurological disorders, including cerebral calcifications, encephalopathy and cerebral atrophy. systemic lupus erythematosus is a representative model for elucidating the mechanism of type-i interferonopathies. in sle, the level of self-dna which is packaged into apoptosis-derived membrane vesicles along with the level of anti-dsdna antibody is increased in the serum of patients (174) . a study revealed increasing levels of isgs (including cgas/sting) as well as the cgamp-detected ratio in peripheral-blood mononuclear cells of sle patients (175) . as innate immune cells, dcs have essential roles in antigen presentation, cytokine secretion, and priming the adaptive response of immune cells (176) . plasmacytoid dcs (pdcs) can internalize and recognize self-dna and they are the main source of type-i ifn in serum during sle (177) . ifnα/β is essential for complete function of immature pdcs (178) . ifnα/β and il-1 can induce mitochondrial oxidative stress in dcs and decrease atp production, which blocks proton-pump function and increases ph of lysosomes. this process inhibits mitochondrial degradation and blocks mtdna clearance, which engages the cgas-sting pathway (31, 179). moreover, monocytes may sense mtdna through cgas-sting and differentiate into dcs (31, 180). neutrophil extracellular traps (nets) are complexes released by neutrophils exposed to stimuli or autoantibody immune complexes. nets comprise extracellularly released chromatin, myeloperoxidase enzymes, and also oxidized mtdna. in lupus-like diseases, nets can be induced by ifnα/β and may play a major part in priming pdcs (181, 182) . all mechanisms stated above contribute to a more aggravated type-iifn response and exacerbate disease. a similar phenomenon can be observed in savi, ataxia telangiectasia (at) and artemis deficiency (183) . however, compared with savi, dcs in sle can prime t-cell maturation significantly and increasing secretion of pro-inflammatory cytokines, such as il-6 and tnfα can also lead to activation of adaptive immunity ( figure 4a) . the cgas-sting pathway can mediate systemic inflammation as well as autoimmune activation. however, it is also involved in the local inflammation of multiple tissues. with regard to ischemic myocardial infarction (mi), cardiac macrophages can sense dying ruptured cells and lead to fatal post-mi cardiac inflammation, which is reversed by ablation of cgas/sting/irf3 (184, 185) . in a non-alcoholic steatohepatitis (nash) model induced by a methionine-and choline-deficient or high-fat diet, lipotoxicity can cause mitochondrial damage and up-regulate sting/irf3 expression in hepatocytes, which in turn promotes lipid accumulation and inhibits glycogen synthesis. all above bring out hepatic inflammation and hepatocytes apoptosis (186) . in this model, mice with deficiency of sting presents alleviated insulin resistance and lower levels of low-density lipoprotein in serum, and also decreased hepatic inflammation and fibrosis/steatosis, in which hepatic macrophages/kupffer cells may take a big part (187, 188) . lipotoxicity can induce p62 to be phosphorylated through the cgas-sting-tbk1 pathway, which causes aggravated protein inclusions in hepatocytes and it indicates that p62 could be a biomarker for nash prognosis (189) . mtdna-dependent inflammation induced by lipotoxicity also occurs in adipose tissue and endothelial cells of blood vessels, which contributes to tissue inflammation, insulin resistance, and cardiovascular diseases (42, 190) . in traumatic brain injury (tbi), local injury initiates breakdown of the blood-brain barrier and global neuroinflammation (191) . sting expression is up-regulated in tbi and can lead to increased expression of pro-inflammatory cytokines and enlargement of secondary injury (192) . reduced autophagy-associated protein expression induced by sting may contribute to the dysfunction of autophagy and dampen the elimination of necrotic tissue, thereby intensifying inflammation (192) . during silicosis, silica can yield cytosolic dsdna release and engage cgas-sting, which activates dcs and macrophages to cause severe lung inflammation. it also leads to death of epithelial cells through the nlrp3 pathway and pulmonary fibrosis (193) . similarly, mtdna release in renal tubule cells has been found to be associated with acute kidney injury by cytotoxic drugs and chronic renal fibrosis (194, 195) . neurodegenerative diseases are correlated with local inflammation (196) . in the central nervous system (cns), microglia is considered to be the main source of cgas-sting-dependent ifn expression (197) . in neurodegenerative diseases, levels of the marker of microglia activation-cluster of differentiation 68 (cd68), and pro-inflammatory cytokines are increased (198) . a significant feature of parkinson's disease (pd) is the neuronal loss of cerebral nuclei (especially dopaminergic neurons in the substantianigra). serine/threonine-protein kinase pink1 and e3 ubiquitin-protein ligase parkin are ubiquitinrelated factors that take part in removing damaged mitochondria by autophagy, and their dysfunction lead to the early onset of pd (199) . parkin −/− and pink1 −/− mice, following exhaustive exercise, show inflammation and loss of dopaminergic neurons, which can be rescued by loss of sting (200) . similarly, at is a genetic disease caused by missense mutation of a dna-repair protein: atm. at patients usually show neurodegenerative defects (especially ataxia) complicated with telangiectasia on their eyes or body, deficiency of adaptive immune cells, and predisposition to cancer (201) . nevertheless, up-regulation of expression of type-i ifn can also be found in at patients and mice, causing them to be prone to autoimmune diseases (36, 183, 202) . this syndrome is related to p53-mediated senescence but also the chronic inflammation mediated by the cgas-sting pathway which engages cytosolic uncombined broken gdna caused by atm dysfunction (127) . in addition, accumulation of cellular mtdna occurs in age-related macular degeneration characterized by retinal pigmented epithelium (rpe) degeneration. this can trigger chronic inflammation by cgas-sting pathway, in which nlrp3 inflammasomes, inflammatory/apoptotic caspases are also involved (203, 204) . with regard to other diseases in which adaptive immune cells prime, cgas-sting has a different role. multiple sclerosis (ms) is a local inflammatory disease of cns. ms is characterized by over-reactive microglia, infiltration of self-reactive t cells, demyelization of nerve fibers and hyperplasia of gliocytes. autoantibodies against proteins expressed in immune-privileged regions of cns also contribute to its pathogenesis (205) . in ms, ifnα/β can attenuate disease severity effectively. this implies a protective role for type-i ifn in cns, which is considered to counteract the pro-inflammatory ifnγ (206) . using experimental autoimmune encephalitis (eae) as a ms model, sting was found to be indispensable for amelioration of type-i ifnmediated neuroinflammation, and it could be induced by a conventional anti-viral drug ganciclovir (207) . ultraviolet (uv) radiation is a factor inversely related to the morbidity of ms (208) . it was found that uv-b irradiation can recruit inflammatory monocytes and produce type-i ifn in a sting-dependent manner (209) . all above indicate that cgas-sting-ifnα/β pathway may have a beneficial effect on some cns inflammatory diseases such as ms. a possible reason for the observed effect above is due to indoleamine 2,3-dioxygenase (ido), which can catabolize tryptophan (trp) oxidatively. trp withdrawal or trpoxidative catabolites can interact with general control non-derepressible 2 (gcn2) and mtor, of which both can control cellular aminoacid metabolism and suppress t helper 1 (t h 1) cells immunity (210) . ifnα/β is a potent ido inducer to suppress proliferation of cd4 + t h 1 cells and promote differentiation of foxp3 + regulatory t (t reg ) cells, which are believed to suppress cnsspecific autoimmunity (210, 211) . in addition, dna released from dying cells can be internalized directly by t cells and sensed by cgas-sting pathway, which leads to enhancement of the t h 2 transcription factor gata3 but suppression of the t h 1 transcription factor t-bet. consequently, this process polarizes naive t cells toward t h 2 differentiation (212). studies mentioned above may (at least in part) explain why the cgas-sting signal is a negative regulator of ms. the inhibitory role of cgas-sting in inflammation is also attributed to its apoptosis-triggering role. in some subtypes of savi and mouse models, apoptosis of blood-vessel endothelial cells or bronchial epithelial cells and leucopenia can be observed (especially t-cell lymphopenia) (166, 169, 170) . when the sting signal is stimulated, apoptosis occurs more frequently in normal or cancerous t cells (119) . also, bone-marrow chimeras and gene-knockout studies have shown that t cells defect in savi are not associated with type-i ifn signaling or cgas (213, 214) . localization of sting at the golgi can cause delay of t-cell mitosis and reduced proliferation independently of irf3 and tbk1 (215) . furthermore, a "upr motif " on the c-terminus of sting can cause er stress and upr, resulting in ca 2+ overloading and t-cell death (82) . a controversial view is that b cells express sting variously and may undergo apoptosis through this way (166, 216, 217) . however, simultaneous signaling by sting and the b-cell antigen receptor can promote b-cell activation and antibody production independently of type-i ifn (217) (figure 4b) . as for some diseases with inflammatory responses involved, the acute phase of pancreatitis causes dying acinar cells to produce free dsdna, which activates cgas-sting signaling in macrophages, and exacerbates inflammation severity (218) . however, in the chronic phase of pancreatitis, cgas-sting activation decreases pancreatic inflammation, which may be mediated by limiting t h 17 response (219). for gut mucosal immunity, transient stimulation of sting could strengthen the function of antigen-presenting cells (apcs) and promote t h 1 and t h 17 immune responses against microbes (220) . chronic sting signaling, however, elicits an il-10 response to control the inflammation and avoids inflammatory enterocolitis such as bowel disease (221) . sting knockout mice present reduced numbers of goblet cells, a decreased ratio of commensal versus harmful bacteria and compromised t reg cells in the gut, making it prone to enterocolitis (222) . chromosomal instability (cin) is an intrinsic feature of cancer, and results spontaneously from errors in chromosome segregation during the mitosis of cancer cells. cin can also be induced manually by radiotherapy or chemotherapy, which causes dsbs. it results in micronuclei formation outside the nucleus, of which rupture brings out irrepressible accumulation of cytosolic self-dna and engages cgas (32, 38, 223). however, normal mitotic processes involve exposure of chromosomal dna to the cytoplasm, but this cannot initiate a substantial inflammatory reaction or apoptosis because nucleosomes can suppress dsdna-cgas binding in a competitive manner (41). an appropriate immune response against tumors via a type-i ifn plays an indispensable part in limiting tumors and prolonging host survival (224) . it was found sting-deficient mice are prone to developing several types of cancer and have poor survival under a tumor burden, whereas stimulation of sting can elicit robust immunity to tumors (225) (226) (227) . a mechanism is many cancer cells expressing cgas can recognize cytosolic dna and produce cgamp to stimulate secretion of type-i ifn through sting (228, 229) . excessive expression of trex1 in cancer cells, which can be induced by radiotherapy, attenuates this progression (228) . cgas-sting can also promote senescence of cancer cells through the p53-p21 pathway (129) . cgas-sting-mediated autophagy contributes to autophagic cell death if mitotic crisis occurs to avoid transformation of cancer cells (121) . melanoma cells can also transfer cgamp produced by them to proximal non-cancerous host cells through gap-junction channels and activate sting in these cells, which contributes to the recruitment of tumor-infiltrating immune cells such as natural killer (nk) cells (51, 230) . expression of the nk cellspecific ligand nkg2d retinoic acid early transcript 1 (rae1) on cancer cells is highly up-regulated by sting once nk cells permeate into tumor tissue (231) . the activation of sting in the endothelium within the tumor microenvironment (tme) could contribute to the remodeling of tumor vasculatures, and may have positive effects on tumor regression (232) . dendritic cells are the main source of type-i ifn in several types of tmes and are dependent on sting signaling (229) . more preferentially than macrophages, infiltrating dcs take up tumor-derived dna or cgamp from dying cell fragments by phagocytosis (27, 29, 129, 226, 233) . moreover, cancer cells can package dna into exosomes and transfer dna to dcs (234) . produced cgamp by cancer cells can also be transferred to dcs through forming gap junction (53) . by activating cgas-sting signal in dcs, cd8α + subtype dcs secret chemokines such as ccl5 and cxcl10 and crossprime infiltrating anti-tumor cd8 + t cells (29, 226, (235) (236) (237) . in contrast, numbers of immune-suppressing cells such as t reg cells, myeloid-derived suppressor monocytes and m2 macrophages have been reported to be decreased (225, 238, 239) . expression of il-15/il-15rα complex is up-regulated in myeloid cells with the help of sting/type-i ifn and promotes tumor regression (240) . tumor cells can evade intrinsic cellular surveillance in different ways. in various cancer cell lines, cgas, sting, tbk1, and irf3 are mutated frequently and their decreased expressions are also related to the high level of methylation (241) . sting expression has been shown to be suppressed by the alternative lengthening of telomeres (alt) pathway, which is responsible for prolonging the telomere length and maintaining the proliferation of tumor cells (242) . a hypoxic environment in tumor cells can lead to accumulation of lactic acid and is associated with the inhibition of tumor-conditional dcs and reduced expression of ifn signaling molecules (243) . in breast cancer, functional up-regulation of expression of human epidermal growth factor receptor 2 (her2), a ligand-independent receptor tyrosine kinase (rtk), can arrest the expression of rac-alpha serine/threonineprotein kinase (akt1) (a key factor in the mtor pathway), which is reported to inhibit the activation of cgas and tbk1 (244, 245) . patients with lung adenocarcinoma have a low probability of survival if they have reduced expression of cgas (132) . thus, expression of the cgas-sting and dna-damage marker histone γh2ax in tumor cells could be considered as independent prognostic factors to predict therapy response and clinical outcome, and could be superior to that of traditional markers like immunogenic cell death and t cells number (246) . however, some scholars have arrived at opposite conclusions. when dsbs occur in cancer cells, cgas can be relocated to the nucleus and obstruct the formation of the parp1-timeless complex, thereby inhibiting homologous recombination repair and maintaining cin, which potentiates tumor evolution (35, 223) . it has also been reported that cgas recognizing cin activates non-canonical nf-κb signaling and potentiates cellular metastasis programs (247) . furthermore, sting −/− mice are resistant to skin carcinogenesis in a 7,12-dimethylbenz(a)anthracene (dmba)-treatment model. it has been demonstrated that when dmba-induced nuclear dna leaks into the cytoplasm, sting can induce chronic inflammatory stimulation that contributes to cancer development (248) . during brain metastasis, cgamp transferred to bystander cells (e.g., astrocytes) can also produce ifnα and tnfα in the tme but, in this context, it will support tumor development and chemoresistance (249) . coordinating with myeloid cells penetrating into the tumor, myeloidderived suppressor cells can also be recruited through the c-c chemokine receptor type 2 (ccr2) (250) . another study found that microparticles yielded by tumor cells can turn macrophages into the m2 type through cgas-sting-tbk1, contrary to previous findings (251) . immune-system interactions with tumor cells are complicated. the effect of cgas-sting on cancer is dependent on the type of tumor, host immune state, activated cell types, therapeutic intervention, and the magnitude of cgas-sting activation. like inflammation generated by cgas-sting, a time-dependent inflammatory anti-tumor response mediated by cgas-sting may be present. temporary activation of cgas-sting in innate immune cells could enhance the anti-tumor effect, whereas sustained activation of cgas-sting might induce immune tolerance of the tumor. more investigations are necessary to ascertain the exact role of cgas-sting in oncology, and elucidate the specific advantages and adverse effects of targeting the cgas-sting pathway in cancer therapy ( figure 5 ). considering the pivotal role of the cgas-sting pathway in infection, inflammation and cancer, positive modulation of the pathway signaling is a promising way to enhance the immune state and restrict microorganisms or heterogeneous cells, whereas negative modulation can control aberrant inflammation. radiotherapy or chemotherapy drugs such as cisplatin or cyclophosphamide can induce dsbs and micronuclei, then trigger the cgas-sting pathway to enhance tumor immunogenicity (252) (253) (254) . in addition, parp inhibitors such as olaparib have promising effects on cancer cells lacking brca2 because of their cooperative dna-repair functions (253) . although cgas activation is inhibited by nucleosomes, taxol can induce mitotic cell-cycle arrest and sustain divided chromatin in the cytosol to activate the cgas-sting pathway slowly, and accumulation of signaling could stimulate apoptosis of cancer cells (41). inhibitors of topoisomerase 1 or 2 used conventionally as chemotherapy drugs trigger minor damage to dna and accumulation of cytosolic dna, which can engage cgas and enhance the anti-tumor or anti-infection responses of cells (255) (256) (257) . cgas-sting is essential on anti-tumor immune checkpoints therapies. for example, blockade of cd47-signal regulatory protein α (sirpα) signaling on dcs can activate nadph oxidase 2 (nox2) and increase the ph in phagosomes along with incomplete degradation of mtdna, which can trigger cgas-sting (129) . sting deficiency in mice abrogates the anti-tumor effect of cd47 blockade (258) . a similar phenomenon also can be seen in anti-programmed cell death-1 (pd-1) therapy (259) . there is greater infiltration of ifnγ + cells and cd8 + t cells and pd-1/pd-1 ligand 1 (pd-l1) expression in tme treated by sting-ligand derivatives (260) . therefore, in several types of tumors, combined administration of a sting agonist and immune-checkpoint antibody could elicit a more curative outcome compared with one therapy alone (238, 261) . viruses can infect cells lacking cgas-sting more effectively, and have higher oncolytic activity compared with virus therapy alone. hence, the use of oncolytic viruses such as talimogene laherparepvec is beneficial for treating tumors with low expression of cgas/sting. sting expression can be regarded as a prognostic measurement for such therapy (262) . some artificial analog molecules of cdns, such as 5,6dimethylxanthenone-4-acetic acid (dmxaa) and 10-carboxymethyl-9 (10h) acridone (cma) can bind the cdn pocket of mouse-specific sting dimers and promote conformational transition of sting from inactive "open" to an active "closed" state (263, 264) . dmxaa showed convincing efficacy in restraining tumors in mice (265) . however, dmxaa is restricted in activating mouse-specific sting but not humanspecific sting, which could be an explanation for the failure of dmxaa in treating 1299 non-small-cell lung cancer patients in a phase-iii clinical trial (nct00662597) (266) . nonetheless, with three substitutions (g230i, q266i, and s162a), human sting can also be induced by dmxaa to undergo conformational transition (264) . another new compound, amidobenzimidazole, has been found to be an agonist of sting without lid closure and has potential therapeutic value (267) . cyclic dinucleotides and their derivates that can stimulate sting directly are candidate adjuvants to restrain tumors. intratumoral administration of c-damp, c-dgmp, or cgamp analogs alone or combined with other adjuvants or tumor antigens have shown anti-tumor effects (259, 268) ; phase-i or ii clinical trials (nct02675439, nct03172936, and nct03937141) of dithio-(rp,rp)-c-damp (known as adu-s100) are ongoing (261) . to avoid the degradation and ensure maximal phagocyte internalization of cdns, endosomolytic nanoparticles have been designed to package and deliver cdns. for example, ph-sensitive nanoparticles (e.g., stingnanoparticles) can release their contents if located in acidic endosomal environments (269) . for treatment of type-i interferonopathies, lessons can be taken from the treatment of canonical autoimmune disease such as sle, but there are several differences. for example, it was found that corticosteroid pulse therapy, γ-immunoglobulins, disease-modifying anti-rheumatic drugs, anti-cd20, and some immunosuppressants (e.g., methotrexate) have limited efficacy against savi (166, 171) . jak inhibitors such as ruxolitinib, tofacitinib and baricitinib that reduce type-i ifn downstream signaling have shown therapeutic value against savi, but further verification of their efficacy is needed (270) . moreover, novel immune therapies, such as anti-ifnα and anti-ifnar immunoglobin, are in clinical trials for sle. these could also be tested against savi in the future (165) . pharmaceutically screening has revealed that some antimalaria drugs, such as suramin, have an inhibitory effect on cgas by blockade of interaction between dna and cgas (271) . in addition, novel small molecules such as ru320521 or g150 can occupy the enzymatic pocket of species-specific cgas to abrogate cgamp synthesis (272, 273) . recently, a study found that aspirin can acetylate cgas at three lysine residues and block cgas activity (274) . with regard to sting, the cyclopeptide astin c can block irf3 recruitment onto the sting signalosome (275) . the molecule h-151 can block the palmitoylation of human-sting (276) . all of these agents are potential candidates for alleviating type-i interferonopathies. the cgas-sting pathway is primarily responsible for the modulation of immune response in cells when facing cytosolic dsdna challenge. moreover, it is complicatedly cross-regulated by other cellular processes or cellular signaling networks. the exact fundamental mechanism of the pathway in cells and the effect on the whole organism in specific states is not completely clear and requires further investigation. in conclusion, the cgas-sting pathway has dichotomous roles in the immune system. in general, cgas-sting-type-i ifn signaling can promote the innate immune response in myeloid cells but alleviate the adaptive immune response exerted by t cells and b cells. cgas shows high expression in apcs such as macrophages and dcs, but sting is expressed in most cells (10) . cgas-sting signaling corresponding to cytoplasmic dsdna in apcs can boost innate and adaptive immunity transiently. in this situation, the dna challenge signal is limited to only macrophages and dcs. their pro-inflammatory and antigenpresenting functions to adaptive immune cells are promoted in the short-term. if the signal spreads to other bystander cells, such as t cells, b cells, local resident cells by means of cgamp transfer, or just aberrant sting activation by gof, it causes apoptosis in bystander cells or adaptive immune cells and immune tolerance in the long-term. therefore, it is reasonable to conclude that the intrinsic function of the cgas-sting pathway is essential for the innate immune system responses of the host immediately after pathogen invasion or abnormal cell appearing. once the challenge persists, the cgas-sting pathway controls the adaptive immune system to avoid chronic, detrimental inflammatory reactions or autoimmune diseases. the inflammatory response exists universally in almost all physiologic and pathologic progressions. cgas-sting is pivotal in modulating cellular inflammation, so it is promising to extend our conception of the cgas-sting pathway onto more diseases with inflammatory responses involved, especially cns-based diseases such as stroke, in which the inflammatory reaction exists but was recognized less. moreover, for targeting the cgas-sting pathway for therapeutic purposes, drugs should be optimized to augment the desirable effect and prevent its unwanted effects. for example, to eliminate tumor cells or infectious agents, agonists of cgas-sting would be a rational option if designed to target apcs exclusively but not t cells or b cells. in this scheme, the antitumor immune response is enhanced while avoiding apoptosis of adaptive immune cells and infiltration of immune suppressor cells. also, most research on the cgas-sting pathway has focused on its ifn-expressing role but overlooked autophagy and cell-death roles, which are also main downstream signaling of the pathway. therefore, some drugs, such as emricasan, are potential apoptosis inhibitors that may have a complementary effect on ameliorating apoptosis of blood-vessel endothelial cells or bronchial epithelial cells, and lymphopenia, in savi. until now, studies of the cgas-sting pathway have been done mainly in the laboratory but it has large space to be explored in clinical or translational fields. additionally more prrs and cellular immune-surveillance pathways may remain to be discovered to piece together the molecular puzzles of the cell. dw drafted the manuscript and drew the figures. wj and jh conceived and revised the review. frontiers in immunology | www.frontiersin.org recognition of endogenous nucleic acids by the innate immune system sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response sting is a direct innate immune sensor of cyclic di-gmp ifi16 is an innate immune sensor for intracellular dna cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects an expression atlas of human primary cells: inference of gene function from coexpression networks bacterial cgas-like enzymes synthesize diverse nucleotide signals structural mechanism of cytosolic dna sensing by cgas structure of human cgas reveals a conserved family of second-messenger enzymes in innate immunity cytosolic rna:dna hybrids activate the cgas-sting axis sequence-specific activation of the dna sensor cgas by y-form dna structures as found in primary hiv-1 cdna cgas produces a 2 -5 -linked cyclic dinucleotide second messenger that activates sting the catalytic mechanism of cyclic gmp-amp synthase (cgas) and implications for innate immunity and inhibition the cytosolic dna sensor cgas forms an oligomeric complex with dna and undergoes switch-like conformational changes in the activation loop cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization structure of the human cgas-dna complex reveals enhanced control of immune surveillance cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders dna-induced liquid phase condensation of cgas activates innate immune signaling human cgas catalytic domain has an additional dna-binding interface that apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production obsessive-compulsive disorder is associated with broad impairments in executive function: a meta-analysis ribonuclease h2 mutations induce a cgas/sting-dependent innate immune response restriction by samhd1 limits cgas/sting-dependent innate and adaptive immune responses to hiv-1 host restriction factor samhd1 limits human t cell leukemia virus type 1 infection of monocytes via sting-mediated apoptosis samhd1 acts at stalled replication forks to prevent interferon induction cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp connexin-dependent transfer of cgamp to phagocytes modulates antiviral responses cancer-cell-intrinsic cgas expression mediates tumor immunogenicity viruses transfer the antiviral second messenger cgamp between cells transmission of innate immune signaling by packaging of cgamp in viral particles cgas-mediated innate immunity spreads intercellularly through hiv-1 envinduced membrane fusion sites priming of dendritic cells by dna-containing extracellular vesicles from activated t cells through antigen-driven contacts cells infected with herpes simplex virus 1 export to uninfected cells exosomes containing sting, viral mrnas, and micrornas slc19a1 is an importer of the immunotransmitter cgamp slc19a1 transports immunoreactive cyclic dinucleotides hydrolysis of 2 3 -cgamp by enpp1 and design of nonhydrolyzable analogs cgas facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity activation by translocation from the er is associated with infection and autoinflammatory disease atg9a controls dsdna-driven dynamic translocation of sting and the innate immune response irhom2 is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting tmed2 potentiates cellular ifn responses to dna viruses by reinforcing mita dimerization and facilitating its trafficking autophagy induction via sting trafficking is a primordial function of the cgas pathway phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation sting specifies irf3 phosphorylation by tbk1 in the cytosolic dna signaling pathway modular architecture of the sting c-terminal tail allows interferon and nf-kappab signaling adaptation structural basis of sting binding with and phosphorylation by tbk1 a conserved plplrt/sd motif of sting mediates the recruitment and activation of tbk1 activation of sting requires palmitoylation at the golgi ubiquitination of sting at lysine 224 controls irf3 activation non-canonical nf-kappab antagonizes sting sensor-mediated dna sensing in radiotherapy immunomodulatory functions of type i interferons dna-binding landscape of irf3, irf5 and irf7 dimers: implications for dimer-specific gene regulation attenuation of cgas-sting signaling is mediated by a p62/sqstm1-dependent autophagy pathway activated by tbk1 positive feedback regulation of type i ifn production by the ifn-inducible dna sensor cgas positive feedback regulation of type i interferon by the interferon-stimulated gene sting sting-mediated disruption of calcium homeostasis chronically activates er stress and primes t cell death sting senses microbial viability to orchestrate stress-mediated autophagy of the endoplasmic reticulum chronic innate immune activation of tbk1 suppresses mtorc1 activity and dysregulates cellular metabolism mtor signaling in growth, metabolism, and disease crosstalk between the cgas dna sensor and beclin-1 autophagy protein shapes innate antimicrobial immune responses a brief history of autophagy from cell biology to physiology and disease autophagy in the renewal, differentiation and homeostasis of immune cells sting directly activates autophagy to tune the innate immune response mycobacterium tuberculosis is protected from nadph oxidase and lc3-associated phagocytosis by the lcp protein cpsa extracellular m. tuberculosis dna targets bacteria for autophagy by activating the host dna-sensing pathway upr, autophagy, and mitochondria crosstalk underlies the er stress response dnase2a deficiency uncovers lysosomal clearance of damaged nuclear dna via autophagy self-consumption: the interplay of autophagy and apoptosis trafficking-mediated sting degradation requires sorting to acidified endolysosomes and can be targeted to enhance anti-tumor response the dna inflammasome in human myeloid cells is initiated by a sting-cell death program upstream of nlrp3 aim2 recognizes cytosolic dsdna and forms a caspase-1-activating inflammasome with asc antagonism of the sting pathway via activation of the aim2 inflammasome by intracellular dna inflammasome activation triggers caspase-1-mediated cleavage of cgas to regulate responses to dna virus infection gasdermin d restrains type i interferon response to cytosolic dna by disrupting ionic homeostasis a noncanonical function of cgamp in inflammasome priming and activation viral dna binding to nlrc3, an inhibitory nucleic acid sensor, unleashes sting, a cyclic dinucleotide receptor that activates type i interferon non-canonical activation of the dna sensing adaptor sting by atm and ifi16 mediates nf-kappab signaling after nuclear dna damage nuclear ifi16 induction of irf-3 signaling during herpesviral infection and degradation of ifi16 by the viral icp0 protein ifi16 is required for dna sensing in human macrophages by promoting production and function of cgamp ifi16 and cgas cooperate in the activation of sting during dna sensing in human keratinocytes cgas-mediated stabilization of ifi16 promotes innate signaling during herpes simplex virus infection viral dna sensors ifi16 and cyclic gmp-amp synthase possess distinct functions in regulating viral gene expression, immune defenses, and apoptotic responses during herpesvirus infection sting-mediated ifi16 degradation negatively controls type i interferon production assembly and localization of toll-like receptor signalling complexes sting requires the adaptor trif to trigger innate immune responses to microbial infection sequential activation of two pathogen-sensing pathways required for type i interferon expression and resistance to an acute dna virus infection cross-regulation of two type i interferon signaling pathways in plasmacytoid dendritic cells controls anti-malaria immunity and host mortality oxidative damage of dna confers resistance to cytosolic nuclease trex1 degradation and potentiates sting-dependent immune sensing mitochondria and cell death: outer membrane permeabilization and beyond mitochondrial inner membrane permeabilisation enables mtdna release during apoptosis apoptotic caspases prevent the induction of type i interferons by mitochondrial dna apoptotic caspases suppress type i interferon production via the cleavage of cgas, mavs, and irf3 signalling strength determines proapoptotic functions of sting puma amplifies necroptosis signaling by activating cytosolic dna sensors autophagic cell death restricts chromosomal instability during replicative crisis induction of necroptotic cell death by viral activation of the rig-i or sting pathway tnfalpha and radioresistant stromal cells are essential for therapeutic efficacy of cyclic dinucleotide sting agonists in nonimmunogenic tumors cellular senescence: aging p16ink4a induces an age-dependent decline in islet regenerative potential autophagy mediates degradation of nuclear lamina extranuclear dna accumulates in aged cells and contributes to senescence and inflammation senescenceassociated secretory phenotypes reveal cell-nonautonomous functions of oncogenic ras and the p53 tumor suppressor cytoplasmic chromatin triggers inflammation in senescence and cancer downregulation of cytoplasmic dnases is implicated in cytoplasmic dna accumulation and sasp in senescent cells innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence cgas is essential for cellular senescence dna-damage-induced type i interferon promotes senescence and inhibits stem cell function bacterial c-di-gmp affects hematopoietic stem/progenitors and their niches through sting a circular rna protects dormant hematopoietic stem cells from dna sensor cgas-mediated exhaustion pqbp1 is a proximal sensor of the cgas-dependent innate response to hiv-1 nono detects the nuclear hiv capsid to promote cgas-mediated innate immune activation p53 induces formation of neat1 lncrna-containing paraspeckles that modulate replication stress response and chemosensitivity hexim1 and neat1 long non-coding rna form a multi-subunit complex that regulates dna-mediated innate immune response stingdependent translation inhibition restricts rna virus replication inhibition of hiv-1 disease progression by contemporaneous hiv-2 infection samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx reshaping of the dendritic cell chromatin landscape and interferon pathways during hiv infection the capsids of hiv-1 and hiv-2 determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hiv-1 activation of innate immunity depends strongly on the intracellular level of trex1 and sensing of incomplete reverse transcription products hiv-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated dna synthesis restriction of hiv-1 and other retroviruses by trim5 hiv-1 evades innate immune recognition through specific cofactor recruitment cyclic di-gmp: second messenger extraordinaire cyclic gmp-amp signalling protects bacteria against viral infection a bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis tuberculosis pathogenesis and immunity mycobacterium tuberculosis differentially activates cgas-and inflammasome-dependent intracellular immune responses through esx-1 brucella abortus triggers a cgas-independent sting pathway to induce host protection that involves guanylate-binding proteins and inflammasome activation beneficial effects of yogasanas and pranayama in limiting the cognitive decline in type 2 diabetes group b streptococcus degrades cyclic-di-amp to modulate stingdependent type i interferon production inhibition of innate immune cytosolic surveillance by an m. tuberculosis phosphodiesterase absence of specific chlamydia trachomatis inclusion membrane proteins triggers premature inclusion membrane lysis and host cell death the chlamydia trachomatis inclusion membrane protein cpos counteracts sting-mediated cellular surveillance and suicide programs yersinia yopj negatively regulates irf3-mediated antibacterial response through disruption of sting-mediated cytosolic dna signaling intracellular bacteria engage a sting-tbk1-mvb12b pathway to enable paracrine cgas-sting signalling sting-dependent type i ifn production inhibits cell-mediated immunity to listeria monocytogenes induction of interferon-stimulated genes by irf3 promotes replication of toxoplasma gondii malaria parasite dna-harbouring vesicles activate cytosolic immune sensors type i interferon in rheumatic diseases activated sting in a vascular and pulmonary syndrome inherited sting-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations stimulator of interferon genes-associated vasculopathy with onset in infancy: a mimic of childhood granulomatosis with polyangiitis severe combined immunodeficiency in stimulator of interferon genes (sting) v154m/wild-type mice sting-associated vasculopathy develops independently of irf3 in mice disease-associated mutations identify a novel region in human sting necessary for the control of type i interferon signaling personalized immunomonitoring uncovers molecular networks that stratify lupus patients aicardi-goutieres syndrome and the type i interferonopathies apoptosisderived membrane vesicles drive the cgas-sting pathway and enhance type i ifn production in systemic lupus erythematosus expression of cyclic gmp-amp synthase in patients with systemic lupus erythematosus. arthritis rheumatol the role of dendritic cells in autoimmunity human plasmacytoid dentritic cells elicit a type i interferon response by sensing dna via the cgas-sting signaling pathway interferon priming is essential for human cd34+ cell-derived plasmacytoid dendritic cell maturation and function interleukin-1beta induces mtdna release to activate innate immune signaling via cgas-sting induction of dendritic cell differentiation by ifn-alpha in systemic lupus erythematosus neutrophil extracellular traps enriched in oxidized mitochondrial dna are interferogenic and contribute to lupus-like disease netting neutrophils are major inducers of type i ifn production in pediatric systemic lupus erythematosus type i ifnrelated netosis in ataxia telangiectasia and artemis deficiency cytosolic dna sensing promotes macrophage transformation and governs myocardial ischemic injury irf3 and type i interferons fuel a fatal response to myocardial infarction activation of the sting-irf3 pathway promotes hepatocyte inflammation, apoptosis and induces metabolic disorders in nonalcoholic fatty liver disease expression of sting is increased in liver tissues from patients with nafld and promotes macrophage-mediated hepatic inflammation and fibrosis in mice sting-mediated inflammation in kupffer cells contributes to progression of nonalcoholic steatohepatitis lipotoxicity induces hepatic protein inclusions through tank binding kinase 1-mediated p62/sequestosome 1 phosphorylation sting-irf3 triggers endothelial inflammation in response to free fatty acid-induced mitochondrial damage in diet-induced obesity priming the inflammatory pump of the cns after traumatic brain injury stingmediated type-i interferons contribute to the neuroinflammatory process and detrimental effects following traumatic brain injury sting-dependent sensing of self-dna drives silica-induced lung inflammation mitochondrial damage and activation of the sting pathway lead to renal inflammation and fibrosis mitochondrial damage causes inflammation via cgas-sting signaling in acute kidney injury how neuroinflammation contributes to neurodegeneration sensing of hsv-1 by the cgas-sting pathway in microglia orchestrates antiviral defence in the cns chronic neurodegeneration induces type i interferon synthesis via sting, shaping microglial phenotype and accelerating disease progression the roles of pink1, parkin, and mitochondrial fidelity in parkinson's disease parkin and pink1 mitigate sting-induced inflammation atm and the molecular pathogenesis of ataxia telangiectasia autoimmune phenomena in ataxia telangiectasia mitochondrial dna has a pro-inflammatory role in amd cgas drives noncanonical-inflammasome activation in age-related macular degeneration multiple sclerosis type i/ii interferon balance in the regulation of brain physiology and pathology activation of the sting-dependent type i interferon response reduces microglial reactivity and neuroinflammation modulation of the immune system by uv radiation: more than just the effects of vitamin d? ultraviolet b irradiation causes stimulator of interferon genes-dependent production of protective type i interferon in mouse skin by recruited inflammatory monocytes. arthritis rheumatol engineering dna nanoparticles as immunomodulatory reagents that activate regulatory t cells activation of the sting adaptor attenuates experimental autoimmune encephalitis nucleic acid sensing by t cells initiates th2 cell differentiation sting-associated lung disease in mice relies on t cells but not type i interferon hierarchy of clinical manifestations in savi n153s and v154m mouse models intrinsic antiproliferative activity of the innate sensor sting in t lymphocytes agonist-mediated activation of sting induces apoptosis in malignant b cells b cell-intrinsic sting signaling triggers cell activation, synergizes with b cell receptor signals, and promotes antibody responses sting signaling promotes inflammation in experimental acute pancreatitis habtezion a. sting signalling protects against chronic pancreatitis by modulating th17 response stingactivating adjuvants elicit a th17 immune response and protect against mycobacterium tuberculosis infection sting-dependent signaling underlies il-10 controlled inflammatory colitis the cytosolic sensor sting is required for intestinal homeostasis and control of inflammation the multifaceted role of chromosomal instability in cancer and its microenvironment immunogenic cell death in cancer and infectious disease sting contributes to antiglioma immunity via triggering type i ifn signals in the tumor microenvironment sting-dependent cytosolic dna sensing mediates innate immune recognition of immunogenic tumors suppression of sting associated with lkb1 loss in kras-driven lung cancer dna exonuclease trex1 regulates radiotherapy-induced tumour immunogenicity growing tumors induce a local sting dependent type i ifn response in dendritic cells tumorderived cgamp triggers a sting-mediated interferon response in nontumor cells to activate the nk cell response rae1 ligands for the nkg2d receptor are regulated by sting-dependent dna sensor pathways in lymphoma sting activation reprograms tumor vasculatures and synergizes with vegfr2 blockade dendritic cells but not macrophages sense tumor mitochondrial dna for cross-priming through signal regulatory protein alpha signaling exosomes shuttle trex1-sensitive ifn-stimulatory dsdna from irradiated cancer cells to dcs host type i ifn signals are required for antitumor cd8+ t cell responses through cd8{alpha}+ dendritic cells sting-dependent cytosolic dna sensing promotes radiation-induced type i interferondependent antitumor immunity in immunogenic tumors targeting dna damage response promotes antitumor immunity through sting-mediated t-cell activation in small cell lung cancer intratumoral sting activation with t-cell checkpoint modulation generates systemic antitumor immunity sting signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion il-15 is a component of the inflammatory milieu in the tumor microenvironment promoting antitumor responses suppression of sting signaling through epigenetic silencing and missense mutation impedes dna damage mediated cytokine production extrachromosomal telomere repeat dna is linked to alt development via cgas-sting dna sensing pathway downregulation of membrane trafficking proteins and lactate conditioning determine loss of dendritic cell function in lung cancer her2 recruits akt1 to disrupt sting signalling and suppress antiviral defence and antitumour immunity akt kinase-mediated checkpoint of cgas dna sensing pathway dna damage predicts prognosis and treatment response in colorectal liver metastases superior to immunogenic cell death and t cells chromosomal instability drives metastasis through a cytosolic dna response inflammation-driven carcinogenesis is mediated through sting carcinomaastrocyte gap junctions promote brain metastasis by cgamp transfer host stingdependent mdsc mobilization drives extrinsic radiation resistance tumor cellderived microparticles polarize m2 tumor-associated macrophages for tumor progression parp inhibition enhances tumor cell-intrinsic immunity in ercc1-deficient non-small cell lung cancer parp inhibitor efficacy depends on cd8(+) t-cell recruitment via intratumoral sting pathway activation in brca-deficient models of triple-negative breast cancer radiotherapy combined with novel sting-targeting oligonucleotides results in regression of established tumors topoisomerase 1 inhibition promotes cyclic gmp-amp synthasedependent antiviral responses topoisomerase ii inhibitors induce dna damage-dependent interferon responses circumventing ebola virus immune evasion cgas/sting axis mediates a topoisomerase ii inhibitor-induced tumor immunogenicity cd47 blockade triggers t cell-mediated destruction of immunogenic tumors sting activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity a sting agonist given with ox40 receptor and pd-l1 modulators primes immunity and reduces tumor growth in tolerized mice magnitude of therapeutic sting activation determines cd8(+) t cellmediated anti-tumor immunity recurrent loss of sting signaling in melanoma correlates with susceptibility to viral oncolysis species-specific detection of the antiviral small-molecule compound cma by sting bindingpocket and lid-region substitutions render human sting sensitive to the species-specific drug dmxaa the sting agonist dmxaa triggers a cooperation between t lymphocytes and myeloid cells that leads to tumor regression randomized phase iii placebo-controlled trial of carboplatin and paclitaxel with or without the vascular disrupting agent vadimezan (asa404) in advanced non-small-cell lung cancer design of amidobenzimidazole sting receptor agonists with systemic activity sting ligand c-di-gmp improves cancer vaccination against metastatic breast cancer endosomolytic polymersomes increase the activity of cyclic dinucleotide sting agonists to enhance cancer immunotherapy efficacy of the janus kinase 1 /2 inhibitor ruxolitinib in the treatment of vasculopathy associated with tmem173-activating mutations in 3 children suramin potently inhibits cgamp synthase, cgas, in thp1 cells to modulate ifnbeta levels small molecule inhibition of cgas reduces interferon expression in primary macrophages from autoimmune mice development of human cgas-specific small-molecule inhibitors for repression of dsdnatriggered interferon expression the cyclopeptide astin c specifically inhibits the innate immune cdn sensor sting targeting sting with covalent small-molecule inhibitors trim56-mediated monoubiquitination of cgas for cytosolic dna sensing the ubiquitin ligase trim56 regulates innate immune responses to intracellular doublestranded dna the e3 ubiquitin ligase rnf185 facilitates the cgas-mediated innate immune response rnf26 temporally regulates virus-triggered type i interferon induction by two distinct mechanisms the e3 ubiquitin ligase amfr and insig1 bridge the activation of tbk1 kinase by modifying the adaptor sting trim14 inhibits cgas degradation mediated by selective autophagy receptor p62 to promote innate immune responses p38 inhibition provides anti-dna virus immunity by regulation of usp21 phosphorylation and sting activation usp18 recruits usp20 to promote innate antiviral response through deubiquitinating sting/mita the deubiquitinase cyld is a specific checkpoint of the sting antiviral signaling pathway sumoylation promotes the stability of the dna sensor cgas and the adaptor sting to regulate the kinetics of response to dna virus senp7 potentiates cgas activation by relieving sumo-mediated inhibition of cytosolic dna sensing g3bp1 promotes dna binding and activation of cgas zcchc3 is a cosensor of cgas for dsdna recognition in innate immune response manganese increases the sensitivity of the cgas-sting pathway for double-stranded dna and is required for the host defense against dna viruses tmem203 is a binding partner and regulator of sting-mediated inflammatory signaling in macrophages the erassociated protein zdhhc1 is a positive regulator of dna virus-triggered, mita/sting-dependent innate immune signaling association of abnormal elevations in ifit3 with overactive cyclic gmp-amp synthase/stimulator of interferon genes signaling in human systemic lupus erythematosus monocytes s6k-sting interaction regulates cytosolic dna-mediated activation of the transcription factor irf3 glycogen synthase kinase 3beta regulates irf3 transcription factor-mediated antiviral response via activation of the kinase tbk1 trim9 short isoform preferentially promotes dna and rna virus-induced production of type i interferon by recruiting gsk3beta to tbk1 lsm14a plays a critical role in antiviral immune responses by regulating mita level in a cell-specific manner trim29 promotes dna virus infections by inhibiting innate immune response trim30alpha is a negative-feedback regulator of the intracellular dna and dna virustriggered response by targeting sting the ubiquitin ligase rnf5 regulates antiviral responses by mediating degradation of the adaptor protein mita usp13 negatively regulates antiviral responses by deubiquitinating sting oligoadenylatesynthetase-family protein oasl inhibits activity of the dna sensor cgas during dna virus infection to limit interferon production interactome and proteome dynamics uncover immune modulatory associations of the pathogen sensing factor cgas sensing of bacterial cyclic dinucleotides by the oxidoreductase recon promotes nf-kappab activation and shapes a proinflammatory antibacterial state the ca(2+) sensor stim1 regulates the type i interferon response by retaining the signaling adaptor sting at the endoplasmic reticulum nitro-fatty acids are formed in response to virus infection and are potent inhibitors of sting palmitoylation and signaling nlrc3, a member of the nlr family of proteins, is a negative regulator of innate immune signaling induced by the dna sensor sting ppm1a regulates antiviral signaling by antagonizing tbk1-mediated sting phosphorylation and aggregation ptpn1/2-mediated dephosphorylation of mita/sting promotes its 20s proteasomal degradation and attenuates innate antiviral response nlrx1 sequesters sting to negatively regulate the interferon response, thereby facilitating the replication of hiv-1 and dna viruses nlrx1 promotes immediate irf1-directed antiviral responses by limiting dsrnaactivated translational inhibition mediated by pkr mitigating sox2-potentiated immune escape of head and neck squamous cell carcinoma with a sting-inducing nanosatellite vaccine mediates hypoxia-induced immunosuppression by repressing cgas nrf2 negatively regulates sting indicating a link between antiviral sensing and metabolic reprogramming kdm5 histone demethylases repress immune response via suppression of sting herpes simplex virus 1 abrogates the cgas/sting-mediated cytosolic dna-sensing pathway via its virion host shutoff protein, ul41 herpes simplex virus 1 tegument protein vp22 abrogates cgas/sting-mediated antiviral innate immunity species-specific deamidation of cgas by herpes simplex virus ul37 protein facilitates viral replication evasion of the sting dna-sensing pathway by vp11/12 of herpes simplex virus 1 herpes simplex virus 1 gamma134.5 protein inhibits sting activation that restricts viral replication human cytomegalovirus protein ul31 inhibits dna sensing of cgas to mediate immune evasion type i interferon production by inactivating the dna sensor cgas without affecting sting essential role of hcmv deubiquitinase in promoting oncogenesis by targeting anti-viral innate immune signaling pathways human cytomegalovirus tegument protein ul82 inhibits sting-mediated signaling to evade antiviral immunity the herpesviral antagonist m152 reveals differential activation of sting-dependent irf and nf-kappab signaling and sting's dual role during mcmv infection human cytomegalovirus-encoded us9 targets mavs and sting signaling to evade type i interferon immune responses inhibition of cgas dna sensing by a herpesvirus virion protein cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas modulation of the cgas-sting dna sensing pathway by gammaherpesviruses sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex poxviruses evade cytosolic sensing through disruption of an mtorc1-mtorc2 regulatory circuit viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling zika virus elicits inflammation to evade antiviral response by cleaving cgas via ns1-caspase-1 axis denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus ns2b protein targets cgas for degradation and prevents mitochondrial dna sensing during infection dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses hepatitis c virus ns4b blocks the interaction of sting and tbk1 to evade host innate immunity hepatitis c virus ns4b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis b virus polymerase disrupts k63-linked ubiquitination of sting to block innate cytosolic dnasensing pathways hiv-2/siv vpx targets a novel functional domain of sting to selectively inhibit cgas-stingmediated nf-kappab signalling we thank k. wood from barrow neurological institute for discussions and editing. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 wan, jiang and hao. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-251939-dvbua4pf authors: nepal, binod title: aids denial in asia: dimensions and roots date: 2007-12-31 journal: health policy doi: 10.1016/j.healthpol.2007.04.011 sha: doc_id: 251939 cord_uid: dvbua4pf abstract aids denial has long been viewed as the obstacle to forging effective response in many asian countries. this article examines the dimensions and roots of this phenomenon. it identifies seven types of views, attitudes, or tendencies that can be described as denial, dissent, disagreements, or doubts. three major factors underlying the aids denial are discussed. these are (1) historical impressions that stds are western diseases, (2) desire of some asian leaders to forge eastern points of view, and (3) long-held negative image towards the peoples or groups who happened to be at the front-line of the population groups exposed to the epidemic. the third factor is the most important source of denial. aids denial is not a new and isolated phenomenon but the one shaped by the global and historical institutions. asian aids denial reflects the authoritarian and moralist grievances arising from the perceived deterioration of traditional moral order. since the late 1980s, well within a decade of the identification of the disease, asia has been warned to be heading towards a devastating aids epidemic. the epidemic has been described as the 'gathering storm' [1] which could create a 'next wave' [2] of infections and deaths in asia pushing the 'continent in peril' [3] and putting the 'economic and social progress in jeopardy' [4] . though the prevalence of the disease in asian regions such as south and southeast asia (0.6%) and east asia (0.1%) is much lower than that in sub-saharan africa (6.1%), almost all the countries have detected the virus in their nationals [5] . yet people who take drugs intravenously and those who sell sex are at the front-line of the population groups hit by the 'storm'. the virus has been detected in the majority of injecting drug users in one or more sites of china, india, indonesia, myanmar, nepal, thailand, and vietnam, and among one-tenth or more sex workers in some sites of cambodia, india, myanmar, nepal, thailand, and vietnam [6, 7] . the incidence of hiv is rising among low-risk female populations in many countries [8] . however, responses to this ongoing crisis have often remained scanty even in the countries which have been speculated to be at the risk of larger epidemics. dwyer et al. observed that although information about devastating impacts of hiv/aids in africa was widely circulated in the region, most countries in asia took no initiative to adopt the measures proven to be effective in controlling the epidemic [9] . there are notable disagreements about the extent, prospect, and prevention of the disease. at the first hand, the control of aids epidemics looks simple, as thailand has shown that the disease can be contained provided that the leaders are committed and focused interventions are put in place [10] . but many other countries have done little to reach the people who are the first to face the 'storm'. a 2003 review showed that only a small fraction of at-risk populations such as injecting drug users, sex workers, and migrants had been reached with the basic prevention services [11] . not much was changed by 2005 [5] . even thailand was no exception given that only a few drug injectors and homosexuals were receiving some outreach services [11] . so, why are the responses to the prospective 'storm' low, slow, and fragmented? why is there little appre-ciation among the leaders that the disease might take a heavy toll if left unchecked? the phenomenon of low, slow, and fragmented responses has been described as the outcome of aids denial. time and again, aids denial has been mentioned as the serious obstacle to controlling aids in asia. the generic label, however, obscures many important dimensions of this challenge. the paper attempts to explore the dimension and roots of the aids denial in asia. the analysis is guided by the social constructionist approach which assumes that the diseases are biological phenomena but they carry socially constructed meaning. widespread stigma and discrimination against people with hiv/aids arise partly from the existing negative public attitude towards these people [12] . the negative public image of hiv/aids and people carrying the virus is not only associated with the nature of the disease but also with the socially constructed meaning or understanding about the risk factors. the extent of stigma varies according to modes of transmission or behaviours perceived to be responsible for the infection and the pre-existing characteristics of the at-risk groups. many different social institutions contribute to the construction of specific public images of the target populations. the people and places at risk of contracting a disease are identified by the epidemiological studies but they do not necessarily become comprehensible to the masses. the public, the media, and the policy-makers begin to develop certain images about the disease and the at-risk people. according to schneider and ingram [13] , 'officials develop maps of target populations based on both the stereotypes they themselves hold and those they believe to prevail among the segments of the public likely to become important to them.' formulation and shifts in policy for a particular group, subpopulation, or community depend on how they are viewed by policy-makers. gauri and liberman contend that where social institutions divide the population groups deeply, elites and common people perceive aids to be a disease of other people outside their community and who are unlikely to mingle with them [14] . when responsible authorities take 'us' and 'they' approach by prejudicially linking the disease with specific sections of the populations, aids policies remain misguided and fragmented. to illustrate, despite very similar socio-demographic setups, response to aids were very different in brazil and south africa; unlike brazil where aids was considered a general threat for the entire population, aids was linked in south africa with the racial issues and aids policy was marred with racial divisions [14] . like aids, the aids denialism is a global phenomenon. this has even tied the hands of the united nations (un) agencies which are looked for leading the global efforts to fight this pandemic. the political declaration adopted on 15 june 2006 by the un general assembly mentioned the term 'vulnerable groups' five times but nowhere specified what they include [15] . this was aimed at avoiding the acknowledgment of the existence of vulnerable groups such as injecting drug users and sex workers. yet much of the international comments on aids denial refer to south africa where the president thabo mbeki questioned the prevailing mainstream views on hiv/aids, and set up the presidential aids advisory panel [16] [17] [18] . inclusion of the so-called dissident scientists in the panel was the most controversial aspect of his initiative and the major cause for severe criticism against him. refuting the established biomedical explanations, mbeki raised doubts about the role of sexual behaviours in driving the epidemic, attributed poverty as a cause for widespread aids deaths in africa, and expressed skepticism to the relevance of antiretroviral therapy. he was, therefore, branded as a 'denialist' and was even considered responsible for 'genocide' for the deaths due to aids in his country [18] . no leader in asia has done anything as controversial as it was done by mbeki. yet asia is not free from denial if the term is understood as encompassing the range of dissenting voices and disagreements. schneider and fassin [16] refer to denial as the 'individual or collective inability to face an intolerable reality by pretending that it does not exist'. there is, however, little clarity as to what the aids denial means in the asian contexts. mention of this term in one or the other context or to accuse governments [cf. 19, 20] is not helpful to understand the full picture of the denial in this region. aids denial is a complex phenomenon manifested in various forms, ways, and contexts. seven major dimensions of denials, doubts, or disagreements about hiv/aids in asian societies are briefly discussed below. for sim-plicity, the term denial has been used here to represent the range of disagreements, controversies, doubts, or dissents. first, a small group of scientists and other professionals have doubted or even denied whether hiv has really been identified or whether it really causes aids. they are mostly from the us or europe but includes a few from asia. a group of the dissidents -leading among them is peter duesberg -argued that hiv is a harmless virus, and that aids is caused by drug abuse, anti-aids drugs, or malnutrition [21, 22] . the other group of dissidents, primarily the perth group, questioned whether hiv was actually identified as a unique retrovirus [23] . in 2000, more than 5000 scientists and professionals signed a statement known as 'the durban declaration' dismissing those dissenting arguments [24] . some of the signatories were from asia. nevertheless, the voices of aids dissidents appear to have no significant influence upon aids policies, as no countries have publicly subscribed to these views. second, aids has long been seen as foreigners' disease. a widely held view in asia in the pastand to a little extent so far -is that aids is a westerner's disease. initial cases of aids identified in some countries of asia belonged to the tourists or citizens returning from the west or the patients receiving the blood imported from the west. the first aids case in indonesia was found in a dutch tourist in bali, a tourist island, in 1987. in china, the first aids case was identified in an argentina-born us citizen and the first four chinese identified with hiv were the haemophiliac patients who received blood imported from the united states (us) [25] . the first aids patient documented in thailand was a thai male returned from the us [26] . these incidents created an illusion about the source and prospect of the disease. third, many asian leaders were apparently confident about the persistence of moral order in their countries. many of them believed that deviant behaviours were absent or rare, and therefore the disease was unlikely to spread to the masses. moralistic and ideological roots of aids denialism in asia will be discussed in next section. to illustrate the illusion of moral order, it is sufficient to quote the then indian health minister: 'ours is a moral society. while tackling aids you [cannot] say you lead licentious lives because [you can use] condoms. i don't think that should be the message. ' [27] fourth, aids has been considered a disease of deviants or isolated groups. when the disease began to appear among the local populations of asia, the virus was initially identified among people who were traditionally considered deviants or immoral. in india, a female sex worker from chennai was the first local person identified with hiv. initial outbreaks of hiv in this country occurred among injecting drug users in manipur, a northeastern state bordering on myanmar [28] and female sex workers in mumbai [7] . likewise, in indonesia, initial cases of hiv infections were found in costal areas frequented by thai fishermen [29] . the first major outbreaks of hiv in china were limited to the injecting drug users mostly from the ethnic minorities in yunnan province [30, 31] . this might have created a false hope that the mainstreams of the society would be insulated from the epidemic. fifth, denial of services to the people exposed to the disease was an inevitable outcome of the perception that individuals contracted the disease owing to their own sinful, deviant, or immoral behaviours. statistics reported by the government agencies showed that until recently only a small fraction of vulnerable groups such as injecting drug users, sex workers, and men who have sex with men were reached with outreach services [11] . even in thailand which has one of a few model programs successful to control the epidemic at the national scale, the interventions have not covered all identified at-risk groups [32] . some critical groups such as injecting drug users, prisoners, men who have sex with men (msm), and immigrants have been deprived of basic prevention and treatment services. therefore, despite growing evidence about the effectiveness, such programs as harm reductions and condom use remain controversial in the region [7] . sixth, statistics on hiv/aids or at-risk groups are considered sensitive items deserving to be hidden. thailand, which developed a model condompromotion program later on, initially suppressed the results of hiv testing among sex workers for the fear that the news of hiv outbreaks would harm the tourism industry. though the country soon took bold steps to publicise the statistics and to insti-tute the much-admired condom-promotion campaign [33] , many other countries in the region continued to show high sensitivity towards the statistics on hiv/aids and the most-at-risk groups. this is one of the reasons behind limited availability and quality of the statistics on the at-risk groups such as injecting drug users, sex workers and their clients, and men who have sex with men in the region [34] [35] [36] . attitude of authorities in several countries of this region is probably reflected in the un theme group's observation about the local governments in china: many local governments do not want to know, or let others know about hiv/aids in their area for fear that it will reflect poorly on the locality and its officials. local governments sometimes suppress information and sometimes even actively oppose research on hiv/aids. [37] finally, sensitivity about the disease is translated into denial of, or disagreement over, the scale of the epidemics. on several occasions, internal and external agencies have seriously doubted one another's estimates. generally, india and china kept questioning the validity of the hiv/aids estimates and relevance of the prevention programs prescribed by the international institutions and western health experts. in 1996, indian officials disagreed with the un estimate of 3 million people living with hiv/aids in the country, and the media even suspected that the 'figures were being inflated by the west to pressure india into accepting vaccine trial and other research on hiv infected people' [38] . a doctor familiar with the aids situation in india, however, warned that 'denial, complacency, and blaming others for the epidemic are the main reasons why hiv has spread so successfully' in the country [19] . similar disagreements erupted in 2004 when richard feachman, the executive director of the global fund to fight aids, tuberculosis, and malaria warned that india had the largest number of people living with hiv/aids in the world, surpassing south africa. he remarked, 'i don't believe in official statistics. india is already in [the] first place' [39] . his arguments were refuted by several senior government officials of india citing that these two countries were not comparable and the gap in hiv prevalence levels were large [40] . likewise, on 13 september 2003, the lancet published a news report entitled 'human rights organization blasts china over hiv/aids cover-up.' human rights watch accused chinese government of being 'in denial' over scale of hiv/aids epidemic. in this report, asian division director of human rights watch, a new york based organization, was quoted as commenting: 'the chinese government has been in denial about the problem for many years', and 'beijing's failure to act decisively in the aids epidemic continues to cost lives and cause incalculable suffering to those living with the virus' [20] . though the aids denial is not so unique to asia, there are certain features specifically conducive for this phenomenon. this section discusses three salient factors: (1) historical impressions that some sexually transmitted diseases (stds) are western diseases, (2) desire of some asian leaders to forge eastern points of view, and (3) long-held negative image towards the peoples or groups who happened to be at the highest risk of contracting the disease. these three factors are, however, interrelated. historically, the well known stds such as syphilis were understood by asians as the westerners' disease. european colonizers were seen as the sources of syphilis into asia. mukherji and chakravarti [41] observed that syphilis, which is believed to have brought to europe by columbian voyagers, was unknown in ancient india until the arrival of portuguese, although gonorrhoea and soft chancre existed since ancient time. they maintained that the absence of any comment about syphilis in sanskrit works would illustrate the absence of this disease in this region. they noted: 'the disease is first mentioned in bhabaprakasa under the name of feringhi roga (portuguese disease) and mercurial preparations are recommended its treatment' [41] . the term feringhi was used in many countries of asia not only to refer to portuguese but any european. in nepal, for example, local name of syphilis is viringi and it seems probable that the name was coined to identify the disease with the feringhi. medical history also indicates that incidence of venereal diseases was probably higher among europeans than asians. according to the 1926 annual report of the public health commissioner with the government of india, rates of hospital admissions for treatment of venereal diseases of british troops (62.1 per 1000) was four times higher than that of indian troops (15.7 per 1000) [41] . an important ideological background to the aids denial stems from the desire of asian leaders to forge an independent view reflecting on the culture, traditions, and philosophies of the east. the thought exaggerates morality, hierarchical relations among the member of the society, community cohesiveness, and extended family systems as unique characteristics of the asian societies. this pattern of thought is sometimes referred, particularly in the east or southeast asia, as the asian values. though the concept of asian values has been criticised as having no factual base [42] , it cannot be denied as having served the authoritarian officials to interpret the aids as a disease of deviants or bad people. this idea imagines asia as unique and denies or denounces behaviours that are considered to be inspired by the western thoughts and promotes oppressive or eliminative approach towards the people who are engaged in those behaviours. the concept of asian value -or exaggeration of morality in particular -is not the only background for aids denial. on several issues, asians have developed opinions different from that of the western scholars, governments, and institutions. the aids denial, which is considered the single most threat to effective aids policy in asia, parallels, to a limited extent, the previous debates on emerging issues of global concern. a relevant example is the great population debate -evolved in the 1950s -about the role of aggressive family planning programs versus role of development for population control in the developing countries [43, 44] . in the 1970s, developing countries questioned the western policy of the uni-focal family planning programs to control population in the third world countries. on some occasions, development was argued as the best contraceptive. for aids, moral order has been seen as the best protection. this paves way for painting negative image of the people who rather need support. the construction of public impression about aids and the at-risk groups began with its initial epidemiological mystery. the disease remained mysterious for some years and societies developed various metaphors reflecting fear, stigma and moralistic misapprehensions [45] . various perceptions about aids and at-risk groups evolved in the west permeated asia well before the disease arrived. in the us, aids was initially found among gays and hence characterized as a 'gay plague' [46] . this prompted the asian policy-makers to dismiss the potential challenge of this epidemic assuming that homosexuality was absent in asia. in the 1980s, aids cases detected in asia were mostly among foreigners, overseas returnees, and female sex workers. until the early 1990s, aids in asia was generally interpreted as the foreigner's disease, particularly westerner's disease, associated with homosexuality and prostitution. for example, in 1990, a journalist observed that the 'indian government's perception that "foreigners" are the principal carriers of hiv does not seem to have changed in recent years' [47] . he added, 'most laws proposed to check the spread of aids are aimed at non-nationals' [47] . similar was the situation in thailand where aids was initially perceived as 'a foreign disease, carried by foreigners and brought from foreign lands', and later as 'a disease of homosexuals' and then as 'a disease of intravenous drug users' [48] . in the philippines, aids was mainly identified with the prostitutes and lower class gays who had contact with foreigners. even the upper class gays were arguing that 'low class gay people should be rounded up for aids testing since they're the only ones now who go around with the foreigners' [49] . in some instances, people from high prevalence neighbouring regions were also blamed and cautioned. for example, when taiwan started to recruit foreign labourers in 1991 to make up its labour shortage, the labourers from south and southeast asia were characterized as the highest risk category who would pass hiv to innocent locals [50] . it has been taking a long time to overcome the misinterpretation that aids is simply a disease of bad people. the groups identified as the high-risk are powerless, unorganized, often from disadvantaged backgrounds, and negatively viewed. so, these groups have been blamed for spreading the disease and considered to be personally accountable for the infections. major messages directed to the negatively viewed powerless groups are that 'they are bad people whose behaviour constitute a problem for others,' and that 'they can expect to be punished unless they change their behaviour or avoid contact with the government' [13] . the negatively viewed powerless at-risk groups mostly included injecting drug users, female sex workers, men who have sex with men, and immigrants. in japan, as the official medical care system excludes undocumented foreign nationals, the immigrants are reluctant to take hiv test and to seek medical care for the fear of deportation [51] . burmese migrants in thailand, especially those who lack work permit, also avoid visiting health facilities because of the fear of deportation [52] . on 24 may 2006, a thai daily, the nation, reported that hiv prevalence among migrants was twice the prevalence in pregnant women, and hence the local public health experts were pointing out migrants as emerging vectors of hiv. marginal population groups such as idus, sex workers, and msms who are the most vulnerable to aids were stigmatized even without aids; the disease has added another burden upon them creating the phenomenon of double stigma [53, 54] . in yunnan, china, for example, drug users carry stigma and are denied participation in the community activities and state sponsored services irrespective of their hiv status [55] . a study from six asian countries identified that the tendency to blame, stigmatize, and discriminate the people vulnerable to, or living with, hiv is realised more clearly in interpersonal contexts such as health facilities but this is grounded in cultural, religious, institutional, and structural frameworks [56] . in some instances, governments use strong negative terms such as social evils to described these people and emphasize punishment rather than support and care [57, 58] . comparatively, aids carries more stigma than do other killer diseases such as severe acute respiratory syndrome and tuberculosis because aids is still seen as the consequence of one's own carelessness [59] . aids denial has many dimensions but the most important one arise from the long-held negative attitude towards the vulnerable populations. there are some variations in the images of aids and at-risk groups across the regions but they are not merely local products; rather they have been shaped by the global and historical institutions as well. the echo has been felt up to the un general assemblies at the international level and down to the individuals at the micro-level. interestingly, no one in asia has taken views as extreme as the south african president mbeki, but there are many instances of disagreements, doubts, and denials that have been affecting the people directly exposed to the pandemic. while indifference, doubts, and blame underpin inaction or slow action in some instances, they have led to adopt negative policies in the other instances. overcoming denials is an important step towards instituting positive and comprehensive responses to aids. strategies can vary among the nations depending on their socio-cultural context and economic and technical ability. a general approach is to tackle pre-existing background stigma towards these vulnerable groups by promoting solidarity and intensifying advocacy. this helps reduce the long-held tendency to see aids as a disease of some careless people. the efforts of local and international networks of people with hiv have been showing some impacts but these institutions should have more than ceremonial recognition. further work is required to improve surveillance, research, and analysis and to better understand the extent and prospect of the epidemics and to show the implications to the society of the continued inactions. such work should be supplemented by conceptual debate on how broader social contexts, not only individual desires, underpin the risk behaviours. in sum, asian aids denial is a reflection of lamentation about the perceived deterioration of traditional moral orders and weakening hold on new generations, rather than the aids per se. when the authorities realise that they need to adapt their views and policies to suit the rapidly changing societies, the phenomenon of aids denial begins to fade. concerted advocacy spearheaded by organized members of the most vulnerable groups can speed up this process. aids in asia: the gathering storm the next wave of hiv/ aids: nigeria aids in asia: a continent in peril economic and social progress in jeopardy: hiv/aids in the asian and pacific region: integrating economic and social concerns, especially hiv/aids, in meeting the needs of the region report on the global aids epidemic hiv/aids surveillance database. us census bureau monitoring the aids pandemic network (map network) report on the global hiv/aids epidemic: 4th global report challenge and response: hiv in asia and the pacific breaking the silence: setting realistic priorities for aids control in less-developed countries unaids, world health organization (who), centers for disease control and prevention (cdc), policy project. coverage of selected services for hiv/aids prevention, care and support in low and middle-income countries in interventions to reduce hiv/aids stigma: what have we learned? social construction of target populations: implications for politics and policy boundary institutions and hiv/aids policy in brazil and south africa political declaration on hiv/aids denial defiance: a socio-political analysis of aids in south africa a synthesis report of the deliberations by the panel of experts invited by the president of the republic of south africa, the honourable mr the embodiment of inequality: aids as a social condition and the historical experience in south africa the aids scare in india could be aid-induced. editorial comments human rights organisation blasts china over hiv/aids cover-up: human rights watch accuses chinese government of being "in denial" over scale of hiv/aids epidemic hiv does not cause aids the aids dilemma: drug diseases blamed on a passenger virus a critique of the montagnier evidence for the hiv/aids hypothesis the durban declaration hiv infection and aids in china 1985 through 1994 acquired immune deficiency syndrome in thailand. a report of two cases india minister vows to beat aids rapid spread of hiv among injecting drug users in north-eastern states of india the current situation of the hiv/aids epidemic in indonesia current status of hiv infection in yunnan province of china hiv infection and aids in china thailand's response to aids: building on success, confronting the future aids and public policy: the lessons and challenges of 'success' in thailand estimates of the number of female sex workers in different regions of the world estimating the number of men who have sex with men in low and middle income countries estimates of injecting drug users at the national and local level in developing and transitional countries, and gender and age distribution un theme group on hiv/aids in china. hiv/aids: china's titanic peril the aids scare in india could be aid-induced india surpasses south africa in aids cases india's response to the hiv epidemic prostitution in india. calcutta: das gupta and company development as freedom famplan: the great debate abates. international family planning perspectives the politics of family planning: issues for the future aids and its metaphors understanding aids: historical interpretations and the limits of biomedical individualism india: less complacency now thailand: the 'foreign' disease philippines: focusing on the hospitality women sexual cultures in east asia: the social construction of sexuality and sexual risk in a time of aids japanese foundation for aids prevention. hiv/aids update sexuality, reproductive health and violence: experiences of migrants from burma in the third phase of hiv pandemic: social consequences of hiv/aids stigma and descrimination and future needs using case vignettes to measure hiv-related stigma among health professionals in china drug abuse, hiv/aids and stigmatisation in a daicommnity in yunnan hiv discrimination: integrating the results from a six-country situational analysis in the asia pacific sex in the city: sexual behaviour, societal change, and stds in saigon uncharted waters: the impact of u.s. policies in vietnam comparative stigma of hiv/aids, sars, and tuberculosis in hong kong this article draws on the research conducted by the author as a phd scholar at the demography and sociology program, the australian national university. the author would like to thank prof. terry hull and dr. zhongwei zhao for their insightful comments on an earlier version of this article. key: cord-257553-479x7av6 authors: kortepeter, mark g.; seaworth, barbara j.; tasker, sybil a.; burgess, timothy h.; coldren, rodney l.; aronson, naomi e. title: health care workers and researchers traveling to developing-world clinical settings: disease transmission risk and mitigation date: 2010-12-01 journal: clin infect dis doi: 10.1086/657115 sha: doc_id: 257553 cord_uid: 479x7av6 with the recent emphasis on funding and training opportunities for global health and humanitarian aid and the increased interest in the field, many health care workers and medical researchers are traveling from resource-replete to resource-limited settings. this type of travel brings unique disease risks not routinely considered for the business or vacationing traveler. this review provides practical advice for this special population of travelers, targeted to specific health care-related risks (needlestick, hemorrhagic fever viruses, severe viral respiratory disease, and tuberculosis), with suggestions for risk mitigation. retrovirals and antibiotics) on hand must all be considered (table 1) . pretravel advice for managing needlestick injuries should start with a discussion of prevention. needlesticks may be more likely to occur in crowded, rushed, and unfamiliar work settings [1] . advise hcws to set up a "sharps container" if one is not available. potential substitutes include an empty container, such as a soda can or plastic laundry detergent bottle. awareness of the disease epidemiology of the region and among potential patients will help in risk assessment should a needlestick injury occur. in addition to human immunodeficiency virus (hiv) and hepatitis viruses, other viruses can be transmitted by needlestick, such as dengue virus and other hemorrhagic fever viruses. other potential disease risks include syphilis, mycobacterial and rickettsial infections, trypanosomiasis, visceral leishmaniasis, and malaria [2] . malaria is particularly notable, because the risk of disease from needlestick may be higher than that of hepatitis viruses or hiv and because fatal and near-fatal cases have occurred as a result of diagnostic delays [3] . knowing the prevalence of drug resistance for needlestick-transmissible endemic diseases is also useful. if a needlestick injury occurs, hcws should note the source patient's region of origin, presenting complaints, history, and physical findings and perform a rapid hiv test or obtain a blood sample for later testing. wash the injury site thoroughly with warm water and soap. caustic antiseptics (eg, bleach and povidone iodide) are not recommended, because they may actually increase the risk of infection as a result of local tissue injury and recruitment of inflammatory cells [4] . a clear plan for follow-up assessments and points of contact is essential. given that diagnostic testing for hepatitis viruses and hiv may be limited at overseas destinations, hcws should consider completing baseline tests for them before travel. hepatitis b virus (hbv) is one of the most transmissible viruses related to needlestick and is generally more common in resource-limited settings than in the united states. the risk to unvaccinated persons is 37%-62% if the source is positive for hepatitis b e antigen (hbeag) and is 23%-37% if the source is negative for hbeag and positive for hepatitis b surface antigen (hbsag). transmission from environmental surfaces can occur even without a cutaneous injury [4] . fortunately, hbv vaccination is highly effective and eliminates this risk for the 90% who respond to the primary series. for those who do not respond, repeat vaccination may protect 30%-50%. before repeating the series, check antibody to hepatitis b core antigen and hbsag to ensure that the patient is not a chronic hbv carrier. deep intramuscular injection with a longer needle may be needed for larger recipients [4] . if serostatus is negative after 6 immunizations, there is no benefit to additional vaccine. seronegative hcws remain at high risk for hepatitis b if exposed, and hepatitis b immunoglobulin (0.06 ml/kg intramuscularly) should be offered after injury and repeated in 1 month [4] . hepatitis b immunoglobulin started within 1 week of exposure provides ∼75% protection [4] . tenofovir and lamivudine have activity against hbv, but there is no guidance for prophylactic use in this setting. consider administration of hepatitis b immunoglobulin before departure for seronegative hcws when concern exists for hbv exposure in a remote setting. passive antibody half-life is 3 weeks, and levels are measurable for ∼2 months [5] . although no prophylaxis for hepatitis c virus (hcv) exposure exists, the needlestick transmission risk is lower (1.8%), and up to 20% of transmitted infections resolve spontaneously. the incubation period is 4-12 weeks. exposed individuals should be monitored with hcv rna testing, if available. immediate treatment is not recommended in light of the significant toxicity and duration of therapy and because some infections will resolve spontaneously, but treatment should be considered if the hcv infection remains established after 12 weeks [6] . most feared is hiv infection after needlestick, but it has the lowest risk of transmission (0.3%). time to initiation of postexposure prophylaxis (pep) is critical. pep is most effective if started immediately and definitely should be started within 3 days of exposure; pep must be continued for 4 weeks [4, 7, 8] . unlike hiv treatment, for which the use of triple-drug combinations is an essential success factor, 3 drugs are unlikely to be significantly better than 2 for pep and paradoxically may be less effective, given that adherence to 3-drug regimens is poorer because of toxicity of the multidrug regimen. zidovudine monotherapy is 79% effective in preventing infection [7] . the world health organization (who) recommends using 2 nucleoside reverse-transcriptase inhibitors from the country's first-line regimen and 3 drugs if the source patient is known to be infected with drug-resistant hiv or if there is 115% resistance in the community [8] . the centers for disease control and prevention (cdc) recommends a 3-drug pep regimen if the source patient is known to be infected with hiv and the source device is a hollow-bore needle or has visible blood contamination. the cdc recommends zidovudine, stavudine, or tenofovir plus emtricitabine or lamivudine. when a third drug is included, both the cdc and who recommend a ritonavirboosted protease inhibitor, not nonnucleoside reverse-transcriptase inhibitors [8, 9] . hcws are strongly encouraged to bring postexposure medications to their destination. follow up for needlestick injury should include a high level of suspicion for any febrile illness in the weeks after the incident; serological tests for hiv, hepatitis, and syphilis at ∼3 months; and adherence support if antiretrovirals are given. hiv rna should be checked if there is an acute febrile illness after an hiv-positive needlestick exposure. if hcv rna measurements are available, they can be monitored at 2, 6, 12, and 24 weeks after a high-risk exposure. viral hemorrhagic fever (vhf) is an acute febrile syndrome, accompanied with a bleeding diathesis, caused by 4 families of single-stranded rna viruses (filoviruses, arenaviruses, bunyaviruses, and flaviviruses) [10, 11] . nosocomial spread and infection of hcws are potential risks but can be mitigated by early recognition and appropriate precautions. the first step in protection is to know which viruses are endemic to the local area, as shown in table 2 [12, 14] . humans are infected via the bite of an arthropod vector, inhalation or ingestion of rodent excretions, or mucosal or percutaneous contact with infected blood or secretions of patients [10] . in underresourced settings, spread of these viruses in the hospital environment is often facilitated by lack of recognition, reuse of equipment (such as contaminated needles), and inconsistent enforcement of basic infection-control practices, frequently because of inadequate supplies. clinical diagnosis of vhf requires an index of suspicion and is challenging, especially in underdeveloped regions, where the broad differential diagnosis of acute febrile illness includes malaria, typhoid, rickettsial illness, and leptospirosis. common presenting symptoms include acute onset of fever, malaise, headache, myalgias, and prostration, frequently accompanied by gastrointestinal complaints. early signs may include pharyngitis, conjunctivitis, cutaneous flushing, or a maculopapular rash. hemorrhagic manifestations may include oozing at intravenous puncture sites, petechiae, purpura, large ecchymoses, or frank hemorrhage; however, manifestations, frequency, and severity of bleeding varies depending on the agent [11, 12] . case fatality rates can range from !1% for rift valley fever to nearly 90% with ebola and marburg fever [15] . specific diagnosis can be made on the basis of detection of virus or viral antigens in serum, plasma, or whole blood by antigen-capture or antibody-detection enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction. viral isolation should be attempted only in a biosafety level 4 containment laboratory, such as those at the cdc or the us army medical research institute of infectious diseases [11] . all hemorrhagic fever viruses except dengue virus are known to infect via artificially produced aerosols in a laboratory environment. potential spread between laboratory animals has raised concern about potential person-to-person airborne transmission [10, 16] . however, the epidemiology of vhf outbreaks indicates that if person-to-person airborne transmission does occur, it is rare [15] . in the 1995 zaire ebola epidemic, infections of hcws declined dramatically after the institution of isolation precautions [17] . however, achieving universal adherence to strict isolation precautions in hot, humid conditions in such environments is a challenge, as demonstrated in a 2000 ebola outbreak in uganda [18] . specific guidance for infection control is available [19] . standard, contact, and droplet precautions should be used in a private room for both inpatient and outpatient settings. although airborne precautions are not required in earlier stages of illness, they should be considered early (1) to eliminate the need for later transfer to an airborne isolation room, (2) for severe pulmonary involvement, and (3) for certain activities that might stimulate coughing or generate aerosols (eg, bronchoscopy and endotracheal intubation) [19, 20] . the who and cdc have developed vhf infection-control recommendations for african health care settings that are applicable to other austere environments [21] . their handbook recommends practical measures for establishing standard precautions for all patients, routine hand washing, and safe handling and disposal of used needles and syringes. upgrading to vhf precautions includes isolating the patient and wearing protective clothing (scrub suit, gown, apron, gloves, mask, headcover, eyewear, and rubber boots). hcw training should occur in advance, and access to patient care areas should be limited to those with a definite need. the cdc identifies 3 levels of risk (casual, close, and high-risk contacts) for potential exposure, along with follow-up recommendations [22] . the only vhf vaccine currently licensed in the united states is for yellow fever. investigational vaccines exist for several other vhfs; however, they are undergoing animal or clinical testing, are only for laboratory workers, or are not generally available [10] . medical countermeasures are limited. in a postexposure situation, expert consultation for treatment or prophylaxis measures is recommended. recently, emergent severe respiratory viral diseases have prompted intensified research and public health concern. a coronavirus causing severe acute respiratory syndrome (sars-cov) spread rapidly in 2003, resulting in a pandemic with frequent nosocomial spread [23] . the reemergence in the human population of avian influenza a(h5n1) in 2003 raised international alarm over a potential novel influenza pandemic. widespread infection in poultry and wild birds continues to cause occasional human cases in southeast asia and the middle east. although human-to-human transmission is limited, the case fatality rate exceeds 60% [24] . most recently, influenza a(h1n1), a reassortment zoonotic influenza virus, emerged in 2009 in north america and spread throughout the world, resulting in a pandemic declaration by june 2009. as of april 2010, 1300,000 confirmed cases (producing at least 17,700 deaths) have been reported from 1200 countries, and the pandemic virus has effectively replaced previously circulating seasonal influenza strains [25] . researchers and hcws face obvious risks for occupational acquisition of respiratory diseases. limited data to quantify the actual risk exist, although compelling anecdotes document the potential danger [26] . protective measures to mitigate risk for those with exposure include contact and respiratory precautions. optimal strategies depend on the nature of the pathogen, its known or suspected transmission routes, and exposure circumstances. transmission of sars-cov appears to occur predominantly through close interactions with infected persons. during the pandemic, transmission to hcws occurred after close, unprotected contact with symptomatic persons and was significantly mitigated once infection-control precautions were implemented; the degree of risk was related to the type and intensity of exposure (endotracheal intubation was significantly associated with contracting sars) [27, 28] . airborne transmission of sars-cov is clearly suggested by modeling studies, retrospective evaluation of nosocomial outbreaks, and anecdotal reports of transmission aboard commercial aircraft [29] [30] [31] . transmission of human influenza viruses occurs via large droplets, direct and indirect contact, and droplet nuclei. the relative contribution of each route is not known and likely varies with the setting and circumstances. there are conflicting opinions regarding the importance of airborne transmission of influenza viruses in humans [32] [33] [34] . direct contact may be more important than inhalation in conditions of high humidity and temperature [35] [36] [37] . frequent and diligent hand hygiene is likely the most important intervention for the interruption of transmission for many respiratory viruses, particularly influenza virus. when clean water is not available in field settings, alcohol-based hand gels should be used. these gels and/or washing with soap and water have demonstrated effectiveness [38] . personnel should be trained in and use protective clothing, gloves, shoe covers, goggles, and masks. the principal question with respect to respiratory protection is whether the mask should be a fitted, high-efficiency, particlefiltering n95 respirator. the answer depends on the relative contribution of aerosols versus large droplets to disease trans-mission. the who cites evidence from observational studies in hospitals suggesting that droplet transmission is responsible for the majority of nosocomially acquired cases to recommend standard plus droplet precautions for persons with seasonal influenza [39] . recent results suggest that surgical masks are effective for preventing seasonal influenza, although some controversy exists [40] . during the 2009 h1n1 pandemic, an institute of medicine panel recommended that hcws use n95 masks, given the uncertainty regarding risks of transmission [41] . in resource-limited settings, priority should be given to risk-reduction interventions focusing on hand hygiene, contact precautions, expedient respiratory protection, and the use of ppe that is effective for aerosol respiratory protection, particularly during exposures likely to generate aerosols (eg, intubation). although heterotypic protection against novel strains and pathogens cannot be expected, influenza vaccination is recommended for individuals involved in respiratory disease research and for hcws caring for such patients, unless contraindicated. vaccination is recommended to decrease the risk of illness due to seasonal influenza virus, reducing the frequency of diagnostic and management dilemmas for those occupationally involved in close contact with individuals with severe respiratory disease, such as that caused by influenza a(h5n1) or sars. prevention of influenza by means of chemoprophylaxis with neuraminidase inhibitors is a potential strategy and may be indicated in certain settings, such as direct contact with sources of avian influenza [39] . in general, tuberculosis is a potential threat to hcws and researchers who travel to work in high-burden countries [42] . approximately 500,000 new cases of multidrug-resistant (mdr) tuberculosis, defined as tuberculosis resistant to isoniazid and rifampin, were reported in 2008, and 1% were treated according to who standards [43] . extensively drug-resistant (xdr) tuberculosis, defined as mdr tuberculosis plus resistance to fluoroquinolones and a second-line injectable (kanamycin, amikacin, or capreomycin), has been reported in 58 countries [44] . mdr and xdr tuberculosis pose special risks. recognition of infectious individuals is difficult because of the lack of laboratory infrastructure for culture and susceptibility testing. treatment of mdr and xdr tuberculosis is prolonged, with significant toxicity and poor outcomes. treatment completion or cure is reported for 46%-87% of mdr cases and for 29%ϫ71% of xdr cases [45] [46] [47] [48] [49] . the epidemiology of drug-resistant tuberculosis is described in the who fourth global report [50] . information is incomplete for some regions, especially africa, but medical literature identifies a serious problem with mdr and xdr tuberculosis there [51, 52] . china represents 25% of the global burden, and india represents 120%. countries of the former soviet union have the highest proportion of drug resistance; 20% of all cases are mdr tuberculosis. other countries with a high proportion of mdr tuberculosis (rates of 13%) are vietnam, thailand, korea, the philippines, peru, and guatemala [50] . because of good infection control, at-risk screening, and treatment of latent tuberculosis, nosocomial transmission is rare in the united states. exposure overseas may occur in locations with uncontrolled transmission and inadequate infection-control practices. exposure is likely not only in hospitals but also in clinics, hospices, prisons, factories, mines, orphanages, and offices [51] . most hcws are not counseled regarding tuberculosis risks before departure, nor are they evaluated on their return. many are unaware of the risk, and if symptoms develop the diagnosis is often delayed, allowing potential transmission to vulnerable populations in us medical facilities. before departure, the risk of tuberculosis at the destination should be assessed. hcws should be screened for latent tuberculosis by an interferon g release assay (igra) or the tuberculin skin test (tst). igras are more specific and are preferred for those with a history of bacille calmette-guérin (bcg) vaccination. if the results of tst and igra are negative, vaccination with bcg may be considered 2-6 months before departure (b.j.s., personal communication). two months after returning, rescreen with an igra. fit testing with either a disposable filtering facepiece respirator or a reusable elastomeric respirator is advised. for those with facial hair, an adequate seal is not possible and a powered air purifying respira-tor is needed. respiratory protection should be brought to the destination. who infection-control guidelines for resource-limited countries suggest measures to limit transmission, including patient cohorting, individual respiratory protection, and natural ventilation [53] . engineering controls (such as negative air pressure) are usually not available; therefore, hcws should minimize the time spent in high-risk areas with poor ventilation when possible. personal respiratory protection is the main option for limiting exposure [54] . treatment of latent tuberculosis is strongly recommended for us hcws. if latent tuberculosis is detected before departure, it is often best to delay treatment until return. the cdc recommends treatment of latent tuberculosis with 9 months of isoniazid or 4 months of rifampin [55] . when latent tuberculosis is diagnosed after travel to high-risk areas, infection with mdr or xdr tuberculosis must be considered. if exposure to mdr or xdr tuberculosis is not reported or is unlikely, standard treatment is indicated. when the risk is significant or definite exposure occurs, treatment based on the suspected or known susceptibility of mdr and xdr tuberculosis exposure can be considered. this approach is often taken in the united states but is based on expert opinion and is controversial. treatment may include 6-12 months of either ethambutol or pyrazinamide with levofloxacin or moxifloxacin [56] . the cdc recommends clinical and radiographic follow-up for 24 months for those with latent tuberculosis likely due to mdr or xdr pathogens, regardless of whether treatment of latent tuberculosis occurs. if tuberculosis develops, rapid identification of resistance is critical. molecular detection of drug resistance is available at the cdc and at some state health departments. consultation with an expert is strongly advised. assistance is available from health departments or the cdc's regional training and medical consultation centers [57] . medical researchers and health care workers who travel to resource-limited settings to conduct patient care or research compose an understudied travel population. the nature of their work, restricted provision of supplies, and potentially limited use of infection-control practices puts them at unique risk for acquiring specific infections. this review provides practical guidance to mitigate the risk of potential occupational infectious diseases transmission in such settings and aspires to raise interest in further study of how to best to protect these individuals. a review of sharps injuries and preventative strategies infection risks following accidental exposure to blood or body fluids in health care workers: a review of pathogens transmitted in published cases occupational plasmodium falciparum malaria following accidental blood exposure: a case, published reports and considerations for post-exposure prophylaxis public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis hepatitis b immune globulin early treatment improves outcomes in acute hepatitis c virus infection: a meta-analysis two drugs or three? balancing efficacy, toxicity, and resistance in postexposure prophylaxis for occupational exposure to hiv joint who/ilo guidelines on post-exposure prophylaxis (pep) to prevent hiv infection updated u.s. public health service guidelines for the management of occupational exposures to hiv and recommendations for post-exposure prophylaxis bichat guidelines for the clinical management of haemorrhagic fever viruses and bioterrorism-related haemorrhagic fever viruses medical aspects of biological warfare viral hemorrhagic fevers as agents of bioterrorism investigations of marburg virus circulation among egyptian fruit bats in southwest uganda transmission of dengue virus without a mosquito vector: nosocomial mucocutaneous transmission and other routes of dengue transmission hemorrhagic fever viruses as biological weapons: medical and public health management transmission of ebola virus (zaire strain) to uninfected control monkeys in a biocontainment laboratory commission de lutte contre les epidémies à kikwit. the reemergence of ebola hemorrhagic fever, democratic republic of the congo outbreak of ebola hemorrhagic fever-uganda interim guidance for managing patients with suspected viral hemorrhagic fever in u update: management of patients with suspected viral hemorrhagic fever-united states world health organization and centers for disease control and prevention. infection control for viral hemorrhagic fevers in the african health care setting management of patients with suspected viral hemorrhagic fever severe acute respiratory syndrome coronavirus as an agent of emerging and re-emerging infection world health organization. cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who world health organization. pandemic (h1n1) 2009-update 95 cluster of cases of severe acute respiratory syndrome among toronto healthcare workers after implementation of infection control precautions: a case series which preventive measures might protect health care workers from sars? evidence of airborne transmission of the severe acute respiratory syndrome virus cluster of sars among medical students exposed to single patient transmission of the severe acute respiratory syndrome on aircraft transmission of influenza a in human beings aerosol transmission of influenza a virus: a review of new studies virus survival as a seasonal factor in influenza and poliomyelitis transmission of influenza virus in temperate zones is predominantly by aerosol, in the tropics by contact: a hypothesis high temperature (30њc) blocks aerosol but not contact transmission of influenza virus influenza virus transmission is dependent on relative humidity and temperature efficacy of soap and water and alcohol-based hand rub preparations against live h1n1 influenza virus on the hands of human volunteers avian influenza guidelines, recommendations, descriptions surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial respiratory protection for healthcare workers in the workplace against novel h1n1 influenza a: a letter report tuberculosis in health care workers in kwazulu-natal, south africa update: tuberculosis facts world health organization. countries that had reported at least one xdr-tb case by time to sputum culture conversion in multidrug-resistant tuberculosis: predictors and relationship to treatment outcome treatment outcome of multidrug-resistant tuberculosis among vietnamese immigrants extensively drug-resistant tuberculosis in california treatment outcomes and long-term survival in patients with extensively drug-resistant tuberculosis multidrug-and extensively drug-resistant tuberculosis anti-tuberculosis drug resistance in the world emergence of increased resistance and extensively drug-resistant tuberculosis despite treatment adherence extensively drug resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and hiv in a rural area of south africa who policy on tb infection control in health-care facilities, congregate settings and households tuberculosis infection control in resource-limited settings in the era of expanding hiv care and treatment targeted tuberculin testing and treatment of latent tuberculosis infection management of persons exposed to multidrug-resistant tuberculosis potential conflicts of interest. all authors: no conflicts. key: cord-264408-vk4lt83x authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: animal models of human viral diseases date: 2017-06-23 journal: animal models for the study of human disease doi: 10.1016/b978-0-12-809468-6.00033-4 sha: doc_id: 264408 cord_uid: vk4lt83x as the threat of exposure to emerging and reemerging viruses within a naïve population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. recent outbreaks of middle east respiratory syndrome corona virus, ebola virus, chikungunya virus, and zika virus illustrate the emerging threats that are encountered. by utilizing animal models in this endeavor, the host response to viruses can be studied in a more complex and integrated context to identify novel drug targets, and assess the efficacy and safety of new products rapidly. this is especially true in the advent and implementation of the fda animal rule. although no one animal model is able to recapitulate all aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to consider prior to an animal study are route of viral exposure, species of animal, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand disease progression, pathogenesis, and immunologic responses to viral infections in humans. furthermore, to test vaccines and medical countermeasures, animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease course. a good animal model of viral infection should allow assay of many parameters of infection, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most studies in small animal models when possible, and studies in nhps to fill in the remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen free (spf), transgenic and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated to humans. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables collection of larger or more frequent blood samples, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains, thus greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic ferrets are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically, the closest species to humans, thus disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns pertaining to experimentation on nhps along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure the fewest number of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, depending on the pathogen, the route of exposure and the dose should mimic the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on viruses for each family that are of the greatest concern for public health worldwide. norovirus, the genus of which norwalk is the prototypic member, is the most common cause of gastroenteritis in the united states (hall et al., 2013) . there are five distinct genogroups (gi-gv) and numerous strains of norwalk virus, including the particularly significant human pathogens gi.1 norwalk virus, gii.2 snow mountain virus, and gii.1 hawaii virus. in developing countries, norwalk virus, also known as "winter vomiting virus," is responsible for approximately 200,000 deaths annually (patel et al., 2008) . a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants (chen et al., 2009; lutgehetmann et al., 2012; turcios-ruiz et al., 2008) . symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation is normally 1-3 days, with symptoms persisting for 2-3 days (koopmans and duizer, 2004) . viral shedding can range from 6 to 55 days in healthy individuals (atmar et al., 2014) . however, longer illness duration can be indicative of immunocompromised status, with the elderly and young having a prolonged state of shedding (harris et al., 2008; rockx et al., 2002) . interestingly, individuals vary greatly in susceptibility to norovirus infection depending on their fucosyl transferase 2 (fut2) allele functionality and histoblood group antigen status, with type a and o individuals susceptible and types ab and b resistant (hutson et al., 2005) . transmission occurs predominately through the oralfecal route with contaminated food and water being a major vector (atmar and estes, 2001; becker et al., 2000; koopmans and duizer, 2004) . vomiting results in airborne dissemination of the virus with areas of 7.8 m 2 being contaminated and subsequent transmission from oral deposition of airborne particles or contact with contaminated fomites, which can remain contaminated for up to 42 days (makison booth, 2014; tung-thompson et al., 2015) . each vomiting event in a classroom setting elevates the risk of norovirus illness among elementary students with proximity correlating with attack rates (evans et al., 2002; marks et al., 2003) . viral titers in emesis and fecal suspensions are as high as 1.2 × 10 7 and 1.6 × 10 11 ges (genomic equivalent copies per milliliter), respectively and the 50% infectious dose is 1320 ges (atmar et al., 2014) . therefore, outbreaks can be extremely difficult to contain. therapeutic intervention consists of rehydration therapy and antiemetic medication (bucardo et al., 2008; moe et al., 2001) . no approved vaccine or therapeutic is available, and development has been challenging given that immunity is short-lived after infection, new strains rapidly evolve and the correlates of protection are not completely understood (chen et al., 2013) . however, one promising strategy utilized a virus-like particle (vlp)-based vaccine that protected or reduced infection by almost 50% in human volunteers (aliabadi et al., 2015; atmar et al., 2011) . given the relatively benign disease in adults, experimental challenge has been carried out on human volunteers (ball et al., 1999; tacket et al., 2000) . viral titers are determined by shedding in feces and sera with histopathology changes monitored by biopsies particularly of the duodenum. the ph of emesis samples collected containing virus is consistent with viral replication in the small intestine with reflux to the stomach (kirby et al., 2016) . additionally, norwalk virus has been shown to bind to duodenal tissue (chan et al., 2011) . however, this type of research is technically difficult and expensive, and thus other models have been developed. a major hindrance to basic research into this pathogen is the lack of permissive cell culture systems or animal models for norwalk virus. nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus are monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity is measured exclusively by assay of viral shedding (rockx et al., 2005) . incidentally, more viruses were needed to create an infection when challenging by the oral route than by the intravenous (iv) route (purcell et al., 2002) . chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route (bok et al., 2011) . although none of the animals developed disease symptoms, viral shedding within the feces was observed within 2-5 days postinfection and lasted anywhere from 17 days to 6 weeks. viremia never occurred and no histopathological changes were detected. the amount and duration of viral shedding was in-line with what is observed upon human infection. as such, chimeric chimpanzee-human antinorovirus neutralizing antibodies have been explored as a possible therapeutic strategy (chen et al., 2013) . a recently identified calicivirus of rhesus origin, named tulane virus, has been used as a surrogate model of infection. unlike norwalk virus, tulane virus can be cultured in cells. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever 2 days postinfection. viral shedding was detected for 8 days. the immune system produced antibodies that dropped in concentration within 38 days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease (sestak et al., 2012) . a murine norovirus has been identified and is closely related to human norwalk virus (karst et al., 2003) . however, clinically the virus presents a different disease. the murine norovirus model does not include observable gastrointestinal clinical signs, possibly in part because rodents lack a vomiting reflex. additionally, mice infected with norovirus develop a persistent infection in contrast to human disease (hsu et al., 2006 (hsu et al., , 2007 khan et al., 2009) . porcine enteric caliciviruses can induce diarrheal disease in young pigs, and an asymptomatic infection in adults (wang et al., 2006 . gnotobiotic pigs can successfully be infected with a passaged clinical norovirus isolate by the oral route. diarrheal disease developed in 74% of the animals and virus was detected in the stool of 44% of the animals. no major histopathological changes or viral persistence was noted (cheetham et al., 2006) . calves are naturally infected with bovine noroviruses (scipioni et al., 2008) . experimental challenge of calves by oral inoculation with a bovine isolate resulted in diarrheal disease 14-16 h postinfection. recovery of virus was achieved after 53.5 and 67 h postinfection (otto et al., 2011) . eastern equine encephalitis virus (eeev), western equine encephalitis virus (weev), and venezuelan equine encephalitis virus (veev) present with near synonymous symptoms. the majorities of human cases are asymptomatic, but can present as a flu-like illness progressing to central nervous system (cns) involvement to include seizures and paralysis. mortality rates vary among the virus, with the highest reported for eev at 36%-75% followed by weev and lastly veev at less than 1% (ayers et al., 1994; griffin, 2007; steele and twenhafel, 2010) . there are currently no licensed vaccines or therapies but a recent phase 1 clinical trial of a veev dna vaccine resulted in veev-neutralizing antibody responses in 100% of the subjects (hannaman et al., 2016) . mouse models have been developed for numerous routes of infection including cutaneous, intranasal (in), intracranial (ic), and aerosol. eeev susceptibility in mouse models is correlated with age, with younger mice being more susceptible than adults. importantly, eeev pathogenesis is dependent on route of infection with delayed progression upon subcutaneous (subq) exposure (honnold et al., 2015) . newborn mice display neuronal damage with rapid disease progression, resulting in death (murphy and whitfield, 1970) . similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, while inoculation via the subq route causes a pantropic infection eventually resulting in encephalitis (liu et al., 1970; morgan, 1941) . a general drawback to the usage of the mouse model is the lack of vascular involvement during the disease course (liu et al., 1970) . after subq inoculation with weev, suckling mice started to show signs of disease by 24 h and died within 48 h (aguilar, 1970) . the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on day 4-5 postinfection. mice were severely ill by day 8 and appeared hunched and dehydrated. death occurred between days 7 and 14 with brain and mesodermal tissues, such as heart, lungs, liver, and kidney involvement (aguilar, 1970; monath et al., 1978) . intracerebral and in routes of infection resulted in a fatal disease that was highly dependent on dose while intradermal (id) and subq inoculations caused only 50% fatality in mice regardless of the amount of virus (liu et al., 1970) . comparing susceptibility of inbred and outbred strains revealed that cd-1, balb/c, a/j, and c57bl6 mice were all highly susceptible to experimental infection via subq inoculation when challenged prior to 10 weeks old with cns involvement and lethality (blakely et al., 2015) . subq/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans (macdonald and johnston, 2000) . virus begins to replicate in the draining lymph nodes at 4 h postinoculation. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis (charles et al., 1995; steele et al., 1998) . aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death (charles et al., 1995; steele and twenhafel, 2010; steele et al., 1998) . in challenge of c3h/hen mice with high dose veev caused high morbidity and mortality (julander et al., 2008b) . viral titers in brain peaked on day 4 postchallenge and remained elevated until animals succumbed on day 9-10 postchallenge. protein cytokine array performed on brains of infected mice showed elevated il-1a, il-1b, il-6, il-12, mcp-1, ifnγ, mip-1a, and rantes levels. this model was used successfully to test antivirals against veev (julander et al., 2008a) . additionally, a veev vaccine inactivated with 1,5-iodonaphthyl azide v3526 protects against both footpad and aerosol challenge with virulent veev in a mouse model (gupta et al., 2016) . guinea pigs and hamsters have also been developed as animal models for eeev studies (paessler et al., 2004; roy et al., 2009) . guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed in brain lesions in the experimentally challenged animals (roy et al., 2009) . subq inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis, and death. in addition, parenchyma necrosis were observed in the liver and lymphoid organs (paessler et al., 2004) . harlan sprague-dawley hamsters develop viremia and progress to respiratory, gastrointestinal, and nervous system involvement when inoculated via subq route. vasculitis and encephalitis were both evident in this model, which mirrors the human disease clinical spectrum (paessler et al., 2004) . weev is highly infectious to guinea pigs and has been utilized for prophylactic screening (sidwell and smee, 2003) . studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases (zlotnik et al., 1972) . cns involvement was evident with intracerebral, intraperitoneal (ip), and id inoculations (zlotnik et al., 1972) . ip inoculation of weev is fatal in guinea pigs regardless of amount of virus inoculum, with the animals exhibiting signs of illness on day 3-4, followed by death on day 5-9 (nalca, unpublished results) . id, im, or iv inoculations of eeev in nhps cause disease, but does not reliably result in neurological symptoms (dupuy and reed, 2012) . intracerebral infection of eeev produces nervous system disease and fatality in monkeys (nathanson et al., 1969) . the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in nhps when they are inoculated by the peripheral route (wyckoff, 1939) . in and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but are less drastic than intracerebral injections (wyckoff, 1939) . the aerosol route of delivery will result in uniformly lethal disease in cynomolgus macaques (reed et al., 2007) . in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed and nhps became moribund and were euthanized between 5-9 days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals (steele and twenhafel, 2010) . similar clinical signs and pathology were observed when common marmosets were infected with eeev by the in route (adams et al., 2008) . both aerosol and in nhp models had similar disease progression and pathology as seen in human disease. very limited studies have been performed with nhps. reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model could be useful for testing of vaccines and therapeutics against weev (reed et al., 2005) . veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at 12-72 h after infection and lasted less than 12 h. secondary fever generally began on day 5 and lasted 3-4 days (gleiser et al., 1961) . veev-infected nhps exhibited mild symptoms, such as anorexia, irritability, diarrhea, and tremors. leukopenia was common in animals exhibiting fever (monath et al., 1974) . supporting the leukopenia, microscopic changes in lymphatic tissues, such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, and focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection (gleiser et al., 1961) . the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day 6 postinfection during the secondary febrile episode. similar to these observations, when cynomolgus macaques were exposed to aerosolized veev, fever, viremia, lymphopenia, and clinical signs of encephalitis were observed but the nhps did not succumb to disease (reed et al., 2004) . a common marmoset model was utilized for comparison studies of south america (sa) and north america (na) strains of eeev (adams et al., 2008) . previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected in with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized 4-5 days after the challenge. although sa strain-infected animals developed viremia, they remained asymptomatic and survived until the end of study. chikungunya virus (chikv) is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics centered within africa and southeast asia (griffin, 2007) . the virus is transmitted by aedes aegypti and aedes albopictus mosquitoes. given the widespread endemicity of aedes mosquitoes, chikv has the potential to spread to previously unaffected areas. this is typified by the emergence of disease reported for the first time in 2005 in the islands of south-west indian ocean, including the french la reunion island, and the appearance in central italy in 2007 (charrel et al., 2007; rezza et al., 2007) . the incubation period following a mosquito bite is 2-5 days, leading to a self-limiting acute phase that lasts 3-4 days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported (powers and logue, 2007) . individuals typically develop a stooped posture due to the pain. for approximately 12% of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of 3 and 18 years old have an increased risk of developing neurological manifestations (arpino et al., 2009) . there is currently no approved vaccine or antiviral. wild-type c57bl/6 adult mice are not permissive to chikv infection by id inoculation. however, it was demonstrated that neonatal mice were susceptible and severity was dependent upon age at infection. six-dayold mice developed paralysis by day 6, and all succumbed by day 12, whereas 50% of 9-day-old mice were able to recover from infection. by 12 days, mice were no longer permissive to disease. symptomatic mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications (couderc et al., 2008; ziegler et al., 2008) . an adult mouse model has been developed by injection of the ventral side of the footpad of c57bl/6j mice. viremia lasted 4-5 days accompanied with foot swelling and noted inflammation of the musculoskeletal tissue morrison et al., 2011) . adult ifnα/βr knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within 3 days. this model was useful in identifying the viral cellular tropism for fibroblasts (couderc et al., 2008) . icr and cd-1 mice can also be utilized as a disease model. neonatal mice inoculated subq with a passaged clinical isolate of chikv developed lethargy, loss of balance, and difficulty walking. mortality was low, 17 and 8% for newborn cd-1 and icr mice, respectively. the remaining mice fully recovered within 6 weeks after infection (ziegler et al., 2008) . a drawback of both the ifnα/βr and cd-1 mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised (teo et al., 2012) . a chronic infection model was developed using recombinant activating gene 1 (rag1 −/− ) knockout mice. in this study, mice inoculated via the footpad lost weight in comparison to the control group. both footpad and subq injected mice developed viremia 5-6 days postinfection, which was detectable up to 28 days postinfection. inflammation was evident in the brain, liver, and lung of the subq inoculated animals at 28-56 days postinfection. despite minimal footpad swelling on day 2 postinfection, on day 14 there was severe muscle damage noted at necropsy, which resolved by day 28 (seymour et al., 2015) . golden hamsters serve as another option for small animal modeling. although hamsters do not appear to develop overt clinical symptoms following subq inoculation, viremia developed in the majority of animals within 1 day postinfection with clearance following from day 3 to 4. histologically, inflammation was noted at the skeletal muscle, fascia, and tendon sheaths of numerous limbs. this study was limited in the number of animals utilized, and more work is needed to further develop the hamster model (bosco-lauth et al., 2015) . nhp models of disease include adult, aged, and pregnant rhesus macaques in addition to cynomolgus macaques (broeckel et al., 2015) . differing routes of infection (subq, iv, and im) have been successfully administered, although there is not a clear understanding of the role that route of transmission plays in subsequent pathogenesis and clinical symptoms. typically, viremia is observed 4-5 days postinfection with a correlation between infectious titer and time to viremia observed in cynomolgus but not rhesus (labadie et al., 2010; messaoudi et al., 2013) . fever began at 1-2 days postinfection and persisted for 2-7 days and 3-7 days in cynomolgus and rhesus, respectively and coincided with rash (chen et al., 2010; labadie et al., 2010; messaoudi et al., 2013) . overall blood chemistries changed in conjunction with initiation of viremia, and returned to baseline 10-15 days postexposure (chen et al., 2010) . cns involvement has been difficult to reproduce in nhp models, although it was reported that high inoculum in cynomolgus did result in meningoencephalitis (labadie et al., 2010) . the nhp models have been utilized to conduct efficacy testing on novel vaccines and therapeutics (broeckel et al., 2015) . dengue virus (denv) is transmitted via the mosquito vectors a. aegypti and a. albopictus (moore and mitchell, 1997) . given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to denv. this results in approximately 50 million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia (gubler, 2002) . it is estimated that there are 20,000 deaths each year due to dengue hemorrhagic fever (dhf) (guzman and kouri, 2002) . there are four distinct serotypes of denv, numbered 1-4, which are capable of causing a wide clinical spectrum that ranges from asymptomatic to severe with the development of dhf (world health organization, 1997) . incubation can range from 3 to 14 days, with the average being 4-7 days. the virus targets dendritic cells and macrophages following a mosquito bite (balsitis et al., 2009) . typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure (world health organization, 1997) . thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis (gregory et al., 2010) . severe disease has a higher propensity to occur upon secondary infection with a different denv serotype (thein et al., 1997) . this is hypothesized to occur due to antibody dependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in order to further progress toward an effective drug or vaccine, small human cohort studies have taken place. however, to provide statistically relevant results, testing must progress in an animal model. in developing an animal model, it is important to note that mosquitoes typically deposit 10 4 -10 6 pfu, and is considered the optimal range during experimental challenge . denv does not naturally replicate effectively in rodent cells, creating the need for mouse-adapted strains, engineered mouse lines, and a variety of inoculation routes to overcome the initial barrier. several laboratory mouse strains including a/j, balb/c, and c57bl/6 are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis (huang et al., 2000; paes et al., 2005; shresta et al., 2004) . a higher dose of an adapted denv strain induced dhf symptoms in both balb/c and c57bl/6 souza et al., 2009) . this model can also yield asymptomatic infections. a mouse-adapted strain of denv 2 introduced into ag129 mice developed vascular leak syndrome similar to the severe disease seen in humans (shresta et al., 2006) . passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased and animals died due to vascular leak syndrome (balsitis et al., 2010) . another mouse-adapted strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability (souza et al., 2009) . ic injection of suckling mice with denv leads to death by paralysis and encephalitis, which is rare in human infection (lee et al., 2015; parida et al., 2002; zhao et al., 2014a) . immunocompromised mice have also been used to gain an understanding of the pathogenesis of denv. the most well-defined model is ag129 which is deficient in ifnα/β and γ receptors and can recapitulate dhf/dss if a mouse-adapted strain is utilized yauch et al., 2009) . scid mice engrafted with human tumor cells develop paralysis upon infection, and thus are not useful for pathogenesis studies (blaney et al., 2002; lin et al., 1998) . df symptoms developed after infection in nod/scid/il2rγko mice engrafted with cd34 + human progenitor cells (mota and rico-hesse, 2011) . rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and labadapted strain of denv 2 (kuruvilla et al., 2007) . a passaged clinical isolate of denv 3 was used to create a model in immunocompetent adult mice. ip injection in c57bl/6j and balb/c caused lethality by day 6-7 postinfection in a dose dependent manner. the first indication of infection was weight loss beginning on day 4 followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, ast and alt, were also observed. viremia was established by day 5. this model mimicked the characteristic symptoms observed in human dhf/dss cases (costa et al., 2012) . vascular leakage was also observed when c57bl/6 were inoculated with denv 2 (st john et al., 2013) . a murine model was developed that utilized infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of denv 2. infected mosquitoes then fed upon the mouse footpad to allow for transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the number of mosquitoes it was exposed to, with 4-5 being optimal. detectable viral rna was in line with historical human infection data. severe thrombocytopenia developed on day 14. this model is notable in that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus (cox et al., 2012) . nhp models have used a subq inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection (marchette et al., 1973) . the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes (marchette et al., 1980) . throughout these preliminary studies, no clinical disease was detected. in order to circumvent this, a higher dose of denv was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day 3 and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed (onlamoon et al., 2010) . a robust antibody response was observed in multiple studies (marchette et al., 1973; onlamoon et al., 2010) . marmosets also mirror human dengue infection, developing fever, leukopenia, and thrombocytopenia following subq inoculation (omatsu et al., 2011 (omatsu et al., , 2012 . nhps are able to produce antibodies similar to those observed during the course of human infection, making them advantageous in studying ade. sequential infection led to a cross-reactive antibody response which has been demonstrated in both humans and mice (midgley et al., 2011) . this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was 3-to 100-fold higher than previously reported, however, no clinical signs were apparent (goncalvez et al., 2007) . the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in 1937 uganda in (smithburn et al., 1940 . after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early 1950s (melnick et al., 1951; taylor et al., 1953) and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia (hayes, 1989) . the virus first reached the western hemisphere in the summer of 1999, during an outbreak involving humans, horses, and birds in the new york city metropolitan area (centers for disease control and prevention, 1999; lanciotti et al., 1999) . since 1999, the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from 3 to 6 days (monath and tsai, 2002) . the mortality rate following neuroinvasive disease ranges from 4% to 11% (asnis et al., 2000; hayes, 1989; hubalek and halouzka, 1999; komar, 2000) . the most severe complications are commonly seen in the elderly, with reported case fatality rates from 4% to 11%. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e., asymptomatic, fever, encephalitis) (pogodina et al., 1983) . thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subq inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia (lieberman et al., 2009; pletnev et al., 2003) . this is mirrored in new zealand white rabbits in that they only develop fever and low levels of viremia following inoculation via footpad (suen et al., 2015) . id inoculation of both marmosets and rhesus macaques did not yield any clinical signs of disease including fever. viremia was detected in both nhp species, but marmosets developed a higher titer for a greater duration than rhesus (verstrepen et al., 2014) . wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and ip routes, resulting in encephalitis and 100% mortality. id route pathogenesis studies indicated that langerhans dendritic cells are the initial viral replication sites in the skin (brown et al., 2007; johnston et al., 1996) . the infected langerhans cells then migrate to lymph nodes and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases (byrne et al., 2001; cunha et al., 2000; diamond et al., 2003; fratkin et al., 2004) . the swiss mouse strain was inoculated ip in order to screen a variety of viral lineages to assess differences in pathogenesis (bingham et al., 2014) . tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared asymptomatic during the first 5 days, became lethargic at approximately day 6, and developed neurologic symptoms between days 7 and 10 . many of the severely affected animals died 7-14 days after infection. viremia was detected in the hamsters within 24 h after infection and persisted for 5-6 days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the previously mentioned monkey experiments by pogodina et al. (1983) , persistent wnv infection was found in the brains of hamsters. zika virus recently came to the forefront of public health concerns with the outbreak in brazil at the end of 2015. the clinical disease spectrum is highly variable with reports of a flu-like illness accompanied by rash, guillan-barre syndrome, and microcephaly in newborns (ramos da silva and gao, 2016) . to date, a correlation between gestational age at which exposure to the virus occurs and severity of microcephaly is not fully understood (brasil et al., 2016) . however, a recent study of pregnant women in columbia found that infection with zika virus during the third trimester was not associated with any obvious structural abnormalities of the fetus (pacheco et al., 2016) . transmission of the virus occurs via the bite from an infected a. aegypti or a. albopictus (ramos da silva and gao, 2016) . other reported routes of exposure include sexual transmission and blood transfusion (cunha et al., 2016; d'ortenzio et al., 2016; hills et al., 2016; mccarthy, 2016) . the emergence of this virus with no approved vaccine or therapy, and few diagnostic options demonstrates the utility of well-characterized animal model development. it was first demonstrated in 1956 that experimentally infected mosquitoes could be used to transmit the virus to mice and nhps (boorman and porterfield, 1956) . a129 mice were susceptible to nonadapted zika virus infection following subq inoculation of the limbs. mice began to lose weight 3 days postinfection and met euthanasia criteria by day 6. microscopic lesions within the brain were noted upon necropsy. in conjunction, viral rna was detected in the blood, brain, ovary, spleen, and liver of the infected mice. wild-type 129sv/ev mice were also challenged with no observable clinical disease. however, viral rna was detected at day 3 postinfection in the blood, ovary and spleen, and then remained at detectable levels in the ovaries and spleen on day 7 (dowall et al., 2016) . footpad inoculation of the virus leads to a fatal disease in ag129 mice by day 7 postinoculation with significant histopathological changes in the brain noted at necropsy (aliota et al., 2016) . ag129 mice were also observed to develop neurologic disease by day 6 postexposure (rossi et al., 2016) . immunocompetent mice are resistant to infection via the subq route (rossi et al., 2016) . recently, a mouse model was identified to verify vertical transmission of the virus. pregnant c57 mice were injected either ip or in utero into the lateral ventricle of the fetal brain. ip inoculation induced transient viremia in the pregnant mice on day 1. viral rna was detected in five out of nine placentas on day 3 postinfection. the virus was able to infect the radial glia cells in the fetal brain and leads to a reduction in the cortical neural progenitors . viral exposure via cerebroventricular space/ lateral ventricle of the fetal brain exhibited small brain size at day 5 postexposure in addition to cortical thinning (cugola et al., 2016; li et al., 2016a) . ifnar1 −/− pregnant mice exposed to the virus had nonviable fetuses. in the same study, wild-type mice were given an anti-ifnar antibody prior to and during infection resulting in detectable virus in the fetal head with mild intrauterine growth restriction (miner et al., 2016) . all of these murine studies will further study of the pathogenesis of vertical transmission and the resulting neurological disorders in conjunction with screening novel countermeasures. nhp studies are currently ongoing for animal model development. numerous viruses from the coronavirus (cov) family exist that infect a wide range of animals. six species have been identified that can infect humans. two of these are alpha coronavirues: hcov-229e and hcov-nl63. four are beta coronavirueses: hcov-oc43, hcov-hku1, hcov-sars, and mers-cov. hcov-229e and hcov-oc43 were first detected in the 1960s from the nasal passages of humans with the "common cold" (gaunt et al., 2010) . hcov-nl63, which was first isolated in 2004, causes upper and lower respiratory infections of varying intensity and has been continuously circulating among humans (van der hoek et al., 2006) . hcov-hku1, first isolated in 2002, has been identified more sporadically but also causes respiratory infections (lau et al., 2006) . a significant portion of common cold infections in humans are caused by coronaviruses. in 2002 and 2012, two human coronaviruses, sars-cov and mers-cov, emerged that caused a great deal of alarm since these infections have resulted in nearly 10 and 40% fatality, respectively (assiri et al., 2013; peiris et al., 2004) . the etiologic agent of severe acute respiratory syndrome (sars), sars-cov, emerged in 2002 as it spread throughout 32 countries in a period of 6 months, with 8437 confirmed infections and 813 deaths (roberts and subbarao, 2006; world health organization, 2003) . no additional cases of community acquired sars-cov infection have been reported since 2004. the natural reservoir of sars-cov is the horseshoe bat and the palm civet is an intermediate host (lau et al., 2005) . the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to 6 days and can be detected in stool, suggesting other modes of transmission are also possible (pearson et al., 2003; rabenau et al., 2005; rota et al., 2003) . sars-cov infection has a 10% case fatality with the majority of cases in people over the age of 15 (peiris et al., 2003; wang et al., 2004) . after an incubation period of 2-10 days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough (drosten et al., 2003) . in some sars cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within 2 weeks and coincides with the induction of high levels of neutralizing antibodies (tan et al., 2004) . in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses (chen and subbarao, 2007; perlman and dandekar, 2005) . infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection (perlman and dandekar, 2005) . virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues (cheng et al., 2004) . sars-cov can replicate in many species, including: dogs, cats, pigs, mice, rats, ferrets, foxes, and monkeys (roper and rehm, 2009) . no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (∼10%), viral replication, and pathology (roberts et al., 2008) . in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models utilize mice, hamsters, ferrets, and nhps. mouse models of sars-cov typically are inoculated by the in route under light anesthesia (roberts et al., 2005) . young, 6-to 8-week-old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinate, with a peak on day 2 and clearance by day 5 postexposure (mcauliffe et al., 2004) . there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis) . in sars-cov infection of c57bl/6 (b6), also yields reduced weight gain and viral k. viral disease replication in the lungs, with a peak on day 3 and clearance by day 9 (glass et al., 2004) . in contrast, balb/c mice 13-14 months of age show weight loss, hunched posture, dehydration, and ruffled fur on day 3-6 postexposure (bisht et al., 2004) . interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. 129s mice and b6 mice show outcomes to sars-cov infection similar to those observed for balb/c mice but have lower titers and less prolonged disease. while the aged mouse model is more frequently used then young mice, it is more difficult to obtain large numbers of mice older than 1 year (table 33 .1). a number of immunocompromised knockout mouse models of in sars-cov infection have also been developed. 129svev mice infected with sars-cov by the in route develop bronchiolitis, with peribronchiolar inflammatory infiltrates and interstitial inflammation in adjacent alveolar septae . viral replication and disease in these mice resolves by day 14 postexposure. beige, cd1 −/− , and rag1 −/− mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs (glass et al., 2004) . stat1 ko mice infected in with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths . the stat1 ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. angiotensin converting enzyme 2 (ace2) and cd209l were identified as cellular receptors for sars-cov, with affinity for the spike (s) protein of the virus (jeffers et al., 2004) . the variations in the ace2 sequence across animal species could partially explain the differences in infection severity (li et al., 2016b; sutton and subbarao, 2015) . since mice in particular have a greater number of sequence differences in ace2, transgenic mice were created that express human ace2 (mccray et al., 2007; netland et al., 2008; yang et al., 2007) . unlike other murine models of sars-cov, mice expressing hace2 had up to 100% mortality, with severity correlating to the level of hace2 expression (tseng et al., 2007) . with high levels of hace2 expression, mice developed a severe lung and brain infection. however, cns k. viral disease infection is only rarely observed in humans infected with sars-cov. syrian golden hamsters (strain lvg) are also susceptible to in exposure of sars-cov. after the administration of 10 3 tcid 50, along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis (roberts et al., 2005) . there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. limited mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred (roberts et al., 2008) . damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal (it) inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge (skowronski et al., 2005) . clinical disease has been observed in several studies excluding one, perhaps due to characteristics of the inoculating virus (kobinger et al., 2007) . sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day 4 postexposure as well as mild alveolar damage in 5%-10% of the lungs, occasionally accompanied by severe pathology within the lungs (martina et al., 2003; ter meulen et al., 2004) . with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or it routes generally results in a very mild infection that resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness but others have rash, lethargy, and respiratory signs and pathology martina et al., 2003; mcauliffe et al., 2004; rowe et al., 2004) . rhesus have little to no disease and only have mild findings upon histopathological analysis (rowe et al., 2004) . agms infected with sars-cov have no overt clinical signs but dad and pneumonitis has been documented (mcauliffe et al., 2004) . viral replication has been detected for up to 10 days in the lungs of agms; however, the infection resolves, and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were involved in the sars-cov outbreak. it and in inoculation of civets with sars-cov results in lethargy, decreased aggressiveness, fever, diarrhea, and conjunctivitis . leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. squirrel monkeys, mustached tamarinds, and common marmosets have not been susceptible to sars-cov infection (greenough et al., 2005; roberts et al., 2008) . vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine, and have included live-attenuated, killed, dna and viral-vectored vaccine strategies (cavanagh, 2003) . an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease (perlman and dandekar, 2005; weiss and scott, 1981) . as such, immune mice had th2type immunopathology upon sars-cov challenge (tseng et al., 2012) . severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported (weingartl et al., 2004) . additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance (clay et al., 2012) . mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. mouse-adapted sars with uniform lethality was developed from 15 serial passages in the lungs of young balb/c mice (mccray et al., 2007; roberts et al., 2007; rockx et al., 2007) . middle east respiratory syndrome (mers-cov) emerged in saudi arabia and is associated with fever, severe lower respiratory tract infection, and oftentimes renal failure (al-tawfiq et al., 2016; omrani et al., 2015) . mers patients can also occasionally manifest with neurological symptoms. mers-cov infection has a high fatality rate. infections in humans can also be asymptomatic. as of october 2015, there were 1589 confirmed cases and 567 deaths (li et al., 2016b) . bats serve as the likely natural reservoir since virus with 100% nucleotide identity to the index case was isolated from egyptian tomb bats (memish et al., 2013) . spread to humans likely comes from infected dromedary camels (adney et al., 2014; azhar et al., 2014) . the host range for mers-cov is dependent on the binding of the viral s protein to the host receptor, which is human dipeptidyl peptidase four (hdpp4), also known as cd26 (raj et al., 2013) . the expression and distribution of dpp4 in the human respiratory tract has recently been well characterized (meyerholz et al., 2016) . interestingly, dpp4 expression is preferentially localized to alveolar regions, perhaps explaining why mers predominantly manifests as an infection of the lower respiratory tract. humans with preexisting pulmonary disease have increased dpp4 expression in alveolar epithelia. small animals typically used for viral disease research, such as mice, hamsters, guinea pigs, and ferrets are naturally nonpermissive to mers-cov infection due to a low binding efficiency of the viral s protein to the host dpp4 (sutton and subbarao, 2015). in contrast the rhesus macaque and common marmoset have complete homology to human dpp4, allowing productive mers-cov infection to occur falzarano et al., 2014; munster et al., 2013; yao et al., 2014) . new zealand white rabbits can be infected with mers-cov, and virus was isolated from the upper respiratory tract, but there were no clinical symptoms or significant histopathological changes (haagmans et al., 2015) . due to the lack of strong binding affinity of the mers-cov s protein to the murine dpp4 receptor, wildtype mice are not susceptible to mers-cov infection. as such, several approaches have been used to create susceptible murine animal models of mers-cov infection by inducing the expression of hdpp4. one approach utilized an adenovirus vector expressing hdpp4 to transduce mice (zhao et al., 2014b) . these mice developed pneumonia but survived mers-cov infection. in mers-cov infection of mice with global expression of hdpp4 resulted in id 50 and ld 50 values of <1 and 10 tcid 50 , respectively (tao et al., 2016) . thus, mers-cov infection of these transgenic mice can be either sublethal or uniformly lethal depending on the dose. inflammatory infiltrates were found in the lungs and brain stems of mice with some focal infiltrates in the liver as well. another strategy uses transgenic mice expressing hdpp4 under either a surfactant protein c or cytokeratin 10 promoter (li et al., 2016b) . in mers-cov infection in these mice resulted in a uniformly lethal disease characterized by alveolar edema and microvascular thrombosis and mononuclear clear cell infiltration in the lungs. the brain stem was also impacted by the infection. dpp4 expression with an ubiquitously expressing promoter from cytomegalovirus also had a uniformly lethal infection with predominant lung and brain involvement, but numerous other tissues were also impacted and contained virus (agrawal et al., 2015) . common marmosets infected with 5.2 × 10 6 tcid 50 (emc-2012) mers-cov by the combined in, oral, ocular, and it routes capitulate the severe disease in human infections (falzarano et al., 2014) . the animals manifested moderate to severe clinical disease, with interstitial infiltration of both lower lung lobes. two of nine animals became moribund between days 4 and 6. viral rna was detected in nasal and throat swabs, various organs, and in the blood of some animals, indicating a systemic infection. histologically, animals showed evidence of acute bronchial interstitial pneumonia as well as other pathological defects. infection of rhesus macaques with mers-cov results in a mild clinical disease characterized by a transient lung infection with pneumonia. rhesus macaques were inoculated with at least 10 7 tcid 50 (emc-2012) mers-cov either by the it route or a combined in, it, oral, and ocular inoculation . the result was a mild respiratory illness including nasal swelling and a short fever with all animals surviving. viral rna was recovered from nasal swab samples and replicating virus was found in lung tissue . mild pathological lesions were found only in the lungs. radiographic imaging of the lungs revealed interstitial infiltrates, which are signs of pneumonia (yao et al., 2014) . interestingly, mer-cov infection is more severe in marmosets compared to rhesus macaques (falzarano et al., 2014) . this is despite the finding that both species have complete homology with humans within the dpp4 domain that interacts with the viral s protein. other host factors influencing disease severity have not yet been identified. transgenic mouse models expressing hdpp4 are ideal for initial development and screening of mers-cov countermeasures, and marmosets can be used for final selection and characterization. filoviridae consists of three genera, ebolavirus and marburgvirus, and a newly discovered group, cuevavirus (kuhn, 2008) . it is thought that various species of bats are the natural host reservoir for these viruses that have lethality rates from 40% to 82% in humans. there is evidence that the egyptian rousette bat (rousettus aegyptiacus) is the natural reservoir for marburgviruses but may not be for ebolaviruses (jones et al., 2015) . marburg virus (marv) first emerged in 1967 in germany when laboratory workers contracted the virus from agms (chlorocebus aerthiops) that were shipped from uganda. ebolaviruses sudan and zaire (sudv and ebov) caused nearly simultaneous outbreaks in 1976 in what is now the democratic republic of congo (drc). the most recent outbreak of ebov in west africa was by far the largest with over 28,000 suspected, probable and confirmed cases and over 11,000 deaths. bundibugyo virus (bdbv) first emerged in 2007 in bundibugyo, uganda with 56 confirmed cases . two other ebolaviruses are known: taï forest (tafv) (previously named cote d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. filovirus disease in humans is a characterized by aberrant innate immunity and a number of clinical symptoms: fever, nausea, vomiting, k. viral disease arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, and anorexia as well as numerous others (mehedi et al., 2011; wauquier et al., 2010) . approximately 10% of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.) (table 33 .2). natural transmission in an epidemic is through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, im, ip, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures . since filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. one issue to take note of in animal model development of filovirus infection is the impact of particle to plaque-forming unit (pfu) ratios on lethality, wherein it is possible that increasing the dose could actually decrease infectivity due to an immunogenic effect produced by inactive virions in the stock. additionally, the plaque assay used to measure live virions in a stock may greatly underestimate the true quantity of infectious virions in a preparation (alfson et al., 2015; smither et al., 2013a) . immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type 1 interferon response (bray, 2001) . however, wild-type inbred mice are susceptible to filovirus that has been mouse adapted (ma) by serial passage in mice (bray et al., 1999) . marv angola was particularly resistant to adaptation, but after 24 serial passages in scid mice, infection caused severe disease in balb/c and c57bl/6 mice when administered in or ip (qiu et al., 2014) . these mice had pathology with some similarities to infection in humans including lymphopenia, thrombocytopenia, liver damage, and viremia. balb/c mice, which are the strain of choice for ip inoculation of ma-ebov, are not susceptible by the aerosol route (bray et al., 1999; zumbrun et al., 2012a) . for aerosol infection of immunocompetent mice, a panel of bxd (balb/c x dba) recombinant inbred strains were screened and one strain, bxd34, was particularly susceptible to airborne ma-ebov, with 100% lethality to low or high doses (approximately 100 or 1000 pfu) ( zumbrun et al., 2012a) . these mice developed weight loss of greater than 15% and succumbed to infection between days 7 and 8 postexposure. the aerosol infection model utilizes a whole-body exposure chamber to expose mice aged 6-8 weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately 1.6 µm and a geometric standard deviation (gsd) of approximately 2.0 for 10 min. another approach uses immunodeficient mouse strains, such as scid, stat1 ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by ip or aerosol routes (bray, 2001; lever et al., 2012; zumbrun et al., 2012a) . mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection (bray et al., 1999) . liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps (zumbrun et al., 2012a) . while most mouse studies have used ma-ebov or ebov, an ip mouse-adapted marv model is also available (warfield et al., 2007 (warfield et al., , 2009 ). ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds (panchal et al., 2012) . recently, a model was created using immunodeficient nsg [nonobese diabetic (nod)/scid/il-2 receptor chain knockout] mice with transplanted human hematopoietic stem cells from umbilical cord blood. these mice were susceptible to lethal wt (nonadapted) ebov by ip and in exposure (ludtke et al., 2015) . the transplanted mice had all of the cellular components of a fully functional adaptive human immune system and upon ebov (brannan et al., 2015; lever et al., 2012) . interestingly, inoculation of infa/br −/− mice with tafv and restv does not result in clinical signs. yet another strategy uses knockout mice lacking possible receptors for filovirus entry, such as niemann-pick c1 and c2 (npc1 and npc2). npc2 (−/−) mice were fully susceptible to infection with ebov but npc1 (−/−) mice were completely resistant (herbert et al., 2015) . hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models (gowen and holbrook, 2008; herbert et al., 2015) . an ip ma-ebov infection model has been developed in syrian hamsters gowen and holbrook, 2008; herbert et al., 2015; tsuda et al., 2011) . this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, utilizes male 5-to 6-week-old syrian hamsters which are infected with 100 ld 50 of ma-ebov. virus is present in tissues and blood collected on day 4 and all animals succumbed to the disease by day 6. infected hamsters had severe coagulopathy and uncontrolled host immune responses, similar to what is observed in primates. (ebihara et al., 2010) guinea pig models of filovirus infection have been developed for ip and aerosol routes using guinea pigadapted ebov (gp-ebov) and marv (gp-marv) (choi et al., 2012; connolly et al., 1999; twenhafel et al., 2015; zumbrun et al., 2012c) . guinea pig models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. a comparison of ip infection of outbred guinea pigs with guinea pig-adapted marv angola and marv ravn revealed similar pathogenesis (cross et al., 2015) . infection with either strain resulted in features of the disease that are similar to what is seen in human and nhp infection, such as viremia, fever, coagulopathy, lymphopenia, elevated liver enzymes (alt and ast), thrombocytopenia, and splenic, gastrointestinal and hepatic lesions. gp-marv-ravn had a delayed disease progression relative to gp-marv-ang. hartley guinea pigs exposed to aerosolized gp-ebov develop lethal interstitial pneumonia. this is in contrast to subq infection of guinea pigs, aerosol ebov challenge of nhps, and natural human infection (twenhafel et al., 2015) . both subq and aerosol exposure of guinea pigs to gp-ebov resulted in only mild lesions in the liver and spleen. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses (100 or 1000 pfu target doses) but lethality drops with low (less than 1000 pfu) presented doses of airborne gp-marv and more protracted disease is seen in some animals (our unpublished observations) (zumbrun et al., 2012c) . weight loss of between 15% and 25% is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day 5, occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day 6 postexposure. blood chemistries (i.e., alt, ast, alkp, and bun) indicating problems with liver and kidney function are also altered late in the disease course. transmission of ebov has been documented from swine to nhps via the respiratory tract (kobinger et al., 2011) . as such, guinea pigs have been used to establish transmission models (wong et al., 2015a,b) . nonexposed guinea pigs were placed in the cages with infected guinea pigs 1 day postexposure to gp-ebov. guinea pigs challenged intanasally were more likely to transmit virus to naive cagemates than those that were exposed by the ip route. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans (dye et al., 2012) . old world primates have been primarily used for development of ip, im, and aerosol models of filovirus infection ( twenhafel et al., 2013) . uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, agms (alves et al., 2010; carrion et al., 2011; davis et al., 1997; hensley et al., 2011a; reed et al., 2007; zumbrun et al., 2012b) . low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. ebov-makona, the strain responsible for the recent large outbreak in west africa, was compared to the "prototype" 1976 ebov strain (marzi et al., 2015) . the disease in cynos was similar for both viruses, but disease progression was delayed for ebov-makona. this delay as well as the lower fatality rate in the 2014 epidemic compared to the 1976 outbreak suggest that ebov-makona is less virulent. the large number of cases in the 2014-15 ebov outbreak brought to light previously underappreciated eye pathology and ocular viral persistence in survivors. while survivors of nhp filovirus infection are infrequent, necrotizing scleritis, conjunctivitis, and other ocular pathology has been observed in ebov-infected animals (alves et al., 2016) . prominent features of the filovirus infections in nhps are onset of fever by day 5 postexposure, viremia, lymphopenia, tachycardia, azotemia, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. petechial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species (zumbrun et al., 2012b) . immunological parameters have been evaluated and t, b, and natural killer cells are greatly diminished as the infection progresses (fernando et al., 2015) . a cytokine storm occurs with rises in ifnγ, tnf, il-6, and ccl2 (fernando et al., 2015) . however, there is also evidence from transcriptional profiling of circulating immune cells that the early immune response is skewed toward a th2 response (connor et al., 2015) . strikingly, animals surviving challenge may have a delay in the production of inflammatory cytokines and chemokines (martins et al., 2015) . clinical disease parameters may have a slightly delayed onset in aerosol models. dyspnea late in infection is a prominent feature of disease after aerosol exposure (zumbrun et al., 2012b) . aerosol filovirus infection of nhps results in early infection of respiratory lymphoid tissues, dendritic cells, alveolar macrophages, blood monocytes, and fibroblastic reticular cells followed by spread to regional lymph nodes then multiple organs (ewers et al., 2016; twenhafel et al., 2013) . a number of pronounced pathology findings include multifocal hepatic necrosis and fibrin accumulation, particularly within the liver and the spleen. for aerosolized marv infection of rhesus, the most significant pathology included destruction of the tracheobronchial and mediastinal lymph nodes (ewers et al., 2016) . lymphocytolysis and lymphoid depletion are also observed (alves et al., 2010) . multilead, surgically implanted telemetry devices are useful in continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection (zumbrun et al., 2012b) . the most recently developed telemetry devices can also aid in plethysmography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. standardized filovirus-infected nhp euthanasia criteria have also been developed to enhance reproducibility for studies that evaluate therapeutic and vaccine countermeasures (warren et al., 2014) . filovirus infection of common marmosets (callithrix jacchus) is also a viable model to study the disease course. respiratory infection of marmosets with marv results in a lethal infection with fever, hemorrhaging, transient rash, disseminated viral infection, increases in liver function enzymes, coagulopathy, hepatitis, and histological lesions particularly in the kidney and liver (smither et al., 2013b) . marmosets are similarly susceptible to infection with ebovkikwit (smither et al., 2015) . thus, ebov or marv infection of marmosets produces features of the disease that are very similar to that of other nhps and humans. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate (rockx et al., 2012) . outbreaks due to nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra virus outbreaks have yet to be reported outside of australia (luby et al., 2009a,b) . hendra was the first member of the genus identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from bat to human. nipah has the distinction of transmission among, although the exact route is unknown (homaira et al., 2010) . the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized as needed for successful transmission (fogarty et al., 2008) . both viruses have a tropism for the neurological and respiratory tracts. the incubation period for hendra virus is 7-17 days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting (hanna et al., 2006) . disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure (playford et al., 2010) . nipah virus has an incubation period of 4 days to 2 weeks (goh et al., 2000) . much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within 24-48 h (lo and rota, 2008). survivors of infection typically make a full recovery; however, 22% suffer permanent sequelae, including persistent convulsions (tan and chua, 2008) . at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because documentation of human cases is at terminal stages. the best small animal model is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died within 4-17 days postinfection. the progression of disease and timeline is highly dependent on dose and route of infection. in inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas ip resulted in tremors and paralysis within 24 h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, with the brain having the highest titer. this model is used for vaccination and passive protection studies (guillaume et al., 2009; rockx et al., 2011; wong et al., 2003) . the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge (torres-velez et al., 2008; williamson et al., 2001) . inoculation with hendra virus via the subq route leads to a generalized vascular disease with 20% mortality. clinical signs were apparent 7-16 days postinfection with death occurring within 2 days of cns involvement. higher inoculum has been associated with development of encephalitis and cns lesions. id and in injection does not lead to disease, although the animals are able to seroconvert upon challenge. the inoculum source does not affect clinical progression. nipah virus challenge only causes disease upon ip injection and results in weight loss and transient fever for 5-7 days. virus was shed through urine and was present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder (hooper et al., 1997) . ferrets infected with hendra or nipah virus display the same clinical disease as seen in the hamster model and human cases (bossart et al., 2009; pallister et al., 2011) . upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within 6-9 days including fever, coughing, and dyspnea. lesions do develop in the ferret's brains, but to a lesser degree than seen in humans. cats have also been utilized as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is 4-8 days and leads to respiratory and neurological symptoms (mungall et al., 2007; johnston et al., 2015; westbury et al., 1996) . this model has proven useful for vaccine efficacy studies. squirrel and agms are representative of the nhp models. for squirrel monkeys, nipah virus is introduced by either the in or iv route and subsequently leads to clinical signs similar to humans, although in challenge results in milder disease. upon challenge, only 50% of animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge (marianneau et al., 2010) . agms are very consistent model of both viruses. it inoculation of the viruses results in 100% mortality, and death within 8.5 and 9-12 days postinfection for hendra and nipah viruses, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis rockx et al., 2010) . the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue, and nipah virus can be located in urine . pigs develop a respiratory disease upon infection with both nipah and hendra viruses (berhane et al., 2008; li et al., 2010; middleton et al., 2002) . oral inoculation does not produce a clinical disease, but subq injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as 4 days postinfection. nipah virus can also be transmitted between pigs. nipah virus was able to induce neurological symptoms in 20% of the pigs, even though virus was present in all neurological tissues regardless of symptoms (weingartl et al., 2005) . within the pig model, it appeared that nipah virus had a greater tropism for the respiratory tract, while hendra for the neurological system. horses are also able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra viruses. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within 2 days (marsh et al., 2011; williamson et al., 1998) . animals died within 4 days postexposure and have interstitial pneumonia with necrosis of alveoli (murray et al., 1995a,b) . virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but are nonpermissive to infection (westbury et al., 1995; wong et al., 2003) . suckling balb/c mice succumb to infection if the virus is inoculated intracranially (mungall et al., 2006) . in exposure with nipah does not induce a clinical disease; however, there is evidence of a subclinical infection in the lungs following euthanasia of the mice (dups et al., 2014) . in addition, a human lung xenograph model in nsg mice demonstrated that the human lung is highly susceptible to nipah viral replication and damage (valbuena et al., 2014) . embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within 4-5 days postinfection (tanimura et al., 2006) . annually, respiratory syncytial virus (rsv) is responsible for the lower respiratory tract infections of 33 million children under the age of 5, which in turn results in 3 million hospitalizations and approximately 200,000 deaths (nair et al., 2010) . within the united states, hospital costs alone amount to over 600 million dollars per year (paramore et al., 2004) . outbreaks are common in the winter (yusuf et al., 2007) . the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and spreads to the lower respiratory tract. incubation for the virus is 2-8 days. rsv is highly virulent leading to very few asymptomatic infections (collins and graham, 2008) . disease manifestations are highly dependent upon the age of the individual. rsv infections in neonates produce nonspecific symptoms including overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting rsv at this age results in an increased chance of developing childhood asthma (wu et al., 2008) . young children develop recurrent wheezing while adults have exacerbation of previously existing respiratory conditions (falsey et al., 2005) . common clinical symptoms are runny nose, sneezing, and coughing accompanied by fever. mortality rates from rsv in hospitalized children are 1%-3% with the greatest burden of disease seen in 3-4 month olds (ruuskanen and ogra, 1993). hematopoietic stem cell transplant patients, solid organ transplant patients, and copd patients are particularly vulnerable to rsv infection and have mortality rates between 7.3% and 13.3% upon infection (anderson et al., 2016) . although there are almost 60 rsv vaccine candidates which are in preclinical and clinical phases, there is no licensed vaccine available and ribavirin usage is not recommended for routine treatment (american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis, 2006; higgins et al., 2016; kim and chang, 2016) . animal models of rsv were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated rsv (fi-rsv) vaccine. this vaccine induced severe respiratory illness in infants whom received the vaccine and were subsequently infected with live virus (kim et al., 1969) . mice can be used to model rsv infection, although a very high in inoculation is needed to achieve clinical symptoms (jafri et al., 2004; stark et al., 2002) . strain choice is crucial to reproducing a physiological relevant response (stokes et al., 2011). age does not affect primary disease manifestations (graham et al., 1988) . however, it does play a role in later sequelae showing increased airway hyperreactivity . primary rsv infection produces increased breathing with airway obstruction (jafri et al., 2004; van schaik et al., 1998) . virus was detected as early as day 3 and reached maximum titer at day 6 postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. a humanized mouse model was recently developed by in inoculation. the challenged mice experienced weight loss and demonstrated a humoral and cellular immune response to the infection (sharma et al., 2016) . cotton rats are useful given that rsv is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after in inoculation (boukhvalova et al., 2009; niewiesk and prince, 2002) . viral replication is 50-to 1000-fold greater in the cotton rat model than mouse model (wyde et al., 1993) . the cotton rats develop mild to moderate bronchiolitis or pneumonia (grieves et al., 2015; prince et al., 1999) . although age does not appear to factor into clinical outcome, it has been reported that older cotton rats tend to take longer to achieve viral clearance. viral loads peak by the 5th day, dropping to below the levels of detection by day 8. the histopathology of the lungs appears similar to that of humans after infection (piazza et al., 1993) . this model has limited use in modeling the human immune response to infection as challenge with the virus induces a th2 response in cotton rats, whereas humans tend to have a response skewed toward th1 (culley et al., 2002; dakhama et al., 2005; ripple et al., 2010) . fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally with rsv via in inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is therefore useful in studying mucosal immunity during infection (gitiban et al., 2005) . ferrets infected by it were found to have detectable rsv in throat swabs up to day 7 postinfection, and positive qpcr up to day 10. immunocompromised ferrets were observed to have higher viral loads accompanied with detectable viral replication in the upper respiratory tract (stittelaar et al., 2016) . chimpanzees are permissive to replication and clinical symptoms of rsv including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms or high levels of viral replication (belshe et al., 1977) . bonnet monkeys were developed an inflammatory response by day 7 with viral rna detected in both bronchial and alveolar cells (simoes et al., 1999) . the chimpanzee model has been proven useful for vaccine studies (hancock et al., 2000; teng et al., 2000) . sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine rsv (meyerholz et al., 2004) . lambs are also susceptible to human respiratory syncytial infection (olivier et al., 2009; sow et al., 2011) . when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after 6 days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within 14 days. rsv replication peaked at 6 days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features with humans (plopper et al., 1983; scheerlinck et al., 2008) . the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; 16 ha and 9 na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within 10 days (taubenberger and morens, 2008) . the rate of infection is estimated at 10% in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately 40,000 people in the united states die each year from seasonal influenza (dushoff et al., 2006) . thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing (braun et al., 2007b) . influenza virus replicates in the upper and lower airways, peaking at approximately 48-h postexposure. infection can be more severe in infants and children under the age of 22, people over the age of 65, or immunocompromised individuals where viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis (barnard, 2009; glezen, 1982) . pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza virus itself, accounting for ∼27% of all influenza associated fatalities (alonso et al., 2003; ison and lee, 2010; speshock et al., 2007) . death, often due to ards can occur as early as 2 days after onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation (taubenberger and morens, 2008) . the h5n1 avian strain of influenza, has lethality rates of around ∼50% (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus the potential for global spread is a major concern (matrosovich et al., 2004; wang et al., 2016) . h7n9 is another avian influenza a strain that infected more than 130 people and was implicated in 37 deaths. approximately 75% of infected people had a known exposure to birds. there is no evidence of sustained spread between humans but these viruses are of great concern for their pandemic potential (zhang et al., 2013) . the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. (2012) . swine are not frequently utilized but are also a potentially useful model for influenza research since they share many similarities to human anatomy, genetics, susceptibility, and pathogenesis (rajao and vincent, 2015). lethality rates can vary with virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, super infection, and sepsis, severe with ards, and neurologic manifestations (barnard, 2009) . also, models can utilize seasonal or avian strains and have been developed to study transmission, important for understanding the potential for more lethal strains, such as h5n1 for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines (gilbert and mcleay, 2008; hagenaars et al., 2008; ortigoza et al., 2012) . inoculation is by the in route, utilizing approximately 60 µl of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with a mmad of <5 µl. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c57bl/6j mice. males and females have equivalent disease but influenza is generally more infectious in younger 2-to 4-week-old (8-10 g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. most inbred laboratory mice lack the mxa gene which is an important part of human innate immune response to influenza infection. the mouse homolog to mxa, mx1 is defective in most inbred mouse strains (staeheli and haller, 1987) . mice with the knocked-in mx1 gene have a 1000-fold higher ld-50 for an influenza a strain (pr8) than wildtype background c57bl/6 mice (grimm et al., 2007) . weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs in influenza infected mice. for more virulent strains, mice may require euthanasia as early as 48 h postexposure, but most mortality occurs from 5 to 12 days postexposure accompanied by decreases in rectal temperature (sidwell and smee, 2000). pulse oximeter readings and measurement of blood gases for oxygen saturation are also used to determine the impact of influenza infection on respiratory function (sidwell et al., 1992) . virus can be isolated from bronchial lavage (bal) fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild to moderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha-1-acidglycoprotein and lung weight also frequently occur. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa 2,3 predominates over sa 2,6 which is why h1, h2, and h3 subtypes usually need to be adapted to mice and h5n1, h2, h6, and h7 viruses do not require adaptation (o'donnell and subbarao, 2011). to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. h5n1 and the 1918 pandemic influenza virus can cause lethal infection in mice without adaptation (gao et al., 1999; taubenberger, 2006) . h5n1 infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia ( dybing et al., 2000; gubareva et al., 1998; lu et al., 1999) . as in humans, the virulence of h5n1 is attributable to damage caused by an overactive host immune response. additionally, mice infected with the 1918 h1n1 influenza virus produce severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia (barnard et al., 2007) . reassortment influenza viruses of the 2009 h1n1 virus and a low-pathogenicity avian h7n3 virus can also induce disease in mice without adaptation . in superinfection models, a sublethal dose of influenza is given to mice followed 7 days later by in inoculation of a sublethal dose of a bacterial strain, such as s. pneumoniae or s. pyogenes (chaussee et al., 2011) . morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to 2 weeks. at least one transmission model has also been developed in mice. with h2n2 influenza, transmission rates of up to 60% among cagemates can be achieved after infection by the aerosol route and cocaging after 24 h (schulman, 1968). rats (f344 and sd) inoculated with rat-adapted h3n2 developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes (daniels et al., 2003) . additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets (lowen et al., 2006) . cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies (eichelberger et al., 2004) . cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted (eichelberger et al., 2006; ottolini et al., 2005) . nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at 24 h postexposure. virus was cleared from the lung by day 3 and from the nares by day 66, but animals had bronchial and alveolar damage, and pneumonia for up to 3 weeks. there is also a s. aureus superinfection model in cotton rats (braun et al., 2007a) . coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans (lambkin et al., 2004; maines et al., 2012; mclaren and butchko, 1978) . like humans, ferrets exclusively express neu5ac, which acts as a receptor for influenza a virus, a feature likely contributing to the susceptibility of ferrets to human-adapted influenza a virus strains (ng et al., 2014) . the glycomic characterization of ferret respiratory tract tissues demonstrated some similarities and some differences to humans in terms of the potential glycan binding sites for the influenza virus (jia et al., 2014) . ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa 2,6 and sa 2,3) within the airways similar to that of humans (van riel et al., 2007) . influenza was first isolated from ferrets infected in with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments (huber et al., 2008; lambkin et al., 2004; maines et al., 2012) . when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by in inoculation of 0.25-0.5 ml containing approximately 10 4 -10 6 egg id 50 dropwise to each nostril. however, a larger inoculum volume of 1.0 ml has also been explored as being more appropriate, yielding more severe and consistent respiratory tract pathology, likely because the larger inoculum is more widely distributed in the lower respiratory tract (moore et al., 2014) . video tracking to assign values to activity levels in ferrets can aid ferret studies, eliminating the need for collection of subjective and arbitrary clinical scores (oh et al., 2015) . viral replication in the upper respiratory tract is typically measured by nasal washes, but virus can also be measured in bronchoalveolar lavage fluid using a noninvasive technique (lee et al., 2014) . influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts 3-5 days for mild to moderately virulent strains (maher and destefano, 2004) . ferrets are more susceptible to influenza a than influenza b strains and are also susceptible to avian influenza h5n1 strains without adaptation (zitzow et al., 2002) . however, the localized immune responses within the respiratory tract of ferrets infected with influenza a and b have been characterized and are similar (carolan et al., 2015) . virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirrors that seen in humans (stark et al., 2013) . nonadapted h1n1, h2n2, and h3n2 have mild to moderate virulence in ferrets. the sequencing of the ferret genome has allowed for the characterization of the ferret host response using rnaseq analysis . distinct signatures were obtained depending on the particular influenza strain to inoculate the ferrets. also helpful is the sequencing and characterization of the influenza ferret infectome during different stages of the infection in naïve or immune ferrets (leon et al., 2013) . since influenza infection is particularly devastating to the elderly population, an aged ferret model of h1n1 influenza infection was developed (paquette et al., 2014) . features associated with increased clinical disease are weakened hemagglutinin antibody generation and attenuated th1 responses. pregnant and breastfeeding women and infants are also susceptible to more severe illness from influenza virus. to study this dynamic, a breastfeeding mother-infant ferret influenza infection model was created (paquette et al., 2015) . notably, the mammary gland itself harbored virus and transcript analysis showed downregulation of milk production genes. in support of the development of therapies, the ferret influenza model for pharmacokinetic/pharmacodynamics studies of antiviral drugs as also been developed (reddy et al., 2015) . critical to this model is ensuring pronounced clinical signs and robust viral replication upon influenza infection. strains of low virulence have predominant replication in the nasal turbinates of ferrets. clinical signs and other disease indicators in ferrets are similar to that of humans with mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia (svitek et al., 2008) . replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract of ferrets correlates with severe disease and lethal outcome. h5n1-infected ferrets develop severe lethargy, greater interferon response, transient lymphopenia, and replication in respiratory tract, brain, and other organs (peng et al., 2012; zitzow et al., 2002) . immunocompromised humans have influenza illness of greater duration and complications. immunocompromised ferrets infected with influenza similarly had prolonged virus shedding (van der vries et al., 2013) . interestingly, antiviral resistance emerged in both humans and ferrets with immunocompromised status infected with influenza. alveolar macrophage depleted of ferrets infected with 2009 pandemic h1n1 influenza also had a more severe disease with greater viral replication in the lungs and greater induction of inflammatory chemokines (kim et al., 2013) . a superinfection model resembling that of mice has been developed by in instillation of influenza in 6-to 8-week-old ferrets followed by in inoculation of s. pneumonia 5 days later (peltola et al., 2006) . this typically resulted in otitis media, sinusitis, and pneumonia. transmission models in ferrets have recently met with worldwide media attention and controversy with regard to the study of h5n1 (enserink, 2013; fouchier et al., 2012; herfst et al., 2012; oh et al., 2014) . in general, some subtypes, such as the 2009 h1n1, can transmit easily through aerosol and respiratory droplets (munster et al., 2009) . of concern, h7n9 isolated from humans was more pathogenic and readily transmissible between ferrets by larger respiratory droplets and smaller particle aerosols (kreijtz et al., 2013; richard et al., 2013; zhang et al., 2013) . h5n1 became transmissible by adopting just four mutations, spreading between ferrets in separate cages (imai et al., 2012) . transmission occurs more readily at the height of pyrexia, but for the 2009 h1n1 in particular, can occur before fever is detected (roberts et al., 2012) . ferret-to-ferret transmission of a mouseadapted influenza b virus has also been demonstrated (kim et al., 2015) . since ferrets can be expensive and cumbersome, influenza infection has been characterized and a transmission model developed in the guinea pig; however, this is a newer model with infrequent utilization thus far (lowen et al., 2014) . old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models which are deficient for h5n1 vaccine studies because there is a lack of correlation with hemagglutination inhibition (murphy et al., 1980) . of old world primates, cynomolgus macaque (macaca fascicularis) is most frequently utilized for studies of vaccines and antiviral drug therapies (stittelaar et al., 2008) . h5n1 and h1n1 1918 infection of cynos is very similar to humans (rimmelzwaan et al., 2001) . cynos develop fever and ards upon in inoculation of h5n1 with necrotizing bronchial interstitial pneumonia . nhps are challenged by multiple routes k. viral disease (ocular, nasal, and tracheal) simultaneously 1 × 10 6 pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h5n1 results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the 1918 h1n1 caused a very high mortality rate due to an aberrant immune response and ards and had more than 50% lethality (humans only had a 1%-3% lethality) (kobasa et al., 2007) . ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains, such as h3n2 (o'donnell and subbarao, 2011) . influenza-infected rhesus macaques represent a mild disease model for vaccine and therapeutic efficacy studies (baas et al., 2006) . host microarray and qrt-pcr proved useful for analysis of infected lung tissues. other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates, such as squirrel and cebus monkeys (baskin et al., 2009) . domestic pig models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h5n1 (isoda et al., 2006; lipatov et al., 2008) . while pigs infected with influenza may have fever, anorexia, and respiratory signs, such as dyspnea and cough, mortality is rare (van der laan et al., 2008) . size and space requirements make this animal difficult to work with, although the development of minipig models may provide an easier to use alternative. cat and dog influenza models have primarily been utilized to study their susceptibility to h5n1 with the thought that these animals could act as sentinels or could serve to transmit the virus to humans (giese et al., 2008; rimmelzwaan et al., 2006) . these models are not generally used to better understand the disease in humans or for testing vaccines or antivirals. rift valley fever virus (rvfv) causes epizootics and human epidemics in africa. rvfv mainly infects livestock, such as sheep, cattle, goats, etc. after 2-4 days incubation period, animals show signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers (balkhy and memish, 2003) . mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans (abu-elyazeed et al., 1996; mundel and gear, 1951) . after 2-to 6-day-incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever (ikegami and makino, 2011; laughlin et al., 1979; peters and linthicum, 1994) . depending on the severity of the disease when the symptoms start, 10%-20% of the hospitalized patients might die in 3-6 days or 12-17 days after the disease onset (ikegami and makino, 2011) . hepatic failure, renal failure or dic, and encephalitis are demonstrated within patients during postmortem examination. live domestic animals especially sheep and goats were used to develop animal models of rvfv (weingartl et al., 2014) . this study indicated that goats were more resistant to the disease compared to sheep. the viremia in goats was lower and had a shorter duration with only some animals developing fever. the susceptibility is influenced by route of infection, breed of animals, the rvfv strain, and growth conditions as well as the passage history. therefore, it might be difficult to establish an animal model with domestic ruminants. mice are one of the most susceptible animal species to rvfv infection. several mouse models including balb/c, ifnar −/− , mbt/pas, 129 and c57bl/6 were exposed to rvfv via parental or aerosol routes of infection (ross et al., 2012) . subq or ip routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice (mims, 1956; smith et al., 2010) . mice start to show signs of decreased activity and ruffled fur by day 2-3 postexposure. immediately following these signs, they become lethargic and generally die 3-6 days postexposure. ocular disease or the hemorrhagic form of the disease has not been observed in mouse models so far (ikegami and makino, 2011) . increased viremia and tissue tropism were reported in mice with (smith et al., 2010) increased liver enzymes and lymphopenia observed in sick mice. aerosolized rvfv causes faster and more severe neuropathy in mice compared to the parental route (dodd et al., 2014; reed et al., 2014) . the liver is a target organ following aerosol exposure and liver failure results in fatality. rats and gerbils are also susceptible to rvfv infection. the rat's susceptibility is dependent on the rat strain utilized for the challenge model and route of exposure. there is also noted age dependence in the susceptibility of rats. while wistar-furth and brown norway strains, and young rats are highly susceptible to rvfv infection, fisher 344, buffalo and lewis strains, and old rats demonstrated resistance to infection via subq route of infection (findlay and howard, 1952; peters and slone, 1982) . similar pathologic changes, such as liver damage and encephalopathy were observed in both rats and mice. the recent study by bales et al. (2012) showed that aerosolized rvfv caused similar disease outcome in wistar-furth and aci rats while lewis rats developed fatal encephalitis which was much more severe than the subq route of infection. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils (anderson et al., 1988) . similar to the rat model, the susceptibility of gerbils was also dependent on age. natural history studies with syrian hamsters indicated that the liver was the target organ with highly elevated alt levels and viral titers (scharton et al., 2015) . lethargy, ruffled fur, and hunched posture were observed in hamsters by day 2 post-subq inoculation and the disease was uniformly lethal by day 2-3 postexposure. this model has been successfully used to test antivirals against rvfv (scharton et al., 2015) . studies thus far showed that rvfv does not cause uniform lethality in a nhp model. ip, in, iv, and aerosol routes have been utilized to develop nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort . monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to humans, such as pathological changes in the liver and hemorrhagic disease. there was no ocular involvement in this model. smith et al. compared iv, in and subq routes of infection in common marmosets and rhesus macaques (peng et al., 2012) . marmosets were more susceptible to rvfv infection than rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subq routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of in challenged marmosets. a recent study by hartman et al. (2014) demonstrated that aerosolized rvfv only caused mild fever in cynomolgus macaques and rhesus macaques, while agms and marmosets had encephalitis and succumbed to disease between days 9 and 11 postexposure. in contrast to other lethal models, the brain was the target organ in agms and marmosets. although no change was observed in ast levels, alp levels were increased in marmosets. little or no change was observed in hepatic enzyme levels in agms. lack of information regarding human disease concerning the aerosol route of exposure makes it difficult to evaluate these animal models. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, appears to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans (ergonul and whitehouse, 2007) . incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts 1-9 days. during the prehemorragic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients (mardani and keshtkar-jahromi, 2007; swanepoel et al., 1989) . over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. (fagbami et al., 1975; nalca and whitehouse, 2007; shepherd et al., 1989; smirnova, 1979) . until recently, the only animal that manifests disease is the newborn mouse. infant mice ip infected with cchfv resulted in fatality around day 8 postinfection (tignor and hanham, 1993) . pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day 6) and the brain (day 7). the recent studies utilizing knockout adult mice were successful to develop a lethal small animal model for cchfv infection (bente et al., 2010; bereczky et al., 2010) . bente et al. infected stat1 knockout mice by the ip route. in this model, after the signs of fever, leukopenia, thrombocytopenia, viremia, elevated liver enzymes and proinflammatory cytokines, mice were moribund and succumbed to disease in 3-5 days postexposure. the second model was developed by using interferon alpha/beta (ifnα/β) receptor knockout mice (ifnar −/− ) (bereczky et al., 2010) . similar observations were made in this model as in the stat1 knockout mouse model. animals were moribund and died 2-4 days after exposure with high viremia levels in liver and spleen. characterization studies with ifnar −/− mice challenged with different routes (ip, in, im, and subq) showed that cchfv causes acute disease with high viral loads, pathology in liver and lymphoid tissues, increased proinflammatory response, severe thrombocytopenia, coagulopathy, and death, all of which are characteristics of human disease . proinflammatory cytokines and chemokines, such as g-csf, ifnγ, cxc-cl10, ccl2 increased dramatically day 3 postchallenge and gm-csf, il-1a, il-1b, il-2, il-6, il-12p70, il-13, il-17, cxcl1, ccl3, ccl5, and tnf-α concentrations were extremely elevated at the time of death/euthanasia. this model is also utilized to test therapeutics, such as ribavirin, arbidol, and t-705 (favipiravir) successfully (oestereich et al., 2014) . experimental vaccines developed for cchf were evaluated in this model provided protection compare to unvaccinated mice (buttigieg et al., 2014; canakoglu et al., 2015, p. 725) . thus, the ifnar −/− mouse model would be a good choice to test medical countermeasures against cchfv, although they have an impaired ifn and immune response phenotype. other laboratory animals, including nhps, show little or no sign of infection or disease when infected with cchfv (nalca and whitehouse, 2007) . butenko et al. utilized agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day 4 postinfection, the animals did not show signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. fagbami et al. (1975) infected two patas monkeys (erythrocebus patas) and one guinea baboon (papio papio) with cchfv. whereas all three animals had low-level viremia between days 1 and 5 after inoculation, only the baboon serum had neutralizing antibody activity on day 137 postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia (rabinovich et al., 1972) and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least 3 months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes (blagoveshchenskaya et al., 1975 (blagoveshchenskaya et al., ). shepherd et al. (1989 infected 11 species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. whereas scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (gerbilliscus brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and mastomys coucha), and syrian hamsters were negative for virus. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed onset of viremia earlier than those infected by the subq or ip routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather rodents (schmaljohn and nichol, 2007) . rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by inhalation of infected rodent saliva, feces, and urine (xu et al., 1985) . human infections can normally be traced to a rural setting with activities, such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention (lednicky, 2003) . the viruses have a tropism for endothelial cells within the microvasculature of the lungs (zaki et al., 1995) . there are two distinct clinical diseases that infection can yield: hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses (nichol, 2001) . hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul (lednicky, 2003) . incubation lasts 2-3 weeks and presents as flu-like in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops which can further progress to shock in approximately 15% patients. overall mortality rate is 7%. infection with dobrava and hantaan viruses are typically linked to development of severe disease. hps was first diagnosed in 1993 within southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus (snv) (centers for disease control and prevention, 1993) . this virus is still the leading cause within north america of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay (padula et al., 2000; stephen et al., 1994) . the first report of hps in maine was recently documented (centers for disease control and prevention, 1993). andes virus (andv) was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans (wells et al., 1997) . the fulminant disease is more lethal than that observed of hfrs with a mortality rate of 40%. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation (peters and khan, 2002) . incubation typically occurs 14-17 days following exposure (young et al., 2000) . unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within 48 h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted (hallin et al., 1996) . syrian golden hamsters are the most widely utilized small animal model for hantavirus infection. hamsters inoculated im with a passaged andes viral strain died within 11 days postinfection. clinical signs did not appear until 24 h prior to death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged snv isolate. no hamsters developed any symptoms during the course of observation. however, an antibody response to the virus that was not dose dependent was determined via elisa. hamsters infected with andv have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day 8 and lasted up to 12 days postinfection. infection of hamsters with andv yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver . normally, snv does not cause a disease in hamsters (wahl-jensen et al., 2007) . but a recent study showed that immunosuppression with dexamethasone and cyclophosphamide in combination causes lethal disease with snv in hamsters (brocato et al., 2014) . the disease was very similar to the disease caused by andv in hamsters. lethal disease can be induced in newborn mice, but does not recapitulate the clinical symptoms observed in human disease (kim and mckee, 1985) . the disease outcome is very much dependent on the age of the mice. younger mice are much more susceptible to virus than the adult mice. adult mice exposed to hanta virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease (seto et al., 2012; wichmann et al., 2002) . knockout mice lacking ifnα/β are highly susceptible to hanta virus infection (muller et al., 1994) . in a study of panel of laboratory strains of mice, c57bl/6 mice were most susceptible to a passaged hanta viral strain injected ip. animals progressed to neurological manifestation including paralyses and convulsions, and succumbed to infection within 24-36 h postinfection. clinical disease was markedly different from that observed in human cases (wichmann et al., 2002) . in a recent study, 2-weekold icr mice was exposed to htnv strain aa57 via the subq route (seto et al., 2012) . mice started to show signs of disease by day 11 postinoculation. piloerection, trembling, hunching, loss of body weight, labored breathing, and severe respiratory disease were observed in mice. studies to develop nhp models were not successful until recently. nhps have been challenged with new world hantaviruses; however, no clinical signs were reported (hooper et al., 2006; mcelroy et al., 2002) . cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease (klingstrom et al., 2002; sironen et al., 2008) . challenge with andv to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within 5 days postinfection. four of six aerosol exposed monkeys and 8 of 11 iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. in a recent study, rhesus macaques were inoculated by the intramuscular route with snv passaged only in deer mice (safronetz et al., 2014) . characteristics of hps disease including rapid onset of respiratory distress, severe pulmonary edema, thrombocytopenia, and leukocytosis were observed in this promising model. viremia was observed 4-10 days prior to respiratory signs of the disease that were observed on days 14-16 postinoculation. with all aspects, this animal model would be very useful to test medical countermeasures against hanta virus. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all of these viruses share common clinical manifestations (mccormick and fisher-hoch, 2002) . lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april (curtis, 2006) . this virus is responsible for 100,000-500,000 infections per year, leading to approximately 5000 deaths (khan et al., 2008) . outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases have sprung up in germany, netherlands, united kingdom, and the united states due to transmission to travelers on commercial airlines (amorosa et al., 2010) . transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex (curtis, 2006) . humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease occurs in 20% of individuals. the incubation period is from 5 to 21 days and initial onset is characterized by flu-like illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease that is permanent for a third of those affected. the overall fatality is about 1%; however, of those admitted to the hospital it is between 15% and 25%. there is no approved vaccine and besides supportive measures, ribavirin is effective only if started within 7 days (mccormick et al., 1986a,b) . the primary animal model used to study lassa fever is the rhesus macaque (jahrling et al., 1980) . aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus mirrored more closely the disease course and histopathology observed in human infection (danes et al., 1963) . iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt (lukashevich et al., 2003) . disease was dose dependent with iv, intramuscular, and subq inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be utilized, and mimic a more natural route of infection (peters et al., 1987) . within this model, the nhp becomes viremic after 4-6 days. clinical manifestations were present by day 7 and death typically occurred within 10-14 days (lukashevich et al., 2004; rodas et al., 2004) . intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns (hensley et al., 2011b) . this pathogenicity is seen in select cases of human lassa fever (cummins et al., 1992; gunther et al., 2001) . a marmoset model has recently been defined utilizing a subq injection of lassa fever virus. virus was initially detected by day 8 and viremia achieved by day 14. liver enzymes were elevated and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases (carrion et al., 2007) . mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is dependent on the mhc background, age of the animal, and inoculation route (salvato et al., 2005) . stat1 knockout mice inoculated ip with both lethal and nonlethal lassa virus strains develop hearing loss accompanied by damage to the inner ear hair cells and auditory nerve (yun et al., 2015) . guinea pig inbred strain 13 was highly susceptible to lassa virus infection. the outbred hartley strain was less susceptible, and thus strain 13 has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus (jahrling et al., 1982) . infection with pichinde virus passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death (lucia et al., 1990; qian et al., 1994) . the guinea pig is an excellent model given that it not only results in similar disease pattern, viral distribution, histopathology, and immune response to humans (connolly et al., 1993; katz and starr, 1990) . infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig models. the virus was injected ip resulting in lethargy and anorexia within 6-7 days. virus was first detected at 3 days, and reached maximum titers within 5 days. neurological symptoms began to appear at the same time, and all animals died by day 9. pneumonitis, pulmonary hemorrhage, and edema were also present (sbrana et al., 2006) . these results were recapitulated with a nonadapted pichinde virus (buchmeier and rawls, 1977; gowen et al., 2005; smee et al., 1993) . the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects 0.6% of the world's population. a greater proportion of infections and deaths occur in subsaharan africa. worldwide, there are approximately 1.8 million deaths per year with over 260,000 being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv-1 and hiv-2, with hiv-2 having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv-1 (de cock et al., 2011) . hiv targets t-helper cells (cd4+), macrophages, dendritic cells (fields et al., 2007) . acute infection occurs 2-4 weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd8+ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd4+ t-cells decline to less than 200 cells/µl; thus cell-mediated immunity becomes impaired and the person is more susceptible to opportunistic infections as well as certain cancers. hiv has a narrow host range likely because the virus is unable to antagonize and evade effector molecules of the interferon response (thippeshappa et al., 2012) . humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of an intact and functional human innate and adaptive immune responses (berges and rowan, 2011) . the scidhu hiv infection model has proven useful, particularly in screening antivirals and therapeutics (denton et al., 2008; melkus et al., 2006) . a number of different humanized mouse models have been developed for the study of hiv, including rag1 −/− γc −/− , rag2 −/− γc −/− , nod/scidγc −/− (hnog), nod/scidγc −/− (hnsg), nod/scid blt, and nod/scidγc −/− (hnsg) blt (karpel et al., 2015; li et al., 2015; shimizu et al., 2015) . cd34+ human stem cells derived from umbilical cord blood or fetal liver are used for humanization (baenziger et al., 2006; watanabe et al., 2007) . hiv-1 infection by ip injection can be successful with as little as 5% peripheral blood engraftment (berges et al., 2006) . vaginal and rectal transmission models have been developed in blt scid hu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv-1 viremia occurs within approximately 7 days postinoculation . in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans (brainard et al., 2009) . importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hivinfected humanized mice and at least some mechanisms of pathogenesis that occur in hiv-infected humans, also occur in the hiv-infected humanized mouse models (baenziger et al., 2006; neff et al., 2011) . the advantage of these models is that these mice are susceptible to hiv infection and thus the impact of drugs on the intended viral targets can be tested. one caveat is that while mice have a "common mucosal immune system," humans do not, due to differences in the distribution of addressins (holmgren and czerkinsky, 2005) . thus, murine mucosal immune responses to hiv do not reflect those of humans. another strategy uses a human cd4-and human ccr5-expressing transgenic luciferase reporter mouse to study hiv-1 pseudovirus entry (gruell et al., 2013) . hiv-1 transgenic (tg) rats are also used to study hiv related pathology, immunopathogenesis, and neuropathology (lentz et al., 2014; reid et al., 2001) . the clinical signs include skin lesions, wasting, respiratory difficulty, and neurological signs. brain volume decreases have been documented and the hiv-1 tg rat is thus used as a model of neuropathology in particular. there are a number of important nhp models for human hiv infection (hessell and haigwood, 2015) . an adaptation of hiv-1 was obtained by four passages in pigtailed macaques transiently depleted of cd8(+) cells during acute infection (hatziioannou et al., 2014) . the resulting disease has several similarities to aids in humans, such as depletion of cd4(+) t-cells (kimata, 2014) . simian immunodeficiency virus (siv) infection of macaques has been widely used as a platform for modeling hiv infection of humans (demberg and robert-guroff, 2015; walker et al., 2015) . importantly, nhps have similar, pharmacokinetics, metabolism, mucosal tcell homing receptors, and vascular addressins to those of humans. thus, while the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero or through breast milk (keele et al., 2009; miller et al., 2005; stone et al., 2009) . there are also macaque models to study the emergence and clinical implications of hiv drug resistance (van rompay et al., 2002) . these models most routinely utilize rhesus macaques (macaca mulatta), cynomolgus macaques (m. fasicularis), and pigtailed macaques (macaca nemestrina). all ages are used, depending on the needs of the study. for instance, use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently employed. female pigtailed macaques have been used to investigate the effect of the menstrual cycle on hiv susceptibility (vishwanathan et al., 2015) . studies are performed in bsl-2 animal laboratories and nhps must be simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied (ebenezer et al., 2012) . exposure in model systems is typically through a single high-dose challenge. iv infection of rhesus macaques with 100 tcid 50 of the highly pathogenic siv/ deltab670 induces aids in most macaques within 5-17 months (mean of 11 months) (fuller et al., 2012) . peak viremia occurs around week 4. aids in such models is often defined as cd4+ t-cells that have dropped to less than 50% of the baseline values. alternatively, repeated low dose challenges are often utilized, depending on the requirements of the model (henning et al., 2014; moldt et al., 2012; reynolds et al., 2012) . since nhps infected with hiv do not develop an infection with a clinical disease course similar to humans, siv or siv/hiv-1 laboratory-engineered chimeric viruses (shivs) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease which progresses to aids, and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv-1 and is more closely related to hiv-2. however, the polymerase region of siv is 60% homologous to that of hiv-1 and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors, thus hiv-1 rt is usually put into siv for such studies (uberla et al., 1995) . sivmac239 is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt, or integrase inhibition (witvrouw et al., 2004) . nhps infected with sivmac239 have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd4+ t-cell depletion. additionally, sivmac239 utilizes the cxcr5 chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry (veazey et al., 2003) . nhps infected with shiv strains, may not develop aids, but these models are useful in testing vaccine efficacy . for example, rt-shivs and env-shivs are useful for testing and evaluation of drugs that may target the envelope or rt, respectively (uberla et al., 1995) . one disadvantage of the highly virulent env-shiv (shiv-89.6 p), is that it uses the cxcr4 coreceptor. of note, env-shivs that do use the cxcr5 coreceptor are less virulent; viremia develops then resolves without further disease progression (humbert et al., 2008) . simian-tropic (st) hiv-1 contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for 3 months, followed by clearance (haigwood, 2009) . a number of routes are utilized for siv or shiv infection of nhps, with iv inoculation the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) 1 month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase susceptibility to infection by atraumatic vaginal instillation (burton et al., 2011) . upon vaginal instillation of 500 tcid 50 of shiv-162p3, peak viremia was seen around 12 days postexposure with greater than 10 7 copies/ml and dropping thereafter to a constant level of 10 4 rna copies/ml at 60 days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac251 (barouch et al., 2012) . an example of an intrarectal model utilized juvenile (2-year-old) pigtailed macaques, challenged intrarectally with 10 4 tcid 50s of siv mne027 to study the pathogenesis related to the virulence factor, vpx (belshan et al., 2012) . here, viremia peaked at approximately 10 days with more than 10 8 copies/ml. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistent high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of haart therapy on virus and t-cells in the male genital tract, adult (3-to 4-year-old) male cynomolgus macaques were intravenously inoculated with 50 aid50s of sivmac251 and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization (moreau et al., 2012) . pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course (abel, 2009) . importantly, mother-to-infant transmission models have also been developed (jayaraman et al., 2004) . pregnant female pigtailed macaques were infected during the second trimester with 100 mid 50 shiv-sf162p3 by the iv route. four of nine infants were infected, one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission as well as the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating, and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and trim alleles), differences between challenge strains, and challenge routes (letvin et al., 2011) . nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the long-term clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade (van rompay et al., 2008) . unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study which used an adenovirus-based vaccine approach (buchbinder et al., 2008) . vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv exposure is more difficult to protect than mucosal exposure and is used as a "worst case scenario." however, efficacy at one mucosal route is usually comparable to other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect (bosch and de sanjose, 2002) . there are over 100 human papillomaviruses, with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately one third of papillomaviruses are transmitted sexually. of these, virulent subtypes, such as hpv-16, hpv-18, hpv-31, hpv-33, and hpv-45 place individuals at high risk for cervical and other cancers. up to 35% of head and neck cancers are caused by hpv-16, particularly oropharyngeal cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no wild-type animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist which use animal papillomaviruses in their natural host or a very closely related species (borzacchiello et al., 2009; brandsma, 1994; campo, 2002) . these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines (rabenau et al., 2005) . wild-type inbred mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, orthotopically transplanted or transgenic, but they are often used to look at immunogenicity of vaccines (jagu et al., 2011; oosterhuis et al., 2011) . transgenic mice used for hpv animal modeling typically express the viral oncogenes e5, e6, e7, or the entire early region of hpv-16 from the keratin 14 promoter which is only active in the basal cells of the mouse epithelium (chow, 2015) . cancers in these models develop upon extended estrogen exposure (maufort et al., 2010; ocadiz-delgado et al., 2009; stelzer et al., 2010; thomas et al., 2011) . transgenic mice with constitutively active wnt/b-catenin signaling in cervical epithelial cells expressing the hpb oncoprotein e7 develop invasive cervical squamous carcinomas (bulut and uren, 2015) . the tumors occur within 6 months approximately 94% of the time. another model uses c57bl/6 mice expressing the hpv16-e7 transgene which are then treated topically with 7,12-dimethylbenz(a)anthracene (dmba) (de azambuja et al., 2014) . these mice developed benign and malignant cutaneous lesions. cervical cancers can also be induced in human cervical cancer xenografts transplanted onto the flanks of athymic mice and serially transplanted thereafter ( hiroshima et al., 2015; siolas and hannon, 2013) . a wild-type immunocompetent rodent model uses m. coucha, which is naturally infected with mastomys natalensis papillomavirus (mnpv) (vinzon et al., 2014) . mnpv induces papillomas, keratoacanthomas, and squamous cell carcinomas and provides a means to study vaccination in an immunocompetent small animal model. wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which is a very closely related species ( breitburd et al., 1997) . in this model, papillomas can range from cutaneous squamous cell carcinomas on one end of spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) causes florid warty lesions in mucosa of the oral cavity within 4-8 weeks postexposure in experimental settings (johnston et al., 2005) . the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv-6 and hpv-11 make this a useful model (nicholls et al., 1999) . these lesions typically spontaneously regress 4-8 weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged 10 weeks to 2 years, with no history of copv, are typically used for these studies. infection is achieved by application of a 10 µl droplet of virus extract to multiple 0.5 cm 2 scarified areas within the mucosa of the upper lip of anesthetized beagles (nicholls et al., 2001) . some investigators have raised concerns that dogs are not a suitable model for high-risk hpv-induced oral cancer (staff, 2015) . bovine papillomavirus (bpv) has a wider host range than most papillomaviruses, infecting the fibroblasts cells of numerous ungulates (campo, 2002) . bpv-4 infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases, such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, rhesus papillomavirus (rhpv), a sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques is very similar to hpv-16 and is associated with the development of cervical cancer ( ostrow et al., 1990; wood et al., 2007) . monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including k. viral disease squirrels (charatan, 2003; khodakevich et al., 1986) . mpxv was discovered during the pox-like disease outbreak among laboratory monkeys (mostly cynomolgus and rhesus macaques) in denmark in 1958. no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until 1970 in zaire (the present drc) with continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of 2003, a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and western hemisphere. among 72 reported cases, 37 human cases were laboratory confirmed during an outbreak (nalca et al., 2005; sejvar et al., 2004) . it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-to-person spread occurs by large respiratory droplets or direct contact (jeézek and fenner, 1988) . most of the clinical features of human monkeypox are very similar to those of ordinary smallpox (breman and arita, 1980) . after a 7-to 21-dayincubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash (di giulio and eckburg, 2004; jeézek and fenner, 1988) . a typical maculopapular rash follows the prodromal period, generally lasting 1-3 days. the average size of the skin lesions are 0.5-1 cm and the progress of lesions follows the order: macules, papules, vesicles, pustules, umblication then scab, and desquamation and lasts typically 2-4 weeks. the fatality rate is 10% among the unvaccinated population and death generally occurs during the 2nd week of the disease (jeézek and fenner, 1988; nalca et al., 2005) . mpxv is highly pathogenic for a variety of laboratory animals and many animal models have been developed by using different species and different routes of exposure (table 33 .3). due to unavailability of variola virus (smallpox) to develop animal models and similar disease manifestations in humans that are similar, mpxv is one of the pox viruses that are utilized very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat1-deficient c57bl/6 mouse, icr mouse, prairie dogs, african dormice, ground squirrels, and gambian pouched rats are highly susceptible to mpxv by different exposure routes (americo et al., 2010; falendysz et al., 2015; hutson et al., 2009; osorio et al., 2009; sbrana et al., 2007; schultz et al., 2009; sergeev et al., 2016; stabenow et al., 2010; tesh et al., 2004; xiao et al., 2005) . cast/eij mice, one of the 38 inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose dependent mortality after in exposure to mpxv. studies with ip route of challenge indicated a 50fold higher susceptibility to mpxv when compared to in route (americo et al., 2010) . scid-balb/c mice were also susceptible to the ip challenge route and the disease resulted in mortality on day 9 postinfection (osorio et al., 2009) . similarly, c57bl/6 stat1 −/− mice were infected in with mpxv and the infection resulted in weight loss and mortality 10 days postexposure. recently sergeev et al. (2016) showed that in challenge of icr mice with mpxv resulted in purulent conjunctivitis, blepharitis, and ruffled fur in these mice although there was no death. the mouse models mentioned here are very promising for screening therapeutics against poxviruses but testing in additional models will be required for advanced development. high doses of the mpxv by ip or in routes caused 100% mortality in 6 days postexposure and 8 days postexposure, respectively, in ground squirrels (tesh et al., 2004) . the disease progressed very quickly and most of the animals were lethargic and moribund by day 5 postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv strains in ground squirrels by the subq route resulted in systemic disease and mortality in 6-11 days postexposure. the disease resembles hemorrhagic smallpox with nosebleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. another study by sergeev et al. (2017) showed that in challenge with mpxv caused fever, lymphadenitis, and skin rash in ground squirrels 7-9 days postexposure. mortality was observed in 40% of the animals 13-22 days postexposure (sergeev et al., 2017) . since mpxv was transmitted by infected prairie dogs in the us outbreak, this animal model has been more thoroughly studied and utilized to test therapeutics and vaccines compared to other small animal models ( hutson et al., 2009; keckler et al., 2011; smith et al., 2011; xiao et al., 2005) . studies using in, ip, and id routes of exposure showed that mpxv was highly infectious to prairie dogs, ip infection with the west african mpxv strain caused a more severe disease and 100% mortality than challenge by the in route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to ip route, the in route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in ip-infected animals (xiao et al., 2005) . hutson et al. (2009) african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and most importantly generalized pox lesions. therefore, this model has the utility for testing therapeutics and vaccines against pox viruses. furthermore, mpxv challenged prairie dogs were used to perform in vivo bioluminescent imaging (bli) studies (falendysz et al., 2015) . bli studies showed real time spread of virus in prairie dogs as well as potential routes for shedding and transmission. the african dormouse is susceptible to mpxv by a footpad injection or in routes (schultz et al., 2009) . mice had decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of 200 and 2000 pfu provided 100% mortality with a mean time to death of 8 days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and lung hemorrhage were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. in a recent study, comparison of the disease pathogenesis was performed by using live bioluminescence imaging in the cast/eij mouse and african dormouse challenged with low dose of mpxv (earl et al., 2015) . following in challenge, mpxv dissemination occurred through the blood or lymphatic system in dormice compared to dissemination that was through the nasal cavity and lungs in cast/eij mice. the disease course was much faster in cast/eij mice (earl et al., 2015) . considering the limited availability of prairie dogs, ground squirrels and african dormice, lack of reagents specific for these species, and not having commercial sources of these species, these small animal models are as attractive for further characterization and vaccine, and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal model for mpxv (edghill-smith et al., 2005; johnson et al., 2011; nalca et al., 2010; stittelaar et al., 2006; zaucha et al., 2001) . during our studies using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day 6 postexposure (nalca et al., 2010) . complete blood count and clinical chemistries showed abnormalities similar to human monkeypox cases with leukocytosis and thrombocytopenia (huhn et al., 2005) . whole blood and throat swabs had viral loads peak around day 10, and in survivors, gradually decrease until day 28 postexposure. since doses of 4 × 10 4 pfu, 1 × 10 5 pfu, or 1 × 10 6 pfu resulted in lethality for 70% of the animals, whereas a dose of 4 × 10 5 pfu resulted in 85% lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, animals succumbed to disease before developing pox lesions. with the low challenge dose, pox lesions were observed but they were few in comparison to the iv model. a recent study also evaluated the cytokine levels in aerosol challenged animals. (tree et al., 2015) . tree et al. (2015) showed that ifnγ, il-1rα, and il-6 increased dramatically on day 8 postexposure the day that death was most likely to occur, and viral dna was detected in most of the tissues. these results support the idea of a cytokine storm causing mortality in monkeypox disease. mpxv causes dose dependent disease in nhps when given by the iv route (johnson et al., 2011) . studies showed that a 1 × 10 7 pfu iv challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an it infection model skips the upper respiratory system and deposits virus into the trachea, delivering the virus directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation. fibrinonecrotic bronchopneumonia was described in animals that received 10 7 pfu of mpxv intratracheally (stittelaar et al., 2006) . although a similar challenge dose of it mpxv infection resulted in a similar viremia in nhps to the aerosol route of infection, the timing of the first peak was delayed by 5 days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately 100-fold lower. this suggests that local replication is more prominent after aerosol delivery compared to the it route. an intrabronchial route of exposure resulted in pneumonia in nhps (johnson et al., 2011) . delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and it infection models, the number of pox lesions was much less than in the iv infection model. a major downside of the iv, it, and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus to the blood stream or to the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting the subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human varv infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b virus (hbv) is one of the most common infections worldwide with over 400 million people chronically infected and 316,000 cases per year of liver cancer due to infection (lee, 1997) . the virus can naturally infect both humans and chimpanzees (guha et al., 2004) . hbv is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture (lai et al., 2003) . the age at which one is infected dictates the risk of developing chronic disease (hyams, 1995) . acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms of hbv infection last for a few weeks before resolving (ganem and prince, 2004) . after this acute phase, lifetime immunity is achieved (wright and lau, 1993) . of those infected, less than 5% will develop the chronic form of the disease. chronicity is the most serious outcome of the disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is 100 times more likely to develop in a chronically infected individual than a noncarrier (beasley, 1988) . the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepatitis virus suggest that x protein may be responsible (spandau and lee, 1988). many individuals are asymptomatic until complications emerge related to chronic hbv carriage. chimpanzees have a unique strain that circulates within the population (hu et al., 2000; . it was found that 3%-6% of all wild-caught animals from africa are positive for hbv antigen ( lander et al., 1972) . natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease (prince, 1972) . to date, chimpanzees are the only reliable method to ensure that plasma vaccines are free from infectious particles (prince and brotman, 2001 ). this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially ideal for these studies given that their immune response to infection directly mirrors humans (nayersina et al., 1993) . recent regulations by the national institute of health (nih) and restrictions to use great apes as animal models forced researches to find alternate models for hbv infection. other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hbv, none develops hepatic lesions or liver damage as noted by monitoring of liver enzymes (pillot, 1990 ). mice are not permissible to infection, and thus numerous transgenic and humanized lines that express hbv proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease (guha et al., 2004) . recently, the entire genome of hbv was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection (huang et al., 2012) . another model that has been developed is hydrodynamic injection of hbv genomes in the liver of mice (liu et al., 1999; yang et al., 2002) . although this model is very stressful to mice and has liver toxicity, it is successfully used to evaluate antivirals against hbv (mccaffrey et al., 2003) . liver chimeric mouse models are an additional set of surrogate models for hbv infection (dandri and lutgehetmann, 2014) . in these models human hepatocytes are integrated into the murine liver parenchyma (allweiss and dandri, 2016) . this model might be used to test antivirals as well as to study the molecular biology of hbv infection. hbv can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses (mason et al., 1982) . the woodchuck hepatitis virus induces hepatocellular carcinoma (summers et al., 1978) . within a population, 65%-75% of all neonatal woodchucks are susceptible to chronic infection (cote et al., 2000) . a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within 3 years of the initial infection in woodchucks, whereas human carcinogenesis takes much longer (gerin et al., 1989) . the acute infection strongly resembles what occurs during the course of human disease. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage (tennant, 2001) . challenge with virus in neonates leads to a chronic infection while adults only develop the acute phase of disease (buendia, 1992) . a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. the marmot himalayan develops an acute hepatitis with a productive infection (lucifora et al., 2010) . hepatitis d virus (hdv) is dependent upon hbv to undergo replication and successful infection in its human host (gerin, 2001) . there are two modes of infection possible between the viruses: coinfection where a person is simultaneously infected or superinfection in which a chronic carrier of hbv is subsequently infected with hdv (purcell et al., 1987) . coinfection leads to a similar disease as seen with hbv alone; however, superinfection can result in chronic hdv infection and severe liver damage (guilhot et al., 1994) . both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d (ponzetto et al., 1991) . a recently published report demonstrated the use of a humanized chimeric upa mouse to study interactions between the two viruses and drug testing (lutgehetmann et al., 2012) new models ranging from nhps to small animals and representing the disease characteristics in humans are necessary to study viral and host factors that drive disease pathogenesis and evaluate medical countermeasures. the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus is a low passage clinical isolate thus animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that occurs in natural disease. in order to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the aforementioned characteristics cannot always be satisfied; however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy fda "animal rule." this rule applies to situations in which vaccine and therapeutic efficacy cannot safely or ethically be tested in humans; thus licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures compared across institutions. this chapter has summarized the best models available for each of the viruses described. the rhesus macaque pediatric siv infection model-a valuable tool in understanding infant hiv-1 pathogenesis and for designing pediatric hiv-1 prevention strategies prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the 1993 outbreak in egypt common marmosets (callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels. emerg generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pathological changes in brain and other target organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus particle-to-pfu ratio of ebola virus influences disease course and survival in cynomolgus macaques progress toward norovirus vaccines: considerations for further development and implementation in potential target populations characterization of lethal zika virus infection in ag129 mice experimental in vitro and in vivo models for the study of human hepatitis b virus infection a model of meningococcal bacteremia after respiratory superinfection in influenza a virus-infected mice middle east respiratory syndrome coronavirus: current situation and travel-associated concerns aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques necrotizing scleritis, conjunctivitis, and other pathologic findings in the left eye and brain of an ebola virus-infected rhesus macaque (macaca mulatta) with apparent recovery and a delayed time of death american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis morbidity and mortality among patients with respiratory syncytial virus infection: a 2-year retrospective review chikungunya and the nervous system: what we do and do not know the west nile virus outbreak of 1999 in new york: the flushing hospital experience hospital outbreak of middle east respiratory syndrome coronavirus diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses norovirus vaccine against experimental human norwalk virus illness determination of the 50% human infectious dose for norwalk virus an epizootic attributable to western equine encephalitis virus infection in emus in texas evidence for camel-to-human transmission of mers coronavirus integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics disseminated and sustained hiv infection in cd34+ cord blood cell-transplanted rag2 −/− gamma c −/− mice choice of inbred rat strain impacts lethality and disease course after respiratory infection with rift valley fever virus rift valley fever: an uninvited zoonosis in the arabian peninsula recombinant norwalk virus-like particles given orally to volunteers: phase i study tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining lethal antibody enhancement of dengue disease in mice is prevented by fc modification animal models for the study of influenza pathogenesis and therapy effect of oral gavage treatment with znal42 and other metallo-ion formulations on influenza a h5n1 and h1n1 virus infections in mice macaque studies of vaccine and microbicide combinations for preventing hiv-1 sexual transmission early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus hepatitis b virus. the major etiology of hepatocellular carcinoma transmission of norwalk virus during football game vpx is critical for sivmne infection of pigtail macaques experimental respiratory syncytial virus infection of four species of primates pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat-1 knockout mouse model crimean-congo hemorrhagic fever virus infection is lethal for adult type i interferon receptor-knockout mice the utility of the new generation of humanized mice to study hiv-1 infection: transmission, prevention, pathogenesis, and treatment hiv-1 infection and cd4 t cell depletion in the humanized rag2 −/− gamma c −/− (rag-hu) mouse model bacterial infections in pigs experimentally infected with nipah virus evaluation of a mouse model for the west nile virus group for the purpose of determining viral pathotypes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice study of susceptibility to crimean hemorrhagic fever (chf) virus in european and long-eared hedgehogs. tezisy konf manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development genetic basis of attenuation of dengue virus type 4 small plaque mutants with restricted replication in suckling mice and in scid mice transplanted with human liver cells chimpanzees as an animal model for human norovirus infection and vaccine development a simple technique for infection of mosquitoes with viruses; transmission of zika virus human papillomavirus research: do we still need animal models? human papillomavirus in cervical cancer development of a hamster model for chikungunya virus infection and pathogenesis a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection the cotton rat model of respiratory viral infections correlates of immunity to filovirus infection filovirus vaccines induction of robust cellular and humoral virusspecific adaptive immune responses in human immunodeficiency virus-infected humanized blt mice animal models of human-papillomavirus-associated oncogenesis interferon alpha/beta receptor-deficient mice as a model for ebola virus disease zika virus outbreak in rio de janeiro, brazil: clinical characterization, epidemiological and virological aspects co-infection of the cotton rat (sigmodon hispidus) with staphylococcus aureus and influenza a virus results in synergistic disease effectiveness of influenza vaccination the role of the type i interferon response in the resistance of mice to filovirus infection a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis the confirmation and maintenance of smallpox eradication a lethal disease model for hantavirus pulmonary syndrome in immunosuppressed syrian hamsters infected with sin nombre virus nonhuman primate models of chikungunya virus infection and disease tissue tropism and neuroinvasion of west nile virus do not differ for two mouse strains with different survival rates pediatric norovirus diarrhea in nicaragua efficacy assessment of a cell-mediated immunity hiv-1 vaccine (the step study): a double-blind, randomised, placebo-controlled, test-of-concept trial variation between strains of hamsters in the lethality of pichinde virus infections hepatitis b viruses and hepatocellular carcinoma generation of k14-e7/n87betacat double transgenic mice as a model of cervical cancer limited or no protection by weakly or nonneutralizing antibodies against vaginal shiv challenge of macaques compared with a strongly neutralizing antibody a novel vaccine against crimean-congo haemorrhagic fever protects 100% of animals against lethal challenge in a mouse model interleukin-1beta but not tumor necrosis factor is involved in west nile virusinduced langerhans cell migration from the skin in c57bl/6 mice animal models of papillomavirus pathogenesis immunization of knock-out alpha/beta interferon receptor mice against high lethal dose of crimean-congo hemorrhagic fever virus with a cell culture based vaccine characterization of the localized immune response in the respiratory tract of ferrets following infection with influenza a and b viruses lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues a small nonhuman primate model for filovirus-induced disease severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus outbreak of acute illness-southwestern united states outbreak of west nile-like viral encephalitis-new york, 1999. mmwr morb. mortal in vitro whole-virus binding of a norovirus genogroup ii genotype 4 strain to cells of the lamina propria and brunner's glands in the human duodenum animal models for studying dengue pathogenesis and therapy us doctors investigate more than 50 possible cases of monkeypox mechanism of neuroinvasion of venezuelan equine encephalitis virus in the mouse chikungunya outbreaks-the globalization of vectorborne diseases inactivated and live, attenuated influenza vaccines protect mice against influenza: streptococcus pyogenes super-infections pathogenesis of a genogroup ii human norovirus in gnotobiotic pigs the immunobiology of sars* induction of tetravalent protective immunity against four dengue serotypes by the tandem domain iii of the envelope protein norovirus infection as a cause of diarrhea-associated benign infantile seizures comparative pathogenesis of epidemic and enzootic chikungunya viruses in a pregnant rhesus macaque model development of norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of norwalk virus gastroenteritis viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome a single sublingual dose of an adenovirus-based vaccine protects against lethal ebola challenge in mice and guinea pigs model systems to study the life cycle of human papillomaviruses and hpv-associated cancers primary severe acute respiratory syndrome coronavirus i nfection limits replication but not lung inflammation upon homologous rechallenge viral and host factors in human respiratory syncytial virus pathogenesis pathogenesis of pichinde virus infection in strain 13 guinea pigs: an immunocytochemical, virologic, and clinical chemistry study pathogenesis of experimental ebola virus infection in guinea pigs transcriptional profiling of the immune response to marburg virus infection the use of a neonatal mouse model to study respiratory syncytial virus infections a model of denv-3 infection that recapitulates severe disease and highlights the importance of ifn-gamma in host resistance to infection effects of age and viral determinants on chronicity as an outcome of experimental woodchuck hepatitis virus infection a mouse model for chikungunya: young age and inefficient type-i interferon signaling are risk factors for severe disease mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice comparison of the pathogenesis of the angola and ravn strains of marburg virus in the outbred guinea pig model the brazilian zika virus strain causes birth defects in experimental models age at first viral infection determines the pattern of t cell-mediated disease during reinfection in adulthood lassa fever encephalopathy: clinical and laboratory findings profound and prolonged lymphocytopenia with west nile encephalitis first complete genome sequence of zika virus (flaviviridae, flavivirus) from an autochthonous transmission in brazil viral haemorrhagic fevers caused by lassa, ebola, and marburg viruses the enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and il-13 production mouse models of hepatitis b and delta virus infection [experimental inhalation infection of monkeys of the macacus cynomolgus and macacus rhesus species with the virus kinetic profile of influenza virus infection in three rat strains pathology of experimental ebola virus infection in african green monkeys. involvement of fibroblastic reticular cells validation of an hpv16-mediated carcinogenesis mouse model middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques selection of unadapted, pathogenic shivs encoding newly transmitted hiv-1 envelope proteins b-cells and the use of non-human primates for evaluation of hiv vaccine candidates antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv-1 in humanized blt mice innate and adaptive immune responses determine protection against disseminated infection by west nile encephalitis virus rift valley fever virus encephalitis is associated with an ineffective systemic immune response and activated t cell infiltration into the cns in an immunocompetent mouse model evidence of sexual transmission of zika virus a susceptible mouse model for zika virus infection identification of a novel coronavirus in patients with severe acute respiratory syndrome subclinical infection without encephalitis in mice following intranasal exposure to nipah virus-malaysia and nipah virus-bangladesh nonhuman primate models of encephalitic alphavirus infection: historical review and future perspectives mortality due to influenza in the united states-an annualized regression approach using multiple-cause mortality data distinct pathogenesis of hong kong-origin h5n1 viruses in mice compared to that of other highly pathogenic h5 avian influenza viruses postexposure antibody prophylaxis protects nonhuman primates from filovirus disease comparative live bioluminescence imaging of monkeypox virus dissemination in a wild-derived inbred mouse (mus musculus castaneus) and outbred african dormouse (graphiurus kelleni) siv-induced impairment of neurovascular repair: a potential role for vegf smallpox vaccine does not protect macaques with aids from a lethal monkeypox virus challenge influenza-induced tachypnea is prevented in immune cotton rats, but cannot be treated with an anti-inflammatory steroid or a neuraminidase inhibitor distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats an outbreak of viral gastroenteritis following environmental contamination at a concert hall natural history of aerosol exposure with marburg virus in rhesus macaques experimental congo virus (ib-an7620) infection in primates further assessment of monkeypox virus infection in gambian pouched rats (cricetomys gambianus) using in vivo bioluminescent imaging respiratory syncytial virus infection in elderly and high-risk adults infection with mers-cov causes lethal pneumonia in the common marmoset immune response to marburg virus angola infection in nonhuman primates fields' virology the susceptibility of rats to rift valley fever in relation to age henipavirus susceptibility to environmental variables pause on avian flu transmission research aetiology: koch's postulates fulfilled for sars virus spinal cord neuropathology in human west nile virus infection therapeutic dna vaccine induces broad t cell responses in the gut and sustained protection from viral rebound and aids in siv-infected rhesus macaques hepatitis b virus infection-natural history and clinical consequences biological heterogeneity, including systemic replication in mice, of h5n1 influenza a virus isolates from humans in hong kong chikungunya virus arthritis in adult wild-type mice epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method development of an acute and highly pathogenic nonhuman primate model of nipah virus infection animal models of hepatitis delta virus infection and disease hepadnavirusinduced liver cancer in woodchucks experimental infection and natural contact exposure of dogs with avian influenza virus (h5n1). emerg megaribavirin aerosol for the treatment of influenza a virus infections in mice discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-pcr chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice studies on the virus of venezuelan equine encephalomyelitis. i. modification by cortisone of the response of the central nervous system of macaca mulatta serious morbidity and mortality associated with influenza epidemics a novel respiratory model of infection with monkeypox virus in cynomolgus macaques clinical features of nipah virus encephalitis among pig farmers in malaysia monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention animal models of highly pathogenic rna viral infections: hemorrhagic fever viruses interferon alfacon-1 protects hamsters from lethal pichinde virus infection primary respiratory syncytial virus infection in mice pneumonitis and multiorgan system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic area-puerto rico acute and chronic airway disease after human respiratory syncytial virus infection in cotton rats (sigmodon hispidus) alphaviruses replication fitness determines high virulence of influenza a virus in mice carrying functional mx1 resistance gene antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal hiv-1 infection in a transgenic mouse model characterization of influenza a/hongkong/156/97 (h5n1) virus in a mouse model and protective effect of zanamivir on h5n1 infection in mice epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century cell culture and animal models of viral hepatitis. part i: hepatitis b expression of the hepatitis delta virus large and small antigens in transgenic mice acute hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model lassa fever encephalopathy: lassa virus in cerebrospinal fluid but not in serum 1, 5-iodonaphthyl azide-inactivated v3526 protects against aerosol challenge with virulent venezuelan equine encephalitis virus dengue: an update pegylated interferonalpha protects type 1 pneumocytes against sars coronavirus infection in macaques asymptomatic middle east respiratory syndrome coronavirus infection in rabbits head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model update on animal models for hiv research norovirus disease in the united states. emerg cardiopulmonary manifestations of hantavirus pulmonary syndrome serum neutralizing antibody titers of seropositive chimpanzees immunized with vaccines coformulated with natural fusion and attachment proteins of respiratory syncytial virus hendra virus infection in a veterinarian a phase 1 clinical trial of a dna vaccine for venezuelan equine encephalitis delivered by intramuscular or intradermal electroporation deaths from norovirus among the elderly, england and wales. emerg aerosolized rift valley fever virus causes fatal encephalitis in african green monkeys and common marmosets hiv-1-induced aids in monkeys west nile fever short communication: viremic control is independent of repeated low-dose shivsf162p3 exposures pathogenesis of marburg hemorrhagic fever in cynomolgus macaques pathogenesis of lassa fever in cynomolgus macaques niemann-pick c1 is essential for ebolavirus replication and pathogenesis in vivo airborne transmission of influenza a/ h5n1 virus between ferrets animal models in hiv-1 protection and therapy advances in rsv vaccine research and development-a global agenda transmission of zika virus through sexual contact with travelers to areas of ongoing transmissioncontinental united states establishment of a patient-derived orthotopic xenograft (pdox) model of her-2-positive cervical cancer expressing the clinical metastatic pattern resolution of primary severe acute respiratory syndromeassociated coronavirus infection requires stat1 mucosal immunity and vaccines nipah virus outbreak with person-to-person transmission in a district of bangladesh eastern equine encephalitis virus in mice i: clinical course and outcome are dependent on route of exposure the lesions of experimental equine morbillivirus disease in cats and guinea pigs a lethal disease model for hantavirus pulmonary syndrome hantaan/ andes virus dna vaccine elicits a broadly cross-reactive neutralizing antibody response in nonhuman primates persistent infection with and serologic cross-reactivity of three novel murine noroviruses molecular characterization of three novel murine noroviruses identification of hepatitis b virus indigenous to chimpanzees manifestation of thrombocytopenia in dengue-2-virusinfected mice transfer of hbv genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice west nile fever-a reemerging mosquito-borne viral disease in europe. emerg live, attenuated influenza virus (laiv) vehicles are strong inducers of immunity toward influenza b virus clinical characteristics of human monkeypox, and risk factors for severe disease shiv-1157i and passaged progeny viruses encoding r5 hiv-1 clade c env cause aids in rhesus monkeys norwalk virus infection associates with secretor status genotyped from sera a prairie dog animal model of systemic orthopoxvirus disease using west african and congo basin strains of monkeypox virus risks of chronicity following acute hepatitis b virus infection: a review the pathogenesis of rift valley fever experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets pathogenicity of a highly pathogenic avian influenza virus, a/chicken/yamaguchi/7/04 (h5n1) in different species of birds and mammals respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice a multimeric l2 vaccine for prevention of animal papillomavirus infections lassa virus infection of rhesus monkeys: pathogenesis and treatment with ribavirin pathogenesis of lassa virus infection in guinea pigs perinatal transmission of shiv-sf162p3 in macaca nemestrina human monkeypox and other poxvirus infections of man cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus glycomic characterization of respiratory tract tissues of ferrets: implications for its use in influenza virus infection studies comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route phenotypic changes in langerhans' cells after infection with arboviruses: a role in the immune response to epidermally acquired viral infection? protection of beagle dogs from mucosal challenge with canine oral papillomavirus by immunization with recombinant adenoviruses expressing codon-optimized early genes detailed analysis of the african green monkey model of nipah virus disease experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera treatment of venezuelan equine encephalitis virus infection with (-)-carbodine c3h/hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection blt humanized mice as a small animal model of hiv infection stat1-dependent innate immunity to a norwalk-like virus pichinde virus infection in strain 13 guinea pigs reduces intestinal protein reflection coefficient with compensation establishment of the black-tailed prairie dog (cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered preexposure in both high-and low-dose monkeypox virus challenges low-dose rectal inoculation of rhesus macaques by sivsme660 or sivmac251 recapitulates human mucosal infection by hiv-1 new opportunities for field research on the pathogenesis and treatment of lassa fever gastrointestinal norovirus infection associated with exacerbation of inflammatory bowel disease isolation of monkeypox virus from wild squirrel infected in nature in hot pursuit of the first vaccine against respiratory syncytial virus pathogenesis of hantaan virus infection in suckling mice: clinical, virologic, and serologic observations respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine the severe pathogenicity of alveolar macrophage-depleted ferrets infected with 2009 pandemic h1n1 influenza virus mouse adaptation of influenza b virus increases replication in the upper respiratory tract and results in droplet transmissibility in ferrets stepping toward a macaque model of hiv-1 induced vomiting as a symptom and transmission risk in norovirus illness: evidence from human challenge studies wild-type puumala hantavirus infection induces cytokines, c-reactive protein, creatinine, and nitric oxide in cynomolgus macaques aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques replication, pathogenicity, shedding, and transmission of zaire ebolavirus in pigs west nile viral encephalitis foodborne viruses: an emerging problem low pathogenic avian influenza a(h7n9) virus causes high mortality in ferrets upon intratracheal challenge: a model to study intervention strategies filoviruses: a compendium of 40 years of epidemiological, clinical, and laboratory studies pathology of human influenza a (h5n1) virus infection in cynomolgus macaques (macaca fascicularis) dengue virus infection and immune response in humanized rag2(-/-)gamma(c) (-/-) (rag-hu) mice chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages viral hepatitis b strong local and systemic protective immunity induced in the ferret model by an intranasal virosome-formulated influenza subunit vaccine origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states antibody to hepatitis-associated antigen. frequency and pattern of response as detected by radioimmunoprecipitation severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coronavirus hku1 and other coronavirus infections in hong kong epidemic rift valley fever in egypt: observations of the spectrum of human illness hantaviruses. a short review hepatitis b virus infection quantitative measurement of influenza virus replication using consecutive bronchoalveolar lavage in the lower respiratory tract of a ferret model characterization of the activity of 2'-c-methylcytidine against dengue virus replication diffusion tensor and volumetric magnetic resonance measures as biomarkers of brain damage in a small animal model of hiv sequencing, annotation, and characterization of the influenza ferret infectome lethality and pathogenesis of airborne infection with filoviruses in a129 alpha/beta −/− interferon receptor-deficient mice experimental inoculation study indicates swine as a potential host for hendra virus early initiation of antiretroviral therapy can functionally control productive hiv-1 infection in humanized-blt mice zika virus disrupts neural progenitor development and leads to microcephaly in mice middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 immunogenicity and protective efficacy of a recombinant subunit west nile virus vaccine in rhesus monkeys study of dengue virus infection in scid mice engrafted with human k562 cells a comparative study of the pathogenesis of western equine and eastern equine encephalomyelitis viral infections in mice by intracerebral and subcutaneous inoculations hydrodynamics-based transfection in animals by systemic administration of plasmid dna the emergence of nipah virus, a highly pathogenic paramyxovirus the guinea pig as a transmission model for human influenza viruses transmission in the guinea pig model a mouse model for the evaluation of pathogenesis and immunity to influenza a (h5n1) viruses isolated from humans transmission of human infection with nipah virus recurrent zoonotic transmission of nipah virus into humans the effect of an arenavirus infection on liver morphology and function hepatitis b virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model ebola virus disease in mice with transplanted human hematopoietic stem cells arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin-6 expression and hepatocyte proliferation humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis detection of hepatitis b virus infection in wild-born chimpanzees (pan troglodytes verus): phylogenetic relationships with human and other primate genotypes proportion of deaths and clinical features in bundibugyo ebola virus infection the ferret: an animal model to study influenza virus local innate immune responses and influenza virus transmission and virulence in ferrets vomiting larry: a simulated vomiting system for assessing environmental contamination from projectile vomiting related to norovirus infection studies on the pathogenesis of dengue infection in monkeys. 3. sequential distribution of virus in primary and heterologous infections studies on dengue 2 virus infection in cyclophosphamide-treated rhesus monkeys crimean-congo hemorrhagic fever experimental infection of squirrel monkeys with nipah virus. emerg a school outbreak of norwalk-like virus: evidence for airborne transmission virology: sars virus infection of cats and ferrets characterization of clinical and immunological parameters during ebola virus infection of rhesus macaques delayed disease progression in cynomolgus macaques infected with ebola virus makona strain. emerg asymmetric replication of duck hepatitis b virus dna in liver cells: free minusstrand dna human and avian influenza viruses target different cell types in cultures of human airway epithelium a role for hpv16 e5 in cervical carcinogenesis replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys inhibition of hepatitis b virus in mice by rna interference zika virus was transmitted by sexual contact in texas, health officials report lassa fever. effective therapy with ribavirin lassa virus hepatitis: a study of fatal lassa fever in humans lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus andes virus infection of cynomolgus macaques regional t-and b-cell responses in influenza-infected ferrets clinical aspects of marburg hemorrhagic fever humanized mice mount specific adaptive and innate immune responses to ebv and tsst-1 isolation from human sera in egypt of a virus apparently identical to west nile virus middle east respiratory syndrome coronavirus in bats, saudi arabia. emerg chikungunya virus infection results in higher and persistent viral replication in aged rhesus macaques due to defects in anti-viral immunity reduced clearance of respiratory syncytial virus infection in a preterm lamb model dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome experimental nipah virus infection in pigs and cats experimental nipah virus infection in pteropid bats (pteropus poliocephalus) an in-depth analysis of original antigenic sin in dengue virus infection propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus rift valley fever virus in mice. i. general features of the infection zika virus infection during pregnancy in mice causes placental damage and fetal demise outbreaks of acute gastroenteritis associated with norwalk-like viruses in campus settings a nonfucosylated variant of the anti-hiv-1 monoclonal antibody b12 has enhanced fcgammariiiamediated antiviral activity in vitro but does not improve protection against mucosal shiv challenge in macaques flaviviruses experimental studies of rhesus monkeys infected with epizootic and enzootic subtypes of venezuelan equine encephalitis virus necrotizing myocarditis in mice infected with western equine encephalitis virus: clinical, electrocardiographic, and histopathologic correlations aedes albopictus in the united states: ten-year presence and public health implications. emerg severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume impact of short-term haart initiated during the chronic stage or shortly post-exposure on siv infection of male genital organs influence of age on susceptibility and on immune response of mice to eastern equine encephalomyelitis virus a mouse model of chikungunya virusinduced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence dengue virus tropism in humanized mice recapitulates human dengue fever functional role of type i and type ii interferons in antiviral defense the occurrence of human cases in johannesburg. s feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine vertical transmission and fetal replication of nipah virus in an experimentally infected cat pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets pneumonia from human coronavirus in a macaque model eastern equine encephalitis virus infection: electron microscopic studies of mouse central nervous system evaluation of three strains of influenza a virus in humans and in owl, cebus, and squirrel monkeys a novel morbillivirus pneumonia of horses and its transmission to humans. emerg a morbillivirus that caused fatal disease in horses and humans global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis cchf infection among animals reemergence of monkeypox: prevalence, diagnostics, and countermeasures experimental infection of cynomolgus macaques (macaca fascicularis) with aerosolized monkeypox virus eastern equine encephalitis. distribution of central nervous system lesions in man and rhesus monkey hla a2 restricted cytotoxic t lymphocyte responses to multiple hepatitis b surface antigen epitopes during hepatitis b virus infection an aptamer-sirna chimera suppresses hiv-1 viral loads and protects from helper cd4(+) t cell decline in humanized mice severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 ferrets exclusively synthesize neu5ac and express naturally humanized influenza a virus receptors field's virology naturally occurring, nonregressing canine oral papillomavirus infection: host immunity, virus characterization, and experimental infection regression of canine oral papillomas is associated with infiltration of cd4+ and cd8+ lymphocytes diversifying animal models: the use of hispid cotton rats (sigmodon hispidus) in infectious diseases induction of focal epithelial hyperplasia in tongue of young bk6-e6/e7 hpv16 transgenic mice the contribution of animal models to the understanding of the host range and virulence of influenza a viruses evaluation of antiviral efficacy of ribavirin, arbidol, and t-705 (favipiravir) in a mouse model for crimean-congo hemorrhagic fever evaluation of oseltamivir prophylaxis regimens for reducing influenza virus infection, transmission and disease severity in a ferret model of household contact a novel video tracking method to evaluate the effect of influenza infection and antiviral treatment on ferret activity human respiratory syncytial virus a2 strain replicates and induces innate immune responses by respiratory epithelia of neonatal lambs common marmoset (callithrix jacchus) as a primate model of dengue virus infection: development of high levels of viraemia and demonstration of protective immunity changes in hematological and serum biochemical parameters in common marmosets (callithrix jacchus) after inoculation with dengue virus middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction dengue virus-induced hemorrhage in a nonhuman primate model preclinical development of highly effective and safe dna vaccines directed against hpv 16 e6 and e7 a novel small molecule inhibitor of influenza a viruses that targets polymerase function and indirectly induces interferon comparison of monkeypox viruses pathogenesis in mice by in vivo imaging a rhesus monkey model for sexual transmission of a papillomavirus isolated from a squamous cell carcinoma infection of calves with bovine norovirus giii. 1 strain jena virus: an experimental model to study the pathogenesis of norovirus infection the cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis zika virus disease in colombia-preliminary report genetic diversity, distribution, and serological features of hantavirus infection in five countries in south america liver injury and viremia in mice infected with dengue-2 virus the hamster as an animal model for eastern equine encephalitis-and its use in studies of virus entrance into the brain a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge identification of an antioxidant small-molecule with broad-spectrum antiviral activity impaired heterologous immunity in aged ferrets during sequential influenza a h1n1 infection influenza transmission in the mother-infant dyad leads to severe disease, mammary gland infection, and pathogenesis by regulating host responses economic impact of respiratory syncytial virus-related illness in the us: an analysis of national databases inhibitory potential of neem (azadirachta indica juss) leaves on dengue virus type-2 replication systematic literature review of role of noroviruses in sporadic gastroenteritis. emerg sars: what have we learned? the severe acute respiratory syndrome severe acute respiratory syndrome bacterial sinusitis and otitis media following influenza virus infection in ferrets neuropathology of h5n1 virus infection in ferrets the draft genome sequence of the ferret (mustela putorius furo) facilitates study of human respiratory disease immunopathogenesis of coronavirus infections: implications for sars hantavirus pulmonary syndrome: the new american hemorrhagic fever rift valley fever inbred rat strains mimic the disparate human response to rift valley fever virus infection experimental studies of arenaviral hemorrhagic fevers experimental rift valley fever in rhesus macaques bovine respiratory syncytial virus protects cotton rats against human respiratory syncytial virus infection human hendra virus encephalitis associated with equine outbreak molecularly engineered live-attenuated chimeric west nile/dengue virus vaccines protect rhesus monkeys from west nile virus structure as revealed by airway dissection. a comparison of mammalian lungs study on west nile virus persistence in monkeys experimental hepatitis delta virus infection in the animal model changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus grune and stratton. hepatitis and blood transfusion perspectives on hepatitis b studies with chimpanzees pulmonary lesions in primary respiratory syncytial virus infection, reinfection, and vaccine-enhanced disease in the cotton rat (sigmodon hispidus) experimental hepatitis delta virus infection in the chimpanzee relative infectivity of hepatitis a virus by the oral and intravenous routes in 2 species of nonhuman primates cardiovascular and pulmonary responses to pichinde virus infection in strain 13 guinea pigs establishment and characterization of a lethal mouse model for the angola strain of marburg virus stability and inactivation of sars coronavirus possibility of extracting hyperimmune gammaglobulin against chf from doneky blood sera dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc swine as a model for influenza a virus infection and immunity zika virus: an update on epidemiology, pathology, molecular biology, and animal model oseltamivir population pharmacokinetics in the ferret: model application for pharmacokinetic/pharmacodynamic study design aerosol exposure to western equine encephalitis virus causes fever and encephalitis in cynomolgus macaques severe encephalitis in cynomolgus macaques exposed to aerosolized eastern equine encephalitis virus differences in aerosolization of rift valley fever virus resulting from choice of inhalation exposure chamber: implications for animal challenge studies an hiv-1 transgenic rat that develops hiv-related pathology and immunologic dysfunction a trivalent recombinant ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous siv challenge infection with chikungunya virus in italy: an outbreak in a temperate region a single dose of an iscom influenza vaccine induces long-lasting protective immunity against homologous challenge infection but fails to protect cynomolgus macaques against distant drift variants of influenza a (h3n2) viruses influenza a virus (h5n1) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts immunomodulation with il-4r alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease animal models for sars aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice transmission of a 2009 h1n1 pandemic influenza virus occurs before fever is detected, in the ferret model experimental norovirus infections in non-human primates synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection recent progress in henipavirus research: molecular biology, genetic diversity, animal models mucosal arenavirus infection of primates can protect them from lethal hemorrhagic fever sars vaccines: where are we? animal models of rift valley fever virus infection characterization of a novel coronavirus associated with severe acute respiratory syndrome macaque model for severe acute respiratory syndrome pathogenesis of aerosolized eastern equine encephalitis virus infection in guinea pigs pathophysiology of hantavirus pulmonary syndrome in rhesus macaques virulence and pathophysiology of the congo basin and west african strains of monkeypox virus in non-human primates arenaviridae. virus taxonomy, viiith report of the international committee on taxonomy of viruses clinical laboratory, virologic, and pathologic changes in hamsters experimentally infected with pirital virus (arenaviridae): a rodent model of lassa fever comparative pathology of north american and central african strains of monkeypox virus in a ground squirrel model of the disease biomedical applications of sheep models: from asthma to vaccines bunyaviruses the use of an animal model to study transmission of influenza virus infection experimental infection of an african dormouse (graphiurus kelleni) with monkeypox virus animal noroviruses human monkeypox infection: a family cluster in the midwestern united states the possibility of using the icr mouse as an animal model to assess antimonkeypox drug efficacy using the ground squirrel (marmota bobak) as an animal model to assess monkeypox drug efficacy experimental inoculation of juvenile rhesus macaques with primate enteric caliciviruses a rodent model of chikungunya virus infection in rag1 −/− mice, with features of persistence, for vaccine safety evaluation respiratory syncytial virus (rsv) pulmonary infection in humanized mice induces human anti-rsv immune responses and pathology viremia and antibody response of small african and laboratory animals to crimean-congo hemorrhagic fever virus infection early activation of natural killer and b cells in response to primary dengue virus infection in a/j mice murine model for dengue virus-induced lethal disease with increased vascular permeability in vitro and in vivo assay systems for study of influenza virus inhibitors viruses of the bunya-and togaviridae families: potential as bioterrorism agents and means of control potential role of immunomodulators for treatment of phlebovirus infections of animals patient-derived tumor xenografts: transforming clinical samples into mouse models treatment of lethal pichinde virus infections in weanling lvg/lak hamsters with ribavirin, ribamidine, selenazofurin, and ampligen a comparative study of the crimean hemorrhagic fever-congo group of viruses the pathogenesis of rift valley fever virus in the mouse model effective antiviral treatment of systemic orthopoxvirus disease: st-246 treatment of prairie dogs infected with monkeypox virus a neurotropic virus isolated from the blood of a native uganda comparison of the plaque assay and 50% tissue culture infectious dose assay as methods for measuring filovirus infectivity experimental respiratory marburg virus haemorrhagic fever infection in the common marmoset experimental respiratory infection of marmosets (callithrix jacchus) with ebola virus kikwit essential role of platelet-activating factor receptor in the pathogenesis of dengue virus infection respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs trans-activation of viral enhancers by the hepatitis b virus x protein filamentous influenza a virus infection predisposes mice to fatal septicemia following superinfection with streptococcus pneumoniae serotype 3 contributions of mast cells and vasoactive products, leukotrienes and chymase, to dengue virus-induced vascular leakage. elife 2, e00481. stabenow correction: a retrospective investigation on canine papillomavirus 1 (cpv1) in oral oncogenesis reveals dogs are not a suitable animal model for high-risk hpv-induced oral cancer clinical profiles associated with influenza disease in the ferret model comparative neurovirulence and tissue tropism of wild-type and attenuated strains of venezuelan equine encephalitis virus administered by aerosol in c3h/hen and balb/c mice a mouse model for human anal cancer first reported cases of hantavirus pulmonary syndrome in canada antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection evaluation of intravenous zanamivir against experimental influenza a (h5n1) virus infection in cynomolgus macaques ferrets as a novel animal model for studying human respiratory syncytial virus infections in immunocompetent and immunocompromised hosts differential pathogenesis of respiratory syncytial virus clinical isolates in balb/c mice limited dissemination of pathogenic siv after vaginal challenge of rhesus monkeys immunized with a live, attenuated lentivirus experimental west nile virus infection in rabbits: an alternative model for studying induction of disease and virus control a virus similar to human hepatitis b virus associated with hepatitis and hepatoma in woodchucks development of animal models against emerging coronaviruses: from sars to mers coronavirus severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction the clinical pathology of crimean-congo hemorrhagic fever human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes nipah virus encephalitis profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers distribution of viral antigens and development of lesions in chicken embryos inoculated with nipah virus characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease the origin and virulence of the 1918 "spanish" influenza virus the pathology of influenza virus infections isolation of west nile virus from culex mosquitoes recombinant respiratory syncytial virus that does not express the ns1 or m2-2 protein is highly attenuated and immunogenic in chimpanzees animal models of hepadnavirus-associated hepatocellular carcinoma mouse models for chikungunya virus: deciphering immune mechanisms responsible for disease and pathology human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets experimental infection of ground squirrels (spermophilus tridecemlineatus) with monkeypox virus. emerg persistent west nile virus infection in the golden hamster: studies on its mechanism and possible implications for other flavivirus infections risk factors in dengue shock syndrome breaking barriers to an aids model with macaque-tropic hiv-1 derivatives dominant role of hpv16 e7 in anal carcinogenesis ribavirin efficacy in an in vivo model of crimean-congo hemorrhagic fever virus (cchf) infection histopathologic and immunohistochemical characterization of nipah virus infection in the guinea pig sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus protective efficacy of a bivalent recombinant vesicular stomatitis virus vaccine in the syrian hamster model of lethal ebola virus infection persistence of human norovirus rt-qpcr signals in simulated gastric fluid outbreak of necrotizing enterocolitis caused by norovirus in a neonatal intensive care unit pathology of experimental aerosol zaire ebolavirus infection in rhesus macaques experimental aerosolized guinea pig-adapted zaire ebolavirus (variant: mayinga) causes lethal pneumonia in guinea pigs animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors a human lung xenograft mouse model of nipah virus infection human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals virulence and reduced fitness of simian immunodeficiency virus with the m184v mutation in reverse transcriptase chronic administration of tenofovir to rhesus macaques from infancy through adulthood and pregnancy: summary of pharmacokinetics and biological and virological effects use of a small molecule ccr5 inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian-human immunodeficiency virus infection experimental infection of rhesus macaques and common marmosets with a european strain of west nile virus protective vaccination against papillomavirus-induced skin tumors under immunocompetent and immunosuppressive conditions: a preclinical study using a natural outbred animal model cataloguing of potential hiv susceptibility factors during the menstrual cycle of pig-tailed macaques by using a systems biology approach temporal analysis of andes virus and sin nombre virus infections of syrian hamsters anti-alpha4 integrin antibody blocks monocyte/macrophage traffic to the heart and decreases cardiac pathology in a siv infection model of aids clinical manifestations, laboratory findings, and treatment outcomes of sars patients. emerg prevalence of noroviruses and sapoviruses in swine of various ages determined by reverse transcription-pcr and microwell hybridization assays porcine enteric caliciviruses: genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology avian influenza viruses, inflammation, and cd8(+) development of a model for marburgvirus based on severe-combined immunodeficiency mice development and characterization of a mouse model for marburg hemorrhagic fever euthanasia assessment in ebola virus infected nonhuman primates hematopoietic stem cell-engrafted nod/scid/ il2rgamma null mice develop human lymphoid systems and induce long-lasting hiv-1 infection with specific humoral immune responses the magnitude of dengue virus ns1 protein secretion is strain dependent and does not correlate with severe pathologies in the mouse infection model human fatal zaire ebolavirus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis immunization with modified vaccinia virus ankarabased recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets invasion of the central nervous system in a porcine host by nipah virus development of a rift valley fever virus viremia challenge model in sheep and goats antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever an unusual hantavirus outbreak in southern argentina: person-to-person transmission? hantavirus pulmonary syndrome study group for patagonia. emerg equine morbillivirus pneumonia: susceptibility of laboratory animals to the virus susceptibility of cats to equine morbillivirus hantaan virus infection causes an acute neurological disease that is fatal in adult laboratory mice a north american h7n3 influenza virus supports reassortment with 2009 pandemic h1n1 and induces disease in mice without prior adaptation transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats a guinea-pig model of hendra virus encephalitis susceptibility of hiv-2, siv and shiv to various anti-hiv-1 compounds: implications for treatment and postexposure prophylaxis a golden hamster model for human acute nipah virus infection ebola virus transmission in guinea pigs intranasal immunization with an adenovirus vaccine protects guinea pigs from ebola virus transmission by infected animals characterization and experimental transmission of an oncogenic papillomavirus in female macaques dengue haemorrhagic fever: diagnosis, treatment, prevention and control, second ed. world health organization, geneva. world health organization clinical aspects of hepatitis b virus infection civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates evidence of a causal role of winter virus infection during infancy in early childhood asthma vertical transmission of zika virus targeting the radial glial cells affects cortex development of offspring mice the antiviral activity of sp-303, a natural polyphenolic polymer, against respiratory syncytial and parainfluenza type 3 viruses in cotton rats experimental infection of prairie dogs with monkeypox virus. emerg epidemiological studies of hemorrhagic fever with renal syndrome: analysis of risk factors and mode of transmission hydrodynamic injection of viral dna: a mouse model of acute hepatitis b virus infection mice transgenic for human angiotensin-converting enzyme 2 provide a model for sars coronavirus infection an animal model of mers produced by infection of rhesus macaques with mers coronavirus a protective role for dengue virus-specific cd8+ t cells the incubation period of hantavirus pulmonary syndrome animal model of sensorineural hearing loss associated with lassa virus infection the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus. epidemiol hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease the pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (macaca fascicularis) hiv-1 infection and pathogenesis in a novel humanized mouse model h7n9 influenza viruses are transmissible in ferrets by respiratory droplet induction of neutralizing antibodies against four serotypes of dengue viruses by mixbiediii, a tetravalent dengue vaccine rapid generation of a mouse model for middle east respiratory syndrome an animal model for studying the pathogenesis of chikungunya virus infection pathogenesis of avian influenza a (h5n1) viruses in ferrets lethal crimean-congo hemorrhagic fever virus infection in interferon alpha/beta receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy the pathogenesis of western equine encephalitis virus (w.e.e.) in adult hamsters with special reference to the long and short term effects on the c.n.s. of the attenuated clone 15 variant development of a murine model for aerosolized filovirus infection using a panel of bxd recombinant inbred mice a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and rhesus macaques characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the us army. key: cord-262232-7ecg1iha authors: crakes, katti r; herrera, carolina; morgan, jessica l; olstad, katie; hessell, ann j; ziprin, paul; liwang, patricia j; dandekar, satya title: efficacy of silk fibroin biomaterial vehicle for in vivo mucosal delivery of griffithsin and protection against hiv and shiv infection ex vivo date: 2020-10-18 journal: j int aids soc doi: 10.1002/jia2.25628 sha: doc_id: 262232 cord_uid: 7ecg1iha introduction: the majority of new hiv infections occur through mucosal transmission. the availability of readily applicable and accessible platforms for anti‐retroviral (arv) delivery is critical for the prevention of hiv acquisition through sexual transmission in both women and men. there is a compelling need for developing new topical delivery systems that have advantages over the pills, gels and rings, which currently fail to guarantee protection against mucosal viral transmission in vulnerable populations due to lack of user compliance. the silk fibroin (sf) platform offers another option that may be better suited to individual circumstances and preferences to increase efficacy through user compliance. the objective of this study was to test safety and efficacy of sf for anti‐hiv drug delivery to mucosal sites and for viral prevention. methods: we formulated a potent hiv inhibitor griffithsin (grft) in a mucoadhesive silk fibroin (sf) drug delivery platform and tested the application in a non‐human primate model in vivo and a pre‐clinical human cervical and colorectal tissue explant model. both vaginal and rectal compartments were assessed in rhesus macaques (mucaca mulatta) that received sf (n = 4), no sf (n = 7) and sf‐grft (n = 11). in this study, we evaluated the composition of local microbiota, inflammatory cytokine production, histopathological changes in the vaginal and rectal compartments and mucosal protection after ex vivo shiv challenge. results: effective grft release and retention in mucosal tissues from the sf‐grft platform resulted in protection against hiv in human cervical and colorectal tissue as well as against shiv challenge in both rhesus macaque vaginal and rectal tissues. mucoadhesion of sf‐grft inserts did not cause any inflammatory responses or changes in local microbiota. conclusions: we demonstrated that in vivo delivery of sf‐grft in rhesus macaques fully protects against shiv challenge ex vivo after two hours of application and is safe to use in both the vaginal and rectal compartments. our study provides support for the development of silk fibroin as a highly promising, user‐friendly hiv prevention modality to address the global disparity in hiv infection. despite the successes of combination anti-retroviral therapy (art) and prevention efforts in controlling the global hiv epidemic, there are major challenges for viral eradication in hivinfected people and the prevention of new hiv infections. of 37.9 million hiv-infected people worldwide, 25.4 million individuals are receiving art which has lowered hiv-associated morbidity and mortality [1] . however, there are still 1.7 million new hiv infections diagnosed per year. preventative healthcare is critical in counteracting the hiv epidemic, particularly within disproportionately affected populations experiencing economic and gender inequities [2] . the gender gap is most notable in areas of sub-saharan africa, where more than half of those living with hiv and those newly infected with hiv are women [1] . in the absence of an effective hiv vaccine, pre-exposure prophylaxis (prep) using anti-retroviral drugs is critical for hiv prevention through sexual transmission [3] , concomitant with several strategies including films [4, 5] , gels [6, 7] , rings [8] [9] [10] and tablets [11] . long-acting injectables including cabotegravir and rilpivirine are being explored [12] , but need careful consideration of pharmacokinetic "tails" to prevent antiviral resistance during the washout period [13, 14] . there is a compelling need for new affordable prevention devices, particularly among young women who have demonstrated modest compliance with pills, gels and rings in the clinical trials [6, 15, 16] . public and private sectors should be engaged in topical prep research with adequate consideration to input from end-users for acceptability and applicability of hiv prevention devices. we have developed a silk fibroin (sf)-based drug delivery platform that can be readily administered into the vaginal and rectal mucosal sites and is easily transportable, or able to be packaged discretely without the need of refrigeration. sf is a biocompatible, biodegradable material made from bombyx mori silkworm cocoons that were developed into a drug carrier of bioactive compounds ranging from small molecules to monoclonal antibodies [17] [18] [19] . the use of sf as a drug delivery system emerged because of its unique tunable b-sheet secondary structure that makes its properties range from easily soluble to crystalline structures nearly impenetrable to water [20] , a desirable characteristic for sustained drug release. other beneficial properties include its mechanical robustness that allows slow protein degradation [21] and thermodynamic stability enabling convenient transport and handling [22] . silk has shown outstanding preclinical properties for a number of treatments, ranging from ocular delivery of drugs [23] to edible coatings of food [24] . aside from its usage in some cases for sutures, the main fda approved clinical use of silk fibroin is the seri surgical scaffold, a surgical mesh for abdominal wall and breast reconstruction [25, 26] . sf formulations can encapsulate several anti-hiv proteins, be stable for more than one year, and mediate quick release or sustained release over the course of one month [22, 27, 28] . considering the need for sustained drug delivery of prep candidates at mucosal sites, sf provides an innovative platform that can be affordably produced, safely administered, and is effective against hiv transmission. griffithsin (grft), a known hiv inhibitor, is a protein lectin that binds high-mannose moieties on glycosylated proteins such as hiv-1 gp120 [29] . grft has broad antiviral activity and inhibits sivmac251 and shiv strains as well as hsv-2, hpv, hepatitis c, coronavirus and japanese encephalitis [30] [31] [32] [33] [34] [35] . in fact, fast-dissolving formulations of grft have been shown to prevent shiv, hsv-2 and hpv infections in vivo [36] . recent studies have shown that grft exerts little to no adverse effects in vitro and in vivo [37] [38] [39] [40] . two clinical trials are evaluating grft gel formulations in rectal and vaginal compartments [41, 42] . the grft formulation in sf is a promising prep candidate because it is effective upon administration and does not require absorption or activation, unlike many antiviral drugs. the solid sf platform provides an alternative to gels, which have been met with inconsistent user adherence [43] . in this study, we evaluated the muco-adhesion and efficacy of sf as a grft delivery vehicle in vaginal and rectal compartments in vivo using non-human primates and in human mucosal tissue explants ex vivo. we sought to produce a sf-based solid dosage form containing a quantity of grft (1 mg) capable of quickly producing inhibitory concentrations (at least 1,000 times the ec 90 value for grft against hiv in vitro) within the small fluid volume available in the macaque vaginal and rectal compartments. effective release of grft from the sf vehicle imparted protection of mucosal tissues from hiv as well as shiv infection ex vivo. thus, sf is a highly promising, userfriendly delivery vehicle for delivering anti-hiv proteins and grft for hiv prevention. 2 | methods 2.1 | griffithsin production griffithsin (grft) was produced in e. coli as previously described [44] and detailed in supporting information. protein concentrations were determined from sequence-based calculated molar extinction coefficients at 280 nm (http://web.expa sy.org/protparam). fluorescently labelled grft was prepared using the alexa fluor â 610 succinimidyl ester (thermofisher scientific) according to the manufacturer's specifications. in brief, 150 lm grft in 20 mm sodium phosphate (ph 7.0) buffer was reacted with a five-fold molar excess of af610 dye for 2 hours. an excess of unreacted af610 dye was removed by dialysis of the labelled protein against buffer (20 mm sodium phosphate, ph 7.0) for at least 24 hours in the dark. silk fibroin (sf) was extracted from bombyx mori silkworm cocoons as described previously [45] and detailed in supporting information. solutions of grft were prepared in 20 mm sodium phosphate buffer and combined with the aqueous silk solution to produce mixtures containing 2.5% (wt./vol.) sf and a final concentration of 1 mg/ml (approximately 68.1 lm) grft or 0.5 mg/ml af610-grft. sets of 1.0 ml solution aliquots were pipetted into sterile 24-well plates, frozen at à 80°c and lyophilized. completely formulated discs were retrieved from the plates with sterile tweezers and applied to test subjects without further alteration. the composition of the final sf-grft discs was selected through iterative testing (supporting information) to produce the platform for delivery into mucosal sites of rhesus macaques that are capable of dissolving within 1 hour or less in the vaginal lumen and quickly releasing inhibitory concentrations of grft into the vaginal and rectal compartments. the silk discs were prepared to retain sufficient mechanical robustness to allow handling and successful placement within these compartments and have an ability to package high concentration of grft (1 mg per disc) for adequate hiv protection in vivo. a total of 22 female rhesus macaques housed at the california national primate center (uc davis, davis, ca) were enrolled in the study as approved by the institutional animal care and use committee (protocol #19743). since the study included vaginal tissue analysis, only female macaques of reproductive age were included with no history of antibiotic treatment in the last 6 months. animals were randomly assigned to three experimental groups: control group macaques without any silk discs (n = 4), sf control group macaques receiving empty silk (n = 7) and sf-grft group macaques receiving silk disc formulation containing griffithsin (n = 11). first, pre-silk swabs were collected at baseline. atraumatic placement of silk discs was performed using a speculum in the vaginal tract and an anoscope in the rectal tract. silk discs were placed in the vagina canal against the cervical os and in the rectal tract approximately 6 cm from the anus. animals were sedated in ventral recumbency for an additional 2 hours (or 1 hour for initial testing) following placement of silk discs to allow for complete dissociation of silk material. after 2 hours, vaginal and rectal secretions at the site of device placement were then collected using a weck-cel spear (beaver visitec). pre-and post-silk placement swabs were used for analysis of vaginal and rectal microbiota, as well as quantitation of grft in fluids. after collection of swabs, vaginal biopsies of approximately 1x3 mm size were obtained using a kevorkian-younge biopsy tool (sklar instruments), whereas pinch rectal biopsies were obtained using an endoscope. biopsies were subsequently processed for use in cytokine measurements and ex vivo viral challenge. the study was conducted between february 2018 and january 2019. to measure grft levels in macaque vaginal and rectal fluids, elisa was performed as previously described [46, 47] and detailed in supporting information. readings from the standard curve in each assay were fit to a four-parameter logistic (4pl) curve and macaque fluid sample concentrations were calculated relative to this fitted curve, with concentrations subsequently multiplied by the appropriate dilution factor. rhesus macaque vaginal/rectal tissue samples and human cervical/colorectal tissues were sectioned into 2 to 3 mm 3 tissue explants comprising both epithelial and muscularis mucosae as described previously [48, 49] . tissue explants were maintained with complete high glucose dmem (containing 10% fetal bovine serum, 2 mm l-glutamine, 100 u/ml of penicillin, 100 lg/ml of streptomycin, 80 mg/ml of gentamicin and 2.5lg/ml of amphotericin b) at 37°c with 5% co 2 . macaque explants: non-polarized vaginal and rectal explants from rhesus macaque tissues were challenged with shiv sf162p3 (10 3 tcid 50 /ml) in the 96 u-bottom well plates for two hours. unchallenged explants served as negative controls. vaginal explants were transferred to a fresh tissue culture and colorectal explants were then transferred onto gelfoam rafts (welbeck pharmaceuticals, uk). explants were cultured for 15 days in the absence of sf-grft. approximately 50% of the supernatants was harvested every 2 to 3 days and replaced with fresh media. supernatants were used for analysis of p27 antigen concentration in culture supernatants at each harvest day by elisa (siv p27 elisa, zeptometrix corporation, buffalo, ny). human explants: surgically resected specimens of human ecto-cervical and colorectal tissues were collected at st. mary's hospital, imperial college healthcare nhs trust, london, uk. all tissues were collected after receiving signed informed consent from all patients through the imperial college healthcare tissue bank approved by research ethics committee wales (iras 17/wa/0161). all patients were hivnegative. non-polarized human tissue explant assays were conducted as described previously [22] , but with modifications to include the use of human cervical and seminal fluids as described in supporting information. sf-grft discs were dissolved in pbs to a concentration of 3 lm and then diluted at required concentrations with tissue culture media for activity testing. ecto-cervical and colorectal explants were incubated with 25% of synthetic cervical mucous (cm) [50] and then with an equal volume of dissolved sf-grft at four different concentrations. explants were then challenged with hiv-1 yu.2 (10 3 tcid 50 /ml) for 2 h at 37°c. virus was pre-treated with an equal volume of 25% seminal fluid (sm) prior to addition to tissue explants exposed to grft. after 2 hours of incubation, explants were washed with pbs and transferred to fresh plates and cultured as described above for macaque explants. the extent of virus replication in tissue explants was determined by measuring the p24 antigen concentration in supernatants (innotest hiv antigen mab elisa, fujirebio europe, belgium). the percentage of inhibition was normalized relative to the p24 values obtained at day 15 of culture for explants not previously exposed to virus (0% infectivity) and for explants infected with virus in the absence of compound (100% infectivity). full-length, replication-competent proviral r5-tropic clade b hiv-1 clone, pyu2 [51, 52] was provided by the nih aids research & reference reagent program (http://www.aidsrea gent.org/). the plasmid was transfected into 293ft cells and the virus was expanded in activated pbmcs for 11 days [53] . viral stock of shiv sf162p3 was expanded in rhesus macaque primary spleen-derived t cells as described in supporting information. tcid 50 was equivalent to 8.1 x 10 8 copies of viral rna per ml of siv gag p27and used for the viral infection of tissue explants ex vivo. cytokines were measured in culture supernatant from unchallenged vaginal and rectal macaque explants after 24 hours of culture. a magnetic multiplex bead immunoassay (r&d systems, minneapolis, mn) was used to detect mcp-1, mip-1b, rantes, ip-10, egf, gm-csf, ifn-c, il-1b, il-1ra, il-2, il-4, il-5, il-6, il-8, il-10, il-15, il-17 and vegf-a on a luminex 200 system (bio-rad, hercules, ca). cytokine levels were normalized against total protein content as measured by a bca protein assay (bio-rad, hercules, ca). silk fibroin discs containing af610-grft were placed for 1 hour in the vaginal and rectal tracts of rhesus macaques. vaginal and rectal tissues were collected and embedded in optimal cutting temperature (oct) compound by snap freezing in isopentane. tissue blocks were stored at à80˚c. tissue sections were fixed in 4% paraformaldehyde (pfa) for 20 minutes and subsequently stained with 4 0 ,6-diamidino-2-phenylindole (dapi) solution. slides were mounted on prolong gold antifade mounting media (thermo fisher) and dried for 24 hours prior to imaging. confocal z-stacks were captured using a leica sp8 sted 3x confocal microscope (leica microsystems, germany) with a white light laser. a 10x and 63x/1.4na oil immersion objective and maximal image size at 1248x1248 pixels were utilized for all acquisitions. z-stacks were performed with a 0.3 lmol/l step size at 1.25x capturing full thickness of each tissue section. vaginal and rectal microbiota were assessed using 16s rrna sequencing as previously reported [54] and described in supporting information. the library was quantified using qpcr followed by 300-bp paired-end sequencing using an illumina miseq instrument (illumina) in the genome center dna technologies core, university of california, davis. demultiplexing, removal of chimeras, rarefication and quality filter of low-abundance sequencing reads were performed by the uc davis host microbe systems biology core facility. analysis of alpha and beta-diversity was performed on r software (vienna, austria). the sequences and metadata reported in this paper have been deposited in ncbi bioproject at https:// www.ncbi.nlm.nih.gov/bioproject (accession: prjna601500). cytokine concentrations and ic 50 values were calculated from sigmoid curve-fits (prism, graphpad). all data presented fulfil the criterion of r 2 > 0.7. ratios between cytokines concentrations in explant cultures from animals dosed with sf or sf-grft discs and cytokines levels from untreated control animals were established. cytokine ratios and ic 50 values were statistically compared using unpaired t test and p values. we compared microbial alpha diversity using observed, chao1, ace, shannon, simpson index as described in the vegan package [55] on r software. we compared beta diversity using the bray-curtis dissimilarity and weighted unifrac, and used pcoa for ordination and clustering with ellipses representing 95% confidence. a permutational multivariate analysis of variance (permanova) was used to test for significant categorical differences across sample time, site, treatment and interactions between time x site, time x treatment, site x treatment and time x site x treatment. the dose-response inhibitory assay was statistically analysed using a two-way anova to test for the effect of biological fluids across various drug concentration. treatment conditions were considered significant at p < 0.05. we developed methodology for generating silk fibroin discs and optimized conditions for encapsulating them with anti-hiv agents for sustained release [22, 27] . in this study, we enhanced the silk fibroin delivery discs with the higher payload of inhibitors. through iterative formulation and testing (table s1, figure s1 ), it was determined that a 1.0 ml volume of 2.5% (wt./vol.) silk fibroin was sufficient to encapsulate 1 mg of grft and generate a silk fibroin biomaterial that was robust to mechanical handling and placement within the vaginal and rectal mucosa in vivo. these sf-grft discs were approximately 1.4 cm diameter with 0.6 cm height (figure s2a ). the morphology of the sf-grft and sf-only discs was evaluated using scanning electron microscopy (sem) and no significant changes were observed in the porous architecture of the discs with grft as compared to the discs without grft ( figure s2b ,c), consistent with our previous observations [22] . however, the mass ratio of grft:sf in the discs is 60x greater in this study, suggesting that the porous micro-architecture of sf is not perturbed by higher loading of grft. ft-ir (fourier transform-infrared) spectroscopic analysis of the protein secondary structural content of the sf-only and sf-grft discs revealed negligible differences, which were dominated by random coil (approximately 35%), with only approximately 23% b-sheet content ( figure s2d) , consistent with the observed high solubility of the discs and their rapid dissolution in fluid ( figure s1 ). sf discs can be produced with a "nonmedicalized" appearance for delivery of grft or other anti-hiv drugs ( figure s2e ). to determine the capacity of grft-encapsulated sf discs for grft release and efficacy of hiv protection, we examined anti-hiv activity of sf-grft discs in human cervical and colorectal tissue explants ex vivo by measuring their susceptibility to hiv challenge. tissue explants treated with sf-grft (containing different concentrations of grft) showed a dose-dependent protection against hiv challenge. the hiv inhibitory capacity was highest at 0.1 lm grft (approximately 1.47 lg/ml), reaching >80% inhibition of hiv infection in both colorectal and cervical explants. since proteinaceous arvs can be prone to changes in oxidation, hydrolysis and biological activity when exposed to fluids in the body, we evaluated grft activity by incubating human cervical and colorectal tissue explants with either cervical mucous (cm) or seminal fluid (sm) prior to a viral challenge. in cervical explants exposed to sm, grft had marginally lower inhibitory capacity following treatment with 0.01 lm grft ( figure 1a ). colorectal explants exposed to cm had modestly lower inhibitory capacity following treatment with 0.01 lm grft ( figure 1b) . these data suggest that the release of grft from sf vehicle and its anti-hiv activity prevailed in the cervical and colon tissues even in the presence of biological fluids. we measured the capacity of the sf-grft vehicle to dispense grft at the mucosal site in vivo, which requires adherence and quick dissolution of the silk scaffold. to assess sf adherence and dissemination of grft at mucosal sites, sf-grft discs were placed in rhesus macaques at the following sites: the vaginal tract (against the cervical os) and the rectal tract (approximately 6 cm proximally) for two hours under sedation. however, mean concentrations of grft in both vaginal and rectal compartments were found to be significantly higher (more than 1,100x for vaginal and 880x for rectal tissues) than the 100x ec 90 value that was previously reported for topical grft dosing in nhps prior to vaginal challenge with shiv sf162 [36] . furthermore, grft detected in vivo was 558-fold higher than in our human cervical explant experiments and 436-fold higher than the maximum inhibition concentration in human colorectal explants (figure 1) . to examine the sf-grft adherence to mucosal tissue, we prepared fluorescently labelled af610-grft in sf discs. vaginal and rectal tissue samples from rhesus macaques were collected one hour following in vivo placement of sf-grft discs. fluorescent imaging of tissue sections by confocal microscopy revealed that grft was well-dispersed along the vaginal epithelium with detectable amounts of grft in the lamina propria (yellow arrow, figure 2b) . a closer view of the vaginal ( figure 2c ) and rectal tissues ( figure 2d ) demonstrated that grft was detectable in both compartments and accumulated largely in the apical lining of the epithelium. our data suggest that sf-grft had effective tissue permeability for a high payload grft delivery to mucosal tissues. the safety profile of sf-grft inserts in rhesus macaques in vivo was assessed by histopathologic evaluation of mucosal tissues and the expression of inflammatory cytokines. to compare changes associated with sf disc placement, we collected tissue samples at the site of sf insert placement and away from the site of sf for each animal. "at the site" refers to the location in which the sf disc was placed, against the cervical os. "away from the site" refers to the site which the biopsy was taken within the vaginal canal away from the cervical os and as close to the vulva as possible, serving as the internal negative control. this approach helped overcome any bias due to an animalto-animal variation within the study, particularly for variations of menstrual cycle stage at time of biopsy. histopathological analysis of vaginal biopsies revealed no significant difference in mucosal epithelium or leucocytic infiltrate between mucosal samples collected at the site of sf disc placement compared to those collected away from the site ( figure 3a, top row) . similar observations were noted in the rectal biopsies following sf placement. all rectal samples were within the normal range as characterized by a low number of mononuclear and neutrophilic infiltrates in the lamina propria, an average crypt length and intact mucosal epithelium ( figure 3a , bottom row). we also determined the levels of inflammatory cytokines in culture supernatants from tissue explants using a multiplex bead immunoassay. exposure to sf alone in the vaginal mucosa induced marginal increases in il-1b and il-1ra, and induced a slight decrease in ip-10 ( figure 3b ), but this pattern of cytokine production was not seen in sf-grft placements which exhibited no changes in the levels of inflammatory cytokines as compared to the controls ( figure 3c ). in addition, sf (figure 3d ) and sf-grft placement ( figure 3e ) in the rectal tract exhibited no changes in cytokine production. we sought to determine the efficacy of in vivo sf-delivered grft to vaginal and rectal mucosal tissues in conferring protection against ex vivo shiv challenge. macaques received either sf (silk) or sf-grft (silk + grft) inserts, followed by a 2-hour incubation period. vaginal and rectal biopsies were collected "at the site" of silk placement and ex vivo challenged with shiv sf162 within one hour of tissue collection. vaginal tissue explants from animals dosed with grft in vivo exhibited effective inhibition of shiv infection ( figure 4a ) which persisted over a 15-day time course (figure 4b ). in contrast, the controls and vaginal explants exposed to sf discs alone were readily infected with shiv as indicated by the high levels of viral p27 levels. similar patterns in shiv inhibition were observed in rectal explants following in vivo exposure to grft discs ( figure 4c ) which lasted over a 15-day time course (figure 4d) . these results highlight the capacity of sf-grft discs to protect both vaginal and rectal compartments against the viral infection. the resident microbiome in the vaginal and rectal tracts is closely associated with the mucosal immune system and may influence susceptibility to hiv infection as previously reported [56, 57] . in addition, the microbial communities at the mucosal sites may also impact the efficacy of topical hiv drugs [58] . we assessed changes in the microbiota composition from the vaginal and rectal samples prior to and following the placement of sf-grft discs by performing bacterial 16s rrna sequencing. no significant changes were observed in the alpha diversity of vaginal microbiota due to the placement of sf or sf-grft discs ( figure 5a) . a comparison of microbial communities at the phyla level showed no significant differences between pre-sf placement and post-sf placement samples ( figure 5b ). weighted pcoa analysis revealed clustering of most samples and slight differences associated with animal-toanimal variation in vaginal microbiomes ( figure 5c ). similarly, no changes were observed in bacterial alpha diversity from rectal samples after sf or sf-grft placement ( figure 5d ). taxonomic analysis of bacterial phyla revealed minor variations in rectal microbiota before and after silk placement likely due to sampling variability. we detected a noticeable variation in relative abundances of epsilonbacteraeota belonging to the helicobacter genus in some of the macaques ( figure 5e ). overall, the increase in relative abundances of epsilonbacteraeota was not a feature of the pcoa analysis, falling within 95% confidence of the weighted unifrac measures ( figure 5f ). bacterial communities in the pre-placement rectal samples clustered with those in post-placement rectal samples independent of sf or grft, indicating that sf and grft did not alter the microbiome in the rectal tract. lastly, we performed a perma-nova to test for categorical variables associated with all vaginal and rectal samples, and no statistical differences were in relation to the time (pre-and post-placement), treatment (ctrl, silk, silk + grft), and interactions between time 9 site (vaginal, rectal), time 9 treatment, site 9 treatment and time 9 site 9 treatment ( figure s3 ). collectively, our data suggest that the safety profile and capacity of sf-grft discs to inhibit shiv infection are independent of microbiome variation in the vaginal and rectal tracts. we report the efficacy of a silk fibroin-based platform for the rapid and sustained mucosal delivery of anti-hiv griffithsin into the vaginal and rectal tissues in vivo and protection against shiv and hiv ex vivo. this non-dribbling, solid delivery platform has great promise for prep due to its efficient mucosal delivery, practicality of its storage and transportation and capacity for a high payload of anti-viral drugs and proteins. oral prep, the most effective hiv prevention method, requires regular intake of hiv medications, which has side effects in hiv-negative people that are not fully realized [59] . since most hiv infections are sexually transmitted, topical prep application is being tested using gels or vaginal rings. there is a need for rapid release of high amounts of antiretroviral molecules into the vaginal and rectal mucosal sites from a user-friendly and easily applicable platform without the need for additional medical assistance. in addition, the formulation should be stable without refrigeration for a prolonged period of time and be easily transported without any leakage. user adherence for hiv protection can be increased by a quick-dissolve film or suppository form that can be easily inserted peri-coitally vaginally or rectally, without a daily dosing of anti-viral medications. it is of great importance that end users such as women and at high-risk adolescents are consulted for their needs and preferences while developing and designing topical prep delivery platforms to achieve better adherence. we sought to identify a delivery platform that can readily package a range of therapeutics, from small molecules to small proteins in sufficient concentrations and be applied to mucosal surfaces for mucosal adherence and rapid drug delivery. it is also desirable to have a delivery vehicle that is itself non-inflammatory and that has sufficient mechanical strength to enable placement in the vaginal or rectal mucosal site. our study shows that silk fibroin discs meet most of these criteria to serve as an effective delivery vehicle for topical application of arvs. the use of insertable gels containing arvs in clinical trials led to moderate success in the caprisa 004 study [7] , and disappointing outcomes in the voice [6] and facts 001 trials, largely due to inconsistent adherence, especially among young women [6, 7, 9, 10, 60] . more recently, vaginal rings or infusions are being studied for the delivery of multiple anti-viral drugs, bnabs or the viral entry inhibitor, 5p12-rantes [61] [62] [63] [64] [65] [66] [67] [68] . however, infusions will require the engagement of medical assistance and may not be easily accessible to women in different settings and geographic locations. vaginal rings have been explored for sustained release, but are not typically used for rapid, on-demand delivery of therapeutics [65] . placement of vaginal rings and infusions may not be easily accessible to women in different settings and geographical locations. a better adoption of a single prevention device is needed for increased acceptance and adherence among young women users [69] [70] [71] . although different studies show some variety among preferences by women, young women in general favoured inserts/films when choosing among several delivery forms because of the decision power for timing of the use and discreteness of the inserts [69] . further development of a vaginal or rectal delivery platform is needed for better adoption, acceptance and adherence by young women as end users [69] [70] [71] . silk discs have the possibility to provide an inexpensive, over-the-counter, on-demand device option to increase user acceptability and adherence. silk discs can be formulated in different shapes and colours for adolescents and young adults ( figure s2e ). based on our findings, quick dissolve silk fibroin discs can be formulated with a single or a combination of hiv drugs or inhibitors at varying concentrations for rapid in vivo delivery into both vaginal/rectal mucosa for hiv prevention. while silk discs are in preclinical studies, grft is being tested currently in two clinical trials [41, 42] . the administration of grft in mice was shown to be safe [37] and it has shown potency against hiv and shiv in rhesus macaque models [36, 40] . we previously reported that sf formulation provides marked stability for grft and maintains its biological activity for one year even at high temperatures (50°c) [22] . in an independent study of grft formulated in carrageenan, it was shown that grft was stable for 6 months at 40°c [ 72] . thus, sf formulations are suitable for ensuring stability and long-term storage of grft, and likely for antiretroviral drugs in geographic locations with varying temperatures. we report that grft can be formulated into quick dissolve sf discs for rapid in vivo delivery into both vaginal/rectal compartments with high mucosal levels and provide protection against shiv infection ex vivo. a fluorophoreconjugated grft was visualized on the apical lining of stratified squamous epithelium of the vaginal tract and columnar epithelium of the rectal tract, suggesting extensive coverage across the mucosal surfaces. lack of any significant inflammatory changes in silk-grft inserted mucosal tissues validate the high safety profiles of sf discs. our findings of grft effects in rhesus macaques are in agreement with grft testing results in mice, which showed no changes in histopathology, blood chemistry or cbc parameters following mucosal or systemic exposure to grft [37] . mucosal inflammation and the composition of local microbiota influence the transmission of hiv infection [73, 74] . we investigated whether animal-to-animal variation in gut microbiome composition among rhesus macaques could be linked to the magnitude of viral infectivity in mucosal tissues. analysis of the composition of vaginal and rectal microbiota, cytokine production associated with sf and sf-grft discs, and levels of viral infection following ex vivo shiv challenge showed lack of any correlations, suggesting that sf-grft discs provided viral protection despite individual variation in vaginal and rectal microbiomes. in addition, placement of sf discs did not alter the microbiome of either compartment, as expected for a short 2-hour time frame. our findings are in the agreement with recent studies of grft in other formulations which showed minimal to no toxicity and marginal effects on the rectal microbiome of rhesus macaques [39] . although the composition of the vaginal microbiome differs between non-human primates and people, some macaques exhibit a vaginal microbial signature similar to that of human bacterial vaginosis, promoting susceptibility to viral infection [75] . though we did not observe changes in the vaginal microbiome associated with sf discs in rhesus macaques, sf formulations have the capacity to encapsulate bacterial strains and could potentially be leveraged to package probiotics for mucosal delivery [76] . the safety profiles and versatility of sf formulations highlight sf-grft as an optimal topical prep candidate for hiv prevention. this study highlights the use of sf as a suitable candidate for prep that holds potential for better adoption and effective hiv prevention among women as end users. sf-grft provided complete protection of human cervical and colorectal tissues against hiv infection ex vivo and remained active against hiv even in the presence of biological fluids. in rhesus macaques, sf-grft discs protected against ex vivo shiv challenge in the vaginal and rectal compartments. sf inserts neither caused cell toxicity nor changes in the vaginal and rectal microbiome. the sf-grft platform warrants further development for in vivo mucoadhesive delivery of a single or combination of anti-viral drugs and proteins, inhibitors of stds and biologically active small molecules. the versatility of sf formulations holds the potential for creating products that protect against hiv as well as enhance mucosal health. the authors declare that they have no competing interests. additional information may be found under the supporting information tab for this article. figure s1 . optimization of the sf disc formulation. figure s2 . characterization of grft-loaded sf discs. figure s3 . permanova results from vaginal and rectal microbial analysis. table s1 . composition of sf discs initially tested data s1. methods and materials. focus on key populations in national hiv strategic plans in the african region effectiveness and safety of oral hiv preexposure prophylaxis for all populations a phase 1 trial to assess the safety, acceptability, pharmacokinetics, and pharmacodynamics of a novel dapivirine vaginal film development and characterization of a vaginal film containing dapivirine, a non-nucleoside reverse transcriptase inhibitor (nnrti), for prevention of hiv-1 sexual transmission tenofovir-based preexposure prophylaxis for hiv infection among african women effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of hiv infection in women safety, acceptability and adherence of dapivirine vaginal ring in a microbicide clinical trial conducted in multiple countries in sub-saharan africa use of a vaginal ring containing dapivirine for hiv-1 prevention in women mtn-025/hope study team. high adherence and sustained impact on hiv-1 incidence: final results of an open-label extension trial of the dapivirine vaginal ring 10th ias conference on hiv science evaluation of rapidly disintegrating vaginal tablets of tenofovir, emtricitabine and their combination for hiv-1 prevention evaluating cabotegravir/rilpivirine long-acting, injectable in the treatment of hiv infection: emerging data and therapeutic potential safety, pharmacokinetics, and immunological activities of multiple intravenous or subcutaneous doses of an anti-hiv monoclonal antibody, vrc01, administered to hiv-uninfected adults: results of a phase 1 randomized trial the promise and pitfalls of long-acting injectable agents for hiv prevention preventing hiv in women-still trying to find their voice methodological challenges in biomedical hiv prevention trials incorporation of proteinase inhibitors into silk-based delivery devices for enhanced control of degradation and drug release modulation of vincristine and doxorubicin binding and release from silk films a review of the emerging role of silk for the treatment of the eye biodegradation of silk biomaterials silk microspheres for encapsulation and controlled release stabilization and sustained release of hiv inhibitors by encapsulation in silk fibroin disks silk hydrogels for sustained ocular delivery of anti-vascular endothelial growth factor (anti-vegf) therapeutics silk fibroin as edible coating for perishable food preservation the biomedical use of silk: past, present, future seri surgical scaffold in 2-stage breast reconstruction: 2-year data from a prospective, multicenter trial sustained release silk fibroin discs: antibody and protein delivery for hiv prevention silk-based biomaterials for sustained drug delivery an antiviral lectin with outstanding therapeutic potential griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae griffithsin has antiviral activity against hepatitis c virus griffithsin protects mice from genital herpes by preventing cell-to-cell spread griffithsin inhibits japanese encephalitis virus infection in vitro and in vivo griffithsin and carrageenan combination to target herpes simplex virus 2 and human papillomavirus griffithsin carrageenan fast dissolving inserts prevent shiv hsv-2 and hpv infections in vivo studies in a murine model confirm the safety of griffithsin and advocate its further development as a microbicide targeting hiv-1 and other enveloped viruses investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate impact of the griffithsin anti-hiv microbicide and placebo gels on the rectal mucosal proteome and microbiome in non-human primates impact of q-griffithsin anti-hiv microbicide gel in non-human primates: in situ analyses of epithelial and immune cell markers in rectal mucosa university of louisville, ky: national institute of allergy and infectious disease a phase 1 trial to evaluate the safety, pharmacokinetics (pk) and pharmacodynamics (pd) of pc-6500 (griffithsin [grft] in a carrageenan gel) in healthy women adherence support approaches in biomedical hiv prevention trials: experiences, insights and future directions from four multisite prevention trials the griffithsin dimer is required for high-potency inhibition of hiv-1: evidence for manipulation of the structure of gp120 as part of the griffithsin dimer mechanism materials fabrication from bombyx mori silk fibroin the role of individual carbohydrate-binding sites in the function of the potent anti-hiv lectin griffithsin isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp blockade of attachment and fusion receptors inhibits hiv-1 infection of human cervical tissue reverse transcriptase inhibitors as potential colorectal microbicides. antimicrob agents chemother synthetic cervical mucus formulation complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replication-competent and -defective viral genomes enhancement of human immunodeficiency virus type 1 infection by the cc-chemokine rantes is independent of the mechanism of virus-cell fusion subclinical cytomegalovirus infection is associated with altered host immunity, gut microbiota, and vaccine responses evaluation of the association between the concentrations of key vaginal bacteria and the increased risk of hiv acquisition in african women from five cohorts: a nested case-control study vaginal bacteria modify hiv tenofovir microbicide efficacy in african women topical inserts: a versatile delivery form for hiv prevention tenofovir 1% vaginal gel for prevention of hiv-1 infection in women in south africa (facts-001): a phase 3, randomised, double-blind, placebo-controlled trial phase i trial of pod-intravaginal rings delivering antiretroviral agents for hiv-1 prevention: rectal drug exposure from vaginal dosing with tenofovir disoproxil fumarate, emtricitabine, and maraviroc broadly neutralizing anti-hiv-1 monoclonal antibodies in the clinic ultra-long-acting tunable biodegradable and removable controlled release implants for drug delivery pharmaceutical approaches to hiv treatment and prevention. advanced therapeutics pharmacokinetics and preliminary safety of pod-intravaginal rings delivering the monoclonal antibody vrc01-n for hiv prophylaxis in a macaque model development and pharmacokinetics of a combination vaginal ring for sustained release of dapivirine and the protein microbicide 5p12-rantes vaginal rings with exposed cores for sustained delivery of the hiv ccr5 inhibitor 5p12-rantes insights for implementation science from 2 multiphased studies with end-users of potential multipurpose prevention technology and hiv prevention products stated product formulation preferences for hiv pre-exposure prophylaxis among women in the voice-d (mtn-003d) study perspectives from young south african and zimbabwean women on attributes of four (placebo) vaginal microbicide delivery forms development of a vaginal fast-dissolving insert combining griffithsin and carrageenan for potential use against sexually transmitted infections lactobacillus-deficient cervicovaginal bacterial communities are associated with increased hiv acquisition in young south african women vaginal microbiome affects hiv risk identification of rhesus macaque genital microbiota by 16s pyrosequencing shows similarities to human bacterial vaginosis: implications for use as an animal model for hiv vaginal infection development of silk fibroin-based beads for immobilized cell fermentations this study was supported by the national institutes of health grants r01ai112011 and r01ai123105 and p51 od011107. krc was supported by nih grant f30 ai150462. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. we thank wilhelm von morgenland and miles christensen at the california national primate research center for technical support and coordination of animal studies. we extend our gratitude to shuang hu for assisting in the tissue explant study. we thank the department of surgery and cancer and the department of obstetrics and gynaecology at st. mary's hospital, imperial college for their assistance in obtaining human tissue. we thank kennedy nguyen and the u.c. merced imaging and microscopy facility (imf) for assistance with sem imaging and the iddrc biological analysis core for luminex plate readers. we thank matthew rolston at the u.c. davis host microbe systems biology core for microbial dna preparations and sequencing analysis. we thank david spencer at the oregon national primate research center for the preparation of the shiv virus stock. key: cord-261382-cyty5noi authors: goffinet, christine title: cellular antiviral factors that target particle infectivity of hiv-1 date: 2016-05-17 journal: curr hiv res doi: 10.2174/1570162x14666151216145521 sha: doc_id: 261382 cord_uid: cyty5noi background: in the past decade, the identification and characterization of antiviral genes with the ability to interfere with virus replication has established cell-intrinsic innate immunity as a third line of antiviral defense in addition to adaptive and classical innate immunity. understanding how cellular factors have evolved to inhibit hiv-1 reveals particularly vulnerable points of the viral replication cycle. many, but not all, antiviral proteins share type i interferon-upregulated expression and sensitivity to viral counteraction or evasion measures. whereas well-established restriction factors interfere with early post-entry steps and release of hiv-1, recent research has revealed a diverse set of proteins that reduce the infectious quality of released particles using individual, to date poorly understood modes of action. these include induction of paucity of mature glycoproteins in nascent virions or self-incorporation into the virus particle, resulting in poor infectiousness of the virion and impaired spread of the infection. conclusion: a better understanding of these newly discovered antiviral factors may open new avenues towards the design of drugs that repress the spread of viruses whose genomes have already integrated. like all intracellularly replicating pathogens, hiv-1 undergoes extensive interplay with cellular machineries and benefits from interactions with essential cofactors which enable and support hiv-1 replication. well-known examples include the hiv-1 receptor cd4, chemokine coreceptors ccr5 and cxcr4, the tat-interacting cyclint1 that enhances processivity of the rna polymerase ii during ltr-mediated viral transcription and the nuclear mrna export factor crm1. on the contrary, eukaryotic cells dispose of a set of antiviral genes, whose expression is often induced or enhanced by type i interferons (ifns), and whose products can effectively interfere with distinct steps of the hiv-1 replication cycle. the impacts of anti-hiv-1 factors acting early post-entry and during virus release include samhd1, apobec3g, mx2 and tetherin, respectively, and are discussed extensively elsewhere. following integration of the proviral dna into the host cell´s chromosome and ltr-driven transcription, newly synthesized viral proteins and the viral genomic rna are transported to the plasma membrane in a coordinated fashion, followed by particle assembly and egress. the composition of these particles is tightly controlled and requires the proper function of viral structural, enzymatic, and accessory proteins, but also cellular factors. a budded virion is confined by a plasma membrane-derived lipid bilayer containing approximately 7-14 trimers of envelope *address correspondence to this author at the institute of experimental virology, twincore, feodor-lynen-str. 7, d-30625 hannover, germany; tel: +49-511-220027-198; fax: +49-511-220027-139; e-mail: christine.goffinet@twincore.de glycoproteins [1, 2] . gag, besides constituting the most abundant protein within virions, is essential in recruiting the viral genomic rnas and other essential proteins, including protease, integrase and reverse transcriptase. of note, a ribosomal frameshifting mechanism ensures incorporation of these enzymatic proteins which are expressed from a gag-pol polyprotein at a low frequency of 5%. incorporated accessory proteins include vpr, vif and nef. a final, protease-mediated series of gag and gag-pol cleavage and rearrangement events renders immature virions fully infectious, resulting in the virons´ interior typical morphological transition from an electron-dense ring lining the surrounding membrane to a cone-shaped appearance in electron microscopy. these rearrangements furthermore trigger the clustering of the sparse env trimers on the virus surface, an event that might contribute to virus maturation and infectiousness [3] . importantly, the right configuration of particles is crucial for assuring a high infectious quality, which is reflected by their ability to efficiently infect new target cells. prevention of apobec3g incorporation by hiv-1 vif provides a compelling example for the fact that modulation of the (cellular or viral) protein composition of nascent virions can have dramatic (positive or negative) effects on the infectiousness of hiv-1 particles. alternatively, alteration of the virus membrane fluidity, restriction of the accessibility of the hiv-1 env glycoprotein to the hiv-1 receptor/coreceptor complex by steric hindrance through an incorporated protein or inhibition of the maturation-induced clustering of hiv-1 env trimers could represent mechanisms of cellular defense that target particle infectivity. despite these theoretical possibilities, until recently no antiviral protein was known that directly reduces particle infectivity. within the cytokine family of interferons, type i and type iii ifns exert antiviral properties, whereas type ii ifns have been considered more immunomodulatory. whereas type iii ifns signal via a specific type iii ifn receptor, which is probably not expressed at high levels on primary hiv-1 target cells in vivo [4] [5] [6] , type i ifns robustly induce upregulated expression of antiviral ifn-stimulated genes (isgs) in cultured primary hiv-1 target cells, including cd4+ t-cells and macrophages. isgs constitute a large group of several hundred genes, from which only a few of them have been analyzed in-depth for potential antiviral activity. this review highlights recent research that identifies processes safeguarding hiv-1 ´s particle infectivity being highly vulnerable. particle infectivity is negatively modulated by a diverse set of cellular proteins, comprising isgs and non-isgs, each of them with an individual mode of action and different sensitivity to viral counteracting strategies. 90k (according to its appearance as a 90 kda species in sds-page)/lectin galectin soluble 3 binding protein (lgals3bp) is a secretory glycoprotein [7] . it was initially identified as a tumor antigen which is upregulated in the context of breast [8] and lung [9] carcinoma. physiological functions of extracellular 90k comprise modulation of cell migration and adhesion by interaction with multiple cell surface receptors, including galectin-3, and modulation of the activity of immune cells [10] . in healthy individuals, 90k protein reaches concentrations up to the µg/ml range in diverse body fluids. since it shares mrna upregulation in cd4+ t-cells of hiv-1 infected individuals [11] and type i ifn-induced expression [10, 12, 13] with established restriction factors of hiv-1, it was included in a functional screen of candidate isgs for antiviral activity (goffinet, unpublished data). 90k acts antiviral against hiv-1 by reducing the particle infectivity of newly produced hiv-1 virions [14] . this defect is accompanied by impaired incorporation of hiv-1 env glycoproteins into virus particles, probably explaining the reduced infectious quality of de novo-synthesized virions, and defective processing of the hiv-1 env gp160 precursor into the mature proteins gp120 and gp41. addition of exogenous, soluble 90k to the producer cell culture fails to inhibit particle infectivity, suggesting that 90k exerts its antiviral effect intracellularly and that outside-in signaling of extracellular 90k is dispensable for its antiviral function. analysis of a set of modified variants of 90k revealed that the two central protein-binding domains btb-poz and ivr are required and sufficient for 90k´s antiviral activity, since deletion of the n-terminal scavenger receptor cysteine rich (srcr)-like domain and the c-terminal domain did not abolish antiviral activity. the exact mode of action of 90k remains elusive to date. experimental data argues against a virion incorporation of 90k. given that 90k and hiv-1 env share subcellular localization and posttranslational maturation in the secretory pathway, and that 90k interferes with hiv-1 env incorporation, a model in which 90k targets hiv-1 env, either via direct or indirect interaction, has been favored. however, co-ip experiments failed so far to provide evidence for a detectable interaction of 90k with hiv-1 env. thus, an indirect scenario, in which 90k inhibits the function or expression of a cellular or a viral factor that is required for efficient hiv-1 env incorporation cannot be excluded. although 90k expression induces a processing defect of the hiv-1 env gp160 precursor, resulting in high cell-associated levels of uncleaved polyprotein and only a small fraction of mature gp120 and gp41, it remains unclear whether the processing defect is the direct cause for poor incorporation of mature glycoproteins or whether these two inhibitory actions can be genetically and/or functionally uncoupled. future work is needed to clarify the exact mode of action of 90k. in human target cells of hiv-1, 90k protein expression is highly cell-type specific. it is readily detectable in primary monocyte-derived macrophages [14] . importantly, sirnamediated knockdown of 90k facilitates the spread of hiv-1 in cultures of primary macrophages and increases the particle infectivity of newly produced virions, indicating that endogenous expression levels of 90k contribute to the control of hiv-1 spread in this important hiv-1 target cell type. in striking contrast, 90k protein is undetectable in primary cd4+ t-cells and in all analyzed immortalized tcell lines, even using very sensitive elisa assays and despite type i ifn-mediated upregulation of 90k mrna, suggesting that 90k expression is tightly repressed at the post-transcriptional level in t-cells. given the specific expression of 90k in macrophages within those hiv-1 target cell types that fully support virus replication, prediction of the in vivo importance of 90k is difficult. expression levels of 90k protein in the serum correlate with viral loads, and high 90k expression levels at the viral set-point are predictive of a negative outcome of the infection [15] [16] [17] . although this seems to contradict a protective role of 90k for the host during hiv-1 infection, a positive correlation of isg expression and viral loads has been long-appreciated [11, 18, 19] . this observation raises the possibility that sustained upregulation of type i ifns during the chronic phase of hiv-1 infection indirectly promotes virusbeneficial effects in addition to the direct antiviral effects of type i ifn-induced isgs which may be protective during acute hiv-1 infection. in addition, it is important to take into consideration that levels of soluble 90k may not necessarily reflect the level of the cell-associated, antiviral protein fraction. along this line, potential hiv-1 evasion mechanisms still need to be defined. keeping the density of glycoprotein molecules on the virus surface low is a property of some viruses that is particularly pronounced for hiv-1, and is thought to provide a selective advantage during immune evasion [20] . it is unclear whether the drawback of reduction of virion infectivity, induced by 90k-mediated paucity of env embedded in particles, is outweighed by improved immune escape. more work is warranted to fully understand the impact of 90k on hiv-1 infection. ifitm proteins are small two-pass transmembrane proteins which are located predominantly at the plasma membranes (ifitm1) or at endosomal and lysosomal membranes (ifitm2 and 3). they belong to the cd225/dispanin protein superfamily, which exists throughout the kingdoms, including bacteria, invertebrates and primates [21] . additional family members, including ifitm5, whose expression is restricted to osteoblasts [22] , and 10 additional, recently discovered human ifitm genes [21] probably do not contribute to inhibition of hiv-1 infection or haven´t yet been tested for antiviral activity, respectively. ifitm1, 2 and 3 are almost ubiquitously expressed and their expression is further upregulated by type i ifns [22, 23] . ifitm proteins contribute to regulation of early development, cell adhesion, and control of cell growth [24] . virologists´ interest in ifitm proteins steeply raised upon the first description of their antiviral activity against influenza a virus and flaviviruses, including west nile virus and dengue virus [22] , indicating a broad antiviral activity of these small factors. further targets of ifitm proteins are coronaviruses, hepatitis c virus, ebola virus and vesicular stomatitis virus (vsv) [23, 25, 26] , as well as bunyaviruses [27] and reoviruses [28] . it appears that expression of ifitm proteins in target cells is sufficient for inhibition of these viruses. specifically, ifitm1, 2 and 3 interfere with glycoprotein-dependent fusion of the viral and endosomal membrane, preventing the delivery of viral genomes to the cytoplasm and resulting in trapping of virions in endosomes and lysosomal degradation [22] . strikingly, a minor snp in human ifitm3, rs12252-c, which leads to expression of an nterminally truncated ifitm3 protein, strongly associates with a poor prognosis during pandemic h1n1/09 influenza a infection [29] . these findings highlight the important contribution of ifitm3 in defending humans against viral pathogenic infections and establish ifitm3 as a bona-fide antiviral factor. the impact of ifitm proteins on single-round hiv-1 infection has been discussed controversially. using rnai, an antiviral activity of ifitm3 was found to be unlikely to exist since no inhibition of cell-free hiv-1 was observed in hela cd4 cells which endogenously express ifitm3 [22] . although ifitm proteins scored positive in a large-scale screen for antiviral activity against hiv-1, the detected antiviral effect must probably be attributed to the vsv-gmediated entry that was employed by the vsv-g/hiv-1 pseudotypes that lacked authentic hiv-1 env [30] . modest consequences of ifitm2 and ifitm3 overexpression and absent antiviral activity of ifitm1 have been reported using a blam-vpr-based hiv-1 fusion assay [31, 32] , suggesting that fusion of cell-free hiv-1 is only weakly affected. in contrast to the moderate effect of ifitm proteins in singleround hiv-1 infection, stable expression of individual ifitm proteins in a t-cell line effectively impedes viral spread in cell culture using a replication-competent hiv-1 [31] [32] [33] [34] [35] , implying more potent effector functions of ifitm proteins in a system in which cell-to-cell transmission predominates. indeed, findings from two independent groups recently shed some light into a potential mechanism [35, 36] . whereas ifitm2 or 3 expressed on membranes of target cells fail to exert antiviral activity in the context of cell-tocell transmission, the antiviral capacity is more obvious when ifitm3 is expressed in the infected donor cells that transmit the virus to ifitm-negative target cells. the antiviral effect potentiates when ifitm is expressed on both donor and target cells. specifically, ifitm proteins are incorporated into membranes of nascent virions and thereby decrease their particle infectivity by impairing fusion [35, 36] , apparently not at the expense of env incorporation and also not in an env-dependent manner [35, 36] . the exact mode of action of virus-associated ifitms, whether virion incorporation of ifitm proteins is governed by specific determinants or occurs randomly, and whether ifitm proteins are incorporated in virus particles of other families, remain yet to be investigated. in the context of other viruses, multiple, partially contradicting models have been debated. it has been suggested that ifitm proteins inhibit the fusogenicity of membranes in which they locate. specifically, ifitm3 may restrict influenza a virus entry by restricting viral membrane hemifusion [37] or by blocking the formation of fusion pores at a post-hemifusion stage [38] . an alternative hypothesis says that ifitm3 disrupts cholesterol homeostasis, leading to accumulation of cholesterol in intracellular compartments [39] . alternatively, viral incorporation of ifitm proteins may cause exclusion and inclusion of yet to be defined specific cellular factors with antiviral activity as a consequence. although evidence for direct viral antagonists of ifitm proteins is lacking, hiv-1 may be able to overcome ifitm-imposed antiviral activity, since evasion mutations in vpu and env were reported after passage of hiv-1 in an ifitm1-expressing tcell line [33] . among the accessory genes of primate lentiviruses, nef displays probably the highest degree of multifunctionality. as evidenced by infections with inactivated nef mutations or deletions in rhesus macaques and humans, an intact nef gene is required in vivo for sustained viremia and development of immunodeficiency [40] [41] [42] . although nef does not exert any enzymatic activity, it fulfills a multitude of functions that culminate in alteration of cell signaling and activation by interfering with the cellular vesicular trafficking machinery. association of nef with membranes is governed by its myristoylation; it is also incorporated in virus particles. downregulation of several cell surface receptors, including cd4 and coreceptors, as well as mhc class i and ii receptors and modulation of transcriptional activity of infected t-cells are established functions of hiv-1 nef. another long-appreciated activity of nef is the enhancement of hiv-1 particle infectivity. production of hiv-1 devoid of an intact nef gene results in impaired particle infectivity, ranging from 3 to 40-fold reduced titers, depending on the producer cell type, target cell type and viral isolate [43, 44] . the most pronounced requirement for nef is seen in lymphoid cells. nef-mediated enhancement of particle infectivity requires its expression in the producer cell, is highly conserved within primate lentiviruses [45] and is maintained during disease progression [46] . the fact that nef is incorporated into virions and cleaved by hiv-1 protease during maturation of particles may support a specific function of virus-incorporated nef that manifests itself early during the next round of infection. its membrane association may be sufficient to result in virion packaging, since nef is also found in orthologous retroviruses when co-expressed in the producer cell. recently, the mechanism of nef-mediated infectivity enhancement has been elucidated [47, 48] . using differential mrna sequencing in different producer cell lines that differ in their requirement for nef for optimal hiv-1 infectivity [47] , and mass spectrometry-based approaches [48] , respectively, members of the serine incorporator (serinc) family were identified as potent cellular inhibitors of retrovirion particle infectivity in the absence of nef. serinc proteins are highly conserved, multi-pass transmembrane proteins expressed predominantly at the plasma membrane with poorly elucidated cellular functions besides that they mediate serine incorporation into phosphatidylserine and sphingolipids [49] . serinc5, which is abundantly expressed in pbmcs, and serinc3 were reported to inhibit hiv-1 in the absence of nef in a dosedependent and synergistic fashion that appears to require serinc incorporation in the virion. serinc5 inhibition acts specifically against env-containing particles, since vsv-g pseudotypes were largely insensitive to serinc5 [47, 48] . nef was shown to counteract serinc activity by downregulating it from the cell surface via clathrindependent endocytosis and sequestering it in endosomes, thus precluding their incorporation into virion particles. interestingly, the prototypic retrovirus mlv has evolved its own antagonist (glyco-gag) to counteract serinc5 [47, 48] , underlining the importance of this antiviral factor. interestingly, expression of serinc3 and serinc5 are not induced or stimulated by type i interferons, in contrast to the targets of most other viral antagonists [47] . the antiviral activity of serinc5 appears to be caused predominantly by post-entry defects, since the detectable slight inhibition of virus-cell fusion does probably not fully account for the far more pronounced infectivity decrease imposed by physiological levels of serinc5 expression [47, 48] . future work is needed to fully understand the antiviral mode of action of serinc proteins, and to decipher whether other enveloped viruses are prone to serinc-mediated inhibition, and how exactly nef counteracts serinc proteins. notably, a better understanding of the serinc-imposed antiviral block to infection and the potential nef-serinc interface might be translated into the design of a small antiviral molecule that interrupts nef-serinc interaction and allows full-blown serinc-mediated hiv-1 inhibition. in the past decade, discovery and characterization of antiviral restriction factors has catapulted our understanding on cell-intrinsic immunity. whereas many antiviral factors were found to target early post-entry steps of the hiv-1 replication cycle, recent findings have uncovered the potential of cellular proteins 90k and members of the ifitm and serinc protein family to reduce the particle infectivity of hiv-1. they act by modifying the essential particle composition or by incorporating into nascent virions, respectively. their existence may, at least partially, contribute to the inherently high frequency of uninfectious hiv-1 particles [50] [51] [52] . extensive investigation is required to fully understand their mode of action and respective virally-encoded antagonistic or evasion strategies, and opens up an exciting new area of research, with potential translation into the design of new antiviral treatment strategies. envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/su), is the primary determinant of su content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus distribution and three-dimensional structure of aids virus envelope spikes maturation-dependent hiv-1 surface protein redistribution revealed by fluorescence nanoscopy characterization of the mouse ifn-lambda ligand-receptor system: ifn-lambdas exhibit antitumor activity against b16 melanoma ifn-lambda (ifnlambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo despite ifn-lambda receptor expression, blood immune cells, but not keratinocytes or melanocytes, have an impaired response to type iii interferons: implications for therapeutic applications of these cytokines cloning and characterization of a human mac-2-binding protein, a new member of the superfamily defined by the macrophage scavenger receptor cysteine-rich domain detection of antigens recognized by a novel monoclonal antibody in tissue and serum from patients with breast cancer identification of a novel serum protein secreted by lung carcinoma cells the secreted tumorassociated antigen 90k is a potent immune stimulator genome-wide mrna expression correlates of viral control in cd4+ t-cells from hiv-1-infected individuals isolation and functional characterization of the human 90k promoter dissecting interferoninduced transcriptional programs in human peripheral blood cells 90k, an interferonstimulated gene product, reduces the infectivity of hiv-1 90k protein: a new predictor marker of disease progression in human immunodeficiency virus infection a 90-kda protein serum marker for the prediction of progression to aids in a cohort of hiv-1+ homosexual men prognostic value of a novel circulating serum 90k antigen in hiv-infected haemophilia patients distinct transcriptional profiles in ex vivo cd4+ and cd8+ t cells are established early in human immunodeficiency virus type 1 infection and are characterized by a chronic interferon response as well as extensive transcriptional changes in cd8+ t cells chronic cd4+ t-cell activation and depletion in human immunodeficiency virus type 1 infection: type i interferon-mediated disruption of t-cell dynamics few and far between: how hiv may be evading antibody avidity the dispanins: a novel gene family of ancient origin that contains 14 human members the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus interferon-induced transmembrane protein-mediated inhibition of host cell entry of ebolaviruses the small interferon-induced transmembrane genes and proteins distinct patterns of ifitmmediated restriction of filoviruses, sars coronavirus, and influenza a virus interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms ifitm-2 and ifitm-3 but not ifitm-1 restrict rift valley fever virus interferoninducible transmembrane protein 3 (ifitm3) restricts reovirus cell entry ifitm3 restricts the morbidity and mortality associated with influenza a diverse range of gene products are effectors of the type i interferon antiviral response the ifitm proteins inhibit hiv-1 infection the n-terminal region of ifitm3 modulates its antiviral activity by regulating ifitm3 cellular localization hiv-1 mutates to evade ifitm1 restriction the c-terminal sequence of ifitm1 regulates its anti-hiv-1 activity ifitm proteins incorporated into hiv-1 virions impair viral fusion and spread ifitm proteins are incorporated onto hiv-1 virion particles and negatively imprint their infectivity ifitm proteins restrict viral membrane hemifusion ifitm3 restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry genomic structure of an attenuated quasi species of hiv-1 from a blood transfusion donor and recipients importance of the nef gene for maintenance of high virus loads and for development of aids brief report: absence of intact nef sequences in a long-term survivor with nonprogressive hiv-1 infection optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene the human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages nef-mediated enhancement of virion infectivity and stimulation of viral replication are fundamental properties of primate lentiviruses modulation of different human immunodeficiency virus type 1 nef functions during progression to aids hiv-1 nef promotes infection by excluding serinc5 from virion incorporation serinc3 and serinc5 restrict hiv-1 infectivity and are counteracted by nef serinc, an activity-regulated protein family, incorporates serine into membrane lipid synthesis quantitation of human immunodeficiency virus type 1 infection kinetics semen-derived amyloid fibrils drastically enhance hiv infection quantification of infectious hiv-1 plasma viral load using a boosted in vitro infection protocol this work was supported by grants from the dfg (collaborative research center 900: microbial persistence and its control), from the helmholtz center of infection research, and from the margarete von wrangell habilitation program to cg. i thank martin das for graphical assistance. the author confirms that this article content has no conflict of interest. key: cord-030427-fn9pfqts authors: huang, feifei; chen, wei-ti; shiu, cheng-shi; sun, wenxiu; toma, lance; luu, binh vinh; ah-yune, judy title: acculturation, hiv-related stigma, stress, and patient-healthcare provider relationships among hiv-infected asian americans: a path analysis date: 2020-08-13 journal: j immigr minor health doi: 10.1007/s10903-020-01068-5 sha: doc_id: 30427 cord_uid: fn9pfqts acculturation may limit hiv-positive asian americans’ active interactions with patient-healthcare providers (hcp) and utilization of hiv healthcare services; however, the specific mediation effect of acculturation still unknown. a bias-corrected factor score path analysis was performed to examine the proposed model of relations among acculturation, stigma, stress, and patient-hcp relationships. a convenience sample of 69 hiv-positive asian americans in san francisco, los angeles, and new york city were recruited and collect data were collected on demographics, hiv-related stigma, stress, and patient-hcp relationships. hiv stigma and stress had a direct, negative effect on patient-hcp relationships. acculturation had a positive total effect on patient-hcp relationships, and was mediated by hiv stigma and stress. a acculturation also had a direct impact on stigma and stress. acculturation, hiv-related stigma, and stress are key elements to achieving good patient-hcp relationships, and provide insights on the design of culturally sensitive interventions to improve patient-hcp relationships. asian americans are one of the fastest growing minority groups in the united states [1] , and they have been disproportionately affected by the hiv epidemic [2] . according to the most recent u.s. census, asian americans represent 5.4% of the total u.s. population at close to 17.2 million [1] . between 2010 and 2016, the asian population in the u.s. grew approximately 17%, yet the rate of hiv diagnosis among this group increased by nearly 70% [2] . in addition, when diagnosed, asian americans are typically already at a late stage of the pre-aids infection and usually go on to develop full-blown aids within a short time [3] . similar to other people living with hiv/aids (plwha), hiv-infected asian americans may also suffer from hiv-related stigma and stress, which in turn influences their patient-healthcare provider (hcp) relationships [4, 5] . the patient-hcp relationship refers to information sharing between patients and providers, shared responsibilities in decision-making processes, and support of patient choices [6] . in asian culture, asian plwha often seek physical and emotional support from hcp when support from friends or family is lacking due to hiv-related shame and stigma [7] . this is unfortunate, since hcp have the knowledge and expertise to treat the disease and instruct patients and family members in how to take care of hiv-related physical and mental symptoms [5] . studies have identified that many plwha have unfavorable patient-hcp relationships [8, 9] . as a result, communication suffers, which leads in turn to poorer quality of care, lower compliance with treatment regimens, decreased the self-efficacy of taking medication [8] , and worse patient satisfaction and quality of life [9] . however, specific research on how to improve the relationships between asian american plwha and hcp to enhance healthcare engagement is limited [4, 10] . in the united states, the highly diverse ethnic asian population includes chinese, filipinos, indians, vietnamese, koreans, taiwanese, and japanese, among others [11] . asian plwha may not only suffer from hiv-related stress and stigma but also have additional stress from acculturation to u.s. customs [12] . acculturation is defined as a process of change that occurs when individuals come from different cultures and interact with the host regions, which is usually developed by following migration, political conquest, or forced relocation [12] . hiv-infected asian americans also face myriad difficulties related to acculturation, such as acculturation stress (e.g. poor english language ability), health access difficulties, stigma, serostatus disclosure issues, limited access to medications, and continuation of family obligations [3, [11] [12] [13] . studies have shown that the level of acculturation is associated with immigrants' stress [12] , stigma, and health-related quality of life [14, 15] . also, for those asian plwha who have a low or moderate acculturation level to u.s. culture, at least one study has shown that stress is significantly mediated by depression symptoms [12] . thus, the level of acculturation can limit asian americans' active interactions with hcp and use of hiv healthcare services [16] . obviously, it is understood that acculturation (i.e. the learned behaviors, beliefs, and attitudes toward the host country) play a crucial role in immigrants' health [17] . several acculturation models describe the acculturation process and its implications for immigrants' well-being. berry's acculturation stress theory addresses the influence of both cultural norms and stress, which is useful in understanding the relationship between acculturation and health-related behaviors [18] . plwha who are immigrants have to face stressful situations such as discrimination and language barriers in the process of adapting to the receiving culture, which makes them highly vulnerable to maladaptive behaviors. furthermore, choi's cultural marginality model states that people emigrating to the united states from non-western cultures may have difficulty adhering to the u.s. medical system, which consequently impacts their well-being and their relationships with hcp [19] . based on this, we conclude that relationships between hcp and immigrants can be influenced by their level of acculturation; however, there is still a lack of understanding as to how the acculturation leverages hiv-stigma, stress, and patient-hcp interactions among asian plwha [4] . to address this gap, we used path analysis to understand the impact of acculturation on the relationships among stigma, stress and hcp for asian immigrants. this will provide insight into potential mechanisms for strengthening and promoting positive interactions between asian americans plwha and hcp. based on the acculturation framework and other reports, we proposed the pathway of acculturation to patient-hcp relationships through selected psychological factors (hiv-related stigma and stress) depicted in fig. 1 [4, 12, 20] . we hypothesized that acculturation can impact all the components of the stigma → stress →hcp pathway and found direct evidence for two (acculturation → stigma and acculturation → stress). acculturation can also indirectly impact hcp through the stigma and stress pathways. the study had two phases: (1) participants completed a 60-min acasi survey that consisted of standardized measures to assess demographics, hiv-related perceived stress, stigma, acculturation, and patient-hcp relationships. these measures have been tested in asian populations and have shown strong reliability and validity over time [6, [21] [22] [23] [24] . participant age, gender, marital status, ethnicity, educational level, working status, immigrant status, years of living with hiv, hiv medication, and recent viral load were collected. this 13-item scale is designed to assess how individuals rate their interaction with their hcp (e.g. "my hiv doctor answers my questions," "my hiv doctor cares about me," and so on). each item was measured on a 4-point scale (1 = always true to 4 = never true). items were reverse-scored and averaged to create a composite, with higher scores representing better patient-hcp relationships [6] . the overall cronbach's alpha reliability estimate for this sample was 0.92. this 21-item scale measures six acculturation issues in asian populations: food preferences, social friend's ethnicity, tv programs and language preferences, music selection, length of residency in the united states, and age at arrival [21, 24] . score ranges are from 1.00 (low acculturation) to 5.00 (high acculturation). for this scale, the overall cronbach's alpha reliability was 0.96. this 40-item scale measures how stigma operates within individuals and how it can affect them. it covers personal stigma, disclosure concerns, negative self-image, and concerns with public attitudes toward people with hiv. each stigma item can be rated using a 4-point likert-type scale (strongly disagree, disagree, agree, or strongly agree), with higher values indicating greater stigma [6, 22] . for this scale, the overall cronbach's alpha reliability was 0.95. this 44-item scale measures perceived stress levels elevate in eight domains: social/psychological problems, sexual relationships, functional problems, social acceptance/rejection issues, work-related issues, family/offspring issues, accessibility to treatment, and treatment outcomes. items are rated on a 5-point likert scale, from "1 = absolutely not stressful" to "5-extremely stressful" [23] . for this scale, the overall cronbach's alpha reliability was 0.97. data analyses were conducted using spss 24.0 (ibm, chicago, il) and the lavaan package in r. in the present study, the data met the assumptions of normality (one sample kolmogrov-smirnov test did not show statistical significance). the continuous variables were expressed as means and standard deviations (sd). categorical variables were expressed as proportions or percentages. pearson's correlation analysis was conducted to examine the relationships among acculturation, hiv-related stress, stigma, and patient-hcp relationships. missing data were replaced using full information maximum likelihood; p < 0.05 was considered significant. due to the sample size in this study, we used bias-corrected factor score path analysis, which has been shown to produce unbiased and efficient estimates in small to moderate sample settings [25] . we tested the pathways in three steps. first, we estimated the variance-covariance matrix of the factor scores along with acculturation, hivrelated stress, stigma, and patient-hcp relationships. second, we corrected the covariance matrix to obtain an estimate of the true covariances. finally, we performed path analysis to test the hypothesized pathway (fig. 1) [26] and obtained unbiased estimates of the true structural path coefficients. the following model-fit indices were used: normed chi-square (χ 2 /df, 1.0 ± 3.0, p > 0.05), root mean squared error of approximation (rmsea < 0.08), comparative fit index (cfi, > 0.9), and tucker-lewis index (tli, > 0.9). to achieve a more parsimonious model, we further removed parameters that did not significantly differ from zero. we also adjusted the estimates by controlling for significant demographic variables: marital status, ethnicity, educational level, working status, immigrant status, and hiv medication (see table 1 ). finally, to investigate the relationships between acculturation and patient-hcp relationships through different pathways, we applied the bootstrap method (repeated 1,000 times) to obtain more stable and valid standard errors of the estimates of the direct and indirect effects of these factors. standardized regression coefficient (β) and p values for β of direct, indirect, and total effects were identified and reported by path analysis. also, to compare the modified pathway with the initial pathway, we applied the akaike information criterion (aic) [27] . demographic characteristics are presented in table 1 . table 2 . the results suggest that acculturation, hiv-related stress, stigma, and patient-hcp relationships are significantly correlated with each other. it was shown by the fit indices that for the proposed model fig. 2 . table 3 summarizes the standardized direct, indirect, and total estimates of the reduced model's paths. according to the model, hiv stigma (β = − 0.154) and stress (β = − 0.203) had a negative direct effect on patient-hcp relationships (p < 0.05 for the model). although acculturation had no significant direct effect on patient-hcp relationships, it still had a positive total effect (β = 0.094), which can be seen to be mediated by hiv stigma (indirect β = − 0.06) and stress (indirect β = − 0.01). moreover, acculturation directly impacted stigma (β = − 0.399) and stress (β = − 0.048). consistent with the hypothesis, 20.30% and 15.40% of the variance in patient-hcp relationships were negatively predicted by hiv-related stress and stigma, respectively. in addition, 39.90% of the variance in hiv-related stigma was negatively predicted by acculturation; 52.60% and 4.80% of the variance in hiv-related stress were positively predicted by stigma and acculturation, respectively. our data and pathways show acculturation to be an important factor in the patient-hcp relationship, which is also echoed by other reports [4, 28] . this is also one of the initial reports to offer pathway evidence that links positive acculturation to patient-hcp relationships in asian americans. coinciding with the explanations provided by berry and choi's models [18, 19] , these hypotheses of the relationships among study variables is supported, except for the direct effect of acculturation and patient-hcp relationships. the present study showed that acculturation is beneficial for patient-hcp relationships to the extent that it decreases perceived stigma and stress in asian american plwha. in other words, for asian american plwha, focusing on enhancing acculturation is warranted, as it is a potential mechanism to reduce hiv-related stigma and stress. consequently, acculturation can enhance patient-hcp relationships. as such, the present study's findings highlight the potential utility of targeting acculturation in interventions aimed at improving patient-hcp relationships. this study confirms the acculturation-stress-patient-hcp relationships pathway. as stipulated by the transactional theory of stress, stress is a result of individuals' appraising a potential stressor as negative and beyond his or her coping resources [29] . the asian american plwhas remained attached to their original cultures while developing connections with the host country [12] , and thus they face the perceived stress of acculturation just as other immigrants do. acculturation stress can include family pressure, the potential for inadvertent disclosure to the public, and other challenges [12, 30] . if asian american plwha perceive acceptance into the dominant culture as a daunting undertaking, the process of integrating to the host country becomes a stressor [12] . on the other hand, if they perceive acceptance into the dominant culture as neutral or positive, and thus manageable, the acculturation process is less stressful and can be seen as a positive occurrence [20] . the results can lead to worse or closer patient-hcp relationships [30] . the hypothesis that the effect of acculturation on patient-hcp relationships is mediated by hiv-related stigma was also sustained. this could be due to the internalization of hiv-related misconceptions from the country of origin, such as the belief that hiv is "dirty" and that hiv is in effect a death penalty [31] . limited english language ability and other acculturation challenges, such as social isolation and lack of self-management, can worsen plwha's own internalized stigma and shame [14] . therefore, their trust of hcp and hiv care in the host country could be jeopardized [32] . the joint united nations programme on hiv/ aids (unaids) has set a 90-90-90 target for all countries to scale up testing and treatment; thus, by 2020, 90% of plwha will know their status; 90% of those diagnosed will be on antiretroviral therapy, and 90% of those receiving antiretroviral medications will be virally suppressed [33] . however, to reach this target, reducing stigma and discrimination are urgently needed [33] . thus, culturally tailored interventions to reduce hiv-related stigma should be tested and implemented, not only to enhance the acculturation process, but also to better engage asian american plwha in managing their own health care. currently, interventions aimed at improving immigrant patients' hcp relationships are lacking, and there are limited studies to support the development of such interventions [4] . thus, the present study could be important for developing a feasible, acceptable and effective, culturally sensitive intervention to improve patient-hcp relationships. this study shows that acculturation-stress and stigma-patient hcp relationship pathways are highly influential. therefore, when designing an intervention to enhance patient-hcp relationships, these factors should be considered, because acculturation, hiv-related stigma and stress are key to achieving therapeutic patient-hcp relationships. due to their typically heavy workload and lack of time to devote to cultural sensitivity, hcp may draw on stereotypical assumptions when they encounter people who are not fluent in english and may be naïve about the u.s. healthcare system [8] . as a [8] . this can lead to care disparities in the areas of both physical and psychological health, diminishing the overall quality of care [4] . thus, supporting providers in their efforts to create mutually beneficial patient-hcp relationships can influence the quality of care and result in better health for plwha [10] . by providing tailored training to hcp on measuring acculturation levels as well as psychosocial needs and barriers, hcp can enhance healthcare engagement among asian american plwha [4, 10] . this study has several limitations. first, the proposed pathway does not explain the majority of variances in patient-hcp relationships, including variances in depression, which were not considered in this model. besides, there might be an overfitting of the pathway due to six covariates that were adjusted for in the pathway: according to the rule of thumb, one covariate per 10 subjects, although we used bias-corrected factor score path analysis, which has been shown to produce unbiased and efficient estimates in small to moderate sample settings. second, the convenience sampling method and a small sample size limits the generalizability of the findings; however, that must be measured against the fact that asian american plwha are one of the hardest-to-reach populations, and this study is one of the pioneering studies in the field. last, this is a cross-sectional study and causality cannot be inferred. therefore, research with time-series and experimental methods is encouraged to further support the hypotheses that follow from this analysis. in this paper, we examined the associations among acculturation, hiv-related stigma, stress, and patient-hcp relationships among asian american plwha. this is a vulnerable group with significant acculturation and psychosocial distress, yet there are limited reports on their experience navigating the u.s. healthcare systems and services. this exploratory study presents that acculturation is beneficial for patient-hcp relationships and that, therefore, it decreases plwha's perceived stigma and stress. interventions designed to decrease hiv-related stress and stigma and support acculturation should be developed in order to enhance patient-hcp relationships. this will improve hiv care and reduce healthcare disparities in asian americans. key facts about asian origin groups in the u diagnoses of hiv infection in the united states and dependent areas revising the american dream: how asian immigrants adjust after an hiv diagnosis effects of individual immigrant attitudes and host culture attitudes on doctor-immigrant patient relationships and communication in canada a structural equation model of patient-healthcare provider relationships and hivinfected patient outcomes in chinese populations relationships between perception of engagement with health care provider and demographic characteristics, health status, and adherence to therapeutic regimen in persons with hiv/aids chinese hiv-positive patients and their healthcare providers: contrasting confucian versus western notions of secrecy and support cross-sectional evaluation of perceived health care provider engagement, self-efficacy, and art adherence in people living with hiv/aids engagement with health care providers affects self-efficacy, self-esteem, medication adherence and quality of life in people living with hiv hiv provider experiences engaging and retaining patients in hiv care and treatment: "a soft place to fall hiv and religion in hiv-infected asians and their families: a qualitative study acculturation and perceived stress in hiv+ immigrants: depression symptomatology in asian and pacific islanders life priorities in the hiv-positive asians: a text-mining analysis in young vs old generation the impact of acculturation on latinos' perceived barriers to hiv primary care pathways to health: an examination of hiv-related stigma, life stressors, depression, and substance use where it falls apart": barriers to retention in hiv care in latino immigrants and migrants acculturation, family cohesion, and mental health among latinos living with hiv on the us-mexico border comparative studies of acculturative stress. international migration review cultural marginality: a concept analysis with implications for immigrant adolescents acculturation, acculturative stress, social status, and well-being among english language proficient immigrants the suinn-lew asian self-identity acculturation scale: an initial report measuring stigma in people with hiv: psychometric assessment of the hiv stigma scale development of the perceived stress scale for people living with hiv/aids in china acculturation and health-related quality of life among vietnamese immigrant women in transnational marriages in taiwan a robust alternative estimator for small to moderate sample sem: bias-corrected factor score path analysis cutoff criteria for fit indexes in covariance structure analysis: conventional criteria versus new alternatives the role of character strengths in depression: a structural equation model communication and culture: predictors of treatment adherence among mexican immigrant patients stress, appraisal, and coping fatigue and sleep disturbance in hiv-positive women: a qualitative and biomedical approach comparison of sexual knowledge, attitude, and behavior between female chinese college students from urban areas and rural areas: a hidden challenge for hiv/ aids control in china stigma gets in my way: factors affecting client-provider communication regarding childbearing among people living with hiv in uganda 90-90-90: an ambitious treatment target to help end the aids epidemic we gratefully acknowledge all the study participants, without them, it is not possible to complete these projects. conflict of interest the authors declare that they have no conflict of interest. key: cord-102905-rlee32x7 authors: leis, jonathan; luan, chi-hao; audia, james e.; dunne, sara f.; heath, carissa m. title: ilaprazole and other novel prazole-based compounds that bind tsg101 inhibit viral budding of hsv-1/2 and hiv from cells date: 2020-05-04 journal: biorxiv doi: 10.1101/2020.05.04.075036 sha: doc_id: 102905 cord_uid: rlee32x7 in many enveloped virus families, including hiv and hsv, a crucial, yet unexploited, step in the viral life cycle is releasing particles from the infected cell membranes. this release process is mediated by host escrt complex proteins, which is recruited by viral structural proteins and provides the mechanical means for membrane scission and subsequent viral budding. the prazole drug, tenatoprazole, was previously shown to bind to escrt complex member tsg101 and quantitatively block the release of infectious hiv-1 from cells in culture. in this report we show that tenatoprazole and a related prazole drug, ilaprazole, effectively block infectious herpes simplex virus (hsv)-1/2 release from vero cells in culture. by electron microscopy, we found that both prazole drugs block the release of hsv particles from the cell nuclear membrane resulting in their accumulation in the nucleus. ilaprazole also quantitatively blocks the release of hiv-1 from 293t cells with an ec50 of 0.8 μm, which is more potent than tenatoprazole. finally, we synthesized and tested multiple novel prazole-based analogs that demonstrate both binding to tsg101 and inhibition of viral egress in the nanomolar range of hiv-1 from 293t cells. our results indicate that prazole-based compounds may represent a class of drugs with potential to be broad-spectrum antiviral agents against multiple enveloped viruses, by interrupting cellular tsg101 interaction with maturing virus, thus blocking the budding process that releases particles from the cell. importance these results provide the basis for the development of drugs that target enveloped virus budding that can be used ultimately to control multiple virus infections in humans. the advent of antibiotics had a major impact on controlling bacterial infections in patients worldwide, with a single drug being used to treat multiple infections. unfortunately, antivirals have not had the same success. there are many contributing factors to this shortcoming, foremost the fact that few mechanisms are shared by different viruses limits targets for a broad-spectrum antiviral. consequently, approved antivirals generally act against individual rather than groups of viruses, limiting a single drug's potential. however, this may change with the finding that multiple classes of enveloped viruses share the same budding mechanism that relies on host-cell endosomal sorting complex required for transport (escrt) proteins (1, 2) . an inhibitor of this pathway could represent a potential broad-spectrum antiviral and have a positive impact on our ability to treat multiple enveloped virus infections with a single therapeutic agent. enveloped viruses bud from the host cell membranes and use the acquired lipid layer as a protective coat that also contains the glycoproteins required for infection of other cells. enveloped viruses do not encode the machinery needed for budding and must recruit host-cell proteins to bud from cells. in hiv, escrt proteins are recruited to virus budding complexes through an interaction between the l-domain (ptapp motifs) in virus structural proteins (3) (4) (5) (6) (7) with cellular protein tsg101 (tumor susceptibility gene 101), a homolog of the e2 ubiquitin conjugating enzyme and a member of the escrt-i complex (6, (8) (9) (10) (11) . tsg101 recruits the cellular escrt-iii complex, which provides the mechanical means for viruses to passage through cell membranes to be released (10, (12) (13) (14) (15) (16) (17) (18) (19) . another enveloped virus family member, herpes simplex virus, hsv, assembles particles in the nucleus and relies on escrt proteins for passage through the nuclear membrane (20) (21) (22) (23) . thus, this pathway may present a common target for treating multiple virus infections. in support of targeting this pathway, a recent seminal discovery in our lab established that an interferoninduced protein, interferon stimulated gene 15 (isg15), specifically targets the escrt proteins in budding complexes to block the release of viruses (1, (24) (25) (26) . this indicates that the human immune system evolved to target the escrt pathway to control infections and supports that this is a natural target. another group identified single-nucleotide polymorphic sites in the 5' region of tsg101, located at positions -183 and +181 relative to the translation start signal, which affect the rate of aids progression among caucasians (27) . these data support the hypothesis that variation in tsg101 affects efficiency of tsg101-mediated release of viral particles from infected cells, altering plasma viral load levels and subsequent disease progression. taken together, these investigations indicate that tsg101 and escrt proteins present a natural antiviral target. currently the prazole family of drugs is best known for their role as proton pump inhibitors (ppis) and a few, namely omeprazole (prilosec), esomeprazole (nexium) and ilaprazole (adiza, noltec, yi li an), are marketed to control symptoms of gastroesophageal reflux disease (gerd) in either the us or abroad. ppis form a covalent bond with the active site of proton pumps, inhibiting their ability to acidify the stomach and reducing symptoms associated with over-acidification (28) . recent reports indicate that drugs from the prazole family, including tenatoprazole and esomeprazole, form a disulfide linkage to tsg101, which results in blocking the release of hiv-1 from cells in culture (5) . other groups recently reported that prazoles may have potential as an antiviral therapeutic in hsv and sars-cov2 in combination with acyclovir or remdesivir, respectively (29, 30) . however, the prazole compound used in these studies, omeprazole, was not potent enough to be predicted to have a therapeutic effect in vivo. this highlights a gap in the ability for current prazoles to have therapeutic potential, and the need for further research on prazoles as antivirals. in the present manuscript, we demonstrate that multiple prazole drugs block the budding of hsv-1 and hsv-2 from vero cells in culture, strengthening the case for the broad-spectrum potential of this mechanism/drugs. most notably, we identified one prazole drug, ilaprazole, which blocks the release of both hiv-1 and hsv-1/2 from cells at an efficiency more potent than reported for tenatoprazole. ilaprazole acts in the low µm range without detectable cell toxicity at inhibitory concentrations. additionally, we designed and synthesized novel prazole analogs that act in the nanomolar range to block virus release, a major step forward in creating a vbi that can be brought to the clinic. to further define the mechanism of action of these prazole drugs on hsv infections, we identified the site of blockage of herpes virus release, which appears to be different from hiv-1. while the blockage to hiv-1 particle release is at the outer cell membrane (5), the prazole drugs appear to first block the passage of the herpes virus through the nuclear membrane. this prevents particles being released into the cytosol, where maturation of their envelope membrane occurs to produce infectious virus. with the prazole-based inhibitors being effective in both hiv and hsv, targeting tsg101 could lead to a broadspectrum antiviral therapy. identification of prazole compounds that bind the uev ubiquitin-binding domain of tsg101. we screened chemical compounds using a fluorescence thermal shift (fts) assay (31, 32) to identify small molecules that bind directly to a truncated form of tsg101 (amino acids 1-145) which contains the ubiquitin e2 variant (uev) ubiquitin-binding domain (fig. 1) . this truncated tsg101, called tsg101-uev, was used because full-length tsg101 has significant solubility issues in aqueous solution. tsg101 is an adaptor protein and thus lacks a readily deployable functional assay, making fts a tractable approach to identify interacting compounds. fts monitors protein thermal denaturation using sypro® orange, a dye which fluoresces when bound to hydrophobic surfaces, which allows monitoring of the changes in hydrophobic surface exposure during protein denaturation (31) . since ligand binding affects protein thermal stability, it can be detected through modulation of protein thermal denaturation (melting) as a shift in melting temperature (tm). tsg101-uev has a well-defined melting curve suitable for fts. we used the fts assay to identify compounds that bind to tsg101-uev. we compared thermal denaturation profile for tsg101-uev in the presence and absence of tenatoprazole and found that it destabilizes the native protein structure, indicating that it binds tsg101-uev ( fig. 1) . we also tested tenatoprazole against proteins unrelated to tsg101, including dhph, eno1, mek4, and did not observe a tm shift (data not shown), indicating that the tm shift of tsg101-uev was due to specific interaction of the prazole compound. this specific binding is consistent with a previous nmr structure in which tenatoprazole forms a covalent disulfide bond to cys73 in the uev domain of the protein (5). this disulfide bond formation can be prevented by including the reducing agent dtt in the assay (fig. s1 ). the addition of dtt abolished the tsg101-uev tm shift caused by the prazoles. therefore, the addition of dtt to the fts assay is a facile means to ascertain if prazole analogs interact with tsg101-uev in a covalent manner. tenatoprazole and esomeprazole were shown to quantitatively inhibit the release of infectious hiv-1 from 293t cells in culture, and it was suggested that these effects may be mediated via changes in viral interaction with tsg101, a key component of the cellular escrt complex (5, 33) . given multiple reports suggesting that herpes viruses also use cellular escrt proteins in their replication process (20) (21) (22) (23) we tested if the tsg101-binding prazole drugs, which blocked budding of hiv-1, would also block the release of herpes viruses from cells. we infected vero cells with hsv-1 and hsv-2 for two hours at a multiplicity of infection (moi) of 0.1 to assay the antiviral activity of tenatoprazole. following infection, cells were treated with different concentrations of tenatoprazole. after 24 or 48 h the media fractions were collected and released virus titers were determined by standard plaque assays (34) . tenatoprazole caused a 3-log drop of hsv-1 and 4-5 log drop of hsv-2 of released virus titer from vero cells in a dose dependent manner (table 1, column 2, 3, and 5) with calculated ec50's ranging from 48-80 µm. total virus titer was also determined to differentiate between virus released into the media and infectious particles present in cell lysate. total infectious virus particles were also reduced by tenatoprazole ( we next imaged herpes virus infected-vero cells using transmission electron microscopy to determine the site of inhibition of release of virus and whether it was similar to observations of hiv-1 release from 293t cells. vero cells grown on glass cover slips were infected with hsv-2 at moi of 0.1 for 2 h and then treated for 24 h with 105 µm tenatoprazole or vehicle control. using electron microscopy, we examined eighty cells with virus particles, and representative images are shown in fig. 2 . in the control, virus particles were in both the nucleus and cytoplasm near the cell surface ( fig. 2a ). in the tenatoprazole-treated cells the cytosol of the intact cells was largely devoid of virus particles (fig. 2b ). instead, we observed large pockets of granular material accumulated in the nucleus and immature virus particles lined on the inside of the nuclear membrane (inset, b). these results suggest that tenatoprazole blocks the passage of herpes virus particles through the nuclear membrane. this result is different from that observed with hiv-1. because tenatoprazole binds tsg101, it suggests that the escrt-i protein complex is involved in transport of hsv-2 through the nuclear membrane. despite the lack of cell toxicity signal at effective tenatoprazole concentrations, the effective concentration is too high for use as a clinical therapy. therefore, more potent analogs are required to explore antiviral therapeutic potential. we set out to identify and test other analogs which were more potent prazole analogs. we began by searching pubchem for analogs of tenatoprazole. we identified and obtained a dozen such compounds from commercial sources and prioritized these for testing based on structural similarities around the sites where tenatoprazole covalently linked to cys73 of tsg101. to this end, tenatoprazole, lansoprazole, rabeprazole, dexlansoprazole, pantoprazole, esomeprazole, 4desmethoxy-omeprazole (an omeprazole analog, 5-methoxy-2[[(3,5-dimethyl-4-methoxy-pridin-2-yl-noxide)methyl]sulfinyl]-1h-benzimidazole; labelled o-omeprazole), omeprazole, and ilaprazole were assessed in the fts assay for their ability to change the tm of tsg101-uev as described above (data not shown). we determined the dose response plots of tsg101 melting temperature shifts caused by these prazole compounds binding to tsg101 (1-145) (fig. 3) . o-omeprazole is the only compound predicted not to form the covalent bond with tsg101, since it has an oxygen linked to a ring nitrogen that is normally a hydrogen in the active prazoles (table 2, right column). as expected, o-omeprazole did not cause a detectable thermal shift. the smallest thermal shift was observed with pantoprazole (gray) and the largest thermal shift was observed with ilaprazole (green). ilaprazole's ability to cause a thermal shift with tsg101 was blocked by the addition of dtt (fig. s1 ), consistent with the idea that the compound forms a disulfide linkage to tsg101. next, we tested the anti-herpes virus activity of these prazole compounds (table 2) . to examine the effects of the compounds on the release of hsv-2 from vero cells, we infected the cells with virus two hours prior to treatment with media containing different concentrations of compound. we incubated the cells for 24 or 48 hours and then collected the cell media fractions. several of the analogs were inactive, including o-omeprazole, pantoprazole, and rabeprazole. we identified a number of active compounds, in which there was a 10-fold spread of inhibition activity against hsv-2, ranging from an ec50 of 140 µm (for esomeprazole) to 3-9 µm (for ilaprazole). thus, we identified prazole analogs that are more potent than tenatoprazole. we provide the structures of prazole compounds tested in this analysis ( table 2 , column 3). of note, ilaprazole contains an additional ring structure compared to tenatoprazole that is predicted to lie in a solvent exposed area of the tsg101 structure that may serve to strengthen the interaction with tsg101. in examining the thermal shift capacity of the prazoles, we found that these roughly correlated to their hsv-2 antiviral activity. this correlation indicates that the fts assay is useful in evaluating structureactivity-relationships (sar) to inform the design of new compounds (fig 3, table 2 ). based on these hsv-2 antiviral assay results, we selected ilaprazole for further antiviral profiling. first, we tested it against hsv-1 (table 3 , columns 2-4). ilaprazole was slightly more effective against hsv-1 than against hsv-2 with ec50 calculations ranging from 0.6-5 µm (table 3) . ilaprazole's potency is a large improvement over tenatoprazole, which inhibited in the high µm range (table 1 & 3) . additionally, ilaprazole was even more effective in inhibiting virus release at 72 h as at 24 h after a single application of the drug (72 h ec50<1µm; compare table 3 , columns 2 & 4). the inhibition caused by tenatoprazole against either virus began to fall off after 48 h (data not shown). we also tested for toxicity in the range of effective concentrations and did not observe cell toxicity using the 96® aqueous one solution cell proliferation assay reagent and wst-1 reagent over a 24 h period ( table 3 , right columns). thus, ilaprazole is more potent and has longer lasting effects than tenatoprazole. we next carried out a transmission electron microscopic examination of cells infected with hsv-1 at a moi 0.1 in the presence and absence of 18 µm ilaprazole to determine if this drug causes the accumulation of virus particles in the nucleus of cells similar to tenatoprazole. without drug, we observe particles in the cytoplasm and in the nucleus (fig 4a & c) , in the presence of drug little or no viral particles are found in the cytoplasm (fig 4b & d) . in both heavily infected cells (fig 4a & b) and mildly infected cells (fig 4c & d) , treatment lead to particles being detected in the nucleus arrayed along the nuclear membrane (fig 4c & d) . this indicates that location of particles in the cell in the presence of drug is independent of the number of particles observed. these results are similar to the effect of tenatoprazole on hsv-2 infected cells (fig. 2) . finally, to confirm the broad-spectrum potential of ilaprazole, we tested whether ilaprazole would inhibit the release of hiv-1 from 293t cells. to this end, cells were transfected with pr9-hiv-1ba-l plasmid and release of virus into the media fraction was detected by monitoring the capsid (ca) protein (p24) via enzyme linked immunosorbent assay analysis (elisa). the drug was tested at concentrations between 0 and 40 µm and the effect of the drug on release of virus assessed (table 4 ). ilaprazole was effective at inhibiting the release of hiv-1 from cells with a calculated ec50 of 0.8 µm. we did not detect toxicity to the cells at the drug concentrations that inhibited the release of hiv-1 over the course of these experiments (data not shown). in an effort to design and identify more potent viral budding inhibitors, we synthesized 53 novel prazolebased analog compounds and assessed these for binding to tsg101-uev using fts. of the compounds screened, eight compounds demonstrated a tm shift greater than or equivalent to that observed with ilaprazole (selected examples, fig 5) indicating that these compounds bind to tsg101. based on the results described above, we are currently assessing the antiviral activity of these compounds in multiple antiviral assays. initial testing of four analog compounds against hiv-1 reveal further increases in potency above that seen with ilaprazole (table 4 ) with ec50 calculations ranging from 14-16 nm. this supports the pursuit of a medicinal chemistry campaign to apply the sar learned from the prazole drugs to identify and develop potent compounds with broad-spectrum antiviral potential. taken together, our results indicate that prazole-based drugs block release of hiv-1 and herpes viruses (hsv-1 and hsv-2), two families of viruses with different replication mechanisms that share the common pathway of tsg101-mediated release of virus particles. interestingly, we were able to identify more potent prazole analogs, in particular ilaprazole as well as novel compounds that are now in development. we found that ilaprazole and our analogs demonstrated antiviral effects significantly more potent, and potentially longer lasting, than other prazoles we tested. we are developing a novel strategy to treat viral infections affecting humans by disrupting a common mechanism used by many enveloped viruses to bud from cells. viral budding inhibitors (vbi) have the potential to be broad-spectrum antiviral therapeutics, potentially being effective against herpes (20-23, (9, 52, 53) . vbis would require testing for antiviral activity towards these different viruses before clinical use but nonetheless present a strong starting point for identifying therapeutics. in this work we demonstrate antiviral activity of prazole compounds, with no detectable cell toxicity at effective concentrations, against two viruses that use different mechanisms for viral replication. of particular note is that the viral genomes are very different, with hiv being rna-based and hsv being dna-based. that one compound works against viruses with such stark difference in viral life cycle types supports that these compounds have potential as a broad-spectrum antiviral agent for current and emerging viruses. this aspect gives this approach advantage over other potential broad-spectrum antivirals, such as remdesivir, which is targeted to rna viruses, limiting its potential as a broadspectrum antiviral (54) . tsg101 binding to the proline-rich ptapp viral l-domains in gag (3, 6, 7, 11, 14, 15) is required for virus particles to be released from cell membranes of infected cells. tsg101 is a member of the escrt-i complex of proteins involved in cell endosomal sorting. the escrt-i complex recruits proteins from the escrt-iii complex with aip1 (19) , which provides the mechanical means for scission of virus particles from cell membranes. thus, blocking the ptapp l-domain sequence from interacting with the host proteins causes the virus budding defect and several lines of independent evidence support this idea. first, drugs targeted to this specific interaction in hiv cause budding defects in cells without detectable off-target effects (5) . second, a research group identified noncoding snps in the 5' region of tsg101, which affect the degree of tsg101-mediated release of viral particles (27) . third, viral infections activate a host innate immunity mechanism, through interferon stimulated gene 15 (isg15), that specifically disrupts virus budding complexes (1) . in response to this immune system defense, many viruses encode enzymes that prevent or reverse isg15 conjugation to cellular proteins to avoid the budding blockade (55) (56) (57) (58) (59) (60) . while the prazole analogs block the release of hiv-1, hsv-1, and hsv-2, the inhibition is manifested in different areas of the cell. the drugs block the release of hiv-1 at the outer cell membrane by preventing pinching of virus particles from the membrane (5). in contrast, herpes viruses appear to be first blocked at the passage of the virus through the nuclear membrane. because the prazole drugs form a covalent bond to tsg101, it strongly suggests that the escrt proteins are important for the herpes virus particles to be released from the nucleus of the cell where they are formed. this is consistent with the recent report by arii et al., (61) that the escrt-iii protein complex mediates herpes virus movement across the nuclear membrane and regulates its integrity. the finding that the prazole drugs cause a significant drop in total infectious herpes viruses can be explained by the trapping of immature particles in the nucleus, which prevents them from migrating into the cytoplasm to exchange enveloped membranes, which makes them infectious. the accumulation of the dense material in the nucleus observed in the electron micrographs suggests that the drug may interfere with normal particle assembly in addition to blocking the release of the particles from the nuclear membrane. the use of prazoles as an antiviral represents an exciting potential case of repurposing existing drugs to act as antiviral agents. currently, omeprazole is marketed as a prodrug for treatment of acid reflux disease. the prodrugs are acid-activated into derivatives that form disulfide linkages with proton pumps (28, 62, 63) . the prodrug, but not the charged sulfonamide derivative, can cross the plasma membrane barrier. the antiviral activity of tenatoprazole has been suggested to be the result of forming a covalent disulfide bond with tsg101 (5). while the binding site for tenatoprazole is near the ubiquitin (ub) binding pocket and not the l-domain binding site, biochemical and confocal imaging data independently demonstrated that tenatoprazole disrupts the binding of tsg101 to the ptapp sequence (5) . while the precise biochemical mechanism remains to be clarified, our fts results support that it may be related to allosteric changes in tsg101 after the drug forms its covalent linkage with cys73. one of our most potent lead compounds is another prazole drug, ilaprazole, which is also marketed for treatment of acid reflux disease in china, india, and south korea (yi li an, adiza, noltec, respectively) indicating that it has reasonable bioavailability and a known clinical safety profile. previous reports did not detect offtarget effects of the prazole drugs affecting tsg101 metabolism inside of cells (5) . the prazoles we tested here also appear to be nontoxic to vero, hela, and 293t cells at the concentrations used to inhibit budding of herpes viruses and hiv-1. a recent report highlighted the potential of prazole compounds to have a therapeutic effect on sars-cov-2 when combined with remdesivir (30) . however, the authors did not definitively identify the mechanism of action of the prazoles and also concluded that the potency of the prazole compound used, omeprazole, is too low to reach therapeutic levels in vivo. a potential mechanism posed by the authors is that the prazoles lead to an increase in lysosomal ph, which is the potential mechanism for lysosomotropic drugs such as chloroquine (64) . in contrast to omeprazole, we hypothesize that ilaprazole and our more potent novel prazole compounds may allow for therapeutic levels to be reached in vivo. in the case of ilaprazole, which is marketed in several asian countries as discussed above, our strong in vitro results lay the foundation for a potential fast-track to broad-spectrum antiviral clinical testing, alone or in combination with other drugs, in these countries. we are currently working to determine if ilaprazole or our novel compounds have activity against sars-cov-2 with or in combination with remdesivir. this would further the potential broad-spectrum antiviral capacity of the prazole compounds described in this report. fts using thermal shift elicited by the small molecule binding effect to protein stability. fts monitors protein thermal denaturation using environment-sensitive dye sypro® orange which fluoresces when bound to hydrophobic surfaces, taking advantage of the changes in hydrophobic surface exposure in protein denaturation. discovery of small molecule binding to target protein utilizes the observation that ligand binding affects protein thermal stability, and therefore can be detected through a shift in the protein's thermal denaturation (melting) temperature (tm). we have employed fts to reveal changes in thermodynamic properties of tsg101 elicited by its interaction with a small molecule. the recombinant tsg101 fragment (amino acids 1-145), prepared as described above in materials and methods (but without label) has a thermal unfolding profile suitable for using fts as a primary screen assay in hts. a fluorescence dye sypro® orange (invitrogen) was used for assay detection. the dye is excited at 473 nm and has a fluorescence emission at 610 nm. the dye binds to hydrophobic regions of a protein that are normally buried in a native protein structure. when a protein is unfolded, the dye interacts with exposed hydrophobic surfaces and the fluorescence intensity increases significantly over that observed in aqueous solution. the tsg101 fragment was premixed at a concentration of 2 μm with a 5x concentration of sypro® orange in hepes buffer (100 mm hepes, 150 mm nacl, ph 7.5). then 10 µl of the protein-dye mix was added to an assay plate and 10 to 50 nanoliters of compound, equal to 10 to 50 µm, were added with an acoustic transfer robot echo550 (labcyte, ca). the plate was shaken to ensure proper mixing, then sealed with an optical seal and centrifuged. the thermal scan was performed from 20 to 90°c with a temperature ramp rate of 0.5°c/min. fluorescence was detected on a real-time pcr machine cfx384 (bio-rad laboratories). comparison of the thermal denaturation profile for tsg101-uev in the presence and absence of tenatoprazole and other prazoles revealed destabilization of the native protein structure, indicating that the compound interacted with tsg101-uev. table 1 . effect of tenatoprazole on hsv-1 and-hsv-2 release from vero cells. tenatoprazole was incubated with vero cells infected with hsv-1 or hsv-2 at a range of concentrations. the virus released into the media fraction at stated times was determined as described in materials and methods. total virus is the amount of virus released from cells plus virus inside of the cells. viability of vero cells incubated with increased concentration of tenatoprazole was determined using the 96® aqueous one solution cell proliferation assay reagent as described in materials and methods. figure 3 . dose-response plots of tsg101 melting temperature shift caused by 10 prazole compounds. different concentrations of prazole compounds were incubated with tsg101 (aa 1-145) and subjected to fluoresecent thermal shift analysis as described in materials and methods. a a table 2 . effect of prazole analogs to inhibit the release of hsv-2 from vero cells. different concentrations of the listed prazole drugs were incubated with hsv-2 infected vero cells for 24 hours and then virus released into the media was quantified by plaque assays. data presented includes the ec50 value (concentration at which virus release is inhibited by 50%). methods are as described in the legend of table 3 . effect of ilaprazole on release of hsv-1 and hsv-2 from vero cells. different concentrations of ilaprazole were incubated for the times indicated with hsv-1 or hsv-2 infected cells similar to that described in the legend to table 1 . virus titer released into the media and total virus was determined. viability of vero cells incubated with increased concentration of tenatoprazole was determined using the 96® aqueous one solution cell proliferation assay reagent as described in materials and methods. titer of hsv-1 in media, 24h titer of hsv-1 in media, 48h titer of hsv-1 in media, 72h titer of hsv-2 in media, 24h total hsv-2 in media + cell lysate, 24h budding of enveloped viruses: interferon-induced isg15-antivirus mechanisms targeting the release process parallels between cytokinesis and retroviral budding: a role for the escrt machinery effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release avian sarcoma virus and human immunodeficiency virus, type 1 use different subsets of escrt proteins to facilitate the budding process tsg101 chaperone function revealed by hiv-1 assembly inhibitors an assembly domain of the rous sarcoma virus gag protein required late in budding fine mapping and characterization of the rous sarcoma virus pr76gag late assembly domain tsg101 can replace nedd4 function in asv gag release but not membrane targeting ubiquitin depletion and dominant-negative vps4 inhibit rhabdovirus budding without affecting alphavirus budding the functionally exchangeable l domains in rsv and hiv-1 gag direct particle release through pathways linked by tsg101 tsg101, the prototype of a class of dominant-negative ubiquitin regulators, binds human immunodeficiency virus type 1 pr55gag: the l domain is a determining of binding nedd4l overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking ptap and ypxl late domains functional role of alix in hiv-1 replication tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding tsg101 control of human immunodeficiency virus type 1 gag trafficking and release divergent retroviral latebudding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins structure and functional interactions of the tsg101 uev domain the protein network of hiv budding aip1/alix is a binding partner for hiv-1 p6 and eiav p9 functioning in virus budding human cytomegalovirus exploits escrt machinery in the process of virion maturation herpes simplex virus type 1 production requires a functional escrt-iii complex but is independent of tsg101 and alix expression herpes simplex virus type 1 cytoplasmic envelopment requires functional vps4 intracellular trafficking and maturation of herpes simplex virus type 1 gb and virus egress require functional biogenesis of multivesicular bodies mechanism of inhibition of retrovirus release from cells by interferon-induced gene isg15 the mechanism of budding of retroviruses from cell membranes the interferon-induced gene isg15 blocks retrovirus release from cells late in the budding process consistent effects of tsg101 genetic variability on multiple outcomes of exposure to human immunodeficiency virus type 1 pharmacokinetics and pharmacodynamics of the proton pump inhibitors omeprazole increases the efficacy of acyclovir against herpes simplex virus type 1 and 2 sars-cov-2 and sars-cov differ in their cell tropism and drug sensitivity profiles ligand screening using fluorescence thermal shift analysis (fts) high-density miniaturized thermal shift assays as a general strategy for drug discovery selective targeting of virus replication by the epstein-barr virus glycoprotein 110 carboxy-terminal tail domain is essential for lytic virus replication functional interaction between the escrt-i component tsg101 and the hsv-1 tegument ubiquitin specific protease the small ring finger protein z drives arenavirus budding: implications for antiviral strategies cellular factors required for lassa virus budding the escrt system is required for hepatitis c virus production vps4 and the escrt-iii complex are required for the release of infectious hepatitis c virus particles small-molecule probes targeting the viral ppxy-host nedd4 interface block egress of a broad range of rna viruses a ppxy motif within the vp40 protein of ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding a host-oriented inhibitor of junin argentine hemorrhagic fever virus egress the multifunctional ebola virus vp40 matrix protein is a promising therapeutic target hiv-1 and ebola virus encode small peptide motifs that recruit tsg101 to sites of particle assembly to facilitate egress involvement of vacuolar protein sorting pathway in ebola virus release independent of tsg101 interaction ebola virus matrix protein vp40 interaction with human cellular factors tsg101 and nedd4 interaction of tsg101 with marburg virus vp40 depends on the pppy motif, but not the pt/sap motif as in the case of ebola virus, and tsg101 plays a critical role in the budding of marburg virus-like particles induced by vp40, np, and gp hepatitis b virus maturation is sensitive to functional inhibition of escrt-iii, vps4, and γ2-adaptin mumps virus matrix, fusion, and nucleocapsid proteins cooperate for efficient production of virus-like particles evidence for a new viral latedomain core sequence, fpiv, necessary for budding of a paramyxovirus requirements for budding of paramyxovirus simian virus 5 virus-like particles l-domain flanking sequences are important for host interactions and efficient budding of vesicular stomatitis virus recombinants ppey motif within the rabies virus (rv) matrix protein is essential for efficient virion release and rv pathogenicity the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus ub surprised: viral ovarian tumor domain proteases remove ubiquitin and isg15 conjugates ovarian tumor domain-containing viral proteases evade ubiquitin-and isg15-dependent innate immune responses antiviral activity of innate immune protein isg15 role of nedd4 and ubiquitination of rous sarcoma virus gag in budding of virus-like particles from cells structural basis for ubiquitin-like isg 15 protein binding to the ns1 protein of influenza b virus: a protein-protein interaction function that is not shared by the corresponding n-terminal domain of the ns1 protein of influenza a virus influenza b virus ns1 protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg15 protein escrt-iii mediates budding across the inner nuclear membrane and regulates its integrity general pharmacological properties of the new proton pump inhibitor restoration of acid secretion following treatment with proton pump inhibitors targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases figure 1 . thermal shift data of tsg101 by lead compound tenatoprazole (n16). the compound caused a dose-dependent shift in the tm for tsg101-uev indicating binding to the key domain of tsg101 as described in materials and methods. supplemental figure fig . s1 effect of adding dtt to the thermal shift of tsg101-uev in the fts assay. the addition of dtt abolishes the tm shift in the fts assay. this is consistent with all of these prazole compounds forming a disulfide bond to tsg101. key: cord-279849-zzkliu76 authors: dapalma, t.; doonan, b.p.; trager, n.m.; kasman, l.m. title: a systematic approach to virus–virus interactions date: 2010-01-20 journal: virus res doi: 10.1016/j.virusres.2010.01.002 sha: doc_id: 279849 cord_uid: zzkliu76 a virus–virus interaction is a measurable difference in the course of infection of one virus as a result of a concurrent or prior infection by a different species or strain of virus. many such interactions have been discovered by chance, yet they have rarely been studied systematically. increasing evidence suggests that virus–virus interactions are common and may be critical to understanding viral pathogenesis in natural hosts. in this review we propose a system for classifying virus–virus interactions by organizing them into three main categories: (1) direct interactions of viral genes or gene products, (2) indirect interactions that result from alterations in the host environment, and (3) immunological interactions. we have so far identified 15 subtypes of interaction and assigned each to one of these categories. it is anticipated that this framework will provide for a more systematic approach to investigating virus–virus interactions, both at the cellular and organismal levels. by design, viral infections in the laboratory almost always occur in the absence of other viruses. while this may be a logical starting point for virus research, viral infections in nature rarely occur in isolation. polymerase chain reaction (pcr)-based techniques have revealed that persistent viral infections are present in all domains of life. archaea can be hosts to several types of phage viruses (snyder et al., 2003) . most environmental and commensal bacterial isolates are infected with one or more phage viruses (ackermann and kropinski, 2007) . yeasts, including endomyces magnusii (pospisek et al., 1994) and even laboratory strains of saccharomyces cerevisiae harbor multiple types of endogenous rna viruses (fujimura and esteban, 2007) . filamentous fungi are similarly affected (nogawa et al., 1993) . phytoplankton are commonly infected by dsdna viruses (bellec et al., 2010) , while both wild and domesticated vascular plants have been shown to carry numerous persistent viral infections, including badnaviruses (kenyon et al., 2008) . finally, genomic sequencing of animals has shown that chromosomes of both invertebrates and vertebrates are permeated with genetic sequences that resemble integrated retroviruses, in various states of viability (gifford and tristem, 2003) . approximately 8% of human genomic dna, for example, is derived from retroviral genomes (tristem, 2000) . however, persistent infections in animals are not limited to retroviruses. the picorna-like nora virus has been detected in all drosophila melanogaster strains tested, both laboratory and wild caught (habayeb et al., 2006) , and life-long herpesvirus infections are well known in humans (chen and hudnall, 2006; pancharoen et al., 2001) as well as marine bivalves, fish, reptiles, birds, and other mammals from mice to elephants (vandevanter et al., 1996) . therefore in any individual, persistent, sometimes congenital, often asymptomatic viral infections provide a background onto which all future viral infections are superimposed. given the now significant evidence for ubiquitous endogenous viral infections in all domains of living things, virus coinfections would seem to be the rule rather than the exception in nature, if not the laboratory. yet, they have rarely been systematically studied for possible effects of one virus on the other, with most documented interactions having been discovered accidently. it is certainly true that coinfection may not result in interactions for all virus species. however, because many viruses induce profound changes in their host, it seems likely that virus-virus interactions are common and may be critical to understanding viral pathogenesis and evolution. therefore, in this review we identify known and potential types of virus-virus interactions (vvis) and organize them into three categories: (1) direct interactions of viral genes or gene products, (2) indirect interactions that result from alterations in the host environment, and (3) a subset of indirect interactions called immunological interactions, unique to organisms equipped with an adaptive immune system. we have so far identified fifteen subtypes of vvi and assigned each to one of the three major categories (table 1) . this framework provides a systematic approach to investigating vvi, both at the cellular and organismal levels. as a first step, it is necessary to define vvi. we define a vvi as a measurable difference in the course of infection of one virus as a result of a concurrent or prior infection by a different species or of course the simple case of a prior viral infection conferring protective immunity against future infections with an immunologically identical virus is well known. but this interaction follows directly from the role of the adaptive immune system, and is therefore a rather uninteresting sort of interaction. a trivial case also exists with virus-induced generalized immunosuppression, as seen with human immunodeficiency virus (hiv)/aids. generalized immunosuppression, regardless of the cause, results in increased replication and pathology by some viruses that have a benign disease course in immunocompetent persons. however, this effect is not specific to viral pathogens. therefore, the discussion of vvi involving virus-induced immunosuppression will be limited here to those in which there is evidence of other viruses impacting the course of infection of the immunosuppressive virus, or evidence of direct interaction of proteins or nucleic acids of the immunosuppressive virus with proteins or nucleic acids of a coinfecting virus (lisco et al., 2009) . these uninformative examples aside, we have collected reports of vvi involving unexpected direct, environmental, and immunological interactions and used them to create what is intended to be a growing online database of these important phenomena. in the sections below, the current subtypes of vvi are each described and illustrated with a few examples. some categories have many more currently known examples than those described here, which are included in the online database. we do not mean to represent either the examples below nor the online list as a complete collection. instead, our aim is to provide the framework to launch a publicly available collection of vvi which allows continuous updates by and for the scientific community. we define a direct vvi as an occurrence in which nucleic acids or proteins of one virus physically interact with the genes or gene products of a coinfecting virus. this definition encompasses six different subtypes of interaction: helper viruses, pseudotype viruses, superinfection exclusion, genomic recombination, embedded viruses, and heterologous transactivation. direct interactions require coinfection of the same cell to take place, but the infections need not take place close in time. in several cases of documented direct interactions described below, cells latently infected with one virus may be infected by the second virus years, or even millenia later if the initial viral genome is incorporated into the germline of the host. a helper-dependent virus is any virus that is replication defective on its own, and therefore requires the gene products of another virus to produce infectious progeny. engineered recombinant helper virus/helper-dependent virus pairs have been developed as safe viral vector technologies (dorigo et al., 2004; marconi et al., 2008) , but several natural pairs of such viruses are also documented. one of the first helper-dependent viruses described was bacteriophage p4, a bacteria-infecting virus that is able to replicate its own genome, but requires the presence of a coinfecting bacteriophage, such as p2, to provide capsid components and cell lysis (shore et al., 1978; six and klug, 1973) . in plants, the carrot mottle virus of the umbravirus genus has been shown to be dependent upon viruses of the luteoviridae family for encapsidation and transmission by aphids (waterhouse and murant, 2008) . a mammalian helper-dependent virus is adeno-associated virus (aav), a replication-defective parvovirus that generally requires a host cell that is coinfected with an adenovirus or herpesvirus in order for virions to be produced and escape the host cell (buller et al., 1981; goncalves, 2005) . since the original description of aav, it has been discovered that genotoxic stress and other factors may also make a host cell permissive for aav progeny production, indicating that aav is not entirely replication defective (meyers et al., 2000; yalkinoglu et al., 1988) . a more clearly defined case is that of hepatitis d virus (hdv), a human pathogen. originally, discovered in the 1970s as a subtype of hepatitis b virus (hbv), further studies led to the understanding that hdv only occurred in hbv infected individuals. although the small viroid-like hdv is able to reproduce its own rna and ribonucleoprotein capsids, it requires the hbv membrane glycoprotein, hepatitis b surface antigen (hbsag) in order to associate with cell membranes and bud as infectious particles (rizzetto, 2009) . therefore, naturally occurring helper-dependent viruses appear to be dominated by non-enveloped viruses that can replicate their genome autonomously and only require a helper virus for packaging and/or release. it should be noted that the interaction between a helperdependent virus and its respective helper virus is not necessarily unidirectional. both aav and p4 significantly inhibit the replication of their helper viruses (barrett et al., 1973; timpe et al., 2006) . in contrast, hdv coinfection with hbv results in increased activity of both viruses, often resulting in a more severe clinical course than hbv alone (rizzetto, 2009 ). virus pseudotyping occurs when two species of virus coinfect a host cell and progeny virions are produced that contain nucleic acid of one parental virus and some structural proteins of the other parental virus. this phenomenon, also called phenotypic mixing, may involve capsid proteins, or in the case of enveloped viruses, only membrane glycoproteins (zavada, 1976) . various bacteriophage, plant viruses, and animal viruses have all been observed to produce phenotypically mixed progeny from coinfections (zavada, 1976) . some coinfections result in pseudotyped virions from both parental genomes, while other interactions result in pseudotyped virions of only one type (certo et al., 1998) . for example coinfection of human syncytiotrophoblasts with human cytomegalovirus and human t-cell leukemia-lymphoma virus (htlv-i) results in htlv-1 capsids within cmv envelopes, but not the reverse (toth et al., 1995) . pseudotyped viruses are distinct from helper-dependent virus interactions since neither virus requires components of the other to complete its replication cycle. envelope glycoproteins are the main receptor binding proteins of most enveloped viruses, and therefore pseudotyped virions often have an expanded host range, able to infect the targets of both parental viruses. two herpesviruses have been shown to produce pseudotyped human immunodeficiency virus (hiv) or htlv-1 virions when coinfected with these retroviruses (heng et al., 1994; toth et al., 1995) . in addition to a hybrid virion morphology, these pseudotyped retroviruses possess an expanded host cell range: herpes-pseudotyped hiv is capable of infecting keratinocytes (heng et al., 1994) . this feature of pseudotyped viruses has been used to advantage in the production of recombinant viral vectors, to expand the range of targets they are able to infect (funke et al., 2009; li et al., 2009; wu et al., 2009 ). a third type of direct vvi is viral superinfection exclusion, which occurs when a primary viral infection induces resistance to subsequent infections by similar viruses. superinfection exclusion is known to occur among bacteriophage, retroviruses, hepadnaviruses, arboviruses, and plant viruses (brindley et al., 2008; geib et al., 2003; mcallister and barrett, 1977; nethe et al., 2005; saumet and lecellier, 2006) . mechanisms of exclusion are diverse and have not been determined in all cases, but all mechanisms described so far depend on direct interaction of products of the primary infection with the secondary infecting virus. for example, the cytoplasmic accumulation of borna disease virus (bdv) nucleocapsid components prevents a subsequent infection of a different bdv strain or arbovirus type through interference with the polymerase of the secondary virus, inhibiting early viral multiplication steps (geib et al., 2003) . the sim protein of bacteriophage p1 appears to block injection of nucleic acid by superinfecting phage at the cell membrane (kliem and dreiseikelmann, 1989) , and the equine infectious anemia virus secretes a soluble protein that masks its cell surface receptor, blocking binding of subsequent viruses (brindley et al., 2008) . in plants, interaction of heterologous viral messenger rna molecules which contain sequence homology induces destruction of both messages by the rna silencing mechanism and inhibition of replication (saumet and lecellier, 2006) . although most examples of superinfection exclusion deal primarily with different strains of the same virus, and inhibition of superinfection is not absolute, better understanding of this vvi has the potential to impact future antiviral development (federico et al., 1996) . coinfections with two or more strains of the same virus species in the same host cell can result in progeny virions that are genetic recombinants of the parental viruses. this phenomenon is arguably the vvi with the most serious consequences for human health. influenza virus coinfections, by virtue of the virus's segmented genome, readily produce recombinant progeny (gardner and shortridge, 1979; nelson et al., 2008) . worldwide surveillance networks monitor the appearance of recombinant influenza viruses as the dramatic antigenic shift that results often produces a virus to which few people have immunity, increasing the risk of a pandemic (nuzzo and lam, 2006) . however, due to the relatively short course of influenza infections, coinfections are relatively uncommon (nelson et al., 2008) . human immunodeficiency virus (hiv) in contrast, is a life-long infection and both coinfections (secondary infection prior to seroconversion) and superinfections are well documented. hiv genomic recombination has been shown to facilitate immune escape (streeck et al., 2008) , evolution of replicationdefective hiv variants (iwabu et al., 2008) , and the spread of drug resistance (burke, 1997) , all of which complicate hiv control. life-long herpesvirus infections also result in homologous recombination between strains in vivo, with similar effects on immune control and drug resistance (chou, 1989; haberland et al., 1999; poole et al., 1999) . more recently, recombination between attenuated poliovirus vaccine strains and virulent wild enterovirus strains has led to regeneration of virulent polioviruses and cases of poliolike paralysis in regions targeted for poliovirus eradication (arita et al., 2005; rakoto-andrianarivelo et al., 2008) . although not as carefully monitored as human pathogens, recombination-driven altered virulence of viral pathogens of crop plants has also been reported (ogawa et al., 2008) . whereas genomic recombination involves nucleic acid transfer between viruses with significant sequence homology and similar if not identical genomic organization, embedded viruses are retroviruses that have integrated themselves into the genomes of unrelated, large dna viruses. presumably, integration is random but only those integrations that leave the dna virus capable of productive infection propagate and are detected. two examples, a moth retroviral element embedded into the genome of autographa californica nuclear polyhedrosis virus (friesen and nissen, 1990) , and reticuloendotheliosis virus embedded in the fowlpox genome (hertig et al., 1997) , illustrate that the effects of embedded viruses can be multifaceted. in both cases, the embedded retrovirus gains an alternative transmission and entry pathway to hosts, but in the case of the retrovirus embedded in the baculovirus, retroviral gene expression is also activated (friesen and nissen, 1990) . and for reasons not yet understood, the fowlpox strains carrying the embedded reticuloendotheliosis virus cause immunosuppression in chickens while reticuloendotheliosis virus-free strains do not (wang et al., 2006) . the final type of direct vvi for which we find documentation, involves transactivation of the genes of one virus species by gene products of a heterologous virus. many viruses encode powerful promoters and transactivating proteins in order to appropriate the cellular transcription machinery for maximum viral gene expression. direct binding and transactivation of a heterologous viral promoter is documented in the case of cytomegalovirus transactivating protein ie2-86 which binds to the −120 to 20 region of the hiv-1 long terminal repeat (yurochko et al., 1999) and in the case of human foamy virus bel1 protein which recognizes and binds to the −158 to −118 region of the hiv-1 long terminal repeat (lee et al., 1992) . epstein-barr virus (ebv) and hepatitis c virus (hcv) coinfection results in significantly higher hcv production than hcv infection alone. it is known that the ebv gene product responsible for enhanced hcv replication is the transcriptional activation protein ebna1 (ebv-encoded nuclear antigen-1) (sugawara et al., 1999) and it therefore seems likely that ebna-1 enhances hcv replication by direct transactivation, however, the targeted genes in hcv have not been identified. it is possible, however, that ebna1 activates hcv genes indirectly, as discussed in more detail below. post-transcriptional heterologous transactivation also takes place. herpes simplex virus protein us11 is an rna binding protein that controls post-transcriptional expression of herpes simplex genes. however, during coinfections it also binds and controls splicing of htlv-1 and hiv-1 transcripts normally controlled by the retroviral proteins rex and rev, respectively (diaz et al., 1996) . viral infections can cause many pathogenic changes in the host. often seen during dual infections is acceleration of disease, because of the compounded nature of the two viral cytopathic effects affecting the host in a negative manner. in this section, indirect vvi resulting from alterations in the host environment created by pre-existing or simultaneous coinfections are explored. five subtypes of indirect environmental vvi are currently recognized: indirect transactivation of genes, breakdown of host physical barriers against infection, altered receptor expression, heterologous activation of antiviral pro-drugs, and modification of the interferon-induced antiviral state. while direct binding and activation of viral transactivating proteins to heterologous viral promoters has been documented, more common are reports of viral infections inducing increased expression or activation of cellular transcription factors, which then act on promoters of coinfecting viruses. for example, hepatitis b virus xprotein transactivates promoters containing kappa-b like enhancer elements, both cellular and viral, but not by binding to the elements itself (twu et al., 1989) . human herpes virus 6 (hhv-6) infection of epstein-barr virus (ebv)-infected cells results in transactivation of the ebv zebra gene, but the transactivation appears to be mediated by cellular transcription factors on the ebv promoter (flamand and menezes, 1996) . the human endogenous retrovirus k (herv-k) ltr is transactivated by hsv-1 protein icp0 via increased binding of cellular transcription factor, ap-1 (kwun et al., 2002) . cmv ie1 protein also transactivates an ltr, the hiv ltr, but does so by inducing increased binding of nf-kappa b to the ltr sequence by an unknown mechanism (kim et al., 1996) . a vvi involving another ebv latency protein, lmp2a, illustrates the potential complexity of indirect virus-virus transactivation interactions. ebv infection, and subsequent lmp2a expression in human cells, results in the activation of a superantigen gene encoded by the long integrated and inactivated human endogenous retrovirus k18 (herv-k18) with serious clinical consequences (hsiao et al., 2006) . however, lmp2a appears to cause gene expression from the remnants of this ancient viral infection by binding to an enhancer 13 kb downstream and transactivating a cellular gene encoded on the opposite strand. transcription of the cellular gene most likely displaces repressors on the herv-k18 promoter sequences, resulting in transcription of its env superantigen gene (hsiao et al., 2009) . in summary, indirect heterologous transactivation is probably the most common vvi, with many interactions yet to be discovered. the diversity of mechanisms they represent and our present incomplete understanding of transcriptional control mechanisms increase the challenge of identifying such interactions. most have been investigated because a coinfection was observed to exacerbate a viral disease. there are potentially other cases of indirect heterologous transactivation that reduce pathology, but are more difficult to detect. viral replication and progeny production are often characterized by cytopathic effects. tissue damage that results can compromise physical barriers within the host, allowing secondary infections to gain access to otherwise protected tissues. this type of vvi has been observed in plants, specifically zucchini squash (cucurbita pepo). some cucumber mosaic virus strains infect zucchini squash plants but only cause localized infections. however, in plants coinfected with cucumber mosaic virus and zucchini yellow mosaic virus, the long distance movement of the cucumber mosaic virus is facilitated and systemic infection of both viruses is observed (choi et al., 2002) . this synergistic effect, which may also be mediated by viral movement proteins (melcher, 2000) , is readily observed by the overall deterioration of the plant as well as by molecular analysis (choi et al., 2002) . examples of this type of vvi also exist for animal viruses. humans infected with herpes simplex viruses 1 or 2 (hsv-1 or -2) have a higher susceptibility for acquisition of hiv, and a higher possibility of transmission of hiv to other persons sheffield et al., 2007) . both of these situations are associated with the ability of hsv-2 to cause open skin lesions and to recruit cd4+ t cells to the sites of these lesions (celum, 2004) . the recruitment of these cells makes more potential host cells available for acquisition of hiv in a herpesvirus lesion than are found in a traumatic lesion. in the case of someone already hiv infected, active hsv coinfection increases the probability of hiv transmission because infected cd4+ t cells are recruited to the open hsv lesion, increasing production of infectious virus at the skin surface (celum, 2004) . another example of barrier compromise allowing increased viral spread involves cytokine mediated tissue damage. atencio et al. showed that newborn balb/c and nih swiss mice coinfected with wild-type polyomavirus (a2 strain) and moloney murine leukemia retrovirus (m-mulv) exhibit growth inhibition and kidney inflammation, whereas either virus alone rarely produced these symptoms. coinfection was found to elevate cytokines il-6, ifn-␥, il-1␤ and il-10 early after infection (7 days) much more than single infections, and may be responsible for the kidney inflammation and runting (atencio et al., 1995) . the density of viral receptors on a prospective host cell is a significant factor in determining whether infection is successful (agnello et al., 1999; li et al., 1999) . human immunodeficiency virus, for example, binds to a complex of cd4 protein and either ccr5 or cxcl4 as its receptor, and it therefore almost exclusively infects human cd4+ t cells. coinfections have been shown to alter the cell types infected by hiv by altering expression of cd4 or the co-receptors ccr5 or cxcl4. human herpes virus 6 (hhv6) has multiple effects in this system. it upregulates cd4 expression on t cells that are already cd4+ increasing their susceptibility to the hiv virus, but it also induces expression of cd4 on the surface of cd8+ t cells making them susceptible to hiv infection as well (lusso et al., 1991) . in addition, hhv-6 coinfection boosts the production of the ccr5 ligand, rantes, which binds to ccr5 and inhibits the complex formation between ccr5 and cd4 needed for hiv to infect cells. exogenous rantes alone, can mimic this inhibitory effect of hhv6 on hiv infection, but it is only inhibitory to hiv strains that utilize ccr5 as a co-receptor, not cxcl4-tropic strains (grivel et al., 2001) . human herpes virus 7 (hhv7) infection also alters cell surface receptor expression in a manner protective against hiv. hhv7 is a t-lymphotrophic virus which also utilizes cd4 as a receptor, and competes directly with hiv for binding sites on host cells. in a host first infected by either hiv or hhv7, cd4 expression on t cells is down-regulated, slowing the spread of a subsequent infection by the other virus (lisco et al., 2007; lusso et al., 1994) . a fourth indirect mechanism by which vvi alter infection outcomes by affecting the host environment is the activation of pro-drugs with antiviral activity. many nucleoside analog antiviral drugs, such as acyclovir, gancyclovir, and famcyclovir, specifically target herpesvirus infected cells because they must be phosphorylated by herpesvirus-encoded kinases or phosphorylases before becoming active. once activated, the drugs can be incorporated into nascent herpesvirus genomes by viral polymerases, where they act as chain terminators, preventing replication. recently, it was shown that acyclovir can be activated by one virus and act on another . hiv, lacking a thymidine kinase is usually unaffected by acyclovir. however, in herpesvirus and hiv dual infected cells, acyclovir decreases the replication of hiv as well as the herpesvirus. acyclovir is phosphorylated by herpesvirus kinases and then moves to directly inhibit hiv reverse transcriptase, having an unintended but beneficial effect for the host . a fifth category of virus-induced change in the host environment that may affect coinfecting viruses involves the innate immune mechanism induced in vertebrates by type i interferons known as the antiviral state. the antiviral state consists of increased expression of a combination of enzymes, which if activated, shut down cellular translation (galligan et al., 2006; staeheli, 1990) . the most critical of these enzymes are pkr and 2 -5 oligoadenylate synthetase (2 -5 oas). protein kinase r (pkr) has multiple roles in a cell, but its role in the antiviral state is to phosphorylate eukaryotic translation initiation factor 2 alpha (eif2␣), inactivating it and shutting down protein synthesis (garcia et al., 2007) . the 2 -5 oas synthesizes unique oligonucleotides which activate rnasel, initiating destruction of cellular and viral rna molecules necessary for translation. activation of both enzymes is dependent on the presence of molecules associated with infection, particularly dsrna, and their activation usually results in cell death (staeheli, 1990) . animals with defective type i interferon signaling pathways, pkr, or 2 -5 oas are much more susceptible to viral infections, indicating the effectiveness of the antiviral state (levin and hahn, 1985) . however, most viruses have also evolved antagonists of pkr and or 2 -5 oas (hengel et al., 2005; langland et al., 2006; levy and garcia-sastre, 2001) . from this information it would seem logical to speculate that an antiviral state antagonist from one virus could benefit a coinfecting virus, and this has been shown to be the case in several in vitro systems. murine cytomegalovirus (mcmv) has two proteins that are known to inhibit pkr, m142 and m143. when these proteins are present and active the virus can readily replicate in the host. in the absence of these two proteins, the antiviral state is activated and mcmv replication is inhibited (budt et al., 2009 ). this antiviral state activation can be overcome by introducing the pkr inhibitors encoded by vaccinia virus (e3l) or herpes simplex virus (icp gamma 34.5) (budt et al., 2009) . a herpes simplex mutant lacking icp gamma 34.5, in turn can be rescued in cv-1 cells by coinfection with the polyomavirus sv40, due to the sv40 large t antigen's inhibitory effect on the antiviral state downstream of icp gamma 34.5 (randazzo et al., 1997) , or by inserting human cytomegalovirus genes for pkr antagonists, trs1 and irs1 (cassady, 2005; shah et al., 2007) . one possible natural example of this type of vvi involves the interaction of hepatitis b and hepatitis d viruses. the hepatitis d genomic rna molecule inhibits pkr-mediated inhibition of translation in a cell free translation system, suggesting that it may protect its helper virus, hbv, from the antiviral state (robertson et al., 1996) . although the known examples of this virus-virus interaction are so far only demonstrated in artificial systems, given the multiple in vitro examples it seems likely that this type of vvi also occurs in nature. as the third main category of vvi, we define a subset of indirect virus-virus interactions that occur only in host species with an adaptive immune system. we set these types of interactions apart, because unlike the other indirect vvi, which are dependent on an overlap of the periods of infection of two viruses, immunological interactions can occur between viral infections that are completely separated in time. this is possible because the adaptive immune system of the host organism is permanently changed by its interaction with a virus, even if that infection is completely eliminated from the host, and is changed in a manner very specific to the species and strain of the infecting virus. at present, four types of immunological vvi have been identified. these include altering the activation state of cellular components of the immune system, and induction of autoimmune responses to self-antigens that cross-react with viral antigens. in addition, the humoral immune response to viral pathogens can unexpectedly give rise to antibodydependent enhancement (ade) of subsequent viral infections. and finally, coinfections, as well as sequential infections, also indirectly interact by re-shaping the t cell memory repertoire such that the immune response induced by one infection can impact the outcome of a subsequent viral infection in an interaction termed heterologous immunity (welsh and selin, 2002) . one means by which a virus may sensitize a host for a subsequent infection is by altering the activation state of potential host cells. hiv infection, for example, is associated with human cytomegalovirus (hcmv) infection in part due to hiv's induction of elevated numbers of activated lymphocytes in certain tissues. since activated lymphocytes are permissive for hcmv infection, this hiv-induced activated cellular state results in a two-to threefold enhancement of hcmv replication in these tissues . another example is seen with lactate dehydrogenaseelevating virus (ldv). ldv stimulates polyclonal b lymphocyte activation, resulting in delayed induction of antibodies needed to control other coinfecting viruses. consequently, friend virus (fv) infection, which is normally asymptomatic in mice due to timely production of neutralizing antibodies, will upon coinfection with ldv propagate and cause symptomatic disease (marques et al., 2008) . the prevalence of viral coinfections may also impact the progression of hiv disease in this manner. specifically, it is not unusual for hiv positive patients to be coinfected with gb virus c (gbv-c) and the persistence of certain genotypes of this virus has been shown to lead to slower hiv progression. (schwarze-zander et al., 2006) . this positive effect for the host appears to be mediated by elevated expression of interferon gamma and the immune cell activation that results. higher viral titers of gb virus c are directly correlated with increased serum interferon gamma, which in turn results in increased numbers of circulating mature dendritic cells that may be controlling the hiv infection (lalle et al., 2008) . sequential viral infections have also been associated with generation of autoimmunity in the host. some viruses are able to break immunological tolerance to "self" by expressing a self-like epitope, but are unable to generate the numbers of autoreactive t cells necessary to trigger an autoimmune response. however, a second infection can, by expanding the autoreactive t cell compartment, cause autoimmunity (welsh and fujinami, 2007) . mice transgenic for a lymphocytic choriomeningitis virus (lcmv) nuclear protein remain healthy when infected with lcmv, but a subsequent infection with either poliovirus or vaccinia virus leads to the development of pancreatic inflammation and autoimmune diabetes (christen et al., 2004; evans et al., 1996) . another example of this type of vvi is associated with enteroviruses such as poliovirus. enterovirus infections have been shown to be a risk factor for several autoimmune diseases including insulin-dependent diabetes mellitus (iddm) (dahlquist et al., 1995; grist et al., 1978; hiltunen et al., 1997; hyoty et al., 1995) . multiple epidemiological studies in humans have established that children that manifest iddm have had more exposures to enteroviruses than healthy subjects (andreoletti et al., 1998; clements et al., 1995; d'alessio, 1992) . interestingly, in countries where a live attenuated polio vaccine which is known to confer cross-protection to enteroviruses (juhela et al., 1998) is used to immunize children, the incidence of iddm is lower than in countries where a killed vaccine that does not induce cross-protection is used. this suggests an association between cross-reactivity of immune responses to different viruses and the development of autoimmune diseases (juhela et al., 1999) . in order to infect animal cells, virus particles usually must bind directly to a specific cell surface molecule in a virus-specific manner (flint et al., 2004) . however, several families of virus are known to take advantage of indirectly binding to the cell surface via crosslinking with antiviral antibodies or virus activated complement components which then bind to host cells bearing fc or complement receptors (takada and kawaoka, 2003) . this process in which increased viral replication is produced by exposure to immune sera, is known as antibody-dependent enhancement (ade) of viral infection. it has been observed in vitro for flaviviruses, coronaviruses and retroviruses (cummings et al., 2005) . mechanisms underlying ade are not fully understood, but seem to include increased efficiency of virus binding to host cells, resulting in higher numbers of infected cells. the most well studied case of ade in humans is dengue hemorrhagic fever (dhf). interestingly, the severe form of dengue illness, which is often fatal, is strongly associated with preexisting heterotypic immunity kliks et al., 1988; sangkawibha et al., 1984) . the presence of non-neutralizing antibodies from a previous dengue infection augment viral growth in vitro (kliks et al., 1989) and in vivo (halstead, 1979) . also, individuals suffering from a secondary dengue virus infection have higher viremia than those with primary infections (vaughn et al., 2000) , and the presence of anti-dengue antibodies in mothers has been associated with the occurrence of dengue hemorrhagic fever in newborns (kliks et al., 1988) . the phenomenon of ade is not unique to dengue virus infections. it has been demonstrated to play a role in west nile virus infections in vitro (peiris and porterfield, 1979) and yellow fever virus in vivo (barrett and gould, 1986) . moreover, infections by two other members of the flavivirus family, the yellow fever virus and the japanese encephalitis virus, have also been shown to be enhanced by ade (gould and buckley, 1989) . this process is also thought to be responsible for the enhanced pathogenicity of viral challenges after vaccination with certain formalin inactivated viral vaccines (porter et al., 1972) , including ones for measles (iankov et al., 2006) , respiratory syncytial virus (ponnuraj et al., 2003) , and rabies (prabhakar and nathanson, 1981) . heterologous immunity gives rise to virus-virus interactions when the outcome of the adaptive immune response to a new viral infection is determined in part by immune memory acquired by the host from prior viral infections. development of a primary adaptive immune response to a pathogen results in an immunological memory, which consists of expanded numbers of long-lived, circulating t and b lymphocytes recognizing epitopes of that specific pathogen (welsh et al., 2004) . due to randomized dna rearrangement processes during the generation of the unique specificities of adaptive immune cells, even genetically identical twins have unique t cell repertoires in their naïve state and therefore show some differences in their responsiveness to the same pathogen. notably, these variations seem to have limited significance for the effectiveness of the immune response to a new infection (welsh et al., 2006) . however, if even a small subset of a t cell memory pool is cross-reactive with antigens of a later encountered pathogen, it will outcompete newly activated t cell clones and dominate that response. that the degree of heterogeneity in the immunological responses between genetically identical hosts could be dramatically influenced in different directions by the history of infections with seemingly unrelated viruses was long unappreciated (welsh and fujinami, 2007; welsh et al., 2006) . however, studies in syngeneic mice have confirmed that the unique identity of memory t cells, raised towards one virus but later activated during an infection with an unrelated heterologous virus, dramatically influence the outcome of the second infection (selin et al., 1996) . this heterologous immunity can result in both beneficial and harmful effects (chen et al., 2001) . in mice, for example, immunity to influenza virus protects the host against vaccinia virus challenge, but enhances the virulence of subsequent cytomegalovirus infections (chen et al., 2003) . while difficult to study in outbred populations, syngeneic animal models allow investigation of immunological memory after heterologous infections. when the diverse viruses lcmv, poliovirus, vaccinia virus, murine cytomegalovirus, and vesicular stomatitis virus were sequentially introduced into syngeneic hosts, the immunological memory against one infection was dramatically altered after each successive infection (selin et al., 1996) . thus, the course of each infection is influenced by the t cell memory pool, and with each infection, the t cell memory to previous encountered agents is modified (welsh and selin, 2002) . the effect of heterologous immunity is seen between many different viruses and disease outcome is dependent on both the nature and the specific order in which the sequentially encountered pathogens were encountered (chen et al., 2003) . although relatively unexplored as a field of study, vvi have already been documented to have significant and unexpected effects on viral disease severity, host range, transmissability, immunopathology, and vaccine effectiveness. increased awareness of the potential for virus-virus interactions and a framework for categorizing different types of interactions as described here, would seem to be necessary steps for achieving better understanding of infectious viral diseases in nature. it has long been noted that many viral infections result in mild disease for most infected individuals, moderate disease for some, and fatal disease for a few. the epidemiology of poliomyelitis, influenza, and the recent west nile virus outbreak in the united states are prime examples of this phenomenon. when occurring in otherwise healthy individuals, these differences in susceptibility have largely been assumed to be governed by cryptic immunological defects, either inherited or acquired (kacprzak-bergman and nowakowska, 2005; trammell and toth, 2008) . while this is likely true in some cases, it is also plausible that some variations in susceptibility are determined by virus-virus interactions. evidence for this is strong in the intensively studied case of hiv/aids (lisco et al., 2009 ). investigation of similar interactions for acute viral infections will be challenging, but may allow better identification and protection of the most vulnerable populations during disease outbreaks. it was not possible, of course, to mention every known virus-virus interaction in this article. rather, the goal was to provide a framework into which all vvi can be organized. an online database has been established (www.musc.edu/vvi/) with the aim of compiling a referenced, searchable, comprehensive list of vvi, actively updated by contributions from the scientific community via moderated forum. we anticipate that the number of vvi subtypes may expand, as during the course of this investigation hints of interactions that seem plausible but are not yet documented were found; for example, stabilization of viral rnas by heterologous viral rna binding proteins. in addition, several cases of vvi described above were discovered simultaneously with a new virus, whose existence was not previously suspected. as a wobble in a star's rotation can indicate the presence of an unseen planet, so the perturbation of a viral replication cycle may indicate the presence of an unknown infection, and a virus-virus interaction. curated list of prokaryotic viruses with fully sequenced genomes hepatitis c virus and other flaviviridae viruses enter cells via low density lipoprotein receptor coxsackie b virus infection and beta cell autoantibodies in newly diagnosed iddm adult patients a sabin 3-derived poliovirus recombinant contained a sequence homologous with indigenous human enterovirus species c in the viral polymerase coding region a model for mixed virus disease: co-infection with moloney murine leukemia virus potentiates runting induced by polyomavirus (a2 strain) in balb/c and nih swiss mice antibody-mediated early death in vivo after infection with yellow fever virus helperdependent bacteriophage p4: a model satellite virus and its implications for animal virology isolation of prasinoviruses of the green unicellular algae ostreococcus spp. on a worldwide geographical scale upregulation of human cytomegalovirus by hiv type 1 in human lymphoid tissue ex vivo an equine infectious anemia virus variant superinfects cells through novel receptor interactions specific inhibition of the pkr-mediated antiviral response by the murine cytomegalovirus proteins m142 and m143 herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication recombination in hiv: an important viral evolutionary strategy a prospective study of dengue infections in bangkok human cytomegalovirus trs1 and irs1 gene products block the double-stranded-rna-activated host protein shutoff response induced by herpes simplex virus type 1 infection genital herpes and human immunodeficiency virus: double trouble the interaction between herpes simplex virus and human immunodeficiency virus nonreciprocal pseudotyping: murine leukemia virus proteins cannot efficiently package spleen necrosis virus-based vector rna memory cd8+ t cells in heterologous antiviral immunity and immunopathology in the lung specific history of heterologous virus infections determines anti-viral immunity and immunopathology in the lung anatomical mapping of human herpesvirus reservoirs of infection systemic movement of a movement-deficient strain of cucumber mosaic virus in zucchini squash is facilitated by a cucurbit-infecting potyvirus reactivation and recombination of multiple cytomegalovirus strains from individual organ donors a viral epitope that mimics a self antigen can accelerate but not initiate autoimmune diabetes coxsackie b virus infection and onset of childhood diabetes dynamic effects of antibody-dependent enhancement on the fitness of viruses a case-control study of group b coxsackievirus immunoglobulin m antibody prevalence and hla-dr antigens in newly diagnosed cases of insulin-dependent diabetes mellitus maternal enteroviral infection during pregnancy as a risk factor for childhood iddm. a populationbased case-control study post-transcriptional transactivation of human retroviral envelope glycoprotein expression by herpes simplex virus us11 protein development of a novel helper-dependent adenovirus-epstein-barr virus hybrid system for the stable transformation of mammalian cells viral infection of transgenic mice expressing a viral protein in oligodendrocytes leads to chronic central nervous system autoimmune disease anti-hiv viral interference induced by retroviral vectors expressing a no producer hiv-1 variant cyclic amp-responsive element-dependent activation of epstein-barr virus zebra promoter by human herpesvirus 6 attachment and entry gene organization and transcription of ted, a lepidopteron retrotranspos on integrated within the baculovirus genome interactions of the rna polymerase with the viral genome at the 5 -and 3 -ends contribute to the 20s rna narnavirus persistence in yeast pseudotyping lentiviral vectors with the wildtype measles virus glycoproteins improves titer and selectivity interferons and viruses: signaling for supremacy the dsrna protein kinase pkr: virus and cell control recombination as a mechanism in the evolution of influenza viruses: a two-year study of ducks in hong kong selective virus resistance conferred by expression of borna disease virus nucleocapsid components the evolution, distribution and diversity of endogenous retroviruses adeno-associated virus: from defective virus to effective vector antibody-dependent enhancement of yellow fever and japanese encephalitis virus neurovirulence enteroviruses in human disease suppression of ccr5-but not cxcr4-tropic hiv-1 in lymphoid tissue by human herpesvirus 6 variation within the glycoprotein b gene of human cytomegalovirus is due to homologous recombination nora virus, a persistent virus in drosophila, defines a new picorna-like virus family in vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody co-infection and synergy of human immunodeficiency virus-1 and herpes simplex virus-1 viruses know it all: new insights into ifn networks field and vaccine strains of fowlpox virus carry integrated sequences from the avian retrovirus, reticuloendotheliosis virus islet cell antibody seroconversion in children is temporally associated with enterovirus infections. childhood diabetes in finland (dime) study group cutting edge: epstein-barr virus transactivates the herv-k18 superantigen by docking to the human complement receptor 2 (cd21) on primary b cells ebv lmp-2a employs a novel mechanism to transactivate the herv-k18 superantigen through its itam a prospective study of the role of coxsackie b and other enterovirus infections in the pathogenesis of iddm immunoglobulin g antibody-mediated enhancement of measles virus infection can bypass the protective antiviral immune response superinfection of defective human immunodeficiency virus type 1 with different subtypes of wild-type virus efficiently produces infectious variants with the initial viral phenotypes by complementation followed by recombination enterovirus infections and enterovirus specific t-cell responses in infancy comparison of enterovirus-specific cellular immunity in two populations of young children vaccinated with inactivated or live poliovirus vaccines influence of genetic factors on the susceptibility to hbv infection, its clinical pictures, and responsiveness to hbv vaccination yams (dioscorea spp.) from the south pacific islands contain many novel badnaviruses: implications for international movement of yam germplasm essential role of nf-kappa b in transactivation of the human immunodeficiency virus long terminal repeat by the human cytomegalovirus 1e1 protein the superimmunity gene sim of bacteriophage p1 causes superinfection exclusion evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever transactivation of the human endogenous retrovirus k long terminal repeat by herpes simplex virus type 1 immediate early protein 0 activation of interferon response genes and of plasmacytoid dendritic cells in hiv-1 positive subjects with gb virus c co-infection inhibition of pkr by rna and dna viruses transactivation of human immunodeficiency virus type 1 long terminal repeat-directed gene expression by the human foamy virus bel1 protein requires a specific dna sequence interferon deficiency syndrome the virus battles: ifn induction of the antiviral state and mechanisms of viral evasion variability of adenovirus receptor density influences gene transfer efficiency and therapeutic response in head and neck cancer immunization with pseudotype baculovirus expressing envelope protein of japanese encephalitis virus elicits protective immunity in mice viral interactions in human lymphoid tissue: human herpesvirus 7 suppresses the replication of ccr5-tropic human immunodeficiency virus type 1 via cd4 modulation coinfecting viruses as determinants of hiv disease acyclovir is activated into a hiv-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues induction of cd4 and susceptibility to hiv-1 infection in human cd8+ t lymphocytes by human herpesvirus 6 cd4 is a critical component of the receptor for human herpesvirus 7: interference with human immunodeficiency virus hsv as a vector in vaccine development and gene therapy b lymphocyte activation by coinfection prevents immune control of friend virus infection superinfection exclusion by bacteriophage t7 the '30k' superfamily of viral movement proteins ubiquitous human adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model multiple reassortment events in the evolutionary history of h1n1 influenza a virus since 1918 retroviral superinfection resistance cloning and characterization of mycovirus double-stranded rna from the plant pathogenic fungus, fusarium solani f. sp. robiniae who issues draft protocol for containing and influenza pandemic the begomoviruses honeysuckle yellow vein mosaic virus and tobacco leaf curl japan virus with dna␤ satellites cause yellow dwarf disease of tomato seroprevalence of epstein-barr virus antibody among children in various age groups in bangkok, thailand. asian pac antibody-mediated enhancement of flavivirus replication in macrophage-like cell lines antibodydependent enhancement, a possible mechanism in augmented pulmonary disease of respiratory syncytial virus in the bonnet monkey model comparison of genetic variability at multiple loci across the genomes of the major subtypes of kaposi's sarcomaassociated herpesvirus reveals evidence for recombination and for two distinct types of open reading frame k15 alleles at the right-hand end the pathogenesis of aleutian disease of mink. ii. enhancement of tissue lesions following the administration of a killed virus vaccine or passive antibody isolation and characterization of the dsrna virus from the yeast endomyces magnusii acute rabies death mediated by antibody reemergence of recombinant vaccine-derived poliovirus outbreak in madagascar herpes simplex virus 1716, an icp 34.5 null mutant, is unable to replicate in cv-1 cells due to a translational block that can be overcome by coinfection with sv40 hepatitis d: thirty years after paradoxical interactions between human delta hepatitis agent rna and the cellular protein kinase pkr risk factors in dengue shock syndrome: a prospective epidemiologic study in rayong thailand. i. the 1980 outbreak anti-viral rna silencing: do we look like plants gb virus c (gbv-c) infection in hepatitis c virus (hcv)/hiv-coinfected patients receiving hcv treatment: importance of the gbv-c genotype reduction of otherwise remarkably stable virus-specific cytotoxic t lymphocyte memory by heterologous viral infections enhanced antiglioma activity of chimeric hcmv/hsv-1 oncolytic viruses effect of genital ulcer disease on hiv-1 coreceptor expression in the female genital tract determination of capsid size by satellite bacteriophage p4 bacteriophage p4: a satellite virus depending on a helper such as prophage p2 viruses of hyperthermophilic interferon-induced proteins and the antiviral state immune-driven recombination and loss of control after hiv superinfection enhancement of hepatitis c virus replication by epstein-barr virus-encoded nuclear antigen 1 antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications effects of adeno-associated virus on adenovirus replication and gene expression during coinfection bidirectional enhancing activities between human t cell leukemia-lymphoma virus type i and human cytomegalovirus in human term syncytiotrophoblast cells cultured in vitro genetic susceptibility and resistance to influenza infection and disease in humans and mice identification and characterization of novel human endogenous retrovirus families by phylogenetic screening of the human genome mapping project database identification of a region within the human immunodeficiency virus type 1 long terminal repeat that is essential for transactivation by the hepatitis b virus gene x dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity evaluation of immune effects of fowlpox vaccine strains and field isolates further evidence on the nature of the dependence of carrot mottle virus on carrot red leaf virus for transmission by aphids pathogenic epitopes, heterologous immunity and vaccine design the privacy of t cell memory to viruses no one is naive: the significance of heterologous t-cell immunity immunological memory to viral infections a pseudotype baculovirus-mediated vaccine confers protective immunity against lethal challenge with h5n1 avian influenza virus in mice and chickens dna amplification of adeno-associated virus as a response to cellular genotoxic stress identification of human cytomegalovirus target sequences in the human immunodeficiency virus long terminal repeat. potential role of ie2-86 binding to sequences between −120 and −20 in promoter transactivation viral pseudotypes and phenotypic mixing key: cord-023841-amfb4jft authors: scherr, johannes title: bewegung und erkrankungen des immunsystems date: 2017 journal: k&#x000f6;rperliche aktivit&#x000e4;t und gesundheit doi: 10.1007/978-3-662-50335-5_17 sha: doc_id: 23841 cord_uid: amfb4jft die effektivität von körperlicher aktivität in der primärals auch sekundärund tertiärprävention ist hinlänglich bekannt. das immunsystem spielt eine entscheidende rolle bei einer vielzahl von erkrankungen, da es durch seine botenfunktion (z. b. durch zytokine) in einer vielzahl der regulationsprozesse mit involviert ist. so kommt es durch moderat-intensive körperliche aktivität zu einer stärkung des immunsystems mit konsekutiv verminderter infektanfälligkeit sowie eher anti-inflammatorischen effekten, wohingegen langandauernde und höher intensive belastungen zu einer schwächung der abwehrfunktion sowie einem pro-inflammatorischen effekt führen. somit stellt eine adäquat dosierte körperliche aktivität eine erfolgversprechende therapieoption bei erkrankungen des infektiologischen formenkreises sowie des immunsystems dar. das chronische erschöpfungssyndrom (chronic fatigue syndrome, cfs) oder auch myalgische enzephalomyelitis (me) ist eine chronische multisystem-erkrankung mit einer prävalenz von ca. 0,3-2,5 % der bevölkerung. hierbei ist bis dato nicht endständig geklärt, ob diese erkrankung ein pathologisch eigenständisches syndrom darstellt (carruthers et al. 2011) , ob die me als eine subform des cfs anzusehen ist (jason et al. 2013 ) oder ob die nichtspezifischen symptome eigentlich auf andere, bisher nicht bekannte erkrankungen zurückzuführen sind. das cfs wurde 1956 erstmalig in england beschrieben, wonach erst nach einem vermehrten auftreten 1988 dieser erkrankung wissenschaftliche beachtung geschenkt wurde (holmes et al. 1988 ). die verwendung unterschiedlicher synonyme (z. b. myalgische enzephalomyelitis) gründet sich in der teilweisen beschreibung unterschiedlicher diagnosekriterien, wobei die am häufigsten verwendeten kriterien von den centers for disease control (cdc) 1994 mit revision 2003 veröffentlicht wurden (fukuda et al. 1994; reeves et al. 2003) . hierbei sind per definitionem zur diagnosestellung 4 der 8 möglichen symptome über einen zeitraum von mindestens sechs monaten notwendig: persistierender kopfschmerz, schlafstörungen, krankheitsgefühl bzw. abgeschlagenheit nach körperlicher aktivität, konzentrationsschwäche, myalgie sowie gelenkschmerzen, schluckbeschwerden und krankhafte schwellung von lymphknoten (lymphadenopathie). in neueren definitionen wird ebenfalls das vorhandensein einer orthostatischen dysregulation als diagnosekriterium gefordert (ganiats 2015) . auch wurde im februar 2015 vom institute of medicine in den usa empfohlen, die termini me/cfs durch den neuen namen systemic exertion intolerance disease (seid) zu ersetzen (iom 2015) . aufgrund der kurzen historie sowie der sich immer wieder ändernden definitionen ist die studienlage in bezug auf cfs bisher sehr gering. so stellen die meisten publikationen präsentationen von fallserien oder studien mit einer kleinen probandenzahl im sinne von pilotstudien dar. die prävalenz für cfs wird in den usa mit 0,3 % angegeben, was einer absolutzahl von ca. 1 million us-bürgern entspricht (reyes et al. 2003) . aufgrund der unterschiedlichen definitionen liegt hier aber eine deutliche schwankungsbreite vor, so dass in anhängigkeit der benutzen kriterien als auch der untersuchten population auch eine prävalenz von 3,3 % möglich sein kann (jason et al. 2012; reeves et al. 2007 ). vor allem aufgrund der neudefinierung der diagnosekriterien dürfte die zahl der an seid erkrankten deutlich ansteigen (jason et al. 2015) . die anzahl der betroffenen gemäß der ursprünglichen definition liegt in deutschland bei ca. 300.000 betroffenen 1 . in der eu sind ca. 1,6 millionen menschen an cfs/ me erkrankt. in bezug auf ein vermehrtes vorkommen in einer ethnizität gibt es aktuell keine hinweise. auch kann das cfs alle altersgruppen betreffen, auch wenn ein altersgipfel zwischen 30 und 45 jahren liegt, wobei frauen zu ca. ¾ betroffen sind (jason et al. 1999 ). wie oben beschrieben, ist der genaue pathomechanismus, der an der entstehung eines cfs/einer me beteiligt ist, bislang nicht geklärt. als eine mögliche ursache wird ein bisher unbekanntes initialereignis diskutiert, welches über den trigger einer immunantwort zu einer dysregulation des immun-und neuroendokrinen systems führt (komaroff u. cho 2011; morris u. maes 2013) . hier konnte bisher kein zusammenhang zu bestimmten proteinen, welche die differenzierung und das wachstum von zellen regulieren, den sog. zytokinen (mit ausnahme von transforming growth factor beta (tgf-b)) mit dem auftreten von cfs nachgewiesen werden (blundell et al. 2015) . auch ein viraler auslöser wie z. b. eine infektiöse mononukleose wurden schon als mögliche pathogenetische ursachen diskutiert. dies begründet darin, dass die meisten patienten mit cfs von einem plötzlichen beginn der symptome nach einem fieberhaften infekt, welcher meist mit einer lymphknotenschwellung (lymphadenopathie) einhergeht, berichten. es konnte jedoch noch kein virus oder ein anderes auslösendes agens identifiziert werden; auch haben nicht alle patienten mit cfs einen vorherigen fieberhaften infekt (komaroff u. cho 2011) . ebenfalls wurde eine erniedrigte periphere sauerstoffaufnahme in die muskelzelle als ursache der belastungsintoleranz und somit ausdruck einer insuffizienten metabolischen adaptation diskutiert (vermeulen u. vermeulen van eck 2014) . aufgrund dieser bisher noch unbekannten pathogenese und hierdurch auch fehlender objektiver klinischer und 17.1 · chronisches erschöpfungssyndrom (myalgische enzephalomyelitis, systemic exertion intolerance disease) laborchemischer parameter kann die diagnosestellung schwierig sein, so dass das cfs eine ausschlussdiagnose darstellt (blundell et al. 2015) . therapie: rolle der bewegung (basiert auf empfehlungen, guidelines, metaanalysen, mit möglichst genauer dosierung) aufgrund der bisher nur unzureichend verstandenen pathogenese sowie der weiterhin diskutierten zugrundeliegenden mechanismen existiert zum aktuellen zeitpunkt noch keine wirklich wissenschaftlich nachgewiesene wirksamkeit eines therapieregimes. hierbei kommen neben symptomatischen auch kausale therapieansätze zur anwendung. letztere basieren vor allem auf pharmakologischen interventionen und kognitiven verhaltenstherapeutischen ansätzen. in bezug auf die medikamentösen therapien wurde bisher die wirksamkeit von immunmodulatoren, antiobiotika sowie antiviralen substanzen untersucht, wobei einschränkend gesagt werden muss, dass die anwendung aller beschriebenen substanzen einen out-oflabel-gebrauch (d. h., eine zulassungsüberschreitende anwendung von medikamenten) darstellt. es konnten hierbei bisher zwei randomisierte und placebokontrollierte studien einen effekt des immunmodulators rintatolimod (als toll-like-receptor 3 (tlr3)-agonist) nachweisen (strayer et al. 2012; strayer et al. 1994) . in studien mit galantamin, hydrocortison, immunglobulin g, valganciclovir, isoprinosin, fluoxetine und verschiedenen komplementärmedizinischen therapeutika konnte bisher kein nachhaltiger nutzen nachgewiesen werden (smith et al. 2015) . als symptomatische behandlungsansätze kommen bisher vor allem nicht-medikamentöse therapien zum einsatz. bei letzteren handelt es sich unter anderem um yoga, bewegungsprogramme, psychologische therapieansätze im sinne einer kognitiven verhaltenstherapie sowie auch um mentalsport und die sog. adaptive-pacing-therapie (apt) . im rahmen der adaptive-pacing-therapie (apt) sollen die betroffenen lernen, die subjektiv zur verfügung stehende energie adäquat so zu nutzen und einzusetzen, dass sie zumindest im stande sind, das wichtigste im alltagsleben selbst erledigen zu können. aufgrund der limitationen der oben genannten »herkömmlichen« therapieansätze erscheinen interventionen mittels körperlicher aktivität bzw. gezieltem training umso wichtiger und auch erfolgsversprechender. jedoch ist auch hier die wissenschaftliche evidenzlage eher gering. so wurden bisher in sehr wenigen studien die effekte von körperlichem training auf verschiedene klinische aspekte des cfs untersucht. als trainingsmethode kamen hier gezielte trainingstherapie mit sukzessiver steigerung der umfänge über die zeit (stufenweise bewegungstherapie, graded exercise therapy (get)), qigong sowie orthostasetraining zur anwendung. in den neueren studien konnte hierbei gezeigt werden, dass die betroffenen, die entweder die stufenweise bewegungstherapie oder die kognitive verhaltenstherapie durchgeführt habe, in bezug auf müdigkeit und physische funktionalität besser abschneiden als betroffene, die an der adaptive-pacing-therapie oder allgemeinärztlicher versorgung alleine teilgenommen haben (white et al. 2011) . in neueren studien konnte ebenfalls eine persistenz dieser effekte über den zeitraum von zweieinhalb jahren nach dem studienbeginn nachgewiesen werden (sharpe et al. 2015) . hierbei kamen in den studien, in denen ein positiver effekt der körperlichen aktivität nachgewiesen werden konnte, jeweils die gleichen bewegungstherapie-schemata zur anwendung (white et al. 2007; fulcher u. white 1997; powell et al. 2001 obwohl es unter der stufenweisen bewegungstherapie auch vorübergehend zu einer symptomverschlechterung kommen kann, würde die abnahme der körperlichen leistungsfähigkeit durch körperliche schonung ebenfalls hierzu führen, so dass eine vermehrte schonung nicht empfohlen bzw. nicht gutgeheißen werden kann. es konnte jedoch bisher nicht in allen studien ein positiver effekt der stufenweisen bewegungstherapie nachgewiesen werden, so dass auch hier noch (vor allem in bezug auf die belastungssteuerung) weitere studien notwendig sind (nunez et al. 2011) . zusammengefasst kann somit aktuell davon ausgegangen werden, dass sich durch gezieltes körperliches training mit adäquater schrittweiser steigerung der belastung sowohl die müdigkeit, die funktionsverluste generell sowie die körperliche leistungsfähigkeit bessern. des weiteren scheint diese behandlungsstrategie neben der kognitiven verhaltenstherapie auch kosteneffektiv in bezug auf das cfs zu sein (mccrone et al. 2012 ). bisher wurden lediglich in einer studie die potenziell negativen effekte im sinne von nebenwirkungen der verschiedenen behandlungsregime untersucht (dougall et al. 2014) . hierbei zeigten sich keine unterschiede zwischen den verschiedenen regimen; vor allem auch keine häufung, weder bei der kognitiven verhaltenstherapie noch bei der stufenweisen bewegungstherapie, so dass diese beiden therapieregime als sicher für cfs-betroffene anzusehen sind. auch eine verschlechterung der klinischen funktionellen beschwerden zeigte sich in diesen beiden regimen am seltensten. es kann hierbei davon ausgegangen werden, dass die beobachteten verschlechterungen auch mehrheitlich den natürlichen verlauf der er-krankung im sinne einer fluktuation und weniger negative reaktionen auf die intervention widerspiegeln. einen negativen einfluss auf das auftreten von nebenwirkungen bzw. verschlechterungen während der behandlung scheinen der schweregrad der erkrankung zu behandlungsbeginn, das vorhandensein einer depressiven störung sowie der bmi zu besitzen. aufgrund der jedoch bisher geringen studienanzahl können auch nur bedingt generalisierte aussagen über nebenwirkungen oder gar kontraindikationen (vor allem auch bei schwerer erkrankten patienten) getroffen werden. gemäß der aktuellen studienlage scheinen sowohl die kognitive verhaltenstherapie als auch die stufenweise bewegungstherapie als therapeutikum geeignet zu sein. letztere bessert vor allem die fatigue sowie die physischen funktionseinschränkungen, so dass diese therapieform vor allem für ambulant führbare patienten geeignet erscheint (larun et al. 2015) . die humanen immundefizienzviren (hiv) hiv-1 und hiv-2 sind vertreter humanpathogener lentiviren in der familie der retroviridae und auslöser der erworbenen immunschwäche aids (acquired immunodeficiency syndrome). sie befallen zellen mit cd4-oberflächenmolekülen, so dass vornehmlich t-helferzellen, aber auch makrophagen, monozyten, epidermale langerhans-zellen und neuronale mikroglia betroffen sind. gemäß falldefinition der europäischen kommission, der who sowie der us-amerikanischen centers for disease control and prevention (cdc) gilt aktuell ein bestätigter positiver hiv-test (gemäß hiv-test-algorithmus mit viertgeneration hiv-1/2 kombinations-immunoassay als suchtest sowie eventuell bei positivem ergebnis gefolgt von einem western-blot-bestätigungstest) als nachweis einer hiv-infektion. nach definition der cdc werden hiv-infektionen anhand der absoluten cd4-zellzahlen (t-helferzellen) in drei verschiedene stadien eingeteilt: stadium 1 (cd4-zellzahl >500 zellen/μl), 2 (cd4-zellzahl 200 bis 499 zellen/ μl) oder 3 (cd4-zellzahl <200 zellen/μl). sollte es zum auftreten einer opportunistischen infektion kommen, ist dies ebenfalls einem stadium 3 gleichzusetzen. sowie gefängnisinsassen anzunehmen (patel et al. 2014 ). die hiv-infektion wird in folgende im weiteren verlauf einer hiv-infektion kommt es zu einer einschränkung der körperlichen leistungsfähigkeit und konsekutiv auch der alltäglichen aktivitäten (zonta et al. 2003; erlandson et al. 2014) . hierbei ist die abnahme der körperlichen leistungsfähigkeit multifaktorieller genese, wobei strukturellen und inflammatorischen myogenen veränderungen, kardiopulmonalen funktionseinschränkungen und physischer dekonditionierung entscheidende rollen zukommen (grau et al.1993; stringer 2000; crystal et al. 2000; johnson et al. 1990 ). auch ist eine hiv-infektion als risikofaktor für eine kardiovaskuläre erkrankung anzusehen. dies liegt zum einen darin begründet, dass sowohl die erkrankung selbst als auch die therapieform der hoch wirksamen anti-retroviralen therapie (haart = »highly active antiretroviral therapy «) zu einer vorzeitigen arteriosklerose führt (di biagio et al. 2012) . der pathomechanismus der daran beteiligten prozesse ist in . abb. 17.1 dargestellt: dies liegt auch daran, dass durch die gute wirksamkeit der haart und damit die transformation der hiv-infektion von einer lebensbedrohlichen zu einer chronischpersistierenden erkrankung die lebenserwartung der hiv-infizierten deutlich gestiegen ist und somit dann auch die folgeerkrankungen bzw. komplikationen sich im laufe der vergangenen jahre und jahrzehnte geändert haben (o'brien et al. 2010 ). trotz der guten wirksamkeit der haart in bezug auf die hiv-infektion ist diese therapieform auch mit mehreren nebenwirkungen in bezug auf den metabolismus (wie z. b. erhöhtes risiko für zentrale fettleibigkeit, glukosetoleranzstörungen/diabetes mellitus, dyslipidämie oder osteoporose) vergesellschaftet, so dass hier ein bewegungstherapeutischer ansatz vielversprechend erscheint. increased atherosclerotic cardiovascular disease positiven effekten auf das immunsystem sind jedoch auch, wie oben beschrieben, die positiven auswirkungen auf die ausdauer, die kraft, die körperkomposition (anstieg der muskelmassen) sowie auf die lebensqualität von bedeutung. in bezug auf die häufigkeit des trainings muss gesagt werden, dass eine gewisse adhärenz/compliance (ca. zwei-bis dreimal pro woche) von wichtigkeit sind, so dass die positiven effekte in vollem umfang zu erreichen sind (perna et al. 1999 ). in den bisher durchgeführten studien konnten die sicherheit wie auch der nutzen von körperlichem training bei hiv-infizierten dargestellt werden. auch bei patienten in fortgeschrittenem stadium scheint es zu keiner negativen beeinflussung der cd4+-zellzahl zu kommen, so dass auch hier die oben genannten positiven effekte überwiegen (gomes neto et al. 2015; stringer et al. 1998; rigsby et al. 1992) , wenngleich hier gesagt werden muss, dass die positiven effekte auf die cd4+-zellzahl wohl vor allem bei patienten im frühen hiv-stadium ausgeprägter zu sein scheinen und in geringerem maße in einem späteren stadium zu finden sind (rigsby et al. 1992 ). körperliche aktivität kann bei hiv-infizierten auf zweierlei wegen zu positiven effekten führen. zum einen kann davon ausgegangen werden, dass die körperliche aktivität positive auswirkungen auf die nebenwirkungen der haart besitzt. da durch diese therapieform vor allem stoffwechselbeeinflussungen zu verzeichnen sind, bietet sich die bewegungsintervention förmlich an. von dieser ist hinreichend bekannt, dass sie sowohl auf die stammbetonte adipositas als auch auf den kohlenhydrat-und lipidstoffwechsel und auf ossäre veränderungen im sinne von osteoporose positive effekte besitzt. zum anderen scheinen die positiven effekte einer bewegungstherapie auf die durch die erkrankung selbst getriggerte vorzeitige arteriosklerose und die zugrundeliegenden inflammatorischen prozesse einen ansatzpunkt zu bieten. hierbei kommt es zu einem direkt durch den hi-virus verursachten schaden des vaskulären endothels sowie begleitend zu einer chronischen inflammation durch die virale hiv-virusreplikation (gomes neto et al. 2015) . somit spielen vor allem die anti-inflammatorischen effekte von moderat-intensiver körperlicher aktivität eine erfolgsversprechende rolle. da eine hiv-infektion jedoch eine erkrankung des immunsystems mit schwächung dessen darstellt, bestehen somit auch befürchtungen, dass bei inadäquater belastung die negativen effekte der körperlichen aktivität die positiven überwiegen. bei adäquat dosierter körperlicher akti-vität (vornehmlich im aeroben intensitätsbereich) ist jedoch von einer stärkung des immunsystems auszugehen (o'brien et al. 2008; gomes neto et al. 2013 ). eine gezielte trainingstherapie bei hiv-patienten ist bis dato recht wenig untersucht, wobei sich anhand der bisherigen studienlage zeigt, dass wohl eine kombination aus ausdauer-zusammen mit krafttraining sowohl physische als auch psychische aspekte verbessert. auch scheint moderat-intensives training einen positiven effekt auf die cd4+-zellzahl und somit auf die erkrankung per se zu besitzen. unter einem infekt der oberen atemwege versteht man eine akute infektionserkrankung der oberen atemwege (d. h., oberhalb des larynx), welche die nase sowie deren nebenhöhlen, den pharynx sowie den larynx betrifft. typischerweise handelt es sich hierbei um tonsillitiden, pharyngitiden, sinusitiden, laryngitiden, otitis media sowie einen grippalen infekt, welcher auch umgangssprachlich als »erkältung« bezeichnet wird. so kommt es vor allem nach anstrengender körperlicher belastung zu einer erhöhten infektanfälligkeit (hellard et al. 2015) . so ist das risiko, an einem urti nach einem anstrengenden wettkampf, wie z. b. einem marathon, zu erkranken, ca. sechsfach erhöht im vergleich zu sportlern, die lediglich trainieren (nieman et al. 1990 ). hierbei scheint es jedoch auch eine gewisse schwelle an dauer/umfang und/oder häufigkeit zu geben, die überschritten werden muss. so konnten bei kürzer andauernden belastungen die oben genannten veränderungen nicht nachgewiesen werden (nieman et al. 1989 ). auch scheint ein guter trainingszustand das erkrankungsrisiko zu reduzieren (nieman et al. 1989 ). die belastungsintensität scheint einen geringeren einfluss auf das immunsystem zu besitzen (diment et al. 2015) . somit kann bei der normalbevölkerung von einem j-förmigen zusammenhang zwischen risiko für das auftreten von infekten und belastungsumfang, -frequenz sowie in geringem maße der intensität ausgegangen werden (nieman 1994) . bei hochleistungssport scheint dieser j-förmigen zusammenhang nicht zu existieren bzw. scheint dieses kollektiv eine erniedrigte anfälligkeit für infekte der oberen atemwege zu besitzen, so dass von einem s-förmigen zusammenhang zwischen infektanfälligkeit und belastungsmodalität ausgegangen werden kann (. abb. 17.2) (malm 2006). ein weiterer zur diskussion stehender ansatz ist eine verschiebung der j-kurve nach rechts zu höheren belastungsintensitäten/-umfängen/-häufigkeiten bzw. abschwächung des einflusses der belastung, was wohl in dem guten trainingszustand von leistungssportlern begründet liegt (. abb. 17.3) (moreira et al. 2009 ). so scheinen hochleistungssportler, welche sich auf einem internationalen leistungsniveau bewegen, eher weniger infektanfällig zu sein (martensson et al. 2014) . ein möglicher umkehrschluss wäre, dass leistungssportler mehr trainieren können, da sie (gemäß des darwinismus) aus genetischer prädisposition mit einem besseren immunsystem ausgestattet und deswegen resistenter gegenüber infekten sind (trammell u. toth 2008) . diese hypothese muss jedoch noch an dem speziellen kollektiv der hochleistungssportler untersucht werden. wie in . abb. 17.4 dargestellt, führt anstrengende körperliche aktivität im blut zu einer reduzierten expression von toll-like receptors, einer erhöhung von il-6 und einer vermehrten katecholamin-und cortisol-produktion. dies hat zur folge, dass es durch eine reduzierte zytokin-produktion von makrophagen und th1-zellen (stellen eine subpopulation der t-helfer-zellen dar) zu einer schwächung der zellvermittelten immunantwort sowie einer geringgradigen inflammationsreaktion kommt (gleeson 2007) . auch kommt es bei lang andauernder höherintensiver körperlicher aktivität zu einem vermehrten oxidativen stress. hierbei ist bekannt, dass oxidativer stress sowohl das angeborene als auch das erworbene immunsystem negativ beeinflusst und somit die infektanfälligkeit erhöhen kann (cannizzo et al. 2011) . hingegen kommt es, wie oben bereits beschrieben, durch moderate bzw. kürzer andauernde aktivität zu einer verbesserten immunabwehr, da im blut sowohl die anzahl von neutrophilen granulozyten und natürlichen killerzellen (als subgruppe der lymphozyten) als auch von toll-like-rezeptoren als auch das immunglobulin a im speichel ansteigen (gleeson 2000) . auch scheint der lysozym-gehalt des speichels, welcher bei grampositiven erregern zu einer lyse der bakterienzellwand führt, durch körperliche aktivität positiv beeinflusst zu sein, so dass hierdurch die unspezifische abwehr des mund-nasen-rachen-raums positiv beeinflusst wird (ligtenberg et al. 2015) . eine detaillierte beschreibung der einflüsse von körperliche aktivität auf das immunsystem ist hier leider nicht möglich, so dass auf weiterführende literatur verwiesen wird (s. hierzu walsh et al. 2011b; dhabhar 2014) . einschränkend muss zu all den oben beschriebenen veränderungen des angeborenen und erworbenen immunsystems (abnahme um ca. 15-25 %) gesagt werden, dass bis zum aktuellen zeitpunkt nicht eindeutig geklärt ist, ob die beobachteten veränderungen durch akute oder chronische körperliche aktivität letztendlich sich auch auf eine veränderte abwehrleistung und somit infektanfälligkeit auswirken. neben infektionen der oberen atemwege kommt es beim erschöpfenden sport jedoch auch zu nicht-infektiösen irritationen der atemwege, welche von der klinik ähnlich wie urtis imponieren, jedoch auf einer lokalen inflammationsreaktion ohne infektiöses pathogen beruhen. so kommt es nur bei ca. 30-40 % der sportler, die urti-symptome selbst angeben, zu einem nachweislichen infektiösen geschehen (spence et al. 2007; cox et al. 2008 ein infekt der oberen luftwege stellt meist eine virale erkrankung (häufig rhinovirus, corona-virus oder rs-virus) dar, die meist nach 3 bis 5 tagen, selten erst nach 2 wochen selbstlimitierend ist. im falle einer erkrankung der oberen atemwege sollte unterschieden werden, ob die symptome lediglich lokal auftreten (d. h., oberhalb des hals/nackens) oder ob eine systemische beeinträchtigung vorliegt. da hier eine imaginäre grenze im bereich des halses/nackens der orientierung zu dienen scheint, hat sich im englischsprachigen raum der begriff »neck check « eingebürgert (harris 2011; purcell 2007) . hierbei gibt es aktuell keine evidenz, dass sich gering-bis moderat-intensive körperliche aktivität für die dauer von 15 bis 30 minuten pro tag negativ auswirkt, wenn die symptome lediglich oberhalb des nackens (wie z. b. rhinorhoe, halsschmerz, schluckbeschwerden, husten) auftreten. im falle eines solchen limitierten auftretens kann die körperliche belastung in abhängigkeit der individuellen verträglichkeit sukzessive gesteigert werden (so lange sich die symptomatik hierunter nicht verschlechtert). sollte es jedoch zum auftreten von symptomen unterhalb des halses (wie z. b. abgeschlagenheit, (mildes) fieber, kopf-, glieder-und muskelschmerzen, erhöhung der entzündungsparameter, tachykardie) kommen, sollte mit belastungsbeginn (auch niedrigintensiv oder nur kurz-andauernd) gewartet werden, bis diese symptome für die dauer von 2 bis 3 tagen ausgeheilt sind (scharhag u. meyer 2014) . neben der normalisierung der klinischen symptomatik sollte auch erst nach normalisierung der inflammationsparameter in der labordiagnostik wieder mit körperlicher belastung begonnen werden. sollten sich hier befunde zeigen, die gegen einen wiederbeginn mit sport sprechen, sollte die situation im zwei-bis dreitägigen intervall re-evaluiert werden (scharhag u. meyer 2014) . als absolute kontraindikationen für körperliches training sind fieber (> 38°c oder 0,5-1°c höher als gewöhnlich) sowie ein unphysiologisch erhöhter ruhepuls (> 10 schläge/ min höher als normal) anzusehen. sollte zu früh oder zu intensiv mit dem training wieder begonnen worden sein, drohen teilweise fatale komplikationen wie beispielsweise eine bakterielle superinfektion oder eine myokarditis. um somit eine zu frühe wiederaufnahme des sports und damit komplikationen zu vermeiden, sollten vor alnach sportfreigabe sollte für den freizeit-wie auch für den leistungssportler das training primär vornehmlich im aeroben intensitätsbereich liegen und, wie oben beschrieben, schrittweise gesteigert werden. sollte sich der aufbau des umfangs komplikationslos darstellen, können im weiteren verlauf auch die intensitäten sukzessive von aerob über aerob-anaerob zu anaeroben anteilen gesteigert werden. sollte die maximale belastbarkeit (sowohl in bezug auf umfang als auch auf intensität) wieder voll gegeben sein (im vergleich zu vor der erkrankung; dies ist meist nach ein bis zwei wochen in abhängigkeit der schwere der erkrankung der fall), kann auch wieder an wettkämpfen (»return to competition «) teilgenommen werden. anhand der aktuellen datenlage kann davon ausgegangen werden, dass regelmäßige, vorwiegend aerobe aktivität das immunsystem stärkt und somit das risiko für infekte der oberen atemwege reduziert. hingegen führen erschöpfende akutbelastungen, welche vor allem lange andauern bzw. in kurzer folge aufeinander folgen, eher zu einer immunsuppression mit konsekutiv erhöhter infektanfälligkeit. streptokokken-angina einfach, bzw. teilweise ist die diagnosestellung schwierig. diese ist jedoch essentiell, da sich die behandlungsregime grundlegend unterscheiden und deswegen eine möglichst frühe und eindeutige diagnosestellung notwendig ist. als komplikation kommt es bei ca. 50 % der im-erkrankten zu einer splenomegalie. die gefahr einer spontanruptur der milz hierunter beträgt (vor allem in den ersten 3 wochen) ca. 0,5 %. aufgrund des erhöhten risikos kann es hierbei auch schon bei leichten traumata zu rupturen kommen, so dass jegliches trauma oder plötzliche intraabdominelle druckerhöhungen nach möglichkeit vermieden werden sollten. die therapieoptionen für die im sind eher limitiert und beschränken sich auf adäquate ruhe, hydration und analgesie sowie symptomatische therapie. sollte ein antibiose aufgrund möglicher bakterieller superinfektionen notwendig sein, sollte hierbei nach möglichkeit sowohl auf penicillin oder aminopenicilline wegen der gefahr eines juckenden, mehrere tage bestehenden exanthems verzichtet werden. als algorithmus zur freigabe bei leistungssportlern in bezug auf sport wird folgendes vorgehen (mit allerdings eher geringem evidenzlevel aufgrund der nur limitiert vorhandenen literaturlage) wie in . abb. 17.6 dargestellt vorgeschlagen. für freizeitsportler sollte mehr zeit eingeplant werden, so dass hier primär der linke pfad der . abb. 17.6 (»sportler außerhalb der saison«) zur anwendung kommen sollte. kontaktsport oder sport mit erhöhung des intraabdominellen drucks sollte im freizeit-, präventionsoder rehabilitationssport frühestens 4 wochen nach normalisierung der milzgröße wiederaufgenommen werden. aufgrund der deutlichen interindividuellen varianz können oben genannte empfehlungen allerdings nur als entscheidungshilfe gesehen werden; die jeweilige entscheidung sollte individuell getroffen werden. eine vollständige ausheilung dauert im mittel ca. 2 bis 3 monate, vereinzelt auch länger. determination of future prevention strategies in elite track and field: analysis of daegu 2011 iaaf championships injuries and illnesses surveillance return to play after infectious mononucleosis chronic fatigue syndrome and circulating cytokines: a systematic review oxidative stress, inflamm-aging and immunosenescence myalgic encephalomyelitis: international consensus criteria clinical and laboratory evaluation of upper respiratory symptoms in elite athletes physical and role functioning among persons with hiv: results from a nationally representative survey effects of stress on immune function: the good, the bad, and the beautiful hiv and accelerated atheroprogression: role of antiretroviral therapy exercise intensity and duration effects on in vivo immunity adverse events and deterioration reported by participants in the pace trial of therapies for chronic fatigue syndrome functional impairment, disability, and frailty in adults aging with hiv-infection the economic burden of non-influenza-related viral respiratory tract infection in the united states the chronic fatigue syndrome: a comprehensive approach to its definition and study. international chronic fatigue syndrome study group randomised controlled trial of graded exercise in patients with the chronic fatigue syndrome redefining the chronic fatigue syndrome mucosal immune responses and risk of respiratory illness in elite athletes immune function in sport and exercise respiratory inflammation and infections in high-performance athletes a systematic review of the effects of different types of therapeutic exercise on physiologic and functional measurements in patients with hiv/aids effects of combined aerobic and resistance exercise on exercise capacity, muscle strength and quality of life in hiv-infected patients: a systematic review and meta-analysis human immunodeficiency virus type 1 infection and myopathy: clinical relevance of zidovudine therapy infectious disease in athletes trainingrelated risk of common illnesses in elite swimmers over a 4-yr period chronic fatigue syndrome: a working case definition beyond myalgic encephalomyelitis/ chronic fatigue syndrome: redefining an illness contrasting case definitions for chronic fatigue syndrome, myalgic encephalomyelitis/chronic fatigue syndrome and myalgic encephalomyelitis contrasting chronic fatigue syndrome versus myalgic encephalomyelitis/ chronic fatigue syndrome a communitybased study of chronic fatigue syndrome reflections on the institute of medicine's systemic exertion intolerance disease physical interventions to interrupt or reduce the spread of respiratory viruses exercise dysfunction in patients seropositive for the human immunodeficiency virus role of infection and neurologic dysfunction in chronic fatigue syndrome hiv, highly active antiretroviral therapy and the heart: a cellular to epidemiological review aerobic exercise training in an aids risk group exercise therapy for chronic fatigue syndrome the effect of physical exercise on salivary secretion of muc5b, amylase and lysozyme susceptibility to infections in elite athletes: the s-curve high training volumes are associated with a low number of self-reported sick days in elite endurance athletes adaptive pacing, cognitive behaviour therapy, graded exercise, and specialist medical care for chronic fatigue syndrome: a cost-effectiveness analysis does exercise increase the risk of upper respiratory tract infections a neuro-immune model of myalgic encephalomyelitis/chronic fatigue syndrome exercise, upper respiratory tract infection, and the immune system infectious episodes in runners before and after a roadrace infectious episodes in runners before and after the los angeles marathon health-related quality of life in patients with chronic fatigue syndrome: group cognitive behavioural therapy and graded exercise versus usual treatment. a randomised controlled trial with 1 year of follow-up aerobic exercise interventions for adults living with hiv/aids effects of progressive resistive exercise in adults living with hiv/aids: systematic review and meta-analysis of randomized trials estimating per-act hiv transmission risk: a systematic review cardiopulmonary and cd4 cell changes in response to exercise training in early symptomatic hiv infection randomised controlled trial of patient education to encourage graded exercise in chronic fatigue syndrome exercise and febrile illnesses prevalence of chronic fatigue syndrome in metropolitan, urban, and rural georgia identification of ambiguities in the 1994 chronic fatigue syndrome research case definition and recommendations for resolution prevalence and incidence of chronic fatigue syndrome in effects of exercise training on men seropositive for the human immunodeficiency virus-1 return to play after acute infectious disease in football players treatment of myalgic encephalomyelitis/chronic fatigue syndrome: a systematic review for a national institutes of health pathways to prevention workshop incidence, etiology, and symptomatology of upper respiratory illness in elite athletes a controlled clinical trial with a specifically configured rna drug, poly(i).poly(c12u), in chronic fatigue syndrome a double-blind, placebo-controlled, randomized, clinical trial of the tlr-3 agonist rintatolimod in severe cases of chronic fatigue syndrome mechanisms of exercise limitation in hiv+ individuals the effect of exercise training on aerobic fitness, immune indices, and quality of life in hiv+ patients genetic susceptibility and resistance to influenza infection and disease in humans and mice decreased oxygen extraction during cardiopulmonary exercise test in patients with chronic fatigue syndrome position statement. part two: maintaining immune health position statement. part one: immune function and exercise comparison of adaptive pacing therapy, cognitive behaviour therapy, graded exercise therapy, and specialist medical care for chronic fatigue syndrome (pace): a randomised trial group pt. protocol for the pace trial: a randomised controlled trial of adaptive pacing, cognitive behaviour therapy, and graded exercise, as supplements to standardised specialist medical care versus standardised specialist medical care alone for patients with the chronic fatigue syndrome/myalgic encephalomyelitis or encephalopathy key: cord-254194-962vynwk authors: galdiero, stefania; falanga, annarita; vitiello, mariateresa; cantisani, marco; marra, veronica; galdiero, massimiliano title: silver nanoparticles as potential antiviral agents date: 2011-10-24 journal: molecules doi: 10.3390/molecules16108894 sha: doc_id: 254194 cord_uid: 962vynwk virus infections pose significant global health challenges, especially in view of the fact that the emergence of resistant viral strains and the adverse side effects associated with prolonged use continue to slow down the application of effective antiviral therapies. this makes imperative the need for the development of safe and potent alternatives to conventional antiviral drugs. in the present scenario, nanoscale materials have emerged as novel antiviral agents for the possibilities offered by their unique chemical and physical properties. silver nanoparticles have mainly been studied for their antimicrobial potential against bacteria, but have also proven to be active against several types of viruses including human imunodeficiency virus, hepatitis b virus, herpes simplex virus, respiratory syncytial virus, and monkey pox virus. the use of metal nanoparticles provides an interesting opportunity for novel antiviral therapies. since metals may attack a broad range of targets in the virus there is a lower possibility to develop resistance as compared to conventional antivirals. the present review focuses on the development of methods for the production of silver nanoparticles and on their use as antiviral therapeutics against pathogenic viruses. viruses represent one of the leading causes of disease and death worldwide. thanks to vaccination programmes, some of the numerous diseases that used to kill many and permanently disable others have been eradicated, such as smallpox in 1979 [1] or have greatly reduced the burden of the disease, such as in the case of the paralytic disease poliomyelitis [2] . however, for some of today's most pressing viral pathogens, there is still no vaccine available. to realize the huge economic impact that several viral diseases cause to the global community, we need only to think to common colds, influenza, various problems due to herpesviruses (from shingles, genital herpes, chickenpox, infectious mononucleosis, up to herpes keratitis, neonatal disseminated infections, or viral encephalitis). other viruses are also able to cause considerable distress and sometimes persistent infections that may lead to cancer or to acquired immunodeficiencies, such as hepatitis viruses (mainly hbv and hcv) or human immunodeficiency virus (hiv). much effort has been expended in attempts to develop vaccines for these diseases, without appreciable success, at least for some of these viruses, namely, hcv, hiv and some herpesviruses. presently, the development of new vaccines for such viruses seems likely to continue to be elusive. together with the risk of emerging or re-emerging viral agents, the field of antiviral compound discovery is very promising. emerging and re-emerging viruses are to be considered a continuing threat to human health because of their amazing ability to adapt to their current host, to switch to a new host and to evolve strategies to escape antiviral measures [3] . viruses can emerge because of changes in the host, the environment, or the vector, and new pathogenic viruses can arise in humans from existing human viruses or from animal viruses. several viral diseases that emerged in the last few decades have now become entrenched in human populations worldwide. the best known examples are: sars coronavirus, west nile virus, monkey pox virus, hantavirus, nipah virus, hendravirus, chikungunya virus, and last but not least, the threat of pandemic influenza viruses, most recently of avian or swine origin. unfortunately the methodological advances that led to their detection have not been matched by equal advances in the ability to prevent or control these diseases. there have been improvements in antiviral therapy, but with a wide margin of ineffectiveness, therefore new antiviral agents are urgently needed to continue the battle between invading viruses and host responses. technological advances have led to the discovery and characterization of molecules required for viral replication and to the development of antiviral agents to inhibit them. most viruses are, indeed, provided by an extraordinary genetic adaptability, which has enabled them to escape antiviral inhibition and in certain cases to regain advantage over the host by mutagenesis that create new viral strains with acquired resistance to most of the antiviral compounds available [3] . the course of viral infections is governed by complex interactions between the virus and the host cellular system. all viruses depend upon a host cell for their protein synthesis. thus, all viruses replicate via a broadly similar sequence of events ( figure 1 ). the virus must first bind to the cell, and then the virus or its genome enters in the cytoplasm. the genome is liberated from the protective capsid and, either in the nucleus or in the cytoplasm, it is transcribed and viral mrna directs protein synthesis, in a generally well regulated fashion. finally, the virus undergoes genome replication and together with viral structural proteins assembles new virions which are then released from the cell. each of the single described phases represents a possible target for inhibition. drugs that target viral attachment or entrance have proved to be very difficult to be discovered. in fact, to date, only one entry inhibitor has been approved by the us food and drug administration (fda). this is enfuvirtide (t-20), a synthetic peptide that targets the hiv gp41 envelope protein to prevent fusion. targeting the early steps of virus entry is a very attractive strategy for therapeutic intervention since the site of action of the inhibitor is likely to be extracellular and therefore relatively accessible; this could be paired by a concomitant action of the same drug on multiple targets to obtain a more effective therapeutic compound. moreover one could expect, in the future, antiviral agents with a broad-spectrum of action against viruses of different families, to be used as first aid compounds against unforeseen viral epidemics or pandemics. due to the outbreak of the emerging infectious diseases caused by different pathogenic viruses and the development of antiviral resistance to classical antiviral drugs, pharmaceutical companies and numerous researchers are seeking new antiviral agents. in the present scenario, nanoscale materials have emerged as novel "antimicrobial agents" due to their high surface area to volume ratio and their unique chemical and physical properties [4, 5] . nanotechnology is an emerging field of applied science and cutting edge technology that utilizes the physico-chemical properties of nanomaterials as a means to control their size, surface area, and shape in order to generate different nanoscale-sized materials. among such materials, metal-based ones seem the most interesting and promising, and represent the subject of the present review. nanotechnology is directly linked with physics, chemistry, biology, material science and medicine. in fact, it finds application in multiple aspects of research and in everyday life such as electronics and new material design. however, its use in medical research is probably one of the fastest growing areas in which the functional mechanisms of nanoparticles and especially metal-based nanoparticles are just beginning to be exploited. nanotechnologies have been used to develop nanoparticle-based targeted drug carriers [6, 7] , rapid pathogen detection [8, 9] , and biomolecular sensing [10] , as well as nanoparticle-based cancer therapies [11, 12] . the use of nanoparticles can be extended to the development of antivirals that act by interfering with viral infection, particularly during attachment and entry. nanoparticles are properly defined as particles with at least one dimension less than 100 nm, and have attracted much attention because of their unique and interesting properties. their singular physical (e.g., plasmonic resonance, fluorescent enhancement) and chemical (e.g., catalytic activity enhancement) properties derive from the high quantity of surface atoms and the high area/volume relation, in fact, as their diameter decreases, the available surface area of the particle itself increases dramatically and as a consequence there is an increase over the original properties of their bulk materials. considering that biological interactions are generally multivalent, the interplay between microbes and host cells often involves multiple copies of receptors and ligands that bind in a coordinated manner, resulting in enhanced specificities, efficiencies, and strengths of such interactions that allow the microbial agent to take possess of the cell under attack. the attachment and entry of viruses into host cells represent a terrific example of such multivalent interactions between viral surface components and cell membrane receptors [13] . interfering with these recognition events, and thereby blocking viral entry into the cells, is one of the most promising strategies being pursued in the development of new antiviral drugs and preventive topical microbicides [14] [15] [16] . in recent years, the use of metal nanoparticles, that may or not have been functionalized on their surface for optimising interactions, is seeing increasing success. the idea of exploiting metals against microorganisms can be considered ancient; in fact, the use of silver was a common expedient for cooking procedures and for preserving water from contamination. the importance of silver for its curative properties has been known for centuries, in fact, silver has been the most extensively studied metal for purpose of fighting infections and preventing food spoilage, and notwithstanding the decline of its use as a consequence of the development of antibiotics, prophylaxis against gonococcal ophthalmia neonatorum with silver ions was considered the standard of care in many countries until the end of the 20th century [17] . silver's mode of action is presumed to be dependent on ag + ions, which strongly inhibit bacterial growth through suppression of respiratory enzymes and electron transport components and through interference with dna functions [18] . therefore, the antibacterial, antifungal and antiviral properties of silver ions and silver compounds have been extensively studied. silver has also been found to be non-toxic to humans at very small concentrations. the microorganisms are unlikely to develop resistance against silver as compared to antibiotics as silver attacks a broad range of targets in the microbes. considering the broad literature that describes silver, as a bulk material, effective against a wide range of pathogens, silver nanoparticles have been analysed and found to be extremely appealing. the silver nanoparticles have also found diverse applications in the form of wound dressings, coatings for medical devices, silver nanoparticles impregnated textile fabrics [19] . the advantage of using silver nanoparticles for impregnation is that there is continuous release of silver ions enhancing its antimicrobial efficacy. the burn wounds treated with silver nanoparticles show better cosmetic appearance and scarless healing [20] . silver nanoparticles have received considerable attention as antimicrobial agents and have been shown to be effective mainly as antibacterial. antimicrobial effectiveness was shown for both gram-positive and gram-negative bacteria [21, 22] . the antibacterial activity of silver nanoparticles was mainly demonstrated by in vitro experiments. activity against methicillin-resistant staphylococcus aureus (mrsa) [23] , escherichia coli [4, 21, 24, 25] , pseudomonas aeruginosa [4] , vibrio cholera [4] and bacillus subtilis [25] has been documented. low concentrations of silver nanoparticles were able to consistently inhibit e. coli [5] while the growth-inhibitory effect on s. aureus was minor. synergistic antimicrobial activity of silver or zinc nanoparticles with ampicillin, penicillin g, amoxicillin, kanamycin, erythromycin, clindamycin, chloramphenicol and vancomycin against s. aureus, e. coli, salmonella typhi and micrococcus luteus was observed [26] [27] [28] . also gold nanoparticles have been exploited as antimicrobial agents, mainly as a tool to deliver other antimicrobials or in order to enhance the photodynamic killing of bacteria [29] . many studies have shown the antimicrobial effects of metal nanoparticles, but the effects of silver nanoparticles against fungal pathogens are mostly unknown; silver nanoparticles, indeed, showed significant antifungal activity against penicillium citrinum [30] , aspergillus niger [30] , trichophyton mentagrophytes [31] and candida albicans [32] . different types of nanomaterials like copper, zinc, titanium [33] , magnesium, gold [34] , alginate [35] and silver have come up in recent years and most of them have proven to be effective against diverse microorganisms. the present review aims at a description of the reported antiviral activities of metal nanoparticles and their production methods, with particular regard to silver nanoparticles. metal nanoparticles have been studied for their antimicrobial potential and have proven to be antibacterial agents against both gram-negative and gram-positive bacteria [4, 5, 21, 26, 36] . theoretically, any metal could be analysed for antiviral activity, however, little effort has been done to determine the interactions of metal nanoparticles with viruses, and only recently some studies have emerged showing that metal nanoparticles can be effective antiviral agents against hiv-1 [37] [38] [39] [40] , hepatitis b virus [41] , respiratory syncytial virus [42] , herpes simplex virus type 1 [43, 44] , monkeypox virus [45] , influenza virus [46] and tacaribe virus [47] . seen the paucity of viruses that have been investigated and the fact that most of the nanoparticles used were made of silver, this section will be instrumental to analyse the inhibitory effect for each single virus (table 1) . acquired immunodeficiency syndrome (aids), the disease caused by hiv, is responsible for over two million deaths per year, among more than 33 million people that are infected. highly active anti-retroviral therapy (haart), a treatment regimen that employs a cocktail of drugs to suppress hiv infection, has significantly improved the quality of life and life expectancy of millions of hiv-infected individuals. numerous hiv-infected individuals are currently treated with haart, and these individuals harbor chronic long-term infection; as a result, hiv eventually develops resistance to these drugs, resulting in a need to change medication regimens and a subsequent increase in the cost of treatment [48] . the replication cycle of hiv-1 is a complex multistep process that depends on both viral and host cell factors. entry into target cells is achieved through fusion of the viral lipid envelope and the cellular plasma membrane [49] . the viral component that acts as a fulcrum for mediating fusion is the trimeric envelope glycoprotein composed of two subunits: gp120, which binds to the cellular receptor, and gp41, which is the subunit bearing the transmembrane segment, and that executes fusion [50] . following gp120 binding to the cellular receptor, cd4, and a subsequent interaction with ccr5 or cxcr4 co-receptors, a conformational change of gp41 leads to membrane fusion and delivery of the capsid to the cytoplasm. soon after entry, the rna is reverse-transcribed into a complementary dna which is converted to a double-stranded dna, and integrated into the cellular genome. the integrated proviral dna is transcribed to generate full-length progeny viral rna and a number of spliced mrna transcripts. transcription and translation, performed by the cellular machinery, result in the synthesis of viral proteins that together with the progeny viral rna are transported to the site of virus particle assembly at the plasma membrane, where the virus gains access to the extracellular milieu upon budding events [51] . elechiguerra et al. [37] were the first to describe the antiviral activity of metal nanoparticles, in fact, they found that silver nanoparticles undergo size-dependent interactions with hiv-1. in their investigations, they explored the possibility that physicochemical properties of nanoparticles may depend on the nanoparticle interactions with a capping agent molecule. for this reason they tested silver nanoparticles with three different surface chemistries: foamy carbon, poly(n-vinyl-2-pyrrolidone) (pvp), and bovine serum albumin (bsa). foamy carbon-coated silver nanoparticles were embedded in a foamy carbon matrix needed to preclude coalescence during their synthesis. pvp-coated nanoparticles were synthesized using glycerine as both reducing agent and solvent. in this method, a metal precursor is dissolved in a liquid polyol in the presence of a capping agent such as pvp [52] . synthesis in aqueous solution was performed for bsa-conjugate silver nanoparticles. interactions of silver nanoparticles with hiv-1 were probed with the aid of high angle annular dark field (haadf) scanning transmission electron microscopy technology. it was possible to obtain sufficient data to determine that the interaction between hiv particles and silver nanoparticles is clearly due to the size of the silver nanoparticle since only nanoparticles within the range of 1-10 nm were able to bind to the virus. in particular, nanoparticles were not randomly attached to the virus, but all the three species of nanoparticles established regular spatial relationships with the viral envelope. the most probable sites for interaction were found to be the sulfur-bearing residues of the gp120 glycoprotein knobs, which being limited in number, may also explain the inability of larger nanoparticles to bind the virus. the capacity of silver nanoparticles to inhibit infectivity of a laboratory-adapted hiv-1 strain at non-cytotoxic concentrations was determined by in vitro assays, and a dose-dependant inhibition of viral infectivity was reported. in particular, bsa-and pvp-coated nanoparticles showed to possess a slightly lower inhibition efficacy, probably because the surface of the nanoparticle is directly bound to and encapsulated by the capping agent. in contrast, the silver nanoparticles released from the carbon matrix have a greater inhibitory effect due to their essentially free surface area. these findings, however, only provide indirect evidence for their proposed mode of interaction through the binding to gp120, therefore, a panel of different in vitro assays was used to elucidate the silver nanoparticles mode of antiviral action against hiv-1 [39] . a luciferase-based assay showed that silver nanoparticles coated with pvp were an effective virucidal agent against cell-free virus (including laboratory strains, clinical isolates, t and m tropic strains, and resistant strains) and cell-associated virus. the concentration of silver nanoparticles at which infectivity was inhibited by 50% (ic50) ranged from 0.44 to 0.91 mg/ml. the observed antiviral effect of silver nanoparticles was due to the nanoparticles, rather than just to the silver ions present in the solution. in fact silver salts exerting antibacterial effect through silver ions, inhibited hiv-1 with a therapeutic index 12 times lower than the one of silver nanoparticles. silver nanoparticles inhibit the initial stages of the hiv-1 infection cycle by blocking adsorption and infectivity in a cell-fusion assay. the inhibitory activity of silver nanoparticles against the gp120-cd4 interaction was also investigated in a competitive gp120-capture elisa, which together with the cell-based fusion assay, showed that silver nanoparticles inhibit hiv-1 infection by blocking viral entry, particularly the gp120-cd4 interaction. besides, silver nanoparticles inhibit post-entry stages of the hiv-1 life cycle, in fact, the antiviral activity was maintained also when the metal nanoparticles were added 12 h after the cell had been infected with hiv. since silver ions can form complexes with electron donor groups containing sulfur, oxygen, or nitrogen that are normally present as thiols or phosphates on amino acids and nucleic acids they might inhibit post-entry stages of infection by blocking hiv-1 proteins other than gp120, or reducing reverse transcription or proviral transcription rates by directly binding to the rna or dna molecules. silver nanoparticles proved to be virucidal to cell-free and cell-associated hiv-1 as judged by viral infectivity assays. hiv infectivity was effectively eliminated following short exposure of isolated virus to silver nanoparticles. these properties make silver nanoparticles a potential broad-spectrum agent not prone to inducing resistance that could be used preventively against a wide variety of circulating hiv-1 strains. pvp-coated silver nanoparticles, being an interesting virucidal candidate drug, have been further investigated as a potential topical vaginal microbicide to prevent transmission of hiv-1 infection [40] using an in vitro human cervical tissue-based organ culture that simulates in vivo conditions [53, 54] . when formulated into a non-spermicidal gel (replens) at a concentration of 0.15 mg/ml, pvp-coated silver nanoparticles were not toxic to the explant, even when the cervical tissues were exposed continuously to the metal for 48 hours, but one minute of pre-treatment of the cervical explant with 0.025 to 0.15 mg/ml of pvp-coated silver nanoparticles prevented the transmission of cell-associated hiv-1 and cell-free hiv-1 isolates. when pre-treatment was carried on for 20 minutes followed by extensive washing the drug conferred almost total protection against hiv-1 transmission for 48 hours, indicating a long-lasting protective effect by the pvp-coated silver nanoparticles in the cervical explants. a different group [38] also reported about the antiviral activity of silver nanoparticles that had been fabricated using hepes buffer. they showed that silver nanoparticles exhibited potent cytoprotective and post-infected anti-hiv-1 activities (at 50 mm a 98% reduction was achieved) toward hut/ccr5 cells in a dose-dependent fashion. similar inhibitory activities were reported for the silver nanoparticles when a citrate solution with nabh 4 as the reducing agent was used, while lower activity was observed for gold nanoparticles (10 nm, fabricated in hepes buffer). the herpesvirus family consists of more than 100 double-stranded dna viruses divided into ï�¡, î² and ï�§ subgroups. only eight herpesviruses are known to commonly infect humans and the remainder are animal herpesviruses infecting a wide variety of animal species. all members of the herpesvirus family cause life-long latent infections and, structurally, all have a linear, double-stranded dna genome packaged into an icosahedral capsid and covered by a lipid envelope with embedded proteins and glycoproteins [55] . symptomatic diseases caused by hsv-1 (prototypic ï�¡-herpesvirus) are generally limited to cold sores of the mouth and keratitis in the eyes, but hsv-1 is capable of causing life-threatening diseases in immunocompromised individuals, including newborns, patients with hiv or patients undergoing immunosuppressive treatment. transmission among humans requires physical contact and after the initial infection, the virus remains latent in neurons, a key feature of ï�¡-herpesviruses [56] . hsv entry into host cells marks the first and possibly most critical step in viral pathogenesis [57] . five viral glycoproteins have been implicated in the viral entry process: gb, gc, gd, gh and gl. all but gc are essential for entry. the initial interaction, or binding to cells, is mediated via interactions of gc and/or gb with heparan sulfate (hs) [58] . the significant reduction of hsv-1 infection in the absence of either viral gc or cell-surface hs [59] point to a key role of gc high-affinity binding to heparan sulfate (hs) on the cell surface. following binding, hsv entry is achieved through fusion of the lipid bilayer of the viral envelope with a host cell membrane. the core fusion machinery is composed by gb and gh/gl [60, 61] , in fact, the complete fusion is only achieved when the three proteins act together. glycoprotein b may act as the premier fusogen [62] [63] [64] , but it seems to need the cooperation of several membranotropic sequences harboured in gh [65] [66] [67] [68] [69] . transcription, replication of viral dna and assembly of progeny capsids take place within the host nucleus, and then there is a complex mechanism for the exit of the newly assembled viruses from the cell [70] . baram-pinto et al. have described in two consecutive works [43, 44] the potential of exploiting metal nanoparticles for viral inhibition. their strategy was proved valid against hsv-1, but was probably intended and may prove useful against other viruses, such as papillomaviruses (hpv) and hiv. in fact, their anti-hsv-1 agents are based on the principle that they mimic hs and may compete for the binding of the virus to the cell. also hiv or hpv use hs as a docking site during infection, therefore the nanoparticles described by baram-pinto et al. may be useful as a broad topical microbicide for sexually-transmitted viral infections. another point of particular interest from their work is the analysis of the significance of the carrier core material, in fact they designed two different metal particles, one made of silver, and the other made with gold, but both with the same coating of mercaptoethane sulfonate (mes) intended to mimic the polysulfonated hs and therefore expected to create a high local concentration of binding molecules for improved inhibitory effect. the silver-(ag-mes) and gold-(au-mes) mes nanoparticles were tested in antiviral assays using the wild-type hsv-1 mcintyre strain. for the inhibition experiments, vero cells and/or virus solutions were treated with ag-mes and au-mes nanoparticles at different time points to analyse the different stages of the viral infection that may be blocked. taken together, their results indicate that sulfonate-capped silver and gold nanoparticles inhibit hsv-1 infections by blocking the attachment and thereby the entrance of the virus into the cells and/or by preventing the cell-to-cell spread of the virus. the inability of soluble mes and unmodified metal nanoparticles to control viral infectivity stressed the importance of spatially oriented functional groups anchored on a nanoparticle core for viral inhibition. at the same time, antiviral activity shown by both ag-mes and au-mes nanoparticles suggest the possibility of using alternative carrier core materials as well. while these results suggest the versatility of the idea of effective viral inhibitions using functionalized nanoparticles, it also indicates that other core materials could also be efficient as long as they are not toxic to the host cells. respiratory syncytial virus (rsv) belongs to the family paramyxoviridae and infects the epithelium of the lungs and the respiratory tract causing serious respiratory disease, especially in children and older people. no vaccine or adequate pharmaceutical compounds are available, underlining the need for the development of future rsv treatments. the rsv genome consists of a single rna molecule of negative-sense rna, which encodes, among others, for two surface glycoproteins, which are exposed on the viral envelope. these glycoproteins are the (g) protein, which serve as a receptor binding protein, and the (f) protein, which is responsible for the fusion between the cell membrane and the viral envelope. as within the name itself of the virus, following infection of cells, the f protein is expressed on the surface of cells and fuse adjacent cells, giving rise to syncytia formation, a well characterised cytopathic effect [71] . sun et al. [42] have utilized silver nanoparticles conjugated to various proteins to study the inhibition of rsv infection in hep-2 cell culture. in their study, the capping agents used for the silver nanoparticles were: (1) poly(n-vinyl-2-pyrrolidone) (pvp); (2) bovine serum albumin (bsa); and (3) a recombinant f protein from rsv (rf 412). the preliminary analysis by transmission electron microscopy yielded interesting results on the interaction between silver nanoparticles with rsv virion particles. bsa-conjugated silver nanoparticles seemed to interact with rsv but without a specific association or spatial arrangement, while rf 412-conjugated silver nanoparticles appeared to be floating freely with no proof of regular attachment. on the other hand, pvp-coated silver nanoparticles were able to bind to the viral surface with a regular spatial arrangement, suggesting a possible interaction with g proteins that are evenly distributed on the envelope of the rsv virion. the hypothesised interpretation for the interaction of pvp-nanoparticles with g proteins is that their small size and uniformity (4-8 nm), compared to the other (bsa and rf 412) coated nanoparticles (3-38 nm) may contribute to the effectiveness of the binding. since toxicity is an imperative issue regarding pharmaceutical compounds, the cytotoxicity of each of the nanoparticle conjugates was established using the trypan blue exclusion assay, and revealed that all of them (bsa-, pvp-and rf 412-silver nanoparticles) showed less than 20% cytotoxicity up to a concentration of 100 î¼g/ml. silver nanoparticles have to be regarded as potentially harmful especially when intended for treating a respiratory disease such as rsv infections. sun et al. [42] have, therefore considered that a saturated surface capping composed of a natural biomolecule (bsa) and a biocompatible chemical (pvp) could be able to mask the pure nano-silver surface and thus would reduce toxicity without hampering efficacy. nevertheless, further studies are needed to validate these in vitro data for the use into the clinical setting, and investigation of the toxic effects and fate of nanoparticles after their deposition in the respiratory tract is mandatory for the future development of anti-rsv silver nanoparticles-based therapeutic compounds. hep-2 cells were infected with rsv mixed with bsa-, pvp-and rf 412-coated silver nanoparticles and infectivity inhibition was evaluated by microscopic examination for syncytia formation and by immunofluorescence microscopy. neither bsa-nor rf 412-coated nanoparticles showed any significant inhibition of rsv infection, while pvp-coated silver nanoparticles inhibited rsv infection by 44%. these results led the authors to conclude that when the silver nanoparticles are conjugated to the pvp protein and mixed with rsv, they bind to the g protein on the viral surface and interfere with viral attachment to the hep-2 cells resulting in the inhibition of viral infection. hepatitis b virus (hbv) is a partially double-stranded dna virus provided with a lipidic envelope coat. hbv has a strong tropism for hepatocytes, and once it has entered the cell, viral particles migrate to the nucleus where the viral genome is completed to form a covalently closed circular dna (cccdna) that serves as the template for the subsequent steps of viral mrna transcription and formation of the pre-genomic rna (pgrna). the pgrna forms the template for the reserve transcription by the viral-encoded reverse-transcriptase that produce new viral genomes [72] . nucleotide (adefovir) and nucleoside (lamivudine, entecavir, and telbivudine) analogue inhibitors, which represent approved pharmaceuticals with direct antiviral activity against hbv, target primarily the viral polymerase reverse-transcriptase. although their effectiveness has been proven, raising drug-resistant hbv strains is fast, therefore limiting the use of these antivirals. lu et al. [41] have analysed monodisperse silver nanoparticles for their ability to inhibit hbv replication. the nanoparticles used in their study were prepared from agno 3 in hepes and measured dimensions of ~10 nm (ag10ns), ~50 nm (ag50ns) and ~800 nm (ag800ns). silver nanoparticles with particle diameters of 800 nm were too toxic for being evaluated as antiviral compound, but 10 nm and 50 nm particles showed only a minor toxicity at the concentration able to inhibit hbv replication. in fact, nanoparticles of both sizes showed potent anti-hbv activities. the ag10ns reached 38% inhibition at 5 âµm and 80% at 50 âµm, while the ag50ns were slightly more active with 53% and 92% at concentration of respectively 5 âµm and 50 âµm. in the same paper by lu et al. [41] , the activity of silver nanoparticles was also compared to 10 nm gold nanoparticles (au10ns) and other silver compounds with silver in different oxidation states, and the overall results showed that the anti-hbv effects of silver nanoparticles are undoubtedly much more pronounced. in conclusion, silver nanoparticles were able to inhibit the production of hbv rna and extracellular virions probably via a specific interaction between the nanoparticles and the double-stranded dna of hbv and/or direct binding with viral particles. the influenza virus is a highly contagious pathogen that causes annual epidemics in the human population, and is much feared for its potential to generate new viruses able to jump to humans from different animal species and causing pandemics. recently, papp et al. [46] have described their studies in which functionalized gold nanoparticles were used to inhibit the influenza virus. this is an orthomyxovirus containing a helical capsid with a genome of eight rna segments. the capsid is covered by a lipid envelope containing mainly two virally-encoded glycoproteins, namely hemoagglutinin (ha) and neuraminidase (na), that forms spiky projections on the surface. the virus binds to the cell plasma membrane through an interaction between ha and sialic acid (sa) residues present on glycoproteins and lipids on the surface of the host cell. this is soon followed by a mechanism of receptor-mediated endocytosis that brings the enveloped virus particle inside the cytoplasm but surrounded by a second lipid bilayer besides the envelope, the endosomal one. inside the late endosome, environment acidification triggers a conformational change of ha, which sets in motion a mechanism of protein (ha) mediated fusion of the endosomal membrane with the viral envelope ending with the release of the nucleoproteins and genome fragments into the cytoplasm [73] . papp et al. [46] strategy was to functionalize gold nanoparticles with sialic acid (sa)-terminated glycerol dendron with the objective to inhibit influenza virus binding to the plasma membrane. gold nanoparticles of different size were produced: one of 2 nm and a second of 14 nm. they found that 2 nm had no inhibitory effect on the hemagglutination, used to test the ability of the influenza virus to bind to a target membrane. on the contrary, the 14 nm gold nanoparticles inhibited hemagglutination at concentrations in the nanomolar range, demonstrating that the activity clearly depends on the particle dimension and the spatial distribution of the interacting ligand/receptor molecules. the same trend, with a more pronounced activity was observed in influenza virus inhibition assays where the sialylated particles of 14 nm size were found to be effective for influenza virus inhibition, whereas the 2 nm analogues did not show significant impact. therefore, they proved that sialic-acid-functionalized gold nanoparticles are able to effectively inhibit viral infection. monkeypox virus (mpv), an orthopoxvirus similar to variola virus, is the causative agent of monkeypox in many species of non-human primates, but it is also a human pathogen with a clinical presentation similar to that of smallpox. mpv is considered a big threat to human life and therefore research is being carried out to develop drugs and therapeutic agents against this virus [74] . different size nanoparticles were produced by plasma gas synthesis and used by rogers et al. [45] in a plaque reduction assay of mpv. the silver nanoparticles used in this work were 25 (ag-np-25), 55 (ag-np-55) and 80 (ag-np-80) nm, and some nanoparticles were also coated with polysaccharide, 10 (ag-ps-10), 25 (ag-ps-25) and 80 (ag-ps-80) nm nanoparticles. these nanoparticles, at concentrations ranging from 12.5 to 100 ï�­g/ml, were evaluated for mpv inhibitory efficacy using a plaque reduction assay. the main results showed that the ag-ps-25 (polysaccharide-coated, 25 nm) and ag-np-55 (non-coated, 55 nm) exerted a significant dose-dependent inhibition of mpv plaque formation, but the mechanism by which this inhibition occurs has not been further investigated. poxviruses enter cells by endocytosis or direct fusion at the plasma membrane, and at least 9 or 10 envelope proteins are involved in the entry mechanism, this is followed by a regulated sequence of events that allow virus replication. many steps in the virus life cycle are still unknown, and this report on the activity of silver nanoparticles is too preliminary to attempt to give a satisfactory explanation of their mechanism of action. probably the silver nanoparticles may intervene in the early steps of binding and penetration by blocking virus-host cell binding by physical obstruction or, if internalised, they can disrupt intracellular pathways important for virus replication. rogers et al. [45] also described that agno 3 was active as a mpv inhibitor, but at the concentration of 100 âµg/ml its toxic effect on vero cells impeded the evaluation of the antiviral activity. interestingly, some of the nanoparticles analysed in the study promoted an increase in the mean number of mpv plaques/well at the highest concentrations used. a potential explanation for these contrasting results could lie in the fact that nanoparticles may tend to aggregate and consequently create on cells areas available for increased contacts between viral particles and the cell membrane, therefore augmenting internalization and plaque formation. however, these data are preliminary and need a more in-depth analysis to draw more significant conclusions. the family arenaviridae is composed of 18 different species of viruses divided into two antigenic groups, the old world and new world (tacaribe complex) groups. the tacaribe complex, in addition to the tacaribe virus (tcrv), includes the viral hemorrhagic fever-inducing viruses junin, machupo, guanarito, and sabia. considering the high transmissibility by person-to-person via the respiratory route, the lack of diagnostic testing, and therapeutic options limited to ribavirin (not a satisfactory efficacy and easily emergence or resistant strains), the arenaviruses are included in the category a list of potential bio-weapons [75] . tcrv is not a human pathogen, but exhibits a close antigenic relationship with junin and guanarito viruses [76] , therefore could serve well as a model virus for arenavirus derived diseases without human pathogenic potential and adequate safety for laboratory manipulation. speshock et al. [47] have recently analysed the activity of two types of silver nanoparticles against tcrv: uncoated (ag-np) and polysaccharide coated silver nanoparticles (ps-ag). they found that when tcrv was treated with 50 î¼g/ml, 25 î¼g/ml and even 10 î¼g/ml of the 10 nm ag-nps significant reduction in the progeny virus titer or no detectable progeny virus was produced. ps-ag particles showed a similar trend but were not as effective, but toxicity was reduced. therefore the polysaccharide coating may indeed protect the cell from the toxic effects of the ag-nps, but it also appears to interfere with the ag-np interaction with tcrv. silver nanoparticles seem to interact with tcrv prior to cellular exposure resulting in a decrease in viral infectivity with 10 and 25 nm ag-nps, therefore, the authors suggested that the silver nanoparticles may bind to virally-encoded membrane glycoproteins. in fact, tcrv glycoproteins are rich in cysteine residues [77] and silver nanoparticles have been shown to bind easily to the thiol groups [78] , which are found in cysteine residues. this interaction can either prevent the internalization of the viral particle by interfering with cellular receptor binding, or may favour the internalization of the silver nanoparticle together with the virus and produce an inhibitory effect on viral replication interfering with the tcrv rna-dependent rna polymerase (l protein). other possible mechanism of action could be related to the fact that the silver nanoparticles bound to the viral glycoproteins may prevent the virus uncoating in the endosome. finally, speshock et al. [47] proved that pre-treatment of the cells with silver nanoparticles had no effect on viral replication, therefore they concluded that the ag-nps could be inactivating the virus prior to entry into the cell. the removal of viruses from water (and the environment in general) is of paramount important for health safety maintenance of our modern society that profoundly relies on water safety for drinking and leisure activities. pathogenic viruses such as adenovirus, norovirus, rotavirus, and hepatitis a commonly occur in both surface and groundwater sources [79] [80] [81] and must be effectively inactivated to provide safe water. titanium dioxide has attracted much attention as a photocatalyst for water treatment, being resistant to corrosion and non-toxic when ingested. the antibacterial properties of tio 2 have been well documented [82] [83] [84] [85] and are attributed to the generation of ros, especially hydroxyl free radicals and hydrogen peroxide [83, 86] . while few studies have investigated the antiviral properties of tio 2 , its potential for inactivating viruses has been demonstrated [84, 87, 88] . however, the inactivation rates obtained in most of these studies were extremely low. for example, cho et al. [84] demonstrated only minor removal of bacteriophage ms2 after 2 h of irradiation using p25 tio 2 suspended at 1 g/l. the inactivation kinetics needs to be greatly improved in order to provide efficient drinking water disinfection. liga et al. [89] have hypothesized a possible synergic mechanism occurring between silver and tio 2 when silver doped titanium dioxide is used for inactivating microorganisms under uv radiation, therefore they demonstrated that silver doping tio 2 greatly enhanced the photocatalytic inactivation of viruses primarily by increasing hydroxyl free radicals production in addition to slightly increasing virus adsorption. silver doping significantly enhanced ms2 inactivation by p25 tio 2 and the inactivation rate increased with silver content. with silver doped tio 2 nanoparticles a considerable removal of ms2 could be obtained in 45 seconds, rendering feasible the goal of achieving virus removal from drinking water using photoreactors exploiting metal nanoparticles. although the continuous evolutions in the field of metal-based nanoparticles for drug delivery, medical imaging, diagnostics, therapeutics and engineering technology, there is a serious lack of information about the impact of metal nanoparticles on human health and environment, probably due to the intrinsic complex nature of nanoparticles that have led to different attitudes on their safety. therefore, an important issue in the use of metal nanoparticles is their potential toxicity. for metal nanoparticles to be effective as antiviral pharmaceuticals, it is imperative to gain a better understanding of their biodistribution/accumulation in living systems. the principal characteristic of metal nanoparticles is their size, which falls in between individual atoms or molecules and the corresponding bulk materials. particle size and surface area can modify the physicochemical properties of the metal material, but can also influence the reactivity of nanoparticles with themselves or with the cellular environment, leading to different modes of cellular uptake and further processing, leading to adverse biological effects in living cells that would not otherwise be possible with the same material in larger forms. in fact, as particle size decreases, some metal nanoparticles show increased toxicity, even if the same material is relatively inert in its bulk form (e.g., ag, au, and cu). apart from size, the biological consequences of metal nanoparticles also depend on chemical composition, surface structure, solubility, shape, and aggregation. all of these parameters can modify cellular uptake, protein binding, translocation to the target site, and most of the biological interactions with the possibility of causing tissue injury. therefore, in terms of safety, the effect of silver nanoparticles is a major consideration: even if they inhibit viral infections, it would not be beneficial if there are adverse effects to humans or animals. a commonly used strategy to reduce a possible toxicity is to use various capping agents to prevent the direct contact of the metal with the cells. potential routes of human exposure to metal nanoparticles used as therapeutic compounds include the gastrointestinal tract, the skin, the lungs, and systemic administration. considering the use of metal nanoparticles from the point of view of a potential antiviral therapy, it is straightforward that the safest and easiest results can be obtained with topical use of nanoparticles as microbicide for direct viral particles inactivation and/or inhibition of the early steps of the viral life cycle, attachment and entry. therefore, the dermal route seems the one of major concern. dermal exposure to metal nanoparticles often takes place when using sunscreen lotions, for example, tio 2 and zno nanoparticles. in healthy skin, the epidermis provides excellent protection against particle spread to the dermis. however, in presence of damaged skin micrometer-size particles gain access to the dermis and regional lymph nodes. a further concern should be the potential of nanoparticles translocation to the brain via the olfactory nerve as a consequence of the vicinity of the nasal mucosa to the olfactory bulb. whether nanoparticles in such tissues have any pathological or clinical significance is uncertain, therefore, more data is needed to properly address the safety concern on the use of metal nanoparticles as pharmaceuticals. several studies have demonstrated the cytotoxic effects of metal nanoparticles [90] [91] [92] [93] , in fact, silver nanoparticles were found to be highly cytotoxic to mammalian cells based on the assessment of mitochondrial function, membrane leakage of lactate dehydrogenase, and abnormal cell morphologies [90] [91] [92] [93] . at a cellular level, metal nanoparticles interact with biological molecules within mammalian cells and can interfere with the antioxidant defence mechanism leading to the generation of reactive oxygen species (ros). such species, in excess, can cause damage to biological components through oxidation of lipids, proteins, and dna. oxidative stress may have a role in the induction or the enhancement of inflammation through upregulation of redox sensitive transcription factors (e.g., nf-îºb), activator protein-1, and kinases involved in inflammation [94] [95] [96] [97] . the generation of reactive oxygen species by cells exposed to silver nanoparticles [91] has been showed in human lung fibroblast and human glioblastoma cells, and as a consequence dna damage and cell cycle abnormalities have been observed. accumulation of silver nanoparticles in various organs (lungs, kidneys, brain, liver, and testes) has been evidenced in animal studies [98] . most of the in vitro studies show dose dependence, in fact, higher doses of silver induce a strongher cellular toxicity. nevertheless, should be considered that in vitro concentrations of nanoparticles are often much higher than the ones used in in vivo experiments, therefore such exposures do not represent a replica of the conditions expected for in vivo exposure. a recent study [99] showed that mice exposed to silver nanoparticles showed minimal pulmonary inflammation or cytotoxicity following subacute exposures, but longer term exposures with higher lung burdens of nanosilver are not investigated, therefore eventual cronic effects may be underscored. this review presents only a brief description of the toxicity derived from the use of metal nanoparticles. a more detailed coverage of the topic is available in recently published reviews [100] [101] [102] [103] . although significant progress has been made to elucidate the mechanism of silver nanomaterial toxicity, a proved consensus on the immediate impact or long term effect on human health is still missing. further research is required to provide the necessary warranties to allow a safely exploitation of the interesting in vitro antiviral properties of silver nanoparticles and their transfer to the clinical setting. nanoparticles are nanoscale clusters of metallic atoms, engineered for some practical purpose, most typically antimicrobial and sterile applications. different wet chemical methods have been used for the synthesis of metallic nanoparticles dispersions. the early methods to produce nanoparticles of noble-metals are still used today and continue to be the standard by which other synthesis methods are compared. the most common methods involve the use of an excess of reducing agents such as sodium citrate [104] or nabh 4 [105] . ayyanppan et al. [106] obtained ag, au, pd and cu nanoparticles by reducing metallic salts in dry ethanol. longenberger et al. [107] produced au, ag and pd metal colloids from air-saturated aqueous solutions of poly(ethylene glycol) (peg). reduction methods can also be used for the production of pt, pd, cu, mi etc., although specific protocols depend on the reduction potential of the source ion [108] . cu and ni are not very stable as the metal particles are easily oxidized requiring strong capping ligands to prevent the oxidation. initially, the reduction of various complexes with metallic ions leads to the formation of atoms, which is followed by agglomeration into oligomeric clusters. controlled synthesis is usually based on a two-step reduction process: in the first step a strong reducing agent is used to produce small particles; in the second step these small particles are enlarged by further reduction with a weaker reducing agent [104] . strong reductants lead to small monodisperse particles, while the generation of larger size particles can be difficult to control. weaker reductants produce slower reduction reactions, but the nanoparticles obtained tend to be more polydisperse in size. different studies reported the enlargement of particles in the secondary step from about 20-45 nm to 120-170 nm [109] . another general method for the production of different metal nanoparticles (au, ag, pt, pd) uses commonly available sugars, e.g., glucose, fructose and sucrose as reducing agents [110] . this approach has several important features: (1) sugars (glucose, fructose, and sucrose) are easily available and are used as reducing agents; (2) upon their exploitation no other stabilizing agent or capping agent is required to stabilize the nanoparticles; (3) sugars are very cheap and biofriendly (4) the nanoparticles can be safely preserved in a essiccator for months and redispersed in aqueous solution whenever required instead of being kept in aqueous solution. an array of other physical and chemical methods have been used to produce nanomaterials. in order to synthesize noble metal nanoparticles of particular shape and size specific methodologies have been formulated, such as ultraviolet irradiation, aerosol technologies, lithography, laser ablation, ultrasonic fields, and photochemical reduction techniques, although they remain expensive and involve the use of hazardous chemicals. therefore, there is a growing concern to develop environment-friendly and sustainable methods. biosynthesis of gold, silver, gold-silver alloy, selenium, tellurium, platinum, palladium, silica, titania, zirconia, quantum dots, magnetite and uraninite nanoparticles by bacteria, actinomycetes, fungi, yeasts and viruses have been reported. however, despite the stability, biological nanoparticles are not monodispersed and the rate of synthesis is slow. to overcome these problems, several factors such as microbial cultivation methods and extraction techniques have to be optimized and factors such as shape, size and nature can be controlled through just modifying ph, temperature and nutrient media composition. owing to the rich biodiversity of microbes, their potential as biological materials for nanoparticle synthesis is yet to be fully explored. the production of metal nanoparticles involves three main steps, including (1) selection of solvent medium; (2) selection of environmentally benign reducing agent; (3) selection of nontoxic substances for the nanoparticles stability [111] . biomineralization is also an attractive technique, being the best nature friendly method of nanoparticle synthesis. in one of the biomimetic approaches towards generation of nanocrystals of silver, reduction of silver ions has been carried out using bacteria and unicellular organisms. the reduction is mediated by means of an enzyme and the presence of the enzyme in the organism has been found to be responsible of the synthesis [112, 113] . therefore in search of a methodology that could provide safer and easier synthesis of metal nanoparticles, it seems that the biogenic synthesis using the filtrated supernatant of different bacterial and fungal cultures is having a considerable impact, where the reduction of metal ions occurs through the release of reductase enzymes into the solution [28, [114] [115] [116] . for an extensive coverage of the biological synthesis of metal nanoparticles by microbes, refer to the recent review by narayanan and sakthivel [117] . in the crusade toward the development of drugs for the therapy of viral diseases, the emergence of resistant viral strains and adverse side effects associated with a prolonged use represent huge obstacles that are difficult to circumvent. therefore, multidisciplinary research efforts, integrated with classical epidemiology and clinical approaches, are crucial for the development of improved antivirals through alternative strategies. nanotechnology has emerged giving the opportunity to re-explore biological properties of known antimicrobial compounds, such as metals, by the manipulation of their sizes. metal nanoparticles, especially the ones produced with silver or gold, have proven to exhibit virucidal activity against a broad-spectrum of viruses, and surely to reduce viral infectivity of cultured cells. in most cases, a direct interaction between the nanoparticle and the virus surface proteins could be demonstrated or hypothesized. the intriguing problem to be solved is to understand the exact site of interaction and how to modify the nanoparticle surface characteristics for a broader and more effective use. besides the direct interaction with viral surface glycoproteins, metal nanoparticles may gain access into the cell and exert their antiviral activity through interactions with the viral genome (dna or rna). furthermore, the intracellular compartment of an infected cell is overcrowded by virally encoded and host cellular factors that are needed to allow viral replication and a proper production of progeny virions. the interaction of metal nanoparticles with these factors, which are the key to an efficient viral replication, may also represent a further mechanism of action ( figure 2 ). most of the published literature describes the antiviral activity of silver or gold nanoparticles against enveloped viruses, with both a dna or an rna genome. considering that one of the main arguments toward the efficacy of the analysed nanoparticles is the fact that they in virtue of their shape and size, can interact with virus particles with a well-defined spatial arrangement, the possibility of metal nanoparticles being active against naked viruses seems appealing. moreover, it has been already proven that both silver and gold nanoparticles may be used as a core material. however, no reports are yet available for the use of other metals, but the future holds many surprises, especially considering that the capping molecules that could be investigated are virtually unlimited. nonetheless, for metal nanoparticles to be used in therapeutic or prophylactic treatment regimens, it is critical to understand the in vivo toxicity and potential for long-term sequelae associated with the exposure to these compounds. additional research is needed to determine how to safely design, use, and dispose products containing metal nanomaterials without creating new risk to humans or the environment. the authors declare no conflict of interest. principles and lessons from the smallpox eradication programme paralytic poliomyelitis: seasoned strategies, disappearing disease mechanisms of viral emergence the bactericidal effect of silver nanoparticles antimicrobial effects of silver nanoparticles a peptide derived from herpes simplex virus type 1 glycoprotein h: membrane translocation and applications to the delivery of quantum dots delivery of nanoparticulate drug delivery systems via the intravenous route for cancer gene therapy dual enlargement of gold nanoparticles: from mechanism to scanometric detection of pathogenic bacteria high-throughput detection and sizing of individual low-index nanoparticles and viruses for pathogen identification array-based sensing with nanoparticles: chemical noses' for sensing biomolecules and cell surfaces a new era for cancer treatment: gold-nanoparticle-mediated thermal therapies nano-oncology: drug delivery, imaging, and sensing virology" fifth edition: virus entry and uncoating virus entry: molecular mechanisms and biomedical applications inhibition of viral-induced membrane fusion by peptides inhibitors of viral entry clinical significance of credã©'s prophylaxis in germany at present antimicrobial effect of surgical masks coated with nanoparticles application of anisotropic silver nanoparticles: multifunctionalization of wool fabric nanosilver as a new generation of nanoproduct in biomedical applications silver nanoparticles as antimicrobial agent: a case study on e. coli as a model for gram-negative bacteria the synthesis of chitosan-based silver nanoparticles and their antibacterial activity silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? a study of the gram-negative bacterium escherichia coli susceptibility constants of escherichia coli and bacillus subtilis to silver and copper nanoparticles synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against staphylococcus aureus and escherichia coli zno nanoparticles enhanced antibacterial activity of ciprofloxacin against staphylococcus aureus and escherichia coli biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria destruction and control of toxoplasma gondii tachyzoites using gold nanosphere/antibody conjugates facile preparation and characterization of highly antimicrobial colloid ag or au nanoparticles antifungal effect of silver nanoparticles on dermatophytes antifungal activity and mode of action of silver nano-particles on candida albicans biosynthesis and characterization of ti/ni bimetallic nanoparticles small molecule-capped gold nanoparticles as potent antibacterial agents that target gram-negative bacteria inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis silver nanoparticles as a new generation of antimicrobials interaction of silver nanoparticles with hiv-1 silver nanoparticles fabricated in hepes buffer exhibit cytoprotective activities toward hiv-1 infected cells mode of antiviral action of silver nanoparticles against hiv-1 pvp-coated silver nanoparticles block the transmission of cell-free and cell-associated hiv-1 in human cervical culture silver nanoparticles inhibit hepatitis b virus replication silver nanoparticles inhibit replication of respiratory sincitial virus inhibition of herpes simplex virus type 1 infection by silver nanoparticles capped with mercaptoethane sulfonate inhibition of hsv-1 attachment, entry, and cell-to-cell spread by functionalized multivalent gold nanoparticles a preliminary assessment of silver nanoparticles inhibition of monkeypox virus plaque formation inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles interaction of silver nanoparticles with tacaribe virus genotypic resistance profiles in antiretroviral-naive hiv-1 infections before and after initiation of first-line haart: impact of polymorphism on resistance to therapy hiv-1 membrane fusion: targets of opportunity aids virus envelope spike structure the retroviruses and their replication electrochemical reduction of noble metal compounds in ethylene glycol development of an in vitro organ culture model to study transmission of hiv-1 in the female genital tract blocking of cell-free and cell-associated hiv-1 transmission through human cervix organ culture with uc781 the family herpesviridae: a brief introduction fields herpes simplex viruses fusing structure and function: a structural view of the herpesvirus entry machinery herpes simplex virus: receptors and ligands for cell entry herpesviruses and heparin sulfate: an intimate relationship in aid of viral entry glycoproteins gb, gd, and ghgl of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a cos cell transfection system entry of herpesviruses into mammalian cells crystal structure of glycoprotein b from herpes simplex virus 1 mutational evidence of internal fusion loops in herpes simplex virus glycoprotein b structural basis of local, ph-dependent conformational changes in glycoprotein b from herpes simplex virus type 1 fusogenic domains in herpes simplex virus type 1 glycoprotein h analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type 1 glycoproteins h and b evidence for a role of the membrane-proximal region of herpes simplex virus type 1 glycoprotein h in membrane fusion and virus inhibition analysis of a membrane interacting region of herpes simplex virus type 1 glycoprotein h the presence of a single n-terminal histidine residue enhances the fusogenic properties of a membranotropic peptide derived from herpes simplex virus type 1 glycoprotein h herpesviruses remodel host membranes for virus egress respiratory syncytial virus and metapneumovirus virology the viruses and their replication. in virology human monkeypox: an emerging zoonotic disease the viruses and their replication properties and characterization of monoclonal antibodies to tacaribe virus the 3' end termini of the tacaribe arenavirus subgenomic rnas antimicrobial effect of silver-impregnated cellulose: potential for antimicrobial therapy occurrence of viruses in us groundwaters detection and quantification of human bocavirus in river water evaluation of public health risks at recreational beaches in lake michigan via detection of enteric viruses and a human-specific bacteriological marker bactericidal activity of tio2 photocatalyst in aqueous media: toward a solar-assisted water disinfection system photocatalitic bactericidal effect of tio 2 thin films: dynamic view of the active oxygen species responsible for the effect different inactivation behaviors of ms-2 phage and escherichia coli in tio 2 photocatalytic disinfection photocatalytic inactivation of escherischia coli. effect of concentration of tio2 and microorganism, nature, and intensity of uv irradiation environmental applications of semiconductor photocatalysis inactivation of microorganisms in a flow-through photoreactor with an immobilized tio 2 layer kinetic evaluation of biocidal activity of titanium dioxide against phage ms2 considering interaction between the phage and photocatalyst particles virus inactivation by silver doped titanium dioxide nanoparticles for drinking water treatment in vitro cytotoxicity of nanoparticles in mammalian germline stem cells cytotoxicity and genotoxicity of silver nanoparticles in human cells in vitro toxicity of silver nanoparticles at noncytotoxic doses to hepg2 human hepatoma cells in vitro toxicity of nanoparticles in brl 3a rat liver cells regulation of nuclear factor-îºb, activator protein-1, and glutathione levels by tumor necrosis factor-î± and dexamethasone in alveolar epithelial cells glutathione, stress responses, and redox signaling in lung inflammation biomedical applications and potential health risks of nanomaterials: molecular mechanisms effects of nanomaterial physicochemical properties on in vivo toxicity twenty-eight-day oral toxicity, genotoxicity, and genderrelated tissue distribution of silver nanoparticles in sprague-dawley rats nanosilver induces minimal lung toxicity or inflammation in a subacute murine inhalation model silver or silver nanoparticles: a hazardous threat to the environment and human health? nanosilver: a nanoproduct in medical application a review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment metal-based nanoparticles and their toxicity assessment adsorption and surface-enhanced raman of dyes on silver and gold sols plasma resonance enhancement of raman scattering by pyridine adsorbed on silver or gold sol particles of size comparable to the excitation wavelength nanoparticles of ag, au, pd, and cu produced by alcohol reduction of the salts formation of metal particles in aqueous solutions by reactions of metal complexes with polymers synthesis, characterization, and stability of dendrimer-encapsulated palladium nanoparticles reproducible preparation of silver sols with small particle size using borohydride reduction: for use as nuclei for preparation of larger particles general method of synthesis for metal nanoparticles completely "green" synthesis and stabilization of metal nanoparticles biosynthesis of silver nanocrystals by bacillus licheniformis biosynthesis of silver and gold nanoparticles using brevibacterium casei fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole biosynthesis of silver nanoparticles from staphylococcus aureus and its antimicrobial activity against mrsa and mrse extracellular synthesis of silver bionanoparticles from aspergillus clavatus and its antimicrobial activity against mrsa and mrse biological synthesis of metal nanoparticles by microbes key: cord-268712-rxdw553c authors: sawyer, alexandra; ayers, susan; field, andy p. title: posttraumatic growth and adjustment among individuals with cancer or hiv/aids: a meta-analysis date: 2010-03-02 journal: clin psychol rev doi: 10.1016/j.cpr.2010.02.004 sha: doc_id: 268712 cord_uid: rxdw553c there is increasing research on posttraumatic growth after life-threatening illnesses such as cancer and hiv/aids, although it is unclear whether growth confers any psychological or physical benefits in such samples. consequently, this meta-analysis explored the relationship between posttraumatic growth and psychological and physical wellbeing in adults diagnosed with cancer or hiv/aids and examined potential moderators of these relationships. analysis of 38 studies (n = 7927) of posttraumatic growth after cancer or hiv/aids revealed that growth was related to increased positive mental health, reduced negative mental health and better subjective physical health. moderators of these relationships included time since the event, age, ethnicity, and type of negative mental health outcome. it is hoped that this synthesis will encourage further examination of the potentially complex relationship between posttraumatic growth and adjustment in individuals living with life-threatening medical conditions. parallel other traumatic stressors in many ways. the diagnosis may be sudden and unexpected, the disease and treatment may pose threats to one's life, and the experience may evoke intense emotional responses of fear and helplessness. at the same time living with a life-threatening illness is not an acute, singular stressful experience, but rather a series of unfolding threats and stressors (cordova, 2008) . cumulatively, these experiences can constitute a traumatic stressor for many individuals with cancer or hiv/aids. experiencing a lifethreatening illness was first recognised as an event that could precipitate posttraumatic stress disorder (ptsd) in the dsm-iv (american psychiatric association [apa], 1994) . rates of ptsd in cancer patients range from 5% to 35% (kangas, henry, & bryant, 2002) and in hiv/aids patients from 30% to 64% (botha, 1996; kelly et al., 1998; martinez, israelski, walker, & koopman, 2002) . over the past decade there has been an important shift in emphasis of research from a nearly exclusive focus on the negative aftermath of such events to consideration of possible positive outcomes (linley, 2003) . researchers have used a number of different terms to describe individuals' reports of benefits in the face of adversity, including posttraumatic growth, adversarial growth, benefit-finding, and thriving. throughout this paper tedeschi, park, and term posttraumatic growth (ptg) will be used to describe a positive change in one's previous level of functioning as a result of the struggle with highly challenging life circumstances. this term differs from resilience, optimism, hardiness, which describe individuals who have adjusted successfully despite adversity (o'leary & ickovics, 1995) , whereas individuals experiencing ptg are transformed by their struggle with adversity. a rapidly increasing literature now testifies to the prevalence of positive life changes and personal growth following cancer and hiv/ aids. equally high rates of positive changes have been reported across both illnesses. between 59% and 83% of people living with hiv/aids have been shown to report positive changes since diagnosis (milam, 2004 (milam, , 2006a siegel & schrimshaw, 2000) . likewise, data suggest that between 60% and 90% of cancer survivors also report positive changes (collins, taylor, & skokan, 1990; fromm, andrykowski, & hunt, 1996; petrie, buick, weinman, & booth, 1999; rieker, edbril, & garnick, 1985) . within the general ptg literature three common categories of growth outcomes have been identified (joseph & linley, 2006; tedeschi et al., 1998) . first, individuals often report that their relationships are enhanced in some way. for example many individuals with cancer or hiv/aids require practical and emotional support, and positive interpersonal experiences may strengthen a person's appreciation of some relationships. second, people change their views of themselves in some way. for example patients may develop a greater sense of personal resilience and strength, an acceptance of their vulnerabilities and limitations, which are typified by a heightened awareness of their own mortality and the fragility of life. third, there are often reports of changes in life philosophy. for example people diagnosed with cancer or hiv/aids are faced with the concern that their disease might progress and shorten their life and these concerns may lead to a shift in priorities and values, and to a different appreciation and approach to day-to-day life. together these positive changes in psychological well-being can lead to a whole new way of living. finally certain changes have been identified specific to individuals facing a serious illness. a recent focus of the ptg research has been the relationship between ptg and health behaviours (milam, 2004; milam, ritt-olsen, & unger, 2004) . luszczynska, sarkar and knoll (2007) found that ptg significantly predicted adherence to antiretroviral therapy in individuals diagnosed with hiv. furthermore, women with breast cancer have described making positive changes in health related behaviours and engaging in more careful cancer surveillance as a result of their experience (sears, stanton, & danoff-burg, 2003) . studies that compare ptg in cancer and hiv/aids patients suggest that growth is experienced in the same multidimensional manner across both illnesses (lechner & weaver, 2009 ). therefore, alongside psychological, interpersonal, and life orientation changes, positive changes in health behaviours may also occur following a life-threatening illness diagnosis. several models have now been proposed regarding the occurrence of ptg. the three most detailed models to date include tedeschi and calhoun's (1995 calhoun's ( , 2004 ) functional descriptive model, organismic valuing theory and christopher's (2004) biopsychosocial-evolutionary theory. although with some variation, most models hypothesize that the experience of a highly stressful or traumatic event violates an individual's basic beliefs about the self and the world and that some type of meaning making or cognitive processing to rebuild these beliefs and goals occurs, resulting in perceptions that one has grown through the process (horowitz, 1986; janoff-bulman, 2004; tedeschi & calhoun, 2004) . although offering different levels of explanation at both the social cognitive and biological evolutionary levels, they are complimentary in that they are underpinned by the notion that people are intrinsically motivated towards growth (joseph & linley, 2006) . an important issue to be addressed in the literature is whether ptg following the diagnosis of a life-threatening illness is associated with psychological and physical benefits (zoellner & maercker, 2006) . however, the current literature is unclear. for example some studies report there is no significant relationship between ptg and distress (cordova, cunningham, carlson, & andryowski, 2001; schulz & mohamed, 2005) , and other studies suggest distress and ptg can co-exist (tomich & helgeson, 2004) . for example barakat, alderfer, and kazak (2006) found that ptg and posttraumatic stress symptoms were positively correlated in adolescent survivors of cancer. however, other studies have reported an inverse relationship between measures of ptg and psychological distress (linley & joseph, 2004; updegraff, taylor, kemeny, & wyatt, 2002; urcuyo, boyers, carver, & antoni, 2005) . therefore, it remains to be established whether the experience of ptg in relation to a life-threatening illness confers any benefit in terms of psychological or physical health. given the discrepant findings on this relationship a systematic integration of the literature is needed, and a meta-analysis is an ideal tool to do this. a previous meta-analysis conducted by helgeson, reynolds, and tomich (2006) investigated the association between ptg and adjustment after a wide range of events such as sexual assault, natural disaster, bereavement, childhood abuse and illness. they found that ptg was related to more positive affect and less depression, but also to more intrusive thoughts about the event. ptg was unrelated to anxiety, distress, quality of life and subjective physical health. as such the aim of the current paper is to present a meta-analysis of the existing literature that will aim to objectively summarize ptg and its relation to adjustment in individuals living with a life threatening illness (cancer or hiv/ aids) and to examine potential moderators of this relationship. one possible explanation for the inconsistency between ptg and adjustment is that the relationship is moderated by other variables. therefore five possible moderators will be examined that might attenuate or accentuate the growth-adjustment relationship. these were chosen because they are commonly assessed within the literature, and have prior empirical and theoretical foundations. the first variable that might moderate the relationship between ptg and adjustment is the length of time since the diagnosis. research and theory suggest that ptg is unlikely to occur shortly after the critical event, but rather takes time to occur and is more likely to be reported in hindsight tedeschi & calhoun, 1995 . therefore it is hypothesized that ptg is associated with positive adjustment when a longer time since the health event has elapsed. three characteristics of the sample will also be examined as moderators: age, gender, and ethnicity. past research has indicated that women (bellizzi, 2004; milam, 2004) , younger participants linley & joseph, 2004; milam, 2004; widows, jacobson, booth-jones, & fields, 2005) , and ethnic minorities are more likely to report ptg. however, it is not clear if and how these individual differences differentially relate to ptg and adjustment . therefore no specific predictions about directionality regarding how these variables might moderate the growth-adjustment relationship will be made. it is also possible that the quality of the study might moderate the relationship between growth and health. for example studies that use a valid measure of growth should reflect actual ptg, and distinguish from other processes such as self-enhancement, positive illusion, and "pseudogrowth" (lechner & antoni, 2004; park & lechner, 2006) . less validated measures may fail to capture ptg, and therefore account for some of the variation in the research. through examination of these moderators it is hoped that the meta-analysis will identify subgroups of adults whose experience of ptg is likely to be positively or negatively related to mental and physical health. in summary, the purpose of the present study is two-fold. primarily it is concerned with estimating the overall effect size of the relationship between ptg following a life threatening illness (cancer or hiv/aids) and various indicators of adjustment. secondly, this analysis hopes to identify the variability amongst studies and explore potential moderators of the growth and adjustment relationship. it is hoped that such a review of the extant literature will lead to an enhanced understanding of the impact of ptg on the adjustment process in individuals living with life-threatening illnesses. a systematic search was conducted to identify studies of ptg in individuals following cancer or hiv/aids. the primary search method for the selection of studies was a review of the psychological and medical literature using the following computerized databases up to october 2009: medline, psycharticles, psychinfo, pubmed, and web of science. relevant key words were used to search for articles within these databases. search terms included key words related to ptg: posttraumatic growth, post-traumatic growth, benefit finding, stress related growth and adversarial growth. these terms were crossed with the following health-related key terms: health, illness, disease, life-threatening, chronic, medical, terminal, cancer, hiv, aids. additional studies were located through the inspection of the reference sections of obtained papers and reviews. relevant journals were also manually searched to locate papers that may not have been identified in the databases. these journals were: psycho-oncology, psychology and health, journal of traumatic stress, british journal of health psychology and journal of consulting and clinical psychology. in addition, active researchers in the field of psychological growth in health samples were contacted to ask for recent papers in the field and for unpublished research to reduce the effect of publication bias. a search of abstracts from relevant conferences was also conducted to locate additional unpublished work in the area. however, no unpublished studies were retrieved. this literature search yielded a preliminary database of 193 published papers. these 193 papers were examined to determine eligibility for inclusion in the meta-analysis. studies had to meet eight criteria for inclusion. first, studies were included only if the sample were adults aged 18 or over. this decision was made because the current literature is unclear whether children or adolescents differentially experience ptg in comparison to adults (ickovics, meade, et al., 2006; ickovics, milan, et al., 2006; milam et al., 2004) , and also only a small number of studies have explored ptg in children and adolescents following illness (too few to include adult vs. child as a moderator variable). this resulted in the exclusion of nine studies. second, the studies had to use a quantitative measure of ptg, which was assessed in relation to a measure of positive psychological adjustment, negative psychological adjustment or physical health. studies that included a purely qualitative assessment of ptg, or papers that were reviews of the literature were excluded from the analysis. this resulted in the exclusion of 87 studies. third, ptg must be measured in cancer or hiv/ aids patients. this criterion resulted in the exclusion of 16 studies. fourth, intervention studies were excluded from the analysis unless they measured ptg at baseline prior to manipulation and effect sizes could be extracted. this resulted in the exclusion of 20 studies. fifth, controlled comparison studies that did not report relevant data for the patient sample were excluded. this resulted in the exclusion of nine studies. longitudinal studies which measured ptg at different time points to adjustment measures were excluded. however, when longitudinal studies reported cross-sectional relationships these were included in the analysis. this resulted in the exclusion of seven studies. studies needed to include the relevant effect sizes (namely the correlation coefficient r) or sufficient statistical information that could be used to compute this statistic. authors of papers with unclear statistical information were contacted to enquire about further information and if this was unable to be provided these papers were excluded from the analysis. 1 only two papers were excluded as a result of this criterion. finally, the authors of five non-english articles were contacted for copies of their papers but these were not provided. of the 193 articles yielded by the literature search 38 studies met all of the requirements for inclusion and were therefore used in the metaanalysis. studies included in the meta-analysis are identified with an asterisk in the reference section and a detailed list of the studies is provided in table 1 . from these papers a number of variables were extracted for analysis: i) sample size, ii) sex composition, iii) ethnicity, iv) mean age, v) time since event, vi) health event vii) adjustment outcome, and viii) effect sizes for these relationships. the methodological quality of each study was also assessed based on a checklist developed by mirza and jenkins (2004) . the five criteria that were assessed were: 1) clear study aims, 2) sample representative of population, 3) clear inclusion and exclusion criteria, 4) validated measure of ptg, and 5) appropriate statistical analysis. the studies were then given a total score of quality with the highest possible being eight (1= yes, 0 = no). table 1 displays the quality scores for each individual study. quality scores ranged from 2-5; however most studies were of good quality with over 50% of studies scoring 4 or more. as expected, the concept of adjustment was operationally defined in a number of ways across individual studies. in our analysis measures were combined and a separate analysis was conducted for positive psychological adjustment, negative psychological adjustment and subjective physical health. psychological adjustment was defined in this paper as the psychological outcome, either positive or negative, following illness. specific adjustment measures associated with each adjustment outcome were also examined as moderators to explore how they might explain variability within the growth-adjustment relationship. these adjustment measures were coded as follows: a) positive psychological adjustment was coded either as psychological health (e.g. positive affect, mental health) or general well-being (e.g. life satisfaction), b) negative psychological adjustment was coded as specific symptoms (e.g. depression, anxiety, ptsd) or general distress, and c) subjective physical health was coded as either general physical health, physical symptoms, or functional ability. to examine the role of possible moderators in the growthadjustment relationship, the following information in each paper was coded and used in the analysis as follows: (i) time since diagnosis was examined as a continuous moderator by using the mean time in months, (ii) sample gender composition was examined as continuous variable coded as percentage of female participants, (iii) sample age was examined as a continuous moderator by using the mean time in years, (iv) it was decided to code ethnicity as a categorical variable, either as b75% white or â�¥75% white, as this strategy minimized data exclusion, and (v) the methodological quality of each study was examined as a continuous moderator. all analyses in this paper were carried out on spss (version 15) using syntax specified in field and gillett (in press) . a separate metaanalysis was carried out for each adjustment outcome. in the present study the correlation coefficient (r) was chosen as the effect size estimate for a number of reasons. first, this was a common metric for which the greatest number of effect sizes could be reported or converted; second, it is easily computed from either chi-square, t, f, and d; and third it is readily interpretable (rosenthal & dimatteo, 2001) . a number of papers reported correlation coefficients only for the subscales of ptg. therefore to guarantee the independence assumption among effect sizes the coefficients were averaged to produce a single effect size associated with overall ptg. when a study did not report the effect size or probability value but stated only the relationship was nonsignificant an effect size of zero was assigned to that relationship. this is a conservative strategy because it generally underestimates the true magnitude of effect sizes (durlak & lipsey, 1991; rosenthal, 1995) . however, this approach is preferable to excluding nonsignificant results from the meta-analysis, because this would result in an overestimation of combined effect sizes (rosenthal, 1995) . the authors of these papers were contacted for further information and there was only one study where an effect size of zero assumed. 2 in meta-analysis two common statistical procedures are used: fixedand random-effect models (hedges, 1992; hedges & vevea, 1998; hunter & schmidt, 2000) . real social science data have been shown to contain variability in effect sizes as the norm, which indicates variable population parameters (field, 2003; field, 2005; field & gillet, in press; hunter & schmidt, 2000) . for this reason, and so the results can be generalized beyond the studies included in the meta-analysis, a random effects model was carried out. hedges and vevea's (1998) method was applied using fisher-transformed correlation coefficients with results reported after the back transformation to the pearson product-moment correlation coefficient (see field, 2005; overton, 1998) . using this method, each effect size is weighted by a value reflecting both the within study variance (1/n â�� 3 for correlation coefficients in which n is the sample size) and the between study variance (ï� 2 ). the exact weight function for each effect size is w * i = 1 n i â��3 +ï� 2 â��1 (see field & gillet, in press for a guide to using hedges and vevea's method). moderator analyses were conducted also using a random-effects general linear model in which each z-transformed effect size can be predicted from the transformed moderator effect (represented by regression coefficient, î²). the moderator effect, î², is estimated using generalised least squared (gls). in both the main analysis and moderator analyses, between study variance was estimated noniteratively (e.g. dersimonian & laird, 1986) . for a technical overview of the gls moderator analysis that we employed see overton (1998) or field and gillet (in press). in any meta-analysis publication bias is a concern. this bias refers to the tendency that the decision to publish a paper is determined by the results of the study (begg, 1994) . for example studies with nonsignificant findings are less likely to be published than those with significant outcomes, which could result in a positive bias within the literature. there are different approaches to estimating publication bias: rosenthal's (1979) fail-safe n, funnel plots and sensitivity analysis. the fail safe n estimates the number of unpublished, nonsignificant studies that would have to exist for the obtained probability value of the population effect size estimate to be rendered nonsignificant. this measure is problematic because its emphasis is on significance testing the population effect size rather than estimating the population effect size itself. therefore, we have chosen to report table 3 stem and leaf plot of effect sizes for negative mental health (rs). .2 3, 4, 5, .1 0, 1, 1, 3, 3, 4, 6, 6, .0 0, 1, 2, 2, 2, 2, 3, 3, 4, 5, 5, 9, 9, 9 â��.0 1, 4, 4, 4, 5, 8, 9, 9 â��.1 0, 0, 1, 1, 1, 2, 2, 3, 3, 3, 7, 7, 9 â��.2 0, 0, 2, 3, 3, 4, 7, 7 â��.3 3, 4, 5, 6 â��.4 2, 4, measures that specifically address bias in the population effect size estimate. first, we produce funnel plots of the effect found in each study against the standard error (light & pillemer, 1984 ). an unbiased sample will show a cloud of data points that is symmetric around the population effect size and has the shape of a funnel (reflecting greater variability in effect sizes from studies with small sample sizes/less precision). second, we performed a sensitivity analysis, which is a method that uses weights to model the process through which the likelihood of a study being published varies (usually based on a criterion such as the significance of a study). we applied the methods proposed by vevea and woods (2005) because they can be applied to relatively small samples of studies such as we have. there were 38 studies included in the meta-analysis; with a total of 7927 participants. sample sizes from individual studies ranged from 52 to 835. 78.0% of the studies focused on individuals with a cancer diagnosis and 21.1% included individuals with a hiv/aids diagnosis. length of time since treatment/diagnosis varied and ranged from 0 to 108 months (m=41.65, sd=31.86). mean age of the sample was 50.66 (sd=9.9). of the studies that provided information on ethnicity, the majority (n=15) included samples predominantly composed of white participants. tables 2-4 graphically represent the effect sizes included in each adjustment meta-analysis by means of a stem and leaf plot. the stem identifies the first digit of an effect size and the leaf identifies the final digit of an effect size. for positive mental health (table 2) , the bulk of effect sizes were in the range of 0 to .26, but the range was quite wide (â��.23 to .49) suggesting the influence of moderator variables. for negative mental health (table 3) , the distribution of effect sizes is relatively symmetrical and is centered around 0 to â��.1. again, the range of effect sizes was quite large (â��.44 to .25) suggesting that moderator variables might usefully explain some of this variability. finally, for physical health (table 4 ) the effect size distribution looks skewed and is centered around 0 to â��.07. three studies appeared to have relatively large positive effect sizes that were inconsistent with the bulk of studies. table 5 shows the individual meta-analyses for each adjustment outcome. ptg was significantly related to higher levels of positive psychological adjustment (ptg explained 1.7% of the variance), lower levels of negative psychological adjustment (ptg explained only 0.3% of the variance), and higher reported levels of physical health (ptg explained 1.4% of the variance). the results suggest considerable variation in effect sizes for the three adjustment outcomes, and it is therefore important to examine factors that moderate these relationships. the funnel plots shown in figs. 2-4 suggest publication bias might be present in the data, as indicated by the non-funnel like and asymmetric distribution of data points around the estimated mean, typical of biased data sets. in particular, for positive mental health ( fig. 1 ) and physical health (fig. 3) , the data cloud is relatively sparse for small studies (the bottom part of the figure). this pattern is indicative of one-tailed publication bias (vevea & woods, 2005) . for negative mental health (fig. 2 ) the cloud is a little sparse around zero for small studies, which indicates two-tailed publication bias (vevea & woods, 2005) . we calculated several publication-bias corrected estimates based on our interpretation of the funnel plots of the overall population effect sizes on positive mental health, negative mental health and physical health. we used vevea and woods ' (2005) weight function model of publication bias to calculate population effect size estimates under different selection bias scenarios. based on the funnel plots, for positive mental health and physical health we assumed moderate (mot) or severe (sot) one-tailed selection bias, and for negative mental health we assumed moderate (mtt) and severe (stt) two-tailed selection bias. the values corrected for selection bias were as follows: for positive mental adjustment, the original population estimate of .13 was reduced to .08 (mot), â��.40 (sot); for negative mental adjustment, the original estimate of â��.05 became â��.05 (mtt) and â��.04 (stt); for physical health the original estimate of .12 became .06 (mot), â��.47 (sot). as such, the estimate of population effect size for negative mental health was unaffected by publication bias. if we assume moderate publication bias, then estimates for positive mental health and physical health were slightly reduced, but if severe publication bias is assumed then the estimates change quite dramatically. as such, our conclusions come with the caveat that if severe publication bias was, in reality, present in the literature then our conclusions would be quite different for positive mental health and physical health outcomes. five moderators that might explain significant amounts of effect size variation for each adjustment outcome were examined. subcategories of each adjustment outcome were also initially explored as moderators. categories of positive psychological adjustment did not significantly moderate the relationship between ptg and positive mental health (p n .05). time emerged as a significant moderator of positive psychological adjustment (î² = .005, p b .001), implying the longer the time since the event, the stronger the relationship between ptg and positive mental health. the age of the sample emerged as a significant moderator (î² = â��.011, p b .01), indicating that samples with younger participants, showed a stronger relationship between ptg and positive adjustment. ethnicity also moderated the relationship between ptg and positive mental health, ï� 2 (1) = 4.77, p b .05, indicating that samples comprised of more than 25% non-white participants demonstrated a stronger relationship between ptg and positive psychological adjustment. gender (î² = .001, p n .05) and quality (î² = .148, p n .05) did not significantly moderate the relationship between ptg and positive psychological adjustment. categories of negative mental health moderated the relationship between ptg and negative psychological adjustment. dummy coding revealed that ptsd symptoms had a stronger negative relationship with ptg in comparison to depression (ï� 2 (1) = 4.29, p b .05), but not in comparison to anxiety (ï� 2 (1) = 0.28, p n .05) and general distress (ï� 2 (1) = 0.18, p n .05). time since the health event, measured in months, moderated negative mental health (î² = â��.003, p b .01), indicating the shorter the time since the event, the stronger the relationship between ptg and negative adjustment. ethnicity was also a significant moderator, ï� 2 (1) = 34.16, p b .001, indicating that samples with more than a 75% white composition demonstrated a stronger negative relationship between ptg and negative adjustment. age also appeared as a moderator (î² = .009, p b .001), indicating that samples with older participants demonstrated a stronger negative relationship between ptg and negative adjustment. quality of the study (î² = .021, p n .05) and participant's gender (î² = .001, p n .05) did not moderate the relationship between growth and negative mental health. categories of physical health did not significantly moderate the relationship between ptg and physical health (p n .05). ethnicity moderated the relationship between ptg and physical health (ï� 2 (1)= 4.75, pb .05), indicating that samples comprised of more than 25% nonwhite participants demonstrated a stronger relationship between ptg and physical health. furthermore, time (î²=.003, pn .05), gender (î²= â��.001, pn .05), age (î²=.003, pn .05), and study quality (î²=â��.013, p=.05) did not significantly moderate the relationship between ptg and physical health. this meta-analytic review summarized the findings from 38 studies examining the association between ptg following cancer or hiv/aids and positive psychological adjustment, negative psychological adjustment, and subjective physical health. despite variability in effect sizes this analysis demonstrated a small positive relationship between ptg and positive mental health. therefore, individuals who perceive ptg following cancer or hiv/aids also report enhanced psychological well-being. furthermore, a small negative relationship was found between ptg and negative mental health. individuals who perceive ptg following cancer or hiv/aids also report reduced symptoms of negative mental health. finally, ptg displayed a small positive relationship with measures of subjective physical health, implying that ptg may also confer some physical benefit. these findings suggest that ptg is associated with positive adaptive consequences, and is therefore an important construct to be studied in clinical and health research. an additional aim of the study was to examine factors that might moderate the relationship between ptg and adjustment, and therefore provide further insight by accounting for variability in effect sizes reported previously. study quality and gender were the only variables that did not moderate the relationship between ptg and outcomes. therefore the implications of these findings are that studies of differing quality do not account for differences in the growth-adjustment relationship and that there are no significant differences between men and women in the growth-outcome relationship. other moderators examined had varying effects on relationships between ptg and different outcomes; each of which will be discussed in turn. subcategories of positive mental health, and subjective physical health did not significantly moderate their relationship with ptg. however, subcategories of negative mental health did moderate the growth-negative mental health relationship. specifically, in comparison to depression, ptsd symptoms showed a stronger negative relationship with ptg. time since the illness emerged as a significant moderator for positive and negative mental health. in the short term, there was a stronger relationship between ptg and negative mental health, but over time there was an increased relationship between ptg and positive mental health. these results are consistent with the results from a previous meta-analysis looking at ptg following a range of traumas . together these findings suggest that in the short-term ptg is influential in reducing negative symptoms, but in the long-term ptg is more instrumental in enhancing positive well-being. this is consistent with tedeschi and calhoun's (1995 calhoun's ( , 2004 ) functional-descriptive model of ptg, which states that the management of emotional distress is essential in the initial stages post-trauma. on the other hand, ptg reported later might reflect more substantive life changes that have positive consequences for quality of life (tomich & helgeson, 2004) . time since the health event did not moderate the relationship between ptg and physical health. age appeared to differentially affect the relationship between ptg and adjustment. younger adults demonstrated a stronger positive relationship between ptg and positive mental health. in comparison older adults displayed a stronger negative relationship between ptg and negative mental health. one explanation is that core beliefs of young people may be more affected than those of older people. for example younger people tend to view the world as less just and less benevolent, and the older groups tend to view the world as luckier and more controllable (calhoun, cann, tedeschi, & mcmillan, 1998) . being diagnosed with cancer or hiv/aids when young might shatter more natural and social rules or beliefs which would generate a greater possibility of reconstructing these core beliefs and therefore promote ptg. another explanation might be that younger people may be more capable and adept at making changes to their lives, which results in enhanced well-being. whereas, older participants may be dealing with other significant life events and be less adaptable compared with younger samples, and therefore ptg may be more useful in reducing and managing distress. age did not act as a significant moderator between ptg and self-reported physical health. ethnicity was a significant moderator of the relationship ptg and all three adjustment measures. specifically, non-white samples displayed a larger effect size for the relationship between ptg and positive mental health and also subjective physical health, compared to samples composed primarily of white participants. in comparison samples composed of predominantly white participants showed a stronger relationship between ptg and negative mental health. this variability may be explained by differences in culture e.g. family, religion, spirituality, which has shown to be important or associated with ptg following stressful life events (milam, 2006a; shaw, joseph, & linley, 2005; tedeschi & calhoun, 1995) . because of these differences, growth in ethnic minority samples may reflect more fundamental and existential changes resulting in enhanced wellbeing. in comparison, growth in predominantly white samples may be used more as a strategy to reduce distress. the results of this study should be interpreted with the following limitations in mind. though the present findings indicate that ptg and positive mental health, negative mental health, and subjective physical health are associated (albeit modestly), only cross-sectional data were included in the analysis, which constrains causal inference. for example it is not clear if ptg leads to better psychological and physical health, or if these factors result in an enhanced perception of ptg. furthermore, even though studies were included in the analysis only if they used a clear measure of ptg the final data set consisted of studies that used varying conceptions of ptg, which could be problematic. for example, past research has indicated that benefit finding and ptg are related but distinct constructs, and might therefore have unique predictors and outcomes (sears et al., 2003) . therefore, future research in the area should ascertain if such constructs are theoretically and empirically interchangeable. the present study did not examine type of illness as a moderator because there were not enough studies of hiv/aids to include cancer vs. hiv/aids as a moderator variable. although research suggests that people with hiv/aids report similar levels and areas of ptg compared to individuals with cancer, there are unique differences between the illnesses, particularly in social responses to individuals with hiv/aids compared to those with cancer (lechner & weaver, 2009) . for example hiv/aids is an infectious disease and people who are hiv positive may face more stigma because of fear, lack of knowledge concerning transmission, and greater perceived accountability (lechner & weaver, 2009 ). this may hinder opportunities for emotional processing and therefore may not facilitate ptg and positive adjustment as readily as cancer and other illnesses. furthermore, meta-analysis, like any other procedure, has its advantages and disadvantages, and this study is no exception. first, where authors of papers reported significant findings but did not include enough statistical information to calculate the effect size, these effect sizes were coded as zero. this is a conservative approach and therefore may have lowered the effect size estimate for each meta-analysis conducted. second, as with many meta-analytic studies, the current findings may over represent those studies that are published and have significant results, preventing the generalization of the current findings to unpublished reports (rosenthal, 1979) . for the overall effects, our publication bias analysis showed that the population effect size estimates were relatively unaffected when corrected for moderate selection bias. this finding gives us some confidence that the results are not idiosyncratic to our sample of studies. however, when correcting for severe publication bias the effect of growth on positive mental adjustment and physical health became strongly negative (the opposite direction to the population effects). although this is a correction for severe publication bias, the current findings should be viewed within the context of these results. despite these limitations, this study has significant implications for research and practice. a weakness in the literature is the lack of consensus between theorists as to whether ptg is best conceptualized as an adaptive coping strategy that people use following a challenging life event, or as an outcome of the struggle with a traumatic event (affleck & tennen, 1996; park & helgeson, 2006; tedeschi & calhoun, 1995 . the findings from this study suggest that shortly after the event ptg may be used as a coping strategy to manage and reduce emotional distress associated with the illness threat. however over time ptg grows and is more significant in enhancing positive well-being. this implies that adjustment to serious illness is an ongoing process that occurs over time tedechi & calhoun, 1995) . as recognized by butler (2007) a challenge of future work is to psychometrically separate these processes so they can be reliably investigated. the results suggest that ptg is associated with a reduction in negative mental health, which was particularly prominent when ptsd symptoms were the outcome. this supports joseph and linley's ( , 2008 conceptualization of how ptg and ptsd relate to each other. traumatic events are thought to shatter assumptions about the self and the world and lead to the symptoms of ptsd. these experiences of reexperiencing, avoidance and arousal are viewed as the cognitive emotional processing of the new trauma related information as individuals search for new meaning in life (joseph & linley, 2008) . as these new meanings are found, and the person's view of themselves and the world is reconstructed, ptg should occur and symptoms of distress should decrease. therefore ptg should be predictive of lower distress, because as people find new meaning they can overcome the cognitive disruption and confusion characterized by ptsd . support for this has been reported by frazier, conlon and glaser (2001) who found that among sexual assault survivors who reported ptg over 12 months were the least distressed. however, joseph and linley (2006) note that this does not mean to imply that the alleviation of distress should automatically lead to the enhancement of growth. according to their organismic valuing theory of growth, ptg should only relate to reduced distress through accommodation (i.e., changing one's global meaning to incorporate the stressor) as opposed to assimilation (i.e., changing one's view of the stressor so that it is consistent with one's global meaning). as such they caution that therapeutic work may impede or disrupt the cognitive processes that are necessary for accommodation and therefore ptg. nonetheless these findings suggest ptg may be a useful target for therapeutic intervention in health care and clinical settings, where the aim is long-term emotional and physical adjustment. psychotherapy for traumatic events such as a serious illness has predominantly focused on the negative effects of trauma, and the goal of therapeutic intervention to promote growth as opposed to alleviate distress will be a major paradigm shift. it is therefore important to raise clinician's awareness of the possibility of positive change. for example, clinicians might recognize the patient's struggle to understand the impact of the illness not only as a posttraumatic response but also as a potential precursor to growth (zoellner & maercker, 2006) . the empirical study of ways to facilitate ptg is in its infancy and only a few intervention studies have included ptg as an endpoint (antoni et al., 2001 penedo et al., 2006) . nonetheless some interventions, which contain techniques aimed at promoting growth, have shown to successfully improve outcomes. for example antoni et al. (2001) found that a psychosocial intervention that taught participants broad cognitive behavioural stress management techniques, served to increase reports of perceived benefits from having had breast cancer, and simultaneously reduced levels of depression. this study demonstrates that ptg can be altered and can be incorporated easily within cognitive behavioural stress-management interventions. however, the findings from the metaanalysis suggest that clinicians should be sensitive to the timing of ptg discussions. for example the present analysis suggests that ptg might be a useful target in the short-term to reduce distress, but in order to enhance well-being ptg should be targeted later on in the adjustment process. however, in agreement with park and helgeson (2006) it is cautioned that large scale interventions to facilitate ptg in cancer and hiv/aids patients should be avoided until researchers understand more about the origins of ptg, the conditions under which ptg is verdical, the best methods to assess ptg, and its relations to psychological and physical health, are fully understood. care should also be taken to avoid imposing an expectation of ptg in the face of serious illness. patients with cancer or hiv/aids often report feeling burdened with the pressure to stay positive and encouraging the identification of positive changes from their illness may be potentially offensive to patients, serve to minimise their experience and lead them to suppress reports of distress (bellizzi & blank, 2006; cordova, 2008) . this meta-analysis of growth in cancer and hiv/aids patients illustrates the promising and exciting nature of this area of research. however, the review also indicates much remains to be learned and highlights areas of research where future work is needed. the present study indicates that in the short term, ptg is associated with a reduction in negative mental health, whereas over longer term, ptg is associated with an enhancement in positive well-being. therefore a clear point of focus is the use of longitudinal studies to further disentangle and clarify the temporal course of this relationship. experimental designs, such as the interventions described earlier, will also help to reveal the causal role of ptg in adjustment and to isolate mechanisms responsible for the effects (algoe & stanton, 2009 ). many of the conclusions reached in this paper regarding moderators of the growth-adjustment relationship are based on theoretical considerations rather than on direct empirical evidence and future studies should attempt to validate and test these hypotheses. moreover, to further explicate the growth-adjustment relationship studies should continue to identify additional mediators and moderators. a particularly relevant moderator to medical populations that should be investigated is the perception of the severity of an illness. a previous meta-analysis found that perceptions of the severity of a traumatic event are related to ptg . as such it might be expected that ptg may have a stronger relationship with psychological well-being and physical health for more subjectively severe illnesses and caution must therefore be taken when generalizing the current findings to less threatening illnesses characteristics and indeed wider trauma populations. the majority of the studies included in the present paper measured ptg so that only positive changes were assessed. this could be problematic because participants may develop a 'response bias' which may lead individuals to over-report ptg, and it may also restrict our characterisation of the life changes that health events may precipitate (tomich & helgeson, 2004) . furthermore, a recent prospective study of severe acute respiratory syndrome (cheng, wong, & tsang, 2006) found that positive associations between ptg and positive well-being are more likely to be found among individuals who perceive benefits from the event, as well as the costs. therefore, examining positive and negative change simultaneously should be considered as a focus of future research investigating ptg and adjustment in health samples. particularly pertinent for this population is the possibility that ptg can serve to improve physical health. although this paper only looked at subjective measures of physical health there is promising preliminary data which suggests that ptg may be related to better physiological functioning. for example cruess et al. (2000) found that among women with breast cancer, cognitive behavioural stress management reduced levels of cortisol through the enhancement of ptg. yet, no studies have addressed possible mechanisms for the relationship between ptg and physical health. a recent model proposed by bower, low, moskowitz, sepah, and epel (2008) suggests that factors often associated with growth such as coping, positive affect and improved relationships, can lead to a state of enhanced allostasis (maintaining stability, or homeostasis, through change, sterling & eyer, 1988) , which buffers against future stress responses. this is a promising model, which merits increased attention in future research. furthermore, the relationship between ptg and health behaviours such as exercise, medication adherence, requires a more detailed examination; particularly regarding how these behaviours might moderate the relationship between ptg and physical health. finally, it is acknowledged that the ways in which ptg is manifested might contain elements that are distinctive to specific cultural environments (calhoun & tedeschi, 2006) . this paper included only three studies conducted in non-western countries and therefore it is clear that there is a need to examine ptg in more diverse ethnic and cultural groups to fully understand the relationship between growth and adjustment. on the basis of this meta-analysis it can be concluded that ptg following cancer or hiv/aids is related to better positive mental health and self-reported physical health, and less negative mental health. this does not preclude that many individuals might experience distress, but rather that ptg is a worthy phenomenon to be studied in clinical and health research. it is hoped that this meta-analysis will encourage further examination of the caveats addressed in this research, so that in the future ptg can perhaps become a viable therapeutic aim in individuals living with a life-threatening illness. construing benefits from adversity: adaptational significance and dispositional underpinnings medical illness and positive life change: can crisis lead to personal life transformation? diagnostic and statistic manual of mental disorders cognitive-behavioural stress management intervention decreases the prevalence of depression and enhances benefit finding among women under treatment for early stage breast cancer posttraumatic growth in adolescent survivors of cancer and their mothers and fathers publication bias expressions of generativity and posttraumatic growth in adult cancer survivors predicting posttraumatic growth in breast cancer survivors positive and negative life changes experienced by survivors of non-hodgkins lymphona posttraumatic stress disorder and illness behaviour in hiv+ patients benefit finding and physical health: positive psychological changes and enhanced allostasis perceptions of positive meaning and vulnerability following breast cancer: predictors and outcomes among long-term breast cancer survivors growing pains: commentary of the field of posttraumatic growth and hobfoll and colleagues recent contribution to it traumatic events and generational differences in assumptions about a just world foundations of posttraumatic growth a path model of the effects of spirituality on depressive symptoms and 24-h urinary-free cortisol in hiv-positive persons perception of benefits and costs during sars outbreak: an 18 month prospective study a broader view of trauma: a bio-psychosocial evolutionary view of the role of the traumatic stress response in the emergence of pathology and/or growth assessing spiritual growth and spiritual decline following a diagnosis of cancer: reliability and validity of the spiritual transformation scale a better world or a shattered vision? changes in life perspectives following victimization facilitating posttraumatic growth following cancer posttraumatic growth following breast cancer: a controlled comparison study breast cancer as trauma: posttraumatic stress and posttraumatic growth cognitive behavioural stress management reduces serum cortisol by enhancing benefit finding among women being treated for early stage breast cancer personal changes, dispositional optimism, and psychological adjustment to bone marrow transplantation meta-analysis in clinical-trials a practitioner's guide to meta-analysis the problem in using fixed-effects models of meta-analysis on realworld data is the meta-analysis of correlation coefficients accurate when population effect sizes vary? how to do a meta-analysis positive and negative life changes following sexual assault positive and negative psychosocial sequelae of bone marrow transplantation: implication for quality of life assessment positive consequences of head and neck cancer meta-analysis fixed-and random-effects models in meta-analysis a meta-analytic review of benefit finding and growth posttraumatic growth in chinese cancer survivors stress response syndromes fixed effects vs. random effects meta-analysis models: implications for cumulative research knowledge urban teens: trauma, posttraumatic growth, and emotional distress among female adolescents psychological resources preotect health: 5-year survival and immune function among hiv-infected women from four us cities psychometric properties of the dutch version of the posttraumatic growth inventory among cancer patients posttraumatic growth: three explanatory models. psychological inquiry, 15 positive adjustment to threatening events: an organismic valuing theory of growth through adversity growth following adversity: theoretical perspectives and implications for clinical practice trauma, recovery, and growth: positive psychological perspectives on posttraumatic stress posttraumatic stress disorder following cancer: a conceptual and clinical review the psychosocial impact of cancer and lupus: a cross validation study that extends the generality of "benefit finding" in cancer patients with chronic disease posttraumatic stress disorder in response to hiv infection psychosocial and sociodemographic correlates of benefit-finding in men treated for localised prostate cancer still stable after all thisâ�¦? temporal comparison in coping with severe and chronic disease posttraumatic growth and group-based interventions for persons dealing with cancer: what have we learned so far? medical illness and positive life change: can crisis lead to personal life transformation? summing up: the science of reviewing research positive adaptation to trauma: wisdom as both process and outcome positive change following trauma and adversity: a review the association of benefit finding to psychosocial and behaviour adaptation among hiv+ men and women received social support, self-efficacy, and finding benefits in disease as predictors of physical functioning and adherence to antiretroviral therapy posttraumatic stress disorder in women attending human immunodeficiency virus outpatient clinics post-traumatic growth in acquired brain injury: a preliminary small scale study posttraumatic growth among hiv/aids patients positive changes attributed to the challenge of hiv/aids posttraumatic growth and hiv disease progression posttraumatic growth among adolescents risk factors, prevalence, and treatment of anxiety and depressive disorders in pakistan: systematic review the psychosocial impact of multiple sclerosis: exploring the patient's perspective. health psychology wellbeing, posttraumatic growth and benefit finding in long-term breast cancer survivors the interaction of post-traumatic growth and post-traumatic stress symptoms in predicting depressive symptoms and quality of life traumatic distress and positive changes in advanced cancer patients personal growth and psychological distress in advanced breast cancer resilience and thriving in response to challenge: an opportunity for a paradigm shift in women's health a comparison of fixed-effects and mixed (random-effects) models for meta-analysis tests of moderator variable effects meaning making and psychological adjustment following cancer: the mediating roles of growth, life meaning and restored just-world beliefs introduction to the special section: growth following highly stressful life events -current status and future directions measurement issues in assessing growth following stressful life experiences a randomized controlled trial of group-based cognitive-behavioral stress management in localized prostate cancer: development of stress management skills improves quality of life and benefit finding positive effects of illness reported by myocardial infarction and breast cancer patients post-traumatic growth after head injury: a long-term follow-up curative testis cancer therapy: psychosocial sequelae the 'file drawer problem' and tolerance for null results writing meta-analytic reviews meta-analysis: recent developments in quantitative methods for literature reviews measuring the meaning of life for patients with incurable cancer: the life evaluation questionnaire (leq) posttraumatic growth and ptsd symptomatology among colorectal cancer survivors: a 3 month longitudinal examination of cognitive processing the report of posttraumatic growth in malaysian cancer patients: relationships with psychological distress and coping strategies turning the tide: benefit finding after cancer surgery changes in finding benefit after cancer surgery and the prediction of well-being one year later the yellow brick road and the emerald city: benefit-finding, positive reappraisal coping, and posttraumatic growth in women with early-stage breast cancer religion, spirituality, and posttraumatic growth perceiving benefits in adversity: stress-related growth in women living with hiv/aids stress-related growth among women living with hiv/aids: examination of an exploratory model allostasis: a new paradigm to explain arousal pathology finding benefit from cancer trauma and transformation: growing in the aftermath of suffering posttraumatic growth: conceptual foundations and empirical evidence posttraumatic growth: positive changes in the aftermath of crisis posttraumatic growth in prostate cancer survivors and their partners is finding something good in the bad always good? benefit finding among women with breast cancer positive and negative effects of hiv-infection in women in low socioeconomic resources finding benefit in breast cancer: relations with personality, coping, and concurrent well-being publication bias in research synthesis: sensitivity analysis using a priori weight functions predictors of posttraumatic growth following bone marrow transplantation for cancer facets of spirituality as predictors of adjustment to cancer: relative contributions of having faith and finding meaning posttraumatic growth in clinical psychology -a critical review and introduction of a two component model key: cord-289443-46w52de3 authors: sironi, manuela; cagliani, rachele; forni, diego; clerici, mario title: evolutionary insights into host–pathogen interactions from mammalian sequence data date: 2015-03-18 journal: nat rev genet doi: 10.1038/nrg3905 sha: doc_id: 289443 cord_uid: 46w52de3 infections are one of the major selective pressures acting on humans, and host-pathogen interactions contribute to shaping the genetic diversity of both organisms. evolutionary genomic studies take advantage of experiments that natural selection has been performing over millennia. in particular, inter-species comparative genomic analyses can highlight the genetic determinants of infection susceptibility or severity. recent examples show how evolution-guided approaches can provide new insights into host–pathogen interactions, ultimately clarifying the basis of host range and explaining the emergence of different diseases. we describe the latest developments in comparative immunology and evolutionary genetics, showing their relevance for understanding the molecular determinants of infection susceptibility in mammals. supplementary information: the online version of this article (doi:10.1038/nrg3905) contains supplementary material, which is available to authorized users. the way in which these analyses have helped to clarify the genetic determinants of species-specific infection and disease, as well as the reasons behind pathogen emergence. although arms races involve both the host and the pathogen, in this review we only focus on genetic diversity in mammalian hosts. host-pathogen genetic conflicts are not confined to mammals (and their pathogens): they drive molecular evolution in most realms of life, including bacterial-bacteriophage systems 5 , plants and their infectious agents 6 , as well as invertebrates and their pests 7, 8 . although we review studies and methods (boxes 1-3) that analyse genetic diversity at the inter-species level, the investigation of intra-species and intra-population signatures of pathogen-driven selection has also provided extremely valuable insight into infectious disease susceptibility, especially in our species. the interested reader is directed towards several recent reviews for more information [9] [10] [11] [12] [13] . the dynamics of host-pathogen interactions a central tenet of the red queen hypothesis is that organisms must continually adapt to survive and thrive in the face of continually evolving opposing organisms. nonetheless, evolution is not all about biotic interactions. at a macroevolutionary level, mixed models of evolution are likely to operate; biotic factors mainly shape species diversity locally and over short time spans, 13 14 comparisons among species take a snapshot of selective events that have been unfolding over long timescales. most of these approaches use extant genetic diversity and phylogenetic relationships among species to infer underlying evolutionary patterns. briefly, inter-species approaches rely on the alignment of orthologous coding sequences, analyse these alignments site-by-site, and at each site determine which, among all possible substitutions, would be non-synonymous (amino acid replacing) or synonymous (non-amino acid replacing) (see the figure) . the observed number of non-synonymous differences per non-synonymous site (dn) and the observed number of synonymous differences per synonymous site (ds) are then estimated. under neutral evolution, the rate at which amino acid replacements accumulate is expected to be comparable to the rate for silent changes and, therefore, dn/ds should be equal to 1 (green codons in the figure). nonetheless, most amino acid replacements are deleterious and, as a consequence, are eliminated by selection; this results in a large preponderance of sites with dn/ds <1, a situation referred to as purifying (or negative) selection (shown in blue in the figure) . conversely, the selective pressure exerted, for instance, by a pathogen, may favour amino acid replacements (for example, changes that modify the sequence and structure of a cellular receptor): in this case, dn/ds may reach values greater than 1, a hallmark of positive (or diversifying) selection (red in the figure) . the figure shows a hypothetical example whereby a virus uses a cellular receptor to infect the host. to prevent viral binding and infection, selection favours variants that modify the sequence and structure of the host receptors; on the other side, the virus adapts to such changes by gaining mutations that keep re-establishing receptor binding. this process fuels a genetic conflict, which is evident at the interaction surfaces. some lineages may be under stronger selective pressure than others and may display lineage-specific selected sites (episodic selection; cyan). in this case the branch of the phylogeny leading to these species may show significant evidence of positive selection . whereas shifts in the physical environment (for example, climate changes and oceanographic and tectonic events) drive evolution at a large scale, across much longer time periods 14 . recently, a new interpretation of the red queen hypothesis was proposed 15 ; the analysis of several phylogenies from different taxa indicated that speciation mostly occurs at a constant rate through rare stochastic events that cause reproductive isolation 15 . this view curtails the role of biotic interactions as major determinants of species diversity 15 . despite these observations, the red queen hypothesis has proven to be an extremely useful framework for the study of host-pathogen interactions. in this context, red queen dynamics can be divided into different types (see ref. 3 for a recent review). frequency-dependent selection, for example, determines allele frequency fluctuations in both host and pathogen populations. in this scenario, rare alleles are favoured by selection (the pathogen, for instance, may be adapted to the most common host genotype and may fail to infect hosts carrying a rare allele), and diversity within populations is maintained. escalatory arms races are another form of selection that usually apply to quantitative or polygenic traits and proceed through recurrent selective sweeps. selection results in an escalation in the phenotypes of both the host (for example, resistance) and the pathogen (for example, virulence). finally, in chase red queen scenarios the host is under pressure to reduce the strength of the interaction through de novo evolution of novelty, whereas the pathogen evolves to tighten the interaction by reducing phenotypic distance. chase scenarios occur when host-pathogen interactions have a complex genetic basis (polygenic); they determine selective sweeps and tend to reduce genetic diversity within populations. over the years, the red queen hypothesis has been supported by the description of rapid rates of evolution in genes involved in genetic conflicts and, in a few instances, by the temporal reconstruction of host-pathogen co-evolution in natural settings 16 . more recently, the development of experimental evolution approaches has allowed its formal testing 17, 18 . although extremely valuable, laboratory-based studies often use an isogenic host population that is infected by one or a few pathogen strains, and such studies only partially recapitulate the complex nature of host-pathogen interactions that occur in real life. for instance, phenotypic plasticity (an environmentally based change in the phenotype) and multiway host-pathogen interactions are common in nature. a remarkable example of phenotypic plasticity is the vertebrate adaptive immune system: through rearrangement and somatic hypermutation, the same genetic arsenal is used to combat a wide array of pathogens and to develop lifelong resistance to some infections. despite the relevance of adaptive immunity for host defence, its action does not preclude pathogen-driven selection at several genes involved in innate immunity or, more generally, in the interaction with pathogens (these represent the focus of this review). as for multiway interactions, these represent the norm: the same host can be infected by multiple pathogens (or even by multiple strains of the same infectious agent) during its lifetime, whereas pathogens differ in their ability to infect one or more host species. thus, multiple host-pathogen interactions might drive the evolution of the same or different molecular systems, blurring the expectations of the red queen hypothesis. finally, hosts with long generation times (such as mammals, which are the focus of this review), evolve at lower rates compared with most of their pathogens and also display smaller population sizes, resulting in an asymmetry of the arms race (although parasites with life cycles involving two or more species may be constrained in their ability to adapt (reviewed in ref. 19) ). even in the presence of a strong selective pressure (for example, a fatal infection), several generations may be required before the molecular signatures of the genetic conflict can be detected in mammalian host genomes 19 . nevertheless, natural selection signatures have been described at several mammalian genes that interact with recently emerged human infectious agents (for example, hiv-1), possibly as a result of the pressure imposed by extinct pathogens or because these agents have established long-lasting interactions with non-human hosts. the 'site models' implemented in the phylogenetic analysis by maximum likelihood (paml) package 91 are widely used to infer positive selection and to identify positively selected sites. these models allow dn/ds to vary from site to site, assuming a constant rate at synonymous sites. data (alignment and phylogenetic tree) are fitted to models that allow (selection models) or do not allow (neutral model) a class of codons to evolve with dn/ds >1. likelihood ratio tests are then applied to determine whether the neutral model can be rejected in favour of the positive selection model. if so, the gene is declared to be positively selected. also, if (and only if) the null hypothesis of neutral selection is rejected, a bayes empirical bayes (beb) approach can be used to detect specific sites targeted by selection (beb calculates the posterior probability that each site belongs to the class with dn/ds >1) 92, 93 . the paml approach implicitly assumes that the strength and direction of natural selection is uniform across all lineages. because this is often not the case, murrell and co-workers recently developed the mixed effects model of evolution (meme, hyphy package) 94 . meme allows the distribution of dn/ds to vary from site to site and from branch to branch; thus, the method has greater power to detect episodic selection, especially if it is confined to a small subset of branches in the phylogeny. a major issue related to these approaches is their extreme sensitivity to errors in sequence (coverage), annotation and alignment. misalignments and incorrect sequence information may result in apparently fast evolutionary rates and thus inflate the false-positive rate [95] [96] [97] . the use of specific alignment algorithms (for example, prank) and filtering procedures (for example, guidance) may partially overcome this problem 98 . likewise, genetic variability that is generated by recombination can be mistaken for positive selection 99 . thus, to limit false positives, alignments should be screened for recombination before running positive selection tests (and, if necessary, split on the basis of recombination breakpoints) or recombination should be incorporated into the model. the accumulation of favourable amino acid-replacing substitutions, which results in more non-synonymous changes than expected under neutrality (dn/ds >1). coronavirus (mers-cov) as a dangerous human pathogen. both ebov and mers-cov are thought to have originated in bats and spread to humans either directly or through an intermediate host. because eids are almost inevitably caused by an existing pathogen that adapts to infect a new host, comparative analyses of different species may help to unveil the genetic and immunological determinants underlying pathogen spillover and infection susceptibility. hiv-1, for example, originated from the crossspecies transmission of the simian immunodeficiency virus siv cpz , which naturally infects chimpanzees 21 . old world monkeys are resistant to hiv-1 infection owing to a post-entry viral block operated by cellular restriction factors. this differential susceptibility to infection was exploited to isolate tripartite motif-containing protein 5 (trim5; also known as trim5α), a major retrovirus restriction factor, from a rhesus macaque cdna library 22 . the protein product of trim5 binds directly to the incoming viral capsid and targets it for disassembly. whereas macaque trim5 is highly efficient against hiv-1, the human protein is not 22 . most species-specific determinants of antiviral activity were mapped to a short amino acid stretch in the so-called b30.2 (or spry) domain of trim5 (ref. 23 ). in primates, this region has evolved under positive selection, and the human lineage shows some of the strongest selection signatures 23 . why then is human trim5 so highly inefficient against hiv-1? possibly because the human gene evolved to fight another retrovirus. in a seminal paper, kaiser and co-workers resurrected an extinct pan troglodytes endogenous retrovirus (pterv1) and showed that the amino acid status of a single residue in the trim5 b30.2 domain modulates its activity against pterv1 and hiv-1, with the gain of restriction for one virus resulting in decreased control of the other one 24 . human trim5 is very active against pterv1, suggesting that our ancestors adapted to fight this virus or some related retrovirus, and this left them (us) unprepared against the hiv-1 epidemic. more recently, several genes identified as hiv-1 host factors were analysed in primates, and evidence emerged of positive selection at five of these (ankyrin repeat domain 30a (ankrd30a), cd4, microtubule-associated protein 4 (map4), nucleoporin 153 kda (nup153) and ran binding protein 2 (ranbp2)) 25 . importantly, most of the positive selection targets in cd4, map4 and nup153 are located in protein regions or domains that are responsible for direct interaction with the virus. the authors suggested that the selective pressure on these genes was exerted by ancient lentiviruses 25, 26 . overall, a number of concepts can be taken from these studies: past infection events may leave a signature that affects the ability of extant species to fight emerging pathogens. evolution may act through trade-offs, whereby changes that are favourable in one specific environment (in this case, the presence of a specific pathogen) may be unfavourable when conditions change. protein regions at the host-pathogen interface are expected to be targeted by the strongest selective pressure. evolutionary studies based on inter-species comparisons allow the identification of molecular determinants of infection susceptibility at single amino acid resolution. mammals display different susceptibility to distinct pathogens, and infection with the same agent can have extremely different outcomes in diverse species (see ref. 27 for a recent review). thus, domestic and wild mammalian (and non-mammalian) species represent natural reservoirs of human pathogens and/or may provide the adaptive environment for pathogen spillover. because host reservoir species and their pathogens often signatures of selection along specific branches can be detected through the so called 'branch-site' models implemented in the phylogenetic analysis by maximum likelihood (paml) package 100 . in analogy to the site models described in box 2, alignment errors result in high false-positive rates when branch-site models are applied 101 ; this issue can be partially mitigated by the use of specific aligners 101 . branch-site models require the phylogeny to be divided into 'foreground' and 'background' branches. a likelihood ratio test is then applied to compare a model that allows positive selection on a class of codons for the foreground branches with a model that does not allow such selection 100 . designation of the foreground branches needs a priori information, possibly based on biological evidence. if no clues are available as to which branches are more likely to have undergone selection, it is still possible to run the analysis by designating each branch of the tree as 'foreground'; this generates a multiple-hypothesis testing problem that must be appropriately corrected 102 . two alternative methods can detect selection at specific lineages without a priori branch partition. the branch site-random effects likelihood (bs-rel) method considers three different evolutionary scenarios (purifying, neutral and diversifying selection) for all branches in a given tree, and each branch is considered independently from the others; the algorithm applies sequential likelihood ratio tests to identify branches with significant evidence of positive selection 103 . the second method, the covarion-like codon model (fitmodel) 104 , allows each site to switch between selective regimes at any time on the phylogeny. thus, switches are not necessarily associated with tree nodes. recently, this approach was shown to be more powerful than the branch-site tests if a priori information is available 105 . both fitmodel and the paml branch-site methods envisage a bayesian approach to identify sites evolving under episodic positive selection. however, extensive simulations revealed that the branch-site approach is accurate but has limited power at detecting sites 106 . this problem has been referred to as the 'selection inference uncertainty principle' -that is, it is difficult to simultaneously infer both the site and the branch that are subject to positive selection 94 . co-evolve for millions of years, evolutionary analyses may help to explain host adaptive events associated with low susceptibility and mild disease outcomes. the most extensive body of knowledge on host-pathogen specificity focuses on viral infections, as the example of trim5 mentioned above testifies, but recent work has also shed new light on bacterial diseases. leptospirosis, one of the most prevalent human bacterial zoonoses worldwide, is caused by bacteria of the leptospira genus. wild rodents are considered to be the main reservoirs for human leptospirosis, but a study of malagasy small mammals indicated that several endemic species of tenrecs and bats are also infected with leptospira species that are markedly specific to their hosts, suggesting long-term adaptation of the bacterium to different hosts 28 . a feature that pathogenic leptospira species share with other bacteria is complement evasion. indeed, these spirochetes have evolved different strategies to elude complementmediated killing; thus, leptospiral immunoglobulin-like (lig) proteins can bind complement factor h (cfh) and c4b-binding protein (c4bp) to mediate complement inactivation at the bacterial surface. a genome-wide analysis of positive selection in six mammalian species indicated that the complement system has been the target of extremely intense selective pressure 29 . similar results were obtained by analysing positively selected genes in the bat myotis brandtii 30 . thus, selection-driven speciesspecific differences at complement genes might explain differential susceptibility to infections. in line with this view, human-specific pathogens such as neisseria gonorrhoeae and neisseria meningitidis bind cfh of human origin, but not cfh from other primates, and a single amino acid change (n1203r) in the chimpanzee molecule restores cfh binding to sialylated gonococci and bacterial killing 31 . several sequenced mammalian genomes are now available; it will be important to study the detailed pattern of molecular evolution at complement genes, with the aim of gaining insight into the determinants of species-specific complement evasion. yersinia pestis provides another remarkable example of differential susceptibility to a bacterial infection. again, rodents act as a natural reservoir for this human pathogen. as with other gramnegative bacteria, lipid a, the biologically active component of y. pestis lipopolysaccharide (lps), is recognized by toll-like receptor 4 (tlr4) and its co-receptor lymphocyte antigen 96 (ly96; also known as md2) (see below). recent data showed that, compared with mouse cells, human cells respond less efficiently to hypoacylated lipid a; this effect is almost entirely due to differences in tlr4 and ly96 sequences, as assessed by the generation of humanized mice 32 . different responsiveness to variably acylated lps from other sources (for example, escherichia coli) had previously been described 33 . starting from this premise, ohto and co-workers 34 solved the crystal structure of the mouse tlr4-ly96-lps and tlr4-ly96-lipid iva (a synthetic tetra-acylated lipid a precursor) complexes and compared them to the human counterparts. structural differences were detected in the interaction of lipid iva with the two mammalian receptors, with some amino acid replacements in ly96 and tlr4 possibly being responsible for the observed differential binding 34 . analysis of tlr4 in mammals revealed that the receptor has evolved adaptively 35 . we mapped positively selected sites onto the structure of the human and mouse complexes and observed that some of these may indeed account for structural differences between humans and mice (fig. 1) . rodents are the most established animal model for human disease, including for susceptibility to infection. in recent years, however, technological advances have made the sequencing of whole genomes a relatively quick and inexpensive process. the genome sequences of non-model mammals that serve as natural reservoirs of human infectious agents are now available, allowing the unprecedented opportunity to exploit these data for molecular evolution studies. bats, for example, are known to host a wide range of viruses that are highly pathogenic to humans 36 . the genomes of six bat species have been sequenced so far, and three of these (m. brandtii, pteropus alecto and myotis davidii) were analysed in detail to unveil the evolutionary history of specific traits 37 . results showed that different families of immune receptors -including killer cell immunoglobulin-like receptors (kirs), killer cell lectin-like receptors (klrs), sialic acid-binding immunoglobulin-like lectins (siglecs) and leukocyte immunoglobulin-like receptors (lilrs) -have expanded or contracted in distinct bat species. also, in these three bat species, as well as in the common ancestor of p. alecto and m. davidii, genes involved in immunity represented preferential targets of positive selection 37 . this is not unexpected: immune-response genes have been shown to have evolved rapidly in most mammalian species analysed to date 9 . thus, although these sequenced bat genomes have not yet provided an explanation as to why bats are tolerant to ebov, for instance, they pave the way for further analyses to test specific hypotheses and/or to address the molecular determinants of host-pathogen interactions. in a recent study, demogines and co-workers 38 showed how this can be accomplished. the authors focused on angiotensinconverting enzyme 2 (ace2), which serves as a receptor for severe acute respiratory syndrome coronavirus (sars-cov) cell entry. in particular, the receptorbinding domain of the viral spike protein is responsible for ace2 binding and is a major determinant of host range 39 . although the human sars epidemic was suggested to have originated from the zoonotic transmission of sars-cov from bats to humans, possibly via an intermediate host (for example, palm civets) 40, 41 , no ace2-binding sars-cov-like virus had been identified in bats when demogines and collaborators started their work 38 . the authors analysed ace2 genes in 11 bat species, and results revealed that the gene evolved adaptively and that the positively selected residues of the bat genes map at the ace2-sars-cov interaction surface (fig. 2) . positive selection localized to a subset of sites or confined to a few species in a phylogeny. these data led to the conclusion that ace2-binding coronaviruses originated in bats 38 . this finding was confirmed in a subsequent study that isolated an ace2-binding sars-like coronavirus from horseshoe bats in china 42 , highlighting the power of evolutionary studies in predicting host range and disease emergence. similarly to sars-cov, mers-cov is thought to have originated in bats and to have spread to humans via an intermediate host, possibly dromedary camels 43 . infection is initiated by binding of the mers-cov spike protein to human dipeptidyl peptidase 4 (dpp4; also known as cd26) 44 . recent data indicate that five amino acids in dpp4 that differ between humans (mers-cov susceptible) and hamsters (non-susceptible) are key determinants for host specificity 45 (fig. 2) . we extended a previous evolutionary analysis of mammalian dpp4 (ref. 46 ): strong evidence of positive selection was found with episodic selection in the vespertilionidae bat family and the panda and ferret branches, as well as in the dog lineage (fig. 2; see supplementary information s1,s2 (box, table)). as shown in fig. 2 , most positively selected sites are located at the dpp4-spike protein interaction surface 47 , and one of these is among those described as binding determinants 45 . thus, as observed for ace2, mers-cov and related viruses (for example, coronavirus hku4) are likely to act as drivers of molecular evolution on mammalian dpp4 genes; it will be especially interesting to evaluate the contribution of positively selected sites in ferrets because these animals are resistant to mers-cov infection. immune responses in mammals are highly coordinated processes involving multiple systems that sense infection, activate antiviral and antimicrobial responses, and trigger adaptive immunity. the evolutionary history of several such systems has been analysed in detail, and below we describe the most recent findings. innate immune receptors. the mammalian immune system is endowed with a repertoire of molecular sensors called pattern-recognition receptors (prrs). these molecules detect pathogen-associated molecular patterns (pamps) and initiate a downstream signalling cascade that culminates in the production of cytokines and antimicrobial factors. the main families of prrs include tlrs, nod-like receptors (nlrs), rig-like receptors (rlrs) and aim2-like receptors (alrs). in the host-pathogen arms race, these molecules represent one of the foremost detection-defence systems; consistently, several studies have reported adaptive evolution at genes encoding mammalian prrs. analyses in primates, rodents and representative mammalian species indicate that positive selection shaped nucleotide diversity at most tlrs, with the strongest pressure acting on tlr4 (refs 35, 48, 49) . similarly to tlr4 (fig. 1) , several positively selected sites in other tlrs are located in pamp-binding regions, raising questions as to whether species-specific host-pathogen co-evolution is occurring, and how these sequence changes translate into differential pamp recognition. in fact, as mentioned above for lps, species-specific differences in ligand binding by tlrs seem to be common and potentially affect the overall immune response to specific pathogens 50 . integration of evolutionary, immunological and genetic studies will be instrumental in the future for medical applications, especially in light of nature reviews | genetics sites that are positively selected in mammals 35 are mapped onto the tlr4 structure (red): several of these flank or correspond (orange) to residues that differ between humans and mice and that surround the phosphate groups of lipid iva (yellow) 34 . if lys367 and arg434 are replaced with the human residues (glu369 and gln436, respectively), the responsiveness of mouse tlr4-ly96 to lipid iva is abolished. b | structures of human cd86 (white; transmembrane and juxtamembrane region) and mir2 (grey; encoded by kaposi sarcoma-associated herpesvirus). cd86 sites that are involved in the interaction and that are positively selected in mammals are shown in red. c | complex of transferrin receptor protein 1 (tfr1) with the surface glycoprotein (gp1) of machupo virus (macv), a rodent arenavirus that can also infect humans through zoonotic transmission. tfr1 residues involved in the interaction with gp1 are in yellow, positively selected sites are in red and positively selected sites that directly interact with gp1 are in orange. the proposed use of tlr ligands as vaccine adjuvants, a step that may require tailoring to distinct species 50 . compared with tlrs, mammalian alrs are much less conserved and more dynamic, with distinct species carrying different sets of functional genes (ranging from 13 in mice to none in some bats) 37, 51 . as a consequence, analysis of several mammals indicated that, with the exception of absent in melanoma 2 (aim2), which is non-functional in several species, no unequivocal orthologues can be inferred for the remaining alr genes. this prevents the application of standard codon-based tests across the entire mammalian phylogeny, although closely related species can be analysed. thus, interferonγ-inducible protein 16 (ifi16) and aim2 were shown to have evolved under positive selection in primates. positively selected sites were observed to mainly localize near to regions or domains involved in dna binding and protein-protein interaction, suggesting modulation of substrate specificity or genetic conflicts with viral inhibitors 52 . positive selection was also described for the three mammalian rlrs (retinoic acid-inducible gene i (rigi; also known as ddx58), melanoma differentiation-associated 5 (mda5; also known as ifih1) and lgp2 (also known as dhx58)), the primate nlr family apoptosis inhibitory protein (naip) and rodent naip2 genes 53, 54 . indeed, as is the case for alrs, rodents have multiple naip paralogues that show widespread evidence of inter-locus recombination. this led to the application of a dn//ds sliding window approach: the naip2 sites evolving with dn/ds >1 were found to be located in the bacterial ligand domain 54 . studies on antiviral restriction factors have been extensive because these molecules represent obvious targets in hostpathogen arms races. specifically, genetic conflicts between host restriction factors and viral components often play out in terms of binding-seeking dynamics (the host factor adapts to bind the viral component) and binding-avoidance dynamics (the virus counter-adapts to avoid binding and restriction by the host factors). the evolutionary history of antiviral restriction factors has been comprehensively reviewed elsewhere [55] [56] [57] , and we only highlight a few recent developments here. the first restriction factor to be identified was the product of the mouse gene friend virus susceptibility 1 (fv1), a protein that protects against murine leukaemia virus (mlv) infection 58 . the origin and evolution of fv1 is extremely interesting: early sequence analysis revealed that it derives from the gag gene of an ancient endogenous retrovirus that is not directly related to mlv 58 . thus, fv1 exemplifies a paradoxical twist of the arms race scenario whereby a viral gene is co-opted by the host to serve an antiviral function (this is not the only instance, see ref. 59) . recent results showed that the fv1 gene was inserted into the mouse genome between 4 million and 7 million years ago, long before the appearance of mlv. thus, the selective pressure exerted by other viruses must have maintained fv1 function and driven its evolution 60 . indeed, analysis of fv1 from wild-type mice indicates that different fv1 products can recognize s2 (box, table) ). genes that evolved from a common ancestral gene through speciation. homologous genes created by a duplication event within the same genome. the observed number of non-synonymous substitutions per non-synonymous site. the observed number of synonymous substitutions per synonymous site. and block multiple genera of retroviruses (for example, equine infectious anaemia virus and feline foamy virus), and a number of positively selected sites in the carboxy-terminal region of fv1 are directly involved in restriction specificity 60 . thus, in a similar way to trim5, fv1 was identified for its ability to restrict an extant virus, but its evolution was driven by different waves of retroviral species, some of which are likely to be extinct. other restriction factors that have been the topic of recent investigation are encoded by two paralogous genes, myxovirus resistance 1 (mx1; also known as mxa) and mx2 (also known as mxb). the protein products of the two genes display high sequence identity but different antiviral specificity. mx1 has broad activity against rna and dna viruses. recently, mitchell and collaborators 61 showed the potential of evolutionary analyses to generate experimentally testable hypotheses on the nature of genetic changes that affect species-specific susceptibility to infection. the authors applied an evolution-guided approach and identified a cluster of positively selected residues in an unstructured surface-exposed mx1 loop (loop 4), which confers antiviral specificity; genetic variation in loop 4 is a major determinant of mx1 antiviral activity against thogoto and avian influenza a viruses, and replacements at a single positively selected site alter the ability of mx1 to restrict these pathogens 61 . more recently, the selection pattern at the mx2 gene, which encodes an antiretroviral effector 62 , was shown to parallel that of mx1, with most selected sites located in loop 4 (ref. 63 ). in mx2, sites selected in the primate lineage were detected outside loop 4, and mx1 also showed evidence of selection in other domains 61, 63 ; these sites are promising candidates for being additional determinants of antiviral activity. antigen presentation and the ensuing t cell activation are central processes in mammalian cellmediated immune response (fig. 3) . therefore, a convenient strategy for pathogens to elude immune surveillance is to hijack the molecular pathways responsible for these processes 64, 65 . in line with the arms race scenario, there is evidence of positive selection at several mammalian genes involved in antigen presentation and the regulation of t cell activation 66, 67 (fig. 3) . the pathogen-driven mechanisms underlying evolution at these genes are likely to be manifold. one mechanism is genetic conflict with a pathogen-encoded component, evidence of which can be seen in the protein cd86. positively selected sites in the transmembrane and juxtamembrane region of cd86 interact with mir2 (fig. 3) , a kaposi sarcomaassociated herpesvirus (kshv) protein that downmodulates cd86 expression 67, 68 . a second mechanism is the use of cell-surface molecules as viral receptors: some adenovirus strains, for example, have been reported to exploit cd80 and cd86 for cellular attachment 69, 70 . a third mechanism is the broadening or tuning of the host's ability to process and present pathogen-derived components. for example, a positively selected site in the carbohydrate-recognition domain of cd207 (also known as langerin; a birbeck granule molecule) affects an amino acid position that is directly involved in the binding of pathogen-derived glycoconjugates 71 . these mechanisms are not mutually exclusive. for example, a plethora of viral pathogens (such as herpes simplex virus 1, human papillomavirus, hiv-1 and kshv) interfere with cd1d trafficking and recycling 72, 73 . as a consequence, the cytoplasmic and transmembrane regions of cd1d display positively selected sites, one of which is within a primate-specific trafficking signal. additional positively selected sites are located in the cd1d extracellular region and flank the t cell receptor interaction surface and the lipid-binding pocket, which suggests that they exert an effect on antigen-binding specificity 67 . finally, we draw attention to one of the few attempts at assessing the part that helminth infections have played as selective pressures for mammals and at integrating epidemiological information into molecular evolutionary approaches. machado and co-workers 74 found evidence of positive selection at the mammalian gene fc fragment of igg, low affinity iiib, receptor (fcgr3b), which is expressed by eosinophils and is involved in the binding of immunoglobulin g (igg)-coated parasites. notably, the authors also tested a specific hypothesis whereby mammalian lineages hosting a wider range of helminth species should show stronger evidence of selection compared with other species (this was accomplished by running the phylogenetic analysis by maximum likelihood (paml) branch-site models with helminth-rich lineages as foreground branches 74 . their hypothesis was verified, providing a plausible explanation for the evolutionary pattern at fcgr3b and suggesting that similar approaches may be used to detect other mammalian genes involved in genetic conflicts with helminth parasites. as exemplified by ace2, host-pathogen interactions are not limited to immune system components. the reasons why genes with no specific defence function may be targeted by the selective pressure imposed by infectious agents are manifold. the best known instances probably refer to gene products that act as incidental receptors for pathogens, as is the case with ace2. other host gene products that engage in genetic conflicts include those that participate in the coagulation cascade and the contact system, which are commonly hijacked by bacterial pathogens to promote tissue invasion or to elude detection by immune cells (see ref. 75 for a review). an alternative possibility is that the host builds a line of defence based on the sequestration of essential micronutrients from the pathogen, a phenomenon known as 'nutritional immunity' . housekeeping genes. incidental receptors are often represented by the products of housekeeping genes, which are typically expressed at high levels by different cell types. among these, the transferrin receptor (tfrc) gene encodes a cell-surface molecule (transferrin receptor protein 1 (tfr1)) that is essential for iron uptake. tfr1 is used as a receptor by mouse mammary tumour virus, canine parvovirus and rodent new figure 3 | genes involved in antigen processing and presentation and t cell regulation are common targets of positive selection in mammals. all pathway components are designated using official gene names (excluding the major histocompatibility complex (mhc) and t cell receptor (tcr)) and are highlighted in red if they are targets of positive selection in mammals or primates 25, 66, 67 . the molecular components of different antigen processing and presentation pathways are shown (details from refs 107, 108) to provide a general overview of the extent of positive selection and to highlight the function of positively selected genes, as most of their protein products directly interact with the antigen. thus, the figure is not meant to show all molecules involved in the process or to convey mechanistic insights. also, some genes may show tissue-specific expression or may be induced under specific circumstances: their products are nonetheless included for the sake of completeness. as for t cell regulatory molecules, the representation does not reflect the stoichiometry of binding (for example, cd28 functions as a dimer). notably, the same molecule may be expressed by different populations of t cells, although here each molecule is shown on one t cell type only (to avoid redundancy). the dashed arrows and '?' indicate steps that lack clear molecular definition or are only inferred. the orange circles, and red and blue shapes at the bottom of the figure represent proteolytic fragments. b2m, β2-microglobulin; blmh, bleomycin hydrolase; calr, calreticulin; cd40lg, cd40 ligand; ctla4, cytotoxic t lymphocyte protein 4; cts, cathepsin; cyb, cytochrome b; erap, endoplasmic reticulum aminopeptidase; havcr2, hepatitis a virus cellular receptor 2; hla-dm, major histocompatibility complex, class ii, dm; icos, inducible t cell co-stimulator; icoslg, icos ligand; ifi30, interferonγ-inducible protein 30; inkt, invariant natural killer t; itcr, invariant tcr; lgmn, legumain; lnpep, leucyl-cystinyl aminopeptidase; ncf, neutrophil cytosol factor; npepps, puromycin-sensitive aminopeptidase (also known as psa); nrd1, nardilysin; pdcd1, programmed cell death 1; pdcd1lg2, programmed cell death 1 ligand 2; pdia3, protein disulfide-isomerase a3; ros, reactive oxygen species; tap, antigen peptide transporter; tapbp, tap-binding protein (also known as tapasin); thop1, thimet oligopeptidase 1; tpp2, tripeptidyl-peptidase 2. the elimination of deleterious amino acid-replacing substitutions, which results in fewer non-synonymous changes than expected under neutrality (dn/ds <1). world arenaviruses. in line with the arms race scenario, tfrc evolved adaptively in rodents and caniforms, and positively selected sites are mainly located in the extracellular domain regions that interact with rodentinfecting arenaviruses (fig. 1) and carnivore-infecting parvoviruses, respectively 76, 77 . interestingly, positive selection at the primate transferrin (tf) gene, which encodes the tfr1 ligand, was also recently described 78 ; in this case, selection is driven by bacteria, not viruses 78 . transferrin is the major circulating iron transporter in mammals and is also thought to participate in nutritional immunity by sequestering iron from bacteria. consistently, most positively selected sites were found to have evolved to counteract binding by bacterial transferrin surface receptors that scavenge host iron 78 . thus, different selective pressures exerted by distinct molecular mechanisms contributed to shaping the evolution of a central homeostatic process -in this case, iron transport in mammals. another housekeeping gene product that acts as a viral receptor is niemann-pick c1 protein (npc1), a sterol transporter located in the membrane of late endosomes and lysosomes. npc1 is expressed by most cell types and is used by filoviruses (such as ebov and marburg virus). evolutionary analysis of mammalian npc1 genes indicated that three positively selected residues are located in the amino-terminal portion of the second npc1 luminal loop; binding of this loop by the ebov glycoprotein (gp) is necessary and sufficient for the viral receptor activity of the sterol transporter 79, 80 (fig. 4) . the second luminal loop of npc1 is also bound with high affinity by the gp encoded by lloviu virus, a bat-derived, ebov-like filovirus 81 . thus, npc1 may represent a universal receptor for filoviruses, and the constant selective pressure exerted by such viruses might have greatly contributed to shaping mammalian genetic diversity at loop 2. these data may have great and immediate practical values. in fact, small molecules that directly target npc1 and disrupt gp binding are regarded as possible therapeutic compounds against ebov [82] [83] [84] (fig. 4) . because mammalian npc1 diversity at the interaction surface is driven by selection, future efforts in this direction are likely to benefit from the incorporation of evolutionary analysis; this would be especially important when testing therapeutic molecules on model organisms and non-human mammals. in humans, mutations in npc1 cause niemann-pick disease type c1, a progressive neurodegenerative condition. this is in line with the central role of this transporter in housekeeping functions; thus purifying selection. is expected to constrain variation in the gene. indeed, the human-mouse dn/ds calculated for the npc1 whole-gene region is definitely lower than 1, as is the case for most genes (fig. 4) . in fact, mammalian npc1 genes show a large preponderance of codons evolving with dn/ds <1, and positive selection is extremely localized in loop 2 (fig. 4) . this specific example illustrates a general concept, whereby molecules involved in central homeostatic processes may be engaged in genetic conflicts with pathogens, although in several instances the sequence space accessible for adaptive mutation without a high fitness cost is expected to be limited. the coagulation cascade and contact system. as anticipated above, several components of the coagulation cascade and contact system evolved adaptively in mammals, most likely as a result of genetic conflicts with bacterial pathogens 85, 86 . for instance, staphylococcus aureus is endowed with an arsenal of proteins that target such systems, including two cysteine proteinases (scpa and sspb) that cleave plasma kininogen at each terminal side of the bradykinin domain to generate kinins, with a consequent increase of vascular leakage 87 . these events are central for bacterial virulence and are linked to the pathogenesis of sepsis. in kininogen 1 (kng1), positively selected sites are located in all domains, with the exception of the highly conserved bradykinin region 85 . one of the positively selected sites defines the n-terminal cleavage site of scpa and sspb, suggesting that sites flanking the bradykinin sequence are evolving to avoid recognition and cleavage by bacterial-encoded proteases. in analogy to the strong purifying selection acting on the bradykinin region, analysis of calculation cascade genes indicated that disease-causing mutations are more likely to occur at sites targeted by purifying selection and are rarer at positively selected sites 86 . again, these observations highlight the coexistence of distinct selective regimes at the same gene regions and exemplify the concept of evolutionary trade-offs. the advent of high-throughput sequencing technologies has allowed for the generation of an unprecedented wealth of genetic data, including the whole-genome sequences of host reservoir species for human pathogens, as well as genetic information for multiple microbial and viral species and strains. moreover, large-scale approaches such as rna interference and mass spectrometry are providing detailed pictures of host-pathogen interactomes 88, 89 . finally, an increasing number of crystal structures of interacting host and pathogen proteins solved in complex are available, allowing the opportunity to determine the structural basis of these interactions to identify regions or amino acids that lie at the host-pathogen contact surface. integration of these data with evolutionary analysis will allow the testing of specific hypotheses, including which species have responded to the pressure exerted by one or more pathogens (see the sars-cov example), which molecules and residues have participated in the arms race and which host-pathogen interacting partners are expected to have co-evolved. these advances are also expected to progressively change evolutionary genetics from a hypothesis-driven to a hypothesis-generating discipline. in this respect, we note that although the arms race scenarios we have described in this review imply some form of host-pathogen co-evolution over time, the nature of the interaction and its dynamics have often been inferred from the observed pattern of variation. indeed, the fact that the same residues that affect specific host-pathogen interactions are targeted by positive selection does not necessarily imply a causal link, and in many instances the specific selective agents underlying molecular adaptations remain to be determined. as shown above, these may well be accounted for by extinct pathogens or by agents that had a major co-evolutionary role in the past but that are now fading away from the landscape of common infections. with a few exceptions 16, 24 , evolutionary studies only investigate extant genetic variation and modern pathogens, with little reconstruction of past events. nevertheless, we do not necessarily need to go back in time: evolutionary analyses can be used as predictive tools to pinpoint which genes and residues are more likely to contribute to present-day host-pathogen interaction and help explain species-specific susceptibility nature reviews | genetics the values for some of the genes discussed in this review are indicated. data were derived from the ensembl biomart database (see further information). b | natural selection acting on mammalian niemann-pick c1 (npc1) genes. npc1 is shown with its predicted membrane topology and protein regions coloured in hues of blue that represent the percentage of negatively selected sites (as detected by the single-likelihood ancestor counting method using datamonkey); the darker the blue, the higher the percentage. the location of three positively selected residues (red) is indicated on the left, and an alignment of the corresponding region is shown on the protein to the right (with red and blue representing positively and negatively selected sites, respectively). the interaction with the glycoprotein (gp; green) of filoviruses (such as ebola virus, marburg virus or lloviu virus) is shown. gp binds npc1 after processing by cellular proteases. ace2, angiotensin-converting enzyme 2; darc, duffy blood group, atypical chemokine receptor; mx1, myxovirus resistance 1; ssd, sterol-sensing domain; tfrc, transferrin receptor; tlr4, toll-like receptor 4. to infection. several studies mentioned above, including those investigating selection at mx1 (ref. 61 ), tfrc (refs 76, 77) , tf 78 and other protein-coding genes 23, 24, 54, 60 , used experimental analyses to show that evolutionary information can indeed be exploited to gain highresolution insight into the molecular determinants of binding affinities at host-pathogen interfaces. the studies of iron transporters 78 hold particular value because the authors analysed the genetic variability of both the host and the pathogen and showed that both parties evolved in response to mutually exerted pressures, in line with the red queen principles. so far, few attempts have been made at integrating evolutionary analyses of host and pathogen interacting partners into a common framework. however, efforts in this direction hold the promise of improving our understanding of the strategies used by both hosts and pathogens to adapt and counter-adapt. in turn, this knowledge has possible biomedical and therapeutic implications, given the ability of different pathogens or distinct strains of the same infectious agent to elude not only natural host defences but also drugs and vaccination strategies. as a final note, we mention that we have exclusively focused on adaptive events involving coding gene regions. nevertheless, several recent studies (see ref. 10 for a review) have highlighted the role of non-coding variants as important determinants of susceptibility to infection within species. thus, host-pathogen conflicts are more than likely to have contributed to adaptive evolution at regulatory elements during speciation. detection of these adaptive events will benefit from the availability of high-throughput techniques (for example, rna sequencing and chromatin immunoprecipitation followed by sequencing) and the development of methodological approaches for dissecting molecular evolution in non-coding regions; notably, recent data have shown the usefulness of a framework similar to dn/ds to analyse the evolutionary history of mammalian transcriptional enhancers 90 . application of this methodology (or extensions thereof) to the study of host-pathogen interactions will provide valuable information on which non-coding sequence changes have been targeted by selection and thus modulate susceptibility to infection or related phenotypes. signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution a new evolutionary law running with the red queen: the role of biotic conflicts in evolution the causes of evolution (longmans, green, & co,1932) revenge of the phages: defeating bacterial defences genomic variability as a driver of plant-pathogen coevolution? mainstreaming caenorhabditis elegans in experimental evolution insights from natural host-parasite interactions: the drosophila model from evolutionary genetics to human immunology: how selection shapes host defence genes population genetic tools for dissecting innate immunity in humans the red queen's long race: human adaptation to pathogen pressure human genome variability, natural selection and infectious diseases natural selection and infectious disease in human populations the red queen and the court jester: species diversity and the role of biotic and abiotic factors through time phylogenies reveal new interpretation of speciation and the red queen host-parasite 'red queen' dynamics archived in pond sediment a matching-allele model explains host resistance to parasites antagonistic coevolution accelerates molecular evolution biological and biomedical implications of the co-evolution of pathogens and their hosts global trends in emerging infectious diseases origins of hiv and the aids pandemic. cold spring harb the cytoplasmic body component trim5α restricts hiv-1 infection in old world monkeys positive selection of primate trim5α identifies a critical species-specific retroviral restriction domain restriction of an extinct retrovirus by the human trim5α antiviral protein positive selection of primate genes that promote hiv-1 replication hiv-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency this is an excellent review highlighting the importance of non-model organisms in understanding zoonotic infections diversification of an emerging pathogen in a biodiversity hotspot: leptospira in endemic small mammals of madagascar patterns of positive selection in six mammalian genomes genome analysis reveals insights into physiology and longevity of the brandt's bat myotis brandtii this work helps to clarify the species specificity of n. gonorrhoeae infection by analysing the binding of sialylated gonococci to human and chimpanzee cfh humanized tlr4/md-2 mice reveal lps recognition differentially impacts susceptibility to yersinia pestis and salmonella enterica lipid a modification systems in gram-negative bacteria structural basis of species-specific endotoxin sensing by innate immune receptor tlr4/md-2 signatures of positive selection in toll-like receptor (tlr) genes in mammals mass extinctions, biodiversity and mitochondrial function: are bats 'special' as reservoirs for emerging viruses? an extremely interesting study providing an overview of the evolutionary history of three bat genomes, with possible implications for immunity-related evidence for ace2-utilizing coronaviruses (covs) related to severe acute respiratory syndrome cov in bats a good example of how evolutionary studies can provide insight into host range and disease emergence retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus a central work showing that dpp4 of human and bat origin acts as a functional receptor for mers-cov host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of middle east respiratory syndrome coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 adaptation and constraint at toll-like receptors in primates contrasted evolutionary histories of two toll-like receptors (tlr4 and tlr7) in wild rodents (murinae) variation matters: tlr structure and species-specific pathogen recognition extensive evolutionary and functional diversity among mammalian aim2-like receptors ancient and recent selective pressures shaped genetic diversity at aim2-like nucleic acid sensors rig-i-like receptors evolved adaptively in mammals, with parallel evolution at lgp2 and rig-i molecular basis for specific recognition of bacterial ligands by naip/nlrc4 inflammasomes rules of engagement: molecular insights from host-virus arms races evolutionary conflicts between viruses and restriction factors shape immunity a cross-species view on viruses positional cloning of the mouse retrovirus restriction gene fv1 paleovirology and virally derived immunity evolution of the retroviral restriction gene fv1: inhibition of non-mlv retroviruses a study in wild mice showing that fv1 antiviral activity is broader than previously thought. it identifies positively selected residues in the c terminus that contribute to antiviral specificity evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor mxa a seminal paper that applies an evolution-guided approach to detect mx1 residues that confer antiviral specificity human mx2 is an interferon-induced post-entry inhibitor of hiv-1 infection evolutionary analysis identifies an mx2 haplotype associated with natural resistance to hiv-1 infection manipulation of costimulatory molecules by intracellular pathogens: veni, vidi, vici!! plos pathog mhc class i antigen presentation: learning from viral evasion strategies an evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection a 175 million year history of t cell regulatory molecules reveals widespread selection, with adaptive evolution of disease alleles the intertransmembrane region of kaposi's sarcoma-associated herpesvirus modulator of immune recognition 2 contributes to b7-2 downregulation the nef protein of hiv-1 induces loss of cell surface costimulatory molecules cd80 and cd86 in apcs members of adenovirus species b utilize cd80 and cd86 as cellular attachment receptors structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site hiding lipid presentation: viral interference with cd1d-restricted invariant natural killer t (inkt) cell activation a threonine-based targeting signal in the human cd1d cytoplasmic tail controls its functional expression evolutionary history of copynumber-variable locus for the low-affinity fcγ receptor: mutation rate, autoimmune disease, and the legacy of helminth infection one of the few studies of helminth-driven selective pressure in mammals that also integrates evolutionary analysis with epidemiological information thrombosis as an intravascular effector of innate immunity dual host-virus arms races shape an essential housekeeping protein an extremely interesting study extending the arms race scenario to a housekeeping protein, the transferrin receptor evolutionary reconstructions of the transferrin receptor of caniforms supports canine parvovirus being a re-emerged and not a novel pathogen in dogs escape from bacterial iron piracy through rapid evolution of transferrin mammalian npc1 genes may undergo positive selection and human polymorphisms associate with type 2 diabetes niemann-pick c1 (npc1)/ npc1-like1 chimeras define sequences critical for npc1's function as a flovirus entry receptor cell entry by a novel european filovirus requires host endosomal cysteine proteases and niemann-pick c1 multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection inhibition of ebola virus infection: identification of niemann-pick c1 as the target by optimization of a chemical probe small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection evolutionary analysis of the contact system indicates that kininogen evolved adaptively in mammals and in human populations positive selection during the evolution of the blood coagulation factors in the context of their disease-causing mutations induction of vascular leakage through release of bradykinin and a novel kinin by cysteine proteinases from staphylococcus aureus viral immune modulators perturb the human molecular network by common and unique strategies genome-wide rnai screen identifies human host factors crucial for influenza virus replication a novel test for selection on cis-regulatory elements reveals positive and negative selection acting on mammalian transcriptional enhancers paml 4: phylogenetic analysis by maximum likelihood accuracy and power of bayes prediction of amino acid sites under positive selection bayes empirical bayes inference of amino acid sites under positive selection detecting individual sites subject to episodic diversifying selection high sensitivity to aligner and high rate of false positives in the estimates of positive selection in the 12 drosophila genomes class of multiple sequence alignment algorithm affects genomic analysis estimates of positive darwinian selection are inflated by errors in sequencing, annotation, and alignment the effects of alignment error and alignment filtering on the sitewise detection of positive selection effect of recombination on the accuracy of the likelihood method for detecting positive selection at amino acid sites evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level the effect of insertions, deletions, and alignment errors on the branch-site test of positive selection multiple hypothesis testing to detect lineages under positive selection that affects only a few sites a random effects branchsite model for detecting episodic diversifying selection modeling the site-specific variation of selection patterns along lineages performance of standard and stochastic branch-site models for detecting positive selection among coding sequences statistical properties of the branch-site test of positive selection towards a systems understanding of mhc class i and mhc class ii antigen presentation cd1 antigen presentation: how it works the authors declare no competing interests. key: cord-263965-i8yutik6 authors: relf, michael v. title: what's old is new! similarities between sars-cov-2 and hiv date: 2020-04-09 journal: j assoc nurses aids care doi: 10.1097/jnc.0000000000000174 sha: doc_id: 263965 cord_uid: i8yutik6 nan today, like so many. what i thought i knew in the morning is often very different by the time i go to bed. i am minimizing my exposure to the media because it is overwhelming and simultaneously frustrating. my days are unfolding very differently than planned. as a nurse and scientist, i value evidence. i believe in the scientific method. i desire accuracy and precision. however, after more than 30 years of being an hiv nurse, i appreciate uncertainty. i also understand how a name stigmatizes populations, creates panic, and allows governments and societies to assign blame. we are hearing a great deal about covid-19 (co 5 corona, vi 5 virus, and d 5 disease) in the popular media. this new disease is impacting so many around the world, as well as how we work, travel, and the global economy. although we are hearing so much about covid-19 (the disease), we are rarely hearing anything about the sars-cov-2 (the official name of the virus that causes covid-19, according to the coronavirdiae study group of the international committee on the taxonomy of viruses; gorbalenya et al., 2020) . according to the world health organization (who), "using the name sars can have unintended consequences in terms of creating unnecessary fear for some populations, especially in asia, which was worst affected by the sars outbreak in 2003" (as cited in hui, 2020) . hence, we are hearing much more about the disease, covid-19, and not much about the virus, sars-cov-2. in time, i anticipate analysts examining the domestic and global response to this pandemic will evaluate if the use of covid-19, in an effort to reduce global anxiety and fear associated with sars, helped or hindered governmental responses and the initial public awareness about the emerging threat. regardless of the name, over the last several weeks, we have witnessed national leaders minimize the risk of the virus. we have been told the situation is "totally under control." we have been told that when the weather gets warmer, "it will miraculously go away." we have been told that "this is the flu." and, we have been told that "we're very close to a vaccine." in reality, the situation is not under control. it is not the flu. it is not going to miraculously go away without global and individual cooperation. and, although a phase i clinical trial was launched on march 16, 2020, to test a vaccine, we are months away, at very best, from having it publicly available if it is found to be efficacious. as i witness the unfolding of the covid-19 pandemic, i continue to think back to the early years of the hiv epidemic. susan sontag's book, aids and its metaphors, keeps coming to mind as i am witnessing similarities between then and now. both hiv and the sars-cov-2 are "seen as an invasion of alien organisms" (p. 9). the alien organism, as described by sontag, "inevitably comes from somewhere else" (p. 47). throughout history, there has been the "german measles", the "french pox", and most recently the "middle east respiratory syndrome"-another coronavirus. in the need to assign geographic blame, hiv was "thought to have started in the "dark continent," then spread to haiti, then to the united states and to europe" (p. 51-52). furthermore, early in the hiv epidemic, we had the "gay plague," "gay cancer," and "grid" or gay-related immune deficiency. the early terminology of the hiv epidemic assigned blame to gay men and downplayed the already existing evidence that other individuals, not just gay men, were also at risk. comparably, in recent weeks, we have heard the sars-cov-2 be referred to as the "wuhan virus," the "chinese virus," and the "kung flu" (hui, 2020) . even the president of the united states has repeatedly used the term, "chinese virus," reinforcing the perception of an alien organism that came from somewhere else. throughout time, the name given to a disease, especially an infectious disease, poses serious negative consequences. for example, the name of disease can "provoke a backlash against members of particular religious or ethnic communities, create unjustified barriers to travel, commerce and trade, and trigger needless slaughtering of food animals. this can have serious consequences for peoples' lives and livelihoods" (world health organization, 2015) . for this exact reason, the who, in may 2015, issued a statement outlining best practices for naming new human infectious diseases (world health organization, 2015) . unfortunately, president trump's referral to the sars-cov-2 as the "chinese virus" is contrary to the 2015 who recommendations and has serious implications, including racism and xenophobia toward asians and asian americans (sandler, 2020) . "just as one might predict for a disease that is not yet fully understood as well as extremely recalcitrant to treatment, the advent of this terrifying new disease, new at least in its epidemic form, has provided a large-scale occasion for the metaphorizing of illness" (sontag, 1989, p. 16) . today, the sars-cov-2 pandemic, like the hiv epidemic of the 1980s and 1990s, is a metamorphosis described as "invading the society, and efforts to reduce mortality … are called a fight, a struggle, a war" (sontag, 1989, p. 10) . yes, we must fight to reduce the burden of covid-19, but we must not fight one another and blame individuals and governments from certain countries or regions for causing this current pandemic. in the united states, we are constantly hearing about getting tested, the availability of test kits, and the delays in getting test results after being tested. metaphorically, as sontag (1989) wrote, "in the older era of artisanal diagnoses, being examined produced an immediate verdict, immediate as the physician's willingness to speak. now an examination means tests. and being tested introduces a time lapse that, given the unavoidably industrial character of competent medical testing, can stretch out for weeks: an agonizing delay for those who think they are awaiting a death sentence or an acquittal" (p. 35). for this contemporary pandemic, we know that testing is critical in limiting the number of new infections. interestingly, everyone wants to be tested for sars-cov-2, or at least that is what is being portrayed in the popular media. however, despite guidelines from cdc in 2006 that recommended hiv testing for everyone between the ages of 13 and 64 years as part of routine health care, we do not see people asking to be tested for hiv. we do not routinely see providers include hiv testing as part of the annual wellness examination. if we did, we would not see one out of seven persons living with hiv not knowing their status. maybe a beneficial outcome of this pandemic will be an increased awareness about the valuable role of routine testing, decreasing the stigma associated with hiv testing for many key populations. in the early 1980s, a common message from the government to alleviate "hysteria" and "frenzy" associated with the aids epidemic was that it "will not spread to 'the general population'" (sontag, 1989, p. 64) . today, however, we are being bombarded with messages related to covid-19 that raise "the disease's metaphorical stature by keeping alive fears of its easy transmissibility, its imminent spread" (sontag, 1989, p. 64 ). while we are learning about this novel coronavirus as the pandemic unfolds, we, as nurses, need to continue to be informed to help reduce the "hysteria" and "frenzy" that seems to be part of any pandemic related to infectious disease. similarly, hiv providers, including nurses and nurse practitioners from around the world, are wondering what to advise persons living with hiv about this contemporary threat. the u.s. centers for disease control and prevention (cdc) has published a great resource document, entitled covid-19: what people with hiv should know (cdc, 18 march 2020), which is available at https://www.cdc.gov/coronavirus/2019-ncov/specific-groups/hiv.html. unfortunately, like hiv, stigma is becoming a part of the discourse associated with covid-19's disease trajectory. we are seeing discrimination toward asians and asian americans, persons who have traveled, and health care providers (cdc, 21 march 2020) . again, the cdc (march 21, 2020) has published a valuable resource document, entitled reducing stigma, associated with this pandemic. please read these documents, share with fellow colleagues, and share with your patients. by the time this editorial is published in the may-june 2020 issue of janac, i know the world will be a different place. i am hopeful that all the health care workers around the world obtained the personal protective equipment needed to safeguard themselves. i am hopeful that self-isolation and shelter in place orders were adhered to so that we flattened the curve. i am hopeful that scientists identified a vaccine that is clinically beneficial. i am hopeful that the increasing numbers of deaths from complications associated with covid-19 were halted. i am hopeful that my friends and colleagues around the world remained strong and healthy. although i will continue to hope, i know that nurses from around the world will continue "to promote health, to prevent illness, to restore health, and to alleviate suffering"-the four fundamental responsibilities of nursing as outlined by the international council of nurses code of ethics (international council of nurses, 2012, p. 1). copyright © 2020 association of nurses in aids care. unauthorized reproduction of this article is prohibited. during these times of uncertainty, i know that nurses are making a difference in every corner of the world. i know that they will continue to do so not only today but also tomorrow, and next week, and next year. i know that as a profession, we have always risen to the occasion and will continue to do so. we, as hiv nurses, know, as stated by florence nightingale, "how very little can be done under the spirit of fear," thus, i know that nurses everywhere, despite fear and uncertainty, will continue to rise to the occasion to care for themselves, each other, and individuals and communities in every corner of the world! with its origins rooted in early african american hymns, and so eloquently spoken by dr. martin luther king so many years ago, i know that "we shall overcome." the author reports no real or perceived vested interests related to this article that could be construed as a conflict of interest. symptoms-testing/reducing-stigma.html?cdc_aa_refval5https% 3a%2f%2fwww.cdc.gov%2fcoronavirus%2f2019-ncov% 2fabout%2frelated-stigma why won't the who call the coronavirus by its name, sars-cov-2? trump abruptly stops calling coronavirus 'chinese virus' at daily press briefing aids and its metaphors who issues best practices for naming new human infectious diseases key: cord-033558-lcgo1tiy authors: schmiedel, stefan; gesenhues, anne title: infektionen, impfungen, reisemedizin date: 2020-10-09 journal: praxisleitfaden allgemeinmedizin doi: 10.1016/b978-3-437-22449-2.00009-5 sha: doc_id: 33558 cord_uid: lcgo1tiy nan mykosen 512 9.5.1 allgemeines 512 9.5.2 candidiasis (soor) 512 9.5.3 systemische mykosen 514 9.5.4 dermatophytosen 514 9.6 protozoeninfektionen 514 9.6.1 toxoplasmose 514 9.6.2 lambliasis (giardiasis) 515 stefan schmiedel und anne gesenhues 9.6.3 sonstige protozoeninfektionen 516 9.7 wurmerkrankungen 517 9.7.1 allgemeines 517 9.7.2 madenwurminfektion (enterobiasis, oxyuriasis) 517 9.7.3 spulwurminfektion (ascariasis) 518 9.7.4 bandwurminfektion (zestoden) 518 9.7.5 echinokokkusinfektion 519 9.7.6 trichinose 520 9.8 sexuell übertragbare krankheiten 520 9.8.1 gonorrhö 520 9.8.2 syphilis (lues) 521 9.8.3 trichomoniasis 522 9.8.4 ulcus molle 523 9.8.5 lymphogranuloma venereum 523 9.8.6 condylomata acuminata 523 9. 9 hiv und aids 524 9.9.1 epidemiologie und übertragungswege 524 9.9.2 labordiagnostik 524 9.9.3 stadieneinteilung und verlauf 527 9.9.4 diagnostik bei hiv-positiven 529 9.9.5 häufige krankheitsbilder bei aids 529 9.9.6 therapie 534 9.9.7 medikamente bei hiv-patienten 539 9.9.8 vorgehen bei kontakt mit hivkontaminiertem material und postexpositionsprophylaxe (pep) 542 9.9.9 präexpositionsprophylaxe (prep) 543 9.10 reisemedizin 544 9.10.1 allgemeines 544 9.10.2 allgemeine empfehlungen zur reisefähigkeit 544 9.10.3 empfehlungen für schwangere 549 9.10.4 empfehlungen für ältere menschen 550 9.10.5 empfehlungen für kinder und säuglinge 551 9.10.6 empfehlungen für chronisch kranke 553 9.10.7 reisevorbereitungen/ prophylaxen 555 9.10.8 reiseimpfungen u. chemoprophylaxe 556 9.10.9 gesundheitsrisiken auf reisen 565 9.10.10 screening nach reiseende 573 9.11 infektionsschutzgesetz 574 9.12 internetadressen 575 9 • biphasisches fieber: temperaturanstieg für 1-2 d auf > 39 °c (erregerausbreitung), abfall der temperatur; 2., meist länger andauernder temperaturanstieg (organbefall), viele virusinf., u. a. influenza (▶ 9.4.4) . sympt. ther., klinikeinweisung die immunisierung durch impfungen ist eine der wichtigsten u. wirksamsten maßnahmen zum schutz vor infektionskrankheiten. mit einer hohen durchimpfungsrate können einzelne krankheitserreger regional o. sogar weltweit ausgerottet werden. immunität gegen erkr. lässt sich passiv (immunglobulingabe), aktiv (tot-o. lebendimpfstoffe) o. komb. beider methoden erreichen. in d besteht keine impfpflicht. impfempfehlungen werden durch die obersten gesundheitsbehörden der länder ausgesprochen. diese öffentliche empfehlung hat im fall von impfschäden eine wichtige bedeutung für die weitere versorgung ( § 20 ifsg). • passive immunisierung durch immunglobuline: vorteil: sofortige schutzwirkung. nachteil: schutz nur von kurzer dauer (4 wo. bis 3 mon.), nur humorale immunität. • aktive immunisierung: der organismus wird mit antigenen von krankheitserregern konfrontiert u. muss selbst eine immunität ausbilden. schutz tritt verzögert ein. • grav.: alle nicht dringend indizierten impfungen (▶ 9.2.4) • bei immunsuppression bzw. immundefekt (tumorther., rheuma, hiv): es gelten die herstellerangaben, die z. t. sehr restriktiv sind; absprache mit behandelndem fa/klinik. seit 2018 aktuelle stiko-empfehlungen bzgl. impfungen bei immundefekten (▶ 9.12) • allergie gegen bestandteile eines impfstoffs (z. b. hühnereiweiß, ▶ 9.2.4) • nach auftreten von ko bei einer impfung besteht bis zur klärung der ursachen eine ki für denselben impfstoff • es gibt keine unzulässig großen abstände zwischen impfungen. jede impfung gilt, sofern der abstand zwischen den einzelnen dosen nicht unterschritten wurde (bsp.: mindestabstand bei grundimmunisierung zwischen 2. u. 3. dosis: 6 mon). auch eine für viele j. unterbrochene grundimmunisierung muss nicht neu begonnen werden. bei impfungen, die nur bis zu einem bestimmten alter empfohlen werden, sollen keine weiteren dosen verabreicht werden, wenn pat. dieses alter überschritten hat. • ist nicht bekannt, wann zuletzt bzw. ob überhaupt eine impfung stattgefun• diphtherie-, pertussis-immunisierung: kinder > 5-6 j., jgl. u. erw.: immunisierung mit reduzierter antigenmenge (erkennbar an der bezeichnung "d" statt "d" u. "ap" statt "ap"). • bei wiederholungsimpfungen i. r. der grundimmunisierung sollte für die weiteren impfungen dasselbe präparat verwendet werden. bei schlechter verträglichkeit bzw. unzureichender immunantwort nach abschluss einer impfserie kann eine impfung mit dem präparat eines anderen herstellers u. u. den gewünschten impferfolg haben. • komb.-impfstoffe helfen, inj. u. impftermine einzusparen; manchmal preiswerter als die summe der jeweiligen monovakzine. umgang mit impfstoffen vor allem lebendimpfstoffe sind temperaturlabil u. müssen vor erwärmung u. gefrieren geschützt werden. alle impfstoffe sind bei 2-8 °c zu lagern; temperatur ständig mit geeichtem min-max-thermometer kontrollieren. impfstoffe, die falsch gelagert o. gefroren wurden, sind zu verwerfen (ggf. hersteller-hotline anrufen). angebrochene amp. sofort verbrauchen, v. a. wg. der gefahr der bakt. kontamination (cave: spritzenabszess). für jede inj. eine neue kanüle verwenden, da an der aufziehkanüle anhaftender impfstoff zu lokalreaktionen der haut führen kann. • • an den ersten tagen nach impfung können subfebrile temperaturen auftreten. bei disposition zu fieberkrämpfen: simultane gabe von antipyretika. • mmr-impfung: zwischen 7. u. 12. d ist eine leichte masernähnliche sympt. mit subfebrilen temperaturen möglich. bei regelrechter anwendung der amtlich zugelassenen impfstoffe extrem selten. • bei verdacht: immunstatus u. ggf. interkurrente inf. zum zeitpunkt der impfung abklären → serum u. ggf. mikrobiolog. proben (serum-stuhlproben) asservieren. • meldung an das örtliche gesundheitsamt u. die arzneimittelkommission der deutschen ärzteschaft, herbert-lewin-platz 1, 10623 berlin, tel. 030-40 04 56-500, fax: 030-40 04 56-555. • impfling bzw. eltern/sorgeberechtigte auf gesetzliche regelungen zur versorgung nach impfschäden hinweisen ( § § 60-64, 66 ifsg). antrag ist beim zuständigen versorgungsamt zu stellen. • immer doppelte dokumentation (in impfausweis des impflings u. in patientenakten in praxis). • möglichst vorhandene dokumente (impfausweis) weiterführen. falls nicht vorliegend: separate bescheinigung möglich. zum späteren nachtrag in impfbücher ist jeder arzt berechtigt. • bei neuausgabe von impfausweisen: who-gerechte formulare bevorzugen ("internationale bescheinigungen über impfungen u. impfbuch", erhältlich beim deutschen grünen kreuz, s. o.). indikations-o. reiseimpfung. masern ▶ 16.8.1. • öffentlich empfohlene impfung bei kindern ab vollendetem 11. lm u. jgl. impfstoff totimpfstoff mit antigenen der meningokokkengruppe b, ab 2. lm 3 impfdosen à 0,5 ml, ab 3. lm 2 impfdosen à 0,5 ml (bexsero®). pertussis-impfung pertussis ▶ 16.8.8. öffentlich empfohlen für sgl. ab vollendetem 2. lm, kinder u. jgl. bis zum 18. lj., personal in päd. u. med. einrichtungen sowie in gemeinschaftseinrichtungen für das vorschulalter, erw. mit kontakt zu kk. einmalig alle gebärfähigen frauen u. kontaktpersonen zu neugeborenen spätestens 4 wo. vor geburt des kindes, wenn letzte impfung > 10 j. u. alle erw. mit der nächsten td-auffrischung! alle erwachsenen sollten ansonsten bei der nächsten fälligen td-imfung diese einmalig als tdap-, bei entsprechender indikation als tdap-ipv-kombinationsimpfung erhalten. impfstoff totimpfstoff. komb.-impfstoffe mit diphtherie, pertussis, polio, hib, hep. b (▶ tab. 9.2) . wg. besserer verträglichkeit gereinigten azellulären impfstoff bevorzugen. impfmodus kinder ab vollendetem 2. lm: 4 impfungen, 3 × 0,5 ml i. m. im abstand von je 4-8 wo., 0,5 ml i. m. 6-10 mon. nach der 3. impfung, 1. auffrischung ab vollendetem 5.-6. lj., 2. auffrischung zw. 9.-17. lj., später nur noch grundimmunisierung aller noch nicht geimpften bzw. komplettierung einer unvollständigen impfserie. alle gebärfähigen frauen u. enge kontaktpersonen zu neugeborenen u. sgl. sollen einmal eine pertussisimpfung erhalten, wenn letzte impfung > 10 j., außerdem alle erw. einmalig. indikation reisen (mittelmeer, orient, fernreisen). kinder ab vollendetem 2. lj. nur aktive immunisierung möglich, keine passivimpfung verfügbar. impfstoff lebendimpfstoff: oralimpfstoff typhoral®, sehr sicherer totimpfstoff typhim vi®. impfmodus oralimpfstoff: typhoral l® an d 1/3/5 je 1 kps., jeweils 1 h vor der mahlzeit; lebendimpfstoff: typhim vi® 1 × 0,5 ml i. m. oder tief s. c. nw gelegentlich übelkeit, durchfall, temperaturerhöhung. schutzwirkung oraler impfstoff: schutz ab 1 wo. nach 3. impfung; schützt nur gegen salmonella typhi, nicht jedoch gegen salmonella paratyphi o. enteritis-salmonellen; schutzrate 40-60 %. parenteraler impfstoff: schutz ab 3. wo p. v., schutzwirkung < 70 %. wiederimpfung nach 3. j. bei weiter bestehendem expositionsrisiko. ki kinder < 3. lm, allg. ki (▶ 9.2.1). besonderheiten antibiotika-ther. u. laxanzieneinnahme gefährden den impferfolg der oralvakazine. beginn einer malariaprophylaxe frühestens 3 d nach letzter einnahme von typhus-impfstoff. indikation seit dez. 2018 durch stiko empfohlene impfung für erw. > 60 lj., außerdem für menschen ab dem 50. lj. mit angeb./erw. immundefekten u. gesundheitlicher gefährdung infolge chronischer grundkrankheiten (z. b. diab. mell., hiv-infektion, ra sle, ced, copd, asthma, chron. niereninsuffizienz). impfstoff adjuvantierter subunit-totimpfstoff (shingrix®). indikation immunstimulation bei rezid. harnwegs-bzw. atemwegsinf. sowie vaginalen inf. (empfehlungsgrad c). impfstoff lyophilisierte extrakte eines spektrums der üblicherweise bei den jeweiligen inf. beteiligten bakterien. atemwegsinf.: z. b. broncho-vaxom® für erw./kinder. hwi: z. b. uro-vaxom®, strovac®. vaginalinf.: z. b. gynatren®. nach möglichkeit sollten impfungen außerhalb der grav. erfolgen. wg. einer theoretisch möglichen gefährdung des fetus keine lebendimpfstoffe anwenden. • unbedenkliche impfungen in der grav.: typhus, tollwut (vitale ind.), polio• antikoagulation/gerinnungsstörungen: gelbfieber u. a. lebendimpfungen thrombozytenabfall 4-6 d p. v.(())). möglichst keine i. m. inj., ggf. auf impfstoffe ausweichen, die s. c. appliziert werden können. • elektive operative eingriffe: 2 wo. abstand zu impfungen wg. möglicher impfreaktionen. • splenektomie: dringlich indizierte impfung: pneumok., hib, meningok. • frühgeborene sollten, unabhängig vom geburtsgewicht, gemäß dem empfohlenen impfalter geimpft werden. impfungen induzieren die hi-virus-replikation um das 3-bis 5-fache. die hiv-konz. erreicht 4-6 wo. p. v. wieder die ausgangswerte. bedeutung für die progression bisher unklar. impfungen unter antiretroviralem schutz durchzuführen, ist eine mögliche vorsichtsmaßnahme. • asympt. hiv-inf.: keine einschränkung. • ausgeprägter hiv-assoziierter immundefekt (< 200/cd4-lymphozyten/μl): nur totimpfstoffe u. passive immunisierung. • hiv-pos. kinder: wg. übertragener mütterlicher ak kann erst im alter von 1 j. endgültig festgestellt werden, ob das kind tatsächlich infiziert wurde. empfehlungen der stiko in dieser situation: für alle impfungen gelten die gleichen ind. u. ki wie für nichtinfizierte. eine allergische reaktion auf eine impfung kann durch den impfstoff selbst o. durch hilfsstoffe im präparat verursacht sein. um immunität abzuklären, zuerst titerbestimmung der entsprechenden ak durchführen. wenn kein erhöhter ak-titer vorliegt, allergolog. abklärung auf hilfsstoffe. hühnereiweiß impfstoffe gegen folgende krankheiten enthalten hühnerprotein (nach abnehmender konz. geordnet): gelbfieber, influenza, masern, mumps, fsme, tollwut. bekannte hühnereiweißallergie vor impfung erfragen bzw. ggf. allergologisch abklären. einteilung der allergischen reaktion in 3 schweregrade: • grad 1: allg. unverträglichkeit von hühnereiern ohne allergische o. anaphylaktische sympt., neg. hauttest: keine gegenanzeigen für impfungen, auch nicht gegen influenza o. gelbfieber • grad 2: im hauttest nachgewiesene, jedoch klin. nicht bedeutsame hühnereiweißallergie: gelbfieber-impfung ist relativ kontraindiziert u. darf nur in dringlichen fällen gegeben werden. impfung gegen influenza in der klinik • grad 3: pat. mit sofortreaktion nach verzehr von hühnereiweiß mit urtikaria, laryngo-/bronchospasmus, rr-abfall. impfung gegen influenza u. gelbfieber kontraindiziert. impfung gegen masern, mumps, fsme u. tollwut (präexpositionell) neomycin u. a. in folgenden impfstoffen enthalten: masern, mumps, röteln, polio (je nach hersteller unterschiedlich). im hinteren abschnitt der roten liste® (rosa seiten) befindet sich ein verzeichnis von notfalldepots, in denen folgende seren u. plasmaderivate aufbewahrt werden: botulismus-antitoxin vom pferd, hep.-b-immunglobulin, polyvalentes immunglobulin, schlangengift-immunserum polyvalent europa, tetanus-immunglobulin, tollwut-immunglobulin, varicella-zoster-immunglobulin. c1-inhibitor, diphtherie-antitoxin vom pferd, obidoxim, röteln-immunglobulin. zusätzlich in der roten liste®: • verzeichnis von informations-u. behandlungszentren für vergiftungen für deutschland u. europa (tab. 3.4) • impfempfehlungen; impfvorschriften für den internationalen reiseverkehr • empfehlungen zur malariaprophylaxe häufige bakt. inf. in der praxis ▶ tab. 9.10, lebensmittelvergiftungen ▶ tab. 9.11, übersicht enteritiserreger ▶ tab. 9.12, übersicht zoonosen ▶ tab. 9.13, neurotrope erreger u. ihre diagnostik ▶ tab. 9.14. krankheitsbilder trachom (serotypen a-c); unspez. genitalinf., neugeborenen-pneumonie, einschlusskörperchen-konjunktivitis (serotypen d-k); lymphogranuloma venereum (serotypen l1-l3; ▶ 9.8.5). erreger synonym pfeiffer-drüsenfieber. erreger severe acute respiratory syndrome coronavirus 2 (sars-cov-2), verursacht covid-19-erkrankung. erstmals identifiziert ende 12/2019 in der chinesischen stadt wuhan (südchinesische provinz hubei). ursprung wird im tierreich vermutet (derzeit verdächtige spezies: u. a. malaiisches schuppentier u. fledermausart "java-hufeisennase"; als ort des primären infektionsursprungs wird der "wuhan huanan großhandelsmarkt für fische und meeresfrüchte" angenommen. bes. bedeutung, weil sie zu einer schweren kindlichen schädigung führen kann. bei primärinf. der schwangeren in 50 % d. f. inf. der frucht. 50-80 % der schwangeren sind seroneg. u. damit gefährdet. primärinf. verläuft bei 75 % der schwangeren inapparent. schädigungsausmaß hängt vom zeitpunkt der inf. ab u. ist im 1. trimenon am größten. bei der geburt haben nur 6-10 % der kinder klin. sympt., die klassische trias "hydrozephalus, retinochorioiditis, zerebrale kalzifikationen" tritt nur in 2 % auf. am häufigsten ist die geburt von subklin. infizierten kindern. in der neugeborenenperiode ggf. fieber, konvulsionen o. prolongierter ikterus. am häufigsten ist latenter verlauf, wobei sich die schädigung erst im kindes-u. jugendalter manifestiert: retinochorioiditis, schielen, taubheit, psychomotorische retardierung, epilepsie. serologisch lässt sich der infektionsstatus von mutter u. kind zuverlässig beurteilen. noch keine obligate unters. i. r. der schwangerenvorsorge (▶ tab. 9.20). erreger enterobius vermicularis (oxyuren), größe 10-12 mm. häufigste wurmerkr. des menschen in mitteleuropa. sitz der würmer im ileum u. kolon, eier werden von den würmern außen (!) am after abgelegt. übertragungswege: • durch kratzen am after digital-orale reinf. • schmierinf. • aufnahme der eier mit kontaminiertem gemüse, salat. präpatenz 5-8 wo. erreger prophylaxe von "kinderkrankheiten" in der umgebung von hiv-positiven (▶ 9.9.6); impfungen bei hiv-pos. kindern (▶ 9.9.6 einteilung nach klin. gesichtspunkten u. anzahl der cd4-zellen/μl (▶ tab. 9.23, ▶ abb. 9.3). cave: virämie und infektiosität zeigen zwei gipfel: am anfang (akute hiv-krankheit) und am ende (terminale aids-krankheit). durch die einführung hoch wirksamer art-regime lässt sich das auftreten vieler aids-definierender erkr. verhindern bzw. hinauszögern. bei verbesserung der immunität unter art kommt es zu deren remission. häufige aids-krankheiten in relation zur cd4-zahl ▶ tab. 9.24, häufige ko bei aids ▶ tab. 9.25. westafrika daneben auch relevanter anteil von hiv-2-inf. (langsamerer krankheitsverlauf als bei hiv-1). doppelinf. mit hiv-1 u. hiv-2 können vorkommen epidemiologie anteil unter den hiv-infizierten in europa: homosexuelle kontakte 50 %, i. v. drogenmissbrauch 10 %, heterosexuelle kontakte ca. 20 %, anzahl zunehmend regional sehr unterschiedlich; gehäuft in ballungsräumen (53 % der inf.). 2015 ca. 84.700 menschen in d mit hiv o. aids, zahl der neuinfektionen in d 2015: ca. 3.200. durch konsequente hiv-überwachung aller blutprodukte seit 1985 sind iatrogene inf unsafe sex" • i. v. drogenmissbrauch mit nadeltausch • von der mutter auf das kind während grav nach initial hoher virämie (viruslast) gleichgewichtszustand (setpoint) aus virusreplikation u. -elimination auf niedrigerem niveau. progression zum stadium aids umso rascher, je höher der setpoint. ohne therapie erkranken innerhalb von 10 j. ca. 50 % der infizierten 16 d p. i.) o. viraler nukleinsäuren (ab 11 d p. i.): zweistufendiagnostik mit such-u. bestätigungstest; inzwischen suchtest elisa der 4. generation verfügbar → sicherer nachweis i. d. r schon nach 6 wo. möglich herzinfarkt innerhalb der letzten 6 mon hyperthyreose • pneumothorax • fehlender druckausgleich bei hno-erkr. (flugreise!) hypertonus > 200/120 mmhg (während des flugs sinkt der systolische druck, der diastolische erhöht sich, deshalb bes. gefahr bei hohen diastolischen werten!) nyha iv) o. instabil. angina pectoris • bei anfallsleiden (relativ, falls starke sedierung u. evtl. begleitung, flug möglich mit druckausgleichsstörungen in der eustachischen röhre: akute otitis media, paukenerguss, tubendysfunktion, akute erkr. der nnh (▶ 23.5.2). cave: barotrauma flugreisetauglichkeit nach operativen eingriffen▶ tab. 9 prophylaxe: nach abklingen der akuten hno-erkr. kann der pat. fliegen, jedoch sollten prophylaktisch einige zeit vor dem start u. bes. ca. 30 min. vor dem sinkflug abschwellende nasentropfen tief in die nase geträufelt werden epileptiker zur ärztlichen beratung gehört beurteilung der fahrtauglichkeit: erkrankungsu. therapiebedingte einschränkungen (z. b. medikamenten-nw) berücksichtigen. nicht nur aus reisemed. sicht, sondern auch aus forensischen gründen pat insulinpflichtige diabetiker dürfen nur in ausnahmefällen fahrzeuge zur fahrgastbeförderung o. lkw (führerscheinklasse c, d) führen (relevant, falls pat hypnotika, sedativa, antiepileptika (▶ 21.8) • zerebrale anfallsleiden fahrtüchtigkeit bei herzrhythmusstörungen richtlinien für pat. nach schwerer ventrikulärer tachykardie o. kammerflimmern: herzinfarktsympt., erste-hilfe-maßnahmen asthmatiker asthmaanfälle können durch klimatische faktoren u. reisestress ausgelöst werden: dosieraerosol immer griffbereit mitführen notfallset mit den im status asthmaticus benötigten medikamenten zusammenstellen bzw stoffwechseleinstellung; falls diese durch zusätzliche erkr. (z. b. inf.) gestört ist, von einer reise mit zeitverschiebungen abraten. durch jetlag-sy. (veränderung der zirkadianen hormone) u. reisestress (evtl. flugangst, hektik → noradrenalin-spiegel ↑) → zusätzliche stoffwechselbeeinträchtigung (kontrainsulinäre hormone ↑) grundsätzlich sollten nur gut geschulte insulinpflichtige diabetiker fernreisen unternehmen andere essgewohnheiten berücksichtigen: z. b. wird in mittelmeerländern selten vor 21 uhr zu abend gegessen → "mediterrane hypoglykämie"; außerdem an mögliche diätprobleme u. mangelnde verfügbarkeit von (diabetiker-)nahrungsmitteln im zielland denken autofahrten nur bei tag u. in begleitung bzw. möglichst alle 2 h pause mit zwischenmahlzeit. auch im auto kohlenhydratvorrat von mind bei zeitverlängerung evtl. zusätzliche insulingabe, bei zeitverkürzung ggf. geringere insulindosis (bz-kontrolle!) beispiel: • stuttgart -los angeles ≙ zeitverlängerung 9 h: plus • los angeles -stuttgart ≙ zeitverkürzung 9 h: minus cave: größte hypoglykämiegefahr während der ersten nächte nach der zeitverschiebung; aufgrund höherer bz-schwankungen häufiger bz messen, um hypoglykämien durch zusätzliche mahlzeiten u. durch evtl. nachspritzen von normalinsulin gegenzusteuern nadeln in ausreichender menge sowie ärztliche bescheinigung für den zoll mitführen (verwechslung mit drogenabhängigen vermeiden) ersatzbatterien mitnehmen, netzspannung kontrollieren (adapter) • reiseapotheke: ▶ tab. 9.34. bes. wichtig sind: breitbandantibiotikum, desinfektionsmittel, verbandmaterial empfehlenswert sind auch instantsuppen u. -bouillon als "durchfallther • verletzungen strikt vermeiden, bes. pat. mit pnp: z. b. strandschuhe mitnehmen. herzschrittmacherpatienten • vorsorglich magnetdetektoren an flughäfen meiden, besser persönlich/ manuell untersuchen lassen • ärztliches attest mitführen, das implantat bestätigt/herzschrittmacherausweis dialysepatienten pat. können über die deutsche dialysegesellschaft niedergelassener ärzte e. v. ein verzeichnis anfordern koch es, brat es, schäl es -oder meide es!"→ schutz vor diarrhö u. a. infektionskrankheiten. eindringlich darauf hinweisen, dass nur strikte einhaltung der verhaltensregeln prophylaktisch wirkt nur abgekocht verwenden. getränke aus verschlossenen flaschen o immer darauf achten, dass der verschluss in restaurants erst am tisch geöffnet wird; in anderen situationen immer selbst öffnen. keine eiswürfel in getränken, keine offenen fruchtsäfte gemüse sehr gut waschen bzw. selbst schälen • nur gut gekochte o. gebratene speisen verzehren, die frisch u. noch warm serviert werden unvollständig gekochte/gebratene o. rohe fische u. meeresfrüchte; generell speisen meiden, die schon längere zeit vor dem verzehr vorbereitet (z. b. pudding) u./o. durch dir kleidung • an kopfbedeckung u. sonnenbrille denken (cave: sonnenstich, hitzschlag, konjunktivitis) auch am strand nicht barfuß gehen (cave: giftige insekten haut bedeckt halten versicherungsschutz private reisekrankenversicherung mit rückholgarantie empfehlenswert • kein baden in süßwasserseen o. -tümpeln, flüssen u. kanälen, auch nicht hände o. füße darin waschen (cave: bilharziose, lambliasis bzw. reisediarrhö durch kontamination mit fäkalien gesundheitsspritzen c-inf. u. a. sexuell übertragbare krankheiten) o. sexuelle abstinenz • aufenthalt in mückensicheren räumen, mit klimaanlage, anwendung von moskitonetzen reiseapotheke • reiseart, reisedauer, zielland, zahl u. alter der mitreisenden (kinder? senioren?) sowie deren vorerkr. u./o. spezielle dispositionen für bestimmte erkr hinweise zur verwendung der einzelnen mittel dem reisenden möglichst schriftlich mitgeben; nicht auf die informationen der beipackzettel verlassen, da diese für laien oft nicht verständlich sind medikamente für reisen in warme länder (z. b. suppositorien für kinder), wenn keine kühlmöglichkeit zu erwarten ist cave: auf impfdokumente o. titer verlassen, nicht auf angaben des pat./reisenden! • obligatorische impfungen für das jeweilige reiseland erfragen dabei reiseroute berücksichtigen; einige länder verlangen impfnachweis schon für zwischenlandung ohne eigentliche einreise impfplan bei reiseimpfungen ▶ tab. 9.36, kurzübersicht: vorkommen u. prophylaxe von tropenkrank falls obligatorische impfungen bei reisenden kontraindiziert sind, muss ein impfbefreiungszeugnis mit unterschrift des arztes u. einem beglaubigungsstempel mitgeführt werden; je nach reiseland in englischer o. französischer sprache u. evtl langzeitreisende sowie pat. mit vorerkr chemoprophylaxe bei erw. ▶ tab. 9.39, bei kindern ▶ tab. 9.40, notfalltherapie ▶ tab. 9.41. aktuelle malariaprophylaxeempfehlungen: ▶ 9 arthemeter/lumefantrin (z. b. riamet®) liegen keine ausreichenden daten vor standby") die in ▶ tab aufgeführten medikamente sind für die notfalltherapie geeignet, wobei die medikation prinzipiell verschieden von der prophylaxe sein soll. die behandlung soll jedoch, wenn möglich, immer im krankenhaus erfolgen, insb. bei rasanter verschlechterung des krankheitsbildes u. bei ind. zur ther. mit chinin (z. b. limptar n®) empfohlene chemoprophylaxe bei kindern resochin ® ab 6 mon. u. 7 kg kg 5 mg/kg kg/wo malarone ® ab 11 kg kg 1 junior-tbl. ≙ ges. 62,5/25 mg pro 10 kg kg/d, max doxy-ct ® ab 8 j. 1,5 mg/kg kg/d bei den meisten mitteln wird die einnahme nach einer mahlzeit ausdrücklich empfohlen. falls innerhalb 1 h erbrochen wird, nochmals einnahme derselben dosis. bei kk u. sgl. konsequente expositionsprophylaxe, behandlung möglichst nur durch arzt mit spezieller erfahrung auswahl der medikamente in abhängigkeit von der zuvor verwendeten chemo 42 einzelne malaria-medikamente mit indikation, nebenwirkungen und kontraindikationen wirkstoff indikation chemoprophylaxe u. ther. in gebieten ohne chloroquin-resistenz, auch bei grav. u. kindern sowie als standby-medikation nw: übelkeit, diarrhö, v. a. zusammen mit alkohol, augenflimmern (wenn längerfristige einnahme, dann spätestens nach 5 j. augenärztliche kontrolle), schwindel ki: retinopathie, g6pd-mangel 2016 in d nicht mehr empfohlen; chemoprophylaxe, keine notfallmed chemoprophylaxe u. ther. für max. 12 wo. u. standby-medikation, bei kindern ab 11 kg kg, zu schwangeren u. stillzeit liegen keine ausreichenden daten vor nw: git-störungen, kopfschmerzen ki: schwere lebererkr chemoprophylaxe in gebieten mit chloroquinu. mefloquin-resistenz (grenzgebiet thailand/kambodscha u. myanmar ≙ burma), in d zur prophylaxe nicht zugelassen nw: schleimhautschäden im ösophagus (deshalb mit viel flüssigkeit einnehmen), git-störungen, fotosensibilisierung. wechselwirkungen mit oralen antidiabetika nur als standby-medikation, keine chemoprophylaxe nw: kopfschmerzen, schwindel, schlafstörungen, palpitationen, git-störungen u. v. a. ki: vorbestehende qt-verlängerungen, z. b. auch durch medikamente. medikamente, die die cytochrom-p-oxidase hemmen bei malaria tertiana (p. vivax) ist im anschluss an die initialther. mit einem der o. g. chemotherapeutika wg. rezidivgefahr eine behandlung mit primaquin (importmedikament) zur eradikation der extraerythrozytären stadien erforderlich • malaria-schnelltests (z. b. binaxnow® malaria) sind nur bei pos ergebnisse möglich) u. ersetzen nicht unters. von blutausstrichen. schnelltests sollten von med alkoholverzicht • nicht lesen • durch zeitverschiebung bei interkontinentalflügen hervorgerufene störung des zirkadianen rhythmus. dadurch beeinträchtigung mentaler u. physiolog. funktionen, z. b. schlaf-u. wachzustand, gedächtnis-u. konzentrationsleistungen, hormonausschüttung • für eine zeitverschiebung ab 2 h werden mind. 24 h benötigt, um jetlag auszugleichen (sog. resynchronisationszeit) verschiebung der schlafphase) sind weniger beeinträchtigend als flüge von west nach ost (zeitverkürzung, häufig überspringen der schlafphase) entsprechend der zeitverschiebung kann reisenden ein verändertes einnahmeschema für medikamente mitgegeben werden, z. b. diabetikern ▶ 9 42 einzelne malaria-medikamente mit indikation nur als standby-medikation, nicht zur chemoprophylaxe nw: kopfschmerzen, schwindel, schlafstörungen, palpitationen, git-störungen u. v. a. ki: vorbestehende qt-verlängerungen, z. b. auch durch medikamente chinin, z. b. limptar n ® ind.: ther. der komplizierten m. tropica, immer im krankenhaus. keine chemoprophylaxe nw: kopfschmerzen, sehstörungen, tinnitus, arrhythmien, git-störungen, bronchospasmus ki: g6pd-mangel, n.-opticus-schaden prophylaxe und therapie anpassung des schlaf-wach-rhythmus schon vor der reise; aus praktischen gründen meist nur für 2-3 h möglich risiken durch natürliche umweltbedingungen sonnen-und uv-licht-exposition trotz zunehmender aufklärung über kurzu. langfristige risiken intensiver sonnenlichtexposition (melanome ▶ 26.10.3, basaliome ▶ 26.10.4, vorzeitige alterung der haut) wird risiko nach wie vor unterschätzt. zu vermeidbaren kurzfristigen folgen intensiver sonnenstrahlung, die das allgemeinbefinden am urlaubsort beeinträchtigen, gehören: polymorphe lichtdermatose sonnenbrand prophylaxe durch konsequenten sonnenschutz mit mind bei sehr hellhäutigen sowie in bestimmten gegenden (gletscher, äquatorialgebiet, australien) u. über die mittagszeit (11-15 uhr) höheren lsf, sunblocker bzw. strikte expositionsvermeidung: langsame u. hauttypgerechte adaptation: morgens o. nachmittags, nie über die mittagszeit schneeblindheit") prophylaxe: möglichst gletscherbrille (seitlich geschlossen) tragen, auf modische brillenformen (z. b. schmetterlingsbrille) verzichten sonnenstich prophylaxe: immer kopfbedeckung tragen risiken durch sport und freizeitaktivitäten aufenthalt in großen höhen/hochgebirge reizschwelle für die höhenanpassung beim gesunden liegt bei ca. 2.500 m ü. m. bei zu schnellem aufstieg über diese höhe hinaus gefahr der akklimatisierungsstörungen (höhenkrankheit/ akute bergkrankheit). entscheidend für die reizschwelle ist die tägl. schlafhöhe (übernachtung) abstieg in tiefere lagen u./o. aufstiegspause mit nachfolgend langsameren aufstiegen. prophylaxe: guter trainingszustand vor antritt der reise (evtl. check-up durchführen); keine zu großen höhenunterschiede tägl. bewältigen, keine gewalttouren; auf ausreichende flüssigkeits-u. salzzufuhr achten; größere touren nur mit erfahrenen bergführern abtransport des pat. in tiefere lagen, sauerstoffgabe; medikamentös: nifedipin p. o. 10-20 mg plus 20 mg in retardform sofort tauchsport verlangt hohes maß an körperl. u. mentaler fitness; minimum-check-up: herz-kreislauf-u. lungenfunktion. bei älteren reisenden evtl. belastungs-ekg. psychische belastbarkeit abschätzen! bei hno-erkr. in der anamnese ist vor einem tauchurlaub ein fachärztliches attest durch hno-arzt voraussetzung erkältungen, allergischen reaktionen); auch keine schleimhautabschwellenden nasentropfen vor tauchgängen anwenden tiefe der tauchgänge darf für mind. 24-48 h danach nicht geflogen werden (genaue zeit wird nach dem letzten tauchgang berechnet, diese berechnung zu lernen, ist bestandteil einer seriösen tauchausbildung) vorkommen: afrika; naher, mittlerer u befällt venengeflecht des kleinen beckens. klin.: blasenbilharziose mit hämorrhagischer zystitis praziquantel (40 mg/kg kg tägl praziquantel (s. o., oft muss höher dosiert u prophylaxe in den entsprechenden gebieten nicht in süßwasserseen u. -tümpeln baden für touristen ist erkr.-risiko niedrig (< 1:1 mio.), da kein o. selten kontakt mit slumbewohnern o. aufenthalt in slums; im falle einer infektion dann erkr aedes aegypti), die in der nähe menschlicher behausungen in wasseransammlungen brüten. nach stich u. eintritt in die blutbahn vermehrung in regionalen lk injektion der konjunktiven typisch. diffuses, stammbetontes erythem am 2.-3. d, danach morbilliformes exanthem am rumpf mit zentripetaler ausbreitung auf kopf u. extremitäten, verbunden mit einem 2. fieberschub. evtl. petechiale blutungen u therapie klinikeinweisung zur diagnosestellung, ggf. intensivmed. maßnahmen. cave: ass wg. vermehrter blutungsneigung, ausweichmittel paracetamol prophylaxe expositionsprophylaxe; haut durch kleidung u. repellenzien schützen. schlafen in räumen mit klimaanlage. keine impfung möglich gelbfieber erreger durch arboviren hervorgerufene u. die stechmücke aedes aegypti sowie haemagogus-arten (moskitos) übertragene erkr. gesamtletalität ca. 20-30 % (who) klinik initialstadium (3 d): plötzlicher beginn mit fieber bis 40 °c, schüttelfrost, starke kopf-u. gliederschmerzen, übelkeit, erbrechen, evtl. relative bradykardie. remissionsstadium: fieberabfall am 3. o. 4. d, evtl. ausheilung. bei schwerem verlauf stadium der organschädigung: hepatitis mit ikterus u. erbrechen, nephritis mit proteinurie, hämorrhagische diathese mit profusen blutungen. ko: leber übersicht tropenvirosen u. a. fieberhafte allgemeinerkr. ▶ tab. 9.43. leitsymptome der häufigsten "importkrankheiten" ▶ tab. 9 diagnostik tropenanamnese bei ungeimpften (o. zu spät geimpften) personen mit typ. klinik, evtl. igm-ak-nachweis gelbfieber" angeben) zur diagnosestellung (ak-nachweis) prophylaxe impfung (▶ 9.2.3) für alle reisen in endemiegebiete (▶ abb. 9.7, ▶ abb. 9.8). cave: viele staaten übertragung vorwiegend fäkal-oral, verunreinigte gewässer, meeresbuchten, aber auch durch blutprodukte (selten). ikz 2-6 wo. vorkommen: ubiquitär in ländern mit schlechten hygienischen verhältnissen klinik im kindesalter (bis ca. 6 j.) meist anikterisch, oft nur git-sympt. bei inf. im erw.-alter lange u. schwere verläufe möglich. cave: bei reisenden > 50 j. ohne immunität häufiger als in jungen jahren fulminante verläufe mit höherer letalität havrix®) für alle fernreisenden; aufgrund der guten verträglichkeit u. der fast 100-proz. schutzwirkung sollte die aktive schutzimpfung einer passiven impfprophylaxe unkomplizierte reisediarrhö betrifft 20-50 % aller fernreisenden erreger 85 % bakt. (meist pathogene e.-coli-stämme), 10 % viral, 5 % protozoen klinik flüssiger o. wässriger stuhl, häufig abdom. krämpfe, übelkeit, blähungen; plötzlicher beginn, milder bis mittelschwerer verlauf über durchschnittlich elotrans®-pulver); in den apotheken tropischer länder sind "oral rehydration salts" (ors) verfügbar, die im angegebenen volumen in abgekochtem wasser aufgelöst werden; auch für diabetiker geeignet • gezuckerten tee trinken, salzgebäck essen • bes. durch exsikkose gefährdet: kk u. sgl. • reduktion der stuhlfrequenz durch motilitätshemmer -loperamid: ab 14 j. 4 mg initial, dann 2 mg nach jedem wässrigen stuhlgang, max. 16 mg/d; kinder 8-13 j. 2 mg initial, dann 2 mg nach jedem wässrigen stuhlgang, max. 8 mg/d prophylaxe strikte einhaltung der allg. verhaltensregeln, bes. kost u. persönliche hygiene (▶ 9.10.6). keine impfung möglich! komplizierte reisediarrhö klinik fieber > 3 d u. evtl ciprofloxacin: 2 × 500 mg/d für 3 d o. azithromycin (zithromax®) 1 × 500 mg/d für 3 d • bei fieber o charakter der reise, dauer u. a.) ist nach längerem tropenaufenthalt unter schwierigen bedingungen angezeigt, z. b. nach arbeitsaufenthalten o. abenteuerreisen (auch wenn der rückkehrende symptomlos ist) u. immer, wenn eines o. mehrere der folgenden sympt. auftritt/auftreten: • unklares fieber immer auch an das versagen der malariaprophylaxe denken. frühzeitig kontakt mit tropenmed bb: auf eosinophilie achten (helminthiasis?) • leberfunktionsparameter • bilharziose-serologie nach aufenthalten in endemiegebieten • stuhlmikroskopie tests zum nachweis von ak gegen viren, bakterien, protozoen, helminthen nur bei konkretem verdacht sinnvoll malaria pat. zur blutabnahme in geeignetes labor schicken, in dem ausreichende erfahrung in anfertigung u. auswertung z. b. eines "dicken tropfens" vorliegt; vorher telefonisch rücksprache nehmen; bei entsprechender klinik immer sofort klinikeinweisung plasmodiensuche immer im edta-blut auf helminthenlarven z. a. einer strongyloidiasis, ggf. serologie • gewebshelminthen-suchtest (wenn 2 der 3 hauptind. zutreffen) indikationen zum serolog wurmerkr. mit gewebestadien): 15 % aller tropenrückkehrer infektionswege -per os: nahrung, schmutzinf., tierkontakte, trinkwasser -perkutan: insektenstiche, süßwasser, boden • unspez. klin. befund: -fieber: schistosoma (katayama-sy., filarien, trichinella, toxocara) -git-beschwerden: schistosoma, strongyloides, toxocara -hautsymptomatik: strongyloides, loa, onchocerca (toxocara) -lungensymptomatik: schistosoma, paragonimus, strongyloides, toxocara alle in § 6 aufgeführten erkr. müssen durch den behandelnden arzt an das zuständige gesundheitsamt übermittelt werden 12) (1) namentlich sind zu melden: krankheitsverdacht, erkr. sowie tod an: • botulismus • cholera • clostridium difficile (erkr./tod bei schwerem verlauf) • covid-19-infektion • diphtherie • humaner spongiformer enzephalopathie, außer familiär-hereditärer formen • akuter virushepatitis nachweis) sowie v. a. u. erkr. an: • über das übliche ausmaß einer impfreaktion hinausgehender gesundheitlicher schädigung durch eine schutzimpfung • mikrobiell bedingter lebensmittelvergiftung o. akuter infektiöser gastroenteritis bei beruflich exponierten personen o. bei zwei o. mehr gleichartiger erkr., bei denen ein epidemischer zusammenhang wahrscheinlich ist o • verletzung eines menschen durch ein tollwutkrankes o. -ansteckungsverdächtiges tier sowie die berührung eines solchen tieres o. tierkörpers 1 hinaus mitzuteilen, wenn personen, die an einer behandlungsbedürftigen lungentuberkulose leiden dem gesundheitsamt ist unverzüglich das gehäufte auftreten nosokomialer infektionen, bei denen ein epidemischer zusammenhang wahrscheinlich ist o. vermutet wird, als ausbruch nichtnamentlich zu melden das auftreten einer bedrohlichen krankheit o. von zwei o. mehr gleichartigen erkr., bei denen ein epidemischer zusammenhang wahrscheinlich ist o. vermutet wird, wenn dies auf eine schwerwiegende gefahr für die allgemeinheit hinweist u. krankheitserreger als ursache in betracht kommen wichtig: zusätzliche meldepflichten einzelner bundesländer beachten! meldung wann und wohin? namentliche meldung muss unverzüglich, spätestens innerhalb von 24 h an das für den aktuellen aufenthaltsort des betroffenen zuständige gesundheitsamt (ga), bei meldung durch diagn. institute an das für den einsender zuständige ga erfolgen. eine meldung darf wg virales hämorrhagisches fieber diese erkr. sollen nur in den sonderisolierstationen in bei erkr. an oder verdacht auf (a: regelung für ausscheider: besuch von gemeinschaftseinrichtungen nach rücksprache mit dem gesundheitsamt möglich): cholera (a) typhus abdominalis; paratyphus; cholera; shigellenruhr bei denen die möglichkeit einer übertragung der krankheitserreger über lebensmittel besteht • ausscheider von: choleravibrionen, ehec kutane lyme-borreliose neuroborreliose mrsa -eine handreichung für hausärzte neuartiges_coronavirus/ ncov.html?cms_box=1&cms_current=covid-19+%28coronavi-rus+sars-cov-2%29&cms_lv2=13490882 -ccdv (chinese center for disease control and prevention aktuelle informationen des rki zu hiv: www.rki.de/de/content/infaz/h/hivaids/hiv_aids.html?cms_box=1&cms_cur-rent=hiv+%28aids%29&cms_lv2=2747670 -deutsche aids-hilfe: www.aidshilfe.de -unaids: www.unaids.org -deutsche aids-gesellschaft (u. a. leitlinien für diagnostik u. ther. der hiv-inf. u. zur cart im erw.-alter u. bei kindern ?__blob=publicationfile&v=17 -reise-, tropenmedizinische informationen, nachrichten, länderdaten, hintergrundinformationen des bernhard-nocht-instituts (hh), u. a. aktuelle informationen zur malaria: www.gesundes-reisen.eu • meldepflicht: meldepflichtige erkr. nach § 6 ifsg • relativ (abhängig von reiseziel, -art, key: cord-252039-732z92dd authors: valdiserri, ronald o.; holtgrave, david r. title: responding to pandemics: what we’ve learned from hiv/aids date: 2020-04-09 journal: aids behav doi: 10.1007/s10461-020-02859-5 sha: doc_id: 252039 cord_uid: 732z92dd nan to state the obvious, we are in the midst of a global pandemic. like sars and mers before it, a new coronavirus (sars-cov-2) that was previously confined to an animal species, has made its way into the human population with devastating results [1] . given the scope of the problem and because at the time of this writing there is no vaccine to prevent infection or proven therapy to treat it, attention is understandably riveted on mitigating the immediate health and economic consequences of covid-19. elected officials, community leaders, health care providers, and scientists are scrambling to respond to the expanding spread of disease in the united states and elsewhere around the world. at a time when society's efforts are necessarily focused on our acute response to this pandemic, it may seem wrongheaded to think about longer-term strategies in response to sars-cov-2. but arguably there is benefit in planning beyond immediate needs and circumstances. said consideration derives from the fact that even after the current wave of this pandemic subsides, we will not have eradicated sars-cov-2. nor will we be spared from future pandemics of other viruses, whether they be respiratory, enteric or bloodborne. what can history teach us about successful responses to past infectious disease pandemics? given the widespread implementation of social distancing (also known by the more accurate designation of "physical distancing") in response to covid-19 disease, public health leaders are deeply interested in the outcomes of these same so-called "nonpharmaceutical interventions" when they were deployed in response to another deadly pandemic of a respiratory virus, the influenza epidemic of 1918-1919 [2] . contemporary analyses support the fact that these interventions, when implemented early and in a sustained manner, were successful in mitigating the consequences of pandemic influenza in the last century [3] . that valuable information is being put to good use during the current wave of sars-cov-2. but what can we say about the public health responses that were undertaken in the years following the 1918-1919 influenza pandemic, after the immediate threat had passed? certainly, there was increased research interest into the causative agent of influenza, leading to the eventual isolation of influenza a from human tissue samples in 1933 [4] . and historians have noted that the lack of a national disease reporting system in the u.s.-which hindered a coordinated response to the influenza outbreak of 1918/1919-was rectified several years later; by 1925 all u.s. states were participating in a national system to report disease [5] . but given the periodicity of influenza, it's come-and-go nature, some experts have opined that our national response to pandemics typically follows a cycle of "panic-neglect-panic-neglect" [6] . sadly, this supposition is supported by patterns of public health funding in the u.s. typically, after a national disease outbreak or other disaster there is an infusion of one-time supplemental funding which generally recedes after the emergency wanes and the attention of legislators turns to other, more immediate issues [7] . when faced with a pandemic of a novel infectious disease, it's understandable that we quickly focus on the necessary biomedical tools required to prevent, diagnose, and treat the offending pathogen. do we have a test that can accurately diagnose infection? how long will it take for scientists to develop an effective vaccine that can prevent disease acquisition? are any drugs available that can cure or safely treat persons once they've become infected? to be sure, each one of these elements-accurate diagnostic test, effective vaccine, successful treatment-is critical but by themselves they do not constitute an adequate pandemic response. why? because, simply stated, a pandemic's impact is not just experienced at the level of the infected individual. by their very nature, pandemics adversely affect large segments of the population, thereby generating negative consequences that spread across social systems and structures. it follows, then, that successful responses to pandemics must include the implementation of societal-level responses and system-wide interventions. to wit, during a pandemic the goal of public health is to protect the health of the entire population, not just to prevent or treat disease among specific individuals. consider the following scenarios. the most accurate diagnostic test will be of little value without a stable system to deliver it in a timely manner to everyone in need-or a sufficiently scaled workforce that's been trained to properly collect and analyze the large influx of specimens. without strong community partnerships to amplify health communication messages coming from public health leaders, panic and misinformation can scuttle the uptake of strategies meant to keep communities safe. for example, a handful of anecdotal reports about negative reactions to a new vaccine could impede community uptake, resulting in substantial missed opportunities for immunization. and even curative treatments will be of little value if large segments of the population in need of them are uninsured or underinsured and unable to afford the therapy [8] . each of these scenarios point out that effective biomedical tools, by themselves, cannot end epidemics. in addition to effective tools and interventions we need surveillance systems to assess and target their need; viable community partnerships across all segments of the public health system to assure that interventions are delivered in a timely, equitable and culturally competent manner; policies, processes and information systems that can continuously monitor the interventions' impact; and a standing public health workforce of sufficient size that is properly equipped and capable of providing these services, in collaboration with other community partners. if one needs proof of the importance of developing and sustaining system-level interventions to meet the ongoing threat of global pandemics, our nation's response to hiv/ aids provides such evidence. to be clear, this comparison is made in reference to our nation's eventual response to hiv-not to its initial response, which was typified largely by denial. early on, most legislators and policy makers ignored the emerging aids epidemic, characterizing it as the consequence of unhealthy behaviors practiced by homosexual men and persons who inject illicit drugs [9] . but a cadre of fearless community activists and farsighted public health leaders were eventually able to push forward a comprehensive agenda that resulted in long term funding for surveillance systems to monitor the spread of disease [10] , community-based systems to test for and prevent hiv infection among the vulnerable [11] and a national system to provide comprehensive medical care, essential support services and needed medications for uninsured and underinsured persons living with hiv-the ryan white hiv/aids program [12] . nor should we overlook america's substantial contribution to fighting the global spread of hiv. in 2003 the united states government implemented pepfar, the largest global health program devoted to a single disease, credited with saving millions of lives globally [13] . true, we have not vanquished hiv from our world, but we have made substantial progress in preventing its spread and improving health outcomes for those who are living with the virus-even in the absence of a vaccine or curative treatment. our national progress has been such that the united states has now set a goal to reduce new hiv infections by 75% come 2025 [14] . without denying the importance of prophylaxis that can prevent the acquisition of hiv [15] or the impact of effective treatments that can reduce viral load such that the risk of sexual transmission is essentially nil [16] , we would not be able to realistically visualize the end of aids in the united states without the continued public investment in systems that are necessary to prevent infection, improve health outcomes for those living with hiv and monitor changes in disease spread and outcome. which brings us back to the point of this commentary. we must not allow ourselves to fall into a cycle of "neglect" by ignoring the threat of future pandemics once this initial wave of sars-cov-2 has passed. our national response to hiv/aids has shown us that in addition to funding the biomedical enterprise so that it can deliver effective treatments (including an eventual vaccine), we must also invest in a strong, stable public health system that is ready to respond when calamities like covid-19 hit [17] . addressing chronic underfunding in state and local health departments is key to that effort [18, 19] . nor should we ignore the vital role that non-governmental organizations-especially community-based organizations-can play when they work in partnership with public health agencies. in the united states and across the world, ngos have been a critical component in successful efforts to prevent and treat hiv [20] . and adequately funded hiv surveillance systems in the u.s.-a rarity among other infectious diseases like hepatitis c virus (hcv)-have provided increasingly detailed information that enable us to quickly identify emerging outbreaks and accurately target prevention and care services to underserved areas. we readily admit that the epidemiology, transmission dynamics and pathogenesis of these two viruses-hiv and sars-cov-2-are vastly different. nor do we mean to infer that the interventions successful in preventing hiv can be directly applied to covid-19 disease. but the principles that we've learned from decades of grappling with the hiv pandemic-in the u.s. and abroad-are relevant in regard to developing successful, long-term responses to protect against other infectious disease pandemics. while maintaining an adequate public health infrastructure may "top the list" of necessities when it comes to protecting population health, other fundamental principles, listed below, are equally important. sustained investment in public health infrastructure is necessary in order to develop, implement and evalu-ate effective system-level interventions in response to emerging and ongoing health threats. approaches to assuring and preserving health must not be limited to the biomedical sciences; we must also actively address the psychosocial influences that affect well-being. affected communities must be actively engaged in identifying and implementing strategies in response to threats to their health and well-being. stigma and fear are constant companions of infectious disease pandemics; proactive steps must be taken to minimize their negative consequences. to reduce health disparities, proactively identify groups and communities at disproportionate risk of developing disease or poor health outcomes and design interventions to reduce these disparities and to promote health equity. in recognition of the interconnected nature of our world, there must be a global component in our response to infectious disease pandemics and this component must be based on the best available information and science. let's be sure to put the hard-won knowledge that we've acquired from our decades-long interaction with the hiv/ aids pandemic into practice as we prepare for the future of covid-19 and other pandemics that have yet to emerge. to paraphrase santayana, failure to learn from our past can only ensure repeated failure in our future. coronavirus has become a pandemic, w.h.o. says. new york times. 2020 the 1918 influenza epidemic in new york city: a review of the public health response nonpharmaceutical interventions implemented by us cities during the 1918-1919 influenza pandemic changing perceptions of pandemic influenza and public health responses how the 1918 flu pandemic revolutionized public health. smithsonian magazine better prepare than reach: reordering public health priorities 100 years after the spanish flu epidemic ready or not: protecting the public's health from diseases, disasters, and bioterrorism. trust for america's health simple, effective but out of reach? public health implications of hcv drugs dawning answers: how the hiv/aids epidemic has helped to strengthen public health dawning answers: how the hiv/aids epidemic has helped to strengthen public health twenty-five years of hiv/ aids-united states health resources and services administration president's emergency plan for aids relief (pepfar) ending the hiv epidemic: a plan for the united states preexposure chemoprophylaxis for hiv prevention in men who have sex with men sexual activity without condoms and risk of hiv transmission in serodifferent couples when the hiv-positive partner is using suppressive antiretroviral therapy developing a financing system to support public health infrastructure america's public health infrastructure needs consistent funding. roll call the impact of chronic underfunding on america's public health system: trends, risks, and recommendations ending hiv in america: not without the power of community key: cord-258167-jqm3qyfm authors: zhou, peng; han, zhenggang; wang, lin-fa; shi, zhengli title: immunogenicity difference between the sars coronavirus and the bat sars-like coronavirus spike (s) proteins date: 2009-09-18 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2009.07.025 sha: doc_id: 258167 cord_uid: jqm3qyfm abstract sars-like coronavirus (sl-cov) in bats have a similar genomic organization to the human sars-cov. their cognate gene products are highly conserved with the exception of the n-terminal region of the s proteins, which have only 63–64% sequence identity. the n-terminal region of coronavirus s protein is responsible for virus–receptor interaction. in this study, the immunogenicity of the sl-cov s protein (ssl) was studied and compared with that of sars-cov (ssars). dna immunization in mice with ssl elicited a high titer of antibodies against hiv-pseudotyped ssl. the sera had low cross-reactivity, but no neutralization activity, for the hiv-pseudotyped ssars. studies using wild bat sera revealed that it is highly likely that the immunodominant epitopes overlap with the major neutralizing sites of the sl-cov s protein. these results demonstrated that sl-cov and sars-cov shared only a limited number of immunogenic epitopes in their s proteins and the major neutralization epitopes are substantially different. this work provides useful information for future development of differential serologic diagnosis and vaccines for coronaviruses with different s protein sequences. searching for the origin of the severe acute respiratory syndrome coronavirus (sars-cov) in wild animals led to the discovery of several sars-like covs (sl-covs) in several horseshoe bats of the genus rhinolophus in china [1, 2] . our previous studies showed that the sl-cov and sars-cov shared similar genomic organization and highly conserved gene products, with the exception of the spike protein (s protein), which had a low sequence identity, especially in the receptor binding domain (rbd) located at the n-terminal region of the s proteins. the difference of sequence in this region was responsible for the failure of the sl-cov s protein (s sl ) to use angiotension converting enzyme 2 (ace2), the known major receptor for sars-cov, as a functional receptor [3] . the sars-cov s protein (s sars ) is responsible for virus entry and induction of neutralizing antibodies, mediated mainly by the rbd at aa 318-510 [4, 5] . it has been demonstrated previously that bat sera from natural infection cross-reacted with, but failed to neutralize sars-cov [1] . therefore, for the development of differential diagnosis and specific vaccines for these viruses, it is necessary and important to have a better understanding of the immunogenicity of s sl , s sars and the difference between s sl and s sars . in this study, the immunogenicity and immunodominant region of s sl was determined using sera generated from dna immunization and naturally infected bats. the data presented here will be useful for future development of diagnostics and vaccines for sars and sars-like coronaviruses. cells. the human cell lines 293t and hela were maintained in dulbecco's modified eagles medium (dmem) supplemented with 10% heated-inactivated fetal calf serum (gibco, usa or sijiqi, china). hela cell lines that stably expressing human ace2 protein have been described in a previous report [6] . preparation of pseudoviruses. the construction of a codon-optimized full-length s gene of sars-cov bj01, bat sl-cov rp3, and chimeric s gene (designated cs 310-518 ), which has the rp3-s gene containing aa 310-518 of bj01 s in replacement of its corresponding region, has been described previously [3] . briefly, 12 lg each of phiv-luc (pnl.4.3.luc.e à r à ) and plasmid pcdna3.1 containing various s genes (or empty vector control) were co-transfected into 2 â 10 6 293t cells in 10 cm dishes by standard calcium phosphate method [6] . the pseudoviruses were purified by ultracentrifugation from cell culture supernatant through a 20% sucrose cushion (10 ml) at 55,000g for 60 min using a ty70 rotor (beckman). the pelleted pseudoviruses were dissolved in 100 ll of pbs and stored at à80°c in aliquots until further use. antisera. six field bat sera showing positive (5 of 6) or negative (1 of 6) cross-reactivity with sars-cov [1] were used in this study. hyperimmune mouse sera were generated by dna immunizations with plasmids pcdna 3.1(+) containing the codon-optimized fulllength s gene of sars-cov bj01, sl-cov rp3, and two chimeric plasmids cs 310-518 and cs 259-518 (see fig. 1 for diagrams). the pcdna 3.1(+) vector was used as a negative control. five groups (five mice per group) of 6-8 week old female balb/c mice were immunized with 30 lg of plasmid dna in 30 ll pbs by in vivo electroporation according to the published method [7] . mice were immunized three times at weeks 0, 3 and 5. sera were collected at week 8. after injection into the right quadriceps muscle, a pair of electrode needles with 5 mm apart was inserted into the muscle to cover the dna injection sites and electric pulses were delivered using an electric pulse generator (electro square porator t830 m; btx, san diego, ca). three pulses of 100 v each, followed by three pulses of the opposite polarity, were delivered to each injection site at a rate of one pulse per second. each pulse lasted for 50 ms. elisa. all elisas were performed under stringent conditions to avoid nonspecific reactions. briefly, 96-well microtiter plates were coated in 0.1 m carbonate buffer (ph 9.3) over night at 4°c with purified pseudoviruses (50-200 ng/well). the plates were washed and blocked with 5% bsa in pbs-0.1% tween 20, and then incubated with bat or mouse sera for 1 h at 37°c. bound antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse igg (lingfei tech.) or goat anti-bat igg (bethyl laboratories, inc.) (dilution at 1:4000). color development was conducted using 3,3,5,5,tetramethylbenzidine (tmb) and the absorbance at 450 nm was determined after the reaction was stopped with 2 m h 2 so 4 . all washes were carried out using pbs-0.1% tween for 5 washes (2 min/wash), and all antibodies were diluted using 0.5% bsa in pbs-0.1% tween. appropriate negative controls were included in every step. neutralization assays. a pseudovirus-based neutralization assay was used to determine the neutralization ability of immunized sera to pseudovirus hiv/bj01-s, and hiv/cs 318-510 . the neutralizing activity of heat-inactivated sera (56°c, 30 min) was determined by mixing 10 ng of pseudovirus (in 30 ll) with diluted antisera (in 30 ll) at 37°c for 1 h. sera-pseudovirus complexes were then mixed with 16 ng polybrene (in 40 ll medium) before they were added to human ace2 expressing hela cells. the infected cells were washed with pbs and lysed (cell culture lysis reagent; promega) at 48 h post infection. the neutralization activity of each antiserum was monitored by measurement of luciferase activity. after the final boost, end-point dilution elisa was used to detect the antibody response against bj01 or rp3-s protein in immunized mice (fig. 2) . other than the negative control, all immunized mice produced antibodies against hiv/rp3-s or hiv/bj01-s pseudovirus, albeit displaying different reactivity against the two pseudovirus antigens. when tested against hiv/rp3-s pseudovirus, antisera generated to the full-length rp3-s had a significantly higher titer than those from the other three groups (fig. 2a) . also, the titers of sera from the two chimeric plasmids cs 310-518 and cs 259-518 were higher than that from the bj01-s construct. on the other hand, when tested against hiv/bj01-s pseudovirus, the antibody titer was lowest for the group immunized with plasmid expressing the full-length rp3-s protein (fig. 2b) . there was little difference in titer among the other three groups. all mouse sera generated using dna immunization were tested for their ability to neutralize the hiv/bj01-s or hiv/cs 318-510 pseudovirus. as shown in fig. 3a , mouse sera from bj01-s, cs 310-518 or cs 259-518 showed strong neutralizing activity to the pseudovirus hiv/bj01-s, whereas the antiserum from rp3-s showed a much weaker neutralizing activity to the same pseudovirus. interestingly, all the sera, except the vector control group, had neutralizing activity to hiv/cs 318-510 (fig. 3b) . however, the rp3-s serum showed neutralizing activity to hiv/cs 318-510 only at high concentrations, indicating the presence of some cross-neutralization epitopes located outside the bj01-s rbd. similar to the results obtained above using mice sera, bat sera naturally infected by sl-cov had a much stronger reactivity towards hiv/rp3-s than the other three pseudoviruses (fig. 4) . knowledge of immunogenicity and immunodominant regions of major viral antigens is important for rational design of effective vaccines and diagnostic tests. sl-covs found in bats are very similar to human isolates of sars-cov in that they have almost identical genomic organization and their gene products share a high level of amino acid sequence identity. we have shown previously that despite substantial antigenic cross-reactivity between bat sera from naturally infected animals and sars-cov, there was no crossneutralization detected [1] . this discrepancy could be explained by the low level of sequence identity shared at the n-terminal domain of the s proteins between the two classes of viruses [1, 2] . the n-terminal region of coronavirus s proteins is known to be responsible for virus attachment to susceptible host cells, hence a major target of neutralizing antibodies [4] . in this study, dna constructs expressing four different s proteins, sars-cov bj01, sl-cov rp3 s and two chimeras, were used to generate hyperimmune sera in mice via dna immunization. the immunogenicity of these proteins was determined using elisa against the s sl and s sars proteins expressed in the form of hiv pseudoviruses. the neutralization activity of the mouse sera was further determined using the hiv/bj01-s and hiv/cs 318-510 pseudovirus. the results obtained clearly demonstrated that mice immunized with bj01-s, cs 310-518 or cs 259-518 generated antibodies with higher antigenic and neutralizing activity to hiv/bj01-s and hiv/cs 318-510 than hiv/rp3-s. vice versa, mice immunized with rp3-s displayed higher reactivity to hiv/rp3-s than hiv/ bj01-s. these results demonstrated that sl-cov and sars-cov shared only a limited number of immunogenic epitopes in their s proteins and the major neutralization epitopes are substantially different among these two viruses. since the rbd for the s sl protein is not known, we were not able to conclude whether the immunodominant regions overlap with the receptor attachment site. for the same reason, we have been unable to identify a suitable susceptible cell line to conduct neutralization tests for the hiv/rp3-s pseudovirus. until such an assay is available, it will be impossible to conduct a detailed comparative study of major neutralization epitopes between the two different viruses. the study conducted with bat sera and four different pseudoviruses provided indirect evidence suggesting a substantial overlap of immunodominant and neutralizing epitopes for the s sl protein. this was best demonstrated by the reactivity of bat sera against pseudovirus hiv/rp3-s and hiv/cs 310-518 , respectively. the replacement of the sequence from aa 310-518 alone almost completely abolished the bat antibody reactivity towards the s sl protein. a variety of sl-cov and other covs found in bats demonstrate that bats are natural reservoir of diverse covs. the high density of bats in habitats provides ample opportunities for recombination, which will in turn increase virus diversity and the chance of spill over into other hosts including humans leading to zoonotic disease outbreaks [3, 9] . recently, it was shown that a synthetic sl-cov containing a very small fragment of the sars-cov s gene was able to infect and cause disease in mice, further highlighting the potential emergence of novel viruses with subtle sequence difference in the s gene [10] . the results obtained from this work would suggest that the current diagnostic tools and candidate vaccines developed for sars-cov are not likely to be specific or effective enough to combat a disease outbreak caused by a sl-cov variant. it is therefore necessary that we continue strategic research in this area to be able to rapidly response to disease outbreaks caused by coronaviruses with similar genetic and pathogenic features, but with different s gene sequences and receptor specificities. ) and one negative bat sera (neg) was determined against sars-cov in a previous study [1] . data are presented as means ± sds. bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine immunogenicity of sars-cov: the receptor-binding domain of s protein is a major target of neutralizing antibodies identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy gene transfer into muscle by electroporation in vivo evidence of the recombinant origin of a bat severe acute respiratory syndrome (sars)-like coronavirus and its implications on the direct ancestor of sars coronavirus synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice we thank mrs. xuefang an of wuhan institute of virology, chinese academy of sciences, china, for technical help with mice immunization. this work was jointly funded by the state key program for basic research grant (2005cb523004) from the chinese ministry of science and technology, the knowledge innovation program key project administered by the chinese academy of sciences (kscx1-yw-r-07). key: cord-260695-qwepi0we authors: postler, thomas s.; pantry, shara n.; desrosiers, ronald c.; ghosh, sankar title: identification and characterization of a long non-coding rna up-regulated during hiv-1 infection date: 2017-11-01 journal: virology doi: 10.1016/j.virol.2017.08.006 sha: doc_id: 260695 cord_uid: qwepi0we long non-coding rnas (lncrnas) are rapidly emerging as important regulators of a diverse array of cellular functions. here, we describe a meta-analysis of two independent rna-seq studies to identify lncrnas that are differentially expressed upon hiv-1 infection. only three lncrna genes exhibited altered expression of ≥2-fold in hiv-1-infected cells. of these, the uncharacterized lncrna linc00173 was chosen for further study. both transcript variants of linc00173 (lnc173 tsv1 and 2) could be detected by qpcr, localized predominantly to the nucleus and were reproducibly up-regulated during infection. knock-out of the linc00173 locus did not have detectable effects on hiv-1 replication. interestingly, however, stimulation of jurkat t cells with pma/ionomycin resulted in a decrease of lnc173 expression, and jurkat cells deficient for lnc173 on average expressed higher levels of specific cytokines than control cells. these data suggest that lnc173 may have a role in the regulation of cytokines in t cells. long non-coding rnas (lncrnas) are defined as rna transcripts that are not translated and are at least 200 nucleotides long. the current gencode release (version 26) has identified 15,787 lncrna genes in the human genome, encoding 27,720 different transcripts, excluding transcribed pseudogenes (https://www.gencodegenes.org/ stats/current.html) . only a few hundred of these lncrnas have been functionally characterized to date, but it has become evident that this class of biomolecules includes central regulators of varied biological processes, such as the regulation of cellular proliferation, immunity, development and even nuclear organization (atianand et al., 2017; chaudhary and lal, 2016; goff and rinn, 2015; perry and ulitsky, 2016; pircher et al., 2014) . based on their genomic context, lncrnas can be grouped into four simple classes: antisense rnas, which are transcribed in the opposite direction of overlapping protein-coding genes; large intergenic noncoding rnas (lincrnas), which are not flanked by other genes in close proximity; intronic lncrnas, which are encoded within an intron of a protein-coding gene; and overlapping lncrnas, which contain a protein-coding gene within a lncrna intron (atianand et al., 2017; spurlock et al., 2016) . more complex classification schemes have also been proposed (chen, 2016) . most lncrnas are thought to be capped, polyadenylated, and spliced using the same cellular machinery as protein-coding transcripts, although distinct differences have been noted (mukherjee et al., 2017; schlackow et al., 2016; ulitsky and bartel, 2013) . depending on their mechanism of action, lncrnas are located in the cytoplasm or in the nucleus. while cytoplasmic lncrnas tend to function as scaffolds for protein complexes or as microrna "sponges", nuclear lncrnas often aid or interfere with transcription in cis or in trans . for instance, nuclear lncrnas have been reported to bind protein regulators of transcription and either guide them to their intended target site or act as a decoy to sequester them (atianand et al., 2017; goff and rinn, 2015) . recent studies have only begun to elucidate the role of lncrnas during viral infections. several viruses have been shown to encode lncrnas in their genome, including both dna and rna viruses (tycowski et al., 2015) . multiple groups have reported that the genome of hiv-1 is transcribed in the antisense direction to yield various lncrna species (kobayashi-ishihara et al., 2012; landry et al., 2007; ludwig et al., 2006; saayman et al., 2014) . while the length of the identified transcripts differed between studies, there is evidence to suggest that at least some of these antisense rnas may interfere with viral transcription by establishing repressive histone methylation at the 5′ ltr (kobayashi-ishihara et al., 2012; saayman et al., 2014; zapata et al., 2017) . beyond virally encoded lncrnas, the expression levels of lncrnas encoded by the host have been shown to be altered upon infection with a variety of viral pathogens (fortes and morris, 2015) . specific examples include influenza a virus (iav), hepatitis c virus, severe acute respiratory syndrome-related coronavirus, adenovirus, herpes simplex virus 1 (hsv-1), human cytomegalovirus, and hiv-1 (carnero et al., 2016; chang et al., 2011; hu et al., 2016; ouyang et al., 2015; peng et al., 2010 peng et al., , 2014 trypsteen et al., 2016; winterling et al., 2014; zhang et al., 2016; zhao et al., 2016) . of the host-encoded lncrnas reported to be differentially expressed upon hiv-1 infection, only nuclear enriched abundant transcript 1 (neat1) and noncoding repressor of nuclear factor of activated t cells (nron) have been functionally characterized (lazar et al., 2016) . neat1 is a nuclear lncrna which stabilizes paraspeckles and is believed thereby to sequester unspliced and singly spliced hiv-1 transcripts (clemson et al., 2009; sasaki et al., 2009; west et al., 2014; zhang et al., 2013; zolotukhin et al., 2003) . of note, neat1 expression has also been shown to enhance the expression of antiviral genes during infection with iav and hsv-1, as well as after poly(i:c) treatment (imamura et al., 2014) . nron, by contrast, is a cytoplasmic lncrna that forms part of a high-molecular-weight complex including nuclear factor of activated t cells (nfat), a transcription factor of central importance to t-cell activation. nron stabilizes the inactive form of nfat, and correspondingly loss of nron results in the hyperactivation of t cells (macian, 2005; sharma et al., 2011; willingham et al., 2005) . a complex interplay of hiv-1 and nron has been described, where nron levels are decreased early during infection by the protein nef and increased by the late gene product vpu (imam et al., 2015) . recently, li et al. (2016) demonstrated that nron specifically induces the degradation of tat and thus contributes to hiv-1 latency. other lncrnas that have been documented to be up-regulated in hiv-1infected cells, such as malat1 and miat, have not been functionally analyzed in the context of infection. most prior reports specifically investigating the effect of hiv-1 on lncrna expression have focused on a small panel of previously characterized lncrnas rather than employing a genome-wide screen. furthermore, there was only limited overlap between the results of these studies, likely reflecting different experimental conditions (imam et al., 2015; zhang et al., 2013) . in this report, we describe a meta-analysis of two independent rna-seq studies of hiv-1-infected cells and show that, unexpectedly, only three lncrnas are differentially expressed in both of these datasets (chang et al., 2011; mohammadi et al., 2013) . of those three candidates, linc00173 exhibits the highest degree of evolutionary conservation. we demonstrate that both transcript variants of linc00173 are indeed detectable in a variety of human cell lines, are located predominantly in the nucleus, and are up-regulated upon infection with hiv-1. while, somewhat surprisingly, loss of linc00173 did not affect the viral replication cycle directly in cell culture, jurkat t cells deficient for linc00173 on average expressed higher levels of a subset of cytokines than control cells, while other cytokines remained unaffected. these data indicate a possible involvement of linc00173 in the regulation of cytokine expression. after filtering out transcripts representing snornas, mirnas and non-coding variants of protein-coding genes, the remaining transcripts were categorized as large intergenic non-coding rnas (lincs), uncharacterized long non-coding rnas (lncrnas), antisense transcripts, pseudogenes, and others (transcripts that do not fall in any of the above categories). the pie chart represents total numbers of transcripts in each category. t.s. postler et al. virology 511 (2017) 30-39 2. results to obtain an unbiased understanding of any changes in lncrna expression during hiv-1 infection, we performed a meta-analysis of two independent rna-seq studies conducted by two different groups using two different virus strains ( supplementary fig. s1 ) (chang et al., 2011; mohammadi et al., 2013) . chang et al. (2011) infected sup-t1 cells with the fully replication-competent hiv-1 isolate lai and performed rna-seq with samples harvested at 12 and 24 h p.i.. using this dataset, we identified non-coding transcripts based on their genbank classification. transcripts with a fold change of ≥ 2 were considered differentially expressed. twelve hours after infection, 21 non-coding transcripts were down-regulated, 3 were up-regulated ( fig. 1a) . at 24 h, 51 transcripts were down-regulated, while 71 had increased expression levels (fig. 1b) . unexpectedly, only 10 transcripts were differentially expressed at both time points (fig. 1c ). next, we filtered out snornas, mirnas, and non-coding transcript variants of protein-coding genes. the remaining transcripts comprised 8 large intergenic non-coding rnas (lincs), 9 uncharacterized lncrnas, 9 antisense transcripts, 16 pseudogenes and 6 other transcripts, such as a mirna polycistron (fig. 1d ). we further filtered these results using the findings of mohammadi et al. (2013) , who infected sup-t1 cells with vsv-g-pseudotyped hiv-1 nl4-3 δenv/egfp and performed rnaseq with samples harvested every 2 h for 24 h. the processed rna-seq data from that study have been made available via the dedicated online platform patterns of expression and analysis of clusters of hiv/host interactions (peachi, http://www.peachi.labtelenti.org). after excluding pseudogenes and the transcripts in the "other" category, we queried the remaining 26 lncrna transcripts from our analysis of the study by chang et al. in peachi. of these, only four were also identified as differentially expressed in peachi (11 were not differentially expressed and 11 were not catalogued). two of those 4 transcripts were variants of one gene, linc00173, identified by genbank as a large intergenic non-coding rna. the remaining 2 transcripts were both antisense rnas, ccdc18-as1 and mcm3ap-as1 (table 1) . we chose to further investigate linc00173 as both of its transcript variants were differentially expressed after hiv-1 infection in the rnaseq studies and it showed the highest degree of conservation across vertebrate species among the 3 candidate genes (table 1) . transcript variant 1 (lnc173 tsv1) is encoded by 2 exons and 1597 nt long, transcript variant 2 (lnc173 tsv2) is encoded by 3 exons and reaches a length of 435 nt. the central section of tsv1 is particularly well conserved across species, with a substantially similar region present even in the chicken genome ( fig. 2a ). rna-seq data from gm78 and k562 cells available in the ucsc genome browser indicate robust transcription and splicing ( fig. 2a ) (kent et al., 2002) . recorded histone modifications in multiple cell types are also consistent with expression, including histone h3k27 acetylation and h3k4 methylation marks near the transcriptional start site ( supplementary fig. s2 ). dnase i hypersensitivity levels suggest an open chromatin conformation at this locus ( supplementary fig. s2 ). all of these data point to active transcription of linc00173. the predicted secondary structures of lnc173 tsv1 and tsv2 are drastically different, reflecting the minimal sequence overlap restricted to the first exon ( fig. 2b , c). 2.3. both transcript variants of lnc173 are detectable in several human cell lines, are polyadenylated and localize predominantly to the nucleus to validate the results of the in silico analysis of lnc173 expression, we isolated rna from 4 human cell lines and probed for the presence of both transcript variants by quantitative rt-pcr (qrt-pcr; fig. 3a -d). tsv1 and tsv2 were readily detectable in jurkat t cells after reverse transcription with random-hexamer or oligo(dt) primers, demonstrating that both transcript variants are expressed and polyadenylated ( fig. 3a ). control samples without reverse transcriptase yielded no signal, confirming specificity of the assay for rna rather than genomic dna. similarly, lnc173 tsv1 and tsv2 were present in the monocyte-derived cell lines u-937 and thp-1, as well as in the renal epithelial cell line hek 293t ( fig. 3b -d). both transcript variants were highly enriched in the nuclear fraction of unstimulated jurkat cells, identifying lnc173 as a nuclear lncrna (fig. 4) . to confirm the rna-seq data by chang et al. and mohammadi et al., we infected hek 293t cells with serial 4-fold dilutions of vsv-gpseudotyped hiv-1 nl4-3 δenv/egfp and performed qrt-pcr with samples harvested 24 h p.i. (zhang et al., 2004) . both lnc173 transcript variants were indeed up-regulated in a dose-dependent manner compared to uninfected control cells. the lncrna malat1, which has also been reported to be induced upon hiv-1 infection, showed a dose-dependent increase of similar magnitude . stat1 mrna levels remained unchanged (fig. 5a ). infection of the monocytic thp-1 cell line mirrored the results of hek 293t infection in magnitude and dose dependence (fig. 5b ). infection of jurkat cells resulted in a reproducible but less pronounced increase in lnc173 expression (fig. 5c) . this was expected, as the efficiency of infection for this cell type ranged only from 50% to 70%, whereas the qrt-pcr table 1 lncrna transcripts with differential expression (fold change ≥ 2) in the rna-seq data sets of both chang et al. (2011) and mohammadi et al. (2013) . refseq id transcript length (nt) analysis reflected the average mrna levels of both uninfected and infected cells (cf. supplementary fig. s3c ). infection efficiency for hek 293t cells, by contrast, reached nearly 100% (cf. supplementary fig. s4b ). to determine the temporal kinetics of lnc173 expression during viral replication, we infected hek 293t cells in a 48-h time course and harvested samples every 12 h. levels of lnc173 tsv1 and tsv2 were increased compared to time-matched controls at 12 h and 24 h p.i., followed by a decline towards control levels. expression levels of malat1, by contrast, continued to increase throughout the entire time course (fig. 5d ). exposure to heat-inactivated viral particles did not result in an increase of lnc173 expression (fig. 5e ). taken together, these data establish that hiv-1 infection does induce an increase in the cellular levels of lnc173 tsv1 and tsv2 in a time-and dose-dependent manner and in multiple cell lines representing several cell types. to determine whether either transcript variant of lnc173 is involved in an important part of the hiv-1 life cycle, we used crispr-cas9mediated deletion to remove the entire linc00173 gene in jurkat cells. specifically, we targeted both ends of the gene with separate guide rnas simultaneously, derived monoclonal lines from the population and identified clones with no remaining copies of linc00173 by pcr. two separate combinations of guide rnas were used, set 4 and set 12 (supplementary table s1 ). a total of 6 lnc173 knock-out (ko) clones was obtained, 2 with set 4 and 4 with set 12. additionally, 4 control clones were derived from jurkat cells transfected with an sgrna-free cas9 vector. the absence of lnc173 transcripts was confirmed by qrt-pcr (fig. 6a , b and supplementary fig. s3a, b) . the residual signal detected with lnc173 ko clone 12-17 was close to the limit of detection but may reflect incomplete deletion of the gene. we then proceeded to systematically probe all parts of the hiv-1 replication cycle. to assess the post-entry steps of replication up to viral gene expression, we infected lnc173 ko cells or controls with vsv-g-pseudotyped hiv-1 nl4-3 δenv/egfp. this construct carries the gene encoding egfp in the open reading frame of the env gene and thus serves as a reliable reporter for the efficiency of reverse transcription, nuclear import, integration and expression of viral gene products (zhang et al., 2004) . a defect at any of these steps would result in a lower number of gfp+ cells and/or a lower level of gfp expression overall, and enhancement would result in the opposite phenotype. as expected with a collection of monoclonal lines derived from a heterogeneous parent population, the percentage of gfp+ cells and the intensity of gfp expression varied considerably between the clones ( supplementary fig. s3c, d) . however, the average values obtained for the 6 lnc173 ko clones and for the 4 control clones were remarkably similar and gave no indication of a defect or enhancement (fig. 6c, d) . analogously, we created two monoclonal hek 293t-derived cell lines with complete deletion of linc00173 (supplementary fig. s4a ). when these lines were infected with vsv-g-pseudotyped hiv-1 nl4-3 δenv/egfp, the percentage of gfp+ cells and gfp levels were also indistinguishable from two control lines ( supplementary fig. s4b, c ). next, we tested whether viral infectivity or particle release might be affected by the absence of lnc173. we produced fully infectious hiv-1 nl4-3 particles in the hek 293t lnc173 ko clone 11-2b3 and compared yield and infectivity to virus produced in hek 293t control clone 1b5. stocks produced from both cell lines yielded similar concentrations of viral particles, demonstrating that virion production and release were unaffected by deletion of linc00173 (supplementary fig. s5a ). when virus from these stocks, normalized for p24 content, was used to infect the tzm-bl reporter cell line to determine infectivity, no difference was detectable (fig. 6e) (kent et al., 2002) . (b-c) prediction for secondary structure of (b) lnc173 transcript variant 1 (tsv1) and (c) lnc173 tsv2. data obtained from lncipedia (www.lncipedia.org) (volders et al., 2015 (volders et al., , 2013 . t.s. postler et al. virology 511 (2017) 30-39 1998). to understand whether the presence of lnc173 influences viral growth over multiple rounds of replication, we infected 4 jurkat lnc173 ko clones and 4 control clones with fully replication-competent hiv-1 nl4-3 and tracked viral replication over a period of three weeks. while results varied between the clones of each group, no clear defect or enhancement was associated with the deletion of linc00173 (fig. 6f) . infection of the same clones with virus produced in lnc173-deficient hek 293t cells yielded a highly similar growth curve ( supplementary fig. s5b ). these results demonstrate that both transcript variants of lnc173 are dispensable for replication of hiv-1 in cell culture. hiv-1 replicates efficiently only in activated cells, and consequently has evolved multiple redundant mechanisms to increase the state of cellular activation in its host cell (alexander et al., 1997; fenard et al., 2005; mcdougal et al., 1985; postler and desrosiers, 2012; simmons et al., 2001; stevenson et al., 1990; zack et al., 1990; zagury et al., 1986) . we therefore investigated whether the increase in lnc173 levels was associated with general t-cell activation. surprisingly, stimulation of jurkat t cells with pma and ionomycin resulted in a marked decrease in the levels of lnc173 tsv1 and tsv2 (fig. 7a) . similarly, rna levels of a previously unknown, likely lnc173 ortholog in the mouse genome decreased upon stimulation with pma and ionomycin in el4 cells, indicating at least some degree of evolutionary conservation of this mechanism (supplementary fig. s6) . interestingly, the lnc173 ko jurkat clones on average expressed higher levels of mrna encoding a subset of cytokines than the cognate control lines. specifically, average mrna levels of ifng, ccl3 and cxcl8 upon stimulation with pma and ionomycin were significantly higher in lnc173 ko clones than in control clones, whereas mrna levels of il2 and tnf did not differ ( fig. 7b and supplementary fig. s7a-e) . due to the central role of ifn-γ in the differentiation of naive t cells into t h 1 cells during the immune response to viral and other intracellular infections, we tested whether the observed increase in ifng mrna translated into an increase in ifn-γ protein levels secreted by lnc173 ko clones (schoenborn and wilson, 2007) . indeed, the average concentration of ifn-γ in the supernatant of lnc173 ko jurkat clones was modestly but reproducibly elevated compared to the average of control clones, consistent with the mrna results ( fig. 7c and supplementary fig. s7f ). although there was considerable variability between the clones of each group, these results raise the intriguing possibility that lnc173 may be involved in the transcriptional regulation of a subset of cytokines. further studies are under way to address this question in detail. of the thousands of lncrnas expressed in the human body, the vast majority remains uncharacterized, in particular in the context of viral infections. until the recently published microarray study by trypsteen et al., all reports investigating the effect of hiv-1 infection on lncrna expression scrutinized only a small panel of previously characterized lncrnas and thus, while yielding important insights, were limited in scope (imam et al., 2015; trypsteen et al., 2016; zhang et al., 2013) . to our knowledge, this is the first study using rna-seq data to address specifically this aspect of the hiv-1 life cycle, although others have touched on the subject (peng et al., 2014) . to minimize the effect of interexperimental variability, we utilized two separate data sets created by two independent groups and employing two different strains of hiv-1 (chang et al., 2011; mohammadi et al., 2013) . the finding that only three lncrnas exhibited differential expression in both studies is somewhat surprising, especially considering that trypsteen et al. (2016) found 328 lncrna-coding genes to be altered (including those identified in this study, namely linc00173, ccdc18-as1 and mcm3ap-as1). the reason for this discrepancy is not clear, but it may stem from the inherent differences between rna-seq and microarray technology. individual lncrna expression levels tend to be lower than mrna levels, and while microarrays afford a lower dynamic range than rna-seq, they provide higher accuracy of quantification for lowabundance transcripts jiang et al., 2011; łabaj et al., 2011; ravasi et al., 2006; toung et al., 2011; xu et al., 2011) . additionally, using two independent data sets is likely to reduce the number of false positives but increase the number of false negatives. linc00173, a previously uncharacterized lncrna, is conserved amongst mammals and at least a partial homolog appears to be present in chickens. as cross-species conservation is often an indicator of functional importance, we chose to further investigate the role of linc00173 during hiv-1 infection. the experimental results presented here clearly demonstrate that its two transcript variants are robustly expressed in various human cell types, in accordance with the available in silico data. consistent with the data mined from the studies by chang et al. (2011) , mohammadi et al. (2013) , and trypsteen et al. (2016) , lnc173 tsv1 and tsv2 are up-regulated during the course of hiv-1 infection. this effect is dose-and, more importantly, timedependent. the observation that lnc173 tsv1 and tsv2 levels increase within the first 12 h after infection, remain increased at 24 h, but then decrease progressively towards baseline at 36 and 48 h, indicates targeted control of expression. nonetheless, loss of the linc00173 locus does not affect any aspect of the hiv-1 replication cycle in cell culture, from entry to particle production. based on their nuclear localization, lnc173 tsv1 and/or tsv2 are likely involved in transcriptional regulation, but their precise role in this process, if any, remains unclear. it is conceivable that lnc173 may contribute to the regulation of viral transcription only under specific conditions, such as entering or exiting viral latencyan intriguing possibility that remains to be explored. t.s. postler et al. virology 511 (2017) 30-39 it is also possible that lnc173 affects the expression or function of proteins that are not directly involved in viral replication on a cellular level but are important on an organismal level, such as cytokines that coordinate the immune response. activation of t cells by exposure to pma and ionomycin, which increases the expression of several cytokines, decreases the levels of both lnc173 transcript variants, opening up the possibility that lnc173 tsv1 and/or tsv2 may be negative regulators of cytokine expression. indeed, upon activation the monoclonal jurkat cell lines deficient for the linc00173 locus on average produced higher mrna levels of several cytokines than cognate controls, lending preliminary support to this hypothesis. ongoing experiments outside the scope of this study will further explore the role of linc00173 in cytokine regulation. in this context, it is also interesting to note that the expression levels of both lnc173 transcript variants are increased during hiv-1 infection but decreased during t-cell activation, even though hiv-1 has evolved several independent mechanisms to induce the activation of host cells (alexander et al., 1997; fenard et al., 2005; postler and desrosiers, 2012; simmons et al., 2001) . this unexpected antagonistic phenotype may imply a specific function of lnc173 to be exploited or blunted by hiv-1. of note, the cytokines exhibiting increased expression in the lnc173 ko jurkat clones include ifn-γ, a cytokine of central importance to the development and maintenance of the adaptive immune response to intracellular pathogens, including viruses (schoenborn and wilson, 2007) . it is therefore tempting to speculate that hiv-1 has evolved the ability to increase levels of lnc173 to impede the immune response. (barrett et al., 2013; chang et al., 2011; edgar et al., 2002) . gene information was analyzed and filtered by individual evaluation of associated refseq identifiers and records. all lncrna candidates identified in this data set were queried in the online platform created for the rna-seq data by mohammadi et al. (2013) , patterns of expression and analysis of clusters of hiv/host interactions (peachi, http://www.peachi.labtelenti.org). to that end, refseq identifiers of candidate genes were matched to corresponding ensembl identifiers (ensembl release 75, www.ensembl.org), which could be entered into peachi (yates et al., 2016) . phylogenetic conservation, expression data, chip-seq data and dnase hypersensitivity data were obtained from publicly accessible tracks on the ucsc genome browser (grch38, hg38; http://genome.ucsc.edu) (kent et al., 2002) . phylocsf score, number of bazzini small orfs and secondary structure prediction were obtained from lncipedia (www. lncipedia.org) (volders et al., 2013 (volders et al., , 2015 . jurkat cells (a kind gift from dr. stephen goff, columbia university), el4 cells, u937 cells and thp-1 cells (both atcc, manassas, va, usa) were maintained in rpmi-1640 medium (gibco/thermo fisher scientific, waltham, ma, usa) supplemented with 10% fetal bovine serum (fbs; atlanta biologicals, flowery branch, ga, usa). hek293t/17 (atcc) and tzm-bl cells (obtained through the nih aids reagent program, division of aids, niaid, nih) were maintained in dmem (gibco/thermo fisher scientific) with 10% fbs (derdeyn et al., 2000; platt et al., 1998) . the plasmids encoding hiv-1 nl4-3 δenv/egfp (pnl4-3-deltae-egfp) and vsv-g (pvsv-g) also were generous gifts from dr. stephen goff (haili zhang et al., 2004) . to produce pseudotyped virions, hek 293t/17 cells were co-transfected with pnl4-3-deltae-egfp and pvsv-g at a mass ratio of 3:1 using fugene 6 (promega, madison, wi, usa). transfection reagent was washed out after 6 h, and virion-containing supernatant was harvested 48 h after transfection. fully replication-competent hiv-1 nl4-3 was produced from plasmid pnl4-3, also obtained through the nih aids reagent program (genbank id af324493) (adachi et al., 1986) . nl4-3 stocks were produced from two monoclonal derivates of hek293t/17, linc00173 knock-out clone 11-2b3 and wild-type control clone 1b5. these cells were transfected using jetprime (polyplus transfection, illkirch, france) and supernatant was harvested after 72 h. the concentration of p24 in fully replication-competent virus stocks was determined by p24 antigen capture assay (advanced bioscience laboratories, rockville, md, usa). to isolate nuclear rna, 10 7 jurkat cells were resuspended in 1 ml phosphate-buffered saline (pbs), 1 ml of lysis buffer c1 (1.28 m sucrose, 40 mm tris-hcl ph 7.5, 20 mm mgcl 2 , 4% triton x-100) and 3 ml ddh 2 o. after incubation on ice for 15 min, cells were centrifuged at 4°c and 580×g for 15 min. supernatant containing the cytoplasmic fraction was discarded. the pellet containing the nuclear fraction was resuspended in 350 µl buffer rlt + β-mercaptoethanol (qiagen, germantown, md, usa). in parallel, 10 7 jurkat cells were resuspended directly in 350 µl buffer rlt + β-mercaptoethanol to obtain whole-cell rna. rna was isolated from both sample sets using the rneasy mini kit (qiagen), following the manufacturer's instructions. this included an on-column dnase i digest during purification. cells were lysed directly in buffer rlt + β-mercaptoethanol and rna was isolated using the rneasy mini kit (qiagen), according to the manufacturer's instructions, with on-column dnase i digest. reverse transcription of usually 1 µg of purified rna into cdna was performed with superscript iii reverse transcriptase and oligo(dt) 12-18 primers, unless noted otherwise. veriquest fast sybr green qpcr master mix was used for qrt-pcr (all thermo fisher scientific). relative cdna levels were determined using the δδc t method (livak and schmittgen, 2001) . see supplementary table s2 for primer sequences. in hek 293t cells, the linc00173 locus was deleted using plasmid pspcas9(bb)-2a-puro (px459), provided by dr. feng zhang (massachusetts institute of technology) through addgene (plasmid error bars indicate standard deviation. (b) mrna levels of the cytokine-encoding genes ifng, ccl3, cxcl8, il2 and tnf after stimulation with 25 ng/ml pma and 500 ng/ml ionomycin for 4 h in lnc173 knock-out and control jurkat cells, as quantified by qrt-pcr. rplp0 was used as reference gene. shown is the average of 6 monoclonal knock-out lines (lnc173 ko) and 4 monoclonal vector control lines, including every data point from 5 to 8 independent experiments. error bars indicate sem. (c) concentration of ifn-γ in the supernatant of lnc173 knock-out and control jurkat cells after stimulation with 25 ng/ml pma and 500 ng/ml ionomycin for 24 h, as determined by elisa. shown is the average of 6 monoclonal knock-out lines (lnc173 ko) and 4 monoclonal vector control lines, including every data point from 5 independent experiments. error bars indicate sem. p-values were determined with welch's t-test. ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05; n.s., not significant. t.s. postler et al. virology 511 (2017) 30-39 #48139) (ran et al., 2013) . specifically, cells were transfected with pairs of plasmids targeting each end of the transcribed sequence, using lipofectamine 2000 (thermo fisher scientific). supplementary table s1 lists the sgrna sequences and combinations used. cells transfected with px459 without an sgrna sequence served as control. successfully transfected cells were selected with 4 µg/ml puromycin for three days. surviving cells were separated into monoclonal cultures by limiting dilution, which were then screened for successful deletion of the linc00173 locus with the primers listed in supplementary table s3 . for deletion of linc00173 in jurkat cells, the same sgrna sequences were introduced into plasmid pspcas9(bb)-2a-gfp (px458), also provided by dr. feng zhang through addgene (plasmid #48138) (ran et al., 2013) . jurkat cells were transfected with the same pairs of plasmids detailed in supplementary table s1 using an amaxa nucleofector ii device and the cell line nucleofector kit v (both from lonza, basel, switzerland). after 48 h, cells with high levels of gfp expression were identified by facs and sorted into monoclonal cultures. screening for successful deletion of linc00173 was performed as described above. hek 293t cells were infected by incubation with vsv-g-pseudotyped hiv-1 nl4-3 δenv/egfp for 4 h at 37°c, while thp-1 and jurkat cells were usually infected by spin inoculation for 2 h at 34°c and 1000×g. polybrene was added at a concentration of 6 µg/ml to all pseudotype infections. after incubation or spin inoculation, supernatant was replaced with fresh medium. unless otherwise noted, cells were harvested 24 h after infection for rna isolation as described above, or 48 h after infection for flow cytometry. the infectivity of virus produced in lnc173-deficient cells or control cells was quantified by infection of tzm-bl reporter cells, essentially as previously described (bixby et al., 2010) . cells were infected in 2-fold serial dilutions with virus amounts normalized for p24 content, each in triplicate. luciferase activity was quantified 48 h after infection using the britelite plus reporter gene assay system (perkinelmer, waltham, ma, usa). monoclonal jurkat cells deficient for lnc173 or controls were infected with virus amounts equivalent to 25 ng p24. supernatant from the infected cells was harvested every 2-4 days and the concentration of p24 was determined by p24 antigen capture assay (advanced bioscience laboratories). to determine the concentration of ifn-γ secreted by lnc173 ko and control jurkat clones, cells were stimulated with 25 ng/ml pma and 500 ng/ml ionomycin for 24 h. clarified supernatant was harvested and ifn-γ concentration was quantified using the ifn gamma human uncoated elisa kit with plates (thermo fisher scientific). production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone a role for natural simian immunodeficiency virus and human immunodeficiency virus type 1 nef alleles in lymphocyte activation immunobiology of long noncoding rnas ncbi geo: archive for functional genomics data sets-update diversity of envelope genes from an uncloned stock of sivmac251 long noncoding rna egot negatively affects the antiviral response and favors hcv replication next-generation sequencing reveals hiv-1-mediated suppression of t cell activation and rna processing and regulation of noncoding rna expression in a cd4+ t cell line long noncoding rnas in the p53 network linking long noncoding rna localization and function an architectural role for a nuclear noncoding rna: neat1 rna is essential for the structure of paraspeckles sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor t-20 is modulated by coreceptor specificity defined by the v3 loop of gp120 the gencode v7 catalog of human long noncoding rnas: analysis of their gene structure, evolution, and expression gene expression omnibus: ncbi gene expression and hybridization array data repository nef is physically recruited into the immunological synapse and potentiates t cell activation early after tcr engagement long noncoding rnas in viral infections linking rna biology to lncrnas gencode: the reference human genome annotation for the encode project functional prediction of differentially expressed lncrnas in hsv-1 infected human foreskin fibroblasts the lncrna nron modulates hiv-1 replication in a nfat-dependent manner and is differentially regulated by early and late viral proteins long noncoding rna neat1-dependent sfpq relocation from promoter region to paraspeckle mediates il8 expression upon immune stimuli synthetic spike-in standards for rna-seq experiments the human genome browser at ucsc hiv-1-encoded antisense rna suppresses viral replication for a prolonged period characterization and improvement of rna-seq precision in quantitative transcript expression profiling detection, characterization and regulation of antisense transcripts in hiv-1 the emerging role of long non-coding rnas in hiv infection long noncoding rna nron contributes to hiv-1 latency by specifically inducing tat protein degradation analysis of relative gene expression data using realtime quantitative pcr and the 2(-delta delta c(t)) method human immunodeficiency virus-type 1 ltr dna contains an intrinsic gene producing antisense rna and protein products nfat proteins: key regulators of t-cell development and function cellular tropism of the human retrovirus htlv-iii/ lav. i. role of t cell activation and expression of the t4 antigen 24 h in the life of hiv-1 in a t cell line integrative classification of human coding and noncoding genes through rna metabolism profiles lncrnas regulate the innate immune response to viral infection unique signatures of long noncoding rna expression in response to virus infection and altered innate immune signaling deep sequencing of hiv infected cells: insights into nascent transcription and host-directed therapy the functions of long noncoding rnas in development and stem cells ribosome-associated ncrnas: an emerging class of translation regulators effects of ccr5 and cd4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1 the cytoplasmic domain of the hiv-1 glycoprotein gp41 induces nf-κb activation through tgf-β-activated kinase 1 genome engineering using the crispr-cas9 system experimental validation of the regulated expression of large numbers of non-coding rnas from the mouse genome an hiv-encoded antisense long noncoding rna epigenetically regulates viral transcription menepsilon/beta noncoding rnas are essential for structural integrity of nuclear paraspeckles distinctive patterns of transcription and rna processing for human lincrnas regulation of interferon-gamma during innate and adaptive immune responses dephosphorylation of the nuclear factor of activated t cells (nfat) transcription factor is regulated by an rna-protein scaffold complex nef triggers a transcriptional program in t cells imitating single-signal t cell activation and inducing hiv virulence mediators biogenesis and transcriptional regulation of long noncoding rnas in the human immune system hiv-1 replication is controlled at the level of t cell activation and proviral integration rna-sequence analysis of human b-cells differential expression of lncrnas during the hiv replication cycle: an underestimated layer in the hiv-host interplay viral noncoding rnas: more surprises lincrnas: genomics, evolution, and mechanisms lncipedia: a database for annotated human lncrna transcript sequences and structures an update on lncipedia: a database for annotated human lncrna sequences the long noncoding rnas neat1 and malat1 bind active chromatin sites a strategy for probing the function of noncoding rnas finds a repressor of nfat evidence for a crucial role of a host non-coding rna in influenza a virus replication inflammation and host response to injury large-scale collaborative research program hiv-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure long-term cultures of htlv-iii -infected t cells: a model of cytopathology of t-cell depletion in aids the human immunodeficiency virus 1 asp rna promotes viral latency by recruiting the polycomb repressor complex 2 and promoting nucleosome assembly novel single-cell-level phenotypic assay for residual drug susceptibility and reduced replication capacity of drugresistant human immunodeficiency virus type 1 transcriptome altered by latent human cytomegalovirus infection on thp-1 cells using rna-seq neat1 long noncoding rna and paraspeckle bodies modulate hiv-1 posttranscriptional expression distinct temporal changes in host cell lncrna expression during the course of an adenovirus infection psf acts through the human immunodeficiency virus type 1 mrna instability elements to regulate virus expression the authors wish to thank drs. yosef sabo and stephen goff (both columbia university) for the generous gifts of their reagents, time and advice. this work was supported by nih grants r01ai068977 (to sg) and r01ai104523 (to rcd), as well as institutional support from columbia university. supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2017.08.006. key: cord-104491-uu2rbtem authors: andiman, warren a. title: where have all the “aids babies” gone? a historical memoir of the pediatric aids epidemic in new haven and its eventual eradication date: 2020-09-30 journal: yale j biol med doi: nan sha: doc_id: 104491 cord_uid: uu2rbtem s.l. was one of our first hiv-positive babies. he was born at yale-new haven hospital (ynhh) in 1982. his mother was a sex worker who also injected drugs. he died at 3½ years following multiple episodes of opportunistic infection and metastatic lymphoma. in the years between 1986 and 1990, 163 hiv-positive mothers gave birth at ynhh. the mother-to-child transmission (mtct) rate was 20 percent. women represented 8 percent of all hiv cases in the us compared with 29 percent in new haven. we had a six times greater proportion of children living with hiv. the mean number of hiv-exposed babies rose annually from 26 (1985-87) to 37 (1988-90). our first team of caregivers comprised a nurse practitioner, a social worker, and me. we were, in time, joined by a growing number of colleagues. enlightened and generous hospital administrators provided us with outpatient space and the promise of continued funding to support additional staff and in 1987, an independent pediatric aids care program. we implemented the proven mtct prevention guidelines articulated in the pediatric aids clinical trials group (pactg) protocol 076 and by 1995, the mtct rate at ynhh fell to 9 percent. since 1996, the mtct rate at ynhh has been zero percent. combination antiretroviral therapy, cart, made its debut in the mid-1990s; five classes of drugs with multiple agents in each were licensed between 2003 and 2013. we designed individual treatment plans for each child and gradually entered an era when our clinic was populated with healthier long-term survivors. our program flourished, based on a multidisciplinary approach which honored interprofessional collaboration. on the morning of january 15, 1985, a jolting banner headline appeared on the front page of the journal-courier (new haven, conn., 1973 -1987 : "candida lawler dies; autopsy due." 2 the newspaper's publishers understood that their readers were already familiar with ms. lawler's story; articles revealing her identity had been published in the new haven register as early as february 1984 [1] . her story received national attention when the cbs television news magazine, 60 minutes, revealed grave concerns about ms. lawler, a 29-year-old woman living with hiv, and whether she and others who were similarly afflicted, should be quarantined. city officials believed that their isolation would stanch the spread of the deadly infection during high risk sexual liaisons with sex workers and sharing syringes during illicit drug use. shawn, ms. lawler's son, 2 was one of our first three "aids 1 babies" and the earliest to whom that label could be applied with certainty. he was born in september 1982, and spent the first 2 months of his life withdrawing from heroin and methadone-drugs that had traversed the placenta. ms. lawler had what are now known as significant risk factors for people living with hiv/aids, including drug use and engagement in sex work. rehabilitation efforts had failed. at the time of her premature death from aids-related pneumonia, the journal-courier mourned the "once vivacious and beautiful woman with long black coal hair…who now appeared aged and emaciated." she might well have been designated new haven's "patient zero," the first publicly-named and shamed aids patient in the elm city. however, a search of the medical records at yale-new haven hospital (ynhh) would have revealed names of at least a dozen additional patients living with hiv. lawler was one among many women living with hiv, but mother-to-child transmission was not yet widely recognized as a possible outcome of maternal infection. at age 2½ months, shawn 2 recovered uneventfully from a bout of bronchiolitis, but was re-admitted 5 months later, lethargic, malnourished, and covered with a disfiguring purplish rash. he had hepatosplenomegaly and marked lymphadenopathy. his discharge diagnoses included disseminated meningococcemia and failureto-thrive. months later, he developed intractable oral thrush and candida diaper dermatitis. he had suffered hair loss, seborrhea, and eczema. he was thinner. these striking clinical signs, particularly his multiple infections and skin diseases, ultimately suggested an immunologic deficit. a comprehensive diagnostic work-up was undertaken. abnormal numbers of "atypical lymphocytes" were found in his blood and he failed to respond to skin test antigens. lymphocyte subset enumeration revealed far too few cd4+t cells, an aberration shared with his mother. we had recently learned that these abnormalities were characteristic of a new disease, referred to by some as "grid," or "gay-related immunodeficiency disorder," "the gay plague." in the early 1980s, the disease spread among men who had sex with men and intravenous drug users, including women. we wondered whether shawn, a toddler, was similarly affected. did ms. lawler pass hiv to her baby during pregnancy, at the time of birth, or perhaps, post-partum through breastfeeding. ms. lawler's blood was send to max essex's lab in boston. he had developed a test for human t-cell leukemia virus iii, htlviii, the name of the virus thought to be the cause of the world's newest immunodeficiency disease. ms. lawler carried antibodies to htlviii. (hiv was the name later assigned by an international congress.) shawn's blood carried the same antibodies. initially, we were unable to determine whether his antibodies were acquired from his mother in utero or were raised actively in response to his own infection. shawn followed an insidious downhill course, common in the early years of the epidemic. there were no antiretrovirals and few drugs to treat some of his multiple infections. at 9 months of age, shawn was admitted to the hospital in respiratory distress. his chest x-ray revealed many large nodules and impressive mediastinal lymphadenopathy. cultures for the usual suspects were negative, but a lung biopsy showed evidence of lymphoid interstitial pneumonitis (lip), an entity only rarely reported in adults and almost never in children. lip was, for the first 10 years of the epidemic, the most common aids-related illness in the pediatric population, an ominous predictor of advanced hiv disease. nevertheless, it responded temporarily to steroids and during his second year of life, shawn gained some weight, was less breathless and more playful. shawn presented with weakness of his left hand and face at age 21 months. a ct scan of his head revealed a mass lesion deep in the midbrain. normal bone marrow was replaced by a b cell lymphoma. several courses of chemo-and radiotherapy stabilized his condition for almost a year, during which time he lived in a crib in an isolation room with other infants infected with hiv. the final event was an unrelenting metastatic infection with mycobacterium avium intracellulare (mac), resulting in grotesque enlargement of his regional lymph nodes, liver, and spleen. standard anti-infective agents and two experimental drugs donated by the cdc, ansamycin and clofazimine (neither of which is currently used), did nothing to change the course of the infection. shawn died at 3½ years of age. hiv-infected 3 babies began to appear regularly in the emergency room and various clinics. the obstetricians were caring for ever-increasing numbers of women testing positive for hiv. in new haven, the mean number of hiv-exposed babies rose annually from 26 to 37 (1988-90) . in fact, 20 percent of all babies born to mothers testing positive for hiv were also testing positive and in immediate need of complex and prolonged interdisciplinary care. we had moved into crisis mode. simultaneous with our need to devise an effective intervention, i had my first triennial leave, a mini-sabbatical during which i could devote myself exclusively to learning more about hiv and aids. previously, i had been involved in research studying the ways in which the epstein-barr virus displayed its pathology in both healthy and immunodeficient children. but now we were witness to the debut of a novel disease caused by a puzzling new virus, one that snared mothers and infants, in addition to men. new diseases weren't being seen that often-a rule that seems to have been broken with some frequency of late (viz. zika, west nile, mers, sars, covid-19). i reasoned that this was my chance to enter a challenging clinical arena on the ground floor, to make this growing plague the center of my academic focus moving forward. descriptions of pediatric aids came to us from the earliest epicenters as graphic retrospective reports. four of the "hot spots" were cities with large indigent populations, people who used drugs, men who had sex with men, and some of their female sex partners: new york city, newark, san francisco, and miami. the majority of babies were infected intrapartum with infectious blood and genital secretions, less so in utero. at the start of my explorations, i sought allies who could show me the ropes, introduce me to a few patients, and teach me some of the relevant clinical science. none of my usual teachers in the pediatrics department could help because they knew no more than i did, having read a few articles and listened to ominous stories on the news. so i made inquiries of colleagues in the department of internal medicine who had been caring for most of the patients. they all came up with the same name, leetha fraulino, aprn. i was surprised to learn that fraulino was a nurse practitioner who had come to ynhh from her previous position as a clinician with the greater new haven visiting nurses association. among her clients in the community were a handful of adult aids patients, some of whom had already spent some time in the hospital and some who were safely managed at home. she recognized the need for a place where the growing number of patients living with hiv could be followed in specialized settings, where they could receive both medical and much-needed psychosocial support. she imagined a place where preventive and supportive care, that included access to good nutrition and decent housing, would forestall the need for visits to the emergency department that frequently ended in moves to the icu. hospital administrators, alarmed by the increasing numbers of uninsured, critically ill patients and the uncompensated costs incurred in their care, agreed to give fraulino access to a few examining rooms in one of the ambulatory clinics for one afternoon a week. she was acquainted with many of her former clients and agreed to have me join her on her daily rounds. we reviewed x-rays, checked the results of laboratory tests, and shared insights we had gathered from the patients with the residents and nurses, including details of each one's social situation, and relationships with significant others and family. having anticipated a surge of hiv-positive babies, fraulino welcomed the chance to have a pediatrician as her ally. we were among the first troops on the front lines, joined in battle against a powerful new foe. i was hooked and my professional life was forever changed. the histories shared by most hiv-positive mothers and their female kinfolk indicated that their babies had been born into "at risk families." about half the mothers had used intravenous drugs which they often shared. some were sex workers and still others reported that they did not consume illicit drugs but had had sexual relationships with men who had. babies born addicted to drugs were immediately transferred to hospital units that were specially equipped to care for these irritable, tremulous, jittery newborns who sometimes spent weeks or months in isolettes at a distance from bright lights and disturbing sounds. they were swaddled and received round-theclock feedings on demand. among a series of impediments, we faced one insurmountable problem: we were not able to discharge hiv-exposed babies to the care of mothers still battling addiction; and experienced foster parents refused to take "aids babies" into their homes for fear of infection. there was only one alternative-the nurses, doctors, and social workers became their de facto guardians. the babies stayed in the hospital for months, sometimes for more than a year. in the early 1980s, we cared for an average of 26 hiv-exposed infants, born each year to hiv-positive mothers. we were obliged to track down a group of persons connected to each index mother, e.g. current and former sex partners, people who injected drugs, and older children. we invited them to meet with us to learn of their infection status. these meetings were long and excruciating. most of the contacts never imagined they might be carrying hiv, complicated by the fact that they might have already spread the virus to unknowing partners. they all needed to be directed to healthcare centers-destinations other than emergency rooms or the community, clinic, or hospital. physician specialists volunteered to help us in the clinics. they knew that aids was a growing epidemic, that they would soon be called upon. they wanted to be well-prepared, knowing they would become the teachers, transmitting their knowledge to the junior troops who were gathering close behind. well-concealed beneath the armor of denial and girded by their selfless efforts, there was sometimes a gnawing worry about the possibility of getting infected with hiv by accidental needlestick, a saliva or vomit-laden splash, perhaps by a urine, fecal, or blood-soaked bed, or maybe a bite or scratch by an agitated patient. (other than by sex or needle-sharing, the risk posed by other behaviors or body fluids was still unknown.) ynhh, the academic epicenter of medical care in southern connecticut, was committed to serving the entire population of new haven and the surrounding towns. its promise of excellent care for all who presented themselves was being challenged by the financial burdens incurred in caring for those who required costly emergency and prolonged intensive care. our team was called upon to educate an already receptive hospital administration. we were invited to present the facts of the epidemic, in all its alarming detail, to the hospital board of trustees. we reminded them that the uncontrolled demand for expensive care could be mitigated by creating a program devoted to longitudinal outpatient care for both adults and children, similar to those for patients with other chronic diseases. the caregivers would forge trusting relationships with their patients in a way that would ensure adherence to care and stimulate the creation of individualized care plans. we would introduce new treatments as soon as they became available. the social workers would focus their efforts on the financial, nutritional, and housing needs of the patients. timely arrangements could be made for in-home nursing care. we would strengthen the education of parents, spouses, friends, and neighbors. our first formidable ally was dr. john fenn, a surgeon and the hospital's chief of staff. he had heard of our work and already learned a bit about the clinical aspects of aids. he knew that the hospital was carrying a heavy burden and that babies and toddlers were beginning to fill isolettes in the nurseries and cribs on the wards. dr. fenn invited himself to make rounds with us a few times a week. he was deeply affected by what he saw and heard. after scrubbing, gowning, and masking, he would sometimes sit on the bed listening to patients' stories. he thanked fraulino and me and commended us for keeping abreast of every patient's status and carried these stories and descriptions of our daily routines to the hospital president, the chairmen of internal medicine, pediatrics, obstetrics and gynecology, and the vice president for nursing. in the wake of these meetings, fraulino and i "walk-in" clinics. those with substance abuse disorders were transferred to drug rehabilitation programs. women were given appointments with gynecologists who would provide access to birth control, cancer screenings, and treatment for sexually transmitted infections. we helped the homeless mothers, rejected by their families, to find housing and food stamps. by the mid-1980s, we had access to reliable antibody assays and both antigen and nucleic acid-based tests that allowed us to measure accurately the present status and arc of the epidemic in children and women of childbearing age. we compared the demographic features of hiv infection in new haven with the state of connecticut and the entire country. we learned that new haven was different! cumulative data through december 1986, revealed that women represented 8 percent of aids cases in the us compared with 29 percent in new haven. similarly, children represented 1 percent of all national cases compared with 6 percent of our local cases. new haven had more than double the proportion of infected drug users compared with the rest of the country. in 1989, the connecticut department of public health reported that one in every 33 african american women giving birth in new haven was hiv-positive, a proportion twice that of all women giving birth in the state, regardless of ethnicity. national statistics revealed that connecticut ranked fourth per capita in the incidence of aids in women and first per capita in the nation in the incidence of aids in children. intravenous drug use by the mother or her partner was the risk behavior that precipitated infection in 87 percent of the babies. in the us, the majority of patients were white (60% white, 38% black and hispanic) whereas in new haven, hiv infection was far more common in people of color (61% black, 14% hispanic, 25% white and other). we lived and worked in a small american city (population estimated 125,000) with a disproportionate onslaught of aids. we were crippled by a dearth of antiretrovirals and effective anti-infectives for many of the opportunistic infections. we needed more clinicians and financial aid to support our efforts. slowly, providers with a multiplicity of skills emerged from their quotidian workplaces and asked fraulino and myself if they could help. june holmes, msw, was one of the first to volunteer. she already knew some of the patients through her work in other parts of the hospital. like fraulino and me, she believed she would be most effective if she could serve our patients even before they were hospitalized and, again, at time of discharge. continuity of care was paramount, starting with the trust built during the first encounter, whether in interviewed on multiple occasions for articles in the new haven register, the new haven advocate, channel 8, the local tv station, and a few radio shows. i remember a few occasions when we chided reporters for having portrayed our patients in a light that may have stoked fear and derision among news consumers. it took some time to sensitize the media and other people of influence. in time, the hospital's board of trustees gave the go-ahead for the "official" creation of the "aids care program." soon the city of new haven and local charitable organizations joined us in common cause. fraulino, now the aids program coordinator and clinical supervisor and b. joyce simpson, rn, mph, once described in great detail the institutional response to the aids epidemic, using ynhh as the paradigm. they reminded us that public teaching institutions are responsible for making subspecialty expertise and stateof-the-art technology accessible to the public and local community needs and providing treatment with new antiretrovirals and anti-infectives. fraulino continued to see all the adults and children with aids-related diagnoses and introduced me to them as the medical director. she assessed their needs and assisted patients and their families to gain access to necessary support services. she participated in discharge planning in conjunction with the nursing and medical staff and developed a long-term plan for each patient. she organized the outpatient clinics and acted as the primary liaison between the inpatient and outpatient settings. in response to a growing patient population, the hospital mounted a second phase of planning. over the course of a year, we were introduced to additional subspecialists, with either adult or pediatric credentials. simpson became our pediatric research nurse and pediatric aids program coordinator. she collected, organized, and analyzed data that fulfilled the specific aims of our various grants. we also welcomed an educator with a graduate degree in public health who oversaw hiv testing and counseling services and two hiv counselors with backgrounds in nursing. the social workers guided families through the onerous process of applying for entitlements, including supplemental security income (ssi) and medicaid. finally, we developed cordial relationships with allies from pediatric and adult psychiatry, neurology, surgery, the chaplain's office, the child life program (evaluates developmental milestones and provides play therapy), nutritional services, respiratory therapy, and pharmacy. the team recognized the importance of establishing collaborative relationships with community-based agencies and organizations. we were living in the midst of a public health crisis and we saw ourselves as the catalysts for change. either individually or in pairs we educated leaders in the visiting nurses association, the methadone maintenance programs, the department of children and met with the same leaders to provide first-hand accounts in all their startling details. we made clear our need for funding and asked for official promises by the hospital and medical school to establish an aids care program, in perpetuity, dedicated to the ongoing outpatient and inpatient care of hiv-infected patients of all ages. we believed that health professionals on the front lines could be effective only with generous private and public support and the backing of two superlative academic institutions-ynhh and yale medical school. at the insistence of two successive chairs of pediatrics, doctors howard pearson and joseph warshaw, the hospital agreed to support half my salary. the pediatrics department would contribute much of the remainder to support my teaching efforts and a small percentage of my ongoing research projects. in a letter written in january 1988, dr. fenn agreed to relieve me of some of my responsibilities in the adult aids effort, by funding two new half-time physician slots. both these physicians would serve the rapidly expanding adult population and i would be allowed to spend more time with the children who were hiv-positive and their families. the hospital also assigned us pediatric clinic space for one, and then two afternoons a week. full time rn and social service positions were added to the mix. dr. fenn penned an official note to dr. warshaw that fully acknowledged the work that fraulino and i had already done. he wrote the following: "the goal in the recent expansion of the program was to allow warren andiman and leetha fraulino some relief from their extraordinary burden and to make sure that the hospital was supplying resources commensurate with the magnitude of the epidemic facing us. i am comfortable that we are doing our part and that we have now provided dr. andiman with the help he most certainly needed and the opportunity to engage in other academic activities." ynhh serves the entire city of new haven, and with the university they are the city's largest employers. as such, the hospital and medical school are obliged to foster cordial relations with the city's leaders and provide them a voice in its stated missions. to keep these leaders apprised of the magnitude and severity of the "aids problem," g. harold welch, the president of the hospital, invited me to meet three city aldermen. after the dinner meeting, he wrote: "the three aldermen who attended are all members of the ynhh board of trustees." the meeting was designed to foster "helpful communication between the city's leaders, their constituents and the hospital. the hope was that the city would take appropriate action in the future." it did, in fact, "take a village" to support our program and keep its accomplishments in the public eye. the media started to take increasing interest in our work and in the changing state of the epidemic. fraulino and i were its attention on the rapid identification and treatment of aids-defining conditions, primarily opportunistic infections, but also certain malignancies, e.g. lymphoma and sarcoma, central nervous system afflictions, including aids encephalopathy, and chronic skin conditions. in the late 1980s, treatments were limited, and cures were unattainable. the relentless pace of disability and disease continued. fifty percent of our hiv-infected children died before the age of 5 years. most of our hiv-positive children had challenging home lives, including difficulties accessing food, transportation, and decent living conditions. their lives were chaotic, lonely, and stressful. some had already lost family members to aids and others faced the possibility of a parent's incarceration or hospitalization. following years of reluctance on the part of "professional" foster parents to bring babies living with hiv into their homes, we joined with the connecticut state department of social services and rolled-out intensive educational programs that featured infection control guidelines. the state increased payments to cover the costs of fostering a fragile, chronically ill child. these inducements encouraged some foster families to care for "aids babies." for the most part, these placements proved to have positive outcomes. there were impressive gains in growth and development and increased adherence to clinic visits and prescription refills. the number of hospital "boarder babies" slowly decreased and by 1989, fell to zero. sometimes the child remained in foster care temporarily, until the mother was released from prison or committed to a drug treatment program. adequate housing was required for family reunification. remarkably, some foster families adopted the children they had nurtured so successfully. the minority of hiv-infected children who reached school age, were consigned to a space in the basement of a municipal building and were "taught" by someone with little or no requisite educational background or training. no classroom in new haven's public schools found space for a child living with hiv. the task of righting this discriminatory practice fell to the pediatric aids care program. our nurses, social workers, and i became community educators whose principal goal was to share the evidence that hiv is not spread by casual contact and that minor incidents of bleeding, spitting, urinating, and even biting, could be managed by the simple application of standard precautions: gloves, soap and water, and the prudent use of diluted household bleach, as needed. at some point, as the abysmally inadequate education of our patients was deemed untenable, i was asked to serve as families (dcf; formerly dcys, department of child and youth services), community aids support groups, like aids project new haven, churches sympathetic to our philosophy of care, connecticut hospice, and other long-term aids residency facilities. the collaborations resulted in improved care and a modest reduction in costs that would otherwise have been borne by the hospital. the pediatric aids epidemic in new haven grew at an alarming rate. along with the hiv-positive babies, who constituted 20 percent of our patient population, we decided early on to continue to follow, temporarily, the 80 percent of babies who had been exposed to the virus but were uninfected (some 130 infants by 1990). we had to guarantee their safe release either to their biological mothers or to foster parents, often close relatives. we found placements for those "new" mothers who required drug treatment and safe housing. the early development of the now independent pediatric program mirrored in many ways the genesis of the adult program. therefore, we were not surprised when a willing group of talented, motivated, and energetic health professionals with diverse skills asked if they could help. we found assignments along the entire spectrum of their training-attendings, residents, post-docs, rns, aprns, social workers, nursing, and medical students. at the beginning, few of these assignments were designated as "official" and none was accompanied by bonuses or special treatment. these were professionals who incorporated their work with hiv-positive children into elective slots or a variety of required outpatient rotations. by 1990, the hospital recognized the need to split the aids care program in two: an adult medicine arm and a pediatric arm. it was obvious that the number of patients needing urgent care was growing too rapidly to remain under the aegis of one medical director who had other clinical, research, and teaching obligations. also, my training as a general pediatrician with expertise in infectious diseases was no longer a good match for the enormous challenge posed by a population of seriously ill adults with life-threatening diseases and chronic conditions. the hospital set out to hire two experienced, academically-trained internists who had already served on the front lines of a city-wide aids epidemic similar to ours. the two saviors, gerald friedland and peter selwyn, by now leaders in the field, and both from montefiore hospital in the bronx, ny, joined the effort in 1990 to lead the adult program. in the absence of an effective antiretroviral agent, the pediatric aids care program initially focused much of 1991. the regimen was comprised of antepartum zdv, given orally 5 times daily, starting at 14 to 34 weeks post-conception and continuing through the remainder of the pregnancy. zdv was also administered to the mother intravenously during labor and delivery. the newborn received oral zdv syrup, beginning at 12 hours postpartum and continuing every 6 hours for the first 6 weeks of life. periodic hiv cultures were done to distinguish between infants infected in utero and those infected intrapartum or postpartum. based on data derived from the 364 mother-infant pairs who completed the study, zdv treatment was associated with a 68 percent reduction in mtct (8% in the treatment group vs 26% in the placebo group). the drug regimen was well-tolerated. when the study results were published, the cdc recommended that the protocol be applied universally to all hiv-positive pregnant mothers. as per the cdc recommendations, we implemented the 076 protocol and by 1995, the mtct rate at ynhh dropped to 9 percent, half the rate we witnessed previously. simpson, our pediatric research nurse made herself available, even at night, to attend the delivery of every hiv-positive mother. she guided the nurses and mothers through the steps of the pactg 076 protocol. she met with each mother and learned about her social support system and living situation. she discovered whether there was money to buy formula, diapers, and clothing. the mother received her first 2-week appointment to the pediatric hiv clinic and a prescription for her child's zdv syrup. before hospital discharge, the nursing staff judged whether the mother could provide a safe home for her baby. the side effects associated with zdv were easily managed, but the inevitable appearance of resistance convinced us that additional antiretrovirals were needed to use in combination with zdv. combination treatment met its earlier success with regimens for tuberculosis and subsequently, for other complex infections and cancer chemotherapy, diseases in which drug resistance had been a significant obstacle to cure. we participated in large multicenter clinical trials assessing the utility of combining non-nucleoside reverse transcription inhibitors (nnrtis), e.g. nevirapine, with a growing number of newer nucleoside reverse transcriptase inhibitors (nrtis) e.g. lamivudine. in time, the protease inhibitors, entry/ fusion inhibitors, and integrase inhibitors were licensed sequentially in the decade between 2003 and 2013. drug side-effects and resistance demanded that we follow the children closely and prepare to intervene with either temporary or permanent alterations in individual drug regimens. in good time, we witnessed varying declines in an expert witness in a complaint brought by the american civil liberties union against the city of new haven. based on the preponderance of the scientific evidence and the recognition that the children were being discriminated against on the basis of physical disability, a clear violation of the american disabilities act, the presiding judge found in favor of the right of hiv-positive schoolchildren to an education equal to their same age peers. as a result, our children gained admission to the city's classrooms with the promise that only the child's teacher, the school nurse, and the principal would be informed of the student's hiv status, an important concession that became operative in instances of accident or emergency. the most basic principle of public health, prevention, had not been fulfilled. we were treating disease but not preventing it. the fda approved the use of zidovudine (zdv, azt) to treat advanced hiv disease in adults. but we learned that zdv, the first in a new class of drugs, reverse transcription inhibitors, provided only short-term survival advantage and its benefits lasted only a few months. despite these disappointments, we convinced some of our pediatric pharmacists to create a syrupy concoction of zdv powder that could be fed by mouth to infants and toddlers. (once a drug is licensed, it can be used off-label if prescribers believe that the benefits outweigh the risks.) we knew of only two adverse reactions, mild anemia and liver dysfunction and monitored both by doing periodic blood tests that led to a change in dose or discontinuation of the drug, if necessary. as in adults, zdv treatment resulted in only temporary clinical improvement-weight gain, greater alertness and activity level, and fewer life-threatening infections. within a few years, the fda approved the use of zdv for children. we were buying time in the hope that more effective antiretroviral agents would arrive. clinical relief is not prevention. fifteen to 20 percent of hiv-positive mothers were still transmitting hiv to their offspring and with some exceptions, the infected babies died before the age of 5 years. nevertheless, we witnessed a veritable miracle in 1994, when the results of the nih-funded study, pactg protocol 076 was published. until that time, strategies for reducing the risk of vertical transmission of hiv were limited to the avoidance of pregnancy altogether or refraining from breastfeeding, which was the most common cause of postnatally-acquired hiv. the double-blind placebo-controlled study, pactg 076, investigating the effects of antiretroviral therapy during pregnancy, was initiated in the parties expressing vehement objections argued that administering hiv tests by way of blood draw to newborns, absent informed consent and expressly against the will of the parents involved, constitutes an illegal search, forbidden under the fourth amendment to the us constitution. the cha filed a motion for a temporary restraining order enjoining the execution and enforcement of the law a mere "scant hours" before the law was scheduled to go into effect. because the judge presiding over the us district court concluded that the plaintiff had not proved the likelihood of success in demonstrating that the statute at issue was unconstitutional, the motion for a temporary restraining order was denied and the law went into effect. despite the law, by 1999 there was only limited evidence that providing antiretroviral therapy to the newborn would lessen the severity of infection, even if there was no prior treatment of the mother during the latter part of pregnancy, labor, and delivery. we now know that early postnatal treatment does lower the viral load in the infant and limits damage to the immature immune system. we also know that the discovery and treatment of hiv infection in a previously untested mother may be lifesaving and nearly always prevents vertical infection in all subsequent births, if the mother continues her treatment. connecticut public act no. 99-2 was the powerful tool we were waiting for. we went into the community and educated physicians who cared for pregnant women. we urged them to become familiar with the new statute and to call us with questions. our nurses constructed detailed protocols for the labor and delivery floors. the mothers were given postpartum appointments to the high risk obstetrics clinic and their infants made visits to the pediatric hiv clinic at 2 weeks of age, while still receiving oral zdv. we had foreknowledge of the impressive preliminary results of the pactg 076 prevention study and had been following its guidelines, even before the study was published. this head start and our intensive educational efforts in the hospital and community resulted in a steady decline in the number of newborns testing hiv-positive. the last infant that tested hiv-positive was born at ynhh in 1996. for each of the past 24 years, we have screened and treated about twenty hiv-positive pregnant women, a significant decrease from earlier decades. none of the babies born to hiv-positive mothers between 1996 and late 2019 were infected. 4 nevertheless, we still care for a few hiv-positive children who have come to us from other parts of the world, mainly africa, and for a handful of adolescent men who have had sex with older hiv-positive men. viral loads and rises in cd4+ t-cell numbers to more normal levels. many of the children felt better and returned to their regular activities, including school attendance. as with adults, combination therapy with an ever-increasing number of agents, altered forever the natural history of pediatric hiv infection, changing it from a rapidly fatal to a chronic, manageable disease, including for some who had survived beyond early childhood. despite these gains, our pre-eminent wish remained unshaken, i.e. to eradicate mtct of hiv, a goal that would prevent the life-long morbidities and suffering that accompany pediatric hiv infection and its attendant limitations and stigma. the pactg 076 protocol, when practiced consistently, proved to be a lifesaver. for all mothers at risk who sought prenatal care and were not already being treated with antiretrovirals, hiv testing was offered. if the test results were positive, simple drug regimens were prescribed along with intensive counseling and admonitions to adhere closely to the instructions for drug compliance. hiv care and obstetric care were linked. when close to term, the mothers were given explicit instructions to appear early in labor, so that zdv could be administered intravenously before their baby's birth. but routine hiv screening was not yet part of obstetric care and it soon became obvious that we were missing some women at risk: those who failed to receive prenatal care and those who did receive care but whose hiv infection status was unknown or not recorded. we posed an audacious question: shouldn't hiv testing be a mandatory part of obstetric care? in the early years, hiv testing was permitted only after a counseling session and signed consent. times change. connecticut public act no. 99-2 became law on october 1, 1999 after much contentious opposition mounted by the connecticut civil liberties union foundation, the connecticut hospital association (cha), and by editorials and harsh opinion pieces in local newspapers. the first part of the law read as follows: [2] ." (2.9%) had been mildly symptomatic or asymptomatic; c). 512 (70%) were seroreverters, i.e. they permanently lost all passively-acquired maternal antibody and were uninfected; d). 12% were deceased and 9.3% were lost to follow-up. once we had optimal practices in place for preventing mtct of hiv and managing the care of children who survived without the burden of lifelong infection, we formally joined the pactg. the pactg (now impaact) is a consortium of academic pediatric institutions charged with testing the efficacy of new antiretrovirals and anti-infectives. we also tested live virus vaccines for their safety in hiv-positive children whose immune function had been restored by antiretrovirals. lastly, we participated in a detailed collaborative natural history study of hiv-infected and hiv-uninfected peers who were exposed to hiv perinatally, i.e. affected but not infected. ultimately, between the years 1994 and 2007, we enrolled scores of children in one or more clinical trials each. the results of these studies meant healthier and more productive lives for thousands of infected and affected hiv-affected children worldwide. among hiv-positive children born prior to 1996, about half, our longer-term survivors, were healthy enough to attend school free of the bane of aids-related conditions. many children were lucky to have found loving foster families who later adopted the children they had raised. the number of "active" patients in in our care fell by 50 percent in the decade ending in 2005. the 51 survivors met with our caregivers every 3 to 4 months. eighty percent ranged in age from 8 to 18 years; 16 percent were older than 18, and 4 percent were younger than 8 years. fifteen children died in the years between 1990 and 1995, half in the hospital and half at home or in hospice. there were only three deaths between 1996 and 2005. among our long-term survivors (lts), adverse medical and social issues are common. their outcomes have ranged from excellent to poor. over 50 percent have maintained undetectable viral loads and cd4+ t-cell counts in the normal range. the number of emergency visits and hospitalizations has fallen precipitously. however, non-adherence to medical regimens, unprotected sexual intercourse, stigmatization, unchecked family discord, depression, and school drop-outs have stymied the realization of the fully successful social and physical outcomes we had hoped for. many families failed to disclose the hiv diagnosis to their children for fear of precipitating untoward psychological repercussions, including long-term stigma. some hiv-positive young after a half dozen years of familiarizing myself with the protean manifestations of pediatric aids, i began participating in research studies that would answer pressing clinical questions. i re-examined the statistics extracted from older studies conducted without benefit of appropriate study design and methodologies. intending to correct these deficiencies, i set myself the task of embarking upon a prospective, longitudinal cohort study to determine the true risk of mtct of hiv, the relationship between the degree of immunodeficiency and the incidence of aids-defining illnesses and survival, and the connection between specific gestational variables and transmission risk. between 1987 and 2007, i was awarded an uninterrupted series of grants and contracts, the first few of which were designed to answer definitively the questions i had enunciated. the earliest of my grants coincided with the inauguration of two private foundations created to support basic and clinical research on hiv and aids: amfar (the american foundation for aids research) and egpaf (the elisabeth glaser pediatric aids foundation). we joined an international alliance of american and european academic centers and our collective data ultimately became the basis for what we now know about the epidemiology, pathogenesis, and natural history of pediatric aids. among the most important of our local and international collaborative findings were the following: i). among babies born to hiv-positive mothers and followed since birth at ynhh between the years 1986 and 2007, the overall risk of transmission of hiv was 9 percent (range 0-20%); ii). the risk of mtct was 20 percent for mothers who received no antiretrovirals during labor and delivery versus 4 percent for mothers who received antenatal and intrapartum antiretrovirals and whose newborns received zdv for the first 6 weeks of life (as per pactg protocol 076); iii). among ynhh mothers who received antiretroviral therapy during pregnancy, the risk of mtct was 6 percent for those whose cd4+t-cell counts were <200 cells/ul versus 0 percent for those mothers with median cd4+ t-cell counts >500/ ul; iv). a meta-analysis of more than a dozen prospective cohort studies (including ours) by the international perinatal hiv group demonstrated that elective c-section before onset of labor and before rupture of membranes, resulted in a decline in the risk of vertical transmission from 19 percent to 10 percent. when c-section was combined with antiretroviral therapy during the third trimester, the risk of mtct of hiv fell further, to 2 percent; v). among 733 hiv-exposed children enrolled in our prospective study (including those enrolled in collaborative studies during which they had received varying lengths and combinations of antiretrovirals), we recorded the range of clinical outcomes in 2007: a). 43 (5.8% had been severely or moderately symptomatic; b). 21 women and men became parents unintentionally, but all their offspring are uninfected (all the pregnant young women complied with their prenatal clinic appointments and took their medications). a minority of our lts have completed high school, college, or job-training and a few have been successfully employed. a handful have been incarcerated on drug charges or acts of violence. one patient, lost to follow-up, died by gunshot. those of us working in resource-rich countries encountered the "coming of age" of the first and largest cohort of hiv-positive children-those born between the early 1980s and the late 1990s. we confronted their need for a major health care transition. data collected by the cdc in 2007, revealed that approximately 24,000 american youth, ages 13-24 years, were living with hiv/aids. this was a 25 percent increase from 2004, attributed to high-risk adolescent sexual behavior and increasing survival of children infected perinatally. developmentally appropriate youth ought to receive care in adult healthcare practices sometime in their early 20s. but there are numerous everyday tasks that represent major hurdles for many teens, in particular adherence to their medical regimens. ultimately, with diligent guidance, most of them progress to a point where they can be safely transitioned. during the years 2006-07, under the guidance of sostena romano aprn, mba, our longest-serving nurse practitioner, and anne murphy, msw, we transitioned 30 patients, ages 15 to 26 years of age, to adult practices: 13 to yale's adult nathan smith clinic, nine to other hospital-based clinics in connecticut, and eight to private practices close to the patients' homes. we have continued this practice as our patients enter late adolescence and young adulthood. the eradication of mtct of hiv in new haven was made possible by the confluence of six nourishing streams: 1). the creation of effective antiretrovirals, anti-infectives, and sensitive diagnostic tests; 2). the flood of grants and contracts from federal, state, local, and private funding agencies; 3). wise and generous support from yale-new haven hospital and the department of pediatrics; 4). the passage in 1999 of connecticut public act no. 99-2; 5). the creation of an effective multidisciplinary approach to care which honors respectful interprofessional collaboration; 6). the spontaneous and enthusiastic union in common cause of doctors, nurses, social workers, students, administrators, and volunteers to serve patients in need, their families and friends, in new haven county and beyond. rethinking complicity in the surveillance of sex workers: policing and prostitution in america's model city special session reduction of maternal-infant transmission of hiv-1 with zidovudine treatment european collaborative study: children born to women with hiv-1 infection: natural history, and rate of transmission a prospective cohort study of children born to hiv-infected mothers. trend in the risk of vertical transmission, mortality, and aids-indicator diseases public act no. 99-2 duration of ruptured membranes and vertical transmission of hiv-1: a meta-analysis from 15 prospective cohort studies update on successes and challenges regarding mother-to-child transmission of hiv-1 transition from pediatric to adult healthcare services for young adults with chronic illnesses: special case of human immunodeficiency virus infection 1 this term was widely used in medical circles and the media in the 1980s to refer to hiv-exposed infants who faced lengthy hospitalizations to ease them through withdrawal from opioids and/or cocaine as they awaited foster home assignments. the term has disappeared now that the rates of vertical transmission of hiv have fallen to negligible numbers. 2 names of patients have been changed to protect their identity. 3 all babies born to hiv-infected mothers are "hiv-positive" until 7 to 15 months of age because they all acquire hiv antibodies transplacentally. but only a fraction are "hiv-infected." eighty percent are "exposed" to hiv and are temporarily hiv-positive, but remain "uninfected." 4 unfortunately, in late 2019, an hiv-positive baby was born at ynhh after a gap of 23 years. the teenage mother received prenatal care at a clinic unaffiliated with ynhh. she was non-compliant with the antiretroviral regimen that was prescribed for her. she was surprised to learn that her newborn baby was infected. the baby received antiretrovirals soon after birth, the mother's regimen was re-started, and the baby's biological father appeared for testing and treatment. all three have been adherent to their various regimens and, as of last report, all are doing well. since the start of 2020 we have followed nine hiv-exposed babies, but no mother-to-child transmissions of hiv have occurred. key: cord-253426-s57wuzyg authors: benkovic, scott; kim, michelle; sin, eric title: 4 cases: hiv and sars‐cov‐2 co‐infection in patients from long island, new york date: 2020-05-19 journal: j med virol doi: 10.1002/jmv.26029 sha: doc_id: 253426 cord_uid: s57wuzyg originating from wuhan, china, the novel coronavirus 2019 (sars‐cov‐2) has been spreading worldwide since the end of 2019. the most common features of these patients include fever, cough, myalgia or fatigue [1]. as of april 16, 2020 the cdc has reported 605,390 cases and 24,582 deaths in the united states. the illness continues to spread through the world infecting people with various different comorbid conditions. presented here are four cases which represent some of the first cases of hiv and sars‐cov‐2 coinfection in long island, new york. these hiv infected patients were compliant with their hiv medication regimen and had robust cd4 t cell counts. the clinical severity ranged from mild to requiring hospitalization. three of the four patients had fever and two had cough. one patient presented with diarrhea, the incidence rate of diarrhea in sars‐cov‐2 infection range from 2% to 50% of cases [4]. one patient had anosmia and aguesia, in a study by moein et al, the 98% of sars‐cov‐2 infected patient experienced some smell dysfunction [5]. one patient required hospitalization however this patient was also infected with influenza a. these cases suggest that uncomplicated cases of sars‐cov‐2 in an hiv infected patient can be managed with self‐isolation at home. this article is protected by copyright. all rights reserved. originating from wuhan, china, the novel coronavirus 2019 (sars-cov-2) has been spreading worldwide since the end of 2019 1 . there has been a reported case of human immunodeficiency virus (hiv) and sars-cov-2 coinfection in a man in wuhan, china 2 , but as of today there have been few reports of coinfection. this letter to the editor seeks to contribute four more cases to the literature. presented here are four cases of coinfection of hiv and sars-cov-2 from long island, new york. their main characteristics are summarized in table i . a 56-years-old male started to feel fatigued as well as noted a decreased in his sense of taste and smell three days after returning to new york from a trip to florida. at his local community physician he was given empiric antibiotics for a presumed sinus infection. although he did not develop fever or respiratory symptoms he became concerned when his symptoms did not resolve after nine days and he went to an urgent care clinic. due to concerns for chemosensory dysfunction in sars-cov-2 infected patients 3 real-time reverse-transcriptase polymerase chain reaction (pt pcr) for the virus was done. sars-cov-2 rt pcr resulted positive three days later. two days after his positive test his symptoms of anosmia and ageusia resolved. this man was diagnosed with hiv in 1995 with a recent cd4 t cell count of 1206 cells/ul and hiv-consisted of emtricitabine, tenofovir alafenamide, dolutegravir and maraviroc. his only other comorbid condition is hyperlipidemia. a 56-years-old male who started to developed subjective fevers and fatigue. 19 days after the initial onset of fatigue he developed a temperature of 102f (38.9c) when he went to urgent care. he had no shortness of breath or cough. his chest x ray was suggestive of pneumonia at which point he was given empiric antibiotics. sars-cov-2 rt pcr resulted positive 2 days later. three days after the positive test result he was asymptomatic. this man was diagnosed with hiv in 1988 with a recent cd4 t cell count of 794 cells/ul and a hiv-1 viral load that was undetectable. his hiv regimen consisted of emtricitabine, tenofovir alafenamide, etravirine and abacavir. his only other comorbid condition consisted of hypertension controlled with lisinopril 10mg daily. a 62-years-old male who had 2 week of non-productive cough and watery bowel movements. he decided to seek medical attention when he developed a temperature of 100.8f (38.2c). at his local emergency room his temperature was 100, blood pressure was 113/65, heart rate was 75, breathing was non-labored and his oxygen saturation was 97% on room air. white blood cell count was 5200 cell/ml, blood urea nitrogen was 19ml/dl, and creatinine was 1.06 mg/dl. chest x-ray did not show any consolidation. he was discharged home with instructions to self-isolate. after discharge his sars-cov-2 rt pcr resulted positive. one week after discharge he no longer has any symptoms. this man was diagnosed with hiv in 1996 with a recent cd4 t cell count 1412 cells/ul and a hiv-1 viral load that was undetectable. his hiv regimen consisted of emtricitabine, tenofovir alafenamide and dolutegravir. his comorbid condition consist of treated hepatitis c, hyperlipidemia on rosvustatin and hypertension on losartan. a 65-years old man presented to urgent care with cough and subjective fever. he was empirically started on oseltamivir 75mg twice a day for 5 days. two days later a test done for influenza a returned positive (influenza a and b rna, qualitative real-time pcr, quest diagnostics). one week after the onset of symptoms, he had completed his course of oseltamivir, however his symptoms did not improve. he went to the emergency room, temperature was 102.9f (39.4c), pulse 83, oxygen saturation 93% on 2 liters nasal cannula, blood pressure was 136/71. he was table table i clinical characteristics and outcome of 4 hiv and covid-19 coinfection clinical features of patients infected with 2019 novel coronavirus in wuhan, china co-infection of sars-cov-2 and hiv in a patient in wuhan city association of chemosensory dysfunction and covid-19 in patients presenting with influenza-like symptoms diarrhea during covid-19 infection: pathogenesis, epidemiology, prevention and management smell dysfunction: a biomarker for covid-19. int forum allergy rhinol a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 cd4 t cell count measured in cells/ul; tenofovir af, tenofovir alafenamide, hld, hyperlipidemia; htn, hypertension, t2dm, type ii diabetes mellitus key: cord-032504-tmohg1mj authors: auld, sara c.; staitieh, bashar s. title: hiv and the tuberculosis “set point”: how hiv impairs alveolar macrophage responses to tuberculosis and sets the stage for progressive disease date: 2020-09-23 journal: retrovirology doi: 10.1186/s12977-020-00540-2 sha: doc_id: 32504 cord_uid: tmohg1mj as hiv has fueled a global resurgence of tuberculosis over the last several decades, there is a growing awareness that hiv-mediated impairments in both innate and adaptive immunity contribute to the heightened risk of tuberculosis in people with hiv. since early immune responses to mycobacterium tuberculosis (mtb) set the stage for subsequent control or progression to active tuberculosis disease, early host–pathogen interactions following mtb infection can be thought of as establishing a mycobacterial “set point,” which we define as the mycobacterial burden at the point of adaptive immune activation. this early immune response is impaired in the context of hiv coinfection, allowing for a higher mycobacterial set point and greater likelihood of progression to active disease with greater bacterial burden. alveolar macrophages, as the first cells to encounter mtb in the lungs, play a critical role in containing mtb growth and establishing the mycobacterial set point. however, a number of key macrophage functions, ranging from pathogen recognition and uptake to phagocytosis and microbial killing, are blunted in hiv coinfection. to date, research evaluating the effects of hiv on the alveolar macrophage response to mtb has been relatively limited, particularly with regard to the critical early events that help to dictate the mycobacterial set point. a greater understanding of alveolar macrophage functions impacted by hiv coinfection will improve our understanding of protective immunity to mtb and may reveal novel pathways amenable to intervention to improve both early immune control of mtb and clinical outcomes for the millions of people worldwide infected with hiv. although tuberculosis incidence declined worldwide over the course of the 20 th century [1] [2] [3] [4] , these gains were quickly reversed with the onset of the hiv epidemic in the 1980s [5] . tuberculosis is now the leading global killer among infectious diseases and the leading cause of death among people with hiv [6] . the profound t cell depletion induced by hiv has long been recognized as a significant driver of the tuberculosis epidemic in people with hiv [7, 8] . however, there is growing awareness that hiv-mediated impairments in innate immunity also contribute to the increased risk of tuberculosis in this vulnerable population. these hiv-mediated impairments in innate immunity are particularly relevant early in mycobacterium tuberculosis (mtb) infection, prior to the onset of adaptive immune responses [9] and, by impairing early control of mtb, set the stage for accelerated progression to active tuberculosis disease. the pattern of early immune control setting the stage for longer term control-and clinically meaningful endpoints-parallels our understanding of the hiv set point. it was first recognized in the late 1990s that the hiv viral load soon after seroconversion, i.e., the hiv set point, is predictive of disease progression [10, 11] . following infection, hiv replicates unchecked until an initial, albeit ineffective, host immune response develops and the set point is established [12] [13] [14] . when viewed through this same lens, early host-pathogen interactions following mtb infection can be thought of as establishing a "mycobacterial set point" (fig. 1) . unlike the set point in hiv, which occurs after an ineffective adaptive immune response, the mycobacterial set point is established before the onset of adaptive immunity. although we currently lack the tools to measure the mycobaterial set point clinically, this theoretical construct provides a framework for understanding early immune responses to mtb, particularly those of the alveolar macrophage. while a multitude of cell types, including recruited interstitial macrophages, dendritic cells, and neutrophils, are infected prior to the onset of adaptive immunity [15] , work by cohen and others has confirmed the importance of the alveolar macrophage to the early mtb response [16] . as we will emphasize in this review, a focus on this critical early period and the mycobacterial set point are useful in understanding the relative impacts, both on a cellular and clinical level, of various host and pathogen impairments that occur in the context of coinfection with mtb and hiv. the concept of a mycobacterial set point also provides us with an overall index of immune health in the context of hiv/mtb coinfection and thereby offers a useful gauge of efficacy when specific pathways are manipulated for therapeutic benefit. host immunity to mtb remains poorly understood, but a focus on the role of alveolar macrophages in establishing a mycobacterial set point and on alterations in macrophage control in the context of hiv coinfection has the potential to yield great insight into some of the earliest immune events in mtb infection. although mtb-and hiv-mediated impairments in pulmonary innate immunity and alveolar macrophage function have been reviewed elsewhere [17, 18] , the impact of these impairments in the context of coinfection has not been well described. in this review, we will explore how hiv coinfection makes alveolar macrophages, the first cells to encounter mtb, more permissive of early mycobacterial growth and is therefore likely to increase the mycobacterial burden at the point at which adapative immunity is activated and the mycobacterial set point is established [16] . by curtailing early immune control and delaying the onset of adaptive immunity, hiv coinfection leads to a higher mycobacterial set point and, consequently, a greater bacterial burden (fig. 1) . in order to demonstrate the utility of the mycobacterial set point framework, we will first review epidemiologic data supporting the importance of innate immunity in people with tuberculosis and hiv coinfection. we will then discuss some of the model systems that are used to study macrophage responses to mtb and hiv and animal model findings in support of a mycobacterial set point. we will then highlight existing data on alveolar macrophage functions-from macrophage phenotype, key cytokines, and oxidative stress, to pathogen recognition and phagocytosis, cell death pathways, and activation fig. 1 graphical depiction of the mycobacterial set point as a theoretical construct, represented as the mycobacterium tuberculosis (mtb) bacterial burden after infection, either alone or in the context of hiv coinfection. after infection, mtb bacterial burden increases until adaptive immunity is activated, thereby establishing the mycobacterial set point. alveolar macrophages, the first cells to encounter mtb, are an important component of the early innate immune response to mtb. as reviewed in the main text, the mycobacterial burden at the point of adaptive immune activation plays a important role in the subsequent control of infection and progression to active tuberculosis disease. in the case of hiv coinfection, impairments in alveolar macrophage functions lead to poor early immune control of mtb which, in turn, leads to a higher mycobacterial set point and greater mtb bacterial burden at the time when adaptive immunity is activated of adaptive immunity-that are impacted by either mtb infection, hiv infection, or coinfection. the examples we provide, which come from a range of cell types and organ systems, including primary alveolar macrophages, monocyte-derived macrophages, and cell lines, are not meant to be exhaustive or comprehensive. rather, our goal is to provoke further inquiry and investigation into this critical area by offering a framework by which to contextualize studies in the field. we will close the review by highlighting several promising avenues for studying the mycobacterial set point in humans, in order to underscore the potential benefit of this concept as a framework for clinically relevant research. from the earliest days of the hiv epidemic it has been clear that tuberculosis outcomes were markedly worse in people with hiv. in a series of nosocomial tuberculosis outbreaks among people with hiv in the early 1990s, the overwhelming majority of patients died, often within a month or two of their diagnosis [19] [20] [21] . these individuals had very advanced hiv, which undoubtedly contributed to their high mortality rates [21] . however, their rapid progression from exposure to active disease, in a time frame prior to the expected onset of adaptive immunity, suggests that impaired innate immunity was likely a contributing factor. similarly, in a study of south african gold miners newly infected with hiv, the risk of tuberculosis disease was found to increase very early in the course of hiv infection, when cd4 counts would still be in a normal range [22] . another study from south africa found that people with hiv on antiretroviral therapy (art) with normal cd4 counts had four times the risk of tuberculosis than people without hiv [23] . likewise, in italy, a setting with a low burden of both hiv and tuberculosis, nearly a third of patients diagnosed with hiv-associated tuberculosis in one study had been receiving art for a median of 27 months, at which point cd4 counts would typically have reached a normal range [24] . taken together, these data offer epidemiologic evidence that defects in both innate and adaptive immunity are likely to contribute to the greater risk of tuberculosis in people with hiv. while these clinical observations cannot establish mechanism or causality, they do provide a rationale for pursuing further basic science and translational studies to better understand the role of innate immunity in controlling mtb infection. over the last century, scientists from successive eras have made remarkable discoveries by bringing the newest available techniques to bear on their investigations of mtb, with various model systems employed over time. while each system has distinct advantages and disadvantages in addressing particular questions, the sheer volume of data derived from the many available systems can make it hard to develop a coherent narrative regarding mtb infection in humans. although a full review of these features of the tuberculosis literature is beyond the scope of this review, we will offer a brief overview of the main characteristics of some of the major models to provide context for the reader [25, 26] . despite their inability to model sophisticated cellular interactions, in vitro systems have long been used as tools for studying mtb [27] . a variety of myeloid cell lines have been employed, and are often helpful in quantifying cytokine and other signaling responses to mtb. in addition, due to the ease with which in vitro systems can be manipulated, studies with primary macrophages from animal models have enabled investigators to dissect the roles of individual proteins and pathways in the mtb response [28, 29] . finally, human primary cells have been used in a variety of ways, including induced pluripotent stem cells, human macrophages, granuloma models, and many more. each system has proven useful in addressing different aspects of the mtb response, but all are hampered by their inability to coordinate the full spectrum of in vivo responses. an additional challenge is the conflation of different types of macrophages, whether from cell-lines, animal models, or humans, as a homogenous population. rather, there is a growing appreciation of the distinctions between tissue-derived and bone-marrow derived macrophages, from their ontogeny to functional capacity and self-maintenance [30] [31] [32] . these distinctions are highly relevant to this review, as bone-marrow derived macrophages do not become polarized in the same manner as alveolar macrophages following mtb exposure, and thus cannot be expected to behave the same [33] . however, given challenges in obtaining primary alveolar macrophages, particularly from humans [34] , many studies are conducted with derived cells. with regards to animal systems, ease of use in handling the zebrafish-mycobacterium marinum model has made it a powerful tool for the study of mtb [35] . several key components of the human response to mtb are present in zebrafish, including the leukotriene a4 hydrolase locus, and it has been used to study everything from treatment strategies for drug-resistant mtb to vaccine development. the mouse model has the virtue of capturing vertebrate, mammalian immune responses in a reproducible, easily scalable system. however, different mouse strains have different levels of susceptibility to mtb and mice do not typically form granulomas or cavities [36] . as a result, the mouse model tends to be suited for studies of cell-mediated immunity and drug development, but less useful for studies of human clinical outcomes [37] . guinea pigs provide another small animal model, with the benefit of recapitulating lung necrosis and other human features, but challenges in measuring their immune responses has historically limited their widespread use [25] . rabbits have also provided an effective model system for mtb research, largely due to the fact that the structural similarities between their lungs and human lungs leads to similar pathology in response to mtb. that said, their larger size and the greater resources required to utilize them has limited their broad applicability [36, 38] . finally, non-human primates, particularly rhesus and cynomolgus macaques, have marked genetic and immunologic similarities to humans and, as such, have yielded significant advances in our understanding of host-pathogen interactions and the potential for vaccineinduced protection in recent years [39, 40] . however, given the vast resources needed to sustain these animals, there are a limited number of research centers capable of conducting non-human primate studies. while in-depth analysis of early immunologic events in human mtb infection remains challenging for the reasons discussed above, poulsen and others established over 50 years ago that tuberculin skin test (tst) conversion in humans, an indicator of the onset of the adaptive immune response, generally occurred by 6 weeks after exposure [41] [42] [43] . poulsen also determined that this period prior to tst conversion is not a time of immunologic quiescence; rather, many individuals had evidence of a robust inflammatory response with fever and elevated inflammatory markers. since those landmark studies, much of our understanding of these early events has relied on data from animal models. experiments in rabbits and guinea pigs by lurie, smith, and harding, established that macrophages provide a niche within which mtb multiplies logarithmically during early infection [44, 45] . others have demonstrated that the onset of adaptive immunity in response to mtb is indeed delayed, arising in part from delayed activation of cd4+ t cells within local lymph nodes [46, 47] . this delay of several weeks, as compared to several days with organisms such as salmonella typhi and listeria monocytogenes, translates into relatively unchecked bacterial growth during the early phase of infection [48, 49] . after this period of logarithmic growth, which has been demonstrated in rabbits, mice, and guinea pigs, the bacteria enter a relatively stationary phase [50] . during this critical time, mtb subverts alveolar macrophage defenses, thereby ensuring prolonged intracellular survival and mycobacterial growth within this pulmonary niche. whether this failure of immunity represents an intrinsic deficiency of macrophages or an insufficient activation of macrophages by t cells remains uncertain [51] . as discussed above, there has been a proliferation of work with non-human primate models in recent years that shed further light on the establishment of the set point, particularly with regards to granuloma formation [52] . studies of macaques with aerosol exposure to mtb have found that early mycobacterial dissemination, as reflected by the development of new granulomas in the first 3-6 weeks following infection (i.e., the period during which the set point is established), is associated with subsequent progression to active disease [53, 54] . conversely, the absence of new granuloma formation, which could be construed as a "low" set point, is associated with immune control and long-term maintenance of latent infection. granuloma size can also stratify the risk of dissemination, with larger granulomas associated with a greater potential for future mycobacterial spread [55] . by linking early immunologic events to clinically meaningful outcomes, these findings underscore the critical importance of early immune control prior to the onset of adaptive immunity. they also provide evidence to support the longstanding belief that progression to tuberculosis disease is determined at the level of the individual granuloma. as the first cell type to encounter mtb, the alveolar macrophage is a key player in the early stages of infection and mtb is known to both cause and benefit from certain alterations in macrophage functionality [56] . several excellent reviews have recently examined the heterogeneity and plasticity of macrophages, in addition to proposing a uniform approach to macrophage nomenclature [31, 32] . alveolar macrophages play myriad roles in the lungs, from housekeeper to innate immune effector, and their role depends heavily on the microenvironment within the alveolar space. for example, in response to inflammatory stimuli such as lipopolysaccharide (lps), alveolar macrophages develop an inflammatory phenotype with increased production of cytokines such as tnf-α and il-6. conversely, in the setting of il-4 exposure, alveolar macrophages shift to an immunosuppressive phenotype and increase production of regulatory cytokines such as tgf-β [31] . although debate continues as to how best to characterize macrophages and how dynamically tissue resident subtypes respond to their local cytokine milieu, it is well known that macrophages respond to different cytokine programs with a variety of functional changes. most experts agree that these changes exist on a spectrum between the classically activated (or m1, or inflammatory) macrophage and an alternatively activated (or m2, or regulatory) macrophage. in the context of mtb infection, the "macrophage polarization ratio, " an indicator of the balance between proinflammatory and anti-inflammatory genes, has been employed to explore how macrophage functionality and subsequent granuloma formation affects the probabilities of mtb control and dissemination (fig. 2) [57] . with this approach, nf-kb signaling was found to be an essential component of the classical activation profile, which, when triggered early, leads to improved clinical tuberculosis outcomes in a nonhuman primate model [57] . alveolar macrophages are also severely impacted by the presence of hiv infection and have been shown to be a reservoir for the virus within the lungs [58] . importantly, coinfection shifts macrophage polarization in a fashion favorable for pathogen survival [59] . both hiv and mtb stimulate a cytokine milieu in the alveolar space that pushes macrophages into an inflammatory phenotype optimized for the uptake of foreign microbes. although these inflammatory cells would ordinarily excel at pathogen killing, the intracellular effects of hiv and mtb render the macrophages less effective and, as a result, permit ongoing pathogen growth [60] . at the same time, there is ample evidence that mtb infection of alveolar macrophages creates a permissive environment for hiv infection and replication, through a variety of mechanisms including increased surface expression of the hiv receptors ccr5 and cxcr4, increased hiv transcription and replication, and induction of nanotubes that promote cell-to-cell hiv viral spread between macrophages [61] [62] [63] [64] . taken together, this synergy between mtb-and hiv-mediated impacts on macrophages enables intracellular growth and survival of both pathogens and, more broadly, fuels the global syndemic of coinfection. macrophage function and polarization is also influenced by immunometabolism, the balance of cellular energy utilization. this represents another growing area of inquiry in which mtb and hiv are likely to have complementary, yet deleterious, effects. mtb infection of alveolar macrophages has been shown to shift cellular metabolism from oxidative phosphorylation to aerobic glycolysis, a shift associated with a more inflammatory, m1-like phenotype [65] . this shift triggers an increase in cellular il-1β production along with a decrease in il-10 production, which, in concert, should increase bacillary killing in vitro. yet, other investigations suggest that persistence of fatty acid oxidation in alveolar macrophages may permit ongoing intracellular mtb growth [66] . at the same time, hiv infection also alters cellular energy balance, oxidative stress levels, and mitochondrial bioenergetics, including for infected macrophages [67] [68] [69] [70] . further study of the combined, net effects of mtb and hiv on alveolar macrophage immunometabolism is needed to expand our understanding of how coinfection impairs early mycobacterial control. macrophage cytokine expression profiles may also vary according to the stage of mtb infection, whereby the secreted protein esat-6 initially skews macrophages towards a m1 phenotype, potentially to facilitate the establishment of a granuloma, and then later to a more permissive m2 phenotype [71] . ifn-γ, a paradigmatic pro-inflammatory cytokine, drives macrophages towards an inflammatory phenotype and, under normal circumstances, is critical for mtb control. however, peripheral blood mononuclear cells isolated from people with hiv have depressed ifn-γ production in response to purified protein derivative (ppd) [72, 73] . il-4, another key cytokine for macrophage polarization, is induced by hiv glycoprotein (gp) 120 [74] . this induction shifts macrophages to an anti-inflammatory phenotype, potentially further reducing early mtb control. in people without hiv, elevated il-4 levels have been associated with progression to tuberculosis disease and the development of pulmonary cavities, thus underscoring the clinical impact of these shifts [75] [76] [77] . interestingly, the relative balance of these cytokines may vary according to the stage of hiv infection, which could translate into differential susceptibility to and control of mtb infection [60] . several key cytokines important for both macrophage polarization and the response to mtb are also altered in the presence of hiv coinfection. for example, gm-csf plays an essential role in cellular control of mtb [78, 79] and its activity is impaired in monocyte-derived macrophages infected with hiv [80] . the gm-csf:m-csf balance is also altered by the presence of mtb [81] and m-csf-driven macrophages are more permissive to mtb replication and dissemination [82] . similarly, interleukin-1 receptor-associated kinase m (irak-m), which is downstream of the gm-csf-dependent transcription factor pu.1, correlates directly with mycobacterial load in human lung tissue and impacts mtb survival [83] . thus, the combined impact of mtb and hiv on the balance of cytokines within the lungs leads to an environment that permits the growth of both pathogens. another avenue ripe for further exploration is the effects of hiv and mtb on redox systems, which can also modulate macrophage function [84] [85] [86] . hiv is known to impair antioxidant defenses in the alveolar macrophage and raise levels of oxidative stress in the lung more generally [18, 87] . the effects in the lung are likely due, at least in part, to glutathione depletion in the airways of people with hiv [88, 89] . mtb, on the other hand, has successfully adapted to hostile intracellular conditions, including high oxidative stress [90, 91] . mtb may also directly contribute to oxidative stress by inducing heme oxygenase (ho)-1, the levels of which directly correlate with treatment outcomes in people with active tuberculosis disease [92] . several studies have demonstrated improved mtb control with glutathione supplementation, including one study using macrophages isolated from people with hiv in which supplementation led to enhanced inflammatory cytokine production and restored macrophage capacity to control mtb growth [89, [93] [94] [95] [96] [97] . of note, the oxidative stress induced by mtb-infected macrophages is also likely to promote hiv reactivation, further complicating the relationship between these two pathogens [98] . an important function of macrophages in the alveolar space is the differentiation of friend versus foe via pathogen recognition and phagocytosis (fig. 3 ) [99] [100] [101] . particularly germane to the recognition of mtb is the mannose receptor, which is downregulated in people living with hiv [102] . hiv also leads to reduced expression and activity of toll-like receptors (tlr), another key receptor for macrophage pathogen recognition [103] [104] [105] . when combined with shifts in tlr-mediated regulation of nitric oxide production induced by mtb [104] , these impairments have the potential to reduce mtb uptake by alveolar macrophages and facilitate prolonged extracellular growth. once a pathogen like mtb has been recognized, it then undergoes phagocytosis [99, 100] . hiv creates favorable conditions for bacteria by impairing phagocytosis of pathogens including streptococcus [106] . the hiv protein nef has also been shown to interfere with phagosome formation by impeding endosomal recruitment and remodeling [107] . of note, impaired phagocytosis has been observed even in alveolar macrophages not directly infected by hiv [58, 108] . while phagocytosis of mtb in the setting of hiv has not specifically been studied, such impairments would enable further extracellular growth [18, 109] . hiv has also been found in macrophage endosomes and is known to impair phagolysosomal fusion in the macrophage [110, 111] . at least some of these impairments may be mediated through the effects of the hivrelated protein transactivator of transcription (tat) [112] . another hiv protein, vpr, has also been shown to perturb phagolysosomal maturation by altering microtubule-dependent trafficking within macrophages [113] . in addition, hiv has been shown to co-opt the rag gtpases, resulting in further disruption of endosomal trafficking [114] . this disruption may facilitate hiv replication and simultaneously assist mtb evasion of lysosomal fusion and killing. il-10 has also been shown to dysregulate the response to mtb in the setting of hiv, at least in part by impairing phagosome maturation [115, 116] . however, in one study of alveolar macrophages from humans with active tuberculosis disease, phagolysosomal acidification was equally disrupted among those with and without hiv, highlighting existing uncertainties about the impacts of mtb and hiv on alveolar macrophage phagocytosis [117] . thus, although further research is needed to define the relevant mechanisms by which hiv impairs the phagocytic response to mtb, the bulk of the existing data suggest that endosomes fail to fuse with acidified lysosomes, thereby promoting mtb survival [118] . alveolar macrophage cell death pathways range from apoptosis to autophagy and necrosis. mtb is known to shift the balance toward necrosis by actively inhibiting apoptosis, thereby allowing the extracellular release of viable mycobacteria from dying alveolar macrophages [119, 120] . similarly, hiv, via the viral proteins tat and glycoprotein-120 (gp-120), stimulates the triggering receptor expressed on myeloid cells-1 (trem-1), thereby inhibiting apoptosis in a p65-dependent manner [121] . hiv may also directly induce autophagy through the nef protein, in order to modulate viral replication and survival, albeit in a manner that may be synergistic with mtb-mediated inhibition of apoptosis [122, 123] . gp-120 also impairs apoptosis-associated killing of pneumococcus within alveolar macrophages, indicating that this impairment may not be specific to mtb [124] . impaired apoptosis of mtb has been tied by other investigators to il-10, which is increased in the bronchoalveolar lavage fluid of people with hiv [125] . additional research is needed to further delineate the impact of these shifts in cell death pathways and to identify whether there are therapeutic opportunities to improve mtb control in patients with and without hiv. one promising recent report found that all-trans retinoic acid was able to promote autophagy of human alveolar macrophages in the setting of mtb infection [126] . the final step required for establishment of the mycobacterial set point is activation of the adaptive arm of the immune system (fig. 3) . mtb has been shown to delay the activation of adaptive immunity in mice by slowing bacterial transport to local lymph nodes [46] . this delay allows for a prolonged period of unchecked bacterial growth within the lungs, with a 10,000-100,000-fold expansion of the mtb pulmonary population during this time. at least one driver of this delay is mtb-mediated inhibition of mhc class ii antigen presentation by dendritic [127] . mtb also manipulates antigen presentation by diverting bacterial proteins through a vesicular export pathway, away from mhc class ii presentation. in the context of hiv, the viral protein nef has been shown to promote the expression of immature, functionally incompetent mhc class ii complexes [128, 129] . recent data have more directly linked these two by demonstrating that hiv-infected dendritic cells have reduced ability to upregulate key costimulatory molecules critical for antigen presentation (e.g., cd40, cd80, and cd86) in the presence of mtb infection [130] . all of this would combine to delay the onset of adapative immunity and further increase the mycobacterial set point. as should be clear from the preceding sections, studies detailing the effects of mtb and hiv on the immune responses within the alveolar space have found an abundance of affected cytokines, pathways, and response elements. although much of the available data suggests ways in which mtb and hiv each make it more likely for the other to gain ground within an individual's lungs, the myriad as-yet-unstudied pathways leave open the distinct possibility that some of their particular effects may cumulatively cancel out those of the other pathogen. it is in this key domain that we believe the concept of a mycobacterial set point most clearly demonstrates its worth. because the set point relates to the mycobacterial burden at the moment in which the adaptive immune system activates, it offers a clinically relevant marker that synthesizes the various effects of coinfections, comorbidities, and therapeutics. as a research tool, then, it will help us understand whether the net effect of a given intervention actually reduces the overall mycobacterial burden, as opposed to fruitfully manipulating a single pathway (with the knowledge that doing so may have deleterious effects on others). fortunately, several novel approaches provide hope for expanding our understanding of these early immunologic events in humans. these tools have the potential to vastly enhance our current knowledge by quantifying the functional impact of coinfection on bacterial burden and early human immune responses, while also offering insight into mechanisms underlying early immune control or escape. the first such tool is 2-deoxy-2-[18f]fluoro-d-glucose ([18f]fdg) positron emission tomography combined with computed tomography (pet-ct), which identifies areas of increased cellular metabolic activity as would occur with an immune response to infection. several macaque studies have leveraged pet-ct imaging to gain insight into early infection by following the progression of individual granulomas after aerosol infection [53, 54, 131, 132] . pet-ct has also been employed to study tuberculosis in humans, including a cohort of 35 people with hiv and latent tuberculosis infection, 10 of whom had pet-avid lesions suspicious for subclinical disease [133] . those with pet activity were significantly more likely to develop active tuberculosis disease during the following 6 months, suggesting metabolic activity on pet imaging may be able to identify patients with ineffective early immune responses to mtb infection. blood-based immunologic signatures represent another potential avenue for studying early infection. recent studies in macaques and humans have identified rna signatures predictive of progression to active tuberculosis disease [134] [135] [136] . notably, the macaque signature was predictive just 3-6 weeks after aerosol infection, providing compelling evidence for the value of these signatures even very early in infection. this signature also identified a novel gene in tuberculosis pathogenesis, prdx2, a member of the reactive oxygen species scavenger system. further studies are warranted to explore the role of prdx2 in early infection, and how that response may be skewed by hiv and other known tuberculosis risk factors. similarly, characterization of mtb-specific t cell responses may provide another means for understanding adaptive immune responses important in early infection. in a recent report of a whole blood assay of t cell responses to 60 mtb antigens, immunogenic mtb antigens were successfully identified across a diverse range of populations and infectious states [137] . longitudinal studies are underway to determine if these t cell signatures correlate with early immune responses-both those that clear infection and those that permit ongoing mtb growth and dissemination. yet another tool for identifying and studying early infection is plasma metabolomics, in which small molecules are identified using high-resolution mass spectrometry. this approach has been successfully employed to identify direct metabolites of mtb, and may be able to identify low levels even early in infection. metabolomics has also been used to identify distinct signatures of the host immune response, whereby investigators were able to distinguish between risk-associated metabolites associated with a higher likelihood of progression to tuberculosis disease and disease-associated metabolites that increased in a time-dependent manner among those with progression to active tuberculosis disease [138] [139] [140] . looking ahead, it will be important to determine whether these metabolic signatures are similarly predictive and valid in people with hiv. across the scientific community, there is overwhelming consensus over the need to understand protective immunity to mtb [55] . as a framework for considering early immunologic responses to mtb, the mycobacterial set point provides a useful construct for understanding the impact of early immune control and clinical outcomes of tuberculosis. given the disproportionate burden of tuberculosis disease and deaths among people with hiv despite widespread art [23] , it is imperative to study the unique impacts of hiv on mtb infection and susceptibility. to date, research evaluating the effects of hiv on the alveolar macrophage response to mtb has been relatively limited, particularly with regard to the critical early events that help to dictate the mycobacterial set point. while we believe that a higher mycobacterial set point is a key contributor to the increased morbidity among people with hiv, additional research is needed to better understand the mechanisms and biological pathways that may be involved in facilitating mycobacterial immune evasion. a greater understanding of the impact of hiv on these earliest responses to mtb may lead to the development of novel interventions and hostdirected therapies to reduce the persistent global burden of tuberculosis in this vulnerable population. treatment of tuberculosis. a historical perspective directly observed therapy for tuberculosis: history of an idea principles of iuatld collaborative tuberculosis progammes tuberculosis therapy: past, present and future pneumocystis pneumonia-los angeles hiv and tuberculosis: a deadly human syndemic the importance of first impressions: early events in mycobacterium tuberculosis infection influence outcome the risk of disease progression is determined during the first year of human immunodeficiency virus type 1 infection initial plasma hiv-1 rna levels and progression to aids in women and men a whole-genome association study of major determinants for host control of hiv-1 host genetic and viral determinants of hiv-1 rna set point among hiv-1 seroconverters from sub-saharan africa estimating the respective contributions of human and viral genetic variation to hiv control beyond macrophages: the diversity of mononuclear cells in tuberculosis alveolar macrophages provide an early mycobacterium tuberculosis niche and initiate dissemination mechanisms of m. tuberculosis immune evasion as challenges to tb vaccine design pulmonary innate immune dysfunction in human immunodeficiency virus centers for disease control and prevention. multidrug-resistant tuberculosis outbreak on an hiv nosocomial transmission of multidrug-resistant tuberculosis among hiv-infected persons-florida an outbreak of multidrug-resistant tuberculosis involving hiv-infected patients of two hospitals in milan, italy. italian multidrug-resistant tuberculosis outbreak study group how soon after infection with hiv does the risk of tuberculosis start to increase? a retrospective cohort study in south african gold miners tuberculosis incidence rates during 8 years of follow-up of an antiretroviral treatment cohort in south africa: comparison with rates in the community tuberculosis in hiv-infected persons in the context of wide availability of highly active antiretroviral therapy animal models of tuberculosis: an overview animal models of cavitation in pulmonary tuberculosis killing mycobacterium tuberculosis in vitro: what model systems can teach us nitric oxide synthase in innate and adaptive immunity: an update large scale comparison of innate responses to viral and bacterial pathogens in mouse and macaque tissue-resident macrophage ontogeny and homeostasis macrophage activation and polarization: nomenclature and experimental guidelines macrophage heterogeneity in tissues: phenotypic diversity and functions differential polarization of alveolar macrophages and bone marrow-derived monocytes following chemically and pathogeninduced chronic lung inflammation human lung immunity against mycobacterium tuberculosis: insights into pathogenesis and protection the zebrafish model of tuberculosis-no lungs needed mouse model of tuberculosis animal models of tuberculosis: lesson learnt anti-vascular endothelial growth factor treatment normalizes tuberculosis granuloma vasculature and improves small molecule delivery prevention of tuberculosis in macaques after intravenous bcg immunization monkey models of tuberculosis: lessons learned some clinical features of tuberculosis. 1. incubation period tuberculous infection in the light of tuberculin matriculation the time-table of tuberculosis resistance to tuberculosis: experimental studies in native and acquired defensive mechanism animal model: experimental airborne tuberculosis in the guinea pig initiation of the adaptive immune response to mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs delayed-type hypersensitivity and cell-mediated immunity in the pathogenesis of tuberculosis tracking salmonella-specific cd4 t cells in vivo reveals a local mucosal response to a disseminated infection organ-specific cd4+ t cell response during listeria monocytogenes infection progressive pulmonary tuberculosis is not due to increasing numbers of viable bacilli in rabbits, mice and guinea pigs, but is due to a continuous host response to mycobacterial products immunity to tuberculosis experimental mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human m. tuberculosis infection early changes by (18)fluorodeoxyglucose positron emission tomography coregistered with computed tomography predict outcome after mycobacterium tuberculosis infection in cynomolgus macaques digitally barcoding mycobacterium tuberculosis reveals in vivo infection dynamics in the macaque model of tuberculosis protective immunity against tuberculosis: what does it look like and how do we find it? intranasal poly-ic treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population macrophage polarization drives granuloma outcome during mycobacterium tuberculosis infection healthy hiv-1-infected individuals on highly active antiretroviral therapy harbor hiv-1 in their alveolar macrophages macrophage polarization: convergence point targeted by mycobacterium tuberculosis and hiv the macrophage in hiv-1 infection: from activation to deactivation? tuberculosis exacerbates hiv-1 infection through il-10/stat3-dependent tunneling nanotube formation in macrophages high turnover of tissue macrophages contributes to tuberculosis reactivation in simian immunodeficiency virus-infected rhesus macaques mycobacterium tuberculosis and hiv coinfection brings fire and fury to macrophages the mtb-hiv syndemic interaction: why treating m. tuberculosis infection may be crucial for hiv-1 eradication cutting edge: mycobacterium tuberculosis induces aerobic glycolysis in human alveolar macrophages that is required for control of intracellular bacillary replication growth of mycobacterium tuberculosis in vivo segregates with host macrophage metabolism and ontogeny emerging role and characterization of immunometabolism: relevance to hiv pathogenesis intracellular expression of tat alters mitochondrial functions in t cells: a potential mechanism to understand mitochondrial damage during hiv-1 replication abnormal immunometabolism and gene accessibility in alveolar macrophages in hiv hiv infection and latency induce a unique metabolic signature in human macrophages mycobacterium tuberculosis virulent factor esat-6 drives macrophage differentiation toward the pro-inflammatory m1 phenotype and subsequently switches it to the anti-inflammatory m2 phenotype depressed t-cell interferon-gamma responses in pulmonary tuberculosis: analysis of underlying mechanisms and modulation with therapy the significance of interferon-γ in hiv-1 pathogenesis, therapy, and prophylaxis hiv-1 gp120 induces il-4 and il-13 release from human fcεri + cells through interaction with the v h 3 region of ige endogenously activated interleukin-4 differentiates disease progressors and non-progressors in tuberculosis susceptible families: a 2-year biomarkers follow-up study increased interleukin-4 production by cd8 and gammadelta t cells in health-care workers is associated with the subsequent development of active tuberculosis increased production of interleukin 4 by cd4+ and cd8+ t cells from patients with tuberculosis is related to the presence of pulmonary cavities role of granulocyte-macrophage colony-stimulating factor production by t cells during mycobacterium tuberculosis infection understanding tuberculosis-analyzing the origin of mycobacterium tuberculosis pathogenicity human immunodeficiency virus type 1 infection inhibits granulocyte-macrophage colony-stimulating factor-induced activation of stat5a in human monocyte-derived macrophages relative levels of m-csf and gm-csf influence the specific generation of macrophage populations during infection with mycobacterium tuberculosis mycobacterium tuberculosis replicates within necrotic human macrophages irak-m alters the polarity of macrophages to facilitate the survival of mycobacterium tuberculosis glutathione and infection mechanisms of control of mycobacterium tuberculosis by nk cells: role of glutathione glutathione levels in antigen-presenting cells modulate th1 versus th2 response patterns oxidative stress during hiv infection: mechanisms and consequences anti-retroviral therapy is associated with decreased alveolar glutathione levels even in healthy hiv-infected individuals glutathione supplementation improves macrophage functions in hiv mycobacterium tuberculosis whib3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response redox biology of tuberculosis pathogenesis mycobacterium tuberculosis induction of heme oxygenase-1 expression is dependent on oxidative stress and reflects treatment outcomes glutathione-redox balance regulates c-rel-driven il-12 production in macrophages: possible implications in antituberculosis immunotherapy evidence for oxidative stress and defective antioxidant response in guinea pigs with tuberculosis glutathione levels and immune responses in tuberculosis patients liposomal glutathione supplementation restores th1 cytokine response to mycobacterium tuberculosis infection in hiv-infected individuals glutathione and adaptive immune responses against mycobacterium tuberculosis infection in healthy and hiv infected individuals mycobacterium tuberculosis reactivates hiv-1 via exosome-mediated resetting of cellular redox potential and bioenergetics. mbio the delivery of endocytosed cargo to lysosomes endosome-lysosome fusion insights into macrophage autophagy in latent tuberculosis infection: role of heat shock protein 16.3 reduced binding and phagocytosis of pneumocystis carinii by alveolar macrophages from persons infected with hiv-1 correlates with mannose receptor downregulation toll-like receptor 2-dependent extracellular signalregulated kinase signaling in mycobacterium tuberculosis-infected macrophages drives anti-inflammatory responses and inhibits th1 polarization of responding t cells toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens human immunodeficiency virus infection alters tumor necrosis factor alpha production via toll-like receptor-dependent pathways in alveolar macrophages and u1 cells the alveolar microenvironment of patients infected with human immunodeficiency virus does not modify alveolar macrophage interactions with streptococcus pneumoniae inhibition of phagocytosis in hiv-1-infected macrophages relies on nef-dependent alteration of focal delivery of recycling compartments small alveolar macrophages are infected preferentially by hiv and exhibit impaired phagocytic function defective phagocytosis by human monocyte/macrophages following hiv-1 infection: underlying mechanisms and modulation by adjunctive cytokine therapy hiv-1 buds and accumulates in "nonacidic" endosomes of macrophages impairment of phagosome-lysosome fusion in hiv-1-infected macrophages hiv-1 tat alters neuronal autophagy by modulating autophagosome fusion to the lysosome: implications for hiv-associated neurocognitive disorders the hiv-1 protein vpr impairs phagosome maturation by controlling microtubule-dependent trafficking hiv-1 enhances mtorc1 activity and repositions lysosomes to the periphery by co-opting rag gtpases hiv-1 infection of macrophages dysregulates innate immune responses to mycobacterium tuberculosis by inhibition of interleukin-10 il-10 blocks phagosome maturation in mycobacterium tuberculosis-infected human macrophages mycobacterium tuberculosis resides in nonacidified vacuoles in endocytically competent alveolar macrophages from patients with tuberculosis and hiv infection endosomal membrane traffic: convergence point targeted by mycobacterium tuberculosis and hiv apoptosis is an innate defense function of macrophages against mycobacterium tuberculosis cell death and autophagy in tuberculosis hiv-related proteins prolong macrophage survival through induction of triggering receptor expressed on myeloid cells-1 human immunodeficiency virus type 1 nef inhibits autophagy through transcription factor eb sequestration mycobacterium tuberculosis and human immunodeficiency virus type 1 cooperatively modulate macrophage apoptosis via toll like receptor 2 and calcium homeostasis hiv gp120 in the lungs of antiretroviral therapy-treated individuals impairs alveolar macrophage responses to pneumococci impaired m. tuberculosismediated apoptosis in alveolar macrophages from hiv + persons: potential role of il-10 and bcl-3 all-trans retinoic acid augments autophagy during intracellular bacterial infection mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo hiv-1 nef impairs mhc class ii antigen presentation and surface expression nef alleles from children with non-progressive hiv-1 infection modulate mhc-ii expression more efficiently than those from rapid progressors hiv interferes with mycobacterium tuberculosis antigen presentation in human dendritic cells analysis of 18fdg pet/ct imaging as a tool for studying mycobacterium tuberculosis infection and treatment in non-human primates sterilization of granulomas is common in active and latent tuberculosis despite within-host variability in bacterial killing characterization of progressive hiv-associated tuberculosis using 2-deoxy-2-[18 f] fluoro-d-glucose positron emission and computed tomography prospective discrimination of controllers from progressors early after low-dose mycobacterium tuberculosis infection of cynomolgus macaques using blood rna signatures four-gene pan-african blood signature predicts progression to tuberculosis a blood rna signature for tuberculosis disease risk: a prospective cohort study a high throughput whole blood assay for analysis of multiple antigenspecific t cell responses in human mycobacterium tuberculosis infection plasma metabolomics in human pulmonary tuberculosis disease: a pilot study high-resolution plasma metabolomics analysis to detect mycobacterium tuberculosis-associated metabolites that distinguish active pulmonary tuberculosis in humans metabolite changes in blood predict the onset of tuberculosis springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank our colleague cheryl l. day, phd, for review, thoughtful discussion, and feedback on this manuscript. sca and bss conceived of the review, drafted and revised the manuscript. both authors read and approved the final manuscript. this work was supported by the following grants: nih/niaid k23 ai134182 key: cord-255683-2eq24jth authors: chen, weizao; zhu, zhongyu; liao, huaxin; quinnan, gerald v.; broder, christopher c.; haynes, barton f.; dimitrov, dimiter s. title: cross-reactive human igm-derived monoclonal antibodies that bind to hiv-1 envelope glycoproteins date: 2010-02-04 journal: viruses doi: 10.3390/v2020547 sha: doc_id: 255683 cord_uid: 2eq24jth elicitation of antibodies with potent and broad neutralizing activity against hiv by immunization remains a challenge. several monoclonal antibodies (mabs) isolated from humans with hiv-1 infection exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. all known broadly neutralizing mabs (bnmabs) are immunoglobulin (ig) gs (iggs) and highly somatically hypermutated which could impede their elicitation. ig ms (igms) are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to iggs. here we describe the identification and characterization of several human igm-derived mabs against hiv-1 which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of 59 healthy donors. these antibodies bound with high affinity to recombinant envelope glycoproteins (gp140s, envs) of hiv-1 isolates from different clades. they enhanced or did not neutralize infection by some of the hiv-1 primary isolates using ccr5 as a coreceptor but neutralized all cxcr4 isolates tested although weakly. one of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. it bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the cd4 binding site. these results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. further studies will show whether such a strategy plays a role in hiv infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. the newly identified mabs could be used as reagents to further characterize conserved non-neutralizing, weakly neutralizing or enhancing epitopes and modify or remove them from candidate vaccine immunogens. the ability of human immunodeficiency virus type 1 (hiv-1) to rapidly generate mutants and evade immune response is the major obstacle for development of protective, prophylactic hiv-1 vaccines. therefore, candidate vaccine immunogens must be capable of eliciting broadly neutralizing antibodies (bnabs) that inhibit viruses from different genetic subtypes. several human monoclonal antibodies (hmabs) such as b12 [1] , x5 [2] , 2g12 [3] , 2f5 and 4e10 [4, 5] exhibit potent and broad hiv-1 neutralizing activity in vitro and can prevent hiv-1 infection in animal models [6] . these bnabs target structures on hiv-1 envelope glycoprotein (env) that are crucial for virus-cell fusion. therefore, envs in various formats are potential candidate immunogens and have been evaluated in animal models and human clinical trials [7] [8] [9] [10] . however, neutralization efficacy of the resulting sera as broad as that by those bnabs has not been achieved by empirically using these envs as immunogens [11] . it has been suggested that characterization of the epitopes of the bnabs could help design vaccine immunogens that would be able to elicit these bnabs or similar antibodies in vivo [12] . although this approach is being vigorously pursued, none of the immunogens designed has yet efficiently elicited neutralizing antibodies with broad specificity. igm is the initial antibody that the host generates when an infectious agent or antigen is encountered. activation of igm-expressing b cells provides impetus for in vivo igm-to-igg isotype switch resulting in production of high-affinity neutralizing or non-neutralizing antibodies. it is therefore important to investigate the human igm repertoire for hiv-1 env-specific antibodies and understand how they interact with hiv-1 envs and impact on viral infection which could help design effective immunogens. previous attempts to select hiv-specific antibodies by use of non-immune libraries have resulted in antibodies with modest neutralizing activity and limited breadth of neutralization [13] . studies by several groups show that human igm antibodies play important roles not only in shaping humoral immunity against hiv-1 but also in inducing cell-mediated response because of their pentameric binding nature as well as their very efficient activity to activate complement. torán et al. [14] indicated that in vivo igm-to-igg isotype switch and affinity maturation may be important for protection and long-term survival in certain hiv-1-infected individuals. sheppard et al. [15] demonstrated that complement-deactivated serum from a healthy volunteer immunized with an hiv-1 clade c gp120 exhibited igm-associated neutralizing activity that was significantly enhanced in the presence of fresh normal human serum as a source of complement. bomsel et al. [16] showed that a human anti-hiv-1 gp41 igm monoclonal antibody blocked the transcytotic route of hiv mucosal transmission in vitro. in this study, we describe identification and characterization of several human igm-derived mabs selected from phage-displayed naive human antibody libraries constructed from healthy donors. these antibodies have high affinity and cross-reactivity with hiv-1 gp120s from different clades, and either neutralize or enhance weakly entry of virions pseudotyped with envs from hiv-1 primary isolates. these data provide additional insights into the possible immune response that hiv-1 infection or viral env-based immunization could elicit and help in the design of candidate vaccine immunogens that elicit potent neutralizers of early transmitted virus. a large (1.5 × 10 10 members) phage-displayed naive human antibody antigen-binding fragment (fab) library (designated m21) was constructed from peripheral blood b cells of 22 healthy donors, spleens of 3 healthy donors, and lymph nodes of 34 healthy donors as described in materials and methods. the phagemid used for library construction, pzyd-n1, was synthesized and briefly described previously [17] . to approximately estimate the sequence diversity of m21, 44 clones were randomly selected from the library and sequenced. no identical sequences of heavy and light chains were found; more than 80% of the clones are productive. of the 44 clones 26 had kappa light chains and the other 18 -lambda light chains. m21 was panned against several hiv-1 envs and human cancer-related antigens; significant enrichment was obtained with all the pannings (data not shown). these results indicate that the quality of the library is likely to be good. to identify human igm-derived antibodies specific for hiv-1 envs, we panned m21 against the gp140 of a clade b isolate, r2 (gp140 r2 ). one of the selected antibody clones, r3h1, contained a tga stop codon at the very beginning of the light chain due to a nucleotide deletion that resulted in a reading-frame shift. the heavy chain variable domain (vh) of r3h1, designated m0, differs from the closest human germline sequence (vh3-23) by mutations distributed in all frameworks (frs) and complementarity determining regions (cdrs) (figure 1a ). m0 contains 15 mutations in nucleotide sequence of the variable (v) region resulting in 9 mutations in amino acid sequence. we corrected the reading frame of the light chain by site-directed mutagenesis and the corrected antibody, designated r3h1m, was expressed at high levels in bacteria as an fab. fab r3h1m specifically bound with relatively high affinity to gp140 r2 (ec 50 , ~60 nm) and the gp140 of a clade c isolate, gxc44 (gp140 gxc44 ) (ec 50 , ~100 nm). because the antibody was initially selected as a functional heavy chain-only antibody, we hypothesized that the binding activity of r3h1m should be attributed mainly to the heavy chain of the antibody and the heavy chain could be paired with light chains derived from different germlines while retaining cross-reactivity with hiv-1 envs. we cloned m0 as an isolated antibody domain and found that it was highly soluble, stable, monomeric, and expressed at high levels in bacteria [18] . in agreement with our hypothesis it specifically bound to gp140 r2 although with low affinity (ec 50 , >1000 nm). we further constructed a light chain-shuffling fab library (3 × 10 7 members) based on the heavy chain of r3h1. to increase the probability for selection of cross-reactive antibodies the library was panned sequentially against gp140 r2 and the gp140 of a clade f isolate, 14/00/4 (gp140 14/00/4 ). five unique clones were identified that contained exclusively lambda light chains ( figure 1b) . they bound to gp140 r2 and gp140 14/00/4 with ec 50 ranging from 2 to 80 nm (table 1 ) while no significant binding to an unrelated antigen, bovine serum albumin (bsa), was observed (data not shown). one of the antibodies, m19, also bound with high affinity to the envs of other three clade b isolates, gp120 bal, gp140 jrfl and gp140 con-s [19] . these results suggest that m19 and possibly the other selected antibodies target conserved epitopes on gp120. to test whether the five newly selected antibodies are capable of neutralizing hiv-1 primary isolates, we used viruses pseudotyped with envs from hiv-1 isolates representing clades a, b and c and using either ccr5 (r5) or cxcr4 (x4) or both (r5x4) as a coreceptor. none of the antibodies exhibited potent broadly neutralizing activity. four antibodies (fabs m19, m19b, m19c and m19d) enhanced infection by bal even at very low antibody concentration while a positive control antibody, m36 [17] , showed efficient neutralization and a negative control antibody, 1a1, did not affect entry ( figure 2a ); m19d enhanced infection by another clade b primary isolate, jrfl, at high concentrations ( figure 2b) . interestingly, the three antibodies that enhanced significantly infection by the bal weakly neutralized iiib which is a tcla clade b x4 isolate (figure 2c ). to find whether the activity of the antibodies is related to antibody size and how the viral infection could be affected by cross-linking of hiv-1 envs, we generated a single-chain fv fragment (scfv) (scfv m19) of m19 and a human igg1 fc-fusion protein (m19fc) of scfv m19; m19 was selected for further characterization because its light chain was relatively less divergent from the germline ( figure 1b) and was the only one which did not contain any somatic mutations in the cdr3 of the light chain. when bal was tested, the enhancing activity of scfv m19 was comparable to that of fab m19 and slightly lower than that of m19fc (data not shown). these data indicate that the antibody access to the epitope was not size-restricted and that the bivalency of the fc fusion protein strengthened the antibody activity probably due to avidity effects. the ability of m19fc to modulate hiv-1 infection in vitro was further tested with six additional clade b isolates and an isolate each from clade a and clade c, respectively ( figure 3) . enhancement was observed with jrcsf (clade b, r5) at very low m19fc concentration and the enhancement was decreased with an increase in antibody concentration. m19fc also conferred slight enhancement of infection by jrfl (clade b, r5) and 92ug037.8 (clade a, r5) at about 200 nm antibody concentration. it neutralized the clade b x4 isolates iiib and nl4-3. it also neutralized r2, which is a clade b r5 isolate, displays some cd4 independence and is moderately neutralization sensitive, as well as gxc44 which is a clade c r5 isolate. no significant antibody activity was observed with the dual tropic clade b isolate 89.6. these results indicate that the antibody activity in terms of neutralization or enhancement varies when different isolates are tested and is not significantly correlated with the coreceptor usage and the cd4 dependency of the viruses in entry. antibody, b12 (a) , and two cd4i antibodies, m36 and m9 (b), in binding to gp120 bal and to gp120 bal -cd4, respectively. an irrelevant human antibody, 1a1, was used as a negative control. to approximately localize the antibody epitopes and begin to elucidate the underlying mechanisms of antibody neutralizing or enhancing activity, we measured the antibody binding to envs from different isolates alone and in complex with cd4 as well as the antibody competition with wellcharacterized antibodies. m19fc bound to a single-chain polypeptide analogue of the hiv-1 gp120-cd4 complex (gp120 bal -cd4) [20] with threefold higher affinity (ec50, ~2.5 nm) than to gp120 bal (ec50, ~10 nm) alone as measured by an elisa (figure 4a ). it did not react with scd4 suggesting that the antibody targeted a structure on gp120 that was outside the cd4-binding site (cd4bs) and could undergo cd4-induced (cd4i) conformational changes. the conformational changes on gp120 bal after cd4 binding were confirmed by a cd4i bnab, m9fc [21] , which exhibited dramatically higher binding to gp120 bal -cd4 than to gp120 bal (figure 4a ). to find out whether the antibody bound to the conventional cd4i epitopes, we used gp140 con-s , which was a synthetic env designed by aligning the consensus env sequences of group m [19] . this env, by design, is composed by shorter v1, v2, v4 and v5 loops and therefore, might expose regions around the variable loops that might contain conserved neutralizing determinants. however, m9 epitope as a representative for cd4i epitopes is still completely hidden on gp140 con-s because m9fc did not bind in the absence of scd4, it did bind with high affinity in the presence of scd4 (figure 4b) . however, m19fc showed comparable binding to gp140 con-s with or without scd4 (figure 4b) suggesting that the antibody does not precisely target cd4i epitopes but other structures on the gp120. although m19fc was not directed against the cd4binding pocket, it strongly competed with the cd4bs bnab, scfv b12, which has binding surface larger than that of cd4 [22] (figure 5a ). m19fc also competed although weakly with two cd4i antibodies, scfv m9 and m36 [17] (figure 5b) . these results suggest that the epitope of m19fc is in very close proximity to the cd4bs and the coreceptor-binding site (corbs) which overlaps with cd4i epitopes on gp120 ( figure 6 ). figure 6 . a schematic representation for the epitope (gray) of m19 on gp120 (blue). the red and white areas denote the cd4bs and the corbs, respectively. the epitope (long dash-dotted circle on the left) of the cd4bs antibody, b12, is indicated in comparison with the cd4-binding area. the dashed circle on the right denotes the corbs for some isolates that could partially overlap with the epitope of m19. to estimate the possibility of rapid elicitation of these antibodies in vivo when an hiv-1 gp120related immunogen is administered, we generated a germline-like scfv of m19 (scfv m19gem) and measured its binding to envs from different isolates in the presence or absence of scd4. no obvious interaction was observed with scfv m19gem when several envs that bound the original m19 were tested (data not shown). previous studies showed that the binding activity of an antibody in the form of fab or scfv could be increased up to thousands of times when it is converted to dimeric formats such as an igg. we therefore fused scfv m19gem to the fc portion of a human igg1 to gain possible avidity effects. still, the fc-fusion protein of scfv m19gem (m19gemfc) did not show measurable binding to the envs with or without scd4 (figure 7) . these results suggest that significant somatic diversification is required for the m19 corresponding germline antibody to achieve recognition of the m19 epitope on gp120. they also indicate that in some individuals certain igm antibodies could undergo somatic hypermutations to relatively high-affinity binders to the env in the absence of hiv-1 infection or immunization with env. figure 7 . binding of the germline-like antibody of m19 to different gp120s in the absence or presence of cd4. the original antibody, m19fc, was used as a positive control. vaccines based on recombinant envs have failed to prevent viral infection in human efficacy trials likely because of their inability to elicit neutralizing antibodies that are broad enough to combat the extremely high variability of the virus. recent success was reported but it is modest and needs to be repeated and further examined [23] . to date only several bnabs have been isolated from hiv-1 patients suggesting difficulties to elicit such antibodies in vivo. however, the presence of epitopes recognized by these bnabs on envs argues that they are still a potential template for vaccine design. different strategies aiming at improving the presentation of neutralizing epitopes are rigorously pursued. analysis of the structures of envs shows that most of their surface is hidden from humoral immune responses by glycosylation and oligomeric occlusion [24, 25] . the cd4bs and the corbs on gp120 are also flanked by loop structures which may further limit the access by antibodies generated by the human immune system [24, 25] . therefore, one strategy focuses on the use of modified envs, in which the variable loops have been deleted [26] [27] [28] [29] or glycosylation removed [30, 31] or both [32] in order to increase the exposure of the neutralizing epitopes. based on the finding that the neutralizing capacity of the antibodies is associated with their ability to bind to native trimeric envs on the virus but does not correlate with binding to isolated monomeric envs, another strategy focuses on preservation or re-construction of the functional trimeric envs [33, 34] . the essential concept is that immunizing with a close mimic of the functional trimer will improve the chances of eliciting neutralizing antibodies. yet another strategy is to use fusion intermediates formed during virus entry [35, 36] or their mimics [37] . hiv-1 entry is initiated by binding of viral gp120 with the receptor cd4 and a coreceptor on the target cell surface. these interactions lead to intermediate env conformations that may include conserved structures useful for vaccine design. however, elicitation of desirable level and breath of neutralizing antibodies with the immunogens generated based on these strategies has not been achieved. while modification of envs based solely on the analysis of the interaction with neutralizing antibodies is not sufficient to create effective vaccines, we propose that the inherent capacity of the human immune system in response to hiv-1 infection should be explored. we have hypothesized that investigation of human igm repertoire for hiv-1 env-specific antibodies and understanding how they react with envs, evolve and impact on viral infection would help design effective hiv-1 immunogens. it has been previously found that igm-based response confers substantial effects on hiv-1 infection which are especially relevant in the context of acute infection [14] [15] [16] . in this study, we describe the identification and characterization of several high-affinity human igm-derived mabs against hiv-1 from phage-displayed naive human antibody libraries constructed from healthy donors. although their potency in vitro appears minor the roles they could play in mediating hiv-1 infection in vivo should not be underestimated. igm antibodies are highly effective in activating complement system to destroy the virus or infected cells. the antibodies selected in this study target highly conserved regions on gp120 which are in very close proximity to the cd4bs and the corbs. because these two regions on gp120 are vital for virus entry and harbor neutralizing epitopes, hiv-1 has evolved strategies such as steric occlusion to protect it from humoral immunity [24, 25] . here we suggest that the possible high immunogenicity of the conserved non-neutralizing epitopes of these antibodies could further divert the immune system from responses to neutralizing epitopes. recent studies [38, 39] showed that crossreactive non-neutralizing antibodies (igms and iggs) were elicited by immunizing mice with recombinant hiv-1 gp140s suggesting the same possibility in humans. because the antibodies we selected are less divergent (totally about 15 amino acid mutations in the v gene products for the heavy and light chains) (figure 1 ) than the known bnabs which all contain extensive somatic mutations (on average >30 mutations) compared to germline antibodies (data not shown), they could be more easily elicited. in addition, these antibodies could directly block the access of some neutralizing antibodies generated by the human immune system, especially those targeting the cd4bs or corbs. this was supported by our finding that m19fc partially reversed the neutralization by a cd4i antibody, m36 [17] (data not shown). these results further support previous propositions to reduce the immunogenicity of unwanted epitopes when gp120/gp140 is used as an immunogen. we found that the closest germline-like antibody to m19 does not bind an env (figure 7) . we observed similar lack of binding of germline-like b12, 2f5 and 2g12 to envs [40] [41] [42] . moreover, we found that there was a lack of or no high-affinity binders to envs in the germline-like antibody repertoire of the human cord blood whereas high-affinity antibodies against other human infectious agents including sars coronavirus and henipaviruses could be easily selected (weizao chen, emily streaker and dimiter s. dimitrov, in preparation). the identified antibodies against sars coronavirus protein s exhibited nanomolar affinity and were very close to germline in sequence. by analyzing the previously identified antibody sequences we found that all of the cross-reactive neutralizing antibodies against hiv-1 were highly diversified from their germline sequences; in contrast, the antibodies against sars coronavirus and henipaviruses had only limited number of mutations [40] [41] [42] . these results indicate that extensive somatic mutations could be required for high-affinity binding to conserved hiv-1 env structures. one could speculate that the maturation pathways for some hiv-1 antibodies are initiated by immunogens that are different from the envs; such immunogens could be used in combination with env-based immunogens to guide the immune system for elicitation of potent bnabs by additional somatic hypermutation and selection. this is in line with our proposition to elicit bnabs by using one or more primary immunogens that are different than envs but can lead to intermediates on the maturation pathways that can be subsequently further somatically hypermutated to matured bnabs [40] [41] [42] . except for the conventional antigen-antibody interaction, gp120s also bind to a small population of human antibodies containing products of mainly vh3 gene family and exhibit superantigen properties by activating the human b cells expressing such antibodies on cellular membrane [43] . the core motif of the superantigen-binding site (sbs) on gp120 is a discontinuous structure spanning the v4 variable domain and the amino-terminal region flanking the c4 constant domain [44] . the determinants on the antibody vh domains critical for gp120 binding are not clear yet while the putative important regions were identified in the fr1 and 3; a modeling study showed that most of the potential contact residues were located on the face of the vhs opposite to the interface for interaction with light chain; other positions in the cdr1 and 2 also influenced the binding [45] . moreover, the gp120-positive antibody vh genes showed >96% similarity to the germlines suggesting that the somatic hypermutations may have adverse effects on gp120 binding [45] . the antibodies described in this study contained vh3-23 gene products ( figure 1a ) and therefore, their high binding could be due to the superantigen-like interactions. however, our results showed that the isolated vh of these antibodies, m0, bound to gp120s with affinity (ec 50 , >1000 nm) much lower than that (ec 50 , 2-80 nm) of the fabs. in addition, the epitope of one (m19) of these antibodies was mapped to a structure in very close proximity to the cd4bs and the corbs on gp120 but not to the defined sbs. these results suggest that the majority, if not all, of the binding activities of the selected antibodies should be contributed by the conventional antigen-antibody recognition. one should note that these antibodies may not represent the native ones because they were selected from phage libraries where the heavy and light chains of the antibodies are randomly recombined. however, previous studies [46] [47] [48] have shown that selection of high-affinity antibodies from such libraries results in antibodies which are identical or very similar to those occurring in the host from which the libraries are made. it has been shown that at least in some systems the antigen selected antibodies such as the autoantibodies against thyroid peroxidase (tpo) from large random phage libraries have the same pairings as those from small libraries where the cognate pairing is preserved and that pairing between light and heavy chain is not promiscuous [49, 50] . the study by chapal et al. [48] directly demonstrates that antibodies derived from combinatorial libraries are likely to represent in vivo pairings, leading to high affinity antibody fragments mimicking the binding of serum autoantibodies to tpo. de wildt et al. [51] analyzed the cognate pairings of heavy and light chain variable domains in 365 human igg+ b cells from peripheral blood and established that the pairings are largely random. the antibodies we selected were originated from large families of genes -heavy chain from vh3 and light chain from vl1 ( figure 1) . importantly, the pairings for two of them, m19 and m19d, have been found in the very limited number (365) of truly human antibodies previously analyzed [51] . moreover, some (e.g., m19 and m19b) of these antibodies are relatively close to their corresponding germlines (figure 1 ). these data suggest the possibility of eliciting such antibodies in vivo during hiv-1 infection or gp120-based immunization. we purchased the 293t cells from atcc. other cell lines and plasmids used for expression of various hiv-1 envs were obtained from the national institutes of health aids research and reference reagent program (arrrp). recombinant gp140s were produced in our laboratories. gp120 bal and the single-chain fusion protein gp120 bal -cd4 were kindly provided by t. fouts (institute of human virology, baltimore; currently at profectus, baltimore, md). horseradish peroxidase (hrp)-conjugated anti-flag tag antibody and hrp-conjugated anti-human igg (fc-specific) antibody were purchased from sigma-aldrich (st. louis). m21 was constructed by using phagemid pzyd-n1 according to the reported protocols [52] . the sources for amplification of antibody gene fragments are commercially purchased poly a+ rnas (bd biosciences, san jose, ca) from peripheral blood b cells of 22 healthy donors, spleens of three donors and lymph nodes of 34 healthy donors, respectively. m2l was used for selection of antibodies against hiv-1 antigens conjugated to magnetic beads (dynabeads m-270 epoxy; dynal inc., new hyde park, ny) as described previously [53] . 5, 5, 2.5 and 1 µg of gp140 r2 were used in the first, second, third and fourth round of panning, respectively. clones that specifically bound to gp140 r2 were identified from the third and fourth round by using monoclonal phage elisa (mpelisa) as described [53] . a light chain-shuffling fab library (3 × 10 7 members) was further constructed based on the heavy chain of r3h1. the light chain repertoire was also harvested from a naive human fab library (5 × 10 9 members) constructed from peripheral blood b cells of 10 healthy donors [53] , in addition to that from m21. the new library was panned sequentially with two gp140s from different clades. for the sequential panning, 5 and 2.5 µg of gp140 r2 were used in the first and third round, respectively; antigens were alternated with 5 and 2.5 µg of gp140 14/00/4 during the second and fourth round, respectively. clones that bound to both gp140 r2 and gp140 14/00/4 were identified from the fourth round by using mpelisa as described [53] . the following primers were used: m36f, 5'-tgg ttt cgc tac cgt ggc cca gcc ggc cca ggt gca gct ggtg-3' (sense); m19fcr: 5'-gtg agt ttt gtc ggg ccc tag gac ggt cag ctt gg-3' (antisense). for construction of scfv, the full-length original or germline scfv gene fragment was synthesized (genscript, piscataway, nj), digested with sfii and cloned into pcomb3x. to generate fc-fusion protein, the scfv gene was pcr (primers m36f and m19fcr) amplified by using the synthetic scfv gene fragment as a template. the new scfv products appended with sfii and apai restriction sites on both sides were digested and cloned into psectagb-fc. the scfv and fab were expressed in e. coli hb2151, as described previously [53] . the bacterial pellet was collected after centrifugation at 5,000 × g for 10 min and resuspended in pbs (ph 7.4) containing 0.5 million-unit polymixin b (sigma-aldrich). after 30 min incubation with rotation at 50 rpm at room temperature, it was centrifuged at 25,000 × g for 25 min at 4 °c. the supernatant was used for purification of scfv and fab by immobilized metal ion affinity chromatography (imac) by using ni-nta resin (qiagen, valencia, ca) according to manufacturer's protocols. fc-fusion proteins were expressed in 293 free style cells. 293fectin (invitrogen, carlsbad, ca) was used to transfect 293 free style cells according to the instructions of the manufacturer. three days post-transfection, the culture supernatant was harvested and used for purification by using nprotein a sepharose 4 fast flow (ge healthcarezcomx, piscataway, nj). binding and competition elisas were performed as described previously [17] . viruses pseudotyped with hiv-1 envs were prepared by cotransfection of 70-80% confluent 293t cells with pnl4-3.luc.e-r-and psv7d constructs encoding hiv-1 envs by using the polyfect transfection reagent (qiagen) according to manufacturer's instruction. pseudotyped viruses were obtained after 48 h by centrifugation and filtration of cell culture through 0.45 µm filters. for neutralization, viruses were mixed with different concentrations of antibodies for 1 h at 37 °c, and then the mixture was added to about 1.5 × 10 4 hos-cd4-ccr5 (used for all r5 and dual tropic viruses) or hos-cd4-cxcr4 cells grown in each well of 96-well plates. luminesence was measured after 48 h by using the bright-glo luciferase assay system (promega, madison, wi) and a lumicount microplate luminometer (turner designs). mean relative light units (rlu) for duplicate wells were determined. relative infectivity (%) was calculated by the following formula: (average rlu of antibody-containing wells/average rlu of virus-only wells) × 100. a major finding of this study is the identification and characterization of several hiv-1-specific human igm-derived mabs and their highly conserved epitopes. such antibodies were rarely investigated and reported. further characterization of these antibodies, their dynamics and epitopes could provide knowledge that in addition to its usefulness for basic understanding of immune responses to hiv-1 could also help in the design of candidate vaccine immunogens that elicit potent neutralizers of early transmitted virus. recognition properties of a panel of human recombinant fab fragments to the cd4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1 broadly crossreactive hiv-1-neutralizing human monoclonal fab selected for binding to gp120-cd4-ccr5 complexes human monoclonal antibody 2g12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1 a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1 broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41 human monoclonal antibodies and engineered antibody domains as hiv-1 entry inhibitors extensively cross-reactive anti-hiv-1 neutralizing antibodies induced by gp140 immunization correlation between immunologic responses to a recombinant glycoprotein 120 vaccine and incidence of hiv-1 infection in a phase 3 hiv-1 preventive vaccine trial a clinically relevant hiv-1 subunit vaccine protects rhesus macaques from in vivo passaged simian-human immunodeficiency virus infection prevention of disease induced by a partially heterologous aids virus in rhesus monkeys by using an adjuvanted multicomponent protein vaccine aiming to induce broadly reactive neutralizing antibody responses with hiv-1 vaccine candidates antibodies, viruses and vaccines a human monoclonal antibody neutralizes diverse hiv-1 isolates by binding a critical gp41 epitope molecular analysis of hiv-1 gp120 antibody response using isotype igm and igg phage display libraries from a long-term non-progressor hiv-1-infected individual a functional human igm response to hiv-1 env after immunization with nyvac hiv c intracellular neutralization of hiv transcytosis across tight epithelial barriers by anti-hiv envelope protein diga or igm human domain antibodies to conserved sterically restricted regions on gp120 as exceptionally potent cross-reactive hiv-1 neutralizers construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain a group m consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype b and c hiv-1 primary viruses expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type 1 gp120-cd4 receptor complex improved breadth and potency of an hiv-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning crystal structure of a neutralizing human igg against hiv-1: a template for vaccine design vaccination with alvac and aidsvax to prevent hiv-1 infection in thailand the antigenic structure of the hiv gp120 envelope glycoprotein structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody the ability of an oligomeric human immunodeficiency virus type 1 (hiv-1) envelope antigen to elicit neutralizing antibodies against primary hiv-1 isolates is improved following partial deletion of the second hypervariable region immunogenicity and ability of variable loop-deleted human immunodeficiency virus type 1 envelope glycoproteins to elicit neutralizing antibodies immunogenicity of dna vaccines expressing human immunodeficiency virus type 1 envelope glycoprotein with and without deletions in the v1/2 and v3 regions selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type 1 envelope influence of n-linked glycans in v4-v5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response role of n-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response modified hiv envelope proteins with enhanced binding to neutralizing monoclonal antibodies immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140 improved elicitation of neutralizing antibodies against primary human immunodeficiency viruses by soluble stabilized envelope glycoprotein trimers crosslinked hiv-1 envelope-cd4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques purified complexes of hiv-1 envelope glycoproteins with cd4 and ccr5(cxcr4): production, characterization and immunogenicity mutagenic stabilization and/or disruption of a cd4-bound state reveals distinct conformations of the human immunodeficiency virus type 1 gp120 envelope glycoprotein broadly reactive monoclonal antibodies to multiple hiv-1 subtype and sivcpz envelope glycoproteins. virology production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins all known cross reactive hiv-1 neutralizing antibodies are highly divergent from germline and their elicitation may require prolonged periods of time germline-like predecessors of broadly neutralizing antibodies lack measurable binding to hiv-1 envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens maturation pathways of cross-reactive hiv-1 neutralizing antibodies immunoglobulin vh3 gene products: natural ligands for hiv gp120 identification of the b cell superantigen-binding site of hiv-1 gp120 structural basis of the gp120 superantigen-binding site on human immunoglobulins influenza virus hemagglutinin-specific antibodies isolated from a combinatorial expression library are closely related to the immune response of the donor original and artificial antibodies thyroid peroxidase autoantibodies obtained from random single chain fv libraries contain the same heavy/light chain combinations as occur in vivo recombinant thyroid peroxidase-specific autoantibodies. i. how diverse is the pool of heavy and light chains in immunoglobulin gene libraries constructed from thyroid tissue-infiltrating plasma cells recombinant thyroid peroxidase-specific autoantibodies. ii. role of individual heavy and light chains in determining epitope recognition analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire construction of a large naive human phage-displayed fab library through one-step cloning potent neutralization of hendra and nipah viruses by human monoclonal antibodies we thank xiaodong xiao in our group for helpful discussion. key: cord-027242-7qq82j2f authors: chrissafidou, angeliki title: infektionen, impfungen, reisemedizin date: 2008-12-11 journal: praxisleitfaden allgemeinmedizin doi: 10.1016/b978-343722442-3.50016-6 sha: doc_id: 27242 cord_uid: 7qq82j2f nan syndrom (sars) 528 9.4.11 vogelgrippe 529 9.5 mykosen 529 9.5.1 allgemeines 530 9.5.2 candidosen (soor) 531 9.5.3 dermatophytosen 531 9.5.4 systemische mykosen 532 9.6 protozoeninfektionen 532 9.6.1 toxoplasmose 533 9.6.2 lambliasis (giardiasis) 534 9.6.3 sonstige protozoeninfektionen 535 9.7 wurmerkrankungen 535 9.7.1 madenwurm-infektion 535 9.7.2 spulwurm-infektion (ascariasis) 536 9.7.3 bandwurm-infektion (zestoden) 537 9.7.4 echinokokkus-infektion 538 9.7.5 trichinose 538 9.8 sexuell ü bertragbare krankheiten 538 9.8.1 gonorrhoe 539 9.8.2 syphilis (lues) 541 9.8.3 trichomoniasis 541 9.8.4 ulcus molle 542 9.8.5 lymphogranuloma venereum 542 9.8.6 condylomata acuminata 543 9. 9 hiv und aids 543 9.9.1 epidemiologie und ü bertragungswege 543 9.9.2 labordiagnostik 546 9.9.3 stadieneinteilung und verlauf 548 9.9.4 diagnostik bei hiv-positiven 548 9.9.5 hä ufige krankheitsbilder bei aids 554 9.9.6 therapie 560 9.9.7 medikamente bei hiv-patienten 564 9.9.8 vorgehen bei kontakt mit hivkontaminiertem material und postexpositionsprophylaxe (pep) 567 9.10 reisemedizin 567 9.10.1 allgemeine empfehlungen zur reisefä higkeit 572 9.10.2 empfehlungen fü r schwangere 573 9.10.3 empfehlungen fü r ä ltere menschen 574 9.10.4 empfehlungen fü r kinder und sä uglinge 575 9.10.5 empfehlungen fü r chronisch kranke 577 9.10.6 reisevorbereitungen/prophylaxen 580 9.10.7 reiseimpfungen und chemoprophylaxe 587 9.10.8 gesundheitsrisiken auf reisen 596 9.10.9 screening nach reiseende 597 9.11 infektionsschutzgesetz 9.1 erhö hung der kö rpertemperatur infolge einer gestö rten wärmeregulation (sollwertverstellung durch pyrogen-oder toxinwirkung, z. b. von makrophagen, tumorzellen, stoffwechselprodukten, bakterienbestandteilen) ; bis 38 c subfebrile temp., bis 38,5 c mäßiges fieber, ü ber 39 c hohes fieber. grunderkr., ggf. facharztü berweisung oder klinikeinweisung bei reduziertem az, bei hohem fieber und vorliegen eines immundefekts (hiv-inf., immunsuppressive ther., z. n. splenektomie), sowie bei fortbestehen des fiebers ü ber 1 wo. hinaus. bei diab. mell . vermehrt bz-kontrollen durch den pat. selbst oder den ha wegen gefahr der bz-entgleisung. fieber unbekannter herkunft, das länger als 2 wo. besteht: 40 % infektionen, 25 % tumoren, 20 % immunologische erkrankungen, 5-10 % seltene ursachen. die immunisierung durch impfungen ist eine der wichtigsten und wirksamsten maßnahmen zum schutz vor infektionskrankheiten. mit einer hohen durchimpfungsrate kö nnen einzelne krankheitserreger regional oder sogar weltweit ausgerottet werden. immunität gegen erkr. lässt sich passiv durch immunglobulingabe, aktiv durch impfung oder kombination beider methoden erreichen. in deutschland besteht keine impfpflicht. impfempfehlungen werden durch die obersten gesundheitsbehö rden der länder ausgesprochen. diese ö ffentliche empfehlung hat im fall von impfschäden wichtige bedeutung fü r die weitere versorgung ( § 20 ifsg). passive immunisierung durch immunglobuline: vorteil der sofortigen schutzwirkung. nachteil: schutz ist nur von kurzer dauer (4 wo. bis 3 mon.), es besteht nur humorale immunität. aktive immunisierung: der organismus wird mit antigenen von krankheitserregern konfrontiert und muss selbst eine immunität ausbilden. der schutz tritt verzö gert ein, ist aber von langer dauer. jede neue antigenzufuhr fü hrt zu einer gedächtnisreaktion mit verstärkung der immunität. totimpfstoffe: abgetö tete krankheitserreger oder aufbereitete antigene; keine impfinfektion mö glich, deshalb auch bei pat. mit immundefekten applizierbar. i. d.r. grundimmunisierung durch 3 inj., meist i. m. (v. a. adsorbatimpfstoffe). lebendimpfstoffe: enthalten attenuierte, aber vermehrungsfähige krankheitserreger r risiko fü r immunsupprimierte und schwangere. zur ausbildung der immunität genü gt i. d.r. 1 impfung. applikation s. c. oder p. o. (je nach impfstoff). hersteller-informationen: in packungsbeilagen, fachinformationen und wissenschaftlichen basis-broschü ren. hersteller kö nnen auch jederzeit telefonisch kontaktiert werden r service-nummern der arzneimittelhersteller finden sich z. b. in der roten liste. behö rdliche informationen: das robert-koch-institut unterhält eine ständige impfkommission (stiko), die impfempfehlungen regelmäßig ü berarbeitet (letzte fassung vom juli 2005). die empfehlungen sind im anhang der "roten liste" abgedruckt. bestellung der impfempfehlungen der stiko bei: robert-koch-institut, kennwort "stiko-empfehlungen", nordufer 20, 13353 berlin, unter fax-nr. 01888-754-2628 oder unter epibull@rki.de. kosten: bis zu 3 exemplare kostenfrei gegen porto von 1,45 3. allgemeiner gesundheitszustand? immunschwäche, -defekt? durchgemachte infektionskrankheiten? frü here impfungen, auftreten von impfreaktionen? gravidität. allergien. wichtig fü r einen lang dauernden impfschutz: der bei der grundimmunisierung erforderliche mindestzeitraum (s. einzelne impfungen) zwischen vorletzter und letzter impfung darf nicht unterschritten werden. es gibt keine unzulässig großen abstände zwischen impfungen. jede impfung gilt. auch eine fü r viele jahre unterbrochene grundimmunisierung muss nicht neu begonnen werden. ist nicht bekannt, wann zuletzt bzw. ob ü berhaupt eine impfung stattgefunden hat, ist dies kein grund, eine grundimmunisierung nicht zu beginnen oder impfungen aufzuschieben oder nicht durchzufü hren. durch zusätzliche impfungen bei bereits bestehendem impfschutz steigt das risiko fü r ko nur bei bcg-impfung (verstärkte lokalreaktion) und pneumokokken-impfung (systemische, allergische reaktionen). lebendimpfstoffe: kö nnen simultan verabreicht werden; werden sie nicht simultan verabreicht, ist ein mindestabstand von 4 wo. zu empfehlen. voraussetzung: vollständiges abklingen der impfreaktion, keine ko. totimpfstoffe (inaktivierte krankheitserreger, d. h. deren antigenbestandteile/ toxoide): keine mindestabstände zu anderen impfungen erforderlich, auch nicht zu solchen mit lebendimpfstoffen. bei wiederholungsimpfungen im rahmen der grundimmunisierung sollte fü r die weiteren impfungen das gleiche präparat verwendet werden. bei schlechter verträglichkeit bzw. unzureichender immunantwort nach abschluss einer impfserie kann eine impfung mit dem präparat eines anderen herstellers u. u. den gewü nschten impferfolg haben. kombinationsimpfstoffe helfen, injektionen und impftermine einzusparen; manchmal sind sie auch preiswerter als die summe der jeweiligen monovakzinen. vor allem lebendimpfstoffe sind temperaturlabil und mü ssen vor erwärmung und gefrieren geschü tzt werden. alle impfstoffe sind bei 2-8 c zu lagern, wobei die temperatur permanent mit einem geeichten minimax-thermometer kontrolliert werden muss. impfstoffe, die falsch gelagert oder eingefroren wurden, sind zu verwerfen. angebrochene amp. sofort verbrauchen, v. a. wegen der gefahr der bakteriellen kontamination (cave: spritzenabszess). fü r die injektion eine neue kanü le verwenden. an der aufziehkanü le anhaftender impfstoff kann zu lokalreaktionen der haut fü hren. bei regelrechter anwendung der amtlich zugelassenen impfstoffe extrem selten. bei verdacht: immunstatus und ggf. interkurrente infektionen zum zeitpunkt der impfung abklären r serum und ggf. mikrobiologische proben (serum-stuhlproben) asservieren. meldung an das ö rtliche gesundheitsamt und die arzneimittelkommission der deutschen ä rzteschaft, herbert-levin-platz 1, 10623 berlin, tel. (030) 40 04 56-5 00, fax (030) 40 04 56-5 55. impfling bzw. eltern/sorgeberechtigte auf gesetzliche regelungen zur versorgung nach impfschäden hinweisen (ifsg § § 60-64, 66). der antrag ist beim zuständigen versorgungsamt zu stellen. immer doppelte dokumentation durchfü hren (in den impfunterlagen des impflings und in den aufzeichnungen des arztes). mö glichst vorhandene dokumente (impfbü cher) weiterfü hren. falls diese nicht vorliegen, kann eine separate bescheinigung ausgestellt werden. zum späteren nachtrag in impfbü cher ist jeder arzt berechtigt. bei neuausgabe von impfbü chern who-gerechte formulare bevorzugen ("internationale bescheinigungen ü ber impfungen und impfbuch", erhältlich beim deutschen grü nen kreuz, s. o.). dokumentiert werden: tag der impfung; art der impfung; bezeichnung des impfstoffs (handelsname); chargen-nummer (sehr wichtig!); impfender arzt. kassenleistung sind alle ö ffentlich empfohlenen impfungen. die einstufung erfolgt durch die stiko und kann sich von bundesland zu bundesland unterscheiden. kosten fü r indikationsimpfungen aufgrund beruflicher risiken werden von der gesetzlich benannten stelle (i. d.r. arbeitgeber) ü bernommen. sonder-und reiseimpfungen mü ssen privat abgerechnet werden. um mit mö glichst wenig injektionen auszukommen, sollten kombinationsimpfstoffe bevorzugt werden. wichtige kombinationsimpfstoffe sind: kostenü bernahme indikationsbezogen durch krankenkassen. indikations-oder reiseimpfung. totimpfstoff; erw.: 20 lg hbsag + 720 hav-antigene; kinder: 10 lg hbsag + 360 hav-antigene. kinderimpfstoff vom 2.-15. lj., erwachsenenimpfstoff ab 16. lj. grundimmunisierung: 3 impfungen, injektion in den m. deltoideus, z. b. twinrix j , 1 ml. schema: 0/4-6 wo./6-12 mon. japan-enzephalitis-impfung indikation sonderimpfung; längerer aufenthalt (4 4 wo.) während der ü bertragungszeit (in den tropen während der regenzeit, in gemäßigten zonen v. a. von spätsommer bis frühherbst) in endemiegebieten (sü d-und ostasien), bevö lkerung in endemiegebieten. totimpfstoff; abgetö tete viren. 3 impfungen von 1,0 ml s. c. an den tagen 0/7/30. kinder 5 3 j. mit 1 /2 dosis. auffrischimpfung nach 1 j. nebenwirkungen 0,5-1 % allergische reaktionen (sofort oder verzö gert 2-3 d p. v.); kopf-und gliederschmerzen, gastrointestinale beschwerden. nach 1-4 j. keine bes., allg. ki ( besonderheiten impfstoff in deutschland nicht zugelassen, kann aber ü ber eine internationale apotheke bezogen werden. impfungen meist nur an gelbfieber-impfstellen. nicht verfü gbar. bei 3-5 % der geimpften um den 9. d p. v. temperaturerhö hung, z. t. mit uncharakteristischem exanthem, "impfmasern" mit mildem verlauf. besonderheiten nach immunglobulin-und serumgaben 4 mon. abstand zur impfung! inkubationsimpfung nur bis 48 h nach exposition sinnvoll. kinder mit asymptomatischer hiv-infektion kö nnen geimpft werden. wenn aktive immunisierung nicht mö glich, unspezifisches gammaglobulin (igg) verabreichen. pat. mit asplenie, geplanter splenektomie, hypogammaglobulinämie, komplementdefekten. sonderimpfung fü r reisen in endemiegebiete (sahelzone, vorderasien, tropisches sü damerika). zur einreise nach saudi-arabien ist eine ac oder acwy-impfung erforderlich, z. b. fü r mekka-pilger. gü ltigkeit: 10 d-3 j. nach der letzten impfung. bei kindern 5 18 mon. ist der impferfolg zweifelhaft. totimpfstoff mit kapselpolysacchariden der meningokokkentypen a, c, w135 und y, z. b. mencevax j acwy. 1 dosis 0,5 ml s. c. verstärkte lokalreaktion, schwellung regionaler lk, kopfschmerzen, temperaturerhö hung. keine bes., allg. ki ( nicht verfü gbar. erfolgt mit rifampicin, z. b. rifa j , rimactan j . asymptomatische meningokokken-träger jeden alters kö nnen zweiterkr. verursachen! deshalb alle personen (einschließlich erw., auch geimpfte personen) im haushalt des erkrankten einbeziehen, falls in der familie mind. ein kind 5 4 j. rifampicin 30-60 min. vor einer mahlzeit einnehmen. orangefärbung von urin, schweiß, tränen und speichel. in 20 % leichte gastrointestinale nw (ü belkeit, erbrechen, durchfall). gravidität; leberschäden. indikation ö ffentlich empfohlene standardimpfung fü r kinder ab vollendetem 2. lebensmon. und jugendliche. erw. ohne impfung, seroneg. exponierte personen (z. b. lehrer). ö ffentlich empfohlen fü r medizinisches personal und personal in der kinderbetreuung. anamnestische angaben ü ber durchgemachte mumps ohne serol. kontrolle nicht verwertbar. vorgesehene impfung zum impftermin in jedem fall durchfü hren, da nw bei impfung nach durchgemachter infektion oder mehrfachen impfungen nicht bekannt. lebendimpfstoff, attenuiertes mumpsvirus. sehr licht-und wärmeempfindlich: kü hlkette! vorzugsweise als kombinationsimpfstoff (mmr;4tab. 9.2). mumpsimpfung kann bei kindern mit asymptomatischer hiv-inf. erfolgen. nutzen von immunglobulinen nicht erwiesen. pertussis-impfung schwere pneumokokken-inf. oder -impfung während der letzten 5 j. bei fortbestehender ind. erw. alle 6 j., kinder 5 10 j. alle 3 j. kinder, die mit konjugatimpfstoff geimpft wurden, erhalten ab dem vollendeten 2. lj. eine impfung mit polysaccharid-impfstoff (mindestabstand zur letzten impfung mit konjugatimpfstoff 2 mon.). bei frü herer wiederimpfung stärkere nw. notwendige aktualisierung des impfstoffs je nach den in europa und nordamerika vertretenen pneumok.-subtypen. nicht verfü gbar. polio49.4.9. laut stiko nur noch sonderimpfung (riegelungsimpfung) bei ausbruch von polio. lebendimpfstoff, enthält impfvirustypen i, ii und iii; kü hlkette! impfmodus grundimmunisierung: 3 orale gaben im abstand von mind. 6 wo. polio49.4.9. seit mai 1998 anstelle des impfstoffs nach sabin als standardimpfung gegen polio fü r kinder ab vollendetem 2. lebensmon. und erwachsene ö ffentlich empfohlen. totimpfstoff mit formolabgetö teten polioviren der typen i, ii und iii. als monovakzine oder in kombinationsimpfstoffen ( 4tab. 9.2). grundimmunisierung mit 2 injektionen (z. b. ipv-virelon j c) s. c. oder i. m. im abstand von 4-6 wo. sowie dritte impfinjektion nach ca. 12 mon. auffrischimpfung alle 10 j. selten verstärkte lokalreaktion. beginn: 2 wo. nach der 2. impfung; konversionsrate: 94-96 %; dauer: individuell verschieden. bei polioexposition evtl. auch vorzeitige auffrischimpfung. keine, auch nicht symptomatische hiv-infektion bei kindern. da eine lokale darmimmunität durch die salk-impfung nicht zustande kommt, fehlt dieser impfung der epidemiologische, seucheneingrenzende effekt. nicht verfü gbar. wenn titer 5 1 : 32. gravidität. postvakzinale kontrolle bei der schwangerenvorsorge. akzidentelle impfung in der gravidität ist kein grund zum schwangerschaftsabbruch; es wurde noch nie die schädigung eines embryos/feten beobachtet. rö teln-prävention bei seroneg. schwangeren bis zur 16. ssw. zuverlässige unterdrü ckung einer infektion nur innerhalb der ersten 4 d nach rö teln-exposition. bei exposition vor der 6. ssw erneute immunglobulingabe nach 4-5 wo. es gibt keine präparate mit spezifischem rö teln-immunglobulin. zur pep bei seroneg. schwangeren human-immunglobulin mit ausreichender rö teln-ak-konz. verwenden, z. b. gamma-venin j , endobulin immuno j , sandoglobulin j . dos. nach angaben des herstellers. im 10. lj., dann alle 10 j. 1 impfdosis (0,5 ml i. m.), vorzugsweise mit td-impfstoff, z. b. td-vaccinol j , "d": diphtherie. bei verletzungen impfung mit tetanus-monovakzine, wenn letzte td-impfung 5 10 j. in zweifelsfällen und bei v. a. unverträglichkeit titerbestimmung. impfschutz, wenn titer 4 0,01 ie antitoxin/ml. gehäuft impf-unverträglichkeiten, wenn titer 4 4 ie antitoxin/ml. keine bes., allg. ki ( 49.2.1). sehr gut verträglicher impfstoff. auch als kombinationsimpfstoffe dt, dpt und td (nach dem 7. lj.). bei antikoagulanzienther. kann das antitoxin tetagam n j auch s. c. angewendet werden. nach verletzung ohne ausreichenden impfschutz simultanimpfung (s. o.). tollwut49.4.8. postexpositionell; präexpositionell ö ffentlich empfohlen fü r risikogruppen (z. b. fö rster, veterinäre). reiseimpfung bei reisen nach sü dostasien und sü damerika. totimpfstoff, human diploid cells (hdc) impfung. wiederimpfung nach 3 j. 1 dosis. immunisierung bei bissverletzungen abhängig vom zeitpunkt der letzten impfung: 5 1 j. r 0-3 d; 1-5 j. r 0-3-7 d; 4 5 j. r entsprechend ungeimpften personen. bei postexpositioneller impfung keine, da erkrankung immer tö dlich verläuft. präexpositionelle impfung: allg. ki ( nach massiver exposition simultanimpfung. typhus49.3.1. reisen ( indikation immunstimulation bei rezid. harnwegs-bzw. atemwegsinf. sowie vaginalen inf. lyophilisierte extrakte eines spektrums der ü blicherweise bei den jeweiligen inf. beteiligten bakterien. atemwegsinf.: nach mö glichkeit sollten impfungen außerhalb der grav. erfolgen. wegen einer theoretisch mö glichen gefährdung des feten (bis auf wenige ausnahmen) keine lebendimpfstoffe anwenden. unbedenkliche impfungen in der grav.: typhus oral, tollwut (vitale ind.), polio-salk, tetanus, hep. a, hep. b, diphtherie, influenza. impfung nur bei sorgfältiger risikoabwägung wegen fehlender erfahrung bei schwangeren: fsme, meningokokken, masern (bei bedarf passive immunisierung), pneumokokken, mumps, japan-enzephalitis, gelbfieber, wenn reise in epidemiegebiet unvermeidlich. kontraindiziert in der grav.: cholera; varizellen und rö teln: bei bedarf passive immunisierung, versehentliche impfung während der grav. keine ind. fü r schwangerschaftsabbruch. eine allergische reaktion auf eine impfung kann durch den impfstoff selbst oder durch hilfsstoffe im präparat verursacht sein. um die immunität abzuklären, zuerst eine titerbestimmung der entsprechenden ak durchfü hren. wenn kein erhö hter ak-titer vorliegt, allergologische abklärung auf hilfsstoffe. impfstoffe gegen folgende krankheiten enthalten hü hnerprotein (nach abnehmender konz. geordnet): gelbfieber, influenza, masern, mumps, fsme, tollwut. v. a. hü hnereiweißallergie vor impfung allergologisch abklären. einteilung der allergischen reaktion in 3 schweregrade. grad 1: allg. unverträglichkeit von hü hnereiern ohne allergische oder anaphylaktische symptome, neg. hauttest: keine gegenanzeigen fü r impfungen, auch nicht gegen influenza oder gelbfieber. grad 2: im hauttest nachgewiesene, jedoch klinisch nicht bedeutsame hü hnereiweißallergie: gelbfieber-impfung ist relativ kontraindiziert und darf nur in dringlichen fällen gegeben werden. impfung gegen influenza in der klinik. grad 3: pat. mit sofortreaktion nach verzehr von hü hnereiweiß mit urtikaria, laryngo-/bronchospasmus, rr-abfall. impfung gegen influenza und gelbfieber kontraindiziert. impfung gegen masern, mumps, fsme und tollwut (präexpositionell) in der klinik. neomycin u. a. in folgenden impfstoffen enthalten: masern, mumps, rö teln, polio (je nach hersteller unterschiedlich). im hinteren abschnitt der roten liste j (rosa seiten), befindet sich ein verzeichnis von notfalldepots, in denen folgende seren und plasmaderivate aufbewahrt werden: botulismus-antitoxin vom pferd, fsme-immunglobulin, hep.-b-immunglobulin, polyvalentes immunglobulin, schlangengift-immunserum polyvalent europa, tetanus-immunglobulin, tollwut-immunglobulin, tollwut-impfstoff, varicella-zoster-immunglobulin. zusätzlich in der roten liste j : verzeichnis von informations-und behandlungszentren fü r vergiftungen für deutschland und europa ( impfempfehlungen; impfvorschriften fü r den internationalen reiseverkehr. empfehlungen zur malariaprophylaxe. tab. 9.13 hä ufige bakterielle infektionen in der praxis angina tonsillaris ( akute bronchitis ( viren ( erkrankung bei vorliegen der folgenden kriterien: akuter krankheitsbeginn, fieber 4 38 c, husten und/oder dyspnoe (atemnot) oder: tod durch unklare akute respiratorische erkrankung. mind. eine der drei folgenden expositionen innerhalb von 7 d vor erkrankungsbeginn: aufenthalt in einem zoonotisch betroffenen gebiet und direktem kontakt mit lebenden oder toten tieren (nur geflü gel, wildvö gel, schweine) oder: tätigkeit auf einer geflü gel-oder schweinefarm, auf der innerhalb der letzten 6 wo. infizierte oder infektionsverdächtige tiere eingestallt waren oder: direkter kontakt zu einem pat. oder seinen sekreten mit klinischem bild und labordiagnostisch nachgewiesener infektion oder laborexposition. mittels virusisolierung und subtypisierung, nukleinsäure-nachweis, z. b. spezifische h5(n1)-pcr und antigennachweis mit monoklonalen h5-oder h7-ak mittels immunfluoreszenztest (ift). grundsätzlich muss der pos. suchtest durch immunoblot-test bestätigt sein. bei definitiv hiv-pos. diagn. stü tzt sich das weitere vorgehen auf ein ungestö rtes vertrauensverhältnis zwischen ha und pat. es dü rfen keine unberechtigten hoffnungen geweckt werden, aber es muss das gefü hl vermittelt werden, dass der ha dem pat. zur seite steht, wenn symptome "kontrolliert" werden mü ssen. im vordergrund der therapie steht die erhaltung der lebensqualität. aufklärung ü ber krankheitsverlauf ( hinweis auf selbsthilfegruppen ( berufliche einschränkungen: offiziell keine, außer bei ü bernahme ins beamtenverhältnis auf lebenszeit. bei ä rzten: von operativer tätigkeit abraten (ü bertragungen durch hiv-pos. operateure sind dokumentiert). kö rperliche ü beranstrengung vermeiden (z. b. leistungssport), keine kampfsportarten wegen des risikos blutender verletzungen, sonst breitensport uneingeschränkt mö glich. prophylaxe von "kinderkrankheiten" in der umgebung von hiv-positiven ( impfungen bei hiv-pos. kindern ( zusätzlich zur medizinischen betreuung der kinder darauf hinweisen, dass die eltern durch aids pflegebedü rftig werden bzw. versterben. kindergarten und schulbesuch: kein erhö htes infektionsrisiko durch ü bliche soziale kontakte, daher keine informationspflicht gegenü ber dritten. die bescheinigung ü ber die freiheit von infektionskrankheiten nach ifsg kann ausgefü llt werden. dennoch ist eine information der kindergarten-bzw. schulleitung am ehesten durch die eltern sinnvoll, um einer gefährdung durch "kinderkrankheiten" begegnen zu können. asymptomatische hiv-inf.: pat. ist klinisch gesund und infektiö s. cd4-zellen meist 4 500/ll (anzahl der cd4-zellen orientiert ü ber das ausmaß des immundefekts). aids-related complex: auftreten von erkr., die keine aids-definierenden erkr. laut kategorie c sind, aber auf eine stö rung der zellulären immunität hinweisen: mund-soor, chron. vulvovaginale candidiasis, fieber 4 38,5 c, diarrhoe 4 4 wo., rezid. herpes simplex oder herpes zoster mehrerer dermatome, periphere neuropathie, bazilläre angiomatose, idiopathische thrombozytopenische purpura. einteilung nach klinischen gesichtspunkten und anzahl der cd4-zellen/ll. bestimmung der cd4-lymphozyten (= t-helfer-zellen) und virusload im rahmen der antiretroviralen ther. ( 49.9.6) sowie bei jeder verschlechterung des gesundheitszustands. bei asymptomatischen pat. ohne antiretrovirale behandlung im ersten halben j. monatlich, um einen trend zu erkennen und um rechtzeitig die ind. fü r antiretrovirale ther. ( 49.9.6) und prophylaxe opportunistischer inf. zu stellen ( 49.9.6). -wenn im ersten halben j. stabile werte, cd4 4 350/ll und virusload 5 30 000/ ml ohne antiretrovirale ther. vorliegen, kontrollen alle 3 mon. -wenn cd4 5 350 bzw. virusload 4 30 000/ml im rahmen des monitorings bei antiretroviraler ther. -resistenzbestimmung vor aufnahme einer behandlung. hä ufige krankheitsbilder bei aids prophylaxe durch konsequenten sonnenschutz mit mind. lsf 12; bei sehr hellhäutigen sowie in bestimmten gegenden (gletscher, ä quatorialgebiet, australien) und ü ber die mittagszeit (11.00-15.00 uhr) hö heren lsf, "sun blocker" bzw. strikte expositionsvermeidung: langsame und hauttypgerechte adaptation: morgens oder nachmittags, nie ü ber die mittagszeit, helle haut nie entblö ßt in die sonne,425.9.1. cave: doxycyclin, johanniskrautpräparate. prophylaxe: mö glichst gletscherbrille (seitlich geschlossen) tragen, auf modische brillenformen (z. b. schmetterlingsbrille) verzichten. prophylaxe: immer kopfbedeckung tragen, gilt v. a. fü r kinder und ältere menschen (cave: glatze). frü he nachtruhe, früher tagesbeginn einige stunden schlafen oder kurzer schlaf ("nickerchen") oder entspannungstechniken sich dem tages lungenpest, virales hämorrhagisches fieber. doppel-pi (2 pi) haben eine gü nstigere pharmakokinetik, z. b. keine nü chterneinnahme erforderlich, und # nw-rate . bei ki gegen pi: kombination aus 2 nrti und einem nichtnukleosidischen reverse-transskriptase-inhibitor (nnrti) mö glich. erprobte kombinationen: 2 nrti und 1 pi: azt/3tc und nfv oder idv oder sqv; z. b. combivir j (azt/3tc) und viracept j (nfv = nelfinavir); 11 tbl./d. 2 nrti und 2 pi: azt/3tc und sqv/rtv oder idv/rtv; z. b. combivir j (azt/ 3tc) und crixivan j (idv = indinavir) plus /d; z. b. combivir j (azt/3tc) und viramune j (nvp = nevirapin); 4 tbl./d. 3 nrti: azt/3tc und abc lamivudin) kö nnen durch zerit j (d4t = stavudin) plus epivir j (3tc = lamivudin) ersetzt werden. cave folgende kombinationen (antagonistische wirkung ! indikationsstellung und durchfü hrung von haart durch ein erfahrenes zen a-oder b-symptome nach cdc 1993) bzw. mit immunthrombozytopenie pat. mit viruslast 4 30 000 kopien/ml plasma pat. mit cd4-zellen 5 350/ll pat. mit relevanter zunahme der viruslast (z. b. mehr als 1 log) pat fakultative indikationen pat. mit viruslast zwischen 10 000 und 30 000 kopien/ml pat. mit 350-500 cd4-zellen/ll pat einzelfällen kann sich bei entsprechender disposition ein diab. mell. manifestieren, bei bekanntem diabetes die stoffwechsellage unter protease-inhibitoren entgleisen protease-inhibitoren mit anderen medikamenten4tab. 9.33 r nicht gleichzeitig verordnen! bz initial, alle 2 wo. und 2 wo. nach ende der pep. hiv-pcr bei grippalem krankheitsbild innerhalb von 4 wo bei beruflicher hiv-exposition im sinne eines unfalls durch die berufsgenossenschaften. bei außerberuflicher hiv-exposition werden die kosten durch die gkv i. d.r. nicht ü bernommen die hausarztpraxis ist meist erste anlaufstelle fü r reisemedizinische fragen. aufgaben des hausarztes einschätzung der reisefähigkeit. beratung ü ber chemoprophylaxe und allg. verhaltensmaßnahmen. durchfü hrung von impfungen untersuchung/screening des zurü ckgekehrten reisenden allgemeine empfehlungen zur reisefä higkeit vor reisemedizinischen maßnahmen/beratungen muss abgeklä rt sein allg. gesundheitszustand/"fitness"? aktuelle, chron., ansteckende erkrankungen? dauermedikation? immunsuppression? gravidität? allergien? impfstatus? kontraindikationen fü r reisen bei der entscheidung, ob ein pat. reisefähig ist, bes. fü r fernreisen, muss zwischen absoluten ki und relativen ki unterschieden werden. relative ki fü r eine reise bedeutet, dass nach risikoabschätzung und beratung durch den arzt sowie sorgfältiger vorbereitung der reise, z. b. medikamentö se und ärztliche notfallversorgung gewährleistet nach magen-darm-blutungen: i. d.r. mindestens 3 wo. fü r pat. mit art. hypertonus 4 200/120 mmhg (während des fluges sinkt der systolische druck, der diastolische erhö ht sich, deshalb bes nyha iv) oder angina pectoris. bei anfallsleiden (relativ, falls starke sedierung und evtl. begleitung, flug möglich) mit druckausgleichsstö rungen in der eustachischen rö hre: akute otitis media, paukenerguss, tubendysfunktion prophylaxe: nach abklingen der akuten hno-erkr. kann der pat. fliegen, jedoch sollten prophylaktisch einige zeit vor dem start und bes. ca. 30 min. vor dem sinkflug abschwellende nasentropfen tief in die nase geträufelt werden (ebenso bei erkältungen und leichter sinusitis) autoreisen keine fahrten unter zeitdruck (puls-und blutdruckerhö hung) hitzestau im auto vermeiden, im sommer keine fahrten um die mittagszeit. nachtfahrten -cave: risiko fü r pat. mit nachtblindheit, katarakt (beratung durch augenarzt) zur ärztlichen beratung gehö rt die beurteilung der fahrtauglichkeit: erkrankungsund therapiebedingte einschränkungen (z. b. medikamenten-nw) sind zu berü cksichtigen. nicht nur aus reisemedizinischer sicht, sondern auch aus forensischen grü nden muss der pat. auf einschränkungen der fahrtauglichkeit hingewiesen werden. dazu gehö ren z. b.: schwere herzrhythmusstö rungen (kreislaufschwäche bei ventrikulärer tachykardie insulinpflichtige diabetiker dü rfen nur in ausnahmefällen fahrzeuge zur fahrgastbeförderung oder lkw (fü hrerscheinklasse ii) fü hren (relevant, falls der pat die akut dekompensieren kö nnen, z. b. schwere neurosen, schizophrenien, chron. progrediente hirnorganische erkr. 4tab. 9.45). cave: nw der meisten mittel, z. b. eingeschränkte fahrtü chtigkeit fü r den notfall kann eine liste wichtiger begriffe bezü glich grav./geburt in englisch oder der jeweiligen sprache des reiselandes nü tzlich sein kontraindikationen fü r reisen in der schwangerschaft relativ (abhä ngig von reiseziel im ersten trimenon und 4 wo. vor der entbindung abortneigung, blutungsneigung. rhesusinkompatibilität in vorausgegangenen schwangerschaften. reisen in epidemiegebiete und in malariagebiete mit chloroquinresistenz. reisen in länder oder gegenden ohne gute ärztliche und fachärztliche versorgung bis zur 32. ssw bestehen meist keine einschränkungen fü r schwangere; mit attest eines arztes kann eine erlaubnis bis zur 36. ssw mö glich sein. genaueres muss die schwangere bei der entsprechenden gesellschaft erfragen fahrten regelmäßig pausen einlegen und fü ße vertreten (verbesserung des venö sen rü ckflusses) verhalten am reiseziel klimatische extrembedingungen vermeiden, kein intensives sonnenbaden (cave: wärme-/kreislaufbelastung und stö rende pigmentflecken) kein schneller aufstieg in große hö hen (ab 2000 m messbarer relativer sauerstoffmangel, d. h. hö hen 4 2000 m ganz meiden einige tage zur akklimatisierung abwarten keine sportarten mit erhö htem traumatisierungsrisiko (z. b. ski alpin, wasserski, tauchen, mannschafts-und kontaktsportarten) bzw. ü bermäßige kö rperliche belastung vermeiden (durchblutungssteigerungen in muskel und haut verringern plazentare durchblutung und sauerstoffversorgung) jedoch ist das krankheitsrisiko erhö ht und die allg. belastbarkeit oft eingeschränkt. dies zeigt auch die verschiebung der erkrankungsursachen im urlaub zugunsten der erkr akute verwirrtheitszustände durch ungewohnte umgebung bei zerebralsklerose. dekompensation einer herzinsuff. oder zerebraler durchblutungsstö rungen, z. b. durch hypoxie im flugzeug oder tropische temperaturen. dehydratation bei kinetosen und diarrhoe kontraindikationen fü r reisen ä lterer menschen absolute und relative ki fü r (fern-)reisen ( 49.10.1) zusätzlich klinisch manifeste zerebralsklerose und schwere inkontinenz dem pat. evtl. ein notfall-set mit den im status asthmaticus benö tigten medikamenten zusammenstellen bzw inf.) gestö rt ist, sollte auf jeden fall von einer reise mit zeitverschiebungen abgeraten werden. durch jet-lag-sy. (veränderung der zirkadianen hormone) und stress der reise (evtl. flugangst, hektik r erhö hter noradrenalin-spiegel) kommt es zur einer zusätzlichen labilisierung des stoffwechsels (kontrainsulinäre hormone "). grundsätzlich sollten nur gut geschulte insulinpflichtige diabetiker fernreisen unternehmen; mö glichst mit angehö rigen oder freunden reisen sport/kö rperliche aktivitäten: kein anstrengender und riskanter sport andere essgewohnheiten berü cksichtigen: z. b. wird in den mittelmeerländern selten vor 21 uhr zu abend gegessen r "mediterrane hypoglykämie"; außerdem an mö gliche diätprobleme und mangelnde verfü gbarkeit von (diabetiker-)nahrungsmitteln im zielland denken längere autofahrten nur bei tag und in begleitung, bzw. mö glichst alle 2 h pause mit zwischenmahlzeit. auch im auto kohlenhydratvorrat von mind. 100 g mitfü hren bei schiffsreisen muss ein antiemetikum griffbereit sein faustregel: 1 /12 der ü blichen dosis des basalen verzö gerungsinsulins plus oder minus pro stunde zeitverschiebung. ab ankunft in der neuen zeitzone: umstellung der uhr und ü bliches schema wie zu hause. beispiel: stuttgart -los angeles = zeitverlängerung 9 h: plus 9 /12. los angeles -stuttgart = zeitverkü rzung 9 h: minus 9 /12 cave: grö ßte hypoglykämiegefahr während der ersten nächte nach der zeitverschiebung; aufgrund der hö heren blutzuckerschwankungen, häufiger bz messen, um hypoglykämien durch zusätzliche mahlzeiten und durch evtl. nachspritzen von normalinsulin gegenzusteuern spritzen und nadeln in ausreichender menge sowie ärztliche bescheinigung fü r den zoll mitfü hren (verwechslung mit drogenabhängigen vermeiden). insulinpumpen: ersatzbatterien mitnehmen, netzspannung kontrollieren. blutzuckermessgerät mitnehmen, um bz tägl hände vor jedem essen mit seife waschen. mö glichst keine gemeinschaftshandtü cher benutzen kleidung an kopfbedeckung und sonnenbrille denken (cave: sonnenstich, hitzschlag, konjunktivitis) mö glichst immer schuhwerk tragen, auch am strand nicht barfuß gehen (cave: giftige insekten flö he (malaria, fsme, borreliose, dengue-fieber,49.10.8): haut bedeckt halten, auch an unterschenkeln und armen; lange hosen oder strü mpfe versicherungsschutz eine private reisekrankenversicherung mit rü ckhol-garantie ist zu empfehlen. fü r privat versicherte: klären, ob die versicherung diese rü ckhol-garantie beinhaltet verhalten im reiseland kein baden in sü ßwasserseen oder -tü mpeln gesundheitsspritzen a. geschlechtskrankheiten) oder sexuelle abstinenz. aufenthalt in mü ckensichern räumen, mit klimaanlage reiseapotheke art und umfang der reiseapotheke wird bestimmt durch reiseart, reisedauer, zielland, zahl und alter der mitreisenden (kinder? senioren?) sowie deren vorerkr. und/oder spezielle dispositionen fü r bestimmte erkr. cave: verschreibung ist keine kassenleistung hinweise zur verwendung der einzelnen mittel dem reisenden mö glichst schriftlich mitgeben; nicht auf die informationen der beipackzettel verlassen, da diese fü r laien oft nicht verständlich sind blähbauch" nicht bei kindern 5 2 j. cave: ileus. lingualtbl. praktisch, falls keine flüssigkeit vorhanden. bei fieber und blut im stuhl: arzt aufsuchen! fieber und schmerzen paracetamol-ratiopharm ass 500 ratio j analgetika-nephropathie; analgetika-asthma cave: bei pat. mit nierenund leberfunktionsstörungen. bei sehr hohem fieber über mehrere tage und starken schmerzen: arzt aufsuchen! kleinkindern nicht mehrtägig und nicht großflächig anwenden sonnenbrand fenistil gel j , divisan j lotion, ass 500 ratio j hautreizung, allergische reaktion prophylaxe ist besser! sonnenschutzmittel mit hohem lsf unruhe nicht bei kindern 5 2 jet-lag/ schlafstörungen pflanzliche schlafmittel nur schlafmittel, falls keine zeit für adäquate adaptation, z. b. kurze geschäftsreisen; einnahme verlängert die synchronisationszeit konjunktivitis augentr gaze/verbandklammern; sicherheitsnadeln instrumente/sonstiges fieberthermometer mit bruchsicherer hülle, kleine schere, fremdkörperpinzette cave: auf impfdokumente oder titer verlassen, nicht auf angaben des pat./reisenden! obligatorische impfungen fü r das jeweilige reiseland erfragen (tropeninstitute, adressen435.1.2) unter berü cksichtigung der reiseroute; einige länder verlangen einen impfnachweis schon fü r eine zwischenlandung ohne eigentliche einreise empfehlenswerte impfungen: abhängig von gesundheitsstatus, zielland, reiseroute und reiseart sowie reisetransportmittel. individuellen impfplan erstellen und durchfü hren. cave: ki fü r impfungen und impfabstände impfbefreiungszeugnis falls obligatorische impfungen bei reisenden kontraindiziert sind, muss ein impfbefreiungszeugnis mit unterschrift des arztes und einem beglaubigungsstempel mitgefü hrt werden; je nach reiseland in englischer oder franzö sischer sprache und evtl expositionsprophylaxe risiko von infektiö sen mü ckenstichen kann verringert werden durch: imprägnierte moskitonetze: z. b. mit permethrin. repellentien. kleidung, die die haut vollständig bedeckt (auch unterarme/unterschenkel/knöchel), v. a. in den dämmerungs-und nachtstunden (transmission findet hauptsächlich während der dämmerung statt). hosen mit insektenspray einsprü hen mü cken stechen auch durch dü nnen stoff chemoprophylaxe in der schwangerschaft bei einer schwangerschaft muss prinzipiell immer eine nutzen-risiko-abwägung erfolgen. proguanil, z. b. paludrine j , und chloroquin, z. b. resochin j , kö nnen in schwangerschaft und stillzeit eingesetzt werden. mefloquin, z. b. lariam j , nicht im 1. trimenon, also erst ab 2. trimenon riamet j , liegen keine ausreichenden daten vor 43 aufgefü hrten medikamente sind fü r die stand-by-notfalltherapie geeignet, wobei die stand-by-medikation prinzipiell verschieden von der prophylaxe sein soll. die behandlung soll jedoch, wenn mö glich, immer im krankenhaus 1-2 tbl./d à 100 mg grenzgebieten thailand/ kambodscha vor bis 4 wo. nach reise * zone a: gebiete ohne chloroquin-resistenz oder ohne plasmodium falciparum. zone b: gebiete mit niedriggradiger chloroquin-resistenz die auswahl richtet sich nach individuellen gesichtspunkten (siehe auch anmerkungen unten) empfohlene chemoprophylaxe bei kindern paludrine j 5 11 mon. 25 mg/d, ab 11 mon. 3 mg/kg kg/d lariam j ab 3 mon. bzw. 5 kg kg 5 mg/kg kg/wo ab 11 kg kg 1 junior tbl. = ges. 62,5/25 mg pro 10 kg kg/d, max doxy v. ct j ab 8 j. 1,5 mg/kg kg/d erfolgen, insbesondere bei rasanter verschlechterung des krankheitsbildes und bei indikation zur therapie mit chinin auswahl der medikamente in abhängigkeit von who-risikoklasse und abstimmung mit der zuvor verwendeten chemoprophylaxe4tab. 9 bei malaria tertiana (p. vivax) und malaria quartana (p. malariae) ist im anschluss an die initialther. mit einem der o. g. chemotherapeutika wegen rezidivgefahr eine behandlung mit primaquin (importmedikament) zur eradikation der extraerythrozytären stadien erforderlich. malaria-schnelltests, z. b. malaquick j , sind nur bei pos. ausfall eine entscheidungshilfe (auch bei hoher parasitämie falsch neg nicht lesen; nach vorn blicken und ein objekt fixieren. kauen (apfel, kaugummi) soll ebenfalls präventiv wirken reaktionsvermögen eingeschränkt, urtikaria, erregungsleitungsstörungen, deshalb nicht bei personen mit hrst und therapie mit chinidin-typ-medikamenten, neurologischpsychiatrische störungen (macht schwere depressionen mit suizidgefahr) nur therapie (keine prophylaxe) unter intensiver ü berwachung. kann zu lebensbedrohlichen hrst führen. deshalb auch kein stand-by mehr, obwohl gut wirksam. nw: ü belkeit, kopfschmerzen, qt-verlängerungen. ki: vorbestehende qt-verlängerungen, bekannte arrhythmien keine chemoprophylaxe reisemedizin 587 dadurch beeinträchtigung mentaler und physiologischer funktionen, z. b. schlaf-und wachzustand, gedächtnis-und konzentrationsleistungen, hormonausschü ttung, darm-und blasenfunktion. fü r eine zeitverschiebung ab 2 h werden mind flü ge von ost nach west (zeitverlängerung, verschiebung der schlafphase) sind weniger beeinträchtigend als flü ge von west nach ost (zeitverkü rzung, häufig ü berspringen der schlafphase) entsprechend der zeitverschiebung kann reisenden ein verändertes einnahmeschema fü r medikamente mitgegeben werden trotz zunehmender aufklärung ü ber kurz-und langfristige risiken intensiver sonnenlichtexposition (melanome425.10.3, basaliome425.10.4, vorzeitige alterung der haut) wird das risiko nach wie vor unterschätzt. zu den vermeidbaren kurzfristigen folgen intensiver sonnenstrahlung, die das allgemeinbefinden am urlaubsort beeinträchtigen sonnenstrahlung in verbindung mit duftstoffen, konservierungsmitteln, fett und emulgatoren in kosmetika und sonnenschutzmitteln. klinik: juckende, evtl abstieg in tiefere lagen und/ oder aufstiegspause mit nachfolgend langsameren aufstiegen. prophylaxe: guter trainingszustand vor antritt der reise (evtl. check-up durchfü hren); keine zu großen hö henunterschiede tägl. bewältigen, keine gewalttouren; auf ausreichende flü ssigkeits-und salzzufuhr achten klinik: hö henlungenö dem mit dyspnoe, rg der atemwege und rapidem leistungsabfall (lebensbedrohlich!) der tauchsport verlangt ein hohes maß an körperlicher und mentaler fitness; minimum-check-up: herz-kreislauf-funktion und lungenfunktion. bei älteren reisenden evtl. belastungs-ekg. psychische belastbarkeit abschätzen! bei hno-erkr aufklärung ü ber dekompressionskrankheit und luftembolie. -psychische stabilität und mentale fitness: es kann unter wasser bei bestimmter disposition zu (lebensgefährlichen) panikreaktionen oder euphorischen ü berreaktionen kommen gefahr: lässt ihre wirkung noch während des tauchgangs nach, ist beim auftauchen der ü berdruckausgleich nicht mehr mö glich. folge: barotraumen in ohren und nnh ( 422.6.3). cave: zusammenhang wird von laien oft nicht verstanden und deshalb nicht befolgt! -vor und nach tauchgängen keinen alkohol trinken abhängig von dauer und tiefe der tauchgänge darf fü r mind. 24-48 h danach nicht geflogen werden (genaue zeit wird nach dem letzten tauchgang berechnet, diese berechnung zu lernen, ist bestandteil einer seriö sen tauchausbildung) infektiö se erkrankungen bilharziose (schistosomiasis) definition durch im menschlichen venensystem (endwirt) lebende pärchenegel (schistosomen) verursacht schistosoma haematobium (afrika, mittl. osten): befällt venengeflecht des kleinen beckens. klinik: blasenbilharziose mit hämorrhagischer zystitis. ko: blasenpapillome, blasenfisteln. diagn.: eier im urin, bei primärinf. serologie. ther.: praziquantel, z. b. biltricide j 3 dosen à 20 mg/kg kg in 4-6 h abstand als 1-tag-behandlung bilanil j 3 dosen à 10 mg/kg kg p. o. als 1-tag-behandlung sü damerika): befällt leber und darm. klinik: darmbilharziose mit ruhrähnlicher kolitis. ko: perirektale abszesse, polypen eiablage im blutgefäßsystem. eier dringen durch die darmwand ins darmlumen. klinik und diagn. wie schistosoma mansoni. ther.: praziquantel (s. o.) für touristen ist das erkrankungsrisiko extrem niedrig, da kein oder selten kontakt mit slumbewohnern oder aufenthalt in slums. nur impfen, falls obligatorisch (einreise nach sansibar und pemba, tansania) typ 2) und das hämorrhagische dengue-fieber (dhf, schwer verlaufend, hohe letalität) sowie das dengue-schock-sy. (dss) hervorrufen. ü bertragung durch tagaktive mü cken (v. a. aedes aegypti), die in der nähe menschlicher behausungen in wasseransammlungen brü ten. nach stich und eintritt in die blutbahn vermehrung in regionalen lk injektion der konjunktiven typisch. diffuses, stammbetontes erythem am 2.-3. d, danach morbilliformes exanthem am rumpf mit zentripetaler ausbreitung auf kopf und extremitäten, verbunden mit einem zweiten fieberschub. evtl. petechiale blutungen und epistaxis. abklingen der symptome nach 5-6 d prophylaxe expositionsprophylaxe; haut durch kleidung und repellentien schü tzen durch arboviren hervorgerufene und die stechmü cke aedes aegypti sowie haemagogus-arten (moskitos) ü bertragene erkr. jährlich ca. 2500-3000 erkrankungsfälle mit einer gesamtletalität von ca. 20-30 % (who). ikz 3-6 d. vorkommen: norden sü damerikas und zentralafrika4abb. 9.6; asien ist frei von gelbfieber! (häufig falschinformation) remissionsstadium: fieberabfall am 3. oder 4. d, evtl. ausheilung. bei schwerem verlauf stadium der organschädigung: hepatitis mit ikterus und erbrechen, nephritis mit proteinurie, hämorrhagische diathese mit profusen blutungen diagnostik tropenanamnese bei ungeimpften (oder zu spät geimpften) personen mit typischer klinik, evtl. igm-ak-nachweis prophylaxe impfung ( 49.2.3); auch empfehlenswert fü r gebiete, fü r die die impfung nicht obligatorisch ist. cave: viele staaten ländern mit schlechten hygienischen verhältnissen; ü bertragung vorwiegend fäkal-oral, verunreinigte gewässer, meerbuchten, aber auch durch blutprodukte (selten). ikz 2-6 wo. vorkommen: ubiquitär in ländern mit schlechten hygienischen verhältnissen klinik im kindesalter (bis ca. 6 j.) meist anikterisch, oft nur gastrointestinale symptome. bei inf. im erwachsenenalter lange und schwere verläufe mö glich. cave: bei reisenden ü ber 50 j. ohne immunität häufiger als in jungen jahren fulminante verläufe mit hö herer letalität havrix j ) fü r alle fernreisenden; aufgrund der guten verträglichkeit und der fast 100 ïgen schutzwirkung sollte die aktive schutzimpfung einer passiven impfprophylaxe (globulin i. m.) vorgezogen werden unkomplizierte reisediarrhoe betrifft 20-50 % aller fernreisenden erreger 65 % bakt. (meist pathogene e.-coli-stämme), 30 % viral, 5 % protozoen klinik flü ssiger oder wässriger stuhl, häufig abdom. krämpfe, ü belkeit, blähungen; plö tzlicher beginn, milder bis mittelschwerer verlauf ü ber durchschnittlich 3-4 d therapie flü ssigkeitssubstitution: orale rehydratationssalze (z. b. elotrans j -pulver); in den apotheken tropischer länder ist "oral rehydration salts" (ors) verfü gbar, dieses im angegebenen volumen abgekochtem wasser auflö sen gezuckerten tee trinken, salzgebäck essen mg initial, dann 2 mg nach jedem wässrigen stuhlgang, max. 16 mg tägl.; kinder 8-13 j. 2 mg initial, dann 2 mg nach jedem wässrigen stuhlgang, max. 8 mg tägl.; bei kindern 5 8 j. imodium j -n lö sung verwenden. -ethacridinlactat 8, z. b. metifex j 200 mg drg.: ab 14. lj. am 1. und 2. krankheitstag jeweils 3 â 1 drg. nach dem essen, ab dem 3. krankheitstag 2 â 1 drg prophylaxe strikte einhaltung der allg. verhaltensregeln, bes. kost und persö nliche hygiene ( im stuhl. cave: malaria ciprofloxacin: 2 â 500 mg/d fü r 3 tage bei fieber oder blutigem stuhl mö glichst auf motilitätshemmer verzichten keine besserung bei fieber oder blutigem stuhl nach 2 d r arzt aufsuchen keine besserung bei wässriger diarrhoe nach 3 d r v. a. lambliasis (häufiger durchfallerreger in warmen ländern) r einmaldosis tinidazol 2 g, z. b. simplotan j charakter der reise, dauer u. a.) ist nach längerem tropenaufenthalt unter schwierigen bedingungen angezeigt, z. b. nach arbeitsaufenthalten oder abenteuerreisen (auch wenn der rü ckkehrende symptomlos ist) und immer, wenn eines oder mehrere der folgenden symptome auftritt: unklares fieber schmerzen beim wasserlassen/blut im urin, genitale affektionen (schmerzen, pruritus, ulzerationen) cave: immer auch an das versagen der malaria-prophylaxe denken. frü hzeitig kontakt mit tropenmediziner aufnehmen bb: auf eosinophilie achten: hinweis fü r helminthiasis. leberfunktionsparameter tests zum nachweis von ak gegen viren, bakterien, protozoen, helminthen bei konkretem verdacht oder unklaren beschwerden. je nach ergebnis: facharztü berweisung zum internisten oder an eine tropenmedizinische klinik malaria pat. zur blutabnahme in geeignetes labor schicken, wo ausreichende erfahrung in anfertigung und auswertung z. b. eines "dicken tropfens" besteht; vorher telefonisch rü cksprache nehmen plasmodiensuche auch im edta-blut mö glich abklä rung einer bluteosinophilie nach tropenaufenthalten stuhluntersuchung auf helminthenlarven zum ausschluss einer strongylose anteil hö her bei langzeitaufenthalten); neg. erregernachweis bei 65 % der tropenrü ckkehrer serum-ige-spiegel 4 1000 iu/ml das gesetz zur bekämpfung und verhü tung von infektionskrankheiten beim menschen (infektionsschutzgesetz, ifsg) regelt aufgaben und koordinierung der zuständigen gesundheitsbehö rden, meldewesen, verhü tung und bekämpfung von infektionskrankheiten (schutzimpfungen, gemeinschaftseinrichtungen, lebensmittelhygiene, wasserhygiene), tätigkeit mit krankheitserregern, entschädigung bei impfschaden, tätigkeitsverbot, bußgeld-und strafbestimmungen. außerdem meldepflichtig (gefahr fü r die allgemeinheit) sind a) das auftreten einer bedrohlichen krankheit oder von mindestens zwei gleichartigen bedrohlichen unbekannten erkrankungen mit wahrscheinlich epidemischem zusammenhang auftreten nosokomialer infektionen mit wahrscheinlich epidemischem zusammenhang verdacht einer außergewö hnlichen impfreaktion oder eines impfschadens zur meldung verpflichtete personen der feststellende arzt, bei stationären einrichtungen der leitende arzt, leiter von heimen oder lagern bei den in tab. 9 der leiter einer pathologisch-anatomischen diagn., wenn ein befund erhoben wird, der mit hoher wahrscheinlichkeit auf eine erkr. oder einen krankheitserreger aus tab. 9.49 o. 9 der leiter des diagn. labors bei krankheitserregern aus tab. 9.49 o. 9 erkrankung an oder verdacht auf (a = regelung fü r ausscheider: besuch von gemeinschaftseinrichtungen nach rü cksprache mit dem gesundheitsamt mö glich): cholera (a) typ b-meningitis; impetigo contagiosa; keuchhusten; ansteckungsfähige lungentuberkulose; lausbefall typhus abdominalis; paratyphus; cholera; shigellenruhr; salmonellose, einer anderen infektiö sen gastroenteritis; virushepatitis a oder e key: cord-032438-cpoalxyd authors: nachega, jean b; fatti, geoffrey; zumla, alimuddin; geng, elvin h title: the where, when, and how of community-based versus clinic-based art delivery in south africa and uganda date: 2020-09-21 journal: lancet glob health doi: 10.1016/s2214-109x(20)30385-5 sha: doc_id: 32438 cord_uid: cpoalxyd nan antiretroviral therapy (art) can suppress hiv plasma rna concentrations and harmful effects of the virus. however, at present, only about 60% of people living with hiv are virally suppressed. 1 therefore, global public health programmes urgently need innovative approaches to improve the rapidity and durability of engaging patients in treatment. in this issue of the lancet global health, ruanne barnabas and colleagues report results of the delivery optimization of antiretroviral therapy (do-art) study, a multicentre, randomised trial comparing community-based art initiation, monitoring, and resupply with use of a hybrid approach (art initiation at the clinic with community monitoring and resupply), and with standard clinicbased art delivery among individuals from south africa and uganda with detectable hiv viral load. 2 the investigators hypothesised that community-based art could overcome logistical barriers, simplify monitoring and art resupply, and increase viral suppression rates, especially among men, enough to make communitybased interventions cost-effective. they found that community-based initiation and treatment significantly increased viral suppression compared with standard clinic-based care among all participants from 63·1% to 73·9%, and among men from 54·3% to 73·2%. the hybrid approach registered smaller but similar effects. the report of the do-art study contributes to a growing body of literature showing that communitybased interventions result in similar or improved patient outcomes compared with clinic-based art delivery. 3, 4 home-based, same-day art initiation integrated with community-based hiv testing improved viral suppression at 12 months in a trial in lesotho. 5 community-based hiv care using differentiated service delivery models, including community-based multimonth art dispensing, have been shown to have favourable outcomes 6 and result in cost savings to both patients and providers. 7 however, most communitybased differentiated service delivery models for art delivery have been developed for patients who are already stable on art, and the most important contribution of the study by barnabas and colleagues is that community-based, same-day art initiation in individuals with elevated viral load was safe and resulted in improved viral suppression after 12 months, particularly among men. with these promising results, the next questions are whether, where, when, and how global public implementers should scale up the do-art approach, particularly among men. the answers, however, depend not only on the rigour or internal validity of the study (for which the investigators should be applauded) but also on the external validity, for which some additional information would be useful. emerging perspectives in implementation research can help position researchers to offer findings that are maximally interpretable in other implementing contexts that differ by geographical (urban vs rural), economic (kenya vs mozambique), and social factors-what do these perspectives suggest for future research that aims to influence execution of the hiv response? mechanisms can inform decisions about scale up. first, an important insight is that the external validity of a finding depends on the mechanism of effect. in this study, more information about how communitybased approaches improved viral suppression would be helpful. if the community-based approach worked through availing art to patients unable or unwilling to get to a clinic, then this approach is most applicable to rural settings, where distance is important. conversely, if patients in the standard of care arm of the trial reached the clinic but did not start art due to lengthy preparation practices, the observed effects might be attenuated since immediate art initiation in the clinic has become the norm over the past 2 years. second, so-called adjunctive practices that accompany study interventions but are not explicitly considered important complicate external validity. in the do-art study, patients starting art in the community also received text message appointment reminders, facilitated rescheduling, follow-up monitoring calls, and potentially more intensive counselling. unless community-based art initiation is universally scaled up concomitantly with these adjunctive practices, the real-world effects might show a drop in art compliance and viral suppression rates compared with trial findings. parsimonious intervention design could improve generalisability. third, measuring and reporting patient preferences is also important. someone living with stigma and near a clinic, for example, might have different preferences for home-based art initiation than someone living far from a clinic. in preference-sensitive interventions such as these, the comparison of interest might not be between community-based and clinic-based art initiation, but rather between a health system that offers both (from which patients could choose) versus a system that only offers one-a concept termed mosaic effectiveness. 8 if preferences are measured, effects stratified by such preferences can inform tailoring of services to the individual. overall, the results from the do-art study extend our public health tool kit and has implications for management of adults with virological failure who are not eligible for differentiated service delivery. further research with longer-term follow-up is required to measure sustained success over time and costeffectiveness to inform policy makers. furthermore, home-based case management could be effective for adolescents in particular, 9 but these patients were not included in the study and whether community-based art initiation and resupply is safe and effective for this population is unknown. the do-art study adds further to the ongoing debate and dialogue on the optimal package of care needed to achieve the third 90 (virological suppression) of the unaids 90-90-90 targets for aids elimination by 2030. 10 jbn is an infectious diseases internist and epidemiologist supported by the us national institutes of health (nih)/national institutes of allergy and infectious diseases (grant number 5u01ai069521; stellenbosch university clinical trial unit of the aids clinical trial group) as well as nih/fogarty international center (grant numbers 1r25tw011217-01; african association for health professions education and research) and 1d43tw010937-01a1 (university of pittsburgh hiv comorbidities research training programme in south africa), and is co-principal investigator of together, an adaptive randomised clinical trial of novel agents for treatment of patients who are high-risk outpatients with covid-19 in south africa supported by the bill & melinda gates foundation. az is a co-principal investigator of the pan-african network on emerging and re-emerging infections funded by the european and developing countries clinical trials partnership and is in receipt of an nih research senior investigator award. we acknowledge the critical unaids. global aids update. 2020 community-based antiretroviral therapy versus standard clinic-based services for hiv in south africa and uganda (do art): a randomised trial community-based interventions to improve and sustain antiretroviral therapy adherence, retention in hiv care and clinical outcomes in low-and middle-income countries for achieving the unaids 90-90-90 targets the effectiveness and cost-effectiveness of community-based support for adolescents receiving antiretroviral treatment: an operational research study in south africa effect of offering same-day art vs usual health facility referral during home-based hiv testing on linkage to care and viral suppression among adults with hiv in lesotho: the cascade randomized clinical trial twelve-month outcomes of communitybased differentiated models of multi-month dispensing of antiretroviral treatment among stable hiv-infected adults in lesotho: a cluster randomized non-inferiority trial economic evaluation of differentiated service delivery models for art service delivery in lesotho: cost to provider and cost to patient mosaic effectiveness: measuring the impact of novel prep methods interventions to improve antiretroviral therapy adherence among adolescents and youth in low-and middleincome countries: a systematic review fast-track-ending the aids epidemic by 2030 key: cord-104162-fe51v2pt authors: zhang, chiyu; forsdyke, donald r. title: potential achilles heels of sars-cov-2 displayed by the base order-dependent component of rna folding energy date: 2020-11-02 journal: biorxiv doi: 10.1101/2020.10.22.343673 sha: doc_id: 104162 cord_uid: fe51v2pt base order, not composition, best reflects local evolutionary pressure for folding of single-stranded nucleic acids. the base order-dependent component of folding energy has revealed a highly conserved region in hiv-1 genomes that associates with rna structure. this corresponds to a packaging signal that is recognized by the nucleocapsid domain of the gag polyprotein. long viewed as a potential hiv-1 “achilles heel,” the signal can be targeted by a recently described antiviral compound (nsc 260594) or by synthetic oligonucleotides. thus, a conserved base-order-rich region of hiv-1 may facilitate therapeutic attack. although sars-cov-2 differs in many respects from hiv-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. this indicates structural invariance (si) sustained by natural selection. while the regions are often also protein-encoding (e.g. nsp3, orf3a), we suggest that their nucleic acid level functions – such as the ribosomal frameshifting element (fse) that facilitates differential expression of 1a and 1ab polyproteins – can be considered potential “achilles heels” for sars-cov-2, perhaps susceptible to therapies like those envisaged for aids. the region of the fse scored well, but higher si scores were obtained in other regions, including those encoding nsp13 and the nucleocapsid (n) protein. composition tends to reflect genome-wide evolutionary pressures. just as a local arrangement of words conveys specific meaning to a text, so base order better reflects local evolutionary pressures. base order is most likely to be conserved when encoding a function critical for survival. assays of the base order-dependent component of the folding energy have shown that a highly conserved region, in otherwise rapidly mutating hiv-1 genomes, associates with an rna structure corresponding, not to a protein-encoding function, but to an rna packaging signal. the latter is specifically recognized by the nucleocapsid domain of the gag polyprotein [8] and is now seen as a potential "achilles heel" of hiv-1 that can be targeted by a recently described antiviral compound (nsc 260594) or by synthetic oligonucleotides [4] . we here report similar highly conserved structural regions of the sars-cov-2 genome, one or more of which should be susceptible to targeting [9, 10] . we identify certain open reading frames (orfs) that, because of their conservation, have so far attracted therapeutic interest mainly related to their functions at the protein level [11, 12] , rather than at the level of the corresponding, yet highly structured, regions of the genome. the ribosomal frameshift element (fse) that is among our results, is attracting attention [10, 13, 14] . yet our analysis, consistent with recent reports [15, 16] , suggests there may be more suitable targets in other regions. these were obtained from the ncbi (bethesda) and gisaid epicov (munich) databases. the wuhan-hu1 sequence (genbank nc_045512.2), deemed taxonomically prototypic [17, 18] , was compared (regarding base substitutions and folding potential) with 381 chinese isolates, 430 italian isolates, and 932 isolates from new york, usa. our "window" starting point was base 1 of the 29,903 base prototype sequence. we refer to windows by their centers. the center of the first 200 base window would be 100. in previous hiv-1 studies the base differences between just two individual sequences sufficed for the tabulation of a statistically significant set of base substitution frequencies [2] . 4 the lower mutation rates of sars-cov-2 strains [11] the energetics of the folding of a single-stranded nucleic acid into a stem-loop structure depend on both the composition and order of its bases. base composition is a distinctive characteristic of a genome or large genome sector. a localized sequence (e.g. a 200 base window), which is rich in the strongly-pairing bases g and c, will tend to have a stable structure simply by virtue of its base composition, rather than of its unique base order. this high gc% value can obscure the contribution of the base order-dependent component of the folding energy, which provides a sensitive indicator of local intraspecies pressures for the conservation of function within a population (i.e. a mutated organism is eliminated by natural selection so no longer can be assayed for function in the population). in contrast, interspecies mutations tend to influence the genome-wide oligonucleotide (k-mer) pressure, of which base composition (gc%) is an indicator. this pressure can act to generate and/or sustain members of emerging species by preventing recombination with parental forms [3, 7, [19] [20] [21] . elimination of this base composition-dependent component facilitates focus on local folding. 5 early studies of rna virus structure by le and maizel [22] were primarily concerned with the statistical significance of rna folding, rather than with distinguishing the relative contributions of base composition and base order. however, with a pipeline between the various programs that were offered by the wisconsin genetics computer group, the base composition and base order-dependent components were separated and individually assessed ("folding of randomized sequence difference" analysis; fors-d analysis). departing from le and maizel, fors-d values (see below) were not divided to yield z-scores, but were simply plotted with statistical error bars [2, 3, 7, 23, 24] . the limits of the latter were generally close to the corresponding fors-d values and, for clarity, are omitted here. a window of 200 bases is moved along a natural sequence in 20 or 50 base steps. a folding program [25] is applied to the sequence in each window to obtain "folding of natural sequence" (fons) values for each window, to which both base composition and base order will have contributed. the four bases in each sequence window are then shuffled to destroy their order while retaining base composition, and folding energy is again determined. this shuffle-and-fold "monte carlo" procedure is repeated ten times and the average (mean) folding value is taken as the approach was employed by others who, rather than shuffling the four bases, favored retaining some base order information. accordingly, they shuffled groups of bases (e.g. the sixteen dinucleotides). following disparagement of the conceptual basis of four base shuffling, which was duly clarified [26] , the validity of single base level shuffling is now generally 6 accepted and is being applied routinely to various viral genomes [15, 16, 27] . the monte carlo procedure can also be simplified to decrease fors-m computational time [28] using support vector machine-based technology [29] . our original software ("bodslp"), written by professor jian-sheng wu [30] , retains the monte carlo approach and was further developed by professor shungao xu as "random fold-scan" for windows-based systems [31] . in addition to assisting the study of infectious viruses and protozoa [32] , fors-d analysis proved fruitful when applied to topics such as speciation [3, 19, 33] , the origin of introns [23, 24, 34] , relating structure to recombination breakpoints and deletions [35, 36] , and relying on a single sequence (rather than alignments) for the determination of positive darwinian selection [37] . however, for a given sequence window, output can follow only from the base order in that window. lost are higher order structures that might occur naturally through long-range interactions [14] . furthermore, if the artificial demarcation of a window happens to cut between the two limbs of a natural stem-loop structure, then lost are what might have been contributed to the folding energetics had a larger window, or a different section point, been chosen. variations in step size will generate differences in windows and hence variations in results. such variations might be less when window margins correspond to natural section points, such as those demarcating an rna transcript. there is also a kinetic aspect, particularly apparent with transcripts, due to the probability that the pattern of early 5' folding will influence later folding. high negative si values indicate structural conservation among a set of genomes. the validity of this approach is supported by prior work with hiv-1. fig. 1 shows application of the index to previously reported data on hiv-1 [2] . here a high negative si index value corresponds 7 to regions recognized as likely to offer achilles heel-like vulnerability [7] . the region around the window centered on 500 nucleotide bases is the focus of recent work [4] [5] [6] . subtype (hxb2) [2, 3] . the region recently recognized as a potential achilles heel (bases 380-640) [4] [5] [6] , has a high negative fors-d value (black triangles) similar to those of the rre (rev response element) and the 3' untranslated region (utr). the si index (continuous red line) indicates highest sequence conservation in the regions of the rna packaging signal and the 3'utr. 2 . 1 . v a r i ation d u e t o b the degree of conservation in sequential windows of members of a set of sequences from china was evaluated as the number of substitutable base positions (polymorphism), relative to the prototype sequence (fig. 3) . this was compared with corresponding fors-d values, the profile of which differed a little from that of fig. 2 due to different step values (see materials and methods). as with some previous studies [23] , there tended to be a reciprocal relationship between the for a more focused view of the reciprocal separation of high negative folding values and corresponding numbers of substitutions (ranging from zero to high positive values) the two were added to provide the "structural invariance" (si) index (fig. 4) . here, despite having some substitutions, orfs nsp13 and nsp3 were preeminent (scoring a region with the highest number of substitutions (see fig. 3 ) centered on base 28900 (scoring +7.6 si units). (for plots of analogous hiv-1 data see fig. 1 ). windows 13400 and 13450 in the fse region scored -12.5 and -13.7 units, respectively. however, there were many more windows with higher negative scores. the si profile for china (fig. 4) was, in broad outline, confirmed with corresponding data later downloaded from italy and new york, usa (fig. 5) . the high negative si indices found in the nsp3, nsp13, orf3a and n regions, were evident with sequences for all three locations. other regions, notably the s region, were also corroborated. the high positive si values, indicating regions likely to have poorly conserved structures, are also corroborated at some locations. a high intraspecies mutation rate for the n protein orf (fig. 3) is also seen when interspecies comparisons are made with other coronavirus species, with implications for early speciation mechanisms [38] . fig. 4 (purple) , with si indices for italy (430 isolates; green) and new york, usa (932 isolates; red). mutation rates of microbial pathogens are generally higher than those of their hosts. while a microbe spreading from host-to-host can "anticipate" that it will face a succession of broadly similar challenges, in the short-term those hosts cannot likewise "anticipate" that new microbial invaders will remain as they were in previous hosts. thus, host immune defenses may be overwhelmed. (in the long-term there is a different scenario related to innate immunity; see below). therapeutic challenges are, first, to locate a conserved, less-variable, part of a pathogen's genome that it will have inherited sequentially from a multiplicity of past generations, and so is likely to carry through to a multiplicity of future generations. second, is to identify the corresponding primary function, be it at the genome, rna transcript, or protein level. third, from this knowledge (that may be incomplete; i.e. function not fully clarified), devise effective pathogen inhibition without imposing deleterious side-effects on the host. viral vulnerability is often assumed to associate with protein-level functions [11, 12] . however, studies of the aids virus have identified genome structure itself as both functional and conserved, so signifying vulnerability [2, 3] (see fig. 1 ). a genomic packaging signal for hiv-1, which is specifically recognized by the nucleocapsid domain of its gag polyprotein, has long been recognized as a potential "achilles heel" [2, 3, 7] so inviting therapeutic exploration [4] [5] [6] . through targeting of specific rna conformations, gag not only influences the assembly of hiv-1 genomic rna into virus particles, but also regulates hiv-1 mrna translation [39] . permit easier switching between regulatory options. indeed, small changes in target rna structure can impede this [6] . thus, unlike most other regions of the hiv-1 genome, mutations here would likely lead to negative darwinian selection at an early stage -hence the high conservation. application of the same bioinformatic technology to the sars-cov-2 virus genome has now revealed similar "achilles heels." 1 4 lacking the chronicity of hiv-1 infection, the genome of sars-cov-2 should have been shaped less by adaptations to counter long-term host immune defenses. it cannot hide within its host genome in latent dna form. yet, the larger sars-cov-2 genome contains many more genes than hiv, which require differential expression according to the stage of infection. even more complex regulatory controls can be envisaged, likely requiring conserved genome conformations at appropriate locations. be they synonymous or non-synonymous, mutations in these structured regions could result in negative selection of the viruses in which they occurredhence high conservation. the ribosome fse located close to base 13468 (figs. 2) would seem to exemplify this [13] , and a potential targeting agent is now available [10]. however, we have here identified other structurally important regions with more base order-dependence and higher degrees of conservation (figs 3-5), that might, either singly or collectively, be better candidates for targeting. when determining folding energy, our approach depends on eliminating contributions of base composition which, as noted, plays an unusual role in the case of the fse. more usually, base composition is a distinctive characteristic of entire genomes or large genome sectors that reflects their underlying oligomer ("k-mer") content. the slow genome-wide accumulation of mutations in oligomer composition (easiest documented as changes in base composition; gc%) can serve to initiate divergence into new species. by preventing that accumulation, potentially diverging organisms can stay within the confines of their species [19] [20] [21] . the presence or absence of synonymous mutations [16, 40] , which affect structure rather than amino acid composition, can have an important role in this process. the primary role of constancy in the base composition-related character is to prevent recombination with allied species (interspecies recombination) while facilitating the intraspecies recombination that can correct mutations, so retaining species individuality [3, 7] . such recombination is initiated by "kissing" interactions between complementary unpaired bases at the tips of stem-loop structures [19] . thus, we are here concerned with localized intraspecies mutations that affect fitness, so making members of a species carrying those mutations liable to natural selection. the mutations 1 5 facilitate within-species evolution rather than divergence into new species. and when that evolution has run its course, some of the polymorphic bases will have become less mutable, so will be deemed "conserved." indeed, mutations of orf nsp3 are high when the sequences of different coronavirus species are compared [41] , yet when, from intraspecies comparisons, mutations (in the form of base substitutions) are scored, they are very low (figs. 3, 4) . our technology (see materials and methods) removes the base composition-dependent component of mutational changes (that relates more to interspecies evolution) and focuses on the base orderdependent component (that relates more to intraspecies evolution). it best reflects localized functions, be they encoding protein or determining the potentiality for folding into higher order structure, of linear, single-stranded, nucleic acid sequences. is conservation necessarily a good indicator of likely therapeutic success? we sought regions that were both high in stem-loop potential and bereft of mutations, following the premise that conserved functions would be best targeted therapeutically, assuming the availability of pathogen-specific therapeutic agents that would not cross-react with hosts. interference with structural nucleic acid level functions would seem less likely to produce unforeseen host sideeffects than with protein-level functions. yet, that possibility remains. indeed, a conserved function in a pathogen could owe that conservation to the pathogen strategy of, whenever possible, mutating to resemble its host. this would make it less vulnerable to host immune defenses. to prevent autoimmunity, the generation of immune cell repertoires involves the negative selection of self-reacting cells so creating "holes" in the repertoire that pathogens can exploit by progressive mutation towards host-self, testing mutational effectiveness a step at a time. this would make it advantageous for the host, during the process of repertoire generation, not only to negatively select immune cells of specificity towards "self," but also to positively select immune cells of specificity towards "near-self." a high level of anti-near-self immune cell clones would constitute a barrier limiting the extent of pathogen mutation towards self. the existence of such positive selection is now generally accepted, with the implication that some pathogen functions might have approached so close to host-self that targeting them therapeutically would result in cross-reactivity [42] . 1 6 this unlikely caveat aside, we deem conservation a reliable indicator that a certain pathogen function is likely to be a suitable target for therapy. attacking a short segment of a pathogen nucleic acid sequence is unlikely to ensnare a similar segment of its host's nucleic acid. in any case, to militate against this, the pathogen specificity of a potential therapeutic agent can be screened against the prototypic human genomic sequence (assuming it is unlikely that patient genomes will significantly depart from this). while prospects for the development of prophylactic vaccines against infection with sars-cov-2 are promising, methods to boost post-infection host immune defenses and to directly target sars-cov-2 are urgently needed. these require a better understanding both of viral interactions with host innate and acquired immune systems [42] , and of viral vulnerabilities. the latter enquiry proceeds in three steps: find specific "achilles heels." design therapies to exploit them. prove their clinical effectiveness. we have here been concerned with the first step. although the bioinformatic technology related to this has long been available [2] , its claim to reveal viral "achilles heels," as promulgated in successive textbook editions [7] , has only recently gained support [4] [5] [6] . this may be because the importance of removing redundant information and analyzing solely the contribution of base order to folding energy, was not fully appreciated. thus, we have here repeated and expanded on past clarifications [26, 30, 31] of the conceptual basis of a technology that has contributed to the understanding of a many biological problems other than viral infections [7] . meanwhile, it is pleasing to note that, with minimal evidence on targeting, progress is being made with the second step [9, 10] . it is hoped that by targeting one or more of the conserved regions in the sars-cov-2 genome here identified, rapid cures will be achieved. authors declare no conflict of interest. low genetic diversity may be an achilles heel of sars-cov-2 reciprocal relationship between stem-loop potential and substitution density in retroviral quasispecies under positive darwinian selection implications of hiv rna structure for recombination, speciation, and the neutralism-selectionism controversy an rna-binding compound that stabilizes the hiv-1 grna packaging signal structure and specifically blocks hiv-1 rna encapsidation the heart of the hiv rna packaging signal? identification of the initial nucleocapsid recognition element in the hiv-1 rna packaging signal evolutionary bioinformatics hiv-1 gag protein with or without p6 specifically dimerizes on the viral rna packaging signal a small interfering rna (sirna) database for sars-cov-2 targeting the sars-cov 2 rna genome with small molecule binders and ribonuclease targeting chimera (ribotac) degraders. acs cent sci example study of a highly conserved sequence motif in nsp3 of sars-cov-2 as a therapeutic target sars-cov-2 and orf3a: nonsynonymous mutations, functional domains, and viral pathogenesis structural and functional conservation of the programmed −1 ribosomal frameshift signal of sars coronavirus 2 (sars-cov-2) the short-and long-range rna-rna interactome of sars-cov-2 an in silico map of the sars-cov-2 rna structurome pervasive rna secondary structure in the genomes of sars-cov-2 and other coronaviruses an evolutionary portrait of the progenitor sars cov 2 and its dominant offshoots in covid 19 pandemic assessing uncertainty in the rooting of the sars-cov-2 phylogeny different biological species "broadcast" their dnas at different (g+c)% "wavelengths success of alignment-free oligonucleotide (k-mer) analysis confirms relative importance of genomes not genes in speciation hybrid sterility can only be primary when acting as a reproductive barrier for sympatric speciation a method for assessing the statistical significance of rna folding conservation of stem-loop potential in introns of snake venom phospholipase a 2 genes: an application of fors-d analysis computer prediction of rna secondary structure calculation of folding energies of single-stranded nucleic acid sequences: conceptual issues scanfold: an approach for genome-wide discovery of local rna structural elements -applications to zika virus and hiv a computational procedure for assessing the significance of rna secondary structure fast and reliable prediction of noncoding rnas evaluation of fors-d analysis: a comparison with the statistically significant stem-loop potential a fors-d analysis software "random_fold_scan" and the influence of different shuffle approaches on fors-d analysis low complexity segments in plasmodium falciparum proteins are primarily nucleic acid level adaptations microsatellites that violate chargaff's second parity rule have base order-dependent asymmetries in the folding energies of complementary dna strands and may not drive speciation a stem-loop "kissing" model for the initiation of recombination and the origin of introns local base order influences the origin of ccr5 deletions mediated by dna slip replication the key role for local base order in the generation of multiple forms of china hiv-1 b'/c intersubtype recombinants positive darwinian selection. does the comparative method rule? codon usage and phenotypic divergences of sars-cov-2 genes human immunodeficiency virus type 1 gag polyprotein modulates its own translation synonymous mutations and the molecular evolution of sars-cov-2 origins a putative role of de-mono-adp-ribosylation of stat1 by the sars-cov-2 nsp3 protein in the cytokine storm syndrome of covid-19 two signal half century: from negative selection of self-reactivity to positive selection of near-self reactivity we thank prof. shungao xu at jiangsu university for software, and ms. le cao and ms.yingying ma at shanghai public health clinical center, fudan university, for their technical support. queen's university hosts forsdyke's webpages. the biorxiv server hosts preprints. key: cord-023669-3ataw6gy authors: masur, henry title: critically ill immunosuppressed host date: 2009-05-15 journal: critical care medicine doi: 10.1016/b978-032304841-5.50056-x sha: doc_id: 23669 cord_uid: 3ataw6gy nan as the population of patients with cancer, organ transplants, vasculitides, and human immunodefi ciency virus (hiv) infection has grown, intensivists are seeing more and more patients with altered immunity. these patients may come to the intensive care unit (icu) because of life-threatening opportunistic infections, or they may develop life-threatening infection while in the icu for an unrelated problem. intensivists must recognize how these patients differ from immunologically normal patients in terms of clinical presentation and management of these infections. this chapter emphasizes the important ways in which immunosuppressed patients differ from immunologically normal individuals in terms of infectious complications. clearly, however, immunosuppressed patients also develop complications from their underlying diseases and the drugs used to treat these underlying processes. these noninfectious complications are not the focus of this chapter but are reviewed in chapter 81. patients who are at increased risk for infectious complications because of a defi ciency in any of their host defense mechanisms are referred to as compromised hosts. patients in icus are almost universally compromised either by virtue of their underlying disease or by virtue of the invasive devices utilized to support and monitor them. patients are termed immunocompromised or immunosuppressed if their defect specifi cally involves immune response. often, patients who have defi cient infl ammatory response (e.g., neutropenia) are grouped into the category of immunocompromised or immunosuppressed, although technically they have a different category of defi cient host response. patients in icus are often immunosuppressed as a result of their underlying disease, therapy, or nutri-tional status. this chapter focuses specifi cally on patients who are immunocompromised or immunosuppressed. the microbial complications that any patient develops are determined by general, nonspecifi c barriers; innate immunity; acquired specifi c immunity; and environmental exposures. nonspecifi c barriers include anatomic barriers such as intact skin and mucous membranes; chemical barriers such as gastric acidity or urine ph; and fl ushing mechanisms such as urinary fl ow or mucociliary transport. organisms that breach these barriers encounter nonspecifi c and innate host factors termed the acute phase response. acute phase responses include trigger molecules and effector molecules. organisms also encounter acquired specifi c immune response systems including mononuclear phagocytes and antibodies. 1 infections that occur may result from normal fl ora that colonize mucosal or cutaneous surfaces. infections may result from abnormal fl ora that have invaded or replaced normal fl ora because of environmental exposures, disrupted barriers, or selective pressure of antimicrobial agents. table 54-1 lists organisms that cause disease when specifi c anatomic defenses are disrupted in individuals with normal microbial fl ora. infections may also result from common defects in the infl ammatory or immunologic systems; examples are detailed in table 54 -2. [1] [2] [3] [4] [5] [6] [7] [8] [9] infl ammatory and immunologic barriers can be disrupted by the primary disease process (e.g., tumor can invade the bone marrow, immunologic abnormalities associated with aplastic anemia or collagen vascular disease can destroy cells either in the bone marrow or the periphery). infl ammatory and immunologic mechanisms can also be disrupted by drugs. cytotoxic drugs, for instance, can reduce neutrophil number and function. certain monoclonal antibodies can destroy lymphocyte populations or interfere with cytokine attachment to receptor sites. some agents such as corticosteroids have multiple effects on neutrophils, lymphocytes, and soluble factors. infections may result from organisms that are usually not pathogenic, but become opportunistic because of poor host defense mechanisms. opportunistic infections are defi ned as those that occur with enhanced frequency or severity in a specifi c patient population compared with a normal patient population. pneumocystis jiroveci, for example, never causes disease in immunologically normal individuals but can cause frequent episodes of pneumonia in certain immunosuppressed patients. candida can cause mild mucosal disease in normal patients receiving antibacterial drugs but causes more frequent and more severe mucositis when patients have impaired cell-mediated immunity. recognition of which host defense mechanisms are disrupted enables the clinician to focus diagnostic, therapeutic, and prophylactic management and optimize patient outcome. for instance, if a patient presents with severe hypoxemia and diffuse pulmonary infi ltrates, a health care provider who recognizes a prior splenectomy as the major predisposition to infection would focus the diagnostic evaluation and the empiric therapy on streptococcus pneumoniae and haemophilus infl uenzae. by contrast, if the patient's major predisposition to infection were hiv infection with a cd4+ t lymphocyte count below 50 cells/µl, the health care provider would focus on pneumocystis jiroveci and s. pneumoniae; if a cytomegalovirus (cmv)-negative patient's major predisposition were a recent allogeneic stem cell transplant from a cmv-positive donor, then cmv would be a prime consideration. [2] [3] [4] [5] [6] [7] [8] [9] immune competence should ideally be measurable by objective laboratory parameters. in fact, the risk for opportunistic infection in patients with hiv infection can be assessed by clinical laboratories with a high degree of accuracy by measuring the number of circulating cd4+ t lymphocytes. 5 the susceptibility of cancer patients to opportunistic bacterial and candida infections can be assessed by measuring the number of circulating neutrophils. 7, 10, 11 the predisposition of patients with certain congenital immunodefi ciencies can be assessed by measuring serum immunoglobulin levels. 12 unfortunately, however, for a large number of immunodefi ciencies, no objective laboratory measures have been validated as predicting the risk of infection. moreover, laboratory measures must be interpreted in context. cd4+ t lymphocyte counts have great prognostic value in patients with hiv infection but not in most other patient populations; neutrophil counts are relevant in all patient populations, but low counts are associated with disrupted mucosal surfaces compared with those with intact mucosa. thus laboratory parameters must be interpreted in the context of the patient's underlying disease-risk is not always easily manageable by measuring one laboratory parameter. most importantly, most patients have multiple overlapping predispositions to infection. knowledge of the infectious complications associated with specifi c diseases, specifi c immune defects, and specifi c laboratory abnormalities is helpful for predicting and managing infectious complications. however, a specifi c diagnosis should be established in each patient: knowledge of the immune defect helps guide empiric therapy or helps determine therapy if a diagnostic procedure is not safe to perform. immunocompromised patients, by defi nition, are susceptible to a broader array of pathogens than immunocompetent patients. understanding the specifi c immune defect can be enormously helpful in understanding the likely location and source of infection. however, the immune defect must be assessed in the context of the specifi c disease: the clinical manifestations of hiv infection, for instance, are quite different from the clinical manifestations of patients with other diseases that alter cellmediated immunity such as lymphoma. the immune defect must also be interpreted with the understanding that predisposition to infection is usually multifactorial: in addition to neutropenia or lymphocyte depletion, patients often have impaired mucosal barriers, poor ciliary function, or breaches in their skin (i.e., from catheters) that can increase their risk of infection. effective management of opportunistic infections requires understanding of several basic tenets of care. 1. diseases may present with subtle symptoms and signs, and patients are predisposed to deteriorate precipitously. because immunocompromised patients may lack infl ammatory and/or immunologic mediators, the clinical manifestations of infections are often less prominent and less impressive than immunocompetent patients with similar complications. thus clinicians must recognize that even subtle changes in skin color, catheter site appearance, chest radiograph, or abdominal examination may warrant an aggressive diagnostic evaluation and early institution of broad-spectrum empiric therapy. although all icu patients demand prompt attention and vigorous diagnostic and therapeutic management, many types of immunosuppres-sion can be associated with especially precipitous clinical deterioration despite their innocuous presentation. 2. fever is not invariably present when patients are infected. although fever is not invariably present in any patient population with infection, immunosuppressed patients are notorious for developing infection in the absence of fever. thus infection must be considered as part of the differential diagnosis among patients with afebrile syndromes that might not appear to be infectious. conversely, patients with fever may not have infection: fever may be a manifestation of the underlying disease, an allergic response to a drug, or an underlying neoplastic or collagen vascular disease. 3. diagnostic evaluation needs to be prompt and defi nitive. as indicated earlier, patients with life-threatening infection may present with subtle symptoms and signs that progress rapidly: these early manifestations merit aggressive attempts to defi ne the anatomy of the lesion and the causative microbial pathogen. because the spectrum of potential pathogens includes a wide array of microorganisms (e.g., viruses, fungi, protozoa, or bacteria), clinicians must be certain that appropriate specimens are obtained and the appropriate microbiologic and histologic tests are ordered to identify common, as well as uncommon or unusual, pathogens. invasive diagnostic techniques such as bronchoalveolar lavage or tissue biopsies should be performed with less hesitancy than in immunologically normal patients. patients often have enhanced risk factors for invasive procedures, such as thrombocytopenia, coagulation factor defi ciencies, or compromised organ function. however, the benefi t of defi nitive diagnosis often outweighs these risks when the procedures are performed by experienced operators. 4. the threshold for initiating broad-spectrum empiric therapy should be low. because patients can deteriorate rapidly and because they are susceptible to such a wide array of microbial pathogens, clinicians should have little hesitation in instituting empiric antimicrobial therapy. this therapy must be directed at the full range of bacterial, fungal, viral, protozoal, and helminthic infections to which patients are predisposed. this therapy should be administered promptly, preferably within an hour of suspecting an infectious process. clinicians should initiate comprehensive regimens: antimicrobial agents can be discontinued or reduced when culture results and clinical events clarify the scenario. 5. foreign bodies and infectious foci should be addressed. patients may need careful imaging to be certain that they do not have an obstructed viscus or localized collection that should be drained. such imaging is appropriate even when signs or symptoms are unimpressive. similarly, patients often have multiple intravascular catheters that may need to be removed, as discussed in chapter 51. 6. consideration should be given to augmenting the immune or infl ammatory response. there may be opportunities to augment immunologic or infl ammatory responses by administering pharmacologic or biologic agents such as granulocyte colony-stimulating factor (g-csf) or intravenous immunoglobulin. [12] [13] [14] [15] eliminating immunosuppressive drugs or reducing the dose can also improve the patient's prognosis. 7. effi cacy and toxicity of therapy should be assessed serially. icu patients characteristically require attentive monitoring to assure the adequacy and safety of therapy. immunocompromised patients often have multiple prior and concurrent insults to their renal and hepatic function, and they often receive multiple drugs that can produce drug-drug interactions. thus monitoring the pharmacokinetics and assessing potential toxicities are especially important in these patient populations. moreover, because response to therapy may be less robust than in immunocompetent patients, antigen titers or pcr titers, as well as serial imaging studies, can be important to assure the adequacy of the management plan. therapy must often be continued longer than in immunologically normal patients. cytotoxic therapy-induced neutropenia is a major predisposition to infection. 7, 11 counts below 500 cells/mm 3 (the total of polymorphonuclear neutrophils and bands) increase susceptibility to infection in a linear fashion (i.e., the lower the neutrophil count, the greater the degree of susceptibility). the absolute neutrophil count is not the only factor that determines susceptibility, however, because some patients with cyclic neutropenias, druginduced neutropenias, or hiv-induced neutropenias, for example, are not nearly as susceptible to infection as are cancer patients receiving cytotoxic therapy. other important contributors to susceptibility, in addition to the absolute neutrophil count, are the duration of neutropenia, the functional capability of neutrophils, the integrity of physical barriers such as the skin and gastrointestinal mucosa, the patient's microbiologic environment (endogenous and exogenous fl ora), and the status of other immune mechanisms. for example, a patient with vancomycin-induced neutropenia during therapy for a staphylococcal infection may not develop any complications if the neutropenia is brief and defense mechanisms are otherwise intact. a patient with hiv-induced neutropenia may have prolonged or even lifelong neutrophil counts below 500/µl yet suffer few serious bacterial complications. 14 the presence of intact physical defense barriers is a major difference compared with cancer patients, whose skin and mucous membranes are disrupted by cytotoxic therapy in which the skin and gastrointestinal tracts are portals of entry for infections that are not controlled by diminished host immunologic or infl ammatory defenses. thus the patient with hiv infection is usually at a much lower risk for a bacterial infection than is a cancer patient, despite a comparable neutrophil count. in the 1960s and 1970s, aerobic gram-negative bacilli such as escherichia coli, klebsiella pneumoniae, and pseudomonas aeruginosa predominated as pathogens in neutropenic patients. anaerobic bacteria and aerobic gram-positive cocci were recognized less commonly. aerobic gram-negative bacillus infections were also associated with a poorer outcome than infections from gram-positive cocci. given the spectrum of pathogenic organisms that were seen in that era, combination therapy was usually advocated. 11,16-24 a number of reasons were proposed to justify combination therapy: (1) broad coverage of potential pathogens; (2) prevention of emergence of resistance; and (3) synergy. in general, these principles are reasonable concepts on which to base a preference for using combination therapeutic regimens. however, no study unequivocally demonstrated that combination therapy provided better outcomes than did monotherapy, assuming that both study arms contained drugs that had activity against the causative organism. in addition, predicting synergy proved diffi cult. 25 in the 1990s the spectrum of causative pathogens in neutropenic patients shifted from a predominance of gram-negative bacilli to a majority of gram-positive cocci including streptococci, staphylococci (including oxacillin-resistant staphylococcus aureus), and enterococci (including vancomycin-resistant enterocci). 20, 24, 26 the development of potent broad-spectrum β-lactam and quinolone drugs in the 1980s and 1990s has provided single agents that can probably provide comparable outcomes to combination therapy when used empirically or specifi cally. in the current era the choice of single or combination regimens is based predominantly on the spectrum of organisms that needs to be covered rather than attempting a strategy of trying to obtain more potency through additive or synergistic combinations. 10, 27 promptly initiating broad-spectrum antibacterial therapy for all cancer patients who are febrile and who are neutropenic (neutrophil count <500/mm 3 ) as a result of cytotoxic chemotherapy is standard practice. 7,10,27 for febrile neutropenic patients who have no apparent source of infection, there is no evidence that the initial antibacterial regimen is any more effective if a broad-spectrum antibacterial regimen consisting of two or more drugs is used instead of a single broad-spectrum antibacterial drug. for stable "low-risk" patients outside the icu, an oral regimen is now considered a reasonable approach. 7, 10, 17 such oral regimens would not be used for inpatients in most circumstances and would not be appropriate for high-risk or unstable patients. 7, 10 antifungal and antiviral drugs are generally not used empirically when neutropenic patients are initially treated unless there is a specifi c reason to have a high suspicion for a fungal or viral process. historically, an infectious cause of fever has been found in about two thirds of febrile, neutropenic cancer patients. when a specifi c causative organism is identifi ed, antimicrobial therapy is modifi ed to include an agent or agents determined to be active by in vitro susceptibility tests and that penetrate to the site of the infection. 10 combination therapy is advocated by some authorities for the specifi c (compared with empiric) therapy of either gram-positive or gram-negative bacteria, although, as noted earlier, there are little data for most pathogens that indicate that a combination regimen produces a better outcome than an appropriate single agent. therapy is generally not narrowed in terms of spectrum, however, because alteration of broad-spectrum coverage to focused therapy has been associated with more complications (e.g., "breakthrough bacteremias") unless the neutropenia resolves. whenever fever persists, therapy has generally been continued during the entire course of neutropenia because cessation of antimicrobial therapy has been associated with recurrent bacteremia resulting from the initial causative organism or a newly identifi ed pathogen. a 10-to 14-day course of antibacterial therapy is usually the minimum recommended if a causative infection is identifi ed. therapy is usually stopped promptly when the neutrophil count exceeds 1000 cells/µml if fever resolves and no source was ever identifi ed. empiric antibacterial therapy has been a successful strategy for reducing morbidity resulting from bacterial processes but has been associated with the emergence of fungal infections, as well as resistant bacterial pathogens. candida and aspergillus organisms, in particular, have become major causes of morbidity and mortality over the past 2 decades. these fungal processes can be diffi cult to diagnose because they are not always associated with detectable fungemia. the emergence of fungi as important pathogens, especially in patients with prolonged neutropenia, has led to the recommendation that empiric antifungal therapy be added to neutropenic patients who do not have an identifi ed bacterial process and who do not defervesce within 4 to 7 days of empiric antibacterial therapy. 7,10 fluconazole or an amphotericin b compound (e.g., liposomal amphotericin b) are often used, although echinocandins or certain other azoles such as voriconazole are being used by some investigators and clinicians. [28] [29] [30] [31] as patients receive chemoprophylaxis with quinolones and/or azoles during periods of intense neutropenia or immunosuppression, breakthrough pathogens are more and more likely to be resistant to the prophylactic agents. 32, 33 thus empiric regimens must be chosen with keen attention to the drugs that patients have received in the recent past, as well as pathogens they have previously been colonized or infected with. 34 patients with fever and neutropenia require aggressive diagnostic efforts to identify the cause of fever so that the appropriate antimicrobial agent is used and appropriate procedures (e.g., surgical drainage, removal of foreign body such as a catheter) can be performed. regular physi-cal examination is necessary to identify sites that merit more focused investigation: with impaired infl ammatory response, fi ndings on examination may be subtle. knowledge of the specifi c immunologic defect is important so that when cultures of blood, sputum, urine, or other appropriate body fl uids or body sites are performed, special microbiologic approaches can be used to detect viruses, fungi, helminths, protozoa, and bacteria. imaging studies are also important because intra-abdominal, intrathoracic, intracerebral, or musculoskeletal processes can be clinically subtle and may not be associated with identifi able organisms in the bloodstream. a growing array of antigen, nucleic acid, and gene detection systems including polymerase chain reaction and microarray gene assays are being investigated to facilitate diagnosis. some antigen or nucleic acid detection systems for blood or other body fl uids can be useful for detecting cryptococcus, histoplasma, hepatitis b and c, hiv, mycobacteria, pneumococci, and legionella. some of these approaches, despite their promising initial reports, are not yet clinically practical because of their level of sensitivity, specifi city, or the cost or expertise required to perform them adequately. careful attention to antimicrobial susceptibility patterns is also important. patients are exposed to repeated courses of antimicrobial agents. patients come into contact with contaminated environments in a variety of health care settings. resistance is no longer an issue exclusively for aerobic gram-negative organisms but is a concern for anaerobes, gram-positive cocci, viruses, fungi, and protozoa. clinicians must recognize that pathogens may be resistant when they are acquired by the patient, or they may become resistant during therapy if there is an inducible resistance mechanism or drug concentrations are not adequate to inhibit or kill the organism. a broad-spectrum agent used as monotherapy for febrile, neutropenic patients should have activity against aerobic gram-positive cocci and aerobic gram-negative bacilli including p. aeruginosa. 7, 10, 19, 35 potential drugs for this indication include certain cephalosporins (e.g., cefepime), carbapenems (e.g., imipenem or meropenem), and βlactam/β-lactamase combination agents (e.g., piperacillintazobactam). ceftazidime is an option chosen by some, but its poor activity against gram-positive cocci has caused some clinicians to use other agents. 18 intensivists must recognize, however, that these monotherapy regimens may not be appropriate in an icu. patients in icus, by defi nition, are either unstable hemodynamically or have a potentially life-threatening process such as diffuse pneumonia or are "fragile" because of concurrent processes. thus combination regimens are preferred by many authorities in icu settings, even though no study clearly documents superior outcomes from such combination regimens. the decade that started in 2000 is an era when microbial resistance is becoming an increasingly important problem for many types of bacteria including aerobic grampositive cocci and anaerobes, as well as aerobic gramnegative bacilli. multiple drug empiric regimens are more likely than monotherapy regimens to include an agent with activity against the offending pathogen(s). thus in a situation in an icu when failure to use an active drug is more likely to be lethal than in other settings, and when enhanced potency is a logical goal, combination therapy is prudent as an initial management strategy. thus adding vancomycin or linezolid or daptomycin for better grampositive coverage, adding a quinolone for better gramnegative bacillus coverage, and adding metronidazole to cefepime would be prudent in this patient population pending results of initial diagnostic studies. of note, however, is that although this strategy is logical, no study has shown convincingly that such an approach improves outcome. 22 a substantial number of febrile, neutropenic patients fail to improve in terms of fever or other manifestations. failure to improve may result from poor immune response, a need for drainage or necessity to remove foreign bodies, the use of drugs without activity against the causative organism, or a noninfectious process including drug allergy (i.e., fever resulting from a drug including an antimicrobial agent). the potential causative processes need to be aggressively reassessed on a regular basis by physical examination, history, cultures, and imaging techniques. most centers add antifungal therapy empirically at day 4 or day 7 of therapy if patients remain febrile. 10, 27, 29, 36, 37 fluconazole, liposomal amphotericin b, caspofungin, or voriconazole may be used: in some situations fl uconazole would be less attractive either because the patient has received fl uconazole prophylaxis or because molds are suspected. 28, 38, 39 the toxicity profi le of amphotericin b, even in its liposomal form, has led many clinicians to prefer voriconazole or one of the echinocandins (i.e., caspofungin, micafungin, or anidulafungin). 30, 40, 41 after empiric antimicrobial therapy is initiated, the optimal duration of therapy is a complex issue that depends on the type and severity of the infectious process and the duration and severity of immunosuppression, especially the neutropenia. if a causative bacterium is identifi ed, a minimum of 7 to 10 days of therapy is generally advocated, with at least 3 to 4 days being administered after neutropenia has resolved. longer courses may be required in certain settings. the duration of antifungal therapy is a complex issue and depends on the specifi c mycosis, the location and extent of disease, and the patient's immune status. 15 this is discussed in chapter 53. the use of combination therapy for fungal diseases remains controversial. 42, 43 a common problem in febrile, neutropenic patients is managing indwelling intravascular lines. [44] [45] [46] in general, these lines can be left in place initially if examination of the site reveals no indication of infection. blood cultures should be drawn through the catheter. although some experts advocate drawing a culture through each port of each catheter, obtaining this many blood cultures is often not feasible. if a patient is hemodynamically unstable and fails to respond promptly to fl uid administration, it is prudent to remove the line in case an infected catheter is the source of the sepsis. failure to remove the foreign body in this situation probably increases the likelihood of an unfavorable outcome. should blood cultures become positive and should the suspicion be high that the catheter is the source, antibacterial therapy may be successful in some settings (e.g., if the pathogen is a bacteria that is relatively sensitive to antibacterial therapy), thus avoiding the need to remove the catheter. situations suggesting that catheter removal is necessary include hemodynamic instability despite aggressive fl uid resuscitation, tunnel infection, or infections resulting from fungi or relatively antibiotic-resistant bacteria such as p. aeruginosa. a major determinant of prognosis is the immunologic status of the patient. prompt return of neutrophil number to normal improves the outcome. the use of g-csf or granulocyte-monocyte colony-stimulating factor (gm-csf), if not contraindicated by the underlying disease, can improve clinical status by hastening the return of neutrophil numbers and function. [12] [13] [14] [15] 47 granulocyte transfusions have not been proved useful in most clinical settings because of the inability to administer a large number of cells with adequate frequency. 48 the manipulation of immune response with cytokines, cytokine inhibitors, or immunoglobulins is the subject of considerable investigation: such interventions may reduce the duration of fever or the incidence of infections when used empirically, but in no setting have they been clearly shown to improve survival when administered after an infection has been documented. an algorithm for managing fever in neutropenic patients is provided in figure 54 -1. table 54 -3 suggests modifi cations of standard empiric regimens in certain common clinical scenarios. given the experience with frequent and severe infectious complications in cancer patients with neutropenia, it has been logical to attempt to prevent infection. 33 most microorganisms causing disease in this patient population arise from endogenous gastrointestinal, cutaneous, or respiratory fl ora. total protected environments probably reduce frequency of infection, but this approach is expensive and inconvenient. trying to prove a consistent benefi cial impact on survival has been diffi cult, and thus such isolation is rarely used anymore. some experts are enthusiastic about placing patients in positive pressure rooms so that pathogens do not enter via particles and droplets from outside the room. this type of isolation has not clearly improved outcome, however, and is not a standard of care. prophylactic bacterial therapy has also been controversial. 32 systemic antibacterial prophylaxis and systemic antifungal prophylaxis have been shown in some studies to reduce the number of infections, but their lack of effect on patient survival, their cost, and their impact on the emergence of resistance have made many clinicians reluctant to use them. selective gastrointestinal decontamination has not consistently improved survival and thus is not recommended by most authorities in the united states. antipneumocystis prophylaxis is, in contrast, highly effective in susceptible populations. prophylaxis for cmv is highly effective in well-defi ned, high-risk patients (e.g., some recipients of organ transplants who are either sero-positive for cmv or who are seronegative but received a graft from a seropositive donor). 2, 4, 49 strategies that reduce the period of immunologic susceptibility (e.g., reduce the duration of neutropenia), such as adding g-csf to a regimen or reducing the intensity of chemotherapeutic regimens, are promising. because so many patients are receiving highly active antiretroviral therapy (haart), opportunistic infections are not complicating the course of hiv infection to the same degree that they did in the 1980s and early 1990s. [50] [51] [52] [53] opportunistic infections continue to occur, however, in three groups of hiv-infected patients: (1) those who are unaware of their hiv status until they develop a clinical syndrome; (2) those who are unable or unwilling to receive appropriate therapy; and (3) those who fail haart and opportunistic infection prophylaxis. although haart has dramatically reduced the incidence of opportunistic infections, a surprisingly large fraction of patients either never respond virologically and immunologically or lose their response within the fi rst 12 to 24 months of therapy. these patients, most of whom have dominant viral quasispecies that are highly resistant to currently licensed antiretroviral drugs, will likely experience immunologic decline over the next few years and will again become more susceptible to opportunistic infections. severe necrotizing mucositis or gingivitis add specifi c antianaerobic agent (e.g., metronidazole, meropenem, imipenem, or piperacillin-tazobactam) plus agent with activity against streptococci; consider acyclovir. ulcerative mucositis or gingivitis add acyclovir and anaerobic coverage. add fl uconazole or caspofungin; consider adding acyclovir. pneumonitis, diffuse or interstitial add trimethoprim-sulfamethoxazole and azithromycin or levofl oxacin or moxifl oxacin (plus broad-spectrum antibiotics if the patient is granulocytopenic). perianal tenderness include anaerobic agents such as metronidazole, imipenem, meropenem, or piperacillin-tazobactam. abdominal involvement add antianaerobic agent (e.g., metronidazole, meropenem, imipenem, or piperacillin-tazobactam). patients with hiv infection develop clinical disease as a result of three basic processes: the direct effect of hiv on specifi c organs (e.g., cardiomyopathy, enteropathy, dementia); immunologically mediated processes (e.g., glomerulonephritis, thrombocytopenia); or opportunistic infections and tumors that are enabled by hiv-induced immunosuppression. hiv appears to cause direct organ damage. 50,53-57 this damage may be mediated by cytokines, lymphocytes, monocytes, or infl ammatory cells. cardiomyopathy, for example, can be a profound and lethal process that can lead to icu admission or complicate other processes. 55 when patients present with or develop pulmonary manifestations such as shortness of breath or diffuse bilateral infi ltrates on chest radiograph, cardiogenic causes must be considered. hiv also causes a diffuse pneumonitis, 56 profound encephalopathy, 54 and a diffuse enteropathy. 57 patients with compatible syndromes need a comprehensive evaluation to look for other specifi c opportunistic infections or tumors, especially those that can be specifi cally treated. in all of the hiv-caused syndromes, hiv as the etiology remains a diagnosis of exclusion. the institution of antiretroviral therapy appears to be benefi cial for patients with susceptible isolates, although data regarding such effects for these hiv-related entities are largely anecdotal. hiv-related thrombocytopenia and anemia appear to be immunologically mediated. 58, 59 both can be severe: platelet counts below 10,000/mm 3 and hemoglobins below 10 g/dl can be seen with the expected complications. these disorders are related to the development of antigenantibody complexes and may improve dramatically with the institution of antiretroviral therapy and a decline in viral load. for thrombocytopenia, intravenous immunoglobulin (or anti rhd antibody), corticosteroids, or splenectomy may also be useful. hemolytic anemia can also be severe: hemoglobin levels below 5 g/dl can be seen. the most prominent manifestations of hiv continue to be the opportunistic infections and tumors that occur as a consequence of hiv-induced immunosuppression. the cd4+ t lymphocyte cell number is a useful marker for predicting the occurrence of opportunistic infections in patients with hiv infection. 5, 9 this relationship of cd4+ t lymphocyte count to the occurrence of opportunistic infection continues to be as valid in the era of haart as it was before the licensing of the fi rst antiretroviral agent, zidovudine, in 1987. [60] [61] [62] figure 54 -2 demonstrates the typical relationship of cd4+ t lymphocyte counts to the occurrence of opportunistic infections. knowledge of this relationship permits the focusing of diagnostic, therapeutic, and prophylactic management. for instance, if a patient with hiv infection and a cd4+ t lymphocyte count of 700 cells/µl presents with diffuse pulmonary infi ltrates, the diagnostic evaluation and empiric antimicrobial regimen should focus on s. pneumoniae; h. infl uenzae; mycoplasma, legionella, and chlamydia organisms, as well as common community-acquired viruses. in contrast, if the same patient had a cd4+ t lymphocyte count of 50 cells/µl, the evaluation and empiric regimen would focus on pneumocystosis and cmv, although the previously mentioned processes that occur at high cd4+ t lymphocyte counts can also occur at lower cd4+ t lymphocyte counts. keeping in mind that cd4+ t lymphocyte counts are useful predictors of susceptibility to infection is important, but they are not perfect. occasionally, patients will develop opportunistic infections at "uncharacteristically" high cd4+ t lymphocyte counts. for instance, 5% to 10% of cases of pneumocystosis occur at cd4+ t lymphocyte counts greater than 200 cells/µl. 61 clinical parameters can provide additional clues; for example, oral candidiasis, a previous opportunistic infection, a prior episode of pneumonia, or high viral load are independent risk factors for the occurrence of pneumocystis jiroveci carinii pneumonia (pcp), and logically for other infections as well. 9 a frequent question is whether an hiv-infected patient's prior cd4+ t lymphocyte count nadir affects the likelihood of an opportunistic infection occurring if haart has stimulated a cd4+ t lymphocyte count rise. specifically, if a patient has a cd4+ t lymphocyte count of 400 cells/µl while receiving haart and that patient's cd4+ t lymphocyte count was 50 cells/µl before haart, is that patient at greater risk for developing an opportunistic infection than another patient whose current cd4+ t lymphocyte count is 400 cells/µl but whose nadir before haart was 250 cells/µl? the data suggest that these two patients have comparable risk (i.e., the current cd4+ t lymphocyte count is the most important predictor of risk and the earlier nadir has only minor infl uence on opportunistic infection susceptibility). 62 in evaluating the differential diagnosis of infectious syndromes in patients with hiv (and in every other patient population as well), geography is an important part of the history. tuberculosis is always a concern because of the extraordinary susceptibility of hiv-infected patients for developing active disease. 63 in many urban settings in the united states, each pulmonary evaluation should include smears and cultures for m. tuberculosis, both to diagnose the appropriate cause of the pulmonary dysfunction and to assist in determining what respiratory precautions are appropriate. in some areas of the country, such as the ohio river valley and indianapolis, histoplasmosis is as common as pneumocystosis in causing diffuse pulmonary infi ltrates. 64 in the southwestern united states, coccidioidomycosis must be recognized as a cause of pulmonary infi ltrates. the clinical presentations of tuberculosis, histoplasmosis, coccidioidomycosis, and other processes such as cmv can be clinically indistinguishable from pcp. thus for patients with pulmonary infi ltrates in an icu, prolonged empiric therapy is discouraged in favor of vigorous efforts to establish a specifi c diagnosis. hiv-infected patients are admitted to icus for several major syndromes: respiratory insuffi ciency, cerebral dysfunction, septic shock, hepatic or renal failure, and drug toxicities. 50 however, patients with hiv infection also come to icus for routine procedures and routine postoperative care. in those situations their management ordinarily requires no extraordinary measures, with two exceptions. first, the staff must be fully aware of how hiv is transmitted, the danger of injuries resulting from sharp objects, and the procedure for managing injuries involving sharp objects contaminated with blood or other biologic fl uids from infected or potentially infected patients. 65 second, drug interactions involving drugs used during procedures and certain antiretroviral drugs can have important clinical consequences. 66, 67 many of the protease inhibitors and the non-nucleoside reverse transcriptase inhibitors that are now the backbone of antiretroviral therapy can inhibit or enhance the metabolism of drugs that depend on the cytochrome p450 system. thus the half-lives of certain analgesics, sedatives, and hypnotics can be prolonged in hiv-infected patients who are taking ritonavir, for example. this pharmacokinetic effect is also relevant for a host of other therapeutic agents used in the icu and may affect their effi cacy or safety. clinicians need to be familiar with these interactions when selecting new therapies for procedures or for clinical entities. patients with hiv infection can develop severe pulmonary dysfunction because of common community-acquired pathogens such as s. pneumonia, legionella, mycoplasma, and chlamydia; adenovirus; infl uenza; or respiratory syncytium virus, as well as other opportunistic viruses and fungi. thus the diagnostic evaluation needs to be comprehensive, emphasizing direct smears of sputum or bronchoalveolar lavage. it is important to recognize that the clinical presentations produced by many causative agents can be similar. for instance, histoplasmosis, tuberculosis, and nonspecifi c interstitial pneumonitis can present identically to pcp. 50, 61, 63, 64, 68 thus although empiric diagnosis and empiric therapy may be reasonable as initial approaches to some patients with hiv infection and mild pneumonitis, such an approach is usually not appropriate for patients in an icu. evaluation of induced sputum is the fi rst step in the diagnostic approach to pcp. sensitivity can be 80% to 95% at many hospitals (at some institutions the yield is considerably lower). 69 specifi city should be 100% in an experienced laboratory. other pathogens, including mycobacteria, fungi, and routine bacteria, can be identifi ed in sputum as well. for intubated patients, respiratory secretions obtained by deep intratracheal suctioning are also likely to be useful, although they have not been as carefully studied as induced sputum. should the diagnosis not be established by evaluation of sputum or intratracheal secretions, bronchoscopy should be performed. bronchoalveolar lavage should diagnose almost 100% of cases of pcp, even if patients have received 7 to 10 days of empiric therapy. 70 a diagnosis of pcp is established by visualizing one or more clusters of organisms. diagnostic criteria for other opportunistic infections are reviewed in chapters 12 and 43. in patients with hiv, cmv merits special mention. culture of sputum or bronchoalveolar lavage does not provide useful information because patients with cd4+ t lymphocyte counts below 100 cells/µl will predictably have cmv present in their secretion independent of whether or not pulmonary disease is present. 71 a diagnosis of cmv pneumonia in this patient population is suggested by cytology and confi rmed by the presence of multiple inclusion bodies in lung tissue obtained by transbronchial or open lung biopsy. similarly, mycobacterium avium complex (mac) and hsv can often be found in respiratory secretions, but these organisms almost never cause pneumonia in patients with hiv infection. in other patient populations they can clearly cause pneumonia, but the dearth of cmv, mac, and hsv pneumonia in this patient population emphasizes the point that it is important to know from published literature what the clinical likelihood is for different microbial processes. fungal pneumonias other than pcp are generally diagnosed by direct microscopy or culture of respiratory secretions (sputum or lavage). candida organisms almost never cause pneumonia in patients with hiv infection. the frequency of cryptococcus, histoplasma, blastomyces, and coccidioides organisms as causes of pneumonia depends on the geographic exposure of the patient. among these mycoses, antigen detection techniques can be useful for fi nding cryptococcus and histoplasma organisms. mycobacteria frequently infect the respiratory tract of patients with hiv infection. as noted earlier, m. avium complex almost never causes pulmonary dysfunction in this patient population. when acid-fast bacilli are seen (as opposed to cultured) in respiratory secretions or tissue, m. tuberculosis is almost always the pathogen; m. kansasii and other mycobacteria less commonly cause disease. screening all patients with acid-fast bacillus smears is important for preventing transmission of tuberculosis and text continued on p. 1126 should be considered as part of a routine respiratory evaluation for patients with radiographic infi ltrates in most areas of the united states. therapy of opportunistic infections is summarized in table 54 -5. 72 while awaiting a specifi c diagnosis, it is reasonable to initiate empiric therapy in patients ill enough to merit admission to an icu. for patients with a cd4+ t lymphocyte count greater than 250 to 300 cells/µl, azithromycin and ceftriaxone or azithromycin and ampicillin-sulbactam would be reasonable choices. for patients with cd4+ t lymphocyte counts below 200 to 250 cells/ µl, levofl oxacin or moxifl oxacin plus trimethoprimsulfamethoxazole or pentamidine plus levofl oxacin or moxifl oxacin would be potential regimens. if pcp is documented, trimethoprim-sulfamethoxazole is always the drug of choice in patients who can tolerate it. table 54-5 lists alternatives for sulfa-intolerant individuals. regardless of which specifi c antipneumocystis regimen is used, corticosteroid therapy is indicated for any patient who presents with an oxygen pressure (po 2 ) below 70 mm hg or an alveolar-arterial gradient higher than 30 mm hg. [73] [74] [75] [76] patients with an initial po 2 lower than 70 mm hg are the subgroup with substantial mortality for whom corticosteroids have been shown to provide a survival benefi t. corticosteroids may provide more rapid and perhaps more complete resolution of pulmonary manifestations in patients who present with better pulmonary function, but survival in this population is so high that clinical trials have not been able to show survival benefi t. some experts are concerned that corticosteroid use will be associated with reactivation of latent infections such as cmv or tuberculosis. however, reactivation of life-threatening infections has not been associated with this corticosteroid regimen. how should a patient with aids-associated pcp be managed if there is no improvement, or if there is deterioration, after 5 to 10 days of therapy? the median time to improvement in clinical variables is 4 to 8 days; therefore, changes in therapy are probably not warranted before 5 to 10 days. at that point the accuracy of the diagnosis should be reassessed: consideration should be given to repeat bronchoscopy with transbronchial biopsy to determine if cmv, fungi, mycobacteria, or a nosocomial bacterial process is present. noninfectious processes such as congestive heart failure or tumor (e.g., kaposi's sarcoma) must also be considered. if pneumocystosis is the only causative process that can be identifi ed, corticosteroids should be added to the regimen if they have not been already. whether switching from one antipneumocystis agent to another or whether adding a second agent is helpful has not been determined by clinical trials. some human pneumocystosis isolates are resistant to sulfonamides, but such testing is available only in a few research centers. most clinicians add parenteral pentamidine to trimethoprim-sulfamethoxazole. parenteral trimetrexate or clindamycin-primaquine could be used as salvage regimens as well. patients who have not improved after 14 to 21 days of therapy with specifi c chemotherapy plus corticosteroids have an exceedingly poor prognosis. should patients with aids-related pcp be intubated and provided with mechanical ventilation? mortality for such patient populations was 70% to 80% in several series in the early 1980s. [77] [78] [79] [80] since that era, supportive care has improved, and treatment modalities for concurrent infectious and noninfectious processes have become more effective. patient selection for ventilatory support is probably also improving. patients who have multiple active opportunistic infections, substantial weight loss, and no response to 14 days of therapy have a worse prognosis than ambulating patients who develop respiratory failure the third day of therapy. thus decisions about icu support for patients with hiv infection and respiratory failure need to be individualized on the basis of a realistic assessment of prognosis, the availability of resources, and the preference of the individual patient. a frequent question for any hiv-infected patient in the icu is whether antiretroviral drugs should be continued or initiated during the critical or life-threatening illness. although there is no specifi c study of various strategies, most authorities discourage the use of antiretroviral drugs in the icu because of drug interactions and drug toxicities. in addition, the initiating haart can be associated with dramatic "immune reconstitution" syndromes that can complicate the process that brought the patient to the icu. [81] [82] [83] finally, almost all antiretroviral drugs that are commercially available are oral: in most situations it is better to discontinue all antiretroviral drugs for a few days or weeks or months rather than risk poor absorption and suboptimal serum levels. the latter would enhance the emergence of drug-resistant hiv. an important cause of admitting hiv-infected patients into the icu is either seizures or altered mental status. either can result from infectious or neoplastic processes caused by meningeal disease or parenchymal involvement. the differential diagnosis of meningeal disease includes pneumococcal and staphylococcal meningitis, cryptococcal meningitis, tuberculous meningitis, and lymphomatous meningitis, as well as involvement from other endemic mycoses and common community-acquired viral and bacterial processes. 24,84 diffuse central nervous system parenchymal disease can be caused by hiv itself, by progressive multifocal leukoencephalopathy, and occasionally by herpes viruses such as cmv or herpes simplex virus. focal mass lesions may be caused by toxoplasmosis or lymphoma. less often, tuberculosis, fungi, conventional bacterial abscesses, nocardia, and other tumors are the cause of focal lesions. these lesions can be diffi cult to distinguish clinically and radiologically. the cd4+ t lymphocyte count can help narrow the differential diagnosis, but csf or brain tissue is usually necessary for defi nitive diagnosis. the routine therapies for many of these processes are outlined in table 54 -5. toxoplasmosis deserves particular mention because of its frequency. [85] [86] [87] toxoplasmosis occurs mainly in patients with hiv infection who have cd4+ t lymphocyte counts below 100 cells/µl, have a positive igg antibody titer against toxoplasma, and who have not been receiving trimethoprim-sulfamethoxazole or dapsone prophylaxis. patients present with altered cognition, focal motor or sensory defi cits, or seizures. lesions may be unifocal or multifocal. they usually enhance with contrast, but this is not invariably true. for patients who fi t the profi le for high risk of toxoplasmosis, and with a compatible presentation, it is reasonable to establish an empiric diagnosis and institute specifi c therapy with sulfadiazine plus pyrimethamine or, for patients unable to tolerate sulfa, clindamycin plus pyrimethamine. corticosteroids may be needed for patients with considerable intracerebral edema or elevated intracranial pressure. antiseizure medication is usually instituted only after a seizure has occurred rather than prophylactically. most patients improve clinically and radiologically within 7 to 10 days. if patients fail to improve, a stereotactic needle biopsy is appropriate, especially because the prevalence of lymphoma is increasing. organisms can be diffi cult to see in brain specimens obtained by this technique. patients with hiv infection develop hypotension resulting from the same types of disorders as with non-hiv infected individuals-sepsis from a primary infection or a wound or device (especially an intravascular access device), fl uid depletion from vomiting or diarrhea, and hemorrhage from a gastrointestinal lesion are examples of common causes. the evaluation of hypotension in a patient with hiv infection must take into account factors particular to this patient population: it is susceptible to opportunistic infections; it undergoes many procedures that can be associated with infectious complications; and it receives an array of drugs, some of which have cardiovascular effects. thus evaluating hypotension in this patient population requires a comprehensive and thorough approach. a differential diagnosis of the major causes is shown in table 54 -6. adrenal function always deserves special attention because several viral processes, fungal and mycobacterial diseases, hiv, and drugs can suppress the adrenal axis and either cause hypotension or exacerbate it. patients with hiv infection typically receive several antimicrobial agents to reduce the likelihood they will acquire opportunistic infections. 9 primary prophylaxis is the term used to indicate strategies that reduce the likelihood of an initial episode of a disease process. secondary prophylaxis is the term used to indicate strategies that prevent recurrences or relapses. chronic suppressive therapy is identical to secondary prophylaxis: this refers to regimens that are continued after the initial therapeutic course to prevent relapses. all patients with hiv infection and cd4+ t lymphocyte counts below 200 cells/µl typically receive antipneumocystis prophylaxis. trimethoprim-sulfamethoxazole is the regimen of choice. patients who actually take this drug have very few breakthroughs of pcp and receive considerable protection against toxoplasmosis and certain routine bacterial infections. alternative regimens include monthly dapsone, weekly dapsone-pyrimethamine, or daily aerosol pentamidine. prophylaxis against m. avium complex is recommended for patients with cd4+ t lymphocyte counts under 100 cells/µl; clarithromycin and azithromycin are currently the drugs of choice. 9 many clinicians also use fl uconazole or acyclovir prophylaxis to reduce the frequency of fungal and viral processes, respectively, although this is not recommended because of issues of cost, pill burden, and the emergence of resistant pathogens. isoniazid prophylaxis is important for any patient with a tuberculin skin test that shows more than 5 mm of induration or a history of substantial recent exposure. 9 transmission of tuberculosis from patients to other patients, from patients to staff, or from staff to patients is an urgent concern in icus. patients with hiv infection are extraordinarily susceptible to tuberculosis. thus an infected patient poses a substantial risk, especially when hospitalized for pneumonia or when undergoing procedures at high risk for producing aerosols such as intubation, bronchoscopy, sputum induction, or aerosol pentamidine treatment. identifying potentially infected patients early and placing them in appropriate isolation until their tuberculosis status is fully examined is important. in many centers, patients with syndromes compatible with pulmonary or upper airway tuberculosis are maintained in isolation at least until three specimens of respiratory secretions have been examined for tuberculosis. hiv-infected health care practitioners need to carefully assess their risk of acquiring tuberculosis by their exposure in the icu. transmission of hiv is an issue that requires attention in the icu. 65 no evidence exists that hiv-infected health care professionals can infect patients, regardless of what procedure they perform, outside of two unusual events. hiv patients pose a risk to health care professionals, however. this risk can be substantially reduced by education, by strict monitoring for compliance with universal precautions, and by having proper equipment. almost all hiv transmission in an occupational setting occurs as a result of injuries involving sharp instruments (e.g., needles, scalpels). the risk of such injuries is about one case of hiv transmission per 250 injuries, but the likelihood of transmission in an individual accident depends on the amount of viremia at the time of the accident (late-stage patients generally have more circulating virus than do early-stage patients) and the nature of the accident. most authorities recommend immediate prophylaxis if a signifi cant injury occurs involving an hiv-infected patient. considerable debate exists over the optimal choice of drugs and the optimal duration of therapy, but it is clear that initiating therapy within a period of hours rather than days is best. many authorities now advocate a haart regimen for any situation when the patient and health care provider determine that therapy is appropriate, and continue that for 4 to 6 weeks. increasingly, icus are caring for organ transplant recipients, either in the period immediately after the procedure or during a crisis that occurs days, weeks, months, or years after engraftment. managing each type of organ transplant recipient has unique features depending on whether bone marrow, kidney, heart, lungs, liver, or other organs are transplanted. 2, 6 laboratory monitoring provides useful predictive information about the status of cellular immunity, humoral immunity, and neutrophil number and function. ultimately, however, clinical experience is necessary with each type of organ transplant and each immunosuppressive regimen to predict the most likely pathogens, when they most characteristically occur in relation to the transplant procedure, and what infl uence each immunosuppressive therapy has. an example of the temporal pattern of infectious complications after bone marrow transplantation is shown in figure 54 -3. 9 although such fi gures are useful conceptually, however, the immunosuppressive regimens are changing rapidly, and such fi gures may be misleading when applied to current transplantation protocols. organ transplant recipients share a complex interaction between immunosuppression and infection. immunosuppression is usually necessary in allogeneic transplantation to permit graft survival. the more potent the immunosuppression, the more likely infection is to occur. strategies that use antimicrobial agents (drugs, vaccines, and other biologic products) aggressively may reduce the risk of and damage from infection in a manner that allows more potent immunosuppression and better graft survival. such approaches may include prophylactic antibacterial and antiretroviral treatment, as well as prompt empiric therapy for emerging febrile episodes. patients receiving hematopoietic stem cell transplantation (hsct) or solid organ transplants are often receiving antimicrobial prophylaxis. acyclovir for hsv, valacyclovir for cmv, fl uconazole for yeast, voriconazole for yeast and molds, trimethoprim-sulfamethoxazole for pcp, and quinolones for bacteria are used in various combinations at different transplant programs. these agents dictate which organisms will break through to cause disease, and what their antibiotic susceptibility patterns will be. several pathogens deserve special mention. cmv is one of the most prominent pathogens for solid organ and bone marrow transplant recipients. [88] [89] [90] most disease is secondary (i.e., disease results from reactivation of a previously acquired, latent infection) in a seropositive organ recipient. in urban areas of the united states, 60% to 70% of the population is seropositive for cmv, and thus 60% to 70% of the transplant recipients will have latent infection that could potentially be reactivated. some cmv seronegative patients acquire primary infections from a cmvinfected organ or from cmv-infected blood or blood products. a few cmv seropositive individuals develop superimposed cmv disease from cmv acquired through a seropositive donor. laboratory monitoring of patients for evidence of cmv disease by using a dna amplifi cation assay, or surveillance of cmv antigen in buffy coat smears, is an important feature in efforts to reduce morbidity and mortality resulting from cmv. 89, [91] [92] [93] [94] [95] intensivists need to understand how to interpret these assays in terms of starting empiric, pre-emptive, or defi nitive therapy. strategies to reduce the frequency of cmv disease with acyclovir, intravenous or oral ganciclovir (or oral valganciclovir), the investigational agent proganciclovir, or immune globulin are used by many programs. cmv disease can cause substantial morbidity and mortality including fever, hypotension, pneumonitis, hepatitis, glomerulitis, enteritis, and allograft injury. the availability of ganciclovir, foscarnet, and cidofovir has enabled these conditions to be treated successfully in many instances, although all three of these drugs are associated with substantial toxicity. whether immune globulin (either immune globulin or specifi c hyperimmune globulin) adds anything to the potency of therapeutic regimens is not clear, although these products are usually administered when they are available. pcp has been reported in recipients of most types of organ transplants. most organ transplant programs use pcp prophylaxis. 6, 33, 96 trimethoprim-sulfamethoxazole is usually the prophylactic agent of choice because it is more effective than other agents, is well tolerated, and reduces the frequency of urinary tract infections and other potential complications (e.g., disease resulting from nocardia, s. pneumoniae, and haemophilus organisms). fungal infections have been common, but the causative pathogens are changing because of changes in prophylactic regimens. with the use of fl uconazole, candida albicans infections became less common. molds, especially aspergillus, became more important pathogens, as did fl uconazole-resistant candida. some programs are now using voriconazole prophylaxis. for such patients, mucormycosis and non-albicans candida are becoming more prominent causes of morbidity. thus clinicians must know what antifungal prophylaxis has been used in order to anticipate which complications will occur. mold infections can be diffi cult to diagnose: serum galactomannan assays can yield specifi c information, but the test has low sensitivity. mold infections almost never cause fungemia. thus diagnosis depends on cultures, which can be highly suggestive if obtained from sources such as bronchoalveolar lavage or biopsy. viral respiratory infections require particular mention because some are treatable and most are transmissible. community-acquired respiratory viruses such as adenoviruses, coronaviruses, or infl uenza can occur in immunocompetent or immunosuppressed patients. when respiratory infections occur in immunocompromised patients, health care professionals need to be certain that a transmissible virus is not the cause because of the potential to infect other patients, families, or hospital staff. of the respiratory infections, rsv deserves special attention in hsct patients. although rsv can, like other community-acquired viruses, cause disease in any patient population, it is especially lethal in solid organ, bone marrow, and stem cell transplants. thus rsv must be specifi cally sought in this patient population, as well as their visitors and health care providers, so that it does not spread to highly susceptible patients. similarly, when caring for immunosuppressed patients, attention to mycobacterium tuberculosis is important because this pathogen can also spread to other patients, families, and hospital staff. with more immigrants in the united states and more patients having travel exposure, m. tuberculosis needs to be considered in the differential diagnosis and specifi cally sought by gene probe, smear, or culture where appropriate. diagnosis and therapy of opportunistic infections and nosocomial infections should follow the guidelines given in chapters 43, 51, and 54. in choosing therapies, attention must be focused on the toxicities of antimicrobial agents and how they infl uence the outcome of the transplanted organ. in addition, drug interactions are important, especially with cyclosporine. drugs that alter hepatic metabolism, such as rifampin, rifabutin, and fl uconazole, can have substantial infl uence on cyclosporine levels and thus need to be used with careful pharmacologic attention. finally, clinicians must recognize that new immunosuppressive regimens and changing prophylactic regimens are changing the spectrum of infectious complications. as mentioned earlier, fungal infections are increasingly likely to be caused by species other than c. albicans: non-albicans candida, fusarium, and rhizopus are recognized with increasing frequency. similarly, prophylaxis with valganciclovir is reducing cmv disease and pushing disease that does occur later and later in relation to the transplant procedure. viruses such as hhv-6 and bk virus are causing disease. thus clinicians need to look for changing spectrum of pathogens, as well as changing manifestations if the morbidity and mortality caused by infection is to be managed optimally. ■ knowledge of a patient's specifi c defects in immunologic and infl ammatory response helps predict which opportunistic pathogens are most likely to occur. ■ icus are increasingly successful in enabling immunosuppressed patients to survive acute crises, especially if the defect in immunologic or infl ammatory function is reversible over time or by replacement therapy. ■ for neutropenic patients, gram-positive cocci are becoming more frequent than gram-negative bacilli as causes of life-threatening illness. ■ resistance to antimicrobial agents is becoming a major problem including bacteria (e.g., vancomycin-resistant enterococci and penicillin-resistant pneumococci), fungi (e.g., fl uconazole-resistant candida organisms), as well as pcp, and viruses (e.g., acyclovir-resistant herpes simplex and ganciclovir-resistant cmv). ■ in neutropenic patients, combination therapy should be considered when treating any life-threatening bacterial process. ■ a substantial fraction of hiv-infected patients with pcprelated respiratory failure can survive mechanical support and be discharged from the hospital. ■ adjunctive corticosteroid therapy is indicated for respiratory failure related to pcp. ■ tuberculosis is a concern in any immunologically abnormal individual with pulmonary disease but is a special concern in hiv-infected patients. tuberculosis in these cases often warrants respiratory isolation until appropriate specimens are evaluated for mycobacteria. ■ organ transplant recipients develop opportunistic infections at relatively predictable points depending on the type of transplantation and the specifi c immunosuppressive regimen used. innate (general or nonspecifi c) host defense mechanisms infections in solid organ transplant recipients infectious diseases associated with complement defi ciencies infection in organ-transplant recipients cd4 counts as predictors of opportunistic pneumonias in human immunodefi ciency virus (hiv) infection recent advances in the diagnosis and management of infection in the organ transplant recipient infections in recipients of hematopoietic stem cell transplantation bacteremia and fungemia in patients with neoplastic disease guidelines for the preventing opportunistic infections among hiv-infected persons-2002 recommendations of the u.s. public health service and the infectious diseases society of america guidelines for the use of antimicrobial agents in neutropenic patients with cancer fever in immunocompromised patients use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodefi ciency committee of the american academy of allergy, asthma and immunology colony-stimulating factors for chemotherapy-induced febrile neutropenia: a meta-analysis of randomized controlled trials neutropenia, neutrophil dysfunction, and bacterial infection in patients with human immunodefi ciency virus disease: the role of granulocyte colony-stimulating factor update of recommendations for the use of white blood cell growth factors: an evidence-based clinical practice guideline monotherapy with meropenem versus combination therapy with ceftazidime plus amikacin as empiric therapy for fever in granulocytopenic patients with cancer. the international antimicrobial therapy cooperative group of the european organization for research and treatment of cancer and the gruppo italiano malattie ematologiche maligne dell'adulto infection program a double-blind comparison of empirical oral and intravenous antibiotic therapy for low-risk febrile patients with neutropenia during cancer chemotherapy monotherapy for fever and neutropenia in cancer patients: a randomized comparison of ceftazidime versus imipenem treatment of febrile neutropenia with cefepime monotherapy additional anti-gram-positive antibiotic treatment for febrile neutropenic cancer patients fever in the pediatric and young adult patient with cancer. a prospective study of 1001 episodes gram-positive infections and the use of vancomycin in 550 episodes of fever and neutropenia ceftazidime monotherapy for empiric treatment of febrile neutropenic patients: a meta-analysis staphylococcus epidermidis: an increasing cause of infection in patients with granulocytopenia antibiotic synergism and response in gram-negative bacteremia in granulocytopenic cancer patients vancomycin-resistant enterococcal infections an evidence-based evaluation of important aspects of empirical antibiotic therapy in febrile neutropenic patients effi cacy of caspofungin against invasive candida or invasive aspergillus infections in neutropenic patients antifungal therapy in patients with fever and neutropeniamore rational and less empirical? caspofungin versus liposomal amphotericin b for empirical antifungal therapy in patients with persistent fever and neutropenia a randomized, double-blind comparative trial evaluating the safety of liposomal amphotericin b versus amphotericin b lipid complex in the empirical treatment of febrile neutropenia. l amph/ablc collaborative study group levofl oxacin to prevent bacterial infection in patients with cancer and neutropenia guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. recommendations of cdc, the infectious disease society of america, and the american society of blood and marrow transplantation breakthrough zygomycosis after voriconazole treatment in recipients of hematopoietic stem-cell transplants beta-lactam antibiotic therapy in febrile granulocytopenic patients. a randomized trial comparing cefoperazone plus piperacillin, ceftazidime plus piperacillin, and imipenem alone risk assessment and riskbased therapeutic strategies in febrile neutropenia randomized, double-blind clinical trial of amphotericin b colloidal dispersion vs. amphotericin b in the empirical treatment of fever and neutropenia voriconazole versus amphotericin b for primary therapy of invasive aspergillosis review of treatment of zygomycosis with posaconazole in a patient with acute myeloid leukemia intravenous and oral itraconazole versus intravenous amphotericin b deoxycholate as empirical antifungal therapy for persistent fever in neutropenic patients with cancer who are receiving broad-spectrum antibacterial therapy. a randomized, controlled trial anidulofungin: a new echinocandin with a novel profi le combination antifungal therapy for invasive aspergillosis treatment of solid organ transplant patients with invasive fungal infections: should a combination of antifungal drugs be used? prevention of intravascular catheter-related infections guidelines for the management of intravascular catheter-related infections guidelines for the prevention of intravascular catheter-related infections eortc guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapyinduced febrile neutropenia in adult patients with lymphomas and solid tumours return of granulocyte transfusions metaanalysis: the effi cacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients intensive care of patients with hiv infection current issues in critical care of the human immunodefi ciency virus-infected patient declining morbidity and mortality among patients with advanced human immunodefi ciency virus infection. hiv outpatient study investigators critical care of immunocompromised patients: human immunodefi ciency virus neurologic manifestations of aids frequency of myocarditis, left ventricular dysfunction and ventricular tachycardia in the acquired immune defi ciency syndrome nonspecifi c interstitial pneumonitis: a common cause of pulmonary disease in the acquired immunodefi ciency syndrome small intestinal structure and function in patients infected with human immunodefi ciency virus (hiv): evidence for hiv-induced enteropathy thrombocytopenia in hiv infection human immunodefi ciency virus-associated autoimmune thrombocytopenic purpura: a review surveillance for aids-defi ning opportunistic illnesses prophylaxis against opportunistic infections in patients with human immunodefi ciency virus infection relations among cd4 lymphocyte count nadir, antiretroviral therapy, and hiv-1 disease progression: results from the eurosida study tuberculosis in patients with human immunodefi ciency virus infection disseminated histoplasmosis in the acquired immune defi ciency syndrome: clinical fi ndings, diagnosis and treatment, and review of the literature public health service guidelines for the management of occupational exposures to hiv and recommendations for postexposure prophylaxis interactions among drugs for hiv and opportunistic infections drug interactions in infectious diseases tuberculosis in patients with human immunodefi ciency virus infection. how often does it mimic pneumocystis carinii pneumonia? diagnosis of pneumocystis carinii pneumonia: improved detection in sputum with use of monoclonal antibodies the diagnosis of pneumocystis carinii pneumonia in patients with the acquired immunodefi ciency syndrome using subsegmental bronchoalveolar lavage lack of clinical utility of cytomegalovirus blood and urine cultures in patients with hiv infection national institute of health-university of california expert panel for corticosteroids as adjunctive therapy for pneumocystis pneumonia: consensus statement on the use of corticosteroids as adjunctive therapy for pneumocystis pneumonia in the acquired immunodefi ciency syndrome corticosteroids prevent early deterioration in patients with moderately severe pneumocystis carinii pneumonia and the acquired immunodefi ciency syndrome (aids) corticosteroids as adjunctive therapy for severe pneumocystis carinii pneumonia in the acquired immunodefi ciency syndrome. a double-blind, placebocontrolled trial a controlled trial of early adjunctive treatment with corticosteroids for pneumocystis carinii pneumonia in the acquired immunodefi ciency syndrome. california collaborative treatment group pneumocystis carinii pneumonia and respiratory failure in aids. improved outcomes and increased use of intensive care units critical care of patients with aids survival and prognostic factors in severe pneumocystis carinii pneumonia requiring mechanical ventilation survival following mechanical ventilation for pneumocystis carinii pneumonia in patients with the acquired immunodefi ciency syndrome: a different perspective incidence and risk factors for immune reconstitution infl ammatory syndrome in an ethnically diverse hiv type 1-infected cohort immune reconstitution syndrome in hiv: validating a case defi nition and identifying clinical predictors in persons initiating antiretroviral therapy incidence and risk factors for immune reconstitution infl ammatory syndrome during highly active antiretroviral therapy infections with cryptococcus neoformans in the acquired immunodefi ciency syndrome evaluation of the policy of empiric treatment of suspected toxoplasma encephalitis in patients with the acquired immunodefi ciency syndrome toxoplasmic encephalitis in patients with the acquired immunodefi ciency syndrome. members of the actg 077p/anrs 009 study team toxoplasmic encephalitis in aids preemptive ganciclovir therapy to prevent cytomegalovirus disease in cytomegalovirus antibody-positive renal transplant recipients. a randomized controlled trial prevention of fungal and hepatitis virus infections in liver transplantation antiviral drugs for cytomegalovirus in transplant recipients: advantages of preemptive therapy randomised trial of effi cacy and safety of oral ganciclovir in the prevention of cytomegalovirus disease in livertransplant recipients. the oral ganciclovir international transplantation study group whole blood real-time quantitative pcr for cytomegalovirus infection follow-up in transplant recipients prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients benefi ts of cytomegalovirus prophylaxis in solid organ transplantation posttransplant microbiological surveillance should prophylaxis for pneumocystis carinii pneumonia in solid organ transplant recipients ever be discontinued? key: cord-022521-r72jtoso authors: miller, tracie l.; cushman, laura l. title: gastrointestinal complications of secondary immunodeficiency syndromes date: 2010-12-27 journal: pediatric gastrointestinal and liver disease doi: 10.1016/b978-1-4377-0774-8.10042-9 sha: doc_id: 22521 cord_uid: r72jtoso nan secondary immunodeficiency syndromes constitute a spectrum of disorders. infections of the gastrointestinal tract pose the greatest risk for children with secondary immunodeficiencies. cellular changes in the gastrointestinal tract (the largest immune organ in the body) that lead to diarrhea and malabsorption, peptic disease, dysmotility, and liver disease are among some of the other disorders of the gastrointestinal tract faced by these children. worldwide, human immunodeficiency virus (hiv-1) infection and malnutrition are by far the most common secondary immunodeficiency states. however, in the united states and other developed countries, severe malnutrition and new cases of perinatal hiv-1 disease are rare because of relatively high standards of living and effective highly active antiretroviral therapies (haart) given to pregnant hiv-infected women that prevent transmission of hiv to the infants. 1 between 2004 and 2005, there were 67 reported cases of perinatally acquired hiv and 4883 new diagnoses of unspecified origin in adolescents 13 to 24 years of age. 2 hiv-infected children and adolescents are now surviving because of effective antiretroviral strategies, yet there is increased horizontal acquisition of hiv in adolescents owing to risky social behaviors. furthermore, children with chronic illness are among the highest population at risk for malnutrition and its sequelae. 3 thus these two disorders serve as models for complications of other secondary immunodeficiency states. the first cases of the acquired immunodeficiency syndrome (aids) were described in the early 1980s. later, in 1984, 4 hiv-1 was determined to be the causative agent, and hiv-1 infection was recognized as a spectrum of disease, ranging from asymptomatic infection to full-blown aids. the aids epidemic claimed an estimated 2 million lives in 2007, and an estimated 2.7 million people acquired hiv-1 in 2007. an estimated 33 million people globally are living with the virus. 5 with the successful preventive strategies of elective cesarean section delivery and chemoprophylaxis of pregnant hiv-1-infected women, the transmission rates plummeted from 15 to 30% to less than 1 to 2% of all hiv-1-infected women delivering infants. 6 the advent of haart in 1996 changed the natural history of hiv-1 in children in many countries. 7 however, the successes of prevention and prophylaxis have not been realized as much in developing countries, where hiv infection continues to increase. for this reason, there are disparate accounts of opportunistic infections and other diseases in regions with high haart accessibility and those with limited haart accessibility. 8 hiv-1 is an rna virus that belongs to the lentivirus family. it has a particular tropism for the cd4 surface antigen of cells, and the binding of hiv-1 to the cd4 receptor initiates the viral cycle. the virus may subsequently replicate within the host cell or, alternatively, the proviral dna within the host cells may remain latent until cellular activation occurs. human t lymphocytes and monocytes-macrophages are the primary cells that are infected with hiv-1, although other cell lines may be infected as well. the net effect is suppression of the immune system and a progressive decline in cd4+ t lymphocytes, which leaves patients susceptible to opportunistic and recurrent bacterial infections. the gastrointestinal tract is the main source of hiv-1 infection when parenteral transmission is excluded. in vertical transmission, hiv-1 is found in the gastrointestinal tract after the fetus swallows infected amniotic fluid, blood, cervical secretions, or breast milk. the virus, inoculated in the gastrointestinal tract, infects the fetus as it enters into the gut-associated lymphoid tissue (galt) through the tonsil or upper intestinal tract. examination of both acute simian immunodeficiency virus (siv) and hiv infection have documented reduced cd4 cell levels in galt prior to a detectable reduction in t cells of the peripheral blood, highlighting the gastrointestinal tract's role and susceptibility. [9] [10] [11] [12] the rates of acquisition of hiv-1 through the gastrointestinal tract are likely related to the quantity of virus in the person transmitting it [13] [14] [15] and the immunologic function and maturity of the patient being infected. mucosal infections with opportunistic infections may increase hiv-1 transmission. mycobacterial infections up-regulate cc chemokine receptor 5 (ccr5) expression in monocytes, which facilitates the entry of ccr5-tropic hiv-1. other factors, such as tumor necrosis factor-α (tnf-α), which is induced by nuclear factor (nf)-кb (which itself is pathogen induced), are potent inducers of hiv-1. 16, 17 cellular routes that potentially can transmit hiv-1 across the gastrointestinal tract include m cells, dendritic cells, and epithelial cells. m cells are specialized epithelial cells that overlie the peyer's patches and transport large macromolecules tracie l. miller • laura l. cushman and microorganisms from the apical surface to the basolateral surface. human transport of hiv-1 by m cells in vivo has not been reported. dendritic cells bind hiv-1 through a dendritic cell-specific adhesion molecule. in vitro studies support the role of dendritic cells in transmitting hiv-1 [18] [19] [20] [21] ; however, the role of the dendritic cell in in vivo transmission of hiv-1 has yet to be determined. epithelial cells express ccr5 and can selectively transfer ccr5-tropic hiv-1. the epithelial cell can transport hiv-1 in vitro from the apical to the basolateral surface. 22, 23 the r5-tropic viruses are transferred in vitro through epithelial cell lines. 24 once transmitted, the lamina propria lymphocytes express ccr5 and cxc4 chemokine receptor 4 (cxcr4), which support hiv-1 replication. 25, 26 early after infection, there is a greater proportion of infected lymphocytes in the lamina propria than in peripheral blood. 27, 28 for the patients actively receiving haart, poles et al. 29 described "cryptic replication" occurring in galt reservoirs in which viral replication is actively taking place at slower rates but hiv-1 rna levels remain undetected in peripheral blood. further, galt contained more than twice as many lymphoid cells (160, 000) than peripheral blood mononuclear cells (70,000) possessing hiv-1 dna with viral replication capacity. there was no significant reduction in these values when the analysis was repeated after 12 months. lymphocytes are able to disseminate the virus to distant sites, with depletion of cd4 cells in the lamina propria 27, 30 and then in the blood. even with aggressive suppression of hiv-1 during the primary stages by highly active antiretroviral agents, cd4 cell depletion is observed in the effector subcompartment gut mucosa when cd4 levels in the peripheral blood have stabilized. 10 as mucosal and peripheral t cells are depleted, monocytes and macrophages become important reservoirs for the virus. the intestinal macrophages do not promote inflammation and do not carry the receptor for ccr5 or cxcr4; however, the blood monocytes are different in their profile and are infected by hiv-1. they are found infected in the blood and thereafter take up residence in the gut. 31 they are stimulated by opportunistic agents and proinflammatory cytokines. 32 recent in vitro studies have implicated the integrin receptor α4β7 on which the hiv-1 envelope binds and transmits signals mediated by an epitope in the v2loop of gp120. this in turn activates lfa-1 and is pivotal in virological synapse formation, allowing rapid cellular dissemination of hiv-1. 33 villous atrophy and gastrointestinal tract dysfunction are coincident with high levels of hiv-1 viral load in the gut. 34 altered epithelial permeability may permit microbial translocation and generalized immune activation leading to localized cytokine production and further replication of hiv. 35 a dysfunctional gastrointestinal tract can produce clinical symptoms that contribute to both morbidity and mortality in children with hiv-1 infection. these symptoms include weight loss, vomiting, diarrhea, and malabsorption (table 42 -1). the advent of antiretroviral treatment induces debilitating effects on mechanisms within the gut that promote chronic hiv infection. mainly, high levels of lipopolysaccharides (lps) are associated with marked systemic immune activation sustaining this infection; antiretroviral therapy decreases levels of lps, promotes cd4+ t cell reconstitution, and may subsequently decrease the systemic immune activation. 36 additional studies examining this relationship stand to offer greater insight into hiv pathogenesis. as mentioned, there are distinct changes in the cellular milieu of the gastrointestinal tract in hiv-1-infected patients. previous studies have shown that activated mucosal t cells play a role in the pathogenesis of enteropathy in the human small intestine 37 and can affect the morphology of the villi and crypts in a manner similar to that seen in patients with hiv-1 infection. the magnitude of viral burden in the gastrointestinal tract is associated with villous blunting and other abnormal morphology. 34 a number of studies in the 1980s associated a distinct enteropathy with hiv-1. 38 diarrhea, weight loss, an abnormally low d-xylose absorption, and steatorrhea, without evidence of intestinal infection, were common findings. jejunal biopsies showed partial villous atrophy with crypt hyperplasia and increased numbers of intraepithelial lymphocytes. this was the first histologic description of a specific pathologic process that occurred in the lamina propria of the small intestine in some patients with hiv-1. others 39 found low-grade small bowel atrophy and maturational defects of enterocytes, supporting an hiv-1 enteropathy characterized by mucosal atrophy with hyporegeneration. however, some investigators have challenged this concept, suggesting that the findings could be attributed to an undiagnosed enteric infection. recently, genotype profiling for genes responsible for endothelial barrier maintenance and metabolic functioning has shown a decreased expression in the presence of increased viral replication in the galt and reduced cd4+ t cell levels. 40 these findings are significant, because they offer an additional modality for evaluating microenvironmental alterations within the gastrointestinal tract of the patient. additional studies will help to determine the efficacy of gene expression profiling in hiv-infected individuals. miller et al. 41 published histologic findings in 43 children with hiv-1 infection. the majority of patients had normal villous architecture, and many of the children with villous blunting had an associated intercurrent enteric infection. distinct features of hyperplasia of the lamina propria and increased intraepithelial lymphocytes were not apparent. bjarnason et al. 42 studied intestinal inflammation and ileal structure and function in patients with a wide spectrum of hiv-1 disease states. hiv-1-infected patients who were minimally symptomatic had normal intestinal absorption and permeability, yet had greater gastrointestinal dysfunction as they progressed to aids. malabsorption of bile acids and vitamin b 12 did not correlate with morphometric analysis of ileal biopsies and was unremarkable in these patients. thus, there was significant mucosal dysfunction with only minor ileal morphologic changes. malabsorption of bile acids may play a pathologic role in patients with aids diarrhea. the absorptive defect of aids enteropathy using a d-xylose kinetic model of proximal absorption was studied 43 and correlated with the results of a schilling test for cobalamin absorption, which measures distal intestinal function. there were minimal histologic abnormalities in both the proximal and distal biopsy sites in patients with diarrhea and no enteric infection. d-xylose absorption was low, and the absorptive defect was more severe and greater than would be expected from the histologic abnormalities found. thus, these findings support the theory that there is little association between histologic characteristics of the small bowel and its absorptive function in patients with hiv-1 infection. most studies do not support a direct role for gastrointestinal malabsorption on growth failure or weight loss. ullrich et al. 39 described gastrointestinal malabsorption in hiv-1-infected patients who had low levels of lactase enzyme in the brush border, crypt death, decreased villous surface area, and decreased mitotic figures per crypt when compared with control patients. in addition, keating et al. 44 described absorptive capacity and intestinal permeability in hiv-1-infected patients. malabsorption was prevalent in all groups of patients with aids, but was not as common in the asymptomatic hiv-1-infected patients. malabsorption correlated with the degree of immune suppression and with body mass index. there were mild decreases in the ratio of jejunal villous height to crypt depth, yet not as severe as the subtotal villous atrophy found in celiac disease. lim et al. 45 found disaccharidase activity decreased proportionately with greater hiv-1 disease severity, although there was no association between disaccharidase levels and weight loss. in addition, mosavi et al. 46 found no correlation between diarrhea and weight loss in hiv-1-positive patients. taylor et al. 47 found mild histologic changes accompanied by severe disaccharidase abnormalities; however, symptoms were severe enough to withdraw lactose in only 25% of the patients. collectively these studies suggest that gastrointestinal malabsorption may be present, but is not always associated with weight loss and diarrhea. formal studies of intestinal absorption in children with hiv-1 are more limited. malabsorption occurs frequently in hiv-1-infected children and may progress with the disease. in one study, 40% of children had nonphysiologic lactose malabsorption and 61% had generalized carbohydrate malabsorption that was not associated with gastrointestinal symptoms or nutritional status. 48 these findings have been confirmed by others. 49 another study in children revealed an association between diarrhea and nutrition. 50 abnormal d-xylose absorption has also been associated with enteric infections in children. 48 fat and protein loss or malabsorption have also been described. sentongo et al. 51 evaluated fat malabsorption and pancreatic exocrine insufficiency using fecal elastase-1 enzyme assay in 44 hiv-1-infected children. hormone-stimulated pancreatic function testing and 72-hour stool and dietary fat sample collection were performed in children with abnormal fecal elastase levels. the prevalence of steatorrhea was 39% and that of pancreatic insufficiency was 0% (95% confidence interval 0 to 9%). there were no associations between steatorrhea and pancreatic insufficiency, growth, hiv-1 rna viral load, cd4 status, or type of antiretroviral therapy. other studies support the absence of association. 52 thus, the clinical significance of steatorrhea in pediatric hiv-1, similar to absorption of other nutrients, is unclear. the etiology of malabsorption in hiv-1 infection is probably multifactorial. the cellular milieu of the lamina propria is altered significantly with hiv-1 infection. 34, 53 the depletion of the cd4 t lymphocytes in the intestinal tract may cause change in the cytokine environment and alter intestinal function. viral load in the intestinal tract may be considerably higher than that measured peripherally, and this can also affect mucosal gastrointestinal structure and function. recently, the hiv-1 tat protein was found to decrease glucose absorption through decreasing the activity of the sodium d-glucose symporter. 54 studies suggesting these hypotheses include that of kotler et al., 55 which looked at intestinal mucosal inflammation in 74 hiv-1-infected individuals. these authors found abnormal histopathology in 69% of the patients, and this finding was associated with altered bowel habits. high tissue p24 antigen levels were observed, and these correlated with more advanced hiv-1 disease. tissue p24 detection was associated with both abnormal bowel habits and mucosal histology. the tissue content of cytokines, including tnf, α-interferon, and interleukin-1β, was higher in hiv-1-infected individuals than in controls, and these increases were independent of intestinal infection. thus, hiv-1 reactivation in the intestinal mucosa could be associated with an inflammatory bowel-like syndrome in the absence of other enteric pathogens. small bowel bacterial overgrowth can be another source of gastrointestinal dysfunction leading to malabsorption. bacterial overgrowth may be due to aids gastropathy, 56, 57 in which the stomach produces only small amounts of hydrogen chloride, allowing bacterial pathogens to escape the acid barrier of the stomach and colonize the duodenum. additionally, iatrogenic hypochlorhydria may be due to the use of acid-blocking agents as treatment for peptic disease. interestingly, some authors have found no relationship between gastric ph and small bowel bacterial colonization and diarrhea in hiv-1-infected patients. 58 enteric pathogens 59 have been associated with enteric dysfunction, as discussed later. with the advent of haart, gastrointestinal symptoms, especially those associated with opportunistic infections, are less common. 60 as viral burden decreases, immunosuppression has less effect on gastrointestinal function. compared to untreated patients, haart-treated patients had greater integrity of intestinal mucosal barrier and decreased villous atrophy. 61 ritonavir, a protease inhibitor, in combination therapy resulted in restoration of gastrointestinal function in 10 children with carbohydrate malabsorption, steatorrhea, protein loss, and iron deficiency. 62 however, one study in adults found similar rates of fat malabsorption in patients taking haart and in those not taking haart. 63 the gastrointestinal tract is a major target for opportunistic infections in hiv-1-infected children. the spectrum of these infections is dependent on hiv-1 disease progression. in developed countries, with improved hiv-1 viral suppression associated with haart, opportunistic infections of the gut and elsewhere are less common. 64 however, immunocompromised children are still at risk for infections with cytomegalovirus (cmv), herpes simplex virus (hsv), cryptosporidium, and microsporidia. previous dogma that much of the diarrhea found in children with hiv-1 infection is not associated with enteric pathogens has been challenged. unusual viral and parasitic infections can be diagnosed as a result of better diagnostic techniques. however, the cause of diarrhea in a significant number of patients with hiv-1 remains undiagnosed. 65 occurrence of opportunistic disease and infection of the gastrointestinal tract in immunocompromised patients relies heavily on the accessibility of haart. as a result, there is great disparity of documented incidence of gastrointestinal infections dependent on access to haart in particular regions. for this reason, we have divided this section into two subsections: gastrointestinal infections in regions with low haart accessibility or in patients with cd4 t-lymphocyte counts less than 200 cells/mm 3 , and gastrointestinal infections in regions with high haart accessibility and successful viral suppression. this does not imply that any of the infections discussed here occur in isolation contingent on haart accessibility, because all hiv-1 patients regardless of haart may encounter these complications. in the post-haart era, it is helpful for a physician to know which backdrop lends itself to specific vulnerabilities. the detection of viral gastrointestinal infections in hiv-1infected children can sometimes be difficult owing to the limitations of diagnostic techniques. the most common gastrointestinal viral pathogen in hiv-1-infected children is cmv. other pathogens, such as hsv, adenovirus, epstein-barr virus, and a variety of other unusual viruses, can also contribute to intestinal dysfunction and diarrhea. hsv infection in an immunocompromised child usually represents reactivation of a latent virus that had been acquired earlier in life. gastrointestinal infection with hsv most commonly involves the esophagus and causes multiple small, discrete ulcers. hsv can also involve other areas of the intestinal tract, including the colon and small bowel. the diagnosis of hsv relies on recognizing the multinucleated intranuclear inclusion bodies (cowdry type a) with a ground-glass appearance and molding of the nuclei. the squamous epithelium is usually infected, although there may also be involvement of intestinal glandular epithelium in the mesenchymal cells. hsv monoclonal antibody staining is confirmatory for the diagnosis. in extensive involvement, there may be transmural necrosis and development of tracheoesophageal fistulas. treatment of hsv and other common gastrointestinal pathogens and their primary sites of involvement are outlined in table 42-2. other herpes viruses have also been detected in the gut of hiv-1-infected individuals. a case report of one 34-year-old hiv-1-infected man with intestinal pseudo-obstruction and disseminated cutaneous herpes zoster revealed positive immunohistochemistry against herpes zoster in a resected portion of the terminal ileum. this area had focal ulceration. the virus was localized to the muscularis propria and myenteric plexi throughout the entire length of the specimen. the authors postulated that the location of the virus in the gut may have been the etiologic factor for the pseudo-obstruction. 66 cytomegalovirus cmv in the immunocompromised child, like hsv, represents reactivation of a latent virus that was acquired in earlier life. cmv is one of the more common viral pathogens of hiv-1infected children. the reported incidence of gastrointestinal involvement in the pre-haart era varied from 4.4% to 52% of patients studied. the incidence rates may have varied based on the techniques of diagnosis. 57 cmv infection is rare in patients with cd4 t-lymphocyte counts greater than 50 cells/mm 3 . 67 cmv may involve any part of the gastrointestinal tract, with an increased incidence in the esophagus or colon. cmv infection usually results in one or two discrete single and large ulcers of the esophagus and colon. lesions may lead to severe gastrointestinal bleeding and hemodynamic instability. cmv inclusion bodies can be discovered incidentally in an asymptomatic patient, and this does not necessarily reflect disease. in patients with upper intestinal cmv disease, there can be dysphagia and upper abdominal symptoms, whereas diarrhea is more common with colitis. the diarrhea can be watery or bloody. children may be systemically ill. 68 the colitis from cmv infection is patchy and can be associated with severe necrotizing colitis and hemorrhage. 69 cmv usually affects the cecum and the right colon. diagnosis is confirmed by endoscopy and biopsy. the histologic appearance of cmv-infected cells is unique (figure 42 -1). these cells are enlarged and contain intranuclear and cytoplasmic inclusion bodies. the nuclear inclusion bodies are acidophilic and are often surrounded by a halo. cytoplasm inclusion bodies are multiple, granular, and often basophilic. cells that are dying may appear smaller and smudged, with poorly defined inclusion bodies. staining for cmv antigen shows that many of the infected cells are endothelial cells with others being perivascular mesenchymal cells. cmv can cause vasculitis because of its target cell population. thus, the spread of cmv occurs with circulating infected endothelial cells. treatment options are outlined in table 42 -2. once haart is established, with decreased viral burden (both hiv-1 and cmv) and improved cd4 counts, cmv treatment may be discontinued without concern for reactivation. 70 infections with other unusual viral pathogens have been described. these include the human papilloma virus and epstein-barr virus, which have been identified in esophageal ulcers of patients with hiv-1. adenovirus of the stomach and colon have also been reported and are often difficult to identify. 71 in the pre-haart era, patients who excreted adenovirus from their gastrointestinal tract had a shorter survival. 72 there are unusual enteric viruses that have been associated with diarrhea in hiv-1-infected children. 73 these viruses, among others, include astrovirus and picobirnavirus. 74 cegielski et al. 75 studied 59 children with hiv-1 infection in tanzania. they looked for enteric viruses identified by electron microscopy of fecal specimens. small round structured viruses (srsvs) were found more frequently in hiv-1-infected children than in uninfected children with chronic diarrhea. rotavirus and coronaviruslike particles were not associated with hiv-1 infection. these authors considered that these srsvs may be associated with hiv-1 infection and could lead to chronic diarrhea in tanzanian children. bacterial infections that involve the gastrointestinal tract of children with hiv-1 infection may be divided into three groups: bacterial overgrowth of normal gut flora; pathogens that can affect immunocompromised children as well as immunocompetent children (salmonella, shigella, campylobacter, clostridium difficile, and aeromonas); and bacterial infections that are more common in immunocompromised children (mycobacterium avium-intracellulare complex; mac). few studies have evaluated bacterial overgrowth in hiv-1-infected children, although gastric hypoacidity has been associated with opportunistic enteric infections and bacterial overgrowth in adult patients with hiv-1. 76 other studies have not found this association. small bowel bacterial overgrowth was not a common finding in a group of 32 hiv-1-infected patients, regardless of the presence of diarrhea, and it was not associated with hypochlorhydria. 58 lactose hydrogen breath testing has shown high baseline readings in children that may indirectly suggest bacterial overgrowth of the small intestinal tract. 48 detection of bacterial overgrowth in the small bowel is usually performed by quantitative duodenal aspirate for bacterial culture, with therapy directed at treating the organisms, which are often anaerobic. common bacterial pathogens include salmonella, shigella, campylobacter, clostridium difficile, and aeromonas. infection with these organisms occurs more frequently in immunocompromised patients. combined morbidity and mortality rates associated with hiv-1 and these bacterial pathogens in developing countries approach 50% in some studies. 77 hiv-1-infected patients with campylobacter infection have higher rates of bacteremia than the general population. deaths from sepsis due to this organism have been reported in severely immunodeficient patients with aids, despite haart. 78 other entities, such as bacterial enteritis, have been described in adults with hiv-1. a study by orenstein and kotler 79 evaluated ileal and colonic biopsies in patients with aids and diarrhea and found bacteria similar to adherent e. coli along the intestinal epithelial border. similar findings were documented by kotler et al., 80 who showed adherent bacteria in 17% of all adult patients with aids. the infection was localized primarily to the cecum and right colon, and three distinct histopathologic patterns of adherence were observed: attachment on effacing lesions, bacteria intercalated between microvilli, and aggregates of bacteria more loosely attached to the damaged epithelium. the bacterial cultures of frozen rectal biopsies yielded e. coli in 12 of the 18 patients. these findings suggest that chronic infection with adherent bacteria can also produce the syndrome of aids-associated diarrhea. in a "look back" evaluation, orenstein and dieterich 71 found that enteropathogenic bacterial infections were overlooked on initial examination and concluded that, for accurate diagnoses, specimens should be evaluated by laboratories with expertise in hiv. intestinal infections with mycobacteria, including mycobacterium tuberculosis, mac, and other atypical mycobacteria, were the most frequently encountered bacterial infections in hiv-1-infected patients in the pre-haart era 81 and became more prevalent in the pre-haart era as patients were living longer with cd4 counts below 200 cells/mm 3 . 82, 83 in the haart era, disseminated mac in colonized patients can be successfully prevented; however, the effects of haart on restoration of cd4 counts do not prevent mac colonization. 84 infection with mac usually occurs in the very late stages of aids in children, when cd4 counts are lower than 200 cells/mm 3 . the most common clinical manifestations of gastrointestinal infections with mac include fever, weight loss, malabsorption, and diarrhea. intestinal obstruction, resulting from lymph node involvement and intussusception; terminal ileitis, which resembles crohn's disease; and refractory gastric ulcers are often found. severe gastrointestinal hemorrhage has also been described. 85 endoscopically, fine white nodules may be seen in the duodenum, or the duodenal mucosa may look velvety and grayish in appearance. segments of the gastrointestinal tract can become infected with mac. histologically, there is a diffuse histiocytic infiltrate in the lamina propria with blunting of the small intestinal villi. these histiocytic infiltrates can be recognized on hematoxylin and eosin staining and on acid-fast stains and are pathognomonic for infection ( figure 42-2) . with the advent of haart, immune reconstitution disease has been described. 86, 87 this is likely an immune reaction in which previously quiescent organisms become active because of the improved immune function associated with haart. this can occur in as many as 25% of patients who respond to haart. 88 lymphadenitis is the most common condition, although abscesses can appear anywhere. severe abdominal complaints may result. appropriate therapies are outlined in table 42 -2, yet this organism is often frustrating to treat. azithromycin 600 mg, when given in combination with ethambutol, is an effective agent for the treatment of disseminated m. avium disease in patients infected with hiv-1. 89 caution must be exercised in administering these multidrug regimens for mac in patients receiving concurrent haart. rifamycins induce cytochrome p450 enzymes and accelerate the metabolism of clarithromycin and hiv-1 protease inhibitors. conversely, clarithromycin inhibits these enzymes, resulting in increased rifabutin toxicity. the net result is treatment regimens that can be extremely difficult to tolerate and manage, especially for sicker patients. clarithromycin and azithromycin must be administered in combination with other agents, such as ethambutol, to prevent the emergence of macrolide resistance. 90 in the early 1980s, cryptosporidiosis was regarded as an aidsdefining disease and an opportunistic intestinal pathogen. it became an important cause of chronic diarrhea, leading to high morbidity and mortality rates in immunocompromised patients. to date, no effective chemotherapy is available. with the introduction of protease inhibitors in haart regimens, the incidence of cryptosporidiosis in patients with aids has declined substantially in developed countries. 91 however, in developing nations, gastrointestinal infection with c. parvum is prevalent and carries high morbidity and mortality rates. 92, 93 although cryptosporidium was initially described in animals, it was first noted to cause an enterocolitis in both immunocompromised and immunocompetent humans in 1976. 94, 95 an intact t-cell response is the primary mechanism that confers protection against this organism; thus, patients with abnormal t-cell function or number are at risk. the spectrum and severity of disease in immunocompromised individuals with cryptosporidiosis correlates with most severe disease found in individuals with defects in the t-cell response. 91 the overall frequency of infection seems to be related to the severity of immunodeficiency and not the specific disorder. 96 cryptosporidium usually affects the gastrointestinal tract, although it has been found in other organs including the biliary tract, 97 pancreas, 98 and respiratory tract. 99 in immunocompetent individuals, the diarrhea is self-limiting, whereas in immunocompromised patients, it may be protracted and associated with significant malabsorption and nutritional compromise. the small intestine is the primary target, although it can occur in any part of the intestinal tract. esophageal cryptosporidiosis has also been described in one child 100 and in adults. clayton et al. 101 described two patterns of enteric cryptosporidiosis. one was accompanied by severe clinical disease with significant malabsorption, with the majority of the organisms found in the proximal small bowel, whereas less severe clinical disease was seen in patients with colonic disease or with infection noted only in the stool. patients with proximal small bowel infection with cryptosporidium showed crypt hyperplasia, villous atrophy, lamina propria inflammatory infiltrates, abnormal d-xylose absorption, greater weight loss, and shorter survival, with greater need for intravenous hydration and hyperalimentation than patients with colonic disease. in other studies, absorption of nutrients showed an inverse correlation with active infection, 102 as shown by altered vitamin b 12 and d-xylose absorption and lactulose and mannitol urinary excretion ratios. intestinal function improved in patients whose oocyte counts were reduced by treatment with paromomycin. symptomatic cryptosporidiosis has been documented in as many as 6.4% of immunocompetent children and 22% of immunodeficient children, whereas in an asymptomatic population, cryptosporidium was found in 4.4% of immunocompetent and 4.8% of immunodeficient children. 103 spiramycin at 100 mg per kg daily for 14 days caused a significant reduction in the shedding of infectious oocysts, and no gastrointestinal symptoms developed in children treated for asymptomatic infection, whereas children who were not treated developed gastrointestinal symptoms. 103 the diagnosis of cryptosporidiosis is made by identifying the organisms in a duodenal aspirate, stool, or tissue sample (biopsies). on hematoxylin and eosin-stained sections, these organisms can be found as rows or clusters of basophilic spherical structures 2 to 4 μm in diameter, attached to the microvillous border of the epithelial cells (figure 42-3) . the tips in the lateral aspect of the villi show the greatest number of organisms in the small intestine. in the colonic epithelium, the crypt and surface epithelial involvement appears equal. cryptosporidium also stain positively with giemsa and negatively with mucous stains. the acid-fast stain on a stool sample is one of the most widely used methods of determining whether a patient has cryptosporidiosis. more recent sensitive and specific methods for diagnosing cryptosporidiosis include fluorescein-labeled igg monoclonal antibodies. 104, 105 treatment of cryptosporidiosis in children with hiv-1 infection is often difficult. the disease can be chronic and protracted with diffuse watery diarrhea and dehydration. several different agents are used to eradicate the organism, with varying success rates. the most effective treatment is to improve immunologic function and nutritional status. with the advent of haart, many children's immune function has been restored with a lower incidence and prevalence of cryptosporidium infection. 106 the introduction of haart in a patient with severe debilitating cryptosporidium infection not only resulted in an increased cd4 count in the peripheral blood and clearance of the organism, but also produced a marked increase in cd4 count in the rectal mucosa on biopsy, suggesting this may have been the main mechanism of clearing the parasite. 107 octreotide therapy of acute and chronic diarrhea, with coincident improvement in nutritional status, eradicated cryptosporidium in one patient. 105, 108 other investigators have used bovine hyperimmune colostrum with benefit. 109, 110 the macrolides, such as azithromycin, have shown some promise in the treatment of cryptosporidium infection. 111, 112 the effect of protease inhibitors as therapy against cryptosporidium has been tested in a cell culture system. 113 nelfinavir moderately inhibited the host cell invasion over a period of 2 hours. indinavir, nelfinavir, and ritonavir inhibited parasite development significantly. the inhibitory effect was increased when the aminoglycoside paromomycin was combined with the protease inhibitors indinavir, ritonavir and, to a lesser extent, saquinavir, compared with the protease inhibitor alone. thus, protease inhibitor therapy may directly (rather than indirectly, through its effects on the immune system) inhibit growth of cryptosporidium. amadi et al. 92 found that a 3-day course of nitazoxanide improved diarrhea, helped eradicate the parasite, and improved mortality in hiv-1-seronegative, but not hiv-1seropositive, children in zambia. treatment with nitazoxanide on immunocompetent patients demonstrated parasitic load reduction, but its effects on immunocompromised patients are not yet palpable. 114 microsporidia are obligate intracellular protozoal parasites that infect a variety of cell types in many different species of animals. these organisms were first described in 1857, when recognized as a cause of disease in nonhuman hosts. 115 the first description of microsporidia (enterocytozoon bieneusi) as a human pathogen was in 1985, and microsporidia have since been described as more common human pathogens. 116 infection with microsporidia typically occurs in patients with severely depressed cd4 t-lymphocyte counts. one of the largest case studies of intestinal microsporidiosis in patients with hiv-1 infection was described by orenstein et al. 117 in 67 adult patients with aids and aids-related complex and chronic nonpathogenic diarrhea. e. bieneusi was diagnosed by electron microscopy in 20 of the patients. jejunal biopsies were more positive than duodenal biopsies. the parasites and spores were clearly visible by light microscopy in 17 of the 21 biopsies. infection was confined to enterocytes located at the tip of the intestinal villus, and the histologic findings included villous atrophy, cell degeneration, necrosis, and sloughing. other investigators [118] [119] [120] found microsporidia in as many as 50% of hiv-1-infected patients with chronic and unexplained diarrhea evaluated in the pre-haart era. e. bieneusi has been documented in 15 to 25% of children with 121 or without 122 diarrhea in developing countries, making it fairly ubiquitous in these regions of the world. other species of microsporidia, including encephalitozoon (septata) intestinalis, can cause significant enteric disease with diarrhea, wasting and malabsorption. encephalitozoon intestinalis differs from enterocytozoon bieneusi in its tendency to disseminate, and it can infect enterocytes as well as macrophages, fibroblasts, and endothelial cells. microsporidia are found with increasing frequency in hiv-1negative patients. 123 infection has been documented in almost every tissue and organ in the body, and in epithelial, mesenchymal, and neural cells. microsporidia can cause inflammation and cell death and a variety of symptoms including shortness of breath, sinusitis, and diarrhea with wasting. if left untreated, microsporidiosis can be a significant cause of mortality. treatments for microsporidia include albendazole, which can relieve clinical symptoms and eliminate microsporidial spores in the feces, especially of the less common pathogen, e. intestinalis. e. bieneusi is more challenging to treat, although therapy with fumagillin or its analogue, tnp-470 (antiangiogenesis agents), has shown promising results. [124] [125] [126] other studies show atovaquone as an effective treatment as well. 127 indirect treatment by improving the immune system with haart has also effectively cleared these organisms. 106, 128, 129 isospora belli is recognized as an opportunistic small bowel pathogen in patients with hiv-1 infection. this organism is most common in tropical and subtropical climates. isosporiasis can be diagnosed by identification of the oocyte in the stool or by biopsy. the diagnosis is critical because, in contrast to cryptosporidiosis or microsporidiosis, the therapy is very effective. i. belli is found within the enterocyte and within the cytoplasm. the organism stains poorly, although the central nucleus, large nucleolus, and perinuclear halo give it a characteristic appearance. the infection produces mucosal atrophy and tissue eosinophilia. a 10-day course of trimethoprim-sulfamethoxazole is effective therapy, and recurrent disease can be prevented by ongoing prophylaxis with this combination drug. 130 ciprofloxacin, although not as effective, is an acceptable alternative for those with sulfa allergies. 130 other therapies for isospora include pyrimethamine, also indicated for patients with sulfa allergies. 131 blastocystis hominis is usually considered a nonpathologic parasite, but it has been described in patients with chronic diarrhea and hiv-1 infection. 132 this organism is more pathogenic in immunocompromised patients and can cause mild, prolonged, or recurrent diarrhea. effective therapy includes diiodohydroxyquinoline 650 mg orally three times daily for 21 days. other protozoan infections that can be found in hiv-1-infected patients are entamoeba histolytica, entamoeba coli, entamoeba hartmanni, endolimax nana, and giardia lamblia in 4% of cases. candidiasis of the gastrointestinal tract is the most common fungal infection in hiv-1-infected children. the esophagus is the primary target of candida, and this infection occurred in the majority of patients during the course of their illness in the pre-haart era. it was also the second most frequent aidsdefining disease, second in prevalence only to pneumocystis jirovecii. patients with candida esophagitis complain of odynophagia or dysphagia and may often have vomiting and recurrent abdominal pain. children often have oral thrush, coincident with more disseminated and invasive candida esophagitis, although the absence of oral thrush does not preclude the diagnosis of candida esophagitis. 41 in one study, oral candidiasis preceded the diagnosis of candida esophagitis in 94% of children. 133 other risk factors include low cd4 count and prior antibiotic use. 133 histopathologically, yeast forms within an intact mucosa confirm invasive disease. this is in contrast to colonization, where the yeast is found overlying intact mucosal surfaces or necrotic tissue. these organisms are best seen with grocott's methenamine silver method or periodic acid-schiff stain. upper gastrointestinal studies are suggestive of candida esophagitis with diffuse mucosal irregularities (figure 42-4) . upper gastrointestinal endoscopy with biopsy and appropriate staining is the most sensitive test for determining invasive candidiasis of the esophagus. candidiasis can also occur in the stomach, as well as the small bowel if the acid barrier has been suppressed either through an intrinsic decrease in gastric acid production or iatrogenically with the use of potent acid blockers. numerous effective therapies have been described to treat candida of the upper gastrointestinal tract, including fluconazole, ketoconazole, and itraconazole. 134, 135 ketoconazole has more hepatic side effects than fluconazole. itraconazole is usually well tolerated and is effective. in severe and invasive disease, either topical or intravenous amphotericin can be used. agents such as oral miconazole and nystatin are not indicated for invasive candida. disseminated histoplasmosis develops in 5% of adult patients with aids in the midwestern region of the united states, and elsewhere. the clinical signs and symptoms related to this infection may be indolent, but left untreated can carry significant morbidity and mortality. 136 the likelihood of disease is higher in patients with cd4 counts under 200 cells/mm 3 . 137 there is enterocolitis associated with infection, and at colonoscopy, plaques, ulcers, pseudopolyps, and skip areas are frequently seen. cryptococcal gastrointestinal disease has been identified in patients with disseminated cryptococcus infection. the esophagus and colon are involved most frequently. p. jirovecii infection of the gastrointestinal tract has also been described. 59 gastrointestinal pneumocystosis develops after hematogenous or lymphatic dissemination from the lungs, or reactivation of latent gastrointestinal infection. the administration of aerosolized pentamidine has increased the risk of developing extrapulmonary spread of p. jirovecii pneumonia. p. jirovecii pneumonia infection can occur throughout the gastrointestinal tract. in the lamina propria there are foamy exudates with p. jirovecii organisms found within them. although more rare, infection of the colon can also cause diarrhea. the effect of haart on rates of infection of the gastrointestinal tract are twofold. first, on a macro level, the advent of haart has led to a dramatic decrease in perinatal transmission of hiv, and therefore the rates of newly infected children have plummeted, 138 with reports of vertical transmission falling between 1 and 2%. 139 second, haart has been successful in immune reconstitution, and therefore in regions with high haart accessibility, there has been a marked decrease in the number of hiv patients presenting with opportunistic infections. these infections have not been eradicated, but the majority of patients adhering to haart are able to achieve cd4+ cell reconstitution and therefore stave these off. patients who sustain chronically low cd4+ t lymphocyte levels in spite of haart accessibility and usage continue to be at risk for the opportunistic infections 140 described in the previous section. when comparing incidence rates of opportunistic infection in the pre-(before january 1, 1997) and post-haart era, there was an overall decrease. 8, 141 specifically: incidence rates of cmv decreased from 1.4 to 0.1; esophageal candidiasis from 0.9 to 0.4; herpes simplex virus from 0.2 to 0; and chronic intestinal and cryptosporidiosis from 1.3 to 0 per 100 persons per year. 141 additionally, nachman et al. 142 reported successful withdrawal of opportunistic infection prophylaxis for a period of 132 weeks in pediatric hiv-positive patients more than 2 years old who had achieved cd4 reconstitution without significant incidence when compared to demographically matched hiv-negative patients. in light of this progress, we have integrated additional immunocompromised patient populations into our discussion of gastrointestinal vulnerabilities. colitis from c. difficile is also more common in the immunosuppressed population owing to chronic antibiotic use and impaired immune system. 143 pulvirenti et al. 144 studied 161 hiv-1-infected patients with c. difficile and found that they had longer hospital stays and more admissions than patients without c. difficile infection, as well as other opportunistic infections such as herpes virus. they found c. difficile-associated diarrhea in 32% of all study patients with diarrhea. however, infection with c. difficile appeared to have little impact on morbidity or mortality. in a 1998, new york state screening study 145 of hospitalized hiv-1-infected patients in the haart era, 2.8% were admitted with a diarrheal diagnosis, with 51.3% of these having a c. difficile infection. thus, even with haart, diarrhea is prevalent and is often associated with identifiable pathogens. c. difficile infection has been reported to be one of the most common bacterial diarrheal pathogens among hiv-infected patients although its rates have decreased with haart. 146 because of the serious complications that are associated with active bacterial enteric infections in immunodeficient children, treatment options are outlined in table 42 -2. h. pylori prevalence is not significantly different between hiv-1-infected patients and hiv-1-negative patients. 147, 148 some investigators have found the seroprevalence of h. pylori to be lower in hiv-1-infected patients, 149 especially as cd4 counts decline with advancing disease. 147 the protection from h. pylori may be a result of frequent antibiotic use or correlated with a more advanced, dysfunctional immune state that results in a decreased inflammatory response to the organism. 150 remission of a high-grade gastric mucosa-associated lymphoid tissue (malt) lymphoma followed h. pylori eradication and haart in a patient with aids. 151 however, a recent study of hivinfected adults showed that 32% of patients with peptic symptoms had h. pylori on biopsy, with those having cd4 counts above 200 cells/mm 3 at a higher risk. 152 treatment of h. pylori in hiv-1-infected children is similar to that in noninfected children, with special attention to drug interactions. in up to 15 to 25% of hiv-1-infected children, the etiology of the diarrhea is unclear. autonomic dysfunction is another potential mechanism of noninfectious diarrhea not previously described. clinically, children with autonomic neuropathy have sweating, urinary retention, and abnormal cardiovascular hemodynamics. it is possible that this autonomic denervation contributes to diarrhea in patients with hiv-1 infection, as suggested by griffin et al. 153 when neuron-specific polyclonal antibodies were applied to jejunal biopsies, there was a significant reduction in axonal density in both villi and pericryptal lamina propria in patients with hiv-1 infection compared with controls, with the greatest reduction in patients with diarrhea. octreotide therapy has shown promising results in some patients. 154 finally, drug side effects should be considered, with many of the antiretroviral therapies causing chronic diarrhea and other gastrointestinal toxicities (table 42-3) . motility problems of the esophagus and stomach have been reported [155] [156] [157] and can be a source of upper gastrointestinal complaints including vomiting, dysphagia, nausea, and dyspepsia. the motility abnormalities may be primary, or they may be secondary to infectious or inflammatory disease of the respective organ. hypertension of the lower esophageal sphincter with incomplete relaxation, esophageal hypocontraction, and nonspecific motility disorders have been described in patients with normal intact esophageal mucosa. 156 gastric emptying, especially in patients with infections or advanced disease, may be delayed, as documented by gastric scintigraphy. 155 however, delayed gastric emptying does not always correlate with upper gastrointestinal symptoms or small bowel motility studies. in adults with hiv-1 infection and minimally advanced disease, gastric emptying of solids was delayed and emptying of liquids accelerated compared with that in controls. no abnormal esophageal motility patterns were found. all patients had a normal endoscopy prior to the motility studies. 157 thus, in the absence of infectious and inflammatory disease in patients with appropriate symptoms, motility studies or empiric trials of prokinetic agents should be considered, with careful consideration of drug interactions. esophageal ulceration can be a result of an intercurrent opportunistic infection. idiopathic oral and esophageal ulcers have been described in both children and adults with hiv-1. 158 these ulcers are characteristically large and may be single or multiple (figure 42-5) . the ulcers are located in the mid to distal esophagus. controversy exists regarding the pathogenesis of these ulcers; some investigators have identified hiv-1 at the ulcer base, 159 whereas others have not. 160 treatment options for these ulcers are limited, but include steroid therapy, with encouraging results, 159 and thalidomide. 161, 162 however, chronic low-dose thalidomide does not prevent recurrence of the oral or esophageal aphthous ulcers. 163 in addition to the potentially teratogenic effects, a significant portion of children receiving thalidomide develop a rash, which precludes use of the drug. significant caution should be exercised when using thalidomide. overall, haart has had a positive impact on esophageal disease occurrence and relapse. 164 the diagnostic approach to the child with hiv-1 or other immunodeficiencies and gastrointestinal symptoms is outlined in table 42 table 42 -2). every effort should be made to correlate timing of the initiation of a drug with onset of symptoms. the clinician should keep in mind that children with active enteric infections may also have secondary problems with malabsorption. if the clinical history and physical examination are suspicious for malabsorption without enteric infection, the next step should include an evaluation of specific nutrient absorption. carbohydrate malabsorption can be detected through lactose breath hydrogen testing, which measures hydrogen production as a response to an oral lactose load. a raised baseline breath hydrogen or early peak of hydrogen production suggests bacterial overgrowth, and appropriate treatment can be initiated. lactose malabsorption results in a level of hydrogen production more than 10 to 15 parts per million over baseline, 60 minutes after ingestion. dietary changes can then be made. d-xylose absorption testing also helps to determine the absorptive capacity of the gastrointestinal tract. d-xylose is an absorbable sugar that does not require active transport for uptake by enterocytes. thus, the d-xylose serum level, after administration of a test dose, reflects the absorptive ability of the gastrointestinal tract and the integrity of the mucosal surface. in younger children, the administered dose is 0.5 g per kg bodyweight, given orally after an overnight fast. in older children and adolescents, the maximum dose is 25 g. a serum level is obtained 1 hour after ingestion. urine samples may be obtained for 5 hours after ingestion as well. plasma citrulline levels correlate with enterocyte mass and function and may be used to indicate gastrointestinal function. 165 fat malabsorption is determined by a 72-hour fecal fat collection. a high-fat diet is administered several days before the collection is initiated and throughout the collection period. an alternative method is to keep a dietary fat intake record during the period of fecal fat collection. the stool is analyzed for total fat content, and the fecal fat is compared with the amount ingested; a coefficient of fat absorption is then calculated. ten percent or more of ingested fat in the stool is considered abnormal. alternatively, a sudan stain may be performed on a random stool sample. this may be helpful as a quick test for fat malabsorption, although it is not so reliable. quantification of fecal elastase may help to determine whether the fat malabsorption is pancreatic in origin. lastly, raised fecal α 1 -antitrypsin levels suggest protein loss from the gut. if noninvasive studies, such as those described above, are not helpful in documenting and determining the etiology of the malabsorption, diarrhea, or vomiting, endoscopy (either upper or lower) with biopsy and appropriate culture of fluid may be useful. miller et al. 41 confirmed histologic abnormalities in 72% of children undergoing upper endoscopy. in 70% of patients in this series, the clinical management of the child was changed because of the endoscopic evaluation. a high diagnostic yield has been supported by other investigators. 166 specific gastrointestinal symptoms are not predictive of abnormal findings at endoscopy; advanced hiv-1 disease stage and an increased number of symptoms seem to be more predictive. 41 histologic studies of the small bowel may aid in determining the degree of the villous blunting, and electron microscopy and special staining for opportunistic pathogens can be performed. quantitative bacterial cultures and parasite evaluation of the duodenal fluid should be obtained when an endoscopy is performed. characteristically, the detection of more than 10 5 organisms per milliliter of duodenal fluid confirms bacterial overgrowth. it is important to obtain both anaerobic and quantitative cultures. however, other studies have shown that endoscopy does not improve the diagnostic yield compared with stool examination in patients with intestinal infection. the only exception is the diagnosis of cmv. 167 an additional study found that flexible sigmoidoscopy was as useful as a full colonoscopy for diagnosing infection. 168 special histologic stains for fungal, mycobacterial, or viral infections did not increase the diagnostic yield over routine hematoxylin and eosin staining. 169 treatment for intestinal infections has been outlined in table 42 -2 and previous sections. therapy for gastrointestinal malabsorption should be directed toward the underlying diagnosis. if clinically symptomatic lactose malabsorption is found, a lactose-free diet should be initiated. compliance may be difficult, because many foods contain lactose. children can limit the effects of dietary lactose by taking exogenous lactase or using lactase-treated milk. there should be careful consideration of calcium and vitamin d intake, because children with hiv-1 infection are susceptible to low bone mineral density. [170] [171] [172] if there is malabsorption of protein and fat, a protein hydrolysate diet should be tried. many of these supplements are poorly tolerated because they are unpalatable. in some circumstances, specialized supplements may need to be administered through a supplemental feeding tube. 173, 174 peptic and motility disorders can be treated as in other, non-hiv-1-infected children, paying careful attention to potential drug interactions with antiretroviral regimens. a variety of other disorders (table 42 -5) can cause secondary immunodeficiencies with effects on the gastrointestinal tract. overall, these disorders are more prevalent than either primary or hiv-1-associated immunodeficiencies. premature infants, children with cancer and associated exposure to immunosuppressant and cytotoxic medications (including children with graft-versus-host disease), and children with protein-losing enteropathy with associated loss of immunoglobulins from the gastrointestinal tract can all be immunodeficient because of the underlying disorder. in general, children with these immunodeficiencies are at risk for many of the same complications that are experienced by children with hiv-1 infection. gastrointestinal tract infections are among the most common problems facing children with other secondary immunodeficiencies. malnutrition is the most common cause of immunodeficiency worldwide. nutritional status and immunity have long been linked in many disease states. before hiv-1 was described, p. carinii (now jirovecii) pneumonia and kaposi's sarcoma, known opportunistic diseases, were first described in otherwise healthy, but malnourished, children and adults in developing nations. 175, 176 this association led investigators to conclude that nutrition alone can affect the immunologic response of an individual. in malnourished children there is a profound involution of lymphoid tissues, including thymic atrophy and diminished paracortical regions of lymph nodes. 177 in young infants and children, protein-calorie malnutrition increases the risk of death by severalfold by increasing the susceptibility to infection. 178 in many countries, the mortality rate increases from 0.5% in children whose weight-for-height percentage of standard is greater than 80%, to 18% in children whose weight-for-height percentage of standard is less than 60%. 179 in other diseases such as cystic fibrosis and cancer, nutritional status has been linked closely to survival and morbidity. malnourished children with leukemia and lymphoma have a higher risk of p. jirovecii pneumonia than children who have normal nutrition. 175 biochemically, protein-calorie malnutrition leads to changes in several aspects of the immune system. cell-mediated immunity, microbial function of phagocytes, complement systems, secretory antibodies, and antibody affinity are consistently impaired in patients with significant malnutrition. additionally, deficiencies of micronutrients, especially zinc and iron, as well as many others, may also have deleterious effects on the immune system. other aspects of immunity that are altered by proteincalorie malnutrition include impaired chemotaxis of neutrophils, decreased lysozyme levels in serum and secretions, and interferon production in antibody response to t-cell-dependent antigens. a child with protein-calorie malnutrition may also have impaired mucosal immunity with lowered concentrations of secretory iga in saliva, nasopharynx, tears, and the gastrointestinal tract compared with well-nourished control children. similar to children and adults with hiv-1 infection, patients with malnutrition have depressed t-cell function not only in the peripheral circulation but also in the intestinal tract. subsequently, plasma cell function and macrophage activity may be impaired, leading to more frequent intestinal infections in children with severe protein-calorie malnutrition. not only does nutrition improve the immunologic functioning of the intestinal tract, but nutrients themselves are trophic and essential for the maintenance of the absorptive capacity of the intestines. in some studies, weight loss greater than 30%, due to other disorders, is associated with a reduction in pancreatic enzyme secretion of over 80%, villous atrophy, and impaired carbohydrate and fat absorption. 180 these disorders are promptly reversed with appropriate nutritional rehabilitation. with villous blunting, antigen uptake can increase, leaving the child at higher risk of enteric infection. the pathogenesis of villous blunting is unclear but may be due to crypt hyperplasia as the primary event with premature sloughing at the villus tip 181 versus loss of enterocytes at the villus tip with resultant proliferation at the crypts. 182 malnutrition and its associated immunodeficiency are of global concern, and researchers have experimented with both dietary regimens and micronutrient supplementation to improve, and perhaps ultimately restore, adequate immunological function. 183, 184 because of the low cost of many micronutrients when compared to pharmacological agents, success in such experimentation could have profound implications for those suffering from malnutrition, as well as other immunocompromised patients. zinc is accepted as a promoter of immune function and consequently has been evaluated in hiv-1 immunocompromised patients. in a south african study that treated patients with 10 mg of zinc (elemental) daily for 6 months and compared them to a control group receiving a placebo, there was a significant difference in patient presentation of diarrhea favoring zinc supplementation. 185 further demonstrating its potential alleviatory effects, canani et al. 186 observed that the transactivating peptide's (tat) secretory mechanism was inhibited by zinc and subsequently prevented diarrhea in pediatric hiv-1 patients. both of these positive outcomes, although documented in hiv-1 patients, present zinc as an additional treatment option for symptoms of malnutrition-related immunodeficiency. some studies have administered multivitamins to hiv-1 patients including adults and children and evaluated effects of specific micronutrients. adequate levels of vitamin a, when bolstered by supplementation in hiv-1 positive and hiv-1 negative children older than 6 months, correlated with reduced mortality and morbidity. 187 although not yet well-documented in children, multivitamin intake of hiv-1 positive adults demonstrated retarded progression of the virus. 187 improved hematologic status, mainly decreased rates of anemia, was observed in women and their children in tanzania who were treated with iron supplements during and after pregnancy. this was marked by an average hemoglobin count that was 0.33 g/dl greater than that of the patient group who did not receive multivitamin treatment. 188 these findings underscore the need for additional randomized control trials in pediatric populations to further understand the role of micronutrient supplementation as a complimentary treatment component. immunosuppressant medications are the mainstay of therapy for many diseases in children with autoimmune disorders, inflammatory bowel disease, chronic pulmonary disease, cancer, and organ transplantation. the best known immunosuppressants include corticosteroids, azathioprine, cyclosporin, tacrolimus, and anti-thymocyte globulin. unfortunately, the effects of these medications are not targeted toward specific organs, but rather indiscriminately suppress immune function throughout the child. thus, several immunologic functions including a decrease in monocyte adherence, neutrophil chemotaxis, and overall suppression of the inflammatory response are present. children are at risk of enteric infections, similar to those described in children with hiv-1 infection. pediatric patients undergoing transplant procedures, specifically solid organ transplant, are at increased risk of acquiring gastrointestinal infections when compared to their healthy counterparts. acutely, these complications present with vomiting, diarrhea, and cramping. the most frequently diagnosed posttransplant infection is cmv, [189] [190] [191] but its onset has been reduced significantly with the administration of prophylactic drugs such as ganciclovir. [189] [190] [191] [192] another aspect of the gastrointestinal tract that renders importance in secondary immunodeficiency is the liver. although data regarding liver complications in pediatric hiv-1 patients are lacking, there are considerable reports on hiv-1 infected adults. hepatitis coinfection, biliary tract disease, and drugderived illness are some of the major complications we discuss in this section. liver complications in secondary immunodeficiency, in their own right, deserve extensive coverage beyond the scope of this chapter, and so this section offers only a snapshot of this complex topic. both hepatitis and hiv infections share similar transmission pathways, and for this reason, it is not surprising that rates of viral coinfections are considerable. hepatitis a is often thought of as the less serious of the hepatitis viruses when treated promptly, and coinfection with hiv does not seem to predispose an individual to adverse outcomes. in 2006, coinfection rates of hepatitis b were reported to be between 5 and 10% in the global hiv population. prolonged hepatitis b infection is one of the greatest concerns of for coinfected patients. 193 hepatitis b virus e antigen (hbeag) is the protein associated with active hepatitis b in patients and is used by clinicians to determine efficacy of medications. for monoinfected hepatitis b patients, proper treatment can result in significant reduction of hbeag in 90 to 95% of patients; however, this success is not mirrored in hiv-coinfected populations. 194 there is also evidence for greater reactivation and replication in hepatitis b-hiv coinfected patients. 193, [195] [196] [197] explanation for this focuses on hiv patients' increased proinflammatory cytokine production and their inability to eliminate hepatocytes infected with hepatitis b. 196, 198 balancing dual treatment for both hepatitis b and hiv viruses presents a unique challenge for clinicians. acquired mutations of hepatitis b render additional treatment considerations, deepening the challenge. resistant hepatitis b strains have been identified, many of them falling under the tyrosine-methionine-aspartate-aspartate category, ymdd. iacomi et al. 199 determined that 28 of 29 hepatitis b-hiv coinfected patients exhibited this specified resistance. lamivudine, once commonly prescribed, has become less effective because of developed resistance. of the various treatment options, only tenofovir is used in hiv treatment and is effective in both the wild-type hepatitis b virus and the resistant ymdd strain, and it is often concomitantly administered with emtricitabine. 200, 201 mothers coinfected with hepatitis c and hiv are more likely to transmit hiv to their children, indicating greater risk of perinatal transmission in coinfected patients. 202, 203 liver disease in coinfected patients may be accelerated when compared to their monoinfected counterparts. pathways that have been proposed to accelerate fibrosis in coinfected patients include direct viral effects, dysregulation of the immune system toward a profibrotic state, and other metabolic pathways that lead to liver toxicity and processes such as steatosis and insulin resistance. 204 giovaninni et al. 203 studied 49 hiv-infected children, 11 of whom were coinfected with hepatitis c. six of the coinfected patients had abnormal alanine aminotransferase (alt) levels. three of the six children had aids, and three had aids-related complex. five children with normal aminotransferase had no detectable viral progression. following acute hepatitis c infection, hepatitis c-hiv coinfected patients are over 20% more likely than monoinfected patients to develop chronic hepatitis c infection. [205] [206] [207] interferon with ribavirin therapy is widespread in chronic hepatitis-c monoinfected patients, and its efficacy in hepatitis c-hiv coinfected patients is yet to be fully realized. 208, 209 when compared to conventional interferon therapy (ifnα-2a), 180 μg/week pegylated interferon (pegifnα-2a) plus 800 mg/day ribavirin demonstrated enhanced histological effects that were also associated with improved virological response in hepatitis c-hiv coinfected patients. 210 end-stage liver failure, cirrhosis, and hepatocellular carcinoma have been observed with greater frequency in hepatitis c-hiv coinfected patients. 205 these findings reflect adult populations and may or may not be indicative of outcomes in pediatric patients. children with acute biliary tract disease may present with rightsided abdominal pain, vomiting, and jaundice. also, elevated serum bile levels may induce pruritus. despite a disproportionate increase in alkaline phosphatase levels, alt levels may or may not be elevated. an ultrasound of the biliary system may be needed when serum sampling is equivocal. biliary tract disease is often obstructive; thus, use of endoscopic retrograde cholangiopancreatography (ercp) will help to identify the obstruction, perhaps provide bile sampling, and even mitigate the obstruction via sphincterotomy. bile sampling may indicate the presence of cmv, cryptosporidium, mycobacterium, or microsporidia, which were all discussed in previous sections. in one study employing sonography, 26 of 41 hiv-infected children displayed hepatobiliary abnormalities, yet there was no evidence of infection. 211 aids cholangiopathy is defined by intraand extrahepatic sclerosing cholangitis and is commonly linked to cmv and cryptosporidium infections. incidence in pediatric hiv populations remains to be thoroughly evaluated. the advent of haart, as discussed previously, has had profound effects on reducing many infections within the gastrointestinal tract. in the liver, however, there has been a negative association between receipt of antiretroviral therapy and development of liver complications, defined as immune restoration hepatitis. 193 serum alt elevation is associated with liver tissue damage and therefore is used to detect liver disease. hepatitis-hiv patients on haart present with elevated alt levels in the blood principally derived from haart-induced hepatotoxicity, resistance to antiretrovirals, and haart noncompliancy. 212 specifically, mitochondrial damage has been implicated as a contributor to liver death, which stems from coinfection and drug treatments. 213, 214 current research aims to determine effective methodology for detecting hepatic mitochondrial toxicity as a means to identify patients at risk of developing liver complications due to treatment. 213 gastrointestinal disorders in children with secondary immunodeficiencies cause considerable comorbidity. infection of the gastrointestinal tract is one the most common complications associated with secondary immunodeficiencies. however, malabsorption, peptic disease, and liver and biliary tract disease are also prevalent. these gastrointestinal complications contribute negatively to the overall clinical outcomes of children who have other chronic medical conditions, and they can often become life-threatening. thus, clinicians should be vigilant and aggressive in the evaluation and treatment of gastrointestinal tract dysfunction in children with secondary immunodeficiencies. incidence of opportunistic and other infections in hiv-infected children in the haart era microbial translocation is a cause of systemic immune activation in chronic hiv infection ritonavir combination therapy restores intestinal function in children with advanced hiv disease plasma citrulline is a biomarker of enterocyte mass and an indicator of parenteral nutrition in hiv-infected patients interactions of nutrition and infection coinfection with hiv-1 and hcv -a one-two punch see expertconsult.com for a complete list of references and the review questions for this chapter reduction of maternal-infant transmission of human immunodeficiency virus type 1 with zidovudine treatment. pediatric aids clinical trials group protocol 076 study group nutrition, child growth, and chronic disease prevention frequent detection and isolation of cytopathic retroviruses (htlv-iii) from patients with aids and at risk for aids aids epidemic update prevention of perinatal hiv transmission: a review of novel strategies efficacy of highly active antiretroviral therapy in hiv-1 infected children incidence of opportunistic and other infections in hiv-infected children in the haart era cd4+ t cell depletion during all stages of hiv disease occurs predominantly in the gastrointestinal tract primary hiv-1 infection is associated with preferential depletion of cd4+ t lymphocytes from effector sites in the gastrointestinal tract viral suppression and immune restoration in the gastrointestinal mucosa of human immunodeficiency virus type 1-infected patients initiating therapy during primary or chronic infection gastrointestinal tract as a major site of cd4+ t cell depletion and viral replication in siv infection transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection a controlled trial of zidovudine in primary human immunodeficiency virus infection prognosis in hiv-1 infection predicted by the quantity of virus in plasma mycobacterium avium complex augments macrophage hiv-1 production and increases ccr5 expression cytomegalovirus induction of tumor necrosis factor-alpha by human monocytes and mucosal macrophages recruitment of hiv and its receptors to dendritic cell-t cell junctions replication of hiv-1 in dendritic cell-derived syncytia at the mucosal surface of the adenoid dc-sign, a dendritic cellspecific hiv-1-binding protein that enhances trans-infection of t cells dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to cd4+ t cells infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp 120 receptor transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier primary intestinal epithelial cells selectively transfer r5 hiv-1 to ccr5+ cells lamina propria lymphocytes, not macrophages, express ccr5 and cxcr4 and are likely target cell for hiv-1 in the intestinal mucosa biological parameters of hiv-1 infection in primary intestinal lymphocytes and macrophages gastrointestinal tract as a major site of cd4+ t cell depletion and viral replication in siv infection peripheral monocyte and naive t-cell recruitment and activation in crohn' s disease lack of decay of hiv-1 in gutassociated lymphoid tissue reservoirs in maximally suppressed individuals rapid mucosal cd4 + t-cell depletion and enteropathy in simian immunodeficiency virus-infected rhesus macaques human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity macrophage functions in hiv-1 infection hiv-1 envelope protein binds to and signals through integrin α4β7, the gut mucosal homing receptor for peripheral t cells macrophage hiv-1 infection and the gastrointestinal tract reservoir microbial translocation is a cause of systemic immune activation in chronic hiv infection disease progression: immune activation, microbes, and a leaky gut evidence that activated mucosal t cells play a role in the pathogenesis of enteropathy in human small intestine enteropathy associated with the acquired immunodeficiency syndrome small intestinal structure and function in patients infected with human immunodeficiency virus (hiv): evidence for hiv-induced enteropathy rapid onset of intestinal epithelial barrier dysfunction in primary human immunodeficiency virus infection is driven by an imbalance between immune response and mucosal repair and regeneration endoscopy of the upper gastrointestinal tract as a diagnostic tool for children with human immunodeficiency infection intestinal inflammation, ileal structure and function in hiv small intestinal human immunodeficiency virus-associated enteropathy: evidence for panintestinal enterocyte dysfunction intestinal absorptive capacity, intestinal permeability and jejunal histology in human immunodeficiency virus and their relation to diarrhea intestinal disaccharidase activity in human immunodeficiency disease lack of correlation between diarrhea and weight loss in hiv-positive outpatients in houston, tx the prevalence and severity of intestinal disaccharidase deficiency in human immunodeficiency virusinfected subjects malnutrition and carbohydrate malabsorption in children with vertically-transmitted human immunodeficiency virus-1 infection italian paediatric intestinal/hiv study group. intestinal malabsorption of hiv-infected children: relationship to diarrhoea, failure to thrive, enteric microorganisms and immune impairment gastrointestinal-dysfunction and disaccharide intolerance in children infected with human immunodeficiency virus association between steatorrhea, growth, and immunologic status in children with perinatally acquired hiv infection pancreatic dysfunction and its association with fat malabsorption in hiv infected children loss of cd4 t lymphocytes in patients infected with human immunodeficiency virus type 1 is more pronounced in the duodenal mucosa than in the peripheral blood inhibitory effect of hiv-1 tat protein on the sodium-d-glucose symporter of human intestinal epithelial cells intestinal mucosal inflammation associated with human immunodeficiency virus infection gastropathy and ketoconazole malabsorption in the acquired immunodeficiency syndrome (aids) intestinal infections in patients with the acquired immunodeficiency syndrome (aids): etiology and response to therapy no relationship between gastric ph, small bowel bacterial colonisation, and diarrhoea in hiv-1 infected patients enteric pathogens associated with gastrointestinal dysfunction in children with hiv infection declining prevalence of opportunistic gastrointestinal disease in the era of combination antiretroviral therapy impairment of the intestinal barrier is evident in untreated but absent in suppressively treated hivinfected patients ritonavir combination therapy restores intestinal function in children with advanced hiv disease hiv-related diarrhea is multifactorial and fat malabsorption is commonly present, independent of haart declining prevalence of opportunistic gastrointestinal disease in the era of combination antiretroviral therapy cd4 counts and enteric infections in a community-based cohort of hiv-infected adults in uganda demonstration of varicella-zoster virus infection in the muscularis propria and myenteric plexi of the colon in an hiv-positive patient with herpes zoster and small bowel pseudoobstruction (ogilvie' s syndrome) laboratory diagnosis of cytomegalovirus (cmv) infections in immunodepressed patients, mainly in patients with aids cytomegalovirusassociated manifestations involving the digestive tract in children with human immunodeficiency virus infection massive lower gastrointestinal hemorrhage caused by cmv disease as a presentation of hiv in an infant viruses causing diarrhoea in aids the histopathology of 103 consecutive colonoscopy biopsies from 82 symptomatic patients with acquired immunodeficiency syndrome: original and look-back diagnoses shorter survival in hiv-positive patients with diarrhoea who excrete adenovirus from the gi tract enteric viruses and diarrhea in hiv-infected patients detection of picobirnavirus in hiv-infected patients with diarrhea in argentina enteric viruses associated with hiv infection in tanzanian children with chronic diarrhea association of gastric hypoacidity with opportunistic enteric infections in 12 patients with aids non-typhoidal salmonella bacteraemia among hiv-infected malawian adults: high mortality and frequent recrudescence enteric and disseminated campylobacter species infection during hiv disease: a persisting but significantly modified association in the haart era diarrheogenic bacterial enteritis in acquired immunodeficiency syndrome: a light and electron microscopy study of 52 cases chronic bacterial enteropathy in patients with aids incidence of mycobacterim avium-intracellulare complex bacteremia in human immunodeficiency virus-positive patients impact of opportunistic disease on survival in patients with hiv infection opportunistic diseases incidence and survival in aids patients who experienced very low cd4+ cell counts in the protease inhibitors era (tupe 3341) low incidence of colonization and no cases of disseminated mycobacterium avium complex infection (dmac) in brazilian aids patients in the haart era severe gastrointestinal hemorrhage due to mycobacterium avium complex in a patient receiving immunosuppressive therapy focal mycobacterial lymphadenitis following initiation of protease-inhibitor therapy in patients with advanced hiv-1 disease treatment of mycobacterium avium complex immune reconstitution disease in hiv-1-infected individuals immune restoration disease after the treatment of immunodeficient hiv-infected patients with highly active antiretroviral therapy a randomized, double-blind trial comparing azithromycin and clarithromycin in the treatment of disseminated mycobacterium avium infection in patients with human immunodeficiency virus risk-benefit assessment of therapies for mycobacterium avium complex infections epidemiology and clinical features of cryptosporidium infection in immunocompromised patients intestinal and systemic infection, hiv, and mortality in zambian children with persistent diarrhea and malnutrition enterocytozoon, and cyclospora infections in pediatric and adult patients with diarrhea in tanzania acute enterocolitis in a human being infected with the protozoon cryptosporidium overwhelming watery diarrhea associated with a cryptosporidium in an immunosuppressed patient a preliminary study of the prevalence of intestinal parasites in immunocompromised patients with and without gastrointestinal manifestations diarrhea and gallbladder hydrops in an immunocompetent child with cryptosporidium infection pancreatitis caused by cryptosporidium parvum in patients with severe immunodeficiency related to hiv infection a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply esophageal cryptosporidiosis in a child with acquired immune deficiency syndrome variation in the enteric distribution of cryptosporidia in acquired immunodeficiency syndrome intestinal function and injury in acquired immunodeficiency-related cryptosporidiosis asymptomatic carriage of intestinal cryptosporidium in immunocompetent and immunodeficient children: a prospective study cryptosporidium and isospora belli infection successful management of intractable cryptosporidial disease with intravenous octreotide, a somatostatin analogue eradication of cryptosporidia and microsporidia following successful antiretroviral therapy rapid increase of mucosal cd4 t cells followed by clearance of intestinal cryptosporidiosis in an aids patient receiving highly active antiretroviral therapy resolution of cryptosporidium infection in an aids patient after improvement of nutritional and immune status with octreotide treatment with hyperimmune colostrum of cryptosporidial diarrhea in aids patients cessation of cryptosporidiumassociated diarrhea in an acquired immunodeficiency patient after treatment with hyperimmune bovine colostrum azithromycin therapy for cryptosporidium parvum infection in 4 children infected with hiv azithromycin as treatment for cryptosporidiosis in human immunodeficiency virus disease effect of antiretroviral protease inhibitors alone, and in combination with paromomycin, on the excystation, invasion and in vitro development of cryptosporidium parvum prevention and treatment of cryptosporidiosis in immunocompromised patients microsporidia: opportunistic pathogens in patients with aids occurrence of a new microsporidian: enterocytozoon bieneusi n.g., n. sp., in the enterocytes of a human patient with aids intestinal microsporidiosis as a cause of diarrhea in human immunodeficiency virus infected patients: a report of 20 cases intestinal microsporidiosis in human immunodeficiency virus-infected patients with chronic unexplained diarrhea: prevalence and clinical and biologic features prevalence of microsporidiosis due to enterocytozoon bieneusi and enterocytozoon (septata) intestinalis among patients with aids-related diarrhea: determination of polymerase chain reaction to the microsporidian small-subunit rrna gene prevalence of intestinal microsporidia in hivinfected individual referred for gastroenterological evaluation intestinal microsporidiosis in hiv-infected children with diarrhea enterocytozoon bieneusi among children with diarrhea attending mulago hospital in uganda diagnostic pathology of microsporidiosis potential efficiency of funagillin in intestinal microsporidiosis due to enterocytozoon bieneusi in patients with hiv infection: results of a drug screening study np 470 is an effective antimicrosporidial agent effects of albendazole, fumagillin and tnp-470 on microsporidial replication in vitro atovaquone is effective treatment for the symptoms of gastrointestinal microsporidiosis in human immunodeficiency virus-1 infected patients therapy for human gastrointestinal microsporidiosis effect of antiretroviral therapy on cryptosporidiosis and microsporidiosis in patients infected with human immunodeficiency virus type 1 trimethoprimsulfamethoxazole compared with ciprofloxacin for treatment and prophylaxis of isospora belli and cyclospora cayetanensis infection in hiv-infected patients. a randomized, controlled trial isospora belli infection: treatment with pyrimethamine etiologies of acute, persistent, and dysenteric diarrheas in adults in bangui, central african republic, in relation to human immunodeficiency virus serostatus esophageal candidiasis in pediatric acquired immunodeficiency syndrome: clinical manifestations and risk factors treatment of fluconazole-refractory oropharyngeal candidiasis with itraconazole oral solution in hivpositive patients safety, pharmacokinetics, and pharmacodynamics of cyclodextrin itraconazole in pediatric patients with oropharyngeal candidiasis gastrointestinal histoplasmosis in patients with aids: case report and review disseminated histoplasmosis in the acquired immune deficiency syndrome: clinical findings, diagnosis and treatment, and review of the literature systematic review of the efficacy of antiretroviral therapies for reducing the risk of mother-to-child transmission of hiv infection antiretrovirals for reducing the risk of mother-to-child transmission of hiv infection pediatric aids clinical trials group protocol 219c team. risk factors for opportunistic illnesses in children with human immunodeficiency virus in the era of highly active antiretroviral therapy trends in opportunistic infections in the pre-and post-highly active antiretroviral therapy eras among hiv-infected children in the perinatal aids collaborative transmission study the rate of serious bacterial infections among hiv-infected children with immune reconstitution who have discontinued opportunistic infection prophylaxis medical diagnoses and procedures associated with clostridium difficile colitis epidemiology and outcome of clostridium difficile infection and diarrhea in hiv infected inpatients hiv and diarrhea in the era of haart: 1998 new york state hospitalizations adult/adolescent spectrum of hiv disease study group. bacterial diarrhea in persons with hiv infection prevalence of helicobacter pylori and endoscopic findings in hiv seropositive patients with upper gastrointestinal tract symptoms at kenyatta national hospital helicobacter pylori in indian hiv infected patients helicobacter pylori infection in an urban african population decreased prevalence of helicobacter pylori infection in hiv patients with aids defining diseases remission of a highgrade gastric mucosa associated lymphoid tissue (malt) lymphoma following helicobacter pylori eradication and highly active antiretroviral therapy in a patient with aids dyspepsia in hiv-infected patients under highly active antiretroviral therapy damage to jejunal intrinsic autonomic nerves in hiv infection effect of octreotide on small intestinal motility in hiv-infected patients with chronic refractory diarrhea delayed gastric emptying in hu man immunodeficiency virus infection: correlation with symptoms, autonomic function, and intestinal motility esophageal motility disorders in hiv patients disturbed gastric motor activity in patients with human immunodeficiency virus infection idiopathic giant esophageal ulcer in an hiv-positive child chronic idiopathic esophageal ulceration in the acquired immunodeficiency syndrome, characterization and treatment with corticosteroids esophageal ulceration in human immunodeficency virus infection use of thalidomide in treatment and maintenance of idiopathic esophageal ulcers in hiv+ individuals thalidomide for the treatment of esophageal aphthous ulcers in patients with human immunodeficiency virus infection. national institute of allergy and infectious disease aids clinical trials group thalidomide in low intermittent doses does not prevent recurrence of human immunodeficiency virus-associated aphthous ulcers natural history of hiv-associated esophageal disease in the era of protease inhibitor therapy plasma citrulline is a biomarker of enterocyte mass and an indicator of parenteral nutrition in hiv-infected patients symptom-specific use of upper gastrointestinal endoscopy in human immunodeficiency virus-infected patients yields high dividends enteric infections and diarrhea in human immunodeficiency virus-infected persons: prospective community-based cohort study. swiss hiv cohort study a prospective study of endoscopy in hiv-associated diarrhea special histologic stains are rarely beneficial for the evaluation of hiv-related gastrointestinal infections predictors of bone mineral density in human immunodeficiency virus-1 infected children decreased bone mineral density with off-label use of tenofovir in children and adolescents infected with human immunodeficiency virus alterations in circulating osteoimmune factors may be responsible for high bone resorption rate in hivinfected children and adolescents gastrostomy tube supplementation for hiv-infected children effect of enteral tube feeding on growth of children with symptomatic human immunodeficiency virus infection immunocompetence of nutritional disorders epidemiologic aspects of the current outbreak of kaposi' s sarcoma and opportunistic infection nutrition, immunity and infection: mechanisms of interactions interactions of nutrition and infection protein-calorie malnutrition: a host determinant for pneumocystis carinii infection nutrition and gastrointestinal disease the gut in malnutrition the enterocyte in celiac disease nutrition and hiv/aids in infants and children in south africa: implications for food-based dietary guidelines micronutrient supplementation in children and adults with hiv infection safety and efficacy of zinc supplementation for children with hiv-1 infection in south africa: a randomised double-blind placebo-controlled trial zinc fights diarrhoea in hiv-1-infected children: in-vitro evidence to link clinical data and pathophysiological mechanism studies of vitamins and minerals and hiv transmission and disease progression multivitamin supplementation improves hematologic status in hiv-infected women and their children in tanzania immunocompromised hosts: perspectives in the treatment and prophylaxis of cytomegalovirus disease in solidorgan transplant recipients current management strategies for the prevention and treatment of cytomegalovirus infection in pediatric transplant recipients ganciclovir: an update of its use in the prevention of cytomegalovirus infection and disease in transplant recipients safety and efficacy of prolonged cytomegalovirus prophylaxis with intravenous ganciclovir in pediatric and young adult lung transplant recipients hepatitis b in the hiv-coinfected patient hepatitis b or hepatitis c and human immunodeficiency virus infection influence of hiv infection on the response to interferon therapy and the long-term outcome of chronic hepatitis b effect of duration of hepatitis b virus infection on the association between human immunodeficiency virus type-1 and hepatitis b viral replication hepatitis b virus infection in the acquired immunodeficiency syndrome adolescent human immunodeficiency virus infection and gastrointestinal disease effect of hiv co-infection on mutation patterns of hbv in patients with lamivudine-resistant chronic hepatitis hepatitis b in hiv patients: what is the current treatment and what are the challenges? diagnosis and management of hepatitis b virus and hiv coinfection hepatitis c in patients with human immunodeficiency virus infection: diagnosis, natural history, meta-analysis of sexual and vertical transmission, and therapeutic issues maternal-infant transmission of hepatitis c virus and hiv infections: a possible interaction coinfection with hiv-1 and hcv -a one-two punch hiv and hepatitis c coinfection protection against persistence of hepatitis c the natural history of hepatitis c virus infection: host, viral, and environmental factors ribavirin in the treatment of chronic hepatitis c peginterferon-α-2a (40kd) plus ribavirin: a review of its use in hepatitis c virus and hiv co-infection histological response to pegifnα-2a (40kd) plus ribavirin in hiv-hepatitis c virus co-infection hepatobiliary abnormalities on sonography in children with hiv infection hiv and chronic hepatitis: co-infection with hiv and hepatitis b virus infection disease-and treatment-related predictors of hepatic mitochondrial dysfunction in chronic hiv infection assessed by non-invasive 13 c-methionine breath test diagnostic alteration of cytochrome oxidase subunit i labeling is associated with severe mitochondropathy in nrti-related hepatotoxicity in hiv patients key: cord-254951-anfkuigj authors: pierre, gashema; uwineza, annette; dzinamarira, tafadzwa title: attendance to hiv antiretroviral collection clinic appointments during covid-19 lockdown. a single center study in kigali, rwanda date: 2020-06-25 journal: aids behav doi: 10.1007/s10461-020-02956-5 sha: doc_id: 254951 cord_uid: anfkuigj nan globally, 78 million people have been infected with hiv and 35 million have died from aids-related illnesses since its inception 39 years ago [1] . in sub-saharan africa an estimated 25.7 million people were living with hiv (plwh), of whom 16.4 million were taking antiretroviral therapy (art) in the year 2018 [1] . in the same year, an estimated 470,000 people died of aids-related illnesses in sub-saharan africa [1] . in rwanda, the ministry of health (moh) led response has contributed to a stable national hiv prevalence of around 3% in the last decade [2] . remarkable progress has been reported in art coverage in rwanda with near universal coverage reported in recent surveys [3, 4] . the pandemic has over 7 million cases with over 400,000 deaths as of 11 june 2020 [5, 6] . an important strategy to curb transmission of the virus has been introduction of national lockdowns. in rwanda, a national lockdown was affected on 21 march 2020 [7] ; becoming the first in africa. the lockdown banned unnecessary movements and visits outside the home and travel between different cities or districts [7] . further, motorcycles, the most common form of transportation for the general population, were banned from ferrying passengers [7] . according to the joint united nations programme on hiv and aids (unaids) and the world health organization (who), a 6-month disruption of antiretroviral therapy (art) during the covid-19 pandemic in sub-saharan africa will increase hiv-related death rate by more than half a million [8] . this is corroborated by five well-described models of hiv epidemics which estimated that a 6-month interruption of supply of art across the whole population of people living with hiv (plwh) on treatment would be expected to lead to an approximately twofold (from 1.87-to 2.80-fold across models) increase in hiv-related deaths over a 1 year period compared to the case with no disruption [9] . given the recent total lockdown in rwanda, this note presents findings from a study aimed to assess attendance to art collection clinic appointments in the period 21 march to 30 april 2020 in kigali, rwanda. we employed a cross sectional study design. the study was conducted at the university teaching hospital of kigali (chuk), art clinic (irb approval: ec/chuk/036/2020). chuk is the largest referral hospital in rwanda. the art clinic provides care and treatment services to approximately 2100 clients. records from all clients with a scheduled follow-up appointment for art collection in the period 21 march to 30 april 2020 were included. in this study, 382 records met the criteria and were available for review. three source records were reviewed for this study namely pharmacy register, client file and linkage register. for each of these sources; clients records are linked by a unique identifier called a tracnet id. the data abstraction form collected information on tracnet id, client demographics (sex; age; residence), who clinical stage, date and result of last viral load test, current regimen, last date for art collection and next date of appointment. two trained health professionals abstracted the relevant data from the source records and performed quality control for accuracy during the period 21-30 may 2020. we used the chi-square test to determine association between categorical dependent and categorical independent variables. a total of 382 participants' data was included in the analysis. 53% were female, 23% were in the age range of 15-24 years, while 27%, 20% and 29% were in the age range 25-34 years, 35-44 years and 45 years or above, respectively. majority (88%) resided in kigali. majority (91%) achieved viral load suppression (vls) (defined as a viral load result of less than 1000 copies/ml) based on last recorded viral load result. less than half (48%) had attended scheduled art collection clinic appointments during the lockdown period of 21 march to 30 april 2020. there was an association between place of residence and attendance status (p = 0.040), 50% staying within kigali attended scheduled art collection clinic appointments during the lockdown period compared to 35% among those living outside kigali. there was an association between who clinical stage and attendance status (p = 0.019), 48% in who clinical stage 1 attended scheduled art collection clinic appointments during the lockdown period compared to 56% and 22% among those in who clinical stage 3 and 4, respectively. there was no association between attendance status with gender (p = 0.159), age (p = 0.660), current drug regimen (p = 0.580), and vls (p = 0.457). additional information about factors associated with attendance status are available in table 1 . it is well-established that without the ability to maintain consistent hiv therapy, some patients face rapid treatment failure. this calls for hiv programs to have appropriate contingency plans wherever crises that hinder plwh from accessing care services may occur [10, 11] . on world aids day in 2016, rwanda launched the national differentiated service delivery model (dsdm) for hiv/aids treatment and service provision; a strategy which includes provision for reducing the frequency of routine healthcare visits for stable, low-risk hiv patients [12] . under the dsdm, stable patients move from monthly to quarterly visits for prescription drug pick-up and from quarterly to biannual clinic visits. this has been working well with ensuring health providers focus care and treatment efforts on higher-risk patients and new patients while stable patients spend less time and money traveling to healthcare facilities, since visits are spaced further apart. while this may be the case, there is need for advance planning to develop programs that can function during a crisis such as a nation-wide lockdown that limits people movement. the lockdown has accentuated the need for a rapid programmatic response to ensure consistent provision of care and treatment services to plwh. further, there is a need to strengthen effective patient tracking for individuals who miss appointments especially during a national crisis. this study has a number of limitations. being a singlecenter study, findings may not be generalized to map the national context. secondly, the factors reported in this study do not show causality as there are prone to a number of confounders. however, findings provide crucial information to warrant further formative research that informs sustainable strategies to ensure continuation of care and treatment services for plwh in similar crisis. factsheet: global aids update viral suppression in a nationwide sample of hiv-infected children on antiretroviral therapy in rwanda world health organization website covid-19) situation dashboard office of the prime minister: announcement on enhanced covid-19 prevention measures sub-saharan africa aids deaths could double due to covid-19 2020 potential effects of disruption to hiv programmes in sub-saharan africa caused by covid-19: results from multiple mathematical models 2020 the consequences of post-election violence on antiretroviral hiv therapy in kenya delivering hiv care in challenging operating environments: the msf experience towards differentiated models of care for settings with multiple basic health care needs team rwanda is one of the first countries to launch the differentiated hiv care model nationally key: cord-048351-4y8ghcpq authors: murdoch, david m; venter, willem df; van rie, annelies; feldman, charles title: immune reconstitution inflammatory syndrome (iris): review of common infectious manifestations and treatment options date: 2007-05-08 journal: aids res ther doi: 10.1186/1742-6405-4-9 sha: doc_id: 48351 cord_uid: 4y8ghcpq the immune reconstitution inflammatory syndrome (iris) in hiv-infected patients initiating antiretroviral therapy (art) results from restored immunity to specific infectious or non-infectious antigens. a paradoxical clinical worsening of a known condition or the appearance of a new condition after initiating therapy characterizes the syndrome. potential mechanisms for the syndrome include a partial recovery of the immune system or exuberant host immunological responses to antigenic stimuli. the overall incidence of iris is unknown, but is dependent on the population studied and its underlying opportunistic infectious burden. the infectious pathogens most frequently implicated in the syndrome are mycobacteria, varicella zoster, herpesviruses, and cytomegalovirus (cmv). no single treatment option exists and depends on the underlying infectious agent and its clinical presentation. prospective cohort studies addressing the optimal screening and treatment of opportunistic infections in patients eligible for art are currently being conducted. these studies will provide evidence for the development of treatment guidelines in order to reduce the burden of iris. we review the available literature on the pathogenesis and epidemiology of iris, and present treatment options for the more common infectious manifestations of this diverse syndrome and for manifestations associated with a high morbidity. since its introduction, art has led to significant declines in aids-associated morbidity and mortality [1] . these benefits are, in part, a result of partial recovery of the immune system, manifested by increases in cd4 + t-lymphocyte counts and decreases in plasma hiv-1 viral loads [2] . after initiation of art, opportunistic infections (oi) and other hiv-related events still occur secondary to a delayed recovery of adequate immunity [3] . some patients initiating art experience unique symptoms during immune system recovery. in these patients, clinical deterioration occurs despite increased cd4 + tlymphocyte counts and decreased plasma hiv-1 viral loads [4] . this clinical deterioration is a result of an inflammatory response or "dysregulation" of the immune system to both intact subclinical pathogens and residual antigens [5] [6] [7] [8] [9] . resulting clinical manifestations of this syndrome are diverse and depend on the infectious or noninfectious agent involved. these manifestations include mycobacterial-induced lymphadenitis [5] , paradoxical tuberculosis reactions [6, 7, 10, 11] , worsening of progressive multifocal leukoencephalopathy (pml) [12] , recurrence of cryptococcosis and pneumocystis jirovecii pneumonia (pcp) [8, [13] [14] [15] [16] , cytomegalovirus (cmv) retinitis [17] , shingles [18] , and viral hepatitis [19] , as well as noninfectious phenomena [20] . because clinical deterioration occurs during immune recovery, this phenomenon has been described as immune restoration disease (ird), immune reconstitution syndrome (irs), and paradoxical reactions. given the role of the host inflammatory response in this syndrome, the term immune reconstitution inflammatory syndrome (iris) has been proposed [21] and has become the most widely used and accepted term to describe the clinical entity. possible infectious and noninfectious etiologies of iris are summarized in table 1 . to date, no prospective therapeutic trials concerning the management of iris have been conducted. all evidence regarding the management of iris in the literature relates to case reports and small case series reporting on management practice. this does not provide reliable evidence regarding either the safety or efficacy of these approaches, but merely guidance regarding the practice of others in managing this difficult condition. in severe cases where the discontinuation of art is a possibility, the potential disadvantages of therapy cessation, such as the development of viral resistance or aids progression, should be considered. despite numerous descriptions of the manifestations of iris, its pathogenesis remains largely speculative. current theories concerning the pathogenesis of the syndrome involve a combination of underlying antigenic burden, the degree of immune restoration following haart, and host genetic susceptibility. these pathogenic mechanisms may interact and likely depend on the underlying burden of infectious or noninfectious agent. whether elicited by an infectious or noninfectious agent, the presence of an antigenic stimulus for development of the syndrome appears necessary. this antigenic stimulus can be intact, "clinically silent" organisms or dead or dying organisms and their residual antigens. iris that occurs as a result of "unmasking" of clinically silent infection is characterized by atypical exuberant inflammation and/or an accelerated clinical presentation suggesting a restoration of antigen-specific immunity. these characteristics differentiate iris from incident opportunistic infections that occur on art as a result of delayed adequate immunity. examples of iris in response to intact organisms include, but are not limited to, the unmasking of latent cryptococcal infection [22] and infection with mycobacterium avium complex (mac) [4, 5, 23, 24] . the most frequently reported iris symptoms in response to previously treated or partially treated infections include reports of clinical worsening and recurrence of clinical manifestations of mycobacterium tuberculosis (tb) and cryptococcal meningitis following initiation of art [6, 7, 10, 13, 16, [25] [26] [27] [28] . in noninfectious causes of iris, autoimmunity to innate antigens plays a likely role in the syndrome. examples include exacerbation of rheumatoid arthritis and other autoimmune diseases [29] . given the role of this antigenic stimulus, the frequency and manifestations of iris in a given population may be determined by the prevalence of opportunistic and non-opportunistic infections to initiation of art. the mechanism receiving the most attention involves the theory that the syndrome is precipitated by the degree of immune restoration following art. in assessing this theory, investigators have examined the association between cd4 cell counts and viral loads and the risk of iris. some studies suggest differences in the baseline cd4 profiles or quantitative viral load at art initiation or their rate of change during haart between iris and non-iris patients [4, [30] [31] [32] [33] [34] , while other studies demonstrate only trends or no significant difference between iris and non-iris patients [7, 35] . these immunological differences between groups have been difficult to verify due to small numbers of iris cases and lack of control groups. an alternative immunological mechanism may involve qualitative changes in lymphocyte function or lymphocyte phenotypic expression. for instance, following art an increase in memory cd4 cell types is observed [36] possibly as a result of redistribution from peripheral lymphoid tissue [37] . this cd4 phenotype is primed to recognize previous antigenic stimuli, and thus may be responsible for manifestations of iris seen soon after art initiation. after this redistribution, naïve t cells increase and are thought to be responsible for the later quantitative increase in cd4 cell counts [38] . these data suggest iris may be due to a combination of both quantitative restoration of immunity as well as qualitative function and phenotypic expression observed soon after the initiation of art. the third purported pathogenic mechanism for iris involves host genetic susceptibility to an exuberant immune response to the infectious or noninfectious anti-genic stimulus upon immune restoration. although evidence is limited, carriage of specific hla alleles suggest associations with the development of iris and specific pathogens [39] . increased levels of interleukin-6 (il-6) in iris patients may explain the exuberant th1 response to mycobacterial antigens in subjects with clinical iris [9, 40] . such genetic predispositions may partially explain why manifestations of iris differ in patients with similar antigenic burden and immunological responses to art. despite numerous descriptions of the infectious and noninfectious causes of iris, the overall incidence of the syndrome itself remains largely unknown. studies to date are often retrospective and focus on specific manifestations of iris, such as tuberculosis-associated iris (tb-iris). in a large retrospective analysis examining all forms of iris, 33/132 (25%) of patients exhibited one or more disease episodes after initiation of art [4] . other cohort analyses examining all manifestations of iris estimate that 17-23% of patients initiating art will develop the syndrome [32] [33] [34] . another large retrospective study reported 32% of patients with m. tuberculosis, m. avium complex, or cryptococcus neoformans coinfection developed iris after initiating art. risk factors identified for the development of iris in one cohort included male sex, a shorter interval between initiating treatment for oi and starting art, a rapid fall in hiv-1 rna after art, and being art-naïve at the time of oi diagnosis [31] . other significant predictors have also included younger age, a lower baseline cd4 cell percentage, a lower cd4 cell count at art initiation, and a lower cd4 to cd8 cell ratio at baseline [4, 32] . it should be noted cohorts differ substantially in study populations and the type of iris (i.e. tb-iris only) examined, making conclusions regarding risk factors for iris difficult. clinical factors associated with the development of iris are presented in table 2 . case reports describing different clinical manifestations of iris continue to appear, expanding the clinical spectrum of the syndrome. because the definition of iris is one of clinical suspicion and disease-specific criteria have yet to be developed, determining the true incidence will be difficult. taken together, these studies suggest iris may affect a substantial proportion of hiv patients initiating art. future epidemiologic and genetic studies conducted within diverse cohorts will be important in determining the importance of host susceptibility and underlying opportunistic infections on the risk of developing iris. in order to aid clinicians in the management of iris, we review the epidemiology, clinical features, and treatment options for the common infectious manifestations of iris. additionally, manifestations associated with significant morbidity and mortality, such as cmv-associated immune recovery vitritis (irv) or immune recovery uveitis (iru), are also reviewed. treatment options and their evidence are presented. until disease specific guidelines are developed for iris, therapy should be based on exist[4, 6, 7, 10, 11, 26, 30-32, 41, 43, 45] rheumatoid arthritis [29] systemic lupus erythematosus (sle) [91] graves disease [92] , autoimmune thyroid disease [93] mycobacterium avium complex [4, 5, 23, 31, [94] [95] [96] sarcoidosis & granulomatous reactions [20, 97] other mycobacteria [4, 56, 57, 98, 99] tattoo ink [100] cytomegalovirus [4, 33, 61, 63] aids-related lymphoma [ [112] molluscum contagiosum & genital warts [32] sinusitis [113] folliculitis [114, 115] ing evidence and individualized according to the severity of presentation. mycobacterium tuberculosis (tb) is among the most frequently reported pathogen associated with iris. narita et al performed the first prospective study to evaluate the incidence of paradoxical responses in patients on tb therapy and subsequently initiated on art. of 33 hiv/tb coinfected patients undergoing dual therapy, 12 (36%) developed paradoxical symptoms [7] . the frequency of symptoms in this group were greater than those observed in hiv-infected controls receiving tb therapy alone, supporting the role of an exaggerated immune system response in the pathogenesis of the syndrome. retrospective studies corroborate the finding that a significant proportion of hiv/tb coinfected patients undergoing haart have symptoms consistent with iris, with estimates ranging from 7-45% [10,26, 30, 35, [41] [42] [43] . the association between a shorter delay between tb treatment initiation and art initiation is an area of debate. while some investigators have found no difference in time from tb therapy to initiation of art between iris and non-iris subjects [30] , others have reported a significant differences between groups [31, 35] . in general, iris occurred in subjects initiated on art within two months of tb therapy initiation [35] . based on these and other data, a decision analysis on art initiation timing in tb patients found the highest rates of iris occurred in patients initiated on art within two months of tb therapy initiation [44] . however, withholding or deferring art until two to six months of tb therapy was associated with higher mortality in scenarios where iris-related mortality was less than 4.6%. future reports from large, prospective observational cohorts may aid in resolving this difficult issue. although consisting primarily of case reports [45, 46] , tb-iris affecting the central nervous system (cns) poses a unique problem. as the availability of art increases in endemic countries, the incidence of cns tb-iris may increase. thus, clinicians should be vigilant in its diagnosis. the commonest clinical manifestations of tb-iris are fever, lymphadenopathy and worsening respiratory symptoms [47] . pulmonary disorders, such as new pulmonary infiltrates, mediastinal lymphadenopathy, and pleural effusions are also common [7] . extrapulmonary presentations are also possible, including disseminated tuberculosis with associated acute renal failure [6] , systemic inflammatory responses (sirs) [48] , and intracranial tuberculomas [45] . pulmonary tb-iris can be diagnosed by transient worsening of chest radiographs, especially if old radiographs are available for comparison. other symptoms are nonspecific, and include persistent fever, weight loss, and worsening respiratory symptoms. abdominal tb-iris can present with nonspecific abdominal pain and obstructive jaundice. in most studies, tb-iris occurs within two months of art initiation [6, 7, 10, 11, 25, 35, 45, 48] . among 43 cases of mtb-associated iris, the median onset of iris was 12-15 days (range 2-114 days), with only four of these cases occurring more than four weeks after the initiation of antiretroviral therapy [7, 10, 25, 26, 30] . these studies suggest the onset of mycobacterial-associated iris is relatively soon after initiation of art, and clinicians should maintain a high level of vigilance during this period. paradoxical cns tb reactions are well described in hivnegative patients, and include expanding intracranial tuberculomas, tuberculous meningitis, and spinal cord lesions [49] [50] [51] . tb-associated cns iris has also been reported in hiv-positive patients [45, 46, 52] . compared to non-cns tb-iris, symptoms tend to occur later, usually 5-10 months after art initiation [45, 50, 52] . crump et al [45] described an hiv-seropositive patient in who developed cervical lymphadenopathy after five weeks of art. five months later, cns symptoms associated with an expanding intracranial tuberculoma appeared after initiation of antituberculous therapy. the significant morbidity in this case illustrates the importance of maintaining a high clinical suspicion for the disease, particularly in endemic areas. treatment for mycobacterial-associated iris depends on the presentation and disease severity. most patients present with non-life threatening presentations which respond to the institution of appropriate antituberculous therapy. however a range of life threatening presentations, such as acute renal failure [6] and acute respiratory distress syndrome (ards) [11] , are described and have significant morbidity and mortality. morbidity and mortality might also be greater in resource-limited settings where limited management options exist. since the pathogenesis of the syndrome is an inflammatory one, systemic corticosteroids or nonsteroidal anti-inflammatory drugs (nsaids) may alleviate symptoms. in studies where therapy for iris was mentioned, the use of corticosteroids was variable [7, 24, 25, 31, 41, 43] and anecdotally effective. therapies ranged from intravenous methylprednisolone 40 mg every 12 hours to prednisone 20-70 mg/day for 5-12 weeks. these practices reflect the lack of evidence from controlled trials for the use of anti-inflammatory agents in iris. a randomized, placebo controlled trial examining doses of prednisone 1.5 mg/kg/day for two weeks followed by 0.75 mg/kg/day for two weeks in mild to moderate tb-iris is currently underway in south africa. until data become available, it is reasonable to administer corticosteroids for severe cases of iris such as tracheal compression due to lymphadenopathy, refractory or debilitating lymphadenitis, or severe respiratory symptoms, such as stridor and ards. interruption of art is rarely necessary but could be considered in life-threatening situations. in hiv-negative patients, adjuvant corticosteroid use in tuberculous meningitis provides evidence of improved survival and decreased neurologic sequelae over standard therapy alone [53, 54] . once other infectious etiologies, have been excluded, standard antituberculous therapy should be initiated or continued as the clinical situation dictates, and a course of corticosteroid therapy should be considered for cns tb-iris. continuation of art is desirable, although its discontinuation may be necessary in unresponsive cases or in those presenting with advanced neurological symptoms. in addition to tb, atypical mycobacteria are also frequently reported as causative pathogens in iris. early observations involving atypical presentations of mycobacterium avium-intracellulare (mac) were first noted with zidovudine therapy [55] . reports of atypical presentations of both mycobacterium tuberculosis (mtb) and mac increased in frequency with the introduction of protease inhibitors and art. in larger cohorts, mac remains the most frequently reported atypical mycobacterium [4, 5, 24] . other atypical mycobacteria rarely associated with iris are referenced in table 1 . in general, mac-associated iris typically presents with lymphadenitis, with or without abscess formation and suppuration [5] . other less common presentations include respiratory failure secondary to acute respiratory distress syndrome (ards) [56] , leprosy [57] , pyomyositis with cutaneous abscesses [23], intra-abdominal disease [58] , and involvement of joints, skin, soft tissues, and spine [58, 59] . several studies have characterized the time of onset of mycobacterium-associated iris. in one study of mac lymphadenitis, the onset of a febrile illness was the first sign of iris and occurred between 6 and 20 days after initiation of antiretroviral therapy [5] . in another study, the median time interval from the start of antiretroviral therapy to the development of mycobacterial lymphadenitis was 17 days (range 7-85 days) [24]. as with tb-iris, evidence for treatment of iris due to atypical mycobacteria are scarce. occasionally, surgical excision of profoundly enlarged nodes or debridement of necrotic areas is anecdotally reported [23, 59] . however, healing is often poor leaving large, persistent sinuses. needle aspiration is another option for enlarged, fluctuant and symptomatic nodes. otherwise, treatment is similar to tb-iris (see mycobacterium tuberculosis iris -treatment). in the pre-art era, cmv retinitis, a vision-threatening disease, carried a high annual incidence and was one of the most significant aids-associated morbidities [60] . after the introduction of haart, jacobson et al described five patients diagnosed with cmv retinitis 4-7 weeks after art initiation. they speculated that an haart-induced inflammatory response may be responsible for unmasking a subclinical infection [17] . in addition to classical cmv retinitis, art led to new clinical manifestations of the infection, termed immune recovery vitritis (irv) or immune recovery uveitis (iru), in patients previously diagnosed with inactive aids-related cmv retinitis [61] . distinct from the minimal intraocular inflammation of classic cmv retinitis, these manifestations exhibit significant posterior segment ocular inflammation thought to be due to the presence of residual cmv antigens or proteins which serve as the antigenic stimulus for the syndrome [62] . clinical manifestations include vision impairment and floaters. in a retrospective cohort, cmv-related iris was common (6/33 of iris cases, or 18%) [4] . in prospective cohorts, symptomatic vitritis occurred in 63% (incidence rate 83 per 100 p-yr) of art responders who carried a previous diagnosis of cmv retinitis but had inactive disease at the onset of antiretroviral therapy. the median time from art initiation to irv was (43 weeks) [63] . another large prospective surveillance study [64] identified 374 patients with a history of cmv retinitis involving 539 eyes. thirtyone of 176 art responders (17.6%) were diagnosed with iru. male gender, use of art, higher cd4 cell counts, and involvement of the posterior retinal pole as factors associated with a reduced risk of developing iru, whereas prior use of intravitreous injections of cidofovir, large retinal lesions, and adequate immune recovery on art were associated with increased risk. the diagnosis of ocular manifestations of iris requires a high level of suspicion. in addition to signs of retinitis, inflammatory symptoms include vitritis, papillitis, and macular edema, resulting in symptoms of loss of visual acuity and floaters in affected eyes. treatment of iris associated cmv retinitis and irv may involve anti-cmv therapy with gancyclovir or valgancyclovir [17, 65] . however, the occurrence of iru in patients receiving anti-cmv therapy draws its use into question [64, 66, 67] . the use of systemic corticosteroids has been successful, and irv may require periocular corticosteroid injections [61, [68] [69] [70] . due to its significant morbidity and varying temporal presentations, clinicians should maintain a high level of vigilance for ocular manifestations of cmv-associated iris. with the introduction of protease inhibitors, increasing rates of herpes zoster were noted in hiv-infected patients. two studies comparing art and non-art patients reported increased incident cases of zoster and rates estimated at 6.2-9.0 cases per 100 person-years, three to five times higher than rates observed in the pre-haart era [18, 71] . while another study [72] reported no difference in overall incidence between haart eras (3.2 cases per 100 person-years), the use of haart was associated with increased odds of developing an incident zoster outbreak (or = 2.19, 95% confidence interval: 1.49 to 3.20). these studies suggest that art may play a role in increasing the risk of zoster, which is reflected in large observational iris cohorts, where dermatomal varicella zoster comprises 9-40% of iris cases [4, 32, 33] . mean onset of disease from art initiation was 5 weeks (range 1-17 weeks) [71] , and no cases occurred before 4 weeks of therapy [18] . both studies identified significant increases in cd8 t cells as a risk factor for developing dermatomal zoster. although complications such as encephalitis, myelitis, cranial and peripheral nerve palsies, and acute retinal necrosis can occur in immunocompromised hiv patients, the vast majority of patients exhibit typical or atypical dermatomal involvement without dissemination or systemic symptoms [18, 71, 73] . a randomized, controlled trial demonstrated oral acyclovir to be effective for dermatomal zoster in hiv-infected patients, facilitating healing and shortening the time of zoster-associated pain [74] . its use in cases of varicella zoster iris appears to be of clinical benefit [18] . the benefit of corticosteroids in combination with acyclovir in acute varicella zoster has been demonstrated in two large randomized, controlled trials. the combination of corticosteroids and acyclovir decreased healing times, improved acute pain, and quality of life, but did not affect the incidence or duration of postherpetic neuralgia [75, 76] . the incidence of postherpetic neuralgia in immunocompetent individuals does not differ significantly from hiv-infected patients, but increases with increasing patient age [77] . successful symptomatic management involving opioids, tricyclic antidepressants, gabapentin, and topical lidocaine patches individually or in combination has been shown to be beneficial [78] [79] [80] [81] [82] and should be attempted in hiv patients with postherpetic neuralgia as a complication of herpes zoster iris. accurate incidence of c. neoformans-associated iris is unknown. it is infrequently reported in overall iris cohorts, and many cases appear as single case reports. a recent study [90] evaluated antifungal combination therapies in the treatment of c. neoformans meningitis in hiv patients. although significant log reductions in colony forming units were observed with all combinations, substantial numbers of patients remained culture positive 2 weeks after therapy. it may be important to delay art until csf sterility can be achieved with effective antifungal combinations such as amphotericin b and flucytosine. however, the exact timing of art and whether attaining csf culture sterility is important in avoiding iris is unknown. this is illustrated by cases of reactivation cryptococcal meningitis described in four patients who had received at least four weeks of antifungal therapy prior to art [13, 22, 83] . it is reasonable to administer systemic corticosteroids to alleviate unresponsive inflammatory effects, as anecdotal benefits have been observed in these patients [21, 84] . furthermore, serial lumbar punctures may be required to manage persistent csf pressure elevations in these patients [85, 86] . although continuation of art has been performed safely [13, 84] , interruption of antiviral therapy may be necessary in severe or unresponsive cases. other less common infectious etiologies, as well as noninfectious etiologies, are listed in table 1 . because these other infectious and non-infectious etiologies are rare, no recommendations exist for their management. while exact estimates of incidence are not yet available, iris in patients initiating art has been firmly established as a significant problem in both high and low income countries. because of wide variation in clinical presentation and the still increasing spectrum of symptoms and etiologies reported, diagnosis remains problematic. fur-thermore, no test is currently available to establish an iris diagnosis. standardized disease-specific clinical criteria for common infectious manifestations of the disease should be developed to: 1) identify risk factors for developing the syndrome and 2) optimize the prevention, management of opportunistic infections. results of trials addressing the optimal timing and duration of treatment of opportunistic infections will assist in developing guidelines for the prevention and management of iris. treatment of iris will remain a clinical challenge due to the variety of clinical presentations and the presence of multiple pathogens capable of causing the syndrome. until a greater understanding of the syndrome is achieved in different regions of the world, clinicians need to remain vigilant when initiating art and individualize therapy according to known treatment options for the specific infectious agent. declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. hiv outpatient study investigators immune reconstitution in hiv infection aids-related opportunistic illnesses occurring after initiation of potent antiretroviral therapy: the swiss hiv cohort study immune restoration disease after the treatment of immunodeficient hiv-infected patients with highly active antiretroviral therapy focal mycobacterial lymphadenitis following initiation of protease-inhibitor therapy in patients with advanced hiv-1 disease acute renal failure on immune reconstitution in an hiv-positive patient with miliary tuberculosis paradoxical worsening of tuberculosis following antiretroviral therapy in patients with aids immune reconstitution syndrome after successful treatment of pneumocystis carinii pneumonia in a man with human immunodeficiency virus type 1 infection levels of il-6 and soluble il-6 receptor are increased in hiv patients with a case of immune reconstitution rheumatoid arthritis determinants of immune reconstitution inflammatory syndrome in hiv type 1-infected patients with tuberculosis after initiation of antiretroviral therapy incidence and risk factors for immune reconstitution inflammatory syndrome during highly active antiretroviral therapy incidence and risk factors for immune reconstitution inflammatory syndrome in an ethnically diverse hiv type 1-infected cohort djurkovic-djakovic o: the prevalence and risk of immune restoration disease in hiv-infected patients treated with highly active antiretroviral therapy immune reconstitution syndrome after highly active antiretroviral therapy in human immunodeficiency virus-infected thai children paradoxical reactions of tuberculosis in patients with the acquired immunodeficiency syndrome who are treated with highly active antiretroviral therapy positive effects of combined antiretroviral therapy on cd4+ t cell homeostasis and function in advanced hiv disease initial increase in blood cd4(+) lymphocytes after hiv antiretroviral therapy reflects redistribution from lymphoid tissues biphasic kinetics of peripheral blood t cells after triple combination therapy in hiv-1 infection: a composite of redistribution and proliferation immune dysfunction and immune restoration disease in hiv patients given highly active antiretroviral therapy explosion of tuberculin-specific th1-responses induces immune restoration syndrome in tuberculosis and hiv co-infected patients incidence of immune reconstitution syndrome in hiv/tuberculosis-coinfected patients after initiation of generic antiretroviral therapy in india paradoxical response to antituberculous therapy in immunocompetent patients and hiv co-infected patients clinical characteristics of iris syndrome in patients with hiv and tuberculosis timing of antiretroviral therapy initiation in tuberculosis patients with aids: a decision analysis military tuberculosis with paradoxical expansion of intracranial tuberculomas complicating human immunodeficiency virus infection in a patient receiving highly active antiretroviral therapy paradoxical reaction during treatment of tuberculous brain abscess in a patient with immune reconstitution disease associated with mycobacterial infections in hiv-infected individuals receiving antiretrovirals systemic inflammatory reaction after starting highly active antiretroviral therapy in aids patients treated for extrapulmonary tuberculosis symptomatic intracranial tuberculoma developing during treatment of tuberculosis: a report of 10 patients and review of the literature clinical spectrum of paradoxical deterioration during antituberculosis therapy in non-hiv multiple intracranial tuberculomas with atypical response to tuberculostatic chemotherapy: literature review and a case report paradoxical presentation of intracranial tuberculomas after chemotherapy in a patient with aids adjunctive corticosteroid therapy for tuberculosis: a critical reappraisal of the literature dexamethasone for the treatment of tuberculous meningitis in adolescents and adults zidovudine-induced restoration of cell-mediated immunity to mycobacteria in immunodeficient hiv-infected patients acute respiratory failure due to mycobacterium kansasii infection: immune reconstitution disease in a patient with aids borderline tuberculoid leprosy: an immune reconstitution phenomenon in a human immunodeficiency virus-infected person nontuberculous mycobacterial immune reconstitution syndrome in hiv-infected patients: spectrum of disease and long-term follow-up localized osteomyelitis due to mycobacterium avium complex in patients with human immunodeficiency virus receiving highly active antiretroviral therapy serious cytomegalovirus disease in the acquired immunodeficiency syndrome (aids) immune recovery vitritis associated with inactive cytomegalovirus retinitis: a new syndrome intraocular viral and immune pathogenesis of immune recovery uveitis in patients with healed cytomegalovirus retinitis incidence of immune recovery vitritis in cytomegalovirus retinitis patients following institution of successful highly active antiretroviral therapy risk of immune recovery uveitis in patients with aids and cytomegalovirus retinitis valganciclovir therapy for immune recovery uveitis complicated by macular edema effect of anti-cytomegalovirus therapy on the incidence of immune recovery uveitis in aids patients with healed cytomegalovirus retinitis the safety of discontinuation of maintenance therapy for cytomegalovirus (cmv) retinitis and incidence of immune recovery uveitis following potent antiretroviral therapy immune recovery uveitis in aids patients with cytomegalovirus retinitis treated with highly active antiretroviral therapy in venezuela treatment of immune recovery vitritis with local steroids immune recovery vitritis and uveitis in aids: clinical predictors, sequelae, and treatment outcomes herpes zoster as an immune reconstitution disease after initiation of combination antiretroviral therapy in patients with human immunodeficiency virus type-1 infection the incidence of, risk factors for, and sequelae of herpes zoster among hiv patients in the highly active antiretroviral therapy era clinical spectrum of herpes zoster in adults infected with human immunodeficiency virus sorivudine versus acyclovir for treatment of dermatomal herpes zoster in human immunodeficiency virus-infected patients: results from a randomized, controlled clinical trial acyclovir with and without prednisone for the treatment of herpes zoster. a randomized, placebo-controlled trial. the national institute of allergy and infectious diseases collaborative antiviral study group a randomized trial of acyclovir for 7 days or 21 days with and without prednisolone for treatment of acute herpes zoster risk factors for postherpetic neuralgia topical lidocaine patch relieves postherpetic neuralgia more effectively than a vehicle topical patch: results of an enriched enrollment study efficacy of oxycodone in neuropathic pain: a randomized trial in postherpetic neuralgia nortriptyline versus amitriptyline in postherpetic neuralgia: a randomized trial treatment of postherpetic neuralgia: an update gabapentin for the treatment of postherpetic neuralgia: a randomized controlled trial relapsing meningitis caused by persistent cryptococcal antigens and immune reconstitution after the initiation of highly active antiretroviral therapy paradoxical recurrent meningitis following therapy of cryptococcal meningitis: an immune reconstitution syndrome after initiation of highly active antiretroviral therapy the role of immune reconstitution inflammatory syndrome in aids-related cryptococcus neoformans disease in the era of highly active antiretroviral therapy raised intracranial pressure complicating cryptococcal meningitis: immune reconstitution inflammatory syndrome or recurrent cryptococcal disease? hiv combination therapy: immune restitution causing cryptococcal lymphadenitis dramatically improved by anti-inflammatory therapy mediastinitis due to cryptococcal infection: a new clinical entity in the haart era case report. recurrence of increased intracranial pressure with antiretroviral therapy in an aids patient with cryptococcal meningitis combination antifungal therapies for hiv-associated cryptococcal meningitis: a randomised trial highly active antiretroviral therapy alopecia universalis and graves' disease in the setting of immune restoration after highly active antiretroviral therapy rheumatic complications of human immunodeficiency virus infection in the era of highly active antiretroviral therapy: emergence of a new syndrome of immune reconstitution and changing patterns of disease mycobacterial cutaneous manifestations: a new sign of immune restoration syndrome in patients with acquired immunodeficiency syndrome treatment of mycobacterium avium complex immune reconstitution disease in hiv-1-infected individuals isolated pulmonary mycobacterium avium complex infection in patients with human immunodeficiency virus infection: case reports and literature review sarcoidosis in a patient with aids: a manifestation of immune restoration syndrome acute bilateral parotitis caused by mycobacterium scrofulaceum: immune reconstitution disease in a patient with aids role of mycobacterium xenopi disease in patients with hiv infection at the time of highly active antiretroviral therapy (haart). comparison with the pre-haart period cutaneous intolerance to tattoos in a patient with human immunodeficiency virus: a manifestation of the immune restoration syndrome immune reconstitution inflammatory syndrome mimicking relapse of aids related lymphoma in patients with hiv 1 infection. leuk lymphoma guillain-barre syndrome associated with immune reconstitution compartmentalization of the immune response in varicella zoster virus immune restoration disease causing transverse myelitis varicella zoster as a manifestation of immune restoration disease in hiv-infected children lymphoid pneumonitis as an immune reconstitution inflammatory syndrome in a patient with cd4 cell recovery after haart initiation immune reconstitution inflammatory syndrome associated with kaposi's sarcoma immune reconstitution inflammatory syndrome in hiv-infected patients with disseminated histoplasmosis hepatitis c virus-associated hepatitis following treatment of hiv-infected patients with hiv protease inhibitors: an immune restoration disease? aids contrast-enhancing progressive multifocal leukoencephalopathy as an immune reconstitution event in aids patients immune reconstitution syndrome associated with parvovirus b19-induced pure red cell aplasia during highly active antiretroviral therapy immune reconstitution syndrome to strongyloides stercoralis infection immune reconstitution disease associated with parasitic infections following antiretroviral treatment immune reconstitution inflammatory syndrome presenting as sinusitis with inflammatory pseudotumor in an hivinfected patient: a case report and review of the literature demodex folliculitis: a skin manifestation of immune reconstitution disease eosinophilic folliculitis: an example of 'immune reconstitution folliculitis none the author(s) declare that they have no competing interests. all authors participated in the drafting of the manuscript. all authors read and approved the final manuscript. http://www.aidsrestherapy.com/content/4/1/9 key: cord-027659-rxbo7b0e authors: bates, imelda; owusu-ofori, shirley title: blood transfusion date: 2020-06-22 journal: manson's tropical diseases doi: 10.1016/b978-1-4160-4470-3.50018-5 sha: doc_id: 27659 cord_uid: rxbo7b0e nan only 39% of the global blood supply is donated in the poorest countries where 82% of the world's population lives. 1 blood transfusion is a vital component of every country's health service (table 14 .1). it can be a life-saving intervention for severe, acute anaemia, but mistakes in the transfusion process can be life-threatening, either immediately or years later through transmission of infectious agents. it is imperative that clinicians have a good understanding of how blood is acquired and prepared for transfusion, and when it should be used, and that governments put in place quality assurance mechanisms to guarantee that blood for transfusion is safe. transfusion medicine is critical to the success of most clinical specialties and should be incorporated into all national health plans and budgets. only 16% of member states meet all the world health organization's (who) recommendations for a national quality blood transfusion system. 1 at the national level the transfusion service should have a director, an advisory committee and clear transfusion policies and strategies (table 14. 2). blood collection, testing and distribution need to be standardized. although centralization of these services may offer the best guarantee of quality, it is often not practical in countries with poorly developed communications and transport infrastructure. in such countries, each hospital organizes its own blood transfusion service and it is then diffi cult to ensure national standardization and quality. hospital-based transfusion services place an enormous burden on laboratory resources and on the families of patients because they are responsible for fi nding suitable blood donors. in a typical district hospital in malawi, the overall cost of the transfusion service, including consumables, proportional amounts for capital equipment, staff time and overheads, was 53% of total laboratory costs and each unit of whole blood cost the laboratory approximately £10 to collect and process. 2 in wealthy countries with nationally or regionally centralized transfusion services, blood donor recruitment, and screening and processing of donated blood, are carried out in purpose-built centres which are separate from the hospitals where the blood is transfused. these centres operate to good manufacturing stan-dards similar to those laid down for the pharmaceutical industry. after donation and exclusion of potentially infected units, the blood is separated into components and fi ltered to remove white cells. computerization enables individual components to be barcoded so they can be tracked back to the original donor. hospitals are profi cient at predicting how much blood they will require and they receive regular consignments through a well-established delivery network. the effi ciency of the system means that one donor centre may provide blood to many hospitals and cover a population of several million. this process is expensive and one unit of blood currently costs over £100. 3 in wealthy countries it is standard practice to optimize the use of each donation of blood by separating it into individual components. these components, which may include plasma, platelets and cryoprecipitate, are prepared by centrifugation using a closed, sterile system. each component has different storage requirements. plasma and cryoprecipitate are kept frozen, red cells are stored at 1-5°c, and platelets at 18-22°c with constant agitation. separation of blood, even into simple components such as cells and plasma, requires equipment and expertise, so in the poorest countries blood components may only be accessible to those living close to a central hospital with blood separation facilities. an unsafe blood supply is costly in both human and economic terms. transfusion of infected blood causes morbidity and mortality in the recipients, and has an economic and emotional impact on their families and communities. those who become infected through blood transfusion are infectious to others and contribute to the spread of disease throughout the wider population. this increases the burden on health services and reduces productive labour. strategies for recruiting blood donors have to balance supply with demand, and yet ensure that the blood is as safe as possible. in general, the safest sources of blood are altruistic voluntary unpaid donors who should be anonymous to the recipient. only 32% of who member states report having at least 90% of their blood supply from voluntary donors, and developing countries have not shown any improvement in recruitment of voluntary donors for several years. 1 in countries without a national transfusion service, each hospital is responsible for fi nding its own donors and processing blood for transfusion. recruiting voluntary donors from the community is expensive and logistically complicated, requiring resources such as a local education programme, dedicated venesection team, vehicles and cold storage. paid donors or 'loan' systems, where family members are responsible for providing blood for their relatives in the hospital, are therefore widespread in poorer countries. cultural taboos and misinformation about donating blood (e.g. 'men will become impotent if they donate blood'; 'hiv can be caught from the blood bag needle') mean that relatives may be reluctant to donate. families are open to exploitation by 'professional donors' who charge a fee to donate in place of a family member. by the time a donor has been found, screened and venesected, and the blood is transfused into the patient, several hours or even days can elapse, especially if blood of a rare group is required. because patients in poorer countries often present late in the course of their disease, severely anaemic patients may die in hospital without ever receiving a blood transfusion. it is unfortunate that in many countries where the majority of transfusions are performed as an emergency and where it is imperative to have a well-stocked blood bank, the 'loan' system, with its inherent delays, predominates. potential 'high-risk' donors, such as commercial sex workers or those having frequent contact with these individuals, intravenous drug abusers, or persons with itinerant or fl uctuating activities such as traders, drivers and military personnel, should be permanently deferred from the donor pool. 4 even in areas where hiv infection rates in the general population are high, donor deferral can be effective in excluding hiv-infected donors. 5 the whole donation process, including tests for hiv and other infections, should be explained to the donor before blood is collected and donors should have the option of knowing the results and receiving counselling. it is imperative that complete confi dentiality is maintained throughout all procedures. infections with organisms that are common in tropical countries, such as hiv-1 and -2, hepatitis a, b, c and d, cytomegalovirus, syphilis, lyme borreliosis, malaria, babesiosis, american trypanosomiasis (chagas' disease) and toxoplasmosis, can all be acquired through blood transfusions. there have also been recent reports of transmission of variant creutzfeldt-jakob disease through blood transfusion and there is a theoretical risk of acquiring severe acute respiratory syndrome (sars) through transfusion of labile blood products. 6, 7 who recommends that all donated blood should be screened for hiv, hepatitis b and syphilis and, where feasible and appropriate, for hepatitis c, malaria and chagas' disease. between 5% and 10% of hiv infections worldwide are thought to have been transmitted through the transfusion of infected blood and blood products. hiv testing of blood donors needs to be highly sensitive, and blood which tests positive should be rejected. before informing the donor of the outcome, all positive results should be confi rmed using a test with a high degree of specifi city. where blood donation is organized locally, the confi rmatory test is often performed at a central laboratory, so there may be delay in informing the donor of the result. malaria can be transmitted by transfusion and has an incubation period of between 7 and 50 days, depending on the species. in areas of low or no malaria transmission, screening for the parasite is important, as recipients are likely to have no immunity. in countries with high malaria transmission, exclusion of parasitaemic donors could result in deferral rates exceeding 30% and consequently would have a major impact on blood supply. 8 it is unclear whether malaria screening is necessary in regions where the disease is common, particularly because most of the blood is given to hospitalized children with malaria who are likely to be receiving antimalarial drugs, or adults who are clinically immune. further research to assess the risks and benefi ts of screening blood for malaria is needed, particularly in relation to pregnant women and patients with hiv infection. screening for hepatitis b surface antigen should be carried out on all donated blood, as hepatitis b-infected blood is almost 100% infectious. fresh blood is potentially infectious for syphilis, but storage at 4°c can inactivate treponema pallidum. globally, the prevalence of hepatitis c, htlv-1 and -2 and chagas' disease is variable and the decision to introduce donor screening for these infections will be based on local assessments of the risks, benefi ts, feasibility and costs. blood should not be separated into components if the residual risk of infection is high, as this will increase the number of potentially infected recipients. in some wealthy countries nucleic acid amplifi cation techniques (nat) have been introduced to improve the safety of blood. although nat may not be cost-effective where infection prevalence is low, it has reduced the residual risk for hiv, hepatitis c virus (hcv) and hepatitis b virus (hbv) infection in germany to 1 in 5 540 000, 1 in 4 400 000 and 1 in 620 000, respectively. 9 blood is usually taken from donors and stored in a blood bank until screening tests for infections have been completed. this system has several drawbacks: potentially infected blood may be mixed up with units that have already been screened, and the whole process of venesection with wastage of blood collection bags is costly. pre-donation screening, by which potential donors are tested for hiv, hepatitis b and possibly hepatitis c at the site of donation before being venesected, may be a more cost-effective way of ensuring safe blood. 10 in wealthy countries the majority of transfusions are planned and carried out electively. by contrast, in poorer countries, and particularly those where the malaria transmission rate is high, most transfusions are given for life-threatening emergencies. in these countries 50-80% of transfusions are administered to children, predominantly for malaria-related anaemia. transfusion can signifi cantly reduce the mortality of children with severe anaemia but it may not have any benefi t unless it is given within the fi rst 2 days of hospital admission. 11 in areas of high hiv prevalence, young children have a relatively low risk of being infected with hiv and potentially have a long life expectancy. however, this is the age group that is predominantly affected by severe malariarelated anaemia and so they are particularly at risk of transfusionacquired hiv infection. 12 pregnant women are the second most common recipients of blood, particularly for haemorrhagic emergencies. 13 other specialities which are signifi cant users of blood are surgery, trauma and general medicine. whether a patient needs a blood transfusion or not is ultimately a clinical decision. emergency transfusions can be life-saving for patients in whom the anaemia has developed too quickly to allow physiological compensation. examples of such emergencies include severe malaria-related anaemia in children, and sudden, severe obstetric bleeding. in contrast, if the anaemia has developed slowly, for example due to hookworm infestation or nutritional defi ciency, patients can generally be managed conservatively by treating the cause of the anaemia and prescribing haematinic replacements. these should be continued for at least 3 months after the haemoglobin has returned to normal, so that body stores can be replenished. it is possible to avoid unnecessary transfusions through the use of clinical transfusion guidelines, and most institutions or organizations have developed guidelines to help clinicians make rational decisions about the use of blood transfusions (table 14. 3). 14 strict enforcement of a transfusion protocol in a malawian hospital reduced the number of transfusions by 75% without any adverse effect on the mortality rate. 15 while the details may vary, the principles underlying most transfusion guidelines are similar and combine a clinical assessment of whether the patient is developing complications of inadequate oxygenation, with measurement of their haemoglobin. the haemoglobin level is used as a surrogate measure for intracellular oxygen concentration. increasingly, transfusion guidelines are making use of evidence which shows that adequate oxygen delivery to the tissues can be achieved at haemoglobin levels that are signifi cantly lower than the normal range. 16 it is easier to develop guidelines than to ensure that they are used in routine practice. implementation of transfusion guidelines is particularly diffi cult if clinicians do not have confi dence in the quality of haemoglobin measurements. it has been shown that when doubtful of the quality of haemoglobin result, clinicians rely entirely on clinical judgement to guide transfusion practice. this may lead to signifi cant numbers of inappropriate transfusions. 17 in a typical district hospital in africa, the cost of providing a unit of blood through the family 'loan' system is approximately 30 times the cost of a quality-assured haemoglobin test. a lack of investment in assuring the quality of a basic but critical test such as haemoglobin measurement can result in a signifi cant waste of resources downstream in the transfusion process, with the additional unnecessary exposure of recipients to the risk of transfusion-related infections. in resource-poor countries, the recommended haemoglobin threshold for transfusions is often well below that which would be accepted in more wealthy countries. for example, american anaesthetists suggest that transfusions are almost always indicated when the haemoglobin level is less than 6 g/dl, 18 whereas in malawi transfusions are recommended for children with haemoglobin levels less than 4 g/dl, provided there are no other clinical complications. 19 complications such as cardiac failure or infection may necessitate transfusion at a higher haemoglobin level. transfusion should be combined with adequate iron and folate replacements and treatment of any underlying conditions that contribute to anaemia, so that a normal haemoglobin count can be achieved during the weeks following transfusion. any transfusion service must be able to guarantee the quality of haemoglobin results. these results are crucial in donor selection and are also used to guide the decision to transfuse patients. although it is the most commonly performed test, accurate haemoglobin estimation is diffi cult to achieve in laboratories without automated blood analysers. 20 the reference technique for haemoglobin measurement is the haemiglobincyanide method. not only does this method need a constant electricity source for the spectrophotometer, but also technicians need arithmetic expertise to calibrate the equipment and automatic pipettes for accurate measurements. in under-resourced countries, district hospitals may use simpler, cheaper and less accurate methods of haemoglobin measurement, many of which are based on visual colour comparisons. while a few individual laboratories in resource-poor countries may be registered with an external system to monitor the quality of laboratory tests, almost no country has a nationwide programme. this means that for many laboratories and their users, the quality of tests, including haemoglobin and those used for screening and determining blood groups, is unknown. complications can occur immediately during transfusion, within a few hours of its completion, or be delayed for many years, as in the case of viral infections. see table 14 .4. transfusion of blood into a recipient who possesses antibodies to the donor's red cells can cause an acute, and occasionally fatal, intravascular haemolysis. this could occur, for example, if group a cells are transfused into a group o recipient who has naturally occurring antibodies to group a cells. the profound haemolysis induces renal vasoconstriction and acute tubular necrosis. treatment involves stopping the transfusion, cardiorespiratory support and inducing a brisk diuresis. in addition to abnormalities indicating renal failure, laboratory fi ndings include haemoglobinuria and haemoglobinaemia. proof of the diagnosis involves rechecking the whole transfusion process including all documentation stages, regrouping the donor and the recipient, and screening for antibodies on red cells with a direct antiglobulin test. these tests are usually available in any hospital laboratory capable of providing a transfusion service. delayed haemolysis has a similar physiological basis to acute intravascular haemolysis but tends to be less severe. the antibody-antigen reaction develops 7-10 days after the transfusion and it is less likely than acute haemolysis to present as a clinical emergency. bacteria can enter the blood bag during venesection or if the bag is perforated at a later stage, perhaps to reduce the volume for a paediatric recipient or during component preparation. gramnegative bacteria, including pseudomonas and yersinia, grow optimally at refrigerator temperatures and infected blood may not necessarily appear abnormal. reactions following infusion of infected blood are often due to endotoxins and may occur several hours after the transfusion has fi nished. although these reactions are rare, they can be severe and fatal. if bacterial contamination is suspected, the transfusion should be stopped and samples from the patient and the blood bag sent to the laboratory for culture. cardiorespiratory support may be needed and broad-spectrum antibiotics should be started immediately and continued until culture results are available. these are episodes of fever (i.e. ≥1°c rise in temperature) and chills for which no other cause can be found. they are due to the recipient's antibodies reacting against antigens present on the donor's white cells or platelets. these reactions are most common in patients who have received multiple transfusions in the past and have therefore been exposed to a broad range of antigens. mild febrile reactions usually respond to simple antipyretics such as paracetamol. more severe reactions may be the fi rst indication of a haemolytic transfusion reaction or bacterial contamination and should be investigated and managed accordingly. these are due to infusion of plasma proteins and manifestations include erythema, rash, pruritus, bronchospasm and anaphylaxis. the transfusion should be stopped and the patient treated with antihistamines. if the reaction is mild and the symptoms and signs completely disappear, the transfusion can be restarted. if this type of mild reaction occurs repeatedly with more than one unit of blood, the red cells can be washed before transfusion. this should only be done if absolutely necessary, as it carries the risk of introducing potentially fatal bacterial infection. severe allergic reactions with evidence of systemic toxicity should be managed as acute anaphylaxis. blood should always be transfused slowly, to avoid overloading the circulation, unless the patient is actively and severely bleeding. overload may be a particular problem when paediatric blood bags are not available, as children may be over-transfused due to miscalculation of the required volume, lack of accurate infusion devices or inadvertent administration of an adult-sized unit of blood. in tropical practice, blood transmission of hepatitis b, hiv-1 and -2, and, in some areas, american trypanosomiasis (chagas' disease) is of particular concern. in general, transfusions are not the major route of transmission of these infections and they may not cause clinical problems until many months or years after the transfusion. four units of blood contain the equivalent of the amount of iron stored in the bone marrow (approximately 1 g). repeated transfusions for chronic haemolytic anaemia, as in thalassaemia major and sickle cell disease, lead to iron deposition in parenchymal cells. eventually, failure of the heart, liver and other organs supersedes. adequate doses of iron chelators, such as injectable desferrioxamine or the newer oral chelator, deferiprone, are able to maintain acceptable iron balance in patients with chronic anaemia receiving regular transfusions. it is not usually necessary to warm blood unless rapid transfusion of large quantities is needed. this may lower the temperature of the sino-atrial node to below 30°c, at which point ventricular fi brillation can occur. if blood needs to be warmed, an electric blood warmer specifi cally designed for the purpose should be used. this keeps the temperature below 38°c, thereby avoiding the haemolysis associated with overheating blood. graft-versus-host disease occurs when donor lymphocytes engraft in an immune-suppressed recipient. the lymphocytes recognize the recipient's bone marrow as foreign and induce aplasia. graftversus-host disease is almost universally fatal and can be prevented by irradiating the donor blood, which inactivates the donor lymphocytes. where blood is in short supply, it is particularly important to ensure that the best anaesthetic and surgical techniques are used, to minimize blood loss during surgery. drugs which improve haemostasis or reduce fi brinolysis, such as aprotinin and cyklokapron, and fi brin sealants, can be effective in reducing perioperative blood loss and hence the need for blood transfusion. cost is a major limiting factor to the use of these therapies in poorer countries, and surgical blood loss is generally not a major contributor to the overall transfusion needs. patients undergoing planned surgery who are likely to require a blood transfusion can have units of their own blood removed and stored prior to surgery for use by themselves only, if signifi cant intraoperative blood loss is anticipated. preoperative autologous donation can reduce the need for allogeneic transfusions by 46-74% 21 but it requires careful organization: the surgeon needs to predict how much blood will be required, the patient has to be fi t enough to withstand removal of one or more units of blood over the weeks preceding the surgery (preoperative haemoglobin will drop by about 1 g/dl), and the surgery must take place within the shelf-life of the blood. as the blood has to be stored in the blood bank there is still a risk that the patient may receive blood which is not his or her own or that the blood may become infected with bacteria during the process. this involves collecting blood lost during the operation and reinfusing it into the patient either during or after surgery. although this technique is practical and safe, and reduces the need for donor blood by 27-53%, 21 it requires specialized equipment and training and may be more expensive than routinely donated blood. 22 normal saline or intravenous replacement fl uids can be used judiciously in acute blood loss, and in certain circumstances may be as effective as whole blood, red cells or plasma. erythropoietin, which stimulates endogenous red cell production, has wellestablished uses in chronic anaemias such as those due to renal failure, cancer and hiv infection. its delayed action makes it unsuitable for use in acute anaemias, the major reason for transfusions in poorer countries. the development of synthetic oxygen carriers, generally perfl uorocarbons, has been fraught with problems and they are not routinely available. 23 in under-resourced countries, especially those with a heavy burden of malaria, the most effective way to avoid the need for transfusions is to reduce the prevalence of anaemia in the community. more studies on the ability and cost of combined interventions such as the provision of bed nets, nutritional supplements and anthelmintic drugs to children to prevent anaemia and reduce transfusion requirements are needed. when resources are very limited, governments may need to make some diffi cult decisions in order to achieve an equitable balance between investing in a transfusion service and public health measures to reduce anaemia. laboratory costs of a hospitalbased blood transfusion service in malawi better blood transfusion safe blood in developing countries excluding blood donors at high risk of hiv infection in a west african city possible transmission of variant creutzfeldt-jakob disease by blood transfusion world health organization. who recommendations on sars and blood safety the risk of malaria transmission by blood transfusion at cotonou human immunodefi ciency virus, hepatitis c and hepatitis b infections among blood donors in germany 2000-2002: risk of virus transmission and the impact of nucleic acid amplifi cation testing predonation screening of blood donors with rapid tests: implementation and effi cacy of a novel approach to blood safety in resource-poor settings effect of blood transfusion on survival among children in a kenyan hospital trends and risk factors of hiv-1 seropositivity among outpatient children anaemia, blood transfusion practices, hiv and mortality among women of reproductive age in western kenya world health organization. blood safety . . . for too few. press release who/25 whd/3 information sheet for clinicians prevention of transfusion-associated hiv transmissions with the use of a transfusion protocol for under 5s electrocardiographic st-segment changes during acute, severe isovolemic hemodilution in humans use of clinical judgement to guide administration of blood transfusions in malawi american society of anesthesiologists task force. practice guidelines for blood component therapy ministry of health and population, malawi. aids control programme. recommended guidelines for the practice of safe blood transfusion in malawi evaluation and costs of different haemoglobin methods for use in district hospitals in malawi autologous transfusion techniques: a systematic review of their effi cacy intra-operative autologous blood management artifi cial o2 carriers: status in 2005 key: cord-262143-s01jrtbb authors: head, michael g; brown, rebecca j; newell, marie-louise; scott, j anthony g; batchelor, james; atun, rifat title: the allocation of us$105 billion in global funding from g20 countries for infectious disease research between 2000 and 2017: a content analysis of investments date: 2020-09-21 journal: lancet glob health doi: 10.1016/s2214-109x(20)30357-0 sha: doc_id: 262143 cord_uid: s01jrtbb background: each year, billions of us$ are spent globally on infectious disease research and development. however, there is little systematic tracking of global research and development. we present research on investments into infectious diseases research from funders in the g20 countries across an 18-year time period spanning 2000–17, comparing amounts invested for different conditions and considering the global burden of disease to identify potential areas of relative underfunding. methods: the study examined research awards made between 2000 and 2017 for infectious disease research from g20-based public and philanthropic funders. we searched research databases using a range of keywords, and open access data were extracted from funder websites. awards were categorised by type of science, specialty, and disease or pathogen. data collected included study title, abstract, award amount, funder, and year. we used descriptive statistics and spearman's correlation coefficient to investigate the association between research investment and disease burden, using global burden of disease 2017 study data. findings: the final 2000–17 dataset included 94 074 awards for infectious disease research, with a sum investment of $104·9 billion (annual range 4·1 billion to 8·4 billion) and a median award size of $257 176 (iqr 62 562–770 661). pre-clinical research received $61·1 billion (58·2%) across 70 337 (74·8%) awards and public health research received $29·5 billion (28·1%) from 19 197 (20·4%) awards. hiv/aids received $42·1 billion (40·1%), tuberculosis received $7·0 billion (6·7%), malaria received $5·6 billion (5·3%), and pneumonia received $3·5 billion (3·3%). funding for ebola virus ($1·2 billion), zika virus ($0·3 billion), influenza ($4·4 billion), and coronavirus ($0·5 billion) was typically highest soon after a high-profile outbreak. there was a general increase in year-on-year investment in infectious disease research between 2000 and 2006, with a decline between 2007 and 2017. funders based in the usa provided $81·6 billion (77·8%). based on funding per 2017 disability-adjusted life years (dalys), hiv/aids received the greatest relative investment ($772 per daly), compared with tuberculosis ($156 per daly), malaria ($125 per daly), and pneumonia ($33 per daly). syphilis and scabies received the least relative investment (both $9 per daly). we observed weak positive correlation (r=0·30) between investment and 2017 disease burden. interpretation: hiv research received the highest amount of investment relative to daly burden. scabies and syphilis received the lowest relative funding. investments for high-threat pathogens (eg, ebola virus and coronavirus) were often reactive and followed outbreaks. we found little evidence that funding is proactively guided by global burden or pandemic risk. our findings show how research investments are allocated and how this relates to disease burden and diseases with pandemic potential. funding: bill & melinda gates foundation. large amounts of funding are allocated to research in infectious diseases each year, 1 spanning pre-clinical science, clinical trials, product development, and public health, including implementation research. these allocations involve numerous stakeholders across the global health community, including funders, researchers, policy makers, and clinicians. however, there is little systematic tracking or detailed analysis of investments into research and development for infectious diseases to support how to make the best funding decisions. 2 nor is there systematic coordination between stakeholders involved in funding research and development, despite efforts such as the who global observatory on health r&d to achieve better coordination. 1 funders differ in their approaches to commissioning research, from the curiosity-driven approaches of the wellcome trust, 3 to the focused data-driven strategies of the bill & melinda gates foundation, 4 which creates a heterogeneous landscape of research priorities. thus, there is a need for an in-depth and comprehensive review of the global research and development landscape to identify what research has taken place, where the research was done, and which institutions were involved in the research. such research on research is crucial for priority setting, informing funding decisions, and improving efficiency in allocating funds. 2 we present research done by the research investments in global health (resin) study group on research investments into infectious diseases from funders in the g20 countries across an 18-year time period spanning 2000-17, comparing amounts invested for different conditions and considering the global burden of disease to identify potential areas of relative underfunding. this study considered research awards related to infectious disease research from 987 public and philanthropic funders in the g20 countries (appendix p 2), made between jan 1, 2000, and dec 31, 2017. the methods used were similar to those described in detail elsewhere. [5] [6] [7] [8] data collected included study title, abstract, award amount, funder, and year. data were manually collated from multiple sources. awards to institutions in the uk between jan 1, 1997, and dec 31, 2013, have been previously analysed. 7, 8 most data (>90%) from 2016 and 2017 were sourced from the uberresearch dimensions database, which includes 4·9 million financial awards across health and nonhealth research and development sectors from 501 global funders. us national institutes of health (nih) data from between jan 1, 2000 and dec 31, 2015, was sourced directly from the project reporter database. other data were sourced from the websites of individual funders, funder databases such as the world report, the uk national research register (a now-archived website owned by the uk department of health), or by contacting the funder directly and requesting data. we applied keyword searches and filters (appendix p 2) to identify studies of human-related infectious disease. awards purely focused on plant pathology or veterinary science were excluded, unless there was a clear zoonotic component. excluded studies were manually reviewed to identify any false negatives. the included financial awards were individually scrutinised to assess their relevance to infection. mgh assessed all financial awards for inclusion and categorised infection-related awards, applying keyword labels as appropriate (appendix pp 2-3). secondary checks on included and excluded awards were made (by rjb and other), as per the study protocol. 7,8 15 000 (15·9%) of 94 074 awards were double checked, with a review of the award inclusion in the dataset and the applied labels indicating the disease areas of the research. where there was disagreement, study information was provided to a third co-author for consensus. research award amounts were adjusted for inflation in original currency and converted to 2017 us$ using the mean exchange rate in the award year. award amounts were missing for 6072 (5·7%) of 94 074 awards, from 13 funders (appendix p 2). in these cases, estimates to our knowledge, this is the first study to describe in depth the global landscape for all infectious disease research from public and philanthropic funders. our study covers 18 years of funding data, so captures long-term time trends and fluctuations. we combined and categorised awards using the classification system developed by the resin study. this strategy allowed us to provide a comprehensive overview of how infectious disease funding has been allocated, and compare findings with the global burden of disease, an important variable to consider when setting research priorities. this information can be used by global health research funders in decision-making processes. our findings show that between 2000 and 2017, hiv received significantly more research funding than similar high-burden diseases such as tuberculosis, malaria, and pneumonia. the usa provides much of the global funding, particularly the us national institute for health. there are several infections that appear neglected compared with their burden of disease, such as syphilis and scabies. thus, the global health community could use our findings to inform discussions, alongside other drivers for research prioritisation. were made using maximum award amounts for that funding stream as per the funder's website, by asking principal investigators for an approximate or exact award amount provided, or by asking in-country researchers who had knowledge of the research and development landscape for typical award amounts. datasets and analyses were circulated to all authors for review and comment. included award types comprised project and programme grants, fellowships, and pump-priming or pilot projects. excluded award types were conference and infrastructure grants and funding focused on operational activities rather than research. labels applied to each award included pathogen, disease areas and specialty (eg, antimicrobial resistance, respiratory, oncology, and paediatrics), and type of science along the research continuum (pre-clinical, phase 1-3 clinical trials, phase 4 and product development research, public health [ focusing on populations], and cross-disciplinary studies across multiple stages of the research continuum). we defined cross-disciplinary as a study that covered more than one stage of the research continuum (for example pre-clinical research that progressed to a phase 1 study). antimicrobial resistance included antibacterial, antiviral, antiparasitic, and antifungal resistance. the diagnostics category included research into screening. sexually-transmitted infections excluded hiv, which had its own category. neglected tropical diseases were based on the who definition (as of oct 23, 2019). 9 burden of disease data were sourced from the global burden of disease study online tool. 10 disease burden data are reported from 2017 for all infectious diseases, and additional examples are presented using hiv/ aids, malaria, tuberculosis, and pneumonia from years 2005 and 2011 (six-year time intervals during the period covered by the investments dataset). measures of disease burden analysed were mortality, disabilityadjusted life years (dalys), and years lived with disability (yld). comparison between awards and their associated observed disease burden were made by calculating investment per mortality, daly, or yld observed. we computed the investment relative to the burden of infection using the following equation: cumulative research investment up to the year of burden measure or number of deaths, dalys, or yld at that timepoint. for example, for assessment of hiv dalys in 2017, the sum of hiv research investment from jan 1, 2000, to dec 31, 2017 ($42·1 billion), was divided by dalys in 2017 (54 446 184), to get an investment per daly metric of $772. descriptive statistics and spearman's correlation coefficient were used to investigate the relationship between research investment and disease burden, using global burden of disease 2017 study data. we used microsoft excel 2016 for data preparation stata se version 16 for data analysis. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the hiv/aids was the pathogen or disease with the greatest amount of funding ($42·1 billion [40·1%]) across 21 403 (22·8%) awards (tables 1, 2). funding for tuberculosis totalled $7·0 billion (6·7%) from 5246 (5·6%) awards, funding for malaria was $5·6 billion (5·3%) from 4437 (4·3%) awards, and funding for pneumonia was $3·5 billion (3·3%) from 2748 (2·9%) awards. funding for ebola virus-related research was $1·2 billion (1·1%); $0·8 billion (68·0%) of all ebola virus-related research investment was awarded between 2014 and 2017, following the high-profile outbreak of ebola virus disease in west africa in 2014. 11 similarly, $0·3 billion of funding was allocated to research on zika virus, of which 96·0% was awarded in 2016 or 2017 after the zika virus epidemic. 12 of the $4·4 billion (4·2%) of funding for influenza, $2·0 billion (45·0%) was awarded in 2006-10, with the highest annual funding amount awarded in 2009 ($0·6 billion [12·8%]). there were global outbreaks of h1n1 influenza in 2005 (so-called bird flu) and 2009 (so-called swine flu). 11 funding for coronavirus-related research was $0·5 billion (0·5%) from 396 (0·4%) grants, with a median award size of $2·0 million (iqr 0·6 million to 2·9 million; , the year after the international sars outbreak, 13 by disease area, $3·8 billion (3·6%) was awarded for antimicrobial resistance from 4845 (5·2%) awards, $4·1 billion (3·9%) was awarded for neglected tropical diseases, $1·1 billion (1·0%) for sepsis, and $1·4 billion (1·3%) for health-care-associated infections. in areas relating to hard-to-reach groups, $2·0 billion (1·9%) was awarded for infections related to drug use and addiction and $0·2 billion (0·2%) for infectious diseases in prison (table 1) . awards for comorbidities and non-communicable diseases included $0·3 billion (0·3%) for mental health and $0·6 billion (0·6%) for cardiovascular disease. funders from the usa provided $81·6 billion (77·8%) of the investment, which covered 42 926 (45·6%) of the awards. within this, the us nih was the largest funder, providing $59·4 billion (56·6% of total us funding) and the greatest number of individual awards (32 967 [35·0%]). the bill & melinda gates foundation provided $9·2 billion (8·8%) in 2317 (2·5%) awards. uk funders provided $8·3 billion (7·9%) in 8358 (8·9%) awards. when the awards had a clear geographical focus, $9·2 billion (8·8%) of the funding was focused on africa and $2·4 billion (2·3%) on asia (appendix p 2). when ranking investment levels on the basis of burden of disease by dalys across 34 infectious diseases (appendix p 2), african trypanosomiasis ($9740 per daly) and genital herpes ($3101 per daly) were ranked first and second, respectively (table 2). hiv/aids ($772 per daly) was ranked eighth, tuberculosis ($156 per daly) was ranked seventeenth, malaria ($125 per daly) was ranked twenty first, enteric infections ($68 per daly) were ranked twenty fourth, and pneumonia ($33 per daly) was ranked twenty eighth. scabies and syphilis were ranked joint last with $9 per daly. when comparing investment for individual infections alongside 2017 dalys, spearman's correlation coefficient was 0·30 (p=0·048), suggesting weak positive correlation between research investment and global burden of disease ( figure 2) . infections within the shaded area of the graph showed a stronger correlation between investment and burden of disease. infections below the shaded area were relatively underinvested, and infections above the shaded area were relatively well-invested, compared with their 2017 dalys burden. when comparing investment by mortality, syphilis ($632 per death) and tetanus ($749 per death) were ranked the lowest of the 27 infections for which mortality data were available (appendix p 2). the highest-ranked infections by investment per death were those for which associated mortality is typically low, specifically chlamydia ($712 076 per death) and african trypanosomiasis ($563 094 per death). hiv was ranked seventh ($44 481 per death), malaria was ranked thirteenth ($9107 per death), tuberculosis was ranked fifteenth ($5936 per death), and pneumonia was ranked twenty fourth ($1392 per death). across different timepoints of the study, hiv-related research consistently received greater investment than did malaria, tuberculosis, or pneumonia (figure 3). pneumonia-related research consistently received far less funding during the study period compared with hiv, tuberculosis, or malaria. by type of science, 35·5% of research funding for hiv was for pre-clinical research, 15·1% for phase 1-3 trials, and 45·9% for public health research (appendix p 9). pneumonia had the greatest proportion of funding allocated to pre-clinical science (55·7%) and the lowest amount for public health research (23·5%) compared with hiv, tuberculosis, and malaria (appendix p 2). in this study, we provide an analysis of $105 billion of research investment as 94 074 public and philanthropic awards for infectious disease research covering the years 2000-17. over half of this investment was for pre-clinical science and over a quarter for public health research. by type of infection, hiv-related research received more than double the investment for tuberculosis, malaria, and pneumonia combined. infections that are relatively less well-funded include some sexually-transmitted infections (syphilis and gonorrhoea) and neglected skin infections, such as scabies. funding for coronavirus-related research was $0·5 billion from 396 grants, of which 95·1% was for preclinical research. however, in 2020 there has been a huge reactive effort to support the response to the covid-19 pandemic, which includes substantial financing for research. 15 as of aug 4, 2020, the resin study has tracked $1·6 billion of global public and philanthropic research funding, which already exceeds the 2017 total investment in hiv research ($1·1 billion; appendix page 2). viral respiratory infections are known to be one of the most likely causes of a pandemic, but despite this and the existing high levels of mortality in young children and older people due to such infections, systems for pneumonia research and advocacy are not well established. 16 confusion over the definition of pneumonia, 17 few experienced researchers to make a strong case to funders, and few high-profile public figures championing the cause have led to pitifully low levels of funding compared with the disease burden. the bill & melinda gates foundation, which is guided by childhood deaths, is the main funder of pneumonia-related research. 5 the metrics used to compare investment by burden of disease are misleading for pathogens such as ebola virus and zika virus, which at first appear to be relatively wellfunded compared with their burden of disease (appendix p 2). however, for these conditions, which are public health emergencies, dalys are not a fair metric to use. outbreaks of this nature are not necessarily high-burden in terms of numbers of cases but are high-risk given the potential for rapid spread to cause widespread outbreaks, an important factor that influences research investment decisions. as illustrated by the evolving covid-19 pandemic, there is a public health need to support outbreak responses and research should very much be part of such a response. such outbreaks create uncertainty and fear, with media promoting a need to do something and urging political circles to respond rapidly. 18, 19 historical funding for coronavirus research was very low, even after the high-profile outbreaks of severe acute respiratory syndrome (sars), due to sars coronavirus, and middle east respiratory syndrome and the potential for the rapid spread of such infections. 20 other analyses highlight how funding for neglected infectious disease research (distinct from neglected tropical diseases) is increasing. 21 our analysis supports this conclusion, for example, showing that research on neglected tropical diseases or with a focus on africa is increasing (appendix p 2). there have been substantial declines in hiv funding, primarily in higher-income settings (and thus not captured under neglected disease definitions). the coalition for epidemic preparedness innovations, founded in 2016, has received substantial research investment from multiple funders to research selected high-threat pathogens. 22 for example, there are several ongoing studies to develop a universal influenza vaccine to reduce pandemic risk, as well as vaccine in development against coronaviruses. 23 antimicrobial resistance, which continues to be a serious worldwide threat, 24 has led to the introduction of the global amr r&d hub with a remit to address challenges and improve coordination and collaboration in global antimicrobial resistance research and development using a one health approach. 25 antimicrobial resistance is also an important contributor to sepsis mortality, which is responsible for 11 million deaths annually with most of the burden in sub-saharan africa, 26 but receives just 1·0% of the funding. research investment analyses can be a valuable audit of a system that has perhaps maximised scientific efficiency through peer review of curiosity driven research and provide a direction for revision of research on under-investigated diseases and subject-based opportunities. the covid-19 pandemic has shown the fragility of national and global infrastructures, and pandemic preparedness will surely be a focus for highprofile global health research stakeholders in years to come. sustainable tracking of how research funding is spent is vital to ensure that all priority areas and knowledge gaps are addressed, 15 and there must be adequate translation of that new knowledge into policy and practice, with findings that can be feasibly adopted in resource-poor settings. multiple factors other than the current and projected burden of disease influence research funding decisions, such as political drivers of decision making (notable in our study given the major funder was the us government), advocacy and lobbying, emergency preparedness for emerging infectious diseases with pandemic potential, and public health research for conflicts and other humanitarian responses. applying a globally recognised label to a disease can be important. who oversees the designated list of neglected tropical diseases, 9 which helps to raise the profile of these conditions and support arguments for research funding. 27 as an example, african trypano somiasis, which has been at the forefront of efforts to tackle neglected tropical diseases, has been described as an extraordinary success story, with a decline in the daly burden by 93% between 2000 and 2017. 28 african trypanosomiasis has received twice the amount of research funding compared with lymphatic filariasis or schistosomiasis, and more than non-neglected tropical diseases, such as meningitis or the respiratory syncytial virus. researchers who study african trypanosomiasis have elimination and eradication in sight, although this will probably require further substantial investments. 28 investment in other neglected areas might help produce similar effective responses, although the type of research investment must be appropriate. for example, our analysis highlighted scabies and syphilis as particularly underfunded. effective treatments are available for scabies, so the most useful research might be around an effective drug supply chain or addressing stigma. other factors beyond the burden of disease also influence the direction and amount of investment. the geographic focus of research investments affects the likelihood of knowledge being translated into policy and practice, particularly in the country or sector where the research was undertaken. 29 it is important to consider where research capacity should be created or enhanced, rather than simply which research areas to prioritise and fund. the uk invests greater resources in former colonies, influenced by historical ties and a shared language. 30 investments in different sectors will also be affected by diplomatic considerations, for example, funding countries seeking cooperation from recipient countries or regions in response to security threats and terrorism. this study has several limitations. there will be missing data, particularly when data could not be accessed from public and philanthropic funders. we propose that the effect of this should not be substantial and should not greatly influence our findings, as the included data relate to 18 of the top 20 leading investors in research. 31, 32 the focus on g20 countries means that funders from countries who are not in this group but are proactive in global health research, such as switzerland and norway, were not included. a key challenge was integrating data that were presented in numerous different formats. future analyses would be simplified considerably if funders could adopt a minimum dataset of required information, perhaps recommended by the who global observatory on health r&d, which would require that applicants add standard labels (eg, the type of science along the research pipeline) to their project at time of submission. applying categories to an award retrospectively is time-consuming and subjective, although errors were reduced by observations from a second author and consensus. automated categorisation based on keyword searches is problematic, since the title and abstract of many awards contain references to diseases that are not the study areas of focus. furthermore, separating out awards for operational or implementation research and activities that are nonresearch based implementation (ie, not generating new knowledge) is a subjective process. our study lacks data from the private sector, particularly concerning tools and products such as vaccines, diagnostics, and therapeutics. the analyses use global burden of disease study data, which are themselves modelled estimates and will on occasions be based on imputations due to missing data, and have been subject to criticism. 33 selection of timepoints for research investments and disease burden will affect our findings as both of these change over time. more than $100 billion has been spent globally on infectious disease research between 2000 and 2017, but this funding does not correlate with current levels of burden or the level of risk posed by infections with pandemic potential. since priority setting for research must consider many different factors, our analysis should be used to support decision making rather than providing clear-cut answers. it is worrying that the funding allocated to infectious disease research is declining during a period in which there are concerns surrounding antimicrobial resistance and the pandemic potential of many pathogens. in conclusion, our findings show where research funding resources are allocated and how this relates to disease burden and diseases with pandemic potential. we anticipate that our results could be an invaluable resource to global health stakeholders (eg, who, research funders, or national governments) who define research strategy and make decisions about the allocation of restricted research and development resources. mgh created the study and led data collection, analysis, and reporting. rjb supported data collection, analysis, and reporting. jb, ra, jags, and m-ln provided contributions to methodology and expert comment and overviews on global health, research investment, and health financing. mgh wrote the first draft. all authors read subsequent drafts and contributed revisions. all authors approved the final manuscript and the and decision to submit for publication. biomedical research; what gets funded where? money and microbes: strengthening research capacity to prevent epidemics how we've defined what success looks like for wellcome's work bill & melinda gates foundation. global health strategy overview sizing up pneumonia investment global funding trends for malaria research in sub-saharan africa: a systematic analysis research investments in global health: a systematic analysis of uk infectious disease research funding and global health metrics uk investments in global infectious disease research 1997-2010: a case study gbd results tool disease control priorities: improving health and reducing poverty ebola research funding: a systematic analysis severe acute respiratory syndrome: historical, epidemiologic, and clinical features mers coronavirus: diagnostics, epidemiology and transmission monitoring investments in coronavirus research and development the missing piece. why continued neglect of pneumonia threatens the achievement of health goals the definition and classification of pneumonia was dr congo's ebola virus outbreak used as a political tool the political economy of the ebola virus disease the lancet t. emerging understandings of 2019-ncov neglected disease research and development: uneven progress coalition for epidemic preparedness innovations towards a universal flu vaccine antimicrobial resistance-a manual for developing national action plans. version 1 global, regional, and national sepsis incidence and mortality, 1990-2017: analysis for the global burden of disease study london declaration on neglected tropical diseases the elimination of human african trypanosomiasis is in sight: report from the third who stakeholders meeting on elimination of gambiense human african trypanosomiasis research, evidence and policymaking: the perspectives of policy actors on improving uptake of evidence in health policy development and implementation in uganda infectious disease research investments follow colonial ties: questionable ethics the 10 largest public and philanthropic funders of health research in the world: what they fund and how they distribute their funds health research funding organizations the evolution of the disability-adjusted life year (daly) we thank all funders who directly or indirectly provided data and those who provided information about and links to the funders. much of the data awarded from 2013 onwards was sourced by accessing the dimensions database, owned by uberresearch (london, uk). we also thank joseph fitchett, for his previous help and support in data collection and analysis and for assisting with secondary checks on included and excluded awards, and pat oxford, who provided support for developing the figures and images. jags is funded by a fellowship from the wellcome trust (214320). the study received funding from the bill & melinda gates foundation (opp1127615). key: cord-259925-g28sx9qu authors: saleemi, mansab ali; ahmad, bilal; benchoula, khaled; vohra, muhammad sufyan; mea, hing jian; chong, pei pei; palanisamy, navindra kumari; wong, eng hwa title: emergence and molecular mechanisms of sars-cov-2 and hiv to target host cells and potential therapeutics date: 2020-10-06 journal: infect genet evol doi: 10.1016/j.meegid.2020.104583 sha: doc_id: 259925 cord_uid: g28sx9qu the emergence of a new coronavirus, in around late december 2019 which had first been reported in wuhan, china has now developed into a massive threat to global public health. the world health organization (who) has named the disease caused by the virus as covid-19 and the virus which is the culprit was renamed from the initial novel respiratory 2019 coronavirus to sars-cov-2. the person-to-person transmission of this virus is ongoing despite drastic public health mitigation measures such as social distancing and movement restrictions implemented in most countries. understanding the source of such an infectious pathogen is crucial to develop a means of avoiding transmission and further to develop therapeutic drugs and vaccines. to identify the etiological source of a novel human pathogen is a dynamic process that needs comprehensive and extensive scientific validations, such as observed in the middle east respiratory syndrome (mers), severe acute respiratory syndrome (sars), and human immunodeficiency virus (hiv) cases. in this context, this review is devoted to understanding the taxonomic characteristics of sars-cov-2 and hiv. herein, we discuss the emergence and molecular mechanisms of both viral infections. nevertheless, no vaccine or therapeutic drug is yet to be approved for the treatment of sars-cov-2, although it is highly likely that new effective medications that target the virus specifically will take years to establish. therefore, this review reflects the latest repurpose of existing antiviral therapeutic drug choices available to combat sars-cov-2. coronaviruses (covs) are a group of enveloped and positive single-stranded rna genome viruses that infect both animals and humans (smith and denison, 2013; xu et al., 2020) . they contain the largest known rna genomes ranging from 27-32 kilobases in length (smith and denison, 2013) . coronavirus-related infections are known to affect the liver, central nervous system (cns), respiratory and gastrointestinal tract of various birds and mammals, including humans (xu et al., 2020) . they are classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (fung et al., 2020) . the novel 2019 coronavirus (2019-ncov) also belongs to the same group of covs. it was named ‗severe acute respiratory syndrome coronavirus 2' (sars-cov-2) by the international committee on taxonomy of viruses (ictv) (fung et al., 2020) . the transmission pattern of sars-cov-2 primarily involves direct close contact with the infected person via nose and mouth secretions, however it can also be transmitted indirectly through contaminated objects or surfaces (world health organization). it was discovered in late december 2019 in wuhan city of china in patients with acute respiratory distress syndrome (ards) symptoms, such as fever, cough, and dyspnea (li et al., 2020; after isolated from the respiratory epithelium (xu et al., 2020) . the genome sequence analysis of sars-cov-2 showed that it belongs to the betacoronavirus genus (β-cov) . the sars-cov-2-related disease was recently named as a novel respiratory 2019 coronavirus disease by the world health organization (who) (xu et al., 2020) . up to date, it is unclear how sars-cov-2 interacts with the host antiviral immunity, hence lessons can be learned from previous studies of other members of the coronavirus family and also human pathogenic viruses, such as human immunodeficiency viruses and severe acute respiratory syndrome (sars) cov known as human covs (hcovs) due to their ability to cause human infections (andersen et al., 2020) . aside from nl63 and 229e; which belong to alphacoronavirus and the remaining five viruses come under the betacoronavirus genus. however, sars-cov, mers-cov, and sars-cov-2 are known to cause severe disease leading to death, whereas 229e, oc43, nl63, and hku1 have only been associated with mild symptoms in infected persons although they are recognized as a potentially zoonotic origins (andersen et al., 2020; corman et al., 2018) . all members of the coronaviridae contain enveloped, positive-stranded spherical rna virions with a length of 120-160 nm and are usually decorated with large visible petal-shaped surface projections known as ‗spikes' when viewed under an electron microscope (singh et al., 2020) . in addition, members of the coronaviridae share common characteristics, which are outlined in (table 1) . within the family, there is also another subfamily such as torovirinae comprising two other genera torovirus and bafinivirus. they are different from coronaviridae mainly due to their different tubular virion shape. the morphology of virion, such as coronavirinae has been unraveled, as illustrated in (fig. 1a) . moreover, the classification of various genera within the subfamily is a little more complicated with high cov recombination frequency, thus necessitating the development of precise criteria proposed over the years (corman et al., 2018) . currently, the primary means of establishing their phylogenetic relationship is through the genomic structure, as shown in (fig. 2) . it is based on these differences that a particular host may be infected by specific genus of the virus, for instance, alpha-and beta-coronavirus infects only mammals and gamma-and delta-coronavirus infects primarily birds and some studies suggest the possibility of the latter genera infecting mammals as well (cui et al., 2019; king et al., 2012; woo et al., 2012) . the specific distinctions between four genera are closely associated with (1) the unique type of nsp1 known as a non-structural rna-binding protein within their genome which may differ in size and sequence and (2) the presence or absence of a commonly shared accessory gene (sheikh et al., 2020) . several examples of viruses within the genera with their unique accessory proteins and genome arrangements can be found in (fig. 3) . the complete sars-cov-2 genome sequencing data had begun to emerge as the real gravity of the situation started to loom over the medical personnels who first encountered this novel coronavirus back in december 2019. their findings have since been published and provided us the information necessary to classify sars-cov-2 into its respective group under the serbecovirus (β-b group) subgenus chan et al., 2020) . the sequence is publicly available on genbank with the accession number mn908947 possessing 30474 nucleotides with close relations to bat sars-like j o u r n a l p r e -p r o o f journal pre-proof coronavirus (cov) isolate bat sl-covzc45 (genbank accession number mg772933) with 89.1% nucleotide identity similarities . in contrast, the origins of hiv have been a subject of debate since its discovery back in 1980s with many theories floating about and evidences not sufficiently conclusive. what is known is that infected individuals were at risk of developing acquired immune deficiency syndrome (aids), which at that time resulted in opportunistic pathogens infecting the individual and due to a pronounced weakened immune system, the individual would succumb eventually to the infection. current data suggest that over the past three decades, hiv has caused more than 35 million deaths worldwide with 35 million living with the virus (pharr et al., 2017) . hiv is classified with other known immunodeficiency viruses, which belong to phylum artveviricota, order ortervirales, and family (vemuri et al., 2020) . besides, three other retroviruses, such as human t-cell lymphotropic virus type 1, 2, 3 (htlv 1, 2, 3) exist within the same subfamily but genus deltaretrovirus also affects the immune system in humans (calattini et al., 2005) . as mentioned earlier, hiv has been categorized into two types; hiv-1 and hiv-2, the former being known as a more pathogenic than the latter, making up the majority of aids cases around the globe, whereas hiv-2 mainly infects individuals in west africa (cloyd et al., 2000) . in general, the most plausible route of evolution can be established by genomic sequence of hiv, while its origin is yet to be understood. in 1986, the genomic analysis found that hiv-2 virus was morphologically similar but genetically different from type 1 and caused more serious aids disease in western africa (jaffar et al., 2004) . another genomic study showed that hiv-2 is firmly associated with the simian immunodeficiency virus (siv) isolated from macaque monkeys (nakayama and shioda, 2015) . since then, the simian close relatives have been further expanded with more and more species of primates in sub-saharan africa found to house similar viruses collectively named siv with special suffixes denoting their species of origin. in general, siv-derived host species have been found to be nonpathogenic, but can become pathogenic once they cross the species (meyerson et al., 2018) ; resulting in the discovery of current pandemic hiv1 strain (group m or main) that is similar to siv cpz in chimpanzees (pan troglodytes) after closer investigation. in addition, the natural host of viruses was identified in cameroon (hahn et al., 2000; keele et al., 2006) . journal pre-proof the morphological structure of hiv is usually spherical, enveloped with 80-100 nm in diameter, and 8 nm surface glycoprotein projections. the internal structure of the virus nucleocapsid, such as a rod or truncated cone-shaped has been shown in (fig. 1b) . hiv viral genome follows the common pattern within the subfamily orthoretrovirinae, which carries two copies with the arrangement order: 5'-gag-pro-pol-env-3', each at approximately 9.3 kb in size. a schematic illustration of the hiv genome is shown in ( into viral envelope precursors with other accessory proteins. in general, the unique sequences of viruses give each genus of a specific viral family its distinguishing characteristics allow its classification into the respective genus, which is lentivirus in case of hiv. in addition to the primary structural genes reported, members of the lentivirus genus also have additional genes, for instance, accessory protein genes vif, vpr, vpu, nef, and regulatory protein genes tat and rev. several additional activities have been ascribed to the resulting proteins transcribed by these genes (secondary activities) which suggest the multifunctional role they play but generally their primary activities are highly conserved (faust et al., 2017) . a summary of these accessory proteins and their role has been shown in (table 2) . atypical pneumonia cases emerged from the zoonotic transmission and showed >95% homology with bat coronaviruses and >70% similarity with the previous sars-cov mcintosh, 2020) . thus, chinese health authorities identified the outbreak as a novel coronavirus (covid-19) . although environmental samples taken from the market tested positive, researchers considered the possibility of human-to-human transmission in other geographical areas after people returned from wuhan celebrating the chinese new year (cny) with their families (wang et al., 2020; chen et al., 2020) . in this context, one study showed a significant correlation between domestic train transportation and the number of cases imported to other provinces in china. besides, non-stop passenger flights were also linked to the number of cases reported abroad, subsequently the cause of this pandemic (rodríguez-morales et al., 2020) . evidence suggests that virus is transmitted by respiratory droplets, direct contact, and airborne transmission. while analysis of confirmed and suspected cases of covid-19 suggested that novel virus could go beyond the respiratory tract (peng et al., 2020) . more interestingly, the study also found that virus can enter the body through eye exposure (li et al., 2020) . notably, several asymptomatic cases have emerged across the globe, arguably the cause of such widespread transmission (bai et al., 2020) . earlier this year, italy declared a state of emergency after registering two cases from wuhan tourists with suspected symptoms of coronavirus after they arrived in rome (giovanetti et al., 2020) . similarly, the usa confirmed the first positive case of sars-cov-2 identified in a woman in her 60's, who had returned from china with good health in mid-january. later, her husband was also tested positive and admitted to the hospital. although he did not travel but was considered to be in close contact with his wife (ghinai et al., 2020) . similarly, a 50year-old female entered the uk from hubei province without previous medical history and no regular medications. after arrival, she developed symptoms of malaise and fever, accompanied by a sore throat and dry cough within three days (lillie et al., 2020) . as some cases show delayed symptoms, the possibility of human-to-human transmission could occur during the asymptomatic incubation period (li et al., 2020; backer et al., 2020) . until an appropriate diagnostic cure is found, researchers have alerted countries to maintain a distance of one meter from infected persons to reduce the risk of transmission (mehraeen et al., 2020) . subsequently, many countries have now introduced movement control orders and lockdowns as preventative measures to curb the spread of the virus (hamzelou, 1981) , urging their citizens to maintain social distance to minimize the spread of the virus. in recent history, another deadly virus called the human immunodeficiency virus (hiv) had emerged, which is the retrovirus responsible for the acquired immunodeficiency syndrome (aids). as a common disease in asia, the concurrence of these diseases could present an alarming problem globally. recently, zhu and colleagues noted that -hiv infected patients need to be regarded as a vulnerable group,‖ after co-infection of covid-19 and hiv was reported in a patient in wuhan, china . previously, hiv-1 emerged in young homosexual men in 1981 (brodie et al., 2004) . five years later, hiv-2 virus was discovered with similar morphology to hiv-1, but antigenically distinct, which increased aids cases reported in patients from western africa in 1986 (clavel et al., 1986) . nonetheless, hiv-2 viral strain was distantly related to hiv-1 and both strains were acquired by humans from non-human primates infected with simian immunodeficiency virus (siv) (heeney et al., 2006) . scientists believed that siv was transmitted to humans after contact with the blood of infected chimpanzees during bushmeat handling. notably, the virus mutated into hiv from its origins and gradually spread from africa to other parts of the world (sharp and hahn, 2011; ayouba et al., 2013; sousa et al., 2017) . thus, confirming the occurrence of both viruses (hiv-1 and hiv-2) as a result of zoonotic transmission from infected primates. in general, human-to-human spread is known to be transmitted from mother to child, and during unprotected sexual intercourse between couples (kassa, 2018) . when hiv infected body fluids, including blood, breast milk, semen, vaginal, and rectal secretion come into contact with mucous membrane in the mouth, rectum, penis, and vagina, it destroys the tissue or injects directly into the bloodstream by a needle or syringe (hladik and mcelrath, 2008; shaw and hunter, 2012) . in summary, it is still unknown whether any identified interrelationship presents between hivs and sars-cov-2. despite the remarkable number of patients infected with hiv and novel covid-19 separately, which extends to cover a wide zone in asia and worldwide. there are very few reports on the sars-cov-2 infection among patients infected with hiv (joob and wiwanitkit, 2020) . it has been observed that hiv targets the specific cd4+ immune cells, including t cells, macrophages, and dendritic cells, resulting in a significant reduction in the number of these immune cells in hiv-infected patients (lane, 2010) . these cd4+ t cells (known as helper cells) are the backbone of the immune system where they activate b cells, macrophage, and cytotoxic t cells to secrete antibodies, destroy ingested microbes and kill the infected cells, respectively (laidlaw, 2016) . journal pre-proof the hiv membrane contains a transmembrane glycoprotein called glycoprotein-41 (gp41) and surface glycoprotein, namely glycoprotein-120 (gp120), which binds to cd4 receptors on the surface of cd4+ cells (pancera et al., 2014) . there are two other chemokine co-receptors known as c-c chemokine receptor 5 (ccr5) and c-c chemokine receptor 4 (cxcr4), which facilitate hiv binding and infusion into cd4+ cells (didigu et al., 2014) . cxcr4 co-receptor is expressed in many peripheral t-cells lymphoma found in the lymphatic sides, including lymph nodes, bone marrow, and spleen (machado et al., 2009) . however, ccr5 is highly expressed on the surface of t cells, macrophages, dendritic cells, eosinophils, and microglia (moore et al., 2004) . the binding of gp120 to cd4 receptor leads to a change in the envelope structure of virus, which allows the co-receptor, either ccr5 or cxcr4 (cxcr4 for t-tropic hiv strains, and ccr5 for m-tropic hiv strains) bind to a specific domain in the gp120. after binding, the n-terminal fusion peptide (gp41) penetrates in the host cell membrane, followed by entry of viral capsid into the cytosol of t cell. after penetration, virus removes and exposes its capsid, which contains the rna genome and its associated enzymes (reverse transcriptase and integrase) into the host cell (naif, 2013) . the virus produces complementary dna (cdna) by transcribing its single-standard rna based on reverse transcriptase activity for producing duplicate strands of viral dna. however, complete viral dna migrates to the nucleus and integrates with dna inside the host cell and unfortunately affects the cellular activity. the integrated dna is used to generate mrna, thus synthesizing the viral proteins and allowing the new virus copy to develop. it has been observed that virus sabotages the cd4+ immune t cells for replication, which leads to increase virus load in the blood and a significant decrease in cd4+ t cells (goodsell, 2015) . interestingly, some researchers found another mechanism of hiv destruction of cd4+ t cells (bolton et al., 2002; yue et al., 2005) . they observed that depletion of cd4+ t cells by apoptosis pathway because of direct hiv infection was only 5-10% of the total cd4+ t cell pool. it has been found that hiv enzyme (integrase) plays a crucial role in the activation of dna-pk sensor of host cell, which activates the apoptotic cascade inside the cell. however, another hiv enzyme (protease) activates the caspase-8 that further triggers the apoptosis of infected cells. besides, specific hiv proteins display on the surface of infected cd4+ t cells, which alarm cd8+ t cells to destroy the infected cd4+ t cells. the immune system reacts by producing antibodies, such as anti-hiv antibodies, which stick to the infected cd4+ t cells and mark their destruction by other immune cells (selliah and finkel, 2001) . however, it has been observed that indirect effect of hiv infection results in majority of cd4+ t cells death, which causes immune deficiency. the direct effect of hiv to cd4+ t cells triggers the expression of interferon-gamma induced protein-16 (ifi-16), followed by the activation of inflammatory cascade inside the infected cell, which ends up with cellular self-j o u r n a l p r e -p r o o f destruction called pyroptosis (doitsh et al., 2014; monroe et al., 2014) . the caspase-1 activated by high expression of ifi-16 triggers the production of cytokines, especially interleukin-1 beta inside the infected cells (lupfer et al., 2015) . the high production of il1-β leads to the creation of inflammatory environment inside the cell, resulting in the initiation of apoptosis (feria et al., 2018) . the infected cells lead to release of interleukin-1 beta (il-1β) to the outside cellular environment, which contributes to the activation of pyroptosis in the non-affected cells, causing a considerable loss of cd4+ t cells (doitsh et al., 2014) . unlike hiv, the exact mechanism how covid-19 induces immune defect is still unknown; however, it could be similar to the previous coronaviruses, including the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). these viruses induce low level of lymphocytes abnormality in blood, or it is known as lymphopenia related to hiv infection. the binding of coronavirus to the host cell required a specific class i viral glycol-transmembrane-protein, such as s protein on the surface of the virus envelope. it needs a specific receptor on the host cells called angiotensin-converting enzyme 2 (ace2) receptor (andersen et al., 2020; dhama et al., 2020) . ace2 is an enzyme spread in many cell membranes of different organs, including lungs, arteries, heart, kidney, and intestines; it plays a significant role in blood pressure regulation. the dramatic reduction in the peripheral cd4+ and cd8+ t cells is one of the symptoms in covid-19 cases related to hiv infection diao et al., 2020) . besides, over circulation of proinflammatory cytokines and chemokines, including interleukins (il-1β, il1ra, il2, il4, il5, il6, il7, il8, il9, il10, il12p70, il13, il15, il17a) , eotaxin, basic fibroblast growth factor (fgf2), granulocyte-colony stimulating factor (gcsf), granulocytemacrophage colony-stimulating factor (gmcsf), interferon-gamma (ifnγ), ifn-γ-inducible protein-c-x-c motif chemokine ligand 10 (cxcl10), interleukins (il-1, il-6, il-8) with interferongamma (ifn-γ), and transforming growth factor-beta (tgf-β) occurred in the acute stage of sars-cov infection triggering the cytokine storm (cameron et al., 2008; huang et al., 2005) . chloroquine is a primary therapeutic drug used to prevent and treat patients infected with malaria. however, the more extended use of chloroquine could produce severe side effects, such as cardiomyopathy (bernstein, 1991; ratliff et al., 1987) and the presence of some reports on macular retinopathy as a minor risk caused by this drug (cubero et al., 1993) . a survey of the patients infected with sars-cov-2 for adverse side effects of chloroquine treatment is yet to be conducted. chloroquine was considered the best therapeutic drug choice available for treating sars-cov-2 infected patients in hospitals. recently, the who has advocated a solidarity clinical trial for covid-19 treatments. the solidarity clinical trial will compare four treatment choices, including chloroquine/hydroxychloroquine, remdesivir, lopinavir/ritonavir, and lopinavir/ritonavir plus interferon beta-1a against standard therapy and determine their relative efficacy against covid-19 (world health organization, meuri). (cockrell et al., 2016) . they found that the enzyme carboxylesterase 1c (ces1c) was deleted and receptor (dipeptidyl peptidase 4, hdpp4) of humanized mers-cov was expressed in order to increase the pharmacokinetics of nucleotide prodrugs. another study conducted by agostini et al. (2018) reported that two mutations were recognized, such as v553l and f276l in the rnadependent rna polymerase gene of the virus after 23 passages of remdesivir drug (agostini et al., 2018) . they also observed that these changes in amino acid reduced the burden of virus and lessened the pathogenesis of sars-cov in mice. in recent months, the prophylactic efficacy of remdesivir was also tested in a rhesus macaque (non-human primate) mers-cov infection model (de wit et al., 2020) . when the treatment with prophylactic remdesivir was started 24 hours before inoculation, mers-cov was found to be inhibited from replicating in the respiratory tissues and prevented from causing clinical disease, which inhibited the development of lung lesions. the same findings were obtained when treatment with remdesivir drug was initiated 12 hours after the virus inoculation (de wit et al., 2020) . the remdesivir therapeutic drug safety data are readily available for humans (mulangu et al., 2019) ; hence, human studies can be conducted to assess the effectiveness of this compound towards novel coronaviruses. the food and drug administration (fda) approved the therapeutics against mers-and sars-cov that have been assessed for antiviral activity. for instance, lopinavir (lpv), a protease inhibitor of hiv-1, was tested to combine with ritonavir (rtv) in order to enhance the half-life of lpv. the combination of these two drugs (lpv/rtv) was found to be more effective in patients against sars-cov and in-vitro study. the calculated ec50 was 4 µg/ml for the fetal rhesus kidney-4 cells (chu et al., 2004) . meanwhile, chan and coworkers reported that lpv/rtv also declined clinical sores, weight loss, and progression of disease in marmosets infected with mers-cov (chan et al., 2015) . however, the antiviral activity of lpv towards mers-cov in the in-vitro study remains controversial. another study conducted by chan et al., (2013) found no optimal ec50 in the vero cells, whereas ec50 was reported to be 8 µm in the huh7 cells in a different study (de wilde et al., 2014) . it was found that infections with mers-cov were mediated by replication of the virus and host inflammatory responses after clinical observations in both humans and animals. those studies had suggested the use of combination therapies including interferons i and ii. however, interferon-beta (ifnb) reduced the replication of mers-cov in the tissue culture and showed the best efficacy with 1.37-17 iu/ml ec50s (chan et al., 2013; hart et al., 2014) . from the study on marmosets infected j o u r n a l p r e -p r o o f with mers-cov, chan et al., (2015) reported the clinical improvement with the use of lpv/rtv with ifnb. also, the clinical randomized control trials were started in saudi arabia in order to determine the efficacy of combinations of therapies, such as ifnb and lpv/rtv in improving the clinical outcomes against mers-cov (arabi et al., 2018) . notably, china has launched another controlled trial to test the effectiveness of ifnα-2b and lpv/rtv in hospitalized sars-cov-2 patients (chictr2000029308). in addition, the therapeutic and prophylactic activities of lpv/rtv-ifnb and remdesivir were compared in a transgenic mouse model infected with mers-cov (sheahan et al., 2020) . it was found that remdesivir reduced lung viral titers, virus replication, and improved the pulmonary function. at the same time, prophylactic combined therapeutics, such as lpv/rtv-ifnb, only resulted in a moderate decline in viral titers and did not produce any effects on other parameters of disease. the combined therapy boosted pulmonary function but did not influence viral replication (sheahan et al., 2020) . thus, it was observed that remdesivir has shown to be effective for treating another antiviral prodrug, ribavirin is a guanosine analogue used to treat many infections caused by viruses, such as hepatitis c respiratory syncytial virus, and patients coinfected with hiv and hepatitis b. in 1980, it was launched to the marketplace for treating the respiratory syncytial virus, especially in children. in most cases, it is used in combination with interferon (ifn). falzarano and co-authors found promising results when ribavirin was combined with ifnα-2b against the rhesus macaque model infected with mers-cov (falzarano et al., 2013) , but there have been some contradictory data on the mers-cov infected patients treated with ifnα2a or ifnβ1 and ribavirin (arabi et al., 2017) . besides, ribavirin also produces adverse side effect such as decreasing haemoglobin concentrations in patient with respiratory disorder that could not be appropriate for use against sar-cov-2. in addition, various other antiviral therapeutic drugs/cocktails, including favipiravir, nitazoxanide, ganciclovir, acyclovir/penciclovir, and the latest fda-approved ivermectin are listed in table 3 . in summary, the repurposing of preexisting antiviral therapeutic drugs is finite, so it is almost inevitable that both new and repurposed drugs will be required to treat covid-19. therapeutic drug choices in response to covid-19 are urgently required and fortunately, some of the preexisting antiviral therapeutics are already progressing into human clinical trials. it is important for pwh to regularly attend their healthcare providers and adhere to therapy (national institute of allergy and infectious disease, 2020). the treatment of pwh can be affected due to stay at home orders enforced by countries worldwide. for instance, several medical practitioners canceled their appointments with hiv suspected people in the usa and switched to telehealth appointments to follow the who guidelines (ives, 2020) . however, telehealth programs are restricted in the wide range of resources that can be given to the customers (siwicki, 2020) , therefore, pwh may not be able to completely access the services needed for hiv therapy. in addition, many pwh patients may not be able to get access to telehealth services for many reasons, such as limited access to technology and lack of knowledge about telehealth that could hinder their therapy progression. pwh are more vulnerable group to contract opportunistic infections, such as tuberculosis, toxoplasmosis, pneumonia, etc. (department of veteran affairs, 2020), than those without immunocompromised systems. hiv-infected people who have suffered from any other disorders may undergo delayed treatment because of covid-19. this can happen in already taxed healthcare system due to hospital overcrowding. moreover, pwh who seek out emergency treatment can face a high risk of experiencing covid-19 among other disorders in the healthcare systems (collins, 2008) . the occurrence of person-to-person transmission for covid-19 is increasing, which means that . they propositioned that persons infected with hiv need to be considered as a vulnerable group for covid-19. nonetheless, no robust evidence of any interrelationship between these two viral infections is yet to be identified. despite the high number of patients infected with hiv as well as the remarkable number of patients with the novel covid-19 disease, which extends to cover a wide zone in asia and worldwide. there are very few reports on the co-infection of hiv and sars-cov. in addition, antiviral therapeutic drugs are broadly used for treating hiv-infected patients, and these drugs have potential to be used against sars-cov-2 (felber et al., 1990; savarino et al., 2003) . thus, the patients infected with hiv who are receiving anti-hiv therapeutic drugs might be at a lower risk for covid-19 compared to the general population. some antiviral therapeutic drugs, including chloroquine, remdesivir, ribavirin, lopinavir/ritonavir, and now fda-approved ivermectin and others (summarized table 3) have the potential to treat patients infected with sars-cov-2. at the same time, more clinical trials are required in order to obtain robust data. also, further studies are urgently required to explore the pathogenicity, mechanisms, and transmission of novel coronavirus. to obtain a better insight of the novel virus, countries should strive to provide more reliable and accurate data by transparency and sharing of data, and more research studies need to be conducted on the reported cases. therefore, countries should continue to work towards developing preventive measures to minimize both the transmission and number of infected patients. in addition to unravelling the uncertainty of the mechanisms of viral replication and host cell entry, which will provide the fundamental knowledge for future research into the development of targeted vaccines and antiviral therapeutic drugs. with continuing efforts to curb the widespread transmission of covid-19 globally, we hope that the novel coronavirus pandemic may alleviate after a few months. in summary, there is an urgent need to develop a new broad-spectrum antiviral therapeutic agent that will be effective to fight against not only the novel respiratory 2019 coronavirus, but also to prepare for a possibly similar virus outbreak in the future. for -if there is any message coming out from the latest outbreaks, it is almost certain that it will happen again.‖ mansab (harrison et al., 2020) j o u r n a l p r e -p r o o f journal pre-proof coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease treatment of middle east respiratory syndrome with a combination of lopinavir/ritonavir and interferon-β1b (miracle trial): statistical analysis plan for a recursive two-stage group sequential randomized controlled trial ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study evidence for continuing cross-species transmission of sivsmm to humans: characterization of a new hiv-2 lineage in rural côte d'ivoire incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china presumed asymptomatic carrier transmission of covid-19 ocular safety of hydroxychloroquine death of cd4+ t-cell lines caused by human immunodeficiency virus type 1 does not depend on caspases or apoptosis aids at 21: media coverage of the hiv epidemic 1981-2002. the nation broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase human immunopathogenesis of severe acute respiratory syndrome (sars) discovery of a new human t-cell lymphotropic virus (htlv-3) in central africa the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro will there be a cure for ebola? epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan implications of the hiv-1 rev dimer structure at 3.2 å resolution for multimeric binding to the rev response element reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) cell death by pyroptosis drives cd4 t-cell depletion in hiv-1 infection treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques making sense of multifunctional proteins: human immunodeficiency virus type 1 accessory and regulatory proteins and connections to transcription the role of tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation feedback regulation of human immunodeficiency virus type 1 expression by the rev protein hiv replication is associated to inflammasomes activation, il-1β, il-18 and caspase-1 expression in galt and peripheral blood targeting the nlrp3 inflammasome in severe covid-19 a tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses innate sensing of hiv-1 assembly by tetherin induces nfκb-dependent proinflammatory responses breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies structure-function relationships in hiv-1 nef first known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the usa the first two cases of 2019-ncov in italy: where they come from quinoline-based antimalarial drugs: a novel class of autophagy inhibitors illustrations of the hiv life cycle severe acute respiratory syndrome-related coronavirus-the species and its viruses, a statement of the coronavirus study group temporal proteomic analysis of hiv infection reveals remodelling of the host phosphoproteome by lentiviral vif variants the coronavirus nucleocapsid protein is adp-ribosylated aids as a zoonosis: scientific and public health implications kaposi's sarcoma and pneumocystis pneumonia among homosexual men--new york city and california interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays coronavirus puts drug repurposing on the fast track origins of hiv and the evolution of resistance to aids setting the stage: host invasion by hiv strategies of development of antiviral agents directed against influenza virus replication clinical features of patients infected with 2019 novel coronavirus in wuhan an interferon-γ-related cytokine storm in sars patients telemedicine surges, fueled by coronavirus fears and shift in payment rules hiv-1 tat reprograms immature dendritic cells to express chemoattractants for activated t cells and macrophages the natural history of hiv-1 and hiv-2 infections in adults in africa: a literature review oseltamivir for influenza in adults and children: systematic review of clinical study reports and summary of regulatory comments complement receptor c5ar1 inhibition reduces pyroptosis in hdpp4-transgenic mice infected with mers-cov sars-cov-2 and hiv mother-to-child transmission of hiv infection and its associated factors in ethiopia: a systematic review and meta-analysis chimpanzee reservoirs of pandemic and nonpandemic hiv-1 cbfβ stabilizes hiv vif to counteract apobec3 at the expense of runx1 target gene expression virus taxonomy. ninth rep int comm taxon viruses pathogenesis of hiv infection: total cd4+ t-cell pool, immune activation, and inflammation the multifaceted role of cd4+ t cells in cd8+ t cell memory lentiviral vif degrades the apobec3z3/apobec3h protein of its mammalian host and is capable of cross-species activity activation of the atr pathway by human immunodeficiency virus type 1 vpr involves its direct binding to chromatin in vivo novel coronavirus disease (covid-19): the first two patients in the uk with person to person transmission early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia gs-5734) protects african green monkeys from nipah virus challenge genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle inflammasome control of viral infection are pangolins the intermediate host of the 2019 novel coronavirus (sars-cov-2)? decreased t cell populations contribute to the increased severity of covid-19 expression and function of t cell homing molecules in hodgkin's lymphoma influenza treatment with oseltamivir outside of labeled recommendations coronavirus genomic rna packaging coronavirus disease 2019 self-care instructions for people not requiring hospitalization for coronavirus disease 2019 (covid-19) species-specific vulnerability of ranbp2 shaped the evolution of siv as it transmitted in african apes a randomized, controlled trial of ebola virus disease therapeutics pathogenesis of hiv infection impact of trim5α in vivo seeing your healthcare provider. hiv gov a structural analysis of m protein in coronavirus assembly and morphology a synthetic serine protease inhibitor, nafamostat mesilate, is a drug potentially applicable to the treatment of ebola virus disease enfuvirtide: a review of its use in the management of hiv infection structure and immune recognition of trimeric pre-fusion hiv-1 env transmission routes of 2019-ncov and controls in dental practice the latest evidence for possible hiv-1 curative strategies a cross-sectional study of the role of hiv/aids knowledge in risky sexual behaviors of adolescents in nigeria diagnosis of chloroquine cardiomyopathy by endomyocardial biopsy going global-travel and the 2019 novel coronavirus nitazoxanide: a first-in-class broad-spectrum antiviral agent nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus differential regulation of nf-κb-mediated proviral and antiviral host gene expression by primate lentiviral nef and vpu proteins effects of chloroquine on viral infections: an old drug against today's diseases coronavirus envelope protein: current knowledge the extended impact of human immunodeficiency virus/aids research biochemical mechanisms of hiv induced t cell apoptosis cold spring harb perspect med origins of hiv and the aids pandemic antiviral drugs against alphaherpesvirus broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov a review of coronavirus disease-2019 (covid-19) telemedicine during covid-19: benefits, limitations, burdens, adaptation. health care news coronaviruses as dna wannabes: a new model for the regulation of rna virus replication fidelity synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein the epidemic emergence of hiv: what novel enabling factors were involved? cytokine storm in covid-19: the current evidence and treatment strategies mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir evolution, distribution, and diversity of immunodeficiency viruses. dyn immune act viral dis a novel coronavirus outbreak of global health concern monitored emergency use of unregistered and experimental interventions (meuri discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavi systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov preferential apoptosis of hiv-1-specific cd4+ t cells hiv-1 vpr induces interferon-stimulated genes in human monocyte-derived macrophages angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target reply to comments on'co-infection of sars-cov-2 and hiv in a patient in wuhan city a novel coronavirus from patients with pneumonia in china the coronavirus replicase human immunodeficiency virus type 1 vpr induces dna replication stress in vitro and in vivo emerging genetic diversity among clinical isolates of sars-cov-2: lessons for today the proximal origin of sars-cov-2 mapping the genomic landscape & diversity of covid-19 based on > 3950 clinical isolates of sars-cov-2: likely origin & transmission dynamics of isolates sequenced in india the authors declare no conflicts of interest coronaviruses cnscentral nervous system sars-cov-2 mers-cov severe acute respiratory syndrome coronavirus 2 middle east respiratory syndrome coronavirus ards acute respiratory distress syndrome who world health organization hivshuman immunodeficiency viruses siv immunodeficiency virus gp41glycoprotein-41 ccr5 c-c chemokine receptor-5 cxcr4 c-c chemokine receptor-4 (ifi)-16interferon-gamma induced protein-16 il-1βinterleukin-1β ace2angiotensin-converting enzyme-2 fgf2basic fibroblast growth factor 2 gcsf granulocyte-colony stimulating factor ifnγ interferon-gamma ip10ifn-γ-inducible protein-10 mcp1monocyte chemoattractant protein 1 mip1amacrophage inflammatory proteins tnf-α tumor necrosis factor-alpha j o u r n a l p r e -p r o o f generally, 5'-utr-replicase-s-m-n-utr-3' (fung et al., 2020; replicase gene overlapped with orfs 1a and 1b that codes two huge polyproteins, pp1a and pp1ab that are processed autoproteolytically into 16 non-structural proteins involved in genome transcription and replication. (sheikh et al., 2020; singh et al., 2020; chan et al., 2020; clavel et al., 1986; zahoor et al., 2014) morphogenesis assembly of virion takes place at the smooth intracellular membranes of the endoplasmic reticulum/early golgi compartments. (masters, 2019) j o u r n a l p r e -p r o o f (geyer et al., 2001; sauter et al., 2015) tat  an hiv transcription factor that supports the generation of full-length viral mrnas. induces transcription and secretion of chemokines which attracts t cells and monocytes increasing infectivity.  (feinberg et al., 1991; izmailova et al., 2003) rev  directs exports of viral messages that are translated into viral proteins, providing genomic rna for packaging and budding virion j o u r n a l p r e -p r o o f key: cord-017412-1avevzya authors: losada, liliana; ghedin, elodie; morris, alison; chu, hong wei; nierman, william c. title: the human lung microbiome date: 2010-10-11 journal: metagenomics of the human body doi: 10.1007/978-1-4419-7089-3_7 sha: doc_id: 17412 cord_uid: 1avevzya the human lower respiratory tract is considered sterile in normal healthy individuals (flanagan et al., 2007; speert, 2006) despite the fact that every day we breathe in multiple microorganisms present in the air and aspirate thousands of organisms from the mouth and nasopharynx. this apparent sterility is maintained by numerous interrelated components of the lung physical structures such as the mucociliary elevator and components of the innate and adaptive immune systems (discussed below) (reviewed in (diamond et al., 2000; gerritsen, 2000)). however, it is possible that the observed sterility might be a result of the laboratory practices applied to study the flora of the lungs. historically, researchers faced with a set of diseases characterized by a changing and largely cryptic lung microbiome have lacked tools to study lung ecology as a whole and have concentrated on familiar, cultivatable candidate pathogens. the human lower respiratory tract is considered sterile in normal healthy individuals (flanagan et al., 2007; speert, 2006) despite the fact that every day we breathe in multiple microorganisms present in the air and aspirate thousands of organisms from the mouth and nasopharynx. this apparent sterility is maintained by numerous interrelated components of the lung physical structures such as the mucociliary elevator and components of the innate and adaptive immune systems (discussed below) (reviewed in (diamond et al., 2000; gerritsen, 2000) ). however, it is possible that the observed sterility might be a result of the laboratory practices applied to study the flora of the lungs. historically, researchers faced with a set of diseases characterized by a changing and largely cryptic lung microbiome have lacked tools to study lung ecology as a whole and have concentrated on familiar, cultivatable candidate pathogens. with the availability of new technologies for cultivation-independent analysis of microbial populations, it is now possible to follow individuals by sampling their lung microbiome sequentially during episodes of disease and recovery in order to identify associations between the lung microbiome and health and disease. any respired contaminating particles or pathogens that evade the lung's physical and immune barriers are usually eliminated by dendritic cells and alveolar macrophages that deliver them into local draining lymph nodes. macrophages kill invading microorganisms while en route to the draining lymph nodes, and in some cases at the nodes themselves (bozza et al., 2002; kirby et al., 2009) . thus, it is not unusual to isolate viable bacteria or fungi from "normal" lung tissue (lass-florl et al., 1999) , and the term "sterile" should be applied with caution. it is perhaps more accurate to say that there is no resident flora that permanently colonizes normal lungs. even normal healthy lungs are not microbe free all the time. lower airway infections by bacteria, viruses, or fungi are among the most prevalent causes of transmissible disease in humans, with two to three million community-acquired (non-hospital-acquired) cases per year in the united states (segreti et al., 2005) . in 2006, the number of deaths attributed to pneumonia (bacterial and viral) and influenza in the united states was 60,000 (gao et al., 2008) www.cdc.gov/ nchs/fastats/deaths.htm). in 2009, nearly 9.3 million new cases of tuberculosis were reported around the world (http://www.who.int/mediacentre/factsheets/ fs104/en/index.html). with proper treatment, the lungs of individuals with these infectious diseases will revert to their normal "sterile" state. little is known about the composition of the microbial population of the upper and lower airways in health or disease. it is likely, given the multiple microorganisms already implicated in chronic lung diseases such as chronic obstructive pulmonary disease (copd) , that there are other undetected organisms and that there are complex relationships between multiple pathogens involved that are not currently understood. a few studies have examined microbial species in limited numbers of normal subjects and patients with various respiratory disorders. one study using 16s rdna clone libraries and microarrays did not detect any bacteria in the lungs of patients without respiratory disease who were briefly intubated for surgery (flanagan et al., 2007) . the same study reported that all patients intubated for longer periods had detectable 16s rdna and that the bacterial diversity present decreased during antibiotic usage. another study used 16s rdna amplification to identify bacterial species in 16 patients with ventilator-associated pneumonia (bahrani-mougeot et al., 2007) . this study identified bacterial pathogens not seen using conventional culture techniques, especially anaerobes, and found that oral bacteria could be detected in the lung. in one study, sputum samples from 25 cystic fibrosis (cf) patients were analyzed using 16s gene profiling and the authors identified an average of 7.2 species present per subject (bittar et al., 2008) . viruses have also been examined in nasal lavages in both asthmatic and normal subjects with cold symptoms using the virochip microarray (kistler et al., 2007) . the microarray technology identified more viruses than conventional culture methods and had excellent sensitivity and specificity compared with pathogen-specific polymerase chain reaction (pcr). an unexpected diversity of human coronavirus and rhinovirus strains was discovered in the subjects. so if the lung is generally sterile, why do some individuals become chronically colonized? what organisms colonize the lungs? those with physically compromised airways or immune system deficiencies are subject to chronic microbial colonization of their airways and to high-frequency episodes of viral, bacterial, or fungal lower respiratory infections. perhaps in no other body site is the direct relationship between disease and microbiome more explicit than in the lungs where there is a distinct and obvious microbial difference between normal and diseased individuals. the lower respiratory tract, composed of the trachea and lungs, is quite different in structure and function from the upper respiratory tract, which is highly colonized by microorganisms. the lungs themselves are divided into different sections according to their function and structures: the bronchi, bronchioles, and alveoli. bronchi and bronchioles are primarily conductive airways surrounded by thick cartilage that allow easy airflow into the parenchyma (or alveolar tissue) of the lungs, where gas exchange occurs. conductive airways are covered in ciliated epithelium interspersed with different types of secretory cells that release mucins, immunomodulatory proteins, surfactants, and proteases. together, the physical and chemical barriers protect against physical and biological damage by establishing a mucociliary elevator, which brings about an upward transport of a mucus stream for the lungs (fraser, 2005) . the secretory cells decrease in proportion from 20 to 30% in the trachea to less than 1% in the distal and alveolar parts of the lungs. in addition to the physicochemical protection provided by cilia and mucus, the epithelium is also protected by several immune cells, including dendritic, langerhans, t lymphocytes, and mast cells, that respond to inhaled antigens establishing a robust immunity (fraser, 2005) . it is thought that in conjunction with the physical barriers provided by the nose and upper respiratory mucosa, these defenses are enough to maintain sterility in the lower respiratory tract. the vast majority of the lung surface epithelium, however, is alveolar. it is estimated that 87% of the total volume of the lungs is alveolar, with only 6% of this being tissue and the remainder gas (stone et al., 1992) . the primary role of this tissue is to carry out gas exchange. the epithelium is mostly a continuous single layer of cells overlying a thin interstitium, which contains numerous capillaries that supply ample blood for gas exchange (fraser, 2005; stone et al., 1992) . unlike the epithelium in the conducting airways, the respiratory epithelium is not ciliated or protected by mucus. instead, it is covered by surfactant proteins that maintain the surface tension for efficient gas exchange. the lack of mucus or secretory cells is compensated by the presence of alveolar macrophages, mast cells, lymphocytes, dendritic cells, and other monocyte-like cells that protect the epithelium from potential pathogens and help maintain sterility. recent world health organisation (who) figures rank lower respiratory diseases second in an assessment of the burden of disease worldwide (http://www.who.int/ respiratory/en/). in 2006, 124, 500 people died in the us due to chronic lower respiratory disease (www.cdc.gov/nchs/fastats/deaths.htm). chronic respiratory diseases include: asthma, copd, cf, and bronchiectasis. these diseases generally lead to impaired clearance and function of the mucociliary elevator and/or the immune protection of the lung. in addition, immune deficiency such as that caused by the human immunodeficiency virus (hiv) also disrupts the typical immune homeostasis in the lungs. without the normal protective barriers, the lungs fall victim to persistent and severe colonization that can ultimately lead to death, particularly for cf patients. as discussed below, the lung microbiome in each of these diseases is very different from normal individuals. the data discussed in the following sections demonstrate a clear link between microbial colonization and severity of disease symptoms. it is unclear, however, what exact role these different microbial populations play in initiating and enhancing the progress of such chronic respiratory diseases. lastly, in some cases, the data hint that some population structures might actually be protective against further decline, but much more research needs to be conducted in this area to make a definitive claim. this chapter discusses the methods for sampling and characterizing the microbiome of the lungs. in addition, it reviews the current status of our understanding of the lung microbiome in asthma, idiopathic bronchiectasis, cf, copd, and during immune deficiency due to hiv infection. several procedures have been developed for sampling the microbial population of the human lung econiche. in order of increasing invasiveness they are sputum induction, bronchoalveolar lavage (bal), bronchial brushing, endobronchial biopsy, and transbronchial biopsy. sputum induction by inhalation of hypertonic saline is a noninvasive method to obtain samples from the lower respiratory tract for cell and microbial analysis (bickerman et al., 1958) . the quality of samples varies and can be scored on the volume of the obtained sputum plugs and the level of salivary contamination as measured by squamous cells observed by microscopy. bal is a procedure in which a bronchoscope is passed through the mouth or nose into the lungs and saline is instilled into a segment of the lung and then recollected for examination (henderson, 1994; reynolds and chretien, 1984) . bal is most commonly used to diagnose infections in both immunocompetent and immunosuppressed patients. bal is the most common procedure for sampling the lower respiratory system microbial colonization/infection status, to sample the components of the epithelial lining fluid, and to determine the protein composition of the airways. it is often used in evaluating the patient's lung immunological status by sampling cells and pathogen levels. bal is an invasive procedure and thus is less ideal for research purposes. bronchial brushing provides access to cells and microbes that are adherent to the luminal surfaces of the lower airways. in this procedure, a flexible fiber optic bronchoscope is used for brushing a targeted lesion or site (fennessy, 1967; zavala et al., 1973) , where induced sputum and bal procedures will allow sampling of cells and microbes that can be washed from the lumen surface, brushing will recover adherent cells (e.g., bronchial epithelial cells) and microbes. recently, brushing techniques have been developed to sample distal lung (i.e., small airway) epithelial cells and associated microbes (ammous et al., 2008) . this technique will enable investigators to further study the microbiome in lung diseases such as copd. endobronchial biopsy involves using the fiber optic bronchoscope to identify appropriate target sites in the lung and obtain large airway tissue samples using inserted alligator forceps, cup forceps, or curette passed through the endoscope's central channel. this procedure poses a higher risk than bal but allows sampling the invasive microbes within the airway tissue (scott et al., 1991; trulock et al., 1992) . transbronchial biopsy, the most invasive of these sampling procedures, is routinely performed for clinical care and allows clinicians and researchers to obtain distal (small) airway tissues as well as alveolar tissues. this procedure has been safely done by several research groups (balzar et al., 2005) and will likely further our understanding of the microbiome in human distal lung tissues, but carries a significant risk of complications. standard microbiological and virologic methods detect only a small proportion of the bacteria and viruses present in various body sites because the great majority of these organisms are uncharacterized or uncultivable. to understand the real diversity, culture-independent methods, such as sequencing, are thus a necessity. sequenced-based identification of microbial species is facilitated by decreased costs of sequencing, and the availability of next-generation sequencing technologies, further enhances the capacity to generate large amounts of data. for the identification of bacterial species within an environment, the amplification of 16s rrna genes (or 16s rdna) using universal primers are useful for diversity characterization because this genetic locus is present in all bacterial species (relman et al., 1991) . the nine hypervariable regions of the 16s rdna can be used for bacterial species identification (chakravorty et al., 2007; rokas et al., 2007) with some regions having better discriminatory value than others. the sequencing and phylogenetic analysis of bacterial 16s rrna derived from microbiome samples has been the primary method used to investigate bacterial diversity in the human body (bik et al., 2006; dekio et al., 2005; eckburg et al., 2005; gao et al., 2007; hugenholtz et al., 1998; hyman et al., 2005; zhang et al., 2006) . these studies have revealed a far higher level of diversity than conventional culture techniques (aly et al., 1976; bik et al., 2006; dekio et al., 2005; kazor et al., 2003; korting et al., 1988; kroes et al., 1999; paster et al., 2001) . these studies revealed that the majority of bacterial sequences correspond to uncultivated species and novel organisms. there was significant intersubject variability and variability between stool and mucosal microbial populations. for example, recent studies by blaser and colleagues at new york university have demonstrated substantial changes in the ratio of the genus streptococcus to propionibacterium in skin samples from healthy persons and in normal skin of patients with psoriasis (ratio = 0.4; n = 2, 649 clones), and from psoriatic lesion samples (ratio = 5.0; n = 1, 314 clones; p = 0.01) (gao et al., 2008) . in a lung study, bacterial diversity was analyzed in the endotrachael aspirates from seven intubated patients colonized with pseudomonas aeruginosa using both sequencing from 16s rrna clone libraries and an oligonucleotide microarray termed as the phylochip (flanagan et al., 2007) . controls were subjects briefly intubated for elective surgery. bacteria were not detected by either method in samples from the controls. sequencing from the clone libraries detected the presence of many orally, nasally, and gastrointestinal associated bacteria including known pathogens. the phylochip detected the same organisms and many additional bacterial groups present at low abundance. following antibiotic therapy, the bacterial populations' diversity decreased and was dominated by a single respiratory pathogen. in six of the seven patients, the dominant species was p. aeruginosa in spite of targeting this organism with antibiotics to which it was reportedly sensitive. the authors hypothesize that the loss of population diversity may directly contribute to pathogenicity, persistence, and development of pneumonia. similarly, amplification of regions from the 18s and internal transcribed spacer (its) regions of the rrna, a conserved fungal gene, allows discrimination of fungal species (fujita et al., 2001; makimura, 2001) . a preliminary study was undertaken to examine the efficacy of a community sequencing method to identify the fungal species in bal lung samples from 23 human subjects. the fungal its1-5.8s-its2 region was amplified and the results showed that 4 of 23 patients (17%) had fungal dna levels that could be reproducibly detected by pcr. the detected fungi included aspergillus fumigatus, candida tropicalis, and penicillium digitatum, among others (denning, unpublished) . these data agree with a culture-based study that showed that 63% of their sample population had evidence of pulmonary fungal colonization (lass-florl et al., 1999) , most commonly with a. fumigatus and other candida species, and also zygomycetes. the results also demonstrate that rrna sequencing is a viable platform for characterization of fungal communities in the human body. although there are no conserved genes that can be targeted for determination of viral diversity, whole genome shotgun of a sample enriched for viruses (such as by filtering) can lead to an effective characterization of viral communities (angly et al., 2006) . hundreds of viral genome sequences can be completed in a single sequencing reaction run using the gs-flx (454/roche) sequencing platform. using this technology and a random priming-based method, referred to as sequence independent single primer amplification (sispa), near-full-length genomes of rna or dna viruses can be sequenced. sispa can be used to sequence known and unknown viral genomes (djikeng et al., 2008) . this viral sequencing methodology can potentially be adapted for the determination of viruses within bal by enrichment using nuclease treatment and filtration followed by taking the extracted total rna and dna through the sispa process followed by sequence comparisons to known viruses. the initial studies of small 16s rrna described above hinted at great diversity within the human microbiome, yet it left important questions unanswered such as the identity of the nondominant community members and their biological roles. the applications of shotgun techniques to the study of the human microbiome (kurokawa et al., 2007; manichanh et al., 2006; zhang et al., 2006) again highlight the extent of microbial diversity associated with the human body while revealing much more of the identity and biology of nonculturable microorganisms. as a result of reduced costs and improved sequencing technologies, it is possible to perform in-depth metagenomic surveys of the human body's microbial diversity beyond the 16s rrna surveys. metagenomics, a term introduced in 1998, describes the functional and sequence-based analysis of total microbial genomes from environmental samples (handelsman et al., 1998) . metagenomics uses techniques that resemble the "whole genome shotgun" approach of single genome sequencing, but it is not limited to a single species. human metagenomics has provided insight into the complex composition of the microbiome of these several body sites, and this information has allowed us to draw tentative conclusions about the relationship between specific microbiomes and health. the human microbiome is composed of multiple "ecological niches", including the mouth (kroes et al., 1999; paster et al., 2001) , esophagus (zhang et al., 2006) , stomach (bik et al., 2006) , intestine , skin (gao et al., 2007) , and vagina (zhou et al., 2004) . our understanding of the overlap and the degree of communication between them is rudimentary at best. perhaps the most extensively studied has been the human gut microbiome where the interaction of the gut microflora, independently or through interaction with the genetic makeup of the host plays a role in obesity, crohn's disease, and ulcerative colitis (frank et al., 2007; gophna et al., 2006; turnbaugh et al., 2006) . asthma is a complex disease characterized by chronic inflammation in the lungs and reversible narrowing of the airways. symptoms include dyspnea, coughing, wheezing, airway hyper-reactivity, chronic eosinophilic atopy, and mucus hyper secretion (busse and lemanske, 2001) . about 20 million people in the us have been diagnosed with asthma; 9 million of them are children. asthma causes 4,000 deaths per year in the us and 11 million exacerbations. asthma is caused by environmental and genetic factors (martinez, 2007) , with asthma attacks resulting from immune responses to inhaled allergens. the majority of asthma exacerbations are caused by viral infections (krishnan et al., 2006) . atypical bacterial infections have also been associated with asthma exacerbations and with chronic asthma (johnston and martin, 2005; martin et al., 2001) . in susceptible individuals, the development of asthma has been associated with bacterial colonization in neonates (bisgaard et al., 2007) and viral and bacterial infections (wu and chu, 2009 ). there is abundant evidence testifying to the importance of microbes to the development and maintenance of asthma. a recent publication using a bacterial gene sequencing method suggests a disordered microbiome in asthmatic airways (hilty et al., 2010) . in the developed world there has been an increased focus on predisposing factors for asthma due to its rapidly increasing prevalence, now affecting up to a quarter of urban children (lilly, 2005) . asthma is known to be caused by environmental and genetic factors (martinez, 2007) . these factors determine asthma severity and how easily it can be treated (martinez, 2007) . many associations with asthma have been detected including exposure to cigarette smoke (thomson et al., 2004) , caesarean section birth relative to natural birth (thavagnanam et al., 2008) , early viral respiratory infections (gold and wright, 2005; harju et al., 2006) , early in life antibiotic use (marra et al., 2006) , and living in the us (gold and wright, 2005) . one theory for the cause of the increase in asthma incidence is the hygiene hypothesis (strachan, 1989) , that the rise in prevalence of asthma is a direct consequence of the success of modern hygienic practices in preventing childhood infections. this hypothesis is supported by numerous studies that have shown that children coming from a less hygienic environment have less asthma and other allergenic diseases (ball et al., 2000; celedon et al., 1999; jarvis et al., 1997) . in addition, alterations in innate immune system genes have been shown to be associated with the inception and development of asthma. these genes include the toll-like receptors and other genes such as mbl, mylk, defb1, jun, inf-î±5, and nos2a reviewed in wu and chu (2009) . asthma exacerbations have long been associated with viral infections (pattemore et al., 1992) . more recently, the use of reverse transcriptase pcr has greatly facilitated the identification of the exacerbation-associated virus. studies using this tool have suggested that 75-80% of asthma exacerbations are caused by virus infections (wark et al., 2002) . rhinovirus (rv) infections during early childhood are associated with the development of asthma, lower respiratory tract infections, and wheezing (jackson et al., 2008; lemanske et al., 2005) . they are also associated with hospitalization for asthma in adults (venarske et al., 2006) . a separate study revealed that patients with allergic asthma infected with rv had increased admissions to hospitals and that dust mite allergen was the primary allergen when these patients were skin tested with a panel of aeroallergens (green et al., 2002) . respiratory syncytial virus (rsv) in infants causes lower respiratory infection leading to pneumonia and bronchiolitis. rsv bronchiolitis is the leading cause of wheezing in infants and young children, and children infected with rsv resulting in bronchiolitis are more likely to develop wheezing and asthma later in childhood (peebles, 2004) . similarly, the human metapneumovirus (hmpv) was first isolated from children in 2001 and has been found to be associated with asthma exacerbations in both children under 5 years of age and adults (foulongne et al., 2006; williams et al., 2004) . mycoplasma pneumoniae and chlamydia pneumoniae are bacteria that attach to airway epithelial cells and cause cell damage. infections by these bacteria have been shown to be associated with asthma exacerbations (johnston and martin, 2005; lieberman et al., 2003; martin et al., 2001) . using a pcr assay, 31 of 55 patients with asthma were positive for either of these bacteria in lung tissue or bal, suggesting that some level of colonization by these bacteria may be common in asthma patients (martin et al., 2001) . studies in a mouse model suggest that preexisting allergic inflammation impairs the ability to upregulate tlr-2 and il-6 in the lungs, leading to decreased clearance of m. pneumoniae and an increase in airway inflammation (kraft et al., 2008) . evidence is accumulating that infections are associated with the induction and development of asthma. first, long-term cohort studies on the development of asthma show that most childhood asthma begins in infancy. the first episode of wheezing begins before the age of 3 and is frequently associated with lower respiratory tract viral infections, usually rsv, but also rv (gern et al., 2000; sigurs et al., 2005) . these infectious episodes and associated wheezing are strong predictors for the development of childhood asthma and atopy (devulapalli et al., 2008; kusel et al., 2007; martinez et al., 1995; singh et al., 2007) . second, many studies have associated viral infections with asthma prevalence in children (devulapalli et al., 2008; jackson et al., 2008; kusel et al., 2007; papadopoulos and kalobatsou, 2007; sigurs et al., 2005; singh et al., 2007; williams et al., 2004) . lastly, wu et al. have provided evidence to suggest that viral infections have a causal role in asthma initiation and development where they show that viral infection during the first 4 months of age is strongly correlated with the development of asthma by age 5 (wu et al., 2008) . only one-third of children with childhood wheezing and asthma, however, will develop persistent asthma symptoms in adulthood (gerritsen, 2002; taylor et al., 2005; vonk et al., 2004) . management of the symptoms with corticosteroid therapy is effective but may not alter the asthma progression (guilbert et al., 2006) . the role of infections in asthma induction and development will likely be shown to be mediated through the effect of these infections on the chronic inflammatory response in the airways of asthmatics. microbial infections can generate either a th2-or a th1-biased response that could exacerbate or attenuate asthma, respectively. in asthmatics, a pro-inflammatory th2 response persists even in the absence of allergens involving cd4+ th2 cells, eosinophils, mast cells, and the th2 cytokines il-4, il-5, il-9, and il-13 (holgate, 2008) . bacterial infections have been shown to contribute to asthma development. in a longitudinal prospective birth cohort study of 411 infants born to mothers with current or previous asthma, neonates colonized in the hypopharyngeal regions with streptococcus pneumoniae, haemophilus influenzae, or moraxella catarrhalis or a combination of these organisms were found to be at increased risk for recurrent wheezing in early childhood and asthma at age 5 (bisgaard et al., 2007) . a protective role for some bacteria has been reported . several studies have found a protective effect of mycobacterial exposure on atopy and airway inflammation (camporota et al., 2003; shirakawa et al., 1997; yang et al., 2002) . these exposures include bacillus calmette-guerin vaccination or heat-killed mycobacterium vaccae. early exposure to bacterial endotoxins may reduce future allergies or asthma (von mutius et al., 2000) , although endotoxins associated with house dust are associated with more asthma symptoms and worse lung function (dales et al., 2006; michel et al., 1996; park et al., 2001) . thus, the role of bacteria in asthma initiation and development appears to be complex. the causative interaction is likely to prove to be the interaction of bacteria and bacterial components in modulating the th1 and th2 innate immune system responses. the characterization of these interactions will be complicated by timing, dose, anatomical site, and duration of the bacterial exposure as well as the host genetic and environmental factors influencing the immune inflammatory response (holt, 1996) . it has recently been demonstrated that patients with severe asthma who are also atopic or sensitized to environmental fungi may benefit from treatment with the antifungal azole itraconazole . this observation has raised questions about the relationships among asthma severity, fungal sensitization, and fungal exposure. the issue is complicated by more than 1.5 million species of fungi that are thought to exist (hawksworth and rossman, 1997 ) and more than 80 species of fungi that have been associated with symptoms of airway allergy (horner et al., 1995) . for one species, a. fumigatus, 20 allergens are thought to participate in human airway allergies (www.allergome.org). determining the clinical relevance of fungal allergens is confounded further by extensive cross-reactivity among fungal allergens (crameri et al., 2009) . fungal allergens can induce a number of different human bronchopulmonary disorders, each with a distinct immune pathogenesis. in allergic bronchopulmonary aspergillosis (abpa), the respiratory system is chronically colonized typically with a. fumigatus. evidence now suggests that severe asthmatics without abpa are more likely to be atopic to fungi than patients with milder disease. the diagnostic label "severe asthma with fungal sensitization (safs)" has recently been applied to this group . in these patients, the fungal sensitization is most commonly a. fumigatus, candida albicans, and penicillium notatum . the association between severe asthma and fungi has been identified in numerous studies. atopy to environmental fungi has been associated with severe asthma (o'driscoll et al., 2005) . many population studies have shown an association between local fungal spore counts and medical emergencies due to asthma exacerbations (atkinson et al., 2006) . furthermore, studies have shown that fungus exposure in fungal-sensitized individuals induces asthma symptoms (malling, 1986; matheson et al., 2005; pulimood et al., 2007; salo et al., 2006; woodcock et al., 2006) . treatment of patients with safs with antifungal drugs has generally led to improvement of asthma symptoms concurrent with improvements in several markers of atopy such as reduced ige values, reduced eosinophils counts, and reductions in the level of dose of oral and systemic steroids required (pasqualotto et al., 2009) . these findings lead to the considerations of the fungal composition of the lung microbiome in asthmatic individuals and indeed in normal individuals. environmental fungi colonize the lungs of otherwise healthy people (lass-florl et al., 1999; okudaira et al., 1977) . these studies were dependent on cultivationbased methods for the detection and identification of these fungi. as a cultivationindependent method, gas chromatography/mass spectroscopy on exhaled breath has revealed the presence of fungus specific biomarkers in patients with cf with and without fungal colonization by a. fumigatus (syhre et al., 2008) . this approach was limited to analyzing for known a. fumigatus markers. the application of sequencing-based approaches for studying the lung microbiome will be essential for revealing the role of fungi in the lung microbiome and the role of the lung microbiome on asthma. cf is the most common inherited lung disease in the world. it is a severe autosomal recessive disease with an incidence of 1:2000 at birth in populations of northwestern european origin, with a mutant gene carrier frequency of 1:23 in these populations. the genetic defect occurs in the cystic fibrosis transmembrane regulator (cftr) protein, which acts to transport chloride across cell membranes. patients with cf are the archetype population with chronic bronchial colonization. symptoms include permanent bacterial colonization of the lower airways, with a formation of a biofilm, fat maldigestion, male infertility, and elevated levels of chloride in the sweat (knowles and durie, 2002) . the thick pulmonary system mucus in cf patients minimizes the effectiveness of the mucociliary elevator in clearing the lung of mucus-trapped microorganisms and other debris. as a consequence, microbes chronically colonize these patients' lungs and they suffer bouts of infection, requiring frequent hospital admission. cultures reveal a wide range of bacteria, including p. aeruginosa, mycobacteria, a. fumigatus, and sometimes viruses. the precise contributions of different microbes to patient morbidity, and the importance of inter-specific interactions remain largely unclear [reviewed in (harrison, 2007) ]. the complexity of this ecosystem is difficult to overstate. as an example of this complexity, the lungs of cf patients contain large numbers of neutrophils that migrate to this location in response to microbial colonization. these neutrophils secrete granule antimicrobial proteins called defensins that kill microbes. by analysis of cf sputum samples, the levels of extracellular defensins are sufficiently abundant that they may damage the airway epithelium (soong et al., 1997) . as another example of this inter-specific complexity, p. aeruginosa in cf lungs produces copious amounts of a tricyclic compound pyocyanin that kills competing microbes and eukaryotic cells. this compound was shown to specifically inactivate a human lung epithelial cell line vacuolar atpase (ran et al., 2003) . a study of the microbiome of the lungs was conducted to explore the hypothesis that organisms not routinely identified by culture occur in the lungs of cf patient airways and may contribute to disease. to test this hypothesis, 16s rrna sequence analysis was performed on bal samples from 42 subjects, 28 cf patients, and 14 other disease controls (harris et al., 2007) . the findings of this analysis were that, for cf subjects, a single rrna type was dominantly represented in the clone libraries prepared from lung microbiome genomic dna. this was not found in the controls. thirteen of the cf subjects' samples contained bacteria not routinely assessed by culture. candidate pathogens were identified in four cf subjects. candidate pathogens were also identified in the controls. this study documented the power of culture-independent molecular techniques to provide a broader view of the airway bacteria than standard clinical culture methods. the cf viral metagenome was explored in a recent study using five cf individuals and five individuals without disease (willner et al., 2009) . in both cohorts, the overall viral diversity was low. the cf bacteriophage communities were highly similar to each other, whereas the non-cf individual had more distinct phage communities. cf eukaryotic viral communities were dominated by a few viruses, including human herpes viruses and retroviruses. the significance of fastidious or noncultivatable organisms in the airway of cf patients is beginning to be explored. application of specific culture conditions to favor the growth of anaerobes coupled with molecular identification techniques have focused attention in cf on bacteria not routinely detected by standard culture and biochemical identification techniques (harris et al., 2007; tunney et al., 2008; worlitzsch et al., 2009) . direct, culture-independent detection techniques have identified much larger numbers of bacterial species in cf airways and have demonstrated the ability to identify likely pathogenic bacteria occurring during exacerbations when routine cultures are negative (harris et al., 2007) . these molecular identification and detection methods have identified bacteria with different antibiotic susceptibilities relative to conventional pathogens and will undoubtedly lead to novel antimicrobial intervention trials in cf (worlitzsch et al., 2009) . similar methodology to detect anaerobes or noncultivatable bacteria has not been applied systematically to patients with idiopathic bronchiectasis. bronchiectasis is characterized by chronic dilation and inflammation of the conducting airways associated with recurring infections (barker, 2002) . it is the pathologic manifestation of several genetic disorders, including cf and primary ciliary dyskinesia (pcd). however, many patients have no identifiable causes. idiopathic bronchiectasis is estimated to affect approximately 110,000 us adults (weycker et al., 2005) . symptoms include cough and chronic sputum production, recurring airway infection, dyspnea, wheezing, and chest pain (barker, 2002) . microbial infections are central to the pathogenesis and progression of disease. much of the research characterizing the composition and significance of the lower airway microbial flora has been done in cf and relatively little is known about the microbial contribution to disease pathogenesis in idiopathic bronchiectasis. however, recent observations suggest a link between the lower airway microbial flora and host disease characteristics. for example, the prevalence of idiopathic bronchiectasis associated with nontuberculous mycobacteria (ntm) appears to be increasing (billinger et al., 2009; marras et al., 2007) . both familial clustering and a characteristic "tall asthenic" phenotype (scoliosis, pectus excavatum, mitral valve abnormalities) in postmenopausal women with bronchiectasis associated with ntm infection (colombo et al., 2009; kim et al., 2008) have been reported. it is unknown whether the age, female sex, and unique body morphotype associations are seen in idiopathic bronchiectasis unassociated with ntm. correlating disease phenotype with microbial flora is dependent upon accurately categorizing the microbial status of the patients. for environmental organisms like ntm, it is important that this categorization include both accurate speciation and determination that the organism likely represents true infection rather than contamination or transient colonization. the american thoracic society and the infectious diseases society of america (ats/idsa) microbiologic diagnostic criteria for pulmonary disease based on sputum specimens call for at least two positive sputum specimens for the same species (kim et al., 2008) . concomitant recovery of filamentous fungi from airway specimens is also common in bronchiectasis, but the pathophysiologic consequences are not known. a recent study in cf patients found that a. fumigatus, like ntm, was commonly present in older patients: 75% of patients aged 16-20 years and in 60% of patients over age 20 (valenza et al., 2008) . amin and colleagues further noted that cf patients who were chronically infected with a. fumigatus (defined as two positive cultures in a given year) had significantly worse airway obstruction as evidenced by a lower forced expiratory volume in one second (fev 1 ) and significantly higher risk of pulmonary exacerbations during subsequent follow-up than patients without a. fumigatus (amin et al., 2009) . this potential negative impact on the course of bronchiectasis and a possible benefit from antifungal treatment for chronic infection in cf have prompted initiation of a multicenter clinical trial of itraconazole in cf patients in canada (amin et al., 2009; shoseyov et al., 2006) . in non-cf bronchiectasis, a recent study suggested that aspergillus is more common in patients infected with ntm than in those without ntm, and that it is commonly associated with fungal lung disease manifestations in ntm-infected patients (kunst et al., 2006) . however, outside the relatively small numbers of idiopathic bronchiectasis patients with allergic bronchopulmonary aspergillosis (abpa) or chronic necrotizing aspergillosis, the pathologic significance of these fungi has not been systematically explored in large numbers of patients and very few data are available for aspergillus species other than fumigatus or filamentous fungi other than aspergillus (kobashi et al., 2006; kunst et al., 2006; raju et al., 2008) . abpa is well described in association with bronchiectasis occurring in asthmatics and patients with cf (malde and greenberger, 2004) . the diagnostic criteria rely on an elevated total ige as well as elevated a. fumigatus-specific ige and igg in the setting of episodic bronchial obstruction, pulmonary infiltrates, and central bronchiectasis. kunst and colleagues assessed the prevalence of positive serologic markers for a. fumigatus [ige by radioallergosorbent test (rast) and precipitins] among idiopathic bronchiectasis patients and found these markers to be commonly present especially in the setting of concomitant ntm disease (kunst et al., 2006) . patients with these serologic markers more commonly had radiographic manifestations suggesting aspergillus-associated disease. while other filamentous fungi such as scedosporium species have been commonly recovered from the airways of both cf and non-cf bronchiectasis patients, the role these fungi play in disease pathogenesis remains controversial (cooley et al., 2007) . while specific ige antibody rast and precipitin assays can be prepared using allergen prepared from the isolated species and correlated with the clinical presentation of allergic bronchopulmonary mycoses, these assays have not been commonly used to characterize the clinical significance of these fungal species recovered from the lower airway (fedorova et al., 2008; lake et al., 1991) . copd is the fourth leading cause of death in the us (petty, 2000) and is expected to rank third in the world by 2020 (lopez and murray, 1998) . despite efforts aimed at smoking cessation, little impact has been made on copd incidence, and current treatments are ineffective in slowing progression of the disease. copd has been defined by the global initiative for chronic obstructive lung disease (gold) as "a disease state characterized by airflow limitation that is not fully reversible". the diagnosis of copd can also encompass those with chronic obstructive bronchiolitis and emphysema. tissue inflammation in copd is characterized by a predominant neutrophil, cd8+ lymphocyte, and macrophage infiltration (keatings et al., 1996; lacoste et al., 1993; o'shaughnessy et al., 1997; saetta et al., 1998) . it has been proposed that the mechanism of tissue damage involves the recruitment and activation of neutrophils, macrophages, and cd8+ t cells with concomitant upregulation of several cellular proteases and inflammatory cytokines. although smoking is clearly the leading risk factor for copd, not all smokers develop disease (buist and connett, 1993) . while smoking can stimulate inflammation in the lungs, smokers with copd have an increased inflammatory response than smokers without copd, and inflammation can persist despite smoking cessation (keatings et al., 1996; lacoste et al., 1993; o'shaughnessy et al., 1997; saetta et al., 1998) . these observations suggest that some other factor or factors contribute to development and perpetuation of the inflammatory response in copd. infection might be one such factor critical in triggering and perpetuating the inflammatory response in copd. the mechanism by which infections might act to promote copd progression has been termed the "vicious circle" hypothesis (sethi, 2000a; sethi and murphy, 2008) . in this scenario, smoking causes structural remodeling that renders smokers more likely to become colonized and/or less able to clear subclinical infection. defects in mucociliary clearance and surfactant abnormalities caused by smoking also contribute to the tendency to develop chronic infection (finley and ladman, 1972; honda et al., 1996; raju et al., 2008; vastag et al., 1985; verra et al., 1995) . once colonization is established, the organism or organisms recruit white blood cells to the lungs, stimulating release of inflammatory cytokines and chemokines as well as proteases. inability to clear the inciting organism perpetuates the cycle, ultimately resulting in tissue destruction, airway thickening, and clinical copd. the most commonly implicated bacteria are h. influenzae, m. catarrhalis, s. pneumoniae, and p. aeruginosa (sethi, 2004) . viruses that seem to be important in copd include adenovirus, influenzae viruses, rhinovirus, respiratory syncytial virus, and human metapneumovirus (mallia et al., 2006; martinello et al., 2006; retamales et al., 2001; seemungal et al., 2001) . these pathogens can be found in patients with copd in the stable state and during exacerbations (sethi, 2004) . the colonization seen in patients with copd is likely playing a role in disease and is not just an innocent bystander. for example, as bacterial load increases, fev 1 falls, and colonization has been associated with greater sputum purulence, increased sputum neutrophils, and increased levels of interleukin (il)-8, tumor necrosis factor (tnf)î±, and neutrophil elastase (obrian et al., 2007; patel et al., 2002; sethi, 2000b; stockley et al., 2000) . exacerbations associated with viruses are more severe and last longer than those without a viral trigger (papi et al., 2006; seemungal et al., 2001) . in addition, exacerbations associated with both bacteria and viruses may be more severe than those associated with single organisms (obrian et al., 2007) , suggesting the usefulness of metagenomic techniques in this disease. colonization with the fungus pneumocystis jirovecii (pc, formerly pneumocystis carinii f. sp. hominis) has recently been implicated in copd pathogenesis. this organism generally causes acute pneumocystis pneumonia (pcp) in patients with immunosuppression such as those infected with hiv, but colonization with the organism occurs in both hiv + and hiv â�� individuals and may be important in copd. colonization with pc is increased in hiv patients with copd and correlates with disease severity (morris et al., 2004b; probst et al., 2000) . animal models also support the role of pc colonization in copd. christensen and colleagues recently reported that in immunocompetent mice, exposure to cigarette smoke and pc colonization resulted in pulmonary function deficits and airspace enlargement characteristic of emphysema (christensen et al., 2008) . in a model of pc colonization in simian/human-immunodeficiency virus (shiv)-infected nonhuman primates (norris et al., 2006) , pc-colonized animals developed airway obstruction and radiographic emphysema while animals infected with shiv alone did not develop these changes (shipley et al., 2010) . although pulmonary infections and neoplasms associated with hiv have decreased since the availability of highly active antiretroviral therapy (haart) (palella et al., 1998) , some pulmonary conditions may actually be increasing in persons with hiv. diseases such as copd, asthma, and bronchiectasis were reported to be increased in those with hiv before the introduction of antiretroviral therapy, and a similar decrease in these conditions as seen in the opportunistic infections has not occurred after antiretroviral treatment of hiv. in fact, in a recent study of hiv + patients, almost 4% of deaths were due to obstructive airway disease in 1998, a threefold increase from the pre-haart era (louie et al., 2002) . before the haart era, hiv + subjects were noted to have an accelerated form of emphysema with significant emphysematous disease seen in subjects less than 40 years old (diaz et al., 1992 (diaz et al., , 2000 . both emphysema and airflow obstruction have been reported in hiv infection. unlike many of the acquired immunodeficiency syndrome (aids)defining opportunistic infections, hiv-associated copd may actually be more common in the current era of hiv as it is frequently reported in those without a history of aids-related pulmonary complications and the now aging hiv + population has a longer exposure to smoking and hiv. given the immunological defects seen with hiv, it is quite possible that hiv + subjects, especially those who smoke, are more prone to develop subclinical pulmonary infections, even if successfully treated with haart. the changes that occur in the lung microbiome have not been studied in hiv, but microbial colonization is a likely factor in the accelerated copd seen in this population. the vicious circle hypothesis of copd could be further worsened in hiv + patients by upregulation of hiv levels in the lung stimulated by pulmonary colonization. several studies have shown that pulmonary infections increase lung levels of hiv. koziel and colleagues reported that hiv rna was detected in 62% of patients with active lung disease compared to 16% of asymptomatic subjects, independent of clinical stage of hiv and serum hiv rna levels (koziel et al., 1999) . the lung appears to be an independent compartment for hiv replication as drug mutations found in bal differ from those in blood (white et al., 2004) . hiv in the lungs is associated with a lymphocytic alveolitis, particularly in those subjects with cd4 cell counts between 200 and 500 cells/î¼l, suggesting that the virus might act independently to stimulate pulmonary inflammation (twigg et al., 1999) . the relationship of hiv pulmonary viral levels, infections, inflammation, and copd has not been examined. pneumocystis colonization is likely important in the pathogenesis of copd in those with hiv as well as in the hivpopulation. in hiv + subjects, the prevalence of colonization is high, particularly if subjects smoke, and colonization is seen even in patients with high cd4 cell counts receiving haart (morris et al., 2004a) . anatomic emphysema is also more common in hiv + patients with pc colonization (morris, unpublished data) . it has recently been shown that pneumocystis colonization in hiv + subjects is associated with worse airway obstruction and an increased likelihood of clinical diagnosis of copd, independent of smoking history and cd4 cell count ). in addition, the shiv-infected nonhuman primates described above serve as a model for the development of copd in the setting of pc colonization and hiv-like immunodeficiency (norris et al., 2006; shipley et al., in press ). microorganisms including bacteria, fungi, and viruses play a central role in development, exacerbation, and progress of lung diseases. even though normal lungs do not have a permanent resident microbiome, diseased lungs are acutely infected and/or chronically colonized. standard laboratory practices have not properly reflected the entirety of the microbiomes, either in health or in disease, and thus newer sequence-based technologies have begun to reveal the true complexity of the lung microbiomes. much more research still needs to be conducted in order to fully understand the microbial burden of the lungs, and how this burden relates to health and disease. it is apparent that conditions that compromise the physical and immune system barriers to lung colonization by microbes result in chronic colonization and recurrent infections. these conditions include chronic inflammation as seen in hiv, asthma, and bronchiecstasis, or physical obstruction observed in cf and bronchiecstasis. the lung microbiomes in each of these conditions has not been properly explored to date, which limits our ability to make definitive conclusions on how to best manage these diseases. our understanding of the fundamental role of viruses in the initial establishment and progress of asthma underscores how little knowledge exists on the role of viral infection in other chronic respiratory diseases. in addition, the fact that some bacterial populations and/or components seem to be protective against further and severe exacerbations in asthma opens the door to questions about the role of microorganisms in protecting against other diseases. the exact nature of this protection is not clearly understood, and great benefit would come from studies that further clarify these intriguing results. furthermore, if some population structures aid in preventing disease progression, it is likely that other population structures may predispose episodes of acute acerbations and progression of the underlying disease condition. a comprehensive understanding of the dynamics of these microbiome interactions would likely result in better, more efficient therapies for these and other respiratory diseases. our developing microbiome analysis technology coupled with our increasing awareness of the potential positive and negative impact of lung population structure on respiratory system health and disease strongly supports the initiation of aggressive projects to characterize the human lung microbiome and its influence on health and disease. bacterial flora in psoriasis the effect of chronic infection with aspergillus fumigatus on lung function and hospitalization in cystic fibrosis patients variability in small airway epithelial gene expression among normal smokers the marine viromes of four oceanic regions temporal associations between daily counts of fungal spores and asthma exacerbations molecular analysis of oral and respiratory bacterial species associated with ventilator-associated pneumonia siblings, day-care attendance, and the risk of asthma and wheezing during childhood relationship of small airway chymase-positive mast cells and lung function in severe asthma an aerosol method of producing bronchial secretions in human subjects: a clinical technic for the detection of lung cancer molecular analysis of the bacterial microbiota in the human stomach nontuberculous mycobacteria-associated lung disease in hospitalized persons childhood asthma after bacterial colonization of the airway in neonates molecular detection of multiple emerging pathogens in sputa from cystic fibrosis patients does helicobacter pylori protect against asthma and allergy dendritic cells transport conidia and hyphae of aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate th responses to the fungus the lung health study. baseline characteristics of randomized participants the effects of mycobacterium vaccae on allergeninduced airway responses in atopic asthma day-care attendance in the first year of life and illnesses of the upper and lower respiratory tract in children with a familial history of atopy a detailed analysis of 16s ribosomal rna gene segments for the diagnosis of pathogenic bacteria pneumocystis murina infection and cigarette smoke exposure interact to cause increased organism burden, development of airspace enlargement, and pulmonary inflammation in mice familial clustering of pulmonary nontuberculous mycobacterial disease infection with scedosporium apiospermum and s. prolificans cross-reactivity among fungal allergens: a clinically relevant phenomenon? airborne endotoxin is associated with respiratory illness in the first 2 years of life detection of potentially novel bacterial components of the human skin microbiota using culture-independent molecular profiling the link between fungi and severe asthma: a summary of the evidence randomized controlled trial of oral antifungal treatment for severe asthma with fungal sensitization: the fungal asthma sensitization trial (fast) study severity of obstructive airways disease by age 2 years predicts asthma at 10 years of age the innate immune response of the respiratory epithelium emphysema-like pulmonary disease associated with human immunodeficiency virus infection increased susceptibility to pulmonary emphysema among hiv-seropositive smokers viral genome sequencing by random priming methods diversity of the human intestinal microbial flora genomic islands in the pathogenic filamentous fungus aspergillus fumigatus transbronchial biopsy of peripheral lung lesions low yield of pulmonary surfactant in cigarette smokers loss of bacterial diversity during antibiotic treatment of intubated patients colonized with pseudomonas aeruginosa human metapneumovirus infection in young children hospitalized with respiratory tract disease molecularphylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases histology and gross anatomy of the respiratory tract multiplex pcr using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains molecular analysis of human forearm superficial skin bacterial biota substantial alterations of the cutaneous bacterial biota in psoriatic lesions relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection host defence mechanisms of the respiratory system follow-up studies of asthma from childhood to adulthood metagenomic analysis of the human distal gut microbiome population disparities in asthma differences between tissue-associated intestinal microfloras of patients with crohn's disease and ulcerative colitis synergism between allergens and viruses and risk of hospital admission with asthma: case-control study long-term inhaled corticosteroids in preschool children at high risk for asthma molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products pathogenic bacteria and viruses in induced sputum or pharyngeal secretions of adults with stable asthma molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis microbial ecology of the cystic fibrosis lung where are all the undescribed fungi bronchoalveolar lavage disordered microbial communities in asthmatic airways pathogenesis of asthma infections and the development of allergy decreased contents of surfactant proteins a and d in bal fluids of healthy smokers fungal allergens impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity microbes on the human vaginal epithelium wheezing rhinovirus illnesses in early life predict asthma development in high-risk children the association of family size with atopy and atopic disease bacterial diversity within the human subgingival crevice nontuberculous mycobacterial disease and aspergillus-related lung disease in bronchiectasis comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes earlylife respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma eosinophilic and neutrophilic inflammation in asthma, chronic bronchitis, and chronic obstructive pulmonary disease allergic bronchopulmonary fungal disease caused by bipolaris and curvularia mp (1999) pulmonary aspergillus colonization in humans and its impact on management of critically ill patients rhinovirus illnesses during infancy predict subsequent childhood wheezing atypical pathogen infection in adults with acute exacerbation of bronchial asthma diversity of asthma: evolving concepts of pathophysiology and lessons from genetics the global burden of disease trends in causes of death among persons with acquired immunodeficiency syndrome in the era of highly active antiretroviral therapy species identification system for dermatophytes based on the dna sequences of nuclear ribosomal internal transcribed spacer 1. nihon ishinkin gakkai zasshi allergic bronchopulmonary aspergillosis an experimental model of rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study diagnosis and immunotherapy of mould allergy. iv. relation between asthma symptoms, spore counts and diagnostic tests reduced diversity of faecal microbiota in crohn's disease revealed by a metagenomic approach does antibiotic exposure during infancy lead to development of asthma?: a systematic review and metaanalysis isolation prevalence of pulmonary nontuberculous mycobacteria in ontario a link between chronic asthma and chronic infection human metapneumovirus and exacerbations of chronic obstructive pulmonary disease asthma and wheezing in the first six years of life. the group health medical associates genes, environments, development and asthma: a reappraisal changes in indoor allergen and fungal levels predict changes in asthma activity among young adults severity of asthma is related to endotoxin in house dust prevalence and clinical predictors of pneumocystis colonization among hiv-infected men association of chronic obstructive pulmonary disease severity and pneumocystis colonization airway obstruction is increased in pneumocystis-colonized human immunodeficiency virus-infected outpatients genomic sequence of the pathogenic and allergenic filamentous fungus aspergillus fumigatus pneumocystis colonization, airway inflammation, and pulmonary function decline in acquired immunodeficiency syndrome mold sensitization is common amongst patients with severe asthma requiring multiple hospital admissions comparison of skin prick tests with specific serum immunoglobulin e in the diagnosis of fungal sensitization in patients with severe asthma inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of cd8+ t lymphocytes with fev1 the effect of elevated temperature on gene transcription and aflatoxin biosynthesis studies on the fungal flora in the lung of human necropsy cases. a critical survey in connection with the pathogenesis of opportunistic fungus infections declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. hiv outpatient study investigators respiratory viruses in childhood asthma infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations house dust endotoxin and wheeze in the first year of life the effects of antifungal therapy on severe asthma with fungal sensitization and allergic bronchopulmonary aspergillosis bacterial diversity in human subgingival plaque relationship between bacterial colonisation and the frequency, character, and severity of copd exacerbations viruses as precipitants of asthma symptoms viral infections, atopy, and asthma: is there a causal relationship? scope of the copd problem in north america: early studies of prevalence and nhanesiii data: basis for early identification and intervention detection of pneumocystis carinii dna in patients with chronic lung diseases epidemic asthma and the role of the fungal mold alternaria alternata gene expression profiles of bronchoalveolar cells in pulmonary tb human targets of pseudomonas aeruginosa pyocyanin the organism causing bacillary angiomatosis, peliosis hepatis, and fever and bacteremia in immunocompromised patients amplification of inflammation in emphysema and its association with latent adenoviral infection respiratory tract fluids: analysis of content and contemporary use in understanding lung diseases what can comparative genomics tell us about species concepts in the genus aspergillus? cd8+ t lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease exposure to alternaria alternata in us homes is associated with asthma symptoms prospective study of transbronchial biopsies in the management of heart-lung and single lung transplant patients respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease principles of antibiotic treatment of community-acquired pneumonia in the outpatient setting bacterial infection and the pathogenesis of copd infectious etiology of acute exacerbations of chronic bronchitis bacteria in exacerbations of chronic obstructive pulmonary disease: phenomenon or epiphenomenon? infection in the pathogenesis and course of chronic obstructive pulmonary disease the inverse association between tuberculin responses and atopic disorder persistent pneumocystis colonization leads to development of chronic obstructive pulmonary disease in a non-human primate model of aids aspergillus bronchitis in cystic fibrosis severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 bronchiolitis to asthma: a review and call for studies of gene-virus interactions in asthma causation purification and characterization of defensins from cystic fibrosis sputum bacterial infections of the lung in normal and immunodeficient patients bronchial inflammation: its relationship to colonizing microbial load and alpha(1)-antitrypsin deficiency distribution of lung cell numbers and volumes between alveolar and nonalveolar tissue hay fever, hygiene, and household size investigation into the production of 2-pentylfuran by aspergillus fumigatus and other respiratory pathogens in vitro and human breath samples asthma in remission: can relapse in early adulthood be predicted at 18 years of age a meta-analysis of the association between caesarean section and childhood asthma asthma and cigarette smoking the role of transbronchial lung biopsy in the treatment of lung transplant recipients. an analysis of 200 consecutive procedures detection of anaerobic bacteria in high numbers in sputum from patients with cystic fibrosis an obesity-associated gut microbiome with increased capacity for energy harvest lymphocytic alveolitis, bronchoalveolar lavage viral load, and outcome in human immunodeficiency virus infection prevalence and antimicrobial susceptibility of microorganisms isolated from sputa of patients with cystic fibrosis mucociliary clearance and airways obstruction in smokers, ex-smokers and normal subjects who never smoked the relationship of rhinovirus-associated asthma hospitalizations with inhaled corticosteroids and smoking ciliary abnormalities in bronchial epithelium of smokers, ex-smokers, and nonsmokers exposure to endotoxin or other bacterial components might protect against the development of atopy childhood factors associated with asthma remission after 30 year follow up neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma prevalence and economic burden of bronchiectasis different resistance mutations can be detected simultaneously in the blood and the lung of hiv-1 infected individuals on antiretroviral therapy human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals fungal contamination of bedding antibiotic-resistant obligate anaerobes during exacerbations of cystic fibrosis patients evidence of a causal role of winter virus infection during infancy in early childhood asthma role of infections in the induction and development of asthma: genetic and inflammatory drivers mycobacterial infection inhibits established allergic inflammatory responses via alteration of cytokine production and vascular cell adhesion molecule-1 expression use of the bronchofiberscope for bronchial brush biopsy. diagnostic results and comparison with other brushing techniques signature patterns revealed by microarray analyses of mice infected with influenza virus a and streptococcus pneumoniae characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods key: cord-018040-k0h5ejjt authors: ilyinskii, p. title: aspects of microparticle utilization for potentiation of novel vaccines: promises and risks date: 2009 journal: silicon versus carbon doi: 10.1007/978-90-481-2523-4_26 sha: doc_id: 18040 cord_uid: k0h5ejjt many recombinant vaccines against novel (hiv, hcv) or ever-changing (influenza) infectious agents require the presence of adjuvants/delivery vehicles to induce strong immune responses. the necessity of their improvement led to the major effort towards development of vaccine delivery systems that are generally particulate (e.g., nanoand microparticles) and have comparable dimensions to the pathogens (viruses or bacteria). the mode of action of these adjuvants is not fully understood but implies the stimulation of the innate or antigen-specific immune responses, and/or the increase of antigen uptake or processing by antigen-presenting cells (apc). moreover, enhancement of adjuvant activity through the use of microand nanoparticulate delivery systems often resulted from the synergistic effects producing immune responses stronger than those elicited by the adjuvant or delivery system alone. among particulate adjuvants, biodegradable microand nanoparticles of poly(d,l-lactide-co-glycoside) (plga) or poly(d,l-lactide) (pla) have been reported to enhance both humoral and cellular immune responses against an encapsulated protein antigen. cationic and anionic polylactide co-glycolide (plg) microparticles have been successfully used to adsorb a variety of agents, which include plasmid dna, recombinant proteins and adjuvant active oligonucleotides and are also currently tested in several vaccine applications. another approach envisions specific targeting of apc, especially peripheral dc and exploitation of particulate systems that are small enough for lymphatic uptake (polystyrene nanobeads). microand nanoparticles offer the possibility of enhancement of their uptake by appropriate cells through manipulation of their surface properties. still, questions regarding toxicity and molecular interaction between microand nano-particles and immune cells, tissues and whole organisms remain to be addressed. these risks and other possible side effects should be assessed in detail especially if mass-production and massive administration of such preparations is to be considered. prevention of infectious disease through vaccination is one of the most important achievements in the history of medicine. its effects on the mortality reduction are enormous and may be compared only to those caused by the establishment of safe water supply [1] . adaptive immunity against infectious disease, i.e. lack of infection in previously exposed individuals has been noted for a long time, most famously by athenian historian thucydides in 5th century bc. it is often said that first recorded attempts of vaccination against infectious disease (smallpox) go back to ad 1000. in china, but they cannot be verified till 16th-17th centuries, when the procedure of variolation started to be used on a reasonably large scale. in the end of xviii pioneering works of jenner and his less-known predecessor jesty on vaccination against smallpox with cowpox paved the way to modern vaccination, whose true founder in a scientific sense was louis pasteur [1] . interestingly, first successful vaccines were launched against two viral diseases, smallpox and rabies. smallpox became first and only infectious disease to be eradicated in 1967-1977 [2] . it is commonly said that jenner's work constitutes the first deliberate use of live virus vector as a vaccine, but we should also note that it was first recorded use of nanotechnology (virion size of a poxvirus is around 200 nm in diameter and 300 nm in length). we should also mention that the problems of unknown side effects, possibility of human infection with new or incompletely inactivated virus and as well as initial difficulties of social acceptance of vaccination that were faced by both jenner and pasteur are not dissimilar from the risks that nanotechnology of vaccination encounters today. in fact, these problems have persisted for more than 200 years and yet many other major diseases have been put under control with the help of vaccination. these include, to name just a few, diphtheria, tetanus, yellow fever, pertussis, poliomyelitis (currently targeted for eradication by who), measles, mumps and rubella. and this list is far from complete. at the same time, it is important to remember that a number of vaccine-related disasters and incidents were caused over this period of time, all of them due to failures in vaccine manufacturing techniques. specifically, during the 1st century of scientific vaccination (post-pasteur) three types of vaccines were generally used: live-attenuated (weakened), killed viruses/bacteria and subunit or extract (chemically or biochemically processed and thus detoxified, the technique that mostly important in anti-toxin vaccination, i.e. against diphtheria). it is thus of no surprise that there were instances of incomplete attenuation, sterilization or detoxification of vaccine products (see [2] for details). while historical role of early-generation vaccines cannot be underestimated, current awareness of health and safety issues resulted in much higher standards of safety and it is true that many past vaccines would not even meet the minimum standards of today. new techniques of vaccine development that may enable a generation of safe, effective and economically feasible vaccines are being actively explored. additionally, it is now clear that successful vaccination against several newly-discovered infectious agents as human immunodeficiency virus (hiv), hepatitis c virus (hcv) or severe acute respiratory syndrome (sars) will require the development of radically new vaccine concepts and products. there are several types of novel vaccines or vaccine components the utilization of which is not mutually exclusive. in fact, their complementary use may be even required in some cases. firstly, there are recombinant molecules: proteins or nucleic acids (na; dna vaccines have been tested extensively over the last decade, rna vaccines are now actively pursued in several experimental systems, but relevant information is scarce at the moment save for rna alphaviral replicons). protein antigens induce immune response directly, while na vectors encode necessary antigens and produce them upon their introduction into organism. secondly, these molecules may be carried or delivered by biological or chemically produced vectors, separately or in combination. these vectors are known to tremendously augment the immune response induced by proteins or nucleic acids used alone. such augmentation is also called adjuvant effect. the size of these vectors is generally within 10-1000 nm and it is a specific mechanism by which our immune system recognizes such particles that underlies their adjuvant potencies (in addition, many carriers protect proteins/na from rapid degradation in vivo and release them into the organism during prolonged periods of time, which also results in higher immunogenicity). needless to say, many details of this mechanism remain to be elucidated, although current vaccine development fashion is definitely of "going small", i.e. toward micro-and nanoscale [3] . in this paper, i will briefly discuss the current state of the vaccine nanotechnology, promises of the new approaches as well as potential risks that these approaches may contain. both recombinant na and protein vaccines have been designed to be safer than traditional vaccines based on whole viruses and bacteria, but their limited immunogenicity has so far hindered their development for clinical use. this led to the sustained research effort aimed at development of specific adjuvants that may make such vaccines more potent without compromising on their safety. currently, only one adjuvant, alum (aluminum hydroxide or aluminum phosphate) is used worldwide in diphtheria, tetanus and hepatitis b vaccines. one of the proposed mechanisms for alum action is generation of small salt particles in which vaccinating agent is trapped and then preferentially taken up by cells of immune system, macrophages and dendritic cells (dc), which, in turn, leads to elevated immune response. multiple attempts to create antigen-bearing particles that will be capable to imitate this suggested avenue of immunogenicity augmentation have been undertaken over the last decade and many of them have been useful. what may be the mechanism of higher immunogenicity exhibited by particulate antigens compared to soluble ones and does the size of these particles matter? adjuvanting effects of micro-and nanoscale particulates are now known to be a consequence of them being of suitable size for uptake by antigen-presenting cells (apc), such as dc and macrophages through phagocytosis or pinocytosis. apc then stimulate t-lymphocytes constituting cellular arm of immune response, which among others is responsible for specific antiviral immunity. this apc-mediated activation of t-cells initiates adaptive immune response. antigens delivered in particulate form are preferentially processed by apc and via so-called mhc class i and ii pathways and then presented to cd8+ (cytotoxic lymphocytes) and cd4+ (t-helpers) t-cells, which first serve as effector cells of immune response and then are instrumental in generation memory t-cells, those that are responsible for the defense of the organism in case of repeated exposure to the infectious agent bearing antigen in question. those particulate antigens that are not directly taken up by dc, but by other cells of the organism (e.g., by muscle cells after intramuscular injection) may also stimulate immune response by two possible pathways. either they eventually either find their way to dc after cell death followed by phagocytosis (if they are stable enough) or they may be processed to the cell surface and then recognized by already activated t-cells thus providing further stimulus for their activation and multiplication. obviously, that particle-driven stability of delivered antigens is a very important part of such an equation. moreover, the surface of micro-or nanoparticle may be further modified to increase their targeting specificity, which can be also attained via simple size variation. it is now clear that different subtypes of dc, those residing in skin and in lymph nodes (ln) are differentially affected by nanoparticles of various size with smaller nanoparticles (10-100 nm) being capable of passive migration to lymph nodes and rapid activation of immature ln-resident dc, while particles of the larger sizes (100-1000 nm) are preferentially uptaked by dc at a vaccine injection site. those skin or mucosal apc residing at those parts of the body where the contact with infectious agents is most likely constitute "the first line of defense" of the organism. it is more than probable that activation of several dc populations done sequentially or simultaneously is a process imperative for generation of potent immune response. moreover, activation of dc does also trigger non-specific immune responses, known as innate (e.g., induction of interferons and other cytokines), which in turn may further potentiate generation of prolonged and strong specific immune response against vaccinating agent. all of these developments made by fundamental research are now actively transposed into the science of vaccine manufacturing. there are two types of vectors of viral origin that have been extensively explored lately, first being viral vectors per se (replication-competent or -deficient) and second, viruslike particles (vlp). viral vectors consist of replicating or non-replicating virus that contains genetic material (in form of dna or rna) encoding the desired antigen. these vectors maintain the ability to penetrate cells and disseminate in the organism and sometimes even replicate, but are rendered harmless by specific changes or mutations. advantages of virally-vectored vaccines include their ease of production, a good safety profile (at least in some cases), ability to potentiate strong immune responses, potential for nasal or epicutaneous delivery and mucosal immunization [3] . they are also more potent than pure dna vaccines (since dna in this case is protected from degradation by viral capsid and directly delivered to virus-susceptible cells), but are especially good if used sometime after initial dna immunization (in so-called prime-boost regimen). adenoviruses are probably among the best-studied viruses and they were heavily exploited for vaccine development over last two decades [4] . there are many promising experimental results using either replication-competent or -incompetent (the latter, by definition, are very safe) adenoviral vectors. however, consistent presence of adenoviruses (the agents of common cold) in human population resulting in pre-exisitng immunity (the ability of human immune system to recognize adenoviral vector and expeditiously remove it from the organism) has somewhat damped the enthusiasm for their clinical use. still, experimental data, especially in the immunization against hiv and influenza were sufficiently promising to enable large-scale clinical trial of adenoviral-based hiv vaccine manufactured by merck (usa). this, as we know, has recently ended in a complete failure. the trial was aborted [5, 6] . importantly, not only this vaccine was not protective, it has apparently also increased risk of hiv infection in vaccinated subjects that were previously exposed to adenovirus (those with pre-existing immunity). notably, we do not currently know the fundamental reason for the latter (and it was not foreseen by anyone based on current immunological knowledge). this example shows that vaccines of novel generation, however promising, may cause side effects of completely unpredictable nature. another type of viral vectors is based on various poxviruses, closely related to vaccinia virus that has been used for many years for vaccination against smallpox. it is known that immunization with vaccinia viruses induces potent immune response against recombinant antigen [7] and many experimental vaccines containing proteins from hiv, influenza, malaria, etc. have been produced and tested. it is important to stress that unmodified live vaccinia virus has never been proposed as delivery vehicle for vaccines aimed at general use (application of such vaccines as anti-cancer therapeutics is outside of this review) since the risks of its utilization would have been enormous. in particular, live vaccinia virus can not be used to vaccinate people with immune deficiencies, suffering from eczema, etc. however, its replication-deficient strain, called modified vaccinia virus ankara (mva) is much safer and still very immunogenic. another approach envisions the utilization of avian poxviruses, e.g. fowlpox or canarypox viruses, which are also incapable of replication in humans, but still immunogenic. similar to adenoviral vectors, the issue of pre-existing immunity should be addressed regarding poxviral-based vaccines since nearly all humans over 40 years old have been vaccinated against smallpox at least once and may therefore respond poorly to new poxviral vaccination. the possibility of adverse effects similar to the one observed in merck anti-hiv vaccine trial can not be discounted as well, although what is true for one group of viruses may not be automatically applied to another. many mva-vectored vaccines have been already developed, but only one of them, against malaria, is now under active clinical investigation, which at the moment appears to be highly successful. in this case prime vaccination with dna molecule is followed by boost immunization with recombinant poxvirus. currently we do not know why poxviral-based vaccine is especially potent against malaria and if these results could be translated into other infectious systems. other replicating virus vectors are based on measles and vesicular stomatitis virus and they suffer from similar drawbacks: prior immunity and safety concerns. the same could be said about two more novel non-replicating viral vectors based on alphaviruses and herpesviruses [4] . it appears that viral vaccine vectors will require considerable breakthrough in order to enable their extensive clinical application. another approach envisions the utilization of vlps. vlps are non-infective virus particles (capsids) consisting of self-assembled viral proteins without na, which mimic the structure of native virions [8] . these particles fall in the general size range of viruses (22-150 nm) [9] , albeit it is fair to say that full viral size range is within 15-400 nm [10] . they also maintain a specific virus-like morphology and are effectively consumed by cells similarly to infective viral particles. they may be formed by a single viral protein or by co-expression of many, in the latter case creating complex virus-like structures. it appears that particulate nature of vlps drives their efficient presentation to the cells of immune system. moreover, those vlps that closely resemble infectious viruses may trigger the same array of antiviral immune responses (e.g., via viral receptor) without causing immune suppression (as living viruses do). vlps can be produced in many expression systems, those of mammalian cells, yeast, bacteria, baculovirus (viruses of insects) and plants. they have been manufactured for many viruses including, but not limited to hiv, hepatitis b virus (hbv), hcv, ebola, influenza, rotavirus, human papillomavirus (hpv) and norwalk virus [8] . several vlpbased vaccines have been licensed for general use, many of them against hbv, which are composed of hbv surface antigen (hbsag), which is a main component of currently used protein-based, alum adjuvant-potentiated vaccine. upon its production in vitro (e.g., in yeast) hbsag protein aggregates in 22 nm particles (single-component vlp), which are both safe and highly immunogenic [11] . in fact, hbsag is now actively pursued as molecular adjuvant on its own ("vlp as platform" approach). fusion of several less immunogenic proteins to hbsag, most notably, influenza m2 protein capable of cross-protective immunity, resulted in significant immunogenicity augmentation [12] . similar data has been reported for hbv vlp containing proteins from the circumsporozoite stage of the malaria plasmodium [13] . improvements of single-protein hbv vlps by inclusion of large and middle viral envelope proteins are also actively sought [9] . the most recently approved vlp vaccine is gardasil that is protective against several types of hpv, which are known to be associated with cervical cancer and genital warts. this vaccine is composed of self-assembled particles of hpv major capsid protein [14] [15] [16] and it also contains an aluminum salt adjuvant. it has been shown to reduce infection of hpv by 90% and is almost 100% effective hpv types 6, 11, 16 and 18. this vaccine if administered early in life is expected to drastically reduce the occurrence of this life threatening disease in women and has generated significant excitement. therefore, it is currently recommended that girls at 9-14 years group age are vaccinated and many u.s. states will likely make this vaccination mandatory. anti-hpv vaccine is clearly the most dramatic example of recent success of recombinant vaccine developers. at the same time, there was a significant fight-back against general utilization of this vaccine stemming from different social groups that we will describe in the final part of this review. several other vlp vaccines are now close to clinical use, those against norwalk virus and rotavirus (both causing acute gastrointestinal infections). rotavirus is the leading cause of diarrhea-related deaths (400-600,000) in children worldwide and clinical trials of vlp-based vaccine against rotavirus has long been advocated [8] . vlp-based vaccine against norwalk virus is now assessed in phase i clinical trials. a variety of other vlp vaccines have been evaluated in preclinical studies include hiv, influenza, hcv, ebola and sars coronavirus [9] . it should be added that non-vaccine nanoparticle vlp applications also exist such as using them as scaffolds to allow assembly of biopolymers (large plant virus-derived vlps are utilized, notably from tobacco and cowpea mosaic viruses), including their utilization as templates to polymerize nanowires as building blocks for semiconductors or manufacture nanowires for the construction of smaller lithium ion batteries [10] . liposomes (phospholipids-based vesicles) are roughly of the similar size as vlp (usually 50-200 nm) and they were used extensively as drug delivery vehicle for nearly four decades. liposomes transport their contents across cellular membranes and release it following fusion with internal cellular compartments called endosomes. thus, they are capable of delivering of encapsulated or adsorbed antigen for vaccine use and were investigated as adjuvants for vaccines against influenza, hiv and malaria, among others. however, it seems that no unmodified liposome-based vaccine possesses an adjuvant effect strong enough to substantiate its clinical development. conversely, modified liposomal vaccines based on viral membrane proteins (virosomes) are sufficiently potent and are approved for use in europe as vaccines against hepatitis a virus (hav) and influenza [17] . immunopotentiating influenza virosomes are liposomes containing main influenza surface protein hemagglutinin intercalated into their membrane. their approximate mean diameter is 120-170 nm. the glycoproteins of influenza virus stabilize the liposomal particles and maintain their receptor-binding actrivities when reconstituted into such protein lipid vesicles and are therefore actively taken up by cells in vivo and actively conveyed to immunocompetent cells [18] . they have an excellent safety profile. other virosomal vaccines produced include not only those against hav, but also combined hav-hbv vaccine in which inactivated hav virions and hbv hbsag cores were covalently coupled to the surface of virosome [18] . adjuvant properties of virosomes can be further increased by integration of costimulatory molecules (immune response mediators and activators) or their specific targeting to particular dc subtype. moreover, they can additionally carry dna or rna and thus provide for multifaceted immune stimulation via different pathways. therefore, virosomes are becoming more and more popular in modern vaccine concepts since they combine the safety and flexibility of subunit vaccines with the biological and immunogenic properties of vlps [10] . another mode of imitating nature is the creation of sometime misleadingly called proteosomes (note the capital "p"), nanoparticles composed of the outer membrane proteins (omps) of neisseria meningitides and other neisseria. proteosome particles are considered to serve as potent vaccine delivery vehicles based on their nanoparticulate (20-800 nm size), vesicle-like nature [19] . in addition, hydrophobicity of proteosomes may inhibit antigen degradation facilitate its uptake and processing by immunocompetent cells. omps have been used successfully in a marketed meningococcal vaccine since 1981 and are considered non-toxic and well-tolerated [3] . meningococcal omps have been safely given parenterally to hundreds of million of children in a haemophilis influenza type b conjugate vaccine [19] . in several cases, a novel adjuvant known as protollin consisting of proteosomes non-covalently complexed with immune stimulant lipopolysaccaride (lps) has been used [20] . proteosome-based influenza vaccines for nasal application have been also generated using this technology and successfully tested and many others (against shigella, hiv, staphylococcal and brucella toxins are under active development). there are other bacteria-based adjuvants, but they may not be called particles in a true sense of words, rather being biomolecules (modified proteins, peptides, components of bacterial wall). it is for their efficient delivery that some of synthetic micro-and nanoparticles can be successfully used. the only nano-sized vaccine adjuvant that is approved for human use (in europe, but not in the u.s.) as a vaccine component is mf59. it was the first new adjuvant to be licensed for human use (in 1997) 70 years after introduction of alum [21] . mf59 is an oil-in-water emulsion composed of <250 nm uniform and stable microparticles made by a drop of oil surrounded by a coat of water droplets held onto oil by surface detergents. specifically, these droplets are formed when squalene (4.3% v/v) and two surfactants, polysorbate 80 (0.5% v/v, tween 80) and sorbitan trioleate (0.5% v/v, span 85) are emulsified in citrate buffer [3] . this oil is obtained from shark liver and is also found in humans as natural metabolite [21] . the strong immunogenicity enhancement of mf59 has been repeatedly demonstrated with some researchers showing an effect even stronger than that of alum. the mechanism of adjuvanticity of mf59 is believed to be through cytokine production, although no precise information is available as of yet. in general, it is thought that adjuvant emulsions potentiate immune responses via formation of antigen depots and stimulation of antibody-producin plasms cells. as noted above, an mf59-adjuvanted influenza vaccine, fluad, is licensed in europe and experimental vaccines for avian influenza have been produced. their strong immunogenicity and protectivity has been demonstrated experimentally. other vaccines strongly potentiated by mf59 include those against hbv, hcv, hiv, herpes simplex virus (hsv), haemophilis influenzae type b and n. meningitides. montanide is a different, water-in-oil emulsion (one modification is a water-in-oil-inwater emulsion). emulsions of montanide51 and 720 are composed of a metabolizable squalene-based oil with a mannide monooleate emulsifier, which augments immunogenicity via formation of antigen depot at the site of injection [3] . while similar to incomplete freund's adjuvant (ifa), a well-known but extremely reactogenic adjuvant that contains a mineral oil and an emulsifying agent, in its physical character, montanide is biodegradable. this eliminates many of the cytotoxic properties inherent for ifa. isa 51 and 720 emulsions have been in phase i and/or ii clinical trials for vaccines against malaria and hiv and various cancers and in most cases they were found to be safe and fairly well-tolerated. another particulate vaccine vehicle is immunostimulating complex (iscom). iscoms are based on triterpenoid saponins (q saponins). they form particles of approximately 40 nm. these are essentially cage-like structures containing protein antigen, cholesterol, phospholipid and the saponin adjuvant quil a or its purified component quil21, which is derived from the aqueous extract from the bark of the south american tree quillaia saponaria molina. these components are held together by hydrophobic interactions. saponins have been tested and used in both human and veterinary medicine for decades and they are known to efficiently induce t-helper responses. they have not been extensively used because of their reactogenicity (quil a composed of more than 23 different saponins is too toxic for human use), although recently semi-synthetic saponins were created that are apparently less toxic [22] . iscom matrix traps the protein antigens (typically hydrophobic membrane proteins) through apolar interactions. a similar vaccine delivery vehicle and adjuvant has also been developed that uses the same material minus the antigen (iscomatrix) and still possesses preferential targeting to apc [23] . a clinical study that compared a classical trivalent subunit influenza vaccine with an iscom adjuvanted version revealed a stronger immune response with the iscom vaccine eliciting rapid antibody responses as well as t helper (cd4+ t-cell) and some ctl (cd8+ t-cell) responses [24] . additional iscom/iscomatrix vaccines have been tested in the clinic for hiv, hsv, hpv and hcv [3] . in all cases, these studies have shown a good safety and tolerability profile in humans as well as effective induction of both humoral and cellular immune responses. still, the actual use of iscoms in human vaccines has been deterred by concerns regarding safety since some saponins are toxic at elevated levels [3, 22] . another completely new mode of biochemical modification of antigen is its attachment to so-called protein aggregating domains that form intracellular inclusions of several hundred nm. such inclusions are then digested via the process called autophagy, which is now known to be linked to effective antigen presentation via mhc class ii pathway. initial data provided evidence of dramatic increase of immunogenicity when a test soluble antigen was attached to such aggregating domain [25] , although this very promising approach is still in the very first stages of possible development. there are several types of synthetic micro-and nanoparticles that are effective in vaccine delivery and potentiation. calcium phosphate microparticles are relatively lessknown. they can be generated by combining (while stirring) of calcium chloride, sodium phosphate and sodium citrate [3] . since calcium phosphate is naturally occurring in the body, these materials are thought not to present a danger of significant side-effects. calcium phosphate microparticles are less than ~1.2 î¼m in diameter (<1,000 nm according to other authors, [26] ). phase i study showed that these microparticles are safe and non-toxic when administered subcutaneously. vaccines utilizing cap, which are currently in preclinical studies include anthrax, hbv, influenza (h5n1 avian and seasonal) and hsv-2 [3] . much better known are polymeric biodegradable microparticles. those used most frequently are poly(d,l-lactide-co-glycolide) (plg/plga) and polylactide (pla) and their copolymers as well as polyorthoesters, polyanhidrids and polycarbonates [27] . microparticles may be loaded by many types of recombinant vaccines, i.e., dna (which is thus protected from degradation), proteins and also other adjuvants of smaller molecular size (of protein, peptide or oligonucleotide nature). these biodegradable, biocompatible polymers have been approved for use in humans (e.g., as sutures, bone implants, screws and implants for sustained drug delivery) and have been extensively studied for use in the formulation of vaccine antigens. plg-based microparticles are the primary candidates for the development of microparticles as vaccine adjuvants since they have been safely used in humans for many years. the direct uptake of biodegradable microparticles into dc has been demonstrated both in vitro and in vivo and it appears that their appropriate size is within 1-3 î¼m range and that cationic microparticles are particularly effective in this regard [28] . in these formulations, antigen can be either entrapped or adsorbed to the surface of the particles. furthermore, these particles can be tailored to degrade over a range of rates, which is especially important since several of those systems degrade rather slowly. thus, additional triggering of their dissolution is necessary for efficient antigen delivery. on a positive side, they can therefore act as a depot from which the encapsulated antigen is gradually released [28] . additionally, polymeric particles may offer high degree of protection to encapsulated antigens delivered orally and nasally and thus subjected to many degrading enzymes residing at these sites of the body. it appears that dcs respond to biomaterials via an innate immune response, which then stimulates an adaptive response to an antigen delivered by polymeric particles. biodegradable and biocompatible micro-and nanoparticles of plga or pla have been reported to enhance both humoral and cellular immune responses against an encapsulated protein antigen. plg nanoparticles can induce systemic antibody titers comparable to those of aluminum salts. it was demonstrated that plg nanoparticles loaded with mpl (an immune stimulant composed of detoxified lipopolysaccharide (lps) from salmonella minnesota) were efficiently taken up by dc. recently, it was shown that the adsorption of antigens at the surface of pla particles also leads to elevated immune response. cationic and anionic plg microparticles have been successfully used to adsorb a variety of agents, including dna, recombinant proteins and adjuvant active oligonucleotides and are also currently tested in several vaccine applications. another approach envisions specific targeting of apc, especially peripheral dc and exploitation of particulate systems that are small enough for lymphatic uptake (polystyrene nanobeads). these are in contrast non-degradable nanoparticles (represented also by gold, latex and silica beads). it appears that solid synthetic, particulate vaccines can induce strong immune responses imitating classical "danger signals" generated by infectious agents and microparticles. in this application 40-50 nm particles preferentially taken by some types of dc are often used and are known to generate potent and broad immune response [23] . moreover, it was demonstrated that for polysterene beads even miniscule difference in size can influence the breadth and type of induced immune response [29] . recently, ultra-small (25 nm) nanoparticle systems were shown to be capable of interstitial-to-lymphatic flow to deliver antigen and adjuvant to ln-resident dc via lymphatic capillaries, whereas 100 nm nanoparticles were only 10% as efficient [30] . also, these ultra-small, ovalbumin-conjugated, polyhydroxylated nanoparticles based on pluronic (a block copolymer of polyethylene glycol and polypropylene glycol) were shown to induce antigen-specific cellular immunity since their surface chemistry has activated complement system, a pathway of innate immunity that serves as a biochemical defense system that clears pathogens nonspecifically, but can also play a role in promoting antigen-specific responses. in general, nanoparticles as vaccine vehicles might have three different advantages over microparticles. most importantly, they have increased surface area for adsorption allowing for a higher antigen/polymer ratio and are also easier to prepare and process. as for their higher immunogenicity, the jury is still out since different groups of investigators are presenting conflicting evidence [31] . it should be also noted that sometimes the utilization of preposition "nano" to describe nearly any type of vaccine component is a bit overdone [3] . since non-degradable nanoparticles may remain in the tissues for extended periods of time, it is thought that it will therefore enhance the time of immobilized antigen presentation and thus augment immunogenicity. gold particles have been frequently described for vaccine delivery both with and without the aid of electroporation, a method which is unlikely to be employed in humans. an alternative approach to delivering dna vaccines employing non-degradable nanoparticles is through particle bombardment also referred to as "gene gun" approach. this is essentially firing the dna-coated gold nanoparticles into the epidermis. while the delivery efficiency of this technique is quite low, only small amounts of dna are required to achieve a significant immune response. this method has been tested for vaccines against hbv, influenza and malaria. furthermore, micro-and nanoparticles offer the possibility of enhancement of their uptake by appropriate cells through manipulation of their surface properties. as delineated above, upon their inoculation, particulate compounds, microspheres or nanoparticles, must reach the secondary lymphoid organs, which are the sites of the immune response. this led to design of novel methods of dermal or transcutaneous vaccination, including the use of micro-and nano-particles to target the skin apc that will then deliver consumed antigens to the ln. finally, pulmonary delivery (which may be especially potent and useful against influenza and other respiratory viruses) requires dry forms of vaccines that are low cost, temperature-tolerant, efficiently aerosolized, and apc-directed. therefore, nanoparticles can play a critical role in the formulation, development and delivery of needle-less pulmonary vaccines and these are now actively pursued as well. additionally, nanoparticles containing vaccines for the oral delivery are also investigated. these particles are 100-200 nm in diameter and are likely needed to be targeted towards a special subtype of apc called m cells that reside within gut-associated lymphoid tissue. it is apparent that novel micro-and nanoparticle-based delivery vehicles are being actively evaluated in many vaccine systems. promising results have been reported from many directions. still, questions regarding toxicity and molecular interaction between micro-and nanoparticles and immune cells, tissues and whole organisms remain to be addressed. there are many regulatory hurdles for new adjuvants and they are there for a reason. one of the greatest hurdles is the sheer size of population that needs to be tested to prove safety of a new adjuvant or vaccine. these numbers have dramatically increased in recent years since it became apparent that some approved drugs have rare serious and even fatal side effects that were not identified because of inadequate sample sizes during their clinical development [23] . during that time the association of adverse reactions with two vaccines resulted in their withdrawal from the market. these are nasal inactivated influenza vaccine associated with increase of cases of bell's palsy and also rotavirus vaccine, the administration of which lead to higher intussusception [23] . the case of adenoviral-based hiv vaccine has been mentioned above. reaction of parts of the society to a new vaccine may be also caused by imaginary side-effects. we have already noted that this is a case with gardasil, a highly efficient anti-hpv vaccine, which is likely to tremendously diminish the incidence of cervical cancer and genital warts. several conservative groups and think-tanks in the u.s. have questioned if such a vaccination will spark more promiscuity in young women. statements like "what message are we sending to our elementary students when we inoculate them for a sexually transmitted disease in the third grade?" or "this means even christian children who are brought up knowing that sexual activity before marriage is a sin would still be forced to be vaccinated against this std or they could not attend school" [32] are not uncommon. but conservatives are not the only ones, who are in opposition to a mandatory vaccination against deadly disease and throughout the history of vaccination such reactions have been noted many times. it was aptly observed that groups fighting against gardasil are very diverse and include christian conservatives, who have long argued that safe sex encourages profligate sex, the growing antivaccine movement, which objects to all school-entry requirements and the parental-rights adherents, incensed by any mandates regarding their children's health [33] . these arguments may seem laughable or medieval to some, but they are very valid to others and it is advisable that the introduction of this great novel product of vaccine technology is done employing all possible precautions and public-relations instruments. unquestionably, there is a lesson to be drawn. no new medicinal technology can be successful unless public is educated and well-informed of it. it is apparent that nanotechnology is currently driving the development of novel vaccines and adjuvants. at the same time, some concerns regarding the toxicity of such small particles have appeared. currently, there is a keen interest in nanotoxicology research since the processing of nanostructures in biological systems could lead to unpredictable and hence unknown toxic effects [34] . one may draw some lesson from the history of polio vaccine when during 1955-1963 more than 98 million americans received one or more of its doses contaminated with polyomavirus sv40, which was then simply unknown being identified and isolated only in 1960. when it became apparent that under specific set of conditions sv40 may cause cancer in laboratory animals, this led to a serious public scare. fortunately, subsequent studies of vaccinated humans over many years have shown that there was no causal relationship between receipt of sv40-contaminated vaccine and cancer. such a completely unforeseen event should not be discounted when we in fact are actively engaged in genetical modification of humans (immunized individual always contains cells of immune system, which genome is partially rearranged in a way that cells of a non-immunized individual are not). other potential problems include high surface area and reactivity of small particles, their involvement in catalytic and oxidative reaction, ability to cross biological membranes as well as slow biodegradability of some materials used for their manufacturing. possibility of all of these being potential toxicity issues are at least partially supported by data describing the effects of pollutants on human health [3] . still, it seems reasonable to anticipate that in the case of vaccines, the infrequent and low-level exposure to nanoparticles that an individual will encounter during immunization is not enough to cause adverse health problems such as those potentially attributed to nanotoxicity effects. with that being said, the development of any novel vaccine adjuvant or delivery platform should undergo all the necessary safety tests. they will also need to withstand public and political scrutiny. recently, regulatory authorities and the general public start to be concerned with products that cross traditional lines, i.e. combine biomaterials with cells, dna or proteins as in non-viral polymeric carriers for vaccines [35] . currently, the most probable reason for the micro-and nano-based vaccines not flooding the market is linked to the cost of their safety testing. necessary clinical trials required for their approval are very long and difficult. unlike animal studies, human trials often require significant waiting period before protection can be analyzed. furthermore, since many vaccines are often administered to healthy individuals, and frequently to infants, it is critical that they are proven safe and well-tolerated in nonhuman primates before entering human trials. these risks and other possible side effects should be assessed in detail especially if mass-production and widespread administration of novel preparations is to be considered. currently, there is an uncertainty about nanoparticle processing in vivo and also near-complete absence of understanding of mechanisns of their interactions with biological systems (although the latter was never necessary in the history of medicine provided that the safety of this or that useful approach is demonstrated experimentally). therefore, continued animal research dealing with nanoparticle in vivo pharmakinetics and tissue distribution, nanotoxicity investigated in the same vein as drug toxicity currently is, as well as human trials for the safety evaluation of the experimental microand nanoparticle-based vaccine regimens will be of immense importance. a short history of vacination vaccines and vaccination in historical perspective nanotechnology in vaccine delivery replicating and non-replicating viral vectors for vaccine development hiv vaccine failure prompts merck to halt trial hiv vaccine may raise risk vaccinia virus: a tool for research and vaccine development virus like particle (vlp) vaccines. in: levine virus-like particles: passport to immune recognition virus-like particles-universal molecular toolboxes hepatitis b vaccine improved design and intranasal delivery of an m2e-based human influenza a vaccine phase i testing of a malaria vaccine composed of hepatitis b virus core particles expressing plasmodium falciparum circumsporozoite epitopes efficacy of a bivalent l1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) l1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase ii efficacy trial sustained efficacy up to 4.5 years of a bivalent l1 virus-like particle vaccine against human papillomavirus types 16 and 18: followup from a randomised control trial recent developments in vaccine delivery systems immunostimulating reconstituted influenza virosomes proteosome tm technology for vaccines and adjuvants protollin: a novel adjuvant for intranasal vaccines mf59 adjuvant emulsion the perfect mix: recent progress in adjuvant research vaccine adjuvants revisited a randomized, double blind study in young healthy adults comparing cell mediated and humoral immune responses induced by influenza iscom vaccines and conventional vaccines adjuvant potential of aggregateforming polyglutamine domains different interactions between isomeric tetrakis-(n-hexadecylpyridiniumyl) porphyrins and cds nanoparticles targeting dendritic cells with biomaterials: developing the next generation of vaccines microparticle-based technologies for vaccines type 1 and 2 immunity following vaccination is influenced by nanoparticle size: formulation of a model vaccine for respiratory syncytial virus exploiting lymphatic transport and complement activation in nanoparticle vaccines a practical approach to the use of nanoparticles for vaccine delivery guard against gardasil who's afraid of gardasil? nation nanotoxicity: the growing need for in vivo study interaction of dendritic cells with biomaterials key: cord-271241-w1q46y63 authors: ruggiero, emanuela; richter, sara n. title: viral g-quadruplexes: new frontiers in virus pathogenesis and antiviral therapy date: 2020-05-18 journal: annu rep med chem doi: 10.1016/bs.armc.2020.04.001 sha: doc_id: 271241 cord_uid: w1q46y63 viruses are the most abundant organisms on our planet, affecting all living beings: some of them are responsible for massive epidemics that concern health, national economies and the overall welfare of societies. although advances in antiviral research have led to successful therapies against several human viruses, still some of them cannot be eradicated from the host and most of them do not have any treatment available. consequently, innovative antiviral therapies are urgently needed. in the past few years, research on g-quadruplexes (g4s) in viruses has boomed, providing powerful evidence for the regulatory role of g4s in key viral steps. comprehensive bioinformatics analyses have traced putative g4-forming sequences in the genome of almost all human viruses, showing that their distribution is statistically significant and their presence highly conserved. since the genomes of viruses are remarkably variable, high conservation rates strongly suggest a crucial role of g4s in the viral replication cycle and evolution, emphasizing the possibility of targeting viral g4s as a new pharmacological approach in antiviral therapy. recent studies have demonstrated the formation and function of g4s in pathogens responsible for serious diseases, such as hiv-1, hepatitis b and c, ebola viruses, to cite a few. in this chapter, we present the state of the art on the structural and functional characterization of viral g4s in rna viruses, dna viruses and retroviruses. we also present the g4 ligands that provide further details on the viral g4 role and which, showing promising antiviral activity, which could be exploited for the development of innovative antiviral agents. viruses, the most abundant organisms on our planet, affect all living beings, including animals, plants, parasites, fungi and bacteria. their subtle ability to develop strategies to evade the host immune system, along with the high mutation and replication rates, has made viruses plague terrestrial ecosystems since their appearance. indeed, virus-borne infectious diseases tend to spread abruptly over wide geographical areas, leading to outbreaks that can dramatically affect health, national economies, and the overall well-being of societies. 1 in the past few decades, pharmaceutical and biotechnological advance has succeeded in developing new therapeutics for the management of different viral diseases: for example, anti-retroviral therapy against the human immunodeficiency virus (hiv), or the pan-genotypic direct-acting antiviral drugs used for hepatitis c (hcv) management. nevertheless, the majority of current therapies are unable to accomplish virus eradication or sustained virological response, therefore the battle against viral pathogens still represents a primary focus of medical research. in addition, the constant onset of new viral pathogens, generally correlated with massive outbreaks, imperils researchers to determine the pathogenic mechanisms of the new viruses, with the ultimate goal to provide new therapeutic approaches in the antiviral field. 2, 3 viruses are obligate intracellular parasites characterized by great diversity in terms of morphology, size, genome nature, and protein composition. a virus particle, also called a virion, consists of the viral genome, which can be either dna or rna in a single-or double-stranded state, surrounded by a protein coat, the capsid. some virus families additionally present an external lipid bilayer called envelope, derived from cellular membranes (fig. 1) . in order to replicate, viruses induce the host cell to synthesize all the essential components necessary to form new virus particles. as a consequence, new virions are assembled in the cell and egress from it to infect other cells, completing their replicative life cycle (fig. 2) . some viruses do not always actively replicate while within the host but remain dormant in defined cell types by integrating their genome into the host dna, or through formation of circular viral genome molecules (episomes). this process is termed viral latency: through it the virus can evade the host immune system, usually establishing a lifelong infection. 4 it is worth noting that each viral class has its own peculiar viral cycle: their details will be described later in this chapter. fig. 1 cartoon representation of a generic virus structure. viruses can be either "naked," i.e., formed by the genome and capsid only, or "enveloped," i.e., with the additional presence of the outer lipid membrane (envelope) and surface proteins, as shown. viruses can be classified according to different parameters, such as genome nature, capsid structure, presence of the envelope, type of infection. the most commonly used system is the baltimore classification, which allocates viruses into seven different classes, according to the nucleic acid type and the resulting mrna synthesis mechanism (table 1) . 5 g-quadruplexes (g4s) have been identified in viruses belonging to almost all groups of the baltimore classification. indeed, in the past few years, research on the biological role of g4s in virology has boomed, providing strong evidence of the relationship between g4s and the regulation of key viral steps. a recent comprehensive bioinformatics analysis traced putative g4-forming sequences (pqss) in all human viruses, showing that their distribution is statistically relevant, making them a distinctive feature of the different virus families. in addition, such pqss are highly conserved, despite the high recombination rates that characterize viruses, suggesting a crucial role for g4s in viral evolution and replication, and corroborating their targeting as a valid pharmacological approach for antiviral therapy. 6 dna viruses include a great variety of viruses in terms of host types, genome length and structure, replication site, replication machinery source and associated diseases. based on their unique characteristics, they can adopt different replication strategies. 7 most of these viruses share the ability to cause latent infections, where the virus remains quiescent in a specific cellular reservoir, in a non-replicative state until reactivation. 8 dsdna viruses have been found to be particularly enriched in pqss. 6 indeed pqs-related functional studies have been reported for most herpesviruses, papillomaviruses and also the hepatitis b virus (fig. 3 ). the herpesviridae family includes a large number of viruses with long linear dsdna genomes: it comprises more than 100 pathogens classified into three subfamilies, alpha-, betaand gammaherpesvirinae, which mainly differ in the host target cells. among all human herpesviruses (hhvs), nine species routinely infect humans, are extremely widespread and responsible for the onset of several different diseases, such as orolabial and genital herpes, chickenpox and shingles, mononucleosis and different types of cancer (table 2) . interestingly, more than 90% of adults are infected with at least one of these. 9 hhvs differ significantly in terms of dna base composition and sequence arrangement; nonetheless, they share many biological properties, including the ability to sustain, after a primary infection, a latent state through maintenance of their genome in the infected organism as an extrachromosomal nuclear episome. 10 during their life cycle, hhvs trigger an intricate chain of events to achieve replication, which involves both cellular and viral structures. briefly, after attachment to the host's cell 11 pharmacological research in the antiherpetic field has been quite successful, since to date, several efficient drugs are available on the market. one of the most efficient antiviral agents is acyclovir, along with its derivatives. these compounds are classified as nucleosides analogues: they block viral replication by being incorporated into the nascent viral dna. they have been widely employed for the treatment of alpha-hhvs and are still considered the first line drugs of choice in the management of these viruses. however, the excessive use of anti-hhvs agents over large periods, especially in immunocompromised patients, has led to the onset of resistant viral strains. moreover, considering that hhvs undergo latency, and that, to date, there is no way to eradicate the virus from the host cells, the development of new compounds with innovative targets and mechanisms of action is urgently needed. 11 genome-wide bioinformatics analysis has revealed remarkable pqs density in all hhv species, mostly in genome regulatory and repeated regions, which are crucial to sustain the episomal form. the enrichment, distribution and conservation rate of these sequences strongly corroborate g4s as functional elements in the regulation of the viral life cycle. 12, 13 in addition, a recent study demonstrated an interesting correlation between g4 sequence composition in hhvs and their hosts, suggesting a possible long-term virus-host co-evolution process. 14 consequently, g4s may represent efficient pharmacological targets, alternative to those aimed at by the current anti-herpetic therapies. the subfamily of alpha-hhvs includes hsv type 1 and 2, and vzv: these are characterized by the establishment of a persistent infection in sensory neuronal cells. most of the studies conducted on the role of g4s in hhvs regulation focused on the hsv-1 virus, as its genome is characterized by 68% gc content, with multiple and highly stable g4-forming sequences. 15 hsv-1 g4s formation has been monitored in infected cells through a g4-specific antibody at different time points post-infection. g4s were found to form in a virus cycle-dependent fashion. during viral replication, viral g4s fold massively in the cell nucleus and localize in different compartments according to the viral genome movements in the cell during the viral cycle. 16 bioinformatics and the following experimental analysis traced conserved pqss in: (i) the promoter region of immediate-early genes of all alpha-hhvs, genes that are expressed at the very early stage of viral infection and encode for regulatory proteins involved in all subsequent viral steps 17 (ii) the terminal and internal repeated regions, which include packaging signals involved in concatemer cleavage during replication 13 (iii) the proximity of recombination breakpoints. 18 most of the identified g4s proved to be exceptionally stable when tested in vitro through biochemical and biophysical assays. employment of known g4 ligands has provided interesting insights in the regulation of hsv-1 g4s, first and foremost the reduction in virus replication, obtained after treatment with either braco-19 (ic 50 $ 8 μm) 15 or a core-extended naphthalene diimide derivative (ic 50 $ 18 nm). 19 both compounds led to a significant reduction in viral dna synthesis and late transcripts production. furthermore, in vitro assays revealed the ability of g4 ligands to improve further hsv-1 g4s stability and their ability to stall polymerase progression. viruses belonging to the betaherpesvirinae subfamily are the human cmv, hhv-6 and hhv-7, which mainly exploit leukocytes as target cells for latency. recent work on this class of viruses has analyzed the regulatory role of g4s in the human cmv and hhv-6. the human cmv is estimated to affect more than 80% of the worldwide population. it has the largest dsdna genome among hhvs ($235 kb), which was found to include a remarkably high number of pqs, distributed among the promoters of immediate-early, early and late genes. the majority of the sequences have been demonstrated to actually fold into g4 structures, and to be stabilized after treatment with two different porphyrinbased g4 ligands, tmpyp4 and nmm. interestingly, in a luciferase reporter system that included different pqss in viral promoters, nmm was shown to suppress the expression of several mrnas and to reduce both intracellular and extracellular viral dna levels. 20 recently, the anti-cmv activity of a polymerase inhibitor cx-5461, a known g4 ligand currently in phase i clinical trial, was also demonstrated. interestingly, the compound induced a 2.0 log reduction in viral titer, together with a significant decrease in the amount of viral dna and pul44, the viral processivity factor: these data indicate that the effect of cx-5461 is exerted at the viral dna replication stage of the viral life cycle. considering that the compound is known to induce a dna damage response (ddr), its antiviral activity may be related to a double g4-related mechanism of action, involving both the viral targets and the activation of cellular stress response pathways. 21 hhv-6 consists of two related viruses known as hhv-6a and 6b, which infect almost 100% of the human population and cause the febrile illness roseola infantum, also called the sixth childhood eruptive disease. the hhv-6 genome is characterized by the presence of variable-length telomere-like repeat regions at its termini, which can integrate by homologous recombination into the human chromosome at the telomeres; however, the mechanisms regulating this process remain poorly understood. considering that telomeres are commonly known to fold into g4s, involvement of these non-canonical arrangements in the regulation of hhv-6 chromosomal integration is plausible. indeed, treatment of hhv-6 infected telomeraseexpressing cells with the g4 ligand braco-19 resulted in a significant reduction in chromosomal integration, likely due to the prevention of telomere end elongation. 22 the human viruses ebv and kshv, classified as gamma-hhvs, establish latency in lymphocytes. they are both associated with the development of lymphomas originating from b cells, especially in immunocompromised patients. ebv is one the most common human viruses in the world, affecting more than 90% of the world's population. its primary infection tends to be generally asymptomatic. however it could lead to infectious mononucleosis and, in the worst case, to various types of cancer. its genome comprises genes encoding for six ebv nuclear proteins that are expressed only in the latent infection and sustain the viral episome maintenance. 23 studies on the characterization and role of g4s in ebv showed that the ebv-encoded nuclear antigen-1 (ebna1), the viral protein pivotal to immune evasion, promotes viral dna replication by interacting with rna g4s and recruiting the cellular origin replication complex. the ebna1 mrna itself is enriched in folded g4s, which behave as cis-acting modulators of viral mrna translation, producing ribosome dissociation. 24,25 ebna1 mrna g4s have also been found to regulate the endogenous presentation of ebna1-specific cd8 + t-cell epitopes, seen in persistent infections. 26 the employment of g4 ligands has helped the investigation of the functional and biochemical characteristics of ebna1. braco-19, for instance, stabilized the viral rna g4 and, in infected lymphocytes, reduced ebv genome copy numbers. it also negatively modulated transcription levels of ebna2 and ebna3a, and inhibited ebna1-dependent dna replication. 24 another g4 ligand, pyridostatin (pds), enhanced the stability of the ebv g4s, reducing ebna1 synthesis level in a concentration-dependent way, both in vitro and in vivo. as a result, ebv-infected cells were found to be less efficiently recognized by virus-specific t cells, although such a mechanism still needs to be elucidated. these data show that g4-mediated inhibition affects processes that are crucial for viral dna replication, 25 reinforcing the importance of alternative dna structures in the regulation of virus processes. the cellular protein nucleolin, which has been widely shown to bind g4s, 27 recognizes and also binds viral ebna1 mrna g4s. as a consequence it downregulates ebna1 expression and antigen presentation, counteracting ebv stealthiness. such interactions can be targeted to modulate ebv progression: indeed the phendc3 g4 ligand was found to prevent nucleolin binding to ebna1 mrna g4s and to increase the endogenous ebna1 levels in ebv-infected b cells and in cells derived from a nasopharyngeal carcinoma. 28 following on from phendc3, variously substituted bis(acylhydrazones) derivatives have also been investigated for their ability to interfere with the nucleolin/ebna1 mrna g4s interaction. two compounds, phendh2 and pydh2, have been found to be more active and less toxic with respect to the lead compound. 29 however, it is worth noting that although many of the tested phendc3 analogues displayed strong in vitro binding and stabilization of rna g4s, only a few of them were able to interfere with ebv immune evasion. this result suggested that the ability to hamper nucleolin/ebna1 interaction might require additional activities, independent from g4 stabilization. indeed, different g4 ligands can generate opposite effects: this is the case of pds, which, in contrast to phendc3, further suppressed ebna1 mrna translation, 25 as also demonstrated by quantitative proximity ligation assay. 30 all these data support the ability of ebna1 g4s to operate as nucleolin-binding platform. a very recent study reported that they also promote nuclear retention, 31 but the relationship between the two processes needs further investigation. notably, ebv nuclear antigens are involved in b-cells transformation, especially ebna1, which is expressed in all ebv-related tumors. 32 therefore, it is tempting to speculate that the nucleolin/ebna1 g4 complex could be involved in virus-related oncogenesis and it might be exploited as a pharmacological target to impair both virus and tumor progression. g4s have also been observed in mrnas of additional gamma-hhv maintenance proteins, known to regulate their self-synthesis, corroborating the structural regulatory role of g4s in the virus system. 25 kshv is the causative agent of kaposi's sarcoma and other lymphoproliferative disorders, such as primary effusion lymphoma and a plasmablastic form of multicentric castleman's disease. it mostly concerns aids patients, as kshv seroprevalence is directly proportional to hiv seropositivity rates. at the moment, there are no treatments for the lytic or latent infections. 33 the kshv genome is arranged in a $ 140 kb long central region, encoding viral genes, flanked by repeated regions (terminal repeats, or trs), which harbor the viral episome unique origin of replication. trs are highly enriched in g/c bases and have been demonstrated to form stable g4s, located both in the forward and reverse strands, and involved in the regulation of episomal replication. stabilization of viral g4s by means of different g4 ligands, phendc3 and tmpyp4, stalled the replication fork at the tr level, with consequent replicative stress and inhibition of kshv dna replication, which resulted in the dramatic reduction (60%) of viral episome copy numbers. 34 in addition, the kshv latency associated nuclear antigen 1 (lana1) protein, which is the master regulatory protein of kshv latency, forms g4 structures in its own mrna, analogous to ebna1. g4 stabilization induced by tmpyp4 treatment downregulated lana1 translation, with consequent reduction of protein expression in kshv infected cells, suggesting that lana1 mrna g4s are involved in the regulation of the translation machinery. interestingly, reduced translation has been found to be strictly related to reduction of antigen presentation, one of the main mechanisms adopted by lana1 to promote immune evasion. 35 altogether, these data confirm that g4s are key regulatory element in the regulation of host-pathogen interaction in the herpesviridae family and support the feasible exploitation of these structures as innovative pharmacological targets in the antiherpetic therapy. among dna viruses, g4s have been also studied in the human papillomavirus (hpv) and the hepatitis b virus (hbv). hpvs include more than 100 characterized viruses, which infect and replicate within epithelial structures, inducing specific hyperplastic lesions. among them, those defined as "high-risk" hpvs can efficiently induce malignant transformation of infected cells, leading to the onset of cervical, anogenital, and several oropharyngeal cancers. notably, high-risk hpvs infections cause almost 99% of cervical cancers and approximately 5% of all cancers worldwide. 36 currently, despite the availability of anti-hpv vaccines, access to effective treatments is limited in many parts of the world; furthermore, no specific anti-hpv drugs are available. g4 forming sequences have been found in 8 out of the 120 identified hpv types, all belonging to the "high risk" hpv subgroup. 37 nevertheless, the majority of the identified sequences are characterized by high polymorphism, 38 which allows the formation of different alternative structures, i.e., hairpins. 39 such dynamism makes hpv g4s difficult to study and, above all, to target; therefore, further in-depth studies will be necessary to disclose the involvement of hpv g4s in the viral life cycle. hbv is a hepatotropic virus, characterized by a partially double-stranded, circular dna genome, which replicates through an rna intermediate, synthesized by a reverse transcriptase. persistent hbv infections are related to serious liver damage, eventually leading to cirrhosis and hepatocellular carcinoma. the pronounced genetic variability associated with hbv determines the existence of 10 genotypes (a-j) which differ in terms of transmissibility, virus loads, response to antiviral therapy, and ability to cause liver disease. hundreds of millions of people worldwide are chronically infected with hbv, and despite the availability of a prophylactic vaccine and antiviral drugs that keep hbv levels under control, full eradication is not yet achievable. treatments are urgently needed, as almost 1 million deaths per year are estimated to be caused by hbv-related cancers and associated diseases. 40 very recently, a g4-forming sequence was identified as a genotypespecific regulator of hbv replication, located in the pres2/s promoter of hbv genotype b. the sequence was shown to fold into an intramolecular hybrid g4 structure, the stabilization of which surprisingly led to enhanced transcription of the pres2/s gene promoter, with consequent production of the hbv surface antigen (hbsag). the positive modulatory effect was demonstrated through the employment of the g4 ligands braco-19 and pds, revealing a yet unknown role for dna secondary structures in the viral context. 41 rna viruses constitute the major part of the global virome, occupying four out of seven of baltimore's classes. they display the highest mutation rates, which often generate multiple complex mixtures of related virus variants, feature responsible for their adaptability to environment selection. the high number of mutations is generated by the rna-dependent rna polymerase that lacks proofreading activity. 42 this enzyme synthesizes rna from the rna template and, therefore, it is essential for replication of most rna viruses. pqss have been abundantly found in both positively-and negativelystranded ssrna viruses, while very few have been observed in dsrna viruses. 6 most of the studies reported so far regard flavi-, filo-and coronaviruses (fig. 4) . the flaviviridae family comprises a large number of (+)-ssrna viruses, the genomes of which serve also as mrna for viral protein translation. more specifically, the whole genome is translated into a unique polyprotein, which subsequently undergoes different cleavages by means of viral and host proteases, to finally provide three structural proteins, namely the pre-membrane/membrane (prm/m), the capsid (c) and the envelope (e) proteins, and seven non-structural (ns) proteins. at both ends of the viral genome, there are two untranslated regions (utr) of different length, which are essential for viral replication and immune modulation and whose activity is strongly related to the secondary structures adopted, mainly stem-loops. 43 flavivirus infections represent a serious global threat because of the continuous outbreaks reported worldwide. pathogenic flaviviruses are mainly transmitted by mosquitoes or ticks and subsequent infections can lead to the onset of severe neurological and non-neurological diseases. the family comprises over 100 virus species, including emerging and re-emerging arboviruses of global significance, such as japanese encephalitis (jev), west nile (wnv), zika (zikv), dengue (denv), yellow fever (yfv), and tick-borne encephalitis (tbev) virus, and also the hepatitis c virus (hcv), which belongs to the hepacivirus genus. albeit commercially available vaccines for some of these viruses have been developed, nowadays no effective specific treatments exist, besides the recent one established against hcv. considering the global threat of flaviviral pandemics, specific treatments are craved for. 44 zikv is transmitted to humans by mosquito bites: in adults, it usually causes mild to no symptoms, while it might be devastating if contracted during pregnancy, as it is related to microcephaly in the unborn child. several g4 forming sequences have been discovered in the positive strand of the zikv genome, while the negative one is devoid of pqss. seven of the identified sequences were found to be surprisingly conserved within more than 50 analyzed flavivirus genomes. given the great viral genome variability, this observation suggests a significant involvement of these pqs in the regulation of the life cycle of these viruses. an additional g4 in the unique 3 0 -utr region is present in zikv: this region is pivotal to viral replication of the negative-sense strand, therefore it could be a target to impair viral progression. 45 hcv affects more than 70 million people worldwide. it can cause both acute and chronic hepatitis, possibly leading to cirrhosis, liver failure and cancer. indeed, hcv is responsible for 25% of liver cancers. the hcv genome, in common with that of all flaviviruses, is organized into a long open reading frame, which produces a unique polyprotein that is processed by proteases to form 10 structural and non-structural proteins. notwithstanding the numerous virus variants emerging constantly in patients, the core protein is the least variable and therefore it represents an attractive antiviral target. 46 in the last decade, the development of direct-acting antiviral agents (daas), a combination of drugs targeting specific nonstructural proteins, has dramatically changed the management of hcv infections, improving sustained virological response by 90%. 47 nevertheless, because of the possible emergence of resistant strains (https:// www.hcvguidelines.org/evaluate/resistance, accessed jan 23, 2020), identification of new targets is always highly valued. bioinformatics and biophysical analysis of hcv g4s has demonstrated the existence of six conserved pqss throughout the whole viral genome, mostly in the negative strand. the one located in the core gene was characterized as the most stable g4. 48 its stabilization with the g4 ligands tmpyp4 and a pds derivative (pdp) promoted viral rna-dependent rna polymerase stalling, with consequent reduction in hcv core protein levels. in vivo analysis revealed that pdp led to g4-mediated antiviral activity in the low micromolar range, by inhibiting intracellular replication of different hcv genotypes. 49 a deeper investigation also showed that the hcv core rna g4 is recognized and bound by the cellular protein nucleolin. this interaction, which has been found to be structure-related, resulted in inhibition of hcv replication in cells. such activity was counteracted by the employment of g4 ligands such as pds and pdp, which competed for the same binding site. tmpyp4 did not produce the same effect, however, this last compound is in general less selective for g4s. 50 moreover, hcv infection promoted nucleolin overexpression, which in turn induced a drastic reduction of hcv total rna levels. conversely, nucleolin silencing was directly correlated with increased hcv rna expression. 48 these results strongly suggest the existence of a host anti-viral immunity mechanism involving g4 structures. additional bioinformatic studies based on a different algorithm showed the presence of a very interesting g4-forming sequence in the stem-loop iiy of the hcv negative strand, precisely in the 3'-utr, which is the initiation site for the (+)-strand replication. the g4 sequence, highly conserved in many different hcv strains, was strongly stabilized by the bisquinolinium derivative phendc3 and as a consequence, rna synthesis was hampered by more than 70%. moreover, in a luciferase system in which luciferase expression was directly related to viral replication, 60% reduction was observed upon treatment with the compound. 51 hcv was also used as the reference rna virus to develop a fluorescence light-up probe, a thioflavine t derivative (tht-ne), for the direct visualization of the native hcv rna genome in living cells. tht-ne showed remarkable specificity for hcv rna g4s with respect to other g4s in different cell models, probably because during infections viral g4s outnumber cellular g4s in order to maximize viral replication. moreover, the hcv replication machinery promotes formation of vesicle-like viral replication complexes that contain most of the newly synthesized viral rna, generating a localized g4 enrichment that improves tht-ne recognition. 52 if this strategy were applicable also to other pathogens, it would represent a striking turning point in pathological research and clinical diagnosis. filoviruses comprises five genera of viruses, including deadly pathogens such as ebola (ebov) and marburg (marv) viruses, which cause fatal hemorrhagic fever in humans and primates, with no cure available thus far. these viruses are characterized by filamentous virions with a $19 kb-long (à) ssrna, which includes seven genes. filoviruses rna replication consists in the generation of a (+)ssrna intermediate, namely the anti-genome, which acts as template for the synthesis of new genomes. studies on the presence of g4s in filovirus genomes has revealed a highly conserved g-rich sequence in the ebov l gene, which encodes for the viral rnadependent rna polymerase and modulates viral replication and transcription. in vitro analysis has demonstrated that this sequence folds into a dynamic parallel g4, stabilized by treatment with the tmpyp4 ligand. interestingly, this compound was able to reduce l gene transcription and to impair replication of the ebov mini-genome, a cell-based method involving firefly luciferase as reporter protein. 53 moreover, induced circular dichroism experiments performed in the presence of the fluorescent probe thiazole orange, revealed the folding into g4s of several sequences derived from original viral isolates of both ebov and marv. 54 additional investigations will be necessary to assess the biological role of the identified sequences in the viral life cycle. the severe acute respiratory syndrome coronavirus (sars-cov) was identified after a massive outbreak in 2003, which led to the death of almost 10% of the affected people. the virus is one of the most pathogenic viruses in humans and it is highly contagious. furthermore, no effective drugs are available against it. sars-cov belongs to the coronaviridae family and has a (+)-ssrna genome of 29.7 kb in length, encoding for 13 viral genes. the nonstructural protein 3, also known as sars unique domain (sud), plays a crucial role in viral replication and transcription. it was demonstrated to preferentially bind g4-forming oligonucleotides that form in the 3 0 -utr of mrnas coding for host-cell proteins implicated in the regulation of several processes, such as apoptosis and signal transduction. for this reason, the sud/g4 interaction has been speculated to be involved in the modulation of genome replication and host cell response to viral infection. 55, 56 in december 2019, a novel coronavirus named sars-cov2 was identified in wuhan, china; it causes the coronavirus disease (covid-19) , which in a minority of people progresses to severe atypical pneumonia that requires hospitalization and has extremely high mortality among the elderly and immunocompromised people. 57 sars-cov-2, being highly contagious, caused a pandemic in the early months of 2020. sars-cov and sars-cov-2 share 86% genome homology: they both infect humans by entering the lower respiratory tract through the angiotensin-converting enzyme 2 (ace2). 58 however, an amino acid composition difference of about 25% was found between the sud of the two viruses, 59 therefore further characterization of the sars-cov-2 sud will be necessary to assess its function and possible interaction with g4 nucleic acids. retroviruses are the most ancient discovered viruses: they are able to infect almost all vertebrates worldwide, causing severe diseases, such as immunodeficiencies, neurological disorders, and cancer. retroviruses are classified into two subfamilies: orthoretrovirinae, including alpha-, beta-, gamma-, delta-, epsilon-retroviruses and lentiviruses; spumavirinae, represented by the genus spumavirus. they possess two copies of (+)-ssrna genome, formed by four coding genes, gag, pro, pol and env, several genes encoding for accessory proteins and two flanking repeat segments followed by untranslated regions. the pol gene encodes for the reverse transcriptase (rt) enzyme, the rna-dependent dna polymerase that allows synthesis of viral dna from the genomic rna. the newly formed dsdna, which presents two identical long terminal repeats (ltrs) at both ends, once integrated into the host's chromosomal dna forms the provirus. the provirus is transcribed and translated to form new virions, or it may remain dormant, i.e., latent: in both cases, it can be no longer eliminated from the host. the ltr located at the 5 0 -end represents the control center for viral gene expression. it is organized in three sections, u3, r, and u5. the u3 region includes binding sites for transcription factors and acts as the unique promoter for retroviral transcription. 60 interestingly, highly conserved pqss have been found in the majority of retrovirus subfamilies, mainly located in the ltrs. biophysics assays have demonstrated the actual ability of these sequences to fold into g4s, which were strongly stabilized by g4 ligands, such as braco-19 and a coreextended ndi, supporting the possibility of targeting the ltr g4s as innovative antiretroviral therapy. 61,62 viruses belonging to the lentiviridae family infect a broad range of mammals: they are responsible for the onset of several neurological and immunological deficiencies, the most known of which is the human acquired immunodeficiency syndrome (aids) caused by hiv-1. related diseases, associated to different lentiviruses, can be observed also in species like simians, felines, equines and cattle. analysis of pqss in these viruses has mainly focused on the promoter region, the 5 0 -ltr, revealing that almost all members of the family share the possibility to fold into g4s. the most intriguing result was the abundance of pqss in relation to species: all lentiviruses affecting primates showed pqss in their 5 0 -ltr. a lower percentage was observed in the bovine lentiviruses, while none, or very low, in the phylogenetically more distant species. these data suggest that the g4 structures are beneficial to the overall virus biology and have consequently been selected for throughout evolution. furthermore, a strong correlation between g4s and the binding site for the transcription factors sp1 and nf-κb was observed in almost all primate lentiviruses, corroborating the key role played by g4s in the regulation of viral transcription. 61 the hiv-1 virus, lead representative of the lentivirus subfamily, is still one of the most serious public health challenges. since its discovery in the early 1980s, successful and significant efforts have been reached in terms of preventions and treatment: to date, almost 38 million people worldwide are affected by the virus, more than half of which are receiving antiretroviral therapy (art), a combination of three or more antiretroviral drugs that reduces virus production and correlated disease progression (https://www. who.int/gho/hiv/en/, accessed jan 23, 2020). nevertheless, there is still no cure to eradicate the virus from the host, and hiv-related deaths continue to impact society and economy: consequently, identification of alternative and effective antiviral targets is urged. g4s perfectly fit into this picture, as they could represent key elements in hiv-1 targeting, both in the lytic and latent state. research on g4s in this virus has been quite productive, on both the viral genomic rna and the integrated provirus (fig. 4) . the hiv-1 genome, as mentioned above, consists in two copies of positive, single-stranded rna that, once synthesized, dimerize in order to be encapsulated into the new virion: this step is critical for viral recombination (fig. 5) . several g-rich sequences have been found in proximity of the dimerization site 63 and in the central portion of the genome, adjacent to a central polypurine tract. 64 both rna regions are associated with dimerization and primer-strand transfer during reverse transcription, events that promote recombination. collected data infer that the g4s in this region might be involved in the regulation of the aforementioned processes, making rna g4s fundamental for the virus. at the 5 0 -utr, specifically in the u3 segment, a highly g-rich sequence, overlapping with three sp1 binding sites, has been found to fold into multiple g4s. these structures provide a contact point for dimerization between the two rna strands, contributing to viral recombination. 65 in detail, three g4s have been identified, u3-ii, u3-iii and u3-iv, which showed remarkable stability and were well recognized by the g4 ligand braco-19, which was able to stall rt progression in vitro, therefore impairing viral dna synthesis and thus viral production. 66 the nucleocapsid protein (ncp7), a viral protein that assists rt by unwinding hiv-1 rna genome secondary structures in order to promote polymerase progression, could counteract such mechanism. ncp7 is known to preferentially bind single-stranded nucleic acids compared to duplex states, and works as a chaperone, as it promotes folding of nucleic acids in their most thermodynamically stable conformations. 67 it was recently demonstrated that ncp7 contributes to rt processivity by recognition and subsequent unfolding of hiv-1 rna g4s. 68 ncp7 has also been reported to interact in vitro with a g4 sequence located in the hiv-1 central dna flap overlapping positive-strand, which forms a tetramolecular g4. 69 the g4s in this region might be involved in the protection of the pre-integrated genome from nuclease degradation. however, the biological outcome of the g4-ncp7 interaction still needs to be elucidated in vivo. regarding the provirus, the ltr u3 promoter sequence is highly enriched in guanines, especially at the sp1 and nf-κb binding sites. 70 in this region, three overlapping g4 structures can form: ltr-ii and ltr-iii that are constitutively formed, and ltr-iv, the folding of which is induced by proteins or ligands binding. 71 ltr-iii and ltr-iv structures, which have been characterized through nmr spectroscopy, showed very peculiar features. ltr-iii g4 folds into a hybrid, three-layered quadruplex, with a conserved duplex stem-loop in the central loop. 72 ltr-iv g4 has a parallel topology with a bulged g-tract at its 3 0 -end. 73 such distinctive structures could be exploited to design specific ligands able to selectively target hiv-1 ltr g4s, overcoming the low selectivity that characterizes currently available g4 ligands. the two ltr g4s are mutually exclusive and induce opposite effects on the ltr promoter activity. ltr-iii g4 stabilization inhibits viral transcription, 71 while ltr-iv g4 hampers this activity. 73 the folding/unfolding of this region is controlled by at least two cellular proteins: nucleolin, which greatly stabilizes the ltr g4s, with a consequent overall silencing effect, 74 and the human ribonucleoprotein a2b1, which in turn unwinds the g4s to promote transcription. 75 these data infer that the fine balance between g4s might be one of the regulatory mechanisms in the regulation of hiv-1 promoter activity. moreover, it has been very recently shown that the ltr transcription machinery also involves another alternative structure, the i-motif, 76 which can form in the provirus negative strand, further supporting the regulatory role of non-canonical nucleic acids structures in virus biology. 77 several g4 ligands have been tested to assess the effect on hiv-1 g4s on the viral promoter and promising stabilizing activity was observed, leading to an overall antiviral effect. braco-19 significantly stabilized the naturally occurring g4s, ltr-ii and ltr-iii, and induced folding of ltr-iv. upon treatment with increasing compound concentrations, ltr promoter activity was reduced by almost 70% when compared to the untreated condition: a mutated non-g4 sequence used as control showed no variation in promoter response and thus confirmed the effect to be g4-related. 71 braco-19 was then tested in various cell lines, using different hiv-1 strains. it exerted promising g4-mediated antiviral activity, in the micromolar range. considering that the g4 ligand also stabilizes hiv-1 rna g4s, the antiviral effect could be ascribed to a dual mechanism of action, both at the pre-and the post-integration level. 66 the development of a new series of core-extended ndi derivatives has significantly improved the anti-hiv-1 activity of the g4 ligands. in fact, these molecules, characterized by exceptional solubility properties, displayed outstanding in vitro stabilizing activity on hiv-1 ltr g4s, with preference for the viral over the human telomeric g4s, used as a cellular reference. most importantly, the series exhibited strong antiviral activity in the low nanomolar range (ic 50 < 25 nm). time-related antiviral assays combined with reporter assays, have confirmed the g4-mediated mechanism of action. 78 further investigation on g4s in the hiv-1 integrated genome have identified three pqss in the nef gene. this encodes for the viral accessory protein nef, a factor involved in the maintenance of a persistent hiv infection in vivo. the nef gene is located at the 3 0 -end of the viral genome, with its coding region that partially overlaps with the 3 0 -ltr. the observed pqss were highly conserved and able to fold into two-tetrads g4s. nef g4s were stabilized in vitro by different g4 ligands, such as tmpyp4, braco-19 and piper. interestingly, employment of tmpyp4 in the tzm-bl reporter cell line, which supports nef-dependent hiv-1 replication, resulted in a dose-dependent inhibition of viral infectivity. 79 altogether, these data support the possibility of exploiting viral g4s as novel anti-hiv-1 targets, possibly in both the lytic and latent infection. indeed, one of the most appealing and interesting aspects about g4s in hiv-1 is their possible involvement in the regulation of viral latency. the virus establishes a state of latent infection in t cells: at this stage, it is not susceptible to antiretroviral drugs. the molecular mechanisms underlying this process are complex and not completely understood; nonetheless some forms of latency entail transcriptional interference, thus the 5 0 -ltr and its g4s might be involved. 80 g4 stabilizing agents have been tested in cells infected with latent hiv-1, where they showed strong antiviral activity, effectively linking g4s to hiv-1 latency. braco-19 in latently infected cells reduced viral titer to undetectable levels, an effect that was still observed after long-term treatment. tmpyp4 administration also blocked viral replication in two lymphocyte t cell lines with established hiv-1 latency. the antiviral activity was found to be coupled with higher apoptosis/death rates, with respect to untreated cells and the effect was enhanced upon association with dna damage repair inhibitors. 81 the involvement of g4s in the regulation of cellular processes, such as transcription, replication, translation and telomere maintenance, has been widely accepted by the scientific community. the development of specific g4 ligands with promising anticancer effects has encouraged identification of new ways to harness g4 modulation in other human diseases, including viral infections. altogether, the data collected in the past few years on g4s in viruses, their interaction with specific ligands and the consequent effect on the modulation of the viral machinery validate the possibility of developing g4-based antiviral drugs. the first aspect to acknowledge is that all the characterized viral g4s are located in highlyconserved genomic regions. in virology, base-conservation analysis is a critical issue, since viruses are characterized by high mutation rates. 82 the limited availability of deposited sequences for many viruses, especially the emerging ones, limits a comprehensive conservation analysis; however, data accumulated so far clearly show that pqss are conserved elements within viruses, thus representing efficient targets in antiviral research. taking into account that both hosts and viruses have genomic sequences able to organize into g4s, one of the most demanding tasks in the design of antiviral g4 ligands is to accomplish selectivity toward viral versus cellular g4s. as a matter of fact, one of the major limitations in the development of these compounds is in respect to the common chemical features that characterize the currently employed g4 ligands: the large flat aromatic core that stacks on the g quartet, increasing discrimination among different g4s; high-molecular weights and positivecharged side chains, which guarantee loops and grooves interaction to the detriment of cellular uptake. 83 indeed, low selectivity and poor druglike properties are major factors in the lack of progression of current g4 ligands beyond phase ii clinical trials. in order to achieve selectivity, deep knowledge about the structural characteristics of each identified g4 is necessary. in fact, loop and groove regions are distinctive marks of the g4 structure and are amenable to selective recognition. structural data on cellular g4/g4 ligand complexes has demonstrated the involvement of the grooves in the stabilization of such interactions, contributing to formation of a compact arrangement that would likely efficiently impair the transcription machinery. 84 it is therefore possible to speculate that enhanced selectivity could be mediated by interaction with loop/groove moieties. to this end, nmr and crystallographic determinations of viral g4s and their complexes with ligands are necessary, in order to allow ad hoc design of new antiviral molecules. to date, only two viral g4 structures have been solved through nmr spectroscopy: the hiv-1 ltr-iii 72 and ltr-iv 73 g4s. molecular docking analysis, coupled to cleavage experiments, has enabled the identification of groove interactions between ltr-iii g4 and the ndi-cu-deta derivative, a highly selective g4-cleaving agent. 85 structure resolution is thus a valid approach for the development of specific compounds. notwithstanding their poor selectivity, most of the g4 ligands tested in viruses have demonstrated promising antiviral activity and, in the majority of cases, the activity was shown to be g4-dependent. one possible explanation for this outcome is the amount of viral g4s, which, during infection, largely exceeds that of cellular g4s, especially during replication, as reported for hsv-1. 16 considering that generally g4s are located in regulatory regions crucial for viral life cycle progression, a combination of abundance of viral g4s and impairment of key viral functions might explain the observed antiviral effect. exploring the effective folding of g4s during different steps could help not only help understand the role of g4s in different viruses, but it would heighten the possibility of targeting them in the most useful and effective way. ultimately, extending this kind of investigation to other viruses would likely lead to a more rapid and successful application of g4 ligands in antiviral research. however, it must be realized that each virus has complex and unique characteristics, therefore investigation of the effective target and mechanism of action of compounds at the molecular level may not be straightforward. the case of the ebv virus is a clear example of this entangled picture: pds impairs ebna1 synthesis, while phendc3 exerts the exact opposite effect. 25, 28 probably, the overall outcome could be ascribed to multiple g4-mediated mechanisms. finally, one of the most exciting applications of g4 ligands in antiviral therapy is the possibility to fight viruses that undergo latency. current available therapies cannot eradicate the latent virus from its host, and consequently infective agents such as hiv and hhvs still cause non-eradicable diseases. in this scenario, a g4-mediated pharmacological approach would affect both the replicating and the latent virus, possibly leading to virus eradication. to this end, the virus-induced manipulation of host chromatin needs to be considered. recently, several studies reported virus-host chromatin interactions and chromatin machinery modulation by viral proteins. 86 epigenetic modifications of viral chromatin and viral transcriptome might be crucial in conveying the virus toward either lytic or latent infection. 87, 88 since g4 formation has been directly linked to the suppressive role of heterochromatin and it seems to occur only in highly transcribed nucleosome-depleted chromatin regions, 89 it would be fascinating to understand how viral g4s affect and could be affected by such a complex mechanism. in conclusion, all the data reported so far on g4s in viruses clearly show that g4 structures are pivotal elements in the regulation of the virus life cycle, both at the lytic and latent levels. g4 ligands can be envisaged as efficient and promising antiviral agents; therefore research on the discovery of new g4-ligand small molecules should be encouraged. the availability of selective molecules will also help to clarify and further understand the role of g4s in the viral life cycle and ultimately in viral pathogenesis. g4-mediated antiviral drugs could represent a striking turning point in the management of viral infectious diseases, especially in people who cannot access immunization, such as immunocompromised and elderly people. finally, the g4-mediated antiviral effects observed in latent systems may lead to cutting-edge therapeutic approaches in the treatment of human deadly malignancies, such as aids, herpes-and hpvrelated cancers. origin of viruses: primordial replicators recruiting capsids from hosts current landscape of antiviral drug discovery novel coronavirus emerging in china-key questions for impact assessment viruses: definition, structure, classification discovery and classification quadruplex forming sequences in the genome of all known human viruses: a comprehensive guide introduction to dna viruses epigenetics and genetics of viral latency herpesviruses: epidemiology, pathogenesis, and interventions herpesviruses: latency and reactivation-viral strategies and host response antiviral therapies for herpesviruses: current agents and new directions genome-wide analysis of g-quadruplexes in herpesvirus genomes pac1 signals of human herpesviruses contain a highly conserved g-quadruplex motif relationship between g-quadruplex sequence composition in viruses and their hosts the herpes simplex virus-1 genome contains multiple clusters of repeated g-quadruplex: implications for the antiviral activity of a g-quadruplex ligand visualization of dna g-quadruplexes in herpes simplex virus 1-infected cells conserved g-quadruplexes regulate the immediate early promoters of human alphaherpesviruses determine the landscape of recombination in hsv-1 a core extended naphtalene diimide g-quadruplex ligand potently inhibits herpes simplex virus 1 replication genome-wide analysis of regulatory g-quadruplexes affecting gene expression in human cytomegalovirus impact of rna polymerase i inhibitor cx-5461 on viral kinase-dependent and -independent cytomegalovirus replication stabilization of telomere g-quadruplexes interferes with human herpesvirus 6a chromosomal integration oncogene among healthy population: an update role for g-quadruplex rna binding by epstein-barr virus nuclear antigen 1 in dna replication and metaphase chromosome attachment quadruplexes regulate epstein-barr virus-encoded nuclear antigen 1 mrna translation constraints on ebna1 synthesis impact on in vivo antigen presentation and early priming of cd8 + t cells binding proteins nucleolin directly mediates epstein-barr virus immune evasion through binding to g-quadruplexes of ebna1 mrna novel cationic bis(acylhydrazones) as modulators of epstein-barr virus immune evasion acting through disruption of interaction between nucleolin and g-quadruplexes of ebna1 mrna cellulo protein-mrna interaction assay to determine the action of g-quadruplex-binding molecules nuclear processing of nascent transcripts determines synthesis of full-length proteins and antigenic peptides epstein-barr virus-associated malignancies: roles of viral oncoproteins in carcinogenesis kaposi sarcoma herpesvirus-associated cancers and related diseases g-quadruplex-interacting compounds alter latent dna replication and episomal persistence of kshv lana and hnrnp a1 regulate the translation of lana mrna through g-quadruplexes human papillomavirus molecular biology and disease association the effect of single nucleotide polymorphisms in g-rich regions of high-risk human papillomaviruses on structural diversity of dna towards understanding of polymorphism of the g-rich region of human papillomavirus type 52 current treatments for chronic hepatitis b virus infections quadruplex motif in an envelope gene promoter regulates transcription and virion secretion in hbv genotype b flavivirus infection-a review of immunopathogenesis, immunological response, and immunodiagnosis current approaches in antiviral drug discovery against the flaviviridae family zika virus genomic rna possesses conserved g-quadruplexes characteristic of the flaviviridae family hcv core protein and virus assembly: what we know without structures hepatitis c virus therapy: no one will be left behind binding of cellular nucleolin with the viral core rna g-quadruplex structure suppresses hcv replication a highly conserved g-rich consensus sequence in hepatitis c virus core gene represents a new anti-hepatitis c sequence and structural selectivity of nucleic acid binding ligands rna synthesis is modulated by g-quadruplex formation in hepatitis c virus negative rna strand lighting up the native viral rna genome with a fluorogenic probe for the live-cell visualization of virus infection chemical targeting of a g-quadruplex rna in the ebola virus l gene ebola virus derived g-quadruplexes: thiazole orange interaction the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes binding macrodomain within the "sars-unique domain" is essential for the activity of the sars-coronavirus replication-transcription complex clinical course and mortality risk of severe covid-19 can we contain the covid-19 outbreak with the same measures as for sars? a tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses effects of retroviruses on host genome function conserved presence of g-quadruplex forming sequences in the long terminal repeat promoter of lentiviruses stable and conserved g-quadruplexes in the long terminal repeat promoter of retroviruses evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic rna mechanism of hiv-1 rna dimerization in the central region of the genome and significance for viral evolution u3 region in the hiv-1 genome adopts a g-quadruplex structure in its rna and dna sequence anti-hiv-1 activity of the g-quadruplex ligand braco-19 hiv-1 nucleocapsid proteins as molecular chaperones for tetramolecular antiparallel g-quadruplex formation hiv-1 nucleocapsid protein unfolds stable rna g-quadruplexes in the viral genome and is inhibited by g-quadruplex ligands quartets direct assembly of hiv-1 nucleocapsid protein along single-stranded dna topology of a dna g-quadruplex structure formed in the hiv-1 promoter: a potential target for anti-hiv drug development a dynamic g-quadruplex region regulates the hiv-1 long terminal repeat promoter major g-quadruplex form of hiv-1 ltr reveals a (3 + 1) folding topology containing a stem-loop structure and possible function of a g-quadruplex in the long terminal repeat of the proviral hiv-1 genome nucleolin stabilizes g-quadruplex structures folded by the ltr promoter and silences hiv-1 viral transcription the cellular protein hnrnp a2/b1 enhances hiv-1 transcription by unfolding ltr promoter g-quadruplexes. sci. rep. 2017 i-motif dna: structure, stability and targeting with ligands a dynamic i-motif with a duplex stem-loop in the long terminal repeat promoter of the hiv-1 proviral genome modulates viral transcription long terminal repeat promoter g-quadruplexes nef coding region: implications for antiviral activity cold spring harb deficiency in dna damage response, a new characteristic of cells infected with latent hiv-1. cell cycle complexities of viral mutation rates the structure and function of dna g-quadruplexes structural basis of dna quadruplex recognition by an acridine drug a catalytic and selective scissoring molecular tool for quadruplex nucleic acids snapshots: chromatin control of viral infection chromatin control of herpes simplex virus lytic and latent infection colocalization of m 6 a and g-quadruplex-forming sequences in viral rna (hiv, zika, hepatitis b, and sv40) suggests topological control of adenosine n 6-methylation g-quadruplex structures mark human regulatory chromatin key: cord-259131-36udb7uc authors: hunegnaw, ruth; mushtaq, zuena; enyindah-asonye, gospel; hoang, tanya; robert-guroff, marjorie title: alveolar macrophage dysfunction and increased pd-1 expression during chronic siv infection of rhesus macaques date: 2019-07-03 journal: front immunol doi: 10.3389/fimmu.2019.01537 sha: doc_id: 259131 cord_uid: 36udb7uc hiv infected individuals have been shown to be pre-disposed to pulmonary infections even while receiving anti-retroviral therapy. alveolar macrophages (ams) play a critical role in lung innate immunity, but contradictory results have been reported regarding their functionality following hiv infection. here, using the siv rhesus macaque model, we document the effect of siv infection on the phenotypic and functional properties of ams. following infection with siv(mac251), ams in bronchoalveolar lavage (bal) sampled over 2to 20-weeks post-infection (wpi) were compared to those in bal samples from naïve macaques. am expression of proinflammatory cytokines tnf-α, il-6, il-1β, and chemokine rantes drastically increased 2-wpi compared to ams of naïve macaques (p < 0.0001 for all), but dropped significantly with progression to chronic infection. phagocytic activity of ams 2-and 4-wpi was elevated compared to ams of naive animals (p = 0.0005, p = 0.0004, respectively) but significantly decreased by 12-wpi (p = 0.0022, p = 0.0019, respectively). by 20-wpi the ability of ams from chronically infected animals to perform siv-specific antibody-dependent phagocytosis (adp) was also diminished (p = 0.028). acute siv infection was associated with increased fcγriii expression which subsequently declined with disease progression. frequency of fcγriii(+) ams showed a strong trend toward correlation with siv-specific adp, and at 2-wpi fcγriii expression negatively correlated with viral load (r = −0.6819; p = 0.0013), suggesting a contribution to viremia control. importantly, pd-1 was found to be expressed on ams and showed a strong trend toward correlation with plasma viral load (r = 0.8266; p = 0.058), indicating that similar to over-expression on t-cells, pd-1 expression on ams may also be associated with disease progression. further, ams predominantly expressed pd-l2, which remained consistent over the course of infection. pd-1 blockade enhanced siv-specific adp by ams from chronic infection indicating that the pd-1/pd-l2 pathway may modulate functional activity of ams at that stage. these findings provide new insight into the dynamics of siv infection leading to am dysfunction and alteration of pulmonary innate immunity. our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in hiv-infected individuals. hiv infected individuals have been shown to be pre-disposed to pulmonary infections even while receiving anti-retroviral therapy. alveolar macrophages (ams) play a critical role in lung innate immunity, but contradictory results have been reported regarding their functionality following hiv infection. here, using the siv rhesus macaque model, we document the effect of siv infection on the phenotypic and functional properties of ams. following infection with siv mac251 , ams in bronchoalveolar lavage (bal) sampled over 2-to 20-weeks post-infection (wpi) were compared to those in bal samples from naïve macaques. am expression of proinflammatory cytokines tnf-α, il-6, il-1β, and chemokine rantes drastically increased 2-wpi compared to ams of naïve macaques (p < 0.0001 for all), but dropped significantly with progression to chronic infection. phagocytic activity of ams 2-and 4-wpi was elevated compared to ams of naive animals (p = 0.0005, p = 0.0004, respectively) but significantly decreased by 12-wpi (p = 0.0022, p = 0.0019, respectively). by 20-wpi the ability of ams from chronically infected animals to perform siv-specific antibody-dependent phagocytosis (adp) was also diminished (p = 0.028). acute siv infection was associated with increased fcγriii expression which subsequently declined with disease progression. frequency of fcγriii + ams showed a strong trend toward correlation with siv-specific adp, and at 2-wpi fcγriii expression negatively correlated with viral load (r = −0.6819; p = 0.0013), suggesting a contribution to viremia control. importantly, pd-1 was found to be expressed on ams and showed a strong trend toward correlation with plasma viral load (r = 0.8266; p = 0.058), indicating that similar to over-expression on t-cells, pd-1 expression on ams may also be associated with disease progression. further, ams predominantly expressed pd-l2, which remained consistent over the course of infection. pd-1 blockade enhanced siv-specific adp by ams from chronic infection indicating that the pd-1/pd-l2 pathway may modulate functional activity of ams at that stage. these findings provide new insight into the dynamics of siv infection leading to am dysfunction and alteration of pulmonary innate immunity. our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in hiv-infected individuals. keywords: alveolar macrophage, siv, rhesus macaque, antibody-dependent phagocytosis, pd-1, fcγriii, bronchoalveolar lavage introduction macrophages are found in tissues of infected individuals and play a continuous role in pathogenesis. although earlier studies reported that macrophages were the major cell type initially infected by hiv (1), more recent studies have shown that cd4+ t cells are preferentially targeted by hiv/siv at the site of transmission (2, 3). however, macrophages still serve as targets for the virus in vivo (4) . they can sustain viral replication, disseminate virus, and serve as a viral reservoir post-infection (5) (6) (7) . cells of the macrophage/monocyte lineage vary greatly in phenotype, longevity, and in phagocytic, immunoregulatory, and secretory properties (8) (9) (10) (11) . macrophages are categorized as classically (m1) or alternatively (m2) activated based on surface markers and functional role (12, 13) . m1 macrophages mediate inflammatory responses against pathogens while m2 macrophages have anti-inflammatory properties, promoting tissue repair and remodeling (14) . alveolar macrophages (am) in the lung uniquely express both m1 and m2 phenotypic markers, indicating ability to quickly respond to pathogens but also prevent immune activation in response to harmless antigens that enter the alveolar lumen (15) . am express cd4 and chemokine receptors making them vulnerable to hiv infection (16) . however, macrophages may be poorly susceptible to hiv induced cytopathic effects. minimal consequences of hiv infection on the macrophage transcriptome were observed (17) ; in contrast expression of hiv nef and gp120 envelope induced macrophage activation (18, 19) . indirect activation can also occur by exposure of uninfected macrophages to viral gene products or cytokines from other infected cells (20) . am are important lung phagocytes (21) , yet am obtained from hiv-infected individuals have shown contradictory results regarding the impact of viral infection on phagocytosis. some studies have shown impaired phagocytosis of opportunistic pathogens by am (22) (23) (24) (25) (26) ; others have reported no change in am phagocytic activity during infection (27, 28) . such variations in outcome may result from differences in length of infection or reliance on the use of monocyte derived macrophages (mdm) and infected cell lines which may not ideally represent clinical situations. macrophages can utilize fcγ receptors to internalize antibody-opsonized virions or infected cells, potentially leading to antibody-mediated clearance of infectious material (29) . antibody-dependent phagocytosis (adp) by am contributes to protection against viral infections such as influenza (30) , west nile (31), adenovirus (32) , and sars coronavirus (33) . however, adp-mediated protection by macrophages against hiv infection has not been observed. here we investigated the dynamics of siv-related changes in am activity and function by sampling bronchoalveolar lavage (bal) from siv infected rhesus macaques during acute and chronic infection. the am response to siv infection consisted of phenotypic changes and alterations in proinflammatory responses, ability to respond to gp120 antigen, and phagocytic activity. fcγriiib expression on am was linked to siv-specific adp and viral control during acute infection. novel results showed increased expression of programmed cell-death-1 (pd-1) on am from chronically infected macaques and positive correlations between pd-1 expressing ams and siv viremia. we believe this is the first report of pd-1 expression on ams of siv infected macaques. our results suggest associations between pd-1 expression, macrophage dysfunction, and lack of viremia control. forty-nine female indian rhesus macaques used in this study were housed in the nci animal facility, bethesda, md, under protocol vb-020. the nci facility is accredited by the association for assessment and accreditation of laboratory animal care international, and its animal care and use committee approved all animal experiments prior to study initiation. standard practices followed recommendations made in the guide for the care and use of laboratory animals of the united states, national institutes of health, and the weatherall report. thirty macaques were used as naïve controls and 19 macaques were challenged weekly intravaginally using a repeated low-dose of siv mac251 (800 tcid 50 ). infection was confirmed for 14 of the macaques with plasma viral loads of ≥50 siv rna copies/ml as assessed by droplet digital pcr (chung et al., manuscript in preparation). the remaining five macaques were infected intrarectally with a single high dose of siv mac251 (4000 tcid 50 ). out of the 19 infected macaques, 9 were euthanized after 2 wpi and the remaining 10 infected macaques were monitored longitudinally for up to 20 wpi. bal samples were obtained using standard techniques (34) . briefly, rhesus macaques were anesthetized, and an endotracheal tube was inserted through which sterile saline solution (10 ml/kg) was instilled. suction was applied to recover the instilled fluid and the lung lavage was collected in sterile conical tubes. cells were pelleted by centrifuging at 1600 rpm for 10 min at 4 • c and washed in 25 ml cold pbs. centrifugation was repeated, and cells were resuspended for counting. cells showed ∼90% viability as determined by trypan blue staining. bal cells were enriched for ams by depleting epcam + , cd2 + , and cd20 + cells. the following reagents were used: rabbit polyclonal anti-epcam (n3c3) (genetex, irvine, ca); cd2 and cd20 microbeads for non-human primate (miltenyi biotec, auburn, ca). depletion was performed using macs cell separation technology according to the manufacturer's instructions (miltenyi biotec, auburn, ca). enriched ams were stimulated for 6 h in 500 µl r-10 media (rpmi + 10% fbs + 5% pen-strep + 5% anti-anti) containing purified native hiv-1 envelope gp120 (advanced bioscience laboratories, inc., rockville, md) at a concentration of 200 nm or lps (thermofisher scientific, waltham, ma) at 500 ng/ml concentration. for experiments assessing cytokine production by pd-1 + ams, cells were stimulated with 10 µg/ml lps in the presence of golgistop (1 µl, bd biosciences) for 18 h prior to intracellular staining. adp activity was measured as previously described (35) . siv mac251 gp120 was biotinylated with a biotin-xx microscale protein labeling kit (life technologies, grand island, ny) and incubated with a 10-fold dilution of 1 µg avidin coated sky blue fluorescent beads (0.8 µm diameter; spherotech, lake forest, il) overnight at 4 • c. enriched ams were plated in a u-bottom 96 well plate at 40,000 cells/well and undiluted bal-f was added. to determine phagocytic activity of am independent of siv specific antibody, bal-f from naïve macaques was used. for siv-specific adp, autologous bal-f from infected macaques was used. the bead-gp120 mixture was brought to a final 50-fold dilution in r-10 media and 50 µl was added to the cells and incubated for 3 h at 37 • c. for pd-1 blocking experiments, bal samples collected from macaques at 20 wpi were enriched for ams as described above and incubated with either 10 µg/ml ultraleaf purified anti-human pd-1 antibody (eh12.2h7, biolegend) or 10 µg/ml migg1 isotype control (sigma-aldrich). cells incubated with naïve bal-f were used for normalization. in all cases, after 3 h of incubation, 70 µl of 2% paraformaldehyde was added for fixation. fluorescent bead uptake was assessed using a bd biosciences lsrii flow cytometer. bead uptake specifically by am was made possible by focusing on cells that were autofluorescent in the fitc channel. the phagocytic score of each sample was calculated by multiplying the frequency of bead-positive cells by the degree of phagocytosis measured as mean fluorescence intensity (mfi) and dividing by 10 4 . values were normalized to background values (cells and beads with either pbs or naïve bal-f) by dividing the phagocytic score of the test sample by the phagocytic score of the background samples. mouse anti-non-human primate or anti-human fluorochromeconjugated monoclonal abs (except where specified otherwise) known to cross-react with rhesus macaque antigens were used in this study. the following antibodies were used for surface staining: bv711 anti-cd4 (l200), bv786 anti-cd45 (d058-1283), buv395 anti-cd3 (sp34-2), buv496 anti-cd16 (3g8), cells were then washed with bd permwash, and incubated with intracellular staining antibodies for 25 min at rt. finally, cells were washed with pbs and staining data were acquired using the 5 laser bd facsymphony (bd biosciences, san jose, ca). fluorescence minus one (fmo) and isotype controls were used to confirm the phenotype and cytokine expressing population of ams ( figure s1 ). data analyses were performed with flowjo (version 10.5, treestar, inc ashland, or) software. antibodies in bal-f were measured as follows: wells of greiner high-binding ½ area 96-well plates were coated overnight at 4 • c with 100 ng/well of siv mac251 gp120 (for determining gp120specific igg) or 50 ng/well of anti-rhesus igg (alpha diagnostics) (for determining total igg) in sodium bicarbonate buffer (ph 9.6) (sigma-aldrich, st. louis, mo). wells were blocked with 200 µl of 1% bsa diluent/blocking solution (kpl) in distilled water for 2 h at rt. bal-f (50 µl) was added and incubated for 1 h at 37 • c. env-specific igg derived from purified serum igg obtained from siv mac251 -infected macaques and quantified as previously described (36) was used to generate a standard curve for env-specific igg. purified rhesus igg (non-human primate reagent resource) was used to generate a standard curve for total igg. plates were washed 5 times with 1x wash solution (kpl). horseradish peroxidase-labeled goat anti-monkey igg (50 µl at a 1:10,000 dilution; alpha diagnostics) was added, and plates were incubated for 1 h at 37 • c. after washing as described above, 50 µl of 3,3 ′ ,5,5 ′ -tetramethylbenzidine (tmb) peroxidase substrate (kpl) was added for 10-20 min at rt. color development was stopped with 50 µl 1 m phosphoric acid (sigma), and plates were read at 450 nm by using a biotek plate reader. siv gp120-specific antibody levels were expressed as ng gp120-specific igg/µg total igg. statistical analysis was performed using one-way anova or 2way anova with the tukey multiple comparisons test as indicated in the figure legends. the wilcoxon matched-pairs signed rank test was also used where indicated. correlation analyses were performed by non-parametric spearman correlations. statistics were generated using graphpad prism. rhesus macaques were infected with siv mac251 and bal was collected at 2, 4, 8, 12, and 20-wpi. nineteen macaques were sampled at 2-wpi and 10 for the subsequent time points. bal samples were also obtained from 30 naïve macaques. ams from bal were characterized by flow cytometry as hla-dr hi , cd11b int , and cd163 + cd206 + (37) (figure 1a) . the mean frequency of ams was >80% of leukocytes found in the lavage samples, consistent with other reports on bal specimens from naive rhesus macaques (37) . no significant change in the frequency of ams was observed over the 20 weeks of infection ( figure 1b) . expression of cd11c and cd14 has been shown to identify am phenotypes with changes in expression occurring during the fibrotic stage of lung disease (38) here, no significant changes in am expression of cd11c or cd14 over the time period studied were observed (figures 1c,d) . we also did not observe changes in the percentage of ams expressing the costimulatory molecules, cd80 and cd86, indicating that any inflammatory responses recorded may not be associated with the expansion of these cell subsets (figures 1e,f) . macrophages secrete proinflammatory cytokines and chemokines in response to hiv infection (39, 40) . however, a clear dynamic of the cytokine and chemokine response by macrophages over the course of hiv/siv infection has not been shown. here we characterized cytokine levels in am collected from siv infected rhesus macaques between 2 and 20-wpi and compared them to levels in ams from naïve macaques. a strong proinflammatory response was seen during the acute phase of infection. expression of tnf-α, il-6, and il-1β in ams was significantly increased by 2-wpi (p < 0.0001 for all; figures 2a-c) . by 4-wpi expression levels were significantly lower. cytokine expression remained low until the 20-wpi time point except for il-6 expression, which increased significantly by 12-wpi compared to 4-wpi ( figure 2b) . the il-6 expression level at 20-wpi was not significantly higher than levels seen in naïve populations. similarly, a higher level of the chemokine rantes was found in ams from macaques at 2-wpi ( figure 2e) . expression decreased to levels comparable to naïve samples by 4-wpi and remained low until the 20-wpi time point (figure 2e ). mip-1α expression did not change over the course of infection ( figure 2d ). overall, we observed a transient increase in the proinflammatory cytokine responses, which decreased to levels comparable to those of naïve macaques during the chronic phase of infection. to assess whether ams were leaning toward an anti-inflammatory role during chronic infection, we evaluated the intracellular expression of il-10 by ams ( figure 2f ). ams play an important role in maintaining an anti-inflammatory environment in the lung (21, 41) . indeed, il-10 expressing ams were detected in naïve animals and the frequency of il-10+ cells was significantly higher compared to that of acutely infected animals ( figure 2f) . interestingly, the percentage of il-10 expressing ams further decreased significantly as infection progressed into the chronic stage ( figure 2f) . thus, siv infection was associated with a significant spike in the am proinflammatory response by 2-wpi, which waned in chronic infection. in addition, the low proinflammatory cytokine response in chronic infection was not associated with an increase in il-10-expressing ams. to investigate am activation, bal cells obtained from naïve and acute and chronically infected macaques at weeks 2, 4, 8, 12, and 20 wpi, were incubated with native gp120 from r5 tropic siv or lps for 6 h, and intracellular expression of mip-1β and il-6 was assessed (figures 3a,b) . both gp120 and lps induced comparable levels of mip-1β and il-6 in ams from naive and acutely-infected animals (figures 3a,b) . however, in chronically infected macaques gp120 did not induce intracellular il-6 or mip-1β expression in ams (figures 3a,b) , and lps did not stimulate expression of either factor to the level induced in naïve animals suggesting a dysfunctional state of the ams (figures 3c,d) . to assess whether ams contribute to clearance of siv, we evaluated their phagocytic activity and ability to perform adp over the course of siv infection using bal samples from 5 naïve animals and 5 siv-infected macaques sampled between 2 and 20-wpi. while the bal samples contained a high percentage of ams (figure 1b) , the cells were further enriched for ams using macs by depleting cd2, cd20, and epcam positive cells ( figure 4a) . to assess phagocytic capacity independent of siv antibody, enriched cells were incubated with siv mac251 gp120-coated fluorescent beads and bal-f from naïve macaques prior to quantification of bead uptake. a significant increase in phagocytic activity was detected during the acute phase, 2 and 4-wpi (figure 4b) . at 8-wpi, phagocytosis decreased and was comparable to naïve cells. activity further decreased significantly at the 12 and 20-wpi time points compared to the acute and naïve time points (figure 4b ). prior to evaluating adp by ams, we assessed siv gp120specific igg in bal-f ( figure 4c) . specific igg was detected at 8-wpi with levels maintained or continuing to rise until 20-wpi. to determine siv-specific adp, ams from naïve macaques or sampled over the course of siv infection were incubated with antigen-coated beads along with autologous bal-f. phagocytosis scores were normalized against the score obtained using naïve bal-f to highlight phagocytic activity associated with the presence of siv-specific antibody ( figure 4d) . significantly higher phagocytic scores were detected at 12-wpi compared to those at naïve and 4-wpi time points, indicating that ams could perform siv-specific adp ( figure 4d) . to further assess adp by ams in chronic infection, phagocytic scores at chronic time points (figure 4d ) were normalized against siv-specific antibody levels ( figure 4c) . results showed that in the presence of comparable antibody levels, ams mediated adp activity at 12-wpi. however, this activity was significantly compromised by 20 week pi ( figure 4e) . thus, am are capable of gp120specific adp for a limited time period during chronic infection. this diminished functional capacity of ams in the chronic stage further highlights the dysfunction observed upon lps and gp120 stimulation (figure 3) . three classes of fcγr are constitutively expressed on ams with an active role in protecting against pathogens in the airway: high-affinity fcγri (cd64) and low-affinity fcγrii (cd32) and fcγriii (cd16) (42) . fcγriii occurs in two isoforms: fcγriiia (expressed mainly on nk cells and macrophages) and fcγriiib (expressed on neutrophils) (43) (44) (45) . infected mdms have shown compromised expression of the γ signaling chain of fcγriiia, possibly leading to altered phagocytic activity (46) . monocytes from chronically infected individuals have also shown reduced expression of fcγriiia and diminished phagocytic activity (47) . however, thus far no correlations have been found between adp and hiv/siv viral load, cd4 count or fcγr expression. given the unique features of ams compared to other tissue macrophages and monocytes, and the importance of phagocytic function in the lung, we investigated whether there was altered fcγr expression on ams over the course of siv infection and any association with adp. bal from naive macaques contained over 80% of fcγri + and fcγrii + ams (figures 5b,c) . both the frequencies and mfi of fcγrii and fcγri remained high and unchanged over the course of infection (figures 5b,c,e,f) . in contrast, the mean frequency of fcγriii + cells was 30% in naïve macaques. at 2-wpi there was a significant increase in fcγriii + am frequency but levels significantly decreased by 8-wpi (figure 5a) . the low frequency of fcγriii + ams was maintained until 20-wpi. a similar increase in am fcγriii mfi was not observed in the acute phase of infection compared to naïve animals ( figure 5d ). however, expression levels were significantly reduced at the 8 and 20-wpi time points compared to levels at 2-wpi ( figure 5d ) in concert with the decreased frequencies observed. the siv-specific adp observed at 12-wpi ( figure 4e ) exhibited a strong trend toward positive correlation with the percentage of fcγriii + ams at the same time point ( figure 5g) . further, the fcγriii mfi at 2-wpi ( figure 5d ) negatively correlated significantly with acute plasma viral load ( figure 5h) . these data suggest that fcγriii plays an important role in siv-specific adp and that its expression is associated with early viral load control. to characterize the dysfunction observed in ams from chronically infected macaques, we looked for parallels with t cell exhaustion. since the initial description of t cell exhaustion in chronic viral infections (48, 49) , common phenotypic and functional properties have been attributed to a dysfunctional state regardless of the type of pathogen. high and prolonged expression of inhibitory receptors are a key feature of t cell exhaustion in chronic viral infections (50) . in particular, overexpression of the pd-1 receptor on cd8 + t-cells plays a major role in t cell dysfunction associated with hiv infection (51) (52) (53) . here, we identified pd-1 + ams in rhesus macaque bal and assessed changes in its expression on ams from 6 macaques over the course of siv infection (figure 6a) . when the frequency of pd-1 + ams was assessed in naive macaques and compared to frequencies in siv infected animals, five out of six siv + macaques showed an increase in frequency (figure 6b) . the pd-1 + am frequency at 20-wpi was significantly different from both naïve and 2-wpi time points: p = 0.0419 and p = 0.0294, respectively. ams at 20-wpi also displayed a significant increase in pd-1 mfi signifying increased expression of the receptor ( figure 6c) . furthermore, the percentage of pd-1 + ams correlated positively with viral load at 20-wpi ( figure 6d) . we also observed a strong trend toward correlation between pd-1 mfi and viral load at 20-wpi (figure 6e) , indicating an association between pd-1 expression in ams and siv disease progression. pd-1 has two well-known ligands via which inhibitory effects have been reported to occur: programmed death -ligand 1 (pd-l1) and programmed death ligand 2 (pd-l2). while pd-l1 has been shown to be expressed broadly in both hematopoeitic and non-hematopoeitic cells, a more restricted expression pattern has been documented for pd-l2, namely antigen presenting cells (apc) (54) . in order to identify a potential role for the pd-1 pathway on am function, we looked for expression of these ligands on ams after siv infection. ams from naïve macaques predominantly expressed high levels of pd-l2 (figure 7b) . this expression decreased modestly, but not significantly, after siv infection and remained consistent thereafter. on the other hand, we observed minimal expression (∼10 fold lower than that of pd-l2) of pd-l1 on ams from naïve macaques ( figure 7a) . further, pd-l1 expression significantly decreased over the course of siv infection. these results indicate that am dysfunction is more likely associated with increased pd-1 expression rather than changes in the expression of its ligands and may occur through the pd-1/pd-l2 pathway. to identify whether pd-1 blockade can rescue am phagocytosis, we examined the phagocytic activity of ams from 7 chronically infected macaques in the presence of pd-1 blocking antibody or mouse igg as control. interestingly, we found higher phagocytic scores in the pd-1 blockade group in 6 out of the 7 animals (p = 0.0312), indicating that pd-1 expression is likely mechanistically linked to impairment of phagocytic activity (figure 7c) . we further assessed cytokine expression of pd-1 expressing ams. we stimulated ams from chronically infected macaques with 10 µg/ml lps and compared cytokine expression between pd-1 + and pd-1 − populations. although not significant, we found lower expression of tnf-α on pd-1 expressing cells compared to the pd-1 − population ( figure 7d) . comparisons with additional cytokines and chemokines (il-6, il-1β and rantes) are not reported here as expression of these cytokines could not be detected in the ams from the infected macaques that were tested. taken together, these data strongly suggest that pd-1 expression on ams during siv infection may be a marker of dysfunction. our results show that in the siv macaque model, the pro-inflammatory response of ams to siv infection peaks in the acute phase of infection and is diminished in the chronic phase as observed following stimulation with lps or gp120. we report that ams from siv infected macaques can perform adp early in infection, but this activity diminishes in the chronic phase. in novel findings, we report an elevated percentage of pd-1 + ams in chronically infected macaques, which positively correlated with viral load. during the same period diminished inflammatory responses and adp were observed. the percentage of ams stayed consistent over the course of siv infection (figure 1b) . viral infections can lead to activation of macrophages and subsequent release of factors that recruit additional cells of the innate and adaptive immune system (55, 56) . the lack of detection of macrophage recruitment to the lung may have resulted from a concurrent lymphocyte recruitment, making the relative number of macrophages appear consistent. indeed, a higher percentage of lymphocytes in bal of hivinfected patients has been reported (57) . here, estimating actual am numbers by multiplying cell count with percentage of ams did not result in significant differences ( figure s2) . including absolute counting beads as part of the initial analysis might have provided more definitive answers. variations in surface marker expression of ams over 20 weeks of siv infection were also not seen, except for changes in fcγriii expression (figures 1, 5) . phenotypic comparisons of ams from the bal of hiv-infected and uninfected individuals also have not shown differences (57) . proinflammatory cytokines il-6 and il-1β, tnf-α, and chemokine rantes was induced in ams during acute infection (figures 2a-e) . the rapid secretion of cytokines and chemokines was expected as the innate immune system initiates lymphocyte recruitment to establish adaptive immunity prior to viral spread. although the hiv/siv infection rate of ams may be low, macrophages respond to exposure to viral particles or virus-derived gene products such as nef, tat, and the gp120 envelope (18, 58) . following the acute phase, the elevated cytokine and chemokine responses by ams were not sustained despite exposure to viral products and likely lps from the gut (59) . further, stimulation with lps or native gp120 ex vivo showed an impaired il-6 and mip-1β response of ams from chronically infected macaques (figure 3) , indicating a potentially compromised inflammatory response against lung pathogens. this novel observation is consistent with data showing inhibition of tnf-α release by macrophages in response to toll-like receptor (tlr)-4 stimulation during hiv infection, even though the expression level of tlr-4 remained unchanged (60) . desensitization of tlr ligands on ams as a result of viral respiratory infections has also been reported (61) . the β-chemokines mip-1α, mip-1β, and rantes can bind to the hiv-1 coreceptor ccr5 indicating a potential protective effect against entry of r5-tropic viruses (62) (63) (64) (65) . the pattern of chemokine expression here shows that any protective effect of β-chemokines expressed by ams is likely limited to the acute phase of infection (figures 2, 3) . the lowering of cytokine and chemokine responses after the acute phase of infection was not associated with increased il-10 expression (figure 2f ). in fact, ams of naïve macaques expressed the highest level of intracellular il-10 ( figure 2f) . some studies have shown no induction of il-10 secretion or mrna in macrophage-tropic hiv-1 infected mdm compared to uninfected cells (66) . others have shown increased il-10 secretion and mrna levels for in vitro hiv-infected pbmc, monocytes and mdms [reviewed in (67) ]. differences in virus tropism or target cells could account for the variations observed. increased levels of il-10 have been detected in the bal-f of hiv infected patients (68) , however the il-10 in these bal samples could have originated from other lymphocytes that upregulate il-10 during hiv infection (69). in sum, our data indicate that siv disease progression is not associated with increased am il-10 expression, suggesting that ams are not switching to a more regulatory phenotype. ams are essential for lung microbial clearance. despite reports that am function is not affected by hiv-1 infection (27, 28) , recent studies have shown that phagocytic activity can be impaired (26, 70) . as viral load and activation status of macrophages can vary over the course of infection, here we tracked the phagocytic activity of ams following siv infection, providing new insights into the dynamics of am function. ams readily internalized antigen coated beads during acute infection, but this ability markedly decreased in chronically infected animals ( figure 4b ). the initial phagocytic activity was non-specific, as no gp120-specific antibody was present, and was likely due to the rise in proinflammatory responses during acute infection (figures 2a-e) . adp was observed in the chronic stage of infection when gp120specific antibody levels increased in bal-f (figures 4c,d) . however, a continued rise in gp120 antibody levels was not associated with increased adp. in fact, decreased adp activity was seen later in chronic infection. therefore, even though the pattern of chemokine expressing ams over the course of siv infection (figures 2a-e) suggested that frequencies returned to naive levels during the chronic phase of infection, the diminished functional activity observed suggested am dysfunction. fcγriii expression was elevated during acute siv infection but decreased as infection progressed (figures 5a,d) . these data are partly in contrast to data from hiv-infected mdms that showed either elevated or no change in fcγr expression [reviewed in (6) ]. the fcγriii isoform expressed in macrophages (fcγriiia) consists of a ligand bindingα-chain associated with disulfide-linked γ-chains (71, 72) . the γ signaling subunit of fcγrs has been shown to be downregulated as a consequence of hiv infection, resulting in aberrant downstream signaling required for phagocytosis (46, 73) . the fcγriii antibody used here recognizes the ligand binding fcγriii α-chain (74); thus the loss of fcγriii expression cannot be explained by downregulation of the γ-chain alone. our data suggest that in spite of an initial increase in the frequency of fcγriii + ams during acute infection (figure 5a) , prolonged hiv infection may lead to diminished expression of fcγriii. this outcome may not have been observed previously as in vitro mdm infection experiments mainly mimic acute stages of infection. nevertheless, although the frequency of fcγriii + ams at 12-wpi had dwindled to <30% of the level observed in naïve animals, a correlative trend between the frequency of fcγriii + ams and adp was observed ( figure 5g) indicating the importance of this receptor in adp activity mediated by ams. furthermore, fcγriii mfi negatively correlated with acute viremia suggesting an important role for fcγriii-mediated phagocytosis in initial viral load control ( figure 5h) . the pd-1/pd-l pathway plays a key role in negative regulation of adaptive immunity in hiv and other viral infections [reviewed in (75) ], but few studies have explored the role of pd-1 in innate immunity, particularly by macrophages. pd-1 expression on t cells following immune activation and its role in t-cell exhaustion when highly expressed during hiv-1 and other chronic viral infections have been described (52, 76). regarding macrophages, pd-1 expression has been linked to diminished ability to clear microbial invasion in septic mice (77) , inhibition of phagocytic activity and tumor immunity (13) , and inability to perform phagocytosis and intracellular killing in patients with tuberculosis (78) . unlike on t cells, pd-1 expression on macrophages in these studies described a single positive population. here, we also identified a single pd-1 + population of ams derived from chronically infected macaques (figures 6c,d) . the macaques were clear of lung infections at the time of bal collection, suggesting pd-1 expression was only associated with siv infection. until now, no direct evidence has implicated pd-1 in am dysfunction. our data show a direct correlation between pd-1 and siv viremia and suggest that in keeping with correlations described with t cells (53) , disease progression can also be associated with pd-1 expression in ams ( figure 6e) . while pd-1 ligands were found to be expressed on ams from naïve macaques, pd-l1 expression levels were low and subsequently declined after siv infection ( figure 7a ). this result was unexpected and in contrast to a study on mdms that showed up-regulation of pd-l1 and pd-l2 after exposure to inactivated hiv virions (79) . however, the alveolar environment is indeed unique and it is unsurprising that am responses to siv infection differ from in vitro exposure of mdms to virions. rodriguez-garcia et al. further indicated differential regulation of pd-l1 and pd-l2 by il-10, whereby presence of il-10 increased pd-l1 expression and its blockade increased pd-l2 expression (79) . analysis of il-10 expression by ams in our study showed a gradual decrease after siv infection and may be one explanation as to why we observed decreased pd-l1 expression ( figure 2c) . pd-l2 expression, however, remained high and did not increase with decreased il-10 expression. future analysis of the factors found in bal-f could provide further insight into the dynamics of pd-l expression on ams. pd-1 blockade experiments have shown enhancement of siv/hiv-specific responses, proliferative ability and cytokine production by exhausted pd-1 high t cells (80) (81) (82) . in keeping with this, blockade of the pd-1/pd-l pathway has been reported to ameliorate phagocytic function in macrophages found in the tumor microenvironment and in active tuberculosis (78, 83) . here, we also found that blockade of pd-1 could significantly improve phagocytic activity further highlighting pd-1 as a factor playing a role in dysfunction of ams ( figure 7c ). in addition, although not significant potentially due to small sample size, we found lower abundance of tnf-α expressing pd-1 + ams compared to the pd-1 − population during chronic infection ( figure 7d) . these data suggested that the presence of pd-1 on ams is likely a factor in any inhibitory role exerted on ams through the pd-1/pd-l pathway. in summary, our longitudinal investigation has provided important new information about the consequences of siv infection on ams, and in novel findings, propose a role for pd-1, a well-recognized inhibitor of adaptive immune responses, on innate immunity against siv infection. all datasets generated for this study are included in the manuscript and/or the supplementary files. forty-nine female indian rhesus macaques used in this study were housed in the nci animal facility, bethesda, md, under protocol vb-020. the nci facility is accredited by the association for assessment and accreditation of laboratory animal care international, and its animal care and use committee approved all animal experiments prior to study initiation. standard practices followed recommendations made in the guide for the care and use of laboratory animals of the united states, national institutes of health, and the weatherall report. therapeutic strategies towards hiv-1 infection in macrophages early events in sexual transmission of hiv and siv and opportunities for interventions th17 cells are preferentially infected very early after vaginal transmission of siv in macaques hiv-1 infection, response to treatment and establishment of viral latency in a novel humanized t cell-only mouse (tom) model macrophages: a crucial reservoir for human immunodeficiency virus in the body the role of monocytes and macrophages in the pathogenesis of hiv-1 infection macrophages sustain hiv replication in vivo independently of t cells bone marrow derived elements and resident microglia in brain inflammation the prolonged lifespan of alveolar macrophages monocytes and macrophages regulate immunity through dynamic networks of survival and cell death the biology of macrophages: i. general principles and properties m-1/m-2 macrophages and the th1/th2 paradigm alternative activation of macrophages the chemokine system in diverse forms of macrophage activation and polarization human alveolar macrophages predominately express combined classical m1 and m2 surface markers in steady state surface cd4 is critical to in vitro hiv infection of human alveolar macrophages hiv-1 infection of macrophages is dependent on evasion of innate immune cellular activation macrophage activation and human immunodeficiency virus infection: hiv replication directs macrophages towards a pro-inflammatory phenotype while previous activation modulates macrophage susceptibility to infection and viral production cell biology of hiv-1 infection of macrophages lymphocytic alveolitis, bronchoalveolar lavage viral load, and outcome in human immunodeficiency virus infection the role of alveolar macrophages in regulation of lung inflammation the effect of hiv infection on phagocytosis and killing of staphylococcus aureus by human pulmonary alveolar macrophages quantitative differences in phagocytosis and degradation of pneumocystis by alveolar macrophages in aids and non-hiv patients in vivo reduced binding and phagocytosis of pneumocystis carinii by alveolar macrophages from persons infected with hiv-1 correlates with mannose receptor downregulation small alveolar macrophages are infected preferentially by hiv and exhibit impaired phagocytic function. small alveolar macrophages are infected preferentially by hiv and exhibit impaired phagocytic function hiv gp120 in the lungs of antiretroviral therapy-treated individuals impairs alveolar macrophage responses to pneumococci opsonic phagocytosis of streptococcus pneumoniae by alveolar macrophages is not impaired in human immunodeficiency virus-infected malawian adults hiv-1 infection does not impair human alveolar macrophage phagocytic function unless combined with cigarette smoking fcgamma receptors as regulators of immune responses alveolar macrophages are critical for broadly-reactive antibodymediated protection against influenza a virus in mice antibody recognition of cell surface-associated ns1 triggers fc-γ receptor-mediated phagocytosis and clearance of west nile virus-infected cells internalization of adenovirus by alveolar macrophages initiates early proinflammatory signaling during acute respiratory tract infection phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected sars coronavirus recovery of alveolar macrophages from rhesus and cynomolgus macaques by lung lavage a robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples nasal dna-mva siv vaccination provides more significant protection from progression to aids than a similar intramuscular vaccination in vivo characterization of alveolar and interstitial lung macrophages in rhesus macaques: implications for understanding lung disease in humans flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung monocyte/macrophage-derived cc chemokines and their modulation by hiv-1 and cytokines: a complex network of interactions influencing viral replication and aids pathogenesis macrophage polarization at the crossroad between hiv-1 infection and cancer development interleukin-10 secretion by alveolar macrophages and monocytes in sarcoidosis quantitative assessment of fc receptor expression and function during in vitro differentiation of human monocytes to macrophages natural killer cell and granulocyte fc gamma receptor iii (cd16) differ in membrane anchor and signal transduction borne von dem ae. the fc-receptor iii of cultured human monocytes. structural similarity with fcriii of natural killer cells and role in the extracellular lysis of sensitized erythrocytes fcγriii (cd16) on human macrophages is a functional product of the fcγriii-2 gene hiv-1 down-modulates γ signaling chain of fcγr in human macrophages: a possible mechanism for inhibition of phagocytosis decreased fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically hiv-1 infected individuals induction and exhaustion of lymphocytic choriomeningitis virusspecific cytotoxic t lymphocytes visualized using soluble tetrameric major histocompatibility complex class i-peptide complexes viral immune evasion due to persistence of activated t cells without effector function redefining chronic viral infection pd-1 is a regulator of virus-specific cd8+ t cell survival in hiv infection upregulation of pd-1 expression on hiv-specific cd8+ t cells leads to reversible immune dysfunction pd-1 expression on hiv-specific t cells is associated with t-cell exhaustion and disease progression the function of programmed cell death 1 and its ligands in regulating autoimmunity and infection alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development the role of macrophages in the development and progression of aids-related non-hodgkin lymphoma the alveolar microenvironment of patients infected with human immunodeficiency virus does not modify alveolar macrophage interactions with streptococcus pneumoniae induction of rapid and extensive betachemokine synthesis in macrophages by human immunodeficiency virus type 1 and gp120, independently of their coreceptor phenotype microbial translocation is a cause of systemic immune activation in chronic hiv infection hiv impairs tnf-α release in response to toll-like receptor 4 stimulation in human macrophages in vitro sustained desensitization to bacterial toll-like receptor ligands after resolutionof respiratory influenza infection interaction of chemokine receptor ccr5 with its ligands: multiple domains for hiv-1 gp120 binding and a single domain for chemokine binding molecular cloning and functional characterization of a novel human cc chemokine receptor (ccr5) for rantes, mip-1β, and mip-1α ld78β, a nonallelic variant of human mip-1α (ld78α), has enhanced receptor interactions and potent hiv suppressive activity c-c chemokine receptor type five (ccr5): an emerging target for the control of hiv infection lack of interleukin 10 expression in monocyte-derived macrophages in response to in vitro infection by hiv type 1 isolates cytokines and hiv-1: interactions and clinical implications tumour necrosis factor (tnf) and tnf-related molecules in hiv-1+ individuals: relationship with in vitro thl/th2-type response hiv-1 decreases nrf2/are activity and phagocytic function in alveolar macrophages a macrophage fcγ receptor and the mast cell receptor for ige share an identical subunit mechanisms for regulating expression of membrane isoforms of fc gamma riii (cd16) the mechanism underlying defective fcγ receptor-mediated phagocytosis by hiv-1-infected human monocyte-derived macrophages the binding epitopes of human cd16 (fc gamma riii) monoclonal antibodies. implications for ligand binding t cell exhaustion pd-1 expression in acute hepatitis c virus (hcv) infection is associated with hcv-specific cd8 exhaustion pd-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis pd-1/pd-l pathway inhibits m.tb-specific cd4+ t-cell functions and phagocytosis of macrophages in active tuberculosis expression of pd-l1 and pd-l2 on human macrophages is upregulated by hiv-1 and differentially modulated by il-10 enhancing siv-specific immunity in vivo by pd-1 blockade pd-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination responsiveness of hiv-specific cd4 t cells to pd-1 blockade pd-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01537/full#supplementary-material key: cord-104490-t42eccng authors: frimpong, shadrack; paintsil, elijah title: a case for girl-child education to prevent and curb the impact of emerging infectious diseases epidemics date: 2020-09-30 journal: yale j biol med doi: nan sha: doc_id: 104490 cord_uid: t42eccng not only do epidemics such as hiv/aids, ebola virus disease (evd), and the current coronavirus disease (covid-19) cause the loss of millions of lives, but they also cost the global economy billions of dollars. consequently, there is an urgent need to formulate interventions that will help control their spread and impact when they emerge. the education of young girls and women is one such historical approach. they are usually the vulnerable targets of disease outbreaks – they are most likely to be vehicles for the spread of epidemics due to their assigned traditional roles in resource-limited countries. based on our work and the work of others on educational interventions, we propose six critical components of a cost-effective and sustainable response to promote girl-child education in resource-limited settings. "study after study has taught us that there is no tool for development more effective than the education of girls. no other policy is as likely to raise economic productivity, lower infant and maternal mortality, or improve nutrition and promote health -including the prevention of hiv/aids." kofi a. annan, former secretary-general, united nations [1] . as it has been the case of hiv/aids and, most recently, the ebola virus disease (evd), the ongoing coro-navirus disease 2019 (covid-19) is a stark reminder of a painful reality: epidemics will continue to be the bane of human existence. consequently, there is an urgent need to critically assess the literature of approaches that have previously mitigated outbreaks and design robust interventions to fight these emerging outbreaks. one such method is the education of young people, especially girls and women. the fact that education is an essential social determinant of health has been well documented [2] . early childhood education provides access to higher-income earning potentials, reducing one's likelihood of getting infected by disease during an epidemic [3] . the benefits of education in improving health outcomes are high in children and young people, who often depend on their parents and guardians for their livelihoods, including financial resources to access education [1, 3] . for instance, a study in ghana found that the higher the level of education a mother has, the better the child's chances of survival [4] . also, according to unicef, young boys and girls who have higher education levels usually have more knowledge about infectious diseases, are less likely to get infected, and tend to adopt behaviors and attitudes that prevent them from being infected [1] . these health-related benefits of education become even more critical in epidemics such as hiv/aids and evd. indeed, several studies from around the world have confirmed that hiv infection rates are at least twice as high among adolescents who drop out of primary school than those who stay in school [3] . the 2014 west african evd outbreak killed over 11,000 people, left many children orphaned, and exposed to malnutrition, hunger, and preventable deaths [5] . deeply affected by the negative repercussions of the lack of quality education, young girls and women in low-and middle-income countries (lmics) usually must contend with power dynamics, traditional roles, and social inequities in communities [1] . while the global community has made significant headway over the years to leverage access to education as a tool to enhance health outcomes for young girls and women, there still exist gaps in addressing health challenges for this vulnerable population in many lmics across the globe [3] . prior educational interventions have often failed to adequately engage the communities in which they operate or are not financially sustainable without long-term donor support [3] . consequently, they crumble during epidemics when donor funding becomes limited, schools close, and teachers and administrators die or become sick. financially sustainable and community-driven educational interventions for young girls can help to address these challenges and improve health outcomes in lmics and help curb epidemics. given the limited resources available for tackling the myriads of global health challenges, we must assess prospective educational interventions in the light of rigorous evidence before implementing them. consequently, we ask: how exactly have global education efforts to improve health performed so far? evidence from published literature sheds some light. in a study of eight sub-saharan african countries, gupta et al. found that females who had eight or more years of schooling were 13% more likely to avoid sex before age 18 compared to their peers with less education [6] . also, in zimbabwe, studies have con-firmed that among 15-18-year-old adolescent girls, those who drop out of school are about five times more likely to have hiv than their colleagues who stay in school [1]. evidence from surveys in malawi, haiti, uganda, and zambia further corroborates these findings by showing a secure link between higher education and fewer sexual partners [7] . the general observation here is that these outcomes are not limited to one country. indeed, kirby et al. also found that, in 11 countries, women with some form of schooling were about five times more likely to have used condoms during sexual encounters than uneducated women in similar backgrounds and settings [8] . these findings underscored global commitments such as the millennium development goals (mdgs) related to hiv/aids, education and girls; the dakar framework for action related to girls' education; and the current sustainable development goals (sdg #4) to promote inclusive and equitable quality education for all [1] . based on these initiatives, many lmics have made commitments to improving girl-child education. in ghana, the early 2000s was the era of media campaigns dubbed "send your girl child to school" [9] . similarly, many other african governments, with the help of international partners, joined these laudable efforts by providing hundreds of millions of dollars to establish schools for girls and to embark on public education programs in rural communities [9] . a natural follow-up question would be; how have these educational commitments and interventions affected health outcomes so far? in a recent systematic review, psaki et al. concluded that, even though investments in schooling may yield positive ripple outcomes for sexual and reproductive health, these effects may not be as pronounced as expected [10] . in a similar light, mensch et al. also concluded in their review that, while improvements in women's educational outcomes such as grade attainment have helped to improve health in several places, the effect may be overestimated [11] . regardless, these two studies highlight the critical roles that educational quality and an intervention's implementation level (community versus national) might play in the synthesis of these outcomes and the conclusions they reported. although there is a wealth of evidence that education is essential for preventive health in the population, the global community failed to meet the mdg #3 of equal access to education for girls by 2015. why then are educational efforts still failing to address the health challenges of young girls and women, as we have observed in the hiv/aids epidemic and, most recently, the evd crisis [12] ? the answer to this question may stem from the challenges of financial sustainability and community engagement that these epidemics expose. this answer corroborates the caveats that mensch et al. (2019) and psaki et al. (2019) highlighted. for instance, during the hiv/aids and recent evd outbreaks, many young girls had to drop out of school to provide and care for their families who were sick and dying, consequently increasing their risk of exposure to these lethal viruses [13] . in these situations, when parents or guardians died, these girls dropped out of school due to default in payment of school fees. in 2003, there were as many as 15 million aids orphans [1], and the 2014 west african evd outbreak left tens of thousands of orphans in its wake [14] . faced with a compounded challenge of affording their nutritional and accommodation needs, they engaged in risky transactional sex as a means of survival [14] . in some cases, these were non-consensual sexual encounters. evidence from the children's ebola recovery assessment of 617 girls in sierra leone found that many young girls who had dropped out of school ended up in ebola-quarantined households where they became subjects of rape and other forms of sexual assault [15] . moreover, the united nations population fund (unfpa) reported that many young girls in the democratic republic of congo encountered rape and sexual attacks from members of armed groups in communities such as north kivu and ituri [16] . beyond issues of gender-based violence and school drop-outs, epidemics also weaken the capacity and quality of educational systems. in many countries that are hard-hit by hiv/aids and evd, many teachers died, and the few that survived also had to suspend their teaching roles to cater for their sick and dying family members [1]. in 2000, at the peak of the hiv/aids crisis, about 815 of primary school teachers (45% of trained teachers) in zambia died from the disease, and close to 8% of the 300 teachers in the central african republic also died [1,17]. these teacher shortages are prevalent in rural areas where schools lose staff. teachers who are affected by disease outbreaks migrate to urban areas for better healthcare for themselves or concerned family members in their care [1] . in malawi, the student-teacher ratio in many schools jumped to about 96 to 1 due to aids-related illnesses [18] . worldwide, 13 out of the 15 countries where over 30% of school-aged girls are out of school, are in sub-saharan africa [19] . yet, this same population of girls remains the most vulnerable during pandemics when schools are closed. in a recent comprehensive report, the malala fund forecasted that there is a high likelihood that more than 10 million secondary school-aged girls in lmics may not return to school after the covid-19 pandemic [20] . there is a need for educational interventions for young girls that can withstand the financial shortfalls and the capacity-weakening effects on educational systems that pandemics cause. an organization that implements such interventions is cocoa360, a nonprofit in rural western ghana which transforms communities by using revenues from existing community resources such as cocoa, to finance educational and health outcomes (figure 1 ). the details concerning cocoa360's model the following are essential ingredients for successful community engagement: (1) community "knocking" -this is the first step in entering a community. use this period to share your concept and vision about the project with community elders, usually, the chief, for their acceptance and input; (2) shared leadership -the community should be involved in the governance of the project right from the start. cocoa360 collaborated with their partner communities to establish a local decision-making body called the village committee (vc) [21] . this group comprises some of the community's respected citizens from diverse religious, ethnic, and occupational backgrounds. the vc serves as a link between cocoa360 and the broader community and ensures that cocoa360's operations, including those at its tarkwa breman girls school and tarkwa breman community clinic, reflect the needs and cultural standards of the community and its members [21] . this alignment is especially important because many such rural communities still do not prioritize girl-child education due to persisting cultural beliefs; (3) community education -the community should be reminded continually of the components, their role in the project's successes, and challenges. such educational efforts will deepen buy-in and ensure the continued success of the program; (4) shared successes -the project's progress should be celebrated with the community in such a way that they know and feel that their contributions matter; and (5) sustainability plan -the community should be involved in formulating strategies that will ensure project longevity. zero tuition costs: efforts to eliminate tuition costs and make education compulsory in many countries have helped increase educational access for young girls, significantly reduced their vulnerability to child marriages, and many sexually transmitted diseases such as hiv/aids. and its impact have already been discussed in a separate publication [21] . herein, we draw on published evidence and our own experiences with cocoa360 to propose six general components (figure 2 ) that should be considered in insulating educational models from epidemics: active community engagement: in a review of interventions that improve girls' education and gender equality, unterhalter et al. highlight the effectiveness of programs that focused on shifting gender norms and encouraging inclusion through community involvement [22] . this finding matches our experience and shared belief that the currency for successful interventions in sub-saharan africa is a strong community engagement. working together with community leaders and members enables external project partners to appreciate cultural nuances and local contexts that need to be considered before, during, and after the implementation process. in many rural communities, community leaders and members coordinate mutual support such as construction labor, contribute resources such as land, and are willing to engage in initiatives that will help financially sustain the intended interventions over the long term. for an education model to be sustainable, it would similarly need to include the inputs and perspectives of the residents and their leaders right from the planning phase, throughout the implementation phase, and post-implementation evaluation efforts as well. cocoa360, a nonprofit organization in rural western ghana, provides an example of how to solicit and successfully engage a community. as a community-based organization, cocoa360 transforms rural communities by using revenues from existing community resources such as cocoa to finance educational and health outcomes [21] . from our experience with community engagement, ysis and systematic reviews has suggested approaches such as conditional cash transfers and bonuses as effective incentives for increasing girls' school enrollment in developing countries [1, 28] . while successful, these approaches rely on external funding to meet non-tuition expenses, which may not always be available. on the other hand, interventions like cocoa360's "farm-for-impact" model seek to consistently keep communities at the forefront of decision making and revenue-generation. consequently, they provide a much better independent pathway to self-support the education of their daughters when donor funding becomes limited or ceases [21] . the process of community-led revenue generation and decision making further imbues a strong sense of communal ownership, which can translate into educational outcomes. for instance, preliminary findings from cocoa360 show that the school attendance rate is 98%, compared to the national rural attendance rate of about 70% for similar schools under the ghana government's free compulsory universal basic education (fcube) program [21] . school-based food and nutrition initiatives: epidemics such as hiv/aids and evd cause many young girls to drop out of school when their parents and guardians are affected or die. consequently, they end up resorting to transactional sex to obtain support for food and accommodation [1]. even for families that remain intact without any deaths during outbreaks, school-based food programs may ease the burden of feeding. this challenge occurs because many families are generally in great need of food during epidemics, as many farmers fall sick and agricultural productivity and output may suffer as a result. indeed, the world food program (wfp) found that in some places, when families were incentivized with food rations for sending their daughters to school, enrollment of girls in school tripled [29] . health-centric and school-based curriculum: the instruction of health habits, hygiene, and sanitary conditions and local cultural contexts and their impact on health behaviors should not just be a footnote in educational programs. instead, they should receive the same attention as math and reading. right from an early age, schools should provide students with age-appropriate health education that will arm them with the knowledge, skills, and tools to take care of their health and be aware of risky health behaviors. in south africa, such health-centric and life skills-based curricula have helped to improve young people's odds of using condoms during sex [30] . synthesis of established evidence also shows that school-based sexual health education has the potential to improve condom use among young people in sub-saharan africa and to reduce the prevalence of sexually transmitted infections such as chlamydia [31] . such comprehensive approaches to education can be achieved by ensuring that for instance, eliminating tuition fees has been found to reduce child marriages in eight countries in sub-saharan africa, including ghana, ethiopia, and rwanda, despite the challenges encountered in the implementation of this policy [23] . additionally, the government of ghana's free compulsory universal basic education (fcube), which aimed to provide tuition-free education for all students in public primary schools, has led to a marked increase in enrollment rates [24] . in countries such as kenya, uganda, and tanzania, eliminating tuition fees has helped to improve school enrollment and student attendance rates, particularly for young girls [3] . in uganda, the government's efforts at easing the burden of tuition led to a 30% increase in girls' enrollment in school with an almost double effect for the poorest economic fifth of girls [3] . in a systematic review of 35 studies from 75 reports, baird et al. also substantiated such findings of the impact of financial incentives on educational outcomes [25] . they found that both conditional and unconditional cash transfer programs improved the likelihood of school attendance and enrollment compared to programs without cash transfers [25] . with that said, it is essential to note that zero tuition costs come at a cost to a nation. in response, several governments have devised ways of domestically financing such interventions via taxation and innovative funding models [26] . public-private partnerships models for funding primary and secondary education should be encouraged. zero non-tuition expenses through community-led revenue generation: as koski et al. have shown, removing the cost of tuition alone would not be enough to reduce child marriage since other barriers to school enrollment may persist [23] . in ghana, despite the fcube, there are still gender disparities in educational access. many young girls are not benefitting from the scheme, and these probably may be due to financial difficulties related to the cost of textbooks, school uniforms, and transportation, which disproportionately plague marginalized households in rural communities [24] . stack et al. found in a study in rural western ghana that in the face of financial challenges, 44.5% of heads of households, typically males, opted for the male child against 27.7% who chose the female child. of these, 26.5% stated that it would depend on the specific circumstances, while 1.3% refused to answer [27] . thus, the contribution of non-tuition expenses towards school attendance is not trivial. community and private initiatives could take this burden off parents and heads of households. a thriving community initiative like this is the cocoa360's "farm-for-impact" model. community members assist with work on a community-run cocoa farm in exchange for tuition-free education and subsidized healthcare. the revenues from the farm are applied to finance educational and healthcare services in rural communities in ghana [21] . evidence from meta-anal-they are theory-driven, address social determinants like social norms, improve cognitive-behavioral skills, train facilitators like teachers, and include schools, families, and communities [32] . safe and protected learning spaces: while schools serve as nurturing grounds for student learning, they can also expose young girls to gender-based violence (gbv) and negatively impact the attainment of educational objectives [33] . several studies have reported a high prevalence of gbv, such as sexual harassment, unsolicited advances, touching, groping, and sexual assaults [34, 35] . for instance, a human rights watch (hrw) study in south african schools revealed that teachers raped young girls in empty classrooms, lavatories, and dormitories [35] . in dodowa, ghana, similar findings of sexual assault of young girls in schools have also been documented [34] . such instances of school-based gbv make schools less attractive places for young girls, who consequently struggle to concentrate on their academics and may, therefore, drop out [36] . for other deterred girls, they may not enroll at all. schools pursuing sustainable educational models should strive to make their learning environments safe for young girls by working with community leaders, teachers, students, and related government stakeholders to implement preventive and punitive measures for perpetrators. evidence from systematic reviews highlights the role of interventions such as life skills training programs and positive peer relationship training approaches to promote self-esteem and to reduce bullying and rape [37] . activities such as drama and arts can also help young people evaluate their gender roles and the steps they can take to ensure that their study spaces are safe and conducive to learning [1]. successfully achieving this goal would require seeing boys as crucial partners to the solution, rather than external opponents. education has proven to be an effective intervention for improving health outcomes and reducing the spread and impact of epidemics. while the global community has made significant progress in leveraging quality girlchild education as a tool to enhance health in lmics, challenges such as financial bottlenecks continue to impede such efforts, especially during outbreaks. sustainable educational models that prioritize active community engagement, abolish tuition and non-tuition expenses, incorporate school-based food programs, infuse health-centric and skill-based curriculum, and promote safe learning spaces are much needed. successful implementation of such efforts would significantly improve educational access and health outcomes for young girls and, consequently, provide long-lasting approaches to fight the spread and impact of epidemics when they emerge. social determinants of health inequalities. the lancet unaids. educate girls, fight aids social factors influencing child health in ghana what was the effect of the west african ebola outbreak on health programme performance, and did programmes recover? public health action sexual initiation among adolescent girls and boys: trends and differentials in sub-saharan africa [internet]. archives of sexual behavior hiv infection and reproductive health in teenage women orphaned and made vulnerable by aids in zimbabwe school-based programs to reduce sexual risk behaviors: a review of effectiveness barriers to school attendance and gender inequality: empirical evidence from a sample of ghanaian schoolchildren. research in comparative and international education causal effects of education on sexual and reproductive health in low and middle-income countries: a systematic review and meta-analysis. ssm popul health evidence for causal links between education and maternal and child health: systematic review ebola crisis: the unequal impact on women and children's health. the lancet global health perception of students on the impact of ebola virus disease conditional cash transfers for education: a comparative analysis between the funder and country. international development, community, and environment (idce) the effects of an ngo development project on the rural community of tarkwa bremen in western ghana impacts of conditional cash transfer programs on educational outcomes in developing countries a meta-analysis. rand labor and population bringing hope to a generation, food aid to help orphans and other vulnerable children. rome: world food programme programming for hiv prevention in south african schools school-based sexual health education interventions to prevent sti/hiv in sub-saharan africa: a systematic review and meta-analysis effective elements of school health promotion across behavioral domains: a systematic review of reviews literature review on the intersection of safe learning environments and educational achievement. u.s. agency for international development scared at school: sexual violence against girls in south african schools a global review of current issues and approaches in policy, programming, and implementation responses to school-related gender-based violence (srgbv) for the education sector school-based interventions to promote adolescent health: a systematic review in low-and middle-income countries of who western pacific region /ijabr_v6(1)16-18.pdf 14. the impact of ebola on education in sierra leone teenage pregnancies in ebola-affected sierra leone [internet]. world vision new ebola outbreak hits women and girls hardest in the democratic republic of the congo education for all -the quality imperative united states agency for international development bureau for africa, office of sustainable development african states' varying progress toward gender equality in education brookings education and covid-19. malala fund re-imagining community development: the cocoa360 model interventions to enhance girls' education and gender equality. education rigorous literature review. department for international development the impact of eliminating primary school tuition fees on child marriage in sub-saharan africa: a quasi-experimental evaluation of policy changes in 8 countries taking education for all goals in sub-saharan africa to the task conditional, unconditional and everything in between a systematic review of the effects of cash transfer programs on schooling outcomes we are grateful to priya bhirgoo for her assistance with helpful comments and edits. key: cord-259233-smmhhroe authors: de armas‐rillo, laura; valera, maría‐soledad; marrero‐hernández, sara; valenzuela‐fernández, agustín title: membrane dynamics associated with viral infection date: 2016-01-28 journal: rev med virol doi: 10.1002/rmv.1872 sha: doc_id: 259233 cord_uid: smmhhroe viral replication and spreading are fundamental events in the viral life cycle, accounting for the assembly and egression of nascent virions, events that are directly associated with viral pathogenesis in target hosts. these processes occur in cellular compartments that are modified by specialized viral proteins, causing a rearrangement of different cell membranes in infected cells and affecting the er, mitochondria, golgi apparatus, vesicles and endosomes, as well as processes such as autophagic membrane flux. in fact, the activation or inhibition of membrane trafficking and other related activities are fundamental to ensure the adequate replication and spreading of certain viruses. in this review, data will be presented that support the key role of membrane dynamics in the viral cycle, especially in terms of the assembly, egression and infection processes. by defining how viruses orchestrate these events it will be possible to understand how they successfully complete their route of infection, establishing viral pathogenesis and provoking disease. © 2015 the authors reviews in medical virology published by john wiley & sons, ltd. viruses are small structures that lack the metabolic pathways and structures necessary to ensure their own survival, relying on their host's machinery to replicate their genome and spread their progeny. accordingly, viruses have developed strategies to enter cells and exploit their structures to replicate. these strategies also serve to evade immune responses, such as those involving toll-like receptors and autophagic-mediated antigen presentation [1] [2] [3] [4] . similarly, viruses use the target cell's main trafficking pathways to ensure their propagation, exploiting the endosome or vesicular compartments by recruiting the clathrin, coatomer protein complex (copi) i and ii (figure 1 ), the endosomal-sorting complex required for transport (escrt) and their accessory proteins (reviewed by [1, 2, 5] : figure 2 ), as well as small guanosine triphosphatases (gtpases) [2] . this is evident during neutrophil-mediated phagocytosis, where microorganisms can be cleared by granule and vesicle secretion [6] . therefore, determining how viruses use and rearrange intracellular organelles during their biological cycle is an important goal that will aid the development of new antiviral strategies, and our understanding figure 1 . viral factories and virus-triggered autophagic membrane flux for replication and egression. some viruses achieve replication by exploiting the cell's membrane transport pathways, thereby generating membrane organelles named viral factories (vfs). these vfs are organised by different viral proteins, and they represent specialized compartments for viral-gene replication, morphogenesis, export, maturation and release. moreover, these compartments also serve to override or evade the immune responses directed against viral genomes. viral proteins can enter secretory pathways by co-translational translocation into the er in order for them to be further transported to the golgi complex, either in vesicles or in a coatomer protein complex (cop) ii-dependent manner. viral complexes formed inside the vfs communicate with vesicles, mitochondria, golgi cisternae and er membranes. this interaction allows viral complexes to be transported through the golgi network to the plasma membrane and it promotes their final release as viral particles. alternatively, some viruses take advantage of the host's autophagic machinery for their own replication and pathogenesis. viruses first initiate the formation of vesicles that bear key autophagic proteins, such as beclin-1 and lc3, capturing portions of membranes from the er and other cytoplasmic elements. this assembly evolves toward an immature double-membrane vesicle (dmv) that serves as an aggresome compartment to recruit viruses or newly formed viral replication complexes. several rna viruses induce the formation of these autophagosome-like vesicles (also referred to as dmvs) to enhance viral replication and non-lytic egression, such as poliovirus and cvb3, hiv-1 and hcv. how these viruses trigger the accumulation of autophagosome-like vesicles and dmvs remains unclear. some theories involve blocking the fusion of nascent autophagosomes with late endosomes and lysosomes, as in the case of hiv-1 nef, which appears to cause autophagosome accumulation by inhibiting their progression towards more mature stages. indeed, autophagosome-like vesicles may represent a trafficking pathway for these viruses, connecting to multivesicular bodies (mvbs), and assuring virus assembly and budding at the cell surface while protecting them from intrinsic antiviral factors and immune responses. the morphogenesis and release of mature and infectious hbv particles also require tsg101 and depend on the escrt-mvb system. under standard conditions the lumen of autophagosomes acidifies after fusion with endosomes that carry vacuolar (h + )-atpase (v-atpase) to form amphisomes. the autophagic membrane flux progresses by fusing with lysosomes in order to form the autolysosome that contains the former's proteinases. poliovirus inhibition of autophagosome formation attenuates viral replication while inhibiting autolysosome formation, and thus, catalytic activity does not affect the virus. however, degradation of cellular triglycerides by autophagy benefits denv replication and autolysosome degradation dampens ifn activation following hcv infection of these pathologies. indeed, there is growing evidence that cell's modify their membranes to defend themselves against pathogens and infection, altering their spatial reorganization and vesicle trafficking. in this review, we focus on the importance of the membrane flux triggered by viruses to achieve replication and egression, and to ensure their propagation. several of the cell's organelles and membrane structures are involved in viral replication and in fact, many viruses use specific cellular compartments to replicate, referred to as viral factories (vfs: figure 1 ). these vfs provide a physical scaffold that brings together elements required for genome replication and morphogenesis [1, 7] . vfs are usually formed by rearranging the host's cell membranes, reorganizing the cytoskeleton and recruiting specific organelles, like mitochondria (reviewed in [8] ). these viral driven events involve the association of replication complexes (rcs) with er derived membranes to form a vf. hence, intracellular membrane dynamics appear to be crucial for viral replication and survival. a well-known example of a vf is that used by vaccinia virus, an enveloped pathogen of the poxvirus family that replicates in the cytoplasm by assembling small rough er-derived cisternae into a microenvironment that resembles a cytoplasmic mini-nucleus for viral replication [9] . similarly, the rcs of togaviruses associate with endocytic membranes, while nodavirus rcs associate with mitochondrial membranes (reviewed in [1] ). thus, specific membrane compartments can be used as vfs by rna viruses to concentrate viral replicases and key cofactors, and ensure efficient viral genome replication [10] . in this context, both rubella virus (rubv), a relevant human teratogenic togaviridae virus [11] , and semliki forest virus (sfv), a member , some viruses attach structural polyproteins to pip 2 -rich membrane regions of the infected cell for further budding and release into the intercellular space. pip 2 confers fluidity to the cell membrane and favours virus-cell fusion. these virions then bind to specific receptors in order to infect the neighbouring target cell at the vs, fusing with its plasma membrane directly or after surfing on actin-structured filopodia, or being internalized by endocytosis as is believed to occur with hiv-1. the vs represents an efficient environment for viral budding. it typically arises in pip 2 -enriched plasma membrane domains, where the membrane of the infected cell is polarized towards the synaptic junction through the movement of vesicles governed by the escrt/alix-tsg101 machinery or by mvbs coordinating the translocation of the mtoc. this scaffolding facilitates subsequent viral infection and spread from the infected to the nearby uninfected cell. in addition, long membrane nanotubes may also form between neighbouring cells, promoting viral protein trafficking. other dynamic membrane events involved in viral infection and spreading are trogocytosis, arf6/pip 2 -mediated membrane dynamics and exosomal transport. trogocytosis involves the exchange of cell surface membrane patches that may contain receptor clusters associated to viral particles, while exosomes are vesicles formed from mvbs that could participate in viral infection and spreading between cells of the alphavirus group of this family [12] , couple their rna synthesis to endosome and lysosome membranes modified by the association of virus specific components. the subsequent fusion of these late endosomes and lysosomes generates cytopathic vacuoles (cpvs) [13, 14] that are lined with small vesicular invaginations or spherules (viral rna replication sites) [13, 14] . cpvs establish complex and reversible contact with endocytotic vesicles through internal membranes interconnected with transport endosomes [8] . for example rubv forms vfs around cpvs via the recruitment of membrane structures from the er cisternae, golgi stacks and mitochondria [8] (figure 1 ). the golgi apparatus is a highly dynamic organelle with a sustained, functional flux of membrane proteins [15] , and it can serve as a morphogenic mould for rubiviruses, coronaviruses, arteriviruses and bunyaviruses [1, 8, 16, 17] . these rubv factories connect viral replication with the assembly and maturation of nascent virions at golgi membranes, contributing to the virus escaping from the host cell's defences [16] . some viruses induce the formation of doublemembrane vesicles (dmvs) and/or autophagosomes for replication [1] [2] [3] [4] 18, 19] , such as the positive rna viruses of the flaviviridae family and nidovirales order [20, 21] (figure 1 ). the rna polymerase of the human poliovirus, a picornaviridae family member responsible for poliomyelitis, can also assemble dmvs [22] . infection by this virus triggers the modification of different intracellular membrane structures and organelles (but not mitochondria), converting them into virus replication vesicles. in fact, poliovirusassociated dmvs resemble autophagosomes, as also described for another picornaviridae family member, coxsackievirus b3 (cvb3 [23, 24] : figure 1 ). autophagosomes are dmvs generated by membrane trafficking and they are related to the catabolic process of autophagy, which involves the degradation of cytoplasmic components within lysosomes [25, 26] . autophagy maintains the organism's homeostasis by sequestering undesired intracellular elements for lysosomal degradation and recycling [25, 26] . viruses often use autophagy to complete their lifecycle and evade immune responses, even though it is based on catalytic pathways [3, 23, 24] . poliovirus, like other positive rna viruses, has evolved the capacity to convert autophagy into a key cellular motor for replication [3, 10, 23] . during autophagy, a cytosolic form of the microtubule-associated protein 1a/1b light chain 3 (lc3-i) conjugates with phosphatidylethanolamine to form lc3-ii and associate with autophagosomal membranes, ultimately producing the degradation of lc3-ii during the late steps of autophagy [27] . conversely, the p62 protein (or sequestosome-1, sqstm1) interacts with ubiquitinated proteins, lc3 and other proteins to ensure the correct degradation of undesired material. lc3-ii augments during active autophagy when p62 is degraded [28, 29] . in this context, poliovirus or cvb3 infection triggers the generation of autophagosomes with a higher lc3-ii/lc3-i ratio and with lc3 foci, structures that support the rna rc without promoting lysosome degradation (evident through p62 stabilization [23, 24] : figure 1 ). however, it is unclear whether these viruses block autophagosome maturation into amphisomes, avoiding autophagosome fusion with endosomes [30] . such events override the appearance of degradative autolysosomes [31] or they may provoke the formation of autophagosome-like structures disconnected from catalytic pathways. it is also thought that these autophagosomes may ultimately serve as a membrane scaffold to permit the egression of nascent virions from infected cells, preventing cell lysis [30] (figure 1 ). all hcv viral genotypes (1a, 1b and 2a), positive rna flaviviruses that are a major cause of chronic liver disease [32], induce autophagosome accumulation [33, 34] . this involves regulation of the unfolded protein response (upr), which relieves er stress and prevents the formation of catalytic autolysosomes by suppressing their fusion with lysosomes [33,34] ( figure 1 ). apparently, the success of viral replication relies on the recruitment of membrane-trafficking proteins to er-derived membrane scaffolds [35] [36] [37] [38] [39] [40] [41] . hence, domain 1 of the non-structural 5a (ns5a) protein and the helicase domain of ns3 are sufficient to achieve efficient dmv formation, which also depends on tightly regulated cis cleavage of the hcv-polyprotein precursor [35] and requires cyclophilin a isomerase activity [36] . ns5a associates with ns5b, a rnadependent rna polymerase, a complex that interacts with vamp (vesicle-associated membrane protein)-associated proteins (vaps) [37,38] and recruits ras-like small gtpases (e.g. rab1, rab5 and rab7), enlarging the viral replication compartment by docking membrane vesicles [39] [40] [41] . this process also regulates autophagy [42], given that hcv-induced autophagosomes support viral replication and the delivery of incoming viral rna to the translation apparatus, and/or the recruitment of cellular factors for translation. however, some controversy still surrounds this issue, autophagosomes can mature into acidic amphisomes in hcv-infected cells [43, 44] , and subsequently fuse with late endosomes or lysosomes [44] (figure 1 ). autophagic membrane flux appears to be necessary to translate the hcv genome, yet it appears to be dispensable once viral infection has begun [45] . moreover, no changes in either p62 or the degradation of long-lived proteins are observed [33], despite the enhanced autolysosome formation in cells expressing hcv replicons [46] . while specific silencing of autophagy genes does not affect viral translation and rna replication, it does apparently alter hcv morphogenesis [47] . however, the silencing of factors critical for autophagosomes formation, like lc3 or atg7, appears to suppress hcv rna replication [48], while hcv replication is apparently potentiated when the upr promotes autophagy [49] . conversely, hcv infection seems to promote autophagy without concomitant stimulation of the upr and autophagy does not appear to be required as a platform for hcv rna replication [50]. thus, doubts remain about the role of autophagy and the upr in hcv replication, although the distinct interactions between autophagy and hcv replication suggest that such membrane flux promotes viral replication. the dengue virus (denv) is a mosquito-borne single positive-stranded rna virus of the flaviviridae family that causes dengue fever [51] . there are five antigenically related but distinct denv virus genotypes (denv-1 to denv-5) [51,52]. like hcv, there is evidence that autophagy may be implicated in denv replication. following cell entry and nucleocapsid uncoating, denv rna is translated into a single polyprotein that passes into the er lumen where the different viral proteins are processed. in fact, denv-2 proteins involved in translation and replication are found in or in close proximity to autophagosomes during viral infection [53, 54] . accordingly, inhibition of autophagosome formation dampens the production of infectious denv-2 particles [53], while stabilizing autophagosomes and/or amphisomes by impeding their fusion with lysosomes enhances viral egression [55] . indeed, denv-3 seems to promote autophagy during early infection [54] , while inhibition of autophagosome formation also dampens the production of infectious denv-3 [54]. hence, denv-2 and -3 appear to interact with the autophagy machinery in a different manner, and while it is conceivable that amphisomes or autophagosomes represent the site of denv-2 translation/replication [54,55], autophagolysosomes could be the crucial site for denv-3 viral replication [54] . the distribution of ns1 or denv-2 and denv-3 double-stranded rna (dsrna) in the different autophagy-associated membrane structures confirms these observations ( figure 1 ). moreover, nascent viral particles are formed and mature in these structures, then travelling through the trans-golgi network (tgn) to egress [56, 57] . remarkably, the precursor membrane (prm) protein of the denv-1-4 genotypes behaves similarly and it is cleaved by the tgn-protease furin in the secretory pathway [58], assuring viral assembly and the infectivity of nascent viral particles [59] . hiv is a single-stranded rna virus (lentivirus genus of the retroviridae family) that causes aids. hiv type 1 (hiv-1) alters the autophagic membrane flux of the host cell's organelles, thereby modulating the intracellular milieu in favour of viral replication and propagation [3] (figure 1 ). when cd4+ t cells, monocytes and dendritic cells (dcs) are infected with hiv-1, autophagic vacuole formation is blocked and the expression of autophagy proteins down-regulated (e.g. lc3 and beclin1 [19, 60] : figure 1 ). remarkably, the hiv-1 protein nef (negative factor) blocks the autophagic flux of membranes, especially during the autolysosome stage of autophagy, resulting in an accumulation of autophagosomes and lc3 in macrophages ( figure 1 ). thus, nef prevents autophagic degradation of hiv-1 biosynthetic intermediates of virions by targeting the lipid class iii phosphatidylinositol 3-kinase (c3-pi3k) complex and by associating with beclin1 (atg6-autophagy-related protein 6 -in yeast). significantly, beclin1 is actually part of the c3-pi3k complex, together with the vacuolar protein sorting-associated proteins 34 (vps34) and 15 (p150). nef therefore alters the sub-cellular distribution of vps34, potentially ensuring the survival of the viral progeny [3, 61] . indeed, nef is thought to promote the appropriate hiv-1 gag membrane localization and processing, thereby facilitating viral cell-to-cell transfer [62] . although the catalytic activity of autophagy appears to be impeded by hiv-1, autophagosome formation or accumulation is still promoted. hence, the hiv-1 gag protein promotes early stages of autophagosome formation by directly interacting with lc3 in macrophages, enhancing hiv-1 yields and gag processing, a critical step in virion assembly and release [61] (figure 1) . notably, newly identified components of the ubiquitin-like conjugation system all seem to be involved in hiv-1 replication (e.g. atg7, atg8-lc3 is its best characterized mammalian homologue-atg12 and atg16l2-responsible for vesicle elongation) [63] . however, it remains unclear how these factors actually affect hiv-1 replication, which occurs in the nucleus of infected cells. moreover, while autophagic vacuoles would appear to be fundamental for hiv-1 morphogenesis and egression, how hiv-1 overrides or uses autophagy to persist remains poorly understood. hence, the infectious capacity of nascent hiv-1 virions depends on the uptake of the viral infectivity factor (vif) during viral budding, a process influenced by histone deacetylase 6 (hdac6), which promotes autophagic clearance of vif [64] . other positive rna viruses exploit the formation of er-derived membrane scaffolds and membrane autophagic flux to replicate (e.g. the norwalk virus), because the membranebound nsp48 protein also binds to vap-a [65] . rna replication may occur in endosomes, lysosomes (togaviruses), peroxisomes and chloroplasts (tombusvirus), or mitochondria (nodaviruses), shielded from immune responses. all positive rna viruses transform cytoplasmic membranes into specialized viral replication sites [10] . the antiviral effect of brefeldin a (bfa), an inhibitor of anterograde er-golgi network membrane dynamics, suggests that membrane trafficking must be active for enterovirus replication, as reported for picornaviruses, poliovirus and coxsackievirus [66, 67] . bfa prevents the membrane flux required to form replication compartments, blocking virus secretion from infected cells [68] by inhibiting adp (adenosine diphosphate)ribosylation factor (arf)-gtp exchange proteins (arf-gefs). this blockade negatively affects copi coat generation at the golgi by diminishing and sequestering arf1-gtp [69] . for several picornaviruses, copii-coated vesicles may provide membranes suitable for replication [70] , although autophagosomes may also contribute at this point [23] (figure 1 ). reovirus and sfv also promote coated-pit formation [71] . moreover, the small gtpase rab7 is soon recruited for sfv internalization when associated to intermediate endosomes [72] , which in turn induces the formation of cpvs that is an important event for viral rna synthesis in target cells [13] . an important biological process common to the recently proposed megavirales order is viral replication within cytoplasmic vfs [73] . giant viruses (also called nucleocytoplasmic large dna viruses-cldvs) belonging to this order are double-stranded dna (dsdna) viruses with a genome and particle size comparable to those of small bacteria [74] . african swine fever virus (asfv; from the asfarviridae family), poxviruses and iridoviruses are the three families of ncldvs that terminate or undergo their entire replication cycle in the cytoplasm [75] [76] [77] . this feature is not observed in herpes viruses or baculoviruses, other large dna viruses of eukaryotes that undergo nuclear dna replication and transcription [78] . giant viruses provoke vf formation in the cytoplasm of infected cells to permit genome replication and morphogenesis [73, 79] . asfv factories are similar to the aggresomes formed at the mtoc (microtubule organizing centre) [80] , and they provoke a rearrangement of the intermediate vimentin cytoskeleton at the mtoc into a star shaped structure that resembles the microtubule aster formed during mitosis, a structure required for late gene expression [81] . together with an asfv chaperone, the hsp70 cell chaperone is recruited to asfv factories, along with mitochondria, facilitating the folding of viral structural proteins like the major capsid protein p72 [82, 83] . nascent asfv virions are formed from vf membranes through the assembly and recruitment of viral proteins in vfs. thus, the viral membranes in vfs may be connected to cellular organelles, particularly given that resident er markers are detected with the viral p17, p54 and pb318l proteins in new viral particles [84] [85] [86] [87] . asfvs are thought to reorganize cell membranes through viral proteins that contain a kde motif, inducing the redistribution of erassociated proteins [88] and the viral p54 protein. the latter is required for the correct vf localization of the membranes and the collapse of the er-derived cisternae [89] . asfv infection is achieved by redistributing membranes from the secretory pathway and tgn [90] . therefore, these common biological features of giant virus replication and virion architecture could reflect a common origin, and the sharing of a large set of ancestral genes [74, 91] . membrane dynamics during viral assembly and budding and infectiousness. during budding, successful infection is achieved by adjustment and distortion of the target cell's plasma membrane [4] . the structural gag polyprotein is common to several retroviruses, like hiv-1 and the murine leukaemia virus (mlv), representing the minimal plasma membrane component required for viral assembly [92] . hiv-1 gag localizes to phosphatidylinositol-4,5-bisphosphate (pip 2 ) rich plasma membrane regions, where pip 2 plays a critical role in hiv-1 virion assembly [93] (figures 1 and 2) . in fact, the matrix viral protein (ma) within the unprocessed hiv-1 gag polypeptide drives gag towards these pip 2 membrane domains [92, 94] in a myristoylation-dependent manner [95] , raft domains where hiv-1 buds [95] [96] [97] . phosphate hydrolysis by polyphosphoinositide 5phosphatase iv (5ptaseiv) diminishes the plasma membrane pip 2 [98] , causing the gag polypeptide to translocate from hiv-1 budding sites at the membrane to cd63 rich compartments, thereby inhibiting viral release [93] . similarly, arf6/q67l expression, a gtp-bound mutant of arf6, alters the trafficking of arf6/pip 2 -associated vesicles, provoking their accumulation in the cytoplasm to where gag is redirected. these complexes lie far from the budding sites at the membrane, thereby dampening virus release [93] . although the assembly of hiv-1 at the cell surface is only partially understood, several key steps in the membrane trafficking of viral proteins have been defined, shedding light on both the viral assembly and budding processes [92, 99] . enveloped viruses like hiv-1, vesicular stomatitis virus (vsv), ebola virus (ebov) and hepatitis b virus (hbv), and other rna and dna viruses, mainly emerge from cells by co-opting the host's escrt machinery [100, 101] , which plays a vital role in cellular abscission and in multivesicular body (mvb) biogenesis (a process by which ubiquitinated misfolded or damaged proteins enter endosomes to be destroyed). in addition, mvbs are important intermediates in endolysosomal transport [102] (figures 1 and 2) . gag activity drives escrt-iii complex formation at the budding site of hiv-1, which binds to and recruits the escrt-i complex and the alg-2 (apoptosis-linked gene 2)interacting protein x (alix). this escrt-iii complex promotes the excision of nascent virions at the cell surface, an event potentially equivalent to the cleavage of intraluminal vesicles from mvbs [103, 104] (figures 1 and 2) . moreover, the tumour susceptibility gene 101 (tsg101) is a subunit of the escrt-i complex that drives viral rna transport and envelope fusion to late endosomes, processes required for infection and rna release [105] ( figure 2) . however, the interaction of viral gag protein with the escrt machinery appears not to be absolutely required for hiv-1 viral budding [106] [107] [108] . nevertheless, interferon-stimulated gene 15 protein (isg-15) inhibits hiv-1 egression by interfering with escrt-iii protein membrane flux during budding [109, 110] . remarkably, morphogenesis and the release of hbv particles also require tsg101 [111] , although this dna virus lacks a viral protein bearing the late (l) domain necessary to interact with the escrtmachinery [101] . however, α-taxilin interacts directly with tsg101 and with the large hbv surface protein (lhbs), thereby recruiting the viral capsids to escrt complexes, thus permitting correct viral formation and egression [111] . therefore, hbv maturation and egression depends on the escrt-mvb system. notably, hbv infected cells also produce large amounts of non-infectious spherical or filamentous envelope particles (svps). these svps are a mixture of lipids and viral surface proteins that accumulate in an er-golgi intermediate compartment (ergic), budding into the lumen and provoking release through the general secretory pathway [112] . many other enveloped rna viruses bud in an escrt-dependent manner [5, 100, 113] , as do most negative-strand non-segmented single-stranded rna (ssrna) viruses, such as rhabdoviruses, filoviruses and most paramyxoviruses, all of which recruit escrts for viral egression [114, 115] . even the budding of negative-strand segmented-ssrna arena viruses involves an escrt-dependent pathway [116, 117] . however, no evidence for the participation of escrts has yet been reported in nipah, measles, hrsv or bornaviridae budding ( [5] ). indeed, the enveloped influenza virus buds in an escrt-independent manner as its matrix protein is devoid of an escrt-binding domain [118, 119] . other viruses are also released from the host's plasma membrane through their mas, such as the newcastle disease virus or vsv. in these cases, bud formation and excision from the membrane are matrix-dependent processes [120, 121] , as for influenza virus. however, much work is still required to determine how membrane dynamics affect the trafficking and assembly of these viruses, particularly in terms of the cellular factors that control the [92, 122] . given all of these findings, membrane dynamics has a crucial influence on the assembly and budding of numerous viruses, and it may represent an important and complex target to limit the viral life cycle. viruses use various cell communication pathways to achieve effective cell-to-cell dissemination [123] . first described for type 1 htlv (htlv-1) [124] , the virological synapse (vs) is a complex structure found at the interface of infected:uninfected cells. viral receptors and the egression machinery accumulate at the vs [125] , making the infection and spread of htlv-1 through t lymphocytes cell-cell dependent. direct cell-to-cell transmission facilitated by the formation of stable cellular junctions has several advantages, including faster replication rates [126] , successful transmigration of infected cells across mucosal barriers [123] and viral protection from host responses. however, such transmission is still to be confirmed for hiv [127, 128] . cell-to-cell spreading of hiv-1 ( figure 2 ) is considered to involve microtubule-mediated polarization and substantial budding, followed by the entry of free viral particles into target cells [129] . thus, it involves pathways that regulate cell-free virus entry by modifying membrane dynamics. in this regard, most hiv-1-infected galt (gut-associated lymphoid tissue) cells in intestinal crypts are infected by concentrated pools of free hiv-1 viral particles in hiv-1-infected humanized mice. fewer infected cells are found in the mucosal regions and the lamina propria, where vs presumably occur [130] , explaining why infection of permissive cells by free viral particles is crucial for hiv-1 replication and pathogenesis in vivo. this is consistent with the recent identification of the key cell signals required for efficient early hiv-1 infection and the establishment of latency in cd4+ t cells [131] [132] [133] [134] [135] [136] . interference with retroviral cell-tocell transmission is not only produced by blocking cytoskeletal motility [137] and depleting membranecholesterol [138] but also, by interfering with arf6governed plasma membrane dynamics. moreover, restricting plasma membrane fluidity caused by altering early hiv-1-triggered phosphatidylinositol-4-phosphate 5-kinase (pi4p5-k) iα activation and the ensuing detrimental effects pip 2 production on hiv-1 transmission [4, 133, 135] . in fact, arf6-coordinated membrane trafficking is required for efficient hiv-1 fusion, entry and infection of cd4+ t lymphocytes [135] (figures 1 and 2) . the flux and turnover of pip 2 -enriched vesicles from the plasma membrane, driven and coordinated by the arf6-gtp/gdp cycle, ensures cell surface membrane regeneration and it allows membrane exchange between the viral and target cell surfaces. this type of membrane trafficking, coupled with enhanced fluidity, is in strong synergy with the key hiv-1/receptor (cd4 and c-x-c or c-c chemokine receptor type 4 or 5-cxcr4 or ccr5), interactions that promote fusion pore formation in target cells. these interactions take place between the non-regenerative hiv-1 viral membrane and the dynamic cell surface membrane, favouring efficient virus-cell fusion, entry and infection, both for the free virus and in the context of the vs [4, 133, 135] (figures 1 and 2) . despite these similarities, some contact-specific events that affect membrane flux should also be considered. during cell-cell hiv transmission, intense viral endocytosis drives entry into neighbouring cells even if they are in contact [139] (figure 2 ). remarkably, biofilm-like structures at the surface of infected cells concentrate htlv-1 viruses for their efficient transmission to target cells [140] and cellular projection is used to transmit pseudo-rabies virus. retroviruses also travel along membrane protrusions that contact adjacent cells and mlv surfs on the filopodia of fibroblasts before entering cells [141] (figure 2 ). hiv-1 also takes advantages of filopodia for cell-tocell transmission [141] , similarly surfing on the narrower membranous nanotubes that connect cells separated no more than 100 μm [142] and facilitating the transfer of viral proteins to the inner side of the membrane. these actin structures extend from hivinfected cells to target cells irrespective of receptorenvelope interactions [142] (figure 2) . the take up of larger membrane invaginations at the vs of connected cells [129] is known as trogocytosis, an event that may also control the extent and stability of the synapse, regulating its duration [143] . hiv particles, like cd4 molecules and other membrane components, are transferred by trogocytosis from uninfected to infected cells in a manner triggered by the hiv-1 envelope (env)/ cd4 [128] (figure 2 ). this mechanism could be very significant and render cells permissive to hiv infection, as recently proposed [144] . the cell-cell contacts and signalling induced by the hiv-1 env complex that occur at the vs can activate autophagic membrane flux, leading to apoptotic cell death of uninfected cd4+ t cells [60, 145] . this lethal autophagy may provoke or enhance immunodeficiency, as observed in vivo where the majority of cd4+ t cells undergoing apoptosis, as well as the peripheral blood and lymph nodes of hiv patients, remain uninfected [146] . simultaneously, autophagy can be suppressed in infected cd4+ t cells [60] , thereby antagonizing env-mediated apoptosis and allowing viral replication to occur in infected cd4+ t cells. in this context, hiv-1 evades immune responses in an hiv-1 env-cd4dependent manner by efficiently impairing autophagy in dcs when early contacts are established [19] . taking into account the role autophagy plays in viral replication, hiv-1 can enhance or suppress autophagy at different stages of its viral cell cycle, favouring persistence and the evasion of immune responses, and therefore, its pathogenesis [19] ( figure 1 ). finally, like other viruses (e.g. cmv), hiv-1 stably associates with professional apcs during infection (such as dcs) to further infect lymphocytes during t-cell scanning or antigen presentation [147] . in fact, hiv-1 enters dcs by exploiting exosomal trafficking during antigen presentation [148] (figure 2 ). this review examines the intracellular trafficking of viruses that occurs in association with cellmembrane structures, some of which may be newly assembled by viruses to ensure their replication and budding. membranes derived from the er, mitochondria, lysosomes and endosomes are sculpted by viruses to generate vfs, acquiring their own functional morphology. these structures help ensure rna replication is accomplished without alerting the host's defence mechanisms. although the importance of membrane dynamics during viral infection has been established, several questions remain unanswered. it remains unclear how proteins from distinct viruses and host cells use the same intracellular membrane compartments or events (e.g. autophagy) to achieve viral replication, without affecting important cellular processes. conversely, it is not clear why viruses replicate in different subcellular membrane compartments, how they move across membranes and which host factors are involved in these events. similarly, we still do not know how these changes in membrane dynamics enable viruses to avoid immune responses. indeed, it remains unclear whether rearranging intracellular organelles enables viruses to escape the anti-replicative activity of natural restriction factors, such as apolipoprotein b mrna-editing enzyme-catalytic, polypeptide-like 3 (apobec3) proteins, tetherin (bst-2/cd317/ hm1.24) or samhd1 (sterile alpha motif (sam) and histidine-aspartate (hd) domain-containing protein 1) for hiv-1 [149] . resolving these issues will help decipher how viruses rearrange membranes during their infection cycle, thereby aiding the design of new antiviral strategies that target these dynamic viral-cell interactions and combat viral infection. these findings may also produce innovations in non-viral gene delivery systems to tackle tumours and immune diseases. new technical developments, such as more powerful microscopy systems [4, 8, 135, 150] , will allow dynamic viral trafficking and egression to be studied in cells with better spatial and temporal resolution. such information will further our understanding of the viral infection process and of how viruses succeed in deceiving the host's immune responses. respectively, spain), unll10-3e-783 (erdf) and fundación cajacanarias, and by the project rd12/0017/0034 integrated in the "plan nacional i + d + i" and co-funded by isciii-"subdirección general de evaluación" and erdf (ris-retic) grants. m-s.v and l.a-r are supported by rd12/0017/ 0034-(ris-retic) and saf2011-24671-fpi-associated grants and fellowships, respectively. we thank dr mark sefton (biomedred sl) for his linguistic revision of the manuscript. we apologize for all research studies and reviews that we have not discussed or cited in this review. we have tried to avoid any and all such omissions but space limitations have surely made this an impossible endeavour. modification of intracellular membrane structures for virus replication viruses and endosome membrane dynamics when autophagy meets viruses: a double-edged sword with functions in defense and offense viral infection: moving through complex and dynamic cell-membrane structures how to get out: ssrna enveloped viruses and membrane fission neutrophil granules and secretory vesicles in inflammation a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication. advances in ultrastructure of the replication sites of positive-strand rna viruses vaccinia virus dna replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei viral rna replication in association with cellular membranes molecular biology of rubella virus biogenesis of the semliki forest virus rna replication complex alphavirus rna replicase is located on the cytoplasmic surface of endosomes and lysosomes rubella virus replication complexes are virus-modified lysosomes microscopic morphology and the origins of the membrane maturation model of golgi apparatus function structural maturation of rubella virus in the golgi complex polymorphism and structural maturation of bunyamwera virus in golgi and post-golgi compartments herpesviruses remodel host membranes for virus egress human immunodeficiency virus-1 inhibition of immunoamphisomes in dendritic cells impairs early innate and adaptive immune responses orf1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex kunjin virus: an australian variant of west nile? oligomeric structures of poliovirus polymerase are important for function subversion of cellular autophagosomal machinery by rna viruses autophagosome supports coxsackievirus b3 replication in host cells maturation of autophagic vacuoles in mammalian cells autophagosome formation: core machinery and adaptations autophagy: process and function p62/ sqstm1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death p62/sqstm1 binds directly to atg8/lc3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. the journal of that which does not degrade you makes you stronger: infectivity of poliovirus depends on vesicle acidification differential role of autophagy in cd4 t cells and macrophages during x4 and r5 hiv-1 infection autophagy pathway intersects with hiv-1 biosynthesis and regulates viral yields in macrophages hiv-1 nef promotes the localization of gag to the cell membrane and facilitates viral cell-to-cell transfer identification of host proteins required for hiv infection through a functional genomic screen the hdac6/apobec3g complex regulates hiv-1 infectiveness by inducing vif autophagic degradation norwalk virus nonstructural protein p48 forms a complex with the snare regulator vap-a and prevents cell surface expression of vesicular stomatitis virus g protein inhibition of poliovirus rna synthesis by brefeldin a gbf1, a guanine nucleotide exchange factor for arf, is crucial for coxsackievirus b3 rna replication inhibition of cellular protein secretion by poliovirus proteins 2b and 3a involvement of cellular membrane traffic proteins in poliovirus replication cellular copii proteins are involved in production of the vesicles that form the poliovirus replication complex endocytosis by random initiation and stabilization of clathrin-coated pits rab7 associates with early endosomes to mediate sorting and transport of semliki forest virus to late endosomes vaccinia-like cytoplasmic replication of the giant mimivirus the origins of giant viruses, virophages and their relatives in host genomes the biochemistry of icosahedral cytoplasmic deoxyviruses role of the host cell nucleus in the replication of african swine fever virus dna frog virus 3 dna replication occurs in two stages viral transcription during autographa californica nuclear polyhedrosis virus infection: a novel rna polymerase induced in infected spodoptera frugiperda cells ultrastructural study of african swine fever virus replication in cultures of swine bone marrow cells aggresomes resemble sites specialized for virus assembly vimentin rearrangement during african swine fever vi-ii a virally encoded chaperone specialized for folding of the major capsid protein of african swine fever virus migration of mitochondria to viral assembly sites in african swine fever virus-infected cells african swine fever virus is wrapped by the endoplasmic reticulum african swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum membrane rupture generates single open membrane sheets during vaccinia virus assembly vaccinia virus lacking a17 induces complex membrane structures composed of open membrane sheets the subcellular distribution of multigene family 110 proteins of african swine fever virus is determined by differences in cterminal kdel endoplasmic reticulum retention motifs mechanism of collapse of endoplasmic reticulum cisternae during african swine fever virus infection african swine fever virus organelle rearrangements. virus megavirales", a proposed new order for eukaryotic nucleocytoplasmic large dna viruses virus assembly and plasma membrane domains: which came first? virus phosphatidylinositol (4,5) bisphosphate regulates hiv-1 gag targeting to the plasma membrane. proceedings of the national academy of sciences of the united states of gag localization and virus-like particle release mediated by the matrix domain of human t-lymphotropic virus type 1 gag are less dependent on phosphatidylinositol-(4,5)-bisphosphate than those mediated by the matrix domain of hiv-1 gag point mutations in the hiv-1 matrix protein turn off the myristyl switch evidence for budding of human immunodeficiency virus type 1 selectively from glycolipidenriched membrane lipid rafts the hiv lipidome: a raft with an unusual composition the isolation and characterization of a cdna encoding phospholipid-specific inositol polyphosphate 5-phosphatase m910119199 m910119199 hiv-1 assembly, budding, and maturation. cold spring harbor perspectives in late budding domains and host proteins in enveloped virus release mechanisms for enveloped virus budding: can some viruses do without an escrt? biogenesis and function of multivesicular bodies the escrt machinery: new functions in viral and cellular biology membrane budding and scission by the escrt machinery: it's all in the neck the escrt-i subunit tsg101 controls endosome-to-cytosol release of viral rna analysis of the assembly function of the human immunodeficiency virus type 1 gag protein nucleocapsid domain functional role of alix in hiv-1 replication human immunodeficiency virus type 1 nucleocapsid p1 confers escrt pathway dependence the interferon-induced gene isg15 blocks retrovirus release from cells late in the budding process budding of enveloped viruses: interferon-induced isg15-antivirus mechanisms targeting the release process identification of alpha-taxilin as an essential factor for the life cycle of hepatitis b virus morphogenesis of hepatitis b virus and its subviral envelope particles viral late domains filovirus assembly and budding rabies virus assembly and budding efficient budding of the tacaribe virus matrix protein z requires the nucleoprotein arenavirus reverse genetics: new approaches for the investigation of arenavirus biology and 158 l. de armas development of antiviral strategies influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles budding of filamentous and nonfilamentous influenza a virus occurs via a vps4 and vps28-independent pathway functional analysis of late-budding domain activity associated with the psap motif within the vesicular stomatitis virus m protein vesicle formation by self-assembly of membranebound matrix proteins into a fluidlike budding domain paramyxovirus glycoprotein incorporation, assembly and budding: a three way dance for infectious particle production avoiding the void: cell-to-cell spread of human viruses spread of htlv-i between lymphocytes by virus-induced polarization of the cytoskeleton retroviral spread by induction of virological synapses quantitation of human immunodeficiency virus type 1 infection kinetics cell-to-cell hiv-1 spread and its implications for immune evasion antigp41 antibodies fail to block early events of virological synapses but inhibit hiv spread between t cells virological synapsemediated spread of human immunodeficiency virus type 1 between t cells is sensitive to entry inhibition stingdependent type i ifn production inhibits cell-mediated immunity to listeria monocytogenes filamin-a regulates actindependent clustering of hiv receptors. nature pi4p5-kinase ialpha is required for efficient hiv-1 entry and infection of t cells moesin is required for hiv-1-induced cd4-cxcr4 interaction, f-actin redistribution, membrane fusion and viral infection in lymphocytes arf6-mediated membrane dynamics to efficiently enter and infect t lymphocytes. molecular gelsolin activity controls efficient early hiv-1 infection requirement for an intact t-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1 human immunodeficiency virus type 1 virological synapse formation in t cells requires lipid raft integrity on the steps of cellto-cell hiv transmission between cd4 t cells biofilm-like extracellular viral assemblies mediate htlv-1 cell-to-cell transmission at virological synapses retroviruses can establish filopodial bridges for efficient cell-to-cell transmission membrane nanotubes physically connect t cells over long distances presenting a novel route for hiv-1 transmission what is trogocytosis and what is its purpose? could cd4 capture by cd8+ t cells play a role in hiv spreading? autophagy is involved in t cell death after binding of hiv-1 envelope proteins to cxcr4 high expression of apo-1 (cd95) on t lymphocytes from human immunodeficiency virus-1-infected children hiv and mature dendritic cells: trojan exosomes riding the trojan horse? exosomes and retroviruses: the chicken or the egg? host factors and hiv-1 replication: clinical evidence and potential therapeutic approaches virus trafficking-learning from single-virus tracking this work and a.v.-f. are supported by the european regional development fund (erdf), saf2008-01729 and saf2011-24671 (micinn and mineco, the authors have no competing interests to declare. key: cord-018070-js9vvsud authors: hayes, anna marie title: human insecurity in the people’s republic of china: the vulnerability of chinese women to hiv/aids date: 2011-10-13 journal: human security doi: 10.1007/978-94-007-1799-2_2 sha: doc_id: 18070 cord_uid: js9vvsud hiv/aids has become one of the world’s leading causes of human insecurity for both men and women. in addition to physiological factors, women’s vulnerability to hiv transmission is primarily fuelled by gender inequality and gender-based discrimination and violence. therefore, women’s vulnerability to hiv transmission is closely linked to issues of empowerment and gender-based power relations. even with this realization however, women are still sometimes overlooked in many hiv/aids prevention and treatment campaigns, such as those in the people’s republic of china (prc), and responses to hiv/aids do not always actively seek to empower women. therefore, a deficiency in women’s human security increases their hiv/aids vulnerability. this chapter examines the intersection of gender inequality and hiv vulnerability as it applies to women in the prc. the unequal status of many women in china, and the privileged position accorded to chinese men, strongly indicates that chinese women face a heightened vulnerability to hiv transmission. while many of these vulnerabilities are similar to women elsewhere in the world and certainly are not unique to china, by overlooking the many social, cultural, economic and political factors that contribute to hiv/aids vulnerability and transmission of the virus, particularly those faced by women, china has a long way to go before chinese women are protected from hiv transmission. given that hiv/aids heightens human insecurity, the stage is set for chinese women (and men) to face an insecure future if the chinese government does not fully implement international best practice, meaning a gendered response, into its overall hiv/aids response. for the purposes of this chapter, the defi nition of human security is aligned closely with that of the united nations which states that human security is both 'freedom from fear' and 'freedom from want', incorporating components such as economic, food, health, environmental, personal, community and political security. this defi nition challenges the more restricted notions of human security and is centred on the principles that human security is a universal concern, its components are interdependent, that it is best achieved through prevention rather than intervention and that it is people-centred (undp 1995 : 232-34) . figures from the ministry of health (moh) show sexual transmission is now the main mode of hiv transmission in the prc, signalling that china has moved into the 'growth period' of its hiv/aids epidemic. at the close of 2009, it was reported that 59% of the total number of hiv/aids cases in china were caused by sexual transmission, with heterosexual transmission accounting for 44.3% of those infections and homosexual transmission accounting for 14.7% (ministry of health of the people's republic of china 2010 : 22) . of the total reported number of hiv/aids cases, 30.8% of plwha in the prc are women demonstrating the vulnerability of women to hiv transmission and increasing the concerns that the country is in a growth period (scawco and unaids 2007 : 4) . also concerning is that since 2005, sexual transmission has been the fastest growing mode of hiv transmission in china (hong et al. 2009 ) . further evidence of china's hiv epidemic moving into its growth period are the country's fi gures on mother-to-child-transmission (mtct). since 2005, it has estimated that mtct of hiv accounted for approximately 1.3-1.5% of the total number of new hiv cases (national center for aids/std prevention and control 2006 : 2; ministry of health of the people's republic of china 2010 : 23). according to the united nations joint program on hiv/aids (unaids) mtct rates in excess of 1% demonstrate that a hiv/aids epidemic meets the criteria for the categorization of a 'generalized epidemic' as mtct highlights rates of hiv among women in the general population who do not belong to any particular high-risk group (gill et al. 2007 ) . therefore, the above results are further evidence that china's overall hiv/aids epidemic has moved into its growth period . if effective prevention and control measures are not introduced prior to or during the growth period, and the country's hiv/ aids epidemic moves into the 'rampant prevalence period', large scale hiv transmission is inevitable. although rates of hiv among the general population remain low in the prc, with rates believed to be between 0.042% and 0.071% (ministry of health of the people's republic of china 2010 : 5), the spread of hiv through heterosexual intercourse among the general population is very much a warning that the numbers of plwha may soon explode across the country. if this occurs, it would make the epidemic very diffi cult to prevent and control and chinese women's vulnerability to hiv transmission will increase substantially, particularly because the prevalence rate of hiv among the general population would increase rapidly. physiologically, women are two to four times more vulnerable to hiv transmission than their male counterparts when engaging in unprotected vaginal intercourse (unaids 2002 : 57) . 2 in addition to physiological risks, women worldwide face a number of vulnerabilities to hiv/aids, deriving from a variety of social, cultural, economic and political factors. a society's gender roles have considerable infl uence on three main areas of hiv/aids vulnerability; accurate sexual and reproductive health knowledge, sexual passivity and aggression, and promiscuity. in addition, 'enabling environments' such as social, cultural, political and economic environments, can all fuel hiv vulnerability among women and these vulnerabilities are largely the result of gender inequality (feinstein and prentice 2000 ) . thus, in order for a state's hiv/aids response to be effective it must include gender-specifi c factors. therefore, women must be recognized as a vulnerable group. however, when asked whether she believed chinese women were particularly vulnerable to hiv transmission, interviewee d (2003, pers. comm., 27 august) , 3 who was the director of a government organization that played a key role in hiv/ aids prevention and treatment, responded that she believed 'women are less vulnerable [than men] to hiv/aids' and that women's vulnerability to hiv/aids largely depended on whether a woman was a sex worker, an intravenous drug user (idu), if she had donated her blood, had a blood transfusion or had used other blood products (interviewee d 2003, pers. comm., 27 august) . this point of view was supported by another interviewee, who was the national programme offi cer for an international non-governmental organization (ingo) responsible for hiv/aids prevention and treatment. in response to the same question, this interviewee stated that 'gender does not play any role [in its hiv/aids policies], and it is not part of mainstream discussions' (interviewee c 2003, pers. comm., 22 august) . these responses are alarming because they ignore the patterns of hiv transmission to women in much of the rest of the world, whereby women in the general population have been found to be extremely vulnerable to hiv transmission in areas with high rates of hiv/aids. 4 however, while interviewee c's organization did not incorporate a gendered response into its overall hiv prevention and treatment programs, her responses indicated that the intersection of gender and hiv vulnerability was being considered (2003, pers. comm., 22 august) . interviewee c believed that gender and economics were important issues in hiv/aids prevention and treatment, even though they were not yet widely recognized or were a part of china's offi cial hiv/aids response and policies. on the issue of what factors increased female vulnerability to hiv transmission, she said: women are vulnerable… for a number of reasons. these include the status of womenpolitical, economic and social status of women. also, the educational level of women is lower than their male counterparts, and unemployment rates are a great deal higher. this has a lot to do with remaining views on the role of women, which follow closely with the traditional stereotypes of women as wives, devoted to house and raising children. they are still seen in this caregiver role. women are also restricted in their access to information, so this also makes them vulnerable because they don't know what hiv/aids is or how to prevent it (interviewee c 2003, pers. comm., 22 august). responses from other interviewees also demonstrated that organizations were aware of the links between gender and hiv vulnerability. when asked what factors he believed increased chinese women's vulnerability to hiv/aids, interviewee e (2003, pers. comm., 27 august), a health specialist for an ingo that included hiv/ aids in its health framework, responded that he felt unemployment and low education levels were key factors because they led to economic insecurity that could lead women to prostitution. he stated that particularly in rural china, female school enrolments were much lower than those of males, and that this adversely affected women's employment opportunities. he also stated that women were further disadvantaged because they were not paid the same as men for equal work. also, because many women had been laid-off from their jobs in northeast china, due to factory closures and cutbacks, many of these women had been forced to enter the sex industry as a means of survival (interviewee e 2003, pers. comm., 27 august) . hence, while both interviewees c and e were aware of the effects gender has on hiv/aids vulnerability, neither their particular organizations, nor any other organizations responsible for hiv/aids prevention and treatment campaigns at the time, incorporated gender into their programmes or education campaigns. interviewee a, who was a senior programme offi cer for an overseas aid agency (2003, pers. comm., 21 august) , offered two possible reasons for this oversight. firstly, she believed a gendered response was not a major component of china's hiv/aids strategy because 'gender issues are generally addressed by the all china women's federation (acwf) or the regional women's commission (rwc)' (interviewee a 2003, pers. comm., 21 august) . secondly, at the time of fi eldwork, ordinary chinese women among the general population were not widely recognized or targeted as being vulnerable to hiv transmission (interviewee a 2003, pers. comm., 21 august). therefore, according to interviewee a, because women among the general population were not regarded as a vulnerable group it made little sense to organizations such as hers to engender their hiv/aids prevention strategies. however, if we consider interviewee a's fi rst response, it would appear that the acwf and rwc have unintentionally 'marginalized' women in discussions on hiv/aids vulnerability among organizations responsible for hiv/aids prevention and treatment campaigns because these organizations do not want to interfere with the role of the acwf and the rwc. this reluctance could in part be because of the historical background of the acwf. this organization was one of the fi rst mass organizations formed by the chinese communist party following the 1949 liberation and was the impetus for the social, legislative and policy changes aimed at improving the status of women in the 1950s. while it was temporarily disbanded during the cultural revolution , the organization was fully reformed in 1978 and since that time it has continued its earlier work on improving the status of women in china through legislative and social change (howell 2003 ) . therefore, the acwf has had a monopoly on women's issues in china for much of china's post-liberation period. while there has been an expansion of women's groups and organizations since the reform and opening of china in 1978, it is signifi cant to note that the acwf has remained an important ally for these groups (lee and regan 2009 ) , and the strongest women's organization in china due to its political legitimacy and sway in the political structure (howell 2003 ) . therefore, interviewee a's reasons for the lack of consideration of gender in offi cial responses to hiv/aids in china demonstrates that there are serious impediments which need to be addressed. it would seem there is a reluctance to either tackle gender issues by non-acwf organizations as they may perceive a turf war confl ict, or simply because they feel that such issues are best handled by the acwf. whatever the reason, because such organizations are reluctant to incorporate gendered responses into their hiv/aids prevention and treatment campaigns women have been marginalized in the mainstream responses to hiv/aids in china even though international experience has repeatedly demonstrated that if they are left unresolved, gender vulnerabilities to hiv transmission fuel hiv epidemics. however, interviewee a did state that should the epidemic move into the 'rampant prevalence' period, these groups (meaning women in the general population not belonging to any of the traditional 'high-risk' groups) would receive more attention from organizations like hers. thus, it would appear some organizations do believe gendered responses may be necessary in the future. nonetheless, the reluctance of international organizations like interviewee a's to incorporate gendered responses was concerning as in many aids-stricken countries, especially those in sub-saharan africa, women among the general population have been found to be at substantial risk of contracting hiv through non-commercial heterosexual intercourse (unaids, unfpa and unifem 2004 : 1-2). as stated above, much of this vulnerability stems from social, cultural, economic and political factors that often refl ect gender inequality and this chapter now turns to an examination of these in the context of women in the prc. accurate knowledge about hiv/aids, including the prevention and transmission of hiv, is an essential part of an adequate response to hiv/aids. however, surveys conducted by some ingos have revealed that as much as 20-40% of china's population has no knowledge of hiv/aids at all (park 2003 : 54; unaids 2002 : 43) . while recent fi gures suggest this has changed and that 'basic' 5 hiv/aids awareness is in the vicinity of between 74.5% and 85.1% for urban and rural residents and migrant workers, there still remain pockets of the population who lack accurate hiv/aids knowledge and awareness (ministry of health of the people's republic of china 2010 : 7). one of the reasons often cited for this lack of knowledge is that due to china's size and regional variances in language dialects, country-wide hiv/ aids education campaigns are diffi cult to conduct. interviewee f (2003, pers. comm., 9 september) , the division director of a government organization that examines hiv/aids related issues, confi rmed this when she stated 'because china is so large… not everyone knows about aids [and] the information has not spread to very remote areas, [therefore] more work will be done there'. these factors seriously complicate the dispersal of accurate hiv/aids knowledge, and are a key area that the chinese government must overcome to effectively respond to china's growing hiv/aids epidemic. however, there also exists a range of other reasons for the poor levels of hiv/aids knowledge in china. the reform and opening period , which began in the late 1970s, loosened societal attitudes and views on sexual issues in china, and this resulted in an increase in premarital sex and societal acceptance of premarital sex (qian et al. 2004 ; wu et al. 2007 ) . a study by the sex sociology institute of the people's university of china found that in people over 40 years of age, 45.7% of men and 24.1% of women reported they had engaged in premarital sex . however, for the 25-29 year old age bracket, rates were signifi cantly higher with 72.2% of men and 46.2% of women reporting they had engaged in premarital sex (xia 2004 : 14) . even though premarital sex is occurring, sex education and the supply of contraceptive devices to young, unmarried people remains a contentious issue. interviewee c (2003, pers. comm., 22 august) stated that sex education was an important topic in china and that there had been much debate over when young people should be taught about sexual health, with some sections of chinese society arguing that the university level was the most appropriate time for formalized sex education . there was also growing debate over abstinence-based sex education as opposed to sex education that promotes 'safer sex', such as using condoms, with the proponents of the abstinence-based sex education style hoping to see a return to 'traditional morality' (xia 2004 : 15) . in the meantime, no real steps have been made in implementing a comprehensive sex education curriculum into chinese schools. as a result, many university students in the prc have 'alarmingly low' levels of aids knowledge and self-perceived risk (wu et al. 2007 : 683) , refl ecting the need for a national sex education curriculum. this is concerning as hiv in china is most prevalent among people in the 20-29 year old age bracket, with this group accounting for 56% of china's overall hiv/aids cases (ma et al. 2009 : 249) . however, these rates of infection are unsurprising when one considers that surveys of university students have found that only 15% of those who were sexually active reported 100% condom use in their sexual encounters during the year prior to the study (ma et al. 2009 : 256) . for many young unmarried people in china, the lack of accurate knowledge on 'safer sex' has been a contributing factor in them engaging in unprotected sex, which easily facilitates the transmission of stis, including hiv, as well as increasing rates of unintended pregnancy. although young unmarried people can access contraceptive services in china, information and advice about contraception is somewhat limited. this is because the national family planning programme only targets married couples for the delivery of contraception information and devices. furthermore, studies conducted in rural and urban shanghai found that when premarital sex resulted in pregnancy, most pregnancies were unintended, and were usually because the couple had not used contraceptives (qian et al. 2004 ) . surveys conducted on confi dence levels when purchasing condoms found that only 44.3% of women compared to 85.5% of men felt confi dent buying condom s (ma et al. 2009 : 257) . clearly, even though premarital sex has become more commonplace and generally more acceptable, a comprehensive sex education programme and easy, more accepted access to reliable barrier methods of contraception are needed if optimum health outcomes are to be achieved. another key factor in this discussion is how condoms are perceived by chinese society. with the resurgence of stis in china, and the need to promote condoms effectively as a means of preventing stis, condoms have undergone a name change from bi yun tao , or 'avoid pregnancy sheath', to an quan tao , or 'safety sheath'. the change refl ects the dual role of condoms in preventing conception, but also as a means of preventing the transmission of stis/hiv (yuan et al. 2003 : 17) . interviewee f acknowledged this change, adding that her organization stressed '…using condoms is benefi cial for men and women, from the point of view of health, because men and women will both benefi t from the[ir] use ' (2003, pers. comm., 9 september) . however, a recent study has reinforced that condoms are still primarily perceived by some university students as pregnancy prevention rather than protection against stis/hiv. the study found that 95% of respondents identifi ed condoms as protection against pregnancy while only 30-41% regarded them as protection against stis/hiv (ma et al. 2009 : 256) . clearly, condoms are a necessary part of an effective hiv prevention strategy so it is imperative that their role in sti prevention is stressed and encouraged in the chinese situation. thus, even with the name change, condom use in china remains low. this is alarming to epidemiologists because of the role of condoms in preventing stis/hiv, particularly for women. a study by xia in 2002 found that 75.1% of male respondents were unwilling to use condoms in their sexual relations because they found them to be 'troublesome', to decrease male sexual pleasure, as well as too expensive. in addition, many unmarried men preferred not to use condoms because they felt their 'sexual and reproductive ability' was proven if their partners became pregnant ( 2004 : 22). furthermore, many men continue to view their sexual relationship with their wife as procreation, whereas sexual pleasure is derived from commercial sex workers. therefore, condoms within marriage are not considered appropriate and they are rarely used, regardless of some men having multiple sexual partners (chen 2008 ) . interviewee b (2003, pers. comm., 22 august), a programme offi cer for an overseas aid agency, believed that women could be vulnerable to stis from their partners because 'there is no such dialogue [safer sex] between husband and wife or partners'. furthermore, she concluded that in south-west china for instance, promotion of condom use in sexual relationships was absolutely necessary because the main route of hiv infection for men there has been idu and for women, it was through heterosexual intercourse 'within the family, within marriage, it's not through commercial sex workers' (interviewee b 2003, pers. comm., 22 august) . increasing condom usage rates among the general population is further complicated because many chinese women use intra-uterine devices (iuds) or have had surgeries such as tubal ligation or hysterectomies to avoid further pregnancies, causing condoms to be viewed as unnecessary. 6 therefore, interviewee b believed that it was imperative condoms be promoted as a sexual health device within marriage and committed relationships in addition to persons engaging in premarital sex or infi delity. while promoting condom use among the general population was also identifi ed as a necessity by interviewees d and c, work is needed on promoting 100% condom use among commercial sex worker s in both their commercial and noncommercial sexual relations. in china, condom use among sex workers is also extremely low with only 10% of sex workers surveyed at various locations reporting that they always used condoms, and close to 50% of sex workers reporting that they had never used a condom with a client (kanabus 2004 ) . this is not surprising considering many sex workers lack adequate hiv/aids information in the fi rst instance and secondly because condom use in sexual exchange generally involves a 'discount' due to both decreased sensitivity and male sexual pleasure. therefore, the economic burden faced when insisting on condom use in the commercial sex exchange means that for many sex workers condom use is not a viable or desirable economic option. in addition, low rates of hiv/aids and sti knowledge has meant that many sex workers do not believe they are at risk of contracting hiv. this clearly indicates a continuing lack of hiv prevention knowledge in an important 'high-risk' group, which increases the likelihood of the transmission of hiv among sex workers and their clients (kanabus 2004 ) . furthermore, other sex workers who wanted to use condoms in the commercial sexual exchange were prevented from doing so because they lacked 'the power to insist on the use of condom s with their clients' . in most instances, it is the client who decides whether or not a condom is used in commercial sexual exchange, again refl ecting the disempowered status sex workers face (kanabus 2004 ) . hence, poor hiv/aids awareness and female disempowerment facilitates such 'high-risk' behaviour, and this situation reinforces the need for widespread public education campaigns to better educate the entire chinese population on hiv/aids prevention and risk. this is particularly pressing considering that commercial sex is the leading contributing factor for the transmission of hiv through heterosexual intercourse (hong et al. 2009 ) . however, such an education campaign must contain accurate and appropriate messages about hiv/ aids and hiv prevention. early state media representations of hiv/aids negatively affected hiv/aids knowledge in china, and how plwha were received by society and by the medical profession. interviewee d (2003, pers. comm., 27 august) stated that when the state media initially discussed hiv/aids, it was portrayed as 'the enemy' and that media reports were largely focused on scaring people about the virus. furthermore, interviewee d believed that these early representations greatly contributed to the stigma and discrimination of plwha in china. this argument was reinforced by dikötter, who claimed that aids was initially described in offi cial discourse as an 'evil from abroad', and that it was widely believed that the 'superior immune system' of the chinese, combined with their 'neo-confucian values', would mean that hiv/aids would not infect the general population but would largely remain limited to homosexual men and sex workers who serviced foreign clients ( 1997 : 78-79). such beliefs became accepted by chinese society and have infl uenced how both the virus and plwha are perceived. in addition to stigma and discrimination , these early representations of hiv/ aids have also led to many people falsely believing that they are not at risk from contracting hiv if they do not belong to one of the above-mentioned groups. this false sense of security was refl ected in the section on 'self-perceived risk of contracting hiv/aids' in a study conducted by the futures group ( 2004 : 17) . this study explored the levels of hiv/aids knowledge among respondents and their attitudes and behaviours towards aids related issues. the study found that 78% of those surveyed believed themselves to be at 'low-risk' of contracting hiv/aids, and that the main reason for such a belief was because very few of the respondents (2%) reported that they knew of a plwha or a person who had died from aids. therefore, because they themselves had not known anyone affected by hiv/aids, their perception of the virus was that it is something that affects the 'other' or that it only affected 'degraded people' (futures group 2004 : 17) . consequently, because they did not fi t either category, they believed themselves to be of little risk of contracting hiv/aids. self-perceived risk has also been skewed by the government's hiv/aids prevention policies, which have long been focused on 'high-risk' groups. it can be argued that current prevention strategies actually put women at risk because they stress partner reduction over condom use as an effective way to avoid hiv transmission. the 'one partner' or 'faithfulness' prevention messages, which teach both men and women to protect themselves against hiv transmission by limiting the number of partners they have to one, has been described by unaids as lulling people into a 'false safety' (unaids 2002 : 44) . surveys that have been conducted in china among traditional 'low-risk' groups such as married women, who do not engage in any of the traditionally recognized 'risky practices' conducive to hiv transmission, have found that most women believe that limiting the number of partners they have to one is much better protection against hiv transmission than using condom s (unaids 2002 : 44) . however, such a measure is dependent upon their spouse having a negative status upon the commencement of the relationship, and not engaging in practices that may cause them to contract hiv for the duration of the sexual relationship. furthermore, the 'one partner' campaign ignores the fact that for many women in the developing world who have contracted hiv/aids through heterosexual intercourse, the source of their transmission was their only sexual partner, usually their husband. 7 often these women were unaware of their husband's hiv+ status, and in other cases, whereby they knew their husband was hiv+, they were unable to say no to sex or insist on condom use due to the unequal gender-based power relations within the relationship. in a study of chinese women who contracted hiv from their husbands, none of the women interviewed were aware of their husband's hiv+ status prior to their own diagnoses. in addition, one husband commented that he felt women 'had little choice if their husbands insisted they do so [have sex], inasmuch as the fact that "we are still married" legitimized their sex' (cited in zhou 2008 : 1119). therefore, the government sponsored campaigns in china that primarily focus on individuals reducing 'risky practices' or limiting the number of sexual partners they have to one, are out of step with reality. instead, hiv/aids prevention campaigns also need to focus on providing easily accessible sexual and reproductive health information for both men and women, and making condoms available and accessible to all sexually active persons. they should also aim to empower women and challenge the negative gender stereotypes and biases attributed to both men and women that heighten their vulnerability to hiv transmission. worldwide, women's vulnerability to hiv/aids is further heightened in societies where women are expected to be passive towards sex. while china was, traditionally, very much a society whereby passivity was expected of women in sexual matters, after 1949 this situation was widely believed to have altered because of mao's proclamations about female equality as well as the belief that the 'smashing' of class 7 surveys conducted in africa reveal that 60-80% of hiv positive women, who contracted hiv from sexual intercourse, reported that their only sexual partner was their husband. another study, which was conducted in india, another region where hiv/aids is growing at an alarming rate, reveals that 91% of hiv positive women surveyed, who had contracted hiv from sexual intercourse, also reported that their only sexual partner was their husband (feinstein and prentice 2000 : 22) . these fi ndings support the results of an earlier study conducted in 1989 which also found the majority of hiv positive women who had contracted hiv through heterosexual intercourse, had also contracted hiv/aids from their only sexual partner, their husband. the researchers in this instance concluded that often 'condom use was more effective in preventing hiv infection than was limiting the number of partners' (berger and vizgirda 1993 : 62). difference would lead to the eradication of outdated sexual stereotypes regarding men and women. however, patriarchal views still permeate chinese society and overall chinese men retain their position of gender privilege, often refl ected in the power dynamics of heterosexual relationships. the one child policy is one of the main factors in increasing female insecurity in contemporary china, particularly within the marital unit. since its introduction, there has been a strong resurgence in son preference , especially in the rural areas where sons play an important role in continuing the family lineage, contributing to the family labour force and their fi lial duty to provide for their parents in old age ( croll et al. 1985 ; croll 2000 ; chan et al. 2002 ) . in some areas of china, the reemergence of son preference has caused there to be strong pressure on women to give birth to a son and failure to do so can lead to domestic violence and even divorce. this is because women are wrongfully blamed over the sex of the child due to poor knowledge on the biological determinants of the sex of the fetus and also because it is believed to be 'the duty of a chinese wife to bear a son to continue the family name' (chan et al. 2002 : 427) . in her discussion of son preference and the resultant violence against women who give birth to daughters, croll provided several accounts of incidences whereby a husband committed acts of domestic violence against his wife after she bore a daughter ( 2000 : 78-80). the acwf has found that reported cases of domestic violence occur in approximately 30% of chinese families, with 32.5% of abused women being beaten around four times per month (xinhua 2000 ) . female suicide rates are also high in the prc, refl ecting female insecurity there. of the total number of female suicides worldwide, 56% occurred in china (renwick 2002 : 383) . in addition, while the urban rate of female suicide is estimated to be in the vicinity of 15.9 per 100,000 women, in rural areas the fi gures have reached 78.3 per 100,000 women, clearly demonstrating a rural/urban divide in female suicide rates in china (hric, cited in renwick 2002 : 383) . a report by the bbc echoed these fi ndings, and stated that many rural women are highly successful in their suicide attempts because they used pesticides and rat poisons to commit suicide (bbc 2002 ) . if gender-based violence such as domestic violence is viewed as an acceptable factor in preserving gender relations in the home, it can substantially increase women's vulnerability to hiv transmission. in societies where there exists 'a cultural ethos that violence is a valid means of solving inter-personal disputes' (whelan 1999 : 11) , such as in the domestic spheres, women may avoid discussing the use of condoms or fi delity issues with their partners for fear of violent response. the fear of violent retribution was identifi ed by women from a range of countries such as guatemala, jamaica and papua new guinea as being the reason why they did not try to negotiate condom usage with their sexual partners (feinstein and prentice 2000 : 24) . women's vulnerability to hiv/aids is heightened by male aggression because it can often be linked to the occurrence of sexual coercion, non-consensual sex and sexual violence against women. for many women, decisions about their sexual behaviour are denied to them because they are forced into sexual intercourse against their will. this is applicable both inside and outside of relationships. in such instances, condom usage is unlikely, so women's vulnerability to hiv transmission is increased (irwin et al. 2003 : 31) . therefore, the accounts given above of high rates of domestic violence and female suicide demonstrate that there are a great number of women in the general population who are facing increased hiv vulnerability due to their unhealthy domestic environment. the traffi c of women in china is also fuelling female vulnerability to hiv transmission, and is most prolifi c in sichuan and guizhou. interviewee a (2003, pers. comm., 21 august) attributed this to the fact that these two provinces 'have a high number of poor farmers and unemployed' so they sell women for fi nancial rewards. interviewee a also stated that the 'traffi c of women in china is generally restricted to marriage, whereas traffi cking outside of china is generally for prostitution'. in saying this, the interviewee believed that the traffi c of women , while bad, would not increase the likelihood of hiv transmission because the women were sold as 'brides', not sex workers (interviewee a 2003, pers. comm., 21 august) . the belief that traffi cked 'brides' are not particularly vulnerable to hiv transmission was also shared by interviewee e (2003, pers. comm., 27 august) . however, this belief demonstrates a serious lack of understanding of human traffi cking as members of the traffi cking gangs sometimes rape the women, regardless of them being traffi cked as 'brides' or as sex workers . furthermore, prospective husbands are sometimes allowed to have intercourse with the women before purchasing them, in order to decide which woman will become their 'bride' and also so that they can bargain the price of the woman ( south china morning post , cited in jaschok and miers 1994 : 264) . clearly, the rape of these women by numerous men, including their 'husband', increases their vulnerability to hiv transmission. in addition, the likelihood that their 'husband' will contract hiv from them or from other women he 'sampled' is also heightened if he does not already carry the virus. hence, it is a very serious misconception that the traffi c of women in china as 'brides' will have little impact on the hiv/aids epidemic there. equally alarming is the view held by some segments of chinese society that 'as long as they [men] have the money, buying a wife or child is their own affair' (li zhongxiu, cited in jaschok and miers 1994 : 265) . again, this kind of attitude illustrates the low status that many women (and children) continue to occupy in chinese society and the patriarchal factors that heighten their vulnerability to hiv/aids. in addition, it is widely predicted that the sale of women in china is set to continue to expand because from a purely economic standpoint, buying a traffi cked bride can be far cheaper than paying a dowry (song, cited in jaschok and miers 1994 : 265) . also, the skewed sex ratios that have resulted from the introduction of the one child policy and resultant resurgence of son preference have seen men far outnumber women in many areas of china (edwards 2000 : 75) . it has been estimated that by 2029, there will be 30 million more males than females in the 20-49 year age bracket due to sex selective abortion and the neglect of girl children in china (chan et al. 2002 : 429) . due to the gender imbalance it is unlikely that these men will be able to fi nd a bride to marry legally and they will increasingly turn to bride traffi cking as the solution, making it a crime that is likely to soar over the next few decades, alongside prostitution. while the chinese government has not ignored this situation, and the public security bureau often detects and arrests human traffi ckers , traffi cking is diffi cult to police as the number of women traffi cked yearly is believed to be in the vicinity of tens of thousands. they are primarily abducted from poor regions in guizhou, sichuan and yunnan provinces (woodman and ho, cited in hughes et al. 1999 ) . further complicating this issue is the fact that many women who have escaped their 'husbands' have not received assistance because some authorities also have sympathy toward men who have been unable to fi nd brides. as a result, women who escape their situation are often returned to their 'husbands' by authorities. villagers also assist in the traffi cking of women either by ignoring the problem, or by helping to buy women. in fact, it has been reported that one remote village collectively purchased women with the intention of soliciting them from their homes (woodman and ho, cited in hughes et al. 1999 ) . the traffi c of women constitutes a serious potential bridge for hiv transmission to the general population and again refl ects a heightened vulnerability, in terms of both hiv transmission and human insecurity, for chinese women. another factor identifi ed by interviewee c as compounding the situation of women's vulnerability to hiv transmission was the migration of rural workers to the cities for work. she stated that although many male migrant workers are married, they leave their wives behind and often engage in 'risky practices' in the cities, such as idu, procuring sex workers, and infi delity. generally, the men return to their homes once a year, during which they engage in sexual activity with their spouse, usually without using condoms. the interviewee further stated that even if the woman may suspect her spouse of having engaged in 'risky practices' while away, many women are unable to insist that their spouse use a condom (interviewee c 2003, pers. comm., 22 august) . in addition, for many rural women, labour migration can heighten their vulnerability to hiv transmission because it removes them from the economic support and protection nets that exist in their home villages, making them easy targets to be lured or forced into prostitution. in fact, a large number of china's sex workers are migrant women (thompson 2003 ) and they sometimes display higher risk behaviour than non-migrant sex workers, in part due to low education levels and poor hiv/aids awareness, which increases their likelihood of contracting hiv (hong et al. 2009 ) . many migrant women have also become the victims of sexual harassment and sexual violence, which also increases their vulnerability to hiv transmission. while conservative estimates suggest the number of sex workers in china is approximately three million to four million (harding 2000 ) , scholars such as professor pan of people's university of beijing believe the fi gure to be much higher when the numbers of women who engage in 'casual or infrequent transactional sex' are included (cited in . it has also been reported by unaids that the public security bureau estimates the number of sex workers in china could be as high as six million (unaids 2002 : 65) . jeffreys states that government authorities in china have called prostitution a 'widespread and growing problem ' ( 2004 : 83) . 8 thus, a conservative estimate of four million sex workers demonstrates that a considerable number of chinese women are vulnerable to hiv infection through prostitution. the illegal nature of prostitution in china is a major barrier to hiv/aids advocacy for sex workers and continues to exacerbate the vulnerable status of sex workers. interviewee d (2003, pers. comm., 27 august) stated that organizations like hers could give hiv prevention information to sex workers, without arresting them, because it was not a government organization. if workers from the government organizations identifi ed sex workers, she stated that they were required to report them because of the illegality of prostitution. after being reported, the identifi ed sex worker would then face detention in a rehabilitation centre. while there are debates in china as to whether or not prostitution should be decriminalized or legalized so as to allow ingos, ngos and government organizations to legally provide sti prevention knowledge and services to sex workers, the rehabilitation system does currently offer an opportunity to reach this vulnerable group with hiv/aids prevention information. however, interviewee d (2003, pers. comm., 27 august) stated that this was not occurring, even though the organization she worked for had been trying to launch programs that linked 'hiv/aids prevention education into the rehabilitation programs of these centres'. furthermore, without adequate help to overcome their economic insecurity, upon release from these centres many women actually returned to prostitution. thus, she stated, an important opportunity was being missed. there is concern however, over the contradiction in the government's response to prostitution, which sees sex workers targeted by interventionist programs, not those soliciting the prostitutes. this obvious gender bias is in part fuelled by the extant view in china that those selling sex always come before those buying sex. this view is problematic in that one could argue that it is demand that drives provision; however at a very basic level it punishes the sex worker as the guilty party in the commercial sex exchange and not the solicitor. it also means that many of the men who solicit prostitutes are left out of the targeted commercial sex hiv prevention strategies although they are clearly a 'high-risk' group (chen 2008 ) . if they are married or have other sexual encounters outside of the commercial sexual exchange, they are also a possible 'bridge' population who have the potential to transmit hiv into the general population. chen ( 2008 ) argues that the failure of the chinese government to adequately respond to this contradiction refl ects that there is an urgent need in china for recognition of the important role men play in safer sexual practice, a responsibility that is continually being thrust onto women. in fact, recent campaigns by the acwf reportedly 'exposed' 27.25 million women across china to hiv/aids prevention and awareness knowledge. however, there was no discussion of if/how men were also given this important knowledge or if they were made aware of how their actions can impact on the hiv vulnerability of their spouses (scawco and unaids 2007 ) . considering the unequal gender-based power relations discussed above, it is unlikely that these types of measures will have much success as men and women both need to be involved in such advocacy programs. to neglect the involvement and importance of men serves to increase women's vulnerability while at the same time proportioning responsibility to women for their own hiv safety, something that is simply unachievable for many women. the failure of the rural health system is also exacerbating the vulnerability of chinese women to hiv/aids. approximately 60% of rural women are now showing symptoms of having untreated rtis or stis (interviewee c 2003, pers. comm., 22 august), both of which increase their susceptibility to hiv through sexual transmission ( jolly and ying 2003 : 2) . the fi gures on stis clearly indicate that behaviours conducive to the transmission of hiv/aids, such as unprotected intercourse, are becoming more widespread. however, rural healthcare facilities continue to be inadequate, and therefore information on hiv/aids and prevention of the virus is not reaching rural men and women (interviewee c 2003, pers. comm., 22 august) . in addition, many rural women are not targeted for information dissemination due to the offi cial belief that these women fall into the 'low-risk' category due to their marital status and individual behaviour. in light of the discussion above, this overly simplistic classifi cation of who is or is not 'at risk' is substantially heightening women's vulnerability to hiv. considering the changing face of china's hiv/aids epidemic in the current era, the failure of the government to have a comprehensive gendered response to the burgeoning aids epidemic in the prc may result in the government ineffectively responding to hiv/aids. chen ( 2008 ) warns that this could lead to an increase in stigma and discrimination against women as their hiv+ rates increase and may further exacerbate women's insecurity. when critiquing the privatization of health care in china, interviewee d (2003, pers. comm., 27 august) stated that prior to the 1990s, the health system in rural china was much better because 'bare foot doctors and health workers were active in even the most remote areas'. however, after the dismantling of government sponsorship, the rural healthcare system was left in ruins, with only expensive private doctors available to meet the health care needs of the rural population. another problem with the provision of health care is that the government does not regulate sti facilities. in fact, some sti clinics are now being rented out to private practitioners. while this can benefi t plwha, because it reduces the possibility of their hiv+ status being leaked, 9 it also causes prices to rise and patient care tends to decline (interviewee c 2003, pers. comm., 22 august) . therefore, the privatization of essential services such as the health industry has seen medical services fall out of the economic reach of many poorer families or individuals. this compounds chinese women's vulnerability to hiv because it limits the ability of both men and women to manage their sexual and reproductive health. another diffi culty identifi ed by interviewee d (2003, pers. comm., 27 august) was that organizations like hers lacked adequate funding both to run programs as well as to support plwha. she stated that because most plwha in china are rural poor, education on lifestyle, proper diet and medication was in vain as 'many [patients] can't follow these instructions because they live in poor conditions in rural china, where their income is just enough to feed their families'. however, with better funding, hiv/aids prevention and treatment campaigns could help plwha with the costs of living and medications, making their lives better (interviewee d 2003 , pers. comm., 27 august). interviewee f (2003 also identifi ed funding as a problem for hiv/aids prevention and treatment. she stated that sometimes her organization wanted to carry out pilot projects, but because they were given insuffi cient funding they were unable to run the projects. clearly, without the funding to properly test pilot programmes or institute nation-wide campaigns, hiv/aids prevention in china will continue to be hindered. on the other hand, the introduction of the 'four frees and one care ' policy in 2003 has seen a positive change in the government's funding response to china's aids epidemic. the 'four frees' provided by the policy include government funded schooling for aids orphans, drug therapy for plwha, prevention of mtct and voluntary counseling and testing (vct). the 'one care' component refers to care and economic assistance for people affl icted with or affected by hiv/aids (cao et al. 2006 : 520) . however, by the government's own admission, the implementation of this policy has been uneven (national center for aids/std prevention and control 2006 : ii), so while it is certainly a step in the right direction, it is not fully operational as it will require a great deal of cooperation between all levels of government and assistance by civil society before it can really meet its objectives. it should be acknowledged that china has been injecting more funds into hiv/aids prevention and treatment strategies since 2003. the 2009 update by unaids and the world health organization reported that there had been a threefold increase in funding by china in the period between 2003 and 2006 demonstrating stronger commitment from the government (unaids and who 2009 ) . the incorporation of civil society into hiv/aids prevention was an issue also identifi ed by interviewee b (2003, pers. comm., 22 august) . she stated that the solitary nature of the moh in combating hiv/aids in china means that it is effectively tackling hiv/aids by itself. while the enabling environments for hiv transmission include diverse fi elds such as employment and public security, interviewee b stated that there was no cooperation between the ministry of public security (mps) and the moh, a point which chen believes has led to; tensions, and sometimes contradictions, between the goals of public policies and the methods of policy practice, such as the confl ict between public security (law enforcement) policies relating to sex work and public health policies for the prevention of hiv/aids ( 2008 : 185). furthermore, issues pertaining to commercial sex workers and idus, which were handled by the mps, also did not involve input from the moh. this is quite possibly the reason why hiv/aids prevention advocacy programs have still not been uniformly implemented in all rehabilitation centres for idus and sex workers . interviewees c and f both believed that education campaigns aimed at the general population could be a valuable way to disperse hiv/aids information. they stated that efforts like the severe acute respiratory syndrome (sars) mass media education campaigns would be a major step in changing people's beliefs about hiv/ aids and increasing knowledge and public awareness of the virus and how it is transmitted. after the cessation of sars, the chinese government did shift its focus to hiv/aids, and education campaigns on hiv/aids have been undertaken. therefore, unlike previous efforts, top leaders in government have demonstrated they are serious in their response to hiv/aids. while this signals a positive change, unfortunately the gender issues that contribute to hiv/aids vulnerability do not appear to be an integral component of these campaigns, so their overall effectiveness is doubtful. in addition, gender inclusive campaigns would also need to be supported with active steps at both the government and grassroots level to reverse the continuing unequal social, political and economic structures that disempowered chinese women, which have been shown to heighten their vulnerability to hiv/ aids. however, beijing's reluctance to support the development of an unrestrained civil society in china makes this unlikely. in the united nations report hiv/aids: china's titanic peril ( 2002 ) , china's political system was identifi ed as possibly the most sensitive obstacle to tackling hiv/aids in the prc. this was because the central government has long appeared uncomfortable with the emergence of organizations that are independent of the government and especially the free fl ow of information that such organizations may facilitate (unaids 2002 : 69-82 ). yet, while the central government may fear that the emergence of civil society could contribute to a breakdown in the chinese communist party's authority in china, civil society participation and the free fl ow of information are not only good governance when responding to hiv/ aids epidemics, but international experience has proven them to be essential elements in a state's response to hiv/aids. therefore, until the central government is willing to allow greater autonomy among the various ngos and ingos operating in china, it is unlikely that the chinese response to hiv/aids will make any real inroads in the prevention of hiv transmission -and even less so in terms of gender-specifi c issues such as the human insecurities that increase women's vulnerability to hiv/aids. when determining women's vulnerability to hiv transmission, female human security , or more aptly their 'insecurity', is an important factor. the status of many women in china, and the privileged position accorded to chinese men, strongly indicates that chinese women face a heightened vulnerability to hiv transmission. while many of these vulnerabilities are similar to women elsewhere in the world and certainly are not unique to china, they attest to the interplay of the unequal status accorded many chinese women due to their sex, their disempowered status within society, unequal gender-based power relations both within the domestic and public arenas, and the patriarchal norms and attitudes that infl uence all of the above. by overlooking the many social, cultural, economic and political factors that contribute to hiv/aids vulnerability and transmission of the virus, particularly those faced by women, china has a long way to go before chinese women are protected from hiv transmission. given that hiv/aids heightens human insecurity, the stage is set for chinese women (and men) to face an insecure future if the chinese government does not fully implement international best practice, meaning a gendered response, into its overall hiv/aids response. hiv hits women hardest prevention of hiv infection in women and children understanding hiv-related stigma and discrimination in a "blameless gender selection in china: its meanings and implication gendering china's strategy against hiv/aids: findings from a research project in guangdong province endangered daughters: discrimination and development in asia china's one-child family policy sex, disease and society: a comparative history of sexually transmitted diseases and hiv/aids in asia and the pacifi c women in the people's republic of china: new challenges to the grand gender narrative gender and aids almanac aids crisis impending: research on knowledge, attitudes and behaviours related to hiv/aids in china demography of hiv/aids in china: a report of the task force on hiv/aids center for strategic and international studies india is new loser in asian aids epidemic aids, bloodheads and cover-ups: the "abc" of henan's aids epidemic hiv/aids-related sexual risks and migratory status among female sex workers in a rural chinese country women's organizations and civil society in china the factbook on global sexual exploitation global aids: myths and facts. tools for fi ghting the aids pandemic women and chinese patriarchy: submission, servitude and escape feminist prostitution debates: are there any sex workers in china? key issues on gender and hiv/aids in china hiv & aids in china why develop and support women's organizations in providing legal aid in china? women's rights, women's organization and legal aid in china behavioural and psychosocial predictors of condom use among university students in eastern china 2005 update on the hiv/aids epidemic and response in china china's secret plague: how one us scientist is struggling to help the government face up to an exploding aids crisis unintended pregnancy and induced abortion among unmarried women in china: a systematic review the 'nameless fever': the hiv/aids pandemic and china's women a joint assessment of hiv/aids prevention, treatment and care in china hiv/aids epidemic in china spreads into the general population china's growing aids epidemic increasingly affects women hiv/aids: china's titanic peril women and hiv/aids: confronting the crisis aids epidemic update redefi ning security: the human dimension gender and hiv/aids: taking stock of research and programmes evolution of china's response to hiv/aids hiv/aids in china ban on family violence urged in china modelling the impact of the legal and policy environment on hiv/aids in china quality of care of reproductive health in china today endangered womanhood: women's experiences with hiv/aids in china the author would like to thank professor donald mcmillen, dr rosemary roberts and an anonymous reviewer for their insightful comments and suggestions. key: cord-031722-n5ja5oqw authors: fields, errol l.; hussen, sophia a.; malebranche, david j. title: mind the gap: hiv prevention among young black men who have sex with men date: 2020-09-10 journal: curr hiv/aids rep doi: 10.1007/s11904-020-00532-z sha: doc_id: 31722 cord_uid: n5ja5oqw purpose of review: young black men who have sex with men (ybmsm) suffer profound health inequities in new hiv diagnoses and clinical outcomes. while the evolution of hiv prevention options has become increasingly biomedical, inequities in access and uptake of these modalities persist. recent findings: studies suggest that while ybmsm display interest and acceptability of varied hiv prevention options, uptake lags due to the lingering effects of intersectional oppression from racism and sexual prejudice, hiv stigma, institutional and provider bias, and unresolved health policy barriers. promising avenues to address these barriers have yet to be fully explored. summary: we have the tools to effectively prevent hiv transmission and acquisition among ybmsm, but we have not yet effectively implemented these tools for this priority population. to end the epidemic, we must tailor and adapt hiv prevention strategies to meet the unique intersecting needs, identities, and social contexts of ybmsm. between 2012 and 2016, hiv diagnoses among adolescents and young adults (aya), defined as individuals aged between 13 and 24 years, increased 6%, whereas hiv diagnoses among older adults declined or stabilized over the same period [1] . in 2018, 21% of the 37,832 new hiv diagnoses in the united states (u.s.) were among aya. moreover, men who have sex with men (msm) made up 92% of cases among aya, of which 51% were young black men who have sex with men (ybmsm). despite an 11% reduction in new hiv diagnoses among ybmsm from 2010 through 2017, racial disparities in hiv incidence and prevalence have persisted, highlighting a need for tailored approaches that match the specificity of the epidemiology and prioritize this vulnerable population. in the past decade, several major advances have emerged in hiv prevention. treatment as prevention (tasp) was introduced with the hiv prevention trials network (hptn) 052 study, which demonstrated that people living with hiv, when treated early with antiretroviral therapy (art) and maintaining viral suppression, markedly reduced their likelihood of transmitting hiv to their heterosexual partners [2] . subsequent studies found that serodifferent same-sex and heterosexual couples demonstrated no new hiv infections over 150,000 condomless sex acts, when the partner living with hiv demonstrated viral suppression on art [3] [4] [5] . this evidence has resulted in the firm conclusion that treatment is prevention, and that those who maintain an undetectable hiv viral load do not transmit hiv to their sexual partners (i.e., undetectable = untransmittable [u=u]) [6] . pre-exposure prophylaxis (prep) has also demonstrated impressive results in reducing hiv transmission, with daily oral pill regimens tenofovir disoproxil/emtricitabine (tdf/ftc) and tenofovir alafenamide/emtricitabine (taf/ftc) reducing the risk of this article is part of the topical collection on the science of prevention hiv acquisition up to 99% with daily adherence, leading to a grade a recommendation from the u.s. prevention services task force (uspstf) [7] [8] [9] [10] [11] . emerging technologies, including long-acting injectable prep (cabotegravir) awaiting fda approval, will further augment and diversify prevention modalities and strategies [12] . dr. anthony fauci, director of the national institute of allergy and infectious diseases, noted in a 2016 op-ed, "we have the tools to end the hiv/aids pandemic… [but] our proven tools have not been implemented adequately or uniformly" [13] . this inequity is apparent in the disparate hiv burden that ybmsm experience. despite the expansion of the hiv prevention toolkit, inadequate access to and/or uptake of these tools among ybmsm threatens to exacerbate rather than eliminate these inequities. the objective of this review is to describe the state of hiv prevention for adolescent (aged 13-17 years) and young adult (aged 18-24 years) ybmsm, including hiv testing and biomedical, behavioral, and structural approaches. we will highlight gaps in implementation of current tools and make recommendations for maximizing prevention strategies for this priority population. hiv testing is an important initial step in both the hiv treatment and prevention cascades. yet studies have described low rates of testing among aya msm in general and among ybmsm in particular. testing rates may be higher among ybmsm compared with young msm in other racial/ethnic subgroups [14] , but relative to their higher relative hiv incidence, testing rates remain insufficient. a number of factors associated with hiv testing among msm have been examined. however, studies have often focused on subgroups defined by age or race/ethnicity and rarely the intersection of these subgroups where ybmsm populations are positioned [14] . further delineations of adolescent (13) (14) (15) (16) (17) and young adult (18) (19) (20) (21) (22) (23) (24) ybmsm communities are rarer still. nevertheless, several factors described in existing studies may be important considerations for ybmsm. national surveys of hiv testing in aya msm report individual and interpersonal level testing barriers including fear of judgment from peers and family, nondisclosure of sexual identity or behavior to parents/guardians or providers, limited sexual health communication with parents/guardians or providers, fear of a positive test result, and low risk perceptions and knowledge of hiv risk [15] . structural level barriers include cost, transportation, limited access to inclusive, comprehensive, youth-specific sexual health services, and lack of inclusive sexual health and hiv-specific health education [16] . smaller surveys and qualitative studies in ybmsm or older adult black msm have described similar barriers including concerns over issues of cost, accuracy, comfort within testing venues, and poor communication from providers related to sexual health [17] [18] [19] . these studies have also described the influence of social contexts (e.g., fear of stigma from families, friends and communities, and the potential consequences of positive test results) on hiv risk perceptions and testing practices [15] . similar barriers have also been noted with newer testing modalities such as home or self-testing [20] . the efficacy of tasp and u=u depends on an intact and robust hiv care continuum including early diagnosis, care linkage, engagement, and sustained viral suppression among people living with hiv. however, ybmsm living with hiv are often less likely to be diagnosed, engaged in care, or virally suppressed compared with other age and race/ethnicity subgroups [21] [22] [23] [24] . several studies have examined barriers and facilitators of treatment engagement, adherence, and viral suppression in this population [21, [25] [26] [27] [28] . among ybmsm living with hiv, barriers to adherence and viral suppression include depressive symptoms and psychological distress [25] , substance use [22, 25] , housing instability, hiv stigma [28] , and being uninsured or underinsured. facilitators to adherence and viral suppression include access to social and tangible support, self-efficacy with communicating with providers, being insured, and higher education level. consequently, due to gaps in the hiv care continuum, hivnegative ybmsm may have sex with individuals in sexual networks with higher rates of undiagnosed/unsuppressed hiv, increasing their risk for hiv acquisition [29] . the prevention benefits of tasp and u=u can only be extended to ybmsm if these gaps in the hiv care continuum are addressed. similarly, the efficacy of oral daily prep depends on care engagement (i.e., prep uptake), medication adherence, and persistence. both tdf/ftc and taf/ftc have shown themselves to be durable and safe options for hiv prevention, being approved for use in adolescents weighing over 35 kg in 2018 and 2019, respectively. however, similar to tasp, issues with awareness, access, provider knowledge and bias, cost, medical distrust, and low risk perception may adversely impact medical engagement and adherence among aya in general and ybmsm in particular [30] . moreover, consent and confidentiality barriers have plagued widespread acceptance of prep among medical and public health communities who would promote prep use [31] , due in part to concerns about adherence with follow-up visits and long-term effects on bone and kidney health [32] . young msm of all ethnicities demonstrate low levels of knowledge, but high levels of acceptability with regard to prep [33] [34] [35] . prep interest, uptake, and usage are particularly low among ybmsm [36] , even in the settings where they are aware of and able to access prep [37, 38] . multilevel and system barriers to prep access exist in families, communities, and medical spaces. pediatric and other medical providers are often limited in their ability to engage aya in general and sexual and gender minority aya in particular around their sexual health [39] [40] [41] [42] [43] . many are also unaware of or unfamiliar with prep, leading to missed opportunities to discuss sexual health and other prevention strategies with ybmsm who may be at risk [44] . provider bias has also been shown to decrease likelihood to prescribe prep to black msm, based on racialized beliefs that more sexual behavior risk compensation will ensue as a result of prep usage among black msm [45] . as a result, black msm have expressed experiencing heightened prep stigma, leading to distrust that affects agency in both medical decision-making and comfort discussing sexuality and behavior with medical providers [27, 46, 47] . specifically, ybmsm and transgender women who have sex with men (tgwsm) express additional stigma surrounding promiscuity assumptions, cost, and other conspiracy beliefs as deterrents from prep use [48] [49] [50] [51] [52] . for those who do initiate prep, it has been found to be effective and well tolerated among adolescent populations, but adherence with quarterly visits may wane over time, particularly among ybmsm [49, 53] . finally, issues related to adolescent ability to consent for hiv prevention services including prep and confidentiality pose significant barriers to prep uptake among aya, including ybmsm. while no jurisdictions currently prohibit minor adolescents from consenting for prep, few have passed statutes that explicitly allow minor consent [54] . where the legal statute is not clear and is subject to interpretation, adolescent access to prep may be limited. young adults may remain covered on their parents' insurance until age 26, which increases their ability to access care but may create a barrier to hiv prevention, prep, and other sexual health services if confidentiality cannot be maintained. an explanation of benefits (eob) and/or bills for laboratory or clinic visit co-pays may be sent to their parents, leading to unintentional disclosure of sexual activity, identity, hiv status, or specific sexual health services/diagnoses. for ybmsm, this may present particularly challenging additions to seeking health care when compounded by the persistent intersectional oppressive forces of racism and sexual prejudice. behavioral interventions targeting behaviors associated with hiv acquisition were once the cornerstone of hiv prevention. however, in today's increasingly biomedical hiv prevention landscape, behavioral considerations are often linked to biomedical modalities-with outcomes that focus instead on enhancing behaviors surrounding hiv testing, linkage, engagement, and adherence with services. traditional behavioral interventions, such as those designed to promote condom use, improve communication with sex partners, or prevent sexually transmitted infections (stis), still have an important role to play in counseling and supporting ybmsm. in fact, behavioral approaches may be even more important in this population due to logistical, financial, policy, and other barriers to obtaining prep, and thus should not be so easily discarded as an irrelevant prevention option. several behavioral hiv prevention interventions have been developed specifically targeting ybmsm, while others may have included ybmsm in their original study populations without restricting participation to that group [55] . these interventions, generally based in established social-behavioral theories (e.g., social cognitive theory), use strategies such as education, social support, and role-playing to decrease frequency of self-reported condomless anal intercourse [56] , increase hiv knowledge, improve hiv/sti-related communication skills, and change attitudes and intentions around condom use [57] . a limitation of behavioral interventions for hiv prevention-regardless of the targeted behavioral outcomes-has often been insufficient consideration of the social context that influences behavior. indeed, behavioral interventions that have been most effective at achieving sustained outcomes in black msm have incorporated and addressed the social determinants that contribute to hiv inequities and disparities experienced by this population. one notable example is many men, many voices (3mv), which was developed by and for black msm to specifically address the impact of racism, stigma, homophobia, familial, cultural, and religious norms on hiv risk behaviors and sexual relationship dynamics [58, 59] . 3mv is a cdc-designated evidence-based intervention that has also been tested specifically among ybmsm and was found to be effective for decreasing condomless sex acts [60] . black msm, including ybmsm, have a higher likelihood of living in neighborhoods containing various psychosocial stressors (e.g., neighborhood-level poverty, crime, drug use). these neighborhood-and community-level factors are associated with increased condomless sex and hiv risk [61] ; thus, structural approaches to address these more distal social determinants could enhance hiv prevention efforts focused on ybmsm. examples of structural approaches have included improving access to quality housing [62] , policy change (e.g., laws related to hiv criminalization or syringe exchange), and economic empowerment (e.g., microfinance or conditional cash transfer interventions, primarily used with adolescent women in lower-middle income country settings) [63, 64] . currently, no such structural hiv preventions have been developed and/or tested specifically for ybmsm. a summary of recommendations for improving effectiveness of hiv prevention services for ybmsm is included in table 1 . universal opt out hiv testing is a component of routine health care maintenance [65] and is recommended annually for sexually active aya msm in pediatric and adolescent primary care settings [66, 67] . however, in addition to the barriers with provider-patient communication described above, relatively low primary engagement among young men [68] (including ybmsm) suggests a need for increased access to testing in community settings. ybmsm prefer school-based and other community settings where hiv testing services are offered in a nonjudgmental, private, and confidential manner [69, 70] . moreover, accessible, trusted, and frequently engaged community settings may be ideal for hiv testing. school-based health clinics [71] , health department sexual health clinics [72] , community-based organizations [73] [74] [75] , and other community spaces may facilitate more open discussion and disclosure of sexual health practices and concerns over traditional medical settings [76] . community locations may also be more conducive to pairing testing resources with peer educators or other evidence-based education and outreach strategies, serving as conduits to primary care through partnering with pediatric and adolescent providers to provide treatment and comprehensive care linkage [77] . hiv and sexual health outreach and education through community spaces where ybmsm feel welcome and comfortable may also increase uptake of hiv-related information, motivation, and behavioral skills and increase their utilization of community resources for hiv testing services [78] in addition to enhancing selfefficacy for discussing sexual health services with medical providers [14] . research on the specific factors associated with hiv testing in both adolescent and young adult black msm is limited. the persistent racial disparities in hiv incidence and prevalence affecting aya black msm [79] despite higher testing rates warrant investigations that stratify study populations according to age and race/ethnicity to identify factors that influence testing in this priority population. additional analysis is also needed to inform the design of testing strategies that account for unique barriers resulting from the intersection of age, race/ethnicity, and sexuality ybmsm often face [80] . given the current emphasis on biomedical aspects of hiv prevention and the limitations in aya health care settings described above, improvements in clinical and cultural competencies related to sexual health are needed to ensure these settings are equipped to provide hiv prevention and other sexual health services to ybmsm [14] . gaps in care of ybmsm and other sexual and gender minority youth have persisted [81] despite numerous policy statements and guidelines from the american academy of pediatrics (aap) [82, 83] and the society for adolescent health and medicine (sahm) [84] . these gaps endure because clinicians are often not sufficiently trained in how to care for racially diverse and lgbtq youth [42, 43] . to ensure that aya providers are consistently well prepared to provide clinically and culturally competent hiv prevention and sexual health care to ybmsm and other lgbtq youth, appropriate health curricula should be integrated into medical education at undergraduate, graduate, and practice levels. current and emerging prevention modalities can only be effective for a particular priority population when implementation processes are informed by members of that group. hiv prevention efforts targeting ybmsm should be developed and implemented based on the voices, perspectives, and priorities of ybmsm themselves [85] . several models of community-informed practices including engaging priority populations in the development of prevention messages and campaigns [86] , incorporation of youth or community advisory boards to inform clinical programming [87] , and integration of peer or near-peer navigators with shared identity or experiences with ybmsm [88] have demonstrated promising effects. while additional programmatic evaluation is needed to determine how to best tailor and implement hiv prevention modalities to ybmsm populations, existing data provides some important insights on what program components are necessary. for instance, employing a holistic health approach, including attention to general wellness, mental health, and substance use as well as youth priorities essential to developmental tasks of adolescents (e.g., achieving education and employment goals, housing, navigating and exploring sexuality [89] , developing healthy romantic and intimate relationships) can serve as a gateway to engaging youth in hiv prevention. holistic approaches with frequent follow-up have been found to be successful in ensuring sustained adherence with prep among ybmsm and should be replicated widely [90] . consideration and appreciation for the unique intersecting identities and experiences of ybmsm is also critical. multileveled approaches that consider larger social/structural issues offer the best way to approach prep specifically, but few programs have fully embraced this reality [26, 89] . a sample of ybmsm ages 18 to 24 stated that family and friends, formal education, television, and the lgbtq community were major sources for hiv prevention information [91] . however, motivation for adopting such information was hampered by apathy, homophobia, and racism. emphasizing the understanding and deconstruction of more proximal social contexts like the desire to embody traditional masculine ideologies [92] could hold the key to more holistic sexual health efforts that umbrella education, interest, and eventual uptake of prep. religion often represents a deterrent to sexual health and hiv prevention due to documented homophobia and sexual prejudice within varied black faith communities [46, 75] . however, emphasizing affirming aspects of religion/ spirituality and their relationship to health access and beliefs, sexual health, and perceived risk for hiv may offer avenues for future exploration [76, 77] . sex work, whether chosen or in the context of food and housing insecurity, is a lived experience not unique to ybmsm, but represents an often-ignored circumstance deserving of consideration within our hiv prevention efforts. finally, leveraging the role of chosen nonbiological families, houses, and other nonheteronormative communities will be key in reaching and addressing the sexual health and hiv prevention needs of ybmsm, many who may suffer displacement from their biological families because of their sexual orientation [78] . medical spaces should also offer a menu of services, both in tailored approaches to hiv prevention (behavioral, biomedical, both) and how it would be best to deliver said servicestraditional brick and mortar approach with scheduled appointment, telehealth consultations, home visits and delivery of meds, or some combination of any of these. moreover, embracing technology in behavioral and biomedical hiv prevention approaches in the form of apps, telemedicine, and other unique interventions is a requirement for youth-based populations [55, 93 •• , 94] . as intergenerational differences may deter communication and uptake of hiv prevention services, it is crucial to ensure that an adequate representation of youth is present for hiv prevention navigation (tasp and prep), and that younger medical staff beyond the medical providers themselves are actively engaged and involved in patient care [95] . a team approach that emphasizes a collaborative and familial environment can provide aya, specifically ybmsm, with the guidance they need to navigate a complicated medical system that is challenging even for adults. the society for adolescent health and medicine recently published a position paper on improving prep access for adolescents and young adults that highlighted three of the most significant policy and structural barriers to aya prep engagement [96 •• ] . first, aya representation in clinical trial research at each stage of prep continuum is limited due to complexities of engaging adolescent minors in research. second, whether adolescents under 18 can consent to prep care is unclear in most jurisdictions and confidentiality issues for aya covered under insurance plans where their parents are the primary policyholders are largely unresolved. third, there is limited financial assistance for expenses beyond prescription costs such as monitoring labs, sti/hiv testing, clinic visits, and other related expenses. these are important gaps for ybmsm and represent policy considerations to consider when tailoring hiv prevention tools to this priority population. we cannot identify what approaches will work for at-risk populations if they are not included in research [97] . while engaging minor adolescents in sexual health research is complex and requires protections be put in place to ensure their safety, there is a growing body of evidence supporting that adolescents can freely consent to research, including research concerning sexual health and hiv prevention [98] [99] [100] . requiring parental permission represents a unique barrier for ybmsm and other lgbtq youth who may not have disclosed their identity or sexual behavior to their parents or otherwise lack parental support. youth have reported being unwilling to participate in hiv-related research that requires parental permission [99, 101, 102] , and parents have also acknowledged that requiring permission can place youth who are not "out" at risk [103] . adolescents do, however, represent a vulnerable population in need of added research protections such as the inclusion of a peer advocate, a strategy that has been endorsed by lgbtq youth [99] . researchers have also called for the inclusion of structured time for youth to reevaluate their decision to participate in a study [104] . youth may also need to be provided with additional information about randomization when participating in trials, and researchers must ensure they understand the distinction between participating in a research project and receiving personalized medical care [101] . finally, the inclusion of youth who have yet to invite their families into their sexual orientation/gender identity requires extra privacy considerations at all stages of research. recruitment or study materials that mention their sexual identities or behaviors may lead to unintentional disclosures if materials are seen by relatives or peers, as may study activities that occur at locations associated with lgbt communities [104] . minor consent laws are decided in states and local jurisdiction, so local hiv, lgbtq and youth advocates, chapters of professional medical and civic associations, and communities should advocate for changes in minor consent laws that will allow adolescents to consent for hiv prevention services, particularly prep. as delivery options for prep continue to expand, ensuring that minor ybmsm can access the full array of hiv prevention options without parental consent will be critical. similarly, for youth covered by their parents' insurance, developing protocols that ensure confidentiality in the nature of sexual health and hiv prevention lab testing is critical. ybmsm must feel comfortable accessing services, knowing that an eob will not disclose their sexual behavior, lab tests, or hiv status to their parents. current policies often require the covered individual to contact their insurer to request changes in either the details provided on the eob or where this information is sent, a process that can be difficult to navigate for many ybmsm and most aya. while patient assistance programs have eased the financial burden for accessing both art and prep medications, ybmsm may still be burdened with the cost of follow-up visits and lab testing. there are several potential sources for financial assistance for these additional costs. some states have developed prep assistance programs to assist with these costs for those at risk for hiv acquisition [105] . both insurance plans and patient assistance programs including those sponsored by pharmaceutical companies should also be approached to include provisions that ensure basic lab services and follow-up visits will be covered, as continued costs for these services serve as a deterrent for ybmsm to stay engaged in care and adherent to medications. ybmsm are at increased susceptibility to hiv partly due to exposure to sexual networks with undiagnosed/unsuppressed hiv and untreated stis that facilitate hiv transmission [106] [107] [108] [109] [110] [111] [112] [113] . interrupting this cycle requires public health diagnosis, prevention, and treatment strategies that prioritize (1) identifying hiv transmission networks; (2) accessing and identifying individuals within these networks; and (3) linking these individuals to effective hiv prevention or treatment options. innovative strategies for identifying transmission networks and individuals within these networks not currently linked to prevention or treatment are needed to reduce hiv risk for ybmsm in these networks. behavioral approaches that focus on prep or art adherence are an important consideration for ensuring effective implementation of biomedical prevention strategies. however, it is also important to consider that prep medications are "indicated in combination with safer sex practices," [115] making the sexual behavioral targets of traditional hiv behavioral interventions continually relevant and important. furthermore, given the challenges with heightened risk and effective implementation of tasp and prep among ybmsm, nonjudgmental behavioral approaches that focus on effective risk mitigation may have even greater importance. whatever the behavioral target, behavioral interventions should also address complexities ybmsm face at the intersection of race, sexuality, and development, with reinforcement of affirming approaches to sexual health that acknowledge and target racial and sexual identification [58, 116] . addressing the distal social determinants of health and the syndemics that contribute to hiv inequities impacting ybmsm are important targets for structural interventions, but have not been explicitly explored in this population. as these inequities persist, the urgency of ongoing hiv burden demands trials of innovative strategies that have been effective in other populations or settings. in addition to what has been previously mentioned, from a societal perspective, we must acknowledge how the pervasive distal scourge of racism relates to hiv prevention, with medical and public health systems working on eradicating discrimination within medical spaces and acknowledging the impact of racism on health inequities among ybmsm [117] . highlighting the intersection of racial and sexual identities is paramount as we know that embracing both is key to wellness among ybmsm [118] . initiatives should also emphasize the roles that gender norms and religious doctrines/beliefs play in the overall health and lived experiences of ybmsm [92, 119] [27, 120] [121, 122]. perhaps most critical for our consideration of distal forces influencing evolving hiv prevention efforts is our framing of sexuality among black men itself, specifically, what sexuality and intimacy means beyond the over-biomedicalized, analytical, and often pathological defining of sexual acts as just sexual networking, condomless sex, and risk for hiv and stis. future hiv prevention models targeting ybmsm should reflect a more contemporary conception of same gender love, intimacy, and pleasure between black men that acknowledges how physical and sexual acts serve as vehicles of emotional connectedness in defiance of lived experiences with racism, sexual prejudice, and other social forces. they are not simply "risky encounters" that require immediate hiv testing, daily pills, or bimonthly injections. creating affirming and nonjudgmental approaches within physical/virtual spaces that reflect these realities for ybmsm is crucial in this equation. effective hiv prevention options have expanded significantly, creating the tools necessary to end the hiv epidemic. ensuring equitable and tailored access to ybmsm is the challenge that lies in front of us. more research and interventions are needed that specifically focus on ybmsm communities. improving medical systems and provider attitudes will be invaluable to enhance clinical spaces that often judge and stigmatize them for simply being who they are. advocating for policy change that facilitates easier access to hiv prevention modalities is essential. the young men who are the focus of this review are not just black or same gender loving individuals. they are not just adolescents and young adults, nor are they simply statistics of an hiv epidemic. their lived experiences embody the intersection of many social identities and external forces. our approaches to hiv prevention must be equally holistic and integrated if we expect our scientific advances to translate to the successful eradication of health inequities suffered by these human beings. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. youth at risk of hiv: the overlooked us hiv prevention crisis antiretroviral therapy for the prevention of hiv-1 transmission viral suppression and hiv transmission in serodiscordant male couples: an international, prospective, observational, cohort study risk of hiv transmission through condomless sex in serodifferent gay couples with the hiv-positive partner taking suppressive antiretroviral therapy (partner): final results of a multicentre, prospective, observational study sexual activity without condoms and risk of hiv transmission in serodifferent couples when the hiv-positive partner is using suppressive antiretroviral therapy hiv viral load and transmissibility of hiv infection: undetectable equals untransmittable preexposure prophylaxis for the prevention of hiv infection: us preventive services task force recommendation statement preexposure prophylaxis for the prevention of hiv infection: evidence report and systematic review for the us preventive services task force tenofovir alafenamide for hiv preexposure prophylaxis tenofovir alafenamide for hiv preexposure prophylaxis preexposure prophylaxis for prevention of hiv acquisition among adolescents: clinical considerations cabotegravir, a new option for prep we have the tools to end the hiv/ aids pandemic. the washington post factors associated with hiv testing in teenage men who have sex with men we hide low rates of human immunodeficiency virus testing among adolescent gay, bisexual, and queer men hiv testing among us high school students and young adults preferences for hiv test characteristics among young, black men who have sex with men (msm) and transgender women: implications for consistent hiv testing from their voices: barriers to hiv testing among black men who have sex with men remain hiv self-testing among young, black msm, and transgender women correlates of self-reported viral suppression among hiv-positive, young, black men who have sex with men participating in a curr hiv/aids rep randomized controlled trial of an internet-based hiv prevention intervention exploring the hiv continuum of care among young black msm viral suppression in adolescents on antiretroviral treatment: review of the literature and critical appraisal of methodological challenges brief report: racial and ethnic disparities in sustained viral suppression and transmission risk potential among persons aged 13-29 years living with diagnosed hiv infection predictors and profiles of antiretroviral therapy adherence among african american adolescents and young adult males living with hiv multiple dimensions of stigma and health related factors among young black men who have sex with men triply cursed': racism, homophobia and hiv-related stigma are barriers to regular hiv testing, treatment adherence and disclosure among young black gay men hiv stigma experienced by young men who have sex with men (msm) living with hiv infection are hiv seroconversions among young men who have sex with men associated with social network proximity to recently or longterm hiv-infected individuals? journal of acquired immune deficiency syndromes (1999) adolescent pre-exposure prophylaxis for hiv prevention: current perspectives advancing hiv biomedical prevention research for at-risk adolescents oral pre-exposure prophylaxis (prep) for hiv prevention in adolescents and young adults attitudes and perceptions of biomedical hiv prevention methods: voices from young men who have sex with men perceptions of and intentions to adopt hiv pre-exposure prophylaxis among black men who have sex with men in los angeles minimal awareness and stalled uptake of preexposure prophylaxis (prep) among at risk, hiv-negative, black men who have sex with men use of pre-exposure prophylaxis (prep) in young men who have sex with men is associated with race, sexual risk behavior and peer network size a young black msm on prep is lost to follow-up and acquires hiv infection: a case to call for improved strategies to support youth adherence and engagement in hiv prevention potential impact of hiv preexposure prophylaxis among black and white adolescent sexual minority males patientprovider communication barriers and facilitators to hiv and sti preventive services for adolescent msm disclosure of same-sex behaviors to healthcare providers and uptake of hiv testing for men who have sex with men: a systematic review the importance of sexual history taking for prep comprehension among young people of color lesbian, gay, bisexual, and transgender-related content in undergraduate medical education health care providers' comfort with and barriers to care of transgender youth a medical care missed opportunity: preexposure prophylaxis and young black men who have sex with men the impact of patient race on clinical decisions related to prescribing hiv pre-exposure prophylaxis (prep): assumptions about sexual risk compensation and implications for access stigma, medical mistrust, and perceived racism may affect prep awareness and uptake in black compared to white gay and bisexual men in jackson, mississippi and boston getting prepared for hiv prevention navigation: young black gay men talk about hiv prevention in the biomedical era stigma and conspiracy beliefs related to pre-exposure prophylaxis (prep) and interest in using prep among black and white men and transgender women who have sex with men challenges in translating prep interest into uptake in an observational study of young black msm experiences of pre-exposure prophylaxis (prep)-related stigma among black msm prep users in los angeles enhancing prep access for black and latino men who have sex with men prep awareness in the context of hiv/aids conspiracy beliefs among black/african american and hispanic/latino msm in three urban us cities safety and feasibility of antiretroviral preexposure prophylaxis for adolescent men who have sex with curr hiv/aids rep men aged 15 to 17 years in the united states minors' access to sti services, state laws and policies enhancing hiv prevention among young men who have sex with men: a systematic review of hiv behavioral interventions for young gay and bisexual men an hiv intervention tailored for black young men who have sex with men in the house ball community org: feasibility and acceptability of delivering an internet intervention to young black men who have sex with men centers for disease c, prevention. evidence-based hiv/std prevention intervention for black men who have sex with men efficacy of an hiv/sti prevention intervention for black men who have sex with men: findings from the manymen, manyvoices (3mv) project reduced sexual risk behaviors among young men of color who have sex with men: findings from the community-based organization behavioral outcomes of many men, many voices (cbop-3mv) project associations between neighborhood problems and sexual behaviors among black men who have sex with men in the deep south: the mari study randomized trial of the effects of housing assistance on the health and risk behaviors of homeless and unstably housed people living with hiv can money prevent the spread of hiv? a review of cash payments for hiv prevention efficacy of a savings-led microfinance intervention to reduce sexual risk for hiv among women engaged in sex work: a randomized clinical trial final recommendation statement: human immunodeficiency virus (hiv) infection: screening. us preventive services task force recommendations for hiv screening of gay, bisexual, and other men who have sex with men-united states bright futures: guidelines for health supervision of infants, children, and adolescents: am acad pediatrics male adolescent use of health care services: where are the boys? school-based hiv/std testing behaviors and motivations among black and hispanic teen msm: results from a formative evaluation hiv testing in nonhealthcare facilities among adolescent msm hiv testing among high school students-united states a call to action planning: local health department and school partnerships for adolescent sexual health hiv and adolescents: guidance for hiv testing and counselling and care for adolescents living with hiv: recommendations for a public health approach and considerations for policy-makers and managers the metropolitan atlanta community adolescent rapid testing initiative study: closing the gaps in hiv care among youth in atlanta hiv testing in nonhealthcare facilities among adolescent msm confidentiality matters but how do we improve implementation in adolescent sexual and reproductive health care? student voices: perspectives on peer-to-peer sexual health education low rates of human immunodeficiency virus testing among adolescent gay, bisexual, and queer men control cfd, prevention. hiv surveillance report the intersection of sociocultural factors and health-related behavior in lesbian, gay, bisexual, and transgender youth: experiences among young black gay males as an example sexual and reproductive health care services in the pediatric setting office-based care for lesbian, gay, bisexual, transgender, and questioning youth ensuring comprehensive care and support for transgender and gender-diverse children and adolescents recommendations for promoting the health and well-being of lesbian, gay, bisexual, and transgender adolescents: a position paper of the society for adolescent health and medicine adolescent lives matter: preventing hiv in adolescents identifying community-informed language to promote hiv pre-exposure prophylaxis (prep) in black lgbtq communities in baltimore brothers building brothers by breaking barriers: development of a resilience-building social capital intervention for young black gay and bisexual men living with hiv what's prep?: peer navigator acceptability among minority msm in washington hiv prevention among diverse young msm: research needs, priorities, and opportunities i am men's health: generating adherence to hiv pre-exposure prophylaxis (prep) in young men of color who have sex with men getting information about hiv prevention: a pilot study i always felt i had to prove my manhood": homosexuality, masculinity, gender role strain, and hiv risk among young black men who have sex with men there's an app for that: using geosocial networking apps to access young black gay, bisexual, and other msm at risk for hiv technology use and preferences for mobile phone-based hiv prevention and treatment among black young men who have sex with men: exploratory research viewpoint: why you should provide hiv pre-exposure prophylaxis (prep) at your college health center hiv pre-exposure prophylaxis medication for adolescents and young adults: a position paper of the society for adolescent health and medicine advancing independent adolescent consent for participation in hiv prevention research minors' and young adults' experiences of the research consent process in a phase ii safety study of preexposure prophylaxis for hiv self-consent for hiv prevention research involving sexual and gender minority youth: reducing barriers through evidence-based ethics the influence of age, health literacy, and affluence on adolescents' capacity to consent to research free testing and prep without outing myself to parents:" motivation to participate in oral and injectable prep clinical trials among adolescent men who have sex with men i won't out myself just to do a survey": sexual and gender minority adolescents' perspectives on the risks and benefits of sex research parent perspectives about sexual minority adolescent participation in research and requirements of parental permission methodological considerations for advancing research on the health and wellbeing of sexual and gender minority youth a multicomponent approach to evaluating a pre-exposure prophylaxis (prep) implementation program in five agencies in new york sexual networks of racially diverse young msm differ in racial homophily but not concurrency individual and network factors associated with racial disparities in hiv among young men who have sex with men: results from the radar cohort study explaining disparities in hiv infection among black and white men who have sex with men: a meta-analysis of hiv risk behaviors the effect of high rates of bacterial sexually transmitted infections on hiv incidence in a cohort of black and white men who have sex with men in the high risk of an hiv diagnosis following a diagnosis of syphilis: a population-level analysis of new york city men human immunodeficiency virus diagnosis after a syphilis, gonorrhea, or repeat diagnosis among males including non-men who have sex with men: what is the incidence? rising rates of hiv infection among young us men who have sex with men a data-driven simulation of hiv spread among young men who have sex with men: the role of age and race mixing, and stis rectal gonorrhea and chlamydia reinfection is associated with increased risk of hiv seroconversion preexposure prophylaxis for the prevention of hiv infection in the united states-2017 update: a clinical practice guideline the associations of resilience and hiv risk behaviors among black gay, bisexual, other men who have sex with men (msm) in the deep south: the mari study the impact of racism on child and adolescent health racial and sexual identities as potential buffers to risky sexual behavior for black gay and bisexual emerging adult men the relationship between gender role conflict and condom use among black msm key: cord-260422-z22t57ju authors: godet, julien; boudier, christian; humbert, nicolas; ivanyi-nagy, roland; darlix, jean-luc; mély, yves title: comparative nucleic acid chaperone properties of the nucleocapsid protein ncp7 and tat protein of hiv-1 date: 2012-06-26 journal: virus res doi: 10.1016/j.virusres.2012.06.021 sha: doc_id: 260422 cord_uid: z22t57ju rna chaperones are proteins able to rearrange nucleic acid structures towards their most stable conformations. in retroviruses, the reverse transcription of the viral rna requires multiple and complex nucleic acid rearrangements that need to be chaperoned. hiv-1 has evolved different viral-encoded proteins with chaperone activity, notably tat and the well described nucleocapsid protein ncp7. we propose here an overview of the recent reports that examine and compare the nucleic acid chaperone properties of tat and ncp7 during reverse transcription to illustrate the variety of mechanisms of action of the nucleic acid chaperone proteins. rna chaperones are proteins that interact with rna molecules to solve the rna folding problem (herschlag, 1995; cristofari and darlix, 2002; schroeder et al., 2004) . rna molecules are synthesized as linear polymers that require appropriate folding to reach their active (native) conformations. along the folding trajectory, rna navigates through a rugged funnel-like landscape biased toward few native structures (fontana et al., 1993; chen and dill, 2000; russell et al., 2002) . intermediate conformations with stabilities close to the native functional states usually coexist such that a fraction of the molecules rapidly folds into their native structures while many others are kinetically trapped in misfolded intermediate conformations ( fig. 1) (thirumalai et al., 1997) . rna molecules are thus prone to adopt stable and persistent alternative secondary structures which have to overpass thermodynamic barriers to correctly fold (treiber and williamson, 1999) . this is why assistance appears to be necessary to reach the active rna conformations. rna chaperones were thus postulated to direct the correct folding of rna molecules and to resolve rna misfoldings (herschlag, 1995) . today's view is that rna chaperones are nucleic acid binding proteins present in all living organisms, including viruses, where they abbreviations: hiv-1, human immunodeficiency virus-type i; ncp7, nucleocapsid protein of hiv-1; zf, zinc finger; na, nucleic acid; tar, transactivation response element; pbs, primer binding site; aa, amino acids. * corresponding author. tel.: +33 03 90 24 42 63; fax: +33 03 90 24 43 12. e-mail address: yves.mely@unistra.fr (y. mély). perform multiple functions (cristofari and darlix, 2002; schroeder et al., 2004) . questioning the structure-function paradigm (wright and dyson, 1999; dyson and wright, 2005) , it was found that the prevalence of functional regions in rna chaperones without a well defined 3d-structure was very high and such intrinsically disordered domains (idd) were proposed to support the chaperone activity (tompa and csermely, 2004; tompa, 2005) . since rna chaperones do not share common sequences, motifs or structures, their identification is hardly predictable, and thus requires adequate assays to establish their chaperoning activity. several in vitro assays are routinely used (cristofari and darlix, 2002; rajkowitsch et al., 2005) and include annealing, helix destabilization, strand displacement (cristofari and darlix, 2002; rajkowitsch et al., 2005 rajkowitsch et al., , 2007 schroeder, 2007a, 2007b; godet and mély, 2010) , cis or trans-splicing (coetzee et al., 1994; zhang et al., 1995; mayer et al., 2002; semrad et al., 2004; belisova et al., 2005; grohman et al., 2007) and hammerhead-ribozyme rna cleavage (bertrand and rossi, 1994; herschlag, 1995) . fewer in vivo assays also exist, such as intron folding trap or transcription antitermination (clodi et al., 1999; lorsch, 2002; rajkowitsch et al., 2005) . the latter tests investigate simultaneously several if not all the components that account for the chaperoning activity. in vitro studies show that rna chaperones function by repetitive transient interactions with rna (cruceanu et al., 2006a; wu et al., 2010; doetsch et al., 2011b) . this property results in transient destabilization of the metastable na conformations and in accelerating the annealing of complementary sequences. the fast binding kinetics is thus likely to be a critical component of the chaperoning activity. monitoring the fast association and dissociation rates of scheme illustrating the rna energy landscape. rna energy landscape is rugged with many local minima that correspond to suboptimal kinetic trap foldings (b and c). rna-chaperones smoothen the landscape by destabilizing metastable conformations and lowering the activation energy between the different conformations, thus directing the folding of rna towards the native conformation. chaperones from na substrates is a challenging issue but none of the currently used assays can yet provide direct measurements of the on-and off-binding rates. indirect evidence of this property has been nevertheless gained from single molecule stretching experiments investigating the chaperone properties of hiv-1 gag and nc (williams et al., 2001; cruceanu et al., 2006a) , and the nucleocapsid protein of the yeast ty3 retrotransposon (chaurasiya et al., 2012) . at the same time, rna chaperones promote molecular aggregation, also known as molecular crowding. rna-chaperones usually present basic domains, ensuring that a significant part of the binding is provided by nonspecific electrostatic interactions. rna chaperones can bind to highly diverse nucleic acid (na) sequences and by so doing can coat na molecules, which is required for function. this has been termed the window of rna chaperone activity (reviewed in darlix et al., 2011) . this binding mode causes the formation of high molecular weight nucleoprotein complexes, where fuzziness appears to be a major trait in addition to molecular crowding (fuxreiter and tompa, 2012; ivanyi-nagy and darlix, 2012) . fast binding kinetics, molecular crowding and fuzziness cause a high local concentration of the partners and a smoother energy landscape resulting in a large increase in the rna folding rate (woodson, 2010) (fig. 1) . multiple functions have been assigned to cellular rnachaperones, such as transcription regulation, rnp assembly, pre-mrna processing, rna nuclear export and translation, and in mirna metabolism. rna-chaperones are also present in the viral world, where they have major roles in virus structure and replication (zúñiga et al., 2009 ). the first reported viral rna-chaperones are the nucleocapsid (nc) proteins of avian and murine retroviruses (prats et al., 1988) . later, a similar chaperone activity was shown for the hiv-1 nc protein (darlix et al., 1990; de rocquigny et al., 1992; barat et al., 1993; dib-hajj et al., 1993; lapadat-tapolsky et al., 1993) . the intense research efforts on hiv/aids benefited to the characterization of chaperones since a large part of our current understanding was gained during the past 20 years from studies on hiv-1 nc protein. other virus-encoded rna chaperones have since been characterized, notably the nucleoprotein n of coronavirus (zúñiga et al., 2007) , the 3ab protein of poliovirus (destefano and titilope, 2006) , the core protein of flaviviridae (cristofari et al., 2004; ivanyi-nagy et al., 2006 sharma et al., 2011) , the nucleoprotein n of hantavirus panganiban, 2005, 2006) or the small delta antigen sdag of hepatitis d virus (huang and wu, 1998; huang et al., 2003 huang et al., , 2004 wang et al., 2003) (for a review see zúñiga et al., 2009) . moreover, viruses are capable of highjacking cellular factors with rna chaperoning activity in the course of viral rna synthesis and translation (zúñiga et al., 2009) . all these examples highlight the variability of chaperone proteins selected through evolution to solve the folding problem and to assist the large nucleic acid rearrangements occurring throughout viral life cycles. here, we review the mode of action of hiv-1 ncp7 in parallel with that of the trans-activator of transcription tat, another potent nucleic acid chaperone encoded by hiv (kuciak et al., 2008; boudier et al., 2010) to illustrate the variety of the mechanisms of action of the nucleic acid chaperone proteins. hiv-1 ncp7 is a small (55 amino acids) basic protein resulting from the protease-mediated cleavage of the pr55gag polyprotein precursor. ncp7 is characterized by two conserved 'cx 2 cx 4 hx 4 c' zinc fingers (zfs) ( fig. 2a) , flanked by small domains rich in basic residues. interestingly the basic zf linker and the n-and c-terminal domains appear to be disordered indicating that ncp7 belongs to the idp family . at the same time, a hydrophobic plateau can form at the top of the zfs in the native protein (morellet et al., 1992 mély et al., 1994) , which is composed of residues val13, phe16, thr24 and ala25 of the proximal zf and residues trp37, gln45 and met46 of the distal zf. this zf plateau plays a key role in the selection of the viral sequences during genomic rna packaging and contributes to ncp7 chaperoning properties (godet and mély, 2010; darlix et al., 2011) . ncp7 can cause nucleic acid destabilization (bernacchi et al., 2002; azoulay et al., 2003; beltz et al., 2003; cosa et al., 2004 cosa et al., , 2006 and can transiently interact with nucleic acids with fast binding and dissociation kinetics (cruceanu et al., 2006a) . ncp7 also activates the annealing of complementary sequences you and mchenry, 1994) and the cleavage of a cognate substrate by a hammerhead ribozyme (bertrand and rossi, 1994; kuciak et al., 2008) , and efficiently rescues the splicing of a group i intron mutant in the t4 phage td gene in vivo (clodi et al., 1999) . the destabilizing activity of ncp7 mainly relies on the zfs (beltz et al., 2005) while the nucleic acid annealing and condensation properties are largely dependent on the basic residues of the disordered regions ( fig. 2a ) stoylov et al., 1997; williams et al., 2001) . the importance of the various ncp7 amino acids can be easily seen on the sequence logo plot resulting from the alignment of 3120 ncp7 hiv sequences of the lanl hiv database (http://www.hiv.lanl.gov/). fig. 2b shows that the information content associated to the amino acids composing the two zfs and the linker are close to their theoretical maximum (log 2 (20) ≈ 4.32 bits). in contrast, the n-terminal domain is less conserved although the positions of the positively charged amino acids are essentially conserved, suggesting that a precise distribution of basic amino acids in the n-terminal and basic linker domains is required for nc function doetsch et al., 2011a) . the gag precursor also exhibits nucleic acid chaperone activity (rein, 2010; wu et al., 2010) . the comparative chaperone activities of recombinant gag (or more precisely gag p6 ) and of the differently processed cleavage products have been investigated. gag can bind nucleic acids with higher affinity than ncp7, while in contrast, the nucleic acid chaperone activity improves greatly as the gag precursor is progressively processed to give ncp15, ncp9 and ncp7 (cruceanu et al., 2006b; rein, 2010; wu et al., 2010) . modification of the chaperone activity of nc during the viral cycle likely accounts for the modifications in the virus structure and function consecutive to the gag cleavage events during maturation (mirambeau et al., 2010) . in particular, critical architectural changes of the viral core result from the nucleoproteic complex condensation as gag is progressively processed (mirambeau et al., 2007) . the progressive decrease of binding affinity as gag is processed is likely ascribed to the loss of the specific high affinity binding mode required for selective vrna encapsidation to favour a less specific and transient binding mode mandatory to facilitate the nucleic acid rearrangements during reverse transcription. the mature ncp7 is characterized by highly dynamic binding properties (cruceanu et al., 2006a) . effective strand annealing activity is notably correlated with nc's ability to rapidly bind and dissociate from nucleic acids. indeed, nc variants with slow on/off rates are poorly efficient in rearranging nucleic acids, even though they are still capable of binding with high affinity to nucleic acids and to aggregate nucleic acids (cruceanu et al., 2006b; stewart-maynard et al., 2008) . in the inner core of the mature viral particle, approximately 1500-2000 copies of ncp7 are coating the genomic rna (briggs et al., 2004; chertova et al., 2006; chen et al., 2009) , thus corresponding to an average occupancy of 5-7 nt per ncp7 molecule. such an occupancy value was found to be required for optimal nc chaperoning activities in vitro (reviewed in darlix et al., 2011) , notably in the annealing reactions taking place during viral dna synthesis by rt. during reverse transcription which occurs in this viral nucleoprotein assemblage called the reverse transcription complex (rtc), ncp7 is thought to direct most of the critical nucleic acid rearrangements, namely primer trnalys,3 annealing to the viral pbs (barat et al., 1989 (barat et al., , 1993 de rocquigny et al., 1992; hargittai et al., 2004) , the first and second dna strand transfers (you and mchenry, 1994; lapadat-tapolsky et al., 1995; auxilien et al., 1999; ramalanjaona et al., 2007; godet et al., 2011) and to assist the formation of the central dna flap (charneau et al., 1994; hameau et al., 2001) . in addition, nc appears to assist the viral dna polymerase activity of rt throughout viral dna synthesis, notably by relieving rt pausing at the initiation step corresponding to ss-cdna synthesis (liang et al., 1998; rong et al., 2001; bampi et al., 2004; liu et al., 2010) , by increasing the polymerization rate and stimulating the rt-rnase h activity (bampi et al., 2006; grohmann et al., 2008) , and by promoting the fidelity of plus-strand priming (jacob and destefano, 2008; post et al., 2009 ) as well as that of (−) and (+) strand dna synthesis (kim et al., 2012) . thus, the overall efficiency of reverse transcription is increased by ncp7 which by forming a condensed but highly mobile ribonucleoproteic complex increases the molecular crowding of the nucleic acids generated during reverse transcription and thus, facilitate their annealing tanchou et al., 1995) . however, direct demonstration of these nucleic acid rearrangements in the viral context is still missing. tat is a small basic protein that has multiple key roles in virus replication and pathogenesis (karn, 1999) . tat is encoded for by two exons and is composed of 101 (clinical isolates) or 86 amino acids (laboratory isolates). the full-length tat is composed of six different regions which composition and functions are briefly described below (fig. 3) . region i (aa 1-20) is acidic and proline rich and is involved in tat-mediated immune suppression (wrenger et al., 1997) . region ii (aa 21-40) contains seven conserved cys residues, where all of them but cys31 are required for transcription transactivation (kuppuswamy et al., 1989; jeang et al., 1999) . these cys residues interact with two zn 2+ ions (frankel et al., 1988a (frankel et al., , 1988b , conferring to tat the property to trigger apoptosis (egelé et al., 2008) . region iii (aa 41-48) represents the highly conserved core that is critical for tat binding to tubulin (chen et al., 2002) , which triggers the mitochondrial pathway of apoptosis and neuronal cytoskeletal changes leading to aids-associated dementia (chen et al., 2002; giacca, 2005) . region iv (aa 49-64) is arg-rich and mediates the binding of tat to the 5 tar sequence (kuppuswamy et al., 1989; betti et al., 2001) . this region also contains the tat nuclear localization signal (vivès et al., 1997) . the c-terminal region contains a glutamine-rich domain (aa 57-72) that contributes to the transactivating activity of tat (kuppuswamy et al., 1989) . this region is also involved in induction of apoptosis in t-lymphocytes upon binding to tubulin (campbell et al., 2004) . finally, region vi (aa 73 to 86 or 101) can interact with the integrin-mediated sites of cellular adhesion and with integrin and fibronectin receptors. it is also involved in the cell penetration properties of tat (barillari et al., 1993) . tat binding to tar rna (trans acting responsive element) activates viral dna transcription initiation and elongation from the 5 ltr promoter (laspia et al., 1989; feinberg et al., 1991) . the tat/tar nucleoprotein complex promotes the recruitment of a series of transcription factors leading to the formation of very active elongating transcription machinery (chun and jeang, 1996; okamoto et al., 1996) . beyond its role in viral dna transcription, tat/rna interactions were reported to influence viral mrna capping and splicing, and translation (chiu et al., 2002; berro et al., 2006; charnay et al., 2009) . moreover, tat could possibly act as a rna silencing suppressor via interactions with dicer, trbp, sirna and mrna (bennasser et al., 2005; bennasser and jeang, 2006) . tat belongs to the idp's family (shojania and o'neil, 2006) . conformational disorder and flexibility may confer to tat its ability to interact with numerous viral and cellular partners (dunker et al., 2005; dosztányi et al., 2006) . together with its nucleic acid binding properties, the disordered nature of tat suggests that this protein is also a nucleic acid chaperone (kuciak et al., 2008) . tat and several tat-derived peptides were found to efficiently activate dna annealing, ribozyme-mediated rna cleavage and rna transsplicing in vitro (kuciak et al., 2008) . tat was also found to induce the displacement of an imperfect dna strand by a perfectly complementary sequence in a dna exchange assay (kuciak et al., 2008) , while no strand-displacement activity was found in assays using complementary rna sequences (doetsch et al., 2011a) , suggesting a nucleation-limited strand exchange activity. the inability of tat to exchange rna strands is likely explained by the limited ability of tat to destabilize rna structures. therefore, tat likely promotes nonspecific nucleic acid annealing reactions when destabilization is not or poorly required. amino acids responsible for the chaperone activity spanned from residues 44 to 61. tat(44-61) was found to be the shortest known sequence with nucleic acid chaperoning activity. interestingly, a panel of alanine-scanning mutations from amino acids 45 to 54 evidenced a striking correlation between the conservation of these amino acids (fig. 3 ) and the positions of the mutations that prevent virus to initiate the natural endogenous reverse transcription (nert) in a cell-free virus supernatant assay (apolloni et al., 2003) . the selection pressure on the central stretch of basic amino acids may thus be related to its implication in reverse transcription. the substantial nucleic acid chaperone properties exhibited by tat may account for its ability to promote the annealing of the primer trna to the viral rna (kameoka et al., 2002) and intervene in the first strand transfer (boudier et al., 2010) and by this way, to stimulate rtion as does ncp7 (harrich et al., 1997; ulich et al., 1999; apolloni et al., 2007) . during reverse transcription, major and complex nucleic acid rearrangements are required to allow the full-length genomic dsdna, also called vdna, to form. all along these steps, the nucleic acid chaperone activity of ncp7 was evidenced to assist and to facilitate the synthesis of the vdna. tat was also found to promote most of these steps in vitro. in the following sections, we will briefly outline the chaperone properties of tat and ncp7 regarding their mechanism to chaperone in vitro the rearrangements of the nucleic acid sequences involved in critical steps of reverse transcription. a. during initiation, the trna lys,3 is placed on the vrna and serves as a primer to be further elongated by rt. b. during the first strand transfer, the minus-strand strong-stop dna is translocated to the 3 end of the viral rna genome, in a reaction mediated by base-pairing of the repeat r sequences at the 3 ends of the rna (containing tar) and cdna (containing ctar) reactants, to allow reverse transcription to resume. c. the second strand transfer involves (1) the synthesis of plus strand strong-stop dna that terminates after copying the 3 end of the trna sequence; (2) trna lys,3 primer removal and (3) the annealing of (−)pbs to the (+)pbs sequence. the annealing of the two complementary pbs dna stem-loops enables rt to complete viral dna synthesis. at the early events of vdna synthesis, the reverse transcriptase (rt) elongates a primer trna annealed to the viral primer-binding site (pbs), a 18-nt conserved sequence in the 5 leader region of the genomic rna, to later synthesize the minus-strand strong-stop cdna (minus ss-cdna) (fig. 4a) . these steps are facilitated by different viral chaperones. 3.1.1. positioning the replication primer trna onto genomic rna during assembly, cellular trna lys isoacceptors are selectively incorporated into virions (jiang et al., 1993; mak and kleiman, 1997; pavon-eternod et al., 2010) . the chaperone properties of the nc domain in pr55gag are thought to facilitate the specific placement of the trna lys,3 primer on the primer binding site (pbs) of the viral rna (cen et al., 1999; feng et al., 1999; cruceanu et al., 2006b; guo et al., 2009; wu et al., 2010) . in vitro, ncp7 directs the annealing of the trna lys,3 primer to the pbs (li et al., 1996) , by facilitating the strand exchange at the level of the trna acceptor stem and by unlocking in the presence of the complementary genomic rna sequence, the highly stable interactions at the level of the t c loop (chan and musier-forsyth, 1997; tisné et al., 2003 tisné et al., , 2004 hargittai et al., 2004; tisné, 2005; barraud et al., 2007) . the kinetics of the trna lys,3 annealing on the pbs sequence follow a nucleation-limited bimolecular reaction. the reaction is enhanced by five orders of magnitude by ncp7, largely due to its ability to strongly promote duplex nucleation (hargittai et al., 2004) . the nc zfs specifically interact with the t c loop. although nc zfs do not promote unwinding of trna lys3 , the truncated form of ncp7 was found to destabilize two base pairs that could serve as nucleation points for the annealing of trnalys3 to the viral rna (tisné et al., 2001) . contrary to nc zfs, a sshs mutant of nc, which lacks the folded zfs, poorly destabilizes the trna tertiary core (hargittai et al., 2001) . it was nevertheless able to anneal more efficiently than ncp7 the trna lys,3 primer onto the pbs (hargittai et al., 2004) . mutants with complete zf deletions are also able to efficiently anneal trna lys,3 primer to the pbs, provided that the basic n-or cterminal domains are present . these zf mutants suggest that the destabilization of the trna core does not appear to be critical in vitro and that multivalent cationic peptides might be sufficient for efficient trna primer annealing to the pbs. this conclusion is also supported by the minimal alterations of the annealing kinetics induced by mutations which alter the secondary or tertiary structure or the stability of the trna (hargittai et al., 2004) and by the greater trna-annealing activity of an n-terminal extended form of ncp7 protein in vitro (roldan et al., 2005) . though the zfs appear dispensable in vitro, it cannot be excluded that as for the second strand transfer (see below), the zfs can induce a specific reaction pathway that is critical for a timely and controlled trna lys,3 /pbs annealing reaction. in full line with its ability to facilitate rna annealing, tat was also reported to increase the efficiency of primer trna placement onto genomic rna. in vitro, tat could even replace ncp7 at this step (kameoka et al., 2002) in a largely electrostatically driven trna annealing promotion. this was confirmed by mutational analysis showing that deletion of the basic domains of tat resulted in the loss of the annealing property (kameoka et al., 2002) . nonetheless, initiation of reverse transcription from tat-annealed trna lys,3 occurred less efficiently than from heat-annealed trna lys,3 (kameoka et al., 2002) . specific formation of the initiation complex is mediated by extended interactions between the hiv-1 rna and trna lys,3 (goldschmidt et al., 2002) . although variable among hiv strains (goldschmidt et al., 2004) , these extended interactions (isel et al., 1996 (isel et al., , 1998 (isel et al., , 1999 (isel et al., , 2010 li et al., 1996; beerens and berkhout, 2002; huthoff et al., 2003) are decisive for the efficiency of the initiation of reverse transcription. formation of the initiation complex requires rearrangements in the 5 utr vrna that may be promoted by the fully processed nc (iwatani et al., 2003; guo et al., 2009) . indeed, trna lys,3 annealed by gag exhibits a strongly reduced ability to initiate reverse transcription and binds less tightly to viral rna than the ncp7-annealed trna lys,3 . this necessary chaperoning of reverse transcription priming thus appears as a key regulatory step which can only be catalysed when a significant amount of ncp7 has been processed. once the primer is placed, the subsequent synthesis of vdna can start. the primer is initially extended by 6 nt in a slow and distributive initiation phase where the dna polymerization is characterized by a rapid dissociation of rt and early kinetic pausing events at positions +3 or +5 nt (isel et al., 1996; lanchy et al., 1996 lanchy et al., , 1998 . ncp7 reduces rt pauses at these positions, although with different efficiencies. interestingly, an additional pause at +1 nt was observed in vitro when the trna was heat-annealed on pbs but not when the annealing reaction was performed using ncp7. in contrast, the pause at +1 position was not affected with zf mutants of nc, suggesting again that all the chaperone properties of the native ncp7 protein are important for promoting an active trnalys,3/vrna initiation complex . on the contrary to the +1 nt pause, nc was not able to help rt to escape the +3 pausing event (liang et al., 1998; rong et al., 2001; liu et al., 2010) . the strong +3 nt pause has been proposed to originate from the template structure and notably from the folding of the a-rich stem-loop located upstream the pbs (liang et al., 1998; liu et al., 2010) . ncp7 induced a partial and transient disruption of the stem secondary structure, as evidenced by a decrease of the high-throughput shape footprinting reactivity and a broadening of the stem end-to-end fret distribution (wilkinson et al., 2008; liu et al., 2010) , but this destabilization appeared nevertheless not sufficient to prevent the +3 nt pause event. in sharp contrast to ncp7, multimerization of gag or gag-related proteins dramatically compromises reverse transcription since the cooperative binding and the slow dissociation rate of the multimerized gag proteins impaired rt processivity (wu et al., 2010) . similarly, full-length two-exon tat (86-or 101-amino acid) but not the truncated oneexon tat (72 amino acid) was found to suppress the elongation of the trna lys,3 /vrna initiation complex (kameoka et al., 2001 (kameoka et al., , 2002 . interestingly, peptides resulting from the cleavage of tat by the hiv-1 protease were able to enhance the synthesis of the (−)ssdna in a natural endogenous reverse transcription assay (apolloni et al., 2003) . thus, as envisioned for gag, the protease cleavage of tat is also needed for promoting primer elongation in vitro. tat was therefore hypothesized as a reverse transcription accessory factor involved in the spatio-temporal control of the reverse transcription. whether or not these findings are biologically relevant, they clearly underline the need for small basic proteins which can bind transiently and remain mobile on the nucleic acid lattice to allow primer extension. taken together, these observations suggest that if trna positioning can be promoted by different partners, primer extension requires a structurally well-defined initiation complex and a dynamic reverse transcription complex, which are likely promoted by the highly dynamic ncp7 (cruceanu et al., 2006a) . initiation of reverse transcription could thus constitute a key regulatory step of which timing is finely regulated by the processing of the gag precursor. this hypothesis is ascertained by observations showing that most of the annealed trna lys,3 in immature extracellular particles are only extended by a few nucleotides (oude essink et al., 1996; huang et al., 1997) . the first strand transfer constitutes a critical step in reverse transcription. this transfer occurs from region r at the 5 -end of the genome to a redundant r region at the 3 -end (fig. 4b) . in hiv-1, the r region consists of two strongly structured hairpins, namely the tar and poly(a) hairpins (baudin et al., 1993; watts et al., 2009) . tar is especially critical for efficient strand transfer (berkhout et al., 2001) . indeed, the antisense ctar dna (further referred as ctar) of the (−)ssdna has to anneal to the complementary tar rna sequence located at the 3 end of the vrna to allow dna synthesis to resume on the acceptor strand (negroni and buc, 2000; basu et al., 2008) . ctar and tar are imperfect stem-loops defined by double-stranded segments separated by numerous conserved bulges, mismatches and an internal loop (baudin et al., 1993) . reacting tar and ctar in vitro does spontaneously lead to the ctar/tar duplex, but at an extremely slow rate (you and mchenry, 1994; godet et al., 2006; vo et al., 2006 vo et al., , 2009 . through its chaperone activity, ncp7 plays a major role in promoting the annealing reaction (tsuchihashi and brown, 1994; you and mchenry, 1994; rein et al., 1998; guo et al., 2000) by enhancing the annealing rate by about 3000-fold at physiological temperature and salt conditions you and mchenry, 1994; driscoll and hughes, 2000; urbaneja et al., 2002) . the stability of tar and ctar local structures appeared of key importance to modulate the strand transfer (wu et al., 2007) . in full line with its ability to bind more transiently and dynamically to nucleic acids than gag, the fully processed ncp7 promotes the first strand transfer more efficiently than gag or gag-derived proteins at low protein concentrations (wu et al., 2010) . whereas the annealing reaction was facilitated as the ncp7 concentration increased, the strand transfer was drastically inhibited in the presence of increasing quantities of gag or gag-derived proteins. as gag and partially processed gag proteins containing the nc domain showed destabilizing and annealing activities almost as effective as ncp7, the inhibition of the strand transfer likely results from a strong restriction of the elongation step, suggesting that gag reduces the ability of rt to traverse the template ('roadblock' mechanism). once again, the ability of ncp7 to remain highly flexible and mobile within the nucleoproteic reverse transcription complex appears critical for reverse transcription to proceed (levin et al., 2005) . detailed mechanistic investigations of the ctar/tar annealing reaction showed that ctar and tar anneal in the absence of ncp7 through formation of an unstable loop-loop interaction that further converts into an extended duplex (vo et al., 2006 (vo et al., , 2009 kanevsky et al., 2011) . ncp7 switches the reaction pathway by directing the hybridization of these sequences through the end of their doublestranded stems, as a consequence of ncp7 ability to destabilize the structure of the ctar stem (godet et al., 2006; vo et al., 2009) . the destabilizing activity of ncp7 induces complex secondary structure fluctuations of the ctar ends cosa et al., 2004 cosa et al., , 2006 , leading to the formation of the open reactive species required for the annealing process. the ncp7-induced mechanistic switch shows that the reaction pathway is selected on the basis of the available reactive intermediates and is governed by the intermediates which require the least bp melting prior annealing (vo et al., 2009) . these observations constitute a nice example of how nucleic acid chaperones may remodel annealing reactions to facilitate the formation of the most stable nucleic acid conformations. the mechanism by which tat and tat-derived peptides activate the annealing of the complementary tar sequences (in the form of dna) was also thoroughly investigated (kuciak et al., 2008; boudier et al., 2010) . like ncp7 or gag, tat(1-86) promotes the hybridization of ctar to tar dna. tat peptides corresponding to the n-terminal acidic domain and to the cys-rich domain are poorly active in annealing. on the contrary, peptides tat(44-61) and tat(48-86) promote tar dna/ctar annealing, tat(44-61) being the most active of the two. this suggests that the basic rna binding domain of tat is critical to promote the annealing reaction. unlike ncp7, neither tat nor tat(44-61) are able to destabilize ctar. tat(44-61) is however able to modify the annealing mechanism of ctar with tar (in a dna/dna annealing context at least) since the analysis of ctar and tar dna mutants clearly evidenced that the reaction is initiated at the bottom of the two reacting species (boudier et al., 2010) . in addition, tat(1-86) and the mature nc were found to show comparable efficiency in promoting the annealing of ctar with the dna equivalent of tar. taken together, data on tat indicates that in the ctar/tar dna reaction, the stimulatory effect of tat on the first strand transfer resembles that of ncp7, though the hybridization reaction is differently nucleated. furthermore, tat and ncp7 were shown to cooperatively activate the annealing of ctar to tar dna, supporting a potent accessory role of tat in the stimulation of the reverse transcription. a second strand transfer reaction is required for reverse transcription to resume. the plus strand transfer involves a sequence of synchronized events (fig. 4c ) consisting in (i) the synthesis of plus strand strong-stop dna that terminates after copying the 3 end of the trna sequence, (ii) the necessary removal of the trna primer and (iii) the subsequent annealing of the minus (−) and plus (+) dna copies at the level of the 18 nt primer binding site (pbs) sequence (basu et al., 2008) . ncp7 chaperoning assistance is involved in many of the steps above described but we will focus here only on the (+)/(−)pbs annealing reaction. the dna pbs sequence folds into a bulged stable 4-bp stem-loop hairpin with a partially ordered pentanucleotide loop and a 4-nt single-strand overhang . using fluorescently labelled (+)pbs with various (−)pbs mutants, the properties of ncp7 and tat on the (+)/(−)pbs hybridization were comparatively investigated. in vitro, (+)pbs can spontaneously anneal to (−)pbs (ramalanjaona et al., 2007) . the pbs(+)/(−) annealing reaction proceeds mainly through the single-strand overhangs of the pbs sequences while nucleation through loop-loop interaction appears negligible in the absence of peptides (fig. 5a) . this was ascribed from a pbs mutational analysis showing that hybridization rates of loop mutants were poorly affected while mutations that decreased the sequence complementarity in the ss overhangs almost completely impaired the reaction (fig. 5b) . in sharp contrast to the nucleic acid sequences involved in the first strand transfer, ncp7 does not melt the stable (−)pbs stem (egelé et al., 2004 (egelé et al., , 2005 . nevertheless, ncp7 strongly promotes the annealing of (+)/(−)pbs stem-loops by increasing the annealing rate by about 60-fold, mainly by accelerating the loop pathway. as a consequence, ncp7 modifies the mechanism of the (+)/(−)pbs annealing reaction by activating the loop-loop kissing pathway that is negligible without ncp7 (ramalanjaona et al., 2007) (fig. 5a and b) . the ability of ncp7 to switch the annealing reaction from the single strand overhang pathway to the loop-loop kissing pathway strongly correlates with the ability of ncp7 to rearrange the pbs loop and to restrict the dynamics of the pbs loop bases (bourbigot et al., 2008; godet et al., 2011) . the latter were investigated using 2-aminopurine, a structural fluorescent probe that minimally affects the folding of the pbs loop and its binding parameters with ncp7. in full line with nmr data (bourbigot et al., 2008) , comparison of the 2ap fluorescence quantum yields in the absence and in the presence of ncp7 clearly evidenced that ncp7 significantly rearranged the pbs loop, notably by decreasing base stacking. time-resolved fluorescence anisotropy also revealed that ncp7 restricts the picosecond to nanosecond dynamics of the pbs loops by limiting the overall flexibility of the loops and freezing the local mobility of the bases where ncp7 is bound. local structural rearrangement and freezing of the local base dynamics of the loop are strictly dependent on the integrity of the zinc finger hydrophobic platform and constitute general features of the destabilizing activity of ncp7 (avilov et al., 2008 (avilov et al., , 2009 godet et al., 2011) . thus, the destabilizing activity of ncp7 is directly responsible for the switch to the loop-loop annealing pathway. as for the first strand transfer, tat(44-61) is able to strongly promote the annealing reaction of (+)/(−)pbs. in the presence of only 2 tat(44-61) molecules per pbs, the annealing rate constant was found six times faster than the one observed for nc(11-55) under saturating concentrations (fig. 5a) to reach a rate close to that observed in the presence of the full-length ncp7. but in sharp contrast to ncp7, tat(44-61) was not able to stimulate the annealing pathway through the loop pathway ( fig. 5a and b) , likely due to its very limited ability to rearrange the pbs loops (unpublished data). tat(44-61) shows striking similarities with sshs 2 nc or sshs 2 nc(11-55), two nc mutants where the cysteines have been substituted by serines to prevent zinc binding and which consequently do not exhibit destabilizing activity. like tat, these two zinc finger mutants do not modify the odn dynamics and structure . thus, tat(44-61) stimulates the (−)/(+)pbs annealing through already available existing pathways in the absence of peptides (i.e. through the ss overhangs). taken together, these data show that tat is able to promote the annealing reaction of the complementary pbs sequences, albeit through a different mechanism from that observed in the presence of ncp7. the activities of tat-derived peptides and ncp7 in the different in vitro models reviewed here are macroscopically very similar. both tat peptides and ncp7 promote the annealing of different complementary sequences, as well as the placement of primer trna on the viral rna. nonetheless, comparison of the mechanism of action of tat and ncp7 evidences striking differences in their ability to chaperone nucleic acid rearrangements. the most important differences result from the inability of tat, unlike nc, to destabilize transiently nucleic acids and modify the local dynamics of the nucleobases. so, even if both proteins are able to aggregate nucleic acids and promote their annealing, the destabilizing activity of ncp7 mediated through its folded zinc fingers is responsible for specific nucleic acid rearrangements and annealing pathways. taken together, these data evidence that chaperoning mechanisms are multiple and if the rna-chaperone concept is relatively straightforward, its expression appears quite diverse. a direct consequence is that no simple or consensual assay exists for evidencing and characterizing the rna chaperone activity. moreover, characterization of the hiv-1 chaperone activities in vivo appears highly challenging due to the fact that the hiv-1 genome encodes for at least three proteins (ncp7, tat and vif) exhibiting redundant na chaperone activities, highlighting the critical necessity of chaperones for viral replication. due to the differential abundance of these proteins along the viral life cycle, it is likely that these proteins exhibit their na chaperoning activities at different steps of viral replication. therefore, spatio-temporal regulation (henriet et al., 2007) and possible cooperativity (boudier et al., 2010) within these different "chaperone solutions" selected to solve the "folding problem" represent very exiting fields to explore. the authors declare no conflict of interest human immunodeficiency virus type 1 protease regulation of tat activity is essential for efficient reverse transcription and replication the hiv-1 tat protein stimulates reverse transcription in vitro role of post-transcriptional modifications of primer trnalys,3 in the fidelity and efficacy of plus strand dna transfer during hiv-1 reverse transcription site-specific characterization of hiv-1 nucleocapsid protein binding to oligonucleotides with two binding sites probing dynamics of hiv-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine destabilization of the hiv-1 complementary sequence of tar by the nucleocapsid protein through activation of conformational fluctuations nucleotide excision repair and template-independent addition by hiv-1 reverse transcriptase in the presence of nucleocapsid protein the chaperoning and assistance roles of the hiv-1 nucleocapsid protein in proviral dna synthesis and maintenance hiv-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer trna analysis of the interactions of hiv1 replication primer trna(lys,3) with nucleocapsid protein and reverse transcriptase the tat protein of human immunodeficiency virus type 1, a growth factor for aids kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the rgd amino acid sequence new insights into the formation of hiv-1 reverse transcription initiation complex strand transfer events during hiv-1 reverse transcription functional sites in the 5 region of human immunodeficiency virus type 1 rna form defined structural domains the trna primer activation signal in the human immunodeficiency virus type 1 genome is important for initiation and processive elongation of reverse transcription rna chaperone activity of protein components of human ro rnps impact of the terminal bulges of hiv-1 ctar dna on its stability and the destabilizing activity of the nucleocapsid protein ncp7 structural determinants of hiv-1 nucleocapsid protein for ctar dna binding and destabilization, and correlation with inhibition of self-primed dna synthesis hiv-1 tat interaction with dicer: requirement for rna evidence that hiv-1 encodes an sirna and a suppressor of rna silencing structural features in the hiv-1 repeat region facilitate strand transfer during reverse transcription hiv-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of tar and its complementary sequence acetylated tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32 facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of hiv-1 and the heterogeneous nuclear ribonucleoprotein a1 characterization of hiv-1 tat proteins mutated in the transactivation domain for prophylactic and therapeutic application the mechanism of hiv-1 tat-directed nucleic acid annealing supports its role in reverse transcription how the hiv-1 nucleocapsid protein binds and destabilises the (−)primer binding site during reverse transcription the stoichiometry of gag protein in hiv-1 the glutamine-rich region of the hiv-1 tat protein is involved in t-cell apoptosis in vitro assembly properties of human immunodeficiency virus type 1 gag protein lacking the p6 domain the role of pr55(gag) in the annealing of trna3lys to human immunodeficiency virus type 1 genomic rna the nucleocapsid protein specifically anneals trnalys-3 onto a noncomplementary primer binding site within the hiv-1 rna genome in vitro mechanism of hiv-1 tat rna translation and its activation by the tat protein hiv-1 reverse transcription a termination step at the center of the genome a single zinc finger optimizes the dna interactions of the nucleocapsid protein of the yeast retrotransposon ty3 hiv-1 tat targets microtubules to induce apoptosis, a process promoted by the pro-apoptotic bcl-2 relative bim rna folding energy landscapes fluorescence fluctuation spectroscopy on viral-like particles reveals variable gag stoichiometry proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages tat stimulates cotranscriptional capping of hiv mrna requirements for rna polymerase ii carboxyl-terminal domain for activated transcription of human retroviruses human t-cell lymphotropic virus i and hiv-1 assaying rna chaperone activity in vivo using a novel rna folding trap escherichia coli proteins, including ribosomal protein s12, facilitate in vitro splicing of phage t4 introns by acting as rna chaperones secondary structure and secondary structure dynamics of dna hairpins complexed with hiv-1 nc protein evidence for non-two-state kinetics in the nucleocapsid protein chaperoned opening of dna hairpins the ubiquitous nature of rna chaperone proteins the hepatitis c virus core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand rna in vitro rapid kinetics of protein-nucleic acid interaction is a major component of hiv-1 nucleocapsid protein's nucleic acid chaperone function nucleic acid binding and chaperone properties of hiv-1 gag and nucleocapsid proteins cis elements and trans-acting factors involved in the rna dimerization of the human immunodeficiency virus hiv-1 flexible nature and specific functions of the hiv-1 nucleocapsid protein first glimpses at structure-function relationships of the nucleocapsid protein of retroviruses trans-activation of the 5 to 3 viral dna strand transfer by nucleocapsid protein during reverse transcription of hiv1 rna viral rna annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers poliovirus protein 3ab displays nucleic acid chaperone and helix-destabilizing activities retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity the rna annealing mechanism of the hiv-1 tat peptide: conversion of the rna into an annealing-competent conformation transient rna-protein interactions in rna folding disorder and sequence repeats in hub proteins and their implications for network evolution human immunodeficiency virus type 1 nucleocapsid protein can prevent self-priming of minus-strand strong stop dna by promoting the annealing of short oligonucleotides to hairpin sequences flexible nets. the roles of intrinsic disorder in protein interaction networks intrinsically unstructured proteins and their functions modulation of microtubule assembly by the hiv-1 tat protein is strongly dependent on zinc binding to tat investigation by fluorescence correlation spectroscopy of the chaperoning interactions of hiv-1 nucleocapsid protein with the viral dna initiation sequences hiv-1 nucleocapsid protein binds to the viral dna initiation sequences and chaperones their kissing interactions the role of tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation the human immunodeficiency virus type 1 gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic rna and placement of trna on the primer binding site rna folding and combinatory landscapes tat protein from human immunodeficiency virus forms a metal-linked dimer dimerization of the tat protein from human immunodeficiency virus: a cysteine-rich peptide mimics the normal metal-linked dimer interface fuzzy complexes: a more stochastic view of protein function hiv-1 tat, apoptosis and the mitochondria: a tubulin link? retrovirology 2 during the early phase of hiv-1 dna synthesis, nucleocapsid protein directs hybridization of the tar complementary sequences via the ends of their doublestranded stem biophysical studies of the nucleic acid chaperone properties of the hiv-1 nucleocapsid protein specific implications of the hiv-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer structural variability of the initiation complex of hiv-1 reverse transcription direct and indirect contributions of rna secondary structure elements to the initiation of hiv-1 reverse transcription probing the mechanisms of dead-box proteins as general rna chaperones: the c-terminal domain of cyt-19 mediates general recognition of rna hiv-1 nucleocapsid traps reverse transcriptase on nucleic acid substrates roles of gag and ncp7 in facilitating trna(lys)(3) annealing to viral rna in human immunodeficiency virus type 1 zinc finger structures in the human immunodeficiency virus type 1 nucleocapsid protein facilitate efficient minus-and plus-strand transfer human immunodeficiency virus type 1 central dna flap: dynamic terminal product of plus-strand displacement dna synthesis catalyzed by reverse transcriptase assisted by nucleocapsid protein hiv-1 nucleocapsid protein zinc finger structures induce trna(lys,3) structural changes but are not critical for primer/template annealing mechanistic insights into the kinetics of hiv-1 nucleocapsid protein-facilitated trna annealing to the primer binding site tat is required for efficient hiv-1 reverse transcription vif is a rna chaperone that could temporally regulate rna dimerization and the early steps of hiv-1 reverse transcription rna chaperones and the rna folding problem primer trna3lys on the viral genome exists in unextended and two-base extended forms within mature human immunodeficiency virus type 1 characterization and application of the selective strand annealing activity of the n terminal domain of hepatitis delta antigen selective strand annealing and selective strand exchange promoted by the n-terminal domain of hepatitis delta antigen identification and characterization of the rna chaperone activity of hepatitis delta antigen peptides on the importance of the primer activation signal for initiation of trna(lys3)-primed reverse transcription of the hiv-1 rna genome initiation of hiv reverse transcription mutational analysis of the trna3lys/hiv-1 rna (primer/template) complex specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer trna3lys structural basis for the specificity of the initiation of hiv-1 reverse transcription fuzziness in the core of the human pathogenic viruses hcv and hiv analysis of hepatitis c virus rna dimerization and core-rna interactions rna chaperoning and intrinsic disorder in the core proteins of flaviviridae efficient initiation of hiv-1 reverse transcription in vitro. requirement for rna sequences downstream of the primer binding site abrogated by nucleocapsid proteindependent primer-template interactions a new role for hiv nucleocapsid protein in modulating the specificity of plus strand priming multifaceted activities of the hiv-1 transactivator of transcription identification of trnas incorporated into wild-type and mutant human immunodeficiency virus type 1 a mechanism for plus-strand transfer enhancement by the hiv-1 nucleocapsid protein during reverse transcription the tat protein of human immunodeficiency virus type 1 (hiv-1) can promote placement of trna primer onto viral rna and suppress later dna polymerization in hiv-1 reverse transcription role for human immunodeficiency virus type 1 tat protein in suppression of viral reverse transcriptase activity during late stages of viral replication structural determinants of tar rna-dna annealing in the absence and presence of hiv-1 nucleocapsid protein tackling tat nucleocapsid protein annealing of a primer-template enhances (+)-strand dna synthesis and fidelity by hiv-1 reverse transcriptase the hiv-1 transcriptional activator tat has potent nucleic acid chaperoning activities in vitro multiple functional domains of tat, the trans-activator of hiv-1, defined by mutational analysis binding and kinetic properties of hiv-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription contacts between reverse transcriptase and the primer strand govern the transition from initiation to elongation of hiv-1 reverse transcription interactions between hiv-1 nucleocapsid protein and viral dna may have important functions in the viral life cycle analysis of the nucleic acid annealing activities of nucleocapsid protein from hiv-1 hiv-1 tat protein increases transcriptional initiation and stabilizes elongation nucleic acid chaperone activity of hiv-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism human immunodeficiency virus type 1 nucleocapsid protein (ncp7) directs specific initiation of minusstrand dna synthesis primed by human trna(lys3) in vitro: studies of viral rna molecules mutated in regions that flank the primer binding site mechanistic studies of early pausing events during initiation of hiv-1 reverse transcription initiation complex dynamics direct the transitions between distinct phases of early hiv reverse transcription rna chaperones exist and dead box proteins get a life primer trnas for reverse transcription folding of the td pre-rna with the help of the rna chaperone stpa spatial proximity of the hiv-1 nucleocapsid protein zinc fingers investigated by time-resolved fluorescence and fluorescence resonance energy transfer the hantavirus nucleocapsid protein recognizes specific features of the viral rna panhandle and is altered in conformation upon rna binding characterization of the rna chaperone activity of hantavirus nucleocapsid protein hiv-1 protease and reverse transcriptase control the architecture of their nucleocapsid partner features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function conformational behaviour of the active and inactive forms of the nucleocapsid ncp7 of hiv-1 studied by 1 h nmr determination of the structure of the nucleocapsid protein ncp7 from the human immunodeficiency virus type 1 by 1 h nmr copy-choice recombination by reverse transcriptases: reshuffling of genetic markers mediated by rna chaperones transactivation by human immunodeficiency virus tat protein requires the c-terminal domain of rna polymerase ii hiv-1 reverse transcriptase discriminates against non-self trna primers profiling non-lysyl trnas in hiv-1 fidelity of plus-strand priming requires the nucleic acid chaperone activity of hiv-1 nucleocapsid protein small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer trna onto genomic rna rna chaperones, rna annealers and rna helicases coupling rna annealing and strand displacement: a fret-based microplate reader assay for rna chaperone activity dissecting rna chaperone activity assays for the rna chaperone activity of proteins investigating the mechanism of the nucleocapsid protein chaperoning of the second strand transfer during hiv-1 dna synthesis nucleic acid chaperone activity of retroviral gag proteins nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: significance for viral replication a hiv-1 minimal gag protein is superior to nucleocapsid at in vitro annealing and exhibits multimerization-induced inhibition of reverse transcription hiv-1 nucleocapsid protein and the secondary structure of the binary complex formed between trna(lys.3) and viral rna template play different roles during initiation of (−) strand dna reverse transcription exploring the folding landscape of a structured rna strategies for rna folding and assembly rna chaperone activity of large ribosomal subunit proteins from escherichia coli analysis of the rna chaperoning activity of the hepatitis c virus core protein on the conserved 3 x region of the viral genome hiv-1 tat is a natively unfolded protein: the solution conformation and dynamics of reduced hiv-1 tat-(1-72) by nmr spectroscopy retroviral nucleocapsid proteins display nonequivalent levels of nucleic acid chaperone activity ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein formation of stable and functional hiv-1 nucleoprotein complexes in vitro kinetic partitioning mechanism as a unifying theme in the folding of biomolecules structural bases of the annealing of primer trna(3lys) to the hiv-1 viral rna heteronuclear nmr studies of the interaction of trna(lys)3 with hiv-1 nucleocapsid protein specific recognition of primer trna lys 3 by hiv-1 nucleocapsid protein: involvement of the zinc fingers and the n-terminal basic extension the annealing mechanism of hiv-1 reverse transcription primer onto the viral genome the interplay between structure and function in intrinsically unstructured proteins the role of structural disorder in the function of rna and protein chaperones exposing the kinetic traps in rna folding dna strand exchange and selective dna annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein functional domains of tat required for efficient human immunodeficiency virus type 1 reverse transcription hiv-1 nucleocapsid protein as a nucleic acid chaperone: spectroscopic study of its helix-destabilizing properties, structural binding specificity, and annealing activity a truncated hiv-1 tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus mechanistic studies of mini-tar rna/dna annealing in the absence and presence of hiv-1 nucleocapsid protein hiv-1 nucleocapsid protein switches the pathway of transactivation response element rna/dna annealing from loop-loop kissing to zipper nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen architecture and secondary structure of an entire hiv-1 rna genome high-throughput shape analysis reveals structures in hiv-1 genomic rna strongly conserved across distinct biological states mechanism for nucleic acid chaperone activity of hiv-1 nucleocapsid protein revealed by single molecule stretching taming free energy landscapes with rna chaperones the n-terminal structure of hiv-1 tat is required for suppression of cd26-dependent t cell growth intrinsically unstructured proteins: re-assessing the protein structure-function paradigm fundamental differences between the nucleic acid chaperone activities of hiv-1 nucleocapsid protein and gag or gag-derived proteins: biological implications effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of hiv-1 nucleocapsid protein human immunodeficiency virus nucleocapsid protein accelerates strand transfer of the terminally redundant sequences involved in reverse transcription escherichia coli protein stpa stimulates self-splicing by promoting rna assembly in vitro role of rna chaperones in virus replication coronavirus nucleocapsid protein is an rna chaperone this work was funded by agence nationale de la recherche (contract anr-2010-blan-1529-01) and agence nationale de recherche sur le sida (anrs). key: cord-254190-bxfne94u authors: tu, wenwei; zheng, jian title: application of humanized mice in immunological research date: 2015-07-07 journal: suppression and regulation of immune responses doi: 10.1007/978-1-4939-3139-2_10 sha: doc_id: 254190 cord_uid: bxfne94u during the past decade, the development of humanized mouse models and their general applications in biomedical research greatly accelerated the translation of outcomes obtained from basic research into potential diagnostic and therapeutic strategies in clinic. in this chapter, we firstly present an overview on the history and current progress of diverse humanized mouse models and then focus on those equipped with reconstituted human immune system. the update advancement in the establishment of humanized immune system mice and their applications in the studies of the development of human immune system and the pathogenesis of multiple human immune-related diseases are intensively reviewed here, while the shortcoming and perspective of these potent tools are discussed as well. as a valuable bridge across the gap between bench work and clinical trial, progressive humanized mouse models will undoubtedly continue to play an indispensable role in the wide area of biomedical research. during the past century, the application of rodent animal models , especially diverse gene-engineered mouse models , provided indispensable platforms and numerous valuable information for the advances in experimental medicine and biological research. however, the gap between species is still the most challenging obstacle for translation of results from rodents to humans . with the great advancement of technology in molecular biology and gene modifi cation, the attempt to establish "humanized" mouse models has made a leap since 1990s [ 1 -3 ] . nowadays, a wide variety of humanized mouse models have been generated and applied in nearly all fi elds of biomedical research [ 4 ] . in this chapter, we briefl y review the history and classifi cation of humanized mouse models and then summarize the current situation and recent advancement of their application in biomedical research, especially in the research of immune-related diseases. in general, the "humanized mice" are composed of three main classes: human gene-expressed transgenic mice ( human genetransgenic mice ), which are modifi ed by gene knock-in or replacement technology to express one or more human specifi c genes; humanized mice carrying human tissue, such as the liver ( humanized liver mice ) in which murine hepatocytes are completely or partly replaced by infused human-original hepatocytes; humanized mice equipped with functional human immune system ( humanized immune system mice ), which are established on immunodefi cient mice by transplanting human immune organs or cells to reconstitute human immune system in mice and thus referred to special "humanized mice." in the following section, we briefl y review the history and current advance of human gene-transgenic mice and humanized liver mice, and then focus on humanized immune system mice. human gene-transgenic mice are closer to gene engineered mice rather than "humanized mice." although the expressions of human gene or protein in transgenic mice provides the platform for studying in vivo role of specifi c human gene or molecule, the value of these data is limited in translational medicine due to the lack of human microenvironment and signal networks in these mice. the most widely used human gene-transgenic mice are human leukocyte antigen (hla)-transgenic mice [ 5 ] . these hlaexpressed transgenic mice represent for a useful tool in studying in vivo tcr-restricted immune responses and thus were adopted in the studies of immune-related diseases during the fi rst 10 years of this century. for example, hla-a0201-transgenic mice were used in inducing cd8 + t cell-restricted type i diabetes (t1d) [ 6 ] and experimental autoimmune encephalitis (eae) [ 7 ] , while hla-drb1-transgenic mice were applied in establishing cd4 + t cellmediated eae [ 8 ], system lupus erythematosus (sle) [ 9 ] and rheumatoid arthritis (ra) models [ 10 ] . more recently, the respective role of hla-dr2 and hla-dq8 in eae [ 11 ] and autoimmune diabetes [ 12 ] was also studied through transgenic mice. meanwhile, transgenic mice with distinct hla subtype expression favor the study of hla-related susceptibility on specifi c diseases, such as eae [ 13 ] , experimental autoimmune uveitis [ 14 ] , arthritis [ 15 -17 ] , allergic bronchopulmonary aspergillosis-like pulmonary responses [ 18 ] , and celiac disease [ 19 ] . although the application of hla-transgenic mice has been reduced due to the simplifi cation of diseases into specialized immune responses, the combination of hla-transgenic technology and reconstitution of human immune system in immunodefi cient mice has re-assigned them vitality in biomedical research, which will be discussed in the next section. other , which had all been blocked by the lack of optimal animal models in "pre-humanized mice time." on the other side, chen et al. tried to stabilize the function of cryopreserved human hepatocytes in immune competent mice through a novel system called "human ectopic artifi cial livers (heals)," which involved juxtacrine and paracrine signal in polymeric scaffolds. they claimed that mice transplanted with heals exhibited persistent normal liver function for weeks and thus provided a window for drugrelated investigation [ 34 ] . however, the effi cacy and value of humanized liver mice in the development of drug are still on debate due to the proposed side effects such as ongoing liver injury caused by transgenic and the infl uences on "normal metabolism" mediated by exogenous treatment [ 35 , 36 ] . apart from these, the potential application of humanized liver mice in immune-related research also deserves further exploration because liver also represents for a critical component of human immune system. the development of humanized immune system mice could be divided into three phases corresponding to the establishment of prkdc scid (protein kinase, dna activated, catalytic polypeptide; severe combined immunodefi ciency) mutation in cb17 mice, the development of nod (non-obese diabetic)-scid mice, and the generation of immunodefi cient mice homozygous for mutation at il (interleukin)-2 receptor γ chain locus [ 2 , 37 ] . each breakthrough mentioned previously signifi cantly improved the engraftment of human immune cells or pluripotent stem cells and stood as milestone on the way to "real humanized mice." the engraftment of multiple human immune components in these mice surpassed conventional human-gene knock-in in breaking the limited viewpoint of studying specifi c molecules under isolated environment. this unique advantage of humanized immune system mice favors their general application in immune-related studies, and opens a window for researchers to observe the interaction among human immune cells in vivo. in the following content, we focus on the characteristics and application of these mouse models and simply refer them as "humanized mice" if not otherwise specifi ed. currently, il2γc −/− mice established on nod/scid and recombination activating gene 2 (rag2) −/− balb/c background were most widely used strains for the reconstitution of human immune system in vivo [ 38 -40 ] . recently, by using bone marrow, liver, thymus (blt) co-transplantation, lavender et al. engrafted high levels of multi-lineage hematopoiesis and organized lymphoid tissues in c57bl/6-rag2 −/− γc −/− cd47 −/− triple-knockout mice. these humanized mice sustained human cell and tissue engraftment as long as 29 weeks post-transplantation without the development of chronic graft-versus-host diseases (gvhd), and thus represented for a new advancement in establishment of humanized mice [ 41 ] . the reconstitution of functional immune system is the key to evaluate the successful establishment of humanized mice. the graft used for reconstituting human immune system includes stem cells [ 42 ] , blt [ 41 ] , and peripheral blood cells [ 43 ] according to specifi c objectives. generally, stem cell and blt transplantation exhibit advantage in establishing stable multi-lineage hematopoietic cells but might need additional treatment for improving development of specifi c cell subpopulations. on the contrary, humanized mice established by peripheral blood cells provide a ready platform for studying the functions of mature immune cells but the length of window appropriate for research is still limited by chronic gvhd and ongoing reduced engraftment. to maximize the potential of humanized mouse model , some progresses have been made recently. firstly, pretreatment or gene-engineering of pluripotent stem cell exhibited satisfactory effects on improving engraftment of immune cells [ 38 , 42 , 44 ] . secondly, human growth factors, cytokines [ 44 , 45 ] or signal regulatory protein alpha (sirpa)expressed [ 46 ] immunodefi cient mice demonstrated superior engraftment for specifi c immune cell subpopulations as well. in the following paragraphs, we briefl y review current status of the reconstitution of specifi c immune cell subpopulations in humanized mice. lymphocytes are most important components of immune system and thus draw a major attention. although human peripheral blood mononuclear cells (pbmc) transplantation led to rapid reconstitution of human lymphocytes in humanized mice, it was found that after initial activation and induction of antibody production, human t cell lymphocytes enter an unresponsiveness status due to loss of human professional antigen-presenting cells (apc), which could be reversed by adoptive transfer of human apc [ 47 ] or activating organ-resident myeloid dendritic cells (dc) through poly(i:c) treating [ 48 ] . meanwhile, stem celltransplanted humanized mice displayed diversifi ed t cell repertoire, but the gap between hla and murine major histocompatibility complex (mhc) molecules prohibited the induction of effi cient t cell-mediated primary immune responses in vivo [ 49 , 50 ]. to overcome these problems, hla-expressed immunodefi cient mice were generated and their effi cacy has been confi rmed [ 51 ] . another concern origins from th1 and th17 immunocompetence in humanized mice [ 52 ] , which supports the utility of their application as surrogate model in transplantation rejection and autoimmunity but might cause some unwanted immune responses against murine tissue antigen as well. distinct from their t cell companion, reconstitution of functional b lymphocytes is generally poor in humanized mice and needed to improve in the future although their primary repertoire were principally unaltered by the differences between mouse and human stromal environments [ 53 ] and their ability to produce antigen-specifi c antibody was partly developed [ 54 ] . as described previously, the reconstitution of myeloid cells not only guarantees immune system intact, but also determines the development and function of both adaptive and innate lymphocytes [ 47 , 48 , 55 ] . unfortunately, monocytes and other myeloid cells usually exhibit immature phenotype and impaired function in humanized mice [ 56 ] , which could be partly rescued by human colony stimulating factor (csf)-1 [ 57 ]. however, the improvements in their survival, differentiation and even migration and residence [ 58 ] are still urgently required. besides leukocytes, other blood components also play important roles during immune response and regulation. recently, hu et al. established the full reconstitution of human platelets in humanized mice after depletion of murine macrophage [ 59 ] , which represents for an interesting attempt in constructing a more "humanized" circulation in mice. in summary, the optimization of humanized mouse model is still on the way and the advances in molecular biology, cellular biology, and system biology will defi nitely bring new era to the development of this useful tool. the applications of humanized mice cover nearly all fi elds of biomedical research and here we concentrate on immune-related studies, especially those aiming at the mechanisms and translational potentials of immune regulation and suppression . we also briefl y summarize the benefi ts brought by these potent models in tumor, infectious diseases, and vaccine studies. cd34 + cd38 lo cd1a − (early t lineage progenitors, etp), and cd34 + cd38 + cd1a + pre-t cells in liver of humanized mice by intrahepatic injection of cd34 + stem cells, establishing a wonderful platform for investigating human t cell development [ 60 ] . however, joo et al. found that human t cells educated by murine mhc in mice without a human thymus differ from normal human t cells marked as higher expression of cd45ro and promyelocytic leukemia zinc fi gure protein (plzf) regardless of similar development stages [ 61 ] . correspondingly, danner et al. generated hla-dr4-expressed nod-rag1 −/− γc −/− mice and demonstrated the critical role of hla class ii molecule for development of functional t cells by infusion with hla-dr-matched human hematopoietic stem cells [ 62 ] . meanwhile, the roles of il-12 [ 63 ] and notch [ 64 ] signals during the development of human cd4 + and cd8 + t cells were evaluated by human hematopoietic stem cell-transplanted mice. moreover, using a human stem cell factor, granulocyte-macrophage colony-stimulating factor (gm-csf) and il-3-expressed nod/scid-γc −/− mice, billerbeck et al. found the increased accumulation of human cd4 + foxp3 + t cells in blood, spleen, bone marrow and liver. most importantly, these cd4 + foxp3 + t cells exhibited potent suppressive capability on t cell proliferation, which made a signifi cant contribution to study of human regulatory t cells (treg) development in vivo [ 65 ] . as described previously, the development of human b cells in humanized mice is relatively weak compared to t cells. in 2011, choi et al. evaluated the effi cacy of busulfan, a chemotherapeutic agent, and claimed that it could effi ciently improve the reconstitution of human specifi c antibody -producing b cells, t cells, macrophage, and even dc from cd34 + cord blood cells with less toxic effects [ 66 ] . on the other hand, kim et al. found that cotransplantation of fetal bone tissue with fetal thymus could facilitate the development and reconstitution of human b cells from fetal liver-derived cd34 + cells together with t cells [ 67 ] . besides adaptive lymphocytes like t and b cells , innate lymphocytes development-related factors were also illustrated in humanized mouse model . as early as in 2008, huntington and di santo made a periodic review on the application of humanized mice in the research of nk cell development [ 68 ] . in 2011, pek et al. further confi rmed the crucial role of il-15 in nk cell development in bone marrow and liver with humanized mouse model [ 69 ] . we believe that more studies in the development of other innate lymphocytes such as nkt, γδ-t cells and innate-like t cells (ilt) will be reported in the near future. myeloid cells are generally regarded as more fragile and diffi cult to survive in "strange environment", which made it attractive and subtle to improve reconstitution of these sensitive cells. addition of human original cytokines such as gm-csf and il-4 was generally accepted as an effi cient way to improve dc maturation [ 70 ] . similarly, the effects of macrophage colony-stimulating factor (m-csf) and fms-related tyrosine kinase (flt)-3 ligand on promoting the development of macrophage [ 71 ] , and cd141 + and cd1c + dc [ 72 ] have also been confi rmed in humanized mouse model respectively. moreover, the development of megakaryocytes was replicated and used as index of dengue virusinfection in humanized mouse model recently [ 73 ] , which also supported the multi-lineage hematopoietic cell development in humanized mice. finally, transplantation of human stem cells from bone marrow of patients with bone marrow failure syndrome into humanized mice provided invaluable tools for evaluating novel gene-targeted therapy before clinical trial [ 74 ] . in summary, the reconstitution of diverse human immune cell populations from their pluripotent progenitors in immunodeficient mice has become a potent platform for investigating the development of human immune system while the next question is how to create a more "humanized" environment in mice for human cells [ 75 ] . the advances in the study of autoimmune diseases in humanized mice, especially those t cell-mediated diseases, are always correlated with development of hla-transgenic technology. in 1999, bachmaier et al. generated a cd4−cd8− double-knockout mice transgenic for human cd4 and hla-dq6 to specifi cally reconstitute the human hla-dq6/cd4 arm in mice and established a dilated cardiomyopathy model [ 76 ] , which was one of the earliest attempt for applying humanized mouse model in the study of autoimmune diseases. using similar strategy, eming et al. established a ra model in a hla-dr4/human cd4/tcr combined transgenic mice with the stimulation of a ra-related human autogenic protein hcgp-39 in 2002 [ 77 ] . however, the lack of human immune system reconstitution in these models constrained their representative for the whole map occurring during autoimmune diseases. on the other side, shultz et al. established t1d model in nod/scid-γc −/− mice by co-transplanting with human stem cell and islet cells [ 78 , 79 ] . importantly, this group pointed out the potential of hla-transgenic immunodefi cient mice in optimization of these models and provided some interesting preliminary data [ 78 ] . soon after, infl ammatory arthritis and type 2 diabetes models were established in hla-transgenic humanized mice by david [ 80 ] and schultz groups [ 81 ] respectively. as we mentioned previously, t cell-mediated immune responses were generally incomplete in humanized mice established on conventional immunodefi cient mice, which usually led to insignifi cant clinical symptoms [ 82 ] and thus limited the application of these models. the involvement of hla not only improves the effi cacy of immune responses, but also provides a platform for study of the relationship between hla subtypes and specifi c diseases susceptibility. nevertheless, the complexity and individuality of hla phenotypes in healthy donors or patients still remain as the biggest challenge in rebuild of physiopathology process in relatively limited hla-expressed humanized mice. due to relatively weak reconstitution of human b cells in humanized mice, the establishment of b cell or antibody -mediated autoimmune diseases seems to be more diffi cult than those t cellmediated autoimmune diseases. kerekov et al. rebuilt the clinical pathogenesis in humanized mice with cells transferred from sle patients and evaluated the potential of b cell-targeted therapy with a chimeric molecule containing a monoclonal antibody against human inhibitory complement receptor type i coupled to a decapeptide that mimic dna antigenicity [ 83 ] . in 2012, another group led by duffi eld recapitulated systemic vasculitis in humanized mice by treating them with anti-proteinase-3 igg isolated from patients [ 84 ] . with the improvement in reconstitution of multiple components of human immune system in humanized mice, it is predictable that the induction of diverse human b cellmediated autoimmune diseases in vivo will be accessible soon. application of humanized mice models in transplantation-related diseases arises as early as the birth of humanized mice but the process is so tortuous till now due to chronic exogenous rejection and ongoing decrease of immune cells . in above three studies, investigators planted solid grafts into immunodefi cient mice before reconstitution of human immune system and induced rejection by infusion of mature human cells. however, the longterm outcome of these models is still not clear. in order to further mimic clinical situation, human cd34 + stem cells were applied in establishing humanized mice. using this strategy, three independent groups reported allogeneic islet transplantation [ 89 ], xenogeneic islet rejection [ 90 ], and xenogeneic skin rejection [ 91 ] models during 2010-2012. unfortunately, insuffi cient development of immune cell populations in these humanized mice still stayed as an obstacle and even led to the failure of rejection [ 89 ] . to solve this problem, some other groups tried to develop a more "mature" human immune system in humanized mice by transplanting human peripheral blood cells. in 2013, our group reported a novel human allogenic gvhd model established on humanized mice reconstituted with human pbmc [ 92 ] . this model reproduced typical clinical process of acute gvhd occurring during allogeneic bone marrow transplantation without apparent interruption of exogenous reactivity. using this model, we evaluated the protective effects of human cd8 + treg induced ex vivo by allogeneic cd40-activated b cells and found that human cd8 + treg could inhibit gvhd and induce long-term tolerance without compromising general immunity and graft-versus-tumor (gvt) activity [ 92 ] . the potent regulatory activity of the cd8 + treg was mainly mediated by the expression of cytotoxic lymphocyte antigen (ctla)-4 on cell surface, while their alloantigen-specifi city and the ability to induce the long-term tolerance favor their clinical application. more importantly, this strategy might reduce clinical dependence on limited hla-match donors and largely improve the survival chance of millions of patients who are waiting for bone marrow transplantation. humanized mouse model undoubtedly brings new hope for transplantation research, but we also need to keep in mind that a lot of questions are still waiting to be answered on this way. as emphasized by brehm and shultz, keys to successful humanized mouse model included available immunodefi cient mouse strains, the choice of tissue to transplant and the specifi c human immune cell population that can be grafted [ 85 ] . besides autoimmune diseases and transplantation-related diseases, humanized mice models are also useful to study some other infl ammatory diseases. in 2002, hammad et al. compared the th2 allergic infl ammation in the lung of humanized mice reconstituted with pbmc. to induce infl ammatory reaction, dcs from home dust mite (hdm)allergic patients or healthy donors were injected intratracheally and mice were then repeated exposed to aerosol of hdm. in contrast to ifn-γ secretion induced in mice receiving normal dcs, those injected with dcs from patients induced il-4 and il-5 production accompanied with the increase of ige production, which represents characteristics of th2 response lymphadenopathy, and other infl ammatory sequelae in humanized mouse model [ 97 ] . in addition to immunopathology study, humanized mouse model was also applied in studying the underlying mechanisms of injury repair. by plating retroviral vector-modifi ed human skin on nude mice and adding human keratinocyte growth factor (kgf) to artifi cial wound in the skin, the re-epithelialization was signifi cantly accelerated [ 98 ] . although this model could not be described as "real" humanized mice because no human immune system was involved in it, this attempt initiated an innovative application of humanized mice. compared to satisfactory reconstitution of circulating blood cells, the successful reconstitution of mucosa immunity in humanized mice is still absent till now. mucosa, especially respiratory and digestive tract surface, plays indispensable role in protection and immune regulation. however, the residence and exchange of immune components in the locus are still diffi cult to rebuild in animal models because the physiological dynamics remains largely unknown the occurrence of humanized mouse model provided a perfect platform for evaluating immunotherapy against tumor. the earliest attempts of inducing antitumor immune responses in humanized mice focused on the generation of specifi c antibody but the outcome varied due to unstable humanization of models [ 104 , 105 ] . in the new century, researchers started to pay more attention on developing complete tumorigenicity, especially metastasis process and its relationship with stromal cells, in immunocompetent humanized mice, and made some signifi cant advances in multiple fi elds like human prostate cancer [ 106 ], mixed-lineage leukemia [ 109 ] . based on these progresses, some novel immunotherapy strategies were evaluated on humanized mouse models, such as inhibitory receptor ig-like transcript (ilt)-3 depletion or blockade in melanoma [ 110 ] and il-15-enhanced nk cell-mediated cytotoxicity against human breast cancer [ 111 ] . recently, our group reported a novel application of pamidronate, a phosphoantigen generally used to treat osteoporosis, in treating epstein-barr virus (ebv)-induced b cell lymphoproliferative disease in humanized mouse model reconstituted with human pbmc [ 112 ] . this "new application of an old drug" was mediated by expanding and activating human vγ9vδ2-t cells, a small cell population of human lymphocytes, which might inspire further exploration of currently available resources. more importantly, the established of donorand tissue-specifi c humanized mouse tumor models will undoubtedly play an indispensable role during the development of individual therapies in the future [ 113 ] . the development of humanized mice represents a milestone in the history of human immunodefi ciency virus (hiv) study. the new generation of humanized mice not only improved our understanding on transmission, latency, and pathogenesis of hiv [ 114 -119 ] , but also provided unprecedented platform for antiviral study. besides further exploration of effi cient virus-specifi c neutralization antibodies [ 120 -125 ] and conventional antiretroviral or antimicrobial therapies [ 126 -128 ] in these models, the effi cacy of vectored immunoprophylaxis [ 129 ] and ccr5-targetd treatment [ 130 -132 ] in preventing hiv transmission were evaluated as well. meanwhile, the crucial roles of hiv-specifi c cd8 + t cells [ 133 , 134 ] and plasmacytoid dc (pdc) [ 135 , 136 ] in the replication of virus and activation of immune responses, and their potentials in targeted therapy were also investigated. other novel immunotherapy assays performed in humanized mouse model included blockade of programmed cell death (pd)-1 receptor [ 137 , 138 ] , engineering hiv-resistant t cells from short-hairpin rna (shrna)-expressing hematopoietic stem/progenitor cells [ 139 ] , and inhibition of hiv replication by a chimera containing an rna aptamer with high binding affi nity to the hiv envelop protein gp120 and virus neutralization properties and a small interfering rna (sirna) triggering sequence-specifi c degradation of hiv rnas [ 140 ] . moreover, a preliminary study on mechanisms underlying viral controlling in hla-b*57 elite controller or suppressor (es) was completed in humanized blt mice and demonstrated that elite suppressors are capable of controlling hiv-1 due to the possession of unique host factors rather than infection with defective virus in vivo [ 141 ] . nowadays, we could even make in-depth study on the cell dynamics in hiv-infected humanized mice model with the help of intravital microscopy [ 142 ] . therefore, it is countable that the future molecular biology will bring more surprise to the efforts of gene therapy against hiv [ 143 ] . except for application in hiv-related studies, humanized mouse model also brought span-new opportunities for other human infectious diseases [ 144 ] , especially those blood-borne pathogencaused diseases such as dengue virus infection [ 145 -149 ] , ebv infection [ 150 -155 ] , hcmv infection [ 156 ] , htlv infection [ 157 ] , and malaria parasite infection [ 158 ] . on the other hand, humanized mouse models for leishmaniasis [ 159 ] , salmonella typhi infection [ 160 , 161 ] , herpesvirus infection [ 162 , 163 ] , mycobacteria infection [ 164 , 165 ] , and group b streptococcus (gbs) infection [ 166 ] have been established. these efforts fi ll in the lacks of suitable animal models for those human-specifi c pathogen-caused diseases and push forward the correlating investigations on development of prevention and treatment , although some technological obstacles like the replication of natural infection and transmission routes are still needed to resolved. in 2011, our group used pbmc-transplanted humanized mouse model to evaluate a novel therapeutic strategy by targeting the host rather than the virus for treating infl uenza virus infection. we demonstrated that aminobisphosphonate can control infl uenza disease through boosting human vγ9vδ2-t cell immunity and this benefi cial effect is active against viruses of varying subtypes and virulence [ 43 ] . nevertheless, differences in the characteristics of molecules, tissues, and organs between human and mice might impair effi ciency of pathogen infection and initiation of specifi c immune responses [ 167 ] . in 2005, lassning et al. increased the susceptibility of mice on human coronavirus by crossing aminopeptidase n (apn), the receptor for human coronavirus (hcov)-229e, and transgenic mice into signal transducer and activator of transcription (stat)-1 null mice [ 168 ] . this work, together with hla-and human cytokines/growth factor-transgenic technology [ 169 ] , provided successful examples for future studying human infectious agents in humanized mice. in the next stage, improvement of versatility and variability of human immune system in humanized mouse model and application of gene-modifi ed pathogens [ 170 ] will defi nitely enhance translational effi ciency of these models. the usage of humanized mice in the development of vaccines targeting human diseases including ebv, hiv-1, dengue virus, infl uenza virus, severe acute respiratory syndrome (sars) corona virus, and carcino-embryonic antigen (cea) has obtained outstanding achievements during the past decade, while the introduction of hla transgenic immunodefi cient mice further accelerated the advancement in this fi eld [ 171 -173 ] . with the improvement of immune cell population reconstitution, more and more novel vaccination protocol will be carried out in humanized mice. compared to conventional mice and non-human primate model, humanized mice exhibit great advantage in translational potential, reproductive capacity and data repeatability, economical and ethical concerns. the increasing applications of diverse humanized mice models in biomedical research during the past two decades significantly improved our understanding on human physiological and pathological, especially immunological process at systemic, cellular, and molecular levels. this further accelerated the development of current translational medicine signifi cantly. nevertheless, there are several major caveats on their development remain to be dealt with, including complete replacement of murine mhc with diversifi ed hla molecules and effi cient methodology to express corresponding growth factors and cytokines at specifi c time and organs [ 174 ] ; how to prolong the maintenance of human engraftment, promote the development of myeloid cells and increase relatively weak quantity and quality of immune cells [ 175 ] ; and the limited development of lymph nodes, inter-organ traffi c of immune cells, and the reconstitution of red blood cells and granulocytes [ 176 ] . in another word, the most important issue is to fi nd the convenient and cost-effective ways to construct appropriate human-like microenvironment including physical structure, intercellular contact and molecular signals transfer in humanized mice. it is foreseeable that knowledge exchange in the age of big data will bring an even more bright future to this advancing tool than ever. analysis of candidate human blood stem cells in "humanized" immune-defi ciency scid mice humanized mice in translational biomedical research mighty mice. scientists are still improving the humanized mouse model but are optimistic about its future role in evaluating aids vaccine candidates humanized mice as a preclinical tool for infectious disease and biomedical research hla transgenic mice as humanized mouse models of disease and immunity prevention of "humanized" diabetogenic cd8 t-cell responses in hla-transgenic nod mice by a multipeptide coupled-cell approach cd8 t cell responses to myelin oligodendrocyte glycoprotein-derived peptides in humanized hla-a*0201 humanized mice for studying human leukocyte integrins in vivo full reconstitution of human platelets in humanized mice after macrophage depletion human t cell development in the liver of humanized nod/scid/ il-2rgamma(null)(nsg) mice generated by intrahepatic injection of cd34(+) human (h) cord blood (cb) cells systemic human t cell developmental processes in humanized mice cotransplanted with human fetal thymus/ liver tissue and hematopoietic stem cells expression of hla class ii molecules in humanized nod. rag1ko.il2rgcko mice is critical for development and function of human t and b cells insuffi cient interleukin-12 signalling favours differentiation of human cd4(+) and cd8(+) t cells into gata-3(+) and gata-3(+) t-bet(+) subsets in humanized mice activation of notch1 promotes development of human cd8(+) single positive t cells in humanized mice development of human cd4+foxp3+ regulatory t cells in human stem cell factor-, granulocytemacrophage colony-stimulating factor-, and interleukin-3-expressing nod-scid il2rgamma(null) humanized mice human b cell development and antibody production in humanized nod/scid/il-2rgamma(null) (nsg) mice conditioned by busulfan co-transplantation of fetal bone tissue facilitates the development and reconstitution in human b cells in humanized nod/scid/il-2rgammanull (nsg) mice humanized immune system (his) mice as a tool to study human nk cell development characterization and il-15 dependence of nk cells in humanized mice gm-csf and il-4 stimulate antibody responses in humanized mice by promoting t, b, and dendritic cell maturation induction of functional human macrophages from bone marrow promonocytes by m-csf in humanized mice flt3-ligand treatment of humanized mice results in the generation of large numbers of cd141+ and cd1c+ dendritic cells in vivo inhibition of megakaryocyte development in the bone marrow underlies dengue virus-induced thrombocytopenia in humanized mice a novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice humanized mice as a model to study human hematopoietic stem cell transplantation generation of humanized mice susceptible to peptideinduced infl ammatory heart disease humanized mice as a model for rheumatoid arthritis humanized nod/ ltsz-scid il2 receptor common gamma chain knockout mice in diabetes research humanized mice for the study of type 1 diabetes and beta cell function role of hla class ii genes in susceptibility/resistance to infl ammatory arthritis: studies with humanized mice humanized mice for the study of type 1 and type 2 diabetes subclinical cns infl ammation as response to a myelin antigen in humanized mice humanized scid mice models of sle anti-proteinase 3 antineutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system targeted activation of human vgamma9vdelta2-t cells controls epstein-barr virus-induced b cell lymphoproliferative disease humanized nod-scid il2rg-/-mice as a preclinical model for cancer research and its potential use for individualized cancer therapies the utility of the new generation of humanized mice to study hiv-1 infection: transmission, prevention, pathogenesis, and treatment generation of hiv latency in humanized blt mice can humanized mice refl ect the complex pathobiology of hiv-associated neurocognitive disorders? blt humanized mice as model to study hiv vaginal transmission hiv-1 infection of hematopoietic progenitor cells in vivo in humanized mice mucosal hiv-1 transmission and prevention strategies in blt humanized mice hiv therapy by a combination of broadly neutralizing antibodies in humanized mice inhibition of in vivo hiv infection in humanized mice by gene therapy of human hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti-hiv antibody inhibitory effect of hiv-specifi c neutralizing iga on mucosal transmission of hiv in humanized mice hiv-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice broadly neutralizing antibodies and viral inducers decrease rebound from hiv-1 latent reservoirs in humanized mice humoral immune responses in humanized blt mice immunized with west nile virus and hiv-1 envelope proteins are largely mediated via human cd5+ b cells long-acting nanoformulated antiretroviral therapy elicits potent antiretroviral and neuroprotective responses in hiv-1-infected humanized mice humanized mice recapitulate key features of hiv-1 infection: a novel concept using long-acting antiretroviral drugs for treating hiv-1 minocycline attenuates hiv-1 infection and suppresses chronic immune activation in humanized nod/ltsz-scidil-2rgamma(null) mice vectored immunoprophylaxis protects humanized mice from mucosal hiv transmission pre-clinical modeling of ccr5 knockout in human hematopoietic stem cells by zinc fi nger nucleases using humanized mice a topical microbicide gel formulation of ccr5 antagonist maraviroc prevents hiv-1 vaginal transmission in humanized rag-hu mice expansion of activated memory cd4+ t cells affects infectivity of ccr5-tropic hiv-1 in humanized nod/ scid/jak3null mice hiv-specifi c cd8(+) t-cell immunity in humanized bone marrow-liver-thymus mice rapid evolution of hiv-1 to functional cd8(+) t cell responses in humanized blt mice effi cient infection, activation, and impairment of pdcs in the bm and peripheral lymphoid organs during early hiv-1 infection in humanized rag2(-)/(-) gamma c(-)/(-) mice in vivo plasmacytoid dendritic cells suppress hiv-1 replication but contribute to hiv-1 induced immunopathogenesis in humanized mice in vivo blockade of the pd-1 receptor suppresses hiv-1 viral loads and improves cd4+ t cell levels in humanized mice pd-1 blockade in chronically hiv-1-infected humanized mice suppresses viral loads engineering hiv-1-resistant t-cells from short-hairpin rnaexpressing hematopoietic stem/progenitor cells in humanized blt mice an aptamer-sirna chimera suppresses hiv-1 viral loads and protects from helper cd4(+) t cell decline in humanized mice hla-b*57 elite suppressor and chronic progressor hiv-1 isolates replicate vigorously and cause cd4+ t cell depletion in humanized blt mice intravital microscopy in blt-humanized mice to study cellular dynamics in hiv infection gene therapy strategies for hiv/aids: preclinical modeling in humanized mice humanized mice for modeling human infectious disease: challenges, progress, and outlook mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice utility of humanized blt mice for analysis of dengue virus infection and antiviral drug testing enhanced humoral and hla-a2-restricted dengue virus-specifi c t-cell responses in humanized blt nsg mice dengue virus infection induces broadly cross-reactive human igm antibodies that recognize intact virions in humanized blt-nsg mice dengue virus tropism in humanized mice recapitulates human dengue fever ebna3b-defi cient ebv promotes b cell lymphomagenesis in humanized mice and is found in human tumors a novel animal model of epstein-barr virus-associated hemophagocytic lymphohistiocytosis in humanized mice epstein-barr virus induces erosive arthritis in humanized mice t cells modulate epstein-barr virus latency phenotypes during infection of humanized mice reproduction of epstein-barr virus infection and pathogenesis in humanized mice adoptive transfer of ebv specifi c cd8+ t cell clones can transiently control ebv infection in humanized mice hcmv infection of humanized mice after transplantation of g-csf-mobilized peripheral blood stem cells from hcmv-seropositive donors adult t-cell leukemia/lymphoma development in htlv-1-infected humanized scid mice of men in mice: the success and promise of humanized mouse models for human malaria parasite infections leishmania major infection in humanized mice induces systemic infection and provokes a nonprotective human immune response humanized nonobese diabetic-scid il2rgammanull mice are susceptible to lethal salmonella typhi infection humanized mice for salmonella typhi infection: new tools for an old problem modeling of human herpesvirus infections in humanized mice human herpesvirus 6a infection and immunopathogenesis in humanized rag2(-)/(-) gammac(-)/(-) mice engrafted human cells generate adaptive immune responses to mycobacterium bovis bcg infection in humanized mice cd4+ cell-dependent granuloma formation in humanized mice infected with mycobacteria humanized mice, a new model to study the infl uence of drug treatment on neonatal sepsis humanized mice for the study of infectious diseases development of a transgenic mouse model susceptible to human coronavirus 229e new generation humanized mice for virus research: comparative aspects and future prospects infectious diseases in humanized mice use of humanized severe combined immunodeficient mice for human vaccine development human immune responses and potential for vaccine assessment in humanized mice broad infl uenza-specifi c cd8+ t-cell responses in humanized mice vaccinated with infl uenza virus vaccines humanized mice: are we there yet? humanized mice: current states and perspectives humanized mice for immune system investigation: progress, promise and challenges this work was supported in part by the area of excellence program supported by the university grants committee of the hong kong sar, china (aoe/m-12/06 and aoe/m-06/08). key: cord-253556-p1y0zeo1 authors: rhodes, scott d.; mann-jackson, lilli; alonzo, jorge; garcia, manuel; tanner, amanda e.; smart, benjamin d.; horridge, danielle n.; dam, cornelius n. van; wilkin, aimee m. title: a rapid qualitative assessment of the impact of the covid-19 pandemic on a racially/ethnically diverse sample of gay, bisexual, and other men who have sex with men living with hiv in the us south date: 2020-08-12 journal: res sq doi: 10.21203/rs.3.rs-57507/v1 sha: doc_id: 253556 cord_uid: p1y0zeo1 persons living with hiv (plwh) may be at increased risk for severe covid-19-related illness. our community-based participatory research partnership collected and analyzed semi-structured interview data to understand the early impact of the covid-19 pandemic on a sample of racially/ethnically diverse gay, bisexual, and other men who have sex with men living with hiv. fifteen cisgender men participated; their mean age was 28. six participants were black/african american, five were spanish-speaking latinx, and four were white. seventeen themes emerged that were categorized into six domains: knowledge and perceptions of covid-19; covid-19 information sources and perceptions of trustworthiness; impact of covid-19 on behaviors, health, and social determinants of health; and general covid-19-related concerns. interventions are needed to ensure that plwh have updated information and adhere to medication regimens, and to reduce the impact of covid-19 on social isolation, economic stability, healthcare access, and other social determinants of health within this vulnerable population. the united states (u.s.) south continues to experience disproportionate hiv rates compared to other regions of the country and has been referred to as the "new" and "latest" u.s. hiv epicenter (1, 2) . southern states account for an estimated 51% of new hiv diagnoses in the u.s. each year, despite having only 38% of the county's overall population. eight of the 10 states with the highest rates of new hiv diagnoses, and nine of the 10 metropolitan statistical areas with the highest rates, are in the south (2) . north carolina (nc) consistently ranks among these top 10 states for hiv diagnoses (3) , and guilford county, located in the piedmont triad region of central nc, has consistently higher rates of hiv than nc and the u.s. overall (4, 5) . guilford county ranks third out of the 100 counties in the state for hiv per 100,000, and its hiv incidence rate is 50% higher than the national rate (6) . coronavirus disease 2019 (covid-19) is a new infectious disease caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which had not previously been reported in humans (7) . manifestations range from asymptomatic infection to severe complications including pneumonia, acute respiratory distress syndrome, coagulopathies, immune system dysregulation, and death. covid-19 is highly contagious and has quickly spread globally. (8, 9) the rst case of covid-19 in nc was identi ed on march 3, 2020, and on march 10, nc declared a state of emergency (10) . given the ongoing rise in community-acquired covid-19 cases in nc, on march 30, a statewide stay-at-home order went into effect that limited activities to those that were considered essential (e.g., health-, safety-, nutrition-, and sanitation-related), banned gatherings of more than ten people, and prioritized social distancing (staying at least 6 feet away from other people) (11) . the nc stay-at-home order was eased on may 8, 2020, with a phased re-opening that continued to limit the types of businesses and institutions that could open and the sizes of gatherings. hiv infection, along with several other immunosuppressing conditions, might increase risk of severe covid-19-related illness (12) . although the underlying mechanism is not fully understood, this risk for plwh may be due to a reduction in lymphocytes, immune system dysregulation, or an increased in ammatory response (13) . comorbidities that are becoming increasingly common among plwh, such as diabetes, chronic kidney disease, chronic obstructive pulmonary disease, and obesity, also increase the risk of developing severe illness from covid-19 (14) . the burden of these risks may not be carried equally among all plwh. communities of color face disproportionate rates of hiv, and black/african american gay, bisexual, and other men who have sex with men (gbmsm) and black/african american transgender women have higher rates of hiv than any other group in the us (15, 16) . in addition, black/african american and latinx plwh have lower rates of care linkage and retention and are less likely to be virally suppressed than white plwh (17) . in addition, persons of color are also experiencing disparities related to covid-19. as of june 2020, black/african american and native american or alaska native persons have covid-19-related hospitalization rates ve times that of non-hispanic white persons, and hispanic or latinx persons have covid-19 diagnosis rates four times those of non-hispanic white persons (18) . these higher rates may be related to living conditions, work circumstances, and health disparities including insurance and healthcare access as well as higher rates of comorbidities that increase the risk of severe illness from covid-19, which are shaped by the larger context of systemic racism and inequalities (16, 19) . therefore, racially/ethnically diverse gbmsm living with hiv are a particularly vulnerable population when considering the risk and impact of covid-19. despite the impact of covid-19 and potential for serious morbidity and mortality, little is known about the pandemic from the perspective of plwh themselves. our long-standing community-based participatory research (cbpr) partnership sought to qualitatively explore the impact of the covid-19 pandemic within a racially/ethnically diverse sample of gbmsm living with hiv. this rapid assessment was conducted by our cbpr partnership in nc that includes racially/ethnically diverse gbmsm living with hiv; representatives from public health departments, hiv service organizations, and clinics that serve plwh; and academic investigators. this partnership has an established history exploring and intervening on the health-related needs and priorities of vulnerable communities (5, (20) (21) (22) . we collected qualitative data from participants who had completed 12 months of participation in and had "graduated" from the wecare intervention. wecare is an evidence-informed intervention that improves hiv care engagement among racially/ethnically diverse gbmsm and transgender women living with hiv by reducing missed hiv care appointments and increasing viral suppression (21, 23, 24) . wecare currently is being conducted in partnership with the regional center for infectious diseases (rcid) in guilford county, nc (21) . rcid is funded by the ryan white hiv/aids program, health resources and services administration (hrsa), to serve low-income plwh who are uninsured or underinsured. rcid provides comprehensive integrated services for plwh, including hiv primary medical care, case management, bridge counseling, nancial assistance, behavioral health care, community outreach nursing, and a dental clinic. the details of the wecare intervention are described elsewhere (21, 23, 24) ; brie y, wecare harnesses social media platforms, including facebook messenger, text messaging (including through applications or "apps" such as whatsapp), and messaging through gps-based mobile apps used for social and sexual networking (e.g., a4a/radar, badoo, grindr, jack'd, and scruff) to improve linkage to and retention in hiv care among plwh. the intervention is targeted to racially/ethnically diverse gbmsm and transgender women living with hiv, tailored to the social media platform preferences of participants, and personalized to each participant's needs (23) . eligibility criteria to participate in wecare include: being age 16 or older, identifying as cisgender male or male-to-female transgender, reporting sex with men, and living with hiv. potential participants are referred to the study by clinic and health department staff. we also advertise the intervention study on facebook through paid targeted advertisements and on other social media platforms, in a southeastern lgbtq (lesbian, gay, bisexual, transgender, and queer) newspaper, and through yers posted within bars, clubs, and coffee shops. furthermore, we recruit participants through word-of-mouth; enrolled participants are encouraged to share information about the study with others in their social networks. if eligible and interested, participants complete written informed consent procedures and enroll. participation in the intervention lasts 12 months, after which participants graduate from wecare. of the graduated wecare participants, we randomly selected and contacted 15 of them to complete an interviewer-administered semi-structured individual interview. the abbreviated interview guide is outlined in table i . demographic questions included some close-ended items; however, most questions in the interview guide were open-ended to allow participants to describe their experiences, perceptions, and attitudes. a trained bilingual interviewer conducted the interviews in english and spanish via telephone between april 23 and may 23, 2020. interviews were recorded and professionally transcribed. constant comparison, an approach to grounded theory, was used to analyze data. constant comparison combines qualitative coding with simultaneous comparison; initial observations are continually re ned throughout data collection and analysis (25). because of the formative nature of this study, we aimed to identify the breadth of experiences, not to quantify them. analysts coded transcripts and developed matrices to identify similarities and differences within and across interviews. based on these matrices, each analyst developed preliminary themes. after preliminary themes were developed, the analysts came together via webex (a web-conferencing platform) to discuss and reconcile nal themes. human subject approval and oversight was provided by wake forest school of medicine institutional review board (irb). fifteen racially/ethnically diverse cisgender men participated; no participants refused to participate. participant mean age was 28. six participants were black/african american, ve were latinx, and four were white; all latinx participants completed their interviews in spanish. one participant self-identi ed as bisexual; all others self-identi ed as gay. eleven participants self-reported being virally suppressed while four reported not knowing whether they were virally suppressed. table ii . participants knew a great deal about covid-19 and its transmission. they were aware of recommended precautions to prevent infection, including wearing a face covering, social distancing, and handwashing. many were aware of the nc stay-at-home order that was in effect at the time of the interview and reported adhering to the order. for example, a participant reported, it's contagious. it affects the respiratory system. it can be fatal in some cases, but some people get through. it's important to take precautions and stay at home like the governor says. (participant [p]13, black/african american, 26 years old) another participant noted, "es un virus muy contagioso, que se transmite de una persona enferma si está muy cerca cuando tose. también, si la persona enferma toca algo, y la persona sana toca la misma super cie, se puede contagiar." ["it is a very contagious virus that is transmitted from a sick person if they are very close when they cough. also, if the sick person touches something, and the healthy person touches the same surface, it can be contagious."] (p4, latinx, 35 years old) covid-19 is perceived as serious, and participants perceive themselves to be susceptible participants reported that covid-19 was serious and that those with compromised immune systems were especially at risk. they were worried about their own increased risks as plwh and noted that hearing about others who had similar characteristics to them being seriously affected also increased their concerns. a participant stated, creo que es muy serio porque nunca en mi vida había visto algo así. afecta a las personas que tienen el sistema inmunológico comprometido como personas con diabetes o con problemas respiratorios, así que es muy serio. uno piensa que porque uno es joven no le va a pasar, pero estamos viendo casos de jóvenes que han muerto por este virus y eso lo hace más grave para mí. [i think it is very serious because i have never seen anything like this in my life. it affects people who have compromised immune systems like people with diabetes or respiratory problems, so it's very serious. you think that because you are young it will not happen to you, but we are seeing cases of young people who have died from this virus and that makes it more serious for me.] (p3, latinx, 28 years old) another participant said, "the coronavirus is making me super cautious. i am just afraid to contract it so i think about it a lot, but i try to stay optimistic about the entire situation." (p14, black/african american, 27 years old) participants also reported worrying about the well-being of friends and family members, particularly those who were older, had comorbidities, or had other risk factors for developing serious illness from covid-19. a participant noted, "pues, estoy muy preocupado por toda mi familia y mi círculo porque no solo me puede afectar a mí, sino a toda mi familia también. participants found available and prevailing information regarding the transmission and prevention of covid-19 to be con icting. social media (e.g., facebook), the internet (e.g., cdc website), and television (e.g., english-and spanishlanguage national and local news) were noted as places that participants obtained information about covid-19. participants also reported that they received information from their workplaces, as guidelines and regulations changed and thus required changes to how work was performed. for example, participants who worked in restaurants noted that they learned about transmission, prevention, and the current state of disease burden from restaurant owners and managers. in addition, participants described friends and family as sources of information about covid-19. a participant reported, "i get info on tv, and i did some research on the internet, read some articles, and heard some through word-of-mouth, like during a meeting at work when we go over protocols and changes that are going into effect because of the coronavirus." (p10, white, 28 years old) another participant agreed saying, "i honestly do not trust anything that the president says." (p14, black/african american, 27 years old) a third participant stated, "no confío mucho porque creo que no le tomaron mucha importancia cuando empezó y por eso estamos con estos números tan altos." ["i do not trust much, because i think they did not take it seriously when it started and that is why we have these high numbers."] (p4, latinx, 35 years old) healthcare providers, including hiv providers, were identi ed in particular as providing timely and trustworthy information about covid-19. a participant stated, "i trust my doctor, especially because i believe they have the best interest at heart and they are the people on the frontline. they are telling us what they understand and what they are seeing from their patients." (p7, black/african american, 23 years old) participants are taking action to reduce their risks participants reported staying home, maintaining social distancing, and cleaning with disinfectants and sanitizers to prevent contracting covid-19. a participant commented, "i have not been out. today is actually the rst time i went out to go to the store. no hugging anyone; i try to stay away from people as much possible or at least six feet." (p6, black/african american, 21 years old) this commitment to prevention was noted by other participants. for instance, a participant stated, "i have been extremely social distancing. i, in fact, haven't left my house; some of my friends do the grocery shopping for me and leave my groceries by the door. i bring them inside, sanitize them, and throw the bags out." (p7, black/african american, 23 years old) further, another participant noted, "guardo mi distancia entre las personas cuando salgo a la tienda, uso desinfectante de alcohol en mis manos, mascarilla cuando salgo afuera, y limpio con desinfectante las áreas que más toco como el refrigerador, las llaves, mi tarjeta de débito, y las mancillas de la puerta." ["i keep my distance from people when i go out to the store, use alcohol disinfectant on my hands, a mask when i go outside, and clean the areas i touch the most with disinfectant, such as the refrigerator, keys, my debit card, and the door handles."] (p3, latinx, 28 years old) participants noted that worries and fears related to covid-19 and behavioral changes that they are making to stay safe are affecting them in both negative and positive ways. a participant noted that he is sleeping less because he is worried about his health, the health and well-being of his friends and family, and the long-term impact of the pandemic locally and globally, reporting, "my sleeping schedule has been changing a lot. it is hard to sleep 8 hours as i used to." (p8, black/african american, 21 years old). another participant commented, "i am working out less to avoid public settings." (p9, white, 23 years old) however, some participants reported positive changes they had made in their health behaviors as a result of staying home and other adaptations related to covid-19 prevention. a participant noted, "i stopped drinking alcohol because i gure that i have bad habits when i am at home alone; because in the rst week or so of the stay home order for the coronavirus, i was drinking a lot and that kind of hurt me." (p11, white, 25 years old). further, another participant shared, "i actually started meditating in the mornings so i can have a good set of mind about all this. and i have been spending more time in the backyard and gardening more than i did before." (p15, white, 27 year old) before the initiation of the covid-19 pandemic, participants were already communicating frequently through social media platforms such as facebook, texting, and gps-based mobile apps. after the pandemic began, however, participants reported increased use of these types of social media platforms. a participant noted, "i use facebook much more now because it is the only way we have to communicate with other people." (p12, black/african american, 21 years old) use of other technology for communication, such as video calls, also increased, as another participant added, "i don't see my friends and family much now, and if i do, we try to facetime on the iphone or call each other on facebook to see them on video. we talk on the phone to catch up. that is the only way we do it to be safe and cautious." (p10, white, 23 years old) participants reported that their mental health was profoundly affected by covid-19 and the necessary precautions required to reduce risks of exposure. a participant noted, "the changes make me feel hopeless and sad. i am just realizing that everyone wants things to go back to normal, but i think a lot of things will change, and lots of polices that are in place will change, like in restaurants and bars. it will never be normal." (p7, black/african american, 23 years old) another participant reported, "i do feel alone, and it kind of reminds me of when i learned that i had hiv." (p15, white, 27 years old) accessing medical care is more di cult participants reported that accessing medical care had become more di cult in the context of covid-19. though as of the time of data collection participants had not experienced interruptions in their hiv care, some had not been able to get other needed care. as a participant reported, "it [covid-19] just makes things harder. i was not able to go to the dentist to take out my wisdom teeth because they do not do anything that is not a priority." (p6, black/african american, 21 years old). participants had also had challenges with virtual appointments (e.g., telemedicine). for example, a participant shared, "for the follow-up appointment i had to see the doctor through zoom, but he couldn't really see me and that made things more challenging, especially because the technology wasn't really working. things are more complicated now." (p14, black/african american, 27 years old) overall, participants reported obtaining hiv medications as needed since the initiation of the pandemic. however, they did report that adhering to medication regimens had become more di cult. a participant shared, "it's been easy to get the medications because the pharmacy sends them, but it has been challenging to take them as i should because i had a routine before. i used to take my medication every day at work, but since i am working from home, i can't follow the same routine." (p15, white, 27 years old) participants reported multiple ways in which covid-19 affected them through social determinants of health. first, some participants noted that they had lost educational opportunities. a participant reported, "i was supposed to have an internship this summer and make money. it's been very stressful, and i am not having a good summer." (p6, black/african american, 21 year old). another participant described losing his job, "i have been laid off from work till further notice, so i have not been working. so it is affecting me that way." (p10, white, 28 years old) participants felt the nancial impact of job losses on other social determinants, such as housing stability. a participant shared, "ya empiezo a tener di cultad; no sé cómo voy a hacer este mes para pagar mi renta." ["i am already having di culty; i don't know what i am going to do this month to pay my rent."] (p5, latinx, 25 years old) participants who had jobs and were able to continue working during the stay-at-home order felt fortunate to be able to maintain an income. however, they also worried about exposure to covid-19 when ful lling roles as essential workers. a participant noted, "i think what makes it challenging is that i work in the public and interact with people every day, and i am not sure where they have been and if they have covid-19 or not. i just think that interacting with people who might have it on a daily basis makes it hard." (p14, black/african american, 27 years old) participants reported a reduction in in-person social support. a participant noted, "it is affecting my interactions with other people; i do not see my family and friends anymore." (p9, white, 23 years old). another remarked on his loneliness saying, "there are times when i think, if i had a boyfriend or a roommate, someone that at least can stay in quarantine with me, that will be better. i mean, i have a cat, but it is just hard sometimes." (p10, white, 28 years old) the economy and its impact on self, families, and friends are concerns participants also reported worrying about the long-term implications of the pandemic for local, regional, and global economies. as a participant shared, my biggest concern is [that] the way that it is happening here will have a devastating impact on the economy, just because it's been so bad. i have such a big fear that this will reach areas of underdeveloped counties where the health system is even weaker than ours, and they will not be able to handle that. in addition, i think that when the economy is not doing well in america, the whole world economy falls after that. (p7, black/african american, 23 years old) participants also worried that states may be re-opening businesses and institutions before the covid-19 pandemic was under control, which could lead to more transmission. as a participant noted, "they are leaving it to the states to reopen or not, so i am worried about its [covid-19] being spread." (p9, white, 23 years old) discussion plwh may be at increased risk for severe covid-19-related illness. in this qualitative study of a sample of racially/ethnically diverse gbmsm living with hiv, we identi ed 17 themes that we grouped into six domains. several ndings deserve further attention. first, participants had high levels of knowledge about the transmission and prevention of covid-19. while participants reported some confusion about con icting and emerging information related to covid-19, they utilized multiple and credible sources to obtain information about the pandemic and risk reduction. this nding could re ect, in part, their past participation in the wecare intervention, which improves health literacy by helping participants identify, evaluate, and use online sources of health information, including cdc and hrsa websites (21, 23, 24) . thus, despite our sample knowing quite a bit about covid-19, many plwh may need support in identifying and accessing correct and updated information to manage their health in light of the dissemination of con icting information regarding covid-19. further, hiv healthcare providers were identi ed as highly trusted sources of information about covid-19. providers must make time to assess covid-19-related information needs, correct misinformation, and support risk reduction among their patients living with hiv. participants did not, however, trust information provided by u.s. president. participants distinguished between the veracity of information about covid-19 provided by the u.s. president and information about covid-19 provided by other government leaders such as dr. anthony fauci, the director of the national institute of allergy and infectious diseases (niaid) and a member of the white house coronavirus task force. it is important to note that this study was conducted early in the covid-19 pandemic, and many participants had not yet navigated medical care, such as a routine o ce visit for hiv care, since the implementation of changes in care delivery related to covid-19. however, some participants reported having non-urgent healthcare visits canceled or postponed, and those participants who had obtained care reported not being satis ed with telemedicine appointments. covid-19 has required acceleration of telemedicine, and much is being learned about how best to provide care using this distance platform (26). as telemedicine becomes more widespread, concerns about con dentiality, access to and quality of care, reliable internet access, and health disparities must be considered. in our research with gbmsm living with hiv in the u.s. south, we have found that most own or have access to smartphones; however, most do not have access to desktop computers or laptops at home. thus, they may not have the requisite technology and/or a private place to participate in telemedicine visits that utilize a video component (21, 23, 24, 27, 28) . transition to telemedicine could potentially reduce hiv care engagement and viral suppression and increase disparities among some of the most vulnerable populations living with hiv. current efforts to improve telemedicine technology and the integration of telemedicine into clinic settings should include targeted efforts to meet the needs of vulnerable populations, including plwh. moreover, while covid-19 did not seem to affect access to hiv medications in this study, the impact of the pandemic affected adherence to medical regimens. interventions may be needed to help plwh strategize how and when they can take their hiv medications given interruption to their routines due to covid-19 (e.g., changes in job and school schedules). plwh may be particularly affected by covid-19 because they may already be experiencing stigma and isolation related to living with hiv (9) . participants noted that living through the covid-19 pandemic felt like when they rst learned of their hiv status, with similar feelings of loneliness and having no one to turn to. they reported limiting their interactions with others to reduce their risk of exposure; however, they subsequently reported feeling alone, isolated, and in need of social support. these feelings can lead to hopelessness and depression and negatively affect medication adherence and health outcomes (29). to address these challenges, interventions may be needed to bolster social support among plwh while maintaining social distancing. given that participants reported using social media at increasing levels since the initiation of the pandemic, approaches that leverage these platforms, such as those used in wecare, may hold particular promise in this context. participants also noted that the covid-19 pandemic resulted in missed educational opportunities, job loss, and nancial hardship. moreover, their worries were broad; they expressed concerns about themselves as well as others who they care about and the impact of the pandemic on the global economy. interventions are needed to address the varied and pervasive harms, health-related and otherwise, caused by the covid-19 pandemic on plwh, and also the underlying social determinants of health that can make plwh more vulnerable to these harms. it is important to note that participants in this study do not re ect the entire population of plwh or plwh in the u.s. south. participants represented a unique sample and may have bene ted from their participation in wecare. further studies are needed with participants that have not participated in such an intervention. we also note the small sample size; however, we purposefully recruited a racial/ethnically diverse sample and terminated data collection when we reached saturation. we also note that we wanted to better understand the breadth of experiences of plwh; future studies should focus on quantifying the experiences identi ed and developing targeted interventions for plwh based on these ndings. this study was designed to be a rst, early step. this study tapped into a small group of racially/ethnically diverse gbmsm living with hiv. it lays the foundation for future research exploring both the immediate and long-term impacts of the covid-19 pandemic among gbmsm living with hiv. additionally, there is profound need for novel interventions to address the impact of the covid-19 pandemic on social isolation, economic stability, access to health care, and other social determinants of health for racially/ethnically diverse gbmsm living with hiv. because increased age is a risk factor for severe illness from covid-19, and nearly half the plwh in the u.s. are over 50 (30), further indicating the potential of severe covid-19-related illness among this population, there is also need for targeted research to understand the impact of the pandemic on older plwh and interventions to support older plwh's needs related to covid-19. these data were collected within the rst three months after the rst covid-19 case was reported in nc and provide important insights about the impact of this rapidly emerging public health issue. since then, new developments have occurred, including a statewide mandate requiring the use of face coverings in public places that went in to effect in nc on june 24, 2020. at the same time, while participants reported engaging in prevention behaviors initially, their adherence with social distancing, face coverings, and use of disinfectants and sanitizers may decrease over time as fatigue sets in. it will be important to build on these early ndings within the continually changing context in terms of covid-19 infection rates, economic impacts, and state and federal government responses. the authors declare that they have no con ict of interest. all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional review board of the wake forest school of medicine and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. this article does not contain any studies with animals performed by any of the authors. informed consent was obtained from all individual participants included in the study. the economy and its impact on self, families, and friends are concerns states may be "opening up" too quickly southern states are now the epicenter of hiv/aids in the us. the washington post centers for disease control and prevention. hiv in the southern united states nc: guilford county department of health & human services social support and other factors associated with hiv testing by hispanic/latino gay, bisexual, and other men who have sex with men in the north carolina hiv/std surveillance report north carolina department of health and human services pathophysiology, transmission, diagnosis, and treatment of coronavirus disease evaluation and treatment coronavirus the burden of covid-19 in people living with hiv: a syndemic perspective north carolina identi es first case of covid-19. raleigh, nc: nc department of health and human services stay-at-home order issued for north carolina. the news & observer evidence used to update the list of underlying medical conditions that increase a person's risk of severe illness from covid-19 clinical outcomes and immunologic characteristics of covid-19 in people with hiv global, regional, and national estimates of the population at increased risk of severe covid-19 due to underlying health conditions in 2020: a modelling study estimating the prevalence of hiv and sexual behaviors among the us transgender population: a systematic review and meta-analysis characterizing the impact of covid-19 on men who have sex with men across the united states in april hiv prevention interventions with adolescents: innovations and challenges in partnerships across the integrated transitions model coronavirus disease 2019 case surveillance -united states assessing differential impacts of covid-19 on black communities cbpr to prevent hiv within ethnic, sexual, and gender minority communities: successes with long-term sustainability preliminary impact of the wecare social media intervention to support health for young men who have sex with men and transgender women with hiv a randomized controlled trial of a culturally congruent intervention to increase condom use and hiv testing among heterosexually active immigrant latino men a social media-based intervention designed to increase hiv care linkage, retention, and health outcomes for racially and ethnically diverse young msm supporting health among young men who have sex with men and transgender women with hiv: lessons learned from key: cord-034714-6e37yylk authors: alleg, manel; solis, morgane; baloglu, seyyid; cotton, françois; kerschen, philippe; bourre, bertrand; ahle, guido; pruvo, jean-pierre; leclerc, xavier; vermersch, patrick; papeix, caroline; maillart, élisabeth; houillier, caroline; chabrot, cécile moluçon; claise, béatrice; malak, sandra; martin-blondel, guillaume; bonneville, fabrice; caulier, alexis; marolleau, jean-pierre; bonnefoy, jérôme tamburini; agape, philippe; kennel, céline; roussel, xavier; chauchet, adrien; de seze, jérôme; fafi-kremer, samira; kremer, stéphane title: progressive multifocal leukoencephalopathy: mri findings in hiv-infected patients are closer to rituximabthan natalizumab-associated pml date: 2020-11-06 journal: eur radiol doi: 10.1007/s00330-020-07362-y sha: doc_id: 34714 cord_uid: 6e37yylk objectives: to compare brain mri findings in progressive multifocal leukoencephalopathy (pml) associated to rituximab and natalizumab treatments and hiv infection. materials and methods: in this retrospective, multicentric study, we analyzed brain mri exams from 72 patients diagnosed with definite pml: 32 after natalizumab treatment, 20 after rituximab treatment, and 20 hiv patients. we compared t2or flair-weighted images, diffusion-weighted images, t2*-weighted images, and contrast enhancement features, as well as lesion distribution, especially gray matter involvement. results: the three pml entities affect u-fibers associated with low signal intensities on t2*-weighted sequences. natalizumab-associated pml showed a punctuate microcystic appearance in or in the vicinity of the main pml lesions, a potential involvement of the cortex, and contrast enhancement. hiv and rituximab-associated pml showed only mild contrast enhancement, punctuate appearance, and cortical involvement. the cd4/cd8 ratio showed a trend to be higher in the natalizumab group, possibly mirroring a more efficient immune response. conclusion: imaging features of rituximab-associated pml are different from those of natalizumab-associated pml and are closer to those observed in hiv-associated pml. key points: • nowadays, pml is emerging as a complication of new effective therapies based on monoclonal antibodies. • natalizumab-associated pml shows more inflammatory signs, a perivascular distribution “the milky way,” and more cortex involvement than rituximaband hiv-associated pml. • mri differences are probably related to higher levels of immunosuppression in hiv patients and those under rituximab therapy. progressive multifocal leukoencephalopathy (pml) is a rare demyelinating disease of the central nervous system caused by reactivation of the jc polyomavirus (jcv). jcv is a ubiquitous polyomavirus, present in nearly 80% of the adult population [1] . primary infection is asymptomatic and leads to viral persistence in infected hosts in different sites such as bone marrow and kidneys. jcv reactivation is triggered by a decrease in immunity [2] , more particularly in cell-mediated immunity [3] . pml incidence increased notably during the 1980s with the hiv pandemic supporting the hypothesis of the essential role played by cell-mediated immunity [1, 4] . nowadays, pml is emerging as a complication of new effective immunosuppressive and immunomodulatory drugs [5] . natalizumab is used as a treatment for multiple sclerosis (ms) and is associated with an increased risk of pml, estimated at 1/100 to 1/10,000 based on three identified risk factors (treatment duration longer than 24 months, anti-jcv antibody index, and prior administration of immunosuppressive therapies) [6] . anti-jcv antibody levels in serum are indeed used to stratify pml risk in anti-jcv antibody-positive ms patients [6] . natalizumab is a monoclonal antibody targeting the alpha-4 integrin that inhibits transmigration of t cells, including cd8 t cells, to and in the central nervous system, which can explain in part its role in pml through a decrease of immune control of jcv in the brain [1] . two other characteristics are that natalizumab forces the migration of hematopoietic stem cells from the bone marrow in which jcv may be persistent and the upward regulation of b cell maturation factors that promote jcv growth [7] . rituximab, an anti-cd20 monoclonal antibody targeting b cell activity and currently used for hematological malignancies and autoimmune disorders, is also associated with a moderately increased risk of pml estimated at 1/32,000 [8] . rituximab acts by depleting b cells and modifying t cell activity, all of which supports a role of rituximab in pml development [9] . knowledge of the mri pattern of pml is essential when this diagnosis is suspected, especially in populations considered at risk, because the prognosis remains poor and is based on early treatment [10] , which consists exclusively so far in restoring the immune system. in the classic form widely described in the hiv population, pml manifests itself as white matter (wm) lesions affecting the u-shaped subcortical fibers with a decreased signal on t1weighted sequences and an increased signal on t2-weighted sequences. on diffusion-weighted images (dwi), the signal is generally increased with restricted diffusion on borders corresponding to active demyelination. low signal on magnetic susceptibility sequences in u-fibers adjacent to the wm lesions of pml has been recently described and appears in relation to intracellular accumulation of iron in macrophages and glial cells during myelin degeneration [15] . those lesions are often not enhanced and have little or no mass effect [11] . previous studies have shown differences in the mri pattern with natalizumab-associated pml (table 1) . indeed, with natalizumab, there are various inflammatory signs such as contrast enhancement and small punctuations in or adjacent to lesions with an increased signal on t2-weighted images and a perivascular distribution called "the milky way" [2, 16, 17] . the lesions have ill-defined boundaries towards wm and sharp boundaries towards gray matter (gm), with cortical gm more frequently involved [18] . a hypothesis to explain these results would be the difference in the level of immunosuppression induced by hiv and natalizumab. in this study, we first aimed to compare the mri presentation of rituximab-associated pml to natalizumab or hivassociated pml, and also analyze biological parameters to assess if the differences in mri could be the consequence of different immunosuppression levels. this retrospective study was approved by our institutional review board. inclusion criteria were (1) a "definite" pml diagnosis according to the american academy of neurology criteria [19] including clinical and imaging-compatible features and detection of jcv dna in the cerebrospinal fluid or in brain tissue by polymerase chain reaction (pcr) or immunohistochemistry; (2) hiv-infected patients or patients treated with immunomodulatory or immunosuppressive drugs such as natalizumab or rituximab, possibly in association with other drugs and whatever the initial illness; and (3) consent was provided for in the hospital's charter. exclusion criteria were patients diagnosed with pml from other causes than hiv infection or associated with other drugs than natalizumab or rituximab and those under 18 years of age. the occurrence of immune reconstitution inflammatory syndrome (iris) or an opportunistic infection was not a cause for exclusion. classical demographic data such as gender, age at the time of diagnosis, and underlying illness were collected, as well as cd4 and cd8 numbers. between 2008 and 2018, 87 patients from 16 centers in france and luxembourg were retrospectively identified (fig. 1 ). of these 87 patients, 72 fulfilled all the inclusion criteria and were therefore included: 32 patients had relapsing-remitting ms and were treated with natalizumab, 20 patients were treated with rituximab (16 had a hematopoietic malignancy, 2 an autoimmune disorder, and 2 an immune deficiency), and 20 patients had an hiv infection. all patients underwent a multisequence mri protocol. due to the study's multicenter design, the explorations were carried out with diverging sequences, magnetic fields strength, acquisition parameters and parameters related to spatial resolution. we chose to define the first brain mri performed at suspicion of pml as the reference examination for all comparisons. all mris were unblinded and reviewed by a junior radiologist (a trainee) with the assistance of an experienced senior neuroradiologist. to compare pml lesions, we described the location (frontal lobe, parietal lobe, occipital lobe, temporal lobe, basal ganglia, supra-or infratentorial or both), involvement of cortex and/or deep gm, distribution (unilobar involving a single lobe, multilobar involving more than one contiguous lobe, widespread involving more than two contiguous lobes or noncontiguous lobes or bilaterality), boundaries (sharp towards wm and gm, ill-defined towards wm and gm), lesion characteristics (aspect on t2-weighted images defined as homogeneous or microcystic corresponding to small lesions in or adjacent to the main pml lesions with an increased signal called the milky way; the presence or absence of a hyposignal on t2*/swi/swan-weighted images, signal on dwi, and statistical analysis was performed using r software, version 3.1, via the gmrc shiny stat application of the strasbourg university hospital (2017) and using the xlstat software in its addinsoft 2016 version. we compared the mri findings between the three groups using the chi-square test and between the nat/ritux, nat/hiv, and ritux/hiv groups using the chi-square test with holm correction. we compared biological data between the three groups using the shapiro test and between the nat/ritux, nat/hiv, and ritux/hiv groups using the student t test with holm correction. median patient age (for all groups) was 49.0 years (range, . in the patient groups, the median age was 42.62 years for the natalizumab group (range, 26-53), 61.5 years for the rituximab group (range, 40-76) and 46.9 for the hiv group (range, 26-64). forty men (55.6%) and 32 women (44.4%) were included in the study: 14 men (43.8%) and 18 women (56.2%) in the natalizumab group, 10 men and 10 women (50% each) in the rituximab group, and 16 men (80%) and 4 women (20%) in the hiv group. the imaging results are summarized in table 2 and table 3 . there was a statistically significant difference in supratentorial involvement between the three groups (78.1% in the natalizumab group, 85% in the rituximab group, and 40% in the hiv group (p = 0.003), especially when comparing the natalizumab group vs the hiv group (p = 0.0269) and the rituximab group vs the hiv group (p = 0.0269) with fewer supratentorial lesion locations in cases of pml occurring in an aids context. concerning gm involvement, there was a significant difference between the three groups for the cortex (p < 0.001) and for the basal ganglia (p = 0.025). post hoc analysis was only contributive for cortex involvement and showed a difference between the natalizumab group vs the hiv/rituximab groups with more cortex involvement in cases of pml that occurred under natalizumab treatment than the other two groups (p = 0.0036 for natalizumab vs rituximab and p = 0.0084 for natalizumab vs hiv). analysis of the edges towards both wm and gm (fig. 2 ) showed significant differences between groups. indeed, lesions were more frequently clearly defined towards gm and ill-defined towards wm in the natalizumab group with no significant difference between the rituximab and hiv groups. the milky way aspect on t2-or flair-weighted images ( fig. 2) was significantly different between the three groups (p = 0.02) and more often seen in the natalizumab group vs the rituximab group (51.6% for natalizumab and 5% for rituximab [p = 0.005]). on dwi, the vast majority of pml lesions had an increased signal (90.9% for natalizumab, 89.5% for rituximab and 100% for hiv). we isolated cases with a peripheral signal in restriction of diffusion (corresponding to a decrease of the signal on adc mapping), called the demyelination front. for this parameter, we observed a significant difference between natalizumab/rituximab (22.7% for natalizumab, 63.2% for rituximab [p = 0.0426]) and natalizumab/hiv (22.7% for natalizumab, 84.2% for hiv, [p < 0.001]) but none between the rituximab and hiv groups. (fig. 3) . lesions with a peripheral contrast enhancement showed a significant difference between the three groups (p = 0.003) and tended fig. 1 flow chart illustrating the patient selection and inclusion process. pml = progressive multifocal leukoencephalopathy, aan = american academy of neurology to predominate in the rituximab group (45% in the rituximab group vs 19.4% in the natalizumab group and 10% in the hiv group). lesions with micronodular enhancement were significantly different between the three groups (p = 0.003), with more cases in the natalizumab group versus the rituximab group (p = 0.0187). there was no difference between natalizumab and hiv groups even if there were more lesions of this type in the natalizumab group (51.6% vs 20% in the hiv group). finally, for distant enhancement, there was a difference between the three groups (p < 0.001), specifically between natalizumab and rituximab (p = 0.0134) and the natalizumab and hiv (p = 0.0358) groups, with more of this type of enhancement in the natalizumab group (fig. 4) . non-enhanced lesions were significantly different between the three groups (p = 0.015), higher in the hiv groups with 80%. cd4/cd8 data were available for 10 patients in the natalizumab group, 12 in the rituximab group, and 15 in the hiv group (fig. 5) this study aims to describe the mri characteristics of pml associated with rituximab and natalizumab and in hiv infection while comparing imaging findings with the level of immunosuppression. we therefore compared cd4/cd8 ratios and found lower ratios in hiv-related pml compared to other groups, as well as a lower cd4/cd8 ratio in the rituximab group compared to the natalizumab group. this suggests that immunosuppression in rituximab is not as severe as in hiv-pm but still stronger than in natalizumab-pml. one of the characteristics of pml under natalizumab described in previous studies [2, 22] is the description of inflammatory signs such as microcystic lesions on t2-weighted images giving a milky way appearance [20, 23] . we found significantly more milky way lesions in the natalizumab group compared to the rituximab group but no significant difference between the natalizumab and hiv groups, even if they tended to predominate in the natalizumab group. the other "inflammatory" sign usually attributed to natalizumab-pml is contrast enhancement, estimated at 35 to 40% of patients in the literature [2] . in the present study, it accounted for 61.3% of the natalizumab group. a difference was observed between the three groups, especially when the enhancement was micronodular, similar to the milky way perivascular distribution predominating in the natalizumab group compared to the rituximab and hiv groups. by inducing a lower degree of immunosuppression compared to hiv, natalizumab-associated pml has more inflammatory signs. this could be due to the mode of action of this drug, which is a more selective immunomodulating agent [17] . we found more supratentorial involvement in drug-related pml than in the hiv-associated form where the brainstem and cerebellum are more likely to be affected. cortical involvement and the typical aspect of the edges (sharp towards gm and ill-defined towards wm) are found preferentially in the natalizumab group, as also found in previous studies [18, 22] . there is no obvious explanation for these characteristics in relation to a difference in the level of immunosuppression. the proportion of hyposignal seen on t2*-or swanweighted images at the cortical-subcortical junction adjacent to pml lesions (fig. 6 ) did not differ between pml groups, in agreement with other studies [15, 21, 24] ; this sign seems nonspecific in pml for the underlying cause of immunosuppression. a demyelination front on diffusion-weighted images is often found with pml in the hiv group and in the rituximab group but more rarely in the natalizumab group. if the demyelination front is related to the active nature of the disease, we can hypothesize that the pml associated with rituximab is more like hiv-pml than natalizumab-pml, possibly due to closer immunosuppression levels [25] . the main limitations of this study are its retrospective and unblinded design as well as the limited number of patients analyzed with complete clinical, biological, and available imaging data. this is explained by the relative rarity of pml even in large referral centers. secondly, due to the multicenter design of the study, the explorations were performed on different mris, both 1.5 t and 3 t and with nonstandardized protocols, and there were some inevitable variations in mri acquisition parameters between patients [26] . regarding the demographic data, we note that the age of the patients differs according to the group, with a tendency to younger patients in hiv and natalizumab groups. this may constitute a selection bias and is explained by the epidemiological data of the pathology studied, with ms and hiv affecting younger people compared to hematological malignancies. fig. 3 in several cases of natalizumab-associated pml showing in (a) on flair-weighted images, we can see gm and especially cortical involvement of the left parietal lobe adjacent to the wm lesion. the left thalamus is also the site of a demyelinating lesion. in b, we observe on t2-weighted images the milky way aspect with microcystic lesions with an increased signal. in c, clear sharp edges towards gm and ill-defined edges towards wm of the demyelinating lesions. in d, e, and f, brain mri from a patient with natalizumab-associated pml showing in (d) a demyelinating right frontal lesion on flair-weighted image with (e) contrast micronodular enhancement in the lesion and (f) remotely in the right temporal lobe. finally, we can see an increased signal on diffusion-weighted images (h) with no rim in restriction on adc mapping (g) mri interpretation was made difficult by the coexistence of inflammatory lesions in the natalizumab group related to ms as well as by the coexistence of opportunistic infections (notably cerebral toxoplasmosis for three patients in the hiv group) and the occurrence of immune reconstitution inflammatory syndrome (in three fig. 4 an example of a hiv-pml case with an infratentorial right cerebellar demyelinating lesion on a. in b, no contrast enhancement of this lesion. diffusion-weighted images and adc mapping of a patient showing an increased signal on diffusion (c) with a rim of demyelination in restriction of diffusion (d) fig. 5 box diagram of the extent of the cd4/cd8 ratio of patients by group patients in the hiv group after initiation of antiretroviral therapy). concerning inclusion criteria, the vast majority of patients in the natalizumab group had already received immunosuppressive treatments before natalizumab treatment was initiated, as had those in the rituximab group, who often had several treatments acting on immunity before or at the same time as rituximab. however, in the present study, we did not make any distinction in the groups according to these possible treatments. their impact on the mri pattern of pml lesions is therefore not excluded. two other factors may have come into play in the mri pattern of pml under rituximab that we did not consider: first, the initial disease of the patient, which could be responsible for the development of pml, especially in the case of hematopoietic malignancies [27] . secondly, the rigorous mri follow-up of patients in the natalizumab group made it possible to detect pml at an earlier stage than in the hiv and rituximab groups. adequate assessment of the quantitative and functional aspects of the global immune response is still a challenge in immunocompromised patients. in hiv-infected patients, the cd4/cd8 ratio is considered to be a good marker of the remaining immune response but may not be as relevant in other immunosuppressed groups such as patients treated with natalizumab or rituximab, irrespective of the underlying disease. the cd4/cd8 ratio does not incorporate the full level of immunosuppression-due to the diversity and complexity of the immune system; in our study, it indirectly and partially reflects the patient's immune status. imaging features of rituximab-associated pml generally look more similar to those of the classically described pml with hiv than those related to natalizumab. those similarities may be explained by a higher level of immunosuppression in hiv patients and patients treated with rituximab compared to patients treated with natalizumab. statistics and biometry nicolas tuzin kindly provided statistical advice for this manuscript. informed consent written informed consent was not required for this study because patient consent was provided for in the hospital's charter. ethical approval institutional review board approval was obtained. • prospective • observational • multicenter study progressive multi-focal leucoencephalopathydriven from rarity to clinical mainstream by iatrogenic immunodeficiency the chameleon of neuroinflammation: magnetic resonance imaging characteristics of natalizumab-associated progressive multifocal leukoencephalopathy progressive multifocal leukoencephalopathy and immune reconstitution inflammatory syndrome (iris) human polyomavirus reactivation: disease pathogenesis and treatment approaches anti-jc virus antibodies: implications for pml risk stratification stratification and monitoring of natalizumab-associated progressive multifocal leukoencephalopathy risk: recommendations from an expert group pathogenesis of progressive multifocal leukoencephalopathy and risks associated with treatments for multiple sclerosis: a decade of lessons learned progressive multifocal leukoencephalopathy and monoclonal antibodies: a review progressive multifocal leukoencephalopathy secondary to rituximab-induced immunosuppression and the presence of john cunningham virus: a case report and literature review outcome and survival of asymptomatic pml in natalizumab-treated ms patients imaging manifestations of progressive multifocal leukoencephalopathy common and uncommon imaging findings in progressive multifocal leukoencephalopathy (pml) with differential diagnostic considerations magnetic resonance imaging pattern in natalizumab-associated progressive multifocal leukoencephalopathy immune reconstitution inflammatory syndrome unmasking or worsening aids-related progressive multifocal leukoencephalopathy: a literature review. front immunol 8:577 low signal intensity in u-fiber identified by susceptibility-weighted imaging in two cases of progressive multifocal leukoencephalopathy natalizumabassociated progressive multifocal leukoencephalopathy in patients with multiple sclerosis: lessons from 28 cases inflammatory natalizumab-associated pml: baseline characteristics, lesion evolution and relation with pml-iris mri pattern in asymptomatic natalizumab-associated pml pml diagnostic criteria: consensus statement from the aan neuroinfectious disease section punctate lesion pattern suggestive of perivascular inflammation in acute natalizumab-associated progressive multifocal leukoencephalopathy: productive jc virus infection or preclinical pml-iris manifestation? brain magnetic susceptibility changes in patients with natalizumab-associated progressive multifocal leukoencephalopathy neuroimaging of natalizumab complications in multiple sclerosis: pml and other associated entities punctate pattern: a promising imaging marker for the diagnosis of natalizumabassociated pml susceptibility-weighted mr imaging hypointense rim in progressive multifocal leukoencephalopathy: the end point of neuroinflammation and a potential outcome predictor progressive multifocal leukoencephalopathy: diffusion-weighted imaging and pathological correlations imaging working group of the observatoire français de la sclérose en plaques. ofsep, a nationwide cohort of people with multiple sclerosis: consensus minimal mri protocol progressive multifocal leukoencephalopathy and anti-cd20 monoclonal antibodies: what do we know after 20 years of rituximab publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments french network for ocular and cns lymphoma (loc) and nord-est neuro-oncoly (neno) organisation.funding the authors state that this work has not received any funding. guarantor the scientific guarantor of this publication is stephane kremer. the authors of this manuscript declare no relationships with any companies whose products or services may be related to the subject matter of the article. key: cord-264884-ydkigome authors: villarreal, luis p. title: the widespread evolutionary significance of viruses date: 2008-07-05 journal: origin and evolution of viruses doi: 10.1016/b978-0-12-374153-0.00021-7 sha: doc_id: 264884 cord_uid: ydkigome in the last 30 years, the study of virus evolution has undergone a transformation. originally concerned with disease and its emergence, virus evolution had not been well integrated into the general study of evolution. this chapter reviews the developments that have brought us to this new appreciation for the general significance of virus evolution to all life. we now know that viruses numerically dominate all habitats of life, especially the oceans. theoretical developments in the 1970s regarding quasispecies, error rates, and error thresholds have yielded many practical insights into virus–host dynamics. the human diseases of hiv-1 and hepatitis c virus cannot be understood without this evolutionary framework. yet recent developments with poliovirus demonstrate that viral fitness can be the result of a consortia, not one fittest type, a basic darwinian concept in evolutionary biology. darwinian principles do apply to viruses, such as with fisher population genetics, but other features, such as reticulated and quasispecies-based evolution distinguish virus evolution from classical studies. the available phylogenetic tools have greatly aided our analysis of virus evolution, but these methods struggle to characterize the role of virus populations. missing from many of these considerations has been the major role played by persisting viruses in stable virus evolution and disease emergence. in many cases, extreme stability is seen with persisting rna viruses. indeed, examples are known in which it is the persistently infected host that has better survival. we have also recently come to appreciate the vast diversity of phage (dna viruses) of prokaryotes as a system that evolves by genetic exchanges across vast populations (chapter 10). this has been proposed to be the “big bang” of biological evolution. in the large dna viruses of aquatic microbes we see surprisingly large, complex and diverse viruses. with both prokaryotic and eukaryotic dna viruses, recombination is the main engine of virus evolution, and virus host co-evolution is common, although not uniform. viral emergence appears to be an unending phenomenon and we can currently witness a selective sweep by retroviruses that infect and become endogenized in koala bears. phylogenetic tools have greatly aided our analysis of virus evolution, but these methods struggle to characterize the role of virus populations. missing from many of these considerations has been the major role played by persisting viruses in stable virus evolution and disease emergence. in many cases, extreme stability is seen with persisting rna viruses. indeed, examples are known in which it is the persistently infected host that has better survival. we have also recently come to appreciate the vast diversity of phage (dna viruses) of prokaryotes as a system that evolves by genetic exchanges across vast populations (chapter 10). this has been proposed to be the " big bang " of biological evolution. in the large dna viruses of aquatic microbes we see surprisingly large, complex and diverse viruses. with both prokaryotic and eukaryotic dna viruses, recombination is the main engine of virus evolution, and virus host co-evolution is common, although not uniform. viral emergence appears to be an unending phenomenon and we can currently witness a selective sweep by retroviruses that infect and become endogenized in koala bears. our understanding of virus evolution has reached a threshold in that it now appears to provide a much broader vista regarding its general signifi cance and infl uence on host evolution. several developments have brought us to this point. one has been the realization that viruses often evolve by processes involving the collective action of a consortia, or quasispecies. and the resulting adaptability and power of such evolution is unmatched by any other genetic entity. much of this volume is dedicated to this issue. in such consortia, the concept of a " wild-type " virus is no longer considered to be the fi ttest type, as the quasispecies itself provides fi tness (see chapters 3 and 4). the quasispecies model resembles population genetics in some ways, but it has led to some signifi cant departures from population genetics, and these departures are very well supported by experiments. another development that has recalibrated our view of the overall signifi cance of viruses is information on the scale and diversity of viruses. viruses are present at a previously unappreciated global level and appear to have affected the evolution of all life on earth. much of this realization has been brought about by the development of metagenomic methods as applied to various habitats. measurements of major habitats (the oceans, soil, extreme environments) have established that our biological world is predominantly viral, in terms of both numbers and diversity ( paul et al. , 2002 ; breitbart et al. , 2003 ; rohwer, 2005a, 2005b ; edwards and rohwer, 2005 ; comeau et al. , 2006 ) . these two developments would seem reason enough to consider virus evolution in a new light. however, there have also been numerous theoretical proposals suggesting viral involvement in some of the very earliest events and major transitions in the evolution of life. we no longer think of viruses as recent agents that escaped from the host chromosomes as run away replicons. viruses now appear very old to us and they relate to and trace all branches of life. the last 30 years have been very active regarding virus evolution. major developments in theory, technology, medicine, and the study of human disease with respect to virus evolution have all occurred. and as we seek to grow and manage various life forms for human use, virus evolution has also had major impact on such efforts. as a science, virus evolution has benefi tted greatly from traditional evolutionary biology. however, since viruses are molecular genetic parasites that are inscrutable by casual observation, our understanding of virus evolution has been dependent upon measuring sequence variation and sequence diversity in a large number of virus genomes. because of this, these small genetic parasites have been the last domain of life to yield their secrets of evolution. and viruses harbor some clearly distinct evolutionary abilities. for one, they are polyphyletic. all major viral lineages have their own distinct origins. they are also difficult if not impossible to defi ne as species and are able to exchange genetic information across normal boundaries. even " dead " and defective viruses can participate in such exchanges, which confuses the defi nitions of fi tness. we now know that viruses can evolve by a consortia process and also exchange information by recombination across vast genetic pools to assemble new mosaic combinations of genes. we thus no longer think of a specifi c genetic lineage in understanding virus evolution, but instead think of a cloud, matrix, or a population as the basis of virus evolution. viruses are inherently fuzzy entities that can differ from their relatives in any specifi c feature. yet even with such fuzziness, it is clear that common themes also link them. patterns of evolution have become clear. diversity and variation are often (but not always) observed. stability and host congruence can also be observed. nevertheless, the evolutionary power of viruses has been learned at a human cost. the application of numerous analytical and phylogenetic tools have provided crucial insights into virus origin and evolution. yet these methods struggle to incorporate the fuzzy nature of viruses and have clear limits, especially regarding quasispecies and high recombination rates. structural biology now also adds tools that extend our vision of virus evolution beyond what can be seen in the genetic sequence. for example, common structural motifs from phage to eukaryotic dna viruses (t4 and herpesvirus) suggest very ancient links in virus evolution that span all domains of life (see below). nevertheless, our analytical methods are currently lacking as we struggle to understand complex genetic mixtures that provide fi tness, reticulated relationships, polyphyletic origins, and virus-host congruence. virus evolution has, for the most part, been considered to be a specifi c, esoteric part of broader evolutionary biology and has been given limited attention in reference works on evolutionary biology (see pagel, 2002 ) . historically, the focus has been on various rna viruses and some dna viruses that cause disease in humans and domesticated animals and plants . however, i have asserted that all life forms must be examined from the perspective of virus evolution ( villarreal, 2005 ) , not just those pathogens that impact on us. how viruses evolve in a more general sense informs us of evolutionary paradigms that have not been previously well understood (especially the evolution of consortia or the dynamics of vast reticulated gene pools). this volume now extends these traditional topics of virus evolution to include the vast virology of the prokaryotic world. in so doing, it illuminates the global consequences viruses have had on all life forms. prior to the 1970s we saw some stunning successes in vaccine control for major viral human diseases, such as polio, measles, mumps, infl uenza, and especially smallpox virus. due to this historic success, american health agencies and educators considered virus disease as a thing of the past, no longer a serious threat. the era of infectious disease that they represented was now one for the historical record, an unfortunate part of human history, or so we thought. in what now seems to have been a clear case of hubris and naivety, we have been humbled by the evolutionary power of viruses, which was woefully underappreciated, even by most virologists. by the end of that decade, the evolution and emergence of hiv-1 permanently changed our views (see chapters 13 and 14) . this has also been followed by a seemingly never-ending series of viral threats as newly emerging viral diseases have come to our attention. hiv-1 provides the only example of a public health situation that has reversed centuries of progress for extending human health and lifespan. it now limits human life expectancy in many parts of the world, especially sub-saharian africa. this development could not have been imagined in the 1970s. we are much less confi dent now about predicting the future of virus evolution and its potential impact on human health. diseases of domesticated microbiological, plant, and animal species have also experienced the trauma of the consequences of emerging viral diseases along with huge losses. however, the human hiv-1 story may not be a fl uke of virology but may be telling us something basic about human and primate evolution. as we sought to understand the origin of this new virus, we have come to appreciate a much broader virus-host story which involves simian immunodefi ciency virus (siv), foamy viruses, and the speciation of old world primates. we have also come to learn that the genomes of these primates show much evidence of past viral interaction and ongoing endogenous retrovirus colonization. the evolution of retroviral endogenization has taken on a much greater signifi cance in basic evolutionary biology. thus it is with great interest that we now study the ongoing endogenization of retroviruses in the koala bear genome (see below). historically, we are compelled to study viruses because they can cause serious disease. new viruses come to our attention also mainly because of disease. it is therefore understandable that most evolutionary biologists mainly think of viruses strictly as agents of disease. these are the products of run away replicons that provide negative selection to host survival. in this light, the application of predator-prey based mathematical models has seemed most appropriate. with such viral disease, variation has long been observed and was initially used for the generation of most vaccines. however, this disease-centric view has also occluded another more prevalent virus-host relationship. for example the emergence of hiv-1 has led us conclude that it likely evolved from various versions of siv. but siv is not pathogenic in its native african primate host. nor does it show the genetic diversifi cation of hiv-1 in these native primate hosts. it is a silent, asymptomatic infection. genomic and metagenomic analysis now allows us to identify many more silent, asymptomatic viruses that would not have previously been observed. we now know of many such viruses that are prevalent in a specifi c host. evolutionary biology must escape the confi nes of disease-centric thinking and seek to understand these relationships as well. in the last ten years i have attempted to provide another view concerning virus-host evolution. i have argued that viruses often attain evolutionary stability by species-specifi c persistence and that such states apply to all domains of life, including prokaryotes. on an evolutionary time-scale, the majority of viral lineages tend to exist as species-specifi c persistent (aka temperate, latent, and chronic) infections in which individual hosts will be colonized by mostly silent (asymptomatic) viruses for the duration of their life . such persistence can have major consequences to the evolution of both virus and host, which also leads us to more directly link virus evolution to broader issues of host evolution. it is from this perspective that we start to clearly see that viruses indeed belong on the tree of life as major participants ( villarreal, 2005 ( villarreal, , 2006 . persisting viruses are not simply agents responsible for destruction of life, but are also agents that create genetic novelty on a vast scale that infl uences all life and promotes symbiosis ( marquez et al. , 2007 ; ryan, 2007 ; villarreal, 2007 ) . the persistent lifestyle of such symbiotic virus-host relationships is not simply a less effi cient, acute infection; nor is it simply a " reservoir " for acute virus (as epidemiologists are prone to assert). neither can it be attributable to concepts of selfi sh dna. persistence represents a major virus life strategy that is both fundamental and highly adapted. it has distinct genetic, fi tness, and evolutionary characteristics that require intimate, host (tissue)-specifi c viral strategies and precise gene functions to attain stable maintenance in the presence of immunity and to allow biologically controlled reactivation. persistence also must resist displacement by similar viruses and competitors. it is virus-host persistence that provides the thread that allows us to link these polyphyletic viral lineages (and their clouds) with the entire tree of life. in turn, this link identifi es a much more fundamental role for viruses in the evolution of host, visible from the very earliest to the most recent events in host evolution. it is from such species-specifi c persistent states that the large majority of acute diseases evolve and emerge by various mechanisms. we know much about virus replication and disease. however, our understanding of the specifi c mechanisms of persistence is generally poor. persistence is a generally silent and inscrutable state, it does not lend itself to in vitro or cell culture experimental models. we are left with but a few examples from which to attempt to extrapolate the possible existence of general relationships. the study of virus evolution thus struggles to incorporate concepts of persistence. another recent and major development in virus evolution is the arrival of various proposals suggesting that viruses have been involved in some major innovation and transition in the evolution of life. in all these proposals, however, it is necessary that the virus in question has attained a stable genomic persistence with its host. these evolutionary events thus seem to be the products of viral-mediated symbiogenesis of host ( ryan, 2007 ; villarreal, 2007 ) . proposals include the possibility that viruses may have originated the dna replication system of all three cellular domains (archaea, bacteria, eukarya) of life ( forterre, 1999 ( forterre, , 2005 ( forterre, , 2006a ( forterre, , 2006b filee et al. , 2003 ; forterre et al. , 2007 ) . the discovery and analysis of the largest dna virus (1200 orf mimivirus), a lytic cytoplasmic virus of amebae (a distant relative of phycodnavirus and poxviruses), has also led to proposals that this virus lineage may represent an ancient fourth domain of life ( raoult et al. , 2004 ; desjardins et al. , 2005 ; claverie et al. , 2006 ) . it is interesting that in an initial structural analysis, the large complex replication centers for mimivirus were confused with the host nucleus ( suzan-monti et al. , 2007 ) . thus, it seems relevant that others have proposed that a distant relative of phycodnaviruses and poxviruses may have originated the eukaryotic nucleus ( villarreal, 1999 ; villarreal and defilippis, 2000 ; bell, 2001 bell, , 2006 takemura, 2001 ) . such proposals, although consistent with various observations, however, remain outside of the consensus of most evolutionary biologists. nevertheless, numerous other observations continue to suggest viral involvement in other major host innovations, such as a viral origin of rag1/2 of the adaptive immune system ( dreyfus et al. , 1999 kapitonov and jurka, 2005 ; fugmann et al. , 2006 ) or the role of endogenous retroviruses (ervs) in the evolution of the placenta ( villarreal and villareal, 1997 ; harris, 1998 ; blond et al. , 2000 ; mi et al. , 2000 ; dupressoir et al. , 2005 ; caceres and thomas, 2006 ) . such possible roles for viruses in host evolution are at odds with accepted views of virus-host relationships, but might be the products of viral symbiogenesis. the metagenomic viral measurements mentioned above for prokaryotic dna viruses, along with the increasing realization that viruses and host can co-evolve, has led to various calls that a viral tree of life needs to be considered and developed ( forterre, 2003 ; villarreal, 2006 ; filee et al. , 2006b ) . a virosphere clearly exists but its nature and boundaries are not so clear. multiple viral origins, their diversity and numerical dominance in distinct and sometimes harsh environments as well as their presence in host genomes suggest that any viral tree of life will be huge, multidimensional, and connected to the host tree of life. as discussed below regarding double-stranded (ds)dna viruses of prokaryotes, they have all the above characteristics. such viruses may represent the big bang of biological novelty. with their unmatched capacity to generate diversity they can function as the mass creators of biological novelty as well as destroyers of most species. surely, such capacity must have had big infl uences on the evolution of life. symbiosis, simply defi ned, is the stable coexistence of two previously separate lineages of organisms. there can be little doubt that many temperate phage can stably colonize a bacterial cell resulting in a stable descendent from two lineages. this is clearly symbiotic. endogenous retroviruses can similarly be found to persist in vertebrate genomes and also appear symbiotic. yet studies of symbiosis seldom consider a role for virus ( villarreal, 2007 ) . how important are viruses in general to evolutionary biology? the core concepts of evolutionary biology were developed well before we had a modern understanding and defi nition of viruses ( luria, 1950 ) . after all, the basic lysogenic model of phage integration was only clarifi ed in 1962 when developed by campbell (see campbell, 2007 ) . that cryptic and defective phage are ubiquitous in the genomes of all prokaryotes is generally considered uninteresting by many in the fi eld of evolutionary biology. i suggest that the seemingly applicable concepts of selfi sh dna effectively derailed any thinking that persisting genetic parasites might have a more germinal role in the evolution of life ( doolittle and sapienza, 1980 ; orgel and crick, 1980 ). yet as outlined above, virus footprints in major evolutionary transitions are clear and a direct role in such events now seems much more plausible. we therefore must seek to defi ning the nature of the virosphere and how its evolution relates to the tree of life. this book represents the fi rst integration of the entire fi eld of virus evolution, including both prokaryotic and eukaryotic life forms. however, because our understanding of virushost relationships remains uneven, the chapters necessarily focus on well-studied models (exemplars). these exemplars also tend to refl ect a historic disease focus (i.e., e. coli , fl owering plants, mouse, humans). it is unfortunate that the silent species-specifi c viruses that tend to exist in stable states with long evolutionary histories seldom provide our examplars. we understand these infections poorly and lack basic defi nitions concerning fi tness or selective advantages. only metagenomics tools now seem able to inform us of their presence, but not their biology. self-organization and the evolution of rna molecules as an origin of biological information are discussed in chapter 1. autocatalytic chemical reactions, such as replication of rna, presents issues such as how to optimize a rugged fi tness landscape yet allow the study of evolution in vitro with rna. rna in vitro reduces genotype-phenotype issues to rna secondary structure and minimal free energy states. this allows both continuity and discontinuity to be measured. these same issues are crucial for the study of rna viruses, whose sites of secondary structure often defi ne replicator identity. these models currently offer the best system to evaluate an early and simple biological world for evolutionary principles (see chapter 3). viruses and viroids with their rna genomes may be the only extant survivors of this pre-dna world. since it was the consideration of error-prone replication that led to the development of the concept of quasispecies (see chapter 2), such models have provided a conceptual foundation which led to several basic concepts. chapter 4 discussed the foundations and various aspects of quasispecies theory. viruses appear to operate close to the error threshold, thus allowing maximum evolutionary exploration ( biebricher and eigen, 2006 ) . however, as presented below, the loss of the " fi ttest " type concept has also led to clear experimental evaluations of consortia-based evolutionary behaviors. such behaviors were not predicted by classical darwinian models. although virologists were initially attracted to quasispecies models, many evolutionary biologists were initially hostile to the application of the quasispecies concept to evolution. it was thought that the classical mathematical models of population biology, as originally developed by wright and fisher, and later applied by kimura and maruyama to asexual haploid populations at the mutationselection balance, had already fully developed the needed models and precluded additional need for the quasispecies concept. the classical models were thus argued to provide adequate mathematical coverage for viruses, including quasispecies and error threshold ( wilke, 2005 ) . however, these two approaches differ fundamentally with regard to the signifi cance of error-prone replication and it was the quasispecies approach that led to the clear experimental establishment that quasispecies selection, per se , is important for viral pathology and fi tness (see below). the development of quasispecies theory to virology does indeed demonstrate distinct differences with population genetics. various phenomena, such as complementation, cooperation, competition, and even defective mediated extinction ( domingo et al. , 2001 , domingo 2006 grande-perez et al. , 2005 ) have been observed, all of which fall outside of the parameters of classical population genetics. viral fi tness has indeed been shown to be due to interaction within a diverse population, and not to the fi ttest or master type. and with rna viruses, error threshold has become a central issue ( biebricher and eigen, 2005 ) . the collective experience has thus made clear the value of theory to biology. many working biologists understand that life seems overly complex and defi es most generalizations. thus they do not always appreciate attempts at general theory. although in reality, biology may indeed often be too complex for accurate theoretical predictions, these theories have nevertheless clearly stimulated crucial concepts and experimental evaluation, and biologists should be encouraged by them. by providing new ways of thinking, entirely new experimental approaches can be developed. the existence of error-prone replication and quasispecies also raises the issue of the conservation of information. how is information stability and higher fi tness attained with such errors? how can genetic complexity be created in such a circumstance? can cooperation (or consortia behavior) result from any of these models? these issues have yet to be resolved. some interesting suggestions, however, have been proposed. one involved cooperative evolution that results from ligated genomes. a model was proposed in 2000 by stadler and schuster in which they considered the dynamics of replicator networks resulting from higher order reactions involving the templated ligation of smaller genomes ( stadler et al. , 2000 ) . although this was based on concepts such as triple-stranded nucleic acids, this clearly has some elements that also resemble the ligation of recombinational processes for dna phage (presented below). a most interesting outcome of these models is that, depending on initial replicator concentrations, permanent coexistence of replicators could result in a cooperative network. such cooperation is a rare outcome for most models and given the conclusion that early dna based life was a " horizontal " consortium, such models are of special interest. the issue of consortia will come up often. consortia selection directly implies cooperation, but cooperation of selfi sh replicators presents dilemmas. replicator networks with interaction functions that give highly non-linear dynamics can result in complex mixtures, with behaviors ranging from survival of the fi ttest to also including attainment of globally stable equilibrium tantamount to permanent coexistence. the fi tness of populations, however, is inherent to current quasispecies concepts in virology. there may also be other ways to explain the genetic origin of cooperation (such as stable persistence involving addiction strategies). the stable persistence of a genetic parasite can compel cooperation and promote the conservation of information (see below). the defi nitions of fi tness with respect to a virus in a natural habitat are far from clear. although the concept of relative replicative fi tness is often applied to lab experiments of virus growth, we know many situations in which virus replication is not maximized in natural settings and many viruses can exist in relatively non-replicative states for long periods. even in the context of an acutely replicating virus in a host organism, the concept of fi tness is clearly conditional, as the virus must replicate through various in vivo habitats that can have opposing selection. as presented below, in vivo models that study fi tness and viral diversity have clearly indicated that diversity per se is important and fi tness is the result of consortia. how do we defi ne such fi tness since the mixture clearly matters? also, how do we defi ne information content or integrity of a consortia? currently, we cannot. in the lab, the viability of a virus is usually measured by the ability to produce plaques. this has been a crucial and main assay for many experimental systems that study virus population. here, the defi nition of fi tness seems direct: plaque formation equals fi tness. various highly useful models have thus been defi ned and developed that depend on these plaque assays (see chapter 4). with this, populations and population growth are defi ned as relative growth of plaque-forming units. however, the concept has always been problematic when considered from a natural virology perspective. plaque formation is clearly not equal to fi tness in natural habitats. there are many examples of highly successful viruses that either plaque poorly or not at all. consider the roughly 100 types of human papillomavirus (hpv), a simple small circular dna virus of epithelia; this does not form plaques in any known system (chapter 18). hpv is clearly fi t, well adapted to, and stable in its human host. in addition, hpv evolution is phylogenetically congruent with their primate host, as are most persistent viral infections. we have yet to understand the definition of fi tness in this situation. in some cases, it seems selection for plaque propagation has clearly resulted in loss of highly conserved genes; such as with the plaque-adapted laboratory strains of cytomegalovirus (cmv). the problem posed by viruses with ineffi cient plaque formation is not limited to dna viruses. many persisting rna viruses also do not plaque well or at all, such as most rna viruses of plants or many insect picorna-like viruses, such as those found in drosophila and bees (which also conserve an extra orf). nor do most persistent infections make lots of virus. low-level persistence, such as hantavirus in rodents, for example, is common ( hart and bennett, 1999 ) . clearly, our simplifying assumptions of viral fi tness and population dynamics cannot apply to these stable evolutionary states. however, if we limit our defi nition of viral fi tness to relative replication or plaque formation we can perform some clear and quantitative evaluations. experimental evaluation forces us to study fi tness by only those defi nitions that we can currently measure. as fi tness appears to be a relativistic and transient concept, depending very much on the tissue, time, place, extant adaptive and innate immunity, and competition, it is likely that we can only measure with any accuracy one aspect of fi tness at any one time. hiv infection of humans shows evidence of this in that the r5 virus is more fi t for transmission and early disease whereas the x4 virus is fi t later during the aids disease phase. clearly conditional, time-dependent issues relate to fi tness defi nitions. however, much more problematic is that we have no theory for viral persistence or its fi tness. we lack specifi c or measurable parameters other than the simple maintenance of genetic material. yet it seems clear that some distinguishing features of persistence can already be recognized. for example, the possible participation of viral defectives (normally considered unfi t), which in numerous circumstances can modify or mediate persistence, would need to be included. clearly, a defective role in persistence would also preclude them from being considered as genetic " junk, " or selfish elements, since they would then matter in measurable ways to the biological outcome of virus persistent infection. persistence also requires an extended duration of infection, not simply maximized replication. in fact, persistence generally requires mechanisms to limit the replication of at least the same virus for at least some time. thus, limited replication must be an essential element for this life strategy. in my judgment, and much like the quasispecies concept, the concept of persistence will eventually be recognized for the fundamental (symbiotic) force it represents in virus evolution. the experimental work of domingo and holland spans the modern assessment of quasispecies theory that occurred in the 1980s and 1990s. these investigators were the chief proponents of this theory, bringing it to the attention of the broader virology community (chapter 4). this work has transformed our thinking and laid the experimental foundations that we now build upon. this current volume is an extension from an earlier book on quasispecies and now encompasses both prokaryotic and eukaryotic viruses . since early experimental phage studies provided the foundations for quasispecies theory ( eigen et al. , 1988 ) , using mathematical descriptions (differential equations) of mutation rates in t-even phage ( luria and delbruck, 1943 ) , this inclusion is appropriate. interestingly, a second early paper measuring replication rates by these same authors also noted the problems of viral interference and defectives ( delbruck, 1945 ) . other early experiments of phage rna polymerase ( batschelet et al. , 1976 ) , especially with rna phage qβ ( domingo et al. , 1978 ) , helped set the stage for the subsequent experiments of the 1980s and 1990s. from the test tube to mouse models to the study of human disease, the work of domingo and colleagues has spanned the entire history of viral quasispecies ( domingo and gomez, 2007 ; chapter 4) . quasispecies deals with the products of error-prone replication. however, it is worth repeating that products of error-prone replication are not behaving in a simple " selfish dna " capacity and are not devoid of biological relevance and phenotype. in their complex populations, they create clear and varied affects on viral adaptability, competition, and fi tness. since quasispecies necessarily involve defective and mutant virus, it is easy (and common) to think of these entities simply as genetic junk ( villarreal, 2005 ( villarreal, , 2006 . defective and even lethal or interfering variation in viral genomes can contribute to adaptability. thus, viruses can clearly adapt as a cloud with a mutant spectra. in addition, unending competition and exclusion, consistent with the red queen hypothesis, has also been observed ( clarke et al. , 1994 ) . the poliovirus-mouse model (see below) in particular has provided a solid experimental system for evaluating the adaptive consequence of quasispecies. it is thus ironic that these same experiments have also made clear that the original simplifying assumptions of the quasispecies ordinary differential as presented by eigen are violated by the resulting quasispecies. these products of error-prone replication do indeed strongly interact with each other in both positive and negative ways and such interactions contribute signifi cantly to the observed fi tness of the population. errors and interaction are important for fi tness. for example, defectives have been reported to mediate extermination of a competing wild-type virus ( grande-perez et al. , 2005 ). complementation has also been observed ( garcia-arriaza et al. , 2004 ) , as has trans -dominant inhibition ( crowder and kirkegaard, 2005 ) . genetic memory of past selection has been shown to be maintained in a minority of the population ( ruiz-jarabo et al. , 2000 ; briones et al. , 2006 ) . such cooperative (consortia) behavior, which can also depend on unfi t or defective members, is at odds with classical darwinian notions regarding survival of the fi ttest. consider, for example, the fi tness of a defective or mutant outside of its role in a quasispecies. such a consideration ignores the very nature of a quasispecies yet it is an issue that has often been posed and experimentally evaluated. we should refrain from thinking of viruses simply as fi t or non-fi t individual types since they clearly exist in populations that provide population-based adaptability. the selection of viral consortia or population raises some fundamental issues for evolutionary biology. this is essentially group selection in which a population, not the fi ttest individual, is selected. this view makes cooperation or interaction of individual genomes a signifi cant component of selection, which is not commonly thought to be a general or accepted process in evolution. yet population selection is no longer a contestable issue in rna virus evolution (see below). i expect that many classical evolutionary biologists might interpret this as evidence that viruses really are an oddity in this feature and are not representative of broader processes of evolutionary biology. furthermore, viral-based group selection may not be limited to quasispecies-based evolution. as presented below, persistent viral infections may also provide population-based selective advantage (see below for the p1 and mouse hepatitis virus (mhv) persistence exemplars; villarreal, 2006 villarreal, , 2007 . since viruses are ancient, numerically dominant, and the most diverse biological entities on earth, no life form can escape exposure to them. all extant life forms have evolved in a viral habitat. thus we should expect that the viral footprints (including defectives) that we now fi nd in all genomes have likely played an active role in their evolution; a role, i would argue, that is fundamental, dynamic, and unending. if we can accept this assertion, we may start to see and appreciate the vast evolutionary power that viruses can bring to bear onto host evolution. we can start to attain a global perspective and appreciation for their ability to assemble genetic function from enormous, complex mixtures of genomes, and select gene sets needed to solve multivariant, temporally dynamic evolutionary problems. we can then seek evidence for the role of viral elements in fundamental host innovation and be open to evaluating the occurrence of viral entities from a constructive perspective and not instinctively dismiss such observations as due to coincidence, " junk, " or selfi sh dna. the advantage of such a perspective is that it will promote the specifi c experimental evaluations that can better assess any constructive role genetic parasites might have played in host evolution. for example, there is much reason to think ervs have played an active role in human evolution (for references see ryan, 2007 ) . the quasispecies concept has provided the foundation for us to understand virus evolution and informed us of the evolutionary power viruses possess. if that power also links to host evolution, then the tree of life becomes enriched by virus, much larger and more dynamic. the recent experimental studies from andino and colleagues using poliovirus in the mouse model should, in my judgment, provide the keystone exemplar regarding the in vivo fi tness of quasispecies (see chapters 6 and 7; vignuzzi et al. , 2006 ) . these studies make clear the importance of quasispecies and error-prone replication. such detailed in vivo experiments were made possible by a long and detailed history of poliovirus studies that has identifi ed the nature of rna polymerase fi delity as well as developed mouse models for the study of pathogenesis. few other virus-host systems could have provided such potential for high resolution. these results also provide the experimental observations that distinguish quasispecies-based evolution from the classical fisher-based population genetics. the general importance of this story for understanding virus evolution thus deserves special emphasis. the very origins of modern animal virology stem from poliovirus studies with the need to develop in vitro cell culture technology in order to grow and evaluate poliovirus and generate variants. the live poliovirus vaccine is of special interest with regards to virus evolution and adaptability. the " live " oral sabin vaccine can be considered to have been a miracle of the practical approach to virology developed in the 1950s ( horaud, 1993 ) in that it was used well before our understanding of the relevant evolutionary theory. the sabin vaccine strain was the result of rodentadapted virus and differs from the neurovirulent mahoney strain by 56 point mutations (in the consensus sequence), although only a small number of these mutations were needed for neurovirulence ( christodoulou et al. , 1990 ) . one of the important neurovirulent mutations was within the rna polymerase gene ( tardy-panit et al. , 1993 ) . however, the signifi cance of this observation took many years to unravel and exploit. in time it became apparent that 3dpol mutants could affect replication fi delity. one poliovirus point mutant, 3dg64s, was shown to have enhanced highfi delity replication and that selective pressure could be designed to increase fi delity in rna polymerase ( pfeiffer and kirkegaard, 2005 ) . another major development was the molecular identifi cation of the poliovirus receptor and the subsequent creation of transgenic mice expressing this receptor, making them susceptible to poliovirus infection. one of these transgenic lines allowed mouse brain infections with neurovirulent versions of poliovirus , and has provided a very useful animal model that allowed the evaluation of viral fi tness in the context of in vivo pathogenesis. although 3dg64s replicates well in culture (with lowered error rate), it was less pathogenic in this mouse model and competed poorly with 3d wild-type virus. it seemed that the decreased viral diversity was less able to generate the variation needed to get past bottlenecks due to multiple selective differences presented in vivo in tissues in the host, such as brain infection ( pfeiffer and kirkegaard, 2006 ). this experimental system also makes clear the greater complexity of fi tness in vivo relative to that typically measured in culture. thus it seems that in vivo there may not be one fi tness but several that cannot be distinguished or individually measured. it is likely that various in vivo barriers require distinct fi tness solutions that tend to create bottlenecks and that the diversity per se is essential to get past such bottlenecks. a population, not a clone or a consensus, appeared more fi t as higher titer infections of 3dg64s also failed to be pathogenic. thus, higher levels of a consensus virus are not equivalent to higher diversity. the relationship between rna polymerase structure, error rates, and ribavirin action is discussed by cameron in chapter 6 and has been the subject of numerous studies ( crotty et al. , 2001 ; vignuzzi et al. , 2005 ) . knowledge of the structure and catalytic mechanism of rna polymerase function has allowed a greatly enhanced level of detail to be considered into what affects error rate (see castro et al. , 2007 ; korneeva and cameron, 2007 ; marcotte et al. , 2007 ) . this has provided insight into the likely action of ribavarin on product fi delity ( harki et al. , 2002 ) . thus, it appeared that even a mutant of rna polymerase with increased fi delity could still generate elevated diversity by various methods. such control of fi delity allowed for the design of control experiments in which the same consensus virus genome could be forced to generate either less or more diverse progeny populations. in no other virus-host system have we attained such detailed insight into issues of error rate as those that were put to such excellent use in the poliovirus-mouse system. how generally important is this poliovirus in vivo quasispecies result? although the poliovirus-mouse system provides us with a fi rm experimental result, it seems likely that the generality of this relationship will be questioned by evolutionary biologists for several reasons. for one, this was observed in a lab constructed model system, which, it could be argued, is not an accurate representation of in vivo virus-host fi tness. also, as mentioned above, group selection is a process that will not readily be accepted as representative by the broader community. is there any evidence that this result with poliovirus indeed represents a general virus-host evolutionary relationship in natural settings? as presented in chapters 13-15, retroviruses and also human hepatitis virus c clearly exist as quasispecies populations that affect disease outcome. in the case of the retroviruses, viral populations show diversity that far exceeds that seen for other rna viruses. in both hiv-1 and hcv there is clear circumstantial evidence for the importance of quasispecies for in vivo disease outcome, drug resistance, and fi tness. in addition, with hcv, cns infection may sometimes result, and such brain infections appear to be mediated by distinct quasispecies ( forton et al. , 2004 ; forton et al. , 2006 ) , reminiscent of the polio virus mouse model. quasispecies memory, as mentioned above, also seems to be an important issue with regard to failure of antiretroviral therapy ( kijak et al. , 2002 ) and it appears that pol gene mutations could also be involved in this ( carobene et al. , 2004 ) . measurements of hiv quasispecies in individual patients indicates that multiple evolutionary patterns can be found in typical individual patients ( casado et al. , 2001 ) , thus mixtures of hiv exist in patients ( bello et al. , 2004 ( bello et al. , , 2005 . and hiv-1 recombination is clearly contributing to diversity ( kijak and mccutchan, 2005 ) . thus, with both hiv-1 and hcv, their capacity to cause human disease is clearly associated with quasispecies compositions that affect fi tness in complex ways. the poliovirus mouse system therefore appears to refl ect quasispecies issues as observed in natural virus-host situations. consideration of retrovirus-host evolution introduces another large issue in evolution: genomic viruses. unlike poliovirus and most rna viruses, retroviruses (e.g. non-lentivirus) have colonized the genomes of animal species in large numbers and represent a large fraction of these genomes. genomic retroviruses are present in vast numbers, most of which are defective and mutant copies. in this genomic colonization they resemble the dsdna viruses of prokaryotes (discussed below) that also colonize all prokaryotes although at a much lower numbers. the human genome has fewer than 26 000 genes, but appears to have 500 000 retroviral-related ltr elements. some of these elements are intact and conserved (human ervs (hervs)) and this genomic population has some clear characteristics of a viral quasispecies. such large amounts of genetic material have previously been dismissed simply as selfi sh or junk dna of no fi tness consequences to the host. however, given the importance of quasispecies mutant genomes for viral fi tness and persistence, we might need to re-evaluate this dismissal. retroviruses are clearly part of the human ancestry thus we should seek to understand, not dismiss their role in human evolution. in contrast to the story above in which polio infection of mouse brain was dependent on the quasispecies resulting from lowered fi delity replication, a different relationship has been proposed for the nidoviruses. these are also positive single-stranded polycistronic rna viruses ( gorbalenya et al. , 2006 ) . this group of virus includes the coronaviruses (e.g. mouse hepatitis virus and sars-associated coronavirus), which are the largest rna viruses known (26-32 kb). it has been proposed that such large genomes have required the adaptation of a high-fi delity rna polymerase in order to increase the error threshold and accommodate large rna genomes. based on the phylogenetics of this polymerase and other rna-processing enzymes, this group of viruses appears to be monophyletic and it is thought that the acquisition of a high-fi delity rna replicase was central to the origin of this lineage. this type of replicase is unique to rna viruses. the monophyletic view stems from an analysis of a small set of conserved genes. overall, however, these larger genomes have many other genes that show no similarities to related viruses. the origins and evolution of these more diverse and numerous genes cannot be currently traced. this is an inherent problem in the analysis of virus evolution: a small selected set of hallmark genes with some similarity are assumed to trace an apparently linear (tree-based) viral lineage whereas the larger number of genes are not included and cannot be traced. if most of rna virus evolution is indeed mediated by a mixed cloud of genomes, any role for mutant mixtures thus becomes obscure. but perhaps there is little else we can currently do given the lack of information. how might we explain the increased fi delity and genome size of the nidoviruses? was there some change in viral adaptation in which quasispecies and generation of mixtures was no longer as important for adaptation? did the need and selection for a larger genome override the use of error to generate adaptability as seen in poliovirus and hiv-1? if so, what selective pressures might have changed this seemingly basic feature? what do we know about the natural biology of these viruses, which might provide some insight into this? unfortunately, the natural distribution and gene functions of the nidoviruses are generally poorly understood. in terms of coronaviruses, numerous mammal and avian species can be infected and the virus will cause acute disease. in several of these acute infections, the virus involved seems to have recently been adapted to the new host from other, often unknown sources. with the recent emergence of the sars virus and human infections, however, much greater attention has been focussed on trying to understand the origin and evolution of this virus. it has recently become clear that there indeed appears to exist an evolutionary stable source of this virus from which adaptation to humans was possible. various bat species have been found to support persistent asymptomatic infections by specifi c versions of sars viruses ( tang et al. , 2006 ; vijaykrishna et al. , 2007 ) . these studies also indicate that there appear to be three different and independent groups of sars viruses in bats. in fact six novel coronaviruses were isolated from six different bat species showing an astonishing diversity in bats. furthermore, phylogenetic analysis indicates that all bat coronaviruses appear to have descended from a common ancestor. only one of these bat groups includes sars and sars-like coronaviruses that adapted to acute human infections. thus, a prevalent and speciesspecifi c persistence of sars viruses is found in particular geographical populations. why is this relationship stable? could the adaptation to a host-specifi c persistence-based basal life strategy provide some explanations for the evolution of the higher fi delity rna replicase of these coronaviruses? as i have argued, persistent viral infection represents the majority of evolutionary stable viral lineages ( villarreal, 2006 ) . however, we have almost no knowledge regarding how these bat sars viruses persist and escape elimination by innate and adaptive immunity and what, if any, role the high-fi delity replicase (or other genes) have in this life strategy. although we cannot yet evaluate natural sars virus persistence in native bat hosts, another coronavirus may be more informative regarding the effects of persistence on host populations. mouse hepatitis virus (mhv) may provide our best exemplar of virus-host relationships and show how the concept of virus addiction relates to population persistence. mhv is the best-studied coronavirus. as a natural and prevalent virus of rodents, mhv is our best natural model of persistent rna virus-host relationships for any mammal. in general, rodents are the most studied non-domestic mammals with regard to natural virus distribution. overall, we know that wild-caught rodents seldom show signs of acute virus infection ( kashuba et al. , 2005 ) . however, asymptomatic virus persistence is ubiquitous in wild rodents ( descoteaux et al. , 1977 ; gannon and carthew, 1980 ; schoondermark-van de ven et al. , 2006 ) , including voles ( descoteaux and mihok, 1986 ) . some fi eld studies have evaluated broader patterns of virus persistence in mice ( singleton et al. , 1993 ; becker et al. , 2007 ) which indicated that wild house mice are highly colonized with mhv (80-100% prevalence). in addition to mhv, mouse cytomegalovirus, mouse parvovirus, mouse thymic virus, and mouse adenovirus are also prevalent. other well-studied mouse viruses, such as lymphocytic choriomeningitis virus (lcmv) and polyomavirus (pyv), were at low natural prevalence. interestingly, some non-native house mice that have colonized isolated islands may lack mhv ( moro et al. , 1999 ) , although most other isolated island populations retain mhv ( moro et al. , 2003 ) . other small mammals have yet to show any viral disease whatsoever (hedgehogs, chinchillas, prairie dogs, gerbils, sugar gliders) ( kashuba et al. , 2005 ) . thus, asymptomatic persistent viral infection is clearly the norm in rodents. yet, in spite of this usual asymptomatic viral persistence, historically, some zoonotic viral disease outbreaks have occasionally been documented in natural populations. one such early outbreak was an epizootic diarrhea that occurred in infant mice ( adams and kraft, 1963 ) . later, it was established that one such infection was due to mouse hepatitis virus ( carthew, 1977 ; ishida et al. , 1978 ) . in spite of this disease outbreak, with mhv, it has since become clear that asymptomatic persistent infections are the norm and are highly stable. yet mhv disease outbreaks, especially in virus-free mouse facilities, are also common and severe. how does mhv attain such stable and prevalent persistence in natural population yet retain the ability to cause disease in naive populations? what maintains the mhv fi tness of natural persistence? it is well known that once mhv is established in a mouse or rat colony it can be very diffi cult to eliminate ( gannon and carthew, 1980 ; lussier and descoteaux, 1986 ) , clearly indicating that stability is rapidly attained and likely genetically programmed by the virus. i propose that these stable evolutionary states of viral persistence are due to a strategy we can call virus addiction ( villarreal, 2005 ) and that mhv can provide the exemplar of such a state. with mhv, only persistently infected mice colonies are protected from the disease that is otherwise caused by the virus. in wild asymptomatic mice, mhv is found mostly as an enteric infection. the cns demyelinated disease that mhv can induce is most observed in newborn pups ( homberger, 1997 ; nash et al. , 2001 ) and once in the brain, mhv can persist in cns with recurring disease ( marten et al. , 2001 ) . this recurring cns disease is also associated with quasispecies (in the s gene) and recombination ( rowe et al. , 1998 ) . the most serious cns disease is in s-gene variant of mhv-4 (jhm), thus as with the poliomouse model, pathogenic fi tness with mhv is also associated with quasispecies. such mhv disease is the bane of all mouse colonies ( knobler et al. , 1982 ) . however, once mhv persistence is attained, the problem to a mouse facility is not due to acute disease, but because immunological measurements are signifi cantly affected by mhv persistence. thus mhv alters mouse molecular identity regarding immunological (t-cell) reactions ( wilberz et al. , 1991 ) . to establish stable asymptomatic persistence, however, mhv needs to infect newborns ( weir et al. , 1987 ) , in which acute disease is prevented due to maternal passive immune antibody transfer ( gustafsson et al. , 1996 ) . being born to immune mothers thus protects against cns disease and promotes enteric (not brain) virus colonization. in addition, it appears that persistence also promotes cross-species transfer ( baric et al. , 1999 ) . mhv persistence may involve genome stability and result in a distinct evolutionary dynamic. asymptomatic persisting infections in a lewis rat, for example, showed no variation in mhv s gene sequence, and no quasispecies as seen in brain infections ( stuhler et al. , 1997 ) . the need to establish stable persistence could then be providing a strong selection for increased genome complexity and stability and might better explain the selection for the enhanced rna polymerase fi delity in nidoviruses. how might such selection operate in natural populations? evolutionary biologists often consider what might differentiate one group from another very similar group in a way that leads to two isolated and distinct populations. consider two hypothetical adjacent hay stacks harboring two mus musculus colonies, one of which is persistently infected with mhv the other which is not. what is the fi tness consequence to the colony harboring mhv relative to its uninfected neighbor? our experience with mhv in mouse breeding colony provides a clear answer. the colony that is persistently infected with mhv will have a distinct advantage over its neighbor as mhv introduced into this uninfected colony will have severe effects on the offspring. eventually, we can expect only the mhv-harboring colony will prevail in both hay stacks. this is a state i have called virus addiction. only mice harboring persistent mhv are protected against the potential pathogenic consequence of acute mhv (or related virus) infection. the population is addicted to the virus. such a state, however, is clearly affecting colonies (or groups) of host, not individuals. an individual either quickly succumbs to the virus infection or, if infected, transmits it to others in the colony. a colony is thus under selection by mhv. to generalize this state, we expect that the persistence of sars in specifi c bat populations would be expected to also affect the fi tness of the corresponding specifi c bat populations. persistence is a more demanding phenotype than acute replication. it requires greater gene complexity to counter host immunity and also to promote self-regulation. thus the enhanced fi delity of rna replication is selected in order to conserve this greater genetic complexity and stability. we know that the high-fi delity rna replication system (including rna pol, helicase, endoribonuclease, and other activities) is also present in an ancient nidovirus relative of coronaviruses, such as fi sh-isolated white bream virus (26 kb rna). i suggest there will also likely be species-specifi c persistent infections with this virus that require this enhanced replication fi delity and maintain this virus in its natural habitat. thus, i suggest, an ancient persistent life strategy could more easily explain the monophyletic character of the nidovirus virus lineage. it is particularly interesting that one of these unique and conserved replication proteins (adp ribose-1-monophosphate) is dispensable for culture growth ( putics et al. , 2005 ) . i suggest it will not be dispensable for persistence. the hiv-1 pandemic is an unfi nished story. hiv-1 represents a real-time biological event in human evolution that confi rms for us the importance of quasispecies and retroviruses to human biology. however, even though its human toll is huge, modern medicine and culture has responded rapidly enough to limit the impact of hiv-1 to the point at which it will not likely be the cause of a selective evolutionary sweep that could have altered human genetic makeup (in contrast to the koala bear endogenization presented below). as described earlier, its amazing adaptability via quasispecies along with extensive recombination contribute directly to hiv-1 ' s diversity and makes it the most dynamic genetic entity ever studied. many studies track the dominant hiv population and fail to examine minority populations. yet it is precisely these minority populations, which evolve independently of the majority population, that can determine drug resistance phenotype and biological outcome ( charpentier et al. , 2004 ; briones et al. , 2006 ; morand-joubert et al. , 2006 ) . clearly, the specifi c makeup of a complex hiv population matters. furthermore, hiv defectives and variants can also have major consequences. in some cases, long-term non-progressors of hiv-1 have shown mixed populations and unusual polymorphism in the early phase of hiv infection, sometimes contributing to long-term non-progression (ltnp) ( alexander et al. , 2000 ) . one population of ltnps was reported to have been colonized by an hiv variant that showed low virus replication and slow or arrested evolution ( bello et al. , 2005 ) . in another case, a stable non-progressor was colonized by a replication incompetent version of hiv-1 ( wang et al. , 2003 ) . some of these non-progressors also appear to resist super-infection . it seems clear that at least in these exceptional situations, non-majority hivs are crucial to the outcome. there is also reason to think that other retroviruses have had a major infl uence on recent primate and human evolution, such as apathogenic persisting foamy virus in primates ( switzer et al. , 2005 ; murray and linial, 2006 ) . human antiretroviral genes seem to have undergone recent adaptations, such as apobec3, which can interfere with exogenous retroviruses (such as mlv and siv) and underwent an expansion in the hominid lineage ( esnault et al. , 2005 ) . it thus seems clear that human and primate evolution has been signifi cantly affected by earlier, prevalent primate retroviruses. another important human-virus quasispecies story that has long been recognized is with hepatitis c virus (hcv), (see chapter 15; domingo and gomez, 2007 ) . hcv seems to have adapted to humans in the recent past, possibly from asymptomatic enteric primate viruses currently found in africa ( smith et al. , 1997 ) . as hcv remains an infection predominantly transmitted by blood, it does not appear to have fully adapted to the tissues of and transmission within its human host. however, like hiv-1, hcv has long been recognized to generate quasispecies in chronically infected people ( martell et al. , 1992 ) and it soon became apparent that the viral quasispecies are affected by and affects the outcome of antiviral therapy hohne et al. , 1994 ; kurosaki et al. , 1994 ; okamoto and mishiro, 1994 ) . thus, successful antiviral therapy is directly correlated with an initial dramatic reduction in genetic diversity. unfortunately, it has become clear that only a minority of hcv-infected individuals will respond favorably to a combination of interferon and ribivarin. thus it seems to be diversity per se and the resulting structure of an hcv quasispecies that has a direct consequence to human health. however, since hcv is less well-adapted to humans compared with hiv-1, it does not pose the same threat to potentially provoke an evolutionary event in human evolution. vsv is a negative-stranded rna virus that has been a very important experimental model and has provided many laboratory measurements regarding quasispecies theory (see chapter 4). using vsv, evidence supporting the red queen hypothesis, involving unending adaptation to greater competition and mueller ' s ratchet has been presented ( clarke et al. , 1994 ; novella et al. , 1995 ; elena et al. , 1996 ) . when vsv was evaluated as an arbovirus, requiring adaptation to alternating and opposing fi tness of insect and mammalian host, it was also apparent that minority quasispecies populations were responsible for maintaining the apparently antagonistic phenotypes ( novella et al. , 1999 ) . thus here too, the consortia character of a quasispecies is clear. yet in natural settings several very different virus-host relationships can be seen with rhabdoviruses. a distant relative of vsv (vhsv) is also known to be responsible for mass die-off of commercially important fi sh ( marty et al. , 2003 ) . this virus infects many teleost species and has shown 100% mortality in many experiments (i.e. with i.p. inoculation). in natural outbreaks, however, it has also shown surprising genetic stability ( einer-jensen et al. , 2006 ) . clearly error-prone rhabdovirus replication must be kept in check by purifying selection in this situation. in contrast, another rhabdovirus, sigma virus of drosophila , is associated with no mortality but is a vertically transmitted persisting virus in specifi c drosophila populations ( fleuriet, 1996 ) . yet in some recent population measurements, sigma virus infected drosophila are expanding for unknown reasons ( fleuriet, 1994 ) . clearly this particular virus-host persistent relationship has some undefi ned selective advantage that operates beyond the lab-based concepts as measured above. other rhabdoviruses also have peculiar host-specifi c relationships, such as bats that tend to support many persistent infections ( badrane and tordo, 2001 ; li et al. , 2005 ) , or birds that seem to be free of almost all rhabdoviruses. clearly, although vsv lab results have been highly informative, we still have much to learn regarding natural settings that affect rhabdovirus adaptation and evolution. another major paradigm for the high rates of negative-strand virus evolution is found with infl uenza virus. due to its history and potential for initiating great human epidemics, it has long held the special interest of evolutionary virologists (see chapter 5; nelson and holmes, 2007 ) . however, this research has not much emphasized the quasispecies character of infl uenza virus evolution. instead, it concentrates on the evolution of the master template or clades of template for the purposes of vaccine development ( webster and govorkova, 2006 ) . the views stemming from this type of evolution have lent themselves well to master template-based phylogenetic analysis and have dominated how many researchers think of virus-host evolution. thus it is curious, given the above emphasis, that the quasispecies character of infl uenza populations often seems of low relevance to issues of acute disease and vaccination, other then to provide a source of diversity. in some situations, viral competitive interference may contribute to drift variation and displacement in antigenic epitopes ( levin et al. , 2004 ). yet outcomes of individual human and bird infections do not seem much affected by specifi c quasispecies structures, as we saw with hiv-1 and hcv. with infl uenza, we are mainly concerned with epidemic human disease. however, by shear numbers of infections and deaths worldwide, it must be admitted that infl uenza virus is really a virus that affects mostly birds. for example, during the 2005 outbreak in china, only 251 humans died whereas 230 million domestic birds died ( smith et al. , 2006 ) . although our concern on the large potential for human disease is understandable, these numbers should inform us of a more basic virushost biology. in this case, infl uenza shows a high affi nity for various birds; migratory water birds in particular can have high prevalence ( wallensten et al. , 2006 ) . some waterfowl, such as wild mallard ducks, have been called the stealth (asymptomatic) carriers of infl uenza h5n1 and free grazing ducks seem to introduce virus into domestic bird populations ( gilbert et al. , 2006 ) . thus waterfowl represent the well-accepted epidemiological concept of a reservoir species ( louz et al. , 2005 ) . but these wild waterfowl, shorebirds, and gulls that are a natural host for avian infl uenza also seem to show a much slower rate of evolution ( spackman et al. , 2005 ) . in contrast, the much higher rate of evolution as seen in chickens and turkeys indicates that these hosts should not be considered as natural reservoirs ( suarez, 2000 ) . in waterfowl, infl uenza infections show several distinctions, such as virus co-infection or virus interference ( sharp et al. , 1997 ) as well as phylogenetically distinguishable waterfowl dendograms, including specifi c m lineages ( makarova et al. , 1998 ; widjaja et al. , 2004 ) . the diverse and stable avian pool of infl uenza virus appears to be ancestral to the infl uenza viruses that infected human populations. the phylogenetic methods that have been adapted from evolutionary biology have been tremendously helpful and have allowed us to trace the seemingly untraceable, virus evolution (see chapter 5). thus, we have often been able to make informed judgments concerning broader patterns of virus evolution and this has become the major tool for the current study of virus evolution, such as infl uenza virus ( nelson and holmes, 2007 ) . infl uenza a, for example can be seen to show extended periods of stasis followed by periods of rapid adaptation that necessitates adaptations in vaccine strategy ( wolf et al. , 2006 ) . however, the evolutionary variation between seemingly similar viruses can be surprisingly large (see above vsv section). for example, the very different phylogenetic behaviors between infl uenza a and measles virus, both acute human respiratory infections due to membrane-bound negative-stranded rna viruses, are striking. the reasons for the maintained genetic stability of measles virus remain poorly understood, but may well involve more complex fi tness associated with systemic infections. phylogenetic methods can also be highly informative regarding the likely origins of viral lineages and possible sources of emergence. for example, the studies of dengue virus by holmes and colleagues suggest that this virus fi rst entered its human host about 1000 years ago, and that sylvatic (african jungle) asymptomatic infection of primates may have provided the origin of this virus that later became a human pathogen ( holmes and twiddy, 2003 ; holmes, 2006 ) . such insight provides valuable clues concerning the likely selective pressures that may lead to the emergence of dengue virus. phylogenetic methods are also highly informative regarding classifi cation and taxonomy relationships and have allowed us to understand viral relationships across broad species defi nitions ( zanotto et al. , 1996 ) . however, phylogenetic approaches necessarily assume the master template is the fi ttest type and that mutations or variants in the rna populations are a source of genetic load that are deleterious and limiting to virus adaptation ( pybus et al. , 2007 ) . such variation is mostly due to " unfi t " mutations, which indicates that a viral cloud is mostly and unfi t consortia. it would seem that such conclusions go against the concept of quasispecies as being fi t per se as described above. in this consideration we see a major weakness of extant phylogenetic methods. they were not developed to access the evolutionary relationship and fi tness of interacting mixtures. nor were they designed to follow the evolution of systems with high rates of recombination between numerous parental templates. we currently lack the analytical tools for such a population analysis. without such tools, however, it seems we can only evaluate those parameters we can defi ne and will remain confused by those we cannot. evolution of a consortia thus provides a new directions for theoretical and laboratory research. we should seek to investigate the mixture, not just its average. another major virus-host system that has been highly studied is the viruses of agricultural plants. our understanding of plant viruses has also been highly infl uenced by disease associated with agricultural domestic species, thus natural virus-plant relationships are much less understood, although some recent fi eld studies are starting to change this situation (see chapter 12). we currently have a rather uneven understanding of broader virus-host relationships and evolution in plants. for example, viruses of the more ancient ferns, if they exist, are essentially unknown. the prevalence and diversity of positivestranded rna viruses in plants is striking. in addition, we are starting to appreciate that virus-virus interactions are also frequently involved, although this issue remains poorly studied. one well-studied family of plant virus are the tobamoviruses of angiosperms (see chapter 11; gibbs, 1999 ) . progenitors of this virus family appear to also be found in algae and fungi consistent with a very long evolutionary history. both high transmission between host and virus-host congruence are observed with these viruses. virus-virus interactions also seem to be important. for example, tobacco mosaic virus (tmv) and tomato golden mosaic virus (tmgv) appear to have shown interactions in australia which have apparently led to the extinction of tmv, but the retention of tmgv with no increase in genetic diversity ( fraile et al. , 1997 ) . plant viruses have also been seen as quasispecies in some but not all settings (see chapter 12; roossinck, 2003 ; roossinck and schneider, 2006 ) . besides the interactions expected for typical viral quasispecies, plants often show evidence of more extensive mixed virus infections. there are, for instance, many examples of satellite viruses that must necessarily interact with other rna viruses of plants. it is also clear that the subviral elements of even a single viral lineage can greatly affect the virus-host relationship. such subviral elements (dis) have been observed to both reduce and intensify disease, and also interact with satellite viruses ( qiu and scholthof, 2001 ) , thus virus-virus interactions are clearly crucial in many situations ( simon et al. , 2004 ) and viral interactions and synergism appear to have led to signifi cant events in plant virus emergence ( fargette et al. , 2006 ) . virus-virus interactions are not limited to plant rna viruses. the ssdna plant geminiviruses also display complex interactions with satellites as well as high diversity in fi eld isolates of east africa ( ndunguru et al. , 2005 ) . thus, plant viruses seem particularly prone to interactions. more recently, virus-mediated symbiosis with respect to host survival has been reported ( roossinck, 2005 ) (discussed below). phylogenetic methods also struggle to address the occurrence of high rates of recombination in viral lineages. such a situation complicates the analysis, creating hardto-defi ne, reticulated trees, although these limitations can be partially overcome by using sliding windows for the analysis. such approaches have allowed surveys of recombination in some viral lineages, such as with the plant potyviruses ( chare and holmes, 2006 ) . however, the rampant recombination and quasispecies generation of hiv-1 makes a quantitative assessment of the virus population problematic. one proposed solution is to use a composition vector method ( gao and qi, 2007 ) . the issue of measuring recombination and tracing evolution in large populations is especially a problem that applies to the dna viruses (phage) of prokaryotes (see below). cells. our perception regarding the overall importance of dna viruses of prokaryotes to the evolution of life on earth has undergone a major shift in recent years. the main realization is that dna phage are the numerically dominant genetic entity in most habitats on earth (mentioned above). in addition, as discussed in chapter 10, it is now clear that some of these viruses are surprisingly complex and that essentially the entire pool of dsdna viruses of prokaryotes may be exchanging dna via recombination at high rates. this would constitute by far the largest common gene pool on earth. historically, the evolution of the dna viruses of prokaryotes has seldom been considered in the broader context of virus evolution or evolutionary biology. although it has long been realized that there are many basic similarities between viruses of bacteria and eukaryotes ( luria et al. , 1959 ) , not until structural studies solved the capsid genes of prokaryotic and eukaryotic viruses did the evolutionary relationships between these viruses become clear. in addition, there have been a number of striking proposals that suggest that dna viruses of prokaryotes may be involved in the origin of several major systems used by cells and that viruses appear to be involved in several major transitions during host evolution. thus we now consider the possibility that these dna phage were fundamental to the origin and evolution of life on earth. it now seems likely that some large dna viruses infecting eubacteria, archaea, and eukaryotes share some common evolutionary histories. it also seems clear that such viruses can link all three domains of life. this realization was not apparent based on phylogenetic sequence conservation, which is absent. it stems from the structure and assembly of virion capsids in which t4 phage, halophage, and the herpesviruses all show clear similarity as well as similarity in replication strategies. in addition, phage prd1 and adenoviruses show similar broad structural and strategic conservation. some biochemical (dna pol family) and genetic similarities (gene order, gene programming) are also apparent, which taken together supports the common origins of these viruses ( hendrix, 1999 ( hendrix, , 2002 hendrix et al. , 1999 hendrix et al. , , 2003 . t4-like viruses in particular seem to represent a major source of global genetic diversity. this giant genetic pool represents a huge potential to affect life ( filee et al. , 2005 ) and the viral genetic creativity represented by this pool would also be vast ( nolan et al. , 2006 ) . since t4-like phage that infect cyanobacteria also encode virus-specifi c type ii photosynthetic core genes, viruses appear able to create the most complex of genes as well ( clokie et al. , 2006 ; sullivan et al. , 2006 ) . as presented in chapter 10, phage are now thought to evolve by distinct and highly mosaic " horizontal " processes of rampant recombination ( hendrix, 2002 ( hendrix, , 2003 . large dna phage appear to be ancient, present before the split of the three main branches of cellular life: bacteria, archaea, and eukarya ( benson et al. , 2004 ) . luca, the last universal common ancestor, would represent the putative cell ancestor prior to this split. however, phyogenetic analysis of common or conserved genes of luca identifi es only about 325 or fewer genes in extant cellular genomes ( mushegian, 1999 ; koonin et al. , 2001 ; mirkin et al. , 2003 ) . ironically, the genes needed for dna replication are not part of this conserved set, calling into question the nature of the fi rst dna-based cell. large-scale " horizontal " transfer seems to have clearly prevailed early in the evolution of dna-based cellular life and it has recently been asserted that luca existed in a highly horizontal " consortia " of cooperative genes that developed the common genetic code ( vetsigian et al. , 2006 ) . since the dna replication proteins in the extant three domains of life have distinct compositions, it has been proposed by forterre that dna viruses and retroviruses were directly involved in the invention of the three extant cellular dna replication systems . according to this view, early cellular life was completely entangled with viral (phage) lineages; hence cells must have evolved from an ancestral " virus " -mediated population not a single genetic lineage. thus the evolution of early life would have clear similarity to the quasispecies (consortia) state of genetic information as seen in rna viruses above. thus the huge creative and adaptive potential of virus would have been directly involved in the very earliest evolution of life. clearly, such conjectures regarding the most ancient events in the evolution of life are hard to substantiate. but, these theories are as viable as any other and deserve serious consideration. in spite of this seemingly unending mosaic exchange in dsdna phage, some phage isolates show surprisingly stable genetic makeup. we now accept that t4-related phage are an important source of the larger global phage genetic diversity and that most such viral genes are novel ( filee et al. , 2006b ; nolan et al. , 2006 ). yet even with t4-like viruses, there can be clear barriers to horizontal gene transfer which promote the evolution of stable viral lineages ( filee et al. , 2006a ) . in t4-type phage, 24 similar core genes could be seen in all genomes, which seem to be inherited in gene blocks that preclude recombination. however, these blocks were not seen in the broader t-even and pseudo t-even genomes. other phage also show surprising genetic stability when repeatedly isolated from similar habitats, such as soil phages of burkholderia ( summer et al. , 2006 ) and bam35 ( saren et al. , 2005 ) as well as some hot spring isolates ( khayat et al. , 2005 ) . this bam35 capsid also identifi es another structural motif mentioned above that is broadly conserved in evolution and shows clear similarity to that capsids found in prd1 and pbcv-1 (discussed below). sh1 also has a clear prd1related capsid, membrane, and genome; thus this halophilic euryarchaeon virus, although showing no sequence similarity to prd1 or any other bacterial phage, is clearly structurally related ( bamford et al. , 2005a ) . it is interesting that overall the viruses of hyperthermophilic crenarchaeota generally show no sequence relationship to phage of bacteria. in addition, the use of the term phage for these viruses can also be questioned as most establish non-lytic chronic infections. many of these crenarchaeota viruses have unique morphologies not found in any other domain of life ( prangishvili et al. , 2006a ( prangishvili et al. , , 2006b ortmann et al. , 2006 ) . some, however, have clear structural and genetic similarity to specifi c phage (i.e. t4). considerations of phage evolution and rampant recombination (especially with t4 and t-even phage) often emphasize the viral lytic lifestyle and host death. in fact this lytic relationship was argued by many early phage researchers to be the fundamental and only character of phage-host relationships in general. we now know, however, that persisting (temperate) phage are also common, some of which have no independent lytic phase. the fundamental model of phage persistence by unique integration into host chromosomes (temperate lysogeny) marks a major development in our understanding of molecular virology and virus-host relationships which was fi rst clarifi ed by campbell in 1962 (see campbell, 2007 . all free-living prokaryotes show the presence of colonized phage in their genomes. both complete and defective genomes of dsdna viruses have been observed in the sequenced dna of all free living prokaryotic genomes ( gelfand and koonin, 1997 ) (exceptions are some intracellular parasites and plastids). thus, the massive genetic diversity and novelty of phage evolution as presented above has a direct conduit into the genetic composition of all prokaryotes via lysogeny. the fi tness and evolutionary consequences of such colonization to the evolution of the host and its virus should be considerable but is in need of theoretical development. fitness of temperate phage, however, is more complicated then that of a lytic virus and, like fi tness of persistence discussed above, cannot be simply described by relative replication or effi cient virion production. here too, successful phage colonization must inherently limit the replication of the same virus. thus, a temperate lifestyle also requires an autoinhibitory capacity. this generally involves an immunity gene set that not only limits self-replication but can also affect replication of other temperate and lytic viruses, i.e. lambda (even as a defective) precludes t4 and other t-even phage. uncolonized hosts are thus susceptible to lysis by highly prevalent acute tailed phage. host fi tness is thus strongly affected by a temperate phage due to its ability to preclude and survive other competing phage. i suggest this situation is similar to the mhv-mouse exemplar above, in that virus-colonized hosts are in a state of " virus addiction " in which persistence is needed to provide protection from the same or similar virus ( villarreal, 2005 ( villarreal, , 2006 . it is well established that most natural populations of bacteria have specifi c patterns of phage colonization, hence the utility of phage typing for strain identifi cation. from this, we can infer that virus-virus competition is a prevalent and major issue regarding the prokaryotic fi tness resulting from a symbiotic temperate phage-host combination. in addition, such virus-host symbiosis can also affect competition with other bacteria. this would be very much like the virus addiction concept outlined above for the mhv examplar. the original observation of a lysogenic process and coining of this term occurred in the 1920s when two pure cultures of bacteria were grown together. it was observed that in some combinations, one strain would lyse the other strain (was lysogenic). later, it became clear that such lysis was mediated by reactivation of temperate phage present in the lysogenic strain, but absent from the non-lysogenic susceptible strain. in this relationship, we see another example of group selection operating on bacterial populations harboring a persistent virus. thus, what host is fi t depends very much on the prevalent viruses it will encounter as well as the viruses that colonize it. bacterial populations that are colonized by the same or similar phage express the appropriate immunity functions and are protected from lysis by the same or similar phage. such a situation has signifi cant implication for the evolution of immunity and group identity for cells. host stability becomes a major fi tness issue for a persistent virus life strategy. it is generally thought that a temperate virus attains a stable colonization of its host by simply integrating into and become one with the host genome. however, there are also clear examples of stable phage persistence that does not integrate and uses other strategies to attain host stability (similar to eukaryotic dna viruses; see below for the p1 phage exemplar of this). like a temperate phage, a host that is colonized by episomal persisting viruses has also been much affected in its evolutionary potential. it is clear that phage can have complex effects on host populations, but these phage themselves often exist in complex and mixed states that can be diffi cult to unravel ( harcombe and bull, 2005 ) . it has been known for some time that the presence of otherwise silent phage can greatly affect the growth of other virus and susceptibility of host. one such silent and common phage that has long been studied is p1. p1 was initially discovered due to its effect on t4 and lambda. however, p1 has been a very interesting model, not because it causes disease or offers potential therapy against bacterial pathogens, but simply because it persists effi ciently as an episome and competes effectively with many other phage ( yarmolinsky, 2004 ) . since it does so without integrating, p1 provides us with one of the only well-studied models that can inform us regarding the molecular strategies and details of how stability in non-genomic persistence is attained. curiously, a main strategy by which p1 attains this stability was inapparent and not suspected after several decades of study. it became apparent only after replication mutations were made that induced self-destruction and uncovered the existence of what came to be called " addiction modules " ( lehnherr et al. , 1993 ) . p1 encodes several gene pairs (toxins/ antitoxins, such as the phd/doc pair) that protect bacteria harboring p1, but kill daughter bacteria that have lost the p1 genome ( gazit and sauer, 1999 ) . this strategy compels colonized e. coli to maintain p1 or die (doc, death on curing). however, these very same addiction systems are also involved in protecting a p1-colonized colony from t4 and lambda infection and will also induce self-destruction when cells are infected by those viruses, protecting the colony (population). p1 also provides an exquisite level of molecular self-identifi cation in that it will recognize a single second copy of its own genome ( yarmolinsky, 2000 ) . what then is the fi tness and evolutionary consequence to e. coli harboring p1? clearly it is major, but mostly host fi tness is affected relative to other viruses. accordingly, when contemplating the amazing complexity of the p1 immunity and how it evolved, yarmolinsky posed the question; " could the byzantine complexity of the controls at immi be the outcome, not of successive host-parasite accommodations, but of competition among related phages? " ( yarmolinsky, 2004 ) . if we answer yes to this question, then we would also conclude that virus-virus interactions and competition in general are major forces in the adaptability and evolution of persisting phage and surviving colonized host. in this light, viral persistence takes on a major role in virus and host evolution. the p1 exemplar has thus provided us the concept of viral addiction that also promotes host group selection. historically, we are biased to think of viruses (and phage) as agents that simply kill their host. some have proposed that the prokaryotic global biomass is phage partitioned into those populations that live and those that die due to viral lysis. from such a perspective, viral novelity would seen of little relevance to host evolution. metagenomic projects as noted above, have sequenced nearly 2 million phage genomes and report that most of these phage genes are unique, not in the database, and likely not derived from host ( edwards and rohwer, 2005 ) . the protein repertoire of sequenced phage indicates that 80% of conserved phage genes are specifi c to phage and show an evolutionary independence from genes of host . this identifi es a massive genetic novelty from virus, which is especially apparent in large dna phage. as just discussed above, however, those hosts that live are also products of phage selection, and persisting temperate phage play a major role in this. such phage colonization allows this massive phage novelty to fi nd its way into host genomes, which allows viral complex gene sets to be applied to novel problems of host adaptation. host novelty can thus be introduced by phage ( comeau and krisch, 2005 ) . that persistence is a major life strategy of phage is confi rmed by the large numbers of genes associated with persistence (i.e. integrases, immunity) observed in metagenomic screens. there is also much practical experience that supports the crucial role of prophage in host evolution. one particularly well-studied system that has been studied for over 50 years is the ongoing evaluation of phage evolution as observed in the dairy industry ( canchaya et al. , 2003 brussow et al. , 2004 ) . the temperate phage analysis of these bacteria follows a long tradition of lambda and e. coli studies ( campbell et al. , 1992 ; canchaya et al. , 2003 canchaya et al. , , 2004 . since lytic phage can severely disrupt dairy fermentation, it was of particular interest to understand and trace their evolution. these studies have led brussow to conclude that much of the more recent dairy bacteria evolution can be considered to have resulted from the action of temperate phage. a similar view applies to e. coli and cyanobacteria. in addition, the ecor collection of 72 sequenced e. coli genomes of medical interest shows that they differ from each other mainly due to patterns of genetic colonization, mostly by prophage, but they also show the presence trna-adjacent defective prophage and plasmid elements that differentiate these strains ( hurtado and rodriguez-valera, 1999 ; mazel et al. , 2000 ; nilsson et al. , 2004 ) . cyanobacteria ( prochlorococcus ) is major model for the study of the origin of the type ii (plant-like) photosynthetic system. since such genes show much evidence of recent and massive horizontal movement, it seem quite likely that prophage are mediators of such transfers, especially as these phage encode their own version of these photosynthetic genes ( lindell et al. , 2004 ; sullivan et al. , 2006 ) . very similar prochlorococcus strains exist in distinct oceanic populations in various habitats known as ecotypes. some think that such ecotypes represent the initial type of genetic variation that leads to speciation. the sequencing of six ecotypes has shown that they are 99% similar to one another, but the genetic variation that distinguishes them is mostly due to patterns of prophage colonization (called phage islands) ( bouman et al. , 2006 ; coleman et al. , 2006 ) . thus in all these prokaryotic models, persisting viruses play a fundamental role in host evolution and host genetic novelty is mostly phage derived. such observations have led some to propose that " war is peace " regarding virus-host evolution ( comeau and krisch, 2005 ) . massive and complex innovation by phage appears to be a major force in the prokaryotic world. prokaryotes are the most adaptable of all cells. if we can accept the above conclusion concerning the role for viruses in the evolution of prokaryotes, we must then ask why such a successful evolutionary strategy was not apparently maintained in eukaryotes? in eukaryotes we see little evidence that largescale integration by dna viruses is an important evolutionary process (although the story with retroviruses is different). why should prokaryotes and eukaryotes differ is such a fundamental way? nevertheless, as noted at the start of this section, we do see good evidence that links the evolution of large dna viruses of prokaryotes to the large dna viruses of eukaryotes. in case we were becoming comfortable with the apparently clear distinctions between rna and dna virus evolution as outlined above (quasispecies vs. domain recombination respectively), the evolution of the parvoviruses informs us that dna viruses can also evolve by a quasispecies process. parvovirus evolution (see chapter 17) can show a sharp contrast to the evolutionary pattern displayed by other small dsdna viruses above (hpv, py). with the emergence of an acute pandemic in domestic dogs and cats (as well as other wild carnivore species), we see what is essentially evolution driven by single point mutations, mostly affecting the capsid genes and host cell receptor binding. this system provides us with one of the better studied examples of the evolutionary dynamics of an emergent viral disease. in addition, in vivo mouse studies with minute virus of mouse (mvm) now make it clear that parvoviruses can behave much like rna viruses, generating quasispecies of diverse progeny that allow a high adaptability for the generation of fi tness and disease in vivo ( lopez-bueno et al. , 2006 ) . this story is very reminiscent of the study of poliovirus in mice mentioned above. human studies with b19 parvovirus are also consistent with high mutation rates ( parsyan et al. , 2007 ; shackelton and holmes, 2006 ) . although not specifi cally addressed in this volume, the viruses of eukaryotic unicellular green algae are of special interest from the perspective of dna virus evolution. these large, complex dsdna membrane-containing icosahedral viruses are abundant in some water habitats ( van etten, 2003 ; ghedin and claverie, 2005 ) . the reason they deserve special attention is that they clearly have many features that are characteristic of both prokaryotic and eukaryotic viruses. they resemble prokaryotic viruses in that their life cycle is clearly phagelike, such as external virion attachment, injection of dna and no pinocytosis. in addition, they also encode many phage-like genes, such as restriction-modifi cation enzymes and homing endonucleases ( filee et al. , 2006c ) . they also resemble eukaryotic viruses in that they have eukaryotic dna replication proteins (dna polymerase beta and pcna; chen and suttle, 1996 ; nagasaki et al. , 2005 ; villarreal and defilippis, 2000 ) as well as many genes associated with eukaryotic signal transduction ( van etten et al. , 2002 ) . thus they represent a clear link between prokaryotic and eukaryotic dna viruses. for example, the dna polymerase of paramecium bursaria chlorella virus (pbcv-1) is the most conserved gene and most closely resembles that found in human herpesvirus and is distantly related to the similar family dna pol encoded by t4. this polymerase is distinct from that of the poxviruses or prd1/adenoviruses (associated with protein-primed dna replication). however, numerous other genes of the phycodnaviruses are similar to some genes found in the mimiviruses (giant dna virus of ameba), including the presence of conserved intenes in the dna pol gene ( ogata et al. , 2005 ) . in view of this it is most curious that in structural similarity, polydnavirus capsids clearly resemble prd1 capsid ( khayat et al. , 2005 ; nandhagopal et al. , 2002 ) . prd1 contains the double-barrel trimer capsid structure that was fi rst observed in adenovirus (for references see saren et al. , 2005 ) . adenovirus also closely resembles prd1 in dna replication strategy (i.e. linear dna with covalently closed ends ( benson et al. , 2004 ; khayat et al. , 2005 ) . the lineage of adenovirus-like dna viruses, however, is thought to be distinct from that herpes and poxviruses and its dna polymerase is clearly distinct from polyndavirus. it is clear that related elements of all these viruses can be found in phycodnaviruses. overall, the phycodnaviruses, like phage, also appear to be creating genes in large numbers and they encode many genes unrelated to their host. what then is the evolutionary relationship that links all of these seemingly distinct viruses? as outlined above, the pattern of evolution of dsdna phage involves lots of exchange by recombination from a vast gene pool. this pool resembles a cloud from which various mosaic subelements and substrategies are assembled to allow viral gene acquisition and novelty ( blum et al. , 2001 ; benson et al. , 2004 ) . does such a distributed pattern of evolution and gene novelty also apply to the phycodnaviruses? recently, another distinct phycodnavirus has been sequenced: coccolithovirus (ehv-86) ( allen et al. , 2006a ( allen et al. , , 2006b conserves only 24 core genes in common with pbcv-1 and is unique to the phycodnaviruses in that it has acquired six dnadep rna polymerase subunit genes, which are absent in all other phycodnaviruses. as rna polymerase is considered a core viral gene function, it is clear that phycodnaviruses can alter some very basic molecular functions during their evolution. oceanic phycodnaviruses are thought to have large infl uence on the free-living populations of eukaryotic algae, such as the termination of algal blooms reported for emilian huxley virus ( martinez et al. , 2007 ; schroeder et al. , 2003 ) . however, not all phycodnaviruses are lytic. another lineage of phycodnaviruses is represented by two viruses of fi lamentous brown algae, esv-1 and firrv-1 ( delaroque et al. , 2003 ) . unlike the lytic phycodnaviruses noted above, these two viruses are " temperate phage " like. that is they exist as silent viruses whose dna is integrated into the germlines of their host. in this, they are unique to all known eukaryotic dna viruses; host chromosome integration is a normal part of their persistent life strategy. esv-1 has a 335 593-bp genome and encodes 231 likely genes ( delaroque et al. , 2001 ) . these genes are mostly unique and only 28 are clearly related to pbcv-1 genes. the gene differences include many replication genes and their gene order is completely different. like the temperate phage-host evolutionary relationship outlined above, it would be most interesting to understand how the integration of these large dna viruses has affected host evolution. thus, the phycodnaviruses appear to represent a basal but diverse viral lineage that has both acute and persistent lifestyle and have some clear relationships to most large eukaryotic dna viruses and many phage. the phycodnavirus exemplar above should leave us with several impressions regarding the nature and evolution of these large and ubiquitous dna viruses of algae, an early eukaryotic host. they show clear linkages by structure and function to both phage and various eukaryotic dna viruses. they also show major variation and novelty in their own genetic composition, including their core genes. in addition, they show clear relationships to distinct and seemingly separate viral lineages (adenoviruses, herpesviruses, poxviruses, iridoviruses). the picture we are left with is that they seem to resemble phage evolution in that they appear to have evolved from a diverse pool that has exchanged many basic viral features and created many new genes. this view, however, contrasts sharply with the work of iyer et al. (2001 iyer et al. ( , 2006 . by considering the small number of conserved genes in four families of eukaryotic dna viruses (poxviruses, asfarviruses, iridoviruses, phycodnaviruses), they suggest that these viruses are monophyletic, evolving from a common nucleo-cytoplasmic large dna virus (ncldv) with an icosahedral capsid. given the above information, i fi nd this view unhelpful and possibly confusing. it has numerous problems. the main problem is that it fails to acknowledge the clear link between prokaryotic and eukaryotic viruses. furthermore, by focussing on a small set of related genes, it represents a traditional perspective as found in evolutionary biology that assumes a common (fi ttest) linear lineage, not a cloud, cooperative, or mosaic pool as the main source of novelty resulting in the matrix pattern of virus evolution. the virosphere is clearly not disconnected from itself, but it is also clearly not a linear or tree-like evolutionary system as suggested above. we must learn to think of virus evolution in its own terms; fuzzy, mixed, reticulated, and cloud-like. as mentioned in the phage section, there have been various publications that suggest a deep evolutionary relationship between the herpesviruses and dsdna viruses of prokaryotes ( rice et al. , 2004 ; khayat et al. , 2005 ; duda et al. , 2006 ; akita et al. , 2007 ) . such enormously distant relationships, however, cannot now be measured by any reliable metric. although herpes-like viruses are found in invertebrates (such as ostreid herpesvirus 1 (oshv-1)) in both lytic and asymptomatic states ( barbosa-solomieu et al. , 2005 ) , our interest in their evolution has been mainly focussed on the vertebrate herpesviruses. vertebrate herpesvirus do tend to show clear sequence conservation that suggests broad patterns of evolution. one interesting feature of this evolution is the apparent link between the biology of the virus and its evolution. a common, but not universal pattern is that of virus and host co-evolution ( mcgeoch et al. , 2000 ( mcgeoch et al. , , 2006 mcgeoch and gatherer, 2005 ) . this trend has maintained several biological characteristics, such as highly species host-and tissue-specifi c persistence (i.e. neuronal and lymphoid persistence). the discovery of hhv-8 has further stimulated studies of herpesvirus evolution in that hhv-8 appears to have undergone much recombination with herpesviruses of related primate lineages ( mcgeoch and davison, 1999 ) . thus recombination seems prevalent in herpesviruses. the apparent link between herpesvirus evolution and recent human evolution, as well as an apparent link to primate retroviral evolution, is fascinating, but of unknown signifi cance ( kung and wood, 1994 ; lacoste et al. , 2000 ) . the herpesviruses lineages will often show the presence of lineage-specifi c genes. many of these genes affect innate and adaptive host functions, whereas others affect host metabolism. when the source of such genes has been contemplated, in contrast to phage, phycodnaviruses, or baculoviruses ( herniou et al. , 2001 ) , it is often proposed that most such herpes genes originate from the host. it is well accepted that the three major lineages of herpesviruses descended from a common ancestor in vertebrates ( mcgeoch et al. , 2006 ) . there have been numerous proposals that most new lineage-specifi c herpesvirus genes have originated from host (see becker and darai, 2000 ) . this includes herpesvirus dutpase ( davison and stow, 2005 ) , and viral chemokines and viral bcl-2 ( nicholas et al. , 1998 ) . in my evaluation of such claims, however, it seems clear that the possibility that there was an ancient viral source of such genes was not considered and cannot now be dismissed. we currently believe that ancient herpesvirus ancestors can be traced to tailed phage ( hendrix, 1999 ; bamford, 2003 ; baker et al. , 2005 ; duda et al. , 2006 ; mcgeoch et al. , 2006 ) . other phage lineages also appear to trace to eukaryotic viruses ( bamford et al. , 2005b ) . within the herpesviruses, the same t-16 icosahedral structure, as well as invertable dna regions are also present in the very distant but much more recognizable oceanic ostreid herpesvirus 1 . given the highly diverse and mosaic nature of large dna virus evolution in prokaryotes and lower eukaryotes described above, it seem quite possible that many other viral genes might also trace far back in virus evolution. consider the example of dutpase in avian and mammalian herpesvirus ( davison and stow, 2005 ; mcgeehan et al. , 2001 ) . the current view requires very complicated gene rearrangements to account for the viral source of this gene from its host. yet we know that diverse dutpases are found in many ancient viral lineages. for example, the ervs present in all vertebrate genomes also conserve dut-pase ( jern et al. , 2005 ) , as do exogenous retroviruses (i.e. lentiviruses) ( mcintosh and haynes, 1996 ) . in fact, since the herpesviruses genes are especially poor in introns, it would seem likely that any herpesviral gene acquisition would necessarily involve a retrovirus via a cdna. the oceans are especially fi lled with large complex dna viruses (such as mimivirus and phycodnavirus, plus numerous relatives of oshv-1) thought to be ancient ancestors of herpesvirus. the phycodnavirus (chlorella virus, pbcv-1) provides a clear bridge between phage and eukaryotic dna viruses. pbcv-1 also encodes a dutpase that has the highly conserved motif iii ( zhang et al. , 2005 ) . many phage are also known to encode dutpases of diverse types, such as b. subtilis (spbeta) ( persson et al. , 2005 ) , and a phage of thermus thermophilus ( naryshkina et al. , 2006 ) . this thermus phage (phiys40) is of special note since its dutpase gene is clearly related to the dutpases of eukaryotic viruses and has a version that has undergone multiple events of recombination from apparently distinct phage, exactly as expected for mosaic phage genes. thus, the origin of new herpesvirus genes might not be so different than that seen in other large dna viruses and a potential ancient source of new genes from these ancestral viruses remains plausible. similar considerations apply to other possible examples of herpesvirus gene capture. for example, the herpes thymidylate synthase (ts) has also been considered to have originated by host gene capture ). yet distinct versions of these genes are also found in different herpesviral lineages, which would necessitate multiple independent " capture " events of different version of host ts genes. ts genes are present in ancient virus sources. for example, bacillus phage beta 22 encodes ts, which also has a self-splicing intron ( bechhofer et al. , 1994 ) . also, phage phikz has a highly conserved ts ( mesyanzhinov et al. , 2002 ), yet this virus lacks a dna polymerase or other replication proteins, clearly indicating that the viral ts genes has a basic viral role. similarly, the cytokines-like genes (such as il-10) as found in poxviruses and herpesviruses appear to have originated in at least three independent events prior to the divergence of mammalian eutherian orders. yet it is still presupposed that they are necessarily the products of host gene capture ( hughes, 2002 ) . comparative genomics supports the idea that the herpesviruse lineages are originating viral genes. a broader phylogenetic analysis of all herpesvirus genomes identifi ed only 17 genes in common to all 30 taxa of herpesvirus . thus only 30 genes appear to be in common to all the herpesviruses. in this analysis, only a few genes of recent origin could be identifi ed as possibly having been transferred between virus and host (e.g. new genes found at tips of phylogenetic dendograms). thus, gene gain in the herpesviruses (as in dna phage and phycodnavirus) is prevalent but the origination of such genes from the host is not prevalent. i suggest that our tendency to assume that new viral genes are usually " stolen " from the host should be revised ( moreira and lopez-garcia, 2005 ). in contrast to the herpesviruses, the poxviruses evolution tend to have little congruence to host evolution (see chapter 19 ). yet, they too show evidence of ancient linkages to other viruses. the replication of poxvirus dna is distinct in that it involves a linear genome with inverted ends that have covalently closed " snapback " dna. the resulting replication structures involve head-to-tail and tail-to-tail intermediates. this replication strategy is very different from that used by the host (and most other dna viruses), but is clearly related to that found in other eukaryotic and prokaryotic viruses. similar replication mechanisms are seen in all poxviruses, as well as african swine fever virus and phycodnaviruses (pbcv-1). this exact replication strategy is also present in archaeal lipothrixviruses (sirv1 and sirv2) which has been proposed to be ancestral to phycodnaviruses and poxviruses ( persson et al. , 2005 ) . a similar replication strategy is also seen with n15 ( lobocka et al. , 1996 ) , an unusual phage of e. coli that persists as a linear dna ( casjens et al. , 2004 ) . conservation of such replication similarities clearly suggests ancestral relationships, but no sequence similarity can be seen between these viruses. the similarity between poxvirus and pbcv-1 dna replication deserves some additional comment. pbvc-1 and herpesvirus have very similar dna polymerase genes, yet differ fundamentally in replication strategy. furthermore, the poxviral dna polymerase gene is very different from that found in the herpesviruses. yet, the pbcv-1 capsid was clearly similar to that of adenoviruses and prd1 phage (and iridovirus capsids). how then do we link poxvirus evolution to other more ancient dna viruses, such as pbcv-1 which has the same dna replication mechanism, but distinct replication proteins? such observations might seem confusing, but they are clearly consistent with mosaic, reticulated evolution of dna viruses. various distinct phage lineages can link in multiple ways to various distinct eukaryotic dna viruses. the concept of a net or matrix rather than a tree is thus a better way to describe the broad topology of dna virus evolution. the issue of gene gain and gene loss is also of central interest to orthopoxvirus evolution. typically, we seek to understand poxviruses evolution from the perspective of pathogenesis, such as the origin of human-specifi c smallpox virus. with the comparative genomics of several orthopoxviruses now possible, we see curious overall patterns of gene loss in their evolution ( randall et al. , 2004 ) . for example, comparing human smallpox to cowpox dna (a rodent virus that is phylogenetically basal to smallpox), we observe an overall diminution of gene content in smallpox virus. several poxviruses seem to have also lost genes relative to cowpoxvirus, especially genes that appear to affect immunity ( hughes and friedman, 2005 ) . i suggest that this evolutionary tendency for gene reduction is associated with a switch from a more demanding species-specifi c persistent life strategy to a less demanding, acute life strategy in a new host. cowpox is a naturally persistent infection in rodents (bank voles) ( feore et al. , 1997 ; chantrey et al. , 1999 ) , which has been called a natural virus reservoir ( hazel et al. , 2000 ) . smallpox is a strictly acute and human-specifi c disease. such gene loss in association with lost persistence could be a general situation and might also explain why clinical isolates of human cytomegalovirus isolates show a strong tendency to delete genes with passage in culture ( davison et al. , 2003 ) . most orthopoxviruses are not phylogenetically congruent with their vertebrate host. host switching and acute replication seem to be relatively common but recent occurrences in their evolution ( babkin and shchelkunov, 2006 ) . the avian poxviruses are not as well studied in this context, but curiously have signifi cantly more complex genomes than the orthopoxviruses ( jarmin et al. , 2006 ) . the entomopoxviruses are even less well understood from both a biological and molecular perspective, although they do conserve 49 genes found in all poxvirus family members ( gubser et al. , 2004 ) . clearly these poxviruses share some degree of evolutionary history. it is most curious that entomopoxviruses have even larger, more diverse and complex genomes than the other poxviruses. why? as insects lack an adaptive immune system (the target of many orthopoxvirus genes), they would seem to present a simpler host for virus adaptation. this group appears to be the most basal phylogenetically, but evolutionary relationships between entomopoxvirus and insect evolution have not been studied. the enotomopoxviruses are particularly prevalent in grasshopper and locust species, often in unapparent states. interestingly, within these viruses we can fi nd examples of major shifts in core replication genes, such as the family of dna pol gene that is used (a shift from dna pol x to dna pol b in two entomopoxvirus lineages). we can recall that the dna pol b gene closely resembles that found in phycodnaviruses (and herpesvirus), but is distinct from that in orthopoxvirus ( zhu, 2003 ) . we also see in the entomopoxviruses some clear links to phage genes, such as t4-like rna ligase found in all entomopoxviruses ( ho and shuman, 2002 ) as well as a lambdalike integrase seen in d1epv ( hashimoto and lawrence, 2005 ) . this integrase in d1epv implies possible integration and persistence, thus it is most signifi cant that d1epv also shows a clear persistent host infection as well as symbiosis and apparent phylogenetic congruence between virus and host. this virus is symbiotic in its parasitoid wasp host in that virus is injected into larval host along with the wasp egg (and also along with a second d1rhv rhabdovirus) and virus is needed for successful host parasitization. this symbiosis is clearly very reminiscent of the genomic polydnaviruses of other parasitoid wasp species. diepv is also expressed in the male poison gland. however, it is unknown if diepv is integrating into the host dna. clearly, d1epv it is part of a complex virus-virus-host symbiotic interaction. the overall evolution of orthopoxviruses contrasts sharply with that of the papillomaviruses as presented by bernard in chapter 18. here, highly species-specifi c and tissuespecifi c host infection are the norm and the viral evolution is typically highly congruent with the host (with some exceptions). the resolution between virus and host can be high, in that human racial and geographical populations, for example, can often be differentiated based on the type of hpv they harbor. yet here too there is evidence of signifi cant shifts in core gene usage early during papillomavirus evolution. in the human and rodent viruses, a highly conserved gene function associated with replication and cell control are the e6 and e7 early genes. in particular, the prb-binding domain of the e7 gene is thought to be central to the biological strategy of the virus. thus, it is most curious that the papillomaviruses of lagomorphs, such as bovine and reindeer papillomavirus, lack an e7 rb-binding domain and instead appear to use e5 or e9 genes for this regulatory function ( narechania et al. , 2004 ) . it seems an early but signifi cant and bifurcating shift occurred in the molecular strategy during the virus-host evolution of this group of viruses for unknown reasons. other small dna viruses (jcv, bkv, py) can also show similar high-resolution host congruence ( shadan and villarreal, 1995 ) . as well as similar curious shifts in basic molecular strategies. for example, the presence of a middle tantigen in mouse virus (a third early gene), but its absence from primate viruses ( gottlieb and villarreal, 2001 ) , clearly differentiates these viral lineages. although the origins of these entire small dna viruses are obscure, and any links to prokaryotic viruses are unknown, it does appear they have tended to retain their overall biological strategy and show a strong tendency for tissue-specifi c (especially kidney) persistence and virus-host congruence. since persistence requires the stable coexistence of a virus and its host, it also fi ts the simple defi nition of symbiosis (the stable living together of two distinct lineages of organisms). viral involvement in symbiosis is a foreign idea to many and possibly presents a fundamentally different view of the role viruses may have in host evolution. a major role for persisting (temperate, cryptic) viruses in the evolution of prokaryotes is no longer a controversial idea. thus, at least in the prokaryotic world, virus persistence can be accepted as adaptive. in eukaryotes, however, viral persistence is seldom considered adaptive. the mhv-mouse exemplar as presented above has suggested how persistence can directly affect host survival. can this be considered an example of symbiosis in the accepted sense? a crowning achievement in the fi eld of symbiosis has been to explain the origin of plastids (chloroplasts, mitochondria) from symbiotic prokaryotes in eukaryotic cytoplasm ( margulis and bermudes, 1985 ) . this idea involves the high adaptability of prokaryotes to provide innovation but would seem not to involve virus in any way. yet here too we can fi nd viral footprints that suggest some involvement. for example, various plastid-specifi c rna and dna polymerases clearly resemble polymerases from t3/t7-like phage ( cermakian et al. , 1996 ; shutt and gray, 2006 ) . other models of symbiosis also show evidence of a viral role, such as the sexual isolation of buchnera ( moran et al. , 2005 ) . another very popular topic in the fi eld of symbiosis is the symbiotic origin of the photosynthetic sea slug, elysa chlorotica . what could be more fascinating than a green sea slug-an animal that can use light for photosynthesis? e. chlorotica eats photosynthetic eukaryotic algae ( vaucheria litorea ) and retains the functional chloroplast from algae for months. here too, however, there lies a viral footprint. this slug harbors an unusual endogenous retrovirus which is expressed in large numbers during sexual reproduction, following which all slugs die via synchronized apoptosis and in which the chloroplasts have accumulated numerous viral particles ( pierce et al. , 1999 ; mondy and pierce, 2003 ) . since there is reason to think gene movement from the algae to the slug genome is involved in this symbiosis, the presence of this retrovirus is a strong candidate to also be involved in symbiogenesis. clearly we should thus investigate retroviral elements as possible symbiotic participants and not dismiss them beforehand as irrelevant or " junk dna " (as is automatically done in many database screens). if viral persistence is a kind of symbiosis, viruses may also mediate the establishment of other symbiotic relationships ( villarreal, 2007 ) . the recent studies by roossinck and colleagues (see chapter 12), in which a persisting virus, a plant, and a fungus were all symbiotically involved in altering the thermal tolerance of the plant could be an example of this ( marquez et al. , 2007 ) . many other virus-host relationships should also be examined for possible symbiosis. for example, placental vertebrate evolution has involved various endogenous retroviruses (i.e. herv-w, herv-frd). intact herv genomes, including env orfs, are important for placental trophoblast fusion (for references see ryan, 2007 ) . some will dismiss this situation as the quirky usurping of a viral gene for host function which is of little general signifi cance. the specifi c erv involved is simply selfi sh and mostly defective genetic material of no general consequence. if so, why is it that in sheep a distinctly different lineage of retrovirus (enjsrv) was also selected to provide a related placental function to a another mammal with signifi cantly different placental reproductive biology? it has been experimentally well established the enjsrv env is essential for sheep embryo implantation ( dunlap et al. , 2006a ( dunlap et al. , , 2006b . enjsrv is the endogenous version of jsrv, a problematic sheep-specifi c retrovirus that induces lung tumors (responsible for the death of dolly, the famous fi rst cloned sheep). the endogenous virus (enjsrv) is present in 20 copies in the sheep genome and all sheep have this virus. sheep genomes also encoded a trans -dominant enjrsv gag that is inhibitory to exogenous jsrv ( mura et al. , 2004 ; oliveira et al. , 2006 ; murcia et al. , 2007 ) . it seems clear that this situation can also be considered from the perspective of viral symbiosis and/or virus addiction in host evolution. we should thus seek to understand why colonization by an erv population might generally provide a good solution to the evolutionary demands of placental biology. there are many other opportunities to examine the potential role of persistent and symbiotic viruses in the evolution of viruses, animals, primates, and humans. for example, as we seek to understand the origins of the adaptive immune system we should pay attention to viral footprints. we can ask, for example, why the major histocompatibility complex (mhc) locus, the most polymorphic, diverse, and rapidly evolving gene set in our chromosome, is so densely colonized with retroviral elements ( andersson et al. , 1998 ) . why is a retrovirus also the basic element of the duplication unit that was thought to have been the progenitor for the expansion of the mhc class i (and ii) genes ( gaudieri et al. , 1997 ; kulski et al. , 1998 kulski et al. , , 2005 ? why do similar herv element (l and 16) also differentiate between human and chimpanzee mhc i ( watkins, 1995 ; kulski et al. , 1999 ) ? what was the role for siv in the evolution of primate mhc ( vogel et al. , 1999 ) ? humans and primates appear to have undergone some signifi cant and relatively recent evolution with regard to their endogenous and exogeneous retroviruses. along these lines, apobec-like genes are basic component of the adaptive immune response but they are also antiretroviral genes that act on retroviral cdna and gag ( ohainle et al. , 2006 ) . the apobec3 antiviral system has expanded recently in humans, but not chimpanzees ( sawyer et al. , 2004 ; ohainle et al. , 2006 ) . why? all african primates support unapparent foamy viruses (and also siv co-infection), but not humans ( murray and linial, 2006 ) . apobec3c is active against foamy viruses ( delebecque et al. , 2006 ) . old world primates also underwent an expansion of ervl colonization (a clear relative of foamy virus) ( sawyer et al. , 2004 ) . was this ervl colonization of relevance to the ancient co-speciation of simian foamy virus and their primate host ( switzer et al. , 2005 ) ? what exactly was the relevance of herv endogenization to human survival and adaptations? curiously, human brain (neocortex) specifically expresses many of these more recent ervs as transcripts ( nakamura et al. , 2003 ; yi et al. , 2004 ) . if we consider these situations as possible examples of virus-mediated symbiosis in human evolution, perhaps they may make more sense of the otherwise confusing role or hervs. as noted, all primates, but especially humans show much evidence of recent endogenization by retroviruses. but these events mostly occurred in our extinct ancestors and we do not see ongoing evidence that any hervs remain active. however, we are currently witnessing a related virus-host evolutionary event of considerable interest. koala bears, native marsupials of australia, are currently experiencing a major epidemic caused by a leukemiainducing retrovirus. as a consequence, they are undergoing massive endogenization by a gammaretrovirus (mlv-related). this virus is similar to gibbon ape leukemia virus, but most likely originated from rodent ancestry ( tarlinton et al. , 2006 ; fiebig et al. , 2006 ) . the expectation is that extinction awaits those koalas that do not adapt or endogenize the retrovirus successfully ( stoye, 2006 ) . this event has the appearances of a retroviral-driven addiction that will result in a genetic variant of koala bear that has acquired a new antiretroviral state. this seems equivalent to the expansion of human apobec3; or perhaps a closer analogy is the endogenization of a suppressive gag as occurred with enjsrv. the surviving koala bears will likely tolerate or be persistently infected with this retrovirus pool. the genome of the species will have undergone considerable (but unpredictable) genetic perturbations and likely contain a large pool of variant and defective retrovirus. however, in so doing, the descendent koalas will likely present a biological hazard to any koala species that remain virus-free (as in virus addiction). currently, one island colony of koalas is suffi ciently isolated to have remained virus-free. this population will henceforth be under persistent threat from populations of endogenized koalas, now favored by group selection. from the very earliest events in evolution of prebiotic replicators to very recent events in human evolution, including the emergence of human-specifi c hiv, we expect viral evolution to show profound effects on the evolution of all life. unlike accepted host evolution, viruses also employ consortia and mixed populations to evolve, sometimes at unprecedented rates. thus viruses have informed us of quasispecies, group dynamics, and group selection in evolution. virus evolution should now be considered as basic science, not just a medical concern. we must acknowledge that the tree of life cannot be properly understood without virus evolution. this book helps to lay the foundation for such understanding. epizootic diarrhea of infant mice: indentifi cation of the etiologic agent the crystal structure of a virus-like particle from the hyperthermophilic archaeon pyrococcus furiosus provides insight into the evolution of viruses unusual polymorphisms in human immunodefi ciency virus type 1 associated with nonprogressive infection a ) genome comparison of two coccolithoviruses b ) evolutionary history of the coccolithoviridae retroelements in the human mhc class ii region host switching in lyssavirus history from the chiroptera to the carnivora orders common ancestry of herpesviruses and tailed dna bacteriophages do viruses form lineages across different domains of life? a ) what does structure tell us about virus evolution? b ) constituents of sh1, a novel lipid-containing virus infecting the halophilic euryarchaeon haloarcula hispanica ostreid herpesvirus 1 (oshv-1) detection among three successive generations of pacifi c oysters ( crassostrea gigas ) persistent infection promotes cross-species transmissibility of mouse hepatitis virus the proportion of revertant and mutant phage in a growing population, as a function of mutation and growth rate an intron in the thymidylate synthase gene of bacillus bacteriophage beta 22: evidence for independent evolution of a gene, its group i intron, and the intron open reading frame serological survey of virus infection among wild house mice ( mus domesticus ) in the uk molecular evolution of viruses, past and present: evolution of viruses by acquisition of cellular rna and dna viral eukaryogenesis: was the ancestor of the nucleus a complex dna virus? sex and the eukaryotic cell cycle is consistent with a viral ancestry for the eukaryotic nucleus coexistence of recent and ancestral nucleotide sequences in viral quasispecies of human immunodefi ciency virus type 1 patients a subset of human immunodefi ciency virus type 1 long-term nonprogressors is characterized by the unique presence of ancestral sequences in the viral population does common architecture reveal a viral lineage spanning all three domains of life? the error threshold what is a quasispecies an envelope glycoprotein of the human endogenous retrovirus herv-w is expressed in the human placenta and fuses cells expressing the type d mammalian retrovirus receptor the genome of the archaeal virus sirv1 has features in common with genomes of eukaryal viruses oceanographic basis of the global surface distribution of prochlorococcus ecotypes a ) here a virus, there a virus, everywhere the same virus? b ) method for discovering novel dna viruses in blood using viral particle selection and shotgun sequencing metagenomic analyses of an uncultured viral community from human feces minority memory genomes can infl uence the evolution of hiv-1 quasispecies in vivo phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion . microbiol the gene of retroviral origin syncytin 1 is specifi c to hominoids and is inactive in old world monkeys phage integration and chromosome structure. a personal history lambdoid phages as elements of bacterial genomes phage as agents of lateral gene transfer the impact of prophages on bacterial chromosomes differences in frequencies of drug resistance-associated mutations in the hiv-1 pol gene of b subtype and bf intersubtype recombinant samples lethal intestinal virus of infant mice is mouse hepatitis virus different evolutionary patterns are found within human immunodefi ciency virus type 1-infected patients the pk02 linear plasmid prophage of klebsiella oxytoca two proton transfers in the transition state for nucleotidyl transfer catalyzed by rna-and dna-dependent rna and dna polymerases sequences homologous to yeast mitochondrial and bacteriophage t3 and t7 rna polymerases are widespread throughout the eukaryotic lineage cowpox: reservoir hosts and geographic range a phylogenetic survey of recombination frequency in plant rna viruses role of minority populations of human immunodefi ciency virus type 1 in the evolution of viral resistance to protease inhibitors extensive recombination among human immunodefi ciency virus type 1 quasispecies makes an important contribution to viral diversity in individual patients evolutionary relationships among large double-stranded dna viruses that infect microalgae and other organisms as inferred from dna polymerase genes the thymidylate synthase gene of hz-1 virus: a gene captured from its lepidopteran host mapping of mutations associated with neurovirulence in monkeys infected with sabin 1 poliovirus revertants selected at high temperature the red queen reigns in the kingdom of rna viruses mimivirus and the emerging concept of " giant " virus transcription of a ' photosynthetic ' t4-type phage during infection of a marine cyanobacterium genomic islands and the ecology and evolution of prochlorococcus war is peacedispatches from the bacterial and phage killing fi elds genetic richness of vibriophages isolated in a coastal environment implications of high rna virus mutation rates: lethal mutagenesis and the antiviral drug ribavirin rna virus error catastrophe: direct molecular test by using ribavirin poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: age-dependent paralysis and a mucosal route of infection trans-dominant inhibition of rna viral replication can slow growth of drug-resistant viruses new genes from old: redeployment of dutpase by herpesviruses the human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome a novel class of herpesvirus with bivalve hosts the complete dna sequence of the ectocarpus siliculosus virus esv-1 genome comparisons of two large phaeoviral genomes and evolutionary implications interference between bacterial viruses: iii. the mutual exclusion effect and the depressor effect restriction of foamy viruses by apobec cytidine deaminases serologic study on the prevalence of murine viruses in a population of wild meadow voles ( microtus pennsylvanicus ) serologic study on the prevalence of murine viruses in fi ve canadian mouse colonies new evolutionary frontiers from unusual virus genomes quasispecies and rna virus evolution: principles and consequences quasispecies: concept and implications for virology quasispecies and its impact on viral hepatitis nucleotide sequence heterogeneity of an rna phage population origin and evolution of viruses selfi sh genes, the phenotype paradigm and genome evolution asymmetric dde (d35e)-like sequences in the rag proteins: implications for v(d)j recombination and retroviral pathogenesis shared architecture of bacteriophage sp01 and herpesvirus capsids a ) ovine endogenous betaretroviruses (enjsrvs) and placental morphogenesis b ) endogenous retroviruses regulate periimplantation placental growth and differentiation syncytin-a and syncytin-b, two fusogenic placentaspecifi c murine envelope genes of retroviral origin conserved in muridae viral metagenomics molecular quasi-species genetic stability of the vhsv consensus sequence of g-gene in diagnostic samples from an acute outbreak evolution of fi tness in experimental populations of vesicular stomatitis virus fluctuation of hepatitis c virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis apobec3g cytidine deaminase inhibits retrotransposition of endogenous retroviruses molecular ecology and emergence of tropical plant viruses the effect of cowpox virus infection on fecundity in bank voles and wood mice transspecies transmission of the endogenous koala retrovirus the role played by viruses in the evolution of their hosts: a view based on informational protein phylogenies marine t4-type bacteriophages, a ubiquitous component of the dark matter of the biosphere a ) a selective barrier to horizontal gene transfer in the t4-type bacteriophages that has preserved a core genome with the viral replication and structural genes b ) t4-type bacteriophages: ubiquitous components of the " dark matter " of the biosphere c ) i am what i eat and i eat what i am: acquisition of bacterial genes by giant viruses female characteristics in the drosophila melanogaster sigma-virus system in natural-populations from languedoc (southern france) polymorphism of the drosophila melanogaster sigma virus system displacement of cellular proteins by functional analogues from plasmids or viruses could explain puzzling phylogenies of many dna informational proteins the great virus comeback-from an evolutionary perspective the two ages of the rna world and the transition to the dna world: a story of viruses and cells a ) the origin of viruses and their possible roles in major evolutionary transitions b ) three rna cells for ribosomal lineages and three dna viruses to replicate their genomes: a hypothesis for the origin of cellular domain luca: the last universal common ancestor origin and evolution of dna topoisomerases identifi cation of unique hepatitis c virus quasispecies in the central nervous system and comparative analysis of internal translational effi ciency of brain, liver and serum variants central nervous system changes in hepatitis c virus infection a century of tobamo virus evolution in an australian population of nicotiana glauca an ancient evolutionary origin of the rag1/2 gene locus prevalence of indigenous viruses in laboratory animal colonies in the united kingdom 1978-1979 whole genome molecular phylogeny of large dsdna viruses using composition vector method evolutionary transition toward defective rnas that are infectious by complementation retroelements and segmental duplications in the generation of diversity within the mhc the doc toxin and phd antidote proteins of the bacteriophage p1 plasmid addiction system form a heterotrimeric complex avoidance of palindromic words in bacterial and archaeal genomes: a close connection with restriction enzymes mimivirus relatives in the sargasso sea evolution and origins of tobamoviruses free-grazing ducks and highly pathogenic avian infl uenza nidovirales: evolving the largest rna virus genome natural biology of polyomavirus middle t antigen . microbiol suppression of viral infectivity through lethal defection poxvirus genomes: a phylogenetic analysis maternal antibodies protect immunoglobulin defi cient neonatal mice from mouse hepatitis virus (mhv)-associated wasting syndrome the viriosphere, diversity and genetic exchange within phage communities impact of phages on two-species bacterial communities synthesis and antiviral evaluation of a mutagenic and non-hydrogen bonding ribonucleoside analogue: 1-beta-d-ribofuranosyl-3-nitropyrrole placental endogenous retrovirus (erv): structural, functional and evolutionary signifi cance hantavirus infections: epidemiology and pathogenesis comparative analysis of selected genes from diachasmimorpha longicaudata entomopoxvirus and other poxviruses a longitudinal study of an endemic disease in its wildlife reservoir: cowpox and wild rodents evolution: the long evolutionary reach of viruses bacteriophages: evolution of the majority . theor bacteriophage genomics bacteriophages with tails: chasing their origins and evolution evolutionary relationships among diverse bacteriophages and prophages: all the world ' s a phage use of whole genome sequence data to infer baculovirus phylogeny bacteriophage t4 rna ligase 2 (gp24.1) exemplifi es a family of rna ligases found in all phylogenetic domains sequence variability in the env-coding region of hepatitis c virus isolated from patients infected during a single source outbreak the evolutionary biology of dengue virus the origin, emergence and evolutionary genetics of dengue virus enterotropic mouse hepatitis virus albert b. sabin and the development of oral poliovaccine origin and evolution of viral interleukin-10 and other dna virus genes with vertebrate homologues poxvirus genome evolution by gene gain and loss accessory dna in the genomes of representatives of the escherichia coli reference collection isolation of mouse hepatitis virus from infant mice with fatal diarrhea common origin of four diverse families of large eukaryotic dna viruses evolutionary genomics of nucleo-cytoplasmic large dna viruses avipoxvirus phylogenetics: identifi cation of a pcr length polymorphism that discriminates between the two major clades use of endogenous retroviral sequences (ervs) and structural markers for retroviral phylogenetic inference and taxonomy rag1 core and v(d)j recombination signal sequences were derived from transib transposons small mammal virology structure of an archaeal virus capsid protein reveals a common ancestry to eukaryotic and bacterial viruses hiv diversity, molecular epidemiology and the role of recombination origin of human immunodefi ciency virus type 1 quasispecies emerging after antiretroviral treatment interruption in patients with therapeutic failure virus persistence and recurring demyelination produced by a temperature-sensitive mutant of mhv-4 horizontal gene transfer in prokaryotes: quantifi cation and classifi cation structurefunction relationships of the viral rna-dependent rna polymerase: fidelity, replication speed and initiation mechanism determined by a residue in the ribose-binding pocket the evolution of mhc diversity by segmental duplication and transposition of retroelements comparison between two human endogenous retrovirus (herv)-rich regions within the major histocompatibility complex ervk9, transposons and the evolution of mhc class i duplicons within the alpha-block of the human and chimpanzee interactions between retroviruses and herpesviruses . singapore; river edge evolution and selection of hepatitis c virus variants in patients with chronic hepatitis c kshv-like herpesviruses in chimps and gorillas plasmid addiction genes of bacteriophage p1: doc, which causes cell death on curing of prophage and phd, which prevents host death when prophage is retained evolution and persistence of infl uenza a and other diseases bats are natural reservoirs of sars-like coronaviruses transfer of photosynthesis genes to and from prochlorococcus viruses protein repertoire of double-stranded dna bacteriophages characterization of the primary immunity region of the escherichia coli linear plasmid prophage n15 parvovirus variation for disease: a difference with rna viruses? cross-species transfer of viruses: implications for the use of viral vectors in biomedical research, gene therapy and as live-virus vaccines bacteriophage: an essay on virus reproduction mutations of bacteria from virus sensitivity to virus resistance virus growth and variation: ninth symposium of the society for general microbiology prevalence of natural virus infections in laboratory mice and rats used in canada different patterns of molecular evolution of infl uenza a viruses in avian and human populations crystal structure of poliovirus 3cd protein: virally encoded protease and precursor to the rna-dependent rna polymerase symbiosis as a mechanism of evolution: status of cell symbiosis theory a virus in a fungus in a plant: three-way symbiosis required for thermal tolerance hepatitis c virus (hcv) circulates as a population of different but closely related genomes: quasispecies nature of hcv genome distribution mhv infection of the cns: mechanisms of immunemediated control molecular dynamics of emiliania huxleyi and cooccurring viruses during two separate mesocosm studies role of disease in abundance of a pacifi c herring ( clupea pallasi ) population . can antibiotic resistance in the ecor collection: integrons and identifi cation of a novel aad gene evolution of the dutpase gene of mammalian and avian herpesviruses the descent of human herpesvirus 8 integrating reptilian herpesviruses into the family herpesviridae toward a comprehensive phylogeny for mammalian and avian herpesviruses topics in herpesvirus genomics and evolution hiv and human endogenous retroviruses: an hypothesis with therapeutic implications the genome of bacteriophage phikz of pseudomonas aeruginosa syncytin is a captive retroviral envelope protein involved in human placental morphogenesis algorithms for computing parsimonious evolutionary scenarios for genome evolution, the last universal common ancestor and dominance of horizontal gene transfer in the evolution of prokaryotes apoptotic-like morphology is associated with annual synchronized death in kleptoplastic sea slugs ( elysia chlorotica ) the players in a mutualistic symbiosis: insects, bacteria, viruses and virulence genes low genetic barrier to large increases in hiv-1 cross-resistance to protease inhibitors during salvage therapy comment on ' the 1.2-megabase genome sequence of mimivirus murine viruses in an island population of introduced house mice and endemic short-tailed mice in western australia pathogens of house mice on arid boullanger island and subantarctic macquarie island late viral interference induced by transdominant gag of an endogenous retrovirus the transdominant endogenous retrovirus enjs56a1 associates with and blocks intracellular traffi cking of jaagsiekte sheep retrovirus gag foamy virus infection in primates the minimal genome concept algal viruses with distinct intraspecies host specifi cities include identical intein elements human endogenous retroviruses with transcriptional potential in the brain the structure and evolution of the major capsid protein of a large, lipidcontaining dna virus lack of the canonical prb-binding domain in the e7 orf of artiodactyl papillomaviruses is associated with the development of fi bropapillomas thermus thermophilus bacteriophage phiys40 genome and proteomic characterization of virions natural history of murine gamma-herpesvirus infection molecular biodiversity of cassava begomoviruses in tanzania: evolution of cassava geminiviruses in africa and evidence for east africa being a center of diversity of cassava geminiviruses the evolution of epidemic infl uenza novel organizational features, captured cellular genes and strain variability within the genome of kshv/hhv8 site-specifi c recombination links the evolution of p2-like coliphages and pathogenic enterobacteria genetic diversity among fi ve t4-like bacteriophages exponential increases of rna virus fi tness during large population transmissions lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells a new example of viral intein in mimivirus adaptive evolution and antiviral activity of the conserved mammalian cytidine deaminase apobec3h genetic heterogeneity of hepatitis c virus in vitro characterization of a koala retrovirus selfi sh dna: the ultimate parasite hot crenarchaeal viruses reveal deep evolutionary connections encyclopedia of evolution identifi cation and genetic diversity of two human parvovirus b19 genotype 3 subtypes marine phage genomics cloning, expression, purifi cation and characterisation of the dutpase encoded by the integrated bacillus subtilis temperate bacteriophage spbeta increased fi delity reduces poliovirus fi tness and virulence under selective pressure in mice bottleneckmediated quasispecies restriction during spread of an rna virus from inoculation site to brain annual viral expression in a sea slug population: life cycle control and symbiotic chloroplast maintenance a ) viruses of the archaea: a unifying view b ) evolutionary genomics of archaeal viruses: unique viral genomes in the third domain of life adp-ribose-1 " -monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture phylogenetic evidence for deleterious mutation load in rna viruses and its contribution to viral evolution defective interfering rnas of a satellite virus structural proteomics of the poxvirus family the 1.2-megabase genome sequence of mimivirus the structure of a thermophilic archaeal virus shows a double-stranded dna viral capsid type that spans all domains of life plant rna virus evolution symbiosis versus competition in plant virus evolution mutant clouds and occupation of sequence space in plant rna viruses quasispecies development by high frequency rna recombination during mhv persistence memory in viral quasispecies viruses as symbionts a snapshot of viral evolution from genome analysis of the tectiviridae family prevalence of naturally occurring viral infections, mycoplasma pulmonis and clostridium piliforme in laboratory rodents in western europe screened from virus succession observed during an emiliania huxleyi bloom phylogenetic evidence for the rapid evolution of human b19 erythrovirus the evolution of small dna viruses of eukaryotes: past and present considerations coinfection of wild ducks by infl uenza a viruses: distribution patterns and biological signifi cance bacteriophage origins of mitochondrial replication and transcription proteins plant virus satellite and defective interfering rnas: new paradigms for a new century prevalence of viral antibodies and helminths in fi eld populations of house mice (mus domesticus) in southeastern australia the origin of hepatitis c virus genotypes emergence and predominance of an h5n1 infl uenza variant in china phylogenetic analyses of type a infl uenza genes in natural reservoir species in north america reveals genetic variation dynamics of autocatalytic replicator networks based on higher-order ligation reactions koala retrovirus: a genome invasion in real time no evidence for quasispecies populations during persistence of the coronavirus mouse hepatitis virus jhm: sequence conservation within the surface glycoprotein gene s in lewis rats evolution of avian infl uenza viruses prevalence and evolution of core photosystem ii genes in marine cyanobacterial viruses and their hosts ultrastructural characterization of the giant volcano-like virus factory of acanthamoeba polyphaga mimivirus ancient co-speciation of simian foamy viruses and primates poxviruses and the origin of the eukaryotic nucleus prevalence and genetic diversity of coronaviruses in bats from a mutation in the rna polymerase of poliovirus type 1 contributes to attenuation in mice retroviral invasion of the koala genome unusual life style of giant chlorella viruses phycodnaviridae-large dna algal viruses collective evolution and the genetic code ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications quasispecies diversity determines pathogenesis through cooperative interactions in a viral population evolutionary insights into the ecology of coronaviruses dna virus contribution to host evolution viruses and the evolution of life how viruses shape the tree of life virus-host symbiosis mediated by persistence a hypothesis for dna viruses as the origin of eukaryotic replication proteins on viruses, sex and motherhood acute and persistent viral life strategies and their relationship to emerging diseases major histocompatibility complex class i genes in primates: co-evolution with pathogens high prevalence of infl uenza a virus in ducks caught during spring migration through sweden first demonstration of a lack of viral sequence evolution in a nonprogressor, defi ning replication-incompetent hiv-1 infection phylogenetic analysis, genome evolution and the rate of gene gain in the herpesviridae the evolution of major histocompatibility class i genes in primates h5n1 infl uenza-continuing evolution and spread elimination of mouse hepatitis virus from a breeding colony by temporary cessation of breeding matrix gene of infl uenza a viruses isolated from wild aquatic birds: ecology and emergence of infl uenza a viruses persistent mhv (mouse hepatitis virus) infection reduces the incidence of diabetes mellitus in non-obese diabetic mice long intervals of stasis punctuated by bursts of positive selection in the seasonal evolution of infl uenza a virus bacteriophage p1 in retrospect and in prospect human endogenous retroviral elements belonging to the herv-s family from human tissues, cancer cells and primates: expression, structure, phylogeny and evolution a reevaluation of the higher taxonomy of viruses based on rna polymerases chlorella virus-encoded deoxyuridine triphosphatases exhibit different temperature optima persistence of extraordinarily low levels of genetically homogeneous human immunodefi ciency virus type 1 in exposed seronegative individuals key: cord-027678-k64whepc authors: chan, kai man; gomersall, charles d title: pneumonia date: 2020-06-22 journal: oh's intensive care manual doi: 10.1016/b978-0-7020-4762-6.00036-9 sha: doc_id: 27678 cord_uid: k64whepc nan • pneumonia can be caused by over 100 organisms. • the relationship between specific clinical features and aetiological organism is insufficiently strong to allow a clinical diagnosis of the causative organism. 2 • early administration of appropriate antibiotics is important. 2 the net result is that the differential diagnosis is wide and treatment should be started before the aetiological agent is known. the differential diagnosis and the likely causative organisms can be narrowed by using epidemiological clues, the most important of which are whether the pneumonia is community-acquired or healthcare-associated and whether the patient is immunocompromised. note that the flora and antibiotic resistance patterns vary from country to country, hospital to hospital and even icu to icu within a hospital and this must be taken into account. evidence-based guidelines have been issued by the british thoracic society, 3 the infectious diseases society of america (idsa) and american thoracic society (ats) 2 and the european respiratory society. 4 links to these and other pneumonia-related guidelines can be found at the following 'link page': http://www. aic.cuhk.edu.hk/web8/pneumonia%20guidelines.htm. an acute infection of the pulmonary parenchyma that is associated with at least some symptoms of acute infection, accompanied by an acute infiltrate on a chest radiograph (cxr), or auscultatory findings consistent with pneumonia (e.g. altered breath sounds, localised crackles) in a patient not hospitalised or residing in a long-term care facility for ≥14 days prior to the onset of symptoms. the overall incidence is 3-40 per 1000 inhabitants per year, with 40-60% requiring hospital admission. overall, 10% of patients are admitted to icu. the overall mortality of hospitalised patient is approximately 10%. 5 pneumonia produces both systemic and respiratory manifestations. common clinical findings include fever, sweats, rigors, cough, sputum production, pleuritic chest pain, dyspnoea, tachypnoea, pleural rub and inspiratory crackles. classic signs of consolidation occur in less than 25% of cases. multi-organ dysfunction or failure may occur depending on the type and severity of pneumonia. the diagnosis of pneumonia may be more difficult in the elderly. although the vast majority of elderly patients with pneumonia have respiratory symptoms and signs, over 50% may also have non-respiratory symptoms and over a third may have no systemic signs of infection. investigations should not delay administration of antibiotics as delays are associated with an increase in mortality. 2 important investigations include: 416 pneumonia 9. urinary legionella antigen. this test is specific (>95%). in patients with severe legionnaires disease sensitivity is 88-100% for l. pneumophilia serogroup 1 (the most commonly reported cause of legionella infection). thus a positive result is virtually single or predominant organism on a gram stain of a fresh sample or a heavy growth on culture of purulent sputum is likely to be the organism responsible. the finding of many polymorphonuclear cells (pmn) with no bacteria in a patient who has not already received antibiotics can reliably exclude infection by most ordinary bacterial pathogens. specimens should be obtained by deep cough and be grossly purulent. ideally the specimen should be obtained before treatment with antimicrobials, if this does not delay administration of antibiotics, and be transported to the laboratory immediately for prompt processing to minimise the chance of missing fastidious organisms (e.g. strep. pneumoniae). acceptable specimens (in patients with normal or raised white blood cell counts) should contain >25 pmn per low-power field (lpf) and <10-25 squamous epithelial cells (sec)/lpf or >10 pmn per sec. these criteria should not be used for mycobacteria and legionella infection. certain organisms are virtually always pathogens when recovered from respiratory secretions (box 36.1). patients with risk factors for tuberculosis (tb) (box 36.2), and particularly those with cough for more than a month, other common symptoms of tb and suggestive radiographic changes, should have sputum examined for acid-fast bacilli. sputum cannot be processed for culture for anaerobes owing to contamination by the endogenous anaerobic flora of the upper respiratory tract. in addition to the factors listed in table 36 .1, foul-smelling sputum, lung abscess and empyema should raise suspicion of anaerobic infection. 8. aspiration of pleural fluid for gram stain, culture, ph and leucocyte count -all patients with a pleural effusion >1 cm thick on a lateral decubitus chest x-ray. contamination and colonisation. pcr assays are more sensitive than culture for mycoplasma and chlamydia species and at least as sensitive for legionella. 7 pcr assays also detect legionella strains other than serogroup 1. the bts guidelines 3 recommend pcr of lower respiratory tract sample or, if unavailable, throat swab for the diagnosis of mycoplasma pneumonia. pcr for chlamydophilia should be performed when invasive respiratory samples were collected from patients with severe community-acquired pneumonia. the role of pcr in diagnosing pcp is mainly limited to non-hiv patients, in whom conventional microscopy and staining of induced sputum and bal have a lower sensitivity than in hiv patients. 9 management general supportive measures intravenous fluids may be required to correct dehydration and provide maintenance fluid. a general approach should be made to organ support with an emphasis on correcting hypoxia. increased mortality among those who do not receive empirical antibiotics that cover the infecting pathogen(s) is well documented. 11 each unit should have its own regimens tailored to the local flora and antibiotic resistance patterns. in the absence of such regimens the regimen outlined in figure 36 other investigations should be considered in patients with risk factors for infection with unusual organisms. bronchoalveolar lavage may be useful in immunocompromised patients, those who fail to respond to antibiotics, or those in whom sputum samples cannot be obtained. 6 molecular diagnosis (e.g. pcr-based methods) has the advantages of quick results (within 3 hours), enhanced sensitivity, independence from organism viability and hence previous antibiotics, and theoretical possibility for determination of antimicrobial susceptibility. 7 of note, it is important to test for genes specific for the organism in question 10 and the sampling site remains important. pcr is most useful when per formed on specimens from a normally sterile site. for example, pcr for pneumococcus is positive in 62% of blood samples from adult patients with confirmed or probable pneumococcal pneumonia, 8 whereas blood cultures are positive in only 37%. for respiratory specimens under most circumstances, interpretation remains problematic due to low specificity related to floral should be modified in the light of risk factors (see table 36 .1). quinolones may be less appropriate in areas with a high prevalence of tb as their use may mask concurrent tb infection. appropriate antimicrobial therapy should be administered within 1 hour of diagnosis. 4, 12 there is controversy regarding the appropriate change to empirical therapy based on microbiological findings. 2, 4 changing to narrower-spectrum antimicrobial cover may result in inadequate treatment of the 5-38% of patients with polymicrobial infection. increasing evidence demonstrates improved outcome with combination antimicrobial as compared with monotherapy, particularly in severely ill patients with bacteraemic pneumococcal pneumonia. 5 odds ratio of death was 1.5 to 6 for monotherapy as compared with combination therapy. benefits were seen only in combination therapy with macrolide as part of the regimen, but not in combination with fluroquinolone regimen. 13 for the treatment of drug-resistant strep. pneumoniae (drsp) the regimens in figure 36 .1 are probably suitable for isolates with a penicillin mic < 4 mg/l. 2 if the mic is ≥4 mg/l an antipneumococcal fluoroquinolone, vancomycin, teicoplanin or linezolid should be given. 4 no clinical trial has specifically addressed this issue. courses as short as 5 days may be sufficient. 14 idsa/ ats guidelines recommend stopping after a minimum of 5 days if the patient is afebrile for 48-72 hours and organ dysfunction has largely resolved. 2 short courses may be suboptimal for patients with bacteraemic s. aureus pneumonia, meningitis or endocarditis complicating pneumonia or infection with less common organisms (e.g. burkholderia pseudomallei or fungi) or pseudomonas aeruginosa. procalcitonin may be useful to guide antibiotic therapy, but not all studies have demonstrated a benefit. 15 this can be assessed subjectively (a response is usually seen within 1-3 days of starting therapy) or objectively on the basis of respiratory symptoms, fever, oxygenation, wbc count, bacteriology, cxr changes, c-reactive protein reduction and procalcitonin reduction of 80-90% from peak value. the average time to defervescence varies with organism, severity and patient age (7 days in elderly patients, 2.5 days in young patients with pneumococcal pneumonia, 6-7 days in bacteraemic patients with pneumococcal pneumonia, 1-2 days in patients with m. pneumoniae pneumonia and 5 days in patients with legionella pneumonia). both blood and sputum cultures are usually negative within 24-48 hours of treatment although p. aeruginosa and m. pneumoniae may persist in the sputum despite effective therapy. cxr changes lag behind clinical changes with the speed of change depending on the organism, the age of the patient and the presence or absence of comorbid illnesses. the cxr of most young or middle-aged patients with bacteraemic pneumococcal pneumonia is clear by 4 weeks, but resolution is slower in elderly patients and patients with underlying illness, extensive pneumonia on presentation or legionella pneumophilia pneumonia. if the patient fails to respond consider the following questions: • has the patient got pneumonia? • are there host factors that explain the failure (e.g. obstruction of bronchus by a foreign body or tumour, inadequate host response)? • has a complication developed (e.g. empyema, superinfection, bronchiolitis obliterans organising pneumonia, metastatic abscess)? • is the right drug being given in an adequate dose by the right route? • is the organism resistant to the drug being given? • are there other organisms? • is the fever a drug fever? useful investigations include computerised tomography (ct) of the chest, thoracocentesis, bronchoalveolar lavage (table 36 .2) and transbronchial or open-lung biopsy. scoring systems have been developed to predict adverse outcome and icu admission including pneumonia severity index (psi), curb-65, crb-65, modified ats major and minor criteria, scap prediction rule, smart-cop, rea-icu index and cap-piro. 16 although they may help identify the sicker patients they should not be used as a sole determinant of icu admission as local admission criteria will be affected by local facilities, both in and outside icu. it should be noted that none of the criteria has been prospectively demonstrated to avoid late transfers or lower mortality. influenza pneumonia may present with severe respiratory failure and multi-organ failure. however the pattern of organ failure appears to vary between strains with h5n1 being associated with a much higher mortality and a higher incidence of multi-organ failure than pandemic h1n1, 17 which itself presented differently to seasonal influenza. in particular, trophism for lower respiratory tract, a higher rate of icu admission 18 and a higher rate of extrapulmonary complications 19 were observed. early initiation of oseltamivir is recommended for critically ill patients although there is no direct evidence of outcome benefit. glucocorticoids do not appear to be useful and may prolong viral replication. 20 bacterial superinfection should be considered, with grampositive cocci being most frequently isolated. 21 failure and cancer) can cause infiltrates on a chest x-ray. identification of the organism responsible is even more difficult than in patients with community-acquired pneumonia owing to the high incidence of oropharyngeal colonisation by gram-negative bacteria. blood cultures are positive in only about 6% of cases of nosocomial pneumonia. ventilator-associated pneumonia (vap) is nosocomial pneumonia arising >48-72 hours after intubation. reported incidence of vap is between 10 and 20% for those receiving mechanical ventilation for more than 48 hours. 22 it is associated with a higher incidence of multi-drug-resistant organisms. 1 nosocomial pneumonia is thought to result from microaspiration of bacteria colonising the upper respiratory tract. other routes of infection include macroaspiration although there are data demonstrating that surgical masks are as effective as n95 (ffp 2) masks in preventing transmission of seasonal influenza in non-icu settings it is important to note that the capacity for airborne transmission (and hence the need for n95 masks) is dependent on the exact characteristics of the organism and the frequency of aerosol-generating procedures so these data should not be extrapolated to other influenza viruses and icu settings. nosocomial pneumonia occurs in 0.5-5% of hospital patients, with a higher incidence in certain groups (e.g. postoperative patients and patients in icu). diagnosis may be difficult: the clinical features of pneumonia are non-specific and many non-infectious conditions (e.g. atelectasis, pulmonary embolus, aspiration, heart table 36 .2 procedure for obtaining microbiological samples using bronchoscopy and protected specimen brushing and/or bronchoalveolar lavage 35, 49 infection control in patients suspected of having a disease that is transmitted by the airborne route (e.g. tuberculosis): • the risk of transmission should be carefully weighed against the benefits of bronchoscopy, which may generate large numbers of airborne particles • perform bronchoscopy in a negative-pressure isolation room • consider the use of a muscle relaxant in ventilated patients, to prevent coughing • staff should wear personal protective equipment, which should include a fit-tested negative-pressure respirator (n95, ffp2 or above) as a minimum; use of a powered air-purifying respirator should be considered suction through the endotracheal tube should be performed before bronchoscopy avoid suction or injection through the working channel of the bronchoscope perform protected specimen brushing before bronchoalveolar lavage management is based on the finding that early treatment with antimicrobials that cover all likely pathogens results in a reduction in morbidity and mortality. 2 the initial selection of antimicrobials is made on the basis of epidemiological clues ( fig. 36.2, table 36 .3). antimicrobials should be administered within 1 hour of diagnosis. 12 the results of microbiological investigations are used to narrow antimicrobial cover later. treatment should be reassessed after 2-3 days or sooner if the patient deteriorates ( fig. 36.3 ). an outline of management based on an invasive approach is given in figure 36 .4. current ats guidelines recommend 7 days' treatment provided the aetiological agent is not p. aeruginosa or other non-lactose fermenter and the patient has a good clinical response with resolution of clinical features of infection. 1 the outcome of patients who receive appropriate initial empirical therapy for ventilator-associated pneumonia for 8 days is similar to those who receive treatment for 15 days. 1 of gastric contents, inhaled aerosols, haematogenous spread, spread from pleural space and direct inoculation from icu personnel. diagnosis is based on time of onset (>48 hours after admission to a healthcare facility 1 ), cxr changes (new or progressive infiltrates) and either clinical features and simple laboratory investigations or the results of quantitative microbiology. using a clinical approach, pneumonia is diagnosed by the finding of a new infiltrate or a change in an infiltrate on chest radiograph and growth of pathogenic organisms from sputum plus one of the following: white-blood-cell (wbc) count greater than 12 × l0 5 /l, core temperature ≥38.3°c, sputum gram stain with scores of more than two on a scale of four of polymorphonuclear leucocytes and bacteria. these are broadly similar to those required in community-acquired pneumonia: • chest x-ray: although studies using a histological diagnosis as the gold standard have demonstrated that pneumonia may be present despite a normal cxr, most definitions of nosocomial pneumonia require the presence of new persistent infiltrates on a cxr. • respiratory secretions: considerable controversy surrounds the issue of whether invasive bronchoscopic sampling (table 36. 2) of respiratory secretions is necessary. whether invasive sampling is employed or tracheal aspirates are used, empirical broad-spectrum antibiotics should be started while results are awaited. the results of microbiological analysis of respiratory secretions are used to either stop antibiotics or narrow the spectrum. 1 although the use of an invasive strategy is associated with a higher likelihood of modification of initial antimicrobials, 23 the effect on important clinical outcome such as mortality, antibiotic-free days, and organ dysfunction is variable. 1 although tracheal aspirates may predominantly reflect the organisms colonising the upper airway, they may be useful in indicating which organisms are not responsible for the pneumonia, thus allowing the antimicrobial cover to be narrowed. 1 the use of dual therapy is not well supported by evidence but it does reduce the probability that the pathogen is resistant to the drugs being given. if an extended spectrum β-lactamase-producing strain or an acinetobacter sp. is suspected a carbapenem should be given. if legionella pneumophilia is suspected use a quinolone. risk factors for mrsa infection in areas with a high incidence of mrsa include diabetes mellitus, head trauma, coma and renal failure. or with enlarged cervical nodes or other manifestations of extrapulmonary disease. clinical disease is seldom found in asymptomatic individuals, even those with strongly positive tuberculin test (heaf grade iii or iv). the outlook for patients with tuberculosis who require icu admission is poor. in one retrospective study the in-hospital mortality for all patients with tuberculosis requiring icu admission was 67% but in those with acute respiratory failure it rose to 81%. 31 the presentation and management of tb in hiv-positive patients are different (see below). identification of mycobacteria multiple 32,33 sputum samples should be collected, preferably on different days, for microscopy for acid-fast bacilli and culture. if sputum is not available bronchial washings taken at bronchoscopy and gastric lavage or aspirate samples should be obtained. gastric aspirates need to be neutralised immediately on collection. bronchoscopy and transbronchial biopsy may be useful in patients with suspected tb but negative sputum smear. pleural biopsy is often helpful and mediastinoscopy is occasionally needed in patients with mediastinal lymphadenopathy. part of any biopsy specimen should always be sent for culture. nucleic acid amplification tests on sputum have sensitivity similar to culture in 1.5% on the 60th day of icu in a recent study using a multicentre high-quality database and incorporating novel statistical methodology to control evolution of severity of illness. 25 several guidelines for prevention of ventilatorassociated pneumonia and hospital-acquired pneumonia have been published. [26] [27] [28] [29] [30] interventions can be divided into general infection control measures and specific measures. general measures include alcoholbased hand disinfection, hospital education programme on infection control, the use of microbiological surveillance and a programme to reduce antibiotic prescription. the major specific recommendations are summarised in table 36 .4. there is no evidence that 'bundles' of recommendations are more effective than the sum of the individual components. the main risk factors are listed in box 36.2. typical clinical features include fever, sweating, weight loss, lassitude, anorexia, cough productive of mucoid or purulent sputum, haemoptysis, chest wall pain, dyspnoea, localised wheeze and apical crackles. patients may also present with unresolved pneumonia, pleural effusions, spontaneous pneumothorax and hoarseness 28, 29 no effect on vap, mainly for staff safety 5. chlorhexidine oral decontamination 27, 29, 30 6. sedation vacation and weaning protocol 27,28 7. judicious use of stress ulcer prophylaxis 27 mortality reduction demonstrated when topical antimicrobials combined with short-course systemic antibiotics, bsac recommended sdd in patients expected to require mechanical ventilation for >48 hours, etf discourage routine use due to concern of emergence of resistant organisms not yet reviewed by guidelines 50 1. high-volume low-pressure ultrathin membrane endotracheal tube cuff with ssd 2. ultrathin membrane cuff with tapered shape and ssd 3. low-volume low-pressure endotracheal tube cuff with ssd 4. balloon device for biofilm removal 5. saline instillation before tracheal suctioning hme = heat moist exchanger; ssd = subglottic secretion drainage. smear-negative patients with pulmonary tuberculosis but have the advantage of a much more rapid result. there is, however, a significant false-negative rate. 32 a normal cxr almost excludes tb (except in hiv-infected patients) but endobronchial lesions may not be apparent and early apical lesions can be missed. common appearances include patchy/nodular shadowing in the upper zones (often bilateral), cavitation, calcification, hilar or mediastinal lymphadenopathy (may cause segmental or lobar collapse), pleural effusion, tuberculomas (dense round or oval shadows) and diffuse fine nodular shadowing throughout the lung fields in miliary tb. inactivity of disease cannot be inferred from the cxr alone. this requires three negative sputum samples and failure of any lesion seen on cxr to progress. cxr appearances in hiv-positive fit-tested negative-pressure respirator (n95, ffp2 or higher). use of a powered air-purifying respirator should be considered when bronchoscopy is being performed. 35 detailed infection control advice can be obtained via the 'link page' (http://www.aic.cuhk. edu.hk/web8/pneumonia%20guidelines.htm.). the lungs are amongst the most frequent target organs for infectious complications in the immunocompromised. the incidence of pneumonia is highest amongst patients with haematological malignancies, bone marrow transplant (bmt) recipients and patients with aids. the speed of progression of pneumonia, the cxr changes ( table 36 .5) and the type of immune defect provide clues to the aetiology. bacterial pneumonias progress rapidly (1-2 days) whereas fungal and protozoal pneumonias are less fulminant (several days to a week or more). viral pneumonias are usually not fulminant, but on occasions may develop quite rapidly. bronchoscopy is a major component of the investigation of these patients. empirical management based on cxr appearances is outlined in table 36 .5. early noninvasive ventilation may improve outcome amongst immunocompromised patients with fever and bilateral infiltrates. 36 pneumocystis jiroveci pneumonia (pcp) 37 the incidence of this common opportunistic infection has fallen substantially in patients with aids who are receiving prophylaxis and effective antiretroviral therapy, with most cases occurring in patients who are not receiving hiv care or among patients with advanced immunosuppression. the onset is usually insidious with dry cough, dyspnoea and fever on a background of fatigue and weight loss. crackles in the chest are rare. approximately 15% of patients have a concurrent cause for respiratory failure (e.g. kaposi sarcoma, tb, bacterial pneumonia). useful investigations are: 1. cxr: classical appearance is diffuse bilateral perihilar interstitial shadowing, but in the early stages this is very subtle and easily missed. the initial cxr is normal in 10%. in a further 10% the changes are atypical with focal consolidation or coarse patchy shadowing. none of the changes are specific for pcp and may be seen in other lung diseases associated with aids. pleural effusions, hilar or mediastinal lymphadenopathy are unusual in pcp but common in mycobacterial infection or kaposi's sarcoma or lymphoma. induced sputum: in this technique the patient inhales nebulised hypertonic saline from an ultrasonic nebuliser. this provokes bronchorrhoea and the patient patients with tb differ from those in non-hiv-infected patients. the decision to initiate anti-tb treatment should be based on level of clinical suspicion, results of afb smear and sometimes mycobacterial culture. if the initial clinical suspicion is strong and the patient is seriously ill attributable to possible tb, treatment should be initiated promptly, sometimes before the result of afb smear. subsequent positivity of afb smear or nucleic acid amplification test provides support to the continuation of treatment. combination chemotherapy consisting of four drugs is necessary for maximal efficacy. treatment is divided into initial phase and continuation phase. the most commonly used initial regimen consists of 8 weeks of rifampicin 600 mg daily (450 mg for patients <50 kg), isoniazid 300 mg daily, pyrazinamide 2 g daily (1.5 g for patients <50 kg) and ethambutol 15 mg/kg daily as initial phase treatment. ethambutol should be used only in patients who have reasonable visual acuity and who are able to appreciate and report visual disturbances. this mandates careful consideration in patients who require heavy sedation. visual acuity and colour perception must be assessed (if ethambutol is to be used) and liver and renal function checked before treatment is started. steroids are recommended for children with endobronchial disease and, possibly, for patients with tuberculous pleural effusions. pyridoxine 10 mg daily should be given to prevent isoniazid-induced neuropathy to those at increased risk (e.g. patients with diabetes mellitus, chronic renal failure or malnutrition or alcoholic or hiv-positive patients). negative afb smear should not delay initial treatment if clinical suspicion remains high. supporting features included chronic cough, weight loss, characteristic chest x-ray findings, emigration from a high-incidence country, no other immediate diagnosis, and positive tuberculin test. patients admitted to an icu with infectious tb or suspected of having active pulmonary tb should be managed in an isolation room with special ventilation characteristics, including negative pressure. patients should be considered infectious if they are coughing or undergoing cough-inducing procedures or if they have positive afb smears and they are not on or have just started chemotherapy, or have a poor clinical or bacteriological response to chemotherapy. 32, 35 patients with non-drug-resistant tb should be non-infectious after 2 weeks of treatment which includes rifampicin and isoniazid. 32 as tb spreads through aerosols it is probably appropriate to isolate patients who are intubated even if only their bronchial washings are smear-positive. staff caring for patients who are smear-positive should wear personal protective equipment including a and/or diuretics (patients often fluid-overloaded). approximately 40% of patients with hiv-related pcp who require mechanical ventilation survive to hospital discharge. 38 initiation of antiretroviral therapy in patients presenting with hiv-related pcp is controversial. the centers for disease control and prevention (cdc) recommend against doing so in the acute phase, but recent data suggest that the outcome may be improved by initiation within the first 4 days of icu admission. 39 this is the most common cause of acute respiratory failure in hiv-positive patients. bacterial pneumonia is more common in hiv-infected patients than in the general population and tends to be more severe. strep. pneumoniae, h. influenza, pseudomonas aeruginosa and s. aureus are the commonest organisms. nocardia and gram negatives should also be considered. atypical pathogens (e.g. legionella) are rare. response to appropriate antibiotics is usually good but may require protracted courses of antibiotics because of high tendency to relapse. patients with severe immunodeficiency (cd4 + t lymphocyte count <100/µl) and a history of pseudomonas infection or bronchiectasis or neutropenia should receive antibiotics that cover p. aeruginosa as well as other gram negatives. the possibility of concurrent pcp or tuberculosis should be excluded. tb may be the initial presentation of aids, particularly in sub-saharan africa. the pattern of tb in hiv patients coughs up material containing cysts and trophozoites. the technique is time-consuming and requires meticulous technique and is less sensitive than bronchoscopy but less invasive. the possibility of concurrent tuberculosis should be considered and steps taken to minimise the risk of spread of infection. the diagnosis in over 90% of cases. specimens should be sent for cytology. transbronchial biopsy is not necessary in most cases. pcr using bronchial lavage specimens may be useful in non-hiv patients with suspected pcp. antipneumocystis treatment should be started as soon as the diagnosis is suspected. treatment of choice is trimethoprim plus sulfamethoxazole (co-trimoxazole) 20 mg/kg/day + 100 mg/kg/day for 3 weeks plus prednisolone 40 mg orally twice daily for 5 days followed by 20 mg twice daily for 5 days and then 20 mg per day until the end of pcp treatment. side-effects of co-trimoxazole are common in hiv patients (nausea, vomiting, skin rash, myelotoxicity). the dose should be reduced by 25% if the wbc count falls. patients who are intolerant of co-trimoxazole should be treated with: • pentamidine 4 mg/kg/day i.v. or • primaquine with clindamycin or • trimetrexate with leucovorin (±oral dapsone). response to treatment is usually excellent, with a response time of 4-7 days. if the patient deteriorates or fails to improve: consider (re-)bronchoscopy (is the diagnosis correct?), treat co-pathogens and consider a short course of high-dose i.v. methylprednisolone fungi are rare but important causes of pneumonia. they can be divided into two main groups based on the immune response required to combat infection with these organisms. histoplasma, blastomycosis, coccidioidomycosis, paracoccidioidomycosis and cryptococcus require specific cell-mediated immunity for their control and thus, in contrast to infections that are controlled by phagocytic activity, the diseases caused by these organisms can occur in otherwise healthy individuals although they cause much more severe illness in patients with impaired cell-mediated immunity (e.g. patients infected with hiv and organ transplant recipients). with the exception of cryptococcus these organisms are rarely seen outside north america. aspergillus and mucor spores are killed by non-immune phagocytes and as a result these fungi rarely result in clinical illness in patients with normal neutrophil numbers and function. this is effectively a combination of the two types of fungal infection in which impaired cell-mediated immunity predisposes to mucosal overgrowth with candida but impaired phagocytic function or numbers is usually required before deep invasion of tissues occurs. primary candida pneumonia (i.e. isolated lung infection) is uncommon 41, 45 and more commonly pulmonary lesions are only one manifestation of disseminated candidiasis. even more common is benign colonisation of the airway with candida. in most reported cases of primary candida pneumonia amphotericin b has been used. in disseminated candidiasis treatment should be directed to treatment of disseminated disease rather than candida pneumonia per se. 45 this is a highly lethal condition in the immunocompromised despite treatment and therefore investigation and treatment should be prompt and aggressive. it is associated with exposure to construction work. definitive diagnosis requires both histological evidence of acute-angle branching, septated non-pigmented hyphae measuring 2-4 µm in width, and cultures yielding aspergillus species from biopsy specimens of involved organs. recovery of aspergillus species from respiratory secretions in immunocompromised, but not immunocompetent, patients may indicate invasive disease with a positive predictive value as high as depends on the degree of immunosuppression. in patients with cd4 + t lymphocytes >350 cells/µl the clinical presentation is similar to tb in non-hiv-infected patients, although extrapulmonary disease is more common. in patients with cd4 + t lymphocytes <350 cells/µl extrapulmonary disease (pleuritis, pericarditis, meningitis) is common. severely immunocompromised patients (cd4 + t lymphocytes <100 cells/µl) may present with severe systemic disease with high fever, rapid progression and systemic sepsis. in these patients lower and middle lobe disease is more common, miliary disease is common and cavitation is less common. sputum smears and culture may be positive even with a normal cxr. response to treatment is usually rapid. management of tb in hiv is complex owing to numerous drug interactions; consultation with an expert in treatment of hiv-related tb should be strongly considered. complex interactions occur between rifamycins (e.g. rifampicin and rifabutin) and protease inhibitors and nonnucleoside reverse transcriptase inhibitors used to treat patients infected with hiv. the choice of rifampicin or rifabutin depends on a number of factors including the unique and synergistic adverse effects for each individual combination of rifampicin and anti-hiv drugs, and consultation with a physician with experience in treating both tb and hiv is advised. 40 idsarecommended dosage adjustment for patients receiving antiretrovirals and rifabutin 37 can be obtained via the 'link page' (http://www.aic.cuhk.edu.hk/web8/ pneumonia%20guidelines.htm.). the optimal time for initiating antiretroviral therapy in patients with tb is controversial. early therapy may decrease hiv disease progression but may be associated with a high incidence of adverse effects and an immune reconstitution reaction. 37 cmv pneumonitis 41, 42 risk of infection is highest following allogeneic stem cell transplantation, followed by lung transplantation, pancreas transplantation and then liver, heart and renal transplantation and advanced aids. if both the recipient and the donor are seronegative then the risk of both infection and disease are negligible. if the recipient is seropositive the risk of infection is approximately 70% but the risk of disease is only 20%, regardless of the serostatus of the donor. however if the recipient is seronegative and the donor is seropositive the risk of disease is 70%. if steroid pulses and antilymphocyte globulin are given for treatment of acute rejection the risk of developing disease is markedly increased. infection may be the result of primary infection or reactivation of latent infection. it is clinically important, but often difficult to distinguish between cmv infection and cmv disease and a definitive diagnosis can be made only histologically. detection of cmv-pp65 antigen in peripheral wbc and detection of cmv dna or rna in the blood by quantitative polymerase chain needle aspiration or, if there is debris within the fluid, drainage using an intercostal drain. the diagnosis is confirmed by aspiration of pus. the mainstay of treatment is drainage either by intercostal drain or by surgical intervention. patients who present before the pus is loculated and a fibrinous peel has formed on the lung can usually be treated by simple drainage. the combination with intrapleural fibrinolysis may be beneficial. optimal surgical management, which consists of decortication (open or thoracoscopic), is indicated if the empyema is more advanced or if simple drainage fails. this is a major procedure and many patients with cardiac or chronic respiratory disease will not tolerate it. alternatives for these patients are instillation of thrombolytics into the pleural space or thoracostomy. antibiotics have only an adjunctive role. broad-spectrum antibiotic regimens with anaerobic cover should be used until the results of microbiological analysis of the aspirated pus are available. all tables and figures are reproduced from icu web (www.aic.cuhk.edu.hk/web8) with permission of the authors. 80-90% in patients with leukaemia or bone marrow transplant recipients. bronchoalveolar lavage with smear, culture and antigen detection has excellent specificity and reasonably good positive predictive value for invasive aspergillosis in immunocompromised patients. although radiological features may give a clue to the diagnosis they are not sufficiently specific to be diagnostic. in acutely ill immunocompromised patients intravenous therapy should be initiated if there is suggestive evidence of invasive aspergillosis while further investigations to confirm or refute the diagnosis are carried out. first-line therapy is voriconazole. 47 echinocandins and amphotericin are alternatives. this may be an uncomplicated effusion that resolves with appropriate treatment of the underlying pneumonia or a complicated effusion that develops into an empyema unless drained. complicated effusions tend to develop 7-14 days after initial fluid formation. they are characterised by increasing pleural fluid volume, continued fever and pleural fluid of low ph (<7.3) that contains a large number of neutrophils and may reveal organisms on gram staining or culture. an outline of management is given in figure 36 .5. collection of pus in the pleural space. follows infection of the structures surrounding the pleural space, including subdiaphragmatic structures, and chest trauma, or may be associated with malignancy. anaerobic bacteria, usually streptococci or gram-negative rods, are responsible for 76% of cases. the diagnosis is usually simple. the patient is usually septic and may have a productive cough and chest pain. the chest x-ray may show features suggestive of a pleural effusion and underlying consolidation but may also show an abscess cavity with a fluid level, in which case ct scanning will be required to distinguish between an abscess and an empyema. ultrasound can be useful to confirm the presence of fluid in the pleural space and to determine whether it can be drained by guidelines for the management of adults with hospital-acquired, ventilatorassociated, and healthcare associated pneumonia infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults pneumonia guidelines committee of the bts standards of care committee. british thoracic society guidelines for the management of community acquired pneumonia in adults: update management of communityacquired pneumonia in adults cdc, and infectious diseases society of america. treatment of tuberculosis the standardization of bronchoscopic techniques for ventilator-associated pneumonia new issues and controversies in the prevention of ventilator-associated pneumonia guidelines for the management of adults with hospital-acquired, ventilatorassociated, and healthcare associated pneumonia infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults pneumonia guidelines committee of the bts standards of care committee. british thoracic society guidelines for the management of community acquired pneumonia in adults: update guidelines for the management of adult lower respiratory tract infections update in community-acquired and nosocomial pneumonia 2009 value of intensive diagnostic microbiological investigation in low-and high-risk patients with community-acquired pneumonia nucleic acid amplification tests for the diagnosis of pneumonia interpreting assays for the detection of streptococcus pneumonia severity of pneumococcal pneumonia associated with genomic bacterial load polymerase chain reaction for diagnosing pneumocystis pneumonia in non-hiv immunocompromised patients with pulmonary infiltrates early mortality in patients with community-acquired pneumonia: causes and risk factors surviving sepsis campaign guidelines for management of severe sepsis and septic shock antibiotics for bacteremic pneumonia: improved outcomes with macrolides but not fluoroquinolones high-dose, short-course levofloxacin for community-acquired pneumonia: a new treatment paradigm an esicm systematic review and meta-analysis of procalcitonin-guided antibiotic therapy algorithms in adult critically ill patients management of communityacquired pneumonia in adults avian influenza (h5n1): implications for intensive care clinical characteristics and 30-day outcomes for influenza a 2009 (h1n1) complications and outcomes of pandemic 2009 influenza a (h1n1) virus infection in hospitalized adults: how do they differ from those in seasonal influenza? corticosteroid treatment in critically ill patients with pandemic influenza a/h1n1 2009 infection analytic strategy using propensity scores bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza a (h1n1) -united states clinical and economic consequences of ventilator-associated pneumonia: a systematic review invasive approaches to the diagnosis of ventilator-associated pneumonia: a meta-analysis ventilator-associated pneumonia and mortality: a systematic review of observational studies attributable mortality of ventilator-associated pneumonia. a reappraisal using causal analysis guidelines for preventing health-careassociated pneumonia 2003. recommendations of cdc and the healthcare infection control practices advisory committee shea/idsa practice recommendation. strategies to prevent ventilator-associated pneumonia in acute care hospitals guidelines for the management of hospital-acquired pneumonia in the uk: report of the working party on hospital-acquired pneumonia of the british society for antimicrobial chemotherapy vap guidelines committee and the canadian critical care trials group. comprehensive evidencebased clinical practice guidelines for ventilatorassociated pneumonia: prevention defining, treating and preventing hospital acquired pneumonia: european perspective active tuberculosis in the medical intensive care unit: a 15-year retrospective analysis national collaborating centre for chronic conditions. tuberculosis. clinical diagnosis and management of tuberculosis and measures for its prevention and control management of tuberculosis in the united states cdc, and infectious diseases society of america. treatment of tuberculosis guidelines for preventing the transmission of mycobacterium tuberculosis in health-care settings noninvasive ventilation in immunosuppressed patients with pulmonary infiltrates, fever, and acute respiratory failure treating opportunistic infections among hiv-infected adults and adolescents: recommendations from cdc, the national institutes of health, and the hiv medicine association/infectious diseases society of america improvements in outcomes of acute respiratory failure for patients with human immunodeficiency virusrelated pneumocystis carinii pneumonia centers for disease control. updated guidelines for the use of rifabutin or rifampicin for the treatment and prevention of tuberculosis among hiv-infected patients taking protease inhibitors or nonnucleoside reverse transcriptase inhibitors the lung in the immunocompromised patient management of cytomegalovirus infection and disease after solid-organ transplantation area under the viraemia curve versus absolute viral load: utility for predicting symptomatic cytomegalovirus infections in kidney transplant patients cytomegalovirus infection in organ-transplant recipients: diagnostic value of pp65 antigen test, qualitative polymerase chain reaction (pcr) and quantitative taqman pcr guidelines for treatment of candidiasis practice guidelines for diseases caused by aspergillus voriconazole versus amphotericin b for primary therapy of invasive aspergillosis the pleural cavity the standardization of bronchoscopic techniques for ventilator-associated pneumonia new issues and controversies in the prevention of ventilator-associated pneumonia key: cord-253182-s60vzf3q authors: fang, evandro f.; xie, chenglong; schenkel, joseph a.; wu, chenkai; long, qian; cui, honghua; aman, yahyah; frank, johannes; liao, jing; zou, huachun; wang, ninie y.; wu, jing; liu, xiaoting; li, tao; fang, yuan; niu, zhangming; yang, guang; hong, jiangshui; wang, qian; chen, guobing; li, jun; chen, hou-zao; kang, lin; su, huanxing; gilmour, brian c.; zhu, xinqiang; jiang, hong; he, na; tao, jun; leng, sean xiao; tong, tanjun; woo, jean title: a research agenda for ageing in china in the 21st century (2nd edition): focusing on basic and translational research, long-term care, policy and social networks date: 2020-09-21 journal: ageing res rev doi: 10.1016/j.arr.2020.101174 sha: doc_id: 253182 cord_uid: s60vzf3q one of the key issues facing public healthcare is the global trend of an increasingly ageing society which continues to present policy makers and caregivers with formidable healthcare and socio-economic challenges. ageing is the primary contributor to a broad spectrum of chronic disorders all associated with a lower quality of life in the elderly. in 2019, the chinese population constituted 18 % of the world population, with 164.5 million chinese citizens aged 65 and above (65+), and 26 million aged 80 or above (80+). china has become an ageing society, and as it continues to age it will continue to exacerbate the burden borne by current family and public healthcare systems. major healthcare challenges involved with caring for the elderly in china include the management of chronic non-communicable diseases (cncds), physical frailty, neurodegenerative diseases, cardiovascular diseases, with emerging challenges such as providing sufficient dental care, combating the rising prevalence of sexually transmitted diseases among nursing home communities, providing support for increased incidences of immune diseases, and the growing necessity to provide palliative care for the elderly. at the governmental level, it is necessary to make long-term strategic plans to respond to the pressures of an ageing society, especially to establish a nationwide, affordable, annual health check system to facilitate early diagnosis and provide access to affordable treatments. china has begun work on several activities to address these issues including the recent completion of the of the ten-year health-care reform project, the implementation of the healthy china 2030 action plan, and the opening of the national clinical research center for geriatric disorders. there are also societal challenges, namely the shift from an extended family system in which the younger provide home care for their elderly family members, to the current trend in which young people are increasingly migrating towards major cities for work, increasing reliance on nursing homes to compensate, especially following the outcomes of the ‘one child policy’ and the ‘empty-nest elderly’ phenomenon. at the individual level, it is important to provide avenues for people to seek and improve their own knowledge of health and disease, to encourage them to seek medical check-ups to prevent/manage illness, and to find ways to promote modifiable health-related behaviors (social activity, exercise, healthy diets, reasonable diet supplements) to enable healthier, happier, longer, and more productive lives in the elderly. finally, at the technological or treatment level, there is a focus on modern technologies to counteract the negative effects of ageing. researchers are striving to produce drugs that can mimic the effects of ‘exercising more, eating less’, while other anti-ageing molecules from molecular gerontologists could help to improve ‘healthspan’ in the elderly. machine learning, ‘big data’, and other novel technologies can also be used to monitor disease patterns at the population level and may be used to inform policy design in the future. collectively, synergies across disciplines on policies, geriatric care, drug development, personal awareness, the use of big data, machine learning and personalized medicine will transform china into a country that enables the most for its elderly, maximizing and celebrating their longevity in the coming decades. this is the 2nd edition of the review paper (fang ef et al., ageing re. rev. 2015). the research agenda in response to rapid population ageing in china has been broad, covering areas including the study of the ageing process itself in laboratory and animal studies, to clinical-level studies of drugs or other treatments for common chronic diseases, and finally policy-level research for the care of the elderly in hospital, community and residential care settings, and its influence on health and social care policies . chinese population statistics taken between 1950-2050 show a reduction in crude death rate (cdr) and total fertility rate (tfr), accompanied by an increase in life expectancy at birth and an expansion of the population aged 65 and above (65+, termed the elderly) (fig. 1a) . as of 2019, the population of mainland china constitutes 18% of global total, with 164.5 million chinese citizens aged 65+, 26 million of whom are 80+. by 2050, it is expected that there will be 1.4 billion chinese, with 365 million aged 65+, a number representing 26.1% of the country's total population (fig. 1b) . furthermore, among this ageing population, 115 million are expected to reach an age of at least 80 and 0.6 million are expected to become centenarians (fig. 1b) . when compared with their counterparts born a decade earlier, the current 80+ generation has reduced annual mortality and disability rates, but has increased cognitive impairment and reduced objective physical performance capacity (zeng et al., 2017b) . to achieve what may be considered a 'healthy ageing society', it is first important to address and prepare for the challenges and issues that are associated with rapidly ageing populations. ageing is the primary driver of most, if not all, chronic diseases, including cancer, cardiovascular diseases, diabetes, and neurodegenerative diseases, particularly alzheimer's disease (ad) and parkinson's disease (pd) (kerr et al., 2017; lautrup et al., 2019; lopez-otin et al., 2013) . the most predominant diseases affecting the elderly in china (65+, data from 2017) include sensory diseases, other non-communicable diseases, digestive diseases, respiratory infections and tuberculosis, skin and subcutaneous diseases, neurological diseases, and musculoskeletal disorders, among others (fig. 1c) . from 1990 to 2017, there were dramatic increases in prevalence of all 21 diseases, excluding a very minor reduction in 'neglected tropical diseases and malaria' (fig. 1c) . the major diseases responsible for death of the elderly in china are cardiovascular diseases, neoplasms, chronic respiratory diseases, and neurological diseases, among others (figs. 1d and 2). compared to their younger counterparts, the elderly population are more fragile, and susceptible to conditions such as cardiovascular diseases, chronic respiratory diseases, diabetes, kidney diseases, unintentional injuries, hiv/aids and sexually transmitted diseases (stds), among others (fig. 2b ). in comparison with data from 1990, new patterns of disease mortality characterize the modern elderly, such as a dramatic reduction in the percentage of death contributed by 'neurological disorders' with an increase in deaths due to hiv/aids and stds (fig. 2b, d) . recognition of the current disease demographics in the elderly in china, and accurate prediction of future trends will enable us to be best prepared for different healthcare needs at different times. in wake of the expanding ageing society in china, and the formidable socio-economic and healthcare challenges, we offer the 2 nd edition of our previously published review . here, we aim to provide an update regarding the situation of the elderly in china using a range of expertise and suggestions from multiple fields which may further propel the exciting and ongoing reforms to china's healthcare system. we hope to explore different ageing care models that can be used to best produce a healthy ageing society (chen, 2009; yip et al., 2019; zhan et al., 2019) . the following sections highlight recent developments in the above areas as well as areas for future research. based on ageing phenotypes and the major disease demographics in the elderly in china (figs. 1 and 2), we chose to focus on frailty (including sarcopenia as an independent subsection), cncds (including cardiovascular disease as an independent subsection), mental health disorders, dental health challenges, elderly infections and immune diseases, as well as hiv, syphilis, and other stds. in view of recent reviews on other grand challenges, including cancer tsoi et al., 2017) , chronic respiratory diseases (zhu et al., 2018) , diabetes and kidney diseases (hu and jia, 2018; wei et al., 2019) , these areas will not explored here. frailty is a biologic syndrome characterized by deteriorating function across a broad spectrum of physiological symptoms (fried et al., 2001) . it can be thought of as a state of vulnerability. some have proposed an index approach to categorize different degrees of frailty; however, these attempts are complicated by the multidimensionality of the underlying causes of frailty, thus creating a dynamic, ever-changing value that is difficult to index (rockwood et al., 2006) . the term physical frailty has been applied to age-related loss of muscle mass and function, that is sarcopenia (detailed in the next section). in recent years, frailty research has increased rapidly in china as a strategy to prevent disability in response to an ageing population (chhetri et al., 2019) . research projects were showcased in the following scientific conferences: the 1 st and 2 nd international china conference on frailty in china, jointly organized by the who collaborating centre on frailty, clinical research and geriatric training at the gerontopole, toulouse, france, the chinese embassy in paris, and the national clinical centre for geriatric diseases, china, and the 4 th asian conference for frailty and sarcopenia in dalian in october 2018, organized by the chinese geriatrics society, beijing institute of geriatrics and gerontology and the chinese health promotion foundation. wide-ranging topics included basic science, epidemiology, definitions and measurements, management, as well as service models. such conferences greatly accelerate basic and clinical research on as well as clinical treatment for frailty. frailty may be used as a population indicator of ageing, and be a useful indicator of a need for treatment. research into prevalence, risk factors, prevention, and incorporation into service delivery models in community, hospital and residential care settings are an important part of the ageing research agenda for china. the importance of recognizing frailty in communitydwelling older people in china has been highlighted in a systematic review and meta-analysis by he et al. (he et al., 2019a) . risk factors for a worsening in frailty among community-living older adults include hospitalizations, older age, previous stroke, lower cognitive function, diabetes and osteoarthritis, while higher socioeconomic status and neighborhood green space were protective factors (lee et al., 2014; yu et al., 2018c) . a comparison of prevalence and incidence of frailty between populations may stimulate further research into prevention strategies and inform government policies. using data from a nationally representative study, wu et al. found that 7% of community-dwelling adults aged 60 years or above were frail in mainland china and the prevalence increased dramatically with age, reaching 32.5% for those aged 85 years or above ). substantial regional disparities exist in the prevalence as well as incidence of frailty in mainland china. for example, the incidence rate of frailty in the northeast was more than double than that in the southeast . furthermore, a comparison of frailty and its contributory factors across three chinese populations (hong kong, urban and rural populations of taiwan) using the ratio of frailty index (fi) to life expectancy (le) as an indicator of compression of morbidity showed higher fi/le in taiwan compared with hong kong. risk factors include low physical activity and living alone . the importance of protein intake to slow the decline in muscle mass and physical function over four years supports the importance of nutrition as an underlying factor for physical frailty .the role of inflammatory cytokines in the pathophysiology of sarcopenia is supported by the finding of slower decline in grip strength for those in the highest quartile of telomere length (woo et al., 2014b) . simple tools for frailty and sarcopenia may be used in a community setting as case finding, without the need for professionally trained personnel (woo et al., 2014a; woo et al., 2015b) . this may represent the first step in the approach to community-based intervention such as group exercises with or without nutritional supplementation for frailty and sarcopenia (yu et al., in press; . how community services may be developed to make frailty as a cornerstone of health and social care systems (woo, 2018) depends on the development of existing community infrastructures. two examples have been described previously: the tai po cadenza hub, and the jockey club e health project, where screening data based on the who integrated care for older people toolkit (who) were collected via ipad, followed by action algorithms for items where action is indicated: e.g., frailty, sarcopenia. this model emphasizes the empowerment of older people and their care-givers, societal-level behavioral changes, and the use of technology in the absence of a low cost primary care system orientated to meeting the needs of older people (woo, 2019) . in the hospital setting, detection of frailty may inform choice of therapies and prognosis, such as mortality and hospitalization in chronic heart failure . closely related to the concept of frailty, sarcopenia is an age-related gradual loss of mass and strength of skeletal muscles resulting in reduced physical performance. major pathological features include a loss of satellite cells and motor neurons, as well as less active neuromuscular junctions (cruz-jentoft et al., 2010) . following the publication of the european consensus group on sarcopenia (cruz-jentoft et al., 2019; cruz-jentoft et al., 2014) , an asian group including chinese researchers formed a panel to arrive at a consensus on the definition of sarcopenia, published in 2014 , and recently updated in 2019. the asian criteria differed from the european consensus definitions. an individual international statistical classification of diseases and related health problems code (m62.84) was assigned to 'sarcopenia' which has stimulated both diagnostic and therapeutic trials worldwide. in china, sarcopenia diagnosis requires some special considerations, including anthropometric and cultural differences. the 2019 guideline of asia working group of sarcopenia (awgs) provides updated guidelines on epidemiology, case-finding, the diagnostic algorithm, measurements of muscle mass, muscle strength and physical performance, and intervention and treatment. the prevalence of sarcopenia is estimated to be between 7.3-12.0% among the general older population and was over 25% in the oldest populations (85+) wang et al., 2018a; woo et al., 2015a; xu et al., 2020, in press; yu et al., 2014) . while old age is the primary risk factor for sarcopenia, other risk factors in the chinese population include household status, lifestyle, physical inactivity, poor nutritional and dental status, and some diseases (osteoporosis, metabolic diseases, etc.). in terms of longer-term clinical outcomes, awgs-defined sarcopenia was significantly associated with increased risks of physical limitations at 4 years, slowness at 7 years, and 10-year mortality, but not of hospitalization wang et al., 2018a; woo et al., 2015a; yu et al., 2014) . interventional strategies for the elderly of china have been the subject of recent research. for instance, an intervention for community-dwelling older adults yielded significant improvements in muscle function based on which when protein was offered as an oral nutritional supplement in combination with resistance exercises (kang et al., 2019) . similar findings were reported in j o u r n a l p r e -p r o o f major behavioral risk factors that are responsible for cncds are prevalent among the elderly in china. nearly 80% of deaths are attributable to unhealthy diet, high blood pressure, smoking, high glucose, air pollution (indoor and outdoor), and physical inactivity (who, 2014) . in china, 56.6% of adults aged 60+ have insufficient dietary balance (daily intake of <400g fruit and vegetables), 22.4% are current smokers, 45% use unclean fuel for cooking, 84% are physically inactive, and 11.4% have harmful alcohol use (who, 2012) . risk factors for major cncds, particularly smoking and alcohol use, are unevenly distributed among older men and women. the prevalence of cigarette smoking is substantially higher among men (41.5%) than women (4.3%). the prevalence of harmful alcohol use among men is more than three times as much as that among women (13.3% vs. 4%). substantial rural-urban disparities in the distribution of risk factors exist among older chinese adults. rural residents have a higher prevalence of smoking (23.7% vs. 19.9%), harmful alcohol use (13.3% vs. 7.5%), insufficient dietary intake (60% vs. 49.5%), and unclean fuel use (89% vs. 7.1%) than those in urban areas, while residents of urban areas have a substantially higher prevalence of physical inactivity than their rural counterparts. 2016, cvd was ranked first in mortality rates, higher than the mortality rates attributed to tumors and other prevalent diseases. studies have shown that in 2013, age-standardized cvd mortality in china was 21% lower than in 1990 (gbd, 2015; zhou et al., 2019) . although the age-standardized cvd mortality rate has declined, the absolute number of cvd deaths is still rising rapidly, and increased by 46% between 1990 and 2013. cvd is a large burden for the chinese healthcare system. with the development of medical technology, and the government's focus on chronic disease management, the problem of cvd in china has improved. however, due to the problems arising from population ageing, cvd still has a great impact on national health. major factors for cvd include hypertension, dyslipidemia, diabetes, air pollution, and excess weight (overweight and obesity). some risk factors are specific to china compared to other countries, however, this is changing as china's economy develops and the population ages. hypertension is an important public health problem in china. the prevalence of hypertension in china among those over the age of 18 is 23.2%, and the number of patients with hypertension in china is estimated to be 245 million . in 2013, 2.5 million deaths were attributed to hypertension in china, accounting for 27.5% of all causes of death (trammell et al., 2016) . with the rapid development of the economy and the ageing population, problems with blood lipid levels in china have gradually increased, and the prevalence of dyslipidemia has increased significantly. the main symptoms of dyslipidemia seen in china are low levels of low-density lipoprotein cholesterol (ldl-c) and hypertriglyceridemia (pan et al., 2016) , while dyslipidemia in the west is characterized by hypercholesterolemia and high levels of ldl-c (toth et al., 2012) . with the change of lifestyle following china's economic development, the number of chinese diabetic patients is growing. overall, 47% of adults in china have diabetes or pre-diabetes, which is slightly lower than the 49-52% in the united states . in recent years, there has been a significant increase in the prevalence of excess weight (bmi: 24.0-27.9kg/m 2 ) and obesity (bmi≥28.0 kg/m 2 ) in chinese residents, as noted over a five-year study period (he et al., 2017b) . the prevalence of combined overweight and obesity among men was 33.8%. air pollution is another important factor leading to cvd. among different particles, pm2.5 (an aerodynamic diameter of 2.5 μm or less ) is most closely related to cvd (brook et al., 2010) . a follow-up study of cohorts of elderly people 65+ in hong kong showed that for every 10 μg /m 3 increase in pm2.5 concentration, the risk of total cvd death increased by 22% (wong et al., 2015) . air pollution is also associated with increased blood pressure. for each 10 μg/m 3 increase in pm2.5 concentration, the per capita systolic blood pressure level increased by 1.30 mmhg, the per capita diastolic blood pressure level increased by 1.04 mmhg, and the risk of hypertension increases by 14% . coronary heart disease, atrial fibrillation (af), heart failure, and atherosclerosis are common forms of cvd. technological developments have allowed for an increase in treatment options and testing methods, including percutaneous coronary intervention (pci), radiofrequency ablation, implantable cardiac defibrillator (icd) and pacemaker implantation. since elderly patients are often associated with more complications, treatment decisions for cvd in elderly patients need to be adjusted individually based on an overall scoring of health. coronary heart disease is a common fatal cvd. for the treatment of coronary heart disease, the number of pci cases has steadily increased in china (zhao et al., 2018b) . the creative study explored antiplatelet treatment options for patients after pci in china, and studies have shown that for patients with low response to antiplatelet drugs after pci, a triple antiplatelet intensive therapy combined with cilostazol is safe and effective (tang et al., 2018) . bleeding events should be paid special attention when administrating dual antiplatelet treatment to acs patients aged 75 and older receiving pci (zhao et al., 2018a) . the risk of all-cause, cardiovascular, and stroke deaths in patients with af is significantly higher than in patients with sinus rhythm . the proportion of chinese patients receiving anticoagulation treatments is low. only 12.7% of patients with af and a chads2 score of 2 or more received anticoagulation treatment . patients with af aged 75+ tend to higher chads2 scores but receive less anticoagulation therapy. the risk of one-year follow-up deaths and adverse events in the elderly is more than doubled compared to other populations yang et al., 2014) . cases of af ablation procedures and icd implantation have steadily increased in china. however, european danish studies suggest that primary prevention through icd implantation has limited benefits in elderly patients with non-ischemic cardiac diseases (kober et al., 2016) . therefore, it is necessary to pay attention to the indications when expanding the population eligible for icd implantation in china. in recent years, the etiology of heart failure in china has changed significantly. the proportion of valvular disease (especially rheumatic valvular disease) has decreased. as china is becoming an ageing society, the number of elderly patients with heart failure has increased. at present, most studies suggest that coronary heart disease is a common cause of heart failure in the elderly, and the proportion of hypertension and pulmonary heart disease in elderly patients with heart failure increases with age. in recent years, the use of diuretics in hospitalized heart failure patients in china has not changed significantly, while the usage rate of digoxin has shown a downward trend. the use of acei, arb, aldosterone receptor antagonists and beta-blockers have shown a significant upward trend . lower extremity atherosclerotic disease (lead) is a common disease in the elderly and an important starting point for systemic atherosclerosis. early detection of lead is of great value in the diagnosis and treatment of systemic atherosclerosis (hiramoto et al., 2018) . to reduce the burden of cvd in china, we recommend interventions directed at altering lifestyles and programs dedicated to the detection and management of risk factors, especially for elderly people. research modeling has shown that if dyslipidemia and hypertension are effectively managed, medical expenses to the tone of $932 billion us from 2016-2030 (stevens et al., 2016) . controlling blood lipids and blood pressure of elderly people over 65 years of age represents the most cost-effective strategy (stevens et al., 2016) . mental health disorders, particularly dementia and depression, are major diseases in the elderly of china. alzheimer's disease international (adi) estimates that over 50 million people worldwide were living with dementia in 2019, and that this figure will rise to 152 million by 2050; the current annual cost of dementia is estimated at 1 trillion us dollars which will be doubled by 2030 (adi, 2019). it is estimated that the number of patients with dementia in china constitutes 25% of the dementia population worldwide, with the prevalence of dementia ranging from 5.14% (95% ci 4.71-5.57, in 2014) to 5.60% (95% ci 3.50-7.60, in 2019) for individuals aged 65+ jia et al., 2014; jia et al., 2020) . the patterns and spread of dementia in china vary geographically and between genders. women are 1.65 times more susceptible than men. western china has a higher prevalence at 7.2%, while central and northern china are lower at 5.2 and 5.5%, respectively, southern china has the lowest prevalence at 4.8%, this variation is possibly due to a variety of reasons including diet, exercise, social networks, healthcare, etc. (chan et al., 2013; jia et al., 2020; wu et al., 2018b) . the incidence of dementia in individuals aged 65+ ranged from 17.7 to 24.0 per 1000 person-years using 10/66 dementia research group criteria, while it was 12.14 per 1000 person-years using dsm-iv criteria (jia et al., 2020; prince et al., 2012; yuan et al., 2016) . while health conditions such as depression, diabetes mellitus, and insomnia correlate with dementia in a global fashion, epidemiological evidence from different regions in china also suggests smoking and heavy alcohol consumption as high risk factors (fan et al., 2019; pei et al., 2014; xue et al., 2019) . depression, a risk factor for dementia, is a common but often neglected disease in the elderly in china . data from a cross-sectional study suggest a prevalence of depression of 39% in the elderly which increases to 45% in the most elderly (yu et al., 2012) . in view of the stigma of mental illness in some areas of china coupled with inadequate health services in rural areas, depression is likely underdiagnosed suggesting the real prevalence may be higher. in addition to its contribution to dementia, depression aggravates the quality of life of the elderly and of their family members, brings the risk of death caused by different reasons, and accordingly is a heavy burden on the society and the healthcare system (zhang and li, 2011) . much effort should be made to address mental health disorders in china, including increasing government investment, the training of more geriatric care professionals with specialties in mental disorders, and raising public awareness, especially in conjunction with more active social activities and exercises. although there have been increased care facilities for citizens 65+ and improved access to health services, the diagnosis and management of dementia and depression are still inadequate, especially in rural areas (jia et al., 2020) . the inclusion of steps to manage dementia in the 13 th five-year plan of the central chinese government marked a major step forwards, and such efforts need to be continued. in view of the insufficiency of medical professionals in regards to mental disorders, especially in rural areas, we recommend increased training to such professionals, and the development of policies to encourage health professionals to work (at least for a short period) in rural areas . in recent years, the public awareness of mental disorders, especially ad, has greatly improved thanks to efforts from social media (e.g., drama shows on ad) and dementia organizations. professional interventions, comprising medicine and combined cognitivepsychological-physical intervention (e.g., family and community support plus playing mahjong and practicing taichi) can mitigate subclinical depression and improve overall mental health (kong et al., 2019; wang et al., 2019c; wong et al., 2014) . although no drug at present is available to cure ad, recent progress on the understanding of ad etiology, such as the involvement of impaired mitophagy and reduced grid-cell-like representations in the human ad brain, along with the development of novel stem cell models, and the use of artificial intelligence (ai), will undoubtedly propel the development of novel drugs (fang, 2019; fang et al., 2019; gilmour et al., 2020; kunz et al., 2015; lin et al., 2018) . the china brain project, covering studies on basic neuroscience, brain diseases, and brain-inspired computing, will greatly benefit the development of novel drugs for different neurological diseases (poo et al., 2016) . while oral health is an important part of the whole body, the prevalence of oral disease is high in the elderly in china, but is largely ignored, while here we focus on dental health. dental caries (tooth decay), periodontal disease and tooth loss in the elderly are issues of global health concern. the burden on healthcare cost and the quality of life of these dental diseases in the elderly remain high . maintaining good dental health is an integral part of healthy ageing. as such, developing effective preventive and therapeutic interventions are needed to protect and enhance dental health and well-being tonetti et al., 2017) . dental caries and periodontal diseases are common oral diseases in the elderly and often lead to tooth loss, edentulism (toothlessness), impaired masticatory function and poor nutrition. according to the 4 th national oral health epidemiological survey (fnohes, 2015 (fnohes, -2016 covering the whole of mainland china, caries and periodontal diseases are highly prevalent in the elderly in china; while the prevalence of caries was above 50% in all age groups (3-5, 12-15, 35-44, 55-64, and 65-74 years) , the rate was 98% in the 65-74 years groups (lu et al., 2018; si et al., 2019) . in adults aged 65-74 years, 90.7% had periodontal diseases, including gingival bleeding (82.6%), dental calculus (90.3%) and a deep periodontal pocket (14.7%) (lu et al., 2018; si et al., 2019) . human oral tissues naturally and gradually degrade with age; a fact also exacerbated by modern lifestyle choices, including the prevalence of sugary diets and a lack of oral hygiene (belibasakis, 2018; lamster et al., 2016) . more specifically, age-dependent changes include a reduction in periodontal support, loss of elastic fibers, and thickening and disorganization of collagen bundles in the connective tissue of the oral mucosa (belibasakis, 2018; lamster et al., 2016; wu et al., 2016b) . severe dental health challenges can cause loss of self-esteem, social difficulties, while also being drivers of common diseases, such as ad, pd, diabetes, and hypertension (belibasakis, 2018; bollero et al., 2017; dominy et al., 2019; lamster et al., 2016) . major risk factors of the high prevalence of dental diseases in the elderly in china include the scarcity of dental health knowledge in the general population, low frequency of daily oral hygiene practices, insufficiency of dental care services, and unhealthy diet habits. daily oral hygiene practices are effective for removing plaque and preventing gingivitis. the average awareness rate of dental health in the chinese elderly was 47.6%, only 30.1% of the elderly brush their teeth twice daily, and a mere 0.8% used dental floss (lu et al., 2018; si et al., 2019; . increased attention to the dental health needs of an ageing population urgently requires combined efforts by relevant stakeholders (lu et al., 2018; si et al., 2019; tonetti et al., 2017; . specifically in the case of older adults, knowledge and competence in oral care, awareness of medical comorbidities and of medications relevant to oral care should all be strengthened. epidemiological surveillance and monitoring of oral diseases and oral healthrelated quality of life in the elderly is needed. oral self-care, access to treatments and preventive services and assuring the affordability of dental care are critical for oral health. looking after teeth and gums by brushing twice a day with fluoride toothpaste and cleaning with dental floss are effective in achieving a good oral health status. likewise, the control of risk factors, such as refraining from the frequent consumption of foods and drink high in sugar, and refraining from smoking, are also important. provisions to expand services to older adults, to meet increasing oral healthcare needs in the ageing population, and to ensure the affordability of dental care should all be emphasized by policymakers. we suggest programmes that promote general oral health education as well as public outreach programmes directed towards the elderly via understandable brochures, and the use of television and other social medias. additionally, it is important to improve the country's dental care infrastructure by training more dentists and oral specialists and ensuring the provision of affordable dental healthcare. it has been well documented that altered immune system components and function are characteristic of ageing and form part of the causes of age-related diseases (nikolich-zugich, 2018) . in ageing, a significant decline in the homeostatic, defensive, and surveillance functions of the immune system is noted. prominent features of the ageing immune system include thymus involution, a decrease in naïve lymphocytes, and an accumulation of memory and senescent lymphocytes; more recently, the concept of 'inflammageing' has been developed (ferrucci and fabbri, 2018) . functionally, impaired immune defense, especially against new antigens for which no memory exists, makes older adults increasingly vulnerable to incident and more severe infections. in addition, a decline in immune surveillance hampers the elimination of premalignant cells, leading to cancer development. older adults also manifest a chronic low-grade inflammatory phenotype (clip), a manifestation of the inflammageing concept, that likely results from uncompensated inhibitory immune regulation and/or an inability to eliminate senescent cells (chen and yung, 2019; chen et al., 2019b) . as such, immune dysregulation is a general feature of ageing. here we provide an update on infectious diseases in the elderly in china. we carried out a comprehensive review on infections in china based on the following public databases: the chinese center for disease control and prevention (ccdc), the data-center of china public health science (ccdc, 2019), and the national bureau of statistics of china (nbsc, 2019). the three most common infectious diseases in 2017 were viral hepatitis, pulmonary tuberculosis (tb) and syphilis (detailed in section 2.8) while the three with the highest mortality rate were aids, tb, and viral hepatitis ( fig. 3a-d) . of note, pulmonary tb was more prevalent than the other two in older adults over 65 years of age (fig. 3c ). generally speaking, infectious diseases are more frequent and deadly in older adults, as seen with the recent 2019-ncov epidemic worldwide huang et al., 2020) ; thus, infectious diseases deserve more attention. viral hepatitis is caused by the hepatitis viruses a, b, c, d, and e (hav, hbv, hcv, hdv, hev) and is prevalent throughout the world, posing a significant threat to human health. china is a highly epidemic area of viral hepatitis with 86.6 million people infected with hbv and 7.6 million infected with hcvas of (who, 2018 . in 2018, there were 1.28 million new cases and 531 deaths among the chinese population (nbsc, 2018) . according to the chinese statutory infectious disease report, viral hepatitis mainly occurred in adults between 20 to 65 years old (83.68%, fig. 3e ). its morbidity in older adults was estimated to be 13.52% in 2016 ( fig. 3e ). however, compared with the morbidity, the mortality of viral hepatitis was higher (28.31%) in the aged population ( fig. 3e ). from the survey, the morbidity and mortality rate of viral hepatitis have ranked in the top five for many years. the morbidity of viral hepatitis was stable in last decade, which is likely due to the wide usage of the hepatitis vaccine ( fig. 3g , h). however, the morbidity of hepatitis in the elderly continues to increase yearly. since most cases of viral hepatitis developed into chronic hepatitis, the lifespan extension seen in china has contributed to a higher number of elderly hepatitis cases. luckily, the mortality of hepatitis has declined in both the aged population and the population at large (fig. 3h ). among the five hepatitis viruses, hdv is rarely detected and is not discussed here. hcv (20.13%) and hev (22.6%) demonstrated high morbidity in the aged population (fig. 3f) . however, the highest mortality is caused by two acute types, hav (60%) and hev (66.7%) (fig. 3f) , indicating a weakened immune responses against acute infection in the elderly. significantly, both the morbidity and mortality of hbv in the aged population were lowest among the four types ( fig. 3f ), further indicating the benefit of the hbv vaccine. however, prophylactic vaccines for hcv and other types of viral hepatitis are still lacking. for patients who have been infected, current treatments are still limited, especially for the elderly patients. one of the reasons for this is the lack of a long-term infection model for use in laboratory conditions (winer et al., 2017) . developing an elderly-representative model would be a useful tool for screening treatment options for those affected by hepatitis diseases. mycobacterium tuberculosis and is typically transmitted through coughs and sneezes. the lack of global tb control is the result of several factors, including hiv coinfection, limited vaccine efficacy, a lack of highly specific and sensitive diagnostic tests, and the rise of multidrug-resistant (mdr) and extensively drug-resistant (xdr) tb strains (venketaraman et al., 2015) . according to the who's 2019 global tuberculosis report, china is ranked third in terms of tb burden when compared with other countries (who, 2019a). pulmonary tuberculosis (p-tb) is the second highest ranked cause of morbidity and mortality among the 28 infectious diseases ranked in 2017 (fig. 3a, b) . however, it is the most frequent infectious disease in the elderly (fig. 3c ). the elderly occupied more than half (54.9%) of all deaths from p-tb (fig. 3e ). in the last decade, the incidence of p-tb has decreased year to year, however, the incidence and mortality rates of p-tb in the elderly remains high in china (fig. 3g , f). there are several reasons for the high incidence and mortality rates of p-tb in the elderly: i) an increasingly ageing population; ii) immune decline; iii) delay of diagnosis and treatment. with the increase of the number of elderly patients, p-tb is rapidly becoming a new public health challenge. several risk factors, such as immune decline, smoking, malnutrition, hiv infection and other chronic diseases, make the elderly susceptible to tb . compared with p-tb in the young, p-tb in the elderly has its own characteristics. elderly patients with p-tb are more contagious than the young, and elderly men are more likely to suffer from tuberculosis than elderly women (lee et al., 2017) . in the elderly, early symptoms of tb are atypical and insidious, and can result in misdiagnosis (rajagopalan, 2001) . furthermore, chronic fibrous cavitation and hematogenous disseminated tb are more common in the elderly population. most elderly patients with p-tb get tb in their youth at which time it is better controlled but, as they age, p-tb can result as immune function declines. moreover, elderly tb patients usually present with several complications, which further complicates diagnosis and treatment (nagu et al., 2017) . all these characteristics have brought special focus on the treatment and diagnosis of tb in the elderly. at present, there are several tb guidelines for high-risk groups (who, 2016b), but few for the elderly. previous studies in the elderly have also focused less on the evaluation of targeted strategies for control and prevention. thus it is necessary to pay more attention in the future to the production of control programs and evaluation of targeted interventions for tb in the elderly. aids is a chronic, potentially life-threatening infectious disease caused by hiv, which was first detected in the united states in 1983 ( barré-sinoussi et al., 1983) . in the last decade, the morbidity and mortality of hiv/aids has increased yearly ( fig. 3g, h) , and it has become the top cause of death by infectious disease in china, including in the elderly (fig. 3b, d) . the morbidity and mortality of hiv/aids in the elderly population is also rising significantly, and notably the mortality in the elderly is much higher than that seen in the young (ccdc, 2016). furthermore, because elderly people have many basic diseases and low awareness of selftesting after hiv infection, the elderly are more likely to already be aids patient at the time of diagnosis of their hiv infection (xing et al., 2014) . a study has shown that 35.5% of newly diagnosed elderly hiv infectors had already developed into the aids stage (liu et al., 2012) . with increasing use and efficacy of antiretroviral therapy for hiv infection, the lifespan of hiv/aids patients has been greatly extended, and more and more hiv/aids patients will enter old age (nizami et al., 2019) . the problem of hiv/aids in the elderly will become increasingly serious in the future. firstly, hiv infection is not commonly checked in the elderly in china upon visit to the hospital, which may lead to uncontrolled disease progression and infection to others. second, the treatment of aged hiv/aids patients may cause more adverse effects, such as cardiovascular disease (hanna et al., 2016; kramer et al., 2009) , ad (brousseau et al., 2009) , and diabetes (guaraldi et al., 2018) . furthermore, cognitive disorders, loneliness, shame and depression may increase the likelihood that they fail to follow their drug regimen, or refuse treatment altogether (greene et al., 2018; vincent et al., 2017) . interestingly, hiv infection is also likely a driver of early ageing, as aids patients age more rapidly than the general healthy population (he et al., 2019b; lin et al., 2019) . to address these problems, the diagnostic process in the aged population should be addressed more cautiously; therapeutic drugs and technologies suitable for the elderly patients should be developed. special attention should also be paid to psychological problems of elderly patients. the hiv epidemic as a sexually transmitted disease will be discussed further below. influenza is an acute viral infection caused by the influenza virus. at present, a total of four types of influenza viruses have been identified, including influenza a, b, c, and d (iav, ibv, icv and idv) (petrova and russell, 2018) . among them, only iav and ibv are able to cause seasonal epidemics and clinical disease. yearly, the extent of the influenza pandemic varies around the world, which causes high morbidity and mortality. because elderly individuals above 65 years of age are immunocompromised and may have preexisting conditions, they are more susceptible to influenza infection and its complications. data accumulated in the last decade showed that the morbidity of influenza has increased in both the general and aged populations (fig. 3g ). like other acute infections, the mortality of influenza in aged patients was higher than in younger population (fig. 3h ). during january 1, 2018 to september 28, 2019, a total of 1626 severe influenza cases were reported in hong kong, among which 1058 patients (65.07%) were over 65 years old (chp, 2019b). in 2018, a total of 5984 influenza cases were reported in macau, among which there were 188 cases were over 65 years (hbgm, 2019a) . however, only a small number of influenza cases acquires laboratory confirmation, as patients usually die of other related illnesses brought on by influenza. thus, the influenza-related mortality rate is greatly underestimated. in 2012, the ccdc estimated that the death rate caused by influenza was 18/100000 in northern china and 11.3/100000 in southern china, and most of the deaths occurred among people aged over 65 years (77.8% in southern cities and 69.6% in the northern) (feng et al., 2012) . the excess mortality of respiratory and circulatory diseases caused by influenza was 12.4/100000 and 8.8/100000, respectively, among which 86% occurred in people aged over 65 years (feng et al., 2012) . pneumonia is an acute respiratory infection that affects the lungs, which is especially deadly in children under 5 years and in the elderly (65+). pneumonia has become one of the major causes of death for the elderly over 65 years. the harm and mortality of pneumonia increases with age. the "2018 china health statistics yearbook" reported that the mortality rate (/100000) of urban residents aged 65-69, 70-74, 75-79, 80-84, and over 85 with pneumonia was 19.63, 34.48, 68.38, 219.07 and 865.53, respectively; and that of rural residents was 11.62, 23. 67, 48.61, 127.90 and 445.93, respectively (nbsc, 2018) . since 2003, pneumonia has been one of the top three causes of death in hong kong (chp, 2019a) . according to statists by the hong kong centre for health protection, the mortality rate of pneumonia was 39/100000 in 2017, with a total of 8032 pneumonia-related deaths. of these cases, 94.68% occurred in people aged over 65 years (chp, 2019c) . in macau, pneumonia also has been cited as one of the top three causes of death for many years (hbgm, 2019b). in summary, old age is known to affect the immune system negatively. immunocompromised elderly adults are more susceptible to common diseases such as influenza and pneumonia, both of which were responsible for many deaths in this age group. in some cases, these infections may lead to complications that then lead to death, and this likely contributes to underreporting, hiding the true effects of influenza and pneumonia. there are multiple methods for improving and maintaining healthy immune function in the elderly: physical activity and exercise are known to enhance the immune system, however effective ranges still need to be established and disseminated (venjatraman and fernandes, 1997) . additionally, the development of vaccines must be prioritized, although challenges exist such as finding suitable mass production methods. perhaps surprisingly, sexually transmitted diseases (stds) are becoming an increasing problem among older age groups. many people aged 50 years or older in china remain sexually active, and the shift towards nursing homes has led to an increase in exposure to possible sexual partners (yang and yan, 2016) . unfortunately, many older adults do not take precautions in their sex life, due to reasons such as a decreased worry about pregnancy (tht_uk, 2018) . high-risk sexual behaviors render them vulnerable to the transmission of hiv and other sexually transmitted diseases (stds), likewise low awareness of the potential risks and low use of sexual health services can result in late diagnosis and treatment of stds among older adults. we here describe the current situation of hiv/aids and other stds in older adults in china, and propose potential preventative measures. as mentioned before the incidence and proportion of older adults in the total number of reported hiv/aids cases is on the rise in china (fig. 3a-d) . the rise in both the number of absolute cases and the proportion of std infections was observed in both genders. the vast majority of cases in older adults resulted from heterosexual copulation, and has brought about an alarming increase in the rate of new infections. for example, in chongqing, the proportion of hiv infections reported in those aged 50 years and older increased dramatically from 27 to 58.4% between 2011 and 2019 (chinanews, 2019)at the same time, the overall number of male cases quadrupled, and the female cases tripled between 2012-2018 (wu, 2019) . among women newly diagnosed with hiv in china between 2010-2016, the proportion of those aged 50 years and older increased from 17.8% in 2010 (2 959/16 603) to 38.1% in 2016 (9 981/26 196) . this proportion is even higher in regions with larger rural populations. in guangxi, 46% of newly reported hiv cases in 2014 were men aged 50+ (hu et al., 2019a) . in addition to the increase in newly reported infection among older adults, people infected with hiv can now survive to an older age, increasing the proportion of advanced-age hiv cases. in addition to hiv other stds are increasing in prevalence among the elderly in china. from 2000 to 2013, the incidence of syphilis in people over 60 years of age increased by over 30%. the proportion of people aged 60 years and older among all syphilis cases was also on the rise, from 8.5% in 2004 to 22.0% in 2013 . between 2008-2016, the incidence of condyloma acuminate in china showed a downward trend, with an average annual decline of 2.2%. however, the incidence rate among people aged 50 and over increased by 4.8% annually (yue et al., 2017) . gonorrhea is not common in the elderly, and china saw an average annual decline of 7.9% in the incidence of gonorrhea. this trend was also seen in older adults (4.5%-10.9%) (gong et al., 2015) . this phenomenon may be related to the short incubation period of gonorrhea, the high self-medication rate of patients, the sensitivity of gonococcal bacteria to antibiotics, and the insignificant clinical symptoms of female patients (wang and ni, 2008) . there are several contributing factors behind hiv/stds transmission in older adults. ageing is associated with various physiological changes in the human body collectively known as frailty. however, physiological changes in sexual function often fail to attract societal attention. male sexual dysfunction and disorders often manifest in the slowing of penile erection, prolonged ejaculation, the dampening of sexual desire, impotence, etc. as women age, their vaginal tissue becomes thinner, drier, and less likely to become fertile. for the above reasons, the use of condoms in the elderly seems to be less important. older women may have less interest in or need for sexual intercourse; however, their male counterparts may continue to be sexually active for a long period of time. cravings for sex combined with loneliness may push men to resort to commercial sex to quench their desire for sex. in rural areas, the hiv prevalence is high among street-based female sex workers and female sex workers working at sex-on-premise venues with low quality of hygiene, such as hairdressing shops. use of condoms and other precautions in these scenarios is likely to be lacking . sexual education in older adults is nearly absent, and it is generally assumed that "age is a condom". embarrassment may discourage older adults from obtaining condoms and other precautions. in a survey in guangxi, although 87.9% of respondents were willing to accept condoms issued free of charge by healthcare services, 64.1% of the respondents were unwilling to take them of their own due to embarrassment (qi and pang, 2012) . despite the growing importance of sexual health among older adults, many of them do not seek health services for sexual problems. in china, data on sexual health in older adults are scarce. existing research focuses mostly on males (jiang, 2016) . few actions have been taken to accommodate older adults' sexual health needs in china. engaging older adults in health program development and policy changes is particularly challenging due to concurrent incidences of disability, frailty, and other comorbidities. conventional top-down strategies are often unappealing and less trusted by the target audience. innovative solutions are needed to develop contextualized sexual health services and ensure that they are inclusive, trusted, and reliable. collectively, hiv/stds are becoming an increasing problem in the elderly in china due to diminished precautions in their sex life, a lack of condom usage, and insufficient sexual education, among other issues. future research focuses should include a) routine sexual healthcare and screening for hiv/stds among older adults, especially those who have highrisk sexual behaviors; b) sexual health education and hiv/stds prevention among older adults; c) late diagnosis of hiv/stds among older adults; and d) healthcare providers' attitude on the sexual health of older adults. modifiable health-related behaviors (hrbs) are key contributors to chronic diseases and early mortality, such that by maintaining a vigorous lifestyle, the processes of frailty, disability, and dementia can be postponed or even prevented (lafortune et al., 2016; rizzuto and fratiglioni, 2014; who, 2019c) . similar public health recommendations for hrbs have been promoted worldwide, namely, refraining from smoking and excessive alcohol consumption, consuming a balanced diet, partaking in regular physical exercise, and maintaining frequent social engagements (who, 2015d ). an international comparison study revealed a large degree of consistency in hrb clustering across six nationally-representative ageing cohorts in the east and west, alongside considerable gender-and country-specific variations (liao et al., 2019b) . particularly, older chinese males were characterized by a much higher probability of being smokers (57%) than their counterparts in japan (28%), korea (38%), usa (17%), uk (15%), and in other european countries (21%~33%) (liao et al., 2019b) . comparable findings have been reported in the who's 2019 report on the global tobacco epidemic, which further indicates that the progress of smoking reduction tends to be noticeably slower in china than the global average (who, 2019d). nevertheless, positive developments of china's concerted tobacco control efforts, such as smoke-free public places, a strengthened ban on tobacco advertising, etc., should be acknowledged (li and galea, 2019) . these smoke-free movements have challenged and hope to gradually change social norms regarding smoking, though they may be less effective among older generations with poor health literacy (hu et al., 2016) . the implementation of the healthy china 2030 action plan provides an opportunity to increase tobacco control (li and galea, 2019) , as well as to address a range of risk factors via a population-based multi-sectoral approach (nhcprc, 2019a) . aiming to enhance the overall health of the chinese population, the plan prioritizes 15 major actions, including the promotion of health literacy, the improvement of nutrition, a new national exercise campaign, more tobacco control measures, the promotion of mental health and environmental health; and specific actions dedicated to four target populations (i.e. women and children, teenagers, older adults, and those undertaking special occupations) and five categories of diseases, i.e. cardiovascular and cerebrovascular diseases, cancer, respiratory diseases (e.g. copd), diabetes, and infectious diseases. besides health-related targets for the health promotion actions for older adults, the importance of building an elderly-friendly and engaging environment is highlighted, which embodies "ageing in place" with humane, equitable and sustainable health and social care resources. social engagement is a key determinant of active ageing (world health organization, 2002) , especially within china's collective cultural background (liao et al., 2019b; liao et al., 2020) . in tandem with physical exercise, social activities may generate health benefits not only for the body but also for the soul. chinese square dancing is a social group-based exercise performed to music in public squares or parks. this low-cost and easy-participation activity is highly popular among middle-aged and retired chinese women, estimated at 100 million participants in 2015 (fang, 2015) . square dancers can meet as often as every day, usually in the early morning or evening after dinner, and sometimes both, upon meeting they organize themselves into rank and file, and exercise for nearly two hours, led by the most proficient dancer (liao et al., 2019a) . as an aerobic exercise accompanied by a dance rhythm, square dancing mobilizes the participants' whole body, improving their balance and cardiopulmonary function (liu and guo, 2013) . it is also cognitively challenging, requiring participants to listen to and process the music, focus on movement and balance, and dance to the rhythm with coordinated body movements (kattenstroth et al., 2010) . moreover, square dancing creates a socially enriched environment for participants to interact with peers, keeping them socially engaged and dispelling loneliness (liao et al., 2019a; liao et al., 2020) . square dancing is a typical example of a grassroots group activity that may serve as inspiration for the design of culturally appropriate health promotion programs for older adults. one possibility is developing similar programs that can be implemented throughout the country, and possibly tailoring them to the local needs and/or cultures. in the past five years, central and local governments in china have made enormous efforts in establishing a multi-dimensional geriatric care system to support healthy ageing in chinese society. more than 30 national policies have been issued to drive the development of this care system, including cross-ministerial policy measures for promoting the growth of elderly services and the integrated development of medical, health and elderly care, through the guiding opinions on advancing the development of age-friendly livable environment (ndrc, 2016) , and the state council opinions on promoting the development of elderly care services (nhcprc, 2019b, c) . following the strategies of the national 5-year plan, provincial and municipal governments have all issued local implementation plans. in places such as shanghai, shandong, jiangsu, zhejiang and guangdong, political will has been accompanied by strong financial support (cnca, 2020). as compared to q3 2014, in q3 2019 there was an additional 2.4 million beds added in public and private nursing homes across china, resulting in a total national supply of 7.55 million beds (mcaprc, 2019a, b) . in 2018, the ministry of civil affairs allocated rmb 2.9 billion (usd 400 million) to support the local expansion of care beds in nursing homes as well as the development of community and home care services. in terms of service utilization, the occupancy rate of nursing home beds is at around 50%, i.e. at any time, there are less than 3.8 million residents in these facilities. 748,000 elderly benefited from nursing care subsidies while 5.22 million benefited from social care subsidies (mcaprc, 2019a, b) . in july 2016, the first national pilot of a long-term care insurance (ltci) program was announced in 15 cities across different regions of china (mhrssprc, 2016) . identification of elderly people with severe care dependency was carried out, and local models of financing care for them in nursing homes, community centers as well as at home were implemented. by june 2019 this pilot program covered a total of 88.54 million people, funding services for 426,000 beneficiaries at rmb 9,200 per year per person (nhsaprc, 2019). while geriatric care system development has attracted strong attention from stakeholders and become a major theme for policy, research and investment, the following challenges need to be understood and addressed before meaningful progress can be made to prepare the country for its rapid entrance into an ageing society. the first challenge is that care needs must be assessed comprehensively and should be subject to regular reassessment in order to develop personalized care plans and identify goals that are aligned among care recipients, providers and payers (who, 2017) . generally, there are currently two types of assessments in use in china: one conducted before admission into nursing homes, the other for entry into the ltci programs. the first type can be quite comprehensive but is often used to decide the charge levels associated with the care service. the second type uses a simple 6-item adl questionnaire and links its results to the funding schemes, e.g. maximum hours of care per month. as the assessment of care needs tends to be one-off and disconnected to care plans or goals (hua, 2019; ma, 2017) , it is difficult to allocate resources dynamically and to analyze care performance or economics. the second challenge involves problems with service capacity. on the one hand, 45% of nursing home beds are left unoccupied and, contrary to international best practice, for the beds that are occupied, only less than 20% are actually utilized by people with severe dependency; on the other hand, according to the national health commission, nearly 188 million seniors have chronic diseases, and 40 million have various levels of disabilities (nhcprc, 2019b, c) . among the over 40 million people with different degrees of care dependency and care needs, under 10% have been served by community and home, and the majority have yet to be cared for (mcaprc, 2019a, b) . some policies have been put in place to attempt to fill the huge gap in caregivers, stating that 10 million more caregivers are needed just to care for the existing group of dependent seniors. however, if the current mainstream model of "replacive care" is not changed, growing care service capacity will only lead to an accelerated rate of care dependency among the high-risk population. additionally, such a model of care is highly unattractive to potential workforce candidates. as a corrective move, the central government has now set a goal to train 2 million more caregivers by 2022 (mcaprc, 2019a, b) . the third challenge is distorted allocation of resources. up until the end of 2018, despite plans to establish a home care-dominant, community-backed and nursing home-supplemented system, investment has remained predominantly in heavy assets, i.e. the development of nursing homes as well as senior-living property projects, resulting in the above-mentioned "oversupply" of care beds (qiao, 2019) . since the 13 th five-year plan, the central government has committed to an annual funding of rmb 1 billion to support innovative pilots of home-or community-based care models (mcaprc, 2017 (mcaprc, -2019 . however, for many local governments, the first and foremost priority when developing local care capacity is to specify land for elderly care use and invest in care facilities construction before or while looking for operators of such facilities. in addition to resistance and reluctance from nursing homes and preexisting policymakers, difficulty in understanding senior population's care needs and evaluating care competency among community and home care providers have prevented financial support schemes from materializing in most parts of china. typical examples of the insufficient support for community and home care service development can be seen in the number of government purchase tenders that fell through without enough qualified bidders. while there is no lack of political will and resources to be invested in further developing the care system, there is an urgent need to pay for access and quality. a value-based resource allocation model focusing on improving population health rather than the current fee-for-service care model would provide china a rare opportunity to benefit from a healthily ageing society (gyurmey and kwiatkowski, 2019; mandal et al., 2017) . to address the above-mentioned challenges and seize the opportunity associated with them, pilots should be designed based on local evidence and should be established in four dimensions. firstly, development of care plans should be focused on individually centered goal based on comprehensive assessments. as highlighted in the latest who icope (integrated care for older persons) package, it is essential for countries and health systems to align the efforts of different stakeholders with a shared care plan that is customized to serve the individuals' priorities and goals. secondly, health and social care resources should be integrated to support the realization of personalized goals of care and, at the population level, to delay and reduce care dependency. rather than further developing passive care capacity to compensate for the increasing need for other fragmented services, devoting resources to the reaching of a consensus among care providers and receivers will serve to empower the population itself, and maximize the pooling of financial and human resources, decreasing the need for an expansion of passive services (who, 2019b). thirdly, the education and training of "integrated care managers" should be developed, whose job would be to work actively in primary care settings to identify care needs and coordinate care resources crucial to achieving societal and individual care goals. mobilizing talents with various backgrounds to understand and operate under the comprehensiveness of geriatric health needs, developing their capability to better communicate and coordinate care efforts across public and private sectors would not only facility the integration of various care services, but make the care work more attractive for those seeking long-term career opportunities (wang and song, 2019) . fourthly, a reform of the payment model used in elderly care services should be carried out, focusing on value rather than volume of care for populations at risk of care dependency. healthcare payments have long been moving from an inefficient, fragmented, fee-for-service model to a value-based capitation or bundled payment model. for geriatric care financing, this reform is likely to develop faster than the reform of payments for healthcare services. setting sustainable goals for care and allocating resources accordingly will be a viable realistic solution to caring for the millions of chinese citizens in need . we recognize the complexity of establishing such a health-oriented care system. for the four dimensions of an integrated care system to be aligned around common goals as discussed above, a pre-requisite should be the interconnectivity of data: linking results across personal health records, assessments of geriatric care needs, and total costs of care, including: social and commercial health insurance payment, out-of-pocket private payment, social welfare payment, as well as other sources of funding for elderly care (threapleton et al., 2017) . palliative care is emerging as a new alternative for hospitalized elderly with life-threatening illness. the who defines palliative care as the prevention and relief of suffering of adult and pediatric patients and their families facing the problems associated with life-threatening illness (including malignant and non-malignant diseases). these problems include physical, psychological, social and spiritual suffering of both patients and their family members the aim of palliative care is to enhance the quality of life, promote dignity and comfort, and may also positively influence the course of illness (who, 2016a). palliative care is the basic skill of medical staff in departments where medical care is provided to end-stage patients (e.g. icus, emergency rooms, geriatric and oncology departments) (ning, 2018) . the 2015 quality of death index survey showed that the death quality of mainland china ranked 71 st out of 80 countries, while taiwan and hong kong ranked 6 th and 22 nd , respectively (eiu, 2015) . while palliative care is widely available in western countries, it is limited in mainland china. according to a report in 2016, only 0.7% (146/22,000) of hospitals offered palliative care services. in china, the proportion of course in palliative medicine at medical schools is relatively low and, often only available as electives for undergraduates or postgraduates (liu and yuan, 2009) . questionnaire data of 5 th year medical students in 2017 and of geriatric nurses in 2016, showed that 58.7% of medical students and 55.7% of nurses had no training or education regarding death or terminal care, and 93% of medical students and 94.3% of nurses had not received any education about hospice and palliative care. thus the need for course education in hospice and palliative care at chinese medical schools is extremely urgent. palliative care is recommended to be introduced early in curative treatments when patients are diagnosed with a life-limiting disease or when the palliative care needs of patients are identified. current palliative care in mainland china is still mainly focused on patients with cancer, with only a few palliative care resources available for other chronic conditions such as copd, hiv and renal failure . therefore, in the future, palliative care should be extended to both patients with cancer, with other life-limiting diseases, and their families. many palliative care guidelines have emphasized that the discussion of advanced decisionmaking among patients and their families should be initiated when patients still possess decision-making capacity (cheng, 2018) . patients in mainland china sometimes fail to grasp or accept the truth of a diagnosis and limited survival time (cheng, 2018) . moreover, according to questionnaire reports from 1,084 patients in 2016, awareness of the concept of advance care planning or advance directives in china is still low (kang et al., 2017) . in mainland china, family members are often held responsible for making decisions for the elderly in their care, despite a lack of knowledge or training, and thus may resort to homeopathic remedies. healthcare professionals generally have to ''respect'' any decision made by the families and try their best to ''save'' patients' lives using many life-sustaining treatments, although they generally hold negative attitudes to useless treatments. such an approach is regarded as an appropriate measure in terms of protecting themselves from medical conflicts. misunderstanding of palliative care as 'giving up on treatment and waiting for the death of the patients' by family members of the patients as well as even by some doctors, should be corrected (hu et al., 2019b; ning, 2018; xiao et al., 2019) . there is an urgent need for the development of hospice and palliative care in china. in recent years, hospice and palliative care have witnessed rapid development. more and more patients, families, and health-care professionals come into contact with the concept and realize the benefit of hospice and palliative care, while more and more educators, organizations, government and other intermediary leaders have paid more attention to the promotion and development of hospice and palliative care. the current trend towards an ageing society poses difficulties due to the additional challenges seen in diseases in the elderly, including longer disease durations, more complications, underwhelming responses to treatment, and poor prognosis, thus in response to this trend, china has established the national clinical research center for geriatric disorders (ncrcgd) (o'meara, 2020; yu et al., 2018a) . funded by the central government, the ncrcgd aims to provide innovative models for the diagnosis, management and further research into geriatric diseases at a national level. the ncrcgd focuses mainly on comprehensive and systematic research into pathogenesis, prevention, diagnosis and treatment of age-related diseases such as ad, pd, and cerebrovascular disease (xwhosp, 2017b, 2019). at the same time, it is committed to building a national elderly medical service network and scientific innovation system by integrating resources of clinical and basic research. for instance, a health data management platform for the elderly could provide a scientific basis for management and decision making. furthermore, through the education and promotion of new theories and technologies of geriatrics to grassroots hospitals, the ncrcgd can build a better medical service system, improving the health of elderly people. the ncrcgd strives to promote the combination of academic and clinical research (xwhosp, 2017b, 2019). research on agerelated diseases has been carried on such various aspects as diagnosis, treatment, and prevention in fields such as immunology and molecular biology (o'meara, 2020). the characterization of the gene pool, a series of research findings and new technologies have been applied at the clinic, which promotes the development of gerontology in the direction of precision medicine for early diagnosis, early prevention, and early treatment. the ncrcgd also serves as an educational harbor to foster the training of geriatricians and promote academic exchange (frailty-china, 2019; xwhosp, 2017b, 2019). the organization also undertakes other social responsibilities, including partnerships with hundreds of institutions across the country, relying on their collaborative research network to successfully carry out a comprehensive assessment of multiple systems for the elderly (xwhosp, 2017a). through a comprehensive assessment of the multiple aspects of the elderly's diseases, fitness, cognition, psychology and society, it may be possible to develop a system for early identification of health imbalances in the elderly that characterize certain diseases, aiding in their early prevention, and helping to reduce the burden of the ageing chinese society, as well as improving the elderly health service system. prospectively, the ncrcgd will also play its essential role in guiding the research and clinical guidelines for elderly people care as well as making more contributions to improve elderly people's quality of life in china. in order to deal with the ongoing boom in the elderly population, the chinese government has put more effort into funding research on ageing and its related diseases in recent decades. during the recent (2019) outbreak of coronavirus in china, older patients with preexisting ageing-related diseases were found to have a much higher casualty rates than younger patients (chen n et al, 2019) , again highlighting the importance of preventing ageing-related diseases. in response to this, along with the growing need for improving the quality of life of the elderly in china, more attention has been placed on the development of pharmacological strategies against ageing, organ degeneration and major ageing-related diseases. in this section, we will discuss recent world-wide progress in pharmacological attempts to improve healthspan, and the significant contributions that chinese researchers have made. calorie restriction (cr) was first demonstrated as an effective way to extend lifespan in rodents (de cabo and mattson, 2019), however the physiological mechanisms behind its anti-ageing effectiveness were not fully understood at the time, and remain uncertain. later studies have suggested that cr might extend lifespan by regulating insulin-like growth factor (igf) and mammalian target of rapamycin (mtor) pathways. metformin is primarily known for treating type 2 diabetes, with its underlying molecular mechanisms leading to the to down-regulation of igf-1 signaling, and the inhibition of cellular proliferation, mitochondrial biogenesis, ros production, dna damage, activity of the mtor pathway, etc. . the anti-ageing effect of metformin is under investigation by the tame (targeting ageing with metformin) trial in the usa. acarbose, an antidiabetic drug, could also disrupt the igf pathway. acarbose has been shown to partially mimic the effects of cr and extend lifespan in mice by controlling blood sugar and slowing carbohydrate digestion (harrison et al., 2019) . a clinical trial on acarbose (clinicaltrials.gov identifier: nct02953093), named study of acarbose in longevity (sail), is in phase 2, and will hopefully shed some light on its pro-longevity effect in humans. mtor is a pivotal nutrition sensor that links cellular metabolism with proliferation, growth and survival by regulating amino acid metabolism, proteostasis, mitochondria dynamics, cellular senescence, etc. (liu and sabatini, 2020) . rapamycin, a well-known inhibitor of mtor, has shown life-extending effects in all model organisms and postpones the onset of age-associated diseases harrison et al., 2009; liu and sabatini, 2020) . despite the promising pro-longevity outcome of using rapamycin in animals, its clinical application in human has been obstructed by growing concern of potential side effects from immunosuppression and hyperglycemia (pallet and legendre, 2013) . whether the dosage can be fine-tuned to avoid these side effects will be the determining factor in whether or not rapamycin becomes a future pro-longevity drug. the application of induced pluripotent stem cells (ipscs) from healthy and pathological ageing individuals (liu et al., 2011) is also propelling further mechanistic studies and translational applications for cr. nicotinamide adenine dinucleotide (nad + ) is a fundamental molecule in human life and health; while there is an age-dependent reduction of nad + , nad + augmentation extends lifespan and improves healthspan in different animal models as well as shows potential to treat different neurodegenerative diseases based on phase i clinical trials (gilmour et al., 2020; lautrup et al., 2019; yoshino et al., 2018) . nad + precursors such as nicotinamide riboside (nr) and nicotinamide mononucleotide (nmn) have emerged as promising approaches for intervention against ageing phenotypes and age-related diseases. supplementation via these precursors can elevate nad + level in vivo and improve glucose metabolism, mitochondria biogenesis, dna repair, neovascularization and neuroprotection . additionally, more than five phase i clinical trials indicate that orally taking nr is well tolerated and able to elevate nad + in the blood (gilmour et al., 2020; lautrup et al., 2019) . several clinical trials are currently operating in parallel, investigating nr's effects on metabolic function in bones (nct03818802), in immunity (nct02812238), and nmn's effect in cardiometabolic function (nct03151239), with others also ongoing. in china, although nad + precursors have become widely available commercially as supplements, clinical trials exploring their disease-treating ability in humans are still lacking. senescent cells accumulate in aged tissues and this accumulation is considered one of the driving forces of ageing. senolytics are a class of molecules specifically designed to induce apoptosis of these senescent cells. clearing senescent cells in mice has been shown to substantially alleviate ageing phenotypes, producing potent therapeutic effects in ageingrelated diseases such as ad (bussian et al., 2018; zhang et al., 2019b) , atherosclerosis (childs et al., 2016) and osteoarthritis (jeon et al., 2017) . in 2016, a joint research team of chinese and american researchers found that the molecule abt263 reduced irradiationinduced senescent bone marrow hematopoietic stem cells (hscs) and muscle stem cells (muscs) in mice . abt263 (a bcl-2 family inhibitor), together with dasatinib (an anticancer drug) and quercetin (an apoptosis inducer) are the most commonly used senolytic drugs. the senolytic cocktail of dasatinib plus quercetin (dq) decreased naturally occurring senescent cells, improved mobility and reduced the risk of mortality . however, a small pilot clinical study using the same dq cocktail in patients with idiopathic pulmonary fibrosis (ipf) reported no change in pulmonary function, frailty index, clinical chemistries and reported health, though the beneficial effects on mobility were still noted (justice et al., 2019) . while clinical trials on senolytic drugs are mainly conducted in the usa, the concept of reducing senescent cells to delay the ageing progress has attracted interest from all over the world. since 2016, the national natural science foundation of china (nsfc) has set up special programs, providing millions of rmb to support research on cellular senescence and organ degeneration. as such, it is recommended that china further expand its investment in senolytics research. targeting the microbiota may also improve age-related diseases, including ad. in 2019, china approved the first domestically invented ad drug, oligomannate (gv-971) (wang et al., 2019d) . considering there has been no new approved anti-ad drugs in the past 17 years, this has been exciting news. despite the potential of these advances, more work is necessary to understand how gv-971 works. additionally, due to the relatively short clinical trial period, further investigation with longer lasting trials is highly recommended. most human trials for potential anti-ageing drug candidates are conducted in patients with certain age-related diseases. despite partial overlap of the pathologies of these diseases, the knowledge from these trials cannot be interpreted as treating ageing itself. therefore, to reach the goal of identifying anti-ageing compounds, a more comprehensive study on disease-free, healthily ageing groups with no obvious health issues is in immediate need. china has the advantage of a large and diverse population, providing an ideal subject pool for this type of study. the knowledge gained from such studies would likely open new avenues to better understand the fundamental aspects of ageing mechanisms, facilitating their treatment. from a public health and policy perspective, it can be seen that continuing research into prevention and management strategies will be important for both non-communicable diseases as well as geriatric syndromes, to ensure that it is not only life expectancy that is increased, but also the quality of life, by promoting independence and reducing reliance on elderly care services. regular monitoring of trends in incidence and case fatalities of common chronic diseases would enable estimates of future disease burdens and guide preventive health policies (chau et al., 2013a; chau et al., 2013b) . in addition, solutions to trends in the occurrence of disability and frailty are also needed (yu et al., 2018b) . such data would inform the design of elder-friendly service delivery models across the whole spectrum, from prevention to primary care, hospital and residential care settings (woo et al., 2013; yu et al., 2019) . currently, hong kong, a special administrative region (sar) of china, has the longest life expectancies in the world for both men and women, such that the need to redesign service models is particularly pressing. by 2064, it is predicted that 33% of the population in hong kong will be aged 65 years and over; 70% will have at least one chronic condition, with an increasing prevalence of disability also predicted (yeoh and lai, 2016) . while the health and social care systems are well developed, there is a mismatch of needs as those with chronic conditions are predominantly managed in the public hospital systems, whereas primary care is predominantly in the private sector. a recent review concluded that better integration of health and social care systems with a primary emphasis on the community could be the best way forward for the ageing population in hong kong (threapleton et al., 2017) , exemplified by the formation of nurse-led district health centers in 2019 (fhb, 2019). other community models with an emphasis on promoting group activities to prevent frailty and aid selfmanagement of chronic diseases have also been developed (cadenza). such developments have the potential to enhance the role of primary healthcare professionals in preventing functional decline (morley et al., 2017) , so that many can retain independence even as life expectancy increases. the who's integrated care for older people (icope), formally launched in october 2019, will form a useful blueprint for policymakers to build on their existing health and social care infrastructure (who, 2015c). experiences of elderly healthcare in the european union (eu) may provide useful tips for the situation in china (table 1 ). in the eu, elderly care is provided in each country based on its own social security system and cultural norms. in most european countries, the family and the state are the main providers of support to older people both in activities of daily living (adl) and in instrumental activities of daily living (iadl) (schmid et al., 2012) . europe is characterized by three types of care provision: 1) 'crowding out', whereby the state largely replaces family care; 2) 'crowding in', whereby the state promotes family care; 3) 'mixed responsibility', whereby both the state and the family take a joint responsibility for care, yet have separate functions (brandt et al., 2009) . in china, family is still the traditional provider for elderly care (wu et al., 2016a) . under current national and social developmental conditions of china, the chinese government encourages a '90/7/3' pattern of eldercare system, namely: 90% of all older people are cared for at home, 7% are cared for in communities, and 3% are cared for in institutions (mayston et al., 2017) . a 'crowd out' system dominates in the nordic countries (denmark, finland, norway, sweden, iceland) , where the government strives to create a comprehensive system of care services in order to reduce the care obligation of the family. in continental european countries such as austria, belgium, france, germany and the netherlands, systems are more mixed in their provision of elderly care, though tend towards a 'crowd out' approach (kasearu and kutsar, 2013) . in the island countries, i.e. the uk and ireland, the system is more mixed, and the private market is the dominant welfare provider, with the government providing two main social care services to older people, one being old age pensions and the other being healthcare . southern european countries (e.g. greece, italy, portugal, spain) have a 'crowd in' system whereby families have more responsibilities for care services to older people (kasearu and kutsar, 2013; wu et al., 2014) . eastern european countries have undergone dramatic political, social and economic changes after the soviet era and experienced a rapid change from 'crowd out' to a 'crowd in' system where family is the main care provider and the government provides basic formal care services (kasearu and kutsar, 2013; wu et al., 2014) . in china, owing to confucian culture and its emphasis on the family, it is taken for granted that the family, most notably adult children, has the responsibility to care for older parents, especially in the rural areas of china, thus older people rely mainly on their children or family for support (chen and silverstein, 2000; wu et al., 2016a) . rapid demographic ageing increases the demand for care in all ageing societies. currently, european countries face the enormous challenge of implementing major reforms to elderly care in order to ensure that the needs of older people can be continuously met in the future (brandt et al., 2009; broese van groenou and de boer, 2016) . to this end, european governments have increasingly relied on informal care in addition to regular and traditional formal care providers from professional home care services, day care units and nursing homes (broese van groenou and de boer, 2016; verbakel et al., 2017) . informal care for older people is generally provided by caregivers from both kin and non-kin groups, including spouses, children, relatives, neighbors, friends, etc. (swinkels et al., 2016) . in europe, around a third of people aged 50 years or older provide informal care to older people. however, shrinking family sizes, the increasing participation of females in the workplace, and rising retirement ages, may pose a drastic challenge to informal care in the future (verbakel et al., 2017) . china is currently facing challenges in its family-based elderly care model due to new family formation, the spread of individualistic values, and frequent internal migration from rural to urban areas encouraged by rapid economic development (wu et al., 2018a) . moreover, china's one-child policy has sped up the process of population ageing by accelerating the change of the fertility rate and, in turn, has weakened the family-based elderly care model in china . in europe, new elderly care arrangements have been gradually developing based on a new combination of family obligations, market provision and public support. in nordic countries, the state, family and market have been changing with regards to their roles in the provision of elderly care, specifically by increasing the provision of publicly funded care services in a forprofit capacity (marketization of elderly care) and increasing the importance of family care (szebehely and meagher, 2018) . in estonia, the idea of community-based support for older people has been increasingly set forth in order to postpone the need for institutional eldercare (tulva et al., 2016) (tulva et al., 2017) . when it comes to the current trend of eldercare in china, marketization has also been discussed to a large extent both at academic and policy levels. the 'public-private-partnership' (ppp) model may improve the efficiency of familybased eldercare. in the 13 th five-year plan for national economic and social development (2016-2020) , the opening-up of the market for elderly care services (e.g. purchase of services by the government) was clearly stated (du and wang, 2016) . elderly care reforms might create new challenges for both europe and china, an important challenge being an increase in inequality in eldercare service utilization among different social groups of older people. older people with higher socioeconomic status will be able purchase private care services whereas those with less social capital will have to rely on more family-based care. in addition, for the chinese government, there is a need to take into account larger inequalities derived from immense resource variations across regions during the development and reform of elderly care services. while mainland china can learn many successful experiences from hong kong, the eu, etc., there remains many unique features that demand the creation of an elderly care system tailored to mainland china. in addition to responding to changes and preparing to adapt to an ageing society at the societal and individual levels, understanding of the mystery of ageing at a molecular level will aid the development of novel strategies to slow ageing and to promote healthy longevity. in the below sub-sections, we will focus on how to use centenarians, the china national genebank database (cngbdb), and ai to further propel ageing research. in china, the numbers of the oldest-old individuals (those aged 80+), near-centenarians (95+), and centenarians are increasing at roughly 10% yearly (fig. 4a-b) , providing unique resources for both basic research and clinical studies. there were 3,384 centenarians in 1953, with the number rising to 35,934 by 2010 (abida and gu, 2008) . based on the un's medium variant projection, by 2050 over a quarter of the global oldest-old population will live in china. as the numbers of the most elderly have expanded, the gender structure of centenarians, the proportion from urban and rural areas, and differences in geographical distribution have formed "three-high" trends in china (data from china's 2010 population census, excluding hong kong, macau and taiwan) . first, there is a gender difference, with 75% of centenarians being female (peng, 2011) (fig. 4a) . data from the 6 th population census of china (2010) reported 27,082 (75.37%) female and 8,852 (24.63%) male centenarians. this could be due to both physiological (e.g., female hormone estrogen) and cultural differences (women often do more housework, pay more attention to healthcare) (austad and fischer, 2016; peng, 2011) . second, urban and rural disparities were clear, wherein more centenarians (56%) live in rural areas, possibly due to a healthier living environment, diet and lifestyle in these regions (cai, 2013; peng, 2011; zeng et al., 2017a) (fig. 4c ). and third, there was geographical difference in the distribution of centenarians. the distribution of longevity areas in china presents several significant characteristics, including province-specific: being majorly in hainan, guangxi, sichuan, yunnan, guangdong and xinjiang, and mostly distributed along river basins, with more centenarians along the pearl and yangtze rivers and the lancang river basins. these characteristics of the area distribution of centenarian suggest that areas beneficial to longevity can be divided into two types: 'natural' and 'economically developed' longevity areas (he et al., 2017; zeng et al., 2017a) (fig. 4d) . studies of centenarians can provide valuable information for early prevention of major diseases, premature ageing, and early death, thus providing the scientific support necessary to cope with the quickly approaching arrival of an ageing society in china. centenarians may represent a prototype of successful ageing. a longitudinal study of a danish 1905 cohort suggests exceptional longevity does not result in excessive levels of disability (christensen et al., 2008) . in fact, some centenarians experience a delayed onset of age-related illnesses (delayers), whereas others did not succumb to any age-related illnesses (escapers) (christensen et al., 2008; hitt et al., 1999) . in addition, one case-control study showed that older individuals had a delayed age of onset of cancer, cardiovascular disease, diabetes mellitus, hypertension and osteoporosis than their respective younger reference groups (ismail et al., 2016) . the china hainan centenarian cohort study (chccs) on 1,002 centenarians is now in progress, focusing primarily on examining biological indicators and medical aspects, and extensively examining psychological and sociological factors (he et al., 2017) . all in all, the study of centenarians is a topic of immense importance for population and health policymakers, as well as for the larger aim to achieve long, healthy lives. state-of-the-art technologies enable the 'big data'-based investigation of the molecular mechanisms of human ageing and its associated diseases, providing unique information for therapies and interventions. the cngbdb is a centralized 'big data' hub of biological data, providing data sharing, knowledge search, computational analysis, management authorization, and visualization services to the global research community. built and maintained by the china national genebank (cngb), cngbdb draws from 3 "banks": the living biobank, the biorepository, and the bioinformatics center, and from 2 "platforms": the digitization platform and the synthesis and editing platform. the research data system of cngbdb integrates molecular data from internal and external sources into nine sub-databases including literature, gene, variation, protein, sequence, project, sample, experiment, and assembly (https://db.cngb.org/news/2/). comparative analyses of species and tissues can identify the molecular causes of ageing phenotypes, corroborate or disprove theories on ageing, and help to understand differences in j o u r n a l p r e -p r o o f the mechanisms of ageing across species. genotype comparisons within a species at the level of individuals and populations can help identify genetic reason for differences in lifespan. this approach may be used to compare populations from different regions of china, or chinese and foreign populations, such as ashkenazi jews and okinawan centenarians in japan, two populations well-known for their longevity, facilitating the discovery of chinese-specific agemodifying genes. the identification of potential life-extending genes eases the design of therapeutics that can mimic the effect of these genes in people without those genes. likewise, treatments can be designed for age-related diseases that result from mutated or nonfunctional genes in specific populations. it is also possible to comparatively analyze gene expression at the tissue level, as tissues age at different speeds. since many age-related diseases, such as ad, occur within a specific tissue, understanding the speed at which tissues age can help chinese gerontologists assess the risk of and help to prevent tissue-specific age-related diseases (wieser et al., 2011) . the advent of 'big data' and machine learning have eased the collection and identification of biomarkers associated with biological age and may allow for the development of personalized clinical diagnostic tools for physicians in the near future (aman et al., 2019) . in the field of medicine, biomarkers refer to measurable indices capable of identifying a condition, state of being, disease, or environmental marker whose presence may reflect a pathophysiological state (naylor, 2003) . the use of biomarkers has been applied to the field of anti-ageing technologies, including the prevention and treatment of age-related disease, and has been used to explore methods to delay or offset the ageing process altogether, and will likely serve as key components to advances in the field (campisi et al., 2019) . in some cases biomarkers may more accurately represent a patient's 'biological age', as opposed to a patient's simple 'chronological age', the former of which is thought to be more clinically relevant (lopez-otin et al., 2013) . the following sections will review three applications of biomarkers at the molecular, individual and societal levels, including current findings as well as potential research directions. as stated above, there are multiple tests that can be used to obtain molecular biomarkers, and several have been validated to some degree by current research. molecular biomarker studies can be roughly separated into 2 classes: smaller scale studies attempting to determine the utility of a given biomarker, and machine learning studies involving thousands of samples with the intention of developing clinical assessment tools. as an example of this, a recent study involving elderly hypertensive patients was used to investigate a wide library of potentially useful biomarkers . here, 416 elderly chinese participants were matched with subjects from a pool of 9,000 normal volunteers. after adjusting for confounding covariate factors, the researchers found that only elevated triglyceride levels were strongly linked to high blood pressure (hong et al., 2017) . while these cross-sectional studies are certainly important for determining which biomarkers should be considered for clinical evaluation, one of the limitations, at least in comparison to machine learning studies, is that they have a low number of samples. in the above examples, most participant groups contained less than 500 people. while this is a surplus number in other medical contexts, one of the advantages of machine learning is its ability to process thousands of samples granting increased accuracy. fittingly, one of its primary uses is to draw meaningful conclusions from mass, simple, cheap, and non-invasive tests. another key limitation is that biomarker assessment studies using these 'smaller' cohorts tend to lack any external validation. perhaps one of the most easily accessible tests that comes to mind is a standard blood test, here the usefulness of machine learning has been demonstrated by putin and colleagues, who designed a modular ensemble of 21 deep neural networks (dnns) of varying depth, structure and optimization for the prediction of human chronological age using a basic blood test (putin et al., 2016) . the team trained the dnns using a collection of over 60,0000 samples from routine blood biochemistry and cell counting assays. the researchers reported that the accuracy of their results provided evidence to suggest that machine learning algorithms could be used to design minimally invasive biomarker tracking methods for ageing that would only improve with greater access to training samples (putin et al., 2016) . another study examined 19 serum biomarkers, and its results were externally validated using a separate data set from the framingham heart study (sebastiani et al., 2017) . such studies highlight the ability of machine learning techniques to infer conclusions from basic samples, and to externally validate such conclusions. still, this was one of the very few studies with this type of external validation and more are needed for clinical application. big data can also be used to assist geriatricians for personalized medicine, defined as a medical approach in which treatment is customized on an individual basis based upon disease subtype, genetics, risk, prognosis, or treatment response using specialized diagnostic tests (frohlich et al., 2018) . for instance, predictive biomarkers for the early detection of certain diseases, may help both patients and doctors to decide on appropriate treatment pathways. in addition, the 'internet of things' refers to the ability of technology to send and receive data via the internet. as wearable/compact technologies become more prevalent (i.e., phone pedometers, pacemakers, insulin trackers) and their data becomes easier to store and share, so too does it become easier to use life-logging data to track individual's wellbeing. unfortunately, while there is great potential for this type of technical approach, there are currently very few cases of applications within clinical practice (frohlich et al., 2018) , with many studies still in an exploratory phase, requiring further research. for example, one study shows that machine learning techniques have significant potential in developing personalized decision support for chronic disease tele-monitoring systems; however, it was noted that the system would be improved with a larger library of comprehensive predictive markers (finkelstein and jeong, 2017) . the use of radiomics, the high-throughput mining of quantitative image features from standard-of-care medical imaging that enables data to be extracted and applied within clinical-decision support systems, has also been proposed, especially within the realm of dementia prevention and detection . these studies have benefited from large sample sizes (>1,000 images) using machine learning. this brings us to the issue of noise reduction, which is crucial for effective use of big data and will enable a more robust extraction of features. given the immense amount of data expected to be handled in future projects, finding ways to store, process, and analyze this data also presents a challenge for future research. at the societal level biomarkers have numerous applications. monitoring population-level biomarkers will likely provide an accurate, real-time view of the health state of a given area. this will allow for targeted interventions catered to suit the specific needs of a population. as stated earlier in this piece, modern medicine has provided for major increases in both quality of life at old age, and life expectancy, however, this can also be considered a potential societal burden. earlier the use of federated systems with respect to online medical records and data sharing was discussed as a potential hurdle to some countries and medical systems in the world. china has recently embraced a centralized health informatics scheme, with over 80% of medical organizations above the county/district level, 27% of town level hospitals and all cdc above the county/district level capable of transmitting real-time reporting on the status of epidemics via the public health information system (zhang et al., 2007) . in the future, the data provided by a centralized medical record system has the capacity to train numerous machine learning algorithms for use with biomarkers. another challenge for population-level biomarker implementation is to select low-cost, minimally invasive testing that can be used at a large scale. with respect to china, great advances have been made in the use of medical informatics within the past 30 years. however one of the hurdles going forward for the country is that much of this investment has been driven by industry and the private sector, and a major priority for the country's future should be to divert resources to academic research (liang et al., 2017) . this is especially true for the poorer members of society, or those without ready access to healthcare, as biomarker are an asset in devising appropriate healthcare plans for populations in need . addressing rural areas may be a challenge both in terms of healthcare delivery and biomarker testing, as these regions may lack sufficient infrastructure for both, posing a challenge for the future . we recommend the use of mobile-equipped information technology services to reach more remote regions. in summary, biomarkers have a great deal of potential for how doctors can prevent, diagnose, and treat illness associated with ageing. while there are many hurdles going forward, the application of machine learning and big data to biomarker research will provide new opportunities to understand ageing at the molecular level, deliver personalized treatment at the individual level, and design influential and effective policy at the societal and population level. since the beginning of the 21 st century, china started to enter a period where it may be classified as an ageing society. at the same time, the compulsory healthcare insurance systems in china has undergone a comprehensive and rapid development, while still emphasizing the ideologies of health equity and social justice . three major health insurance schemes have been launched, achieving near-universal coverage in a short time, which gained early appraisal by emulating the goal of the provision of affordable and equitable basic healthcare for all by 2020 (yip et al., 2012) . after the establishment of the urban employee basic medical insurance (uebmi) in 1998, the chinese government implemented the new rural cooperative medical scheme (ncms) for rural residents in 2003, and the urban residents basic medical insurance (urbmi) for urban residents without employment in 2007. as a result, social health insurance coverage increased from 29.7 to 87.9% between 2003 and 2008, and further to 95.7% by 2011, and has been stable since (meng et al., 2012) . in order to further reform the fragmented health insurance system, the latter two of these schemes were combined into the basic medical insurance for rural and urban residents in early 2016, with a target of making the system less complicated, but more equitable for various social groups. in the past 10 years of the new round of healthcare reform beginning in 2009, the chinese government dramatically increased financial investment, with half of all investment in the form of funded premium subsidies for residents to be covered by the social health insurance system (yip et al., 2019) . universal coverage has since led to improved access to and utilization of healthcare (meng et al., 2019) , decreased the prevalence of catastrophic health expenditure (yip et al., 2019) , and reduced out-of-pocket expenditure as a proportion of total health expenditure, especially for vulnerable groups, including older adults (xu and mills, 2019) . however, the social health insurance system in china still faces the dual challenge of population ageing (demand) and inefficient delivery on the side of the healthcare system (supply), raising both health expenditures and individual disease burden. out-of-pocket expenditure as a proportion of disposable personal income increased from 4.98% in urban regions and 5.17% in rural areas in 2008 to 5.59% in 2017 (xu and mills, 2019) . concretely speaking, population ageing addressed the increasing health and social care needs of older people. according to the report on the fifth national health services survey, the prevalence of non-communicable disease had increased more than 20% between 2003-2012, from 50.1 to 71.8%, while the inpatient rate rose from 7.6 to 17.9% in between 2003-2013. the outpatient rate also increased to 49.7% in 2013 (nhfpc, 2015) . the reimbursement of social health insurance improved rapidly and accounted for 42% of total health expenditure in 2017, though while out-of-pocket payments dropped from 60% in 2000 to 28% to 2017, financial protection and services packages were insufficient for the elderly (meng et al., 2019) , especially for those in rural regions. reported a three times-higher risk of catastrophic health expenditure among the old population in rural regions . in 2013, expenditures on hospitalization for older people in urban areas were reimbursed 64% by social health insurance and 53% were covered for their rural counterparts (who, 2015b). regional disparity in health benefits for the elderly with insurance aside, a problem of inequity among different health insurance schemes on health outcomes for older adults is still a great challenge. uebmi recipients were found to have better physical and psychological health outcomes compared to those with urbmi or ncms insurance. this demonstrates a transformation in health insurance reform from an emphasis on the opportunity-oriented health equity measured by coverage and healthcare accessibility to stressing outcome-based equity composed of health outcomes for the elderly, namely "outcome-based health equity", giving priority to disadvantaged groups . in terms of supply-side deficiency and unsatisfied progress in the past 10 years, gaps in the public hospital and pharmaceutical reform have tremendously limited the effectiveness of social health insurance reform, even though the public hospital has removed mark-ups for drug sales, adjusted pricing mechanism, reformed provider payment systems and changed governance structures at the county level (yip et al., 2019) . the hospital-centered health delivery system has induced the growth of both health expenditure and health insurance expenses, which worsened the control of non-communicable diseases and health outcome improvement in ageing society. unexpectedly, the usage of outpatient and inpatient services in primary health facilities declined from 62% and 29% in 2009 to 54% and 18% in 2017 . due to the lack of qualified long-term care facilities, the length of hospitalization was longer for the old population aged 60 and over (who, 2015b), demanding higher expenditure input to cope while wasting health resources. we advocate the immediate application of an integrated health and social care-oriented, particularly in community settings, with the objective of increasing affordability and improving the quality of care for older people. population ageing, family structure shift, and migration, were three major challenges limiting the efficacy of traditional informal care provided by families and their networks. a large proportion of older people with functional disability or dementia will continue to create enormous challenges for an immature long-term care system in china . it was estimated about 40 million (19.5% of the elderly) older people had some sort of functional disability by 2015, among which 12.4 million (6.05% of old population) had a serious status of disability (nhfpc, 2015) . at the same time, china has become the largest country in the world to have over 9.5 million people with dementia (jia et al., 2020) . in response to the increasing need for social care of older people with disability, the central government of china has implemented a pilot practice of ltci policies in 15 cities, while some local governments were also encouraged by the central government to initiate county-level pilot experiments on ltci since 2016, in hopes of stimulating the growth of long-term care providers (luo and zhan, 2018) . most ltci schemes were based on the social health insurance system, though these pilots had distinct and diverse eligibility conditions, premium contributions, need assessment instruments, and benefit packages. the reason for carrying out a pilot practice rather than fully implementing a uniform nation-wide scheme reflected the complexity of ltci, and a worry about cost escalation noted ltci introduction in the more mature ageing societies of japan and germany. after two years of practice, a few evaluations were conducted to estimate the outcome of these pilot practices, identifying a host of problems. there are several characteristics and unique features present in the chinese ltci scheme. at first, coverage was narrow and limited only to older people with the most serious degrees of disability, and excluded older adults with dementia due to security issues and lower quality of care skills. by the end of 2017, less than 2% of the older population in the pilot cities were covered by ltci plans in qingdao (zhu and osterle, 2019) , which was the first city to launch the ltci scheme in china, a higher proportion of those 65 years and older was achieved in the ltci practice of the mature ageing populations in germany (11.7%) and japan (12.8%) (oecd, 2013) . secondly, the need assessment tools used by each pilot city were fragmented and biased. some pilot cities or counties only employed the barthel index to measure physically functional disability, but did not measure cognitive function with any scales, thus leading to the exclusion of older people with dementia from ltci coverage. more seriously, the results of need assessment were not applied to long-term care service provision, but only used as a "gate keeper" for receiving the fixed benefit package. assessment tools should be transformed from simple to comprehensive, from a physically oriented test to a multi-dimension health status one, even from health assessment to service assessment. thirdly, in most pilot plans, long-term care was provided by designated institutions through a contract, and a homeand-community-based caregiver was paid by the insurance scheme, however reimbursements were limited such that a large proportion of costs was still paid out-of-pocket by service users themselves, and unmet needs were still high among the disabled elderly . in addition, the inequality in access to long-term care services between advantaged and vulnerable elderly was enlarged. in most pilot schemes, higher numbers of benefit packages were allocated to insured groups living in nursing homes or receiving formal care than to those living at home receiving informal care. retired people with uebmi had higher affordability and preferred to live in the institutions and received higher reimbursements from insurance. rural residents could not access good quality long-term care facilities, and received fewer benefits. the inequality that remains in ltci practices highlights how policy reform ought to reevaluate and reconstruct the currently fragmented schemes and direct more attention to the disabled elderly with lower socio-economic status and without financial or family support. although china attempted the ltci scheme, its most urgent priority was to establish a unified meanstest public budget system to cover the most vulnerable social groups regardless of their living locations. through lu et al. (2017) 's projection, an investment as small as 0.25% of gdp (equivalent to about 1.25% of fiscal revenue) would greatly benefit the frail elderly and those with serious problems of functional disability and/or poor financial status . in 2016, the central committee of the communist party of china and the state council issued the healthy china 2030 plan. corresponding with the health-related sustainable development goal (sdg), this is a national mid-term and long-term strategic plan for moving towards universal health coverage and improving health equity, with emphases on health coverage for the whole life circle, including healthy ageing (prc, 2019). in 2018, china made a major restructuring of national healthcare governance. the national health commission (originally called the ministry of health) administers and regulates the healthcare delivery system and include two new areas of responsibility: elderly care and tobacco control (yip et al., 2019) . in addition, the national healthcare security administration was established. it is in charge of administering essential health insurances (urban employee basic health insurance, urban-rural resident basic health insurance, which integrated the original urban resident basic health insurance and new cooperative medical scheme) and medical assistance for the poor and vulnerable groups as well as deciding on pricing and drug procurement. rapid ageing and an alarming increase in non-communicable diseases (ncds) have arisen as major health concerns in china marten et al., 2014) . in 2009, the national basic public health service program was established, which included health management for elderly people, patients with major ncds (hypertension and diabetes), among others (nhfpc, 2017) . the program is financed by government funds, and the government's per capita allocation increased from 15 to 55 rmb between 2009 (nhfpc, 2017 . china's ongoing healthcare system reform prioritizes transforming hospital-centered treatment care to integrated and continued care through a tiered healthcare delivery system (meng et al., 2019) . a tiered healthcare delivery model defines the functions at each health facility level, and coordinates care across levels. a common model is that hospitals lead medical alliances to deliver integrated care, and provide support and training to strengthen primary health services wang et al., 2018b; wbwho, 2019) . in addition, residents are able to register with a family doctor team who provide preventive and basic healthcare as well as referral services. the government target is universal registration by 2020 (nhc, 2016) . china has made good progress in improving equal access to healthcare and financial risk protection for socially vulnerable people over the past decade (fu et al., 2018; meng et al., 2019; yip et al., 2019) , but challenges remain. there is a lack of qualified primary healthcare providers who are able to serve as gatekeepers, and the quality of primary healthcare is poorly characterized meng et al., 2019) . previous studies reported very low proportions of blood pressure and blood glucose control among patients with hypertension and diabetes seeking care from primary health facilities, and common over-prescription of antibiotics su et al., 2017; wang et al., 2014) . patients persistently bypass primary health facilities and seek perceived good quality of care in high level hospitals, despite many patients complaining of high medical costs and long wait times . on the other hand, most hospitals still largely rely on fee-for-service payment and tie doctors' salary to the hospital revenue generation, which gives hospitals an incentive to attract and retain patients rather than shifting them primary healthcare. overuse and overprovision of health services are common in china (meng et al., 2019; yip et al., 2019) . consequently, health expenditure has continued to escalate, a trend which threatens the long-term financial sustainability of basic health insurance schemes. the efficiency in using health resources is low (meng et al., 2019; yip et al., 2019) . as china continues to progress as an ageing society, strengthening primary healthcare system to provide integrated care will be fundamental to meet growing health needs and obtain the best value from existent health resources. it is difficult to shift from treatment-based intensive care to population-based preventive care and health management while perverse financial incentive for hospitals are not controlled or eliminated. this requires effective collaboration across related sectors led by a strong coordinating authority and needs to bridge policy dialogue to ensure health in all policies workable and achievable. china's prolonged demographic shift has led to decreased fertility, elevated sex ratios, rapid ageing, fast urbanization and major geographic redistributions (peng, 2011) , an interdisciplinary collaborative approach is necessary to prepare and face the challenges as society continues to age. we present a summary on ways to achieve a healthy ageing society in china at societal, individual, and molecular levels (fig. 5) . breaking knowledge gaps and eliminating boundaries among different sectors to further integrate and synergize different healthcarerelated parties at societal, individual, and molecular levels will optimize the outputs of the chinese healthcare system. the chinese government has adopted a positive stance to investment across the whole spectrum of ageing policies, medical education and training, basic ageing and geriatric research, prevention, primary care, and hospital and residential care. it is necessary to establish updated ageing policies on retirement age, to incentivize employment of the elderly, to encourage lifelong learning, and to invest in senior volunteer programmes (yeoh and lai, 2016) . the education and practice of geriatric medicine has been and will continue to be enhanced, including to further increase the teaching of geriatrics-related subjects in medical school, to design high-quality residency and fellowship programs, and to further integrate geriatric principles into general clinical practice (yu et al., 2018a) . the establishment of the national alliance of ncrcgd has been highly appreciated and welcomed and it will continue to serve as a national platform to educate and train geriatricians. while the achievement of healthy ageing and longevity is emerging as an important task for china as in many other countries, this may also be accompanied by socio-economic challenges. one of the major concern is that creating a society where healthy and active ageing and longevity are taken for granted may lead to a swelling of the elderly in the workforce, leading to limitations in job availability for the young, and proving to be a potent economic issue. countries like china and south korea are entering a society of population ageing, showing low birth rates and increased life expectancy, which changes the whole economy korea, 2017) . population ageing will likely have several macro-economic effects, touching various domains such as overall industrial structure, current account and inflation, output growth, household finance, labor markets, consumption, and even fiscal and monetary policy (korea, 2017) . it is therefore imperative to give a comprehensive assessment of population ageing in view of its effects on society and the economy in the long-term, to provide evidence to inform future policies. while a challenging task, some suggested responses to the early-emerging changes that could be taken to offset the effects of the ageing society include promoting the production of larger families in the young, finding ways to ensure jobs remain for the young should the elderly be able to continue longer in their positions, along with more general preparations on transform to an elderly-friendly society. it is delightful to witness the progress of basic and translational ageing research in china, as supported by increases in the number of grants and funding opportunities, as well as by rising numbers of high profile publications and discoveries (he et al., 2019c) . joint efforts from the government and stakeholders of each and every sector should be encouraged to nurture an elderly-friendly society, of most import are reforming the social support system to support china's ageing society, and the introduction of health service/investment interventions aimed at reducing inequalities in health among older people in china. we suggest current research focus on basic and translational gerontology to improve healthy longevity in the elderly, and on developing an integrated and affordable health and social care delivery system to meet the complex needs of a growing elderly population, and to finally transform china into an ageenabling country where well-being and healthy longevity can be celebrated for decades to come. in response to the ageing society and in order to improve the quality of life of the elderly in china, strategies at societal, individual, and cellular levels are presented, detailed in the text. the art of traditional chinese paper cutting '松鶴延年' (may you enjoy longevity like the pines and the cranes, whereby both considered long living in chinese culture) was from shutterstock with paid standard license. p53-dependent and p53-independent activation of autophagy by arf the nad(+)-mitophagy axis in healthy longevity and in artificial intelligence-based clinical applications sex differences in lifespan isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) metformin as a tool to target aging microbiological changes of the ageing oral cavity oral health and implant therapy in parkinson's patients: review intergenerational help and care in europe providing informal care in a changing society particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the dementia with features of alzheimer's disease and hiv-associated dementia in an elderly man with aids clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline cadenza china's new demographic reality: learning from the 2010 census from discoveries in ageing research to therapeutics for healthy ageing aids database infectious diseases epidemiology of alzheimer's disease and other forms of dementia in china associations of dietary protein intake on subsequent decline in muscle mass and physical functions over four years in ambulant older chinese people trends in hip fracture incidence and mortality in chinese population from hong kong 2001-09 trends in ischaemic heart disease hospitalisation and case fatality in the hong kong chinese population 2000-2009: a secondary analysis temporal and spatial distribution characteristics of hiv-infected and aids patients aged 15 years and over in china from meta-inflammageing at the crossroad of geroscience disparities in hiv and syphilis prevalence and risk factors between older male clients with and without steady sex partners in southwestern rural china sarcopenia in asia: consensus report of the asian working group for sarcopenia epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study current situation and progress toward the 2030 health-related sustainable development goals in china: a systematic analysis cancer statistics in china intergenerational social support and the psychological well-being of older parents in china chronic low-grade inflammatory phenotype (clip) and senescent immune dysregulation launch of the health-care reform plan in china advance care planning in chinese seniors: cultural perspectives prevention of disability in the frail chinese older population senescent intimal foam cells are deleterious at all stages of atherosclerosis proportion of people aged 50 years and older in hiv-infected cases doubled in 9 years age-standardised death rates by leading causes of death flu express number of deaths by leading causes of death by sex by exceptional longevity does not result in excessive levels of disability national and local government policies at sarcopenia: european consensus on definition and diagnosis: report of the european working group on sarcopenia in older people sarcopenia: revised european consensus on definition and diagnosis prevalence of and interventions for sarcopenia in ageing adults: a systematic review effects of intermittent fasting on health, aging, and disease porphyromonas gingivalis in alzheimer's disease brains: evidence for disease causation and treatment with small-molecule inhibitors population ageing and the development of social care service systems for older persons in china the 2015 quality of death indexranking palliative care across the worldan economist intelligence unit study, commissioned by the lien foundationkey findings infographic (the economist intelligence unit sleep duration and the risk of dementia: a systematic review and meta-analysis of prospective cohort studies mitophagy and nad(+) inhibit alzheimer disease mitophagy inhibits amyloid-beta and tau pathology and reverses cognitive deficits in models of alzheimer's disease nuclear dna damage signalling to mitochondria in ageing a research agenda for aging in china in the 21st century report on square-dancing in china influenza-associated mortality in temperate and subtropical chinese cities inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty district health centres machine learning approaches to personalize early prediction of asthma exacerbations frailty asia-china conference from hype to reality: data science enabling personalized medicine research in health policy making in china: out-of-pocket payments in healthy china 2030 global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the global burden of disease study targeting nad(+) in translational research to relieve diseases and conditions of metabolic stress and ageing epidemiological characteristics and trends of chinese syphilis from loneliness in older adults living with hiv the increasing burden and complexity of multi-morbidity and polypharmacy in geriatric hiv patients: a cross sectional study of people aged 65 -74 years and more than 75 years program of all-inclusive care for the elderly (pace): integrating health and social care since 1973 trends in cardiovascular disease mortality among persons with hiv in acarbose improves health and lifespan in aging het3 mice rapamycin fed late in life extends lifespan in genetically heterogeneous mice annual report of notifiable diseases statistical yearbook prevalence and risk factors for frailty among community-dwelling older people in china: a systematic review and meta-analysis hiv and aging in mainland china: implications for control and prevention research basic and translational aging research in china: present and future interventions for lower extremity peripheral artery disease centenarians: the older you get, the healthier you have been association of demographic, lifestyle factors and serum biomarkers with hypertension in elderly chinese people diabetes in china: epidemiology and genetic risk factors and their clinical utility in personalized medication who framework convention on tobacco control in china: barriers, challenges and recommendations vitamin d3 activates the autolysosomal degradation function against helicobacter pylori through the pdia3 receptor in gastric epithelial cells association between sleep duration and sarcopenia among community-dwelling older adults: a cross-sectional study psychometric properties of the chinese mainland version of the palliative care spiritual care competency scale (pcsccs-m) in nursing: a cross-sectional study analysis on the problems and countermeasures of the needs assessment of long-term care insurance in china clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet prevalence of mental disorders in china: a cross-sectional epidemiological study compression of morbidity is observed across cohorts with exceptional longevity local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment the prevalence of dementia in urban and rural areas of china dementia in china: epidemiology, clinical management, and research advances analysis of risk factors for male sexual dysfunction senolytics in idiopathic pulmonary fibrosis: results from a first-in-human, open-label effects of whey protein nutritional supplement on muscle function among community-dwelling frail older people: a multicenter study in china attitudes toward advance directives among patients and their family members in china intergenerational solidarity in families: interplay between the family and the state superior sensory, motor, and cognitive performance in elderly individuals with multi-year dancing activities alzheimer's disease: cellular and molecular mechanisms defibrillator implantation in patients with nonischemic systolic heart failure treating depression with tai chi: state of the art and future perspectives population aging: impacts and policy imperatives metabolic abnormalities, antiretroviral therapy and cardiovascular disease in elderly patients with hiv reduced grid-cell-like representations in adults at genetic risk for alzheimer's disease behavioural risk factors in mid-life associated with successful ageing, disability, dementia and frailty in later life: a rapid systematic review the aging mouth: differentiating normal aging from disease nad+ in brain ageing and neurodegenerative disorders treatment delay and fatal outcomes of pulmonary tuberculosis in advanced age: a retrospective nationwide cohort study transitions in frailty states among community-living older adults and their associated factors effect of an integrated payment system on the direct economic burden and readmission of rural cerebral infarction inpatients: evidence from anhui, china the prevalence, incidence, management and risks of atrial fibrillation in an elderly chinese population: a prospective study healthy china 2030: an opportunity for tobacco control the primary health-care system in china catastrophic health expenditure and rural household impoverishment in china: what role does the new cooperative health insurance scheme play? development of medical informatics in china over the past 30 years from a conference perspective and a sino-american comparison personal and social environmental correlates of square dancing habits in chinese middle-aged and older adults living in communities similarities and differences in health-related behavior clustering among older adults in eastern and western countries: a latent class analysis of global aging cohorts multiyear square dancing is associated with superior mental processing capacity but not memory in middle-aged and older chinese women: a cross-sectional propensity score matching analysis age-specific associations between hiv infection and carotid artery intima-media thickness in china: a cross-sectional evaluation of baseline data from the chart cohort long-term effects of ambient pm2.5 on hypertension and blood pressure and attributable risk among older chinese adults apoe4 causes widespread molecular and cellular alterations associated with alzheimer's disease phenotypes in human ipsc-derived brain cell types recapitulation of premature ageing with ipscs from hutchinson-gilford progeria syndrome mtor at the nexus of nutrition, growth, ageing and disease emerging hiv epidemic among older adults in nanning, china construction of palliative care training contents in china: a delphi study comparative research of middel-aged women fitness effects of taiji quan and sqaure dance intergenerational transfers and informal care for disabled elderly persons in china: evidence from charls the reform of the medical welfare system and health equity for the elderly in china: a study in zhejiang outcome-based health equity across different social health insurance schemes for the elderly in china prevalence of multiple chronic conditions among medicare beneficiaries, united states the hallmarks of aging ageing, dental caries and periodontal diseases a budget proposal for china's public long-term care policy the 4th national oral health survey in the mainland of china: background and methodology crossing the river by feeling for the stones: contesting models of marketization and the development of china's long-term care services a comparative study on the needs assessment and rating assessment of long-term care insurance at home and abroad: taking qingdao and japan as examples (in chinese) realigning the incentive system for china's primary healthcare providers value-based contracting innovated medicare advantage healthcare delivery and improved survival an assessment of progress towards universal health coverage in brazil a journey without maps-understanding the costs of caring for dependent suggestions on further expanding the supply and promoting the consumption of elderly care services the 1st, 2nd 3rd, 4th circular of the central government on supporting the pilot areas of home-based and community-based elderly care service reform what can we learn from china's health system reform? trends in access to health services and financial protection in china between 2003 and 2011: a cross-sectional study ministry of human resources and social security of the people's republic of china integrated care: enhancing the role of the primary healthcare professional in preventing functional decline: a systematic review tuberculosis among the elderly in tanzania: disease presentation and initial response to treatment china statistical yearbook the guiding opinions on advancing the development of age-friendly livable environment by the state council healthcare reform office, national health and family planning commission, national development reform commission healthy china action plan (2019-2030) by the national health commission of the people's republic of china national health commission of the people's republic of china the state council opinions on promoting the development of elderly care services center for health statistics and information, nhfpc. an analysis report of national health services survey in china national health and family planning commission. national basic public health services guideline official paper: implementation of national basic public health services in 2018, by the national health and family planning commission of the people's republic of china the twilight of immunity: emerging concepts in aging of the immune system hospice and palliative care in mainland china: history, current status and challenges trends in mortality from human immunodeficiency virus infection, 1984-2016: an autopsy-based study how health research will support china's ageing population health at a glance: oecd indicators adverse events associated with mtor inhibitors the prevalence, awareness, treatment and control of dyslipidemia among adults in china dementia studies in chinese populations china's demographic history and future challenges the evolution of seasonal influenza viruses china brain project: basic neuroscience, brain diseases, and brain-inspired computing the chinese government: healthy china dementia incidence and mortality in middle-income countries, and associations with indicators of cognitive reserve: a 10/66 dementia research group populationbased cohort study deep biomarkers of human aging: application of deep neural networks to biomarker development a survey of aids cognitive attitudes among middle-aged and elderly men in high-risk areas why is old-care industry unable to be prosperous? (in chinese) tuberculosis and aging_ a global health problem lifestyle factors related to mortality and survival: a mini-review development of pharmacotherapies for the treatment of sarcopenia gendered support to older parents: do welfare states matter? biomarker signatures of aging comparison of the clinical features and outcomes in two age-groups of elderly patients with atrial fibrillation oral health status of chinese residents and suggestions for prevention and treatment strategies estimating the future burden of cardiovascular disease and the value of lipid and blood pressure control therapies in china availability, cost, and prescription patterns of antihypertensive medications in primary healthcare in china: a nationwide cross-sectional survey trends in the informal and formal home-care use of older adults in the netherlands between nordic eldercare: weak universalism becoming weaker randomized comparisons of double-dose clopidogrel or adjunctive cilostazol versus standard dual antiplatelet in patients with high posttreatment platelet reactivity: results of the creative trial integrated care for older populations and its implementation facilitators and barriers: a rapid scoping review designing healthcare for the most common chronic condition--multimorbidity dental caries and periodontal diseases in the ageing population: call to action to protect and enhance oral health and well-being as an essential component of healthy ageing -consensus report of group 4 of the joint efp/orca workshop on the boundaries between caries and periodontal diseases prevalence of lipid abnormalities in the united states: the national health and nutrition examination survey nicotinamide riboside is a major nad+ precursor vitamin in cow milk cancer burden with ageing population in urban regions in china: projection on cancer registry data from world health organization promising community-based practices to postpone the need for institutional elderly care: a descriptive study of community developers in estonia. east-west studies exercise, immunity and aging mycobacterium tuberculosis informal care in europe: findings from the european social survey (2014) special module on the social determinants of health hivrelated shame and health-related quality of life among older, hiv-positive adults association between depressive symptoms and sarcopenia in older chinese community-dwelling individuals gray matter age prediction as a biomarker for risk of dementia use and prescription of antibiotics in primary healthcare settings in china prevalence and ethnic pattern of diabetes and prediabetes in china in 2013 melancholy or mahjong? diversity, frequency, type, and rural-urban divide of social participation and depression in middle-and old-aged chinese: a fixed-effects analysis sodium oligomannate therapeutically remodels gut microbiota and suppresses gut bacterial amino acids-shaped neuroinflammation to inhibit alzheimer's disease progression people-centred integrated care in urban china epidemiological characteristics of legally reported sexually transmitted diseases in china from international development of integrated care systems status of hypertension in china: results from the china hypertension survey world bank and world health organization. healthy china: deepening health reform in china: building high-quality and value-based service delivery prevalence of kidney damage in chinese elderly: a large-scale population-based study control smcfd, prevention. study on global ageing and adult health (sage) wave 1: china national report china country assessment report on ageing and health. geneva: world health organization world report on ageing and health world report on ageing and health (geneva: world health organization) who guidelines on tuberculosis epidemiological estimates for viral hepatitis in the western pacific global tuberculosis report 2019 the icope package of tools risk reduction of cognitive decline and dementia: who guidelines (geneva: world health organization who report on the global tobacco epidemic, 2019: offer help to quit tobacco use (geneva: world health organization) computational biology for ageing long-term hepatitis b infection in a scalable hepatic co-culture system satellite-based estimates of long-term exposure to fine particles and association with mortality in elderly hong kong residents the well-being of community-dwelling near-centenarians and centenarians in hong kong: a qualitative study challenges of population ageing: putting frailty as a cornerstone of health and social care systems combating frailty and sarcopenia in aging populations: switching to a more positive paradigm elder-friendly service delivery models validating the sarc-f: a suitable community screening tool for sarcopenia? defining sarcopenia in terms of incident adverse outcomes telomere length is associated with decline in grip strength in older persons aged 65 years and over active ageing: a policy framework prevalence and correlates of frailty among community-dwelling chinese older adults: the china health and retirement longitudinal study progress of intervention work for high-risk groups in 2018 and work requirements for 2019 associations between quality of relationships and life satisfaction of older mothers in estonia suicide prevention in old age in china 2002-2017: the linkage to the mipaa suicide among older people in relation to their subjective and objective well-being in different european regions effect of aging on periodontal inflammation, microbial colonization, and disease susceptibility prevalence of dementia in mainland china, hong kong and taiwan: an updated systematic review and meta-analysis qualitative study on perceived dignity of cancer patients undergoing chemotherapy in china hiv/aids epidemic among older adults in china during 2005-2012: results from trend and spatial analysis 10 years of china's comprehensive health reform: a systems perspective factors associated with oral health service utilization among adults and older adults in china senolytics improve physical function and increase lifespan in old age sarcopenia in community-dwelling oldest old is associated with disability and poor physical function. the journal of nutrition, health & aging incidence of frailty among community-dwelling older adults: a nationally representative profile in china diabetes mellitus and risks of cognitive impairment and dementia: a systematic review and meta-analysis of 144 prospective studies china national geriatrics clinical medical research center-clinical and scientific research collaboration alliance established in beijing opening of the national clinical research center for geriatric disorders (xuanwu hospital site introduciton of the national clinical research center for geriatric demographic and psychosocial correlates of sexual activity in older chinese people financing institutional long-term care for the elderly in china: a policy evaluation of new models impact of frailty on mortality and hospitalization in chronic heart failure: a systematic review and meta-analysis risk factors and incidence of stroke and mace in chinese atrial fibrillation patients presenting to emergency departments: a national wide database analysis prevalence and patterns of multimorbidity in a nationally representative sample of older chinese: results from charls an investment for the celebration of aging 10 years of health-care reform in china: progress and gaps in universal health coverage early appraisal of china's huge and complex health-care reforms nad(+) intermediates: the biology and therapeutic potential of nmn and nr prevalence and correlates of depressive symptoms in chinese older adults: a population-based study geriatric medicine in china: the past, present, and future a comparison of health expectancies over 10 years: implications for elderly service needs in hong kong incremental predictive value of sarcopenia for incident fracture in an elderly chinese cohort: results from the osteoporotic fractures in men (mros) study effects of a multi-component frailty prevention program in pre-frail community-dwelling older persons: a randomized controlled trial trajectories of frailty among chinese older people in hong kong between 2001 and 2012: an age-period-cohort analysis frailty and its contributory factors in older adults: a comparison of two asian regions (hong kong and taiwan) is neighborhood green space associated with less frailty? evidence from the mr. and ms. os (hong kong) study incidence of dementia and subtypes: a cohort study in four regions in china epidemiological characteristics of condyloma acuminata in china's std surveillance sites from demographics, phenotypic health characteristics and genetic analysis of centenarians in china survival, disabilities in activities of daily living, and physical and cognitive functioning among the oldest-old in china: a cohort study bridging the gp gap: nurse practitioners in china gender and marital status differences in depressive symptoms among elderly adults: the roles of family support and friend support prevalence and risk factors of active pulmonary tuberculosis among elderly people in china: a population based cross-sectional study baseline characteristics and management of patients with atrial fibrillation/flutter in the emergency department: results of a prospective, multicentre registry in china senolytic therapy alleviates abeta-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an alzheimer's disease model an investigation into health informatics and related standards in china management, and outcomes of patients hospitalized for heart failure in china: results from the china heart failure (china-hf) registry epidemiology of cardiovascular disease in china: current features and implications in-hospital outcomes of dual loading antiplatelet therapy in patients 75 years and older with acute coronary syndrome undergoing percutaneous coronary intervention: findings from the ccc-acs (improving care for cardiovascular disease in china-acute coronary syndrome) project percutaneous coronary intervention in patients with acute coronary syndrome in chinese military hospitals, 2011-2014: a retrospective observational study of a national registry mortality, morbidity, and risk factors in china and its provinces, 1990-2017: a systematic analysis for the global burden of disease study disease burden of copd in china: a systematic review effects of exercise and nutrition supplementation in community-dwelling older chinese people with sarcopenia: a randomized controlled trial china's policy experimentation on long-term care insurance: implications for access for more details). c-d. demographics showing the major diseases affected to (c), and death in (d) the elderly in china between 1990 (yellow) and 2017 (green). data source: global burden of disease study figure 2. morbidity and mortality of selected diseases by age in (e) disability-adjusted life years (dalys) (millions) in the elderly (65+) in china in 2017. (per 10000 people) of viral hepatitis, pulmonary tuberculosis (p.tb), influenza, hiv infection and aids from 2004 to 2016 were extracted and presented as general (open) or aged (65 years or older, closed) population ), the 6 th population census of china (2010) and projection (2020-2050) of china's national bureau of statistics. c. women account for 75% of the total number of centenarians in china and the proportion of rural centenarians is far higher than that of urban area. data source: china's 2010 population census, excluding hong kong, macau and taiwan area. the map was made by r. d. geographical distribution of the relative number of centenarians. the proportion of centenarians in china's total population (centenarian ratio) has a significant regional imbalance the authors acknowledge the valuable work of the many investigators whose published articles they were unable to cite owing to space limitations. the authors thank dr. vilhelm bohr at the nia and university of copenhagen for reading of the manuscript. e.f. key: cord-252167-2zxw3sh8 authors: poteat, tonia c; baral, stefan title: celebrating the struggle against homophobia, transphobia and biphobia as central to ending hiv transmission by 2030 date: 2020-05-14 journal: j int aids soc doi: 10.1002/jia2.25532 sha: doc_id: 252167 cord_uid: 2zxw3sh8 nan sixteen years ago, louis georges tin, advocate for black and lesbian, gay, bisexual, transgender and intersex (lgbti) rights, launched an appeal for universal recognition of may 17 as the international day against homophobia to honour the world health organization's decision to remove homosexuality from their list of mental disorders in 1990 [1] . over time, the movement has grown and made explicit its relevance to all people with diverse sexual orientations, gender identities, gender expression and sex characteristics. may 17 is now commemorated as the international day against homophobia, transphobia and biphobia (idahot) in more than 130 countries, including 37 where same-sex acts are illegal [2] . this year's theme "breaking the silence" calls for an end to the stigma and violence that drive shame, increase hiv vulnerability, and hinder access to and uptake of hiv prevention and care services [3] . as the world grapples with the covid-19 pandemic, the novel coronavirus is highlighting existing disparities with disproportionate deaths among populations made most vulnerable by structural violence and discrimination, including lgbti people. at the same time, some state actors are misusing emergency powers enacted to fight the pandemic to target lgbti communities with structural and physical violence [4] . this year's idahot theme calls on us to speak out against this violence, not only because freedom from violence is a universal human right but also because doing so is essential to the goal of ending hiv as a pandemic by 2030 [5, 6] . in 2020, the status of protective and punitive laws affecting the sexual, reproductive and human rights of lgbti communities around the world is dynamic [7] . some countries have decriminalized same-sex practices, whereas others have reinforced criminalization; some countries have increased restrictions on organizations serving lgbti groups; others have increased constitutional rights, whereas others have further restricted them; and finally, some countries have enacted specific protections for lgbti people from discrimination and from sexual orientation and gender identity change efforts or "conversion therapy, " whereas others have backtracked those same protections. while more resource-constrained settings tend to have more restrictive legal contexts secondary to colonialism and ongoing neocolonialism, notably, there is no clear division by geography, income level or development index that separates countries where lgbti rights are advancing and those where there have been setbacks. moreover, there is no connection between rights contexts for lgbti individuals and whether the country in which they are a citizen are signatories of the un declaration of human rights, which was intended to define fundamental human rights to be universally protected [8] . to clarify international principles specific to sexual orientation and gender identity, the yogyakarta principles were originally developed in 2006 [9, 10] . the goal of these principles was to further articulate legal standards to protect the health and wellbeing of lgbti communities around the world. the yogyakarta principles were updated in 2017 with what was called the yogyakarta principles plus 10 (yp+10), which focused on principles and obligations specific to sexual orientation, gender identity, gender expression and sex characteristics [11] . yp+10 reinforced that all lgbti people have a right to simply live free of criminalization for who they are and who they love. importantly, yp+10 reinforced the links between these rights and health consistent with the who definition as more than the absence of disease but attaining physical and mental wellbeing. there are data supporting the harmful effects of stigmas against lgbti people at every step of the cascade limiting achieving coverage of evidence-based hiv prevention strategies, testing approaches, linkage to treatment for those living with hiv and sustained viral suppression. indeed, many of the innovations in hiv were developed to specifically mitigate the harmful effects of these intersecting stigmas including implementation strategies such as hiv self-testing, app-based surveillance and service delivery strategies, and outreach-based linkage and treatment services. and while many of those innovations have been successful in improving outcomes, they do not change the underlying constructs which reinforce individual hiv risks. often, we see the language of non-heteronormative practices such as anal sex as being "risky. " however, consensual anal sex is a healthy sexual practice. "risk" is introduced in the context of serodifferent sexual partners having condomless anal sex where one is viraemic. and the introduction of that risk is a failure at many levels including limited capacity in health centres to provide sex-positive education for clients about anal sex, limited lgbti-focused community-based organizations to support outreach, limited availability of condom and appropriate lubricant choices, and of course intersecting stigmas in health centres challenging testing and linkage to treatment for those living with hiv. the international community tends to focus stigma mitigation efforts on the last piece of this-stigma in the health centre [12] . indeed, to truly overcome disproportionate hiv-related risks among lgbti communities around the world means a fundamental shift in how we conceptualize sex and love from risk mitigation to celebration. combatting stigma, discrimination and violence against sexual and gender minorities requires action at multiple levels and across many sectors. laws and policies that penalize same-sex relationships and consensual sexual practices, prohibit marriage between consenting adults, and criminalize gender identity and expression should be repealed in keeping with universal human rights standards. sexual orientation and gender identity change efforts have been shown to cause significant harm, especially for lgbti youth, and should not be sanctioned by any accrediting body or government [13, 14] . anti-stigma interventions using participatory theatre, professional training and other modalities have been effective in reducing interpersonal stigma and healthcare stigma that underpin experiences of lgbti violence [15] [16] [17] . ensuring that healthcare workers are trained to provide welcoming and competent care for lgbt people is key to engaging key populations effectively in hiv prevention and care and to promoting an effective human rights-based response to the covid-19 pandemic. importantly, researchers and implementers must continue to reach and engage lgbti communities as partners in efforts to develop, test and implement acceptable, effective stigma-reducing, anti-violence interventions needed to truly end new hiv infections by 2030. international day against homophobia, transphobia, and biphobia condemns misuse and abuse of emergency powers to target marginalized and vulnerable populations abuse and discrimination: key factors militating against control of hiv/aids among the lgbti sector ending violence and discrimination against lesbian, gay, bisexual, transgender, and intersex people trans and intersex association, mendos lr. state-sponsored homophobia 2019. geneva: ilga2019 universal declaration of human rights statesponsored homophobia. a world survey of laws prohibiting same sex activity between consenting adults yogyakarta principles: applying existing human rights norms to sexual orientation and gender identity characterizing cross-culturally relevant metrics of stigma among men who have sex with men across eight sub-saharan african countries and the united states association between recalled exposure to gender identity conversion efforts and psychological distress and suicide attempts among transgender adults parent-initiated sexual orientation change efforts with lgbt adolescents: implications for young adult mental health and adjustment implementation science and stigma reduction interventions in low-and middle-income countries: a systematic review changing hearts and minds: results from a multi-country gender and sexual diversity training exploring the potential of participatory theatre to reduce stigma and promote health equity for lesbian, gay, bisexual, and transgender (lgbt) people in swaziland and lesotho key: cord-022168-qautse9a authors: liu, li; morrow, matthew p.; bagarazzi, mark title: clinical use of dna vaccines date: 2017-07-25 journal: handbook of electroporation doi: 10.1007/978-3-319-32886-7_106 sha: doc_id: 22168 cord_uid: qautse9a owing to their unique advantages in simplicity, safety, scalability, and possibility of repeated administrations, dna vaccines represent an appealing and competitive immunization approach for a wide array of conditions, including but not limited to infectious diseases and cancer immunotherapy. despite the exciting efficacy observed in preclinical studies, dna vaccines have faced challenges in inducing strong immune responses in humans. this unexpected poor immunogenicity has severely hampered the translation of dna vaccines from investigational medications to licensed products. to overcome this obstacle, tremendous efforts have been made to improve antigen expression and enhance immunogenicity. among these endeavors, in vivo dna electroporation (ep) has proved to be a breakthrough technology capable of mediating efficient dna uptake and resulting in enhanced antigen expression and vaccine immunogenicity. ep-mediated dna delivery has become one of the major platforms used in clinical trials to evaluate dna vaccines in humans. in this chapter, in addition to ep delivery, other progress made in dna vaccine development including plasmid optimization, antigen design, and immunologic adjuvants is also reviewed. finally, the use of dna vaccines in the context of clinical trials for infectious diseases and cancer immunotherapy is summarized. specifically, the strategies that allow dna vaccines to overcome antigenic diversity for viral infection and break immune tolerance for cancer therapy are explored. based on the advantages of dna vaccines and the immense progress, led by the electroporation-mediated vaccine delivery, dna vaccines appear to have the potential to fundamentally transform the vaccine field, providing important benefits for preventing and curing diseases. in 1990, wolff and colleagues first demonstrated that long-term gene expression could be achieved simply by direct injection of plasmid dna into mouse skeletal muscle (wolff et al. 1990 ). two years later, tang et al. reported that in vivo delivery of plasmid dna encoding human growth hormone (hgh) resulted in the production of detectable levels of hgh in host mice. importantly, the inoculated mice developed anti-hgh antibodies, suggesting the immunological use of dna (tang et al. 1992) . these pioneering observations engendered great enthusiasm for using dna immunization against infectious diseases, as well as in the applications of gene therapy. the immunological potential of dna was confirmed by several independent preclinical studies demonstrating that dna immunization could result in protection from infectious diseases in immunized animals (moss 2009 ). the action of dna immunization involves intramuscular or subcutaneous injection of a dna plasmid encoding the antigen of interest expressed under the control of a eukaryotic promoter. upon administration of the plasmid dna, the in vivo expressed antigen can stimulate the host immune system to elicit a response. as a third generation platform, dna vaccination has distinct advantages over other vaccine approaches. dna vaccines can induce both humoral and cellular immune responses making them distinct from conventional inactivated vaccines and subunit vaccines. in addition, plasmid dna has a number of unmethylated cpg motifs, due to its bacterial origin, that are capable of stimulating innate immune responses via toll-like receptor 9 (tlr9). the ability to activate all arms of the immune system (b cells, t h cells, and cytotoxic t cells) means the dna vaccine platform has the potential for not only prophylactic but also therapeutic applications. unlike the traditional prophylactic vaccines that prevent infections from occurring, therapeutic dna vaccines can be used as an immunotherapy to treat chronic viral infections and cancers by direct killing of latently infected cells and mutated tumor cells. dna vaccines have also been shown to have a benign safety profile. the injected plasmid only encodes a selected gene from a pathogen, eliminating risk of pathogenic infection or reversion to virulence, a safety concern that is associated with live attenuated vaccines. importantly, as plasmid dna vaccines are noninfectious, the elicited immune responses are solely specific to the antigen encoded by the transgene rather than to the plasmid itself. as a result, no anti-dna vector responses have been observed, thereby allowing for repeated vaccine administration without weakening the specific immune response. this feature is critical for the prime and boost regimen, a commonly used approach for enhancing vaccine immunogenicity. another advantage that makes dna vaccines a competitive option is its ease of design and manufacture. the advancement of gene synthesis and recombinant dna technology allows for flexible and rapid alterations of the expressed immunogen. large-scale production of plasmids can be completed within a short period of time, making dna vaccines particularly suitable for responding to pandemic outbreaks. finally, dna vaccines are more stable and resistant to temperature extremes than other vaccine forms which are of great benefit for storage, transport, and distribution especially in resource-limited regions. despite all the positive characteristics and the superb efficacy observed in preclinical studies, dna vaccines have been slow to demonstrate similar success in humans. the unforeseen poor immunogenicity observed in most early clinical trials was disappointing and poses a formidable challenge for dna vaccine licensure. however, several dna vaccines have been approved for animal health. the first one, developed by fort dodge laboratories against west nile virus infection in horses was approved by the us department of agriculture (usda) in 2005. a second dna vaccine (apex-ihn, novartis animal health) was licensed in canada for prevention of hematopoietic necrosis virus infection in farmed salmon and trout. in 2010, a therapeutic cancer vaccine (oncept, merial) designed to treat dogs with melanoma was approved by the usda. finally, in 2008, an injectable dna plasmid (lifetide sw 5, vgx animal health) encoding porcine growth hormone releasing hormone (ghrh) was licensed in australia as a gene therapy for use in breeding age sows to increase the number of piglets in litters. the approval of these plasmids as commercial products in veterinary practice is very encouraging and sheds lights on human dna vaccine development. for example, the licensed therapeutic vaccine for canine malignant melanoma was developed by using human tyrosinase (a melanoma associated antigen) as the immunogen to immunize dogs. the human tyrosinase elicited a strong immune response against dog melanoma and significantly prolonged the lives of dogs with advanced disease (bergman et al. 2003) . the success of this vaccine supported the concept of using a xenogeneic protein to break central immune tolerance for cancer vaccine development (liu 2011) . additional insight gained from the success of dna vaccines in veterinary practice is that the dna scaling-up might not be a potential hindrance for dna vaccine being effective in humans. it has been extrapolated that an impractically high dose of dna vaccine would be required for use in human subjects in order to reproduce the vaccine efficacy obtained in mice (tregoning and kinnear 2014) . however, the clinical benefits observed in large animals, including horses and pigs, suggest that the size of humans may not be the reason for the various disappointing clinical results (liu 2011) . in light of the aforementioned advantages of dna vaccines and encouraged by their success in veterinary medicine, great efforts have been made to translate dna immunization into the clinic, with novel strategies being explored to enhance immunogenicity. since the first human study in 1998 (calarota et al. 1998) , numerous clinical trials with dna vaccines targeting a wide spectrum of diseases have been performed or are currently underway. in the following sections, various aspects related to dna vaccine development including its working mechanisms, route of delivery, advances being made in optimizing dna vaccine immunogenicity, and their usage in the applications of infectious diseases and cancer treatment will be addressed. upon intramuscular or intradermal injection, plasmid dna is internalized by myocytes or keratinocytes located at the site of vaccine administration. the plasmid encoded antigen is then expressed intracellularly using host cell machinery. however, since myocytes and keratinocytes are not professional antigen presenting cells (apc) and do not express co-stimulatory molecules, they are unable to productively stimulate cytotoxic t lymphocytes (ctl) activation. in order to prime a cellular response, the intracellularly produced antigens must be released into extracellular matrix either by secretion or by cell lysis. the released antigens are then engulfed by apcs and loaded onto major histocompatibility complex (mhc) class i molecules leading to activation of cd8 + t cell. this uptake mechanism is different from that which occurs with exogenous proteins and is thus termed as cross-priming (liu 2011) . dna plasmids can also directly transfect apcs that have migrated to the injection site. after being expressed in apcs, the antigens are cleaved to peptides and uploaded onto mhc i or ii molecules, leading to activation of both cd8 + ctls and cd4 + t h cells. additionally, antigens released from myocytes and keratinocytes can be captured by b lymphocytes leading to activation of humoral immunity. in addition to the stimulation of the adaptive immune system, dna plasmids can also trigger activation of innate immunity. plasmids derived from bacteria contain hypomethylated cpg dinucleotides motifs, which are immunostimulatory and rarely seen in eukaryotes. the cpg motifs bind to toll-like receptor 9 (tlr9), a transmembrane protein found on a number of immune cells including dendritic cells (dc), b cells, and natural killer (nk) cells. activation of tlr9 leads to a cascade of pro-inflammatory responses and results in the production of various cytokines including type i interferon (ifn), interleukin (il)-12, il-18, and tumor necrosis factor-alpha (tnf-α). in addition to cpg-tlr9 pathway, the double-stranded structure of plasmids can be detected by dna sensors in cytosol, which triggers activation of the stimulator of interferon genes (sting)/tank-binding kinase 1 (tbk1) pathway, leading to type i interferon (ifn) production. historically, various administration routes including intramuscular, intradermal, subcutaneous, intranasal, vaginal, oral, and intravenous route have been tested (liu 2011; tregoning and kinnear 2014) in order to elicit a desired immune response from dna immunization. the selection of the appropriate delivery method is dependent on multiple factors including optimum expression, immunogenicity, feasibility, and tolerability. among the considered delivery approaches, intramuscular (i.m.) injection is the principal route of choice for dna vaccination due to the fact that skeletal muscle is readily accessible and highly effective in taking up dna. furthermore, myocytes are energy rich and postmitotic cells capable of sustaining long-term transgene expression for eliciting durable immunity (aurisicchio et al. 2013; tregoning and kinnear 2014) . in addition to i.m. injection, intradermal (i.d.) delivery is also considered to be an effective pathway for vaccination not only because of its greater ease of accessibility but also the prominent presence of large amount of immune cells in the dermis including dendritic cells, langerhans cells, and migrating lymphocytes. both i.m. and i.d. are two major administration routes used in preclinical studies and clinical trials for dna delivery. some studies suggested that the nature of the immune response could be driven by the vaccine administration route. for example, due to the enrichment of antigen presenting cells in skin, the i.d. route is believed to preferentially induce cellular immune responses as a result of direct transfection of apcs. on the other hand, i.m. injection equally induces both humoral and cellular immunity via direct transfection and by the crosspriming mechanism. however, in a recent clinical trial where an hiv-specific dna vaccine was delivered via either an i.m., or i.d. route, no differences in induced immune responses were observed (enama et al. 2014 ). after administration, the plasmid dna must successfully overcome a series of barriers before mounting the desired immune response. compared to viral vectors, uptake of naked dna by somatic cells is inefficient; most of the i.m.-injected dna does not actually transfect cells (liu 2011) . after entering the cell, plasmid dna needs to translocate to the nucleus where dna transcription occurs. however, the efficiency of intracellular movement is very low, with less than 0.1% of plasmid dna that enters the cytosol is eventually transcribed (tregoning and kinnear 2014) . moreover, prior to arrival at the nucleus, dna in the cytosol is subject to degradation by nuclease digestion. finally, during the process of protein synthesis, mrna translation efficiency can also be modulated by factors such as the presence of a kozak sequence and codon usage. to overcome these obstacles, several approaches focusing on augmenting dna uptake, maximizing protein expression, and enhancing antigen immunogenicity have been developed and tested in clinical trials. several dna delivery methods including gene gun, jet injection, electroporation (ep), cationic polymers, and liposomes have been used in clinical trials. given that the inclusion of extra chemical components may delay the licensure of the vaccine due to concerns over the inflammatory profile of the agents, the device-mediated delivery is considered to be a more universal and preferred approach for enhancing dna uptake (tregoning and kinnear 2014) . among the device-mediated approaches, ep has the highest transfection efficiency and has achieved the most remarkable progress in dna vaccine delivery (aurisicchio et al. 2013; best et al. 2009 ). the ep technology was initially devised for in vitro transfection (see also ▶ chap. 23, "gene delivery by electroporation in vitro: mechanisms"). the mechanism of ep involves using controlled electric pulses to open transient pores on cell membrane through which the negatively charged dna molecules gain access to the cytoplasm. the cell membrane is permeabilized for only a short period of time and rapidly closes after electrical treatment terminates, returning the cell to its original state. ep has been proven to be a very powerful technology, resulting in 100-1000-fold increases in plasmid uptake and subsequent antigen expression compared to naked dna vaccines (aurisicchio et al. 2013 ). furthermore, ep has the additional benefit of causing tissue damage that can lead to local inflammation and release of cytokines (fioretti et al. 2010) . this adjuvant effect of ep consequently promotes the induction of innate and adaptive immune responses, potentiating the immunogenicity of the delivered dna. the ep technology has been safe and well tolerated (bagarazzi et al. 2012) with the most common minor side effects observed in clinical trials being local injection-site pain and discomfort associated with short muscle contraction triggered by electric pulses. it is believed that ep represents the most promising technology in dna delivery (aurisicchio et al. 2013) . recently, the application of i.m. injection followed by ep delivery has become one of the major platforms used in clinical trials to evaluate dna vaccines in humans. however, it should be noted that ep technology has intrinsic limitations. the ep approach requires penetration with several sharp needle-like electrodes into the subject's body followed by introduction of transient electric pulses. as a result, the ep procedure is less convenient, more invasive, and sometimes intimidating, potentially reducing patient compliance compared to simple i.m. injection. furthermore, the ep procedure demands developing practical and affordable devices and providing adequately trained health professionals to administer the vaccine. the immune responses to dna vaccines have been shown to be dependent on antigen expression (tregoning and kinnear 2014) . therefore, optimizing antigen expression is key to improving vaccine performance. plasmid codon optimization is one such approach to augment expression. it refers to changing the antigen coding sequence, via synonymous substitutions, from the wild type to a selected sequence that has the most abundant trnas in the cytosol of transfected cells. through codon optimization, adverse rare codons are avoided and secondary structures in the mrna are minimized, therefore, protein expression can be increased severalfold. in addition to codon optimization, various manipulations of the dna sequence can be performed to modulate antigen intracellular processing and immune response priming. for example, adding nuclear localization signals to the plasmid helps direct dna to the nucleus for transcription. for the purpose of introducing cellular immunity, ubiquitin coding sequence can be incorporated in the dna vaccine to enhance protein degradation and antigen peptide production in the proteasome (leifert et al. 2004 ). if humoral immunity is desired, the antigen coding region can be modified by adding a leader sequence capable of guiding the expressed antigen so that it undergoes proper folding in the endoplasmic reticulum (er), export through the secretory pathway, and release into extracellular matrix for further presentation to b lymphocytes and apcs (fioretti et al. 2010 ). due to its bacterial origin, plasmid dna carries a naturally occurring adjuvant. co-injection of antigen-encoding plasmid with an empty vector carrying no insert has been shown to induce stronger immune responses than using the antigen-encoding plasmid alone (liu 2011) . the enhanced immunogenicity was attributed to the adjuvant effect of the hypomethylated cpg oligodinucleotides that can bind to tlr9, thereby stimulating innate immunity and creating an inflammatory milieu for boosting the adaptive immune response against the encoded antigen (liu 2011) . in an attempt to mimic this adjuvant effect, additional cpg motifs have been used in dna vaccine platforms resulting in enhanced immune responses. alternatively, instead of using incorporated cpg sequence to trigger tlr activation, co-administration of additional plasmid expressing ligands for tlr7/8/9 or their signaling molecules has been shown to improve the immunogenicity of dna vaccines (kwissa et al. 2007) . one disadvantage of using the inflammatory adjuvants is that they boost the vaccine immunogenicity in a sequential fashion, i.e., innate immunity activation followed by adaptive immunity activation. it is possible that the acute inflammation induced by the adjuvant may result in clearance of the transfected cells before sufficient antigen is expressed (tregoning and kinnear 2014) . to avoid this timing issue, genetic adjuvants have been investigated to enhance vaccine immunogenicity. genetic adjuvants refer to immunoregulatory cytokines and proteins encoded by additional plasmids. the most commonly used genetic adjuvants include il-2, il-12, il-15, granulocyte-macrophage colony-stimulating factor (gm-csf), intercellular adhesion molecule 1 (icam-i), cd86, and heat shock protein 70 (hsp70) (fioretti et al. 2010; tregoning and kinnear 2014) . the plasmid encoding the genetic adjuvant is normally delivered by co-injection with the plasmid encoding the vaccine. alternatively, the adjuvant transgene can be tailored and encoded in cis with the antigen coding sequence in the same dna vector as well (fioretti et al. 2010) . in this way, the peak expression of the genetic adjuvant will match antigen expression, leading to a synergistic response. moreover, the effect tends to be more specific than tlr-type adjuvants, which can be reactogenic (tregoning and kinnear 2014) . conventional licensed influenza (flu) vaccines have traditionally been made by growing the recommended seasonal vaccine strains in eggs followed by inactivation or attenuation. the inactivated or attenuated virus in the vaccine promotes an immune response that can prevent infection from identical or very similar viruses. however, due to antigenic diversity, the annually recommended flu vaccine can become ineffective if new strains emerge. in order to avoid frequent composition adjustment for emerging influenza strains, new vaccine platforms capable of inducing a universal immune response against a broad spectrum of influenza strains are greatly needed. viral surface proteins are the primary targets for prophylactic vaccine development, because of their crucial role in initiation of infection and their prominent display on the virion surface. however, due to host immune pressure, these surface proteins are constantly evolving and have high degrees of variability, making it difficult to develop a universal vaccine. thanks to the versatility and ease of rapid alteration of the expressed immunogen, a dna vaccine strategy has great potential in overcoming influenza antigenic diversity. for example, a novel synthetic consensus sequence technology has been developed to broaden the effectiveness of the vaccine to cover variant strains of influenza displaying antigenically altered hemagglutinin (ha). specifically, using the combination of extensive genetic data and a computer algorithm, the gene sequences encoding the ha protein of a large number of circulating influenza strains were analyzed. subsequently, a new arrangement of the dna sequence is created resulting in an average gene sequence for the ha protein. the synthetic consensus dna sequence is substantially similar to but different from those encoded by naturally occurring circulating variants. upon delivery of the synthetic consensus plasmid dna, the in vivo expressed, artificial, and unmatched ha led to generation of neutralizing antibodies capable of recognizing a wide variety of related strains (choo et al. 2010) . the ability of synthetic consensus plasmid-generated h5n1 dna vaccine in cross-protection has been tested in ferrets, in which the sera derived from immunized animals exhibited protective neutralization against all four subclades of h5n1; this protection level had not been attained previously with strain-matched influenza vaccine candidates (choo et al. 2010) . a synthetic consensus dna vaccine targeting h5 and h1 variant strains of influenza has been tested in a phase i clinical trial (nct01405885). it is well known that the vaccine-induced influenza neutralizing antibodies invariably target the globular head region of influenza hemagglutinin (ha) and are largely strain specific. on the other hand, a localized region in the ha stem was found to be structurally conserved among type a influenza viruses; antibodies specific to this region are broadly neutralizing. although the ha stem-specific antibodies have been identified in humans, it has not been possible to specifically elicit them through vaccination. a prime-boost strategy involving dna vaccination has been developed and shown to be able to induce broadly neutralizing antibodies (wei et al. 2010) . specifically, vaccination with plasmid dna encoding h1n1 influenza hemagglutinin and boosting with seasonal vaccine or replication-defective adenovirus 5 vector encoding ha stimulated the production of broadly neutralizing antibodies specific to the conserved stem region. this prime-boost combination increased the neutralization of diverse h1n1 strains dating from 1934 to 2007 and offered cross-protection against divergent h1n1 viruses in mice and ferrets (wei et al. 2010 ). based on these results, the prime-boost strategy was further tested in a human study (nct00776711) to evaluate its ability in stimulating broadly neutralizing antibodies (ledgerwood et al. 2011) . it was found that h5 dna priming with a 24-week monovalent inactivated vaccine (miv) boost interval induced protective hemagglutination inhibition (hai) titers in 81% of individuals. moreover, as compared to another phase i trial (nct01086657), where an h5n1 miv vaccine was used as the priming immunization (homologous prime-boost), dna-miv heterologous prime boost approach demonstrated a stronger ability in eliciting humoral immunity as evidenced by a more than fourfold increase in hai titers (ledgerwood et al. 2011) . the results obtained from these studies suggested that dna vaccine prime followed by a miv or viral vector boost is a promising approach for the development of a universal influenza vaccine for humans. in addition to surface proteins, viral antigens that are inaccessible to antibodies, such as nucleocapsid protein (np) and matrix protein (m), can also be included as the immunogen in dna vaccines. generally, these internal structural proteins are highly conserved among variant strains. ctls raised against these conserved internal proteins can confer cross-protection against diverse related strains. for example, in a preclinical study, a dna vaccine targeting the np from influenza h1n1 strain elicited broadly effective ctls that provided cross-protection against challenge from a h3n2 strain arising three decades later. these findings suggest that immune responses to both surface and internal viral proteins will provide optimal protection. therefore, in order to obtain the greatest efficacy, it is conceptually reasonable to include multiple plasmids in an influenza dna vaccine cocktail to induce antibody to the ha protein and cytotoxic immunity to the viral np and m protein. in the case of influenza, the traditional manufacturing process for the licensed flu vaccines is labor intensive and time consuming, which impedes an imperative response against outbreak crisis. this occurred in 2009, when a swine-origin h1n1 influenza caused a global pandemic. the licensed pandemic vaccine in the form of killed viruses could not be released until a half year after the world health organization (who) announcement of the global outbreak. in contrast, by virtue of its simplicity in manufacturing, an investigational pandemic h1 dna vaccine was quickly made available 3 months earlier than the licensed pandemic vaccine. when tested in a phase i clinical trial (nct00973895), the h1 dna vaccine elicited both humoral and cellular immunity against the pandemic strain. the immune response could be further boosted by the licensed pandemic vaccine when it became available (crank et al. 2015) . the results derived from this phase i clinical trial suggests that dna vaccines are of particular value in a pandemic setting to control the outbreak of emerging infectious disease. since its initial identification in early 1980s, the scientific community has worked diligently towards a safe and efficacious vaccine that renders sterilizing immunity against hiv infection. however, despite the tremendous efforts made during the past more than three decades, a successful hiv vaccine remains elusive. historically, for a large number of infectious diseases, the live attenuated or killed whole virus proved to be the first and second best approach in successful vaccine development (gallo 2005) . however, neither of them can be included in hiv vaccines due to the safety concerns that attenuated hiv would cause aids and one cannot be certain that all virus particles would be completely inactivated. to date, only two subunit vaccines, namely, the hepatitis b virus (hbv) vaccine and the human papillomavirus (hpv) vaccines are approved for clinical use. however, similar success has not been reproduced for other viral diseases including hiv infection. it was noted that the hbv recombinant protein is different from many other viral recombinant proteins in regards to its ability to form particles, a feature that may render hbv recombinant protein super immunogenic (liu 2011) . nevertheless, the field remains far less experienced with the recombinant subunit vaccine approach. hence, it has been reasoned that for a long period of time, the formulation of hiv vaccines might be limited to hiv proteins or their dna form (gallo 2005) . the first clinical trial of dna vaccination against hiv was performed in 1998, in which the infected patients were immunized with plasmid constructs encoding the nef, rev, or tat regulatory protein. the immunization resulted in detectable but transient-specific cytotoxicity in eight of nine patients immunized (calarota et al. 1998 ). since then, clinical studies using improved formulation and delivery strategies have been conducted. in several phase i trials, in an effort to improve the immunogenicity, additional plasmids encoding il12 or il15 were given together with dna vaccine encoding hiv env, gag, and pol proteins. the inclusion of these molecular adjuvants led to an increase in magnitude and breath of cellular and humoral immunity (felber et al. 2014) . recently, several vaccines using dna as a prime in combination with different boosts have also been evaluated. the boosts used in these trials were either viral vectors (recombinant modified vaccinia ankara (rmva), recombinant adenovirus (rade), or recombinant vesicular stomatitis virus (rvsv))expressing env or in the form of a purified recombinant gp120 protein (felber et al. 2014 ). the rationale of using a dna prime and protein boost approach is based on the observations that dna vaccination is suited to eliciting strong cellular immunity, whereas protein immunization preferentially induces antibody response (felber et al. 2014) . from 2003 to 2006, a large-scale efficacy trial carried out in thailand (rv144 trial) used a similar prime-boost schedule, in which the priming dna (hiv env, gag, and pro gene) was introduced by a canarypox vector rather than by a naked dna vaccine. although the dna uptake efficiency mediated by viral vectors could be higher than naked dna, one potential disadvantage of using viral vector is that the preexisting anti-vector immunity may dampen the effectiveness of the priming immunization, an issue that can be perceived from the failed step (also referred to as merck v520-023) trial. nonetheless, until now the rv144 trial is the only one to demonstrate a transient and modest protection benefit. notably, the limited protection appeared to be correlated with the humoral responses. nevertheless, a consensus agreement has been reached in the field that an effective hiv vaccine should afford both humoral and cellular immunity whereby neutralizing antibody blocks virus entry at the sites of infection, and ctls eliminates any infiltrating infection (felber et al. 2014 ). an additional application of dna vaccines for hiv is the potential for use as therapy. using the simian immunodeficiency virus (siv)/macaque model, a therapeutic dna vaccine induced potent cellular responses so that the viral rebound was not observed for many months after cessation of the anti-retroviral therapy (art) (felber et al. 2014) . similarly, up to tenfold reduction in viremia and delay in the kinetics of viral rebound has been observed in some human studies testing the therapeutic dna vaccines (mylvaganam et al. 2015) . however, the clinical benefit of these positive results is still unclear. it has been hypothesized that to optimally boost the preexisting hiv-specific immune responses, therapeutic vaccines need to be combined with other therapeutic approaches such as check point inhibitors (mylvaganam et al. 2015) . the application of dna vaccination is not limited to influenza and hiv infection. to date, there are more than 10 viral diseases for which dna vaccines have entered clinical trials. a list of clinical trials targeting other viral diseases is summarized in table 1 . among these trials, a therapeutic dna vaccine targeting cytomegalovirus (cmv) reactivation in cmv-seropositive hematopoietic stem cell transplant recipients has entered the pivotal phase iii study (nct01877655) in 2013. this vaccine consists of two plasmids expressing cmv antigens glycoprotein b (gb) and phosphoprotein 65 (pp65). in a previous phase ii trial (nct01903928), this cmv dna vaccine provided initial evidence of the safety and revealed significant reduction in viral load and extended period of time before the onset of viremia (smith et al. 2013) . it has been thought for some time that t lymphocytes act as sentinels in recognizing and eliminating continuously arising neoplastic cells. indirect evidence supporting the notion that immunotherapy could be a useful alternate treatment for cancers comes from the observations that the incidence of some malignancies, such as kaposi's sarcoma, non-hodgkin's lymphoma, and invasive cervical cancer, was increased significantly in immunocompromised patients. the presence of cd8 + and t h cells in the tumor microenvironment was also found to be a favorable prognostic factor for many different cancers. moreover, tumor infiltrating t lymphocytes (tils) have been successfully used in adoptive t cell transfer for treatment of metastatic melanoma and resulted in tumor regression. thus, one effective approach for developing cancer immunotherapies is to activate the immune system through vaccination to generate cytotoxic t lymphocytes capable of eliminating tumor cells. dna immunization is of particular interest for developing therapeutic cancer vaccines based on the generation of t cells. ideally, the vaccination-activated t cells should be able to recognize the tumor-associated antigens (taas) and initiate targeted killing of malignant cells. however, most tumors are caused by loss of growth control in normal tissues and thus express self-antigens that are either not recognized by or are only weakly reactive to the immune system. this phenomenon is described as immune tolerance, an inherent mechanism to prevent autoimmunity in which the immune system attacks the host's own cells and tissues. therefore, one key element to improve dna vaccine efficacy is to formulate a vaccine with an immunogenic cancer antigen so that it can prime t cells for immune responses. to date, a large number of cancer antigens have been identified. based on their expression pattern, cancer antigens can be classified into four categories. the first group consists of antigens that are known as cancer-testis (ct) antigens. these antigens are only expressed on particular tumor cells and immune privileged germ line tissues but not on normal adult cells. melanoma-associated antigen (mage) and new york esophageal squamous cell carcinoma 1(ny-eso-1) are examples of this group. the second class of antigens is a large and diverse group that includes any protein found at increased levels in tumors compared with normal healthy cells and tissues. these antigens are termed as overexpressed self-proteins. representative members for this family are prostate-specific membrane antigen (psma), human telomerase reverse transcriptase (htert), and human epidermal growth factor receptor 2 (her2/neu). antigens that are specific to a certain type of tissue and shared between tumors and normal tissue of origin are called differentiation antigens. prototypes in this class include gp100, tyrosinase, and melan-a/melanoma antigen recognized by t cells 1(mart-1), which are molecules expressed by both melanoma and normal melanocytes. finally, antigens that are found exclusively on tumor cells but not on any normal tissues are called tumor-specific antigens (tsa). the unique tumor antigens are generated by somatic mutations as a result of aberrant gene expression. the mutated proteins usually play an important role during the process of cancer development and thus are selected to survive (yang et al. 2014) . because tumor-specific antigens are only present on tumor cells, they are readily recognized by the host immune system and are ideal targets for cancer vaccine development. however, incorporating tumor-specific antigens in the vaccine design is complicated by the fact that the same tumor type can be induced by various distinct point mutations, making it difficult to identify a broadly applicable tumorspecific antigen for a vaccine (yang et al. 2014 ). viral antigens carried by oncogenic viruses such as hpv and hbv represent another class of tumor-specific antigen. for example, the two hpv viral oncoproteins, e6 and e7, are required for the induction and maintenance of cellular transformation and are consistently co-expressed in hpv-associated cancers. their exclusive presence on virus-infected cells and their nature of foreign origin renders vaccines targeting these proteins less vulnerable to the central immune tolerance and the undesired "on-target off-tumor" cytotoxicity. although cancer antigens have the potential to activate the immune system resulting in tumor regression, their application in human cancer vaccination is hampered by their inherent poor immunogenicity. most cancer antigens are tumor related rather than tumor specific. the unresponsiveness to self-antigens caused by immune tolerance is a major roadblock for cancer vaccine development. thus, there is a pressing need for innovative cancer vaccine design so that immune tolerance can be circumvented. one way to induce immunity against a tissue specific differentiation antigen on cancer cells is to vaccinate with a xenogeneic version of antigen. as the name suggests, a xenogeneic antigen is homologous to the cancer antigen but originates from a species foreign to the host. xenogeneic dna vaccines incorporate plasmid dna encoding for a protein homologous between the two species. ideally, xenoantigens should be sufficiently different from self-antigens in order to be immunogenic; they must, on the other hand, also preserve an optimal homology range with self-proteins to secure the cross-reactivity of the induced immune responses. the most striking example of using xenogeneic antigen to break immune tolerance is evidenced by the licensed canine melanoma dna vaccine, a therapeutic agent for advanced melanoma (bergman et al. 2003) . this vaccine consists of dna encoding a human version of tyrosinase, which enabled the dog's immune system to break tolerance to the dog tyrosinase and mount an effective response. the concept of using xenoantigens to enhance vaccine immunogenicity is also supported by a recent preclinical study in which mice immunized with human p53 (hp53) dna vaccine protected against challenge with murine colon cancer mc38 while those immunized with mouse p53 (mp53 dna) were not (soong et al. 2013) . in a therapeutic model, established mc38 tumors were also well controlled by treatment with hp53 dna therapy in tumor bearing mice compared to mp53 dna. mice vaccinated with hp53 dna plasmid exhibited an increase in mp53-specific cd8+ t-cells compared to vaccination with mp53 dna (soong et al. 2013) . associative recognition is another strategic antigen design aimed at breaking central immune tolerance. the rationale of this approach is to introduce additional gene coding sequences to a dna vaccine by which a fusion protein containing the cancer antigen and the associated peptide is expressed. the peptide moiety has epitopes that are highly immunogenic to t h cells, which help induce cd8 + cytotoxic t cell and cd4 + helper t cell responses against the weakly immunogenic selfantigen. for example, tetanus toxin (tt) contains epitopes that avidly bind to t helper cells. in several preclinical studies, tt-fused antigen has been used in dna vaccines to significantly enhance the immunogenicity of various cancer antigens including c-myb, pas domain containing 1(pasd1), and ny-eso-1. these studies demonstrate the potential of dna vaccines incorporating tt epitopes in order to enhance immunogenicity (yang et al. 2014) . breaking central immune tolerance is critical for cancer vaccine efficacy. however, breaking tolerance may raise the risk of autoimmunity. many cancer antigens are also expressed on normal cells, albeit at very low level. enhancing antigen immunogenicity should not be at the expense of damaging normal tissues. the detrimental "on-target off-tumor" side effect has been reported in cancer treatment using adoptive transfer of genetically engineered t cells. until now, strategies used to break immune tolerance have not been shown to trigger harmful autoimmune attacks against normal tissues. however, more studies are needed to further evaluate the safety of breaking immune tolerance. it also should be noted that breaking immune tolerance represents only one of the greatest obstacles faced by scientists developing effective cancer vaccines. other factors in the peripheral immunosuppressive networks including, but not limited to, myeloid-derived suppressor cells (mdsc), regulatory t cells (t reg ), inhibitory cytokines, and immune exhaustion also profoundly affect the efficacy of dna vaccination. therefore, in agreement with the notion that most effective cancer therapies are multimodal, clinical development of cancer vaccines may ultimately be part of polytherapy (e.g., vaccine + checkpoint blockade mabs) to counteract central and local immunosuppressive mechanisms. numerous therapeutic dna vaccines are currently being evaluated in clinical trials to assess their translational potential. these vaccines target a wide variety of malignancies including cervical cancer, colorectal cancer, prostate cancer, breast cancer, head and neck cancer, melanoma, merkel cell carcinoma, and leukemia. a summary of these trials including their targeted cancer antigens, strategies used to break the immune tolerance, and trial stages is shown in table 2 . the tert antigen encoded by the dna vaccine was synthesized and codon-optimized. two immunogenic mutations (r589y and d1005y) were incorporated into the htert sequence to assist in breaking tolerance to date, the most successful and encouraging outcomes of using dna vaccine in the clinical setting were obtained from treatment of malignant diseases where the etiological agent is of foreign viral origin, such as the human papillomavirus (hpv), as these viral agents can readily induce a strong immune response against cancerous cells harboring viral antigens. in the case of hpv, more than 100 strains have been identified with most of them causing asymptomatic infections. however, oncogenic high-risk hpv types (e.g., hpv16, 18, 31, 33, and 45) have been shown to be the etiological agents for cervical, penile, vulvar, vaginal, anal, and oropharyngeal cancers. particularly, hpv16 and 18 have been regarded as the genotypes most closely associated with cervical intraepithelial neoplasia (cin) and cervical cancers. the hpv genome encodes two classes of proteins including the early (e) and late (l) proteins. the late proteins (l1 and l2) are structural components of viral capsid, whereas, the early proteins (e1, e2, e4, e5, e6, and e7) are regulatory proteins participating in dna replication, transcriptional regulation, cell transformation, and viral assembly. among these viral proteins, e6 and e7 can inactivate tumor suppressor p53 and retinoblastoma (rb) protein, respectively, thereby preventing cellular apoptosis, promoting cell proliferation, and contributing to progression to intraepithelial neoplasia and carcinoma. moreover, e6 and e7 expression in hpv-infected cells is constitutive and irrespective of infection stage. in contrast, after primary infection, the expression of several early (e2, e4, and e5) and late (l1 and l2) proteins becomes invisible as a result of the deletion of the viral genes upon viral integration into host genome (lin et al. 2010) . therefore, owing to their constitutive expression in hpv-associated malignancies and their crucial role in tumor pathogenesis, the e6 and e7 proteins are ideal choices of hpv antigens for developing therapeutic vaccines against hpv-associated malignancies. indeed, several dna vaccines targeting the hpv16/18 e6 and e7 proteins have been tested in clinical trials and demonstrated impressive efficacy as evidenced by regression of high grade cin to normal tissue and clearance of viral infection. during the last few years, great progress has been made in the field of dna vaccination. the advancements in plasmid construction, antigen design, delivery device optimization, and use of immunologic adjuvants have greatly enhanced the immunogenicity and efficacy of dna vaccines. among these developments, electroporation represents the most remarkable progress, which exerts the greatest impact on dna vaccine efficacy. recently, a hpv16/18-specific dna vaccine delivered by ep demonstrated clinical benefit in a phase iib clinical trial in the form of regression of cervical intraepithelial neoplasia (cin2/3). this encouraging result has led to resurgence of the platform. it is promising that with further improvement, dna immunization will bring revolutionary transformation to the vaccine field. to that end, scientists are working diligently towards the first licensure of dna vaccines. cancer vaccination by electro-gene-transfer immunotherapy against hpv16/18 generates potent th1 and cytotoxic cellular immune responses long-term survival of dogs with advanced malignant melanoma after dna vaccination with xenogeneic human tyrosinase: a phase i trial administration of hpv dna vaccine via electroporation elicits the strongest cd8+ t cell immune responses compared to intramuscular injection and intradermal gene gun delivery cellular cytotoxic response induced by dna vaccination in hiv-1-infected patients dna-based influenza vaccines: evaluating their potential to provide universal protection phase 1 study of pandemic h1 dna vaccine in healthy adults phase i randomized clinical trial of vrc dna and rad5 hiv-1 vaccine delivery by intramuscular (i.m.), subcutaneous (s.c.) and intradermal (i.d.) administration (vrc 011) hiv dna vaccine: stepwise improvements make a difference dna vaccines: developing new strategies against cancer the end or the beginning of the drive to an hiv-preventive vaccine: a view from over 20 years adjuvanting a dna vaccine with a tlr9 ligand plus flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus dna priming and influenza vaccine immunogenicity: two phase 1 open label randomised clinical trials targeting plasmid-encoded proteins to the antigen presentation pathways therapeutic hpv dna vaccines dna vaccines: an historical perspective and view to the future prospects for control of emerging infectious diseases with plasmid dna vaccines hiv therapeutic vaccines: moving towards a functional cure clinical development of a cytomegalovirus dna vaccine: from product concept to pivotal phase 3 trial xenogeneic human p53 dna vaccination by electroporation breaks immune tolerance to control murine tumors expressing mouse p53 genetic immunization is a simple method for eliciting an immune response using plasmids as dna vaccines for infectious diseases induction of broadly neutralizing h1n1 influenza antibodies by vaccination direct gene transfer into mouse muscle in vivo dna vaccine for cancer immunotherapy ▶ gene delivery by electroporation in vitro: mechanisms key: cord-019964-9leljj8j authors: nan title: recent research in infectious disease date: 2005-01-22 journal: j infect doi: 10.1016/j.jinf.2004.11.005 sha: doc_id: 19964 cord_uid: 9leljj8j nan value was 83.8%, and negative predictive value was 98.6%. the sensitivity of the assay was significantly higher than that of antepartum culture (54%). furthermore, many clinicians use a risk-based approach to gbs colonization by treating high-risk women with empiric antibiotics without waiting for culture results. the assay was also significantly more sensitive than the risk-factor approach (94% versus 42%) with regard to predicting intrapartum status. the quick and accurate results obtained with the idi-strep b assay during labor suggest that its use would lead to reduced rates of neonatal gbs colonization and the more judicious use of antibiotics. 2004;39:1129-35. how positive blood culture results are reported has an impact on outcomes of bsi st. louis (md consult)-many patients with bloodstream infection (bsi) do not receive adequate therapy, even after the final microbiologic report is available. the method of reporting microbiologic information to the physician has a significant impact on whether and how this information is used, according to the october 15 clinical infectious diseases. dr. emilio bouza and colleagues of hospital general universitario 'gregorio marañón' in madrid evaluated data from 197 bsis. microbiologic results were reported in 3 different ways. in 109 cases (group a), the physician was informed by telephone of the results and given a written report after the definitive results were known. in 99 cases (group b), physicians also received a written report at the bedside, along with written therapeutic recommendations. in 89 cases (group c), physicians also received clinical advice orally. during empiric treatment, 27.7% of patients received inappropriate treatment, and 13.7% received no treatment. these patients had significantly longer hospital stays (mean, 27.2 versus 19.4 days) and a higher risk of having clostridium difficile-associated diarrhoea (8.3% versus 1.9%), infection-related death (23.3% versus 13.6%), and all-cause mortality (30.8% versus 19.4%) than patients whose empiric therapy was appropriate. after the reports were received, therapy was changed for 52.3% of patients in group b and 53.1% in group c but for none of the patients in group a. consequently, the proportion of days during which patients received adequate treatment was significantly smaller in group a than in groups b and c (66.3% versus 92.1% and 91.2%, respectively). group a also received significantly fewer defined daily doses of appropriate antibiotics (16.4 versus 22.2 and 20.7 days) . the duration of hospitalization, mortality rates after reports were received, and costs also tended to be lower in groups b and c. these results suggest that written and/or oralalert reports providing clinical advice in addition to standard reports will improve the management of patients with bsi. dr. lynda anne szczech and colleagues of the women's interagency hiv study recruited 2,038 hiv-positive from october 1994 through november 1995. differences in the development of a new acquired immunodeficiency virus-defining illness (adi) or death were compared according to renal status and haart status. at baseline, 287 patients (14.1%) had proteinuria. these patients had significantly lower cd4c lymphocyte counts and higher viral loads than patients without proteinuria, both at baseline and before starting haart. before haart was widespread, proteinuria was associated with a significantly increased risk of developing a new adi (hazard ratio [hr], 1.37) and death (hr, 1.35). an elevated creatinine level was also a significant independent risk factor for mortality (hr, 1.72 per decrease in the inverse creatinine level). however, even after haart was initiated, proteinuria was a significant independent predictor of mortality (hr, 2.07). an elevated creatinine level was also a significant predictor of new-onset adi and death (hrs, 1.54 and 1.96 per decrease in the inverse creatinine level, respectively. whether the kidney modulates hiv disease or whether these findings merely reflect the impact of greater comorbidity is not known. what is clear, however, is that hiv-positive women with these findings need improved monitoring and more aggressive treatment. genetic and transmission analysis of helicobacter pylori strains within a family raymond j, thiberge j-m, chevalier c, kalach n, bergeret m, labigne a, et al. to look for evidence of intrafamilial infection, we isolated 107 helicobacter pylori clones from biopsied specimens taken from both parents and four children. we compared the sequences of two housekeeping genes (hspa and glmm) from these clones with those of 131 unrelated strains from patients living in different geographic regions. strain relationships within the family were determined by analyzing allelic variation at both loci and building phylogenetic trees and by using multilocus sequence typing. both hspa-and glmm-based phylogenetic trees showed east asian and african branches. all samples from family members showed natural mixed infection. identical alleles found in some strains isolated from the children and parents, but not in the strains isolated from unrelated patients, demonstrated that strains have circulated within the family. several mechanisms, such as point mutations, intragenic recombination, and introduction of foreign (african) alleles, were shown to enhance strain diversity within the family. increasing reports of the appearance of novel nonmultiresistant methicillin-resistant staphylococcus aureus mrsa (mrsa) strains in the community and of the spread of hospital mrsa strains into the community are cause for public health concern. we conducted two national surveys of unique isolates of s. aureus from clinical specimens collected from nonhospitalized patients commencing in 2000 and 2002, respectively. a total of 11.7% of 2,498 isolates from 2000 and 15.4% of 2,486 isolates from 2002 were mrsa. approximately 54% of the mrsa isolates were nonmultiresistant (resistant to less than three of nine antibiotics) in both surveys. the majority of multiresistant mrsa isolates in both surveys belonged to two strains (strains aus-2 and aus-3), as determined by pulsed-field gel electrophoresis (pfge) and resistogram typing. the 3 aus-2 isolates and 10 of the 11 aus-3 isolates selected for multilocus sequence typing (mlst) and staphylococcal chromosomal cassette mec (sccmec) analysis were st239-mrsa-iii (where st is the sequence type) and thus belonged to the same clone as the eastern australian mrsa strain of the 1980s, which spread internationally. four predominant clones of novel nonmultiresistant mrsa were identified by pfge, mlst, and sccmec analysis: st22-mrsa-iv (strain emrsa-15), st1-mrsa-iv (strain wa-1), st30-mrsa-iv (strain swp), and st93-mrsa-iv (strain queensland). the last three clones are associated with community acquisition. a total of 14 sts were identified in the surveys, including six unique clones of novel nonmultiresistant mrsa, namely, sts 73, 93, 129, 75, and 80slv and a new st. sccmec types iv and v were present in diverse genetic backgrounds. these findings provide support for the acquisition of sccmec by multiple lineages of s. aureus. they also confirm that both hospital and community strains of mrsa are now common in nonhospitalized patients throughout australia. use of multiple nucleic acid amplification tests to define the infected-patient 'gold standard' in clinical trials of new diagnostic tests for chlamydia trachomatis infections martin dh, nsuami m, schachter j, hook ew 3rd, ferrero d, quinn tc, gaydos c nucleic acid amplification tests (naats) can be used to define the infected-patient 'gold standard' for the purpose of designing studies of the performance of chlamydia trachomatis diagnostic tests. it is unclear how many test results run by different naats and what combinations of specimens comprise the best infected-patient gold standard. we approached this question with data from a large study of the performance of a new naat. data were available from three endocervical swabs and a urine specimen collected from each of 1,412 women and tested by three different naats. results from all three assays were used equally in a rotating fashion to define the infected-patient gold standard. multiple different infectedpatient gold standards for estimating swab and urine specimen sensitivity and specificity for one naat method were created by varying the number and combinations of swab and urine comparator results with two different naats, the effect of changing the infected-patient gold standard definition was determined by constructing receiver-operator-like curves with calculated sensitivities and specificities for each test. the one-positive-of-two-results or two-positive-oftwo-results (same or two different assays) infected-patient gold standard definitions produced low sensitivity and low specificity estimates, respectively. if four comparator naat results were used, the any-three-positive-offour-results definition or the at-least-one-specimen-positive-by-each-of-two-comparator-assays definition appeared to provide better combinations of sensitivity and specificity estimates. the any-two-positive-out-of-three-results definition resulted in estimates that were as good as produced with the former two definitions. this analytic approach provides a means of clearly visualizing the effects of changing naat-based infected-patient gold standards and should be helpful in designing future studies of new c. trachomatis diagnostic tests. prospective study of human metapneumovirus infection in children less than 3 years of age konig b, konig w, arnold r, werchau h, ihorst g, forster j most lower respiratory tract infections (lrtis) in children under the age of 3 years are due to respiratory syncytial virus (rsv). epidemiological, host, and viral factors eventually account for the severity of lrtis, but they do not completely explain it. human metapneumovirus (hmpv) was recently identified in children with lrtis. in a population-based prospective multicenter study (the pri.de study, conducted in germany over 2 years), we tested 3,369 nasopharyngeal secretions from children younger than 3 years of age with lrtis for rsv a and b, influenza viruses (ivs) a and b, and parainfluenza viruses (pivs) 1 to 3. of the children requiring intensive care (nz85), 18% had hmpv infections, and 60% of these children were infected with hmpv in combination with rsv. we did not detect hmpv in a randomly selected subset of rsv-positive nasopharyngeal secretions (nz120) from children not requiring intensive care support. hmpv was detected in !1% of virus-negative samples from patients without intensive care support (nz620). our data support the hypothesis that coinfections with rsv and hmpv are more severe than infections with either rsv or hmpv alone, at least in children younger than 3 years of age. candida parapsilosis is an important cause of bloodstream infections in the health care setting. we investigated a large c. parapsilosis outbreak occurring in a community hospital and conducted a case-control study to determine the risk factors for infection. we identified 22 cases of bloodstream infection with c. parapsilosis: 15 confirmed and 7 possible. the factors associated with an increased risk of infection included hospitalization in the intensive care unit (adjusted odds ratio, 16.4; 95% confidence interval, 1.8 to 148.1) and receipt of total parenteral nutrition (adjusted odds ratio, 9.2; 95% confidence interval, 0.9 to 98.1). samples for surveillance cultures were obtained from health care worker hands, central venous catheter insertion sites, and medical devices. twenty-six percent of the health care workers surveyed demonstrated hand colonization with c. parapsilosis, and one hand isolate was highly related to all case-patient isolates by tests with the dna probe cp3-13. outbreak strain isolates also demonstrated reduced susceptibilities to fluconazole and voriconazole. this largest known reported outbreak of c. parapsilosis bloodstream infections in adults resulted from an interplay of host, environment, and pathogen factors. recommendations for control measures focused on improving hand hygiene compliance. heikkinen t, silvennoinen h, peltola v, ziegler t, vainionpaa r, vuorinen t, kainulainen l, puhakka t, jartti t, toikka p, lehtinen p, routi t, juven t background: influenza vaccination of healthy children is encouraged because children are frequently hospitalized for influenza-attributable illnesses. however, most children with influenza are treated as outpatients, and scarce data are available on the burden of influenza in these children. methods: we performed a prospective study of respiratory infections in preenrolled cohorts of children %13 years old during 2 consecutive respiratory seasons (2231 child-seasons of follow-up). at any sign of respiratory infection, we examined the children and obtained a nasal swab for the detection of influenza. the parents filled out daily symptom diaries. of all the enrollees, 94% remained active participants in the study. results: the average annual rate of influenza was highest (179 cases/1000 children) among children !3 years old. acute otitis media developed as a complication of influenza in 39.7% of children !3 years old. for every 100 influenza-infected children !3 years old, there were 195 days of parental work loss (mean duration, 3.2 days). influenza causes a substantial burden of illness on outpatient children and their families. vaccination of children !3 years old might be beneficial for reducing the direct and indirect costs of influenza in children. moscicki ab, ellenberg jh, crowley-nowick p, darragh tm, xu j, fahrat s background: the risk of developing the human papillomavirus (hpv)-associated precancer high-grade squamous intraepithelial lesion (hsil) in human immunodeficiency virus (hiv)-infected adolescents is unknown. we examined the risk of developing hsil among adolescents with and without hiv infection. methods: hiv-infected (nz172) and-uninfected (nz84) girls aged 13-18 years who were participating in a multicenter study of primarily horizontally acquired hiv infections in adolescents (reaching for excellence in adolescent health care) and who did not have hsil on cytologic examination at study entry or at the first follow-up visit were followed at 6-month intervals. hiv-uninfected girls were recruited for comparison in a 2:1 ratio (hiv infected: hiv uninfected). the primary outcome was cytologic diagnosis of hsil confirmed by expert review. results: incidence of hsil by the end of follow-up was higher for hiv-infected girls than for hiv-uninfected girls (21.5% vs. 4.8%, respectively). in multivariate analysis, use of hormonal (either estrogen/progesterone oral combination or medroxyprogesterone acetate intramuscular) contraceptives, high cervical mucous concentrations of interleukin (il)-12, a positive hpv test, and persistent low-grade squamous intraepithelial lesion (lsil) were significantly associated with the development of hsil. conclusions: the incidence of hsil was alarmingly high in hivinfected adolescent girls. however, when other predictors were considered in multivariate analysis, hiv status was not retained in the model. the heightened risk for hsil associated with persistent lsil underscores the need to closely monitor hiv-infected adolescents with lsil. the risk for hsil associated with high concentrations of il-12 may be suggestive of a local immune dysregulation. the role of hormonal contraception as a risk factor deserves further investigation. little is known about the epidemiologic profile of trichomoniasis in men and its relationship to human immunodeficiency virus (hiv) infection. among men presenting for care for symptomatic sexually transmitted infections (stis) in malawi, trichomoniasis is not considered for first-line treatment. methods: we conducted a cross-sectional survey of 1187 men attending either a dermatology or sti outpatient clinic in the capital of malawi. men were interviewed, and the etiologies of the stis were determined. results: at the sti clinic (nz756 men), we identified 150 men (20%) with trichomonas vaginalis infection, 358 men (47%) with hiv infection, and 335 men (44%) with neisseria gonorrhoeae infection. at the dermatology clinic (nz431 men), we identified 54 (13%), 118 (27%), and 2 (0.5%) men, respectively. at both clinics, a lower education level and reporting never having used a condom were predictive of t. vaginalis infection. only at the dermatology clinic was older age associated with infection, and only at the sti clinic were marital, genital ulcer disease, and hiv-infection status associated with t. vaginalis infection. at the sti clinic, urethral symptoms attributable to trichomoniasis were more severe among hiv-positive men than among hiv-negative men. conclusions: given its high prevalence and the increased risk for hiv transmission, t. vaginalis infection should be reconsidered for inclusion in the malawi stitreatment regimen for men. 2004; 190(8):1448-55. pmid: 15378437 [pubmed-in process] role of human neutrophil peptide-1 as a possible adjunct to antituberculosis chemotherapy kalita a, verma i, khuller gk we report the role of human neutrophil peptide (hnp)-1 as an adjunct to antituberculosis (anti-tb) drugs. the combination of hnp-1, isoniazid, and rifampicin was evaluated against mycobacterium tuberculosis h(37)rv in vitro, ex vivo, and in vivo, and synergism was observed on the basis of reductions in minimum inhibitory concentrations (mics) of these agents. in vitro results revealed o1log unit reductions even when hnp-1 and anti-tb drugs were used at 1/16 mics. this combination was also found to be bactericidal against intracellular mycobacteria even at 1/8 mics of hnp-1 and drugs. hnp-1 used in conjunction with anti-tb drugs resulted in significant clearance of bacterial load from lungs, liver, and spleen of infected, compared with control animals. the effective therapeutic dosage of drugs could be reduced to half by supplementing hnp-1 in the therapeutic schedule. these results clearly suggest that hnp-1 can be used as adjunct chemotherapy with conventional drugs against tb. in this phase iii trial, dr. jo-anne h. van burik and the national institute of allergy and infectious diseases mycoses study group studied 882 children and adults undergoing hsct. about half were randomized to receive micafungin (50 mg; 1 mg/kg for patients !50 kg), whereas the others received fluconazole (400 mg; 8 mg/kg for patients !50 kg). drugs were administered once a day via infusion during the neutropenic phase of hsct (i.e., before engraftment), and they were continued until neutropenia resolved or day 42 after hsct, whichever came earlier, or until fungal infection developed. time to treatment success was also significantly shorter with micafungin. no excess toxicity occurred with micafungin as compared with fluconazole, and fewer patients receiving micafungin dropped out of the study due to drug-related adverse events (4.2% versus 7.2%, pz0.058). these results suggest that micafungin is an effective alternative to fluconazole for antifungal prophylaxis in neutropenic patients during hsct. the authors obtained baseline blood samples from 174 girls 12 to 15 years old (76% african-american; 24% white). seropositivity for hsv-1, hsv-2, and cmv was also assessed 3 years later. at baseline, 41% of subjects (71 girls) had a history of sexual activity; this proportion increased to 73% (127 girls) by the 3-year follow-up. overall, seropositivity for cmv increased from 71% to 81%, seropositivity for hsv-1 increased from 44% to 49%, and seropositivity for hsv-2 doubled, from 7% to 14%. among sexually experienced girls, hsv-2 seropositivity increased from 15% to 19%. these increases correspond with attack rates of 13.8 cases of cmv per 100 person-years and 3.2 cases of hsv-1 per 100 person-years in the entire cohort. among sexually experienced girls, hsv-2 attack rates were 4.4 cases per 100 person-years of sexual activity. at follow-up, generalized estimating equation modeling identified 4 factors significantly associated with hiv-2 seropositivity: it also saves money, according to a new study. dr. kent k. hu and colleagues of veterans affairs puget sound health care system in seattle, wash, and other institutions discuss their findings in the november 15 clinical infectious diseases. the authors developed a decision analysis model based on hospitalized patients most likely to require a cvc for 2 to 10 days (i.e., those in intensive care units, with immunosuppression, or receiving total parenteral nutrition). cvcs were inserted according to either msb techniques (person inserting the catheter must wear a head cap, face mask, sterile body gown, and sterile gloves and use a full-size sterile drape around the site) or in accordance with lessstringent measures (only sterile gloves and a small regional sterile drape). in the base-case analysis, the use of msbs actually lowered costs by $252 as compared with less-stringent infection control measures ($369 versus $621 per catheter inserted). the incidences of catheter-related bsi (2.8% versus 5.3%), catheter colonization (2.9% versus 5.5%), and death (0.4% versus 0.8%) were also lower with msbs. during sensitivity analyses-even over a wide range of clinical and economic assumptions-the use of msbs resulted in improved patient safety. these data suggest that the improved management of infection with the use of msbs also pays off by lowering medical costs. thus, even though msbs are sometime cumbersome and time consuming, the authors suggest that they should be routinely used during cvc insertion. 2004;39:1441-5. hypogonadism is a risk factor for gynecomastia in hiv-positive men st. louis (md consult)-some studies suggest antiretroviral therapy is a risk factor for gynecomastia in men with human immunodeficiency virus (hiv) infection. however, no association has ever been found between the duration of exposure to any antiretroviral drug and the development of gynecomastia. instead, it appears hypogonadism is a predisposing factor to gynecomastia in hiv-infected men, according to the november 15 clinical infectious diseases. dr. alejandra biglia and colleagues of the university of barcelona in spain and other institutions studied 2,275 men with hiv infection. clinical examination with confirmatory sonography showed 40 men, or 1.8%, had gynecomastia. this is similar to the prevalence of gynecomastia in the general population. these 40 cases were matched with 44 men with hiv but not gynecomastia, and clinical, demographic, and laboratory features were extensively compared between the 2 groups. the mean free testosterone index was significantly lower in the cases than in the controls (42.6% vs. 58.0%). hypogonadism was the strongest independent predictor of gynecomastia (adjusted odds ratio [or] 7.6). other factors increasing the risk of gynecomastia included hepatitis c virus infection (adjusted or 6.1) and lipoatrophy (adjusted or 5.6). furthermore, gynecomastia resolved in many patients with persistent gynecomastia after transdermal or intramuscular testosterone administration, and in fact improved without any intervention in a substantial proportion of men. the authors also compared antiretroviral drug use between the cases and controls. significantly more cases were taking stavudine (73% vs. 41%) or efavirenz (33% vs. 14%), but significantly more controls were taking zidovudine (34% vs. 10%). however, the duration of exposure to each drug did not differ significantly between cases and controls, which further argues against antiretroviral therapy as the cause of gynecomastia in hivpositive men. the authors conclude gynecomastia in hivinfected men appears to be related more to hypogonadism than to adverse effects of antiretroviral therapy. if gynecomastia proves to be related to hypogonadism, then hormone treatments may be able to reverse this lipodystrophy, and further study is warranted. clin infect dis 2004;39:1514-9. taira av, neukermans cp, sanders gd human papillomavirus (hpv) has been implicated as the primary etiologic agent of cervical cancer. potential vaccines against high-risk hpv types are in clinical trials. we evaluated vaccination programs with a vaccine against hpv-16 and hpv-18. we developed disease transmission models that estimated hpv prevalence and infection rates for the population overall, by age group, by level of sexual activity within each age group, and by sex. data were based on clinical trials and published and unpublished sources. an hpv-16/18 vaccine for 12year-old girls would reduce cohort cervical cancer cases by 61.8%, with a cost-effectiveness ratio of $14,583 per quality-adjusted life year (qaly). including male participants in a vaccine rollout would further reduce cervical cancer cases by 2.2% at an incremental cost-effectiveness ratio of $442,039/qaly compared to female-only vaccination. vaccination against hpv-16 and hpv-18 can be cost-effective, although including male participants in a vaccination program is generally not costeffective, compared to female-only vaccination. yokoe ds, noskin ga, cunningham sm, zuccotti g, plaskett t, fraser vj, et al. we evaluated antimicrobial exposure, discharge diagnoses, or both to identify surgical site infections (ssi). this retrospective cohort study in 13 hospitals involved weighted, random samples of records from 8,739 coronary artery bypass graft (cabg) procedures, 7,399 cesarean deliveries, and 6,175 breast procedures. we compared routine surveillance to detection through inpatient antimicrobial exposure (s9 days for cabg, s2 days for cesareans, and s6 days for breast procedures), discharge diagnoses, or both. together, all methods identified ssi after 7.4% of cabg, 5.0% of cesareans, and 2.0% of breast procedures. antimicrobial exposure had the highest sensitivity, 88% to 91%, compared with routine surveillance, 38% to 64%. diagnosis codes improved sensitivity of detection of antimicrobial exposure after cesareans. record review confirmed ssi after 31% to 38% of procedures that met antimicrobial surveillance criteria. sufficient antimicrobial exposure days, together with diagnosis codes for cesareans, identified more postoperative ssi than routine surveillance methods. this screening method was efficient, readily standardized, and suitable for most hospitals. miner al, sands ke, yokoe ds, freedman j, thompson k, livingston jm, et al. we investigated using administrative claims data to identify surgical site infections (ssi) after breast surgery and cesarean section. postoperative diagnosis codes, procedure codes, and pharmacy information were automatically scanned and used to identify claims suggestive of ssi ('indicators') among 426 (22%) of 1,943 breast procedures and 474 (10%) of 4,859 cesarean sections. for 104 breast procedures with indicators explained in available medical records, ssi were confirmed for 37%, and some infection criteria were present for another 27%. among 204 cesarean sections, ssi were confirmed for 40%, and some criteria were met for 27%. the extrapolated infection rates of 2.8% for breast procedures and 3.1% for cesarean section were similar to those reported by the national nosocomial infection surveillance program but differ in representing predominantly outpatient infections. claims data may complement other data sources for identification of surgical site infections following breast surgery and cesarean section. several studies have shown that nasopharyngeal sampling is more sensitive than oropharyngeal sampling for the detection of pneumococcal carriage in children. the data for adults are limited and conflicting. this study was part of a larger study of pneumococcal carriage on the navajo and white mountain apache reservation following a clinical trial of a seven-valent pneumococcal conjugate vaccine. persons aged 18 years and older living in households with children enrolled in the vaccine trial were eligible. we collected both nasopharyngeal and oropharyngeal specimens by passing a flexible calcium alginate wire swab either nasally to the posterior nasopharynx or orally to the posterior oropharynx. swabs were placed in skim milktryptone-glucose-glycerin medium and frozen at k708c. pneumococcal isolation was performed by standard techniques. analyses were based on specimens collected from 1,994 adults living in 1,054 households. nasopharyngeal specimens (11.1%; 95% confidence interval [ci], 9.8 and 12.6%) were significantly more likely to grow pneumococci than were oropharyngeal specimens (5.8%; 95% ci, 4.8 to 6.9%) (p!0.0001). few persons had pneumococcal growth from both specimens (1.7%). therefore, both tests together were more likely to identify pneumococcal carriage (15.2%; 95% ci, 13.7 to 16.9%) than either test alone. although we found that nasopharyngeal sampling was more sensitive than oropharyngeal sampling, nasopharyngeal sampling alone would have underestimated the prevalence of pneumococcal carriage in this adult population. sampling both sites may give more accurate results than sampling either site alone in studies of pneumococcal carriage in adults. microbiol 2004; 42(11):4974-6. pmid: 15528682 [pubmed-in process] effects of intrapartum penicillin prophylaxis on intestinal bacterial colonization in infants jaureguy f, carton m, panel p, foucaud p, butel mj, doucet-populaire f early-onset group b streptococcal (gbs) infections remain a leading cause of morbidity and mortality in infants. to prevent the vertical transmission of gbs and neonatal gbs infection, guidelines recommend intrapartum penicillin or amoxicillin prophylaxis. this intrapartum antibiotic prophylaxis (iap) is suspected to favor colonization by antibiotic-resistant bacteria. however, the effects of this prophylaxis on the patterns of acquisition of gastrointestinal bacterial flora in infants have never been studied. we collected stool samples from 3-day-old infants born to mothers who received intrapartum amoxicillin (antibiotic-exposed group; nz25) and to untreated mothers (non-antibiotic-exposed group; nz25). the groups were matched for factors known to affect intestinal microbial colonization: gestational age, type of delivery, and type of feeding. qualitative and quantitative differential analyses of the bacterial flora in stool samples were performed. similar numbers of infants in the nonantibiotic-exposed and antibiotic-exposed groups were colonized by aerobic bacteria and amoxicillinresistant enterobacteria (75 and 77%, respectively) (pz0.79). in contrast, significantly fewer infants in the antibiotic-exposed group than in the nonantibiotic-exposed group were colonized by anaerobic bacteria, especially clostridium (12 and 40%, respectively) (p!0.05). regarding intestinal bacterial colonization, the differences between antibiotic-exposed and non-antibiotic-exposed infants were remarkably few. the only statistically significant effect was the reduced initial bacterial colonization by clostridium in the antibioticexposed group. in our study, the use of iap did not favor colonization by beta-lactam-resistant bacteria. however, further evaluations are required to highlight the potential risks of the widespread use of antibiotics to prevent early-onset gbs infection. microbiol 2004; 42(11):5184-8. pmid: 15528713 [pubmed-in process] usefulness of routine epicardial pacing wire culture for early prediction of poststernotomy mediastinitis mekontso-dessap a, honore s, kirsch m, houel r, loisance d, brun-buisson c poststernotomy mediastinitis (psm) is one of the most serious complications of cardiac surgery, and its associated morbidity and mortality demand early recognition for emergency therapy. in this study, we investigated the usefulness of epicardial pacing wire (epw) cultures for the prediction of psm. among 2,200 patients who underwent a cardiac surgical procedure at our hospital between 1 january 1999 and 31 december 2001, 82 (3.7%) had psm; staphylococcus aureus was the organism (45.1%) most frequently isolated at the time of surgical debridement. epws from 1,607 (73.0%) patients, 73 (4.5%) of whom developed psm, were cultured. epw cultures from 466 (29.0%) were positive, most often (74.9%) for coagulase-negative staphylococci. epw cultures were truly positive in 26 cases, truly negative in 1,106 cases, falsely positive in 428 cases, and falsely negative in 47 cases (with sterile cultures in 35 cases and a culture positive for an organism different from that isolated at the time of debridement in 12 cases). epw culture had a positive predictive value of only 5.7% and a high negative predictive value (95.9%) for the diagnosis of psm, with an accuracy of 70.4%. however, the likelihood ratio of positive (1.27) and negative (0.89) tests indicated only small changes in pretest-to-posttest probability. therefore, a strategy of routine culture of epws to predict psm seems questionable. dual-probe assay for rapid detection of drug-resistant mycobacterium tuberculosis by real-time pcr wada t, maeda s, tamaru a, imai s, hase a, kobayashi k mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in mycobacterium tuberculosis. for rapid detection of drug-resistant m. tuberculosis in clinical specimens, a simple and applicable method is needed. eight taqman minor groove binder (mgb) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). the target of six mgb probes was the rpob gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpob gene, and one probe was designed as a tuberculosis (tb) control outside the rpob gene hot-spot. we also designed probes to examine codon 315 of katg and codon 306 of embb for mutations associated with resistance to isoniazid and ethambutol, respectively. our system was m. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. detection limits in direct and preamplified analyses were 250 and 10 fg of genomic dna, respectively. the system could detect mutations of the rpob, katg, and embb genes in dnas extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary tb. this system was much faster (3 h from dna preparation) than conventional drug susceptibility testing (3 weeks). results from the dual-mgbprobe assay were consistent with dna sequencing. because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drugresistant m. tuberculosis in clinical laboratories. to define methicillin-resistant staphylococcus aureus (mrsa) reservoirs in the community and their population dynamics, we studied the molecular epidemiology of a random sample (nz490) from a collection of 2154 inpatient and outpatient mrsa isolates during a 7-year period in san francisco. we noted a progressive replacement of type ii staphylococcal chromosomal cassette (scc)mec-bearing isolates with type iv sccmec-bearing isolates, which coincided with o4-fold increase in methicillin resistance between 1998 and 2002. type iv sccmec-bearing isolates involved in the increase in methicillin resistance belonged to 4 molecular genotypes. these 4 genotypes were associated predominantly with community-onset disease, rather than hospital-or long-term-care facilityonset disease (76.9% vs. 19.4% vs. 3.7%; pz0.0005), suggesting that they are not feral descendants of hospital isolates. the longitudinal results linked the dramatic increase in mrsa infections to an expanding community reservoir of mrsa genotypes with intrinsic community survival advantage. the outbreak of monkeypox in the midwestern united states during june 2003 marks the first documented human infection in the western hemisphere. consistent with those in outbreaks in africa, most cases in this outbreak were associated with febrile rash illness. we describe a cluster of monkeypox in a family with a spectrum of clinical illness, including encephalitis, and outline the laboratory confirmation of monkeypox. methods: standardized patient information was collected by questionnaire and medical chart review; all cases described were laboratory confirmed. laboratory methods included nucleic acid detection, viral culture, serologic testing, histopathologic evaluation, and immunohistochemical testing. results: of 3 family members with monkeypox, 2 had rash illness only, and 1 required hospitalization for severe encephalitis. the family member with the mildest clinical course had previously received smallpox vaccination. diagnostic testing by both polymerase chain reaction and culture revealed infectious monkeypox virus in skin lesions of all 3 patients; 2 patients had orthopoxvirus detected by immunohistochemistry in skin lesions. the patient with encephalitis had orthopoxvirus-reactive immunoglobulin m (igm) in cerebrospinal fluid. all patients had detectable igm responses to orthopoxvirus antigens. conclusions: these 3 patients illustrate a spectrum of clinical illness with monkeypox despite a common source of exposure; manifestation and severity of illness may be affected by age and prior smallpox vaccination. we report that monkeypox, in addition to causing febrile rash illness, causes severe neurologic infection, and we discuss the use of novel laboratory tests for its diagnosis. the proportion of infected cells to total cells was measured in serial breast milk samples collected from 291 hiv-1-infected women in nairobi, kenya, by use of real-time dna polymerase chain reaction amplification of bmcs. the number of infected bmcs per million cells was associated with levels of cell-free viral rna in breast milk previous studies demonstrated that the concentration of bmcs varies throughout lactation, and we used these data to transform infected bmcs per million cells to infected bmcs per milliliter. the estimated concentration of infected bmcs per milliliter was higher in colostrum or early milk than in mature milk (p!0.001) factors influencing increases in cd4 cell counts of hiv-positive persons receiving long-term highly active antiretroviral therapy smith cj, sabin ca, youle ms, kinloch-de loes s, lampe fc, madge s, cropley i, johnson ma, phillips an background: highly active antiretroviral therapy (haart) results in an improvement in immunologic function. we sought to investigate the factors associated with increases in cd4 cell count among human immunodeficiency virus (hiv)-positive antiretroviral-naive patients starting haart.methods: five hundred ninety-six subjects were followed for a median of 2.5 years (interquartile range, 1.0-4.0 years). factors associated with changes in cd4 cell counts in the first 3 months of haart and from 3 months onwards were analyzed.results: after 6, 12, and 24 months of haart, the median increases in cd4 cell counts were 114, 181, and 248 cells/mm(3), respectively; 84%, 84%, and 80% of subjects had a virus load of !400 copies/ml during the same periods. white ethnicity, higher pre-haart virus load, and lower pre-haart cd4 and cd8 cell counts were associated with greater increases in cd4 cell counts during the first 3 months of haart. from 3 months onward, a greater cumulative proportion of time spent with virus load !400 copies/ml was associated with a more favorable change in cd4 cell count (an average increase of 5.2 cells/mm(3)/year [95% confidence interval [ci], 3.8-6.7 cells/mm (3)/year] for each extra 10% cumulative time spent with a virus load !400 copies/ml) (p!0.0001). for every 100 cells/mm(3) higher in baseline cd4 cell count, the increase was 6 cells/mm(3)/year less (95% ci, 2-11 cells/mm(3)/year) (pz0.02). sex, risk group, age, and haart regimen were not associated with increases in cd4 cell counts.conclusions: these findings emphasize the importance of maintaining virological suppression and suggest other factors that influence long-term cd4 cell response. 2004; 190(10):1860-8. pmid: 15499544 [pubmed-in process] association of levels of hiv-1-infected breast milk cells and risk of mother-tochild transmission rousseau cm, nduati rw, richardson ba, john-stewart gc, mbori-ngacha da, kreiss jk, overbaugh j understanding how the level of human immunodeficiency virus type 1 (hiv-1)-infected breast milk cells (bmcs) affects hiv transmission via breastfeeding can shed light on the mechanism of key: cord-029423-o24dthlk authors: iwuji, collins c.; churchill, duncan; bremner, stephen; perry, nicky; to, ye; lambert, debbie; bruce, chloe; waters, laura; orkin, chloe; geretti, anna maria title: a phase iv randomised, open-label pilot study to evaluate switching from protease-inhibitor based regimen to bictegravir/emtricitabine/tenofovir alafenamide single tablet regimen in integrase inhibitor-naïve, virologically suppressed hiv-1 infected adults harbouring drug resistance mutations (pibik study): study protocol for a randomised trial date: 2020-07-20 journal: bmc infect dis doi: 10.1186/s12879-020-05240-y sha: doc_id: 29423 cord_uid: o24dthlk background: currently recommended boosted protease-inhibitor (bpi) regimens may be associated with increased risk of cardiovascular or chronic kidney diseases; in addition, boosted regimens are particularly associated with drug-drug interactions. since both cardiovascular and renal disease, and polypharmacy, are common in ageing people with hiv, there is a need for alternative efficacious regimens. bpi-based regimens are often the treatment of choice for individuals with pre-treatment or treatment-acquired resistance but it is plausible that carefully selected hiv-positive individuals with drug resistance, who are virologically suppressed on their current bpi regimen, could maintain virological efficacy when switched to bictegravir, emtricitabine and tenofovir alafenamide (b/f/taf) fixed dose combination (fdc). methods/design: a phase iv, investigator-initiated, multicentre, open label pilot, randomised two-arm study to assess the safety and efficacy of switching from bpi regimen to b/f/taf single tablet regimen in integrase inhibitor-naïve, virologically suppressed adults with hiv-1 infection harbouring drug resistance mutations. eligible individuals will either continue on their bpi regimen or switch to b/f/taf fdc. after 24 weeks, all participants in the bpi arm will be switched to b/f/taf and followed for a further 24 weeks and all participants will be followed for 48 weeks. the primary efficacy endpoint is the proportion of participants with hiv-1 rna < 50 copies/ml at week 24 using pure virologic response whilst the secondary efficacy endpoint is the proportion of participants with hiv-1 rna < 50 copies/ml at week 48. other secondary outcome measures include between arm comparisons of drug resistance at virological failure, safety and tolerability and patient-reported outcome measures. discussion: we aim to provide preliminary evidence of the efficacy of switching to b/f/taf in patients with virological suppression on a bpi-based regimen who harbour select drug resistance mutations. trial registration: isrctn 44453201, registered 19 june 2019 and eudract 2018–004732-30. boosted darunavir and atazanavir are recommended as preferred boosted protease inhibitors (bpi) in the british hiv association treatment guidelines. however, boosted darunavir was associated with increased cardiovascular risk in a large prospective observational study [1] . although boosted atazanavir has not been associated with increased cardiovascular risk [2] , cohort studies indicate an increased risk of chronic kidney disease [3] . in clinical trials, atazanavir recipients experienced higher discontinuation rates from adverse events than darunavir [2, 4] which were driven by hyperbilirubinaemia and renal events. ageing of people living with hiv is resulting in increasing prevalence of cardiovascular and renal diseases; in addition, polypharmacy is common due to comorbidities, pointing to an additional limitation for bpi due to their particularly high potential for drug-drug interactions [5, 6] . there is evidence from clinical trials that switching from virologically suppressive bpi-based antiretroviral therapy (art) to regimens based on the newer strand transfer integrase inhibitors, bictegravir [7] and dolutegravir [8] is safe and efficacious. these studies however excluded individuals known to harbour hiv-1 drug resistance. it is well established that hiv-positive individuals that have either pre-treatment drug resistance (pdr) or limited drug resistance following failure of first-line art achieve virological suppression on regimens comprising a bpi plus two nucleos(t)ide reverse transcriptase inhibitor (nrtis) [9, 10] . in contrast, in the switchmrk study, switching virologically suppressed individuals from a bpi to the integrase inhibitor raltegravir resulted in an increased risk of virological failure relative to individuals maintained on the bpi. a post-hoc analysis suggested that this effect might be mediated by prior virological failure compromising the activity of the nrti backbone [11] . however, evidence indicates that second-generation instis, including bictegravir and dolutegravir, have an improved barrier to resistance relative to first-generation compounds and may overcome both pdr and limited treatment-associated drug resistance. in the dawning study, a second line switch to a regimen comprising the insti dolutegravir plus 2 nrtis, where at least one nrti was predicted to be fully active based on resistance analysis at the time screening, was superior to a bpi plus 2 nrtis at 24 weeks in patients failing first line art [12] . dawn ing study suggests second-generation insti like dolutegravir and by extension bictegravir are likely to be successful in switch strategies in the presence of either pdr or treatment-acquired drug resistance, including m184v/i or thymidine analogue mutations (tams). a small, open label, single arm study switched 37 patients (54% on a bpi-based regimen) harbouring the lamivudine (3tc) and emtricitabine (ftc) associated mutation m184v/i to the fixed dose combination (fdc) of elvitegravir/cobiscitat/emtricitabine/ tenofovir alafenamide with no virological failures observed after 12 weeks of follow up [13] . another study investigated whether efficacy was maintained following a switch to bictegravir/emtricitabine/tenofovir alafenamide fdc (b/f/ taf) in individuals suppressed on either a pi-based regimen or the fdc of dolutegravir/abacavir/lamivudine (dtg/abc/3tc). amongst the 572 participants on b/f/ taf, 532 (93%) achieved virologic suppression (vl < 50 copies/ml), with missing virologic data accounting for the majority of the remaining. of the 572 participants, 405 (71%) had baseline resistance data available determined by both historical genotypes and baseline proviral dna sequence; 52 (13%) of whom had nrti associated resistance mutation present. 35/36 (97%) of those patients with archived f/taf resistance mutations maintained hiv-1 rna suppression through week 48 [14] . it should be noted that in virologically suppressed patients it is often possible to recover proviral sequences from cellular reservoirs in peripheral blood [14] . the presence of archived drug resistance mutations identified by sequencing proviral dna is not necessarily reflective of the full range of resistant variants that may have emerged in an individual. furthermore, it does not necessarily correlate with an increased risk of failure as integrated provirus is often defective [15] and reactivation of a particular virus is likely to be a stochastic event. in studies that examine drug activity in the presence of archived drug resistance, duration of follow up is crucial because the likelihood that a particular latent virus carrying a certain mutation would reactivate may increase with time, and the levels of adherence to art over time play a key modulating role. in the single arm switch study referred to earlier, drug resistance sequencing based on proviral dna missed half of m184v/i mutation present in historical genotype [13] . taken together these studies suggest that b/f/taf may be effective in maintaining virological suppression in patients with historical evidence of drug resistance mutations. in light of this, we hypothesize that switching hiv-positive patients who harbour selected drug resistance mutations and are virologically suppressed on bpi regimen to b/f/taf will maintain virological efficacy over 24 weeks. the pibik trial is a phase iv, investigator-initiated, prospective, multicentre, open label pilot, randomised two arm study to assess the safety and efficacy of switching from a bpi-based regimen to b/f/taf single tablet regimen in insti-naïve, virologically suppressed hiv-1 infected adults harbouring drug resistance mutations. the allocation ratio is 1:1 (fig. 1 ). subjects will be enrolled from seven sexual health clinics in england. these are: clinical staff in the hiv department will identify potential participants by any of the following methods: review of a clinic records/database, pre-identification of patients attending for routine care, review of notes during routine clinical follow up and posters/advertisements in the clinics to inform patients of the study. clinical staff identifying patients will be members of the direct care team or research nurses or doctors working within the same hiv multidisciplinary team. a medically qualified doctor on the study delegation log will confirm eligibility. anonymised information on participants who are not randomised / registered for consort (16) reporting will include the reason, if they are not eligible for trial participation, or if they are eligible but declined. potentially eligible participants will be invited to attend for an appointment, having been provided with a participant information sheet (pis). adequate time will be allowed for questions and to consider the study before agreeing to participate. the investigator or designee will provide adequate explanation of the aims, methods, objectives and potential hazards of the study. it will also be explained to the individual that they are free to refuse or withdraw from the study for any reason without fig. 1 trial design detriment to their future care or treatment. the investigator can decide to withdraw a subject from the study for urgent medical reasons or repeated non-compliance with the study protocol. once randomised, withdrawn subjects may be replaced if considered necessary by the chief investigator. 18 years and above on a bpi-based art regimen with documented hiv-1 rna < 50 copies/ml for at least 6 months on current regimen and at screening (a switch from tenofovir disoproxil fumarate (tdf) to tenofovir alafenamide (taf), lamivudine (3tc) to emtricitabine (ftc), or splitting co-formulated tablets to their individual component or vice versa will not be considered true regimen changes) must have a historical genotype eligible drug resistance mutations in historical genotype include at least one of the following: a single repeat of a laboratory screening test will be allowed for test results that are unexpected based on documented prior laboratory results. provides written, informed consent to participate is willing to comply with the protocol requirements if a woman and of childbearing potential and are willing to continue practicing one of the following: o must be using effective birth control methods, that is has an expected failure rate of < 1% per year and willing to continue practicing these birth control measures during the trial and for at least 30 days after the end of the trial. effective methods include iud, combined pill, contraceptive injection, implant, ius, contraceptive vaginal ring, contraceptive patches etc. o must be truly abstinent from penile-vaginal intercourse from 2 weeks prior to administration of study drug, throughout the study, and for at least 30 days after the end of the trial (when this is in line with the preferred and usual lifestyle of the subject.) [periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods), and withdrawal are not acceptable methods of contraception]. women who are postmenopausal for least 2 years, women with a total hysterectomy, and women who have a tubal ligation are considered of nonchildbearing potential. if male, and sexually-active with female partners of child bearing potential, is using effective barrier contraception, and willing to continue using this during the trial and for at least 30 days after the end of the trial. o presence of any of the following mutations: k65r/n/e o presence of multidrug resistance mutations: t69ins, q151m with or without a62v, v75i, f77l, f116y o three or more tams (m41l, d67n, k70r, l210w, t215f/y, or k219q/e/n/r) individuals experiencing decompensated cirrhosis (e.g., ascites, encephalopathy, or variceal bleeding) an opportunistic illness within the 30 days prior to screening active tuberculosis infection have been treated with immunosuppressant therapies or chemotherapeutic agents within 3 months of study screening, or expected to receive these agents or systemic steroids during the study (e.g., corticosteroids, immunoglobulins, and other immune-or cytokine-based therapies) current alcohol or substance use judged by the investigator to potentially interfere with patients' adherence to study procedure. a history of malignancy of less than 5 years or ongoing malignancy (including untreated carcinoma in-situ) other than cutaneous kaposi's sarcoma (ks), basal cell carcinoma, or resected, non-invasive cutaneous squamous carcinoma. individuals with biopsyconfirmed cutaneous ks are eligible but must not have received any systemic therapy for ks within 30 days of day 1 and are not anticipated to require systemic therapy during the study. active, serious infections (other than hiv 1 infection) requiring parenteral antibiotic or antifungal therapy within 30 days prior to day 1 (except if the parenteral therapy is for syphilis infection) any other clinical condition or prior therapy that will, in the opinion of the investigator, make the patient ineligible any known allergies to the excipients of b/f/taf fdc females who are pregnant (as confirmed by positive urine pregnancy test) women who are breastfeeding women of childbearing age not using any reliable form of contraception (e.g. intrauterine device/ intrauterine system, long-acting contraceptive injection) acute hepatitis in the 30 days prior to study entry, anyone with hepatitis c (hcv) who is likely to need direct acting antivirals in study any concomitant medications that cannot be administered with taf (i.e strong inducers of pglycoprotein) or bictegravir (dofetilide, rifampins) the investigational medicinal product in this trial is bik-tarvy® comprising fdc of b/f/taf. each film-coated tablet contains bictegravir sodium equivalent to 50 mg of bictegravir, 200 mg of emtricitabine, and tenofovir alafenamide fumarate equivalent to 25 mg of tenofovir alafenamide. eligible individuals will either continue on their bpi regimen (arm 1) or switch to b/f/taf fdc (arm 2). after 24 weeks, all participants in the bpi arm will be switched to b/f/taf arm and will be followed up for a further 24 weeks whilst those immediately switched to b/f/taf at baseline will be followed up for 48 weeks as illustrated in fig. 1 . a participant is free to withdraw from the study at any time. in addition, the investigator may decide, for reasons of medical prudence, to withdraw a participant. if a participant discontinues study medication dosing, every attempt would be made to keep the participant in the study and continue to perform the required studyrelated procedures and follow-up procedures. if this is not possible or acceptable to the subject or investigator, the participant may be withdrawn from the study. study medication may also be discontinued in the following instances: if the participant withdraws his/her consent. if the investigator considers in the interest of the subject (i.e. intercurrent illness, unacceptable toxicity) that it is best for them to withdraw their consent. the participant fails to comply with the protocol requirements or fails to cooperate with the investigator. pregnancy during the course of the study. the date and reasons for the withdrawal will be clearly stated on the participant's ecrf and source document. every attempt should be made to arrange follow up visits for participants who are withdrawn from the trial (including where individuals fall pregnant). participant adherence b/f/taf and bpi will be assessed through: pill counting at each visit by a research team member and recording of the number of pills returned self-report using a visual analogue scale (vas) participants will bring in all pill bottles at each study visit. the total number of pills remaining at each visit will be counted and, then returned to the participant to take until the bottle is finished. the percentage of compliance for each participant will be calculated. participants will also be asked be asked to self-report their level of adherence at each visit using the vas in which they would be asked to indicate their level of adherence in the previous 30 days on a scale ranging from 0 to 100% in which 0 represents no pill taken and 100 represents every single dose had been taken. where adherence is < 80%, this will lead to likely withdrawal from the study although this will be at the discretion of the investigator. the primary endpoint is the proportion of participants with hiv-1 rna < 50 copies/ml at week 24 using pure virologic response (pvr). pure virologic response is defined as follows: we considered a number of sample size scenarios bearing in mind the pilot nature of the study (table 1) . we will perform a futility analysis at 24 weeks when assessing the primary outcome. at 24 weeks, with 98 participants in the trial, we will have 80% power for 10% significance to conclude non-inferiority of the b/f/taf arm assuming a non-inferiority margin of 13% and viral suppression in 90% of participants in both arms. for the study to continue beyond 24 weeks, we need 90% (45/50) of the individuals randomised to the b/f/ taf arm to be suppressed with the lower limit of the confidence interval to just lie above 80%. recruiting 50 participants per arm would achieve this at the 90% confidence level. the sample size required decreases as the level of confidence in our estimates decreases. we have allowed 12 months to recruit 100 participants over the seven sites involved in the trial. this requires recruitment of 1-2 participants per month per site which is deemed feasible. each site has been allocated a target of 14-15 subjects. in the case of slow enrolment, additional sites will be offered participation in the study. the web-based sealed envelope™ system will be used to allocate individuals randomly to arm 1 and arm 2. the statistician will provide the randomisation list. each study site will be provided with a randomisation guide. the sealed envelope™ system will randomise subjects within 8 strata as shown in table 2 . the purpose of the stratification is to balance the treatment arms on important prognostic factors such as: the bpi used in the subject's baseline regimen (atazanavir or darunavir) use of lipid lowering therapy at study day 1 (yes/no) number baseline mutations of the nrti class (< 2 vs. ≥2) investigators randomise patients by completing an onscreen form with patient details, stratification factors, inclusion and exclusion criteria. investigators are immediately shown the treatment allocation. trial managers have real-time access to recruitment statistics and are notified by email of every new randomisation. the randomisation application conforms to the requirements of fda 21 cfr part 11, electronic records; electronic signatures and ich gcp. no-one can delete records from the randomisation database, so that all randomisations have to be accounted for. audit log files detailing all activity on the randomisation system are available to the trial manager. neither the investigators nor the participants will be blinded to the treatment allocation. the presence of resistance mutations will be determined using historical genotype results obtained in local laboratories in those eligible according to the inclusion and exclusion criteria. we would obtain more information on resistance mutations by sequencing cellassociated hiv-1 dna in peripheral blood mononuclear cells prior to commencing b/f/taf. the results of proviral dna sequence will not be available in real time and will not be used to inform treatment decisions but will further the understanding of the clinical importance of archived resistance mutations, if any, in individuals developing virological failure. the baseline visit will not exceed 30 days after the screening visit. follow up of participants will continue until all participants have accrued 48 weeks from their baseline visits. individuals who have completed week 48 visit will be followed up 30 days post cessation of trial treatment via a telephone call or a standard of care clinical appointment for performance of the following assessments; adverse events and symptoms review, hiv associated conditions, concomitant medications and for women of child bearing potential (wocbp), confirmation that contraception has been used in the previous 30 days. study procedures, screening, randomisation and safety monitoring will be according to attached visit schedule in table 3 . individuals with virological failure defined as a rebound in hiv-1 rna ≥ 50 copies/ml, which is subsequently confirmed at the following scheduled or unscheduled visit. following the initial detection of virological rebound, subjects will be asked to return to the clinic for a scheduled or unscheduled blood draw (2 to 3 weeks after the date of the first measured rebound) for repeat viral load testing. if virological rebound is confirmed and the hiv-1 rna is ≥200 copies/ml, the blood sample from the confirmation visit will be the primary sample used for hiv-1 genotypic testing. after a participant's first post-baseline resistance test, additional testing will be conducted on a case-by-case basis. any participant may be discontinued at the investigator's discretion or per local treatment guidelines. if no resistance is detected from the genotype, the participant may remain on study drugs and a repeat hiv-1 rna measurement should be performed (2 to 3 weeks after date of test with hiv-1 rna ≥ 50 copies/ml). investigators should carefully evaluate the benefits and risks of remaining on study drug for each individual participant and document this assessment in the on-site medical record. data on patient reported outcome measures will be collected using the hiv-si and the psqi. the hiv-si is a validated, self-administered 20-item health-state questionnaire for use in clinical care and research amongst people living with hiv (plhiv)in order to identify and address common and bothersome symptoms associated with hiv treatment and disease [16] . the instrument is considered to be the gold standard in contemporary hiv-symptom research [17] . respondents will be asked about their experience with each 20 symptoms during the past 4 weeks using a 5-point likert scale. response options and scores are as follows: 0) i don't have this symptom, 1) i have this symptom and it doesn't bother me, 2) i have this symptom and it bothers me a little, 3) i have this symptom and it bothers me, 4) i have this symptom and it bothers me a lot. the pittsburgh sleep quality index (psqi) is a selfrated questionnaire which assesses sleep quality and disturbances over a 30-day recall period [18] . nineteen individual items generate seven component scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction. it uses a likert scale in with the following scores: 0) not during the past month, 1) less than once a week, 2) once or twice a week, 3) three or more times a week. the sum of scores for these seven components yields one global score (0 to 21). a total score of 5 or greater is indicative of poor sleep quality. participants with early study termination from whatever cause will undergo the assessments outlined in table 3 . a source data worksheet will be created to capture all the relevant information and will be filed as source documentation in the participants' notes. questionnaires and self-report vas scores will also be completed by the patients. a source data agreement will be completed prior to recruitment commencing to ensure all parties are aware which documents constitute source data. data from the source will be entered onto the electronic case report form (ecrf) on the web-based macro™ electronic data capture system. data entered will be checked by the data manager in accordance with the data management plan (supplementary appendix) and queries raised to the clinical sites via macro when appropriate. clinical sites will be responsible for the entry of data into the ecrf. patient data will be entered using study number only and no patient identifiable data will be seen by the data management team. direct access will be granted to authorised representatives from the sponsor, host institution and the regulatory authorities when appropriate to permit trial-related monitoring, audits and inspections. archiving will be the responsibility of the sponsor and all documentation will be archived for 25 years, with the exception of the health records which will be kept in accordance with uk law and local policy. the sponsor named archivist will be responsible for ensuring the documentation is prepared in line with the relevant sponsor standard operating procedures. all randomised patients who received at least one dose of the study medication will be included in the analysis. summary statistics will be presented by trial arm using median with interquartile ranges for continuous variables with skewed distributions or mean with standard deviations for normally distributed variables. categorical variables will be summarised using frequencies and proportions. non-inferiority for the futility analyses will be concluded if the lower bound of a two-sided 90% ci for the difference in proportions (b/f/taf minus boosted pi) of patients with plasma hiv-1 rna < 50 copies/ml at week 24 is greater than − 13%. primary efficacy endpoint proportions of individuals with hiv rna < 50 copies/ml at 24 weeks will be estimated using pvr. the percentage of participants with pvr for hiv-1 rna cut-off at 50 copies/ml at week 24 will be summarized. differences between trial arms will be estimated together with 95% confidence intervals. secondary efficacy endpoint proportions of individuals with hiv rna < 50 copies/ml at 48 weeks will be estimated using pvr. the percentage of participants with pvr for hiv-1 rna cut-off at 50 copies/ml at week 48 will be summarized. differences between trial arms will be estimated, together with 95% confidence intervals. the proportion of patients with hiv-1 rna < 50 copies/ml at weeks 24 and 48 using pvr in those with any archived resistance detected in proviral dna will be estimated. the proportion of patients with any emergent drug resistance following virological rebound will be estimated. the analysis of the following secondary safety outcomes will be presented as the estimated difference and 95% confidence interval between arms from baseline to 24 and 48 weeks mean total cholesterol, ldl, hdl and triglycerides will be estimated. mean hba1c mean weight and bmi laboratory and clinical adverse events will be described and summarised using percentages. treatment differences for patient reported outcomes using the hiv-si module and the psqi will be compared by using the prevalence of symptoms reported by each method and presented with 95% confidence intervals. consistent with prior analyses on hiv-si [19, 20] we would dichotomise symptoms into not bothersome (scores of 0 or 1) or bothersome (scores of 2, 3 and 4). the overall bothersome symptom count at baseline will be generated by counting the number of individual symptoms scored as bothersome. reported poor sleep quality scores on the psqi will be summarised by the seven components as well as the global scores by arm. the global scores will be dichotomised into poor sleep quality (score of < 5) and good sleep quality (≥5) and an exact 95% binomial confidence interval presented for the difference in prevalence between arms. . in the event of missing data, only available data will be included in the analyses and missing data will be quantified but not imputed. for missing data relating to primary and secondary efficacy endpoints, using the principle of pvr, the last known measured viral loads will be carried forward if data is missing at the 24 and 48 week time points. a three-member independent data safety monitoring board (dsmb) has been established comprising specialists in clinical infectious diseases, hiv medicine and a clinical trial statistician. the role of the dsmb will be to safeguard the interest of the trial participants, assess safety and efficacy of the intervention and to monitor the overall conduct of the trial. the dsmb should receive and review the progress and accruing data of the trial and provide advice on the conduct of the trial to the trial steering committee (tsc). the dsmb would perform an interim review of the trial's progress including updated figures on recruitment, data quality, main outcomes and safety data and will have responsibility on the decision whether to stop or continue the trial. the dsmb will meet six-monthly but the frequency of meetings may depend on recruitment rates or other trial events. further details on the dsmb charter can be found in the supplementary appendix. information on all adverse events (aes), adverse reactions (ars), serious adverse reactions (sars), suspected unexpected serious adverse reactions (susars) and serious adverse events (saes) will be documented in the case report forms. aes, saes, ars, sars and susars may be directly observed, reported spontaneously by the participant or by questioning the participant at each study visit. these will be followed up until they are resolved or the participant's participation in the study ends (i.e. until the final crf is completed for that participant). any untoward event that may occur subsequent to the reporting period that the investigator assesses as related to the study drug medication will also be reported as an adverse event. the adverse event reporting period will be from consent until the participant's final study visit. after informed consent, but prior to initiation of study treatment, all saes and adverse events related to protocol-mandated procedures would be reported on the crfs. following initiation of study treatment, all aes, regardless of cause or relationship until 30 days post cessation of trial treatment would be reported on the crfs. in addition, all serious adverse events assessed by the investigator as related to the investigational medication would continue to be followed even after participation in the study is over. such events would be followed until resolution, or until no further change can reasonably be expected. this study is registered with the international standard randomised controlled trials number registry (44453201) and with the european union drug regulating authorities clinical trials database (2018-004732-30). the main study findings will be reported in accordance the consolidated standards of reporting trials (consort) statement [21] . the study received ethical approval from the health research authority (19/lo/ 0905) and will be conducted in accordance with the declaration of helsinki. written, informed consent will be sought from participants by an appropriate member of the research team identified on the delegation log and this is mandatory prior to any study procedures. participants would be made aware that they may not continue to be prescribed b/f/taf after the end of the trial unless they are eligible according to nhs england prescribing criteria for tenofovir alafenamide. in this situation, participants would be switched to alternative efficacious art combination decided by the local principal investigator. all investigators and trial site staff will comply with the requirements of the general data protection regulation 2018 (gdpr) [22] with regards to the collection, storage, processing and disclosure of personal information and will uphold the act's core principles. personal information will be collected, kept secure, and maintained. this will involve the creation of coded, depersonalised data where the participant's identifying information is replaced by an unrelated sequence of characters, secure maintenance of the data and the linking code in separate locations using encrypted digital files within password protected folders and storage media and limiting access to the minimum number of individuals necessary for quality control, audit, and analysis. the confidentiality of data will also be preserved when the data are transmitted to sponsors and co-investigators by using only pseudonymised codes rather than personal identifiable information. trial data will be stored for 25 years and the principal investigator at site is the data custodian. it is plausible that carefully selected hiv-positive individuals with pre-treatment or treatment-acquired resistance who are virologically suppressed on their current pi-based regimen could maintain virological efficacy when switched to b/f/taf fdc. the hypothesis is based on a consideration of each component of b/f/taf. taf, like the earlier version tenofovir disoproxil fumarate (tdf), is a prodrug of tenofovir. however, taf yields 5-fold higher intracellular concentrations of the active moiety tenofovir diphosphate (tfv-dp) in hiv target cells than tdf, despite much lower plasma drug concentration [23] .. since taf and tdf produce the same active metabolite, they have similar resistance profiles but it could be proposed that the higher intracellular concentrations of tfv-dp yielded by taf could be beneficial in resistant viral isolates [24] . furthermore, the selective conversion of taf to tfv-dp within hiv target cells and lower levels in plasma is associated with less renal and bone toxicity [25] . hiv-1 strains harbouring the m184v/i mutation, which causes high-level resistance to 3tc and ftc, display increased or restored susceptibility to tenofovir [26] . m184v/i mutants also display a loss of fitness that reduces their replication capacity and may account for the partial residual activity of 3tc in the presence of the mutation. in art-experienced individuals who developed virological failure whilst treated with either zidovudine or stavudine, the stepwise accumulation of tams resulted in increasing resistance to tenofovir, with three or more tams being associated with markedly reduced tenofovir susceptibility [27] . this cross resistance to tenofovir is more marked for the tam-1 pathway of mutations (m41l, l210w, and t215y) than the tam-2 pathway of mutations (d67n, k70r, k219/e/n/q/r, and t215f). as a result, we have allowed a maximum of 2 tams when assessing eligibility for inclusion in the trial. the revertant mutations t215s/c/d/e/i/v/n/a/l which arise from viruses that once harboured t215y/f do not directly impact nrti susceptibility [28] . however, both in vitro and in vivo, the effect of tams on tenofovir is partially reversed by the presence of the m184v/i mutation [29, 30] with 3tc maintaining residual activity in viruses harbouring this mutation [31] . bictegravir has potent in vitro activity against laboratory strains and clinical isolates of hiv-1, a higher genetic barrier to resistance development than raltegravir (ral) and elvitegravir (evg), and a statistically improved resistance profile compared to ral, evg, and dtg against a set of patient derived insti-resistant viral isolates [32] . in the earnest [33] , and mobidip [31] studies, bpi given with nrti in individuals with previous virological failure and predicted limited nrti activity due to resistance (mainly m184v/i and tams) achieved high rates of virological suppression. in the dawning study [12] , dtg a high genetic barrier insti, demonstrated superior virological efficacy over a bpi. hence there is a strong scientific plausibility for bictegravir demonstrating high rates of virological efficacy in the presence of a limited pattern nrti mutations when switching patients from bpi regimen to b/f/taf. we do not foresee a challenge to recruiting the 100 participants required for this trial, however recruitment will be monitored closely and if sluggish, we would activate additional sites for participation in the study. since all sites will be utilising standardised study documents and procedures, the number of study sites should not affect data quality. furthermore, all sites will be closely monitored for compliance with the study protocol. the trial started enrolling participants on 16 september 2019 and it is anticipated that enrolment will continue until september 2020. cardiovascular disease and use of contemporary protease inhibitors: the d:a:d international prospective multicohort study metabolic effects of darunavir/ritonavir versus atazanavir/ritonavir in treatment-naive, hiv type 1-infected subjects over 48 weeks development and validation of a risk score for chronic kidney disease in hiv infection using prospective cohort data from the d:a:d study efficacy and tolerability of 3 nonnucleoside reverse transcriptase inhibitor-sparing antiretroviral regimens for treatment-naive volunteers infected with hiv-1: a randomized, controlled equivalence trial combination antiretroviral therapy and the risk of myocardial infarction contemporary protease inhibitors and cardiovascular risk efficacy and safety of switching to fixed-dose bictegravir, emtricitabine, and tenofovir alafenamide from boosted protease inhibitor-based regimens in virologically suppressed adults with hiv-1: 48 week results of a randomised, open-label, multicentre, phase 3, non-inferiority trial switching from a ritonavir-boosted protease inhibitor to a dolutegravirbased regimen for maintenance of hiv viral suppression in patients with high cardiovascular risk second-line antiretroviral treatment successfully resuppresses drug-resistant hiv-1 after first-line failure: prospective cohort in sub-saharan africa effectiveness of protease inhibitor/nucleos(t)ide reverse transcriptase inhibitor-based second-line antiretroviral therapy for the treatment of human immunodeficiency virus type 1 infection in sub-saharan africa: a systematic review and meta-analysis switch to a raltegravir-based regimen versus continuation of a lopinavir-ritonavir-based regimen in stable hiv-infected patients with suppressed viraemia (switchmrk 1 and 2): two multicentre, double-blind, randomised controlled trials superior efficacy of dolutegravir (dtg) plus 2 nucleoside reverse transcriptase inhibitors (nrtis) compared with lopinavir/ritonavir (lpv/r) plus 2 nrtis in second-line treatment: 48-week data from the dawning study a phase 3b open-label pilot study to evaluate switching to elvitegravir/ cobicistat/emtricitabine/tenofovir alafenamide (e/c/f/taf) single-tablet regimen in virologically-suppressed hiv-1 infected adults harboring the nrti resistance mutation m184v and/or m184i (gs-us-292-1824): week 24 results resistance analyses of bictegravir/emtricitabine/tenofovir alafenamide switch studies defective proviruses rapidly accumulate during acute hiv-1 infection development and validation of a self-completed hiv symptom index patient reported outcome instruments used in clinical trials of hiv-infected adults on nnrti-based therapy: a 10-year review the pittsburgh sleep quality index: a new instrument for psychiatric practice and research patientreported symptoms over 48 weeks among participants in randomized, doubleblind, phase iii non-inferiority trials of adults with hiv on co-formulated bictegravir, emtricitabine, and tenofovir alafenamide versus co-formulated abacavir, dolutegravir, and lamivudine vacs project t. patient-reported symptoms on the antiretroviral regimen efavirenz/ emtricitabine/tenofovir statement: extension to randomised pilot and feasibility trials guide to the general data protection regulation (gdpr) antiviral activity, safety, and pharmacokinetics/pharmacodynamics of tenofovir alafenamide as 10-day monotherapy in hiv-1-positive adults characterization of hiv-1 resistance to tenofovir alafenamide in vitro tenofovir alafenamide: a novel prodrug of tenofovir for the treatment of human immunodeficiency virus relationships among various nucleoside resistance-conferring mutations in the reverse transcriptase of hiv-1 genotypic and phenotypic predictors of the magnitude of response to tenofovir disoproxil fumarate treatment in antiretroviral-experienced patients stanford university hiv drug resistance database. marvel on rt mutations at position tenofovir resistance and resensitization phenotypic susceptibilities to tenofovir in a large panel of clinically derived human immunodeficiency virus type 1 isolates boosted protease inhibitor monotherapy versus boosted protease inhibitor plus lamivudine dual therapy as second-line maintenance treatment for hiv-1-infected patients in sub-saharan africa (anrs12 286/ mobidip): a multicentre, randomised, parallel, open-label, superiority trial antiviral activity of bictegravir (gs-9883), a novel potent hiv-1 integrase strand transfer inhibitor with an improved resistance profile assessment of second-line antiretroviral regimens for hiv therapy in africa publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank all the members of the pibik study group in all the participating sites, the data safety and monitoring board, the trial steering committee and the research and enterprise department of the university of sussex. received: 6 april 2020 accepted: 7 july 2020 supplementary information accompanies this paper at https://doi.org/10. 1186/s12879-020-05240-y.additional file 1. authors' contributions ci conceived the study and drafted the manuscript. dc, co, lw, sb, np, amg contributed to the design of the study and assisted in drafting the manuscript. dl, cb developed the data management plan and data collection tools for the study and assisted in drafting the manuscript. yt is responsible for the coordination of the study and contributed to the manuscript draft. ci obtained funding for the study. all authors have read the final manuscript and give approval for it to be published. availability of data and materials not applicable. this trial protocol has been approved by the health research authority, uk (19/lo/0905). all patients in the trial will provide written informed consent to participate. competing interests ci has received honoraria, support to attend conferences and research funding (paid to university of sussex) from gilead sciences. dc has received honoraria and support to attend conferences from gilead sciences and viiv. lw has received speaker/advisory fees or conference support from gilead, viiv, janssen, msd, cipla and mylan. co has been a recipient of grants, speaker's bureau and travel sponsorship from gilead, msd, viiv, gsk and janssen. np has received honoraria for services rendered to gilead sciences. amg receives personal consultancy fees from roche pharma, provides consulting services to gilead sciences, janssen, and viiv (paid to the university of liverpool) and is the recipient of research funding from gilead, roche pharma, and viiv (paid to the university of liverpool). all other authors declare that they have no competing interests. key: cord-262017-utvy0i8l authors: tobar vega, pool; erramilli, shruti; lee, eugene title: talaromyces marneffei laboratory cross reactivity with histoplasma and blastomyces urinary antigen date: 2019-06-21 journal: int j infect dis doi: 10.1016/j.ijid.2019.06.018 sha: doc_id: 262017 cord_uid: utvy0i8l talaromyces marneffei is a fungal opportunistic infection usually seen in immunocompromised patients from eastern countries. in the us when examining hiv-patients for suspected fungal infections, laboratory serological tests guide therapy until cultures are available. we present the case of a 35-year-old hiv patient originally from thailand in which urine lab results were positive for blastomyces and histoplasma antigen, but biopsy showed t. marneffei. concomitantly the patient presented with hyponatremia which was deemed to be from siadh. we present the first case of a patient with t. marneffei cross reactivity with blastomyces, histoplasma and siadh due to pulmonary disease. endemic to southeast asia, east asia and china, talaromyces marneffei is a dimorphic fungus capable of causing systemic fungal infections in immunocompromised patients (supparatpinyo et al., 1994) . since its discovery in the 1950s, the majority of cases have been documented in hiv patients with low cd4 counts. in northern thailand, t. marneffei is the fourth most prevalent opportunistic infection in this population (chariyalertsak et al., 2001) . clinical manifestations include fever, malaise, lymphadenopathy, cough, and hepatosplenomegaly (wu et al., 2008) . while the frequency of t. marneffei infection has decreased with the advent of retroviral therapy, if left untreated the infection frequently leads to respiratory failure with a poor prognosis. in the u.s. patients with hiv infection usually undergo testing for endemic fungal infections such as blastomyces, histoplasma, coccidioides and paracoccidioides. indirect serological results help to make faster decisions given that cultures take several days or weeks to grow. clinical and geographic context plays a particularly important role because some of these tests have been shown to have cross-reactivities. pulmonary infection either by fungi, bacteria or virus has been observed to cause concomitant hyponatremia, with inappropriate levels of antidiuretic hormone (siadh) often found as the underlying etiology. the exact mechanism is not understood but hypoxemia and hypercapnia are thought to play an important role in the pathophysiology (rose et al., 1984) . in the following, we describe a t. marneffei infection with unusual laboratory and clinical characteristics. the patient was a 35-year-old male from thailand who presented with generalized weakness and fever. his past medical history was relevant for hiv infection (since age 21) on haart (bictegravir, emtricitabine & tenofovir alafenamide). two weeks prior to his admission, he had travelled to chicago, las vegas and utah. during this time, he developed a productive cough with blood-tinged sputum, subjective fever, chills, and anorexia with associated weight loss. on physical exam, he was noted to have multiple erythematous, raised, scaly/crusted lesions on the face, neck and abdomen (figure 1 hiv viral load was 3.12 * 10 6 copies/ml and cd4 count was 8 cells/ml. right upper quadrant ultrasound revealed hepatomegaly and a chest x-ray reported bilateral peri-tracheal soft densities up to 3 cm in diameter, interstitial markings, and bilateral pulmonary nodules. chest ct scan without contrast showed patchy pulmonary densities and multiple peri-hilar nodules ( figure 2 ). ct scan with contrast of the head and neck did not reveal acute intracranial abnormalities but did show cervical lymphadenopathy. gram stain smear and culture of the sputum were negative. respiratory viral panel including influenza, parainfluenza, coronavirus and rsv was negative. legionella urine antigen was negative. bacterial blood cultures and gram stain were negative. quantiferon gold and three sputum samples for afb/culture were negative for tuberculosis. serological testing for cryptococci and blastomyces were negative. however, urine antigen testing for both blastomyces and histoplasma were positive. finally, a biopsy of one of the cutaneous lesions demonstrated dermal and subcutaneous neutrophil and histiocyte infiltrate with the presence of intracellular yeast, findings which were consistent with t. marneffei. t. marneffei infections typically manifest in severely immunocompromised patients. current guidelines recommend that in hiv patients from endemic countries with a cd4 count <100 cells/ml, primary preventive therapy with itraconazole should be initiated (panel on opportunistic infections in hiv-infected adults and adolescents, 2019). clinical manifestations appear to vary depending on the severity and underlying etiology of immune compromise in the patient, differing between hiv vs. non-hiv causes such as malignancies or transplant patients. fever, splenomegaly, anemia, transaminitis, and absence of leukocytosis seem to be more frequently found in hiv-positive patients (kawila et al., 2013) . in our case, clinical findings included fever, neck lymphadenopathy, and respiratory symptoms. laboratory work demonstrated transaminitis along with a cd4 count of 8 cells/ml. an initial laboratory test for endemic fungi can guide initial treatment towards early antifungal medication. however, crossreactivity between antigens in various fungal infection detection tests is well-documented, and cross reactions between histoplasma and blastomyces antigens are the most common (wheat et al., 1986) . others have been described, such as that of histoplasma antigen in patients with sporotrichosis (assi et al., 2011) . in our case, urine antigen testing results for both histoplasma and blastomyces were positive, serum testing was negative. it is of note that the sensitivities of the blastomyces and histoplasma antigen detection test in urine are approximately 80% and 89% respectively. specificity is around 90% for both tests, usually having to rule out each other as the main confounder (cunningham et al., 2015; frost and novicki, 2015) . hiv status can affect tests based on antibody detection. since the tests used to guide therapy are based on antigen detection, sensitivity is unlikely to be affected by hiv infection. these infections in their disseminated forms will receive amphotericin-b with itraconazole. however, differentiation is important given that blastomycosis is treated for a year versus talaromyces which is treated for 12 weeks (sirisanthana et al., 1998; saccente and woods, 2010) . siadh is an exclusion diagnosis that requires an extensive work up to rule out other etiologies including adrenal insufficiency, thyroid disease, and volume depletion (shu et al., 2018) . adh is produced on the paraventricular thalamic nucleus and thus classically this syndrome is observed after neurological insults that cause an excess in adh. nevertheless, it has been observed that respiratory tract infections can cause inadequate adh secretion and these are the most common infections in hiv patients. increase in the a-a gradient and hypoxia/hypercapniainduced adh secretion are some of the non-osmotic mechanisms thought to trigger elevations in adh in this population. it has been proposed that hypercapnic acidosis and hypoxemia induce central release of vasopressin through peripheral chemoreceptors and baroreceptors stimulation respectively (rose et al., 1984; dreyfuss et al., 1988) . tuberculosis, cryptosporidium, plasmodium infections and pneumocystis pneumonia have been previously reported as pulmonary infections causing siadh. it is of note that hiv by itself could contribute to siadh. but the mechanism underlying this infection is usually mediated by hiv induced thyroid and adrenal insufficiency. in our case, the patient had increased urinary sodium, decreased serum osmolality, normal cortisol and tsh levels and absence of neurological affect, leaving the pulmonary fungal infection as one of the explanations for inadequate adh secretion. in conclusion, laboratory work up for endemic fungal infection can have false positive results with infections such as talaromyces. this cross reactivity is especially important when assessing patients from endemic countries. manifestations of t. marneffei infection are diverse, and disease description is limited due to the small number of cases. a novel manifestation observed in our patient was the presence of siadh likely secondary to the respiratory talaromyces infection. to our knowledge, this is the first case reporting systemic mycosis due totalaromyces marneffei with associated hyponatremia secondary to siadh and cross-reactivity with blastomyces and histoplasma in urine antigen testing. cross-reactivity in the histoplasma antigen enzyme immunoassay caused by sporotrichosis clinical presentation and risk behaviors of patients with acquired immunodeficiency syndrome in thailand, 1994-1998: regional variation and temporal trends sensitivity and specificity of histoplasma antigen detection by enzyme immunoassay acute infectious pneumonia is accompanied by a latent vasopressin-dependent impairment of renal water excretion blastomyces antigen detection for diagnosis and management of blastomycosis panel on opportunistic infections in hiv-infected adults and adolescents. guidelines for the prevention and treatment of opportunistic infections in hiv-infected adults and adolescents: recommendations from the centers for disease control and prevention, the national institutes of health, and the hiv medicine association of the infectious diseases society of america antidiuresis and vasopressin release with hypoxemia and hypercapnia in conscious dogs clinical and laboratory update on blastomycosis hiv/aids-related hyponatremia: an old but still serious problem amphotericin b and itraconazole for treatment of disseminated penicillium marneffei infection in human immunodeficiency virus-infected patients disseminated penicillium marneffei infection in southeast asia evaluation of cross-reactions in histoplasma capsulatum serologic tests clinical presentations and outcomes of penicillium marneffei infections: a series from aknowledgement is given to dr saad, peguy who proof read this manuscript. consent was obtained from patient for the publication of this manuscript.consent was obtained from patient for the publication of this manuscript. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors declare no conflict of interest. key: cord-017782-dtveihrj authors: fong, i. w. title: litigations for hiv related complications date: 2010-11-30 journal: medico-legal issues in infectious diseases doi: 10.1007/978-1-4419-8053-3_13 sha: doc_id: 17782 cord_uid: dtveihrj in 1992, a 27-year-old male with same sex exposure requested human immuno-deficiency virus (hiv) testing anonymously at a walk-in clinic. he was advised that the test (hiv serology) was positive and he requested a repeat test (anonymously) 1 month later, which was also reported as being positive. about 2 years later, he was assessed by a general practitioner for symptoms of depression and continued medical care. at that time, investigations revealed a cd4 t-cell count of about 700 cells/ul. sometime in 1996 a repeat blood test revealed a cd4 cell count just <500 cells/ul. no consultation to an infectious diseases specialist or hiv clinic was made. the gp(general practitioner) then initiated a regimen consisting of didanosine, lamivudine, and saquinavir for hiv infection. at that time, testing for hiv viral load was not generally available to the medical community, but became procurable in 1997. initially, the patient tolerated the regimen well and over the next 3 years his cd4 cell count was maintained above 600–700 cells/ul and the hiv viral load remained undetectable (<50 copies). however, the patient started to show morphologic changes of moderate facial and peripheral lipoatrophy, developed mild sensory peripheral neuropathy, and increased liver enzymes attributable to fatty liver, and elevations of the fasting serum glucose. in the summer of 2000, although the cd4 cell count remained stable, the hiv viral load was reported as being over 7,000 copies/ul. at this time, the patient was referred to a university hospital hiv clinic. load of >7,000 copies/ul came to the conclusion that there was either a mix-up in the blood specimen samples or error in the labeling or reporting. efforts to verify or clarify the initial hiv serology were unsuccessful as no permanent records were kept for anonymous hiv serology results. the patient (plaintiff ) initiated litigation against the gp (defendant) for medical malpractice. specific charges were: (1) the gp should have repeated the hiv serology to confirm that the plaintiff was hiv infected, (2) the defendant was negligent in starting treatment for hiv infection without proof of disease, (3) the physician lacked knowledge of hiv infection and should have referred the patient to a specialist or hiv clinic, (4) treatment of toxic medications were given for several years without any clear indication, and (5) the gp did not adequately inform the patient on the pros and cons of therapy, nor explain the potential toxicities and side-effects. financial compensation by the plaintiff was sought for psychological suffering over the years with the false impression that he was hiv infected, and physical suffering from the side effects of the medications and the need to take unnecessary large amounts of pills for several years. the side effects had affected his social life and left a permanent physical stigma, and also adversely affected his performance at work (due to absenteeism from adverse events). the latter had resulted in his inability to perform at a high level and thus retarded his progress in his career path. all these effects have indirectly affected his earning ability over 3-4 years, and also future earning capacity. the present aids pandemic is caused by the hiv-1 strain and hiv-2 is predominantly found in west and central africa but is rare in developed nations. seroconversion after exposure usually occurs within 2 weeks to 3 months, but occasionally may take 12 months or longer. 1 delayed or protracted time for seroconversion may be seen especially in immunosuppressed subjects. 2 usually by 6 months after exposure, seroconversion should occur in 95% or more of cases. 3 a period of viremia and antigenemia without detectable antibodies occurs within 4-6 weeks of initial hiv infection. at this phase, high levels of plasma p24 antigen or viral rna can be detected, and the viremia and antigenemia decline to very low levels coinciding with seroconversion. detection of antibodies to hiv remains the most cost-effective and commonly used method to prove hiv infection. enzyme-linked immunoassay (elisa) is the most commonly used assay to test for hiv-1 and hiv-2 because of its low cost, standardized procedure, reliability, and rapid turnaround. 1 for experienced laboratories under optimal conditions (commonly licensed kits) the sensitivity and specificity of the elisa are both 99%. 1 false negative reactions can occur in infected persons early in the course before seroconversion and in immunosuppressed patients. false positive elisa results can occur for various reasons, including human error, variability in the test kits, hemodialysis, auto-immune disease, multiple myeloma, hemophilia, alcohol hepatitis, positive rapid plasma regain (rpr) test, and for unknown reasons (idiopathic). 1 the elisa uses hiv antigen to bind igg hiv antibodies in the test sample. the western blot test (wb) is the most commonly used confirmatory test for the presence of hiv specific antibodies. compared to elisa, the wb is more expensive, time-consuming and requires more technical expertise to interpret. false negative wb can also occur in the very early phase of hiv infection before development of antibodies. false positive reactions can occur in auto-immune disorders, polyclonal gammopathies, hyperbilirubinemia, subjects with human leukocyte antigen (hla) antibodies, and healthy individuals. in low risk populations, the chance of false-positive reaction of elisa and wb combined is extremely low -1 in 135,000. 4 the probability of another test being false positive in the same person tested at another time for both tests would be 1:135,000 â 135,000 or 1 in 18 billion chance. although the polymerase chain reaction (pcr) can be used to detect the hiv genome before antibody production, the pcr is highly prone to contamination with nucleic acids, which causes many false-positive reactions and therefore has not been recommended for diagnostic purposes. these pcr tests are thus mainly used for serial measurements of plasma hiv-1 rna for quantitation over the range of 75-500,000 rna copies/ml to monitor progress and response to therapy. in high risk populations, detection of hiv-dna by pcr has been found to have falsepositive rates of 2-3.4%. 5, 6 data from bayer on the versant hiv-1 rna 3.0 assay (bdna) found that all 22 of 912 false-positive samples quantitated were 1,000 copies/ml or less (personal communication with dr. r. ziermann from bayer). the main issue in this case is related to acceptance of a patient's history of a serious disease (from a test performed elsewhere) without verifying the results. although physicians commonly accept the history of a patient's underlying illness as valid, treatment for a disorder with potentially toxic agents should always require verification of the diagnosis. it could therefore be argued that the gp was remiss in instituting a cocktail of medications without having a confirmed copy of the test result for hiv infection. this is particularly damaging for an asymptomatic subject with no history of opportunistic infection or clinical evidence of aids complication. moreover, a cd4 count cannot be used as a surrogate marker for the diagnosis of hiv infection. although the cd4 + t lymphocyte quantitative count is a very useful and standard test to monitor patients for progression of hiv disease or response to therapy, it can be low in many conditions. the normal cd4 + t lymphocyte count usually averages 8.0-10.5 â 10 8 cells/l (800-1,050/mm 3 ), but the range of normality (2 standard deviations of the mean) is quite wide (500-1,400 cells/mm 3 ). 7 about 80% of the normal blood lymphocytes are t lymphocytes and nearly twothirds of blood t lymphocytes are cd4 + (helper) lymphocytes and most patients with lymphocytopenia have reduction in absolute number of cd4 + t lymphocytes. 8 there are many conditions that can be associated with lymphocytopenia and lower than normal cd4 + lymphocyte count. although hiv infection is the most common viral infection associated with cd4 + lymphocytopenia, other viral infections can transiently decrease cd4 + cell counts (including measles, corona viruses and others). 8 the list of conditions associated with cd4 + lymphocytopenia (besides viral infections) include bacterial and fungal sepsis (including tuberculosis), major surgery, recent trauma or hemorrhage, malignancy, glucocorticoid use, cytotoxic chemotherapy, radiotherapy, auto-immune diseases, nutritional deficiencies, organ transplantation, acquired common variable immunodeficiency, and idiopathic cd4 + lymphocytopenia. 8 furthermore, it is common to observe biologic variations in the absolute cd4 + cell count even in hiv infected subjects without any other factors. a healthy adult may have, at some time, transient decrease in cd4 + cell count below 500. whether the plaintiff's cd4 + cell count decline was due to viral upper respiratory tract infection or other causes was not clear. in hiv-infected patients, the t lymphocytes decline by 4% per year for every log 10 hiv rna copies/ml in the plasma. 7 currently, the optimal time to start antiretroviral therapy (art) for asymptomatic hiv patients is not clear. there is consensus that patients with aids complications or symptomatic disease should be started on art. there is still controversy as to the optimal time to initiate art in asymptomatic patients. some guidelines recommend considering starting art below 350 cells/mm 3 and others recently <500 cells/mm 3 , but there are no randomized controlled trials to provide a clear answer. how can we resolve the issue of two hiv serology tests taken at separate times in the same subject being false positive? there are several possibilities, none of which can be proven in or out of court. it is possible, that since blood samples taken in the clinic were labeled with a code number to provide anonymity, that the samples were mislabeled and originated from a truly hiv infected subject. however, the chance of that occurring twice in a row would be extremely low or unlucky. it is also possible that the plaintiff suffered from a mental disorder or delusion (such as munchausen's syndrome) and imagined that he had a positive hiv serology. there was no indication of a psychiatric disorder from the gps office records. rare false claims of a medical disease (including hiv infection) may be encountered under unusual conditions where the person can expect some form of material gain, i.e., financial, improved living conditions, sympathetic reduction in sentences for criminal offenses, etc. none of these appeared evident from review of the records. feigned hiv infection has been reported in malingering patients [9] [10] [11] and in young women with psychosocial disorders with history of prolonged sexual, physical and emotional abuse. 12 a retrospective study from an hiv clinic in a municipal hospital identified seven patients with fictitious hiv infections, six of whom had a history of illicit narcotic abuse. 13 a survey of ten other local hospitals found that known cases of alleged (fictitious) hiv infection occurred at eight of the hospitals but only one of the ten hospitals routinely documented hiv infections before initiating care. 13 in a specialist hiv unit in central london over a 5-year period, 12 patients (1.7% of admissions) with feigned hiv/aids were identified. 14 a young man, aged 36 years, presented to a new family physician (fp) in 1994 with symptoms of 15 lb weight loss, chronic diarrhea for 3 weeks and night sweats. he was found to be unwell, with evidence of significant weight loss from wasting, oral thrush, and oral hairy leukoplakia. an hiv serology performed was positive (screen and confirmatory), and his cd4 + lymphocyte count was 80 cells/mm 3 . he was started on antiretrovirals and prophylaxis for pneumocystic pneumonia. the patient was a practicing homosexual with multiple partners (none of whom were known hiv infected) and he used condoms sometimes, but inconsistently for sexual encounters. he had no known past medical illness and claimed to have previously tested negative for hiv infection in 1988. he claimed to have requested an hiv test in 1990 but there was no record of this in his previous fp records. an hiv serology was performed in the summer of 1993 (which was positive), but the patient was never informed of the results and apparently was lost to follow-up. the records subsequently in 2000 indicated that the young man was attending an hiv clinic regularly with no opportunistic infection and was clinically stable on a combination of art with a stable cd4 + lymphocyte count of 160 cells/mm 3 and undetectable hiv (<50 copies). the patient in 2000 initiated lawsuit against his original fp for medical malpractice. the charges against the physician were that he failed to perform an hiv test in 1990 despite the plaintiff's request and he was negligent in failing to notify the plaintiff of the result of the hiv serology in 1993. these acts of negligence by the defendant resulted in delay in the diagnosis and treatment, thus allowing his hiv infection to progress to aids. furthermore, failure on the part of the defendant had resulted in missed opportunities to start earlier treatment, and the delay in initiation of art resulted in a decrease in his expected life span and affected the quality of his life. the defendant countered that there was no record of the plaintiff ever requesting an hiv test in 1990. furthermore, the plaintiff never kept the appointment after the positive hiv serology in 1993 to be notified of the result. moreover, since the plaintiff was subsequently lost to follow-up, he never had a chance to counsel him on hiv disease or institute treatment. following acute hiv infection, about 50-70% of subjects develop clinical symptoms of variable severity, from mild flu-like illness to aseptic meningitis. 15 there is also evidence that severity and duration of clinical acute illness of a primary infection is of prognostic importance. the risk of developing aids within 3 years of seroconversion in subjects who were asymptomatic or had mild illness was only 10% versus 78% (eight times greater) in those with seroconversion illness of at least 14 days. 16 peak viral replication soon after infection occurs in 2-4 weeks and levels of virus can exceed >10 7 copies/ml in plasma. this is associated with a dramatic drop in circulating cd4 + lymphocytes, then a slowing of t-cell loss, and rebound by 9-12 weeks. this rebound of cd4 + cells corresponds to a decline in viral load, which reaches a steady state (set point), which is variable by 9-12 weeks. clinical progression of hiv disease has been tied to a set point level with lower levels associated with better prognosis. 17 at 1 year after seroconversion, patients demonstrate a fall of about 349 cd4 + cells/mm 3 (mean baseline 999 cells/mm 3 ), followed by a more gradual decline in cd4 + cells in the later period of infection. 18 there is usually a variable period of 8-10 years span before patients develop aids (about 50%). the typical hiv-infected person shows a progressive decline of cd4 + lymphocytes (50-100 cells/year) over time. long-term non-progression or elite controllers represent <5% of hiv-infected subjects who maintain relatively normal cd4 + cell count and very low or immeasurable viral load for 8 years to decades without therapy. 19 this is a heterogenous group of elite controllers whose benign course may result from robust immune responses against hiv, or defective poorly replicative virus secondary to deletion of the nef gene. 20, 21 there is evidence that host factors that influence the course of hiv disease are correlated to polymorphisms dominating the hla region, with class i polymorphism dominating the hla associations. 22 there is also evidence from studies in the united states and europe that hla-b57 and hla b-27 are strongly associated with long term survival or non-progression. 22 not all long-term non-progressors are "elite or viremic controllers" (patients with undetectable hiv or plasma hiv rna levels of 50-2,000 copies/ml). these patients with cd4 + cell counts of >500 cells/mm 3 for at least 10 years most often had hiv rna levels of >2,000 copies/ml, but had significantly lower hiv rna than subjects with typical progression. thus plasma set point hiv rna levels explain <50% of the variability in rates of clinical progression. 24 the chemokine receptor 5 (ccr5) protein serves as a co-receptor on cd4 + lymphocytes for certain strains of hiv-1. homozygosity for a 32-base pair deletion allele (ccr5d32) protects against hiv infection (1% of caucasians) and heterozygosity (individuals with one allele) show a decreased progression to aids. 25 there is also evidence that co-infection with gb virus (gbv-c), a flavivirus not known to cause disease (in subjects with gbv-c viremia), have slower progression and slower decrease in cd4 + cell counts than those without gbv-c infection. 23 although the reasons for this protective effect are unclear, there is evidence that gbv-c inhibits hiv replication in peripheral blood mononuclear cells in vitro, and since gbv-c infects cd4 + cells, this may compete with the hiv for target cells for infection. 23 a minority of patients with hiv infection can rapidly progress to aids within 1-3 years of their infection. this may be related to host genetic factors and age at the time of infection and other extrinsic conditions. older age at the time of infection (>25 years) has been associated with faster progression of the disease in hemophiliacs and older homosexuals. 23 concomitant co-infection with cytomegalovirus (cmv) has been associated with rapid progression to aids in hemophiliacs and others. 26, 27 co-infection with htlv-i may increase the risk for development of aids while htlv-2 can delay the progression of disease. 23, 28, 29 active tuberculosis can also enhance hiv replication and cause rapid progression to aids. 30 however, although treatment of active tuberculosis for 6 months is associated with increased cd4 + cell count, it does not markedly affect the hiv viral loads. 31 the role of hepatitis c-virus (hcv) co-infection on the progression of hiv disease has been conflicting, with some studies showing more rapid progression, but others have found no effect on the development of aids. 23 the clade or strain of hiv-1 may play a role in the course of the disease. clade d of hiv-1 is associated with faster progression to death in africa than clade-a and b. 32 women in senegal infected with c, d or g hiv clade were eight times more likely to develop aids than those infected with clade a subtype (the predominant sub-type). 33 infection with multiple strains of hiv-1 (more common in women) have also been associated with faster disease progression. 34 socio-economic factors such as poverty, homelessness, drug and alcohol abuse, and black race play indirect roles in the prognosis and disease progression of hiv infection, primarily through lower access to medical management, delay in instituting antiretrovirals and poorer compliance with medications. although a previous study found that alcohol and psychoactive drugs did not accelerate hiv disease, 35 there is in vitro evidence that alcohol, cocaine and narcotics can impair the immune response to hiv-1 and allow enhanced replication in peripheral blood mononuclear cells. 23 a case report of rapid progression to aids within a year of infection has also been attributed to alcoholism. 36 it would appear that the plaintiff became infected with hiv sometime between 1988 (reported hiv-negative) and 1993 (first noted hiv-positive). however, by 1994 he had progressed to symptomatic aids. thus, his course was more rapid than usual hiv infected individuals were, and especially as no other conditions or factors were recognized that could accelerate his course of disease. however, the failure of the fp to notify the patient of his hiv-positive status before recognition of his condition was only 1 year. would an earlier diagnosis by 1 year and assuming institution of art then, affected the outcome as to lifespan and quality of life? appropriate treatment a year earlier with art would likely have aborted or ameliorated his symptomatic disease, of weight loss, diarrhea, and malaise. however, it is less clear whether his expected lifespan would be any greater. if we assume that over the preceding year his cd4 + cell count probably declined by 50-100 cells/mm 3 , then even at that time he would have already progressed to aids (cd4 + cell count <200 cells/mm 3 ). there is reasonable good cumulated evidence that starting therapy when the cd4 + cell count is very low (<200 cells/ mm 3 ) is associated with less chance of immune reconstitution and greater risk of opportunistic complications than those started on art when the cd4 + cell count was >200 cells/mm 3 . the optimum cd4 + cell count for initiating art has not been well established although recent large observational cohort studies (retrospective and prospective ) and suggests better outcomes for hiv infected patients receiving earlier art with cd4 + cell count !350 cells/mm 3 or >500 cells/mm 3 ; the data however is flawed and controversial. 37, 38 lack of randomization in these studies could result in significant biases as motivated, health-conscious individuals would likely do better that those less motivated. it is not clear in these studies as to the cause of excess mortality in those not accepting treatment. for instance, it would be expected and predictable to find excess mortality (from any disease) in marginalized people (homeless, alcoholics, drug abusers), who are less likely to start art, which may be unrelated to hiv complications, such as suicides, homicide, accidents, drug overdose, liver failure or other diseases more prevalent in these groups (diabetes mellitus, cardiovascular disease, chronic lung disease and cancer). the defendant denied the plaintiffs claim that earlier hiv test (in 1990) was requested. it could be argued, however, that the fp should have been doing regular hiv serology in an individual that belongs to a high-risk group (with the patient's consent). the center for disease control and prevention (cdc) estimate that nearly 50% of men who have sex with men (msm) with hiv infection are unaware of their status. the cdc national behavior surveillance system of high-risk venue-based recruitment found 25% of msm tested to be infected with hiv, and nearly 50% of the hiv infected individuals were unaware of their infection. 39 in new york city, the hiv incidence rate among msm was 2.3%, with 52% of those infected being unaware of their hiv seropositivity. 39 it is estimated that 21% of hiv-infected people in the us who are unaware of their infection may account for up to 52% of new infections. cdc hiv testing guidelines recommend annual testing for high-risk populations (including msm), and since 2006 have recommended universal opt-out hiv screening in all health care settings. 40 thus, the plaintiff could argue that the defendant fell below the standard of care by not recommending and performing annual hiv tests. furthermore, if he were found to be hiv seropositive earlier, (by 1990 or before) with careful monitoring and institution of art before his cd4 + cell count fell <350 cells/mm 3 , his quality of life and life expectancy would be greater. 41 what is the effect of life expectancy with late treatment initiation for hiv disease? in a recent study using a state-transition model of hiv disease, the projected life expectancy of hiv uninfected and hiv infected persons with similar risk profiles were compared. 41 those with hiv infection lost 11.92 years of life if they received care concordant with guidelines and late treatment initiation resulted in 2.60 additional years of life lost (greatest for hispanics [3.90 years]). 41 an infectious disease (id) specialist/internist was consulted to assess a 41-year-old male with mild pancytopenia and a past history of bilateral pneumonia the year before. the patient had a history of multiple sexual contacts with prostitutes 5 years prior and had refused hiv testing the year before when he developed pneumonia (which was suspected to be pneumocystic pneumonia [pcp]). at this office visit, he agreed to an hiv test and a cd4 + cell count. the patient was called for a return appointment to discuss the results of the test a month later, but this appointment was cancelled by the patient for personal reasons. the blood test revealed the patient was hiv seropositive with a very low cd4 + cell count of 5 cells/mm 3 , but the results were not given over the phone or by mail. thus, the subject remained unaware of his hiv status and severe immune deficiency. about 3 months later, the patient attended an optician for blurred vision and he was referred to a hospital er for an ophthalmologist consultation. he was briefly assessed by the attending er physician, but due to the long waiting period pending full eye assessment, he left prematurely. the patient arranged an appointment with the id specialist in the er of the suburban community hospital. the subject was told of his hiv status and a brief retinal examination (without pupillary dilatation) by the id physician revealed no abnormality. an appointment was arranged for another office visit to the id specialist to discuss hiv therapy in 2 weeks. one week later the subject returned to the er with respiratory symptoms and poor vision. he was admitted as possible pcp under the care of the id physician, but no eye examination was performed. a week after his admission to hospital, a neurologist who was consulted found very poor vision with light perception only in the right eye and finger counting on the left eye. fundoscopy revealed bilateral chorioretinitis and ophthalmology consultation was requested, but treatment of cmv retinitis was only instituted 2 days later. his course was complicated by retinal detachment secondary to cmv retinitis with almost complete blindness in the right eye and severe visual impairment of the left eye -legally blind. malpractice litigation was brought by the patient against the id physician and the admitting er physician of the hospital. the charges against the id consultant were: (1) failure to notify the plaintiff of his hiv status and seriousness of his condition, (2) failure to do a proper eye examination or refer him to an ophthalmologist when he was first seen in the er a week before his admission, (3) failure to do a fundoscopy or arrange urgent ophthalmology consultation on admission to the hospital, (4) delay in starting appropriate treatment for cmv retinitis even after the neurologist findings were consistent with the diagnosis. the claim filed against the er physician was for neglect in performing an eye examination, despite the patient's symptoms of poor vision and failure to require an urgent ophthalmology consultation. that prompt recognition of cmv retinitis and immediate institution of antiviral therapy could have resulted in better visual result. failure of the id physician to inform the plaintiff of the seriousness of his condition, even by phone, could have resulted in prevention of visual loss and admission to hospital if treatment with art and pcp prophylaxis were started 3 months before his hospital admission. the defendant (id specialist) countered that it was the plaintiff who canceled the follow-up appointment for counseling on his condition, and it was neither his policy nor the recommended standard to discuss these issues on the phone. therefore, failure to initiate earlier art before the aids complications was due to the fault of the plaintiff. furthermore, his eye examination performed at the first er visit revealed no abnormalities. visual complaints in hiv infected persons can be unrelated (as in normal people) or related directly to complications of aids or indirectly due to medications. ocular manifestations are common in people with aids, and before the advent of highly active art (haart), the majority of patients with aids developed some ocular involvement at some time. 42 the most frequent ocular abnormality was usually silent or asymptomatic and occurred in nearly 50% of aids patients before the era of haart -hiv microangiopathy, consisting of cotton wool exudates, and less frequently hemorrhages. 42 occasionally hiv retinopathy could present with visual impairment from larger branch vein or central retinal vein occlusion. the most dreaded ocular complications of aids were from opportunistic ocular infections (cmv retinitis, herpes zoster (vzv) retinitis, or herpes zoster ophthalmicus, toxoplasma retinitis and ocular syphilis), or neoplasm (kaposi sarcoma of the lids and conjunctivae, and orbital or intraorbital lymphoma). 42 cmv retinitis is the most frequent sight threatening ocular complication of aids, occurring in the late stages when the cd4 + lymphocyte counts <50 cells/ml. in the pre-haart era cmv retinitis occurred in 30% of patients with aids, and the number of new cases has dramatically fallen since widespread use of haart by 55-95% (average 80%). 42 the incidence of cmv retinitis among patients with cd4 + cell count <100 cells/ml was 10% per year and for many patients with cd4 + cell count <50 cells/ml, it was 20% per year. symptoms of cmv retinitis include floaters, flashing lights, loss of visual field, or visual loss. in the early stages with small peripheral retinal lesions patients can be asymptomatic and 13-15% of persons with cd4 + cell count 50 cells/ml have asymptomatic cmv retinitis. 43 lesions adjacent to the optic nerve or fovea (posterior pole of the retina or macula) are immediately vision threatening. the retina has been divided into three zones for clinical assessment of risk to vision. zone 1 lies within 1,500 mm from the edge of the optic nerve, zone 2 extends from the edge of zone 1 to the equator of the eye, and zone 3 extends from the equator to the pars plana (pigmented posterior zone of the ciliary body). see fig. 13 .1 for the schematic diagram of the zones of the retina. lesions of zone 1 are immediately sight-threatening and require urgent treatment, whereas lesions of zones 2-3 may be observed for short periods of time without risk of loss of visual acuity. 42 the mean time for progression of peripheral lesions without treatment was found to be 22 days (enlargement to uninvolved retina by !750 mm in width). 44 the complications of untreated or delayed treatment of cmv retinitis include impaired vision to blindness, secondary to progressive retinitis with hemorrhages, scarring and retinal detachment. in the pre-haart era, retinal detachment in cmv retinitis occurred in 25% at 6 months and in 50-60% at 1 year. 42 the diagnosis of cmv retinitis can be made reliably by an experienced ophthalmologist by dilated direct or indirect ophthalmoscopy. examination of the fundus through an undilated pupil is inadequate to diagnose or exclude cmv retinitis as only 10% of the retina can be evaluated. 42 the aim of treatment with anti-cmv drugs (ganciclovir intravenously or oral valganciclovir) is to arrest progression of the disease, prevent further spread, and preserve vision. treatment with anti-cmv agents does not eradicate the virus but delays progression and relapse, until immunity can be restored by haart. anti-cmv therapy, half the dose after induction for 3 weeks, can be discontinued once the cd4 + cell count is >100-150 cells/ml for 6 months. some experts advocate intravitreal injection or ganciclovir implant in addition to systemic therapy for zone 1 cmv retinitis to avoid loss of vision. 42 for persons recently discovered to be hiv-seropositive, it is ideal to give the results in person confidentially, and at the same time counsel the patient on the disease. however, there are several options available if the individual were reluctant to return for follow-up appointment (or cancels the appointment). the results could be forwarded to the fp and notify him or her of the patient's cancelation plus need for counseling, close monitoring, and for initiating art or pcp prophylaxis depending on the cd4 + cell count. if the subject has no fp, then the person can be notified of the results by letter or phone, or through the public health department. although some physicians are reluctant to discuss confidential issues on the phone, there is no edict against this practice. however, confidentiality needs to be maintained and the identification of the person on the phone should be verified. this has become a common practice with financial institutions and even lawyers who discuss medicolegal cases with medical experts on the phone. since the defendant knew the plaintiff had advanced hiv disease (aids) as indicated by the very low cd4 + cell count, it was mandatory that the patient be made aware of the seriousness of his condition as soon as possible by one of the above mechanisms. his failure to impart this information to the plaintiff directly or indirectly could be considered negligence by the court. failure of the defendant to perform a dilated ophthalmoscopy or arrange for urgent ophthalmology consultation when the plaintiff initially presented with impaired vision also falls below the standard of practice in an hiv infected individual with a cd4 + cell count of <50 cells/ ml. the physician ought to have known that cmv retinitis was a main concern, and could be sight threatening and that examination by un-dilated fundoscopy would be insensitive and inaccurate. based on the evidence presented, it could be argued by the plaintiff's lawyer that had the patient been notified earlier of the seriousness of his condition and accepted treatment with haart 3 months before his hospital admission, it is likely that he would have had a better quality of life and preservation of his vision. although counsel for the defendant may counter that the plaintiff should be responsible for his own health (as he canceled the follow-up appointment), there were several avenues available to the defendant to ensure that the patient became aware of his serious illness, and he failed to utilize any of them. whether or not a court may consider these failures as human errors from oversight in a busy medical practice and not medical malpractice would be difficult to predict. review of testing for human immunodeficiency virus low sensitivity of elisa testing in early hiv infection duration of human immunodeficiency virus infection before detection of antibody measurement of the false positive rate in a screening program for human immunodeficiency virus infection detection of human immunodeficiency virus dna using polymerase chain reaction in a well-characterized group of homosexual and bisexual men concordance of polymerase chain reaction with human immunodeficiency virus antibody detection immunology of hiv infection william's hematology, 7 th edition fraudulent aids factitious aids the baron has aids: a case of fictitious human immunodeficiency virus infection and review factitious hiv syndrome in young women factitious hiv infection: the importance of documenting infection feigned hiv infection/aids: malingering and munchausen's syndrome primary human immunodeficiency virus type i infection: review of pathogenesis and early treatment intervention in humans and animal retrovirus infection clinical course of primary hiv infection prognosis in hiv infection predicted by the quantity of virus in plasma cd4 + lymphocyte cell enumeration for prediction of clinical course of human immunodeficiency virus disease immunopathogenic mechanisms of hiv infection immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of hiv-1 brief report: absence of intact nef sequence in a long-term survivor with non-progressive hiv-1 infection consistent associations of class i and ii and transporter gene products with progression of human immunodeficiency virus type i infection in homosexual men overall features of hiv pathogenesis prognosis for long term survival natural control of hiv-1 replication and long-term non-progression: overlapping but distinct phenotypes genetic restriction of hiv-1 infection and progression to aids by a deletion allele of the ckr5 structural gene cytomegalovirus infection and progression towards aids in hemophiliacs with human immunodeficiency virus infection cytomegalovirus seroconversion as a cofactor for progression to aids progression to aids in homosexual men coinfected with hiv-1 and htlv-1 in trinidad htlv-i/ii seropositivity and death from aids among hiv-i seropositive intravenous drug users influence of tuberculosis on human immunodeficiency virus (hiv-i): enhanced cytokine expression and elevated b2-microglobulin in hiv-i associated tuberculosis human immunodeficiency virus-i rna levels and cd4 lymphocyte counts during treatment for active tuberculosis in south african patients different rates of disease progression of hiv type i infection in tanzania based on infecting subtype human immunodeficiency virus type i subtypes differ in disease progression infection with multiple human immunodeficiency virus type i variant is associated with faster disease progression no evidence for a role of alcohol or other psychoactive drugs in accelerating immunodeficiency in hiv-i positive individuals alcoholism and rapid progression to aids after seroconversion effect of early versus deferred antiretroviral therapy for hiv on survival timing of initiation of antiretroviral therapy in aids-free hiv-i infected patients: a collaborative analysis of 18 hiv cohort studies hiv prevalence, unrecognized infection and hiv testing among men who have sex with men -five us cities sexually transmitted diseases treatment guidelines early versus standard antiretroviral therapy for hiv-infected adults in haiti cytomegalovirus retinitis and low cd4 + t-lymphocyte counts intravenous cidofovir for peripheral cytomegalovirus retinitis in patients with aids what lessons can we learn from these cases? l all patients with self-reported hiv-seropositive status should be verified by repeating the test. l physicians should pay more careful attention to patients' symptoms and complaints and act with reasonable promptness. l deal with patients' complaints as you would want to be done to yourself or relatives. key: cord-015936-4fwkf8fn authors: nan title: subject index, volumes 123-130 date: 2005-11-04 journal: j virol methods doi: 10.1016/s0166-0934(05)00346-0 sha: doc_id: 15936 cord_uid: 4fwkf8fn nan hiv; 2 ltr circles; new marker (125) 11 hiv antigen/antibody combined assay; hiv; serology; hiv-1 p24 antigen assay (127) (127) (128) (128) 67 yeast expression; pichia pastoris; sars-cov; n protein (130) 83 enzyme analysis; flaviviruses; rt-pcr key: cord-010203-dt9m596i authors: hellen, christopher u.t.; wimmer, eckard title: viral proteases as targets for chemotherapeutic intervention date: 2004-08-26 journal: curr opin biotechnol doi: 10.1016/0958-1669(92)90010-g sha: doc_id: 10203 cord_uid: dt9m596i many viruses encode proteinases that are essential for infectivity, and are consequently attractive chemotherapeutic targets. the biochemistry and structure of the human immunodeficiency virus proteinase have been characterized extensively, and potent peptide-mimetic inhibitors have been developed. techniques and strategies used to improve the efficiency of these compounds are likely to be applicable to other viral proteinases. many human and animal viruses encode proteinases that play important roles at different stages in the infection cycle, including separation of functionally different domains from a precursor polyprotein (enabling cleavage products to be transported to different cellular compartments) and regulation of a variety of events in viral replication, such as uncoating, activation of replicative enzymes and morphogenesis [1] . these proteinases are essential for virus infectivity, and have therefore come to be seen as attractive targets for chemotherapeutic intervention, particularly because they have unusual cleavage specificities that differ from those of host proteases. proteinases are encoded by all retroviruses, including hiv and human t-cell leukemia virus and have been identified in a growing number of dna and positivesense rna viruses. these include adenoviruses and herpesviruses [2",3"] as well as picornaviruses, flaviviruses (such as dengue and yellow fever viruses), pestiviruses and the related hepatitis c virus. a number of these viruses cause diseases of medical or veterinary importance that are not amenable to conventional preventive or prophylactic measures, and proteinase inhibition therefore represents a valid alternative therapeutic approach. many viral proteinases have only been identified recently, and characterization is consequently in its early stages. to illustrate potential strategies in the analysis of viral proteinases, and in the design and development of inhibitors we shall therefore focus on the picornavirus and retrovirus proteinases, as they have elicited the greatest academic and industrial interest, and as a result, have been characterized in detail. hiv-1 has the genetic organization 5'-gag-pol-env-3' that is typical of retroviruses. the gag and pol genes encode inner structural, and replicative proteins respectively, and are translated as polyproteins that are cleaved at eight sites by the proteinase (pr) ( fig. 1 and table 1 ). these polyproteins are transported to the plasma membrane and cleavage occurs after budding of immature particles, resulting in morphological changes associated with virion maturation. the substrate specificity of hiv-1 pr is puzzling in that pr catalyzes specific cleavage at a small number of polyprotein sites that show no apparent sequence conservation. analysis of viral and non-viral substrates suggests that no subsite has absolute specificity, and that a combination of moderate interactions may be sufficient to confer catalytic specificity [4"]. heterogeneity in the composition of viral polyprotein cleavage sites probably plays a role in determining the rate, and consequently the order, of cleavage at different sites. a proteinase-deficient hiv mutant produced noninfectious immature virions containing unprocessed polyprotein [5] , an observation that is crucial to the consideration of pr as a therapeutic target. hiv-1 pr is a c 2 symmetric homodimeric aspartyl proteinase that consists of two identical 99-amino-acid subunits. their termini interdigitate at the dimer interface, but otherwise the topology of hiv-1 pr is similar to that of pepsin-like aspartyl proteinases [6"']. techniques for the large scale purification of recombinant hiv-1 pr, and for the routine assay of its proteolytic activity are fundamental prerequisites for the development of inhibitors, and various methods have been reported [7"']. the successful design of substratebased inhibitors of other aspartyl proteinases, such as pepsin and renin, suggested the strategy of designing analogous peptide-mimetic inhibitors of hiv-1 pr by incorporating non-hydrolyzable 'transition-state' mimics into substrate analogues [7"',8",9-]. this approach is based on a key step in aspartyl proteinase catalysis: generation of a tetrahedral diol by hydration of a trigonal amide (fig. 2a) . the high (millimolar) k m values of peptide substrates indicate that potent (i.e. nanomolar) pr inhibitors must incorporate structural features that significantly increase their binding affinity. peptide-based inhibitors have several disadvantages, including vulnerability to degradative enzymes, rapid clearance, and poor oral absorption. these problems are commonly addressed by minimizing size and peptide-like character of promising lead compounds. the first reported pr inhibitor was pepstatin [10], a diagnostic inhibitor of aspartyl proteinases. this weak inhibitor contains two statine residues that embody transition state analogue i (fig. 2b ). to identify more potent inhibitors, dreyer et al. [11] compared the effectiveness as pr inhibitors of five different classes of dipeptide isosteres inserted into a consensus heptapeptide template. heptapeptides (p4-p3' or p3-p4') are the shortest substrates that are cleaved efficiently by pr. statine-based (i), reduced amide (ii) and phosphinate (iii) transition state analogues exhibited modest potency, but placement of phe-gly hydroxyethylene dipeptide isosteres (iv) into the consensus template yielded compounds that inhibited hiv pr at nanomolar concentrations in vitro and prevented polyprotein processing, virion maturation and viral spread at 25-100btm in cell culture. truncation and extensive structure-activity analysis at the p1, pi" and p2' positions led to the identification of highly potent (subnanomolar) pr inhibitors based on dihydroxyethylene (v) [12-] and hydroxyethylene (iv) [13"] isostere transition state analogues. potency was enhanced by incorporating residues that stabilize the extended inhibitor structure, presumably due to optimized hydrogenbonding in the substrate binding cleft. for example, the p2' and p3' residues (leu-phe) can effectively be replaced by various substituted aminobenzocycloalkanes [14" ]. the pi' position can accept side-chains unrelated to natural amino acids, allowing modifications to be made that enhance solubility and thus cell penetration [15", 16-] . such substitutions may reduce binding affinity (k i) but the enhanced solubility may nevertheless result in a net increase in antiviral activity. the ability to cleave the amino terminus to proline distinguishes hiv pr from non-viral aspartyl proteinases. hydroxyethylamine (vi) structures that readily accomodate the prolyl imino acid have been incorporated into a number of potent inhibitors [17,18",19,20" ]. modification of a protected tripeptide incorporating this structure by substituting the imino residue decahydroisoquinoline at the pi' position yielded highly potent inhibitors of pr in vitro and in cell culture, such as the compound ro 31-8959 [19,21" ]. this inhibitor was expected to have considerable selectivity, and indeed it inhibited human aspartyl proteinases such as gastricsin, renin and pepsin by less than 50% at a concentration of 10pm. typical ic90 values for ro 31-8959 (5-30 nm) are 1000-fold below its cytotoxic concentration in uninfected host cells. recent reports indicate that a 600 mg oral dose every 8 hours is sufficient to maintain the mean human plasma concentration at about 70nm. the potency of ro 31-8959 is strongly dependent on tight binding by the p2 and particularly the p3 substituents, whereas binding of a second class of hydroxyethylamine inhibitors (which contain a noncyclic, secondary amine in place of the decahydroisoquinoline residue) [22" ] is less dependent on these interactions, and this second class binds more tightly in the pi"-p2' region. peptide substrates are inherently asymmetric and the pr dimer must therefore lose its perfect c 2 symmetry during catalysis. symmetry is permissible for inhibitors, however, and might even improve binding affinity and selectivity over endogenous aspartyl proteinases. these considerations have led to the design of a series-of diaminoalcohol-and diaminodiolbased inhibitors with c 2 (vii) or pseudo-c 2 symmetry [23,24"-26" ]. these inhibitors are potent even at subnanomolar concentrations and highly selective in vitro, but most have suffered from poor solubility, leading to modest potency in cell culture. strategies to circumvent this deficiency and thus enhance activity in cell culture have included modification of terminal residues and their linkage groups to increase solubility. these interactions include extensive van der waal's contacts with residues that define the hydrophobic 82-82' binding pockets, and a hydrogen bonding system that sandwiches the inhibitor strand between the catalytic cleft and the flaps. the hydroxyl groups of type v, vi and vii inhibitors form hydrogen bonds with both catalytic aspartates. significantly, all complexes contain a tetrahedrally coordinated active-site water molecule, which bridges two flap residues and two inhibitor carbonyl groups, prompting suggestions that an improved inhibitor would contain a functional replacement for the water [24",27] . the similarities in the extended conformation of all inhibitors, as well as in the induced conformational changes that they cause in the enzyme, indicate that it is possible to model and improve peptidic inhibitors on the basis of these known structures. an alternative approach to discover novel templates for the design of non-peptide inhibitors is to search three-dimensional structure databases for molecules with a shape that is complementary to the active-site cleft. to date, this approach has led to identification of the antipsychotic agent haloperidol as a weak pr inhibitor [29] . the picornaviridae are a family of small icosahedral viruses that includes the etiological agents of several important human and animal diseases. it consists of five genera, including rhinovirus (the common cold virus) and enterovirus (e.g. poliovirus and hepatitis a virus). picornaviruses have a positive-sense monopartite rna genome that encodes a single large polyprotein. it is processed by three different proteolytic activities which can each be regarded as serving a distinct function ( fig. 3 and table 2 ) [30" ]. the initial event in this cascade is cleavage by 2ap r° at its own amino terminus, separating the p~ structural protein precursor from the nascent polyprotein. secondly, functional proteins are released from the p1 and p2-p3 (non-structural) protein precursors by 3cp r° or its precursors. finally, maturation cleavage of the vp0 capsid protein occurs on encapsidation of viral rna to yield infectious virus particles. in addition to their role in viral replication, the 2a and 3c proteinases of poliovirus (and by implication, of other picornaviruses) are responsible for aspects of the dramatic inhibition of host cell rna and protein synthesis that occurs on infection. the 2a proteinase is involved in degradation of the eukaryotic initiation factor elf~fy, which is correlated with shut-off of cap-dependent translation [31] , and 3cp ro inactivates transcription factor iiic, inhibiting polymerase iii transcription [32" ]. sequence alignment and inhibitor studies suggested that both 2a and 3c proteinases are related structurally to trypsin-like serine proteinases, with the noat all eight sites within the polyprotein (fig. 3) . sites in other picornaviruses are slightly more heterogenous. poliovirus 2apro cleaves tyr-gly dipeptides at the p1-2a junction and within the three-dimensional polymerase, but although all corresponding sites in other picornaviruses have a gly residue at the pi' position, various residues occur at the p1 position. aliphatic residues occur at the p4 positions of most 2ap r° and 3cp r° sites. mutagenesis and peptide cleavage experiments indicate that cleavage site recognition depends on a minimum substrate length (six residues for 3cp r°) and the presence of specific residues at positions that differ according to both the virus and the proteinase [38,39",40",41"43" ]. there are additional conformational determinants of recognition of cleavage sites within polyproteins, so the large (millimolar) k m values of peptide substrates may reflect their greater conformational freedom. potential peptidemimetic inhibitors are likely to exhibit similar flexibility, and must therefore be conformationally constrained and incorporate structural features that increase their binding affinity. the lack of absolute specificity at most subsites, and the requirement for peptide substrates to extend to the p4 position indicates that the substrate binding clefts of 3cp r° and probably 2ap r° are capable of extensive hydrogen bond interactions with such inhibitors. however, only a few inhibitors of 3cp ro have been reported [44",45] . proteases are encoded by several dna viruses and numerous rna viruses in addition to the picornaviruses and retrovimses discussed above. although they are all potential targets for chemotherapeutic intervention, significant progress in inhibitor development has only been reported for hiv-1 pr. in the few years since its identification, the structure of pr and numerous inhibitor complexes have been determined, and highly potent peptidemimetic inhibitors have been developed. knowledge of the strategies used in enhancing the potency and specificity of pr inhibitors, and in overcoming the inherent limitations of peptide-based inhibitors is likely to prove invaluable in the development of peptidemimetic inhibitors of other viral proteinases. heinrikson rl: the complexities of aids: an assessment of the hiv protease as a therapeutic target. chem today 1991, 9:6-27. an account of the recent progress in substrate-based inhibitor design is complemented by a concise summary of the structure of hiv pr and a clear exposition of its application to structure-based inhibitor design. norbeck :1225-1228. the first of" series of disclosures [i3"-15",16"'] from a group at merck illustrating strategies to selectively modify termini and side-chain residues to increase potency, reduce size and peptidic character, and to increase solubility (and hence antiviral activity) of hydroxyethylene isostere-based inhibitors. this paper describes the effect of modification of the pi', p2' and p3' residues. benzocycloalkyl amines as novel c-termini for hiv-1 protease inhibitors hiv-1 protease inhibitors based on hydroxyethylene dipeptide isosteres: an investigation into the role of the pi' side chain on structure-activity systematic modification of the p]' residue of the lead inhibitors described in [13",14"] established that side chains unrelated to natural amino acids are tolerated at this position, permitting substitutions that increase solubility and cell penetration synthesis and antiviral activity of a series of hiv-1 protease inhibitors with functionality tethered to the p1 or pi' substituents: x-ray crystal structure assisted design computer-assisted molecular modelling was used to design derivatives of the lead inhibitor l-685,434 [14"] with increased cell penetration and antiviral potency. an x-ray crystal structure of the hydroxyethylamine analogues of the p17/p24 substrate cleavage site are tight-binding inhibitors of the hiv protease effect of hydroxyl group configuration in hydroxyethylamine dipeptide isosteres on hiv protease inhibition. evidence for multiple binding modes in a series of hydroxyethylamine isostere inhibitors, the preferred diastereomeric configuration of an essential hydroxyl group depended on both the length and nature of the peptide framework. this hydroxyl group is hydrogen bonded to the two catalytic asp residues rational design of peptide-based hiv proteinase inhibitors novel binding mode of highly potent hiv-proteinase inhibitors incorporating the (r)-hydroxyethylamine isostere binding of the hydroxyethylamine inhibitor ro 31-8959 described in na: effects of a specific inhibitor of hiv proteinase (ro 31-8959) on virus maturation in a chronically infected promonocytic cell line (u1) antiviral activity of the potent hiv inhibitor ro 31-8959 is sufficient to inhibit acute and chronic infections. the low toxicity of this compound renders it a highly promising antiviral agent in aids chemotherapy a series of potent hiv-1 protease inhibitors containing a hydroxyethyl secondary amine transition state isostere: synthesis, enzyme inhibition, and antiviral activity a novel subclass of potent hydroxyethylamine inhibitors containing a secondary amine isostere in place of the cyclic amine of ro 31-8959 show differences in structure-activity relationships and in binding mode structure-based c a symmetric inhibitors of tiiv protease design, activity and 2.8a struc-lure of a c2 symmetric inhibitor complexed to hlv-1 design of c 2 symmetric inhibitors exploiting the perfect symmetry of hiv pr revealed by x-ray crystallography x-ray crystal structure of the hiv protease complex with l-700,417, an inhibitor with pseudo-c 2 symmetry an illustration of the application of x-ray crystallography to rational drug design, which in this instance revealed unoptimized hydrogen bonding and several water-mediated pr-inhibitor interactions antiviral and pharmokinetic properties of c 2 symmetric inhibitors of the human immunodeficiency virus type 1 protease this paper illustrates the difficulties in reconciling the competing demands on pr inhibitors for tight hydrophobic interactions with pr subsites, and aqueous solubility required for bioavailability and in vivo efficacy x-ray crystallographic structure of a complex between a synthetic protease of human immunodeficiency virus 1 and a substrate-based hydroxyethyalmine inhibitor structure at 2.5a resolution of chemically synthesized human immunodeficiency virus type 1 protease complexed with a hydroxyethylene-based inhibitor structure-based design of nonpeptide inhibitors specific for the htmmal immunodeficiency virus 1 maturation of poliovirus capsid proteins a concise review of the role of three distinct proteolytic activities in the release of capsid proteins from the poliovims polyprotein and their subsequent assembly into virions poliovirus protease 2a induces cleavage of eulkaryotic initiation factor 4f polypeptide p220 poliovirus proteinase 3c converts an active form of transcription factor hic to an inactive form: a mechanism for inhibition of host cell polymerase ill transcription by poliovirus the severe inhibition of rna polymerase iii-mediated transcription in polio-infected cells is a result of inactivation of transcription factor iiic by the proteolytic activity of 3cp r° site-directed mutagenesis of the putative catalytic triad of poliovirus 3c proteinase site-directed mutagenesis experiments suggest that the catalytic triad of polio 3cp ro (his40, glu71, cys149) structurally resembles trypsinlike serine proteinases but differs significantly in its constituent residues analysis of putative active site residues of the poliovirus 3c protease experiments designed to evaluate two conflicting structural models of polio 3cp ro suggest that glu71 is a constituent residue of the catalytic triad whereas asp85 is involved in polyprotein substrate recognition characterization of poliovirus 2a proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor p220 polio 2apro containing a cysl09ser substitution within the putative his20, asp38, cysl09 catalytic triad retains significant autocatalytic activity expression and characterization of recombinant hepatitis a virus 3c protease a colorimetric assay was used to characterize cleavage by purified hepatitis a virus 3cp to putative papainrelated thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi, c~-and coronaviruses computer-assisted analysis was used to identify the first viral proteirlases that are distantly related to papain-like thiol proteinases role for the p4 amino acid residue in substrate utilization by the pouovirus 3cd proteinase ba: a rapid method for determination of endoproteinase substrate specificity: specificity of the 3c proteinase from hepatitis a virus a novel and rapid technique was used to demonstrate that hepatitis a vires 3cp ro has a strong preference for smali residues (ala, ser, gly) at the pl' position, but has little specificity at p2'. 41. weidner jr, dunn bm: development of synthetic peptide substrates for the poliovirus 3c proteinase a high performance liquid chromatography assay reveals that polio 3cp ro has a strong preference for a proline p2' residue, and a continuous fluorescence assay is reported determinants of substrate recognition by poliovirus 2a proteinase positions are strict determinants of substrate recognition by polio 2apro, but p2', pi' and p3 positions are broadly tolerant of substitution. substrate requirements for cleavage in trans are more stringent than for cleavage in cis hepatitis a virus 3c proteinase substrate specificity hepatitis a virus 3cp ro has strong preferences for residues at p4 and pa positions, and differs in specificity from enteroviral 3c proteinases structure and stereochemistry of thysananone: a novel human rhinovirus 3c protease inhibitor from thyanophora penicilloides. telrahedron lett a novel non-peptide (naphthoquinone) inhibitor of rhinovirus 3c proteinase spiro lndoline beta-lactams, inhibitors of poliovirus and rhinovirus 3c-proteinases key: cord-018646-fqy82sm6 authors: huremović, damir title: brief history of pandemics (pandemics throughout history) date: 2019-05-16 journal: psychiatry of pandemics doi: 10.1007/978-3-030-15346-5_2 sha: doc_id: 18646 cord_uid: fqy82sm6 intermittent outbreaks of infectious diseases have had profound and lasting effects on societies throughout history. those events have powerfully shaped the economic, political, and social aspects of human civilization, with their effects often lasting for centuries. epidemic outbreaks have defined some of the basic tenets of modern medicine, pushing the scientific community to develop principles of epidemiology, prevention, immunization, and antimicrobial treatments. this chapter outlines some of the most notable outbreaks that took place in human history and are relevant for a better understanding of the rest of the material. starting with religious texts, which heavily reference plagues, this chapter establishes the fundamentals for our understanding of the scope, social, medical, and psychological impact that some pandemics effected on civilization, including the black death (a plague outbreak from the fourteenth century), the spanish flu of 1918, and the more recent outbreaks in the twenty-first century, including sars, ebola, and zika. given to ways plagues affected the individual and group psychology of afflicted societies. this includes the unexamined ways pandemic outbreaks might have shaped the specialty of psychiatry; psychoanalysis was gaining recognition as an established treatment within medical community at the time the last great pandemic was making global rounds a century ago. there is a single word that can serve as a fitting point of departure for our brief journey through the history of pandemics -that word is the plague. stemming from doric greek word plaga (strike, blow), the word plague is a polyseme, used interchangeably to describe a particular, virulent contagious febrile disease caused by yersinia pestis, as a general term for any epidemic disease causing a high rate of mortality, or more widely, as a metaphor for any sudden outbreak of a disastrous evil or affliction [4] . this term in greek can refer to any kind of sickness; in latin, the terms are plaga and pestis (fig. 2.1 ). perhaps the best-known examples of plagues ever recorded are those referred to in the religious scriptures that serve as foundations to abrahamic religions, starting with the old testament. book of exodus, chapters 7 through 11, mentions a series of ten plagues to strike the egyptians before the israelites, held in captivity by the pharaoh, the ruler of egypt, are finally released. some of those loosely defined plagues are likely occurrences of elements, but at least a few of them are clearly of infectious nature. lice, diseased livestock, boils, and possible deaths of firstborn likely describe a variety of infectious diseases, zoonoses, and parasitoses [5] . similar plagues were described and referred to in islamic tradition in chapter 7 of the qur'an (surat al-a'raf, v. 133) [6] . throughout the biblical context, pandemic outbreaks are the bookends of human existence, considered both a part of nascent human societies, and a part of the very ending of humanity. in the apocalypse or the book of revelation, chapter 16, seven bowls of god's wrath will be poured on the earth by angels, again some of the bowls containing plagues likely infectious in nature: "so the first angel went and poured out his bowl on the earth, and harmful and painful sores came upon the people who bore the mark of the beast" (revelation 16:2). those events, regardless of factual evidence, deeply shaped human history, and continue to be commemorated in religious practices throughout the world. as we will see, the beliefs associated with those fundamental accounts have been rooted in societal responses to pandemics in western societies and continue to shape public sentiment and perception of current and future outbreaks. examined through the lens of abrahamic spiritual context, serious infectious outbreaks can often be interpreted as a "divine punishment for sins" (of the entire society or its outcast segments) or, in its eschatological iteration, as events heralding the "end of days" (i.e., the end of the world). throughout known, predominantly western history, there have been recorded processions of pandemics that each shaped our history and our society, inclusive of shaping the very basic principles of modern health sciences. what follows is an outline of major pandemic outbreaks throughout recorded history extending into the twenty-first century. the athenian plague of 430 b.c. the athenian plague is a historically documented event that occurred in 430-26 b.c. during the peloponnesian war, fought between city-states of athens and sparta. the historic account of the athenian plague is provided by thucydides, who survived the plague himself and described it in his history of the peloponnesian war [7] . the athenian plague originated in ethiopia, and from there, it spread throughout egypt and greece. initial symptoms of the plague included headaches, conjunctivitis, a rash covering the body, and fever. the victims would then cough up blood, and suffer from extremely painful stomach cramping, followed by vomiting and attacks of "ineffectual retching" [7] . infected individuals would generally die by the seventh or eighth day. those who survived this stage might suffer from partial paralysis, amnesia, or blindness for the rest of their lives. doctors and other caregivers frequently caught the disease, and died with those whom they had been attempting to heal. the despair caused by the plague within the city led the people to be indifferent to the laws of men and gods, and many cast themselves into self-indulgence [8] . because of wartime overcrowding in the city of athens, the plague spread quickly, killing tens of thousands, including pericles, athens' beloved leader. with the fall of civic duty and religion, superstition reigned, especially in the recollection of old oracles [7] . the plague of athens affected a majority of the inhabitants of the overcrowded city-state and claimed lives of more than 25% of the population [9] . the cause of the athenian plague of 430 b.c. has not been clearly determined, but many diseases, including bubonic plague, have been ruled out as possibilities [10] . while typhoid fever figures prominently as a probable culprit, a recent theory, postulated by olson and some other epidemiologists and classicists, considers the cause of the athenian plague to be ebola virus hemorrhagic fever [11] . while hippocrates is thought to have been a contemporary of the plague of athens, even possibly treating the afflicted as a young physician, he had not left known accounts of the outbreak [12] . it was another outbreak that occurred a couple of centuries later that was documented and recorded by contemporary physicians of the time. the outbreak was known as the antonine plague of 165-180 ad and the physician documenting it was galen; this outbreak is also known as the plague of galen [13] . the antonine plague occurred in the roman empire during the reign of marcus aurelius (161-180 a.d.) and its cause is thought to be smallpox [14] . it was brought into the empire by soldiers returning from seleucia, and before it abated, it had affected asia minor, egypt, greece, and italy. unlike the plague of athens, which affected a geographically limited region, the antonine plague spread across the vast territory of the entire roman empire, because the empire was an economically and politically integrated, cohesive society occupying wide swaths of the territory [15] . the plague destroyed as much as one-third of the population in some areas, and decimated the roman army, claiming the life of marcus aurelius himself [13] . the impact of the plague on the roman empire was severe, weakening its military and economic supremacy. the antonine plague affected ancient roman traditions, leading to a renewal of spirituality and religiousness, creating the conditions for spreading of new religions, including christianity. the antonine plague may well have created the conditions for the decline of the roman empire and, afterwards, for its fall in the west in the fifth century ad [13] . the justinian plague was a "real plague" pandemic (i.e., caused by yersinia pestis) that originated in mid-sixth century ad either in ethiopia, moving through egypt, or in the central asian steppes, where it then traveled along the caravan trading routes. from one of these two locations, the pestilence quickly spread throughout the roman world and beyond. like most pandemics, the justinian plague generally followed trading routes providing an "exchange of infections as well as of goods," and therefore, was especially brutal to coastal cities. military movement at the time also contributed to spreading the disease from asia minor to africa and italy, and further to western europe. described in detail by procopius, john of ephesus, and evagrius, the justinian epidemic is the earliest clearly documented example of the actual (bubonic) plague outbreak [16] . during the plague, many victims experienced hallucinations prior to the outbreak of illness. the first symptoms of the plague followed closely behind; they included fever and fatigue. soon afterwards, buboes appeared in the groin area or armpits, or occasionally beside the ears. from this point, the disease progressed rapidly; infected individuals usually died within days. infected individuals would enter a delirious, lethargic state, and would not wish to eat or drink. following this stage, the victims would be "seized by madness," causing great difficulties to those who attempted to care for them [17] . many people died painfully when their buboes gangrened; others died vomiting blood. there were also cases, however, in which the buboes grew to great size, and then ruptured and suppurated. in such cases, the patient would usually recover, having to live with withered thighs and tongues, classic aftereffects of the plague. doctors, noticing this trend and not knowing how else to fight the disease, sometimes lanced the buboes of those infected to discover that carbuncles had formed. those individuals who did survive infection usually had to live with ''withered thighs and tongues'', the stigmata of survivors. emperor justinian contracted the plague himself, but did not succumb [18] . within a short time, all gravesites were beyond capacity, and the living resorted to throwing the bodies of victims out into the streets or piling them along the seashore to rot. the empire addressed this problem by digging huge pits and collecting the corpses there. although those pits reportedly held 70,000 corpses each, they soon overflowed [17] . bodies were then placed inside the towers in the walls, causing a stench pervading the entire city. streets were deserted, and all trade was abandoned. staple foods became scarce and people died of starvation as well as of the disease itself [17] . the byzantine empire was a sophisticated society in its time and many of the advanced public policies and institutions that existed at that time were also greatly affected. as the tax base shrank and the economic output decreased, the empire forced the survivors to shoulder the tax burden [19] . byzantine army suffered in particular, being unable to fill its ranks and carry out military campaigns, and ultimately failing to retake rome for the empire. after the initial outbreak in 541, repetitions of the plague established permanent cycles of infection. by 600, it is possible that the population of the empire had been reduced by 40%. in the city of constantinople itself, it is possible that this figure exceeded 50 % [17] . at this point in history, christian tradition enters the realm of interpreting and understanding the events of this nature [20] . drawing on the eschatological narrative of the book of revelations, plague and other misfortunes are seen and explained as a "punishment for sins," or retribution for the induction of "god's wrath" [21] . this interpretation of the plague will reappear during the black death and play a much more central role throughout affected societies in europe. meanwhile, as the well-established byzantine empire experienced major challenges and weakening of its physical, economic, and cultural infrastructure during this outbreak, the nomadic arab tribes, moving through sparsely populated areas and practicing a form of protective isolation, were setting a stage for the rapid expansion of islam [22, 23] . the black death "the plague" was a global outbreak of bubonic plague that originated in china in 1334, arrived in europe in 1347, following the silk road. within 50 years of its reign, by 1400, [24] it reduced the global population from 450 million to below 350 million, possibly below 300 million, with the pandemic killing as many as 150 million. some estimates claim that the black death claimed up to 60% of lives in europe at that time [25] . starting in china, it spread through central asia and northern india following the established trading route known as the silk road. the plague reached europe in sicily in 1347. within 5 years, it had spread to the virtually entire continent, moving onto russia and the middle east. in its first wave, it claimed 25 million lives [24] . the course and symptoms of the bubonic plague were dramatic and terrifying. boccaccio, one of the many artistic contemporaries of the plague, described it as follows: in men and women alike it first betrayed itself by the emergence of certain tumours in the groin or armpits, some of which grew as large as a common apple, others as an egg...from the two said parts of the body this deadly gavocciolo soon began to propagate and spread itself in all directions indifferently; after which the form of the malady began to change, black spots or livid making their appearance in many cases on the arm or the thigh or elsewhere, now few and large, now minute and numerous. as the gavocciolo had been and still was an infallible token of approaching death, such also were these spots on whomsoever they showed themselves [26] . indeed, the mortality of untreated bubonic plague is close to 70%, usually within 8 days, while the mortality of untreated pneumonic plague approaches 95%. treated with antibiotics, mortality drops to around 11% [27] . at the time, scientific authorities were at a loss regarding the cause of the affliction. the first official report blamed an alignment of three planets from 1345 for causing a "great pestilence in the air" [28] . it was followed by a more generally accepted miasma theory, an interpretation that blamed bad air. it was not until the late xix century that the black death was understood for what it was -a massive yersinia pestis pandemic [29] . this strain of yersinia tends to infect and overflow the guts of oriental rat fleas (xenopsylla cheopis) forcing them to regurgitate concentrated bacteria into the host while feeding. such infected hosts then transmit the disease further and can infect humans -bubonic plague [30] . humans can transmit the disease by droplets, leading to pneumonic plague. the mortality of the black death varied between regions, sometimes skipping sparsely populated rural areas, but then exacting its toll from the densely populated urban areas, where population perished in excess of 50, sometimes 60% [31] . in the vacuum of a reasonable explanation for a catastrophe of such proportions, people turned to religion, invoking patron saints, the virgin mary, or joining the processions of flagellants whipping themselves with nail embedded scourges and incanting hymns and prayers as they passed from town to town [32] . the general interpretation in predominantly catholic europe, as in the case of justinian plague, centered on the divine "punishment for sins." it then sought to identify those individuals and groups who were the "gravest sinners against god," frequently singling out minorities or women. jews in europe were commonly targeted, accused of "poisoning the wells" and entire communities persecuted and killed. non-catholic christians (e.g., cathars) were also blamed as "heretics" and experienced a similar fate [33] . in other, non-christian parts of the world affected by the plague, a similar sentiment prevailed. in cairo, the sultan put in place a law prohibiting women from making public appearances as they may tempt men into sin [34] . for bewildered and terrified societies, the only remedies were inhalation of aromatic vapors from flowers or camphor. soon, there was a shortage of doctors which led to a proliferation of quacks selling useless cures and amulets and other adornments that claimed to offer magical protection [35] . entire neighborhoods, sometimes entire towns, were wiped out or settlements abandoned. crops could not be harvested, traveling and trade became curtailed, and food and manufactured goods became short. the plague broke down the normal divisions between the upper and lower classes and led to the emergence of a new middle class. the shortage of labor in the long run encouraged innovation of labor-saving technologies, leading to higher productivity [2] . the effects of such a large-scale shared experience on the population of europe influenced all forms of art throughout the period, as evidenced by works by renowned artists, such as chaucer, boccaccio, or petrarch. the deep, lingering wake of the plague is evidenced in the rise of danse macabre (dance of the death) in visual arts and religious scripts [36] , its horrors perhaps most chillingly depicted by paintings titled the triumph of death (fig. 2. 2) [37] . the plague made several encore rounds through europe in the following centuries, occasionally decimating towns and entire societies, but never with the same intensity as the black death [2] . with the breakdown of societal structure and its infrastructures, many professions, notably that of medical doctors, were severely affected. many towns throughout europe lost their providers to plague or to fear thereof. in order to address this shortage in times of austere need, many municipalities contracted young doctors from whatever ranks were available to perform the duty of the plague doctor (medico della peste) [38] . venice was among the first citystates to establish dedicated practitioners to deal with the issue of plague in 1348. their principal task, besides taking care of people with the plague, was to record in public records the deaths due to the plague [39] . in certain european cities like florence and perugia, plague doctors were the only ones allowed to perform autopsies to help determine the cause of death and managed to learn a lot about human anatomy. among the most notable plague doctors of their time were nostradamus, paracelsus, and ambrois pare [40] . the character of the plague doctor was drawing from experiences from ancient cultures that had dealt with contagious diseases, medieval societies observed the connection between the passage of time and the eruption of symptoms, noting that, after a period of observation, individuals who had not developed symptoms of the illness would likely not be affected and, more importantly, would not spread the disease upon entering the city. to that end, they started instituting mandatory isolation. the first known quarantine was enacted in ragusa (city-state of dubrovnik) in 1377, where all arrivals had to spend 30 days on a nearby island of lokrum before entering the city. this period of 30 days (trentine) was later extended to 40 days (quarenta giorni or quarantine) [42] . the institution of quarantine was one of the rarely effective measures that took place during the black death and its use quickly spread throughout europe. quarantine remains in effect in the present time as a highly regulated, nationally and internationally governed public health measure available to combat contagions [43] . the spanish flu pandemic in the first decades of the twentieth century was the first true global pandemic and the first one that occurred in the setting of modern medicine, with specialties such as infectious diseases and epidemiology studying the nature of the illness and the course of the pandemic as it unfolded. it is also, as of this time, the last true global pandemic with devastating consequences for societies across the globe [44] . it was caused by the h1n1 strain of the influenza virus, [45] a strain that had an encore outbreak in the early years of the twenty-first century. despite advances in epidemiology and public health, both at the time and in subsequent decades, the true origin of spanish flu remains unknown, despite its name. as possible sources of origin, cited are the usa, china, spain, france, or austria. these uncertainties are perpetuated by the circumstances of the spanish flu -it took place in the middle of world war i, with significant censorships in place, and with fairly advanced modes of transportation, including intercontinental travel [44] . within months, the deadly h1n1 strain of influenza virus had spread to every corner of the world. in addition to europe, where massive military movements and overcrowding contributed to massive spread, this virus devastated the usa, asia, africa, and the pacific islands. the mortality rate of spanish flu ranged between 10% and 20%. with over a quarter of the global population contracting that flu at some point, the death toll was immense -well over 50 million, possibly 100 million dead. it killed more individuals in a year than the black death had killed in a century [46] . this pandemic, unusually, tended to mortally affect mostly young and previously healthy individuals. this is likely due to its triggering a cytokine storm, which overwhelms and demolishes the immune system. by august of 1918, the virus had mutated to a much more virulent and deadlier form, returning to kill many of those who avoided it during the first wave [47] . spanish flu had an immense influence on our civilization. some authors (price) even point out that it may have tipped the outcome of world war i, as it affected armies of germany and the austrian-hungarian empire earlier and more virulently than their allied opponents (fig. 2.4) [48] . many notable politicians, artists, and scientists were either affected by the flu or succumbed to it. many survived and went on to have distinguished careers in arts and politics (e.g., walt disney, greta garbo, raymond chandler, franz kafka, edward munch, franklin delano roosevelt, and woodrow wilson). many did not; this pandemic counted as its victims, among others, outstanding painters like gustav klimt and egon schiele [49] , and acclaimed poets like guillaume apollinaire. it also claimed the life of sigmund freud's fifth child -sophie halberstadt-freud. this pandemic was also the first one where the longlingering effects could be observed and quantified. a study of us census data from 1960 to 1980 found that the children born to women exposed to the pandemic had more physical ailments and a lower lifetime income than those born a few months earlier or later. a 2006 study in the journal of political economy found that "cohorts in utero during the pandemic displayed reduced educational attainment, increased rates of physical disability, lower income, lower socioeconomic status, and higher transfer payments compared with other birth cohorts" [50] . despite its immense effect on the global civilization, spanish flu started to fade quickly from the public and scientific attention, establishing a precedent for the future pandemics, and leading some historians (crosby) to call it the "forgotten pandemic" [51] . one of the explanations for this treatment of the pandemic may lie in the fact that it peaked and waned rapidly, over a period of 9 months before it even could get adequate media coverage. another reason may be in the fact that the pandemic was overshadowed by more significant historical events, such as the culmination and the ending of world war i. a third explanation may be that this is how societies deal with such rapidly spreading pandemicsat first with great interest, horror, and panic, and then, as soon as they start to subside, with dispassionate disinterest. hiv/aids is a slowly progressing global pandemic cascading through decades of time, different continents, and different populations, bringing new challenges with every new iteration and for every new group it affected. it started in the early 1980s in the usa, causing significant public concern as hiv at the time inevitably progressed to aids and ultimately, to death. the initial expansion of hiv was marked by its spread predominantly among the gay population and by high mortality, leading to marked social isolation and stigma. hiv affects about 40 million people globally (prevalence rate: 0.79%) and has killed almost the same number of people since 1981 [52] . it causes about one million deaths a year worldwide (down from nearly two million in 2005) [53] . while it represents a global public health phenomenon, the hiv epidemic is particularly alarming in some sub-saharan african countries (botswana, lesotho, and swaziland), where the prevalence tops 25% [54] . in the usa, about 1.2 million people live with hiv and about 12,000 die every year (down from over 40,000 per year in the late 1990s). hiv in the usa disproportionately affects gay population, transgendered women, and african-americans [55] . being a fairly slowly spreading pandemic, hiv has received formidable public health attention, both by national and by international administrations and pharmaceuticals. advances in treatment (protease inhibitors and anti-retrovirals) have turned hiv into a chronic condition that can be managed by medications. it is a rare infectious disease that has managed to attract the focus of mental health which, in turn, resulted in a solid volume of works on mental health and hiv [56] . by studying the mental health of hiv, we can begin to understand some of the challenges generally associated with infectious diseases. we know, for example, that the lifetime prevalence rate for depression in hiv individuals is, at 22%, more than twice the prevalence rate in general population [57] . we understand how depression in hiv individuals shows association with substance abuse and that issues of stigma, guilt, and shame affect the outlook for hiv patients, including their own adherence to life-saving treatments [58] . we know about medical treatments of depression in hiv and we have studies in psychotherapy for patients with hiv. some of those approaches can be very useful in treating patients in the context of a pandemic. given the contrast between the chronicity of the hiv and the acuity of a potential pandemic, most of those approaches cannot be simply translated from mental health approach to hiv and used for patients in a rapidly advancing outbreak or a pandemic. smallpox was a highly contagious disease for which edward jenner developed the world's first vaccine in 1798. caused by the variola virus, it was a highly contagious disease with prominent skin eruptions (pustules) and mortality of about 30%. it may have been responsible for hundreds of millions of fatalities in the twentieth century alone. due to the wellcoordinated global effort starting in 1967 under the leadership of donald henderson, smallpox was eradicated within a decade of undertaking the eradication on a global scale [59] . the smallpox outbreak in the former yugoslavia in 1972 was a far cry from even an epidemic, let alone a pandemic, but it illustrated the challenges associated with a rapidly spreading, highly contagious illness in a modern world. it started with a pilgrim returning from the middle east, who developed fever and skin eruptions. since a case of smallpox had not been seen in the region for over 30 years, physicians failed to correctly diagnose the illness and nine healthcare providers ended among 38 cases infected by the index case and first fatality [60] . socialist yugoslavia at the time declared martial law and introduced mandatory revaccination. entire villages and neighborhoods were cordoned off (cordon sanitaire is a measure of putting entire geographic regions in quarantine). about 10,000 individuals who may have come into contact with the infected were placed in an actual quarantine. borders were closed, and all non-essential travel was suspended. within 2 weeks, the entire population of yugoslavia was revaccinated (about 18 million people at the time). during the outbreak, 175 cases were identified, with 35 fatalities. due to prompt and massive response, however, the disease was eradicated and the society returned to normal within 2 months [60] . this event has proven to be a useful model for working out scenarios ("dark winter") [61] for responses to an outbreak of a highly contagious disease, both as a natural occurrence [62] and as an act of bioterrorism [63] . severe acute respiratory syndrome (sars) was the first outbreak in the twenty-first century that managed to get public attention. caused by the sars corona virus (sars-cov), it started in china and affected fewer than 10,000 individuals, mainly in china and hong kong, but also in other countries, including 251 cases in canada (toronto) [64] . the severity of respiratory symptoms and mortality rate of about 10% caused a global public health concern. due to the vigilance of public health systems worldwide, the outbreak was contained by mid-2003 [65] . this outbreak was among the first acute outbreaks that had mental health aspects studied in the process and in the aftermath, in various part of the world and in different societies, yielding valuable data on effects of an acute infectious outbreak on affected individuals, families, and the entire communities, including the mental health issues facing healthcare providers [66] . some of the valuable insights into the mental health of patients in isolation, survivors of the severe illness, or psychological sequelae of working with such patients were researched during the sars outbreak. "swine flu" or h1n1/09 pandemic the 2009 h1n1 pandemic was a reprise of the "spanish flu" pandemic from 1918, but with far less devastating consequences. suspected as a re-assortment of bird, swine, and human flu viruses, it was colloquially known as the "swine flu" [67] . it started in mexico in april of 2009 and reached pandemic proportions within weeks [68] . it began to taper off toward the end of the year and by may of 2010, it was declared over. it infected over 10% of the global population (lower than expected), with a death toll estimated varying from 20,000 to over 500,000 [69] . although its death rate was ultimately lower than the regular influenza death rates, at the time it was perceived as very threatening because it disproportionately affected previously healthy young adults, often quickly leading to severe respiratory compromise. a possible explanation for this phenomenon (in addition to the "cytokine storm" applicable to the 1918 h1n1 outbreak) is attributed to older adults having immunity due to a similar h1n1 outbreak in the 1970s [70] . this pandemic also resulted in some valuable data studying and analyzing the mental health aspects of the outbreak. it was among the first outbreaks where policy reports included mental health as an aspect of preparedness and mitigation policy efforts. this outbreak of h1n1 was notable for dissonance between the public sentiment about the outbreak and the public health steps recommended and undertaken by who and national health institutions. general public sentiment was that of alarm caused by who releases and warnings, but it quickly turned to discontent and mistrust when the initial grim outlook of the outbreak failed to materialize [71] . health agencies were accused of creating panic ("panicdemic") and peddling unproven vaccines to boost the pharmaceutical companies (in 2009, some extra $1,5 billion worth of h1n1 vaccines were purchased and administered in the usa) [72] . this outbreak illustrated how difficult it may be to gauge and manage public expectations and public sentiments in the effort to mobilize a response. it also demonstrated how distilling descriptions of the impact of a complex public health threat like a pandemic into a single term like "mild," "moderate," or "severe" can potentially be misleading and, ultimately, of little use in public health approach [73] . ebola virus, endemic to central and west africa, with fruit bats serving as a likely reservoir, appeared in an outbreak in a remote village in guinea in december 2013. spreading mostly within families, it reached sierra leone and liberia, where it managed to generate considerable outbreaks over the following months, with over 28,000 cases and over 11,000 fatalities. a very small number of cases were registered in nigeria and mali, but those outbreaks were quickly contained [74] . ebola outbreak, which happened to be the largest outbreak of ebola infection to date, gained global notoriety after a passenger from liberia fell ill and died in texas in september of 2014, infecting two nurses caring for him, and leading to a significant public concern over a possible ebola outbreak in the usa [75] . this led to a significant public health and military effort to address the outbreak and help contain it on site (operation united assistance) [76, 77] . zika virus was a little known, dormant virus found in rhesus monkeys in uganda. prior to 2014, the only known outbreak among humans was recorded in micronesia in 2007. the virus was then identified in brazil in 2015, after an outbreak of a mild illness causing a flat pinkish rash, bloodshot eyes, fever, joint pain and headaches, resembling dengue. it is a mosquitoborne disease (aedes aegypti), but it can be sexually transmitted. despite its mild course, which initially made it unremarkable form the public health perspective, infection with zika can cause guillain-barre syndrome in its wake in adults and, more tragically, cause severe microcephalia in unborn children of infected mothers (a risk of about 1%) [78] . in brazil, in 2015, for example, there were 2400 birth defects and 29 infant deaths due to suspected zika infection [79] . zika outbreak is an illustrative case of the context of global transmission; it was transferred from micronesia, across the pacific, to brazil, whence it continued to spread [78] . it is also a case of a modern media pandemic; it featured prominently in the social media. in early 2016, zika was being mentioned 50 times a minute in twitter posts. social media were used to disseminate information, to educate, or to communicate concerns [80] . its presence in social media, perhaps for the first time in history, allowed social researchers to study the public sentiment, also known as the emotional epidemiology (ofri), in real time [81] . while both public health institutions and the general public voiced their concern with the outbreak, scientists and officials sought to provide educational aspect, while concerned public was trying to have their emotional concerns addressed. it is indicative that 4 out of 5 posts on zika on social media were accurate; yet, those that were "trending" and gaining popularity were posts with inaccurate content (now colloquially referred to as the "fake news") [82] . this is a phenomenon that requires significant attention in preparing for future outbreaks because it may hold a key not only to preparedness, but also to execution of public health plans that may involve quarantine and immunization. since 2016, zika has continued to spread throughout south america, central america, the caribbean, and several states within the usa. it remains a significant public health concern, as there is no vaccine and the only reliable way to avoid the risk for the offspring is to avoid areas where zika was identified or to postpone pregnancy should travel to or living in affected areas be unavoidable [78] . disease x disease x is not, as of yet, an actual disease caused by a known agent, but a speculated source of the next pandemic that could have devastating effects on humanity. knowing the scope of deleterious effects a pandemic outbreak can have on humankind, in the wake of the ebola outbreak, the world health organization (who) decided to dedicate formidable resources to identifying, studying, and combating possible future outbreaks. it does so in the form of an r&d blueprint, though devising its global strategy and preparedness plan that allows the rapid activation of r&d activities during epidemics [83] . r&d blueprint maintains and updates a list of so-called identified priority diseases. this list is updated at regular intervals and, as of 2018, it includes diseases such as ebola and marburg virus diseases, lassa fever, middle east respiratory syndrome coronavirus (mers-cov) and severe acute respiratory syndrome (sars), nipah and henipa virus diseases, zika, and others [84] . for each disease identified, an r&d roadmap is created, followed by target product profiles (i.e., immunizations, treatment, and regulatory framework). those efforts are important to help us combat a dangerous outbreak of any of the abovementioned diseases, but also to fend off disease x. since disease x is a hypothetical entity, brought by a yet unknown pathogen that could cause a serious international pandemic, the r&d blueprint explicitly seeks to enable cross-cutting r&d preparedness that is also relevant for both existing culprits and the unknown future "disease x" as much as possible. who utilizes this r&d blueprint vehicle to assemble and deploy a broad global coalition of experts who regularly contribute to the blueprint and who come from several medical, scientific, and regulatory backgrounds. its advisory group, at the time, does not include mental health specialists [85] . the black death: the greatest catastrophe ever the great leveler: violence and the history of inequality from the stone age to the twenty-first century. chapter 10: the black death mortality risk and survival in the aftermath of the medieval black death an epidemiologic analysis of the ten plagues of egypt the noble qur'an surah thucydides' description of the great plague the plague of athens: epidemiology and paleopathology the thucydides syndrome: a new hypothesis for the cause of the plague of athens the thucydides syndrome: ebola déjà vu? (or ebola reemergent?). emerg infect dis hippocrates of kos, the father of clinical medicine, and asclepiades of bithynia, the father of molecular medicine the antonine plague and the decline of the roman empire the plague under marcus aurelius and the decline and fall of the roman empire the antonine plague: a global pestilence in the ii century d justinian's plague (541-542 ce) loeb library of the greek and roman classics justinian's flea: plague, empire, and the birth of europe plague in the ancient world: a study from thucydides to justinian ecclesiastical history (ad431-594), trans the attitude of the secular historians of the age of justinian towards the classical past the justinian plague (part one) influence of the epidemic on the rise of the islamic empire mortality risk and survival in the aftermath of the medieval black death death and miasma in victorian london: an obstinate belief adaptive strategies of yersinia pestis to persist during inter-epizootic and epizootic periods the black death 1346-1353: the complete history medieval europe: a short history black death. simon and schuster the black death the air of history (part ii) medicine in the middle ages mixed metaphors. the danse macabre in medieval and early modern europe the theme of death in italian art: the triumph of death daily life during the black death communities and crisis: bologna during the black death nostradamus: the new revelations. barnes & noble books images of plague and pestilence: iconography and iconology the origin of quarantine lessons from the history of quarantine, from plague to influenza a. emerging infectious diseases cdc: remembering the 1918 influenza pandemic molecular virology: was the 1918 flu avian in origin? plagues & wars: the 'spanish flu' pandemic as a lesson from history pandemic versus epidemic influenza mortality: a pattern of changing age distribution contagion and chaos expressionist portraits is the 1918 influenza pandemic over? long-term effects of in utero influenza exposure in the post-1940 u.s. population america's forgotten pandemic: the influenza of 1918 the spread, treatment, and prevention of hiv-1: evolution of a global pandemic estimates of global, regional, and national incidence, prevalence, and mortality of hiv, 1980-2015: the global burden of disease study 2015 academy of consultation-liaison psychiatry, hiv psychiatry bibliography meta-analysis of the relationship between hiv infection and risk for depressive disorders cognitive behavioural therapy for adherence and depression in patients with hiv: a three-arm randomised controlled trial the last major outbreak of smallpox (yugoslavia, 1972): the importance of historical reminders shining light on "dark winter evaluating public health responses to reintroduced smallpox via dynamic, socially structured, and spatially distributed metapopulation models extracting key information from historical data to quantify the transmission dynamics of smallpox responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management summary of probable sars cases with onset of illness from 1 was sars a mental health catastrophe? gen hosp psychiatry geographic dependence, surveillance, and origins of the 2009 influenza a (h1n1) virus in new theory, swine flu started in asia, not mexico. the new york times estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study risk factors for hospitalisation and poor outcome with pandemic a/ h1n1 influenza: united kingdom first wave assessing the severity of the novel influenza a/ h1n1 pandemic doctors rake in billions battling h1n1 flu by dalia fahmy. abc news reflections on pandemic (h1n1) 2009 and the international response the emergence of ebola as a global health security threat: from 'lessons learned' to coordinated multilateral containment efforts overview, control strategies, and lessons learned in the cdc response to the 2014-2016 ebola epidemic ebola outbreak in west africa military ebola mission in liberia coming to an end zika: the origin and spread of a mosquito-borne virus the microcephaly epidemic and zika virus: building knowledge in epidemiology propagating and debunking conspiracy theories on twitter during the 2015-2016 zika virus outbreak the emotional epidemiology of h1n1 influenza vaccination spreading the (fake) news: exploring health messages on social media and the implications for health professionals using a case study who: r&d blueprint, about the r&d blueprint who: r&d blueprint, scientific advisory group members key: cord-256786-7gca01lr authors: bartilotti‐matos, f; davies, p. title: pearls and pitfalls: two contrasting hiv diagnoses in the covid‐19 era and the case for screening date: 2020-08-13 journal: j med virol doi: 10.1002/jmv.26428 sha: doc_id: 256786 cord_uid: 7gca01lr the risk of coronavirus disease 2019 (covid‐19) for people living with hiv (plwh) is poorly understood. the vast majority of reported cases of covid‐19/hiv co‐infection consists of those with an established hiv diagnosis who are on anti‐retroviral therapy (art). better knowledge of the effects of covid19 on hiv patients who are art naïve is required. two cases of previously undiagnosed hiv presenting to secondary care with respiratory symptoms are detailed in this series, with a view to extrapolate lessons on blood borne virus (bbv) screening in the covid‐19 era. this article is protected by copyright. all rights reserved. two cases of previously undiagnosed hiv presenting to secondary care with respiratory symptoms are detailed in this series, with a view to extrapolate lessons on blood borne virus (bbv) screening in the covid-19 era. the first patient, a fit and well white scottish 38-year-old man, with a body mass index (bmi) of 24.6, presented after five days of dyspnoea and a productive cough. his temperature was 38 o c and spo2 was 89% on room-air. investigations were remarkable for a lymphocyte count of 0.7x10 9 /l, a c-reactive protein (crp) of 242mg/l and a chest radiograph demonstrating subtle bibasal consolidation. subsequently, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was present on polymerase chain reaction (pcr) from a viral swab. he rapidly deteriorated with progressive respiratory failure requiring intubation and ventilation. whilst intubated, a routine bbv screen on day four of admission demonstrated hiv seropositivity. there were no discernible clinical or lifestyle risk factors for this diagnosis bar disease severity. at diagnosis, the viral load was 6.35log10 and cd4 + was 220cells/mm 3 . art (emtricitabine/tenofovir and dolutegravir) and co-trimoxazole, for pneumocystis jiroveci pneumonia prophylaxis (pjp -pcr negative), were commenced via nasogastric tube. he went on to make a full recovery and was discharged on day 17 of admission. the second patient was a white scottish 51-year-old man with a bmi of 23.6. he had a background of herpes zoster, weight loss, oral candidiasis and pernicious anaemia. he presented with dyspnoea and diarrhoea. examination was unremarkable save oral candidiasis. he was afebrile, tachycardiac at 120bpm, mildly dyspnoeic at 23bpm and oxygen saturations were 96% on air. bilateral consolidation on chest radiograph was reported as indeterminate accepted article for covid-19. blood investigations demonstrated a lymphocyte count of 0.4x10 9 , a crp of 27mg/l and d-dimer of 720ng/ml. it was felt that covid-19 was the likely diagnosis. a computed tomography pulmonary angiogram (ctpa) was performed, ruling out a pulmonary embolus but demonstrating bilateral ground glass changes that were reported as atypical for covid-19. despite these findings and two negative swabs for sars-cov2 pcr, covid-19 remained the clinical diagnosis until day-six of admission when a bbv screen confirmed hiv seropositivity with a viral load of 5.28log10 and cd4 + 25cells/mm 3 . a subsequent sputum sample was positive for pjp on pcr for which he was started on treatment dose co-trimoxazole. he was started on art (emtricitabine/tenofovir and raltegravir) prior to discharge. he was discharged on day 19 with follow-up by infectious diseases. the current scientific consensus is that plwh receiving treatment are at no greater risk of severe covid-19. 1, 2, 3, 4 however, the evidence base in the published literature pertains to stable patients with a supressed viral load. to the best of our knowledge only seven patients diagnosed with hiv at presentation with acute covid-19 have been reported, see table one. 5, 6 there is an inference of a higher disease severity and younger age at presentation, however conventional risk factors such as obesity or respiratory disease impart significant variability. 15, 16 case one is the first to our knowledge requiring intubation. these cases confer salient lessons. firstly, the hiv and covid-19 co-infection was diagnosed in the absence of risk factors, prompted by high disease severity in an atypically young and fit patient. on the second case, a classic presentation of pjp, a covid-19 mimic, was initially missed in the context of the pandemic, due to similarities in clinical presentation, radiographic changes, and blood parameters. there are approximately 7,500 patients living with undiagnosed hiv in the uk. 7 they are considered immunosuppressed, under the assumption that this may correlate with a severe covid-19 phenotype. 8 previous case series have observed a non-significant correlation between a low cd4 + count and increased disease severity (table one), but this is inconsistent. 9 without other risk factors for covid-19, the severe presentation in case one supports this assertion. this also support previous findings of a lower age requiring hospitalisation. 2, 3, 8, 14 larger studies in high prevalence settings are required to explore these associations further. the british hiv association (bhiva) recommends hiv screening for community acquired pneumonia presentations. 11 however, there is currently no extension of this to patients with covid-19. a systematic review published in july 2020 states that hiv testing should be offered to anyone presenting with a clinical picture of viral pneumonia. 3 active patient screening is required to extend our knowledge on the role of hiv as a risk factor for covid-19. undiagnosed plwh represent a vulnerable patient group and advocating routine bbv screening is essential for those requiring hospitalisation with covid-19. the additional benefit of this would be prompting the early diagnosis of mimics such as pjp, seen in the second case, which conveys a high mortality and requires aggressive treatment. capacity in virology laboratories may preclude mass testing, however it should be advocated for in high-prevalence areas and in younger patients with atypically severe presentations. there may be a role for point of care hiv-antibody tests depending on local epidemiology and resources. there are no current or potential conflicts of interest. this article is protected by copyright. all rights reserved. written informed consent was obtained for both patients. francisca bartilotti matos and peter davies both conceived the idea of this correspondence and participated equally to its writing. we had full access to all the data in the study and had final responsibility to submit it for application. there have been no funding sources. none to declare. table table 1 -a summary of notable case series published to date of hiv/ covid-19 co-infections. basic demographics included country of origin and clinical characteristics of the cases. in addition to a precis of the main findings of the article. clinical characteristics and outcomes in people living with hiv hospitalized for covid-19 covid-19 in people living with human immunodeficiency virus: a case series of 33 patients coronavirus disease 2019 (covid-19) outcomes in hiv/aids patients: a systematic review clinical features and outcomes of hiv patients with coronavirus disease 2019 covid-19 in patients with hiv: clinical case series recovery from covid-19 in two patients with coexisted hiv infection hiv in the united kingdom: towards zero 2030: 2019 report description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort clinical features and outcome of hiv/sars-cov-2 coinfected patients in the bronx hospitalized patients with covid-19 and human immunodeficiency virus: a case series uk national guidelines for hiv testing the spanish hiv/covid-19 collaboration. incidence and severity of covid-19 in hiv positive persons receiving antiretroviral treatment a case of sars-cov-2 infection in an untreated hiv patient in tokyo, japan the characteristics of two patients coinfected with sars-cov-2 and hiv in wuhan hiv/sars-cov-2 coinfected patients in istanbul, turkey covid-19 in 3 people living with hiv in the united kingdom one case of coronavirus disease 2019 (covid-19) in a patient co-infected by hiv with a low cd4 + t-cell count key: cord-010175-p2py9wau authors: winter, harland; chang, tien-lan title: gastrointestinal and nutritional problems in children with immunodeficiency and aids date: 1996-04-01 journal: pediatr clin north am doi: 10.1016/s0031-3955(05)70421-1 sha: doc_id: 10175 cord_uid: p2py9wau nan harland winter, md, and tien-lan chang, md children acquire human immunodeficiency virus (hiv) either perinatally from an infected mother (vertical transmission) or from infected blood or blood products. the number of children infected following a blood transfusion has dropped markedly following the institution of rigorous screening protocols for blood donors in the mid-1980s. by the early 1990s, more than 95% of newly diagnosed hiv-infected children acquired the disease via vertical infection.= the world health organization estimates that more than 10 million people throughout the world are infected with hiv (table 1) . three million of these individuals are women, most of whom are fewer than 40 years of age, whereas 500,000 of them are children. 37 thus, heterosexual transmission of hiv is the most common means of acquiring the infection when viewed from a worldwide perspective. hiv, a single-stranded rna lentivirus, infects cells that express a receptor capable of binding to the envelope glycoprotein (gp), gp 120. t lymphocytes and monocytes or macrophages that are cd4-positive are the primary targets of the virus, but reports suggesting that other cells in the gastrointestinal tract can be infected have led investigators to data from world health organization: the hiv-aids pandemic: 1993 overview. geneva: who/ gpngnp193. 1, 1993. speculate that gastrointestinal symptoms may be related to epithelial cell infection with hiv-1. fox and colleagues9 reported that hiv-1 infection of the gastrointestinal tract was limited to the lymphoid elements of the lamina propria; other investigators believe that, because intestinal epithelial cell line cultures became infected in the laboratory,1y epithelial cells were infected in vivo in hiv-infected adults.'o, the characteristics of the mucosal immune system most likely have a significant role in the pathobiology of hiv-1 disease in children; however, mucosal immune function has not been studied specifically in hiv-infected children and, thus, pediatricians are left to speculate that observations made in the adult hiv-infected population are relevant to children. table 2 summarizes gastrointestinal mucosal immunologic changes that occur in hivinfected individuals. vertical transmission occurs in approximately 30% of hiv-infected pregnant women who do not take antiretroviral therapy during pregnancy. the observations that transmission is increased in women who were symptomatic or who had more advanced aids27 and that zidovudine therapy given during pregnancy reduces perinatal transmission3 suggest that viral burden is an important factor in vertical transmission; however, the effects of maternal nutritional status, micronutrient deficiency, or acute infection on viral replication are difficult to evaluate. in addition, most hiv-infected women in africa, asia, and south america breast-feed their infants. this additional means by which infants can possibly become infected complicates assessment of factors contributing to transmission. in africa, the percentage of postnatal transmission is approximately 50y0.~~ nevertheless, the morbidity and mortality caused by formula feeding in countries where potable water is a premium and safe infant formula is not readily available seem to be greater than the risk of acquiring hiv-1 from breast milk. the current recommendation is for the hiv-exposed infant to have formula feeding if and only if safe the deterioration of the immune system and mucosal immune systems results in cellular and humoral immunoregulatory deficiencies. in the gastrointestinal tract, hiv-infected lymphocytes could migrate from the lymphoid aggregates through the mesenteric nodes, the thoracic duct, and into the circulation. following selection by receptors on high endothelial venules, these infected cells then migrate home to the lamina propria, whereby in situ hybridization isolated hiv-infected cells can be identified (fig. 1 ). most evidence 'supports the hypothesis that deterioration of mucosal immune function results in bacterial overgrowth; increased production of bacterial products, such as endotoxin; activation of mucosal lymphocytes with increased cytokine production; and probable interaction between immunoregulatory elements and epithelial cell function (fig. 2) . although the reasons for early development of lactose intolerance and malabsorption are not known, substances involved in immune regulation also may interact with intestinal epithe-lial cells, resulting in dysfunction. hiv also may have a role in the genesis of intestinal dysfunction, but data are not available. clearly, enteric infections begin to occur at the time when immune function is deteriorating (fig. 3) . the contribution of chronic intestinal infection to immune dysfunction, malabsorption, and malnutrition suggests that all of these factors are interrelated (fig. 4) . one of the more important determinants of survival for the hivinfected child is the health status of the mother. in studies from africa, if an hiv-infected mother is symptomatic or dies, her hiv-infected infant is at increased risk for chronic diarrhea partially because of the resulting reliance on formula.31 chronic diarrhea in the hiv-infected child is an important prognostic variable for predicting malnutrition and death. because of the availability of safe formula in north america and europe, the relationship between maternal health and infant survival is not as obvious. nevertheless, a chronically ill mother has an obvious negative impact on infant growth and development, particularly if no additional support is available, such as respite and day care programs designed to enrich infants' psychosocial development and nutritional status. in hiv-infected children, nausea and vomiting can be caused by infectious diseases, such as helicobacter pylori or cytomegalovirus, medications, or central nervous system disorders. in a child with nausea, anorexia may be the presenting manifestation because she or he is not able to verbalize the sensation. in these individuals, refusal to chew or eat may be caused by gingival disease or painful lesions of candida in the mouth. in many children, an identifiable agent or pathogen may not be found despite a thorough search. some of the therapeutic agents that have been implicated as causes of nausea and vomiting are as follows: altered mental status or developmental delay should alert the clinician to the possibility of central nervous system disease, such as encephalopathy caused by hiv, or pathogens, such as toxoplasmosis. lymphoproliferative disorders in the central nervous system are rare in the pediatric population; however, lymphoma of the gastrointestinal tract can cause splenomegaly resulting in compression of the stomach and early satiety. evaluation of hiv-infected children with anorexia, nausea, or vomiting should begin with a careful history, social history, physical examination, and neurologic evaluation. an upper gastrointestinal radiograph is not reliable enough to establish or rule out mucosal disease. for this reason, endoscopic evaluation is frequently necessary in children with persistent symptoms and normal hepatobiliary and pancreatic tests. mucosal biopsies may identify an enteric pathogen or inflammation that can be treated with a specific agent. if no cause can be found, symptoms can be managed with phenothiazine derivatives, such as triethylperazine maleate (torecan), prochlorperazine (compazine), or promethazine (phenergan). other agents for which anecdotal treatment experience exists in children include: benzquinamide (emete-con); trimethobenzamide hydrochloride (tigan); hydroxyzine (vistaril or atarax); metoclopramide (reglan); cisapride (propulsid); and scopolamine (transdermscop); dronabinal (marinol). if treatment fails to relieve the symptoms, re-evaluation should be considered. difficulty in swallowing (dysphagia) or pain with swallowing (odynophagia) in children can be caused by oral lesions that can be identified by careful inspection of the mouth. stomatitis caused by 2',3'-dideoxycytidine 5'-triphosphate (ddc), herpes simplex or candida is treatable if the diagnosis is established. when oral lesions are present, coexistent esophagitis should be suspected. in contrast, if the mouth is free of lesions, esophagitis cannot be ruled out. candida and cytomegalovirus are the most common infectious agents causing esophagitis. dysphagia and odynophagia in hiv-infected children are more commonly associated with candida than with cytomegalovirus. children who are taking h, antagonists seem to be at increased risk for developing candida esophagitis. medications, such as zidovudine, have been reported to cause esophageal ulceration if, when swallowed, they do not reach the stomach.6 treatment for specific causes of oral or esophageal lesions is summarized in table 3 . although enteric pathogens are frequently identified as the cause the incidence of of diarrhea and weight loss in hiv-infected enteric infection in hiv-infected children seems to be lower,4o and the relationship between diarrhea, enteric pathogens, and growth retardation is not as clearly understood. in figure 4 , the interrelationship between malabsorption, malnutrition, immune deficiency, and enteric infection is depicted. enteric infection results in intestinal injury and malabsorption, which, if not compensated by additional nutrient support, results in nutritional deficiency. the development of malnutrition causes immune deficiency, which is characterized by a defect in t-cell function that is similar to the defect caused by hiv disease. defective t-cell function results in increased susceptibility to enteric infection, and the circle is completed. hiv can interact at any of the stages of this cycle. in theory, intestinal absorption can be altered by modifying enterocyte function through immune modulators. by increasing apoptosis, hiv could cause premature senescence of enterocytes and decrease brushborder expression of disaccharidases and peptidases. some of these same agents, such as the cytokine, tumor necrosis factor-a), are upregulated by hiv infection, affect intermediate metabolism, and cause malnutrition by increasing nutrient requirements. the effects of hiv on the immune system are well known and result in immunocompromise and increased susceptibility to opportunistic infection. similar immunoregulatory abnormalities probably occur in the mucosal immune system, resulting in enteric infection. thus, hiv interacts at many levels to potentiate the development of malabsorption, malnutrition, immune deficiency, and enteric infection. giardia zarnbzia causes watery diarrhea, abdominal distention, and crampy abdominal pain.4, 22, 31 metronidazole or furazolidone is effective therapy and eradicates the organism in more than 70% of infected individuals. giardia zambzia does not occur more frequently in hivinfected children than in the general population, but retreatment may be necessary in the immunocompromised host. crypfosporidium parvum causes an acute, self-limited diarrheal illness in the immunocompetent host, but in the immunodeficient child with hiv disease, the infection causes a secretory diarrhea that is chronic and debilitating. the organism usually can be identified in the stool by immunofluorescent techniques or by kinyoun carbolfuchsin stain.7 in hiv-infected children in the united states, the incidence of cryptosporidiosis is lower than that reported in africa and south america.2, 4, 33 crypfosporidium can infect the small intestine, colon, gallbladder, biliary tract, and pancreatic duct. no therapy is consistently effective in eradicating the organism, but octreotide is reported to decrease stool reports of the beneficial effects of hyperimmune bovine colostrum suggest that this form of passive immunotherapy may be effective in hiv-infected individual^.^^ other enteric parasitic infections, including isospora bezzi and microsporidium, are rarely identified in hiv-infected children; however, blastocysfis hominis, a protozoan whose role as an enteric pathogen is still debated, may be more prevalent in hiv-infected children with diarrhea than in hiv-negative ~h i l d r e n .~ bacteria are an important cause of diarrhea throughout the world and for this reason contribute to the list of identifiable pathogens found in hiv-infected children. in africa, pathogenic strains of escherickia coli were identified in over three fourths of hiv-infected children. 22 the risk for other bacterial enteric infections is not known for hiv-infected children, but the incidence of salmonella, skigella, campylobacter, yersinia, and clostridium difficile do not seem to be increased in hiv-infected children. the incidence of helicobacter pylori may be decreased in hivinfected chi1dren.l the most serious enteric bacterial infection is mycobacterium avium-intrace2lulare, which causes a multisystemic infection involving the lungs, liver, mesenteric lymph nodes, gastrointestinal tract, and bone marrow in the most severely immunocompromised hosts with cd4 counts less than 50 cells/mm3. acidfast bacilli can be identified in the jejunal mucosa or grown from stool or blood. the most common gastrointestinal symptoms of m avium-intracellulare are abdominal pain and diarrhea, and neither responds dramatically to therapeutic intervention. combinations of medications chosen from clarithromycin, ethambutol, ciprofloxacin, amikacin, rifampin, clofazamine, and azithromycin have been tried.14 rotavirus is the viral agent that most frequently causes chronic diarrhea. in the immunocompromised child, rotaviral diarrhea can be severe, persistent, and difficult to distinguish from other agents causing secretory diarrhea. diagnosis is established by identification of rotavirus in the stool using an enzyme-linked immunoassay. enterally administered serum immunoglobulin is effective therapy,i5 but little published data exist on the treatment for rotavirus in hiv-infected children. other viral pathogens, such as adenoviruses, can cause diarrhea but also are associated with systemic infection and fulminant hepatitis. cytomegalovirus usually causes an asymptomatic enteric infection, but some individuals develop focal ulcerations in the colon or jejunum and present with bloody diarrhea and abdominal pain. gastrointestinal bleeding is unusual in hiv-infected children, but, when present, it may be caused by focal ulcerations in the colon, stomach, small intestine, or esophagus from cytomegalovirus-induced disease. merely culturing cytomegalovirus from the intestinal mucosa does not establish a link between diarrhea and the infection. histologic evidence of mucosal injury is necessary. ganciclovir and foscarnet are used to treat cytomegalovirus-induced intestinal disease in children with active symptoms. bone marrow suppression is the main serious side effect. many children with hiv disease develop lactose intolerance earlier than predicted by genetic predisp~sition.'~ nevertheless, these lactoseintolerant children do not seem to have an increased probability for growth retardation or diarrheal disease. the impact of lactose malabsorption on the nutritional health of hiv-infected children is unclear; however, children who have decreased absorption of the carbohydrate d-xylose have an increased incidence of harboring an enteric pathogen. 17 to evaluate hiv-infected children with chronic, nonbloody diarrhea, stool analysis for bacterial, viral, and parasitic infection should be performed. blood and polymorphonuclear leukocytes in the stool are indicative of colitis and should prompt evaluation of the colonic mucosa. if no enteric pathogen is identified, functional tests, such as lactose breath hydrogen and d-xylose absorption, may be useful in guiding nutritional therapy. the most beneficial diagnostic test is an upper endoscopy with biopsy. in addition to routine histology, mucosal biopsies of any focal lesions should be tested for bacterial, fungal, and viral culture and analyzed via electron microscopy. because mycobacterium and cytomegalovirus may not be detectable during endoscopic evaluation, surveillance biopsies of the jejunum should be evaluated by electron microscopy and culture. despite these diagnostic studies, enteric pathogens frequently are not identified in many hiv-infected children with diarrhea. hiv-infected children with abdominal pain should be evaluated for enteric infection, especially if they have diarrhea. fever and abdominal pain are symptoms that can indicate the presence of mycobacterium. association of these symptoms with the ingestion of milk should alert the clinician to the possibility of lactose intolerance, but for many children with lactase deficiency, the relationship is not evident. in addition, pancreatitis in the hiv-infected child is a serious and debilitating illness. not only do these children experience crampy abdominal pain, but the association with meals results in decreased caloric intake and increases the potential for malnutrition. lipase seems to be an early and sensitive marker for pancreatitis in the pediatric population.18 medications such as ddi and ddc are associated with pancreatitis, which may develop following many manths of therapy.39 other medications including pentamidine, trimethoprim-sulfamethoxazole, and dapsone have been implicated as causes of pancreatitis in children. the development of pancreatitis is an ominous event, and in one published study, the mean survival of children with pancreatitis was 8 months following the diagnosis.ls because of the guarded outcome, decisions to perform additional diagnostic tests should be made after much discussion with the health care team. if a dilated pancreatic duct is identified by ultrasonography, the indication for endoscopic retrograde cholangiopancreatography should be based on quality-of-life issues. although strictures of the pancreatic duct could contribute to the symptoms, if therapeutic intervention is not feasible, invasive diagnostic studies should not be performed. although the majority of hiv-infected children have hepatomegaly, few experience severe hepatocellular dysfunction; fibrosis; or cirrhosis that results in coagulopathy, ascites, varices, or hepatic failure. many of the medications used to treat complications of hiv disease cause hepatocellular injury or cholestasis; however, infectious agents, such as hepatitis b, that cause hepatocellular injury by immune mechanisms have milder clinical courses in immunodeficient hosts.z4 preservation of immune function in hiv-infected children could account for the apparent increase in chronic active hepatitis in the pediatric population compared with the incidence in although abnormalities in liver function tests are not diagnostic, they are beneficial as screening procedures. elevated transaminases are caused by infectious agents, medications, or nutritional deficiency and malnutrition. when the transaminases exceed four times normal, viral disease or a drug-induced hepatitis should be suspected. m avium in tracellulare, hepatic pneumocysfis carinii, fungal-induced hepatitis, cytomegalovirus, or extrahepatic biliary tract obstruction cause elevation of alkaline phosphatase. liver biopsy is necessary to identify hepatic pathogens and should be considered in a child presenting with either fever and elevated liver function tests or a focal hepatic lesion. therapeutic intervention is available for some of the viral agents that cause hepatitis, but most infectious disorders in immunodeficient hosts do not respond favorably to treatment. wasting of body mass is one of the more serious manifestations of hiv disease. in adults, the decline in lean body mass correlates with decreased quality of life and eventual death.s, l3 in children with aids, growth failure and failure to thrive have been recognized symptoms from the beginning of the epidemic.28 infants born to hiv-infected mothers seem to weigh less by 3 months of life and to be shorter by 6 months of life when compared with hiv-exposed, but noninfected infants. in long-term survivors more than 8 years of age, lean body mass wasting and short stature are common clinical features. the etiology of these derangements in growth is multifactorial, possibly including deranged metabolism, malabsorption, or decreased nutrient intake. the mechanism for the catabolic process is not known, but futile cycling of energy substrates, protein wasting, or hypermetabolism mediated by cytokines such as tnf, interleukin (1l)-1, il-6, and the interferons may contribute to the problem. the initial assessment of hiv-infected children with failure to thrive is directed at determination of caloric intake, nutrient losses, and metabolic requirements. if caloric intake is diminished, the reason for anorexia should be determined. nausea, abdominal pain, oral lesions, depression, despair, or lack of access to food need to be evaluated by the health care team. nutrient losses caused by diarrhea and malabsorption may contribute to increased nutrient requirements. enhanced metabolic requirements from febrile illnesses, recurrent infection, or from hiv replication may result in weight loss. anti-retroviral therapy can result in weight gain shortly after starting therapy. 23 counseling and oral supplements are the first steps in nutritional treatment for children with weight loss or decreased lean body mass. providing increased calories and protein may reverse the loss, but most children require additional measures of support. although nasogastric tube feeding is simple and effective for short-term management, the adverse effect on quality of life and the increased possibility of sinus disease are limiting factors. in children requiring nutritional supplementation lasting greater than 2 weeks, endoscopic placement of a gastrostomy tube button increases compliance and tolerance. as many as 150% recommended daily allowance for calories may be required to achieve weight gain in hiv-infected children. newly developed one-step gastrostomy buttons permit endoscopic insertion of devices that do not limit activity and provide access for nutritional support. despite providing sufficient nutrition to gain weight, enteral supplementation16 and gastrostomy tube feedings" do not increase lean body mass in hiv-infected children. similarly, appetite stimulants, such as megestrol acetate, a progesterone derivative, and dronabinol, a tetrahydrocannabinol derivative, do not increase lean body mass in adults infected with hiv. promising data in adults suggest that mammalian cell-derived recombinant human growth hormone therapy results in weight gain and anabolism as measured by stool nitrogen, urine nitrogen, and potassium excretion.20 if valid in the pediatric population, growth hormone could prove to be an effective treatment for failure to thrive by increasing lean body mass. anecdotal experience implicates specific vitamin deficiencies as contributing to the nutritional problems of hiv-infected children. in regions in which vitamin deficiency is endemic, it is not surprising to see the problem amplified in hiv-infected children. decreased vitamin a causes diminished t-cell response to mitogens and antigens, atrophy of lymphoid tissue,3â° and is associated with increased maternal-child transmission.z9 supplementation of vitamin a seems to increase cd4 + cells, boost antibody response, and decrease morbidity and mortality from other infectious diseases.8 the effect of vitamin a supplementation on the health of hiv-infected children in the united states is not known. other vitamins, including vitamins d, e, 8, (thiamine), b2 (riboflavin), niacin, b6, bi2, folic acid, c, and carnitine, have been evaluated in various populations of hiv-infected individuals, and although abnormalities can be demonstrated for some vitamins, deficiencies related to the generalized state of malnutrition and not specifically to hiv-induced disease are difficult to prove beyond a reasonable doubt. similarly, deficiencies of iron, zinc, and selenium have been described in hiv-infected individuals. although these minerals have an important role in immunoregulathe redundancy of the immune system to provide protection against infection suggests that by the time the system begins to fail, no single cause can be found to correct the problem. for this reason, supplementation with a single therapeutic nutrient intervention can improve laboratory phenomena, but rarely impacts on a patient's quality of life or immunoregulatory defects. patients with primary immunodeficiency disorders frequently experience gastrointestinal problems in association with other clinical manifestations of systemic disease. the respiratory and gastrointestinal tracts are exposed to the environment and, in response, have developed complex systems to protect their mucosal surfaces from pathogens. antibody production, cell-mediated immune function, complement, and phagocytic function act together to prevent infection and uncontrolled inflammation. in the gastrointestinal tract, enteric pathogens and chronic inflammatory bowel disease are the two major clinical aspects of primary immune deficiency. surprisingly, individuals with identical deficiencies may not experience similar gastrointestinal symptoms. for example, children with immunoglobulin a deficiency may be asymptomatic or may have chronic diarrhea associated with chronic intestinal inflammation disease. in general, children with t-cell defects seem to have a higher incidence of chronic gastrointestinal problems compared with children with antibody defiqiency syndromes, complement defects, or disorders of phagocytic function. table 4 lists the common primary immunodeficiencies together with the gastrointestinal manifestations commonly associated with each disorder. immunodeficient children pose a challenge to clinicians because of the interrelationship between infectious disease, metabolism, gastrointestinal tract function, psychosocial problems, and immune function. the interplay between these factors is not always clear, and frequently the best course of therapy is obscured because of an inability to determine which factors have the greatest impact on child health. to optimize therapeutic intervention, a multidisciplinary health care team must be involved with the management of children and their families. heiicobacter pylari in children with acquired immunodeficiency syndrome disseminated histoplasmosis as the acquired immunodeficiency syndrome-defining illness in an infant intestinal parasites and hiv infection in tanzanian children with chronic diarrhea centers for disease control: zidovudine for the prevention of hiv transmission from mother to infant significance of altered nutritional status in acquired immune deficiency syndrome (aids) esophageal ulceration induced by zidovudine aids, zinc deficiency and thymic hormone failure et a1 vitamin a supplementation and child mortality: a meta-analysis detection of hiv-1 rna in the lamina propria of patients with aids and gastrointestinal disease human immunodeficiency virus infection of enterocytes and mononuclear cells in human jejunal tissue effect of enteral tube feeding on growth of children with symptomatic human immunodeficiency virus infection italian multicentre study: epidemiology and clinical features of pediatric hjv infection: results from an italian multicentric study on 544 children bosy composition studies in patients with the acquired immunodeficiency syndrome recommendations on prophylaxis and therapy for disseminated mycobacterium avium complex disease in patients infected with the human immunodeficiency virus benefit of oral immune globulin therapy in patients with immunodeficiency and chronic diarrhea growth and body composition in children infected with the human immunodeficiency virus-1 malnutrition and carbohydrate malabsorption in children with vertically transmitted human immunodeficiency virus 1 infection et a1 pancreatitis in pediatric human immunodeficiency virus infection hiv replication and persistence in human gastrointestinal cells cultured in vitro anabolic effects of recombinant human growth hormone in patients with wasting associated with human immunodeficiency virus infection human immunodeficiency virus detection in bowel epithelium from patients with gastrointestinal symptoms diarrhea among african children born to human immunodeficiency virus-infected mothers: clinical, microbiologic and epidemiologic features effect of continuous intravenous infusion of zidovudine (azt) in chldren with symptomatic hiv infection hepatic involvement in patients with human immunodeficiency virus infection: discrepancies between aids patients and those with earlier stages of infection pediatric acquired immunodeficiency syndrome: special considerations for developing nations efficacy of octreotide in the management of chronic diarrhea in aids perinatal transmission of the human immunodeficiency virus type 1 to infants of seropositive women in zaire survival in children with perinatally acquired human immunodeficiency virus type 1 infection chlehanaw j d maternal vitamin a deficiency and mother-tochild transmission of hiv-1 altered t cell subset proportions in vitamin a deficient children a prospective study of diarrhea and hiv-1 infection among 429 zairian infants chronic active hepatitis in a child with human immunodeficiency virus infection survival experience of 789 children with acquired immunodeficiency syndrome gastrointestinal symptoms in patients infected with human immunodeficiency virus: relevance of infective agents isolated from gastrointestinal tract et a1 cessation of cryptosporidium-associated diarrhea in an acquired immunodeficiency syndrome patient after treatment with hyperimmune bovine colostrum postnatal transmission of human immunodeficiency virus type 1 from mother to infant. a prospective cohort study in kigali, rwanda statement on breast feeding and hiv. who/ unicef consultative meeting of world health organization: the hiv/aids pandemic: 1993 overview. geneva: who/ gpa/cnp/93 long-term toxicity/activity profile of 2',3'-dideoxyinosine in aids or aids-related complex gastrointestinal dysfunction and disaccharide intolerance in children infected with human immunodeficiency virus key: cord-011155-zraqyx78 authors: reif, lindsey k.; abrams, elaine j.; arpadi, stephen; elul, batya; mcnairy, margaret l.; fitzgerald, daniel w.; kuhn, louise title: interventions to improve antiretroviral therapy adherence among adolescents and youth in lowand middle-income countries: a systematic review 2015–2019 date: 2020-03-09 journal: aids behav doi: 10.1007/s10461-020-02822-4 sha: doc_id: 11155 cord_uid: zraqyx78 adolescents and youth living with hiv have poorer antiretroviral treatment (art) adherence and viral suppression outcomes than all other age groups. effective interventions promoting adherence are urgently needed. we reviewed and synthesized recent literature on interventions to improve art adherence among this vulnerable population. we focus on studies conducted in lowand middle-income countries (lmic) where the adolescent and youth hiv burden is greatest. articles published between september 2015 and january 2019 were identified through pubmed. inclusion criteria were: [1] included participants ages 10–24 years; [2] assessed the efficacy of an intervention to improve art adherence; [3] reported an art adherence measurement or viral load; [4] conducted in a lmic. articles were reviewed for study population characteristics, intervention type, study design, outcomes measured, and intervention effect. strength of each study’s evidence was evaluated according to an adapted world health organization grade system. articles meeting all inclusion criteria except being conducted in an lmic were reviewed for results and potential transportability to a lmic setting. of 108 articles identified, 7 met criteria for inclusion. three evaluated patient-level interventions and four evaluated health services interventions. of the patient-level interventions, two were experimental designs and one was a retrospective cohort study. none of these interventions improved art adherence or viral suppression. of the four health services interventions, two targeted stable patients and reduced the amount of time spent in the clinic or grouped patients together for bi-monthly meetings, and two targeted patients newly diagnosed with hiv or not yet deemed clinically stable and augmented clinical care with home-based case-management. the two studies targeting stable patients used retrospective cohort designs and found that adolescents and youth were less likely to maintain viral suppression than children or adults. the two studies targeting patients not yet deemed clinically stable included one experimental and one retrospective cohort design and showed improved art adherence and viral suppression outcomes. art adherence and viral suppression outcomes remain a major challenge among adolescents and youth. intensive home-based case management models of care hold promise for improving outcomes in this population and warrant further research. adolescents and youth, 10 to 24 years of age, represent a growing proportion of people living with hiv around the world and have worse outcomes than all other age groups [1] [2] [3] [4] [5] [6] . in recent years, aids-related deaths among adolescents and youth increased by 50% while they have decreased among all other age groups [7] . in 2018, 510,000 young people between the ages of 10 to 24 years were newly-infected with hiv, 40% of whom were between 10 and 19 years of age [8] . in addition to heterosexual transmission, a generation of children infected with hiv perinatally are now aging into adolescence, adding to the burden of disease in this age group. adequate adherence to an antiretroviral therapy (art) regimen leading to viral suppression is essential for an adolescents' own health and well-being, and to reduce further hiv transmission. yet, adolescents and youth have poor adherence to drug regimens for many chronic illnesses [9] [10] [11] [12] . adherence to art is further complicated by hivrelated stigma [13] [14] [15] [16] . the period of adolescence and youth is characterized as a time of great physiological and psychological growth and development [1, 17] , increased desire for independence from parents [18] , and increased risk-taking [19] , adding another layer of complexity. during this developmental stage, initiation of sexual activity is common and may include early pregnancy [20] . adolescents and youth also lack financial autonomy, are prone to peer pressure, and lack problem-solving skills [21] [22] [23] [24] [25] . further, in resource-limited settings, external factors including poverty, food scarcity, and hiv-related stigma acutely influence art adherence and hiv outcomes [26] [27] [28] [29] [30] [31] [32] [33] . major barriers to art adherence for adolescents and youth can be divided into 3 categories: patient-level factors (e.g. socioeconomic status, stigma) [34, 35] , health services factors (e.g. clinic waiting times, drug availability, quality of care) [36, 37] , and medication factors (e.g. dosing, high pill burden, side effects) [3, 38, 39] . much of the research on art adherence among young people has focused on identifying and estimating the prevalence of these barriers [29, 33, 38, 40, 41] . a comprehensive review of the literature between 2003 and 2015 identified 10 studies which evaluated interventions to improve adherence in adolescents in developed countries [42] . effective interventions included daily interactive text reminders for dosing [43, 44] , and computer-driven support programs [45] . however, none of the studies included were conducted in a low-or middle-income country (lmic) [46] , areas where the global hiv epidemic is centered. further, most of these studies were descriptive reports or pilot studies with small sample sizes and thus had insufficient power to detect meaningful effects. given the critical need to identify effective approaches to improve outcomes among adolescents and youth living with hiv, we evaluated and synthesized the recent published literature on research conducted in a lmic aimed at improving art adherence in this population. we searched the pubmed database for english language articles which evaluated interventions to improve art adherence among adolescents and youth living with hiv, conducted in a lmic, and published between september 2015 and january 2019 using the search terms indicated in fig. 1 [47] . we then manually reviewed the references sections of relevant articles. records were managed using endnote and duplicates were removed manually. one reviewer (lr) conducted the primary search and articles selected for inclusion were approved by all authors. articles selected met the following criteria: (1) included adolescents and youth ages 10-24 years; (2) evaluated an intervention to improve art adherence; (3) included an art adherence measurement [(((((((structural) or ((behavioral))) and intervention)) and ((((((art) or hiv medication) or haart)) and ((adherence) or persistence)) and hiv))) and (((young adult) or adolescent) or adolescence) or youth))] intervention not conducted in a lmic (n=4) review or protocol paper (n=25) articles included in quantitative review (n=7) outcome (self-report, pill count, or medication event monitoring systems (mems)) or viral load (vl) as a proxy for an adherence measurement; (4) conducted in a lmic. eligible manuscripts were not restricted by study design and included studies which evaluated structural, behavioral, or health services-related interventions aimed at improving art adherence. each of the articles meeting our inclusion criteria was reviewed for characteristics of the study population (e.g. age, time on art, health status at baseline), type of intervention evaluated, study design, outcome measured (art adherence or vl), and intervention efficacy. the strength of each study's evidence was evaluated according to an adapted grade system utilized by the world health organization [48] . this system classifies studies into four levels based on study design, analysis plan, and existence of comparison groups. high quality evidence (level 4) are randomized control trials (rct) with statistical testing comparing groups. moderate quality evidence (level 3) are prospective cohort studies with statistical testing comparing groups. low quality evidence (level 2) are retrospective or descriptive studies with statistical testing of between or within group comparisons; and very low quality evidence (level 1) are studies without statistical comparisons. due to the small selection of studies and diversity of outcome measures, a meta-analysis was not conducted to synthesize the effect size across studies. we reviewed the seven included studies to determine interventions with the most potential for impact and assessed areas in need of further research and evidence. the pubmed search identified 1229 articles, of which 1121 were excluded based on review of the abstract. a full text review was completed on 108 articles. seven studies met the inclusion criteria. reasons for exclusion are listed in fig. 1 ; if an article was excluded for failing to meet more than one criteria, it is listed under the first exclusion category as prioritized in the criteria above. the most common reason for exclusion was only including participants outside the age range of interest (10-24 years) or reporting outcomes which were not disaggregated so as to discern results among adolescents and youth (i.e. results reported for ages 18-29 years) . the seven studies meeting our inclusion criteria are described in table 1 . all were conducted in sub-saharan africa and included adolescents living with hiv who were aware of their hiv status and were receiving hiv care at a health facility. the median sample size was 702 (range 94-96,706). we also reviewed and report the results of 4 articles that were excluded because the studies were not conducted in a lmic. we briefly summarize the results of these studies here and their potential transportability to an lmic setting, but did not include them in the main review since our primary goal was to identify interventions which would be directly applicable in resource-limited settings where the global hiv burden among adolescents and youth is greatest. studies either focused on: (1) patient-level interventions, i.e. interventions implemented at the individual level in addition to standard hiv care (n = 3); or (2) health services interventions which re-structured the way hiv care was provided, also called 'models of hiv care' [49, 50] (n = 4). in the first patient-level intervention, a once-weekly sms text message was designed to check-in with participants about their general well-being [51] . one study arm received this weekly check-in message but could not respond to the sender. the second arm received the same message and could respond to the sender. the third study arm received standard care, i.e. no messages. the second study evaluated an economic intervention in which a savings account was established which could be used for small business development or education, i.e. school fees or lunches to address financial-related barriers to art adherence [52] . in the third study, three consecutive monthly individual intensive adherence counseling sessions were provided to participants. the goal of the sessions was to identify adherence barriers and develop individualized plans to address them [53] . four studies evaluated health services interventions which are categorized as either less intensive (n = 2) or more intensive (n = 2) models of care (fig. 2 ). the two less intensive models of care reduced the amount of time stable participants spent in the clinic. in the first, multi-month art prescriptions allowed participants to come to the clinic less frequently ranging from every other month and some only twice per year for medication refills and clinical check-ups [54] . in the second, a group-based model of care, patients formed groups of 25-30 participants and met every other month for group counseling, brief check-ups, and distribution of art refills [55] . groups were facilitated by a lay health worker and nurse and met at a health facility or community venue. two studies of more intensive models of care targeted newly diagnosed adolescents initiating art or those who had not yet been defined as adherent or stable. these two models of care augmented regular care with additional support through home-based case management by a community [56] or peer counselor [57] . in the first, a community-based support worker (i.e. lay health worker) made weekly home visits to provide additional individualized adherence support. as participants became stable, frequency of home visits decreased to monthly and then quarterly, but increased again in the event of any clinic visit lapses [56) . in the second, peer counselors (i.e. trained adolescents and youth ages 18 to 24 years living with hiv) referred to as 'community adolescent treatment supporters' provided adherence counseling and psychosocial support via weekly home visits [57] . of the three studies which evaluated patient-level interventions, two used experimental designs and one entailed a retrospective cohort study. the study evaluating text messaging used a 3-arm rct to compare text messaging and text messaging with the option to respond to standard care (no text messaging). the primary outcome was mean art adherence over the 48-week study period which was measured using medication event monitoring systems (mems) and defined as the ratio of recorded bottle openings to the number of prescribed bottle openings [51] . eligible patients were 15 to 22 years of age, in care at an hiv care facility, and prescribed art. the study evaluating a participant savings account used a 2-arm rct comparing the intervention to standard care. the primary outcome was vl < 40 copies/ µl 24 months after study enrollment [52] . eligible patients were ages 10 to 16 years of age, in care at an hiv care facility, and prescribed art. the study evaluating monthly intensive adherence counseling sessions used a retrospective cohort design and no comparison group. the primary outcome was vl < 1000 copies/µl measured within 180 days of completing the 3 sessions [53] . eligible patients were ages 10 to 19 years of age, in care at an hiv care facility, prescribed art and had a vl > 1000 copies/µl. none of the interventions in the three studies significantly improved art adherence or vl outcomes in their primary analyses (all grade level 4). sms text messaging did not significantly improve art adherence over a 48-week period. mean adherence (percentage of prescribed doses taken) was 64% in the group that received text messages (p = 0.27 compared to control), 61% in the group that received text messages with the option to respond (p = 0.15 compared to control), and 67% in the control group [51] . the study comparing participant savings accounts to standard care also showed no statistically significant difference in viral if a study included children and/or adults, we report the total number of adolescents and youth included, the age group specified, and results specific to this age group suppression between arms. the proportion of participants with a vl < 40 copies/µl at 24 months did not significantly differ by arm − 65.9% in the intervention arm and 63.4% in the control arm [52] . the observational study of intensive adherence counseling also observed low rates of vl suppression post-counseling. among 192 adolescents included in the study, 117 (60%) had a repeat vl measurement after 3 counseling sessions and 34/117 (29%) achieved a vl < 1000 copies/µl [53] . the four studies evaluating health services interventions included three retrospective cohort studies and one rct. the study evaluating multi-month art prescriptions was a retrospective cohort study and used programmatic data to report outcomes of children and adolescents who transitioned to this less intensive model of care after they were deemed clinically stable and art adherent, defined as having an improving cd4 + cell count or cd4% or a vl < 400 copies/µl and pharmacy pill count > 95%. the primary outcome − vl < 400 copies/µl-was compared between age groups (< 1 year, 1-4 years, 5-9 years, 10-14 years, and 15-19 years) [54] . the group-based intervention study evaluating community-based adherence clubs was a retrospective cohort study among patients who self-reported art adherence, had been on art for > 12 months, and had 2 consecutive vl measurements < 400 copies/µl. outcomes were maintaining a vl < 400 copies/µl and viral rebound at 12 months from enrollment and were compared between age groups (16-24 years and ≥ 25 years) [55] . since eligibility for both of these studies required established art adherence and clinical stability, these evaluations aimed to determine if adolescents and youth could maintain a stable status after shifting to a less intensive model of care. the two studies of health service interventions which evaluated a more intensive model of care-home-based case management in addition to regular clinical care -included one retrospective cohort study and one rct. the study evaluating the community-based support worker intervention examined a retrospective cohort of adolescents and youth ages 10 to 24 years who were newly diagnosed with hiv and were art-naïve. outcomes included mean medication possession ratio (ratio of days of dispensed medication to days between pharmacy visits) and vl < 400 copies/µl assessed at 3 and 5 years from initiating art. outcomes were compared to participants who did not receive support over the same 5 year study period [56] . the study evaluating community adolescent treatment supporters was a two-arm rct among adolescents ages 10 to 15 years who were in care at an hiv care facility and prescribed art. participants were randomized to the treatment supporter intervention or standard care and the primary outcome-self-reported art adherence-was compared by study arm [57] . both studies of less intensive models of care-multi-month art prescriptions [54] and group-based care [55] -showed a relatively high proportion of adolescents and youth maintained a vl < 400 copies/µl over study follow-up (75% and 97.2%, respectively) (grade level 2). however in both studies, compared to children and adults, adolescents and youth were at higher risk for viral rebound (i.e. vl > 400 copies/µl). in the study evaluating multi-month prescriptions, at baseline approximately 85% of children (ages 1-9 years) and 80% of adolescents (ages 10-19 years) had a vl < 400 copies/µl. over 60 months of follow-up with annual vl measurements, this proportion remained steady fig. 2 types of health services interventions among children, but decreased among adolescents to approximately 75% [54] . in the study of community-based adherence clubs, the proportion of adolescents (ages 16-24 years) retained in care at 12 months was significantly lower than that observed among adults (ages ≥ 25 years) − 90.9% vs. 94.1%, respectively (p = 0.022). among those retained in care, the proportion of participants with vl < 400 copies/ µl was similar − 97.2% of adolescents and 98.0% of adults (p = 0.194)-but in adjusted analyses, adolescents were at significantly higher risk of experiencing viral rebound compared to adults (adjusted hazard ratio: 2.24; 95% ci 1.0-5.04) [55] . the two studies examining intensified models of carehome-based case management [56, 57] -were the sole studies included in our review which improved art adherence and viral suppression. the community-based support worker intervention which involved home visits by a lay health support worker, found that 5 years from initiating art, 81.2% of participants receiving the support intervention achieved a vl < 400 copies/µl compared to 62.8% among those not receiving support (adjusted odds ratio: 0.24; 95% ci: 0.06-1.03; p = 0.055) (grade level 4). in the rct examining the community adolescent treatment supporter intervention which involved home visits from a peer counselor also living with hiv, 12-months from study enrollment, 71.8% of participants in the intervention arm self-reported art adherence compared to 39.3% receiving standard care (p < 0.05) (grade level 3). there were four additional studies which met inclusion criteria but were conducted in high income settings. two studies evaluated intensive individual or group counseling in the united states. the positive strategies to enhance problem-solving skills (steps) intervention consisted of five onehour counseling sessions rooted in cognitive-behavioral theory and motivational interviewing skills administered by a master's or doctoral-level clinician [58] . in a pilot rct including participants self-reporting < 90% adherence at baseline, 14 participants were randomized to the steps intervention or standard care. after 4 months, mean art adherence among participants randomized to steps increased by 13%, and decreased by 26% among participants randomized to standard care, measured by mems data. no statistical comparison was conducted for this pilot rct. the accept intervention was a group-based educational intervention including topics on stigma, disclosure, healthy relationships, and life planning [59] . in an rct including 103 adolescents and youth newly diagnosed with hiv, participants in the accept intervention arm had a 2.33 greater likelihood of self-reported art adherence at 12 months than those in the control arm (p = 0.005). two studies evaluating technology-driven interventions showed statistically significant improvements in vl suppression. in a prospective cohort study conducted in argentina, an intervention of twice-monthly private messaging through social media was evaluated among adolescents and youth with 2 consecutive vl measurements > 1000 copies/µl. twenty-two participants were enrolled and at 32 weeks, 64% achieved a vl < 1000 copies/µl [60] . an rct conducted in the united states included 66 participants ages 14-26 years with a detectable vl at baseline and evaluated an iphone game designed around themes of 'fighting' or 'destroying' hiv in the body by taking art. after 16-weeks, among participants who had newly initiated art, the decrease in mean log vl of participants in the intervention arm (3.63-0.93) was significantly greater than the decrease in mean log vl of participants in the control arm (3.94-1.53) (p = 0.04) [61] . this systematic review identified seven studies published between 2015 and 2019 which evaluated a patient-level or health service intervention to improve art adherence among adolescents and youth living with hiv ages 10-24 years in a lmic. this expands upon a prior review of the same topic examining studies published between 2003 and 2015 [42] , none of which were conducted in a lmic. among the seven studies in our review, three employed experimental designs appropriately powered to detect intervention effects, and five included a vl outcome, an objective biomarker of adherence [31] . the increase in the number and location of studies observed compared to the earlier review indicates a positive shift of focus to identify interventions to improve outcomes in regions with the greatest hiv burden. additional high-quality evidence should be emerging soon from four ongoing rcts we identified, two from sub-saharan africa [62] [63] [64] [65] . the outcomes of the interventions evaluated indicate that art adherence and viral suppression among adolescents and youth remain a major challenge. none of the three studies evaluating patient-level interventions, including two rcts contributing high-quality evidence, showed improvement in either outcome. adolescents and youth face a variety of barriers to art adherence, which evolve as they develop physically, emotionally, and socially. interventions designed to target a single, specific challenge, such as forgetfulness, may be insufficient. for example, the text message intervention was very narrow in scope-no additional counseling was done via text message and the messages were only sent weekly, so did not serve as a daily reminder for taking art medication. further research on combination interventions such as incorporating text messaging within a larger package of services is warranted, particularly as such combination intervention strategies have been effective among adults [66, 67] . the majority of studies reviewed focused on how hiv services were delivered to adolescents or youth [54] [55] [56] [57] . the two studies on less intensive models of care indicated that adolescents and youth may be less likely to remain clinically stable as they were more likely to experience viral rebound than children or adults in both studies. additional studies show that among adolescents and youth who do achieve vl suppression, only 50% maintain this for one year [68] . given adolescence itself is a dynamic period marked by constant development and changes to risk factors, and adolescents and youth are known to have suboptimal viral suppression outcomes, the concept of 'stable' adolescents and youth may be misleading. conversely, the more intensive hiv care models were the most effective interventions. home-based case management interventions, which provided additional counseling to identify and address specific barriers which may not be recognized in the clinical setting, showed improved adherence and viral suppression outcomes. individualized care and treatment within a larger health system may be the most efficient way to identify multiple or evolving challenges and then respond with a custom combination strategy. for example, if an adolescent or youth faces family discrimination, inability to miss school for clinic appointments, and lack of a trusted clinician relationship, the case-management intervention can target this specific barrier combination -family and social isolation, structural challenges of clinic-based care, and poor provider relationship [69, 70] . several methodological limitations in the studies included should be noted. while community adolescent treatment supporters significantly improved outcomes [57] , art adherence was ascertained through self-report which may be subject to social desirability bias artificially leading to a positive effect. more objective adherence measures such as mems, drug levels or vl outcomes could avoid bias. the community-based support worker intervention also showed improved outcomes [56] , but assignment to the intervention was not randomized. patients received the intervention based on availability of support workers in the area, thus unmeasured confounding cannot be ruled out. for example, support workers may have only been available in more developed or easier-to-access locations. further, as this intervention was evaluated using existing clinical data in a retrospective cohort design, data on the quality and consistency of intervention implementation is lacking, including frequency of home visits, type of support provided, and content of counseling. lastly, a majority of studies report results just 1-year from implementing the intervention [50-52, 54, 56] . evaluation of an intervention's effect over a longer period, particularly for new models of hiv care, could provide information on the long-term durability of an intervention on participant outcomes. an additional limitation is the inability to identify adolescents and youth who have adequate art adherence but are not virally suppressed, likely because of the presence of art drug resistance. for participants who are art-naïve or who have changed art regimens, vl outcomes can accurately reflect improved art adherence, but those who may have acquired art drug resistance are unlikely to be vl suppressed even with improved art adherence [71] . four of the seven studies in this review only report vl outcomes and not an art adherence measurement and only one is limited to art-naïve participants [56] . reporting the art regimen prescribed could begin to address this limitation since research shows certain regimens are more likely to produce resistance than others [72, 73] . only one study in this review included data on participants' prescribed art regimen [53] . some interventions implemented in a high income setting improved outcomes among adolescents and youth [58] [59] [60] [61] and may be feasibly adapted for use in resource-limited settings. those utilizing social media or smart phone applications show promise given rapidly increasing access to internet and mobile phones among young people in lmics [74] . conversely, implementing interventions involving cognitive behavioral theory and motivational interviewing in highburden resource-limited settings is unlikely due to the high level of counselor training (i.e. implemented by phd-level practitioner) and amount of time required for individualized counseling. medication-related barriers-side-effects and ease of daily dosing-are critical aspects of adherence and require simplification. we did not include studies which address such barriers through use of alternative regimens or formulations. use of more potent and/or better tolerated art regimens such as integrase inhibitors, long-acting and injectable regimens [75] [76] [77] have shown promise among adults and should be examined among adolescents and youth. other medication-related interventions among adolescents and youth such as weekends off regimens have been shown to be non-inferior [78] , and single-tablet regimens have achieved significantly higher vl suppression rates than multi-tablet regimens [79] . medication-related interventions implemented in parallel with health services interventions could significantly and sustainably impact adolescent and youth art adherence. art adherence and viral suppression outcomes remain a major challenge among adolescents and youth living with hiv in lmics. recent studies of interventions to improve art adherence among this population show inconsistent effects, highlighting the need for additional, innovative approaches which specifically target the needs of adolescents and youth. to date, individually-targeted interventions have not shown significant effects on adherence, but health services interventions which enhance clinic-based care with home-based care, appear promising. there is a clear need for appropriately powered studies examining combination interventions. standardizing the key outcomes applied across these studies can streamline limited resources, maximize the impact of research, and yield effective interventions to improve health outcomes for adolescents and youth living with hiv. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. contemporary issues on the epidemiology and antiretroviral adherence of hiv-infected adolescents in sub-saharan africa: a narrative review high rates of virological failure and drug resistance in perinatally hiv-1-infected children and adolescents receiving lifelong antiretroviral therapy in routine clinics in togo antiretroviral adherence issues among hiv-positive adolescents and young adults high attrition before and after art initiation among youth (15-24 years of age) enrolled in hiv care antiretroviral therapy adherence and retention in care in middle-income and low-income countries: current status of knowledge and research priorities adherence to protease inhibitor therapy and outcomes in patients with hiv infection 2015-child ren-adole scent s-and-aids-stati stica l-updat e-execu tive-summa ry_244.pdf. accessed 15 unicef. adolescent hiv prevention. unicef data interventions for enhancing medication adherence how to detect and manage low patient compliance in chronic illness effectiveness and content analysis of interventions to enhance medication adherence in hypertension: a systematic review and meta-analysis protocol interventions for enhancing medication adherence patient adherence to hiv medication regimens: a review of published and abstract reports depressive symptoms, neurocognitive impairment, and adherence to highly active antiretroviral therapy among hiv-infected persons correlates and predictors of adherence to highly active antiretroviral therapy: overview of published literature medication adherence among hiv+ adults: effects of cognitive dysfunction and regimen complexity everyone has a secret they keep close to their hearts': challenges faced by adolescents living with hiv infection at the kenyan coast social cognitive development during adolescence examining the link between adolescent brain development and risk taking from a social-developmental perspective determinants of adolescent pregnancy in sub-saharan africa: a systematic review depression and stigma in high-risk youth living with hiv: a multi-site study examining the relationship between psychological distress and adherence to anti-retroviral therapy among ugandan adolescents living with hiv psychosocial challenges and protective influences for socio-emotional coping of hiv+ adolescents in south africa: a qualitative investigation neurodevelopment in perinatally hiv-infected children: a concern for adolescence impact of hiv severity on cognitive and adaptive functioning during childhood and adolescence forget about forgetting: structural barriers and severe non-adherence to antiretroviral therapy qualitative analysis of barriers and facilitators encountered by hiv patients in an art adherence programme patient-reported barriers to adherence to antiretroviral therapy: a systematic review and meta-analysis facilitators and barriers to antiretroviral therapy adherence among adolescents in ghana the effectiveness of counseling, material support and/or nutritional supplementation on improving adherence to anti-retroviral therapy and clinical outcomes among hiv patients: a systematic review of quantitative evidence protocol viral suppression in adolescents on antiretroviral treatment: review of the literature and critical appraisal of methodological challenges living situation affects adherence to combination antiretroviral therapy in hiv-infected adolescents in rwanda: a qualitative study if i have nothing to eat, i get angry and push the pills bottle away from me": a qualitative study of patient determinants of adherence to antiretroviral therapy in the democratic republic of congo patientrelated risks for nonadherence to antiretroviral therapy among hiv-infected youth in the united states: a study of prevalence and interactions contributions of disease severity, psychosocial factors, and cognition to behavioral functioning in us youth perinatally exposed to hiv system-level factors as predictors of adherence to clinical appointment schedules in antiretroviral therapy in cambodia factors associated with adherence to antiretroviral therapy among adolescents living with hiv/aids in low-and middle-income countries: a systematic review barriers to haart adherence among human immunodeficiency virus-infected adolescents antiretroviral medication adherence among the reach hiv-infected adolescent cohort in the usa barriers to medication adherence in hiv-infected children and youth based on selfand caregiver report mapping patient-identified barriers and facilitators to retention in hiv care and antiretroviral therapy adherence to andersen's behavioral model antiretroviral therapy adherence enhancing interventions for adolescents and young adults 13-24 years of age: a review of the evidence base improving adherence to antiretroviral therapy for youth living with hiv/aids: a pilot study using personalized, interactive, daily text message reminders the use of cell phone support for non-adherent hiv-infected youth and young adults: an initial randomized and controlled intervention trial motivational enhancement system for adherence (mesa): pilot randomized trial of a brief computer-delivered prevention intervention for youth initiating antiretroviral treatment the world by income and region. the world bank preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015: elaboration and explanation antiretroviral therapy for hiv infection in infants and children: towards universal access: recommendations for a public health approach. geneva: world health organization a toolkit for health facilities: differentiated care for hiv and tuberculosis reimagining hiv service delivery: the role of differentiated care from prevention to suppression text messaging for improving antiretroviral therapy adherence: no effects after 1 year in a randomized controlled trial among adolescents and young adults does economic strengthening improve viral suppression among adolescents living with hiv? results from a cluster randomized trial in uganda low hiv viral suppression rates following the intensive adherence counseling (iac) program for children and adolescents with viral failure in public health facilities in uganda multi-month prescription of antiretroviral therapy amongst children and adolescents: experiences from the baylor international pediatric aids initiative (bipai) in six african countries implementation and operational research: community-based adherence clubs for the management of stable antiretroviral therapy patients in cape town, south africa: a cohort study the effectiveness and costeffectiveness of community-based support for adolescents receiving antiretroviral treatment: an operational research study in south africa effectiveness of community adolescent treatment supporters (cats) interventions in improving linkage and retention in care, adherence to art and psychosocial well-being: a randomised trial among adolescents living with hiv in rural zimbabwe positive strategies to enhance problem-solving skills (steps): a pilot randomized, controlled trial of a multicomponent, technology-enhanced, customizable antiretroviral adherence intervention for hiv-infected adolescents and young adults project accept: evaluation of a group-based intervention to improve engagement in care for youth newly diagnosed with hiv utility of mobile communication devices as a tool to improve adherence to antiretroviral treatment in hiv-infected children and young adults in argentina enhancing health among youth living with hiv using an iphone game adaptive antiretroviral therapy adherence interventions for youth living with hiv through text message and cell phone support with and without incentives: protocol for a sequential multiple assignment randomized trial (smart) evaluating a multi-component, community-based program to improve adherence and retention in care among adolescents living with hiv in zimbabwe: study protocol for a cluster randomized controlled trial positive steps -a randomized controlled efficacy trial of an adaptive intervention for strengthening adherence to antiretroviral hiv treatment among youth: study protocol conditional economic incentives and motivational interviewing to improve adolescents' retention in hiv care and adherence to antiretroviral therapy in southeast nigeria: study protocol for a cluster randomised trial a combination intervention strategy to improve linkage to and retention in hiv care following diagnosis in mozambique: a cluster-randomized study effectiveness of a combination strategy for linkage and retention in adult hiv care in swaziland: the link4health cluster randomized trial longitudinal antiretroviral adherence among adolescents infected with human immunodeficiency virus metropolitan atlanta community adolescent rapid testing initiative: the impact of motivational interviewing and intensive case management on the psychosocial and clinical care outcomes of adolescents and young adults with hiv she makes me feel that i'm not alone": linkage to care specialists provide social support to people living with hiv hiv treatment adherence, drug resistance, virologic failure: evolving concepts an evaluation of elvitegravir plus cobicistat plus tenofovir alafenamide plus emtricitabine as a single-tablet regimen for the treatment of hiv in children and adolescents dolutegravir plus abacavir-lamivudine for the treatment of hiv-1 infection meet us on the phone: mobile phone programs for adolescent sexual and reproductive health in lowto-middle income countries experiences with long acting injectable art: a qualitative study among plhiv participating in a phase ii study of cabotegravir + rilpivirine (latte-2) in the united states and spain longacting intramuscular cabotegravir and rilpivirine in adults with hiv-1 infection (latte-2): 96-week results of a randomised, open-label, phase 2b, non-inferiority trial safety and tolerability of long-acting cabotegravir injections in hiv-uninfected men (eclair): a multicentre, double-blind, randomised, placebocontrolled, phase 2a trial weekends-off efavirenzbased antiretroviral therapy in hiv-infected children, adolescents, and young adults (breather): a randomised, open-label, noninferiority, phase 2/3 trial uptake and virological outcomes of single-versus multi-tablet antiretroviral regimens among treatment-naive youth in the hiv research network key: cord-026112-58sa5z03 authors: dehghani-dehej, farzaneh; hosseini, zinat; mortazkar, poupak; khanaliha, khadijeh; esghaei, maryam; fakhim, atousa; bokharaei-salim, farah title: prevalence of hcv and/or hbv coinfection in iranian hiv-infected patients date: 2020-04-24 journal: nan doi: 10.2217/fvl-2019-0066 sha: doc_id: 26112 cord_uid: 58sa5z03 aim: hiv-infected patients risk coinfection with hbv and hcv. this study aimed to investigate molecular epidemiology of hbv and hcv coinfection in iranian hiv-infected individuals. materials & methods: in this cross-sectional study, serological markers of hbv and hcv infection (hepatitis b surface antigen [hbsag], hepatitis b e-antigen [hbeag], hepatitis b e-antibody [hbeab] and hepatitis b core antibody [hbcab]) and anti-hcv antibodies [anti-hcv abs] were tested in 198 iranian hiv-infected patients. from plasma, hbv viral load was determined using cobas taqman 48, and hcv-rna was detected by reverse transcriptase-nested pcr. results: 85 out of 198 (42.9%) patients were anti-hcv ab positive and 42/198 (21.2%) had detectable hcv-rna. eight (4.0%) had traceable hbv-dna. all these patients were infected by hbv genotype d. 55 (27.8%) were hbcab positive. nine (4.4%) were hbsag and anti-hcv ab positive. conclusion: none were hiv-rna/hcv-rna/hbv-dna positive, 21.2% were hiv-rna/hcv-rna positive and 4.0% were hiv-rna/hbv-dna positive. therefore, studies on diagnosing these infections in hiv-infected individuals may be valuable. epidemiology of the infection [11, 12] . hepatitis c has a global impact in terms of mortality and morbidity with over 70 million people infected all around the world [13] . according to studies in iran, the prevalence of hcv infection is nearly 0.5% (1.0% in men and 0.1% in women) [14, 15] . for hcv infection and the liver damage associated with it, the leading cause of mortality and morbidity is among hiv-infected patients. according to available evidence, hiv/hcv-coinfected patients are at higher risk for liver cirrhosis and hepatocellular carcinoma (hcc) [16, 17] . given that the transmission routes of hiv, hbv and hcv viruses are common, these infections can occur simultaneously. worldwide, nearly 40 million people are living with hiv, about 2.6 million people are infected with hbv and about 2.8 million people are hcv-infected [2] . hiv infection intensifies natural history of hbv infection, which can lead to an increase in rates of hbv persistence, relapse of hbv (resurgence of hepatitis b surface antigen [hbsag] , hepatitis b e-antigen [hbeag] or hbv-dna) and considerable clinical disease. previous studies of the hbv/hiv coinfection have shown that hiv leads to a lack of protective immunity against hbv, increased risk of cirrhosis and hcc and liver-related mortality [18, 19] . the effect of antiretroviral therapies (arts) on the natural history of hbv-related disease have been different, in some studies, it leads to recovery from hbv infection and in other studies, with relapse of hepatitis b [18, 20] . the death rate in hiv-positive patients decreased after taking combination arts, but only in those with hiv/hbv or hiv/hcv coinfection. the mortality rate is high due to liver damage. hiv/hbv-coinfected people have a higher rate of progression to liver fibrosis, cirrhosis, hcc, less clearance of hbsag and occult hbv infections (obi) are more frequent in these patients [13] . therefore, it seems that screening for hbv infection in the hiv-infected individuals should be done. testing hbsag, hbeag and ab and determining hbv viral load are an essential part of hbv infection assessment in hiv/hbv-coinfected patients. obviously, determination of cd4 counts and hiv viral load are necessary along with the antiretroviral drug-resistant response [21] . according to evidence, hcv/hiv-coinfected patients are at higher risk for cirrhosis and hcc [22] . hiv infection exacerbates natural history of hcv infection. hcv-rna loads in these patients are higher and clearance of hepatitis c viremia after acute infection in hiv-positive patients are less, and liver diseases in these patients show more progress than patients with hiv infection alone [20] . it is known that hbv and hcv infections have been associated with various clinical manifestations in people with hiv infection including impaired immune response during arts, and also increased susceptibility to artsrelated liver toxicity [23] . therefore, prior to the administration of art, patients should be tested for the presence of these infections. the aim for this study is to investigate the prevalence of hcv and/or hbv coinfection in iranian hiv-infected individuals. from september 2015 to june 2018, 198 consecutive iranian hiv-positive individuals who were referred to hospitals affiliated with iran university of medical sciences (iums), tehran, iran, were entered to this study. the research was approved by the iums' ethical committee, and all of the studied population were informed about this survey, and a written informed consent was obtained from all the subjects and also from parents of hiv-infected children in this cross-sectional study. collection of the specimens 5 ml of the patient's blood was taken from each participant into an edta-containing vacutainer tube. after separation of the plasma by centrifugation (5 min at 3000 rpm), plasma was stored at -80 • c until analysis. plasma specimens from ten individuals who were infected with hcv, and ten subjects who were infected with hbv were used as positive controls, and also plasma samples from ten healthy blood donors were used as negative controls for the experiments. serologic tests by enzyme immunoassay serological markers of hbv and hcv infection such as hbsag, hbeag, hepatitis b e-antibody (hbeab), hepatitis b core antibody (hbcab) and anti-hepatitis c virus antibodies (anti-hcv) abs were tested by the commercial enzyme immunoassay kits (dia. pro, milano, italy), according to the manufacturer's protocols. hbv viral load was assessed in 500 μl of the studied subjects plasma specimens using the high pure dna extraction kit and cobas taqman 48 kit (roche diagnostics, ca, usa) according to the manufacturer's procedure [24] . this test is a real-time pcr assay that is based on dual-labeled hybridization probe that targets two regions (precore and core) of hbv. the detection limit of the cobas taqman 48 kit is 6 to >1 × 10 8 iu/ml [25] . the hbv genotyping was determined in hbv-dna-positive specimens using the inno-lipa™ hbv kit (innogenetics, ghent, belgium) according to the manufacturer's protocols [26] . hcv detection by reverse transcriptase-nested pcr method & hcv genotyping with restriction fragment length polymorphism assay to detect genomic hcv-rna in the plasma samples of studied subjects, the viral rna was isolated from 140 μl of plasma using the qiaamp viral rna isolation kit (qiagen gmbh, hilden, germany) based on the manufacturer's procedure. the quantity and quality of the extracted rna was evaluated using the nanodrop™ spectrophotometer (thermo fisher scientific, wilmington, nc, usa) [27] . the hcv-rna was detected in extracted rna of plasma samples by the reverse transcriptase-nested pcr (rt-nested pcr) assay using two sets of primers for the 5non-translated region (5 -ntr) of hcv, as previously described in detail [28, 29] . the amplified pcr products of subjects' samples, negative and positive control specimens, and 100 bp dna size marker were electrophoresed on a 2.2% gel agarose and then stained with syber green and visualized by a uv transilluminator. the genotyping of hcv was determined in hcv-positive samples with restriction fragment length polymorphism (rflp) assay, based on a protocol that previously described in detail [28, 29] . the statistical analysis was performed using spss software version 20 (spss inc., il, usa). the kolmogorov-smirnov test was conducted to determine the quantitative variables' normality. the analysis of continuous variables was done using kruskal-wallis and one-way analysis of variance (anova) tests. the statistical differences between the two groups were evaluated by fisher's exact test and chi-square test when appropriate. p-values <0.05 were considered statistically significant. from september 2015 to june 2018, a total of 198 hiv-infected individuals (anti-hiv abs and hiv-rna positive) were enrolled in this cross-sectional study. the mean age of subjects was 35.3 ± 13.5 years (a range of 1-68 years old). out of 198 studied individuals, 126 (63.6%) were male. complete information of the demographic, laboratory and epidemiological characteristics were presented in table 1 . a significant association was observed between the sex of the participants and alanine aminotransferase (alt), aspartate aminotransferase (ast) level, anti-hcv abs, hcv-rna (p < 0.001) in plasma samples, and also in epidemiological parameters such as history of having unprotected sex, history of imprisonment, injection drug users (idus), idu sexual partners, history of tattooing, history of needle stick (p < 0.001) and history of transfusion (p = 0.034) ( table 1) . a significant relationship was observed between coinfection with hcv or hbv in hiv-infected patients and cd4 + t-cell count (p = 0.044), in other words, cd4 + t-cell count was very low in patients with these coinfections. a strong association was observed between the sex of the participants and level of education (p = 0.042) and marital status (p < 0.001) ( table 2) . 85 (42.9%) of studied subjects were positive for anti-hcv abs in plasma samples; and 42 (21.2%) had detectable hcv-rna in the plasma ( table 1 ). the hcv genotyping was performed using rflp assay for hcv-rna-positive specimens, and the results of hcv genotyping are presented in table 3 . eight (4.0%) of the studied cases had detectable hbv-dna in the plasma samples ( table 1 ). the hbv genotyping was carried out for these samples by the inno-lipa hbv kit. all these patients were infected by hbv genotype d. 55 (27.8%) of the participants were hbcab positive, and a strong relationship was observed between the sex of the studied participants and anti-hbcab in plasma specimens (p < 0.001) ( table 1) . this survey demonstrated that none of the iranian hiv-infected individuals were hiv-rna/hcv-rna/hbv-dna positive simultaneously, 21.2% were hiv-rna/hcv-rna positive and 4.0% were hiv-rna/hbv-dna positive. no significant association was observed between the hiv viral load in coinfected patients and monoinfected patients (p = 0.054). nine (4.4%) of the studied hiv-infected patients were hbsag and anti-hcv ab positive. all the information about demographic and laboratory parameters of these patients are presented in table 4 . despite the existence of successful prevention and treatment methods, the simultaneous infection of hiv, hbv and hcv is still a worldwide health issue. with the use of antiretroviral medicines and longer life expectancy in 10 hiv-infected patients, the complications of this chronic disease and its intersection with other viral infections are more evident [30] . in iran, the prevalence of hiv and other blood-related viral infections, such as hcv is relatively low in the general population [31] . the present survey was conducted on 198 individuals who were infected with hiv to investigate the molecular epidemiology of hcv/hbv coinfection in these individuals. this study showed that none of the hiv-infected people were hiv-rna/hcv-rna/hbv-dna positive simultaneously, 4.0% were hiv-rna/hbv-dna positive and 21.2% were hiv-rna/hcv-rna positive. hcv infection is more common in people infected with hiv than in hiv-negative individuals [32] . the rate of hcv/hiv coinfection is different around the world and is heavily dependent on geographical location, socioeconomic conditions of that particular location and high-risk groups [32] . nearly 37 million people are infected with hiv so far and about 70 million people all over the world are infected with hcv [2, 13] . approximately 2,278,400 people have hiv/hcv coinfection in the world, and about 62% of them are people that have been infected through injecting drugs [32] . the present study showed that approximately 81 (40.9%) of the patients are those who have injected drugs and about 46 (23.2%) of them are those who had idu sexual partner (that includes only women) ( table 1 ). the epidemiologic parameters such as injection drug abuse, needle sharing, tattooing, history of imprisonment and history of having unprotected sex revealed higher prevalence of hcv-coinfection in these patients compared with monoinfected cases. in the coinfected patients, the level of liver enzymes alt/ast was significantly high. while the first way of hiv transmission in iran is from sharing injection needles among idus [33] , hiv transmission cases in idus has begun to decline from 2005, a trend that has continued so far, and today hiv transmission through unprotected sexual contact is increasing (36.8%) [34, 35] . in this study the number of idus was 81 (40.9%). since the transmission of hcv by sexual contact is a rare phenomenon, the number of people coinfected with hcv/hiv is expected to decrease in the future [3, 36] . in the current study, 75 (37.9%) of the hiv-positive patients had a history of unprotected sex and probably the hiv virus in these patients transferred through unprotected sex. perhaps this is the reason for the decrease in the number of patients with hcv/hiv coinfection (21.2%) compared with previous reports [37, 38] . of course, in recent years, the increase in the transmission of hcv in males that have sexual intercourse with other males has been seen due to high-risk sexual behaviors. in addition, there have been cases of hcv spontaneous clearance in people who inject drugs (pwid) after art [39, 40] . the hcv genotyping on the plasma specimens showed a prevalence for subtypes, 1a (40.5%), 1b (16.7%) and 3a (33.3%). in four samples, the divergence genotype detected a mixed infection of two subtypes (1a/3a-1ab/3a) ( [41] [42] [43] . according to various reports from around the world, it seems that more research is required in this field with a wider population. like hcv infection, all hiv-infected patients should be screened for hbv infection. cirrhosis, hcc and hepatotoxicity after arts are the effects of hbv/hiv coinfection [44] . hbv vaccination should be done in all hiv individuals with hbv-negative laboratory tests. similar to hcv, hcc occurs in hbv/hiv-coinfected patients without cirrhosis [45] . hbv viral load is higher in hiv/hbv-coinfected patients than hbv-monoinfected individuals [46] . the present study found that 55 (27.8%) of these patients have anti-hbcab and 25 (12.6%) of them have hbsag. in some people, antibodies of the hbv core can be detected without anti-hbsag and hbeag [47] . our study confirms previous reports that male subjects are at high risk of developing hbv infection. typical methods for the transmission of both hbv and hiv are the sexual pathway and injection drug abuse [48] , while transmission of hcv by sexual pathway is unusual and given that in recent years the pathway for hiv transmission has changed, the prevalence of hcv is changing, but a significant change in the prevalence of hbv is unlikely to occur [49] . in a study on blood donors with an nat test in tehran, the incidence and residual risk for hiv was lower than those in developed countries, whereas hbv and hcv was higher compared to developed countries. in the iranian population, hiv infection is lower than the other countries, and screening tests are effective for blood donors. in the case of hcv, an increased incidence of hcv infection has been observed in the iranian society and in blood donors in recent years; this may be due to the highest number of idus in iran compared to other middle eastern countries [50] . incidence and high residual risk in iran indicates the nature of the endemic hbv virus. due to the launch of hbv vaccination in iran in 1993, we have to wait for the effects of the vaccination among the iranian population. based on that study, blood donors should have a more accurate technique similar to the accurate nat-screening techniques [51] . in patients with coinfection of hiv with hcv and/or chronic hbv, progressive liver fibrosis, cirrhosis and hcc can occur and coinfection of hiv with hbv and/or hcv can affect the management of hiv infection and complicate it [44, 52] . therefore, it is best to identify infection of hepatitis as quickly as possible. the result of this study revealed that none of the participants were hiv-rna/hcv-rna/hbv-dna positive simultaneously. to the best of our knowledge, the current survey is the first research that has analyzed the presence of the molecular epidemiology of hcv/hbv coinfection in iranian hiv-infected individuals; therefore, the results of this study cannot be compared with the result of other iranian research. there have been reports of coinfection with hbv and hcv in hiv-positive people, for example, 0.5% in singapore [53] , 0.62% in germany [54] and 1.7% in serbia [55] . it seems that further research focusing on this issue is needed. although there are studies that indicate seroprevalence of hiv/hcv/hbv coinfection in iran. for example, bakhti et al. found that 8% of hiv-infected individuals are coinfected with hcv/hbv (hbsag and anti-hcv ab positive), and in a meta-analysis, bagheri amiri et al. reported that coinfection of hiv/hbv/hiv was close to zero in the general population, street children and healthcare workers, while it peaked to 1.25% in pwid [56] . this study revealed that in iranian hiv-infected individuals, 4.4% of the individuals are seropositive for hbv/hcv (hbsag and anti-hcv ab positive). according to a previous study, prevalence of cirrhosis in hiv/hbv/hcv triple-infected patients was higher than hiv/hbv-or hiv/hcv-coinfected individuals [57] . therefore, prevention programs for hiv/hbv/hcv coinfection are in need of development. this study reveals that there is a high prevalence of hcv infection (21.2%) in hiv-infected individuals (hiv-rna/hcv-rna positive), as well as 4% of these people infected with hbv (hiv-rna/hbv-dna positive). also, the result of this survey highlighted that none of the hiv-infected subjects were hcv-rna/hbv-dna positive simultaneously. therefore, it seems that in hiv-positive patients, in addition to routine diagnosis of all the authors of this article are very grateful to those who volunteered to participate in this research. the current research was funded by research deputy of iran university of medical sciences (iums), tehran, iran with grant number 8921215087. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. ethical approval for this research was obtained from the local ethics committee of iran university of medical sciences (iums), tehran, iran, that is accordance with helsinki declaration (ethical code: ir.iums.fmd.rec 1396. 8921215087). all of the volunteers participating in this study were informed about this research prior to their enrollment. in addition, for investigations involving human subjects, informed consent has been obtained from the participants involved. hiv diagnosis and treatment through advanced technologies world health organization global health observatory current diagnostic methods for hiv interference of apoptosis by hepatitis b virus characterization of hepatitis b virus with complex structural variations hiv-hepatitis b virus coinfection: epidemiology, pathogenesis, and treatment global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity hepatitis b: screening, awareness, and the need to treat hepatitis b virus infection in the general population of iran: an updated systematic review and meta-analysis hepatitis c virus (hcv) genotyping by annealing reverse transcription-pcr products with genotype-specific capture probes identification of a novel hepatitis c virus genotype from punjab, india: expanding classification of hepatitis c virus into 8 genotypes molecular epidemiology of hepatitis c virus in cambodia during 2016-2017 hepatitis b, hepatitis c, and mortality among hiv-positive individuals seroprevalence of hepatitis c virus: the first population-based study from iran the impact of ifn-γ gene polymorphisms on spontaneous clearance of hcv infection in fars province, southern of iran the presence of autoantibodies to cytoplasmic rod and ring particles in the serum of patients with chronic hepatitis c virus infection hepatocellular carcinoma after sustained virological response with interferon-free regimens in hiv/hepatitis c virus-coinfected patients hiv-1, hepatitis b virus, and risk of liver-related mortality in the multicenter cohort study (macs) hepatitis b and long-term hiv outcomes in co-infected haart recipients survival in patients with hiv infection and viral hepatitis b or c: a cohort study management of hiv and hepatitis virus coinfection human immunodeficiency virus/hepatitis c virus (hcv) co-infected patients with cirrhosis are no longer at higher risk for hepatocellular carcinoma or end-stage liver disease as compared to hcv mono-infected patients liver fibrosis and hepatitis b coinfection among art naive hiv-infected patients at a tertiary level hospital in northwestern tanzania: a cross-sectional study detection of hepatitis b virus covalently closed circular dna in the plasma of iranian hbeag-negative patients with chronic hepatitis b detection of hbv genome in the plasma and peripheral blood mononuclear cells of iranian hbsag negative patients with hiv infection: occult hbv infection distribution of hepatitis b virus genotypes in azerbaijani patients with chronic hepatitis b infection prevalence of jc polyomavirus large t antigen sequences among iranian patients with central nervous system tumors hepatitis c genotypes in finland determined by rflp occult hepatitis c virus infection in iranian patients with cryptogenic liver disease trends in underlying causes of death in people with hiv from co-infection of human immunodeficiency virus, hepatitis c and hepatitis b virus among injection drug users in drop in centers prevalence and burden of hcv co-infection in people living with hiv: a global systematic review and meta-analysis trend of hiv/aids prevalence and related interventions administered in prisons of iran − 13years' experience antiretroviral drug resistance mutations in naïve islamic republic of iran aids progress report hiv-1 genetic diversity and transmitted drug resistance frequency among iranian treatment-naive, sexually infected individuals epidemiological distribution and genotype characterization of the hepatitis c virus among hiv patients in kashan high prevalence of hcv coinfection in hiv-infected individuals in shiraz, islamic republic of iran sexually transmitted hepatitis c infection: the new epidemic in msm? occasional spontaneous clearance of chronic hepatitis c virus in hiv-infected individuals hiv and hcv coinfection: prevalence, associated factors and genotype characterization in the midwest region of brazil prevalence of hepatitis c virus (hcv) coinfection in hiv infected individuals in south india and characterization of hcv genotypes detection and analysis of hepatitis c virus in hiv-infected patients in the guangxi province of china long-term liver diseases after initiation of antiretroviral therapy in hiv-infected patients with and without hbv or hcv coinfection risk of hepatocellular carcinoma across a biological gradient of serum hepatitis b virus dna level hiv-hepatitis b virus co-infection: epidemiology, pathogenesis and treatment isolated antibody to hepatitis b core antigen in human immunodeficiency virus type-1− infected individuals hepatitis c, hepatitis b, and human immunodeficiency virus infections among non-intravenous drug-using patients attending clinics for sexually transmitted diseases epidemiology of hepatocellular carcinoma: new trends prevalence and trend of hepatitis c virus infection among blood donors in iran: a systematic review and meta-analysis incidence and residual risk of hiv, hbv and hcv infections among blood donors in tehran hepatic decompensation in antiretroviral-treated patients co-infected with hiv and hepatitis c virus compared with hepatitis c virus-monoinfected patients: a cohort study factors associated with hepatitis b and c co-infection among hiv-infected patients in singapore daclatasvir plus sofosbuvir, with or without ribavirin, in real-world patients with hiv-hcv coinfection and advanced liver disease comparison of demographic, epidemiological, immunological, and clinical characteristics of patients with hiv mono-infection versus patients co-infected with hcv or/and hbv: a serbian cohort study hbv and hcv coinfection prevalence in iran-a systematic review and meta-analysis hepatic decompensation in patients with hiv/hepatitis b virus (hbv)/hepatitis c virus (hcv) triple infection versus hiv/hcv coinfection and the effect of anti-hbv nucleos(t)ide therapy key: cord-048467-1dus0u4m authors: civaner, murat; arda, berna title: can "presumed consent" justify the duty to treat infectious diseases? an analysis date: 2008-03-06 journal: bmc infect dis doi: 10.1186/1471-2334-8-29 sha: doc_id: 48467 cord_uid: 1dus0u4m background: aids, sars, and the recent epidemics of the avian-flu have all served to remind us the debate over the limits of the moral duty to care. it is important to first consider the question of whether or not the "duty to treat" might be subject to contextual constraints. the purpose of this study was to investigate the opinions and beliefs held by both physicians and dentists regarding the occupational risks of infectious diseases, and to analyze the argument that the notion of "presumed consent" on the part of professionals may be grounds for supporting the duty to treat. methods: for this cross-sectional survey, the study population was selected from among physicians and dentists in ankara. all of the 373 participants were given a self-administered questionnaire. results: in total, 79.6% of the participants said that they either had some degree of knowledge about the risks when they chose their profession or that they learned of the risks later during their education and training. of the participants, 5.2% said that they would not have chosen this profession if they had been informed of the risks. it was found that 57% of the participants believed that there is a standard level of risk, and 52% of the participants stated that certain diseases would exceed the level of acceptable risk unless specific protective measures were implemented. conclusion: if we use the presumed consent argument to establish the duty of the hcw to provide care, we are confronted with problems ranging over the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the near-impossibility of being able to evaluate retrospectively whether every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases. our findings suggest that another problem can be added to the list: one-fifth of the participants in this study either lacked adequate knowledge of the occupational risks when they chose the medical profession or were not sufficiently informed of these risks during their faculty education and training. furthermore, in terms of the moral duty to provide care, it seems that most hcws are more concerned about the availability of protective measures than about whether they had been informed of a particular risk beforehand. for all these reasons, the presumed consent argument is not persuasive enough, and cannot be used to justify the duty to provide care. it is therefore more useful to emphasize justifications other than presumed consent when defining the duty of hcws to provide care, such as the social contract between society and the medical profession and the fact that hcws have a greater ability to provide medical aid. in the course of providing health care service, health care workers (hcws) are continually exposed to many workrelated health risks. one of these risks is the exposure to infectious diseases. these diseases can include the flu, aids, tuberculosis, and hepatitis, and can be transmitted through physical contact, exposure to contaminated blood, or via the respiratory system. and, needless to say, such risks do indeed at times prove fatal. the consequences of occupational exposure to pathogens are not limited solely to bodily infections. each year, thousands of hcws are adversely affected by psychological trauma stemming from months of anxiously awaiting the results of serological tests, tests made necessary due to potential infection incidents. the anxiety experienced by hcws is related to the perception of risk from the incident and the resulting infection that may occur, and by the worry of what the reactions of others might be, such as colleagues, family, and friends, all who have to be informed. during this uncertain waiting period hcws will frequently experience intrusive thoughts, problems concentrating, difficulty sleeping, frequent loss of temper, and a decrease in sexual desire, which can act as a catalyst to exacerbate any pre-existing and unresolved emotional issues [1] . and if it turns out that the health care worker has indeed been infected by one of these contagions, the serious personal consequences to that health care worker can include the postponement of childbearing, damaged personal relationships, having to alter sexual practices, experiencing the side effects of prophylactic drugs, chronic disabilities, loss of employment, denial of worker compensation claims, possible need for a liver transplant, and premature death [2] . aids, sars, and the recent epidemics of the avian-flu have all served to remind us of the occupational risks faced everyday by hcws; the result being that the recent appearance of these diseases has forced this issue onto the common agenda and helped to spark renewed interest in the debate over the limits of the moral duty to treat. in seeking an answer to this question it is useful to have an understanding of the occupational risks faced by hcws as well as an understanding of the attitudes of hcws to these risks. for example, studies conducted in various countries have shown that, especially when there was a risk of being infected with aids, hcws may refuse to treat a patient on the grounds that there is a risk of being infected by this patient [3] [4] [5] [6] [7] [8] [9] [10] [11] . and despite the fact that the hepatitis viruses are transmitted more easily than hiv, it is the fear of being infected with hiv that causes many hcws to experience the greatest amount of stress and anxiety [12] . in a study which compared the relative risks of transmission of both hbv and hiv, the reasons for physicians' underlying fears of particular contagions were also inves-tigated and described. [13] . according to the study, people initially percieve the risk to be greater when there is a high likelihood of death involved with infection (as with hiv) even though there may be less risk of infection, as opposed to when there is a higher risk of infection but a lower risk of death involved with that infection (as with hbv). additionally, since the likelihood of sexually transmitting hbv between heterosexual partners is less than that of transmitting hiv, the consequences of hbv infection are again percieved to be less severe than the consequences of hiv infection. in this way, the hazards posed by hbv infection conflict less with the obligation to protect family members from harm. it was also found to be important that there is less of a stigma attached to having hbv than there is to having hiv. and, finally, the fact that there is a vaccine for hbv infection, which is more than 90 percent effective (for vaccinated hcws the risk of death from infection is reduced by a factor of nearly twenty), also was found to greatly influence the perceptions of the physicians. additionally, factors other than a fear of the contagion can contribute to the reluctance to treat a particular patient. some physicians and dentists express concern that if it is discovered that they treat patients with aids, then those patients who don't have hiv may shun their practice. still, other physicians insist they do not know enough about hiv infection and are too busy to learn [14] . another reason for which a physician may refuse to treat hiv-positive patients is that the physician feels they have a duty to protect their other patients, basing their reasoning on the principle of "first do not harm". by treating hiv-positive patients they claim that they may potentially be putting their other patients at risk for infection [15] . furthermore, as has been reported, there is always the possibility that when a hcw is able to reject the patient based on a more benign excuse, for example if the patient does not have enough money, it is even easier, and all the more likely, for treatment to be refused, even though this refusal was done in the interest of protecting the physical health of the individual health care provider [14] . in the literature, most studies have concentrated primarily on the attitudes and rationale behind the refusal to treat. before one can set out to effectively explore the attitudes of hcws however, it is important to first consider the question of whether or not the "duty to treat" might be subject to contextual constraints, such as providing health care to a patient suffering from an infectious disease which may be particularly contagious or for which adequate treatment measures may not yet be available. clark, in his article about physicians' duty to treat, claimed that there are three reasons in which such a duty is grounded [16] : "... since the ability to render aid is greater, the obligation to assist is (...) elevated. second, by consideration of daniels' argument that by freely joining a profession designed to combat disease, one consents to some standard of risk, and third, by realizing that the profession has flourished due to socially negotiated promises to be available in such times of duress." in his article "duty to treat or right to refuse", daniels argues that when a person chooses a career in a particular profession, it must be understood by all parties that this individual has both accepted and is willing to take the risks that are inherent to that profession [14] : "consent is crucial where obligations to take risks exist in various occupations or professions. for example, we assume that in choosing their careers, undergoing the training involved, and agreeing to follow the codes and practices regulating their work, firefighters and police have given consent to facing the significant risks they are obliged to take. there are strong parallels to medicine. people who enter medical fields clearly had alternatives. there is a general understanding that physicians face an increased risk of contagion from disease, an understanding refined during schooling and training." daniels proposes, however, that some situations can exceed the standard level of risk (slr) [14] : "for example, it is common to screen new house staff and nurses in medical centers to determine whether any individuals face special risks of contagion, such as immunosuppression or pregnancy. those at high risk may then be asked to avoid certain treatment situations, materials, or hospital areas.(...) protecting immunosuppressed providers is reasonable "risk management", a measure taken to reduce bad outcomes. but such special protection supports the claim that only standard risks are included in the duty to treat.(...) some nosocomial risks clearly take us beyond what duty requires." it is perhaps more illustrative if this argument (from this point on, this statement will simply be referred to as the "presumed consent") is written in classic form: health care services should be provided to patients who have a contagious disease. contracting an infectious disease while providing health care services to a patient with a contagious disease is an occupational risk. it is generally assumed that by joining the health care profession physicians have given their consent to be exposed to an increased risk of disease contagion. this assumption is based on the following facts: a. there is a general understanding that physicians face an increased risk of contagion from disease, an understanding refined during schooling and training. b. people who enter medical fields clearly had alternatives. some nosocomial risks clearly take us beyond what duty requires. there is a moral duty to treat patients who have a contagious disease so long as the risk to the hcw is below the slr. if we are to accept this argument, then the pressing question becomes how to determine and define the risks which are deemed to be standard and acceptable versus those which are believed to exceed and, indeed, outweigh the duty of the health care provider to treat. in order to begin to answer this question, it will be useful to investigate the nature of the choice (and all that goes along with making it) that an individual makes when they decide to enter a particular profession. for instance, how wise is it to assume that at the time of choosing their future profession the hcw was fully aware of the risks involved with such work? perhaps they were not made aware of the risks until their education and training. furthermore, if they were aware of, and fully appreciated, the risks prior to deciding on a particular profession, would they have even chosen that profession in the first place? and, finally, how is the slr to be determined, and which of the infectious diseases would then exceed this slr? in order to effectively analyze the presumed consent argument it is necessary to have an awareness of the diverse opinions and beliefs of hcws and, also, to understand their different motives and backgrounds. additionally, knowledge of what hcws feel about the risk concept and of how they feel about their duty to treat patients with contagious diseases can also be of great value to educators as they plan their curricula and it can be used by the authorities in charge of health care systems in order to better organize their services. the purpose of this study was to analyze whether or not the third premise grounds the duty to treat, namely, "it is generally assumed that by joining the health care profession physicians have given their consent to be exposed to an increased risk of disease contagion". in order to carry out this analysis, the opinions and beliefs of physicians and dentists regarding the occupational risks of infectious diseases were investigated; and, by extension, the argument that the notion of "presumed consent" may be grounds for supporting the hcws' duty to treat was also analyzed. for this cross-sectional survey, the study population was selected from among physicians and dentists in ankara, the capital of turkey. a self-administered questionnaire designed to assess the beliefs and opinions of the participants regarding the occupational risks of infectious diseases was used. this questionnaire was also used to obtain the socio-demographic information of the participants. the 17 items on the questionnaire were developed by reviewing previous studies in the literature [1, [3] [4] [5] [6] [7] [8] [9] [10] . a draft of the questionnaire was distributed to experienced health care professionals and later revised based on their criticism and suggestions. in both of the universities in which this study was conducted there are ethics committees which had been established for the purpose of determining the ethical appropriateness of pharmaceutical trials using humans; since our study involved only the use of a questionnaire and not an experimental drug, we did not apply for approval from either of these ethics committees. instead, written permission to carry out the study was granted by the dean of the faculty of medicine and by the chief manager of university hospitals. in addition, all of the potential participants were fully informed about the aim and structure of the study. furthermore, potential volunteers were all made aware that participation was strictly voluntary and that all of the answers they provide would be done so anonymously. the questionnaire was administered to a total of 373 health care workers: all of the 236 physicians who work in surgical specialties at the ankara university ibn-i sina hospital and to all of the 137 dentists in the gazi university faculty of dentistry. dentists were included in this study because, aside from being hcws themselves, there are a number of studies in the literature which show that dentists, citing various reasons, may also refuse to treat patients with contagious diseases. and, in order to better assess the fact on which the third premise of presumed consent is based, we decided to include only professional health care workers, instead of students and others who might still be in the process of deciding whether or not to currently enter the field. in total there were 230 participants, 101 physicians and 129 dentists, who completed the questionnaire, for an overall response rate of 61.7%. the questionnaire was later sent back to the non-respondents one month after the first survey, and 28 of these were completed and returned to us. the mean age of the participants was 33.8 â± 9.6 years, while 56.5% were male and 43.5% were female. additionally, the average amount of time that they had been working in the medical profession was found to be 8.5 years (min. 0, max. 40). all of the data was collected anonymously. the difference between the two groups, physicians and dentists, was compared using the chi-square test, with a p-value of <0.05 accepted as statistically significant. all analyses were carried out using spss 11.5. of the hcws surveyed in this study, roughly half stated that they understood that by choosing their profession they would be exposing themselves to an increased risk of contracting contagious disease (55.2%). and at the time of entering the faculty, 24.4% of the participants expressed that they were unaware of any increased risks; however, they later learned of these risks during their education and training. in other words, 79.6% of the participants stated that they had known about the risks either at the time they chose their profession or that they had later learned of the risks during their training and education. additionally, 6.5% of the participants answered that they had only come to realize the kinds of risks they would face after starting to work. the percentage of participants who claimed that if they had been aware of the risks earlier they would not have chosen to enter or continue in the medical profession was 5.2%. table 1 are statements which physicians and dentists chose as best reflecting their personal opinions regarding the occupational risks of infectious disease. in general, the physicians, prior to their education and training, were significantly more aware of the potential risks associated with their profession than were the dentists (p < 0.05). a significantly higher percentage of the dentists however, stated that they only learned of the occupational risks of dentistry during their education and training (p < 0.05). there was no statistically significant difference between the two groups in terms of the other opinions questioned. the participants were also asked whether or not they agree with the argument "when people choose, and continue to practice, the medical or dentistry profession, they are then required to accept all of the occupational risks resulting from the infectious diseases they might confront". the aim of this question was to determine whether or not the hcws each have their own individual working-definition for the slr. of the participants, 57.4% believed that there is such a level. 52.2% felt that certain diseases would exceed the level of acceptable risk unless specific protective measures were implemented, and 5.2% said that some diseases were always beyond the slr, no matter what precautions might be taken. no statistically significant difference was found between the physicians and the dentists. listed in table 2 are the diseases which, under certain circumstances, were cited as potentially exceeding the slr. among the participants who stated that there would be a slr for providing health care to the patients of specific diseases unless protective measures were implemented, aids and hepatitis c and b were the most frequently cited of these diseases (71.7%, 64.2%, and 56.7%, respectively). the participants who felt that some diseases would always exceed a slr expressed, that hepatitis b, tuberculosis, and bacterial meningitis always would go beyond the slr (41.7%, same for all). according to these participants, the occupational risk of potentially being infected with hiv is paramount to all other risks. percentage-wise, aids was the most frequently mentioned disease that would exceed the slr, more so than sars. all of the participants who answered that some diseases would be beyond the slr were then asked what criteria they used to make their determination. the most com-monly expressed criteria, in order, regarding the diseases, were the likelihood of transmission, whether or not protective measures are available, and whether or not immunization is possible (66.7%, 65.2%, and 58.3%, respectively). the distribution of these criteria among the physicians and dentists can be seen in table 3 . physicians expressed significantly more often than dentists that if there was no immunization or treatment available for a particular disease, then that disease would exceed the slr (p < 0.01). in terms of other criteria, there were no significant differences observed between the two groups. the primary aim of this paper is to evaluate the claim that presumed consent may constitute grounds for the moral duty to treat. the presumed consent argument is valid, because its conclusion should logically be accepted if its premises are taken into account. to analyze the soundness of the argument we carried out a survey investigating the opinions of hcws about the occupational risks of infectious diseases. in total, 79.6% of the participants said that they either had some degree of knowledge about the risks when they chose their profession or that they learned of the risks later during their education and training. in other words, one fifth of the participants either lacked adequate knowledge about the occupational risks when they chose their profession or were not sufficiently informed of these risks during their faculty education and training. this means that the assumption stated in premise 3 may be wrong for an important proportion of health care workers. it seems reasonable to suggest that the words "there is a general understanding" would be misleading if used to characterize a social concept of which the applicability and, indeed, the very existence, are yet to be established by sociological studies. it is also useful to discuss the other problems associated with presumed consent; in particular, the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the nearimpossibility of being able to evaluate, in retrospect, whether or not every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases. if we are to use the presumed consent argument, then the findings of this study indicate that when a new epidemic of a contagious disease occurs, or when the medical profession is confronted with a disease for which no immunization or treatment options are available, some hcws are not bound by the duty to treat according to the presumed consent argument. this seems potentially problematic and demands serious consideration. how appropriate is it to describe the healthcare provider's responsibility and duty as stemming from their 'consent'? to address this question, it is helpful to reflect on the conditions required for an individual to be able to give consent that is well-informed. for an hcw's consent to be informed, the following should first be explained to them: (a) the risk posed by each of the contagious diseases known at that given time, (b) commonly agreed criteria and definitions of situations that would surpass the slr, and (c) the fact that there will always be a degree of uncertainty involved with working in the medical profession, as new risks may emerge at any point during one's professional life. if not necessarily when they choose their profession, then at least after being given the relevant knowledge during education and training, the person's choice should be regarded as informed. it should therefore be ensured that hcws are acquainted with each new and emerging risk, and with any methods of prevention developed during or after their education and training. if a person's choice is to be confidently regarded as informed, it is imperative that these conditions be met. of course, the question now becomes: how possible is it to satisfy all these conditions? it is quite easy to imagine more than one answer to this question, but one thing is for sure: any thoughtful answer would acknowledge that choice is determined both by factors that are under the control of the individual and by factors that are not. personal factors such as educational status, perception of the world and ambitions all influence an individual's choice of profession strongly. nevertheless, factors outside the individual's control also play a large role in determining that choice. the environment in which the person grew up -their family life, the jobs of their parents, their community, social class and cultureall contribute to forming that individual's background, which (needless to say) has a very large influence on the opportunities and choices available to them. even though a person may not have been sufficiently informed when they chose their profession, it can be argued that during their education and training they will learn all relevant knowledge about the occupational risks associated with working in the medical profession. if so, it is fair to assume that when this individual begins to work after graduation they will be willing to confront any of those risks. in theory at least, it can be presumed that every student who passes their exams and goes on to graduate from the faculty is informed of the risks; so it can be argued that all hcws who are currently active in their profession have consented to accept the risks posed by all the contagious diseases known at the time of their graduation. of course, the diverse factors that determine the quality of education, such as the particular educational methods used, the course content, the abilities and knowledge of the instructors, role-models, and the personal features and motivations of students, are all potential sources of variation. but for the sake of argument, let us assume that a standardized education program is implemented throughout all medical schools. if that were the situation, then the argument that the individual has been made aware of the occupational risks during education would be true to the extent that the education program addressed those risks sufficiently. nevertheless, it would be hard to claim that presumed consent is valid for every individual. for many people, a degree in medicine is very costly, both financially and in terms of time and energy. under such circumstances, it is difficult for a person to quit their schooling despite the awareness they gain of the occupational risks involved. individuals might feel pressured and confused by the two options confronting them: on the one hand, dropping out of medicine and forfeiting all the time, effort and money spent on schooling towards that aim; and on the other, reluctantly accepting the occupational risks, which may look frightening to the individual at that moment. of course, it is important to remember that the person chose the medical profession in the first place, and numerous positive and beneficial elements are associated with working within it, which may ultimately serve to temper and override the individual's fear of the risks. an additional source of pressure may be that, for whatever reasons, switching educational tracks is too difficult; it may appear too daunting or be financially untenable. it seems likely that in the end the individual will choose to continue with their education and embark on a career in medicine, despite hesitation and fear of the risks. it is difficult to describe a decision made under conditions of such uncertainty and stress as 'informed'. as can be seen, we do not choose our profession from among a wide array of possibilities spread out in front of us by thoroughly researching each one so that we are fully informed of its nature; everybody's options are different and it is a large and difficult task to make oneself sufficiently informed of them. moreover, as described above, the decision to quit medical school can be quite difficult: on one side of the dilemma there are occupational risks that must be accepted regardless of misgivings on the part of the individual; on the other side, very influential factors pressure the individual to continue their medical education. thus, the claim that "people who enter medical fields clearly had alternatives" is debatable and sometimes even doubtful. in theory it sounds right; nobody has to be a physician. but in practice, having alternatives does not mean that all our decisions are made freely or autonomously. theoretically, the fundamental problem with the presumed consent argument is that it cannot explain why there is always some degree of uncertainty about the occupational risks of working in the medical profession, particularly stemming from new and emerging diseases. quite simply, if there was little or no knowledge of a risk at the time the individual became informed and gave their (tacit) consent, then this individual never accepted the risk, implicitly or otherwise, because it was unknown at the time the individual was informed. from a historical perspective, it is possible to see that while the medical profession was once concerned only with treating diseases, its vocational responsibility came in time to include preventative, promotive and rehabilitative healthcare services. as the notions of human rights and patient rights have developed and become widespread, perceptions about the health profession have changed at the community level. diseases that once killed millions of people can now be treated with a simple medicament, but today we are faced with new and challenging diseases unheard of in the past. as a result, the continuous cycle of changespurred on by greater knowledge and technological advances and confrontations with new and untreatable diseases -serves to alter the identity and nature of the medical profession, and out of all this arises a constant degree of uncertainty. this characteristic uncertainty is present both when the individual chooses the profession and throughout their education and training periodand, indeed, for the entirety of their professional career. it is therefore not possible for someone to be fully informed when they choose their profession, nor is it possible for them to become fully informed during their education and training; nevertheless, the person should be informed about the uncertainty involved with working in the medical profession. in the light of this uncertainty, answers such as "if i had known i would not have chosen it" are not very meaningful, because there is no way to anticipate all the potential risks one might encounter in the course of a professional life. the only sure thing amid the uncertainty is that diseases such as sars and avian 'flu will always continue to emerge. so far in the discussion, the difficulties of satisfying the conditions needed to validate the presumed consent argument have been described. it seems virtually impossible to fulfill all these conditions satisfactorily. at this point it is important to discuss two particular problems regarding the argument itself. first, it seems nearly impossible to evaluate individually whether the choice made by every hcw to enter the medical profession was informed. the only way to do that would be laboriously to ask each hcw whether they were informed when they decided on the health care profession. even then, irrespective of whether the person's answer to this question reflects the truth, the only thing that could be learned from such a broad and extensive interrogation would be the individual's perception, not their actual knowledge. by extension, we could not question the participants' level of knowledge in this study, but rather how they perceive their level of knowledge. because it is futile to seek objectivity in people's perceptions, it would not be sound to use those perceptions to determine whether the hcw's choice was informed, and thus whether they have taken on the duty to treat. and if these perceptions are regarded as subjective, as they should be, then it would be very difficult to develop a set of standard criteria that could be used to establish whether a hcw has been informed. this would also complicate efforts to reach a consensus on forming criteria by which various levels of risk could be defined universally. unfortunately, such difficulties can only hamper efforts to protect the right of every patient to receive the best treatment available. the second problem is that there is very likely to be more discrimination against patients with certain diseases, as hcws use this argument to justify their refusal to treat those diseases. the world medical association's declaration of geneva, which states the basic moral values of the medical profession, specifies that there should be no discrimination, regardless of the circumstances: "i will not permit considerations of age, disease or disability, creed, ethnic origin, gender, nationality, political affiliation, race, sexual orientation, or social standing to intervene between my duty and my patient" [17] . however, the physicians' right to choose their patients is described by the same organization in another policy called "twelve principles of provision of health care in any national health care system" [18] : "any health care system should allow the patient to consult the physician of his choice, and the physician to treat only patients of his choice, without the rights of either being affected in any way." for regulations at the national level, it is generally held that if there is an urgent need for medical care [19, 20] , or if no other physician is available to whom the patient can apply [21, 22] , this right would be negated and the available physician must treat the patient. if we look at these obligations in reverse, we can see that the physician might refuse to provide health services to the patient if there is no urgent need for medical care and/or if another physician is around to whom the patient can be referred. besides, the physician might also refuse the patient on the grounds that their prejudices may adversely affect the advice or treatment that they provide [20] . in actuality, this flexibility was written into the regulations in order to ensure that the patient receives the highest quality care possible; but as can be seen, it could also be abused. and if the presence or absence of consent is used as a criterion to define the duty to treat, in addition the existing flexibilities, then a mechanism would be created by which hcws may freely, and perhaps excessively, discriminate among patients. to summarize, considering all the points discussed above, the soundness of the presumed consent argument can be doubted. therefore, it should not be claimed that there is a duty to treat on the basis of the presumed consent argument, as the argument itself is not persuasive. it is also important to note that 5.2% of our participants said that they would not have chosen this profession if they had been informed of the risks. in other words, most of those hcws who claimed that they were uninformed of the occupational risks when they entered the faculty, or were not fully informed of them during their education period, stated that they still would have chosen the medical profession even if they had been more aware of the risks. this finding tells us that, generally speaking, hcws place relatively little importance on being informed beforehand. further support for these findings comes from the answers given by the participants to the other questions. nearly half said that there is no slr, and the other half felt that the diseases they evaluate would not surpass slr if the appropriate protective measures are available. also, table 2 indicates that at least 28.3% of the participants thought that none of the diseases listed in that table would exceed the slr regardless of circumstances. it can therefore be concluded that a large majority of the hcws place more emphasis on their working conditions than on being informed beforehand. in addition, the criteria most commonly stated by the participants for determining the slr were the likelihood of transmission of a disease, whether protective measures are available and whether immunization is possible. each of these criteria is related to protecting the hcw from infection, not to the treatment or the effects of a particular disease. this means that as long as protective measures are available, the hcws would regard a given disease as below the slr, so it has nothing to do with being informed beforehand. besides, the only disease used as an example in this study that could be claimed to exceed the slr was sars; aids, hepatitis c, hepatitis b, tuberculosis and bacterial meningitis all fell below the slr according to these criteria. nevertheless, only 30.9% of the participants suggested that sars would surpass the slr. to put this into perspective, sars was not even observed until 2003, and the research in the present study was conducted in 2004 and 2005. therefore, most participants in this study were not aware of sars when they chose the medical profession, nor were they ever informed of it during education or training. they nonetheless felt that the duty to treat pertained even to patients with sars. all of this suggests that factors more useful and relevant than presumed consent influence the decision of hcws to choose and continue in the medical profession; these factors may include the social contract between society and the medical profession, and the greater ability of hcws to provide medical care [16] . it is these factors that should be investigated and emphasized when defining a moral duty to treat. this study could be limited by several factors. the first limitation could be due to a socially desirable response bias; some participants might have given what they perceived as the 'right' answers to the questions rather than the answers that reflect their opinion or belief. in order to address this concern, future studies could benefit by using qualitative methods, which provide more reliable results about the motives and opinions of participants. also, this study was not prospective, so recall bias might have affected the responses of the participants. furthermore, the extent to which the results of this study are applicable to hcws such as nurses or physicians who work in internal specialties is uncertain. future studies that include other hcws as participants may broaden our understanding of the beliefs and opinions of hcws, thereby allowing us to state our claims and shape our arguments more precisely. finally, it should be mentioned that the response rate for this study (61.7%) was slightly lower than is generally expected for a survey. nevertheless, despite all these methodological limitations, we believe that our findings support our conclusion about the persuasiveness of the presumed consent argument. if we use the presumed consent argument to establish the duty of the hcw to provide care, we are confronted with problems ranging over the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the near-impossibility of being able to evaluate retrospectively whether every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases. our findings suggest that another problem can be added to the list: one-fifth of the participants in this study either lacked adequate knowledge of the occupational risks when they chose the medical profession or were not sufficiently informed of these risks during their faculty education and training. as we stated above, in order for a candidate hcw to be informed literally, three items should be explained to them: (a) the risk posed by each of the contagious diseases known at that given time, (b) commonly agreed criteria and definitions of situations that would surpass the slr, and (c) the fact that there will always be a degree of uncertainty involved with working in the medical profession, as new risks may emerge at any point during one's professional life. in this study it has been shown that at least some hcws may not be informed of (a). also, it is not currently possible to inform hcws of (b) since there are no widely-agreed criteria and definitions to allow for a universally accepted slr; and there is currently no standard education for all hcws to ensure that (c) is satisfied. considering this in addition to the problems mentioned above, the third premise of the presumed consent argument appears implausible and, consequently, the duty to treat cannot be grounded persuasively on the consent assumption. it is therefore more useful to emphasize justifications other than presumed consent when defining the duty of hcws to provide care, such as the social contract between society and the medical profession and the fact that hcws have a greater ability to provide medical aid. furthermore, in terms of the moral duty to provide care, it seems that most hcws are more concerned about the availability of protective measures than about whether they had been informed of a particular risk beforehand. it seems important that further research be carried out to improve understanding of the opinions and perceptions of hcws and the basis of their definitions, as this information could prove very helpful in defining a duty to treat that can be effectively put into practice. it is also important that a well-organized ongoing educational program that is needs-based and easily accessible be provided to hcws at both the graduate and postgraduate levels. in particular, this program must be continuously updated regarding aids and other diseases that may cause the hcws to behave discriminatively towards patients, even though these diseases are below the slr. such continuing medical education is the best answer to the justification "when i chose the profession/when i graduated, this disease did not exist!" for refusing treatment. emphasizing the social role of hcws, and educating them about the professional obligations derived from the social contract betweeen the profession and the wider social order, would further reduce that kind of reasoning. in addition, stricter standards for the duty to provide care should established by determining the criteria for a slr and identifying the situations and conditions that would exceed this slr. each of these measures could serve to remind hcws that they have a moral responsibility, as individual hcws, to be aware of professional obligations and to act as responsible members of the profession. moreover, the working environment of hcws should be provided with preventative measures that can be applied both generally and specifically and should emphasize their use. for a circumstance in which a preventative measure has been developed for a disease but is not available for treating a particular case, it would not be easy to justify the claim that there is an undeniable duty to provide care at that moment. international health care worker safety center: annual number of occupational percutaneous injuries and mucocutaneous exposures to blood or potentially infective biological substance knowledge, practices and attitudes towards hiv positive and aids patients among dental auxiliaries. east african medical journal do p: knowledge, attitudes, and practices among physicians on hiv/aids in quang ninh, vietnam. aids patient care and stds knowledge, attitudes, and practices regarding sexually transmitted infections among general practitioners and medical specialists in karachi survey of hiv/aids knowledge and attitudes of kuwaiti family physicians. family practice an investigation of dentists knowledge, attitudes and practices towards hiv+ and patients with other bloodborne viruses in south cheshire knowledge and attitudes of japanese dental health care workers towards hivrelated disease a knowledge, attitudes, beliefs and practices (kabp) survey on hiv infection and aids among doctors and dental surgeons in singapore oral care of hiv infected patients: the knowledge and attitudes of irish dentists discriminatory attitudes and practices by health workers toward patients with hiv/aids in nigeria the role of psychosocial assessment and support in occupational exposure management guidelines for prevention of transmission of human immunodeficiency virus and hepatitis b virus to health-care and public-safety workers duty to treat or right to refuse? hastings cent rep semmelweis revisited: the ethics of infection prevention among health care workers in harm's way: ama physicians and the duty to treat the world medical association: declaration of geneva the world medical association: twelve principles of provision of health care in any national health care system general medical council: good medical practice college of physicians and surgeons of alberta: physician/patient relationships -cpsa guideline turkish medical association: turkish medical association professional code of ethics right to refuse work becomes another sars issue we would like to thank to prof. huma ã�mã¼rlã¼, vice dean, for her helps to the survey's administration in gazi university faculty of dentistry, assoc. prof. alp ergã¶r for his comments on the first draft of the field study, and mr. anthony clark for his kind effort to polish the language of the text. as dr. singer says: "there is a threshold beyond which health care workers aren't obliged to take personal risks. we don't expect firefighters to jump into a burning pit, or police officers to throw themselves in front of a bullet. how health care workers define this threshold is an intensely personal decision. ... but obviously, it has serious implications for our collective response to a problem like sars." [23] . it is clear that to rely upon the presumed consent argument to define the duty to treat will not make our collective response to potential epidemics such as sars or avian 'flu any more effective or robust. hcws: health care workers; slr: standard level of risk the author(s) declare that they have no competing interests. mc and ba have contributed equally to the conception, design and writing of the manuscript. all authors read and approved the final manuscript. the questions referred to:â�¢ whether the participant knew when they chose their profession that they would have an increased risk of being infected by a contagious disease?â�¢ if the participant did not know of this occupational risk, whether or not they later learned of it during their training?â�¢ if they still have not been formally made aware of the risks, do they think that they have enough knowledge about the current risks that they face?â�¢ if they had known about the risks earlier, would they still have chosen this particular profession?â�¢ when somebody chooses to be a physician or a dentist, are they obligated to accept all of the occupational risks regarding infectious diseases?â�¢ if not, what criteria must we use to determine that a particular disease is below the slr?â�¢ which diseases are below the slr?publish with bio med central and every scientist can read your work free of charge the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/8/29/prepub key: cord-020778-4jslid14 authors: el sayed, khalid a. title: natural products as antiviral agents date: 2007-09-02 journal: nan doi: 10.1016/s1572-5995(00)80051-4 sha: doc_id: 20778 cord_uid: 4jslid14 since the ancient times, natural products have served as a major source of drugs. about fifty percent of today's pharmaceutical drugs are derived from natural origin. interest in natural products as a source of new drugs is growing due to many factors that will be discussed in this article. viruses have been resistant to therapy or prophylaxis longer than any other form of life. currently, there are only few drugs available for the cure of viral diseases including acyclovir which is modeled on a natural product parent. in order to combat viruses which have devastating effects on humans, animals, insects, crop plants, fungi and bacteria, many research efforts have been devoted for the discovery of new antiviral natural products. recent analysis of the number and sources of antiviral agents reported mainly in the annual reports of medicinal chemistry from 1984 to 1995 indicated that seven out of ten synthetic agents approved by fda between 1983-1994, are modeled on a natural product parent. it has been estimated that only 5-15% of the approximately 250,000 species of higher plants have been systematically investigated for the presence of bioactive compounds while the potential of the marine environment has barely been tapped. the aim of this review is to provide an overview on the central role of natural products in the discovery and development of new antiviral drugs by displaying 340 structures of plant, marine and microbial origin that show promising in vitro antiviral activity. since the ancient times, natural products have served as a major sourc e of drugs. abou t fift y percen t o f today' s pharmaceutica l drug s ar e derive d from natura l origi n [1] . th e growin g interes t i n natura l product s a s a source o f ne w drug s ca n b e attribute d t o man y factor s includin g urgen t therapeutic needs , th e wid e rang e o f bot h chemica l structure s an d biological activitie s o f natura l secondar y metabolites , th e adequac y o f bioactive natura l product s a s biochemica l an d molecula r probes , th e development o f recen t technique s t o accuratel y detect , isolat e an d structurally.characterize th e bioactiv e natura l product s an d advance s i n solving th e deman d fo r suppl y o f comple x natura l product s [1] . historically, th e majorit y o f th e natura l product-base d drug s includin g cyclosporine, paclitaxel and camptothecin derivatives were first discovered by traditiona l cell-base d in vitro assay s befor e thei r rea l molecula r biological targets were identified [2] . these cellular biological responses of natural products are likely to be associated wit h the inherent properties of secondary metabolites for the defense of their producing organisms [2] , infectious vira l disease s remai n a worldwid e problem . viruse s hav e been resistant to therapy or prophylaxis longer than any other form o f lif e due to their nature because they totally depend on the cells they infect fo r their multiplication an d survival. this peculiar characteristic has rendered the developmen t o f effectiv e antivira l chemotherapeuti c agent s ver y difficult. currently , there are only few drugs available for the cure of viral diseases includin g acyclovi r (1) , the know n antiherpeti c dru g whic h i s modeled o n a natura l produc t parent . i n orde r t o comba t viruse s whic h have devastatin g effect s o n humans , animals , insects , cro p plants , fung i and bacteria, many research efforts hav e been devoted for the discovery of new antiviral natural products. although the search for naturally occurrin g products whic h ca n interfer e wit h vira l infection s bega n wit h th e successful isolation of antibiotics from microorganism s but it has not been as intensive a s that of synthetic antiviral agent s [3] . this is mainly du e to the tendenc y o f most virologists wh o adop t a rational desig n o f antivira l agents rathe r tha n towar d empiricis m especiall y wit h th e progres s i n knowledge o f vira l replicatio n [3] . moreover, ther e ar e som e problem s arising from the screening of crude extracts, as well as with the purificatio n and identificatio n o f the antivira l component s fro m thes e crud e extracts . these problems became less intense with the recent advances in differen t chromatographic an d spectroscopi c technologies . man y natura l an d synthetic compound s wer e foun d t o sho w in vitro antivira l activit y bu t were much les s effective whe n tested in vivo. thi s could be attributed t o difficulty i n dru g transportatio n t o th e cell s o f th e infecte d tissu e especially i f thes e tissue s becom e inflamme d du e t o infection . man y antivirally activ e compound s ar e to o toxi c fo r therapeuti c applications . however, natura l product s remai n th e bes t resourc e fo r chemicall y diversed ne w lea d entitie s tha t coul d serv e fo r futur e developmen t a s potent and safe antiviral agents. recent analysis of the number and sources of antivira l agent s reporte d mainl y i n th e annua l report s o f medicina l chemistry fro m 198 4 t o 199 5 indicate d tha t seve n ou t ofte n syntheti c agents approve d b y fd a betwee n 1983-1994 , ar e modele d o n a natura l product paren t [4] . thes e drug s are : famciclovi r (2) , ganciclovi r (3) , sorivudine (6) , zidovudin e (7) , didanosin e (8) , zalcitabin e (9 ) an d stavudine (10) [4] . the aim of this review is to provide an overview on the central role of natural products in the discovery and development of new antiviral drugs. the original latin meaning of "virus" is "poison", "venom" or "slime" [5] . the word "virus " was also used figuratively i n the sense of "virulen t or bitter feeling", "stench" or "offensive odor " [3] . in the late 1800s, the term "virus" was bestowed on a newly discovered class of pathogens, smaller than bacteria being studied by louis pasteur and others of that era [6] . as late as 1907 , "virus" was defined a s "the poison of an infectious diseas e especially foun d i n th e secretio n o r tissue s o f a n individua l o r anima l suffering fro m infectious diseases [5] . in the early decades of the twentieth century, viruses were identified a s infectious agent s that were filterabl e and invisibl e i n the ligh t microscop e whic h superficiall y distinguishe d them from most familiar microorganisms [5] . today, viruses are defined as noncellular infectiou s agent s that vary in size, morphology, complexity , host range and how they affect thei r hosts [7] . however, they share three main characteristics in common: a) a virus consists of a genome, either rna o r dn a cor e (it s geneti c material ) whic h i s surrounde d b y a protective protei n shell . frequentl y thi s shel l i s enclose d insid e a n envelope (capsid) that contains both proteins and lipids, b) a virus can be replicated (multiplied ) onl y afte r it s genetic materia l enter s a host cell . viruses are absolutely dependen t o n the host cells' energy-yieldin g an d protein-synthesizing machinerie s an d henc e the y ar e parasite s a t th e genetic level, c) a virus's multiplication cycle includes the separation of its genomes from its protective shells as an initial step [7] . when a virus is outside the host cell, it is considered no more alive than a chromosome [6] . the interval between successive mitosis of the individual cell is divided into three periods [7] : 1-th e gl perio d precedes dna replication. its average duration is 12 hours. 2-th e s period during which dna replicates. its average duration is 8 hours. 3-th e g2 period i n which the cell prepares for th e next mitosis. its average duration is 4 hours. rna an d protei n ar e no t synthesize d whil e mitosi s proceeds , i.e. , during th e metaphas e whic h i s betwee n 0 2 an d g l period s bu t ar e otherwise synthesize d throughou t th e multiplicatio n cycl e [7] . nongrowing cell s are usually arreste d i n the gl period ; the resting stat e i s referred t o a s go. under norma l grov^h conditions , cells o f a growing culture multiply in an unsynchronized manner, hence cells at all stages of the cycle are present. the aging of cells starts after abou t 50 passages by d slowing their growth rate. the amount of time they spend in go after eac h mitosis gradually increases . the chromosomal complemen t change s fro m normal diploi d t o aneuploid pattern , supernumerar y chromosome s an d i t finally fragmente d an d th e cel l dies . malignan t tissue s giv e ris e t o aneuploid cel l line s tha t hav e infinit e lif e span s an d ar e know n a s continuous cell lines. the main feature of normal animal cell is its compartmentalization [7] . the dna o f the animal cel l i s restricted to the nucleus at all cell cycle stage s except durin g metaphas e whe n no nucleus exists . the synthesi s o f rn a occurs i n the nucleu s an d mos t o f i t remains there , but messenge r rn a and transfe r rn a migrat e t o th e cytoplasm . ribosoma l rn a i s synthesized i n th e nucleolus ; th e tw o ribosoma l subunit s ar e partl y assembled in the nucleolus and nucleus then migrate to the cytoplasm. all protein synthesi s procee d i n th e cytoplasm . th e mitochondria , whic h i s located onl y i n th e cytoplasm , contain s dna-s , rna -an d proteinsynthesizing system s of their own [7] . viruses replicate in different ways . in all cases, the viral dna or rna i s copied repeatedly. viral proteins are synthesized inside a suitable host cell where man y ne w vira l particle s ar e assemble d [6] . generall y viruse s replicate through the following stage s [3, 6, 7] , whic h ar e incorporate d int o th e host' s plasm a membrane. 4-thes e viral nucleic acids, enzymes and capsid proteins are assembled into ne w vira l particle s (genomes ) togethe r wit h thei r associate d rna or dna polymerase. 5-th e newly formed viral particles are released from the infected cell. viruses usually replicat e by lytic or temperate pathways. in the lytic pathway, stages 1-4 from above proceeds quickly and the virus is released as the host cells undergo lysis, ruptures and dies after los s of its contents. in temperat e pathways , th e viru s doe s no t kil l th e hos t cel l bu t th e infection enter s a period of latency, in which viral genes remain inactive inside th e hos t cell . i n som e case s o f latenc y th e vira l gene s becom e integrated into the host's dna, replicated along with it and passed along to all daughter cells. in time, damage to the dna or some other event may activate transcription of the viral genes therefore new viral particles can be produced an d infected cell s are destroyed [6] . proposed targets of some specific antivira l chemotherap y ar e illustrate d i n figur e 1 and ca n b e summarized as [3] : 1-attachmen t (adsorption) of the viral particle to the host cell. 2-penetratio n of the host cell by infectious viral particles. 3-particle s uncoating, release and transport o f viral nucleic acid and core proteins. 4-nuclei c acid polymerase release and/or activation. 5-translatio n of m-rna to polypeptides which are early proteins. 6-transcriptio n of m-rna. 7-replicatio n of nucleic acids. 8-protei n synthesis (late proteins). 9-vira l polypeptides cleavage into useful polypeptides for maturation. 10-morphogenesi s and assemblage of viral capsids and precursors. 11-encapsidatio n of nucleic acid. 12-envelopment. 13-release. proteins represen t th e mai n vira l component . protein s ar e th e sol e constituent o f capsids, the major componen t of envelopes and also they are associated with the nucleic acids of many viruses as core proteins [7] . viral proteins have a wide range of molecular weight ranging from 10,000-150,000 daltons. viral proteins also vary in number, some viruses posses as few as three species while others contains up to 50 protein species. all members o f th e sam e viru s famil y displa y almos t th e sam e highl y characteristic electrophoretic protein patterns [7] . glycoproteins: vira l envelope s usuall y contai n glycopotein s i n th e form o f oligomeric spike s or projections. th e carbohydrate moietie s of glycoproteins are formed o f oligosaccharide (10-15 monosaccharide units) which are linked to the polypeptide backbone through n-and o-glycosidi c bonds involvin g asparagin e an d serin e o r threonine , respectively . thei r main component s are : galactos e an d galactosamine , glucos e an d glucoseamine, fucose , mannos e an d siali c aci d whic h alway s occupie s a terminal position [7] . example of some viral proteins with specialized functions are : hemagglutinins: man y anima l viruse s (e.g. , ortho -an d paramyxoviruses) agglutinate the red blood cells of certain animal species. this mean s tha t thes e re d cell s contai n receptor s fo r certai n surfac e components of viral particles that act as cell attachment proteins which are glycoproteins and known as hemagglutinins. viral hemagglutinins could be used in their quantitative measurement [7] . enzymes: anima l vira l particle s ofte n contai n enzyme s (tabl e 1) . these enzymes are virus-specific. i n addition to the enzymes summarize d in tabl e 1 , viruse s ofte n contai n othe r enzymes . amon g the m ar e th e enzymes tha t modif y bot h end s o f m-rn a molecule s synthesize d b y their cappin g enzyme s an d poly(a ) polymerases . protei n kinases , deoxyribonucleases, dna-dependen t phosphohydrolase s an d topoisomerases are also often present in viruses [7] . homeostasis of cell numbers in multicellular organisms is maintained by a balance betwee n cel l proliferatio n an d physiologi c (programmed ) cel l death. apoptosis is a process by which cells undergo physiologic death in response t o a stimulu s an d i t i s a predictabl e serie s o f morphologicall y defined events . it is divided int o two stages namely, the breakdown of the nucleus an d alteratio n o f th e cel l shap e an d th e plasm a membran e permeability. th e consequence s o f apoptosi s ar e th e fragmentatio n o f nuclear dna , the zeiosi s (boiling ) o f the cytoplas m associate d wit h th e blebbing and increased granularity of the plasma membrane and fracturin g of the cel l int o subcellula r dna-containin g apoptoti c bodies . apoptosi s process i s different fro m necroti c cell death by involvement o f lysosoma l enzyme leakage into the cytoplasm, the swelling of the cell and the actual rupture of the plasma membrane. necrosis is often induce d by agents that affect membran e integrity , generalize d protei n synthesis , o r energ y metabolism [9] . apoptosis ca n b e induced*b y a variet y o f stimuli , e.g. , steroids, cytokines, dna-damaging agents , growth factor withdrawa l an d in case o f t or b cells, antigen-receptor engagement . apoptosi s i s also a mechanism b y whic h cytotoxi c lymphocyte s kil l thei r targets . man y viruses ca n induc e apoptosi s i n infecte d cell s whil e man y othe r viruse s especially transformin g viruses , ca n inhibi t apoptosi s an d allo w fo r cel l transformation. th e nuclea r change s durin g apoptosi s induc e chromati n [6] . viruses caus e severa l hundre d infectiou s disease s t o man y plant s afte r successfully penetratin g their cell walls, reducing the yield of a variety o f crops includin g tobacco , potatoes , tomatoes , a s wel l a s man y othe r vegetables, inducin g seriou s economi c damages . som e insect s tha t fee d plants assis t i n vira l infection . vira l particle s ma y b e clingin g t o thes e insects' piercing or sucking devices and when these devices penetrate plant cells, infectio n occurs . many anima l viruse s infec t human s an d animal s causin g severa l seriou s diseases. tabl e 2 present s a summar y o f som e anima l viruse s an d th e diseases they induce. viroids ar e plant pathogen s whic h consis t o f naked strand s o r circle s o f rna with no protein coat. viroids are mere snippets of genes smaller than the smalles t know n vira l dn a o r rn a molecul e an d the y ca n hav e damaging effect s o n citrus , avocados , potatoe s an d othe r cro p plants . apparently, enzyme s alread y presen t i n a hos t cel l synthesiz e viroi d rna then use this new viroid rna as a template for building new viroids. some unidentified infectiou s agents cause some rare fatal diseases of the nervous system including scrapie in sheep and kuru and crutzfeldt-jaco b (mad cow ) diseas e i n humans . probabl y thes e disease s ar e cause d b y infectious protei n particles , tentativel y name d prions . prion s migh t b e synthesized accordin g t o informatio n i n mutate d genes . researcher s studying scrapie , hav e isolate d th e gen e codin g fo r altere d form s o f a protein in infected cells [6] . viruses are either measured as infectious units, i.e., in terms of their ability to infect, multipl y an d produce progeny or as viral particles, regardless of their function a s infectious agents [7] . titration mean s th e measuremen t o f the amoun t o f viru s i n terms o f th e number of infectious unites per unit volume. plaque formation [7 ] monolayers of susceptible cells are inoculated with small aliquots of serial dilutions o f th e viru s suspensio n t o be titrated. wheneve r vira l particle s infect cells, progeny virus particles are produced, released and immediately infect adjoinin g cells . this process i s repeated until after 2-1 2 incubatio n days o r more . area s o f infecte d cell s develo p plaque s tha t ca n b e see n with a naked eye. agar is frequently incorporate d i n the medium to ensure that th e liberate d progen y viru s particle s i n th e mediu m d o no t diffus e away an d initiat e separat e o r secondar y plaques . the infecte d cell s mus t differ i n some recognizable manner from non infected ones , i.e., they must be completely destroyed, become detached from the surface o n which they grow o r possess stainin g properties differen t fro m thos e o f normal cells . the mos t commo n metho d t o visualize plaques i s to apply neutra l re d o r crystal violet to the infected cel l monolayers and then counting the number of no n staine d area s [7] . titer s ar e expresse d i n term s o f numbe r o f plaque-forming unit s (pfu ) pe r milliliter . ther e i s a linea r relationshi p (linear dose-response curve ) between the amount of virus and the numbe r of plaque s produce d whic h indicate s tha t eac h plaqu e i s produced b y a single vira l particle . th e viru s progeny i n eac h plaqu e ar e clones . viru s stocks derived fro m singl e plaques are named "plaqu e purified " whic h i s important i n isolatin g pur e viru s strains . plaqu e formatio n i s th e mos t desirable metho d o f viral titration becaus e i t is economic an d technicall y simple. however, not all viruses can be measured thi s way due to lack of host cells that can develop the desired cytopathic effects (cpe) . many anima l viruse s ge t adsorbe d b y red bloo d cell s (rbcs ) o f variou s animal species. each viral particle is a multivalent, i.e., it can adsorb more than on e cel l a t a time . i n practice , th e maximu m numbe r o f cell s wit h which any particular virus can combine is two since rbcs are bigger than viral particles. in a virus-cell mixture in which the number of cells exceeds the number of viral particles, the small number o f cell dimer that may b e formed i s generally undetectable . if the number o f viral particles exceed s the number of cells, a lattice of agglutinated cells is formed tha t settles out d in a characteristic readily distinguishable manner from th e settling pattern exhibited b y unagglutinate d cell s [7] . hemagglutinatio n assa y i s th e determination o f the virus that will exactly agglutinate a standard numbe r of rbcs. because the number of viral particles required fo r this is readily calculated (slightly higher than the number of cells), hemagglutination is a highly accurate and rapid assay. in vitro antiviral screening assays [11, 12] the vira l infectivit y i n culture d cell s i s determine d durin g viru s multiplication i n the presenc e o f a singl e teste d compoun d o r extrac t o r after extracellular incubation. current antiviral chemotherapy [13, 14] research i n antiviral chemotherapy starte d around early 1950' s when the search fo r anticance r drug s revealed severa l new compounds tha t inhibi t viral dn a synthesis , e.g. , th e pyrimidine analo g idoxuridin e whic h wa s later approve d a s a topica l treatmen t fo r herpe s keratitis . sinc e then , research effort s wer e focused o n both purine and pyrimidine nucleosid e analogs [13] . with the emergence of aids epidemic, research on antiviral generally an d specificall y anti-hi v becam e highes t priority . man y o f these retrovirus proteins have been purified and characterized for the sake of designing drugs that would selectively inhibit some critical enzymes of hiv such as reverse transcriptase and protease which are required for the final packaging of this virus particle. 1-zidovudin e (7 ) (previousl y azidothymidine , azt ) i s a deoxythymidine analo g tha t als o requires anaboli c phosphorylatio n for activation . i t competitivel y inhibit s deoxythymidin e triphosphate fo r th e rt . i t als o act s a s a chai n terminato r i n th e synthesis of pro viral dna. it is active against hiv-1, hiv-2 and the human t cell lymphotropi c viruses . resistance to 7 occurs due to mutation in rt gene. didanosine (8 ) i s a syntheti c analo g o f deoxyadenosine . i t i s anabolically activated to 2,3-dideoxyadenosine-5-triphosphate which inhibits viral replication as 7. resistance is typically associated with mutation at codon 74. zalcitabine (9) is a pyrimidine nucleoside that inhibits replication of hiv-1 in a similar mechanism to 7. mutation at codon 65 induces resistance which is associated with the decrease in susceptibility to 8 and 9. 4-stavudin e (10) is a thymidine analog that also requires a metabolic activation as that of 7. it is active against hiv-1. 5-lamivudin e (11) is a nucleoside analog which in vitro inhibits hiv-1 and hbv . it inhibits hiv-rt and shows synergistic effec t wit h 7 against hiv-1 . it requires metabolic phosphorylation a s that of 7. high level of resistance is developed by mutation at codon 184. 1-indinavi r is a specific inhibitor of hiv-1 protease which is essential for th e production o f mature and infectious virions . it i s currently clinically approved for treatment of hiv-1 infections. 2-ritonavi r is an inhibitor of hiv protease with high bioavailability. it is metabolized by the hepatic p450 cytochrome oxidase system and hence suffers from several drug interactions. 3-saquinavi r is a synthetic peptide-like analog that inhibits the activity of hiv-1 protease and prevents the cleavage of viral polyproteins. 3-ribaviri n [13 ] i s a guanosin e analo g tha t i s intracellularl y phosphorylated b y the host cell's enzymes . despite its mechanism is not yet fully elucidated , it apparently interferes with the synthesis of guanosin e triphosphat e t o inhibi t cappin g o f vira l mrn a an d some viral rna-dependent polymerases. its triphosphate derivative inhibits the replication o f a wide range o f rna and dna viruses including influenz a a and b , parainfluenza, respirator y syncytia l virus (rsv), paramyxovirus, hcv and hiv-1. four basi c approache s ar e conducte d fo r plan t selectio n fo r antivira l screening assays : 1 -rando m collectio n o f plant s followe d b y mas s screening. 2-ethnomedical approach. 3-literature-based follow up of the existing leads. 4-chemotaxonomic approac h [12] . the second and third approaches ar e th e mos t favore d one s becaus e o f thei r cost-effectiv e applicability. the selection based on folkloric use proved five times higher percentage o f active leads than other approaches. the random approach usually affords mor e novel compounds with antiviral activity. combining ethnomedical, phytochemical an d taxonomical approache s is considered the best compromise. different cel l culture-base d assay s ar e currentl y availabl e an d ca n b e successfully applie d fo r plan t extract s an d pur e compounds . antivira l agents tha t interfer e wit h on e o r mor e vira l biosyntheti c dynami c processes are good candidates as clinically useful drugs . virucidal agents that extracellularl y inactivat e viru s infectivit y ar e rathe r candidate s a s antiseptics. th e ke y factor s tha t determin e th e selectio n o f th e assa y system are : simplicity , accuracy , reproducibility , selectivit y an d specificity [12] . after evaluatio n o f th e antivira l potenc y o f a teste d compound along with its cytotoxicity, the therapeutic index in a given viral system i s calculated. th e therapeutic inde x i s defined a s a ratio o f the maximum drug concentration at which 50% of the normal cells grovrth is inhibited to the minimum drug concentration at which 50% (sometimes 90 or 99%) of the virus is inhibited. the relative potency of a new antiviral agent should be compared with an existing approved drug. in vivo testing of any new in vitro active antiviral agen t i s considered th e key ste p befor e an y huma n clinica l trials . thi s mode l shoul d predic with an animal's metabolic processes. 4-provin g tha t the compoun d wil l resist an d will no t adversel y affec t the immune system [12] . two usefu l anima l model s ar e usuall y employed : heterologu s o r homologus. in heterologus systems, a disease is induced by a virus from a n animal origi n i n an experimenta l anima l tha t mimic s th e huma n disease . several review s hav e bee n publishe d dealin g wit h natura l productsderived antivira l compound s [11, 12, [16] [17] [18] [19] [20] [21] [22] [23] . presently, there ar e only tw o plant-derived compounds under clinical development [2] . (-f)-calanolide a (12) i s a c2 2 coumari n isolate d fro m th e malaysia n rainfores t tree , calophyllum langigerum b y th e u.s . national cance r institut e [2] . i t shows a poten t hiv-r t inhibitor y activit y [2] . in vitro studie s o f 1 2 demonstrated activit y agains t hiv-1 includin g az t an d othe r nonnucleoside r t inhibitors-resistan t strains . it als o show s synergisti c anti -hiv activity i n combination with nucleoside rt inhibitors: 7, 8 and 9 [2] . to overcom e th e difficult y o f suppl y o f 12 , its tota l chemica l synthesi s was accomplishe d [2] . i n jun e 1997 , clinica l developmen t o f 1 2 wa s started as a potential drug for treatment of aids. a single -center 7-mont h u.s. phas e l a clinica l tria l o f 1 2 wa s starte d t o asses s it s safet y an d tolerability [2] , sp-30 3 (13 ) i s a mixtur e o f natura l oligomeri c proanthocyanidins u p to a molecular weigh t 2100 daltons. it is isolated from th e late x o f a latin america n plan t croton lechleri [2] , it show s potent in vitro activity against hsv and other varieties of dna and rna viruses. virend, whic h i s the topical formulatio n o f 13 , is evaluated i n phase ii clinical trials for the treatment of genital herpes in combination with acyclovir. these trials were later suspended as they proved virend to have no additional benefit over using oral acyclovir alone. provir, the oral formulation o f sf-sob, is proved to be safe and well tolerated in phase i trials but ineffective i n phase ii for the treatment of rsv since there was no adequate absorption by patients. however, provir was proved effectiv e in symptomati c treatmen t o f traveler' s diarrhe a throug h restoratio n o f normal bowel function and prevention of recurrences [2] . alkaloids are heterogeneous group of compounds linked by the common possession o f a basi c nature , containin g on e o r mor e nitroge n atom s usually in combinations part of heterocyclic system [11] , their precursors are usually amino acids and they exert certain biological activities. many alkaloids are also foun d i n animals and humans where they coul d exer t a profound pharmacologica l activit y [11] . tabl e 1 illustrate s variou s alkaloids with activity against many animal viruses. lycorine (16), pretazettine (17) apqrphing; oliverine (18), pgnzqphgnanthriding: chelidonine (21) schumannificine (29 ) flavqiioid: opium; morphine (34) , codeine (35), papaverine (36 ) phenantl iroquinozolizidine: cryptopleurine (37 ) pipgriding; 1-deoxynojirimycin (38) , l -deoxymannojirimycin (39) , a-homonojirimycin (40 ) protoberberine: berberine (41) , columbamin e 1 (42) , palmatine (43) pvrrolizidine: 1 austra l ine (44 ) ouinoline/isoquinoline: table 4 summarizes the antiviral activity of plant and some non-plant carbohydrates. chromones, furanocoumarins an d flavonoids ar e common constituent s i n many plan t families . coumarin s ar e specifically abundan t i n the familie s rutaceae an d umbellifera e [11] . the yield coul d sometime s reac h u p t o 1% o f the dr y plant weight . tabl e 5 illustrates various antivira l activitie s of chromones, coumarins and flavonoids. lignans ar e widesprea d secondar y metabolite s i n plant kingdom . the y occur in many parts of plants especially wood, resin and bark trees [11] . they are also found i n many roots, leaves, flowers, fruits an d seeds [61] . there is an evidence that lignans play a major rol e in plant-plant, plantinsect an d plant-fungu s interaction s [11] . the chemica l structure s o f t e r m i l i g n a n ( 1 0 4 ) , t h a n n i l i g n a n ( 1 0 5 ) , anolignan (106 ) justicidin a (107) [89] lignans ar e divers e an d comple x despit e the y ar e essentiall y dimer s o f phenylpropanoid unit s (c6-c3 ) linked by the central carbons of their sid e chains [11] . presently, there are six lignan subgroups : butane derivatives , lignanolicles (butanolides) , monoepoxylignan s (tetrahydrofura n derivatives), bisepoxylignans (3,7-dioxabicyclo(3.3.0)-octane derivatives) , cyclolignans (tetrahydronaphthalenes ) an d cyclolignan s base d o n naphthalene [11] . phenolics, benzoquinones , naphthoquinones , anthraquinones and phenylpropanoids ar e abundant secondary metabolite s in plants. table 6 illustrates the reported antivira l activitie s of these plan t secondary metabolites. tannins ar e phenoli c compound s tha t ar e abundan t i n plan t kingdom . basically, ther e ar e tw o type s o f tannins . hydrolysabl e typ e whic h usually consists of simple phenolic acids, e.g., gallic acid, which is linked to sugar . th e condense d typ e i s simila r t o flavonoids . th e know n medicinal tannin-containin g plan t lemo n bal m (melissa officinalis, labiatae) is extensively studied as antiviral agent [11] . leave s of this plant contain abou t 5% , dry weight o f tannins which ar e mainly constructe d from caffei c acid . a cream containing 1 % of a specially prepare d dried extract from lemon balm leaves has been introduced to the german market for loca l therap y o f herpes infection o f the ski n [90] . the effect o f this cream i n th e topica l treatmen t i s statisticall y significan t a s proven b y clinical studies [90] . this is a decisive indication that constituents and /or extracts of plants could serve as useful lead s for developing the antiviral drugs for the future. terpenoids are also abundant secondary metabolites condensed tannins: catgghinjg aci d (156 ) condense d t a n n i n s , p a v e t a n n i n s , cinnamtannins. 1 galloy l catechi n an d epicatechin . procyanidin b 2 (157), [29] in plant world. terpenoids are essentially derived from the basic 5-carbons isoprene unit . thes e ar e classifie d into : monoterpene s (cio) , sesquiterpenes (ci5) , diterpene s (c20) , triterpenes , sterols , saponins . about seve n hundre d polyacetylene s hav e bee n isolate d s o fa r mainl y from plant s belongin g t o th e famil y o f asteraceae , umbellifera e an d campanulaceae [11] . polyacetylenes occu r principally a s straight chai n polyines, allenes , phenyl, thiophenyl , thioether an d spiroketal-enoethe r derivatives in a quite high yield. thiophenes and related sulfur compounds are usuall y groupe d togethe r wit h th e polyacetylene s becaus e o f thei r common biosyntheti c pathway s [11] . few plant-derive d lactones , butenolides an d phospholipid s sho w antivira l activity . th e antivira l activities o f thiophenes , polyacetylenes , lactones , butenolide s an d phospholipids from plant origin are reported in table 8. thiarubine a (190) thiophene-a (191) phenylheptatriyne ( [11] . additional proteins (pap-ii an d paps) were found i n relative smaller amounts. subsequently, other rips were found i n other plants and exhibit similar antiviral activity, e.g., tritin fro m triticum aestivum seed , geloni n fro m gelonium multiflorum seed , momordin fro m momordica charantia seed , sapori n fro m saponaria officinalis seed , dianthin fro m dianthus caryophyllus leaf , tricosanthi n from trichosanthes kirilowii [11] , bryodin 2 from bryonia dioica [129 ] and bougainvillea antivira l protei n i (ba p i ) fro m bougainvillea spectabilis roo t [130] . some varieties of nicotiana glutinosa produce a protein called avf which afford som e protectio n agains t tm v b y restrictin g it s lesion s i n a n analogy manne r t o interferon s [11] . av f i s a glycoprotei n whic h i s terminally phosphorylate d wit h 22,00 0 dalton s molecula r weight . th e mechanism of action of avf is not yet established. meliacin i s a n antivira l glycopeptid e o f molecula r weigh t 5000-600 0 daltons, isolated from th e leaves of melia azedarach (meliacea ) [11, 134] . its aprotinine i s anothe r plant-derive d antivira l polypeptid e whic h specifically inhibi t myxoviruse s especiall y influenz a a . aportinin e i s known to be a protease inhibitor . it acts by interferin g wit h the essentia l step of cleavage of the precursor hao into subunit polypeptides and hence prevents the viral infection [11] . many plant-derive d di -and tri-peptides wer e proved to be active agains t hsv an d measle s viru s (mv) . thes e peptide s consis t mainl y o f carbobenzoxy derivative s o f phenylalanin e [11] . cationi c peptide s ar e used as nature's antibiotics, being produced i n response to an infection i n virtually mos t organism s includin g plant s an d insects . cationi c peptide s and protein s ar e no w proceedin g throug h clinica l trial s a s topica l antibiotics and antiendotoxins [136] . several thousan d plan t extract s hav e bee n show n t o posses s in vitro antiviral activit y wit h littl e overla p i n specie s betwee n studies . i n mos t cases, th e assa y method s ar e designe d t o detec t virucidal , prophylacti c [11] [11] [11] [11] [11] [11] [11] [11] [11] [11] [11] [11] [11] [11] [11] [11] activities an d t o defin e extract s tha t interfer e w^it h vira l replicatio n i n cultured cells . aqueous an d organi c extract s have generall y bee n prove d equally fruitfu l an d hence i t is not feasible t o asser t that an y on e metho d of extractio n i s preferable . furthe r characterizatio n o f th e activ e constituents in these active extracts should reveal some useful compounds . many o f activ e extract s ma y tur n ou t t o b e identica l o r relate d t o th e previously describe d structure classes. yet, there also may be a possibility for som e nove l phytochemicals . tabl e 9 summarize s som e o f th e mos t active extracts in the literature. with marin e specie s comprisin g approximatel y on e hal f o f tota l globa l biodiversity fo r whic h estimate s rang e betwee n 3-50 0 x 10^ specie s o f prokaryote and eukaryote organisms. the marine macrofauna represent s a broader rang e of taxonomic diversit y than found i n terrestrial evironmen t [150] . with a typical eukaryote possessing 50,000 genes, the global marine macrofauna ar e the source of 2.5 x 10^^ -1.5 x 101^ primary products an d an associate d extensiv e rang e o f secondar y metabolite s [150] . presently, only fe w thousan d nove l compound s fro m marin e origi n hav e bee n identified. thes e compounds have been revealed unique in chemical and pharmacological terms . however, onl y fe w promising therapeuti c lead s many marine-derived peptides, alkaloids, proteins, nucleosides and other a^-containing compound s were show n to be active agains t severa l vira l species. table 10 illustrates these activities. the antiviral activities of various marine-derived terpenoids, steroids and carotenoids are summarized in table 11 . halogenated cyclohexadienone (245) sggqviitgrpgngs: avarol (246) [188] [189] [190] [191] [192] [193] [194] [195] [196] [197] [198] [199] [200] [201] [202] [203] [204] ( table 11 ) . contd.. [217] . a polysaccharide from the green marine alga ulva lactuca inhibited the reproduction of many human and avian influenza viruses . acid hydrolysi s of thi s polysaccharid e reveale d th e presenc e o f arabinose , xylose , rhamnose, galactose , mannos e an d glucos e i n rati o o f 1:1:9:5:2:5:16 , respectively along with an unidentified suga r [218] . treatment o f laminari n isolate d fro m th e marin e alg a laminaria cichorioides b y endo-p-l,3-glucanas e fro m marin e invertebrate s transformed p-l,3:l,6-gluca n int o a highl y efficien t preparatio n agains t tobacco mosai c virus named antivi r [219] . polysaccharides, compose d o f mannose, galactose, glucose, uronic acid and sulfate group s (7-8% wt/wt) was obtained from the marine microalga cochlodinium polykrikoides sho w potent inhibitor y activitie s against : influenz a viruse s typ e a an d b , respiratory syncytia l viruse s typ e a an d b , hiv-1 , hsv-1 an d parainfluenza viruse s typ e 2 and 3 . no cytotoxicit y no r inhibitio n t o th e blood coagulation were observed up to 10 0 |lig/ml [220] . an unusua l sulfate d mannos e homopolysaccharide , isolate d fro m th e pacific tunicat e didemnum molle show s in vitro anti-hi v activity . th e nmr dat a of this polysaccharide reveal s that it consists o f a sequence o f 2,3-disulfated mannos e unit s joined throug h p (1,6 ) glycosidi c linkage s [221] . a natural sulfate d mucopolysaccharid e (oku40) , extracted fro m a marine plan t dinoflagellata an d a n artificia l sulfate d polysaccharid e (0ku41), was prepared fro m a marine pseudomonas, displaye d antivira l activities agains t hiv-1 an d -2 , zidovudine-resistan t hiv-1 , hsv-1 , influenza viruse s a an d b , respiratory syncytia l viru s an d measle s viru s without displayin g cytotoxicit y o r inhibition o f blood coagulatio n o f host cells [222] . table 1 2 illustrates the reported antiviral activities of the common marin e secondary metabolite s polyacetylenes , quinones/pyrones , macrolide s an d prostaglandins. the human dream of finding a n antiviral antibiotic from microbial origin which selectively affects the viral but not the host cell in a similar manner to th e regula r antibiotic s ha s no t becom e a reality . however , severa l microbial-derived metabolite s sho w promisin g activity . th e us e o f microbes t o modif y syntheti c compound s o r t o accomplis h specifi c desired reactio n i s commo n i n pharmaceutica l industry . adenin e arabinoside (ara a, 200) , which is approved for clinical investigation, is also produce d b y a streptomyces s p [19] . example s o f secondar y metabolites fro m microbia l origi n tha t sho w antivira l activitie s ar e presented below. rifamycins ar e microbial-derived macrolide s that were isolated i n 195 7 from the actinomycete streptomyces mediterranei, obtained from the soil of the pine forests of southern france [18] . of these, rifamycin b (306) is the leas t toxic . additio n o f diethylbarbituri c aci d t o th e fermentatio n medium result s i n th e productio n o f 30 6 only . rifampici n i s th e c3hydazone semisyntheti c derivativ e o f rifamycins . rifamycin s sho w in vitro and in vivo anti-poxyviruses, e.g., vv activities [18] . these activities are apparently du e to inhibition of the early step of viral morphogenesi s which affect s th e assembl y o f immatur e vira l particles . the inhibitor y activity of rifamycins o n retroviruses is also reported [18] . many natural and semisyntheti c rifamycin s inhibi t th e virio n rna-dependen t dn a polymerase (rt ) [18] . rifamycin b (306) wa s reported activ e agains t murine sarcom a viru s (msv ) du e t o it s rt , focu s formatio n an d cel l transformation inhibitor y activities [18] . rifamycin antibiotics also inhibit the rt of rauuscher leukemia virus, preventing its leukomogenic activity [18] . streptovaricin b (307), tolypomycin an d geldanamycin ar e examples of ansamycins whic h ar e chemicall y relate d t o rifamycins . th e streptovaricins an d tolypomycin s resembl e rifamycins i n rt-inhibitor y activity [18] . streptovaricin b inhibits the replication o f poxviruses by inhibiting earl y stage s o f mrn a synthesi s [18] . inhibitio n o f focu s formation of msv by streptovaricins is also reported [18] . gliotoxin (308) is a fungal metabolite , isolated from aspergillus terreus and foun d t o hav e antibacterial , antitumo r an d antifunga l effect s [18] . gliotoxin acetate inhibited the cpe of poliovirus in monkey kidney cell cultures due to the early stag e inhibition o f rna viral replication [18] . gliotoxin also inhibits influenza virus-induced rna polymerase. arantonins are related compounds, isolated from arachniotus aureus dixidi aspergillus terreus and show rt inhibitory activity [19] . the activity is attributed to the epidithiapiperazinedione moiety, as the case of 308. sporidesmin distamycin a (309 ) i s a n oligopeptide , isolate d fro m streptomyces distallicus that inhibits transcription and replication of dna viruses along with its other related semisynthetic analogs [18] . example of dna viruses inhibited by this group are vaccinia virus and hsv-1. distamycin a also shows inhibitory activity for rt of retroviruses [18] . daunomycin (310 ) and doxorubicin (311 ) are anthracycline glycosides , isolated from streptomyces peucetius [18] . these compounds are used in cancer chemotherapy du e to their ability to bind dna. both compounds inhibit rt and production of murine leukemia virus [18] . actinomycin d is a peptide antibiotic, produced by streptomyces parvulu. it interacts with cellular dna and inhibits the replication of mammalian viruses tha t depen d o n cellula r functions , e.g. , rabie s viru s [18] . mithramycin i s a relate d compoun d tha t inhibit s influenz a an d pseudorabies viruse s probabl y du e t o inhibitio n o f hos t cel l rn a polymerase ii [18] . cordyceptin (313 ) i s a fermentation produc t o f th e fung i aspergillus nidulans and cordyceps militaris. cordyceptin inhibit s the synthesis of mrna an d hence the replication o f both rna an d dna viruses [18] . [18] . toyocamycin (314 ) i s anothe r adenosin e derivativ e produce d b y streptomyces toyocaensis [18] . this compoun d selectivel y inhibit s th e ribosomal rn a synthesi s i n fibroblasts . th e synthesi s o f adenovirusspecific mrn a is also inhibited by 314 [18] . sinefungin i s a n adenin e derivative , isolate d fro m streptomyces griseolus an d i t effectivel y inhibit s v v mrn a (guanine-7 -jmethyltransferase an d hence inhibits methylation of its mrn a [18] . bilayer structure . filipin show s activity agains t vsv and to less degree against influenza and rauscher leukemia virions [18] . amphotericin b is another related antifungal antibiotic. its methyl ester is active against hsv-1 and -2, vv, sv and vsv with less cytotoxicity and improved water solubility [18] . aphidicolin i s a tetracycli c diterpenoid , produce d b y th e fungu s cephalosporium aphilicola. it has the ability to inhibit hsv-1, -2, vv and herpetic keratitis of rabbit [19] . the mechanism of this compound is not yet established. cytochalasin b (316 ) i s a metabolite o f the mol d helminthosporium dermatoideum [19] . it inhibits hexose transport i n cells and hence i t is used fo r th e stud y o f virus-specifi c glycoprotei n synthesis . i t show s potent inhibitor y activit y agains t hsv-1 an d -2 apparentl y du e t o th e inhibition of viral glycosylation [19] . cytochalasin d is a closely related compound tha t show s activit y agains t adenoviru s bu t i t enhance s th e infectivity of poliovirus and parainfluenza [19] . it is a phenolic acid, isolated from a penicillium sp. and shows inhibitory activity against hsv-1 and -2, vv, semliki forest, influenza a viruses and coxsackievirus. its effect i s probably due to cytotoxicity [19] . additional antivira l microbial-derived metabolite s are summarized i n table 13 . there i s a n urgen t nee d t o identif y nove l activ e chemotype s a s lea d fo r effective antivira l chemotherapy . recen t year s hav e witnesse d grea t advances in this area. the enormity of natural products as antiviral agent s started t o b e expresse d i n th e are a o f antivira l chemotherapy . thi s i s represented b y the fda's approva l fo r clinica l investigatio n o f two plantderived compounds, in addition to one compound of marine origin and one microbial-derived compound . ou t o f te n syntheti c approve d drug s between 1983-1994 , seve n wer e modeled o n a natural produc t pare n [4] . the developmen t o f recen t technique s t o dereplicate , accuratel y detect , isolate, structurall y defin e an d automate d assa y th e bioactiv e natura l products will result in more lead of antiviral agents. it has been estimate d that onl y 5-15 % o f th e approximatel y 250 , 00 0 specie s o f highe r plant s have bee n systematicall y investigate d fo r th e presenc e o f bioactiv e compounds while the potential of the marine environment has barely bee n tapped [4] . consequently , natura l product s represen t potentia l antivira l leading resources for imaginative discoverers. natural product s a s a resource fo r new drugs recent natural products based drug development: a pharamaceutical industry perspective plant products as potential antiviral agents natural products in drug discovery and development the virus a history of the concept virology apoptosis i n viral infections. i n apoptosis: the molecular basis of cell death marburg an d ebol a viruses . i n antivira l compound s fro m plants plan t substance s a s antiviral agents basi c & clinica l pharmacology antiviral agents : characteristic activit y spectru m dependin g o n the molecula r targe t wit h whic h the y interact interferon-induce d antivira l action s an d thei r regulation. i n va n hoof , l . plan t substance s a s antiviral agents biorgani c marin e chemistry antivira l agent s fro m natura l sources chemotherapy o f viral infection . natura l products natura l product s a s antivira l agents anti-huma n immunodeficienc y virus (anti-hiv ) natura l product s wit h specia l emphasi s o n hi v revers e transcriptase inhibitors antivira l substances screening o f highe r plant s fo r plant antivira l agents . iii. isolation o f alkaloid s from clivia miniata rege l (amaryllidaceae) antiherpe s viru s activit y o f aporphine alkaloids biologica l an d phytochemical evaluatio n o f plants . v . isolatio n o f tw o cytotoxi c alkaloid s from chelidonium majus effect o f benzo[c]phenanthidin e alkaloids on reverse transcriptase and their binding property to nucleic acids evaluatio n o f natura l product s a s inhibitors o f huma n immunodeficienc y viru s type-1 (hiv-1 ) revers e transcriptase anti-hi v an d cytotoxic alkaloid s fro m buchenavia capitata antivira l component s of ophiorrhiza mungo tetrahydroxyoctahydroindolizidine alkaloid , fro m seed s o f castanospermum anstrale castanospermin e i n alexia species inhibitio n o f mammalian digestiv e disaccharide s b y polyhydrox y alkaloids anti-hi v michellamine s fro m ancistrocladus korupensis michellamin e b , a novel plan t alkaloid, inhibit s huma n immunodeficienc y virus-induce d cel l killin g b y a t least two distinct mechanisms a n activ e antiviral alkaloi d fro m boehmeria cylinderica (l. ) sw . (urticaceae) a-homono-jirimycin [2,6-dideoxy-2,6-imino-dglycerol-l-heptitol ] fro m omphalea diandra l. : isolatio n an d glucosidas e inhibition enzym e inhibition . viii : mod e o f inhibitio n o f revers e transcriptaseactivity b y analogues, isomers and related alkaloids o f coralyne inhibitor y natural products . 26 . quinolin e alkaloid s fro m euodia oxburghiana antiherpes viru s actio n o f atropine activit y i n vitro of mgn-3, an activated arabinoxyla n from ric e bran myeloblastosi s an d huma n immunodeficiency viru s revers e transcriptas e inhibitors , sulfate d polysaccharides extracte d fro m se a algae antimicrob glycoprotein of human immunodeficiency typ e 1 bind s sulfated polysaccharide s and cd4-derived syntheti c peptides reverse transcriptas e isolate d fro m th e malaysia n tree , callophyllum inophyllum linn e. a-(l-3)-d-mannose-specifi c plan t lectins are markedly inhibitor y to human immunodeficiency viru s an d cytomegaloviru s infection s in vitro anti-huma n immunodeficiency viru s phenolic s from licorice the calanolides , a novel hiv-inhibitor y clas s o f coumari n derivative s fro m the tropical rainfores t tre e callophyllum langigerum specifi c inhibitio n o f th e revers e transcriptas e o f huma n immunodeficiency viru s typ e 1 an d th e chimeri c enzyme s o f huma n immunodeficiency viru s typ e 1 an d 2 b y non -nucleosid e inhibitors specifi c inhibition o f huma n immunodeficienc y viru s typ e 1 reverse transcriptas e mediated by soulattrolide, a coumarin isolate d from the latex of calophyllum teysmannii studie s of the plantaginis herba. 9. inhibitory effects o f flavonoids from plantago specie s on hiv reverese transcriptase activity thre e dimensiona l quantitativ e structure-activit y relationshi p (qsar) o f hi v integras e inhibitors : a comparativ e molecula r fiel d analysi s activit y o f som e flavonoids agains t viruses antivira l activit y o f flavone s an d flavans antiviral activit y o f 3-methoxyflavones , i n "plan t flavonoid s i n biolog y an d medicine: biochemical , pharmacologica l an d structure-activit y relationships " cody ne w antivira l compound s i n "advances i n viru s research antivira l avtivit y o f 5,6,7-. chemother, trimethoxyflavon e an d its potentiation o f the antiherpes activit y o f acyclovir differentia l inhibitory effect s o f variou s her b extract s o n th e activitie s o f revers e transcriptase an d variou s deoxyribonuclei c aci d (dna ) polymerase isolation an d structur e o f woodorien , a ne w glucosid e havin g antivira l activity fro m woodwardia orientalis mechanisti c evaluatio n o f ne w plant-derive d compounds tha t inhibi t hiv -revers e transcriptase biologica l activitie s o f lignans secondar y metabolite s fro m plant s a s antiretroviral agents : promisin g lead structure s fo r anti-hi v drug s o f th e future a n investigatio n o f th e antivira l activit y o f podophyllum peltatum studie s o n th e pharmacologica l activitiesof amazonian euphorbaceae assesmen t o f th e anti-hi v activit y o f a pin e cone isolate nove l i n vitr o anti-hi v activ e agents arctigeni n a s a lea d structur e fo r inhibitor s o f huma n immunodeficiency viru s type-1 integrase tw o ne w lignan s with activit y agains t infuenz a viru s fro m th e medicina l plan t rhinacanthus nasutus new anti-hiv , antimalarial an d antifungal compound s from terminalia bellerica antivira l activit y o f lignan s an d their glycosides from justiciaprocumbens selective inhibition o f human immunodeficienc y virus type-1 synthesis and anti-hiv activit y o f l,r-dideoxy-gossypo l and related compounds vergleic h de r antivirale n aktivita t vo n oxidierte r kaffiisaure un d hydrokaffeesaure gege n herpesvirus hominis typ i und typ 2 in vitro antiviral caffeoy l ester s fro m spondias mombin anti-aid s agents , 1 . isolatio n an d characterization o f fou r new tetragalloylquini c acid s a s a new clas s o f hi v reverse transcriptas e inhibitor s fro m tanni c acid anti -aids agents, 2. inhibitory effect s o f tannins on hiv reverse transcriptase an d hiv replication i n h9 lymphocyte cells th e peltatols , nove l hiv-inhibitor y catecho l derivative s from pothomorphe peltata nove l phloroglucinol s fro m th e plan t melicope sessiliflora (rutaceae) cytotoxi c an d antiherpetic activit y o f phloroglucino l derivative s fro m mallotus japonicus (euphorbiaceae) structur e o f euglobal-gl , -g 2 an d -g 3 fro m eucalyptus gerandis. thre e new inhibitor s of epstein-barr virus activation structur e o f syzygiol : a skin-tumo r promotio n inhibitor hiv-revers e transcriptas e inhibitor s o f eucalyptus globulus hiv-inhibitor y an d cytotoxic oligostilbenes fro m th e leaves of hopea malibato inactivation o f envelope d viruse s b y anthraquinone s extracte d fro m plants anthraquinone s a s a new clas s o f antivira l agents agains t huma n immunodeficienc y virus light-induce d acidificatio n b y the antiviral agen t hypericin a poten t nove l hiv-inhibitor y naphthoquinon e trime r from a concospermum sp antivira l phenylpropanoi d glycoside s from th e medicinal plan t markhamia lutea secondar y metabolite s from plant s a s antiretroviral agents : promisin g lead structure for anti-hlv drugs of the future purification an d characterization o f antiviral substance s from th e bud of syzygium aromatica antiviral substance s from the root of paeonia species euagitannin s a s activ e constituent s o f medicinal plants structure an d antiherpetic activit y amon g th e tannins antiviral euagitannin s fro m spondias mombin phytochemistry inhibitor y effect s o f tannin s o n revers e transcriptas e fro m rn a tumo r virus anti-hiv activit y o f euagitannins comparativ e studies of the inhibitory propertie s of antibiotics o n human immunodeficienc y virus and avaian myeloblastosi s virus reverse transcriptas e an d cellular dn a polymerase viru s inhibitio n b y tea , caffein e an d tanni c acid plant antivira l agents . vii. antiviral an d antibacterial proanthocyanidin s fro m the bark of pavetta owariensis a-typ e proanthocyanidin s fro m ste m bar k o f pavetta owariensis dimeri c an d trimeri c proanthocyanidin s possessin g a doubl y linke d structur e fro m pavetta owariensis antivira l activit y o f tanni n fro m th e pericarp of punica granatum l . against genital herpes virus in vitro effects o f condensed tannin s and related compounds on reverse transcriptase inhibitory effects o f tannic acid sulfate an d related sulfate s o n infectivity, cytopathi c effec t an d gian t cel l formatio n o f huma n immunodeficiency vms in vitro antiviral activity o f calcium elenolate antivira l agents o f plant origin . ii . antiviral activit y o f scopaduli c aci d b derivatives th e effec t o f a^-tetrahydrocannabino l o n hepes simplex viru s replication antiviral agent s o f plant origin . iii . scopadulin , a nove l tetracycli c diterpen e fro m scoparia dulcis inactivation o f measel s viru s an d herpes simplex viru s b y saiko-saponins structur e o f tw o antivira l triterpen e saponin s fro m anagallis arvensis in vitro activity of dammar resi n triterpenoid effect o f saponins from anagallis arvensis on experimenta l herpes simplex keratiti s i n rabbits preliminar y studie s o f antivira l activit y o f triterpenoi d saponins : relationships betwee n thei r chemical structure s an d antiviral activity antivira l activitie s o f glycerrhizin an d it s modifie d compound s agains t huma n immunodeficienc y virus typ e 1 (hiv-1) an d herpes simplex typ e 1 (hsv-1) in vitro cytotoxi c saponin s fro m ne w zealan d myrsine specie s triterpenoi d saponin s fro m maesa lanceolata triterpenoid saponin s a s anti-hi v principle s from fruit s o f gleditsia japonica an d gymnocladus chinensis an d a structur e activit y correlation anti-hi v triterpen e acid s from geum japonicum betulinic aci d an d platani c aci d a s anti-hi v principle s from syzigum claviflorum an d the anti-hiv activit y o f structurall y relate d triterpenoids betulini c aci d derivatives : a ne w class of human immunodeficienc y viru s type 1 specific inhibitor s with a new mode of action betulinic aci d derivatives : a ne w clas s o f specifi c inhibitor s o f huma n immunodeficiency viru s type 1 entry antivira l activit y o f dammarane saponin s agains t herpes simplex viru s i anti-aids agents. 30. anti-hiv activit y o f oleanoli c acid, pomoli c aci d an d structurall y relate d triterpenoids nigranoi c acid , a triterpenoid fro m schisandra sphaerandra tha t inhibit s hiv-1 revers e transcriptase alkylidene bicyclic butenolide with antiviral activity and its p-glucopyranosid e from homalium cochinchinensis molecular similarit y o f anti-hi v phospholipids bryodi n 2 a ribosome-inactivatin g protein fro m th e plan t bryonia dioica, 1990 , us patent a n antivira l protei n from bougainvillea spectabilis roots ; purificatio n an d characterization protei n toxin s an d thei r us e i n cel l biology expression characteristic s o f pokeweed antivira l proteins (paps) : two distinct type s of proteins action s o f pokewee d antiviral protei n o n virus-infecte d protoplasts plan t immunomo-dulator s fo r terminatio n o f unwante d pregnanc y an d fo r contraception an d reproductive health inhibitio n o f foo t an d mout h viru s (fmdv) uncoating by a plant-derived peptid e isolate d from melia azedarach l . leaves. argent th e therapeuti c potentia l o f cationic peptides nee m see d oi l inhibit s aphi d transmission o f potat o viru s y t o pepper antiinfectiv e activit y o f a plan t preparatio n from geranium sanguineum l in vitro anti-influenz a viru s activit y o f a plan t preparation fro m geranium sanguineum l phyllanthus amarus suppresse s hepatiti s b viru s b y interruptin g interactio n betwee n hb v enhance r i an d cellula r transcription factors th e plant s of th e genu s phyllanthus a s a potentia l sourc e o f new drugs a revie w of th e plant s o f th e genu s phyllanthus: thei r chemistry , pharmacolog y an d therapeutic potential nontoxi c therapeuti c extract s o f larrea tridentata in vitro activity o f extracts of persea americana leave s on acyclovir-resistan t an d phosphonoaceti c resistan t herpes simplex virus antiviral activit y o f a n extrac t fro m leave s o f th e tropica l plan t acanthospermum hispidum interaction b y various plant extracts anti-herpeti c activit y o f variou s medicina l plan t extracts stud y o f stapelia pregnane s an d veratrum alkaloids anti-hi v activit y o f alkalin e extract s o f rooibo s te a leaves progres s i n th e acquisitio n o f ne w marine -derived anticance r compounds : developmen t o f ecteinascidin-74 3 (et-743) pharmacologicall y activ e compound s fro m marin e invertebrates: drugs from the sea marin e product s a s a sourc e o f antivira l dru g leads . drug development research antiviral an d antitumor compound s fro m tunicates structure s of th e didemnins , antivira l an d antitumo r depsipeptide s fro m a caribbea n tunicate antivira l an d antitumo r compound s fro m a caribbea n tunicate bioactiv e peptides from a marine mollus k elysia rufescens an d its algal diet bryopsis sp callipeltin a , an anti-hiv cycli c depsipeptid e from th e new caledonia n lithistid a spong e callipelta sp eudistomin s c ; e ; k an d l ; poten t antivira l compound s containing nove l oxathiazepin e rin g fro m caribbea n tunicat e eudistoma olivaceum bromotopsenti n an d dihydroxybromotopsentin: antivira l and antitumor bis(indolyl) imidazoles from caribbean deep-se a sponge s o f th e famil y halichon d fro m a sponge dercitus sp a ne w guanidinostyren e fro m th e cora l tubastrea aurea nove l antivira l an d antimicrobia l compounds from th e spong e acarnus erithacus (d e laubenfles) revised structure s of polyandrocarpidines antivira l an d antibacteria l bromopyrrol s from agelas coniferin and hal o derivative s thereof bioactiv e compounds from aquati c an d terrestria l sources mycalamid e a , a n antiviral compoun d from a new zealan d spong e o f th e genu s mycale antivira l an d antitumor agent s from a ne w zealan d sponge , mycale sp . 2 . structure s an d solution conformation s o f mycalamides a and b isolatio n an d structur e elucidation of onnamide a, a new metabolite of a marine sponge, theonella sp ptilomycalin a : a novel polycyclic guanidine alkaloid o f marine origin new antiviral an d cytotoxic compound s fro m th e spong e cramb e crambe bioactiv e bisoxazole s from a marine sponge antiviral indolocarbazole s from a blue-green alg a belongin g t o th e nostocaceae aplidiasphingosine , a n antimicrobia l an d antitumo r terpenoid from a n aplidiiim sp . (marine tunicate) nove l alkaloid s fro m th e spong e batzella sp. : inhibitors o f hiv gpl20-human cd 4 binding b-carboline s fro m th e blue-gree n alga dichothrix baueriana alkaloid s fro m antarcti c spong e kirkpatrickia varialosa. par t 1 : variolin b , a new antitumo r an d antivira l compound unusua l re d pigmen t fro m th e spong e trikentrion loeve, anti-hiv-1 metabolite contributio n t o th e stud y o f marin e products . xxxii. the nucleosides o f sponges antivira l agent s from a gorgonian a proto n nuclea r magnetic resonance study of the antihypertensive and antiviral protein bds-1 from th e se a anemon e anemonia sulcata: sequentia l an d stereospecifi c resonance assignmen t and secondary structure 2-(l-chloro-2-hydroxyethyl)-4,4-dimethylcyclohexa-2,5-dienone : a precursor of 4,5-dimethylbenzo[i]furan from the red alga desmia hornemanni inhibitio n o f the replicatio n of etiologi c agen t o f acquire d immun e deficienc y syndrom e (huma n t -lymphotropic retroviru s /lymphadenopathy-associate d virus ) b y avaro l an d avarone antivira l chamigren e derivative , pc t internationa l application w o 8 6 03 sesquiterpenoid isocyanid e purificatio n from a marine sponge an d its use as a neoplasm inhibitor , virucide and fungicide a n antivira l sesquiterpen e hydroquinone from the marine sponge strongylophora hartmani thre e new sesquiterpen e hydroquin-ones from marine origin frondosin s a an d d , hiv -inhibitory sesquiterpen e hydroquinon e derivative s from euryspongia sp cytotoxic and antiviral diterpene from a caribbean deep water marine sponge, spongia sp ne w antiinflammator y an d antiviral diterpenoid s fro m a marine octocoral o f the genus solenopodium brianthei n v , a new cytotoxic an d antiviral diterpen e isolate d from briareum asbestinum reiswigin s a an d b , nove l antiviral diterpene s fro m a dee p wate r sponge bioactive norsesterterpene 1,2-dioxane s froma thia sponge , mycale sp variabilin an d relate d compounds from a sponge genus sarcotragus venustatriol : a ne w antiviral triterpene tetracycli c ether from laurencia venusta holothurinosides : ne w antitumo r no n sulphated triterpenoi d glycoside s from the sea cucumber holothuriaforskalii biologically activ e saponin s an d saponin-lik e compound s fro m starfis h an d brittle-stars new secosteroids from an undescribed gorgonia n o f the genu s muricella hiv-inhibitor y natura l products . 11 . comparative studie s of sulfated sterol s from marine invertebrates weinbersterol disulfate s a and b , antivira l steroi d sulfate s fro m th e spong e petrosia weinbergi new antivira l stero l disulfat e ortho esters fro m th e marin e spong e petrosia weinbergi meroterpene s fro m cystoseira usneoides ii cucumaria-xanthin s a , b and c from the se a cucumber cucumaria japonica caracterisation chimiqu e e t activit e virostatiqu e in vitro vis d u viru s d e la fevere jaune quelques carraghenanes extrait s d'algues roug e senegalaises polysaccharides a s antivira l agents : antiviral activit y o f carrageenan antivira l carbohydrate s from marin e re d algae antiviral and anticoagulant activity of polysaccharides from marine brown algae antivira l activitie s o f sulfate d derivative s o f a fucosamine -containing polysaccharide o f marine bacterial origin inhibitor y effec t o f sulfate d derivative protectio n o f tobacc o plant s against tobacc o mosai c viru s b y b-l,3;l,6-gluca n "antivir " obtaine d b y enzymatic transformatio n o f laminarin in vitro antiviral activitie s o f sulfated polysaccharide s fro m a marine microalga {cochlodinium polykrikoides) against huma n immunodeficienc y viru s an d othe r envelope d viruses a sulfate d mannose polysaccharid e wit h anti-hi v activit y fro m th e pacifi c tunicat e didemnum molle brominate d polyacetyleni c avid s fro m th e marin e spong e xestospongia muta: inhibitor s of hiv protease petrosynol an d petrosoli c acid, tw o nove l natura l inhibitor s o f th e revers e transcriptas e o f huma n immunodeficiency viru s from petrosia sp absolute stereo -chemistr y o f onchitriol s i and ii mod e o f inhibitio n o f hi v revers e transcriptase b y 2-hexaprenylhydroquinone, a novel genera l inhibito r of rnaand dna-directed dna polymerases antibioti c an d antitumo r misakinolid e composition an d thei r derivatives . pc t internationa l applicatio n w o 8 8 00 a ne w inhibito r o f huma n cytomegaloviru s proteas e fro m streptomyces sp a nove l antivira l agents whic h inhibit s th e endouncleas e o f influenz a viruses sattabacin s an d sattazolins : new biologicall y activ e compound s with antivira l propertie s extracte d from a bacillus sp ne w antivira l antibiotics , kistamicin s a an d b . i . taxonomy , production, isolation , physico -chemica l propertie s an d biological activities isolatio n an d synthesi s o f caprolactin s a and b , ne w caprolactam s from a marin e bacterium structures o f ne w polyacetylen e triglyceride s an d indolocarbazole s fro m th e myxomycetes lycogala epidendrum antibiotic s fro m glidin g bacteri a xlvii. thiangazole : a novel inhibito r o f hiv-1 fro m polyangium sp fluvirucin s al , a2 , bl , b2 , b3 , b 4 an d b5 , ne w antibiotics activ e agains t influenz a a virus. i. production, isolation , chemica l properties an d biological activities structura l studie s o f mm46115 , a nove l tetroni c acid containin g macrolid e wit h antivira l an d antibacteria l activit y isolate d from actinomadur a pelletieri antibiotic s fro m glidin g bacteria . xliii . phenoxan : a nove l inhibitor o f hiv-1 infectio n i n cel l culture s from polygoniu m sp. , strai n p i v019 (myxobacteria) structur e an d biologica l activity a new peptid e antibiotic . production, isolation , an d propertie s o f lanthiopeptin manufactur e o f diaminobutyri c aci d homopolymer wit h streptoalloteichu s fo r us e a s antibioti c an d virucide derivatives of oxetanocin: oxetanocins h, x and g and 2-aminooxetanocin a a ne w adenosin e deaminase inhibitor containing chlorine. production, isolation and properties a novel specific inhibito r against reverse transcriptase structur e o f virantmycin , a nove l antivira l antibiotic studie s o n sf-1836 c substance . meiji seika kenkyu nempo a new antiherpetic agent, ah-1763 lia, produced by streptomyces cyaneus strai n no. 1763 structur e o f sc h 68631 : a new hepatiti s c virus proteinas e inhibito r fro m streptomyces sp pradimici n antibiotics an d thei r manufactur e wit h actinomadura new antivira l antibiotics , cycloviracins b l an d b2.1. production, isolation , physico-chemica l propertie s an d biologica l activities the structure s o f quartromicin s al , a 2 an d a3 : novel macrocycli c antivira l antibiotics possessin g fou r tetroni c aci d moieties antibiotic s from basidiomycetes . xxxix. podoscyphic acid , a new inhibito r of avian myeloblastosi s viru s an d moloney murin e leukemi a viru s revers e transcriptas e fro m a podoscypha species key: cord-017885-cz19y60u authors: maziarz, eileen k.; perfect, john r. title: cryptococcosis date: 2014-11-24 journal: diagnosis and treatment of fungal infections doi: 10.1007/978-3-319-13090-3_15 sha: doc_id: 17885 cord_uid: cz19y60u cryptococcosis is an infectious disease caused by the encapsulated fungi cryptococcus neoformans and cryptococcus gattii. once a relatively uncommon cause of human disease, cryptococcal infection can develop in apparently immunocompetent hosts and has emerged as an important opportunistic infection in humans over the past several decades as immunocompromised populations expand in the setting of hiv/aids, organ transplantation, malignancies, and treatment for other conditions. clinical manifestations are myriad but pulmonary and central nervous system (cns) infections are the most common. improvements in diagnostic testing and standardized approaches to antifungal therapy, when available, have made considerable impact in the management of this infection. while the widespread use of highly active antiretroviral therapy (haart) has improved the outcome of cryptococcosis in many hiv-infected patients, cryptococcosis remains an entity of considerable morbidity and mortality in many parts of the world, and restoration of host immunity can present management challenges that require individualized management. as immunocompromised populations continue to expand, it is likely that cryptococcosis will remain an important opportunistic fungal infection of humans requiring ongoing investigation. cryptococcosis is an infectious disease with a wide range of clinical presentations caused by pathogenic encapsulated yeasts in the genus cryptococcus. currently, there are two species of these fungi that commonly cause disease in humans: cryptococcus neoformans, which causes cryptococcosis in both immunocompetent and immunocompromised hosts, and cryptococcus gattii, which is primarily a pathogen in apparently immunocompetent patients but can also cause disease in the immunocompromised. c. neoformans was first identified as a human pathogen in 1894 by two german physicians, otto busse and abraham buschke, when they described a circular yeast-like microorganism in a lesion on the tibia of a woman; the microorganism was initially named saccharomyces hominis [1] . the name c. neoformans has been consistently adopted in both the mycology and medical literature since 1950 [2] . in the mid-1970s, when kwon-chung discovered two mating types of c. neoformans that produced fertile basidiospores, the organisms were subsequently separated into two varieties, var. neoformans (serotypes a and d) and var. gattii (serotypes b and c). these two varieties were recently separated into two species, c. neoformans and c. gattii, based on their genetic background and phylogenetic diversity, as proposed by kwon-chung in 2002 [3] . it is possible, as more molecular information is gathered from genome sequencing, that c. neoformans var. neoformans (serotype d) and c. neoformans var. grubii (serotype a) will be divided into separate species as well as other cryptic species. the incidence of cryptococcosis began to rise in the late 1970s. early case reports of cryptococcal infections were primarily associated with cancer, autoimmune diseases, organ transplantation, and receipt of corticosteroids as these immunocompromised populations expanded [4] . a major surge in new cases of cryptococcosis occurred during the first two decades of the hiv pandemic, when cryptococcal infection was an important opportunistic infection (oi) in all parts of the world. furthermore, around 2000, c. gattii strains (previously geographically restricted to tropical and subtropical regions) caused a localized outbreak of cryptococcosis in apparently immunocompetent individuals on vancouver island [5] . this has increased recognition that these fungi can exploit new geographical environments and cause disease in both immunocompromised and apparently immunocompetent hosts. despite the development of highly active antiretroviral therapy (haart), which has decreased the rate of hiv-related cryptococcosis in developed countries, the burden of cryptococcal infection is still very high in developing countries and in those individuals without access to health care. it has been estimated that there are a million cases per year with more than 600,000 deaths due to cryptococcosis worldwide [6] . the life cycle of c. neoformans and c. gattii involve asexual (yeast) and sexual (basidiospores/hyphae) forms. the asexual form is the encapsulated yeast that reproduces by narrow-based budding and is found most commonly in clinical specimens, whereas the sexual stage, which exists in one of two mating types, "alpha" or "a," is observed only under certain conditions, resulting in meiosis to form basidiospores. the vast majority of clinical infections and environmental isolates are caused by "alpha" mating-type locus strains. since the sexual stage of c. neoformans and c. gattii has been described, their teleomorphs were named filobasidiella neoformans and filobasidiella bacillospora, respectively. c. neoformans and c. gattii usually appear as white-tocream, opaque, and mucoid colonies that grow to several millimeters in diameter on the most routine agar within 48-72 h. with some strains, a few colonies occasionally develop sectors with different pigmentation or different morphologies (i.e., wrinkled, smooth, mucoid). both cryptococcal species will grow readily on most fungal culture media without cycloheximide at 30-37 °c in aerobic conditions. however, c. neoformans is generally more thermotolerant than c. gattii, and, within this species, serotype a is generally more tolerant than serotype d strains. in addition to their ability to grow at 37 °c, the yeast produce a thick shedding polysaccharide capsule, melanin pigments, and the enzymes urease and phospholipase, which allow cryptococcus to be readily identified from other yeasts. these are also considered to be yeast virulence factors. cryptococcosis was considered an uncommon infection prior to the aids epidemic, associated with malignancies, organ transplantation, and certain immunosuppressive treatments. beginning in the early 1980s, the incidence of cryptococcosis increased significantly and between 6 and 10 % of persons with aids developed cryptococcosis [7, 8] . in fact, hiv/aids was found to be associated with 80 % of cryptococcosis cases worldwide. cryptococcal infection is a major oi in hiv-infected patients as the cd4 + cell count falls below 100 cells/µl. following widespread implementation of haart, the incidence of cryptococcosis among patients with hiv/aids has fallen significantly in most developed nations. the incidence of cryptococcal infection in persons not infected with hiv has remained stable during this time. moreover, in developing nations with limited access to haart, the prevalence of and morbidity and mortality associated with cryptococcosis remains unacceptably high, accounting for up to 600,000 deaths per year. besides hiv infection, other risk factors for acquiring cryptococcal infections include many conditions that result in an immunocompromised status (table 15. 2). although both c. neoformans and c. gattii can cause cryptococcosis in apparently normal hosts, the percentage of c. gattii infections causing disease in such patients is significantly higher than for c. neoformans. c. neoformans is found throughout the world in association with excreta from certain birds such as pigeons and in tree hollows. c. gattii is commonly associated with several species of eucalyptus and other trees [9] . while the link between the environmental source of infection and cryptococcosis cases is not precise, there is evidence to suggest an increased risk of cryptococcosis and asymptomatic cryptococcal antigenemia following intense bird exposures. recently, there has been a strong link between the c. gattii outbreak in humans on vancouver island and common environmental yeast exposures. although these fungi can be detected in endobronchial specimens from humans without disease (colonization), clinicians should be alert for subclinical disease or potential for disease when cryptococcus is isolated from any clinical specimen. approximately 95 % of cryptococcal infections are caused by serotype a strains ( c. neoformans var. grubii) with the remaining 4-5 % of infections caused by serotype d ( c. neoformans var. neoformans) or serotype b and c strains ( c. gattii). whereas c. neoformans serotype a is found worldwide, serotypes b and c are found primarily in (table 15. 3) [10] . in australia and new zealand, serotypes b and c caused up to 15 % of all cases of cryptococcosis cases in one study, but serotype a remains the predominant serotype even in these endemic areas [11] . to date, only c. gattii strains have been reported to cause a widespread defined outbreak of disease [5] . cryptococcosis occurs primarily by inhalation of the infectious propagules, either dehydrated (poorly encapsulated) yeasts or basidiospores, into pulmonary alveoli. direct inoculation into tissue due to trauma can be a portal of entry in occasional cases and, potentially, yeast may enter through the gastrointestinal tract. after the yeasts are inhaled into the lungs of a susceptible host, they encounter alveolar macrophages, and other inflammatory cells are recruited through release of cytokines and chemokines such as interleukin (il)-12, il-18, monocyte chemotactic protein (mcp)-1, and macrophage inflammatory protein (mip)-1α. cryptococcal infection primarily involves granulomatous inflammation, which is a result of a helper t cell (th1) response with cytokines including tumor necrosis factor, interferon-γ, and il-2 [12] . in many circumstances, the yeasts remain dormant (yet viable) in hilar lymph nodes or pulmonary foci of an asymptomatic individual for years and then disseminate outside those complexes when local immunity is suppressed, similar to that which is observed in cases of reactivation tuberculosis or histoplasmosis [10] . in a patient with severely compromised cellular immunity, the yeasts reactivate and proliferate at the site of infection and then disseminate to other sites causing progressive clinical symptoms. recent advances in the molecular biology of cryptococcus have confirmed several virulence factors. the three classical virulence factors of c. neoformans include: capsule formation, melanin pigment production, and the ability to grow well at 37 °c [9, 12] . the prominent antiphagocytic polysaccharide capsule, which is composed of glucuronoxylomannan (gxm), is unique to cryptococcus species and is considered an essential virulence factor that has multiple effects on host immunity. in addition, c. neoformans possesses an enzyme that catalyzes the conversion of diphenolic compounds to form melanin, which may have a biological role to protect the yeasts from host oxidative stresses and which may partially explain the organism's neurotropism. finally, its ability to grow at 37 °c is a basic part of the virulence composite for most of the human pathogenic fungi including cryptococcus, as molecular studies have linked high-temperature growth with certain signaling pathways and enzymes that this yeast has acquired or adapted over time in order to enhance its pathogenicity. other virulence factors include phospholipase and urease production and multiple enzymes associated with protection against oxidative stresses. c. neoformans and c. gattii have a predilection for establishing clinical disease in the lungs and central nervous system (cns). other organs that may be involved in cryptococcosis include skin, prostate, eyes, bone, and blood [2, 8, 10, 13] . in fact, this yeast may cause disease in any organ of the human body, and widely disseminated cryptococcal infection can affect multiple organs in severely immunosuppressed patients (table 15 .4). the respiratory tract serves as the most important portal of entry for this yeast, and thus there are many clinical manifestations of pulmonary cryptococcosis, ranging from asymptomatic transient or chronic colonization of the airways or simply a pulmonary nodule on radiograph to life-threatening fungal pneumonia with acute respiratory distress syndrome (ards) [2, 8] . in a normal host with cryptococcal infection, asymptomatic pulmonary cryptococcosis can occur in about one third of patients with pulmonary infection and patients may present to care with only an abnormal chest radiograph. the most common radiologic findings of cryptococcosis include well-defined single or multiple noncalcified nodules ( fig. 15 .1) and pulmonary infiltrates ( fig. 15. 2), but other less frequent radiographic findings include pleural effusions, hilar lymphadenopathy, and lung cavitation. patients with pulmonary cryptococcosis can present with symptoms of acute onset of fever, productive cough, respiratory distress, chest pain, and weight loss [14] . the outbreak of c. gattii infections in vancouver island included several cases of severe symptomatic pulmonary cryptococcosis in apparently immunocompetent individuals. in an immunocompromised patient, especially those with hiv infection, cryptococcal pneumonia is usually symptomatic and can progress rapidly to ards, even in the absence of cns involvement. most immunocompromised patients with cryptococcal in-fection, however, present with cns rather than pulmonary symptoms. in fact, more than 90 % of hiv/aids patients with cryptococcal infection already have cns cryptococcosis at the time of diagnosis, many of whom will have a paucity of respiratory complaints. the findings in chest radiographs of immunocompromised patients with pulmonary cryptococcosis are the same as those in immunocompetent patients, but alveolar and interstitial infiltrates tend to be more frequent and imaging can mimic pneumocystis pneumonia. accelerated presentations of cryptococcal pneumonia are more common among immunocompromised patients. in pulmonary cryptococcosis, if the infection is confined to the lung, serum cryptococcal polysaccharide antigen is usually negative. however, while a positive serum polysaccharide antigen may indicate the dissemination of the yeast from the lung, it does not confirm cns involvement. in immunocompromised individuals with pulmonary cryptococcosis, a lumbar puncture to rule out cns disease should be considered regardless of the patient's symptoms or serum polysaccharide antigen test results. the only setting in which screening a lumbar puncture may not necessarily need to be performed in a patient with cryptococcus isolated from the lung is in the asymptomatic, immunocompetent patient with disease that appears to be limited to the lungs. clinical manifestations of cns cryptococcosis include headache, fever, cranial neuropathy, alteration of consciousness, lethargy, memory loss, and signs of meningeal irritation [2, 8] . these findings are usually present for several weeks and therefore cause a clinical syndrome of subacute meningitis or meningoencephalitis. however, on some occasions, patients can present more acutely or lack typical features including headache. in hiv-infected patients with cns cryptococcosis, the burden of fungal organisms in the cns is usually high. therefore, these patients may have a shorter onset of signs and symptoms, higher cerebrospinal fluid (csf) polysaccharide antigen titers and intracranial pressures (icps), and slower csf sterilization after starting antifungal treatment. different species may produce differences in clinical manifestations. for instance, one species may have a predilection to cause disease in brain parenchyma rather than the meninges. in certain areas of the world, c. gattii tends to cause cerebral cryptococcomas ( fig. 15 .3) and/or hydrocephalus with or without large pulmonary mass lesions more frequently than c. neoformans. these patients with brain parenchymal involvement usually have high icp and cranial neuropathies, and respond poorly to antifungal therapy. cutaneous infections are the third most common clinical manifestations of cryptococcosis. patients can manifest several types of skin lesions. one common skin lesion is a papule or maculopapular rash with central ulceration that may be described as "molluscum contagiosum-like." these lesions are indistinguishable from those due to other fungal infections including histoplasma capsulatum, coccidioides immitis, and penicillium marneffei. other cutaneous lesions of cryptococcosis include acneiform lesions, purpura, vesicles, nodules, abscesses, ulcers ( fig. 15.4) , granulomas, pustules, plaques, draining sinus, and cellulitis. because there are many skin manifestations in cryptococcosis that mimic other infectious as well as malignant conditions, skin biopsy with culture and histopathology are essential for definitive diagnosis. skin lesions of cryptococcosis usually represent disseminated cryptococcal infection. primary cutaneous cryptococcosis is very rare and is usually associated with skin injury and direct inoculation of the yeasts. solid organ transplant (sot) recipients on tacrolimus seem to be more likely to develop skin, soft-tissue, and osteoarticular infections due to cryptococcus [15] . tacrolimus has anti-cryptococcal activity at high temperatures, but loses this activity as environmental temperatures decrease; this may in part explain the increased frequency of cutaneous cryptococcosis in these patients. despite this series of patients, however, the most common site of disseminated infection in sot recipients still remains the cns, including patients receiving tacrolimus. prostatic cryptococcosis is usually asymptomatic, and the prostate gland is considered to be a sanctuary site for this yeast. the prostate may serve as an important reservoir for relapse of cryptococcosis in patients with a high fungal burden [16] . latent c. neoformans infection has even been recognized to disseminate to the bloodstream during urological surgery on the prostate [17] . cultures of urine or seminal fluid may still be positive for cryptococcus after initial antifungal treatment of cryptococcal meningitis in aids patients [18] , strongly supporting the need for prolonged antifungal treatment to clear the prostate in these severely immunocompromised patients. in the early reports of cryptococcal meningitis before the aids epidemic, ocular signs and symptoms were noted in approximately 45 % of cases [19] . the most common manifestations were ocular palsies and papilledema. in the present hiv era, several other manifestations of ocular cryptococcosis have been identified, including the presence of extensive retinal lesions with or without vitritis, which can lead to irreversible blindness. furthermore, catastrophic loss of vision without evidence for endophthalmitis has also been reported [20] . visual loss may be due to one of two pathogenic processes. the first is caused by infiltration of the optic nerve with the yeasts, producing rapid visual loss with few effective treatments. the second is due to increased icp and compression of the ophthalmic artery. in this setting, patients have slower visual loss and treatment with serial lumbar punctures or ventricular shunts can prevent or slow down visual loss. in addition to lung, cns, skin, prostate, and eye, c. neoformans can cause disease in many other organs (table 15 .4). cryptococcemia can occur in severely immunosuppressed patients but rarely causes endocarditis. bone involvement of cryptococcosis typically presents as one or more circumscribed osteolytic lesions in any bone of the body, occasionally associated with "cold" soft-tissue abscesses, and has been associated with sarcoidosis. bone marrow infiltration can be observed in severely immunocompromised hosts. cryptococcal peritonitis [21] and cryptococcuria are also reported in several case series. any organ of the human body can be a site of cryptococcal infections. there are several methods used for the diagnosis of cryptococcosis. these techniques include direct examination of the fungus in body fluids, histopathology of infected tissues, serological studies, and culture of body fluids or tissues. molecular methods, while available, are not currently used in routine clinical practice. the most rapid method for diagnosis of cryptococcal meningitis is direct microscopic examination for encapsulated yeasts by an india ink preparation of csf. cryptococcus can be visualized as a globular, encapsulated yeast cell with or without budding, ranging in size from 5 to 20 µm in diameter. it is easily distinguished in a colloidal medium of india ink when mixed with csf ( fig. 15.5 ). approximately 1-5 ml of specimen is recommended for use in the india ink preparation. india ink examination can detect encapsulated yeasts in a csf specimen with a threshold between 10 3 and 10 4 colony-forming units of yeasts/ml of fluid. the sensitivity of the india ink preparation technique is 30-50 % in non-aids-related cryptococcal meningitis and up to 80 % in aids-related disease. some false-positive results can be found from intact lymphocytes, myelin globules, fat droplets, and other tissue cells. also, dead yeast cells can remain in the csf and be visualized by india ink preparation for varying periods of time during and after appropriate antifungal treatment. this is a limitation of direct microscopy of csf during the management of cryptococcal meningitis [22] . cryptococcus can be identified by histological staining of tissues from lung, skin, bone marrow, brain, or other organs [23] . histopathological staining of centrifuged csf sediment is more sensitive for rapid diagnosis of cryptococcal meningitis than the india ink method [24] . peritoneal fluid from chronic ambulatory peritoneal dialysis, seminal fluid, bronchial wash, or bronchoalveolar lavage fluid can also be used for cytology preparations in the diagnosis of cryptococcal infections, whereas india ink preparations from these body fluids are difficult to interpret [25, 26] . fine needle aspiration for cytology of peripheral lymph nodes, adrenal glands, or vitreous aspiration; percutaneous transthoracic biopsy under real-time ultrasound guidance; or video-assisted thoracoscopic lung biopsy on pulmonary nodules, masses, or infiltrative lesions can be used for obtaining tissues for cytology/histopathology [27] . a variety of positive staining methods have been described to demonstrate the yeast cells in tissue or fluids, ranging from the nonspecific papanicolaou or hematoxylin and eosin stains to the more specific fungal stains such as calcofluor, which binds fungal chitin, or gomori methenamine silver (gms), which stains the fungal cell wall [2, 25] ( fig. 15.6 ). several stains can identify the polysaccharide capsular material surrounding the yeasts. these stains can be especially useful in presumptively identifying cryptococcus when the organism does not grow or cultures are not obtained. they include mayer's mucicarmine, periodic acid-schiff (pas), and alcian blue stains [2] . the fontana-masson stain appears to identify melanin in the yeast cell wall. the fungus is observed as a yeast that reproduces by formation of narrow-based budding with a prominent capsule. gram stain is not optimal for identification of this yeast, but may show diagnosis of cryptococcosis has improved significantly over the past several decades with the development of serological tests for cryptococcal polysaccharide antigen and/ or antibody. use of serum cryptococcal antibodies for diagnosis of cryptococcosis has not been adopted. in contrast, detection of cryptococcal capsular polysaccharide antigen in serum or body fluids by a latex agglutination (la) technique has been robust in its performance and is considered the gold standard diagnostic test for serological diagnosis of cryptococcosis. this test uses latex particles coated with polyclonal cryptococcal capsular antibodies or anti-gxm monoclonal antibodies and has overall sensitivities and specificities of 93-100 % and 93-98 %, respectively [28, 29] . the false-positive rate of cryptococcal capsular polysaccharide antigen testing is 0-0.4 % [30] . the majority of false-positive results can be explained by technical error (improper boiling/treatment), presence of rheumatoid factor or interference proteins, and infections with trichosporon beigelii [31] or some bacterial species [32] . however, most of the false-positive results of la testing for cryptococcal polysaccharide antigen have initial reciprocal titers of 8 or less [28] . therefore, results of such low titers must be carefully interpreted within the clinical context. falsenegative results of the la test for cryptococcal polysaccharide antigen in cryptococcal meningitis are unusual but can be seen due to a prozone effect, and, therefore, high-risk negative specimens should be diluted and retested [33] . low fungal burden, as in chronic low-grade cryptococcal meningitis or in the very early stages of cryptococcal infection, and improper storage of patient sera can also cause false-negative results in la cryptococcal polysaccharide antigen tests [34] . enzyme immunoassays (eias) for detection and quantification of cryptococcal polysaccharide antigen of all four serotypes of c. neoformans in sera and csf have been developed to detect the major component of the polysaccharide capsule, gxm, with sensitivities and specificities of 85.2-99 and 97 %, respectively [28, 35] . this methodology is automated and overcomes some of the practical limitations of la testing. previous studies have compared eia and la assays and revealed no significant difference between these testing methods. eia for cryptococcal polysaccharide antigen does not give discrepant results with rheumatoid factor or serum macroglobulins and is not affected by prozone reactions. both la and eia testing have been rigorously studied and are recommended for use in both serum and csf samples. recently, a lateral flow assay (lfa) was introduced in the diagnostic repertoire for cryptococcal infection and is food and drug administration (fda) approved for use in serum and csf. the semiquantitative lfa offers many advantages over other serological methods, including rapid turnaround (approximately 15 minutes), minimal requirements for specialized laboratory infrastructure, stability at room temperature, and low cost [36] . the lfa has been evaluated against both eia and culture, with sensitivities of 96-100 % for serum and plasma and 70-94 % for urine samples [36] [37] [38] [39] . this assay has good performance across a broad range of clinical settings, including resource-limited settings and among cohorts with low burden of hiv infection and high rates of c. gattii infection, for which some eia and la tests are known to be insensitive [36] [37] [38] [39] [40] . the satisfactory performance of lfa combined with established cost-effectiveness and practical advantages of this approach support its use as a point-of-care testing (including preemptive screening of high-risk patients) in resource-limited settings [36, 37, 41] . although the presence of cryptococcal polysaccharide antigen in serum is undoubtedly suggestive for dissemination of cryptococcal infection outside the lung, the precise value of cryptococcal polysaccharide antigen for diagnosis of nondisseminated pulmonary cryptococcosis remains less certain. generally speaking, detectable cryptococcal antigen in serum should make clinicians consider that infection is now also located outside the lung. in a high-risk patient with clinical symptoms suggestive of meningitis, identification of cryptococcal antigen in csf or serum is rapid, specific, noninvasive, and virtually diagnostic of meningoencephalitis or disseminated cryptococcosis even when the india ink examination or culture is negative [42, 43] . the la test for serum cryptococcal polysaccharide antigen is widely used for detecting cryptococcal infection in patients with aids, as an initial screening test for those with fever of unclear etiologies or neurological symptoms. in some patients, it may represent the only means of achieving an etiologic diagnosis of invasive cryptococcosis or early diagnosis prior to cns involvement. likely because of its sensitivity, the detection of cryptococcal polysaccharide antigen in the serum may precede clinically obvious disseminated cryptococcal disease ("isolated cryptococcal polysaccharidemia") in severely immunosuppressed patients [44] [45] [46] . the management of these cases, in which there is a positive serum antigen and other nonspecific clinical findings in hiv-infected patients with negative fluid or tissue cultures, is uncertain. persons of high risk with isolated cryptococcal antigenemia probably do benefit from antifungal therapy to prevent or delay the development of overt cryptococcosis [44] . generally, positive serum antigen tests at titers of 1:4 or more strongly suggest cryptococcal infections in these patients. baseline cryptococcal polysaccharide antigen titers in serum and csf may carry prognostic significance in patients with cryptococcal meningitis [47] . a study in hiv-related acute cryptococcal meningitis indicated that a baseline titer of csf cryptococcal polysaccharide antigen of 1:1024 or greater was a predictor of death during systemic antifungal treatment [48] . after initiation of systemic antifungal therapy, patients may respond to treatment and titers of cryptococcal polysaccharide antigen fall. similarly, a rise in csf cryptococcal polysaccharide antigen titers during suppressive therapy has been associated with relapse [49] . however, it is important to emphasize that the use of changing antigen titers to make therapeutic decision should be done with caution, as titers may not be equivalent across different serological modalities [39] . the kinetics of polysaccharide elimination remains unclear and, despite the accuracy of commercial kits for general diagnosis, the accuracy of specific titers can vary from kit to kit even from the same clinical specimen. cryptococcus can be easily grown from biologic samples such as csf, sputum, and skin biopsy on routine fungal and bacterial culture media. colonies can usually be observed on solid agar plates after 48-72 h of incubation at 30-35 °c in aerobic conditions. antibacterial agents, preferably chloramphenicol, can be added to the media when bacterial contamination is considered. the yeast, however, do not grow in the presence of cycloheximide at the con-centration used in selective fungal isolation media (25 µg/ ml). despite relatively rapid growth for most strains, cultures should be held for 3-4 weeks before discarding, particularly for patients already receiving antifungal treatment. conversely, cultures may be negative despite positive microscopic examinations (india ink) due to nonviable yeast cells, which may persist for a prolonged period of time at the site of infection. positive blood cultures are frequently reported in aids patients and may actually be the first positive test for cryptococcal infection in a febrile high-risk patient. c. neoformans colonies will appear on routine fungal media as opaque, white, creamy colonies that may turn orange-tan or brown after prolonged incubation. the mucoid appearance of the colony is related to the capsule size around the yeasts. cryptococcus does not routinely produce hyphae or pseudohyphae, or ferment sugars, but is able to assimilate inositol and hydrolyze urea [50] . c. neoformans and c. gattii have the ability to use galactose, maltose, galactitol, and sucrose [50] . there are special media such as canavanine-glycine-bromthymol blue (cgb) agar that can be used to differentiate c. gattii strains from c. neoformans strains [51] . a number of molecular techniques have been developed for identification of cryptococcal species from biological specimens including single and multiplex polymerase chain reaction (pcr) fingerprinting, random amplified polymorphic dna (rapd), pcr restriction fragment length polymorphism (rflp) analysis, multi-locus sequence typing (mlst), and matrix-assisted laser desorption ionization time-of-flight (maldi-tof) mass spectrometry [52] [53] [54] [55] [56] [57] [58] . these highly sensitive and specific methods have been evaluated with a variety of biologic samples [59] and can rapidly identify to the species and subspecies/genotypic level, including identification of recognized and novel strains within geographical niches [60] . while the expense and specialized techniques required of these methods preclude widespread use in clinical practice, their use in larger-scale investigations will continue to enhance our understanding of the epidemiology, pathogenesis, and nuances of antifungal management, as well as identify microevolution of different strains [61] . the infectious diseases society of america clinical practice guidelines for the management of cryptococcal disease (summarized in tables 15.5, 15.6), updated in 2010, provide a suitable framework for therapeutic decision making [62] . the updated guidelines provide detailed recommendations for specific "at-risk" populations and address different management strategies based on host, site of infection, and potential complications of cryptococcal infection. while subtle nuances exist based on host and site of infection, general principles for the management of cryptococcal infection can provide the cornerstone of a treatment plan in most cases. primary regimen ambd (0.7-1 mg/kg/day) plus flucytosine (5-fc; 100 mg/kg/day) 2 weeks liposomal amb (3-4 mg/kg/day) or amb lipid complex (ablc; 5 mg/kg/day) pls 5-fc(100 mg/kg/day) for patients predisposed to renal dysfunction 2 weeks alternative regimens b 4-6 weeks ambd (0.7-1 mg/kg/day) or liposomal amb c (3-4 mg/kg/day) or ablc (5 mg/kg/day) if flucytosine-intolerant 2 weeks ambd (0.7-1 mg/kg/day) plus fluconazole (800 mg/day) 6 weeks fluconazole (> 800 mg/day, preferably 1200 mg/day) plus 5-fc (100 mg/kg/day) 10 6-12 months ablc amb lipid complex, amb amphotericin b, 5-fc flucytosine a initiate haart 2-10 weeks after beginning antifungal regimen. shorter duration (i.e., 2 weeks) of induction therapy can be considered for certain low-risk patients b can be considered as alternative regimen in circumstances in which primary regimen not available but are not encouraged as equivalent substitutes c liposomal amphotericin can be safely administered in doses as high as 6 mg/kg/day d after 1 year of therapy, if successful response to arvs (cd4 count > 100 and viral load low or undetectable for > 3 months), discontinuation of antifungal therapy can be considered. consider reinstitution if cd4 count falls below 100 e consider stepwise de-escalation of immunosuppressive regimen if allograft function permits f if csf culture remains positive at 2 weeks of therapy or initial presentation with neurologic complications, longer therapy preferred g caution due to concomitant calcineurin inhibitor use amphotericin b deoxycholate (ambd) remains the foundation of treatment for disseminated cryptococcosis and severe cryptococcal infection. a standard induction dose of 0.7-1 mg/kg/day is recommended. liposomal amphotericin b (ambisome) at 3-6 mg/kg/day has become a preferred alternative treatment with similar outcomes to that of ambd but with less nephrotoxicity and is specifically recommended for primary induction in organ transplant patients as well as patients at risk for renal dysfunction [62] [63] [64] . higher doses of ambd have been shown to be more rapidly fungicidal [65, 66] . flucytosine (5-fc) is primarily used in combination therapy with ambd for first-line therapy in cryptococcal meningitis or severe pulmonary cryptococcosis at a dosage of 100 mg/kg/day in divided doses in patients with normal renal function [67, 68] . the combination of ambd and 5-fc represents the most potent fungicidal regimen with more rapid sterilization of csf cultures at 2 weeks as demonstrated by multiple studies [66, 67, 69] . early studies from hiv infection demonstrated increased rates of csf sterilization and fewer relapses with the combination of ambd and 5-fc followed by itraconazole maintenance [67] . this initial combination regimen has since been compared against multiple alternatives, with the superiority of its fungicidal activity consis-tently confirmed [69] . similar results have been observed among the most severe cases of cryptococcal infection [66, 70] . early mycological failure (as defined by persistently positive csf cultures at day 14) has for many years been associated with late treatment failure and poor outcome [71] , and lack of 5-fc has been independently associated with both early [72] and late [70] mycological failure. the improved fungicidal activity of combination therapy with ambd plus 5-fc has been shown to translate into a direct survival benefit compared with ambd monotherapy, with improved survival at 10 weeks lasting up to 6 months [66] . 5-fc should be dose-adjusted for renal dysfunction, with therapeutic monitoring performed 3-5 days after initiation of therapy, to maintain 2-h post-dose levels under 100 µg/ml (goal , to reduce its primary side effect of bone marrow suppression [73] . though combination induction therapy with ambd and 5-fc remains the recommended standard of care for severe cryptococcosis including cryptococcal meningitis, limited availability of 5-fc in resource-limited settings presents significant challenges for managing patients in areas where the disease burden and mortality rates are highest. alternative combination therapies have been investigated, the most efficacious of which has been ambd (0.7 mg/kg/day) plus fluconazole (800 mg/day), which has demonstrated improved rates of a composite end point of csf culture negativity, neurological improvement, and survival compared with ambd alone or in combination with lower doses of fluconazole [74] . fluconazole (at doses of 800-1200 mg/ day) in combination with ambd (standard dosing) has been shown to demonstrate similar rates of fungal clearance from csf as standard ambd plus 5-fc in a randomized study performed in hiv-infected patients in south africa [75] and offers a potential viable option for effective initial therapy in settings where access to 5-fc is limited. whether the survival benefit observed with ambd plus 5-fc will be observed with this regimen remains uncertain. additional alternative regimens for primary therapy are available in the guidelines but their use is not encouraged based on limited data on the success of these regimens [76] . use of fluconazole in the absence of a polyene is not recommended given the fungistatic nature of this drug, poor success, higher relapse rates, and increased resistance in relapse when used as monotherapy for induction [62, 77] . however, in areas without access to ambd, high doses (1200 mg/day) of fluconazole should be commenced. csf sampling should be performed to rule out cns involvement b csf sampling can be considered but not required in the absence of neurological symptoms or high serum cryptococcal antigen c if successful response to arvs (cd4 count > 100 and viral load low or undetectable for > 3 months) and stable serum cryptococcal antigen, can consider discontinuation of antifungal therapy after 12 months of therapy a three-stage regimen of induction/consolidation/maintenance is employed in the treatment of cryptococcal meningitis in all patients, irrespective of host risk factors [62, 67] . in hiv-infected patients, the initial induction treatment usually begins with combination therapy with ambd plus 5-fc for at least 2 weeks as above, followed by consolidation treatment with fluconazole 400-800 mg/day for 8 weeks in patients who have demonstrated favorable response. following consolidation, a long-term suppressive/maintenance phase is commenced with oral fluconazole, 200-400 mg given once daily. this has been demonstrated to effectively reduce rates of relapse from ~ 40 % to less than 5 % in the pre-haart era [78] . secondary prophylaxis can be discontinued after 1-2 years of antifungal therapy in patients who respond to haart with rise in cd4 + cell counts to greater than 100 cells/µl and decline in viral load (hiv rna) to undetectable levels for at least 3 months [62, 79, 80] . itraconazole can be used as an alternative consolidation treatment for cryptococcosis, but first-line therapy is with fluconazole. despite its poor csf penetration and inconsistent oral bioavailability, itraconazole has been successfully used in the treatment of cryptococcal meningitis [81] ; however, it has been shown to be inferior to fluconazole during the suppression phase [82] and requires therapeutic drug monitoring due to its poor bioavailability. newer triazoles including posaconazole and voriconazole are not specifically incorporated into practice guidelines but are active against cryptococcal isolates in vitro and have been shown to demonstrate moderate efficacy in patients with refractory disease [83, 84] . in patients with hiv-associated cryptococcal infection, haart has a major impact on long-term prognosis. however, given concerns regarding immune reconstitution inflammatory syndrome (iris), the optimum timing for haart initiation in the setting of ois has been a subject of much debate. early retrospective studies suggested an increased risk of iris among hiv-infected patients initiated on haart early after the diagnosis of an oi [85, 86] . more contemporary studies have demonstrated conflicting results regarding outcomes of cryptococcal infection based on timing of haart initiation [87] [88] [89] [90] [91] . the cryptococcal optimal art timing (coat) study provides the best evidence for current recommendations regarding timing of haart initiation in patients with cryptococcal meningitis [92] . haart-naïve patients were randomized to receive immediate (within 48 h) or deferred (greater than four weeks) haart following a minimum of 7 days of antifungal therapy with ambd and high-dose fluconazole. this trial was stopped early after interim analyses suggested poorer early survival among patients receiving immediate haart (55 % vs. 70 %, p = 0.03), particularly among patients with altered mentation and low csf white blood cell count. although a trend toward increased rates of and earlier iris was observed in the immediate haart group, this was not statistically significant. the above data support recommendations to delay initiation of haart in patients with cryptococcal meningitis for a minimum of 4 weeks after starting antifungal therapy (potentially longer if the primary regimen does not include ambd) and after demonstration of a sustained clinical response to antifungal therapy [62, 93] . interruption of haart and/or corticosteroid treatment may be used to control symptoms if severe cryptococcal iris occurs. organ transplant recipients with cns cryptococcal infection are managed similar to hiv-infected patients, with the exception of preferential use of lipid formulations of amphotericin b to limit nephrotoxicity [62] . the principles of induction, consolidation, and maintenance therapy remain the same. repeat csf sampling at 2 weeks is recommended in this population and a longer course of induction therapy should be pursued if csf cultures remain positive at 2 weeks, as this scenario is associated with increased 6-month mortality [94] . unlike hiv-infected patients, relapse rates among organ transplant recipients are quite low (~ 1.3 %), such that a shorter course of maintenance therapy with fluconazole (6-12 months) can be pursued following standard consolidation [62, 94] . drug interactions between fluconazole and immunosuppressive agents should be anticipated due to fluconazole-induced cyp3a4 inhibition, and preemptive adjustment (reduction) in calcineurin inhibitors should be made. management of immunosuppression in the setting of cryptococcal infection requires recognition of the increased risk of iris associated with abrupt withdrawal or reduction of immunosuppression in organ transplant recipients with increased rates of allograft loss reported in some patients [95] [96] [97] . stepwise reduction in immunosuppression is recommended, though the approach should be individualized for each patient. screening for hiv and cd4 lymphopenia is recommended among patients who present with cryptococcosis without apparent risk factors [62] . very little prospective data are available on the management of cryptococcal infection among this heterogeneous group of "apparently immunocompetent" patients lacking classical risk factors for cryptococcosis. what is known is based on early studies that included a heterogeneous mix of patients and were performed prior to acceptance of the standard algorithm of induction, consolidation, and maintenance therapy and higher-dose polyene therapy [68] . recommendations for longer induction therapy (4 weeks or more) in this population are based on the recognition of poorer outcomes and higher mortality rates in this group of patients in both early [68, 98] and contemporary [99] studies. an additional 2 weeks of therapy should be considered if 5-fc is not included in the induction regimen [62] . recommendations for consolidation and maintenance parallel those for hiv-infected and transplant patients, and reflect early reports of relapse rates approaching 30 % within the 1st year prior to introduction of consolidation and maintenance antifungal therapy [62, 68] . criteria for stopping treatment in these patients include resolution of symptoms, generally following at least 1 year of suppressive therapy. patients may have prolonged positive cryptococcal polysaccharide antigen tests and/or slightly abnormal csf findings for months during successful therapy, and if there are concerns about cure, follow-up csf culture should be considered. just as host factors influence management approaches for cryptococcal infection, site of infection also matters. airway colonization in a nonimmunosuppressed individual poses a low risk for invasive pulmonary infection (and dissemination) and treatment can be deferred. some experts would still favor treatment with fluconazole in this scenario, given the relative benign nature of this therapy. however, among immunocompromised patients with isolated pulmonary cryptococcosis, treatment is recommended to prevent dissemination [62] . it should be emphasized that a thorough evaluation to rule out systemic disease/dissemination is warranted in this group of patients to provide optimal treatment. this includes blood and csf cultures as well as serum and csf cryptococcal antigen testing. if the results of the above evaluation are negative, symptoms are mild, and there is no evidence of diffuse pulmonary infiltrates or ards, oral fluconazole (400 mg/day) is recommended for 6-12 months. however, in any patient in whom cryptococcemia is identified, symptoms are severe, ards is present, or csf examination reveals asymptomatic cns involvement, treatment for cryptococcal meningitis is recommended [62] . cerebral cryptococcomas often can be managed with prolonged antifungal therapy without need for surgical removal unless mass effect or other evidence of obstruction is identified. at least 6 weeks of induction therapy with ambd plus 5-fc, followed by 6-18 months of consolidation therapy with fluconazole (400-800 mg/day), is recommended for management. surgery should be considered for large lesions (> 3 cm) or the presence of obstructive hydrocephalus [62] . localized infection of extrapulmonary nonmeningeal sites can occasionally occur with direct inoculation, but more commonly represents disseminated infection. suspicion for the latter must be maintained when cryptococcus is identified from a sterile body site, as management strategies will differ if disseminated disease is present. consultation with ophthalmology is indicated in cases of cryptococcal eye disease [62] . restoration of pathogen-specific immunity as a result of haart or following reduction of immunosuppression in sot recipients can result in a destructive inflammatory response known as the immune reconstitution inflammatory syndrome (iris). iris is best characterized in association with c. neoformans infection of the cns, particularly among hiv-infected patients, and is associated with significant morbidity and mortality [85, 86, 88, 89, [100] [101] [102] [103] [104] [105] [106] [107] [108] . proposed criteria for iris include onset of symptoms within 12 months of haart initiation (with concomitant cd4 + recovery) [109] . in addition, iris is estimated to occur in 5-11 % of sot recipients with cryptococcal infection and has been associated with an increased risk of allograft failure [95, [110] [111] [112] [113] [114] ; cryptococcal iris may also be observed in non-hiv-infected, nontransplant patients [115] . clinical features of cryptococcal iris are similar to cryptococcal infection, most commonly presenting as cns disease, although lymphadenitis, pneumonitis, multifocal disease, soft-tissue involvement, and mediastinitis have all been reported [109, 116] . meningeal disease is the most frequent and most serious presentation [109] ; aseptic meningitis with associated intracranial hypertension and csf pleocytosis is most commonly observed [100, 102, 103, 105, 106, 108] . a hallmark histopathologic finding is suppurative or necrotic granulomatous inflammation with yeast seen in tissues despite negative tissue cultures [95, 112, 116, 117] . the presence of a positive csf culture in cases of suspected cryptococcal iris should raise suspicion for direct antifungal failure or resistance, particularly in settings where fluconazole therapy is widely used as the standard of care [88] . cryptococcal iris represents unchecked reversal of a th2 (anti-inflammatory) to th1 (pro-inflammatory) immune response in the setting of immune reconstitution [118] . prospective cohort studies of haart-naïve individuals indicate that an ineffectual host immune response to initial infection is associated with a greater likelihood of future iris [105] . a three-phase theory of cryptococcal immune reconstitution has been postulated, marked by: (1) failure of antigen clearance due to inappropriate th2 response; (2) lack of effector response despite inflammatory signaling; and, ultimately, (3) vigorous pro-inflammatory responses (both th1 and th17) to residual antigen, recognized clinically as iris [100] . there are no reliable diagnostic tests for iris, and establishing the diagnosis presents a considerable challenge [101, 119] . the differential diagnosis includes progressive disease due to persistent immune deficiency, failure of antimicrobial therapy (due to resistance or nonadherence), coinfection with other ois, and drug toxicity. a high index of suspicion is necessary for recognizing atypical presentations or manifestations at distant sites. nevertheless, distinguishing between disease progression related to ongoing immune deficiency and clinical deterioration due to restoration of host immunity has important management implications. csf analyses and biomarkers may be useful in distinguishing between relapse and iris. prospective studies have demonstrated that csf opening pressure [89] and wbc count [100, 105] at the time of an iris event are significantly higher than baseline values for individual patients, and higher csf opening pressures can distinguish iris from relapsed infection [102] . treatment options for cryptococcal iris are based largely on expert opinion [62] . implicit in management is ensuring the efficacy of antifungal therapy, particularly in settings where access to ambd may be limited and fluconazole resistance may account for recurrent meningitis episodes [120, 121] . in the absence of disease relapse or direct antifungal resistance, modification of antimicrobial therapy is not indicated [62] . once the diagnosis of iris is suspected, consideration of disease severity is warranted. a significant proportion of minor cases will improve without specific treatment [86, 88, 108] . corticosteroids have been shown to reduce the need for hospitalization and to improve short-term quality of life and functional status without increased risk of complications in paradoxical tuberculosis (tb)-associated iris [122] ; the role of corticosteroids in cryptococcal iris, however, is not as well established and should be reserved for life-threatening cases, particularly in light of their association with increased mortality in one study [123] . other antiinflammatory agents have been used in cryptococcal iris, but the number of patients treated with any of these agents is too small to draw substantive conclusions [86, 124, 125] . other management strategies, including therapeutic lumbar drainage in the setting of intracranial hypertension [62, 122, 126] and, at times, surgical drainage of suppurative lymph nodes [116, 117] , are important adjunctive therapies that may be considered in severe disease. although no controlled studies have been performed, continuation of haart in the setting of iris is recommended and has been performed safely without adverse effects in several studies [87, 88, 103, 119, 127] . similarly, withdrawal or reduction of immunosuppressive agents is standard practice in managing infectious complications in sot recipients [111] . given the putative risk of iris with abrupt withdrawal or discontinuation of immunosuppressive agents in these patients, gradual de-escalation during the initiation of antifungal therapy is advised to reduce the risk of future iris [95, 111, 112] . persistent and relapsed infection must be distinguished from iris, as management strategies will differ significantly. persistent disease can be defined as persistently positive csf cultures after 1 month of antifungal therapy, whereas relapse requires new clinical signs and symptoms and repeat positive cultures (at same or distant sites) after initial improvement and fungal sterilization [62] . surrogate markers, including biochemical parameters, india ink staining, and cryptococcal antigen titers, are insufficient to define relapse or alter antifungal therapy. general recommendations for management in these cases include resumption of induction therapy, often for a longer duration and at increased dosages, if tolerable, and pursuance of antifungal susceptibility testing [62] . while routine in vitro susceptibility testing of cryptococcal isolates at the time of initial therapy is not recommended, there is a role for testing in cases of suspected relapse or persistent infection [62] . it is generally recognized that primary antifungal resistance to most agents is rare, although reduced susceptibility to 5-fc has been observed in untreated patients [128] and echinocandins have no reliable activity against this yeast. reduced susceptibility to fluconazole has been described in cases of culture-positive relapsed cryptococcal meningitis associated with prior fluconazole therapy [77, 129, 130] (table 15 .6). along with the optimization of antifungal therapy, management of increased icp is critically important. elevated icp is correlated with overall fungal burden, and is thought to be due to csf outflow obstruction by clumped yeast forms [131] . an icp of 250 mm h 2 o or greater is considered elevated and is associated with increased morbidity and mortality [123] . persistently elevated icp after 2 weeks of treatment is associated with poorer clinical responses among patients with hiv-associated cryptococcal meningitis [62] . intracranial imaging should be performed prior to lumbar puncture if impaired mentation or focal neurologic deficits are present. a baseline measurement of csf pressure should be obtained in all patients with suspected cryptococcal meningitis. aggressive attempts to control increased icp should occur, if elevated and if there are signs/symptoms to suggest increased icp (headache, mental status changes, and new focal neurological findings). treatment options for managing acutely elevated icp include repeated lumbar punctures (daily until pressure and symptoms are stable for > 2 days), lumbar drain insertion, ventriculostomy, or ventriculoperitoneal (vp) shunt (table 15 .7) [123] . medical treatments such as corticosteroids (unless there is a component of iris), mannitol, and acetazolamide have been used in some cases, but are generally not recommended for use in management of increased icp in cryptococcal meningitis [132] . some patients may develop symptoms of obstructive hydrocephalus necessitating the placement of a permanent vp shunt during the first 1-2 years of treatment, and occasionally at initial presentation. sterilization of csf is not required prior to placement of a vp shunt, which can be inserted once a patient is receiving the appropriate antifungal therapy [133] . prevention of cryptococcal disease can be achieved by use of haart in hiv-infected patients. fluconazole prophylaxis has been shown to be effective for preventing cryptococcosis in aids patients with persistently low cd4 + cell counts (below 100 cells/µl) [134, 135] , but due to concerns regarding antifungal resistance, this approach is not currently recommended, and haart remains the best strategy for the prevention of cryptococcal disease in this population. routine screening for cryptococcal infection and/or prophylaxis are not recommended in sot recipients, even when immunosuppression is augmented in patients with previously (appropriately) treated infection [136] . although cryptococcal gxm-tetanus toxoid conjugate vaccine and specific monoclonal antibodies to cryptococci have been developed, clinical trials have not been initiated to determine their usefulness in human subjects [137, 138] . one hundred years ago: the history of cryptococcosis in greifswald. medical mycology in the nineteenth century cryptococcus neoformans proposal to conserve the name cryptococcus gattii against c. hondurianus and c. bacillisporus cryptococcosis in the era of aids-100 years after the discovery of cryptococcus neoformans a rare genotype of cryptococcus gattii caused the cryptococcosis outbreak on vancouver island (british columbia, canada) estimation of the current global burden of cryptococcal meningitis among persons living with hiv/aids cryptococcosis: population-based multistate active surveillance and risk factors in human immunodeficiency virus-infected persons. cryptococcal active surveillance group genetics of cryptococcus neoformans dolin r, editors. mandell, douglas, and bennett's principles and practice of infectious diseases epidemiology and host-and variety-dependent characteristics of infection due to cryptococcus neoformans in australia and new zealand. australasian cryptococcal study group cryptococcus neoformans: a sugar-coated killer with designer genes the wide spectrum of cryptococcal infections the spectrum of pulmonary cryptococcosis clinical spectrum of invasive cryptococcosis in liver transplant recipients receiving tacrolimus richman dd. persistent cryptococcus neoformans infection of the prostate after successful treatment of meningitis. california collaborative treatment group disseminated cryptococcosis after transurethral resection of the prostate persistence of cryptococcus neoformans in seminal fluid and urine under itraconazole treatment. the urogenital tract (prostate) as a niche for cryptococcus neoformans ophthalmologic complications of cryptococcal meningitis catastrophic visual loss due to cryptococcus neoformans meningitis cryptococcus neoformans peritonitis in a patient with alcoholic cirrhosis: case report and review of the literature prognostic factors in cryptococcal meningitis. a study in 111 cases histopathology of cryptococcosis and other fungal infections in patients with acquired immunodeficiency syndrome rapid diagnosis of cryptococcal meningitis by microscopic examination of centrifuged cerebrospinal fluid sediment cytologic diagnosis of cryptococcus neoformans in hiv-positive patients utility of bronchoscopic sampling techniques for cryptococcal disease in diagnosis of pulmonary cryptococcosis by ultrasound guided percutaneous aspiration comparison of commercial kits for detection of cryptococcal antigen comparison of three commercial cryptococcal latex kits for detection of cryptococcal antigen detection of cryptococcal antigen. comparison of two latex agglutination tests detection of a trichosporon beigelii antigen cross-reactive with cryptococcus neoformans capsular polysaccharides in serum from a patient with disseminated trichosporon infection cross-reactivity between stomatococcus mucilaginosus and latex agglutination for cryptococcal antigen false-negative cryptococcal antigen test detection of cryptococcus neoformans antigen in body fluids by latex particle agglutination comparison of the premier cryptococcal antigen enzyme immunoassay and the latex agglutination assay for detection of cryptococcal antigens evaluation of a novel pointof-care cryptococcal antigen test on serum, plasma and urine from patients with hiv-associated cryptococcal meningitis evaluation of a newly developed lateral flow immunoassay for the diagnosis of cryptococcus clinical utility of the cryptococcal antigen lateral flow assay in a diagnostic mycology laboratory comparison of four assays for the detection of cryptococcal antigen large-scale evaluation of the immuno-mycologics lateral flow and enzyme-linked immunoassays for detection of cryptococcal antigen in serum and cerebrospinal fluid cost effectiveness of cryptococcal antigen screening as a strategy to prevent cryptococcal meningitis in south africa infections with cryptococcus neoformans in the acquired immunodeficiency syndrome cryptococcal meningitis in non-hiv-infected patients serum cryptococcal antigen in patients with aids systematic screening of cryptococcal antigenemia in hiv-positive adults in uganda screening for cryptococcal antigenemia in patients accessing an antiretroviral treatment program in south africa serology of human cryptococcosis comparison of amphotericin b with fluconazole in the treatment of acute aids-associated cryptococcal meningitis. the niaid mycoses study group and the aids clinical trials group measurement of cryptococcal antigen in serum and cerebrospinal fluid: value in the management of aids-associated cryptococcal meningitis philadelphia: churchill livingstone the biochemical basis for the distinction between the two cryptococcus neoformans varieties with cgb medium cryptococcus species identification by multiplex pcr development of a singleplex pcr assay for rapid identification and differentiation of cryptococcus neoformas var. grubii, cryptococcus neoformans var. neoformans, cryptococcus gattii, and hybrids identification by random amplification of polymorphic dna (rapd) of a common molecular type of c. neoformans var. neoformans in patients with aids or other immunosuppressive conditions simultaneous identification of molecular and mating types within the cryptococcus species complex by pcr-rflp analysis consensus multi-locus sequence typing scheme for cryptococcus neoformans and cryptococcus gattii maldi-tof ms enables the rapid identification of the major molecular types within the cryptococcus neoformans/c. gattii species complex matrixassisted laser desorption ionization-time of flight mass spectrometry-based method for discrimination between molecular types of cryptococcus neoformans and cryptococcus gattii detection of cryptococcus by conventional and molecular methods multilocus sequence typing reveals three genetically distinct subpopulations of cryptococcus neoformans var. grubii (serotype a), including a unique population in botswana genotyping and antifungal susceptibility testing of cryptococcus neoformans isolates from cameroonian hiv-positive adult patients clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious disease society of america liposomal amphotericin b (ambisome) compared with amphotericin b both followed by oral fluconazole in the treatment of aids-associated cryptococcal meningitis comparison of 2 doses of liposomal amphotericin b and conventional amphotericin b deoxycholate for treatment of aids-associated acute cryptococcal meningitis: a randomized, double-blind clinical trial of efficacy and safety high-dose amphotericin b with flucytosine for the treatment of cryptococcal meningitis in hiv-infected patients: a randomized trial combination antifungal therapy for cryptococcal meningitis treatment of cryptococcal meningitis associated with the acquired immunodeficiency syndrome. national institute of allergy and infectious diseases mycoses study group and aids clinical trials group treatment of cryptococcal meningitis with combination amphotericin b and flucytosine for four as compared with six weeks combination antifungal therapies for hiv-associated cryptococcal meningitis: a randomised trial major role for amphotericin-flucytosine combination in severe cryptococcosis early mycological treatment failure in aids-associated cryptococcal meningitis determinants of disease presentation and outcome during cryptococcosis: the crypto a/d study antimicrobial therapy and vaccines a phase ii randomized trial of amphotericin b alone or combined with fluconazole in the treatment of hiv-associated cryptococcal meningitis comparison of the early fungicidal activity of high-dose fluconazole, voriconazole, and flucytosine as second-line drugs given in combination with amphotericin b for the treatment of hiv-associated cryptococcal meningitis combination flucytosine and high-dose fluconazole compared with fluconazole monotherapy for the treatment of cryptococcal meningitis: a randomized trial in malawi symptomatic relapse of hiv-associated cryptococcal meningitis after initial fluconazole monotherapy: the role of fluconazole resistance and immune reconstitution a placebo-controlled trial of maintenance therapy with fluconazole after treatment for cryptococcal meningitis in the acquired immunodeficiency syndrome discontinuation of secondary prophylaxis for cryptococcal meningitis in human immunodeficiency virus-infected patients treated with haart: a prospective, multicenter, randomized study discontinuation of maintenance therapy for cryptococcal meningitis in patients with aids treated with haart: an international observational study itraconazole therapy for cryptococcal meningitis and cryptococcosis a comparison of itraconazole versus fluconazole as maintenance therapy for aidsassociated cryptococcal meningitis. national institute of allergy and infectious diseases mycoses study group voriconazole treatment for less-common, emerging or refractory fungal infections activity of posaconazole in the treatment of central nervous system fungal infections incidence and risk factors for immune reconstitution inflammatory syndrome during haart incidence and risk factors of immune reconstitution inflammatory syndrome complicating hiv-associated cryptococcosis in france early haart reduces aids progression/death in individuals with acute opportunistic infections: a multicenter randomized strategy trial immune reconstitution inflammatory syndrome in hiv-associated cryptococcal meningitis: a prospective study cryptococcal immune reconstitution inflammatory syndrome after haart in aids patients with cryptococcal meningitis: a prospective multicenter study mbuagbaw l optimal timing for haart initiation in patients with hiv infection and concurrent cryptococcal meningitis early versus delayed initiation of haart for concurrent hiv infection and cryptococcal meningitis in sub-saharan africa haart initiation within the first 2 weeks of cryptococcal meningitis is associated with higher mortality: a multisite randomized trial world health organization. rapid advice: diagnosis, prevention and management of cryptococcal disease in hiv-infected adults, adolescents and children. geneva: world health organization antifungal management practices and evolution of infection in organ transplant recipients with cryptococcus neoformans infection allograft loss in renal transplant recipients with cryptococcus neoformans associated immune reconstitution syndrome an immune reconstitution syndrome-like illness associated with cryptococcus neoformans infection in organ transplant recipients cellulitis revealing a cryptococcosis-related immune reconstitution inflammatory syndrome in a renal allograft recipient a comparison of amphotericin b alone and combined with flucytosine in the treatment of cryptococcal meningitis comparison of temporal trends of three groups with cryptococcosis: hivinfected, solid organ transplant and hiv-negative/non-tranpslant clinical features and serum biomarkers in hiv immune reconstitution inflammatory syndrome after cryptococcal meningitis: a prospective cohort study defining immune reconstitution inflammatory syndrome: evaluation of expert opinion versus 2 case definitions in a south african cohort the role of immune reconstitution inflammatory syndrome in aids-related cryptococcus neoformans disease in the era of haart timing of cryptococcal immune reconstitution inflammatory syndrome after haart in patients with aids and cryptococcal meningitis outcomes of cryptococcal meningitis in uganda before and after the availability of haart paucity of initial cerebrospinal fluid inflammation in cryptococcal meningitis is associated with subsequent immune reconstitution inflammatory syndrome immune reconstitution inflammatory syndrome (iris) associated with cryptococcus neoformans infection in aids patients intramedullary abscess resulting from disseminated cryptococcosis despite immune restoration in a patient with aids cryptococcal immune reconstitution inflammatory syndrome: report of four cases in three patients and review of the literature cryptococcal immune reconstitution inflammatory syndrome in hiv-1-infected individuals: proposed clinical case definitions th17 cells and il-17 receptor signaling are essential for mucosal host defense against oral candidiasis opportunistic infection-associated immune reconstitution syndrome in transplant recipients cellulitis revealing a cryptococcosis-related immune reconstitution inflammatory syndrome in a renal allograft recipient immune reconstitution syndrome after voriconazole treatment for cryptococcal meningitis in a liver transplant recipient novel immune regulatory pathways and their role in immune reconstitution syndrome in organ transplant recipients with invasive mycoses the poor prognosis of central nervous system cryptococcosis among nonimmunosuppressed patients: a call for better disease recognition and evaluation of adjuncts to antifungal therapy mediastinitis due to cryptococcal infection: a new clinical entity in the haart era hiv combination therapy: immune restitution causing cryptococcal lymphadenitis dramatically improved by anti-inflammatory therapy immunological profiles of immune restoration disease presenting as mycobacterial lymphadenitis and cryptococcal meningitis tuberculosis-associated immune reconstitution inflammatory syndrome: case definitions for use in resource-limited settings symptomatic relapse of hiv-associated cryptococcal meningitis after initial fluconazole monotherapy: the role of fluconazole resistance and immune reconstitution independent association between rate of clearance of infection and clinical outcome of hiv-associated cryptococcal meningitis: analysis of a combined cohort of 262 patients randomized placebocontrolled trial of prednisone for paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome diagnosis and management of increased intracranial pressure in patients with aids and cryptococcal meningitis. the niaid mycoses study group and aids cooperative treatment groups cryptococcal immune reconstitution syndrome during steroid withdrawal treated with hydroxychloroquine treatment of hiv-related inflammatory cerebral cryptococcoma with adalimumab paradoxical immune reconstitution inflammatory syndrome associated with previous cryptococcus neoformans infection in an hiv-positive patient requiring neurosurgical intervention aids: immune reconstitution inflammatory syndrome: a reappraisal flucytosine primary resistance in candida species and cryptococcus neoformans correlation of in vitro azole susceptibility testing with in vivo response in a murine model of cryptococcal meningitis correlation of fluconazole mics with clinical outcome in cryptococcal infection elevated cerebrospinal fluid pressures in patients with cryptococcal meningitis and acquired immunodeficiency syndrome a randomized, double-blind, placebo-controlled trial of acetazolamide for the treatment of elevated intracranial pressure in cryptococcal meningitis treatment of hydrocephalus secondary to cryptococcal meningitis by use of shunting primary prophylaxis with fluconazole against systemic fungal infections in hiv-positive patients a multicentre, randomized, double-blind, placebo-controlled trial of primary cryptococcal meningitis prophylaxis in hiv-infected patients with severe immune deficiency cryptococcosis in solid organ transplant recipients: current state of the science cryptococcus neoformans serotype a glucuronoxylomannan protein conjugate vaccines: synthesis, characterization, and immunogenicity therapeutic efficacy of monoclonal antibodies to cryptococcus neoformans glucuronoxylomannan alone and in combination with amphotericin b. antimicrob agents chemother suggested reading casadevall a, perfect jr. cryptococcus neoformans combination antifungal therapy for cryptococcal meningitis large-scale evaluation of the immuno-mycologics lateral flow and enzyme-linked immunoassays for detection of cryptococcal antigen in serum and cerebrospinal fluid a rare genotype of cryptococcus gattii caused the cryptococcosis outbreak on vancouver island (british columbia, canada) evaluation of a newly developed lateral flow immunoassay for the diagnosis of cryptococcus cryptococcosis in the era of aids-100 years after the discovery of cryptococcus neoformans discontinuation of maintenance therapy for cryptococcal meningitis in patients with aids treated with haart: an international observational study dolin r, editors. mandell, douglas, and bennett's principles and practice of infectious diseases clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious disease society of america measurement of cryptococcal antigen in serum and cerebrospinal fluid: value in the management of aids-associated cryptococcal meningitis comparison of amphotericin b with fluconazole in the treatment of acute aids-associated cryptococcal meningitis. the niaid mycoses study group and the aids clinical trials group key: cord-260071-z29b30sd authors: zhong, yu; yoshinaka, yoshiyuki; takeda, tadahiro; shimizu, noriko; yoshizaki, sayaka; inagaki, yoshio; matsuda, shinobu; honda, gisho; fujii, nobutaka; yamamoto, naoki title: highly potent anti-hiv-1 activity isolated from fermented polygonum tinctorium aiton date: 2005-03-27 journal: antiviral res doi: 10.1016/j.antiviral.2005.02.003 sha: doc_id: 260071 cord_uid: z29b30sd a water-soluble extract of fermented polygonum tinctorium aiton (polygonaceae) called sukumo, exhibited a potent inhibitory activity against hiv type 1 in vitro. the extract potently suppressed acute hiv-1 (iii(b)) infection in mt-4 cells with ec(50) values of 0.5 μg/ml but exhibited low cytotoxicity to mt-4 cells even at a high concentration (cc(50) > 1000 μg/ml). it also inhibited giant cell formation in co-cultures of hiv-infected cells and uninfected molt-4 cells. sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. there was a good correlation between the extract's anti-hiv-1 activity and its inhibitory effects on hiv-1 binding. it also suppressed replication of herpes simplex virus type 1 in vero cells with an ec(50) of 11.56 μg/ml. on the other hand, there was no appreciable activity against influenza a virus, poliovirus or sars corona virus when tested at concentrations ranging from 3.2–400 μg/ml as shown by microscopic image analysis for cytopathic effect (cpe). physico-chemical studies revealed that the anti-hiv activity in the extract was essentially maintained after boiling at 100 °c in 1n hcl or 1n naoh, and after treatment with 100 mm naio(4). the inhibitory activity of the extract was also not reduced after pronase digestion. the active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of 10,000–50,000. one of the logical targets of the viral life cycle at which to inhibit hiv-1 replication is the step in the process where the infectious virion enters its host cell (moore and stevenson, 2000; lin et al., 2002) . therefore, the identification of hiv entry inhibitors, which can serve as novel anti-hiv drugs, is urgently needed. retroviral infection is initiated by the attachment of the virion to the cell surface, which even occurs before glycoproteins on the viral envelope interact with specific receptors on the host cell to trigger fusion. a great variety of polyanionic compounds have been described which act as virus adsorption inhibitors. this class of compounds also comprises the cosalane analogues, containing the polycarboxylate pharmacophore, as well as the sulfated polysaccharides extracted from sea algae (nakashima et al., 1987a (nakashima et al., , 1992 santhosh et al., 2001; witvrouw and de clercq, 1997) . all of these compounds are assumed to exert their anti-hiv activity by shielding the positively charged sites in the v3 loop region of the viral gp120 envelope glycoprotein, and interrupting virus attachment to the negatively charged heparan sulfate proteoglycans on cell surface, and inhibiting the specific binding to the cd4 receptor of cd4 + cells. some of these compounds can also interfere with later events in receptor-mediated fusion by virtue of attachment to gp120. these compounds probably do not penetrate into cells because of their mass and highly anionic charge, but rather, act as antiviral agents by impeding the attachment and subsequent entry of virus particles into the cell. a number of sulfated polysaccharides, including dextran sulfate and heparin, have been reported to have potential as antiviral drugs, since they inhibit the replication of a variety of viruses in vitro (baba et al., 1988; bartolini et al., 2003; nakashima et al., 1989; ylisastigui et al., 2000) . the extent of inhibition appeared to be dependent on both the viral strain and host cell type. dextran sulfate interferes with the association of gp120 with cxcr4 while having no detectable effect on gp120-cd4. the interaction between polyanions and x4 or x4r5 gp120 was readily detectable, whereas weak or undetectable binding was observed with r5 gp120 (moulard et al., 2000) . cosalanes inhibited the binding of gp120 to cd4 as well as the fusion of the viral envelope with the cell membrane and is more potent against r5 hiv-1 rf in cem-ss cells than against vs x4 hiv-1 iiib in mt-4 cells (santhosh et al., 2001) . polygonum tinctorium has been used extensively in chinese and japanese folk medicine for the treatment of many infectious diseases and is believed to have effects such as detoxification, anti-pyrexia and anti-nociception. extracted constituents of this medicinal plant, such as tryptanthrin, has been shown to possess anti-fungal, cancer chemopreventive and anti-bacterial activities (honda and tabata, 1979; koya-miyata et al., 2001; kataoka et al., 2001; miyake et al., 2003) , while pigment (ptp) has an anti-anaphylactic activity (kim et al., 1998) . in this study, we report for the first time the potent anti-hiv-1 and hsv-1 activity of an aqueous extract from the fermented leaves of polygonum tinctorium (sukumo). this extract was found to be highly selective against hiv-1 and hsv-1 in vitro. sukumo extract suppresses production of hiv-1 by inhibiting the viral entry process through binding to the virus envelope and thus preventing hiv-induced syncytium formation with an exceedingly broad therapeutic window. based on the results of physico-chemical analysis of the anti-viral active factor, it is putatively a novel polyanionic high-molecular-weight compound containing a phenolic substructure in aqueous extract of sukumo. sukumo was collected from the leaves of polygonum tinctorium (tokushima, japan) and fermented for 3 months, which was provided and identified by dr. matsuda. voucher specimens were deposited at the institute of hemorheological function of food co. ltd., hyogo, japan. sukumo powder (100 g) was refluxed three times with 99.9% ethanol, then with water (1 l). the aqueous solution was clarified by filtering through a 0.2 m filter. the high-molecular compounds were precipitated from the aqueous extracts of sukumo by 66.6% ethanol, which were collected by centrifugation (10,000 rpm, 30 min) in a yield of 26.8% (26.8 g). anti-hiv activity of the sukumo extract was tested and stored at 4 • c before use. mt-4, molt-4 cells and molt-4 cells chronically infected with the hiv-1 (iii b ) strain (molt-4/iiib) were cultured in rpmi-1640 medium supplemented with 10% fetal bovine serum (fbs) (cansera international inc., canada) and antibiotics (100 g/ml penicillin/100 g/ml streptomycin). 293t, vero and stably expressing cd4-ccr5 of hos cells were maintained in dulbecco's modified eagle's medium (dmem) containing the same supplements. x4/hiv-1 (iii b ) was prepared by propagation in molt-4/iiib cells. hiv-1 molecular clones of the x4 hiv-1 strain nl4-3 and the r5 hiv-1 strain jrcsf were prepared by transfection of 293t cells with nl4-3 or jrcsf plasmids carrying full-length proviral dna. the culture supernatants were clarified by 0.45 m filters and frozen at −80 • c. herpes simplex virus type 1 was propagated in vero cells (kindly provided by dr. shaku). cell-free virus stock was prepared by sonication of hsv-infected vero cells in 9% skim milk and stored at −80 • c until use. to determine the anti-hiv-1 activity and cytotoxicity of the sukumo extract, mt-4 cells were either infected with hiv-1 (iii b ) strains at a multiplicity of infection (moi) of 0.01 or un-infected (mock infection). cell viability was quantified with mtt (dojindo, kumamoto, japan) assay for mt-4 cells. ec 50 values were calculated in infected cells for the anti-hiv-1 effect and cc 50 values were calculated in un-infected cells for drug cytotoxicity (ichiyama et al., 2003) . peripheral blood mononuclear cells (pbmcs) from hiv-1-seronegative donors were isolated by ficoll-hypaque density gradient centrifugation. pha (1 g/ml, sigma-aldrich)/il-2 (100 u/ml, shionogi, osaka, japan) activated pbmcs were infected for 2 h with 20 ng of hiv-1 p24 gag (x4/nl4-3 or r5/jrcsf) in the presence or absence of the sukumo extract (0.64-400 g/ml), washed three times with pbs and cultured for 7 days in rpmi-1640 medium/10% fbs plus 100 u/ml il-2 with or without the sukumo extract. hiv-1 p24 gag of culture supernatant was determined by automated enzyme-linked immunosorbent assay (eilsa) (fuji rebio inc., tokyo, japan). in this assay the p24 antigen from zeptometrix (buffalo, new york) was used as the standard. chronically hiv-1 (iii b )-infected molt-4 cells were co-cultured with hiv non-infected molt-4 cells (ratio = 1:1) at 37 • c for 1 day in the presence of a test compound at graded concentrations. cell-cell fusion was analyzed by confocal microscopic assessment of syncytium formation. sukumo extract were also tested as an inhibitor of hsv-1 replication in vero cells using a standard plaque assay. two hour after treatment with sukumo extract, vero cells were subjected to a 1-h infection with about 50 pfu of hsv-1 in the absence or presence of serially diluted sukumo extract and then cultured with 199 medium supplemented with 1% fbs, human ␥-globulins (164 g/ml, sigma-aldrich) and antibiotics, with or without compounds cultured for 4 days. the cells were stained with giemsa solution. the numbers of viral plaques were calculated as a percentage of the tested control in order to determine the percent inhibition. the ec 50 value is the concentration of compound that inhibits viral replication by 50% relative to control. human mt-4 cells (4 × 10 5 ) were suspended in fresh medium in the presence or absence of various concentrations of sukumo extract at 37 • c for 1 h. after washing, the cells were incubated with hiv-1 nl4-3 (200 ng of p24 gag) for 2 h on ice or at 37 • c in the absence or presence of extract. the cells were washed with pbs/2% fbs and the pellet resuspended with 500 l of lysis buffer (pbs containing 5% tritonx-100 and 1% bsa). levels of p24 gag were quantified by an automated elisa system. mt-4 or hos/ccr5 cells (5 × 10 5 ) were pretreated with normal human igg (zymed, south san francisco) at 0.1 mg/ml in pbs containing 2% fbs buffer for 30 min on ice to block the fc receptors and then were treated by anti-cxcr4 antibodies (5 g/ml, 12g5. r&d systems inc.) or anti-ccr5 antibodies (3.5 g/ml, 2d7. biosciences-pharmingen, san diego) in the presence or absence of sukumo extract at 37 • c for 2 h. cells were washed with pbs/2% fbs and strained with fitc-conjugated anti-mouse igg (0.02 mg/ml, american qualex) for 30 min on ice. pretreated mt-4 cells also were stained with fitc-conjugated anti-human cd4 or monoclonal mouse antibodies of isotype igg (negative control for flow cytometry) (1:50 dilution, dako). the cells were fixed in 1% paraformaldehyde-pbs solution and analyzed on facs calibur (becton dickinson), a flow cytometer with cellquest software (becton dickinson). pseudotyping vesicular stomatitis virus protein g (vsv-g) onto hiv cores from an env-defective reporter virus was carried out as follows. plasmid dna (10 g) encoding envelope from vsv-g was co-transfected with pnl-e env(−) nef(−) (20 g) into 293t cells using the calcium phosphate method. the virus titer was determined based on the level of p24 gag. time course assays were conducted to determine which steps in viral infection (entry and post-entry) were inhibited by sukumo extract. three treatment schedules were applied for hiv-1 infection with 293t cells, which were infected with pseudotyped hiv-1/vsv-g viruses (5 ng/ml of p24 gag). the amount of p24 gag in culture supernatant was determined to assess hiv-1 replication. sukumo extract was used at serial concentrations in a range of 0.16-100 g/ml and added at different times. the levels of p24 gag were determined after 3 days of incubation by an auto-elisa system. the binding assay was used to determine the affinity of sukumo extract for virions by viral replication assay. separation of sukumo extract and virus was carried out on a chromatography of gel filtration system with a column of sephacryl s-500 (1 by 18 cm) (amersham pharmacia biotech, sweden). the column was equilibrated and the compounds were eluted from column with pbs. the samples were separated into the following three samples; sukumo extract control: up to 150 l of sukumo extract (16 mg/ml), with 450 l of rpmi-1640 medium containing 10% fbs added; virus control: up to 150 l of pbs, with 450 l of cultured supernatant containing hiv-1 nl4-3 added; sukumo extract-virus mixture: up to 150 l of sukumo extract, with 450 l of cultured supernatant containing hiv-1 nl4-3 added. a volume of 500 l of each sample was injected onto the analytical column after incubation at 37 • c for 1 h and one fraction of eluant was collected (1 ml) on ice. the elution peak of the sukumo extract control fractions at a wavelength of 492 nm and anti-hiv-1 iiib activity by mtt assay were monitored to determine the elution position of sukumo extract (fig. 4a ). the levels of p24 gag in the eluted fractions were measured with auto-elisa to determine the elution position of virus (fig. 4b ). viral infectivity was analyzed for the eluted fractions of the virus control and sukumo extract-virus mixture; the selected fractions 6 and 7 were clarified by 0.2 m filter. mt-4 cells (4 × 10 5 /ml) were infected by a mixture of the eluted fractions. two hours after infection, the cells were washed and added to fresh rpmi-1640/10% fbs medium, cultured for 4 days and the p24 gag of supernatant was measured. anion exchange chromatography was carried out on a deae-sephacel column, which had been equilibrated with phosphate buffer (ph 7.2). the bound sample was eluted by stepwise increases of the nacl concentrations in phosphate buffer. the eluted fractions were analyzed for anti-hiv activity using the mtt assay method. the sukumo extract was also separated with 15% sds-page. the gel was stained by silver reagent and cut as described in fig. 6b . sukumo was re-extracted with rpmi-1640 medium from the sds-gel fractions and collected supernatants for anti-hiv-1 activity. the anti-hiv-1 activity of sukumo extract was first investigated by conventional mtt assay using mt-4 cells. sukumo extract completely inhibited hiv-1 (iii b strain) replication in mt-4 cells at a concentration as low as 3.9 g/ml. its 50 and 90% effective concentrations (ec 50 and ec 90 ) were 0.5494 and 2.1378 g/ml, respectively. the 50% cytotoxic concentration (cc 50 ) was found to be >1000 g/ml (fig. 1a) , and the selectivity index (ratio of cc 50 to ec 50 ) of sukumo extract was >1820, indicating that this compounds is very potent and selective. sukumo extract was also evaluated for the inhibition of wild-type herpes simplex virus-1 replication in infected vero cells, using a standard viral plaque assay (fig. 1b) . sukumo extract, and exhibited anti-viral activity with an ec 50 value of 11.56 g/ml. however, no inhibitory activity was observed against influenza a virus, poliovirus and sars virus when sukumo extract was tested at concentrations ranging from 3.2 to 400 g/ml (data not shown). sukumo extract inhibited a variety of hiv-1 isolates, including a laboratory adapted isolate iiib strain, laboratory molecular clones x4 type nl4-3 and r5 type jrcsf in a variety of cells, including molt-4, jurkat, pm1, and cd4-ccr5 expressing hos cells (data not shown). the inhibitory activity of sukumo extract against x4 hiv-1 (nl4-3) and r5 hiv-1 (jrcsf) replication in pbmcs was also demonstrated pha-stimulated pbmcs were infected for 2 h at 37 • c in the absence or presence of 0.64-400 g/ml sukumo extract followed by washing. 1 × 10 6 /ml infected cells per well were seeded in 24-well plate and were incubated for 7 days in the absence or presence of appropriate concentrations of compound. quantity of hiv-1 p24 gag was measured by auto-elisa system. by p24 assay of culture supernatants of the cells infected with the viruses exhibiting ec 50 values of 12.02 and 11.5 g/ml, respectively (fig. 2) . the p24 gag levels of untreated samples of hiv-1 nl4-3 and jrcsf were 27.914 and 14.096 ng/ml, respectively. hiv-1 replication in mt-4 cells appeared to be more sensitive to sukumo extract than in pbmcs. in the various steps of the hiv-1 life cycle, we next investigated at which step sukumo extract exerts its effect as an hiv-1 antagonist. to determine whether the viral binding to cells is a target of sukumo extract, a binding assay was carried out to measure the effect of sukumo extract on virions/cell surface interactions. mt-4 cells were mixed with x4 virus nl4-3 on ice for 2 h, and then the cells washed to remove the unbound viruses. the results demonstrate that sukumo extract blocked virus-cell binding with an ec 50 of 2.02 g/ml (fig. 3a) . the inhibitory effect of sukumo extract on hiv-1 entry to cells was also studied in mt-4 cells. cells were incubated with the same virus at 37 • c for 2 h, treated with trypsin to remove bound virions, and then the intracellular p24 gag of hiv-1 was measured. sukumo extract inhibited viral entry with an ec 50 of 1.84 g/ml (fig. 3a) . the binding and entry of hiv-1 nl4-3 in mt-4 cells was efficiently inhibited by sukumo extract in a dose-dependent manner. a similar result was also observed with r5 type hiv-1 jrcsf on hos/cd4-ccr5 cells (data not shown). these experiments show that there is a good correlation between the anti-hiv activity and the inhibitory activity against virus binding/entry induced by the sukumo extract. sukumo extract also completely prevented syncytium formation through co-culture of molt-4 and hiv-1-converted molt-4 cells at a concentration of 25 g/ml and efficiently prevented it even at 3.125 g/ml (fig. 3b) . these data indicate that sukumo extract exerted its effect at an initial step of hiv-1 infection, such as viral entry and membrane fusion in the target cells. we then analyzed changes in cd4 and cxcr4 expression on mt-4 cells and ccr5 expression on hos/cd4-ccr5 cells upon treatment with different concentrations of sukumo extract. only a high concentration of sukumo extract (200 g/ml) caused down-expression of cd4 (68.87% of control). when sukumo extract was used at the concentrations of 200, 25 and 3.125 g/ml, the levels of cxcr4 expressed were only 15.33, 40.64 and 62.81% of control, respectively. in contrast, the levels of ccr5 expression on the surface of hos cells were 73.25, 82.01 and 90.6% of control at the same concentration range (fig. 3c) . to determine the sukumo extract and hiv-1 interaction, we applied a chromatographical analysis using a sephacryl s-500 for separation of the virus particles and the sukumo extract based on differential molecular size. when sukumo extract was fractionated, the main anti-hiv-1 activity was eluted in fractions 10-14, as revealed by the mtt assay the eluted fractions 6 and 7 were selected and infected into mt-4 cells for 2 h at 37 • c. after washing, the cells were incubated for 4 days and p24 gag of culture supernatant was measured by auto-elisa. (fig. 4a) . on the other hand, hiv-1 was eluted in fractions 6 and 7 as shown by p24 assay (fig. 4b left) . when an excess amount of sukumo extract was mixed with hiv-1 and separated with sephacryl, the viral peak was detected in fractions 6 and 7 once again (fig. 4b right) , while anti-hiv activity was still observed in fraction 10-14 (data not shown). to see whether these fractions contained an infective capacity of hiv-1, the amount of p24 gag was assessed in the supernatant of mt-4 cells after infection. we used the same volume (150 l) of eluted fractions 6 and 7 from viral control, which contained 0.53 and 0.69 ng of p24 antigen, or those from the sukumo extract-virus mixture which contained 10.24 and 1.78 ng of p24 antigen to infect 4 × 10 5 mt-4 cells, respectively. as shown in fig. 4c , fractions 6 and 7 obtained from the viral control exhibited high hiv-1 activity (161.35 and 226.32 ng/ml in p24 level) 4 days after infection while fractions 6 and 7 from the sukumo extract-virus mixture had p24 levels as low as 21.6 and 13.76 ng/ml, respectively. these results strongly suggests that the sukumo extract specifically bound to viral particles and was efficiently trapped by viral particles so that viral infectivity was significantly abrogated due to the blockage of entry into the cells. although all the data provided evidence that hiv-1 entry could be a primary anti-viral target of sukumo extract, there still remained the possibility that sukumo extract exerts its effect on a late step of viral replication. to address this, a time course assay was performed using a single cycle infection with vsv-g pseudotyped hiv-1 and 293t cells. p24 gag in the supernatant was measured 3 days post-infection. the result showed that a dose-dependent anti-viral activity of sukumo extract was observed when it was added at the time b (entry) step. in contrast, the inhibition was not seen when sukumo extract was added at the time a (pretreatment) or time c step (post-entry) at any concentrations studied (range 0.16-100 g/ml) (fig. 5) . a similar result was also observed when hos/cd4-ccr5 cells were used (data not shown). these results suggest that sukumo extract does affect an early step, not a post-entry step, of the viral life cycle. based on these studies, we conclude that there is persuasive evidene that sukumo extract is a binding inhibitor that interferes with virion/cells interactions and that this inhibition is likely mediated through binding to the hiv-1 viral envelope. the anti-viral factor was extracted from sukumo using organic solvent and water. inhibitory activity was found in the aqueous extract. crude sukumo extract was fractionated by deae-sephacel column chromatography. the main fractions which had anti-viral activity was eluted from the column by 1.0-2.0 m nacl (fig. 6a) . the sukumo extract was also separated by using sds-page and anti-hiv-1 activity was detected in fractions 3-7 of the sds-gel extracts corresponding to a molecular weight of 10,000-50,000 (fig. 6b) . gas chromatography analysis of the acid hydrolysates of the sukumo extract revealed the carbohydrate contents of ara:xyl:man:gal:glc were 5.1:1:2.1:3.3:2.6 ( table 1) and sds-page/pas staining yielded a bright red band (zacharius et al., 1969 ) (data not shown). elemental analysis revealed that the sulfer content is 1.14% in the fig. 5 . effect of sukumo extract on vsv-g pseudotyped hiv-1 replication. 293t cells were infected with the hiv-1 nl-e strain lacking env and nef with vsv-g envelope of pesudotyped virus. 0.16-100 g/ml sukumo extract was used and anti-hiv-1 activity was determined 3 days later by measuring p24 gag. treatment a (a pre-entry step): the cells were incubated with sukumo extract for 2 h at 37 • c and washed before exposure to virus, and then the cells were infected and incubated in the absence of sukumo extract. treatment b (an entry step): the cells were exposed to virus in the presence of sukumo extract for 2 h, then both sukumo extract and unabsorbed viruses were removed by washing. the cells were further incubated in the absence of sukumo extract. treatment c (a post-entry step): the cells were infected with virus for 2 h, unabsorbed virus were removed and further incubated in presence of sukumo extract. sukumo extract ( table 1 ). the anti-viral factor in sukumo extract was stable under a wide range of ph conditions. as shown in table 2 , the anti-viral activity of sukumo extract was not reduced after treatment with 6n h 2 so 4 , 1n hcl or naio 4 , but the value of ec 50 (1.095 g/ml) was somewhat decreased when it was treated with 1n naoh. the activity was also not lost after being heated at 121 • c for 20 min and was not inactivated by protease (trypsin, proteinase k and pronase) digestion. finally, we addressed whether the anti-hiv-1 activities of sukumo extract, heparin and dextran sulfate were abrogated after they were treated with acid. when sukumo extract was boiled at 100 • c in 6n h 2 so 4 and 1n hcl for 6 h the 50% effective concentration (ec 50 ) against hiv-1 was essentially unaffected. in sharp contrast, when heparin was boiled at 100 • c in 6n h 2 so 4 for 6 h or dextran sulfate in 1n hcl for 2 h, the 50% effective concentrations against hiv-1 were >940 and >1000 g/ml by mtt assay, while those of the untreated samples were 8.2095 and 0.7707 g/ml, respectively (table 2) . sukumo extract potently and selectively inhibited hiv-1 replication in vitro. the compound was also evaluated for activity against various virus species with or without an envelope including vesicular stomatitis virus g protein enveloped hiv-1 pseudotyped type virus. whereas sukumo extract was active against herpes simplex virus, it was devoid of any activity against influenza a virus, sars virus and a non-enveloped poliovirus. based on the current knowledge of hiv, several stages of the viral life cycle are potentially vulnerable to inhibitors. these can be divided into the entry steps and post-entry steps. in this study, we have demonstrated by several different techniques that sukumo extract inhibits the hiv-1 infectious process at the cell entry step. the data presented in fig. 3 indicate that sukumo extract is able to block viral binding to target cells and inhibits virus-induced cell-cell fusion. furthermore, a time-course experiment showed that the full protective activity of sukumo extract was achieved when the compound was present during the 2-h virus adsorption period, but none of the effect was seen when the compound was incubated with the cells prior to viral infection. also, the extract did not suppress the viral replication after the virus had entered the cells. thus, sukumo extract interferes with an early event of the virus replication cycle, most presumably the viral adsorption step. two classes of cell surface molecules, cd4 and chemokine receptors, as well as ccr5 or cxcr4, are often viewed as hiv coreceptors which mediate hiv-1 entry. we found that the down-modulation of hiv-1 receptor cd4 or co-receptor ccr5 in target cells was induced by the sukumo extract. however, the inhibitory activity was rather weak. in addition, this activity of sukumo extract was lost if the cells were washed prior to addition of antibody, indicating that the compounds can only weakly associate with the cell surface. therefore, the results cannot perfectly explain why the sukumo extract is able to block virus entry of hiv so efficiently, especially the r5 hiv-1 virus. the effect of sukumo extract on the viral binding process was assessed directly, using a chromatography method (fig. 4) . the results show that sukumo extract was bound to hiv-1 and was separated along with the larger virus particle fraction from a gel filtration column. from this study we hypothesized that sukumo extract exerts its anti-hiv activity by binding to the viral envelope glycoprotein. this results in prevention of virus attachment to the cell surface receptor or co-receptor, whereby interference with early adsorption and entry into the hiv replicative cycle. these findings are consistent with the hypothesis that sukumo extract interferes with virions rather than cell function. it might also explain why sukumo extract is less toxic to target cells in vitro. the biochemical features of water extract of sukumo prepared from polygonum tinctorium that selectively inhibited the replication of hiv-1 were studied. the anti-viral activity was extracted from sukumo in a variety of ways, using water and organic solvents (hexane, chloroform, acetone and ethanol). inhibitory activity was found in the aqueous extracts, whereas the extracts by organic solvents did not show any anti-hiv activity. indigo, a staining ingredient and tryptanthrin, a low molecular weight component from polygonum tinctorium, also did not exihibit any anti-hiv activity (data not shown). the main fraction of anti-hiv activity was eluted from the deae-sephacel column, a negative ion-exchange column at higher molar (1.0-2.0) nacl. this result indicate that the active factor(s) is highly anionic. it was also confirmed that the anti-hiv compound(s) consist of phenolic substructure by fecl 3 -k 3 fe(cn) 6 staining (barton et al., 1952 ) (data not shown) and a polysaccharide containing sulfur atom by sugar analysis and elemental analysis, respectively ( table 1) . the factor was estimated to be a high molecular weight compound of 10,000-50,000 by sephadex g-75 gel-filtration analysis (data not shown) and an sds-gel of sukumo extract (fig. 6b) . no protein was detected in the water extract of sukumo with sds-page/silver staining. the data confirm our observation that the inhibitory activity of sukumo extract was not inactivated by protease digestion or heating at 121 • c for 20 min. furthermore, boiling of the sukumo extract in the presence of 1n hcl, 6n h 2 so 4 and 1n naoh for 6 h did not result in any loss of this activity. similarly, it was not inactivated by naio 4 treatment (nakashima et al., 1987b) , which breaks down carbohydrates (table 2 ). this suggests that the sugar backbone is not essential for the anti-hiv activity of the sukumo extract. the pharmaceutical value of the sukumo extract is likely to be further enhanced by its stability over a wide range of ph values, as shown by the heating at 121 • c 20 min and treatment with acid and alkaline conditions. since the anti-hiv-1 activity of sukumo was higher than that of fresh leaves (data not shown), the possibility that the active substances were derived from bacteria could not be excluded. we compared difference between representative sulfated polysaccharides and sukumo extract for their susceptibility to acid treatment. the anti-hiv-1 effect was clearly abrogated by this treatment in the case of dextran sulfate and heparin, but not sukumo extract ( table 2 ). the anti-hiv-1 activity of heparin was completely destroyed by 6n h 2 so 4 treatment as in the case of 1n hcl treatment of dextran sulfate. unlike epigallocatechin gallate, a polyphenolic substance from green tea (suzutani et al., 2003; yamaguchi et al., 2002) , sukumo extract did not exert any anti-hiv-1 activity on the post-virus entry process. further work on the characterization of the sukumo extract and its potency as an anti-viral candidate drug is in progress. mechanism of inhibitory effect of dextran sulfate and heparin on replication of human immunodeficiency virus in vitro susceptibility to highly sulphated glycosaminoglycans of human immunodeficiency virus type 1 replication in peripheral blood lymphocytes and monocyte-derived macrophages cell cultures paper chromatography of phenolic substances isolation of antifungal principle tryptathrin, from strobilanthes cusia o. kuntze a duodenally absorbable cxc chemokine receptor 4 antagonist, krh-1636, exhibits a potent and selective anti-hiv-1 activity antibacterial action of tryptanthrin and kaempferol, isolated from the indigo plant (polygonum tinctorium lour.), against helicobacter pylori-infected mongolian gerbils inhibition of mast celldependent anaphylactic reactions by the pigment of polygonum tinctorium (chung-dae) in rats prevention of azoxymethane-induced intestinal tumors by a crude ethyl acetateextract and tryptanthrin extracted from polygonum tinctorium lour cell surface ccr5 density determines the postentry efficiency of r5 hiv-1 infection promoting effect of kaempferol on the differentiation and mineralization of murine pre-osteoblastic cell line mc3t3-e1 new targets for inhibitors of hiv-1 replication selective interactions of polyanions with basic surfaces on human immunodeficiency virus type 1 gp120 purification and characterization of an avian myeloblastosis and human immunodeficiency virus reverse transcriptase inhibitor, sulfated polysaccharides extracted from sea algae. antimicrob antiretroviral activity in a marine red alga: reverse transcriptase inhibition by an aqueous extract of schizymenia pacifica anti-hiv activity of dextran sulphate as determined under different experimental conditions inhibition of human immunodeficiency viral replication by tannins and related compounds correlation of anti-hiv activity with anion spacing in a series of cosalane analogues with extended polycarboxylate pharmacophores anti-herpesvirus activity of an extract of ribes nigrum l sulfated polysaccharides extracted from sea algae as potential antiviral drugs inhibitory effects of (−)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (hiv-1) soluble glycosaminoglycans do not potentiate rantes antiviral activity on the infection of primary macrophages by human immunodeficiency virus type 1 glycoprotein staining following electrophoresis on acrylamide gels the authors thank dr. fumio shaku for performing the infection of hsv-1 experiments and gift of wild-type herpes simplex virus 1 and vero cells. this work was supported by grants from the ministry of education, science, and culture and the ministry of health, labor, and welfare of japan. the manuscript was reviewed prior to submission by pacific edit. key: cord-018440-qugmnolo authors: nan title: when a diagnosis is reportable date: 2008 journal: evidence-based medical ethics doi: 10.1007/978-1-60327-246-9_16 sha: doc_id: 18440 cord_uid: qugmnolo nan scott was admitted for further evaluation and treatment by the medical team. his wife was asked to step out of the room while the medical resident asked scott some further questions. the resident told scott that she was concerned that he had primary syphilis based on his ulceration and adenopathy and stated he should be tested for that as well as for hiv, given his thrush. when she pressed scott for information about his sexual history he initially hesitated, but then acknowledged that he had in fact had sexual relations with a female prostitute, "but only once." he consented to tests for hiv-1 antibodies and syphilis, but begged the resident "please don't tell my wife." both tests came back positive shortly thereafter. when scott learned the news, he admitted to the medical resident that he has also had sexual relations with a woman at his office. he again implored that his test results not be shared with his wife or his other sexual partners. questions for thought and discussion: what are the obligations of the medical resident for reporting the positive hiv and syphilis test results? who do these results get reported to? who notifies the sexual partners of patients who test positive for sexually transmitted infections? question for thought and discussion: when should patient confidentiality be broken, even if it is against the patient's wishes? the advent of antiretroviral therapy has led to improved survival rates for those infected with hiv. despite national campaigns designed to encourage people to get tested for hiv, around one-quarter of those infected are unaware of their diagnosis and many persons are diagnosed at an advanced stage of disease. in a 2005 report from the centers for disease control and prevention (cdc) it was estimated that 39 percent of patients diagnosed with hiv are subsequently diagnosed with aids in less than 12 months. among aids-defining illnesses (see table 12 .1), esophageal candidiasis is one of relatively high incidence. identifying patients with hiv infection prior to their developing an hiv-related illness would be beneficial and allow antiretroviral treatment to be initiated earlier. one way in which early diagnosis of hiv infection may be better achieved is through more aggressive screening at the primary care provider level. in one study by gao, et al. it was found that only 28 percent of the over 3,000 patients surveyed were asked about sexually transmitted infections (stis) at their last routine health visit. from 1990 to 2000, rates of primary and secondary syphilis declined a sharp 89.7 percent; however, a resurgence has been seen in the subsequent years. the greatest increases have been in men, particularly those having sex with other men (msm). according to one study by ciesielski and boghani, msm who have syphilis are 10 times more likely than heterosexual males with syphilis to be coinfected with hiv. the chancres caused by syphilis disrupt the natural barriers against hiv infection and increase susceptibility to hiv infection by up to five times. stis such as syphilis are indicative of risk-taking behavior that is associated with increased transmission of hiv. based on 2005 data, the cdc reports that, in adult and adolescent males, aids is transmitted via sexual contact in men who have sex with men (msm) in about 59 percent of cases. about 8 percent is due to high risk heterosexual contact. although scott is married to a woman and claims to have had high risk heterosexual encounters, he should be specifically questioned about having past sexual relations with other men. msm is a grouping which includes persons who self-identify as gay, bisexual, or heterosexual and, hence, includes men that may also have sexual relations with women. sociocultural stigmatization of homosexuality, particularly in certain minority groups, may prevent some msm from self-identifying as gay or bisexual. some men who proclaim themselves to be heterosexual may admit to being "on the down low," an expression referring to those men who occasionally also have sex with other men. nonjudgmentally asking a patient "do you have sex with men, women, or both?" is probably the best way to obtain such sexual history information during the medical interview. by law licensed physicians and other health care providers must report cases, or even suspected cases, of certain medical conditions and communicable diseases that they come upon in practice to their local health department (for those in north carolina, please see table 12 .2). cases must be reported in a timely fashion to help prevent epidemic spread of infectious diseases or agents of biological warfare. the list of reportable diseases, mechanism for reporting, and timing of the filed report are established by state and local public health departments, in association with the united states department of health and human services (hhs). health care practitioners are encouraged to familiarize themselves with the individual laws and guidelines in their state(s) of practice. in many states, once the hhs is notified of a reportable disease such as confirmed hiv infection, a health investigator representing that state's hhs will become the law involved in the case. the investigator will meet with the patient and encourage them to seek medical attention and to use abstinence or barrier methods to help prevent spread of the virus. they will also attempt to determine who the patient may have, through sufficient contact, exposed to the disease. although they are strongly encouraged to do so, the patient is not required by law to divulge all sexual contacts to the investigator. if a patient has not personally notified their partners of their risk for hiv infection, the investigator may then contact all divulged sexual partners for the need to pursue testing and potential treatment for hiv. this is done in a way that does not disclose who may have exposed them to the virus. under the health insurance portability and accountability act of 1996 (hipaa), patient confidentiality laws mandate that scott's diagnoses of hiv and syphilis must be kept confidential, per his request, from all people who are not at risk for contracting these diseases from him. however, when a patient's known disease presents an imminent threat to the health or life of others, then confidentiality must be broken. similar to the precedent established in the case of tarasoff v. regents of the university of california, there is a duty to protect the safety of an intended victim that supersedes the duty to maintain a patient's right to confidentiality. anyone who has sexual relations with or engages in other risk behaviors with scott is at risk for contracting hiv and syphilis, and protecting the health and welfare of those contact persons takes precedent over maintaining scott's confidentiality. additionally, if scott knowingly exposes others to hiv, this may be considered a crime in some states, although individual state laws are variable regarding what qualifies as criminal activity in this regard. health care practitioners should specifically learn the law of their state of practice regarding this issue. due to variability in state laws regarding mandatory reporting of hiv status by patients to persons with occupational exposure to bodily fluids (e.g., dental hygienists, nurses, physicians, etc.), the use of universal contact precautions is always prudent. the principle of respect for dignity requires respect for scott's emotions, his relationships, and his privacy. scott is naturally upset about the possibility that his diagnosis of syphilis and hiv will be shared with his wife and he is worried about the effect this knowledge may have on their relationship. to protect scott's relationship with his wife and his privacy, it appears that scott's diagnosis should remain confidential and his wife should not be informed. however, none of the principles of health care ethics is absolute and there are situations in which each of the principles may be overridden by more important considerations. in this case the principle of respect for dignity and the medical confidentiality that it requires are outweighed by the need to protect others in society from grave and foreseeable harm. this conclusion can be justified by comparing the harms that would be done and the harms that would be prevented by violating confidentiality in this case. scott will be harmed emotionally. he may feel angry and betrayed. his relationship with his wife may be ruined and may even end. on the other hand, if scott's wife is informed, then she can get tested for syphilis and hiv, seek treatment if she has been infected, and if not infected, she can take steps to protect herself from becoming so. the harm prevented, then, is the harm of serious and life-threatening illness. the same conclusion may be reached using the principles of beneficence and non-maleficence, considering the harms done and the harms prevented for scott, alone. informing scott's wife of her exposure to syphilis and hiv means the loss of scott's privacy and possibly his marriage, but it also prevents the emotional pain of knowing he has infected his wife with a deadly disease. comparing these harms, it could be argued that notifying scott's wife is in his best interests, as well as hers. the same applies to notifying scott's other sexual partners. communicable diseases present a case in which individual privacy and medical confidentiality are overridden by public health concerns. this is the moral basis for the current reporting, counseling, and notification program carried out by local public health departments. once scott's diagnosis is reported, he will meet with a health investigator who will record the names of his contacts and notify them of their exposure to syphilis and hiv. most states use "confidential" hiv testing, which means that contacts will not be told who has been diagnosed as hiv seropositive and has subsequently named them as contacts at risk for infection. scott should be informed that his diagnosis will be reported to the public health department and that he will be asked to meet with an investigator and give the names of his sexual contacts. ideally, scott would name his wife as a sexual contact and she would be notified along with his other contacts. however, it could be argued that it would be better for scott's wife (in terms of time for tests and treatment or protection) and better for him (in terms of the future of their relationship) if scott told her himself about his diagnosis and her risk. with this in mind, the medical resident could counsel scott to inform his wife as soon as possible. scott needs to be aware that, if they stay together for any length of time, his wife will likely come to know that he is very sick and she may guess the cause of his illness based on his symptoms and treatments. if scott refuses to tell his wife, the medical resident could intervene on the wife's behalf by informing the public health department that scott is married, thereby guaranteeing she will be notified by the health investigator. now that the evidence-based medicine, legal precedent, and relevant ethical principles for this case have been reviewed, formulate a strategy to address the ethical conflicts in this case. if necessary, perform additional research into local and state laws and hospital regulations. consider delving further into the background medical literature to assist with making sound therapeutic decisions. devise a treatment approach that addresses the needs of the patient and his family, that is both ethically and medically sound, and that is culturally competent. ensure that the strategy employs fair and appropriate utilization of medical resources, and that the approach is practical and feasible within the limits of the medical system at large. work out a clear and professional way to communicate the proposal to the patient and his family. attempt to foresee challenges that may arise in conveying or implementing the plan. determine what follow-up will be necessary to ensure that the chosen strategy remains successful for the patient in the long-term. reflect on how the knowledge and skills learned from this case can be used to improve the care of patients that may be encountered in future practice. in this case a patient is diagnosed with two reportable infectious diseases and patient confidentiality must be broken to maintain the health and safety of those who have come into contact with him. how much does fear of "exposure" via broken confidentiality impact a patient's initiative to get tested for diseases such as hiv? should hiv testing ever be anonymous or confidential? is public health more important than an individual's health and rights? ethics and human rights committee, american college of physicians estimated hiv prevalence in the united states at the end of 2003. national hiv prevention conference the incidence of aids-defining illnesses in 4,883 patients with human immunodeficiency virus infection cases of hiv infection and aids in the united states missed opportunities to assess sexually transmitted diseases in u.s. adults during routine medical checkups hiv infection among men with infectious syphilis in chicago key: cord-023884-etkhrgxp authors: meremikwu, martin; ezedinachi, emmanuel; ehiri, john e. title: malaria in women and children date: 2009-05-18 journal: maternal and child health doi: 10.1007/b106524_12 sha: doc_id: 23884 cord_uid: etkhrgxp after reading this chapter and answering the discussion questions that follow, you should be able to: explain the global burden of malaria, discuss its clinical manifestations, and appraise its health impact on women and children. analyze the mechanisms and consequences of malaria and hiv co-infection and discuss current treatment, control and prevention strategies. describe the challenges posed by vector resistance to insecticides, parasite resistance to antimalarials, climate change, wars/conflicts, and hiv/aids to malaria control and prevention efforts. evaluate social, cultural, and economic limitations of community-based programs for malaria control and prevention. malaria is caused by plasmodium, a protozoan parasite transmitted through the bite of infected female anopheline mosquitoes. the four species of plasmodium known to cause malaria in humans are p. falciparum, p. malariae, p. ovale, and p. vivax. plasmodium falciparum is the most virulent of these species and is responsible for most cases of malaria infections and malaria deaths in sub-saharan africa. plasmodium vivax, the second most common species of the malaria parasite, is more prevalent in asia and is rarely associated with acute complications of malaria or fatality. box 12.1 presents definitions of some of the most commonly used terms in malaria epidemiology. figure 12 .1 shows the global distribution of malaria transmission risk. malaria transmission occurs in africa, asia, and the americas, but sub-saharan africa bears over 80% of the global burden of malaria mortality (ehiri et al. 2004) . malaria is still a major public health problem in parts of southeast asia with foci of high p. falciparum transmission and high incidence of multidrug resistance. more than 40 species of anopheles mosquitoes transmit malaria. anopheles gambiae, which is the most efficient and resilient vector, is the predominant vector in most parts of tropical africa, where it finds adequate rainfall, temperature, and humidity to support its breeding. figure 12 .2 provides an illustration of the life cycle of plasmodium in the human and in the mosquito vector. spleen rates (percentage of the population with palpably enlarged spleen at any given time) and parasite rates (percentage of the population with malaria parasites in peripheral blood film at any given time) are traditionally used as malariometric indices to determine whether or not malaria is endemic in a given area. the entomologic inoculation rate (eir) is believed to be a better measure of malaria transmission and risk of infection than spleen or parasite rates. however, it is more difficult to assess. eir is the product of human biting rates (the number of mosquitoes biting a person over a given period of time) and the sporozoite rate (the proportion of vectors with sporozoites in the salivary glands) ). rupture and release the sporozoites, which then migrate into the mosquito's salivary glands, ready for injection into the human host. parasitemia: the presence of parasites in the blood. the term can also be used to express the quantity of parasites in the blood (e.g., 'a parasitemia of 2%''). phagocyte: a type of white blood cell that can engulf and destroy foreign organisms, cells and particles. platelets: small, irregularly-shaped bodies in the blood that contain granules. these cells are important components of the blood coagulation (clotting) system. presumptive treatment: treatment of clinically suspected cases without, or prior to, results from confirmatory laboratory tests. protozoan: single-celled organism that can perform all necessary functions of metabolism and reproduction. some protozoa are free-living, while others, including malaria parasites, parasitize other organisms for their nutrients and life cycle. residual insecticide spraying: treatment of houses by spraying insecticides that have residual efficacy (i.e., that continue to affect mosquitoes for several months). residual insecticide spraying aims to kills mosquitoes when they come to rest on the walls, usually after a blood meal. resistance: the ability of an organism to develop strains that are impervious to specific threats to their existence. schizogony: asexual reproductive stage of malaria parasites. in red blood cells, schizogony entails development of a single trophozoite into numerous merozoites. a similar process happens in infected liver cells. schizont: a developmental form of the malaria parasite that contains many merozoites. schizonts are seen in the liver-stage and blood-stage parasites. sequelae: morbid conditions following as a consequence of a disease. severe malaria: occurs when p. falciparum infections (often in persons who have no immunity to malaria or whose immunity has decreased) are complicated by serious organ failures or abnormalities in the patient's blood or metabolism, resulting in cerebral malaria, with abnormal behavior, impairment of consciousness, seizures, coma, or other neurologic abnormalities, severe anemia due to hemolysis (destruction of the red blood cells), hemoglobinuria (hemoglobin in the urine) due to hemolysis, pulmonary edema (fluid buildup in the lungs) or acute respiratory distress syndrome (ards), which may occur even after the parasite counts have decreased in response to treatment, abnormalities in blood coagulation and thrombocytopenia (decrease in blood platelets), cardiovascular collapse, shock, acute kidney failure, hyperparasitemia, where more than 5% of the red blood cells are infected by malaria parasites, metabolic acidosis (excessive acidity in the blood and tissue fluids), often in association with hypoglycemia (low blood glucose). splenomegaly: enlargement of the spleen, found in some malaria patients. splenomegaly can be used to measure malaria endemicity during surveys (e.g., in communities or in school children). sporozoite rate: the proportion of female anopheline mosquitoes of a particular species that have sporozoites in their salivary glands (as seen by dissection), or that are positive in immunologic tests to detect sporozoite antigens. sporozoite: a stage in the life cycle of the malaria parasite. sporozoites are produced in the mosquito and migrate to the mosquito's salivary glands. they can be inoculated into a human host when the mosquito takes a blood meal on the human. in the human, the sporozoites enter liver cells malaria transmission can be perennial (occurring throughout the year), high, intense, and/or stable, low, unstable, and seasonal. high stable transmission of mostly p. falciparum associated with high incidence of severe illness and mortality among preschool children is the predominant pattern of malaria in most of sub-saharan africa. the average malaria incidence rates across several parts of africa with high transmission are estimated at 1.4 per persons per year, 0.59 per persons per year, and 0.11 persons per year for age groups <5 years, 5-14 years, and !15 years, respectively (snow et al. 2003) . in low transmission areas, incidence rates of malaria are much lower, and differ only marginally between the young and older age groups. malaria epidemics are more likely to occur in areas with seasonal and unstable transmission. figure 12 .3 illustrates the various pathways by which malaria contributes to poverty, under-development, malnutrition, and maternal and infant mortality. some 300-500 million malaria episodes occur annually. children under 5 years of age in sub-saharan africa and women who are pregnant for the first or second time bear the heaviest burden of malaria morbidity and mortality. an estimated 250 million episodes of clinical malaria occur in young sub-saharan african children annually. about 1 million cases are cerebral malaria, 4 million cases are severe anemia, and approximately 1 million result in death. estimates of malaria mortality show wide variation. a review of the literature on this subject shows that the number of deaths due to malaria in african children aged less than 5 years where they develop into the next stage of the malaria parasite life cycle (the liver stage or exoerythrocytic stage). stable malaria: a situation where the rate of malaria transmission is high without any marked fluctuation over years though seasonal fluctuations occur. strain: a genetic variant within a species. sulfadoxine-pyrimethamine: a drug used against malaria. trophozoite: a developmental form during the blood stage of malaria parasites. after merozoites have invaded the red blood cell, they develop into trophozoites (sometimes, early trophozoites are called ''rings'' or ''ring stage parasites''); trophozoites develop into schizonts. uncomplicated malaria: the classical, (but rarely observed) uncomplicated malaria attack that lasts 6-10 hours. it consists of a cold stage (sensation of cold, shivering), a hot stage (fever, headaches, vomiting, seizures in young children), and finally a sweating stage (sweats, return to normal temperature, tiredness). the classical (but infrequently observed) uncomplicated malaria attacks occur every second day with the ''tertian'' parasites (p. falciparum, p. vivax, and p. ovale) and every third day with the ''quartan'' parasite (p. malariae). more commonly, the patient presents with a combination of symptoms that include fever, chills, sweats, headaches, nausea and vomiting, body aches, general malaise. unstable malaria: a situation where the rate of malaria transmission changes from year to year. vaccine: a preparation that stimulates an immune response that can prevent an infection or create resistance to an infection. vector: an organism (e.g., anopheles mosquitoes) that transmits an infectious agent (e.g. malaria parasites) from one host to the other (e.g., humans). source: malaria glossary -centers for disease control and prevention http://www.cdc.gov/malaria/glossary.htm could be between 625,000 and 1,824,000 annually (breman et al. 2004) . about 250,000 of those that survive develop sequelae from neurological complications of p. falciparum malaria. pregnant women are more vulnerable to adverse consequences of malaria than other adults. an estimated 10 million infections occur in pregnant women annually, resulting in 500,000 cases of severe maternal anemia and 500,000 low birth weight babies (greenwood et al. 2005) . in malaria-endemic countries of africa, up to 40% of all outpatient clinic visits and between 20 and 50% of all hospital admissions are due to malaria (who 2003) . although the incidence of uncomplicated malaria is lower in adolescents aged 10-19 years than younger school aged and preschool children, the burden of malaria in this age group could be substantial in areas with high and stable transmission. a recent review of the epidemiology and pattern of malaria in adolescents estimates the clinical malaria rate in african adolescents aged 10-20 years to be 0.252 attacks per adolescent per year (lalloo et al. 2006) . results of analyses based on rainfall and temperature data and geographic information system (gis) population databases in areas with high and stable malaria transmission put the yearly estimate of the number of malaria attacks in children aged 0-4 years, 5-9 years, and 10-14 years at 81.3 million, 16.0 million, and 13.4 million, respectively. the clinical pattern and deleterious consequences of malaria infection vary, depending on the level of acquired malaria immunity of the individual and the pattern of malaria transmission in an area. in areas with high and stable malaria transmission, resident adults and older children acquire sufficient partial immunity to reduce the risk of severe and fatal malaria but younger children and pregnant women remain vulnerable to severe and complicated malaria. malaria infection may be asymptomatic or symptomatic. the majority of malaria infections in areas where transmission is high and stable are asymptomatic. even when malaria infection is asymptomatic, it is believed that the high prevalence of low parasitemic and asymptomatic malaria infections contribute to the high prevalence of mild and moderate childhood anemia. in these settings, young children who are less immune to the disease are more likely to have clinical malaria following infections. the common symptoms of uncomplicated malaria are fever, poor appetite, aches, malaise, nausea, and vomiting. uncomplicated malaria is the most common reason for which children and adults use the health service in sub-saharan africa. uncomplicated malaria accounts for about 40 and 30% of outpatient attendance and hospital admissions, respectively. malaria is also a leading cause of absenteeism and poor performance at work and school. uncomplicated malaria is rarely fatal when treated promptly with effective antimalarial drugs. in preschool children, delayed treatment or failure to treat uncomplicated falciparum malaria could lead to rapid disease progression to severe and potentially fatal malaria within a period often less than 48 h from onset of illness. plasmodium falciparum causes severe malaria through complex processes that involve immunological substances known as cytokines (john et al. 2000) leading to impaired perfusion and damage to tissues and organs. these pathological changes lead to clinical and laboratory features that are characteristic of severe and complicated malaria, namely cerebral malaria that is associated with impaired consciousness, repeated convulsions, severe malarial anemia, hypoglycemia, respiratory distress, and circulatory collapse. children that die from malaria would have one or more of these signs. the risk of death is higher in patients with multiple signs (schellenberg et al. 1999) . case fatality rate of complicated falciparum malaria is 10-50%. about 10-17% of those that survive cerebral malaria have residual neurological problems such as dyskinesia, cortical blindness, seizures, and learning disorders (meremikwu et al.1997 ). most of these disorders are resolved within 6 months but about 2% persist for longer periods of time causing varying degrees of disability and impaired intellectual development (murphy and breman 2001) . anemia childhood anemia in low-income countries is caused by multiple factors including poor nutrition, malaria, intestinal parasites, hiv/aids, and inherited blood disorders (e.g., glucose-6-phosphate dehydrogenase (g-6-p-d) deficiency and sickle cell disease). in areas with high transmission, malaria is the leading etiological factor for anemia. the processes by which malaria causes anemia are not yet fully understood; however, malaria-related toxins and immunological factors are believed to cause increased hemolysis, increased splenic clearance of infected and uninfected red blood cells, and impaired production of red blood cells in the bone marrow (dyserythropoeisis). in areas of africa with high malaria transmission, surveys have shown high prevalence rates of anemia (hemoglobin <11 g/dl) among infants and children under 5 years of age (as high as 50-80% in several areas). most of these cases of anemia go unnoticed and untreated because they are mild and cause no symptoms. although children with mild and chronic anemia do not feel distinct symptoms of illness, mild anemia is associated with chronic debility. it can cause such adverse effects as reduced activity and impaired cognition and learning. these chronic effects of malarial anemia in concert with malaria-related school absenteeism and neurological complications from cerebral malaria, adversely affect childhood development and education in sub-saharan africa (mung'alaodera et al. 2004) . severe anemia (hemoglobin <5 g/dl) is a common acute complication of falciparum malaria. it is responsible for high case fatality and often follows massive hemolysis from a single episode of falciparum malaria. repeated episodes or poorly treated episodes of uncomplicated malaria are fairly common pathways to severe anemia in infants and young children who are residents of areas with high and stable malaria transmission. in many communities in africa where there are high levels of p. falciparum resistance to chloroquine and sulphadoxine-pyrimethamine, the continued use of failed drugs has resulted in an increase in the incidence of severe malarial anemia. case fatality from severe malarial anemia varies from 1% in treated cases to over 30% when associated with other complications of falciparum malaria, especially respiratory distress and deep coma (john et al. 2000) . many more children with life-threatening severe malaria anemia do not have access to formal health care where adequate treatment and blood transfusion are possible. this indicates that overall case fatality from severe malarial anemia is likely to be much higher than reported. blood transfusion for severe malaria-related anemia accounts for a remarkable proportion of new pediatric hiv infections in africa (crawley and nahlen 2004) . given the multifactorial nature of the etiology of childhood anemia, interventions to prevent or treat it should involve several approaches. for instance, mass de-worming of children and micronutrient supplementation programs are interventions that have the potential to reduce the burden of childhood anemia in developing countries (briand et al. 2007 ). insecticide-treated nets, chemoprophylaxis, and intermittent preventive treatment are malariaspecific interventions that have been shown to significantly reduce morbidity and mortality from malaria-related anemia (briand et al. 2007 ). malaria is a leading cause of hemolytic and vasoocclusive crisis in african children and adolescents with sickle cell disease. sickle cell disease is the most common inherited hematological disease among africans. the prevalence of the sickle cell trait (heterozygous inheritance on an abnormal and a normal gene) can be as high as 25-40% in some parts of africa with 1-3% affected by the disorder (inheritance of a pair of abnormal gene). a paradoxical relationship exists between the sickle cell gene and malaria. the sickle gene is believed to confer some measure of protection against malaria to those with the trait (one abnormal gene); however, it is a leading cause of morbidity and mortality among those with the disorder (two abnormal genes). two other notable chronic effects of malaria in children and adolescents include malarial nephropathy and hyperactive malarial splenomegaly. malarial nephropathy results from gradual damage of kidney cells by an antigen-antibody complex that is caused by previous malarial infection. there are no reliable data on the magnitude of renal morbidity which are caused by this malaria-induced pathology. however, it is believed that the problem is substantial. hyperactive malarial splenomegaly (also called tropical splenomegaly syndrome) is another chronic, but less common presentation of malaria among children and adolescents in the tropics. this condition is characterized by an enlarged spleen, high levels of malarial immunoglobulin (igm), sinusoidal lymphocyte infiltration, and resolution with prolonged antimalarial therapy. plasmodium falciparum and p. vivax are known to cause significant effects on maternal and child health during pregnancy. plasmodium falciparum exerts the worst effects among all the species of malaria parasite. in sub-saharan africa, the transmission of p. falciparum is predominantly high and intense with high levels of morbidity and mortality among infants and pregnant women. the major consequences of malaria infection during pregnancy are clinical episodes of malaria, maternal anemia (hemoglobin concentration <11 g/dl), or severe anemia (hemoglobin concentration <8 g/dl), placental parasitemia, intrauterine growth retardation, preterm births, and low birth weight. table 12 .1 shows the contribution of malaria to adverse maternal and child health outcomes. malaria in pregnancy is estimated to account for up to 25% of cases of severe anemia, 10-20% of babies born with low birth weight, and 5-10% of neonatal and infant deaths are due to malariainduced lbw (greenwood et al. 2005) . the effect of malaria in pregnancy is influenced by the level of malaria immunity acquired by the mother before pregnancy. this depends on the pattern and intensity of malaria transmission. the parasite species, the number of previous pregnancies, and the presence of human immunodeficiency virus (hiv) also remarkably impact malaria morbidity and mortality during pregnancy. in areas with high and stable malaria transmission, the prevalence and intensity of p. falciparum parasitemia are higher in pregnant women than in non-pregnant women. the majority of malaria infections in pregnant women living in high transmission areas are asymptomatic because of immunity acquired from repeated exposure to malaria before pregnancy. the adverse consequences of malaria during pregnancy in areas of high transmission are anemia, placental malaria, intrauterine growth retardation, and low birth weight. in areas of low or unstable transmission, acquired malaria immunity is low in all age groups. pregnant women with malaria in this area are vulnerable to severe manifestation of the disease including cerebral malaria. the evidence that malaria and hiv co-infection increases morbidity associated with both conditions has been confirmed by several studies (snow et al. 2003) . impact of the complex interaction between malaria and hiv appears to be most profound in pregnancy and children. hiv infection in pregnancy is known to increase the risk of malaria infection (population attributable risk (par), 10-27%), maternal anemia (par, 12-15%), and low birth weight (par, 11-38%) (steketee et al. 2001) . the mechanism by which hiv infection alters malaria morbidity is not well understood. it is believed to be due to systemic and placental immunologic changes that are induced by hiv. in a rwandan cohort study that included 228 hiv-positive and 229 hiv-negative participants, the incidence of malaria was almost twice as high in the hiv-positive group (6.2 per 100 women-months) than in the hiv-negative group (3.5 per 100 women-months) (ladner et al. 2002) . a review of studies on malaria and hiv coinfection shows that hiv infection in pregnancy significantly increases the risk of peripheral and placental malaria parasitemia. malaria in pregnant women infected by hiv is more likely to cause higher parasite densities, febrile illness, severe anemia, and low birth weight than malaria in those without hiv infection (snow et al. 2003) . in the absence of hiv infection, the deleterious effects of malaria in pregnancy, notably low birth weight and maternal anemia, were significantly worse in those pregnant for the first or second time than in those who have been pregnant for three or more times (ter kuile et al. 2004 ). with hiv co-infection, the pattern of malaria morbidity is similar across all categories of pregnant women (ter kuile et al. 2004) . a review of studies in areas of sub-saharan africa with high and stable malaria transmission shows that hiv-1 infection and clinically diagnosed aids increased the incidence of malaria 1.2-fold and 2fold, respectively (korenromp et al. 2005) . in these high transmission areas, hiv-1 infection in children increased hospitalization for malaria and malaria case fatality 6-fold and 9.8-fold, respectively. at the same time in low transmission areas, the incidence of severe malaria and malaria case fatality increased 2.7-fold and 3.6-fold, respectively. the effect of hiv on malaria incidence is worse in hiv patients with lower cd4 counts. in adult patients living in high malaria transmission areas, hiv increased the malaria incidence 1.2-fold, 3-fold, and 5-fold when cd4 counts were !500, 200-499, and <200/ml, respectively (korenromp et al. 2005) . the increase in morbidity and mortality associated with hiv and malaria co-infection, both of which are highly prevalent in most parts of sub-saharan africa, calls for more focused research in this area and for integration of service delivery. one way of achieving greater impact is the integration of malaria and hiv/aids control activities within maternal and child health programs. achieving high coverage of insecticide-treated bed nets (itns) use and prompt access to treatment with artemisininbased combination treatments (acts) would contribute to the reduction in the morbidity and source: who-afro (2004) mortality attributable to hiv co-infection with malaria in high transmission areas. in areas of low intensity and unstable transmission, widespread and effective indoor residual spraying combined with effective treatment using artemisinin-based combination therapy (act) is cost-effective and has been shown to significantly reduce malaria morbidity and mortality (snow et al. 2003) . the following section provides a summary of the three-pronged approach to malaria control recommended by the world health organization's malaria control program (who 2005) . indoor residual spraying, environmental management to eliminate mosquito breeding sites, and use of larvicides are known to be effective in reducing malaria when used in combination. aerial and terrestrial spraying of insecticides is used in parts of south america and asia to control malaria. this intervention strategy is cost intensive and low in effectiveness. it is therefore, not an appropriate control measure for sub-saharan africa given the complex terrains and weak economies of these malaria-endemic countries. insecticide-treated bed nets (itn) have been shown by studies in a variety of settings to be effective in reducing the incidence of clinical malaria by half and fatalities by about a third (snow et al. 2003) . population coverage for itn in most parts of africa remains low (<20%). the low re-treatment rate at the expiration of the usual period of potency (6 months) was a major challenge, even in areas that achieved high itn coverage. the development and widespread deployment of factory-treated nets with lifelong protective effects (llins) has eliminated the need to re-treat insecticide-treated nets. the persisting challenge is how to improve access to itns by poor women and children who need to be protected from severe and fatal malaria. the global fund for tuberculosis aids and malaria is providing funding to countries in endemic low and middleincome countries to support this intervention. a systematic review of randomized controlled trials conducted in africa showed that itns used in pregnancy compared to ''no nets'' significantly reduced the risk of placental malaria in all pregnancies (relative risk 0.79, 95% confidence interval 0.63-0.98). the review also showed that itns significantly reduced the risk of low birth weight (relative risk 0.77, 95% ci 0.61-0.98) and fetal loss in the first to fourth pregnancy (relative risk 0.67, 95% ci 0.47-0.97). however, this was not the case in women with more than four previous pregnancies (gamble et al. 2006) . in a large randomized controlled trial in communities with intense and perennial malaria transmission, itn use significantly reduced the risk of severe malaria anemia, placental malaria, and low birth weight among those pregnant for the first to fourth time, but not in those pregnant for five or more times (ter kuile et al. 2003) . the adherence to itn use in pregnancy was shown to be significantly lower in adolescent and young women, who are most at risk for the deleterious consequences of malaria (browne et al. 2001 ). this observation and the known risk of higher malaria morbidity associated with first pregnancy (involving mostly adolescent women) make it necessary to specially target this age group for intervention. in summary, the limited risk assessments undertaken so far with regard to the safety of itns suggest that they are relatively safe. however, a cautionary note regarding the need to monitor the health effects of long-term exposure to insecticides in resourcepoor settings has been presented by ehiri et al. (2004) . although the use of mosquito nets is not new, mass use of itns as a population-based malaria control tool is a relatively new technology, and some uncertainty remains about the potential for problems as their use expands (hirsch et al. 2002) . prompt treatment of malaria with efficacious and affordable antimalarials is a key component of the global malaria control strategy. the emergence and spread of malaria parasites (especially p. falciparum) resistant to the commonly used affordable antimalarials, like chloroquine (cq) and sulphadoxine-pyrimethamine (sp), hampered malaria control in africa and has deteriorated the malaria situation on the continent. the emergence of these multidrug-resistant malaria parasites led to the adoption of combination treatment options as the gold standards for treating malaria. the who (2006) recommends that the ideal drug combination should contain two drugs that are individually effective against the blood stages of the parasite and use completely different mechanisms to kill the parasite. based on results from several well-conducted studies, the who recommended that combinations that contain artemisinin (a drug derived from the chinese plant a. annua l.) or its derivatives and another structurally unrelated and more slowly acting drug provide the best therapeutic effects and are safe. this category of drug combinations is collectively known as artemisinin-based combination treatments (acts). the advantages of artemisinin-based combination treatments (acts) have been outlined by the who to include the following (who 2006 monotherapy with artemisinin derivatives requires multiple doses given for 7 days due to their characteristic short half-life. the other key advantage of artemisinin containing combination treatments (acts) is the shortened duration of treatment (3 days), with expected improvement in patient compliance to treatment. if the partner drug is effective, acts ensure prompt recovery and high cure rates. they are generally well tolerated. replacing the older failing or failed monotherapies with effective drugs will reduce morbidity and mortality. the challenge, however, remains how to deliver these drugs to the people that need them. implementation of this policy would put significant cost burdens on national malaria control programs. however, the costs of failing to change, such as an increase in childhood deaths and high cost of hospitalization, make it a necessary and cost-effective program. affordability of acts is a major issue affecting their effective deployment in malaria control programs in sub-saharan africa. acts are generally too expensive for most people in low-income settings where malaria is endemic. while drugs such as chloroquine and sulphadoxine-pyrimethamine (sp), which were previously used for treating uncomplicated malaria, cost only a few us cents, the new acts cost about $2-$3.5 and even higher when not discounted. international efforts to address this issue championed by the roll back malaria (rbm) partnership have yielded some positive results, especially through the global fund for tuberculosis, aids and malaria (brundtland 2002) . however, huge gaps still exist. unfortunately access to prompt treatment with effective antimalarial drugs remains very low in many sub-saharan countries, leading to the persistence of high malaria mortality rates. the reasons for poor access to treatment are mainly due to weak health systems that are poorly patronized by the populace and a lack of funds to procure and effectively deliver expensive artemisinin-based combination treatment (acts). acts are necessary since high levels of p. falciparum resistance have rendered chloroquine and sulphadoxine-pyrimethamine ineffective. these were the cheaper treatment options that have been used for several decades. most children who become ill with malaria in these areas are usually treated at home with poor quality or inappropriately administered medicines that were purchased from local, often untrained drug vendors. antimalarial treatment policies, adopted by each country, depend on the epidemiology of the disease, including patterns of transmission, drug resistance, political environment, and economic context. the adoption of acts in sub-saharan africa was preceded by establishment of local evidence on the effectiveness of existing first-and second-line drugs which have demonstrated consistently high treatment failure rates due to parasite resistance (snow et al. 2003) . the who (2006) also recommends that countries developing antimalarial treatment policies should strive to ensure that all populations at risk have access to prompt treatment with safe, good quality, effective, affordable, and acceptable antimalarial drugs and there is rational use of antimalarial drugs in order to prevent the emergence and spread of drug resistance induced by unduly high selection drug pressure on mutant malaria parasites. delivery of effective and safe antimalarial treatment to poor rural populations and those in difficult, hard-to-reach settings poses enormous challenges to malaria control programs in africa. in many endemic countries, the formal health system is weak. often the health system consists of a few ill-equipped health facilities run by inadequately trained and/or poorly motivated health personnel. the proportion of the people that access these services is so low that successful malaria treatment programs in africa would be impossible without community-based delivery mechanisms including adequately trained and equipped informal community-based providers and caregivers who provide treatment and preventive services as close as possible to where people live and work. delivering community health care such as malaria treatment services through primary healthcare centers has long been identified a big challenge by jeffery (1984) as summarized in box 12.2. a careful appraisal of these factors in the context of the current situation of malaria control efforts in most endemic countries in sub-saharan africa shows situations that are as pertinent today as they were over two decades ago when they were highlighted by jeffery (1984) . home management of malaria (hmm), the strategy currently recommended by the who (mendie et al. 2003) as an effective community delivery mechanism for antimalarial treatment, is likely to address some of the limitations highlighted in box 12.2. the hmm strategy entails educating community health workers, volunteers, mothers, and caregivers to recognize symptoms of malaria and treat with appropriate antimalarial drugs (mendie et al. 2003) . its goal is to ensure early recognition and prompt and appropriate response to malarial illness in under-5 children in the home and community by enabling health workers, mothers, and caregivers to recognize malarial illness early and take appropriate action. the who hmm strategy consists of four strategic components: 1. ensure access to effective and good-quality antimalarial drugs (preferably pre-packed) at community level. 2. ensure that community drug or service providers (e.g., patent medicine vendors, volunteer village health workers, community health extension workers) have necessary skills and knowledge to manage malaria. limitation of outreach capacity to geographically remote areas, and particularly to nomadic populations inability to make efficient use of community resources (both human and material) difficulty in achieving full community acceptance of chemotherapy insufficient training of local health workers lack of understanding of health problems and their solutions potential ineffectiveness of the curative drug or drug dosage used, usually through the emergence of parasite resistance to the drug undesirable side-effects of the drug source: jeffery (1984) 3. ensure an effective communication strategy to enable caregivers to recognize malarial illness early and take appropriate action. 4. ensure good mechanisms for supervision, monitoring, and communication activities. as shown in chapter 27, integrated management of childhood illness (community imci) also addresses both preventive and curative aspects of malaria control by seeking to improve community and family practices. giving prophylactic antimalarial drugs to prevent malaria is a routine practice for non-immune persons visiting malaria-endemic areas. malaria prophylaxis refers to daily or weekly administration of antimalarial drugs at a dose that is usually smaller than the therapeutic doses with a view to preventing clinical malaria. intermittent preventive treatment (ipt) refers to full therapeutic doses of an antimalarial given at specified time points to presumptively cure asymptomatic malaria and prevent clinical malaria or such other adverse consequences as anemia or placental malaria. usually, sulphadoxine-pyrimethamine (sp) is used for ipt as it requires a single dose and has a long half-life. the rationale is that intermittent treatment is likely to have fewer adverse events than prophylaxis because it is taken less often, and it is easier to deliver through clinics, reducing poor adherence with self-administration. chloroquine was the most widely used drug for malaria prophylaxis in pregnancy. the high prevalence of resistant strains, and the fact that most women adhered poorly to the weekly regimen required to achieve beneficial effects, rendered chloroquine chemoprophylaxis ineffective for malaria control in pregnancy. meta-analysis included in a cochrane systematic review of randomized controlled trials showed that ipt with sulphadoxine-pyrimethamine significantly reduced the risk of severe maternal anemia (relative risk 0.60, 95% ci 0.50-0.78; 2,243 participants), placental malaria (relative risk 0.35, 95% ci 0.27-0.47; 1,232 participants), and low birth weight (relative risk 0.58, 95% ci 0.43-0.78; 1,399 participants) in women who were pregnant for the first or second time (garner and guâ¨lmezoglu 2006) . ipt with sulphadoxine-pyrimethamine (sp) along with consistent use of itns are currently recommended as cost-effective and evidence-based interventions to prevent the deleterious effects of malaria in pregnancy and to reduce the associated maternal and infant morbidity and mortality. almost all the 35 countries in africa with stable malaria transmission are already implementing intermittent preventive treatment in pregnancy (iptp) with sp (vallely et al. 2007 ). one of the key challenges with implementation of ipt is the high rate of parasite resistance to sp, and the lack of a safe and effective alternative to this antimalarial. in most parts of africa sp failure exceeds 20% and surveillance data on the trends are lacking in most cases. the effectiveness of this intervention in areas with high sp failure rates is yet to be adequately studied. the suggestion that two and three doses of sp, respectively, should be used in areas with sp resistance <30 and 30-50% remains to be validated by robust research data. the continued use of ipt with sp in areas where sp resistance exceeds 50% also needs to be justified by research. there is also the problem of how to handle malaria co-infection with hiv in areas with high prevalence of hiv. a third dose of sp for ipt has been recommended for areas with high hiv prevalence but there is a need to monitor impact on such outcomes as severe anemia and low birth weight, and to study possible drug interactions in those receiving anti-retroviral treatment. in malaria-endemic communities, use of antimalarial drugs for prophylaxis or intermittent preventive treatment (ipt) is recommended for only pregnant women and special vulnerable groups such as children with sickle cell disease. several randomized controlled trials in malariaendemic communities have shown consistently that malaria prophylaxis and intermittent preventive treatment of infants (ipti) and young children are effective. a cochrane systematic review and meta-analysis (meremikwu et al. 2005) showed that receiving antimalarial drugs as prophylaxis or intermittent treatment reduced the incidence of clinical malaria episodes and severe anemia by about 50% in preschool children living in malaria-endemic communities. two main reasons are commonly given for discouraging widespread use of malaria chemoprophylaxis in preschool children in endemic communities. the first reason is the concern that giving malaria prophylaxis to infants and young children living in malaria-endemic areas will delay or minimize their chances to acquire protective immunity and result in a rebound rise in the incidence of severe morbidity and mortality later in life. the second reason is that poor compliance to weekly antimalarial drug prophylaxis could induce drug pressure and selection of mutant resistant strains of p. falciparum. intermittent preventive treatment of infants (ipti) with treatment doses of sp under direct observation at the time of routine immunization offers a better programmatic option, since it eliminates the problem of non-compliance and is expected to have little or no adverse effect or interfere with the child's ability to acquire malarial immunity. a major challenge to implementation of ipti, among others, is the rising incidence of sp resistance which is the principal drug currently used for this intervention. there has also been a concern about the possible interaction between sp and the routine infant vaccines but this has not been supported by any strong evidence. timely and efficient deployment of efficacious vaccines is widely accepted as an effective child survival strategy. the development of a successful malaria vaccine especially against p. falciparum would contribute remarkably to reduction of the unacceptably high childhood death from malaria. unfortunately decades of efforts at vaccine development have yet to meet this expected public health success. developing vaccines against parasitic infections poses greater challenges than developing vaccines for virus and bacterial infections because of their more complex nature and larger genomes. the multiple stages of the malaria parasite and the different proteins they express pose additional challenges to the development of a potent malaria vaccine. an allstage malaria vaccine capable of inhibiting growth or killing all of these different stages of malaria poses a complex challenge. researchers involved in development of malaria vaccine devote their efforts to three key strategies that target the preerythrocytic and erythrocytic stages of the life cycle in humans (fig. 12. 2), and vaccines that induce antibodies in humans that can kill or prevent development of viable sexual forms ingested by the mosquito vectors. the pre-erythrocytic stage vaccines aim to prevent sporozoites (the stage of plasmodium that mosquitoes pass to humans) from invading and developing in the liver, while an asexual erythrocytic stage vaccine limits the invasion of erythrocytes or prevents their multiplication in the erythrocytes. the complete mapping of the p. falciparum genome with a better understanding of the organism at sub-cellular and molecular levels coupled with recent advances in genomic and proteomic science has led to a remarkable increase in the number of candidate malaria vaccines. there is no time better than the present to scale up support for malaria vaccine research and development. the goal of most of the initial efforts of malaria vaccine development is complete prevention of the disease with the hope of eliminating malaria. the disappointing results of early malaria vaccine trials appear to have diminished this enthusiasm. should efforts to develop a malaria vaccine capable of completely preventing clinical malaria fail, most public health experts and vaccine researchers advocate the goal of making malaria vaccines that ameliorate the severity of the disease and reduce the level of fatality. in africa, where pregnant women and children bear the greatest burden of severe malaria, such a vaccine will be a significant addition to maternal and child health services and will help to reduce the burden of childhood disability attributable to cerebral malaria. the opportunities provided by better research tools and a better understanding of the plasmodium and anopheles genome make the prospects and possibilities of a malaria vaccine better today than ever before. funding for malaria vaccine development and field trials has increased in recent years. however, it is still far short of the expected investment, given its huge potential for improving child survival and contributing to achievement of the millennium development goals (mdg). the inadequacies of health information systems and vital registration processes in most parts of sub-saharan africa make it difficult to obtain reliable records of malaria mortality. facility-based records of deaths, when available, are not representative of the situation in the larger population given that the majority of sick children do not use health facilities and most deaths occur outside the formal health facilities. most of the available mortality data from malaria-endemic areas are estimates and prospective mortality data from demographic surveillance systems validated by verbal autopsies . the inefficiency of health information systems and vital registration processes in sub-saharan african countries makes it difficult to obtain sufficient and timely information to track the performance of malaria control programs. the malaria situation globally deteriorated in the past three decades. this resulted in increased malaria-related morbidity and mortality, especially in sub-saharan africa where emergence and spread of multidrug-resistant malaria parasites and breakdown of malaria control programs were the leading reasons, among others (korenromp et al. 2003) . greenwood et al. (2005) have given an elaborate summary of the factors believed to have contributed to the deterioration of the global malaria situation in box 12.3. while the discovery of additional malaria control measures such as a highly effective malaria vaccine should be expected to increase the gains of malaria control efforts, several appraisals and overviews of global malaria control efforts agree that the key reasons for the recent decline in the gains of malaria control efforts have not been the lack of effective malaria control measures. there is consensus that the four technical elements of the global malaria control strategy (box 12.4) affirmed by the international ministerial conference held in amsterdam under the auspices of the world health organization in 1992 have been essentially effective in the years preceding and succeeding the amsterdam conference. careful study of malaria control scenarios (mostly in sub-saharan africa) that have failed, or achieved only minimal success with these same strategies, shows that these control programs lacked the pre-conditions for effectiveness of the global strategy (box 12.5) as also outlined in the amsterdam ministerial conference on malaria. when the rbm strategy was established in 1998, it was in response to these deficiencies. the rbm is a partnership between the who, other un agencies, bilateral aid agencies, nongovernmental organizations, and governments of climate instability: drought and floods increased malaria transmission in different epidemiological circumstances global warming may have led to increased malaria transmission especially in some highland areas civil disturbances and unrest have resulted in the collapse of malaria control programs and refugee situations with attendant effects on malaria transmission across epidemiological areas and increased risk of epidemics changes and increase in travel patterns within endemic areas and from non-endemic areas to endemic areas putting many non-immune people at risk hiv increases susceptibility to malaria and increases the burden on the health service emergence and spread of drug resistant p. falciparum has been a key reason for deterioration of malaria situation in especially africa and southeast asia insecticide resistance: resistance to pyrethroids used for treated bed-nets has emerged in anopheles gambiae (in west africa) and anopheles funestus (in southern africa). high vector resistance to anopheles funestus diminished the use of ddt for household spraying in southern africa. source: greenwood et al. (2005) malaria-endemic countries. the rbm has a longterm goal of reducing malaria morbidity and mortality by at least half by 2010. rbm was not meant to be a new malaria control strategy but rather an organized global effort to facilitate the effective implementation of the global control strategy. the evidence that large-scale and effective use of itns can reduce the incidence of malaria and malaria-related deaths is both strong and consistent (lengeler 2000) . insecticide-treated mosquito nets (itns) can reduce all-cause childhood mortality by about a fifth; with about 6 lives saved for every 1,000 preschool children protected with itn (lengeler 2000) . it is estimated that full itn coverage in sub-saharan africa could prevent 370,000 child deaths per year (lengeler 2000) . insecticidetreated nets are cost-effective, but endemic poverty and inadequate sensitization of people in malariaendemic areas remain the major reasons for low use (snow et al. 2003) . the cost-effectiveness of itns (us $19-85 per disability-adjusted life year (daly)) is similar to most childhood vaccines (who 2003) . when community coverage is high, itns not only protect those who sleep under them, but also those in the same dwelling (the home effect) and those living nearby (the community effect) (snow et al. 2003) . the year 2005 marked the end of the target set by african heads of state to achieve at least 60% access to prompt and effective treatment of malaria and 60% itn coverage for under-5 children and pregnant women. however, most countries in sub-saharan africa fell far short of these targets. it was also in the same year that rbm set the landmark target of halving malaria mortality by 2010. appraisal of malaria control efforts at the end of 2005 uniformly indicated that resources available for procurement of malaria control commodities (acts, itn, and diagnostic kits) were grossly inadequate. the appraisal also showed that malaria control personnel at national and regional levels was inadequately equipped. donors and governments should develop effective mechanisms to monitor the access that children, adolescents, pregnant women, and children in difficult circumstances have to evidence-based to provide early diagnosis and treatment to plan and implement selective, multiple and sustainable preventive measures: including vector control; personal protection (notably insecticide treated nets); and chemoprophylaxis/intermittent preventive treatment. to detect early, contain or prevent epidemics; to conduct focused research, and to regularly assess the country's malaria situation, including ecological, social and economic determinants of the disease. source: who (1992) political will: sustained political commitment from all levels and sectors of government integration: malaria control should be an integral part of health systems, and be coordinated with relevant development programs in non-health sectors community participation: communities should be full partners in malaria control activities resource mobilization: adequate human and financial resources should be mobilized source: who (1992) treatment and preventive interventions for malaria. donor funds specifically tagged to providing resources and infrastructure for effective management of severe and complicated malaria have been grossly inadequate. supportive care for women and children with severe malaria is grossly impeded by weak health systems in malariaendemic countries. funds meant for providing adequate infrastructure and personnel for managing severe malaria should be tagged to bilateral and multilateral health system support grants. 1 globally, women, children, and adolescents in sub-saharan africa are known to bear the greatest burden of malaria morbidity and mortality. list any six factors most peculiar to the region that account for this high burden. 2 list five consequences of malaria infection in children and pregnant women. 3 an integrated approach is advocated as an efficient and cost-effective strategy for the management of malaria co-infection with hiv/aids. briefly discuss what you understand by integrated management and describe how such an integrated approach might be operationalized in practice. 4 what are artemisinin-based combination treatments (acts) and what are the advantages of their use in the treatment of malaria? 5 what are the challenges of community delivery of malaria treatment through existing primary healthcare systems? is home treatment of malaria a better option? discuss the reasons for your position. 6 list six factors that contribute to the worsening of the global problem of malaria? how can these be addressed? what should be the role of roll back malaria initiative in global malaria control? conquering the intolerable burden of malaria: what's new, what's needed: summary intermittent preventive treatment for the prevention of malaria during pregnancy in high transmission areas the impact of insecticide-treated bednets on malaria and anemia in pregnancy in kassena-nankana district, ghana: a randomized controlled trial external evaluation of roll back malaria prevention and treatment of malaria in young african children mass use of insecticide-treated bednets in malaria endemic poor countries: public health concerns and remedies insecticide-treated nets for preventing malaria in pregnancy drugs for preventing malaria in pregnant women programmatic environmental assessment for insecticide-treated materials in usaid activities in sub-saharan africa. agency for international development (usaid), office of sustainable development the role of chemotherapy in malaria control through primary healthcare: constraints and future prospects cytokine responses to plasmodium falciparum liver-stage antigen 1 vary in rainy and dry seasons in highland kenya measurement of trends in childhood malaria mortality in africa: an assessment of progress toward targets based on verbal autopsy malaria attributable to the hiv-1 epidemic, sub-saharan africa hiv infection, malaria, and pregnancy: a prospective cohort study in kigali malaria in adolescence: burden of disease, consequences, and opportunities for intervention insecticide-treated bednets and curtains for preventing malaria effective delivery methods for malaria treatment. reducing malaria's burden: evidence of effectiveness for decision makers the pattern of neurological sequelae of childhood cerebral malaria among survivors in calabar chemoprophylaxis and intermittent treatment for preventing malaria in children the burden of the neuro-cognitive impairment associated with plasmodium falciparum malaria in sub-saharan africa gaps in childhood malaria burden in africa: cerebral malaria, neurological sequelae, anemia, respiratory distress, hypoglycemia, and complications of pregnancy african children with malaria in areas of intense plasmodium falciparum transmission: features on admission to the hospital and risk factors for death the public health burden of plasmodium falciparum malaria in africa: deriving the numbers. working paper 11, disease control priorities project the burden of malaria in pregnancy in malaria-endemic areas reduction of malaria during pregnancy by permethrintreated bed nets in an area of intense perennial malaria transmission in western kenya the burden of co-infection with human immunodeficiency virus type 1 and malaria in pregnant women in sub-saharan africa intermittent preventive treatment for malaria in pregnancy in africa: what's new, what's needed world health organization (1992) ministerial conference on malaria cost-effectiveness of social marketing of insecticide-treated nets for malaria control in the united republic of tanzania a strategic framework for malaria prevention and control during pregnancy in the african region. who document: afr/mal/04/01. brazzaville: world health organization regional office for africa world health organization guidelines for the treatment of malaria. who/htm/mal/2006.1108. geneva: world health organizagtion international travel and health: situation as of 1 key: cord-029450-4rnrq78l authors: prattichizzo, francesco; bonafè, massimiliano; giuliani, angelica; costantini, andrea; storci, gianluca; sabbatinelli, jacopo; olivieri, fabiola title: response to: letter to the editor on “bonafè m, prattichizzo f, giuliani a, storci g, sabbatinelli j, olivieri f. inflamm-aging: why older men are the most susceptible to sars-cov-2 complicated outcomes. cytokine growth factor rev” by eugenia quiros-roldan, giorgio biasiotto and isabella zanella date: 2020-07-18 journal: cytokine growth factor rev doi: 10.1016/j.cytogfr.2020.07.013 sha: doc_id: 29450 cord_uid: 4rnrq78l nan dear editor, we read with vivid interest the commentary by quiros-roldan and colleagues [1] , which suggests that the framework we proposed to explain the high rate of mortality of coronavirus disease 19 in elderly and age-related disease (ard)-affected populations [2] might not apply to hivinfected patients, despite the pervasive role of inflamm-aging and immune-senescence in this setting of patients. preliminary data on an italian cohort suggest that only a minor portion of hiv-positive patients with probable sars-cov-2 infection died, which yields a low case-fatality rate if compared to other highrisk populations [3] . similarly, data on a large chinese cohort and another case series both reported a low incidence and a mild severity of sars-cov-2 infection in hiv-infected patients [4, 5] . nevertheless, another patient cohort in spain conveyed a rate of infection in hiv-positive individuals similar or slightly higher (but lower when including suspected cases) compared to the general population [6] . in this study, overall mortality was lower than that reported in the general population, but double in the age range from 50 to 59 years, with a higher age-adjusted mortality. of note, the mean age of all the four hiv-positive cohorts was lower compared to other reports showing data of sars-cov-2 infection in the general population [7] . finally, the prevalence of patients with a more severe form of covid-19 was higher in hiv-positive subjects compared to the general population [6] . accumulating data suggest that the covid-19 cases suffering the worst outcome are those with reduced count and functional exhaustion of t lymphocytes, as well as those affected by the cytokine release syndrome, i.e. the cytokine storm [8] . notably, the majority of hiv-positive patients enrolled in the four abovementioned studies had next-to-normal cd4 cell counts, while one of the studies showed that those with low recent cd4 cell counts suffered from increased disease severity than individuals with proper immunity [6] . in addition, also viral load was well controlled in the majority of tested patients (as it is observed in clinical practice), suggesting that the eventual pro-inflammatory drift promoted by hiv might be limited. of note, no data regarding markers of t cell exhaustion were j o u r n a l p r e -p r o o f reported in any of studies conducted thus far, while only one reported circulating levels of interleukin-6 (il-6) [6] . on the basis of the literature above, we posit that testing for t-cell senescence markers (e.g. pd-1, tim-3, ctla-4, and tigit) [9] and measuring major inflammatory markers in hiv-sars-cov-2 co-infected patients is mandatory to disentangle the relevance of immune senescence and inflamm-aging in the covid-19 outcome in this specific population. nevertheless, a protective role of multiple anti-retroviral drugs cannot be excluded at this stage. indeed, while preliminary findings suggest a lack of activity of tenofovir or protease inhibitors on sars-cov-2 viral lifecycle [3] [4] [5] [6] , the low number of patients studied so far bars prevents any exhaustive analysis. in regard to this issue, preliminary data from a non-randomized clinical trial suggest a non-significant decrease in mortality in covid-19 patients treated with the protease inhibitor lopinavir/ritonavir [10] . finally, the peculiar immune make-up of hiv-positive patients is worth to be carefully considered. indeed, hiv patients have a complex remodelling in interferon (ifn) responses, with an early increase in ifn-α and a delayed, systemic rise of ifn-γ [11] . in turn, these two mediators have been suggested to halt (with a variable degree of success) the infection or the replication of sars-cov, the virus responsible for the previous outbreaks of sars [12] [13] [14] . to this respect, it would be interesting to explore if patients with diseases characterized by high levels of type i ifns, e.g. systemic lupus erythematosus, or treated with recombinant type i ifns are protected to some extent from sars-cov-2 infection. of note, interferon-α2b improves outcome in rhesus macaques infected with mers-cov, another member of the coronavirus family [15] . more recently, it was reported that the detrimental hyper-inflammatory response to sars-cov-2 infection is tightly coupled with an impaired capability to trigger type i ifns [16] . a randomized controlled trial evaluating the efficacy of a triple antiviral therapy with combined interferon beta-1b, ribavirin, and lopinavir-ritonavir on covid-19 patients proved the superiority of the triple antiviral therapy compared to lopinavirritonavir alone in terms of improved clinical recovery and faster rate of viral clearance, with a striking suppression of il-6 levels in the interferon arm of the study after 48 hours of treatment [17] . similarly, the results of an on-going trial testing the safety and the efficacy of interferon-α2b in patients with j o u r n a l p r e -p r o o f covid-19 may help to clarify the potential beneficial effects of type i interferon on covid-19 (clinicaltrials.gov, nct04293887). in this regard, it is worth remembering that type i ifns not only exert anti-viral activity, but inhibit the activation of the inflammatory response, thus preventing destructive local phenomena and systemic life-threatening outcomes [16, 18] . another, alternative hypothesis is that a "mild" state of immunosuppression (but with no overt signs of inflamm-aging) may eventually be helpful to limit the sars-cov-2 induced damage, a framework that is currently being explored [19] . more broadly, the development of a signature properly summarizing the inflamm-aging/immune senescence status is being demonstrated hard to develop, given the emerging complexity and heterogeneity of the aging process and in particular of the two abovementioned components [20] . on the other side, this appears the best approach at present to predict which patients will most likely suffer of sars-cov-2 consequences, given that aging-related characteristics (e.g. age and obesity) or surrogate markers of aging (il-6, lymphocyte count) represent the best tools to foresee mortality in this setting [21] [22] [23] . overall, the interesting observations provided by quiros-roldan and colleagues support the notion that much research is needed to better disentangle the underpinning of the diverse outcomes observed among different patients infected with sars-cov-2. however, their reasoning also reminds us that, while several features of immune senescence and inflamm-aging are recapitulated in hiv patients, these phenomena are much more complex than that those occurring following a chronic viral infection. in other words, inflamm-aging and immune senescence occur in a complex context, where the aging immune system interacts with an aging body that undergoes complex metabolic reshaping and remodelling. this consideration should prompt further research effort aimed at better characterizing the pillars of aging and, generally, the aging process itself, in order to develop diverse strategies beyond vaccination and anti-viral drugs to protect the most exposed part of the society against the ongoing, but also eventual, future pandemic. all authors: no conflict. inflamm-aging: why older men are the most susceptible to sars-cov-2 complicated outcomes inflamm-aging: why older men are the most susceptible to sars-cov-2 complicated outcomes clinical features and outcomes of hiv patients with coronavirus disease 2019 a survey for covid among hiv/aids patients in two districts of covid-19 in people living with human immunodeficiency virus: a case series of 33 patients description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients targeting t-cell senescence and cytokine storm with rapamycin to prevent severe progression in covid-19 induction of a striking systemic cytokine cascade prior to peak viremia in acute human immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis b and c virus infections increased sensitivity of sars-coronavirus to a combination of human type i and type ii interferons potent inhibition of sars-associated coronavirus (scov) infection and replication by type i interferons (ifn-alpha/beta) but not by type ii interferon (ifn-gamma) synergistic inhibition of sars-coronavirus replication by type i and type ii ifn treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques imbalanced host response to sars-cov-2 drives development of covid-19 triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial ribosomal dna instability: an evolutionary conserved fuel for inflammaging the immunology of covid-19: is immune modulation an option for treatment? cellular senescence, immunosenescence and hiv predictors of mortality in hospitalized covid-19 patients: a systematic review and meta-analysis severe obesity, increasing age and male sex are independently associated with worse in-hospital outcomes, and higher in-hospital mortality why is hyperglycaemia worsening covid-19 and its prognosis? key: cord-010845-pakh49dy authors: isiguzo, godsent; du bruyn, elsa; howlett, patrick; ntsekhe, mpiko title: diagnosis and management of tuberculous pericarditis: what is new? date: 2020-01-15 journal: curr cardiol rep doi: 10.1007/s11886-020-1254-1 sha: doc_id: 10845 cord_uid: pakh49dy purpose of review: this review provides an update on the immunopathogenesis of tuberculous pericarditis (tbp), investigations to confirm tuberculous etiology, the limitations of anti-tuberculous therapy (att), and recent efficacy trials. recent findings: a profibrotic immune response characterizes tbp, with low levels of acsdkp, high levels of γ-interferon and il-10 in the pericardium, and high levels of tgf-β and il-10 in the blood. these findings may have implications for future therapeutic targets. despite advances in nucleic acid amplification approaches, these tests remain disappointing for tbp. trials of corticosteroids and colchicine have had mixed results, with no impact on mortality, evidence of a reduction in rates of constrictive pericarditis and potential harm in those with advanced hiv. small studies suggest that att penetrates the pericardium poorly. given that there is a close association between high bacillary burden and mortality, a rethink about the optimal drug doses and duration may be required. summary: the high mortality and morbidity from tbp despite use of anti-tuberculous drugs call for researches targeting host-directed immunological determinants of treatment outcome. there is also a need for the identification of steps in clinical management where interventions are needed to improve outcomes. electronic supplementary material: the online version of this article (10.1007/s11886-020-1254-1) contains supplementary material, which is available to authorized users. although there has been some encouraging progress in the field of tuberculosis, such as the promising recent outcome of a phase 2b placebo-controlled trial of the m72/as01 e tb vaccine [1••] , a great deal remains to be done. the statement is particularly true for tuberculous pericarditis (tbp), where lack of understanding of this severe form of extra-pulmonary tuberculosis (eptb) has hampered discovery and translation of cost-effective, rapid diagnostic tests and host-directed therapies able to prevent its debilitating complications and associated mortality. the presence of hiv contributes considerably to the complexity of the disease process-both at the immunological and clinical levels. tb causes more deaths than any other infectious disease globally with the who estimating that there were 1.3 million tb deaths in hiv-uninfected individuals and 300,000 hiv-tb co-infected deaths in 2017 alone. this may be an underestimate of hiv co-infected tb deaths, as post-mortem studies of hiv-infected individuals in regions where tb is endemic have indicated that 45.8% of tb cases are undiagnosed at the time of death, and 87.9% of all deaths were due to disseminated tb [2] . the actual incidence of tbp is variable depending on the degree of tb endemicity of the region. in a case series from spain, for example, only 4% of 294 admissions for the acute pericardial disease were due to tbp [3] . in africa, pericardial effusions are predominantly due to tb, ranging from 64.9 to 70% in various reports [4, 5] . the hiv pandemic contributes to the high burden of tbp seen especially in southern africa, as illustrated by a study that included 84 hiv-infected individuals with effusive pericarditis-only 3 of these individuals had a diagnosis other than tb [5] . there is very little new data to inform the current epidemiology of tbp in children. however, considering that eptb accounts for 21-44% of all tb in children, it is likely an under-recognized problem [6] . a recent prospective observational study from papua new guinea reported a case fatality rate of 25% (4 out of 16) in children with tbp [7] . the most recently published retrospective observational study of children with tbp reported only one death out of 30 children, with the cause of death being ascribed to disseminated tb. the authors acknowledge that actual mortality might have been underestimated due to loss to follow-up [8] . the study reported that 10% of children developed constrictive pericarditis. this is in contrast to an earlier retrospective study of 44 pediatric tbp cases which reported no deaths and 5 cases (20%) of constrictive pericarditis at follow-up [9] . there is a need for further research in pediatric tbp, especially considering the disproportionate impact of eptb on this vulnerable population. there has been some progress in our understanding of the pathogenesis of tbp, particularly as it relates to the disease in hiv-infected hosts. mycobacterium tuberculosis (mtb) bacilli can enter the pericardium by retrograde lymphatic spread, hematogenous dissemination, or, uncommonly, by direct contiguous spread from adjacent infected structures such as the lungs, pleura, and spine [10] . the most common mechanism of mtb spread in hiv co-infected individuals is via hematogenous dissemination [11] , whereas in hiv-uninfected tbp patients, pericardial fluid (pcf) lymphocytes are predominantly cd4+ effector memory t cells and the pcf lymphocytes in hiv-infected patients are dominated by cd8+ t cells [12, 13] . hiv viral load is higher in pcf than in plasma of hiv-positive tbp patients and inversely correlates with the proportion of pcf cd4+ t cells [13] . the cellular influx into the pcf is accompanied by a range of predominantly proinflammatory and pro-fibrotic mediators (e.g., interferon-γ, interleukin (il)-10, il-1β, il-6, il-8, interferon-γ induced protein, and tumor necrosis factor (tnf)), that accumulate in the pericardial fluid [14, 15] , and low levels of the antifibrotic tetra peptide n-acetylseryl-aspartyl-lysyl proline (acsdkp) [16] . acsdkp is broken down by angiotensinconverting enzyme [17] , suggesting a novel role for ace inhibitors for tbp to prevent pericardial fibrosis. finally, recent reports suggest that among both hiv-infected and hivuninfected patients with culture-positive pericardial fluid, mtb bacillary loads are as high as 3.91 log 10 cfu/ml (range 0.5-8.96), with bacillary loads over 5.53 log 10 cfu/ml being significantly associated with mortality [18] . these findings are significant as they challenge the commonly accepted concept that tbp is predominantly a paucibacillary condition driven by an intense delayed-type hypersensitivity response to tb antigens [10] . in at least some subsets of patients, tbp is, in fact, multibacillary with significant implications for both outcomes and ensuring that current drug regimens are effective at penetrating the pericardium to do their work. there are four pathological phases of tbp that correspond with the various clinical manifestations observed (table 1 ) [11] . two broad mechanisms drive the clinical presentation of pericarditis: fluid accumulation within the pericardium which compresses the heart chambers throughout the cardiac cycle impeding both cardiac filling and cardiac contraction (tamponade); or thickening of the pericardium with minimum or absent effusion resulting in impairment of cardiac filling in diastole (constriction). the predominant clinical manifestation of tbp is the syndrome of heart failure regardless of mechanism although a relatively small proportion of patients with tamponade may also have hemodynamic compromise with hypotension tachycardia and shock. new registries and trials suggest that the most common clinical presentation of tbp is as effusive pericarditis (79.5%) [19] corresponding to stage 2. constrictive pericarditis, which corresponds to stages 3 and 4, is one of the most severe sequelae of tbp (fig. 1 ). before the widespread use of att and routine evacuation of the pericardium, constrictive pericarditis was found in 30-60% of patients [3, 20] , whereas more modern prospective studies report rates of 5-25% [21••]. it occurs less frequently in those with hiv infection [22] . some patients present with a combination of both tamponade and constrictive physiology believed to be a stage between 2 and 3 (effusive-constrictive) and highlighting that despite the discussion of these as separate clinical entities, they may overlap in clinical practice [19, 23] . the most challenging aspect of the diagnosis of tbp remains to be establishing a tuberculous etiology. despite best efforts, 15-20% of the pericardial disease are never diagnosed [24] , reflecting the overall paucity in reliable, cost-effective new diagnostic tests that can rapidly aid clinical decision-making. adenosine deaminase (ada) remains the most widely used biochemical test even though it is limited as a rule in test [25] , and the yields from fluid microscopy and culture remain both low and time-consuming. the result is that the practice in many high tb burden regions has been to treat for tb empirically [19] despite observational evidence that empiric therapy is associated with higher morbidity and mortality [26] . for ease of diagnosis in tb endemic countries, some authors have developed criteria for diagnosis of definitive and probable tbp [27] based on clinical and laboratory findings (table 2) . pericardiocentesis, when feasible (effusion > 1 cm), remains an essential part of the approach to patients with suspected tbp. it provides not only therapeutic but also diagnostic benefit [28] . a typical finding from the aspirated fluid is a protein-rich lymphocytic exudate that is frequently macroscopically bloodstained. light's criteria remain the most helpful to distinguish between transudates (usually heart failure) and exudates [29] . culture of pericardial fluid remains the most widely used diagnostic test for tbp with sensitivity ranging between 53 and 75%; however, it takes at least 3 weeks to yield results [30] . there have been no new applicable technologies or methods to increase the reduced yield of pericardial fluid smear for acid-fast bacilli (afb); however, the high specificity of positive microscopy may justify its continued use [31] . concomitant hiv infection does seem to affect the yield of pericardial fluid culture. a recent study involving predominantly hiv-infected participants (72%), and making use of liquid culture media only, detected culture positivity in 46 out of 131 (35%) of pericardial fluid specimens [31] . where there is diagnostic doubt despite biochemical ada and fluid culture, or where the pericardial fluid is difficult to obtain or in the research setting, a biopsy of the pericardium or epicardium may be warranted [32•, 33] . however, there are no new techniques or tools to increase the yield from histological analysis, which remains 10-64% [27] . what is new is that problems with obtaining representative pericardial biopsy samples could potentially be overcome by using percutaneous pericardioscopy. a small pilot study using the technique demonstrated a diagnostic yield of 60% using afb staining of biopsy samples, with an additional 13.3% being diagnosed by identification of granulomatous inflammation on histology [30] . xpert mtb/rif and xpert mtb/rif ultra are cartridgebased nucleic acid amplification tests that can detect mtb and simultaneously test for rifampicin resistance in under 2 h. in a study assessing the accuracy of xpert mtb/rif in tbp and other forms of extrapulmonary tb, the sensitivity was 63.8%, with high specificity (100%) [34] . elsewhere, use of xpert mtb/rif on pericardial biopsy samples increased the sensitivity to 78% [35••] . these results do not show much improvement from much older studies on polymerase chain reaction (pcr)-based techniques [36] . finally, it is notable that the use of xpert mtb/rif in combination with ada does increase the sensitivity and overall accuracy of the diagnosis of tbp and could be considered a diagnostic option where there is major diagnostic uncertainty [37] . the associated costs of such a strategy are high, perhaps explaining the lack of widespread uptake of such a strategy among clinicians. gamma-interferon (ifn-γ) is produced in large amounts by both cd-4+ and cd-8+ t lymphocytes in the setting of tbp, which has been demonstrated to be highly accurate as a diagnostic biomarker since it was first evaluated [38] [39] [40] . a recent meta-analysis of four published studies found an overall sensitivity (97%) and specificity (99%) of pcf ifn-γ to diagnose tbp, in addition to a positive likelihood ratio of 187 and negative likelihood ratio of 0.03, which provides strong evidence that pcf ifn-γ could be used as a stand-alone test to diagnose or exclude tbp [41••] . despite its clear superiority, ifn-γ is not a widely available commercial test in diagnostic laboratories in endemic areas due to high cost. tuberculin skin test (tst) and interferon-gamma release assays (igras) are of limited value in areas of high tb endemicity as positivity of either merely reflects exposure to mtb antigens and cannot accurately discern active tb disease [42] . furthermore, the purified protein derivative (ppd) used by the tst can yield false-positive results in individuals vaccinated with bacilli calmette-guerin (bcg) and in those who have been sensitized to nontuberculous mycobacteria (ntm). the treatment of tbp is aimed at achieving three goals: killing and control of active mtb; relief of the cardiac compression and adverse hemodynamic sequelae (tamponade and heart failure); and the prevention of complications of maladaptive pericardial remodeling and healing, including constrictive pericarditis. the goal of effective killing and control of mtb using four anti-tuberculous drug regimens (rifampicin, isoniazid, ethambutol, and pyrazinamide) for a minimum of 6 months has been the standard of care for decades in the absence of evidence of comparative effectiveness. this is important because of recent findings from a small pharmacokinetic and pharmacodynamic study, which demonstrated that there is low penetration of anti-tb drugs in the pericardial space over the first 24 h following drug administration. the median peak pericardial fluid rifampicin concentrations were significantly lower than the minimum inhibitory concentration (mic) [43] . in the same study, pyrazinamide peak concentration was 40 times lower than the ph adjusted mic. isoniazid was the only drug found at adequate concentrations in the pericardial fluid of patients with tbp. these findings suggest that there is an urgent need to investigate higher doses or alternative drug regimens in patients with tbp, in whom poor drug penetration may contribute to the poor outcomes observed. this is particularly true considering recent evidence of the close relationship between the burden of bacillary load within the pericardium and mortality [18] . echocardiographic-or fluoroscopic-guided needle pericardiocentesis remains the treatment of choice to evacuate the pericardium of compressive pericardial fluid and alleviate cardiac tamponade. the european society of cardiology (esc) working group on myocardial and pericardial diseases have developed a triage system based on the opinion of experts which can be used to guide clinical decisionmaking in the management of patients with cardiac tamponade [44] . there are still no studies comparing routine evacuation of the pericardium and evacuation only for clinical tamponade. however, the recent characterization of the pericardial fluid with toxic proinflammatory and profibrotic mediators has added to the list of theoretical benefits the manual removal of these mediators to prevent short-and long-term damage to the pericardium. there is also good circumstantial evidence that in recent prospective studies with high rates of pericardiocentesis [21, 45, 46] , the rates of constrictive pericarditis are lower (5-15%) than the rates (17-30%) quoted from eras when there was less aggressive use of the intervention [3, 20] . the goal of preventing pericardial fibrosis and constrictive pericarditis is of importance in sub-saharan african. this is because the syndrome is an important and relatively common cause of heart failure in the region [47] , where the facilities, expertise, and experience needed to perform definitive surgical pericardiectomy are virtually non-existent [48] . interventions for the prevention of constrictive pericarditis which have been tested or are being tested include corticosteroids, mycobacterium indicus pranii, colchicine, and fibrinolytic therapy. four randomized controlled trials [21••, 46, 49, 50] have evaluated the role of intrapericardial and oral corticosteroids for the prevention of progression to constriction. in a recent cochrane review of the 4 randomized trials [51] , analysis of the cumulative data suggested that there was low certainty of evidence for a 20% reduction in all-cause mortality (risk ratio (rr) 0.80, 95% confidence interval (ci) 0.59-1.09; 660 participants), compared with those who did not receive steroids in whom a significant reduction in pericarditis-related deaths was reported (rr 0.39, 95% ci 0.19-0.80; 660 participants). however, among hiv-positive participants, in the large and most robust of the studies, no evidence for a reduction in mortality was found with a possible increase in the rates of hiv/aids-related malignancy in those with advanced hiv but who were not yet on art at enrolment [52••] . the same study showed a reduction in the risk of constrictive pericarditis and rehospitalizations in participants on corticosteroids independent of hiv status [21••] . based on these findings, the european society of cardiology pericardial disease guidelines recommend that it may be reasonable to use adjunctive corticosteroids at a tapering dose of 120 mg of prednisolone over 6 weeks in patients with tbp without hiv infection, and to avoid their use in hiv-infected individuals because of the increased risk of malignancy [28] . the use of m. indicus pranii immunotherapy was evaluated in the same rct by factorial design and was found to be neutral on all efficacy outcomes in hiv-negative participants, with increased malignancy in the subset with advanced hiv but not on art [21••] . in an underpowered south african study, investigators tested whether the positive impact of the drug colchicine on preventing complications in patients with idiopathic pericarditis [53] could be extended to patients with tbp. they tested the hypothesis that colchicine taken daily for 6 weeks may reduce the rates of constrictive pericarditis relative to a placebo control arm receiving "usual care." the results were neutral, with no benefit or harm found [54] . finally, intrapericardial fibrinolysis is currently under investigation in the second investigation of the management of pericarditis (impi-2) trial (https://clinicaltrials.gov/ct2/show/ nct02673879). the rationale for its use is based on its theoretical ability to break up loculated fibrin strands allowing for complete evacuation of the pericardium and reduction in mediators of fibrosis; and 60 years of experience from case reports and small case series where success rates have been high [55••] . the multicenter trial is currently in its feasibility phase. tuberculous pericarditis remains a major cause of cardiovascular death and disability in tb endemic regions of the world, particularly where the prevalence of hiv is high. historically, contributors to this high morbidity and mortality have included the following: little progress in our understanding of the immunopathogenesis of the disease in order to identify potential targets for therapy; failure to develop a relatively inexpensive, rapid, accurate, and widely available test that can be used out in the field at the point of contact in patients with the suspected disease; a better understanding of the determinants and drivers of post-tuberculous fibrosis and constrictive pericarditis; more detailed understanding of the pharmacokinetic and pharmacodynamics of anti-tuberculous drug in both plasma and the pericardium and its impact on short-and long-term outcomes. in this review, we have highlighted areas where there have been significant developments, such as a more detailed study of the immune pathways, cells, and cytokine mediators of the response to tb; those such as diagnosis of tuberculous etiology, and interventions to reduce mortality and morbidity where progress has been elusive; and described those areas where there may be opportunities to turn the tide on this burdensome disease. conflict of interest godsent isiguzo, elsa du bruyn, patrick howlett, and mpiko ntsekhe declare that they have no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. the article provides hope for eradication of tuberculosis by the finding that m72/as01e provided 54.0% protection for m. tuberculosis-infected adults against active pulmonary tuberculosis disease prevalence of tuberculosis in post-mortem studies of hiv-infected adults and children in resource-limited settings: a systematic review and meta-analysis tuberculous pericarditis: ten year experience with a prospective protocol for diagnosis and treatment pericarditis-a five year study in the african epidemiology of pericardial effusions at a large academic hospital in south africa global epidemiology of childhood tuberculosis the burden of presumed tuberculosis in hospitalized children in a resource-limited setting in papua new guinea: a prospective observational study tuberculous pericardial effusions in children tuberculous pericarditis in children: a review of 44 cases tuberculous pericarditis tuberculous pericarditis with and without hiv characterization of the immunological features of tuberculous pericardial effusions in hiv positive and hiv negative patients in contrast with non-tuberculous effusions hiv-1 infection alters cd4+ memory t-cell phenotype at the site of disease in extrapulmonary tuberculosis effect of prednisolone on inflammatory markers in pericardial tuberculosis: a pilot study a compartmentalized profibrotic immune response characterizes pericardial tuberculosis, irrespective of hiv-1 infection ac-sdkp (n-acetyl-seryl-aspartyl-lysyl-proline) and galectin-3 levels in tuberculous pericardial effusion: implications for pathogenesis and prevention of pericardial constriction the effects of angiotensin converting enzyme inhibitors (ace-i) on human nacetyl-seryl-aspartyl-lysyl-proline (ac-sdkp) levels: a systematic review and meta-analysis tuberculous pericarditis is multibacillary and bacterial burden drives high mortality clinical characteristics and initial management of patients with tuberculous pericarditis in the hiv era: the investigation of the management of pericarditis in africa (impi africa) registry experience with pericarditis at groote schuur hospital, cape town: an analysis of one hundred and sixty cases studied over a six-year period 1121-30 this landmark rct was the first in africa to investigate the place of steriod in tuberculous pericarditis. the results show that among hiv negative patients with tbp hiv infection is associated with a lower incidence of constriction in presumed tuberculous pericarditis: a prospective observational study a modern approach to tuberculous pericarditis epidemiology of pericardial diseases in africa: a systematic scoping review diagnostic accuracy of adenosine deaminase for tuberculous pericarditis: a meta-analysis mortality in patients treated for tuberculous pericarditis in sub-saharan africa tuberculous pericarditis esc guidelines for the diagnosis and management of pericardial diseases: the task force for the diagnosis and management of pericardial diseases of the european society of cardiology (esc) endorsed by: the european association for cardio-thoracic surgery (eacts) pleural effusions an approach to the patient with suspected pericardial disease determinants of pcr performance (xpert mtb/ rif), including bacterial load and inhibition, for tb diagnosis using specimens from different body compartments one of the issues that delay early commencement of treatment in tbp is definitive diagnosis. the authors of the article introduce use of a pericardioscope for etiology of large pericardial effusions diagnostic accuracy of quantitative pcr (xpert mtb/rif) for tuberculous pericarditis compared to adenosine deaminase and unstimulated interferon-γ in a high burden setting: a prospective study ): e0188704. the world health organization has identified development of point of care diagnostic tools as the key in improvement of diagnosis and outcome in tuberculosis. the authors of this article show the high sensitivity of xpert mtb/rif in tbp. however, its non-superiority compared with older pcr is a drawback and calls for further investigations comparison of pcr, culture, and histopathology for diagnosis of tuberculous pericarditis diagnostic accuracy of quantitative pcr (xpert mtb/rif) for tuberculous pericarditis compared to adenosine deaminase and unstimulated interferon-gamma in a high burden setting: a prospective study diagnostic value of interferon-gamma release assays on pericardial effusion for diagnosis of tuberculous pericarditis the use of adenosine deaminase and interferongamma as diagnostic tools for tuberculous pericarditis tuberculous pericardial effusion: analysis of commonly used diagnostic methods the authors highlight the role of interferon gamma as a stand-alone test to exclude or diagnose tbp, which is a potential step in rapid diagnosis interferon-gamma release assays for the diagnosis of latent tuberculosis infection in hiv-infected individuals: a systematic review and meta-analysis poor penetration of antibiotics into pericardium in pericardial tuberculosis triage strategy for urgent management of cardiac tamponade: a position statement of the european society of cardiology working group on myocardial and pericardial diseases a "vanishing", tuberculous, pericardial effusion the management of tuberculous pericardial effusion: experience in 233 consecutive patients the causes, treatment, and outcome of acute heart failure in 1006 africans from 9 countries cardiac surgery capacity in sub-saharan africa: quo vadis? controlled trial of prednisolone as adjuvant in treatment of tuberculous constrictive pericarditis in transkei management of tuberculous constrictive pericarditis and tuberculous pericardial effusion in transkei: results at 10 years followup interventions for treating tuberculous pericarditis rationale and design of the investigation of the management of pericarditis (impi) trial: a 2 x 2 factorial randomized double-blind multicenter trial of adjunctive prednisolone and mycobacterium w immunotherapy in tuberculous pericarditis colchicine for recurrent pericarditis (corp): a randomized trial a prospective investigation into the effect of colchicine on tuberculous pericarditis the authors of this article posit that fibrin deposition could be central in the development of constrictive pericarditis and a potential target in its prevention cardiovascular magnetic resonance characterisation of pericardial and myocardial involvement in patients with tuberculous pericardial constriction with and without hiv co-infection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-025628-9611eglg authors: bonagura, vincent robert; rosenthal, david walter title: infections that cause secondary immune deficiency date: 2020-05-29 journal: stiehm's immune deficiencies doi: 10.1016/b978-0-12-816768-7.00049-1 sha: doc_id: 25628 cord_uid: 9611eglg discriminating between patients with microbial infections that cause secondary immune effects from those with the same infection who have primary immune deficiency disease can be difficult. there are many microbes that temporarily “stun” innate and/or adaptive immunity during a primary infection. the common result of temporary inhibition, or permanent depletion of host immunity during some primary infections is the development of superinfections of other microbes that cause significant morbidity, and on occasion, mortality. in addition, microbes that cause persistent infection, such as the human immune deficiency virus, deplete effective immunity over time and can also lead to secondary infections with other microbes ultimately leading to death if not appropriately treated. in some cases, such as influenza virus infection, mortality can be dramatic due in large part to acquired secondary bacterial infections. many microbes manipulate host immunity and thereby inhibit the ability of patients to combat secondary microbial infections. the overall survival of patients primarily infected with some viruses, parasites, or bacteria, is closely linked to the ability of secondary infections to take advantage of the immune modulation induced by the primary infection. herein we discuss some of the secondary immune defects caused by select viruses (measles, influenza, hiv1, htlv), parasites, (leishmania, malaria), and bacteria (bordetella pertussis). furthermore, the genetic susceptibility of a given host to become infected, or develop severe disease, also determines whether an infected individual who becomes infected with a secondary microbe survives. future studies are needed to explore not only the immunomodulation caused by select microbes, but also the repertoire of immune responses expressed by infected individuals in order to predict clinical outcomes of these infections. thus, understanding the delicate balance that exists between “immune altering” microbes that suppress immune responses during primary infections leading to severe secondary infections versus those infections found in patients with inborn errors of innate or adaptive immunity, is essential in predicting the clinical outcome and the appropriate treatment for these two different patient populations. immune deficiencies can be subdivided into two categories: primary or secondary. this chapter describes secondary defects related to infection, and other forms of secondary immunodeficiency are described elsewhere in this book. some microbes manipulate or exhaust effector immune responses, leading to secondary infections by other microbes. while the manipulation or exhaustion of immune responses is an important defense mechanism of some microbes to evade effective immune clearance, it is also likely that the genetic immune response repertoire that programs how an infected individual will respond to these microbes contributes significantly to whether the resulting immune responses will protect against, or facilitate, superinfection by other microbes. in this chapter, we discuss how some microbes manipulate or, in the case of the human immune deficiency virus (hiv), exhaust protective innate and/or adaptive immunity, ultimately leading to severe microbial superinfections by other infectious organisms that cause significant morbidity and, on occasion, mortality from secondary infections. a critical component of host defense includes the expression of immunosuppressive cytokines. il-10, and tgf-b, and the generation of regulatory t cells that can express these cytokines serve to limit the immune-mediated damage related to host defense. these same elements also cause secondary immune deficiency by suppressing or blocking effector th1-like responses. these immunoregulatory elements, generated in response to the original infection, as a consequence, can lead to secondary immune deficiency and the development of severe or fatal infection with other microbes. some of the microbes that cause temporary or permanent changes in immune responses to other organisms are shown in table 49 .1 (see below). in addition, fig. 49 .1 shows the functional diversity of cd4 þ t cells that has evolved to control the microbiome present on mucosal surfaces. in concert with the repertoire of these t cells, and the cytokines and chemokines that they express, the continuum of macrophages ( fig. 49. 2) 1 and their respective cytokine/chemokine repertoires expressed in response to microbial infection, form the critical immune response elements that are required to mount and maintain an effective immune response against these microbes. the amount of immunomodulatory cytokines, such as il-10, expressed during infection contributes to the development of secondary infections during and after recovery from the primary infection. alteration in the balance between immunity and immune suppression is in part, based on how much il-10 is expressed during a given infection. il-10 promoter polymorphisms that control high, intermediate, or low expression of il-10 2e4 during and after microbial infection may influence the development of resistance or susceptibility to secondary microbial infection. excessive il-10 levels defined by a given host's il-10 genotype could predispose an individual to develop secondary infections by blocking appropriate pro-inflammatory responses. in contrast, low il-10 levels, also defined by a given host's genotype, could protect against secondary infection, at the expense of limiting collateral tissue damage and t cell memory that higher il-10 expression would support. 5 taken together, the successful balance of cellular innate and adaptive immune responses, and the cytokine/chemokine repertoires that they express during a primary infection to an infectious microbe, can temporarily or permanently "hijack" effective immune responses that are necessary to prevent other infections by microbes it is often difficult to distinguish secondary immune deficiency in a normal individual with a significant initial primary infection, where the offending microbe temporarily subverts or paralyzes the adaptive immune response to other microbes, from a patient with primary immune deficiency disease (pidd) who presents with a serious initial infection (box 49.1). several clues help distinguish between an initial infection in these different patient populations. 6e8 among these clues that patients with pidd are more likely to have are: patients with normal immune systems who become infected with viruses, bacteria, or parasites known to temporarily subvert or paralyze adaptive immunity without the above history are more likely to have secondary immune defects caused by microbes that prevent appropriate immunity to other organisms. thus, the challenge for clinicians is to distinguish between patients with pid versus those with microbe-induced, secondary immune deficiency at the time of first presentation of a serious infection. in this review, we address the patient population with secondary, infection driven immune compromise, in contrast to patients with pidd who are the focus of the remainder of this textbook. sepsis with a wide variety of organisms causes a profound secondary immune deficiency. it is not an uncommon scenario to have to rule out a pidd in the intensive care setting where both the underlying disease and medications may alter the immunologic findings. sepsis is one of the more common causes of secondary lymphopenia (box 49.1). furthermore, there are both acute disruptions of immunologic function and longer term findings that are poorly characterized that are seen after trauma and burn injuries as well. in fact, increased mortality persists for years after an episode of sepsis. immunologic findings occurring with acute sepsis include: examples of microbes that cause temporary or long-term secondary immune deficiency 9 shows some of the microbes that are known to induce secondary immune deficiency, the cells that they affect, and the pathogens causing secondary infection. in some cases, secondary immune deficiency occurs as a result of a cytokine storm. some infections known to cause a cytokine storm are shown in box 49.2. below, we describe examples of how some of these microbes suppress immunity and thereby cause secondary immune deficiency, predisposing patients to secondary microbial infections. measles virus (mv) continues to cause child morbidity and mortality worldwide, despite the availability and use of an effective live attenuated measles vaccine. 10e14 part of the reason why control of mv continues to be elusive is that it is highly contagious for susceptible individuals and there are difficulties with vaccine delivery. mv infection begins in the respiratory tract, spreads systemically in lymphoid, epithelial and endothelial cells, and ultimately infects multiple organs, 15 causing a characteristic fever, rash, and conjunctivitis 10e14 days after respiratory infection ( fig. 49.3a and b). high fever, rhinorrhea and conjunctivitis typically precede the rash and the rash migrates from the head and neck to the hands and feet over 3e4 days. many of these manifestations are caused by the immune response made to mv, and commonly this response clears mv in infected tissues and prevents re-infection for life (fig. 49 .3c and d). however, mv infection can cause several weeks of immune suppression after resolution in select individuals. this is the primary cause of measles-associated deaths: mv-induced secondary infection. 16 although vaccination against measles is very high in the united states, 92.7% of children aged 19e35 months were vaccinated in 2017, 17 there are pockets of unvaccinated people who are susceptible to local outbreaks of measles. since "herd immunity" is primarily effective when the vaccination rate is around 96%, 18 vaccination levels below this level leave children and adults at risk for primary mv infection and secondary microbial infections. begin approximately 10 days after infection, with prodromal symptoms of fever, conjunctivitis, and oral koplik's spots, followed by a maculopapular rash for 3e5 days. (c) the rash represents adaptive immune responses with infiltration of cd4 þ and cd8 þ t cells into sites of mv replication and clearance. there is a rapid activation, expansion, and then contraction of mv-specific cd8 þ t cells. cd4 þ t cell responses appear at the same time, but activation is prolonged. mv-specific igm appears with the rash, and this is commonly used for diagnostic purposes. this is followed by the sustained mv-specific igg synthesis. immune suppression is evident during acute disease and for many weeks after recovery. (d) cytokines and chemokines produced during infection are found in plasma in elevated amounts. early, il-8 is increased. during the rash, ifn-g and il-2 are g and il-2 are produced by activated t h 1-like, cd4 þ and cd8 þ t cells. after rash resolution, t h 2-like t cells and regulatory cd4 þ t cells produce il-4, il-10, and il-13. measles was the first virus clearly identified to cause increased susceptibility to other microbial secondary infections. most often, measles-associated deaths are caused by severe, overwhelming pneumonia and diarrhea. 16 suppression of delayed hypersensitivity has been identified in tuberculin-sensitized individuals many weeks after complete resolution of mv infection (fig. 49 .3c). 19 furthermore, several weeks after successful mv recovery, increased susceptibility to other infections has been reported, and t cell function and in vitro proliferation of t cells in response to mitogens has been shown to be markedly decreased (fig. 49.4a and b) . 1, 20, 21 immunosuppression occurs during a period of intense immune activation that occurs during the onset of the mv rash and anti-mv immune responses (fig. 49.3c and d) . lymphopenia, skewing of th2-like chemokine polarized responses, and suppression of lymphocyte proliferation have also been documented (fig. 49.3d ). mv infection causes decreases in t and b cells in the blood during the mv rash period. 22e25 altered trafficking and increased apoptosis of mv-infected and uninfected lymphocytes contribute to the development of lymphopenia. 22,26e30 while lymphocyte numbers rapidly return to normal in the blood after the rash resolves, immunologic abnormalities persist. 21, 22, 31, 32 immune suppression, th2 cytokine polarization of cd4 þ t cells, and treg induction have been associated with indirect immunosuppression caused by mv infection. 33, 34 mv infection is also associated with suppression of il-12 expression, lymphocyte cd30 expression, and il-4, il-10, and il-13 expression after rash resolution. 35e37 reduction of il-12 production reduces t cell expression of type i cytokines, particularly ifn-g 10,32 (fig. 49.3d ). it is possible that mv interacts with the complement regulatory molecule cd46 in polarizing th2-like cytokine production, causing activation of signaling cascades that modify cell function, although this interaction is not firmly established. 38, 39 the mv-cd46 interaction may alter innate immunity by selectively downregulating receptor expression. 40e46 this would increase susceptibility to complement-mediated lysis of mv-infected cells, and decrease antigen presenting cell production of il-12 47, 48 and crosslinking of cd46 on t cells, leading to the induction of regulatory cd4 þ t cells and enhanced il-10 levels. 49 these interactions would induce th2-like polarization that would favor b cell maturation, provide lifelong mv antibody memory, and protect against mv reinfection. this polarization, however, would also depress apc activation and th1-like responses to new pathogens. mv suppresses pbmc proliferation to mitogens after mv resolution, and this continues for several weeks (fig. 49.4b ). 20,31 il-2 supplementation can improve, but not fully restore, this responsiveness. this suggests that defective il-2 expression is in part responsible for this proliferative defect. 50 cell cycle arrest in g1 after in vitro infection with mv is a recognized cause of hyporesponsiveness to mitogens. 42,51e53 mv rna can persist in pbmcs for months after mv resolution 54, 55 and may reduce mitogen proliferation, although this has not been established. the receptor used by wildtype mv to infect cells, cd150, is a dual function co-receptor for lymphocyte activation, and enhances ifn-g expression. 56e58 however, mv binding to cd150 can also downregulate receptor expression. 59,60 t cell signaling through the mv glycoprotein complex of h and f1-f2 in the membranes of virions or mv-infected cells 61e65 may also contribute to immunosuppression. this inhibitory signal prevents t cell s-phase entry for several days, and is independent of cell death, membrane fusion, soluble inhibitor production, or t cell infection. 52,61,62,65e67 thus, there is a delay in cell cycle progression and an accumulation of t cells in the g0/g1 phase. 52, 66, 67 the mechanism by which h/f1-f2 suppresses mitogen-induced proliferation is unknown, but it is associated with mv-induced interference of t cell activation of phosphoinositide 3-kinase (pi3k) in t cells, or il-2 receptor ligation. 68 il-2 added to mv-treated cells activates signal transducer and activator of transcription 3 (stat3) but fails to activate akt kinase, which is required for cell cycle progression. 69 the modulatory effects of mv with glycoprotein complexes, and the downstream consequences of this interaction, have recently been summarized. 10,68e70 while the relevance of these processes to the in vivo suppression of t cell lymphoproliferation remains to be identified. the combination of the established mechanisms leading to post-mv infection immunosuppression, and those that remain to be elucidated, cause, in select individuals, severe and on occasion, fatal secondary infection with other microbes. a key epidemiologic factor in measles-related deaths is vitamin a deficiency. in the developing world, the world health organization recommends vitamin a supplementation. studies have demonstrated improved outcomes and suggest an effect on the mucosal barrier and also on improved t cell function though by an undefined mechanism. three world-wide (pandemic) outbreaks of influenza virus (iv) occurred in the 20th century, in 1918, 1957, and 1968. they are now known to represent three different antigenic subtypes of influenza a virus: h1n1, h2n2, and h3n2, respectively. 71 the 1918 "spanish flu" epidemic claimed an estimated 50 million lives and 20%e40% of the worldwide population became ill; approximately 675,000 americans died during this epidemic. 72 since then, iv infections have caused more than 20,000 deaths yearly in each of the 20 epidemics from 1957 to 1991, 73 and greater than 40,000 deaths occurred in each of the more recent epidemics, thus, iv is a formidable pathogen. 74 while influenza-related mortality can in part be attributed to direct effects on the respiratory system, many of the deaths associated with iv infection are caused by increases in susceptibility to secondary bacterial pneumonia. 75 in fact, it has been suggested that the major mortality and morbidity resulting from iv infection may be caused by secondary bacterial infection that is associated with the inhibition of neutrophil function. 73 the bacteria commonly causing pneumonia in the most severely ill influenza patients are streptococcus(s.) pneumoniae, staphylococcus aureus, and haemophilus influenzae. together, iv infection itself, and secondary bacterial pneumonia, were the most common causes of infectious death in the united states in 2002. 76 research, clinical, and epidemiological studies show that there is a positive correlation between the increase in morbidity and mortality during influenza epidemics and pandemics, and an increase in secondary s. pneumoniae infection. 75, 77, 78 it has been hypothesized that influenza infection alters neutrophil function, thereby reducing the effectiveness of phagocytemediated killing of bacteria. an alternative hypothesis is that the tissue damage caused by iv alters the epithelial surface of the respiratory tract, thereby exposing different surface receptors to which s. pneumoniae adhere, and/or increasing the affinity of s. pneumoniae for its receptors, which may result in increased growth and decreased neutrophil killing of s. pneumoniae in the respiratory tract. support for the former hypothesis comes from reports using both in vitro and in vivo models of influenza infection. 79e90 neutrophils are important in resistance to s. pneumoniae infection independent of an influenza infection. 91 influenza a virus alters the three major properties of the neutrophil that are crucial for bacterial clearance, namely chemotactic responsiveness, phagocytosis, and intracellular killing. this alteration of neutrophil function likely increases the susceptibility of an influenza-infected patient to s. pneumoniae infection because of decreased phagocytosis and killing of these bacteria by neutrophils. 80, 84, 89, 92 in humans, iv was reported to alter cell function by interacting with g proteins. 81, 90, 93 human neutrophils express monomeric and trimeric g proteins that have critical roles in activation and regulation of various signal pathways, leading to chemotaxis and metabolic function. 94, 95 susceptibility to s. pneumoniae in mice is greatest at six days after influenza infection, consistent with the clinical findings. 96e99 influenza-induced tissue damage is also greatest six days after influenza infection, as is adherence of s. pneumoniae to murine influenza-infected tracheas. 100 influenza-induced neutrophil dysfunction is also greatest six days after influenza infection, 101 and murine mortality secondary to s. pneumoniae infection is greatest seven days after infection. although neutrophils accumulate in the lungs of mice infected with influenza by day six, they do not function in resistance to s. pneumoniae. thus, it is likely that bactericidal function of lung neutrophils is suppressed, making these influenza-infected mice susceptible to s. pneumoniae. effective neutrophil phagocytosis and killing of s. pneumoniae is necessary to eliminate s. pneumoniae from the lungs. 102 neutrophils are affected within 30 min of in vitro influenza infection, 83, 84, 87, 89, 103 with some effects seen as rapidly as 5 min after incubation with iv. changes in infected neutrophils include decreased protein phosphorylation, accelerated apoptosis, decreased respiratory burst activity, an altered cytoskeleton, depressed bactericidal capacity identified by release of reactive oxygen species, decreased chemotactic ability, decreased adherence, decreased release of lactoferrin into phagosomes, and inhibition of lysosomeephagosome fusion. 81, 82, 84, 87, 89, 92, 103 in addition, the effects of influenza virus infection on neutrophil function are not limited to the lungs, indicating that these effects are systemic in nature. 89, 90 influenza infection also increases susceptibility to s. pneumoniae by increasing cytokine production (tnf, ifn-g, mcp-1, il-10, il-6) in the lung. 75 il-10 is also elevated in post-influenza pneumococcal pneumonia, leading to increased susceptibility to s. pneumoniae long after an influenza infection has been resolved. 104 susceptibility remains at least 14 days after a primary influenza infection, and is thought to be mediated by il-10 inhibition of neutrophil function, resulting in increased bacterial growth in the lungs leading to mortality. 104 of note, viral influenza neuraminidase increases s. pneumoniae adherence in the lungs by cleaving sialic acid residues, and exposing receptors to which s. pneumoniae can adhere. 78 thus, both neutrophildependent and -independent mechanisms cause increased susceptibility to secondary s. pneumoniae infection after a primary influenza infection. novel therapies that can restore neutrophil function caused by iv infection may be helpful. 73, 75 human immune deficiency virus: long-term immunosuppression hiv is a double-stranded, enveloped rna retrovirus from the genus lentivirus within the subfamily retroviridae, with a tropism for human cd4 þ cells, including t cells and macrophages. 105 two hiv types have been identified, hiv-1 and hiv-2, and both cause similar but not identical human disease. the hiv genome contains three structural genes (gag, pol, and env) and six regulatory genes (tat, rev, nef, vif, vpr, and vpu). these proteins, their function, and their role in forming the viral particle have recently been summarized. 105 the env protein is cleaved to produce two envelope proteins, gp120 and gp41, which are involved in hiv binding to cd4 and the chemokine receptors cxcr4 and ccr5 on cell surfaces. 105 tat, nef, and rev proteins play a role in downregulating classical mhc class i molecules on the surface of hiv-infected t cells. 106e108 in the case of tat, hla-c and hla-e are spared. 109 the classical hla presenting molecule hla-c on antigen presenting cells is not as potent in presenting viral peptides to t cells similar to the non-classical mhc class i molecule hla-e. however, the non-classical mhc class i molecule hla-e, similar to hla-a, hla-b, and hla-c, suppress nk function. this viral strategy to evade immune surveillance serves two functions: (1) prevent cd8 þ t cells from recognizing hiv peptides presented by class i mhc molecules, and (2) prevent nk cell recognition and activation because hla-c and hla-e remain on hiv-infected cells leading to inhibition of nk killing of class i mhc expressing target cells. the nef protein also downregulates cd4 expression on the surface of hiv-infected cells, which is a co-receptor of the t cell receptor participating in t cell activation. 110 this facilitates hiv-infected cell escape from immune surveillance. following hiv gp120 protein binding to cd4 and ccr5 molecules on target cells, hiv-infected cells migrate to the lymph nodes, where initial replication and infection of nearby cd4 þ t cells occurs. 111 during acute hiv infection, gut-associated lymphoid tissues, predominantly memory cd4 þ t cells, are severely depleted. there is high hiv viremia and immune activation. 112, 113 hiv induces t cell lymphopenia through several mechanisms: hiv-induced apoptosis; a viral cytopathic effect; non-specific immune activation-induced apoptosis; and cytotoxicity of hiv-infected cells. 105 autophagy is also induced by hiv env protein in uninfected t cells. 114 in addition, shedding of gp120 molecules by hiv triggers a series of events that cause the adaptive immune system to become less effective by altering the normal balance of immunoregulatory th1 and th2 t cells. impaired function of hiv-infected macrophages and dendritic cells contributes to the failure of effective innate and adaptive immune responses to secondary infection. the acute and latency phase of hiv infection is shown in fig. 49 .5. 115, 116 without combined antiretroviral therapy (carv), cd4 þ t cell counts progressively decrease, and the host usually succumbs to infections with opportunistic organisms that occur because of hiv-induced secondary immune deficiency. specific anti-hiv, cd4 þ , and cd8 þ t cells, and neutralizing anti-hiv antibodies develop, however, these responses are eventually overcome by viral escape strategies. 105 patients can present with constitutional symptoms, such as fever, weight loss, diarrhea, lymphadenopathy, secondary opportunistic infections, and viral skin infections, heralding the presence of an immunocompromised immune system. peripheral cd4 þ t cell counts of <200 cells/ml herald the development of aids. this t cell depletion predisposes patients to develop opportunistic infections including, but not limited to, cytomegalovirus, herpes simplex, varicella zoster virus, pneumocystis jirovecii pneumonia, histoplasmosis, toxoplasmosis, coccidioidomycosis, cryptosporidium, nocardia, mycobacterium avium complex, salmonella, and toxoplasma gondii. if hiv-infected patients do not receive antiretroviral treatment, repeated infections with these opportunistic secondary infections ultimately lead to death. thus, hiv infection exhausts the adaptive immune system, leading to chronic depletion of cd4 þ t cells and immune dysfunction of the multiple effector immune responses that are required to prevent secondary bacterial, viral, fungal, and protozoan infections. human t-lymphotropic viruses (htlv) are complex type c retroviruses with a capsid that contains two simple rna strands with associated reverse transcriptase and integrase enzymes which are essential for insertion of the virus into the host genome. 117 several types of htlv have been identified (htlv-1, htlv-2, htlv-3, htlv-4). while hiv-1 was previously called htlv-3, htlv-3 is a different virus. 117 similar to other retroviruses, htlv-1 contains gag, pro/pol and env genes that have structural and functional roles. in addition, the px region codes for regulatory proteins (such as transactivator protein, tax, and the helix basic zipper protein, hbz) essential for viral transcription, and inhibition of signal transduction pathways, such as nfkb (leading to decreased socs1), and ap-1. this leads to htlv-1 inducing cytotoxic t cells that can kill virus-infected cells. 118 the htlv-1 tax gene can decrease th1-like antiviral signaling pathways both by modulating the suppressor of cytokine signaling 1 (socs1) 119 and also through the aryl hydrocarbon receptor protein (aip) that binds to interferon regulatory factor 7 (irf7), thus decreasing type i interferon (ifn-a/b) expression. 120 htlv-1 modifies the behavior of cd4 þ t cells and alters their cytokine production. htlv-1 is clinically associated with adult t cell leukemia/lymphoma (atl), and tropical spastic paraparesis/htlv-1-associated myelopathy (pet/ham) 121 and can cause autoimmune disease, such as rheumatoid arthritis, systemic lupus erythematosis, and sjögren's syndrome. 117e120 the development of the htlv-1 induced autoimmunity is thought to rely on molecular mimicry, and htlv-1 induced t cell immunosuppression is caused by direct infection of cd4 þ t cells. cd4 þ t cells that have altered function and cytokine production. tax is the primary inducer of clonal infected t cell expansion, and genetic instability. 117 tax expression promotes t cell activation, proliferation, and resistance to apoptosis. 122 clinically, patients infected with htlv1 have increased infections and increased autoimmunity, particularly rheumatoid arthritis and sjogren syndrome. the autoimmunity correlates somewhat with viral load, however, there are immunologic mechanisms that probably facilitate breaks in tolerance leading to autoimmunity. in addition, natural killer (nk) cells from individuals with pet/ham have decreased expression of the activating receptor nkp30. 123 this likely results in decreased nk cell activation, leading to decreased ability of nk cells to kill virusinfected cells. htlv infection is clinically associated with an increased risk of disseminated strongyloidiasis, 124 schistosomiasis, 125 likely due to high levels of ifn-g, decreasing the production of th2-like cytokines (il-4, il-5, il-13) and ige essential to contain these parasitic infections. taken together, htlv induces cytotoxic t cells to kill virus-infected cells, alters cd4 þ t cell function and cytokine production and it decreases nk cell activation leading to susceptibility to subsequent disseminated parasitic infections. many viral infections have been associated with bm failure or hyperproliferative syndromes. table 49 .2 summarizes some of the human viral infections that cause self-resolving or persistent bm suppression. 126 however, the specific pathogenic mechanisms that underlie the reported virally induced bm manifestations have not been fully characterized. nonetheless, many different viruses generate the same pathological condition. this suggests that a common underlying mechanism(s), specifically virologic or immunologic are responsible for the clinical pathologic outcome. certain virus can also lead to different pathological manifestations in different patients heralding a genetic basis for aberrant immune activation in bm failure. there are 4 different mechanisms by which viruses can affect hematopoietic stem and progenitor cells (hspc). these mechanisms include: (1) direct viral infection, (2) viral recognition by hspcs and indirect effects, (3) inflammatory mediators, and (4) changes in the bm microenvironment. taken together, many types of viruses can affect hematopoiesis acutely (parvovirus b19, dengue), transiently, permanently, chronically (cmv, hiv), systemically (hiv), locally (influenza), directly, or indirectly, causing disruption of the hematopoietic process. 126 parasites that hijack immune responses to other microbes leishmaniasis: temporary immune suppression vl is fatal if not treated, and is caused by leishmania donovani, leishmania infantum, and leishmania chagasi. 129, 130 immunity to leishmaniasis is mediated by the cellular and humoral arms of the mammalian immune system: the innate system (by neutrophils, macrophages, and dendritic cells) and by adaptive (t cells) responses. 131 sand-fly bites cause minimal tissue damage, but the bite promotes neutrophil recruitment. 132, 133 parasites can survive for a time within neutrophils, and eventually they parasitize neutrophils. viable parasites are engulfed by macrophages or dendritic cells. inside the macrophages, promastigotes change into amastigotes and they reproduce by binary fission, ultimately rupturing the macrophage and releasing amastigotes into the blood. leishmania modulates the normal antimicrobial mechanisms of the macrophages. they increase membrane fluidity and thereby disrupt lipid rafts and apc antigen presentation as well as lysosomal fusion. 134 leishmania-infected apcs interact with t cells, and together they express cytokines and chemokines that begin to "hijack" the immune system, which enables these parasites to survive. 135 cell-mediated immunity is the principle host defense against leishmania species. t h 1-like cells primed mainly by apcs and il-12 expression are major effector responses to this microbe. 136e139 the dichotomy between t h 1-like protection (ifn-g, il-2, and tnf) and th2like disease progression (il-10, il-4) in mice has been shown to be essential in cl infection, 140 although this paradigm is less clear in humans infected with vl. 141, 142 the immunopathology of vl is complex, and involves cellular and genetic factors that confer disease susceptibility, versus resistance to leishmania species. 143 persistent vl correlates with a chronic th2-like (il-10, il-4, il-5, and il-13) response to l. donovani 142 and increased serum il-10 expression. 144e147 il-10 is crucial in establishing and maintaining t h 2-dependent chronic suppression of th1-like mediated immunity in cl. 148e150 and inhibits amastigote killing. 150,151 il-10 also inhibits the production of ifn-g. 152 cellular immunity impairment correlates with active disease progression secondary to il-10 expression, independent of ifn-g levels. 153 in murine cl, cd4 þ cd25 þ foxp3 þ natural tregs are a major il-10 source and are crucial for parasite survival. 154 however, il-10 is also derived from non-tregs. 146, 155 this suggests that any therapeutic approach to treat leishmania infection will require species-specific manipulation of the immune responses made to these parasites. for example, the potent t h 2-like cytokine il-4 is critical in cl, but not in vl. 141 thus, modulating il-4 expression may be helpful in vc, but not in the treatment of cl. 156 resolution of human and murine vl depends on the production of th1-like cytokines. 139,140,147,157e160 il-12 and ifn-g are crucial in controlling parasite growth and development. 157, 161 while il-10 suppresses host immunity and helps parasite survival, it is the antidote to inflammation that reduces tissue damage caused by exaggerated inflammation. 162 since ifn-g -producing cells can also produce small amounts of il-10, this may act as a negative feedback in controlling tissue damage 142, 146, 163, 164 and supporting immune memory. tnf stimulates ifn-g -induced expression of nitric oxide by apcs that can kill vl parasites. 165e170 th17-like cells that produce il-17 are pro-inflammatory, and stimulate il-6, tnfa, and chemokine expression. however, l. donovani strongly induces il-17 and il-22, and enhances the secretion of these cytokines that protect this parasite. 171 cd8 þ cytotoxic t cells are also important in controlling cl and vl. 172 cd4 þ and cd8 þ t cells, and their pro-inflammatory cytokines, are required to control vl parasite proliferation and leishmaniacidal activity. 138,161,173e176 however, in canine vl, antibodies are insufficient in providing protection in the absence of -proinflammatory t cell responses. 132 thus, in addition to protective t cell responses, other t cell subsets, including tregs and th17 cells, confer either susceptibility or resistance to animal verses human vc. other cells may also be important in the resolution of these infections, thus further investigation is required in designing vaccines or treatments for these diseases. greater than 20% of cl patients have reported secondary bacterial infections. 177 s. aureus, coagulase-negative staphylococcus, escherichia coli, proteus, and klebsiella have been cultured from lesions (listed in decreasing frequency). however, the incidence of these infections is higher in ulcerated compared to non-ulcerated lesions. thus, both disruption of the skin, and possibly immunosuppression caused by cl, likely play roles in the development of secondary bacterial infection in cl, leading to severe or lethal bacterial infection. 177 increased treg numbers in cl have been linked to activation/reactivation of latent microbial infections (mycobacterium tuberculosis, toxoplasma, herpes viruses) that cause significant morbidity and mortality, possibly as a result of immunosuppression and other factors, although how activation/reactivation of dormant infections occurs remains unknown. 178 thus, leishmania infection causes secondary infections, and activation/reactivation of dormant infections that can lead to severe or lethal outcomes through multiple mechanisms. 178 l. donovani infection can also clinically resemble and cause hemaphagocytic lymphohistiocytosis (hlh) and successful treatment of leishmaniasis can lead to the complete resolution of hlh symptoms. 179, 180 these reports open the question as to whether hlh patients should be prescreened and aggressively treated for visceral leishmania infection prior to instituting stem cell transplantation for hlh when from endemic areas. 179 vl has been associated with significant secondary infections. these may be confounded by hlh where neutropenia is common. infections ranging from sepsis to urinary tract infections occur in a significant subset of patients with vl. the most common pathogens causing sepsis are s. aureus, klebsialla pneumonia, and pseudomonas aeruginosa. common laboratory features include neutropenia, eosinopenia and leukopenia. co-infection with hiv alters the clinical presentation with more lung and gastrointestinal involvement. taken together, the innate response (professional apcs) is altered by leishmaniasis infection, leading to polarized adaptive immune responses that support or block persistent infection by leishmania organisms. this subverts or supports effective immunity to these parasites, and in patients with persistent infection, the th2-like/treg bias may predispose patients infected with leishmania species to develop or reactivate lethal secondary superinfections, and the development of hlh-like clinical symptoms. malarial organisms are parasites with a major public health impact world-wide. there were an estimated 219 million cases of malaria (range 154e289 million) and 660,000 deaths (range 610,000e971,000) in 2010. 181 plasmodium species cause malaria and can induce immunosuppression in infected individuals 182 resulting in increased susceptibility to secondary infections, such as non-typhoidal salmonella, 183 herpes zoster virus, 184 hepatitis b virus, 185 moloney leukemia virus, 186 and epstein-barr virus reactivation. 187 efficacy of heterologous vaccines can also be suppressed in malaria-infected patients, 188 further documenting the immunosuppressive effects of malaria infection. malaria parasites inhibit dc maturation 189 via uptake of malaria pigment hemozoin (hz) from parasitized rbcs. 190 dc inhibition reduces t cell expansion, cytokine production, and migration into b cell follicles. 191 the effect on dc changes occurs during the different phases of this infection. although dc function is impaired immediately following the initial burst of parasitemia, t and b cell responses to heterologous antigens change dynamically during the course of infection. 191 t cell proliferation, and effector function and migration are suppressed, and b cells do not expand or produce antibodies. hz, prevents the formation of stable, long-lasting cellecell contacts between t cells and dcs impairing costimulatory activity. 192 nonetheless, there is also a t cells functional defect. 188 taken together, impairment of both innate and adaptive signaling induced by malarial infection/hz pigment skews the immune response toward tolerance instead of immunity, and this subversion of effective immunity leads to secondary infection with other organisms as a consequence of this parasitic infection. invasive infections in patients with malaria are due to s. pneumoniae, h. influenzae type b, s. aureus, e. coli and other gram-negative bacteria. risk factors include younger age, recent malaria infection, severe anemia, splenomegaly, hiv-co-infection and severe malnutrition. some of this susceptibility seems to reflect functional asplenia and impaired barrier function. bordetella pertussis: temporary immunosuppression b. pertussis, the causative agent of whooping cough, is an infection that can be fatal in infants, but in older children, adolescents, and adults usually causes a chronic cough of varying severity that generally persists long after clearance of the infection. this bacterial infection of the airways is an important cause of infant death world-wide, and continues to be a public health concern even in countries with high vaccination coverage. while estimates of the incidence of new cases of pertussis vary, 193, 194 pertussis infection world-wide varies between 16 million and 50 million cases yearly, 95% of which are in developing countries. 193, 194 in addition, between 195,000 and 300,000 deaths, mostly of young infants who were either unvaccinated or incompletely vaccinated, occur yearly. 193, 194 for several decades, infant immunization programs using pertussis vaccines of high efficiency have been highly successful in preventing severe pertussis in infants. however, pertussis continues to be a significant health issue world-wide. following an incubation period of 9e10 days (range 6e20 days) patients develop catarrhal symptoms including cough, and over 1e2 weeks develop coughing paroxysms that end in the characteristic "whoop" associated with this infection. although the cause of the characteristic "whoop" associated with b. pertussis infection remains unresolved, a significant body of knowledge has accumulated to explain how b. pertussis induces immune suppression, evasion, and modulation, thereby leading to a poor clinical outcome. fig. 49 . 6 195 gives an overview of several b. pertussis virulence factors that have immunomodulatory properties, the cells that they target, and the mechanisms thought to be involved in the induction of immune dysregulation caused by this infection. it has been shown that b. pertussis-derived filamentous hemagglutinin (fha) is the major surface structure that mediates adherence of b. pertussis to host cells, primarily to cilia of airway ciliated epithelium. however, multiple factors likely contribute to this binding process, 196 including secreted adenylate cyclase toxin (act), which enhances fha adherence to lung epithelial cells. 197 fha also has immunosuppressive and modulatory activities by its ability to induce fha-specific tregs that secrete il-10 and suppress t h 1-like responses to b. pertussis. 198 anti-fha antibodies can also reduce phagocytosis of these bacteria by human neutrophils. 199 act, a secreted toxin, targets host phagocytic cells by binding complement receptor 3 (cr3; cd11b/cd18). 200, 201 act-deficient mutants of b. pertussis are more efficiently phagocytosed by human neutrophils, 202 and in mice, lower bacterial loads are found in the respiratory tract. 203 secreted act upregulates major histocompatibility complex class ii mhc and co-stimulatory molecule expression on dendritic cells (dcs). act also prevents maturity, and thereby decreases their proinflammatory cytokine production. 204e206 tracheal cytotoxin (tct) is also expressed at relatively high levels by b. pertussis, and is a disaccharide-tetrapeptide fragment of peptidoglycan. purified tct, in synergy with lipopolysaccharide (lps), damages ciliated airway epithelial cells through production of il-1a and nitric oxide, 207 causing deleterious effects on neutrophils. 208 pertussis toxin (pt), uniquely produced by b. pertussis, is another secreted toxin expressed by this bacterium. pt adp ribosylates several heterotrimeric g proteins in mammalian cells, has long been known to disrupt signaling pathways with a wide range of downstream effects, 209 and can cause immunosuppression. pt causes lymphocytosis. 210 pt-deficient mutant strains show reduce levels of airway infection 24 h after inoculation because pt delays neutrophil recruitment and influx to the airways, 203, 211, 212 reduces anti-b. pertussis antibody-induced bacterial clearance, 211 depletes airway macrophages 213 enhancing infection. 214 since adp ribosylation of airway macrophage g proteins by pt has been shown to be long-lasting, and correlates with active infection-promoting longevity of this microbe. 212 this evidence supports the concept that the effects of pt on host cells in the airway may be particularly long-lived. pt also exerts multiple suppressive effects on the immune system beyond innate immune cells, including suppression of serum antibody responses to b. pertussis antigens following infection, 214e216 reduction of major histocompatibility complex class ii expression on human monocytes, 217 and modulation of dendritic cell expression of surface markers. 218 b. pertussis infection causes immunomodulation of toll-like receptor (tlr4), 219 which recognizes the lipopolysaccharide found on b. pertussis and gram-negative bacteria. 220,221 tlr4 signaling triggered by b. pertussis induces il-10, which can inhibit inflammatory responses, and limit airway inflammation, 220 and can synergize with act to induce il-10. 206 in addition, tlr4-dependent responses induced by b. pertussis drive lower levels of inflammatory cytokines. 222 thus, pt promotes b. pertussis infection by multiple mechanisms through its effects on innate immunity in the initial stages of disease in naïve individuals. this reduces adaptive immune responses during the initial infection, and promotes re-infection in partially immune individuals. the consequences of these suppressive effects on both the innate and adaptive immune responses induced by b. pertussis infection can cause secondary infection, typically pneumonia, which is the major cause of fatal outcome from this infection. 223 infants are at highest risk of all complications related to b. pertussis infection but all ages have relatively high rates of secondary complications. approximately 50% of infants exhibit apnea of greater or lesser duration and a quarter develop pneumonia. roughly 5% have otitis media and 1% develop seizures. in adults, the most common secondary effect is pneumonia occurring in about 5%. distinguishing patients with secondary immune deficiency disease caused by a primary infection in an individual with an "intact" immune system from patients with an underlying pidd is often a challenge. recognizing primary infections that can cause secondary immunodeficiencies is an important factor in discrimination between these two groups of patients. clinical immunologists need to always consider whether an acute, serious infection is causing a secondary primary infection in an "immunologically intact" individual, or is the herald of an underlying defect in a genetically defined immunocompromised patient with pidd. the long term treatment and clinical outcomes of these two different patient populations is linked to understanding how severe primary infections cause disease in these two different settings. macrophage polarization in bacterial infections interindividual variations in constitutive interleukin-10 messenger rna and protein levels and their association with genetic polymorphisms interleukin 10 secretion in relation to human il-10 locus haplotypes microsatellite alleles and single nucleotide polymorphisms (snp) combine to form four major haplotype families at the human interleukin-10 (il-10) locus interleukin-10 and the interleukin-10 receptor attending to warning signs of primary immunodeficiency diseases across the range of clinical practice ten warning signs of primary immunodeficiency: a new paradigm is needed for the 21st century how effective are the 6 european society of immunodeficiency warning signs for primary immunodeficiency disease? t-cell response to bacterial agents measles virus-induced suppression of immune responses measles in europe: an epidemiological assessment unacceptably high mortality related to measles epidemics in niger, nigeria, and chad the dynamics of measles in sub-saharan africa large measles epidemic in switzerland from 2006 to 2009: consequences for the elimination of measles in europe acute measles in patients with and without neurological involvement: distribution of measles virus antigen and rna factors associated with fatal cases of measles. a retrospective autopsy study measles j frequently asked questions about measles in u.s j cdc. center for disease control substandard vaccination compliance and the 2015 measles outbreak prospective study of the magnitude and duration of changes in tuberculin reactivity during uncomplicated and complicated measles cellular immune responses during complicated and uncomplicated measles virus infections of man cytokine production in vitro and the lymphoproliferative defect of natural measles virus infection functional and phenotypic changes in circulating lymphocytes from hospitalized zambian children with measles dendritic cells process exogenous viral proteins and virus-like particles for class i presentation to cd8þ cytotoxic t lymphocytes t-lymphocyte subpopulations in relation to immunosuppression in measles and varicella nonhuman primate models of measles apoptosis as a cause of death in measles virus-infected cells the dual-function cd150 receptor subfamily: the viral attraction the sap and slam families in immune responses and x-linked lymphoproliferative disease measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and t cells measles virus nucleoprotein induces cell-proliferation arrest and apoptosis through ntail-nr and ncore-fcgammariib1 interactions, respectively measles virus infection in rhesus macaques: altered immune responses and comparison of the virulence of six different virus strains immunologic abnormalities accompanying acute and chronic viral infections differential cd4 t cell activation in measles pathogenesis of measles virus infection: an hypothesis for altered immune responses cytokine imbalance after measles virus infection has no correlation with immune suppression metabolic effects of acute measles in chronically malnourished nigerian children -4) and cd30 expression/release during measles infection measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (cdw150) but not cd46 as a cellular receptor differential receptor usage by measles virus strains functional modulation of human macrophages through cd46 (measles virus receptor): production of il-12 p40 and nitric oxide in association with recruitment of protein-tyrosine phosphatase shp-1 to cd46 identification of a cytoplasmic tyr-x-x-leu motif essential for down regulation of the human cell receptor cd46 in persistent measles virus infection measles virus haemagglutinin induces down-regulation of gp57/67, a molecule involved in virus binding the cytoplasmic domains of complement regulatory protein cd46 interact with multiple kinases in macrophages polymorphic expression of cd46 protein isoforms due to tissue-specific rna splicing cell-to-cell contact via measles virus haemagglutinin-cd46 interaction triggers cd46 downregulation receptor usage and differential downregulation of cd46 by measles virus wild-type and vaccine strains mechanism of suppression of cell-mediated immunity by measles virus measles virus infects human dendritic cells and blocks their allostimulatory properties for cd4þ t cells activation of human cd4þ cells with cd3 and cd46 induces a tregulatory cell 1 phenotype in vitro studies of the role of monocytes in the immunosuppression associated with natural measles virus infections suppression of t lymphocyte function by measles virus is due to cell cycle arrest in g1 cell cycle arrest rather than apoptosis is associated with measles virus contact-mediated immunosuppression in vitro measles virus inhibits mitogen-induced t cell proliferation but does not directly perturb the t cell activation process inside the cell prolonged measles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction slow clearance of measles virus rna after acute infection a novel receptor involved in t-cell activation engagement of the signaling lymphocytic activation molecule (slam) on activated t cells results in il-2-independent, cyclosporin a-sensitive t cell proliferation and ifn-gamma production signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with th1 cytokine patterns slam) is a receptor for measles virus but is not involved in viral contact-mediated proliferation inhibition mechanism of cd150 (slam) down regulation from the host cell surface by measles virus hemagglutinin protein interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppression in vitro measles virus exploits dendritic cells to suppress cd4þ t-cell proliferation via expression of surface viral glycoproteins independently of t-cell trans-infection measles virus-induced promotion of dendritic cell maturation by soluble mediators does not overcome the immunosuppressive activity of viral glycoproteins on the cell surface induction of maturation of human blood dendritic cell precursors by measles virus is associated with immunosuppression proteolytic cleavage of the fusion protein but not membrane fusion is required for measles virus-induced immunosuppression in vitro measles virus-induced immunosuppression in cotton rats is associated with cell cycle retardation in uninfected lymphocytes measles virus-induced immunosuppression in vitro is associated with deregulation of g1 cell cycle control proteins measles virus interacts with and alters signal transduction in t-cell lipid rafts disruption of akt kinase activation is important for immunosuppression induced by measles virus measles virus induces expression of sip110, a constitutively membrane clustered lipid phosphatase, which inhibits t cell proliferation influenza pandemics of the 20th century pandemic flu history the ability of polymorphonuclear leukocyte priming agents to overcome influenza a virus-induced cell dysfunction influenza vaccination levels in selected states -behavior risk factor surveillance system both influenza-induced neutrophil dysfunction and neutrophil-independent mechanisms contribute to increased susceptibility to a secondary streptococcus pneumoniae infection deaths: leading causes for surveillance and impact of influenza in the united states role of neuraminidase in lethal synergism between influenza virus and streptococcus pneumoniae influenza a virus alters structural and biochemical functions of the neutrophil cytoskeleton effects of infection with influenza virus on the function of polymorphonuclear leukocytes alterations in cell protein phosphorylation in human neutrophils exposed to influenza a virus. a possible mechanism for depressed cellular end-stage functions virus-induced neutrophil dysfunction: role in the pathogenesis of bacterial infections characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses depression of monocyte and polymorphonuclear leukocyte oxidative metabolism and bactericidal capacity by influenza a virus depression of neutrophil function induced by viruses and its role in secondary microbial infections influenza a virus-induced polymorphonuclear leukocyte dysfunction in the pathogenesis of experimental pneumococcal otitis media inhibition of neutrophil lysosome-phagosome fusion associated with influenza virus infection in vitro. role in depressed bactericidal activity effect of priming polymorphonuclear leukocytes with cytokines (granulocyte-macrophage colony-stimulating factor [gm-csf] and g-csf) on the host resistance to streptococcus pneumoniae in chinchillas infected with influenza a virus influenza a virus-induced polymorphonuclear leukocyte dysfunction polymorphonuclear leukocyte dysfunction during influenza virus infection in chinchillas the importance of neutrophils in resistance to pneumococcal pneumonia in adult and neonatal mice influenza a virus accelerates neutrophil apoptosis and markedly potentiates apoptotic effects of bacteria g-protein alteractions in polymorphonuclear leukocytes (pmnl) induced by influenza a virus (iav) guanine nucleotide binding properties of rap1 purified from human neutrophils mechanism of gm-csf stimulation of neutrophils facilitated expansion of pneumococcal colonization from the nose to the lower respiratory tract in mice preinfected with influenza virus lethal synergism between influenza virus and streptococcus pneumoniae: characterization of a mouse model and the role of platelet-activating factor receptor impact of influenza and other community-acquired viruses combined infections in mice with influenza virus and diplococcus pneumoniae adherence of type i streptococcus pneumoniae to tracheal epithelium of mice infected with influenza a/pr8 virus tracheal function during influenza infections the host response to invasion by streptococcus pneumoniae: protection and the pathogenesis to tissue damage apoptosis: a mechanism of cell killing by influenza a and b viruses il-10 is an important mediator of the enhanced susceptibility to pneumococcal pneumonia after influenza infection secondary immunodeficiencies, including hiv infection hiv-1 nef-induced down-regulation of mhc class i requires ap-1 and clathrin but not pacs-1 and is impeded by ap-2 virus evasion of mhc class i molecule presentation rev activity determines sensitivity of hiv-1-infected primary t cells to ctl killing the selective downregulation of class i major histocompatibility complex proteins by hiv-1 protects hivinfected cells from nk cells hiv nef-mediated cd4 down-regulation is adaptor protein complex 2 dependent viral reservoirs, residual viremia, and the potential of highly active antiretroviral therapy to eradicate hiv infection cd4þ t cell depletion during all stages of hiv disease occurs predominantly in the gastrointestinal tract elevations in il-10, tnf-alpha, and ifn-gamma from the earliest point of hiv type 1 infection autophagy is involved in t cell death after binding of hiv-1 envelope proteins to cxcr4 the immunopathogenesis of human immunodeficiency virus infection htlv-1, immune response and autoimmunity natural history of adult t-cell leukemia/lymphoma and approaches to therapy human t cell leukemia virus type 1 tax inhibits innate antiviral signaling via nf-kb-dependent induction of socs17 aryl hydrocarbon receptor interacting protein targets irf7 to suppress antiviral signaling and the induction of type i interferon* tropical spastic paraparesis and htlv-1 associated myelopathy: clinical, epidemiological, virological and therapeutic aspects molecular biology of human t cell leukemia virus functional capacity of natural killer cells in htlv-1 associated myelopathy/tropical spastic paraparesis (ham/tsp) patients atypical clinical presentation of strongyloidiasis in a patient co-infected with human t cell lymphotrophic virus type i htlv-1 modifies the clinical and immunological response to schistosomiasis impact of viral infections on hematopoiesis: from beneficial to detrimental effects on bone marrow output immunity to visceral leishmaniasis using genetically defined live-attenuated parasites leishmaniasis: current situation and new perspectives visceral leishmaniasis: what are the needs for diagnosis, treatment and control? leishmaniasis vaccine: where are we today? cutting edge: neutrophil granulocyte serves as a vector for leishmania entry into macrophages in vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies leishmania donovani affects antigen presentation of macrophage by disrupting lipid rafts the immune response to leishmania: mechanisms of parasite control and evasion interleukin-12 restores interferon-gamma production and cytotoxic responses in visceral leishmaniasis il-10 and il-12 are the main regulatory cytokines in visceral leishmaniasis neutralization of il-12 demonstrates the existence of discrete organ-specific phases in the control of leishmania donovani il-12 enhances th1-type responses in human leishmania donovani infections genetically modified live attenuated parasites as vaccines for leishmaniasis t helper (h)1/th2 and leishmania: paradox rather than paradigm interleukin-10 and the pathogenesis of human visceral leishmaniasis cytokines and their stats in cutaneous and visceral leishmaniasis il-10 mediates susceptibility to leishmania donovani infection determinants of response to interleukin-10 receptor blockade immunotherapy in experimental visceral leishmaniasis splenic accumulation of il-10 mrna in t cells distinct from cd4þcd25þ (foxp3) regulatory t cells in human visceral leishmaniasis splenic cytokine responses in indian kala-azar before and after treatment two types of mouse t helper cell. iv. th2 clones secrete a factor that inhibits cytokine production by th1 clones the role of il-10 in crossregulation of th1 and th2 responses immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase c-mediated signal transduction events interleukin-10 and interleukin-4 inhibit intracellular killing of leishmania infantum and leishmania major by human macrophages by decreasing nitric oxide generation homology of cytokine synthesis inhibitory factor (il-10) to the epstein-barr virus gene bcrfi potential role for interleukin-10 in the immunosuppression associated with kala azar cd4þcd25þ regulatory t cells control leishmania major persistence and immunity distinct roles for il-6 and il-12p40 in mediating protection against leishmania donovani and the expansion of il-10þ cd4þ t cells endogenous il-4 is necessary for effective drug therapy against visceral leishmaniasis induction of host protective th1 immune response by chemokines in leishmania donovaniinfected balb/c mice functional paradox in host-pathogen interaction dictates the fate of parasites intracellular replication-deficient leishmania donovani induces long lasting protective immunity against visceral leishmaniasis complete cure of experimental visceral leishmaniasis with amphotericin b in stearylamine-bearing cationic liposomes involves down-regulation of il-10 and favorable t cell responses endogenous interleukin-12 regulates acquired resistance in experimental visceral leishmaniasis the two faces of interleukin 10 in human infectious diseases conventional t-bet(þ)foxp3(-) th1 cells are the major source of host-protective regulatory il-10 during intracellular protozoan infection cd4(þ)cd25(-)foxp3(-) th1 cells are the source of il-10-mediated immune suppression in chronic cutaneous leishmaniasis visceral leishmaniasis in mice devoid of tumor necrosis factor and response to treatment role and effect of tnf-alpha in experimental visceral leishmaniasis tumor necrosis factor-alpha synergizes with ifn-gamma in mediating killing of leishmania major through the induction of nitric oxide role of chemokines in regulation of immunity against leishmaniasis balancing immunity and pathology in visceral leishmaniasis chemokines in host-parasite interactions in leishmaniasis il-17 and il-22 are associated with protection against human kala azar caused by leishmania donovani cd8 cytotoxic t cells in cutaneous leishmaniasis adoptive immunotherapy against experimental visceral leishmaniasis with cd8þ t cells requires the presence of cognate antigen experimental visceral leishmaniasis: role of endogenous ifngamma in host defense and tissue granulomatous response immunization with a recombinant stage-regulated surface protein from leishmania donovani induces protection against visceral leishmaniasis role of l3t4þ and lyt-2þ cells in experimental visceral leishmaniasis isolation of bacteria causing secondary bacterial infection in the lesions of cutaneous leishmaniasis role for cd4(þ) cd25(þ) regulatory t cells in reactivation of persistent leishmaniasis and control of concomitant immunity leishmania in hlh: a rare finding with significant treatment implications case of hemophagocytic lymphohistiocytosis with leishmaniasis immunodeficiencies in parasitic diseases plasmodium falciparum malaria and salmonella infections in gambian children herpes zoster in children following malaria association of hepatitis b surface antigen carriage with severe malaria in gambian children depression of immune response to moloney leukaemia virus by malarial infection exposure to holoendemic malaria results in elevated epstein-barr virus loads in children impairment of the immune response to vaccination after acute malaria dendritic cells can initiate protective immune responses against malaria phagocytosis of the malarial pigment, hemozoin, impairs expression of major histocompatibility complex class ii antigen, cd54, and cd11c in human monocytes suppression of adaptive immunity to heterologous antigens during plasmodium infection through hemozoin-induced failure of dendritic cell function malaria impairs t cell clustering and immune priming despite normal signal 1 from dendritic cells cdc -pertussis: pertussis in other countries immunomodulation in the pathogenesis of bordetella pertussis infection and bordetella bronchiseptica adherence to cilia is mediated by multiple adhesin factors and blocked by surfactant protein a adenylate cyclase influences filamentous haemagglutininmediated attachment of bordetella pertussis to epithelial alveolar cells pathogen-specific t regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective t helper type 1 responses by bordetella pertussis phagocytosis of bordetella pertussis incubated with convalescent serum bordetella adenylate cyclase toxin: a swift saboteur of host defense pore-forming and enzymatic activities of bordetella pertussis adenylate cyclase toxin synergize in promoting lysis of monocytes influence of cr3 (cd11b/cd18) expression on phagocytosis of bordetella pertussis by human neutrophils pertussis toxin and adenylate cyclase toxin provide a one-two punch for establishment of bordetella pertussis infection of the respiratory tract pertussis toxin and the adenylate cyclase toxin from bordetella pertussis activate human monocyte-derived dendritic cells and dominantly inhibit cytokine production through a camp-dependent pathway adenylate cyclase toxin from bordetella pertussis synergizes with lipopolysaccharide to promote innate interleukin-10 production and enhances the induction of th2 and regulatory t cells bordetella type iii secretion and adenylate cyclase toxin synergize to drive dendritic cells into a semimature state synergistic epithelial responses to endotoxin and a naturally occurring muramyl peptide effect of tracheal cytotoxin from bordetella pertussis on human neutrophil function in vitro pertussis toxin in the analysis of receptor mechanisms biological activities of crystalline pertussigen from bordetella pertussis pertussis toxin inhibits neutrophil recruitment to delay antibody-mediated clearance of bordetella pertussis pertussis toxin plays an early role in respiratory tract colonization by liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications pertussis toxin targets airway macrophages to promote bordetella pertussis infection of the respiratory tract suppression of serum antibody responses by pertussis toxin after respiratory tract colonization by bordetella pertussis and identification of an immunodominant lipoprotein homologous and heterologous protection after single intranasal administration of live attenuated recombinant bordetella pertussis bordetella pertussis infection of primary human monocytes alters hla-dr influence of pertussis toxin on cd1a isoform expression in human dendritic cells toll-like receptor 4-mediated innate il-10 activates antigen-specific regulatory t cells and confers resistance to bordetella pertussis by inhibiting inflammatory pathology host genetics of bordetella pertussis infection in mice: significance of toll-like receptor 4 in genetic susceptibility and pathobiology comparative toll-like receptor 4-mediated innate host defense to bordetella infection cytokine storms in infectious diseases pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology new fronts emerge in the influenza cytokine storm molecular pathogenesis of viral hemorrhagic fever immune-mediated cytokine storm and its role in severe dengue virus-induced pathogenesis, vaccine development, and diagnosis of novel h7n9 avian influenza a virus in humans: a systemic literature review cytokine storm combined with humoral immune response defect in fatal hemorrhagic fever with renal syndrome case lipoxin a4, a 5-lipoxygenase pathway metabolite, modulates immune response during acute respiratory tularemia molecular pathogenesis of ehrlichia chaffeensis infection evidence that lipopolisaccharide may contribute to the cytokine storm and cellular activation in patients with visceral leishmaniasis key: cord-022348-w7z97wir authors: sola, monica; wain-hobson, simon title: drift and conservatism in rna virus evolution: are they adapting or merely changing? date: 2007-09-02 journal: origin and evolution of viruses doi: 10.1016/b978-012220360-2/50007-6 sha: doc_id: 22348 cord_uid: w7z97wir this chapter argues that the vast majority of genetic changes or mutations fixed by rna viruses are essentially neutral or nearly neutral in character. in molecular evolution one of the remarkable observations has been the uniformity of the molecular clock. an analysis of proteins derived from complete potyvirus genomes, positive-stranded rna viruses, yielded highly significant linear relationships. these analyses indicate that viral protein diversification is essentially a smooth process, the major parameter being the nature of the protein more than the ecological niche it finds itself in. synonymous changes are invariably more frequent than nonsynonymous changes. positive selection exploits a small proportion of genetic variants, while functional sequence space is sufficiently dense, allowing viable solutions to be found. although evolution has connotations of change, what has always counted is natural selection or adaptation. it is the only force for the genesis of a novel replicon. there is no such thing as a perfect machine. accordingly, nucleic acid polymerization is inevitably error-prone. yet the notoriety and abundance of rna viruses attests to their great success as intracellular parasites. indeed some estimates suggest that 80% of viruses have rna genomes. it follows that replication without proofreading can be a successful strategy. there is a price to pay, however. manfred eigen was the first to point out that without proofreading there is a limit on the size of rna genomes. obviously, if the mutation rate is too high, any rna virus will collapse under mutation pressure. as it happens, rna viral genomes are up to 32 kb long while mutation rates are 1-2 per genome per cycle or less. possibly, rna viruses and retroviruses have simply not invested in proofreading, in which case mutations represent an inevitable genetic noise, to be tolerated or eliminated. hence there would be no loss of fitness, fixed mutations being neutral. a corollary of this would be that the intrinsic life style of a virus is set in its genes. the alternative is to suppose that most fixed mutations are beneficial to the virus in allowing it to keep ahead of the host and/or host population. by this token variation is an integral part of the viral modus vivendi. the twin requirements of a successful virus are replication and transmission. under the rubric replication, a virus could vary to increase its fitness, exploit different target cells or evade adaptive immune responses. in terms of transmission, variation might allow a virus to overcome herd immunity. these two scenarios emphasize the two sides of the molecular evolution debate; one highlights neutrality while the other puts a premium on positive selection. purifying or negative selection is ever operative -a poor replicon invariably goes asunder. through rounds of error and trial, positive selection is the only means of creating a novel replicon. so long as the ecological niche occupied doesn't change, the virus doesn't need to change, purifying selection being sufficient to ensure existence. this raises an important issue: we know that, over the time that we are living and loving, as well as doing experiments, writing papers and reviewing, humans are not evolving. ernst mayr noted that "the brain of 100 000 years ago is the same brain that is now able to design computers" (mayr, 1997) . positive fitness selection among mammals is effectively inoperative over our lifetimes. and certainly since we have known about hiv and aids. how is it that vertebrates, invertebrates, plants, fungi and bacteria, all species with a low genomic mutation rate, can control viruses which mutate so much faster-sometimes by a factor of 106 (holland et al., 1982; gojobori and yokoyama, 1985; domingo et al., 1996) . yet they do. we come to the basic question-to what extent is genetic variation exploited by an rnavirus, if at all? and if so, what is the virus adapting to? the answer invariably given to the second question is "the adaptive immune system" (seibert et al., 1995) . yet apart from the vertebrates none of the other groups mentioned above mounts antigen-specific immune responses. this chapter will argue that most fixed mutations are neutral. in molecular evolution one of the remarkable observations has been the uniformity of the molecular clock. although there has been intense debate as to what molecular clocks mean and quite how far they deviate from null hypotheses, fibronectin fixes mutations faster than alpha-or beta-globin, which do so faster than cytochrome c, etc. rates of amino acid fixation are intrinsic to different proteins. yet some viruses give rise to persistent infections, others to sequential acute infections. all succumb to the vagaries of transmission bottlenecks. how many rounds of infection are necessary to fix mutations? for example, the tremendous dynamics of viral replication have been described. whether it be hiv, hbv or hcv, plasma viral turnover is of the order of 108-10 '2 virions per day (ho et al., 1995; wei et al., 1995; nowak et al., 1996; zeuzem et al., 1998) . between 10% and 90% of plasma virus is cleared. in the case of hiv this can involve more than 200 rounds of sequential replication per year (wain-hobson, 1993a,b; ho et al., 1995; pelletier et al., 1995; wei et al., 1995) . many of these variables and unknowns can be removed by comparing the fixation of aminoacid substitutions in pairs of viral proteins from two genomes. if one assumes that the two gene fragments remain linked, through the hellfire of ay immune responses and bottlenecking inherent in transmission, relative degrees of fixation should be attainable. note that, so long as frequent recombination between highly divergent genomes is not in evidence, this assumption should be valid. this procedure is outlined in figure 6 .1. the first example is taken from the vast primate immunodeficiency virus database (lanl, 1998) . when normalized to the p66 reverse transcriptase product designated rt, amino acid sequence divergence for p17 gag, p24 gag, integrase, vif, gp120, the ectodomain of gp41 and nef all reveal highly significant linear relationships ( figure 6 .2, table 6 .1). the relative rates vary by a factor of two or more. why the hypervariable gp120 protein shows a relatively low degree of change with respect to the reverse (henikoff and henikoff, 1993) . it is well established that protein sequence comparisons are more informative when weighted for genetic and structural biases in amino acid replacements. in the blosum weight matrices series, the actual matrix that was used depends on how similar the sequences to be aligned are. different matrices work differently at each evolutionary distance. for a given virus, different protein sequence sets were compared to a given reference such as rt in the case of hiv/siv. n indicates the number of independent two-by-two comparisons. the data were checked for the possibility that a rogue genome strongly influenced the data. only in the case of the inoviridae were there insufficient complete sequences, six in fact, to yield satisfying analyses. instead all pairwise comparisons were made, hence the data points reflect dependent data (#). the form of the linear regressions are given where y and x refer to the first and second protein listed in the column "paired proteins". the correlation coefficients r were highly significant in all cases, the corresponding probabilities being: + < 0.02;" < 0.005; *< 0.001. .2 graphical representation of paired divergence for orthologous proteins taken from complete hiv-1, hiv-2 and siv genome sequences, y = different proteins, x = p66 sequence of the reverse transcriptase (rt). x and y values correspond to blosum-corrected fractional divergence. only non-overlapping regions were taken into account. the straight lines were obtained by linear regression analysis. their characteristics are given in table 6 .1. transcriptase (rt) can be explained by gap stripping, which eliminates the hypervariable regions. consequently the gp120 data effectively reflects the conserved regions. the linearity, even out to considerable differences, indicates that multiple substitutions and back mutations, which must be occurring, do so to comparable degrees. although these data were derived from completely sequenced primate immunodeficiency viral genomes, analyses on larger data sets, such as p17 gag/p24 gag or gp120/gp41, yielded relative values that differed from those given in table 6 .1 by at most 14%. the absence of points far from the linear regression substan-tiates the assumption that recombination between highly divergent genomes is rare. this does not preclude recombination between closely related genomes. the linear regressions passed close to the origin in nearly all cases. only for nef was there some deviation, suggesting that nef was saturating to a different extent from all other proteins. however, as linear correlations involving nef data were always statistically significant, this trend may be fortuitous. note that the data cover the earliest phase, intrapatient variation (generally <10%), continuing smoothly to cover interclade, intertype and finally interspecies comparisons. yet this in spite of different environments-that of an individual's immune system, different immune systems stigmatized by highly polymorphic hla, and finally differences between humans, chimpanzees, mandrills and african green monkeys accumulated over 30 million years. the same forces were uppermost during all stages of diversification. it is remarkable that the very different proteins, such as gp120 and the gp41 ectodomain (surface glycoproteins), p17 gag and p24 gag (structural), rt and integrase (enzymes) and nef and vif (cytoplasmic), all yield linear relationships ( figure 6 .2), as though fixation was an intrinsic property of the protein. applying the same analysis to complete rhinoviral genomes yielded comparable results, i.e. highly significant linear relationships for vp1, vp2 and vp3 (capsid proteins), p2a and 3c (proteases), p2c (cytoplasmic proteins involved in membrane reorganization) compared with the rna-dependent rna polymerase (3d) as reference ( figure 6 .3, table 6 .1). hence figure 6 .2 does not represent some quirk of primate lentiviruses. of course, vertebrate viruses have a redoubtable adversary in the host adaptive immune system. the swiftness of secondary responses is reminder enough. an analysis of proteins derived from complete potyvirus genomes, positive-stranded rna viruses, yielded highly significant linear relationships (table 6 .1). a number of revealing points can be made. firstly, the linear relationships hold out to very large blosum distances (0.9). secondly, potyviruses infect a wide variety of different plants, as their florid names betray. finally, the linear relationships cannot result from adaptive immune pressure because plants are devoid of adaptive immune systems. they only have powerful innate immune responses. unfortunately there are insufficient insect rna viral sequences to allow a comparable study. however, a glance at a few beetle nodavirus capsid sequences (dasgupta et al., 1984; dasgupta and sgro, 1989) shows extensive genetic variation with a majority of synonymous base substitutions, typical of most comparisons of mammalian viral sequences (see below). for the time being there doesn't seem to be anything obviously different about insect virus sequence variation. although insects do not mount adaptive immune responses, the breadth and complexity of their innate immune systems is salutary (brey and hultmark, 1998) . a final example is afforded by the inoviruses, bacteriophages of the fd group, which includes m13. although dna viruses fix mutations at a slower rate than rna viruses, they too show linear relationships among comparisons of their i, ii, iii and iv proteins (table 6 .1). and of course bacteria are devoid of adaptive immunity as well. whether the comparisons were between capsid proteins versus enzymes, or secretory versus cytoplasmic molecules, significant linear relationships were obtained for pairwise comparisons in amino acid variation in all cases. such proteins are vastly different in their threedimensional folds and functions. some are "seen" by humoral immunity, others are not. for the plant viruses and bacteriophages, only innate immunity is operative. it is as though the rate of amino acid sequence accumulation is an intrinsic feature of the protein, reminiscent of the differing slopes for the accumulation of substitutions by alpha-globin and cytochrome c already alluded to. of course pairwise comparisons of these two proteins from differing organisms would yield a straight line going through the origin in a manner typical of figures 6.2 and 6.3. hence it is fairly safe to assume that, for viral proteins too, amino acid substitutions are accumulated smoothly over time. indeed, this has been shown explicitly for a number of proteins from a varied group of viruses, including the influenza a, coronaviruses, hiv and herpes viruses (hayashida et al., 1985; gojobori et al., 1990; querat et al., 1990; villaverde et al., 1991; elena et al., 1992; sanchez et al., 1992; mcgeoch et al., 1995; yang et al., 1995; leitner et al., 1997; plikat et al., 1997) . the above analyses indicate that viral protein diversification is essentially a smooth process, the major parameter being the nature of the protein more than the ecological niche it finds itself in. the simplest hypothesis to explain the smoothness of protein sequence diversification is that the majority of fixed amino acid substitutions are neutral, being accumulated at rates intrinsic to each protein. this is not to say that positive selection is inoperative, merely that the majority of fixed substitutions are essentially neutral, so much so that it does not strongly distort the data from a linear relationship expected for genetic drift. in other words, neither the impact of different environments nor the ferocity of the adaptive immune response has much to do with fixation of most substitutions. this is important for the one-dimensional man in all of us sequencers who see all mutations and ask questions about genotype and phenotype -usually about genotype. a short aside is necessary here. it is interesting that in a few areas of rna virology much has been made of escape from the adaptive immune response, particularly cytotoxic t lymphocytes, so leading to persistence (nowak and mcmichael, 1995; mcmichael and phillips, 1997) . however, it is not at all obvious that this is the case (wain-hobson, 1996) . it must not be forgotten that it is possible to vaccinate against a number of rna viruses such as measles, polio and yellow fever. be that as it may, many dna viruses, intracellular bacteria and parasites persist. in these cases de novo genetic variation arising from point mutations is too slow a means to thwart an adaptive immune response. for example, after 1700 generations, under experimental conditions whereby muller's ratchet was operative, s. typhimurium accumulated mutations such that only 1% of the 444 lineages tested had suffered an obvious loss of fitness (anderson and hughes, 1996) . that this number of generations could be achieved within as little as 45 days gives an idea of the time necessary to generate a mutation affecting fitness. this is more than enough time to make a vigorous immune response. some inklings of immune system escape for the herpes virus ebv (de campos-lima et al., 1993 came to nought (burrows et al., 1996; khanna et al., 1997) . when antigenic variation is in evidence among dnabased microbes, it invariably results from the use of cassettes and multicopy genes rather than point mutations resulting from dna replication. and of course such complex systems could have only come about by natural selection. finally de novo genetic variation of an rna virus has never been suggested or shown to be necessary for the course of an acute infection. for a virus to persist thanks to genetic variation the phenomenon of epitope escape must be strongly in evidence by the time of seroconversion, generally 5-6 weeks. yet such data are not forthcoming, and not for want of trying. when viruses do play tricks with the immune system it is invariably by way of specific viral gene products that interfere with the mechanics of adaptive and innate immunity (ploegh, 1998) . in the clear cases where genetic variation is exploited by rna viruses, it is used to overcome barriers to transmission set up by the host population, e.g. herd immunity. the obvious example is influenza a virus antigenic variation in mammals. another way of assessing the contribution of positive selection to sequence variation is to compare the relative proportions of synonymous (ks) and non-synonymous (k) base substitutions per site. a ka/k s ratio of less than 1 indicates that purifying selection is uppermost, while a ratio more than i is taken as evidence of an excess of positive selection. comparisons for hiv proteins from different isolates have yielded the same result (myers and korber, 1994) . some mileage was made out of the fact that this ratio increased with increasing distance of sivs with respect to hiv-1, which in turn led to a discussion of siv pathogenesis (shpaer and mullins, 1993) . however, this may reflect a lack of adequate correction for multiple hits. this effect is illustrated by a comparison of the set of 72 orthologous proteins encoded by herpes simplex viruses 1 and 2 (hsv-1 and hsv-2; figure 6 .4a). the more divergent the protein sequence, the greater the ka/k s ratio. that some proteins fix substitutions faster than others is no surprise. yet as figure 6 .4b shows, the k s values change little as they are near to saturation. when k is small, k s > k. this suggests that reliable interpretation of ka/k s ratios is possible only when the degree of nucleic acid divergence is small. now this is the realm of viral quasispecies rendered accessible by pcr. hiv studies abound, reflecting both the phenomenal degree dolan et al., 1998) . a. ka/k s ratio as a function of uncorrected percentage amino acid sequence divergence (linear regression was ka/k s = 1.25 divergence + 0.04, r = 0.87 (p < 0.001)). b. individual k s and k a variation with percentage divergence (k s = 0.53 divergence + 0.35 and k a -0.76 divergence -0.03 with correlation coefficients of 0.54 and 0.97 respectively, p < 0.001 for both). note how at small degrees of divergence, k>>k a decreases as divergence increases. basically, k s is approaching saturation, being uncorrected for multiple and/or back mutations. of sequence variation and its importance as a pathogen, so we'll stick to some such examples that are illustrative. concerning ka/k s ratios for hiv gene segments, widely varying conclusions have been published supporting all sides (meyerhans et al., 1989 pelletier et al., 1995; wolinsky et al., 1996; leigh brown, 1997; price et al., 1998) , so much so that three comments are in order. firstly, many studies have used small numbers of sequences and substitutions and even regions as small as nonameric hla class-irestricted epitopes. in such cases statistical analyses are essential to test the significance of the distribution of synonymous and non-syn-onymous substitutions. this is particularly important as the point substitution matrix is highly biased (pelletier et al., 1995; plikat et al., 1997) . it turns out that when the proportions are so analysed the distributions are rarely significantly different from the neutral hypothesis (leigh brown, 1997) . secondly, the method for counting substitutions is highly variable, ranging from two-by-two comparisons, scoring the number of altered sites in a data set, to phylogenetic reconstruction. this latter method reflects more closely the process of genetic diversification. when so analysed, almost all of the data sets indicated proportions of synonymous to non-synonymous substitutions indistinguishable from that suggested by genetic drift and/or purifying selection (pelletier et al., 1995; plikat et al., 1997) . thirdly, prudence is called for. the fact that obviously defective sequences can be identified, occasionally accounting for large fractions of the sample (martins et al., 1991; gao et al., 1992) , indicates that not all genomes have undergone the rigours of selection (nietfield et al., 1995) . indeed, in peripheral blood, hiv is invariably lurking as a silent provirus within a resting memory t-cell. such t-cells have half lives of 3 months or more (michie et al., 1992) . hence it would be erroneous to interpret findings based on a single or clustered samples (price et al., 1997) . only when the above caveats are borne in mind is there any hope of discerning how hiv accumulates mutations. when these issues are attended to, purifying selection is dominant (pelletier et al., 1995; leigh brown, 1997; plikat et al., 1997) . one must not deny that positive selection is operative, merely that it is hard to pinpoint when looking at full-length sequences. indeed it is like looking for the proverbial needle in a haystack. in the context of ka/ks-type analyses, the two classic cases in the literature are the hla class i and ii molecules and influenza a haemagglutinin (hughes, 1998; hughes and nei, 1988; ina and gojobori, 1994) . the peptide contact residues of both class i and ii molecules have been under tremendous positive selection. changes in the five antigenic sites on the flu a haemagglutinin help the virus overcome herd immunity set up during previous flu epidemics. consequently, finding ka/k s > i in these regions was, in some ways, a pyrrhic victory because the papers needed experimental data to identify the positively selected segments in the first place. more recently endo et al. (1996) have screened the sequence data bases for proteins in which ka/k s > 1. of 3595 homologous gene groups screened, covering about 20 000 sequences, only 17 groups came up positive, of which two were encoded by rna virusesthe equine infectious anaemia virus envelope proteins and the reovirus g1 (outer capsid) proteins. the former case is intriguing as there is no obvious correlation between sequence changes and neutralizing antibodies (carpenter et al., 1987) . the authors noted that, when a comparable ka/k s analysis was restricted to small segments, the number of protein groups scoring positive rose to 5% (endo et al., 1996) . despite the explanatory power of these ratios, the number of identifiable cases of positively selected segments is small indeed. these numbers would probably shrink were phylogenic reconstruction used. to summarize the section, synonymous changes are invariably more frequent than nonsynonymous changes. positive selection may be operative in the evolution of viral protein sequences. when it is, it apparently exploits only a small fraction of mutants. the two rates touted by evolutionary-minded virologists are the mutation rate and the mutation fixation rate. the first describes the rate of genesis of mutations, the second attempts to describe their fixation within the population sampled over a period of time. in the case where all substitutions are neutral, the mutation rate (m) equals the fixation rate (f) per round of replication. it appears that such a situation applies to the evolution of parts of the siv and hiv-1 genomes over 1-3 years (pelletier et al., 1995; plikat et al., 1997) . if fixation rates are measured over one year, then f = n.m, where n is the annual number of consecutive rounds of replication. it is simple to show that several hundred rounds of sequential replication are required (wain-hobson, 1993b; pelletier et al., 1995) . given that the proviral load of an hiv-l-positive patient (~107-10 9) changes by less than a factor of 10 over 5 years or more, and given the assumption that an infected cell produces sufficient virus to generate two productively infected cells, then annual production would be something akin to 2 200 , or 10 6~ which is impossible. clearly even a productive burst size of 2 is too large (wain-hobson, 1993a,b) . this must be reduced to 1.1 to achieve a realistic proviral load (1.12~176 note that the real value for the effective burst size must be even lower, as proviral load is turning over more slowly than once a day. yet to explain the temporal increase in proviral load, the productive burst size must be 2 or more. thus the calculation reveals massive destruction of infected cells, precisely what was to be expected from immensely powerful innate and adaptive immune responses. when purifying selection is in evidence, some additional factor must be introduced to couple the fixation and mutation rates. as the accumulation of most substitutions proceeds in a protein-specific linear manner for small degrees of divergence, the above equation can be modified to f -p-n. m, where 1 > p > 0 is a constant indicating the degree of negative selection. note immediately that, as p < 1, more rounds of replication are needed to produce the same percentage amino acid fixation. a corollary is an even greater degree of destruction of infected cells. consider the example of a virus that is fixing substitutions only slowly, about 10 -5 per site per year, something like the ebola virus glycoprotein. the mutation rate for ebola is not known but is probably around 10 -4 per site per cycle (drake, 1993) . hence p.n ~10 -1. what is the value of n? most mammalian viruses replicate within 24 h, while obviously outside of a body they do not replicate. consequently a value of n = 50-200 is probably not unreasonable. accordingly p; 2 x 10 -3 to 5 x 10-4. this means that most mutations generated are deleterious. of those that are fixed, most are neutral, as has been discussed above. the last two sentences describe a profoundly conservative strategy-rna viruses are seen merely to replicate far more than giving rise to genetically distinct, even exotic, siblings. what a stultifying picture, in contrast to the shock-horror of tabloid newspaper virology and that atmospheric, yet profoundly ambiguous term, emerging viruses. conservative perhaps, but is there any suggestion that viruses are more or less so than other replicons? like extrapolation, choosing examples can be problematic. however let's consider one example, the eukaryotic and retroviral aspartic proteases (doolittle et al., 1989) . the former exist as a monomer with two homologous domains, while the retroviral counterpart functions as a homodimer. despite these differences the folding patterns are almost identical, meaning that the enzymes may be considered orthologous. between humans and chickens there is approximately 38% amino acid divergence among typical aspartic proteases ( figure 6 .5). the hiv-1 and hiv-2 proteases differ by a little more, 52%. no one would doubt the considerable differences in design, metabolism and lifestyle separating us and chickens. on either side of the hiv protease coding region one finds differences: hiv-1 is vpx-vpu ⧠while hiv-2 is the opposite, i.e. vpx+vpu-; there are differences in the size and activities of the tat gene product; the ltrs are subtly different. yet both replicate in the same cells in vivo, produce the same disease, albeit with different kinetics: hiv-2 infection progresses more slowly. if these differences are esteemed too substantial, consider the 28% divergence between the hiv and chimpanzee siv proteases. these two viruses are isogenic. pig and human chromosomal aspartic proteases may differ by around 17%, the differences between these two species being, george orwell apart, obvious to all. even by this crude example, the aids viruses would seem to be more conservative than mammals in their evolution. the same argument pertains to the rhinoviral p2a and 3c serine proteases (figure 6 .5). this conclusion is even more surprising when it is realized that hiv is fixing mutations at a rate of 10-2-10 -3 per base per year. by contrast, mammals are fixing mutations approximately one million times less rapidly, i.e. approximately 10-8-10 -9 per base per year (gojobori and yokoyama, 1987) . however, the generation times of the two are vastly different, about i day for hiv and about 15-25 years for humans. normalizing for this yields a 100-fold higher fixation rate per generation for hiv than for humans. amalgamating this with the preceding paragraph, we see that hiv is not only evolving qualitatively in a conservative manner, but it is doing so despite a 100-fold greater propensity to accommodate change. the same arguments go for almost all rna viruses and retroviruses. why is this? although they mutate rapidly, their hosts are effectively invariant in an evolutionary sense. probably sticking to the niche is all that matters, which is no mean task given the strength of innate and adaptive antiviral immune responses. john maynard smith's argument was simply put. for organisms with a base substitution rate of less than i per genome per cycle, he reasoned that all intermediates linking any two sequences must be viable, otherwise the lineage would go extinct. the example used was self explanatory: word --+ wore--+ gore -+ gone ~ gene (maynard smith, 1970) . the same is true for viruses, even though their mutation rates are 6 orders higher; the rate for a given protein is still less than 1 substitution per cycle. even for rather stable viruses like ebola/marburg and human t-cell leukaemia virus type 1 and 2 (htlv-1/-2), the number of intermediates is huge. while the enormity of sequence space is basically impossible to comprehend, the amount accessible to a virus remains vast. for the lineage to exist, the probability of finding a viable mutant must be at least 1/population size within the host. imagine a stem-loop structure. any replacement of a g:c base pair must proceed by a single substitution, given that the probability of a double mutation is approximately 10 -4 that of a single mutation. let substitution of a g:c pair pass by a g:u intermediate, finishing up as a:u. although g:u mismatches are the most stable of all mismatches, they are less so than either a g:c or an a:u pair. there are two scenarios: either the g:u substitution is of so little consequence that it is fixed per se, in which case there would be no selection pressure to complete the process to a:u. alternatively, the g:u substitution is sufficiently deleterious for selection of a secondary mutation to occur from a pool of variants, so completing the process. yet the g:u intermediate cannot be so debilitating otherwise the process would have little chance of going to completion. note also that if the fitness difference is small with respect to the g:c or a:u forms, more rounds of replication are necessary to achieve fixation of g:u to a:u. a corollary is that there must be a range within which fitness variation is tolerated. this is reminiscent of nearly neutral theories of evolution and their extension to rna viruses (chao, 1997; ohta, 1997) . note also that from a theoretical perspective the same secondary structure can be found in all parts of sequence space with easy connectivity (schuster, 1995; schuster et al., 1997) . figure 6 .6 shows a number of variations on an hiv stem-loop structure, crucial for ribosomal frameshifting between the gag and pol open reading frames. there have been substitutions at positions 1, 2, 5, 8 and 11 and even an opening up of the loop. all come from viable strains, yet the environment in which these structures are operative, the human ribosome, is invariant. if the changes are all neutral the situation is formally comparable to the steady accumulation of amino acid substitutions. however, if the intermediates are less fit, it has to be understood how they can survive long enough in the face of a plethora of competitors, approximately 1/mutation rate or about 10 000 for hiv. the latter is probably the case as there are hiv-1 genomes with c:g to u:a substitutions at positions 5 and 8 ( figure 6 .6). extensions of nearly neutral theory would fit these findings well (chao, 1997; ohta, 1997) . that there are many solutions to this stem-loop problem is clear. if hiv-2 is brought into the picture, the remarkable plurality of solutions is further emphasized ( figure 6 .6). degeneracy in solutions found by viruses is revealed by some interesting experiments on viral revertants. the initial lesions substantially inactivated the virus. yet with a bit of patience, sometimes more than 6 months, replicationcompetent variants that were not back mutations were identified (klaver and berkhout, 1994; olsthoorn et al., 1994; berkhout et al., 1997; willey et al., 1988; escarmis et al., 1999) . as the frequencies of mutation and back mutation are not equivalent, such findings are, perhaps, not surprising. what they show is the range of possible solutions adjacent to that created by the experimentalist. loss of fitness can be achieved hiv-2 gag-pol figure 6.6 "shifty" rna stem-loop structures from hiv-1 m, n and o group strains as well as from hiv-2 rod. this structure is part of the information that instructs the ribosome to shift from the gag open reading frame to that of pol. in addition to the hairpin is a heptameric sequence (underlined). frameshifting occur within the gag uua codon within the heptamer and continues agg.gaa etc.* highlights differences in nucleotide sequences compared with the m reference strain lai. by sequential plaquing of rna viruses, the socalled miiller's ratchet experiment, which has been analysed at the genetic level for fmdv . different lesions characterized different lineages. recent work was aimed at characterizing the molecular basis of fitness recovery following large population passage. not one solution was found but a variety, even in parallel experiments (escarmis et al., 1999) . this reveals the impact of chance in fitness selection on a finite population of variants, which is trivially small given the immensity of sequence space. another example of degeneracy in viable solutions is the isolation of functional ribozymes from randomly synthesized rna (bartel and szostak, 1993; ekland et al., 1995) . from a pool of approximately 1014 variants, through repeated rounds of positive selection, it was estimated that the frequency of the ribozyme was of the order of 10 -8, which is small indeed. yet 10-8.1014=106 . even erring by four orders of magnitude, 100 distinct ribozymes could well have been present in the initial pool. although the sequence space occupied may well represent a tiny proportion of that possible for a rna molecule of length n, the space is so large that the number of viable solutions is large, large enough to permit a plethora of parallel solutions to the same problem. these experiments, ribozyme from dust, are cases in plurality. further evidence of the large proportion of viable solutions in protein sequence space comes from in vitro mutagenesis. for example bacteriophage t4 lysozyme can absorb large numbers of substitutions (rennell et al., 1991) with very few sites resisting replacement (figure 6.7) . other examples include the lymphokine, interleukin 3, in which some forms with enhanced characteristics were noted (olins et al., 1995; klein et al., 1997) . with modern mutagenic methods allowing mutation rates of 0.1 per base per site or less, hypermutants of the e. coli r67 dihydrofolate reductase (dhfr) were found by random sequencing of as little as 30 clones (martinez et al., 1996) . whatever the mutation bias, mutants with 3-5 amino acid replacements within the 78-residue protein could be attained (figure 6 .8). other mutagenesis studies sought enzymes with enhanced catalytic constants or chemical stability. for subtilisin e variants with enhanced features for two parameters could be identified from a relatively small population of randomly mutagenized molecules (kucher and arnold, 1997) . these data indicate that functional sequence space is probably far more dense than hitherto thought. most of the above examples concern maintenance or enhancement of function. an interest-ing example was recently afforded by engineering cyclophilin into a proline-specific endopeptidase (qu6m6neur et al., 1998) . the proline binding pocket of cyclophilin was modified such that a single amino acid change (a91s) generated a novel serine endopeptidase with a 101~ proficiency with respect to cyclophilin. addition of two further substitutions (f104h and n106d) generated a serine-aspartic-acid-histidine catalytic triad, the hallmark of serine proteases. the final enzyme proficiency was 3.5 x 1011 mol/1, typical of many natural enzymes. this shows the interconnectedness of sequence spaces for two functionally very different proteins. if sequence space were sparsely populated, the probability of observing such phenomena would be small. many viruses recombine, and via molecular biology more can be made, some of which are tremendously useful research tools, such as the shivs, chimeras between siv and hiv ( figure 6 .9). although many groups have tried to recombine naturally hiv-1 with hiv-2 or siv, none has succeeded. natural and artificial recombination represent major jumps in sequence space. that one can observe such genomes means that the new site in functional sequence space must be only a few mutations figure 6.7 systematic amino acid replacement of bacteriophage t4 lysozyme residues. amber stop codons were engineered singly into each residue apart from the initiator methionine. the plasmids were used to transform 13 suppressor strains. of the resulting 2015 single amino acid substitutions, 328 were found to be sufficiently deleterious to inhibit plaque formation. more than half (55%) of the positions in the protein tolerated all substitutions examined. the side chains of residues that were refractory to substitution were generally inaccessible to solvent. the catalytic residues are glu11 and asp20. adapted from rennell et al., 1991 . the e. coli r67 plasmid. all were trimethoprim resistant. only differences with respect to the parent sequence are shown. a representation of the three-dimensional structure is shown above. adapted from martinez et al., 1996, with permission. from a reasonably viable solution, otherwise it would take too long to generate large numbers of cycles and, along with them, mutants. the ferocity of innate and adaptive immunity must never be forgotten. off on an apparent tangent, the phylogeny of geoffrey chaucer's the canterbury tales was recently analysed by programs tried and tested for nucleic acid sequences. the authors used 850 lines from 58 fifteenth-century manuscripts (barbrook et al., 1998) . apart from the fact that it appears that chaucer did not leave a final version but some annotated working copy, the radiation in medieval english space is fascinating. all the versions are viable and "phenotypically" equivalent even though the "genotypes" are not so. it is ironic that william caxton's first printed edition was far removed from the original. (n.b., printers merely make fewer errors than scribes, tantamount to adding a 3' exonuclease domain to an rna polymerase). given the inevitability of mutation, is it possible that over the aeons natural selection has selected for proteins that are robust, those that are capable of absorbing endless substitutions? for if amino acid substitutions were very difficult to fix, huge populations would need to be explored before change could be accommodated. recently the unstructured n-terminal segment of the e. coli r67 dhfr was shown to stabilize amino acid substitutions in a non-functional miniprotein devoid of this segment (figure 6 .10; martinez et al., 1996) . while the mechanism by which this occurs is unknown, it suggests that there may be parts of proteins, even multiple or discontinuous segments, that may help the protein accommodate inevitable change. formally it can be seen that such figure 6.9 genetic organization of naturally occurring hiv-1 and siv recombinants and unnatural, genetically engineered, siv-hiv-1 chimeras called shivs. segments are hatched according to stain origin. references are hiv-1 mal and hiv-1 ibng (gao et al., 1996) , hiv-1 92rw009.6 (gao et al., 1998) , sivagm sab-1 (jin et al., 1994) and shivsbg (dunn et al., 1996) . proteins would have both short-and long-term selective advantages, for they would permit the generation of larger populations of relatively viable variants as well as buffering the lineage against the effects of bottlenecking. what fraction of amino acid residues is necessary for function? answer -very few. a few examples taken from among the primate immunodeficiency viruses are typical. almost all these viruses infect the same target cell using the membrane proteins cd4 and ccr5. primary hiv-1 isolates use the chemokine receptor ccr5 and rarely the homologous molecule cxcr4, which differs by 81% in its extracellular domains. yet two substitutions in the viral envelope protein gp120 are sufficient to allow use of the cxcr4 molecule (hwang et al., 1991) . curiously, the ccr5 chemokine receptor homologue, us28, encoded by human cytomegalovirus, can be used by hiv-1 despite the fact that us28 and ccr5 differ by 88% in the same extracellular regions . clearly only a small set of residues are necessary for docking . another example is afforded by the vpu protein, which is unique to hiv-1 and the chim-panzee virus sivcpz (huet et al., 1990) . vpu is a small protein inserted into the endoplasmic reticulum, tucked well away from humoral immunity. despite an average amino acid sequence difference of 0.5% among orthologous human and chimpanzee proteins, hiv/sivcpz vpu divergence is almost beyond reliable sequence alignment (figure 6 .11): an n-terminal hydrophobic membrane anchor and a couple of perfectly conserved serine residues, which are phosphorylated, and that's about it. among hiv-1 strains, or between sivcpz sequences, the situation was a little better. yet the necessity of keeping vpu is beyond doubt. a fine final example concerns the hiv/siv rev proteins. these small nuclear proteins are crucial to viral replication. despite this, only 5 residues are perfectly conserved. the situation has been taken beyond the limit, at least ex vivo, in that the htlv-1 rex protein can functionally complement for hiv-1 rev (rimsky et al., 1989) , despite the fact that they are completely different proteins. the above is reminiscent of what is known about enzymes and surface recognition. provided the protein fold is maintained, only a small fraction of residues actually contribute to function, a point made recently in two reviews on rna viral proteases (ryan and flint, 1997; ryan et al., 1998) . insertions and deletions are generally less than 2-3 residues in length and confined to turns, loops and coils (pascarella and argos, 1992) . if globular proteins or at least domains are, to a first approximation, taken as spheres, then the surface area is the least for any volume. if amino acids are equally viewed as smaller, closely packed spheres, then a minimum number will be exposed on the surface, ready to partake in recognition and function. the molecular biologist frequently thinks like an engineer who can redesign from scratch. yet replicons have been constrained by a series of historical events representing variations on a founding theme. while they are fit enough to survive, are they the best possible? this question is salutary, for we live in a society that is more and more competitive and, thanks to global communications, knows about the most successful athletes or businessmen worldwide. yet who can remember the name of any olympic athlete who came in fourth? is not fourth best in any large population remarkable? how good are viruses as machines? once again let us look at some examples from hiv-1. reverse transcription feeds on cytoplasmic dntps. yet supplementing the culture milieu with deoxycytidine -which is scavenged and phosphorylated to the triphosphate-substan-tially increased viral replication (meyerhans et al., 1994) . it is known that good expression of a foreign protein is frequently compromised by inappropriate codon usage. by redesigning codon usage of the jellyfish (aequorea victoria) green fluorescent protein gene to correspond to that typical of mammalian genes, greatly improved expression was achieved in mammalian cells (haas et al., 1996) . the same group engineered codon usage of the hiv-1 gp120 glycoprotein gene segment to correspond to that of the abundantly expressed human thy-1 surface antigen. again expression was greatly improved (haas et al., 1996) . the coup de grace came with the reciprocal experiment-engineering thy-1 gene codon usage to correspond to that of gp120. thy-1 surface expression was greatly reduced (haas et al., 1996) . since hiv-1 was first sequenced, it has been known that its codon usage is highly biased (wain-hobson et al., 1985; bronson and anderson, 1994) . something is clearly overriding maximal envelope expression. furthermore, gp120 codon usage is similar for all other hiv-1 genes whether they be structural or regulatory. for that matter, codon usage is comparable for most lentiviruses bronson and anderson, 1994) . it was possible to show via dna vaccination that codon-engineered gp120 elicited stronger immune responses in mice than the normal counterpart (andre et al., 1998) . might this finding suggest that the optimum is actually away from mass production? yet if there is a shadow of reality in this thesis, it indicates that fitness optima in vivo may not necessarily parallel the expectations of fitness based on ex-vivo models. in this context note also that htlv-1 infects exactly the same cell as hiv, yet its codon usage is very different from that of hiv and the thy-1 gene (seiki et al., 1983) . if fitness optimization were ever operative in vivo, then one would predict steady increases in virulence for those viruses that do not set up herd immunity. at some point a plateau would be reached. yet the higgledy-piggledy way by which virulent strains come and go suggests that this is not so. some might use the word stochastic. whatever. if fitness selection can be overridden and we don't have a good theory for it, then we're in a sorry state. there is abundant evidence that, as a good first approximation, rna viruses ex vivo perform as expected from the quasi-species model (holland et al., 1982; eigen and biebricher, 1988; duarte et al., 1992; clarke et al., 1993; eigen, 1993; novella et al., 1995; domingo et al., 1996; quer et al., 1996; domingo and holland, 1997) , which is fitness dominated. problems arise transposing it to the in vivo situation, notably: 9 first and foremost: how does one determine fitness in vivo? should such measurements score intrahost viral titres or transmission probabilities from an index case? (if a virus doesn't spread it's dead.) for outbred populations, is it in fact virulence? 9 second: host innate immunity is hugely powerful, a fact leading rolf zinkernagel to remark with typical aplomb that in terms of immunity "an inferferon receptor knock-out mouse is a 1% mouse" (huang et al., 1993; van den broek et al., 1995a,b ). yet the enhanced susceptibility of scid humans or various knock-out mice to infections indicates the part played by adaptive immunity. for example, influenza a can persist in scid children (rocha et al., 1991) . how are innate and adaptive immune responses coupled and how are they influenced by genetic polymorphisms? 9 third: with acquired immunity rising by day 3 in an acute infection, the virus is replicating in the face of a predator whose amplitude is increasing. 9 fourth: immune responses are densitydependent. that is, the more the virus replicates the stronger the immune response. if the relationship were simply linear one could see how a virus might be able to keep just ahead, given a short lag in the immune response time. but if it were non-linear? indeed it must be so, otherwise it would not be possible to resolve an acute infection. it is not easy to discern where optimal viral fitness would lie. 9 fifth: the wrath of combined immune responses is such that there is massive viral turnover. for the three best known cases, hiv, hbv and hcv, between 108 and 1012 virions are turning over daily, representing between 10% and 90% of the whole (ho et al., 1995; wei et al., 1995; nowak et al., 1996; zeuzem et al., 1998) . indeed, these are probably underestimates, given beautiful data from the late 1950s and 1960s showing that, for a variety of rna viruses, plasma titres decay with a half-life of 15-20 min, whether the animal be immunologically naive or primed (mimms, 1959; nathanson and harrington, 1967) . from this one may conclude that any viral population is unlikely to be in equilibrium. and if a population is not in equilibrium, fitness selection is compromised. 9 sixth: a glance at any histology slide or textbook is a salient reminder of spatial discontinuities over distances of one or two cell diameters. for example the hugely delocalized immune system is characterized by a multitude of different lymphoid organs, a myriad of subtly different susceptible cell types, and a m616e of membrane molecules. the exquisite spatial heterogeneity of hiv within the epidermis and splenic white pulps has been described (cheynier et al., 1994 (cheynier et al., , 1998 sala et al., 1994) . the same seems to be true for hcv-infected liver (martell et al., 1992) . for hpv infiltration of skin, spatial discontinuities and gradients are also apparent. discontinuities reduce the possibilities for competition and hence selection of the fitter forms. indeed the m~iller's ratchet experiment and clonal heterogeneity are the most vivid expressions of this. 9 seventh:muchhasbeenmadeofprivilegedsites and viral reservoirs. basically this is reminding us of the fact that immune surveillance is modulated in some organs like the brain. there are some suggestions that cytotoxic t-cells have difficulty infiltrating the kidney. viral reservoirs undermine fitness selection. 9 eighth: in the case of the immunodeficiency viruses, antigenic stimulation of infected yet resting memory t-cells means that variants may become amplified for reasons that have nothing to do with the fitness of the variant (cheynier et al., 1994 (cheynier et al., , 1998 ). mayr again: "wherever one looks in nature, one finds uniqueness" (mayr, 1997) . as mentioned, the cardinal difference between the behaviour of rna viruses ex vivo and in vivo is the existence of spatial discontinuities. for replicons, cloning is the ultimate separation. it allows a variant to break away from dominating competitors, disrupts or uncouples a fitter variant locked in competitive exclusion (de la torre and holland, 1990) . the effect of bottlenecking on fitness, as well as the m(iller's ratchet experiments, have been described (chao, 1990; duarte et al., 1992; novella et al., 1995; escarmis et al., 1996) . transmission frequently involves massive bottlenecking, and is very much an exercise in cloning. all this should not surprise because allopatric speciation is omnipresent in the origin of species, darwin's galapagos finches being an obvious example. given the non-equilibrium structure of viral variants, vastly restricted population sizes in respect to sequence space, founder effects in vivo take on great importance. while answers for some of these issues seem far away, constraints on fitness selection cannot be so strong that a chain of infections becomes a mfiller's ratchet experiment. yet is that correct? in the experiments with phage ~6, vsv and fmdv, most of the lineages resulted in decreased fitness. yet for some there were no changes, while for a few there were even increases in the fitness vectors (chao, 1990; duarte et al., 1992; escarmis et al., 1996) . could symptomatic infections reflect bottleneck transmission of those fitter clones with asymptomatic (subclinical) infection representing fitness-compromised clones? analysis of rna viruses ex vivo is analogous to the study of bacteria in chemostats. fitness selection dominates. yet there is a world of difference between bacterial strains so selected and natural isolates. one of the observations frequently made upon isolation of pathogenic bacteria is the loss of bacterial virulence determinants (miller et al., 1989) . indeed, ex-vivo passage of rna viruses has been used to select for attenuated strains used in vaccination. a virus must replicate sufficiently within a host to permit infection of another susceptible host. if the new host is of the same species, differences between the two are minimal-a small degree of polymorphism being inevitable in outbred populations. given that viruses with a small coding capacity interact particularly intimately with the host-cell machinery, it follows that infection of a host from a related species has a greater probability of succeeding if the cellular machinery is comparable. indeed, the closer the two species, the greater the probability. in turn, if the virus gets a toehold and can generate a quasispecies, then only few mutations would probably be necessary to adapt to the new niche. yet species is a difficult word. what might a viral species be? martin (1993) wrote a fascinating review on the number of extinct primate species estimated from the fossil record. depending on the emergence time of primates of modern aspect, he was able to estimate the total number that existed as 5500-6500. the present number of 200 primates species would thus represent about 3.4-3.8%. more importantly from our viewpoint was his calculation of the average survival time of fossil primate species as a mere 1 million years (martin, 1993) . given that rna viruses are fixing mutations approximately i million times faster than mammals (holland et al., 1982; gojobori and yokoyama, 1985; , a viral species would become extinct after approximately 1 year! immediately the annual influenza a strain comes to mind. yet rabies, polio and htlv-1 have arguably been around for millennia. clearly the word "species", when taken from primatology, cannot apply to the viral world. frogs provide a more interesting example. they have been around for several hundred millions of years, and members of some lineages can interbreed despite 75 million years separation. naturally, their protein sequences have not stood still during that time (wilson et al., 1977) . enough is conserved to allow breeding. maybe the primate picture has undue weight in our appreciation of virology. phenotype can be maintained despite changes in genotypeobvious to a biologist. as usual, holland wasn't far from the mark when he wrote: as human populations continue to grow exponentially, the number of ecological niches for human rna virus evolution grows apace and new human virus outbreaks will likely increase apace. most new human viruses will be unre-markable -that is they will generally resemble old ones. inevitably, some will be quite remarkable, and quite undesirable. when discussing rna virus evolution, to call an outbreak (such as aids) remarkable is merely to state that it is of lower probability than an unremarkable outbreak. new viruses can and do emerge but on a scale that is probably 15-20 logs less than the number of viral mutants generated up to that defining moment (wain-hobson, 1993 ). they will result from a small number of mutations and a dose of reproductive isolation. the above has attempted to show that the vast majority of genetic changes fixed by rna viruses are essentially neutral or nearly neutral in character. positive selection exploits a small proportion of genetic variants, while functional sequence space is sufficiently dense, allowing viable solutions to be found. although evolution has connotations of change, what has always counted is natural selection or adaptation. it is the only force for the genesis of a novel replicon. once adapted to its niche, there is no need to change. in such circumstances an rna i i i ii i ii i bacteria, archaea, yeast figure 6.12 latitude in microbial genome sizes. rna viruses and retroviruses are confined to one log variation in size (3 to --32 kb). by contrast, dna viruses span more than 2.5 logs going from the single-stranded porcine circulovirus (1.8 kb) to chlorella virus (~330 kb, encoding at least 12 dna endonuclease/methyltransferase genes; zhang et al., 1998) and bacteriophage g (--670 kb). the distinction between phage dna and a plasmid has often proven difficult (waldor and mekalanos, 1996) . as can be seen, the genome size of the largest dna viruses overlaps the smallest intracellular bacteria such as mycoplasmas (580 and 816 kb) and is not too far short of autonomous bacteria such as haemophilus influenzae (1.83 mb). virus would no longer be adapting, even though it could be changing. why is the evolution of rna viruses so conservative? why do they mutate rapidly yet remain phenotypically stable? the lack of proofreading proscribes the genesis of large genomes, restricting their genome sizes to a 1 log range (figure 6.12) . among the smallest rna and retroviruses are ms2 and hepatitis b virus, both about 3 kb, while the largest are the coronaviruses at 32 kb or more. most of their proteins are structural or regulatory and take up the largest part of the coding capacity of the virus. additional proteins broadening the range of interactions with the host cell, or rendering the replicon more autonomous, are relatively few. large, gene-sized duplications that may contribute to diversification and novel phenotypes are rare, reducing the exploration of new horizons. thus, evolution of rna viruses is probably conservative because they cannot shuffle domains so generating new combinations. that the information capacity of rna viral genomes is limited by a lack of proofreading is neither here nor there, for they are remarkably successful parasites. rna viruses change far more than they adapt. muller's ratchet decreases fitness of a dna-based microbe increased immune response elicited by dna vaccination with a synthetic gp120 sequence with optimized codon usage the phylogeny of the canterbury tales isolation of new ribozymes from a large pool of random sequences forced evolution of a regulatory rna helix in the hiv-1 genome role of the first and third extracellular domains of cxcr-4 in human immunodeficiency virus coreceptor activity molecular mechanisms of immune responses in insects nucleotide composition as a driving force in the evolution of retroviruses unusually high frequency of epstein-barr virus genetic variants in papua new guinea that can escape cytotoxic t-cell recognition: implications for virus evolution role of host immune response in selection of equine infectous anemia virus variants fitness of rna virus decreased by muller's ratchet evolution of sex and the molecular clock in rna viruses hiv and t-cell expansion in splenic white pulps is accompanied by infiltration of hiv-specific cytotoxic t-lymphocytes antigenic stimulation by bcg as an in vivo driving force for siv replication and dissemination genetic bottlenecks and population passages cause profound fitness differences in rna viruses nucleotide sequences of three nodavirus rna2's: the messengers for their coat protein precursors primary and secondary structure of black beetle virus rna2, the genomic messenger for bbv coat protein precursor hla-a11 epitope loss isolates of epstein-barr virus from a highly al1+ population t cell responses and virus evolution: loss of hla all-restricted ctl epitopes in epstein-barr virus isolates from highly all-positive populations by selective mutation of anchor residues rna virus quasispecies populations can suppress vastly superior mutant progeny the genome sequence of herpes simplex virus type 2 rna viral mutations and fitness for survival basic concepts in rna virus evolution origins and evolutionary relationships of retroviruses rates of spontaneous mutations among rna viruses rapid fitness losses in mammalian rna virus clones due to muller's ratchet high viral load and cd4 lymphopenia in rhesus and cynomolgus macaques infected by a chimeric primate lentivirus constructed using the env, rev, tat, and vpu genes from hiv-1 lai the viral quasispecies sequence space and quasispecies distribution structurally complex and highly active rna ligases derived from random rna sequences does the vp1 gene of foot-and-mouth disease virus behave as a molecular clock? largescale search for genes on which positive selection may operate genetic lesions associated with muller's ratchet in an rna virus multiple molecular pathways for fitness recovery of an rna virus dibilitated by operation of miiller's ratchet determining divergence times with a protein clock: update and reevaluation human infection by genetically diverse siv-sm related hiv-2 in west africa the heterosexual human immunodeficiency virus type 1 epidemic in thailand is caused by an intersubtype (a/e) recombinant of african origin a comprehensive panel of near-fulllength clones and reference sequences for non-subtype b isolates of human immunodeficiency virus type 1 rates of evolution of the retroviral oncogene of moloney murine sarcoma virus and of its cellular homologues molecular evolutionary rates of oncogenes molecular clock of viral evolution, and the neutral theory codon usage limitation in the expression of hiv-1 envelope glycoprotein evolution of influenza virus genes performance evaluation of amino acid substitution matrices rapid turnover of plasma virions and cd4 lymphocytes in hiv-1 infection rapid evolution of rna genomes rna virus populations as quasispecies immune response in mice that lack the interferon-gamma receptor genetic organization of a chimpanzee lentivirus related to hiv-1 protein phylogenies provide evidence of a radical discontinuity between arthropod and vertebrate immune systems pattern of nucleotide substitution at major histocompatibility complex class i loci reveals overdominant selection identification of the envelope v3 loop as the primary determinant of cell tropism in hiv-1 statistical analysis of nucleotide sequences of the hemagglutinin gene of human influenza a viruses mosaic genome structure of simian immunodeficiency virus from west african green monkeys the role of cytotoxic t-lymphocytes in the evolution of genetically stable viruses evolution of a disrupted tar rna hairpin structure in the hiv-1 virus the receptor binding site of human interleukin-3 defined by mutagenesis and molecular modeling directed evolution of enzyme catalysts analysis of hiv-1 env gene sequences reveals evidence for a low effective number in the viral population tempo and mode of nucleotide substitutions in gag and env gene fragments in human immunodeficiency virus type 1 populations with a known transmission history molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses escape of human immunodeficiency virus from immune control hepatitis c virus (hcv) circulates as a population of different but closely related genomes: quasispecies nature of hcv genome distribution primate origins: plugging the gaps exploring the functional robustness of an enzyme by in vitro evolution independent fluctuation of human immunodeficiency virus type 1 rev and gp41 quasispecies in vivo natural selection and the concept of a protein space this is biology temporal fluctuations in hiv quasispecies in vivo are not reflected by sequential hiv isolations in vivo persistence of a hiv-l-encoded hla-b27-restricted cytotoxic t-lymphocyte epitope despite specific in vitro reactivity restriction and enhancement of human immunodeficiency virus type 1 replication by modulation of intracellular deoxynucleoside triphosphate pools lifespan of human lymphocyte subsets defined by cd45 isoforms coordinate regulation and sensory transduction in the control of bacterial virulence the response of mice to large intravenous injections of ectromelia virus. i. the fate of injected virus experimental infection of monkeys with langat virus sequence constraints and recognition by ctl of an hla-b27-restricted hiv-1 gag epitope size of genetic bottlenecks leading to virus fitness loss is determined by mean initial population fitness how hiv defeats the immune system viral dynamics in hepatitis b virus infection the meaning of near-neutrality at coding and non-coding regions saturation mutagenesis of human interleukin-3 leeway and constraints in the forced evolution of a regulatory rna helix analysis of insertions/deletions in protein structures the tempo and mode of siv quasispecies development in vivo calls for massive viral replication and clearance identification of a chemokine receptor encoded by human cytomegalovirus as a cofactor for hiv-1 entry genetic drift can dominate short-term human immunodeficiency virus type 1 nef quasispecies evolution in vivo viral strategies of immune evasion positive selection of hiv-1 cytotoxic t lymphocyte escape variants during primary infection antigen-specific release of beta-chemokines by anti-hiv-1 cytotoxic t lymphocytes engineering cyclophilin into a proline-specific endopeptidase reproducible nonlinear population dynamics and critical points during replicative competitions of rna virus quasispecies nucleotide sequence analysis of sa-omvv, a visna-related ovine lentivirus: phylogenetic history of lentiviruses systematic mutation of bacteriophage t4 lysozyme trans-dominant inactivation of htlv-i and hiv-1 gene expression by mutation of the htlv-i rex transactivator antigenic and genetic variation in influenza a (hin1) virus isolates recovered from a persistently infected immunodeficient child virus-encoded proteinases of the picornavirus super-group virus-encoded proteinases of the flaviviridae spatial discontinuities in human immunodeficiency virus type 1 quasispecies derived from epidermal langerhans cells of a patient with aids and evidence for double infection genetic evolution and tropism of transmissible gastroenteritis coronaviruses how to search for rna structures. theoretical concepts in evolutionary biotechnology rna structures and folding. from conventional to new issues in structure predictions natural selection on the gag, pol, and env genes of human immunodeficiency virus 1 (hiv-1) human adult t-cell leukemia virus: complete nucleotide sequence of the provirus genome intergrated in leukemia cell dna rates of amino acid change in the envelope protein correlate with pathogenicity of primate lentiviruses nucleotide sequence of the visna lentivirus: relationship to the aids virus antiviral defense in mice lacking both alpha/beta and gamma interferon receptors immune defence in mice lacking type i and/or type ii interferon receptors fixation of mutations at the vp1 gene of footand-mouth disease virus. can quasispecies define a transient molecular clock? the fastest genome evolution ever described: hiv variation in situ viral burden in aids running the gamut of retroviral variation nucleotide sequence of the aids virus lysogenic conversion by a filamentous phage encoding cholera toxin viral dynamics in human immunodeficiency virus type i infection in vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity biochemical evolution adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection molecular evolution of the hepatitis b virus genome quantification of the initial decline of serum hepatitis c virus rna and response to interferon alfa chlorella virus ny-2a encodes at least 12 dna endonuclease / methyltransferase genes we would like to thank past and present members of the laboratory and numerous colleagues for endless discussions over the years. mark mascolini needs a special word of thanks for painstakingly going through the manuscript. this laboratory is supported by grants from the institut pasteur and the agence nationale pour la recherche sur le sida. key: cord-011064-1d3v87km authors: roskin, krishna m.; jackson, katherine j. l.; lee, ji-yeun; hoh, ramona a.; joshi, shilpa a.; hwang, kwan-ki; bonsignori, mattia; pedroza-pacheco, isabela; liao, hua-xin; moody, m. anthony; fire, andrew z.; borrow, persephone; haynes, barton f.; boyd, scott d. title: aberrant b cell repertoire selection associated with hiv neutralizing antibody breadth date: 2020-01-20 journal: nat immunol doi: 10.1038/s41590-019-0581-0 sha: doc_id: 11064 cord_uid: 1d3v87km a goal of hiv vaccine development is to elicit antibodies with neutralizing breadth. broadly neutralizing antibodies (bnabs) to hiv often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. a subset of hiv-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. we sequenced antibody heavy-chain repertoires in a large cohort of hiv-infected individuals with bnab responses or no neutralization breadth and uninfected controls, identifying consistent features of bnab repertoires, encompassing thousands of b cell clones per individual, with correlated t cell phenotypes. these repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. our data indicate that the development of numerous b cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of hiv neutralizing antibody breadth. nabs against hiv are able to recognize the envelope glycoproteins of diverse viral variants, often by targeting conserved epitopes that play important functional roles in the viral life cycle. after years of chronic infection, between 10% and 50% of hivinfected individuals develop antibodies with neutralizing breadth in their serum, depending on the criteria used to define breadth 1 . many hiv bnab lineages have stereotyped genetic and structural features, such as high frequencies of somatic hypermutation (shm) and long heavy-chain complementarity-determining region 3 (cdr-h3) sequences. these sequence features are rare in healthy human antibody repertoires, but contribute to targeting vulnerable epitope sites on the hiv envelope glycoprotein 2 . understanding the mechanisms contributing to bnab formation is a priority for hiv vaccine development: vaccines eliciting bnab-like antibodies could potentially protect against a wide range of hiv strains. why do some hiv-infected individuals generate neutralizing antibody breadth, while others do not? it is possible that all individuals who are infected have a similar potential for producing bnabs, but that formation of these antibodies is rate-limited by very rare stochastic events such as formation of naive b cells with particular cdr-h3 sequences, or the acquisition of particular somatic mutations during affinity maturation. in this model, bnab lineages in each patient would be unusual outliers from the rest of their antibody repertoire. alternatively, some infected individuals could be predisposed to generating bnab-like antibodies due to systematic differences in their b cell repertoire formation or selection processes. in such patients, bnabs would arise from a pool of many antibody lineages with similar features. evaluation of germline allelic variants in antibody heavy-chain variable gene segments found no correlation with bnab production 3 . however, other features of repertoire formation such as v(d)j segment recombination frequencies, cdr-h3 junction production, shm frequency or differences in b cell selection could also affect the likelihood of producing bnab-like antibodies. factors such as the t cell populations providing help to b cells; regulatory t (t reg ) cell populations that may control autoreactive b cells; or the mixtures and frequencies of hiv viral variants in the patient, could also lead to systematic differences in neutralizing antibody breadth between patients. here, we analyze antibody heavy-chain (igh) gene repertoires in 96 patients with chronic hiv infection (46 patients with extensive neutralizing breath (bnab) and 50 patients without neutralizing breadth (nonab)) and 43 hiv-uninfected controls from the same geographical regions, to assess whether the two hiv-infected groups show systematic differences in antibody repertoire formation or selection. our results based on extensive sequencing of each patient's igh repertoire indicate that hiv-infected individuals show significant perturbations of their antibody repertoires, including suppressed average shm frequencies and increased frequencies of b cells expressing antibodies with features associated with autoreactivity. bnab individuals each have hundreds to thousands of antibody lineages with long cdr-h3s and very high shm frequencies. in contrast, nonab subjects and hiv-uninfected individuals appear to select against antibody lineages with long cdr-h3 and high shm frequencies. further, we note that patient b cell and t cell phenotypes are linked, with a negative correlation seen between ctla-4 + t reg cells and bnab patient cdr-h3 lengths. an unrelated chronic viral infection, human cytomegalovirus, shows no evidence of eliciting the igh repertoire deviations seen in bnab hiv-infected individuals. our results indicate that production of hiv neutralizing antibody breadth is associated with specific systematic differences in individual b cell repertoire formation and selection. igg shm in hiv-infected bnab and nonab individuals. we first evaluated shm frequencies in igh repertoires encoding each antibody isotype and subclass, to assess the effects of hiv infection, as well as to test for differences between bnab and nonab individuals. bnab individuals were those whose serum showed the broadest inhibition of a panel of 12 hiv isolates in a pseudovirus infection assay, with 95% inhibition of at least seven isolates (see methods). hiv-infected individuals showed overall decreases in mean shm frequencies in igh expressed as igg but not in other isotypes, compared to uninfected individuals ( fig. 1a and supplementary fig. 1a ). bnab individuals, however, had mean igg shm frequencies closer to those of uninfected people. notably, the most highly mutated antibodies (represented here by the shm value of the 99th percentile in the distribution of shm frequencies in each participant) were more mutated in igg subtypes of bnab individuals compared to both uninfected controls and nonab individuals ( fig. 1b and supplementary fig. 1b) . the 99th percentile shm values of igg in bnab individuals approach the shm frequencies of well-described bnab monoclonal antibodies isolated in previous studies (fig. 1c) , and are higher than the shm of non-neutralizing antibodies or those elicited by vaccination in previous studies ( supplementary fig. 1c ). we noted that the standard deviation of shm values for the clones within each individual was also higher in igg subtypes in bnab individuals compared to nonab or hiv-uninfected individuals ( supplementary fig. 1d ). the antibody clonal lineages at the 99th percentile of mutation or higher, represent an average of 790 distinct clones (range 287-1,390) in each bnab individual, rather than small numbers of outlier clones. frequencies of insertions and deletions (indels) in the antibody variable regions have been previously shown to be correlated with shm frequency 4 . here, insertion frequency in ig genes correlated with shm frequency and was decreased in all hiv-infected individuals compared to uninfected individuals, but deletion rates in igg1 in bnab individuals were higher, even than in uninfected individuals ( supplementary fig. 1e ). iga antibodies behaved differently from the other isotypes, having increased indel rates in hiv-infected individuals compared to uninfected individuals, even though their shm frequencies were similar to those of uninfected controls. these results highlight further differences in igg shm or selection processes compared to the other isotypes in antibody repertoires of patients with hiv infection. antibodies with long cdr-h3 regions have been previously reported to be enriched for autoreactive or polyreactive sequences, although this is not the only determinant of antibody autoreactivity 5 . we found that igg subtype antibodies in both bnab and nonab individuals had longer cdr-h3s compared to uninfected individuals, as previously reported ( fig. 2a and supplementary fig. 2a) 6 . the fraction of very long cdr-h3 (>19 amino acids) was also higher in igg subtypes hiv-infected individuals compared to uninfected controls but did not differ between bnab and nonab individuals ( fig. 2b and supplementary fig. 2b ). clones with >19 amino acids in their cdr-h3s represented thousands of distinct lineages in each participant (mean of 8,548 for bnab individuals, 7,540 for nonab individuals and 7,891 for uninfected individuals). to evaluate whether these cdr-h3 length differences could be attributed to the early steps of b cell antigen receptor (bcr) rearrangement in the bone marrow, we sequenced igh rearrangements from a genomic dna template and analyzed the sequences of unproductive rearrangements (the remnants of unsuccessful first attempts by the b cell precursor to rearrange the igh locus). nonfunctional rearrangement cdr-h3s were longer than productive ones but surprisingly, all hiv-infected individuals had shorter nonproductive cdr-h3s than uninfected individuals, with bnab individuals having the shortest nonproductive cdr-h3s of all (fig. 2c) . therefore, the increased proportion of igg antibodies with cdr-h3 lengths exceeding 19 amino acids in hiv-infected individuals does not arise during bcr repertoire formation but is a consequence of subsequent b cell selection. long cdr-h3s and high shm in bnab igg repertoires. given the observed differences in shm and cdr-h3 features between antibody repertoires in hiv-infected individuals and those of uninfected individuals, we sought to determine whether any interactions between antibody characteristics were specific to bnab individuals, as many known bnab antibody lineage types require both long cdr-h3 and high shm frequencies in their development. we plotted the average cdr-h3 lengths for antibodies at the 70th, 80th, 90th, 95th and 99th or higher percentiles of shm frequency in each patient's repertoires of each antibody isotype ( fig. 3 and supplementary fig. 2c ). bnab-producing individuals showed a significant increase in cdr-h3 lengths in the most-mutated fractions of their igg repertoires, in contrast to nonab or uninfected individuals. therefore, bnab producers are distinct from nonab individuals in the selection or persistence of b cells expressing antibodies with long cdr-h3 regions, particularly when these are highly somatically mutated. previous studies have measured frequencies of gp120 + b cells in bnab patient blood to be in the order of 30 per 30,000 (0.1%) igg + memory b cells and bnabs to be 1-4 per 30,000 (0.003-0.01%) igg + memory b cells, suggesting that the shm and cdr-h3 differences that we observe between bnab and nonab individuals are not due solely to hiv-specific gp120-binding b cell lineages [7] [8] [9] [10] . decreased ctla-4 + t regs in bnab individuals with long cdr-h3. we evaluated whether the shm frequencies or cdr-h3 lengths in bnab, nonab or uninfected individuals were related to hiv viral load or frequencies of cd4 + t cells or other t cell subsets. average shm frequencies in igg antibodies within the nonab group were correlated with their cd4 + t cell frequencies, but this was not the case for the bnab or uninfected individuals, suggesting that nonab individuals alone show a dependence on cd4 + t cell frequencies for maintaining shm (supplementary fig 3a) . as expected, both bnab and nonab individuals had lower cd4 + t cell frequencies than uninfected individuals ( supplementary fig. 3b ) and as previously reported, bnab individuals had higher viral loads than nonab individuals, although these average differences between groups were not sufficient to predict bnab status ( supplementary fig. 3c) 11 . the 99th percentile igg shm frequencies were positively correlated with cd4 + t cell frequencies and were negatively correlated with higher viral loads in nonab individuals, but not in bnab individuals ( supplementary fig. 3d ,e). the data suggest that bnab producers respond differently to their immunological environment (viral load and frequencies of cd4 + t cells; supplementary fig. 3f ) and achieve very high shm frequencies in numerous clones despite having lower cd4 + t cell frequencies than healthy individuals. we next examined t cell subsets of known helper or regulatory functions. resting memory follicular helper (t fh ) cell (cxcr3 − pd-1 + within cd4 + cxcr5 + ) frequencies in the blood were not correlated with 99th percentile igg shm frequencies ( supplementary fig. 4a ). given that the shm frequencies in the most highly mutated igg fractions were higher in bnab individuals than in nonab or healthy individuals, generation of very high shm frequencies was not limited by cd4 + t cell or memory cd4 + t fh cell frequencies in the bnab individuals. there were no significant relationships between the frequency of regulatory cd4 + t reg cells (cd25 + foxp3 + cd4 + cells) and extreme (99th percentile) igg shm frequencies (fig. 4a ) or cdr-h3 lengths in any subject group (fig. 4b) . circulating t follicular regulatory (t fr ) cells (cd25 + foxp3 + within cxcr5 + cd4 + t cells) showed no correlation with cdr-h3 lengths ( supplementary fig. 4b ), and only a weak negative correlation with the 99th percentile igg shm frequencies in both bnab and nonab individuals ( supplementary fig. 4c ). the 99th percentile igg shm frequencies had no relationship to the proportion of t reg cells expressing ctla-4 ( fig. 4c) , but there was a significant negative correlation between the frequency of ctla-4 + t reg cells and mean cdr-h3 lengths in bnab individuals (p = 0.0016; fig. 4d ). ctla-4 is an activation marker in cd4 + t cells, and plays an important role in the control of humoral responses by cd4 + t reg and t fr cells 12 . hiv viral loads were usually higher in bnab individuals than nonab individuals ( supplementary fig. 3d ). while this analysis cannot prove causation, and it is possible that these t cell populations are coordinately regulated with the b cell repertoires in study participants rather than directly affecting the b cells, the bnab patients with the lowest frequencies of ctla-4 + t reg cells had the longest cdr-h3s. the length of the longest cdr-h3s (99th percentile) in bnab individuals showed a similar negative correlation with ctla-4 + t reg cell frequencies. we observed a weak but nonsignificant correlation between ctla-4 + t reg cell frequencies and patient viral loads in bnab individuals ( supplementary fig. 4d ). bnab ighv4-34 usage and selection against autoreactivity. most known bnabs use a restricted set of ighv gene segments. we examined ighv segment usage frequency in the 1% most-mutated clones of each individual, focusing on segments such as ighv4-34, which possesses natural autoreactivity in the unmutated state, to determine whether there were differences that might predispose them to forming bnabs (fig. 5a-c and supplementary fig. 5a ). after bonferroni multiple hypothesis correction, we identified a statistically significant difference in the usage of ighv4-34 between groups, in igg, igm and igd antibodies, but not in unproductive igh rearrangements or in the iga compartments. hiv-infected individuals used ighv4-34 more than uninfected controls and bnab individuals used ighv4-34 more frequently than other groups. this difference was most evident in the most-mutated clones in each individual ( fig. 5c and supplementary fig. 5b) . b cells expressing antibodies using ighv4-34 convey information about the immune system's control of autoreactive clones. unmutated ighv4-34 encodes a tyrosine residue at codon 26 (in the imgt numbering system) at the junction between framework 1 and cdr-h1 that contributes to a hydrophobic patch that binds poly-n-acetyllactosamine carbohydrate self-antigens (known as 'big i' and 'little i' antigens) on red blood cells 13, 14 . shm that alters codon 26 can remove self-reactivity and is very common in b cells that use ighv4-34 (ref. 13 ). hiv-infected individuals have decreased codon 26 mutation frequencies compared to uninfected individuals, when normalized for total antibody mutation frequencies, suggesting that hiv infection is associated with a permissive state in which potentially autoreactive clones that would otherwise be eliminated can persist (fig. 5d ). previous studies with relatively small cohorts have reported lower bcr sequence diversity in individuals with hiv as compared to uninfected controls; however, it can be challenging to distinguish between true decreases in species richness versus the presence of some larger clones 15 . by sequencing replicate igh libraries from six independent genomic dna (gdna) template aliquots for each individual, we calculated a normalized 'clonality score' equivalent to the probability that randomly selected reads from two replicate igh libraries from a sample will be from the same b cell clone. unrearranged ighv gene segments do not appear in these gdna libraries as they are too far upstream of the ighj gene segments to give pcr amplicons. individuals who were hiv-infected had elevated clonality compared to individuals who were hiv-uninfected (fig. 6) . in all groups, shm frequency was increased in expanded clones compared to those showing no evidence of clonal expansion ( supplementary fig. 6 ). there was no difference between bnab and nonab individuals in this measure, even when differences in cd4 + t cell counts and viral loads were included in the analysis (supplementary fig. 3 and ref. 11 ). we hypothesized that igh repertoire differences as well as antigendriven selection would give rise to distinctive common or 'convergent' rearrangements distinguishing individuals who were hiv-infected from those who were hiv-uninfected and bnab from nonab individuals. such convergent sequences could be useful for diagnostic purposes, identifying b cell repertoires that have been shaped by exposure to hiv and those that give rise to bnab antibody types. some convergent sequences would be expected to be hiv-specific, while others could be polyreactive, autoreactive or specific for other antigens. we searched for shared igh sequences that were informative for distinguishing individuals who were hiv-infected from those who were hiv-uninfected. individuals were scored based on the number of such shared sequences that they demonstrated. receiver operating characteristic (roc) plots showed that this classification scheme was effective for distinguishing individuals with hiv infection from those who were uninfected, achieving a >90% true positive rate with a <10% false positive rate, with an area under the curve (auc) of 0.95 (fig. 7a) . the same approach, attempting to distinguish bnab individuals from nonab individuals performed less well, with an auc of 0.72, but showed some discrimination between these groups. selection of convergent clonotypes for these classifications was subjected to cross validation to minimize overfitting of the classifier, but we did not empirically test the binding specificity of the convergent antibodies identified. when we searched for the sequences of known monoclonal bnabs that have been previously isolated from a few of the individuals in the bnab patient cohort in this study, using a threshold of 80% nucleotide identity in cdr-h3, equal cdr-h3 length and the same ighv and ighj gene segment usage, we identified bnabs ch01, ch02, ch03 and ch04 in the igh repertoire data from individual 0219; bnabs dh511.1/2/3/6/7p/8p and dh511.4/5/10p in individual ch0210; and ch105 in individual ch0505 (refs. 9, 16, 17 ). at a cdr-h3 identity threshold of 75%, we also identified bnabs ch103, ch104 and ch106 in individual ch505 (ref. 17 ). we did not find convergent antibodies to the known bnab lineages in other broad-neutralizing individuals at these cdr-h3 identity thresholds. we infer that the antibody lineages responsible for hiv neutralizing breadth in these other individuals do not share high sequence identity with known bnabs. to further evaluate the ability of igh repertoire data to classify hiv infection status and neutralizing breadth, we combined the presence or absence of convergent igh sequences with general repertoire statistics listed in table 1 : mean igg mutation level; 99th percentile igg mutation level; mean igg, igm and igd cdr-h3 lengths; fraction of 'long' (>19 amino acids) igg, igm and igd cdr-h3s; mean nonfunctional cdr-h3 length from gdna libraries; and usage of ighv4-34 in the 1% most-mutated igg sequences, in a random forest model (fig. 7b ) with the same cross-validation scheme as above 18 . while this more complex model yielded only a small improvement on the already accurate classification of hiv infection status (auc of 0.96), the addition of repertoire statistics markedly improved the categorization of bnab and nonab individuals (auc of 0.89). cytomegalovirus minimally alters igh repertoires. finally, we assessed whether the igh repertoire features differing with hiv infection state and neutralizing breadth were specific to hiv, by considering an unrelated chronic viral infection, human cytomegalovirus (cmv), a herpesvirus causing a lifelong persistent infection that is highly prevalent in many populations and almost ubiquitous in hiv-infected cohorts. we sequenced igh repertoires in hivseronegative blood donors, of whom n = 52 were cmv-seropositive and n = 61 were cmv-seronegative. individuals who were cmvseropositive compared to those who were cmv-seronegative showed none of the igh repertoire features seen in individuals who were infected with hiv and bnab individuals, such as overall shm differences in igg isotypes ( supplementary fig. 7a) , 99th percentile shm values in igg isotypes ( supplementary fig. 7b) , cdr-h3 lengths ( supplementary fig. 7c ) or cdr-h3 fraction longer than 19 amino acids ( supplementary fig. 7d) , cdr-h3 lengths in progressively higher-shm fractions of the igg repertoires ( supplementary fig. 7e ) or ighv4-34 usage (supplementary fig. 7f) . these results indicate that the hiv-associated or bnab-associated igh repertoire features we observed in the hiv-infected indviduals are not a generic consequence of all chronic viral infections. many human immune responses show heterogeneity, such as when memory of previous exposures shapes responses to new influenza viral strains 19 or host genetic background influences hepatitis b vaccination responses 20, 21 . it has been unclear whether an individual's hiv neutralizing antibody breadth depends on rare stochastic events giving rise to very few bnab clones or whether bnab producers have systematic bcr repertoire differences leading to many potential bnab lineages. our data favor the latter possibility, showing numerous lineages with high shm and long cdr-h3s in bnab individuals. notably, the most highly mutated fraction of iggs in bnab individuals have higher shm frequencies than those of nonab or uninfected individuals and number in the hundreds to thousands per individual, rather than representing rare outlier clones. human pre-or pro-b cells with long cdr-h3s are removed from the repertoire before the naive b cell stage, and antigen-experienced b cell populations are further selected to have shorter cdr-h3s compared to the naive repertoire 5, 22 . we found that cdr-h3s in unselected, unproductive igh in hiv-infected individuals were shorter than in uninfected controls, similarly to findings in common variable immune deficiency 22 (cvid) and potentially reflecting altered v(d)j recombination in the bone marrow. in contrast, cdr-h3s in productive igg antibodies were longer in hiv-infected individuals compared to uninfected individuals, showing a strong selection for longer cdr-h3s. however, only the bnab individuals showed selection favoring long cdr-h3 in the most highly mutated fractions of their antibody repertoires. these findings do not exclude a major role for the course of the viral infection and the number and variety of hiv antigenic variants that arise in each patient in driving antibody repertoire phenotypes. bnab producers had higher viral loads, suggesting that greater antigen drive could contribute to bnab lineage development 23 . previous estimates of hiv broadly neutralizing or gp120 + b cells in bnab individuals ranged from 0.003-0.1% of memory igg + b cells, indicating that these cells are unlikely to account for the differences in shm and cdr-h3 values that we observed between bnab and nonab individuals [7] [8] [9] [10] . however, gp120 + and bnab b cell measurements may underestimate frequencies of cells specific for other unknown hiv antigenic variants in participants. from t cell phenotyping data previously published 11 , we identified t cell correlates of bcr repertoire features in individuals who were infected with hiv. shm frequencies in bnab individuals were not correlated with total cd4 + t cell or memory t fh frequencies in the blood. ctla-4 + t reg frequencies correlated with cdr-h3 lengths in bnab producers, opening the possibility that these cells may be involved, either directly or indirectly, in controlling cdr-h3 lengths in the maturing bcr repertoire of bnab individuals. alternatively, increased ctla-4 expression in t reg cells in this context may primarily indicate that higher levels of t reg activation are present under conditions that also lead to shorter cdr-h3s. transient modulation of immune checkpoints such as the ctla-4 pathway during hiv vaccination has been proposed as a new approach to enable hiv-infected individuals to generate bnabs. any attempt to test this idea would need to include strategies to minimize autoimmune side effects, such as those seen in cancer immunotherapy trials 24, 25 . our analysis raises the hypothesis that ctla-4 blockade could lead to hiv-specific antibodies with longer cdr-h3s. antibodies using unmutated ighv4-34 are implicated in cold agglutinin disease, an autoimmune hemolytic anemia associated with hiv infection and other immune disorders 26, 27 . igg in hivinfected individuals used ighv4-34 at higher frequencies than in uninfected controls, particularly in bnab individuals. shm of codon 26 in ighv4-34 decreases this autoreactivity, a process termed 'clonal redemption' and is seen at high frequencies in hivinfected individuals 13 . serum reactivity with an anti-idiotype antibody that binds unmutated ighv4-34 has been loosely correlated with broad hiv neutralization 28 . we found that bnab producers show significantly lower shm frequencies of codon 26 in ighv4-34 gene segments compared to uninfected controls, suggesting that the lack of selection against autoreactive antibodies is associated with the development of hiv neutralizing breadth, consistent with previous reports of bnab producers showing various autoantibodies in their sera 29, 30 . in summary, our evidence points to the loss of normal selection against b cells expressing igg with long cdr-h3 regions and high shm frequencies, and a general loss of selection against autoreactive antibodies, in the development of hiv neutralizing breadth. ctla-4 + t reg cells show a strong negative correlation with cdr-h3 lengths in the bcr repertoires of bnab producers, suggesting that this cell population, or ctla-4 pathway manipulation more generally, could be targets for future strategies to improve neutralizing breadth generated by hiv vaccination. a major topic of current study is the way that differences in the hiv viral populations, such as the diversity, concentrations or epitope features of viral subpopulations, may affect the b cell phenotypes we observe. hiv-infected individuals produce characteristic 'convergent' clones with highly similar sequences detected in two or more individuals that are rarely detected in healthy people. these convergent clones indicate whether an individual is infected by hiv, although we have not determined whether all such clones are specific to hiv. we also find other clones that more weakly classify an individual's hiv neutralizing breadth. the accuracy of bnab classification is enhanced in models that incorporate additional repertoire characteristics, but convergent clones alone are sufficient to predict hiv infection status accurately. as sequence data accumulate for human antibodies specific for various pathogens, vaccines and other antigens, it may become possible to use antibody repertoire data to predict an individual's responses to future immune stimuli and to improve vaccination strategies for challenging pathogens such as hiv. any methods, additional references, nature research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41590-019-0581-0. methods patient cohorts. participants were recruited in a multisite study of acute and chronic hiv-1 infection that included an uninfected control arm, as previously reported 11 . the study was approved by the duke medicine institutional review board as well as the ethics boards of the local sites. all participants provided informed consent. on average, participants in the chronic arm of the study were followed for >400 d. patients defined clinically as virus controllers were excluded from the study. participants who were infected with hiv and uninfected controls were predominantly african and regions of origin of these individuals are described 11 . matching of nonneutralizing hiv-infected controls to broadly neutralizing hiv-infected individuals was carried out using propensity scorematching with age, sex and country of origin as input variables, and resulted in a nonneutralizing set of control individuals that did not significantly differ in age, sex or country of origin compared to the broadly neutralizing individuals. bnab patient status was determined by serological measurement of the reciprocal inhibitory dilution for 50% reduction of infection (id 50 ) for a panel of 12 hiv isolates in the tzm-bl pseudovirus neutralization assay, a measure of hiv neutralization based on decreases in expression of a tat-regulated firefly luciferase (luc) reporter gene after one round of infection with env-pseudotyped viruses 31 . to be categorized as a bnab individual, the participant's principal component score (pc1) from the neutralization panel data was required to exceed a value of 3 (representing the top 14% of neutralizers) and their id 50 score was required to be at least 95 for 7 of the 12 viral isolates in the panel 11 . all nonneutralizing hivinfected controls had pc1 scores <0.1 and did not exceed an id 50 threshold of 95 for any hiv isolate in the serological panel. for comparison of cmv-seropositive and cmv-seronegative participant igh repertoires in supplementary fig. 7 , a separate cohort of 113 healthy adult blood donors from the stanford blood center was used. all participants provided informed consent. these participants were negative for pathogen-screening testing for hiv, hcv, hbv, htlv, west nile virus, tppa (syphilis) and trypanosoma cruzi, and had no symptoms of acute illness or diseases, including malignancies. participant ages ranged from 17-87 years (median: 52 years and mean: 49 years). sequence data from these participants are reported by nielsen et al. 32 . for analysis of circulating cd4 + t reg and t fr cells, monoclonal antibodies used for surface staining included cd3 pe-texas red (s4.1; life technologies), cd4 apc-h7 (sk3), cd25 pe-cy7 (2a3), cxcr5 af488 (rf8b2) and pd-1 bv421(eh12.1) (all from bd biosciences). pbmcs were stained for 15 min at 37 °c before washing and staining for 10 min at 20 °c with live/dead fixable aqua dead cell stain (life technologies). the foxp3 fix/perm kit (ebioscience) was then used to fix and permeabilize cells at 20 °c for 15 min before staining with foxp3 apc (pch101) and ctla-4 percp-efluor710 (14d3) (both from ebioscience) at 20 °c for 45 min, after which they were washed twice with permeabilization buffer. flow cytometry data were acquired with a cyan adp analyzer (beckman coulter) or lsr fortessa x-20 analyzer (bd biosciences) with standard filter sets and data were analyzed using flowjo software (v.8.8.6; treestar). the t cell flow cytometry data were previously published 11 and details of the gating strategies used for identification of cell subsets are described there. heparinized whole-blood samples (5 to 8 ml) were collected and pbmcs were isolated by centrifugation of diluted blood over histopaque-1077 (sigma-aldrich) or ficoll-paque (ge healthcare). gdna was isolated via column purification (qiagen), magnetic bead-based isolation (magna pure, roche life science) or centrifugation-based purification (archivepure, 5 prime). rna was isolated using the allprep dna/rna kit (qiagen). pcr amplification of cdna for igh library sequencing. for rna-derived antibody isotype libraries, cdna was synthesized with superscript ii reverse transcriptase (thermo fisher scientific) with random hexamer primers. templates were amplified by pcr using isotype-specific primers located in the first exon of the constant (c1) regions (supplementary table 2 ) and biomed-2 ighv primers in the framework 1 (fr1) region (supplementary table 4) 22, 33 . these primers also encoded approximately half of the illumina linker sequences needed for cluster generation and sequencing on the miseq instrument. sample identity was encoded by eight-nucleotide multiplex identifier barcodes in each primer. for illumina cluster recognition, four randomized nucleotides were encoded in the primers immediately after the illumina linker sequence in the constant region primers. each antibody isotype for each sample was amplified in a separate pcr reaction to prevent formation of cross-isotype chimeric pcr products. pcr was carried out with amplitaq gold (thermo fisher scientific) following the manufacturer's instructions using a program of: 94 °c for 7 min, 35 igh sequence processing and analysis. sequences with exact matches to ighv and ighj (gdna) or isotype constant region (for cdna-derived data) barcodes were assigned to the corresponding samples and replicate libraries. barcodes and primer sequences were trimmed. matches to gene-specific primer sequences allowed for a one-nucleotide mismatch. trimmed sequences had their v, d and j gene segments and junctional bases annotated with igblast 1.3.0 (ref. 34 ) and a sequence database of germline gene segments from the international immunogenetics information system (imgt) 35 . annotation of antibody isotype and subtype of rna-derived sequences was accomplished by looking for perfect matches between constant regions from the imgt database and sequences upstream of the constant region primer. cdr-h3 sequences were identified on the basis of the conserved cysteine-104 and the motif downstream of the conserved tryptophan-118 residue 36 . non-igh artifactual sequences and those with poor ighv matches (ighv segment match bit score less than 140) were removed from the data. the python scripts and r code used for secondary analyses of the data can be provided on request. in total, 25,507,784 sequences from hiv-infected individuals (mean: 265,706 per individual) and 8,393,313 sequences from healthy controls (mean: 195,193 per individual) were obtained and analyzed. for the cmv cohort analysis, at total of 36,720,077 sequences from individuals who were cmv-seropositive and 45,429,593 sequences from those who were cmv-seronegative was obtained and analyzed. inference of clonal lineages. sequences were clustered into putative clonal lineages using single-linkage clustering. briefly, the process starts with all sequences in their own lineages and iteratively, two lineages are merged if any two reads, one from each lineage, satisfy the following four criteria: (1) come from the same individual; (2) share the same v and j segment annotations (not including allele designation); (3) have equal cdr-h3 length; and (4) cdr-h3 nucleotide sequences match with 90% or more identity. the process ends when there are no lineages satisfying the criteria. this clonal inference procedure produced 6,821,567 clones from hiv-infected individuals (mean: 710,578 per person) and 2,400,986 clones from healthy controls (mean: 66,349 per person). the mean number of reads per lineage was 3.68. to calculate a summary measure of the contribution of clonally expanded b cells to the repertoire of each individual, normalizing for sequencing depth in each individual, we calculated a clonality score as: p ijk;i≠k n ij´nik p jk;j≠k t j´tk where n ij and n ik are the copy numbers of clone i observed in independent replicate pcr libraries, j and k, generated from independent aliquots of template dna and articles nature immunology t j and t k are total read numbers in the corresponding replicate libraries. the log base 10 of this score is shown in the figures. to determine antibody amino acid sequences that distinguish individuals with and without hiv infection or bnab from nonab-producers, we clustered igh amino acid sequences to identify convergent antibodies, that is antibodies elicited by different subjects with similar igh sequences. heavy-chain sequences annotated with the same v and j segment (not considering alleles) and the same cdr-h3 length were clustered based on 90% cdr-h3 amino acid sequence similarity. partitioning of sequences by v, j and cdr-h3 length was performed using custom software, while the cdr-h3 amino acid sequence clustering was performed using cd-hit 37 with options -c 0.90 -l 4 -s 0 -g 1 -b 1. for discriminating individuals with and without hiv infection, clusters were selected as informative if (1) they contained sequences from at least three individuals; (2) 80% of subjects with the sequences were hiv-infected; and (3) the cluster had an expected mutual information of 0.06 bits or greater 38 . for discriminating bnab from nonab-producers, clusters were selected if (1) they contained sequences from at least three individuals; (2) 80% of individuals with the sequences were bnab producers; and (3) the cluster had an expected mutual information of 0.05 bits or greater. a lower threshold was used to separate bnab from nonab-producers due to fewer individuals in this comparison. classifying individuals with predictive clusters. for each of the predictive clusters, a consensus cdr-h3 amino acid sequence was calculated by taking the most common amino acid at each position, where each cluster member was weighted by the number of participants with that sequence. a new individual was scored by co-clustering their annotated igh sequences with the consensus sequences of the predictive clusters. this clustering was performed using the same method as detailed above. the new individual was assigned a score equal to the number of predictive clusters that have sequences from the individual co-clustering with the sequences of the cluster. to test the classification accuracy of this prediction method, we performed fourfold cross validation. subject groups (individuals who were infected with hiv versus those who were not or bnab producers versus nonab-producers) were separated into four sets using stratified random sampling. the first three sets were used to select predictive igh sequences as described above. the selected sequences were then used to score the fourth set as described above. to give an accurate estimate of the generalization error of this method, this process was repeated for 160 partitions. features in the random forest classifier 39 included: the mean and 99th percentile mutation level of the igg compartment; the mean cdr-h3 length and fraction of long cdr-h3s (>19 amino acids) of the igg, igm, igd and out-of-frame sequences; clonality score; fraction of igg sequences using ighv4-34 and the presence or absence of the convergent igh sequences from the previous section. the same cross-validation scheme as above was used here. class probabilities were used to calculate the roc curve and auc. bioinformatical and statistical analysis. analysis of heavy-chain data was performed with custom python 40 code using pandas 41 and numpy 42 . statistical tests and generation of plots was performed in r 43 using rstudio 44 , ggplot 45 and plyr 46 . p values were calculated by using the two-sided wilcoxon mann-whitney u-test. box-whisker plots show the median (horizontal line), interquartile range (box) and 1.5-times the interquartile range (whiskers). prevalence of broadly neutralizing antibody responses during chronic hiv-1 infection hiv-1 neutralizing antibodies: understanding nature's pathways ability to develop broadly neutralizing hiv-1 antibodies is not restricted by the germline ig gene repertoire immunoglobulin gene insertions and deletions in the affinity maturation of hiv-1 broadly reactive neutralizing antibodies predominant autoantibody production by early human b cell precursors low frequency of broadly neutralizing hiv antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations maturation pathway from germline to broad hiv-1 neutralizer of a cd4-mimic antibody staged induction of hiv-1 glycan-dependent broadly neutralizing antibodies analysis of a clonal lineage of hiv-1 envelope v2/v3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors two distinct broadly neutralizing antibody specificities of different clonal lineages in a single hiv-1-infected donor: implications for vaccine design immune perturbations in hiv-1-infected individuals who make broadly neutralizing antibodies the coinhibitory receptor ctla-4 controls b cell responses by modulating t follicular helper, t follicular regulatory, and t regulatory cells clonal redemption of autoantibodies by somatic hypermutation away from self-reactivity during human immunization the i binding specificity of human vh4-34 (vh4-21) encoded antibodies is determined by both vh framework region 1 and complementarity determining region 3 dynamics of immunoglobulin sequence diversity in hiv-1 infected individuals potent and broad hiv-neutralizing antibodies in memory b cells and plasma co-evolution of a broadly neutralizing hiv-1 antibody and founder virus random decision forests antibody landscapes after influenza virus infection or vaccination host genetic factors and vaccine-induced immunity to hepatitis b virus infection differential genetic determination of immune responsiveness to hepatitis b surface antigen and to hepatitis a virus: a vaccination study in twins igh sequences in common variable immune deficiency reveal altered b cell development and selection the neutralization breadth of hiv-1 develops incrementally over four years and is associated with cd4 + t cell decline and high viral load during acute infection hiv-host interactions: implications for vaccine design immune related adverse events associated with anti-ctla-4 antibodies: systematic review and meta-analysis presence and significance of cold agglutinins in patients with hiv infection persistent cold agglutinins in aids and related disorders 9g4 autoreactivity is increased in hiv-infected patients and correlates with hiv broadly neutralizing serum activity cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv-1 antibodies polyreactivity and autoreactivity among hiv-1 antibodies tiered categorization of a diverse panel of hiv-1 env pseudoviruses for assessment of neutralizing antibodies shaping of infant b cell receptor repertoires by environmental factors and infectious disease design and standardization of pcr primers and protocols for detection of clonal immunoglobulin and t-cell receptor gene recombinations in suspect lymphoproliferations: report of the biomed-2 concerted action bmh4-ct98-3936 igblast: an immunoglobulin variable domain sequence analysis tool imgt, the international immunogenetics information system 25 years on a new clustering of antibody cdr loop conformations cd-hit: accelerated for clustering the next-generation sequencing data introduction to information retrieval classification and regression by randomforest python language reference v.2.7 (python software foundation data structures for statistical computing in python python for scientific computing r: a language and environment for statistical computing (r foundation for statistical computing rstudio: integrated development environment for r (rstudio elegant graphics for data analysis the split-apply-combine strategy for data analysis reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the b cell heavy-chain sequences analyzed here are available through the short read archive. data from the hiv cohort can be found in the bioproject prjna486667. data from cmv-seropositive and seronegative healthy controls are deposited as bioproject prjna491287. we gratefully acknowledge the participants who volunteered for this study. support for this work was provided by grants from the national institutes of health, national institute of allergy and infectious diseases, division of aids; um-1 grant for the duke center for hiv/aids vaccine immunology-immunogen discovery ai100645; national institutes of health grant nos. r21-ai100696, chavi-ai0678501, r01ai127877 and r01ai130398; and mrc programme grant no. mr/ k012037. a patent application related to computational methods used in this paper is in preparation by k.m.r. and s.d.b. supplementary information is available for this paper at https://doi.org/10.1038/ s41590-019-0581-0.correspondence and requests for materials should be addressed to b.f.h. or s.d.b.peer review information l. a. dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.reprints and permissions information is available at www.nature.com/reprints. key: cord-015376-z739ifu5 authors: savarino, andrea; buonavoglia, canio; norelli, sandro; trani, livia di; cassone, antonio title: potential therapies for coronaviruses date: 2006-08-31 journal: expert opin ther pat doi: 10.1517/13543776.16.9.1269 sha: doc_id: 15376 cord_uid: z739ifu5 coronavirus replication offers several attractive targets for chemotherapy. these include: viral entry (inhibited by chloroquine and peptides); viral rna (targeted by antisense approaches/rnai); the main protease 3clpro (inhibited by peptidic molecules such as hiv-1 protease inhibitors and miscellaneous compounds); the accessory protease(s) plpro(s) (inhibited by zinc ions); rna-dependent rna polymerase (inhibited by aurintricarboxylic acid and antisense approaches); and helicase (inhibited by bananins). chloroquine and hiv-1 protease inhibitors (with well-known toxicity profiles) should be considered for clinical tests if severe acute respiratory syndrome (sars) re-emerges; however, there are other attractive compounds. lessons should be learnt from aids research for choosing the best strategies. coronaviruses were isolated from chickens in 1937 and were long considered to be important pathogenic agents in animals, producing seasonal cold or mild gastrointestinal infections in humans. it was not until the outbreak of the severe acute respiratory syndrome (sars) during 2002/2003 that extensive efforts went into the research for specific anticoronavirus therapies. the emergence of the sars coronavirus (sars-cov), capable of killing ∼ 900 people in a few months, clearly indicated that more resources had to be put into coronavirus research. in the 3 years after 2003, more potential anticoronaviral compounds have been patented than in the previous decades of coronavirus research. here, the authors review the published patents/patent applications on the most relevant compounds/approaches for potential anticoronaviral therapies, focusing on those approaches targeting one specific step of the coronavirus life cycle as they are more likely to be used as lead compounds for further drug development. in case the reader needs information on an anticoronavirus drug that has been left 'orphan' of a molecular target, they are directed to excellent reviews in [1, 2] . finally, the authors highlight those compounds that are likely to have an immediate clinical application in case sars re-emerges. 1. introduction 2. principal drug targets and specific inhibitors 3. expert opinion s glycoprotein contacts the tip of one lobe of ace2. it does not contact the other lobe, nor does it occlude the peptidase active site. binding of the s protein to ace2 is not altered by the addition of a specific ace2 inhibitor [9] . these crystallographic data suggest that specific sars-cov inhibitors could be developed to block the ace2/s protein interaction and these sars-cov inhibitors should be different from ace2 inhibitors blocking the peptidase activity (inhibitors of the ace2 enzymatic activity have been patented by millennium pharmaceuticals, inc. [101] ). the atomic details at the interface between the two proteins described by li et al. [9] give important information for virtual screening of antiviral compounds. alanine-scanning mutagenesis analysis indicated that, in ace2, charged amino acids between residues 22 -57 were important, k26 and d30, in particular [10] . a soluble form of ace2 has been described for potential sars treatment [101] . although it may be applied as a decoy for neutralising circulating viruses, this approach could hardly find a clinical application, as suggested by lessons learnt from aids research. a similar approach, that is, the use of soluble cd4, the main receptor for hiv-1, was postulated for the treatment of hiv-1/aids. despite the in vitro data being encouraging [11] , soluble cd4 has not found a clinical application in ∼ 20 years. specific inhibitors of the s glycoprotein/ace2 interaction have been produced using isolated portions of the s glycoprotein [102] . using a system in which a lentivirus expressing green fluorescent protein and pseudotyped with the sars-cov s glycoprotein infects 293t cells stably expressing ace2, the inventors detected potent antiviral activity of a peptide comprising amino acids 318 -510 of the s glycoprotein (ic 50 < 10 nm). approaches using peptides in order to prevent viral entry are encouraged by results from aids research. indeed, proof-of-concept has been provided that systemic administration of a peptide from a highly conserved region of the hiv-1 viral envelope glycoprotein gp41, significantly impacts on viral load [12] . an easy strategy to prevent the attachment of a virus to a cell is the use of neutralising antibodies. monoclonal antibodies directed to the s glycoprotein of sars-cov have been produced [103] . these antibodies inhibit the interaction of the virus with ace2. this strategy may be applied in passive immunotherapy or immunoprophylaxis after exposition to sars-cov, and, based on the affinity of these antibodies with the s1 region responsible for binding ace2, this invention might be exploited in screening assays for anti-sars-cov compounds. compounds binding the s glycoprotein at the site for interaction with ace2 should also prevent the interaction with the antibodies. another strategy to elicit neutralising antibodies is the use of specific vaccines [104] . however, the description of vaccination strategies would go beyond the scope of this review, which deals with therapeutic approaches. another group of strategies against coronaviruses is aimed at targeting the cellular receptor rather than the virus itself. receptor down-modulation is postulated to be a valuable approach to limit the entry of coronaviruses inside target cells [105] . an invention uses double-and single-stranded oligomeric antisense compounds, particularly single-or double-stranded oligonucleotides (rna or rna-like), and single-stranded oligonucleotides (dna or dna-like) for use in modulating expression of aminopeptidase n [105] . multiple mechanisms exist by which short synthetic oligonucleotides can be used to modulate gene expression in mammalian cells. these include degradation by rnase h or rna interference (rnai; an excellent review on these mechanisms can be found in [3] ). an advantage of this approach would be the extreme selectivity due to the oligonucleotide match requirement, while a disadvantage would be the extreme complexity in the administration of these molecules. furthermore, it is not yet known what would be the effect of ace2 down-modulation in humans. the most promising approach to limit the entry of a coronavirus into target cells is probably represented by an old antimalarial drug, chloroquine (1), which has the advantage of being a small molecule and, therefore, presenting no difficulties in administration. past studies have supported the idea that chloroquine might inhibit coronavirus replication. cells infected with hcov-229e and treated with nocodazole (a microtubule depolymerising agent that blocks transport from early to late endosomes) produced decreased amounts of hcov-229e antigens [13] . this result indicated that endosomal transport is needed for hcov-229e infection and cells treated with chloroquine expressed decreased amounts of hcov-229e antigens [13] . meanwhile, unpublished data obtained from the authors' group showed that chloroquine potently inhibited replication of ccov at therapeutically achievable concentrations (figure 2) . as: i) chloroquine is also endowed with anti-inflammatory properties (reviewed in [14] ); and ii) clinical worsening of individuals with sars in week 2 is apparently related to immunopathological damage [15] , some of the authors suggested that chloroquine be considered in the treatment of sars and tested against sars-cov [14] . the authors' hypothesis was confirmed in two independent in vitro studies. researchers at the belgian catholic university of leuven found that chloroquine inhibited sars-cov replication with an ec 50 value of 8.8 ± 1.2 µm, within the range of blood concentrations achievable during antimalarial treatment [16] . the dose-inducing 50% cytostatic activity was much higher, that is, 261.3 ± 14.5 µm. time-of-addition experiments indicated that chloroquine affected an early stage of sars-cov replication [16] . researchers at the centers for disease control and prevention (atlanta, ga, usa) reported potent anti-sars-cov effects of chloroquine in vitro, attributable to a deficit in the glycosylation of the sars-cov receptor ace2 [17] . again, the antiviral drug concentrations were not cytotoxic. these results await confirmation in animal models. as discussed by van ranst et al., chloroquine is given prophylactically to people travelling to malaria-endemic areas. if sars re-emerges, chloroquine can be of great importance as prophylactic medication for people living in and travelling to the affected area. chloroquine is ubiquitously available, of low cost and easy to administer. the drug is generally well-tolerated and may also be administered to pregnant women. however, one disadvantage could be the possibility of retinopathy in cases of chronic exposure to the drug, such as those of prolonged prophylactic use. nevertheless, this side effect is reversible at the early stages and, thus, can be prevented by regular examination of the fundus. the use of quinoline compounds, including chloroquine, in combination with a multivitamin preparation has been claimed to treat different viral infections including those caused by coronaviruses [106] . patent [107] discloses an invention consisting of an adhesive patch containing an essential oil and an antimicrobial compound, such as chloroquine, in order to filter and kill pathogens such as coronaviruses entering the respiratory tract. given the mechanism of action of chloroquine (acting on cells rather than on the coronavirus itself ), this invention will not result in the anticoronavirus effect of chloroquine. the genomic rna of coronaviruses contains a capped, polyadenylated, single-stranded, positive-sense genomic rna that is 27 -32 kb in length and is the largest known rna virus genome. coronavirus-infected cells contain a characteristic 3′ coterminal and nested mrna. the mrna has a capped leader sequence consisting of < 70 nucleotides that is derived from the 5′ end of the genome. a non-translated region (utr) of 200 -400 nucleotides follows this leader sequence. another utr at the 3′ end of the rna genome is followed by a polya tail of variable length. both the 3′-and 5′-utrs are important in rna replication and transcription, as is the transcription-regulatory sequence, which is a typical feature of coronaviruses. this short motif is usually near the beginning of each open reading frame (orf) and the 3′ end of the leader sequence. moreover, the consensus sequence, 5′-cuaaac-3′, is found immediately in front of the s protein, and membrane (m) protein genes and orf 10 [3]. gene organisation in most coronaviruses follows the same pattern of genes coding for polyproteins 1a and 1b, s, m, envelope protein and nucleocapsid protein (figure 1) . some group ii coronaviruses possess a haemagglutinin esterase. coronavirus genomes also contain a variety of additional orfs that encode 2 -4 nonstructural proteins (nsps) with no known function; these genes are not conserved among coronaviruses. whereas the genes encoding the functional proteins are directly translated from genomic rna, production of the structural proteins requires the synthesis of specific mrnas by the functional proteins (figure 1 ) [3] . both the genomic and the mrnas can be targeted by the rna interference technique described in the previous section [3] . the use of small interfering rnas (sirnas) has been claimed for the treatment of sars in humans [108] . an advantage of this approach would be the expectedly low toxicity due to the extreme selectivity of coronavirus inhibition. again, a disadvantage could be the complexity in the administration of these molecules. in this context, encouraging results are derived from the sars-cov-infected rhesus macaque model. intranasal administration of chemically synthesised sirna duplexes resulted in the reduction of: i) sars-cov infection-induced fever; ii) sars-cov viral levels; and iii) acute diffuse alveoli damage. macaques did not show any toxic effects [18] . following cell infection, the viral replicase gene (fundamental for genomic rna replication and structural protein production) is translated directly from the viral genome [19] . autocatalytic processing by two types of proteases, which are part of the replicase polyprotein, releases nsps [20] [21] [22] . these form a membrane-bound rna replication complex [23, 24] . one of the two types of coronaviral proteases, that is, the 3c-like protease (3clpro), resides in nsp5 and, after autocleavage, releases the downstream replicase subunits [21] . 3clpro is responsible for cleavages at most polyprotein processing sites and, thus, is termed the 'main' protease, whereas coronaviral plpro(s) cleave only at the three n-terminal polyprotein processing sites and, thus, are termed 'accessory' protease(s). 3clpro is a cysteine protease. in the active site of sars-cov 3clpro, cys145 and a his41 form a catalytic dyad [25] . cysteine proteases usually display a catalytic triad consisting of serine, histidine and aspartate; however, the 3d structure of sars-cov 3clpro shows no aspartate residue in the vicinity of cys145 and his41. sars-cov 3clpro is a homodimer with each monomer comprising three domains [25] . the first two domains of 3clpro show a chymotrypsin-like folding with a root mean square deviation from bovine α-chymotrypsin of only 2.6 å [22] . the folding of the first two domains is responsible for the catalytic reaction, whereas the third domain is α-helical and plays a critical role in enzyme dimerisation [26] . the chymotrypsin-like folding of 3clpro is intriguing because the overall sequence identity between the 3clpro and α-chymotrypsin is quite . chymotrypsin-folding of 3clpro raised the hypothesis that 3clpro was originally a chymotrypsin-like serine protease that later evolved into a cysteine protease [27] . the potential usefulness of 3clpro as a drug target is supported by: i) its fundamental role in coronavirus replication; ii) its well defined 3d structure; and iii) preliminary clinical observation indicating that drugs cross-targeting this enzyme, that is, the hiv-1 protease inhibitors (hiv-1 pis; 2 -6) produced some clinical benefits in patients treated with ifns and ribavirin. the most thoroughly studied group of drugs targeting 3clpro are represented by hiv-1 pis. clinical, virological, biochemical and bioinformatic data concur in suggesting 3clpro as the main target for the anticoronaviral effect of hiv-1 pis, although robust data providing a causal link between 3clpro inhibition and impairment of sars-cov replication are still lacking. the story of hiv-1 pis as a potential sars treatment began during the 2003 sars outbreak with the empirical administration to affected individuals of the antiretroviral preparation kaletra (abbott), containing lopinavir (2) and ritonavir (3) at a ratio of 4:1 (w/w) [28] . a retrospective matched cohort study of chan et al. [29] found that the addition of lopinavir/ritonavir to standard initial treatment protocols was associated with a reduction in the overall death rate (2.3%) and intubation rate (0%) when compared with a matched cohort who received standard treatment (15.6 and 11.0%, respectively, p < 0.05), and with a lower necessity of methylprednisolone at high doses. in another study [30] , chu et al. evaluated the effects of kaletra in association with ribavirin, a drug that causes mutations in the rnas of many viruses. the adverse clinical outcome (acute respiratory distress syndrome or death) was significantly lower in the treatment group than in the historical controls treated with ribavirin alone (2.4 versus 28.8%, p < 0.001) at day 21 after the onset of symptoms. the clinical effects of antiretroviral drugs on sars were initially attributed to non-specific anti-inflammatory effects, but in vitro studies later on showed that some members of the hiv-1 pi class could indeed exert direct antiviral effects against sars-cov [31] . the hiv-1 pi nelfinavir (4; 10 µm), but not ritonavir (3), significantly and efficiently inhibited viral antigen expression (measured by immunofluorescence), the production of virions (measured by real-time reverse transcriptase-polymerase chain reaction [rt-pcr]) and the cytopathic effect (measured by the methyl tetrazolium assay) in vero e6 cells infected with sars-cov at a multiplicity of infection of 0.01. moreover, time-of-addition experiments showed that nelfinavir inhibited sars-cov replication at a postentry step and that lopinavir (3; 6.4 µm) inhibited the cytopathic effect of sars-cov (measured by plaque-reduction assay) in fetal rhesus kidney-4 (frhk-4) cells [30, 31] . of note, the effects of lopinavir were synergistic to those of ribavirin, a drug shown to be otherwise ineffective in the treatment of sars [30] . therefore, chu et al. attribute to this antiviral synergism the clinical benefits observed in individuals with sars and treated with ribavirin plus kaletra [30] . chen et al. [32] screened the effects of different antiviral compounds against ten clinical isolates of sars-cov. these compounds included the nucleosidic reverse transcriptase inhibitors (nrtis) zidovudine and stavudine, the non-nucleosidic reverse transcriptase inhibitor (nnrti) nevirapine, and the pis ritonavir and lopinavir. again, only lopinavir resulted in exerting detectable effects against sars-cov. the ec 50 value of lopinavir was in the range of 1.6 -6.4, and 6.4 -12.8 µm at 48 and 72 h postinfection respectively, in the frhk-4 cell line [32] . however, they warn that the antiviral effects are variable and cell line-dependent. for example, the ec 50 value of lopinavir against the prototype sars-cov strain 39849 was in the range of 3.2 -6.4 µm in frhk-4 cells, and 6.4 -12.8 µm in vero cells [32] . therefore, it is likely that the antiviral effects of some anti-sars compounds reported in the literature have been exaggerated or underestimated depending on the assay and cell line used by the different groups. in this regard, the example of ribavirin is interesting. some report that it is an inhibitor of sars-cov replication, whereas others report that it exerts its antiviral effects only at concentrations too high to be reached in vivo [30, 32, 33] . the latter view is also supported by clinical data. the limited experimental evidence available for an inhibitory effect of pis on 3clpro is supported by computational simulations revealing that the catalytic site of 3clpro allows the docking of several hiv-1 pis. clinically used anti-hiv-1 pis only partially fill the binding cavity of 3clpro, but a recent bioinformatic study [34] indicates stable ligand-protein interactions. among three well-known hiv-1 pis, that is, indinavir (5), saquinavir (6) and ritonavir (3), only ritonavir was found to display significant hydrogen bonding with 3clpro. these observations are in line with previous calculations of other groups using 3clpros of sars-cov and tgev [35, 36] . among the various compounds tested, zhang et al. [36] indicated ritonavir as the compound with the highest binding affinity (k i = 5.6 x 10 -25 m). these authors attribute to the other kaletra component, lopinavir, a k i value of 8.7 x 10 -20 m [34] . instead, enwitheesuk et al. [35] attribute a k i value of 10 -7 m to ritonavir. more recent calculations attribute a k i value of 10 -5 m to ritonavir [34] . this k i value is more realistic than those previously reported given that hiv-1 pis, at the highest concentrations tested (i.e., 10 µm), have been determined to inhibit the 3c-like protease only partially [31] . one advantage of hiv-1 pis is that these are among the very few drugs for which clinical evidence for potential usefulness in the treatment of sars is provided, although it is still very limited. a randomised trial would be needed to validate these results if sars were to return. a disadvantage of kaletra might be found in the adverse effects on lipid metabolism and by the high costs of these molecules. there is no patent specifically devoted to claiming the use of hiv-1 pis as sars-cov inhibitors. however, one of the previously quoted patents [107] claims the use by inhalation of different compounds, including all principal hiv-1 pis, expert opin. ther. patents (2006) 16(9) against different respiratory infections, such as those caused by coronaviruses. administration of these compounds by inhalation should allow the achievement of local drug concentrations sufficient to inhibit viral replication and to avoid systemic side effects. other peptidic inhibitors of 3clpro are being developed. different peptidic inhibitors of 3clpro, such as compounds 7 -10, have been described together with procedures for their synthesis [109] . published application [110] describes methods for the synthesis of other sars-cov 3clpro inhibitors. biological data on the activity of some compounds against sars-cov are also supplied. compound 11 was found to have an ec 50 value of 2.1 µg/ml and ic 50 value of > 50 µg/ml, which gave a selectivity index (si) of > 24. compound of example 24 (12) was found to have an ec 50 value of 0.02 µg/ml and ic 50 value of > 10 µg/ml, suggesting a potentially high therapeutic index. the same compound inhibited 100% of the cytopathic effect of sars-cov at 1 µm, with no detectable toxicity in cell cultures. for two other compounds, only the data on inhibition of the viral cytopathic effect are provided. at 1 µm, compound 13 inhibited 100% of the cytopathic effect of sars-cov, whereas compound 14 inhibited it by 94.5%. not enough data are available on this group of drugs to draw further conclusions; however, the in vitro data seem to be encouraging. based on the structural similarity between the 3c protease of rhinoviruses and coronaviral 3clpro, the use of the rhinovirus inhibitors, such as compounds 15 -23, against sars-cov has been proposed by pfizer and agouron pharmaceuticals [111, 112] . these compounds contain the ethyl-acrylate ester moiety as terminal warhead and, thus, act as michael acceptors. according to docking studies conducted by the inventors, the nucleophilic carbons in position 2,3 should covalently bind to the active site of sars-cov 3clpro, thus inactivating it irreversibly. other michael acceptor inhibitors of 3clpro (24 -44) are described in pfizer's patent application [111] and similar compounds, such as 45 and 46, have been claimed by agouron pharma [113] . although the inventors describe the procedures for testing these compounds in infected cell cultures, only molecular docking data are provided. other inhibitors presenting an acrylate group have been described and include a natural substance, namely, cinanserin (47) [114] . in general, nucleophilic protease inhibitors, due to their theoretical capacity to covalently inactivate 3clpro, are expected to potently inhibit sars-cov in vivo. however, the safety of these molecules should be carefully tested in cell cultures and animal models, given the potential toxicity of nucleophilic molecules. other miscellaneous inhibitors of 3clpro have been described, such as dicyclic or multicyclic compounds 48 -72 [115] , and molecules presenting the backbone (73) [116] . these chemically diverse molecules are in too early a phase of testing to allow any conclusions to be drawn. invention [117] describes boron-containing 3clpro inhibitors. these inhibitors are possible protease inhibitors and may have the backbones (74 -88), wherein the r 1-4 moieties can be h or different substituents, including aromatic cyclic compounds or heterocycles; x, y and u are various linker groups, and q is represented by different moieties, including ketones, alkanes, alkenes or cyclic compounds. the inventors chose to use boron-containing compounds to inhibit coronavirus 3clpro based on the use of boric acid and various boronic acids as inhibitors of β-lactamases (proteases and β-lactamases have in common the capacity of hydrolysing an amide bond). these compounds are effective in inhibiting 3clpro in enzymatic assays, but no biological data from infected cells are provided. again, it is too early to draw any conclusions about these particular molecules, but the use of boron-containing compounds to inhibit viral proteases seems to be interesting in light of recent crystallographic data using the hiv-1 protease [37] . the processing of the amino-proximal nsps is carried out by one or two paralogous protease domains within nsp3, the largest of the nsps [21] and they are defined by homology to the papain-like fold [20] and constitute the peptidase family c16 [38] . although mutational analyses supported the presence of a cys-his catalytic dyad [21, 39] , it is now clear that these enzymes also have a catalytic triad cys-his-asp [40] . most coronaviruses harbor two such papain-like protease sequences (plpro), pl1pro and pl2pro, whereas scov and the avian infectious bronchitis coronavirus (ibv) utilise only one, which is equivalent to pl2pro [41] . pl2pro may cleave down-and upstream of nsp3 [38, 41] , but only upstream cleavages were associated with pl1pro [21, 42, 43] . in actual fact, coronaviral plpros, although built on the common papain-like scaffold, look structurally more like ubuquitin-specific proteases (usps, merops family c19) than like papain (merops family c1) [38, 40] . coronaviral plpros have a two-domain organisation with a (circularly permutated) zn-finger domain nested in the middle of the papain-like fold, precisely as in the case of usps [20, 40] . plpros have been recently shown to possess deubiquitinylating activity [42] . this might be a means by which coronaviral proteins may escape the proteasome degradation pathway, thus limiting their antigenic presentation on hla class 1 molecules and avoiding the immune responses. these recent observations strengthen the hypothesis that plpro(s) may be an important drug target for therapeutic interventions. although substances inhibiting sars-cov plppro have been described [43] , no patents or patent applications have been published yet on the search engines consulted. it is hoped that the recent resolution of its structure [42] will help in the development of new effective inhibitors of coronavirus replication. interestingly, zinc ions inhibited the protease activity potently with an ic 50 value of 1.3 µm [43] . the inhibition is specific because other divalent metals, such as mg, mn, ca, ni and co, had no effect on the activity of sars-cov plp2 at 10 µm (data not shown). therefore, plpro inhibitors patentable in the future are likely to contain zinc. one of the proteins released by the proteolytic activity of the 3clpro main protease is nsp12, acting as a rna-dependent rna polymerase (rdrp; nsp12). coronaviral rdrp catalyses the production of new full-length genomic rnas to be packaged into virions as well as a nested set of monocistronic mrnas that encode all structural proteins. these activities require the formation of intermediate negative-sense rna (figure 1) . due to its pivotal role in viral rna synthesis and, consequently, in protein synthesis and genome duplication, the 106 kda rdrp represents an attractive target for anti-sars therapy [44] . however, there is a lack of structural and biochemical information on any coronavirus rdrp, and structural predictions are complicated by the fact that the coronavirus rdrps are significantly diverged from cellular and viral rna polymerases. recently, a structure model was built for the catalytic domain of the sars-cov rdrp [45] . the model provides first insights into the active site of the protein and also enables conclusions to be drawn about the properties of potential nucleoside analogue-inhibitors of coronavirus rdrps. thus, it was proposed that potential nucleoside analogue-inhibitors should contain groups at their 2′ and 3′ positions that are capable of making hydrogen bonding interactions with rdrp residues 623 and 691. clearly, direct structural information is highly desirable for the development of effective inhibitors of this key enzyme. a patent application claims the use of aurintricarboxylic acid (ata; 89) as an inhibitor of sars-cov rdrp [118] . according to the inventors, ata inhibits sars-cov replication in vero cells by ≥ 100-fold at 0.4 mg/ml and by > 1000-fold at 0.8 mg/ml. a significant inhibition of s glycoprotein production was observed in sars-cov-infected cells treated with ata as a result of inhibition of viral protein synthesis. no detectable toxicity was observed in uninfected vero cells treated with similar concentrations of ata. however, ata is quite a non-specific inhibitor of different dna/rna polymerases. thus, the potential usefulness of ata per se should be considered with caution, given the past experience in testing this compound as a hiv-1 integrase inhibitor. despite the initial encouraging results, this compound was abandoned due to insufficient specificity for hiv-1 integrase inhibition. in the case of coronaviruses, research on this compound should not be abandoned, given the paucity of reported rdrp inhibitors. for example, ata could serve as a valuable lead for the development of specific inhibitors of coronaviral rdrp. sars-cov rdrp could also be inhibited by oligonucleotides and mimics thereof [119] . the appropriate sequence of bases (indicated as bx in the following structural formulae) may be created by regular oligonucleotides or oligonucleotide-like molecules. these oligonucleotide-like molecules might have multiple applications. modified oligonucleotides and their mimics have been designed to prevent degradation by rnases before they come into contact with the viral rnas, and increase hybridisation properties or lipophilicity. the last of these properties facilitates the transition of these molecules through the plasma membrane. modifications aimed at preventing degradation by rnases include reversibly protected oxygens or substituents in position 2′ (90), and dicyclic substituents of the ribose (91). to increase the binding properties to a target rna, the use of tricyclic heterocyclic moieties, such as compound 92, has been taken into account. peptide nucleic acids (93) using the uncharged amide bond instead of the phosphate diester linking adjacent riboses could not only increase hybridisation properties, but also facilitates the passage of the molecule through the cellular membrane. morpholino-based oligomeric compounds (94) are non-ionic mimics of oligonucleotides more likely to pass through the cellular membrane and less likely to form complexes with proteins as compared with their oligonucleotide counterparts. the same invention takes into account the use of cyclohexenyl nucleic acids (cena). these molecules have the general formula of compound 95 where the furanose ring is replaced with a cyclohexenyl ring. in general, the incorporation of cena monomers into a dna chain increases the stability of a dna/rna hybrid. apart from their potential for rnai strategies, these molecules might be used for forming complexes with the viral positive-sense rna, thus hindering its transcription/duplication. the inventors are aware of the potential difficulties with the systemic administration of these molecules, and consider the inhalation route as a possible way of administering the molecules to humans. another protein released by the proteolytic activity of 3clpro is nsp13, a ntpase-helicase (hel) that specifically unwinds polynucleotide duplexes with a 5′ to 3′ polarity. hels from hcov-229e and sars-cov have very similar properties: a 5′ to 3′ polarity of unwinding; and a stimulation of atpase activity by single-stranded polynucleotides. the ability of hel to hydrolyse a wide variety of ribonucleosidetriphosphates (ntps) and deoxyribonucleosidetriphosphates (dntps) is consistent with the triphosphate moiety forming the basis of the recognition factor for binding and hydrolysis. it is possible that this lack of selectivity in ntp hydrolysis also enables the helicase to act as a broad rna 5′-triphosphatase in viral 5′ capping. the basal atpase activity of coronaviral helicase is stimulated by most polynucleotides 15-to 25-fold, with the exception of poly(g) and poly(dg). a significant variation in strength of homopolynucleotide binding was reported, suggesting that the sars-cov helicase may possess elements of secondary structure/sequence recognition capability. it has been hypothesised that this specificity may play a role in the localisation of a putative rdrp-helicase complex to selected genomic locations during subgenomic rna synthesis, augmenting proposed base-pairing factors. the 5′ to 3′ direction of unwinding suggests that the helicase may aid the replicative process by disrupting secondary structure or displacing bound proteins and annealed rna fragments, putative roles that will require experimental validation. coronaviral hel possesses a cystein-rich putative metal binding domain at the n-terminal portion that forms mutational analysis in the related equine anaemia virus and has been implicated in subgenomic mrna synthesis, genome replication and virion biogenesis [46] . hel is thought to be essential for nidovirus viability and, therefore, is a potential target for the development of anti-sars drugs. interestingly, hel was reported to be inhibited by the adamantine/pyridoxal conjugates, bananins (95 -98) [47] . unfortunately, the authors found no published patents or patent applications regarding these compounds in the search engines consulted. in case no specific patents exist, bananins may serve as valuable lead compounds for patentable hel inhibitors. many compounds with possible anticoronavirus activity have been researched, especially after the major sars outbreak in 2003. some of these compounds deserve particular attention because they are drugs that are already registered for other uses in humans. these drugs include the antimalarial chloroquine and hiv-1 pis. the antihelmynthic compound, niclosamide (96) is one such drug; however, its molecular target has not been defined yet [48] . drugs with well-known and long-studied toxicity profiles represent interesting compounds that might be immediately tested in clinical trials in case sars re-emerges. the claims covered by the patents contain novel and interesting applications of existing drugs. these include drug administration by inhalation. this route of administration could of course reduce the systemic side effects of the drugs and allow an increase in local concentrations. increased local concentrations could be particularly important in an anticoronavirus use of drugs designed for other disease conditions. the existence of such patents will probably allow the industrial development of new ways of administering old drugs. on the other hand, the patents/applications on the use of very promising compounds, such as chloroquine or hiv-1 pis, in coronavirus infections do not cover all of their possible applications. for the uses of chloroquine and hiv-1 pis not covered by the existing patents/applications, these drugs might be sold at a lower price, which could be of particular interest for resource-poor countries. as for chloroquine, its use as a prophylactic weapon for residents in, or travellers to, sars-affected areas (encouraged by the several-decade long use of this drug in antimalarial prophylaxis) is, to the authors' knowledge, not covered by any patents. the same goes for the use of hiv-1 pis as a systemic treatment for in addition, many patents cover the use of brand new agents inhibiting validated drug targets within the coronavirus life cycle. these compounds will possibly represent new therapeutic weapons if they prove to have satisfactory toxicity profiles and sufficient anticoronavirus activity in animal models. alternatively, they may represent interesting drug leads. of note, no patents or patent applications have been published yet on potential inhibitors of plppro(s) or helicase. as these enzymes seem to be novel and extremely interesting drug targets due to their multiple functions, it will be necessary in the near future to fill this gap by developing more specific inhibitors. finally, lessons for the choice of the best antiviral strategies should be learnt from the successes and failures of a quarter-century of research against another viral infection, such as hiv-1/aids. in part granted by the italian ministry of health-iss-rome under a specific sars-project. identification of critical determinants on ace2 for sars-cov entry and development of a potent entry inhibitor blocking of hiv-1 infectivity by a soluble, secreted form of the cd4 antigen potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry human coronavirus hcov-229e enters susceptible cells via the endocytic pathway effects of chloroquine on viral infections: an old drug against today's diseases? a review providing the immunological grounds for a potential use of chloroquine in the clinical management of sars clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine this article describes the first experiments showing an anti-sars-cov effect of chloroquine chloroquine is a potent inhibitor of sars coronavirus infection and spread this research article provides the mechanisms of sars-cov inhibition by chloroquine using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque the coronavirus replicase binding site-based classification of coronaviral papain-like proteases virus-encoded proteinases and proteolytic processing in the nidovirales intracellular localization and protein interactions of the gene 1 protein p28 during mouse hepatitis virus replication identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins viral rna replication in association with cellular membranes coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors a novel auto-cleavage assay for studying mutational effects of the active site of severe acute respiratory syndrome coronavirus 3c-like protease medical treatment of viral pneumonia including sars in immunocompetent adult treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds severe acute respiratory syndrome-related coronavirus is inhibited by ifn-α expanding the frontiers of existing antiviral drugs: possible effects of hiv-1 protease inhibitors against sars and avian influenza this paper reviews the information available on the use of hiv-1 protease inhibitors against sars-cov and provides the results of a molecular docking study identifying inhibitors of the sars coronavirus proteinase this bioinformatic analysis of drug/enzyme docking provides interesting insights on the use of hiv-1 protease inhibitors as lead compounds for development of 3clpro inhibitors old drugs as lead compounds for a new disease? binding analysis of sars coronavirus main proteinase with hiv, psychotic and parasite drugs an interesting bioinformatic approach evaluating the fitness for the 3clpro active site of compounds already approved for use in humans from nonpeptide toward noncarbon protease inhibitors: metallacarboranes as specific and potent inhibitors of hiv protease the peptidase database mechanisms and enzymes involved in sars coronavirus genome expression severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme papain-like protease(s) (plpro) -papain-like protease 2 (plp2) from severe acute respiratory syndrome coronavirus (sars-cov): expression, purification, characterization, and inhibition molecular biology of severe acute respiratory syndrome coronavirus molecular model of sars coronavirus polymerase: implications for biochemical functions and drug design the only molecular model available for coronaviral rdrp. important for the design of specific inhibitors the severe acute respiratory syndrome (sars) coronavirus ntpase/helicase belongs to a distinct class of 5' to 3' viral helicases this paper provides detailed information on ntpase/helicase the adamantane-derived bananins are potent inhibitors of the helicase activities and replication of sars coronavirus inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers. key: cord-255690-xc4bxin4 authors: rolain, jean-marc; colson, philippe; raoult, didier title: recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century date: 2007-07-16 journal: int j antimicrob agents doi: 10.1016/j.ijantimicag.2007.05.015 sha: doc_id: 255690 cord_uid: xc4bxin4 chloroquine (cq) and its hydroxyl analogue hydroxychloroquine (hcq) are weak bases with a half-century long use as antimalarial agents. apart from this antimalarial activity, cq and hcq have gained interest in the field of other infectious diseases. one of the most interesting mechanisms of action is that cq leads to alkalinisation of acid vesicles that inhibit the growth of several intracellular bacteria and fungi. the proof of concept of this effect was first used to restore intracellular ph allowing antibiotic efficacy for coxiella burnetii, the agent of q fever, and doxycycline plus hcq is now the reference treatment for chronic q fever. there is also strong evidence of a similar effect in vitro against tropheryma whipplei, the agent of whipple's disease, and a clinical trial is in progress. other bacteria and fungi multiply in an acidic environment and encouraging in vitro data suggest that this concept may be generalised for all intracellular organisms that multiply in an acidic environment. for viruses, cq led to inhibition of uncoating and/or alteration of post-translational modifications of newly synthesised proteins, especially inhibition of glycosylation. these effects have been well described in vitro for many viruses, with human immunodeficiency virus (hiv) being the most studied. preliminary in vivo clinical trials suggest that cq alone or in combination with antiretroviral drugs might represent an interesting way to treat hiv infection. in conclusion, our review re-emphasises the paradigm that activities mediated by lysosomotropic agents may offer an interesting weapon to face present and future infectious diseases worldwide. cq is a 9-aminoquinoline known since 1934, which emerged during the first part of the 20th century as an effective quinine substitute and the drug of choice against malaria [1] . it proved to be among the most successful antimalarial drugs on a worldwide scale owing to its wide deployment coinciding with the geographical distribution of plasmodium and its high intrinsic antiparasitic efficacy and low toxicity. concomitant with a gradual decrease in its use for therapy and prophylaxis of plasmodium-induced disease worldwide, related to the emergence of cq-resistant parasites, cq and its hydroxyl analogue hcq have gained interest in the field of other infectious diseases [2] . the mechanism of action of cq is multiple, differing according to the pathogen, and has not been well elucidated for all microorganisms. cq and hcq enter cells as non-protonated forms where they become protonated according to the henderson-hasselbach law, i.e. in a manner inversely proportional to the ph [2] . they therefore concentrate within acidic organelles, including endosomes, lysosomes and golgi vesicles, in which they increase the ph [3] . over the last decade, two main mechanisms of action of cq have been well described, i.e. alkalinisation of acid vesicles in cells infected by intracellular bacteria and fungi, and alteration of post-translational modifications of newly synthesised proteins in cells infected by viruses. the proof of concept of the use of cq as an anti-infectious agent, other than an antimalarial agent, has been fully demonstrated for the first time in vitro and in vivo with the model of chronic q fever. this concept was initially based on cellular biology findings, mainly by manipulation of the ph of acidic vacuoles where coxiella burnetii, the agent of q fever, live and multiply. demonstration of a negative effect on growth of c. burnetii by lysosomotropic agents [4] was the first step, followed by demonstration that alkalinising c. burnetii-containing vacuoles could restore the intracellular activity of antibiotics ( fig. 1 ) [5] . this paradigm was later used to demonstrate the clinical efficacy of an association of doxycycline and hcq in the treatment of chronic q fever endocarditis and this regimen is now the reference treatment and is to date the only model of confirmed clinical efficacy ( fig. 1 ) [6] . there is strong evidence of such an effect in other intracellular bacteria, especially for tropheryma whipplei, the agent of whipple's disease, which multiply in phagosomes, since agents that increase the intravacuolar ph decrease bacterial viability [7] . by analogy with c. burnetii, hcq restores the intracellular activity of doxycycline in vitro [8] and a clinical trial using this regimen is under evaluation. several other bacteria and fungi live and multiply in acidic vacuoles and preliminary in vitro data are encouraging for the usefulness of cq in such infections. for viruses, cq led to an inhibition of low-ph-dependent entry steps or alteration of post-translational modifications of newly synthesised proteins, especially via inhibition of glycosylation. these effects have been well described in vitro for many viruses, with human immunodeficiency virus type-1 (hiv-1) being the most studied. moreover, preliminary in vivo clinical trials have suggested that cq alone or in combination with antiretroviral drugs might represent an interesting way to treat hiv infection. here we review available in vitro and in vivo data on the effects of cq/hcq on bacterial, fungal and viral infections, with the concept that manipulation of the intracellular ph in cells and modification of glycosylation of proteins by lysosomotropic agents instead of antimicrobial compounds is a powerful approach as new therapeutic strategies for the prevention and therapeutic management of several infectious diseases, including some of great public health concern worldwide. cq/hcq also have anti-inflammatory properties, however these will not be discussed in this review. the intracellular location of several bacteria and fungi has been known for decades as a critical point to explain failure of antibiotic treatment to eradicate these pathogens from host cells [9] . intracellular pathogens evade the first-line antimicrobial defence, which includes attack by phagocytes (fig. 2 ). after being internalised by the cell, usually there is formation of a phagosome that rapidly fuses with lysosomes. the bacteria are then killed by oxygen-dependent and oxygenindependent killing mechanisms, which leads to acidification of the phagolysosome (ph 4.5) and acidic activation of lysosomal enzymes. intracellular pathogens may evade this lysosomal pathway by several mechanisms: (i) lifestyle in lysosome free-cells such as erythrocytes (bartonella spp.); (ii) escape from the phagosome before fusion with lysosomes and multiplication in the cytosol (rickettsia and shigella) (fig. 2) ; (iii) inhibition of phagolysosomal fusion and multiplication in the phagosome (chlamydia, ehrlichia, legionella, salmonella, yersinia, brucella, mycobacterium, francisella, histoplasma capsulatum and aspergillus fumigatus) (fig. 2) ; and (iv) survival and multiplication in phagolysosomes (c. burnetii, t. whipplei, staphylococcus aureus, candida albicans and cryptococcus neoformans) (fig. 2) [5, 9, 10] . there are many arguments suggesting that the low ph environment within phagosomal compartments of the cell is critical for many intracellular pathogens to access cellular iron for growth [11, 12] . cq treatment of different cells, including macrophages, could inhibit the growth of several of these intracellular bacteria by neutralising the phagolysosomal ph (table 1 ; figs. 1 and 2). thus, two main mechanisms can explain intracellular bacterial inhibition by cq: ph-dependent iron deprivation [11] ; and direct toxicity by increasing the phagolysosomal ph, which is harmful for the growth of several intracellular pathogens such as c. burnetii [13] and t. whipplei [7] . coxiella burnetii is the agent of q fever and is a strict intracellular bacterium that is able to survive in phagolysosomes where a low ph (ph 4.5) is necessary for its metabolism [4, 72, 73] . q fever includes acute manifestations (mainly pneumonitis and hepatitis) and chronic forms (mainly endocarditis) [74] . usually a regimen of doxycycline 200 mg per day for 3 weeks is recommended for patients with acute q fever. presently, this treatment of acute q fever is not sufficient to prevent the development of chronic q fever [75] . thus, reliable antibiotic therapy for chronic infection is a more challenging problem since antibiotics are not bac-tericidal in vitro against c. burnetii [74] . when the ph of c. burnetii-containing phagolysosomes was raised using basic lysosomotropic agents such as cq, methylamine and ammonium chloride, bacterial multiplication was inhibited, showing a direct negative effect on growth by lysosomotropic agents, including cq [4, 13] . an original killing assay model developed by maurin et al. [5] demonstrated that doxycycline, pefloxacin and rifampicin did not show any significant bactericidal activity. conversely, it was shown that in vitro intracellular antibiotic activity was restored and was correlated with modification of the ph by lysosomotropic agents (fig. 3) . the lack of bactericidal activity was probably due to inactivation by the low ph of the phagolysosomes in which c. burnetii survives. addition of a lysosomotropic alkalinising agent, i.e. cq, to antibiotics improved the activities of doxycycline and pefloxacin, which then became bactericidal [5, 13] . the model in which bacteria actively multiply only at acidic ph is a good paradigm to demonstrate that lack of bactericidal effect of antibiotics is due to intraphagolysosomal acidity. these in vitro findings have now been evaluated in many in vivo studies of the treatment of patients with chronic q fever endocarditis using a combination of doxycycline and hcq (at 1 g/ml in serum) for 18-36 months. indeed, prior to this treatment, patients were treated with a long-term table 1 bacteria, fungi and viruses inhibited by chloroquine and/or hydroxychloroquine (in vitro data) reference fungi reference virus reference coxiella burnetii [5, 13] histoplasma capsulatum [24] hiv [2, [29] [30] [31] [32] ] tropheryma whipplei [7, 8] cryptococcus neoformans [15, 25] sars-cov [33, 34] legionella pneumophila [11] paracoccidioides brasiliensis [26] influenza viruses [35] [36] [37] [38] francisella tularensis [12] penicillium marneffei [15, 27] flavivirus, including yellow fever virus [39] mycobacterium tuberculosis [14] aspergillus fumigatus [28] rubella virus [ tetracycline and quinolone regimen for at least 4 years, with a high percentage of relapses [6] . this regimen was compared with a combination of doxycycline and hydroxychloroquine sulphate in patients suffering from q fever endocarditis [6] . of 14 patients treated with a doxycycline and quinolone combination, 1 died, 7 relapsed (3 were re-treated and 4 switched to the new regimen), 1 was still being treated and 5 were considered cured using this regimen only. the mean duration of therapy for cure in this group was 55 months (median 60 months) [6] . twenty-one patients received the doxycycline and hcq regimen: 1 patient died of a surgical complication, 2 were still being treated, 17 were cured and 1 was currently being evaluated. two patients treated for 12 months but none of the patients treated for >18 months relapsed [6] . the mean duration of treatment in this group was 31 months (median 26 months). this regimen allowed a reduction in the duration of therapy to 18 months for many patients and also reduced the relapse rate to <5% [6] . this regimen is now the current therapy for the treatment of chronic q fever. cq used at therapeutic dosages may have some deleterious effects, including the risk of retinopathy, necessitating a regular ophthalmological examination [74] . cq levels in serum should be monitored to ensure that they are maintained at 1 ± 0.2 mg/l. similarly, an hiv-infected patient with q fever endocarditis was successfully treated with valvular replacement and a combination of doxycycline and hcq [76] . this combined treatment is probably also indicated in cases of c. burnetii vascular graft infection, as reported recently [77] . similarly, a retrospective study of patients diagnosed as having q fever during 1985-2000 evaluated the risk of developing endocarditis according to the regimen of antibiotics given to the patients [75] . when these regimens were compared, 6 (75%) of 8 patients who did not receive treatment developed a chronic infection, 5 of 10 developed a chronic infection when receiving doxycycline alone for 2 weeks to 6 months, and none of the 12 who received doxycycline and hcq for 1-15 months developed chronic infection. the regimen containing hcq was found to be significantly superior in preventing q fever endocarditis compared with doxycycline alone (p = 0.009) [75] . no significant differences were found between treatment with doxycycline alone and no treatment [75] . it is now established that development of q fever endocarditis may be prevented by searching for minor valvulopathies with echocardiography following diagnosis of acute q fever [78] and by treatment with a combination of doxycycline and hcq for 1 year [79] . finally, four cases of q fever osteoarticular infection (two tenosynovitis and two spondylodiscitis complicated by paravertebral abscess) were eventually cured using the combination of doxycycline and hcq [80] . whipple's disease was invariably fatal before the advent of antibiotics. however, current therapeutic recommendations are not based on therapeutic trials or adjusted according to the susceptibility of t. whipplei to various antimicrobial agents, since the bacteria was only isolated in 2000 [81] . following isolation of the bacteria, it has been shown that vacuole acidification is critical to the survival of t. whipplei in phagosomes, since agents that increase the intravacuolar ph decrease bacterial viability ( fig. 1) [7] . by analogy with c. burnetii, we have demonstrated that doxycycline alone was not bactericidal against t. whipplei in an in vitro cell model and that alkalinisation with hcq may restore activity [8] . a regimen based on this observation (doxycycline and hcq) has thus far been the only successful bactericidal regimen against t. whipplei in vitro. whether this regimen will work in a general clinical setting remains to be established, but it has been successful in four of our patients: two with classic whipple's disease and two with blood culture-negative endocarditis [82] . staphylococcus aureus is a facultative intracellular bacterium that resides within phagolysosomes [83] [84] [85] [86] . the intracellular location of certain strains of s. aureus serves as a reservoir of bacteria that is thought to be important in therapy of recurrent infections in humans and in chronic staphylococcal mastitis in dairy cows [87] . although aminoglycosides are bactericidal for extracellular staphylococci, they are ineffective in reducing the intracellular form of the microorganism [88] . it was hypothesised that diminished susceptibility of intracellular s. aureus may be related to the acidic ph within phagolysosomes [88] . it has been demonstrated that alkalinising s. aureus-containing vacuoles could restore the intracellular activity of aminoglycosides [89] . furthermore, intracellular killing of s. aureus correlated well with increased lysosomal ph due to lysosomotropic alkalinising agents [89] . recently, it has been demonstrated that cq and ammonium chloride significantly enhanced intracellular killing by levofloxacin [22] . the bactericidal activity of levofloxacin was partially restored when the ph was neutralised from 5.0 to 7.4 [22] . the bactericidal activity of moxifloxacin, abolished in the intracellular salt medium, was partially restored when the ph was raised from 5.0 to 7.4 [22] . similarly, alkalinisation of phagolysosomes significantly enhanced intracellular killing by moxifloxacin [90] . in a model of bovine mastitis due to s. aureus, it has been clearly demonstrated that low intraphagolysosomal ph affects the ability of an antibiotic to kill intracellular bacteria, since the activity of rifampicin was enhanced at ph 5.0 [91] . similar reasoning probably explains why rifampicin, which both penetrates within eukaryotic cells [92] and is more active at acidic ph [87] , displays bactericidal activity against intracellular s. aureus [93] . legionella pneumophila is a strict intracellular bacterium that multiplies in human mononuclear phagocytes and is responsible for legionnaire's disease [94] . cellular iron metabolism is of critical importance to l. pneumophila since its multiplication is dependent upon the availability of intracellular iron. it has been demonstrated that cq and ammonium chloride inhibit the intracellular multiplication of l. pneumophila by limiting the availability of iron to the bacterium [11] . thus, cq may interfere with intracellular iron metabolism by recycling iron from ferritin by blocking degradation of ferritin by acid proteases [11] . francisella tularensis bv. tularensis and f. tularensis bv. palearctica are facultative intracellular bacteria responsible for tularaemia. it has been demonstrated that f. tularensis finds a successful niche for replication in an acidified vacuole where iron is concentrated [12] . growth of f. tularensis in murine macrophages has been shown to be dramatically inhibited in vitro by cq in a dose-dependent manner [12] . intracellular localisation in an acidic vesicle, which facilitates the availability of iron essential for francisella growth, is a survival tactic of this bacterium and iron depletion is one mechanism that macrophages use to inhibit its growth [12] . cq has been reported to inhibit the intracellular multiplication of mycobacterium tuberculosis both in human monocyte-derived macrophages and mouse peritoneal macrophages [11] . it seems possible that cq inhibits m. tuberculosis intracellular multiplication by raising intracellular ph and limiting the availability of iron to this bacterium, as it does for l. pneumophila. similarly, it has been demonstrated that addition of cq results in a significant reduction of the intracellular growth of mycobacterium avium in bone marrow-derived macrophages [11] . in vitro activity of cq and/or hcq has been demonstrated for other bacteria, including salmonella enterica serovar typhi, escherichia coli, bacillus anthracis, bacillus subtilis, borrelia burgdorferi, brucella abortus and listeria monocytogenes (table 1; fig. 1 ). these bacteria may be good candidates for clinical use of cq/hcq. recent in vitro studies indicate that cq may also have interesting activity against fungal diseases ( fig. 1 ; table 1) , including mainly h. capsulatum [95] and c. neoformans [96] . intracellular h. capsulatum is adapted to survive within the mammalian phagolysosome and resides within a membrane-bound phagosome that does not fully acidify. histoplasma capsulatum is able to maintain a phagosomal ph of 6.5 [97] by inhibition of phagolysosomal fusion [98] and by expression of a unique endogenous h + -atpase that buffers the phagosomal ph [99] . it has been demonstrated that cq induces an antihistoplasmal state in macrophages by restricting the ph-dependent release of iron within the phagolysosome [24] . similarly, cq has been shown to kill c. neoformans, but by a mechanism independent of iron deprivation [15, 25] . indeed, unlike h. capsulatum, c. neoformans is able to maintain a phagolysosome milieu at ca. ph 5.1 [100] and addition of cq increases the phagolysosomal ph allowing inhibition of growth at alkaline ph [25] . it has been reported that a. fumigatus has the ability to inhibit fusion of the phagosome with the lysosome and that cq may increase killing of this fungus in macrophages by a phdependent mechanism [28] . cq has been shown to inhibit the intramacrophagic growth of penicillium marneffei [15] , an opportunistic fungus that causes disseminated infection in acquired immune deficiency syndrome (aids) patients by increasing the intravacuolar ph and disrupting ph-dependent metabolic processes [27] . the decrease in the intracellular iron concentration results in impaired functionality of several cellular enzymes with a subsequent deleterious effect on critical steps such as replication of cellular dna or gene expression [27] . finally, the effect of cq on multiplication of paracoccidioides brasiliensis has been studied in human monocytes and in a murine paracoccidioidomycosis model [26] . cq was demonstrated to be able to kill p. brasiliensis grown in human monocytes. the effect of cq was reversed by fenta, an iron compound that is soluble at neutral to alkaline ph, but not by holotransferrin, which releases iron only in an acidic environment. thus, cq inhibits p. brasiliensis survival in human monocytes by iron deprivation [26] . (table 1) 4.1. mechanisms of antiviral activity (fig. 4) the ph increase induced by cq/hcq within acidic organelles, including endosomes, lysosomes and golgi vesicles, is involved in its antiviral activity via two main mechanisms. first, these drugs might be responsible for inhibition of viruses requiring a ph-dependent step for entry into their host cell. indeed, many viruses have a low-ph-dependent conformational change that triggers fusion, penetration and/or uncoating, and for these viruses endocytosis is crucial due to acidification that occurs within the endosomal pathway [101] . thus, in this mechanism the antiviral effect is dependent on the extent to which the virus uses endosomes for entry [59] . for instance, shibata et al. [35] found that cq might prevent the uncoating of influenza b virus by increasing the lysosomal ph above the critical value required for inducing fusion between the virus envelope and the lysosomal membrane. cq was also found to inhibit uncoating of the hepatitis a virus (hav) [42] . second, cq/hcq might inhibit post-translational modifications of the virus envelope glycoproteins by proteases and glycosyltransferases within the trans-golgi network and endoplasmic vesicles. indeed, some of these enzymes require a low ph for their activity and cq/hcq might therefore lead to decreased viral infectivity through impaired envelope maturation. flaviviridae are examples of viruses for which cq could act as an antiviral by inhibiting their envelope maturation pathway through alteration of the proteolytic processing of the prm protein [39] . also, a putative mechanism for anti-hiv-1 activity is through alteration of the glycosylation pattern and amino acid charge within several regions of the gp120 viral envelope protein [2, 102] . for instance, a reduction in the number of potential n-linked glycosylation sites within the v3 region of gp120, which might provide for altered immune escape and broadening of the antibody repertoire, has been observed [102] . savarino et al. [2] previously found that cq decreased the infectivity of newly produced hiv-1 as well as the ability of hiv-1-infected cells to form syncitia, and this was associated with structural changes in gp120. recent data further suggest that cq may be responsible for inhibition of the biosynthesis of sialic acid. indeed, it was recently observed that this drug inhibited cellular enzymes involved in sialic acid biosynthesis [103] . this might represent a major antiviral mechanism, since sialic acids are a component of hiv-1 envelope glycoproteins. cq/hcq may also have indirect antiviral effects. indeed, cq was found to be effective in preventing the spread of severe acute respiratory syndrome (sars)-associated coronavirus (cov) in cell culture by interfering with terminal glycosylation of the cellular receptor, angiotensin-converting enzyme 2 (ace2) [33] ; and sialic acids, biosynthesis of which might be inhibited by cq/hcq, are component of receptors of sars-cov and orthomyxoviruses [36] . in addition to these two main mechanisms, other possible mechanisms such as immunomodulatory and antiinflammatory properties have been suggested, however these will not be discussed in the present review. in vitro activity of hcq/cq has been reported for a wide range of viruses, most frequently in experiments aiming to study the cycle replication pathways using cq, especially the mechanism by which viruses penetrate host cells (table 1 ; fig. 4 ). inhibitory concentrations of cq fell within the 0.5-10 mol/l range, depending on antiviral cq doses, the viruses that were targeted and the assays used for assessment of the antiviral effect. importantly, these concentrations are in the range that is clinically achieved in plasma during malaria therapy, varying from 1.6 mol/l to 12.5 mol/l [104] . the most studied in vitro effect of cq/hcq has been against hiv. moreover, in vivo studies quasi-exclusively concerned hiv-1. this is probably due to the high morbidity and mortality related to hiv-1 worldwide and the need for low-cost antiretroviral therapies in resource-poor countries. cq/hcq have largely been shown to inhibit hiv replication in vitro. importantly, this inhibition was observed in several cell line models, but also in lymphocytes and mono-cytes from peripheral blood [29] . an anti-hiv effect has been demonstrated either in the presence of high concentrations of cq/hcq prior to infection of hiv-1-permissive cells [29, 30, 102] or during incubation of hiv-infected cells with cq concentrations similar to those found in peripheral blood from individuals chronically treated with cq [31] . thus, these data suggest both a preventive and curative effect of this drug against hiv. the concentration inhibiting 50% of viral replication (ic 50 ) ranged between 1 mol/l and 10 mol/l for various hiv strains and culture cells in the studies of savarino et al. [2] . boelaert et al. [105] described an additional inhibition of hiv-1 replication with hydroxyurea plus didanosine (ddi) or zidovudine, with a cq ic 50 of 0.4-0.9 mol/l for the cell lines and 0.2-0.9 mol/l for the primary cells; no cq-induced toxicity or apoptosis was noted. interestingly, according to in vitro data, cq/hcq also appears to be active against hiv-2, which mostly circulates in west africa, as well as against hiv-1 of different subtypes [2] . this deserves attention since hiv-2 strains have established or suggested natural resistance to several antiretroviral drugs, such as non-nucleoside reverse transcriptase inhibitors and likely some protease inhibitors (pis) [106] . anti-hiv-1 activity of cq/hcq has been observed in a few in vivo studies since 1995 [107] [108] [109] [110] [111] . two small, phase ii, randomised, double-blind studies, including 40 and 72 patients with cd4 cell counts of 200-500 cells/mm 3 , compared reduction of plasma hiv-1 rna levels in individuals treated with hcq versus either placebo or zidovudine [107, 108] . in both trials, more than two-thirds of patients were antiretroviral-naive. in the first trial, 8 weeks of treatment with 800 mg hcq per day resulted in a significant mean 0.6 log 10 reduction of hiv-1 load (p = 0.022), whereas no significant decrease was observed in the placebo arm (20 patients in each arm) [107] . concomitantly, the percentage of cd4 + lymphocytes remained stable in the hcq group, whereas it significantly decreased in the placebo arm (p = 0.032). in the second trial, 35 and 37 patients received hcq or zidovudine, respectively, for 16 weeks [108] . hiv-1 load was significantly reduced in both groups (by 0.4 log 10 copies/ml and 0.6 log 10 copies/ml, respectively) and, interestingly, 0 of 35 patients in the hcq group versus 8 of 37 patients in the zidovudine group showed an increase in hiv-1 rna levels. two other recent non-controlled studies have been reported. in singapore, 22 patients with hiv-1 load <100 000 copies/ml and cd4 cell count >150 cells/mm 3 received hcq (200 mg), hydroxyurea and ddi twice daily for 48 weeks, resulting in a 1.3 log 10 decrease in plasma hiv-1 rna levels [109] . hiv-1 rna levels were further reduced compared with baseline (mean decrease 1.6 log 10 copies/ml) in all 14 patients who completed a 144-week course of therapy, with drug resistance mutations detected in 4 patients at this time point [110] . in another study in india, 18 patients with cd4 counts >350/mm 3 received lamivudine, hydroxyurea and cq (250 mg) twice daily for 6 months [111] . hiv-1 load reduction was significant (−2.0 log 10 ), reaching undetectable levels in 10 patients, and the median rise in cd4 count was 78 cells/mm 3 . altogether, these in vivo data in patients with non-severe immunosuppression (cd4 cell count >200/mm 3 ) at least suggest that hiv-1 resistance to cq/hcq alone or in combination with antiretroviral drugs might not develop easily [36] . in contrast, addition of cq to a zidovudine and ddi regimen provided no significant improvement in viro-immunological parameters in 21 hiv-1-infected children in a recent study from thailand [112] . it is interesting to note that a 243-fold accumulation of cq in colostrum cells of african mothers taking 100 mg of cq per day has been observed. this suggests that this drug could be potentially active as an adjuvant to post-natal antiretroviral prophylaxis of mother-to-child transmission by decreasing hiv-1 load in milk in geographical areas where vertical transmission is of great concern [113,114]. in several settings, cq/hcq should be used in combination with other antiviral drugs, questioning whether associations may be additive or synergistic. the additive effect of cq and zidovudine has been shown, and might also exist in association with ddi or hydroxyurea [105] . furthermore, the combined effect of cq and pis is synergistic in a dose-independent manner [2] . savarino et al. showed that cq in combination with pis carries out a combined inhibitory effect on p-glycoprotein and multidrug resistance protein 1, which is involved in efflux of pis, a major class of antiretrovirals. interestingly, the synergism between cq and pis was associated with a decreased threshold of susceptibility to pis in resistant isolates [2] . of note, cq is a major substrate of cytochrome cyp3a4 [116] , and nevirapine (a non-nucleoside hiv-1 reverse transcriptase inhibitor) and hiv pis are well recognised cyp3a4 inducers and inhibitors, respectively [115] . thus, these latter drugs might subsequently decrease or increase the levels/effects of cq. it has been shown that endosomal transport is needed for human coronavirus hcov-229e and that cells treated with cq displayed reduced expression of viral antigens [114] . more recently, cq was found to have strong antiviral effects on sars-cov infection in cell cultures when they were treated either before or after exposure to the virus (even 3-5 h following infection), suggesting both prophylactic and therapeutic effects [33, 34] . in keyaerts et al.'s study [34] , cq inhibited viral replication with a 50% effective concentration (ec 50 ) of 8.8 mol/l. of note, the dose inducing 50% cytostatic activity was much higher (261.3 mol/l). potential mechanisms of action of cq against coronaviruses are through underglycosylation of ace2, which has been identified as a functional cellular receptor of sars-cov spike protein [33, 117] . alteration by cq of the sars-cov spike protein is controversial [33, 118] . a ph-related reduction of the transduction of sars-cov pseudotype viruses has also been suggested [33, 119, 120] . of note, biot et al. [121] recently reported the design and synthesis of hydroxyferroquine derivatives with antimalarial, anti-hiv and anti-sars-cov activities. the activity of cq against orthomyxoviridae (influenza a and b viruses) has been described for several decades [35, 37, 122] and in vitro assays on avian influenza virus strains are ongoing [36] . shibata et al.'s [35] results suggested that cq prevents the uncoating of influenza b virus. ooi et al. [38] found that the ic 50 values of cq against influenza a viruses h1n1 and h3n2 were 3.6 mol/l and 0.84 mol/l, respectively. in a recent study, blanchard et al. [45] found that pretreating target cells with cq inhibited hepatitis c virus (hcv) clone jfh-1 propagation in cell culture, which suggests that hcv, like flaviviruses and pestiviruses, enters cells through clathrin-mediated endocytosis and fusion within an acidic endosomal compartment. cq was also found to be active against hepatitis b virus (hbv) and duck hbv [43, 44, 123] , contrasting with another study in which infection of human hepatocyte cultures with hbv was found to be unaffected by cq [124] . seven patients with histologically proven chronic active hepatitis b have been treated with 150-450 mg of cq for a median of 12 months [125] . in all patients, alanine aminotransferase (alt) returned to normal values and prothrombin time improved. interestingly, alt increased in three patients following inadvertent cq withdrawal and returned to prior levels on re-administration. in four patients, a repeat liver biopsy 1 year later revealed inactive cirrhosis. recently, an enhancement by cq of human cd8 + t-cell cell response to hbv antigen has been observed [126] . inhibition by cq of hepatitis a virus (hav) uncoating and replication has also been described [42, 60] . a major advantage of cq/hcq is their limited and preventable toxicity. long experience of the use of these drugs in the treatment of malaria has already demonstrated the safety of short-term administration to humans. moreover, cq/hcq have been widely used for chronic administration in rheumatic diseases, chronic q fever and for antimalarial prophylaxis for up to several years with only a low incidence of adverse effects even during pregnancy [114, [127] [128] [129] . the main adverse effect reported in long-term administration of these drugs was macular retinopathy due to the cumulative dose, which could be prevented with regular visual monitoring during the course of treatment [6, 130] . in summary, cq/hcq have several advantages as antimicrobial agents, including multiple potential mechanisms and a broad spectrum of activity at clinically achievable plasma concentrations, together with well known and limited toxicity and low cost. two major concepts have emerged to explain the activity of cq/hcq, namely alkalinisation of phagolysosomes for intracellular bacteria and fungi, and inhibition of entry steps and protein glycosylation for viruses. the pioneer and the only model of an infectious disease that could be treated by manipulation of intracellular ph by a lysosomotropic agent was chronic q fever. this review re-emphasises that the c. burnetii paradigm and activities mediated by lysosomotropic agents could be generalised for other intracellular pathogens living in acidic vacuoles or that require a low ph for multiplication. this may offer an interesting weapon to face present and future infectious diseases worldwide. funding: none. competing interests: none declared. ethical approval: not required. chloroquine-resistant malaria anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors 4-aminoquinolines-past, present, and future: a chemical perspective biochemical stratagem for obligate parasitism of eukaryotic cells by coxiella burnetii phagolysosomal alkalinization and the bactericidal effect of antibiotics: the coxiella burnetii paradigm treatment of q fever endocarditis: comparison of two regimens containing doxycycline and ofloxacin or hydroxychloroquine survival of tropheryma whipplei, the agent of whipple's disease, requires phagosome acidification antibiotic susceptibility of tropheryma whipplei in mrc5 cells optimum treatment of intracellular infection intracellular organisms chloroquine inhibits the intracellular multiplication of legionella pneumophila by limiting the availability of iron. a potential new mechanism for the therapeutic effect of chloroquine against intracellular pathogens growth of francisella tularensis lvs in macrophages: the acidic intracellular compartment provides essential iron required for growth bactericidal effect of doxycycline associated with lysosomotropic agents on coxiella burnetii in p388d1 cells inhibition of tubercle bacilli in cultured human macrophages by chloroquine used alone and in combination with streptomycin, isoniazid, pyrazinamide, and two metabolites of vitamin d3 experimental results on chloroquine and aids-related opportunistic infections inhibition of the salmonella typhi oral vaccine strain, ty21a, by mefloquine and chloroquine the effect of ph on the inhibitory activity of chloroquine against escherichia coli chloroquine enhances survival in bacillus anthracis intoxication the preferential inhibition of bacillus subtilis spore outgrowth by chloroquine an in vitro study of the susceptibility of mobile and cystic forms of borrelia burgdorferi to hydroxychloroquine effect of endocytic and metabolic inhibitors on the internalization and intracellular growth of brucella abortus in vero cells factors influencing the intracellular activity of fluoroquinolones: a study using levofloxacin in a staphylococcus aureus thp-1 monocyte model inhibition of rab5a exchange activity is a key step for listeria monocytogenes survival chloroquine induces human macrophage killing of histoplasma capsulatum by limiting the availability of intracellular iron and is therapeutic in a murine model of histoplasmosis chloroquine induces human mononuclear phagocytes to inhibit and kill cryptococcus neoformans by a mechanism independent of iron deprivation chloroquine inhibits paracoccidioides brasiliensis survival within human monocytes by limiting the availability of intracellular iron inhibition of intramacrophage growth of penicillium marneffei by 4-aminoquinolines pksp-dependent reduction of phagolysosome fusion and intracellular kill of aspergillus fumigatus conidia by human monocyte-derived macrophages inhibition of human immunodeficiency virus type 1 replication by hydroxychloroquine in t cells and monocytes weak bases affect late stages of mayaro virus replication cycle in vertebrate cells chloroquine inhibits hiv-1 replication in human peripheral blood lymphocytes inhibition of hiv-1 replication by hydroxychloroquine: mechanism of action and comparison with zidovudine chloroquine is a potent inhibitor of sars coronavirus infection and spread in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine mechanism of uncoating of influenza b virus in mdck cells: action of chloroquine new insights into the antiviral effects of chloroquine antihistaminics, local anesthetics, and other amines as antiviral agents in vitro inhibition of human influenza a virus replication by chloroquine acidotropic amines inhibit proteolytic processing of flavivirus prm protein pathway of rubella virus infectious entry into vero cells rubella virus: mechanism of attenuation in the vaccine strain (hpv77) examination of potential inhibitors of hepatitis a virus uncoating inhibition of duck hepatitis b virus infection by lysosomotropic agents antiviral strategies in chronic hepatitis b virus infection: ii. inhibition of duck hepatitis b virus in vitro using conventional antiviral agents and supercoiled-dna active compounds hepatitis c virus entry depends on clathrin-mediated endocytosis early events in arenavirus replication are sensitive to lysosomotropic compounds mechanism of lymphocytic choriomeningitis virus entry into cells ammonium chloride and chloroquine inhibit rabies virus infection in neuroblastoma cells processing and presentation of cell-associated varicella-zoster virus antigens by human monocytes respiratory syncytial virus inhibits granulocyte apoptosis through a phosphatidylinositol 3-kinase and nf-b-dependent mechanism effects of lysosomotropic weak bases on infection of bhk-21 cells by sindbis virus mechanism of enhancement of the antiviral action of interferon against herpes simplex virus-1 by chloroquine inhibition of multiplication of herpes simplex virus type 1 by ammonium chloride and chloroquine epstein-barr virus enters b cells and epithelial cells by different routes entry of poliovirus type 1 and mouse elberfeld (me) virus into hep-2 cells: receptor-mediated endocytosis and endosomal or lysosomal uncoating chloroquine induces empty capsid formation during poliovirus eclipse mechanism of entry into the cytosol of poliovirus type 1: requirement for low ph endoproteolytic activation of newcastle disease virus fusion proteins requires an intracellular acidic environment mechanism of borna disease virus entry into cells inhibition of vesicular stomatitis virus infection by spike glycoprotein. evidence for an intracellular, g proteinrequiring step attenuation of recombinant vesicular stomatitis viruses encoding mutant glycoproteins demonstrate a critical role for maintaining a high ph threshold for membrane fusion in viral fitness inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine studies on the mechanism of entry of vaccinia virus in animal cells inhibition of murine rna tumor virus replication and oncogenesis by chloroquine early steps in fmdv replication: further analysis on the effects of chloroquine weak bases affect late stages of mayaro virus replication cycle in vertebrate cells entry of feline calicivirus is dependent on clathrin-mediated endocytosis and acidification in endosomes effect of chloroquine on african swine fever virus infection t cell proliferative response to bovine leukaemia virus (blv): identification of t cell epitopes on the major core protein (p24) in blv-infected cattle with normal haematological values infectious entry pathway for canine parvovirus cytoplasmic trafficking of minute virus of mice: low-ph requirement, routing to late endosomes, and proteasome interaction ph dependence of the coxiella burnetii glutamate transport system coxiella burnetii: the 'query' fever bacterium. a model of immune subversion by a strictly intracellular microorganism risks factors and prevention of q fever endocarditis q fever endocarditis in hiv-infected patient coxiella burnetii vascular graft infection endocarditis after acute q fever in patients with previously undiagnosed valvulopathies from acute q fever to endocarditis: serological follow-up strategy q fever osteoarticular infection: four new cases and a review of the literature cultivation of the bacillus of whipple's disease whipple's disease the survival of staphylococci within human leukocytes intracellular survival of staphylococci the influence of extracellular and phagolysosomal ph changes on the bactericidal activity of bovine neutrophils against staphylococcus aureus modification of interactions between neutrophils and staphylococci by lysosomotropic week bases activity of antibiotics against staphylococcus aureus within polymorphonuclear neutrophils effect of low intraphagolysosomal ph on antimicrobial activity of antibiotics against ingested staphylococci phagolysosomal alkalinization and intracellular killing of staphylococcus aureus by amikacin factors compromising the activity of moxifloxacin against intracellular staphylococcus aureus evaluation of antibiotic effectiveness against staphylococcus aureus surviving within the bovine mammary gland macrophage intracellular distribution and activity of antibiotics killing of intraleukocytic staphylococcus aureus by rifampin: in vitro and in vivo studies chloroquine and the fungal phagosome determination of the ph of the cryptococcus neoformans vacuole histoplasma capsulatum modulates the acidification of phagolysosomes regulation of the macrophage vacuolar atpase and phagosome-lysosome fusion by histoplasma capsulatum cloning and sequence analysis of an h(+)-atpase-encoding gene from the human dimorphic pathogen histoplasma capsulatum cryptococcus neoformans resides in an acidic phagolysosome of human macrophages dissecting virus entry via endocytosis effect of chloroquine on reducing hiv-1 replication in vitro and the dc-sign mediated transfer of virus to cd4+ t-lymphocytes kinetic mechanism of quinone oxidoreductase 2 and its inhibition by the antimalarial quinolines clinical pharmacokinetics and metabolism of chloroquine. focus on recent advancements the additive in vitro anti-hiv-1 effect of chloroquine, when combined with zidovudine and hydroxyurea susceptibility of hiv-2, siv and shiv to various anti-hiv-1 compounds: implications for treatment and postexposure prophylaxis hydroxychloroquine treatment of patients with human immunodeficiency virus type 1 comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type 1 hydroxychloroquine, hydroxycarbamide, and didanosine as economic treatment for hiv-1 hydroxyurea and didanosine as initial therapy for hiv-infected patients with low viral load: safety, efficacy and resistance profile after 144 weeks low cost anti-retroviral options: chloroquine based arv regimen combined with hydroxyurea and lamivudine: a new economical triple therapy therapeutic potential of chloroquine added to zidovudine plus didanosine for hiv-1 infected children mother-tochild transmission of hiv-1: timing and implications for prevention effects of chloroquine on viral infections: an old drug against today's diseases? drug interactions of hiv protease inhibitors in vitro metabolism of chloroquine: identification of cyp2c8, cyp3a4, and cyp2d6 as the main isoforms catalyzing n-desethylchloroquine formation angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein characterization of severe acute respiratory syndromeassociated coronavirus (sars-cov) spike glycoprotein-mediated viral entry ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities infectious cell entry mechanism of influenza virus inhibition of hepatitis b dna polymerase by intercalating agents ph-independent uptake of hepatitis b virus in primary human hepatocytes treatment of chronic active hepatitis b (cah b) with chloroquine: a preliminary report chloroquine enhances human cd8+ t cell responses against soluble antigens in vivo correlation between serum levels of doxycycline and serology evolution in patients treated for coxiella burnetii endocarditis correlation between ratio of serum doxycycline concentration to mic and rapid decline of antibody levels during treatment of q fever endocarditis ocular toxicity and antenatal exposure to chloroquine or hydroxychloroquine for rheumatic diseases ophthalmologic considerations and testing in patients receiving long-term antimalarial therapy key: cord-015958-68dmza13 authors: ceccherini-nelli, luca title: globalizzazione in medicina: l’emergenza hiv date: 2007 journal: effetti, potenzialit&#x000e0; e limiti della globalizzazione doi: 10.1007/978-88-470-0609-6_8 sha: doc_id: 15958 cord_uid: 68dmza13 l’ottimismo generato dalle migliori condizioni di vita (cibo e acqua più sani, migliori sistemi di raccolta rifiuti e di discarica, nuove conoscenze nella biologia e nella medicina capaci di consentire lo sviluppo e l’uso diffuso di vaccini, la produzione di antinfettivi e di antiparassitari più sicuri ed efficaci) che avevano portato nel mondo occidentale all’allungamento dell’aspettativa di vita da una media di 46,5 anni nel 1950 a 65 anni nel 2002 (51 anni per i redditi bassi, 78 per gli alti), negli anni ottanta si era già esaurito per l’emergenza di agenti infettivi “nuovi” (non riconosciuti prima) e per la riemergenza di altri già noti, a causa sia di fattori determinati dall’agente infettante stesso, quali l’acquisizione della capacità di salto di specie o la formazione di varianti farmacoresistenti, che di fattori determinati dall’ospite, quali: 1) manovre invasive iatrogene responsabili di infezioni ospedaliere; 2) cambiamenti climatici capaci di favorire il diffondersi di parassiti vettori di infezione e alterazioni degli ecosistemi (con prevalenza incontrollata di predatori o di prede); 3) esplosione demografica con ripercussioni importanti sulle tecnologie industriali di produzione alimentare, sullo sviluppo economico-urbanistico tumultuoso, sulle migrazioni di rifugiati; 4) promiscuità sessuale e turismo sessuale; 5) tossicodipendenza, e infine 6) spostamenti delle persone e delle merci che sono sempre stati fonte di diffusione degli agenti infettivi, ma che avevano raggiunto livelli di quantità e frequenza impensabili precedentemente [1] (vedi anche i capitoli pubblicati altrove in questo volume). l'ottimismo generato dalle migliori condizioni di vita (cibo e acqua più sani, migliori sistemi di raccolta rifiuti e di discarica, nuove conoscenze nella biologia e nella medicina capaci di consentire lo sviluppo e l'uso diffuso di vaccini, la produzione di antinfettivi e di antiparassitari più sicuri ed efficaci) che avevano portato nel mondo occidentale all'allungamento dell'aspettativa di vita da una media di 46,5 anni nel 1950 a 65 anni nel 2002 (51 anni per i redditi bassi, 78 per gli alti), negli anni ottanta si era già esaurito per l'emergenza di agenti infettivi "nuovi" (non riconosciuti prima) e per la riemergenza di altri già noti, a causa sia di fattori determinati dall'agente infettante stesso, quali l'acquisizione della capacità di salto di specie o la formazione di varianti farmacoresistenti, che di fattori determinati dall'ospite, quali: 1) manovre invasive iatrogene responsabili di infezioni ospedaliere; 2) cambiamenti climatici capaci di favorire il diffondersi di parassiti vettori di infezione e alterazioni degli ecosistemi (con prevalenza incontrollata di predatori o di prede); 3) esplosione demografica con ripercussioni importanti sulle tecnologie industriali di produzione alimentare, sullo sviluppo economico-urbanistico tumultuoso, sulle migrazioni di rifugiati; 4) promiscuità sessuale e turismo sessuale; 5) tossicodipendenza, e infine 6) spostamenti delle persone e delle merci che sono sempre stati fonte di diffusione degli agenti infettivi, ma che avevano raggiunto livelli di quantità e frequenza impensabili precedentemente [1] (vedi anche i capitoli pubblicati altrove in questo volume). nel secondo decennio l'infezione aveva raggiunto i 35 milioni di infetti nel mondo mentre si sensibilizzava l'opinione pubblica per la prevenzione e per la cooperazione (furono istituiti organismi nazionali e internazionali di intervento specifico), mentre venivano sviluppati nuovi farmaci e si preparavano regimi terapeutici combinatori più efficaci, capaci di limitare la progressione della malattia e la trasmissione materno-fetale, mentre continuava lo sforzo congiunto dei ricercatori per lo sviluppo del vaccino attraverso la caratterizzazione molecolare fine del virus, la caratterizzazione del rapporto virus-cellula e virus-ospite. soltanto alla fine degli anni novanta, l'analisi di un campione di sangue di un maschio adulto bantu di kinshasa, repubblica democratica del congo, prelevato nel 1959, risultato vicino per analisi filogenetica della sequenza nucleotidica al nodo di origine di differenziazione di tutti i maggiori gruppi di hiv, permise di stabilire in maniera definitiva [4] che all'origine dei ceppi noti di hiv ci fosse stato un comune progenitore diffuso negli anni quaranta-cinquanta e che il salto di specie fosse avvenuto uno o due decenni prima con almeno tre ingressi indipendenti del virus hiv-1 dallo scimpanzè all'uomo, ciascuno dei quali avrebbe poi generato un gruppo filogenetico autonomo: m (main, di maggior successo evolutivo), o (outlier), n (non-m, non-o). il gruppo m, ancora in continua evoluzione incontrollata, è costituito oggi da 9 cladi (a-d, f-h, j e k) e da più di 13 ricombinanti fra i sottotipi, i crf (circulating recombinant forms) originati dalla ricombinazione pretrascrizionale dei cladi, ciascuno con caratteristiche biologiche proprie tendenti alla maggior patogenicità e alla farmacoresistenza. il clade b è ancora il più comune in nord america, europa ed australia; ma la prevalenza dei ceppi non-b è aumentata progressivamente negli stati uniti, cuba, francia, spagna, svizzera, ma soprattutto nel canada, belgio, portogallo e nei paesi scandinavi (dove rappresentano oggi più del 40%) in relazione all'immigrazione e ai viaggi in aree endemiche [5] . in questo periodo di tempo sono state studiate ulteriormente le caratteristiche replicative virali (l'entità giornaliera di produzione e di eliminazione), le caratteristiche patogenetiche (il danno immunologico virale e la capacità rigenerativa dei substrati cellulari dell'ospite che vi si oppone, il sequestro virale nei compartimenti tissutali), la variabilità virale (tasso di mutazione trascrizionale indotta sia dalla trascrittasi inversa virale che dalla ricombinazione pretrascrizionale); infine sono stati caratterizzati i parametri di variabilità virale all'interno dei quali il virus continua a dare danno all'ospite senza soccombere alla pressione selettiva immunologica e farmacologica [6] . nel terzo decennio dell'infezione si sono ottenute nuove conoscenze sull'interazione virus/sistema immune [7] [8] [9] . sono stati sviluppati nuovi antivirali specifici: attualmente sono disponibili 19 farmaci, di quattro tipi diversi, che hanno come bersaglio tre passaggi essenziali nella replicazione virale hiv: gli analoghi nucleotidici e non nucleotidici che agiscono come terminatori di catena nella trascrizione inversa dell'rna infettante in dna intermedio di replicazione; gli inibitori delle proteasi che bloccano la maturazione delle proteine virali precursori in proteine virali funzionali; gli inibitori dell'ingresso virale che impediscono la penetrazione del virus nelle cel-lule bersaglio [10] . il loro costo non è alla portata dei paesi in corso di sviluppo, soprattutto per l'esigenza di usarli in combinazione. e la loro efficacia è stata verificata soltanto sui ceppi b, prevalenti nei paesi occidentali dove sono stati sviluppati. si continuano a identificare nuove varianti virali e nuovi focolai geografici di varianti virali: questo rende conto di un dinamismo nella variabilità virale di complessità crescente; la ricombinazione fra varianti risulta avere un ruolo sempre maggiore nel generare diversità, specialmente nei punti geografici "caldi" dove le diverse forme genetiche si incontrano. anche la superinfezione è importante nella diversificazione e nella propagazione delle varianti. oggi si riconosce lo sviluppo di diversità virale e di progenie locali a partire dai punti geografici in cui sono stati introdotti i mutanti in nuove popolazioni. la definizione delle caratteristiche di queste varianti e la comprensione di come queste si generino in risposta alla reattività immune e/o alla terapia antivirale è importante per lo sviluppo di vaccini. siamo agli inizi della comprensione, tramite la epidemiologia molecolare, delle implicazioni della diversità globale dell'hiv [11] [12] [13] . questa enorme variabilità resta nel terzo decennio di sviluppo dell'infezione una sfida per la determinazione della carica virale nelle aree dove il virus è più diversificato, per l'analisi della resistenza farmacologica e per lo sviluppo di vaccini [14, 15] . anche il volto della malattia è cambiato: sono presenti grandi focolai epidemici in tutto il mondo, soprattutto nei paesi in via di sviluppo, dove la trasmissione è prevalentemente eterosessuale, ma rilevanti focolai di infezione sono presenti anche nei paesi più industrializzati, dove sono colpiti i più svantaggiati. il quadro è attualmente definito dai seguenti numeri [16] : 28 milioni di morti per aids, più di 40-45 milioni di infetti, 14 milioni di orfani. prima causa di malattia e morte al mondo fra i soggetti di età compresa fra i 15-59 anni. solo nel 2003 si calcolano 3 milioni di morti, 5 milioni di nuovi infetti di cui 800000 bambini. più del 90% delle persone infette vivono nei paesi in via di sviluppo, dove le poche risorse vanno a sostegno della sopravvivenza, non della diagnosi o della terapia e tanto meno della prevenzione dell'infezione. soltanto nell'africa sub-sahariana, al 2003 si stimano più di 26 milioni di persone infette, con 3,2 milioni di nuovi infetti, per via eterosessuale; l'aids qui rappresenta più del 60% di tutte le cause di morte, e ha ridotto alla metà l'aspettativa di vita; qui senza trattamento farmacologico i pazienti in aids muoiono nel 100% dei casi, con un tempo di sopravvvenza spesso inferiore a un anno; si calcola che 4,1 milioni di pazienti nell'africa sub-sahariana abbiano bisogno di trattamento, ma che solo il 2% di essi ne abbia accesso; e che qualora l'accesso al trattamento antivirale fosse dispo-nibile, la sua efficacia potrebbe essere compromessa dallo stile di vita, dalle limitate conoscenze e dallo scarso rapporto fiduciario medico-paziente come dimostrato in altri paesi in via di sviluppo [17] . il 70% dei soggetti hiv-positivi di tutto il mondo vive nell'africa sub-sahariana, nonostante questa contenga solo l'11% della popolazione globale. ne consegue che è necessario aumentare drasticamente la capacità di prevenzione, di trattamento e di cura per ridurre il danno che l'hiv/aids procura in questa parte del mondo. i primi segni di impatto positivo ci sono nelle zone urbane dell'uganda, dove la prevalenza dell'infezione di hiv neonatale è declinata progressivamente nell'ultimo decennio dal 25-30% del 1991 al 15% del 1996, all'11% del 2000 [18, 19] . per il quarto decennio dell'infezione, che terminerà nel 2010, la proiezione è di 50-75 milioni di infetti [20] nonostante le conoscenze sviluppate, nonostante la terapia, capace di rallentare efficacemente, quando disponibile, la progressione di malattia verso l'exitus, nonostante i tentativi di sviluppare vaccini innovativi. l'aids colpisce i giovani nella loro vita produttiva e ne dimezza l'aspettativa di vita: questo ha e avrà ripercussioni gravi sull'economia mondiale. per questo il programma delle nazioni unite unaids ha sviluppato un piano dettagliato per limitare il danno della pandemia e provvedere per il trattamento antivirale per 3 milioni di persone infette con hiv nei paesi in corso di sviluppo, nel 2005 [21] ; l'efficacia di questo programma è in corso di valutazione. in un quadro così tragico si sono sviluppati anche aspetti relativamente positivi che emergono dalla pandemia e si riflettono sui miglioramenti dei sistemi sanitari e sulla loro efficacia. le vittime dell'epidemia e i loro interessi sono molto più presi sul serio di prima sia nelle misure di contenimento dell'infezione che nei trials clinici per la sperimentazione di farmaci (sia nella loro programmazione che nella sperimentazione che nella rendicontazione). la strategia di contenimento e di controllo iniziale della diffusione della pandemia aveva segregato ulteriormente i gruppi a comportamento a rischio e li aveva fatti scomparire dalla rilevabilità per lo stigma associato all'infezione; l'attuale strategia di trattamento include la cooperazione e l'inclusione sociale. era inizialmente in discussione la frequenza a scuola dei bambini infetti, o il lavoro, particolarmente nella sanità, di adulti infetti; era inoltre in discussione il prevalere dei diritti alla privacy dei malati nei confronti dei partners non infetti; e il diritto della madre alla riservatezza piuttosto che il diritto del nascituro di essere trattato farmacologicamente per non nascere infetto. oggi è acquisito che la salute deve essere legata al contesto ambientale, economico e politico. non era così una volta: la necessità stessa di un costo politico di farmaci salvavita nei paesi più a rischio e in via di sviluppo ha portato a battaglie legali impegnative dei più svantaggiati contro le industrie farmaceutiche consorziate che sono state costrette a una maggior solidarietà. l'aids ha insegnato che le malattie di un paese possono influenzare la salute e la stabilità globale mondiale. ha impegnato una vasta parte della comunità scientifica internazionale in molti settori della ricerca, non solo virologica, ma anche immunologica, clinica e molecolare. ha contribuito allo sviluppo della tecnologia di sviluppo farmaceutico, vaccinale, delle conoscenze della fisiologia e della patologia umana, compresa quella tumorale (i tumori associati ad aids: linfomi, sarcomi e carcinomi, sono di natura virale e correlano con il grado di immunocompromissione), delle strategie di trattamento e di prevenzione. ha insegnato che ampliare il più possibile l'accesso alla diagnosi significa anche far prendere coscienza dell'infezione e contribuire alla prevenzione della diffusione; ampliare il più possibile l'accesso alla terapia significa, da una parte, maggior benessere e maggior produttività sociale, e, dall'altra, minor carica virale e minor rischio di trasmissione orizzontale e verticale. per i paesi in via di sviluppo sono attualmente allo studio sistemi semplici di diagnosi e di monitoraggio, mentre si cerca la semplificazione dei regimi terapeutici per ottenere maggior aderenza clinica al trattamento. si cerca così di migliorare la prevenzione, il trattamento e di affrontare molte delle questioni che portano alla diffusione dell'infezione: la povertà, l'ineguaglianza sociale e lo stigma. sono nate diverse organizzazioni locali, nazionali e internazionali per contribuire alla lotta all'aids, e offrire una risposta globale adeguata alla diffusione globale dell'infezione. l'unità operativa complessa di virologia dell'università degli studi di pisa è responsabile, tra l'altro, della diagnosi e del follow-up molecolare della variabilità genetica dell'hiv indotta dal sistema immunitario e dalla terapia antivirale specifica nell'area geografico/sanitaria di sua competenza, dove i ceppi hiv di tipo b e non-b, sia puri che in forma ricombinante, risultano rilevabili e a diffusione locale. questa unità operativa può insegnare quanto fa localmente e, quindi, contribuire all'avanzamento delle conoscenze là dove c'è bisogno; e, come contropartita, può imparare il tipo di evoluzione molecolare che ci si può attendere localmente studiando i ceppi di hiv di zone evolutivamente avanzate, ad esempio nella repubblica centro-africana, che è parte dell'area geografica iniziale dell'infezione da hiv ed è rimasta un crogiuolo di tutti i ceppi e di molte varianti. a sostegno delle iniziative di una nostra ex-allieva della facoltà di medicina e chirurgia dell'università di pisa, specialista in malattie infettive, che successivamente ha preso i voti come suora carmelitana di s. teresa, si è costituito un gruppo omogeneo, interessato a offrire ai paesi africani supporto economico, scientifico, logistico, medico-chirurgico e di laboratorio. il gruppo si è trasformato in comitato e, recentemente, con il progredire dell'interesse per l'iniziativa, in onlus, denominato: noi per l'africa -onlus: progetto per la realizzazione di un ospedale a bossemptélé, nella repubblica centrafricana, a 295 km dalla capitale, prefettura di ouham-pendé (11160 abitanti, circa il 20-25% dei quali hivpositivo, con aspettativa di vita media ridotta del 50% dalla malnutrizione, dalle malattie infettive e dalle scarse prospettive economiche e sociali), nel terreno (20000 m 2 circa) concesso all'onlus dalla missione cattolica delle suore carmelitane di s. teresa che è presente nell'area da circa 40 anni. l'associazione noi per l'africa-onlus non ha fini di lucro ed ha lo scopo di promuovere la raccolta di fondi da destinare alla promozione, rea-lizzazione e gestione di strutture, infrastrutture socio-sanitarie, educative e culturali nei paesi in via di sviluppo, con particolare riferimento ai paesi del continente africano [22] . il progetto si propone, nell'ambito dell'infezione da hiv i seguenti obiettivi: 1) creare un centro di screening volontario per l'hiv dove sia possibile eseguire sia la diagnosi di laboratorio che il followup molecolare della variabilità genetica virale, 2) sviluppare le attività di prevenzione della trasmissione dell'hiv dalla madre al bambino, 3) assicurare la prevenzione e il corretto trattamento delle infezioni opportuniste e delle infezioni sessualmente trasmissibili, 4) favorire l'accesso e il monitoraggio del trattamento antiretrovirale. tutto questo verrà svolto nel rispetto dei protocolli nazionali, che vedono nella lotta all'aids l'obiettivo primario, ma che mancano delle risorse essenziali per raggiungerlo. le degenze pediatriche e il centro nutrizionale, il laboratorio convenzionale e di biologia molecolare, il blocco operatorio con i letti per l'ospedalizzazione dei bambini e degli adulti rappresentano un salto di qualità nei servizi sanitari attualmente esistenti localmente, ai quali possono accedere gli abitanti di bossemptélé e dei centri limitrofi. grazie ai contributi di diversi enti, il centro è attualmente in fase avanzata di costruzione dell'intero complesso (prevista per il dicembre 2006, circa 1400 m 2 ): si propone di offrire supporto per la diagnosi e il follow-up di varie malattie infettive e di dare accesso al trattamento secondo gli standard europei sia antivirale hiv che anti-infettivo/parassitario a 5000 persone entro il 2008. questa iniziativa rappresenta il nostro tentativo di dare una risposta non solo locale a una infezione virale così generalizzata e grave. nel mondo globalizzato e interconnesso soltanto nell'ultimo decennio diverse infezioni virali si sono avvicendate, competendo con l'hiv per l'attenzione scientifica, medica, economica e sociale e beneficiando delle conoscenze, metodologie di rilevazione, organizzazione sanitaria, sociale ed economica acquisite nella battaglia contro l'aids; sono sia nuove identificazioni ( il virus sin nombre, nuovo hantavirus della famiglia bunyiavirideae, causa febbre emorragica, complicazioni renali e polmonite essudativa con altissima mortalità; si trasmette per contatto diretto, senza vettori, da roditori infetti (topo di campagna), per areosol di loro urine/feci. il nuovo sierotipo, emerso nel 1993, ha interessato, per scarsità di condizioni igieniche, gli indiani navajo negli stati uniti causando ostruzione polmonare di tipo essudativo [23] . il virus sabia nuovo arenavirus della famiglia delle arenaviride, è capace di dare infezioni persistenti asintomatiche nei roditori e acute gravi nella specie umana (febbre emorragica e coagulazione intravasale disseminata) che si infetta per scarsa igiene e alterazioni ambientali (sviluppo economico-urbanistico tumultuoso) che portano a contatto umano i roditori nelle zone rurali del brasile [24] . i virus hendra e nipah, strettamente correlati, sono nuovi membri della famiglia paramixovirideae, genere henipavirus. l'hendra, è stato riconosciuto in australia nel 1994, in seguito a infezione di 13 cavalli e del loro fantino [25] morti per sindrome respiratoria acuta; è stato isolato in altri focolai fatali equini e umani e successivamente isolato da pipistrelli fruttivori (volpi volanti) non manifestamente malati. la trasmissione fra i cavalli potrebbe essere dovuta al rilascio di virus con le secrezioni nasali e con le urine; non è stata evidenziata trasmissione interumana. nipah, riconosciuto fra il settembre 1998 e l'aprile 1999 durante una massiccia devastazione di fattorie di maiali in malesia, si è diffuso a singapore a causa del trasporto alimentare di maiali infetti (e delle nuove tecnologie industriali di produzione alimentare). ha causato solo febbre con sintomi respiratori nella maggior parte degli animali, con il 5-15% di mortalità. l'uomo si è infetta-to professionalmente (massima incidenza fra gli allevatori di suini) manifestando sindrome respiratoria e neurologica con coma e morte nel 40% dei casi (105 decessi, 265 persone infette); alcuni soggetti sono recidivati e morti quando erano già in corso di guarigione. il focolaio epidemico è stato controllato con il blocco delle esportazioni e con il sacrificio di più di un milione di maiali e con un grave danno economico [26] . data la somiglianza genetica con il virus hendra è stato cercato e trovato nelle volpi volanti in australia, malesia, bangladesh e cambogia che sembrano essere il serbatoio naturale dell'infezione. il virus hhv-8, gamma herpesvirus, famiglia herpesvirideae, è stato identificato molecolarmente nel 1995 come l'agente causale del sarcoma di kaposi, un sarcoma già noto da secoli nel bacino del mediterraneo e nell'africa, ma che si è diffuso per via sessuale in maniera epidemica con la pandemia di hiv; di recente è stata riconosciuta la potenziale trasmissione virale con i trapianti [27] . l'agente prionico, nella nuova variante dell'encefalite spongiforme o variante della malattia di creutzfeldt-jacob (v-cj), associata all'ingestione di carni di bovini affetti da encefalopatia spongiforme bovina (mucca pazza) da parte di soggetti di età media, all'inizio dei sintomi, di 26 anni, con degenerazione neurologica a evoluzione fatale in 14 mesi circa. è un tipico esempio di introduzione nella specie umana, nel 1996, di un nuovo patogeno per alterazioni tecnologiche di produzione industriale animale (vedi capitolo 6). l'infuenza aviaria, ortomixovirus, famiglia ortomixovirideae, infetta varie specie di uccelli sia acquatici che terrestri, ha dimostrato frequenti passaggi nell'uomo per salto di specie, dal 1997, con flusso continuo nei due sensi e rischio di pandemia umana reale. un'altra pandemia influenzale (dopo le documentate pandemie: "spagnola" nel 1918, "asiatica" nel 1957, "honk kong" nel 1968, "russa" nel 1977) sembra sempre più inevitabile dal momento che ceppi molto patogeni e capaci di rapida evoluzione molecolare, gli h5n1, si stanno radicando nella popolazione aviaria e circolano insieme, nelle popolazioni asiatiche, a ceppi di influenza umana, creando (nell'uomo, negli uccelli acquatici o nel pollame) le premesse per mutazioni e/o riassortimento genetico virale che può dar luogo a un ceppo altamente diffusibile [28] . il numero di volatili coinvolti nell'emergenza di focolai di infezione virale è aumentato più di 100 volte : dai 23 milioni di esemplari nel periodo 1959-1998 a più di 200 milioni nel periodo 1999-2005. focolai epidemici di vaste dimensioni si sono verificati per il superamento virale delle comuni barriere di bio-sicurezza e per l'ingresso del virus proveniente da allevamenti rurali e semi-intensivi in circuiti commerciali del-l'allevamento e del trasporto di animali vivi. globalmente diverse centinaia di milioni di polli sono morti di infezione o per la prevenzione della diffusione dell'infezione e più di 160 persone sono risultate infette, con più del 50% di mortalità [29] . il metapneumovirus, membro della famiglia paramixovirideae, strettamente correlato con il pneumovirus aviario, è emerso per le migliorate capacità diagnostiche piuttosto che per aumento di diffusione e/o salto di specie. è stato identificato nel 2001 in olanda come causa di infezione respiratoria acuta in bambini ospedalizzati. è risultato capace di esacerbare l'asma bronchiale e causare malattia severa in anziani, infanti, immunocompromessi con pneumopatie di base, in europa, nord america, asia, australia [30] . il virus sars-cov, membro della famiglia coronavirideae, genere coronavirus, causa una zoonosi, per salto di specie, altamente diffusibile che ha interessato l'uomo con una nuova sintomatologia definita da: sintomi respiratori, infiltrati polmonari radiologicamente evidenti e contatto primario o secondario con viaggiatore da un'area coinvolta dall'infezione. ha interessato il personale ospedaliero (20% delle infezioni) e i membri familiari in stretto contatto, fino ad interessare complessivamente più di 8000 casi in 30 paesi diversi in 9 mesi (fra il novembre 2002 e luglio 2003) [31] , causando un allarme globale oms per il tasso di mortalità che è risultato compreso fra il 7-17%, con punte del 50% negli ultrasessantenni specialmente se diabetici e cardiopatici. ha stranamente interessato relativamente poco i bambini; ha mostrato accelerazioni nella diffusione per contatto diretto in ambienti chiusi ospedalieri, abitativi, in mezzi di trasporto e in luoghi di riunione, attraverso aerosol ambientali e fomiti (lenzuola, coperte, abiti). la sua origine riconosciuta è legata a una o più specie di animali selvatici: civette palmari dell'himalaya, procioni, furetti, gatti domestici. può ancora riemergere nei mesi invernali, ritrasmesso dal serbatoio naturale all'uomo e propagarsi nuovamente in ospedali o laboratori. il virus del vaiolo della scimmia (monkeypox), malattia delle foreste pluviali dell'africa centrale e occidentale, è stato causa di morte nell'1-10% degli infetti, soprattutto nei bambini nella repubblica congo, da dove il virus è stato identificato nel 1970 e dichiarato eradicato nel 2001. è comparso improvvisamente nei cani della prateria e nell'uomo negli stati uniti nel corso del 2003, si presume in seguito all'importazione di 800 piccoli mammiferi domestici roditori originari del ghana: scoiattoli, porcospini, ratti giganti, varietà di topi [32] . i filovirus marburg ed ebola (dei due l'ultimo è il più frequentemente riemerso) sono diffusibili per contatto diretto, tramite fluidi biologici ed aerosol sia da carcasse di primati non umani che per trasmissione interumana (sia in ambienti rurali che ospedalieri); sono rapidamente fatali, nel 50-100% dei casi per febbre emorragica, così da estinguere il focolaio di infezione per esaurimento di nuovi casi di trasmissione. diversi focolai epidemici d'infezione (dodici focolai) sono stati documentati nell'africa sub-sahariana, ma anche in europa (in germania nel 1967, quando fu isolato per la prima volta, negli stati uniti, nel 1989, in italia nel 1992); il rischio di riemergenza è continuo [33] . il virus toscana, phlebovirus della famiglia bunyiavirideae, identificato nel 1971 in italia, nella regione toscana, dove causa più del 50% dei casi di meningite-meningoencefalite nella provincia di siena (più frequentemente ad andamento benigno), in zone collinari infestate da flebotomi vettori è attualmente in espansione territoriale in tutto il bacino del mediterraneo [34] . il virus west nile, genere flavivirus della famiglia flavivirideae, riconosciuto nel 1997 in israele, è comparso nel 1999 a new york trasportato da uccelli nelle loro rotte migratorie; è risultato disseminato localmente dalle zanzare, dai cavalli, da altri mammiferi e dall'uomo. si è diffuso molto rapidamente in africa, medio oriente, romania, russia, nelle americhe, in canada, nel messico. nel solo 2002, negli stati uniti si sono registrati 4100 nuovi casi, 3000 (73%) dei quali neuroinvasivi, con 284 morti. è risultato trasmesso nei paesi occidentali oltre che dalle zanzare anche con l'allattamento, le trasfusioni e i trapianti d'organo [35] . la malattia umana è caratterizzata da una malattia leggera, autolimitante simile al dengue. inizia come febbre e mialgia; in una modesta percentuale di pazienti, soprattutto anziani e immunocompromessi, progredisce in una forma più severa con coinvolgimento del snc: encefalite e meningite. il tasso di mortalità è risultato intorno al 10%, ma i sopravvissuti dimostrano alterazioni neurologiche permanenti. il virus dengue, genere flavivirus della famiglia delle flavivirideae, causa un'infezione che può essere asintomatica, severa o fatale per sindrome emorragica e shock; è trasmesso da zanzare, con una diffusione paragonabile a quella della malaria, presente endemicamente in africa, nelle americhe, nel medio oriente, in asia, nella costa del pacifico occidentale. si calcola che infetti 50 milioni di nuovi soggetti annualmente [35] e che 2,5 miliardi di persone vivano in aree a rischio di trasmissione epidemica; in italia si riscontrano 40-80 casi annui di dengue in soggetti che hanno viaggiato nelle zone endemiche. il virus chikungunya, alphavirus della famiglia togavirideae, si trasmette tramite zanzare; causa artropatia virale; è una malattia tipicamente tropicale che in molte zone del pianeta convive con il dengue. nota da numerosi decenni in africa tropicale e asia sud-orientale è divenuta recentemente endemica anche nei paesi e nelle isole dell'oceano indiano (india, malaysia, la reunion, madagascar, indonesia, mauritius, mayotte, seychelles, comore) e successivamente è comparsa in maniera sporadica in europa (anche in italia) in pazienti che hanno viaggiato nei paesi nei quali l'infezione è endemica. ha causato allarme oms nel marzo 2005 quando ha interessato in maniera massiccia l'isola di la reunion (200000 persone sospette di infezione, 3115 casi sintomatici, fra cui 31 medici sentinella presenti nel paese) poichè oltre all'artropatia sono comparsi disordini neurologici, decessi, infezioni congenite e casi di sospetta trasmissione via trasfusione [37] . cambiamenti ecologici, aumento dei viaggi e della temperatura globale sono alla base dell'aumento della diffusione e della prevalenza di insetti vettori di infezioni virali da phlebovirus, flavivirus ed alfavirus, che possono sviluppare pandemie in una popolazione globale suscettibile di infezione. il virus hbov, nuovo membro del genere bocavirus, famiglia delle parvovirideae, è stato descritto in infezioni respiratorie gravi dell'infanzia in svezia nel 2005 [38] e successivamente in australia, giappone, sud corea, sud africa, in europa (francia) e in giordania, in associazione con altre infezioni respiratorie umane. la sua organizzazione genomica e la sua sequenza nucleotidica indica alta omologia con il parvovirus bovino e canino 1. la presenza del virus e di numerose varianti in aree non contigue indica una diffusione precedente alla sua identificazione. vecchi e nuovi agenti virali contribuiscono al quadro già complesso della medicina globalizzata; le lezioni che la pandemia di hiv avrà saputo impartire saranno preziose per identificarli, monitorarli, trattarli con nuovi antivirali specifici, possibilmente prevenirli con vaccini innovativi. non sembra che lo sviluppo scientifico e tecnologico da solo sarà in grado di debellare vecchie e nuove potenziali pandemie; sarà inevitabile che l'uomo si confronti comunque con molte delle condizioni, note da tempo [39] , che contribuiscono alla diffusione delle infezioni virali e che sono stati chiamate "traguardi di sviluppo per il millennio" (millennium development goals) che includono per tutti: risorse economiche adeguate, educazione avanzata, uguaglianza fra i sessi, mancanza di carestie e di deterioramento dell'ambiente, acqua sana e accessibile. questi traguardi sono stati definiti fino al 2015, per ciascun'area del globo e sono da monitorare perché avanzi lo sviluppo e la povertà sia ridotta in tutto il mondo; perché migliorino i sistemi sanitari nazionali ed internazionali di intervento contro le infezioni, perché i sistemi di vigilanza, rilevazione e comunicazione in tempo reale siano sempre più efficienti nel trasferire informazioni preziose e condivisibili alle persone a rischio, con il necessario contributo interdisciplinare clinico, igenistico, veterinario e di ricerca scientifica e applicativa. world health report 2003 -shaping the future. geneva: world health organization aids as a zoonosis: scientific and public health implications the discovery of hiv as the cause of aids an african hiv-1 sequence from 1959 and implications for the origin of the epidemic prevalence of drugresistant hiv-1 variants in untreated individuals in europe: implications for clinical management viral dynamics in human immunodeficiency virus type 1 infection pathogenesis of hiv infection: what the virus spares is as important as what it destroys immune control of hiv: the obstacles of hla and viral diversity apoptosis of uninfected cells induced by hiv envelope glycoproteins antiviral drug discovery and development: where chemistry meets with biomedicine replicative homeostasis: a fundamental mechanism mediating selective viral replication and escape mutation replicative homeostasis ii: influence of polymerase fidelity on rna virus quasispecies biology: implications for immune recognition, viral autoimmunity and other "virus receptor" diseases hiv drug-resistant strains as epidemiologic sentinels emerging infectious diseases molecular epidemiology of hiv-1 variants in the global aids pandemic: an update density-dependent decay in hiv-1 dynamics 3°ias conference on aids pathogenesis and treatment adherence to antiretroviral therapy: a qualitative study with physicians from rio de janeiro, brazil the spread of hiv-1 in africa: sexual contact patterns and the predicted demographic impact of aids the next wave of hiv/aids the world health organization strategy (2003) the who and unaids global initiative to provide antiretroviral therapy to 3 million people with hiv/aids by the end of hantavirus pulmonary syndrome: a zebra worth knowing a morbillivirus that caused fatal disease in horses and humans nipah virus outbreak in malaysia post-transplant kaposi sarcoma originates from the seeding of donor-derived progenitors are we ready for pandemic influenza? writing committee of the world health organization (who) consultation on human influenza a/h5. avian influenza a (h5n1) infection in humans a newly discovered human pneumovirus isolated from young children with respiratory tract disease cumulative number of reported cases of severe acute respiratory syndrome (sars) geneva the detection of monkeypox in humans in the western hemisphere ebola virus antibody prevalence in dogs and human risk transmission of west nile virus from an organ donor to four transplant recipients epidemic dengue/dengue hemorrhagic fever as a public health, social, and economic problem in the 21st century chikungunya virus: its recent spread to the southern indian ocean and reunion island cloning of a human parvovirus by molecular screening of respiratory tract samples lederberg j (eds) for the committee on emerging microbial threats to health in the 21st century, board on global health, institute of medicine (2003) microbial threats to health: emergence, detection, and response key: cord-021382-10omkpwl authors: abdul-khaliq, catherine title: development of a united kingdom national external quality assessment scheme (uk neqas) for hiv point of care testing date: 2011-03-17 journal: nan doi: 10.1093/biohorizons/hzr004 sha: doc_id: 21382 cord_uid: 10omkpwl the use of hiv point of care tests (pocts) is increasing rapidly in both laboratory and other settings. these tests are often performed by non-laboratory trained staff. at the present time there are no external quality assessment (eqa) providers in the uk offering proficiency testing schemes for hiv point of care testing. the aim of this study is to develop such an eqa scheme. firstly, a selection of the most widely used pocts was selected and their performance assessed using existing hiv-positive serology eqa specimens. all assays produced the correct results however intensity of results observed for the same specimen differed greatly between poct devices. in addition the effect of various sub-groups of hiv-1 serum samples on the hiv poct assays was investigated and no difference between the results on the pocts was observed. ultimately four serum specimens, two hiv-1 and one hiv-2 positive, one hiv negative, were chosen and sent to nhs laboratories and sexual health clinics for testing as part of a pilot eqa scheme for hiv poct. results were excellent with 97% of participants reporting correct results (n= 20). the study highlighted a lack of awareness of eqa particularly in non-laboratory settings, although recommendations (iso 22870:2006) are in place for the users of such devices. in conclusion, the need for eqa for providers of point of care testing is an integral part of ensuring reliability of results and quality of care for the patient. in 2009, the health protection agency studied the number of people being tested for hiv at sexual health clinics and not returning for their diagnosis. the report produced 1 revealed that the number of infected individuals in the uk at the end of 2008 was estimated to be 83 000 with approximately 22 400 individuals unaware of their infection (fig. 1) . many patients remain anonymous at sexual health clinics and it is therefore impossible to trace them and inform them of their test result. this obviously has a severe impact on the risk of hiv transmission. it has been shown in several studies such as that by darnell one of the priority areas for the unaids framework for 2009-2011 is to reduce the transmission of hiv. testing for hiv at the point of care may increase the number of adults willing to take a test as the results are produced at the same time as testing. this would therefore reduce the risk of transmission and allow anti-viral treatment to start earlier. 3 point of care tests (pocts) are investigations carried out near to or at the patient's side. they are rapid immunochromatographic, flow through or particle agglutination assays that provide results immediately or within minutes depending on the test device. only a small volume of patient sample is required to perform the test, often a drop of blood from a finger prick. 4 the sample formats for use in pocts vary between devices but can be serum, plasma, whole blood or saliva. the majority of poct devices approved for diagnostic use involve hiv-recombinant antigens and/or synthetic peptides bound to the membrane in the poct device, the sample is applied to the membrane and if antibodies are present they form a complex with the membrane-bound antigen that induces a coloured reaction. in addition to the detection of hiv antibodies, new fourth-generation pocts can also detect the presence of hiv p24 antigen; antibodies to p24 antigen are membrane bound and detect p24 antigen in the sample. these tests reduce the time to diagnosis as hiv infection can be detected before seroconversion. 5 studies show that the use of pocts for the detection of hiv is increasing rapidly in both laboratory and other clinical settings, especially in developing countries. for example, following the introduction of pocts in malawi the number of people receiving an hiv test annually rose from 5000 to 40 000 and the number of those tested who remained to receive results increased from 69% to 99.7%. 4 hiv pocts are often performed by non-laboratory trained individuals such as nurses. 6 the results of these tests are interpreted by the user, which is very subjective and relies heavily on user expertise and knowledge of the test principle. 4 adequate training and quality assurance procedures should be in place, as with any other assay, to ensure reliable results. 7 external quality assessment (eqa) has an important role in the quality assurance measures of every laboratory or clinic as participation in an eqa scheme allows the user to monitor their own quality assurance procedures and to readily identify and remedy any problems with internal quality control measures. it demonstrates to patients and other professionals a commitment to quality. 8 participating in an eqa scheme contributes to a laboratory's accreditation status awarded by the appropriate governing body, for instance the clinical pathology accreditation. although participation in an eqa scheme is not compulsory for sexual health clinics or laboratories, unless they wish to gain accreditation, a bulletin by the medicine and healthcare products regulatory agency and a document by the international organization for standardization (iso) relating to point of care testing (en iso 22870:2006) state that users of pocts should participate in an appropriate eqa scheme. eqa involves the analysis of simulated specimens designed to reflect the characteristics of a clinical sample. simulated specimens of known but undisclosed content are sent at regular intervals often monthly or quarterly to participating laboratories. the participants then test the samples using their own routine procedures and report their results to the eqa provider. the results of all participants are analysed and a report produced that shows a participants own result and the anonymized results of all other participants in the scheme. the report also includes a cumulative assessment of the participant's performance allowing them to monitor the performance over a period of time. the united kingdom national external quality assessment service (uk neqas) for microbiology is the leading external quality assessment (eqa) provider in the uk. they provide eqa schemes in all disciplines related to microbiology to diagnostic laboratories across uk and worldwide. bioscience horizons † volume 4 † number 1 † at the time of the study there were no eqa providers in the uk offering proficiency testing schemes for hiv point of care testing. given that the use of hiv pocts is rising rapidly in the uk and worldwide, the study proposed that eqa of hiv poct providers is beneficial to ensure quality, reliable results for patients especially in primary care settings where no supporting laboratory is available. in september 2009, uk neqas for microbiology published results of an hiv point of care testing questionnaire sent to 293 genito urinary medicine clinics across the uk. 9 the questionnaire sought information on whether pocts were used, frequency of use, assay type and sample format used, e.g. whole blood, serum, oral fluid, etc. the intention of this project was to develop and distribute a pilot hiv point of care testing eqa scheme to interested participants in laboratories and other clinical settings. for this purpose, the various kits available on the market for hiv point of care testing were used to demonstrate performance on existing uk neqas hiv serology samples. the focus was on the detection of anti-hiv antibodies and possibly the detection of hiv p24 antigen for fourth-generation pocts. the effect of various sub-types of hiv serum samples on the performance of hiv poct assays was also determined. feedback from the uk neqas questionnaire sent to sexual health clinics across the uk identified current users of hiv pocts and the devices employed. a selection of the most commonly used hiv poct devices were included in this investigation namely determine hiv-1/2 ab (third generation), insti hiv-1/2 test and the oraquick rapid hiv-1/2 antibody test. in addition to the above tests, the determine hiv-1/2 ag/ab combo fourth-generation poct was included for testing (this became available in spring 2009). the performance of these kits was investigated using two different sets of uk neqas hiv serology specimens. plasma for eqa was provided by the nhs blood and transplant from living donors. all donations received by uk neqas were provided without patient identifiers. eqa material, from living donors, is exempt from the human tissue act 2004 and ethical approval was not required. the first set of specimens had been previously characterized by the genscreen ultra hiv-1/2 eia and inno-lia line assay to determine hiv group 1 or 2 and based on these results 20 specimens (16 hiv-1 and 4 hiv-2) were selected for testing. selection was determined by hiv strain and the band profiles revealed on the line assay; a range of differing profiles were selected. nineteen of the 20 hiv-positive specimens had been previously diluted using negative sera with dilutions varying from 1:10 to 1:117 and one specimen was not diluted. an additional specimen confirmed negative for hiv antibodies, hepatitis b surface antigen and hepatitis c antibodies was also included for testing. all specimens were re-tested using genscreen ultra hiv-1/2 ag/ab eia, these results were used as a reference for poct results. the second set of specimens had previously been characterized by molecular methods so the sub-group and viral load had been established; five specimens of sub-groups b, c, g and crf02_ag were selected to test poct performance across sub-groups. all specimens had been heat inactivated at 568c for 30 min and viral inactivation was fully completed by adding 1% tween 80. the specimens were tested according to the manufacturer's instructions for all poct kits and reactive results were scored on the basis of colour intensity, 4þ strongest to þ/2 weakest. an additional investigation was performed where the second set of specimens were re-tested as 1 in 70 dilutions, the specimens were diluted using serum confirmed negative for hiv antibodies, hepatitis b surface antigen and hepatitis c antibodies. the poct signals produced by the diluted specimens were compared with the initial signals produced from the testing of the same specimen undiluted. current users of hiv pocts in sexual health clinics as well as participants of the uk neqas hiv serology scheme that use hiv pocts were invited to participate in the pilot eqa scheme for hiv point of care testing via letter. those wishing to participate were asked to respond within a given time. those who did not respond within this time limit were contacted via telephone to identify the reason for not responding and provide them with another opportunity to participate. forty-six invitation letters were sent and 20 chose to participate in the scheme, 10 nhs laboratories and 10 sexual health clinics. the participating sexual health clinics were given a unique identifying number (identifier) to ensure the results they reported remained confidential and anonymous. current participants of the hiv serology scheme were already assigned with a unique identifier. four eqa serology specimens were distributed to each pilot scheme participant along with a reply form for the reporting of results. instructions and safety information were also included. all returned results were analysed to determine performance with each specimen and the kits used. performance of poct kits was investigated using a first set of specimens fully characterized by enzyme immunoassays (eias) and line assays. all specimens correctly showed reactive results for hiv-1 and hiv-2 antibody with all pocts. however 7 of 20 specimens showed weak reactive results for hiv antibodies on both insti hiv 1/2 and oraquick ...................................................................................................................................................................................................................................... rapid hiv-1/2 antibody kits, including 3 of 16 hiv-1 and all four hiv-2 specimens. all specimens tested negative for hiv p24 antigen. all results were valid as control lines were clearly observed in all tests. accuracy of the results obtained were assured as re-testing of the specimens using the genscreen eia matched those from the previous characterization and the results obtained from an outside reference laboratory. the variation in signal strength of the pocts depicted in fig. 2 was typical of the variation observed in this study for all specimens on the different pocts. both determine pocts produced very clear reactive results with every specimen. all signals for the determine tests were graded at 3þ or above (fig. 2) . for the insti hiv-1/2 test device an example of a 2þ signal can be seen for specimen 3, a 1þ signal for specimen 4 and a þ/2 signal for specimen 2. an investigation into the effect of hiv-1 subtypes on poct results was performed using a second set of specimens fully characterized by molecular methods. all hiv-1 sub-type specimens produced clear reactive signals on the pocts. no effect was observed due to differences in sub-type. interestingly, the reactive signals observed were overall much stronger than the signals observed with the first set of specimens (fig. 3) . it was anticipated that the differences in signal strength observed may be due to dilution of the specimens. an additional investigation was performed where the second set of specimens were re-tested as 1 in 70 dilutions. the results were compared against the initial signals produced from the testing of the same specimen undiluted (table 1) . there was little difference observed with determine tests as signals were all very clear whether the specimen was neat or diluted, this was expected due to the performance of the tests throughout the study. an effect on signal strength was observed with some of the insti hiv-1/2 and oraquick rapid hiv-1/2 antibody test results, a 4-fold decrease in some cases (table 1) . however, a clear correlation between signal strength and dilution factor could not be made. in order to select the most suitable specimens for distribution in the pilot scheme, the results of the poct investigations were evaluated and the serology specimens selected were those from the first set that produced the strongest reactive signals across all poct devices. based on these evaluations three serology specimens, two hiv-1 and one hiv-2-positive were selected. additionally, a serology specimen confirmed negative for hiv selected from uk neqas stocks was also included for distribution in the pilot scheme. feedback from the uk neqas hiv poct questionnaire identified users of hiv pocts in sexual health clinics. these ...................................................................................................................................................................................................................................... clinics as well as current participants of the uk neqas hiv serology scheme that use hiv pocts were invited to participate in the pilot scheme. overall 20 responses were received and 18 sets of results were returned. two sets of results were pending as the participants were awaiting test kits (fig. 4) . the four eqa serology specimens were distributed to each pilot scheme participant along with a reply form for the reporting of their results. all returned results were analysed to determine performance with each specimen and the kits used. the most popular kits used were determine hiv-1/2 ag/ab, determine hiv-1/2 ab and insti hiv-1/2 ab (fig. 5) . three participants chose to test the samples using a combination of kits, namely determine hiv 1/2 ab and unigold (n ¼ 1), determine hiv 1/2 and immunocomb ii hiv 1/2 bispot (n ¼ 1) and hiv determine and insti hiv-1/2 (n ¼ 1). overall performance was excellent in this pilot scheme with 97% of participants reporting the correct results. one participant reported an indeterminate result for specimen 0216 using the insti hiv-1/2 ab test and one participant reported hiv p24 antigen positive for the negative specimen 0217 using determine hiv-1/2 ag/ab combo test (fig. 6 ). the investigation into the performance of the pocts using the first set of hiv serology specimens revealed that all pocts showed correct results. the reactive results with the insti hiv-1/2 and oraquick rapid hiv-1/2 antibody kits were very weak in some cases and could lead to difficulties in interpretation whilst the determine kits produced clear reactive results. it seems that in some cases dilution of the specimens did have an effect on the intensity of reactive signals observed with the insti hiv-1/2 and oraquick rapid hiv-1/2 antibody results. although a clear correlation between the dilution factor and signal intensity could not be made, the effect dilution of the sera may have on poct results should be taken into account when selecting specimens for future distributions. the investigation into the effects of various sub-types of hiv-1 on the pocts showed very strong reactive results on all devices regardless of sub-type. however, a limited panel of sub-types were tested and only from within hiv-1 group m. increasing immigration has introduced greater incidence of hiv-1 circulating recombinant forms (crf), non-b sub-types, hiv-1 group o and hiv-2 to other parts of world where they are not usually encountered. 11 a study at king's college and st. thomas hospitals showed that the prevalence of newly acquired infections with hiv-1 non-b subtypes is increasing in the uk and europe, 42 out of 183 individuals included in the study infected with hiv-1 non-b sub-type had acquired their infection in the uk. 12 this poses a challenge to the poct devices largely based on hiv-1 sub-group b antigens and peptides. uk neqas for microbiology provides eqa schemes worldwide including parts of africa where the prevalence of non-b sub-groups and hiv-2 is higher. further studies should assess all subtypes with the aim of incorporating current circulating strains into future distributions. no specimens included in the study were positive for hiv p24 antigen. it was anticipated that the use of determine hiv-1/2 ag/ab combo will increase as at the time of the study it was already employed by 24% of the participants. it was recommended that p24-antigen-positive material is sourced for future investigations. it may prove difficult to source antigen-positive material as blood would need to be donated from recently infected individuals before seroconversion has occurred or from patients who have progressed to late stage aids when levels of p24 antigen increase as antibody levels diminish. it may also be possible to spike existing uk neqas specimens with p24 antigen as recombinant p24 antigen is commercially available. however, this may also prove to be difficult in practice as antibodies present in the serum sample would form complexes with p24 antigen upon addition rendering it undetectable by the pocts. participant performance in this pilot scheme was excellent. constructive feedback was received from those who participated, although overall there was a lack of awareness of eqa schemes within sexual health clinics and the role the schemes had in this setting. it became apparent that, in most cases, the clinics had never encountered, or even heard of eqa before and were unaware how this type of assessment would benefit them. upon explaining the purpose and benefits of eqa, more interest was shown and participating clinic numbers rose from 3 to 10. personally contacting the clinics was a very time-consuming process as it proved difficult to make contact with the appropriate people especially when some clinics were only open once or twice a week. in some cases those contacted were unaware of who would give consent for participation and were unwilling to help any further. often telephone messages and follow-up emails were left unanswered. it was accepted that a lack of awareness of eqa was the main reason for the low number of participating sexual health clinics however correspondence with those who chose not to participate also highlighted other reasons for not participating. cost implications were a concern for those clinics operating as charities, even though the pilot was free of charge they had to take into account the costs of kits involved in testing. some clinics were interested in the scheme but did not participate due to lack of staff and time as certain clinics only operate 1 or 2 days a week they felt eqa was another burden on their heavy workload. in a minority of cases, a general lack of interest in eqa was encountered where the clinics in question felt that their own internal quality assurance measures were sufficient. for future pilot studies, it is recommended that further promotion of the scheme be carried out at the local and management level. it would be important to target higher management levels of primary care trusts, charities and organizations involved in sexual health such as the terrence higgins trust or the british association for sexual health and hiv to further promote the benefits of eqa and make them aware of the recommendations from the medicines and healthcare products regulatory agency and from the international organization for standardization. if needed, an eqa scheme may be developed that also provided quality and technical training. the term point of care infers that the test is carried out at the patient's side to action care immediately. however, it does have a wider use and so it is important that an eqa provider identifies the users of pocts and how they are used in all types of settings. for example, it is not necessary to perform these tests in a laboratory and so testing can be encountered in settings such as sexual health clinics, in developing countries where charitable organizations visit remote villages and test the whole population or in hospitals or healthcare settings in cases of accidental exposure. it is possible that these tests are performed due to ease of use rather than as a point of care or rapid test and it is often the case that the patients sample is referred to a laboratory for confirmatory testing, which delays the result for the patient. in the uk there are now recommendations for hiv pocts to be used for screening in many settings. sir liam donaldson, the uks chief medical officer, and professor christine beasley, the uks chief nursing officer, have requested that hiv testing be carried out in all healthcare settings where appropriate. 13 this includes general practices, hospital emergency departments and other departments within hospitals where hiv-positive patients unaware of their infection status may present with diseases common to aids and hiv, for example thoracic departments where cases of pneumonia are encountered. it also recommends hiv screening using rapid assays for all new patients taken on by gp surgeries. labour wards are also recommended users of hiv pocts in order to screen women in labour who have not yet been tested during routine ante-natal appointments. further research should ascertain the extent to which hiv pocts are used in these settings in order to identify possible eqa participants. at this current time where there is no cure or vaccine for hiv, preventing its transmission is the only way to reduce the number of new infections. hiv remains a significant threat to public health in the uk and worldwide however early diagnosis, that can be improved with the use of point of care testing, together with effective treatment means that those affected are living longer and have a decreased risk of transmitting the infection. this study concludes that the need for eqa for providers of this testing is an integral part of ensuring reliability of results and quality of care for the patient. author biography c.a. gained a bsc (hons) in biomedical science in 2010 and hopes to embark on a successful and rewarding career in diagnostic microbiology. her more immediate aspirations are to complete the ibms specialist portfolio and gain an msc in medical microbiology. hiv in the united kingdom evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products aids the biological basis point-of-care rapid tests for hiv antibodies could the new hiv combined p24 antigen and antibody assays replace p24 antigen specific assays? point-of-care testing in microbiology-the advantages and disadvantages of immunochromatographic test strips rapid human immunodeficiency virus test quality assurance practices and outcomes among testing sites affiliated with 17 public health departments united kingdom national external quality assessment service for microbiology directory feedback from hiv point of care testing questionnaire performance of the oraquick rapid antibody test for diagnosis of human immunodeficiency virus type 1 infection in patients with various levels of exposure to highly active antiretroviral therapy rapid assay for simultaneous detection and differentiation of immunoglobulin g antibodies to human immunodeficiency virus type 1 (hiv-1) group m, hiv-1 group o, and hiv-2 evidence for onward transmission of hiv-1 non-b subtype strains in the united kingdom improving the detection and diagnosis of hiv in non-hiv specialties including primary care key: cord-020101-5rib7pe8 authors: nan title: cumulative author index for 2008 date: 2008-11-17 journal: virus res doi: 10.1016/s0168-1702(08)00367-5 sha: doc_id: 20101 cord_uid: 5rib7pe8 nan dettori, g., see medici, m.c. (137) 163 devi, s., see osman, o. (135) murine leukemia virus reverse transcriptase: structural comparison with hiv-1 reverse transcriptase the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus detection of ovine herpesvirus 2 major capsid gene transcripts as an indicator of virus replication in shedding sheep and clinically affected animals genetic characterization of equine influenza viruses isolated in italy between a new living cell-based assay system for monitoring genome-length hepatitis c virus rna replication unraveling the puzzle of human anellovirus infections by comparison with avian infections with the chicken anemia virus the contribution of feathers in the spread of chicken anemia virus cloning and subcellular localization of the phosphoprotein and nucleocapsid proteins of potato yellow dwarf virus, type species of the genus nucleorhabdovirus the p26 gene of the autographa californica nucleopolyhedrovirus: timing of transcription, and cellular localization and dimerization of product complete genomic sequence of turkey coronavirus recombinant l and p protein complex of rinderpest virus catalyses mrna synthesis in vitro molecular divergence of grapevine virus a (gva) variants associated with shiraz disease in south africa sequence analysis of a reovirus isolated from the winter moth operophtera brumata (lepidoptera: geometridae) and its parasitoid wasp phobocampe tempestiva (hymenoptera: ichneumonidae sars coronavirus replicase proteins in pathogenesis virus-induced gene silencing in medicago truncatula and lathyrus odorata evaluating the 3c-like protease activity of sars-coronavirus: recommendations for standardized assays for drug discovery hbx modulates iron regulatory protein 1-mediated iron metabolism via reactive oxygen species pathogenetic mechanisms of severe acute respiratory syndrome detection of a novel circovirus in mute swans (cygnus olor) by using nested broadspectrum pcr chimaeric hiv-1 subtype c gag molecules with large in-frame c-terminal polypeptide fusions form virus-like particles cross-species recombination in the haemagglutinin gene of canine distemper virus sapovirus-like particles derived from polyprotein cauliflower mosaic virus gene vi product n-terminus contains regions involved in resistance-breakage, self-association and interactions with movement protein adenovirus vector induced innate immune responses: impact upon efficacy and toxicity in gene therapy and vaccine applications interfering with cellular signaling pathways enhances sensitization to combined sodium butyrate and gcv treatment in ebv-positive tumor cells evidence for recombination between pcv2a and pcv2b in the field retroviral reverse transcriptases (other than those of hiv-1 and murine leukemia virus): a comparison of their molecular and biochemical properties mitochondrial plasmids of sugar beet amplified via rolling circle method detected during curtovirus screening appearance of intratypic recombination of enterovirus 71 in taiwan from the circulation of subgenogroups b5 and c5 of enterovirus 71 in taiwan from in vitro replication of bamboo mosaic virus satellite characterization of the interaction of domain iii of the envelope protein of dengue virus with putative receptors from cho cells intrahost evolution of envelope glycoprotein and orfa sequences after experimental infection of cats with a molecular clone and a biological isolate of feline immunodeficiency virus the sars-coronavirus plnc domain of nsp3 as a replication/ transcription scaffolding protein limited compatibility between the rna polymerase components of influenza virus type a and b serotype-specificity of recombinant fusion proteins containing domain iii of dengue virus very virulent infectious bursal disease virus isolated from wild birds in korea: epidemiological implications genetic analysis and evaluation of the reassortment of influenza b viruses isolated in taiwan during the enhanced immune responses of mice inoculated recombinant adenoviruses expressing gp5 by fusion with gp3 and/or gp4 of prrs virus effect of antiviral treatment and host susceptibility on positive selection in hepatitis c virus novel hiv-1 reverse transcriptase inhibitors synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast saccharomyces cerevisiae evolutionary analyses of european h1n2 swine influenza a virus by placing timestamps on the multiple reassortment events hepatitis b pregenomic rna splicing-the products, the regulatory mechanisms and its biological significance human papillomavirus 16 e6, l1, l2 and e2 gene variants in cervical lesion progression pathology and hematology of the caribbean spiny lobster experimentally infected with panulirus argus virus tomato leaf curl virus satellite dna as a gene silencing vector activated by helper virus infection down-regulation of sclerotinia sclerotiorum gene expression in response to infection with sclerotinia sclerotiorum debilitation-associated rna virus presence of p1b and absence of hc-pro in squash vein yellowing virus suggests a general feature of the genus ipomovirus in the family potyviridae the truncated virus-like particles of c6/36 cell densovirus: implications for the assembly mechanism of brevidensovirus tubulovesicular structures are a consistent (and unexplained) finding in the brains of humans with prion diseases interferon antagonist function of japanese encephalitis virus ns4a and its interaction with dead-box rna helicase identification of novel viral interleukin-10 isoforms of human cytomegalovirus ad169 cross-reactive and serospecific epitopes of nucleocapsid proteins of three hantaviruses: prospects for new diagnostic tools genetic diversity of the vp1 gene of duck hepatitis virus type i (dhv-i) isolates from southeast china is related to isolate attenuation the major tegument structural protein vp22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection mutational events during the primary propagation and consecutive passages of hepatitis e virus strain je03-1760f in cell culture the n protein of tomato spotted wilt virus (tswv) is associated with the induction of programmed cell death (pcd) in capsicum chinense plants evolutionary relationships of virus species belonging to a distinct lineage within the ampelovirus genus a novel genomic constellation (g10p[3]) of group a rotavirus detected from buffalo calves in northern india phylogeny of lagos bat virus: challenges for lyssavirus taxonomy dc-sign enhances infection of cells with glycosylated west nile virus in vitro and virus replication in human dendritic cells induces production of different modulation of cellular transcription by adenovirus 5, ⌬e1/e3 adenovirus and helper-dependent vectors involvement of cytoskeleton in junín virus entry hiv-1 reverse transcriptase inhibitor resistance mutations and fitness: a view from the clinic and ex vivo genomic characterization of novel marine vesiviruses from steller sea lions restricted quasispecies variation following infection with the gb virus b molecular characterization of vp4, vp6 and vp7 genes of a rare g8p[14] rotavirus strain detected in an infant with gastroenteritis in italy retroviral reverse transcription mechanisms of resistance to nucleoside analogue inhibitors of hiv-1 reverse transcriptase truncation of cytoplasmic tail of eiav env increases the pathogenic necrosis characterization of russian rabies virus vaccine strain rv-97 bacteriophage preparation inhibition of reactive oxygen species generation by endotoxin inhibition of autographa californica nucleopolyhedrovirus (acnpv) polyhedrin gene expression by dnazyme knockout of its serine/threonine kinase (pk1) gene serine/threonine kinase (pk-1) is a component of autographa californica multiple nucleopolyhedrovirus (acmnpv) very late gene transcription complex and it phosphorylates a 102 kda polypeptide of the complex amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus phosphorylation of the tgbp1 movement protein of potato virus x by a nicotiana tabacum ck2-like activity comparative genomics of serotype asia 1 foot-and-mouth disease virus isolates from india sampled over the last two decades low dna htlv-2 proviral load among women in são paulo city antiviral potentials of medicinal plants a molecular epidemiological study of rabies in puerto rico the latent membrane protein 1 (lmp1) encoded by epstein-barr virus induces expression of the putative oncogene bcl-3 sars coronavirus accessory proteins hepatitis b viruses: reverse transcription a different way increase in proto-oncogene mrna transcript levels in bovine lymphoid cells infected with a cytopathic type 2 bovine viral diarrhea virus plantibody-mediated inhibition of the potato leafroll virus p1 protein reduces virus accumulation interferon ␤1-a and selective anti-5ht 2a receptor antagonists inhibit infection of human glial cells by jc virus the begomoviruses honeysuckle yellow vein mosaic virus and tobacco leaf curl japan virus with dna␤ satellites cause yellow dwarf disease of tomato genetic structure of a population of potato virus y inducing potato tuber necrotic ringspot disease in japan molecular characterisation and phylogenetic analysis of chronic bee paralysis virus inhibition of west nile virus replication in cells stably transfected with vector-based shrna expression system complete genome sequence analysis of dengue virus type 2 isolated in detecting molecular adaptation at individual codons in the glycoprotein gene of the geographically diversified infectious hematopoietic necrosis virus positive natural selection in the evolution of human metapneumovirus attachment glycoprotein sphingomyelin induces structural alteration in canine parvovirus capsid late steps of parvoviral infection induce changes in cell morphology molecular cloning and sequence analysis of the duck enteritis virus ul5 gene the interaction between kshv rta and cellular rbp-j and their subsequent dna binding are not sufficient for activation of modulation of hepatitis b virus replication by expression of polymerasesurface fusion protein through splicing: implications for viral persistence seroprevalence and genetic evolutions of swine influenza viruses under vaccination pressure in korean swine herds the cycle for a siphoviridae-like phage (vhs1) of vibrio harveyi is dependent on the physiological state of the host expression and biochemical characterization of nsp2 cysteine protease of chikungunya virus effect of sirna mediated suppression of signaling lymphocyte activation molecule on replication of peste des petits ruminants virus in vitro prevalence and molecular characterization of wu/ki polyomaviruses isolated from pediatric patients with respiratory disease in functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association genetic variation of hepatitis c virus in a cohort of injection heroin users in wuhan identification of a conserved linear b-cell epitope at the n-terminus of the e2 glycoprotein of classical swine fever virus by phage-displayed random peptide library lópez-galíndez, an hiv-1 215v mutant shows increased phenotypic resistance to d4t hiv-1 p17 binds heparan sulfate proteoglycans to activated cd4 ϩ t cells up-regulation of murid herpesvirus 4 orf50 by hypoxia: possible implication for virus reactivation from latency effective inhibition of japanese encephalitis virus replication by small interfering rnas targeting the ns5 gene size reversion of a truncated dna␤ associated with tobacco curly shoot virus f gene recombination between genotype ii and vii newcastle disease virus growth of tick-borne encephalitis virus (european subtype) in cell lines from vector and non-vector ticks dna recognition properties of the cell-to-cell movement protein (mp) of soybean isolate of mungbean yellow mosaic india virus emerging g9 rotavirus strains in the northwest of china implications of recombination for hiv diversity induction of apoptosis in vero cells by newcastle disease virus requires viral replication, de-novo protein synthesis and caspase activation subcellular localization of the triple gene block proteins encoded by a foveavirus infecting grapevines phylogenetic analysis of the ns5 gene of dengue viruses structural basis for drug resistance mechanisms for non-nucleoside inhibitors of hiv reverse transcriptase importance of cholesterol for infection of cells by transmissible gastroenteritis virus animal models and vaccines for sars-cov infection comparative full genome analysis revealed e1: a226v shift in oropouche virus entry into hela cells involves clathrin and requires endosomal acidification molecular evidence for polyphyletic origin of human immunodeficiency virus type 1 subtype c in transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells rnase h activity: structure, specificity, and function in reverse transcription a single nucleotide change in hop stunt viroid modulates citrus cachexia symptoms sequence analysis of mrna transcripts encoding jembrana disease virus tat-1 in vivo isolation of a type 3 vaccine-derived poliovirus (vdpv) from an iranian child with x-linked agammaglobulinemia a review of studies on animal reservoirs of the sars coronavirus genetic analysis of dengue 3 virus subtype iii 5ј and 3ј non-coding regions allopurinol, an inhibitor of purine catabolism, enhances susceptibility of tobacco to tobacco mosaic virus effects of acp26 on in vitro and in vivo productivity, pathogenesis and virulence of autographa californica multiple nucleopolyhedrovirus sars-cov replication and pathogenesis in an in vitro model of the human conducting airway epithelium rna transcription analysis and completion of the genome sequence of yellow head nidovirus mechanisms of inhibition of hiv replication by non-nucleoside reverse transcriptase inhibitors acute non-cytopathic bovine viral diarrhea virus infection induces pronounced type i interferon response in pregnant cows and fetuses extracellular vesicles containing virus-encoded membrane proteins are a byproduct of infection with modified vaccinia virus ankara analysis of jembrana disease virus mrna transcripts produced during acute infection demonstrates a complex transcription pattern complete sequence of the genome of avian paramyxovirus type 2 (strain yucaipa) and comparison with other paramyxoviruses cloning and sequencing of capsid protein of indian isolate of extra small virus from macrobrachium rosenbergii a member of a new genus in the potyviridae infects rubus molecular epidemiology of rabies in indonesia key: cord-017629-fuv157f1 authors: de groot, anne s.; moise, leonard; mcmurry, julie a.; martin, william title: epitope-based immunome-derived vaccines: a strategy for improved design and safety date: 2008-07-31 journal: clinical applications of immunomics doi: 10.1007/978-0-387-79208-8_3 sha: doc_id: 17629 cord_uid: fuv157f1 vaccine science has extended beyond genomics to proteomics and has come to also encompass ‘immunomics,’ the study of the universe of pathogen-derived or neoplasm-derived peptides that interface with b and t cells of the host immune system. it has been theorized that effective vaccines can be developed using the minimum essential subset of t cell and b cell epitopes that comprise the ‘immunome.’ researchers are therefore using bioinformatics sequence analysis tools, epitope-mapping tools, microarrays, and high-throughput immunology assays to discover the minimal essential components of the immunome. when these minimal components, or epitopes, are packaged with adjuvants in an appropriate delivery vehicle, the complete package comprises an epitope-based immunome-derived vaccine. such vaccines may have a significant advantage over conventional vaccines, as the careful selection of the components may diminish undesired side effects such as have been observed with whole pathogen and protein subunit vaccines. this chapter will review the pre-clinical and anticipated clinical development of computer-driven vaccine design and the validation of epitope-based immunome-derived vaccines in animal models; it will also include an overview of heterologous immunity and other emerging issues that will need to be addressed by vaccines of all types in the future. the availability of immunome-mining tools has fueled the design and development of vaccines by a process that has come to be termed 'reverse vaccinology,' 'vaccinomics,' 'immunome-derived vaccine' (idv) design, or 'genome-derived vaccine' design (rappuoli and covacci 2003; petrovsky and brusic 2002; pederson 1999; de groot and martin 2003; doytchinova, taylor and flower 2003) . this vaccine concept is based on the identification of a minimal set of antigens that induce a competent immune response to a pathogen or neoplasm. recognition of antigens occurs through the presentation of b cell and t cell epitopes derived from the antigen, in the correct immunological milieu. in its minimal form, an idv would contain only adjuvanated b cell and t cell epitopes in delivery vehicles such as liposomes. when these minimal components are packaged in an appropriate delivery vehicle, the complete package comprises an idv. compared to traditional vaccines, idvs have the potential to be safer and more effective since the vaccine focuses the protective immune response on the most essential antigenic elements of the pathogen/neoplasm. a number of idvs have been tested in clinical trials (elliott 2008; gahery et al. 2006; asj¨o et al. 2002; kran et al. 2004) . because epitope-based idvs are generally considered to be safe, when compared to other vectored or attenuated live vaccines, many have progressed rapidly from pre-clinical concept into clinical trials. in the cancer vaccine field, where epitope-based vaccines are well-established, many such vaccines are currently in phase i/ii clinical trials (pietersz, pouniotis and apostolopoulos 2006) . this chapter will review the development and validation of idvs; it will also provide a step-by-step guide to develop idv using validated immunoinformatics tools. these tools have the potential for dramatically accelerating the development of new and improved vaccines for the emerging and existing infectious diseases. whole-antigen-based idvs will be covered briefly; however, the main focus will be on epitope-based idvs and a description of the immunoinformatics tools that have been developed to accelerate the pre-clinical phase of vaccine discovery. so as to illustrate the process of pre-clinical vaccine development using these tools, two epitope-based idvs case studies will be presented: (i) a genome-derived vaccine for tularemia and (ii) an epitope-based hpv vaccine for adjunctive treatment of cancer. application of molecular biology techniques led to the sequencing of genomes and improved definition of the proteome (expressed proteins). even though the fundamental concept of the 'immunome' (the subset of fragments of expressed proteins that interface with the host immune system) is relatively well accepted, many important questions remain. due to genetic variation and poorly understood determinants of antigen processing, it has become clear that immunomes can vary substantially from one host to the next, even when major histocompatibility complex (mhc) and antibody germline genes are shared. the extent of the overlap between immunomes (in different hosts) and the general size of the immunome-representing epitopes derived from a particular pathogen or cancer remain to be determined. recently published studies are beginning to address these questions, partly due to the availability of algorithms that facilitate the identification of epitopes from whole genomes. the size of the immunome has been puzzling vaccinologists for decades. certainly, there are examples in the literature that 'a single epitope protects.' for example, crowe et al. (2006) recently found that immunization with a single th epitope provided a one-log reduction in influenza viral titers early in infection. a single epitope has also been shown to protect against viral disease in woodchucks (menne et al. 1997) , and multiple single epitopes have protected mice against an array of pathogens (an and whitton 1997 and olsen et al. 2000) . a more diverse set of t cell epitopes appear to be critical to immune response to vaccinia (in mice): 49 class i mhc epitopes were shown to contribute to the large majority of the cd8+ t cell immune response (moutaftsi et al. 2006) . remarkably, it was found that 49 of these predicted epitopes (derived from more than 175,000 candidates) represented over 90% of the vaccinia-specific cd8 t cell repertoire. how this number might be extrapolated to humans is unknown, but the result is relevant because 49 epitopes is few enough to be easily packaged and delivered in a vaccine. an epitope-based idv for genetically diverse populations of humans will almost certainly require more than that number of epitopes, particularly if the vaccine is intended to protect against complex bacteria or viruses, or against solid tumors presenting variable antigenic profiles. harnessing the power of immunoinformatics accelerates the tasks of defining the immunome and of identifying and developing new vaccines for human diseases. the task of developing an epitope-based idv can be deconstructed into a series of achievable steps. after selecting a target organism, the next step in the immunome-to-vaccine process is to identify, from within the target genome, a set of potentially antigenic genes/proteins. these sequences can then be screened using a variety of in silico, in vitro, and in vivo mechanisms or a combination of methods. in the case of small viral genomes, it may be possible to include the entire genome in the search universe. in the case of larger bacterial and viral pathogens, the traditional vaccine targets include surface proteins and secreted proteins (which can easily be found using in silico screening programs), toxins (which can be identified through homology matching), and virulence factors (which can be identified through the use of comparative genomics). however, many other types of proteins may also be worthy of consideration: in particular, proteins expressed in high amounts (such as viral capsid proteins), proteins overexpressed during growth or replication, or proteins overexpressed in response to stress conditions. for cancer vaccines, comparisons between cancerous and normal tissue can uncover cancer-specific genes. one approach involves the use of mrna derived from cancerous and normal tissue to probe dna microarrays, followed by selection of genes that are upregulated in cancerous and not in normal tissue (mathiassen et al. 2001; sepkowitz 2001) . using 'reverse vaccinology', a term recently coined by rino rappuoli, the immune memory of subjects who have successfully encountered and defeated a pathogen can be interrogated to identify the primary targets of a natural immune response. rappuoli and colleagues identified novel vaccine targets using in silico techniques to screen the target genome for 'surface protein-like' sequences. candidate proteins were then expressed in escherichia coli and screened against human sera isolated from pathogen-exposed subjects. reactive proteins were deemed relevant to immune response (pizza et al. 2000) . a related approach for discovering candidate vaccine antigens involves analyzing the target pathogen's proteome in silico, using t cell epitope mapping tools. putative t cell epitopes identified can then be screened against peripheral blood mononuclear cells (pbmc) isolated from human subjects who have been infected with the target pathogen (or who have the target cancer). t cell reaction to a particular peptide epitope, typically measured by elisa or elispot assay, implies that the protein from which the peptide was derived, expressed, processed, and presented to the immune system in the course of a 'natural' immune response. using this method, measuring immune response to an epitope reveals a protein antigen. our group describes this approach as 'fishing for antigens using epitopes as bait.' other common in vitro techniques used to identify expressed or overexpressed genes/proteins include: 2d sds-page, (kaufmann et al. 1992; sonnenberg and belisle 1997; hernychova et al. 2001) , mass spectrometry (tomlinson, jameson and naylor 1996) , and/or tandem mass spectrometry (barnea et al. 2002) . in the context of vaccines against infectious diseases, it may be prudent to exclude proteins that are highly conserved across species; such proteins (including housekeeping genes) may cross-react with unrelated avirulent organisms or with self-proteins to which there may be pre-existing tolerance. however, conservation across species should not be confused with conservation within species variants. sequence conservation within species variants is a highly desirable trait for vaccine components and one that the idv approach is particularly well suited to harness. rna viruses in particular (hcv, hiv, coronaviruses) are highly variable pathogens. in these cases, selecting epitopes that are conserved across variants or subtypes may allow for the development of a more broadly applicable vaccine. alternatively, single proteins that are relatively well conserved, when compared to the balance of the target pathogen, can be selected as vaccine candidates. recently, for example, one team has developed a hexon-epitope vaccine that may be effective against a range of adenoviruses, across serotypes (leen et al. 2008) . once critical antigens have been identified, the next steps in epitope-based idv development are to select epitopes and confirm their immunogenicity. once the adaptive immune system has been engaged, a humoral, or antibodybased response forms the first line of defense against most viral and bacterial pathogens. antibodies recognize b cell epitopes composed of either linear peptide sequences or conformational determinants, which are present only in the three-dimensional form of the antigen. several b cell epitope prediction tools, such as 3dex and cep, have been proposed and are in the process of being refined (enshell-seijffers et al. 2003; schreiber et al. 2005; kulkarni et al. 2005) . unfortunately, the computational resources and modeling complexity required to predict b cell epitopes are enormous. this complexity is due in part to the inherent flexibility in the complementarity determining regions (cdr) of the antibody and in part due to glycosylation, and other post-translational modifications can result in modification of b cell epitopes. although accurate b cell epitope mapping tools remain elusive, the selection of potent b cell antigens can be accelerated using t cell epitope mapping tools. when considering b cell antigens as potential subunit vaccines, it may be important to also consider their t cell epitope content since the quality and kinetics of the antibody response is dependent upon the presence of t help. b cell antigens, which contain a significant t help, may outperform b cell antigens lacking cognate help. and in some cases, an identified t cell epitope may contain a b cell epitope. although different epitopes activate t and b cells, it has been widely reported that b cell epitopes have been shown to co-localize near, or overlap, class ii (th, cd4+) epitopes (graham et al. 1989; rajnavolgyi et al. 1999 ). the adaptive immune system's second line of defense is the t lymphocyte. class i-restricted cytotoxic t cells (cd8+ ctl) directly engage and attack infected host cells. class ii-restricted t helper cells mediate the growth and differentiation of both t effector cells and antibody-producing b lymphocytes. both class i and class ii t cells carry out their roles in response to t cell epitopes, small linear fragments derived from protein antigens, displayed on the surface of antigen-presenting cells (apc) by various alleles of mhc. while b cells and antibodies generally recognize epitopes on surface proteins only, t cells recognize epitopes derived from a variety of proteins. once taken up by apc, antigenic proteins are broken down by digestive enzymes. during this process very large numbers of peptide fragments are released. any one of these fragments could be a t cell epitope, but only about 2% of all the fragments generated can implant themselves in the binding groove of the mhc molecule and be presented on the surface of the apc. one of the critical determinants of t cell epitope immunogenicity is the strength of epitope binding to mhc molecules (lazarski et al. 2005) . peptides binding with higher affinity are more likely to be selected by mhc molecules and to be displayed on the cell surface where they can be recognized by t lymphocytes. using a variety of methods including frequency analysis, support vector machines, hidden markov models, and neural networks, researchers have developed highly accurate tools for modeling the mhc-peptide interface and for accurately predicting t cell epitopes. for a review of t cell epitope mapping tools, see de groot and berzofsky (2004) and the accompanying issue of methods. what all these tools have in common is an ability to quickly screen large volumes of genomic sequences for putative epitopes; this preliminary screen reduces the search space dramatically, typically by at least 20-fold. the ability to accurately predict t cell epitopes from raw genomic data is fundamental to the development of an idv. however, even a highly accurate prediction is still only a prediction. before including predicted epitopes in a candidate vaccine, it is important to validate their immunogenicity in vitro and in vivo. once identified, peptides representing the selected epitopes are then synthesized. hla binding assays can be used to assess whether peptides derived from immunoinformatics analysis can bind to either mhc class i or class ii by measuring the affinities of predicted epitope sequences for the hla alleles in vitro. in vitro evaluation of mhc binding can be performed by quantifying the ability of exogenously added peptides to compete with a fluorescently labeled known mhc ligand (steere et al. 2006 ) and can be adapted for high throughput (mcmurry et al. 2007a) . epivax routinely uses these high-throughput hla binding assays to confirm epitope predictions in vitro. a concordance between hla binding and immunogenicity is often observed (mcmurry et al. 2005 ). peptides are used to measure t cell responses in vitro; they can be of variable lengths (9-25). peptides presented in the context of class i mhc are generally limited to 9 or 10 amino acids in length, although some processing is believed to occur during the t cell assay and so 15-mers are also used for class i assays. in contrast with class i epitopes, which are short and fit tightly in the bounded mhc molecule, class ii (t helper) epitopes lie within an open-ended groove in the mhc ii. as such, a class ii epitope can shift within the groove, thereby accommodating mhc of various haplotypes. the only limit on the size of the peptide is its ability to remain in a linear conformation in the open-ended groove. both mhc class i-and mhc class ii-restricted epitopes (targeting cd4+ and cd8+ t cells, respectively) are believed to be important for the development of effective vaccines. cd4+ t helper cells enhance and amplify cytotoxic t cell (ctl) immune responses and have been shown to be important in the development of cd8+ t cell memory to a range of pathogens (ahlers et al. 2001) . ctls generally play a role in the containment of viral and bacterial infection (plotnicky et al. 2003) , and the prevalence of ctls usually correlates with the rate of pathogen clearance. like peptides, whole antigens too can be used to measure t cell responses in vitro. the recognition of these antigens requires the presence of an apc that is capable of processing and presenting peptides derived from the antigen. if blood from exposed individuals is available, the peptides validated as mhc ligands in binding assays can be tested for their reactivity with t cells, serum, or both. a positive immune response (as measured by elisa, elispot, or intracellular cytokine staining) should be interpreted as a sign that the parent protein interfaces with host immune response in the course of natural infection or disease. following confirmation, the peptides that stimulate a response can be considered vaccine candidates themselves or can be used to select the entire protein for use in a subunit vaccine. these candidates can then be incorporated into a vaccine delivery vehicle with an appropriate adjuvant. elisa and elispot are related methods for detecting t cell responses by the measurement of cytokines secreted by the t cells (gamma interferon, il-2, and il-4 are examples). the expansion (proliferation) of t cells in response to stimulation by peptide:mhc can be measured by (1) the dilution of a fluorescent dye in subsequent generations of cells (cfse) and (2) the incorporation of a radioactive label in the proliferating cell's dna (tritiated thymidine incorporation assay). fluorescence activated cell sorting (facs) and intracellular cytokine staining (ics) are the most precise methodologies available for measuring and defining t cell response. for example, t cells that respond to a particular epitope can be directly labeled using tetramers (comprising mhc class ii: peptide complexes). labeled cells can then be sorted and counted, and the phenotype of t cells that respond to the antigen can be determined using cell surface markers and ics (tobery et al. 2006 ). factors extrinsic to processing, such as the cytokine milieu induced in response to a particular component of a vaccine (krieg et al. 1998) or pathogen (ghosh et al. 1998) , also play a role in the conditioning of the immune response. thus, t cell epitopes may be necessary to drive immune response, but are not sufficient. co-stimulatory molecules that provide a second signal, the right cytokine milieu and other factors directing the nature (th1 vs. th2) of the immune response, are also crucial (shahinian et al. 1993; kuchroo et al. 1995) . adjuvants provide this added 'boost' in the context of vaccines. the same range of delivery vehicles that exist for conventional vaccines can be used for the development of idvs and epitope-based idvs. for example, idvs and epitope-based idvs can be formulated and delivered as pseudo-proteins or peptides in a carrier vehicle such as a liposome or viral-like protein (vlp); alternatively, the sequence of the idv antigens or epitope string can be inserted into a viral or bacterial vector such as adenovirus or salmonella; alternatively, a dna vaccine construct encoding the antigen(s) or epitopes can be developed. the choice of adjuvants for use in humans is relatively extensive and each adjuvant has advantages and disadvantages. the advantages and disadvantages of each type of vaccine delivery vehicle and adjuvant here is beyond the scope of this chapter; readers are referred to a review by fraser et al. (2007) . the next step in the development of epitope-driven idv is to determine whether immunization provides competent immune response. the idv or epitope-based idv is administered and immune responses to the components are evaluated following immunization. even though a range of animal models are used for the evaluation of vaccines, results from immunogenicity studies in these models should be interpreted with caution. although their functions may be similar, the mhc of mice, rodents, and non-human primates differ from human mhc (known as hla in the context of human immune response) at the amino acid level and these differences effect which epitopes can be presented. this helps to explain why different strains of mice (balb/c, c57bl/6) have different immune responses to pathogens as well as vaccines for those pathogens (klitgaard et al. 2006) . in particular, epitope-based vaccines that are developed using predicted human t cell epitope mapping tools can be tested only in murine models that are hla transgenic. fortunately, a number of transgenic mouse strains that express the most common hla a, hla b and hla dr, molecules have been developed. t cell responses in these mice correlate directly with t cell responses observed in infected/vaccinated humans (man et al. 1995; shirai et al. 1995) . hla transgenic mice are now routinely used to assay and optimize (human) epitopedriven vaccines in pre-clinical studies (ishioka et al. 1999; charo et al. 2001; livingston et al. 2001) . despite the limited number of hla class ii alleles for which tg mice have been developed, comparisons of immunogenicity can be done to a high degree of accuracy in the mouse model for selected hla class ii alleles (hla dr 0101, 0301, 0401, 1501). unfortunately, it appears these mice may have difficulty breeding due to poorly understood consequences of their transgenic heritage, limiting the use of this important model system. the final step in the development of any vaccine is experimental validation of the immunogenicity and protective efficacy of computationally selected antigens. currently, a series of experimental vaccines have shown efficacy in animal models and several idvs are being tested in clinical studies. in the context of infectious disease, genome-derived vaccines that have progressed furthest along the vaccine development pipeline are generally based on whole proteins rather than epitopes (rappuoli and covacci 2003; pizza et al. 2000) . however, epitope-based idvs are currently being developed for a range of infectious diseases by the authors' laboratory and by many others (depla et al. 2008 ). while it is common knowledge that subunit-based vaccines can protect against infection, similar success with epitope-based approaches is not as widely known. in addition to the studies previously cited, immunization of balb/c mice with three doses of a peptide construct containing an h-2(d)-restricted cytotoxic t lymphocyte (ctl) epitope from a murine malaria parasite induced both t cell proliferation and a peptide-specific ctl response mediating nitricoxide-dependent elimination of malaria-infected hepatocytes in vitro, as well as partial protection of balb/c mice against sporozoite challenge (franke et al. 2000) . in a separate study, immunization of balb/c and cba mice with measles virus ctl epitopes resulted in the induction of epitope-specific ctl responses and conferred some protection against encephalitis following intracerebral challenge with a lethal dose of virus (schadeck et al. 1999) . these are just a few successful examples of many studies carried out in animal models; however, translation to prevention of disease in humans has been difficult to achieve. in contrast with whole-protein subunit vaccines, idvs and epitope-based idvs have taken longer to make the transition from animal model to the clinic, mainly due to the novelty of the concept and perhaps unfounded concerns that epitopes are not sufficient for the generation of effective immune response. cancer therapy is an exception to this rule. as previously described, eptiopebased idvs have been evaluated in the context of therapy against chronic infection or cancer (ueda et al. 2004; valmori et al. 2003) . in the cancer vaccine field, where the concept of epitope-driven vaccines is well established, many more peptide vaccines have successfully passed pre-clinical tests and are currently in phase i/ii clinical trials (pietersz, pouniotis and apostolopoulos 2006) . new approaches are emerging, which may improve the success rate and, indeed, the results from recent clinical trials prove the principle. one approach is to identify epitopes that are unique to the tumor (prostate, lung, colon) and to pre-screen the patient for response to the peptide. this approach, called personalized vaccination, takes into account the diversity of ctl epitope recognition among patients. whereas the response rates to classical (non-personalized) peptide vaccines have been disappointing, responses to personalized vaccines (in a phase i trial, conducted in japan) have been as high as 11.1% in the advanced cancers and equal to or more than 20% in malignant glioma and cervical cancers, respectively (itoh and yamada 2006) . it is noteworthy that just a few epitope-driven vaccines against viral and microbial pathogens have reached the stage of phase i or ii efficacy trials in humans. for example, bionor immuno's hiv p24 gag peptide vaccine (vacc-4x) was demonstrated to be safe and well tolerated in phase i trials (asj¨o et al. 2002) and dose-dependent and immunogenic in phase ii trials in norway (kran et al. 2004) . similarly, nardin's epitope-based vaccine for malaria is moving along the clinical trial pathway (nardin et al. 2000) . in this section, we describe the immunomics tools developed and used by the epivax vaccine development group in recent collaborations with dr. steve gregory of lifespan, dr. ousmane koita of the university of bamako, mali and dr. david weiner of university of pennsylvania. t cell epitopes are linear peptides that bind to mhc molecules. binding is mediated by the interactions between the r-groups of the amino acids in the peptide ligand and the pockets on the floor of the mhc binding groove. because the mhc:peptide interaction is well characterized, pattern-matching algorithms can be used to screen protein sequences for peptides that will bind mhc. the authors of this report currently use the epimatrix system, a suite of epitope-mapping tools that has been validated by more than a decade of use in selecting putative epitopes for in vitro and in vivo studies (see references (de groot et al. 1997; bond et al. 2001; dong et al. 2004; mcmurry et al. 2005; koita et al. 2006) . the epimatrix algorithm is based on a set of class i and class ii hla matrices wherein individual frequencies of all 20 amino acids (aa) in each hla pocket position are applied to the prediction of overlapping 9-and 10-mer peptides. in a typical analysis, protein antigens are parsed into overlapping 9-mer frames where each 9-mer overlaps the last by eight amino acids. each 9-mer is then scored for predicted binding affinity to one or more class i or class ii hla alleles. in order to compare potential epitopes across multiple hla alleles, epimatrix raw scores are converted to a normalized 'z' scale. peptides scoring above 1.64 on the epimatrix 'z' scale (typically the top 5% of any given sample) are likely to be mhc ligands. since class ii epitopes can be promiscuous, our approach to the prediction of class ii epitopes is to estimate the binding potential of each frame with respect to each of a panel of eight common class ii alleles (drb1*0101, *0301, *0401, *0701, *0801, *1101, *1301, and *1501). taken together, these alleles 'cover' the genetic backgrounds of most humans worldwide (southwood et al. 1998 ) and they also represent the predominant types of 'pockets' for the most common mhc. recently, the epimatrix system has been utilized to measure the potential immunogenicity of whole proteins. in this context, epimatrix assesses the aggregate epitope density of a given protein with respect to the aggregate epitope density of a set of randomly generated pseudo-protein sequences of similar size (de groot 2006) . by correcting for the size and expected epitope density, the potential immunogenicity candidate vaccine antigens can be directly compared. further immunogenicity of low scoring proteins may be enhanced by modifying the immunogenic region amino acid sequence so that it contains more t cell epitopes (see illustration of this approach in the hpv vaccine section, below). peptides predicted to bind to multiple hla alleles are known as promiscuous t cell epitopes. the clustimer algorithm is used to scan the output produced by the epimatrix and identifies the polypeptides predicted to bind to an unusually large number of hla alleles. briefly, the scores of each analyzed 9-mer are aggregated. high-scoring 9-mers are then extended at the n-and c-terminal flanks until the predicted epitope density of the promiscuous epitope falls below a given threshold value. this particular approach to mapping epitopes has also been useful for discovering 'epibars,' which may be a signature feature of highly immunogenic, promiscuous class ii epitopes. an example of a promiscuous t cell cluster containing an epibar (tetanus toxin 830-844) is shown in fig. 1 . a single t cell epitope 'cluster' usually ranges from 9 to about 25 amino acids in length and can contain anywhere from 4 to 40 binding motifs. using epimatrix as described above and clustimer, scores above 10 and, in particular, scores above 15 indicate significant immunogenic potential (de groot 2006) . note the horizontal bar of high z scores at position 308 in fig. 3 . having observed this 'epibar' pattern to be characteristic of promiscuous epitopes, the authors have integrated the pattern into the prospective selection of clusters. promiscuous epitopes also exist, to a certain degree, for class i alleles. some laboratories have demonstrated cross-presentation of peptides within hla 'superfamilies' (such as the a3 superfamily: a11, a3, a31, a33, and a68) described by . the authors have confirmed cross-mhc binding and presentation to t cells in our hiv vaccine studies ). one limitation of conventional vaccination, and to a lesser extent natural infection, is that the immune system focuses strongly on the most mutable immunogen of the virus -typically, the viral envelope. in the case of hiv and other viruses, vaccination with more conserved, subdominant epitopes has been shown to circumvent this hierarchy and potentiate cross-strain protection (ostrowski et al. 2002; nara and lin 2005) . in like manner, a conserved t helper-directed vaccine may provide a more 'democratic' way of stimulating immune response, increasing the number targets for t cell recognition, thereby providing t help to antibody response despite potential viral variability (santra et al. 2002; subbramanian et al. 2003; scherle and gerhard 1988; scherle and gerhard 1986; russell and liew 1979; johansson et al 1987) . the genetic variability of some pathogens constitutes a significant challenge to the efforts to design a vaccine driven by cellular immune response de groot et al. 2002) . the authors have been involved in developing an hiv-1 vaccine that includes highly conserved (cross-clade) t cell epitopes. the conservatrix algorithm, developed for this application, parses input sequences into component strings (the lengths of the strings may be determined ) indicates the potential of a 9-mer frame to bind to a given hla allele. all z scores in the top 5% (>1.64) are considered 'hits'. though not hits, scores in the top 10% are considered elevated; scores below 10% are masked for simplicity. frames containing four or more alleles scoring above 1.64 are colloquially referred to as 'epibars' (see frames 831:yikanskfi and 832: ikanskfig). this band-like pattern is characteristic of promiscuous epitopes. the tetanus toxin peptide scores are extremely high for all eight alleles in epimatrix; the deviation compared to expectation is + 23.82 by the operator) and then searches the input dataset for matching segments. conservatrix may be used to compare strings derived from different strains of the same organism (hepatitis c, for example, or hiv) or to search a given sequence for a user-supplied target sequence. target sequences may be input as specific sequences or as coded patterns. thus, the operator can use 'wild cards', allowing for one or more of the amino acid residues in any given peptide sequence to be any amino acid [x], or a limited set of amino acids such as [l, v] . results of each analysis are stored in a database and may be browsed or exported to another program for analysis. by selecting highly conserved epitopes, regardless of their distance from the ancestral hiv-1 genome, we have identified sequences conserved for structural and functional reasons and are therefore less likely to be modified in the course of further evolution of hiv-1 (peyerl et al. 2004; koibuchi et al. 2005 ). the problem of virus variability also significantly complicates the selection of epitopes that have a population-coverage advantage; such epitopes are termed 'clustered,' 'superfamily,' or 'promiscuous.' to address this problem, the authors developed epiassembler ) to identify sets of overlapping, conserved, and promiscuously immunogenic epitopes and assemble them into extended immunogenic consensus sequences (ics) (see fig. 2 ). in theory, proper processing and presentation of these sequences would allow for the presentation of highly conserved peptides in the context of more than one mhc. the resulting peptide is not a 'pseudosequence' as such, since each constituent epitope occurs in its corresponding position in the native protein. thus, while the full-length 'immunogenic consensus sequence' is not necessarily found in any one variant sequence, the peptide is more representative of the sequence universe. in the case of hiv, for example, we used the ics approach to design a peptide-based vaccine. while the full-composite ics peptides happen to be exactly conserved in a few individual strains of hiv, each peptide represents a significant percentage of circulating strains, because every constituent overlapping epitope is conserved in a large number (range 893 to 2,254) of individual hiv-1 strains. as compared with immunogenic consensus sequences, randomly selected counterparts, on average, contain half as many binding motifs and cover a third fewer isolates. to develop vaccines of equivalent antigenic 'payload,' using conventional methods would be prohibitively expensive as it would require including multiple different variants of each antigen. this approach has been useful for identifying highly immunogenic epitopes for hiv vaccine design . by focusing on conserved, mhc-promiscuous t helper epitopes, the ics approach has the potential to efficiently overcome the genetic variability of both virus and host. one of the advantages of idv is that it is possible to omit deleteriously crossreactive epitopes. perhaps, the most famous example of an adverse effect due to cross-reactivity with self was observed following vaccination for lyme disease with the osp a protein. the vaccine has been recently re-engineered with the cross-reactive epitope removed (willett et al. 2004 ). in the context of our own work, peptides selected for in vitro evaluation are evaluated for homology with human proteins by blasting the sequences against the human sequence database at genbank (http://www.ncbi.nlm.nih.gov/). blastimer automates the process of submitting sequences to the websites featuring search engines such as the blast engine at ncbi (www.ncbi.nlm.nih. gov/blast). by default, blastimer blasts sequences against all non-redundant x x x x x x fig. 2 the ics assembly operation was performed using epiassembler (bill martin, epivax, 2004) . left panel: each variant strain is first analyzed and a highly conserved, putatively promiscuous 9-mer is chosen as the core peptide. mismatches with the selected epitope sequences are represented with the letter x. right panel: additional epitopes are then identified, which overlap with the natural n-and c-terminal flanking regions of the core 9-mer epitope. the overlap length requirement is decreased by one amino acid iteratively until reaching a minimum of three overlapping amino acids. if more than one suitable overlap is identified, the overlapping peptide with the higher overall epimatrix rank is selected. this process is repeated using the extended peptide as the new core sequence. the cycle can be repeated for the length of an entire protein or can be truncated when the peptide reaches a length that can be easily produced synthetically genbank cds translations, pdb, swissprot, pir, and prf. blastimer assesses the homology between the submitted sequence and the sequence of proteins of other organisms. patent blast, on the other hand, targets a database of sequences gleaned from patents. users of either program may control all of the submission options available to interactive users at ncbi. in both cases, results are recorded in a database and can be browsed, exported, or summarized and rendered in a report format. according to the authors' standard practice, any peptide that shares greater than 80% identity with peptides contained in the human proteome is eliminated from consideration in a vaccine. a number of methods for enhancing epitope-based vaccines have been described and implemented (thomson et al. 1998; rodriguez and whitton 2000) . one approach is to align the epitopes in a protein or dna vaccine construct as a 'string of beads' without any intervening sequences or 'spacers' in a dna plasmid encoding the individual epitopes . however, the lack of 'natural flanking sequences' -has raised concern that their proteolytic processing may be compromised, and that junctional epitopes, peptides other that the specific peptides of interest, may be generated as a result of processing (godkin et al. 2001) . to address this concern, the authors developed vaccine-cad (see fig. 3 fig. 3 vaccine-cad, illustrated with three sample epitopes represented by the words 'create,' 'new,' and 'epitopes.' the default arrangement of the words results in unintended sequences, represented by the words 'eaten' and 'ewe,' at the junctions between the intended epitopes. reiterative modifications in the arrangement of the epitopes results in the development of a sequence that has no 'pseudoepitopes' (new epitopes that were not intended) at the junctions of the juxtaposed epitopes junctional epitopes, the insertion of spacers and breakers, the requirements for secretion or processing tags, and the evaluation of epitope strings for potential homologies to human protein fragments. an important component in the epitope-driven vaccine process is the selection of epitopes from the regions of pathogens that are presented by mhc molecules for t cell recognition. 'mhc binding motifs,' first identified by r¨otzschke and falk, are the patterns of amino acids in peptides that are known to promote the binding of peptides containing these patterns to the mhc molecules on the surface of apcs rotzschke et al. 1991) . different mhc molecules have different binding motifs, limiting the set of mhc ligands that can be presented in the context of any given mhc. while consideration of hla alleles may lead to concern about the selection of epitopes for broad coverage of populations, gulukota and delisi (1996) and sette and sidney (1998) have demonstrated that epitope-based vaccines that contain epitopes restricted by selected 'supertype' hla can provide the broadest possible coverage of the human population. furthermore, recent studies by brander and walker indicate that there may be even greater flexibility in the binding of epitopes to mhc than previously recognized; this is also consistent with the data recently presented by frahm et al. (2004) . the inclusion of 'promiscuous epitopes' -epitopes that are recognized in the context of more than one mhc (paina-bordignon et al. 1989; de groot et al. 1991; sette and sidney 1998) in epitope-driven vaccines may therefore overcome the challenge of genetic restriction of immune response. in addition, the repertoire of possible mhc-restricted epitopes recognized by an individual's t cells has been shown to be quite variable, even between hla-matched individuals (jameson, cruz and ennis 1998; gianfrani et al. 2000; betts et al. 2001 ). aggregatrix, a new algorithm that was recently developed at epivax, iteratively searches for the combination of epitopes that achieves maximal cross-clade representation. the authors performed this analysis for our hiv epitopes, as shown in fig. 4 , evaluating each hla-a2-restricted hiv peptide individually and the set in aggregate for coverage of hiv-1 strains by year (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) , by country of origin, and by clade. as can be seen in this figure, the set of highly conserved a2-restricted peptides we have tested and confirmed in elispot assays covered between 54% and 86% of strains in a given year, between 33 and 100% of strains in a given country, and between 0% and 100% of strains in a given clade. the hla a2 peptides cover 85, 78, 78, and 80% of clades a, b, c, and d, respectively. this is a remarkable breadth of coverage for a limited set of coverage of gaia hiv b7 peptides--individually and in aggregate-across time, countries, and clades fig. 4 gaia hiv a2 peptides -individually and in aggregate -percentage coverage of strains by year, country, and clade. each row of the matrix denotes a specific peptide; the peptide's protein of origin is included within the peptide id. each column of the matrix denotes a specific year, country, or clade, grouped as indicated. the percentage coverage of strains is represented on a color gradient, with warm tones indicating values above 50% and cool tones indicating values below 50%. the bottom row of the matrix shows the percentage of each protein set that contains at least one peptide from our pool. black boxes indicate that no isolates of the protein are available for that year, clade, or country. the bottom row represents the aggregate percent coverage for the epitope set. each cell of the matrix represents the percentage coverage per peptide, except for the bottom row cells, which represent the aggregate percentage coverage for the peptide set. column headers are listed here for space considerations: left to right, the year columns are 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, and 2005 ; aggregate coverage of strains by year ranges from 47% (2000) hla a2 epitopes, given the well-known ability of hiv to mutate away from hla (nguyen et al. 2004; iversen et al. 2006) . in studies of immune response to therapeutic proteins, the authors have observed that subject-to-subject variation in t cell response closely relates to (1) subject hla type and (2) the number of protein-derived peptides that match the subjects hla. to describe this relationship, the authors developed a metric that may be useful in the development of epitope-based vaccines or in the clinical assessment of immune response to vaccines, called the 'individualized t cell epitope measure' or item. the item can be calculated for each subject who is responding to a given epitope by summing the epimatrix z-scores for each positive peptide for each hla allele in a given subject's haplotype, thus: item score ¼ ½epimatrix score of peptide for hla type 1 þ ½epimatrix score of peptide for hla type 2 þ . . . the same calculation can be performed for larger peptides and proteins by summing all of the scores for the subject's hla type. this calculated score allows for the individualized potential immunogenicity to be predicted, based on the number of putative epitopes contained in a protein that might be presented to their t cells, based on their hla haplotype. using this score, it is possible analyze the contribution of haplotype to the corresponding t cell response. in our prospective evaluations, significant correlations were found between the ifn-gamma response to a given antigen and the item scores for individual subjects (for data published in koren et al. (2007) r= 0.69, p= 0.0134). in addition, correlations between the item score and patient hla were also observed for their antibody titers. further evaluations of this method for predicting individual responses to vaccines and therapeutic proteins are in progress. defining the immunome by identifying t cell epitopes and confirming their immunogenicity is but the first step of vaccine development. a number of conditions extrinsic to the mhc-ligand interaction may influence the final composition of the epitope ensemble. for example, whether or not a predicted epitope is confirmed is related to: (1) the quantitative expression of the source protein; (2) the number of different epitopes derived from this protein, which are presented on the surface of the apc (wherry et al. 1999) ; (3) the amino acids that flank the epitope (bergmann et al. 1994; shastri, serwold and gonzalez 1995; livingston et al. 2001) ; and (4) proper cleavage and trimming by proteolytic enzymes in the proteasome and processing pathways (van kaer et al. 1994; york et al. 1999; chen et al. 2001; toes et al. 2001) . for example, it is very likely that end-to-end epitope presentation, such as was used in the design of the oxavi hiv gag/epitope vaccine, impaired the presentation of the epitopes in immunogenicity studies. vaccine-cad also takes into account the role of flanking residues: studies conducted in murine models have demonstrated that residues flanking an mhc class i epitope strongly influence the delivery of the intact epitope to tap following proteasome degradation (thomson et al. 1995; hozhutter, frommel and kloetzel 1999; mo et al. 1999 ). in addition, livingston et al. (2001) have tested a standard spacer sequence (-gpgpg-) for vaccine constructs consisting of mhc-ii-restricted, th-cell epitopes; the use of this spacer disrupts junctional epitopes that might compete for degradation or for mhc binding (both g and p are unusual carboxy-terminal anchors for a peptide that binds to class ii mhc). this approach has been used for constructs with up to 20 epitopes, in assays where responses were detected to the majority of epitopes (livingston et al. 2001). 6 methods of confirming idv 6.1 two case studies francisella tularensis is a zoonotic bacterium. it is endemic to certain communities such as martha's vineyard, massachusetts, usa, where it is known as rabbit fever. tularemia represents a potentially dangerous biological weapon owing to its high degree of infectivity, ease of dissemination, and capacity to cause severe illness. despite several decades of research, no vaccine for tularemia is licensed for public use. for a review of tularemia vaccines, see mcmurry et al., 2007b. we have been actively developing an epitope-based tularemia vaccine combining computational immunology with in vitro and in vivo validation (mcmurry et al 2007a) . the starting point of our vaccine was the fully annotated f. tularensis subsp. tularensis (schus4) genome published in by larsson et al. (2005) . a prototype vaccine containing only class ii epitopes has been tested in challenge studies in hla drb1*0101 transgenic mice. for this vaccine, the epimatrix algorithm was utilized to identify highly promiscuous t cell epitopes within the tularemia genome. twenty-five class ii-restricted epitopes were selected, synthesized, and screened in vitro using a recombinant soluble hla class ii competition-binding assay (described above). peptides that bound with high affinity were then tested ex vivo in elispot assays with blood obtained from f. tularensis subsp. tularensis-exposed individuals. search-light analysis was also performed on supernatants derived from human cell culture stimulated with peptide using a panel of nine cytokines. forty-two percent of peptides bound to drb*0101 and are likely to bind to several other alleles (in that they were predicted using the clustimer algorithm). elispot assays showed positive ifn-gamma responses to 21/25 individual peptides and to peptide pools in nearly all of the 23 human study. the number of epitopes recognized per subject ranged from 1 to 17 and averaged 4 per subject; not every peptide was tested for every subject. peptides that elicit a robust memory response, as evaluated by these various assays, were incorporated into a vaccine construct and tested in challenge studies with hla transgenic mice as a possible vaccine against tularemia. immunogenicity studies in hla transgenic drb1*0101 mice were performed using the multi-class ii-epitope dna constructs and/or peptides representing the epitopes, and t cell responses were evaluated in ifn-gamma elispots. hla dr1 transgenic mice were challenged with five times the ld50 of f. tularensis lvs; 57% of vaccinated mice survived, all non-vaccinated mice died (manuscript in preparation). this result demonstrates the potential for a genome-derived epitope-based vaccine to protection from a class a bioterror pathogen. importantly, the protection we observed is accounted for by only 10 of the 14 schus4 epitopes in the vaccine, as they were conserved in the lvs challenge strain. the schus4-specific epitopes that were found to be significantly immunogenic would further contribute to protection in a schus4 challenge. this result is consistent with other findings that a limited set of epitopes may be sufficient to induce a protective immune response (moutaftsi et al. 2006) . studies using schus4 (the wild type tularemia) are planned. the results obtained to date appear to indicate that vaccine design that originates with the whole genome may lead to the development of a protective epitope-based vaccine. cervical cancer is the second leading cause of death afflicting women worldwide; 40% of cervical patients develop persistent, recurrent, or widely metastatic disease. while a preventive vaccine now exists for hpv, there is a need for a therapeutic vaccine to treat existing cases of hpv, especially in resource-poor areas where access to the preventive vaccine is limited. cellular immune responses are believed to be critical for effective immune response to cancer; accordingly, epivax is pursuing the development of an 'immunotherapeutic vaccine' which would focus on the proteins primarily expressed either before or during carcinogenesis (e1 and e2, e6, and e7). in order to maximize immunogenicity across hpv subtypes, our strategy involves analyzing variant strains of hpv protein sequences, using (1) epimatrix to identify class i and class ii hla motif matches, (2) conservatrix to identify those motif matches that are conserved, and (3) epi-assembler to weave together the conserved immunogenic sequences into a full immunogenic consensus sequence (ics) antigen. a full ics vaccine antigen would retain the fundamental structure of its naturally occurring counterparts; however, it will contain more and better epitopes than would occur in any such one counterpart. the authors have identified five conserved epitopes in e1 and e2, which have stimulated significant responses in elispot ifn-g assays. in the proposed vaccine, these epitopes and others will be incorporated in their natural context within the proteins, which could be delivered as dna, proteins, or a primeboost combination. by preserving the natural flanking regions surrounding our epitopes, we hope to retain, in large part, the natural processes surrounding hpv protein degradation, transport, and presentation as they occur during natural infections. the ics approach described here is the same as that illustrated in fig. 5 , but extending over the full natural length of the protein. for a more in-depth review of a similar approach being pursued for influenza, see mcmurry et al. (2008) . the epivax hpv vaccine illustrates yet another aspect of vaccine design: 'megatope' proteins, re-engineered to increase the epitope content. this approach already had some success (okazaki et al. 2006) . in the case of variable viruses such as hcv, influenza, and hiv, one limitation of conventional vaccination, and of natural infection, is that the immune system often focuses strongly on the most mutable immunogens. idvs can be constructed from alternative antigens, which are more conserved or more protective, circumventing this problem (russell and liew 1979; scherle and gerhard 1986; scherle and gerhard 1988; santra et al. 2002; subbramanian et al. 2003) . amino-acid sequences of ns3 proteins from available hpv isolates identification of immunogenic and conserved t-cell epitopes using conservatrix, epimatrix. (epitopes represented by open symbols) ns3 protein is optimized to incorporate the most conserved immunogenic epitopes in their natural context within the protein. illustration of our novel strategy to generate an hpv vaccine candidate. the putative epitopes identified during this analysis will be combined to form several consensus sequences, which retain the fundamental structure of these hpv proteins but which also contain an unnaturally large number of conserved t cell epitopes in addition, broadening the t cell repertoire might make it possible to impair viral escape and decrease viral loads sufficiently to disrupt transmission. epitope-driven vaccines also offer distinct advantages over vaccines encoding whole protein antigens, since epitopes are safe and can be packaged into relatively small delivery vehicles. the epitope-driven approach offers platform independence: a delivery vehicle (peptide, dna, multi-epitope construct) can be modified or selected midway into the development process. multiple conserved epitopes, in addition to augmenting the efficacy of a preventive vaccine, could provide a broad and universal cellular immunity, known to be crucial for containment of infection, although perhaps ineffective for protection against infection. despite these advantages, there are a number of reasons that a given pathogen-directed, epitope-based vaccine might fail to reach clinical trials or protect humans: (1) the limited number of epitopes expressed by the vaccine (i.e., poor payload quantity); (2) limited conservation of epitopes (leading to limited coverage of variant clinical isolates) (3) the limited hla population coverage (i.e., poor payload quality); (4) suboptimal vaccine delivery; and/or (5) the dearth of suitable animal models. in addition, the concept of epitope-driven vaccines is relatively novel. complete genome sequences have been available for only a little more than a decade now and the tools to process the data for vaccine design are only newer. experimental validation needed to push forward these vaccines into clinical trials is now emerging and promises to enable epitope-based vaccines to claim a prominent place in the vaccine world. the technologies needed to identify immunostimulatory antigens and epitopes from pathogen genomes are already well developed. the principle focus of future research in this area will likely be in fine-tuning these technologies and expanding them to tailor immune responses in individuals. for example, development of epitope mapping algorithms for dq and dp class ii hla alleles will make it possible to completely characterize immunomes. this information will make it possible to generate comprehensive individual t cell epitope measures (item) based on an individual's hla genetic make-up and allow researchers to identify a priori clinically important epitopes and screen clinical cohorts for subjects that are more likely to develop targeted immune responses. furthermore, genome-mapping tools that are currently available are not yet useful for discovering b cell epitopes, whether from proteins or from nonprotein components such as carbohydrates or lipid antigens. immunoinformatics tools that are currently available cannot be used to accurately predict conformational (b cell) epitopes that interact with antibody, although such tools are being refined (enshell-seijffers et al. 2003) . thus, the immunogens identified using in silico approaches must be evaluated in vitro and also in appropriate challenge models, prior to progressing to vaccine trials. protective immune response probably also involves some engagement of the innate immune system; it has been impossible to differentiate between effective and non-protective epitopes. cytokine milieu may affect the outcome of immunization; thus, a limited number of toll-receptor agonists (imler and hoffmann 2001) have been identified and these are under study in conjunction with idvs. in the future, toll-receptor signaling 'pathogen-associated molecular patterns' (pamps) might also be modeled and selected using immunoinformatics tools. besides antigen identification, the success of idvs relies heavily on delivery technologies. these areas continue to independently mature and provide important lessons to epitope-based vaccine design. the major areas of research to watch include biological macromolecule (including cytokines), lipopeptide, and polysaccharide adjuvants and particulate (liposomes, exosomes, virosomes, nanoparticles) and cell-based delivery systems. the development of safe and effective vaccines against emerging infectious diseases such as influenza, both seasonal and pandemic, hiv, and tb, in addition to cancers associated with infectious pathogens such as hbv and hcv, is an urgent and achievable public health priority. in addition, vaccines for the prevention and treatment of cancer hold enormous promise for human health. the threat of bioterrorism following the events of september 11, 2001, provided vaccinologists with a persuasive argument for more rapid development of vaccines against viral and bacterial pathogens that are now included on the nih category a-c biopathogen list (http://www3.niaid.nih.gov/topics/ biodefenserelated/biodefense/research/cata.htm). 'emerging infectious diseases' were added to the vaccine wish list following the outbreak of severe acute respiratory syndrome (sars) in guangdong china in 2002. indeed, only a few months following the publication of the sars-coronavirus (sars-cov) genome (marra et al. 2003; rota et al. 2003) , researchers began to map vaccine components using new bioinformatics and immunoinformatics tools, coupled with improved immunology techniques and specialized animal models. new vaccines based on this approach are currently being evaluated in animal models, less than a year from the start of the epidemic. future vaccine approaches may need to move away from 'whole' protein vaccines for a wide range of reasons. multiple antigen or epitope vaccinations such as the approach illustrated here could be one way to elicit the sort of strong th1 response necessary to pathogens following infection, in the context of a therapeutic vaccine. this approach could also be useful for a wide range of pathogens for which genomes have been partially or completely mapped. as described in this chapter, our group is actively pursuing the development of epitope-driven vaccines for hiv koita et al. 2006) , franciscella tularensis, helicobacter pylori, and smallpox. we have progressed from genome-derived epitope mapping to challenge studies in less than one year for some of these vaccine development programs. epitope-based and whole antigen idvs are now just beginning to enter clinical trials, but this relative disadvantage may be cured with the tincture of time. one reason for the relative paucity of idvs in clinical development is that the immunoinformatics tools for developing these vaccines have really only evolved in the last 10 to 15 years. the average length of time to develop a vaccine may be 20 years or more. while immunoinformatics tools are useful for accelerating the discovery and pre-clinical stage of vaccine development, testing vaccines in animal models and developing clinical trials is a lengthy process. it is likely that idv and epitope-based idv will begin to enter clinical trials and emerge on the market in greater numbers in 5 to 10 years. high-affinity t helper epitope induces complementary helper and apc polarization, increased ctl, and protection against viral infection quantitative and qualitative analyses of the immune responses induced by a multivalent minigene dna vaccine a multivalent minigene vaccine, containing b-cell, cytotoxic t-lymphocyte, and th epitopes from several microbes, induces appropriate responses in vivo and confers protection against more than one pathogen phase i trial of a therapeutic hiv type 1 vaccine, vacc-4x, in hiv type 1-infected individuals with or without antiretroviral therapy analysis of endogenous peptides bound by soluble mhc class i molecules: a novel approach for identifying tumor-specific antigens differential effects of flanking residues on presentation of epitopes from chimeric peptides analysis of total human immunodeficiency virus (hiv)-specific cd4(+) and cd8(+) t cell responses: relationship to viral load in untreated hiv infection an hla-directed molecular and bioinformatics approach identifies new hla-a11 hiv-1 subtype e cytotoxic t lymphocyte epitopes in hiv-1-infected thais dna immunization of hla transgenic mice with a plasmid expressing mycobacterial heat shock protein 65 results in hla class i-and ii-restricted t cell responses that can be augmented by cytokines immunoproteasomes shape immunodominance hierarchies of antiviral cd8(+) t cells at the levels of t cell repertoire and presentation of viral antigens uneven distribution of mhc class ii epitopes within the influenza virus immunomics: discovering new targets for vaccine and therapeutics from genome to vaccine -new immunoinformatics tools for vaccine design from immunome to vaccine: epitope mapping and vaccine design tools engineering immunogenic consensus t helper epitopes for a cross-clade hiv vaccine from genome to vaccine: in silico predictions, ex vivo verification human immunodeficiency virus reverse transcriptase t helper epitopes identified in mice and humans: correlation with a cytotoxic t cell epitope an interactive web site providing major histocompatibility ligand predictions: application to hiv research hiv vaccine development by computer assisted design: the gaia vaccine immunoinformatics: mining genomes for vaccine components rational design of a multiepitope vaccine encoding t-lymphocyte epitopes for treatment of chronic hepatitis b virus infections hla-a2-restricted cd8+-cytotoxic-t cell responses to novel epitopes in mycobacterium tuberculosis superoxide dismutase, alanine dehydrogenase, and glutamine synthetase proteomics in vaccinology and immunobiology: an informatics perspective of the immunone phase i trial of a cd8+ t cell peptide epitope-based vaccine for infectious mononucleosis the mapping and reconstitution of a conformational discontinuous b-cell epitope of hiv-1 allele-specific motifs revealed by sequencing of self-peptides eluted from mhc molecules consistent cytotoxic-t-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities a subdominant cd8(+) cytotoxic t lymphocyte (ctl) epitope from the plasmodium yoelii circumsporozoite protein induces ctls that eliminate infected hepatocytes from culture improving vaccines by incorporating immunological coadjuvants new cd4+ and cd8+ t cell responses induced in chronically hiv type-1-infected patients after immunizations with an hiv type 1 lipopeptide vaccine lipoarabinomannan induced cytotoxic effects in human mononuclear cells human memory ctl response specific for influenza a virus is broad and multispecific naturally processed hla class ii peptides reveal highly conserved immunogenic flanking region sequence preferences that reflect antigen processing rather than peptide-mhc interactions the structural requirements for class ii (i-ad)-restricted t cell recognition of influenza hemaglglutinin: b cell epitopes define t cell epitopes hla allele selection for designing peptide vaccines construction of a francisella tularensis two-dimensional electrophoresis protein database a theoretical approach towards the identification of cleavage determining amino acid motifs of the 20s proteasome toll receptors in innate immunity utilization of mhc class i transgenic mice for development of minigene dna vaccines encoding multiple hla-restricted ctl epitopes personalized peptide vaccines: a new therapeutic modality for cancer conflicting selective forces affect t cell receptor contacts in an immunodominant human immunodeficiency virus epitope human cytotoxic t-lymphocyte repertoire to influenza a viruses antigen-presenting b cells and helper t cells cooperatively mediate intravirionic antigenic competition between influenza a virus surface glycoproteins tuberculosis and leprosy: attempts to identify t-cell antigens of potential value for vaccine design reduced susceptibility of recombinant polyclonal antibodies to inhibitory anti-variable domain antibody responses limited sequence evolution within persistently targeted cd8 epitopes in chronic human immunodeficiency virus type 1 infection confirmation of immunogenic consensus sequence hiv-1 t cell epitopes in bamako clinical validation of the ''in silico'' prediction of immunogenicity of a human recombinant therapeutic protein hla-and dose-dependent immunogenicity of a peptide-based hiv-1 immunotherapy candidate (vacc-4x) the role of cpg dinucleotides in dna vaccines b7-1 and b7-2 costimulatory molecule activate differentially the th1/th2 developmental pathways: application to autoimmune disease therapy cep: a conformational epitope prediction server the complete genome sequence of francisella tularensis, the causative agent of tularemia the kinetic stability of mhc class ii peptide complexes is a key parameter that dictates immunodominance identification of hexon-specific cd4 and cd8 t-cell epitopes for vaccine and monotherapie optimization of epitope processing enhances immunogenicity of multiepitope dna vaccines definition of a human t-cell epitope from influenza a non-structural protein 1 using hla-a2.1 transgenic mice tumorassociated antigens identified by mrna expression profiling induce protective anti-tumor immunity diversity of francisella tularensis schu4 antigens recognized by t lymphocytes after natural infections in humans: identification of candidate epitopes for inclusion in a rationally designed tularemia vaccine a call to cellular and humoral arms: enlisting cognate t cell help to develop broad-spectrum vaccines against influenza a tularemia vaccinesan overview analyzing mycobacterium tuberculosis proteomes for candidate vaccine epitopes characterization of t-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a t-cell epitope distinct proteolytic processes generate the c and n termini of mhc class i-binding peptides a consensus epitope prediction approach identifies the breadth of murine t(cd8+)-cell responses to vaccinia virus hiv-1: the confounding variables of virus neutralization synthetic malaria peptide vaccine elicits high levels of antibodies in vaccines of defined hla genotypes frequent human leukocyte antigen class i alleles are associated with higher viral load among hiv type 1 seroconverters in thailand epitope enhancement of a cd4 hiv epitope toward the development of the next generation hiv vaccine efficient protection against mycobacterium tuberculosis by vaccination with a single subdominant epitope from the esat-6 antigen identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp5 ectodomain universally immunogenic t cell epitopes: promiscuous recognition by t cells the immunome computational immunology: the coming of age fitness costs limit viral escape from cytotoxic t lymphocytes at a structurally constrained epitope design of peptide-based vaccines for cancer identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing the immunodominant influenza matrix t cell epitope recognized in human induces influenza protection in hla-a2/k(b) transgenic mice a repetitive sequence of ebstein-barr virus nuclear antigen 6 comprises overlapping t cell epitopes which induce hla-dr restricted cd4+ t lymphocytes reverse vaccinology and genomics enhancing dna immunization exact prediction of natural t cell epitope t cells primed by influenza virion internal components can cooperate in the antibody response to haemagglutinin prior vaccination increases the epitopic breadth of the cytotoxic t-lymphocyte response that evolves in rhesus monkeys following a simian-human immunodeficiency virus infection ctl epitopes identified with a defective recombinant adenovirus expressing measles virus nucleoprotein and evaluation of their protective capacity in mice functional analysis of influenza-specific helper t cell clones in vivo. t cells specific for internal viral proteins provide cognate help for b cell responses to hemagglutinin differential ability of b cells specific for external vs. internal influenza virus proteins to respond to help from influenza virus-specific t-cell clones in vivo 3d-epitope-explorer (3dex): localization of conformational epitopes within three-dimensional structures of proteins tuberculosis control in the 21st century hla supertypes and supermotifs: a functional perspective on hla polymorphism differential t cell costimulatory requirements in cd28-deficient mice presentation of endogenous peptide/mhc class i complexes is profoundly influenced by specific c-terminal flanking residues ctl responses of hla-a2.1-transgenic mice specific for hepatitis c viral peptides predict epitopes for ctl of humans carrying hla-a2.1 definition of mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, n-terminal amino acid sequencing, and electrospray mass spectrometry several common hla-dr types share largely overlapping peptide binding repertoires antibiotic-refractory lyme arthritis is associated with hla-dr molecules that bind a borrelia burgdorferi peptide magnitude and diversity of cytotoxic-t-lymphocyte responses elicited by multiepitope dna vaccination in rhesus monkeys targeting a polyepitope protein incorporating multiple class ii-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by cd4+ cytotoxic t lymphocytes: a novel approach to vaccine design minimal epitoeps expressedin arecombinant polyepitope protein are processed and presented to cd8+ t cells : implications for vaccine design a comparison of standard immunogenicity assays for monitoring hiv type 1 gagspecific t cell responses in ad5 hiv type 1 gag vaccinated human subjects discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products strategy for isolating and sequencing biologically derived mhc class i peptides dendritic cell-based immunotherapy of cancer with carcinoembryonic antigen-derived, hla-a24-restricted ctl epitope: clinical outcomes of 18 patients with metastatic gastrointestinal or lung adenocarcinomas simultaneous cd8+ t cell responses to multiple tumor antigen epitopes in a multipeptide melanoma vaccine altered peptidase and viral-specific t cell response in lmp2 mutant mice the induction of virusspecific ctl as a function of increasing epitope expression: responses rise steadily until excessively high levels of epitope are attained an effective secondgeneration outer surface protein a-derived lyme vaccine that eliminates a potentially autoreactive t cell epitope proteolysis and class i major histocompatibility complex antigen presentation key: cord-013336-42thiglv authors: wang, cheng; wang, ya-jie; tucker, joseph d.; xiong, ming-zhou; fu, hong-yun; smith, m. kumi; tang, wei-ming; ong, jason j.; zheng, he-ping; yang, bin title: correlates of hiv self-testing among female sex workers in china: implications for expanding hiv screening date: 2020-10-22 journal: infect dis poverty doi: 10.1186/s40249-020-00765-5 sha: doc_id: 13336 cord_uid: 42thiglv background: human immunodeficiency virus (hiv) self-testing may help improve test uptake among female sex workers. china has implemented many hiv self-testing programs among men who have sex with men, creating an opportunity for promotion among female sex workers. however, there is a limited literature on examining hiv self-testing among female sex workers. this study aimed to examine hiv self-testing experiences and its determinants among female sex workers in china. methods: a venue-based, cross-sectional study was conducted among chinese female sex workers in 2019. participants completed a survey including social-demographic characteristics, sexual behaviors, and hiv self-testing history, the distribution of which were analyzed using descriptive analysis. multivariable logistic regression was conducted to identify associations with hiv self-testing. results: among 1287 chinese female sex workers, 1072 (83.3%, 95% confidence interval [ci] 81.2–85.3%) had ever tested for hiv, and 103 (8.0%, 95% ci 6.6–9.6%) had ever used hiv self-testing. more than half reported that the self-test was their first hiv test (59.2%, 61/103), around one-fifth reported hiv self-testing results influenced the price of sex (21.4%, 22/103). a minority of individuals reported ever experiencing pressure to undertake hiv self-testing (6.8%, 7/103). after adjusting for covariates, hiv self-testing was positively associated with receiving anal sex in the past month (adjusted odds ratio [aor] = 2.2, 95% ci 1.4–3.5), using drugs before or during sex (aor = 2.8, 95% ci 1.8–4.5), injecting drugs in the past 6 months (aor = 2.6, 95% ci 1.2–6.0), being diagnosed with other sexually transmitted infections (aor = 1.6, 95% ci 1.0–2.5), tested for other sexually transmitted infections in the past six months (aor = 3.4, 95% ci 2.1–5.5), ever tested in the hospital (aor = 3.4, 95% ci 2.0–5.6), and ever tested in the community (aor = 1.5, 95% ci 1.2–1.9). conclusions: our findings suggest that hiv self-testing could expand overall hiv testing uptake, increase hiv testing frequency, reach sub-groups of high-risk female sex workers and has limited potential harms among female sex workers. hiv self-testing should be incorporated among chinese female sex workers as a complement to facility-based hiv testing services. (sti) acquisition [2] . the world health organization recommends frequent hiv testing in female sex workers to increase engagement in hiv prevention services and reduce risk of onward transmission [3] . however, hiv testing uptake remains low among female sex workers in low-and middle-income countries (lmic) [4, 5] . in china, studies suggest that approximately half of female sex workers remain unaware of their hiv serostatus [6, 7] . although facility-based hiv testing have helped increase testing coverage among female sex workers in china [7, 8] , many barriers persist. these include concerns about confidentiality of status [9] , lack of privacy [7] , fear of social stigma and condemnation [10] , lack of health care providers and inconvenient testing systems [11] . self-testing for hiv may help improve test uptake among female sex workers. self-testing is the process whereby a person collects a specimen, performs the test, and interprets the result themselves. studies in female sex workers in uganda [12] , kenya [13] , malawi [14] , and zambia [15] have shown that hiv self-testing can increase the frequency of hiv testing and safer sex practices, decrease stigma associated with hiv testing, provide a user-friendly, rapid, accurate and private setting of testing, as well as an opportunity for decentralized hiv testing and alternative service delivery models. however, there have been few studies examining hiv self-testing among female sex workers in countries outside of sub-saharan africa, including china [16] [17] [18] . currently, a total of 59 countries globally have policies supporting using hiv self-testing among key populations [19] . in china, highly sensitive and specific hiv self-test kits are available through community-based organizations or e-commerce platforms [11] . china has implemented many hiv self-testing programs and gained experience among men who have sex with men (msm) [20, 21] , creating an opportunity to promote hiv self-testing among female sex workers. the aim of this study was to examine hiv self-testing experiences and its determinants among female sex workers in china. a venue-based, cross-sectional study was conducted in eight cities (beijing, tianjin, shenzhen, kunming, jiaozhou, yunfu, xiangyang and longnan) within seven provinces in china between august 17 and october 17, 2019. these eight cities were selected based on local capacity and the availability of ongoing public health outreach programs for female sex workers. we partnered with eight local female sex workers community-based organizations (cbo) in those eight cities with experience of conducting female sex workers outreach programs including condom promotion, sexual health education, hiv and syphilis rapid testing and counseling, and linkage to care (accompaniment to clinical services for infected individuals). prior to this study, a mapping of the sex work venues was performed by local cbo in each study site according to geographic area and type of venue. a convenience sampling method was used to recruit female sex workers in selected venues in each city. we categorized the sex work venues into high tier and low tier based on the clientele's socioeconomic status [22] . low tier venues include foot bathing shops, hair salons or barber shops, massage parlors, roadside restaurants, roadside shops, guesthouses, streets or public outdoor places. high tier venues include karaoke bars, hotels, sauna, and nightclub. at each site, at least 30% of participants were lowtier sex workers. the inclusion criteria for this study were as follows: born biologically as a female; aged 18 or above; exchanged sex at least once for money/goods in the past three months; willing to participate and complete the survey. the survey questionnaire was created on wenjuanxing (changsha haoxing information technology co., ltd., changsha, china) based on discussions with local cbo stakeholders, policy makers and international hiv experts. we also piloted the survey with 50 volunteer female sex workers. the purpose of this formative research was to ensure the survey was simple to complete and consistent with our written survey content. this pilot data was not included in the final analysis. in the formal survey, each questionnaire was selfadministered by eligible participants with the help of outreach workers. participants would receive united states dollars (usd) 2 on completion of the study. the questionnaires submitted within five minutes were deemed invalid based on our pilot testing prior to the study that a minimum time of five minutes were required to respond all the survey items. social-demographic and sexual behavior characteristics included: age, marital status, place of residence, annual income, education, time of providing commercial sex in the current location, number of cities worked for selling sex, number of clients served in the past month, charge for vaginal sex, whether condoms were used consistently when engaged in commercial sex in the past month, illicit substance use, sti testing history. consistent condom use in the past month was defined as always using a condom during commercial sex. self-testing history for hiv included location where the kit was obtained, self-test results, post-test healthcare seeking behaviors, change in testing frequency after the first use of a self-test kit, experienced pressure from selftesting, whether giving or selling a self-test kit to a client, influences of performing self-testing on sex exchange. categories of pressure included physical violence, threats of violence, verbal abuse, psychological pressure, excessive control of activities, withholding of household resources, and threats to end a relationship [23] . descriptive analysis was conducted to describe the distribution of the sample regarding background characteristics, substance use, sexual behaviors, hiv and syphilis testing. univariable and multivariable logistic regression was conducted to explore socio-demographic and behavioral variables associated with hiv self-testing. in the multivariable model we adjusted for age, legal marital status, educational attainment, and annual income. all analyses were conducted on sas (v9.2, sas institute inc., cary, nc). this study was approved by the dermatology hospital of southern medical university (2019017). a verbal informed consent was obtained from all the participants who agreed to participate in this study. overall, 1443 women met the inclusion criteria. eightyone individuals declined to participant the study, and 77 completed the questionnaire less than five minutes. finally, a total number of 1287 women completed the survey. among those participants, 1072 (83.3%) had ever tested for hiv, and 103 (8.0%) had ever used hiv self-testing. most participants were between 18 and 35 years old (53.8%), from low tier venues (57.3%), married (43.8%), had a junior high school degree (45.2%), and had an annual income between usd 5001 and usd 14 000 (56.3%). the majority were employed (54.4%), residing in the province where the study was done (51.4%) and working in current location over one year (50.51%). the socio-demographic characteristics of respondents who ever used hiv self-testing were comparable to women who never used hiv self-testing (table 1) . of 1287 individuals, the median number of clients served in the past month was 22 (interquartile range [iqr]: 12-56). the median amount of payment received for vaginal sex was usd 20 (iqr: 15-45). and 42.1% (542/1287) reported using condoms consistently when engaged in commercial vaginal sex in the past month. for oral sex, 58.3% (750/1287) reported providing oral sex in the past month, of whom only 16% (120/750) reported using condom consistently. for anal sex, 18.9% (243/1287) reported receiving anal sex in the past month and 41.6% (101/243) reported using condom consistently. the vast majority of women (78.4%, 1009/1287) reported having never used drugs before or during sex. almost three-fifth of women (59.7%, 768/1287) reported bulk purchasing of condoms, and a small proportion of individuals (5.2%, 67/1287) have experience of bulk purchasing hiv self-testing kits ( table 2) . 9%, 3/103 ). the majority sought care following reactive/indeterminate hiv self-testing result (81.8%, 9/11). among those individuals who sought care after hiv self-testing, most women sought care within two weeks (66.7%, 6/9), and either at a specialty sexually transmitted infection (sti) clinic or in a health facility run by the centers for disease control (table 3) . among individuals who had hiv self-tested, around one-fifth reported hiv self-testing results influenced the price of sex (21.4%, 22/103). a minority of individuals reported ever experiencing pressure to undertake hiv self-testing (6.8%, 7/103). the most common pressure was psychological pressure (2.9%, 3/103), followed by threatened to end a relationship (2.9%, 3/103) ( table 3) . a majority of hiv self-testers reported some difficulties in performing hiv self-testing (71.84%, 74/103). pricking fingers (59.5%, 44/74) or using collection tube to collect blood (50.0%, 37/74) were the most commonly reported difficulty. the most commonly reported reasons for using hiv self-testing were that they wanted to know their infection status (55.3%, 57/103), and they recently had high risk contact (54.4%, 56/103). the most common reason for not performing self-testing was that they had never heard of hiv self-testing before (42.6%, 504/1184) (additional file 1: table s1 ). in the multivariable model adjusted for age, legal marital status, educational attainment and annual income, the following factors were positively correlated with hiv self-testing: receiving anal sex in the past month (adjusted odds ratio [aor] = 2.2, 95% ci 1.4-3.5), using drug before or during sex (aor = 2.8, 95% ci 1. 8-4.5) , injecting drugs in the past six months (aor = 2.6, 95% ci 1.2-6.0), being diagnosed with other stis (aor = 1.6, 95% ci 1.0-2.5), tested for other stis in the past 6 months (aor = 3.4, 95% ci 2.1-5.5), ever tested in the hospital (aor = 3.4, 95% ci 2.0-5.6), and ever tested in the community (aor = 1.5, 95% ci 1.2-1.9) ( table 4 ). female sex workers are at high risk of hiv acquisition and transmission [24] . our study suggests that self-testing for hiv could expand overall testing uptake, increase testing frequency, and has limited potential harms among female sex workers. this study expands the literature by focusing on hiv self-testing among female sex workers, including women from multiple provinces, and exploring associated benefits and harms. findings from this study can help inform hiv self-testing interventions among female sex workers. our study suggests that few female sex workers in china have performed hiv self-testing. this is consistent with rates of hiv self-testing reported in malawi [14] , zimbabwe [15] , uganda [12] , and kenya [13] . since the world health organization released guidelines recommending hiv self-testing among under-served and high-risk populations in 2016 [25] , many studies in the sub-saharan africa have shown that hiv self-testing has a good acceptability and feasibility for female sex workers [12, 14, 15, 26] . studies have suggested that adding hiv self-testing to existing community-based testing and counseling services among female sex workers is acceptable, cost-effective and efficient to improve linkage to care [15, 26, 27] . china should take more effort to explore strategies integrating hiv self-testing to existing facility-based testing services. additionally, in the context of wide online availability of hiv self-testing kits in china [11] , our data could help to inform interventions. our study found low frequency of physical violence or other types of violence related to hiv self-testing among female sex workers. the frequency of physical violence associated with hiv self-testing is comparable to that reported in zambia [15] and kenya [13] . intimate partner violence is common and could be exacerbated by self-testing services that inadvertently allow a sex worker manager or partner to influence testing behaviors [13, 28] . our results suggest that self-testing is not associated with physical violence, although further research and attention are needed. we found that self-testing for hiv can effectively reach high-risk female sex workers, and facilitate higher frequency testing. in our study, hiv self-testing was correlated with receiving commercial anal sex in the past month and drug use before or during sex. this finding is consistent with studies conducted among men who have sex with men in china [21] and france [29] . additionally, our study found that a large proportion of hiv selftesters reported having never been tested for hiv before the self-testing, and approximately one-third of hiv selftesters reported increasing testing uptake after initial use of hiv self-testing. this suggests hiv self-testing has the potential to increase the frequency of hiv testing among female sex workers, specifically among individuals not reached by provider-based strategies [11, 30] . our study has several limitations. first, the survey captured a convenience sample of female sex workers population in china, likely resulting in selection bias. although the convenience sampling method can generally be implemented more easily, faster and with fewer resources [31] , the study sample might not be representative of the population as a whole, which limits the statistical inference and generalizations [32] . second, this study was conducted among female sex workers in cities with relatively high involvement in hiv prevention programs. the results of this study may not be generalizable to female sex workers in cities that have fewer hiv prevention programs. third, all the data were collected through self-report, which may be prone to information bias. although self-testing for hiv may be effective in expanding female sex workers testing uptake and frequency, several challenges remain. individuals with a reactive result require a clinic visit for confirmation of infection, which can be inconvenient, and create a barrier for female sex workers to linkage to care [33] . female sex workers tend to be high degree of mobility and vulnerable to criminality [34] , highlighting the need of social support for training, counseling, and ancillary self-testing services [15] . adequate linkages to counseling, treatment, and care for hiv self-testers who test positive, and test quality assurance remain essential [35] . innovative approaches are needed to improve hiv testing uptake among female sex workers. our findings suggest that hiv self-testing has the potential to expand overall testing uptake, enable more frequent testing, reach sub-groups of high-risk female sex workers, and has limited potential harms among female sex workers. self-testing for hiv should be incorporated among chinese female sex workers as a complement to facilitybased testing services. future studies to explore effective mode of promoting hiv self-testing among female sex workers are warranted. supplementary information accompanies this paper at https ://doi. org/10.1186/s4024 9-020-00765 -5. additional file 1: table s1 . difficulties and reasons for performing hiv self-testing among chinese female sex workers. hiv: human immunodeficiency virus; sti: sexually transmitted infections; lmic: low-and middle-income countries; ci: confidence interval; cor: crude odd ratio; aor: adjusted odd ratio; iqr: interquartile range. unaids report on the global aids epidemic need for intervention services for promotion of condom use by female sex workers to consider size of entertainment venues: a cross-sectional study world health organization. consolidated guidelines on hiv testing. geneva: world health organization africans in south china face social and health barriers joint united nations programme on hiv/aids; 2016 the hiv, syphilis, and hcv epidemics among female sex workers in china: results from a serial cross-sectional study between the neglected health of international migrant workers in the covid-19 epidemic analysis of situation and influencing factors of providing hiv rapid testing service by community based organizations expanding syphilis testing: a scoping review of syphilis testing interventions among key populations convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year • at bmc, research is always in progress. learn more biomedcentral.com/submissions ready to submit your research ready to submit your research ? choose bmc incarcerated sex workers and hiv prevention in china: social suffering and social justice countermeasures syphilis self-testing: a nationwide pragmatic study among men who have sex with men in china direct provision versus facility collection of hiv self-tests among female sex workers in uganda: a cluster-randomized controlled health systems trial promoting male partner hiv testing and safer sexual decision making through secondary distribution of self-tests by hiv-negative female sex workers and women receiving antenatal and post-partum care in kenya: a cohort study hiv self-testing services for female sex workers, malawi and zimbabwe hiv self-testing among female sex workers in zambia: a cluster randomized controlled trial attitudes and acceptability on hiv self-testing among key populations: a literature review scaling up hiv self-testing in sub-saharan africa: a review of technology, policy and evidence hiv self-testing in pune, india: perspectives and recommendations of female sex workers and peer educators global output of research on the health of international migrant workers from acceptability and feasibility of a social entrepreneurship testing model to promote hiv self-testing and linkage to care among men who have sex with men hiv self-testing among online msm in china: implications for expanding hiv testing among key populations the prevalences of neisseria gonorrhoeae and chlamydia trachomatis infections among female sex workers in china pressured hiv testing "in the name of love": a mixed methods analysis of pressured hiv testing among men who have sex with men in china hiv infection among female sex workers in concentrated and high prevalence epidemics: why a structural determinants framework is needed world health organization. guidelines on hiv self-testing and partner notification: supplement to consolidated guidelines on hiv testing services. geneva: world health organization cost-effectiveness of community-based human immunodeficiency virus self-testing in the impact and cost-effectiveness of community-based hiv self-testing in sub-saharan africa: a health economic and modelling analysis acceptability of woman-delivered hiv self-testing to the male partner, and additional interventions: a qualitative study of antenatal care participants in malawi access to and use of unauthorised online hiv self-tests by internet-using french-speaking men who have sex with men usage of purchased self-tests for hiv and sexually transmitted infections in amsterdam, the netherlands: results of populationbased and serial cross-sectional studies among the general population and sexual risk groups a comparison of respondent-driven and venue-based sampling of female sex workers in liuzhou sampling studies to estimate the hiv prevalence rate in female commercial sex workers hiv testing in a high-incidence population: is antibody testing alone good enough? addressing vulnerabilities of female sex workers in an hiv prevention intervention in mumbai and thane: experiences from the aastha project a review of self-testing for hiv: research and policy priorities in a new era of hiv prevention publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we are very grateful to all the participants who participated in this study. authors' contributions cw, mx and by developed the initial concept for the manuscript. cw drafted an initial draft. yw conducted the statistical analysis. the remaining authors (jdt, hf, wt, mks, jjo, hz) edited and contributed content to the final draft. all authors have read and approved the final manuscript. all authors read and approved the final manuscript. this work was supported by guangdong medical research foundation (a2019402). the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. not applicable. not applicable. the authors declare no conflicts of interest. key: cord-034269-gqy61g8l authors: abayneh, kinfe; mengistie, bizatu; oljira, lemessa; tiruye, getahun title: clients’ satisfaction with services for prevention of mother-to-child transmission of hiv in public health facilities in diredawa city, eastern ethiopia date: 2020-10-21 journal: hiv aids (auckl) doi: 10.2147/hiv.s264854 sha: doc_id: 34269 cord_uid: gqy61g8l background: ethiopia has a very high burden of hiv infection among children, contracted from their mothers, and nearly two-thirds of pregnant women do not receive prevention of mother-to-child transmission (pmtct) services. ensuring clients’ satisfaction with pmtct services is one of the bases to scale up service utilization and mitigate mtct of hiv. however, in ethiopia, particularly in the study area, evidence related to clients’ satisfaction with pmtct services is scanty. methods: a facility-based cross-sectional study was conducted among women attending antenatal care in diredawa city. systematic random sampling was used to select 517 study participants. interviewer-administered structured and pretested questionnaires were used to collect data. statistical significance was regarded as p≤0.05 with a 95% ci. results: client satisfaction with pmtct services was 82.2% (95% ci 66.4%–94.3). receiving the service from a hospital (aor 2.34; 95% ci 1.5, 3.98), no formal education (aor 2.53, 95% ci 1.52–4.2), primary education (aor 2.17 95% ci 1.17–4.04), receiving preand post-hiv test counseling from the same provider (aor 4.93, 95% ci 2.98–7.17), gestational age above first trimester (aor 1.74, 95% ci 1.12–2.71), and waiting time ≤15 minutes (aor 2.31, 95% ci 1.28–4.16) were positively associated with client satisfaction with pmtct services. conclusion: client satisfaction with pmtct services is relatively high. receiving the service from a hospital, no formal education or only primary education, gestational age above first trimester, getting preand post-hiv test counseling from the same provider, and waiting time ≤15 minutes to receive services were factors associated with client satisfaction. a greater number of skilled pmtct-service providers would improve service quality and hasten its delivery. furthermore, providing mentoring and supportive supervision of health centers with pmtct programs and keeping the same provider in posttest counseling is also mandatory. mother-to-child transmission (mtct) of hiv can occur during pregnancy, labor, childbirth, and breastfeeding. 1, 2 globally, approximately 38 million people are currently living with hiv, and tens of millions of people have died due to aids-related causes since the beginning of the epidemic. 3 although there have been significant declines in new infections since the mid-1990s, there were still about 1.7 million new infections in 2019. 4 hiv remains a leading cause of death globally among women of reproductive age. however, aids-related deaths have declined, due in part to antiretroviral treatment scaling up, 5 and aids-related death had been reduced to 690,000 in 2019, a 37% decrease from 1.1 million in 2010. 5, 6 about 80% of pregnant women living with hiv were receiving antiretroviral treatment in the year 2017. this can be considered a significant improvement compared to 2010, when only 51% had access to the treatment. 7 despite this significant progress, 740,000 women of reproductive age became hiv-positive. nearly three-quarters of these women live in 23 countries. the vast majority are in sub-saharan africa and are classified as high priority for the prevention of mtct (pmtct), as reported by the joint united nations programme on hiv/ aids. 7, 8 pregnant women and children aged <15 years in sub-saharan africa account for around two-thirds of the world's hiv infections, and this in turn has serious complications for children born to positive women. 9 in this regard, in 2019 it was estimated that around 150,000 children were newly infected with hiv, 10 of which >90% of them were infected through mtct. 11, 12 in the absence of treatment, about half these infected children will die before their second birthday. 11 although there is progress in increasing availability of free antenatal care (anc) and pmtct programs in sub-saharan africa, the rate of mtct of hiv is still 15%-40%. 13 ethiopia is one of the east african countries to have been highly affected by hiv. 14, 15 as a 2016 report indicates, around 20,000 people have died due to the virus. 14, 16 similarly, ethiopia is also among the ten countries in the world with the highest number of children infected by mtct, 17 and the rate of mtct of hiv, including breastfeeding, is about 15.9%. 16 accordingly, in 2007 ethiopia started universal screening of pregnant women for hiv, and in a majority of cases hiv testing during pregnancy is provided as part of anc. 2 however, progress is not satisfactory: only two-thirds of pregnant women had an anc visit, 18 below the who goal for hiv screening (80%). 11 though universal screening was adopted, the 2012-2013 report showed that even among mothers who had attended anc, more than a third had not been tested for hiv, 19 thus representing a significant missed opportunity for provision of pmtct services. 19, 20 the federal ministry of health also recognized and accepted the vision of an hiv-free generation endorsed by unaids to reduce hiv mtct to <2% by 2020. 2, 21 to achieve this goal, hiv screening during pregnancy should be raised to >95%. 22 thus, ensuring client satisfaction while delivering pmtct services has significant benefit in optimizing uptake, adherence, and retention throughout care for better outcomes. 23, 24 strengthening the linkage of pmtct services within maternal, newborn, child health, sexual and reproductive health, and family-planning services in health facilities is one of the strategies for addressing pmtct targets. 25 client satisfaction with pmtct services is an indicator of quality of service and is one of the measures of desired outcomes in health care, 25, 26 as it influences service utilization. 27 despite assessing and addressing client satisfaction being of paramount importance in scaling up pmtct-service utilization, there is scant proof concerning magnitude and factors associated with client satisfaction with pmtct services, particularly in diredawa city, eastern ethiopia. this study was conducted in diredawa city, eastern ethiopia, which is located 515 km from the capital city addis ababa. the population of the city is estimated to be 388,279. of this population, 194,770 (50.1%) are male and 193,509 (49.8%) are female. 28 this facility-based cross-sectional study was conducted from january to february 2018. all women attending anc in public health facilities of diredawa city were the source population. women who visited the selected health facilities for anc services were included as the study population. a single population-proportion formula was used to determine sample size: assumption of a 95% significance level, 4% margin of error, 71.2% client satisfaction with pmtct services in public health facilities, 26 and a 5% non-response rate. the final sample size was determined to be 517. there are ten public health facilities in diredawa city, and of these, five public health facilities were selected by lottery. proportional allocation was used to select the sample from anc units of selected health facilities. finally, systematic random sampling was applied to recruit study participants. the study purposively selected and observed 15 clients-provider counseling sessions in both pretest and posttest hiv/aidscounseling services. we used a standardized questionnaire adapted from a unaids best-practice tool that focuses on hiv/aids-counseling services to measure client satisfaction. 26, 29 structured questionnaires containing sociodemographic and obstetric characteristics of respondents, comfort with counselors' approaches, privacy during counseling, topics of discussion during hiv counseling, and understandability of messages during anc was used to measure a more nuanced understanding of client satisfaction. 23, 26, [30] [31] [32] furthermore, serviceprovision processes were also observed for 15 client-counseling sessions using a checklist. 23, 25, 26, 33 interviewer-administered questionnaires were prepared first in english and then translated into amharic and afan oromo as per the mother tongue of the participants and translated back into english for consistency by language experts. pretesting was done on 5% of the total sampled population at hiwot fana specialized university hospital, then corrections and modification of the tool were carried out accordingly. five diploma midwives and two supervisors not working in the selected health facilities were involved as data collectors. exit interviews of clients were done by trained data collectors. the investigators and supervisors closely followed the data-collection process throughout the data-collection period. each day, data collected were checked for completeness and corrective measures taken accordingly. these comprised a package of services that included primary prevention of hiv in women, prevention of unintended pregnancy among hiv-infected women, interventions to reduce transmission from hiv-infected pregnant and lactating women to their children, and care and support of women, children, and families infected and affected by hiv. 11 this was defined as client perception of the service or product in terms of meeting or even exceeding their expectations. 23 client satisfaction was measured using the questionnaire with a 14-item scale. we used a fivepoint likert-scale instrument, in which 5 denoted very satisfied and 1 very dissatisfied. 26 satisfaction level was assessed using a five-pont likert scale (1 very dissatisfied, 2 dissatisfied, 3 neutral, 4 satisfied, and 5 very satisfied). this refers to the period for which clients were expected to wait before receiving services. data were first coded, then double-entered on epidata version 3.1 and exported into spss version 21 for analysis. for the purpose of analysis, the five-item likert scale was changed to a binary outcome variable (satisfied and dissatisfied). accordingly, those who responded "very satisfied" and "satisfied" were categorized as satisfied clients and those who responded "neutral", "dissatisfied", and "very dissatisfied" were categorized as unsatisfied. 26 descriptive statistics were used to analyze each variable, and results are summarized in the tables. hosmer-lemeshow and omnibus goodness-of-fit tests were used to assess whether necessary assumptions for the application of logistic regression met. variables with p-value ≤0.25 on bivariate logistic regression were considered in the multivariable model. crude odds ratios (cors) and adjusted odds ratios (aors) with 95% ci were calculated, and variables with p-value ≤0.05 on multivariable analysis were deemed statistically significant. ethical clearance was obtained from the institute of health research ethical review committee of haramaya university college of health and medical sciences. permission letters were obtained from diredawa health bureau and each of public health facilities prior to the study. voluntary written and signed consent was obtained from each study participant after informing them of the objectives of the study, confidentiality, right to withdrawal, benefits, and risks. this study was conducted in accordance with the declaration of helsinki. a total of 516 pregnant women were involved in the study, making a response rate of 99.8%. more than half (52.9%) of the participants were hospital attendants and their mean age was 24.74±4.3 years. a majority (94.6%) were married, and 83.5% were urban residents. two-thirds (65.5%) of the study participants were housewives (table 1) . the average gestational age of current pregnancy was 25.7 weeks. more than half (54.5%) of the study participants were multigravida, and a majority (88.3%) had come primarily for anc follow-up. almost all (98%) had heard about the presence of pmtct services before they came to the health facility for anc, and their main source of information for pmtct services was health-care providers (72%). the duration of the counseling sessions was 5-60 minutes, with an average of 20 minutes. regarding counseling services, more than half (56.2%) of the participants received both pretest and posttest hiv counseling from the same counselor. during the counseling sessions, a majority (87%) did not face a language barrier ( table 2) . concerning client responses on the benefit of pmtct services, almost all (99%) participants found that pmtct-service counseling was beneficial and 86.1% liked the discussion on hiv/aids. most (97.1%) were happy with the opening hours of the health facilities. similarly, most (96%) were happy with the sessions and recommended the importance of the services to others. over half (59.4%) of the participants had come to the health facilities on others' recommendations, and only 3.9% of the participants preferred it that hiv/aids would not be discussed during their antenatal visit. fifteen counseling sessions were observed during pre-and posttesting. in all of the 15 observations that were made, the counselor had received the women in a welcoming manner and created a trusting or supportive rapport. it was observed that the counselor listened to women's ideas and concerns in 12 of the 15 sessions and invited to ask questions in six. the counselors also attempted to respond to each of the questions raised. in all observed sessions, such counselor qualities as avoiding judgment or disapproval, treating the women with empathy, dignity, and respect, using language that could be easily understood by the women, and maintaining privacy were seen. counselors checked to be sure that the women understood the information provided for all 15 observed sessions. in all pretest counseling sessions, it was observed that counselors introduced and oriented the session for the women, prepared them well for the hiv test, explored options for reducing risk, and assessed them for possible risks. posttest counseling for negative results was performed similarly in all the observed sessions. negative hiv-test results were provided, riskreduction plans negotiated, support for risk-reduction most (96.6) of the participants were satisfied or very satisfied with the provider's greeting. the same proportion were satisfied or very satisfied with the adequacy of time for counseling and counselors' respect. however, more than half (55.0%) were dissatisfied with the availability of laboratory services when needed (table 3 ). of the 516 study participants, 424 (82.2%, 95% ci 66.4%-94.3%) clients were satisfied with the pmtct component of hiv services. the final multivariable logistic regression analysis showed that receiving counseling services from a hospital increased the odds of client satisfaction compared to those who received these from a health center (aor 2.34, 95% ci 1. (table 4 ). in this study, 82.2% of clients were satisfied with pmtct services, in line with a finding reported from sebeta, ethiopia (80.7%), 34 but lower than findings reported in tanzania (92%) 33 and hadiya, ethiopia (90%). 35 this discrepancy might be due to differences in study settings. this study was conducted with urban clients, who are more aware of the option of treatment available, as they are frontline for information about pmtct-related issues. as such, this could make them less likely to be involved in counseling sessions. on the other hand, clients living in rural settings might not have adequate knowledge on what is expected from counselors during sessions or might not be aware of the unavailability of laboratory services, making them easily satisfied. 23, 35 in contrast, higher prevalence of client satisfaction was identified in this study than studies in adama (74.7%) 26 and dodoma, tanzania (75.2%). 23 this high proportion might be due to differences in study period or geographical variation. ethiopia has taken many steps to resolve some of the barriers facing women when seeking maternity services. from these measures, the inclusion of prenatal, delivery, and postnatal care were listed as services free of charge. 36 however, there are serious constraints on the continuous supply of equipment or precursors for laboratory investigation, which causes clients not to get the necessary services or exposes them to charges for these services. as such, unavailability of laboratory services will profoundly lower clients' perceived quality of care or satisfaction. 37 similarly, this study revealed that more than half (55.0%) of the participants were not satisfied with the availability of certain laboratory services. the present study found that client satisfaction with pmtct services was nearly twice as times common among respondents with no formal and primary education dovepress than those with secondary education and above. likewise, a similar finding was found in dodoma, tanzania. 23 this might be due to educated clients being more aware of pmtct-related information or educated groups being less satisfied than less educated groups, because of higher-quality service expectations. moreover, participants with higher education may inappropriately consider themselves as more knowledgeable and at lower risk of infection and thus fail to participate actively in counseling sessions. 38 the finding of this study revealed that the odds of client satisfaction with pmtct services were 2.34 times higher among participants who accessed the services from a hospital than their counterparts. this may be the impact of unavailability of necessary laboratory services at health centers. similarly, participants in this study were not satisfied with laboratory services. this is due to lack of adequate supplies and skilled personnel to provide appropriate services in health centers compared to hospitals. 35 gestational age was significantly associated with client satisfaction with pmtct services. the odds of being satisfied with pmtct services were 1.74 times higher among participants with gestational age above first trimester than their counterparts. this was in agreement with a study conducted in addis ababa, ethiopia. 39 a possible explanation might be that clients in their first trimester usually have yet to be exposed to pmtct services. therefore, this unfamiliarity with the services might limit their interaction with providers and hence, hinder their full participation in the services. client satisfaction with pmtct services was 1.45 times more common among participants who waited for ≤15 minutes than their counterparts. this result was congruent with studies carried out in nigeria 31 and tanzania. 23 this might be due to the fact that clients usually relate waiting time to the kind of service they are going to receive or clients liking to receive a service with minimal waiting time, making them satisfied without considering the quality of services. hiv-testing and -counseling guidelines for pmtct recommend that both pretest and posttest counseling be offered by the same counselor. keeping the same provider in pre-and post-hiv-test counseling helps to secure client confidentiality and privacy and ensure client satisfaction.-40,41 receiving pre-and post-hiv test counseling from the same provider is positively associated with client satisfaction with pmtct services. 23, 26, 31, 39 this study used a validated and standardized questionnaire that had been tested and revised to assess client satisfaction with pmtct services, and we also observed specific client-provider interaction while delivering services. however, the scope is limited in addressing all factors that can probably affect client satisfaction with pmtct services. client satisfaction with pmtct services was relatively high. however, more than half of the participants were not satisfied with availability of laboratory services. no formal education, primary education, receiving services from a hospital, with the same counselor in pretest and posttest hiv counseling, and gestational age above first trimester were positively associated with client satisfaction with pmtct services. furthermore,waiting time ≤15 minutes was positively associated with pmtct-service satisfaction. therefore, mentoring and supportive supervision of health centers with pmtct programs, ensuring availability of supplies and equipments for necessary laboratory investigations, and providing pre-and posttest hiv counseling by the same provider would improve clients' pmtct satisfaction. moreover, there should be an increase in the number of skilled pmtct-service providers to further optimize quality in general and speed up service delivery in particular. all related data have been presented within the manuscript. the data set supporting the conclusions of this article is available from the authors upon reasonable request. federal democratic republic of ethiopia. country progress report on the hiv response aids prevention and control office federal ministry of health. guidelines for prevention of mother to child transmission of hiv in ethiopia seizing the moment, tackling entrenched inequalities to end epidemics unaids report on the global aids epidemic shows that 2020 targets will not be met because of deeply unequal success covid-19 risks blowing hiv progress way off course a spotlight on adolescent girls and young women aids info), miles to go: global aids update joint united nations programme on hiv/aids. start free stay free aids free 2017 progress report hiv infection and aids in sub-saharan africa: current status, challenges and opportunities global and regional trends: while there has been promising progress in the hiv response, children continue to be affected by the epidemic pmtct strategic vision 2010-2015: preventing mother-tochild transmission of hiv to reach the ungass and millennium development goals who. who recommendations on the diagnosis of hiv infection in infants and children why is mother to child transmission (mtct) of hiv a continual threat to new-borns in sub-saharan africa (ssa) global burden of disease of hiv-associated cryptococcal meningitis: an updated analysis hiv and aids in east and southern africa regional overview global aids monitoring, spectrum estimates (unaids/who) and who hiv country intelligence mother to child transmission of hiv and associated factors among hiv exposed infants at public health facilities, dessie town ethiopian demographic health survey country progress report on the hiv response barriers and facilitating factors to the uptake of antiretroviral drugs for prevention of mother-to-child transmission of hiv in sub-saharan africa: a systematic review world health organization. global guidance on criteria and processes for validation: elimination of mother-to-child transmission of hiv and syphilis clients' satisfaction with services for prevention of mother-to-child transmission of hiv in dodoma rural district antiretroviral drugs in the cupboard are not enough: the impact of health systems' performance on mother-to-child transmission of hiv quality of prevention of mother to child transmission (pmtct) of hiv services in public hospitals of hadiya zone, southern ethiopia prevention of mother-to-child transmission (pmtct) of hiv services in adama town, ethiopia: clients' satisfaction and challenges experienced by service providers hiv testing during pregnancy for prevention of mother-to-child transmission of hiv in ethiopia use of unaids tools to evaluate hiv voluntary counselling and testing services for mineworkers in south africa utilization of pmtct services and associated factors among pregnant women attending antenatal clinics in addis ababa assessment of clients' satisfaction with the pmtct counselling service in benin city, edo state, nigeria prevention of mother to child transmission of hiv/aids: service utilization and associated factors among selected public health facilities in ethiopia predictors of patient dissatisfaction with services for prevention of mother-to-child transmission of hiv in dar es salaam utilization of prevention of mother-to-child transmission of hiv services and associated factors among antenatal care attending mothers in sebeta town, central ethiopia quality of pmtct services in gebretsadiq shawo memorial hospital user fees and maternity services in ethiopia assessment of client satisfaction in labor and delivery services at a maternity referral hospital in ethiopia acceptance of hiv testing among pregnant women in we would like to acknowledge haramaya university for their technical and financial support of this study. next, our gratitude goes to all data collectors and supervisors. last but not least, thanks to all anc attendees who participated in the study. without them, this research would not have been realized. the authors declare no conflicts of interest in this work. key: cord-010699-mfe1oajn authors: suehiro, tamy taianne; damke, gabrielle marconi zago ferreira; damke, edilson; de azevedo ramos, paloma luana rodrigues; de andrade pereira silva, marcela; pelloso, sandra marisa; huh, warner k.; franco, ricardo argemiro fonseca; da silva, vânia ramos sela; scarinci, isabel cristina; consolaro, marcia edilaine lopes title: cervical and oral human papillomavirus infection in women living with human immunodeficiency virus (hiv) and matched hiv-negative controls in brazil date: 2020-05-11 journal: infect agent cancer doi: 10.1186/s13027-020-00301-y sha: doc_id: 10699 cord_uid: mfe1oajn background: despite the demonstrated role of human papillomavirus (hpv) in the etiology of cervical cancer and the strong evidence suggesting the importance of hpv in the development of oropharyngeal cancer, several aspects of the interrelationship between hpv infection in both body sites remain unknown, specifically in female human immunodeficiency virus (hiv)-positive (hiv+) patients. we aimed to assess the prevalence, distribution, and concordance of cervical and oral hpv in hiv+ women and matched hiv-negative (hiv-) controls in brazil. material and methods: cervical and endocervical samples for cytological screening and hpv detection and oral samples were collected from 115 hiv+ women using highly active antiretroviral therapy (haart) and 139 hiv-matched controls (hiv-) in maringá city, brazil. risk factors were assessed using a standardized questionnaire, and the data regarding hiv infection were obtained from the patients’ medical records. hpv detection and typing were performed using the kit multiplex xgen multi hpv chip hs12. results: hiv infection was well controlled in this cohort, but women who exhibited detectable hiv loads were significantly associated with hpv-positive status overall (p = 0.03) and in cervical mucosa (p = 0.01). hiv+ women had significantly more abnormal cytological findings (p = 0.04) than hivwomen. of the 115 hiv+ women, 48.7% were positive for cervical and/or oral hpv dna; of the 139 hivwomen, 41% were positive for cervical and/or oral hpv (p = 0.25). both hiv+ and hivwomen had a statistically higher prevalence of cervical hpv infection than oral infection. the concurrent hpv infection in two anatomical sites was similar in hiv+ and hivwomen; however, hpv type concordance was not observed. hpv type distribution was different between the anatomical sites in both groups, and hiv+ women presented less common types, mainly in oral mucosa. conclusion: our data support the importance of testing hpv infection in hiv+ women, even when the hiv infection is well controlled. prospective studies are required to better understand the natural history of hpv infection in both anatomical sites, specifically in hiv+ women. the association between persistent high-risk human papillomavirus (hrhpv), squamous cervical cancer (cc), and some vaginal and anal cancers has been well-established [1] [2] [3] . additionally, recent data demonstrated that hpv is also associated with a subset of head and neck cancers (hncs), including a worldwide range~20-80% of oropharyngeal cancers (opcs) [4, 5] . individuals living with human immunodeficiency virus (hiv+) are more susceptible to infection, less likely to clear the virus and have a higher risk of hpv-related cancers than hiv-negative (hiv-) individuals [6, 7] . although the incidence of overall cancer has decreased in hiv+ individuals with the advent of highly active antiretroviral therapy (haart), hpvrelated cc risk is higher among hiv+ individuals [8, 9] . moreover, in hiv+ women, cc tends to respond poorly to recommended therapies, becomes more aggressive, and may have a worse prognosis [6] . with the increase in survival of hiv+ women due to haart, progression of oncogenic viral infection into malignancy may result in an increased incidence of hpv-associated oropharyngeal, genital, and anal cancers [8] [9] [10] . currently, the hiv/acquired immunodeficiency syndrome (aids) pandemic impacts the poorest and the youngest in low-resource settings [11] . hiv+ young women are key populations at high risk for developing hpv-related cancers, particularly in low-and middle-income countries such as brazil [12, 13] . with the recent approval of the 9valent hpv vaccine, the risks of persistent hpv infection and hpv-related cervical precancerous lesions and malignancies are expected to decrease significantly [14] . with the appropriate vaccination rate thresholds, this vaccine provides a logical rationale for increasing screening intervals considering the anticipated decrease in the burden of disease [15] . therefore, understanding the specific prevalence, distribution and concordance of cervical and oral hpv in at-risk women in specific geographic locations will provide further insight into the effectiveness of this new vaccine against oral hpv infection, which has not been proven yet. considering the high burden of hpv-related cancers among hiv+ women and the possible effectiveness of a 9-valent hpv vaccine [16] , it is critical to understand the prevalence and types of hpv infections in oral and cervical mucosa in hiv+ women (and matched controls). however, while several studies on hiv+ women have reported cervical, oral, or anal hpv type distribution [17] [18] [19] [20] [21] [22] [23] , only a few studies have addressed concurrent cervical and oral hpv prevalence [24] [25] [26] . despite the demonstrated role of hpv in the etiology of cc and the strong evidence suggesting the importance of hpv in the development of opc [27] [28] [29] [30] , several aspects of the interrelationship between oral and cervical infections remain unknown, specifically in hiv+ female patients. the present study aimed to assess the prevalence, distribution, and concordance of cervical and oral hpv in hiv+ women and matched hiv-controls in the southern region of brazil, a geographic area with a high incidence of hiv and cc. participants included 115 hiv+ women receiving haart and 139 hiv-aged 19 to 66 years who attended the specialized assistance service (sae) for sexually transmitted diseases (std/aids in maringá, southern brazil) from september 2017 to may 2018. women with the following characteristics were included in this study: women with confirmed hiv/aids diagnosis using two different methods were included in the hiv+ group and women with two hiv/aids negative results using two different methods were included in the hiv-group. exclusion criteria include: women with previous hysterectomy, pregnant, younger than 19 years, and women with no history of sexual intercourse. of the 778 hiv+ women enrolled in the sae, 324 were eligible for the study. the sample size was calculated with a hpv cervical prevalence of 50% in hiv+ women [19] , a 95% confidence interval (ci), and an error estimate of 5%. with an increase of 10% for possible participant losses, the total sample size was fixed at 138 randomly selected women. sae also provides other services and hiv screening. therefore, to obtain a comparable sample, 138 matched controls from the list of patients served by the sae were identified. the controls were matched by age. written informed consent was obtained from 254 women, and based on their hiv serology status, participants were assigned to the hiv+ (n = 115) or hiv-group (n = 139). participants were interviewed using a standardized questionnaire to obtain demographic information (e.g., age, educational attainment, household income, race/ethnicity); tobacco and alcohol use/abuse; obstetric and gynecologic history (e.g., nursing staff contacted all women, administered the questionnaire, and collected the cervical and oropharyngeal samples. ecto−/endocervical samples were collected using an ayre's spatula and cytobrush for cervical cytology and polymerase chain reaction (pcr) for hpv; the samples for hpv testing were stored in thinprep® pap test solution. the conventional cytological smears were sent to the clinical cytology laboratory at state university of maringá (uem) and were graded according to the bethesda system [31] . full-mouth oral/oropharyngeal scraping, including the cheeks, tongue, palate, tonsils, and oropharynx, was performed using a sterile brush with soft bristles; samples were stored in thinprep® pap test solution. moreover, a gargle sample was obtained by having the participant gargle with 10 ml of 0.9% sterile saline for a total of 30 s (10s, rinse; 5 s, gargle; 10s rinse; 5 s, gargle), collected in a sterile cup and stored at − 4°c [20, 32] . all oral/oropharyngeal samples will be referred as "oral samples." detection and typing of hpv were performed using the kit multiplex xgen multi hpv chip hs12 (mobius life science), following the manufacturer's instructions. ). an additional hpv universal probe was used to detect other, non-specified types of hpv. samples with invalid outcomes were retested, and the second result was considered definitive. women were considered positive for oropharyngeal hpv if one of the samples, oral and/ or scraping, was positive for hpv. furthermore, women were considered negative for oropharyngeal hpv if their oral and scraping samples were negative for hpv. statistical analyses were performed using the graph-pad prism 6.0 (san diego, california, usa) software. all variables were expressed as absolute and relative frequencies. for univariate analysis (unadjusted odds ratios [ors]), categorical variables were compared with hpv infection using the chi-squared and fisher's exact test. crude ors and 95% confidence intervals (cis) were calculated. a p-value < 0.05 was considered significant. a total of 254 (115 hiv+ and 139 hiv-) women were included in the study, with all 254 cervical and 508 oral samples (oral scraping and gargle) having sufficient dna for hpv assessment. all 254 (100%) participants had conclusive hpv test results from both anatomical sites. hiv diagnosis (years) yes 93 (80.9) --haart, highly active antiretroviral therapy; or = odds ratio; ci = confidence interval demographic and clinical characteristics of the two groups of patients are presented in table 1 . the median age was 42.17 ± 10.18 years old for hiv+ women and 41.4 ± 12.31 years old for hiv-women. compared to hiv-women, hiv+ women were significantly more likely to have less than 8 years of schooling (p = 0.0003), non-white skin color (p = 0.009), their first sexual intercourse at < 18 years old (p = 0.04), more than two sexual partners (p = 0.004 for 2-7 partners and p = 0.007 for > 7 partners), and a higher number of parities (p = 0.0006 for 1-2 parities and p 0.0001 for ≥3 parities), and were less likely to report screening for cc within the past three years (p = 0.01). hiv-women were, however, more likely to be a widowed (p = 0.004). most hiv+ women presented excellent control of the hiv infection based on their compliance with haart (80.9%), preserved cd4+ t lymphocyte count (82.6% with > 350 cells/mm 3 ), and suppressed current viral loads (86.1% undetectable). additionally, most of the hiv+ women had had documented hiv infections for < 5 years (63.5%) ( table 1) . cytology showed no signs of malignancy in most women from both groups. overall, 13.0% of hiv+ women and 5.0% of hiv-women presented abnormal cytological findings (p = 0.04). atypical squamous cells of undetermined significance (asc-us) were observed in 2.6% of hiv+ women and in 1.4% of hiv-women (p = 0.66); lowgrade squamous intraepithelial lesions (lsil) were observed in 7.8% of hiv+ women and in 2.8% of hivwomen (p = 0.15); high-grade squamous intraepithelial lesions (hsil) were observed in 2.6% of hiv+ women and 0.8% of hiv-women (p = 0.33). overall, 56 (48.7%) of the 115 hiv+ women were positive for cervical and/or oral hpv dna, while 57 (41%) of the 139 hiv-women were positive for cervical and/or oral hpv (p = 0.25). both hiv+ and hiv-women had a statistically higher prevalence of cervical hpv infection than oral infection, including higher rates of hrhpv, lrhpv, universal hpv, and infection by multiple hpv types in cervical samples compared to oral ones, as shown in table 2 . hpv dna was detected in oral samples from 17 (14.8%) hiv+ women and 13 (9.4%) hiv-women (p = 0.24) ( table 3) . multiple hpv infections were detected in five samples (4.4%) from hiv+ women and in four samples (2.9%) from hiv-women (table 2 ). statistical analysis did not reveal an association between hiv+ status and the presence of hpv dna in the oral mucosa (p = 0.83). fifty-one (44.3%) hiv+ women and 52 (37.4%) hivwomen had hpv-positive cervical samples (p = 0.48) ( table 3) . multiple hpv types were detected in 28 (24.3%) of hiv+ and in 29 (20.9%) of hiv-patients (table 2) . there was no significant difference between the hiv+ and hiv-women with regard to hpv types. to better understand the association between cervical and oral hpv infection, the prevalence of concurrent hpv infection in this population was investigated. eight (7.0%) hiv+ women and 7 (5.0%) hiv-women had concurrent hpv infection in their cervical and oral samples (p = 0.6) ( table 3) . concordance between hpv types in the cervical and oral samples was not observed in either group. in the hiv+ group, the most frequent cervical hrhpv types observed were hpv18, hpv45, and hpv58 (14.8% each). in the oral site, the most prevalent hrhpv was hpv39 (33.3%) followed by hpv18, hpv45, hpv52, and hpv68 (16.6% each). the most prevalent cervical lrhpv found in this group was hpv6 (17.5%), followed by hpv61 (12.5%), and in oral mucosa, the most prevalent lrhpv was also hpv6 (28.6%), followed by hpv62 and hpv81 (21.4%) (fig. 1) . in the hiv-group, the most prevalent hrhpv in the cervical mucosa was hpv18 (14.2%), followed by hpv16 and hpv68 (11.9% each). in the oral site, hpv51 and hpv66 were the most prevalent hrhpv (33.3% each). the most prevalent lrhpvs in the cervical site were hpv81 (29.0%) and hpv54 and hpv70 (16.1% each), and the most prevalent lrhpv in the oral site was hpv6 (30.0%), followed by hpv43 (20%) (fig. 2) . hiv+ women with recent detectable hiv loads were significantly associated with hpv-positive status overall (or = 3.75; ci = 1.22-11.10; p = 0.03) and in cervical mucosa (or = 4.61; ci = 1.50-13.66; p = 0.01) ( table 4) . when the characteristics of the hiv+ women were analyzed in relation to hpv status, only current smoking was associated with overall (or = 3.80; ci = 1.22-10.42; p = 0.01) and cervical hpv-positive status (or = 3.94; ci = 1.31-11.76; p = 0.01 for both), as shown in table 5 . in the present study, we aimed to determine the hpv prevalence, distribution, and type concordance between cervical and oral samples of hiv+ women and hivmatched controls in the southern region of brazil, a geographic area with high incidences of hiv and cc. our data demonstrated that hiv+ and hiv-women had a similar and high hpv prevalence in cervical and in oral sites. however, hiv+ women had higher prevalence of abnormal cytological findings than hiv-women. hpv type distribution was different between the anatomical sites within both groups, and hiv+ women commonly presented with narrower hpv type distributions, mainly in the oral mucosa. finally, the frequency of concurrent hpv infection in both sites was low, and hpv type concordance was not observed. our results showed that the prevalence of cervical hpv infection was 44.3% in hiv+ women and 37.4% in hiv-women. studies across different populations present varying rates of hpv infection: .1% in hiv+ women and 44.9-86.5% in hiv-women in the cervical site [20, 24, 25, 33] . the high prevalence of hpv in hiv-negative women in this study can be explained, at least in part, by the fact they were recruited from a specialized assistance service (sae) for sexually transmitted diseases, and were therefore at higher risk for sexually transmitted infections, including hpv. additionally, the rates of oral hpv infection in hiv+ women and hiv-control were 14.8 and 9.4%, respectively. other studies have shown that the prevalence of hpv infection in the oral site can vary significantly (12-68.5% in hiv+ women and 2-31.4% in hiv-women) [20, 24, 33] . although hiv infections were well controlled and cytology results showed no malignancies, abnormal cytological findings were significantly higher in hiv+ women than in hiv-women. these data are consistent with the results of the previous studies showing that hiv+ women are less likely to eliminate the virus, have subsequent persistent hrhpv infections and have higher risk of developing precancerous lesions and malignancies, even with the appropriate use of antiretroviral therapy, than hiv-women [8, 9, 19, 34, 35] . our results demonstrated no significant difference in hpv detection in the uterine cervix or oral mucosa between hiv+ women and hiv-women; however, hpv infection was significantly higher in the cervical mucosa than in the oral site in both groups. these findings are consistent with previous studies suggesting that the natural history of hpv infection varies by anatomical site, and a higher prevalence of hpv infection is observed in the cervical mucosa than in the oral mucosa in hiv+ women [36] [37] [38] . this can be explained, at least in part, by the evidence that the oral cavity is a hostile environment for the establishment of infectious agents due to the presence of both mechanical and molecular mechanisms related to digestion [39] . more specifically, fakhry et al. [39] conducted a study that directly compared the local immunologic profiles of the oral cavities and cervices of healthy women using paired secretion specimens. this study showed that the oral cavity contained significantly higher concentrations of immunoregulatory factors that were related to the adaptive and cellmediated immune response than the cervix, which may in part explain the significantly lower burden of sexually transmitted infections such as chlamydia trachomatis, hpv, and hiv-1 in the oral cavity than in the cervix. according to the authors, these findings provide additional information to better understand the differences in the etiology and natural history of pathogenic agents that are capable of colonizing both the oral cavity and female reproductive tract. the most prevalent hpv types in the cervical samples from both hiv+ and hiv-women were hrhpv18 and hrhpv58; however, hrhpv45 and hrhpv16 were frequently detected in hiv+ women and hiv-women, respectively. prevalence studies around the world have shown that hrhpv types 16, 18, 31, 33, 35, 52 , and 58 are the most commonly detected hrhpvs in cc, with hrhpv16 being the most common in all populations, with the exception of hiv+ people [1] . data have consistently shown that hiv+ women are frequently more infected with other hrhpv types than hrhpv16 and hrhpv18, such as hrhpv52 and hrhpv58 [40] . the high prevalence of non-vaccine hrhpv types of 2-valent and 4-valent vaccines in the cervical and oral mucosa found in our study suggests that the 9-valent hpv vaccine is significantly required, which is considered important to reduce the risk of developing hpv-related cancers, specifically in the hiv+ population. unlike the distribution of hpv types in cervical samples, hpv type concordance between the hiv+ and hiv-groups was not observed in oral samples. hrhpv39 was the most common hrhpv detected in oral samples of hiv+ women, followed by hrhpv18, hrhpv45, hrhpv52, and hrhpv68, with these types being also frequently found in cervical samples in the same group. however, in hiv-women, the oral hrhpv types observed were totally different, with hrhpv51 and hrhpv66 being the most frequently detected hrhpv. current evidence has shown that hrhpv16 and hrhpv18 contribute to the majority (approximately 85%) of hnc cases worldwide, while the remaining cancers are caused by hrhpv33, hrhpv35, hrhpv52, hrhpv45, hrhpv39, and hrhpv58 [41, 42] . examination of concurrent hpv infections in different anatomical sites has been limited. hence, we concurrently investigated the prevalence of hpv infection and hpv type distribution in cervical and oral sites to better understand their clinical significance. simultaneous hpv cervical and oral infection was low in both hiv+ and hiv-women. moreover, hpv type concordance was not observed, a finding consistent with previous studies [20, 24] . taken together, these observations suggest that various hpv types are more likely to occur in a different anatomical sites, and/or that the two anatomical sites can clear certain hpv types or distinct exposures, while allowing other hpv types to cause persistent oral and cervical hpv infections. prior investigations have demonstrated that engagement in some high-risk behaviors may facilitate hpv infection and act as a cofactor for viral persistence, contributing to cancer progression in the oral and cervical mucosa. in the present study, we found that current smoking was associated with overall and cervical hpv infection in hiv+ women. several studies have already demonstrated that smoking status acts as a predictor and cofactor in cervical hpv infection, which is possibly due to the alteration of the mucosa cells, making them more susceptible to infection, changing the immune mediators, causing dna damage, and promoting the integration of hpv dna into the host genome [42] [43] [44] [45] . our study demonstrated that hiv+ and hiv-women in southern brazil had high hpv prevalence in cervical and oral sites. in this population, hiv+ women had more abnormal cytological findings than hiv-women. this study provides important epidemiological data about the potential risk of developing cc. furthermore, the high prevalence of non-vaccine hrhpv types of the 2-valent and 4-valent vaccines in the cervical and oral mucosa of hiv+ and mainly in hiv-women found in our study highlights the importance of the 9-valent vaccine. concurrent hpv infection in both sites was uncommon, and hpv type concordance was not observed, likely reflecting the differences in the risk factors and the natural history of hpv infection at the two anatomical sites. these findings confirm the prevalence of hpv infection in hiv+ women. prospective studies are required to better understand the natural history of hpv infection in both anatomical sites, specifically in hiv+ women, and the impact of vaccination programs in at-risk groups. a review of human carcinogens. iarc monogr eval carcinog risks to humans. france human papillomavirus-related diseases in hivinfected individuals the human papillomavirus type 16 e6 oncoprotein activates mtorc1 signaling and increases protein synthesis hpv-associated head and neck cancer: a virus-related cancer epidemic incidence and risk factors of hpv-related and hpv-unrelated head and neck squamous cell carcinoma in hiv-infected individuals prevalence of human papillomavirus genotypes in hiv-1-infected women in human papillomavirus-related disease in people with hiv the impact of haart on hpv-related cervical disease hiv-positive women have higher risk of human papilloma virus infection, precancerous lesions, and cervical cancer human papillomavirus-related cancers among people living with aids in puerto rico fact sheet on adolescent health. geneva adolescent girls and young women: key populations for hiv epidemic control a scoping review of prevalence, incidence and risk factors for hiv infection amongst young people in brazil potential impact of a 9-valent hpv vaccine in hpv-related cervical disease in 4 emerging countries (brazil, mexico, india and china) impact of hpv vaccination and cervical screening on cervical cancer elimination: a comparative modelling analysis in 78 low-income and lower-middle-income countries a 9-valent hpv vaccine against infection and intraepithelial neoplasia in women human papilloma virus types in the oral and cervical mucosa of hiv-positive south african women prior to antiretroviral therapy epidemiological data of different human papillomavirus genotypes in cervical specimens of hiv-1-infected women without history of cervical pathology risk factors for cervical hpv infection and genotypes distribution in hiv-infected south brazilian women oral and cervical hpv infection in hiv-positive and hiv-negative women attending a sexual health clinic in sao paulo higher prevalence of oral human papillomavirus infection in hiv-positive than hiv-negative thai men and women prevalence and distribution of cervical high-risk human papillomavirus and cytological abnormalities in women living with hiv in denmark-the shade high-risk human papillomavirus genotypes distribution in a cohort of hivpositive women living in europe: epidemiological implication for vaccination against human papillomavirus relationship between prevalent oral and cervical human papillomavirus infections in human immunodeficiency virus-positive and -negative women cervical and oral human papillomavirus types in hiv-1 positive and negative women with cervical disease in south africa prevalence and concordance of human papillomavirus infection at multiple anatomic sites among hiv-infected women from chennai hpv involvement in head and neck cancers: comprehensive assessment of biomarkers in 3680 patients human papillomavirus-related oropharyngeal cancer hpv in head and neck cancer-30 years of history. recent results cancer res fortschritte der krebsforsch prog dans les rech sur le cancer hpv in head and neck carcinomas: different hpv profiles in oropharyngeal carcinomas -why? bethesda system for cervical-vaginal cytology risk factors for acquisition and clearance of oral human papillomavirus infection among hiv-infected and hiv-uninfected adults prevalence of cervical, oral, and anal human papillomavirus infection in women living with hiv in denmark-the shade cohort study high prevalence and incidence of hpv-related anal cancer precursor lesions in hiv-positive women in the late haart era the occurrence of human papilloma virus (hpv)-related cancers in human immunodeficiency virus (hiv)-infected women compliant with highly active antiretroviral therapy (haart) sixmonth natural history of oral versus cervical human papillomavirus infection natural history of anal vs oral hpv infection in hiv-infected men and women hpv infection, cervical abnormalities, and cancer in hiv-infected women in mumbai, india: 12-month follow-up comparison of the immune microenvironment of the oral cavity and cervix in healthy women women with hiv are more commonly infected with non-16 and-18 high-risk hpv types hpv dna, e6/e7 mrna, and p16ink4a detection in head and neck cancers: a systematic review and meta-analysis worldwide burden of cancer attributable to hpv by site, country and hpv type carcinoma of the cervix and tobacco smoking: collaborative reanalysis of individual data on 13,541 women with carcinoma of the cervix and 23,017 women without carcinoma of the cervix from 23 epidemiological studies response to therapy and outcomes in oropharyngeal cancer are associated with biomarkers including human papillomavirus, epidermal growth factor receptor, gender, and smoking differential effect of smoking on gene expression in head and neck cancer patients all the authors contributed to the manuscript. tts, gmzfd, ed, and melc searched the literature and prepared the manuscript. smp collected the biological samples from the women. tts and melc wrote the manuscript. melc, wh, rf, vrss, smp, llrar, maps, and ics participated in the research design and execution. tts and ed performed the statistical analysis. gmzfd, ed, wh, raaf, vrss, smp, and ics were involved in revising the manuscript to include critically important intellectual content. melc revised the final version of the manuscript and provided information and suggestions. all the authors read and approved the final draft of the manuscript. this work was supported through a research grant from the nci (p30ca013148-43s2). the funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.availability of data and materials all data are included in the manuscript. the study involves human participants. this study was approved by the local ethics committee ( each author gives consent for publication. the authors declare that they have no competing interests.author details 1 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-021966-5m21bsrw authors: shaw, alan r.; feinberg, mark b. title: vaccines date: 2009-05-15 journal: clinical immunology doi: 10.1016/b978-0-323-04404-2.10092-2 sha: doc_id: 21966 cord_uid: 5m21bsrw nan vaccines represent one of the most effective and cost-effective medical and public health achievements of all time. 1 worldwide, vaccination programs are currently estimated to save over 3 million lives each year. in addition to having such a major beneficial impact on vaccine-preventable disease morbidity and mortality, the direct and indirect impacts of vaccination programs translate into economic savings of many billions of dollars each year. in what is considered to be one of the most significant medical successes of all time, a collaborative and comprehensive vaccination campaign against smallpox resulted in the global eradication of the disease in 1979. 2 similarly, efforts to eradicate poliomyelitis have made tremendous progress in reducing the global disease burden, and will hopefully soon overcome certain residual societal and programmatic obstacles to provide the second successful example of elimination of a major health threat by vaccination. concerted global efforts to provide measles vaccine have resulted in the control and elimination of the disease in many countries, including substantial reductions in mortality in a number of developing countries where the residual disease burden is greatest. these and other examples provide clear evidence of the power of vaccines in favorably manipulating host immunity to confer dramatic public health benefits, at both the individual and population level. as vaccines are administered to healthy individuals (often to entire age cohorts or populations), to prevent diseases caused by infectious agents to which they might be exposed in the future, they differ in important ways from pharmacologic agents that are used to treat individuals in whom a disease process is already manifest (or who display predispositions to disease). for this reason, vaccines are unique in the way that they impact on societies and in the way that societal commitment to vaccination determines their ultimate impact. as a result, vaccination efforts provide an informative window on challenges that need to be successfully navigated at the interface between scientific opportunity and societal capacity and commitment. indeed, current limitations in realizing the full global potential of available vaccines relate more to existing inadequacies in health care financing and infrastructure (especially as they are manifest in developing countries), and the relative value that societies place on disease prevention, than they do to any inherent biological limitations of vaccines themselves. fortunately, recent acceleration of new vaccine introductions in developing countries through public and private initiatives to build immunization infrastructure and provide funding of vaccine purchase offers hope that vaccines will one day be equitably available to all who need them. 3 the importance of vaccines extends beyond their use as public health tools to include their role as drivers of immunologic discovery. the history of vaccine development is rich with immunologic insights that emerged from careful observations of how diseases spread in populations and how such spread differs in disease-naïve and experienced populations, as well as of how innovative experimental approaches revealed fundamental aspects of immune system function. the general concept of immunity induced by prior exposure to a disease (including its specificity and potential lifelong duration) was appreciated by the ancient greeks. use of the word 'immunity' itself dates to the 14th century when it was applied to describe the relative susceptibility and resistance of populations to plague. the subsequent successes of edward jenner and louis pasteur in the development of effective smallpox and fowl cholera immunization strategies, respectively, provided a foundation for modern immunology; pasteur himself coined the term 'vaccine' in recognition of jenner's use of vaccinia virus. jenner's smallpox immunization studies also provided early experimental support for the concept of immune memory. pasteur's efforts provided the first demonstration of the attenuation of pathogens by their propagation in culture (or by passage in nonnatural animal hosts), while robert koch demonstrated that killed pathogens could also engender immunity. the discovery of bacterial exotoxins by emile roux and alexandre yersin facilitated the discovery of antibodies and their potential use in passive immunotherapy with antitoxin antibodies by emil von behring and shibasaburo kitasato. these discoveries enabled the development of active immunization against diphtheria and tetanus using toxin-antitoxin mixtures. paul ehrlich's development of accurate methods for antibody quantitation made passive immunotherapy and active toxin-antitoxin immunization far more reliable and effective, and provided a stimulus for significant advances in immunologic theory. in each of these instances, vaccine development illuminated central mechanisms of immune system biology. vaccine development today has transitioned from an approach that was once largely empirical to one that is based on the hypothesis-driven application of techniques in molecular biology and immunology. evidence for this synergy can be seen in recent studies of vaccine-elicited immune responses to illuminate primary and memory t-and b-cell responses in humans, as well as the strong discovery stimulus provided by ongoing efforts to develop new vaccines for major infectious diseases for which vaccines are not currently available. vaccine development today faces a number of significant challenges. there exist tremendous public health needs to address major well-known pandemic diseases, including acquired immunodeficiency syndrome (aids), tuberculosis, and malaria, for which no vaccines currently exist and for which natural immunity does not provide a helpful guide for vaccine development. furthermore, there exists a need to confront effectively newly emerging and re-emerging diseases, ranging from the well-known, but constantly changing, threats from influenza pandemics to the appearance of previously unknown zoonotic infections such as the coronavirus that causes severe acute respiratory syndrome (sars). with changes in population density, mobility, and social constructs, along with alterations in the global climate, ecological circumstances, and the proximity of humans to animal reservoirs for previously confined infectious agents, the concept of new infectious agents entering human populations and spreading rapidly around the world is no longer novel. in confronting prevalent or newly emerging diseases, vaccines are looked to as the most promising line of defense. however, the speed at which new infectious disease threats have been shown to emerge and spread, and the fact that the pathogens that now need to be confronted may display tremendous genetic variability (e.g., human immunodeficiency virus (hiv)) or an identity that cannot be predicted in advance (e.g., avian influenza or agents like sars) places unprecedented demands on the vaccine development process. in addition to these new challenges, there remain unmet needs in the derivation of vaccines that can achieve the greatest public health benefit. these needs include the development of new ways to achieve more effective vaccine-elicited immune responses in neonates whose immune systems are immature (or are impacted by maternal antibodies) (chapter 32) and in the elderly whose immune system function may be waning as a result of immune senescence (chapter 33). fortunately, the scientific foundation provided by basic and applied immunology and the use of new methods for pathogen identification, antigen discovery, vaccine production, adjuvant development, and novel vector derivation afford important opportunities for vaccine development and additionally present the possibility of improving on natural immunity. success in vaccine development will be predicated on continuing the historical synergy between advances in vaccine technology and basic immunologic discovery. toward that end, this chapter focuses on preventive vaccines for infectious diseases and how they are developed. although current routine vaccine recommendations are reviewed, given the active state of new vaccine introduction and evolving vaccine recommendations, as well as differences in recommendations in different countries, readers are encouraged to refer to up-to-date national resources for the most current information. while vaccine approaches are being actively explored to modify beneficially malignant and immunologic diseases (autoimmunity and allergy), these are beyond the scope of the current discussion. n impact of vaccination programs n unlike other medical interventions, vaccines confer benefits to both individuals and populations. 4, 5 while individuals may be protected from infection or disease by vaccine-induced immune responses, decreasing the number of susceptible hosts in a population also helps break the chain of transmission that pathogens require to spread and persist in human populations by induction of 'herd immunity. ' the benefits of herd immunity depend on achieving sufficiently high immunization rates in a population to impact pathogen transmission dynamics (including the potential for extinction of ongoing interhost transmission). the requisite level of vaccination coverage of a population needed to compromise pathogen spread significantly varies between pathogens, and is influenced both by vaccine efficacy (and its duration) and by the reproductive characteristics and infectiousness of the pathogen. analysis of the impact of vaccination programs in the usa provides an example of the beneficial impact of vaccines when used routinely and when high coverage levels are achieved. 6 as shown in tables 92.1 and 92.2, vaccination programs in the usa dramatically decreased the annual morbidity of many vaccine-preventable diseases. in many instances, the disease burden from several vaccine-preventable diseases of childhood has been reduced by over 99% since vaccine introduction (e.g., diphtheria, tetanus, measles, mumps, rubella, and polio). the somewhat lower rate of decline of pertussis (the annual morbidity of which has been reduced by a nonetheless impressive 83%) relates to the limited duration of vaccine-induced immunity, which is estimated to wane within 5-10 years after childhood vaccination. it is anticipated that recent availability of pertussis booster vaccines for use in adolescents and adults will lead to significant further declines in pertussis morbidity. even for diseases targeted by vaccines that have been in widespread use for less time (< 10 years), impressive decreases in disease morbidity have been seen (e.g., varicella, hepatitis a, and pneumococcal disease). in a notable recent demonstration of the population benefits of vaccines, introduction of the 7-valent pneumococcal conjugate vaccine resulted in a decrease of 73% in disease morbidity in children under 5 years of age within the first 5 years of its introduction. interestingly, the rate of meningitis and bloodstream infections caused by antibiotic-resistant streptococcus pneumoniae also fell by 81% in this age group. in a striking related finding illustrating how vaccines can impact pathogen transmission dynamics, rates of antibiotic-resistant pneumococcal infections also declined by 49% in individuals over the age of 65 who had not received the vaccine. thus, direct protection by vaccination of children who represent a reservoir of infection provided, via herd immunity, significant indirect benefits to those who did not themselves receive the vaccine. in addition to their benefits in preventing disease morbidity and mortality, routine vaccination programs are also impressively cost-effective. evaluation in the usa of the impact of ten vaccines routinely given as part of the childhood immunization schedule (diphtheria, tetanus, pertussis, haemophilus influenzae b (hib), polio; measles, mumps, rubella, hepatitis b and varicella) found that more than 14 million cases of disease and more than 33 500 deaths were averted over the lifetime of the immunized birth cohort of children. 7 when the cost of the vaccination program was compared to the economic impact of diseases prevented, these vaccines alone are estimated to save nearly $10 billion each year. when including indirect economic benefits (such as the time parents take off from work to care for sick children), the annual savings to society exceed $40 billion. when 30 preventive services were ranked based on clinically preventable disease burden and cost-effectiveness, childhood immunization received the highest score. 8 progress in the development of new vaccines accelerated significantly towards the end of the 20th century, with the development of vaccines against diseases that were not previously preventable by vaccination, but also with the development of improved versions of existing vaccines. thus, the number of diseases that can be prevented by vaccines included in the us centers for disease control and prevention's (cdc) routine childhood and adolescent immunization schedules grew from seven in 1985 to 16 in 2007 (table 92 .3 and fig. 92.1) . moreover, in the past several years, new vaccines have been introduced for adolescents and young adults (e.g., pertussis booster (tdap), meningococcal conjugate, and human papillomavirus (hpv) vaccines), and older adults (e.g., tdap and zoster vaccines) have shown that the value of vaccines extends across the human lifespan (figs 92.1 and 92.2). new combination vaccines have been developed to increase the simplicity and acceptability of vaccination regimens, as well as to improve overall compliance with the recommended series of vaccines. such combinations include either those that contain multiple inactivated or recombinant antigens (such as a combination diphtheria, pertussis, tetanus, hib, and hepatitis b vaccine) or multiple live attenuated viruses (such as a combination measles, mumps, rubella, and varicella vaccine (mmrv)). the development of a combination vaccine is often more complicated than simply combining individual antigens, for when antigens are administered in combination, immunologic interference is sometimes seen. this necessitates titration of antigen combinations (and in the case of combinations of inactivated and/or recombinant antigens, adjuvant selection) to achieve immune responses that are not inferior to each of the antigens administered individually. despite their readily demonstrable public health impact, the value of vaccines is often not appreciated, for when vaccine programs are successful the diseases that they cause become less prevalent and may disappear. however, to prevent resurgence of an infectious disease that has been brought under control, vaccination programs need to be continued. the difficulties facing current efforts to eradicate poliomyelitis have demonstrated that failure to maintain high immunization coverage rates can lead to prompt re-emergence and spread of the disease. even in developed countries, maintenance of strong immunization programs with high degree of coverage is needed where infectious diseases can travel with remarkable speed -and do so even before the extent of spread is evident. n principles of immunization n the terms vaccination and immunization are often used interchangeably. however, vaccination specifically refers to efforts to induce protective immune responses by administration of a vaccine, whereas immunization more generically refers to interventions -either active or passive -that seek to confer immune protection. active immunization describes the induction of immune responses by administration of a specific antigen or antigens, while passive immunization involves the administration of exogenous immunologically active substances (historically, antibodies present in sera obtained from immune individuals or animals) to confer temporary protection from an infectious pathogen or toxin. although the approaches for passive immunization waned in the later half of the 20th century, the advent and increasing robustness of monoclonal antibody technology have led to a resurgence of interest in passive immunization. vaccines seek to engender immune responses similar to those that confer immunity to re-infection in individuals who experience (and survive) natural infection with a given pathogen. in lieu of formal demonstration of a specific type of antibody or cellular immune response that contributes to prevention or accelerated clearance of an infection, most often vaccine efficacy is demonstrated first in the course of a placebo-controlled trial. in some instances, specific immune effector mechanisms, such as a specific level or type of antibody response, can be identified that correlate with immune protection. in this case, the 'correlate of immunity' provides a benchmark against which similar vaccines can be compared. in the case of most inactivated vaccines, subunit vaccines, and recombinant vaccines that produce antibody responses, but generally meager cd8 t-cell responses, it is likely that humoral immune responses are the primary or sole protective immune mechanism. in the case of live attenuated vaccines that induce both cellular and humoral immune responses against the pathogen, it is likely that both arms of the immune system act in concert to confer immunity. however, the actual mechanisms of immune protection induced by either a natural infection or a vaccine are generally not understood in detail for many infectious diseases. similarly, although vaccines depend on the induction of immunologic memory, the magnitude, character, and duration of immune memory differ between vaccines, as can the actual mechanism of immune protection. for certain vaccines, such as those that protect against bacterial diseases induced via production of toxins (e.g., diphtheria or tetanus), protection induced by toxoid-based vaccines is clearly dependent on persistent antibody (igg) and memory b-cell responses, ensuring that sufficient antitoxin antibodies are present at the time of toxin exposure to inactivate and clear the toxin. in other cases, such as long-lived protection against hepatitis b, if sufficient levels of antibodies are achieved in the initial immunization period, even hosts who may with time lose detectable levels of antibody responses remain protected. 9 in this instance, given the relatively long incubation period of hepatitis b, memory antiviral b-cell responses induced by the vaccine can be activated, facilitating neutralization and clearance of the infection before clinical disease is manifest. although it is popularly believed that vaccines confer protection by inducing 'sterilizing immunity' -wherein an infectious agent is blocked from even infecting one cell in an exposed host -this is clearly not the case for a number of vaccines. for example, the inactivated poliovirus and live attenuated rotavirus vaccines do not prevent some degree of local replication of their pathogenic counterparts in the gastrointestinal tract of exposed hosts. however, they are both effective in preventing clinical disease. in the case of poliovirus vaccine, this is mediated by elicitation of antibody responses that block dissemination of the infection to the central nervous system; while in the case of rotavirus, as yet unidentified immune effectors limit local virus replication so that significant gastrointestinal damage does not occur following infection. 10, 11 the major types of vaccines licensed for use include live attenuated organisms, killed or inactivated organisms, subunit vaccines consisting of purified (or partially purified) components of an organism, and subunit vaccines produced by recombinant dna technologies. the use of live attenuated vaccines dates back to the early work of jenner and pasteur on smallpox and fowl cholera vaccines, respectively. 12, 13 the fundamental concept of live attenuated vaccines is to mimic the effective host immune responses that follow natural infections. most live attenuated vaccines currently in use were derived by propagation of initially pathogenic organisms in culture on cells from different (nonhuman) species, or at nonphysiologic temperatures, for prolonged periods. driving pathogen evolution in culture to select for variants adapted to growth in heterologous cell types ex vivo often leads to the derivation of pathogen variants that grow poorly in vivo in humans and are unable to cause clinical symptoms. vaccines developed via this approach include those used to prevent a number of viral and bacterial infections, including yellow fever, measles, mumps, rubella, polio (the 'sabin vaccine'), varicella-zoster (used both for the prevention of chickenpox and shingles) and rotavirus (one version of the available vaccines), tuberculosis, and cholera. more recent technologies being applied to live attenuated vaccine development include the application of reverse genetic strategies ( fig. 92 .3) and those involving genetic reassortment with attenuated viral variants, as have been used to develop polyvalent live attenuated vaccines against influenza and rotavirus ( fig. 92.4) . 10, 11 the live attenuated vaccines currently in use are highly efficacious (> 90%) and protection is frequently durable. the efficacy of many live attenuated vaccines likely reflects the ability of the attenuated vaccine to replicate within vaccinated hosts, and to expose the immune system to pathogen-derived antigens in a manner that closely resembles the nature, location, and effects of natural infection. because live attenuated vaccines replicate within immunized individuals, they can induce both cellular (cd4 and cd8) and humoral (b-cell) effector responses and immunologic memory. in addition, as the live attenuated vaccines likely activate the host innate system in a manner similar to their pathogenic parents, they provide inherent adjuvant effects in augmenting adaptive immune responses. a key consideration in the development of any live attenuated vaccine relates to the relative balance between the ability to induce sufficient immune responses in vivo to confer protection (often associated with level of preserved replicative ability in vivo), and the ability to cause symptoms (which may also relate to the extent of in vivo replication). as such, an effective but also safe and well-tolerated vaccine needs to strike a specific balance between level of attenuation and level of immunogenicity. in addition, depending on the nature and number of genetic mutations responsible for the attenuated phenotype, a potential risk of reversion to a pathogenic form exists for certain vaccines. for most live attenuated vaccines, this has not been observed to be a problem in clinical practice -likely because the attenuating mutations are sufficiently numerous or genetically stable. one vaccine where reversion to pathogenic form was seen involved specific components of the live attenuated oral poliovirus vaccine (opv; the 'sabin vaccine'). in this instance, vaccine reversion to wild-type was shown to lead rarely to cases of paralytic polio (approximately one case per million doses administered). 14 based on these observations and the elimination of endogenous polio transmission in many developed countries, the inactivated polio vaccine (ipv; the 'salk vaccine') was substituted for opv. however, in light of a favorable cost-benefit ratio, high degree of efficacy, and ease of administration, opv continues to be the mainstay of polio vaccination efforts in developing countries. the use of physical or chemical methods to kill or otherwise inactivate a pathogenic organism represents a second major approach to vaccine production. 15, 16 in most cases, treatment with chemical agents such as β-propiolactone and formaldehyde is used to eliminate pathogen infectivity. while this approach has the benefit of presenting most of a pathogen's antigenic repertoire to the immune system of the immunized host, it can only be used in instances where the inactivated pathogen does not possess constituents that would confer significant toxicity. vaccines based on killed pathogens are believed to exert their protective effects via elicitation of pathogen-neutralizing antibodies and the induction of memory b-cell responses (likely in concert with cd4 t-cell memory). however, because inactivated pathogens cannot accomplish de novo synthesis of pathogen-derived gene products in antigen-presenting cells (apcs), they do not typically induce cd8 t-cell responses (chapter 6). in addition, killed vaccines are generally less immunogenic than live attenuated vaccines. as a result, they are commonly administered with an adjuvant (most often alum: see section on adjuvants, below) to augment their immunogenicity. a number of viral and bacterial vaccines currently in use are killed/inactivated vaccines, including whole-cell bordetella pertussis vaccine and the influenza virus, rabies virus, and hepatitis a virus vaccines. a number of bacteria produce toxins that represent the major pathogenic components responsible for disease in infected humans. examples include corynebacterium diphtheriae and clostridium tetani. detoxified versions of these toxins are referred to as 'toxoids,' and represent the purified components of vaccines preventing diphtheria and tetanus, respectively. toxoids have historically been produced by chemical inactivation of toxins, but more recently, genetic inactivation via targeted mutagenesis has been employed. the acellular pertussis vaccine is also a purified subunit vaccine composed of a defined set of protein constituents prepared from cultured bordetella pertussis. the mechanism of immune protection conferred by purified subunit vaccines is the antibody response elicited by vaccination. antibodies directed against the capsular polysaccharides present on encapsulated bacteria also confer protective immunity in a number of important instances by inducing antibodies that exert opsonophagocytic effects (promoting phagocytosis of antibody-coated bacteria) and, in some instances, bactericidal effects. 17 initial successful vaccine efforts against streptococcus pneumoniae and neisseria meningitidis utilized purified preparations of capsular polysaccharides. although such purified polysaccharides can induce protective levels of antibody responses in adults, they are poorly immunogenic in children under 2 years of age (as a function of the relative immaturity of their immune systems). in addition, t-independent antibody responses elicited by purified capsular polysaccharides are less durable than those that are produced in the presence of cd4 t-cell help. as a means of both augmenting antibody responses against polysaccharide antigens in young children and facilitating their persistence, the development of conjugate vaccines represented an important advance. 18 in this approach, purified polysaccharides are chemically conjugated to a carrier protein (such as diphtheria toxoid or an outer-membrane protein complex (ompc) derived from n. meningitidis). the carrier protein augments cd4 t-cell helper responses to the polysaccharide antigens, and enables elicitation of durable protective antibody responses even in young children. polysaccharideconjugate vaccines have been produced that protect against haemophilus influenzae b, streptococcus pneumoniae, and n. meningitidis infections. if two such segmented viruses with different genetic characteristics are used to infect one cell, the progeny viruses from this mixed infection will carry a range of mixtures of the genes of the two parent viruses. using either genetic or immunologic screening methods, reassorted viruses carrying the precise gene composition of interest can be selected. this approach has recently been employed to generate live attenuated vaccines against rotavirus and influenza virus. the strategy for generation of the pentavalent bovine-human reassortment rotavirus vaccine is shown above. rotaviruses have a segmented double-stranded rna genome comprising 11 independent rna elements. the outer shell of the virus comprises two proteins vp4 and vp7 that are involved in cell binding and entry and that specify the viral serotype (p type for vp4 and g type for vp7). vp4 and vp7 also represent the targets of virus-neutralizing antibodies. the pentavalent bovine-human rotavirus vaccine was generated by a 'modified jennerian' approach in which the bovine rotavirus wc3 (which is attenuated in humans as a result of host range restriction) serves as the gene donor for the backbone on to which gene segments encoding four common human rotavirus g types (g1-4) as well as one very common p type (p8) (derived from individual rotavirus isolates) were reassorted via a process of cell co-infection and subsequent selection of the recombinant viruses with the desired composition of bovine and human gene segments. an analogous genetic reassortment approach has also been used to generate live attenuated influenza vaccines. in this instance, three attenuated 'cold-adapted' viral strains (two a types and one type b) are used in co-infections in tissue culture with recent circulating wild-type influenza strains to derive vaccine strains that include the two relevant hemagglutinin (ha) and neuraminidase (na)-encoding gene segments admixed with the six 'backbone' genes from the attenuated master donor virus for use in annual influenza vaccines. the first recombinant vaccine developed, the recombinant hepatitis b surface antigen (hbsag) prepared in yeast, was developed in hopes of avoiding safety concerns related to the plasma-derived hbsag vaccine. 19 the knowledge that immune sera could provide protection by passive immunization of naïve hosts, and that purified inactivated plasmaderived hbsag vaccine could elicit protective antibodies, laid the groundwork for development of this recombinant vaccine. 20 the recombinant vaccine, when combined with adjuvant (alum), elicits favorable immune responses, is highly efficacious and is well tolerated -all features that recombinant vaccines are now expected to deliver. the second recombinant vaccine developed targeted prevention of borrelia burgdorferi infection (the cause of lyme disease), and was based on a purified recombinant version of the ospa protein. this vaccine, although conferring some degree of efficacy, faced implementation challenges, and was not widely embraced. as a result, it was withdrawn from the market. more recently, recombinant technology-derived purified subunit vaccines have been developed that consist of virus-like particles (vlps) that self-assemble when the l1 protein of hpv is produced in isolation of other viral proteins ( fig. 92 .5). 21 the l1 protein is the target of virusneutralizing antibodies and vaccines consisting of a mixture of types 16 and 18 (the cause of ~70% of cases of cervical cancer) and 6 and 11 (the cause of ~90% of cases of genital warts) or of hpv types 16 and 18 alone have been shown to be highly efficacious and well tolerated. 22 interestingly, hpv vlps induce antibody responses that exceed those that follow natural hpv infections. 23 in light of these successes, and the power and versatility of recombinant antigen production methods, a major proportion of new vaccine development efforts involves the use of protein subunit vaccines produced by recombinant technologies. vaccines produced by this method are those that depend largely or exclusively on the induction of antibodies against individual or a selected subset of pathogen proteins. because a number of proteins produced in isolation by recombinant methods have been observed to elicit lower immune responses than do natural infections or live attenuated vaccines, the development and use of adjuvants to optimize recombinant vaccine immunogenicity represent an important parallel area for future exploration. n vaccine development and evaluation n as a necessary prelude to clinical evaluation of candidate vaccines in humans, extensive preclinical research and development activities are undertaken to establish that the vaccine candidate has the desired properties. toward this end, a number of key issues need to be addressed. first, animal studies must show that the vaccine candidate raises the desired type and magnitude of immune response against the infectious agent. second, the vaccine needs to protect animals against death or disease in an appropriate challenge model, when feasible. ideally, in the course of these studies, a specific type or level of immune response, referred to as a correlate of immune protection, can be identified. third, the vaccine should be relatively free of serious discernible toxicities and side effects in animals when administered by the route intended for humans. fourth, it is necessary to demonstrate that the vaccine can be produced in a consistent manner by a process that is consistent with the current good manufacturing practices (cgmp) process by which the first clinical trial materials will be produced (www.fda.gov/cber/gdlns/indcgmp.pdf ). bioengineered l1 proteins (5) l1 pentamer self-assembled virus-like particle in specific instances, vlps can be produced via a process of self-assembly of individual viral capsid proteins produced by recombinant dna methods in cell culture systems. this approach has a number of attractive aspects, including the ability to produce vlps that accurately display conformationally correct epitopes recognized by neutralizing antibodies and the absence of pathogen-derived nucleic acids. in addition, recombinant vlps have been employed to derive safe and effective vaccines for pathogens, such as hepatitis b virus (hbv) and human papillomavirus (hpv), that cannot be grown in culture (and are thus refractory to standard vaccine approaches of attenuation or inactivation). the generation of the vlps that comprises newly developed hpv vaccines is shown. the hpv l1 proteins (which represent the major capsid protein and target of virus-neutralizing, protective antibodies), derived from hpv types of interest (e.g., types 16, 18, 6, and 11) are produced via recombinant methods. under appropriate conditions, individual bioengineered l1 proteins first self-assemble into pentamers, and then into vlps that are comprised of 72 pentamers and that are almost identical, both morphologically and antigenically, to infectious hpv virus particles. vlps prepared from individual hpv types are then combined with specific adjuvants to prepare the final vaccine products. even before preclinical studies are completed, vaccine developers typically begin an initial dialog with regulatory authorities (such as the food and drug administration (fda) or the european medicines agency (emea)) to set expectations about what will be necessary and sufficient for advancement to clinical studies in humans (www.fda.gov/cber/ genetherapy/isct092506sh.pdf ). phase i studies primarily focus on detailed assessment of the safety and tolerability of a vaccine, but evaluation of its immunogenicity is also frequently conducted. generally, a phase i study includes fewer than 100 healthy volunteers divided unequally between those who receive vaccine or placebo (2 or 3 vaccinees per placebo recipient). phase i studies typically employ escalating doses of the candidate vaccine, with a dose range progressively increasing in steps of three-to fivefold often being used. blood samples are taken at prescribed intervals and analyzed for laboratory evidence of potential toxicity, as well as for evidence of vaccine-elicited immune responses. a phase i study is considered successful if it demonstrates that the candidate vaccine is well tolerated or identifies any immediate safety concerns that will need to be closely monitored in potential future clinical studies. ideally, phase i studies also provide an initial indication of the optimal dose level and number of doses required. a phase ii study typically includes several hundred to a few thousand volunteers (randomized between vaccine and placebo) and can assume two general design types. phase iia studies provide additional safety data on a larger number of individuals of the intended age who receive the intended vaccine dose (who are more representative of the general population intended for vaccine use than the very healthy individuals included in the phase i study), as well as provide additional data on vaccine immunogenicity. even larger phase iib studies can provide additional data on vaccine safety and immunogenicity in subjects generally representative of those for whom the vaccine might be recommended, but importantly, also provide the first opportunity to address to answer the question, 'does this vaccine work in humans?' the size of a phase iib study needed to detect a signal of vaccine efficacy depends on the attack rate of the infection being targeted by the vaccine. the development of new vaccines depends on the convergence of public health need, biological plausibility, and practical feasibility. vaccine development programs are influenced by multiple considerations, including: >> what are the major unmet medical and public health needs today? >> what is known about the natural history and pathogenic mechanisms of the infection of interest? >> is immunity to a given antigen associated with protection against disease following re-exposure in the context of natural infection? >> if natural immunity capable of preventing re-infection follows an initial infection with the pathogen, can a specific host immune effector mechanism (e.g., antibody, cd8 t-cell) be identified as the likely agent (or 'correlate') of immune protection? if so, can a threshold level of this specific immune correlate needed for protection from re-infection be defined? >> can the pathogen be grown in culture? if so, does the pathogen cause such a life-threatening disease that an attenuated version of the virus would face an impossible barrier for demonstration of safety? >> can a specific antigen (or antigens) be identified that represents the target of protective host immune responses? >> if the protective immune response is mediated by antibodies, can the target antigen (be it a protein or polysaccharide) be produced in scalable quantities in a form that mimics its native structure so that it can effectively elicit antibody responses that can block the key functional role(s) of the target molecule in the pathogen lifecycle or otherwise lead to the clearance of an incipient pathogen infection? >> having chosen an antigen and presentation system, what is the best way to produce it on a large scale? choices will be limited by the nature of the antigen and delivery system, but definition of an optimal system for producing the vaccine (prokaryotes like escherichia coli, or diverse eukaryotic hosts including yeast, insect cells, plants, or cultured plant cells, mammalian cells) is a central consideration. >> what is the most effective way to present the antigens of the pathogen of interest to the immune system? modern molecular biology and biochemistry have provided numerous options for vaccine immunogen presentation, including recombinant proteins (and recombinant virus-like particles (vlps)), synthetic proteins, protein-polysaccharide conjugates, and gene delivery systems (recombinant viral vectors, or dna vaccines) >> is the antigen of interest sufficiently immunogenic on its own, or is augmentation of the desired immune response by conjugation to a specific carrier or addition of an adjuvant necessary to elicit a sufficient and sufficiently durable immune response in individuals in the target population for vaccination? >> what types of potential safety concerns can be anticipated for the vaccine in question? >> what is the attack rate of the infection in the general population? if the infection occurs relatively rarely in an overall population, can a subset of the population be identified that has a higher risk of infection so as to accelerate the achievement of statistically significant protection? is this subset sufficiently similar to the rest of the population to enable extrapolation of the clinical results to the broader target population as a whole? >> what tests to evaluate vaccine immunogenicity will need to carried out on clinical samples obtained from participants in the clinical trials? will measurement of antibody titers, t-cell responses, pathogen presence and quantity, pathogen serotype, and any other parameter peculiar to the disease in question represent the primary criteria for vaccine effect? development and validation of theses tests represent an essential component for the feasibility and success of a vaccine clinical study phase ii studies also present the first opportunity to identify a potential laboratory immunological correlate of protection from disease -if nature and prior experience have not already done so. in order to do so, the placebo recipients in the phase ii trial must experience a sufficient number of cases of disease while vaccine recipients need to exhibit significant evidence of decreased risk of infection or disease. in addition, immunological measurements in the vaccinees need to capture the relevant protective immune responses (e.g., the type and level of antibody and/or cellular immune response that predict protection) and measure them with sufficient precision and reliability. if laboratory measurements of immunity correlate with vaccine protection, subsequent refinements of the vaccine, its adjuvant, its manufacturing process, or its regimen may be assessed by simple immunogenicity studies, rather than repeating efficacy studies. once efficacy is established for a vaccine, it is very difficult to carry out a double-blinded, placebo-controlled efficacy study. vaccines that have been shown to be immunogenic and well tolerated in phase ii studies can then advance to pivotal phase iii studies required for vaccine licensure by regulatory authorities. phase iii studies are intended to expand further the safety database in a larger number of individuals (who are representative of the specific populations for which the vaccine will ultimately be used), establish definitive evidence of protective efficacy, and to establish clinical consistency of the vaccine made by the process run in the facility intended for licensure and commercialization (www.fda.gov/cber/genetherapy/ isct092506jcr.htm). typically, phase iii studies include 10 000 or more subjects in a blinded, placebo-controlled design. this size trial allows the identification of less frequent safety events. it also provides an opportunity to capture data on health care utilization, cost, and impact of the vaccine on these parameters. as a new vaccine will ultimately be included in a vaccine program where multiple vaccines may be administered at the same time, it is also necessary to conduct concomitantuse studies. the developer of the new vaccine must show that the new vaccine does not impact on the immunogenicity of the existing vaccines, and that the existing vaccines do not impact on the immunogenicity of the new vaccine. in contrast to drugs, where licensure by the fda is the primary determinant of how a new product is implemented in medical practice, vaccine use in the usa includes an additional process that evaluates how best to employ a new vaccine to optimize its implementation and public health impact. the us cdc has responsibility for making recommendations about the use of licensed vaccines, and it relies on its advisory committee on immunization practices (acip) for guidance. the acip considers several aspects in addition to a vaccine's safety and efficacy, including the anticipated costeffectiveness and practical feasibility of potential alternative vaccine deployment strategies and consideration of how a new vaccine may be successfully implemented in clinical practice to achieve the greatest public health impact. once the cdc has received, reviewed, and accepted the recommendation of the acip, the recommendation is published in its final official form in morbidity and mortality weekly report (mmwr; www.cdc.gov/nip/publications/acip-list.htm). the recommended immunization schedule for children and adolescents ( fig. 92.1) is updated on an annual basis and can be accessed at www.cdc. gov/nip/recs/child-schedule.htm. the recommended adult immunization schedule (fig. 92. 2) is also updated on an annual basis and can be accessed at www.cdc.gov/nip/recs/adult-schedule.htm. the recommended adult immunization schedule includes information concerning use in special populations (such as health care workers and pregnant women) and individuals with specific conditions associated with altered or impaired immune function (such as individuals with congenital and acquired immunodeficiency syndromes, recipients of immunosuppressive therapies, malignancies, asplenia, liver disease, and renal disease). readers are encouraged to check to ensure that they are following current recommendations. pregnancy registries currently exist for four vaccines in the usa. health care professionals are encouraged to report exposures of pregnant women to the appropriate registry: hbv vaccine (800-670-6126), hpv vaccine (800-986-8999), meningococcal vaccine (800-822-2463), and varicella vaccine (800-986-8999). n vaccine safety n unlike drugs that are utilized to treat individuals suffering from a given disease state, vaccines are administered to normal, healthy infants, adolescents, and adults. consequently, standards for the safety and tolerability of vaccines are set at a very high level. when developing a new vaccine, a graded process of clinical studies is employed that involves increasingly larger numbers of volunteers and that typically progresses from individuals who are selected to be free of any identifiable health problems to those who are selected to be representative of the overall population for whom the vaccine is being developed. if phase i studies reveal no evidence of safety concerns and the desired evidence of immunogenicity, a major focus of the series of larger randomized double-blind, phase ii placebo-controlled studies that are then conducted is to explore the safety and tolerability of a vaccine in increasingly vulnerable populations (such as those who may have identified pre-existing health problems or asymptomatic abnormalities detected on screening laboratory studies). reflecting the importance of documenting the safety of a new vaccine, phase iii studies to assess the safety and efficacy of a new vaccine now typically involve large numbers of volunteers. indeed, as a result of needing to provide evidence for safety, it is now common to have the size of the phase iii trial be significantly larger than would be necessary to document vaccine efficacy. the ability of a study to identify an increased risk of any given adverse event with sufficient statistical power is directly related to the size of the population in the study. as a general rule, a study of 300-400 subjects is needed to measure the risk of an event that happens in one out of 100 individuals. for one in 1000, 3000-4000 subjects are needed. even in studies of this size, very rare events may not be identified, and if a specific safety concern exists substantially larger trials may be needed. the recent experience with the development of rotavirus vaccines provides an illustrative example of the importance placed on documenting vaccine safety. 24 rotavirus is an important cause of serious gastroenteritis in infants and young children, and the associated diarrhea and vomiting can lead to life-threatening dehydration. in developing countries where health care resources and effective rehydration options are limited, over 600 000 infants die of rotavirus gastroenteritis each year. 25 given the global importance of rotavirus gastroenteritis, the first licensure of an orally administered rotavirus vaccine in 1998 was a very welcome advance. however, as the vaccine entered routine pediatric practice, it was recognized that a low, but increased incidence of intestinal intussusception was seen after the first and second doses (with about one case of intussusception seen per 10 000 vaccinees.) 26 upon recognition of this association, the vaccine was withdrawn from the market. 27 with the evident public health need for a safe and effective rotavirus vaccine, it was hoped that alternative rotavirus vaccines then in development (both oral vaccines based either on a combination of bovine-human reassortant viruses (fig. 92.4) or an attenuated human rotavirus strain) might differ from the first licensed rotavirus vaccine and not result in an increased rate of intussusception. however, to demonstrate that these alternative rotavirus vaccines were safe, and that an increased risk of intussusception was not inherent to rotavirus vaccines as a class, very large-scale safety studies were required. toward this end, the safety of each of these vaccines was evaluated in studies involving about 70 000 infants -just to evaluate whether the rate of intussusception in vaccinees was discernibly increased compared to the normal background rates seen in the placebo recipients. 10, 11 fortunately, both vaccines were found to be well tolerated and no increase in intussusception was observed in vaccine as compared to placebo recipients. in light of the documented efficacy of these vaccines determined in earlier and significantly smaller phase iii trials, both have now been licensed in a number of countries. however, even with the large phase iii studies conducted for these newer rotavirus vaccines, they will still be studied in large postlicensure active surveillance safety studies and closely monitored in active and passive vaccine safety surveillance systems (see below). following vaccine licensure, safety is tracked via a number of means, including both active and passive surveillance studies of adverse events. active surveillance includes phase iv postmarketing studies of vaccine safety in larger populations in real-world use. formal postmarketing studies can include tens of thousands of individuals or more. an alternative type of postmarketing safety study is carried out by the us fda and the cdc within the context of the vaccine adverse event reporting system (vaers) database (www.vaers.hhs.gov or by telephone: 800-822-7967). the vaers database accepts spontaneous reports of adverse experiences from health care providers, patients, parents, vaccine manufacturers, and other sources. 28 the best use of the vaers database is to identify signals in a population that may appear following the introduction of a new vaccine. a newer vaccine safety surveillance system, known as the vaccine safety database (vsd), has been developed by the cdc in cooperation with seven large health maintenance organizations (hmos) around the usa. 29 the vsd contains the complete medical records of all the members from the participating hmos, and the information used to populate the database is entered by health care professionals using relatively consistent terminology, improving the quality, uniformity, and usefulness of the data. particularly important is that the vsd construct allows comprehensive epidemiological analyses to determine if the incidence rate of a specific adverse event is higher among vaccinees than nonvaccinees. in addition to vaers and the vsd, the cdc has also created a clinical immunization safety assessment network that reviews patterns of clinical syndromes that may follow vaccination. while the safety profile of a vaccine can be relatively well defined through the efforts described above, confidence in vaccination programs has often been challenged by public perceptions, either real or unsubstantiated, about vaccine safety. in some instances, specific vaccines have been associated with increased incidence of a specific adverse experience, such as the association between the first-generation rotavirus vaccine and an increased risk of intussusception following vaccination. however, a number of other safety concerns that have emerged are not supported by scientific evidence. an example of this can be found in the case of concerns about the association of whole-cell pertussis vaccines with permanent brain damage -concerns that were later shown to be unfounded. nevertheless, public concerns about the safety of the whole-cell pertussis vaccine resulted in decreased levels of pertussis vaccination coverage that were soon followed by epidemics of whooping cough in the uk and japan. 30 another example is the allegation that certain vaccines, such as the combination measles, mumps, rubella (mmr) vaccine, are associated with autism. highlighting how perceptions of temporal association can give rise to public concerns, mmr vaccines are generally given around 1 year of age, and autism is generally diagnosed in the second year of life. although the alleged causal association between mmr and autism has been refuted by thorough scientific analyses, reports in the popular media in the uk resulted in a dramatic drop in vaccination rates, followed by an increased rate of new infections. 31, 32 n vaccines not yet available n although an impressive armamentarium of vaccines is now available, safe and effective vaccines have yet to be developed for a number of very important infectious diseases. the reasons underlying the lack of effective vaccines for an array of important pathogens include biological considerations, safety concerns, and practical constraints. of these, the biological considerations are often the most important barrier. as discussed above, vaccines have been successfully developed for pathogens whose natural infections give rise to natural immunity wherein the infected host (at least those who survive initial infection) is no longer susceptible to re-infection (such as measles, yellow fever virus, or smallpox) or who experiences significantly less severe clinical sequelae upon re-infection (such as rotavirus). in instances where natural immunity follows natural infection, not only is a precedent for immune protection established, but the nature of protective host responses can be studied, providing a correlate of protection to guide vaccine development efforts. however, for many of the pathogens for which vaccines remain elusive, natural immunity does not follow natural infection. in the absence of natural immunity, not only is a precedent for successful immune containment lacking, but no potential correlates of protection are available to inform vaccine development. in some instances where natural immunity does not follow natural infection, persistent infections are established and maintained by active virus replication that cannot be controlled or cleared by host immune responses (such as hiv and hepatitis c). alternatively, other pathogens are able to persist in the host through establishment, via diverse mechanisms, of latent infections that are resistant to host immune clearance (such as tuberculosis or herpes viruses (such as herpes simplex virus (hsv) or epstein-barr virus (ebv))). in other instances, even when the host is cleared of an infection via drug vaccines not yet available treatment, the host remains susceptible to re-infection and disease in the future (such as malaria). although different pathogens have evolved diverse strategies for evasion of host immune responses -ranging from manifestation of tremendous genetic diversity and propensity for immune escape; to sequestration of critical structural domains that might be susceptible to antibody neutralization; to the utilization of specific mechanisms to evade host innate and adaptive immune effectors -the common end result is frustration of vaccine development. while failure of host clearance of an infection is a common theme underlying the lack of vaccines, additional obstacles to vaccine development include other immunologically related considerations as well as both practical and safety considerations. examples of immunologically related obstacles include instances where prior exposure to a given pathogen predisposes the host to more severe disease manifestations upon re-infection (as has been proposed in the case of dengue virus) or where earlier vaccine development efforts inadvertently lead to severe adverse events following infection with the targeted pathogen (such as respiratory syncytial virus (rsv )). in each of these cases, the adverse events that follow a secondary immune exposure are believed to be the result of immunopathologic responses that result from the nature of the immune response elicited by the initial exposure to pathogen-derived antigens (by either infection or vaccination). given that the mechanisms underlying these immunopathologic processes are incompletely understood, the development of vaccines that are highly immunogenic but not similarly inclined to elicit immunemediated adverse consequences represents a substantial challenge (especially given the very high expectations for vaccine safety). an additional immunologically related challenge relates to the observation that certain organisms encode antigens that resemble constituents of the human host. for example, in the case of neisseria meningitidis group b, the bacterial polysaccharide resembles those found on certain human cell lineages, thus raising concerns about whether polysaccharide-based vaccines successfully developed for group b n. meningitidis might yield undesirable autoimmune responses. 33 an additional distinct, but important, practical barrier to new vaccine development relates to the prevention of diseases that are threats to pregnant women or their offspring (where immunization of the pregnant woman might be able to protect the neonate). although a number of inactivated vaccines are either routinely recommended for use in pregnant women (e.g., inactivated influenza vaccine) or can be used in pregnant women for pre-or postexposure prophylaxis for those at risk of infection (e.g., inactivated hepatitis a vaccine and recombinant hbsag vaccine), the development of new vaccines specifically for use in pregnant women or the study of new vaccines in pregnant women has been impeded by concerns arising from potential litigation that might follow the appearance of a congenital abnormality in a child born to a mother who was vaccinated while pregnant. 34 given the 2-3% prevalence of congenital abnormalities, the practical difficulties in proving the safety of a new vaccine specifically administered to pregnant women, and the current litigious environment surrounding vaccines, the development of new vaccines to address important infections of pregnant women and their neonates (e.g., group b streptococcus: gbs) faces significant challenges. there remain a number of important infectious diseases for which no effective preventive vaccines exist. below, we list the major 'missing' vaccines, comment on why they are not yet available, and highlight the major approaches currently being explored to develop them. at the end of 2006, an estimated 40 million people were living with hiv infection, and in the preceding year approximately 4.5 million people became newly infected, and approximately 3 million individuals died of aids. as the most promising biomedical intervention to contain the aids pandemic, the development of an hiv vaccine is a top global health priority. yet, hiv infection represents a vexing challenge to vaccine development. 35, 36 hiv infection does not result in clearance of the virus due to a host immune response. following infection of target cells, the genome of hiv -a retrovirus -is transcribed into a dna copy via the action of reverse transcriptase. the newly formed dna copy of the hiv genome then integrates into the host cell chromosomes (referred to as a provirus) as a requisite step in the viral lifecycle. once integrated into the chromosome of an infected cell, the hiv provirus can alternatively be actively transcribed, leading to the synthesis of viral mrnas and subsequently to production of new virus particles, or it can remain in a transcriptionally silent, functionally latent state in a small percentage of infected cells. as infected cells harboring latent hiv proviruses do not produce hiv protein antigens, they cannot be recognized by host antiviral immune responses and can thereby persist undetected. upon subsequent activation of latently infected cells at some later time, viral rna transcription can be coincidently activated leading to production of progeny virions. as hiv targets activated cd4 t cells for infection and consequent depletion, the host's ability to mount both hiv-specific and non-hivspecific immune responses is progressively impaired. the ability of the host to clear hiv infection is further complicated by the extensive genetic diversity of virus populations that emerge, and progressively diverge, within infected individuals as a function of a replicative cycle that is accomplished by the inherently error-prone reverse transcriptase and the numerous cycles of replication that occur in infected individuals. as a result of these influences, genetically diverse populations of hiv variants are established in infected persons that facilitate the outgrowth of genetic variants that can escape from selective pressures -be they effective host cellular or humoral immune responses, or the inhibitory effects of antiretroviral drugs. 37 an extraordinary degree of genetic diversity is also manifest in the hiv variants seen in different individuals and in different geographic regions. as successful vaccines for other infectious agents have historically had to protect against pathogens exhibiting only limited genetic diversity, hiv represents an unprecedented challenge. as many successful vaccines protecting against viral infections are predicated on the induction of neutralizing antibody responses against the viral surface proteins that mediate attachment to and entry into target cells, significant efforts have focused on the potential of the hiv surface envelope (env) glycoprotein, gp120, to elicit infectionneutralizing antibodies. 38 unfortunately, hiv gp120 is highly resistant to the action of antibodies by virtue of its heavy glycosylation and its native conformation that shields functionally critical structural domains from antibody binding. as a result, candidate gp120-based vaccines have failed to elicit meaningful levels of neutralizing antibodies in immunized human volunteers and have not protected from hiv infection in two large phase iii studies. given the inability, to date, of candidate hiv env-based vaccines to elicit appreciable levels of neutralizing antibodies, current vaccine strategies are largely focused on the induction of cd8 cytotoxic t-cell responses against the more constrained and conserved antigens, such as vaccines not yet available gag, pol, and nef. it has been hypothesized that induction of high levels of hiv-specific cd8 t-cell responses prior to infection may not prevent infection, but may enable infected individuals to control virus replication better. should this hypothesis be valid, individuals immunized with such vaccines may exhibit lower levels of ongoing hiv replication, progress to aids more slowly, and potentially be less likely to transmit hiv infection to others. much of this work involves vectored gene delivery systems (such as adenoviral vectors, described below). however, the recently announced results of a phase iib 'test of concept study' 39 failed to demonstrate a beneficial effect on either prevention of infection or reduction of viral load among volunteers who received the vaccine despite the induction of appreciable levels of hiv-specific ctl responses by the recombinant adenovirus-based vaccine employed. while this study result does not, in and of itself, refute the 'ctl hypothesis', it represents a significant disappointment for the aids vaccine research effort, and raises important questions about the ability of vaccine-elicited cell mediated immune responses to favorably alter the outcome of hiv infection. 39a there are also efforts under way to utilize the recently solved three-dimensional structure of the hiv env glycoprotein to guide the derivation of nonnative structures that might serve as better immunogens to elicit broadly cross-reactive neutralizing antibodies. in addition, relatively conserved and functionally essential sequences of the extracellular domain of the hiv transmembrane env protein, gp41, are being explored as immunogens to elicit broadly neutralizing antibodies. malaria is the world's most common vector-borne disease -estimated to cause approximately 500 million clinical cases and 2 million deaths annually. 40, 41 the disease hits hardest in africa, and is especially severe in children under 5 years of age. in addition to direct morbidity and mortality, malaria is responsible for debilitating illness with enormous social and economic consequences. of the four malaria-associated protozoal species, plasmodium flaciparum and p. vivax represent the two major agents. these parasites have a three-stage lifecycle taking place both within the mosquito, and in the liver and blood of the infected host, and each cycle is largely distinct from the others from an immunological perspective. as a result of the multiple strategies for evasion of host immune response that the parasite has evolved, parasite replication proceeds at high levels despite active host immune responses. 42, 43 either as a result of these specific immune evasion strategies or the inability of the infected human host to mount immune responses that clear the parasite, prior infection does not protect an individual from repeated subsequent infections. although the severity of disease is often attenuated following repeated infection, the mechanism of disease modulation is incompletely understood, and the limited relative immunity engendered by prior infection is easily lost if an individual leaves a malaria-endemic region. as such, the limited impact and duration of host immune responses to malaria parasites suggest that any successful vaccine strategy will need to do far better than natural immune responses -a high bar for efforts to develop an effective vaccine. roughly two dozen antigens have been cloned and tested as potential vaccine immunogens, and with a few exceptions the results have been disappointing. 44 one antigen, the circumsporozoite antigen, presented as a fusion with hbsag (rts,s), has shown modest promise in human studies. 45 this vaccine is now undergoing larger-scale clinical efficacy testing to determine if the magnitude of protection would justify largescale implementation efforts. should 'proof of concept' be supported in these studies, but the absolute magnitude of efficacy be insufficient, future efforts will likely focus on the identification of an appropriate adjuvant to improve the magnitude and duration of immune responses. alternative approaches include immunization with irradiated or genetically attenuated sporozoites. 46 definition of novel antigens expressed at specific stages of the parasite lifecycle, and evaluation of combinations of multiple parasite antigens. mycobacterium tuberculosis is an intracellular mycobacterial pathogen that represents one of the world's most common and most serious infectious diseases. 47 over 2 billion people are believed to harbor latent m. tuberculosis infections, and approximately 8 million active cases of tuberculosis and over 2 million deaths occur each year. furthermore, the interface of hiv infection and its attendant immune system damage both increases the severity of m. tuberculosis infection and increases the infectiousness of infected individuals. the emergence and dissemination of m. tuberculosis isolates that are resistant to multiple antimicrobial drugs represent a growing public health threat. however, while the need for a vaccine to prevent tuberculosis is clear, a significant number of challenges face vaccine development efforts. 48 most individuals infected with m. tuberculosis can control the acute phase of mycobacterial replication, and mount vigorous innate and adaptive immune responses to the infection. however, the infection is often not cleared by the host's immune response, and the mycobacteria are able to persist and multiply within vacuoles inside macrophages. longterm latency is established in fibrotic cysts in the lung. the recrudescence and dissemination of m. tuberculosis occur at a later time in a number of infected individuals, likely as a result of waning host immune control. although the ability of m. tuberculosis to persist despite active innate and adaptive immune responses represents a major challenge to vaccine development, the fact that most individuals can contain (if not clear) m. tuberculosis infection suggests that a vaccine that can alter the course of the natural infection by limiting early dissemination and decreasing the risk of later recrudescence could provide major public health benefits. efforts to develop a vaccine against tuberculosis date back many decades. bacille calmette-guérin (better known as bcg), based on mycobacterium bovis, was first introduced in 1921. 49 currently, bcg is provided as a component of the routine expanded programme for immunization (epi) schedule and is administered to a significant majority of the world's children. although some protective efficacy (50-80%) has been reported against miliary infection and m. tuberculosis meningitis in children, conflicting results have been obtained in different studies regarding the ability of bcg to protect against pulmonary tuberculosis in adults. one explanation for the overall limited efficacy of bcg emerges from formal genome sequencing studies that have disclosed significant differences between m. tuberculosis and of the vaccine strain of bcg. the variability in the results of bcg efficacy studies in different populations and geographies may derive from variations in the geographic prevalence of cross-reactive mycobacterial species (that may themselves confer partial protection), or the fact that bcg vaccines used throughout the world do not represent a homogenous preparation -with the root strain of bcg having been widely distributed and passaged extensively under diverse conditions. vaccine efforts against tuberculosis have primarily focused on the evaluation of specific mycobacterial antigens (e.g., esat6, ag85, and hsp60) that have been tested as vaccines in animal models with variable success. 50, 51 some of these strategies are now being advanced into human clinical trials. an alternative strategy is based on improving the performance of the bcg vaccine by insertion of genes encoding specific potential protective antigens that it normally lacks. in addition, the development of auxotrophic mutants of m. tuberculosis is being explored as a potential immunogenic and specifically attenuated live vaccine. the determination of the sequence of the m. tuberculosis genome nearly a decade ago helped identify numerous previously unknown gene products, and increased the repertoire of antigens to be evaluated for their ability to induce protective immune responses. 52 the pathogen sequence is also being used to elucidate virulence determinants and thereby help guide efforts to attenuate m. tuberculosis rationally. together with influenza virus, rsv and piv account for a substantial majority of pediatric upper respiratory illness and consequent acute otitis media. a variety of influenza vaccines are licensed for pediatric use, but vaccines to prevent infection with the paromyxoviruses rsv and piv remain elusive. a significant impediment to vaccine development for rsv and piv traces back to unanticipated untoward results obtained in clinical studies of inactivated rsv vaccines in the early 1960s. 53 these early-generation rsv vaccines -based on cultured virus that had been inactivated with formalin -raised a potent antibody response in immunized children. however, on subsequent natural exposure to rsv, vaccine recipients exhibited more frequent and significantly more severe lower respiratory tract rsv infections than did unimmunized children. as a similar phenomenon was also seen with a formalin-inactivated measles vaccine in the same era, a common immunopathologic mechanism may be operative. 54 while the mechanism of exacerbation of rsv disease by the early inactivated vaccines is incompletely understood, it has been suggested that chemical inactivation of rsv and measles resulted in modification of a critical neutralizing structure on the surfaces of these viruses, thereby limiting the induction of the most potent neutralizing antibodies and favoring nonneutralizing and potentially immunopathologic antibody responses. (passive protection against rsv is available for premature infants in the form of monoclonal antibodies that target the rsv f protein (one of the viral envelope glycoproteins); certain anti-rsv antibody responses can clearly mediate protective as opposed to deleterious effects. 46 ) alternatively, or in addition, it has been proposed that inactivated rsv vaccines may have preferentially induced a th2-type immune response when a th1-type response may be needed to effect protection of the lower respiratory tract from rsv infection and damage. while excellent live attenuated measles vaccines have been developed, rsv and piv have so far resisted the approach used for measles and mumps (these are all members of the paramyxoviridae family of viruses). based on the successful precedent provided by the live attenuated measles vaccine, an attenuated or reverse genetics-engineered rsv is considered the most promising approach. however, stable attenuation of rsv has been difficult to achieve and vaccine safety concerns result in their cautious advancement through clinical evaluation. 55 effective vaccines for meningococcus types a, c, y, and w135 are available as straight capsular polysaccharides and as conjugated polysaccharides. 56 the group b polysaccharide shares chemical similarity with a shorter sugar found on the surface of neuronal tissue. 57 while it is possible to make highly immunogenic conjugates with the group b polysaccharide, theoretical concerns about cross-reactivity with self antigens has impeded the development of this type of vaccine. current work centers on a handful of relatively well-conserved surface proteins of meningococcus. gbs is a common component of the flora of the female genital tract, and transfer to the neonate is the cause of severe infections that are fatal or have serious sequelae. 58 short-course intrapartum antibiotics are recommended for culture-positive women, and this approach has cut the incidence of neonatal infections by about two-thirds, thus reducing somewhat the urgency of vaccine development. however, short-course antibiotics could ultimately drive the emergence of antibiotic-resistant gbs. candidate vaccines have been shown to elicit a protective response. 34 however, aside from a reduced market, the main impediment to development of a gbs vaccine is concern over vaccination of pregnant women or women of childbearing age. any birth defect might be attributed to the vaccine, and in a litiginous society, this would be problematic for a vaccine producer. prior to the advent of effective polymerase chain reaction methods for screening blood donations, hcv was a significant cause of transfusionrelated hepatitis. currently, transmission of hcv among the normal population is quite low; transmission among injection drug users remains high. hcv is another pathogen where infection does not typically result in an immune response that clears the infection. however, a minority of hcv patients do spontaneously clear their infection, suggesting that an appropriate immune response could do the job. current vaccine work is concentrated on vectored gene delivery vaccines, primarily adenoviruses, intended to raise antiviral cytotoxic t-cell responses. 59 with the exception of the live attenuated varicella-zoster virus (vzv) vaccine used for the primary prevention of chickenpox and reactivation of latent vzv infections (the cause of shingles and postherpetic neuralgia in older individuals), there are no other vaccines available for use in humans to prevent infection with members of the herpes virus family. 60 hsv types 1 and 2 cause recurrent vesicular eruptions "above or below the belt," respectively. like other herpes viruses, hsv infections are not cleared by the immune system and the virus can persist, remaining in a latent state that is functionally inaccessible to immune recognition and clearance. in addition, like other herpes viruses, hsv encodes a number of gene products that promote evasion of host immune responses. recent attempts to make hsv2 vaccines have used virus glycoproteins produced by recombinant dna methods. a recent clinical efficacy trial of this vaccine approach showed partial protection of women, but not men, who were seronegative for hsv1. 61 the reasons for this curious result are not clear, but efforts to develop this type of vaccine continue. in addition, a number of preclinical studies are exploring the ability of cell-mediated immune responses to hsv antigens induced by recombinant vaccine vectors (e.g., adenoviruses: see novel vaccine vectors, below) to prevent or ameliorate hsv infections. genetically engineered attenuated hsv variants have also been studied in experimental animal models. it is not clear when these new strategies may advance to clinical evaluation in humans. another herpes virus, cmv is a very common infection in humans, with 50-80% of individuals being infected by adulthood. cmv is a cause of severe infections in neonates, causing debilitating neurological sequelae. following initial infection, cmv persists in infected humans, despite the fact that anti-cmv antibodies are present and that a very sizeable proportion of the overall host cd4 and cd8 immune responses are specific for cmv antigens. ongoing virus persistence and replication in the face of active host immune responses are likely explained by cmv's sophisticated repertoire of host immune evasion functions (including those that inhibit antigen presentation mechanisms and immune effector responses). for these reasons, to be successful, vaccine development efforts will need to elicit immune responses that are significantly more effective than the quantitatively impressive, but functionally limited, immune responses that are generated in the course of natural cmv infections. live attenuated vaccines have been investigated sporadically since the 1970s. 62 an attenuated strain, the towne strain, showed some effect, but was judged to be insufficiently immunogenic. hybrids of the attenuated towne strain and the virulent toledo strain remain in development. recent work has included recombinant dna (rdna)-derived proteins (via either dna vaccine approaches or recombinant viral vectors, such as attenuated poxviral vectors). 63, 64 ebv is a herpes virus that represents the causative agent of infectious mononucleosis and is widespread among the human population. in concert with incompletely understood environmental (and perhaps additional host) factors, ebv is also etiologically associated with burkitt's lymphoma. the ability of ebv to establish persistent infections in humans (along with latent infections at the cellular level) despite readily detectable antiviral immune responses suggests that, like other herpes viruses, the development of effective ebv vaccine will likely be challenging. ebv vaccines have been in development since the 1980s with the coat protein, gp220/350, as the most common vaccine antigen studied. 65 dengue fever virus is a mosquito-borne flavivirus (the virus family that includes japanese encephalitis virus and yellow fever virus -for which successful vaccines exist). dengue virus is endemic in a substantial portion of tropical and subtropical areas and causes febrile disease as well as hemorrhagic fever. there are four distinct serotypes of dengue fever virus. prior infection with one serotype has been implicated in predisposing for more severe disease following infection with a second dengue fever virus serotype, although the evidence supporting this concept has been questioned and the underlying pathogenic mechanisms are incompletely understood. 66 the processes of vaccine development have changed significantly in recent years -a process facilitated by substantial improvements in understanding of human immune system function, as well as the advent of powerful new technologies for vaccine development. as a result of these advances, vaccine development is now commonly pursued in a hypothesis-driven manner and is a far less empiric pursuit than in the past. however, at the same time, the infectious diseases for which no effective vaccines currently exist represent more challenging targets than those diseases that have yielded to vaccine development efforts in the past. furthermore, global changes that influence the emergence and rate of spread of infectious diseases place unprecedented challenges on the productivity and pace of new vaccine development efforts. vaccines not yet available >> the challenge of responding rapidly and effectively, with powerful new technologies, to newly emerging infectionsincluding those that haven't been seen in humans before (e.g., severe acute respiratory syndrome (sars)) or for which novel antigenic variants are anticipated but cannot be predicted (e.g., pandemic influenza) >> maximizing the value of innovative new approaches while ensuring the safety of new vaccines so derived one hypothesis proposes that antibodies against the initial infecting serotype bind to the surface of virus particles of the novel infecting serotype, but do not neutralize the infection. in a process referred to as "immune enhancement" of infection, still infectious complexes of antibody virus particles are then envisioned to be preferentially taken up by cells of the reticuloendothelial system that represent primary target cells for virus replication. although the veracity of this hypothesis in not established, it does present certain theoretical concerns about what type of antibody responses will need to be induced by vaccines to exert beneficial rather than detrimental effects. the general belief is that a vaccine providing equivalent immunity against all four serotypes will be required. vaccines based on inactivated virus, engineered chimeric viruses based on the yellow fever virus vaccine platform, engineered deletion mutant viruses, and rdna-derived proteins are in various stages of development. 67 n new antigen discovery methods n historically, vaccine antigens were not discovered in the literal sense. rather, whole organisms were inactivated by either heat or chemistry or organisms were attenuated by forcing growth in nonphysiological conditions. the entire antigenic repertoire of the organism was delivered to the immune system. the isolation of tetanus, diphtheria, and pertussis toxins, along with chemical detoxification schemes, allowed the production of more refined vaccines. the isolation and purification of polysaccharide capsules from a range of important bacterial pathogens enabled the development of additional vaccines. with the advent of molecular biology in the late 1970s, a new set of tools allowed a more directed approach for the discovery of pathogen virulence factors, vaccine antigen discovery, and vaccine development. the tools of molecular biology enabled for the first time the development of vaccines against pathogens that could not be propagated in culture, including the successful development of recombinant hbv and hpv vaccines. development of these vaccines was enabled by clinical and animal model studies showing that antibodies directed against a specific viral target antigen (e.g., the hbv surface antigen or the hpv l1 protein) were implicated in protection. in addition, molecular biologic approaches enabled the derivation of fully recombinant vaccine antigens (such as those developed using a limited set of defined antigens of bordetella pertussis), including genetically modified versions of bacterial toxins that maintain their proper antigenic structures but are no longer toxic. however, for many of the pathogens for which vaccines do not currently exist, application of these recombinant dna technology-enabled strategies are insufficient due to incomplete understanding of the pathogen antigens that would elicit a protective host immune response. as such, the development of additional techniques to discover protective antigens was needed. fortunately, several important technological advances that facilitate discovery of previously unknown protective antigens from even very complex microorganisms have opened a new era in vaccine development. the earliest rdna technology-enabled methods of antigen discovery involved the expression of individual pathogen-derived gene products (or fragments thereof ) in bacterial hosts (typically escherichia coli) using rdna expression vectors. here, the genome of a pathogen is broken up, and the fragments are inserted into a plasmid or a viral vector, typically a lambda bacteriophage. 68 colonies, or plaques, are spread on a membrane, allowed to grow, and hopefully express the cloned gene fragments. the ability of these recombinant gene products (now isolated in individual colonies) to react with antibodies present in the serum of individuals who had recovered from infection with that pathogen could then be directly assessed. antibodies present in the immune sera are assessed for their ability to identify antigens by immunochemical reactivity. in this way, the entire genome of a given pathogen could be scanned for potential immunoreactivity. 69 such reactivity would both indicate the in vivo expression of that gene product, as well as document its antigenicity. however, additional studies are needed to demonstrate whether antibody responses against a newly defined antigen have any protective potential. to document the ability of an antigen to elicit protective immune responses, it is necessary to immunize an experimental animal (most commonly, mice) and then, following experimental pathogen challenge, evaluate infection outcomes in immunized versus nonimmunized animals. as this approach has most often been used to identify antigens recognized by host humoral responses, sera from animals immunized with a candidate antigen can then be transferred to a naïve host to provide evidence that the antibody response to the antigen represents the relevant agent of immune protection. more recently, as dna sequencing became more efficient and scaleable, determining the entire sequence of the genomes of viruses, bacteria, and parasites has become routine, 70, 71 allowing identification of previously unknown genes (and predicted gene products) that can be evaluated as vaccine immunogens. scanning the entire pathogen genome via specific computer analysis programs, genes that exhibit specific characteristics can be identified (e.g., predicted expression on the cell surface by virtue of possession of a leader sequence for secretion or membrane anchor sequences). 72 in addition, the relative conservation of the gene within the pathogen population can be determined by assessment of gene sequences from multiple distinct isolates. once potential vaccine antigens are identified, each candidate gene is expressed in an appropriate rdna system, and the protein product is tested in an animal model. 73 the first bacterial genome sequenced in its entirety was that of haemophilus influenzae, marking the beginning of a new approach to vaccine antigen discovery. 74 since this initial bacterial genome sequence determination, genomic sequencing of pathogens has advanced exponentially. over 300 bacterial genomes have now been sequenced, and hundreds more are currently in process. genome-based antigen discovery is being applied to a wide range of bacteria, including streptococci, pneumococci, staphylococci and chlamydia, as well as nonbacterial pathogens such as plasmodium falciparum. 75 an alternate, promising approach to novel antigen discovery has been built on technological advances in proteomics. 76, 77 these advances include development of high-resolution two-dimensional gel electrophoresis techniques and mass spectrometry methods that enable separation, identification, and purification of individual proteins from the complex mixture of proteins expressed by a pathogen. in proteomic analyses, a small culture of bacteria, preferably taken directly from an infected person, or otherwise grown in physiologically similar conditions, is subjected to physical or enzymatic treatment with specific proteases to generate peptide fragments that are then fractionated by a micro-high-performance liquid chromatography method and sequenced by molecular mass-by-mass spectrometry. an overlapping set of peptides of approximately 8-10 amino acids is sufficient to identify an antigen and provides the means to find the gene. although proteomic analysis is, in some ways, more involved than genomic analysis, it provides an important new approach to antigen identification, and offers a direct way to document that the specific protein identified is actually expressed by the pathogen (including, for example, demonstration that a protein of interest is expressed on the external surface of the pathogen). 78, 79 a combination of proteomic and serologic methods to select potential novel vaccine immunogens, called serological proteome analysis, or serpa, 80, 81 can be used to screen the pathogen proteome for expressed proteins that are recognized by antibodies present in sera obtained from individuals who have recovered from an infection with the pathogen. the proteomic approach to antigen discovery has been applied to identify novel vaccine candidates for a number of human pathogens, including helicobacter pylori, chlamydia pneumoniae, staphylococcus aureus, bacillus anthracis, haemophilus influenzae, and plasmodium falciparum. as in new genomic methods for antigen discovery, having identified a gene encoding a candidate antigen by proteomic methods, it is then necessary to show that an immune response of the desired type can be raised against the protein. in addition, it is necessary to show that immune responses elicited following immunization with the candidate antigen engender some degree of protection. often, pure protein antigens produced by recombinant methods are not very immunogenic. as such, many of the emerging recombinant vaccines so produced will likely require enhancement of their immunogenicity by means of an adjuvant. the term 'adjuvant' (derived from the latin adjuvare, to help) refers to any substance added as a component of a vaccine preparation -in addition to the vaccine antigens themselves -that improves the immunological response to the antigen. as such, 'adjuvant' is a catch-all term including a broad range of molecular entities that act via diverse -and, in a number of instances, yet to be elucidated -pathways. until recently, most adjuvants were derived empirically and the mechanisms by which they augmented immune responses were unknown . as a result, there were few, if any, principles available to guide the improvement of known adjuvants or the development of new ones. however, recent advances in understanding of the mechanisms by which dendritic cells sense the presence of pathogens and their constituents, and translate this information to shape the quantity, quality, and durability of host cellular and humoral adaptive immune responses, have transformed adjuvant discovery and optimization. what was once a process of trial and error now represents an area of hypothesis-driven research and mechanism-based discovery. a particularly promising advance emerged from the discovery that pathogen sensing by the innate immune system is mediated by recognition of specific pathogen-associated molecular patterns (pamps) by pathogen recognition receptors (prrs) such as the toll-like receptors (tlrs) that are expressed on dendritic cells (dcs) and other hematolymphoid and some epithelial cells 82 (chapter 3). the pathogen-derived pamps recognized by tlrs consist of structures that are found only in or on pathogens (including bacteria, viruses, and parasites) and are not part of normal vertebrate biology. following binding of a specific pamp to a specific prr, a specific cellular activation and response cascade is triggered that can directly confront an intruding pathogen and/or lead to the activation of specific host adaptive immune response mechanisms. these breakthroughs in basic immunology have been readily translated into what can now be considered the science of adjuvant biology. 83, 84 such progress has occurred at an especially opportune time as new vaccine development strategies have transitioned from traditional approaches using attenuated or killed pathogens to highly defined and purified recombinant proteins (so-called "subunit" vaccines) or nonreplicating vectored antigens. although these newer approaches are promis-ing from the perspective of vaccine safety and the opportunity they afford to design the structures of vaccine immunogens, recombinant or synthetic vaccines are often inherently less immunogenic than traditional vaccines based on attenuated live viruses or intact killed organisms. in the context of current vaccine development and regulatory approval processes, an adjuvant is developed as part of a vaccine, not as an independent product. consequently, there are currently no adjuvants licensed by regulatory authorities as stand-alone products. most contemporary efforts to develop novel adjuvants are focused on the targeted activation of tlrs that are expressed on specific cells critical for the generation of innate and adaptive host responses to specific pathogens. 85 a family consisting of 10 distinct tlrs has been identified to date in humans (chapter 3). tlrs are expressed in a number of innate immune cells, including dcs, macrophages, neutrophils, endothelial cells, and fibroblasts. given the importance of dcs as critical antigen-presenting cells, most studies of the biology of tlr signaling have focused on these cells. different tlrs are expressed on distinct subpopulations of dcs, and, depending on the tlr, in distinct cellular compartments. tlrs expressed on the surface of human myeloid dcs include tlr2 (which is heterodimerized with tlr 1 or 6), as well as tlrs 4, 5, 6, and 10 ( fig. 92.6 ), while these same cells express tlrs 3 and 8 within endoplasmic reticulum (er) and phagolysosomes. plasmacytoid dcs (pdcs) express tlr7 and 9 within er/phagolysosomes. the tlrs expressed on the cell surface are primarily activated by pamps encountered in the extracellular environment, while tlrs expressed in the er/phagolysosomes are activated by pamps (including viral pathogen-derived rna or dna) that tend to be routed through these endosomal compartments. activation of tlrs on innate immune cells leads to their production of specific cytokines, as well as their expression of co-stimulatory molecules, leading to induction of adaptive immune responses. given that different dcs express different tlrs, and that signaling via different tlrs results in the expression of a distinct pattern of cytokines, it is believed that activation of specific tlrs can variously favor the induction of th1-or th2-biased immune responses, or can differentially augment either direct or cross-presentation pathways for antigen presentation ( fig. 92.6 ). although most data on induction of specific types of immune responses by engagement of specific tlrs have emerged from murine studies (and have not yet been validated in humans), the ability to tailor an adjuvant preparation to achieve a desired type of immune response with a specific vaccine immunogen is a promising notion. naturally occurring ligands for tlrs include lipopolysaccharide (lps) from bacterial cell walls (recognized by tlr4), triacyl lipopeptides (recognized by tlrs 1 + 2), diacyl lipopeptides (recognized by tlrs 1 + 6), peptidoglycan (recognized by tlr2), flagellin (the monomer that makes up flagella, recognized by tlr5), singlestranded rna (recognized by tlr7), double-stranded rna (recognized by tlr3), and unmethylated dna containing the dinucleotide pair cpg 86 (recognized by tlr9). based on these insights, a variety of approaches to develop adjuvants predicated to activation of specific tlr pathways are being actively pursued. one interesting aspect of adjuvant development is how it is revealing the mechanisms of action of adjuvants that were originally identified via a process of trial and error, as well as delineating important aspects by which certain empirically derived vaccines are able to induce high-level, long-lasting immune responses. one illustrative example can be found in the case of complete freund's adjuvant (cfa), which has long served as the benchmark for laboratory studies of adjuvants. cfa is a mixed emulsion of mineral oil, mannide monooleate, and killed mycobacteria. however, it is far too reactogenic for use in humans, causing significant pain and abcesses at the site of injection -reactions that would be exacerbated if cfa were to be used repeatedly. an alternative preparation termed incomplete freund's adjuvant (ifa) lacks the mycobacterial component, but it too is associated with injection site reactions that are severe enough to limit its use to experimental therapeutic cancer vaccines. although cfa's toxicity precludes its use as a vaccine adjuvant in humans, many of its constituents (including liposaccharides, dna, and specific bacterial cell wall components) are now understood to exert their adjuvant effects on vaccine-induced immune responses via engagement of specific tlrs. similarly, the live attenuated mycobacterium bovis strain, bcg, long widely employed as a vaccine for the prevention of tuberculosis, includes cell wall, peptidoglycan, and dna components that activate specific tlrs. interestingly, the highly effective yellow fever vaccine 17d has been shown to activate multiple tlrs as part of its induction of antiviral immune responses. 87 it is quite likely that other live attenuated viruses that transiently replicate in immunized hosts also activate innate immune responses via engagement of tlrs. in yet another example involving a nonreplicating vaccine immunogen, one version of the hib polysaccharide conjugate vaccines now licensed for use in children for the prevention of invasive hib disease includes the meningococcal outer-membrane protein complex (ompc) as its protein carrier. ompc conjugates have favorable immunogenic properties that correlate with the ability of ompc to activate dcs via tlr2. 88 hundreds of different adjuvant formulations have been tested in animal models, and a few have been advanced into human studies. with a few α α (7) toll-receptor agonists. alum, the classical adjuvant most often used in vaccines in humans, includes a range of salts of aluminum precipitated under basic conditions, usually aluminum sulfate mixed with sodium or potassium hydroxide plus a variable amount of phosphate. 89 the relative proportions will determine the size, charge, and solubility of alum. the composition of alum used as an adjuvant varies in currently available vaccines and may influence vaccine immunogenicity. alum is utilized as an adjuvant in many of the currently available vaccines composed of inactivated toxins or recombinant proteins (live attenuated vaccines do not include alum or other adjuvants). alum serves two main purposes as an adjuvant. first, it acts as an antigen depot. vaccine antigens adsorb to alum and elute from it following injection into the host. second, alum acts a mild irritant, causing the recruitment of leukocytes necessary for generation of an immune response to the site of injection. adsorption of antigens on to alum routinely improves immunogenicity, particularly the antibody response. alum does not typically enhance cd8 t-cell responses. alum has been a component of many vaccines for decades and has an excellent safety record. as new adjuvants are developed, alum may remain as a component of combination adjuvant mixtures (as is the case with some newer adjuvants now approaching clinical use), or it may eventually be supplanted by other agents that more effectively provide favorable depot and local inflammatory responses to accentuate host immune responses. using lipids with polar head groups (e.g., triglycerides) and differing types of hydrophobic tails, one can form either micelles (spheres) or multilamellar sheets in aqueous environments. 90 under the right conditions, antigens can be incorporated into the spheres or between layers of the sheets, providing a potential slow-release depot system. immunopotentiators such as qs21 or detoxified lps derivatives (such as monophosphoryl lipid a (mpl)) may be added to the lipid mix. 45 iscoms are a proprietary form of liposomes made of cholesterol, saponins from quillaia bark (various members of the qs-x family of triterpene glycosides), and phospholipids that form cage-like structures into which antigens can be entrapped or intercalated. 86 iscom complexes may provide a depot function, as well as facilitate the delivery, uptake, and processing of vaccine immunogens by apcs. purified influenza virus hemagglutinin (ha) and neuraminidase mixed with phosphatidyl choline and phosphatidyl ethanolamine (polar lipids) will form empty particles that have the surface properties of influenza virus. adding an antigen in solution before mixing the lipids results in the incorporation of the antigen inside the particle. this provides a vehicle for delivering antigens to the interior of a cell, via the influenza ha membrane fusion process, thereby enabling antigen processing and presentation via both major histocompatibility complex (mhc) class 1 and 2 pathways. 91 emulsions numerous oil-in-water and water-in-oil emulsions have been tested as adjuvants. one such emulsion, mf59, is used in a licensed influenza vaccine. mf59 consists of squalane, a metabolizable shark oil and two surfactants, polyoxyethylene sorbitan monooleate and sorbitan trioleate, in an oil-in-water emulsion. 92 cytokines are host-produced immunomodulators that regulate immune cell action (chapter 10). several cytokines are being tested as potential vaccine adjuvants, including granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin-2 (il-2), and il-12. of the defined tlr agonists being explored as vaccine adjuvants, lps and its partially detoxified form, mpl, which activate tlr 4, have been most thoroughly explored in clinical trials. with evidence of enhanced ability to increase the percentage of individuals responding with protective antibody levels to hepatitis b as compared to a standard hepatitis b vaccine, one hepatitis b vaccine that employs an adjuvant formulation (termed as04) consisting of a combination of alum and mpl 93 has been licensed for use in high-risk individuals. in addition, a vaccine against hpv that uses the same adjuvant formulation may be licensed soon, and candidate hsv-1 and malaria vaccines currently being studied in latestage clinical trials also include this alum-mpl combination adjuvant. a wide variety of tlr9-specific agonists consisting of oligodeoxynucleotides containing unmethylated cpg motifs (cpg-odn) are being evaluated in preclinical studies. these cpg-odns resemble bacterial dna, modified to include a phosphorothioate backbone to increase their stability. two cpg-odn adjuvants have been evaluated in recent phase i and ii trials and shown to increase the timing and magnitude of induction of protective antibody levels, as well as the proportion of responding individuals, to recombinant hbsag vaccine as compared with the current commercially available version of the vaccine. 94 one of these cpg-odn adjuvants also elicits protective antibody responses in immunized hiv-infected individuals who had previously failed to respond to the hepatitis b vaccine. this approach is now being studied as a way of inducing protective immune responses to hepatitis b earlier after initiation of the vaccination regimen or with fewer doses of the vaccine. in addition to the cpg-odn-based tlr9 adjuvants described above, small chemical compounds with structures that resemble nucleic acid bases have been identified that activate tlr7 (e.g., imiquimod) or both tlr7 and 8 (e.g., resiquimod). these compounds are being evaluated as vaccine adjuvants in preclinical studies. flagellin, a tlr5 agonist, is also being explored as an adjuvant. recently, attention has also been focused on coupling, rather than mixing, tlr agonists to antigens. cpg oligonucleotides conjugated to antigens have been tested in preclinical studies of hepatitis b vaccines 95 and in human clinical trials for treatment of allergy. 96 ligands for tlr7/8 have been coupled to hiv antigens, 97 and the ligand for tlr5 (flagellin) has been fused to a variety of antigens. 98, 99 in some instances, coupling a tlr ligand to an antigen resulted in a substantial improvement of the immune response compared to mixtures -potentially the result of enabling the antigen and the tlr ligand to co-locate in the same dc compartments. numerous preclinical studies have confirmed that many natural and synthetic tlr agonists possess adjuvant activity. importantly, early human clinical trials of tlr-predicated adjuvants have supported the promise of this approach to mechanism-based strategies to augment vaccine immunogenicity. an important challenge is to define the most potent and best-tolerated variants, and to define rules by which activation of specific tlr pathways might translate into predictable augmentation of desired types of immune responses. it is hoped that general rules will emerge to suggest which of an increasing number of novel adjuvants in development performs best with which type of vaccine immunogen, and if results obtained with a specific type of immunogen-adjuvant combination can be extrapolated to predict the likelihood of enhanced immunogenicity with other vaccines. although beyond the reach of available experimental results, the ability to tailor, titrate, and otherwise optimize immune responses to vaccines by manipulation of specific tlr pathways appears a realistic future possibility. however, important challenges remain. in particular, a primary challenge for nextgeneration adjuvant development is finding a combination that retains immunopotentiating action while minimizing vaccine-associated adverse experiences. short-term adverse experiences, such as local injection site reactions, represent undesirable side effects that may disqualify candidate adjuvants early in clinical development. however, given that vaccines are administered to healthy people to prevent potential future infectious diseases, the potential for rarer adverse experiences (such as autoimmunity) that may only be manifest with much longer latency from the time of vaccine adjuvant administration will undoubtedly be important considerations for use in prophylactic vaccines. as induction of cell-mediated immune responses is considered an important component of vaccine strategies for many diseases for which no vaccines are currently available (many of which are caused by intracellular pathogens), there is a need to develop safe and readily scalable approaches to elicit durable cd8 t-cell responses in immunized humans. further, given the critical role that cd4 t cells play in induction, differentiation, and maintenance of cd8 t-cell responses, any such novel vaccine strategy will likely also require appropriate cd4 tcell responses. as elicitation of cd8 t-cell responses against a foreign antigen usually depends on the de novo expression of the antigen within a host cell and its subsequent processing and presentation via class i mhc pathways (chapter 6), most novel vaccine strategies are predicated on the need to achieve synthesis of pathogen-derived antigens within apcs of immunized human hosts. with an increasing appreciation of the role that cross-presentation pathways can play in elicitation of class i-restricted cd8 t-cell responses, such de novo antigen synthesis may not need to occur within apcs themselves (which may be an advantage for potential vaccine delivery strategies that do not directly target apcs). one of the many attractive attributes of effective live attenuated vaccines is their ability to recapitulate (to various degrees) many of the processes that lead to the generation of potent immune responses following natural infection. these processes include the fact that replication of all viruses depends on gaining access to host cells for genome replication and for the synthesis of essential components of virus particles that permit further propagation of the infection within and between hosts. one immunologic benefit of this requirement is that de novo synthesis of viral gene products within infected cells provides a key opportunity for viral antigen presentation (via mhc class i pathways) and elicitation of antiviral cellular immune responses. along with the processing and presentation of intact virus proteins via mhc class ii pathways leading to production of antiviral antibody responses, live attenuated viral vaccines have a strong track record for induction of broad cellular and humoral immune responses that likely both contribute to conferring protective immunity. however, despite their track record of success, it is likely that few, if any, new live attenuated viral vaccines will be derived in a manner that resembles previous successful efforts (e.g., the empiric derivation of live attenuated polio, yellow fever, or varicella-zoster vaccines). important reasons for this change include the desire for safe and well-characterized vaccines whose mechanisms of attenuation are defined and that can be monitored in the course of vaccine production and use. indeed, most of the recently developed live attenuated vaccines were derived using new approaches for genetic reassortment (fig. 92.4) wherein genome segments encoding pathogen-derived antigens of interest are recombined with a common set of genome segments that carry attenuating mutations (derived either by use of attenuating viral passage under specific conditions in cell culture (e.g., the cold-adapted influenza vaccine or use of a virus obtained from a nonhuman host that is itself inherently unable to replicate to high levels in humans, e.g., the reassortant rotavirus vaccine prepared via genetic reassortment between human and bovine rotavirus strains (fig. 92.4) 100 ). although such approaches have proven successful, they are limited in that they can only be applied to homologous viruses (e.g., those derived from the same virus type) whose genomes are segmented and capable of ready genetic reassortment in culture, or to viruses that can be manipulated by reverse genetics. in response to the desire to produce vaccines that can safely and reliably elicit desired immune responses, especially t-cell responses, several approaches are being explored to develop novel vector systems that permit the expression of pathogen-derived antigens. as many of these approaches are based on viruses distinct from the viral pathogen targeted for induction of host immune responses, the inserted pathogen-derived gene products are expressed via recombinant methods as heterologous antigens. alternatively, in nonviral expression systems, such as dna vaccines, the pathogen-derived antigen is expressed in isolation and does not depend on virus-mediated antigen delivery to apcs following host inoculation. collectively, such recombinant heterologous expression systems are commonly referred to as 'vaccine vectors.' in some instances, such recombinant vectors express only a specific antigen (in the case of dna vaccines or certain viral vectors, e.g., adenovirus), while in others both the inserted pathogen-derived antigens and antigens encoded by the viral vector 'backbone' are expressed (e.g., poxvirus vectors). most new approaches employ expression systems that are inherently nonreplicating (e.g., dna vaccines) or that employ viral vectors that can replicate at high levels in tissue culture but not in vivo (e.g., complemented adenovirus deletion variants or host range-restricted poxviruses). while numerous approaches are being pursued to develop novel vaccine vectors, they will all need to meet certain common criteria to emerge as vaccine approaches applicable for widespread use. in particular, any successful approach must be safe in healthy and immunodeficient humans (given their increased representation in the population as a result of hiv infection and therapeutic immunosuppression), desirably immunogenic (including in individuals who may have been previously exposed to the virus from which the vector was derived, e.g., vaccinia or adenovirus), and able to be produced in large quantities and in a stable manner. a limited number of vaccine vector approaches now being pursued are likely to meet these criteria. should successful approaches emerge, there will likely be interest in applying them for use in vaccines targeting diverse pathogens. thus, while definition of promising, broadly applicable, vaccine vector approaches may help simplify certain aspects of vaccine regulatory review and manufacture, they may also present challenges to prioritize use for specific applications should administration of a given vaccine vector on one occasion compromise or preclude successful administration at a later time. nevertheless, the development of novel vaccine vectors is laying essential groundwork for the development of next-generation vaccines. several novel vaccine vectors currently being studied in preclinical studies and human clinical trials are described below, all of which depend on the delivery and expression of a candidate pathogen-derived gene sequence. in a number of ways, dna vaccines represent the simplest approach to deliver pathogen-derived genes. viral vectors similarly serve to deliver pathogen gene sequences to host apcs, either directly or indirectly, but do so in a manner that depends on and takes advantage of the lifecycle and tropism of the virus that is being adapted to express the exogenous pathogen gene products. the ability of purified plasmid dna containing heterologous antigens expressed under the control of eukaryotic transcriptional regulatory and rna processing signals to elicit immune responses when injected into experimental animals was discovered serendipitously. 101 however, since the initial, quite surprising, description, the development of so-called dna vaccines has become an active area of preclinical and clinical vaccine development. 102 reasons for this enthusiasm include the attractive simplicity and facile preparation of vectors that encode only the defined antigen of interest (which can itself be manipulated via recombinant methods to assume a desired configuration), a reasonably straightforward method for vaccine production, and the inherent stability to temperature (which is much greater than most currently live or subunit vaccines). although the dna vector is most commonly injected intramuscularly, the generation of specific immune responses depends on the uptake of the vector dna by apcs followed by the expression, processing, and presentation of vector-encoded antigens. as tissue and tissue fluids present a hostile environment for purified dna, and the process of dna uptake by apcs appears to be relatively inefficient, much of the dose of injected dna is degraded before it can be reached by an apc that can initiate the desired immune response. most dna vaccine research has been pursued in mice, although studies have now been performed in numerous animal species. studies have usually utilized intramuscular injection of vaccine vector dna, but various intradermal and transdermal approaches have also been explored. murine studies have shown that administration of antigen-encoding plasmid dna can elicit appreciable cellular and humoral immune responses that may confer protection against experimental challenge. however, translation of these promising results in animal models to humans has proven frustrating. while dna vaccines have been generally well tolerated in immunized volunteers, in most human studies of dna vaccines, administration of even substantial quantities of dna vaccine vectors has elicited relatively low-level immune responses. it is not yet known whether these disappointing results reflects fundamental differences in the immunogenic behavior of dna vaccines in humans and mice, or the fact that the dna doses administered to humans do not match those administered to mice (dna per weight of the immunized host). given the substantial size differences between humans and mice, it would likely be impractical (for reasons of both vaccine supply and the actual process of administration of sufficiently high doses of dna) to administer the relative murine dose to humans. as such, a variety of approaches are being explored to prolong dna survival in tissue, promote more efficient targeting of dna to apcs, or to develop novel adjuvants that might specifically amplify immune responses to dna vaccines. 103, 104 dna vaccines are currently being used as candidate preventive vaccines for a wide variety of infectious diseases, including hiv, tuberculosis, malaria, and cmv. poxviruses represent the family of viruses that are physically the largest viruses and that possess the largest genomes. much of the poxvirus genome encodes gene products that serve to evade host immune responses, and that are not required for virus replication in tissue culture. further, facile techniques for the insertion and deletion of specific viral genes have been developed. the ability to accommodate sizeable foreign gene inserts is, in part, a function of the large size of the poxvirus genome (and the large packaging capacity of poxvirus virions). as a result of these favorable attributes, poxviruses have been utilized extensively in laboratory studies of virus biology, recombinant protein production, and host immune responses. 105 although poxviruses encode multiple gene products that help the virus evade host immune responses, they are, nevertheless, potent immunogens. studies of individuals immunized decades ago with vaccinia virus (in the course of smallpox eradication efforts) have shown that this virus induces long-lasting memory t-and b-cell immune responses. in contrast to most of the other viral vectors currently being developed, poxviruses can replicate readily in culture and do not require an engineered host cell to support propagation ex vivo. one important limitation of all poxvirus vectors developed to date is that, given the large size of the poxvirus genomes and the multitude of gene products they naturally express, even large inserts derived from foreign pathogens of interest will present only a minority of the vaccine vector antigens delivered to and recognized by the host immune system. to be effective, approaches to focus immune responses on the antigen of interest will need to be developed. toward this end, a variety of so-called 'prime-boost' 106 approaches are being explored where the host immune response is primed with one type of recombinant vaccine vector (such as a dna vaccine or adenovirus vector) and then boosted with subsequent delivery of poxvirus vectors encoding the same antigen. in this manner, immune responses to antigens of interest have been significantly augmented in a number of preclinical studies. vaccinia virus represents the prototypic vaccine vector. this virus is the same one that was employed in the successful smallpox eradication campaign, and has been used as a laboratory tool for decades. however, given current high expectations for vaccine safety, and the increased number of immunodeficient individuals present in the population (as a result of the new antigen discovery methods emergence of the hiv pandemic and the increased use of immunosuppressive therapies in clinical medicine) at high risk of serious adverse events, and potentially fatal consequences, from vaccinia immunization, the original vaccinia strains used in smallpox eradication efforts are not considered safe for general use. however, studies of vaccinia-based vaccine vectors have provided a strong basic foundation for research on other more highly attenuated poxvirus variants. modified vaccinia ankara (mva) is an attenuated vaccinia virus that was originally derived by prolonged passage of a vaccinia virus isolate on chicken embryo fibroblasts in culture. in the course of extensive passage in culture, a viral variant emerged that had fortuitously deleted large sections of the viral genome, including those that encode important poxvirus immune evasion genes and those that determine the ability of the virus to replicate on cells obtained from different animal species. specifically, while mva grows well on chicken cells, it cannot replicate in human cells in culture or in vivo, conferring an inherent safety feature. mva was safely administered to over 100 000 individuals at high risk of adverse consequence for vaccinia immunization toward the end of the smallpox eradication effort. more recently, it has garnered renewed interest as a potential safer smallpox vaccine in the wake of concerns about bioterrorism threats. even though mva cannot replicate in mammalian cells, the virus demonstrates favorable immunogenic properties. mva has been used as a vector expressing genes for a wide variety of genes, including hiv and malaria antigens either alone or, as described above, in 'prime-boost' regimens, where mva has been administered following initial priming immunizations with other vaccine vectors. a concerted effort is under way to improve further the performance of mva by manipulating a series of poxvirus genes that dampen the human immune response to the virus (and to any antigens inserted in it). 107 avipox is a family of poxviruses that infect birds and cause respiratory diseases in poultry. canarypox, a member of the avipox group, has been adapted as a vaccine vector. canarypox replicates well on avian cells in culture but cannot replicate on human cells in culture or in humans in vivo. as a result, canarypox, like mva, provides an interesting vector system with inherent safety features. 108 canarypox vectors carrying hiv genes have been tested in several clinical studies, either alone, or in 'prime-boost' regimens following priming with adenovirus vectors and recombinant protein antigens. in a large ongoing phase iii hiv vaccine trial, a recombinant canarypox vector is being used as a priming vector, followed by boosting with a recombinant version of the hiv gp120 surface env protein. to date, the results from human clinical trials of canarypox vectors have been disappointing, with only low-level specific immune responses generated in human volunteers. 109 adenoviruses, one of the common causes of upper respiratory and gastrointestinal infections, have seen extensive use in clinical trials and were one of the first gene therapy vectors. 110 most adenovirus vectors currently being studied in preclinical and clinical settings are disabled by deletion of the early e1 genes that are necessary for replication in an immunized host. most adenovirus vaccine vectors developed have used the well-characterized and readily produced adenovirus serotype 5 (ad5) as the vector 'backbone.' disabled adenovirus vectors are grown in cells that express the e1 genes artificially inserted into the cell's genome. 111 once these disabled vectors, encoding a heterologous pathogen-derived antigen of interest, enter a cell, the pathogen gene product is expressed, processed, and presented by host apcs. as adenoviruses can directly infect dendritic cells, they promise to provide efficient vaccine vectors. robust antibody and cd8 t-cell responses to heterologous antigen genes expressed by adenovirus vectors have been observed in preclinical animal models. furthermore, in early-phase human clinical trials, adenovirus vectors have been generally well tolerated, and proven to be the most effective of any recombinant vector system studied to date in eliciting high-level cd8 t-cell responses. the main potential drawback to widespread use of adenovirus vectors in humans is that, depending on the adenovirus type and the geographic location, variable levels of pre-existing immunity are found in humans as a result of prior naturally acquired adenovirus infections. high levels of antibody against the adenovirus vector might blunt the immunogenicity and efficacy of an adenovirus vector-based vaccine, but it remains to be seen if this will be a significant limitation. 112 should pre-existing immunity to adenovirus vectors derived from epidemiologically prevalent serotypes (e.g., ad5) limit vaccine immunogenicity, current efforts to develop vaccine vectors based on serotypes that are rare in human populations or novel adenovirus vectors specifically designed to avoid preexisting antibody responses may yield effective alternative approaches. adenovirus vectors are currently used in clinical trials for vaccines against hiv, 113 malaria, influenza, and a range of other pathogens. alphaviruses are rna viruses that cause zoonotic diseases, such as venezuelan equine encephalitis. these viruses do not normally circulate in humans, so immunity to these viruses is quite rare in humans. alphaviruses have a strategy for overexpressing the proteins that make up the virion by making a separate subgenomic rna specifically encoding these gene products. current recombinant alphavirus vaccine vector strategies take advantage of this subgenomic transcript, replacing the viral genes with selected genes for other antigens, but maintaining the signals for translation and protein production. in addition, through use of genetic complementation, it is possible to generate virus particles that only contain this heterologous antigen-encoding expression cassette. such virus particles can efficiently mediate infection of host cells, but because they lack other alphavirus genes needed for virus replication cannot spread beyond the initial target cell infected. 114, 115 alphavirus vectors rival the adenoviruses in efficiency of protein production in tissue culture and have induced robust antibody and t-cell responses in preclinical studies. 116 one current limitation of the alphavirus vector system is the difficulty of scaling the production system; however, this is a technical matter that should be addressable. in addition, ample safety data will be needed before widespread use of alphavirus vaccines achieves endorsement by regulatory authorities for use in healthy populations. adeno-associated viruses (aav ) belong to a family of single-stranded dna viruses (parvoviruses) that include the b19 parvovirus that causes a rash in children known as 'fifth disease' (measles, mumps, rubella, and varicella make up the first four). aav is transmitted in conjunction with adenovirus infection, and is not known to cause any significant disease. it is poorly immunogenic in the course of natural infections. 117 aav can integrate into the genome of the infected cell, usually in a particular place on chromosome 19, although integration does not appear to be efficient or site-specific when replicationdefective adenoviruses of the type being developed as vaccine vectors are used. the propensity for chromosomal integration and poor immune response to the virus made aav a good candidate for gene therapy; cells with an integrated viral genome could deliver a gene product for a long time without the immune system killing the infected cell. recently, efforts have been made to adapt replicationdefective aav as a vaccine vector. although encouraging results have been reported in preclinical studies, phase i studies in humans have demonstrated disappointing immunogenicity. n summary n the challenges to optimizing the full public health potential of existing vaccines largely relate to programmatic considerations. in contrast, the terrible impact of infectious diseases that cannot now be prevented by vaccines (such as the 'big three' killers of hiv, tuberculosis, and malaria) pose direct challenges to the scientific community to develop new generations of vaccines that overcome the largely biological obstacles to control and elimination of these diseases. the nature of the challenges posed by such pathogens necessitates that future vaccine efforts will not simply recapitulate the immune responses engendered by natural infection (as has been the premise of traditional vaccine development efforts), but rather, substantially improve upon them. as the development of vaccines to prevent infections with the so-far refractory pathogens is pursued, improved understanding of the immune response to natural infection, as well as delineation of the reasons why host immune responses fail either to clear incipient infections or prevent future new ones, will be essential. fortunately, early empiric approaches have now been replaced with hypothesis-driven strategies enabled by improved insight into the functioning of the human immune system, as well as new technologies, including higher-resolution tools to describe and quantitate pathogen-specific immune responses; novel methods for antigen discovery and targeted optimization of immunogenicity; the development of new, mechanism-based adjuvants; and the advent of innovative methods for vaccine vector-mediated antigen delivery. thus, although the challenges may be vexing, the scientific and technical foundations on which vaccine development efforts rest have never been stronger. n references n for many important infectious diseases for which no vaccines are currently available, successful derivation of effective vaccines will depend on improving upon natural immunity, especially in those instances where natural immunity does not follow natural infection (such as human immunodeficiency virus (hiv) and malaria) or where safety concerns limit the development of specific protective antigens (neisseria meningitidis group b). in addition, for other pathogens that typically manifest significant genetic (and antigenic) diversity (such as influenza, hiv, and bacteria such as streptococcus pneumoniae), a need exists to develop novel vaccines that can protect against a wide range of variants with a limited number of vaccine immunogens. towards these ends, a number of new approaches, enabled by new vaccine technologies, are being pursued, including: >> targeted alteration of protective antigens to increase their ability to elicit protective immune responses (e.g., efforts to alter the structure of the hiv env glycoproteins gp120 and gp41 so that they elicit higher-level, more potent, neutralizing antibody responses than their native counterparts) >> the development of synthetic consensus antigens able to elicit broader immune responses than would sequences obtained from individual pathogen isolates (e.g., efforts to develop consensus immunogens able to elicit cytotoxic t-lymphocyte (ctl) responses against genetically diverse hiv-1 variants) >> techniques for new antigen discovery to identify novel conserved antigens within otherwise genetically diverse pathogens (e.g., streptococcus pneumoniae) or those for which currently known protective antigens cannot be developed as vaccines (e.g., neisseria meningitidis group b) >> the use of novel adjuvants or vaccine vectors to enable generation of higher-level and/or more functional immune responses to pathogen antigens by vaccination than are seen following natural infection, and to enable high-level, fully functional memory immune responses to be activated at the time of initial infection (e.g., efforts to elicit high-level hivspecific or hepatitis c virus-specific ctls by recombinant viral vectors) >> use of novel methods to shift relative immunodominance of specific pathogen gene products to increase the immunogenicity of conserved antigens from otherwise diverse pathogen genomes that are typically poorly immunogenic in the course of natural infections (e.g., efforts to augment the antibody response to the influenza a virus m2 protein via the use of potent adjuvants or conjugation to immunogenic carrier proteins) ten great public health achievements -united states principles and lessons from the smallpox eradication programme advance market commitments for vaccines against neglected diseases: estimating costs and effectiveness mass vaccination: when and why immunisation and herd immunity standards for immunization practice for vaccines in children and adults economic evaluation of the 7-vaccine routine childhood immunization schedule in the united states priorities among recommended clinical preventive services development of an anti-core lipopolysaccharide vaccine for the prevention and treatment of sepsis safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis safety and efficacy of a pentavalent human-bovine (wc3) reassortant rotavirus vaccine the history of the smallpox vaccine grease, anthraxgate, and kennel cough: a revisionist history of early veterinary vaccines polio eradication: the opv paradox review of current preclinical testing strategies for bacterial vaccines standardization of acellular pertussis vaccines vaccines against polysaccharide antigens conjugate vaccines newer directions in vaccine development and utilization the development of novel hepatitis b vaccines papillomavirus l1 major capsid protein self-assembles into virus-like particles that are highly immunogenic quadrivalent vaccine against human papillomavirus to prevent anogenital diseases immunologic responses following administration of a vaccine targeting human papillomavirus types 6, 11, 16, and 18 the rotavirus vaccine saga global illness and deaths caused by rotavirus disease in children effect of rotavirus vaccination programme on trends in admission of infants to hospital for intussusception rotashield: the ill-fated rhesus-human reassortant rotavirus vaccine a review of the vaccine adverse event reporting system database vaccine safety surveillance using large linked databases: opportunities, hazards and proposed guidelines permanent brain damage and pertussis vaccination: is the end of the saga in sight? absence of detectable measles virus genome sequence in blood of autistic children who have had their mmr vaccination during the routine childhood immunization schedule of uk the mmr vaccination and autism controversy in united kingdom 1998-2005: inevitable community outrage or a failure of risk communication? molecular mimicry of host structures by lipooligosaccharides of neisseria meningitidis: characterization of sialylated and nonsialylated lacto-n-neotetraose (galbeta1-4glcnacbeta1-3galbeta1-4glc) structures in lipooligosaccharides using monoclonal antibodies and specific lectins prospects for prevention of childhood infections by maternal immunization an hiv vaccine -evolving concepts prospects for an aids vaccine: three big questions, no easy answers update of the drug resistance mutations in hiv-1: 2004 hiv gp120 vaccine aidsvax -vaxgen, hiv vaccine aidsvax -vaxgen recent advances in the development of hiv-1 vaccines using replication-incompetent adenovirus vectors one step forward, two steps back -will there ever be an aids vaccine? child mortality and malaria transmission intensity in africa the past, present and future of childhood malaria mortality in africa the immune response to plasmodium falciparum malaria longevity of the immune response and memory to blood-stage malaria infection malaria vaccines in development irradiated sporozoite vaccine induces hla-b8-restricted cytotoxic t lymphocyte responses against two overlapping epitopes of the plasmodium falciparum sporozoite surface protein 2 global epidemiology of tuberculosis advances in tuberculosis vaccine strategies modern vaccines. mycobacterial diseases tuberculosis vaccines: current progress preclinical testing of new vaccines for tuberculosis: a comprehensive review tuberculosis: from genome to vaccine respiratory virus immunization. i. a field trial of two inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory syncytial virus vaccine sensitizing versus immunizing properties of inactivated measles vaccine recombinant bovine/ human parainfluenza virus type 3 (b/hpiv3) expressing the respiratory syncytial virus (rsv) g and f proteins can be used to achieve simultaneous mucosal immunization against rsv and hpiv3 the new meningococcal conjugate vaccine. a profile of its safety, efficacy, and indications for use antigenic similarities between brain components and bacteria causing meningitis. implications for vaccine development and pathogenesis prevention of perinatal group b streptococcal disease. revised guidelines from cdc current status of a hepatitis c vaccine: encouraging results but significant challenges ahead prophylactic vaccine strategies and the potential of therapeutic vaccines against herpes simplex virus clinical trials of prophylactic and therapeutic herpes simplex virus vaccines cytomegalovirus vaccine prepared in wi-38 a canarypox vector expressing cytomegalovirus (cmv) glycoprotein b primes for antibody responses to a live attenuated cmv vaccine (towne) vaccine strategies against human cytomegalovirus infection phase i/ii studies to evaluate safety and immunogenicity of a recombinant gp350 epstein-barr virus vaccine in healthy adults understanding dengue pathogenesis: implications for vaccine design prospects for a dengue virus vaccine a method for the screening of fusion protein expression by lambda-gt11 recombinant clones without the preparation of lysogens antibody screening of bacteriophage lambda gt-11 dna expression libraries vaccinology at the beginning of the 21st century reverse vaccinology, a genome-based approach to vaccine development the pan-genome: towards a knowledge-based discovery of novel targets for vaccines and antibacterials genomics and proteomics in reverse vaccines haemophilus influence: the impact of whole genome sequencing on microbiology genome sequence of the human malaria parasite plasmodium falciparum bacterial proteomics and vaccine development proteomics approaches towards antigen discovery and vaccine development contribution of mass spectrometry-based proteomics to immunology structural proteomics and computational analysis of a deadly pathogen: combating mycobacterium tuberculosis from multiple fronts identification of in vivo-expressed immunogenic proteins by serological proteome analysis of the bacillus anthracis secretome identification of tumor antigens in renal cell carcinoma by serological proteome analysis innate immune recognition on regulation of phagosome maturation and antigen presentation translating innate immunity into immunological memory: implications for vaccine development innate immune induction of the adaptive immune response liposomes and iscoms understanding the role of innate immunity in the mechanism of action of the live attenuated yellow fever vaccine 17d haemophilus influenzae type bouter membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor 2 (tlr2) and requires the presence of tlr2 for optimal immunogenicity structure and properties of aluminum-containing adjuvants immunoactivating potential of multilamellar liposome vesicles (mlv) in murine popliteal lymph node (pln) test adjuvant and antigen delivery properties of virosomes safety and immunogenicity of nonadjuvanted and mf59-adjuvanted influenza a/h9n2 vaccine preparations new hepatitis b vaccine formulated with an improved adjuvant system cpg 7909 adjuvant improves hepatitis b virus vaccine seroprotection in antiretroviral-treated hivinfected adults testing of cpg-optimized protein and dna vaccines against the hepatitis b virus in chimpanzees for immunogenicity and protection from challenge immunostimulatory sequences of dna and conjugates in the treatment of allergic rhinitis hiv gag protein conjugated to a toll-like receptor 7/8 agonist improves the magnitude and quality of th1 and cd8 + t cell responses in nonhuman primates vaccination with recombinant fusion proteins incorporating toll-like receptor ligands induces rapid cellular and humoral immunity a west nile virus recombinant protein vaccine that coactivates innate and adaptive immunity the development of multivalent bovine rotavirus (strain wc3) reassortant vaccine for infants intramuscular injection of plasmid dna heterologous protection against influenza by injection of dna encoding a viral protein dna vaccines: recent developments and future possibilities from plasmids to protection: a review of dna vaccines against infectious diseases poxvirus-based vaccine candidates for hiv: two decades of experience with special emphasis on canarypox vectors drug evaluation: dna/mva primeboost hiv vaccine improving recombinant mva immune responses: potentiation of the immune responses to hiv-1 with mva and dna vectors expressing env and the cytokines il-12 and ifn-gamma applications of canarypox (alvac) vectors in human and veterinary vaccination high-dose recombinant canarypox vaccine expressing hiv-1 protein, in seronegative human subjects biology of adenovirus and its use as a vector for gene therapy adenovirus-based expression vectors and recombinant vaccines established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. potential for immunosuppression and vector engineering to overcome barriers of immunity attenuation of simian immunodeficiency virus sivmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing gag venezuelan equine encephalitis virus vectors expressing hiv-1 proteins: vector design strategies for improved vaccine efficacy an alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector recombinant alphaviruses as vectors for anti-tumour and anti-microbial immunotherapy immune responses to adeno-associated virus and its recombinant vectors key: cord-027860-s97hdhh6 authors: zeimet, anthony; mcbride, david r.; basilan, richard; roland, william e.; mccrary, david; hoonmo, koo title: infectious diseases date: 2020-06-22 journal: textbook of family medicine doi: 10.1016/b978-1-4377-1160-8.10016-8 sha: doc_id: 27860 cord_uid: s97hdhh6 nan infections of the upper respiratory tract accounted for more than 36 million ambulatory medical visits in 2005, according to the national ambulatory medical care survey (cherry et al., 2007) . although a large percentage of these infections are viral in origin, antibiotics are still prescribed for more than 50% of patients with acute respiratory tract infection (arti). acute bronchitis, in the arti category, is defined as a respiratory infection in which cough is the predominant symptom and there is no evidence of pneumonia. antibiotics are often prescribed despite limited evidence that they shorten the duration of acute bronchitis. with increasing incidence of antibiotic resistance, bronchitis allows physicians to practice "prescriptive restraint" and to provide supportive therapy. consider using the phrase "chest cold" to help patients understand the viral and benign nature of this infection. chronic bronchitis is one of the manifestations of chronic obstructive pulmonary disease (copd) and is defined clinically as cough and sputum production on most days for 3 months annually for 2 years. chronic bronchitis is thought to be primarily inflammatory in origin, although infection may be associated with acute exacerbations; with increased sputum production and worsening dyspnea, antibiotics have proved effective in acute episodes. however, systemic corticosteroids are the mainstay of copd exacerbation management. the patient with acute bronchitis presents with cough, often productive. patients may report clear or colored mucus in association with the presumed diagnosis of acute bronchitis. despite what many patients believe, the color of sputum, even purulent sputum, is not predictive of bacterial infection. the cough of bronchitis can last up to 4 weeks, sometimes even longer. typically, acute bronchitis is associated with other manifestations of infection, such as malaise and fever. respiratory viruses are thought to cause the majority of cases of acute bronchitis. influenza a and b, parainfluenza, respiratory syncytial virus (rsv), coronavirus, adenovirus, and rhinovirus are common pathogens in the viral category. clues to a specific virus may be found in the patient history; for example, rsv might be considered when there is household exposure to infected children. influenza typically presents with sudden onset of symptoms, including fever, myalgias, cough, and sore throat. neuraminidase inhibitors are modestly effective in shortening the duration of influenza in ambulatory and healthy patients (by about 1 day), if initiated in the first 48 hours of illness. the resistance patterns of influenza a and b have shifted in the last several years and may vary based on yearly viral strains. influenza b has remained, as of 2010, sensitive to zanamivir (relenza) and oseltamivir (tamiflu). currently circulating strains of influenza a, both h1n1 and h3n2, and influenza b have generally remained sensitive to both oseltamivir and zanamivir (fiore et al., 2011) . family physicians are advised to consider restraint in the prescribing of these agents, since resistance is of great concern. yearly influenza immunization and cough etiquette and hygiene are likely the most useful techniques for influenza management. studies have identified other pathogens, such as mycoplasma pneumoniae and chlamydophila pneumoniae, in a small minority of cases of clinical acute upper respiratory illness with cough as the predominant symptom. no significant benefit has been found in treating these infections with antibiotics. an exception in the treatment of acute bronchitis-like illness with antibiotics is when confirmed or probable bordetella pertussis is present. early treatment with a macrolide antibiotic and patient isolation will likely decrease coughing paroxysms and limit spread of disease (braman, 2006) . although common upper respiratory bacterial pathogens, such as moraxella (branhamella) catarrhalis, streptococcus pneumoniae, and haemophilus influenzae, may be isolated from patients with acute bronchitis, their relevance is questionable because these bacteria can be present in the respiratory tract of healthy individuals. obtaining sputum for culture when bronchitis is the diagnosis generally is not useful. antibiotics may offer a modest benefit in the treatment of acute bronchitis, with many studies showing no statistical significance in the outcome of treated versus not-treated groups. measures of function, such as duration of illness, loss of work, and limitation of activity, have not shown clinically significant improvement in those with acute bronchitis taking antibiotics. coupled with cost and the potential for side effects, the use of antibiotics for acute bronchitis is not recommended. if a provider decides to use an antibiotic in a specific patient situation, narrow-spectrum respiratory agents are preferred, such as a first-generation macrolide or doxycycline. treating the symptom of cough in acute bronchitis is an important concern for patients. in adults with acute bronchitis with signs of airway obstruction, evidenced by wheezing on examination or decreased peak expiratory flow rate, beta-2 agonists may be helpful in alleviating cough. these agents are not helpful for children with acute cough or adults with cough and no evidence of airway obstruction. side effects of tremor and an anxious feeling must be weighed against this benefit. patients often are primarily interested in alleviating symptoms caused by respiratory illness. unfortunately, there is mixed evidence for the use of over-the-counter (otc) and prescription cough medications. dextromethorphan and codeine may be somewhat effective, although they have not been evaluated in randomized, double-blinded, placebo-controlled trials for acute bronchitis. combination first-generation antihistamine-decongestant products may be effective for the cough associated with colds. naproxen showed efficacy against cough in one upper respiratory model study (sperber et al., 1992) . guaifenesin acts as an expectorant and may have some effect on cough by its mucus-thinning properties. community-acquired pneumonia (cap) is defined as an acute infection of the pulmonary parenchyma and, along with influenza, is the seventh leading cause of death in the united states. fever, cough, sputum production, pleuritic chest pain, and dyspnea are common symptoms of cap. nausea, vomiting, and diarrhea also may occur, and in elderly patients, cap may present with mental status changes. although its absence usually makes pneumonia less likely, fever can be absent in the elderly patient. other physical examination findings include an elevated respiratory rate, conversational dyspnea, tachycardia, and rales. egophony and dullness to percussion may be noted with focal consolidation. typical laboratory findings include leukocytosis. the diagnosis of pneumonia is based on the presence of symptoms and the presence of an infiltrate on chest radiograph. if infiltrate is not present, consider obtaining a chest tomography scan (which has higher sensitivity) to rule in or rule out cap. if negative, other diagnoses should be considered. the most common microbiologic agent of pneumonia is often not isolated (table 16 -1). furthermore, studies have shown that bacteriologic causes of pneumonia cannot be determined by radiographic appearance (i.e., "typical" vs. "atypical"). in the proper clinical setting, certain clinical microbes should be considered because they can affect treatment considerations and epidemiologic studies. these include legionella spp., influenza a and b, and communityacquired methicillin-resistant staphylococcus aureus (mrsa). certain diagnostic tests are performed based on clinical setting. blood cultures are not routinely done in the outpatient setting but should always be done if the patient is being admitted to the hospital, ideally before antibiotics are given. the use of gram stain and sputum culture remains controversial but can provide more evidence of a bacterial cause (e.g., many pmns). if sputum cultures are being obtained, it is recommended that the physician have the patient expectorate directly into a specimen cup and have it sent immediately for processing. this can increase the yield of isolating streptococcus pneumoniae among antibiotics for the treatment of bronchitis is not recommended because of the cost, potential for side effects, and lack of clinical benefit (braman, 2006; smith et al., 2009 ) (sor: a). in the treatment of bordetella pertussis, early administration of a macrolide antibiotic and patient isolation will likely decrease coughing paroxysms and limit spread of disease (braman, 2006) (sor: a). in adults with acute bronchitis with signs of airway obstruction, as evidenced by wheezing on examination or decreased peak expiratory flow rate, beta-2 agonists may be helpful in alleviating cough (braman, 2006) (sor: b). for acute exacerbation of copd associated with purulent sputum and increased shortness of breath, treatment with antibiotics decreases mortality by 77% and treatment failure by 53% (ram et al., 2009 ) (sor: a). 210 other respiratory pathogens. other tests include urine antigen tests for s. pneumoniae, legionella pneumophila serogroup 1, and nasal swab for influenza a and b. in young children, rsv, adenovirus, and parainfluenza in addition to influenza are common causes. nasal swab for rsv and influenza can be rapidly done, but the other causes can be determined with viral cultures, serology, enzyme-linked immunosorbent assay (elisa), and polymerase chain reaction (pcr), although results usually are received after resolution of the acute symptoms. perhaps the most important decision for clinicians is to determine the location of treatment. the american thoracic society (ats) and the infectious diseases society of america (idsa) recommend use of the pneumonia severity index (psi), which uses 20 variables to risk-stratify the patient into five mortality classes, or the curb-65, which measures five clinical variables in this decision making. the curb-65 may be the easiest and most convenient to use at the site of decision making. a score of 0 or 1 indicates treatment as an outpatient; a score of 2 requires hospital admission to the general medical ward; and a score of 3 or more indicates admission to an intensive care unit (icu) (box 16-1). treatment of cap should be targeted toward the most likely etiology (table 16 -2). outpatient therapy for patients who have no comorbidities and have not received antibiotics within the last 3 months includes doxycycline or a macrolide antibiotic. use of a fluoroquinolone antibiotic (levofloxacin or moxifloxacin) should be reserved for patients with more complicated pneumonia and those requiring hospitalization. patients who have comorbid conditions or recent antibiotic exposure, or who will be hospitalized, should receive a respiratory fluoroquinolone or combination therapy with a betalactam drug plus a macrolide, for 48 to 72 hours after fever abates (usually 5-7 days' total therapy). if an organism is isolated, therapy may be narrowed to cover the causative agent. the clinician should consider longer therapy and appropriate antibiotics to cover for infection by less common organisms such as staphylococcus aureus or pseudomonas aeruginosa. if the patient has no more than one abnormal value (temperature <37.8° c, heart rate <100, respiratory rate <24, sbp >90, o 2 saturation >90%, po 2 >60 on room air) and the patient is able to maintain oral intake and has a normal mental status, the clinician can safely switch to oral therapy and discharge the patient from the hospital. unless the etiology of the pneumonia is known, the physician should switch to oral antibiotics in the same class as the intravenous antibiotics used. the u.s. preventive services task force (uspstf) along with idsa and ats recommend annual influenza vaccinations to those over 50 years of age, those who are (or who reside with those who are) at high risk for influenza complications, and all health care workers. furthermore, the pneumococcal vaccine should be given to all those over age 65. smoking cessation is also important and should be discussed at each clinic visit. • concerns about development of resistant seasonal and h1n1 swine-derived influenza virus should be considered in the decision to administer antiviral medications to healthy patients with these infections. • the abrupt onset of fever with chills, headache, malaise, myalgias, arthralgias, and rigors during "flu season" is sufficient to diagnose influenza. • prevention of influenza is generally with vaccination. influenza deserves special mention because it is an important cause of pneumonitis and can precede a bacterial pneumonia. influenza viruses are medium-sized enveloped ribonucleic acid (rna) viruses that consist of a lipid bilayer with matrix proteins with spiked surface projections of glycoproteins (hemagglutinins, neuraminidase) on the outer surface ( figure 16-1) . both influenza a and influenza b have eight segmented pieces of single-stranded rna. the only difference between influenza a and b is that b does not have an m2 ion channel. hemagglutinins, three types of which typically infect humans (h1, h2, h3), bind to respiratory epithelial cells and allow fusion with the host cell. neuraminidase, consisting of two types (n1, n2), allows release of virus from the infected cells. a unique aspect of influenza is that antigenic variation occurs annually. antigenic shift is caused by a genetic reassortment between animal and human influenza strains, producing a novel virus that generally causes the worldwide pandemics. influenza viruses circulate mostly among humans, birds, and swine. sometimes; a human strain and an animal strain can intermingle and create a new, unique virus. this is what happened during spring 2009, heralding the most recent pandemic and creating "novel h1n1 influenza" (swine influenza). genotype analysis • score 0 or 1: outpatient treatment • score 2 : inpatient treatment on a general medical floor • score >3: inpatient treatment in an intensive care unit bun, blood urea nitrogen. locally adapted guidelines should be implemented to improve the processing of care variables and relevant clinical outcomes in pneumonia (mandell et al., 2007) (sor: b) . objective criteria or scores should always be supplemented with physician determination of subjective factors, including the patient's ability to take oral medication safely and reliably and the availability of outpatient support resources (sor: b). for patients with curb-65 score of 2 or higher, more intensive treatment (i.e., hospitalization or, where appropriate and available, intensive in-home health care services) is usually warranted (sor: c). of this strain determined that components came from an influenza virus circulating among swine herds in north america that combined with a virus circulating among ill swine in eurasia, creating a new influenza strain capable of causing disease in humans. because this virus had not previously infected humans, it had the potential to cause widespread morbidity and mortality worldwide. during pandemics, the u.s. centers for disease control and prevention (cdc) estimates an additional 10,000 to 40,000 deaths caused by influenza. although higher than in nonpandemic years, mortality was significantly less than initially predicted in 2009. no recent antibiotic therapy a respiratory fluoroquinolone alone or an advanced macrolide plus a β-lactam † † an advanced macrolide plus a β-lactam, or a respiratory fluoroquinolone alone (regimen selected will depend on nature of recent antibiotic therapy) intensive care unit (icu) a β-lactam † † plus either an advanced macrolide or a respiratory fluoroquinolone pseudomonas infection is not an issue but patient has a β-lactam allergy a respiratory fluoroquinolone, with or without clindamycin pseudomonas infection is an issue ‡ ‡ (cystic fibrosis, impaired host defenses) either (1) copd, chronic obstructive pulmonary disease; mrsa, methicillin-resistant staphylococcus aureus. * azithromycin or clarithromycin. † that is, the patient was given a course of antibiotic(s) for treatment of any infection within the past 3 months, excluding the current episode of infection. such treatment is a risk factor for drug-resistant streptococcus pneumoniae and possibly for infection with gram-negative bacilli. depending on the class of antibiotics recently given, one or another of the suggested options may be selected. recent use of a fluoroquinolone should dictate selection of a nonfluoroquinolone regimen, and vice versa. ‡moxifloxacin, levofloxacin, or gemifloxacin. § dosage: 1 g orally (po) three times daily (tid). ¶ dosage: 2 g po twice daily (bid). ** high-dose amoxicillin (1 g tid), high-dose amoxicillin-clavulanate (2 g bid), cefpodoxime, cefprozil, or cefuroxime. † † cefotaxime, ceftriaxone, ampicillin-sulbactam, or ertapenem. ‡ ‡the antipseudomonal agents chosen reflect this concern. risk factors for pseudomonas infection include severe structural lung disease (e.g., bronchiectasis) and recent antibiotic therapy, health care-associated exposures or stay in hospital (especially in the icu). for patients with cap in the icu, coverage for s. pneumoniae and legionella species must always be considered. piperacillin-tazobactam, imipenem, meropenem, and cefepime are excellent β-lactams and are adequate for most s. pneumoniae and h. influenzae infections. they may be preferred when there is concern for relatively unusual cap pathogens, such as p. aeruginosa, klebsiella spp., and other gram-negative bacteria. § § piperacillin, piperacillin-tazobactam, imipenem, meropenem, or cefepime. ## data suggest that older adults receiving aminoglycosides have worse outcomes. ¶ ¶ dosage for hospitalized patients, 750 mg/day. the abrupt onset of fever, along with chills, headache, malaise, myalgias, arthralgias, and rigors during "flu season," is sufficient to diagnose influenza. as the fever resolves, a dry cough and nasal discharge predominate. a rapid nasal swab or viral cultures can be used to confirm the diagnosis of influenza but is rarely needed. in fact, the sensitivity of these rapid tests can range from 50% to 70%, so a negative test does not rule out influenza. the primary care physician needs to determine if the patient has influenza or the common cold, because symptoms of both illnesses generally overlap (table 16-3) . treatment of influenza is generally not necessary because it is usually a self-limiting condition. treatment should be reserved for those with comorbidities who present within 48 hours of symptom onset. neuraminidase inhibitors (zanamivir and oseltamivir) prevent the release of virus from the respiratory epithelium and are approved for both influenza a and influenza b. the m2 inhibitors (amantadine and rimantadine) are approved by the u.s. food and drug administration (fda) for the treatment of influenza a because these drugs block the m2 ion protein channel, preventing fusion of the virus to host cell membrane (influenza b has no m2 ion channel). the use of m2 inhibitors is limited because of increasing resistance among influenza a viruses, as well as causing central nervous system (cns) problems that are usually exacerbated in elderly persons, who are more likely to seek treatment for influenza (table 16 -4). the major complication of influenza is a secondary bacterial pneumonia or exacerbation of underlying copd. initial improvement in clinical symptoms followed by deterioration usually suggests a secondary bacterial pneumonia, which can usually be confirmed with a chest radiograph showing an infiltrate. other, less common complications of influenza include myositis, myocarditis, pericarditis, transverse myelitis, encephalitis, and guillain-barré syndrome. prevention of influenza is generally with vaccination. box 16-2 outlines patients at risk for influenza complications who should be vaccinated yearly. although anyone wanting an influenza vaccine should be vaccinated, during periods of vaccine shortage, high-risk groups have priority. a well-matched vaccine can prevent influenza among 70% to 90% of adults and decrease work absenteeism. conversely, a poorly matched vaccine only prevents influenza in 50% of healthy adults. proper hand hygiene and covering one's cough are two additional important components in preventing the spread of influenza virus. • population-based vaccination programs have been highly effective in decreasing the incidence of many viral infections. • acyclovir can be used in adults and children with varicella to decrease symptoms if given in the first 48 hours after rash onset, but its benefit must be weighed against its cost and the possibility of development of viral resistance. • antiviral medications should be considered to decrease the incidence of postherpetic neuralgia, particularly in older patients. early treatment (within 48 hours of onset of symptoms) with oseltamivir or zanamivir is recommended for influenza a (jefferson et al., 2006) (sor: a). use of oseltamivir and zanamivir is not recommended for patients with uncomplicated influenza with symptoms for more than 48 hours (kaiser and hayden, 1999) (sor: a). oseltamivir and zanamivir may be used to reduce viral shedding in hospitalized patients or to treat influenza pneumonia (sor: c). (from treanor jj: influenza viruses, including avian influenza and swine influenza. in mandell gl, bennett je, dolin rd (eds) . mandell, douglas, and bennett's principles and practices of infectious diseases, 7th ed. philadelphia, churchill livingstone, 2010, p 2266.) • measles has had a resurgence in recent years and should be suspected when a patient presents with cough, coryza, conjunctivitis, and head-to-toe rash. • epstein-barr virus and cytomegalovirus infections are generally not clinically distinguishable, and their treatment is primarily supportive. vaccinations have dramatically decreased the incidence of a number of historically common viral infections; smallpox has been eradicated through widespread vaccination. however, recent outbreaks of measles and mumps on college campuses underscore the need to remain vigilant in administering vaccines at the population level, even though no vaccine is available for many common viruses. varicella is one of the classic viral exanthems of childhood. before routine vaccination, having chickenpox was one of childhood's "rites of passage." the virus, a herpesvirus (human herpesvirus 3), is effectively transmitted, causing outbreaks in schools and households. patients with primary varicella present with fever, headache, and sore throat. generally within 1 to 2 days of onset of symptoms, a papulovesicular rash erupts diffusely. the classic description of the chickenpox lesion is "a dewdrop on a rose petal," suggesting a central vesicle on an erythematous base. lesions continue to appear for 5 to 7 days. all lesions going from papule to vesicle to crusted lesion takes about 2 weeks. patients are considered to be infectious, primarily through respiratory secretions, during the 2 days before symptoms appear and until all lesions are crusted. treatment of varicella is generally supportive. control of spread may be a concern in group-living environments such as schools or residence halls. isolation of the infected patient away from those susceptible to varicella infection is standard practice. acyclovir can be started within the first 24 hours after rash eruption to achieve an attenuation of the infectious course. in children, this means a decrease in the duration of fever by about 1 day and a decrease in the number of lesions (swingler, 2010) . in adults, acyclovir decreases rash duration and the number of lesions, although the results are less significant than for children. adult dosing of acyclovir for varicella is 800 mg five times daily. the marginal benefit must be weighed against the possible development of resistance at a population level and the cost of the medication. complications of varicella can include secondary infection of skin lesions, pneumonitis, encephalitis, and dehydration from vomiting and diarrhea. varicella is prevented primarily through administration of vaccine. the vaccine is highly effective in children, with recommended dosing at 12 to 15 months with a second dose at 4 to 6 years. varicella is now included in a measles-mumpsrubella (mmr) vaccine, which can be given between 12 months and 12 years of age. the varicella vaccine is a live, attenuated virus and should not be given to certain immunocompromised patients. the vaccine can also be administered to exposed immunocompetent contacts, although the benefit is clearer for children than adults. severely immunocompromised patients exposed to varicella (particularly those with advanced hiv disease) may be given high-dose acyclovir to prevent development of disease. herpes zoster is a reactivation of the neurotropic varicella virus, typically in a dermatomal distribution. this is more common in elderly or immunocompromised patients but can occur in healthy people as well. patients with zoster may note generalized malaise, hyperesthesia, numbness, tingling, and pain in the skin before development of a rash. the appearance of the rash is the same as for chickenpox, although most often isolated to a unilateral dermatome. the diagnosis of herpes zoster is clinical based on the history and the classic appearance of the rash. in immunocompromised patients, however, the rash may not be dermatomally isolated. when the diagnosis is unclear, viral culture can be obtained from the base of a lesion. antiviral medications are likely to decrease the incidence of postherpetic neuralgia and are recommended, particularly in elderly patients (wareham, 2010) . valacyclovir (1 g three times daily) or famciclovir (500 mg every 8 hours) for 7 days is likely more effective than acyclovir in achieving this result. either drug should be started as soon after the diagnosis as possible, preferably within 48 to 72 hours of rash onset. when patients have established postherpetic neuralgia, gabapentin and tricyclic antidepressants are helpful in alleviating the pain. the rash of zoster is infectious to the touch. patients should be advised to keep the rash covered until all the lesions have crusted. zoster of the trigeminal nerve can extend to the eye and warrants immediate ophthalmologic intervention. a vaccine to prevent herpes zoster in adults was released in 2006. the zoster vaccine differs from the varicella vaccine in that the amount of attenuated virus is 14 times higher in the zoster vaccine. the vaccine decreases the incidence of zoster by 50%. it is recommended for administration by the american academy of family physicians (aafp) to adults over age 60, regardless of prior varicella or zoster history. although generally well tolerated, the vaccine is somewhat costly. in 2008, more measles cases were reported than in any other year since 1997 (cdc, 2010) . measles is the "first disease" of childhood from the history of medicine. in adults, measles infection may be acquired in the face of waning immunity from remote immunization. a booster dose of mmr vaccine is recommended before college entry. clinically, measles presents with cough, coryza (nasal irritation and congestion), and conjunctivitis. fever is common several days before the onset of the rash. the rash of measles typically spreads from head to toe and has an erythematous, papular appearance with a "sandpaper" feeling. koplik's spots are erythematous papules with a bluish center on the oral mucosa and appear early in measles. measles is highly contagious through droplets. lymphopenia and neutropenia are common laboratory findings with measles infection. complications of measles include primary infections such as pneumonia, gastroenteritis, encephalitis, and the rare subacute sclerosing panencephalitis. secondary infections such as otitis media, pneumonia, and adenitis may also occur. treatment is supportive, and the implications of measles infection are primarily in the public health realm. patients with measles should be isolated for at least 4 days after the appearance of the rash. it is important to recognize that patients are contagious for 2 days before the development of symptoms. careful verification of immunization status for close contacts is essential. clinical infectious mononucleosis is a common infection in adolescents and early adults. the clinical syndrome is most often caused by epstein-barr virus (ebv), although cytomegalovirus (cmv) may also be the source in this clinical syndrome, which includes fever, exudative tonsillitis, adenopathy (often including posterior cervical or occipital nodes), and fatigue. ebv is transmitted in oral secretions and may be transmitted sexually as well. b cells are infected with ebv either directly or after contact with epithelial cells, resulting in diffuse lymphoid enlargement. the diagnosis of infectious mononucleosis is made by recognizing the clinical symptoms of fever, pharyngitis, and adenopathy along with the laboratory findings of greater than 50% lymphocytes with 10% or more atypical lymphocytes (hoagland, 1952) . also, a positive serologic test for heterophile antibody assists the family physician in the diagnosis. to differentiate ebv from cmv mononucleosis, serology (igg and igm) may be obtained. results of these tests are generally not available in time to have a significant benefit clinically. splenic enlargement as part of this lymphoid hypertrophy can lead to splenic rupture (0.1% risk) (dommerby et al., 1986) . athletes with infectious mononucleosis must be managed carefully to avoid their participation in sports that could result in abdominal trauma. other risks associated with infectious mononucleosis include upper airway obstruction, asymptomatic transaminase elevation, thrombocytopenia, and rash after the administration of ampicillin or amoxicillin. routinely obtaining transaminase levels in patients without clinical hepatitis is of little value and can increase the overall cost of management. treatment of infectious mononucleosis is largely supportive. patients should be instructed to treat fever with antipyretics, rest, and expect symptom duration of 2 to 4 weeks, although symptoms can last for several months. the use of steroids, such as prednisone, has shown limited benefit. data suggest an initial benefit 12 hours after steroid administration, although this is lost within several days (candy and hotopf, 2006) . combination of steroid and an antiviral (valacyclovir) may have some positive effect on fatigue. • the most common presentation of tuberculosis is pulmonary disease. • tuberculosis is diagnosed by acid-fast bacilli smears and cultures. • standard first-line agents to treat tb are isoniazid, rifampin, pyrazinamide, and ethambutol. • high-risk patients with a positive purified protein derivative skin test or quantiferon-tb gold test should be treated for latent tb infection. • the current recommendation for first-line treatment for latent tb is 9 months of oral isoniazid. tuberculosis skin testing should be interpreted without regard to bacille calmette-guérin (bcg) history, because bcg is administered in areas where tb is endemic and bcg does not provide complete protection from tb infection. tuberculosis (tb) is a disease that has plagued humans since antiquity, with evidence of spinal tb in neolithic and early egyptian remains. at present, tb affects approximately one third of the world's population. tb is the world's second most common cause of death from infectious disease after human immunodeficiency virus or acquired immunodeficiency syndrome (hiv/aids). tuberculosis is caused by mycobacterium tuberculosis, an acid-fast bacillus. tb is acquired by inhalation of respiratory droplets. these respiratory droplets are spread by coughing. brief contact carries little risk for acquiring tb, and infection generally does not occur in open air; open-air sanatoriums were the cornerstone of tb treatment before antimicrobial therapy. in the united states, tb incidence rates have been on the decline since 1992, coinciding with the control of hivinduced aids by antiretroviral therapy. however, tb remains prevalent in certain high-risk groups (i.e. immigrants, iv drug use, homeless persons). most cases of tb are in people age 15 to 49 years. tb in elderly persons is generally caused by a reactivation of latent infection acquired in the remote past, whereas tb in young children indicates ongoing active transmission in the community. infection in children is more likely to progress to active tb and disseminated disease. persons with hiv infection have a disproportionately higher risk for acquiring tb than the general population. tuberculosis is most frequently manifested clinically as pulmonary disease, but it can involve any organ. extrapulmonary tb accounts for about 20% of disease in hiv-seronegative persons but is more common in hiv-seropositive persons. pulmonary tb typically manifest with fever, night sweats, chronic cough, sputum production, hemoptysis, anorexia, and weight loss. chest radiographs in patients with pulmonary tb typically reveal upper-lobe cavitary lesions and can reveal infiltrates or nodular lesions, as well as lymphadenopathy ( figure 16 -2). tb in the setting of advanced hiv co-infection does not generally manifest in the typical manner (table 16-5) . acyclovir started within the first 24 hours after varicella rash eruption can attenuate the infectious course, decreasing duration of fever by 1 day and reducing the number of lesions (sor: a). administration of varicella vaccine to a susceptible child within 3 days of exposure will likely modify or prevent disease (macartney and mcintyre, 2008) the diagnosis of pulmonary tb is made by the demonstration of acid-fast bacilli (afb) in sputum and the growth of m. tuberculosis in culture. these patients typically have an abnormal chest radiograph, as previously described. m. tuberculosis is a slow-growing bacterium, and cultures can take up to 6 weeks to grow. a pcr assay developed for m. tuberculosis can be run on afb smear-positive sputum to hasten the diagnosis of pulmonary tb. a positive pcr on afb-positive sputum is diagnostic of pulmonary tb, but a negative test does not rule out the diagnosis. patients with afb positive smears from sputum samples should be started on anti-tb therapy while awaiting results of pcr and cultures. the treatment of tb always uses multiple agents with anti-tb activity. single agents should never be used. the standard first-line agents are isoniazid (inh), rifampin (rif), pyrazinamide (pza), and ethambutol (emb) (figure 16 -3 and table 16-6). if administered, inh should be given with pyridoxine (vitamin b 6 ; 25-50 mg orally daily) to prevent neuropathy. treatment of active pulmonary tb is generally for 6 months regardless of hiv status, but treatment may need to be extended in certain situations. directly observed therapy (dot) is the preferred mechanism of administration to ensure compliance. many local county and state health departments have systems for dot. treatment of hiv-seropositive patients with tb who are receiving an antiretroviral (arv) regimen that contains a protease inhibitor is complicated by the latter's interaction with rifamycins (particularly rifampin). management of such patients should be coordinated with an infectious diseases specialist, who also should manage drug-resistant tb treatment. in the united states, latent tuberculosis infection (ltbi) is the most prevalent form of tuberculosis. ltbi is the term given to patients with a positive purified protein derivative (ppd) skin test without evidence of active tb. ppd has been used for more than 100 years and relies on delayed-type hypersensitivity (dth) to m. tuberculosis cellular proteins. early late figure 16 -3 treatment algorithm for tuberculosis. patients in whom tb is proved or strongly suspected should have treatment initiated with isoniazid, rifampin, pyrazinamide, and ethambutol for the initial 2 months. a repeat smear and culture should be performed when 2 months of treatment has been completed. if cavities were seen on the initial chest radiograph or the acid-fast smear is positive at completion of 2 months of treatment, the continuation phase of treatment should consist of isoniazid and rifampin daily or twice weekly for 4 months to complete a total of 6 months of treatment. if cavitation was present on the initial chest radiograph and the culture at completion of 2 months' therapy is positive, the continuation phase should be lengthened to 7 months (total of 9 months of treatment). if the patient has hiv infection and the cd4+ cell count is less than 100/mm 3 , the continuation phase should consist of daily or three-times-weekly isoniazid and rifampin. in hiv-uninfected patients having no cavitation on chest radiograph and negative acid-fast smears at completion of 2 months of treatment, the continuation phase may consist of either once-weekly isoniazid and rifapentine, or daily or twice-weekly isoniazid and rifampin, to complete a total of 6 months (bottom). patients receiving isoniazid and rifapentine, and whose 2-month cultures are positive, should have treatment extended by an additional 3 months (total of 9 months). *emb may be discontinued when results of drug susceptibility testing indicate no drug resistance. †pza may be discontinued after it has been taken for 2 months (56 doses). ‡rpt should not be used in hiv-infected patients with tb or in patients with extrapulmonary tb. therapy should be extended to 9 months if 2-month culture is positive. afb, acid-fast bacilli; cxr, chest radiograph (x-ray); emb, ethambutol; inh, isoniazid; pza, pyrazinamide; rif, rifampin; rpt, rifapentine. because ppd relies on dth, any factor that reduces the dth affects the host response to ppd. the most common clinical example is use of corticosteroids, which blunt the dth response and can complicate ppd interpretation. therefore, ppd testing should not be performed while a patient is taking corticosteroids. also, tb testing should be targeted to those with higher risk of infection and should not routinely be done in those with low risk (ats/cdc, 2000) . the ppd can also give false-positive results in patients with previous bacille calmette-guérin (bcg) vaccination or with infection by other mycobacterial infections. in the united states, this may cause difficulties in testing immigrants from countries who routinely use bcg vaccination programs. however, previous bcg vaccination should not change the interpretation of the ppd or willingness to treat such individuals accordingly. ‡when dot is used, drugs may be given 5 days per week and the necessary number of doses adjusted accordingly. although there are no studies that compare five with seven daily doses, extensive experience indicates this would be an effective practice. § patients with cavitation on initial chest radiograph and positive cultures on completion of 2 months of therapy should receive a 7-month (31 weeks, either 217 doses [daily] or 62 doses [twice weekly]) continuation phase. ¶ five-days-a-week administration is always given by dot. rating for 5 day per week regimens is aiii. ¶ ¶ not recommended for hiv-infected patients with cd4+ cell counts <100 cells/μl. ** options 1c and 2b should be used only in hiv-negative patients who have negative sputum smears at completion of 2 months of therapy and who do not have cavitation on initial chest radiograph. for patients started on this regimen and found to have a positive culture from the 2-month specimen, treatment should be extended an extra 3 months. the dth response can wane over time. to overcome this problem, nonreacting patients may undergo repeat ppd 1 week after their initial ppd. the diagnosis of ltbi is made by interpretation of a ppd and by ascertaining the patient's risk factors for progression to active tb if left untreated . interpretation of the ppd should be based on the area of induration and not the area of surrounding erythema. persons whose ppds have converted from negative to positive within 2 years are presumed to have been infected recently. the decision to use ppd means treating the patient for ltbi if the ppd test is positive. patients at increased risk for progression to active tb include those who have been recently infected (recent ppd converters); patients who are hiv seropositive; patients who have silicosis, diabetes, or chronic renal failure (including those receiving hemodialysis); solid-organ transplant recipients; patients with gastrectomy or jejunoileal bypass or head and neck cancer; injection drug users; patients with chest radiograph evidence of prior tb; and patients who weigh at least 5% less than ideal body weight. patients taking chronic corticosteroid therapy and those who are to receive tumor necrosis factor alpha (tnf-α) blockers (e.g., infliximab) are also at risk. patients taking corticosteroids also have higher risk of progression to active tb with larger doses and longer courses of corticosteroids. standard therapy for ltbi is inh, 300 mg orally daily for 9 months, regardless of hiv status. again, inh should always be administered with pyridoxine to prevent neuropathy. to overcome the false-positive results and confusion of ppd testing in certain populations, newer interferon-gamma (ifn-γ) release assays such as the quantiferon-tb gold (qft-g) test have been developed to detect latent m. tuberculosis. qft-g quantifies the release of ifn-γ from lymphocytes of the host's blood in response to three m. tuberculosis target antigens that are absent from bcg and most other nontuberculous mycobacterium spp. the advantages of using qft-g include one-time blood testing without the need for followup visit, no triggering of amnestic responses, and possibly more specific response to m. tuberculosis. however, qtf-g use in immunocompromised or anergic patients is limited, with indeterminate results. some studies also show discordant results in individuals tested with both ppd and qtf-g. in general, qtf-g may be used in all circumstances in which the ppd is used. however, whether the qtf-g is truly more specific or sensitive than the ppd in latent or active tb is yet to be determined. • the u.s. preventive services task force recommends "highintensity" behavioral counseling to at-risk adults and adolescents to prevent sexually transmitted infections. • be specific in addressing patients' sexual practices so as to provide appropriate prevention advice. hiv-positive persons recent contacts of tuberculosis patients fibrotic changes on chest radiography consistent with prior tuberculosis patients with organ transplants and other immunosuppressed patients (receiving equivalent of ≥15 mg/day of prednisone for at least 1 month) development in the primary prevention of stis is immunization against human papillomavirus (hpv). the vaccine can prevent infection with certain strains of hpv that cause cervical cancer and genital warts. trials are ongoing to determine the effectiveness of daily arv therapy in preventing transmission of hiv. vaccination investigation is ongoing for herpes simplex, chlamydia trachomatis, and hiv. this breadth of research effort holds promise for the future in the prevention of stis. the uspstf recommends "high-intensity" behavioral counseling to at-risk adults and adolescents to prevent stis. highintensity counseling involves multiple sessions and often is delivered to groups of patients. unfortunately, this type of intervention has limitations in its practicality for population-based delivery. no risk of harm was discovered in the delivery of counseling for sti prevention. vaccination is the most important form of primary prevention of common infectious diseases. two vaccines are currently on the market for hpv prevention-one that protects against four viral subtypes (6, 11, 16, 18) and is licensed for use in males and females 9 to 26 years of age, and the other against two subtypes (16,18), licensed for females 10 to 25 years of age. hepatitis b is a sexually transmitted infection, and immunization is recommended for adolescents who have not been previously inoculated. this is a requirement in many states for school entry. hepatitis a can be transmitted by oro-anal sexual contact, and vaccination should be offered to patients who are contemplating engaging in this sexual practice. recommendations surrounding the use of barrier methods for sti prevention should be tailored to the sex practices of the client. for example, a percentage of women use anal sex as a method of birth control but may not consider the need for condom use with this practice. the question, "do you regularly use condoms?" has little relevance to infection control for many sexual practices. evidence supports the advice to use barrier methods of latex or other approved material in a manner that prevents the exchange of blood and body fluids in decreasing stis. condoms confer a 30% risk reduction for herpes simplex and up to an 80% risk reduction for hiv, when used correctly (weller and davis-beaty, 2002; martin et al., 2009) . the secondary prevention of stis is achieved through direct and nonjudgmental patient assessment and screening and avoiding assumptions about patient sexual practices. screening is a tool to prevent the inadvertent spread of infection as well as the sequelae of undetected disease. infectious genital ulcers are associated with herpes simplex virus (hsv), syphilis, chancroid, lymphogranuloma venereum, and granuloma inguinale. hsv is by far the most common, affecting 50 million people in the united states. hsv-1 and hsv-2 are chronic, neurotropic viral infections that enter through epithelium and come to rest in the dorsal root ganglia. therefore, infection leads to lifetime presence of the virus, but the clinical manifestation of this condition is variable. a small percentage of those with serologic evidence of hsv-2 (10%-25%) have had symptoms of clinical herpes infection. in addition, patients with hsv infection can shed the virus in the absence of symptoms, creating a prime opportunity for spread. herpes simplex outbreak may be followed by a prodrome of malaise, fever, and regional lymphadenopathy before the appearance of grouped vesicles on an erythematous base. the vesicles are typically quickly broken and become ulcerated in appearance, with each vesicle usually less than several millimeters in size. true first-time infections tend to present more severely than secondary presentations of previously infected individuals, with a prodrome present in 80% of cases. the lesions can be in any location around the genitals or rectum, on the proximal thighs and buttocks, inside the vagina, and in and around the mouth. the lesions are most who to screen? often painful, particularly when on mucosal surfaces, or itchy. in women, herpes simplex can present with cervicitislike symptoms with bleeding and discharge and cervical ulcerations on examination, or simply mucopurulent cervicitis. herpetic lesions around the urethra tend to be extremely painful and can make urination difficult. rectal hsv can be confused with irritation, perianal fissure, and even candidiasis because of its often beefy-red appearance and itching. vesicles typically appear 6 days after infection and can last up to 2 weeks in an initial infection. subsequent outbreaks tend to have a shorter duration and to be less uncomfortable for patients. confirmation of infection is helpful, but the diagnosis can be made primarily on the clinical appearance of the exanthema. vigorous sample collection from an ulcer (which the patient may not appreciate) to be sent for pcr identification and typing is the most readily available method of laboratory diagnosis. serum antibody testing is not useful in the initial hsv diagnosis because antibody levels will not be appreciable early in infection. the appearance of convalescent immunoglobulin g (igg) and igm levels several weeks after a suspected outbreak might help to support the diagnosis of hsv infection. the value of screening for hsv immunity is debatable and should generally not be recommended for asymptomatic individuals. in addition, the uspstf recommends against screening asymptomatic pregnant women for hsv to prevent transmission to the newborn. given that many patients with hsv infection never manifest symptoms, the value of knowing that one is hsv seropositive is questionable. in addition, hsv-1 and hsv-2, although classically oral and genital, respectively, can "mix and match" based on sexual practices. it is often confusing for asymptomatic individuals to know that they have hsv antibody (do i have cold sores? do i have genital herpes? how should this change the way i live my life?). in monogamous couples with one partner known to be hsv positive and the other with unknown status, testing of the latter may indicate suppressive therapy in the seropositive partner if the other is found to be negative. regular barrier method use decreases transmission of herpes in both men and women, with patients using condoms 100% of the time having a 30% reduction in hsv acquisition from those who never use condoms (martin et al., 2009) . serodiscordant couples may also decrease transmission through antiviral suppressive therapy to the hsv-positive partner (table 16-8) . syphilis is a spirochetal infection that has resurged since 2001, the nadir year since 1996. syphilis infection rates are highest in men who have sex with men. syphilis is much less common than the other stis, with an infection rate of 5.6 per 100,000 population in the united states (vs. 496 per 100,000 for chlamydia). syphilis presents in several stages. the primary phase of syphilis is a painless ulcer called a chancre (figure 16-4) . the chancre may be visible on the genitals, although it can also be inside the vagina, mouth, or rectum, making it difficult to find. this lesion will appear within 3 weeks of transmission and will last for several weeks untreated. the secondary phase of infection is disseminated and involves a diffuse macular rash, typically with palm and sole lesions, generalized lymphadenopathy, fever, and condyloma latum (smooth, moist lesions on genitals without cauliflower appearance of condyloma acuminatum). tertiary syphilis is often asymptomatic but affects the heart, eyes, and auditory system and can be associated with gumma formation. gummas are soft, granulomatous growths in organs that can cause mechanical obstruction and weakening of blood vessel walls. latent infection often involves the cns. diagnosis of primary syphilis is challenging. the test of choice is darkfield microscopy, which is not readily available. direct fluorescent (monoclonal) antibody (dfa) testing may be available. antibody tests for syphilis, such as the rapid plasma reagin (rpr) and the less frequently used venereal disease research laboratories (vdrl), are often not positive early in infection and thus cannot be used to rule out primary syphilis based on a single reading. treponemal antigen testing (eia) may be available in some laboratories. the fluorescent treponemal antibody absorption (fta-abs) test may also be negative in the early infection. direct pcr for primary syphilis lesions has been tested but is not yet fda approved. a physician may choose to treat presumptively if a painless chancre and risk factors are present and may then do a convalescent rpr test in 1 to 2 weeks to confirm the infection by the appearance of a positive reaction. one would expect a fourfold change in titer of either test to indicate the presence of disease. primary and secondary syphilis are treated with a single injection of penicillin g, 2.5 million units. other regimens do not have proven effectiveness but can be used in the penicillin-allergic patient, including doxycycline, 100 mg twice daily for 14 days; ceftriaxone, 500 mg to 1 g intramuscularly (im) daily for 8 to 10 days; or azithromycin, 2 g as a single oral dose, although resistance to azithromycin has been observed. patients treated for primary syphilis should have periodic clinical follow-up and serologic testing to determine a fourfold decrease in rpr reactivity within 6 months. latent syphilis can be either early, meaning infection within the last year, or late, meaning infection beyond a year. early latent syphilis is treated with a single injection of penicillin g, 2.4 million units. syphilis of late latency or unknown duration is treated with three injections of penicillin g, 2.4 million units, in 3 consecutive weeks. for penicillin-allergic patients, doxycycline, 100 mg twice daily for 28 days, is required. those with latent syphilis should have ophthalmic examination as well as evaluation for vascular gumma formation. suspected neurologic involvement of latent syphilis must be evaluated with cerebrospinal fluid (csf) examination and treatment with aqueous penicillin g, 3-4 million units intravenously (iv) every 4 hours for 10 to 14 days. partners of patients with newly diagnosed syphilis are at risk for infection. partners within 90 days of a diagnosis of primary syphilis should be tested, but treated presumptively even if serologic testing is negative. for partners prior to 90 days before diagnosis, serology is generally reliable in detecting presence of infection and may guide treatment. patients with secondary syphilis should inform partners within 6 months before diagnosis, or 12 months for those diagnosed with tertiary syphilis (table 16 -9). chancroid may occur in regional outbreaks and presents with a painful genital ulcer and suppurative regional adenopathy. herpes and syphilis should both be ruled out in the patient suspected of having chancroid infection. chancroid is caused by haemophilus ducreyi and there is currently no fda approved test to directly detect this organism. treatment with azithromycin (1 g as single dose), ceftriaxone (250 mg im as a single dose), ciprofloxacin (500 mg twice daily for 3 days), or erythromycin (500 mg three times daily for 7 days) are all alternatives (table 16 -10). it may be necessary to perform incision and drainage on fluctuant inguinal nodes. patients should be reexamined in 1 to 2 weeks to ensure healing of the primary ulcer(s) and resolution of the adenopathy. partners who had contact with the infected patient starting 10 days before development of the patient's symptoms should be treated, regardless of the presence of symptoms. less common ulcerating stis include lymphogranuloma venereum (lgv) and granuloma inguinale ( figure 16 -5). lgv causes regional adenopathy and often an ulcer at the point of entry. rectal lgv may cause a proctocolitis with anal pain, discharge, bleeding, and diarrhea. lgv is caused by chlamydia trachomatis serotypes and can be detected by testing swabbed material from open lesions or aspirates from lymph nodes with culture, dfa, or nucleic acid detection. treatment is noted above (table 16-10) . granuloma inguinale, caused by klebsiella granulomatis, is rare in the united states and causes progressive ulcerative disease of the genitals. a second sti category includes those causing the clinical presentation of vaginal discharge, pelvic pain, dyspareunia, and dysuria in women and penile discharge and dysuria in men, as well as possible rectal pain and discharge in men and women. of this group, chlamydia trachomatis is the most common, causing 1.2 million infections in the united states in 2008 (cdc, 2009 ). in fact, chlamydia is the most frequently reported reportable infection. the majority of women with chlamydia infection are without symptoms. many men are asymptomatic as well. regular screening for chlamydia, as recommended by the uspstf, can significantly reduce the incidence of pelvic inflammatory disease (pid), one of the most serious sequelae of untreated infection. in women with untreated chlamydia infection, in addition to pid, tubo-ovarian abscess, tubal scarring and ectopic pregnancy, and infertility can all result. as previously mentioned, regular screening is currently recommended for all sexually active women under age 24, all pregnant women under 24, and at-risk pregnant and nonpregnant women over 24. chlamydia testing can be performed on several liquid-based papanicolaou (pap) tests. endocervical swabs for nucleic acid amplification are acceptable when a conventional pap smear is being used. given the recent liberalization of recommendations about pap testing for women under 21 years of age, urine nucleic acid amplification is a readily available alternative for chlamydia testing. this can easily be done at a contraceptive counseling clinic. urine testing is also an acceptable method of testing for men, in addition to a urethral swab. rectal chlamydia infection can occur in individuals who practice receptive anal intercourse. an fda-approved method of testing should be used for screening and diagnosis of this infection. asymptomatic chlamydia infection is treated with either a single dose of azithromycin, 1 g orally, the drug of choice, or doxycycline, 100 mg twice daily, for 7 days (table 16-11) . patient-delivered partner therapy (pdpt), the practice of dispensing treatment to diagnosed patients to treat their partner(s), has proved effective in reducing reinfection rates and further spread of infection. ept is legally allowable in 21 states and potentially allowable in another 21. chlamydia infection may present symptomatically in men or women with symptoms of dysuria and with discharge and with pelvic pain and dyspareunia in women. the discharge of c. trachomatis, versus that of neisseria gonorrhoeae, is said to be more mucoid than purulent, although this characteristic is not specific enough to provide diagnostic accuracy. symptomatic chlamydia, without evidence of pid, is treated the same as asymptomatic infection. many practitioners will treat presumptively for chlamydia and gonorrhea in patients who present with the symptoms previously mentioned while they wait for confirmatory testing. neisseria gonorrhoeae infection may be asymptomatic in both men and women. the current uspstf recommendation is for screening women at risk. men with penile gonorrhea typically present with purulent penile discharge and dysuria with n. gonorrhoeae infection. mucopurulent discharge, dysuria, pelvic pain, and dyspareunia are typical symptoms in women. in patients who engage in anal intercourse, anal discharge, rectal pain, and bleeding can be presenting symptoms. gonococcal pharyngitis is within the differential of exudative pharyngitis in sexually active patients. when symptomatic, throat pain, tonsillar exudates, and anterior cervical adenopathy may be present. testing for gonorrhea can be done using liquid-based pap technologies, cervical or urethral swabs, or urine for nucleic acid amplification. in men with visible discharge, a gram stain with white blood cells (wbcs) and gram-positive intracellular diplococci has a high degree of sensitivity. culture testing may be preferred for suspected pharyngeal and rectal specimens pending fda approval of other methods. again, physicians may opt to treat patients with mucopurulent cervicitis or urethritis presumptively for gonorrhea and chlamydia while waiting for confirmatory testing. fluoroquinolone therapy is no longer recommended because of widespread resistance (table 16-11) . because reinfection with gonorrhea is common for several months after treatment, it may be advisable to retest patients with confirmed gonorrhea in the 3 months after treatment. similarly, stis may be an indicator of risk behavior, and a complete risk history and testing for other stis is advisable if not completed at the initial visit. in male patients with symptomatic urethritis, a causative agent may not be identified, a situation often referred to as nongonococcal urethritis (ngu). technically, chlamydia is included in this category. organisms such as ureaplasma urealyticum and mycoplasma genitalium may be the cause and may be difficult to detect. treatment for these infections is the same as for symptomatic chlamydia, with azithromycin or doxycycline (table 16 -11). it is recommended that partners of patients with ngu should be evaluated and treated. in some cases, testing of partners may detect a specific organism as the cause of infection (e.g., chlamydia). trichomonas vaginalis causes vaginitis in women, who may have a stereotypic frothy, green, and foul-smelling discharge. many women are asymptomatic with trichomoniasis. in addition to causing asymptomatic infection in men, t. vaginalis may cause urethritis. this organism may be suspected in men when patients have repeated treatment failures and no other explanation for symptoms. microscopic examination of vaginal discharge is 60% to 70% sensitive in women. a first voided urine specimen or urethral swab for microscopic exam may be helpful in identifying the protozoa. culture for trichomonas, which requires a special medium, may be necessary to identify this infection accurately in men. trichomonas is effectively treated with a single 2-g dose of metronidazole (table 16-11) . for non-sti causes of vaginal discharge, see the online discussion at www.expertconsult.com. pelvic inflammatory disease can be caused by a number of organisms, including chlamydia, and presents with pelvic pain and discharge. findings that contribute to the diagnosis of pid include fever greater than 101° f, cervical or vaginal mucopurulent discharge, abundant wbcs on saline preparation of vaginal discharge, elevated erythrocyte sedimentation rate (esr), elevated c-reactive protein (crp), and evidence of n. gonorrhoeae or c. trachomatis infection. hospitalization with parenteral antibiotics may be necessary in pregnant patients, patients in whom surgical emergency cannot be ruled out, those who do not respond to oral treatment, those who cannot tolerate oral treatment, and patients who have severe illness or tubo-ovarian abscess. when treating pid parenterally, improvement of symptoms for 24 hours may prompt a change to oral therapy (table 16-12) . conversely, if oral therapy is not producing significant improvement within 2 to 3 days, admission for parenteral therapy may be necessary. patient awareness of human papillomavirus infection has greatly increased in recent years, in large part related to the patient-directed advertising of the hpv vaccine. hpv is likely the most common sti. thirty types of hpv can infect the genital area, some causing genital warts, some causing malignancies of the genital organs, and most being asymptomatic. the gross categories most often used are "high risk" (most often types 16 and 18) and "low risk" (types 6 and 11) hpv infection, the former more often associated with genital cancer. prevention of hpv infection and cervical cancer was revolutionized with the release of the hpv vaccine, which is effective in reducing the incidence of hpv-associated disease. currently, two vaccines are licensed in the united states. gardasil (merck), released in 2006, includes protection against viral types 6, 11, 16, and 18. it is approved for the prevention of vulvar and vaginal cancer and for the prevention of cervical cancer, cervical dysplasia, and genital warts in females age 9 to 26. the vaccine was recently approved for males of the same age range for the prevention of genital warts. more recently, cervarix (glaxosmithkline) was approved for the prevention of cervical cancer and cervical dysplasia from hpv types 16 and 18 in women age 10 to 25. ideally, the vaccine should be administered before initiation of sexual activity to prevent initial acquisition of these hpv types. patients who are already sexually active may also receive the vaccine. the transmission of hpv to men decreases with consistent condom use, from 53.9% in men who never use condoms to 37.9% in men who "always" use them. unfortunately, hpv can infect skin that is not covered by the use of traditional barrier methods (nielson et al., 2010) . male circumcision may decrease the transmission of hpv. patients have many questions about hpv, in particular about screening for asymptomatic infection. hpv infection occurs with high frequency in the sexually active population; up to 50% or more of sexually active individuals have hpv at some point in their life. in addition, hpv is effectively transmitted, even if contact does not involve genital-togenital touching (i.e., manual stimulation can transmit the virus). again, most hpv infections are without symptoms and resolve spontaneously through eradication by the intact immune system. for all these reasons, screening for the mere presence of hpv infection has minimal utility. there is no treatment for asymptomatic hpv infection. the most common presentation of hpv infection is in the context of an abnormal pap smear. hpv is directly linked to cervical dysplasia. for women over age 21 and under 35, hpv testing with high-risk viral detection is common. the presence of high-risk hpv informs further management of the pap result. it is currently recommended that women over 35 be automatically tested for high-risk hpv infection at the pap smear. patients may present with visible warts, or these may be detected at routine or sti screening. genital warts are often cosmetically unacceptable to patients, even though they are infrequently functionally problematic. in some circumstances, wart burden can be high enough to cause physical discomfort or relative obstruction of the vagina or rectum. vulvovaginal candidiasis and bacterial vaginosis are generally not thought to be sexually transmitted, although they are often in the differential diagnosis of sexually transmitted infection (sti). both these infections likely are related to changes in the vaginal ph and the normal flora distribution. it is not always clear which of these factors is primary and which is secondary, because at diagnosis, both ph and normal vaginal flora will often be abnormal. vulvovaginal candidiasis is a common infection causing typically white, curdlike discharge, itching, and sometimes dysuria. the causative organism is usually candida albicans but can be other candida spp. antibiotics can alter normal vaginal flora, so the recent use of antibiotics may predispose women to candidiasis. physical examination may reveal erythematous external genitalia as well as external and internal white, clumping discharge. usually, no distinctive odor is associated with vaginal yeast. wet preparation of vaginal specimen or treatment with potassium hydroxide (koh) may reveal branching pseudohyphae and yeast. when ph is performed, it should be directly on the vaginal discharge and not on the saline-diluted specimen because the saline will alter the ph of the specimen. typically, the ph of yeast discharge is less than 4.5 (normal vaginal ph,3.8-4.5). bacterial vaginosis (bv) is the most common cause of infectious vaginal discharge (spence and melville, 2007) . many different organisms are associated with the diagnosis of bv, including gardnerella vaginalis and mycoplasma hominis. women with bv may report discharge, vaginal irritation, vaginal odor, and at times, dysuria. findings of bv are often detected during a normal screening pap smear or pelvic examination. physical findings may reveal signs of vaginal irritation. the discharge is usually thin and gray. an amine (fishy) odor may be produced with the application of koh. the finding of clue cells, or epithelial cells with adherent bacteria, under saline preparation microscopy and a decrease in normal lactobacilli are common findings. the amsel criteria are useful in bv diagnosis; other scoring systems (e.g., nugent criteria) have been used but require gram staining. the specific amsel criteria are (1) milky, homogeneous, adherent discharge; (2) discharge ph greater than 4.5; (3) positive whiff test (fishy smell with addition of koh); and (4) at least 20% clue cells on microscopic examination. if three of the four criteria are present, the likelihood of bv is 90%. in routine vaginal examination and bimanual examination for patients with vaginal discharge, signs and symptoms of vaginitis are poor predictors of the microbiologic cause of infection (schaaf et al., 1990) . the clinical examination and office testing, in fact, are fair predictors of the true cause of infection (lowe et al., 2009 ). many patients with vaginal discharge will use over-the-counter preparations before consulting a physician, which can delay correct diagnosis of the etiology of symptoms. patient-collected, low vaginal swabs may be as useful as provider-collected specimen in making a diagnosis for the patient with vaginal discharge. the purpose of bimanual examination is to evaluate for signs of pelvic inflammatory disease and is not necessary in the low-risk patient with vaginal discharge. treatment of asymptomatic bv or vaginal yeast is not necessary in the nonpregnant patient or usually is not needed to test or treat partners of patients with isolated yeast or bv. when infection is recurrent, particularly when a woman's male partner is uncircumcised, treatment of the male partner for carriage of either infection may be warranted. options for treatment of recurrent infections are presented in etable 16-1. the treatment of warts is destructive and may serve to stimulate an immune response to the hpv-infected cells, which are typically "above" the surveillance mechanisms of the immune system in the epidermis. office methods of treatment include cryotherapy and trichloroacetic acid or podophyllin resin application. patients may apply podofilox 0.5% solution or gel or imiquimod 5% cream (table 16-13) . for more extensive cases of warts or intra-anal or intravaginal infections that are difficult to treat using the previous methods, surgical techniques may be necessary to achieve resolution. untreated, warts may resolve spontaneously, remain the same, or worsen. patients with pediculosis pubis, or pubic lice, most often present with pruritus or with visible nits. pubic lice are visible on inspection of the pubic area, as are nits, which are adherent to the hair shaft. partners of patients with pubic lice should also be treated to prevent reinfection. linens and clothing should be laundered or dry-cleaned or kept in a closed plastic container or bag for 72 hours. scabies diagnosis can be challenging. again, patients present with itching that can be anywhere on the body, although often in the genital area or on the buttocks when infection is sexual in origin. the pruritus associated with sarcoptes scabiei is a result of sensitization to the mite droppings underneath the skin as the mite burrows. the classic "burrow" or linear papular eruption is not always present. scraping of lesions with microscopic examination may be performed to identify the mite. as with pediculosis, close contacts should be treated. linens and clothing should be laundered or dry-cleaned or isolated in plastic containers for 72 hours. the pruritus-associated with scabies can take several weeks to resolve after treatment. patients living in group settings (dormitories or apartments) may reinfect one another as a result of inadequate primary treatment of all contacts ( cryotherapy trichloroacetic acid (tca): small amount applied until wart whitens podophyllin resin, 10% to 25% all these may be repeated every 1 to 2 weeks until warts are resolved. podofilox 0.5% solution or gel applied twice daily for 3 days, followed by 4 days of no therapy. imiquimod 5% cream applied once daily at bedtime three times a week for up to 16 weeks; washed off 6 to 10 hours after application. urinary tract infection (uti) is defined as significant bacteriuria in the presence of symptoms. uti accounts for a significant number of emergency department visits; an estimated 20% of women experience a uti in their lifetime. the urinary tract is normally sterile. uncomplicated uti involves the urinary bladder in a host without underlying renal or neurologic disease. the bladder mucosa is invaded, most often by enteric coliform bacteria (e.g., e. coli) that ascend into the bladder via the urethra. sexual intercourse can promote this migration, and cystitis is common in otherwise healthy young women. frequent and complete voiding has been associated with a reduction in the incidence of uti. complicated uti occurs in the setting of underlying structural, medical, or neurologic disease. signs and symptoms of a uti include dysuria, frequency, urgency, nocturia, enuresis, incontinence, urethral pain, suprapubic pain, low back pain, and hematuria. fever is unusual. up to 30% of patients with symptoms of cystitis have a smoldering pyelonephritis, especially when symptoms have been present for more than 1 week. a patient with pyelonephritis usually appears ill, with fever, sweating, and prostration, along with costovertebral angle (flank) tenderness in most cases. the differential diagnosis of uncomplicated uti includes use of diuretics or caffeine, interstitial cystitis, vaginitis, pregnancy, pelvic mass, pid, and benign prostatic hypertrophy (bph). if a uti is suspected, the initial test of choice is urinalysis, although with classic signs and symptoms of infection in women, this test is not always necessary. pyuria, as indicated by a positive result on the leukocyte esterase dip test, is found in the majority of patients with uti. the presence of urinary nitrites is fairly specific for uti. the combination of positive leukocyte esterase and nitrites improves sensitivity. on urine microscopy, levels of pyuria as low as two to five leukocytes per high-power field (2-5 wbcs/hpf) in a centrifuged specimen are significant in the female patient with appropriate symptoms, as is the presence of bacteriuria. urine culture and sensitivity are not needed in simple utis. cultures should be done in patients with recurrent utis, patients with pyelonephritis, and pregnant patients. antibiotic therapy can be given in a 3-day regimen for young, sexually active women. a 7-to 10-day course of antibiotics should be used in pregnant patients and patients with complicated utis. all the drugs listed in table 16 -15 can be used in a 3-day or 7-to 10-day course. clinical practice guidelines that include telephone assessment and treatment have shown a decrease in unnecessary laboratory utilization while maintaining quality of care (saint et al., 1999) . trimethoprim-sulfamethoxazole (tmp-smx) has been a mainstay of uti therapy, but in some localities, resistance of e. coli to tmp-smx is 20% (mehnert-kay, 2005) . if a urine culture is done and the organism is resistant to the drug prescribed, a change in antibiotics is indicated only if the patient is still symptomatic. for symptomatic treatment, a bladder anesthetic can be used, such as phenazopyridine (pyridium), 200 mg three times daily for 2 days. patients should be warned that this produces an orange tinge in tears and urine. patients should also be instructed to increase fluid intake. pyelonephritis is suggested by a failure of a short course of antibiotics. signs and symptoms of pyelonephritis include shaking chills and fever higher than 38.5° c (101.3° f), flank pain, malaise, urinary frequency and burning, and costover-tebral angle tenderness. the infection can produce septic shock. a patient who is unable to tolerate oral intake should be hospitalized and given empiric iv antibiotics aimed at broad-spectrum gram-negative coverage, such as third-generation cephalosporins, fluoroquinolones, or aminoglycosides, while awaiting results of blood and urine cultures. a 14-day course of antibiotic therapy (iv or po) is recommended. although the most common bacterial infection during pregnancy, the incidence of uti in pregnancy is similar to that reported in sexually active nonpregnant women of childbearing age. up to 40% of pregnant women with tmp-smx, 160/800 mg q12h trimethoprim, 100 mg q12h fluoroquinolones ‡ ciprofloxacin, 100-250 mg q12h ciprofloxacin xr, 500 mg qd gatifloxacin, 200 mg qd levofloxacin, 250 mg qd nitrofurantoin monohydrate/macrocrystals, 100 mg q12h nitrofurantoin macrocrystals, 50-100 mg qid amoxicillin, 250 mg q8h or 500 mg q12h cephalexin, 250 mg q6h, or other cephalosporin consider 7-day regimen. amoxicillin, 250 mg q8h or 500 mg q12h nitrofurantoin monohydrate/macrocrystals, 100 mg q12h nitrofurantoin macrocrystals, 50-100 mg qid cephalexin, 250 mg q6h, or other cephalosporin tmp-smx, 160/800 mg q12h male gender, diabetes, symptoms for 7 days, recent antimicrobial use, age > 65 tmp-smx, § 160/800 mg q12h fluoroquinolones, as per 3-day regimens cephalexin, 250 mg q6h, or other cephalosporin consider 7-day regimen. from hooton tm, stamm we. diagnosis and treatment of uncomplicated urinary tract infection. infect dis north am 1997;11:551. tmp-smx, trimethoprim-sulfamethoxazole; qd, every day; q12h, every 12 hours; q6h, every 6 hours; q8h, every 8 hours; qid; four times daily. * treatments listed to be prescribed before etiologic agent is known (gram stain may help); therapy can be modified when cause is identified. † characteristic pathogens are escherichia coli (85%-90%) and staphylococcus saprophyticus (5%-15%); other organisms account for less than 5% of cases and include proteus mirabilis, klebsiella pneumoniae, and enterococcus spp. ‡fluoroquinolones should not be used in pregnancy. § although classified as pregnancy category c, tmp-smx is widely used; however, avoid its use in the first and second trimesters. untreated bacteriuria in the first trimester develop acute pyelonephritis later in pregnancy. premature births and perinatal mortality are increased in pregnancies complicated by uti. therefore, in pregnant women, asymptomatic bacteriuria should be actively sought and aggressively treated with at least one urinalysis, preferably toward the end of the first trimester. nitrofurantoin, ampicillin, and the cephalosporins have been used most extensively in pregnancy and are the regimens of choice for treating asymptomatic or minimally symptomatic uti. tmp-smx should be avoided in the first trimester because of possible teratogenic effects and should be avoided near term because of a possible role in the development of kernicterus. fluoroquinolones are avoided because of possible adverse effects on fetal cartilage development. for pregnant women with overt pyelonephritis, admission to the hospital for parenteral therapy should be the standard of care; beta-lactam agents with or without aminoglycosides are the cornerstone of therapy. prevention of uti, including pyelonephritis, can be accomplished during pregnancy with nitrofurantoin or cephalexin taken prophylactically after coitus or at bedtime without relation to coitus. such prophylaxis should be considered for patients who have had acute pyelonephritis during pregnancy, patients with bacteriuria during pregnancy who have had a recurrence after a course of treatment, and patients who had recurrent uti before pregnancy that required prophylaxis. catheter-associated utis are associated with increased mortality and costs. risk factors for catheter-associated utis include the duration of catheterization, lack of systemic antibiotic therapy, female gender, age older than 50 years, and azotemia. to help prevent infection, urinary catheters should be avoided when possible and used only as long as needed. the catheter should be inserted with strict aseptic technique by trained persons, and a closed system should be used at all times. treatment of catheter-associated uti depends on the clinical circumstances. symptomatic patients (e.g., those with fever, chills, dyspnea, and hypotension) require immediate antibiotic therapy along with removal and replacement of the urinary catheter if it has been in place for a week or longer. in an asymptomatic patient, therapy should be postponed until the catheter can be removed. patients with long-term indwelling catheters seldom become symptomatic unless the catheter is obstructed or is eroding through the bladder mucosa. in patients who do become symptomatic, appropriate antibiotics should be administered and the catheter changed. therapy for asymptomatic catheterized patients leads to the selection of increasingly antibiotic-resistant bacteria. recurrence of uncomplicated cystitis in reproductive-age women is common, and some form of preventive strategy is indicated if three or more symptomatic episodes occur in 1 year. however, risk factors specific to women with recurrent cystitis have received little study (sen, 2008) . several antimicrobial strategies are available, but before initiating therapy, the patient should try such simple interventions as voiding immediately after sexual intercourse and using a contraceptive method other than a diaphragm and spermicide. ingestion of cranberry juice has been shown to be effective in decreasing bacteriuria with pyuria, but not bacteriuria alone or symptomatic uti, in an elderly population. cranberry juice may be effective for preventing uti in young, otherwise healthy women. if simple nondrug measures are ineffective, continuous or postcoital-if the infections are temporally related to intercourse-low-dose antimicrobial prophylaxis with tmp-smx, a fluoroquinolone, or nitrofurantoin should be considered. typically, a prophylactic regimen is initially prescribed for 6 months and then discontinued. if the infections recur, the prophylactic program can be instituted for a longer period. an alternative approach to antimicrobial prophylaxis for women with less frequent recurrences (<4 a year) is to supply tmp-smx or a fluoroquinolone and allow the patient to self-medicate with short-course therapy at the first symptoms of infection. a minority of patients have relapsing uti, as evidenced by finding the same bacterial strain within 2 weeks after completion of antimicrobial therapy. two factors can contribute to the pathogenesis of relapsing infection in women: (1) deep tissue infection of the kidney that is suppressed but not eradicated by a 14-day course of antibiotics and (2) structural abnormality of the urinary tract, particularly calculi. patients with true relapsing utis should undergo renal ultrasound, intravenous pyelogram (ivp), or voiding cystourethrogram, and longer-term therapy should be considered. urinary tract infection is one of the most common infections of childhood. factors predisposing to uti include taking broad-spectrum antibiotics (e.g., amoxicillin, cephalexin), which are likely to alter gastrointestinal and periurethral flora; incomplete bladder emptying or infrequent voiding; voiding dysfunction; and constipation. uti in young children serves as a marker for abnormalities of the urinary tract. imaging of the urinary tract is recommended in every febrile infant or young child with a first uti to identify children with abnormalities that predispose to renal damage. imaging should consist of urinary tract ultrasonography to detect dilation of the renal parenchyma. voiding cystourethrography is often ordered but does not appear to improve clinical outcomes in uncomplicated utis (alper and curry, 2005) . a common complication of uti in men is prostatitis. bacterial prostatitis is usually caused by the same gram-negative bacilli that cause uti in female patients; 80% or more of such infections are caused by escherichia coli. the pathogenesis of this condition is poorly understood. antibacterial substances in prostatic secretions probably protect against such infections. a national institutes of health (nih) expert consensus panel has recommended classifying prostatitis into three syndromes: acute bacterial prostatitis, chronic bacterial prostatitis, and chronic pelvic pain syndrome (cpps). acute bacterial prostatitis is a febrile illness characterized by chills, dysuria, urinary frequency and urgency, and pain in the perineum, back, or pelvis. the bladder outlet can be obstructed. on physical examination, the prostate is found to be enlarged, tender, and indurated. pyuria is present, and urine cultures generally grow e. coli or another typical uropathogen. chronic bacterial prostatitis is a clinically more occult disease and may be manifested only as recurrent bacteriuria or variable low-grade fever with back or pelvic discomfort. urinary symptoms usually relate to the reintroduction of infection into the bladder, with both pyuria and bacteriuria. a chronic prostatic focus is the most common cause of recurrent uti in men. cpps is the diagnosis for the large group of men who present with minimal signs on physical examination but have a variety of irritative or obstructive voiding symptoms; perineal, pelvic, or back pain; and sexual dysfunction. these men can be divided into those with and those without inflammation (defined as >10 wbcs/hpf in expressed prostatic secretions). the etiology and appropriate management in these patients, regardless of inflammatory status, is unknown. • laboratory findings in acute tick-borne infection often include a normal or low wbc count, thrombocytopenia, hyponatremia, and elevated liver enzymes. • doxycycline is the drug of choice for patients with rmsf. • appropriate antibiotic treatment should be initiated immediately with strong suspicion of ehrlichiosis. • if left untreated, lyme disease can progress to cognitive disorders, sleep disturbance, fatigue, and personality changes. in the united states, more vector-borne diseases are transmitted by ticks than by any other agent. tick-borne diseases can result from infection with pathogens that include bacteria, rickettsiae, viruses, and protozoa. most tick-borne diseases are transmitted during the spring and summer months when ticks are active. a knowledge of which species of tick is endemic in an area can help narrow the diagnosis (table 16-16) . rocky mountain spotted fever (rmsf) is the most severe and most often reported rickettsial illness in the united states. it is caused by rickettsia rickettsii, a species of bacteria that is spread to humans by ixodid (hard) ticks (figure 16-6) . initial signs and symptoms include sudden onset of fever, headache, and muscle pain, followed by development of rash. the disease can be difficult to diagnose in the early stage. rmsf is most common among males and children. risk factors are frequent exposure to dogs and living near wooded areas or areas with high grass. the presentation of rsmf is nonspecific, following an incubation of about 5 to 10 days after a tick bite. initial symptoms can include fever, nausea, vomiting, severe headache, muscle pain, and lack of appetite. later signs and symptoms include rash, abdominal pain, joint pain, and diarrhea. the rash first appears 2 to 5 days after the onset of fever. most often it begins as small, flat, pink, nonitchy spots on the wrists, forearms, and ankles. the characteristic red spotted rash of rmsf is usually not seen until the sixth day or later after onset of symptoms. as many as 10% to 15% of patients never develop a rash (figure 16-7) . no widely available laboratory assay provides rapid confirmation of early rmsf, although commercial pcr testing is available. therefore, treatment decisions should be based on epidemiologic and clinical clues. treatment should never be delayed while waiting for confirmation by laboratory results. routine clinical laboratory findings suggestive of rmsf include normal wbc count, thrombocytopenia, hyponatremia, and elevated liver enzyme levels. serologic assays are the most often used methods for confirming cases of rmsf. doxycycline is the drug of choice for patients with rmsf. therapy is continued for at least 3 days after fever subsides and until there is unequivocal evidence of clinical improvement, generally for a minimum total course of 5 to 10 days. tetracyclines are usually not the preferred drug for use in pregnant women. whereas chloramphenicol is typically the preferred treatment for rmsf during pregnancy, care must be used when administering chloramphenicol late during the third trimester of pregnancy because of risks associated with gray baby syndrome. three species of ehrlichia in the united states are known to cause disease in humans. ehrlichia chaffeensis, the cause of human monocytic ehrlichiosis, occurs primarily in southeastern and south-central regions and is primarily transmitted by the lone star tick, amblyomma americanum ( figure 16-8) . human granulocytic ehrlichiosis is caused by anaplasma phagocytophila or anaplasma equi and is transmitted by ixodes ticks. ehrlichia ewingii is the most recently recognized human pathogen, with cases reported in immunocompromised patients in missouri, oklahoma, and tennessee. after an incubation period of about 5 to 10 days following the tick bite, initial symptoms generally include fever, pregnant women should be screened for asymptomatic bacteriuria in the first trimester of pregnancy (wadland and plante, 1989) (sor: a). pregnant women who have asymptomatic bacteriuria should be treated with antimicrobial therapy for 3 to 7 days (nicolle et al., 2005) (sor: b) . pyuria accompanying asymptomatic bacteriuria should not be treated with antimicrobial therapy (nicolle, 2003) (sor: c1). a 3-day course of tmp-smx (bactrim, septra) is recommended as empiric therapy of uncomplicated utis in women, in regions where the rate of resistant e. coli is less than 20% (warren et al., 1999) (sor: c). fluoroquinolones are not recommended as first-line treatment of uncomplicated utis, to preserve their effectiveness for complicated utis (warren et al., 1999) (sor: c). a randomized, placebo-controlled trial of 150 women over 12 months found that cranberry juice and cranberry extract tablets significantly decreased the number of patients having at least one symptomatic uti per year (stothers, 2002) appropriate antibiotic treatment should be initiated immediately when there is a strong suspicion of ehrlichiosis on the basis of clinical and epidemiologic findings. the treatment recommendations are the same as for rocky mountain spotted fever. rifampin has been used successfully in a limited number of pregnant women with documented ehrlichiosis. babesiosis is caused by hemoprotozoan parasites of the genus babesia. the white-footed deer mouse is the main reservoir in the united states, and the vector is ixodes ticks. most infections are probably asymptomatic. manifestations of disease include fever, chills, sweating, myalgias, fatigue, hepatosplenomegaly, and hemolytic anemia. symptoms typically occur after an incubation period of 1 to 4 weeks and can last several weeks. the disease is more severe in immunosuppressed, splenectomized, or elderly patients. diagnosis can be made by microscopic examination of thick and thin blood smears stained with giemsa, looking for the parasite in red blood cells (rbcs). options for treatment include clindamycin plus quinine or atovaquone plus azithromycin. lyme disease is caused by the spirochetal bacterium borrelia burgdorferi. ixodes ticks are responsible for transmitting lyme disease bacteria to humans. in the united states, lyme disease is mostly localized to states in the northeastern, mid-atlantic, and upper north-central regions, as well as northwestern california. lyme disease most often manifests with a characteristic bull's-eye rash (erythema migrans) accompanied by nonspecific symptoms such as fever, malaise, fatigue, headache, muscle aches, and joint aches (figure 16-9) . lyme disease spirochetes disseminate from the site of the tick bite, causing multiple (secondary) erythema migrans lesions. other manifestations of dissemination include lymphocytic meningitis, cranial neuropathy (especially facial nerve palsy), radiculoneuritis, migratory joint and muscle pains, myocarditis, and transient atrioventricular blocks of varying degree. if left untreated, the disease can progress to intermittent swelling and pain of one or a few joints (usually large weight-bearing joints such as the knee), cognitive disorders, sleep disturbance, fatigue, and personality changes. the diagnosis is based primarily on clinical findings, and it is often appropriate to treat patients with early disease solely on the basis of objective signs and a known exposure. serologic testing may provide valuable supportive diagnostic information in patients with endemic exposure and objective clinical findings that suggest later-stage disseminated lyme disease. treatment for 3 to 4 weeks with doxycycline or amoxicillin is generally effective in early disease. cefuroxime axetil or erythromycin can be used for persons allergic to penicillin or who cannot take tetracyclines. later disease, particularly with objective neurologic manifestations, can require treatment with intravenous ceftriaxone or penicillin for 4 weeks or more, depending on disease severity. tularemia is caused by francisella tularensis, one of the most infectious pathogenic bacteria known. most cases in the united states occur in south-central and western states. humans can become infected through diverse environmental exposures, including bites by infected arthropods; handling infectious animal tissues or fluids; direct contact with or ingestion of contaminated food, water, or soil; and inhalation of infective aerosols. inhaled f. tularensis causes pleuropneumonitis. some exposures contaminate the eye, resulting in ocular tularemia; penetrate broken skin, resulting in ulceroglandular or glandular disease; or cause oropharyngeal disease with cervical lymphadenitis. untreated, bacilli inoculated into skin or mucous membranes multiply, spread to regional lymph nodes, multiply further, and then can disseminate to organs throughout the body. the onset of tularemia is usually abrupt, with fever, headache, chills and rigors, generalized body aches, coryza, and sore throat. a dry or slightly productive cough and substernal pain or tightness often occur with or without objective signs of pneumonia. nausea, vomiting, and diarrhea can occur. sweats, fever, chills, progressive weakness, malaise, anorexia, and weight loss characterize continuing illness. rapid diagnostic testing for tularemia is not widely available. respiratory secretions and blood for culture should be collected in suspected patients and the laboratory alerted to the need for special diagnostic and safety procedures. streptomycin (1 g im bid for 10 days) is the drug of choice, and gentamicin is an acceptable alternative. tetracyclines and chloramphenicol can also be used. colorado tick fever is an acute viral infection transmitted by the bite of the dermacentor andersoni tick (figure 16-10) . the disease is limited to the western united states and is most prevalent from march to september. symptoms start about 3 to 6 days after the tick bite. fever continues for 3 days, stops, and then recurs 1 to 3 days later for another few days. other symptoms include excessive sweating, muscle aches, joint stiffness, headache, photophobia, nausea, vomiting, weakness, and an occasional faint rash. routine blood tests might show a low wbc count, mildly elevated liver function, and mildly elevated creatine phosphokinase (cpk). diagnosis is confirmed by testing blood for complement fixation immunofluorescent antibody staining to colorado tick virus. treatment is removal of the tick and treatment of symptoms. physicians should advise patients who walk or hike in tickinfested areas to tuck long pants into socks to protect the legs and wear shoes and long-sleeved shirts. ticks show up on white or light colors better than dark colors, making them easier to remove from clothing. if attached, ticks should be removed immediately by using a tweezers, pulling carefully and steadily. insect repellents such as deet, alone or in combination with permethrin, may be helpful. • most cases of cellulitis are caused by staphylococci or streptococci, but other causes should be considered by clinical situation. • physicians must rule out more ominous causes of skin inflammation, such as necrotizing fasciitis and pyomyositis, when considering cellulitis. • edema-associated cellulitis is best treated by mobilizing edema fluid. cellulitis is an acute, spreading inflammation of the derma and subcutaneous issue. patients complain of tenderness, warmth, swelling, and spreading erythema. in contrast to erysipelas, cellulitis usually lacks sharp demarcation at the border. factors that predispose to cellulitis include trauma, an underlying skin lesion (furuncle, ulcer), or a complication arising from a wound, ulcer, or dermatosis. occasionally, cellulitis results from a blood-borne infection that metastasizes to the skin. pain and erythema usually develop within several days and are often associated with malaise, fever, and chills. the area involved is often extensive, red, hot, and swollen. patchy involvement with skip lesions can be seen. regional lymphadenopathy is common, and bacteremia can occur. several clinical entities resemble cellulitis, including pyoderma gangrenosum, gout, and insect bites. necrotizing fasciitis and gas gangrene are surgical emergencies. given that the predominant organism involved in most cases of cellulitis is a grampositive coccus, clinical history and morphology on physical examination usually suffice in the diagnosis and treatment of cellulitis. a history of freshwater exposure may implicate aeromonas hydrophila as the causative organism; saltwater appropriate antibiotic therapy should be initiated immediately when there is suspicion of rocky mountain spotted fever, ehrlichiosis, or relapsing fever rather than waiting for laboratory confirmation (bratton and corey, 2005; spach et al., 1993) (sor: c). treatment with doxycycline (vibramycin) or tetracycline is recommended for rmsf, lyme disease, ehrlichiosis, and relapsing fever (bratton and corey, 2005; spach et al., 1993) (sor: c). recommended actions to prevent tick-borne disease include avoidance of tick-infested areas; wearing long pants and tucking the pant legs into socks; applying diethyltoluamide (deet) insect repellents; using bed nets when camping; and carefully inspecting oneself frequently while in an at-risk area (bratton and corey, 2005; spach et al., 1993) (sor: c). antibiotic prophylaxis is not routinely recommended for a tick bite to prevent lyme disease, unless the risk of infection is high (wormser et al., 2006) (sor: b). recommended treatment for suspected tularemia is streptomycin or gentamicin given empirically before evidence of laboratory confirmation (bratton and corey, 2005; spach et al., 1993) (sor: c). exposure suggests vibrio spp. cellulitis in a patient with liver disease and shellfish ingestion moves vibrio vulnificans to the top of the differential. patients with soft tissue infection should have blood drawn for laboratory testing if signs and symptoms of systemic toxicity are present (e.g., fever or hypothermia, tachycardia, hypotension). laboratory testing should include blood culture and drug susceptibility tests; wbc count with differential; and measurement of creatinine, bicarbonate, cpk, and crp levels. hospitalization should be considered for patients with hypotension or an elevated creatinine level, low serum bicarbonate level, elevated cpk level (i.e., 2-3 times upper limit of normal), marked left shift, or crp level greater than 13 mg/l (123.8 nmol/l). gram stain with culture and culture of needle aspiration or punch biopsy specimens should be performed to determine a definitive etiology, and a surgical consult should be considered for inspection, exploration, and drainage. findings that may signal potentially severe, deep, soft tissue infection and that may require emergent surgical evaluation include cutaneous hemorrhage, gas in the tissue, pain disproportionate to physical findings, rapid progression, skin anesthesia, skin sloughing, and violaceous bullae. radiologic studies may be helpful if abscess or osteomyelitis is a possibility. ultrasonography is helpful in detecting a subcutaneous collection of fluid. magnetic resonance imaging (mri) is also useful in differentiating cellulitis from necrotizing fasciitis. the diagnosis of necrotizing cellulitis is by direct surgical examination or by frozen pathology sections. empiric antibiotics for immunocompetent patients with cellulitis should be targeted toward gram-positive cocci (table 16-17) . broader coverage should be initiated for diabetic patients to include gram-positive aerobes, gram-negative aerobes, and anaerobes. patients who present with severe infection or whose infection is progressing despite empiric antibiotic therapy should be treated more aggressively; the treatment strategy should be based on results of appropriate gram stain, culture, and drug susceptibility analysis. in the case of staphylococcus aureus, the physician should assume that the organism is resistant, and agents effective against mrsa, such as vancomycin, linezolid (zyvox), or daptomycin (cubicin), should be used. the antibiotic may be switched from an intravenous drug to an oral drug when fever has subsided and the skin lesion begins to resolve, usually in 3 to 5 days. the total duration of therapy should be 7 to 14 days. longer duration may be required if the response is slow or is associated with abscess, tissue necrosis, or underlying skin processes (infected ulcers or wounds). treatment of cellulitis should include elevation and immobilization to decrease swelling. patients with interdigital dermatophytic infections should be treated with a concomitant topical antifungal applied once or twice daily. topical antifungals can also help reduce the risk of recurrence of the cellulitis. support stockings, good skin hygiene, and prompt treatment of tinea pedis helps with prevention of cellulitis in patients with peripheral edema, who are predisposed to recurrence. in patients who continue to have frequent episodes of cellulitis or erysipelas, prophylactic treatment with penicillin v, 250 mg or 500 mg orally twice daily, or erythromycin, 250 mg once or twice daily (for penicillin-allergic patients), may be indicated. • the majority of furuncles and carbuncles are caused by staphylococcus spp., increasingly, community-acquired methicillin-resistant s. aureus. • drainage of pus is of primary importance in treating skin and soft tissue infections. • culture of sstis is important in guiding antibiotic treatment when initial measures of drainage are not effective. • for recurrent boils, consider referral to infectious disease specialist, possibly to eradicate carriage state. furuncles, or boils, are infections of the skin and soft tissue usually associated with a hair follicle. carbuncles are an extension of this skin and soft tissue infection continuum and involve more of the surrounding and subcutaneous tissue. the broad category skin and soft tissue infections (sstis) is used to describe this continuum that includes furuncles and carbuncles. sstis are common in both healthy and immunocompromised patients and likely initiate with some breach of the skin integrity, such as irritation of hair follicles from friction or microscopic trauma to the skin. up to 74% of furuncles and carbuncles are caused by community-acquired methicillin-resistant staphylococcus aureus (ca-mrsa) (cdc, 2010). other potential causative organisms include nonresistant staphylococcus spp. and streptococcus spp. it has become increasingly important to obtain culture of a lesion to direct antibiotic coverage given the increase in ca-mrsa. there is no reliable historical or examination element that will distinguish a ca-mrsa from a methicillin-sensitive staphylococcal skin lesion. stereotypically, patients report ca-mrsa lesions starting like a spider bite. furuncles and carbuncles can occur anywhere on the body, although the axillae, groin, and buttocks are particularly common sites. in addition, practices that cause skin trauma (e.g., shaving, waxing) are often noted in patients with these sstis. fever and malaise are uncommon with milder lesions but become more frequent with the increasing scope of localized infection. of primary importance in the management of carbuncles and furuncles is facilitation of drainage of any purulent material. with smaller lesions, this may be accomplished by heat application by the patient at home. as lesions increase in size and fluctuance, surgical drainage is essential to facilitate resolution of an ssti. it is important to consider culture penicillin, given parenterally or orally depending on clinical severity, is the treatment of choice for erysipelas (sor: a). for cellulitis, a penicillinase-resistant semisynthetic penicillin (amoxicillin/clavulanate) or a first-generation cephalosporin should be selected, unless streptococci or staphylococci resistant to these agents are common in the community (sor: a). for suspected mrsa skin infections, oral treatment options include trimethoprim-sulfamethoxazole, clindamycin, and doxycycline of purulent material when performing incision and drainage in the event that the patient fails to improve and antibiotic coverage becomes necessary. cure rates of lesions with drainage alone exceed 90%. careful follow-up after drainage is essential to ensure clinical improvement; daily dressing changes in the office after surgical drainage is effective. the addition of postdrainage antibiotics has not shown much added benefit. to prevent the spread of infection to others who come into contact with the patient recovering from an ssti, an occlusive dressing to prevent leakage of lesion fluid and careful hygiene are indicated. there is no evidence that extensive cleaning of common spaces (e.g., locker rooms) prevents the spread of ssti-causing bacteria more than routine cleaning measures. towels and soiled clothing should be laundered in hot water, and any common equipment should be cleaned per manufacturer recommendations. when lesions do not respond to heat, or when lesions are larger yet not amenable to drainage, antibiotics may be used. reasonable first-line antibiotic coverage for nonfluctuant lesions may include dicloxacillin, first-or secondgeneration cephalosporins, macrolides, or clindamycin. in patients with suspected ca-mrsa, better choices include tmp-smx, tetracycline, or clindamycin. it is important to note that up to 50% of ca-mrsa species will be resistant to clindamycin, particularly if the patient has been treated with other antibiotics in the previous weeks to months . oral administration of these antibiotics is acceptable in the nontoxic patient. patient signs and symptoms that would warrant hospital admission include fever or hypothermia, tachycardia, or hypotension as signs of sepsis and lesions greater than 5 cm in size (table 16-18) . for patients with recurrent sstis, evaluation for the presence of nasal carriage with a nasal culture is indicated. the value of eradication of bacterial carriage is unclear. referral for infectious disease specialist evaluation may be indicated to guide decision making in the patient with recurrent furuncles and carbuncles. • the existence, severity, and extent of infection, as well as vascular status, neuropathy, and glycemic control, should be assessed in patients with a diabetic foot infection. • visible bone and palpable bone on probing suggest underlying osteomyelitis in patients with a diabetic foot infection. • before an infected wound of a diabetic foot infection is cultured, any overlying necrotic debris should be removed to eliminate surface contamination and to provide more accurate results. patients with diabetes are prone to skin ulcers caused by neuropathy, vascular insufficiency, and diminished neutrophil function. minor wounds can be secondarily infected, leading to ulcer formation. these ulcers often have extensive undermining with necrotic tissues and are often close to the anus, thus promoting an environment suitable for multiple species of microorganisms, including anaerobes. diabetic foot infections range in severity from superficial paronychia to deep infection involving bone. non-limb-threatening infections involve superficial ulcers with minimal cellulitis (<2 cm from portal of entry), no signs of systemic toxicity, and no significant ischemia in the limb. cure rates of fluctuant skin lesions with drainage alone is over 90%. postdrainage antibiotics do not significantly improve outcomes rajendran et al., 2007) (sor: a). trimethoprim-sulfamethoxazole (tmp-smx), clindamycin, and tetracycline are first-choice antibiotics when ca-mrsa is suspected. up to 50% of ca-mrsa species will be resistant to clindamycin, particularly in the patient previously treated with other antibiotics (sor: c). subcutaneous tissues, and prominent ischemia. infection in patients who have recently received antibiotics or who have deep, limb-threatening infection or chronic wounds are usually caused by a mixture of aerobic gram-positive, aerobic gram-negative (e.g., escherichia coli, proteus spp., klebsiella spp.), and anaerobic organisms (e.g., bacteroides, clostridium, peptococcus, and peptostreptococcus spp.) . surgery is necessary to unroof encrusted areas, and the wounds need to be examined and probed to determine the extent of the infection and check for bone involvement (dinh et al., 2008) . debridement or drainage should be promptly performed. deep wound cultures should be obtained if possible. if deep culture is not feasible, gram stain and culture from the curettage of the base of the ulcer or from purulent exudates may be needed to guide antibiotic therapy (figure 16-11) . plain radiography of the foot is indicated for detection of osteomyelitis, foreign bodies, and soft tissue gas. when plain radiography is negative but osteomyelitis is clinically suspected, radionuclide scan or mri should be performed. mri provides more accurate information regarding the extent of the infectious process. the presence of peripheral artery disease and neuropathy should be assessed. the antibiotic regimen should be based on meaningful bacteriologic data. however, the initial regimen for a previously untreated patient with non-limb-threatening infection should focus on s. aureus and streptococci. mild infections may be treated with dicloxacillin or cephalexin for 2 weeks. amoxicillin/clavulanate may be used if polymicrobial infection is suspected. if msra is suspected, oral treatment options include tmp-smx or doxycycline. for limb-threatening infections, broad-spectrum antibiotics are recommended for coverage of group b streptococci, other streptococci, enterobacteriaceae, anaerobic gram-positive cocci, and bacteroides spp. treatment regimens include ampicillin-sulbactam or ertapenem (invanz), clindamycin plus a third-generation cephalosporin, and clindamycin plus ciprofloxacin. intravenous vancomycin should be added if mrsa infection is suspected. ciprofloxacin as a single agent is not recommended. in addition to antibiotic treatment, good glycemic control should be obtained and open wounds gently packed with sterile gauze moistened with ¼-strength povidone-iodine (betadine) solution. edema should be reduced by bed rest, elevation, and diuretic therapy as indicated. for prevention of diabetic foot ulcers, all patients with diabetes should have an annual foot examination that includes assessment for anatomic deformities, skin breaks, nail disorders, loss of protection sensation, diminished arterial supply, and inappropriate footwear. • the use of prophylactic antibiotics may be necessary in the initial management of bite wounds, particularly if the bite is on the hand or face or from a cat. • first-generation cephalosporins (e.g., cephalexin) are not effective as monotherapy for bite wounds because of resistance issues. • avoid primary wound closure in the management of bite wounds. it is estimated that bites account for 800,000 medical visits annually in the united states, making up 1% of emergency department visits. bite wounds consist of lacerations, evulsions, punctures, and scratches. the microbiology of bite wounds is generally polymicrobial, with an array of potential bacteria from the environment, the victim's skin flora, and the biter's oral flora. dog bites account for approximately 80% of all animal bites requiring medical attention, in which 85% are provoked attacks. most dog bites occur on the distal extremities, but children tend to sustain facial bites. patients who present for medical attention are often concerned about the care of disfiguring wounds or the need for appropriate vaccination (i.e., tetanus, rabies). however, up to 30% of medically treated wounds may become infected. these wounds are often contaminated with multiple strains of aerobic and anaerobic bacteria. local signs of infection with erythema, edema, pain, and purulent drainage are common with animal bite wounds. although the most frequently isolated pathogen related to dog and cat bite wounds is pasteurella multocida, the array of potential organisms is much greater. anaerobes such as bacteroides tectum, prevotella spp., fusobacteria, and peptostreptococci can be isolated from animal bite wounds 75% of the time, mostly from wounds with abscess formation. capnocytophaga canimorsus has also been associated with fatal infection from fulminant sepsis in asplenic patients. wounds inflicted by cats are often scratches or tiny punctures located on the extremity and are likely to become infected and lead to abscess formation. in the united states, venomous snakes bite approximately 8000 people yearly. envenomation in such snakebites account for the majority of morbidity and mortality associated with such bites. however, infection of soft tissue structures may also occur as a result of oral flora from the snake, which tends to be fecal in nature because live prey usually defecate in the snake's mouth with their ingestion. human bites are not uncommon, especially in children. human bites have a higher complication and infection rate than do animal bites. human bite wounds most often affect the hand and fingers and in some cases may present as "love routine wound swabs and cultures of material from sinus tracts are unreliable and strongly discouraged in the management of diabetic foot infection (pellizzer et al., 2001; senneville et al., 2006) nips" to the breast and genital areas. self-inflicted bites often include wounds of the lip and tissues surrounding the nail, such as paronychia. also included in this are clenched-fist injuries or "fight bites," which result in small lacerations to the knuckles when striking a person in the mouth. normal human oral flora, rather than skin flora, is the source of most bacteria isolated from human bite wound cultures (viridans streptococci, eikenella corrodens). management of bite wounds is the same as for any other wound: good wound care in the form of adequate irrigation and debridement of nonviable tissue as needed (table 1619) . bite wounds in general do not require primary closure, but after adequate irrigation and debridement, wounds may be approximated and closed by delayed primary or secondary intention. an exception to this rule may include bite wounds to the face. general wound management measures such as tetanus toxoid administration should also be employed. bite wounds involving the hands should be evaluated by a hand surgeon, given the risk of adjacent tendon sheath, bone, or joint involvement and the dire consequences if such structures are involved. the transmission of rabies through the bites of domestic pets in the united states and developed countries is rare. in fact, the dog strain of rabies is considered eliminated in the u.s. dog population, and cat bites are often managed through observation of the animal, without the immediate need for rabies postexposure treatment (pet). however, wild mammal exposure, especially bat, skunk, or raccoon, often warrants pet, which involves thorough cleaning of the bite wound, ideally with povidone-iodine solution, along with rabies immune globulin given at the wound site and rabies vaccine given on days 0, 3, 7, and 14. bite wounds should be considered contaminated wounds from presentation, given the oral microbial flora of humans and animals, and most patients should probably receive antibiotics early. empiric antibiotics are used to eradicate oral flora inoculated from the mouth of the biter, whether human or animal, into the wound. all moderate to severe animal bite wounds, or wounds that have an associated crush injury or that are close to a bone or joint, should be considered contaminated with potential pathogens, and these patients should receive 3 to 5 days of "prophylactic" antimicrobial therapy. gram stains with culture of bite wounds are specific but not sensitive indicators of bacterial growth. nonetheless, gram stain can be used to help guide initial empiric antibiotic therapy. amoxicillin-clavulanic acid (amoxicillin-clavulanate; augmentin) or penicillin plus a penicillinase-resistant penicillin are normally first-line agents for empiric therapy directed at bite wounds. first-generation cephalosporins (e.g., cephalexin) are not effective as monotherapy because of resistance of some anaerobic bacteria and e. corrodens. a 5-to 10-day course of antibiotics is usually adequate for infections limited to the soft tissue, and a minimum of 3 weeks of therapy is required for infections involving joints or bones. close follow-up is required in all bites to ensure adequate healing. of special consideration in human bite wounds is the potential for spread of viral pathogens, most notably hepatitis b virus (hbv) and hiv, if the source person is positive. hbv exposure in this setting should be handled in the same manner as other exposures, with administration of hbig and hbv vaccination. with regard to hiv, cdc guidelines for managing nonoccupational hiv exposure recommend handling each case individually in consultation with an infectious diseases specialist. • the diagnosis of osteomyelitis is based on radiographic findings (plain radiograph or mri) showing bony destruction along with histologic analysis and culture results. • chronic osteomyelitis is not an emergency, and antibiotics can be safely withheld until an etiologic diagnosis is established. • diabetic foot infections require a careful evaluation to assess perfusion and vascular supply, and corrective measures should be undertaken to reestablish adequate perfusion if necessary. • in diabetic foot ulcers, if one can probe to bone, the patient most likely has osteomyelitis. • orthopedic hardware infections are best managed in conjunction with an infectious diseases specialist and orthopedic surgeon. osteomyelitis is defined as progressive, inflammation leading to destruction of the bone, usually secondary to an infectious agent. bacteria can enter bone through hematogenous seeding or a contiguous focus after trauma, implantation of a foreign device, or a local soft tissue infection. acute osteomyelitis is defined as infection that evolves over a few weeks. chronic osteomyelitis implies persistent infection of several weeks to months. hematogenous osteomyelitis occurs primarily in children within the metaphyses of long bones (tibia and femur) and vertebrae in adults. in addition to local signs of inflammation and infection, patients generally have various systemic signs, including fever, irritability, and lethargy. physical findings include tenderness over involved area and decreased range of motion in adjacent joints. chronic osteomyelitis usually occurs in adults, caused by an open injury to bone and surrounding soft tissue. erythema, drainage around area, and bone pain are usually present on physical examination. systemic symptoms occur less frequently. the diagnosis of osteomyelitis is based on the clinical picture and supporting laboratory and radiologic findings. leukocytosis and elevations in crp and esr may use of antibiotic prophylactic after bites of the hand reduces the incidence of infection (medeiros and saconato, 2005) (sor: b) . antibiotic prophylaxis after bites by humans reduces incidence of infection (sor: c). animal bite: ascertain the type of animal, whether the bite was provoked or unprovoked, and the situation/environment in which the bite occurred. if the species can be rabid, locate the animal for 10 days' observation or sacrifice. patient: obtain information on antimicrobial allergies, current medications, splenectomy, mastectomy, liver disease, and immunosuppression. record a diagram of the wound with the location, type, and depth of injury; range of motion; possibility of joint penetration; presence of edema or crush injury; nerve and tendon function; signs of infection; and odor of exudate. infected wounds should be cultured and a gram stain performed. anaerobic cultures should be obtained in the presence of abscesses, sepsis, serious cellulitis, devitalized tissue, or foul odor of the exudate. small tears and infected punctures should be cultured with a minitipped (nasopharyngeal) swab. copious amounts of normal saline should be used for irrigation. puncture wounds should be irrigated with a "high-pressure jet" from a 20-ml syringe and an 18-gauge needle or catheter tip. devitalized or necrotic tissue should be cautiously debrided. debris and foreign bodies should be removed. radiographs should be obtained if fracture or bone penetration is possible to provide a baseline for future osteomyelitis. wound closure may be necessary for selected, fresh, uninfected wounds, especially facial wounds, but primary wound closure is not usually indicated. wound edges should be approximated with adhesive strips in selected cases. prophylaxis: consider prophylaxis (1) for moderate to severe injury less than 8 hours old, especially if edema or crush injury is present; (2) if bone or joint penetration is possible; (3) for hand wounds; (4) for immunocompromised patients (including those with mastectomy, liver disease, or steroid therapy); (5) if the wound is adjacent to prosthetic joint; and (6) if the wound is in the genital area. coverage should include pasteurella multocida, staphylococcus aureus, and anaerobes. treatment: cover p. multocida, s. aureus, and anaerobes. use oral medication if the patient is seen early after a bite and only mild to moderate signs of infection are present. the following can be considered for cat or dog bites in adults: • first choice: amoxicillin/clavulanic acid, 875/125 mg bid or 500/125 mg tid with food. • penicillin allergy: no alternative treatment for animal bites has been established for penicillin-allergic patients. the following regimens can be considered for adults: 1. clindamycin (300 mg po qid) plus either levofloxacin (500 mg po daily) or trimethoprim-sulfamethoxazole (2 double-strength tablets po bid). 2. doxycycline, 100 mg po bid. 3. moxifloxacin, 400 mg po daily. 4. in the highly penicillin-allergic pregnant patient, macrolides have been used, but the wounds must be watched carefully. on emergency department discharge, a single starting dose of parenteral antibiotic, such as ertapenem (1 g), may be useful in selected cases. if hospitalization or closely monitored outpatient follow-up is required, intravenous agents should be used. current choices include ampicillin/sulbactam and cefoxitin. the rising incidence of community-acquired s. aureus isolates that are methicillin resistant and therefore resistant to the drugs recommended here emphasizes the importance of susceptibility-testing any s. aureus isolates. indications include fever, sepsis, spread of cellulitis, significant edema or crush injury, loss of function, a compromised host, and patient noncompliance. give tetanus booster (td; tetanus and diphtheria toxoids for adults) if original three-dose series has been given but none in the past 5 years. adults who have not received acellular pertussis vaccine (tdap), should be given this instead of td. give a primary series and tetanus immune globulin if the patient was never immunized. rabies vaccine (on days 0, 3, 7, 14, and 28) with hyperimmune globulin may be required, depending on the type of animal, ability to observe the animal, and locality. elevation may be required if any edema is present. lack of elevation is a common cause of therapeutic failure. be seen but can also be normal. blood cultures may be positive in up to half of children with acute osteomyelitis. if plain radiographs show bone destruction and inflammation; the diagnosis of osteomyelitis is confirmed. typical findings on plain-radiographs will include osteolysis, periosteal reaction, and sequestra (segments of necrotic bone separated from living bone by granulation tissue). findings seen on plain radiographs usually denote a process that has been ongoing for at least 2 weeks. bone scintigraphy (bone scan) is often performed on patients with suspected osteomyelitis; however, sensitivity is quite low, and a negative result can offer false reassurance to the physician, so its routine use is not recommended. if the plain-radiographs are negative but the suspicion for osteomyelitis is still high, an mri scan should be considered. once the diagnosis of osteomyelitis has been made, the next step is to obtain an etiologic diagnosis. histopathologic and microbiologic examination of bone is the "gold standard." cultures of sinus tracts are not reliable for identifying the causative organism. common causative bacteriologic organisms in neonates include staphylococcus aureus, group b streptococci, and escherichia coli. later in life, s. aureus is most common, and in elderly persons, gram-negative organisms such as pseudomonas aeruginosa and serratia spp. have increased incidence. empiric antibiotics are rarely required for chronic disease but are often necessary for acute osteomyelitis. ideally, surgical debridement of all necrotic tissue and inflammatory debris (pus) should be undertaken and multiple surgical cultures with bone histology samples obtained. antimicrobial therapy will be dictated by test results. generally, treatment is for 4 to 6 weeks. with the exception of the fluoroquinolone class of antibiotics, which achieve high serum levels with oral administration, bone antibiotic levels cannot exceed the minimum inhibitory concentration (mic) for the infecting organism; therefore, antibiotics must be given intravenously. this underscores the importance in obtaining a bacterial diagnosis so that the appropriate antibiotic can be used for the duration of treatment. acute osteomyelitis is usually readily curable; however, chronic osteomyelitis is generally more refractory to therapy and requires repeat debridement and antibiotic courses. patients with uncontrolled diabetes are at increased risk for development of osteomyelitis, especially in the presence of neuropathy or venous or arterial insufficiency. s. aureus and beta-hemolytic streptococci are the predominant organisms, although other gram-positive or gram-negative aerobic or anaerobic bacteria may also be seen. plain radiographs should be the initial test to evaluate for the presence of osteomyelitis, followed by mri if negative. if there is a draining sinus, the "probe to bone" test should be performed with a sterile probe; if bone is palpated, the diagnosis of osteomyelitis is highly likely. further evaluation of the diabetic patient should be to assess for vascular insufficiency with the use of ankle-brachial indices and transcutaneous oximetry. if significant compromise is found, arteriography followed by revascularization should be undertaken. surgical debridement is again the cornerstone of treatment, along with antibiotics directed toward the causative microorganism. infections secondary to orthopedic hardware devices have become common problems with the increasing incidence of hip, knee, and shoulder replacement surgeries. also, patients with traumatic injury resulting in a fracture often have hardware implanted to stabilize the bone. these patients present in one of the three following ways: 1. early: symptoms develop less than 3 months after surgery and have an acute presentation with pain, erythema, and warmth, usually caused by s. aureus and gram-negative bacilli. 2. delayed: symptoms develop 3 to 24 months after surgery, generally with subtle signs of infection, including implant loosening and persistent pain, and usually caused by less virulent organisms such as coagulasenegative staphylococci and propionibacterium acnes. 3. late: symptoms develop 24 months after surgery and are usually caused by hematogenous seeding from skin, dental, respiratory, and urinary infections. treatment requires debridement of the surrounding tissue and hardware removal, although this cannot always be done in patients with bone instability. it is recommended that treatment follow-up should occur at 24 hours and perhaps 48 hours for outpatients. reporting the incident to a local health department may be required. from goldstein ejc. bites. in mandell gl, bennett je, dolin rd (eds). mandell, douglas, and bennett's principles and practice of infectious diseases, 7th ed. philadelphia, churchill livingstone--elsevier, 2010. po, orally; bid, twice daily; tid, three times daily; qid, four times daily. of these infections be done in conjunction with an infectious diseases specialist working with the orthopedic surgeon. septic arthritis is defined as infection within the joint space of two bones. the major causative organisms include s. aureus and in the sexually promiscuous individual, neisseria gonorrhoeae. intravenous drug users are likely to develop septic arthritis within unusual joints (e.g., sternoclavicular, sacroiliac). rheumatoid arthritis, presence of joint prostheses, and steroid use are predisposing factors for development of septic arthritis. diagnosis is usually based on clinical presentation of a warm, swollen joint with limitation in range of motion. a joint aspiration should be completed and the synovial fluid sent for gram stain with culture, wbc count with differential, and crystal analysis to rule out gout and pseudogout. blood cultures should also be drawn before initiation of antibiotics. gonococcal arthritis usually presents as an acute arthritis involving one or more joints in a sexually active individual. two thirds of patients have dermatitis with one or multiple, usually asymptomatic, lesions that progress from macular to papular and finally vesicular or pustular. joint fluid, urethral, and rectal cultures should also be obtained. treatment is generally with a third-generation cephalosporin intravenously until improvement, followed by oral therapy to complete a 1-week course of therapy. treatment of nongonococcal arthritis requires proper draining of the infected joint. this is often done surgically, although repeat needle drainage may also be successful if the joint is easily accessible. treatment generally depends on the gram stain and includes a third-generation cephalosporin, with the addition of vancomycin if gram-positive cocci in clusters are seen. duration of therapy is 3 to 4 weeks. • a comprehensive history and physical examination with laboratory and radiologic evaluation are important in the workup for fever of unknown origin (fuo). • if routine information is unrevealing, more specific testing for fuo is undertaken based on the patient's age, travel history, and disease process to develop a differential diagnosis. • the serum ferritin level (often elevated with malignancy) and naproxen test (reduces fever with malignancy) may be helpful in determining an underlying malignant process. • initiation of empiric antibiotics should be done only in specific fuo situations to prevent skewing culture results, thus maximizing isolation of the causative organism. patients who have a persistent fever despite workup are generally classified as having a "fever of unknown origin" (fuo). in 1961, petersdorf and beeson described 100 patients with persistent fever, otherwise known as fever of unknown origin. they introduced the standard, classic definition of fuo: fever higher than 38.3° c (101° f) on several occasions, persisting without diagnosis for at least 3 weeks, with 1 week of investigational study in the hospital setting. with advancing technology, this definition has been revised to allow for more than two outpatient visits, or 3 days if investigation is in the hospital setting. most patients with fuo have chronic or subacute symptoms and can be safely evaluated in the outpatient setting, with a median time to diagnosis of 40 days. the differential diagnosis of fuo is quite broad and extensive. determining an etiologic diagnosis of an fuo depends on generating a differential diagnosis compatible with the patient's history and physical examination. the principal disease categories for fuo include infection (30% overall), neoplasms (18%), collagen vascular diseases (12%), and miscellaneous (14%) (box 16-5). because of this broad differential, a newer classification system divides fuo into four groups: classic, nosocomial, neutropenic, and hiv associated, which helps narrow the differential diagnosis. furthermore, classic fuo can be broken down into three subgroups: infants and children, elderly, and travelers. despite an extensive workup, the etiologic diagnosis usually remains elusive in 7% to 30% of patients (box 16-4) . the diagnostic workup of fuo should begin with a thorough history and physical examination, including documentation of the fever. the patient may provide a diary noting the date and time of fever. routine noninvasive investigations are recommended in all patients before diagnosing fuo (box 16-6). acute febrile illness is never called an fuo. the patient's medication profile is reviewed because numerous drugs can be the cause. if unrevealing, a workup is initiated based on the differential diagnosis for the patient's age, travel history, geographic location, and disease process. dukes criteria for infective endocarditis have 99% specificity in patients with fuo. when the initial investigations are not helpful in identifying a cause, imaging should be considered, such as computed tomography (ct) scans of the chest, abdomen, and pelvis; ct may reveal an abscess or suggest an underlying malignancy. an elevated serum ferritin level can suggest a neoplasm or myeloproliferative disorder and, if normal, greatly decreases the chance that the patient has an underlying malignancy. lower-extremity doppler ultrasound should be considered in the sedentary or obese patient to rule out deep venous thrombosis. a temporal artery biopsy should be considered in the elderly patient to rule out temporal arteritis. liver biopsy has a high diagnostic yield with minimal toxicity, whereas bone marrow cultures usually have a low yield and should be considered only in special situations. empiric therapy with antibiotics is rarely appropriate for the patient with fuo. a diagnosis is essential to guide treatment of osteomyelitis requires surgical debridement followed by a 4-to 6-week course of intravenous antibiotic therapy (sor: c). septic arthritis is usually caused by a gonococcus in a sexually active adult, and use of a third-generation cephalosporin is the mainstay of therapy (sor: a). nongonococcal arthritis should be treated with surgical debridement or repeated needle aspirations, with a third-generation cephalosporin and vancomycin if gram-positive cocci are seen (goldenberg, 1998) (sor: b). treatment, and use of antibiotics may delay determining a causative infectious agent. the naproxen test (naprosyn; 375 mg po every 12 hours for 3 days) is helpful in determining if the fever is secondary to infection or malignancy. a dramatic decrease in the patient's temperature during the test generally indicates a malignant focus, whereas minimal or no response indicates an infectious etiology. the prognosis of fuo depends on the etiologic category. undiagnosed fuo has a very favorable outcome. patients in whom diagnostic investigations fail to identify an etiology should be followed clinically with serial history reviews and physical examinations until the fever resolves or new diagnostic clues are found. connective tissue diseases are more prominent. infections: malaria, hepatitis, pneumonia/bronchitis, uti/pyelonephritis, dysentery, dengue fever, enteric fever, tb, rickettsial infection, acute human immunodeficiency virus (hiv) infection, amebic liver abscess. postoperative urinary and respiratory tract instrumentation; use of intravascular devices; drug therapy; immobilization. septic thrombophlebitis, pulmonary embolus, clostridium difficile colitis, drug fever. fungal: 40% susceptible to empiric antifungals, 5% will be resistant to empiric therapy. bacterial: 10% not responding to empiric antimicrobial therapy and usually with cryptic focus. unusual pathogens: 5% will be toxoplasmosis (toxoplasma gondii) reactivation, atypical mycobacterial, tb, fastidious pathogens (legionella, mycoplasma, chlamydophila). viral: 5% of causes (hsv, cmv, ebv, hhv-6, vzv, rsv, influenza, parainfluenza). other: 10% will be transplant related (e.g., gvhd) following stem cell transplant, 25% will be undefined. infections: mycobacterium avium complex (mac), pneumocystis carinii pneumonia (pcp), cytomegalovirus (cmv), histoplasmosis, viral (hcv, hbv, adenovirus, hsv esophagitis, vzv encephalitis), tb, other fungi, cerebral toxoplasmosis, disseminated cryptosporidiosis. neoplasms: lymphoma, kaposi's sarcoma. other: drug fever, castleman's disease. hsv, herpes simplex virus; ebv, epstein-barr virus; hhv, human herpesvirus; vzv, varicella-zoster virus; rsv, respiratory syncytial virus; gvhd, graft-versus-host disease; hcv, hepatitis c virus; hbv, hepatitis b virus. abscesses: hepatic, subhepatic, gallbladder, subphrenic, splenic, periappendiceal, perinephric, pelvic, and other sites. granulomatous: extrapulmonary and miliary tuberculosis, atypical mycobacterial infection, fungal infection. intravascular: catheter-related endocarditis, meningococcemia, gonococcemia, listeria, brucella, rat-bite fever, relapsing fever. viral, rickettsial, and chlamydial: infectious mononucleosis, cytomegalovirus, human immunodeficiency virus, hepatitis, q fever, psittacosis. parasitic: extraintestinal amebiasis, malaria, toxoplasmosis. collagen vascular diseases: rheumatic fever, systemic lupus erythematosus, rheumatoid arthritis (particularly still's disease), vasculitis (all types). granulomatous: sarcoidosis, granulomatous hepatitis, crohn's disease. tissue injury: pulmonary emboli, sickle cell disease, hemolytic anemia. familial mediterranean fever fabry's disease cyclic neutropenia intra-abdominal infections may either be uncomplicated (limited to the gut lumen, such as gastroenteritis or colitis) or complicated (extending through to the peritoneum) . the clinical presentation of complicated intra-abdominal infections can range from mild symptoms such as nausea, mild abdominal pain, and cramping to lifethreatening septic shock. clinical findings result from local or diffuse inflammation with or without abscess formation. fever and abdominal pain are typically present, with additional symptoms depending on the organ involved. elderly and immunocompromised patients present with atypical, usually milder symptoms. imaging studies form an important adjunct to diagnosis. management involves empiric antibiotic coverage for bowel flora-mainly streptococci, enterococci, enteric gram-negative rods, and anaerobes-as well as controlling the source of infection, usually through surgery. • spontaneous bacterial peritonitis usually occurs in the setting of ascites and chronic liver disease. • spontaneous bacterial peritonitis is a diagnosis of exclusion. • ascitic fluid culture yield improves with inoculation into blood culture bottles at bedside. spontaneous bacterial peritonitis (sbp) is a form of infectious peritonitis without a surgically correctable cause and is therefore a diagnosis of exclusion. the route of infection in sbp is usually not apparent and is often presumed to be hematogenous, lymphogenous, by transmural migration through an intact gut wall from the intestinal lumen, or in women, from the vagina via the fallopian tubes (levison and bush, 2010) . sbp occurs in the setting of ascites in most cases, and it is particularly common in patients with cirrhosis. in pediatric populations, those with postnecrotic cirrhosis or nephrotic syndrome are more often affected. in adults, almost 70% of patients who develop sbp have child-pugh class c liver disease, and 10% to 30% of hospitalized patients with cirrhosis and ascites have sbp (mowat and stanley, 2001) . sbp is almost always caused by a single organism, typically enteric gram-negative rods, most often e. coli, followed by klebsiella pneumoniae. gram-positive cocci account for about 25% of episodes of sbp, and streptococci are isolated most often. sbp caused by anaerobes is rare. growth of more than one organism should raise the suspicion of secondary peritonitis. signs and symptoms of sbp are subtle and require a high index of suspicion. fever greater than 100° f (38° c) is the most common presenting sign, occurring in 50% to 80% of cases. abdominal pain, nausea, vomiting, and diarrhea are usually present. peritoneal signs (abdominal tenderness or rebound tenderness) are common but may be absent in patients with ascites. in adults, mental status changes may also occur. sbp is often confused with acute appendicitis in children. in adults, sbp should be suspected in any patient with previously stable chronic liver disease who undergoes acute decompensation in clinical status. spontaneous bacterial peritonitis is diagnosed by analysis of ascitic fluid obtained by abdominal paracentesis. infection has been typically defined as an ascitic fluid wbc count higher than 250 cells/mm 3 , which is considered diagnostic even when the culture of the ascitic fluid is negative. in cases where bloody fluid is obtained ("traumatic paracentesis"), the wbc count should be corrected by 1 wbc per 250 rbcs/mm 3 . the use of bedside dipstick for leukocyte esterase has a high false-negative rate and is not recommended (nguyen-khac et al., 2008) . ascitic fluid culture yield can be increased by inoculating blood culture bottles with 10 ml of ascitic fluid at the bedside. blood cultures should also be obtained as part of the workup. after the diagnosis of peritonitis is established, secondary peritonitis should be ruled out. ct of the abdomen with oral and intravenous contrast can help direct the surgeon to a particular source of infection, as opposed to doing a full exploratory laparotomy. a high ascitic fluid total protein (>1 g/dl) or amylase level is suggestive of secondary peritonitis. the treatment of choice is generally a third-generation cephalosporin such as cefotaxime (2 g iv every 8-12 hours) or ceftriaxone (2 g iv once daily). patients who have an ascitic fluid wbc count higher than 250 cells/mm 3 should be given empiric intravenous antibiotics without delay. oral amoxicillin-clavulanic acid can be used for mild, uncomplicated cases (navasa et al., 1996) . duration of treatment varies diagnosis of fuo may be assisted by the dukes criteria for endocarditis, ct scan of the abdomen, nuclear scanning with a technetiumbased isotope, and liver biopsy (mourad et al., 2003) (sor: b) . routine bone marrow cultures are not recommended in the fuo workup (mourad et al., 2003) (sor: b) . empiric antibiotics should be initiated only in specific situations, to avoid skewing culture results and thus maximizing potential isolation of the causative organism (mourad et al., 2003) (sor: b). from 5 to 14 days depending on clinical response. patients usually respond to appropriate antibiotic therapy within 48 to 72 hours; otherwise, a repeat paracentesis should be performed. if the ascitic fluid wbc count does not decrease by more than 25%, alternative diagnoses should be considered. prophylaxis with a fluoroquinolone or trimethoprim-sulfamethoxazole should be considered, particularly in high-risk patients (garcia-tsao and lim, 2009 • bacterial meningitis is life threatening and requires urgent medical attention and treatment. • viral encephalitis should be treated with acyclovir until herpes simplex virus is ruled out. • most brain abscesses are caused by streptococci and staphylococcus aureus. • the cns infections most likely to be encountered in clinical practice include meningitis, encephalitis, and abscess. • all cns infections can be difficult to diagnose, and a high index of suspicion by the health care provider is sometimes indicated to ensure patient survival. • mri is the most sensitive neuroimaging test for encephalitis. • acyclovir should be started immediately and continued until hsv pcr testing is obtained. meningitis can be acute, subacute, or chronic. in otherwise healthy children, the three most common organisms causing acute bacterial meningitis are streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae type b (hib). isolation of an organism other than these three organisms from the csf of a child older than 2 months always requires an explanation or evaluation for unusual host susceptibility. children with cochlear implants, asplenia, hiv infection, or csf leak from basilar skull or cribriform fracture are at greater risk for pneumococcal meningitis. deficiencies in terminal components of complement lead to greater risk for meningococcal infection (saez-llorens and mccracken, 2008) . in adults, the common etiologic agents of acute meningitis include s. pneumoniae, n. meningitidis, and listeria monocytogenes. patients with acute meningitis most often present with fever, headache, meningismus, and altered mental status. infants can present with nonspecific symptoms such as inconsolable crying, irritability, nausea, vomiting, and diarrhea. lethargy, anorexia, and grunting respirations indicate a critically ill infant. older children may complain of headache, vomiting, back pain, myalgia, and photophobia; may be confused or disoriented; and may verbalize specifically that the neck is stiff or sore. seizures are noted in up to 20% to 30% of children before hospital admission or early in the course of the illness. in contrast, patients with subacute or chronic meningitis may have the same symptoms with a much more gradual onset, lower fever, and associated lethargy and disability. mycobacterium tuberculosis, treponema pallidum (syphilis), borrelia burgdorferi (lyme disease), and fungi (e.g., cryptococcus neoformans, coccidioides spp.) are the most common agents (tunkel et al., 2010) . physical examination should look for papilledema, middle ear and sinus infections, petechiae (common with n. meningitidis), nuchal rigidity, and in infants, a bulging fontanel. blood cultures should be taken. a lumbar puncture (lp) for csf analysis should be done as soon as possible. a brain ct scan before lp is not necessary if the patient has no evidence of immunocompromise, cns disease, new seizure, papilledema, altered consciousness, or focal neurologic deficit, and if a subarachnoid hemorrhage is not suspected. if neuroimaging is necessary, blood cultures should be taken and antibiotics given before the study; a delay in administration of antibiotics leads to a worse outcome. csf should be sent for cell count, wbc differential, glucose, protein, and gram stain with culture. acid-fast bacilli stain and cryptococcal antigen may be obtained when indicated. empiric antibiotics for the initial treatment of bacterial meningitis are listed in table 16 -20, but these should be tailored to the isolated organisms whenever possible. adjunctive dexamethasone is recommended for children and infants with hib meningitis, but not if they have already received antibiotics. in adults, adjunctive dexamethasone is recommended for pneumococcal meningitis (tunkel et al., 2004) . close contacts of patients with n. meningitidis should receive rifampin, 20 mg/kg (not to exceed 600 mg) twice daily for 2 days, or ciprofloxacin, 500 mg as a single dose, or ceftriaxone, 250 mg im as a single dose. unimmunized persons exposed to h. influenzae meningitis should receive rifampin (turkel et al., 2010) . pregnant women should not receive rifampin or doxycycline. a repeat lp should be done if no clinical response is seen after 48 hours of appropriate antibiotic therapy, particularly for patients with resistant pneumococcal disease and those who received dexamethasone. neonates with gram-negative bacilli and patients with ventriculoperitoneal (vp) shunts require documentation of csf sterility. the duration of antimicrobial therapy is 7 days for patients with n. meningitidis or hib, 10 to 14 days for pneumococcal meningitis, and 14 to 21 days for streptococcus agalactiae. spontaneous bacterial peritonitis is treated with third-generation cephalosporins (cefotaxime or ceftriaxone), with ampicillin-sulbactam, fluoroquinolones, or carbapenems as alternative agents (solomkin et al., 2010) (sor: b) . patients with diffuse peritonitis should undergo an emergency surgical procedure as soon as possible, even if ongoing measures to restore physiologic stability need to be continued during the procedure (sor: b). viral meningitis viral meningitis manifests similar to bacterial meningitis, although its course is rarely aggressive. the diagnostic process and examination are similar to those for bacterial meningitis. viral meningitis is usually caused by enteroviruses, hsv, mumps virus, and hiv. along with the signs of meningitis, signs that suggest a viral etiology include genital lesions (hsv-2), diarrhea, or a maculopapular rash (enteroviruses). diagnosis is made by the history, examination, and csf results. early in the course, the csf might show predominantly neutrophils that can resemble bacterial meningitis. treatment is symptomatic. suppressive therapy should be offered to patients with recurrent hsv meningitis. although encephalitis can also be caused by bacteria and fungi, the great majority of cases are caused by viruses. herpes simplex accounts for 10% of cases. patients present with fever, acute decreased level of consciousness, and occasionally, seizures and language, memory, or behavior disturbances. mri is the most sensitive neuroimaging test for encephalitis and might show temporal lobe inflammation in early hsv encephalitis. csf studies and electroencephalography (eeg) are also recommended for all patients with encephalitis. herpes simplex pcr should be done, and acyclovir should be given immediately until hsv encephalitis is ruled out. during late summer and early fall, doxycycline should be considered to cover for tick-borne illnesses, and testing should include the mosquito-borne encephalitides such as west nile, st. louis, eastern equine, and western equine. treatment depends on the suspected etiologic agent but is generally supportive (tunkel et al., 2008) . a brain abscess is a focal, intracerebral infection that develops into a collection of pus surrounded by a well-vascularized capsule. although fungi and protozoa (particularly toxoplasma) can also cause brain abscesses, bacterial causes are much more common. streptococci are found in 70% of bacterial abscesses and are usually from oropharyngeal infection or infective endocarditis, whereas staphylococcus aureus accounts for 10% to 20% of isolates and is more often found after trauma. community-associated mrsa strains have been increasing. enteric gram-negative bacilli (e.g., e. coli; proteus, klebsiella, and pseudomonas spp.) are isolated in 23% to 33% of patients, often in patients with ear infection, septicemia, or immunocompromise and those who have had neurosurgical procedures. most clinical symptoms are caused by the size and location of the abscess rather than the systemic signs of an infection. headache is the most common complaint and may be accompanied by fever, mental status changes, evidence of increased intracranial pressure (nausea, vomiting, papilledema), or focal neurologic deficits. diagnosis is usually made by ct scan with iv contrast showing the characteristic hypodense center with a peripheral uniform ring enhancement, with or without a surrounding area of brain edema. mri is becoming the preferred imaging modality because of increased sensitivity, particularly for detecting satellite lesions. additional testing depends on risk factors and the likely underlying source of infection and may include blood cultures, chest imaging, testing for hiv and antibodies to toxoplasma, and transesophageal echogram. empiric therapy typically involves vancomycin, ceftriaxone, and metronidazole. optimal management also includes surgical drainage for most abscesses, both to find an etiologic microorganism and to improve chances of cure (turkel, 2010) . • most acute diarrheal illness is viral and can be managed symptomatically and with appropriate attention to hydration. • travelers' diarrhea is usually caused by diarrheogenic escherichia coli. • the infection in travelers' diarrhea is usually self-limited. • antibiotics may shorten the duration of diarrhea by 1 to 3 days. • the most common cause of antibiotic-associated diarrhea is clostridium difficile. • treatment of antibiotic-associated diarrhea involves discontinuing the offending agent, if possible. adjunctive dexamethasone is recommended for children and infants with h. influenzae type b meningitis, but not if they have already received antibiotics (tunkel et al., 2004) (sor: a). in adults, adjunctive dexamethasone is recommended for pneumococcal meningitis (tunkel et al., 2004) (sor: b). diarrhea is a common presenting complaint in the primary care physician's office. not all causes of diarrhea are infectious, and not all infectious causes of diarrhea require specific antibiotic therapy. diarrhea remains a major cause of morbidity and mortality, particularly for children in the developing world. diarrhea is an alteration of normal bowel function, characterized by an increase in the water content, volume, or frequency of stools. acute diarrhea is typically defined as present less than 14 days, and diarrhea is considered chronic when symptoms persist longer than 30 days (figure 16-12 ). infectious diarrhea seen in the primary care physician's office is most frequently caused by viruses. a number of viral agents can cause diarrheal illness (box 16-7). rotaviruses are the principal enteric pathogens in children less than 5 years of age and the most important cause of hospitalization and infant mortality related to diarrheal illnesses. noroviruses evaluate severity and duration obtain history and physical examination [1] [2] [3] [4] [5] treat dehydration report suspected outbreaks 6 check all that apply: 7 are the most common cause of food-borne disease worldwide. viral gastroenteritis is usually an acute self-limited illness, referred to as the "stomach flu." enteric viruses are easily spread by fecal-oral transmission, through contamination of food and water, fomites, and person-to-person spread. secondary attack rates can be high. nausea and vomiting are the most prominent symptoms of viral gastroenteritis. diarrhea, fever, headache, and constitutional symptoms may also be experienced. these viral infections can occur at any time during the year, but tend to occur more often in the winter. there is no specific therapy. treatment is supportive, with particular emphasis on adequate replacement of fluids and electrolytes. if rehydration can be accomplished enterally, it is preferred. both the pentavalent bovine-human reassortment (rv5) and the oral, live-attenuated monovalent (rv1) rotavirus vaccines are effective for prevention of severe gastroenteritis. the rv5 vaccine series is recommended for children at ages 2, 4, and 6 months, whereas the rv1 vaccine should be administered to children 2 and 4 months of age. approximately 40% of travelers to developing regions of the world will develop diarrhea. bacteria are responsible for approximately 80% of diarrhea acquired by travelers. other important causes include viruses and parasites. the onset of the majority of cases of travelers' diarrhea is usually within 5 to 15 days after arrival. the presentation is typically a noninflammatory, nonbloody diarrhea associated with abdominal discomfort, fever, nausea, or vomiting. the duration is usually 1 to 5 days. enterotoxigenic e. coli is responsible for approximately 30% of travelers' diarrhea. enteroaggregative e. coli is the second most common bacterial agent and causes 20% of cases. salmonella, shigella, and campylobacter spp. are less often detected but are important causes of dysentery, particularly in asia and africa. dysentery is severe inflammatory diarrhea manifested by fever and bloody stools. most cases of travelers' diarrhea are self-limited, but chronic postinfectious irritable bowel syndrome may occur in up to 10% of those who experience diarrhea. prevention of travelers' diarrhea is an important component of pretravel counseling for high-risk countries. food should be boiled, cooked, or peeled and water boiled to avoid consumption of fecal contamination. if a person develops travelers' diarrhea, a short course of antibiotics with rifaximin, ciprofloxacin, or azithromycin can shorten the duration of illness by 1 to 3 days. antibiotic therapy is recommended for persons with bloody diarrhea or fever. rifaximin, a nonabsorbed antibiotic, is not effective against invasive pathogens and should not be administered for dysentery. ciprofloxacin or azithromycin should be used for dysenteric symptoms based on local antimicrobial susceptibilities. antibiotics are frequently prescribed in the primary care physician's office for a variety of infections. unfortunately, antibiotics can alter the normal host microflora that can be protective against other infections. antibiotic effects on the normal gastrointestinal tract microbiome can lead to antibiotic-associated diarrhea, which causes significant morbidity and mortality. administration of antibiotics usually precedes symptoms of antibiotic-associated diarrhea by about 1 week but can be as distant as 2 or 3 months. strong associations with clindamycin (cleocin), cephalosporins, penicillins, and fluoroquinolones have been demonstrated, but any antibiotic can lead to antibiotic-associated diarrhea. the most important cause of antibiotic-associated diarrhea is clostridium difficile, an anaerobic, gram-positive, spore-forming rod. c. difficile is implicated as the cause in up to 25% of antibiotic-associated diarrhea cases, in 50% to 75% of antibiotic-associated colitis cases, and in more than 90% of antibiotic-associated pseudomembranous colitis cases. risk factors for c. difficile diarrhea include antibiotics, health care exposure (recent stay in hospitals or long-term care facilities), older age (>60), and comorbid conditions. the clinical presentation of c. difficile colitis is usually diarrhea, abdominal pain or cramping, and fever in a patient who recently received antibiotics. leukocytosis is common and may be profound; levels can be consistent with leukemoid reaction. a rare but potentially fatal complication is toxic megacolon. toxic megacolon manifests as acute colonic dilation to a diameter greater than 6 cm, associated with systemic toxicity and the absence of mechanical obstruction. with its high associated mortality, any patient who develops toxic megacolon requires immediate surgical evaluation for possible colectomy. diagnosis of c. difficile diarrhea is achieved by demonstration of c. difficile toxin a or b in the stool by enzyme immunoassay (eia) or cell culture cytotoxicity assay in a symptomatic patient with a previous history of antibiotic use. asymptomatic patients should not be tested. with the improved sensitivities of these diagnostic assays, one stool sample is usually sufficient to test for c. difficile, unless symptoms recur. test of cure after therapy with repeat stool for c. difficile toxin is not recommended because stools may remain positive for c. difficile toxin despite clinical resolution. endoscopy can demonstrate pseudomembranes in the colon. pseudomembranes are diagnostic of c. difficile infection, but are often not present. endoscopy may only reveal the presence of nonspecific colitis. clostridium difficile colitis is treated by discontinuing the offending agent(s) if possible and initiating antibiotic therapy (box 16-8). antimotility agents should be avoided. oral metronidazole (flagyl), 500 mg three times daily for 10 to 14 days, is recommended for mild-moderate c. difficile diarrhea. severe diarrhea should be treated with oral vancomycin. oral vancomycin is currently not recommended for all patients with c. difficile diarrhea because of concerns for the promotion of vancomycin-resistant enterococci (vre) and its expense. about 10% to 20% of patients experience relapse in travelers' diarrhea, in which enterotoxigenic e. coli or other bacterial pathogens are likely causes, prompt treatment with a fluoroquinolone, azithromycin, or rifaximin or, in children, azithromycin 10 mg/kg/day once daily can reduce the duration of an illness from 3 to 5 days to 1 to 2 days (dupont, 2010) (sor: a). after therapy. for relapse, a repeat course of the original c. difficile treatment should be administered. patients who have mild to moderate cases without volume depletion or systemic toxicity can be treated as outpatients. discussions of the following infections can be found online at www.expertconsult.com: • infectious viral hepatitis • endocarditis treat mild-moderate c. difficile diarrhea with metronidazole (zar et al., 2007) evidence-based reviews of the diagnosis and treatment of many common clinical problems. www.mdcalc.com/curb-65-severity-score-community-acquired-pneumonia curb-65 score calculator to determine the severity of communityacquired pneumonia and need for hospitalization. the complete reference list is available online at www.expertconsult.com. anthony zeimet hepatitis is defined as inflammation of the liver that is commonly induced by viruses that include the hepatitis viruses a through e, which will be the focus of this discussion. other viruses that can induce hepatitis include epstein-barr virus (ebv), cytomegalovirus (cmv), herpes simplex virus (hsv), varicella zoster virus (vzv), adenovirus, and coxsackievirus. various medications and alcohol abuse are two important nonviral causes. most infectious causes of hepatitis are self-limiting; however, hepatitis b and c viruses can cause a chronic infection that may lead to cirrhosis and eventual liver failure, as well as hepatocellular carcinoma. hepatitis a virus (hav) and hepatitis e virus (hev) are spread by the fecal-oral route and only cause an acute infection. hepatitis b, c, and d viruses (hbv, hcv, hdv) are spread through the blood and have an acute form of disease that sometimes can become chronic. the clinical presentation of hepatitis is clinically indistinguishable. asymptomatic infections are more common than symptomatic infection. symptoms generally include right upper quadrant (ruq) abdominal pain, anorexia, nausea, vomiting, diarrhea, dark-colored urine, pale stools, and generalized malaise; patients may notice a yellow hue to their skin or eyes. pruritus is common, caused by deposition of bilirubin in the skin. the physical examination generally reveals jaundice and sclera icterus in addition to ruq pain. hepatomegaly is seen in 85% and splenomegaly in 15% of patients with hepatitis. liver function tests reveal elevated levels of aspartate transaminase (ast), alanine transaminase (alt), and bilirubin, and to a lesser extent, alkaline phosphatase (alp). hepatitis a virus is the most common cause of viral hepatitis worldwide. poor hygiene practices in both the industrial and the developing world account for its prevalence. in the united states, hav is common among lower socioeconomic groups, daycare attendants and workers, men who have sex with men (msm), and illicit drug users. hepatitis a is often acquired by travelers to endemic areas. the incubation period is 15 to 45 days (mean, 30 days). hav is highly contagious, and peak fecal shedding generally occurs at the onset of illness in most infected patients. viremia averages 30 to 90 days. hav infection manifests as an acute, self-limited illness, with the prodromal symptoms lasting about a week before the onset of jaundice. jaundice generally resolves after 2 weeks, and most patients recover. fulminant hepatic failure is possible but extremely rare. diagnosis of acute hav infection is made by demonstration of anti-hav immunoglobulin m (igm) in the patient's serum. this may be negative if the patient presents early, and repeat testing may be necessary if hav is strongly suspected. anti-hav igg in the serum indicates remote infection or immunization (efig 16-1). treatment is primarily supportive, except in patients with fulminant liver failure, who may require a liver transplant. vaccination should be administered to all patients who are seronegative and to persons at increased risk for acquiring hav, including those about to travel to endemic areas, patients with chronic liver disease or receiving clotting factor concentrates, msm, hiv-positive patients, and illicit drug users. certain areas of the united states now require mandatory vaccination of children as well as those who work in the restaurant industry. the vaccine is safe and highly efficacious and is given as a two-dose series at 0 and at 6 to 12 months. passive immunization with immune globulin is recommended for those exposed to the virus by a known contact, including household and sexual contacts, and those who are traveling to an endemic area for less than 4 weeks but never vaccinated. any person who receives immune globulin should also start the vaccination series. hepatitis b virus infection can be acute or chronic. about 40,000 people die from acute hbv infection annually, and 500,000 die of cirrhosis and hepatocellular carcinoma caused by chronic infection. about 400 million people worldwide are living with chronic hbv infection. in the united states, an estimated 1.25 million residents have chronic hbv infection, with 4000 to 5500 deaths each year. significant burdens of disease are seen in asia, pacific islands, sub-saharan africa, amazon basin, and eastern europe. most adults with acute hbv will clear the virus, with less than 5% progressing to chronic infection. chronic infection will develop in almost all children infected perinatally and in 50% of those who become infected at 1 to 5 years of age. hbv is transmitted through exchange of body fluids, sexually and perinatally. in the united states, most hbv cases are acquired during adolescence and early adulthood with onset of sexual activity, experimentation with drug use, and sometimes occupational exposure. fever, polyarthralgia, rash, and a serum sickness-like illness are features of hbv infection in addition to jaundice and may be seen in association with polyarteritis nodosa. clinicians have the most difficulty in interpreting the various serologic tests for diagnosis of hepatitis b (etable 16-3) . the mean incubation period is 60 to 70 days, with a range of 30 of 180 days after infection. diagnosis of acute infection can be detected by obtaining hbv surface antigen (hbsag), which can appear as early as 1 week after exposure but generally by day 50. in a patient strongly suspected to have hbv infection, the clinician can consider checking the hbv dna viral load; which can be detected as early as 1 week after exposure. eventually the patient will develop an anti-hbv surface antibody, which indicates recovery from the illness. the other viral serologies for hbv are rarely obtained in acute illness. in chronic hbv infection, there are three major phases of infection: 1. immune tolerant. active viral replication in the liver with high levels of hbv dna levels but essentially normal or minimal elevation of ast and alt. most patients eventually progress to the next stage. 2. immune active. more robust liver inflammation with alt elevation, and liver biopsy shows inflammation with or without fibrosis. hbv early antigen (hbeag) is detected along with hbsag. 3. inactive carrier state. as patients enter this phase, they clear the hbeag and develop anti-hbe antibody and have undetectable or low levels of hbv dna, with normalization of alt and liver inflammation. if patients become hbsag negative, they then develop anti-hbs and have resolved their infection; otherwise, they are considered a chronic carrier. treatment of acute hbv is primarily supportive. in the last decade, however, there have been significant advances in the treatment of chronic hbv infection. the use of interferon has long been the mainstay of treatment and has a defined, limited course but is generally poorly tolerated. with the advent of the hiv/aids epidemic and research into treatment of hiv disease, antiviral medications are now starting to replace interferon as the preferred treatment option for hbv patients. nucleoside/nucleotide analogs such as lamivudine, adefovir, entecavir, tenofovir, and telbivudine are generally given for long-term, indefinite therapy to prevent progression of liver disease and development of hepatocellular carcinoma. any patient with chronic hbv infection should be referred to an infectious diseases specialist or a hepatologist to determine the appropriate treatment course. universal vaccination of newborns and infants is routine in the united states since 1991, and the incidence of hbv infection has declined. during primary care visits, the vaccination status of any adult or adolescent born before 1991 should be reviewed and the vaccine offered. the vaccine requires three doses given at 0, 1, and 6 months. an unvaccinated person or neonate who is exposed to the body fluids of a hbv-infected individual should start the vaccination series in addition to receiving the hepatitis b immune globulin (hbig). hepatitis c virus infection is the most common cause of chronic viral hepatitis in the united states. hcv does have an acute form of infection but is usually subclinical and rarely diagnosed. the cdc estimates that there are more than 2.7 million people with hcv infection. hcv is generally transmitted parenterally, as in injection drug users who share needles. before 1992, those who received a blood transfusion may have contracted hcv. sexual transmission acute hbv has been reported in monogamous couples, with one partner who has hcv infection and the other without infection who eventually acquires the virus. this occurs in 3% to 5% of couples and represents a rare mode of transmission. because the most common mode of acquisition is sharing needles, any patient who is hcv positive should be screened for hiv because these two infections often occur together (ebox 16-1). the diagnosis of acute hcv infection can be made by obtaining a hcv rna viral load; although this is rarely done because the initial infection is subclinical. chronic disease is generally discovered by a positive anti-hcv antibody along with an elevated hcv rna viral load. hcv genotype should also be obtained in any positive individual, because this has important prognostic factors with regard to therapy, with genotype 1a and 1b the predominant type in the united states and unfortunately having a poor response to therapy. as with hbv, chronic hcv infection can lead to cirrhosis and the development of hepatocellular carcinoma. treatment consists of 24 to 48 weeks of interferon and ribavirin therapy. any patient being considered for therapy should be referred to an infectious diseases specialist or hepatologist. a liver biopsy is often needed to determine appropriate treatment candidates. also known as the hepatitis delta antigen virus, hdv is a defective virus that requires the presence of hbv to be infectious. hdv should be suspected in any patient with chronic hbv who develops acute hepatitis. hepatitis d is endemic in the mediterranean, balkans, africa, middle east, and amazon basin. diagnosis is made through an anti-hdv antibody in the presence of someone with positive hbsag or anti-hb core antibody igm or igg. treatment is supportive. any person vaccinated against hbv cannot become infected with hdv. similar to hav infection, hev is spread by the fecal-oral route. hev only has an acute form and does not progress to chronic infection. most reported epidemics have been related to consumption of contaminated drinking water. hev is endemic to southeast and central asia, north africa, middle east, mexico, brazil, venezuela, and cuba. hepatitis e can be considered a cause of infectious hepatitis in the united states in the traveler returning from an endemic area. the incubation period is 40 days. infection is of major concern during pregnancy, which can cause death in late pregnancy. diagnosis is made by demonstration of anti-hev antibody in serum. treatment is supportive. • endocarditis prophylaxis is now recommended solely for patients at high risk of a complicated course with a more narrow range of cardiac conditions. • routine prophylaxis for gi and gu procedures is no longer recommended. • the duke criteria represent a reliable scoring system for diagnosing endocarditis. • echocardiography is indicated to confirm suspected endocarditis. bacterial endocarditis is one of the most feared infections; although uncommon, it carries high morbidity and mortality. increase in antibiotic resistance among bacteria causing this infection has created challenges for effective treatment. the fundamental view of the american heart association (aha) in preventing infective endocarditis has shifted in recent years. views on pathophysiology have not changed substantially, but it is now recognized ebox 16-1 persons for whom hepatitis c virus (hcv) screening is recommended persons who have injected illicit drugs in the recent and remote past, including those who injected only once and do not consider themselves to be drug users. persons with conditions associated with a high prevalence of hcv infection, including: persons with human immunodeficiency virus (hiv) infection persons with hemophilia who received clotting factor concentrates before 1987 persons who have ever received hemodialysis persons with unexplained abnormal transaminase (aminotransferase) levels prior recipients of transfusions or organ transplants before july 1992, including: persons who were notified that they had received blood from a donor who later tested positive for hcv infection persons who received a transfusion of blood or blood products persons who received an organ transplant children born to hcv-infected mothers health care, emergency medical, and public safety workers after a needle stick injury or mucosal exposure to hcv-positive blood current sexual partners of hcv-infected persons * modified from centers for disease control and prevention. recommendations for prevention and control of hepatitis c virus (hcv) infection and hcv-related chronic disease. mmwr 1998;47(rr):1-39. *although the prevalence of infection is low, a negative test in the partner provides reassurance, making testing of sexual partners of benefit in clinical practice. • universal vaccination of infants with hepatitis b vaccine reduces the risk of acute hepatitis, chronic carrier state, and complications of chronic infection and may be more effective than selective vaccination of high-risk individuals (lee et al., 2006) (sor: a). • as part of a comprehensive health evaluation, all persons should be screened for behaviors that place them at high risk for hepatitis c infection (ghany et al., 2009 ) (sor: b). • liver biopsy may be considered in patients with chronic hcv infection to determine fibrosis stage for prognostic purposes or to make a treatment decision (ghany et al., 2009 ) (sor: b). that cumulative daily episodes of bacteremia likely carry more risk than the transient bacteremia caused by dental procedures. infective endocarditis likely begins with turbulent flow and damaged endothelium around heart valves, which allow platelet aggregation and thrombus formation, causing a "nonbacterial thrombotic endocarditis" (wilson et al., 2007) . the presence of bacteremia then allows this vegetation to become seeded with infection. bacterial "adhesins" are present to a greater degree in some species and allow for more effective attachment to the injured area of endothelium. with high concentrations of bacteria in the mouth, vagina, gi tract, and perhaps gu system, antibiotic prophylaxis was initiated when these anatomic locations were manipulated. recommendations for infective endocarditis prevention changed in 2007-2008, with aha recognizing more likely benefit from providing adequate population-based dental care and good oral hygiene, and thus less significant ongoing bacteremia at home in brushing, flossing, and "toothpicking," than in providing antibiotic prophylaxis to patients undergoing a dental procedure. no prospective rct has shown that dental prophylaxis prevents infective endocarditis. with recognition of the risk associated with administration of antibiotics (gi upset, diarrhea, rash, anaphylaxis) and the risk of contributing to increasing antibiotic resistance, versus the likely negligible benefit, aha has substantially changed its advice on this long-held practice. a preexisting cardiac condition produces a predisposition to the development of infective endocarditis (ebox 16-2). for example, those who have valve replacement for infection of an infected native valve carry a lifetime risk of 630 per 100,000 patient-years. the risk in the general population without known heart disease is 5 per 100,000 patient-years. more concerning, however, is the risk to a given patient of poor outcome if the patient develops endocarditis, which drives current aha recommendations. those with an infected mechanical valve have a mortality rate of about 20%, versus 5% or less for patients with an infected native valve (wilson et al., 2007) . a summary of current recommendations for endocarditis prophylaxis is provided in etable 16-4. of note, gi and gu procedures have been removed from those for which antibiotics are recommended, unless those systems are actively infected at the time of the procedure. the same is true for skin and soft tissue procedures, in that only infected tissue would warrant antibiotics to prevent infective endocarditis. it is still recommended to provide prophylaxis for respiratory tract procedures, if the respiratory wall will be invaded through biopsy or the procedure. in addition, respiratory procedures to treat infections (e.g., empyema) should be combined with antibiotic administration (nishimura et al., 2008) . antibiotic regimens for prophylaxis for dental procedures are still based primarily on synthetic penicillins as their cornerstone. this is with recognition that streptococcus viridans is both a mouth floral inhabitant and a common agent causing infective endocarditis. with other procedures, antibiotics should be targeted to bacterial pathogens causing any active infection in the system being manipulated. ebox 16-2 cardiac conditions associated with the highest risk of adverse outcome from endocarditis for which prophylaxis with dental procedures is reasonable prosthetic cardiac valve or prosthetic material used for cardiac valve repair previous ie congenital heart disease (chd) * unrepaired cyanotic chd, including palliative shunts and conduits completely repaired congenital heart defect with prosthetic material or device, whether placed by surgery or by catheter intervention, during the first 6 months of the procedure † repaired congenital heart defect with residual defects at the site or adjacent to the site of a prosthetic patch or prosthetic device (which inhibit endothelialization) cardiac transplantation recipients who develop cardiac valvulopathy *except for the conditions listed above, antibiotic prophylaxis is no longer recommended for any other form of chd. †prophylaxis is reasonable because endothelialization of prosthetic material occurs within 6 months after the procedure oral antibiotic william osler discussed "malignant endocarditis" in 1885 and its great diagnostic challenge. in 2009 the modified duke criteria remains a reliable tool for assessing patients with endocarditis. endocarditis is suspected in febrile patients without an obvious source, in those with recent bacteremia (including iv drug use), in those with underlying cardiac predisposition, and perhaps in patients with the clinical finding of a new cardiac murmur. in establishing a diagnosis of infective endocarditis, a patient is considered to have definite disease if two major or one major and three minor or five minor criteria are present. possible disease is defined as one major and one minor or three minor criteria (ebox 16-3). pathologic specimens showing changes consistent with endocarditis would make a definitive diagnosis. echocardiography is indicated in making the diagnosis of infective endocarditis. transthoracic echocardiography (tte) is helpful if vegetations are seen, although size of the patient and other disease (e.g., copd) may limit the ability of tte to view the cardiac valves adequately. if tte is negative and suspicion remains, transesophageal echocardiography (tee) is indicated. tte may be more widely available, depending on regional and institutional variation, and should be used rather than delaying this diagnostic test. bacteria present within valvular vegetations are often less metabolically active, which partly explains the requirement for longer courses of antibiotics for this type of infection. clearly, therapy for endocarditis should be targeted at the organism identified on blood culture, if any. the counting of antibiotic days should begin when the blood culture becomes negative and not at the start of the particular agent. recommendations for antibiotic use in infectious endocarditis are highly variable and based on the presence or absence of synthetic valvular material and the infectious agent (etable 16-5). generally speaking, a minimum of 4 weeks of iv antibiotics is indicated. in cases of resistant organisms, up to 8 weeks may be required. in either case, synergistic use of agents such as gentamicin may be indicated for the first several weeks of treatment, which then can be discontinued. the ability of a given patient to complete this course at home versus in a health care facility is dependent on the dosing frequency of the antibiotic, availability of inhome nursing services, and the type of intravenous access through which the antibiotic will be delivered. at the completion of endocarditis therapy, echocardiography should be repeated to re-assess the function of the valve(s) in question. valvular dysfunction at the completion of therapy is a good indication that the patient will need valve replacement in the future. there are circumstances, like the development of congestive heart failure in the face of endocarditis, in which primary surgery is indicated. typical microorganisms consistent with ie from 2 separate blood cultures: viridans streptococci, streptococcus bovis, hacek group, staphylococcus aureus; or community-acquired enterococci in the absence of a primary focus; or microorganisms consistent with ie from persistently positive blood cultures, defined as follows: at least 2 positive cultures of blood samples drawn >12 hours apart; or all of 3 or a majority of ≥4 separate cultures of blood (with first and last sample drawn at least 1 hour apart). single positive blood culture for coxiella burnetii or anti-phase 1 igg antibody titer >1:800 echocardiogram positive for ie (tee recommended for patients with prosthetic valves, rated at least "possible ie" by clinical criteria, or complicated ie [paravalvular abscess]; tte as first test in other patients) defined as follows: oscillating intracardiac mass on valve or supporting structures, in the path of regurgitant jets, or on implanted material in the absence of an alternative anatomic explanation; or abscess; or new partial dehiscence of prosthetic valve; new valvular regurgitation (worsening or changing or preexisting murmur not sufficient) predisposition, predisposing heart condition, or idu fever, temperature >38° c vascular phenomena, major arterial emboli, septic pulmonary infarcts, mycotic aneurysm, intracranial hemorrhage, conjunctival hemorrhages, and janeway's lesions immunologic phenomena: glomerulonephritis, osler's nodes, roth's spots, and rheumatoid factor microbiologic evidence: positive blood culture but does not meet a major criterion as noted above * or serological evidence of active infection with organism consistent with ie echocardiographic minor criteria eliminated echocardiography should be performed in all patients with suspected infective endocarditis (baddour et al., 2005) there is no evidence that antibiotic prophylaxis is effective or ineffective for preventing infectious endocarditis after dental procedures in patients at risk (chung, 2009 ) (sor: c). regimen dosage * and route duration (wk) chronic cough due to acute bronchitis: accp evidencebased clinical practice guidelines interim recommendations for the use of influenza antiviral medications in the setting of oseltamivir resistance among circulating influenza a (h1n1) viruses, 2008-09 influenza season national ambulatory medical care survey: 2005 summary short-course antibiotic treatment in acute exacerbations of chronic bronchitis and copd: meta-analysis of doubleblind studies antibiotics for exacerbations of chronic obstructive pulmonary disease antibiotics for acute bronchitis effects of naproxen on experimental rhinovirus colds: a randomized, double-blind, controlled trial infectious diseases society-american thoracic society consensus guidelines on management of community-acquired pneumonia in adults emergence of a novel swineorigin influenza a (h1n1) virus in humans acute pneumonia influenza. in: schlossberg d, ed. clinical infectious disease seasonal influenza in adults and children: diagnosis, treatment, chemoprophylaxis, and institutional outbreak management. clinical practice guidelines of the infectious diseases society of america antivirals for influenza in healthy adults: systematic review hospitalizing influenza in adults infectious diseases society-american thoracic society consensus guidelines on management of community-acquired pneumonia in adults influenza viruses, including avian influenza and swine influenza steroids for symptom control in infectious mononucleosis hepatosplenomegaly in infectious mononucleosis, assessed by ultrasonic scanning infectious mononucleosis vaccines for post-exposure prophylaxis against varicella (chickenpox) in children and adults centers for disease control and prevention (ats/cdc). targeted tuberculin testing and treatment of latent tuberculosis infection centers for disease control and prevention. extensively drug-resistant tuber culosis-united states short-course therapy with rifampin plus isoniazid, compared with standard therapy with isoniazid, for latent tuberculosis infection: a meta-analysis genital warts expedited partner therapy in the management of sexually transmitted diseases. atlanta: us department of health and human services center for disease control and prevention. sexually transmitted disease surveillance. division of std prevention accuracy of the clinical diagnosis of vaginitis compared with a dna probe laboratory standard a pooled analysis of the effect of condoms in preventing hsv-2 acquisition consistent condom use is associated with lower prevalence of human papillomavirus infection in men the limited value of signs and symptoms in the diagnosis of vaginal infections cervical cancer condom effectiveness in reducing heterosexual hiv transmission sexually transmitted diseases treatment guidelines genitourinary infections urinary tract infection in children diagnosis and management of uncomplicated urinary tract infections american geriatric society. guidelines for the diagnosis and treatment of asymptomatic bacteriuria in adults asymptomatic bacteriuria: when to screen and when to treat the effectiveness of a clinical practice guideline for the management of presumed uncomplicated urinary tract infection in women recurrent cystitis in non-pregnant women a randomized trial to evaluate effectiveness and cost effectiveness of naturopathic cranberry products as prophylaxis against urinary tract infection in women screening for asymptomatic bacteriuria in pregnancy: a decision and cost analysis guidelines for antimicrobial treatment of uncomplicated acute bacterial cystitis and acute pyelonephritis in women tick-borne infections tick-borne diseases tick-borne diseases in the united states the clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis practice guidelines for the diagnosis and management of skin and soft tissue infections furuncles and carbuncles centers for disease control and prevention. health-care-associated methicillin-resistant staphylococcus aureus (mrsa) double-blind, placebo-controlled trial of cephalexin for treatment of uncomplicated skin abscesses in a population at risk for community-acquired methicillin-resistant staphylococcus aureus infection practice guidelines for the diagnosis and management of skin and soft-tissue infections bacteriological study of diabetic foot infections diabetic foot infection diagnostic accuracy of the physical examination and imaging tests for osteomyelitis underlying diabetic foot ulcers: meta-analysis diagnosis and treatment of diabetic foot infections deep tissue biopsy vs. superficial swab culture monitoring in the microbiological assessment of limb-threatening diabetic foot infection culture of percutaneous bone biopsy specimens for diagnosis of diabetic foot osteomyelitis: concordance with ulcer swab cultures bite infections antibiotic prophylaxis for mammalian bites (cochrane review) sexually transmitted diseases treatment guidelines fever of unknown origin a comprehensive evidence-based approach to fever of unknown origin fever of unexplained origin: report on 100 cases complicated intra-abdominal infections; spontaneous bacterial peritonitis; and secondary bacterial peritonitis and intra-abdominal abscesses members of veterans affairs hepatitis c resource center program. management and treatment of patients with cirrhosis and portal hypertension: recommendations from the department of veterans affairs hepatitis c resource center program and the national hepatitis c program mandell, douglas, and b ennett's principles and practices of infectious diseases spontaneous bacterial peritonitis: diagnosis, treatment and prevention review article: the utility of reagent strips in the diagnosis of infected ascites in cirrhotic patients randomized, comparative study of oral ofloxacin versus intravenous cefotaxime in spontaneous bacterial peritonitis diagnosis and management of complicated intra-abdominal infection in adults and children. guidelines by the surgical infection society and the infectious diseases society of america acute bacterial meningitis beyond the neonatal period brain abscess practice guidelines for the management of bacterial meningitis the management of encephalitis: clinical practice guidelines of the infectious disease society of america acute meningitis guidelines on acute infectious diarrhea in adults. the practice parameters committee of the american college of gastroenterology a comparison of vancomycin and metronidazole for the treatment of clostridium difficileassociated diarrhea, stratified by disease severity acute cholecystitis nett's principles and practices of infectious diseases diagnosis and management of complicated intra-abdominal infection in adults and children: guidelines by the surgical infection society and the infectious diseases society of america infectious viral hepatitis national institutes of health consensus development conference statement: management of hepatitis b acute viral hepatitis hepatitis b virus infection chronic viral hepatitis chronic hepatitis diagnosis, management and treatment of hepatitis c: an update acute viral hepatitis hepatitis c virus infection effect of hepatitis b immunisation in newborn infants of mothers positive for hepatitis b surface antigen: systematic review and meta-analysis chronic hepatitis b: update infective endocarditis: diagnosis, antimicrobial therapy, and complications prescription of antibiotics for prophylaxis to prevent bacterial endocarditis experience with a oncedaily aminoglycoside program administered to 2184 adult patients acc/aha 2008 guideline update on valvular heart disease: focused update on infective endocarditis prevention of infective endocarditis: guidelines from the american health association. circulation key: cord-014608-g3p19coe authors: nan title: pneumococcal colonization and carriage date: 2014-12-01 journal: pneumonia (nathan) doi: 10.1007/bf03399438 sha: doc_id: 14608 cord_uid: g3p19coe nan homologous recombination is one of the main evolutionary forces affecting streptococcus pneumoniae. the highly recombinogenic nature of this species allows introduction of genetic material with selective advantages in carriage, the state that is a prerequisite for the development of pneumococcal invasive diseases. to study the impact of recombination on the evolution of a carriage pneumococcal population, whole genome sequencing was used to characterize 3,085 pneumococcal carriage isolates from a 2.4 km 2 thai refugee camp collected over a 3-year period. this high sampling density allowed us to characterized genetic exchanges in the pneumococcal population at a high resolution. recombination 'hotspots' showed remarkable consistency between lineages, some of which were associated with drug resistance. temporal trends in recombination at these sites reflected changes in antibiotic consumption, suggesting recombination facilitates adaptation to changing selection pressures. the highest frequencies of receipt and donation of dna fragments exchanged through homologous recombination were observed in non-encapsulated lineages, implying a potential role in diversification and adaptation of the overall population played by these non-vaccine target lineages. these findings expand our understanding of pneumococcal population in carriage and help inform the design of future intervention strategies. d. bogaert 1 1 the netherlands individuals differ markedly in their susceptibility to and clinical presentation of respiratory infections, despite that most young children and many adults are colonized with potential bacterial pathogens like streptococcus pneumoniae. the reasons for these individual differences are not yet fully understood, but clearly multifactorial. besides pathogen-related (virulence)-factors, host-related factors like immune-status and genetic background and environmental factors, a possible fourth factor might be of relevance, i.e. the commensal community of bacteria 'hosting' these potential pathogens. the collective genomes of these commensal inhabitants are referred to as the human microbiome. this microbiome contains highly complexity communities of bacteria, which differ between individuals and even more between niches. the human microbiome in general has shown crucial for an appropriate development of our immune system and our mucosal barriers, and for prevention of pathogen adherence and expansion. we studied the development and composition of microbiota of the upper respiratory tract in different age-groups and in relation to environmental and disease characteristics. we observed highly complex and nichespecific communities of bacteria. even within the upper respiratory tract, the microbiota composition differs depending on the exact anatomical location. furthermore, the composition of respiratory microbiota varies with host and environmental factors like age, season, infant feeding, and viral presence. moreover, our data suggest a correlation between the presence and abundance of specific bacteria and stability of microbiota, as well as susceptibility to respiratory infections. finally, clear evidence has been found for patterns of bacterial interactions within the respiratory microbiota, with a central role played by streptococci. no conflict of interest pneumonia 2014 volume 3 isppd-9 / pneumonia 2014 mar 9-13;3: oral poster abstracts although cohort studies have indicated associations between streptococcus pneumoniae and other microbes in the nasopharynx, they do not allow statements on causality. here a unique experimental human pneumococcal carriage model is used to study whether the nasopharyngeal microbiome is perturbed by pneumococcal exposure and whether a specific composition determines subsequent establishment of pneumococcal carriage. healthy adult volunteers were assessed for pneumococcal carriage by culture of nasal wash samples (nws). those without naturally acquired pneumococcal carriage received an experimental intranasal pneumococcal challenge with serotype 6b or 23f. the composition of the nasopharyngeal microbiome was longitudinally studied by 16s rdna pyrosequencing on nws collected before and 2, 7 and 14 days after challenge. among the 40 selected volunteers 10 were natural carriers and 30 were experimentally challenged. microbiome composition data were attained for 117 nws. by principal component analysis of the individual nasopharyngeal communities before challenge we identified 5 distinct microbiome profiles. natural pneumococcal carriage was particularly common in one of these profiles (p = 0.005). a more diverse microbiome (p = 0.034) and low presence of corynebacterium spp. (p = 0.046) prior to challenge were associated with establishment of pneumococcal carriage. whereas the nasopharyngeal microbiome was not perturbed after exposure to serotype 6b, its diversity increased upon exposure to serotype 23f (p = 0.004). five nasopharyngeal microbiome profiles were identified with different natural pneumococcal carriage rates. a more diverse microbiome and low presence of corynebacterium spp. prior to challenge were associated with establishment of pneumococcal carriage. perturbation of the nasopharyngeal microbiome upon pneumococcal exposure seems to be strain dependent. r. das 1 , m. larose 1 , r. bucala 2 , j.n. weiser 1 1 internal medicine, university of pennsylvania school of medicine, philadelphia, usa; 2 internal medicine, yale school of medicine, new haven, usa human genetic polymorphisms associated with reduced expression of macrophage migration inhibitory factor (mif) have been linked to the risk of community-acquired pneumonia (cap). since streptococcus pneumoniae is a leading cause of cap and nasal carriage is a precursor to invasive disease, we explored the role of mif in pneumococcal colonization using a mouse model. pneumococcal colonization led to local and systemic mif production. mice deficient in mif (mif -/-) were prone to higher density and more prolonged colonization compared to wild-type. the delayed clearance in mif -/mice correlated with reduced recruitment of nasopharyngeal macrophages. the upregulation and positive feedback provided by monocyte chemotactic protein-1 (mcp-1 or ccl2) was impaired in the mif -/mice. in vitro, pneumococcal infection of macrophages induced transcription and release of mif, processes dependent on bacterial expression of the pore-forming toxin, pneumolysin, and induction of map kinase phosphorylation. a point mutation in pneumolysin, which eliminates pore-formation, was sufficient to abrogate macrophage production of mif. correspondingly, nasal colonization with pneumolysin-deficient bacteria led to decreased local mif upregulation and reduced macrophage recruitment. prior work has demonstrated that pneumolysin-deficient bacteria show delayed clearance of colonization; this effect was eliminated in mif -/animals. downstream of primary clearance, mif -/animals also demonstrated reduced anti-pneumococcal antibody production and reduced ability to clear secondary colonization. finally, delivery of mif to the nasopharynx restored macrophage recruitment and pneumococcal clearance. our work suggests that mif is important for innate and adaptive immunity to pneumococcal colonization and could be a contributing factor in the genetic susceptibility to cap. no conflict of interest pneumonia 2014 volume 3 background and aims: data on the nasopharyngeal carriage prevalence of streptococcus pneumoniae across age groups are important to help predict the impact of introducing pneumococcal conjugate vaccines (pcvs) into routine vaccination programmes, given their important indirect effect. yet most carriage studies are conducted in children <5 y only. we investigated whether carriage rates in young children could be used to infer those in older age groups. methods: we conducted a systematic review of studies providing carriage estimates across age groups in healthy populations not previously exposed to pcv, using medline and embase. we used bayesian linear meta-regression background: the impact of 7-valent pneumococcal conjugate vaccine (pcv) in developed countries was enhanced by indirect protection of unvaccinated individuals, mediated by reduced nasopharyngeal carriage of vaccineserotype pneumococci. the potential indirect protection of 10-valent pcv (pcv10) in a developing country setting is unknown. we sought to estimate the effectiveness of programmatic introduction of pcv10 in kenya against carriage of vaccine-serotype pneumococci and non-typeable haemophilus influenzae (nthi). methods: pcv10 was introduced into the infant vaccination program in kenya in january 2011, accompanied by a catch-up campaign in kilifi county for children <5 years. we conducted annual cross-sectional studies of carriage among an age-stratified, random population sample in the 2 years before and 2 years after pcv10 introduction. approximately 500 individuals were enrolled each year. carriage prevalence ratios in the 2 years following pcv10 introduction compared to baseline were determined by log-binomial regression. results: among children aged <5 years the prevalence of vaccine-serotype, non-vaccine-serotype and nthi carriage was 0.34, 0.41 and 0.54, respectively, at baseline and 0.13, 0.57 and 0.40, respectively, after pcv10 introduction. adjusted prevalence ratios were 0.36 (95%cis: 0.26-0.51), 1.37 (1.13-1.64 ) and 0.62 (0.52-0.75), respectively. among individuals ≥5 years, the adjusted prevalence ratios for vaccine-serotype pneumococci and nthi were 0.34 (95%cis: 0.18-0.62) and 0.71 (0.56-0.89), respectively. conclusion: following programmatic use of pcv10 in kilifi, carriage of vaccine-serotypes was reduced by 64% in children <5 years and 66% in older individuals. these findings suggest that pcv10 introduction in africa will have a substantial indirect effect on invasive pneumococcal disease. transwell experiments in preliminary experiments. conclusion: results suggest s. salivarius-mediated inhibition of pneumococcal adherence is not megaplasmiddependant, and requires s. salivarius contact with host cells. as such, inhibition of pneumococcal adherence is likely to be mediated by competition for binding, rather than by megaplasmid-encoded bacteriocins. this work may facilitate the development of novel strategies for the prevention of pneumococcal colonisation and disease. isppd-9 / pneumonia 2014 mar 9-13; 3:1-286 m. daana 1 , g. rahav 2 , h. jaber 2 , a. hamdan 3 , a. thalji 4 , f. jaar 5 , a. goral 2 , e. glazer 2 , w. awida 2 , m. raz 6 , g. regev-yochay 2 1 primary care, maccabi healthcare services, jerusalem, israel; 2 infectious disease unit, sheba medical center, ramat gan, israel; 3 primary care, private clinic, nabulus, palestine (via israel); 4 primary care, private clinic, ramalla, palestine (via israel); 5 primary care, private clinic, bethlehem, palestine (via israel); 6 jerusalem-hashfela dis., maccabi healthcare services, modiin, israel background: type1 pilus proteins are potential targets of future protein-based vaccines. yet, piliated strains were reported to decrease soon after pcv implementation. here we report a differential effect of pcv7 on carriage of non-piliated strains in a population-based study comparing a vaccinated vs. nonvaccinated population before and after pcv7 implementation. methods: consecutive annual cross-sectional surveys of sp carriage among children <3.5y were performed from 2009 (pre-pcv) to 2011 (post-pcv). palestinian children from a) ramalla, nabulus and bethlehem living under palestinian-authority's (pa) health policy, where pcv7 was not yet implemented and b) east-jerusalem (ej) where pcv7 was implemented (7/2009). clinical data were collected and sp/serotype/antibiotic susceptibilities were identified. presence of type1 pilus was determined by rrgc pcr. results: a total of 983 and 1772 children from ej and pa were screened. sp carriage was ~30% and was not affected by vaccination policy (after adjusting for age/region/year/recent antibiotic treatment). carriage of vt7 strains decreased significantly following pcv implementation (52% to 22%, p < 0.001). the decrease was mainly of nonpiliated vt strains, while piliated-vt strains did not decrease. piliated strains consisted 50% of all vt in 2009 vs. 80% in 2011, (p < 0.001). in addition, piliated non-vt13 strains emerged, from 1% (2009) to 25% (2011). the emerging nonvt13 piliated serotypes were 35b, 11a and 19b. pcv implementation was associated with increased carriage of piliated strains (adjusted odds ratio (aor): 2.6, 95%ci: 1.2-6.4). conclusion: the differential effect of pcv7 on non-piliated strains suggests that type1 pilus confers an intrinsic advantage for colonization and that pilus proteins are attractive vaccine targets. background and aims: in april 2010 the 7-valent pneumococcal conjugate vaccine (pcv7) was replaced by the 13-valent pcv. we investigated pneumococcal carriage in children eligible for pcv7 or pcv13 and their household contacts. methods: eligible families in hertfordshire and gloucester were identified and a nasopharyngeal swab obtained from consenting household members between july 2012 and march 2013. samples were cultured for streptococcus pneumoniae and serotyped by standard methods. for each serotype the ratio of its prevalence in invasive pneumococcal disease (ipd) to its carriage prevalence (case:carrier ratio, ccr) was calculated. results were compared with previous carriage studies in 2001/2 and 2008/9, before and after pcv7 introduction. results: 217 households were included. among <5 year-olds 47.7% (95% confidence interval 41.8-53.5) were carrying a pneumococcus compared with 51.0% in 2008/9 and 48.4% in 2001/2. the odds of carrying a pcv7 serotype was significantly reduced in 2008/9 and 2012/3 relative to 2001/2, while the odds of carrying any of the extra six pcv13 serotypes increased after pcv7 introduction but declined significantly after pcv13 introduction. the ccrs for the frequently-carried serotypes were relatively low, with the highest ccr observed for serotypes 7f, 19a, 3, 8, and 33f . across the three carriage studies, ccr estimates were stable for nearly all serotypes. conclusion: carriage of pcv13-only serotypes has rapidly reduced post-pcv13 introduction in both vaccinated and unvaccinated individuals with a continued decline in transmission of pcv7 serotypes. carriage rates in children remain unchanged, but the low ccrs of replacing serotypes should further reduce overall ipd across all age groups. background: meningitis continues to be associated with high mortality. in papua new guinea (png), streptococcus pneumoniae is now the most important cause of meningitis. where introduced, pneumococcal conjugate vaccines have helped to reduce disease burden but due to limited serotype coverage these vaccines do not protect against all pneumococcal lineages and may contribute to serotype replacement. serotype-independent protein-based vaccines incorporating several pneumococcal proteins present in all pneumococcal strains may be more favourable. methods: focusing on serotypes 2, 5 and 7 (common causes of meningitis in png), we characterised 40 invasive pneumococcal isolates from meningitis cases and 22 carriage isolates from png to identify dominating clonal complexes. results: multi-locus sequence typing (mlst) from 3 main serotype clusters revealed sequence types (sts) common in eastern europe, suggesting importation of these lineages. png pspa types correlated with the serotypes. serotype 2, 5 and 7b/c were associated with the pspa haplotypes 7, 1 and 11, respectively. no sequence variation within the pspa sequences of a clonal complex was observed. broad cross-reactivity was observed when 9 pneumococcal isolates from png with different pspa types were tested for binding with monoclonal antibodies specific for semiconserved pspa sequences. 9/9 and 7/9 strains were recognized by pspa and stkp-specific monoclonal antibodies, respectively. conclusion: we have identified dominating clonal complexes with limited genetic diversity within the pneumococcal population of colonisation and disease isolates in the eastern highlands of png. more studies incorporating mlst and pspa typing are needed to shed light on the country-wide diversity of pneumococci in png. background and aims: while there are several studies addressing pneumococcal colonization of the upper respiratory tract in young children, less is known about carriage in the elderly. this work aimed to evaluate the patterns of streptococcus pneumoniae carriage in adults over 60 years of age in portugal. methods: between april 2010 and december 2012, nasopharyngeal and oropharyngeal swabs were obtained from adults over 60 years of age, living in an urban area (n = 1,945) or in a rural area (n = 1,416). pneumococci were isolated by routine procedures, serotyped, genotyped (mlst), and antibiotyped. associations between carriage, socio-demographic and clinical data were analyzed by multiple logistic regression. results: carriage was higher in the rural area than in the urban area (3.4% vs 1.4%, respectively, p < 0.001). overall, the carriage rate was 2.3%. multiple logistic regression identified as risk factors for pneumococcal carriage, living in the rural area (or=2.0, 95%ci:1.2-3.5), being at a nursing home (or=2.0, 95%ci:1.1-3.6) and smoking (or=4.4, 95%ci:1.9-9.2). in total, 77 pneumococci were isolated, representing 26 serotypes and 40 sequence types (sts). the most prevalent serotypes were 19a (13.0%), 6c (9.1%), 22f (9.1%), 23a (9.1%), and 35f (7.8%). most isolates (93.5%) had previously described sts. non-susceptibility to penicillin was found in 11.7% of the isolates and resistance to macrolides in 19.5%; 15.6% of all isolates were multidrug resistant. conclusion: pneumococcal carriage in the elderly is low and there is a high serotype and genotype diversity. living in the rural area, being at a nursing home and smoking were risk factors for pneumococcal colonization. background: in developing countries like india, hiv infected children access antiretrovirals late, when their cd4 count drops <350. hiv viral load, not cd4 count may be more accurate in predicting risk of np colonization and ipd in these children. objective: to determine the association between hiv viral load and pneumococcal colonization. methods: np swabs were taken at 8 week intervals 4 times across 6 months from hiv infected children in a pneumonia prevention study. information on age, art status, cd4 count, viral load, and weight for age z-score was collected in pneumococcal vaccine naïve hiv infected children. results: out of 47 children, 16 had no pneumococcal colonization over six months (group-1), 31 (66%) had at least one episode of colonization (group-2). 9/47 (19%) of children were in art, 5 in group-1 and 4 in group-2. the mean age of children in both groups was 5. mean cd4 count was comparable 684 vs 703 in group-1 and group-2 (p = 0.89). mean waz-score was -2.9 vs. -3.09 (p = 0.71). children in group-2 had a mean viral load of 153,515 almost twice that of group-1, 80,050. two children had viral loads of 1 x10 6 in group-2 whereas the highest viral load in group-1 was 4x10 5 . conclusion: hiv infected children not qualifying for art but with high viral loads have increased risk of colonization with pneumococcus. access to pneumococcal vaccines for this high risk group in developing countries is important. background: hiv infected adults and children are at disproportionately high risk of invasive disease from streptococcus pneumoniae. pneumococcal conjugate vaccines (pcvs) are effective in hiv infected individuals but are not yet available in government programs. objective: to determine baseline carriage of streptococcus pneumoniae in vaccine naïve mother child pairs affected by hiv, and dynamics of transmission over the course of six months. methods: we are conducting an interventional study on the effect of the pneumococcal conjugate vaccine, pcv-13, and the hib conjugate vaccine on nasopharyngeal carriage, of these vaccine preventable pathogens, in families affected by hiv, in west bengal. four nasopharyngeal samples were collected from pneumococcal vaccine naïve, hiv infected children ages 2-15 years, and their mothers, at two month intervals over the course of six months. calcium alginate swabs were collected and placed in stgg media and processed in the microbiology laboratory using standardized protocols. isppd-9 / pneumonia 2014 mar 9-13; 3:1-286 results: 404 nasopharyngeal swabs have been collected from hiv infected children and 375 swabs from their mothers. in children 100/404 (24.7%) swabs grew pneumococcus and 19/375 (5%) swabs in adults were positive for pneumococcus. fifty children have completed six months of follow-up. in this group, 35/50 (70%) children had at least one out of four swabs grow pneumococcus over the course of six months. initial results show that in 31/50 children (62%), pneumococcal carriage disappeared within 2 months. in 4/50 (8%), carriage persisted for > 2 months, and in one case for > 4 months. background: pneumococcal colonization in nasopharynx (np) is known to precede pneumonia, and np density is suggested to correlate with disease severity. we studied the correlation between pneumococcal dna density in np, and anti-cps immunoglobulins (ig) levels to homologous serotypes, in adult community-acquired pneumonia (cap). serotypes were grouped according to case fatality rates (cfr; weinberger, 2004) . methods: in a prospective study of 235 adult cap patients, admission sera were collected, and culture on blood, sputum, np-aspirate (npa) and np-swab were performed. quantitative pcr on npa for pneumococcal dna (spn9802) densities (log-10 dna copies/ml) were performed. anti-cps ig-titres to homologous serotypes were measured by optical density and compared (%od) with pooled sera from post-vaccinated adults. the vaccination frequency was very low. results: of 68 cap patients with np pneumococcal dna, 47 patients with culture-positive pneumococcal aetiology had admission ig-titres determined. no overall correlation between colonization rates and ig-titres was noted (table). in cases with medium/low cfr serotypes, colonization densities were significantly higher in those with high, than low, ig-titres. no difference in duration from onset of illness to admission was noted between serotype categories. conclusion: in pneumococcal cap, high colonization densities and high admission ig-titres correlated well for serotypes associated with medium/low cfr. thus, a significant bacterial load in np may be necessary for pneumonia development, in patients colonized with these serotypes. mean log-10 copies/ml (standard deviation) p value background: optochin (op) and bile solubility tests (bst) have good specificity when applied to isolates from clinical specimens which have a high pretest probability of identification of pneumococci. the same tests may have different analytical specificity for nasopharyngeal isolates as the upper respiratory tract is a reservoir for both pneumococci and non-pneumococcal streptococci. non-pneumococcal confounding of op and bst is known. objectives: we applied lyta pcr and sequencing to nasophayngeal isolates of 'pneumococci' as reference methods for identification from nasophayngeal swab cultures. methods: nasopharyngeal swabs were collected from well children 3 months to 5y of age from karachi, pakistan as part of a pneumococcal carriage study to evaluate pcv-10 impact. swabs were collected in stgg media and subcultured on sheep blood agar (sba). isolates with suggestive colony morphology (draughtsman or mucoid, alpha hemolytic) were selected for op and bst. 40 pneumococcal isolates were selected for study on the basis of difficulty in identification (small op zones or discrepancy in op and bst). pneumococcal autolysin gene lyta was detected isppd-9 / pneumonia 2014 mar 9-13; 3:1-286 by pcr, followed by sequencing (macrogen inc.) as reference identification methods. blast analysis by appending sequences to ncbi database using clustalw2 is ongoing. results: of 40 isolates selected, 12 isolates had discrepant op and bst results (op resistant but bile-soluble). lyta was detected in 29 of 40 isolates (72.5%). results from sequencing of the pcr product are under evaluation by blast analysis. conclusion: results obtained indicate that suggestive colony morphology, op and bst used together may still confound pneumococcal identification from nasopharyngeal samples. background: due to the significantly increased risk of invasive disease, hiv infected adults and children are an important group to access pneumococcal conjugate vaccines. nasopharyngeal colonization precedes invasive disease; determining the pneumococcal serotypes colonizing the nasopharynx of hiv infected individuals is important in the design of new pneumococcal vaccines. objective: to determine pneumococcal serotypes from nasopharyngeal samples in vaccine naïve hiv infected mothers and children. we are conducting an interventional study looking at the effect of the pneumococcal conjugate vaccine, pcv-13, on nasopharyngeal carriage in families affected by hiv in west bengal. here we report baseline serotypes from 43 isolates taken from vaccine naive mothers and children infected with hiv. samples were collected using calcium alginate swabs and stored in stgg media. these were processed using standard microbiologic methods and pneumococcal isolates were then serotyped at a reference laboratory. results: serotype results for 43 isolates from 35 children and 8 mothers are available. 4 out of 8 strains in mothers are vaccine represented strains-3, 19a, 4. three strains in mothers are not vaccine represented-13, 6c, 34. in children 12 out of 35 serotypes are vaccine represented--14, 19a, 19f, 3, 6a, 6b, 9v. the most common serotype was 19a (5/43) and 13 (6/43). 16/43 (37%) of isolates are represented in pcv-13. serotypes that are not vaccine represented include10a, 11, 11a, 12, 15a, 15c, 18a, 20, 24f, 29, 31, 36, 38 (one each) , 34 (2 isolates), 6c (3 isolates), and 13 (6 isolates). background and aims: insight into bacterial colonization patterns of potential pathogens and commensals in the nasopharynx might elucidate healthy and/or susceptible conditions for development of respiratory disease. we therefore studied the dynamics of microbiota profiles over time in young children. methods: we characterized the consecutive nasopharyngeal microbiota profiles of 60 healthy children at the ages of 6 weeks, and 6, 12 and 24 months by 16s gs-flx-titanium-pyrosequencing, and analyzed the consecutive profiles by spectral co-regularized clustering and biomarker detection algorithms. results: overall, we identified 6 distinct microbiota profiles represented by the dominant genera moraxella, haemophilus, streptococcus, or staphylococcus, a combination of dolosigranulum and corynebacterium, plus cluster-specific low abundant biomarker bacteria. we observed specific patterns of change over time, with more stable patterns marked by early presence and high abundance of the moraxella and corynebacterium/ dolosigranulum clusters, and less stable profiles marked by high abundance of the haemophilus or streptococcusdominated clusters. the streptococcus-dominated profile was additionally shaped by high abundance of prevotella, gemella, bacteroidetes (unclassified), acinetobacter and porphyromonas, and observed in on average 15% of the samples, decreasing from 20% in 6-week-olds to 7% in 24-months-olds. together with the haemophilus-dominated profile, the streptococcus-dominated profile was associated with increased frequency of parent-reported respiratory infections. the current study enabled us to gain insight in the dynamic nature of nasoparyngeal microbiota in infants. our results suggest that the composition of early-life microbiota is associated with long-term stability and may predict susceptibility to disease. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 background and aims: the fiji pneumococcal project (fipp) evaluated a reduced dose pcv7 primary series in infancy, followed by the 23vpps at 12 months of age. children receiving 0 or 1 pcv7 dose were given a catch-up dose at 2 years of age. immune hyporesponsiveness was observed in children aged 18 months who were given 23vpps, compared with those who were not. here we assess the long-term impact of 23vpps vaccination on carriage rates and densities of pneumococci and other common bacterial species. we also compared differences in carriage of pneumococcal serotypes, and examined differences between the two main ethnic populations. methods: nasopharyngeal swabs (n = 194) were collected from a subset of healthy fipp participants now 5-7 years old. pneumococci were identified by standard culture-based methods and serotyped by latex agglutination/ quellung. carriage rates and densities of pneumococci, haemophilus influenzae, staphylococcus aureus and moraxella catarrhalis were determined by quantitative pcr. results: preliminary analysis found there were no differences in pneumococcal carriage rates (40 vs. 52%) or vaccine-type carriage (20 vs. 17%) for 23vpps recipients compared with non-recipients. similar results were found for pneumococcal densities. analysis of the impact of 23vpps vaccination on carriage of other species is ongoing. indigenous fijian children had significantly higher carriage rates of pneumococci (57 vs. 14%), h. influenzae (63 vs. 24%) and m. catarrhalis (86 vs. 41%), and lower carriage of s. aureus (23 vs. 41%) compared to indo-fijian children. conclusion: initial analysis shows 23vpps receipt at 12 months has no long-term impact on pneumococcal carriage rate or density. background: in july 2011 the expanded program of immunization (epi) program of the gambia replaced pvc7 with pcv13. we used this opportunity to determine the impact of replacing pcv7 with pcv13 on the prevalence of pneumococcal nasopharyngeal carriage. methods: healthy gambian infants who had received 3 doses of a pcv were recruited. nasopharyngeal swabs were collected from infants and their mothers during cross-sectional surveys undertaken in april-june 2011 (pcv7 group) and in april-june 2012 (pcv13 group). pneumococci were isolated following standardized methods and serotyped by latex agglutination. results: 339 and 350 mother/infants pairs were recruited in the pcv7 and pcv13 vaccinated groups respectively. the overall pneumococcal carriage rate in infants was similar in the two groups (85.8% and 84.3%, p = 0.902). carriage of pneumococci of pcv7 and pcv13 vaccine types (vt) was lower in the pcv13 than in the pcv7 vaccine recipients [4.9% versus 9.4% (p = 0.025) for pcv7-vt and 18.0% versus 33.3.0% (p < 0.001) for pcv13-vt, respectively]. the prevalence of carriage of the six additional serotypes included in the pcv13 and not in the pcv7 vaccine was 13.4% in the pcv13 and 23.9% in the pcv7 recipients respectively (p = 0.001). however, there was a significant increase in non-typable pneumococci among pcv13 compared to pcv7 vaccinated children (0.6% versus 6.6%, p = 0.005). prevalence of carriage in mothers was similar in both surveys for all study endpoints. conclusion: replacing pcv7 by pcv13 decreased the prevalence of pneumococcal carriage of vaccine type pneumococci in vaccinated gambian infants one year after introduction of pcv13 but not in their mothers background: 7-valent pneumococcal vaccine (pcv7) was implemented for infants in the dutch national immunization program (nip) in 2006, and replaced by pcv10 in 2011. eradication of vaccine serotype from carriage and emergence of non-vaccine serotypes was observed, with particularly high peaks in serotype 19a carriage 4.5 years after pcv7-implementation (spijkerman,2012) . this suggests a vaccine-induced disbalance among serotypes circulating in children. methods: we performed a cross-sectional study 6.5 years after pcv-implementation (2012/13), studying pneumococcal carriage in 330 pcv10-vaccinated 11-month-old children and 330 pcv7-vaccinated 24-month-old children. present results were compared with carriage data in identical age groups from before, and from 3 and 4.5 years after pcv7-implementation (n ≈ 330/group). bootstrap calculations were performed to investigate changes in diversity of serotypes carried (hanage,2010) . results: in 2012/13, carriage of vaccine-type pneumococci had almost completely disappeared, but overall pneumococcal carriage was similar to the pre-vaccination carriage rates. after 6.5 years, distribution of nonvaccine serotypes was more similar to pre-pcv7 data, indicating a more evenly distributed ranking, and no serotype was exceeding a carriage rate of 10%. between 2010 and 2012/13, the high carriage rates of serotype 19a had declined from 12% to 9% in 11-month-old children and from 14% to 8% in 24-month-old children. conclusion: our data suggest a new balance in non-vaccine serotype carriage emerging in vaccinated children 6.5 years after the first pcv implementation in the dutch nip. carriage of serotype 19a declined more prominently in pcv7 vaccinated children and not in pcv10 vaccinated children, and is more likely to a newly achieved balance rather than cross-protection by pcv10. center for american indian health, johns hopkins bloomberg school for public health, baltimore md, usa; 2 emory university, rollins school of public health, atlanta ga, usa background: pneumococcal conjugate vaccines (pcvs) reduce vaccine serotype pneumococcal nasopharyngeal (np) colonization prevalence and density in vaccinated children. establishing the relationship between the child's colonization density and the colonization status of close contacts may partly explain the mechanism of pcv indirect effects. methods: american indian households participated in a continuous, cross-sectional np colonization study (january 2010-april 2012). an np specimen was collected from each participant and pneumococcus isolated by culture. a subset of np specimens from households comprised of a child <8 years of age, mother and father were selected for subsequent testing by lyta quantitative pcr. pneumcococcal colonization of >105 colony forming units (cfu)/ml was categorized as dense. multilevel logistic regression analysis measured odds of parental colonization by child's density accounting for intra-household correlation. results: 223 families were identified for this analysis (n = 669 participants); 28% of participants (n = 189) were positive for pneumococcus. children were more frequently colonized than their parents (53% vs. 16%; p<0.01) and more densely colonized than their parents (10 5.4 vs. 10 4.6 cfu/ml; p < 0.01). adjusting for the study site where subjects were sampled and differences between american indian tribes, parents living with a densely colonized child had a greater odds of being colonized (odds ratio (or): 2.6, p < 0.01); mothers were not more likely to be colonized than fathers (or: 1.09, p=0.3). conclusion: dense pneumococccal colonization of children is associated with a greater likelihood of parental colonization. reducing strain-specific colonization density among children through pcv could reduce colonization and risk of disease in family members and other close contacts. background and aims: nasopharyngeal (np) carriage of streptococcus pneumonia is an important risk factor for invasive pneumococcal disease. this study characterizes np carriage rates and loads of s. pneumoniae in the first 12 weeks of life in a rural cohort in the gambia. methods: 480 np swabs were collected from 120 infants at monthly intervals from within minutes of birth to 3 months. quantitative pcr targeting the autolysin gene (lyta) was performed on the dna extracts from the swabs. results: preliminary results from 76 newborns showed a significant increase in pneumococcal carriage (p < 0.01) with 32 % at birth, 59% at one month, 68% at two months and 85% at three months. the mean pneumococcal load also increased significantly from 1.09x10 1 cells/ml at birth, increasing to 1.56x10 3 cells/ml, 1.72x10 3 cells/ ml and 1.80x10 4 cells/ml at one, two and three months respectively, (x 2 (3) = 84.103, p < 0.01). logistic regression analysis revealed that birth weight >3.1kg (odds ratio (or) 1.72, 95%ci 1.04-2.84, p = 0.03), health centre rather than hospital births (or 0.38, 95%ci 0.18-0.78, p < 0.01), and having 1-3 older siblings (or 2.1, 95%ci 1.07-4.16, p = 0.03) all significantly increased pneumococcal carriage. however gender had no significant effect (or 1.21, 95%ci 0.75-1.94, p=0.44). after adjusting for birth weight, place of birth and number of siblings, only place of birth and having 1-3 siblings remain significant, (or 0.38, 95%ci 0.18-0.80, p = 0.01) and (or 2.13, 95%ci 1.00-4.56, p = 0.05) respectively. conclusion: both pneumococcal carriage and load increase significantly within weeks of birth which could implicate susceptibility to neonatal infections. this data is important for future studies looking at natural pneumococcal protection dynamics in neonates and young infants. bacterial pneumonia due to streptococcus pneumoniae remains the leading cause of hospital admission in hiv infected individuals. pneumococcal conjugate vaccine (pcv) are effective in hiv infected individuals for the prevention of invasive disease from this pathogens; however, it is not yet available through government programs in india. studies looking at the impact of these vaccines on nasopharyngeal colonization in families affected by hiv can potentially inform health policy in india. we describe the design for our ongoing study looking at the impact of pneumococcal conjugate vaccine in families affected by hiv being carried out in west bengal. organizing a community based clinical study depends much on cultural expectations and socio-political scenario of the sites involved. we describe our experience with different tools used in the consent and follow up process in our study u. devi 1 , v. malik 1 , j. mahanta 1 1 bacteriology, regional medical research centre north east region(icmr), dibrugarh, india background & aims: sequence types prevailing in northeast india are not known. we present sequence types currently completed. methods: dna was extracted using phenol-chloroform method. primer pairs used for amplifying the seven housekeeping genes namely aroe, gdh, gki, recp, spi, xpt and ddl were obtained from those listed in mlst website (http://spn.mlst.net/). amplicons were sequenced on both sides (commercially) and sequences were edited using genious pro v 5.4.6. the core sequences were uploaded to pneumococcus database for allele and st identification. results: table below isppd-9 / pneumonia 2014 mar 9-13;3:1-286 background and aims: although indirect effects of pneumococcal conjugate vaccination (pcv) have been welldocumented in adults, there are few data regarding the herd effects in young infants. this age group are too young to be vaccinated with pcv but are at high risk for severe bacterial infections. fiji introduced pcv10 in 2012. the impact of 10valent pneumococcal conjugate vaccine (pcv10) introduction on nasopharyngeal carriage is being monitored as part of the new vaccine evaluation project. the indirect effects of pcv10 introduction will be measured in young infants (5-8 wk) through annual carriage surveys (pre-and post-pcv10 introduction). methods: nasopharyngeal swabs were collected from 500 healthy infants aged 5-8 wk before, and one year after, pcv10 introduction. swabs were stored in stgg and frozen at -80°c until analysis. streptococcus pneumoniae, staphylococcus aureus, and haemophilus influenzae were detected using quantitative pcr, and pneumococcal molecular serotyping performed by microarray. results: preliminary analysis of the pre-pcv10 swabs found carriage rates of 30% for s. pneumoniae, 23% for s. aureus, and 27% for h. influenzae. serotyping by microarray found that 40% of pneumococci identified were pcv10 serotypes. we are currently examining swabs taken one year post-pcv10 introduction to investigate potential herd immune effects on nasopharyngeal carriage in this age group. conclusion: our studies in fiji provide a unique opportunity for investigating pcv impact in young, unvaccinated infants. data will help to evaluate potential herd effects on carriage of pneumococcal serotypes and other pathogens in young infants in a low-income country setting. background and aims: detection of multiple serotypes of streptococcus pneumoniae is necessary to understand the effects of pneumococcal conjugate vaccination in carriage studies. this study aimed to compare serotyping by latex agglutination and microarray within the context of a 7-valent pneumococcal conjugate vaccine (pcv-7) trial. methods: nasopharyngeal swabs (nps) were collected from 22 newborns at regular intervals up to 12 months of age. all children received at least three doses of pcv-7. s. pneumoniae was identified by conventional microbiology techniques. s. pneumoniae isolates were serotyped using the latex agglutination and microarray sweep method. results: microarray detected 2, 3 and 4 serotypes in 28%, 11% and 3% of 157 nps respectively, compared to 7%, 1% and 0% by latex agglutination (p < 0.001). although a common serogroup was detected in 94% of nps by both methods, latex agglutination was more likely to miss co-carried low abundance serotypes. for instance, in one nps both methods detected serotype 6a/b, however, latex agglutination did not detect serotypes 37, 16f and an nt which had relative abundance of 8%, 6% and 3% by microarray respectively. in four other nps collected from the same subject at consecutive timepoints, serotype 16f was present but appeared to be cleared from carriage by latex agglutination. the predominant serotypes detected by latex agglutination were 19a(10%) and 13(9%) while by microarray they were 13(8%) and 19a(7%). conclusion: we report improved detection of multiple serotypes using microarray method over latex agglutination. highly sensitive molecular techniques should be employed in post-vaccine implementation carriage studies. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 background and aims: monitoring the dynamics of pneumococcal carriage makes it possible to evaluate the epidemiological characteristics of streptococcus pneumoniae disease and the theoretical coverage offered by pneumococcal vaccines. it has been demonstrated that the nasopharyngeal (np) sampling of respiratory secretions is superior to oropharyngeal (op) sampling for identifying pneumococci carried by younger children, but adult data are conflicting and there are no studies of adolescents. methods: in order to compare the efficiency of op and np sampling in identifying and quantifying s. pneumoniae carriage in healthy adolescents, two swab samples were obtained from 530 adolescents aged 15-19 years, the first taken from the posterior pharyngeal wall through the mouth (op) and the second through the nose (np). bacterial genomic dna was tested for the autolysin-a (lyta) and wzg (cpsa) genes of s. pneumoniae in order to evaluate pneumococcal carrier status. positive cases were serotyped. results: s. pneumoniae was identified in 35.8% of the op swabs and 3.5% of the np swabs (p < 0.0001). the serotypes included in the 13-valent pneumococcal conjugate vaccine (pcv13) were found in all but two op samples (98.9%) and only 64.7% of the np samples (p <0.0001). the most frequently identified pcv13 serotype in both groups was 19f, followed by serotypes 5 and 9v. conclusion: op sampling appeared significantly more effective than np sampling in identifying and characterising pneumococcal carrier status in adolescents. this suggests that op sampling should be used when evaluating the dynamics of pneumococcal carriage among adolescents and the theoretical coverage offered by pcv13. background: hiv-infected children are at high risk for pneumococcal disease. antimicrobial-resistant streptococcus pneumoniae can lead to poor clinical outcomes and increased cost of medical care. we examined antimicrobial susceptibility among nasopharyngeal pneumococcal isolates obtained from hiv-infected children in mozambique before the introduction of the 10-valent pneumococcal conjugate vaccine (pcv10) in april 2013. methods: between october 2012 and march 2013, we enrolled hiv-infected children age <5 years presenting for routine care at seven hiv clinics in 3 sites, including maputo (urban-south), nampula (urban-north), and manhi?a (rural-south). a single nasopharyngeal swab was obtained and cultured following broth enrichment. susceptibility to commonly-used antibiotics was evaluated for pneumococcal isolates using broth dilution susceptibility tests according to definitions from the 2013 clinical laboratory standard institute guidelines. serotyping was performed using quellung reaction. results: a total of 343 isolates were obtained from 336 hiv-infected children. overall, 304 (88.6%) isolates were resistant to co-trimoxazole, 56 (16.3%) to erythromycin, 23 (6.7%) to chloramphenicol, and 1 (0.3%) to amoxicillin. none were resistant to ceftriaxone or penicillin. three isolates had intermediate resistance to penicillin (mic=4); all 3 were pcv10 serotypes. the proportion of resistant isolates that were serotypes covered by pcv10 for cotrimoxazole, erythromycin, chloramphenicol and amoxicillin was 51.6%, 97.6%, 72.7% and 100% respectively. conclusion: given the low prevalence of β-lactam resistance, those agents continue to be useful first-line antibiotics for treating pneumococcal disease in hiv-infected children in mozambique. pcv10 introduction will likely lead to an important decline in the circulation of resistant pneumococci in this population. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 background and aims: we have previously reported that experimental human carriage was an immunizing and protective event. now, in volunteers inoculated with pneumococcus, we characterize the kinetics of circulating antigen-specific plasma and memory b cell populations in blood and further characterize their phenotype in blood and in the lung (bronchoalveolar lavage (bal)). method: healthy volunteers were inoculated with a 6b pneumococcal strain. blood samples were collected at days 0, 7, 14 and 35 post inoculation for elispot analyses. flow cytometric analysis were performed using b cells purified with anti-cd19 microbeads. cells were labeled for surface phenotype and cellular antigen-specificity determined using biotinylated capsular polysaccharide (ps) 6b labeled with anti-biotin-fitc. results: we evaluated igg and iga secreting plasma and memory b cells to ps6b, whole cell pneumococcus, pspa, pspc and flu in 96 samples collected from 25 volunteers (15 carriers and 10 non-carriers). preliminary analysis revealed a transient increase in the numbers of circulating plasma cells peaking at day 7 for ps6b and at day 14 for pspa. carriage increased b1a but not b1b cells in blood at day 21. we are now analyzing the impact of carriage upon b cell populations in the lung. conclusion: while b1a cells produce serum antibody, b1b cells are involved in long-term protection against bacterial pathogens such as pneumococcus. our results suggest that although pneumococcal nasal exposure is immunizing and elicits antibodies, this response may be restricted to the b1a and not the b1b subset. acknowledgements: the bmgf gce and mrc-fapesp background and aims: respiratory viral infections may affect bacterial colonisation of the human nasopharynx and efficiency of transmission. the particular interaction between influenza and streptococcus pneumoniae (sp) has received particular attention. we investigated whether trivalent live attenuated influenza vaccine (laiv) influences nasal carriage and density of sp in healthy children. methods: 151 children were recruited into a randomised controlled stepped-wedge study to start laiv (2 doses, one month apart) either at an initial study visit or one month later, with nasopharyngeal swabs taken 7 and 28 days after each dose, allowing comparisons both between vaccinated and unvaccinated children and pre-and postvaccine. swabs, stored in stgg, were analysed using real-time pcr (rt-pcr) for lyta. results: at 7 days post-vaccination s.pneumoniae was detected (ct<35) in 69.2% children and 66.2% of controls (p = 0.706) with similar density in the two groups at both day 7 and 28 (p =0.50 and 0.46, respectively). likewise, when values pre-and both 7 and 28 days post-first vaccine dose were compared in all children studied, no differences in colonisation rates were shown. conclusion: laiv did not cause detectable changes in rates or density of nasal carriage with pneumococcus, measured by pcr, in this population, among whom colonisation rates were high. it seems possible that while infection with wild type influenza and other respiratory viral infections may affect bacterial colonisation, the largely sub-clinical infection caused by attenuated vaccine viruses do not. isppd-9 / pneumonia 2014 mar 9-13;3:1-286 b. morales-aza 1 , p. sikora-liszka 1 , l. januario 2 , f. rodrigues 2 , a. finn 1 1 cellular and molecular medicine, university of bristol, bristol, united kingdom; 2 infectious diseases unit & emergency service, hospital pediatrico de coimbra, coimbra, portugal background and aims: studies comparing rates and/or densities of streptococcus pneumoniae (sp) colonisation in children with respiratory infections and in health are conflicting. we tested the hypothesis that they are unchanged in acute otitis media with spontaneous otorrhoea (aomso) . methods: in february-march 2011 we swabbed 516 children attending nurseries(an) and 104 with aomso. nasopharyngeal swabs were stored at -80°c in stgg broth and 50µl cultured using standard techniques. sp, haemophilus influenzae (hi) and moraxella catarrhalis (mc) were identified and densities assessed by scoring numbers of colonies (0-5 where 5 is >100). results: 80% of the children with aomso attended nurseries. by univariate analysis, rates of colonisation and mean densities did not differ apart from mc density. by multivariate analysis (adjusting for age), colonisation rates and densities for both hi and mc were lower in aomso. the mean total number of bacterial species identified was similar in the two groups (1.7vs1.8; p =0.674). conclusion: children with aomso did not have higher rates or densities of sp but significantly lower densities of both hi and mc were seen. this relative imbalance between species in otitis may point to the ecological conditions predisposing to disease. pneumococcal conjugate vaccines (pcv) were implemented from 2006 in the uk. colonisation precedes disease therefore carriage can be an early indicator of epidemiological changes. circulating pneumococci were highly characterised to assess vaccine impact and generate data informative to future vaccine policy and formulation. nasopharyngeal swabs were collected from children of four years of age and under each winter from 2006/7 to 2010/11. pneumococci were isolated using conventional microbiology. next generation sequencing was utilised to comprehensively type isolates and determine sequence variation in numerous genes important to pneumococcal typing and virulence using a single method. carriage remained stable. conversely pcv7 and pcv13-unique vaccine serotypes (vt) significantly decreased with concomitant increases in non-vaccine serotypes (nvt). significant decreases for vt 6a, 6b, 19f and 23f and increases for nvt 21, 23b, 33f and 35f were detected. significant changes were observed for associated genotypes. novel non-synonymous sequence variation was observed to be common in capsular defining genes within serotypes, particularly for serogroups 19 and 6. newly described serotypes 6d and 6g sequences were detected. ten novel pneumolysin protein alleles were observed. pneumolysin sequences associated with non-pneumococcal streptococci were detected in pneumococci and vice-versa. serotype replacement resulted in increased nvt colonisation, which could progress to disease cases. future control of pneumococcal disease through vaccination will require response to replacement, targeting prevalent replacing serotypes or broader acting pneumococcal vaccines. however strategies should account for potential target diversity and consequent specificity. whole genomes offer an invaluable resource for documenting sequence variability of genes throughout the genome. background and aims: the 13-valent pneumococcal conjugate vaccine (pcv13) replaced the 7-valent pneumococcal conjugate vaccine (pcv7) in the alaska childhood immunization schedule in 2010. we assessed the indirect effect of pcv13 on pneumococcal colonization and disease in alaskan adults. methods: to determine pneumococcal colonization, we recruited a convenience sample of residents of all ages from 8 rural villages and children aged <5 years at 2 urban pediatric clinics annually during 2008-2012; we determined their pcv13 vaccination status and obtained nasopharyngeal swab specimens. pneumococci were identified/serotyped by standard laboratory methods. we identified invasive pneumococcal disease (ipd) cases, defined as streptococcus pneumoniae cultured from a sterile site, through conducting statewide surveillance. results: during 2008-2012, we recruited 4,310 children aged <5 years (89% with >1 dose pcv13 in 2011-2012) and 8,336 adults aged >18 years. overall pneumococcal carriage among rural and urban children <5 years and adults >18 years remained stable (mean/year: 66%, 35%, and 14%, respectively). from 2008 through 2012, pcv13 serotype carriage declined among children <5 years (13%-4%, p-value for trend <0.01) and adults >18 years (4%-1%, p <0.01). during 2011-2012, 6/943 (<1%) adults >50 years were colonized by pcv13 serotypes. pcv13-ipd rates among alaskan adults >18 years declined from 8.2/100,000 persons in 2008 to 5.2/100,000 in 2012 (p-value <0.01). conclusion: pediatric pcv13 vaccination provided indirect protection against vaccine-type colonization and ipd to unvaccinated adults. potential benefits to adults aged >50 years from routine pcv13 vaccination appears to be small because pcv13 serotype carriage is low and pcv13-ipd rates are declining due to indirect protection. exposure to streptococcus pneumoniae in the nasopharynx is thought to eventually lead to protective immunity. the role of anti-protein serum antibodies in mediating this immunity is unclear but vaccines incorporating pneumococcal proteins are being developed. we investigated the immunogenicity of individual proteins following pneumococcal colonisation in kenyan and burmese infants to establish whether antigen immunodominance is similar in diverse communities. nasopharyngeal swabs to detect pneumococcal colonisation and serum samples at various time points were obtained in two separate studies from burmese refugee and kenyan infants. sera was analysed for igg to 27 pneumococcal proteins using multiplex electrochemiluminescence and titres compared between the cohorts. prevalence of nasopharyngeal colonisation was compared at 18 and 36 weeks. statistical analysis was performed on log-transformed data using r. significance was defined of p-value less than 0.05. cross sectional prevalence of carriage at 18 weeks was 83% and 81% and at 36 weeks was 81% and 86% in the kenyan and burmese cohorts, respectively. when comparing igg to individual proteins, the top 8 immuno-dominant proteins were identical between the two cohorts. burmese infants had significantly higher birth igg titres than the kenyan cohort to 22/27 antigens. however the ratio of 9m:birth igg titres were significantly higher for the kenyan cohort whose titres at 9 months were higher to all antigens (15/27 significantly). despite differences in the kinetics of igg production after birth, similar antigens were immunodominant in these two diverse populations. this data suggests that vaccines based on protein antigens are likely to have universal value. the immune response to colonisation is important in the pathogenesis of streptococcus pneumoniae but the role of anti-protein antibodies is unclear. to assess this we studied the relationship between anti-protein serum igg, salivary iga (sal-iga) and pneumococcal colonisation among american indian children and adults. adults and children (mean age 4y3m) had nasopharyngeal swabs taken monthly for 6 months to detect colonisation and saliva and serum collected at months 0 and 6. individuals who became colonized at any time were age matched to those never colonized. igg and sal-iga titres to 27 pneumococcal proteins were measured using multiplexed electrochemiluminescence. overall for the entire cohort, adults had higher igg and sal-iga than children for 24/27 proteins. at entry to the study, only igg to pneumolysin correlated with protection against pneumococcal acquisition, and this only in adults. in adults who became colonised an increase in sal-iga to 15/27 proteins with no significant changes in igg was seen. in contrast children acquiring pneumococcus had higher igg titres to 15/27 proteins at month 6 compared with month 0, but no changes in sal-iga. there were no changes in antibody titres during the study in uncolonised adults or children. pneumococcal colonisation stimulated different patterns of immune responses in adults and children. previous antigen experience and the maturity of the immune system appears critical in determining the serological outcome of pneumococcal acquisition in the nasopharynx. of the proteins studied, only anti-pneumolysin serum responses correlated with protection against pneumococcal acquisition, and this was only observed in adults. introduction: streptococcus pneumoniae (sp) vaccination induces general and local immune response. according to our preliminary results, in vaccinated children, reduction in penicillin resistance in nasopharyngeal isolates have been found. as the further step of the study, for confirmation of phenotype difference, the serotype distribution has been investigated. aim: the study was conducted to assess the serotype distribution in nasopharyngeal carriage in pediatric population vaccinated with a conjugate 13-valent vaccine (pcv13) as compared to non-vaccinated children. methods: all sp isolates obtained during the randomized, prospective study has been performed to assess the serotype distribution of sp strains isolated from carriage among 750 children aged 17 months-60 months of age. the study group consisted of 359 fully, age appropriately immunized children by using pcv13 within the official vaccination program in the city of kielce (schedule -at 3, 5, 14 months of life) and 391 children not vaccinated against pneumococci living in the same region (the city of ostrowiec). nasopharyngeal swabs were obtained by trained medical personnel, cultured in certified laboratory, sp isolates have been tested by using the quellung method in reference laboratory according to standard previously described procedure. results: the prevalence of pneumococcal carriage was similar in vaccinated and non-vaccinated children (28,4% vs 27,6% respectively). the serotype analysis in vaccinated carriers showed only 5% serotypes covered by the pcv13 in comparison to 57% in non-vaccinated population (p < 0.0001). conclusion: vaccination with pcv13 has significant impact on the reduction of vaccine covered serotypes in sp carriers and correlates with penicillin resistance of these isolates. introduction: pneumococci commonly colonize the nasopharynx of children. iceland introduced pneumococcal conjugate vaccine (pcv-10) in the childhood vaccination program in 2011. the aim was to investigate changes in serotype prevalence and antimicrobial susceptibility before vaccinated children attend the day care centres. material and methods: nasopharyngeal samples were collected annually from healthy children age 2-6 years, attending fifteen day care centres in the greater reykjavík area during 2009-2013. the children´s parents answered a questionnaire about antibiotic use during the 6 months before the sampling. results: the number of children sampled annually varied between 420 and 516. the mean carriage rate of pneumococci was between 56% and 72%, decreasing significantly with age (for 2013, each additional year of age: odds ratio (or)=0.81, p = 0.016). the proportion of children carrying penicillin non-susceptible pneumococci (pnsp) was 5-8%, also decreasing with age (for 2013, each additional year of age: or=0.61, p = 0.026). the pnsp was highest among children receiving antibiotics up to 30 days prior to sampling (for 2013: or=2.6, p = 0.026). the serotype proportions varied considerably from year to year for most of the serotypes. the most common types were 6b (13%) we investigated the impact of vaccination on nasopharyngeal (np) carriage and lower airway infection (lai) in these children, many of whom receive long-term azithromycin therapy. methods: children were enrolled, and np swabs and bronchoalveolar lavage (bal) fluid were collected under anaesthetic, stored at -80 o c and processed, as previously described. lai was defined as >10 4 cfu/ml bal fluid. data were recorded on vaccines administered since birth. azithromycin resistance was defined as mic>0.5 mg/l. results: from 2007 to 2013, 155 indigenous children aged 5.2 to 154.6 (median 27.1) months with hrct-confirmed bronchiectasis were enrolled; 89 (57%) were male. there were no significant differences in np carriage or lai between vaccine groups (table). isppd-9 / pneumonia 2014 mar 9-13;3:1-286 †azir, azithromycin-resistant spn strain(s) as % children (80% of resistant strains had mic≥16mg/l); *vaccine serotypes in bold, azir serotypes in italics. there are currently insufficient data to draw conclusions regarding vaccine impact. temporal trends and the emergence of multi-resistant non-vaccine serotypes (e.g. 15a) also need to be considered. pcv studies are ongoing. pneumococcal carriage prevalence at baseline did not differ by individuals receiving art during follow-up or not (21.0% vs. 20.6%, p = 0.99). individuals who received art had higher pneumococcal carriage than individuals who did not receive art (25.5% vs. 19.6%, p = 0.03), particularly for serotypes not included in pcv13 (15.9% vs. 9.5% p = 0.004). following adjustment, increased odds of carriage were still observed for individuals on art, but results were non-significant (all serotypes: or1.36, 95%ci 0.85-2.17, p = 0.20; non-pcv13 serotypes: or1.90, 95%ci 0.98-3.68, p = 0.06). conclusion: pneumococcal carriage in hiv-positive adults in malawi remained high despite start of art, with a tendency to increased carriage of non-pcv13 serotypes. hiv-positive adults on art remain an important reservoir for pneumococcal diversity. we investigated whether this decrease was reflected in pneumococcal carriage dynamics, by studying seasonal and long-term patterns in pneumococcal carriage in karonga, northern malawi, over a three-year period. methods: mother/infant pairs were recruited in a longitudinal study investigating the effect of maternal hiv-status on infant pneumococcal acquisition. nasopharyngeal swabs were collected monthly for one year from infants, mothers and siblings <5 years of age. samples were collected between january 2009 and november 2011, before introduction of pneumococcal conjugate vaccine. we used generalized additive mixed models to examine seasonal and long-term trends in pneumococcal carriage incidence, adjusted for age and maternal hiv-status. results: in total, 185 mother/infant pairs and 140 siblings were included in the study. pneumococcal incidence was seasonal, with the highest incidence in the cold season (p < 0.001). a decreasing trend in pneumococcal incidence was observed in all age groups over the three-year study period (p < 0.001). for serotype 19a an upward trend was observed for infants (p <0.001) and siblings (p = 0.01). in infants, pneumococcal incidence decreased from 49.3% (95%ci 42.4-56.2%) in the cold season in 2009 to 38.4% (95%ci 32.6-44.5%) and 22.1% (95%ci 14.2-31.8%) in the same months in 2010 and 2011. conclusion: before introduction of pneumococcal vaccination in malawi, pneumococcal carriage incidence dramatically decreased during the study period. seasonal and long-term trends need to be taken into account when evaluating vaccine effectiveness, in particular when a before-after design is used. background: monitoring nasopharyngeal carriage complements invasive pneumococcal disease surveillance and provides information on circulating serotypes. we report the impact of 13-valent pneumococcal conjugate vaccine (13vpcv) on nasopharyngeal carriage in western australian aboriginal people after 13vpcv replaced 7vpcv in the national immunization program in july 2011. methods: nasopharyngeal swabs (nps) were collected opportunistically from aboriginal children and adults living in urban, rural and remote areas of western australia three years before and two years after 13vpcv was given to aboriginal children in a 2-4-6-month schedule. specimens were cultured using selective media and pneumococci serotyped using the quellung reaction. results: among 823 nps cultured post-introduction of 13vpcv, the pneumococcal carriage rate in children <5yrs was 65% and 31% in people ≥5yrs compared with 72% and 35%, respectively, in 1500 ns collected pre-introduction of 13vpcv. the most common serotypes following introduction of 13vpcv were 16f (9%), 19f (9%), 11a (7.5%) 6c (7.5%), and 19a (7%) in children <5yrs and 19f (9%), 10a (6%), 15b (6%), 6a (5%) and 6c (5%) in older people. carriage of the six additional 13vpcv serotypes declined from 18.3% to 15.0% in children <5yrs. conclusion: overall pneumococcal carriage rates in west australian aboriginal people remain high with limited decline post-13vpcv introduction. three of the most commonly carried serotypes in children <5yrs are not covered by pcv13 while serotype 19f (included in pcvs since 2001) continues to be commonly carried. ongoing surveillance is needed to follow the longer term impact of 13vpcv, identify emerging serotypes and inform future vaccine development. n. iwanaga 1 , s. nakamura 1 , k. oshima 1 , t. kajihara 1 , t. takazono 1 , y. imamura 1 , k. yanagihara 2 , k. izumikawa 1 , t. sunazuka 3 , s. omura 3 , s. kohno 1 1 department of molecular microbiology and immunology, nagasaki university graduate school of biomedical sciences, nagasaki, japan; 2 department of laboratory medicine, nagasaki university hospital, nagasaki, japan; 3 kitasato institute for life sciences, kitasato university, tokyo, japan background and aims: streptococcus pneumoniae (the pneumococcus) colonizes the mucosal surfaces of the human upper respiratory tract (urt), and occasionally leads to invasive disease. regulation of nasopharyngeal colonization could be a candidate for preventing pneumococcal infections. we investigated whether macrolides, which have variant immunomodulatory effects, could promote the clearance of pneumococcal colonization via immunomodulation. methods: peritoneal macrophages extracted from mice were pretreated with clarithromycin (cam) and azithromycin (azm). the levels of cytokines in the supernatant were determined by elisa and cytokine array, and intracellular chemokine analysis was performed by flow cytometry. the cell signaling pathway was also analyzed by westernblotting and gene silencing assay. the macrolides-mediated pneumococcal clearance was examined by using intranasal bacterial challenge in mice. em900 (a novel 12-membered non-antibiotic macrolides but has an immunomodulatory effect) was orally administered throughout this experiment. urt lavages were analyzed for the kinetics of colonization, the cellular response and inflammatory cytokines. results: the production and mrna expression of ccl2 of peritoneal macrophages were significantly induced by cam and azm treatment in a dose-dependent manner. intracellular ccl2 was also detected by flow cytometry. westernblotting showed phosphorylation of nf-kb 15 minutes after macrolides-treatment. the density of pneumococcus in urt was significantly decreased in em900 treated mice compared to untreated mice at 14 days after pneumococcal inoculation. coincidentally, the recruitment of macrophages and ccl2 mrna expression in nasal cavity were significantly increased by administration of em900. conclusion: immunomodulatory action of macrolides promotes the clearance of pneumococcal nasopharyngeal colonization by inducing the accumulation of macrophages to urt. background and aims: nasopharyngeal colonisation is the initial step in pathogenesis of pneumococcal diseases, including otitis media. pneumococcal vaccines are less effective against otitis media than they are against systemic pneumococcal infections. previous studies have suggested that streptococcus salivarius, a commercial probiotic originally isolated from the throat, could be a potential pharyngeal probiotic. we investigated the effects of s. salivarius on pneumococcal colonisation and otitis media by using in vitro and in vivo models of infection. methods: high (5 to 10 times more than pneumococci), medium (approximately equal) or low (5 to 10 times less) numbers of s. salivarius were added before, with, or after pneumococci (pre-, co-, and post-administration respectively) to human epithelial ccl-23 cells in vitro. the percent colonisation of pneumococci was determined after 3 h incubation. in experiments using infant mice, repeated pernasal administration of s. salivarius (~10 7 colony forming units (cfu)/dose) was used as an intervention in a model of pneumococcal colonisation and otitis media. bthere was a time-dependent (pre-administration more effective than co-and post-administration) and dosedependent (high numbers more effective than medium or low numbers) inhibition of pneumococcal adherence to ccl-23 cells by s. salivarius. however, s. salivarius colonised the nasopharynx of mice poorly, and did not alter pneumococcal colonisation levels or the development of otitis media in vivo. conclusion: our data indicated that s. salivarius can inhibit pneumococcal colonisation in vitro, but not in infant mice. further well-designed human trials are warranted to evaluate s. salivarius as a potential probiotic to reduce otitis media. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 background: using nasopharyngeal carriage as a marker of vaccine impact, pneumococcal colonisation and its relation to invasive disease and demographic attributes were examined in children, their parents, and older adults in the uk following the introduction of pcv7 and prior to pcv13. methods: nasopharyngeal swabs were collected, between november 2010 and september 2011, from children 25-55 months of age who had previously received three doses of pcv7, their parents, and adults aged 65 years and over. pneumococcal serotyping was performed according to who guidelines with non-typeable isolates further analysed by comparative genomic hybridisation (cgh) microarray. surveillance of invasive pneumococcal disease (ipd) in england and wales was conducted throughout the corresponding time period. results: pneumococcus was isolated from 47% of children, 9% of parents and 2.2% of older adults. for the corresponding groups the proportion of serotypes covered by pcv7 were 1.5%, 0% and 15.4%, with a further 20%, 44.4% and 7.7% coverage added by those in pcv13. in each group the proportion of ipd due to the serotypes covered by pcv7 were 1.0%, 7.4% and 5.1% with a further 65.3%, 42.1% and 61.4% attributed to those in pcv13. a significant correlation between contact with children and pneumococcal carriage in older adults was noted. conclusion: in this population the rate of carriage is highest in children and those exposed to them. direct vaccine impact is exemplified by the low carriage and ipd rates of pcv7 serotypes in vaccinated children, whilst the indirect effects of herd protection are implied by similar observations in unvaccinated parents and older adults. background and aims: kenya introduced 10-valent pneumococcal conjugate vaccine (pcv10) for infants at 6, 10, and 14 weeks in february 2011 without catch-up vaccination for older children. we evaluated pcv10 impact on pneumococcal carriage among children living in a crowded, informal settlement. methods: we conducted cross-sectional carriage surveys during october to december in 2009, before vaccine introduction, and 2012, after vaccine introduction, among randomly-selected children <5 years old in kibera, an urban slum in nairobi. vaccination histories were verified. nasopharyngeal swabs were placed in stgg media, and specimens were cultured for streptococcus pneumoniae isolation after broth enrichment. pneumococcal isolates were serotyped by multiplex-pcr and quellung reaction. results: among 545 children in 2009 and 309 in 2012, median ages were 2.3 and 1.0 years, respectively. the proportion of children <5 years colonized with pneumococci was similar in 2009 and 2012 (90% and 92%, respectively). among all children, pcv10 serotypes were found in 38% in 2009 compared to 17% in 2012 (percent change, -54%; p < 0.001). serotypes with the greatest reductions were 19f (percent change, -67%) and 9v (percent change, -65%). pcv10 serotypes decreased by 57% in infants <12 months (p < 0.001) and 59% in children 1-4 years (p < 0.001). in a subset of unvaccinated children 1-4 years old (n = 85), pcv10 serotypes decreased by 61% (p < 0.001). conclusion: less than 2 years after pcv10 introduction, declines in pcv10-type colonization were substantial even in children not eligible for vaccination. our data suggest that herd effects are seen early in a program without catchup vaccination. introduction: the nasopharynx of children is the main habitat of pneumococci. the difference in pneumococcal carriage between healthy children and children with upper respiratory tract infections (urti) is usually speculative. the aim of the study was to compare serotypes and antibiotic susceptibilities of pneumococci in healthy carriage and children with urti. material and methods: a comparison was made between isolates collected during studies of pneumococcal carriage of children attending day care centres in 2009-2012 (dcc group), and nasopharyngeal clinical samples from children with upper respiratory tract infections (cs group) submitted to the microbiology laboratory at landspitalinn during the same period. comparisons were made between children of the same age. results: there were 1281 isolates from children in the dcc group and 770 in the cs group. the most common serotypes were 23f, 6a, 19f, 6b and 19f, 6a, 23f, 6b in the dcc and cs group respectively. the difference was significant for 19f (p < 0.0001), 23f (p = 0.02) and non-typable (p < 0.0001) isolates. penicillin non-susceptible pneumococci were more common in the cs group (265 isolates, 34%) compared to the dcc group (171 isolates, 14%), the difference being significant in the age groups 1.5-1.9 years (p = 0.03), 2-2.9 years (p = 0.001) and 4-4.9 years of age (p = 0.02). multi-resistant streptococcus pneumoniae was also more often found in the cs group (33% vs. 14%). conclusion: there was a significant difference in carriage of the main serotypes between the two study groups and a significantly higher resistance rate in the cs group. isppd-9 / pneumonia 2014 mar 9-13;3:1-286 background and aims: the carriage of streptococcus pneumoniae in the nasopahrynx of children has been shown to precede disease and serves as a source of transmission in a community. we carried out the present study to determine the culture positivity and antibiotic susceptibility pattern of s. pneumoniae from nasopharynx of children. methods: as a part of community based acute respiratory infection (ari) surveillance in villages of ballabhgarh block of haryana, north india. naso-oropharyngeal swabs were collected from 1378 children suffering from ari (alri-542, auri-836) and 253 controls in the age group 0-10 years from aug 2012 to aug 2013. a total of 202 urine samples (alri-177 and 25 controls) were also collected from these children to screen for pneumococcal antigen by binaxnow. ethical approval was obtained from the institutional review committee. results: a total of 68 children out of 1378 (5%) having ari, were culture positive for s.pneumoniae in nasopharyngeal swabs. age wise distribution of s.pneumoniae culture positivity was 0-2 years -alri-28/316; auri-13/214, 2-5 years -alri-5/159, auri-3/32 and 5-10 years -alri-3/67, auri-7/302. among controls, 9 were also positive in 0-2 yrs. all isolates were sensitive to penicillin & resistant to co-trimoxazole. erythromycin resistance was seen in 18%. of the 202 urine samples positivity for pneumococcal antigen was alri-88/177 and control-12/25 with age wise distribution as 2-5yrs-alri-46/88, control-10/16. the study showed that pneumococcal positivity among children was high in our community especially in the age groups of 0-2 years. all strains remain penicillin sensitive and cotrimoxazole resistant. background and aims: nasopharyngeal (np) carriage of pneumococcus predisposes young children to invasive pneumococcal disease. this study characterizes the impact of the seven-valent pneumococcal conjugate vaccine (pcv7) on pneumococcal carriage and the microbiome in the first year of life. methods: np swabs were collected from 102 subjects from birth to twelve months at regular intervals. g1 (n = 39) and g2 (n = 30) subjects were from pcv7 naive communities and g3 (n = 39) subjects were from widely vaccinated communities. g1 subjects received pcv7 after 8 months while g2 and g3 subjects were vaccinated at 2, 3 and 4 months. microbiology techniques were used to culture and serotype pneumococci. microbiome analysis was conducted by bar-coded 16s-rrna gene pyrosequencing. results: overall pneumococcal carriage was 79% (95%ci:75 -82%), 75% (95%ci:71-79%) and 82% (95%ci:79-85%) for g1, g2 and g3 participants respectively. the prevalence of pcv7 serotypes was 29% (95%ci:24-34%) among the g1 subjects, significantly higher than among subjects from g2 14% (95%ci:11-18%) and g3 5% (95%ci:3-7%) p < 0.001. the most common pcv7 serotypes were 19f (4%), 14 (4%) and 23f (3%) while the predominant non-vaccine serotypes were 19a (13%), 13 (4%), 35b (4%) and 15b (3%) among all the children. in the first eight months of life, there were 6-fold more significant changes at the genus level (including lactococcus and staphylococcus) among g1 compared to g3 subjects. conclusion: early exposure to pcv7 does not reduce pneumococcal carriage but alters the carriage of vaccine serotypes and impacts the development of the microbiome which has important implications for the long-term effectiveness of pcv. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 j.y.r. lai 1 , r. marsh 1 , h. smith-vaughan 1 background and aims: bacterial viability can be affected by storage and transport conditions prior to processing. in remote settings, temporary storage conditions before transport to a laboratory may be necessary. this study aims to determine optimal storage conditions for maintaining pneumococcus and nontypable haemophilus influenzae (nthi) viability prior to processing or long-term storage at -80°c. methods: the study was conducted in two phases: i) a controlled in vitro experiment using four pneumococcal serotypes and one nthi to determine bacterial survival at varying concentrations in different storage conditions (liquid nitrogen (ln2) vapour shipper, dry ice, domestic freezer, domestic fridge, ice bricks, crushed ice) prior to -80°c storage; ii) investigating the impact of the different storage conditions on the viability of pneumococcus and nthi using nasopharyngeal (np) swabs. results: the in vitro experiments showed no significant difference between survival of different pneumococcal serotypes subject to different storage conditions up to 24 hours after inoculation. placement of isolates directly into a ln2 vapour shipper led to approximately 30% loss of nthi viability; findings suggest that freezing at -20°c may be necessary before ln2 shipper storage. conclusion: transport conditions for np specimens need to account for the storage and transport needs of individual bacteria of interest, and will depend on time required for temporary storage. for studies requiring a ln2 vapour shipper, specimens may need prior freezing at -20°c to maximise viability of nthi. methods: children were eligible if they were resident in a participating remote indigenous community between september 2008 and december 2012 and were 0 to <6 years of age. this analysis compares np carriage in pcv7and phid-cv10-vaccinated children (>=2 doses) < 36 months of age. results: carriage of either pneumococcus (spn) or non-typeable h. influenzae (nthi) was ~90%; around 35% pneumococci were penicillin non-susceptible and 16% azithromycin resistant. conclusion: pneumococcal and nthi carriage is almost universal in young indigenous children, regardless of vaccine type. replacement non-susceptible non-pcv types now dominate. post-booster analyses are warranted. broader vaccine protection from an early age is urgently needed. phid-cv10 serotypes 15% 11% conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 background: otitis media (om) in high risk children is caused by co-infection with pneumococcus (spn) and nontypeable haemophilus influenzae (nthi). prevenar7 (pcv7) has not had a significant impact on tympanic membrane perforation (tmp) rates of 20% by 6 months in aboriginal children in the northern territory. an early, mixed vaccine schedule which maximises potential activity against diverse pathogens may be beneficial. methods: a randomised controlled trial (rct) of i) 10-valent pneumococcal h. influenzae protein d conjugate vaccine synflorix (phid-cv10) versus ii) 13-valent pneumococcal conjugate vaccine (pcv13) (at 2,4,6 months) versus iii) an early combination schedule of phid-cv10 at 1,2 and 4 months with additional pcv13 at 6 months. allocation is concealed and primary outcome (immunogenicity at 7 months of age) is assessed blind. estimated sample size is 339. nasopharyngeal (np) carriage and om are secondary outcomes. results: eighty seven infants have been randomised, 3 have withdrawn. overall prevalence of om, tmp and np carriage at 1, 2, 4, 6 and 7 mo is shown in table 1 . conclusion: high risk populations may benefit from variation of standard vaccine schedules but implementation challenges necessitate high quality evidence to support such variations. this trial provides first rct data on om, immune response and carriage in phid-cv10 and pcv13 vaccinees, and will evaluate a novel early combined option for high risk children. background and aims: the impact of conjugate pneumococcal vaccines alters the dynamics of streptococcus pneumoniae serotypes carriage necessitating surveillance of disease. prospective surveillance of pneumococcal carriage was conducted from april 2009 in a university-tertiary care hospital in hong kong sar. all paediatric patients hospitalized with with respiratory illness and/or fever with a nasopharygeal aspiration performed were included. methods: nasopharyngeal aspirates (npa) collected in to a modified transport medium were analyzed using routine microbiological procedures. micro-broth based methodology (clsi) was used to obtain the mic of the isolates while sequential-multiplex pcr (cdc/atlanta) were used for serotyping. results: data was analyzed for 6-monthly intervals; april-september and october-march. of the 10,909 npa samples, 1359 (12.5%) yielded s. pneumoniae. over the 7 time periods, a significant reduction in the pneumococcal isolation rates (p < 0.007, chi square for trend) was identified. of the 1291 isolates available for further analysis, the commonest serotypes were 19f (14%), 6b and 6c (9.8% each).percentage of serotypes/ groups included in the pcv 7 ranged from 50.2% in april-sept 2009 to 24.2% in oct-2011-march 2012, whilst those included in the pcv13 ranged from 66.5% to 40.4% for the same time periods (p < 0.005, chi square for trend). the overall cefotaxime non-susceptibility rate at meningitis and non-meningitis breakpoints (clsi) were 29.4% and 12.9% respectively. isppd-9 / pneumonia 2014 mar 9-13; 3:1-286 conclusion: pneumococcal carriage rates among hospitalized children showed a significant reduction with the introduction of pneumococcal vaccination in hong kong. however, continuous monitoring is needed to identify long term trends that may come with serotype replacement. background: streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus are potentially pathogenic bacteria which colonize the nasopharynx (np), with hiv-infected children at greater risk for invasive disease. pneumococcal conjugate vaccine (pcv) reduces np-colonization by the vaccine-serotypes, but may affect the equilibrium of np-colonization by other bacteria in healthy children. we compare the bacterial np-colonization between hiv-infected (n = 321) and hiv-uninfected (n = 243) children vaccinated with 7-valent pcv at 6, 10 and 14 weeks of age. methods: children in soweto, south africa, had np-swabs for bacterial culture taken prior to the three pcv-doses (visits 1-3) and at 19.5, 39, 47 and 67 weeks of age (visits 4-7). swabs were cultured for bacteria by standard methods and pneumococcal serotyping done by the quellung method. results: prevalence of vaccine-serotype colonization was similar between hiv-infected and hiv-uninfected children, except visit-6 (p = 0.019); figure. overall pneumococcal (visits 1-4) and non-vaccine serotype (visits 1-5) colonization were lower in hiv-infected children. hiv-infected children had lower prevalence of s. aureus colonization at visit-1 (p < 0001), but higher prevalence from visit-4 onward (p < 0.05); whereas h. influenzae colonization was greater among hiv-uninfected from visit-2 (p < 0.05). conclusion: although hiv-infected children had a similar prevalence of vaccine-serotype colonization compared to hiv-uninfected children, they had a lower prevalence of non-vaccine serotype and h. influenzae colonization, but higher prevalence of s. aureus colonization. background and aims: up to one million children die every year due to pneumococcal disease. streptococcus pneumoniae infections increased in both incidence and severity in persons with congenital or acquired immunodeficiency. there is lack of information on the impact of pneumococcal vaccine on children older than those covered by the expanded program of immunization (epi) vaccine schedule and in those infected with hiv, as successful immunity is dependent on mounting a sufficient immune response to the vaccine. this study aimed to (1) determine the number and proportion of children carrying new (not present at baseline) vaccine serotypes of s. pneumoniae isolated from nasopharyngeal swabs at 6±2 months post vaccination in recipients of prevenar13® compared with those given hib vaccine. (2) to determine the serum antibody response (>4-fold and geometric mean concentration) to pneumococcal vaccine serotypes at 3±1 months after vaccination. method: this was a double blinded crossover randomised controlled trial of the efficacy of prevenar13® in preventing acquisition of nasopharyngeal carriage of s. pneumoniae among hiv infected children aged 1-14 years. eligible participants were randomised into prevenar13® or hib vaccines each given at baseline and 3±1 months later. nasopharyngeal and serum samples were collected at baseline and 3±1 months later. results: we have enrolled 73 under five children, the overall pneumococcal isolation rate at baseline was 71% (n = 73) and 73% (n = 68) after two doses. serotyping is still done but results will be available at the conference. conclusion: pneumococcal colonisation is common in children with hiv/aids, vaccination may reduce it but replacement colonisation may occur. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 background and aims: acute otitis media (aom) is one of the most common infections in children. there are several bacterial species among principal aom-associated pathogens but the leading role is usually attributed to streptococcus pneumoniae. this study was performed to clarify etiology of bacterial aom and compare it with nasopharyngeal colonization. methods: we examined middle ear fluid (mef) obtained from 310 patients with aom who were ≤60 month old (age median, 28 months) at five pediatric hospitals in moscow (russia). in parallel, in 305 patients nasopharyngeal swab was collected. serotyping was performed by specific antisera from statens serum institute (ssi) (denmark). results: culture-positive aom was observed in 39% (121/310) of patients. s. pneumoniae was isolated in 55%, staphylococcus pyogenes in 22%, staphylococcus aureus in 14%, haemophilus influenzae in 12%, and moraxella catarrhalis in 8% of culture-positive patients. among 67 s. pneumoniae mef isolates, 15 different serotypes were identified but 6 leading serotypes contributed to >75% of the distribution (19f, n = 17; 3, n = 12; 6b, n = 9; 14, n = 5; 19a, n = 5; 23f, n = 4). mef and nasopharyngeal serotypes were the same in >90% (32/35) of cases. in a patient with aom, isolation of s. pneumoniae or s. pyogenes from the nasopharynx increased the probability of aom of that particular etiology to 60% or 75%, respectively. s. pneumoniae isolates displayed reduced susceptibility or resistance to penicillin (29%), macrolides (26%), co-trimoxazole (50%). the leading bacterial pathogen in children with aom was s. pneumoniae. available pneumococcal polysaccharide conjugate vaccines (pcv) cover from >90% (pcv-13) to 61% (pcv-7 and pcv-10) of the aomassociated serotypes. background and aims: pneumococcal infections remain a major medical problem associated with high morbidity and mortality. moreover, the resistance of streptococcus pneumoniae to conventional antibiotics is constantly growing. implementation of pneumococcal conjugate vaccines (pcvs) has dramatically reduced the incidence of the vaccine type-associated pneumococcal diseases in many countries. however, information on seroepidemiology of s. pneumoniae in russia is limited. methods: we performed serotyping and antibiotic susceptibility testing on 867 noninvasive pneumococcal isolates prospectively collected in 2009-2013 from children (median age 3.5 years) who sought medical care at five pediatric hospitals in moscow. the isolates were recovered from nasopharynx (71.2%), middle ear fluid (14.3%), and lower respiratory tract specimens (13.6%). results: in total, 45 different serotypes were identified. the leading serotypes included serotypes 19f (21.6%), 6b (12.8%), 23f (10.1%), 14 (9.1%), 6a (8.3%), and 3 (7.6%). the frequency of the pcv-7, -10, and -13 serotypes was 58.2%, 59.8%, 78%, respectively. the rate of the multidrug resistant pneumococci (mdr, i.e. resistant to >=3 antimicrobials) was 21.5%. the majority of the mdr isolates had serotype 6b, 14, 19a, and 19f . penicillin nonsusceptibility displayed 27.5% of the isolates. the resistance rate to erythromycin was 26.1%. among the examined erythromycin-resistant strains, 54% had erm(b) gene and 13% had mef gene as a single resistance determinant, whereas both determinants were found in 31% of these strains. conclusions: our data predict a good coverage of the circulating s. pneumoniae by the pcvs and could be useful for evaluating serotype distribution in support of pcv introduction in russia. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 background: invasive pneumococcal diseases (ipd) are responsible for almost 1 million deaths per year among children under five years of age. streptococcus pneumoniae, one of the main causative agents, has over 90 serotypes yet the current conjugate vaccine only includes 13 serotypes (pcv13). pneumococcal proteins are conserved across all serotypes and therefore hold potential as vaccine candidates. aims: this study aimed to investigate the transfer of antibodies against major pneumococcal antigens from pregnant mothers to their infants, and to correlate antibody concentrations with nasopharyngeal carriage (npc). methods: we analysed plasma from mother's (up to 3 months post-birth) and infants (cord blood only) to determine antibody concentrations and avidity to recombinant pneumolysin (rply), pneumococcal surface protein a (pspa) and choline-binding protein a (cbpa) using an in-house igg elisa. results: antibody transfer was antigen-dependent with significantly higher levels of antibodies against pspa and cbpa in mothers compared to cord blood but similar levels for rply. pspa and cbpa were better transferred than rply from mother to child (p = 0.0001 and p = 0.0024 respectively). antibodies to pspa and cbpa but not rply were retained up to 3 months in mothers with no significant difference in the avidity index at any time-point. conclusion: our pilot data shows that transfer of antibodies against major pneumococcal proteins from mother to child is antigen-dependent. further work will explore the relationship with npc. this information together with current studies on infant antibody development will help to elucidate levels of antibodies required for protection against npc and invasive disease. background and aims: nowadays, pneumococcus is the major cause for meningitis, with a mortality rate of 15%-40%. meningitis survivors may suffer from severe sequelae such as learning impairment, deafness, mental retardation, hydrocephalus and more. meningitis is characterized by an acute inflammation. we hypothesize that functional alterations in the brain may result from immune independent pathways since treatment with dexamethasone, in addition to the antibiotic treatment, did not improve therapy outcome. previous studies in our laboratory identified a group of cell-wall adhesins and their target molecules in lung derived epithelial cells. currently we study the possible involvement of those adhesin in streptococcus pneumoniae interaction with brain derived neural cells. methods: the ability of the recombinant adhesin to interfere in s. pneumoniae adhesion to human u251 glioblastoma multiforme and nsc-34, a hybrid of mouse neuroblastoma and embryonic mouse motor neurons, cells was studied. using confocal microscopy, the existence of the previously identified receptors in those cells was analyzed. results: under conditions in which the bacteria do not affect survival, wu2 strain adhered to u251 and nsc-34 cells. rgts, rptsa, rfba and rnox were found to inhibit bacteria adhesion to the cells. previously identified putative target molecules such as bmper, mmrn1, eps 1, pcdh19 and intβ4 were found to be expressed in both cell lines. conclusion: s. pneumoniae gts, ptsa, fba and nox protein were found to mediate bacterial adhesion to neural cells. moreover, previously identified target molecules to bacterial adhesins were found to reside in the neural cells. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 preliminary results of risk factors for pneumococcal carriage in young infants and children in fiji prior to pcv10 are shown. methods: 499 healthy 5-8 week old infants and 500 healthy 12-23 month old children participated in a crosssectional survey documenting risk factors for pneumococcal nasopharyngeal carriage. nasopharyngeal swabs were collected using standard methods. pneumococci in young infant swabs were identified by lyta quantitative pcr (qpcr). dominant α-haemolytic morphology, and an example of each morphological variant in children's swabs, were identified as pneumococci using standard methods. risk factors were assessed by multivariate logistic regression. results: for young infants, univariate analysis showed rural residence and living with other young children to be significant risk factors for carriage. after controlling for confounding, only rural residence was associated with carriage (odds ratio (or) 4.02, 95% ci 1.85 -8.73, p < 0.001). for children, univariate analysis showed coryza, cough, living with > 2 children, birth by vaginal delivery, and being indigenous fijian to be risk factors for carriage. after controlling for confounding, carriage was associated with being indigenous fijian (or 2.51, 95% ci 1.70 -3.71, p < 0.001), and birth by vaginal delivery (or 2.17, 95% ci 1.14 -4.16, p < 0.024). conclusion: preliminary findings suggest carriage risk factors may differ by age. however, results are preliminary and final analyses will be presented at the meeting. pneumococcal nasopharyngeal colonization is a pre-requisite for pneumococcal disease (pd); hiv-infection increases the risk of pd. we investigated pneumococcal colonization and serotypes acquisition in perinatally hiv-exposed children. 333 hiv-infected and 491 hiv-exposed-uninfected (heu) children who were randomized to isoniazid or placebo (p1041, tb-prophylaxis study), had 6 schedule visits between 6-42 months of age at which nasopharyngeal swabs were collected for pneumococcus serotyping. no pneumococcal vaccine was given. at study entry, 81% of the hiv-infected children were asymptomatic, 4% moderately symptomatic and 1% severely symptomatic for aids. hiv-infected children were less likely to be colonized with 13-valent pneumococcal conjugate vaccine (pcv13) serotypes than heu at 6 months of age (31.5% vs. 43.3%; p = 0.03), however, no differences in colonization were observed at the other visits. over the 36 months study period pneumococcal colonization increased in both hiv-infected and heu children from 45.1% to 76.9% (p-value for trend < 0.001) and from 57.1% to 60.2%, respectively (p-value = 0.014). the rate of overall new serotype acquisition was lower among hiv-infected than heu children (47% vs. 52%; p = 0.008), while pcv13-serotypes acquisition was similar in both groups (33-36%) and non-pcv13 serotypes were acquired less frequently by hiv-infected (11.0%) compared to heu (14%; p=0.009). acquisition of non-typeable pneumococcus was more common in hiv-infected (4%) than in heu (2%; p = 0.033). pcv13-serotypes new acquisition was common in our population and similar rates were observed in hiv-infected and heu children. despite a greater susceptibility to pd in hiv-infected children, overall and non-pcv13 acquisition rates were lower in hiv-infected compared to hiv-uninfected children. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 the 7-valent pneumoccocal conjugate vaccine (cv7) was introduced into the south african immunization program in 2009 as a three-dose schedule at 6 weeks, 14 weeks and booster at 9 months age. we studied the rate of new serotypes acquisitions in children receiving this schedule and compared it to a historical cohort of pcv-unvaccinated children. children aged 5-8 weeks were enrolled from december/09 to april/10, while the historical cohort was enrolled in 2007. participants had nasopharyngeal swabs done on seven occasions between one-month following the second pcv7 dose up until two years, with the same schedule undertaken among the historical cohort. swabs were cultured for pneumococcus. a total of 1551 nasopharyngeal swabs from 236 pcv7-vaccinated children were collected during the study, from which 1059 (68.3%) were positive for pneumococcus. the prevalence of pneumococcal colonization peaked at 41 weeks of age (n = 175/228, 76.8%) and ranged between 63-69% thereafter. overall colonization by pcv7-serotypes was 21.7%. during the first two years of life pcv7-vaccinated children acquired pneumococci less frequently than unvaccinated children (46.2% vs. 55.9%, p < 0.001), especially pcv7-serotypes (11.3% vs. 25.0%, p < 0.001). non-vaccine serotypes were acquired more often by pcv7-vaccinated compared to -unvaccinated children (32.4% vs. 27.4%, p = 0.018). acquisition of non-typeable pneumococci was similar between pcv7-vaccinated and -unvaccinated children (2.6% vs. 3.5%, p = 0.24, respectively). this study demonstrates that the 2+1 schedule implemented in south africa has resulted in a reduced risk of vaccine-serotype acquisition among pcv7-vaccinated children. as such, there is expected to be reduced transmission and consequently indirect protection against pneumococcal disease among unvaccinated individuals in south africa. s.a. nzenze 1 , a. von gottberg 2 , t. shiri 1 , l. de gouveia 2 , a. violari 3 , m.c. nunes 1 , s.a. madhi 2 1 background and aims: central australian aboriginal communities have amongst the highest reported rates of respiratory infections worldwide. there are however no community level data on the prevalence of respiratory symptoms and respiratory pathogens that may be associated with those symptoms. our aim was to address this knowledge gap. methods: we convenience sampled every individual who presented for any reason to a remote community clinic over a three week period in a non-epidemic respiratory season. demographic and clinical data were obtained. anterior nasal swabs were collected and tested for adenovirus, respiratory syncytial virus (rsv), influenza virus, parainfluenza viruses (pivs) types 1,2 3, human metapneumovirus (hmpv), rhinoviruses (rvs), coronaviruses (hcov) (oc43, 229e,nl63 + hku1), bocavirus, ki and wu polymaviruses (ki and wupyv), streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, staphylococcus aureus (including methicillin-resistant staphylococcus aureus (mrsa)), and bordatella pertussis using real-time pcr methods. results: 140 participants were enrolled, contributing 153 study visits and 150 nasal specimens; 21% were aged < 5 years. a respiratory symptom was present in 74 (48.4%) of all episodes. at least one virus was present in 50% of episodes with a specimen, at least one bacterium in 80% of episodes and viral-bacterial coinfection was present in 28.7%. respiratory symptoms were 2.4 times (95%ci 1.4 -4.3) more likely in episodes of viral-bacterial coinfection than those without. conclusions: the prevalence of both symptoms and pathogens are high this population. interventions to reduce the burden of respiratory disease in this population will require community wide approaches to reduce infection and carriage in both children and adults. background and aims: aboriginal children in remote regions of central australia have the highest yet reported rates of hospitalised, world health organisation (who) defined, radiologically-confirmed pneumonia. we aimed to describe the respiratory viruses and bacteria detected in deep nasal swabs associated with hospitalised pneumonia in these children. methods: datasets from two studies of hospitalised pneumonia in central australian aboriginal children aged less than 5 years were combined. chest xrays were assessed for consolidation according to the who protocol and by a paediatric pulmonologist (pp). deep nasal swabs collected on admission were analysed by pcr for 6 bacteria (streptococcus pneumoniae, haemophilus influenzae, staphylococcus aureus, moraxella catarrhalis, chlamydia pneumoniae and mycoplasma pneumoniae) and 17 viruses (human rhinoviruses, adenovirus, human metapneumovirus, respiratory syncytial viruses a and b, influenza viruses a and b, parainfluenza types 1, 2 and 3, bocavirus, wu and ki polyomaviruses and human coronaviruses huk1, oc43, nl63 and 229e). results: complete clinical, radiological and laboratory data were available on 103 children. fifty percent (n = 51) had pp-confirmed lobar consolidation and 28% (n = 29) had consolidation as per the who protocol. at least one bacterium was identified in 92.2% of all children, at least one virus in 42.7% and both viruses and bacteria were detected in 39.8%. there were no statistically confirmed differences between lobar and non-lobar pneumonia with respect to any organism. conclusion: there appears to be no clear relationship between nasal detection of respiratory viruses and bacteria and the presence of radiologically-confirmed lobar consolidation in aboriginal children hospitalised with pneumonia. introduction: upper respiratory tract mucosal surfaces are primary sites of pneumococcal infections. pneumococcal vaccines may induce production of salivary iga and igg. mucosal antibody responses to pneumococcal vaccines were investigated in a pneumococcal conjugate vaccine (pcv7) trial in papua new guinea. methods: children were randomized to receive pcv7 in a 0-1-2 (neonatal) or 1-2-3-month (infant) schedule or no pcv7. all children received pneumococcal polysaccharide vaccine (ppv) at age 9 months. saliva was collected at ages 1, 2, 3, 4, 9, 10 and 18 months. salivary iga and igg concentrations to all pcv7 serotypes and ppv serotypes 1, 3, 5, 7f, 19a were measured in 420 samples (60 children) using a multiplex fluorescent bead-based assay. results: pcv7 had little impact on salivary iga before 9 months but geometric mean titres (gmts) for salivary igg to serotypes 4, 18c and 19f were higher at ages 2-4 months in pcv7 recipients than in controls. at ages 9 and 10 months, gmts for pcv7-serotype-specific iga were 100-1778 and 891-6607 ng/ml, respectively; in pcv7-primed children and 78-1479 and 74-3020 ng/ml in unprimed children. equivalent ranges for salivary igg were 38-723 and 478-12928 ng/ml in pcv-primed children and 27-214 and 40-300 ng/ml in unprimed children. average mean fold increases (mfis) in pcv7-serotype-specific iga 1 month post-ppv were 7.6, 5.5 and 2.6 in neonatal, infant and control groups, respectively and 20.4, 12.0 and 1.3 in neonatal, infant and control groups for pcv7-serotype-specific igg. conclusion: pcv7 generally primes mucosal immune responses for boosting by ppv at 9 months. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 streptococcus pneumoniae causes serious illnesses such as pneumonia, meningitis, and acute otitis media. a main antigenic determinant of s. pneumoniae is polysaccharide (ps), which shows a poor immunogenicity as a t cell independent antigen. in this study, we applied cationic liposome for s.pneumoniae ps and examined immune responses such as mucosal iga, systemic igg and t-cell response. mucosal immunity is one of the important issues because the pathogen enters the host through the respiratory mucosa. a vaccine inducing the production of protective secretory iga, as well as systemic igg, would be desirable. the cationic liposomal ps showed higher respiratory mucosal iga compared to pneumococcal conjugate vaccine. and cationic liposome brought the t-cell dependent immunity against pneumococcal polysaccharide showing considerable high t-cells proliferation compared to ps only and high interleukin-4 (th2 marker) production. and the cationic liposome showed a depot effect at the injection site, which could induce a prolonged presentation of the incorporated antigen. all of the results were induced with a formulation composed of ps and liposome without any additional carrier proteins. these results suggest that cationic liposomal ps could be useful for immune responses against a polysaccharidebased s. pneumoniae vaccine. s.s. park 1 , s. pyo 1 , d. rhee 1 1 caseinolytic protease l (clpl) is a member of hsp 100 family, found mostly in gram-positive bacteria. here we report the characterization of recombinant clpl protein, a major heat shock protein (hsp) in streptococcus pneumoniae (pneumococcus) in vitro. previously, we characterized clpl with 6-his tag at the n-terminal on chaperone and atpase activities. however, n-terminal his-tag affected clpl functions. therefore, clpl was rechracterized without his-tag. clpl has adp and atp hydrolase activity by using either mg 2+ or mn 2+ , but mn 2+ stimulates atp hydrolase activity than mg 2+ . mn 2+ increases atp hydrolase activity as well as refolding and heat aggregation prevention activities. however, clpl does not seem to require auxiliary factors (dnakje system) for chaperone activity. clpl forms hexamer in the presence of adp, atp, and atp-γ-s. mutagenesis studies on the two walker a motifs (k127a, t128a and k458a, t459a), displayed that two nucleotide binding domains are involved in chaperone and atp hydrolase activities and hexamerizaion. taken together, pneumococcal clpl is a mn-dependent auxiliary-factor independent chaperone. s. pelton 1 , k. shea 2 , k. hsu 3 , a. loughlin 3 1 pediatrics and epidemiology, boston universithy schools of medicine and public health, boston, usa; 2 epidemiology, boston universithy school of public health, boston, usa; 3 pediatrics, boston universithy school of medicine, boston, usa background: changes in prevalence of nasopharyngeal (np) carriage of nonvaccine pneumococcal serotypes (nvst) may herald increases in ipd caused by these serotypes and potential erosion of vaccine effectiveness. objective: to evaluate changes in pneumococcal serotypes (all, nvst, pcv7, and pcv13 unique) in the np following substitution of pcv13 for pcv7 in children <2 years with catch-up through 5 years. methods: demographic information and np cultures were obtained from 5,307 children <5 years seen in primary care between 7. 1.2010 and 6.30.2013 . all isolates of streptococcus pneumoniae (sp) were serotyped by quellung reaction with type-specific antisera. results: the overall prevalence of sp carriage was 22.8%, and was lowest during summer months. no significant changes in the carriage prevalence of all sp, nvst, or pcv7 serotypes were observed. carriage of pcv13 unique serotypes declined over time (figure 1 ). streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. the pneumococcal nasopharyngeal carriage preceded and is a source of the spread of disease to others pneumococcal. indonesia is the country with the highest incidence of pneumonia in southeast asia, and lombok island in western part of indonesia has a highest pneumonia incidence. methods and results: pneumococcal nasopharyngeal isolate has been done in 1200 healthy children aged 2 months to 5 years of 5 health centre in central lombok, lombok island. on those isolates, optochin test, pcr assay, and sensitivity to antibiotics were performed. the prevalence of s. pneumoniae carrier rate was 46.17%, with distribution in less than 2 years of age and above were 26.8% and 48.8%, respectively. most commonly serotypes were 6a/b, 19f, 23f, 15b/c, and 19a. all serotypes were susceptible to majority of antibiotics, except to cotrimoxazole (36 %). overall of isolates belonged to strains covered by 50,1 % for pcv10 and 55,3 % for pcv13. we found 74.7% of the subjects exposed to passive smoking and 74.7% have pneumococcal positive isolate conclusion: the prevalence of nasopharyngeal carriage of s. pneumoniae is very high and most commonly serotypes found are quite similar to those reported ten years ago in the same area. approximately a half of carriages were covered by either pcv10 or pcv13. the serotypes distribution should be conducted to other areas to achieve the overall figure of pneumococcal pharyngeal isolate coverage by pcv background: invasive disease due to streptococcus pneumoniae is one of the leading cause of death in children under 5 years of age. nasopharyngeal colonization precedes the invasive disease in some children. factors influencing nasopharyngeal colonization by s. pneumoniae would probably help in prevention of serious illness such as pneumonia, meningitis and septicaemia. aims: to study the demographic profile of children with nasopharyngeal colonisation of s. pneumoniae. methods: a total of 500 children between 6 months to 5 years were included in the study. a detailed questionnaire was filled for each child to delineate the vaccination status, underlying risk factors and socioeconomic status. nasopharyngeal swabs were obtained from children following strict protocols and were plated immediately in the appropriate culture plates. results: out of the 500 children, 45 had grown s. pneumoniae in the nasopharyngeal swab culture. majority was males, less than 2 years of age and belonged to families with a lower income. other risk factors that were identified were average ventilation in the house hold and over crowding in a few of them. majority had received hib vaccine. smoking, duration of breast feeding, attending day care and predisposing respiratory infection or use of antibiotics did not influence nasopharyngeal colonization. conclusion: nasopharyngeal carriage of s. pneumoniae is common in male children less than 2 years of age belonging to families with a lower income. background and aims: acute respiratory tract infection (ari) is a leading cause of morbidity and mortality in young children. streptococcus pneumoniae is the most common bacterial pathogen of pneumonia in children. we used ari surveillance platform to identify common bacterial pathogens associated with acute respiratory symptoms in children below 10 years. methods: a dynamic cohort of children (population at inception 2754) in four villages of ballabgarh was kept under weekly surveillance from aug'12 to aug'13. all children were screened weekly for presence of any respiratory symptom (cough, sore throat, ear ache/discharge, nasal discharge/congestion, shortness of breath/respiratory difficulty). these ari cases were further assessed by nurses and classified into acute upper respiratory tract infection (auri) and acute lower respiratory tract infection (alri) as per who guidelines. age-matched healthy controls from same village were also selected. naso-pharyngeal swabs were taken from all-alri, controls and 5% of auri. primary cultures were done on blood agar and chocolate agar at aiims. results: in 2666 child years of surveillance in last one year -408 alri, 793 auri and 243 control naso-pharyngeal samples were taken. s. pneumoniae was found positive in 61 samples (alri-32;7.8%, auri-20;2.5%, control-9;3.7%), haemophilus influenzae positive in 21 samples (alri-13;3.2%, auri-7;0.9%, control -1;0.4%) and staphylococcus aureus positive in 6 samples (alri-5;1.2%, auri-1;0.1%, control -nil). the incidence of s. pneumoniae, h. influenzae and s. aureus in children with alri was 12.0 (95%ci:8.3-16.7), 4.9 (95%ci:2.7-8.1) and 1.9 (95%ci:0.7-4.2) per 1000 child years respectively in the population. conclusion: s. pneumoniae was found to be the most common bacterial respiratory pathogen followed by h. influenzae and s. aureus in children with alri in this community. conclusion: pneumococcal nasopharyngeal colonization has changed profoundly since the introduction of conjugate vaccines and overall colonization by pneumococcus has declined in recent years. by 2012, non-vaccine serotypes have nearly completely replaced vaccine serotypes. the impact on clinical disease remains to be seen. results: 827 pneumococcal isolates were collected over these 3 periods; an overall pneumococcal carriage rate of 63%. prevalence rates varied from 51% in 2004 (188/370 samples) to 66% in 2008 (145/220 samples) and 68% in isppd-9 / pneumonia 2014 mar 9-13; 3:1-286 2012 (494/723 samples) . also the prevalence of individual serotypes/groups covered by pcv13 varied considerably on the 3 time points with a natural increase in prevalence of serotype 1 (0 to 13%), 4 (0 to 2%) and 14 (6 to 12%) and a natural decrease of serotype 18c (1 to 0.3%). serotypes/groups 6 and 23f remained stable (approx. 30%) whereas non-vaccine serotypes 16, 34 and 38 emerged. the theoretical pcv13 coverage varied in time between 66% and 87%. conclusions: overall pneumococcal carriage rates, serotypes distribution and theoretical vaccine coverage were highly variable between 2004 and 2012. we conclude that surveillance of pneumococcal carriage at a single timepoint can under-or overestimate the long-term impact of pcv-13 in high-risk populations. background and aims: portugal is one of the few countries where pcvs are widely used despite not being part of the national immunization program (nip) nor being reimbursed. we evaluated the evolution of serotypes carried by young children over a 17-year period, spanning the pre-vaccine era up to the early-pcv13 era. methods: nasopharyngeal swabs were obtained from children (0-6 years) attending day-care in lisbon/oeiras area in yearly cross-sectional studies conducted between 1996-2012. pneumococci were isolated and serotyped. pcv use was extracted from the child's immunization bulletin. results: 8,313 swabs were obtained (average 593/year). carriage remained stable (average 61.5%). use of pcv7 gradually increased to >70% between 2007-2010. pcv10 use was low (≤5%). pcv13 use was 63% by 2012 (among those born after 2009). major shifts have occurred in circulating serotypes (figure) . pcv7 types have declined substantially but are still in circulation. in 2012, the 6-extra pcv13 types and non-pcv13 types (including 6c) accounted for ~10% and ~75% of all isolates, respectively. in 2012, the 6-extra pcv13 types were significantly less frequent among pcv13 vaccinated than among pcv13 non-vaccinated carriers (2.9% vs 19.6%, p<0.001). isppd-9 / pneumonia 2014 mar 9-13; 3:1-286 conclusion: significant changes in carried serotypes have been occurring although not as dramatic as those reported in countries that introduced pcvs in the nip. whether these changes will be sustained is uncertain. health authorities should consider universal introduction of pcvs in portugal. objective: to provide epidemiological data on streptococcus pneumoniae carriage in high risk population, this study focuses on the serotype distribution and antibiotics susceptibility of s. pneumoniae carriage in hiv-infected children in jakarta, indonesia. materials and findings: nasopharyngeal swabs were collected from 90 hiv sero-positive children age 4 to 144 months. s. pneumoniae was identified by conventional and molecular methods. serotyping, antibiotics susceptibility test, and sequence type were performed by sequential multiplex pcr, the disk diffusion method and mlst respectively. we identified s. pneumoniae in 42 children (46.7%). serotype 19f was identified most commonly (n = 8, 19.0 %) followed by 9a and 6a/b (n = 4, 8.5%), 23f (3 isolates), 9v, 35b, 11a (two isolates each) and serotypes 18, 12f, 15b/c, 3 and 35f (single isolates each). antibiotic susceptibility was observed for chloramphenicol (85.4%), erythromycin (73.2%), clindamycin (78.0%)) and sulphamethoxazole-trimethoprim (39.0%). furthermore, multidrugs resistant isolates of serotype 19f belonging to the st320 clone (3 isolates) and the st263 clone (2 isolates) were identified. conclusion: our study gives insight into prevalence and serotype distribution of s. pneumoniae strains among young hiv patients in indonesla. these findings may help to decide upon potential preventive strategies targeting invasive pneumococcal disease in indonesia. background: invasive disease due to streptococcus pneumonia is one of the commonest causes of death in children. some pneumococcal serotypes that colonizes nasopharynx of healthy children can lead to invasive disease. it is important to identify the serotypes colonizing the nasopharynx to enable prevention by way of vaccination. aims: to determine the serotype profile of s. pneumonia isolated from healthy children. methods: a total of 500 children between 6 months to 5 years were included. a detailed questionnaire was filled to delineate the vaccination status, underlying risk factors and socioeconomic status. nasopharyngeal swabs were taken as per protocol and plated immediately.. serotyping was done using statens kit. results: 45/500 grew s. pneumoniae. 6b was the commonest serotype identified (16/45), most of them were infants with overcrowding and improper ventilation.6 children who had 23f serotype were > 2 years of age and overcrowding was again found to be a significant risk factor. 19f was seen in 5 children and there was no significant risk factor associated. serotypes 14, 4,18c, 19v and nontypeable were seen in 3, 3, 1, 1, and 10 children respectively. factors like duration of breast feeding, smoking and previous respiratory infection did not have any influence on the carriage rate. conclusion: nasopharyngeal carriage was found to be common in healthy children without any significant risk factors. serotype 6b was the commonest serotype isolated. background and aims: we recently published a set of updated world health organization (who) standard methods for detecting pneumococcal carriage, including sample collection, transport and storage, pneumococcal identification and serotyping. methods: we identified key changes in the 2013 revised recommendations compared with the 2003 guidelines. results: a single nasopharyngeal swab in stgg medium remains the sample of choice. important changes are: new recommendations or major changes specimen type document swab quality collect nasopharyngeal and oropharyngeal swabs from adults. calcium alginate, rayon, dacron or nylon swabs are acceptable for culture-based studies. swabs of inert material (e.g. nylon or dacron) should be used for molecular methods. flocked swabs have many desirable characteristics. swab collection, transport and storage 1 ml stgg medium. freeze at -80°c as soon as possible, no 'safe limit' for 4°c storage, store at -20°c for days (not weeks). culture 10 µl of specimen, record if larger volume. coba and cna plates acceptable alternatives to gentamicin-blood agar. recording semi-quantitative growth is optional at least one α-haemolytic colony (representing the dominant morphology). if other colonies are tested, results should be analysed separately. real-time lyta is the current best molecular identification approach. optochin non-susceptible isolates should be tested for bile solubility. 'wet' or 'dry' quellung method is gold-standard. latex agglutination acceptable, other methods may be suitable. important areas for future research will be described. there are important differences between the 2003 and 2013 guidelines. adherence to standard methods will reduce variability when conducting pneumococcal carriage studies. background and aims: the impact of viral upper respiratory tract infection (urti) on pneumococcal load in the human nasopharynx is uncertain. we hypothesised that acquisition of a viral urti would be associated with an increase in nasopharyngeal pneumococcal concentration in children <5 years, followed by a subsequent decrease. methods: all members of 47 households from kilifi district, coastal kenya, had nasopharyngeal swabs (nps) collected twice weekly during jan-june 2010 and tested by a respiratory multiplex pcr. swabs from children <5 years old collected two weeks before, during and four weeks after a first episode of respiratory syncytial virus (rsv) or rhinovirus with urti symptoms were also tested by quantitative real-time pcr for lyta and alu targets as a measure of pneumococcal load per μg of human dna present in the swab transport medium. results: fifty-three viral episodes were available for study. pneumococcal carriage was universal among study participants and co-infecting viruses were present in 35% of swabs. the geometric mean increase in pneumococcal concentration with acquisition of viral infection was 4-fold, p<0.001, and remained so when the analysis was restricted to swabs without co-infecting viruses. pneumococcal concentration fell 0.6-fold after viral urti but without statistical significance, p=0.144. conclusion: the modest increase in pneumococcal concentration may be an important contributing factor to the development of bacterial pneumonia or invasive pneumococcal disease among children with preceding viral respiratory tract infections at a population level. the impact on population level transmission of pneumococcal carriage during viral urti is potentially even greater. with the worldwide introduction of pneumococcal conjugate vaccine (pcv), it is imperative that carriage serotypes in children are determined in areas of vaccine introduction. pcv10 was introduced in pakistan in april 2013. methods: from two districts of sindh (karachi and matiari) 698 nasopharyngeal swabs were collected from children aged 3 months to 5 years in january 2013 (before pcv-10 introduction). swabs were subcultured on sheep blood agar and isolates with suggestive colony morphology were tested for optochin sensitivity. we found that 561 (80.4%) of the specimens yielded pneumococci. a sampling of 100 isolates, representing 100 different swabs, were shipped to the centers for disease control and prevention(cdc), atlanta ga, for serotyping using sequential real time triplexed-pcr reactions according to published cdc protocols. results: fifty-three of 100 isolates have been serotyped. of these 34 %(n=18) were 6a/b/c/d, 20.8% (n=11) were 23f, 15.%1(n=8) were 19a, 7.5%(n=4) were 19f, 7.5%(n=4) were 18c/f/b/a, 7.5%(n=4) were 9v/a, 5.7%(n=3) were 14 and 1.9%(n=1) were determined to be serotype 4. serotype determination of remaining 47 isolates is ongoing. resolution of common serogroups into individual serotypes will be carried out by quellung at cdc. conclusion: this is the first report of nasopharyngeal pneumococcal serotypes from pakistan. nasopharyngeal carriage rate among children in lower sindh is high. data obtained so far reveals high prevalence of vaccine serotypes (all either pcv10 or pcv10-related). complete serotyping of all 561 pneumococcal isolates is critical to confirm these early observations. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 k.m. shea 1 , a. rabbani 1 , s.i. pelton 2 1 epidemiology, boston university school of public health, boston, usa; 2 pediatrics, boston university school of medicine, boston, usa background: children with certain chronic diseases are at increased risk for ipd, but it is not known whether this is because they are more likely to be colonized with streptococcus pneumoniae, or because they are more likely to develop disease once colonization has occurred. methods: we compared the rate of s. pneumoniae colonization in children <5 years with underlying comorbidities to the rate in children without comorbidities. demographic information and nasopharyngeal specimens were collected from 2,441 children <5 years seen in primary care at a large urban u.s. hospital after introduction of pcv13, from july 2010-july 2013. standard microbiologic methods were used to characterize pneumococcal serotypes, and patient records were used to ascertain comorbidities. results: one third (n=937) of our study population had ≥1 comorbidity. among 2,211 recorded comorbidities, wheezing was the most common (61.6%), followed by sickle cell haemoglobinopathies (12.3%), prematurity (12.2%), and chronic heart disease (7.0%). children with a diagnosis of wheeze or asthma in their clinical record had a higher rate of colonization than children without a wheeze or asthma diagnosis (26% vs. 21%, p=0.054). although not statistically significant, children with other chronic lung (22% vs. 18%) and kidney diseases (30% vs. 22%) also had higher rates of s. pneumoniae colonization. conclusion: children with medical record evidence of wheeze appear to have increased rates of s. pneumoniae colonization. future analyses will provide insight into whether this association persists after controlling for covariates such as age and vaccination. funding: aspire award in pediatric vaccine research from pfizer, inc. background: nasopharyngeal carriage is a precursor for pneumococcal disease and can be useful for evaluating pneumococcal conjugate vaccine (pcv) impact. we studied pre-pcv pneumococcal carriage among hiv-infected and -uninfected children in mozambique. methods: between october 2012 and march 2013, we enrolled hiv-infected children age <5 years presenting for routine care at seven hiv clinics in 3 sites, including maputo (urban-south), nampula (urban-north), and manhiça (rural-south). we also enrolled a random sample of hiv-uninfected children <5 years old from a demographic surveillance site in manhiça. a single nasopharyngeal swab was obtained and cultured following broth enrichment. pneumococcal isolates were serotyped by quellung reaction. (13%), 23f (13%), 6a (9%), 6b (6%), 19a (5%), 13 (5%) and 14 (5%) were most common; serotype could not be determined for 36 (6%). the proportion of isolates included in the 10-and 13-valent vaccines (pcv10 and pcv13) was 47% and 64%, respectively, with no significant differences by hiv status. conclusion: pneumococcal carriage was common, with little variation by geographic region or hiv status. serotype coverage of pcv10, which was introduced in mozambique in april 2013, was lower than that of pcv13. ongoing carriage studies will show whether pcv10 will have similar benefits for hiv-infected and -uninfected children. background and aims: determining nasopharyngeal carriage of pneumococcal serotypes is a valuable epidemiological surveillance tool. however, current serotyping methodologies are limited in their ability to detect multiple serotype carriage, thus underestimating the complexity of carriage by missing rare or low abundant serotypes. the health protection agency conducted a longitudinal nasopharyngeal carriage study (a) of 489 individuals in england over 10 months pre-pcv7 in 2001/2 and a cross-sectional study (b) of 400 individuals post-pcv7in 2009. conventional culture and serotyping identified 932 and 125 swabs containing pneumococci respectively. the swabs were stored at -70 o c in skimmed milk, tryptone, glucose, glycerol broth (stgg). the present study used a dna microarray to re-examine a sample of swabs previously yielding a single serotype to assess the prevalence of co-colonisation with multiple serotypes. methods: 410/932 pneumococcal-positive swabs from study a and 115/125 from study b were re-examined using a molecular serotyping dna microarray (hinds et al). results: 17% samples contained multiple serotypes: 14% contained two, 2% three and 0.4% four serotypes respectively. a further 13% samples contained "serotype variants" where cps loci did not match known serotypes; possibly indicating non-typable pneumococci, novel serotypes or closely related streptococcus spp. conclusion: the dna microarray detected 17% co-colonisation in a subset of swabs from previous carriage studies, despite long-term storage at -70 o c for several years. conventional methods had only detected 0.16% co-colonisation. emergence of non-vaccine serotypes post-conjugate vaccine introduction may partly result from unmasking rather than serotype replacement. co-colonisation will have a significant impact on future modeling studies. background: the cholesterol-dependent pore-forming toxin pneumolysin is a key virulence factor of streptococcus pneumoniae. it has been shown to be the cause of apoptosis and complement activation, as well as cause damage to tissues in the lung and brain. detection of pneumolysin by tlr4 has been described; however reports are conflicting, depending on the model and bacterial strain used. there are very few reports about the role of tlr4 in the detection of pneumolysin in human primary immune cells. methods: human monocyte-derived dendritic cells (dcs) were treated with sirna to tlr4 and infected with unencapsulated serotype 4 pneumococci (t4r), the pneumolysin mutant (t4rδply) or treated with highly purified recombinant pneumolysin. the secretion of the pro-inflammatory cytokine il-12p70 was measured in the cell culture supernatant of infected dcs. results: the pneumolysin mutant t4rδply induces il-12p70 secretion levels in dcs which are up to 2000 times higher than the levels induced by t4r. the il-12p70 secretion in response to both strains is dependent on the uptake of the bacteria by dcs and does not require tlr4. recombinant pneumolysin, in contrast, induces tlr4dependent secretion of il-12p70. conclusion: we do not currently understand the complex signaling effects of pneumolysin on dcs. our results indicate that the stimulation with purified toxin has different effects on dcs than in the context of infection with whole bacteria. we hypothesize that pneumolysin has an inhibitory function in the context of bacterial infections and we are setting out on investigating this effect with our future studies. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 introduction: because children are the main transmitters of pneumococci, carriage studies focussing on the young provide good information on the circulating serotypes. in 2011, the 13-valent pneumococcal conjugate vaccine (pcv13) replaced the 7-valent vaccine (pcv7), which was used since 2006 for childhood immunisation in norway. we assessed vaccine-induced changes in carriage among children younger than 6 years by comparing the pcv13 sample with a pre-vaccine and pcv7 sample. methods: a cross-sectional sample with questionnaire data and nasopharyngeal swabs was obtained in autumn 2013 from children attending day-care centres in and near oslo, the norwegian capital. all isolates were serotyped and antimicrobial-susceptibility was tested. results were compared to samples obtained in autumn 2006 (611 swabs, 539 isolates) and autumn 2008 (602 swabs, 563 isolates). results: we obtained 367 swabs with 244 isolates (carriage-prevalence 62% [95%ci 54-70]) but continue sampling until ≥800 swabs are obtained. carriage was highest among the youngest (<24 months; 80% [95%ci 67-89]). in the pre-vaccine and pcv7 samples, the overall prevalence was 78% [95%ci 73-82] and 80% . the prevalence of pcv13-serotypes decreased from 540/1000 to 316/1000 to 93/1000 children, respectively. the decrease was observed in all age-groups. while after pcv7 introduction the prevalence of non-vaccine serotypes increased from 336/1000 to 606/1000, after switching to pcv13, the prevalence remained constant (569/1000). non-pcv serotypes 21, 35f, 11a and 23a have become the dominant serotypes. conclusion: preliminary results indicate decreased carriage of vaccine serotypes after switching from pcv7 to pcv13, with no increase in carriage of non-vaccine serotypes. background: three leading pathogens responsible for bacteremic pneumonia in hiv infected children are streptococcus pneumoniae, staphylococcus aureus, and haemophilus influenzae. bacterial interference, or competition amongst organisms in the nasopharyngeal space, is well documented. studies investigating dynamics of these associations in hiv infected mother and children are not well reported. aim: to investigate bacterial associations of s. pneumoniae, s. aureus, and h. influenzae in the nasopharynx of mother and children with hiv infection methods: four nasopharyngeal samples were collected from pneumococcal vaccine naïve, hiv infected children ages 2-15 years, and their mothers, at two month intervals over the course of six months. calcium alginate swabs were collected and placed in stgg media and processed in the microbiology laboratory using standardized protocols. we have collected about 404 from children and 375 from mother, till now. overall dual carriage with pneumococcus and s. aureus was found to be 13/404 in children and 3/375 in mothers (all hiv infected). similarly, dual carriage with pneumococcus and h.influenzae was found to be 25/404 in children and 2/375 in mothers. a positive association was seen between pneumococcus and h. influenzae [odds ratio (for mother): 13.84; 95% ci : 1.06-126.82; odds ratio (for children): 5.62; 95% ci : 2.74-11.66]. any association between pneumococcus and s. aureus was not found in hiv infected mother or children [odds ratio (for mother) : 1.29; 95% ci: 0.23-4.78; odds ratio (for children) : 0.67; 95% ci: 0.32-1.33]. no conflict of interest isppd-9 / pneumonia 2014 mar 9-13;3:1-286 the perch study group 1 1 international vaccine access center, johns hopkins bloomberg school of public health, baltimore, usa background: low specificity of definitions for pneumococcal pneumonia (pp) limits evaluation of pneumococcal conjugate vaccine (pcv) impact. we investigated the usefulness of absent streptococcus pneumoniae carriage in assessing pp. methods: perch studies severe/very severe hospitalized pneumonia among children aged 28d-59m. the presence of nasopharyngeal (np) carriage by pcr (lyta) or culture was calculated among cases using microbiologicallyconfirmed pneumococcal pneumonia (mcpp), who-defined alveolar consolidation (cxr-ac), and cxr-ac or other infiltrate plus elevated c-reactive protein (crp) as outcome measures. odds ratios, adjusted for site and age, were calculated. *south africa only npv of pneumococcal carriage for mcpp was relatively high (table) . npv for the cxr-ac outcomes were lower. stratifying by prior antibiotic use and pcv status had little impact on findings. npv decreases as prevalence of pp increases and specificity decreases (figure). figure: negative predictive value of carriage as a function of specificity and prevalence of pneumococcal pneumonia among childhood cases of severe/very servere hospitalized isppd-9 / pneumonia 2014 mar 9-13;3: conclusion: absence of pneumococcal carriage might increase specificity of pp definitions which might be applicable in pcv impact evaluations. however, the lower npvs for cxr-ac and for cxr-ac/other infiltrate plus elevated crp need further clarification. pediatrics, queen silvia's children's hospital sahlgrenska university, gothenburg, sweden; 2 pediatrics, sahlgrenska university hospital, gothenburg, sweden background and aims: the serotype distribution of pneumococci has changed after large-scale use of pneumococcal conjugate vaccines (pcv). data indicate that serotypes also changed over time before availability of pcv. the aim of this study was to compare serotypes of pneumococci obtained from normally sterile body fluids in 2 swedish studies with strains isolated during the 1970s + 1980s and during 1998 -2001, i.e. long before any pcv was available. methods: 215 strains were serotyped in the old and 836 strains in the new study. serotyping was performed with the capsular swelling test. results: the 7 most common serotypes in the first study were in descending order 3, 4, 14, 7f, 23f, 6a and 6b and in the new study 1, 7f, 9v, 14, 4, 12f and 6b. significant increases were seen in serotypes 1 (2 -14 %), 9v (2 -9 %) and 12 f (<1 -6 %). serotype 3 decreased significantly (12 -4 %). serotype 19a, which has increased significantly in the us after widespread use of pcv7 and in 2 countries before pcv7 was introduced, (israel, korea), remained unchanged in the present comparison (3.7 and 3.1 %). conclusions: major changes in the serotype distribution occurred in sweden (as in many other countries) before any pcv was available. changes in serotype distribution may therefore in some cases be due to natural changes and not only to replacement of vaccine types with nonvaccine types. studies have demonstrated that carriage is a necessary precondition for invasive pneumococcal disease (ipd). we assessed pneumococcal np colonization prevalence in india and in neighboring countries to assess if there were systematic differences. methods: we reviewed published and unpublished studies reporting on pneumococcal colonization prevalence among healthy children <15 years of age in india and neighboring countries from 1993 to 2013. study characteristics, colonization prevalence, and antibiotic resistance were systematically abstracted. we compared agespecific colonization prevalence in india to other countries. results: we identified 15 pneumococcal np colonization studies from india and 21 from surrounding countries. in all studies, colonization prevalence increased immediately following birth and peaked between 5-9 months of age; peak prevalence for studies in india (range: 46-77%) was similar to other countries (42-85%). colonization prevalence did not decline markedly (>30%) in any studies during age 1-10 years, except one india study with a rapid decrease after age 7 months. pneumococcal np colonization prevalence after age 1 year in india (range 20-57%) overlapped with prevalence in other countries (28-83%). data were insufficient to determine whether antimicrobial resistance or antibiotic use explained variation in colonization between studies. conclusions: pediatric pneumococcal np colonization prevalence in india is similar to surrounding countries. risk factor prevalence for np colonization was not reported and may explain colonization variation between studies. pneumococcal colonization prevalence varies between studies but does not systematically vary between countries in south and southeast asia. isppd-9 / pneumonia 2014 mar 9-13;3:1-286 pediatric immunology and infectious diseases, wilhelmina children's hospital university medical center utrecht, utrecht, netherlands; 2 centre for immunology of infectious diseases and vaccines, national institute for public health and the environment (rivm), bilthoven, netherlands; 3 diagnostic microbiology, regional laboratory of public health, haarlem, netherlands background and aims: the gold standard for streptococcus pneumoniae carriage detection is the conventional culture of a nasopharyngeal swab. saliva however, has a history of being one of the most sensitive methods in surveillances on pneumococcal colonisation (heffron, 1939) . here, we compared the sensitivity of nasopharyngeal swabs and saliva samples from pcv7-vaccinated 24-month-old children tested for s. pneumoniae and nasopharyngeal serotype carriage using conventional and molecular diagnostic methods. methods: nasopharyngeal and saliva samples were simultaneously collected from 289 asymptomatic 24-month-old children, cultured and pneumococcal strains were serotyped by quellung method. dna extracted from harvests of all bacterial growth on pneumococcus-selective medium was tested for s. pneumoniae and serotypes present using quantitative-pcr (qpcr) targeting species-specific genes lyta (carvalho, 2007) and piaa and sequences specific for subset of serotypes (azzari, 2010). results: altogether 240 (85%) of 289 24-month-old children were identified as carriers by any method. molecular detection of s. pneumoniae in culture-enriched nasopharyngeal samples had highest sensitivity (73%) followed by qpcr of cultured saliva (60%) and conventional culture of nasopharyngeal swabs (57%). isolation of live pneumococci from saliva generally failed due to abundant polymicrobial growth. for the subset of serotypes targeted by qpcr (1, background and aims: saliva has a history as being one of the most sensitive methods for streptococcus pneumoniae carriage detection (heffron, 1939) . here, we applied molecular diagnostic methods to study pneumococcal carriage in saliva from schoolchildren. methods: saliva was collected from 50 students (aged 5 to 10 years) of a rural school near utrecht, transported to the lab on wet ice, cultured and the remaining volume stored frozen. cultures were inspected for s. pneumoniae colonies and then all bacterial growth was harvested. dna extracted from raw and culture-enriched samples was tested for the presence of s. pneumoniae specific genes lyta and piaa using quantitative-pcr (qpcr) and considered positive when both genes had c t values below 40. sample serotype composition was determined in dna extracted from culture-enriched saliva samples using qpcr (azzari, 2010) and by analysing sequences generated by conventional pcr (carvalho, 2010; carvalho 2013) . results: two children (4%) were culture-positive for s. pneumoniae. thirty-two (64%) children were qpcr-positive for s. pneumoniae in raw saliva and 44 (88%) in culture-enriched samples. using molecular methods to determine sample serotype composition, we detected 83 pneumococcal strains of 22 serotypes in 40 of 50 samples from carriers, with 26 carriers (59% of 44) positive for 2 to 6 serotypes. conclusion: conventional culture detection of s. pneumoniae in saliva is extremely difficult due to saliva's polymicrobial nature. these limitations were addressed by combining culture-enrichment and sensitive molecular methods, resulting in more than ten-fold higher rates of pneumococcal carriage and high rates of co-colonisation detected in schoolchildren. pneumococcal colonization and carriage comparison of the 2003 and 2013 world health organization (who) guidelines for detecting upper respiratory carriage of streptococcus pneumoniae department of infectious disease surveillance and control, national institute for health and welfare portugal; 10 epidemiology and demography, kemri-wellcome trust research programme, kilifi, kenya; 11 vaccine programme unit, national institute for health and welfare key: cord-004031-sw60qbbj authors: aylward, ryan e.; van der merwe, elizabeth; pazi, sisa; van niekerk, minette; ensor, jason; baker, debbie; freercks, robert j. title: risk factors and outcomes of acute kidney injury in south african critically ill adults: a prospective cohort study date: 2019-12-10 journal: bmc nephrol doi: 10.1186/s12882-019-1620-7 sha: doc_id: 4031 cord_uid: sw60qbbj background: there is a marked paucity of data concerning aki in sub-saharan africa, where there is a substantial burden of trauma and hiv. methods: prospective data was collected on all patients admitted to a multi-disciplinary icu in south africa during 2017. development of aki (before or during icu admission) was recorded and renal recovery 90 days after icu discharge was determined. results: of 849 admissions, the mean age was 42.5 years and mean saps 3 score was 48.1. comorbidities included hypertension (30.5%), hiv (32.6%), diabetes (13.3%), ckd (7.8%) and active tuberculosis (6.2%). the most common reason for admission was trauma (26%). aki developed in 497 (58.5%). male gender, illness severity, length of stay, vasopressor drugs and sepsis were independently associated with aki. aki was associated with a higher in-hospital mortality rate of 31.8% vs 7.23% in those without aki. age, active tuberculosis, higher saps 3 score, mechanical ventilation, vasopressor support and sepsis were associated with an increased adjusted odds ratio for death. hiv was not independently associated with aki or hospital mortality. ckd developed in 14 of 110 (12.7%) patients with stage 3 aki; none were dialysis-dependent. conclusions: in this large prospective multidisciplinary icu cohort of younger patients, aki was common, often associated with trauma in addition to traditional risk factors and was associated with good functional renal recovery at 90 days in most survivors. although the hiv prevalence was high and associated with higher mortality, this was related to the severity of illness and not to hiv status per se. acute kidney injury (aki) is commonly encountered in the intensive care unit (icu) [1] , but with a widely variable reported incidence due to non-standardization of its definition [2] . regardless of the definition used, aki is a well-recognized independent risk factor for mortality, is associated with substantial morbidity and is a current major cause for global concern [3] [4] [5] [6] . furthermore, aki requiring dialysis is now recognized as a risk factor for end stage kidney disease in the long term [7, 8] and is associated with poor long-term quality of life after icu discharge [9, 10] . aki has been well characterized in high income (hi) countries and appears to be increasing in incidence [5, [11] [12] [13] . however, there is a marked paucity of data from african icu's concerning the incidence, aetiology and effect of aki on mortality and functional renal recovery, where the prevalence of hiv and trauma is high and where resources are often limited [6, 14, 15] . renal replacement therapy is an expensive [16] and scarce resource in south africa [17] and in particular, in the rest of sub-saharan africa [18, 19] . the international society of nephrology has boldly called for 0by25: the elimination of preventable deaths from aki by 2025 [20] . timely diagnosis and prevention remain the most important strategies. accordingly, understanding the epidemiology of aki in lower and middle income (lmi) countries must be a key step in tackling this problem. our aim therefore, was to describe the epidemiology of aki in patients admitted to our south african multidisciplinary icu, where there is currently a high prevalence of hiv and traumaassociated admissions. we sought to characterize the factors associated with the development of aki, the effect of hiv on aki as well as survival and to report on the 90-day renal function outcome of all who developed aki. an observational prospective design was used. cohorts were divided into those who did and did not develop aki (prior to and/or after admission to the icu) as well as by hiv status where known. all patients older than 12 years admitted to the livingstone hospital icu between 3 january 2017 and 3 january 2018 were included. patients who died within 6 h of being admitted to icu, those who were brain-dead awaiting organ harvesting, and patients with known or presumed end stage kidney disease were excluded from the study. the livingstone hospital adult icu is a tertiary service, closed, multi-disciplinary 16-bed unit serving a catchment area of 1.6 million people. the hospital is government-funded and is located in the nelson mandela bay metropole in south africa where 1.15 million people live in an urban setting, 12.3% of whom live in informal shack dwellings and 36.6% of whom are unemployed. the balance of 450,000 people live in surrounding rural areas within a radius of 250 km [21] . full time consultant supervision is provided by two intensivists and two nephrologists. the provision of dialysis is also government-funded and not restricted within the icu or by hiv status. however, general prognosis and current resource limitations are taken into consideration prior to the admission of any patient to the icu [22] . the modality of dialysis is chosen by the treating consultant based on clinical status with a preference for intermittent haemodialysis or sustained low efficiency daily dialysis (sledd) due to cost constraints. continuous renal replacement therapy (crrt) is reserved for severely haemodynamically unstable patients and for those with raised intracranial pressure. referring disciplines include medicine, trauma, general surgery, urology, neurosurgery, orthopaedics, obstetrics and gynaecology. elective cardiology and cardio-thoracic surgery have their own dedicated icu. obstetrics also has their own high care although patients with advanced organ dysfunction are referred to our unit. aki was diagnosed and staged according to the kidney diseases improving global outcomes (kdigo) definition [23] . a normal serum creatinine not older than 90 days was assumed to be the baseline where available, as recommended [24] . the cause of aki was determined by the treating intensivist/nephrologist and more than one cause could be assigned. aki was recorded as resolved once creatinine improved to the known or presumed baseline. if renal function had not recovered by hospital discharge, patients were followed up for at least 90 days following icu discharge or until renal recovery. patients who had not recovered their renal function by this time were deemed to have chronic kidney disease (ckd). approval for the study (protocol number: 067/2016) was granted by the walter sisulu university human research ethics unit. since we were conducting a nonexperimental study that would not influence clinical decision-making or patient management, the need for study participant consent was waived by the ethics unit. demographic data including age, sex, race and details related to co-morbidities were recorded. for patients known with hiv, a premorbid cd4 count and viral load was recorded, where available. the simplified acute physiology score 3 (saps 3) [25] was calculated within the first hour of icu admission. the sequential organ failure assessment (sofa) [26] was calculated 24 h after admission and every third day thereafter, or sooner if the patient's condition deteriorated. vasopressor and mechanical ventilation requirements were also recorded. cause of aki, renal replacement modality and creatinine on admission, peak and discharge were recorded for patients in the aki cohort. sepsis was defined using sepsis-3 criteria [27] . data were exported from the research electronic data capture (redcap) hosted at the university of cape town [28] and analyzed with rstudio, 2017 (version 3.4.2). hypothesis tests were considered significant if the two-sided p-value < 0.05. continuous data were tested for normality using the kolmogorov-smirnov, shapiro-wilk, anderson-darling and pearson's chi-squared tests. normally distributed data are reported as means (standard deviation) and skewed data as medians (interquartile range). discrete data are presented as numbers (percentages). the student's t-test and the mann-whiteney u test were used to compare continuous data and the chisquare and fischer's exact test were used for discrete data, as appropriate. missing outcome data (n = 12) were analysed using multiple imputation. hazard ratios for mortality by aki and hiv status were calculated using the cox proportional hazards model. multivariate logistic-regression models were used to determine associations of developing aki and dying. variables by bivariate analysis with an alpha level < 0.1 between aki and non-aki cohorts as well as known predictors for aki (age, sex, hypertension, active malignancy and admission saps 3 score) were included in the model. kdigo stages 1, 2 and 3 were compared to patients who did not develop aki as reference. a total of 875 patients were admitted to the icu during the study period and 26 were excluded from the analysis; fig. 1 details the reasons for exclusion. vital status after icu discharge could not be established for 12 patients due to in-patient transfers to other hospitals and the unavailability of further records; six were in the aki cohort, and outcomes were imputed. in-hospital mortality was 21.6% while mortality in icu was 13.4%. the most common diagnoses admitted were: assault (14%), motor and pedestrian vehicle accidents (12%), acute abdomen (10%), pneumonia (6%; including cases later identified as tuberculosis) and self-inflicted drug/toxin overdose (3.5%). admissions included surgical emergencies (n = 451, 53.1%), medical emergencies (n = 261, 30.7%), surgical elective cases (n = 110, 13%) and obstetric emergencies (n = 27, 3.2%). table 1 shows the baseline characteristics of all patients admitted to the icu and by aki status. aki developed in 497 patients (58.5%), 80.5% of whom were diagnosed with aki on admission to the icu. the maximum kdigo stage was stage 1 in 138 (27.7%), stage 2 in 152 (30.6%) and stage 3 in 207 (41.7%) patients respectively. figure 2 shows the main causes identified for developing aki. herbal ingestion was only documented in 4 patients and 12 patients were exposed to antituberculous drugs (rifampicin and isoniazid). of the 105 hiv positive patients who developed aki, 24 had received tenofovir prior to aki diagnosis. histology was obtained in situations where the cause of aki was not clear or the clinical course of the patient was unclear. kidney biopsies were performed in 5 patients (1% of the aki cohort), only 1 of whom had hiv which showed interstitial hiv associated nephropathy. the others were in hiv negative subjects; 3 showed features of mesangiocapillary glomerulonephritis, 1 of which was crescentic and 1 had features of ascending pyelonephritis. risk factors associated with aki are presented in table 2 . on multivariate analysis, male gender, increased sofa and/or saps 3 score, increased length of stay, the need for vasopressor drugs and eighty-eight patients were dialyzed (42.5% of patients with stage 3 aki, 18.0% of the cohort with aki and 10.4% of the entire cohort), 67.0% of whom were fig. 3 ). the most common indications for dialysis initiation were life-threatening hyperkalaemia (44.3%), uraemic symptoms such as encephalopathy or seizures (34.1%) and refractory metabolic acidosis (34.1%). the development of aki was associated with a higher in-hospital mortality rate of 31.8% compared to 7.2% in those without aki (hazards ratio 4.07, 95% ci 2.66; 6.21; logrank p < 0.001 (fig. 4) ). further, the odds of dying increased stepwise with increasing kdigo aki stage (fig. 5) in the aki cohort, an increased adjusted odds ratio for death was observed with increasing age, active tuberculosis, higher saps score, receipt of mechanical ventilation, receipt of vasopressor support and in those with sepsis (table 3) . hiv infection was associated with worse survival in those with aki (fig. 6) . however, on multivariate analysis, hiv was not found to be an independent risk factor for mortality (table 3) . patients with hiv and aki were more severely ill on admission with mean saps 3 score (sd) of 58.2 (15.9) versus 53.3 (13.8), p = 0.007 and sofa (iqr) of 8 (4; 11) versus 6 (3; 9), p = 0.01. they also had a higher likelihood of having active tuberculosis [or (95%ci) = 3.55 (1.42; 9.36), p = 0.008]. the median premorbid cd4 count was significantly lower in those with aki than without (204.5 cells/microliter versus 404 cells/microliter, p = 0.025) but viral suppression was similar in both groups (viral load log 2.0 vs 2.5 respectively, p = 0.234). although icu length of stay was less in hiv patients with aki (median icu days 3 vs 6, p = 0.002), the proportion of patients with hiv who died within the first 24 h was higher at 10.5% compared to 2.6% in those who were hiv negative, p = 0.009. this is the largest prospective african study of aki in critically ill adults with an hiv seropositive rate of 32.6%. similar studies include reports from morocco (n = 97) [29] , the democratic republic of congo (drc, n = 476) [30] and egypt (n = 532) [31] . however, only the drc study reported hiv prevalence, which was low at 2.9%. consistent with other lmi countries, patients were younger than in hi country cohorts (mean age 40.6 years) with lower comorbidity rates [1, 18, 19, [29] [30] [31] [32] , mostly of black african ethnicity, and were representative of the local community that we serve [21] . as in other african [29] [30] [31] and hi country [1] icubased studies, aki was very common in our cohort and affected nearly two thirds (58.5%) of all patients admitted to the icu. aki was associated with male gender, higher severity of illness, more sepsis, longer icu stay and the need for vasopressors. pre-existing ckd was negatively associated with aki but reflects an admission bias against very ill patients with ckd to the unit due to a lack of resources to continue with chronic renal replacement therapy in most. increased age, severity of illness, sepsis, mechanical ventilation, the use of vasopressors and the presence of tuberculosis were independently associated with mortality in those with aki. furthermore, increasing stage of aki showed a stepwise increase in the risk of mortality. although our unit admits emergency medical as well as elective surgical cases, just over a quarter of all admissions were trauma related, reflecting the alarmingly high levels of interpersonal violence and road traffic accidents prevalent in south africa. in 2010, interpersonal violence and road injury combined were the second leading cause of death and disability adjusted life years lost in south africa, after hiv which was the leading cause [33] . consequently, a large proportion of aki was attributable to hypovolaemic shock (55.1%) and rhabdomyolysis (24.8%) in keeping with the degree of trauma-related admissions. two recent retrospective studies in south africa also highlighted aki in trauma victims as a major contributor to morbidity and mortality [34, 35] . this reflects a major change in the epidemiology of aki in south africa where older reports have not highlighted trauma as a major cause of aki [14, 15, 36] and has important public health implications for health administrators. sepsis was also a common precipitant of aki at 40.6% which is similar to that reported in other lmi country studies in the critically ill [1, 31, 37] . herbal and traditional medicine use is not a prominent cause of aki in our region compared with prior reports from other regions [38, 39] . tropical diseases such as malaria are not endemic in our region and are therefore also not common precipitants of aki. severe aki (kdigo stage 3) was common, affecting 41.4% of all those with aki and 24.4% of all admissions. the number of patients dialyzed (10.4% of all admissions) was slightly lower than in the large acute kidney injury-epidemiologic prospective investigation (aki-epi) study where 13.5% of all admissions were dialyzed [1] and may be explained by local practice to usually delay the initiation of dialysis until more classic indications exist. notwithstanding this fact, most patients requiring dialysis were initiated early (within 24 h of icu admission) reflecting the advanced state of organ dysfunction at admission and late presentation that is common in lmi countries [32] . although the aki-epi study was multinational, only 34 patients from africa were included. continuous renal replacement therapies are available in our center but cost in excess of 10 times more than intermittent dialysis. as such, crrt is reserved for specific indications and the rate of crrt use was much lower in our study at 13.6% compared to 75.2% in the aki-epi study. of those who developed aki and survived, a significant proportion of patients with stage 3 aki did not recover renal function fully (12.7%). south africa has a very high hiv burden of 7.9 million people living with hiv as well as the largest number of people on antiretroviral therapy in the world [40] . the hiv seropositive rate of 32.6% in our cohort is consistent with the known background prevalence of hiv in our province of 25.2% in adults aged 15-49 when measured in 2018 [40] . this is vastly different to a retrospective study in a south african medical icu in 2004 [41] where only three patients (6.5%) in the aki cohort were hiv positive. in our study, active tuberculosis was very common, affecting 1 in 16 of all admissions. this is likely in part due to the high prevalence of hiv, but also due to the high reported background incidence of tuberculosis in the community of 1095 cases/100 000 per year [42] . whilst tuberculosis was not associated with aki per se, it was independently associated with mortality. many cases of active tuberculosis were diagnosed during icu admission through microbiological means and many were not the primary cause of admission. hiv infection was associated with higher mortality, as well as the presence of sepsis and active tuberculosis. it was also associated with increased severity of illness and the need for emergency admission. length of stay was shorter for those with hiv, but reflects earlier mortality in those with increased severity of illness. as in other studies in the post-haart era [36, 37, [43] [44] [45] , it would appear from our data that traditional predictors of mortality such as higher severity of illness are implicated in predicting mortality and not hiv status, cd4 count, viral suppression nor the use of haart. proportionally few (22.9%) hiv positive patients that developed aki were receiving tenofovir-based haart at the time of icu admission. although there was little difference in viral suppression between groups, the cd4 count was significantly lower in the group that developed aki thereby placing patients at risk for the immune reconstitution inflammatory syndrome (iris) [46] . while tenofovir has been shown to be nephrotoxic [47] , we hypothesize that the pathogenesis of aki in at least some of our hiv patients was related to the often recent initiation of haart with the development of an unmasking tuberculosis-associated iris [48] and consequent tuberculosis sepsis syndrome with associated aki. this has previously been reported [49] and is likely to be under-recognized. this study needs to be viewed in the context of its limitations. we may have underestimated the incidence of aki since we were unable to reliably utilize the kdigo urine output criterion for diagnosis as patients admitted to the icu were not always weighed or catheterised. secondly, 43.9% of all patients admitted to the icu were not tested for hiv. all patients who were able to consent were encouraged to have an hiv test; however, patients that were moribund or confused were not tested for hiv without indication or consent. on the other hand, to our knowledge, this is the largest published prospective cohort of critically ill adults with hiv and aki [50] . the study was inclusive of all major disciplines with the exception of cardiothoracics and the loss to follow up was low at 1.4%. standardised criteria for the diagnosis of all stages of aki were used and 90-day renal recovery data was obtained. in this large prospective multidisciplinary icu cohort of younger patients in a lmi country with a high hiv prevalence and many trauma related admissions, aki was frequently encountered, and was associated with a high mortality, but good functional renal recovery in most survivors. while hiv infection was associated with higher mortality, this was due to increased severity of illness, not hiv status per se. epidemiology of acute kidney injury in critically ill patients: the multinational aki-epi study timing and outcome of aki in critically ill patients varies with the definition used and the addition of urine output criteria incidence, outcomes, and comparisons across definitions of aki in hospitalized individuals small acute increases in serum creatinine are associated with decreased long-term survival in the critically ill acute kidney injury: an increasing global concern raising awareness of acute kidney injury: a global perspective of a silent killer five-year risk of end-stage renal disease among intensive care patients surviving dialysis-requiring acute kidney injury: a nationwide cohort study acute kidney injury and chronic kidney disease as interconnected syndromes six-month outcome in acute kidney injury requiring renal replacement therapy in the icu: a multicentre prospective study health-related quality-of-life among survivors of acute kidney injury in the intensive care unit: a systematic review the intensive care medicine agenda on acute kidney injury intensity of continuous renal-replacement therapy in critically ill patients intensity of renal support in critically ill patients with acute kidney injury epidemiology of acute kidney injury in africa nephrology in africa-not yet uhuru cost and mortality associated with postoperative acute kidney injury an effective approach to chronic kidney disease in south africa. south african medical journal =. suid-afrikaanse tydskrif vir geneeskunde acute kidney injury among adult patients with sepsis in a low-income country: clinical patterns and short-term outcomes clinical characteristics and 30-day outcomes of intermittent hemodialysis for acute kidney injury in an african intensive care unit international society of nephrology's 0by25 initiative for acute kidney injury (zero preventable deaths by 2025): a human rights case for nephrology provincial profile: eastern cape the critical care society of southern africa consensus guideline on icu triage and rationing (conictri) kdoqi us commentary on the 2012 kdigo clinical practice guideline for acute kidney injury choice of reference serum creatinine in defining acute kidney injury saps 3--from evaluation of the patient to evaluation of the intensive care unit. part 2: development of a prognostic model for hospital mortality at icu admission the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine developing a new definition and assessing new clinical criteria for septic shock: for the third international consensus definitions for sepsis and septic shock (sepsis-3) research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support epidemiology of acute kidney injury in moroccan medical intensive care patients: a regional prospective, observational study acute kidney injury is a powerful independent predictor of mortality in critically ill patients: a multicenter prospective cohort study from kinshasa, the democratic republic of congo risk, predictors, and outcomes of acute kidney injury in patients admitted to intensive care units in egypt the contrasting characteristics of acute kidney injury in developed and developing countries health and health care in south africa-20 years after mandela acute kidney injury on presentation to a major trauma service is associated with poor outcomes a prospective study of the demographics, management and outcome of patients with acute kidney injury in cape town renal failure in hiv-positive patients-a south african experience acute dialysis in hiv-positive patients in cape town nephrotoxins in africa acute kidney injury associated with the use of traditional medicines the fifth south african national hiv prevalence, incidence, behaviour and communication survey acute renal failure in the medical icu still predictive of high mortality. south african medical journal =. suid-afrikaanse tydskrif vir geneeskunde nationwide and regional incidence of microbiologically confirmed pulmonary tuberculosis in south africa, 2004-12: a time series analysis acute kidney injury among hivinfected patients admitted to the intensive care unit hiv-positive patients in the intensive care unit: a retrospective audit. south african medical journal =. suid-afrikaanse tydskrif vir geneeskunde clinical characteristics, outcomes and risk factors for death among critically ill patients with hiv-related acute kidney injury a practical approach to the diagnosis and management of paradoxical tuberculosis immune reconstitution inflammatory syndrome tenofovir nephrotoxicity: acute tubular necrosis with distinctive clinical, pathological, and mitochondrial abnormalities tuberculosis-associated immune reconstitution inflammatory syndrome and unmasking of tuberculosis by antiretroviral therapy acute kidney disease due to immune reconstitution inflammatory syndrome in an hiv-infected patient with tuberculosis the outcome of hiv-positive patients admitted to intensive care units with acute kidney injury springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank all the rotating doctors who helped collect data. noline van vuuren was invaluable in data checking and cleaning. authors' contributions ra wrote the protocol, collected and cleaned data and wrote the manuscript. evdm assisted with the protocol, consultancy and manuscript writing. sp cleaned data and performed the statistical analysis. mvn collected and cleaned data. je assisted with consultancy, following up patients in the renal unit and appraisal of the final manuscript. db assisted with data collection, consultancy and appraisal of the final manuscript. rf assisted with the protocol, consultancy, manuscript writing and follow up of patients in the renal unit. all authors approved the final version of the manuscript. the authors gratefully acknowledge roche south africa for an unrestricted research grant that was used to fund data capturing, analysis, presentation and publication of the study (ref: sa/nonp/1812/0050). the funder did not contribute to the design, conduct of the study, analysis or interpretation of results. not applicable. the authors declare that they have no competing interests.author details 1 adult critical care unit, livingstone hospital, port elizabeth, south africa. key: cord-017831-anadq4j9 authors: lai, yi-horng title: network analysis of comorbidities: case study of hiv/aids in taiwan date: 2015-07-30 journal: multidisciplinary social networks research doi: 10.1007/978-3-662-48319-0_14 sha: doc_id: 17831 cord_uid: anadq4j9 comorbidities are the presence of one or more additional disorders or diseases co-occurring with a primary disease or disorder. the purpose of this study is to identify diseases that co-occur with hiv/aids and analyze the gender differences. data was collected from 536 hiv/aids admission medical records out of 1,377,469 admission medical records from 1997 to 2010 in taiwan. in this study, the comorbidity relationships are presented in the phenotypic disease network (pdn), and φ-correlation is used to measure the distance between two diseases on the network. the results show that there is a high correlation in the following pairs/triad of diseases: human immunodeficiency virus infection with specified conditions (042) and pneumocystosis pneumonia (1363), human immunodeficiency virus infection with specified malignant neoplasms (0422) and kaposi’s sarcoma of other specified sites (1768), human immunodeficiency virus acquired immunodeficiency syndrome, and unspecified (0429) and progressive multifocal leukoencephalopathy (0463), and lastly, human immunodeficiency virus infection with specified infections (0420), meningoencephalitis due to toxoplasmosis (1300), and human immunodeficiency virus infection specified infections causing other specified infections (0421). the human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids) epidemic is one of the most important and crucial public health risks facing governments and civil societies in the world. adolescents were at the center of the pandemic in terms of transmission, impact, and potential for changing the attitudes and behaviors that underlie this disease. therefore, hiv/aids prevention has become a priority all over the world. the first hiv/aids case in taiwan was reported in 1984. as of the end of 2013, the total number of hiv/aids cases had been accumulated to 26,475. faced with this serious situation, taiwan's centers for disease control worked with other departments and dedicated a tremendous amount of effort and resources to introduce harm reduction programs. total reported cases dropped in 2006, which was the first trend reversal since 1984. in 2008 and thereafter, the epidemic took a turn; infections mainly occurred through sexual encounter [1] . there are no clear boundaries between many diseases, as diseases can have multiple causes and can be related in several dimensions. from a genetic perspective, a pair of diseases can be related because they have both been associated with the same gene, whereas from a proteomic perspective, diseases can be related because diseaseassociated proteins act on the same pathway [2] . during the past half-decade, several resources have been constructed to help understand the entangled origins of many diseases. many of these resources have been presented as networks in which interactions between disease-associated genes, proteins, and expression patterns have been summarized. goh, cusick, valle, barton, vidal, and barabási created a network of mendelian gene-disease associations by connecting diseases that have been associated with the same genes [3] . besides, more and more studies have applied the network approach in diseases, such as neurodegenerative diseases [4] , infertility etiologies [5] , sars, and hiv/aids [6] . a comorbidity relationship exists between two diseases whenever they affect the same individual substantially more than chance alone. in the past, comorbidities have been used extensively to construct synthetic scales for mortality prediction [7, 8] , yet their utility exceed their current use. studying the structure defined by entire sets of comorbidities might help the understanding of many biological and medical questions from a perspective that is complementary to other approaches. for example, a recent study built a comorbidity network in an attempt to elucidate neurological diseases' common genetic origins [9] . heretofore, however, neither this data nor the data necessary to explore relationships between all diseases is currently available to the research community. this present study decided to provide this data in the form of a phenotypic disease network (pdn) that includes all diseases recorded in the medical claims. additionally, this study illustrates how a pdn can be used to study disease progression from a network perspective by interpreting the pdn as the landscape where disease progression occurs and shows how the network can be used to study phenotypic differences between patients of different demographic backgrounds. this study indicates the directionality of disease progression, as observed in our dataset, and finds out that more central disease in the pdn are more likely to occur after other diseases and that more peripheral diseases tend to precede other illnesses. in order to guide hiv/aids-related diseases prevention program, this study conducted the pdn of hiv/aids to explore the relationship between hiv/aids and other diseases. the objective of this study is to identify diseases that are highly correlated with hiv/aids, and discuss gender differences. the national health insurance (nhi) program was initiated in taiwan in 1995 and covers nearly all residents. in 1999, the bureau of nhi began to release all claims data in electronic form to the public under the national health insurance research database (nhird) project. the structure of the claim files is described in detail on the nhird website and in other publications [10]. nhird offers reliable, systematic, and complete data for disease detection. the datasets contained only the visit files, including dates, medical care facilities and specialties, patients' genders, dates of birth, and the four major diagnoses coded in the international classification of disease, 9th revision, clinical modification (icd-9-cm) format [10, 11] . in total, the icd-9-cm classification consists of 657 different categories at the 3 digit level and 16,459 categories at 5 digits. human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids) is coded 042 as human immunodeficiency virus (hiv) infection disease, 0420 as human immunodeficiency virus infection with specified infections, 0421 as specified infections causing other specified infections, 0422 as human immunodeficiency virus infection with specified malignant neoplasms, and 0429 as acquired immunodeficiency syndrome, and unspecified. to protect privacy, the data on patient identities and institutions had been scrambled cryptographically. the visit files in this study represented 1,377,469 admission activities within the nhi from 1997 to 2010. demographically, the data set consists of 1,377,469 admission medical records from 1,000,000 patients. of all these patients, 50.93% were females, 48.93 were males, 20.67% were over 71 years of age, and 536 persons were diagnosed with hiv/aids (table 1) . these hiv/aids admission medical records included 45 females (8.40%) and 491 males (91.60%). of all the 536 hiv/aids records, 461 (86.01%) had major diagnoses coded 042, 21 (3.92%) had major diagnoses coded 0420, 3 (.56%) had primary diagnoses coded 0421, and 7 (8.77%) had major diagnoses coded 0429 (table 2 ). there are several limitations to the current study. first, although data gathered from nhird is comprehensive and reliable, there are still some mistakes that the system couldn't find, such as code entry errors. these errors may be carried into data y.-h. lai pre-processing, and it is beyond the control of this study. second, the data was not upto-date. although future researchers are still recommended to apply the method of this study to analyze the characteristics of patients for the purpose of disease prevention, changes of medical treatments and other factors should be considered. third, this study does not have a global sample, so there might be limits to replicate the findings of this study in all the other countries. it might, however, be generalized to other ethnic chinese population due to the similarity in genes and physiology. to measure the comorbidity relationships, it is necessary to quantify the strength of comorbidities by introducing a notion of distance between two diseases. a difficulty of this approach is that different statistical distance measures have biases that over-or under-estimate the relationships between rare or prevalent diseases. these biases are important given that the number of times a particular disease is diagnosed, such as its prevalence, follows a heavy tailed distribution [2] , meaning that while most diseases are rarely diagnosed, a few diseases have been diagnosed in a large fraction of the population. in this study, the φ-correlation is used to quantify the distance between two diseases. the φ-correlation, which is pearson's correlation for binary variables, can be expressed mathematically as [2, 12] : where c ij is the number of patients affected by both diseases, n is the total number of patients in the population and p i and p j are the prevalence of diseases i and j. the distribution of φ values representing all disease pairs where c ij >0 is presented in fig 1, 3 and 5. this research utilizes data from nhird to obtain the four major diagnoses codes of all patients. this study calculates φ-correlation with equation 1. pajek 4.03 program was used to compute the compute the degree of centrality and betweenness of each node and the path value (φ-correlation). this study is focused on the path between each disease as network and the correlation as the value of line (path weight), and they could be affected by different populations, which indicates differences in gender for each population. it can be summarized the set of all comorbidity associations between all diseases expressed in the study population by constructing a phenotypic disease network (pdn). in the pdn, nodes are disease phenotypes identified by unique icd9 codes, and links phenotypes that show significant comorbidity according to the measures introduced above. in principle, the number of disease-disease associations in the pdn is proportional to the square of the number of phenotypes, yet many of these associations are either not strong or are not statistically significant [2] . the structure of the pdn can be explored by focusing on the strongest and the most significant of these associations. the pdn can be seen as a network of the phenotypic space. this network allows people to understand the relationship between illnesses. the distribution of φ-values representing all disease pairs is presented in figure 1 . most of them are between .000 and .005. a discussion on the confidence interval and statistical significance of these measures can be found in hidalgo, blumm, barabási, and christakis's study, and φ-correlation > .06 is statistically significant [2] . in figure 2 , nodes are diseases; links are correlations. node color identifies the icd9 category; node size is proportional to disease prevalence. link color indicates correlation strength. the pdn built using φ-correlation. all statistically significant links where φ-correlation>.06 are shown here. human immunodeficiency virus infection with specified conditions (042) and pneumocystosis pneumonia (1363), cryptococcal meningitis (3210), candidiasis of mouth (1120), unspecified secondary syphilis (0919), kaposi's sarcoma of unspecified (1769), cryptococcosis (1175), kaposi's sarcoma of palate (1762), neurosyphilis, unspecified (0949), kaposi's sarcoma of lung (1764), kaposi's sarcoma of lymph nodes (1765), and late syphilis, latent (096) are highly correlated. those φ-correlations are all above .06. the relationship between human immunodeficiency virus infection with specified conditions (042) and pneumocystosis pneumonia (1363) is the strongest. pneumocystis pneumonia is a form of pneumonia, caused by the yeast-like fungus pneumocystis jirovecii. pneumocystis pneumonia is not commonly found in the lungs of healthy people, but, being a source of opportunistic infection, it can cause a lung infection in people with a weak immune system [13] . human immunodeficiency virus infection with specified infections (0420) and human immunodeficiency virus infection specified infections causing other specified infections (0421), kaschin-beck disease (7160), pneumocystosis (1363), meningoencephalitis due to toxoplasmosis (1300), falciparum malaria (0840), kaposi's sarcoma of other specified sites (1768), with specified malignant neoplasms (0422) are highly correlated. those φ-correlations are all above .06. y.-h. lai the φ-correlation of human immunodeficiency virus infection with specified infections (0420) and meningoencephalitis due to toxoplasmosis (1300) is highest. toxoplasmosis is a parasitic disease caused by the protozoan toxoplasma gondii. the parasite infects most genera of warm-blooded animals, including humans, but the primary host is the felid family. infection occurs by eating infected meat, particularly swine products. by ingesting water, soil, or food that has come into contact with infected animals' fecal matter [14] . besides, human immunodeficiency virus infection with specified infections (0420) and specified infections causing other specified infections (0421) is highly correlated, and the φ-correlation is .11. human immunodeficiency and kaposi's sarcoma of sk specified infections (0420) highly correlated. those φthe φ-correlation of hu lignant neoplasms (0422) a highest. human immunodeficien specified (0429) and prog correlated. those φ-correlat the distribution of w va most of them are between . in pdns for females (figure 4 ), human immunodeficiency virus infection with specified conditions (042) and cryptococcal meningitis (3210), kaposi's sarcoma of unspecified (1769), and pneumocystosis (1363) are highly correlated. human immunodeficiency virus acquired immunodeficiency syndrome, unspecified (0429) and other cerebellar ataxia (3343), and progressive multifocal leukoencephalopathy (0463) are highly correlated. those φ-correlations are all above .06. the distribution of w values representing all disease pairs is presented as fig. 5 . most of them are between .000 and .005. in pdns for males ( figure 6 ), human immunodeficiency virus infection with specified conditions (042) and unspecified secondary syphilis (0919), cytomegaloviral disease (0785), kaposi's sarcoma of palate (1762), amebic liver abscess (0063), kaposi's sarcoma of lung (1764), kaposi's sarcoma of lymph nodes (1765), cryptococcosis (1175), late syphilis, latent (096), candidiasis of mouth (1120), cryptococcal meningitis (3210), neurosyphilis, unspecified (0949), kaposi's sarcoma of unspecified (1769), and pneumocystosis (1363) are highly correlated. those φ-correlations are all above .06. the φ-correlation of human immunodeficiency virus infection with specified conditions (042) with pneumocystosis (1363) is highest. human immunodeficiency virus infection with specified infections (0420) and specified infections causing other specified infections (0421), meningoencephalitis due to toxoplasmosis (1300), pneumocystosis (1363), kaschin-beck disease (7160), kaposi's sarcoma of other specified sites (1768), with specified malignant neoplasms (0422), and falciparum malaria (0840) are highly correlation. those φ-correlations are all above .06. the φ-correlation of specified infections causing other specified infections (0421) with human immunodeficiency virus infection with specified infections (0420) is highest. human immunodeficiency virus acquired immunodeficiency syndrome, unspecified (0429) and hiv infection, unspecified (0449) is highly correlation, and the φcorrelation is .09. through the pdn, this paper has identified the diseases that are associated with hiv/aids. it could be showed that the pdn has a complex structure where some diseases are highly connected while others are barely connected at all. while not conclusive, these observations can explain the observation that more connected diseases are seen to be more lethal, as patients developing highly connected diseases are more likely those at an advanced stage of disease, which can be reached through multiple paths in the pdn. the findings suggest that human immunodeficiency virus infection with specified conditions (042) and pneumocystosis pneumonia is highly correlated (1363). this result is consistent with aliouat-denis, chabé, demanche, aliouat el, viscogliosi, guillot, delhaes, and dei-cas's study [13] . pneumocystis pneumonia is especially seen in people with cancer undergoing chemotherapy, hiv/aids, and the use of medications that suppress the immune system. human immunodeficiency virus infection with specified infections (0420) and meningoencephalitis due to toxoplasmosis (1300) is highly correlation. besides, it is also highly correlation with human immunodeficiency virus infection specified infections causing other specified infections (0421). the result is the same as dubey, hill, jones, hightower, kirkland, roberts, marcet, lehmann, vianna, miska, sreekumar, kwok, shen, and gamble''s study [14] . human immunodeficiency virus infection with specified malignant neoplasms (0422) and kaposi's sarcoma of other specified sites (1768) is highly correlation. the result is the same as holmes, hawson, liu, friedman, khiabanian, and rabadan's study [15] . since patients infected with hiv/aids have a high risk of developing kaposi sarcoma, the prevention of this malignant disease should be prioritized. human immunodeficiency virus acquired immunodeficiency syndrome, and unspecified (0429) and progressive multifocal leukoencephalopathy (0463) is highly correlation. the result is the same as casado, corral, garcía, martinez-san millán, navas, moreno, and moreno's study [16] and sano, nakano, omoto, takao, ikeda, oga, nakamichi, saijo, maoka, sano, kawai, kanda [17] . progressive multifocal leukoencephalopathy is a usually fatal viral disease characterized by progressive damage or inflammation of the white matter of the brain at multiple locations. it is caused by the jc virus, which is normally present and kept under control by the immune system. jc virus is harmless except in cases of weakened immune systems. progressive multifocal leukoencephalopathy occurs almost exclusively in patients with severe immune deficiency, most commonly among patients with acquired immune deficiency syndrome, but people on chronic immunosuppressive medications including chemotherapy are also at increased risk of progressive multifocal leukoencephalopathy [16, 17] . for females, human immunodeficiency virus infection with specified conditions (042) and cryptococcal meningitis (3210), kaposi's sarcoma of unspecified (1769), and pneumocystosis (1363) are highly correlation. human immunodeficiency virus acquired immunodeficiency syndrome, unspecified (0429) and other cerebellar ataxia (3343), and progressive multifocal leukoencephalopathy (0463) are highly correlation. for males, human immunodeficiency virus infection with specified conditions (042) and pneumocystosis (1363) is highly correlation. human immunodeficiency virus infection with specified infections (0420) and meningoencephalitis due to toxoplasmosis (1300), and falciparum malaria (0840) are highly correlation. human immunodeficiency virus infection with specified malignant neoplasms (0422) and kaposi's sarcoma of other specified sites (1768) is highly correlation. human immunodeficiency virus acquired immunodeficiency syndrome, unspecified (0429) and hiv infection, unspecified (0449) is highly correlation. exploring comorbidities from a network perspective could help determine whether differences in the comorbidity patterns expressed in different populations indicate differences in races, country, or socioeconomic status for each population. here this study show as an initially stage that there are differences in the strength of comorbidities measured for patients of different gender. the pdn could be the starting point of studies exploring these and related questions. communicable diseases & prevention-hiv/aids, health topics a dynamic network approach for the study of human phenotypes the human disease network a network approach to clinical intervention in neurodegenerative diseases infertility etiologies are genetically and clinically linked with other diseases in single meta-diseases network-based analysis of comorbidities risk during an infection: sars and hiv case studies chronic conditions and risk of in-hospital death improved comorbidity adjustment for predicting mortality in medicare populations probing genetic overlap among complex human phenotypes international classification of diseases, ninth revision the phi-coefficient, the tetrachoric correlation coefficient, and the pearson-yule debate pneumocystis species, co-evolution and pathogenic power. infection prevalence of viable toxoplasma gondii in beef, chicken, and pork from retail meat stores in the united states: risk assessment to consumers discovering disease associations by integrating electronic clinical data and medical literature continued declining incidence and improved survival of progressive multifocal leukoencephalopathy in hiv/aids patients in the current era rituximab-associated progressive multifocal leukoencephalopathy derived from non-hodgkin lymphoma: neuropathological findings and results of mefloquine treatment acknowledgements. this study is based in part on data from the national health insurance research database provided by the bureau of national health insurance, department of health and managed by national health research institutes (nhri). the interpretation and conclusions contained herein do not represent those of bureau of national health insurance, department of health or national health research institutes. key: cord-006331-s2qf98lj authors: spiridonova, v. a. title: molecular recognition elements: dna/rna-aptamers to proteins date: 2010-05-23 journal: biochem mosc suppl b biomed chem doi: 10.1134/s1990750810020046 sha: doc_id: 6331 cord_uid: s2qf98lj the review summarizes data on dna/rna aptamers, a novel class of molecular recognition elements. special attention is paid to the aptamers to proteins involved into pathogenesis of wide spread human diseases. these include aptamers to serine proteases, cytokines, influenza viral proteins, immune deficiency virus protein and nucleic acid binding proteins. high affinity and specific binding of aptamers to particular protein targets make them attractive as direct protein inhibitors. they can inhibit pathogenic proteins and data presented here demonstrate that the idea that nucleic acid aptamers can regulate (inhibit) activity of protein targets has been transformed from the stage of basic developments into the stage of realization of practical tasks. during the last decade a significant break has been achieved in the use of basic knowledge on dna in applied studies. the development of highly technolog ical analytic methods employing immobilized dna is one of rapidly developing directions. the major achievement of microarray technology (i.e. dna chips) consists in possibility of the use of various dna libraries amplified by polymerase chain reaction (pcr) for the development of sets of dna sequences. using hybridization these sets can rapidly analyze and compare sequences of thousands genes, their mutant forms, dna polymorphism and to discover new genes. the second direction consists in the develop ment of irrational design of nucleic acids for studies of nucleic acid protein recognition. in 1990, two labs (the gold and szoztak laboratories; usa) indepen dently developed the selex method (systematic evolution of ligands by exponential enrichment) [1, 2] . using this method it is possible to isolate targeted nucleic acid molecules (aptamers) from the large set of individual molecules (more than 10 18 ) known as the combinatorial library. aptamers are small single stranded dna or rna molecules of 40⎯100 nucle otide residues in length with rather complex three dimensional structure. such complex structure deter mines aptamer ability to bind various molecules including proteins. thus, such complex process of biosynthesis of protein recognizing elements, antibod ies, which nature has been naturally creating for thou sands years, is now modeled in vitro. selection begins with generation of a large rna library with fixed 5' and 3' ends and a degenerated region of 30-60 nucleotides in length (fig. 1) . such library contains 10 14 -10 15 variants of rna molecules, which are folded in complex 3d structure. the library is incubated with a protein and rna molecules bound to the protein target are separated from unbound rna molecules. the bound rna molecules are separated from proteins and then amplified by means of reverse transcriptase and pcr to obtain a new pool of mole cules with increased affinity. the procedure is usually repeated 10-15 times until maximal number of aptamers exhibiting affinity to the target will be clearly detectable in the enriched fraction. aptamers are then cloned (usually into a bacterial vector) and sequenced. in the case of dna the selection also begins with dna library in which the randomized region is flanked by fixed sequences at 5' and 3' ends. to pro duce single stranded dna molecules either asymmet ric pcr or one of primers carries a biotinylated tag, which helps to separate one dna strand from another on streptavidin columns are used. many reviews on detailed description of all steps of this method have been published to date [3] [4] [5] [6] [7] [8] [9] [10] . now, the idea that nucleic acid aptamers can regulate (inhibit) activity of protein targets has been transformed from the stage of basic developments into the stage of realization of practical tasks. in this review the major attention is paid to the aptamers to proteins involved into patho genesis of wide spread human diseases. tuer and gold proposed to use combinatorial rna libraries for creation of rna ligands selectively bound to t4 dna polymerase [1] . rna ligands were named as aptamers by ellington and szoztak [2] . they also introduced a name of this method, selex. now convincing evidence exists that aptamers are a new effective group of therapeutics, which may represent the scheme illustrating the selex method for preparation of dna and rna aptamers. an initial randomized dna library is transformed into single stranded dna (ssdna) and then introduced into binding reaction with a protein, pre immo bilized onto a column. rna is obtained by transcription of the initial library; the latter carries introduced promoter of t7 rna polymerase. rna molecules are also exposed for binding with the protein immobilized onto the column. after removal of unbound molecules, dna/rna molecules that bind to the immobilized protein are separated from this protein by phenol chlo roform treatment and then subjected to alcohol sedimentation. rna molecules are used for cdna preparation by means of reverse transcriptase. all resultant molecules are then transformed into double stranded dna (dsdna). in the case of dna ssdna prepared using asymmetric pcr is used again for repeated binding. rna molecules are subjected for transcription and resultant molecules are used for binding with the immobilized protein. the selection includes 10⎯15 rounds and this yields an enriched fraction of aptamers. the next step includes cloning into a plasmid vector and sequencing of resultant sequences. an important tool against many diseases. the drug macugen has already been approved by us fda (food and drug administration) for the treatment of age related macular degeneration (amd). some aptamers are now in different stages of pre and clini cal trials [11, 12] . highly specific (antibody like) recognition and binding of aptamers to their protein targets make them attractive therapeutics. aptamers (as well as antibod ies) are folded into complex three dimensional struc tures and form hairpins and loops. the range of disso ciation constants characterizing binding of dna and rna aptamers to their protein targets varies from nanomolar to subnanomolar levels. aptamers can dis criminate related proteins consisting of the same structural domains [13] [14] [15] [16] . it should be noted that the use of 1000 fold excess of aptamer doses in animal models employed in preclinical trials and in therapeu tic applications in humans did not cause allergic reac tions [11, 17] . studies on biocompatibility and phar macokinetics of aptamers and investigations of various modifications of aptamer structures have been per formed for their further applications as drugs [12] . nuclease degradation is the major problem that complicates manipulations with oligonucleotides. protection against nonspecific action of nucleases during selection includes modification of pyrimidine nucleotides at ribose c2' (amino and fluoro deriva tives) [18] [19] [20] , use of liposomes as carriers [21] , post selection ribose c2' hydroxyl modifications by intro ducing methyl, allyl, amino groups, etc. [22] [23] [24] . the molecular mass of short polynucleotides is 8-14 kda; this corresponds to 25-40 nucleotides. such a small size facilitates rapid renal filtration within a few minutes. aptamer modification by conjugation to polyethylene glycol or other agents and their attach ment to the liposome surface prolonged the period of aptamer action [21, 25] . usually, aptamers exhibit high specificity towards their targets and this should be taken into consider ation in preclinical trials on animal models. however, protein orthologs may decrease efficacy of such com pounds. nevertheless, an improved selection process named as "toggle selex" seems to overcome this problem. toggle selex has been proposed for rna libraries incubated with the same protein from human plasma. during the follwing rounds of selection it was also exposed to binding with the animal ortholog used in subsequent preclinical trials [26] . studies on pharmacokinetics of aptamers subjected to modifications should be accompanied by analysis of their excretion from the body. this is important because in addition to the main disease patients may have multiple dysfunctions including renal insuffi ciency. for regulation of aptamer activity rusconi pro posed the antidote proof of concept (the method of rational design) [27] . using watson crick base pairing he designed an antidote structure as a complementary sequence that bound to the aptamer, altered its struc the mechanism of antidote action in the pair aptamer antidote (modified from [26] ). the antidote is an oligonucleotide of 15 nucleotides in length; it forms an inactive complex with the aptamer to factor ixa involved into the blood coagulation cas cade. possessing a sequence complementary to the aptamer, the antidote forms a complex with the aptamer and thus alters its 3d structure. antidote inactive form ture and, thus, prevented its complex formation with the protein target (fig. 2) . the proposed concept gave a unique possibility of aptamer regulation because it allows to control administration of an aptamer based drug in any clinical use. considering aptamers as direct protein inhibitors it is very attractive to use them for studies of inhibitory mechanisms and also for therapeutic application. pos sibility of aptamer preparation for any class of biomol ecules allows to evaluate importance of their use and increases areas of their applications. advantage of aptamers maintaining their activity in multicellular organisms significantly facilitates preclinical trials, saves time and reduces costs required for antibody preparation on animal models. finally, design of an antidote that control aptamer activity possibly repre sents the most important contribution to the develop ment of nuclei acid therapy because it can control drug dosage and, thus, determine required safety. it should be noted that in addition to biomedical studies aptam ers are also used as a recognizing elements in microar ray based biosensors; this is another important and logic continuation of the development of the dna chip technology. this review summarizes current knowledge on aptamers to various proteins, their affinity to protein targets; it describes inhibitory properties of aptamers and information about preclinical and clinical trials. 2.1. thrombin thrombin is a key protein in blood clotting process. this serine protease is generated during a cascade of proteolytic reactions initiated by epithelial damage. thrombin is produced from prothrombin by factor xa. active thrombin catalyzes the reaction of fibrinogen conversion into fibrin, which forms a fibrin matrix for the thrombus by "capturing" blood cells [28] . throm bin also activates platelets via interaction with their par receptors and regulates expression of some sub strates and activation molecules such as p selectin [29, 30] . problem of hemostasis requires creation of such thrombin inhibitor, which would be specific for blood clotting process, does not cause allergic reaction and effectively regulates this process. an anti thrombin aptamer was one of the first therapeutic aptamers obtained by the selex method. the single stranded dna aptamer was isolated from a pool of ~10 13 oligo nucleotide sequences containing a 60 nucleotide ran domized region [31] . the 5 round selection resulted in identification of aptamers forming a complex with thrombin, which was characterized by the k d values ranged from 25 to 200 nm. these aptamers were based on the 15 nucleotide sequence: dggttggtgtg gttgg (15tba). the aptamer increased time required for clot formation from 25 to 170 s in vitro and from 25 to 43 s in human blood plasma. the 15tba structure was investigated by means of nmr analysis, which included the study of 15tba alone, in the complex with thrombin and the study of aptamer binding with the anion binding site of thrombin, exosite 1 [32] [33] [34] [35] . 15tba forms a complex compact tertiary structure known as g quadruplex (fig. 3 ). other laboratories also used the selex method to perform aptamer selection to thrombin [36, 37] . 15tba also influenced platelet aggregation stimulated by thrombin. thrombin also caused proteolytic activa tion of platelet par 1 receptor and its aptamer inhib ited this activation in a dose dependent manner [38] . the anticoagulant activity of the aptamer was tested on monkeys. the prothrombin time (pt) increased by 1.7 fold in 10 min and returned to base line 10 min after aptamer administration. 15tba also inhibited platelet aggregation and prolonged platelet activation induced by thrombin [39] . the aptamer was also investigated using an anticoagulation model of extracorporeal circulation in sheep. the pt values reached 40⎯45 s (versus 21.7 s of the baseline level), whereas control pt remained close to the baseline. in the other experiment 15tba was investigated using a the structure of the 15tba aptamer to thrombin, which inhibits fibrinogen hydrolyzing activity. 15tba has the oligonucleotide sequence dggttggtgtggt tgg. the g quadruplex structure is a structure forming element for dna. eight of nine guanines form two planar g quartets with three loops; the loop tgt located in the center and two symmetrical loops tt. the presence of octa coordinating calcium ion and stacking interaction between g quartets of the duplex determine maintenance of the g quadruplex. calcium ion is located between par allel planes of the g quadruplex. cardiopulmonary bypass (cpb) model. the study included examination of anticoagulation activity, pharmacokinetics and renal clearance of the aptamer [40] . animals were subdivided into two groups: one group received injections of heparin (300 u/kg) and protamine in boluses and was used as control of activ ity by cpb. the second group of animals received aptamer infusion (0.3-0.5 mg/kg per 1 min). these animals were characterized by increased pt, activated partial thromboplastin time (aptt), and activated clotting time (act), which then returned to the base line after infusion [39] [40] [41] . in the pharmacokinetic studies using the cpb model, the elimination half life of the aptamer was 1.9 min; however, during the 60 min infusion this parameter increased up to 7.7 min. these results sug gested that the aptamer would function in the animal model and that unmodified dna aptamers were rap idly eliminated from the body. now this dna aptamer is under preclinical trials by archemix corp. for subsequent trials in humans. anti thrombin rna aptamers were obtained using the library with a 30 nucleotide randomized sequence [42] . the anti thrombin aptamers were iso lated after 12 rounds of selection. the enriched frac tion was then cloned in the plasmid puc18 and sequenced; this yielded two classes of aptamers. the conservative motif in 22 clones of the class i rnas was represented by the sequence uccggaucgaag uuaguaggcgga inside a variable zone. one of the best anti thrombin aptamers was characterized by the k d value of 9.3 ± 1.0 nm. members of the second class exhibited lower affinity (the k d value of 155 ± 9.0 nm). competitive analysis with heparin and hirudin dem onstrated that heparin but not hirudin displaced the rna aptamer from its complex with thrombin. this suggests affinity of this aptamer to thrombin exosite ii. however, tests on functional activity in animal models have not been performed. in preclinical trials highly specific aptamers to human proteins may demonstrate lowered affinity to protein orthologs in animal models. to overcome this problem so called "toggle" approach has been pro posed: 2' fluoro rna aptamers were incubated with a mixture of human and porcine thrombin during the first round and then porcine and human thrombin were alternatively used in subsequent rounds of selection [43] . after 13 rounds of selection clones with the con servative sequence gggaacaaagcugaaguac uuaccc have been found; they exhibited cross reac tivity with porcine and human thrombin. the complex with human thrombin was characterized by the disso ciation constant k d of 2.8 ± 0.7 nm and the complex with porcine thrombin had the k d value of 83 ± 3 pm. the aptamer increased clotting time (thrombin con centration was 10 nm) of blood plasma from 11.6 ± 0.2 to 22.6 ± 1.4 s. in porcine plasma tog 25 increase clot ting time from 15.7 ± 0.7 to 61.9 ± 1.2 s. improvement of thrombin dependent platelet aggregation by the aptamer occurred in the dose dependent manner. the higher effect was achieved using porcine platelets: a 10 fold excess of tog 25 inhibited thrombin activity by 90% [26, 43] . factor viia (fviia) is a trypsin like protease involved into the coagulation cascade. in combination with the tissue factor (tf) fviia plays a critical role in thrombin formation and thus promotes active clot for mation. aptamers to fviia have been isolated from an rna library using the selex method. these aptam ers inhibited activation of factor x (inactive precursor in the coagulation cascade) by fviia [15] . after 16 rounds of selection from the 2' amino modified rna library the isolated aptamers formed a complex with fviia characterized by the k d value of 11.3 ± 1.3 nm. specificity of some aptamers was investigated in bind ing reactions with other protein factors (fxia and fxa). the micromolar range of k d values determined for these complexes suggested nonspecific binding of these aptamers with protein factors. addition of the anti fviia aptamer inhibited an initial rate of fx acti vation by about 95%. experiments on dilution of the reaction mixture revealed a dose dependent mode of inhibition. the aptamer prolonged clotting time up to 175% in the pt test. factor ixa (fixa) is a serine protease that plays an important role in formation of critical mass of throm bin required for coagulation. the complex tf/fviia performs proteolytic cleavage of the protein factor fix into its active form fixa; the latter binds to fviiia on the platelet surface and activates factor fx to fxa, which catalyzes conversion of prothrombin into thrombin [28] . rusconi et al. performed rna selection to fixa; after eight rounds of selection they found an aptamer, which bound to fixa with the k d value of 0.65 ± 0.2 nm and exhibited 5000 fold higher affinity to fixa compared with fviia, fxa, fxia and activated protein c [20] . a truncated version of this aptamer (9.3t) maintained high affinity to fixa (k d of 0.58 ± 0.1 nm) and totally inhibited fx hydrolysis by the enzyme complex. the anticoagulation activity of 9.3t was evaluated using activated partial thromboplastin time (aptt). the aptamer increased clotting time in the dose dependent manner and caused a several fold increase in aptt. in continuation of the antidote theory rus coni obtained an rna antidote, which caused revers ible inhibition of 9.3 t thus creating a drug/antidote pair for the anticoagulation therapy. using the com plementary base pairing principle the second rna oligonucleotide complementary to the 9.3t aptamer was created. after administration of the antidote nucleotide the anticoagulation activity of the anti fixa aptamer changed in 10 min and this effect per sisted for over 5 hours [16] . almost in 5% of 12 million people receiving heparin therapy heparin induced thrombocytopenia (hit) is developed after one year [28] and this is the reason for cessation of the heparin therapy. patients who need repeated anticoagulant therapy receive hemodialysis, which complicates patient's life. to prolong the effect of the anti fixa aptamer in vivo rusconi c.p. et al. prepared a cholesterol deriva tive (ch 9.3t), which exhibited high affinity (k d = 5.3 ± 1.1 nm) and anticoagulant activity [27] . tests on por cine and mouse plasma have shown the same efficacy of the animal models as in the case of human plasma. using the porcine anticoagulant system the aptamer increased pt and aptt comparable with its effects on pt and aptt in human plasma samples. there was significant difference between the modified and initial aptamers: the cholesterol moiety increased half life of ch 9.3t to 60-90 min (versus 5-10 min for 5-10 min). the antidote 5 2 c neutralized more than 95% of the aptamer effect within 10 min in animal models [27] . the antithrombotic effect of the aptamer was investigated in mouse thrombosis model, which was induced by administration of ferric chloride to the carotid artery; mice were pretreated with ch 9.3t or a functionally inactive aptamer with scrambled nucle otide sequence (negative control). all the mice in the negative control group developed an occlusive throm bus in 8.1 ± 0.1 min. in the aptamer treated group 80% of mice maintained clear normal carotid artery blood flow during 30 min (time required for the occlu sive thrombus formation ≥24.4 min) [27] . the effect of the 5 2c antidote was accessed using the model of active bleeding (tail transection). mice were pre treated with ch 9.3t or an aptamer with scrambled sequence and the tail was cut 1 h after the treatment. blood losses were measured for 15 min after tail transection. animals treated with ch 9.3t exhibited significantly more blood loss (176 ± 23.7 μl) compared with controls (48 ± 17.8 μl). administration of the 5 2c immediately after tail transection prevented hem orrhage in the aptamer treated animals (blood loss was 54.5 ± 13.6 μl) [27] . the biopharmaceutical company regado bio sciences continues studies on the fixa aptamer anti dote pair named as reg1, which is under first stage of clinical trials. hepatitis c virus (hcv) is a major cause of both sporadic and viral hepatites differed from hepatitis a or b [44] . the nonstructural protein 3 (ns3) is a serine pro tease that exhibits protease and helicase activity and represents a good target for inhibition of hcv. aptamer selection was performed using a library car rying a 120 nucleotide randomized region and after 6 rounds of selection two aptamers inhibiting protease and helicase activities were obtained [45] . for identi fication of the aptamer demonstrating affinity to the active site of ns3 subsequent selection was performed using a truncated polypeptide δns3. using an rna library with a 30 randomized region authors per formed 9 rounds of selection and identified 45 clones, which bound δns3 [46] . according to their nucleotide sequences aptamers were subdivided into three families. they all contained a conservative region ga(a/u)ugggac. these aptamers formed a complex with δns3 with the k d value of 10 nm, caused 90% inhibition of protease activity of the δns3 peptide and full sized ns3 bound to a maltose binding protein (mbp ns3). in vivo hcv proteins are processed by ns3 and ns4a cofac tor. for modeling of physiological conditions the aptamer effect on ns3 activity was tested in the pres ence of the p41 peptide, which caused a sevenfold increase of mbp ns3 activity. under these conditions the aptamer inhibited mbp ns3 activity by 70% [46] . human neutrophil elastase (hne) is involved in various inflammatory diseases, including acute respi ratory distress syndrome (ards), septic shock, arthritis, and ischemia reperfusion injury [47] . a covalent inhibitor of hne, a diphenyl phosphate derivative of valine (valp), was coupled to an rna library to enhance the binding of the inhibitor with hne [47] . ten rounds of selection yielded an rna aptamer conjugated to the dna:valp substrate (rna 10.11: dna:valp). the aptamer demonstrated bind ing to hne (k d = 71 nm) and enzyme inhibition (k i = 5 nm) in vitro. in contrast to the rna aptamer 10.11 or the substrate dna:valp administered separately the aptamer modified with the substrate inhibited hne ex vivo in the rat model of ards [48] . the same group also performed a valyl phosphonate: dna library selection to find more potent hne inhibitors. authors used a single enantiomer form of the valyl phospho nate, which was compared with a racemic mixture. inhibitor selection was performed using purified elastase and also secreted elastase in the presence of neutrophils [49] . after 18 rounds of selection the aptamer ed45, which inhibited hne, was found. the aptamer was truncated to a 42 mer, named nx21909, and tested in a rat model of lung inflammation. a 40 nmol dose of nx2109 inhibited neutrophil infiltration by 53% in the lung of rat in vivo [49] . 3.1. vegf angiogenesis plays a central role in various physio logical and pathological processes. vegf (vascular endothelial growth factor) is one of the best character ized growth factors; it is involved into initial steps of angiogenesis and represents one of the most promising targets for anticancer therapy [50] . an increased vegf level associated with angio genesis was observed during tumor growth and metastases, premature aging and age related degener ation of tissues [50] [51] [52] . using the selex method ruckman performed 12 rounds of selection cycles and isolated aptamers to human vegf 165 with k d of 50 pm in a 2' f pyrimi dine rna library [19, 20, 53⎯55] . for increased sta bility against nucleases two aptamers were additionally modified by the 2' o position [55] . these aptamers were characterized by the k d values of 49 and 130 pm; they were specific to vegf 165 and did not bind to related proteins: vegf 121 and placenta growth factor plgf 129 . the aptamers to vegf 165 inhibited the bind ing of vegf 165 to its receptors, flt 1 and kdr (kinase domain receptor). using 125 i labeled vegf 165 inhibition of receptor binding was evaluated: theic 50 values for aptamer competition with the flt 1 receptor and kdr were ranged from 50-300 and 2-60 pm, respectively [55] . therapeutic potential of the aptamers to vegf was evaluated by the miles assay representing simple and rapid means of monitoring the ability of aptamers to inhibit the activity of vegf 165 in vivo. it is assessed as vascular wall permeability in animal models. the test was performed using adult guinea pigs and the most effective aptamer inhibited vascular permeability by 58% at 1 μm [55] . pharmacokinetics of the 2 fluoro pyrimidine and 2' o methyl purine aptamer to vegf called as nx 1838 has been investigated in monkeys. during intravenous administration of this aptamer as a conjugate with a 40 kda polyethylene glycol was characterized by half life of 9.3 h and a clearance rate of 6.2 ml/h. subcuta neous administration resulted in 80% absorption into the tissues within 8-12 h [25] . preclinical and clinical trial of nx 1838 also called as macugen was performed by eyetech pharmaceuti cals inc for the treatment of age related macular degeneration in diabetic patients [11, 12] . the synthetic aptamer nx 1838 was also investi gated in the rat model of angiogenesis. these studies confirmed a significant inhibition by 80% of angio genesis by means of vegf in the presence of this aptamer. the 1a phase of clinical trials did not reveal any significant complications after a single adminis tration of the drug. in addition 80% of patients dem onstrated stable improvement during observation for 3 months after injections, 27% of patients demon strated a threefold improvement of vision among dia betic patients (etdrs) [10] . clinical trials (phase 2) have shown that multiple macugen administration with or without photody namic therapy (pdt) did not cause any serious impairments; moreover 87.5% of patients demon strated stable vision improvement and 25% of patients demonstrated significant improvement evaluated using the etdrs system (early treatment for dia betic retinopathy study). during the third phase of clinical trials macugen (under the commercial name pegaptanib) was used as the only drug every 6 weeks over a period of 48 weeks at a dose of 0.3, 1 and 3 mg intravitreously [56] . all three groups of patients demonstrated significant improvement of vision. the severe loss of visual activ ity determined as the loss of 30 letters of visual acuity reduced from 22 to 10% in the group receiving 0.3 mg of macugen. in addition, 33% of patients receiving this dose maintained their visual acuity or gained acu ity (versus 23% of control group). no antibodies against macugen were found. eytech in cooperation with pfizer obtained fda approval for the use of macugen for the treatment of amd. these first results demonstrate that aptamers may be effective drug prep arations. it is known that the intensive tumor development is accompanied by neovascularization and therefore an aptamer to vegf was used for inhibition of tumor vascularization (and tumor growth). the aptamer isolated and optimized by ruckman et al. was tested in a mouse model of wilmis tumor (the most common malignant tumor of the kidneys in chil dren). tumor was implanted into a mouse kidney and its growth was maintained for one week, and then the aptamer to vegf (200 μg) or vehicle (phosphate buff ered saline) were administered for experimental and control mice respectively, daily for 5 weeks [57] . after decapitation of animals authors observed that tumors weighed 84% less in treated versus control animals. lung metastases were seen only in 20% of the aptamer treated animals (versus 60% of animals from the control group). the aptamers tested in the murine nephroblastoma exhibited the decrease in tumor growth by 53% compared with control [57] . the increase in bfgf correlates with appearance of various diseases including retinopathy, rheumatoid arthritis, leukemia [58] . jellinek and co authors used a 2' amino pyrimi dine derivative rna library and performed 11 rounds of aptamer selection [58] . they found an aptamer named as m21a, which exhibited binding to bfgf with k d of 0.35 nm; a competitive binding study revealed that it competed for bfgf binding with unfractionated heparin and low molecular weight hep arin. the inhibitory activity of m21a was also investi gated using chinese hamster ovary (cho) cells: the aptamer bound to its target with the k d values of 1-3 nm [59] . the effect of m21a on the endothelial cell motility was also investigated using the migration of endothelial cells to a denuded area in bovine aortic cells where endogenous bfgf is essential for activity. at concentrations > 50 nm the aptamer inhibited cell migration in a dose dependent manner (as compared with control). the rna aptamer inhibited bfgf bind ing to its cell receptor [20] . platelet derived growth factor (pdgf) is a mito gen composed of two homologous (a and/or b) chains linked by three disulfide bonds; this dimeric protein is involved into wound healing and progression of vari ous diseases including atherosclerosis and glomerulo nephritis. many tumor cell lines produce and secrete pdgf [60] . a dna selection in vitro against human recombi nant pdgf ab was performed and after 12 rounds dna aptamers characterized by k d of 50 pm were iso lated. three aptamers effectively inhibited pdgf bb binding to pdgf α and β receptors with the k i value of 1 nm. the anti pdgf aptamers also inhibited mitogenic effects of pdgf on cells expressing pdgf β receptors with k i of 2.5 nm [61] . one aptamer termed 36t was truncated, 2'o methyl, 2' fluoro modified and capped at the 3' end (to increase resistance to nucleases) and conjugated to 40 kda peg (to increase its lifetime in blood circula tion) [62] . the modified aptamer exhibited high affin ity binding to the human protein (k d = 100 pm); it was tested using a rat glomerulonephritis. in this model intravenous administration of this aptamer (2.2 mg/kg) twice a day decreased mitoses by 64% on day 6 and by 78% on day 9. animals treated with this aptamer were characterized by a decreased mono cyte/macrophage index and glomerular matrix over production. control animals received a scrambled sequenced oligonucleotide or peg for 6 days [63] . interferon γ (ifn γ)exhibits various immunoregu latory effects. although its antiproliferative effect is less pronounced than in ifn α and ifn β, ifn γ is the most potent activator of macrophages and the inducer of expression of mhc class ii molecules [58] . in healthy nervous tissue ifn γ is almost absent, how ever, during inflammatory processes in the nervous system and in multiple sclerosis it is overproduced. ifn γ secretion can result in inflammatory and autoim mune diseases. rna selection using 2 fluoropyrimi dine and 2 aminopyrimidine rna or a mixture of these two modifications were screened for aptamers that inhibited receptor binding of ifn γ [64] . the resultant aptamer, 2' amino 30 had a k d value for its complex with receptor of 2.7 nm. in the culture of a549 cells it inhibited receptor binding of ifn γ with k i value of 10 nm. this aptamer also inhibited induction of the mhc complex regulated by ifn γ and icam 1 expression with ic 50 values of 700 and 200 nm, respectively [64] . endothelial receptor tyrosine kinase tie2 plays an important role in vascular wall stability. angiopoietin 2 (ang 2) is a natural antagonist, which is obviously expressed only during active angiogenesis (e.g. tumor growth) [65] . for investigation of ang 2 by aptamers 11 rounds of rna selection were performed and rna molecules exhibiting specific binding to ang 2 were iso lated. one aptamer demonstrated high affinity to ang 2 (k d = 3.1 nm); it did not bind to ang 1 (k d > 1 μm). this aptamer was truncated to 41 mer (k d = 2.2 nm) and the truncated aptamer inhibited ang 2 function in a cell culture and in a rat model, where it significantly inhibited neovascularization by 40% [65] . influenza is one of the most widespread disease in the world. control of this disease includes active cam paigns of vaccination, use of drugs blocking neuro minidase action. the use of aptamers helps to block virus binding to cell receptors. binding of isolated dna aptamers to viral hemagglutinins blocked virus penetration into cells [66, 67] . extracellular domains of influenza hemagglutinin cause agglutination of blood cells (mainly erythro cytes). hemagglutinin determines virus binding to cells. neuraminidase is responsible for : 1) ability of a viral particle to penetrate into the host cell; 2) ability of viral particles to leave host cells after reproduction. two dna aptamers were obtained to the hemaggluti nin peptide (residues 91-261), which is responsible for binding of an oligosaccharide component of cell receptors. the aptamer a22 exhibited high binding activity and blocked agglutination of chicken red blood cells [66] . the effect of a22 was confirmed by microscopic studies; they revealed preservation of cell structure compared with control preparations, in which damage of cell structure in the presence of influenza virus was observed. the same authors iso lated two rna aptamers to hemagglutinin using an rna library containing a 30 nucleotide randomized region. a predicted secondary structure in 73 bases long included nucleotides of the randomized region and also constant sequences of the flanking region. one of the aptamers exhibited binding to hemaggluti nin with the k d value of 2.9 nm. the affinity of this aptamer was 15 fold higher than that of a monoclonal antibody to this protein. moreover, the rna aptamer allowed to discriminate hemagglutinins isolated from two various strains [67] . binding proteins 5.1. tat protein therapeutic applicability of aptamers has been undertaken during studies of hiv replication. human immunodeficiency viruses hiv 1 and hiv 2 that belong to a lentivirus class selectively affect t helpers. a regulatory tat protein activates viral replication. the tar element (of 60 nucleotides long) presented in all predicted viral transcripts is required for func tioning of tat protein. it was proposed to express the 60 nucleotide tar sequence to capture tat protein into an rna decoy [68, 69] . tar rna, hiv tran script, was expressed in cem ss cells. the rna decoy inhibited hiv 1 replication over 99% in vitro. inhibition of viral replication in cd4+ cells proceeded at the high level tar aptamer expression from the trna promoter. changes in the nucleotide sequence of hair pins or loop in the structure of the tar aptamer abol ished the ability of the tar aptamer to inhibit hiv rep lication [68, 70] . the selex library consisted of a randomized sequence of 120 nucleotides was used for selection of aptamers to tat proteins. after 11 rounds of selection a truncated variant of an rna aptamer named as rna tat was obtained; it formed a complex with the tat protein with k d of 120 pm. this 37 mer rna aptamer inhibited hiv 1 in vitro and decreased viral replica tion by 70% in a cell culture [71] . other groups also found rna decoys, which bound tat protein and inhibited hiv 1 [72, 73] . no significant incompatibility between tar and tat interactions of hiv 1 and hiv 2 belonging to var ious subfamilies were found. however, although tat 1 could transactivate hiv 2 through tar 2, tat 2 did not interact with tar 1 in hiv 1 [74] . the viral protein rev promotes transportation of partially spliced rna molecules to cytoplasm, where they provide synthesis of usual retroviral products. the rre (rev responsive element) element composed of about 234 nucleotides forms a complex three dimen sional structure, to which rev protein binds. using a trna promoter for expression of the rre element, the major rev binding site in hiv 1, overexpression of this construct has been estimated in the cells. expres sion of the chimeric trna rre aptamer caused inhi bition of viral replication by more than 90% [75] . the aptamers passed phase 1 clinical trials, in which this construct was transduced in vitro to cd34+ cells obtained from bone marrow of a hiv 1 infected sub jects followed by subsequent reinfusion into these sub jects. aptamer administration did not cause adverse effects, however, a rather low level of rre gene expression was observed possibly due to inappropriate conditions for gene transfer [76] . reverse transcriptase (rt) was the first target for the development of the selex method for hiv ther apy. tuerk and gold published the pioneer paper on selex. they used rt as the target for isolation of rna ligands, inhibiting hiv replication [1] . after 9 rounds selection from rna populations random ized at 32 positions authors isolated rna that specifi cally bound to hiv rt and inhibited activity of this enzyme [77] . the sructure of the rna aptamer was subsequently characterized and experiments per formed on cells indicated inhibition of hiv 1 replica tion by 90-99% [78] . in addition, aptamer expressing t cells completely blocked the spread of hiv in cul ture [79] . proliferation of myocardial and vascular cells is a central problem in the development of such cardiovas cular diseases as hyperplasia, atherosclerosis, malig nant tumors [80, 81] . e2f plays a central role in regu lation of cell proliferation. this factor exhibits highly specific binding to a double stranded dna containing eight base pairs tttcgcgc. the constructed 14 mer dna aptamer containing a sequences for e2f binding was tested for inhibition of e2f activity [82] . in vascular smooth muscle cells (vsmc) stimulated by e2f the 14 mer oligonucleotide (odn) inhibited vsmc proliferation and expression of the genes c myc and cdc2, controlling cell cycle, and proliferating cell nuclear antigen (pcna). in vivo the 14 mer odn was transfected to rats with experimental carotid injury and this markedly suppressed the fibrosis for mation compared with nontransfected arterial seg ments. furthermore, this inhibition continued up to 8 weeks after a single transfection [81] . transfer of an e2f decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo. using the selex method other aptamers to e2f were also prepared; they inhib ited dna binding activity of this protein [82] . these interesting results prompted dzau's group to test the e2f aptamer in humans in order to determine whether it can limit intimal hyperplasia during intra venous administration [83, 84] . the e2f aptamer was delivered to the infra inguinal vein by transfection. cell transfection efficiency was 89%, expression of c myc and pcna reduced by 73 and 70%, respectively, com pared with the control group. after 12 months in the group of patients treated with the e2f aptamers fewer occlusions were registered compared with control. the e2f dna aptamer is now evaluated by cor genetch inc., in a phase iii study to estimate its effi cacy at limiting coronary and peripheral vascular damages. this aptamer is very close to clinical appli cation. the e2f aptamer was also used for evaluation of long term protection from neointimal hyperplasia and atherosclerosis [85] . hypercholesterolemic rabbits were treated with intravenous injections of e2f aptamer or scrambled oligonucleotide. after 6 months (when animals were put on a cholesterol containing diet) the e2f aptamer treated group of animals was free of plaque whereas animals treated with the scram bled oligonucleotide and also control animals had extensive plaque formation [85] . finally, selex was used to obtain an rna aptamer that would bind and inhibit e2f. insertion of the e2f aptamer into a trna expression cassette yielded rnas exhibited effective inhibition of e2f1 binding to dna [86] . to test the ability of the e1 rna aptamer to block proliferation, human fibro blasts were treated with e1 rna aptamer and prolifer ation was then induced. the rna aptamer inhibited s phase by 80% compared with control [86] . thus, natural and in vitro selected aptamer can act as prolif eration inhibitors. transcription factor nf κb activates genes involved into inflammatory processes and synthesis of cytokines, interferons, mhc proteins, growth factors, and cell adhesion molecules, which play a central role in infarctions and various ischemic pathologies [87] . it is also required for hiv 1 gene expression and regula tion of cell tumors. a double stranded dna aptamer exhibiting high affinity binding to nf κb and named as a "natural decoy" was investigated in vivo using a cardiac ischemic/reperfusion model and a significant effect in inhibiting this injury was observed [88] . in a rat model of thrombosis animals transfected with the nf κb aptamer showed improved recovery of coro nary flow (97% versus 61% in control) 3 days after transfection [89] . the aptamer treated group demon strated a lower percentage of neutrophil adhesion to endothelial cells (38% versus 81%) and a lower level of interleukin 8 (109 versus 210 ng/mg) as compared with control [89] . a fluorescent labeled aptamer to nf κb was investigated in a murine model of nephritis, where it blocked glomerular inflammation and expression of the inflammatory markers il 1α, il 1β, il 6, icam 2, vcam 1 [90] . using the selex method an rna aptamer was also genetade against the p50 subunit of nf kb. four teen rounds of selection yielded the rna aptamer, exhibiting high affinity binding to p50 and inhibition of nf kb binding to dna by preventing protein dimerization [91] . work with aptamers has important advantages over antibodies: ⎯in clinical practice aptamers may be applicable in the same fields where antibodies are already used for treatment, but in contrast to antibodies aptamers are non immunogenic; ⎯aptamers exhibit the same high affinity to their protein targets as antibodies; ⎯aptamers can bind and penetrate to a pathologi cal nidus faster than antibodies; ⎯aptamer antidotes may be developed and they can control activity of the administered aptamer. the selex method originally developed for nucleic acid binding proteins is now actively used for studies of proteins, which lack natural complexes. the method is applicable for manipulations with individ ual proteins and for work with cell cultures. proc. natl. acad. sci. usa trombozy v kardiologii. mekhanismy razvitiya i vozmozhnosti terapii (thromboses in cardiology. mechanisms of develop ment and therapeutic capacities) proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa, 1992 proc. natl. acad. sci. usa proc. natl. acad. sci. usa key: cord-016313-n4ewq0pt authors: baranyi, lajos; dropulic, boro title: advances in lentiviral vector-based cell therapy with mesenchymal stem cells date: 2012-09-27 journal: mesenchymal stem cell therapy doi: 10.1007/978-1-62703-200-1_14 sha: doc_id: 16313 cord_uid: n4ewq0pt the field of possible application of mesenchymal stem cells in medicine and research expanded tremendously with the advent of improved lentiviral-vectors capable of inserting stable copies of genes of interest and expressing proteins or biologically active rna species ad libitum, performing delicate gene editing or active gene silencing or serving as advanced drug delivery systems utilized in ex vivo cell therapy. the combination of these two fields has created a number of new areas of research in the landscape of modern medicine which are now extensively studied and discussed here. these areas include tissue engineering, tissue repair, wound healing and tissue implants, anticancer therapies, angiogenesis, myocardial infarction and repair as well as understanding and treating acute lung damage and injury. in addition, genetically modified, tagged mscs are being intensively deployed in research and therapeutic attempts of the various ailments of the central nervous system including parkinson’s disease, alzheimer’s disease, various phases of acute ischemia and trauma. the emergence of new and important data for type ii diabetes research is being followed with treatment suggestions and studies of senescence to find novel applications for genetically engineered mscs. we find in ­general that genetically modified mscs are at the cusp of breaking through from basic research into the next phase of clinical trials. stem cells, including the mesenchymal stem cells (mscs), are very close manifestations of plato's imagery of the shadows on the cave wall, since they are dif fi cult to study outside their intimate interactions with their microenvironment [ 288 ] . our observation methods change their responses and characteristics [ 9, 74, 82, 103, 145, 252 ] , as in quantum physics, when the observation changes the observed. with that caveat, we can admire the rapid development in stem cell research. alas, the dif fi culties in research are faithfully re fl ected in the confusion in the nomenclature used for describing and classifying stem cells, including the classes of stem cells of mesodermal origin. the recent de fi nition of mscs by dominici states that mscs are a stromal cell type, possessing the following characteristics and markers: plastic adherence in cell culture, speci fi c surface antigen expression of cd105(+), cd90(+), cd73(+), cd34(−), cd45(−), cd11b(−) or cd14(−), cd19(−) or cd79a(−), hla-dr1(−) and multi-lineage in vitro differentiation potential (osteogenic, chondrogenic, and adipogenic) [ 59 ] . however, this de fi nition would neatly exclude cd34+ hematopoietic stem cells (hscs ), while one also could argue that the hematopoietic stem cells are just a specialized subclass of the mesenchymal progenitors [ 180 ] . another subset of mscs that express hyaluronan (cd44), an adhesion molecule important for stem cell homing [ 14, 125, 281 ] , would also be excluded, but their perivascular equivalents could be considered to be true mpcs [ 180 ] . it becomes even more complicated if we include the results of (stem) cell reprogramming, when more or less differentiated cell types are regressed into less differentiated, pluripotent cell types [ 91, 185, 192, 239 ] , providing us with a never ending stream of novel biomarkers, more often represented by whole proteome analysis [ 1, 203 ] . time will tell, what are the biomarkers and criteria for properly characterizing the particular stem cell populations, but there is a functional de fi nition lingering around as a fi rm conceptual handle on the idea of cell plasticity of which stem cells are prominent representatives [ 21, 22 ] . the plasticity indicates the ability of matured or not fully differentiated cells to differentiate into novel cell types, or more accurately, it describes the existence of cells specialized into becoming progenitors of differentiated cells while sustaining their own type and maturation level. combining this with the embryology and origins of cell lineages from the three primordial "dermata" (ecto-, endo-, and mesoderm) provides us with a useful and generalized de fi nition of mscs being the pluripotent, self-renewing stromal cells of mesenchymal origin and allowing us to determine the speci fi c biomarkers later, at our convenience, as the state of affairs in mesenchymal stem cell biology progresses and solidi fi es. there is no doubt that we will fi nd the appropriate placement for the specialized subtypes as well as the proper and practical placement of some of the induced pluripotent cells (ipcs) in the realm of mscs. regardless of the exactitude of the classi fi cation, the (omni-) presence of the mesenchymal cell lineages in all of the organs and tissues [ 11, 102, 116, 132, 180 ] renders them good candidates not only for general stem cell therapy [ 22, 73, 102, 235 ] , but even more promising is the potential use of mscs for gene therapy [ 35, 45, 176, 196 ] , cell reprogramming [ 9, 36, 252 ] , delivery of bioactive molecules [ 163, 196 ] , and tissue engineering [ 4, 82, 153 ] . in addition, a number of new issues are arising from the results describing the importance of stem cells in inducing and sustaining the malignant phenotype and the potential therapeutic targeting of a wide range of the elusive cancer stem cell types [ 27, 183, 217, 244 ] . the genetic modi fi cation of mscs and associated cell types with lentiviral vectors opens their application beyond reliance upon the innate properties of the cells. expression of proteins that can modulate their biology or therapeutic properties enormously expands their utility for therapy. the lack of a crisp de fi nition of all the stem cell types affects targeting of lentiviral vectors to speci fi c subsets of stem cells. however, recent successful efforts in the pseudotyping of lentiviral vector is a step in the right direction. the use of the vsvg pseudotype expanded the tropism of the lentiviral vectors and, as a result, practically any cell type can be targeted and the narrowing of the tropisms by developing novel vector-pseudotypes will be addressed. the emergence of single chain antibodies as pseudotype indicates that we can expect a rapid expansion of this technology in the near future and will result in a precise tool for studying cell lineages. we focus and limit our review on the recent progress made in stem cell research using lentiviral vector-based gene delivery, a method that is emerging as the safest and most effective way to modify (stem) cells permanently or temporarily, if using non-integrating versions of the novel generations of lentiviral vectors, both of which have clear potential for a wide range of research application in preclinical studies as well as therapeutic applications. the most commonly used lentiviral vector framework is hiv-1 based although hiv2, siv (simian immunode fi ciency virus ), fiv (feline immunode fi ciency virus) have been successfully tested ; see review by dropulic [ 62 ] . the native hiv-1 is a human pathogen; but it had been modi fi ed to eliminate pathogenicity and increase safety before considering it as a broadly available tool for gene transfers. typically, lentiviral vector are generated by trans complementation , a process that separates the essential components of hiv (the genes encoding gag-pol , rev , and env ) into separate plasmids, which lack the packaging signal and , therefore, can never end up in a packaged vector unless they appear in a recombinant sequence [ 63 ] . the components (tat , vif, etc.) responsible for pathogenicity by upregulation of transcription [ 54 ] and export of genomic rna to the cytoplasm have been successively removed from the constructs. the potential for a recombination event is minimized, and for all practical purposes avoided, by carefully editing the genes and using codon degeneracy to reduce the chances of recombination with the wild-type virus. these separate plasmids are used to co-transfect a packaging cell, typically hek293, along with a payload plasmid that carries the packaging signal necessary for starting the envelope formation and encapsidation of the mrna that carries the payload gene (s) as well as the 5 ¢ and 3 ¢ long terminal repeats (ltr s) necessary for integration into the transcriptionally active regions of the host chromosomes. packaging is a delicate process, which ensures that with the rna, appropriate trnalys, protease, integrase, and reverse transcriptase enzymes are carried by the vector with the packaging elements necessary for successful cell entry, reverse transcription of vector rna to dna, transport of that dna into the nucleus, and the permanent integration of the dna into host chromosome. the env gene encodes the protein gp160 that is cleaved into trimer-forming gp120, which appears as spikes decorating the vector particle; and gp41, that carries a transmembrane region and a carboxy terminal sub-domain that interacts with the nucleocapsid within the envelope. the n-terminal domain has a fusogenic domain that facilitates cell entry by fusing the outer membrane of the vector with the cell membrane. a region further down to from the amino terminal region also binds to gp120 which in turn binds to primary hiv-1 receptors on the target cd4+ of t lymphocytes. this property if left unmodi fi ed would signi fi cantly limit the usability of the lentiviral vector, as very few cells types can be directly infected by hiv-1. pseudotyping overcomes this limitation and permits targeting to any mammalian cell. pseudotyping essentially replaces the original hiv env gene with a corresponding molecule from other viruses and carries over the cell-targeting speci fi city (i.e., the tropism of the virus) and obviates some of the safety concerns related to gp120 [ 258 ] . the list of successful pseudotypes and cell tropism is rather lengthy [ 18, 76-79, 105, 222 ] and growing. the most successful pseudotype so far uses the env from vesicular stomatitis virus (vsvg) that successfully broadens the tropism to cells in the brain, kidney, and liver amongst other. it extends to mesenchymal (stem) cells, even those in nondividing (resting) state [ 77, 136, 280 ] . filovirus env pseudotypes shift the tropism to a more limited set of cells, airway epithelial and endothelial cells [ 130, 148 ] . baculovirus gp64 and hepatitis c virus e1 and e2 pseudotypes redirect the vectors toward liver cells targeting their respective receptor, cd81 (tetraspanin) [ 19 ] . rabies virus env has been shown to ef fi ciently retarget the vectors to neuronal cells [ 162, 262 ] . rd114 env pseudotyped lentiviral vector show preference for hematopoietic cellular compartment [ 20, 56, 85, 115, 221, 268 ] . however, some applications require targeting a speci fi c cell type, which is not necessarily covered by the available pseudotypes listed above. in those cases, new targeting methods have been developed to further tighten the tropism of lentiviral vector by co-expressing cell-speci fi c coreceptors that recognize one of the cell-type speci fi c markers. the payload plasmid components providing the backbone for the transfer vector in the early hiv vectors were composed of a 5 ¢ ltr, followed by a major splice donor site, a packaging signal site encompassing the packaging signal components of the 5 ¢ region of gag (necessary for high ef fi ciency packaging and high vector titer) and a deletion of the rest of the gag gene. deletion of the u3 region from the 3 ¢ ltr promoters became also possible by relaying on constructing genes with their own promoter(s). the latest generation of lentiviral vectors carry an additional safety element, the self-inactivating ltr (sin lentiviral vector , [ 114 ] ) replacing the ltr with an hiv-independent promoter from cytomegalovirus (cmv). in these vectors the ltrs are modi fi ed in a way that upon integration they lose their intrinsic promoter ability reducing genotoxic potential. in addition the irreversible changes that occur during integration diminish the ability to mobilize after integration and to recombine with other elements to form a full-fl edged, replication capable virus [ 25, 279 ] . the formal proof of increased safety is still lacking, ironically, because of the inability to create and detect rcl capable viruses from lentiviral vector-treated cells [ 25 ] , indicating that this risk is mainly theoretical. the removal of rre and the associated splice donor and acceptor elements results in signi fi cant loss of transduction ef fi ciency of the vector [ 127 ] , while adding a 100 nucleotide central polypurine tract (central dna fl ap) restores the transduction ef fi ciency by improving the reverse transcription and nuclear transport ef fi ciency [ 47, 283 ] . the woodchuck hepatitis virus transcriptional regulatory element (wpre ) is another widely used regulatory element added to the lentiviral vector backbone to stabilize the transcription transgene mrna levels and improve transgene expression [ 292 ] . however, an open reading frame of the oncogenic whv-x element has been found within the native wpre sequence [ 128 ] , so the sequence has been modi fi ed to remove the translation start site [ 282 ] . further optimization continues to improve the safety of lentiviral vector, such as isolating the integrated vector dna to prevent translation beyond the vector boundaries by adding isolating elements. h owever, the insulators have themselves proven to be genotoxic in some instances, and no proof has emerged that such isolators are truly needed [ 100 ] . gene switches such as tet -on and -off have been added to subsequent generations of lentiviral vector and proven to be highly functional, operating with very low leakage [ 194, 260 ] . the cre-lox system has also been successfully implemented in the lentiviral context allowing high ef fi ciency engineering and sophisticated, site-speci fi c recombination techniques including the delivery and irreversible switching by small hairpin rna (shrna ) expression [ 135 ] , a tool extensively used in gene function analysis [ 37, 193 ] . a major concern regarding the safety of lentiviral vectors has been the potential genotoxicity resulting in oncogenesis, as observed previously during clinical trials for treating x-linked severe combined immunode fi ciency (x-scid) with transplanted hscs treated with murine oncoretroviral vectors carrying the gamma chain of il2r genes [96] [97] [98] [99] . the preferential insertion of oncoretroviral vectors in proximity of the lmo2 proto oncogene and the subsequent constitutive activation of the proto-oncogen driven by the enhancer element (ltr) in the vector resulted in uncontrolled cell proliferation. however, in a series of studies comparing oncoretroviruses and lentiviral vector, it has been shown that while the oncoretroviral vectors trigger a dose dependent acceleration of cancer onset in a mouse transplantation model sensitive to cancer-triggering genetic changes (cdkna2−/−), lentiviral vectors lacked such activity [ 172, 175 ] even though the vector integration rate was signi fi cantly higher. this important observation implying that a low level of insertional mutagenicity has been con fi rmed independently by several groups indicating a favorable safety pro fi le for lentiviral vectors while emphasizing the importance of vector design and avoidance of strong enhancers in the vectors [ 32, 169, 171 ] . recent clinical trials have supported the good safety pro fi le of lentiviral vectors . there have been no oncogenic effects reported in any of trials using lentiviral vectors to date [ 30, 83, 123, 143, 161, 195, 210, 276 ] . gene silencing by small interfering rna (rnai) is based on duplex formation between the mrna and a short complementary micro rna or small inhibitory rna, each having the ability to interfere with the protein synthesis and downregulate the expression levels of the targeted protein. a major problem with the inhibitory rna technologies is the short half-life and delivery of the rnai. this can be resolved using lentiviral vectors encoding arti fi cial genes with appropriate micro rna sequences that can be integrated into the host cell dna and ef fi ciently transcribed into primary micro rna that utilizes the natural intracellular processing by microprocessor complex formation with drosha to form small hairpin rna (shrna) in the nucleus. the exported mirna is subsequently cleaved by dicer and produces the complex forming inhibitory rnai. the process is rather complex, but ef fi cient to produce a signi fi cant blockade of protein expression that may be incomplete but readily achieves signi fi cant reduction, that is adequate for gaining insight into the function of the targeted protein and ef fi cient enough for phase i and ii clinical trials, though no therapeutic use has been approved by the fda. it is interesting to note that mscs are capable of secreting cholesterol-rich phospholipid microparticles encapsulating mirna, and therefore have the potential to facilitate intercellular communication and act as regulatory agents in their microenvironment [ 38 ] . an hematopoietic or general pluripotent stem cells are often selected targets for rna interference-based interventions and one of the promising efforts deal with creating arti fi cial virus resistance genes and virus resistant somatic cells. preventing hiv infection by reconstituting the immune system with such stem cell-derived virus-resistant progeny has been used as model system with signi fi cant clinical relevance [ 121 ] . the idea is that an ef fi cient hiv infection requires virus entry through the cd4 surface antigen and one or more virus co-receptors, among which ccr5 has been shown to play an essential role in the case of r5 tropic viral strains involved in primary hiv infection. clinical data indicate that ccr5 de fi ciency or certain mutations in this co-receptor protect the infected individuals from the onset of full blown aids, and the hope is that the arti fi cial knockdown of ccr5 using gene therapy and rnai will achieve similar protection [ 8, 57, 121, 146, 233 ] . the relative inef fi ciency of the ccr5 suppression remains a signi fi cant issue, but major improvement and complete knockdown of ccr5 have been achieved with somewhat longer (28 base instead of 23) shrna [ 7 ] . mesenchymal stem cell research is taking full advantage of the shrna techniques by characterizing the subtle, and not so subtle, changes induced by individual gene knockdowns. it is a long held view that mechanical stresses and mechanical characteristics of stem cells, as well as the microenvironment, can affect stem cell proliferation and differentiation. lentiviral vectors are excellent and ef fi cient targeting tools for these stem cells, even resting ones, and can deliver the shrna without causing major changes and stress that would otherwise change the stem cells on its own. chowdhury et al. studied the spreading response of mscs and showed that myosin ii , f-actin , src, or cdc42 were essential for cell spreading and changes in the mechanical characteristics ("softening") of the stem cells led directly to the downregulation of the oct3/4 gene. this indicates the possibility that small mechanical events may affect the embryo and developing tissues and even transplanted stem cells [ 41 ] . another area of ef fi cient use of lentiviral vectors and rnai technology in stem cell research is the production of transgenic embryos which carry knockdown genes. production of transgenic embryos is highly ef fi cient, and if the fertilized egg is transduced at a single cell stage, the entire germ line is affected, or partial chimerism can be achieved if multicellular embryos are treated with lentiviral vectors. an example of such a study is that by wang et al., in which they showed that the knockdown of runx1 in embryonal tissues and mscs by lentiviral vector-delivered interfering rna blocked chondrogenesis in limb buds [ 257 ] . the technique has been shown to be very ef fi cient for transgenesis, as high as 44% average rate of germ-line transmission can be achieved [ 227 ] , providing a new source of gene-modi fi ed mscs. a recent comprehensive review of the use of naturally occurring regulatory mirna technology in mesenchymal stem cell research has been written by guo et al. [ 93 ] , indicating that stem cells have discrete and distinct expression pro fi les that can account for intrinsic stem cell properties such as self-renewal and pluripotency, a property that cannot longer be overlooked by experts dealing with mscs. the accumulating data indicate that the progenitors and terminally differentiated mesenchymal cells can be tracked and de fi ned by function-related mirnas in addition to the already established sets of surface markers. the mirnas already identi fi ed affect osteogenic differentiation, chondric differentiation, adipogenic differentiation, myogenic differentiation, neuronal differentiation, wound healing, and replicative senescence. these advances open a wide array of possibilities to direct the differentiation patterns of the stem cell population temporarily by using non-integrating lentiviral vectors that are automatically lost from dividing cell populations and lead to the natural disappearance of control signal after a few cell division but potentially giving a push to the original stem cell population to develop in a preferred direction. extensive progress has been made in regards to the elucidation of the hedgehog signaling pathway in mscs using rna interference delivered with lentiviral vectors. the data suggest that at least some of the elements indeed act through the regulatory mirna network, by downregulating the cellular mirna levels. the data, however, also suggest signi fi cant off-target effects of the interfering rna molecules and indicate that we are a long way from the potential clinical use of the elucidated networks [ 124 ] . an ingenious method was devised by hu et al., to prepare the brain for traumatic interventions (surgery, extensive stem cell transplantations, etc.) by downregulating the cerebral matrix metalloproteinase 9 (mmp9) using lentiviral vector and mmp-9 shrna 2 weeks before the trauma. the knockdown of mmp-9 with the shrna proved to be an effective way to preserve the blood-brain barrier, and they achieved signi fi cant reduction of brain infarction volumes, reduction of brain water content and evans blue/igg extravasation (measure of edema formation) as well as a reduction in the neurobehavioral de fi cit in their rat brain trauma model [ 108 ] implying a potential for improved protocols for traumatic brain interventions needed for more extensive type of intracranial stem cell implantations. as mentioned earlier, lentiviral vectors provide a very ef fi cient method for generating transgenic embryos, signi fi cantly reducing the need for the generation of a high number of embryos to establish new sources of gene-modi fi ed stem cell lines, embryonic, or other [ 227 ] . the lentiviral technology is able to deliver a payload of 6-8 kb very ef fi ciently, but payloads of 10kb can be handled and delivery of 12-13 kb is possible, at a cost of lower ef fi ciency. this payload-carrying capacity allows the delivery of very large genes such as the gene encoding blood clothing factor viii, a 2,351 amino acid long protein together with its stabilizer, the von willebrand factor (2,813 amino acids in its native form) simultaneously or, one may need to use domain-engineered and shortened version of both; similarly it can be used to deliver all three chains of an igm molecule in a single, tri-cistronic complex. the implication is that the lentiviral vector system has suf fi cient payload capacity to deliver a number of relevant genes together with several supporting molecules envisioned for highly complex gene therapy scenarios currently outside the scope of monogenic gene therapy as practiced today. it may be used to target diseases with multi-gene disorders such as high blood pressure, arthritis, or diabetes in the future. zinc-fi nger nucleases (zfns) have the remarkable ability to (a) bind to a speci fi c location in the double-stranded dna; (b) break the double-stranded dna at that speci fi c location and, if an endogenous repair template is provided, (c) initiate homology-directed repair, restoring the integrity of the newly edited double-stranded dna. as their name implies, there is a speci fi c dna-binding part of this class of enzymes that consists of a tandem repeat of dna-binding zinc-fi nger motifs, hence the dna binding speci fi city and a catalytic domain, foki . for dna cleavage to occur, foki has to dimerize, one on the sense and the other on the antisense strand, while the zinc-fi nger domains attach to the right target half site and the left target half site. upon binding, a nick with a 5 ¢ overhang is initiated by foki between the target sites and the homology-directed dna repair mechanism is activated. what makes this con fi guration useful is that the spacer between the two target half sites can be several hundreds or even thousands of base pairs long and by providing a template for the activated repair mechanism, a novel dna sequence of equal length can be introduced into the dna; see a recent reviews by caroll [ 29 ] and others [ 50, 101, 117, 134, 246 ] . fundamentally, two factors determine the ef fi cacy of the dna editing or repair that the technology allows. the fi rst is the speci fi city of the zinc-fi nger binding, which also determines the length of the spacer and the proper speci fi city and uniqueness of the binding site and allows the minimization of the off-target effects that may be introduced by similar sites far away from the desired and targeted locus [ 101 ] . huge efforts are being made to tailor the zinc-fi nger nucleases for particular applications and improving the selectivity by successfully engineering the dna-binding speci fi city of the binding domain [ 3, 101, 158, 201, 220 ] . the second factor is the ef fi cient delivery of the zfns and the template dna by vectors. while the early attempts relied on retroviral vectors, adeno and adeno-associated vectors, and even baculovirus vectors, the recent advances in the fi eld clearly indicate that the lentiviral delivery system is considered to be a safer and more ef fi cacious route. as high as 50% conversion rate can be achieved with lentiviral delivery in a variety of cell lines and human embryonic stem cells [ 154 ] as compared with the earlier best rates of 18% with other methods in human and other species [ 3, 101, 197, 198, 201, 220, 245, 264 ] . one of the many holy grails of medicine, the ability to replace diseased tissue or even entire organs, seems to be hovering at the not too distant horizon. there is rapid progress in a wide range of areas, but at the center of the solution is almost always biocompatible scaffolding that is populated with a wide variety of cells. the strategically positioned cells fi nd their place within the 3d structure, propagate, differentiate, and fi ll the available space, while producing a structure that can replace or enhance the damaged tissue in the form of various implants or prosthetics. as for scaffolding, the options are quite numerous, including those obtained from cadavers or live organs (animal or human origin), by removing the cells while preserving the fi brous tissue that maintains the basic morphology of the organ. alternatively, a scaffold can be printed with various 3d printers [ 126, 159, 214-216, 229, 255, 261 ] . processed cartilage can also result in scaffold and it can be used to rebuild and regrow an implantable ear, nose, or cartilage for trachea reconstruction [ 174 ] . the culture, expansion, and differentiation of human mscs into arti fi cial tissues represent a very complex series of events and lentiviral vectors often serve as excellent research tools for marking, visualizing, and tracking the process [ 253 ] , or modifying the gene or protein expression patterns [ 68 ] . a number of tissue engineering attempts have reached the clinic and lentiviral vector have played various roles in the advancement of the technology. a very promising technology is the use of these scaffolded arti fi cial tissues employing mscs and lentiviral vectors for delivering biologics for prolonged times. van damme succinctly described the potential of these arti fi cal tissues built on scaffolds and providing arti fi cial implants for drug delivery. lentiviral vectors were used to transduce mesenchymal cells to express green fl uorescent protein (gfp) or fviii. expression was superior compared to oncoretroviral transduction, showing consistently higher transduction rates and expression remained high for several months post-transduction. the transduced cells retained their stem/progenitor cell properties, and they were still capable of differentiating along adipogenic and osteogenic lineages in vitro, while maintaining high gfp and fviii expression levels. implantation of lentiviral vector-transduced human bone marrow mesenchymal cells using collagen scaffolds into immunode fi cient mice resulted in ef fi cient engraftment of gene-engineered cells and provided sites for transgene-expression in vivo. in addition to the bone marrow-derived stem cells, adipose tissue-derived mesenchymal stem cells have been shown to be amenable to populate implantable scaffolds and retain the potential to differentiate into osteogenic cells. some of these scaffolds have been engineered for use in reconstructing craniofacial bone defects. lentiviral vector have been used to deliver fl uorescent proteins to track cells during manipulation such as osteogenic differentiation. the gfp-marked stem cells and their progeny remained fl uorescent over the 8 weeks of the study period. the gfp-marked stem cells were successfully induced into osteogenic cells both in monolayers and threedimensional scaffolds. quanti fi cation showed no decrease in staining of the osteoinduced stem cells indicating the ef fi ciency and durability of the labeling [ 256 ] . tissue engineered vascular grafts built on bilayered elastomeric poly (ester-urethane) urea scaffolds and seeded with pericytes have shown promise in the past. however, in vitro endothelialization is still an issue for the use of these types of grafts. doebis et al. reported in 2006 enhanced endothelialization using allogeneic endothelial cells or their precursors, expressing recombinant anti-alpha-mhc i single chain antibody to prevent rejection. the recombinant antibody was delivered ef fi ciently ex vivo using lentiviral vector, and has signi fi cantly reduced the mhc-1 expression levels as well as the killing of allogeneic cells by mhc-1 speci fi c cd* + t cells [ 58 ] . the results suggest that these allogeneic cells may provide a suitable alternative supply for the lining of vascular prostheses. endothelial cells and their precursors are attractive targets for gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. there have been a few reports which show lentiviral vector-mediated gene transfer ef fi ciency. sacoda and colleagues compared the effectiveness of lentiviral vector compared to adeno and oncoretroviral vectors. bovine aortic endothelial cells (baecs) were infected, in vitro, with these viral vectors. transduction ef fi ciency of beta-gal gene transfer in baecs by adenovirus, lentiviral vector, or retrovirus at a multiplicity of infection (moi) of 10 (determined on hela cells) was 69 ± 11, 33 ± 8, or 22 ± 6% respectively. at higher moi [ 50 ] both adenovirus and lentiviral vectors achieved an almost 100% transduction rate. however, retroviral vectors showed only 48 ± 6% at moi 50 and no increase at moi 100. the percentage of beta-gal positive cells decreased rapidly at longer passage of cells after being transduced by adenovirus. in contrast, lentiviral vector and retrovirus vectors mediated transductions showed sustained higher percentage of positive cells. furthermore, the transductions by lentiviral vectors had no signi fi cant effect on viability of baecs suggesting that for long-term cell therapy the lentiviral vectors have overall the best features [ 219 ] . expressing il10 in similar settings in the early, initiation phase, also inhibited and delayed the onset of the rejection process [ 287 ] . one of such cases in which the performance of the endothelial cells may need to be boosted is to increase the resistance to ischemia-reperfusion injury of the vascularized transplants and implants or normal tissues undergoing prolonged surgery. this is a condition which occurs too frequently and is responsible for devastating tissue injury caused by systemic activation of the complement system. lentiviral vectors can be used to force the over-expression of the anti-apoptotic gene, bcl-xl and indeed, it has shown signi fi cant protection from early apoptotic loss of vascular endothelial cells [ 286 ] . recently, tooth tissue engineering has attracted more and more attention. stem cellbased tissue engineering is thought to be a promising way to replace a missing tooth. the potential mscs for tooth regeneration mainly include stem cells from human exfoliated deciduous teeth (sheds), adult dental pulp stem cells (dpscs), stem cells from the apical part of the papilla (scaps), stem cells from the dental follicle (dfscs), periodontal ligament stem cells (pdlscs), and bone marrowderived mscs (bmscs). a recent review by peng et al. shows promising progress [ 190 ] . however, in practice, tissues other than bone marrow can serve as stem cell donors, including adipose tissue, periodontal ligament, and pulp for oral tissue regeneration [ 206 ] . the experimental data suggest that not only the stem cells ex vivo, but cells in the osteogenic tissue are amenable to direct transduction by lentiviral vector [ 259 ] . this opens up the periodontal reconstruction interventions to the bene fi cial effects of gene therapy enhancing the wound healing and improving engraftment by expressing growth promoters at low and slowly decreasing concentrations. estrela published an excellent review on the potential of mscs in regeneration of dental tissues [ 68 ] and rodrigues-loza reviewed the mesenchymal cell types recovered from dental tissues [ 208 ] . other data clearly show that the primary osteogenic cells are ef fi ciently transduced by lentiviral vector, and that their infusion into the mandible is a feasible method for locally delivering dna to primary osteogenic and bone cells in rat models [ 259 ] , indicating that future applications in vivo dental implant enhancement, using dental scaffolding, bone healing, and tooth regeneration may be feasible. recent efforts extend toward engineering dental repair by changing the expression of growth factors and bone morphogenic proteins leading to dentin formation, as discussed in a 2011 review by casagrande [ 31 ] and which seem to be amenable to cell therapy efforts with nonintegrating lentiviral vector. one of the tissues that is often injured but that presents dif fi culties when it comes to healing and repairs is the tendon. enhancing the healing process by in situ overexpression of helper factors such as il10 could reduce recuperation time and perhaps improve the quality of the repair. richetti et al. reported promising results in a murine model of patellar tendon injury after direct injection of an il10 transgene using lentiviral vector. although the tendons showed no obvious histological difference, the il-10-treated groups had superior mechanical characteristics by day 42 [ 205 ] . although the mechanism of wound healing in tendons is not yet understood, the involvement of mscs is suspected and delivery of additional factors that partake in healing process is discussed by meyerose and ashlan [ 13, 163 ] . recent fi ndings by shamis and colleagues [ 230 ] demonstrated that embryonic stem cells could be directed to speci fi ed and alternative mesenchymal cell fates whose function could be distinguished in engineered human skin equivalents. lentiviral shrna-mediated knockdown of hepatocyte growth factor (hgf) resulted in a dramatic decrease of hgf secretion from cell lines (edk cells) that led to a marked reduction in their ability to promote keratinocyte proliferation and reepithelialization of cutaneous wounds. in contrast, h9-mscs demonstrated features of mscs but not those of dermal fi broblasts, as they underwent multilineage differentiation in monolayer culture, but were unable to support epithelial tissue development and repair and produced signi fi cantly lower levels of hgf. characterization of these induced mesenchymal cells in 3d, engineered human skin equivalents demonstrated the utility of this tissue platform to predict the functional properties of stem cell-derived fi broblasts before their therapeutic use in reconstructive skin transplantation and wound healing. inhibition of hyper-keratinization by expressing a mutant form of tcgf beta3 that has lost its binding site for latency-associated peptide, reduced the re-epithelialization density and fi broblast/myo fi broblast trans-differentiation within the wound area [ 251 ] in a mouse skin wounding model. the expression of this mutated gene was achieved by injecting lentiviral vectors encoding the muttcgf beta3, into the regenerating tissue and the changes induced by this intervention predict a signi fi cant decrease in keloid formation and provide a potential model for preventing the painful dis fi gurement that follows the abnormally strong skin remodeling and scar tissue formation that oftentimes accompanies wound healing. the data indicate that future stem cell therapy with carefully designed interventions for patients prone to scar tissue formation could fi nd wide spread application. one of the causes of erectile dysfunction is the damaged penile cavernous smooth muscle cells (smcs) and sinus endothelial cells. song reports that it may be feasible to restore these cells by applying mscs to penile cavernous ecs or smcs. for this purpose immortalized (via lentiviral vector encoding v-myc) human bone marrow mesenchymal stem cell line b10 cells were transplanted into the cavernosum of sprague-dawley rats and harvested 2 weeks later. the expression of cd31, von willebrand factor (vwf), smooth muscle cell actin (sma), calponin, and desmin was determined immunohistochemically in rat penile cavernosum. multipotency of b10 to adipogenic, osteogenic, or chondrogenic differentiation was found. expression of endothelial cell-speci fi c markers (cd31 or vwf protein) and expression of smooth muscle cell-speci fi c markers (calponin, sma, or desmin protein) were demonstrated in grafted b10 cells indicating that human mscs may be a good candidates in the treatment of penile cavernosum injury [ 238 ] . angiogenesis requires the presence and active involvement of mscs and therefore mscs are ready to be recruited into the areas when there is a need for novel blood vessels: the in fl amed, hypoxic, tumor infested locations. gehmert et al. described an interesting model to study the migration of mscs. in their work, immunode fi cient mice were engrafted with human breast cancer cells (4t1) in the left mammary pad. a day later, the mice were injected ip with luciferase-labeled adipose tissue-derived mscs (using lentiviral vector technology). the mscs were found to rapidly migrate into the tumor, con fi rming the previous observations that mscs can be found within the tumor stroma and vasculature, even if the in fl ammation is not present, as the immunode fi cient mice lacked the in fl ammation signaling pathway. based on this result, it can be suggested that mscs can be attracted solely by the cytokines produced by the tumor. however, the power of in fl ammation has been clearly demonstrated in control animals, which received e. coli injections at contralateral locations and attracted all the mscs leaving the tumor implant msc-free [ 84 ] . elucidating the migratory mechanisms of the mscs seems to be an important step toward fi nding a delivery system to in fl ammatory sites and fi nding the conditions for clear migration into established tumors. even the simple marking of tumor tissue with fl uorescent proteins (such as gfp) holds important promise for surgeons, as delineating a breast cancer in situ during surgery would be possible by applying uv light and tracing the contours of the tumor. the technique already allows sophisticated molecular imaging combined with stem cell therapy [ 254 ] . wang and colleagues used the ability of mscs to differentiate into endothelial cells in vivo to establish whether the differentiated mscs persist in vivo and to determine if this potential persistence contributes to functional improvement after experimental myocardial infarction. they generated a lentiviral vector encoding two distinct reporter genes, one driven by a constitutive murine stem cell virus promoter and the other driven by an endothelial-speci fi c tie-2 promoter. the endothelial speci fi city of the lentiviral vector was validated by its expression in endothelial cells but not in undifferentiated stem cells. the lentivirus-transduced mscs were injected into peri-infarct are as of the hearts of severe combined immunede fi cient mice. persistence of injected cells was tracked by bioluminescence imaging (bli) and veri fi ed by immunohistochemical staining. the bli signal from the endothelial-speci fi c reporter revealed that the stem cells differentiated into endothelial cells 48 h after injection. however, both the constitutive and endothelial-speci fi c signals disappeared by day 50. nonetheless, the improvement in left ventricle ejection fraction with therapy persisted for up to 6 months. immunohistochemical staining showed that stem cell-derived endothelial cells integrated into endogenous cd31+ vessels. furthermore, stem cell-transplanted hearts had more cd31+ vessels and a lesser degree of cardiac fi brosis compared with the controls at 6 months. increased angiogenesis and decreased fi brosis were associated with cardiac functional improvement. similarly mscs double-marked with gfp-lentiviral vector and superparamagnetic iron oxide could be followed by mri for up to 8 months in a porcine model of infraction and revascularization [ 274 ] . endothelial cells respond to mild injurious stimuli by upregulating anti-apoptotic gene expression to maintain endothelial integrity. ec dysfunction and apoptosis resulting from ischemia/reperfusion injury may contribute to chronic allograft rejection. under optimized conditions for lentiviral vector transduction of rat aortic endothelial cells (raec) the delivery of the anti-apoptotic gene, bcl-xl, via lentiviral vector, protects raec from apoptotic death. the authors con fi rmed the damaging effect of the reperfusion phase. endogenous bax expression increased with i/r injury, whereas endogenous bcl-xl remained constant. raec transduced with lentiviral vector expressing bcl-xl were protected from early apoptosis caused by i/r injury, correlating with reduced cytochrome c release into the cytosol. this protective effect may be attributed to altering the balance of pro-and anti-apoptotic proteins, resulting in sequestration of the harmful bax protein, and may open up new strategies for controlling chronic allograft rejection [ 286 ] . inhibition of na+/h+ exchanger 1 (nhe1 ) reduces cardiac ischemia-reperfusion (i/r) injury as well as cardiac hypertrophy and cardiac failure. although the mechanisms underlying these nhe1-mediated effects suggest delay of mitochondrial permeability transition pore (mptp) opening, and reduction of mitochondrial-derived superoxide production, the possibility of nhe1 blockade targeting mitochondria has been incompletely explored. a short-hairpin rna sequence mediating speci fi c knock down of nhe1 expression was incorporated into a lentiviral vector (shrna-nhe1) and transduced into the rat myocardium. nhe1 expression of mitochondrial lysates revealed that shrna-nhe1 transductions reduced mitochondrial nhe1 (mnhe1) by approximately 60%, supporting the expression of nhe1 in mitochondria membranes. electron microscopy studies corroborate the presence of nhe1 in heart mitochondria. immunostaining of rat cardiomyocytes also suggests colocalization of nhe1 with the mitochondrial marker cytochrome c oxidase. to examine the functional role of mnhe1, mitochondrial suspensions were exposed to increasing concentrations of cacl 2 to induce mptp opening and consequently, rat heart mitochondrial swelling. shrna-nhe1 transduction reduced the cacl 2 -induced mitochondrial swelling by 64 ± 4%. whereas the nhe1 inhibitor hoe-642 (10 m m) decreased mitochondrial ca 2+ -induced swelling by only 37 ± 6. because mitochondria from rats injected with shrna-nhe1 present a high threshold for mptp formation, the bene fi cial effects of nhe1 inhibition in i/r resulting from mitochondrial targeting should be considered as a future target for cell therapy [ 250 ] oxidative stress is important in a number of pathologies, including cardiovascular diseases, such as atherosclerosis and cardiac ischemia-reperfusion injury. an important mechanism for adaptation to oxidative stress is the induction of genes through the antioxidant response element (are) which regulates the expression of antioxidant and cryoprotective genes via the transcription factor nrf2 (nuclear factor e2-related factor 2). as nrf2-regulated genes are induced during oxidant stress, occurring for example in reperfusion after ischemia, hurttila et al. took a novel approach to exploit are for the development of oxidative stress-inducible gene therapy vectors. to this end, one, two, or three are-containing regions from human nad(p)h: quinone oxidoreductase-1, glutamate-cysteine ligase modi fi er subunit and mouse heme oxygenase-1 were cloned into a vector expressing luciferase under a minimal sv40 promoter. the construct, which was the most responsive to areinducing agents, was chosen for further studies in which a lentiviral vector was produced for an ef fi cient transfer to endothelial cells. heme oxygenase-1 (ho-1), which has well-characterized anti-in fl ammatory properties, was used as the therapeutic transgene. in human endothelial cells, are-driven ho-1 overexpression inhibited nuclear factor-kappa b activation and subsequent vascular cell adhesion molecule-1 expression induced by tumor necrosis factor-alpha. they concluded that the are element is a promising alternative for the development of oxidative stressinducible gene therapy vectors [ 111 ] . progenitor cell therapy is a potential new treatment option for ischemic conditions in the myocardium and skeletal muscles. however, it remains unclear whether umbilical cord blood (ucb)-derived progenitor cells can be therapeutic in ischemic muscles and if yes, whether the ex vivo gene transfer can be used for improving the effect. the use of lentiviral vector led to ef fi cient transduction of both ucb-derived hscs and mscs resulting in long-term transgene expression. moreover, it did not alter the differentiation potential of either hscs or mscs. in addition, the therapeutic potential of cd133+ and msc progenitor cells transduced ex vivo with lentiviral vector encoding the mature form of vascular endothelial growth factor d (vegf-d ) or the enhanced green fl uorescent protein (egfp) marker gene achieved permanent gene expression. the transplantation of the progenitor cells into nude mice serving as mouse model of skeletal muscle ischemia enhanced the regeneration of ischemic muscles, but notably, without a detectable long-term engraftment of either cd133+ or msc progenitor cells. the results show that rather than directly participating in angiogenesis or skeletal myogenesis, the ucb-derived progenitor cells indirectly enhance the regenerative capacity of skeletal muscle after acute ischemic injury. however, rather counter-intuitively, the vegf-d gene transfer into the progenitor cells did not improve the therapeutic effect in ischemic muscles [ 131 ] . another cell type with improved adult stem cell functions has been discovered and cells have been isolated from the peripheral blood of young children. this clonally expandable, telomerase expressing progenitor cell type is distinct from hematopoietic or mesenchymal stromal cells and resembles that of embryonic multipotent mesoangioblasts. cell numbers and the proliferative capacity correlate with donor age, and express the pluripotency markers klf4 , c-myc , as well as low levels of oct3/4, but lack sox2 . overexpression of sox2 by lentiviral transduction of sox2 (sox-mabs) enhances pluripotency and facilitates differentiation to cardiovascular lineages. furthermore, the number of smooth muscle actin positive cells was higher in sox-mabs. in addition, pluripotency of sox-mabs was shown in a mouse model by demonstrating the generation of endodermal and ectodermal progenies and injection of sox-mabs into nude mice after acute myocardial infarction resulted in improved cardiac function compared to mice treated with control cells (cmabs). furthermore, cell therapy with sox-mabs resulted in an increased number of differentiated cardiomyocytes, endothelial cells, and smooth muscle cells in vivo [ 133 ] . mesenchymal stem cell therapy emerges as a viable therapy in the context of acute lung injury /acute respiratory distress syndrome and chronic disorders, such as lung fi brosis and chronic obstructive pulmonary disease. there is evidence for bene fi cial effects of mscs on lung development, repair, and remodeling. the engraftment in the injured lung does not occur easily, but several studies report that paracrine factors can be effective in reducing in fl ammation and promoting tissue repair. mscs release several growth factors and anti-in fl ammatory cytokines that regulate endothelial and epithelial permeability and reduce the severity of in fl ammation, as reviewed by arboreau et al. [ 2 ] , suggesting that carefully controlled expression of these factors using transduced stem cells could enhance the bene fi cial effects of the mesenchymal stem cell therapy. this may be a risky proposal, however, since constitutive expression of tgf beta /tgf alpha in epithelial mscs generated breast cancer stem cells [ 12 ] . acute respiratory distress syndrome (ards) is a crippling disease with no effective therapy, and characterized by progressive lung damage followed by dyspnea. mscs have been proposed as a new therapeutic modality for ards because the stem cells can attenuate in fl ammation and repair the damaged tissue by differentiating into several cell types. the bene fi cial effect of the stem cells is still a minor mystery, as it is known that macrophages participate in the development of ards and that mscs can only weekly modulate macrophage function. the chemokine ccl2 is a potent inducer of macrophage recruitment and activation, and its expression is elevated in patients with ards. a set of mscs have been generated by transducing the cells with a lentiviral vector expressing 7nd, a dominant-negative inhibitor of ccl2, expecting enhanced therapeutic function of the mscs if the hypothesis is valid. the transduction was effective, and the stem cells produced a large amount of 7nd. after inducing lung injury by bleomycin treatment, the iv-injected mscs readily migrated into the site of injury as con fi rmed by immunostaining 24 h postinjection. this fi nding suggests that mscs could work as a drug delivery tool. mice treated with 7nd-expressing mscs showed signi fi cantly milder weight loss, suffered less severe lung injury, lower collagen content, lesser accumulation of in fl ammatory cells and in fl ammatory mediators, and ultimately showed signi fi cant gains in survival [ 218 ] . no evidence of 7nd-mesencymal stem cell-induced toxicity was observed during or after treatment. thus, inhibiting the effects of macrophages may greatly enhance the ability of mscs to affect lung repair in ards. direct transduction of lung tissues for gene therapy has always been an attractive proposal. the reoccurring problem, however, is that the airways are far less accessible to vector particles than hoped for and the depth of penetration of inhaled substrate ends in the branches which are larger than 100 m m in diameter [ 48, 263 ] . an attractive alternative delivery of gene therapy components could be the intrapleural injection of mscs. to enable tracking, the cells were labeled with green fl uorescent protein (gfp) using a lentiviral vector, and were found readily attached to the pleura of sprague-dawley rats. the isolated and recovered cells preserved the typical mesenchymal stem cell phenotype and could differentiate into adipocytes, osteoblasts, and chondroblasts in vitro. the highest number of the labeled cells was found to be adhered to the mediastinal pleura, but no labeled cells were detected in the lung parenchyma or other tissues/organs, such as the liver, kidney, spleen, and mesenterium, a remarkable compartmentalization of a stem cell transplant [ 200 ] . alzheimer's disease (ad) is one of the most devastating conditions and its prevalence is still rising paralleling the increase of average life expectancy. a hallmark of the disease is the accumulation of amyloid plaques and extensive neurodegeneration in the context of an intracerebral in fl ammation, leading to progressive dementia. over the years, a tripartite set of goals crystallized, when the potential treatments of ad were considered: (a) stop the progression of the disease by reducing/reversing the plaque formation; (b) stop the neurodegeneration that seems to be a consequence of both internal changes (neuro fi brillary tangle formation and related issues) and changes external to the cells, related to plaque formation and degeneration of the neuronal microenvironment; and (c) recover neurological function by replenishing the lost neuronal compartment [ 71, 81, 94, 122, 152, 188, 291 ] . interestingly, mscs and stem cell therapy are increasingly considered a potentially important part of the toolset to achieve these goals. the symptoms that are collectively categorized as ad often have different backgrounds, some of which seem to have roots implying genetic causes, such as improper processing of beta amyloid peptide. consequently, a disease-modifying therapeutic approach in alzheimer's disease aims to reduce the accumulation of neurotoxic beta amyloid aggregation peptides. habish et al. report new fi ndings for a potential autologous stem cell-based strategy for delivery of enzymatic activities against beta amyloid formation in the brain. f-spondin and neprilysin (cd10), genes expressed in adult mscs, are known to be involved in the formation and degradation of beta amyloid peptides, respectively. coincubation of the converted mscs with hek-293 cells stably expressing amyloid precursor protein (app) lead to a signi fi cant cell dose-dependent decrease of amyloid peptide release and deposition, indicating that mscs might be useful for delivering antiamyloid activity to treat ad [ 95 ] . this direction of research is gaining new momentum from the discovery of a new beta amyloid secretase and the tremendous progress gained in recent years in the fi eld of amyloid formation, its contribution to neurodegenerative diseases [ 122 ] and allowing new gene therapies to be conceived and tested. one effort has utilized human umbilical cord blood-derived mscs (hucb-mscs) which were transplanted into amyloid precursor protein and presenilin1 double-transgenic mice. this experiment resulted in signi fi cantly improved spatial learning and a decrease in memory decline. furthermore, beta amyloid peptide deposition, beta-secretase 1 (bace-1 ) levels, and the hyper-phosphorylation of the tau proteins were dramatically reduced in hucb-msc transplanted app/ps1 mice. interestingly, these effects were associated with reversal of disease-associated microglial neuroin fl ammation, as evidenced by decreased microglia-induced pro-in fl ammatory cytokines, reduction in the number of alternatively activated microglia , and decrease in anti-in fl ammatory cytokines. combining these fi ndings with the potential cell therapy targeting, these mscs are expected to produce a sustained neuroprotective effect by establishing a feed-forward loop engaging the alternative activation of microglia, thereby ameliorating disease pathophysiology and reversing the cognitive decline associated with amyloid deposition [ 139 ] . peng and colleagues report additional details on the use of lentivirus-expressed sirna as a method to ameliorate alzheimer disease neuropathology in app transgenic mice by reducing the levels of beta-site app cleaving enzyme 1, or bace1 [ 189 ] . a series of experiments demonstrated the potential of neural stem cells transduced by a multigenic lentiviral vector stably expressing recombinant human nerve growth factor in relevant amounts to exploit their ability for therapeutic applications. the multigenic lentiviral vector contained a tricistronic cassette to express simultaneously up to three independent genes: (1) rhngf (beta subunit); (2) egfp (enhanced green fl uorescent protein); and (3) neo (r) (neomycin antibiotic resistance gene). lentiviral vectors were released in culture media and subsequently used to transduce mouse stem cells. remarkably, the subsequent test revealed that engineered nscs were all positive for egfp and after 30 passages in vitro engineered cells maintained their multipotentiality to differentiate into neurons, astrocytes, and oligodendrocytes. furthermore, it was found that rhngf-stem cell-derived neurons expressed choline acetyltransferase and displayed an enhanced axonal growth. the stem cells showed an altered sphere forming frequency either in rhngf-nsc or in both groups of control nsc. lentivirus-mediated rhngf gene transfer into nsc was achieved without changes in the expression of neural differentiation markers, like microtubule-associated protein 2 (map2) (a/b), glial fi brillary acidic protein (gfap) and chondroitin sulfate proteoglycan [ 34 ] . secreted rhngf increased axonal sprouting by rhngf-nsc-derived neurons, which was associated with chat expression. rhngf-nscs may prospectively be a good candidate for the treatment of neurodegenerative diseases. a protein that has been shown to promote app accumulation is beta-secretase (beta-site app cleaving enzyme 1, or bace1). typically, a marked increase in the level of bace1 is found in the cerebrospinal fl uid of those affected with alzheimer's disease. through in vivo studies using app transgenic mice, it has been demonstrated that decreasing the expression of bace1 via lentiviral vector delivery of bace1 sirna has the potential for signi fi cantly reducing the cleavage of app, the accumulation of these products, and the consequent neurodegeneration. as such, lentiviral-expressed sirna against bace1 is a therapeutic possibility in the treatment of ad. neprilysin has recently been implicated as a major extracellular beta amyloid degrading enzyme in the brain. a unilateral intracerebral injection of a lentiviral vector expressing human neprilysin (lenti-nep) was tested in transgenic mouse models of amyloidosis reduced amyloid-beta deposits by half relative to untreated mice, indicating that neprilysin may have a role in alzheimer's disease treatment. that said, a more ef fi cient delivery system is likely required, a property that a neprilysin expressing stem cell could potentially provide [ 160 ] . gene transfer to the central nervous system provides a powerful methodology for the study of gene function and gene-environment interactions in vivo, in addition to a vehicle for the delivery of therapeutic transgenes for gene therapy. research has been signi fi cantly aided by successfully targeting speci fi c regions of brain, and for parkinson's disease, the substantia nigra. the key to success is the ease of pseudotyping lentiviral vectors, which makes it possible to change the patterns of tropism. cannon et al. used isogenic lentiviral vector particles encoding a gfp reporter and pseudotyped with envelope glycoproteins derived from vesicular stomatitis virus (vsv), mokola virus (mv), lymphocytic choriomeningitis virus (lcmv), or moloney murine leukemia virus (mulv). adult, male lewis rats were injected unilaterally with stereotactic infusions of vector into the substantia nigra. three weeks later, patterns of viral transduction were determined by immunohistological detection of gfp. different pseudotypes gave rise to different sites of transgene expression. vsv and mv pseudotypes transduced midbrain neurons, including a subset of nigral dopaminergic neurons. in contrast, lcmv-and mulv-pseudotyped lentiviral vector resulted in transgene expression exclusively in astrocytes. the restricted transduction of astroglial cells was not explained by the cellular distribution of receptors previously shown to mediate entry of lcmv or mulv. the availability of neuronal and astrocyte-targeting vectors will allow dissociation of cell autonomous and cell nonautonomous functions of key gene products in vivo. similar tissue and cell-speci fi c patterns can be achieved in stem cells using cell/tissue-speci fi c promoters and mirna [ 43, 79, 86, 177, 186, 199, 213, 232, 242, 266, 290 ] . multipotent mesenchymal stromal cells have raised great interest for brain cell therapy due to their ease of isolation from bone marrow, their immunomodulatory and tissue repair capacities, their ability to differentiate into neuronal-like cells, and for their ability to secrete a variety of growth factors and chemokines. a subpopulation of human mscs, the marrow-isolated adult multilineage inducible (miami) cells, when combined with pharmacologically active microcarriers (pams) have shown great promise in a rat model of parkinson's disease. pams are biodegradable and non-cytotoxic poly (lactic-co-glycolic acid) microspheres, coated by a biomimetic surface and releasing a therapeutic protein, which acts on the cells conveyed on their surface and on their microenvironment. in this study, pams were coated with laminin and designed to release neurotrophin 3, which stimulate the neuronal-like differentiation of miami cells and promotes neuronal survival. after adhesion of dopaminergic-induced (di)-miami cells to pams in vitro, the complexes were grafted in the partially dopaminergic-deafferented striatum of rats, which led to a strong reduction of the amphetamine-induced rotational behavior together with protection/repair of the nigrostriatal pathway. these effects were correlated with the increased survival of di-miami cells that secreted a wide range of growth factors and chemokines. moreover, the observed increased expression of tyrosine hydroxylase by cells transplanted with pams may contribute to this functional recovery [ 52 ] and provide an excellent new delivery system for genetically modi fi ed/enhanced cells into substantia nigra. lewy body disease is a heterogeneous group of neurodegenerative disorders characterized by alpha-synuclein accumulation and includes gradually worsening dementia with lewy bodies (dlb) accumulating in neurons followed by advanced parkinson's disease (pd). recent evidence suggests that impairment of the lysosomal pathways (i.e., autophagy) involved in alpha-synuclein clearance might play an important role. for this reason, the expression levels of members of the autophagy pathway in brains of patients with dlb and alzheimer's disease and in alpha-synuclein transgenic mice were examined by immunoblot analysis. in dlb cases, the levels of mtor were elevated and atg7 were reduced compared to controls and ad. levels of other components of the autophagy pathway such as atg5, atg10, atg12, and beclin-1 were not different in dlb compared to controls. in dlb brains, mtor was more abundant in neurons displaying alpha-synuclein accumulation. these neurons also showed abnormal expression of lysosomal markers such as lc3, and ultrastructural analysis revealed the presence of malformed autophagosomes in abundance. similar alterations were observed in the brains of alpha-synuclein transgenic mice. intracerebral infusion of rapamycin, an inhibitor of mtor, or injection of a lentiviral vector expressing atg7 resulted in reduced accumulation of alphasynuclein in transgenic mice and amelioration of associated neurodegenerative alterations supporting the notion that defects in the autophagy pathway, and more speci fi cally in mtor and atg7, are associated with neurodegeneration. this supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects using genetically altered stem cells [ 44, 270 ] . although the advances in parkinson's disease research to date are signi fi cant, the lack of clinical use of genetically modi fi ed cells is a bit surprising and may indicate an oversight and underuse of the advanced tools provided by the combination of stem cells and lentiviral vectors. lasting cerebral ischemia is a frequent (~80%) consequence of stroke and, as a result, most of the stroke research is focusing on ameliorating the devastating consequences of ischemic events: endothelial damage, neurodegeneration, and breakdown of the blood-brain barrier (bbb) leading to dif fi cult-to-treat cerebral edema [ 108 ] . data indicate that transplantation of human umbilical cord stem cells helps to protect ischemic brain [ 149 ] , and the protection is partially attributed to cytokines and protective factors produced by these stem cells [ 10, 149 ] . another promising fi nding is that the mesenchymal and neuronal stem cells preserve their ability to differentiate into glial and neuronal cells [ 51, 119, 149, 231, 249, 275 ] . various studies on focal cerebral ischemic models have implicated the direct activation and expression of matrix metalloproteinases (mmps), especially mmp-9, as a key orchestrator of bbb disruption. moreover, studies have shown that mmp-9 sirna can protect the bbb from ischemia/reperfusion injury. one study investigated the neuroprotective role of a lentiviral vector-mediated mmp-9 shrna following focal cerebral ischemia [ 108 ] , indicating that it is possible to deliver mmp-9 inhibitors by genetically enhanced stem cells. this study also showed the ability to deliver the target deeper into the affected area normally not accessible by direct lentiviral vector infusion. the forerunner of such interventions is a study testing the hypothesis that transplantation of human neurotrophin-3 (hnt-3) over-expressing neural stem cells into rat striatum after a severe focal ischemia would promote functional recovery. the rat neural stem cells were transduced with a flag-tagged hnt-3 gene in a lentiviral vector. the stem cells were transplanted into the striatum ipsilateral to the injury of adult rats 7 days after 2 h occlusion of the middle cerebral artery from 3 days to 2 weeks after transplantation. the modi fi ed cells (nscs-hnt3, as de fi ned by flag immuno fl uorescence staining) that survived the transplantation procedures could secrete signi fi cantly higher levels of neurotrophin-3 protein in the graft sites than controls ( p < 0.001). furthermore, the rats that accepted nscs-hnt3 exhibited enhanced functional recovery on neurological and behavioral tests, compared with control animals transplanted with saline or untransduced stem cells, indicating that they might have value for enhancing functional recovery after stroke [ 285 ] . recovery from ischemic events is slow and rather unpredictable. however, there seem to be new therapeutical opportunities that could enhance the process such as using vegf-induction therapy [ 16 ] . there is accumulating evidence indicating that vegf has direct neuroprotective effects on various cultured neurons of the central nervous system. interestingly, in vivo vegf controls the correct migration of facial branchiomotor neurons in the developing hindbrain and stimulates the proliferation of neural stem cells in enriched environments and after cerebral ischemia. on the other hand, transgenic mice expressing reduced levels of vegf develop late-onset motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis (als). also, reduced levels of vegf have been implicated in a polyglutamine-induced model of motor neuron degeneration. intracerebroventricular delivery of recombinant vegf protein delays disease onset and prolongs survival of als rats, whereas intramuscular administration of a vegf-expressing lentiviral vector increases the life expectancy of als mice by as much as 30%. deciphering the precise role of vegf at the neurovascular interface promises to uncover new insights into the development and pathology of the nervous system and should be helpful to the design of novel strategies to treat (motor) neurodegenerative disorders [ 137 ] . vegf-expressing mscs have also been found bene fi cial in parkinson's disease [ 16, 271 ] . the development of lentiviral particles engineered for macrolide-responsive human vascular endothelial growth factor 121 (vegf121) expression will bring closer the in vivo use of inducible growth factor cell therapies, expressing the factors only in ischemic conditions using hypoxia-inducible erythropoietin promoter [ 6 ] . alternatively, the inducible vegf121 promoter system also compared favorably with isogenic streptogramin-and tetracycline-responsive con fi gurations and showed excellent growth-factor fi ne-tuning following transduction into a variety of mammalian cell lines and different human primary cells. chicken embryos transduced for macrolide-controlled vegf121 production can be fi ne-tuned to prime a dose-dependent neovascularization [ 168 ] . expression of survivin (svv) using an sin lentiviral vector carrying vascular endothelial growth factor further improved the expression of vegf and basic fi broblast growth factor in male sprague-dawley rats under hypoxic conditions. the in vivo experiment that produced this observation consisted of three groups of rats, one receiving intravenous injection of 500 m l of phosphate-buffered saline without cells (control group) and two groups administered the same volume solution with either three million gfp-mscs (group gfp) or svv/gfp-mscs (group svv). all animals were submitted to 2 h middle cerebral artery occlusion followed by reperfusion. modi fi cation with svv further increased secretion of both factors. the survival of the transplanted cells in the svv group was 1.3-fold higher at 4 days after transplantation and 3.4-fold higher at 14 days after transplantation, respectively, when compared with group gfp and reduced the cerebral infarct volume by 5.2% at 4 days after stroke and improved post-stroke neurological function at 14 days after transplantation. modi fi cation with svv could further enhance the therapeutic effects of mscs possibly through improving the mscs survival capacity and upregulating the expression of the protective cytokines in the ischemic tissue [ 151 ] . the identi fi cation of the genes differentially regulated by ischemia will lead to an improved understanding of cell death pathways such as those involved in the neuronal loss observed following a stroke. furthermore, the characterization of such pathways could facilitate the identi fi cation of novel targets for stroke therapy. one such novel approach was the ampli fi cation of the differential gene expression patterns in a primary neuronal model of stroke, by employing a lentiviral vector system to speci fi cally bias the transcriptional activation of hypoxically regulated genes. over-expression of the hypoxia-induced transcription factor subunits hif -1 alpha and hif-2 alpha elevated hypoxia-mediated transcription of many known hif-regulated genes well above control levels. furthermore, many potentially novel hif-regulated genes were discovered that were not previously identi fi ed as hypoxically regulated. most of the identi fi ed novel genes were activated by a combination of hif-2 alpha over-expression and hypoxic insult. these included several genes with particular importance in cell survival pathways and of potential therapeutic value. hypoxic induction of hif-2 alpha may therefore be a critical factor in mediating protective responses against ischemic injury. further investigation of the genes identi fi ed in this study may provide increased understanding of the neuronal response to hypoxia and may uncover novel therapeutic targets for the treatment of cerebral ischemia [ 202 ] and the genes need to be considered as useful targets in future mesenchymal stem cell therapies. however, the use of hypoxia-induced gene therapy has to be evaluated carefully in the light of recent provocative observations indicating that the hypoxic phenotype contributes to appearance of highly malignant cancer forms from the initial epithelial-mesenchymal transition to the ultimate organotropic colonization, and that can potentially be regulated by hypoxia, suggesting a master regulator role of hypoxia and hifs in metastasis [ 6, 155 ] . furthermore, modulation of cancer stem cell self-renewal by hifs may also contribute to the hypoxia-regulated metastasis program. the hypoxia-induced metastatic phenotype may be one of the reasons for the modest ef fi cacy of anti-angiogenic therapies and may well explain the provocative fi ndings that anti-angiogenic therapy increased metastasis in preclinical models [ 155 ] . the image of a wheelchair-bound superman exempli fi ed for all of us the tragedy that affects many of the victims of traumatic spinal cord injury and motivated research into protecting and restoring spinal-cord functionality beyond and above the usual efforts. the results are promising on many fronts [ 236 ] . on one hand, the intervertebral disk , cartilage, and bone injuries that threaten the integrity of the spinal cord can be almost completely healed and the healing can be facilitated and enhanced by stem cell therapies in most of the experimental models. the treatment often includes stem cells engineered with lentiviral gene transfer for enhancing and promoting wound healing and tissue restoration [ 13, 87, 89, 109, 247 ] . signi fi cant success has been achieved by expressing bone morphogenic proteins in the injured tissue [ 80 ] and observations that mechanical stimulation has a multiplying effect in bone regeneration will hopefully carry the research into clinical trials [ 140 ] sooner than later. probably, the fi rst trials will be done in well-designed spinal surgery, allowing even risky interventions, currently not practiced [ 13, 88, 163 ] . the progress is signi fi cantly slower when it comes to restoring the functionality of severed spinal cord, but successful demonstration that mscs migrate into the site of injury and differentiate into proper cell types needed for the healing [ 224 ] predicts potential breakthroughs. in this set of experiment, mesenchymal cells were labeled with green fl uorescent protein using lentiviral vector, were injected into the subarachnoid space, and their migration and differentiation was observed. cells were found on the surface of the injured spinal cord parenchyma, in deeper area of the perivascular spaces and some of them had been found deeply integrated into the parenchyma. immunostaining for nestin demonstrated that some gfp-positive cells differentiated into neural stem cells and mature neurons or glial cells in situ. lentiviral vectors pseudotyped with rabies env were successfully used to deliver genes into spinal cord and site of injury and showed successful retrograde transfer into deeper areas, indicating that gene therapy is possible and factors necessary for further differentiation of stem cells can be delivered [ 224, 241 ] . further advances in pseudotyping with rabies virus glycoprotein has a promise for more ef fi cient motor neuro-speci fi c delivery of transgenes and restoration of neuronal functions. as the examples indicate above, mscs have been recognized as promising delivery vehicles for gene therapy in the cns. a particularly unmet need is delivery of compounds that could help patients suffering from a particularly aggressive form of cancer, gliomas. a glimpse into a possible future can be gained from experiments in which stem cells were used to evaluate the antitumor effect of cytosine deaminase (cd) in a rat c6 glioma model . lentiviral vectors expressing cd and enhanced green fl uorescent protein (egfp) were constructed and transduced into rat mscs which were intracranially injected alone or in combination with c6 glioma cells supported by unlabeled parental mscs. the presence and effect of the engineered stem cells were then correlated with the possible effects on tumor growth, tumor cell apoptosis, tumor size, and rat survival in the presence of 5-fl uorocytosine (5-fc). fei et al. found that the cd/egfp cells were largely localized at the junction of the tumor with normal tissue. the mean survival time of rats co-injected with c6 glioma cells and mscs-cd/egfp cells was signi fi cantly extended to 45.9 days with tumor size reduction when compared with rats injected with c6 glioma cells alone surviving an average of 15.3 days, or those co-injected with c6 glioma cells and parental cells surviving only for 16.0 days. in addition, data suggest that msc-cd/egfpmediated gene therapy promoted tumor cell apoptosis in rat c6 gliomas [ 72 ] . without going into detail, hypoxia-induced genes seem to play an important role in the fate of mscs and require further studies, as modifying and preconditioning as well as changing their effects temporarily by gene therapy indicates a plethora of important insights into the potential use of this complicated class of stem cells in tumor therapy [ 142, 147, 150, 155, 267, 272 ] , and we expect rapid progress in this area in the near future. the rational is that tumor cells have signi fi cantly altered metabolism with a shift toward the anaerobic pathway and changes in the respective gene expression patterns providing novel targets and delivery methods for cancer therapy. transplantation of hscs to correct a series of lysosomal storage diseases and peroxisomal disorders has almost 25 years of history and involves over 20 diseases [ 23 ] . however, the success was limited to only a small subclass of diseases such as hurler syndrome, x-ald, and infantile krabbe disease. detailed studies are now available suggesting that hematopoietic stem cells are suitable only for a carefully selected cases, leaving open the fi eld for a more versatile mesenchymal stem cell therapy, especially those instances having neurological symptoms [ 69 ] . bone marrow-derived mscs are another promising platform for cell-and gene-based treatment of inherited and acquired disorders including a whole range of lysosomal storage diseases. several animal models exist to run preclinical studies [ 164 ] . human mscs distribute widely in a murine xenotransplantation model, and the human stem cells are amenable to lentiviral vector-mediated transduction to obtain expression of therapeutic levels of enzyme in xenotransplantation models of human disease (non-obese diabetic severe combined immunode fi cient mucopolysaccharidosis type vii [nod-scid mpsvii]) [ 164 ] . transduced mscs persisted in the animals that underwent transplantation and comparable numbers of donor mscs were detected at 2 and 4 months after transplantation. the level of circulating enzymes were suf fi cient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues providing additional evidence that transduced human mscs retain their normal traf fi cking ability in vivo and persist for at least 4 months, while able to deliver therapeutic levels of proteins in an authentic xenotransplantation model of human disease. similar results have been reported by muller and colleagues, who were able to restore aryl sulfatase and beta galactosidase levels in genetically de fi cient bone marrow mscs, and showed that untransduced cells from patients with metachromatic leukodystrophy , who are asa de fi cient, took up a substantial amount of asa that was released into the media from mscs [ 173 ] , an important milestone for future attempts to try stem cell therapy of metachromatic leukodystrophy. gm1 ganglyosiosys was successfully treated with mscs in a mouse beta-galactosidase knockout model indicating that autologous transplantation may be feasible using lentiviral-transduced mscs [ 228 ] . fabry disease affects an estimated 1 in 40,000-60,000 males, and far less frequently females. it is an inherited lysosomal disorder caused by a de fi ciency of alpha-galactosidase a (alpha-gal a). the systemic accumulation of globotriaosylceramide (gb3) results in gradual tissue deterioration leading to organ failure. there is a limited mouse model of the disease showing gb3 accumulation in an alpha-gal a-de fi cient mouse model. however, most of the important clinical manifestations are absent and the lack of relevant large animal model hinders the development of proper cell therapy. when compared to the human alpha-gal a, the porcine alphagal a showed a high level of homology in the coding regions. cell lysate and supernatants from fabry patient-derived fi broblasts transduced with a lentiviral vector carrying the porcine alpha-gal a cdna (lv/porcine alpha-gal a) showed high levels of alpha-gal a activity, and its enzymological stability was similar to that of human alpha-gal a. even more importantly, uptake of secreted porcine alpha-gal a by non-transduced cells was observed. furthermore, gb3 accumulation was reduced in fabry patient-derived fi broblasts transduced with the lv/porcine alpha-gal a. the fi nding that the porcine version of the gene is also x-linked (x22q) provides hope that a large animal (porcine) model of fabry disease can be constructed in the near future for use in testing a novel application of cell therapy using mscs [ 278 ] . the success of such model and eventually the feasibility of the treatment depends on the "bystander phenomenon ," i.e., the transduced mesenchymal cells intended for delivering the enzyme secrete the enzyme in abundance, but the defective cells in their microenvironment also must be able to take up the enzyme and utilize it. to facilitate the uptake, a fusion protein between gb3 and hiv tat protein has been made [ 104 ] . if successful, the range of enzyme replacement therapy approach could widen signi fi cantly. the data published by higuchi et al. indicate that indeed the tat's ability to penetrate the cell membrane was maintained in the recombinant fusion protein and it enhanced the enzyme uptake, as expected. since the different manifestations of the disease produce problems in different organs (brain, kidney, and heart), it seems to imply that mscs will be the best candidates for this enzyme replacement therapy as the earlier attempts to perform enzyme replacement therapy in mouse model showed insuf fi cient ef fi ciency [ 277 ] . the enormity of the problems posed by diabetes is re fl ected by the statistics published on the nih website ( http://diabetes.niddk.nih.gov/dm/pubs/statistics/#dd ). by the age of 65, almost one in four americans suffers from diabetes. the at-risk population of prediabetics is 37% of the population older than 20 years. the sheer number of patients indicates that restoring glucose metabolism by pancreas or pancreatic islet transplantation, even in the most severe cases, is just impractical if not impossible. the low engraftment rate makes the prospects of such treatment even worse, especially, as there never will be a suf fi cient number of donors. that leaves the stem cell technology as the major source of hope for solving the relevant issues in recovering regulated insulin production and glucose regulation functionality in diabetes. a large number of clinical trials using mscs are under way [ 75, 106, 120 ] , and a rather confusing sets of stem cell markers are listed in these studies indicating that there is a plurality of stem cells residing in different tissues, all of which have the potential to help pancreatic tissue regeneration. not surprisingly, the most obvious source of these stem cells could be the pancreas itself, from which the resting stem cells can be isolated, reactivated, and expanded by variety of stimulants. the data are still being evaluated, and need further con fi rmation, reproduction, and lineage tracing. the currently available datasets could not fi rmly substantiate the claims when using different markers ( carbonic anhydrase ii vs. hepatocyte nuclear factor 1 beta ) [ 61, 113, 237, 243 ] . since then, neurogenin 3 also was considered as a marker for endocrine type differentiation of proto beta cells [ 273 ] , leaving the subject as to whether well-de fi ned adult beta islet cell progenitors truly exist in signi fi cant numbers rather murky. the phenomenon of in vitro trans-differentiation of the acinar cells into beta cells upon exposure to egf, lif, notch1-inhibitors [ 15 ] looks promising, and recently zhou and colleagues added a more extensive study on in vivo reprogramming of adult pancreatic exocrine cells into beta cells [ 289 ] . however, the reported ef fi ciency was low and the progenitor cells remained elusive. this left the fi eld searching for other sources, including mscs from bone marrow, liver, intestine, and neural tissue (reviewed by efrat [64] [65] [66] and jones [ 118 ] ), that are capable of trans-differentiating into insulin-producing beta cells. with the available results, their ultimate hope was that these cells could be used to seed the pancreas with new sets of insulin producing islands. since lineage tracing was often omitted and the reproducibility of the results remained unsettled, the fi eld, despite its high importance, seems to be somewhat in shambles [ 106, 107 ] , ready for deployment of the novel, lentiviral vector supported techniques. szabat et al. report a signi fi cant set of results on beta-cell maturation using lentiviral vector-based lineage-study examining a novel pdx1/ins1 dual fl uorescent reporter vector. they con fi rmed that individual adult human and mouse beta-cells exist in at least two differentiation states, distinguishable by the activation of the ins1 promoter. they performed real-time imaging of the maturation of individual cultured beta-cells and followed the kinetics of the maturation process in primary human and mouse beta-cells and collected gene expression pro fi ling data as well. the gene expression pro fi ling of facs puri fi ed immature pdx1+ /ins1 (low) cells and mature pdx1 (high)/ins1 (high ) cells from cultures of human islets, mouse islets, and min6 cells revealed that pdx1+/ins1 (low) cells are enriched for expression of multiple genes associated with beta-cell development/progenitor cells, proliferation, apoptosis, as well as genes coding for other islet cell hormones such as glucagon [ 240 ] . it turns out that trans-differentiation can be successfully performed using mafa. mafa is a leucine zipper transcription factor from the maf family that can be activated by p38 map kinase. this protein is a known pancreatic transcriptional factor controlling the beta-cell-speci fi c transcription of the insulin gene [ 40 ] . expressing it using lentiviral vectors in placenta-derived multipotent stem cells (pdmscs) that constitutively expressed oct-4 and nanog resulted in signi fi cantly upregulated expression of a series of pancreatic development-related genes (sox17 , foxa2 , pdx1, and ngn3 ), similar to that of native pancreas and islet tissues. mafa increased the expression levels of the mrnas of nkx2.2 , glut2 , insulin , glucagons, and somatostatin , and further facilitated the differentiation of pdmscs into insulin + cells. importantly, the expression of mafa in pdmscs xenotransplanted into immunocompromised mice improved the restoration of blood insulin levels to control values and greatly prolonged the survival of graft cells in immunocompromised mice with stz-induced diabetes [ 40 ] . another successful lineage analysis and monitoring the induced trans-differentiation was reported by cheng et al., in which a relatively abundant epithelial cell source, fetal human pancreas, was used to assess the proliferation potential, changes in lineage markers during culture, and capacity for generating insulin-expressing beta cells from fetal epithelial cells. the fetal epithelial cells readily formed primary pancreatic progenitor cultures, although their replication capacity was rather limited. this was overcome by introduction and expression of htert (human telomerase reverse transcriptase) which greatly enhanced cellular replication in vitro. however, during culture the htert-modi fi ed pancreatic progenitor cells switched their phenotype gaining additional mesodermal properties. this phenotypic switching was inhibited when a pancreas-duodenal homeobox (pdx)-1 transgene was expressed with a lentiviral vector, along with inductive signaling through activin a and serum deprivation. this restored endocrine properties of htertmodi fi ed cells in vitro and were able to express insulin in vivo in immunode fi cient mouse model [ 39 ] . the complexities of these result indicate that a sophisticated multi-gene cell therapies may be needed to solve the issues of proper modulation of transdifferentiation pathways. other strategies using a lentiviral vector-based approach to achieve beta-cell proliferation through the beta-cell-speci fi c activation of the hepatocyte growth factor (hgf)/cmet signaling pathway are also being explored. one of these methodologies is based on the beta-cell-speci fi c expression of a ligand-inducible, chimeric receptor (f36vcmet ), under transcriptional control of the promoter from the human insulin gene, and its ability to induce hgf/cmet signaling in the presence of a synthetic ligand (ap20187) and result in speci fi c proliferation of human pancreatic beta-cells [ 182 ] . the selective, regulated beta-cell expansion may help to increase the availability of cells for transplantation in patients with advanced diabetes. these recent studies show that rapid progress may be achieved in this fi eld and lentiviral vectors may provide the necessary tools to analyze the issues. however, some of the notable efforts are made to avoid stem cell therapy altogether in certain types of diabetes. instead, choosing a more direct route, applying in vivo gene therapy for expressing insulin gene in cell types other than beta cells. ren et al. successfully restored near normal insulin levels for 500 days by expressing insulin in resting liver cells transduced with lentiviral vector using a rat diabetes model [ 204 ] . although monogenic disease appears to be the most obvious human diseases to treat with gene therapy, since they are caused by a single-gene defect, the progress in clinical studies has thus far been rather limited. explanations for the lack of success include inef fi ciency of transductions in vivo, dangers posed by vectors, the failure to permanently correct the gene defect in suf fi cient number of cells, or the rapid turnover of cells. alternative approaches therefore involve the search for and use of stem cell populations and depleting the active stem cell compartments ablation using cytostatic drugs to give chance if increased engraftment by transplanted stem cells. combining the versatility and availability of mscs, their ability to engraft, the use of autologous instead of allogeneic sources for safe transplantation, and the fact that the stem cell population can be expanded in vitro allows highly ef fi cient ex vivo gene therapy relying on latest generation of lentiviral vectors. cystic fi brosis (cf) is caused by a mutation in the gene for the cystic fi brosis transmembrane conductance regulator protein (cftr). the mutant form of the protein causes severe defect in mucus metabolism in the lungs and intestinal track that deteriorates into a life-long, deadly disease. cf is theoretically amenable to gene therapy. in spite of intensive research and a large number of clinical trials in the last 18 years, little practical success can be shown for treating cystic fi brosis [ 191 ] . the explanations include the fact that the deeper regions in the inner surface of the lung are not accessible to direct inhalation and direct treatment [ 48, 263 ] . in light of this fi nding, stem cells remain the most promising delivery vehicles. castellani et al. reviewed the recent attempts to identify lung-or bone marrow-derived populations of stem cells or progenitor cells and application of such cells, allogenic or gene-corrected autologous cells, to colonize the airways, while differentiating into functional respiratory columnar epithelial cells [ 33 ] . when the reporter gene expression was analyzed in trachea-lungs and bronchoalveolar lavage, 0.4-5.5% of stem cells survived in injured airways, but no stem cells survived in control, healthy airway, or in the epithelial lining fl uid [ 138 ] . the most successful approaches thus far appear to be obtained with bone marrow-derived mscs, although the trans-differentiation rate thus far has been limited to below 10-14% [ 26 ] . as an alternative, the proven multipotent nature of bronchoalveolar stem cells isolated from lung tissue may provide another promising approach for stem cell therapy. some additional improvement is expected from more ef fi cient targeting of lentiviral vectors. mitomo and colleagues built a sendai virus env-pseudotyped siv lentiviral vector that can be manufactured at high enough titer and is capable of transducing respiratory epithelium of the murine nose in vivo at levels that may be relevant for achieving clinical bene fi t to cystic fi brosis patients [ 167 ] . availability of novel cystic fi brosis gene-carrying stem cell lines derived from placental mesenchymal cells certainly will help to speed up the research [ 53 ] . however, much more needs to be known about the normal differentiation and functioning of the airway's basal cells and the differentiation and lineages of stem cells to have more ef fi cient treatment options both for gene therapy and for stem cell therapy [ 207 ] . we expect that the intensity of research and push for clinical trials will remain high as the outline of directions will become clearer. also the methods to derive respiratory cell types from stem cells will remain a critical piece [ 181 ] . this disease is an x-linked recessive disorder caused by a mutation in the dystrophin gene that destabilizes muscle cell membranes and causes muscle dystrophy in approximately 1 of 3,600 boys. the musculoskeletal abnormalities deteriorate to a fatal level and the average life expectancy is no more than 25 years, even with high quality care. the research is facilitated by the availability of dystrophin-de fi cient transgenic mice (mdx-mice ) and double knockout (utrophin/dystrophin-de fi cient mice) that can be used as experimental disease models [ 144, 269 ] . human immortalized pluripotent cell lines expressing the mutant dystrophin gene are also available [ 187 ] . the ability of mscs to differentiate into muscle cells places them on the top of the list of candidates that could be used to treat duchenne muscular dystrophy [ 157 ] . lentiviral vectors have been used in this fi eld for conditional immortalization of human cells for basic biologic studies. cudre-maroux et al. demonstrated that the lentiviral vector-mediated transduction of immortalizing genes into human primary cells is an ef fi cient method for obtaining such cell lines. for duchenne muscular dystrophy, the muscle satellite cell model was used to examine the impact of the transduced genes on the genotypic and phenotypic characteristics of the immortalized cells. the most commonly used immortalizing gene, the sv40 large t antigen (t-ag), was extremely ef fi cient at inducing the continuous growth of primary myoblasts, but the resulting cells rapidly accumulated major chromosomal aberrations and exhibited profound phenotypic changes. in contrast, the constitutive expression of telomerase and bmi -1 in satellite cells from a control individual and from a patient suffering from duchenne's muscular dystrophy yielded cell lines that remained diploid and conserved their growth factor dependence for proliferation. however, despite the absence of detectable cytogenetic abnormalities, clones derived from satellite cells of a control individual exhibited a differentiation block in vitro. in contrast, a duchenne-derived cell line exhibited all the phenotypic characteristics of its primary parent, including an ability to differentiate fully into myotubes when placed in proper culture conditions. this cell line should constitute a useful reagent for a wide range of studies aimed at this disease [ 46 ] . a realistic source of stem cells would be the adipose tissue-derived stem cells that can be enhanced for muscle repair. forced expression of myod using lentiviral vector in vitro strongly induced myogenic differentiation, while the adipogenic differentiation was inhibited. moreover, myod-expressing human multipotent adipose-derived stem cells had the capacity to fuse with dmd myoblasts and can restore dystrophin expression. importantly, transplantation of these modi fi ed human, multipotent, adipose-derived stem cells into injured muscles of immuno-depressed rag2(−/−)gam-mac(−/−) mice resulted in a substantial increase in the number of human multipotent adipose-derived stem cell-derived fi bers [ 92 ] . goncalves and colleagues went a step further and devised a technique to monitor the fusion events necessary for myoblast formation by using an elaborate bipartite genetic switch that relays on recombinaseinducible genetic switch that is activated after two cell types, one of which expresses cre and the other the rest of the elements with loxp1 sites, that switch on only upon fusion. this provides a sensitive tool to study the lineages and process of myocyte fusion in transgenic system [ 90 ] . ikemoto et al. used high transduction-ef fi ciency lentiviral vector-mediated gene transfer into freshly isolated autologous satellite cells. freshly isolated cells have better myogenic capability than satellite cell-derived myoblasts, and expansion of the satellite cells does not affect their regenerative potential. the transduced cells successfully regenerated the targeted muscle groups in mdx mice [ 112 ] . however, the vsvg pseudotyped lentiviral vector are inferior in transducing nondividing murine cells, and shunchang et al. demonstrated that by pseudotyping with feline immunode fi ciency virus env better transduction rates can be achieved [ 234 ] . we include this disease because of the challenges researchers faced in attempts to use cell-based therapies. granulomatous disease is a rather rare x-linked immunode fi ciency disorder caused by mutations in the cybb gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (nadph)-oxidase catalytic subunit gp91(phox). earlier attempts to restore the gene function with oncoretroviral vectors failed due to (a) gene silencing common for retroviral inserts, (b) high risk of genotoxicity that these oncoretroviral vectors pose [ 165 ] , and (c) low transduction ef fi ciency and inability to target appropriate cell lineage and practice differentiation-restricted gene expression [ 17 ] . the solution for the compound problem seems to lie in using a safer and more ef fi ciently targeting lentiviral vector system [ 17, 223 ] . it has been demonstrated and repeatedly con fi rmed that by using lentiviral vector it is possible to transduce hscs as well as differentiated neutrophiles from patients with x-linked chronic granulomatous disease (x-cgd) and correct the x-cgd-phenotype in the nod/scid model. the lentivector was a vsv-gpseudotyped, third-generation, self-inactivating (sin) lentivector encoding gp91 (phox). lentiviral vector ef fi ciently transduced cd34+ peripheral blood stem cells under ex vivo conditions nonpermissive for cell division and resulted in 54% of the cells expressing gp91 (phox). lentivector also achieved signi fi cant correction of differentiated human x-cgd neutrophils arising in vivo in nod/scid mice that underwent transplantation (20% and 2.4%, respectively). thus, third-generation sin lentivector-gp91 (phox) performs well as assessed in human x-cgd neutrophils differentiating in vivo, and the studies suggest that the nod/scid model is generally applicable for in vivo study of therapies evaluated in human blood cells expressing a speci fi c disease phenotype [ 165, 209, 226 ] . however, long-term solution can be expected from transducing hscs, and not the fully differentiated neutrophils that have only limited lifespan left and the lack of genotoxicity safety of the third-generation sin v lentiviral vector seem to address these requirements perfectly. wilson's disease is a genetic disease caused by a spectrum of mutations in the atp7b gene , whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. zhang and colleagues reported in 2011 an attempt to restore the normal phenotype by directed hepatocyte differentiation from human-induced pluripotent stem cells. the phenotype correction was achieved by chaperone drug curcumin that can reverse the functional defect in vitro in the case of the r778l chinese hotspot mutation in the atp7b gene. they propose this model system for correcting the gene using lentiviral technology [ 284 ] . atp7b gene seems to be relevant for the wider mesenchymal stem cell fi eld as over-expressing it protects mscs form copper toxicity . this in turn could be used as a selection advantage of transduced mscs over the non-transduced ones in copper-rich environment for enriching the transduced mesenchymal cell compartment before transplantation [ 225 ] . the image of the "fountain of youth" represents a mirage deeply engraved in the human psyche and expresses our fear and resentment of one of the inevitabilities of life: if we are lucky, we will get old, decrepit, suffer a lot from series of painful, chronic diseases, and fi nally succumb. the irony is that those who we consider unlucky, die young, but are saved from the long lasting predicaments of aging. certainly, the intricacies of the factors leading to longevity, or the lack thereof, keep generations of stem cell researchers awake and busy and for good reasons. a model for aging has been found in the condition known as progeria , or more precisely the hutchinson-gilford progeria syndrome , a rare disease affecting children of both sexes and which is caused by a mutant prelaminin a gene, encoding the lamin a-processing enzyme. prelaminin a that retains a farnesyl group, subsequently expressing its abnormal form, progerin. progerin in turn is anchored to the nuclear membrane and destabilizes the nucleus, limiting the ability of cells to divide and leading to premature cell death. unlike other accelerated aging diseases affecting dna repair (werner's and cockayne's syndrome), progerin may play role in normal aging process [ 211 ] and its production is slowly turned on in cells that have uncapped chromosomes, i.e., have truncated telomeres, resulting in premature depletion of stem cell compartments. see the popular ucsf website for details: http://www.ucsf.edu/news/2011/10/10766/aging-disease-children-sheds-lightnormal-aging . additional upregulation of multiple genes in major in fl ammatory pathways indicated an activated in fl ammatory response in progeria patients. this response has also been associated with normal aging, emphasizing the importance of studying progeria to increase the understanding of the normal aging process [ 178 ] . the progressing disease shows a pattern of tissue and organ degeneration that correlates with the depletion of a variety of stem cell compartments, a correlation fi rst pointed out by favreau [ 70 ] . the insight into the role of stem cell depletion in progeria accumulated rapidly in the last couple of years [ 170, 178, 179, 211 ] . this leads to the establishment of an animal model by creating zmpste24 knockout mice [ 67 ] in 2008, which showed premature senescence and progeroid symptoms. with the role of stem cells in aging and in progeria, the doors opened for studying stem cell renewal via dedifferentiation. autologous or heterologous transfer of native or lentivector-enhanced cells [ 129, 184, 212, 265 ] are being actively considered as a possible interventions to slow down progeria as well as natural aging [ 28 ] . however, in both cases, the changes are systemic and murine gene therapy data indicate that the therapy in lysosomal storage disease models, affecting large segments of the body, is more ef fi cient if done at an earliest possible age [ 28, 129 ] . this may have something to do with the limited availability of the microenvironment for the modi fi ed or transplanted stem cells. for this, the preexisting ones, even when "old" and malfunctioning, already occupy the microenvironment appropriate for stem cells. we already know that wound sites create new sites and attract mscs [ 5 ] . also, it is possible that cancer growth is able to generate and maintain an appropriate microenvironment for cancer stem cells [ 24 ] as well as mscs [ 42 ] (potentially for use as anticancer agents) but the normal tissue, even in aging, seems to be resilient in accepting externally provided stem cells. experiments are under way to create arti fi cial microenvironments using nanotechnology to deliver stem cells that produce therapeutical factors [ 52 ] , and 3d scaffolding mimicking bone marrow niches are being designed for similar purpose [ 55 ] and lentiviral vector are often used to deliver the genes of interest [ 60, 141, 166, 248 ] . virxsys pioneered the use of lentiviral vectors in phase i clinical trials to deliver antisense hiv genes as an antisense rna therapy for aids [ 110, 156 ] . this established an initial safety pro fi le for the ex vivo use of lentiviral vectors (see http:// clinicatrials.gov : identi fi er vrx496-usa-05-002). this phase i trial demonstrated the safety and tolerability of a single dose of approximately ten billion autologous hiv infected cd4+ t cells transduced with the lentivector vrx496 carrying a 937-base antisense targeting the hiv envelope. these encouraging results have led to design of a phase ii clinical trial to evaluate the safety, tolerability, and biological activity of four or eight repeated infusions of fi ve to ten billion autologous vrx496modi fi ed hiv+, cd4+ t cells. a major obstacle to completing this phase ii trial was manufacturing enough cells to administer multiple infusions in patients. for this study the safety issues were cleared successfully, opening the way for more extensive use of lentiviral vectors in clinical trials. currently, 16 lentiviral clinical trials are listed at the clinicaltrials.gov website. all of these trials are in early stage, phase i or ii. three of these trials have not yet started and 11 trials are still recruiting patients. most of the clinical trials are focused on hematopoietic stem cells, which are outside of the scope of this work. one trial targets netherton syndrome (clinicaltrials.gov: identi fi er: nct01545323) and attempts to restore lekti serine protease levels in an affected 5 cm 2 skin area, a proof of principle study that has the potential to utilize mesenchymal stem cells in the future. we witnessed a tremendous progress in characterizing and understanding stem cells, the factors needed for maintaining the stem cell phenotype as well as changing it in a predictable mode forcing the mesenchymal stem cells into various differentiation pathways. this progress provides a test bed for a higher level of bioengineering when the genetic buildup of the stem cells is changed to achieve well-de fi ned therapeutical goals. the overview of the recent literature presents a long list of "proof of concept experiments" in which tantalizing possibilities are validated as things that can be accomplished in a wide range of fi elds representing different pathologies: from the debilitating alzheimer's, parkinson's diseases; various traumatic neuronal injuries, diabetes, neuronal and cardiac ischemia; to agerelated tissue degeneration and tissue engineering or delivery of biologics for therapeutical purposes. in parallel, lentiviral vectors are becoming highly valued tools in this tedious work as they are highly ef fi cient vehicles for gene delivery to mark cells, express genes of interest, proteins, and various inhibitory rna species in stem cells. consequently these stem cells, especially the various forms of mscs, have been shown to be highly effective in delivering the targeted genes to dif fi cultto-reach tissues, including the cns. manipulating genes and gene expression, gene transfer has been made safe and ef fi cient by the recent progress in lentivector technology and merged successfully with the stem cell technology. this fi eld has reached an advanced stage, at which it has become feasible to use them safely in a clinical environment. more and more researchers as well as clinicians are becoming familiar with the power of these technologies both for ex vivo and in vivo cell therapy. it does not take a prophet to predict that advanced stem cell therapy has gaining a strong foothold, and even though a tremendous amount of work is needed to be done for it to become a everyday intervention, it is here to stay and will become a routine treatment for the next generation of patients. biological characteristics of stem cells from foetal, cord blood and extraembryonic tissues mechanisms of cellular therapy in respiratory diseases arti fi cial dna cutters for dna manipulation and genome engineering tissue engineering technologies: just a quick note about transplantation of bioengineered donor trachea and augmentation cystoplasty by de novo engineered bladder tissue wound microenvironment sequesters adipose-derived stem cells in a murine model of reconstructive surgery in the setting of concurrent distant malignancy neuroprotective effect of combined hypoxia-induced vegf and bone marrowderived mesenchymal stem cell treatment complete knockdown of ccr5 by lentiviral vector-expressed sirnas and protection of transgenic macrophages against hiv-1 infection speci fi c transduction of hiv-susceptible cells for ccr5 knockdown and resistance to hiv infection: a novel method for targeted gene therapy and intracellular immunization stem cells and reprogramming: breaking the epigenetic barrier? mesenchymal stem cell conditioned media attenuates in vitro and ex vivo myocardial reperfusion injury non-skin mesenchymal cell types support epidermal regeneration in a mesenchymal stem cell or myo fi broblast phenotype-independent manner tgf{beta}/ tnf{alpha}-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype genetically engineered mesenchymal stem cells: applications in spine therapy hyaluronan and mesenchymal stem cells: from germ layer to cartilage and bone notch signaling as gatekeeper of rat acinar-to-beta-cell conversion in vitro transplantation of human bone marrow-derived mesenchymal stem cells promotes will there be a live-attenuated hiv vaccine available for human safety trials by the year 2000? interview by gordon nary toward modeling the bone marrow niche using scaffold-based 3d culture systems transduction of human hematopoietic stem cells by lentiviral vectors pseudotyped with the rd114-tr chimeric envelope glycoprotein rna-based gene therapy for hiv with lentiviral vector-modi fi ed cd34(+) cells in patients undergoing transplantation for aids-related lymphoma an anti-major histocompatibility complex class i intrabody protects endothelial cells from an attack by immune mediators minimal criteria for de fi ning multipotent mesenchymal stromal cells. the international society for cellular therapy position statement vascular targeting and antiangiogenesis agents induce drug resistance effector grp78 within the tumor microenvironment adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation lentiviral vectors: their molecular design, safety, and use in laboratory and preclinical research a thirdgeneration lentivirus vector with a conditional packaging system cell-based therapy for insulin-dependent diabetes mellitus cell therapy approaches for the treatment of diabetes beta-cell replacement for insulin-dependent diabetes mellitus nuclear envelope defects cause stem cell dysfunction in premature-aging mice mesenchymal stem cells in the dental tissues: perspectives for tissue regeneration novel treatment for neuronopathic lysosomal storage diseases-cell therapy/gene therapy expression of a mutant lamin a that causes emery-dreifuss muscular dystrophy inhibits in vitro differentiation of c2c12 myoblasts nanotherapeutics for alzheimer's disease (ad): past, present and future the antitumor effect of mesenchymal stem cells transduced with a lentiviral vector expressing cytosine deaminase in a rat glioma model dual origin of mesenchymal stem cells contributing to organ growth and repair hematopoietic stromal cells and megakaryocyte development bone marrow-derived stem cell transplantation for the treatment of insulin-dependent diabetes a novel lentivector targets gene transfer into hhsc in marrow from patients with bm-failure-syndrome and in vivo in humanized mice advances in the fi eld of lentivector-based transduction of t and b lymphocytes for gene therapy measles virus glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both slam and cd46 entry receptors strategies for targeting lentiviral vectors enhancement of posterolateral lumbar spine fusion using low-dose rhbmp-2 and cultured marrow stromal cells peptides for therapy and diagnosis of alzheimer's disease mesenchymal stem cells in cartilage repair: state of the art and methods to monitor cell growth, differentiation and cartilage regeneration development of lentiviral gene therapy for wiskott aldrich syndrome limitation of in vivo models investigating angiogenesis in breast cancer rd114-pseudotyped retroviral vectors kill cancer cells by syncytium formation and enhance the cytotoxic effect of the tk/gcv gene therapy strategy tissue-speci fi c shrna delivery: a novel approach for gene therapy in cancer cervical motion preservation using mesenchymal progenitor cells and pentosan polysulfate, a novel chondrogenic agent: preliminary study in an ovine model potential applications for using stem cells in spine surgery cervical interbody fusion is enhanced by allogeneic mesenchymal precursor cells in an ovine model rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity methods for making induced pluripotent stem cells: reprogramming a la carte enhancement of myogenic and muscle repair capacities of human adipose-derived stem cells with forced expression of myod the role of micrornas in self-renewal and differentiation of mesenchymal stem cells strategies, development, and pitfalls of therapeutic options for alzheimer's disease ef fi cient processing of alzheimer's disease amyloid-beta peptides by neuroectodermally converted mesenchymal stem cells gene therapy of x-linked severe combined immunode fi ciency insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of scid-x1 a serious adverse event after successful gene therapy for x-linked severe combined immunode fi ciency optimized lentiviral vector design improves titer and transgene expression of vectors containing the chicken betaglobin locus hs4 insulator element zinc-fi nger nuclease based genome surgery: it's all about speci fi city role of mesenchymal stromal cells in solid organ transplantation immunomodulatory effect of mesenchymal stem cells alpha-galactosidase a-tat fusion enhances storage reduction in hearts and kidneys of fabry mice transgenic rabbit production with simian immunode fi ciency virus-derived lentiviral vector the quest for tissue stem cells in the pancreas and other organs, and their application in beta-cell replacement lineage tracing evidence for transdifferentiation of acinar to duct cells and plasticity of human pancreas lentivirus-mediated transfer of mmp-9 shrna provides neuroprotection following focal ischemic brain injury in rats stem cellbased approaches for intervertebral disc regeneration ef fi cient lentiviral vector-mediated control of hiv-1 replication in cd4 lymphocytes from diverse hiv + infected patients grouped according to cd4 count and viral load oxidative stress-inducible lentiviral vectors for gene therapy autologous transplantation of sm/c-2.6(+) satellite cells transduced with micro-dystrophin cs1 cdna by lentiviral vector into mdx mice carbonic anhydrase ii-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth self-inactivating lentiviral vectors with u3 and u5 modi fi cations speci fi c and stable gene transfer to human embryonic stem cells using pseudotyped lentiviral vectors functional differences between mesenchymal stem cell populations are re fl ected by their transcriptome an update on targeted gene repair in mammalian cells: methods and mechanisms cell-based treatments for diabetes mesenchymal stem cells for the treatment of neurodegenerative disease human placenta-derived mesenchymal stem cells and islet-like cell clusters generated from these cells as a novel source for stem cell therapy in diabetes generation of hiv-1 resistant and functional macrophages from hematopoietic stem cell-derived induced pluripotent stem cells identi fi cation and biology of beta-secretase safety considerations in vector development hedgehog signaling, epithelial-to-mesenchymal transition and mirna (review) directing stem cell homing biosurface engineering through ink jet printing minimal requirement for a lentivirus vector based on human immunode fi ciency virus type 1 potential oncogene activity of the woodchuck hepatitis post-transcriptional regulatory element (wpre) neonatal gene therapy of mps i mice by intravenous injection of a lentiviral vector filovirus-pseudotyped lentiviral vector can ef fi ciently and stably transduce airway epithelia in vivo umbilical cord blood-derived progenitor cells enhance muscle regeneration in mouse hindlimb ischemia model resident vascular progenitor cells: an emerging role for nonterminally differentiated vessel-resident cells in vascular biology sox2 transduction enhances cardiovascular repair capacity of blood-derived mesoangioblasts methods in mammalian cell line engineering: from random mutagenesis to sequence-speci fi c approaches cre/loxp recombination system and gene targeting human mesenchymal stem cells (hmscs) expressing truncated soluble vascular endothelial growth factor receptor (tsflk-1) following lentiviral-mediated gene transfer inhibit growth of burkitt's lymphoma in a murine model vegf at the neurovascular interface: therapeutic implications for motor neuron disease developing cell therapy techniques for respiratory disease: intratracheal delivery of genetically engineered stem cells in a murine model of airway injury human umbilical cord blood-derived mesenchymal stem cells improve neuropathology and cognitive impairment in an alzheimer's disease mouse model through modulation of neuroin fl ammation cell therapy for bone regeneration-bench to bedside survival and neuronal differentiation of mesenchymal stem cells transplanted into the rodent brain are dependent upon microenvironment hypoxia preconditioned mesenchymal stem cells improve vascular and skeletal muscle fi ber regeneration after ischemia through a wnt4-dependent pathway gene transfer in humans using a conditionally replicating lentiviral vector improved motor function in dko mice by intravenous transplantation of bone marrow-derived mesenchymal stromal cells hypoxia and hypoxia inducible factors in cancer stem cell maintenance inhibition of hiv-1 infection by a unique short hairpin rna to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction mesenchymal stromal cells expressing heme oxygenase-1 reverse pulmonary hypertension human fetal trachea-scid mouse xenografts: ef fi cacy of vesicular stomatitis virus-g pseudotyped lentiviral-mediated gene transfer human umbilical mesenchymal stem cells promote recovery after ischemic stroke hypoxia-inducible factor-1alpha is essential for hypoxia-induced mesenchymal stem cell mobilization into the peripheral blood effects of transplantation with bone marrow-derived mesenchymal stem cells modi fi ed by survivin on experimental stroke in rats neuro fi lamentopathy in neurodegenerative diseases arti fi cial cell microencapsulated stem cells in regenerative medicine, tissue engineering and cell therapy gene editing in human stem cells using zinc fi nger nucleases and integrase-defective lentiviral vector delivery hypoxia and hypoxia-inducible factors: master regulators of metastasis antisensemediated inhibition of human immunode fi ciency virus (hiv) replication by use of an hiv type 1-based vector results in severely attenuated mutants incapable of developing resistance sources of adult mesenchymal stem cells applicable for musculoskeletal applications -a systematic review of the literature design, engineering, and characterization of zinc fi nger nucleases biomimetic mineral-organic composite scaffolds with controlled internal architecture neprilysin gene transfer reduces human amyloid pathology in transgenic mice recent advances in lentiviral vector development and applications rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery mesenchymal stem cells for the sustained in vivo delivery of bioactive factors lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease site-speci fi c gene insertion mediated by a cre-loxpcarrying lentiviral vector targeting the tumor and its microenvironment by a dual-function decoy met receptor toward gene therapy for cystic fi brosis using a lentivirus pseudotyped with sendai virus envelopes in vivo transduction of hiv-1-derived lentiviral particles engineered for macrolide-adjustable transgene expression insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors barrier-to-autointegration factor in fl uences speci fi c histone modi fi cations the genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of hsc gene therapy hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration in vitro analysis of multipotent mesenchymal stromal cells as potential cellular therapeutics in neurometabolic diseases in pediatric patients in situ tissue engineering for tracheal reconstruction using a luminar remodeling type of arti fi cial trachea inserting optimism into gene therapy ex vivo gene transfer and correction for cell-based therapies a dual speci fi city promoter system combining cell cycle-regulated and tissue-speci fi c transcriptional control dedifferentiation rescues senescence of progeria cells but only while pluripotent in vitro pathological modelling using patient-speci fi c induced pluripotent stem cells: the case of progeria origin of hematopoietic progenitors during embryogenesis deriving respiratory cell types from stem cells regulated expansion of human pancreatic beta-cells targeting the perpetrator: breast cancer stem cell therapeutics ef fi ciency of lentiviral transduction during development in normal and rd mice reprogramming to pluripotency: stepwise resetting of the epigenetic landscape targeting of therapeutic gene expression to the liver by using liver-type pyruvate kinase proximal promoter and the sv40 viral enhancer active in multiple cell types disease-speci fi c induced pluripotent stem cells glial cells in (patho)physiology lentivirus-expressed sirna vectors against alzheimer disease mesenchymal stem cells and tooth engineering stem cell therapy for cystic fi brosis: current status and future prospects progress in understanding reprogramming to the induced pluripotent state conditional mutagenesis in mice: the cre/loxp recombination system lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study lentiviral vectors approach the clinic but fall back: national institutes of health recombinant dna advisory committee review of a fi rst clinical protocol for use of a lentiviral vector mesenchymal stem cells as therapeutics and vehicles for gene and drug delivery plant biotechnology: zinc fi ngers on target chimeric nucleases stimulate gene targeting in human cells identi fi cation of the sensory neuron speci fi c regulatory region for the mouse gene encoding the voltage-gated sodium channel nav1.8 intrapleural delivery of mesenchymal stem cells: a novel potential treatment for pleural diseases zinc-fi nger nucleases for somatic gene therapy: the next frontier identi fi cation of potential stroke targets by lentiviral vector mediated overexpression of hif-1 alpha and hif-2 alpha in a primary neuronal model of hypoxia de fi ning pluripotent stem cells through quantitative proteomic analysis longterm correction of diabetes in rats after lentiviral hepatic insulin gene therapy effect of interleukin-10 overexpression on the properties of healing tendon in a murine patellar tendon model stem cell technologies for tissue regeneration in dentistry airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling mesenchymal stem cells derived from dental tissues third-generation, self-inactivating gp91(phox) lentivector corrects the oxidase defect in nod/scid mouse-repopulating peripheral blood-mobilized cd34+ cells from patients with x-linked chronic granulomatous disease current development of lentiviral-mediated gene transfer stem cell depletion in hutchinson-gilford progeria syndrome human-induced pluripotent stem cells produced under xeno-free conditions tissue speci fi city of enhancer and promoter activities of a herv-k(hml-2) ltr porous titanium scaffolds fabricated using a rapid prototyping and powder metallurgy technique collagen scaffolds reinforced with biomimetic composite nano-sized carbonate-substituted hydroxyapatite crystals and shaped by rapid prototyping to contain internal microchannels novel collagen scaffolds with prede fi ned internal morphology made by solid freeform fabrication gastric carcinogenesis and the cancer stem cell hypothesis mesenchymal stem cells stably transduced with a dominantnegative inhibitor of ccl2 greatly attenuate bleomycin-induced lung damage lentiviral vector-mediated gene transfer to endotherial cells compared with adenoviral and retroviral vectors engineering zinc fi nger nucleases for targeted mutagenesis of zebra fi sh lentiviral vectors pseudotyped with a modi fi ed rd114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and cd34+ cells derived from human and nonhuman primates targeting retroviral and lentiviral vectors biochemical correction of x-cgd by a novel chimeric promoter regulating high levels of transgene expression in myeloid cells migration of mesenchymal stem cells through cerebrospinal fl uid into injured spinal cord tissue overexpressed atp7b protects mesenchymal stem cells from toxic copper lentivirus-mediated gene transfer of gp91phox corrects chronic granulomatous disease (cgd) phenotype in human x-cgd cells genotypic features of lentivirus transgenic mice intracerebral cell transplantation therapy for murine gm1 gangliosidosis indirect rapid prototyping of biphasic calcium phosphate scaffolds as bone substitutes: in fl uence of phase composition, macroporosity and pore geometry on mechanical properties fibroblasts derived from human embryonic stem cells direct development and repair of 3d human skin equivalents mesenchymal stem cell transplantation modulates neuroin fl ammation in focal cerebral ischemia: contribution of fractalkine and il-5 superior tissue-speci fi c expression from tyrosinase and prostate-speci fi c antigen promoters/enhancers in helper-dependent compared with fi rstgeneration adenoviral vectors a highly ef fi cient short hairpin rna potently down-regulates ccr5 expression in systemic lymphoid organs in the hu-blt mouse model expression of truncated dystrophin cdnas mediated by a lentiviral vector mscs: biological characteristics, clinical applications and their outstanding concerns feasibility of a stem cell therapy for intervertebral disc degeneration pancreatic exocrine duct cells give rise to insulinproducing beta cells during embryogenesis but not after birth potential differentiation of human mesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells induced pluripotent cancer cells: progress and application kinetics and genomic pro fi ling of adult human and mouse beta-cell maturation cervical spinal cord delivery of a rabies g protein pseudotyped lentiviral vector in the sod-1 transgenic mouse. invited submission from the joint section meeting on disorders of the spine and peripheral nerves micromanipulating viral-based therapeutics growth and regeneration of adult beta cells does not involve specialized progenitors tumor-initiating and -propagating cells: cells that we would like to identify and control highly ef fi cient endogenous human gene correction using designed zinc-fi nger nucleases genome editing with engineered zinc fi nger nucleases mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte formation development of an in vitro model for the simultaneous study of the ef fi cacy and hematotoxicity of antileukemic compounds mesenchymal stem cell transplantation changes the gene expression pro fi le of the neonatal ischemic brain silencing of cardiac mitochondrial nhe1 prevents mitochondrial permeability transition pore opening gene delivery of a mutant tgfbeta3 reduces markers of scar tissue formation after cutaneous wounding induced pluripotent stem cells: fundamentals and applications of the reprogramming process and its rami fi cations on regenerative medicine transcriptional pro fi ling of human mesenchymal stem cells transduced with reporter genes for imaging molecular imaging of mesenchymal stem cell: mechanistic insight into cardiac repair after experimental myocardial infarction the engineering of patient-speci fi c, anatomically shaped, digits in vitro osteogenic differentiation of adipose stem cells after lentiviral transduction with green fl uorescent protein runx1/aml1/ cbfa2 mediates onset of mesenchymal cell differentiation toward chondrogenesis targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with vsv, ebola, mokola, lcmv, or mulv envelope proteins local gene transfer to calci fi ed tissue cells using prolonged infusion of a lentiviral vector harnessing hiv for therapy, basic research and biotechnology macroarchitectures in spinal cord scaffold implants in fl uence regeneration transduction patterns of pseudotyped lentiviral vectors in the nervous system establishment of an erythroid cell line from primary cd36+ erythroid progenitor cells targeted genome editing across species using zfns and talens in vivo gene transfer into adult stem cells in unconditioned mice by in situ delivery of a lentiviral vector combinatorial control of suicide gene expression by tissue-speci fi c promoter and microrna regulation for cancer therapy effect of hypoxia on the gene pro fi le of human bone marrow-derived mesenchymal stem cells the in fl uence of semen-derived enhancer of virus infection on the ef fi ciency of retroviral gene transfer microdystrophin delivery in dystrophin-de fi cient (mdx) mice by genetically-corrected syngeneic mscs transplantation long-term ef fi cacy and safety of human umbilical cord mesenchymal stromal cells in rotenone-induced hemiparkinsonian rats vegf-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for parkinson's disease mesenchymal stem cells promote cardiomyocyte hypertrophy in vitro through hypoxia-induced paracrine mechanisms beta cells can be generated from endogenous progenitors in injured adult mouse pancreas magnetic resonance evaluation of transplanted mesenchymal stem cells after myocardial infarction in swine transplantation of adipose tissue-derived stem cells for treatment of focal cerebral ischemia current advances in retroviral gene therapy correction of cardiac abnormalities in fabry mice by direct intraventricular injection of a recombinant lentiviral vector that engineers expression of alpha-galactosidase a sequencing and characterization of the porcine alpha-galactosidase a gene: towards the generation of a porcine model for fabry disease self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb glycosaminoglycans and pdgf signaling in mesenchymal cells validation of a mutated pre sequence allowing high and sustained transgene expression while abrogating whv-x protein synthesis: application to the gene therapy of was the hiv-1 dna fl ap stimulates hiv vector-mediated cell transduction in the brain rescue of atp7b function in hepatocyte-like cells from wilson's disease induced pluripotent stem cells using gene therapy or the chaperone drug curcumin transplantation of neural stem cells modi fi ed by human neurotrophin-3 promotes functional recovery after transient focal cerebral ischemia in rats lentiviral-mediated overexpression of bcl-xl protects primary endothelial cells from ischemia/reperfusion injury-induced apoptosis lentivirus-mediated gene transfer of viral interleukin-10 delays but does not prevent cardiac allograft rejection loss of proliferation and differentiation capacity of aged human periodontal ligament stem cells and rejuvenation by exposure to the young extrinsic environment in vivo reprogramming of adult pancreatic exocrine cells to beta-cells organ-speci fi c expression of the lacz gene controlled by the opsin promoter after intravenous gene administration in adult mice neurovascular pathways to neurodegeneration in alzheimer's disease and other disorders woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors key: cord-011903-zqt6vu6d authors: duby, zoe; mcclinton appollis, tracy; jonas, kim; maruping, kealeboga; dietrich, janan; lovette, ashleigh; kuo, caroline; vanleeuw, lieve; mathews, catherine title: “as a young pregnant girl… the challenges you face”: exploring the intersection between mental health and sexual and reproductive health amongst adolescent girls and young women in south africa date: 2020-07-18 journal: aids behav doi: 10.1007/s10461-020-02974-3 sha: doc_id: 11903 cord_uid: zqt6vu6d in south africa, adolescent girls and young women (agyw) are at risk of poor mental health, hiv infection and early pregnancy. poor mental health in agyw is associated with increased sexual risk behaviours, and impeded hiv testing and care. using in-depth interviews and focus group discussions, we explored subjective experiences of mental health and sexual and reproductive health (srh) amongst 237 agyw aged 15–24 years in five south african districts. respondents shared narratives of stress, emotional isolation, feelings of depression, and suicidal ideation, interconnected with hiv, pregnancy and violence in relationships. findings show that agyw in south africa face a range of mental health stressors and lack sufficient support, which intersect with srh challenges to heighten their vulnerability. framed within the syndemic theory, our findings suggest that south african agyw’s vulnerability towards early pregnancy, hiv infection and poor mental health are bidirectional and interconnected. considering the overlaps and interactions between mental health and srh amongst agyw, it is critical that mental health components are integrated into srh interventions. poor mental health, including depressive disorders and stress, contributes significantly to the burden of disease in south africa, and other parts of sub-saharan africa, and is also associated with negative sexual and reproductive health (srh) outcomes for women, such as 'unintended' or early pregnancy, and increased risk behaviours for hiv [1] [2] [3] . researchers in the field of women's health have highlighted the need to further explore the syndemic interactions between psychosocial vulnerability, mental health, hiv infection, and poor srh outcomes [4] . in south africa's hiv epidemic, the largest in the world, a quarter of all new infections occur amongst adolescent girls and young women (agyw) aged 15-24, three times as high as their male counterparts [5] . as with hiv, south africa also has high rates of teenage pregnancy; in 2016, 16% of females aged 15-18 years had begun childbearing [6] . agyw in south africa are more susceptible to depressive symptoms than their male counterparts, and are likely to remain underdiagnosed and untreated [7] [8] [9] . thus, their vulnerability lies at a biological, social, and environmental nexus. the onset of depression in particular, but other mental health problems as well, can coincide with other developmental milestones such as sexual debut and escalated risks for hiv infection. estimates suggest that approximately three quarters of mental health comorbidities that affect adults across the life course emerge during adolescence and young adulthood [7] . adolescents' mental health status can have profound impacts on their future health, social, and economic circumstances as adults, particularly in contexts of poverty and vulnerability [10] . the development of poor mental health outcomes during this period is influenced by neurological, hormonal, and physical changes associated with puberty, combined with changes in adolescents' social environments [11] . evidence from south africa and other countries in the sub-saharan african region show that age-specific risk factors for depression and anxiety disorders include lower socio-economic status, lack of social capital and support, substance use, and exposure to violence and traumatic events [12] . adolescents growing up in the context of socio-economically adverse communities are faced with a range of additional psychosocial and health risks that may evoke stress and negatively affect their mental health; these risks include exposure to hiv, substance use, violence, and other stressors [13] . poverty has been shown to be associated with heightened vulnerability to experiencing poor mental health, including mood and anxiety disorders [13] . in addition to age-related factors and socio-economic factors, gender-related factors, including sexual and reproductive biology, also play a role in contributing to mental health risks. adolescent pregnancy poses a significant mental health burden, predisposing agyw to adverse mental health outcomes, with depression and anxiety being the most common [14] . in resource-deprived settings in sub-saharan africa, pregnancy amongst agyw is associated with adverse mental health outcomes and psychosocial stresses including stigma and discrimination [14] . in the south african context, pregnancy may exacerbate existing social and contextual stressors, adding additional stressors such as interpersonal relationship challenges, regret around 'unintended' pregnancies, and depression [15] . globally, suicide is the second leading cause of mortality among females aged 10-24 years; with low and middle income countries accounting for over 75% of global suicide deaths [11] . rates of suicidal ideation, defined as the thought of killing oneself, are highest among adolescents on the african continent, with hiv as a contributing factor [11] . the syndemic theory of health refers to the clustering of risk factors, or co-occurring and intersecting epidemics embedded in the particular social context in which an individual is situated, which combine and interact to create vulnerability to health outcomes that are worse than any one risk factor alone would cause [16, 17] . by focusing on the 'biosocial complex', the interconnected and cooccurring health issues, as well as the social and environmental factors that promote and enhance negative health outcomes, the syndemic theory can help to explain the way in which risk behaviours which lead to negative srh outcomes, namely hiv infection and 'unintended' pregnancy, are situated within co-occurring and interacting psychosocial health conditions, including psychological distress and poor mental health [9, 18, 19] . the immense physical, neurocognitive, mental, and social changes that occur during adolescence not only affect mental health, but also influence sexual behaviour; during this period of transitioning to adulthood, adolescents are at increased risk of hiv infection [18] . the association between depressive symptoms and decreased sexual agency and decision-making power in agyw are compounded by low self-esteem; in turn these are associated with increased risk behaviours, including increased susceptibility to pressure to have sex, comfort seeking, condomless sex, transactional sex, trans-generational sex, substance use, and 'unintended' pregnancy [7, 8, [19] [20] [21] . some of the mechanisms through which poor mental health symptoms influence sexual risk include substance use, maladaptive coping mechanisms to deal with stress, and impaired decision-making, indicating poor mental health as a prospective predictor of sexual risk [22] . depressive symptoms in agyw have also been correlated with a lack of ability to withstand social pressure, including peer pressure to engage in risky behaviours, a tendency to be more subservient and less assertive in sexual relationships, as well as with being more vulnerable to intimate partner violence and abuse [8, 19] . in addition to the links between depression and increased sexual risk taking, depression is also associated with impeded health seeking behaviour, including hiv testing [23] . considering the overlaps and interactions between mental health and srh amongst agyw is critical. greater insight into the lived subjective experience of depression and stress, and how these are linked to srh outcomes is needed. there appears to be a gap in the literature pertaining to the ways in which mental health and psychosocial risks, including depression and stress, intersect and overlap with srh related factors such as distress caused by 'unintended' pregnancy, material/emotional stressors of having a child, or social stigma [16] . various studies explore depression amongst hiv positive women, but there has been little exploration of mental health issues that arise due to, or co-occur with, srh outcomes. although mental health was not an initial focus of the research, upon qualitative enquiry, the significance of poor mental health outcomes impacting on sexual and reproductive health practices emerged as a salient theme, warranting closer examination. we examined agyw's narratives and conceptualizations of their own mental health, that of peers, and of the surrounding emotional and psychosocial support context in order to explore the ways in which these factors might interact with sexual health outcomes. this research formed part of the herstory study, which evaluated a comprehensive combination hiv prevention intervention for agyw implemented in ten priority districts in south africa from 2016 to 2019, funded by the global fund (https ://www.samrc .ac.za/intra mural -resea rch-units /healt hsyst ems-herst ory). included in the analysis for this paper are qualitative data from 63 in-depth interviews (idis) and 24 focus group discussions (fgds) conducted between august 2018 and march 2019 in five south african districts, with a total of 237 agyw aged 15-24 years. of the 237 agyw, 63 were from the western cape (wc), 50 from kwazulu-natal (kzn), 41 from mpumalanga (mpu), 35 from the north west (nw), and 48 from the eastern cape (ec). participant recruitment took place in selected schools and communities through liaising with school staff and/or intervention implementers in order to identify eligible participants, arrange interviews, and secure appropriate venues. a brief demographic questionnaire was also administered. idis (20-40 min) and fgds (40-90 min) were conducted in english, isizulu, isixhosa, setswana, or siswati, by one of two lead interviewers, accompanied by a research assistant, all female, and all of whom had received training on the study protocol, design, research tools, and human subject research ethics. semi-structured topic guides comprised of open-ended questions and probes guided discussions. included in the topic guides were questions relating to sources of social/emotional support; agyw were asked who they talk to or seek support from when experiencing emotional/relationship/school/health/srh concerns or challenges. mental health was not a specific focus of the study but arose in response to these. the research team engaged in an on-going reflective process of note-taking and debriefing discussions, which formed part of the collaborative interpretation discussions and analysis process. informed consent was obtained from all participants 18 years and older. written assent with written guardian consent was obtained for those younger than 18 years. participants were provided with a zar 50.00 (approximately us$ 3.00) supermarket voucher, transport reimbursement, and refreshments. the study protocol and research tools were approved by the south african medical research council research ethics committee, and by the associate director for science in the center for global health in the centers for disease control and prevention. the research team received training on the study protocol and procedures for reporting and managing social harms and adverse events, as outlined in human subject research ethical guidelines. during data collection, private-sector social workers were procured to assist with ensuring access to social support services for participants who needed psychosocial support. audio recordings of idis and fgds were transcribed verbatim into their original language, reviewed by the researcher who conducted the interviewer for accuracy, translated into english and re-reviewed by the interviewer. data analysis followed a thematic approach, in which a pre-determined deductive codebook underwent cyclical review and refinement [24] [25] [26] . collaborative interpretation by the research team, comprising the two interviewers who were also co-investigators, along with four other co-investigators, included individual data immersion and familiarisation, repeated deep readings of transcripts, documentation of reflective thoughts, and sharing growing insights about the research topic during regular team discussions. the codebook was entered into nvivo 12 software, which was used to organise and label relevant text from the transcripts. as concepts and themes emerged, the team collaboratively reviewed them, returning to the data, and refining themes. weekly research meetings were held throughout the data collection and analysis phases allowing for team debriefing and examination of how thoughts and ideas were evolving as they engaged with the data. three feedback workshops were held with 32 agyw aged 15-24 at three of the study sites, some of whom had previously participated in idis and fgds, and some who had not. the objective of these workshops was to review and discuss the preliminary analysis and interpretations, ensure accurate and appropriate interpretation of the data, clarify misunderstandings, and confirm findings and interpretations. during the workshops, the research team summarised and presented key themes and findings to the participants, who were then invited to give feedback, discuss their interpretation of the findings, and expand or elaborate on themes. facilitated discussions on each theme were captured through notes and audio recordings, transcribed and reviewed, and included in the overall analysis. amongst the 237 agyw respondents aged 15-24, the mean age was 17.4 years. of these, 99% (n = 235/237) selfreported to have been assigned female at birth. amongst the agyw respondents, 97% (n = 227/234) self-identified their gender as female, with two identifying as transgender, and three as gender-variant. most, 86% (n = 202/235) of agyw self-identified as heterosexual/straight, 4% (n = 9) as homosexual/gay/lesbian, and 7% (n = 17) as bisexual. for reporting of language spoken at home, the top three languages were isixhosa (39%, n = 92/237), isizulu (25%, n = 59/237), and siswati (15%, n = 35/237). overall, 18% (n = 41/232) of the agyw reported to have had a pregnancy. emergent themes in the qualitative data included agyw narratives and perceptions of depression, stress, and suicide. in the accounts of agyw, poor mental health, including depression and suicidal risk were linked to sexual/ romantic relationship challenges, early pregnancy and child-bearing, parenting responsibilities, experiences of violence/abuse, hiv status, and lack of emotional support. suicide risk emerged as a salient theme and was associated with discovery of pregnancy or an hiv positive status, low self-esteem, and a lack of anyone to trust or confide in. in general, agyw voiced a need for increased access to support, and additional information on mental health. the findings presented below are arranged into key thematic areas that emerged during analysis. illustrative quotations are excerpts from english transcripts or translations; in brackets are details of the respondents' site and sample group. in selected excerpts, original language terms/words have been included in italicised brackets for the purpose of illustrating the exact words/language used by participants relating to key concepts associated with mental health. the rationale for this is that often concepts such as "depression" have been framed in a universal/western way, without attention to contextual specificity. where qualitative research uses translations, there is a danger of the original meaning and concept getting lost in the translation process, as translators seek to find 'equivalent' terms [26] . suicidal ideation emerged as salient theme across provinces, despite there being no specific question probes relating to suicide. according to agyw, issues such as self-harm and having suicidal tendencies were common amongst their peers. one participant expressed hesitancy using the diagnosis of 'depression', but described self-harming and suicidal ideation: "there are girls, i don't want to say 'depressed', but who do things like self-harming, some attempt suicide" (15) (16) (17) (18) ec) . agyw made links between low self-esteem and self-worth, and lacking a sense of belonging, with suicidal ideation: "most girls… have a low self-esteem… feel as though they don't belong in this world. that's why people commit suicide. i used to have that… mentality… suicidal thoughts because of people" (19) (20) (21) (22) (23) (24) nw) . illustrating the link between srh and mental health, feelings of emotional isolation leading to suicidal ideation were exacerbated in the case of hiv positive or pregnant agyw who feel unable to access support: "this thing of suicide is becoming popular now, even here at school… especially when girls are pregnant or hiv positive, because they can't share it with anyone, they don't trust anyone" (15-18 years, wc). the sense of having no one to trust or confide in, and seek emotional support from, resulted in agyw feeling emotionally isolated, fostering suicidal ideation: "we don't share our sexual and personal life things… we keep it to ourselves, then some of us commit suicide (sizigcinia kuthi, abanye bethu ke baphela sebezibulala)" (15-18 years, wc, isixhosa). suicide was linked to feelings of isolation after an hiv positive diagnosis: "(when) the nurse told her that is she is hiv positive, she didn't know who to tell… so she took a rope and hanged herself because she had no one to talk to" (15-18 years, wc). the discovery of being pregnant was also described as a difficult emotional event. agyw in the older age group, 19-24 years, described personal experiences with suicidal ideation in this situation: "when i found out i was preg-nant… that was very difficult, i even thought about sui-cide… it was tough (kwabanzima kakhulu, ngangicabanga ngisho ukuyibulala, ya kwaku tough)" (19-24 years, kzn, isizulu). additional links between mental health and srh were apparent in the narratives of suicidal ideation in relation to the stress of teenage pregnancy, compounded by fear of hiv: "as a young pregnant girl… the challenges you face… maybe you will find out that he (baby's father) is hiv-positive… those are challenges that can be a problem and you end up committing suicide… a better solution is to kill yourself (yizona ngqinamba lezo ezingaba inkinga ugcine usu… usuzibula… i solutions kuncono ukuthi uzibulale)" (19-24 years, kzn, isizulu). respondents suggested that due to social stigma attached to teenage pregnancy, pregnant agyw fear being judged and gossiped about: "pregnant girls feel sad… some even contemplate suicide (azive efuna ukuzibulala)… because of hearing unpleasant things about their life being spoken by other people. (15-18 years, wc, isixhosa); "pregnant girls are teased, and then they drop out of school, they don't fin-ish… here at school… we gossip about each other in the toilets" (15-18 years, wc). parents' attitudes towards their daughters' romantic and sexual behaviour prevented agyw from accessing support: "like most girls, i got pregnant at an early age. some girls resort to committing suicide (ezibulala) or just run away from home because they cannot face their parents" (15-18 years, wc, isixhosa). getting involved in transactional relationships, compounded by a sense of shame and fear of social judgement, also led to depression and suicidal ideation: "most girls in the community, they get into those (transactional) relationships, to a point that it damages them… they end up being depressed… 'why are you doing this and that to me in front of people?'… they end up like that and they end up trying to commit suicide… 'he embarrassed me in front of people, tomorrow how will people look at me?'" (19-24 years, nw). the emotional 'burden' of teenage pregnancy was described as a key contributing factor to poor mental health: "they say having a child is a good thing, but as a teenager it is a burden, it's difficult to cope" (15-18 years, ec). financial, material and relationship insecurity added stress to pregnancy: "the baby's father has denied the baby, there will be stress of how you are going to support the baby, because the (social) grant is not enough" (19-24 years, kzn). those agyw who had experienced unexpected discovery of pregnancies described their stress related to being rejected by families, kicked out of school or from home. one participant described her concerns after finding out she was pregnant in grade 10: "i was confused and didn't know what to do… (i told my boyfriend) my dad is strict… i will be chased away from home" (19-24 years, kzn). those agyw who became pregnant with casual sex partners, or who were not in committed relationships described the stress and unhappiness they experienced. one young woman described how she wanted to terminate her pregnancy but was told it was already too late to do so, and how this unwanted pregnancy caused her stress: "i had stress… i only realised when i was 4 months 2 days that i was pregnant… if i had realised this earlier, i was going to do an abortion… then i asked the doctor 'is there any other way i can do an abortion?'… he then said 'it's either you die… i will not allow you to risk an abortion'…[sigh] i was not ready to have a child at that time… i knew how my situation was… the guy i was dating, i was just dating him for fun. i did not see myself having a child with him, or to have future with him… that was why i was going to abort this baby… i did not want the child… everything failed… i did not eat, i had stress… the one who impregnated me was staying in a shack" (19-24 years, nw). a lack of emotional support from partners/fathers of children also contributed to stress and depression amongst young mothers: "where does the stress go? …to me… i'm always watching this child, he cries the whole day and i don't know why… i'm holding him, gave him his bottle, he continues to cry, i don't know why he is crying… you call him (baby's father)… (but) he doesn't take any action… i become depressed and it affects the child" (19-24 years, nw). being a single parent was described as difficult and stressful: "if you are a single mother, there is nothing nice… (you) have love for your baby but that's it. everything else is not nice… it's difficult to raise the child" (19-24 years, nw). the feeling that former dreams and aspirations for the future were shattered by unexpected pregnancies heightened feelings of hopelessness and depression: "it's not going to be dark forever, things will be right… but what i can say? …to be pregnant unexpectedly is not good at all… life is not good… especially if… you had plans and maybe life does not go the way you had planned… i am speechless… for me now, life is not good… it's not good… tough times…" (19-24 years, nw). lacking a supportive social environment negatively impacted on mental health and self-esteem: "when people are discouraging me… i get very sad… i'm trying… i'm telling them that… and they say 'you cannot do that… you're weak'… it makes me angry, but… i don't defend myself" (19-24 years, nw). some agyw suggested that they tried to cope without sharing their problems with anyone: "i keep my problems to myself… i talk to no one… i keep to myself and own it, i don't make my problem someone else's… if ever i have something troubling me i will keep it to myself… eventually i will be fine (ndizade ndibe right)" (19-24 years, ec, isixhosa). the lack of emotional support for dealing with traumatic life events, including grief over the death of a loved one, was present in agyw narratives: "when i think about something that happened in the past my heart becomes sore (intliziyo yam ibabuhlungu)… (like) when i think about my mother… she passed away… there is nobody (i talk to at home)… i don't feel free talking to them… i don't speak to anyone at school (either)" (15-18 years, ec, isixhosa). a minority of agyw were vocal about receiving emotional support at home: "i know i am loved at home and they show me that they love me because they care for me and stuff" (19-24 years, ec). sexual and romantic relationships with violent and controlling partners were also described by some of the agyw, who ended up living in a state of fear: "if i have made friends… and we want to go out as girls, then he (boyfriend) will refuse and beat you. even when you make a minor mis-take… he will beat you, and you end up afraid… you now live in fear… when happy, it doesn't last for long… sometimes he will take out his anger on you even when you did nothing… but you continue to love him even when friends try to talk some sense to you, but you will continue staying and loving him because you are afraid of him and not at liberty to do your own things" (19-24 years, mpu). refusal to have sex with a partner also led to violence: "sometimes, it happens that he wants to sleep with you, and you don't want to, then he gets angry and he beats you" (19-24 years, kzn). those agyw who had experienced intimate partner violence explained their reluctance to disclose to her family and friends: "in most times, you keep quiet and when they ask you 'what happened, why are you hurt?' you just tell them that you got hurt, you turned and bumped into a wall" (19-24 years, kzn) . experiencing violence negatively impacted agyw self-esteem and self-worth: "it has to do with how you perceive yourself, he sees me as not good enough then maybe you will find that boyfriend that hits you, he is the one that you want to stay with because you think where else will you find another boyfriend? …when he hits you that means this person does not see any value on you he beats you, abuses you… physically you will be injured obviously because that will hurt you… and she will think that she is not good enough" (15-18 years, ec). pregnancy increased agyw dependence on partners, even when they are violent: "my friend is pregnant… (her boyfriend) beats her… in her pregnancy the guy did not care for her and he was beating her saying the child is not his" (19-24 years, ec). our study did not initially set out to examine mental health amongst agyw, but narratives around depression, stress and suicide became salient, as did evidence of their interconnection with sexual and reproductive health. feelings of stress, anxiety and not being able to cope, even to the point of suicide ideation, were associated with hiv status, unexpected discovery of pregnancy, and parenting responsibilities. violence in relationships, a lack of emotional support from family and partners, and financial insecurity interact to exacerbate agyw vulnerability to poor mental health and srh outcomes. agyw in our study who had been pregnant, shared narratives of negative emotions they had experienced on discovering their pregnancy, leading to depression and suicidal ideation. the social causation hypothesis theory posits that stressful circumstances or events increase an individual's susceptibility to manifesting or experiencing mental health problems [13] . it would therefore make sense that the emotional aspects related to the discovery of an unexpected pregnancy or an hiv positive status would act as a stressor and have potentially negative mental health outcomes [16] . it is likely that after encountering a stressor, adolescents will experience stress and symptoms of depression and anxiety [13] . the stress related to the discovery of an unexpected pregnancy is compounded by the shame and social stigmatisation of teenage pregnancy, and the ensuring social isolation from family and community increases the risk for psychological distress [7] . the framing of pregnancy during adolescence as a social problem means that pregnant teens receive limited social support, which in turn is linked to poor mental health outcomes [2] . the stress related to the discovery of an unexpected pregnancy is heightened further in the case of a dual discovery of being hiv positive [27] . the presence of depression, anxiety, and post-traumatic stress disorder in hiv-positive individuals is related to diagnosis and disclosure, and hiv-positive women experiencing 'unintended' pregnancy are at high risk for antenatal depression [27, 28] . agyw in our study described a worrying trend of suicidal ideation. thoughts about suicide narrated by agyw were related to unexpected discovery of pregnancy and its consequences, hiv diagnosis, and feelings of emotional isolation. suicide was described as "the best solution" to situations of stress created by the discovery of unexpected discovery of pregnancy or hiv positive status, lack of material/financial or emotional support for young mothers, and feelings of victimisation as a result of gossip or judgement. adolescents faced with multiple stressors may experience a sense of being overwhelmed and unable to cope, and view suicide as an escape [13] . agyw in our study associated low self-esteem and low self-worth with depression and suicidal ideation. negative self-cognitions and low self-worth are associated with depression, and positive self-esteem is a critical component of emotional well-being [29] . adolescents' ability to manage their stress symptoms or address the stressor they encounter may be pivotal to protecting their mental health state, as it may buffer the impact of experienced stress on mental health [13] . additionally, self-esteem and social support are amongst the 'protective assets' associated with improved srh outcomes [30] . in our study, agyw expressed feeling emotionally isolated, lacking people who they felt they could trust and confide in without fear of judgement or recrimination. emotional isolation and lack of support, especially when faced with stressors such as the discovery of an unexpected pregnancy or an hiv diagnosis, negatively impacts mental health. the emotionally distressing aspects of unexpected discovery of pregnancy, or an hiv diagnosis, combined with a lack of social support, contribute to the high rates of depression amongst agyw [16] . agyw in our study described additional stress related to teenage pregnancy and child-bearing relating to concern around the ability to support a baby financially, especially when there was a lack of material and/or emotional support from the father of the child. agyw in south africa, particularly those in resource-constrained or violent households, face a variety of personal and structural challenges, linked to disempowerment and psychological distress more broadly; an 'unintended' pregnancy can compound pre-existing social and economic vulnerabilities, and result in heightened feelings of stress and unhappiness [31] . socio-economic disadvantage compounds other stressors in an adolescents' life, and when co-occurring with pregnancy or hiv, can lead to poor mental health outcomes and a lack of utilisation of health care services [2, 13] . one limitation of this study was that the framing and concept of 'unintended' pregnancy was not investigated in more depth. for this reason, we avoid using the term 'unintended', and instead use the words of agyw respondents themselves when describing their unexpected discovery of a pregnancy, rather than objectively categorising the pregnancies as 'unintended'. in addition, social desirability bias relating to the stigma and shame around mental health may have resulted in a lack of disclosure from agyw regarding their own feelings of depression or suicide ideation. interpreting our findings within a syndemic theory framework is helpful, in order to describe the integration of sociocultural, psychological, and physiological factors that combine to shape agyw vulnerabilities and experiences [32] . our findings suggest that south african adolescent girls and young women's vulnerability towards early pregnancy, hiv infection and poor mental health are bidirectional and interconnected. the social context in which south african agyw are situated, as described by respondents in our study, is characterised by a lack of social support, economic insecurity, and stigma, and serves to exacerbate the gendered and age-related vulnerabilities of this population. this interaction of socio-cultural, economic, structural, gendered, age-related and biological factors increase south african agyw's heightened risk of negative srh outcomes, cooccurring with psychological distress and poor mental health [33] . in line with the syndemic theory suggesting synergistic interactions between epidemics, and the interconnectedness and clustering of psychosocial conditions such as 'unintended' pregnancies, psychosocial distress, and hiv infection in agyw, there is a need for comprehensive hiv prevention programming inclusive of mental health support [34] . it is clear that interventions aiming to reduce rates of teenage pregnancy and reduce hiv acquisition amongst agyw in south africa, need to incorporate mental health components [35] . recommendations have been made for integrating mental health care into care for patients with chronic non-communicable diseases, as well as communicable diseases such as hiv [10] , but few recommendations for integrating mental health into srh delivery exist [36] . the links between mental health, hiv status, and 'unintended' pregnancy, exacerbate the need to strengthen the integration of routine mental health screening in srh and hiv programming in order to enhance the health outcomes amongst agyw [12] . addressing underlying mental health risks may be an important additional strategy to promote sexual risk reduction, and behavioural interventions which are able to improve mental health are also more effective in preventing negative sexual health outcomes such as hiv infection [22] . the indication of a dual burden of psychological distress and sexual risk behaviours suggests that screening for mental health disorders should be integrated into srh services [22] . despite the evidence of intersecting epidemics, mental health screening is not standard in hiv prevention and care settings and has not been added to the hiv care cascade. combination interventions inclusive of psychological and behavioural components may be able to achieve greater reductions in sexual risk behaviour among adolescents, as incorporating psychological health interventions appears to be a critical part of any comprehensive strategy for mitigating hiv risk [21] . mental health services targeted at agyw, especially those that are hiv positive and/or pregnant, need to be integrated into srh services, especially those that aim to be "youth-friendly"; prevention, diagnosis and management of depressive symptoms should also be included in the package of comprehensive services [14, 19] . early mental health screening could help catch agyw who might not yet be diagnostically clinical depressed. given the evidence, it is likely that agyw have overlapping epidemics that are clinically significant. practical recommendations for improving mental health care delivery to agyw include improving mental health, advocacy, decentralization of services, taskshifting and on-the-job training [12, 37] . the way in which mental health issues, such as stress and depression, are defined and conceptualised differs across settings and socio-cultural contexts, and interventions needs to be contextually relevant [1] . in addition to contextual and conceptual equivalence, linguistic equivalence of terms related to mental health should be taken into account. the words "depression" and "anxiety" do not have direct equivalents in some south african languages, with psychological distress, including depression, described behaviourally rather than cognitively, and expressed and/or experienced somatically, or situated within the relational domain [29, 38] . in addition, the term "unintended pregnancy" is problematic, with traditional measurements dichotomously classifying pregnancies as intended or not, based on a woman's intentions before she became pregnant [39, 40] . "unplanned pregnancies" likewise have been defined as pregnancies which occur when a woman is using contraception or did not desire pregnancy as an outcome [39] . the key problem with these binary classifications of pregnancies as unintended/intended, planned/unplanned, is that they fail to consider the complexity of intention, motivation and desire [40] . the construct of 'pregnancy acceptability' may be a more useful way to taking into account the complexity of pregnancy intentions, in the context of women's lived experiences, emotions, relationships, and socio-economic circumstances [40] . a woman's emotions, levels of preparedness, and acceptance of a pregnancy are likely to change in reaction to external factors, which in turn influence health outcomes [40] . the biomedical/clinical understandings and definitions of "depression" and "unintended pregnancy" may not always capture an individual's subjective experiences and articulation of these states [41] , and in order for any intervention to be successful, there is a need to be sensitive and reflective of the reality of people's lives, with an understanding of the language that agyw use to describe their lived experiences of depression and pregnancy, which is why qualitative research such as the findings we present here, is much needed [40] . indeed, agyw in south africa construct "depression" and pregnancy is a complex phenomenon manifesting in a variety of emotions, thoughts, and behaviours; finding ways to surface contextually congruent understandings of sexual and reproductive health, and mental health can inform the development of interventions that are contextually and population relevant [29] . mental health and srh interventions and services need to be contextually appropriate and reflective of the reality of people's lives. screening tools need to take into account the diversity of understandings of emotional suffering and distress, using appropriate terms, language and concepts. it is evident that agyw in south africa face substantial social adversities and related mental health challenges due to a range of srh, social, economic, environmental, physiological and interpersonal factors. building on previous research that has found associations between depressive symptoms and psychological distress related to pregnancy, combined with a lack of social support amongst south african women [16] , our findings provide rich descriptive data on the lived reality of the interconnected psychosocial risks including stress, emotional isolation, feelings of depression and suicidal ideation, with 'unintended' pregnancy and hiv that agyw in south africa face, from their own perspectives. framing these interconnections within the syndemic framework can help to inform interventions that seek to address agyw risk. as psychological distress is associated with increased risk behaviours, it is critical that efforts to address early pregnancy and hiv infection amongst agyw incorporate mental health components. interventions to improve emotional wellbeing and coping mechanisms for agyw are needed in order to improve sexual and reproductive health outcomes; indeed, in a context where hiv, stis, early pregnancy are common, it is all the more important to have such interventions integrated into srh services and part of largescale programmes for agyw. understanding the context of mental health is crucial in order to design and implement effective mental health programming, and to provide appropriate psycho-social support to young women, and in turn, address sexual and reproductive health challenges. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. building resilient families: developing family interventions for preventing adolescent depression and hiv in low resource settings social support among hiv-positive and hiv-negative adolescents in umlazi, south africa: changes in family and partner relationships during pregnancy and the postpartum period burden of non-communicable diseases in sub-saharan africa, 1990-2017: results from the global burden of disease study the intersection of hiv, social vulnerability, and reproductive health: analysis of women living with hiv in rio de janeiro south african national hiv prevalence, incidence, behaviour and communication survey south african medical research council, icf. south africa demographic and health survey 2016. pretoria: national department of health high prevalence of depression symptomology among adolescents in soweto, south africa associated with being female and cofactors relating to hiv transmission gender differences in depression and smoking among youth in cape town mental health and hiv sexual risk behavior among patrons of alcohol serving venues in cape town, south africa an evaluation of the health system costs of mental health services and programmes in south africa. cape town: alan j flisher centre for public mental health suicidal thoughts and behaviour among south african adolescents living with hiv_ can social support buffer the impact of stigma? prevalence and correlates of depression and anxiety symptoms among out-of-school adolescent girls and young women in tanzania: a cross-sectional study stress and coping: considering the influence of psychological strengths on the mental health of at-risk south african adolescents. child care pract depression and its psychosocial risk factors in pregnant kenyan adolescents: a cross-sectional study in a community health centre of nairobi it's better for me to drink, at least the stress is going away": perspectives on alcohol use during pregnancy among south african women attending drinking establishments mapping a syndemic of psychosocial risks during pregnancy using network analysis the syndemic effects of intimate partner violence, hiv/aids, and substance abuse on depression among low-income urban women understanding the role played by parents, culture and the school curriculum in socializing young women on sexual health issues in rural south african communities associations between depressive symptoms, sexual behaviour and relationship characteristics: a prospective cohort study of young women and men in the eastern cape, south africa mental health and hiv sexual risk behaviour among university of limpopo students psychological and behavioral interventions to reduce hiv risk: evidence from a randomized control trial among orphaned and vulnerable adolescents in south africa anxiety and depression strongly associated with sexual risk behaviors among networks of young men in dar es salaam depression and anxiety as risk factors for delayed careseeking behavior in human immunodeficiency virus-infected individuals in south africa qualitative data analysis for health services research: developing taxonomy, themes, and theory thematic analysis: striving to meet the trustworthiness criteria theme development in qualitative content analysis and thematic analysis double disclosure bind: complexities of communicating an hiv diagnosis in the context of unintended pregnancy in durban what is the relevance of mental health to hiv/aids care and treatment programs in developing countries? a systematic review you get angry inside yourself": lowincome adolescent south african girls' subjective experience of depression mentoring interventions and the impact of protective assets on the reproductive health of adolescent girls and young women this baby came up and then he said, 'i give up!': the interplay between unintended pregnancy, sexual partnership dynamics and social support and the impact on women's well-being in beyond comorbidity: a critical perspective of syndemic depression and diabetes in cross-cultural contexts syndemics and the biosocial conception of health a syndemic of psychosocial and mental health problems in liberia: examining the link to transactional sex among young pregnant women mental health system costs, resources and constraints in south africa: a national survey integrating mental health into south africa's health system: current status and way forward umthente uhlaba usamila-the 3rd south african national youth risk behaviour survey measuring depression and anxiety in sub-saharan africa the measurement and meaning of unintended pregnancy it's not planned, but is it okay? the acceptability of unplanned pregnancy among young people. women's health issues a feminist phenomenological description of depression in low-income south african women we would like to acknowledge and thank the adolescent girls and young women, and other participants who agreed to make themselves available to take part in this research, and share their views, opinions and experiences with us. the combination hiv prevention interventions were funded by the global fund to fight aids, tb and malaria, and implemented in 10 districts in south africa by a range of government departments and civil society organisations that were appointed by the organisations responsible for the management of the agyw programme: western cape department of health, kwazulu-natal treasury, kheth'impilo, soul city institute for social justice, and the networking hiv and aids community of southern africa (nacosa). the programme was aligned with the she conquers campaign and was implemented with support from the south african national aids council (sanac) through the country coordinating mechanism (ccm) and the ccm secretariat. key: cord-010310-jqh75340 authors: nan title: next generation technology for epidemic prevention and control: data-driven contact tracking date: 2018-12-24 journal: ieee access doi: 10.1109/access.2018.2882915 sha: doc_id: 10310 cord_uid: jqh75340 contact tracking is one of the key technologies in prevention and control of infectious diseases. in the face of a sudden infectious disease outbreak, contact tracking systems can help medical professionals quickly locate and isolate infected persons and high-risk individuals, preventing further spread and a large-scale outbreak of infectious disease. furthermore, the transmission networks of infectious diseases established using contact tracking technology can aid in the visualization of actual virus transmission paths, which enables simulations and predictions of the transmission process, assessment of the outbreak trend, and further development and deployment of more effective prevention and control strategies. exploring effective contact tracking methods will be significant. governments, academics, and industries have all given extensive attention to this goal. in this paper, we review the developments and challenges of current contact tracing technologies regarding individual and group contact from both static and dynamic perspectives, including static individual contact tracing, dynamic individual contact tracing, static group contact tracing, and dynamic group contact tracing. with the purpose of providing useful reference and inspiration for researchers and practitioners in related fields, directions in multi-view contact tracing, multi-scale contact tracing, and ai-based contact tracing are provided for next-generation technologies for epidemic prevention and control. outbreaks of infectious diseases could cause huge losses in human lives. the spanish pandemic in 1918 led to over 20 million deaths [1] . as of 2014, approximately 3.3 billion people worldwide are at risk of malaria, and every 60 seconds one patient dies due to malaria infection [2] . the death rate of tuberculosis has exceeded aids, becoming the most deadly infectious disease in the world. about 80% of people in south africa have latent tuberculosis and there were 450,000 cases of tuberculosis in 2013 alone [3] . along with the serious threat to human lives, infectious diseases also bring huge economic losses. statistically, malaria causes an economic loss of 12 billion us dollars every year in african countries [4] . seasonal influenza in the us causes an annual economic burden of 87 billion us dollars [7] . the developments in vaccines and drugs have enabled us to combat infectious diseases and have greatly reduced the harm brought to human society. however, the sudden emerging infectious diseases caused by the drug resistance and the inherent variability of viruses still remains to be a serious global problem that often leaves us in an unprepared and vulnerable situation. for example, the h1n1 flu in 2009 mutated to become the h7n9 flu in 2014 and the h3n2 flu in 2015. the h3n2 virus began to spread through contact networks in hong kong in january 2015, and 122 lives were taken in just one month [8] . the death toll rose to 591 after three months. thus, in the fight against various kinds of infectious diseases, relying solely on vaccine development is far from enough. a more effective ''active prevention and control'' method is desperately needed so as to rapidly detect and block figure 1. the spatial distribution of h7n9 cumulative cases in the early stage of the outbreak in mainland china in 2013 [10] . in this case, infected cases are recorded with the information of location and time, while the contact information dominating transmission remains unknown. the transmission paths of new infectious diseases, detaining the disease to a minimum spread until its eradication. many infectious diseases are transmitted through personto-person ''contact''. in computational epidemiology, a contact is simply defined either as a direct physical interaction (e.g. sexual contact) or proximity interaction (e.g. to people being within 1m of each other, or being in the same room) [9] . human contact interactions constitutes a ''contact network'' of virus transmission. in this network, nodes represent individuals, and links represent contact relationships. the structure of the contact network significantly affects the spatiotemporal patterns of virus spread. for example, in the case of respiratory infections that spread through droplets, interactions like face-to-face communication, shaking hands, crowd gathering, and sharing vehicles enable the spread of diseases and increase the possibilities of transmission from infected to susceptible persons. tracking the contact interactions of individuals can effectively restore the ''invisible'' virus transmission paths, quickly locate and isolate high-risk individuals who were in contact with infected persons, and can aid in quantitative analysis of the transmission paths, processes, and trends of the infectious diseases, all leading to the development of corresponding effective epidemic control strategies. the biggest obstacle in contact tracking is obtaining data that directly describes contact behaviors. because contact interactions between individuals are diverse and often subtle, they are difficult to be directly observed and recorded. in other words, it is hard to obtain first-hand high-quality data for contact tracking. when a disease is spreading, the impact of the disease could be observed, instead of the underlying direct interactions between individuals. for example, during the outbreak of h7n9 bird flu, it is difficult to identify who were infected due to contacts with certain infected people. as shown in fig. 1 , only new h7n9 cases and the number of deaths in different time and space can be observed. many epidemiology scholars and computer scientists have conducted research on how to accurately capture individuals' contact behavior data as well as how to indirectly infer the contact network from other data sources. many methods have been proposed, most of which utilize intelligence data analytics related technologies, such as intelligent sensing, network modeling and analysis, data visualization, multi-source heterogeneous data mining, data-driven reverse engineering, machine learning, and multi-agent simulation, among others. based on the granularity of contact modeling, the existing methods can be classified into four categories: static individual contact tracking, dynamic individual contact tracing, static group contact tracking, and dynamic group contact tracking. each of these methods are described and discussed separately in following sections. individual contact tracking records fine-grained ''individualto-individual'' contact information, such as contact time, location, frequency, duration, etc. the most common ways to gather contact information are non-automatic methods, e.g., offline and online questionnaires [11] , [12] , [24] , and automatic methods, e.g., mobile phone, wireless sensors, rfid, and gps [5] , [16] , [18] , [21] . offline questionnaire has been used for many years in some counties to trace sexual contacts of sexually transmissible infections (stis), particularly for hiv [31] . in recent 30 years, hiv has killed more than 30 million people. currently, there are still 2 million newly infected hiv individuals and half of them will be died every year [32] , [33] . to reveal the spread patterns of hiv infection and aids in the u.s., fay et al. analyzed the patterns of same-gender sexual contact among men using the data developed from a national sample survey in 1970 and 1988, respectively. they found that at least 20.3 percent of adult men have had sexual contact with another man in life, and never-married men are more likely to have same-gender sexual contacts [34] . similarly, merino et al. [35] sampled 294 homosexual men from colombia as volunteers to answer questionnaires on sexual practices. analysis of these questionnaires suggests two significant risk factors for hiv-1 infection: 1) having sexual contact with foreign visitors; 2) having more than ten homosexual partners. they suggest that the spread of hiv-1 infections should be monitored at the international level and more attention should be paid to these subgroups with high transmission rates. in general, most of us tacitly approve that unsafe sexual behavior would be more prevalent among individuals with optimal viral suppression. in the swiss cohort study on april 1, 2000, wolf et al. [36] investigated the unsafe behavior among 4948 hiv-infected individuals by selfreported questionnaire. however, after adjustment for covariate, it reported that unsafe sex is associated with other factors, e.g., gender, age, ethnicity, status of partner, having occasional partners, living alone, etc. in recent years, researchers designed questionnaires to measure the validity and reliability of sexual abstinence and avoidance of highrisk situations. for example, najarkolaei et al. [37] sampled 348 female undergraduate students from tehran university, iran, and assessed the validity and reliability of the designed sex, behavioral abstinence, and avoidance of high-risk situation. zhang et al. [38] surveyed 250 hiv-positive persons on their socio-demographic characteristics and sexual behavior, and traced hiv infection status of 431 persons who had heterosexual contact with the hiv carriers. among these 431 persons, 59 were hiv-positive, i.e., the secondary attack rate was 13.7%. therefore, they appeal to improve the knowledge about hiv/aids, enhance psychological education, and promote the use of condom, so as to suppress the transmission of hiv. in addition to hiv, offline questionnaire has also been used to trace the contact between individuals to investigate other infectious diseases such as chlamydia trachomatis infection, zika, and flu. to seek the view of patients with chlamydia trachomatis infection on legislation impinging on their sexual behavior, an investigation was performed on 192 patients at std clinics in stockholm, sweden in 1997. during the past 6 months, men (40%) were more likely to have sexual intercourse with occasional partners than women (21%), and the mean number of men and women was 2.3 and 2.2, respectively [39] . the zika virus is primarily transmitted by aedes species mosquitoes. however, by reviewing the travel experience and sexual intercourse of infected individuals in the us, researchers confirmed that there were 14 cases of zika virus infection were transmitted by sexual contact [40] . for instance, a person in texas was getting infected with the zika virus after sexual contact with someone who had acquired the infection while travelling abroad [41] . in 2012, molinari et al. [7] investigated the contact interactions of 1,074 students in a high school through questionnaires. a local campus contact network was established based on information such as the length of contact time and contact frequency. the outbreak process of flu was then simulated based on this established contact network. they found that the classroom is the location with the most campus contact and that class break and lunch break are the times with the most campus contact. offline questionnaire is an efficient way to trace private contact interactions such as sexual practice between individuals. however, it needs to find target participants one by one within a specific region, which is time consuming and needs more physical labor. moreover, data collected by this method is usually time delayed and incomplete. with the purpose of collecting more timely and low-priced data of various kinds of contact behaviors, online questionnaires such as online survey and web-based survey have been extensively applied. in 1998, a national online survey was constructed in 1690 adolescent males, using computer-assisted self-interviewing (audio-casi) technology. comparing with traditional selfadministered questionnaire, the prevalence of male-male sex with intravenous drug users estimated by audio-casi was higher by more than 3% [42] . influenza-like illness (ili) outbreaks on a large scale every year in many countries, recording and detecting ili are important public health problems. flutracking, a weekly web-based survey of ili in australia, has been used to record the past and current influenza immunization status of participants in winter influenza seasons for many years [43] . it only takes the participants less than 15 seconds to complete the survey, including documenting symptoms, influenza vaccination status, and mobility behaviors such as time off work or normal duties. in 2008, the peak week detected by flutracking was 31 august, which was contemporaneous with that in other influenza surveillance systems [44] . for the first three years being applied, the participants increased from 394 to 982 and 4,872 in 2006, 2007, and 2008, respectively, due to its convenience in completing the survey and its accuracy in detecting the peak week of influenza activity. flutracking also provides vaccine effectiveness analysis by investigating the status of vaccinated and unvaccinated participants. in 2012, the ili weekly incidence peaked in mid-july in the unvaccinated group, 1 month earlier than vaccinated group confirmed by national influenza laboratory [45] . in recent years, by cooperating with the health department, organizational email systems, and social media, flutracing gained over 1000 new participants each year by sending invitations from existing participants. as a result, the number of online participants in flutracing has exceeded 26,000 in 2016 [43] . contact information collected through an online questionnaire is more timely and low-priced than offline questionnaire. however, it still cannot record real-time contact information, and, moreover, contact information collected online sometimes inaccurate or even false [46] . because people on the internet are usually anonymous, which is incapable to verify the information of their real name, age, place of residence, etc. individual contact information obtained through offline or online questionnaires is usually time delayed, incomplete, and inaccurate. with the aim to collect dynamic, complete, and accurate individual contact information, some researchers began to use mobile phone, wireless sensors, rfid, and gps devices to track individual contact behaviors. in recent years, the application of mobile phones has become increasingly universal, providing a convenient way to record real time location information [58] , [59] . in 2011, yoneki [16] developed the first contact pattern discovery software, fluphone, which could automatically detect and record the contact behavior between users by mobile phone. the researchers collected the contact information of 36 users on the cambridge university campus with this software and established the contact network between different users at different times. then, they simulated an influenza outbreak on this network using a seir model. in view that the large power consumption of gps and bluetooth resulted in short standby time of mobile phones, yoneki and crowcroft [17] further developed a new contact tracking application, epimap, using wearable sensors, which had lower power consumption and longer standby time. epimap thoughtfully transmits and stores data by satellite, as many high-risk areas are in developing countries where there often are not enough wireless communication facilities to support contact tracking. wearable wireless sensors can record individuals' contact events such as time, location, and duration continuously and accurately, and gradually becomes a useful tool for collecting high-precision contact data in small areas [20] . it has been applied to discover contact patterns in various kinds of social settings such as hospitals and campuses. for example, mit media lab researchers nathan eagle and alex pentland proposed the reality mining method as early as 2006. this method suggests the use of wearable wireless sensors to record people's daily activities [13] , [14] . they developed an experimental system to record the activities of several mit students in a teaching building over time, and then established a small social network describing their contact relationships [15] . salathã© et al. [18] collected the contact interactions of 788 students in a high school in the united states for one day using wireless sensors, and they established an individual-based campus contact network. it was found that the campus contact network had high density connectivity and a small-world structure [19] . however, it is costly to trace contact interactions using wearable wireless sensors, especially when the number of individuals being monitored is large. moreover, people wearing wearable wireless sensors are very conspicuous, participants are unwilling to wear such devices due to privacy concerns especially for patients. radio frequency identification (rfid) is a non-contact automatic identification technology, by which the contact behavior can be recorded when individuals carrying a small non-contact chip getting closer. in 2009, olguin et al. [15] collected 16,000 contacts among 119 people (including medical staff, children in critical condition, and nursing staff) in a children's hospital in the united states using radiofrequency identification devices (rfid), and established a contact structure for the hospital. similarly, yoneki [16] collected students' contact data from a french primary school using radiofrequency identification devices. more recently, in october 2016, ibm researchers kurien and shingirirai from africa labs invented a radio tag designed to extend tracker working distance, and implemented it in tracking tuberculosis ( fig. 2) [6] . each tag contains a tiny sensor, figure 2. an ibm researcher holds a micro-radio tuberculosis tracker [6] . in october 2016, ibm researchers from johannesburg, south africa, released their latest research update: using cheap radio tags to anonymously track the contact transmission paths of tuberculosis. this study is an important step for ibm in helping who eliminate tuberculosis. a storage device, and a battery. radio tags can communicate with each other, allowing individuals' contact interactions to be recorded when two tags are in close proximity. the contact data collected by the radio tags is presented in a three-dimensional visualization system. using of the intelligent data analysis method provided by the system, medical staff can view the spatiotemporal distribution of tuberculosis patients in real time, track the transmission paths of tuberculosis, and find high-risk populations. because of the high cost of tuberculosis vaccines, contact data can also aid in the determination of high-priority vaccinations. however, the traditionally used radiofrequency tracker has a limited transmission and receiving range and only works within a small area. gps (global positioning system) has the capability of long-distance positioning, which has been widely used for tracing indoor and outdoor mobility behaviors and physical activities [55] , [56] . with the aged tendency of population, tracing mobility behavior is critical for measuring, describing, and comparing mobility patterns in older adults. for example, hirsch et al. [47] investigated the mobility patterns using gps tracing data collected from 95 older adults in vancouver, canada, with the goal of understanding neighborhood influences on older adults' health. they found that participants who were younger tend to drive more frequently and live far from their neighborhoods. gps devices have also been used for tracing physical activities of adolescents in school and other social settings [48] [50] . for instance, rodriguez et al. [49] sampled 293 adolescent females in minneapolis and san diego, usa, and traced their physical activity and sedentary behaviors by gps every 60s in different settings. physical activities were more likely to occur in parks, schools, and places with high population density during weekend, less to occur in places with roads and food outlets. besides, tracing animals in the sea or on the land using gps devices can obtain detailed spatiotemporal data regarding the movement patterns. for instance, dujon et al. [51] traced a green turtle travelling more than 300002km across the indian ocean and obtained more than 450,2000 locations. moreover, by tracing the whole-body motion dynamics of a cheetah using gps devices, patel et al. [52] illuminated the factors that influence performance in legged animals. although detailed individual contact information can be collected through non-automatic methods, e.g., offline and online questionnaire, and automatic methods, e.g., mobile phone, wearable wireless sensors, rfid, and gps devices. these methods are mostly limited to small-scale population experiments due to high cost and short range collection. they have not been applied to large areas or large-scale contact behavior studies. group contact tracing captures contact interactions of human beings with similar characteristics (e.g., age, occupation, hobbies) in different social settings from the macroscopic level. static group contact behavior can be traced by large-scale questionnaire and simulated by multi-agent models. dynamic group contact behavior can be inferred by data mining method like tensor deconvolution. in recent years, a composite group model that can characterize population heterogeneity and model epidemic spreading dynamics, overcoming the difficulty of obtaining fine-grained individuals' data has attracted much attention. such models not only simulate the transmission process, but also depict the contact structure of a larger population. the composite group model divides the population into several meta-populations by age or spatial location, so that individuals within a meta-population have similar biological characteristics (such as susceptibility, infectivity, latent period, and recovery period). then, the process of epidemic transmission can be modeled using group contact interactions among meta-populations instead of individuals' contact interactions [23] . based on this model, the infection and spread of epidemics can be described as a reaction-diffusion process. ''reaction'' characterizes the process of individual infection within a meta-population. ''diffusion'' characterizes the transfer process of epidemic diseases between different meta-populations through the group contact structure (fig. 3 ). in addition, there is a practical significance in establishing contact networks for composite groups because control strategies for epidemic diseases are usually oriented towards composite populations, for example, vaccination groups are usually sectioned by age when planning vaccine allocation strategies. [23] . the diffusion process is illustrated from a macroscopic perspective, i.e., the transmission among different meta-populations, whereas the reaction process is illustrated from a microscopic perspective, i.e., the individual infection within a meta-population. the composite group contact network was first established using questionnaires. in 2008, mossong et al. [24] conducted the polymod research project in europe, in which they organized a wide-range survey on contact behaviors, involving volume 7, 2019 7,290 participants from eight european countries. a total of 97,904 contact records were collected. they found that contact interactions have significant spatial heterogeneity, with most individual contacts occurring at home (23%), work (21%), school (14%), places of entertainment (16%), and while using transportation (3%). further, contact structures under different scenarios have obvious differences. there are some age-related contact patterns: in many scenarios (such as in schools), individuals are more likely to contact people of similar age; most of the contact between children and their parents occurs at home, while most contacts for adults occur in workplaces. the researchers thus divided the population into several meta-populations, establishing a composite group model based on age. interaction probabilities between different age groups were estimated according to questionnaire data (fig. 4) , and a contact network based on composite groups was established. the simulation method based on multi-agent models is also applied to the establishment of contact networks. this generally involves combining the questionnaire survey with population census data to establish the contact structure of composite groups. iozzi et al. [9] modeled a virtual society with the characteristics of italian society based on questionnaire data from 55,773 people. human daily migration behaviors were simulated by a virtual community, and a contact matrix of the composite group was obtained. based on this matrix, the outbreak process of italian b19 (human parvovirus) was successfully simulated. similarly, eubank et al. [26] simulated the movement of individuals within a city by large-scale agent system, and then modeled a group contact structure based on their simulation. the data they used included population census data, land usage, population migration, and other daily behavior data. constructing contact matrix for meta-population requires large-scale even nationwide questionnaire survey, which is quite costly and time delayed. multi-agent method simulates human mobility behaviors in the virtual world based on the contact matrix constructed using the data from the real world of the past [22] . it doesn't consider the changes of existing contact patterns caused by human self-awareness and epidemic-control strategies in the future. most of the above studies focus on static properties of contact behaviors, such as the contact object (who is contacting), scene (where this contact happens), frequency, and duration. in other words, the aforementioned studies assume that the contact patterns of the individual remain stationary. however, contact interactions usually change with time, and show different temporal and spatial patterns. for example, contact interactions can change periodically with the weather and season, vary significantly between workdays, weekends, and holidays, and may be adjusted in response to the threat of an epidemic disease and during the outbreak by reducing travel or wearing face masks [26] , [27] . additionally, governmentimposed epidemic-control strategies can significantly change individuals' contact patterns [17] , [23] , [24] , [30] . for example, during the outbreak of h1n1 flu in hong kong in 2009, interventions, such as flight reductions, school closures, and vaccination efforts, significantly altered individuals' contact interactions [27] [29] . dynamic contacts between individuals are more difficult to be observed and recorded than static contacts because of the limitations of existing contact tracing methods. offline and online questionnaires are incapable of recording real-time contact information, and usually time delayed to receive feedback from participants. automatic contact tracing methods such as mobile phone, wearable wireless sensor, rfid, and gps devices can collect continuous mobility information [53] , [60] . however, all these methods are limited to monitoring mobility behaviors for small-scale population, due to the large consumption of power, short range of positioning, high cost of money, etc. besides, most people cannot be expected to agree to have their dynamic contact interactions monitored in real time because of privacy issue. for example, wearing a tracker can also be equated to declaring one's self an infectious disease patient. tuberculosis patients in african countries are branded with social prejudice, making wearing an identifier a sensitive issue [6] . in light of these obstacles, a new path that does not ''directly'' capture and record individuals' dynamic contact behaviors, but ''indirectly'' infers the dynamic contact model of a large-scale population from other readily available data sources must be found. infectious disease surveillance, like that depicted in fig. 1 , expands everyday with the vast applications of information technology in the medical field. surveillance data record spatiotemporal information related to the spread of infectious diseases, which is the result of the spread model acting on the real contact network, as shown in fig. 5(a) . such surveillance data can be regarded as an external manifestation of the implicit contact network, suggesting that the dynamic contact network could be ''inversely'' inferred from infectious disease surveillance data, as shown in fig. 5(b) . essentially, this is a complex inverse engineering problem: using the observed dynamics phenomenon to determine the dynamic structure that leads to the phenomenon. in other words, determining time-dependent contact interactions using the timedependent spread trend of infectious diseases. based on the idea of inverse engineering, yang et al. [25] proposed a novel modeling and inference method for constructing a dynamic contact network based on tensor model. they described the spatiotemporal patterns of composite group contacts as a tensor, modeled the inference of the dynamic contact network as low-rank tensor reconstruction problem, and proposed a tensor deconvolution based inference method by fusing compression perception, sparse tensor decomposition and epidemic propagation models. this method makes it possible to determine the dynamic contact network of the large-scale composite group from population census data and surveillance data of many epidemic diseases. using this method, composite group dynamic contact networks for hong kong and taiwan were established using population census data and surveillance data of a variety of infectious diseases (such as h1n1, influenza, measles, mumps, etc.) for these two areas. the temporal and spatial evolution patterns of individuals' dynamic contact interactions were analyzed. based on the established dynamic contact network, they further studied the spread law, and prevention and control strategies of h1n1 epidemic disease. they arrived at two important conclusions: (1) in the h1n1 outbreak in hong kong in 2009, if the beginning of the new semester was delayed two to six weeks, the total number of infections would have been reduced by 10% to 25%; (2) the best strategy for prevention and control of h1n1 spread is vaccination of school-age children in the first few days of the new semester. contact tracking based on intelligent information processing technology represents an active prevention and control strategy for infectious diseases. its main functions are to achieve early detection and timely intervention of infectious diseases. research on contact tracking methods not only expands the options for preventing and controlling infectious diseases, but also further improves people's understanding of their own contact behaviors. contact tracking has become an increasingly mature datadriven technology for disease prevention and control, evolving from individual tracking to group tracking. individual tracking attempts to capture more detailed contact interactions for accurate locating of infected patients and high-risk susceptible populations. traditional offline questionnaire is a practical method for tracing private contact interactions between individuals such as sexual practice. however, it is quite costly and time delayed to find target participants and receive feedback from them. comparatively speaking, online questionnaire serves a low-priced way to collect feedback from participants timely. however, it cannot record the time exactly when contact occurs. meanwhile, offline and online questionnaires sometimes provide inaccurate information of human mobility. for example, klous et al. [46] surveyed 870 participants in a rural in the netherlands using questionnaire and gps logger, respectively. investigations on walking, biking, and motorized transport duration showed that time spent in walking and biking based on questionnaire was strongly overestimated. the use of automatic contact tracing methods enabled researchers to obtain continuous and accurate individual contact information, e.g., time, location, duration, etc. mobile phone and wireless sensors were widely used to trace mobility behaviors of students in campus and patients in hospital. then, small-scale contact network within the tracing regions can be constructed and the diffusion process of infectious volume 7, 2019 disease such as influenza can be simulated and analyzed in detail. however, the use of mobile phone is limited to tracing short-term contact behavior because of large power consumption of gps and bluetooth. wearable wireless sensors can only be applied to small-scale population due to its high cost and privacy concerns. rfid devices are convenient carrying which solves privacy concerns very well, but it only can be used for short range collection. gps device has the advantage of long-distance positioning. however, it is costly to capture indoor mobility behaviors due to the requirement of communication stations [57] . all these automatic contact tracing methods have not been used for studies of large-scale individual contact so far. group tracking replaces individual contacts with the contact probability of meta-populations, which, to some extent, overcomes the obstacles of individual tracking. using a contact matrix of meta-population, contact patterns regarding people with similar features can be depicted from the macroscopic level. however, the contact matrix is usually constructed using the data collected from a nationwide questionnaire, which is static and can only represent the contact patterns of the past. to explore dynamic contact patterns of meta-population, a data-driven ai (artificial intelligence) method was adopted, i.e., tensor deconvolution [25] . based on this method a dynamic evolutionary model of the group contact was constructed and dynamic contact patterns were inferred inversely through insights into the time-dependent nature of the infectious disease surveillance data. nevertheless, it should be noted that although it can characterize a wider range of dynamic contact behaviors, it cannot be used to accurately locate unique contact events because of the coarse granularity of the captured contact behaviors. exploring social contact patterns for epidemic prevention and control is an every promising research direction, and some potential future development directions are illustrated as follows. a. multi-view contact tracing data obtained from different views can give expressions to different patterns of mobility behaviors. for example, offline and online questionnaire can accurately record contact events occurred in places that individuals frequently visited [53] . gps devices can record indoor and outdoor contact events happened occasionally [54] . heterogeneous contact network constructed by various kinds of information can provide a new way for analyzing and simulating the spread of epidemics. therefore, tracing mobility behaviors and analyzing contact patterns from multi-views to get new insight into what heterogeneous contact patterns like will be a new direction in the future. existing studies focus on either individual level or group level contact tracing, presenting independent contact patterns from microscopic and macroscopic scales, respectively. however, group contact patterns are formed by collaborative behaviors of individual mobility, while individual mobility behaviors can be influenced by others in the same group. revealing hidden interactions between individual contact and group contact will be helpful to identify influential individuals as sentries for disease monitoring. therefore, discovering hidden interactions from multi-scale contact patterns that tunneling individual contact and group contact will be a new opportunity for early epidemic detection. dynamic mobility behaviors lead to complex contact patterns, which are usually hidden and cannot be directly traced by non-automatic or automatic methods. a better way to infer dynamic contact patterns is adopting ai-based methods using heterogeneous real-world data. existing studies such as tensor deconvolution consider the combination of contact probabilities within real-world social settings like school, home, and workplace as linear [25] . however, hidden dynamic contact patterns within these social settings could be more complicated than linear models can characterize. therefore, exploring advanced ai-based contact tracing methods, e.g., multi-view learning [61] [64] , deep learning [65] , broad learning [66] , etc., will be the next generation technology for epidemic prevention and control. in this paper, we introduced current studies on contact tracing and its applications in epidemic prevention and control. this paper covered 2 research directions, i.e., individual contact and group contact, which were introduced from both static and dynamic aspects. non-automatic tracing methods like offline and online questionnaires record static individual contact information, while automatic tracing methods like mobile phone, wearable wireless sensor, rfid, and gps devices collect dynamic contact events. static group contact patterns can be depicted by a coarse granularity contact matrix constructed by large-scale questionnaire data, dynamic contact patterns, however, can only be inversely inferred using data-driven ai technologies. both individual and group contact tracing are promising research directions and filled with challenges, especially for dynamic contact tracing. collecting contact data from multi-views and analyzing contact patterns from multi-scale mobility interactions will be new directions in the future. moreover, exploring advanced ai-based contact tracing methods using heterogeneous and multi-source data will provide new opportunities for epidemic prevention and control. hechang chen received the m.s. degree from the college of computer science and technology, jilin university, in 2014, where he is currently pursuing the ph.d. degree. he was enrolled in the university of illinois at chicago as a joint training ph.d. student from 2015 to 2016. his current research interests include heterogenous data mining and complex network modeling with applications to computational epidemiology. bo yang received the b.s., m.s., and ph.d. degrees in computer science from jilin university in 1997, 2000, and 2003, respectively. he is currently a professor with the college of computer science and technology, jilin university. he is currently the director of the key laboratory of symbolic computation and knowledge engineer, ministry of education, china. his current research interests include data mining, complex/social network modeling and analysis, and multi-agent systems. he is currently working on the topics of discovering structural and dynamical patterns from large-scale and time-evolving networks with applications to web intelligence, recommender systems, and early detection/control of infectious events. he has published over 100 articles in international journals, including ieee tkde, ieee tpami, acm tweb, dke, jaamas, and kbs, and international conferences, including ijcai, aaai, icdm, wi, pakdd, and asonam. he has served as an associated editor and a peer reviewer for international journals, including {web intelligence} and served as the pc chair and an spc or pc member for international conferences, including ksem, ijcai, and aamas. updating the accounts: global mortality of the 1918-1920 'spanish' influenza pandemic plasmodium ovale: a case of notso-benign tertian malaria the global burden of respiratory disease impact of the large-scale deployment of artemether/lumefantrine on the malaria disease burden in africa: case studies of south africa, zambia and ethiopia high-resolution measurements of face-to-face contact patterns in a primary school tracking tuberculosis in south africa the annual impact of seasonal influenza in the us: measuring disease burden and costs updated situation of influenza activity in hong kong little italy: an agent-based approach to the estimation of contact patterns-fitting predicted matrices to serological data the tencent news what types of contacts are important for the spread of infections? using contact survey data to explore european mixing patterns estimating within-school contact networks to understand influenza transmission reality mining: sensing complex social systems time-critical social mobilization capturing individual and group behavior with wearable sensor fluphone study: virtual disease spread using haggle epimap: towards quantifying contact networks and modelling the spread of infections in developing countries a high-resolution human contact network for infectious disease transmission collective dynamics of small world networks close encounters in a pediatric ward: measuring face-toface proximity and mixing patterns with wearable sensors computing urban traffic congestions by incorporating sparse gps probe data and social media data modeling human mobility responses to the large-scale spreading of infectious diseases modelling dynamical processes in complex socio-technical systems social contacts and mixing patterns relevant to the spread of infectious diseases characterizing and discovering spatiotemporal social contact patterns for healthcare outbreaks in realistic urban social networks skip the trip: air travelers' behavioral responses to pandemic influenza the effect of risk perception on the 2009 h1n1 pandemic influenza dynamics quantifying social distancing arising from pandemic influenza behavioral responses to epidemics in an online experiment: using virtual diseases to study human behavior an evaluation of an express testing service for sexually transmissible infections in low-risk clients without complications hiv/aids: 30 years of progress and future challenges reflections on 30 years of aids prevalence and patterns of same-gender sexual contact among men hiv-1, sexual practices, and contact with foreigners in homosexual men in colombia, south america prevalence of unsafe sexual behavior among hiv-infected individuals: the swiss hiv cohort study sexual behavioral abstine hiv/aids questionnaire: validation study of an iranian questionnaire study on the risk of hiv transmission by heterosexual contact and the correlation factors a survey of patients with chlamydia trachomatis infection: sexual behaviour and perceptions about contact tracing transmission of zika virus through sexual contact with travelers to areas of ongoing transmission-continental united states zika virus was transmitted by sexual contact in texas, health officials report adolescent sexual behavior, drug use, and violence: increased reporting with computer survey technology insights from flutracking: thirteen tips to growing a web-based participatory surveillance system flutracking: a weekly australian community online survey of influenza-like illness in flutracking weekly online community survey of influenza-like illness annual report mobility assessment of a rural population in the netherlands using gps measurements generating gps activity spaces that shed light upon the mobility habits of older adults: a descriptive analysis what can global positioning systems tell us about the contribution of different types of urban greenspace to children's physical activity? out and about: association of the built environment with physical activity behaviors of adolescent females a study of community design, greenness, and physical activity in children using satellite, gps and accelerometer data the accuracy of fastloc-gps locations and implications for animal tracking tracking the cheetah tail using animal-borne cameras, gps, and an imu examining the spatial congruence between data obtained with a novel activity location questionnaire, continuous gps tracking, and prompted recall surveys using global positioning systems in health research: a practical approach to data collection and processing mobile sensing in environmental health and neighborhood research feasibility and acceptability of global positioning system (gps) methods to study the spatial contexts of substance use and sexual risk behaviors among young men who have sex with men in new york city: a p18 cohort sub-study strengths and weaknesses of global positioning system (gps) data-loggers and semi-structured interviews for capturing fine-scale human mobility: findings from iquitos using mobile phone data to study dynamics of rural-urban mobility inferencing human spatiotemporal mobility in greater maputo via mobile phone big data mining gps tracking in neighborhood and health studies: a step forward for environmental exposure assessment, a step backward for causal inference? multi-view clustering with graph embedding for connectome analysis a self-organizing tensor architecture for multi-view clustering mmrate: inferring multi-aspect diffusion networks with multi-pattern cascades inferring diffusion networks with sparse cascades by structure transfer partially observable reinforcement learning for sustainable active surveillance broad learning: an emerging area in social network analysis key: cord-017887-pj6pal35 authors: ouyang, bo; dong, ying; chou, james j. title: structural and functional properties of viral membrane proteins date: 2018-06-29 journal: advances in membrane proteins doi: 10.1007/978-981-13-0532-0_6 sha: doc_id: 17887 cord_uid: pj6pal35 viruses have developed a large variety of transmembrane proteins to carry out their infectious cycles. some of these proteins are simply anchored to membrane via transmembrane helices. others, however, adopt more interesting structures to perform tasks such as mediating membrane fusion and forming ion-permeating channels. due to the dynamic or plastic nature shown by many of the viral membrane proteins, structural and mechanistic understanding of these proteins has lagged behind their counterparts in prokaryotes and eukaryotes. this chapter provides an overview of the use of nmr spectroscopy to unveil the transmembrane and membrane-proximal regions of viral membrane proteins, as well as their interactions with potential therapeutics. we will focus our discussion on two classes of transmembrane (tm) proteins encoded by viruses, viroporins (or viral channels) and membrane fusion proteins, as these proteins have been sought after as antiviral targets and they often exhibit peculiar structural features not seen in other membrane proteins. as implied by their name, the viroporin proteins can form channel-like structures in lipid bilayer that permeate ions or solutes. now over a dozen viroporins from various sources have been characterized, covering a wide range of ion substrates including h + , k + , na + , ca 2+ , and cl − , as well as larger substrates such as rna. the exact function of ion channel activity in many viruses is not yet known, though their roles have been implicated in entry, virus assembly and virus release. the function of membrane fusion proteins is much better defined, that is, to enable viral entry by mediating virus-host membrane fusion. the viral fusion proteins have large extramembrane domains for receptor recognition and for grabbing onto the host cell membrane, but the function of their tm and membrane-proximal regions have been elusive. functional mutagenesis studies have suggested that, at least in the cases of hiv-1 and influenza a viruses, the tm domains (tmds) of fusion proteins are not merely membrane anchors, but play important roles in membrane fusion and viral infectivity. apart from the channels and fusion proteins, some viruses have developed enzymatic domains anchored to the membrane, e.g., the polymerases of the hepatitis c virus and the neurominidase of the influenza viruses. in these cases, the tmds are believed to only serve as membrane anchors and thus will not be discussed in this chapter. viroporins serve highly diverse functions in viral infection cycles (illustrated in fig. 6.1 ). early studies of viroporins have focused on only a few systems including 2b of poliovirus, 6k of togavirus, and m2 from influenza a virus (am2). the 2b is a well-known viroporin that can modify membrane permeability and allow the passage of ions and small molecules during later stages of viral infection [1] [2] [3] . the 6k is a small, hydrophobic acylated protein that has been shown to form cation-selective channels when inserted into lipid bilayers [4] . the 6k is proposed to be involved in virus budding, but the exact role of 6k in the viral life cycle is poorly characterized [5] [6] [7] . the am2 proton channel is the most studied viroporins because (1) it is the drug target of the first anti-flu drug (amantadine or rimantadine) and (2) it is one of the smallest proton channels with proton selectivity motif not found in any of the proton channels from other organisms [8] [9] [10] . since the definition of viroporin has been put forward, more and more viroporins have been classified (table 6 .1). the influenza b virus encodes the bm2 proton channel that is a functional homolog of the am2 but cannot be blocked by the adamantane family of drugs [11] . the human immunodeficiency virus type 1 (hiv-1) vpu and the hepatitis c virus (hcv) p7 have been shown to form oligomeric channels to conduct cations [12] [13] [14] [15] [16] . the 2b protein from the enterovirus 71 (ev71), however, displayed altered channel activity, as it conducts anions (e.g., cl − ) instead of cations [17] . paramecium bursaria chlorella virus 1 (pbcv-1) has a viral k + channel named kcv that contains the canonical potassium selectivity filter [18] and was proposed to adopt similar structure as kcsa [19] . in addition to the ion channels, the vp4 from rhinovirus can form pores that (1) . virion attaches directly host cell receptors on the membrane (2) . and subsequently enters the cell by receptor-mediated endocytosis (3) . acidification of the endosomal vesicles triggers conformational changes in the virion, resulting in fusion between the viral and the endosomal membranes, allowing the release of the nucleocapsid into the cytoplasm. the viral rna is then released into the cytoplasm and presented to the endoplasmic reticulum (er) (4) . at the er, viral rna is translated into protein that is processed by viral and host proteases (5) . after the viral replication complex is synthesized, rna synthesis begins by the transcription of an antisense viral rna followed by the amplification of viral rna (6) . the newly synthesized rna is subsequently packaged by capsid protein, forming a nucleocapsid (7) . virus assembly occurs on the surface of the er when the nucleocapsid buds into the er lumen, then transported through the golgi, where acidification induces virus maturation (8) . mature viral particles are released in the neutral ph of the extracellular millieu (a) viral entry, which acidifies the virion interior immediately after endocytosis and facilitates rna release (b) dissipation of the proton gradient in the golgi and the trans-golgi network. the viroporins influenza a virus (iav) matrix protein 2 (m2) and hepatitis c virus (hcv) p7 equilibrate the proton concentration with the cytosol to reduce the acidification of vesicular acidic compartments (c) viroporins play an essential role in assembly, budding and release of the viral particles (d) alteration of cellular ca 2+ homeostasis. calcium uptake by the mitochondria can lead to dissipation of the inner-mitochondrial-membrane potential (δψm), permeabilization of the outer mitochondrial membrane and, finally, the release of cytochrome c. in the cytosol, cytochrome c promotes the subsequent formation of apoptosome that is involved in apoptosis allow single-stranded viral rna to cross the membrane [20, 21] , further expanding the list of substrates that viroporins transport. probably the best-defined roles of viroporins are that of the m2 proton channels of influenza a and b viruses, both of which involve equilibrating ph between compartments. one important role of the m2 is to equilibrate ph across the viral membrane during viral entry, which acidifies the virion interior immediately after endocytosis and facilitates rna release [22, 23] (fig. 6.1a ). another role of m2 is to equilibrate ph across the trans-golgi membrane of the infected cells during viral maturation, which preserves the pre-fusion form of the hemagglutinin fusion protein through de-acidification of the golgi lumen [24] (fig. 6.1b) . the roles of other cation-selective viroporins are less clear. one of the general roles proposed for viroporin is to depolarize either the er or plasma membrane to facilitate membrane curvature formation during virus budding [25, 26] (fig. 6.1c ). in the case of hcv, for example, the p7-mediated channel activity has been reported to facilitate virus release [27, 28] . another general role for viroporin is simply to cause cation leakage, which would induce cellular stress and programmed cell death [29] (fig. 6.1d ). in addition to ion permeation, several viroporins are known to participate in viral assembly through interaction with other proteins. for example, the influenza am2 and bm2 proteins both have significant cytoplasmic domains that are believed to recruit matrix proteins m1 during virus assembly [30] [31] [32] . the hcv p7 protein has also been reported to interact with other non-structural proteins such as ns2, and this interaction appears to be crucial for the production of infectious hcv particles [33, 34] . pore formation indicated but a defined oligomeric state either does not exist or is not characterized enveloped viruses infect host cells by the fusion of viral and cellular membrane. depending on the type of the virus, fusion can occur either with the host plasma membrane or with the endosomal membrane. the membrane fusion must occur without consuming energy; it is catalyzed by major conformational changes of specialized envelope proteins generally known as viral fusion proteins. despite their diversity, all known viral fusion proteins convert from a prefusion state (dimers or trimers, depending on the class), through a key intermediate state (extended conformation), to a postfusion state (a trimer of hairpins) that brings the fusion peptide, attached to the target membrane, and the tm domain, attached to the viral membrane, into close proximity thereby facilitating the fusion of viral and target membranes ( fig. 6 .2a) [35] . three distinct classes of viral fusion proteins (exemplified in fig. 6 .2b) have been defined based on structural criteria [36] . the class i includes fusion proteins from some of the best characterized human pathogens, such as influenza and hiv-1. these proteins are trimers of a single chain precursor that requires a proteolytic cleavage to generate two fragments. for example, the influenza a hemagglutinin (ha) contains the n-terminal fragment ha1 and c-terminal fragment ha2 [37] , and the hiv-1 envelope protein is cleaved into the gp120 and gp41 fragments [38] . the fusogenic fragment gp41 bears a hydrophobic fusion peptide capped by the outer receptor binding fragment gp120 [39] . receptor binding accompanies cap protein release, allowing major conformational changes in the gp41 that ultimately leads to fusion [40] . in the case of influenza, the cap ha1 is released by low ph of the endosome after endocytosis [41] . the class ii fusion proteins are found on flaviviruses, alphaviruses, and bunyaviruses (e in flaviviruses, e1 in alphaviruses, gn and gc in bunyaviruses) [42, 43] . although these fusion proteins are architecturally different from the class i fusion proteins, i.e., class ii fusion proteins are mostly made of β-sheets as opposed to the helical structures of the class i fusion proteins, they operate on the same physical principle. their fusion peptides are formed as loops at the tips of β-strands that serve as an anchor to insert into the host membrane after endocytosis. unlike class i proteins, which remain trimeric, their conformational change involves a change in oligomeric state from pre-fusion dimers to post-fusion trimers [44, 45] . the reduced ph of the endosome induces a cascade of subsequent refolding and trimerization events that achieve membrane fusion [46] . members of the class iii include g protein of vsv, gb of herpes simplex virus and epstein-barr virus (ebv), and gp64 of the insect-cell baculovirus [47] . the class iii fusion proteins combine some of the features of class i and ii fusion proteins [48] . each protomer is composed of five domains that contain a central trimeric coiled-coil, a hallmark of class i proteins in their postfusion states, three of the domains are predominantly made of β-sheets and their fusion peptide more closely resemble those of class ii proteins in that their fusion loops are found at the tips of extended β-strands [49] . due to the essential roles of viral fusion proteins during infection, they have long been pursued as targets for antiviral intervention. for example, the approved small molecule drug arbidol is used as a broad-spectrum inhibitor of influenza a and b virus, as well as hepatitis c virus [50, 51] . unlike many other broad-spectrum antivirals, arbidol has an established mechanism of action against the has in influenza a and b viruses that involves the inhibition of virus-mediated membrane fusion and thus viral entry [50] . in addition, enfuvirtide (t20, fuzeon) is an approved peptide drug that blocks the hiv-1 entry [52] ; it is derived from the c-terminal heptad repeat 2 region of the hiv-1 gp41 envelope glycoprotein, designated the c-peptide, that disrupts membrane fusion by competing with an intra-molecular interaction that is important for the refolding of gp41 during membrane fusion [53] . apart from being therapeutic targets, another important medical use of the fusion proteins is vaccine development. viral fusion proteins are usually presented on the virion surface with high copy numbers, and thus are popular antigens for eliciting broadspectrum neutralizing antibodies. for example, the native conformations of the the n terminus of ha1 and the c-terminus of the ha2 ectodomain are labeled. blue arrow: position of fusion peptides inserted near threefold axis in prefusion form. only ha2 is shown on the bottom. the n-terminus (blue arrow; note that the fusion peptide is not part of the structure shown) and c-terminus of the blue-colored subunit are indicated class ii: dengue virus type 2 e protein (1oan and 1ok8). the radial ("top") view shows just the "stem-less" ectodomain (1oan). colors: domain i: magenta; domain ii: cyan; domain iii: yellow; stem: cyan; colors for domains i, ii and iii are the same in the postfusion representation. a dashed cyan arrow on the postfusion trimer shows where the stem emerges from domain iii. red asterisks: fusion loops class iii: vsv g ectodomain (2j6j and 2cmz). the three chains are in gold, sea green, and magenta. dashed lines show the location of a disordered, c-terminal segment that connects the folded protein to the transmembrane anchor. only the magenta-subunit c-terminal segment is shown on the bottom. the curved gold arrow indicates that in the transition from the conformation on the top to the conformation on the bottom, the domain bearing the fusion loops flips over by about 180° to engage the host-cell membrane. red asterisks: fusion loops hiv-1 envelope glycoprotein have being pursued intensely as immunogens for b-cell based vaccine development [54, 55] . for most single-pass tm proteins, the role of their tmds beyond membrane anchoring is unresolved, partly because of the difficulty in structural studies: they are notorious for resisting crystallization and due to their small sizes, unfeasible for visualization by cryo-electron microscopy (em). there have been only a few examples of crystal structures of the small tmds, including the tmd of the am2 [56, 57] and the more recent crystal structure of the tm helix dimer of the glycophorin a protein [58] . as cryo-em is rapidly approaching atomic resolution, it is becoming increasingly used to examine the structures of the much larger viral fusion proteins containing the tmd. most notably, a recent cryo-em study of the hiv-1 envelope glycoprotein (env) including the tmd achieved a high resolution structure of the prefusion state of the hiv-1 env [59] . this study, however, showed that the tmd and the membrane-proximal regions of the env are disordered, possibly due to incompatibility between the membrane-associated regions of the env and the detergent used to solubilize the env. in this chapter we mainly focus on the use of nmr to study the tm and membrane-proximal regions of viral membrane proteins, including methods for structure determination and for investigating drug binding. the first challenge of nmr studies is preparing sufficient quantities of isotope labeled proteins and this is not trivial for small and hydrophobic proteins. a robust approach is to force highlevel expression of the hydrophobic peptides, which are toxic to cells, into inclusion bodies. in our experience, the most effective implementation of this approach is to express the peptide as a c-terminal fusion to the trple protein (which drives inclusion body formation) in the pmm-lr6 vector [60] [61] [62] (fig. 6 .3a). the trple has an n-terminal 9×his tag to permit nickel affinity purification. a methionine between the trple and the peptide is added for cleavage by cyanogen bromide so that the peptide can be released from the trple. purification of the peptide involves (1) purifying the trple-peptide fusion protein from the inclusion bodies by nickel affinity, (2) cleavage by cyanogen bromide of the fusion protein in formic acid, and (3) separation of the peptide from trple or fusion protein by reverse-phase hplc. while the above protocol is quite general for hydrophobic peptides, it requires that the peptides have no methionine in the middle of the peptide sequence. if the native methionines cannot be mutated, an alternative method is to use soluble fusion domains (e.g., the maltose binding protein) with specific protease cleavage site for releasing the peptide. the purified hydrophobic peptides can be reconstituted in any membrane mimetic media, including detergent micelles, bicelles, and lipid nanodiscs. while nanodiscs are the closest mimic of native membrane, we find that the lipid/detergent bicelle system is a good compromise between having the capacity to provide a near lipid bilayer environment and generating good nmr spectra. previous studies on the bicelle system with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dmpc) as lipid and (1,2-dihexanoyl-sn-glycero-3-phosphocholine (dhpc) as detergent have shown that when the molar ratio of lipid to detergent (q) > 0.5, the assembly reaches the ideal bicelle condition in which the lipid and detergents are well segregated [63, 64] . at q = 0.5, for example, the estimated diameter of the lipid bilayer region of the bicelle disc is ~45 å according to the equation describing bicelle assembly [64] [65] [66] , and this size is sufficiently large to accommodate small tmds. remarkably, even bicelles of such size could generate nmr spectra of sufficiently high quality to enable full-scale structure determination, as was demonstrated for the structure fig. 6 .3a schematic of hydrophobic peptide sample preparation using hcv p7 as an example 1. trple plasmid with targeted peptide is transformed into and bl21(de3) competent cells for expression 2. the cells were grown in m9 media 3. cells were collected by centrifugation and lysed by sonication 4. after lysis, the inclusion bodies and membranes were collected by centrifugation and dissolved in 6 m guanadine buffer. insoluble aggregates were removed by centrifugation. the supernatant was then subjected to ni-nta purification. 5. the protein was eluted using 400 mm imidazole 6. cyanogen bromide cleavage followed by dialysis in water 7. the protein was further separated by reverse-phase hplc or ni-nta 8. the lyophilized peptide was then dissolved in detergent and refolded by dialysis against the nmr buffer 9. nmr experiments were performed at spectrometers determination of the trimeric tmds of the fas death receptor [67] and hiv-1 env [68] . here we provide an example of bicelle reconstitution from a previous study on the tmd of hiv-1 env [68] . the hydrophobic peptides (lyophilized) are first completely dissolved in strong organic solvent (e.g., hexafluoro-isopropanal) with calculated amount of dmpc. the solution is slowly dried to a thin film under nitrogen stream. the dried film is then dissolved in 8 m urea containing calculated amount of dhpc. the denaturant is removed by dialysis, during which the dmpc: dhpc ratio is monitored by 1d nmr, and the loss of dhpc during dialysis is compensated by further addition of dhpc. owing to the small size of most of the tmds of viral membrane proteins, structure determination by nmr is normally straightforward. probably the biggest challenge is measuring inter-protomer restraints in cases of oligomer complexes. in particular, structure determination of symmetric oligomers faces the challenge of measuring noe-based 1 h-1 h distances between structurally equivalent protomers having the same nmr peaks, which are needed as inter-protomer restraints. a proven way to solve this problem is using a mixed sample in which half of the protomers are ( 15 n, 2 h)-labeled and the other half 13 c-labelled, for measuring, exclusively, noes between the 15 n-attached 1 h of one protomer and 13 c-attached 1 h of the neighboring protomer [60, 69] (example from the tmd of the fas receptor shown in fig. 6 .3b). once the trimer solution is established, conventional 15 n-and 13 c-edited noe experiments can then be used to collect self-consistent backbone-sidechain and sidechain-sidechain noe restraints. in addition to structure determination, an important aspect of small tmds is interaction with membrane. the bicelle system allows accurate determination of the position of tmd in the bilayer region of the bicelles using a solvent paramagnetic relaxation enhancement (pre) analysis [63] . in this method, titration of the water-soluble and membrane-inaccessible paramagnetic agent, gd-dota (gadolinium (iii) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate), is used to generate the solvent pre. this approach is based on the notion that if the bicelle is sufficiently wide, the lateral solvent pre becomes negligible, thus allowing the use of measurable solvent pre to probe residue-specific depth immersion of the protein along the bicelle normal. at each titration point, a 2d 1 h-15 n correlation spectrum is recorded to measure residue-specific pre. for each of the residues, the pre titration curve is fitted to exponential decay to derive the residue-specific pre amplitude (pre amp ). the pre amp vs. (residue position) along the trimer symmetry axis (parallel to the bilayer normal) is then analyzed using the sigmoidal fitting method [63] to determine the tmd region that resides at the center of the bilayer as well as the thickness of the bilayer around the protein. for studying small molecule drug binding to viral membrane proteins, chemical shift perturbation (csp) induced by drug titration is a convenient probe for identifying the approximate binding site. but, cautions need to be taken due to complications associated with membrane-mimetic media. first, many small molecule inhibitors are hydrophobic and preferentially partition into detergent micelles or lipid/detergent bicelles, and thus the csp can be induced simply by alteration of the micelle/bicelle environment. second, in addition to csp caused by close ligand contact, csp could arise from changes in conformation and/or dynamics. hence, the more direct noe data is preferred wherever feasible. the simplest and the most sensitive approach for collecting protein-drug noe is using proteins that are 15 n-labeled and completely deuterated so that noe between the protein backbone amide protons and drug protons could be measured unambiguously (fig. 6.3c ). once the binding site has been unambiguously spotted, further contacts between the drug and protein sidechains can be measured using conventional 13 c-edited methyl or aromatic noesy. details of these experiments have been described in studies that identified the drug binding sites in the influenza am2 and hcv p7 channels [60, 70, 71] . structural studies of viroporins have been challenging because these small membrane proteins are typically dynamic and very hydrophobic. in the past 10 years, multiple biophysical techniques including solution nmr, solid-state nmr, x-ray crystallography, and em have been used to gradually fill the structural gaps. taking the influenza am2 for example, there is now structural information in crystal and solution states, in lipid bilayer, under different phs, and bound to different small molecules. these complementary structural data allow elucidation of the functional mechanism from different view angles. the am2 of influenza a and the bm2 of influenza b are 97-and 109-residues single-pass membrane proteins, respectively, that form homotetramers in membrane [72] [73] [74] [75] [76] . the sequences consist of three domains: an extracellular n-terminal domain, a transmembrane domain (tmd) and an intracellular c-terminal domain. these domains arrange into different structures. the only homologous sequence between the two proteins is the hxxxw sequence motif of the tmds that is essential for channel activity. the tm region of the am2 contains residues 24-46. the unstructured extracellular segment of am2 is relatively conserved and has been sought after as a vaccine epitope [77] [78] [79] [80] [81] . bm2 has a similar sized tmd (residues 4-33), but a much larger c-terminal cytoplasmic domain [32, 82] . the bm2 cytoplasmic region also assembles into oligomers which are important for recruiting the matrix proteins to the cell surface during the viral assembly [30, 31, 83, 84] . the first high-resolution structures of the am2 channel were reported concurrently by solution nmr spectroscopy and x-ray crystallography in 2008 [56, 62] . subsequent x-ray and solution nmr studies also determined structures under different conditions [57] and with different drug resistance mutations [85, 86] . moreover, structural characterization by solid-state nmr studies generated the channel structures in the lipid bilayer environment [87] [88] [89] . the above am2 structures solved under different conditions show significantly different conformations ( fig. 6.4a) , suggesting that the tetrameric assembly of the am2 is quite dynamic and is sensitive to the reconstitution environment. the structural plasticity observed in am2 is likely present in many other viral membrane proteins, which could explain why structural study of this type of proteins has been so difficult. despite the differences, the experimental structures converged to a common mode of channel assembly: a left-handed four-helix bundle forms the channel pore, and that tetramerization of the four tm helices is further stabilized by intermolecular contacts between c-terminal amphipathic helices flanking the tmd [62, 88] . this mode of assembly places the "h" and "w" of the hxxxw sequence motif inside the channel (fig. 6.4a) . four imidazole rings of his37, which are ph-gating features and are essential in transporting protons, are packed closely inside the pore (fig. 6.4b) . moreover, packing of the trp41 indoles creates a channel gate, which occludes the c-terminal end of the pore. the structure of the bm2 channel was solved using solution nmr methods [32] . although the overall assembly of the bm2 tmd is similar to that of the am2 tmd, i.e., both show left-handed helical packing, the two differ substantially in details ( fig. 6.4c) . unlike the am2, the bm2 channel shows strong coiled-coil characteristics with regular heptad repeats [8] . in the bm2 structure, the two heptad repeats show that positions a and d are occupied mostly by hydrophilic residues such as serines and his19; positions g and e are occupied by hydrophobic leucines and phenylalanines, respectively, to allow for peripheral hydrophobic interactions between the leucines and phenylalanines (fig. 6.4d ). this arrangement for coiled-coil assembly in membrane allows the tm segment to form a stable tetramer by itself and is the opposite to that of water-soluble coiled-coil tetramer, in which positions a and d are typically hydrophobic residues and positions g and e are polar residues [90] . the histidine (his19) and tryptophan (trp23) of the hxxxw motif are also pore-lining in the inverse coiled-coil assembly in membrane. the cytoplasmic domain of the bm2 also oligomerizes into a left-handed coiled-coil tetramer, but it is water-soluble and shows strong bipolar distribution in the surface charges. this charged domain supports the interaction with the m1 matrix protein [32] . the structural arrangement of the histidine and tryptophan inside the pore of the am2 and bm2 suggests that the two residues play an essential role in the channel selectivity and channel gating (fig. 6.4e ). functional mutagenesis [91] and nmr measurements [92] showed that proton transport across the membrane through the am2 channel involves cycles of histidine protonation and deprotonation, and that the histidines serve as proton shuttling devices. not all histidines can be protonated at the same time in the narrow channel, as protonation of one histidine would increase the energy barrier for the protonation of another histidine. indeed, multiple pka values (8.2, 6.3 and one below 5.0) have been detected in the am2 [91, 93] . therefore, our current understanding of the proton conduction mechanism is that the minimally required unit for ph-dependent proton conduction in the am2 and bm2 is the his-trp complex. it was proposed in ref. [93] that in the non-conductive state two pairs of histidines in the tetramer each share one proton, which explains structures of the influenza proton channels and mechanism of proton conduction. the many structures of the influenza m2 channel. the pdb codes 2rlf and 2kwx represent the solution nmr structures of the wildtype and the v27a mutant determined using residues 18-60. the 3c9j and 3lbw are crystal structures of the tm domain (residues 22-46) determined at ph 7.3 and ph 6.5, respectively. the structures 2l0j and 2kqt were obtained using solid-state nmr using protein constructs that encompass residues 22-62 and residues 22-46, respectively the high pk a ~8.2. lowering ph results in the protonation of the third histidine from the n-terminal side, which, in turn, disrupts the two histidine dimers and leads to the proton conductive state. it was also proposed that protonation of histidines leads to cation-π interactions between the histidine and tryptophan [94, 95] . the remaining question to be addressed is how does the third protonation affect the tryptophan conformation, allowing proton to be relayed to the c-terminal side of the tryptophan gate [91] . the viroporin p7 encoded by the hcv genome is a 63-residue protein that oligomerizes in membrane to form cation-selective channels [15, 16] , with higher selectivity for ca 2+ than k + /na + [96, 97] . the channel activity of p7 is important for the assembly and release of infectious viruses, although the molecular mechanism of this function remains unknown [27, 28] . as in the case of am2, structural characterization of p7 was confronted with challenges of coping with the hydrophobic and dynamic nature of the protein. earlier nmr studies of p7 under conditions that support the monomeric state of the protein showed that p7 has three tm helical segments: two in the n-terminal half of the sequence and one near the c-terminus [97, fig. 6 .4b isolated view of the pore-lining histidine (magenta) and tryptophan (cyan) sidechains in m2 and bm2 channels. images are from the high resolution crystal structure (3lbw) and nmr structure (2rlf) of m2 and nmr structure of bm2 (2kix) 98] . although the monomeric state should not conduct ions, it could be involved in interacting with the ns2 protein during virus assembly [33, 34] . the oligomeric form of p7 was first examined using single-particle em, which showed that p7 from hcv genotype 2a (jfh-1 strain) assembles into hexamers in 1,2-diheptanoylsnglycero-3-phosphocholine (dh 7 pc) micelles and the complex adopts a flowerlike shape that does not resemble any of the known ion channel structures in the database [99] . later, a more detailed structure of the p7 hexamer was determined by solution nmr using p7 from genotype 5a (euh1480 strain) reconstituted in dodecylphosphocholine (dpc) micelles [60] . consistent with the flower-shaped em images, the nmr structure shows a funnel-like architecture with six minimalist chains, each containing three helical segments: h1, h2, and h3. the h1 and h2 form the narrow and wide regions of the funnel-shaped cavity, respectively, and the h3 helices wrap the channel peripheral by interacting with h1 and h2 (fig. 6.5a) . the assembly strategy adopted by p7 differs significantly from those known channels from bacteria and eukaryotes. ion channels typically have two essential features: (1) pore elements that support selective ion dehydration; (2) a gate or constriction that prevents non-specific permeation, but can open in response to regulating factor such as ph, voltage, ligand, or the ion of selection itself [100, 101] . the channel interior of p7 has a number of strongly conserved residues that are likely candidates to serve the above functions. one suspect is asn9, which forms a ring of carboxamide near the narrow end of the channel (fig. 6.5b) . residue 9 is asparagine in all strains except being substituted with histidine in genotype 2 viruses. formation of a ring of carboxylates or carboxamides has been a recurring theme in prokaryotic and eukaryotic channels that have selectivity for divalent cations. for examples, the cora mg 2+ channel has a pentameric ring of asparagines [102] , and the calcium release-activated calcium (crac) channel orai has a hexameric ring of aspartic acids [103] . these channels all have strong selectivity for divalent cations, although they can also conduct monovalent cations such as na + and k + . in addition to the asn9 ring, residues near the hinge between h1 and h2 (ser12, asn16, trp21) are in an arrangement that may also bind in addition to what appears to be the cation selectivity ring near the narrow, n-terminal exit of the channel, the wider, c-terminal entrance of the channel is decorated with a conserved ring of arginines or lysines (fig. 6.5b) . placement of a positively charged ring at the entrance was anticipated because it may repel cations. but an earlier study reported that a designed tm barrel with internal argininehistidine dyads forms efficient cation selective channels [104] because the immobile arginines can recruit mobile anions, which in turn facilitate cations to diffuse through the pore. it is interesting to note that a highly basic region containing arg155, lys159 and lys163 in the pore was also found in the crac orai structure [103] . one possible role of these basic residues in cation selective channels is binding and obstructing anions while allowing cations to diffuse into the pore. this mechanism would be consistent with the observation that replacing arg35 with negatively charged aspartic acid largely abrogated conductance [60] . there are still many unanswered questions. does the nmr structure, solved in the absence of ca 2+ and inhibitors, represent the open or closed state of the channel (if the two states exist)? how strong is the ca 2+ selectivity of p7? or was p7 developed as a general, unspecific cation channel for the purpose of dissipating membrane potential? for example, another recently study reported p7 activity in dissipating proton gradients within cell membrane compartments [105] . it is unclear what ion flux mediated by p7 plays a dominant role in the hcv life cycle. from a structural perspective, the funnel-like architecture is formed with multiple helical segments connected by hinges and short loops and we believe this flexibility can afford the dynamic opening and closing of the tip of the channel. probably the most intriguing aspect of small molecule interaction with viroporin is the finding that the adamantane derivatives amantadine and rimantadine have inhibitory effect on multiple viroporins including the influenza am2 and hcv p7. amantadine (symadine) or rimantadine (flumadine) was the first licensed drug for treating influenza infections [106] . in fact, the compound also played critical roles in the early days of functional characterization of the am2 channel [107] [108] [109] . the bm2 channel is a functional and structural homolog of am2 but is not sensitive to the adamantane family of drugs [11] . remarkably, the hcv p7 channel structure is completely different but showed detectable, though not strong, sensitivity to rimantadine [15, 110] . the mechanism of how amantadine/rimantadine inhibit the am2 channel has been elusive for quite some time. previous confusion came mainly from the multiple binding sites that have been observed experimentally. in a crystallographic study of the am2 tmd (residues in the presence of amantadine, the drug density was found inside the channel near residue ser31, but at structural resolution of 3.5 å, it was difficult to confirm the position of amantadine binding [56] . at the same time, however, a solution nmr study of a longer version of am2 (residues showed that rimantadine binds to an external, lipid-facing pocket around residue asp44 between adjacent tm helices [62] . the two different binding sites obviously suggest very different mechanisms of inhibition. one is the drug directly blocking the channel passage, and the other is the drug binding to the external site allosterically favors the closed state of the channel. subsequent solid-state nmr measurements of the am2 in lipid bilayer showed that both sites exist, with higher affinity for the internal site [87] . the strongest evidence that the internal site is the primary site of drug action came from functional studies using an am2-bm2 chimera protein [111, 112] . in this study, the authors constructed a chimera of m2 variants from influenza a and b viruses that contains only internal site showed that the chimera channel is still amantadine sensitive, indicating that the internal site is the primary site of drug inhibition. later, the complex structure of the tmd of the chimera with rimantadine was determined by solution nmr, providing a detailed view of the drug binding inside the channel pore [70] . the drug binding site consists of eight methyl groups of the m2 tetramer (two from each subunit: val27 c γ1 h 3 and ala30 c β h 3 ) that form a deep internal hydrophobic pocket surrounding the adamantane cage of the drug (fig. 6.6a ). the structure also shows that the nitrogen of the rimantadine amino group may form a hydrogen bond with the backbone carbonyl oxygen of ala30 of one of the four subunits. the terminal methyl group of the rimantadine is in the middle of the pore, facing the open space in the channel around the gly34 position. the structure also shows that rimantadine binding is slightly tilted: its vertical axis is on average ~20° from the c4 symmetry axis of the channel. the tilt angle is consistent with the amantadine tilt in the m2 channel observed with solid-state nmr spectroscopy [87] . in addition to blocking the am2 channel, the amantadine and its derivatives have also been shown to pose some inhibitory effects on the p7 channel conductance [15, fig. 6.6 the amantadine and rimantadine binding sites in the m2 and p7 channels (a) the precise nmr structure of the am2-bm2 chimeric channel with rimantadine determined in dhpc micelles and at ph 7.5. left panel: detailed illustration of the methyl groups (in green) that interact with the adamantane cage. right panel: surface representation for showing the hydrophobic pocket that fits the drug snuggly. one of the four subunits is omitted for drug visibility (b) the amantadine or rimantadine binding site of the p7 channel determined by nmr in dpc micelles and at ph 6.5. left panel: the drug binds to six equivalent hydrophobic pockets of the p7 channel. right panel: a close view of amantadine docked into the binding pocket as determined using nmr noe restraints (c) comparison between adamantane binding sites of influenza m2 and hcv p7 channels top: the internal pocket that wraps around rimantadine in the am2-bm2 chimeric channel. the am2-bm2 chimeric channel is a well-behaved model system wth its n-terminal half is from influenza a m2 protein (sensitive to amantadine or rimantadine inhibition) and its c-terminal half is from influenza bm2 protein (insensitive to amantadine or rimantadine). on the right panel, one subunit of the tetrameric complex is removed to unveil the channel interior bottom: amantadine binds to the peripheral pockets between the h2 and h3 helices; and a representative pocket among six equivalent pockets in the p7 hexamer 113 ]. the physical binding sites of amantadine and rimantadine have been identified in p7 of genotype 5a using intermolecular noe experiments [60] . the noe data revealed that amantadine or rimantadine binds to six equivalent hydrophobic pockets (due to the six-fold symmetry of the p7 channel) between the pore-forming and peripheral helices (fig. 6.6b) . in each site, leu52, leu53, and leu56 from h3 and val25, val26, and phe20 from h2 appear to form a hydrophobic pocket that wraps around the adamantane cage of the drug. the amino group of amantadine or rimantadine is facing the largely hydrophilic channel lumen. an important property of the drug binding site is that it consists of elements from different helical segments and from different monomers. as rationalized above, permeation of cations through the p7 channel may depend on opening the narrow end of the funnel, which in turn depends on the reorientation of the helical segments. the binding of adamantane derivatives to the pocket may inhibit channel activity allosterically by causing the channel to close. indeed, an nmr relaxation dispersion study showed that residues at the h1-h2 hinge (phe19) and the narrow end of the cavity (val7, leu8) experienced substantial chemical exchanges (k ex~1 000 ± 79 s −1 and ~10% excited state). this data is consistent with movements of the h1 helices that cause the tip of the funnel to open and close. more importantly, addition of rimantadine slowed down motion at the tip of the channel, as relaxation dispersion curve for val7, which has significant chemical exchange in the apo state, is completely flat in the drug-bound state [114] . the dispersion curve of phe19 is also significantly flatter, and individual curve fit yielded k ex value of 67 ± 182 s −1 . clearly, rimantadine binding makes the channel less dynamic. therefore, the rimantadine may thus act as a "molecular wedge" that prevents the dynamic "breathing" of the channel required for ion conduction. comparing the amantadine or rimantadine binding mode of hcv p7 to that of influenza am2 shows two fundamentally different mechanisms of drug inhibition. in the case of am2, one drug binds to one channel. drug binding inhibits proton transport by directly blocking the channel passage; it also prevents channel from opening. in the case of p7, amantadine and rimantadine are clearly too small to block the channel. they instead bind to six equivalent sites outside of the channel cavity, which can afford up to six drugs per channel. if rimantadine binding to this site is relevant to inhibition, as crudely suggested by previous functional mutagenesis, drug binding to these sites inhibits cation conduction with an allosteric mechanism, possibly by stabilizing the closed state of the channel. although the mechanisms of drug inhibition may be completely different, the structural bases that govern drug-binding affinity for am2 and p7 are actually similar, and they involve hydrophobicity and size of the pocket, and position of the drug amino group (fig. 6.6c ). in the case of the am2 tetramer, the drug adamantane cage fits snuggly in a hydrophobic pocket formed by eight methyl groups from val27 and ala30 (two from each subunit), while the drug amino group forms polar contact with the backbone oxygen of ala30 and points to the polar region of the channel cavity. for the p7 channel, the adamantane cage is in contact with ten methyl groups and an aromatic group from the protein. these hydrophobic groups form a deep hydrophobic pocket that also matches closely the size of the adamantane cage. the amino group of amantadine or rimantadine points to the channel lumen; it is in position to form polar contacts with the electronegative groups such as backbone carbonyl of residues 15-17. hence, having a greasy pocket for the adamantane cage and a nearby electronegative group to interact with the amino group may be a general requirement for amantadine or rimantadine binding. previous functional mutagenesis studies have suggested that the tmds of viral fusion proteins are not only limited to the function of membrane anchoring, but also involved in other functions such as membrane fusion or assembly of the fusion protein on the viral membrane. for example, sequence analysis of the ha from mutant viruses and site-specific mutagenesis of the fusion peptide identified a group of mutations in the n-terminal half of the influenza ha tmd severely affected membrane fusion (the hemifusion to pore formation) [115] [116] [117] . in the case of hiv-1, multiple lines of evidences suggest that the tmd of gp41 is not merely a membrane anchor, but plays critical roles in membrane fusion and viral infectivity [118] [119] [120] [121] [122] . the amino acid sequence of gp41 tmd is also highly interesting. there is a gly rich motif in the tmd, which suggests some sort of oligomerization. even more peculiar is the presence of a conserved arginine in the middle of the predicted tm region. unlike the structural biology of viroporins, there is essentially no structural information of tmds of viral fusion proteins except for that of the tmd of hiv-1 env. hence, we focus on the discussion on the trimeric membrane anchor of the tmd of the hiv-1 envelope spike. hiv-1 envelope spike [env; trimeric (gp160) 3 , cleaved to (gp120/gp41) 3 ] is a type i membrane protein that fuses viral and host cell membranes to initiate viral infection [123] . the gp120 and gp41 are the receptor recognition and membrane fusion proteins, respectively. conformational changes in gp120 when triggered by binding to receptor (cd4) and co-receptor (e.g., ccr5 or cxcr4) lead to a cascade of refolding events in gp41 (similar to those illustrated in fig. 6.2a) , and ultimately to membrane fusion [39, [124] [125] [126] . the mature and functional env spikes, (gp120/gp41) 3 , are the sole antigens on the virion surface and thus important candidates for vaccine development [127, 128] . the native prefusion conformation of hiv-1 env is recognized by most broadly neutralizing antibodies (bnabs) [129] [130] [131] and it is generally believed to have the potential to induce such antibody responses. thus, the native conformation of env spikes on the surface of virions is extremely important to immunogen design in b-cell based vaccine development. a vast amount of structures of the ectodomain (ecd) of the gp120/gp41 complex have been determined [39, 124, 125, [132] [133] [134] [135] [136] [137] [138] [139] , but relatively little is known about the tm and membrane proximal regions of gp41 due to challenges of preserving the native-like folding of these regions in membrane mimetic media. it has been shown that truncations in the cytoplasmic tail (ct) of gp41 could alter the antigenic surface of the env ecd on the opposite side of the membrane [129] , suggesting that the ecd, the tmd, and the membrane proximal regions are conformationally coupled, and thus the conformational stability of the tmd is an important consideration for immunogen design in b-cell based hiv-1 vaccine development. recently, the nmr structure of the gp41 tmd has been solved in bicelles (made of dmpc lipid and dhpc detergent; q = 0.5) [68] using a gp41 fragment (residues 677-716) from a clade d hiv-1 isolate 92ug024.2, designated gp41 hiv1d (677-716). the tmd forms a well-structured trimer, almost helical all the way from the n-to the c-terminal end. it shows two peculiar features not seen with other known oligomeric tm helices (tmhs) (fig. 6.7a) . one unusual feature is that the tmd trimer appears to be stabilized by two separate packing modes (fig. 6.7c) . the n-terminal half encompassing the gxxxg motif forms a coiled-coil trimer, whereas the c-terminal half is held together by a network of polar contacts, which we named the hydrophilic core. for the n-terminal coiledcoil, the helical wheel representation of the trimer clearly indicates packing of hydrophobic residues such as ile and val at the "a" and "d" positions of the heptad motif, forming a hydrophobic core. the gxxxg is a well-known motif that drives tmh dimerization [58, 140, 141] . in the classic example of the glycophorin a tmd dimer structure, the two glycines of one tmh allow close packing with the gxxxg face of another tmh, resulting in a very strong tmh dimeric complex [58, 140] . there has been no previous report, however, of the gxxxg involvement in tmh trimerization. in the coiled-coil region of the hiv-1 env tmd, only g690 is involved in the trimer assembly, i.e., its small sidechain allows a close vdw contact with v689 of the adjacent tmh. the other glycine, g694, is on the periphery of the trimer facing outwards and its mutation to alanine or valine has essentially no effect on tmd trimerization, as well as env functions [68] . therefore, the key difference in the structural role of the gxxxg motif between the glycophorin a tmd dimer and hiv-1 env tmd trimer is that both the glycines are required for forming intermonomer contacts in the dimer, while only one glycine is important for the trimeric assembly. tm segments of many viral fusion proteins contain a gxxxg motif or "smallxxxsmall" motifs ("small" refers to residues with a small side chain, such as, glycine, alanine, serine or cysteine) [142] [143] [144] , suggesting that oligomerization of their tmds may be a common property. for example, recent biochemical evidence has shown that the tmds of hepatitis c virus envelope glycoproteins e1 and e2 form stable dimers or trimers that are also resistant to sds [143] . no highresolution structure of any tmd oligomer from other viral fusion proteins has been reported, due to technical challenges for structural studies of such constructs in the context of lipid bilayer. the nmr structure of the hiv-1 env tmd may provide some clues for how other viral fusion proteins oligomerize in the membrane. another peculiar feature is the presence of three copies of arginine (r696) near the middle of the tmhs (fig. 6.7b) , suggesting three unbalanced charges in the hydrophobic core of the membrane if the arg remains protonated. in the nmr structure, the tips of the long sidechains of these arginines are facing lipids and each of them is surrounded by three hydrophobic residues (l692, l695, and i697). it is also interesting to note that r696 occupies a "d" position in the coiled-coil, with its cβ facing inwards to the trimer interface. precise physical basis of how r696 is accommodated in the highly hydrophobic environment remains unclear, but the underlying mechanism must tolerate a lys residue, which is present in some viral isolates at this position. it is interesting to mention that the noe experiment for the r696 epsilon protons showed clear water noe even with a short noe mixing time of 60 ms, suggesting that the arg is somehow hydrated in the membrane. a positively-charged arg or lys in the tm segment of viral fusion protein is present in some related enveloped viruses, including simian immunodeficiency virus, caprine arthritis and encephalitis virus, equine infectious anemia virus, visna virus, and foamy virus, as well as in hepatitis c virus [145] [146] [147] [148] [149] [150] , but absent in many others. it is therefore not a prerequisite for viral membrane fusion in general [121] . functional mutagenesis indicated that the r696a mutant of hiv-1 env showed some defect in cell-cell fusion, but it could be fully compensated by high env expression [68] . moreover, this mutant has wildtype viral infectivity. in vitro infectivity and cell-cell fusion do not, however, mimic all the conditions under which the virus moves from one cell to another in an infected individual, nor are those assays particularly sensitive to physiologically relevant kinetic parameters. the structures of viroporins discussed above are all substantially different from any of the channel structures found in prokaryotes and eukaryotes, and are thus clean structural targets for developing antiviral compounds. the available structures also suggest that viroporins generally adopt minimalist architecture and can possess structural features compatible with selective ion transport. the viral channels, however, may not be as functionally robust or specifically regulated as some of their counterparts in prokayrotes and eukaryotes, because viruses often do not need intricate regulation of channel activities during their infection cycles. having minimalist structure also makes viroporins fragile and sensitive to the membrane environment. this is reflected by the conformational variations observed for the am2 channel in different reconstitution media. therefore, it remains important to examine these viroporins in more native environments, e.g., lipid bilayer and full-length proteins. as solid-state nmr continues to improve spectral resolution [87, 89] , obtaining detailed structures of viroporins in liposomes should in principle be feasible in the near future. alternatively, the ideal bicelle system can be explored with solution nmr to revisit some of the viroporin structures determined previously in detergent micelles. future establishment of the nmr systems for viroporins under more native conditions would certainly provide versatile and effective platforms for investigating inhibitor binding. apart from the progress in structure determination, major challenges lie ahead in the functional aspects of viroporin research. the precise functional roles of many viroporins are still unclear. moreover, due to the lack of robust single-channel recording setups suitable for viroporins, the ion conductance properties of most viroporins have not been fully characterized. therefore, better definition of the functional roles and channel properties of viroporins will certainly draw greater enthusiasm for developing therapeutics that target this interesting family of membrane channels. the example from hiv-1 env suggests that tmds of viral fusion proteins can adopt very interesting structures, and the distinct structural features certainly allude to their roles in viral fusion protein assembly and incorporation into the envelope, as well as in the process of membrane fusion. the nmr structural study of the hiv-1 gp41 tmd in bicelles with q = 0.5 [68] demonstrates that the structures of most of the viral fusion protein tmds can in principle be determined in essentially lipid bilayer environment using modern nmr techniques. moreover, the combined use of ideal bicelles (q ≥ 0.5) and solvent paramagnetic relaxation enhancement measurements can be used to determine the membrane partition of the tmds in the bilayer region of the bicelles [63] . we believe the next major challenge lies in understanding the roles of the fusion protein tmds in the fusion mechanism. in the cases of the flu ha and hiv gp41, for example, new experiments need to be designed to address questions such as, "does the tmd interact with the fusion domain in the hemifusion stage in which the two domains are presumably in close proximity?" and "does the tmd play a role in facilitating the conversion from the hemifusion to the pore formation?" understanding the structural properties and conformational stability of the tmd may also have far reaching implication to vaccine development and this has been recognized at least for hiv-1. as mentioned above, truncations in the ct of the env could drastically alter the sensitivity of the env ecd to the known trimer-specific bnabs [129] . the continuous structure from the n-to c-terminal ends of the env tmd, as observed by nmr, suggests that the env ecd can be structurally coupled via the tmd. indeed, it has also been shown that the env tmd can also modulate the antigenic structure of the ecd in a cell-cell fusion assay and a pseudovirusbased neutralization assay [68] . the results show direct correlation that more disrupted tmd trimer led to less inhibition or neutralization by the trimer-specific bnabs that target only the ecd. the results suggest that the stability of the tmd trimer is an important consideration when designing immunogens for hiv vaccines. the hiv env tmd is, however, not directly linked to the env ecd. another important segment known as the membrane-proximal external region (mper) is the direct link that connects the tmd to the ecd, and thus it could in principle also affect the antigenic properties of the env ecd. the mper sequence is extremely conserved with five absolutely conserved tryptophans, suggesting that the mper probably also has interesting structural features. the conformation of the mper attached to the tmd trimer in a lipid bilayer environment is still unknown (fig. 6.7d) . previous nmr studies of an isolated mper peptide in detergent micelle suggested that the mper is monomeric and folds into a kinked helix with many hydrophobic residues embedded in the micelles [151, 152] . this of course would have a rather negative implication for the mper as a vaccine epitope because antibodies that bind to the mper must dig it out of the lipids, which could be accompanied with polyreactivities. the env tmd appears to be well structured and assembled strongly to trimers. hence, the structure of the mper when connected to the trimeric tmd and in the context of a lipid bilayer remains to be characterized. plasma membrane-porating domain in poliovirus 2b protein. a short peptide mimics viroporin activity cell permeabilization by poliovirus 2b viroporin triggers bystander permeabilization in neighbouring cells through a mechanism involving gap junctions poliovirus 2b insertion into lipid monolayers and pore formation in vesicles modulated by anionic phospholipids alphavirus 6k proteins form ion channels semliki forest virus 6k protein modifies membrane permeability after inducible expression in escherichia coli cells discovery of frameshifting in alphavirus 6k resolves a 20-year enigma the alphavirus 6k protein activates endogenous ionic conductances when expressed in xenopus oocytes influenza m2 proton channels structural and dynamic mechanisms for the function and inhibition of the m2 proton channel from influenza a virus m2 protein from influenza a: from multiple structures to biophysical and functional insights influenza b virus bm2 protein has ion channel activity that conducts protons across membranes identification of an ion channel activity of the vpu transmembrane domain and its involvement in the regulation of virus release from hiv-1-infected cells correlation of the structural and functional domains in the membrane protein vpu from hiv-1 channel activity of a viral transmembrane peptide in micro-blms: vpu(1-32) from hiv-1 the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug the hepatitis c virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives dids blocks a chloride-dependent current that is mediated by the 2b protein of enterovirus 71 a potassium channel protein encoded by chlorella virus pbcv-1 the viral potassium channel kcv: structural and functional features rna transfer from poliovirus 135s particles across membranes is mediated by long umbilical connectors capsid protein vp4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (m1) promotes export and inhibits import unpacking the incoming influenza virus influence of amantadine resistance mutations on the ph regulatory function of the m2 protein of influenza a viruses viroporins: structure and biological functions chlorovirus-mediated membrane depolarization of chlorella alters secondary active transport of solutes hepatitis c virus p7 and ns2 proteins are essential for production of infectious virus hepatitis c virus p7 protein is crucial for assembly and release of infectious virions viroporins from rna viruses induce caspase-dependent apoptosis the influenza virus m2 protein cytoplasmic tail interacts with the m1 protein and influences virus assembly at the site of virus budding cytoplasmic domain of influenza b virus bm2 protein plays critical roles in production of infectious virus solution structure and functional analysis of the influenza b proton channel identification of specific regions in hepatitis c virus core, ns2 and ns5a that genetically interact with p7 and co-ordinate infectious virus production subcellular localization and function of an epitope-tagged p7 viroporin in hepatitis c virus-producing cells mechanism of membrane fusion by viral envelope proteins structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme structure of influenza haemagglutinin at the ph of membrane fusion hiv-1 envelope glycoprotein biosynthesis, trafficking, and incorporation core structure of gp41 from the hiv envelope glycoprotein mechanism of membrane fusion by viral envelope proteins intermonomer interactions in hemagglutinin subunits ha1 and ha2 affecting hemagglutinin stability and influenza virus infectivity class ii virus membrane fusion proteins class ii fusion proteins structure of the dengue virus envelope protein after membrane fusion crystal structure of dengue virus type 1 envelope protein in the postfusion conformation and its implications for membrane fusion characterization of a membraneassociated trimeric low-ph-induced form of the class ii viral fusion protein e from tick-borne encephalitis virus and its crystallization class iii viral membrane fusion proteins characterization of ebv gb indicates properties of both class i and class ii viral fusion proteins class iii viral membrane fusion proteins structural basis of influenza virus fusion inhibition by the antiviral drug arbidol arbidol as a broad-spectrum antiviral: an update improved pharmacological and structural properties of hiv fusion inhibitor ap3 over enfuvirtide: highlighting advantages of artificial peptide strategy enfuvirtide: the first therapy to inhibit the entry of hiv-1 into host cd4 lymphocytes b-cell-lineage immunogen design in vaccine development with hiv-1 as a case study b cell responses to hiv-1 infection and vaccination: pathways to preventing infection structural basis for the function and inhibition of an influenza virus proton channel structure and mechanism of proton transport through the transmembrane tetrameric m2 protein bundle of the influenza a virus crystal structure of the glycophorin a transmembrane dimer in lipidic cubic phase cryo-em structure of a native, fully glycosylated, cleaved hiv-1 envelope trimer unusual architecture of the p7 channel from hepatitis c virus the structural basis for intramembrane assembly of an activating immunoreceptor complex structure and mechanism of the m2 proton channel of influenza a virus optimal bicelle size q for solution nmr studies of the protein transmembrane partition structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state nmr magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules structural basis and functional role of intramembrane trimerization of the fas/ cd95 death receptor structural basis for membrane anchoring of hiv-1 envelope spike the structure of phospholamban pentamer reveals a channel-like architecture in membranes structural investigation of rimantadine inhibition of the am2-bm2 chimera channel of influenza viruses structural basis of interaction between the hepatitis c virus p7 channel and its blocker hexamethylene amiloride influenza virus m2 protein is an integral membrane protein expressed on the infected-cell surface structural characteristics of the m2 protein of influenza a viruses: evidence that it forms a tetrameric channel influenza virus m2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds influenza b virus bm2 protein is an oligomeric integral membrane protein expressed at the cell surface the m2 proton channels of influenza a and b viruses a universal influenza a vaccine based on the extracellular domain of the m2 protein influenza a vaccine based on the extracellular domain of m2: weak protection mediated via antibody-dependent nk cell activity an influenza a vaccine based on tetrameric ectodomain of matrix protein 2 enhanced influenza virus-like particle vaccines containing the extracellular domain of matrix protein 2 and a toll-like receptor ligand prokaryote-expressed m2e protein improves h9n2 influenza vaccine efficacy and protection against lethal influenza a virus in mice solid-state nmr investigation of the conformation, proton conduction, and hydration of the influenza b virus m2 transmembrane proton channel distinct domains of the influenza a virus m2 protein cytoplasmic tail mediate binding to the m1 protein and facilitate infectious virus production influenza b virus bm2 protein is a crucial component for incorporation of viral ribonucleoprotein complex into virions during virus assembly mechanism of drug inhibition and drug resistance of influenza a m2 channel solution nmr structure of the v27a drug resistant mutant of influenza a m2 channel structure of the amantadine binding site of influenza m2 proton channels in lipid bilayers insight into the mechanism of the influenza a proton channel from a structure in a lipid bilayer structure and mechanism of the influenza a m218-60 dimer of dimers a switch between two-, three-, and four-stranded coiled coils in gcn4 leucine zipper mutants kinetic analysis of the m2 proton conduction of the influenza virus mechanisms of proton conduction and gating in influenza m2 proton channels from solid-state nmr histidines, heart of the hydrogen ion channel from influenza a virus: toward an understanding of conductance and proton selectivity comparison of the activities of bm2 protein and its h19 and w23 mutants of influenza b virus with activities of m2 protein and its h37 and w41 mutants of influenza a virus interactions between histidine and tryptophan residues in the bm2 proton channel from influenza b virus cation-selective ion channels formed by p7 of hepatitis c virus are blocked by hexamethylene amiloride nmr structure and ion channel activity of the p7 protein from hepatitis c virus secondary structure, dynamics, and architecture of the p7 membrane protein from hepatitis c virus by nmr spectroscopy the 3-dimensional structure of a hepatitis c virus p7 ion channel by electron microscopy principles of selective ion transport in channels and pumps structural basis for the coupling between activation and inactivation gates in k(+) channels crystal structure of the cora mg2+ transporter crystal structure of the calcium release-activated calcium channel orai synthetic multifunctional pores: deletion and inversion of anion/cation selectivity using pm and ph two different conformations in hepatitis c virus p7 protein account for proton transport and dye release antiviral activity of 1-adamantanamine (amantadine) the molecular basis of the specific antiinfluenza action of amantadine influenza virus m2 protein has ion channel activity ion channel activity of influenza a virus m2 protein: characterization of the amantadine block genotype-dependent sensitivity of hepatitis c virus to inhibitors of the p7 ion channel functional studies indicate amantadine binds to the pore of the influenza a virus m2 proton-selective ion channel an amantadine-sensitive chimeric bm2 ion channel of influenza b virus has implications for the mechanism of drug inhibition a conserved basic loop in hepatitis c virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria transverse relaxation dispersion of the p7 membrane channel from hepatitis c virus reveals conformational breathing fusion mutants of the influenza virus hemagglutinin glycoprotein a point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin changes in the transmembrane region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein affect membrane fusion mutations in the membrane-spanning domain of the human immunodeficiency virus envelope glycoprotein that affect fusion activity role of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein in cell-cell fusion and virus infection conserved arginine residue in the membrane-spanning domain of hiv-1 gp41 is required for efficient membrane fusion the transmembrane domain of hiv-1 gp41 inhibits t-cell activation by targeting multiple t-cell receptor complex components through its gxxxg motif viral membrane fusion atomic structure of the ectodomain from hiv-1 gp41 structure and immune recognition of trimeric pre-fusion hiv-1 env hiv entry and its inhibition antibody neutralization and escape by hiv-1 rapid evolution of the neutralizing antibody response to hiv type 1 infection hiv-1 envelope. effect of the cytoplasmic domain on antigenic characteristics of hiv-1 envelope glycoprotein hiv-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120 a next-generation cleaved, soluble hiv-1 env trimer, bg505 sosip.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies structure of an unliganded simian immunodeficiency virus gp120 core structures of hiv-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody structure of a v3-containing hiv-1 gp120 core atomic structure of a thermostable subdomain of hiv-1 gp41 three-dimensional solution structure of the 44kda ectodomain of siv gp41 crystal structure of a soluble cleaved hiv-1 envelope trimer cryo-em structure of a fully glycosylated soluble cleaved hiv-1 envelope trimer a transmembrane helix dimer: structure and implications spatial structure of the dimeric transmembrane domain of the growth factor receptor erbb2 presumably corresponding to the receptor active state interaction and conformational dynamics of membrane-spanning protein helices hepatitis c virus envelope glycoprotein e1 forms trimers at the surface of the virion the transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus g protein mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus nucleotide sequence of the visna lentivirus: relationship to the aids virus an evolutionarily conserved positively charged amino acid in the putative membrane-spanning domain of the foamy virus envelope protein controls fusion activity contribution of the charged residues of hepatitis c virus glycoprotein e2 transmembrane domain to the functions of the e1e2 heterodimer hiv-1 broadly neutralizing antibody extracts its epitope from a kinked gp41 ectodomain region on the viral membrane antibody mechanics on a membrane-bound hiv segment essential for gp41-targeted viral neutralization the active oligomeric state of the minimalistic influenza virus m2 ion channel is a tetramer the oligomeric state of the active bm2 ion channel protein of influenza b virus evidence for the formation of a heptameric ion channel complex by the hepatitis c virus p7 protein in vitro oligomerization state and supramolecular structure of the hiv-1 vpu protein transmembrane segment in phospholipid bilayers the human immunodeficiency virus type 1 vpu protein enhances membrane permeability severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release analysis of sars-cov e protein ion channel activity by tuning the protein and lipid charge viral weapons of membrane destruction: variable modes of membrane penetration by non-enveloped viruses viroporin-mediated membrane permeabilization. pore formation by nonstructural poliovirus 2b protein the hpv-16 e5 protein represses expression of stress pathway genes xbp-1 and cox-2 in genital keratinocytes hpv-16 e5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside gm1 at the plasma membrane of cervical cells the human papillomavirus type 16 (hpv-16) e5 protein sensitizes human keratinocytes to apoptosis induced by osmotic stress viral encoded potassium ion channel is a structural protein in the chlorovirus paramecium bursaria chlorella virus-1 (pbcv-1) virion key: cord-021481-tvs1pnib authors: singh, gatikrushna; heng, xiao; boris-lawrie, kathleen title: cellular rna helicases support early and late events in retroviral replication date: 2018-08-17 journal: retrovirus-cell interactions doi: 10.1016/b978-0-12-811185-7.00007-8 sha: doc_id: 21481 cord_uid: tvs1pnib retroviruses commandeer cell rna helicases (rhs). cell rhs are necessary for early and late events in retrovirus replication. the provirus is adopted by the cell-endogenous nuclear and cytoplasmic gene expression types of machinery. whereas retroviruses engender the supportive activity of cell rhs, other rna viruses provoke theantiviral role of this superfamily of conserved proteins. in this chapter, we contrast retrovirus reliance on host rna helicases to support their replication cycle, with the virus-encoded helicaseactivity utilized by rna viruses in cytoplasmic factories. ironically, rhs are agonists to retroviruses and antagonists to other rna viruses. essential for viral rna replication, electrostatic binding to rna and assist in packaging of the viral rna to virions (kadare and haenni, 1997; kwong et al., 2005; ranji and boris-lawrie, 2010) . essential for cleavage of host and viral proteins (kwong et al., 2005) . essential for viral replication with membrane and rna-binding properties (kwong et al., 2005) . essential for atpase activity and interact with other elements of the viral replication apparatus (ranji and boris-lawrie, 2010) . hepeviridae hepatitis e virus orf1 encodes several nonstructural proteins required for hev replication and protein processing (kadare and haenni, 1997) . togaviridae (alphavirus) semliki forest virus essential for inhibition of cellular transcription by inducing rapid degradation of rpb1, a catalytic subunit of the rnapii complex (kadare and haenni, 1997; kwong et al., 2005) . nidovirales sars-coronavirus, human coronavirus 229e pathogenicity determinant; essential for nascent rna hydrolysis to yield 5 ′ -diphosphate rna (kwong et al., 2005) . interact and recruit host cell replication proteins to viral origin, including dna polymerase alpha and replication protein a for viral replication (kwong et al., 2005) . phosphohydrolase and helicase activities for virus replication (kwong et al., 2005; ranji and boris-lawrie, 2010) . arteriviridae equine arteritis virus important for early step of mrna transcription by stabilizing subgenomic rna synthesis (tijms et al., 2007) . viruses replicating in cytoplasmic factories maintain virus-encoded rna helicases (table 7 .1), which may antagonize the antiviral activity of cellular rna helicases (kadare and haenni, 1997; kwong et al., 2005; ranji and boris-lawrie, 2010) . since the advent of "omics technology," the roles of rna helicases in cell biology have been avidly characterized (table 7 .2). in the context of retroviruses, particularly hiv-1, candidate rna helicase cofactors were identified by tandem-affinity chromatography-coupled proteomics using tagged retroviral proteins and genome-wide screens using rna helicase-directed sirna (brass et al., 2008; konig et al., 2008; jäger et al., 2012) . overlap in the screens and validation of select rna helicase activities has produced an initial comprehensive picture of the retrovirus-rna helicase interface ( fig. 7.1) . whereas rna helicases support a diversity of activities in cells and the antiviral response to pathogens, a consistent theme is binding and rearranging of nucleotide-nucleotide pairings. at least 85 human proteins encode the core helicase domain, which is designated by highly conserved amino acid motifs, e.g., dead, deih, or dexh (x = any). despite the extreme conservation of the core helicase domain, rna helicases operate in all compartments of eukaryotic cells and exhibit diverse activities that are attributable to ancillary domains. rna helicases shuttle between the nucleus and cytoplasm and participate in mrna biogenesis, nucleus-cytoplasm transport, pioneer-round translation, cotranslational decay, and steady-state translation by contributing to the formation of dynamic ribonucleoprotein complexes (rnps) (fig. 7 .1 and table 7 .2). rigi and related rna helicases demonstrate affinity for replication intermediates of rna viruses that reproduce in the cytoplasm and activate innate response. the rna helicase superfamily (sf) is categorized into three subfamilies (sf 1, 2, and 3) according to combination of invariant amino acids in the core helicase motif: asp-glu (de) with two other conserved amino acids; asp-glu-ala-asp, abbreviated as dead, deah, or dexh (x = any) residues in the core motif. besides the namesake domain (dead box, ddx or dhx proteins), ancillary domains in rna helicase sf members vary widely and engender interaction with specific substrate rnas and protein cofactors ( fig. 7.2) . functionally conserved across the plant and animal kingdoms, the helicase core promotes native rna folding by rearranging of duplex nucleotide substrates. by an allosteric mechanism, nucleotide triphosphate (ntp) binding at the helicase core induces conformational change, followed by ntp hydrolysis (fairman-williams et al., 2010) . versatile as an rh is, rearranging activity is common without ntp-dependent helicase activity. changing components of rna-protein complexes, designated rnpase activity, is associated with rna binding, rather than ntp hydrolysis, and is categorized a chaperone activity. structurally, the catalytic helicase core within each sf is almost identical, consisting of two similar recombination protein reca domains with highly conserved residues that coordinate binding and hydrolysis of the ntp. the helicase domains share nine sequence motifs: q is solely present in dead box helicase; grouping i, also called walker a: grouping ia, ib; ii, also called walker b; tethers rna polymerase ii to chromatin-bound dnabinding proteins (e.g., brca1, p300/cbp beta, p16ink) (nakajima et al., 1997) . component of 7sk rnp that regulates availability of pcaf and p-tef (van herreweghe et al., 2007) . binds and is necessary for translation of select rnas: 5 ′ utr of jund proto-oncogene, (hartman et al., 2006) type i collagen mrnas (manojlovic and stefanovic, 2012) . tethers lin28 to polysomes (jin et al., 2011) . rearrangement of rna secondary structure attributed to atp hydrolysis. spry domain facilitates association with cellular rnps for translational initiation, nuclear export, ribosome, and spliceosome assembly (fang et al., 2005 (fang et al., , 2004 . essential for crm1-dependent nuclear export of viral rna, remodel of exporting mrnps (yedavalli et al., 2004) . interacts with nf-kb to suppress p65-mediated transcription (xiang et al., 2016) . associated with eif4e-dependent mrna translation (soto-rifo et al., 2013) . bind gc-rich sequences, modulate alternative splicing during cell differentiation . coregulators of estrogen and androgen signaling pathway . essential for 5 ′ to 3 ′ rna unwinding activity and factor for nonsense-mediated mrna decay (nmd) (gregersen et al., 2014) . nucleocytoplasmic shuttling rh that is enriched in mitochondria; associates with mitoribosome (lessel et al., 2018) . cofactor of zap in nmd (ye et al., 2010) . associates with cytoplasmic face of nuclear pore complex, remodel of exporting mrnps (diot et al., 2016) . contributes atpase and helicase activity essential for spliceosome assembly (wisskirchen et al., 2011) . modulates the coupling of mrna splicing with nucleocytoplasmic transport of mature mrnas (delaleau and borden, 2015) . groupings iii and iv, v, and vi. sf 1 and 2 members contain conserved walker a and b motifs in the active site providing ntpase binding and hydrolysis activity, respectively (del campo et al., 2009) . whereas nonspecific rna binding is attributable to core helicase domain, specific recognition of cognate rna is achievable by double-stranded rnabinding domains (dsrbds), described below. retroviruses, by mimicking 5 ′ utr features in cognate cell-derived mrna targets, commandeer rna helicases to promote native folding of cis-acting rna elements. retrovirus rna becoming cloaked in cellular rnps is advantageous to evade surveillance and recognition of this type of virus infection. retroviral antagonism of innate response remains a controversial area intertwined with biology of restriction factors. introns figure 7.1 critical events in hiv-1 replication are supported by rna helicases early steps in hiv-1 replication involve reverse transcription in the cytosol of capsid-associated viral rna (n = 2) to double-stranded cdna that transits the nuclear pore and integrates into the host chromosome (blue lines) to form a provirus (red line surrounded by black dotted line) with the involvement of at least two rhs (dhx9, ddx19a). late events involve provirus transcription by rna polymerase ii and associated rhs (dhx9, dhx30, ddx5, ddx17); rna processing by ribonucleoprotein components (i.e., upf1, uap56); rev oligomerization at the rev responsive element (rre) of intron-containing hiv rna fostered by ddx1 and upf1. rev-dependent rrecontaining transcripts undergo crm1-dependent nuclear export assisted by ddx3 on cytoplasmic face of the nuclear pore. completely spliced viral transcripts are transported by nxf1/nxt1 nuclear export receptor fostered by dbp5 positioned on the inner side of the nuclear membrane. polysomal cbp80-bound hiv rna is associated with dhx9; polysomal eif4e-bound hiv rna associated with ddx3, eif4g, eif4a. upf1 rna surveillance cofactors, ddx17 and dhx30, influence hiv rna dimerization; dimer rna packaging with viral structural/enzymatic proteins is modulated by mov10, ddx24, and dhx30 in context of stoichiometric amounts of dhx9 (n = 2). current understanding of rna helicase activities in retroviruses has been propelled by results of genome-wide screens. tandem-affinity purification by liang and colleagues used epitope-tagged viral proteins and mass spectrophotometry in the first proteomic screen for hiv-associated candidate rna helicases. the tandem-affinity purification of epitope-tagged hiv gag in cells isolated gag binding partners that included dhx9, rna helicase gu(a)/ddx36, ddx18, ddx24 (roy et al., 2006) . affinity purification of hiv proteins and mass spectrometry interaction statistics identified ddx6, ddx20, and ddx49 (jäger et al., 2012) . genome-wide short interfering rna (sirna) screens to identify host proteins involved in hiv replication identified eight helicases including dhx9, dhx30, ddx3, and ddx1 (brass et al., 2008; konig et al., 2008) . williams et al. performed a sirna library screening targeting 59 cellular helicases (williams et al., 2015) . hela cells were cotransfected by specific sirna to target the helicase and to generate vesicular stomatitis virus g glycoprotein (vsv-g) pseudotyped hiv-1. the culture supernatants were analyzed for virion production by gag p24 elisa, and the infectivity of progeny virions was determined by luciferase reporter assay. hiv infectivity was significantly reduced by downregulation of ddx3, ddx5, ddx10, ddx11, ddx17, ddx18, ddx21, ddx24, ddx28, and ddx52 and dhx9; the results indicating direct or indirect activity of the helicase in facilitating virion infectivity (lorgeoux et al., 2012; williams et al., 2015) . ensuing investigations validated selected rna helicases that promote human retrovirus replication (hiv-1, human t-cell lymphotropic virus type 1) by ectopic overexpression and downregulation by rna interference and crispr technology in cell-based assays, biophysical approaches, and traditional biochemical approaches. an ultimate goal for virologists and structural biologists alike is antiviral targeting of pertinent rna helicases. a daunting task because retroviral rna biology is intertwined with nuclear rna helicases at every step, genetic, biophysical, and structural studies have defined rna helicases important to early and late steps in hiv replication. the deepening knowledge of rna helicase ancillary domains and cognate viral nucleotide-nucleotide pairings subject to helicase unwinding is critical information to eventually segregate retroviral rnp targets from cellular rnps necessary to cell viability. the following sections trace the steps in retroviral replication in context of cell biology and prospective antiviral activity of select rna helicases (table 7 .3). cellular rna helicases are sitting at the center of current research to elucidate the composition and function of retroviral rnps in retrovirus biology. a number of studies have demonstrated that exogenous overexpression of rna helicases and/or downregulation by rna interference diminishes the abundance and infectivity of progeny hiv-1, often in cell type-specific manner (e.g., mov10, dhx9, dhx30). these observations have been attributed to aberrant reverse transcription of the incoming genomic rna in an unseemly relationship to the posttranscriptional life of primary retroviral transcripts. a key to interpreting rna helicase activity in reverse transcription of retroviral rna has been the determination of stoichiometry in virions and cognate viral rna structure, as determined for dhx9 boeras et al., 2016) . in 2006, roy et al. identified hiv-1 virions deficient in dhx9 were poorly infectious and poorly executed reverse transcription. downregulation of dhx9 by sirna in producer cells did not affect viral genomic rna dimerization or packaging, but those virions were poorly infectious, as determined by activation of a galactosidase indicator (magi) in hela-t4 cells (roy et al., 2006) . similar virion infectivity loss was also observed by infection of primary cells (bolinger et al., 2010) . the trna lys3 -viral rna complexes extracted from dhx9-deficient virions were less efficient in base extension in a primer extension assay (roy et al., 2006; xing et al., 2011) . in addition to reverse transcription initiation, the strand transfer step was also impaired in the absence of dhx9 (roy et al., 2006) . importantly, a gain-of-function experiment demonstrated that exogenously expressed dhx9 packaged into hiv-1 virions rescues . promotes initiation phase of hiv-1 reverse transcription (roy et al., 2006) . binds to topoisomerase ii alpha complex that remodels chromatin structure for hiv-1 integration (raghavendra et al., 2010) . modulates hiv-1 preintegration complex activity through host factors ledgf/p75 and integrase interactor 1 (lorgeoux et al., 2012) . mediates the association of creb-binding protein (cbp) with rna pol ii to promote hiv-1 transcription nakajima et al., 1997) . binds to hiv-1 tar rna through double-standard rnabinding domains (dsrbds) . promotes recruitment of pcaf and p-tefb to tar/tatbound transcription complex . dbp5/ddx19a ddx1 ddx3 ddx5 and ddx17 promotes nuclear export of the unspliced rna of avian sarcoma virus through two direct repeats in 3 ′ utr (leblanc et al., 2007) . facilitates rev oligomerization at rev responsive element (rre), enhancing crm1-dependent nuclear export of hiv-1 transcripts (fang et al., 2005) . facilitates remodel and release of rev/rre-dependent hiv rnps from nuclear pore (yedavalli et al., 2004) . interact with hiv-1 rev protein; may modulate rev subcellular localization between nucleolus, nucleoplasm, and cytoplasm (williams et al., 2015) . stabilizes viral unspliced rna in nuclear and cytoplasmic rnps; necessary to gag protein production (ajamian et al., 2008) . mov10/upf-1 like facilitates hiv-1 rna stability; binds to nucleocapsid (nc) region of gag and packaged into viral particle (abudu et al., 2012) . reverse transcription initiation when the endogenous dhx9 has been silenced (xing et al., 2012) . dhx9 associates with hiv-1 gag and is recruited into virus particles in an rna-dependent manner (roy et al., 2006) . in 2012, the observed stoichiometry of dhx9 in virions was determined proportional to the genomic rna, n = 2 (sharma and boris-lawrie, 2012). recently, isothermal titration calorimetry confirmed that the dimeric hiv-1 5 ′ utr conformation binds one dhx9 molecule per rna strand, in a manner independent of binding to the nucleocapsid (nc) domain of gag. nuclear magnetic resonance spectra using a deuteriumlabeling approach resolved the structure of the dimeric 5 ′ utr in complex with the n-terminal dsrbds of dhx9. the structure of the large molecular mass complex was found to be dependent on dhx9 binding to a double-stranded region of the primer-binding site (pbs) segment of the 5 ′ utr. these results are consistent with measurement of dhx9-depleted virus particles of reduced level of trna lys3 that annealed onto the pbs (roy et al., 2006) . in the viral rna, translation of viral mrna eif4a/ddx2 ddx3 dhx9 component of eif4f translation initiation complex; atpdependent unwinding of 5 ′ utr structure (sonenberg, 1993) . recruited to eif4e-bound hiv rnp, associated with eif4f translation activity (soto-rifo et al., 2013) . facilitates viral protein synthesis of hiv-1 and htlv-1 by increasing the polysome loading (bolinger et al., 2010; hartman et al., 2006) . binds the 5 ′ utr of hiv-1, htlv-1, spleen necrosis virus, bovine leukemia virus, mason pfizer monkey virus, feline leukemia virus and promote rev/rreindependent hiv gag production (bolinger et al., 2007) . promotes dimerization in-solution; recruited to virion by binding the contiguous 5 ′ utr in sequences between the primer activation sequence and primer-binding site (boeras et al., 2016) . regulates production of gag and inhibits genomic rna packaging and infectivity . promotes dimerization of rous sarcoma virus 5 ′ utr in-solution (stake et al., 2015) . a single a-to-c substitution was sufficient to disrupt the conformation of the pbs segment and attenuate virion infectivity in cells. therefore, the contribution of dhx9 to virion infectivity is attributable to its structure-dependent binding at the pbs segment of the hiv 5 ′ utr during virus assembly (boeras et al., 2016) , consistent with prior data positing dhx9 fosters conformations favoring the action of viral reverse transcriptase (xing et al., 2011) . validated cellular cofactors in reverse transcription are few: trna synthetase and cognate trna and dhx9 that together engender pbs conformation for trna annealing and extension by reverse transcriptase. proteomics results indicate dhx9 is also a component of hiv preintegration complex, conceivably activating chromatin to initiate the integration process (raghavendra et al., 2010) . once integrated, nuclear rh associates with provirus, supporting transcription and viral rna processing, export, translation, dimerization, and genomic rnp assembly. the intimate association between the provirus and cellular rnps mimics that of a cellular gene, and conceivably those nuclear marks shield viral genomes in newly infected cells from recognition by dhx58/rigi and other innate sensors that detect cytoplasmic viral replication intermediates and trigger interferon response. the up-frameshift protein 1 (upf1)-like sf helicase member mov10 has a deag helicase motif and atp-dependent 5 ′ to 3 ′ directional rna helicase activity (gregersen et al., 2014) . mov10 is associated with gag nucleocapsid rna-binding domain and is detectable in hiv virions (abudu et al., 2012) , but its activity is controversial. biochemically, mov10 interacts with rev in an rna-independent manner, and rev/rev response element-dependent nuclear export was reduced by dominant-negative helicase mutant ( fig. 7.1 ). exogenous expression of mov10 diminishes production of virions and those virions exhibit deficient hiv reverse transcription and poor infectivity, positing mov10 as an antiviral factor (goodier et al., 2012) . in unresolved controversy, independent research identified sirna downregulation of endogenous mov10 did not change virion production and considered endogenous mov10 acts as cofactor (arjan-odedra et al., 2012) . in aggregate, modulation of mov10 incurs an imbalance in hiv rnp activity, consistent with indirect and possibly beneficial effect on biogenesis of infectious hiv (huang et al., 2015) . dhx9 is an essential gene product and mouse knockout is lethal to embryos. the dhx9 gene lays in the 1q25 tumor susceptibility locus associated with neoplasms of prostate, lung, and breast. roles for dhx9 in tumor biology involve dysregulation of dna damage response related to gene expression and dna genotoxic stress. a nucleocytoplasmic shuttling deih helicase, dhx9 bridges chromatin-bound transcription coactivator proteins (e.g., ep300/cbp) to rna polymerase ii (table 7 .2). the tethering requires dhx9's n-terminus and flanking residues of a minimal transactivation domain nakajima et al., 1997) . by connecting coactivators with rna polymerase ii through a dhx9 bridge, this rna helicase activates transcription of specific genes. susceptible coactivators, in addition to cbp, include nfkb p65, brca1 breast cancer-specific tumor suppressor brca1, the zic2 protein, ews-fli1 oncoprotein, p16ink4a tumor suppressor and multidrug resistance 1 protein (mdr1). moreover, dhx9 is component of a 7sk storage rnp that sequesters the positive transcription elongation factor b (p-tefb), a rate-limiting cofactor of tfiih that regulates efficiency transcription elongation of many genes, including hiv (van herreweghe et al., 2007) . because of limited p-tefb, rna polymerase ii stalling of hiv transcription complex occurs within the r region of the hiv long terminal repeat yielding truncated transcripts of ∼100 nucleotides that are identified as tat transactivation responsive element, tar (table 7 .2). tat/tar interaction facilitates 5 ′ end capping of nascent rna necessary to recruitment of nuclear cap-binding proteins cbp80/cbp20 (zhou et al., 2003) . in the absence of tat, dhx9 exhibits biochemical affinity for tar, an observation that may explain dhx9 overexpression activates transcription of hiv tar-luc reporter rna . overexpression of dhx9 is posited to tip 7sk-p-tefb equilibrium toward dissociation of p-tefb, ultimately facilitating tat/tar transactivation of hiv. released from 7sk rnp, p-tefb stimulates hiv tat/tar-dependent transcriptional elongation through a mechanism that involves both phosphorylation of the c-terminal domain of rna polymerase ii and by interfering with the activity of negative elongation factors (table 7 .3). speculation is dhx9 loading to tar during basal transcription modulates 5 ′ utr rna conformation and the retention of cbp80 in later stages of hiv expression (see below). dhx9 overexpression increased gag protein and virion production in hek293 cells and altered balanced splicing of primary hiv rna (tang et al., 1999) . moving beyond exogenous expression assays that disrupt the balance between rnp components, rna interference proved invaluable to modulate an essential gene product like dhx9. validation studies in cos cells demonstrated sirna downregulation of this rh reduced gag, vif, rev, and nef rna expression (bolinger et al., 2010; roy et al., 2006) . cos, a simian fibroblast line, did not recapitulate reduced hiv steady-state hiv rna and instead demonstrated modulation of gag rna accumulation in polysomes that is proportional to reduction in viral protein production (hartman et al., 2006) . in contrast to results in hek293 cells, dhx9 downregulation in cos cells did not reduce either hiv rna abundance or nucleocytoplasmic transport, positing perturbation in rnp components between the transformed cells (hartman et al., 2006) . paralogs, dhx30 and dhx9, have nearly identical amino acid conservation but accumulate in different subcellular compartments (fig. 7.2) . dhx9 accumulates in nucleus and dhx30 accumulates in mitochondrion. attributable to truncation of one dsrbd at n-terminus and ∼100 arginine-rich amino acids at the c-terminus, dhx30 functions in the assembly of the large mitochondrial ribosomal mutation of dhx30 reduces mitochondrial localization (lessel et al., 2018) . overexpression of dhx30 indicates a supportive role in hiv replication: dhx30 overexpression increases production of viral gag proteins and yield of virus particles by 2-to 3-fold and severely inhibits the packaging of hiv-1 rna into virus particles, upregulating defective interfering particles by 10-fold , consistent with change in rnp dynamics. zhou et al. (2008) determined dhx30 interacts with the hiv-1 5 ′ utr, potentially catalyzing native refolding important for genomic rna dimerization similar to dhx9. in-solution assays with recombinant proteins demonstrated dhx9 and dhx30 promoted dimerization of the hiv 5 ′ utr. dhx30, but not dhx9, fostered dimerization of the rous sarcoma virus (rsv) 5 ′ utr (stake et al., 2015) . this trend follows the observation that the gallus gallus genome encodes dhx30, but not dhx9, and suggests that dhx30 supports rnp dynamics attributable to higher order conformation of the avian retrovirus 5 ′ utr and possibly distinct chaperone activity of rsv gag nucleocapsid (stake et al., 2015) . two decades ago, study of genetically simple retrovirus mason-pfizer monkey virus (mpmv) uncovered existence of the cis-acting constitutive transport element (cte) and its necessity for cytoplasmic expression of simian retrovirus unspliced rna (srv1). synthetic cte transcripts and cell rna-binding proteins were incubated with hela cell lysates and in stringent conditions coisolating rna-binding proteins were identified, e.g., dhx9 and dhx9cofactor hap95; aly/ref and rh uap56/ddx39b cofactors that tether the nxf1-nxt1 complex (also known as tap nuclear export receptor). the study of cte/tap activity significantly expanded knowledge of nucleocytoplasmic transport of cell mrnas and regulation by many virus rnas. ten years ago and in context rsv, a genetically simple avian retrovirus, ddx19b/yeast dbp5 was determined to facilitate nuclear export of genome-length unspliced rna (leblanc et al., 2007) . drawing on extensive knowledge of the nuclear pore complex in yeast, dbp5 is positioned on the cytoplasmic face of the nuclear pore complex and catalyzes disassembly of the export rnp ( fig. 7.1) . whereas srv1/mpmv cte connects tap nuclear export receptor, genetically more complex retroviruses encode a cis-acting viral cte-like element (rre) and viral transacting protein (rev) to activate nuclear export of unspliced viral rna. yeast two-hybrid screening identified rev binding rh, ddx1, and ddx3 (fang et al., 2004) . reporter assays and rna export measurements validated these host cofactors facilitate rev/rre-dependent gene expression. ddx1 is a dead box family member containing spry domain and interacting with poly(a) rna. both yeast and mammalian two-hybrid assays demonstrated strong interaction of ddx1 helicase with hiv-1 rev protein. ddx1 (amino acids 189-333) directly interacts with the nuclear inhibitory signal of rev at amino acids 10-24. downregulation of ddx1 reduces the rev/rredependent luciferase reporter activity and also impairs the nuclear export of unspliced mrna (edgcomb et al., 2012; fang et al., 2005) . ddx3 is a nucleocytoplasmic shuttling protein that localizes to the cytoplasmic side of the nuclear pore ( fig. 7.1) . yedavalli et al. demonstrated sirnamediated downregulation of ddx3 reduces rev/rre/crm1-mediated hiv-1 rna export. based on the transdominant activity of ddx3 atpase mutant, ddx3 was postulated to facilitate the final release from the nuclear pore of the crm1-bound rev/rre rnp (yedavalli et al., 2004) . mechanistically, ddx3 is posited in binding crm1 molecules bound to the exposed nuclear export signal (nes) of rre-bound rev oligomers expelling crm1/rev/rre-containing hiv-1 rna cargo into the cytoplasm (lorgeoux et al., 2012) . in this remodel of the hiv rnp, ddx3 activity is similar in helicase-driven release by dbp5/ nfx1/nxf1 nuclear export receptor activity of mrna cargo (fig. 7.1 ). ddx1 and ddx3 coordinate rev/rre-dependent gene expression. ddx1 binding to rre modulates rre conformation, promotes binding of monomeric rev, resulting in rev oligomerization (lamichhane et al., 2017) . rev independently interacts with nuclear export receptor crm1 through the leucinerich nes (cullen, 2003) , tethering the rre/rev-crm1 rnp with affinity for ddx3 at the nuclear pore (lorgeoux et al., 2012) (fig. 7.1) . roles of other candidate rh are poorly understood, specifically ddx5, ddx17, ddx21, dhx36, and dhx9, in rre/rev-mediated nuclear export (zhou et al., 2013) . in retrospect, an obvious yet unanticipated precedent came to light through characterizing rnps deposited at exon-exon junctions; nuclear history of metazoan mrna facilitates its translational activity in the cytoplasm (le hir et al., 2001) . these lessons involve rna helicase exchanges that retroviruses sometimes recapitulate from activity of cellular transcripts, i.e., upf1/staufen (dugre-brisson et al., 2005) , dhx9/jund (hartman et al., 2006) , dhx36 (rhau)/guanine-quadruplex-rich rna (chalupníková et al., 2008) (table 7 .2). recruited to nascent mrnas in the nucleus, these rhs remain associated in cytoplasmic rnps that become polysomes or rna granules that are translationally-incompetent, dramatically distinct fates attributable to distinct rnp compositions. upf1, also known as ddx39b, is an rna-binding atpase that contains zn-coordinating finger motifs, acidic motifs, and basic amino acid clusters, and it is a well-established cofactor in the cotranslational process of nonsensemediated decay, or nmd (nicholson et al., 2010) . a component of the exon junction complex that binds nascent rna, upf1 shuttles between the nucleus and cytoplasm using the crm1 pathway, and activates rna surveillance at exon-exon junctions through a series of phosphorylation events during pioneerround translation. in association with nuclear cap-binding proteins cbp80/ cbp20, upf1 is a sentinel in the process of nmd. nmd parses nonsense-free mrnas from nonsense-containing transcripts that have potential to translate polypeptide deleterious to the cell. upf1, upf2, and upf3 activate cotranslational rna decay when scanning ribosome encounter a stop codon that is aberrantly positioned within an open reading frame. however, upf1 bound to retrovirus unspliced rnas is responsible for alternative activity: subverting nmd at the premature stop codon laying within the polymerase open reading frame of unspliced transcripts (fig. 7.1) . despite its canonical role in activating nmd, upf1/dhx39a stabilizes hiv unspliced rna that harbors a premature stop codon and lacks exon junction complex. upf1 stabilizes primary retroviral rna in both nucleoplasm and cytoplasm. upf1 downregulation by sirna decreases hiv rna and thereby diminishes the synthesis of virion structural proteins. while residual levels of virions are observed, they are poorly infectious, positing beneficial role of upf1 in balancing hiv rnp compositions (ajamian et al., 2008) . the molecular switch triggering cap exchange is poorly understood. canonically, nuclear cap-binding proteins cbp80/cbp20 are replaced by the cytoplasmic cap-binding protein, eif4e. eif4e is considered the rate-limiting factor for steady-state translation and engages eif4g and the helicase eif4a to form the eif4f mrnp. historically, translation control has been viewed through the prism of eif4e and the requisite rna helicase activity of eif4a. discovered decades ago, eif4a provides critical unwinding activity to 5 ′ utr secondary structures, recruits the 40s ribosomal subunit, and sends the activated ribosome scanning the 5 ′ utr (sonenberg, 1993) . recent studies used antibodies to the nuclear cap-binding protein cbp80 and the cytoplasmic counterpart eif4e to isolate hiv mrnps from the cytoplasm . hiv-specific rt-qpcr assessing relative abundance of hiv transcripts confirmed the completely spliced nef transcript was in the eif4e rnp, at levels similar to the cellular rna control. by comparison, unspliced hiv rna was poorly enriched in the eif4 rnp and was instead retained in the cbp80-rnp. despite the retention of nuclear cap-binding proteins, viral protein synthesis remained robust. polysome analysis verified gag was translated from the cbp80-rnp, a noncanonical rnp associated with pioneer-round translation. rna granules are attributed to downregulation of eif4e activity during oxidative stress, heat shock, amino acid deprivation, or other physiological stress. mouland et al., (2000) characterized downregulation of eif4e activity and hiv rna accumulation in rna granules. in 2013, soto-rifo et al., identified ddx3-bound hiv rna accumulating with eif4g and pabpc1 in cytoplasmic granules comprised of translation-incompetent mrnps (soto-rifo et al., 2013) . physiological downregulation of eif4e did not shut down hiv protein synthesis and hiv rna remained on polysomes and associated with dhx9 (fritz, singh, boris-lawrie, in preparation). ongoing studies evaluate the possibility that cbp80-dhx9 rnps contribute an alternative strategy for cap-dependent translation. rna helicases serve as drug targets in a variety of settings. in peripheral blood mononuclear cells (pbmcs), a small molecule inhibitor of ddx3 efficiently reduced hiv replication (radi et al., 2012) . given the importance of several cellular rna helicases in retrovirus replication, targeted disruption of rna helicases may attenuate latent provirus reactivation. the prospects in this setting are small molecules targeting cognate rnas, particularly viral rna elements. a prospect buoyed by growth in knowledge of structural biology, critical information is complete understanding of three-dimensional features of rna helicases and cognate rna. a stealth approach, similar in principle to highly active antiretroviral therapy, is targeting two or more rna helicase-viral rna interfaces necessary to distinct events in the viral replication cycle. the potential increases to curb emergence of drug-resistant viruses by leveraging essential rna helicase activities in cellular cofactor rnas. in closing, retroviruses use cellular gene expression machinery, maintaining the interaction with host rna helicases at every step of viral replication: reverse transcription and integration to form the provirus; transcription of the provirus and posttranscriptional regulation; and dimerization and packaging of the genomic rnp ( fig. 7.1) . reconciling the beneficial activity of shuttling rna helicases to retroviruses with the antiviral activity of cytosolic helicases that oppose other rna viruses remains to be fully understood. from the vantage point of a retrovirus, rna helicases are cofactors required to transcribe provirus dna, stimulate transcriptional elongation and rna processing, and facilitate the nucleocytoplasmic transport of retroviral rnps to become translated. from the vantage point of a cell, rna helicases are antiviral sensors, detecting nonself-rna in the cytoplasm and triggering an antiviral state. however, retroviruses circumvent these cellular rna sensors, instead co-opting nuclear rna helicases and using them in a manner analogous to cellular transcripts, dissipating the restrictive activity of rna helicases that actively thwarts cytoplasmic replication of many other rna viruses. identification of molecular determinants from moloney leukemia virus 10 homolog (mov10) protein for virion packaging and anti-hiv-1 activity unexpected roles for upf1 in hiv-1 rna metabolism and translation dual roles of rna helicase a in creb-dependent transcription endogenous mov10 inhibits the retrotransposition of endogenous retroelements but not the replication of exogenous retroviruses dhx9/rha binding to the pbs-segment of the genomic rna during hiv-1 assembly bolsters virion infectivity rna helicase a modulates translation of hiv-1 and infectivity of progeny virions rna helicase a interacts with divergent lymphotropic retroviruses and promotes translation of human t-cell leukemia virus type 1 identification of host proteins required for hiv infection through a functional genomic screen recruitment of the rna helicase rhau to stress granules via a unique rna-binding domain nuclear mrna export: insights from virology rna helicases ddx5 and ddx17 dynamically orchestrate transcription, mirna, and splicing programs in cell differentiation unwinding by local strand separation is critical for the function of dead-box proteins as rna chaperones multiple export mechanisms for mrnas influenza a virus polymerase recruits the rna helicase ddx19 to promote the nuclear export of viral mrnas interaction of staufen1 with the 5 ′ end of mrna facilitates translation of these rnas ddx1 is an rna-dependent atpase involved in hiv-1 rev function and virus replication sf1 and sf2 helicases: family matters the rna helicase ddx1 is involved in restricted hiv-1 rev function in human astrocytes a dead box protein facilitates hiv-1 replication as a cellular co-factor of rev a role of rna helicase a in cis-acting transactivation response elementmediated transcriptional regulation of human immunodeficiency virus type 1 mov10 rna helicase is a potent inhibitor of retrotransposition in cells mov10 is a 5 ′ to 3 ′ rna helicase contributing to upf1 mrna target degradation by translocation along 3 ′ utrs rna helicase a is necessary for translation of selected messenger rnas rna helicase mov10 functions as a co-factor of hiv-1 rev to facilitate rev/rre-dependent nuclear export of viral mrnas global landscape of hiv-human protein complexes evidence that lin28 stimulates translation by recruiting rna helicase a to polysomes virus-encoded rna helicases global analysis of host-pathogen interactions that regulate early-stage hiv-1 replication viral and cellular rna helicases as antiviral targets a dead-box protein acts through rna to promote hiv-1 rev-rre assembly the exon-exon junction complex provides a binding platform for factors involved in mrna export and nonsense-mediated mrna decay tap and dbp5, but not gag, are involved in dr-mediated nuclear export of unspliced rous sarcoma virus rna erratum: de novo missense mutations in dhx30 impair global translation and cause a neurodevelopmental disorder from promoting to inhibiting: diverse roles of helicases in hiv-1 replication a novel role of rna helicase a in regulation of translation of type i collagen mrnas the doublestranded rna-binding protein staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic rna encapsidation rna helicase a mediates association of cbp with rna polymerase ii nonsense-mediated mrna decay in human cells: mechanistic insights, functions beyond quality control and the double-life of nmd factors discovery of the first small molecule inhibitor of human ddx3 specifically designed to target the rna binding site: towards the next generation hiv-1 inhibitors identification of host proteins associated with hiv-1 preintegration complexes isolated from infected cd4+ cells rna helicases: emerging roles in viral replication and the host innate response association of rna helicase a with human immunodeficiency virus type 1 particles the ddx5 and ddx17 rna helicases are cornerstones in the complex regulatory array of steroid hormone-signaling pathways determination of host rna helicases activity in viral replication thriving under stress: selective translation of hiv-1 structural protein mrna during vpr-mediated impairment of eif4e translation activity remarks on the mechanism of ribosome binding to eukaryotic mrnas the dead-box helicase ddx3 substitutes for the cap-binding protein eif4e to promote compartmentalized translation initiation of the hiv-1 genomic rna hiv-1 and two avian retroviral 5 ′ untranslated regions bind orthologous human and chicken rna binding proteins the carboxyl terminus of rna helicase a contains a bidirectional nuclear transport domain arterivirus subgenomic mrna synthesis and virion biogenesis depend on the multifunctional nsp1 autoprotease identification of rna helicases in human immunodeficiency virus 1 (hiv-1) replication -a targeted small interfering rna library screen using pseudotyped and wt hiv-1 the cellular rna helicase uap56 is required for prevention of double-stranded rna formation during influenza a virus infection coordinate roles of gag and rna helicase a in promoting the annealing of formula to hiv-1 rna in vitro and in vivo analysis of the interaction between rna helicase a and hiv-1 rna dexh-box protein dhx30 is required for optimal function of the zinc-finger antiviral protein requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function the tat/tardependent phosphorylation of rna polymerase ii c-terminal domain stimulates cotranscriptional capping of hiv-1 mrna ddx5 facilitates hiv-1 replication as a cellular co-factor of rev the packaging of human immunodeficiency virus type 1 rna is restricted by overexpression of an rna helicase dhx30 key: cord-016718-cxn1ewfw authors: anderson, virginia title: performing interventions: the politics and theatre of china’s aids crisis in the early twenty-first century date: 2017-11-08 journal: viral dramaturgies doi: 10.1007/978-3-319-70317-6_9 sha: doc_id: 16718 cord_uid: cxn1ewfw theatrical productions attest to a radical shift in chinese governmental policy and public awareness of the aids epidemic at the dawn of the twenty-first century; state-subsidised theatre worked directly with the government to contain the transmission of hiv. produced by two of the country’s most elite cultural institutions, the shanghai dramatic arts center and the beijing people’s art theatre respectively, the dying kiss (shengsi zhiwen) in 2003 and student zhao ping (zhao ping tongxue) in 2005 represented a sea change in the political response to the epidemic while documenting public perceptions towards people living with hiv and aids in china. marking policy change, they reflect experiences that capture a society transitioning from denial to confrontation at the dawn of the twenty-first century. it was sixteen years after china's first aids case was identified that the nation's government made hiv a high national priority by publicly acknowledging the severity of the threat the virus posed to its population in 2001 (wu et al. 2007: 679) . theatre at high-profile institutions was to serve as a powerful tool for prevention, stigma reduction and national image preservation. government-appointed aids ambassador and stage and screen celebrity pu cunxin communicated a vision for theatre's utility: "we can't leave the arts out of the fight against aids but the arts must disseminate knowledge in an artistically superior waynot just spread education and propaganda. the power of real art is to move people, to reflect people's experiences" (pu 2005) . 3 these two high profile theatres provided a literal stage for the demonstration of government-sponsored efforts to turn the tide of the epidemic in china. such a partnership was explicitly financial as well as ideological: the dying kiss was co-produced by the shanghai municipal health bureau and the shanghai dramatic arts centre and student zhao ping was coproduced by the china youth fund for the prevention of aids and gobon guilin latex (a point discussed later in this essay). 4 prior to these productions, government officials and the government-controlled media kept the virus separate from promoted views of chinese nationalism, resulting in intense stigma for people living with hiv or aids. with new policies and such visible cultural leadership, a new commitment to hiv and aids prevention and care was palpable in the early twenty-first century. in this chapter, i first establish an historical context for these plays through a discussion of the epidemic's evolution in china through the end of the twentieth century. i then consider the modes of production and the sometimes subtle political content of each play in relation to shifts in governmental policy (with which both content and production are intertwined). rooting my work in historical and dramaturgical analysis as well as interviews with artistic contributors, health workers and activists, i argue for the significance of the dying kiss and student zhao ping as embodiments of government-fueled popular perceptions of hiv and aids in china at the start of the twenty-first century. despite their short runs, these productions established an important precedent for theatre to address stigma and to affect governmental policy and even china's international standing as a leader in the global effort to address the epidemic. early in the new millennium the goal of many theatre artists who addressed the epidemic in china was to alert audiences to its immediacy, to keep them from fearing it and to reduce isolation and shame endured by affected individuals. the need for such goals becomes clear as one considers the cultivation of stigma over the preceding decades. the trajectory of the aids epidemic in china is often divided into three stages (shao 2001; huang 2013) . yiming shao, chief expert on aids for the chinese center for disease control and prevention, describes the 'introductory phase' as between roughly 1985 and 1988, when a popular perception of aids as a foreign disease was established. during this period, a small number of cases involving foreigners and chinese citizens who had been traveling internationally was identified in coastal cities. yanzhong huang, senior fellow for global health for the council on foreign relations, assesses the impact of these early cases on the perception of the growing crisis: the initial statistics reinforced the myth that hiv/aids was not so much a public health problem as a social ill confined to western countries. like their us counterparts, chinese scientists and public health officials were initially convinced that hiv/aids spread mainly through homosexuality and promiscuity (xinhua july 22, 1987) . believing that both behaviors were 'illegal and contrary to chinese morality' and therefore limited in china, senior health officials were confident that the aids epidemic was unlikely to occur within their borders. (2013: 86-87) the second, 'slow' or 'regional' phase of the epidemic, is attributed to the period between 1989 and 1994 and describes the steady increase in incidence and the appearance of the disease within border regions of the country. it is often marked by the identification of 146 hiv-positive intravenous drug users within china's southwest yunnan province. hiv spread widely during this phase, reaching the majority of china's provinces and adding several hundred cases each year. the geographical site of the next turning point lies in the central henan province, where the practice of selling blood for cash was widespread in the mid-1990s and hiv infection in the region was recorded at over 50 per cent of the population (rosenthal sept. 16, 2001a) . by 1995, hiv incidence climbed into the thousands. the third, 'nationwide', phase of the epidemic paved the way for undeniable recognition in the new millennium at about the same time. by 1998, hiv or aids cases were reported in all thirty-one provinces; china documented its first case of mother-to-child transmission, and sexual transmission of the disease had rapidly increased. still, intravenous drug use and commercial blood and plasma donation accounted for the majority of transmissions during this phase, at 43.9 per cent and 24.1 per cent of the estimated number of hiv infections, respectively (china ministry of health 2004). the government estimates that in just four years, between 1999 and 2003, the number of people living in china with hiv increased from 500,000 to 840,000 (thompson 2005: 4) . during this pivotal period in the first years of the new millennium, theatre artists directly engaged with new governmental policies, cultural identity, and social realities for people living with hiv or aids. their productions serve as time capsules of a radical shift in the nation's response to the epidemic. by world aids day 2001, the chinese news media were covering the epidemic in force. in addition to interviews with people living with hiv and widely-distributed public service announcements, a fictional television 'soap opera about a businessman who contracts the aids virus [sic] after a one-night stand with a prostitute [sic], featuring some of china's most popular actors' called if i have tomorrow aired during prime time on china central tic (cctv) (rosenthal 5 december 2001b) . 5 pan guiyu, vice minister of the sfpc, promoted if i have tomorrow by saying that it 'not only introduces to the public scientific knowledge about aids, but also sets a good example of the correct attitude people should hold toward the aids patients [sic] and their deadly disease. [it] explores the theme of aids from social, family, ethic [sic] and moral perspectives, cautioning people to keep away from aids and calling for social concern for aids patients' (state council 2001) . foreshadowing the theatre about to emerge, government leaders invested in the power of entertainment to bring issues surrounding hiv to the public's attention. even through this first programme of its kind, conservative morality was linked to a promoted national identity. in a series for the new york times, elisabeth rosenthal documented the direct link between an increase in news media coverage and the government's evolving position on the epidemic, concluding: 'in a country where all news media are state-owned and content is more or less controlled, the burst of interest [in hiv and aids] clearly reflects a government decision to allow greater discussion of an epidemic that is growing rapidly, but has previously received only intermittent attention by the media' (5 december 2001b). the changes occurred quickly; in 1999, a short public-service announcement promoting condom use was pulled from television by officials who worried that it was 'too risqué', but only two years later safer sex was openly discussed on the radio. even with more open public discussion, stigma remained and news coverage at the turn of the millennium still reinforced early perceptions of hiv and aids as a problem linked to foreigners; people living with hiv or aids featured on television had generally contracted hiv overseas and concealed their identity by wearing sunglasses or turning their backs to the camera (rosenthal 5 december 2001b) . social discrimination was widespread, manifest through well-documented isolation, the loss of resources and services, verbal stigma, secondary stigma endured by family members and fellow villagers, and even self-discriminating behaviour (cao et al. 2006; hardee et al. 2009 ). despite this increase in reporting, an official shift in policy did not occur until 2003, described by meghan laslocky for pbs as 'the tipping year for china with regard to recognition of aids'. in an iconographic watershed moment, prime minister wen publicly shook the hand of a person living with hiv. laslocky writes: many say it took a televised handshake for china to wake up. on world aids day 2003, prime minister wen jiabao shook the hand of an hivpositive person, and a close-up of their joined hands was broadcast around the country. finally, with by some estimates one million aids victims [sic] in henan province alone, silence was no longer an option, and the country's leaders were scrambling to come up with policies to show that they had a plan. (laslocky 2007) that year, a new administration headed by president hu jintao, prime minister wen jiabao and vice premier and the then health minister wu yi put the implementation of evidence-based hiv policies high on the national agenda (sun et al. 2010 ) and announced a new national aids control policy, 'four frees and one care' (free treatment, free voluntary counseling and testing (vct), free prevention of mother to child transmission (pmct) and free schooling for aids orphans, as well as provision of social relief for hiv patients). 6 however, such high-level policy making could not in and of itself prompt deep cultural change; after a photo of president hu jintao shaking hands with a person living with hiv was published, the man's daughter was expelled from school because of her father's serostatus (wan 2014) . the impression of government leadership was important, however, and two plays at high-profile state theatres captured this dramatic shift in policy and served as a tool for prevention, stigma reduction and national image preservation. while government spokespeople frequently stated that compassion was needed for all people living with hiv, dramaturgically, the dying kiss is set up to emphasise the protagonist's 'innocence' (in contrast to the 'guilt' of others). even as the play educates audiences about hiv prevention and stigma, protagonist xiao lu is portrayed as a life-saver, a devoted son, a loving brother, a dedicated fiancé and a citizen unfairly discriminated against. for these reasons, producers at the shanghai dramatic arts center were optimistic when they sought funding from the city's board of health to fund additional performances beyond the planned six. nonetheless, the request was denied with the rationale that, with its limited reach, theatre was not a good model for raising awareness (yu 2005) . the board did, however, supply printed educational materials to distribute to audiences. this act demonstrates conflict among government leaders: while some stress that formal educational materials cannot adequately capture the human dimension of the epidemic (pu 2005) , not everyone was willing to step away from traditional modes of education and prevention, even during this period of increased awareness and hiv and aids policy reform. nevertheless, the internationally acclaimed theatre is a flagship of modern china's culture and to produce the play on this topic in the first place was a demonstration of commitment to the issue. the banner atop the shanghai dramatic arts center production program for the dying kiss declares the play to be 'the first play about aids in china'. while this description is not quite accurate, 8 the production remains remarkable for its full-length treatment of the emotional experience of a chinese person living with hiv and the embodiment of popular conceptions of hiv and aids. the play, written by li rong and li shengying and directed by terence chang, is based on actual events that were documented by journalist tu qiao in a monthly newspaper column and her subsequent book entitled a century's sorrows (2000) . it depicts the development of a trusting relationship between the journalist and xiao lu, a person living with hiv. the dying kiss is a zhuxuanlu, or main melody play, one that reinforces government-supported values. claire conceison explores the complexity of the 'main melody' campaign in chinese spoken drama, describing a 'complicated dialectic with which the government was able to exert control over theatre workers while at the same time theatre workers were able to manipulate this control to their own advantage' (1994). these nuances are captured within the dying kiss ( fig. 9.1) . the opening stage directions begin to correct the popular misunderstanding that hiv is an illness coming from beyond china's borders by indicating that taiwanese singer luo dayou's classic love song 'my hand passing through your black hair' plays on set, and 'suddenly brings people to an intangible world that seems far away but is actually very close' (li and li 2003 : 1, emphasis added). the time is now and the location is a corner of the city. 9 journalist xu qian serves as the audience's intended surrogate in the play; through her relationship with xiao lu, the audience member who believes that they are far removed from the virus develops a personal relationship with someone living with hiv. the documentation of early twenty-first century popular understandings of hiv and aids abound from the first moment, sometimes with graphic, ignorant language. upon learning that xu qian is going to interview a person living with hiv, her friend wa wa sets three rules for interaction: she must not shake hands with her interview subject, she must keep three feet away during the interview and she must refrain from talking over forty-five minutes. xu qian responds to her friend's demands with an assurance that includes a grotesque description of the physical manifestation of hiv, in line with popular misconceptions: i understand wa wa, because everyone would have the same reaction. ai zi! wow! the untreatable disease! aids! … it eats your healthy cells, and swallows your entire body. your beauty and figure all turn into rotting meat, a disgusting pile of rotting meat. i am not reading you the poetry of shakespeare, i am interviewing such a person. (li and li 2003: 2) although xu qian defies her friend and shakes the hand of xiao lu, who is described as 'a handsome man', afterwards she 'secretly looks at her palm and wipes it on her pants' (ibid.: 3). blame and stigma endured by people living with hiv are dominant themes throughout the play. echoing the sentiments of pu cunxin and the government's public stance on hiv and aids, xiao lu addresses his sense of social isolation in stark language and graphic imagery: even if people did get the disease because of their dissolute lifestyle, i do not want you to look at them that way. their lives are already hard enough and now they need to endure everyone's disdainful look. they are like rats curling up in the city's corner waiting to die, and in the morning the cleaner would throw them into the garbage truck. who doesn't want to live healthily and happily? (li and li 2003: 3) this vivid and dehumanising comparison to rats contributes to stigma even as the play's producers sought to lessen it. nevertheless, it captures contemporary popular fears. herein lies the complexity of the dying kiss as a signal of governmental change; it calls for compassion, but employs language that underscores fears and revulsion. a nationalistic view of china is reinforced through heroic action, gender dynamics, and echoes of the long-promoted idea that hiv and aids perme-ated china's borders from abroad; much is made of the idea that xiao lu acquired hiv while in thailand. the protagonist is chinese, and national pride is evoked as transmission is portrayed through valiant action: xiao lu saved the life of a co-worker when an industrial sewing machine punctured her hand. when he gallantly freed her, it fell down upon him, puncturing his finger (21-22). the gender dynamics in this scenario are significant. as alicia leung argues (2003) , at the time of this production, china 'maintained a high degree of control over gender construction in order to legitimize its historical achievement of revolution and liberation, … this is derived from the core philosophy confucianism in which human role relations are cultivated and developed within a male-centered world' (359). therefore, xiao lu was upholding core, nationalist values when he contracted hiv (while overseas) and is thereby presented as one of the play's 'innocent victims'. what the play does manage to achieve is to show discrimination in medical care in china, from references to nurses hiding from people living with hiv (4) to xiao lu's decision to seek treatment overseas (9). the play contrasts the blame, social stigma and harsh treatment in china with compassion and care at a treatment centre in thailand. 10 a late scene between xiao lu and his doctor serves as a direct critique of the national response while providing a vision of the future (that aligns with the new governmental policies): director cai: i should apologize to you. … do you remember how i distrusted and doubted you when we met for the first time? xiao lu: that was normal. everyone would think like that. director cai: xiao lu, it is not right to think in that way, especially as medical personnel. no matter how this person got aids, he deserves our sympathy and needs our devoted care. any prejudice would just hurt them more deeply, which fails to help people fight the plague by weakening their fighting capacity. … i wonder why a patient tries so hard to try to prove his innocence? doesn't it reflect prejudice ingrained in our values? while people are suspecting and despising each other, aids becomes increasingly rampant, devouring our land and our lives! i think that only when we create a better recovery? environment and cultural? atmosphere, can we help patients to fight disease and make a miracle. in a sense, every patient is a soldier who fights in the frontline. (li and li 2003: 24) as the scene draws to a close, director cai and xu qian (our audience surrogate) reflect on how much they've learned from xiao lu. still, such a critique of china was to be made only within a larger critique of foreign value systems. drawing on this notion of blame and guilt associated with hiv and aids, the play provides an indictment of capitalism and perceived western values while emphasising a commitment to core values of chinese nationalism. perhaps no scene documents this denunciation with greater clarity than a flashback to 'heaven's home', the treatment facility in thailand where xiao lu receives care. making a strong contrast with the hospitals of china, xiao lu describes his new surroundings to the audience: 'here people are absolutely equal. here, the doctors and nurses are kind hearted like angels. they gave me comfort and the newest treatment' (18). 11 there, xiao lu meets a monk, whose lived buddhist values stand in sharp contrast to xiao lu's father's claimed christianity. the monk's story serves as a critique of capitalist values, further emphasising that some-antinationalist-behaviours make people 'deserve' to have hiv. the monk used to be a government official with a 'loving, sweet wife, a smart son in high school'. he then grew jealous of friends who had more money and beautiful women and quit his government job to go into business: 'at that time, bosses were flying all over the sky, ceos were prevailing on the land. when you yell boss or ceo on the street, nine out of ten people would turn around. it's worth nothing.' he made a lot of money (19). he reveals that he contracted hiv through infidelity and that, 'without knowing, i brought the disease to my wife' and his son has since cut off contact with him. now, devoted to his spiritual life, he explains: i know i'm going to hell. then why don't i use my last time to make friends and contribute to the society to build a better afterlife? … i will use buddha's great wisdom to light your fire of life, use buddha's endless power to ignite your courage to conquer the disease, and use buddha's forgiveness to make clear that you need to cherish your every day, alive. (20) this emphasis on society instead of the individual reflects core chinese cultural values. varying degrees of guilt and innocence and a commitment to nationalism emerge through this story. if the monk had not succumbed to capitalist desires, he would still be working for the government with a happy wife and son in further embodiment of cultural values. another play captured nationalistic tensions surrounding hiv and aids at the beginning of the twenty-first century, this time written and directed by one of the leading theatre artists in china, tian qinxin. student zhao ping focuses on the cultural clashes between a younger generation of chinese college students and older, more conservative professors and police officers. its development history captures the conflict between art expressing china's youth culture and official restrictions. in addition to its place in china's official response to the hiv and aids epidemic, student zhao ping is noteworthy within the context of tian's career in that, as she herself observes, it is not like much of her other work (tian 2005a ). the play is left out of most critical discussions of the acclaimed writer/director who is perhaps best known for her passionate and visually inspired presentations of huaju, or spoken drama. the piece began its development when tian was approached by representatives of the china youth development foundation to write a play with themes about the devastating impact of aids and its prevention among young people. tian knew of the prejudice and stigma surrounding hiv and aids and felt as though, for the newest generation, the increasing incidence of hiv was an educational problem, and not only for those who tested positive. she asked; 'who is paying attention to the people who are in fear? i wanted to explore this question. this play looks at the educational problem in this context' (tian 2005a) . brechtian in form and aim, the structure was the rehearsal of a play, calling for actors to play multiple roles even as they shifted between characters and themselves and provided commentary on the action. there were three different levels of interaction: actor to actor, actor to character and character to character, enabling brief moments of audience interaction like that in augusto boal's forum theatre. in these moments, this technique was used for audience empowerment and social change at the grass-roots level. the play begins with the sexual harassment of zhao ping by four western foreigners (played by actors wearing face masks, according to the stage directions; tian 2005b: 2) at an international hotel in beijing. presumed to be acting as a sex worker, zhao ping is reported to the police by hotel security. informed of the charge, university faculty and administration debate how prostitution and their (unnamed) university policy could co-exist. police officers join the meeting and share information from their interrogation: zhao ping has multiple boyfriends and 'if she has a foreign boyfriend, she should get her blood tested' (4). the university officials blame zhao ping's behaviour on her education and her professor xuedong comes under fire. it is revealed to the audience that zhao ping and xuedong have been involved romantically. when zhao ping is convicted of prostitution, university officials fail her final paper, the last requirement for graduation. without telling anyone where she is going, zhao ping then disappears. two months later, a friend of zhao ping tells xuedong that zhao ping had emailed her and shared that one of her boyfriends was diagnosed with aids. concerned about his own status, xuedong goes to the hospital to be tested. following his blood test, he moves 'into another dimension' (16). tian qinxin pointed to this scene as the crux of the play; as an artist, she was most interested in the emotions surrounding hiv and aids and, for her, 'the most interesting time is the period of waiting for results and the fear people experience. it doesn't matter what the results are, but whoever that person is, they need to have friends supporting them' (tian 2005a) . xuedong describes how he feels 'as if my spirit has left my body'. while unnamed characters offer reassurances about current treatment options and prognoses, zhao ping and her friend express contrary ideas: 'it cannot be cured', 'you would need a huge sum of money. average people cannot afford it,' and 'even flu vaccines change every year. viruses change' (tian 2005b: 15) . tian qinxin maintains that the fear xuedong experiences is an opportunity for education (2005a); he is enveloped in fear, creating an aura of tension within which the actors switch in and out of character to speak frankly about the disease and risky behaviour directly with the audience through improvisation (tian 2005b: 16) . as the educational discussion ends, the audience returns to the fictional world in which a police officer reports that neither the foreigners' claims of prostitution at the hotel nor zhao ping's claims of harassment are considered reliable and, following review of security footage, zhao ping is exonerated. concluding the play, zhao ping, now attending an us university, exchanges emails with xuedong through which she reveals that she does not have aids: she made that up as, indeed, she had made up her boyfriends. her manipulation of authority figures leaves the audience questioning what might be trusted in their own relationships. taken in its entirety, student zhao ping is less a play about hiv and aids and more about generational conflict and gender construction, but this improvised scene, broken out of the fictional narrative, serves an important purpose: ensuring direct, frank conversation about hiv with the audience. that it does so while promoting a specific brand of condoms is an issue i discuss later (fig. 9.2) . produced by the national theatre company of china, the play was marketed as a fundraiser for aids orphans but tian emphasised its importance for a particular, wider demographic: the rising youth generation within the overall population. the generational divide was captured in the press and described as emotions caught in fierce collision; the construction of gender roles is held up for interrogation and zhao ping's agency stands in sharp contrast with her teacher's view that women are 'born irrational, selfless, and passive' (tian 2005b: 5) . theatre critic faye wong observes of the play, 'compared with the physical aspects of the disease, the ideological roots of the virus are more horrible ' (2005) . tian describes how these ideas found manifestation in her process: before making the play, i interviewed some college students and professors about their views on aids. we do not cover drugs or moving scenes of caring for aids patients in this play, because television already tells this aspect of the epidemic extensively and vividly. theatre cannot surpass television on this. we strove to present the collision of thought of people from different ages behind the sexual confusion of aids. (cited in wong 2005) this emphasis on sexuality and individuality may appear markedly progressive and, certainly, compared to the traditional conservative morality in the dying kiss, it is. however, the play was sponsored by gobon guilin latex, the foremost condom manufacturer in china (see global business 2005; zou et al. 2012) , which promoted its brand not only by providing condoms for the audience in a gift bag along with informational brochures, but for conspicuous product placement within the play's final moments: xuedong: i should not preach to the audiences, but people should not have a promiscuous life. zhao ping: if you can't do that, i think, at least be safe. xuedong: the "yezhanpai" is pretty good everyone: we should use "yezhanpai" produced by guilin gaobang. (tian 2005b: 17) such a corporation would have a financial interest in promoting (safe) sexual freedom, partnering theatre and industry in efforts to contain the epidemic. furthermore, the market was auspicious: with the increased awareness and action concerning hiv by the government and in the popular media, condom sales were increasing dramatically ('global business' 2005) . while promoting discussion of hiv, the play was also promoting a new ideology concerning sexual freedom. this cultural shift is apparent within governmental regulation of the play. when tian sought official approval of the script from the cultural bureau in beijing prior to production, she was told that, while the subject of hiv was important, the script's sexual content was a problem. tian was flabbergasted that she had been commissioned to write a play about aids for an increasingly sexually active generation but was not to address sex, a potent means of hiv transmission. she said: 'there's no way we could present this story to this generation without talking about sex' (2005a). she sought and found a loophole in the approval system. during my interview with her, she emphasised that this was a time of significant cultural reform; 'there was a socialist system for performance approval and a new, special socialism category' through which the play was finally approved (tian 2005a) . like the dying kiss, tian's play reinforces the idea that hiv and aids are associated with foreigners. indeed, in order to torment her lover, zhao ping plays off the cultural imagination by telling xuedong that she may have contracted hiv from her foreign boyfriend. despite the progressive depiction of zhao ping's sexuality, the play presents the police as a conservative moral authority. while they conclude that zhao ping had not been engaging in prostitution and so should not face punitive actions from the university, therein lies the condemnation of sex workers. similarly, zhao ping had lied about having a foreign lover, suggesting that her own serostatus might be different if in fact she had had a foreign lover. negotiating national identity during an international epidemic both legitimising and challenging the early chinese cultural assignation of aids as a foreign disease, these productions comingle with concurrent policy to acknowledge the reality of the disease in china. the dying kiss and student zhao ping reflected the government's evolving stance concerning the virus. these plays protected the honour of chinese national identity by portraying the disease as something entering chinese society from outside the country's borders. the plays addressed in this study mark policy change, reflecting experiences that capture a society transitioning from denial to confrontation at the dawn of the twenty-first century. since the time these plays were produced few theatrical productions in china have approached the topic of hiv and aids explicitly, while television and feature films continue to offer representation. 12 however, stigma endures (kazar and wang 2014) and it remains to be seen how the performing arts in china might productively intervene. this research would not have been possible without the generous scholarly support and tireless translation work of dr claire conceison, the financial assistance of the then tufts university provost jamshed barucha, and the enthusiastic research and translation assistance of connecticut college students qingmei (cleo) han and especially shuhan zhang. an early version of this chapter benefitted from generous feedback from participants in the 'theatre and national/ with hiv through such facilities, removed from central society, perhaps most notably the wat phrabat nampu temple, which began as an aids hospice in 1992 and continues its service today. see wright et al. (2009) . 12. notably, one star-studded film, love for life (gu et al. 2011) , is set in the 1990s instead of contemporary china. documentaries such as the blood of yingzhou district (lennon and yang 2007) , the epic of central plains (ai 2007), together (zhao 2010) understanding hiv-related stigma and discrimination in a "blameless a joint assessment report of hiv/aids prevention, treatment and care in china the main melody campaign in chinese spoken drama 26 companies of the global business coalition on hiv/aids (gbc) announce immediate commitments to fight aids in china hiv and aids stigma and discrimination in china: results from a national survey celebrity philanthropy in china: the political critique of pu cunxin's aids heroism governing health in contemporary china aids demolition brigades" clear path for property developer the spread of aids in china: a slow acknowledgment of the 'capitalism loving disease the blood of yingzhou district feminism in transition: chinese culture, ideology and the development of the women's movement in china xin ru zhi shui shengsi zhiwen beijing: beijing people's art theatre and oly café blood and tears: a chinese family's ordeal in a nation in denial of aids suddenly, aids makes the news in china chinese national identity and national identity gaps in east asia aids epidemic at age 25 and control efforts in china state council information office and the china international publishing group (cipg) evolution of information-driven hiv/aids policies in china hiv carriers stage plays in beijing china confronts aids interview with author (c. conceison, trans.). national theatre company of china zhao ping tongxue = student zhao ping shiji zhitong: zhongguo songfen aizibingren wanquan jilu [a century's sorrows: a complete record of a person with aids from china interview with shuhan zhang discrimination against people with hiv/aids in china modern asian theatre and performance student zhao ping" is a psa for people born in the 80s hospice and palliative care in southeast asia: a review of developments and challenges in malaysia, thailand and the philippines evolution of china's response to hiv/aids. the lancet together. new york: dgenerate films chinese cultural values and chinese language pedagogy (doctoral dissertation) condom use in china: prevalence, policies, issues and barriers key: cord-034036-1wigu3i3 authors: yu, changhao; zhou, dong; jiang, weijia; mu, jie title: current epidemiological and etiological characteristics and treatment of seizures or epilepsy in patients with hiv infection date: 2020-10-21 journal: acta epileptologica doi: 10.1186/s42494-020-00028-8 sha: doc_id: 34036 cord_uid: 1wigu3i3 seizures or epilepsy is one of the common serious complications in patients with advanced human immunodeficiency virus (hiv) infection or diagnosed with immune deficiency syndrome, with higher incidence and prevalence than in the general population. generalized seizures are the most common type in the patients. opportunistic infections are a stereotypical predisposing factor for seizures in hiv patients, but a variety of pathogenic factors can also be found in these patients, such as metabolic perturbation and drug-drug interactions. the diagnostic criteria for seizures in these patients are the same as those in the general population. as hiv patients with seizures need to take both antivirals and antiepileptic drugs, the risk of drug-drug interactions is greatly increased, and the side effects of drugs may also become more prominent. at present, most experience in antiepileptic drug usage has come from the general population, and there is still a lack of guidance of antiepileptic drug use in special groups such as the hiv-infected people. unlike the old-generation drugs that involve metabolisms through cyp450, the first-line antiepileptic drugs usually bypass cyp450, thus having less drug-drug interactions. in this review, we summarize the recent research progress on the above-mentioned widely discussed topics and make a prospect on future research direction. patients living with advanced human immunodeficiency virus (hiv) infection or diagnosed with acquired immune deficiency syndrome (aids) usually have complications related to the central nervous system (cns), which may include psychiatric disorders, encephalitis, cerebrovascular diseases, etc. [1] [2] [3] . seizures or epilepsy is a serious type of complication. patients with witnessed seizures need long-term periodic medication control, making it a challenge for clinical drug management. in pediatric patients living with hiv, seizures or epilepsy may be concurrent with brain development delay or neuron-cognitive impairment [4] . besides, hiv-seropositive patients with documented seizures are at a higher risk of developing other cns syndromes, compared with the hiv-negative patients [5] . among these patients, opportunistic infection (oi) as a result of deep immunosuppression is the most frequent cause; however, there is a considerable proportion of patients with no predominant causes, suffered from hiv encephalopathy (hive). another part of patients has seizure relapses due to the interactions between antiepileptic drugs (aeds) and antiretroviral drugs (arvs), which makes seizure improperly controlled. in addition, the decades of development on treatment options clinically proven to be effective and widely accepted for seizures and epilepsy arise from populations sufferring only from seizures or epilepsy. targeted research or treatment plans for hiv-seropositive patients with documented seizures are needed. epidemiological research has advanced our understanding of the distribution characteristics of seizures or epilepsy in the hiv+ population. this may help formulate new medication protocols for hiv+ patients with epilepsy. this review updates research information from the epidemiological characteristics of seizures and epilepsy in the hiv-seropositive population to the causes and then the therapeutic protocols. we also review the survival status and medication methods of hiv+ patients with documented seizures or epilepsy, in order to provide clinical decision-makers with the latest progress to refer to. the prevalence and incidence of seizures in the hiv+ population the incidence and prevalence of seizures are higher in the hiv-infected population than in the general population [6] [7] [8] [9] . as demonstrated in previous research, the incidence and prevalence of seizures in the general population range 0.4-1.0% [10] and 0.05% [11] , respectively, while those in the hiv+ patients (including the patients with documented seizures prior to hiv infection and patients with newly-onset seizures [nos] after the infection of hiv) are 1.8-19.8% and 2-19.8%, respectively [7, 8] . these two statistical indicators varies among different countries/regions or longitudinally in time. studies from centers in africa peculiarly tended to report a higher prevalence or incidence, while the developed countries usually reported lower incidence. a retrospective study from ireland showed that the incidence of seizures is estimated to be doubled in the hiv+ population than in the local general population (with the incidence of seizures is 2.4%) [7] . however, after a 5-year retrospective study, kim et al. from korea found the incidence of seizures in hiv+ patients was 3.0%, compared with 0.24% in the general population [12] . samia et al. from south africa reported both prevalence and incidence of seizures in the included hivinfected children as 7.6% [13] . another study from south africa reported a 23% rate of history of seizure in 227 involved hiv+ children [14] . this situation may be due to the pandemic of aids in africa, where many children were afflicted through the maternal-neonatal transmission. what's worth noticing is that reports from india also revealed a relatively higher distribution of seizures in hiv+ patients. sinha et al. reported an overall 20% proportion of nos in a local hospital [15] . further, studies carried out in around 1990 reported a higher incidence. wong et al. and holtzman et al. reported the incidence of nos in new york to be 11 and 18%, respectively [16, 17] . before the invention of highly active antiretroviral therapy in1996, patients were more likely to suffer from complications caused by increased virus load. gender does not seem to influence the prevalence or incidence. studies have shown that the sex ratio was similar between the enrolled hiv+ population and patients with seizures [7, 13, 18, 19] . this suggests that, in some studies where the nos occurrence was 3-10 times higher in male patients than in female patients, the imbalance in the sex ratio of recruited hiv+ patients might explain the different occurrence [6, 12, 16] . however, whether gender plays a role in the susceptibility still needs more rigorous research to prove. at present, there is no obvious evidence that the age factor could promote or reduce the occurrence of seizures in hiv+ patients to a certain extent. although children are more vulnerable to cns infections than adults, affected by the heterogeneity between different studies, it cannot be asserted that the hiv+ children are more prone to seizures than adults. notably, studies in different regions showed that the mean age of patients at nos occurrence was 30-40 years old [7, 19, 20] . as for the prevalence or incidence of epilepsy in the hiv+ population, most studies only reported the prevalence or incidence of seizures in detail, and only a few reported recurrent rates and followed up to the diagnosis of epilepsy. from the three studies reported so far, the proportion of hiv+ patients who had seizures and were subsequently diagnosed with epilepsy was very high (37/38, 21/27, and 32/52) [6, 13, 14] . it can be considered that the epidemiological characteristics of epilepsy in hiv+ patients may be similar to seizures. seizures are reported to emerge at different stages of hiv infection, and are manifested as the initial clinical symptom in some patients after hiv infection. for example, some patients are hospitalized for seizure occurrence and screened to be hiv-seropositive afterward. under most circumstances, patients are at the advanced stage of infection, when the oi becomes a common event, from which seizures arise. and it seems that the more advanced stage of hiv infection, the greater the likelihood of seizures to occur. indeed, many patients inflicted seizures after the diagnosis of aids [16] . satishchandra et al. discovered that seizures were the presenting manifestation in one of five cases in their series [21] . intriguingly, the majority of seizures in hiv+ patients in their study were still present at the stage of advanced infection. from the statistical data, seizures are unlikely to be conceived of as an exceptional complication of advanced hiv infection, but they have an adverse impact on the quality of life. according to the epilepsy definition by international league against epilepsy (ilae) in 2014 [22] and seizures classification in 2017 [23] , most seizures in hiv-infected patients are generalized. the proportion of generalized seizures in patients with nos ranged from 35.3% [12] to 67.3% [14] . the epilepsy type could not be demonstrated in hivinfected patients because of a lack of studies. some studies have reported the existence of status epilepticus (se) in their hiv-infected participants, with a proportion from 0.175 to 4.34% [7, 12, 14, 20] , while in a metaanalysis published in 2017, status epilepticus had a prevalence of 12.6/100,000 among general populations [24] . this suggests that hiv may be an underlying risk factor for status epilepticus. however, since the etiological evidence of patients with status epilepticus has not been stated, the possibility of other causes cannot be ruled out. to figure out the relationship between hiv infection and status epilepticus, further research is needed. on the whole, the etiology of seizures in hiv-infected patients comes from hiv itself and other causes induced by hiv infection. the drug-drug interactions are also responsible for the recurrence of seizures, and they will be discussed in detail in the following therapeutic sections. even dating back to 30 years ago, the ois were also reported as the most dominating cause of seizures in hivinfected patients [17] . hiv+ patients are more susceptible to pathogenic microbes such as toxoplasmosis, cryptococcus species, tuberculosis, and john cunningham virus (jcv), four most common pathogens for oi, due to the immunosuppression. typically, it is when the cd4 count is lower than 100 cells/μl that these pathogens cause encephalitis [25] . nevertheless, tuberculosis is an exception that can cause tuberculous meningitis (tbm) at any level of cd4 count as long as there is immune suppression [26] . following the widely-adopted highly active antiretroviral treatment (haart), both the incidence and mortality of these ois have declined. according to a most updated meta-analysis, ois account for nearly 50% of the causative factors in approximately 68% of hiv+ patients experiencing nos with conclusive causes, with toxoplasmosis diagnosed in the majority (21%), followed by progressive multifocal leukoencephalopathy (pml) and cryptococcal meningitis (14% for each) [9] . this paper focuses on several regular infectious pathogens and non-infectious agents. infection with toxoplasma gondii, an obligate intracellular parasite, is the cause of toxoplasmosis. domestic cats are the only known definitive hosts of t. gondii and often lead to the indirect infection of the parasite in humans through food contamination with spore-forming oocysts. t. gondii is one of the most common infectious agents responsible for newly-onset seizures in hiv seropositive patients [18, 27, 28] . they are often afflicted with focal seizures caused by mass lesions. ct or mri scan often shows single or multiple nodular lesions. however, even if no hypointense lesions could be seen, some silent lesions still revealed themselves at autopsy. csf biochemical examination is a more reliable choice for diagnosis than serologic assays, and a study reported a sensitivity of 75% with the use of b1 gene nested pcr [29] . however, the lumbar puncture cannot be administered to patients with increased intracranial pressure due to the high risk of cerebral hernia; in this circumstance, how to diagnose and when to decide to start treatment usually rely on empirical experience. the routine protocol for hiv+ patients is a long-term consecutive inhibition therapy until continuous antiretroviral therapy raises the cd4 count over 200 cells/μl. and a standard therapeutic plan is the co-administration of pyrimethamine, sulfadiazine, and folinic acid. besides, a prospective study from cameroon concluded that newly-onset seizures in these patients have a benign clinical course and favorable prognosis, thus not requiring aeds [30] . it is convinced that cryptococcal meningitis can be caused by the spread of fungal pulmonary infections. it is the second most common hiv-associated oi among adults in the sub-saharan africa [31] , and the global prevalence of cryptococcal infection has been estimated to be 6% in hiv+ patients [32] . according to a prospective study, in 28% of the hiv-infected cryptococcal meningitis participants, the generalized seizures were regularly witnessed [19] . csf analysis is a piece of important evidence for diagnosis. the opening pressure often appears to be greater than 25 cmh 2 o [33] . csf cultures are definitive standards, but it may take weeks to reveal a positive result. the india ink assay can assist diagnosis, but a negative result cannot deny the possibilities of positive results. results of imageology may be nonspecific due to the nonmeningitic neurologic complications of hiv, such as atrophy in hive patients. the prognosis of such patients is correlated with the csf opening pressure. patients with seizures tended to have higher opening pressure, which has an inverse relationship with the occurrences of seizure attacks. for the treatment of hiv+ patients, an adequate treatment course at the beginning is recommended. the administration of amphotericin b should not be interrupted until the csf culture turns negative. and a consistent maintenance therapy using fluconazole should follow the initial therapy as far as cd4 count is over 100 cells/μl [34] . in patients with nos, cautions should be given to aed applications due to the potential drug-drug interactions and the latent impairment in renal clearance [19] . therefore, it is more practicable to screen asymptomatic cryptococci infection among hiv-seropositive patients, thus facilitating prompt prevention and treatment. the seizure is usually secondary to tbm, with previous studies reporting an incidence of 11.3-39.1% [35, 36] and a high possibility to reoccur. the co-infection with hiv is a strong risk factor for progression into active tuberculosis, seizures may occur not only during the active phase. tbm can present with seizures in 10-16.3% of patients [37, 38] . the type of seizures can be generalized, or focal with or without secondary generalized seizures. the contrast-enhanced mri is more helpful than ct in confirming seizures in patients with tbm [39] . the outcomes of background slowing and epileptiform discharges in eeg and hyponatremia in csf laboratory tests suggest a high risk of seizure occurrence [40, 41] . patients with non-single seizures often have a poor prognosis. because some aeds and antituberculosis drugs have drug interactions [42] , it is controversial whether to administer aed in patients with single seizure. under this condition, cortical involvement and epileptiform discharges are important reference indicators [36] . pml is a demyelinating disease of the cns characterized by extensive lesions ascribed to the infection of oligodendrocytes by the jc virus. it occurs almost exclusively in patients with immunosuppression, and its prevalence in hiv-infected individuals is around 4-5% [43, 44] . a considerable amount of studies have described pml as a crucial cause of seizures in such a population, and the incidence of seizures was found to be 18% [4, 12, 45, 46] . the type of seizures can also be focal or generalized. pcr of csf is a reliable tool for diagnosis. neuroimaging often shows single or multiple fusion lesions without mass effect. nevertheless, patients presenting with seizures are inclined to have demyelinating lesions in the immediate vicinity of the cortex. usually, treatments have satisfying effects on seizures. no noticeable pathological factors could be found among a considerable percentage of hiv+ patients [6, 13] . more and more researchers tend to presume that hive is a consequence of the direct impact of hiv on the cns [21, 47] . an early form of aseptic, hive develops within days to weeks after hiv infection [48] . some researchers found microglial nodules and multinucleated giant cells in the brains of patients at autopsy and believed that this may be the evidence that hiv induces central neurological conditions [16] . patients infected with hiv are prone to serum electrolyte distributions, such as hyponatremia, hypomagnesemia, and hypocalcemia. it is well known that hypomagnesemia can induce seizures. and a crosssectional survey showed that hyponatremia is also a risk factor for seizures with decreased serum sodium levels, resulting in an ascending risk [49] . although electrolyte disturbances are common among hiv+ patients with seizures, only one study clearly stated that two patients had seizures as a consequence of hyponatremia and hypomagnesemia, respectively [20] . hyponatremia is likely to play an important role in inducing seizures. as most patients may have more conspicuous etiology presented (such as oi), the electrolyte disturbances are easily to be neglected. as we all know, hiv and hiv-related factors are high risk factors for stroke. it has been reported that hiv infection increases the risks of both ischemic and hemorrhagic stroke and cns ois also significantly increase the risk of stroke [50] . stroke can lead to acute seizures or post-stroke epilepsy. but few reports have addressed hiv+ patients who developed seizures due to stroke. this may be related to the age distribution of the patients included in cohorts, or may be masked by a more conspicuous cause. the diagnosis of epileptic seizures and epilepsy among hiv+ patients is still based on the distinctive clinical manifestations accounted by witnesses, combined with eeg and features of radiological examinations. the clinical manifestations consist of loss of consciousness (seizures, transients, stereotypes, or repetitive), limb rigidity, locked jaw, gaze, biting the tongue, mouth foaming, and hypermobility. currently, the diagnosis of epilepsy follows the diagnostic criteria established by ilae in 2014 and the classification system by ilae in 2017 [22, 23] , as follows: 1) at least two unprovoked (or reflex) seizures occurring > 24 h apart; 2) one unprovoked (or reflex) seizure and a probability of further seizures similar to the general recurrence risk (at least 60%) after two unprovoked seizures, occurring over the next 10 years; 3) diagnosis of an epilepsy syndrome. the general medical treatment goal of seizures is to control acute symptoms and prevent a recurrence. due to the immunosuppression effect that hiv has on the host, and the frequent combined symptoms such as wasting syndrome in patients, the prevention of seizure and treatment of epilepsy often become a long-term process. therefore, this population is routinely treated medically. surgical therapies or vagus nerve stimulation should be considered if medication-resistant epilepsy exists and is estimated to be effective. surgical treatment is beyond the scope of this article. the following will discuss the aed usage in hiv+ patients. as is specified by the national institute for health and care excellence [51] , aed selection should be rationalized depending on the type of seizures and the different phases and duration of seizures. if patients are in the acute seizure attack phase, the first aim is to control recurrence and reduce complications. intravenous injection of benzodiazepines (such as diazepam) should be given. assessment of a nos is important. if it lasts less than 20 min, status epilepticus is beyond consideration, so oral aeds and treatment of the cause are recommended. unless there is a risk of developing status epilepticus, treatment for the first unprovoked seizure is not advised [52] . any seizure that becomes prolonged or recurrent seizures without recovering consciousness between the seizures can be considered to have progressed to status epilepticus. benzodiazepines (such as lorazepam, diazepam, or midazolam intravenous injection) are still the first-line drugs for initial status epilepticus [53] . when a nos occurs, the decision on how long to take aed treatment is largely related to the risk of recurrence. patients with a 60% risk of recurrence as abnormal eeg or brain lesions should consider the regular course of treatment with aeds. because of the high recurrence rate of seizure in hiv-infected patients, some scholars have suggested that long-term medication plans should be customized once nos appear [21] . however, there are also objections, suggesting short-term aed treatment, on the grounds of avoiding potential side effects of drug and the aed-arv interactions [7] . older-generation aeds often have a drug interaction with arv, especially in the case of joint use of enzymeinducing aeds (carbamazepine, phenobarbital, phenytoin) and nonnucleoside reverse transcriptase inhibitor (nnrti or efavirenz for example) and/or protease inhibitor (pi). this results in either an increase in serum levels of aed or arv, which will increase the risk of potential drug toxicology; or a decrease in serum level, which will lead to virologic failure or seizure recurrence. a study has shown that the combined use of carbamazepine and efavirenz reduces the serum concentration of efavirenz [54] . another study showed that the half-life of nevirapine in patients treated with phenytoin sodium was significantly reduced [55] . this is because this generation of aed is mainly oxidized and metabolized by the cyp450 enzyme system, but efavirenz is both an inhibitor and an inducer of cyp3a4. some aeds are also partially (valproic acid, phenobarbital, or oxcarbazepine) or completely (lamotrigine) eliminated through conjugation. as pis are becoming well-received among hiv+ patients, these two classes of drugs also interact mutually. it has been reported that when combined, lopinavir/ritonavir and atazanavir/ritonavir both lead to a significant decrease in the bioavailability of lamotrigine [56] . however, efavirenz is unlikely to interfere with aeds and make seizure easier to recur; aeds that are mainly dependent on conjugation degradation generally do not affect arvs and result in virologic failure, at the regular dosage of course [42] . as for the first-choice aeds, like levetiracetam, lacosamide, gabapentin, and pregabalin, a common feature of them is that they are neither metabolized via the cyp450 enzyme system nor rely on conjugation to eliminate. the main route of clearance of these aeds is renal excretion. though having no significant drug interactions with arvs [57, 58] , they still face restrictions in application. levetiracetam, which is reported to have the least aed-arv interactions, often causes intolerable neuropsychiatric side effects [59, 60] . and deliberate assessment of the tolerance of patients with impaired renal function should be performed when gabapentin or pregabalin is administered. when conditions are not qualified to use the recommended first-line drugs, valproate can be used instead. in the human body, more than 50% of valproate is oxidized by cyp450, and the remaining is eliminated by conjugation. but so far, there is no clear evidence that its combined use with arv can cause virologic failure. as valproate has dual inhibiting activities on cyp and uridine glucuronyl transferases (ugt), its combined use with arv (zidovudine) or pi (lopinavir/ritonavir) has been reported to increase valproate serum levels [61] . in vitro experiments have shown that valproate stimulates hiv replication [62] , but this conclusion has not been confirmed in vivo. on the contrary, a study reported a significant reduction of latent hiv in resting t cells in 3 of 4 patients when valproate was combined with haar t [63] . therefore, valproate is a promising option when first-line aeds are not available. how to prevent seizure recurrence in hiv+ patients can be viewed from another perspective. since ois account for most noss, managing to reduce the viral load and increase cd4 count are also effective ways to decrease the risk of seizure recurrence. a cohort study found that the incidence decreased by about 1% after art administration [64] . another strictly controlled retrospective case-control study showed that earlier implementation of combination antiretroviral therapy (cart) was likely to prevent hiv-infected children from eventually developing epilepsy [65] . a study even suggested that it was safe to apply more than one aeds in hiv+ patients currently treated by cart, as it was helpful to restore the host's immunity, as long as its cumulative dosage was under an adaptable level [66] . based on the above discussion, it is prospective to have a new therapeutic conception as co-administered aed and arv to offset mutual drawbacks without an ascending drug toxicology risk. compared to adults, children with hiv infection are more sensitive to medication dose and are more vulnerable to side effects [67, 68] . children are at a stage of growth and development, and are susceptible to the developmental delay and even cognitive developmental delay due to the adverse effects of hiv on the brain. therefore, therapeutic protocols in children should keep balance between the efficacy and side effects of aeds and arvs. at present, there is no worldwide guideline on the optimal medication for children, and the department of health in south africa recommends sodium valproate as a first-line drug [13] . but what is worth noting is that in some poor-resource regions, the old generation of aeds is the only choice to reach. as mentioned above, performing cart at the correct time can reduce the risk of seizures, which is of vital importance for prognosis in hiv-infected children. as the immune system of children is not well-developed, hiv infection at this stage may deteriorate the immune system and result in seizures. studies showed that there was no significant difference in the percentage of cognitive impairment between children with early and delayed administration of cart [65, 69] . instead, children who deferred the treatment are prone to developmental delays and even cognition obstacles due to hive or other cns diseases complicated by hiv [70] . this suggests that the benefits of early implementation of cart may outweigh the more cumulative drug toxicity from longer use. and given the special situation in africa, some scholars have suggested that art should be carried out once children have any cns complications [71, 72] . over the decades, enormous amounts of research have been carried out on the prevalence and incidence of seizure and epilepsy among hiv-infected populations. however, it is a challenging task to precisely describe the distribution of seizures and epilepsy among the hiv+ population, due to the following four reasons. 1) up to now, nearly all the relevant studies are retrospective instead of being prospective, which may inappropriately include the cases who had an undocumented seizure before hiv infection, thereby exaggerating the incidence of nos. 2) most of these studies were performed in selected cases from one or two local medical establishments, and large-scale cross-sectional investigation is imperatively needed. the available findings may have geographic limitations and lack broader geographical representation. for example, the conclusions drawn from neurological medical centers may show a higher prevalence than from the general hospitals, because more refractory patients are referred to the former. results of local hospitals may underestimate the prevalence as it is difficult to guarantee the representativeness of the sample patients for the whole hiv-infected population. 3) many of the previous studies focused on the incidence or prevalence of seizures but neglected the development of epilepsy. in the past 20 years, the diagnostic and classification criteria of epilepsy and seizures have changed several times. for some quite early studies, it is hard to determine whether the diagnosis was based on internationally recognized diagnostic standards and whether the diagnosis at that time could be classified into the corresponding revised diagnostic entries nowadays. 4) in some studies reporting epilepsy prevalence and incidence, the diagnostic standards was not clarified. as these studies used a retrospective setting, the authenticity of the data may be questionable. the frequent types of epilepsy and seizures are also controversial. it is well known that the seizures caused by acute cns infections secondary to hivimmunological suppression are mostly focal, but what's the paradox is that generalized seizures dominate in a large number of reports [6, 9, 13, 18, 21, 72] . this may be explained by the possibility that the secondary generalized seizures were mistakenly regarded as generalized seizures, that is, the initial focal symptoms were not witnessed. this contradiction also reflects a pervasive retrospective bias that cannot be ignored in these retrospective studies. suggestion on this section: we appeal for more prospective studies in the future to eliminate the retrospective bias. before describing the occurrence of seizures or epilepsy, a statement of the diagnostic procedure (or obedience of the acknowledged diagnostic criteria) should be delivered. and it is advisable to distinguish the chronological order between nos and hiv infection when making medical records. however, when the time point of hiv infection cannot be determined, the art start time is recommended as an alternative. although hive has been largely reported in numerous studies, it remains doubtful whether hive is as frequent as reported. according to the centers for disease control and prevention, hive cannot be diagnosed unless the patient meets at least one of its criteria for ≥2 months [73] . only few studies stated that the diagnosis of hive strictly followed the standard [14] . most literature merely attributed hive to seizures in patients with no obvious etiology. considering that these studies were mainly retrospective, the incidence of hive may have been exaggerated. suggestion on this section: the majority of hiv+ patients with seizures are often affected by several etiological factors, and these risk factors may reinforce each other. it is recommended that researchers carefully check for potential causes that could be missed. for prospective studies, a well-recognized diagnostic criterion should be used. for retrospective studies, hiv infection cannot be blindly attributed to patients with no obvious pathogenic factors. the current mainstream aed guidelines are based on the results of clinical trials in the general population, rather than being specific to the hiv+ population. seizures in hiv+ patients are mostly a consequence of ois. it is common to administer multiple drugs for different pathological conditions at the same time, considering the patients' antidote competence, drug interactions, toxicology, and side effects. sometimes even giving up medication for a certain etiological agent should be considered. suggestion on this section: we call for cohort studies that specifically include hiv+ epilepsy patients. choosing drugs with different metabolic pathways may decrease the likelihood of drug-drug interactions. in summary, this paper discuss the clinical characteristics and treatment protocols of hiv+ patients with seizures or epilepsy. as a frequent serious complication of hiv infection often caused by ois, seizures have a prevalence of 2-19.8% and incidence of 1.8-19.8% among the hiv-infected population, and mostly appeared in a generalized type. the contemporary medication therapy for seizures or epilepsy is welldeveloped, but largely based on the general population. future therapeutic studies in hiv+ patients with seizures are urgently desired. psychiatric disorders among people living with hiv/aids in iran: prevalence, severity, service utilization and unmet mental health needs hiv-1-associated central nervous system dysfunction hiv infection and risk of cardiovascular diseases beyond coronary artery disease neuroaids in children association of hiv infection with epilepsy and other comorbid conditions frequency of seizures and epilepsy in neurological hiv-infected patients seizures in hiv: the case for special consideration new-onset seizures among hiv infected drug naïve patients from south india prevalence and incidence of new-onset seizures and epilepsy in patients with human immunodeficiency virus (hiv): systematic review and meta-analysis estimating prevalence, incidence, and disease-relatedmortality for patientswith epilepsy in managed care organizations systematic review and meta-analysis of incidence studies of epilepsy and unprovoked seizures clinical features of seizures in patients with human immunodeficiency virus infection prevalence of seizures in children infected with human immunodeficiency virus seizures in children with hiv infection in south africa: a retrospective case control study new-onset seizures among hiv infected drug naive patients from south india seizures in human immunodeficiency virus infection new-onset seizures associated with human immunodeficiency virus infection: causation and clinical features in 100 cases prospective analysis of seizures occurring in human immunodeficiency virus type-1 infection seizures in human immunodeficiency virus-associated cryptococcal meningitis: predictors and outcomes prospective study of new-onset seizures in patients with human immunodeficiency virus infection: etiologic and clinical aspects seizures in hiv-seropositive individuals: nimhans experience and review ilae official report: a practical clinical definition of epilepsy operational classification of seizure types by the international league against epilepsy: position paper of the ilae commission for classification and terminology status epilepticus-related etiology, incidence and mortality: a meta-analysis safe treatment of seizures in the setting of hiv/aids tuberculous meningitis: diagnosis and treatment overview incidence of epilepsy and brain toxoplasmosis in hospital mario rivas, honduras aids and epilepsy detection of toxoplasma gondii in cerebrospinal fluid from aids patients by nested pcr and rapid identification of type i allele at b1 gene by rflp analysis clinical features of seizures in hiv patients with toxoplasma encephalitis: 32nd international epilepsy congress barcelona, spain 2nd -6th costeffective diagnostic checklists for meningitis in resource-limited settings global burden of disease of hiv-associated cryptococcal meningitis: an updated analysis relationship of cerebrospinal fluid pressure, fungal burden and outcome in patients with cryptococcal meningitis undergoing serial lumbar punctures clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america adult tuberculous meningitis in qatar: a descriptive retrospective study from its referral center new-onset seizures in adults with tuberculous meningitis during long-term follow-up: characteristics, functional outcomes and risk factors tuberculous meningitis tuberculous meningitis in adults: review of 61 cases central nervous system tuberculosis : etiology, clinical manifestations and neuroradiological features eeg changes in tuberculous meningitis: a clinicoradiological correlation seizures in tuberculous meningitis antiepileptic drug interactions -principles and clinical implications progressive multifocal leukoencephalopathy: what's new? mapping the history and current situation of research on john cunningham virus -a bibliometric analysis aids and epilepsy seizures and their outcome in progressive multifocal leukoencephalopathy new onset seizures in hiv-infected patients without intracranial mass lesions or meningitis--a clinical, radiological and spect scan study a retrospective clinical, laboratory and outcome analysis in 43 cases of acute aseptic meningitis hyponatremia and risk of seizures: a retrospective cross-sectional study association of hiv and opportunistic infections with incident stroke: a nationwide populationbased cohort study in taiwan epilepsies: diagnosis and management. london: national institute for health and care excellence diagnosis and treatment of the first epileptic seizure: guidelines of the italian league against epilepsy a comparison of lorazepam, diazepam, and placebo for the treatment of outof-hospital status epilepticus pharmacokinetic interaction between efavirenz and carbamazepine after multiple-dose administration in healthy subjects brief report: enzyme inducers reduce elimination half-life after a single dose of nevirapine in healthy women lopinavir/ritonavir reduces lamotrigine plasma concentrations in healthy subjects pregabalin pharmacology and its relevance to clinical practice special populations: the management of seizures in hiv-positive patients medical management of epileptic seizures: challenges and solutions pharmacokinetic interaction between zidovudine and valproic acid in patients infected with human immunodeficiency virus valpoic acid reduces the intracellular level of glutathione and stimulates human immunodeficiency virus depletion of latent hiv-1 infection in vivo: a proof-of-concept study neurologic disorders incidence in hiv+ vs hiv-men: multicenter aids cohort study early antiretroviral therapy is protective against epilepsy in children with human immunodeficiency virus infection in botswana clinical outcomes and immune benefits of anti-epileptic drug therapy in hiv/aids lamotrigine-associated rash: risk/benefit considerations in adults and children adverse cutaneous reactions associated with the newest antiretroviral drugs in patients with human immunodeficiency virus infection cognitive function and neurodevelopmental outcomes in hiv-infected children older than 1 year of age randomized to early versus deferred antiretroviral therapy: the predict neurodevelopmental study neurocognitive functioning in pediatric human immunodeficiency virus infection: effects of combined therapy hiv-1-related encephalopathy in infants compared with children and adults. french pediatric hiv infection study and the seroco group neurologic and neurobehavioral sequelae in children with human immunodeficiency virus (hiv-1) infection revised classification system for human immunodeficiency virus infection in children less than 13 years of age not applicable. key: cord-021668-33zfio0u authors: tyring, stephen k. title: syndromal tropical dermatology date: 2009-05-15 journal: tropical dermatology doi: 10.1016/b978-0-443-06790-7.50005-3 sha: doc_id: 21668 cord_uid: 33zfio0u nan stephen k. tyring with increasing numbers of persons from industrialized, temperate countries traveling and/or working in tropical lands, there is a marked need for physicians to be able to diagnose accurately and treat tropical diseases with mucocutaneous manifestations. while some studies demonstrate that approximately one-third to two-thirds of travelers returning from tropical countries experience some health problem, diarrhea is the most prevalent complaint. mucocutaneous problems, however, are among the top five health complaints of the returned traveler, and comprise 10-15% of health concerns of persons returning from the tropics. 1 during international conflicts, soldiers from north america, europe, and australia are often required to serve in tropical lands and sometimes develop diseases not familiar to physicians of their home countries. this was the case for french soldiers serving in vietnam in the 1950s and american soldiers serving there in the 1960s and 1970s. recently hundreds of american and allied troops serving in iraq and afghanistan have developed "baghdad boils," i.e., leishmaniasis, transmitted by sand fly bites ( fig. 1.1) . likewise, millions of persons from tropical countries now live and work in temperate lands and may present with medical problems with which the physician is not familiar. whereas the cutaneous problems in the returned traveler are frequently the result of infectious diseases, skin diseases of non-infectious etiologies usually predominate. such non-infectious sources of skin problems include excessive sun exposure, cutaneous reactions to medications taken for prophylaxis (including phototoxic reactions) or exposures to marine, freshwater or other irritants. furthermore, whether it is the traveler or the immigrant presenting to the physician, many cutaneous complaints are unrelated to the person's travel or national origin, but are the same conditions seen daily in every physician's office. therefore, the physician should not ignore the common sources of dermatological problems while searching for an exotic etiology. another, somewhat recent source of patients with tropical skin diseases are adoptees who frequently originate in central america or southeast asia. these children could be infected with organisms having a long incubation period that may not have been detected by physical examinations and not preventable by available vaccines. tropical infections in temperate lands, however, are not totally unique to travelers. for example, the outbreak of monkeypox in wisconsin, usa, in 2003 was a result of prairie dogs acquiring the virus from gambian rats housed in adjacent cages in pet stores. the prairie dogs then transmitted the infection to humans who had never been near the usual range of monkeypox, i.e., central africa. occasionally, the patient with a tropical disease is neither the traveler nor exposed to an animal carrying an infectious agent. the carrier may be a friend or relative who is a returned traveler who has acquired a tropical infection and who has not yet developed signs or symptoms. this possibility has recently been given much attention due to the potential spread of severe acute respiratory syndrome (sars) or avian influenza virus. on the other hand, the contaminated food may have originated in a tropical or subtropic area, such as when oysters from the gulf of mexico are shipped to the midwest usa and are consumed raw. the resulting vibrio vulnificus or hepatitis a infection thus produces gastrointestinal and cutaneous manifestations in persons who may not have visited the source of the shellfish. therefore, it is always important to ask about new pets, changes in diet, or any other change in persons with a suspected tropical disease. on the other hand, the traveler may have purchased non-consumable items which are the source of their dermatoses. for example, animal skins used for rugs or blankets may be the source of anthrax. a non-infectious cause may include nickel-containing jewelry, to which the patient has developed contact dermatitis. whereas travelers naturally fear large carnivores while on camera safari or sharks and a variety of other aquatic animals while swimming or diving, it must be remembered that the animal (indirectly) responsible for most morbidity and mortality is the mosquito (i.e., malaria, dengue, etc.) ( fig. 1.2 ). an example of a mosquito-borne disease that was considered primarily "tropical" in the recent past but is now relatively common in much of north america is infection with the west nile virus ( fig. 1.3 ). sometimes the skin findings on physical examination are not the reason for the physician visit or even the patient's complaint. such skin findings may be cultural, such as tattoos, scarification, or the result of the use of kava or of chewing betel nuts. some cultural practices, however, would be considered abuse in industrialized countries, but are widely accepted religious/cultural practices in certain lands. an example of such practice is female circumcision, which is practiced in many countries in sub-saharan africa. on the other hand, the skin changes may be much more benign, transient, and may even be the result of previous therapies, such as cupping and coining, widely practiced by immigrants from southeast asia. considerations for deciding the differential diagnosis of cutaneous manifestations of tropical diseases and/or of diseases acquired while traveling must be based not only on the type of lesions and systemic symptoms but also on the patient's history of travel. because the incubation period of various infectious diseases differs widely, it is important to know when the patient traveled. for frequent travelers, the history may become complex if they report having visited many destinations within the past few months. because vectors differ with the climate, the season of travel is also noteworthy. even in a tropical country where the temperature is always hot or warm, there may be a dry season and a rainy season. because seasons are reversed north and south of the equator, it is important to know the season at the destination. the duration of the stay is significant, not only because it increases the chance of acquiring an infectious disease but also because it tells the physician if the person was in the tropics during the incubation period of the suspected disease. whether the visitor was only in an urban environment or also in a rural area is relevant. whereas a sexually transmitted disease could be acquired in either location, an arbovirus or a zoonosis might be more likely in a rural situation. the altitude of the destination could provide a clue to the etiology of the skin condition, as could the type of sleeping condition. for example, a sexually transmitted disease could easily be acquired in a five-star hotel, but an infection transmitted by a flea, louse, or mite would be more likely in someone who slept on the ground and/or in a tent. the type and preparation of food and drink consumed by the traveler would not only help explain gastrointestinal symptoms; it could also be a clue to cutaneous signs, i.e., unsafe drinking water or milk or raw or undercooked meat, fish, or shellfish. a list of the patient's current and recent medications can be very useful and should include prescription drugs, illicit drugs, and herbal remedies, because the source of the cutaneous problem may not be directly related to the travel destination, but rather may be due to medications taken to prevent travel-related illnesses. for example, many antimalarials, such as chloroquine, mefloquine, proguanil, quinine, and halofantrine, can cause cutaneous reactions, and chloroquine, doxycycline, and quinine can cause photosensitivity. interestingly, chloroquine can worsen psoriasis. a number of agents taken to treat or prevent diarrhea can also cause cutaneous reactions, such as quinolones (ciprofloxacin, ofloxacin, sparfloxacin, levofloxacin), furazolidone, metronidazole, trimethoprim-sulfamethoxazole and bismuth sulfate; quinolones are particularly likely to produce photosensitivity. antihelmintic medications, such as ivermectin, albendazole, and diethylcarbamazine, can also produce pruritus and rash. even diethyltoluamide (deet), used to prevent arthropod bites, can cause an irritant dermatitis when used in high concentrations. because many medications in tropical countries are sold over the counter and/or have different trade names than in industrialized lands, patients are not always certain what they received if treated during their travel. likewise, an injection or transfusion given in a tropical country might also carry an increased risk of contamination. a similar risk might be taken by having acupuncture, tattoos, or body piercing in tropical lands, but these procedures can be hazardous even in industrialized countries, because the first intervention is occasionally done by non-medical personnel and the other two are almost never done by medically trained persons. a history of pre-travel vaccinations and/or immunoglobulins would be useful for possible exclusion of certain suspected etiologies. for example, if the yellow fever vaccine and the hepatitis a and/or b vaccine series were administered in sufficient time before the travel, it is less likely that these viruses were the source of the medical complaint. the traveler's occupational or recreational exposure to dirt, water, or animals can be an important component of the history. an animal bite or scratch should be easy to remember, but the bite of many arthropods may not even be noticed until after a cutaneous reaction has appeared and the responsible fly, mite, or flea has moved on to the next victim. exposure to some animals may be more indirect. for example, the spelunker (cave-explorer) may inhale aerosolized bat guano and develop rabies without ever touching a bat. a history of swimming, boating, or surfing can be a clue to an aquatic/marine etiology. such fresh-or brackish-water activities may increase the risk of infection with schistosomiasis or with free-living ameba, while marine activities may be associated with jellyfish stings, contact with the venomous spines of certain fish, or irritant dermatitis from fire coral. a preexisting skin abrasion or laceration or a puncture wound from a sea urchin or sting ray may result in a secondary bacterial infection. thus, a complete medical and travel history and physical examination is imperative in helping to narrow the differential diagnoses in the returned traveler, the immigrant, or the adoptee with a tropical origin. the qualitative and quantitative nature of the skin lesions are very important and are discussed in detail later in this chapter. specific attention must be given to the age of the patient as well as to the person who is immunocompromised due to human immunodeficiency virus (hiv), internal malignancy, organ transplantation, or other iatrogenic source of immunocompromise. blood tests, e.g., liver/kidney function tests, complete blood counts with differentials, urinalysis, skin scraping, biopsy, and/or culture are often necessary to confirm the diagnosis. a recent example of the importance of knowing both the patient's national origin and their immune status was seen when an hiv-seropositive man from myanmar presented with the first case of penicillium marneffei reported from houston, tx ( fig. 1.4) . many viral diseases that were not considered "tropical" 50 years ago are now much more frequently seen in immigrants from tropical countries or in travelers who did not receive their recommended childhood vaccines. three common examples are measles, rubella, and hepatitis b. until the 1960s measles and rubella were very common sources of infection in temperate countries, but in the twenty-first century they have become rare in industrialized countries, except for imported cases. worldwide, however, almost one million children die of measles annually ( fig. 1 .5) and rubella still causes many congenital abnormalities. measles is still the number one vaccine-preventable killer of children in the world. morbidity and mortality are often the result of secondary bacterial infections developing in malnourished infants with measles ( fig. 1.6 ). in east asia, sub-saharan africa, and many other parts of the tropical world, hepatitis b is very common and a major source of morbidity and mortality. although measles, rubella, and hepatitis b should not be a problem in the immunized traveler, many travelers have not received the proper immunizations because they or their parents had unfounded concerns about the safety of the vaccines. this problem continues to grow as more people reach child-bearing age without ever knowing anyone who has suffered from the childhood diseases common in the first half of the 20th century. therefore, they do not understand that the approved vaccines are a million-fold safer than the diseases they are designed to prevent. sexually transmitted diseases (stds) should be considered at the top of the differential diagnoses when a patient presents with syndromal tropical dermatology genital lesions and/or urogenital discharge. [2] [3] [4] although many of the same considerations would be true whether the patient was a recent traveler or not, certain factors should be given attention in travelers: ■ was the person traveling without his/her spouse/family and therefore outside his/her usual social structure? ■ did the person travel to countries where sex workers are readily available? while sex workers are available in most parts of the world, legally or illegally, the traveler might be less likely to acquire an std in mecca during a haj than in amsterdam, bangkok, or nairobi, where sex workers are very prevalent. ■ did the person attend parties where large amounts or alcohol and/or drugs were consumed (e.g., "spring break" in the usa)? ■ if the traveler is strongly suspected of having an std, did he/she visit a destination where chancroid, granuloma inguinale (gi), or lymphogranuloma venereum (lgv) (l serovars of chlamydia trachomatis) is prevalent? if so, the diagnostic tests and therapy might need to be expanded beyond those under consideration for stds acquired in temperate lands. when one std is confirmed, there is an increased possibility of acquisition of additional stds. not only is this the case because the source partner(s) may have had multiple stds, but also because having certain stds makes a person more susceptible to other stds. the best example of this phenomenon is the twoto fivefold greater risk of acquiring hiv if the person with a genital ulcer disease (gud) has sex with an hiv-positive individual. the reasons for this increased risk include the reduced epithelial barrier in all guds as well as the infiltrate of cd4+ cells in certain guds such as genital herpes. these cd4+ cells are the targets for hiv infection. genital herpes is the most prevalent gud in industrialized countries. in fact, the centers for disease control and prevention estimate that there are 45 million herpes simplex virus type 2 (hsv-2)-seropositive persons in the usa. in the tropics, chancroid has been the most frequently diagnosed gud, followed by syphilis and genital herpes, but the last two diseases are becoming more prevalent in certain tropical countries. depending on the travel destination, lgv and gi must also be considered. the dates and duration of travel are important components of the history because the primary clinical presentation of all these guds ranges between 2-3 days (genital herpes and chancroid) and 4 weeks (syphilis and gi). currently, the world health organization (who) estimates that there are 46 million hiv-seropositive persons in the world. many of these people have gud, which may be changed both qualitatively and quantitatively by hiv. therefore the traveler may have a "non-classical" presentation of gud. in addition, it should be remembered that the signs and symptoms of gud can also appear on the perianal area/buttock or in or around the mouth. other locations are possible, but less likely. in general, however, multiple, painful, usually bilateral vesicles which progress to ulcers on skin or start as ulcers on mucous membranes, then heal over after 3-4 without therapy or within 2-3 weeks with antiviral therapy, are consistent with genital herpes. because most true primary cases of genital herpes recur, a history of multiple recurrences of the vesicles or ulcers is highly consistent with genital herpes. this diagnosis can be confirmed by viral culture or serology. in the absence of these tools, a useful test is the tzanck smear, which usually demonstrates multinucleated giant cells in herpetic lesions, but is of low sensitivity and specificity. genital herpes, however, can present many diagnostic dilemmas because the first recognized clinical occurrence is often not the result of a recent infection but rather represents a first-episode, non-primary outbreak. whereas a true primary outbreak of genital herpes is usually consistent with acquisition of the virus 2 days to 2 weeks previously, a first-episode, non-primary outbreak may be consistent with an infection any time in the past. in this case, the patient's recent travel history may be of less importance than his/her sexual encounters of the most distant past. although syphilis is much more common in many developing countries than in the usa, western europe or australia, the lack of travel certainly does not exclude syphilis. this diagnosis should be suspected when the patient presents with a single, non-tender, genital, perianal, or lip ulcer associated with non-tender lymphadenopathy. while chancroid is uncommonly reported in industrialized countries, it is very common in the tropics. it is usually characterized by one or more painful genital ulcers and painful syndromal tropical dermatology lymphadenopathy. in lgv the primary lesion is usually very transient and is often not seen. the clinical presentation is usually that of tender inguinal lymphadenopathy, sometimes with a suppurating bubo. the diagnosis of gi is very rarely made outside the tropics. the presentation is usually that of one or more non-tender genital ulcers with inguinal swelling. if any of these bacterial guds is suspected, the appropriate diagnostic tests must be initiated, i.e., serology for syphilis and lgv, culture for chancroid and lgv, tissue examination for gi, and the appropriate antibiotic started. whereas a history of multiple recurrences of genital vesicles or ulcers would be consistent with genital herpes, a more difficult scenario is represented by the patient who reports a single outbreak of non-specific genital signs and symptoms that are resolved by the clinic visit. a western blot or type-specific serological test for hsv-2 would determine whether the person was infected with this virus, but it would not be definitive proof that that hsv-2 was responsible for the resolved outbreak. for example, a hsv-2 serologically positive person may acquire syphilis, but the genital ulcer may resolve without therapy, or with inadequate treatment, before the clinic visit at home. thus, a careful history may reveal the need for serology for hsv-2 as well as for syphilis. because hiv can be acquired concomitantly with or subsequently to these guds, but not produce genital manifestations, hiv testing should be conducted as well. although many patients may be hesitant to admit sexual activity that puts them at risk for stds, others will worry about these activities following travel (or any time) and ask to be tested for "everything." if the sexual encounter with a new partner has been very recent, the serological test may be falsenegative because serology for syphilis, hiv, or hsv-2 may require weeks to become positive in the majority of persons after initial infection. patients who ignore their primary genital lesions because of denial or difficulty finding medical care during their travels may believe that the problem is gone because the lesion has resolved. if syphilis is the cause of the gud, it may reappear weeks or months later as non-genital cutaneous manifestations in the form of secondary (or tertiary) syphilis ( fig. 1.7) . a careful history regarding the primary lesion may lead to the appropriate diagnostic tests and therapy. some stds may not produce any genital signs or symptoms and the disease may be diagnosed long after the travel (or the non-travel acquisition), making it more difficult to find the source of the infection. although over 90% of hiv-seropositive persons eventually develop indirect mucocutaneous manifestations of infection, the primary rash of seroconversion (if present) is not noticed by most patients. therefore, the diagnosis is usually made when the patient develops systemic signs and symptoms (fever, chills, diarrhea, weight loss, lymphadenopathy) and/or develops one or more of the opportunistic infections, neoplasms, or inflammatory skin problems frequently seen in hiv patients. similar to hiv, primary infection with hepatitis b rarely produces genital lesions. diagnosis is usually made long after infection due to systemic symptoms or non-specific skin changes such as jaundice. it is noteworthy that hepatitis b is the only std for which a prophylactic vaccine is available. therefore, a history of successful hepatitis b vaccination makes this diagnosis less likely. although the pustules of disseminated gonococcemia are distinctive, the consequences of untreated neisseria gonorrhoeae (gonococcus) are usually pelvic inflammatory disease, epididymitis, proctitis, pharyngitis, or conjunctivitis. pelvic inflammatory disease can also be caused by chlamydia trachomatis (non-l serovars), mycoplasma hominis, or various anaerobic bacteria. the non-l serovars of c. trachomatis can also cause epididymitis, proctitis, and conjunctivitis. pharyngitis can also be due to hsv-2 or entamoeba histolytica. the initial presentation of gc, c. trachomatis (non-l serovars), ureaplasma urealyticum, mycoplasma genitalium, trichomonas vaginalis, or even hsv-2 may be urethritis. vaginal discharge can be caused by any of these organisms as well as candida albicans, gardnerella vaginalis, peptostreptococci, bacteroides spp. or mobiluncus spp. these organisms can usually be diagnosed by smear, wet-mount, dna detection, serology, or culture. antimicrobial therapy is usually initiated based on the physical examination and smear or wet-mount and modified, as needed, when other laboratory studies are completed. infection with human papillomaviruses (hpv) is one of the most common stds in the world, but the clinical implications of the infection vary widely. there are over 20 hpv types that can cause genital lesions, but most infections do not result in any visible lesions. because the incubation period of hpvs can be months, or even years, if and when genital lesions do develop, it is often very difficult for the patient to determine the source partner. therefore, it is usually a challenge for the physician to relate hpv lesions to travel, especially recent travel. non-oncogenic genital hpv, such as types 6 and 11, result in condyloma acuminatum, which can be treated with cytodestructive therapy, surgery, or with the immune response modifier, imiquimod. oncogenic genital hpv, such as types 16 and 18, can result in anogenital cancer, the most prevalent of which is cervical cancer. cervical cancer is the second most prevalent cancer killer of women in the world and sexually transmitted diseases over 99% of all cervical cancer is caused by hpv. most cervical cancer deaths are in tropical countries, making hpv one of the world's deadliest tropical diseases (although rarely listed with the other major tropical diseases). there are many reasons why more cervical cancer deaths occur in tropical countries. first, in industrialized countries most women receive regular pap smears, which result in early detection and subsequent therapy of cervical abnormalities, thus reducing progression to cervical cancer. if cancer is detected, surgery, radiation therapy, or chemotherapy is available. in most tropical countries, regular pap smears are not the standard of care. therefore, cervical cancer is often detected too late for successful intervention, even if it is available. second, there appears to be a genetic susceptibility that allows oncogenic hpv to progress to malignancy. this genetic susceptibility appears to be more prevalent in certain tropical countries. the rarity of male circumcision in many tropical countries appears to be a risk factor for the development of cervical cancer in these males' partners. third, most of the world's estimated 46 million hiv-seropositive individuals live in tropical countries where no antiretroviral therapy is available. not only is cervical cancer an acquired immunodeficiency syndrome (aids)-defining illness in hiv seropositive women, the same hpv can cause anal cancer, which is a major problem in homosexual men with hiv. molluscum contagiosum (mc) is a poxvirus that can be sexually transmitted, resulting in wart-like lesions on the genitalia. in contrast to hpv, however, mc does not progress to malignancy. like condyloma acuminatum, however, mc can be treated with cytodestructive therapy, surgery, or imiquimod. ectoparasites such as scabies, sarcoptes scabiei, and pubic lice, phthirus pubis, can be sexually transmitted. in contrast to many stds, however, these ectoparasites can be easily treated with topical medications such as lindane or permethrin. like all stds, if the sexual partner is not treated concomitantly, reinfection is common. the most common cause of fever after tropical travel is malaria, which usually does not have specific cutaneous manifestations. dengue fever is the second most common cause of fever in the traveler and does have somewhat specific cutaneous manifestations, making dengue fever the leading cause of fever with rash in the traveler returning from a tropical destination ( fig. 1.8) . other common causes of fever and rash include hepatitis viruses, rickettsia, and some enteric fevers. it should always be kept in mind, however, that fever in the returned traveler may not be due to exposure during travel. for example, the fatigue of travel, i.e., jet lag, may make one more susceptible to influenza or other common infections in temperate lands. when both fever and rash are seen, the time between travel and onset of signs and symptoms becomes increasingly important. if travel preceded fever and rash by less than 1-2 weeks, considerations should include anthrax, dengue fever, diphtheria, ehrlichiosis, hemorrhagic fever viruses, leptospirosis, lyme disease, measles, meningococcal infections, plague, rickettsia, toxoplasmosis, trichinosis, tularemia, typhoid fever, and yellow fever. if the period between travel and fever/rash is up to a month, the list should be expanded to include hepatitis viruses (a, c, and e), hiv, rubella, schistosomiasis and trypanosomiasis. if at least 3 months separate travel from fever/rash, the following infections should be considered: bartonellosis, filariasis, gnathostomiasis, hepatitis viruses (b and c), histoplasmosis, hiv, leishmaniasis, lyme disease, melioidosis, penicilliosis, syphilis, trypanosomiasis, and tuberculosis. in each case, however, the nature of the fever, the type of rash, the destination of the travel, and any other symptoms must be considered. because many infections producing fever with rash can be rapidly fatal and/or easily spread, it is imperative immediately to initiate diagnostic tests and antimicrobial therapy for the presumed cause of the infection. such infections include anthrax, bartonellosis, candida (macronodules), diphtheria, disseminated gonorrhoeae (papules and pustules over joints), hepatitis viruses, leptospirosis, meningitis (asymmetrical, scattered, petechiae, and purpura), plague, pseudomonas (ecthyma gangrenosum), relapsing fevers, rickettsia (scattered petechiae and purpura), staphylococcus (osler's nodes, diffuse toxic erythema), streptococcus (janeway lesions, diffuse toxic erythema), strongyloides (migratory petechiae and purpura), syphilis, tuberculosis, typhoid fever (rose spots) ( fig. 1.9 ), various gram-negative bacteria (i.e., peripheral gangrene), vibrio (especially v. vulnificus), and viral hemorrhagic fevers (petechiae, purpura, hemorrhage). eosinophilia may be due to diverse processes, such as allergic, neoplastic, and infectious diseases. 5 although an allergic reaction could easily result from an exposure during travel, eosinophilia in the returned traveler may have nothing directly to do with the travel. on the other hand, it may be due to an infectious process or to a drug taken for prophylaxis or therapy during travel. if an infection is the cause of the eosinophilia, it is usually a parasitic disease, especially due to a helminth. only a few viral, bacterial, or fungal diseases are associated with both rash and eosinophilia, e.g., streptococcal (i.e., scarlet fever), tuberculosis, hiv, and coccidioidomycosis. protozoa only rarely provoke eosinophilia. the principal helminth that causes eosinophilia is strongyloides. when strongyloides is disseminated such as in the hyperinfection syndrome, skin lesions such as urticaria, papules, vesicles, petechiae, and migratory serpiginous lesions become common, especially if the patient is given systemic corticosteroids (because strongyloides was not considered). pruritic, erythematous papules can be seen as a result of schistosomal cercariae, as in swimmer's itch. eosinophils may be seen in the skin biopsy as well as in the blood. pruritic lesions of the skin and subcutaneous tissues are commonly associated with eosinophilia in onchocerciasis. lymphangitis, orchitis, and epididymitis are also commonly observed. in loiasis, fever and eosinophilia are typically seen. migratory lesions, especially angioedema, are usually erythematous and pruritic. likewise, gnathostomiasis produces recurrent edema after ingestion of raw fish. the skin lesions are usually erythematous, pruritic, and/or painful. drug hypersensitivity is a relatively common cause of eosinophilia and may be associated with non-specific skin changes such as urticaria and/or phototoxic reactions. although most drugs that cause eosinophilia may not be taken for purposes related to traveling, increased sun exposure during travel may make the problem clinically apparent. because antibiotics may be taken for prophylaxis or therapy more frequently during traveling, they should be given careful consideration when eosinophilia is detected. such antibiotics include penicillins, cephalosporins, quinolones, isoniazid, rifampin, and trimethoprim-sulfamethoxazole. non-specific cutaneous manifestations of tropical diseases may include pruritus and urticaria. frequently, more specific signs may accompany pruritus and urticaria, which are useful in narrowing the differential diagnoses. if eosinophilia is found with the pruritus and urticaria, helminthic infections should be considered. therefore, consideration should be given to trichinellosis, strongyloidiasis, schistosomiasis, onchocerciasis, loiasis, hookworms, gnathostomiasis, dracunculiasis, and cutaneous larva migrans. pinworms, as well as protozoa such as amebiasis, giardiasis, and trypanosomiasis, are less likely to produce eosinophilia. pruritus and urticaria are possible with spirochetes such as borrelia (e.g., relapsing fevers), spirillum (e.g., rat-bite fever) and treponema (i.e., syphilis and pinta). yersinia (e.g., plague) is another bacteria that produces pruritus and urticaria, which can be present before buboes form. the hepatitis viruses (e.g., a, b, and c) can produce pruritus and urticaria, as can a number of ectoparasites and biting arthropods, e.g., ticks, scabies, bedbugs, lice, fleas, mites, and flies. [6] [7] [8] [9] [10] jaundice although hepatitis viruses can produce pruritus and urticaria, jaundice is a more specific indication that the problem has a hepatitic etiology. not only can all the hepatitis viruses (a-e) produce jaundice, other tropical viruses also do so commonly, e.g., yellow fever and rift valley fever. less frequently, dengue and epstein-barr viruses can cause jaundice, as can such bacteria as leptospira (i.e., leptospirosis), coxiella (i.e., q fever) and treponema (i.e., syphilis). protozoa, such as malaria, and drug reactions can also be responsible. although vesicles and bullae can appear as a result of contact dermatitis or drug eruption, including photodermatitis and photoexacerbated drug eruptions as well as toxic epidermal necrolysis, many cases represent the early stages of a viral or bacterial infection. the most common viral etiology in the traveler or non-traveler includes the herpesviruses, especially herpes simplex virus 1 and 2, as well as varicella-zoster virus, both primary varicella and herpes zoster. measles and many enteroviruses (e.g., hand, foot, and mouth disease) can present with vesicles, as can certain alphaviruses. a number of poxviruses, such as vaccinia, variola, orf, tanapox, and monkeypox, can produce vesicles. less commonly, vesicles comprise an early stage of certain bacterial diseases such as vibrio vulnificus, bacillus anthracis, brucella spp., mycobacteria tuberculosis, mycoplasma spp., rickettsia akaru and staphylococcus (bullous impetigo). other organisms such as fungi that cause tinea pedis, protozoa (e.g., leishmania brasiliensis), and helminths (e.g., necator americanus) can occasionally cause vesicles. a wide variety of infectious and non-infectious etiologies are related to both macules and to papules. almost any of the vesicular diseases listed above may initiate first as a macule, then a papule, before becoming a vesicle. a number of drugs, arthropod bites (e.g., mosquito or flea) and infestations (e.g., scabies and other mites) commonly cause macules and/or papules. the range of terrestrial, freshwater, and marine contactants can elicit these cutaneous reactions, as can a spectrum of drugs. viral etiologies include hiv, as in the hiv seroconversion syndrome, epstein-barr virus (infectious mononucleosis), human herpesvirus 6 (roseola), parvovirus b-19 (fifth disease), measles, rubella, and various hemorrhagic fever viruses. many bacteria can be responsible, such as rickettsia, bacillus anthracis, spirochetes (spirillum, leptospira, borrelia, treponema), coxiella burnetii, yersinia pestis, salmonella typhi, bartonella bacilliformis, and brucella. histoplasmosis and coccidioidomycosis are fungal diseases commonly associated with ulcers and other specific skin lesions use in bioterrorism. the best-recognized etiology of a noninfectious eschar is a bite from a brown recluse spider. petechiae and purpura can result from adverse reactions to a number of drugs. the most important infectious cause of petechiae and/or ecchymoses with fever is meningococcemia, which has a high rate of morbidity and mortality and is widespread throughout the tropical world and found sporadically in industrialized countries. other bacterial causes include borrelia, burkholderia, enterococcus, haemophilus, leptospira, pseudomonas, rickettsia, streptobacillis, treponema, vibrio, and yersinia. a number of hemorrhagic fever viruses can cause petechial or purpuric lesions, but the most prevalent viral causes are enteroviruses, cytomegalovirus, dengue, and yellow fever. protozoal diseases (e.g., malaria and toxoplasmosis) and helminths (e.g., trichinellosis) can also induce this clinical presentation. changes in pigmentation can be seen after a variety of medications, many of which are taken for prophylaxis or therapy related to travel. these agents include a spectrum of drugs such as antibiotics, antidiarrheals, anthelmintics, and antimalarials, many of which can also elicit photosensitization. a number of infectious agents can also alter pigmentation. leishmaniasis, pinta, and tinea versicolor may be associated with hypopigmentation or hyperpigmentation. leprosy, onchocerciasis, syphilis, and yaws are more often associated with hypopigmentation. erythrasma, hiv, and loiasis are more frequently causes of hyperpigmentation. with the exception of the movements of larvae of myiasis, mucocutaneous migratory lesions are usually due to infections with helminths. the best-recognized example is cutaneous larval migrans, but migratory lesions can also be due to dracunculiasis, fascioliasis, gnathostomiasis ( fig. 1.10) , hookworms, loiasis, paragonamiasis, sparganosis, or strongyloidiasis. in conclusion, the differential diagnoses of mucocutaneous lesions in the returned traveler, immigrant, or adoptee should be based on the morphology of the lesions, but the patient's symptoms, general medical, and exposure history must all be considered. [11] [12] [13] [14] [15] [16] [17] the physical examination and laboratory results must be integrated with the patient's vaccination and medication record. the travel destination(s), travel duration, living, work/recreation conditions, food and drink ingestion, and activities while traveling must all be taken into consideration. it must not be forgotten, however, that many mucocutaneous problems in the returned traveler or the immigrant/adoptee are not related to the travel or the country of origin but can be the same disorders seen daily in patients who have never left their local communities. health problems after travel to developing countries an overview of sexually transmitted diseases. part i (bacterial stds) an overview of sexually transmitted diseases. part ii (viral stds) an overview of sexually transmitted diseases. part iii. sexually transmitted diseases in hiv infected patients ulcers and other specific skin lesions 11 dermatoses associated with travel to tropical countries: a prospective study of the diagnosis and management of 269 patients presenting to a tropical disease unit cutaneous manifestations of intestinal helminthic infections parasitic infections of the skin skin problems in returning travelers dermatologic manifestations of parasitic disease the travel and tropical medicine manual kaposi's sarcoma and other manifestations of human herpesvirus 8 mucocutaneous manifestations of viral diseases tropical dermatology: viral tropical diseases lyme borreliosis dermatologic infectious diseases in international travelers macules and/or papules. certain protozoa such as toxoplasma gondii and leishmania can also induce these types of lesions. among helminths, hookworm disease, strongyloidiasis, and onchocerciasis can be associated with macules and/or papules. although otherwise similar, papules are usually less than 0.5-1.0 cm, while nodules are larger than 0.5-1.0 cm in diameter. except for certain poxviruses which cause orf and milker's nodules, as well as warts and malignancies induced by hpv, viruses rarely form nodules. on the other hand, all subcutaneous and systemic mycoses can induce nodules. bacterial causes of nodules include bartonella (verruga peruana and cat-scratch disease), buckholderia mallei (glanders), calymmatobacterium granulomatis (gi), chlamydia trachomatis (lgv), klebsiella rhinoscleromatis (rhinoscleroma), leptospira autumnalis (leptospirosis), mycobacteria spp. (atypical mycobacteria, cutaneous tuberculosis, leprosy, etc.), nocardia brasiliensis (and other bacterial causes of mycetoma) and treponema pallidum (bejel, yaws). protozoan causes of nodules include amebiasis, leishmaniasis, and trypanosomiasis. almost all helminthic infections that have mucocutaneous manifestations can induce nodules, e.g., coenurosis, cysticercosis, dirofilariasis, dracunculiasis, echinococcosis, filariasis, gnathostomiasis, loiasis, onchocerciasis, paragonimiasis, schistosomiasis, sparganosis, and visceral larval migrans. if the helminthic nodule contains sufficient fluid, it will produce a cyst. cysts can be seen in helminthic infections such as coenurosis, echinococcosis, filariasis, gnathostomiasis, loiasis, and onchocerciasis. there are also arthropod causes of nodules such as myiasis, scabies, tick granulomas, and tungiasis. whereas ulcers can form as a result of breakdown of previously normal skin, they frequently develop from nodules after inflammation destroys the epidermis and papillary layer of the dermis. herpes simplex virus is a very common cause of ulcers in both tropical and temperate regions of the world. other causes of gud are bacterial, e.g., chancroid, gi, lgv, and primary syphilis. other bacterial diseases that commonly cause ulcers include anthrax, bacterial mycetomas, diphtheria, glanders, melioidosis, mycobacterial diseases (e.g., buruli ulcer, leprosy, tuberculosis), plague, rickettsia, tropical ulcers, tularemia, and yaws. a number of fungi can form nodules that break down into ulcers, or they can induce ulcers from systemic spread: blastomycosis, chromomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, lobomycosis, mycetomas, paracoccidioidomycosis, penicilliosis, and sporotrichosis. the most common helminthic cause of cutaneous ulcers is dracunculiasis, when the worm erupts from the skin. two protozoan diseases cause ulcers -amebiasis and leishmaniasis. arthropod causes of ulcers include myiasis and tungiasis. many bites (e.g., brown recluse spiders and various snakes), stings (e.g., insect, jellyfish, and scorpion) or venomous spines of various fish can also induce ulcers. an eschar can be seen in both temperate and tropical lands due to pseudomonas aeruginosa (i.e., ecthyma gangrenosum), but the most common causes of eschars are rickettsia. eschars due to anthrax can be seen in persons who work with animal skins, but anthrax has received much attention recently due to its potential key: cord-005882-iodfgzjf authors: kaufmann, stefan h e; mcmichael, andrew j title: annulling a dangerous liaison: vaccination strategies against aids and tuberculosis date: 2005-04-05 journal: nat med doi: 10.1038/nm1221 sha: doc_id: 5882 cord_uid: iodfgzjf human immunodeficiency virus (hiv) and mycobacterium tuberculosis annually cause 3 million and 2 million deaths, respectively. last year, 600,000 individuals, doubly infected with hiv and m. tuberculosis, died. since world war i, approximately 150 million people have succumbed to these two infections—more total deaths than in all wars in the last 2,000 years. although the perceived threats of new infections such as sars, new variant creutzfeldt-jakob disease and anthrax are real, these outbreaks have caused less than 1,000 deaths globally, a death toll aids and tuberculosis exact every 2 h. in 2003, 40 million people were infected with hiv, 2 billion with m. tuberculosis, and 15 million with both. last year, 5 million and 50 million were newly infected with hiv or m. tuberculosis, respectively, with 2 million new double infections. better control measures are urgently needed. immunodeficiency. with new antiretroviral drugs, hiv infection can be controlled, but not eradicated. treatment, however, is expensive and less than 5% of individuals infected with hiv in developing countries have access to effective treatment. anti-hiv drugs target hiv reverse transcriptase, protease, integrase or the coiled coil domain of gp41. used in combinations of three or more, they are very effective at treating hiv infection: patients with advanced aids have shown dramatic albeit incomplete recovery of cd4 + t cell counts and immune function, though sometimes the revitalized immune response causes severe problems by reacting to previously silent infecting pathogens. yet even when virus loads have become undetectable for several years, cessation of drugs results in rapid rebound of virus and return to the pretreatment levels 4 . this implies that patients need to take these drugs for life. but because of serious side effects and expense, indefinite treatment is not possible for most of the world. drug resistance by virus mutation is well described 5 and although triple therapy reduces the risk substantially, imperfect adherence to treatment regimes may result in selection of resistant virus. as is the case for tuberculosis, this could limit treatment options in the near future. transmission, however, can be reduced by 'safer sex' practices. such behavior changes may account for the reduction in prevalence of hiv-1 infection from >20% to 8% in uganda over 10 years 6 . in south africa, more than 10% of the 40 million inhabitants are infected with hiv and more than 5% suffer from active tuberculosis. in this country, more than half of all individuals with tuberculosis have concurrent hiv infection. immunodeficiency caused by hiv infection increases the risk of developing tuberculosis dramatically-10% of double-infected individuals will develop active tuberculosis within a year of coinfection, as compared to the 10% lifelong risk in individuals infected with m. tuberculosis alone. despite the availability of effective chemotherapy to cure tuberculosis and the successful development of antiretroviral drugs that control hiv, vaccines provide the only realistic hope of effective prevention. tuberculosis most frequently develops in the lung (fig. 1) . once tubercle bacilli have reached the lung alveoli, they are taken up by alveolar macrophages and probably dendritic cells (dcs) in the lung interstitium (fig. 1) . m. tuberculosis is protected by a recalcitrant cell wall rich in waxes and other glycolipids 7 , which is responsible for the unique staining characteristics of these microbes and contributes significantly to resistance against host defense. infected macrophages and dcs, therefore, do not destroy their bacterial prey and thus serve as a mobile habitat, transporting the microbes to the lung parenchyma and eventually to the draining lymph nodes 8, 9 . infected macrophages attract monocytes and a lesion develops. dcs in the lymph nodes present mycobacterial antigens to t cells. at this early stage of infection, secreted proteins such as early secreted antigen for t cells (esat-6) and antigen 85 (ag85) presumably serve as dominant antigens for cd4 + t cells, which accumulate in great numbers in lesions early after infection. at later stages of infection, cd8 + t cells also become stimulated 10 . in addition, unconventional t cells are activated, including t cells expressing a γδ t cell receptor with specificity for small phosphorylated ligands and t cells with specificity for glycolipids, which are presented by major histocompatibility complex (mhc) class i-like molecules of the cd1 family [11] [12] [13] [14] . studies in nonhuman primates support a role for γδ t cells in protective immunity against tuberculosis 13 . the cd1-restricted t cells presumably evolved as a result of the evolutionary cross-talk between mycobacteria and the mammalian host, as they are specific for glycolipids abundant in mycobacteria but not found in other microbial pathogens 11 . antigen-specific t cells induce the formation of a granuloma around infected macrophages, primarily composed of monocytederived macrophages (some of which transform into multinucleated giant cells), cd4 + t cells and an outer ring of cd8 + t cells. at later stages, the granuloma is surrounded by a fibrotic wall and lymphoid follicular structures develop in the vicinity, probably orchestrating the local immune response 15 . (i) immediate eradication of m. tuberculosis by the pulmonary immune system. this alternative is rare to absent. (ii) infection transforms into tuberculosis. this frequently occurs in immunodeficient individuals, with the notable example of hiv infection increasing the risk of developing tuberculosis 800-fold. (iii) infection does not transform into disease because m. tuberculosis is contained inside granulomas. in the diseased individual, m. tuberculosis is no longer contained because caseation of the lesion results in dissemination and transmission of m. tuberculosis. after inhalation, m. tuberculosis is engulfed by alveolar macrophages and dcs. in draining lymph nodes, these cells present mycobacterial antigens to different t cell populations. antigen presentation probably involves cross-priming, allowing transfer of mycobacterial antigens from infected macrophages to dendritic cells. antigen-specific cd4 + t cells, cd8 + t cells, γδt cells and cd1-restricted t cells participate in protection. most importantly, macrophages are activated by ifn-γ and tnf-α. in addition, t cells may kill mycobacteria present in macrophages by means of perforin and granulysin. in the granulomatous lesion, macrophages are activated by t lymphocytes through production of type i cytokines, notably interferon (ifn)-γ and tumor necrosis factor (tnf)-α 16 . ifn-γ activates the capacity to control m. tuberculosis in macrophages 17, 18 . moreover, ifn-γ and tnf-α are instrumental in walling off m. tuberculosis inside granulomatous lesions. the importance of these cytokines in the containment of mycobacteria is well illustrated by the observation that individuals with deficient ifn-γ signaling suffer from rapid onset of mycobacterial diseases, and that treatment with antibodies that neutralize tnf-α in individuals with rheumatoid arthritis reactivates tuberculosis in those with latent infection 19 . the granuloma persists for years and efficiently contains tubercle bacilli as long as the individual remains immunocompetent. the granuloma deprives the arrested mycobacteria of oxygen and nutrients and as a direct consequence, the microbes survive, probably in a state of dormancy [20] [21] [22] [23] . the gene expression profile of m. tuberculosis is altered in response to the restricted growth conditions in the granulomatous lesion. it is highly probable that some of these dormancy-related gene products serve as the major antigens of dormant m. tuberculosis and hence represent promising candidate antigens for a postexposure vaccine aimed at preventing reactivation tuberculosis. the best-known dormancy antigen is the α-crystallin, a small heat-shock protein (hsp) also termed hspx 23 . the mechanisms that determine latency and disease outbreak in tuberculosis remain elusive. although the risk of reactivation is understood to be determined by both host and microbial factors in addition to environmental components, virtually nothing is known about the responsible genes. increasing epidemiologic evidence that strains of the m. tuberculosis beijing/w genotype family, many of which are of the mdr type, are spreading throughout the world suggests that this family of m. tuberculosis evolved against the evolutionary pressure of bcg vaccination and chemotherapy 24, 25 . a careful histologic analysis of lung autopsy material performed from 284 cadavers more than 100 years ago in zürich showed almost 100% m. tuberculosis infection in individuals over 21 years of age, who had died of unrelated reasons 26 . we can assume similarly high incidences nowadays for individuals living in tuberculosis 'hot spots' (e.g., prisons in some countries) and infection rates between 10% and 50% in areas with high m. tuberculosis incidences. how and why disease outbreak occurs after a period of latency are unclear 27, 28 . increased risk of tuberculosis as a result of unique environmental and behavioral factors such as alcohol abuse or dietary iron overload emphasizes the importance of these components, but again provides little explanation of specific control mechanisms 29 . a considerable proportion of individuals with tuberculosis develop disease within the first year after infection. moreover, superinfection as well as reinfection after successful tuberculosis chemotherapy have been described 30 . it is probable that these individuals develop an insufficient immune response against natural infection with m. tuberculosis because of genetic host susceptibility, immune suppression by the pathogen or both. elucidation of the genetic mechanisms underlying susceptibility and protective immune mechanisms in resistant individuals that prevent disease outbreak in face of ongoing infection, as well as identification of the pathogen genes that promote transformation of latent infection into active tuberculosis, will facilitate rational design of a postexposure vaccine 27, 28, 31 . of equal importance will be the careful analysis of the efficacy of bcg vaccination. bcg was found to protect against adult tuberculosis in the uk and against leprosy in malawi, but did not provide any protection in the world health organization trial in south india 32 . overall, a 50% protective efficacy has been estimated for bcg 33 . although environmental factors, notably frequent infection with atypical mycobacteria, probably have a role, contribution of genetic host factors should be assessed more carefully. it is possible that a small proportion of bcgvaccinated individuals develops a long-lasting immune response against tuberculosis, whereas the majority does not. because any new vaccine has to protect individuals who do not mount an appropriate immune response to natural infection with m. tuberculosis or to vaccination with bcg, the susceptible and resistant target populations and the criteria that distinguish them from each other need to be defined carefully. vaccination strategies against tuberculosis subunit vaccines given alone or in addition to bcg. new subunit vaccines against m. tuberculosis comprise antigen-adjuvant formulations, naked dna vaccine constructs or recombinant carriers expressing antigen ( table 1) 34 . they are mainly based on protein antigens. unfortunately, reliable rules for defining protective antigens for t cells do not exist. most vaccine antigens investigated thus far are early produced secreted antigens such as ag85, a shared antigen with bcg, and esat-6, which is an antigen unique to m. tuberculosis (fig. 2) 35, 36 . such antigens are probably adequate for pre-exposure vaccination. for individuals latently infected with m. tuberculosis, a postexposure vaccine composed of dormancyrelated antigens such as hspx seems more suitable (fig. 2) [21] [22] [23] . a recent screen has assessed the protective efficacy in mice of more than 30 antigens that are differentially expressed in the proteomes of m. tuberculosis versus bcg 37 . from these, only one antigen was identified that induced a robust protection similar to that of bcg, arguing against a unique role in protection of proteins exclusively present in the proteome of m. tuberculosis. although proteins will constitute the major antigens of subunit vaccines, glycolipid antigens can be included in these vaccine formulations. such glycolipids could serve both as antigen in the context of cd1 and as adjuvant for toll-like receptors (tlrs) 11 . generally, subunit vaccines crucially depend on appropriate adjuvants 38 that stimulate t helper type 1 (t h 1) immune responses by the different t cell populations required for protection against tuberculosis. naked dna constructs can stimulate cd4 + and cd8 + t cells. although they have proven their high immunogenicity in small-rodent animal models, human studies are thus far mostly disappointing. but new carrier systems such as polylactide glycolyde microparticles may facilitate application of naked dna vaccines in humans 39 . recombinant bacterial and recombinant viral carriers expressing defined mycobacterial antigens have been constructed and their vaccine efficacy against tuberculosis have been determined in experimental animal models. these vectors induce potent cd8 + and cd4 + t h 1 responses. the most advanced vaccine of this type is recombinant modified vaccinia virus ankara (r-mva) expressing ag85, which has already entered phase 1 clinical trials 40, 41 . vaccination with mtb 72f protein in as02a adjuvant or in the form of naked dna induces protective immunity in both mice and guinea pigs 42 72f-as02a vaccine formulation has now entered a phase 1 clinical trial in healthy m. tuberculosis-uninfected volunteers ( table 2) . another protein-based vaccine comprising a fusion protein of esat-6 and ag85 in a mixture of oligodeoxynucleotides and a polycationic peptide (ic31) as an adjuvant has shown promising results in experimental animals and hence is aimed at entering a phase 1 clinical trial later in 2005 ( table 2) 36 . generally heterologous prime-boost schemes are composed of a relatively simple first component (e.g., dna) that primes a focused immune response, and a second boost vaccine that induces an inflammatory response (e.g., using a recombinant virus) to magnify the initial response. to achieve a proper prime-boost effect, shared antigens are required for the subunit boost vaccination. in tuberculosis, however, heterologous prime-boost vaccination schemes starting with a bcg prime followed by a subunit boost are considered most promising, not only for scientific reasons but also because bcg vaccination of newborns cannot be given up prematurely. the recently discovered antigen rv3407 is encoded in the bcg genome but is undetectable in its proteome 37 . the increased protection seen in bcg-primed mice after boost vaccination with naked dna encoding rv3407 indicates that this protein is an important antigen of m. tuberculosis that is suboptimally expressed in bcg. similarly, boost with mtb 72f in as02a adjuvant increased protection induced by bcg prime 43 . the r-mva expressing ag85 induced substantial protection both when given alone or as booster vaccination on top of bcg prime 41 . a recent clinical phase 1 trial showed that r-mva-ag85 induced ag85-specific ifn-γ-secreting t cells ( table 2) 40 . more profound effects were observed in the bcg prime-r-mva-ag85 boost group, underlining the importance of heterologous prime-boost vaccination in the rational delineation of future tuberculosis vaccination strategies. vaccine efficacy data of mutant m. tuberculosis are still limited. during the early stage of infection, m. tuberculosis replicates actively. accordingly, m. tuberculosis knockout mutants, in which synthesis of essential amino acids is prohibited, will not grow in lungs of experimentally infected mice. typically, these are auxotrophic mutants that die of starvation in the absence of the required nutrients ( table 1) 44, 45 . a second group of m. tuberculosis knockout mutants show reduced growth in lungs of experimentally infected mice, but persist at a lower level than wild-type microorganisms. these mutations include deficient regulatory proteins, such as the phop/phoq two-component regulatory system and a variety of mutants with deficient lipid metabolism or transport 46, 47 . although genes of this group include virulence factors, it is also possible that the gene products are needed for persistence rather than harming the host directly. m. tuberculosis knockout mutants deficient in genes expressed during dormancy replicate in the lungs of experimental mice in an unconstrained manner early after infection, but cease to grow at later stages because of their impaired dormancy gene program. one of the best described genes of this group is that which encodes isocitrate lyase 48 . m. tuberculosis knockout mutants, which grow as well as wild-type microorganisms but induce weaker pathology, lack true virulence factors. some knockout mutants behave like wild-type m. tuberculosis in the lung but do not disseminate to other organs. the heparin-binding hemagglutinin adhesion molecule seems to participate in the dissemination of m. tuberculosis to extrapulmonary sites and hence qualifies as a member of this group 49 . factors that facilitate dissemination to other tissues should be deleted in a viable vaccine in order to focus the response on the lymph nodes. a recently defined group of m. tuberculosis knockout mutants comprises strains that grow as well as wild-type microorganisms in normal mice, but do not persist in mouse knockout mutants with defined immunodeficiency 50 . vaccine candidates based on m. tuberculosis knockout mutants will probably face strong objections against use in human clinical trials largely because of the genetic stability of the mutants. reversion is best prevented by having two independent chromosomal gene deletions. the advantage of m. tuberculosis knockout mutants is their profound stimulation of all t cell populations relevant to protective immunity. on the other hand, m. tuberculosis is known to impair the host immune response. therefore, it is probably not sufficient to reduce the virulence of m. tuberculosis knockout mutants. additional genetic modifications improving the immune response and reducing pathology will be required. although these vaccines will have a long way to go, a rationally designed m. tuberculosis mutant lacking a defined set of virulence genes in the long run could have great potential as a tuberculosis vaccine. as compared to m. tuberculosis, the current vaccine bcg lacks approximately 130 genes that are clustered in 16 regions of difference (rd) 51 and are at least in part involved in pathogenicity and persistence. reintroduction of selected genes may increase immunogenicity, antigenicity or both of bcg without reverting it to a pathogen ( table 1) . improved immunogenicity results in the stimulation of a profound immune response. antigenicity can be improved by introduction of additional antigens or by overexpression of antigens expressed at suboptimal level. pym et al. have introduced the entire rd1 region of m. tuberculosis into bcg, comprising at least 11 genes 52 . although it remains to be clarified whether the recombinant bcg (r-bcg) expressing rd1 genes is endowed with improved immunogenicity, antigenicity or both, it was claimed that this vaccine candidate provided slightly better protection than parental bcg in mice. not unexpectedly, however, this r-bcg candidate showed higher virulence in immunocompromised mice as compared to wild-type bcg. reintroduction of genes missing in bcg that do not confer virulence might increase vaccine efficacy of bcg without decreasing its safety. horwitz et al. introduced the gene encoding ag85 into bcg, an abundant secreted protein in m. tuberculosis 53 . although the gene is also expressed in bcg, it was assumed that overexpression of this immunodominant antigen would increase protective immune responses. indeed, in guinea pigs, r-bcg expressing ag85 induced substantially higher protection than parental bcg against tuberculosis. this vaccine was at least as safe as bcg and has recently entered a phase 1 clinical trial ( table 2) . the r-bcg vaccine candidates with higher immunogenicity comprise strains that express human cytokines such as ifn-γ, ifn-α, interleukin (il)-2, il-12 and granulocyte macrophage colony stimulating factor (gm-csf) [54] [55] [56] . however, these cytokine-secreting r-bcg have yet to prove superior protection in animal models against tuberculosis, as they have not yet been compared to parental strains. another approach to improving immunogenicity of bcg takes advantage of the biological activity of listeriolysin (hly) 57 . in its natural host, listeria monocytogenes, this cytolysin forms pores in the phagosomal membrane, promoting listerial egression into the cytosol 58 . in the cytosol, listerial antigens are readily introduced into the mhc class i pathway, causing preferential stimulation of cd8 + t cells. compelling evidence suggests that bcg is insufficiently equipped for stimulating cd8 + t cells, whereas cd4 + t cell stimulation is satisfactory 59 , the gene encoding hly was introduced into bcg to improve cd8 + t cell stimulation. indeed, such vaccine constructs induced better protection than parental bcg in mice (grode, l. et al; unpublished data). production of this vaccine under good manufacturing practices (gmp) conditions has been initiated and this candidate is aimed at entering a phase 1 clinical trial in 2005 ( table 2) . the use of subunit vaccines is based on the assumption that one or a few protective antigens and t cell clones will suffice for efficacious protection 16 . in contrast, attenuated viable vaccines utilize the whole spectrum of expressed antigens to stimulate an optimal combination of t cell subtypes. genetically engineered viable vaccines are therefore best suited as replacements for bcg. despite previous reluctance, a recent expert group meeting has strongly advocated development of viable recombinant vaccines against tuberculosis because they are the most potent stimulators of protective immune responses that perform better than bcg in experimental animal models 60 . as an essential requirement, any new viable vaccine should be at least as safe as bcg in immunocompetent and safer than bcg in immunocompromised individuals. cross-reactive immune responses against atypical mycobacteria might prematurely eradicate an improved r-bcg or attenuated recombinant m. tuberculosis and thereby impair efficacy of genetically engineered viable vaccines. although subunit vaccines are probably not affected by the preexisting cross-reactive immunity to environmental bacteria, generally immunity induced by subunit vaccines is short-lived. given the complementary strength of both types of new vaccine candidates, a heterologous prime-boost regimen comprising a prime with a viable vaccine candidate superior to bcg and a boost with a subunit vaccine candidate will probably be the most promising combination to ensure long-lasting efficacy. hiv is a member of the lentivirus subgroup of the retrovirus family, so named because of a long period of clinical latency after infection. its envelope is derived from cell membrane and expresses the glycoproteins gp120 (responsible for virus attachment to cells) and gp41 (responsible for membrane fusion). internally, matrix (gag p17) and capsid (gag p24) proteins enclose the viral rna. other viral proteins include the reverse transcriptase, protease, integrase and regulatory proteins nef, tat, rev, vif, vpr and vpu. the virus infects by attaching gp120 to the cd4 molecule and either the chemokine receptor ccr5 or cxcr4 present on t cells 61 . gp41 mediates fusion and in the cytosol the rna is reverse transcribed and the preintegration complex moves to the nucleus, where the cdna is integrated as provirus into host dna in activated cells. gene expression is normally immediate; first regulatory and then structural proteins are made, regulated by tat and rev proteins. new virus particles bud from the surface of infected cells about 24 h after infection. virus is shed for a further day or so before the cell dies 62 . neutralizing antibodies eventually appear, although too late to prevent infection, and are easily evaded by viral mutation. t cell immunity seems to control the infection over several years but with a dynamic process of immune selection and escape occurring 63 . cd4 + t cells are both deleted and functionally impaired early 64 , with hiv-specific t cells preferentially infected and depleted 65 . however, in some cells (e.g., long-lived memory t cells and macrophages), gene expression is delayed and the infection becomes latent. the progressive loss of cd4 + t cells leads to clinical immunodeficiency, which is manifested by opportunistic infections and, often, reactivation of tuberculosis. these infections rapidly cause death if untreated. hiv infects cd4 + t cells and monocytes and macrophages. the virus is cytopathic in activated t cells, but less so in macrophages, and not at all in latently infected cells with integrated provirus. these different forms of infection present problems for vaccines. those that stimulate and maintain high levels of neutralizing antibodies could prevent initial infection, but once that has occurred, it is almost impossible to eliminate hiv. under prolonged antiretroviral drug therapy, virus is eliminated from the activated t cells that express the enzymes targeted by the drugs, but not from cells in which virus turns over slowly or is latent 66 . thus if an hiv vaccine does not prevent infection, it is unlikely to eliminate the virus but may be able to control the ongoing infection and prevent disease. there is no known protective immunity against hiv, in the sense that virus is never eradicated from those infected so that it is impossible to find people protected by previous infection, as is the case for many other viruses, such as measles, mumps and influenza. this poses serious problems for vaccine development, although it is not unique (epstein-barr virus is another such example). adult monkeys infected with attenuated siv (e.g., with deletions in nef) do not become sick, and are protected from challenge with pathogenic siv 67 . this protection is probably, but not certainly, immunological 68 . some highly hiv-exposed individuals resist hiv infection; others develop t cell responses to hiv 69 but not serum antibodies, and their immunity depends on continuing exposure to the virus 70 . this may represent immune resistance, although it is hard to prove. another clue may come from the rarity of superinfection in people infected with hiv who are repeatedly exposed to further infection. although there are elegant studies of superinfection 71 , it is suspected that it is rare; if so, its rarity probably reflects immune protection. the spread of hiv-1 infection around the world in 20 years has been alarming. the worst epidemics are in sub-saharan africa, where more than 50% of young adults are infected. mortality from hiv infection without drug treatment is close to 100%, although infected people usually survive from 5 to 10 years before developing aids. the introduction of effective antiretroviral drugs in high-income countries has reduced mortality without reducing infection. hiv is the most variable virus known. there are six major subtypes, a, bc, d, e, f and g, which are 'ancient' (in the hiv context of 30-50 years) and have distinct geographical distribution (fig. 3) 72 . the complexity is even greater because of recombinations between subtypes and within subtypes. each subtype differs by >20% in amino-acid sequence, so that t cells specific for one subtype are unlikely to cross-react substantially with the others; each epitope of 9-15 amino acids will probably have two or more mutations, and this level of variability is known to adversely affect t cell recognition 73 . although there are a few cross-reactive t cell epitopes, as well as conserved segments of hiv, it may be necessary to match any virus subtype with that of the circulating viruses in the population to be vaccinated. it is less certain that subtype-specific vaccines will be necessary for stimulating neutralizing antibodies, although sequence variability is known to be a major problem. a further complication is the variability within each subtype and within each individual. it is clear that both antibodies and cd8 + t cells are very effective at selecting virus escape mutants [74] [75] [76] [77] . as human leukocyte antigen (hla) type controls the epitopes recognized by t cells, it is not surprising that hla type influences virus sequence in individuals as a consequence of t cell-driven escape 78 . however, the mutant virus may be less fit and the escape can offer advantages to the host. the constraints on the virus probably account for the overall conservation of virus sequence in primary infection, before the immune response evolves. therefore, vaccines that focus on conserved consensus sequences may have the best chance, at minimum selecting slightly less fit viruses during primary infection. a t cell vaccine that permits infection and induces selection of escape mutants could undermine vaccine effectiveness, as has been seen in macaques 79 . but selection of less fit virus has also been observed in macaques in whom siv infection remains controlled, so this could favor the host 80 . challenges. for hiv there are two major challenges: to find (i) immunogens that can stimulate broadly cross-reacting neutralizing antibodies and (ii) immunogens that stimulate high levels of persisting cd8 + and cd4 + t cells. both humoral and cellular immunity may be needed, but they require different types of immunogens; eventually vaccines could be mixed to achieve both (fig. 4) . a prophylactic vaccine will be given months or years before exposure and, hopefully, prevent or ameliorate infection. an antibody-inducing vaccine could prevent infection 81 , but as indicated above, a vaccine that stimulates t cell immunity cannot stop virus infecting cells. the latter vaccination might enable the host to suppress the infection for long periods and prevent disease. thus, macaques vaccinated to stimulate t cell immunity against siv became infected when challenged with virus, but had very low virus loads and experienced little effect on their health as compared to unvaccinated controls [82] [83] [84] . because it is almost impossible to eliminate hiv infection, this may be the best that can be achieved. however, many chronic virus infections as well as latent m. tuberculosis infection are controlled effectively in the long term by cellular immunity without causing disease. hiv-2 does not cause disease in more than 80% of those infected in west africa 85 , suggesting that the immune response and the virus are in balance in the infected but healthy individual. at present there is no realistic candidate hiv vaccine. it was thought that gp120 preparations would stimulate neutralizing antibodies, and they do against tissue culture-adapted hiv strains. however, all such vaccines have failed to neutralize primary virus isolates and one tested in two large efficacy trials failed to protect (nam, http://www.aidsmap.com/). now the aim is to design hiv envelope protein immunogens that will stimulate protective antibodies. unfortunately the dice are loaded against this: (i) the envelope is heavily glycosylated, making it largely nonimmunogenic; (ii) it is conformationally variable, so that the most sensitive site, the chemokine receptor binding site, is not exposed unless cd4 + has bound; (iii) the cd4 + binding site is deeply recessed and hard to access by antibodies; (iv) besides the carbohydrates, crucial parts of the gp120 surface are protected by hypervariable loops, which can and do vary by mutation at no cost to the virus, thereby escaping neutralizing antibodies 86, 87 . real novelty in immunogen design is needed to get around these problems. as an alternative, much effort is being made to create vaccines that stimulate t cell immunity, particularly cd8 + t cells. in mice, several approaches comprising plasmid dna and various recombinant viruses and recombinant bacteria have given promising results [88] [89] [90] . also particulate proteins and peptide-pulsed dcs can efficiently stimulate cd8 + t cells 91, 92 . but these findings are not easily transferable to primates. dna vaccines stimulate only weak responses in humans 93 and recombinant viruses give mixed results. the poxviruses (canary pox, the attenuated vaccinia virus (nyvac) and mva) induce weak primary responses 94, 95 , possibly because the recombinant immunogen is swamped by more than 200 poxvirus proteins. also, these viruses may be too attenuated, with little or no proliferation after infection. however, they may be better at boosting t cell responses that have already been primed (e.g., bcg; table 1 ). the most promising results to date come from recombinant adenovirus 5, which has stimulated strong and sustained t cell responses, but its applicability is constrained by the high frequency of antibodies to this virus in most human populations. alternate candidates in trial include other strains of adenovirus, adeno-associated virus, alphaviruses and r-bcg as vectors for hiv. as for tuberculosis, heterologous prime-boost regimes offer enhanced t cell immune responses 96, 97 and have been observed in macaques and humans 94 . it seems that in humans, recombinant adenoviruses are good at both priming and boosting, whereas recombinant poxviruses such as mva are better at boosting than priming (mcmichael, a.j., goonetilleke, n., guimaeres-walker, a., dorrell, l., yan, h., hanke, t., unpublished data). vaccine-induced t cell responses should include both cd4 + and cd8 + t cells, even though the former are targets for the virus. cd4 + t cell help is important for optimal cd8 + t cell responses 98-101 -an issue of particular importance to hiv vaccine development because natural infection results in impaired cd4 + t cell immunity. protein immunogens typically elicit antibody responses and t helper cell responses, but the latter may be of the t h 2 type, especially when alum is used as an adjuvant 102 ; t h 2 responses have little or no antiviral activity. live intracellular infections generally elicit t h 1 responses and cd8 + t cell responses, hence the use of live attenuated vectors to mimic a virus infection. these deliver the required immunogen into the cytosol, from which it can enter the mhc class i antigen presentation pathway directly, or indirectly when the cell dies and virus protein is taken up by dcs for 'cross-priming' of cd8 + t cells [103] [104] [105] [106] . people who are homozygous for the δ32 variant of ccr5 do not express an intact chemokine receptor and are resistant to hiv infection 107 . immune resistance is suspected for people who are highly exposed, yet uninfected. many generate cd8 + and/or cd4 + t cell responses, but without any serum antibody 69 . it is not certain that the t cell responses are responsible for resistance. if they are, it is encouraging because the levels of t cell response seen in current vaccine trials could be sufficient. hiv enters the host through (usually genital) mucosa. hiv infects, or attaches to, mucosal langerhans cells first and then migrates to local lymphoid sites 108 an hiv vaccine should stimulate b and t cell as well as mucosal and innate immunity. live attenuated hiv probably provokes all of these, but because it will set up a persistent infection, there are insoluble safety issues that render its use unlikely in humans. therefore, it may be necessary to build a set of vaccines to stimulate each component separately and administer them in combination. if control equivalent to that of chronic epstein-barr virus or cytomegalovirus or latent m. tuberculosis infection could be achieved for hiv, with reduced or negligible further transmission, it would represent a considerable advance. measuring the efficacy of the vaccine would be problematic, being dependent on reduced virus load at set point, the time at around 6 months after primary infection when virus level stabilizes. the set-point level is inversely correlated with time of survival. only neutralizing antibodies, stimulated by vaccines, offer any real chance of sterile immunity and such vaccines are a long way off. a prophylactic vaccine for hiv is badly needed. unless current trials of vaginal microbicides are unexpectedly successful, there is no other way of controlling the infection and both antibody-and t cell-inducing vaccines need a total rethink. the most advanced of these vaccines is the merck recombinant adenovirus 5 (ad5) vaccine that is about to enter clinical trials. but even if successful, a different vector will be needed to deal with the problem of pre-existing antibody immunity to ad5. the merck trial may, however, indicate whether vaccines that stimulate t cell immunity offer any benefit, and that type of news is to be welcomed. could aids and tuberculosis vaccines be given together? probably, but this may be decades away. in parts of africa, bcg is given at birth. combining bcg with an aids vaccine, with a boost at puberty, may be feasible in the distant future. preclinical studies: tuberculosis and aids vaccine testing. most tuberculosis vaccine candidates have been tested in mice or guinea pigs. studies in mice take advantage of the in-depth knowledge of the mouse immune system, whereas guinea pigs have the advantage of developing a pathology that highly resembles human tuberculosis. these experimental animal studies form the starting point of the tedious pipeline leading to a new vaccine candidate 110 . the us national institutes of health, through their preclinical tuberculosis screening program, and the european union, through its tbvac integrated project in the framework program 6, sponsor vaccine testing in experimental animals under standardized conditions. thus far, the two programs utilize different screening procedures, which need to be harmonized in order to achieve comparability of different vaccine candidates. however animal models cannot unequivocally predict the efficacy of a vaccine candidate against tuberculosis or hiv and immunogenicity results in mice often do not predict responses in humans. although nonhuman primate studies can be bypassed before phase 1 and phase 2 clinical trials, they may become necessary before embarking on phase 3 trials. phase 1 and phase 2 clinical trials should also be exploited for determination of immunological correlates of protection (i.e., markers that unequivocally predict protective efficacy of a vaccine candidate). currently, ifn-γ produced by t cells with specificity for defined antigens is probably the best marker of protection 111 for tuberculosis, but this is much less clear for hiv. in the most impressive vaccine protections studies, using attenuated siv as a vaccine it is not known what confers protection 68, 112 and the ifn-γ elispot assay for t cells does not correlate with protection in vaccine-protected macaques 68 . additional markers are required. the siv challenge model in macaques has been very useful for hiv vaccine development 67 , but there are limitations. in these experiments, the virus challenge dose is always large, compared to repeated lowdose exposures in humans. in addition, the vaccine is nearly always homologous to the challenge virus, giving good chances of success that are unrealistic in humans. attempts are being made to introduce more representative challenges 113 . finally the siv or siv-hiv hybrid (shiv) challenges may be slightly misleading; it seems easier to protect against the aggressive hybrid virus shiv 89.6p compared to the less virulent siv mac 239. it is unclear which will be closer to an hiv exposure in humans. despite these problems, the existing data suggest vaccineinduced protection is possible. therapeutic vaccination for hiv is rarely discussed, but must be considered as recent studies in macaques and humans [114] [115] [116] give it some credibility. in humans, therapeutic vaccination would probably be used to stimulate t cell responses in hiv-infected people whose virus was well controlled by antiretroviral drugs, with the aim of terminating antiretroviral therapy (art) once the t cell levels were boosted. the rationale is that t cells, particularly cd8 + t cells, are known to be effective in long-term control of the virus in the absence of drug treatment, but t cell responses decline in patients on effective art 117 . when art is stopped, virus rebounds to pretreatment levels 4 . if the t cell levels were already boosted, this might lead to lower virus levels at the new set point. at its best, this treatment could enable withdrawal of art for prolonged periods and offer new options in low-income countries in particular. patients whose virus was controlled in this way might also be less likely to transmit virus. clinical trials. the earliest vaccine trials are small and are concerned with vaccine safety and immunogenicity. then immunogenicity is optimized by finding the best vaccine dose and route of delivery. finally, the vaccine efficacy trials involve several thousand volunteers who are at high risk for infection. translation of vaccine candidates from bench science into clinical trials is relatively straightforward scientifically but complicated logistically and extremely expensive (box 1). phase 1 trials can be done in the country of origin of the vaccine, but increasingly good sites are being established in developing countries and several countries have hiv vaccine trial experience. in contrast, experience with tuberculosis vaccine trials is marginal and currently only a few phase 1 trials have been initiated. two phase 3 trials of vaxgen gp120 in 10,000 people showed clearly that the aids vaccine candidate offered no protection (e.g., reported at http://www.hivandhepatitis.com/ vaccines/022503a.html). a phase 2b trial has now been proposed before 3. here, between 1,000 and 2,000 high-risk volunteers are given placebo or vaccine and all are followed for evidence of infection. once 30 trial participants are infected, a detailed analysis of the relationship between infection and immunity is made. in the case of aids, virus load, subtype and t cell and humoral immune responses are determined and correlated and the degree of protection ascertained. it is arguable that such an approach might have obtained a result for the vaxgen trial in a shorter time at lower cost. it would be worthwhile to consider a similar strategy for tuberculosis vaccine efficacy trials. vaccine recipients for phase 1 trials are volunteers who are at negligible risk of infection. nevertheless, they have to be counseled about the implications of testing, which adds some burden to all parties. it has proved relatively easy to recruit such volunteers in the uk and in kenya and uganda for aids vaccine testing. for efficacy trials, phase 2b and phase 3, it will be necessary to work with individuals with high risk for hiv infection. sex-worker cohorts are usually too small and follow up can be difficult. in a high-risk region in africa, the annual incidence may be 1-10%, but the implementation of trials inevitably raises awareness and the best possible advice must be given to volunteers, particularly concerning condom use. therefore, it should be expected that incidence might decrease by half. this is important to realize in determining statistical powers before embarking on the trial. the population of high-risk volunteers is less well circumscribed for tuberculosis than for aids. there are major ethical issues in efficacy trials. double blinding and placebo controls are essential, but a difficult question is what to offer in terms of chemotherapy to trial participants in low-income countries such as africa. it is now generally agreed that the best possible (rather than best regional) therapy should be offered to all trial participants who become infected; the unresolved questions are for how long and when, given that drugs may not be required for 5-10 years. although it is desirable to insist on the highest safety standards for any vaccine candidate as defined by the fda and the emea it may be worthwhile to carefully assess whether risk-benefit relationships differ for countries with low or high tuberculosis or aids prevalence. side effects of new vaccine candidates may be tolerable for a vaccine that can prevent tuberculosis or aids mortality and morbidity in countries where incidences are skyrocketing, but not for countries with low tuberculosis or aids prevalence 118 . hiv and tuberculosis vaccine trials will probably need multiple sites. a trial involving 2,000 volunteers with detailed prescreening, individual counseling and careful follow up will probably necessitate at least 20 sites. although in theory such sites would have the infrastructure and could also conduct trials for both tuberculosis and hiv, it is unlikely that this would be possible at the same time because of high cost, a requirement for multiple vaccine groups and complicated data analysis. aids and tuberculosis rampage most freely in low-income countries where the total annual budget for public health is below us $5 per capita [119] [120] [121] [122] . currently available therapeutic regimes exceed such budgets by several fold. similarly, vaccine development from the bench to the field consumes huge amounts of financial resources. consequently development of vaccines against tuberculosis and aids will be most successful as joint public-private enterprise with support from governmental and nongovernmental funding organizations and philanthropic foundations. such a consortium would be best equipped to bring forward vaccine development by a combination of 'push' and 'pull' programs. although support for preclinical research will be best directed by classical push programs, incentives should be made available for alternative approaches 118 . the removal of roadblocks serves as a good example for a support strategy emphasizing innovation and not restrictive in the experimental approach. unconventional research areas, such as therapeutic hiv-1 and tuberculosis vaccination, could be further stimulated by pull programs rewarding a vaccine candidate that proves successful figure 4 the major components of the immune response can be stimulated by different types of vaccine. inactivated vaccine t cell immunity innate immunity • intellectual property. for regulatory and manufacturing reasons, vaccines that go forward into trials must be protected as intellectual property. this ensures that sponsors, indemnifiers, gmp producers, etc. know what they are dealing with and that others will not be working unlicensed with the same product. • gmp production. preparation of a vaccine to 'good manufacturing practice' quality is essential for the regulators and ensures that each batch is identical and can be tracked. this is expensive (e.g., $100,000-200,000 for mva) and requires outsourcing. the manufacturer provides detailed documentation of processes and purity. • toxicity testing. this is required before submission to the us food and drug administration (fda) or the european medicines agency (emea). normally this requires acute toxicity and studies of distribution and persistence of vaccine at distant and critical sites after immunization. dose schedules should be the same as used for the trial, even though the animals are smaller and a detailed report is needed. • regulatory submission to the national or international authority (fda, emea, medicines and healthcare products regulatory agency, etc.). these follow standard procedures but require very detailed information including full protocols, standard operating procedures of assays, details of safety monitoring, definition of endpoints, statistical calculations, details of data handling. • ethical approval. this connects to regulatory submission. besides all protocols, details of informed consent, explanation to volunteers, details of volunteer recruiting and letters to family doctor explaining the trials are required. • other approvals may be needed from the gene therapy advisory committee (in the uk) and local and national safety committees for handling recombinant organisms. in preclinical studies. at the transition from the preclinical to the clinical phase, pull programs providing incentives for the market-ready final product would be most appropriate 118 . these include tax reduction for vaccine production, secured future markets in developing countries, a tiered price system, and reduced interest rates or debt release by the world bank for vaccine purchase and distribution. in the absence of such incentives, even the best vaccine candidate may fail to reach the desired goal of unrestricted distribution in countries affected most by aids and tuberculosis. lack of financial incentive may provide some unique opportunities for vaccine development against aids and tuberculosis. notably, lowto-absent competition for financial profits can promote comparative clinical trials under the umbrella of governmental, nongovernmental or philanthropic organizations either alone or together with the aim first to select the most promising candidates, and second to proceed by 'learning by doing' (i.e., continuous vaccine improvement of candidates in iterative clinical trial processes). analysis of the immune responses of vaccine trial participants may provide new information that can be further explored in experimental animal models and help to define the next clinical trial step. strong efforts are required to harmonize vaccine trials and accompanying vaccine efficacy testing. it is rewarding that several major organizations including the us national institutes of health though their intramural and extramural programs, the bill and melinda gates foundation through aeras for tuberculosis and their vaccine enterprise for aids as well as the european union through their european & developing countries clinical trials partnerships program have committed themselves to bring vaccines against aids and tuberculosis from the bench to the field. vaccination strategies against these two diseases need to be integrated as soon as possible, considering the intimate interdependence of the two deadly pathogens and the consequences of their liaison. obviously, numerous hurdles need to be overcome. yet, even if only partially protective vaccines can be developed, return on investment will be enormous. this is not only true for the most impoverished countries, for which a rapid solution is vital, but also for industrialized countries, which may suffer significantly from global economic and social regressions and further weakening of unstable states. global tuberculosis control: surveillance, planning, financing (world health organization is the development of a new tuberculosis vaccine possible? die schutzimpfung gegen tuberkulose mit "bcg immune control of hiv-1 after early treatment of acute infection guidelines for using antiretroviral agents among hiv-infected adults and adolescents can population differences explain the contrasting results of the mwanza, rakai, and masaka hiv/sexually transmitted disease intervention trials?: a modeling study a waxy tale by mycobacterium tuberculosis intracelluar models of mycobacterium tuberculosis infection mycobacterium tuberculosis: the indigestible microbe cd8 t cells in tuberculosis cd1 and tuberculosis gamma/delta and other unconventional t lymphocytes: what do they see and what do they do? adaptive immune response of v gamma 2v delta 2(+) t cells during mycobacterial infections mycobacterial phosphatidylinositol mannoside is a natural antigen for cd1d-restricted t cells human tuberculous granulomas induce peripheral lymphoid folliclelike structures to orchestrate local host defence in the lung how can immunology contribute to the control of tuberculosis? disseminated tuberculosis in interferon gamma gene-disrupted mice an essential role for interferon gamma in resistance to mycobacterium tuberculosis infection infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase iii trial. attract study group the immunological aspects of latency in tuberculosis mycobacterium bovis bcg response regulator essential for hypoxic dormancy inhibition of respiration by nitric oxide induces a mycobacterium tuberculosis dormancy program regulation of the mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin global dissemination of the mycobacterium tuberculosis w-beijing family strains worldwide occurrence of beijing/w strains of mycobacterium tuberculosis: a systematic review localisation und ausheilung der tuberculose. archiv für pathologische anatomie und genetic dissection of immunity to mycobacteria: the human model genetics of susceptibility to human infectious disease iron and microbial infection exogenous reinfection as a cause of recurrent tuberculosis after curative treatment survival perspectives from the world's most successful pathogen, mycobacterium tuberculosis the bcg story -lessons from the past and implications for the future efficacy of bcg vaccine in the prevention of tuberculosis. metaanalysis of the published literature selecting the components for a safe and efficient tuberculosis subunit vaccine-recent progress and post-genomic insights immunogenicity and protective efficacy of a tuberculosis dna vaccine protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and esat-6 application of mycobacterial proteomics to vaccine design: improved protection by mycobacterium bovis bcg prime-rv3407 dna boost vaccination against tuberculosis novel vaccination strategies microparticles as vaccine adjuvants and delivery systems recombinant modified vaccinia virus ankara expressing antigen 85a boosts bcg-primed and naturally acquired antimycobacterial immunity in humans enhanced immunogenicity of cd4(+) t-cell responses and protective efficacy of a dna-modified vaccinia virus ankara prime-boost vaccination regimen for murine tuberculosis differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, mtb72f, delivered as naked dna or recombinant protein the protective effect of the mycobacterium bovis bcg vaccine is increased by coadministration with the mycobacterium tuberculosis 72-kilodalton fusion polyprotein mtb72f in m. tuberculosis-infected guinea pigs auxotrophic vaccines for tuberculosis characterization of auxotrophic mutants of mycobacterium tuberculosis and their potential as vaccine candidates an essential role for phop in mycobacterium tuberculosis virulence complex lipid determine tissue specific replication of mycobacterium tuberculosis in mice persistence of mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase the heparin-binding haemagglutinin of m. tuberculosis is required for extrapulmonary dissemination identification of mycobacterium tuberculosis counterimmune (cim) mutants in immunodeficient mice by differential screening comparative genomics of bcg vaccines by whole-genome dna microarray recombinant bcg exporting esat-6 confers enhanced protection against tuberculosis recombinant bacillus calmette-guerin (bcg) vaccines expressing the mycobacterium tuberculosis 30-kda major secretory protein induce greater protective immunity against tuberculosis than conventional bcg vaccines in a highly susceptible animal model manipulation and potentiation of antimycobacterial immunity using recombinant bacille calmette-guerin strains that secrete cytokines bacille calmette-guerin (bcg)-associated inflammation and fibrosis: modulation by recombinant bcg expressing interferon-gamma (ifn-gamma) recombinant bacille calmette-guerin (bcg) expressing human interferon-alpha 2b demonstrates enhanced immunogenicity mycobacterium bovis bacille calmette-guerin strains secreting listeriolysin of listeria monocytogenes molecular determinants of listeria monocytogenes pathogenesis improved protection by recombinant bcg. microbes infect new live mycobacterial vaccines: the geneva consensus on essential steps towards clinical development recent advances in understanding the molecular mechanisms of hiv-1 entry and fusion: revisiting current targets and considering new options for therapeutic intervention hiv-1 dynamics in vivo: implications for therapy escape of human immunodeficiency virus from immune control hiv-1 viremia prevents the establishment of interleukin 2-producing hiv-specific memory cd4+ t cells endowed with proliferative capacity hiv preferentially infects hiv-specific cd4+ t cells the challenge of viral reservoirs in hiv-1 infection protective effects of a live attenuated siv vaccine with a deletion in the nef gene prospects for an aids vaccine cytotoxic t cell responses to multiple conserved hiv epitopes in hiv-resistant prostitutes in nairobi late seroconversion in hiv-resistant nairobi prostitutes despite preexisting hiv-specific cd8+ responses hiv-1 superinfection despite broad cd8+ t-cell responses containing replication of the primary virus timing the ancestor of the hiv-1 pandemic strains t cell cross-reactivity and conformational changes during tcr engagement hiv escape: there and back again envelope-constrained neutralization-sensitive hiv-1 after heterosexual transmission hiv and siv ctl escape: implications for vaccine design rapid evolution of the neutralizing antibody response to hiv type 1 infection evidence of hiv-1 adaptation to hla-restricted immune responses at a population level eventual aids vaccine failure in a rhesus monkey by viral escape from cytotoxic t lymphocytes reversion of ctl escape-variant immunodeficiency viruses in vivo transfer of neutralizing igg to macaques 6 h but not 24 h after shiv infection confers sterilizing protection: implications for hiv-1 vaccine development control of a mucosal challenge and prevention of aids by a multiprotein dna/mva vaccine control of viremia and prevention of clinical aids in rhesus monkeys by cytokine-augmented dna vaccination replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity plasma viral load, cd4 cell percentage, hla and survival of hiv-1, hiv-2, and dually infected gambian patients hiv vaccine design and the neutralizing antibody problem the antigenic structure of the hiv gp120 envelope glycoprotein humoral and cell-mediated immune responses to live recombinant bcg-hiv vaccines vaccine developments a recombinant vector derived from the host range-restricted and highly attenuated mva strain of vaccinia virus stimulates protective immunity in mice to influenza virus priming of human immunodeficiency virus type 1 (hiv-1)-specific cd8+ t cell responses by dendritic cells loaded with hiv-1 proteins hiv vaccine strategies safety and immune responses to a dna-based human immunodeficiency virus (hiv) type i env/rev vaccine in hiv-infected recipients: follow-up data enhanced t-cell immunogenicity of plasmid dna vaccines boosted by recombinant modified vaccinia virus ankara in humans hla class i serotypes and cytotoxic t-lymphocyte responses among human immunodeficiency virus-1-uninfected thai volunteers immunized with alvac-hiv in combination with monomeric gp120 or oligomeric gp160 protein boosting enhancement of mhc class i-restricted peptide-specific t cell induction by a dna prime/mva boost vaccination regime enhanced immunogenicity for cd8+ t cell induction and complete protective efficacy of malaria dna vaccination by boosting with modified vaccinia virus ankara cd4+ t cells are required for the maintenance, not programming, of memory cd8+ t cells after acute infection requirement for cd4 t cell help in generating functional cd8 t cell memory a conditioned dendritic cell can be a temporal bridge between a cd4+ t-helper and a t-killer cell reduced functional capacity of cd8+ t cells expanded by post-exposure vaccination of gamma-herpesvirusinfected cd4-deficient mice in interleukin-4-deficient mice, alum not only generates t helper 1 responses equivalent to freund's complete adjuvant, but continues to induce t helper 2 cytokine production cd8+ t cell cross-priming via transfer of proteasome substrates antigen bias in t cell cross-priming type i interferons promote cross-priming: more functions for old cytokines cross-priming of cd8+ t cells stimulated by virus-induced type i interferon homozygous defect in hiv-1 coreceptor accounts for resistance of some multiply-exposed individuals to hiv-1 infection the pathogenesis of sexual mucosal transmission and early stages of infection: obstacles and a narrow window of opportunity for prevention cd4+ t cell depletion during all stages of hiv disease occurs predominantly in the gastrointestinal tract tuberculosis and the tubercle bacillus interferon-gamma assays in the immunodiagnosis of tuberculosis: a systematic review mechanisms of protection induced by attenuated simian immunodeficiency virus repeated low-dose mucosal simian immunodeficiency virus sivmac239 challenge results in the same viral and immunological kinetics as high-dose challenge: a model for the evaluation of vaccine efficacy in nonhuman primates viremia control following antiretroviral treatment and therapeutic immunization during primary siv251 infection of macaques therapeutic dendritic-cell vaccine for chronic hiv-1 infection vaccination of macaques with long-standing sivmac251 infection lowers the viral set point after cessation of antiretroviral therapy decay kinetics of human immunodeficiency virus-specific effector cytotoxic t lymphocytes after combination antiretroviral therapy creating incentives for pharmaceutical research on neglected diseases world health organization. global atlas of infectious diseases report on the global aids epidemic world health organization. the world health report 2004 -changing history hiv-1/aids and the control of other infectious diseases in africa methylation-dependent t cell immunity to mycobacterium tuberculosis heparin-binding hemagglutinin therapy of tuberculosis in mice by dna vaccination boosting vaccine for tuberculosis the authors declare that they have no competing financial interests. volume the sentence "in south africa, more than 10% of the 40 million inhabitants are infected with hiv and more than 5% suffer from active tuberculosis" should read "more than 0.5% suffer from active tuberculosis."in table 2 two sentences in this article appeared incorrectly. on page 1164, in the eighth paragraph, the third sentence should read "to the u937-transplanted scid mice, 1 mg of either sg/17, yn907 or vehicle was injected intravenously, and 20 mg of either arac or normal saline was injected intraperitoneally on day 7 of transplantation." on page 1164, in the eighth paragraph, the fourth sentence should read "to nod-scid mice transplanted with patients' leukemic cells, 2 mg of either sg/17 or yn907 was injected intravenously, and 40 mg of either arac or normal saline was injected intraperioneally on days 3 and 4 of transplantation." key: cord-016829-37i1bn9m authors: nan title: bilateral and multilateral financing of hiv/aids programs: the world bank, the international monetary fund, the global fund, bilateral donors and the private sector date: 2008 journal: global lessons from the aids pandemic doi: 10.1007/978-3-540-78392-3_7 sha: doc_id: 16829 cord_uid: 37i1bn9m this chapter examines the operations of the world bank (a multilateral development institution), the international monetary fund (a multilateral financial institution) and the global fund to fight aids, tuberculosis and malaria (a multilateral fundraising and financing institution) to fight hiv/aids. we also examine the role of bilateral donors and the private sector in financing the fight against hiv/aids. we examine the relationships among bilateral donors and international organizations, what distinguishes their roles in the global hiv/aids pandemic and the extent to which their activities overlap. in addition, we consider how funding strategies and parameters may affect the effectiveness of aids funding in preventing transmission and providing treatment. the following series of tables (7.1-7.7) show the funding requirements for hiv/aids and break down those requirements by area in which the funding is needed. table 7 .8 contrasts the actual funding available and the funding gap. (in chap. 4, we considered the potential economic impact on recipient countries of while the increase in funding for hiv/aids is a positive development, the multiplicity of donors has created problems regarding overlapping actions and conflicting conditions attached to the aid. in 2005, over one hundred ministers, heads of agencies and other senior officials endorsed the paris declaration on aid effectiveness, an international agreement to increase efforts in harmonization, alignment and managing aid for results with a set of monitorable actions and indicators (oecd 2005) . also in 2005, the unaids global task team on improving aids coordination among multilateral institutions and international donors (gtt) published a set of recommendations on how countries and multilateral institutions can streamline their aids-related activities. many of these recommendations are aimed at improving the coordination of the aids-related activities of multilateral organizations and minimizing overlap. the recommendations build upon the principles of the "three ones" -the three principles for coordinated response at the country level: (1) one agreed hiv/aids action framework that provides the basis for coordinating the work of all parties; (2) one national aids coordinating authority, with a broad based multi-sector mandate; and (3) one ing to a 2006 unaids report, more than 40% of national aids plans were not serving as the framework for contributions by donors (unaids 2006) . more than 40% of national aids plans were not evaluated for cost or provided sufficient clarity on inputs and outputs, with the result that donors preferred to engage more poor alignment of donor strategies with national efforts and poor harmonization among donor procedures for aid management are significant impediments to achieving universal access to treatment for hiv/aids, because of the number and diversity of organizations that have become involved in addressing the pandemic. duplication of work and high transaction costs by individual agencies, together directly with countries (sidibe et al., 2006) . with the absence of important learning exchanges, economies of scale and synergies, impedes the speed, quantity, and quality of the response to the pandemic (sidibe et al., 2006) . thus, while pursuing affordable access to medicine and expanding medical services infrastructure are both essential elements for expanding access to treatment, they form a two-legged stool. the harmonization of donor and governance processes represents a key third element in the efforts to expand access to information and treatment. donald kaberuka, president of the african development bank, has noted that in the late 1980s, 22 members of the development assistance committee of the oecd (oecd/dac) accounted for 95% of total aid to developing countries. twenty years later, aid to developing countries is delivered through more than 150 multilateral agencies, 33 bilateral members of the oecd/dac and at least 10 non-dac governments. as a result, some developing countries have more than 700 active projects and receive more than 400 missions a year, each with its own spectives, more resources, greater innovation and competition, which could reduce costs and improve program delivery. however, this has not been the result thus far (kaberuka 2007) . figure 7 .4 shows the sources of the estimated and projected funding for the aids response from 2005 to 2007. government ministries, not just the health ministry) (patel 2004) . however, even where the host government has donor coordination mechanisms in place, coordination is made more difficult when individual donors come with pre-established priorities, without involving host countries in the setting of priorities (dekay 2004) . one clear example is pepfar. it has established abstinence-only as a priority, and denied funding for needle exchange programs, which are priorities for the us government that do not necessarily coincide with the priorities of host governments (see chap. 8 for details). in addition to the multiplicity of players, donors and host governments with different interests, ideologies, demands and expectations make coordination more difficult for host countries. when donors are driven by their own national ideologies and interests, at the expense of scientifically proven approaches, they undermine coordination and local leadership. moreover, when donors focus on civil society, they may undermine adherence to a national hiv/aids framework (mwale 2004 ). there has also been a proliferation of recipients. figure 7 .5 shows the distribution of entities that participated in the preparation of funding proposals for round four of the global fund. with respect to the lack of donor coordination, some argue that the source of the problem is the host government, which may fear losing control over the agenda to a well-coordinated group of donors, rather than the donors themselves. the center for global development has analyzed the policies and practices of pepfar, the global fund and the world bank's map in mozambique, uganda and zambia, and compared these systems against six key funding practices that can help donors support the national aids response in a manner consistent with the aid effectiveness principles of the paris declaration. these best practices are: donor commitments and disbursements can diverge. table 7 .9 shows that, in the case of some bilateral donors, commitments have exceeded disbursements, while for (oecd 2006) . others the reverse is true. in most cases, the actual disbursements lag commitments since its foundation since the end of the world war ii, the world bank has grown in size and importance. in 2008, it provides financial and technical assistance to developing countries through two development institutions owned by 185 member countries -the international bank for reconstruction and development (ibrd) and the international development association (ida). the world bank's current mission is to reduce global poverty and to improve living standards. the ibrd working with the government; building local capacity; keeping funding flexible; ing data. this study made the following recommendations to all three donors to increase the effectiveness of aid: (1) jointly coordinate and plan activities to support the national aids plan; (2) assist the host government in tracking total national aids funds; (3) focus on building and measuring capacity; (4) develop strategies with host governments and other donors to ensure financial sustainability; and (5) strengthen financial data collection and disclosure. the study also offered specific recommendations for how each donor can improve its own prorecommendations are addressed further below. focuses on middle income and creditworthy poor countries, while ida focuses on the poorest countries. together they provide low-interest loans, interest-free credit and grants to developing countries for education, health, infrastructure, communications and other development programs. the world bank's traditional loan package integrates loans with analytic and advisory services. the world bank's research program supports studies on a range of development issues (http://web. worldbank.org). the world bank provides loans to national governments to assist in the implementation of national hiv/aids programs. the bank also does research and provides assistance to make national aids programs more effective. the world bank's first hiv/aids-related project was in 1988. between 1988 and 2005, the bank the world bank organizes its aids-related activities globally, through its "global hiv/aids program of action" and six regional hiv/aids programs: (1) sub-saharan africa, (2) east asia and the pacific, (3) europe and central asia, (4) latin america and the caribbean, (5) middle east and north africa, and (6) south asia. the purpose of this organizational structure is to have a global focus gram to increase the effectiveness of aid (oomman et al., 2007) . these specific selecting appropriate recipients; making the money move; and collecting and shar-7 bilateral and multilateral financing of hiv/aids programs loaned over usd 2.5 billion to finance national hiv/aids programs. the world bank's programs seek to focus support where national programs need it the most. the world bank has adopted the "three ones principles", noted above. the world bank monitors and evaluates its programs to ensure hiv/aids programs are context-specific. the world bank also seeks to integrate hiv/aids issues into the broader development planning process. the world bank recognizes the need to make all of the key players part of each national strategy -governments, the private sector, community and civil society organizations, people living with hiv/aids, ngos and international organizations (world bank 2005a). the bank's program of action focuses on five areas: (1) sustained funding for hiv/aids programs and health systems; (2) better national hiv/aids planning, focused on high-risk groups and locations; (3) accelerating implementation of national hiv/aids plans by overcoming administrative constraints that delay the use of new funding; (4) building country monitoring and evaluation systems and that takes regional differences into account and to define the world bank's role in relation to other multilateral and international actors. the overall goals of these programs are prevention and treatment, taking into account local transmission patterns and sources of infection, in order to avoid a mismatch between funding and sustained funding is crucial. once people with hiv begin taking medication, they be monitored on an ongoing basis. if the underlying funding is not sustained, and more infectious and develop drug-resistant viruses. thus, the suspension of treatment is not only inhumane, but also makes prevention and treatment more difficult. resources are limited and need to be used effectively. targeting tripartite preventiontreatment-human rights strategies at high-risk groups has proved effective in countries like brazil (see chap. 2). targeting high-risk groups is a key component of an effective national hiv/aids strategy, particularly when those groups have been marginalized through discrimination. while funding for hiv/aids programs has increased, there remains an "implementation gap" between the available funding and its deployment. this is due to impediments to quick implementation: a lack of skilled personnel; unpredictable or conditional funding; burdensome disbursement and procurement processes; government reluctance to contract out implementation to civil society or the private sector; and multiple systems of management, monitoring and evaluation to meet different donor requirements. to address this problem, the world bank is simplifying its own processes and procedures, strengthening its implementation advisory service and coordinating un, global fund and world bank actions through the global joint problem-solving and implementation support team (gist) (world bank 2005a). the global aids monitoring and evaluation support team (gamet) seeks to build a well-functioning monitoring and evaluation system in each country, to enhance the impact of national programs. gamet provides field support to build country systems and capacity, as well as training and guidelines (world bank 2005a). the world bank's main focus has shifted from getting resources to countries to ensuring that more impact evaluations are done of bank-supported hiv/aids et al., 2006) . the scope of the world bank's operations is quite wide, covering a wide range of development issues in a large number of countries, in addition to its work related to hiv/aids. projects with an hiv/aids component of more than usd one million, in 60 countries. these projects include health sector support and reform, prevention and control ( http://siteresources.worldbank.org / inthivaids / projects /20385466/ gramsupport. programs, human resources development and education and national aids prowas designed to help countries intensify and expand their multi-sector national financing06-24-04.xls) some of these projects are conducted as part of the responses to the hiv epidemic, to dramatically increase access to hiv prevention, in rwanda the world bank used map community grants for hiv initiatives to fund over 100 civil society organizations to provide preventive, medical and support services for people living with hiv. in 2002, rwanda was among the ten countries most severely affected by hiv and had recently emerged from a genocide/war. the usd 30.5 million grant became effective on 11 august, 2003 and was fully committed and almost fully disbursed by the end of 2006. the world bank board of directors approved additional financing of usd 10 million on 2 sustainability. building, and monitoring and evaluation of programs. just over 10% paid for hiv/ one part of this project was to help female sex workers to find different sources of income. several factors contributed to the success of this project. first, the project engaged local champions to mobilize people (in this case, the deputy mayor in charge of social welfare on the district hiv/aids commission became personally engaged in designing the program design). second, to break the aids-poverty cycle, poverty mitigation accompanied preventive measures (in this case, new sources of income provided a basis for further, community-based economic development projects). third, the project sought to empower women to benefit the whole family (in this case, empowering them to provide better education and health care for their children). fourth, ongoing support was provided to sustain the success of the project (in this case, to prevent the women returning to prostitution when challenges arise) (world bank 2007) . however, as we will see in chap. 9, programs for sex workers that focus on alternative employment have been criticized for contributing to their stigmatization and failing to address the needs of those who continue to earn their income from sex work. another part of the rwanda map project provided orphans and vulnerable children with sewing machines and training as tailors so that they could start a clothes-manufacturing association. this program combined information about hiv and aids, reproductive health and life skills with income generating activities. the children then served as role models and sources of information on hiv and aids for other vulnerable children. the experience with this program suggests three key factors for success. first, establish solidarity among participants, in this case by bringing the children together to find solutions to their problems and design their own interventions. second, enable beneficiaries to be role models for behavioral change. third, address poverty and vulnerability in order to make aids prevention effective (world bank 2007). in yet another part of the map project, the rwanda national youth council (cnjr) created a voucher system for expanding access to hiv testing for youths aged 10-24 (almost 40% of the population). the rationale behind promoting testing is that enabling people to learn their hiv status is a first critical step in changing behavior. the cnjr trained 230 peer educators in behavioral change communication and reached out to youth through anti-aids clubs and sports and cultural activities. the voucher system enabled youths to go to local health facilities on designated days, minimizing waiting times. the map funding was used to pay for these services. this approach was more cost-effective (usd 2 per person) than using mobile units to reach these youths (usd 10 per person). it also enhanced the returns on global fund investments in facility-based services. in the first 4 months almost 70,000 youths were tested. about 6% tested hiv positive. the three main lessons from the cnjr campaign were (1) hiv testing is critical to modifying sexual behavior and expanding condom use; (2) better knowledge leads to greater empathy and solidarity with people living with hiv; and (3) awareness campaigns foster a culture of responsibility, trust and faithfulness among young couples (world bank 2007) . the rwandan government developed a treatment plan with the support of the clinton foundation. a user fee policy with a sliding scale results in most rwandans receiving free care because they live below the poverty line. the rwandan government, the world bank, the clinton foundation, the global fund, us centers for disease control and prevention (cdc) and the us government/pepfar worked together to design, implement and monitor the treatment program. different sites were funded by different donors (the world bank, the global fund and the us government/pepfar). patients receiving treatment increased from 870 patients at seven sites at the end of 2002 to roughly 32,000 patients at 130 sites national procurement system, the world bank and the global fund finance generic drugs and the us government/pepfar pays for brand name drugs. the clinton foundation has helped to lower the prices paid for drugs and diagnostics. pepfar funded the "tracnet" system, which uses mobile phones to transmit information. it tracks the number of patients on treatment and the drugs dispensed nation-wide, to manage the treatment program and supplies efficiently and to avoid drug shortages that would lead to interruptions in treatment. interruptions in treatment could diminish the effectiveness of treatment by creating resistance to the drugs being used and raise costs by requiring the use of other drugs and medical treatments. performance-based contracting and bonuses for health care staff expanded key hiv services rapidly in a relatively short time (for example, the number of hiv tests performed by staff) (world bank 2007). the world bank has also addressed the hiv/aids epidemic in central america on a regional basis. four of the six countries in latin america with the highest hiv prevalence are in central america. two central american countries have prevalence rates above 1%, which is a key threshold for an hiv epidemic to run out of control unless prevention efforts are improved among high-risk groups, such as commercial sex workers, men who have sex with men and prisoners (see chap. 2). the adult hiv prevalence rates are as follows (in alphabetical order): costa rica (0.6%); el salvador (0.6%); honduras (1.6%); guatemala (1%); nicaragua (0.2%) and panama (0.9%). by 2010, the adult hiv prevalence rate may reach 2% in the region. hiv transmission in central america is primarily associated with heterosexual sex (with the exception of costa rica), as in africa and the caribbean (world bank 2006) . the world bank has developed a model to help governments to allocate resources efficiently to prevent the maximum number of new infections (world bank 2003) . in central america, the world bank also studied the extent of discrimination and stigmatization and identified areas where changes in general legislation or hiv/aids laws are necessary (world bank 2006). economist jagdish bhagwati has taken the world bank to task for not having a sufficiently narrow focus. in particular, he has opined that the world does not need a global think tank staffed by 8,500 people and that the world bank should not be involved in intellectual programs for countries, like india, that have sufficient resources to handle their own affairs. thus, in his view, the world bank should be cut to 300 staff to focus on a good intellectual program for countries that need it (bbc news 2007a). easterly (2006) has accused the world bank of wasting resources on inefficient bureaucracies and for having a late response to the hiv/aids epidemic (it had only implemented one project by 1993 and only completed ten by 1998). in a comparative study of us and world bank funding for hiv/aids between 1995 and 1999, smith (2007) found that, although there is a positive correlation between the incidence of hiv/aids and poverty and the likelihood of receiving aid from the world bank, the us government is more responsive to the incidence of hiv/aids and poverty with its foreign aid donations. moreover, the world bank allocates more money to countries with political systems that are less likely to use the funds for public goods like education, treatment and health care, and does not do as well as the united states in targeting aid to countries most in need of funding (smith 2007 ). it is difficult to assess the success of the world bank's hiv/aids programs, since they are ongoing, diverse and numerous. however, the world bank's current programs have incorporated several features that suggest that they will be effective: following "the three ones", ongoing support, monitoring and evaluation of programs for effectiveness, sharing information regarding the factors that contribute to the success of individual projects and working with other multilateral and bilateral donors to reduce overlap. the world bank has undertaken or commissioned three reviews of its hiv/aids work (world bank 2005a). the "interim review of the multi-country hiv/aids program for africa", was done in early 2004. this review listed the following key barriers and challenges: (1) many national hiv/aids plans are not strategic, and are poorly prioritized; (2) prevention, care and treatment efforts are too small, and coverage is too low; (3) management and implementation constraints hamper action; (4) health systems are weak and overwhelmed, particularly with efforts to expand access to treatment; (5) the effort to expand antiretroviral (arv) treatment raises difficult issues of equity, sustainability and adherence; (6) prevention remains inadequate, regardless of the stage of the epidemic in a given country; (7) stigma and discrimination, denial and silence persist, to the point that some people would rather die than let others know they are hiv positive; and (8) donors sometimes create additional problems for countries, for example in tanzania, where program managers spend more time meeting the needs of visiting donors than implementing the programs. the world bank's operations evaluation department (oed) has assessed the development effectiveness of the world bank's country-level hiv/aids assisimpact of the aids epidemic) (world bank 2005b). the oed's assessment found more focused and cost-effective way. it has helped raise political commitment, create or strengthen aids institutions, enlist ngos, and prioritize activities. the oed's assessment also found that political commitment and capacity had been overestimated and needs continuous assessment. moreover, failure to reach people with the highest risk behaviors likely had reduced the efficiency and impact of assistance. the effectiveness of the national response had been hampered by weak monitoring and evaluation and the failure to ensure that country-based research as a result of its assessment, the oed made several recommendations for the future work of the world bank in the hiv/aids epidemic. first, it should help governments use human and financial resources more efficiently and effectively. second, the world bank should help governments to be more strategic and selective, and to prioritize activities that will have the greatest impact. third, it should work to strengthen national institutions for managing and implementing the long-run response, particularly in the health sector. fourth, it needs to improve the local evidence base for decision-making and create incentives to ensure that program 7 bilateral and multilateral financing of hiv/aids programs tance (defined as policy dialogue, analytical work and lending to reduce the scope or that the world bank's assistance has induced governments to act earlier or in a is focused on priorities areas. decisions are guided by relevant local evidence and rigorous analytical work. wilson has concluded that the world bank's map programs must place far greater emphasis on improved surveillance, research and analysis in order to design more intelligent maps that take into account prevalence and transmission patterns, the distinction between generalized and concentrated epidemics, the effect of stigma and community values on treatment programs and the role of political leadership in national aids strategies. the money must follow the epidemic by focusing primarily on normative and behavioral change in the general population in generalized epidemics and on high coverage of vulnerable groups in concentrated epidemics. in generalized epidemics, emphasis must shift from knowledge and awareness to normative change. in more concentrated epidemics, the world bank needs greater focus on groups and areas where transmission is occurring (wilson 2007) . a 2007 survey revealed that more than a third of patients on hiv medication in sub-saharan africa die or discontinue their treatment within 2 years of starting; only 61.6% of all patients were still receiving medication. the study found that many were too late taking up arv drugs and died within a few months of commencing treatment. for some it was impractical to travel to distant clinics. the researchers also found evidence that, in cases where patients had to pay for arvs, some stopped treatment. some people also suffered from stigma: in some workplaces, people are not able to carry their arvs and take their arvs freely at workplaces. retention rates between individual arv programs varied widely across africa. one program in south africa retained as many as 85% of their patients after 2 years while another in uganda retained only 46% of patients after the same period of time (bbc news 2007b). the world bank was a member of the gtt. the agreements reached by the gtt, including the division of labor, are reflected in the world bank's program of action (world bank 2005a). however, as noted above, more work is needed to resolve the problems created by the proliferation of donors. in its 2007 study of funding practices, the center for global development made the following recommendations to the world bank regarding map: (1) focus resources on building government capacity; (2) become a knowledge bank, with a focus on prevention; (3) make the transition to the use of existing government systems; (4) increase individual disbursement amounts; and (5) publicly disdominique strauss-kahn, who became the director general of the imf on 1 november 2007, has noted the need for the imf to adapt to a new global economic order (to better reflect the rise of emerging market economies and the interests of less developed ones, plus the need to better regulate globalization) and to downsize or die. in strauss-kahn's view, the imf needs to reach out to other multilateral organizations, in particular the world bank. the imf also needs to mend fences in a meaningful way with regions of the world that felt hard done by during close data (oomman et al., 2007) . the imf is an international organization that was established to promote international monetary cooperation, exchange stability and orderly exchange arrangements; to foster economic growth and high levels of employment; and to provide temporary financial assistance to countries to help ease balance of payments adjustment. it has 185 member countries. the imf is to be guided in all its policies and decisions by the purposes set forth in article i of the articles of agreement of the international monetary fund. article i sets out these purposes as follows: 1. to promote international monetary cooperation through a permanent institution which provides the machinery for consultation and collaboration on international monetary problems. 2. to facilitate the expansion and balanced growth of international trade, and to contribute thereby to the promotion and maintenance of high levels of employment and real income and to the development of the productive resources of all members as primary objectives of economic policy. 3. to promote exchange stability, to maintain orderly exchange arrangements among members, and to avoid competitive exchange depreciation. 4. to assist in the establishment of a multilateral system of payments in respect of current transactions between members and in the elimination of foreign exchange restrictions which hamper the growth of world trade. the fund temporarily available to them under adequate safeguards, thus providing them with opportunity to correct maladjustments in their balance of payments without resorting to measures destructive of national or international prosperity. 6. in accordance with the above, to shorten the duration and lessen the degree of disequilibrium in the international balances of payments of members. since inception the imf's purposes have remained the same, but its operations have developed over time, particularly with respect to surveillance of public expenditure management systems, financial assistance and technical assistance. the imf focuses on three kinds of operations. first, surveillance operations monithe imf also provides emergency assistance to support recovery from natural disthe imf can allocate additional spending to hiv/aids as part of national poverty-reduction strategies. the imf also provides advice to countries on the macroeconomic impact of hiv/aids and how to manage inflows of foreign aid. reduction strategy papers, which provide the operational basis for imf and world bank loans to low-income countries and fo r debt relief under the heavily indebted poor countries (hipc) initiative (imf 2005) . unlike development banks, the imf does not lend for specific projects. thus, unlike the world bank and the global fund, the imf does not have projects specifically aimed at hiv/aids. rather, hiv/aids issues are addressed as part of the imf's general operations. the research of the imf on hiv/aids has been focused primarily on its macroeconomic impact. with respect to hiv/aids, imf (and world bank) policies have been criticized for not considering the impact of structural adjustment requirements (austerity making debt repayment a higher priority than health care, thereby undermining the ability of national governments to finance health care. peter lurie et al. (1995) argued that export-oriented, structural-adjustment policies were undermining hiv/aids control by contributing to social changes that favor the spread of hiv in the developing world, through increased mobility, migration, urbanization and dislocation of family units. tor economic and financial developments and provide policy advice, aimed especially at crisis prevention. second, the imf also lends to countries with balance of payments difficulties, to provide temporary financing and to support policies aimed aimed at poverty reduction. third, the imf provides countries with technical assis-at correcting the underlying problems. loans to low-income countries are also tance and training. the imf also conducts economic research and gathers statistics shocks facility. non-concessional loans are subject to the imf' s market-related related to these three types of operations. an imf loan usually stipulates the specific policies and measures a country has to implement to resolve its balance interest rate, which is revised weekly to take account of changes in short-term in-of payments problem. low-income countries may borrow at a concessional interest rate through the poverty reduction and growth facility and the exogenous country-level hiv prevention and treatment programs form part of many poverty asters and conflicts, in some cases at concessional interest rates (http://www.imf.org). terest rates in major international money markets. large loans carry a surcharge. measures) on the spread of hiv/aids. the imf has also been criticized for hanlon (1999) noted that the imf required the mozambican government and other african governments to reduce spending in the 1990s without decreasing repayments of its debts, thereby contributing to cuts in spending on health services that increased corruption in hospitals. the international community later recognized that much of africa's debt could not be paid and agreed to cancel some debts. this led to the hipc initiative in 1996. however, hanlon reported that with the hipc mozambique would still be paying back usd 100 million a year, because the world bank and imf agreed to cancel only the part of the debt that mozambique was not paying. the hipc only canceled the uncollectable debt. thus, mozambique was paying usd 275,000 per day in debt service, but only usd 100,000 per day for its entire health service. in 2003, african ministers of finance, planning and economic development warned that the enhanced hipc initiative was not delivering long-term debt sustainability. they recommended that the imf impose fewer structural conditions and provide for outcomes-based conditions where appropriate. the ministers also urged the imf and world bank, bilateral partners and the african development bank to avoid "cross-conditionalities" that impede access to resources. they recommended that to provide greater fiscal flexibility, the imf should also analyze the linkages, trade-offs and policy choices required to attain the millennium development goals (which include addressing the hiv/aids pandemic). they also proposed that evaluating exogenous shocks should be a standard feature of imf discussions with member states, and that access to loans should be extended to countries suffering from exceptional exogenous shocks such as the onslaught of imf and world bank to consider revising the eligibility criteria for assistance to middle-income countries affected by the aids epidemic, and to find ways of ensuring that countries could expand expenditure on health and social welfare without violating conditions that impose limits on public spending (eca 2003) . the imf restricted aid to mozambique's budget in order to control inflation and required donors' aid to fund more projects outside the state budget, contrary to the policy of many donors (hanlon 2006) . the imf resident representative mozambique argued that it was difficult for the government to be certain about the level of donor payments in the medium term, and it might not be prudent to make medium term expenditure commitments (such as the hiring of health personnel) given the uncertainty about whether the money would still be available over the next budget cycle. the imf representative recognized the need to improve the design of the imf fiscal target taking into account that donor aid has increasingly moved away from lending to support capital investment projects, and toward direct budget support for hiring personnel and purchasing medicine (perone 2006) . this story highlights the ongoing importance of policy coordination on the ground between the imf, donors and national governments and the need for sustainable funding from donors. the global fund was created to raise and disburse additional private and public sector funds for the fight against aids, tuberculosis and malaria. as of october 2007, the global fund had committed usd 8.4 billion in 136 countries. it provides around two thirds of all international financing for tuberculosis and malaria and close to a quarter of the global resources for aids. as we saw in chap. 4, malaria prevention is a cost-effective means to prevent hiv/aids in areas with high malaria prevalence, because of the impact of life-expectancy on incentives to change sexual behavior to reduce the risk of hiv/aids. the prevalence of hiv/aids also has a direct impact on the spread of extensively drug-resistant tuberculosis (xdr-tb). people living with aids are especially susceptible to xdr-tb because of their depressed immune systems, increasing the risk that xdr-tb will spread rapidly in sub-saharan africa (picard 2007). in turn, an xdr-tb epidemic could reduce life expectancy, thereby hampering hiv/aids prevention. thus, it is important to attack all three diseases together. there are two key differences between the global fund and the world bank. first, the global fund is a hands-off operation that focuses on financing rather than implementation, whereas the world bank is very involved in the implementation of its programs. second, the global fund has a much narrower scope of operations with respect to the issues that it tackles, being restricted to aids, tuberculosis and malaria. the world bank has a much broader development agenda, albeit one that incorporates health care in general, and hiv/aids in particular, into its development programs. section ii of the framework document of the global fund sets out its purpose in the following terms: the purpose of the fund is to attract, manage and disburse additional sustainable and significant contribution to the reduction of infections, illness and death, thereby mitigating the impact caused by hiv/aids, section ii of the framework document clarifies that the fund's mandate is to finance prevention, treatment, care and support in an integrated and balanced way, not to implement such programs. the framework document provides further that the global fund "should make use of existing international mechanisms and health plans". in making its funding decisions, the global fund is to focus on best 7.4 the global fund: a fundraising and financing institution 2003). resources through a new public-private partnership that will make a reduction as part of the millennium development goals (global fund tuberculosis and malaria in countries in need, and contributing to poverty expansion of government/private/ngo partnerships. the global fund is required to support programs that reflect "national ownership", by focusing upon the technical quality of proposals, while leaving the design of programs and priorities to partners within recipient countries. "country coordinating mechanisms" (ccms) are country-level partnerships that develop and submit grant proposals to the global fund based on priority needs at the national level. after grant approval, they oversee progress during implementation. ccms include representatives from the public and private sectors, including governments, multilateral or bilateral agencies, non-governmental organizations, academic institutions, private businesses and people living with the diseases. the proposals that are supported by the global fund must be consistent with international law and agreements, respecting intellectual property rights laws, such as trips. at the same time, the programs should encourage efforts to make quality drugs and products available at the lowest possible prices and give priority to the most affected countries and communities, and to those countries most at risk. the framework document provides specifically for programs that aim to eliminate the stigmatization of and discrimination against those infected and affected by hiv/aids, especially for women, children and vulnerable groups. the framework document requires the global fund to use, wherever possible, existing monitoring and evaluation mechanisms. monitoring at the country level is country-driven, but also linked to the fund's monitoring and evaluation system at a global level. grantees need to be: (1) accountable to donors for the use of funds and achievement of results; (2) responsive to developing countries; and (3) responsive to the needs of those infected and directly affected by the three diseases. the framework document contemplates the following options for who oversees the process of monitoring both global and local program progress on behalf of the global fund board: global fund secretariat; ad hoc monitoring and evaluation working group; the world bank's operations evaluation department; a un agency; existing mechanisms (unaids, stop tb, roll back malaria); an independent monitoring and evaluation oversight committee appointed by the global fund board; or a third party (for example, an accounting firm or university). the world bank serves as the trustee for the global fund. the trustee has primary responsibility for financial accountability, including: (1) collection, investment, and management of funds; (2) disbursement of funds to national-level entities, on the instruction of the global fund board; (3) reporting to stakeholders on the financial management of the fund and the allocation of fund resources; and (4) independent audits. local funding agents are organizations (mainly multinational audit firms) that the global fund contracts to provide the secretariat with the information used to practices and performance, linking resources to the achievement of clear, measurable and sustainable results. another focus is on the creation, development and report on the capacity of the agencies that implement funded programs and on program results and make recommendations regarding future funding. the outfor global fund offices in the relevant countries, thereby enabling the global fund to have a leaner administration and to set up more rapidly in a country (euro the framework document requires the highest priority to be given to proposals from countries and regions with the greatest need, based on the highest burden of disease and the least financial resources. these are identified as including sub-saharan africa, currently the region most affected, as well as some countries within the caribbean, asia-pacific, latin america and central and eastern europe. the criteria for identifying these proposals include: (1) disease burden for hiv, tb and/or malaria; (2) poverty indicators, such as per capita gnp and the un human such as recent disease trends, size of population at risk, prevalence of risk factors, extent of cross-border and internal migration, conflict, or natural disaster; (4) political commitment, as indicated by contribution to the financing of the proposal, public spending on health, existence of supportive national policies or the presence of a national counterpart in the proposal; (5) existence of a country coordination mechanism, which consists of an inclusive collaborative partnership, with all relevant partners engaged in planning, decision-making and implementation. the framework document requires the global fund to provide grants to public, private and non-governmental programs for the prevention, treatment, care and support of the infected and directly affected, which may include: increased access to health services; provision of critical health products (for example, bed nets; condoms; antiretroviral, anti-tb and anti-malarial drugs; treatment for sexually transmitted infections; laboratory supplies and materials; and diagnostic kits); training of personnel and community health workers; behavioral change and outreach; and community-based programs, including care for the sick and orphans. technical review panels, made up of independent, impartial teams of experts appointed by the global fund board, review grant proposals, based on criteria set by the board, and make recommendations to the board for final decision. the global fund's technical evaluation reference group (an independent advisory body) was charged with overseeing a five-year evaluation on the operations of the global fund, during 2006-2008. the first area of evaluation is "organizational efficiency", which analyzes whether the global fund is efficient and effective in fulfilling its core principles, including acting as a financial instrument rather than implementation agency and furthering country ownership. the second area of evaluation is "partnership environment", which considers how effective a striking feature of the global fund's very user-friendly web site is the global fund evaluation library (http://www.theglobalfund.org/en/links_resources/ brary/). this web page makes both internal and external evaluations of the global fund readily available and has the stated aim of improving the global fund's delivery of key services for hiv/aids, tuberculosis and malaria by stimulating open debate and evaluation of the fund. this novel method of inviting and inciting critical evaluations, in order to improve performance, stands in contrast to the world bank and imf approaches to external criticism. the global fund has been criticized for funding drugs without strengthening the underlying health systems. in response, the global fund opened the possibility of funding health system improvements, but this was not used often and rarely covered staff remuneration. the world bank has proposed leaving the strengthening of health systems up to the world bank, in order to avoid overlap. however, the world bank itself has been criticized for poor performance in supporting and reinforcing public health services, particularly given its role, together with the international monetary fund, in maintaining limits on public health expenditures and wages. the global fund, with its narrow funding mandate, has also been criticized for not placing more conditions on the use of the resources provided and for giving into technical advisors from the world bank and bilateral donors who oppose the exceptional nature of aids (philips 2007) . and efficient the global fund partnership system is in supporting hiv, malaria and tb programs at the global and country level. the third area of evaluation is "health impact", which studies the global fund's contribution to reducing the burden of the three diseases. fig. 7.12 global fund, sector of recipients (2001) (2002) (2003) (2004) (2005) (2006) the global fund evaluates program performance after 2 years. the center for global development analyzed data on 134 of the first 140 global fund grants evaluated in 2006. due to the subjective nature of the evaluation process, this analysis should be seen as an analysis of evaluation scores rather than of the actual performance of the programs. moreover, the results were not intended to be used to influence the distribution of funding, but rather to allocate resources for oversight and risk management. the programs that scored lowest had government agencies as principal recipients, had a large amount of funding, were focused on malaria, had weak initial proposals or were evaluated by the accounting firm kpmg. countries with a high number of doctors per head, high measles immunization rates, few health-sector donors and high disease-prevalence rates had higher evaluation scores. poor countries, those with small government budget deficits and those that have or have had socialist governments also received higher scores. programs in which a government was the principal recipient received significantly lower scores than those with civil society, private sector or multilateral recipients. malaria programs were 12.9% less likely than hiv/aids or tuberculosis programs to receive an a. in addition, evaluation scores were slightly higher in countries with higher prevalence rates for the target disease of the program (radelet countries with stronger health systems and larger numbers of trained health workers were more likely to have successful programs. this suggests that the global fund needs to ensure greater oversight in countries with weaker systems and capacity, and underscores the importance of building strong health systems rather than focusing narrowly on short-term targets. however, evaluation scores were lower in programs where there were many other donors. this may have been due to the greater administrative and management burden placed on recipients when there are multiple donors. alternatively, it may indicate that the incentives for strong performance are weaker when recipients have many funding alternatives the global fund's country coordination mechanism has been criticized for focusing on government actors and not including people with hiv/aids (gonzalves 2004; mckai 2004) . faith-based organizations in host countries have also not been included in country coordination mechanisms to the degree that they would like, often due to a lack of information from governments, a lack of access to guidelines for funding proposals and a need for assistance in preparing funding the center for global development convened a high-level independent working group to help the new executive director of the global fund define the major tasks that require attention, and provided specific recommendations for action. the working group consisted of 21 members, including relevant experts in government, civil society organizations, foundations, academia, private sector companies and disease-based partnership initiatives. some of the working group's key recommendations were: (1) convene a "heads of agencies group" with the director general of the who, the executive director of unaids, and the president of the proposals (lee et al., 2002) . ( radelet and siddiqi, 2007) . world bank to jointly tackle key problems in technical assistance, procurement, monitoring, evaluation and other key issues; (2) work with other agencies to develop an information market for technical assistance, building on existing mechanisms with unaids, the stop tb partnership and roll back malaria; (3) provide early warning information on country programs to recipients, ccm members, governments, international partners and key ngo groups so that actions can be taken quickly to get programs back on track; (4) commission a management audit of the secretariat, consider hiring additional portfolio managers and consider shifting from one portfolio manager per country to teams of two or three working across countries; (5) hire a full-time professional fund raising team, led by a senior professional and comprised of experts with diverse skills to work with traditional, non-traditional and private sector donors; (6) make the executive director a nonvoting member of the board so that the experiences and insights of the executive director and the secretariat are more fully reflected in board discussions (radelet 2006) . in its 2007 study of funding practices, the center for global development made gaps; (2) re-examine strategies to build local capacity; (3) simplify procedures for a 2007 external evaluation of the global fund's local funding agent system evaluation found that the system needs: (1) greater emphasis on health program skills (since few local funding agents have staff with health backgrounds); (2) to implement a quality assurance system to verify the adequacy of local funding agent methods (for example, with respect to documentation of audits); (3) to provide more complete capacity assessments of the organizations that implement programs; (4) to increase the use of in-country partnerships; (5) to implement a comprehensive performance evaluation system for local funding agents; and (6) management process (euro health group 2007 ). an internal evaluation made similar findings: (1) that there were gaps in local funding agent documentation that failed to meet professional auditing standards; (2) adherence to specific auditing standards needed to be integrated as a requirement in the tendering process; (3) a lack of a consistent management approach; (4) that the global fund secretariat should introduce a systematic performance evaluation system for local funding agents and a handbook; (5) that capacity assessment should extend to sub-recipients, not just principal recipients of funding; (6) the health program skills and experience of local funding agents was inadequate; and (7) that local funding agents needed to develop partnerships with other health-sector players through networking, to have greater access to health sector intelligence and improve coordination with other players (technical evaluation reference group 2007). the united states government accountability office (gao) also conducts reviews of the global fund, since the united states contributes funds. in 2007, the gao review of 80 grant disbursements and 45 grant renewal decisions found that good performers; and (4) publicly disclose data (oomman et al., 2007) . 7 bilateral and multilateral financing of hiv/aids programs the following recommendations to the global fund: (1) keep the focus on funding concluded that the system should be maintained, but needs to be improved. the to create an operational manual or handbook to govern the local funding agent bilateral governmental donations are an important source of financing for hiv/aids programs. in 2005, the resources available from all sources totaled usd 8.3 billion, out of which g7/ec and other donor government commitments for hiv/aids totaled roughly usd 4.3 billion. of these donor government commitments, usd 3.5 billion were bilateral commitments and usd 810 million were contributions to the global fund. the g7/ec commitments were 85% of the total donor govern-they were well documented and that documentation had improved since 2005. the gao also noted that the global fund has begun developing a risk assessment framework to improve the identification of risks that may affect grant implementation, having found that an earlier risk assessment model was inadequate. however, the gao also found that the lack of a mandatory system of local funding agent performance assessment limits the global fund's ability to determine the quality of local funding agent monitoring and reporting (gao 2007). as we noted earlier, commitments and disbursements are not the same thing. for example, while the united states accounted for 54.4% of bilateral commit-7.13 and 7.14). many donors prefer to channel funding through bilateral programs (81% of g7/ ments). however, preferences vary from one donor country to the next. for example, at on end of the spectrum, the uk made 93% of its commitments through bilateral ec commitments), rather than the multilateral channel (19% of g7/ec commitchannels and 7% through the global fund. at the other end of the spectrum, italy made 20% of its commitments through bilateral channels and 80% through the global fund. canada was in the middle, with 46% of its commitments through bi-2006). engaging the private sector in hiv/aids prevention and treatment is increasingly becoming a major focus for governments, advocates and public health practitioners. the private sector, through domestic firms or the foreign direct investment of multinational firms, is a significant source of financing for the prevention and treatment of hiv/aids. private charitable foundations have also become an important source of funding in the fight against hiv/aids. two issues regarding private sector donors provoke controversy. one is the issue of donations of goods and services in kind by the private sector. critics argue that such donations impede the ability of recipient governments to build their own infrastructure. supporters argue that such donations give a boost to local efforts to build infrastructure, provided they take place in close cooperation with recipient governments. a second issue is donor reliability and sustainability, particularly with respect to donations from the private sector (although this issue is also raised regarding other sources of funding). here, the concern is that host governments will be unwilling to integrate funding for hiv/aids into general revenues that support the overall health infrastructure (for example, salaries for medical personnel) unless there is some assurance that the funding will be ongoing (mckinnell 2004) . while the participation of the private sector in addressing the hiv/aids pandemic is very important, its efforts need to be coordinated with the other players (governments, donors and ngos) and the roles of the various players need to be well defined. for example, oxfam has criticized developed countries and the world bank for undermining governments' ability to deliver public services by advocating inappropriate private sector projects in health. oxfam acknowledges that the private sector has a role to play, but argues the private sector cannot provide services on the necessary scale, geared to the needs of all citizens (oxfam 2006) . companies have an incentive to invest in hiv/aids programs due to the impact of the pandemic on enterprise performance. this impact depends on worker attrition due to sickness and death, the corresponding costs to the firm for providing health and sickness benefits, replacement costs to obtain new workers and the 7.6 the role of the private sector in funding prevention and treatment 249 lateral channels and 54% through the global fund (see fig. 7 .15) (kates and lief, same time, the health of a country's population may affect inflows of foreign hiv/aids pandemic thus creates a catch-22 situation. hiv/aids has clear effects on a company's workforce and its customer base. the literature suggests that skilled workers are most likely to contract the virus, waterhousecoopers survey found that only 39% of organizations in eastern vention among employees and their families and providing access to treatment. for example, in botswana an early and innovative program by debswana, a diamond mining company that is the country's largest non-government employer, made it the first company to provide free anti-retroviral treatment to employees and their spouses (center for global development 2005). however, as with multilateral organizations such as the world bank, the imf, the global fund and the un, it is important to have an overarching framework in order to ensure the adoption of best practices by individual firms and to minimize overlap between the private sector and the other players that are involved in addressing the pandemic. in this regard, there have been some important national and global initiatives. an example of a national initiative is the commercial market strategies (cms) project, which was a 5-year usaid-funded project (1998) (1999) (2000) (2001) (2002) (2003) . cms's aids treatment brochure articulated the business case for providing arvs to employees as part of their health care benefits. the key global initiative is the global business coalition on hiv/aids (gbc), which has 220 members, headquartered in thirty countries, employing over eleven million people in more than 200 countries and is supported almost entirely by its member companies. the gbc and booz allen hamilton conducted a baseline survey and interview program to establish a basis to look at the scope and depth of the response being made by the global business community. the study highlighted variations in business response by region, industry and enterprise scale. the study found that business increasingly sees hiv/aids as a strategic as well as a social responsibility issue, and manages programs and resources based on bottom line impact (gbc and booz, allen, hamilton 2006) . the data source was the expertise and experiences of 75 gbc member companies across 17 industries that were surveyed in april 2006 and 30 companies who participated in a detailed interview program. the baseline showed business response in the form of an index, on a scale of 0-10. the index was calculated from the number of companies active in each of 10 global business hiv/aids categories of best practice aids standard (bpas), each with five levels of action. the impact of hiv/aids on worker productivity (ramachandran et al., 2005) . at the and that these are extremely difficult to replace. customers, suppliers and inves-direct investment to low-and middle-income countries (alsan et al., 2004) . the tors in a company are also likely to be affected by hiv/aids, and this effect is expected to increase as the virus spreads (bloom et al., 2001) . however, a price-on an individual basis, firms can have a tremendous impact in promoting pre-africa had hiv/aids policies in place (pricewaterhousecoopers 2003). 75 surveyed companies had an average index score of 4.5. this was equivalent to being active in more than 8 of 10 categories with two actions underway in each. the most active 25% scored 7.5 and the least active 25% scored 1.4. this variation was primarily due to perceived business needs and the length of time the companies had been addressing the hiv/aids issue. of the 10 bpas categories there are two in which the business response was particularly strong -prevention initiaoil) (gbc and booz, allen, hamilton 2006). were twice as likely to fully subsidize treatment for employees in high prevalence ments, suggesting that program design and follow up could be enhanced. with the survey found that developing and implementing a company hiv/aids prothe gbc survey had several key findings regarding ways to improve the global business community's response to hiv/aids. first, there is a need to develop strategies to work closely with suppliers and business associates to expand the network of business engagement. second, there is a need to partner with ngos, community, and local government to develop and fund programs and initiatives with greater reach. third, companies should extend confidential testing and treatment programs, including monitoring of testing participation rates, access to viral load tests and, in high prevalence areas, treatment for dependents and post employment. fourth, companies should focus on balanced prevention and treatment programs that target behavior change in conjunction with treatment. fifth, there is a need to increase the role of business in advocacy and to extend programs into emerging markets. in particular, ceos and senior management should provide leadership to dispel myths and stigma, break down workplace barriers and influence community change. in this regard, the gbc study concluded that the private sector can enhance their response to hiv/aids by treating it like any other disease and integrating responses into broader packages supporting health and well-being. employment contracts and health benefits packages should include hiv as a normal component rather than an exception. rather than isolating hiv/aids, interventions should be part of a comprehensive health system for employees and the broader community (gbc and booz, allen, hamilton 2006) . the gbc study recommends that an hiv/aids response be a core component of an overall business strategy, whether the response takes the form of cause-related marketing, co-investments with governments on hiv programs or comprehensive overall market leader and will also help sustain markets and serve a need in countries like india and china, with rapidly growing young, sexually active middleclass populations (gbc and booz, allen, hamilton 2006). in addition to direct company participation in prevention and treatment programs, there have been efforts to create market-based incentives for the private sector to donate more funds to combating hiv/aids. the most notable example is product red, an economic initiative designed to deliver a sustainable flow of private sector money to the global fund to fight aids, tuberculosis and malaria. this initiative was announced at the world economic forum annual meeting in 2006 by bono and bobby shriver. with product red, the world's leading companies made a commitment to channel a portion of their profits from sales of speciallydesigned products to the global fund to support aids programs for women and children in africa. another market-based initiative for the private sector is "social marketing". population services international (psi), a nonprofit organization based in washingpanies seeking to fight hiv/aids in the workplace. psi, a large aid contractor, uses commercial marketing strategies to promote health products, services and healthy behavior in low-income and vulnerable populations in 70 developing countries. products and services are sold at subsidized prices rather than being provided free of charge. the logic behind social marketing is that charging money will enhance the perceived value of products and services, increase the likelihood needs to be charged for hiv treatment in order for the recipients to see "value" in the drugs has been discredited. arata kochi, the director of the world health organization's malaria program, has concluded that the social marketing approach is not an effective means to expand the use of mosquito nets to prevent malaria. using the social marketing approach, mosquito nets were sold through local shops at subsidized prices, with donors underwriting the losses and paying consultants to come up with brand names and to advertise the nets. the social marketing approach was also used to distribute condoms and oral rehydration salts. however, it was revealed that the united states agency for international development (usaid) was spending 95% of its malaria budget on consultants and 5% on goods like nets, drugs and insecticide. experiences in kenya helped to persuade the who to change its policy. a 5-year study of 40 health districts revealed that a psi social marketing scheme for malaria nets only increased coverage from 7% of the population to about 21% between 2002 and 2006. in contrast, when the health ministry got a grant from the global fund that allowed it to hand out 3.4 million free nets in 2 weeks, coverage rose to 67%, and distribution became more equitable. under social marketing, the richest of the poor had 38% coverage, while the poorest of the poor had only 15%. after the distribution of free nets, they were about equal and deaths of children dropped 44%. free distribution was also cheaper. with consultant fees, transportation, advertising and shipping, social marketing added about usd 10 to the cost of each net beyond the usd 5 to usd 7 that manufacturers charged. even with payments to volunteers, the added cost of free distribution was only about usd 1.25 mã©decins sans frontiã¨res (msf) has criticized donors for proposing to continue having patients make a financial contribution to the cost of arv treatment in populations where incomes are barely adequate for people to feed themselves (mã©decins sans frontiã¨res 2005) . aids patients need to receive treatment their entire lives without interruption in order to avoid death or creating drug-resistant mutations of hiv. requiring patients (who do not have enough money for food) to pay for treatment reduces the effectiveness of arv treatment, reduces adherence and decreases survival rates. even where arv treatment is free of charge, where countries define aids care narrowly many direct treatment costs must be covered by patients. for example, the high cost of laboratory tests can deter patients from 7.6 the role of the private sector in funding prevention and treatment ton, dc., created a comprehensive corporate aids prevention program for comof use, and motivate commercial sector involvement. however, the notion that fee 253 per net (kyama and mcneil, 2007) . monitoring the effectiveness and side effects of arv treatment. a related problem is the cost of treatment for opportunistic infections (such as pneumonia), which may bankrupt patients before they even start arv treatment. other costs, such as consultation or hospital fees, can also constitute a barrier to treatment for patients in developing countries. as a result, msf has recommended that major international donors such as the global fund, pepfar, and the world bank should require recipients to provide arv treatment and other essential elements of aids care without patient contributions, and to slightly increase funding to include these complementary costs (philips 2007) . the world bank president, former us trade representative robert zoellick, has called on the bank's 185 member nations to give the private sector a bigger role in development (reuters 2007) . with respect to the world bank's aids programs, as well as other health-related issues, careful thought will have to be given to the appropriate role for the private sector, particularly the failure of social marketing to achieve adequate and equitable coverage in poor populations. the experience of the private sector thus far indicates that the business response to the aids pandemic needs to be standardized and coordinated with the activities of other players, notably national governments, ngos and multilateral institutions. while some companies limit their response to donations of money, goods and services, others have taken a more proactive approach by implementing prevention and treatment programs for their employees and, in some cases, the dependents of employees. this proactive approach is particularly useful in areas with high prevalence rates and poor public health care infrastructure. global multinational companies are in a position to use their influence with suppliers and other business associates to expand the use of best practices, particularly with respect to prevention and treatment programs. in this regard, the gbc plan to review the state of business and aids annually and to use the results of these assessments to adapt the best practice aids standard is to be commended. the gbc study notes that companies have addressed child labor, wage equity and occupational safety through their supply chains, including distributors, business associates and small and medium enterprises. however, this network of global suppliers has been a weak link in the aids response. if tapped, the reach of employee and community networks could make a tremendous impact in mobilizing communities around hiv prevention and treatment. similarly, enhanced supply chain practices promoting youth education and gender equity can help to address underlying social factors that contribute to the spread of hiv/aids (gbc and booz, allen, hamilton 2006). the further standardization of best practices would be a useful next step. the international standards organization (iso) certification process that is currently used to improve business processes could serve as a model. the iso has specific standards for the automotive industry and has specific standards for environmental processes. while these standards are voluntary, many multinational companies require their suppliers to be iso-certified. the creation of specific iso standards for hiv/aids policies, or for company health policies more generally, would be a private charitable foundations have become a significant source of funding, research and political leadership in fighting hiv/aids. in this section, we examine the hiv/aids activities of three us foundations: the bill & melinda gates foundation, the kaiser family foundation and the william j. clinton foundation. it is notable that these foundations have largely avoided overlap in the nature of their operations related to hiv/aids, by specializing in addressing different needs. the first two foundations have also collaborated closely. useful vehicle for standardizing best practices and expanding the adoption of best practices to suppliers. as with iso, the standards could be voluntary, but major multinationals could require their suppliers to get certified in order to qualify as suppliers. as of 31 december 2006, the bill & melinda gates foundation had net assets totaling usd 30 billion, which it uses to fund global development and global health initiatives. the mission of the foundation's global health program is to encourage the development of lifesaving medical advances and to help ensure they reach the people who are disproportionately affected. the foundation is guided by the belief that all lives, no matter where they are lived, have equal value. with respect to global health, the foundation focuses its funding in two main areas: (1) access to existing vaccines, drugs and other tools to fight diseases common in developing countries; and (2) research to develop health solutions that are effective, affordable and practical. in developing countries, the foundation supports efforts to prevent and treat diseases and conditions that meet three criteria: (1) they cause widespread illness and death in developing countries; (2) they represent the greatest inequities in health between developed and developing countries; and (3) they receive inadequate attention and funding. hiv/aids is one of the foundation's priority diseases. to slow the global spread of hiv, the foundation supports the development of vaccines and other tools and strategies with the potential to prevent tens of millions of infections and deaths. the foundation also funds comprehensive initiatives that include both prevention and treatment (http://www.gatesfoundation. org/globalhealth/). the bill & melinda gates foundation's primary focus with respect to hiv/aids is on prevention. illustrative examples of the programs funded by the foundation are: (1) avahan (which means call to action in sanskrit), an aids prevention initiative established in india in 2003, to which it has committed usd 258 million; (2) the global hiv prevention working group, an international panel of experts; and (3) the global hiv vaccine enterprise. through the avahan program, the foundation is working to expand access to effective prevention in the six states with india's highest infection rates and along the nation's major trucking routes. the working group is an international panel of more than 50 leading public health experts, clinicians, researchers and people affected by hiv/aids. it is coconvened by the henry j. kaiser family foundation and the bill & melinda gates foundation. the global hiv prevention working group has developed reports and other materials about critical hiv prevention issues, such as scaling up the combined use of proven prevention strategies (male circumcision, aids education, condoms, hiv testing and prevention of mother-to-child transmission) in order to cut the number of projected new infections to 2015 in half (global hiv prevention working group 2007). the global hiv vaccine enterprise is an international alliance of independent organizations dedicated to accelerating the development of an hiv/aids vaccine through collaborative research efforts. the bill & melinda gates foundation serves as interim chair and funds vaccine research. in july 2006, the bill & melinda gates foundation funded 16 grants totaling usd 287 million to create an international network of collaborative research consortia focused on accelerating the pace of hiv vaccine development (http://www.gatesfoundation.org). the kaiser family foundation is a non-profit, private operating foundation focusing on the major health care issues facing the united states, but has a growing role in global health. the foundation has an endowment of over half a billion dollars and an operating budget of over usd 40 million per year. unlike grant-making foundations, kaiser develops and runs its own research and communications programs, sometimes in partnership with other non-profit research organizations or major media companies. this foundation focuses on three areas of activity: (1) producing policy analysis and research; (2) operating a large-scale health news and information service on the web and a series of specialized websites; and (3) developing and helping to run large-scale public health information campaigns in the united states and around the world, through direct partnerships with major media companies. the current focus of the latter is on hiv/aids, with an emphasis on reaching young people (http://www.kff.org/about/index2.cfm). the william j. clinton foundation is president bill clinton's vehicle for strengthening the capacity of people in the united states and throughout the world to meet the challenges of global interdependence. one of the foundation's main initiatives is focused on hiv/aids. in 2007, the foundation helped to negotiate major price reductions for 66 developing countries for 16 medicines critical to fighting hiv/ aids, in an agreement with pharmaceutical manufacturers (see chap. 9). these countries together have 90% of aids cases in the developing world. the foundation's hiv/aids initiative (chai) has also involved political leadership from president clinton to reduce fear and ignorance of the disease and to discourage discrimination. donations are the primary source of revenue for the foundation, making up usd 109,730,002 of the usd 112,687,167 in revenue for 2006 (http:// www.clintonfoundation.org). the clinton foundation also funds access to treatment and information. it has committed usd 38 million to fund a 3-year program to ensure all hiv-positive children in kenya receive treatment. the funds would pay for a public awareness campaign and purchase anti-retroviral drugs to rapidly scale up the number of infected children under treatment from 2009 to 2010. half all hiv-positive children in kenya die before their second birthday; 50% of those die because they are not provided with treatment. anti-retroviral therapy would increase the life expectancy to 27 years. of an estimated 102,000 hiv-positive children, 60,000 are in need of treatment and only 13,000 are on life-saving anti-retroviral therapy. a survey commissioned by the kenyan ministry of medical services showed that 61% of mothers and caregivers were unaware of the availability of hiv testing for children and that only 12% had taken their children for hiv testing (ndegwa 2007) . there is also collaboration among private firms, private foundations and host governments. for example, an innovative model for fighting hiv/aids in africa is being piloted in botswana through a public-private partnership involving the government of botswana, the bill & melinda gates foundation and merck & co., inc. the partnership is intended to help botswana achieve an "aids-free generation by 2016" by expanding prevention, supporting treatment, increasing counseling and testing and empowering communities (center for global development and merck 2007). the main focus of this chapter has been on multilateral financial institutions. however, it is important to note the role of other financial donors, notably bilateral governmental arrangements. private actors and ngos also make significant contributions to addressing the global aids pandemic. as important as it is to marshal the resources of numerous governmental and non-governmental agencies, the multiplicity of donors involved in the aids epidemic, together with a multiplicity of donor requirements, is a significant issue in seeking to expand prevention and treatment programs. national political leadership is a crucial part of any hiv/aids strategy. in many countries, statements made by politicians who make decisions on resource allocation have been appalling. in south africa, tshabalala-msimang, the health minister, shares south african president mbeki's rejection of the international scientific consensus that aids is caused by the hiv. the health minister has also criticized the use of standard anti-retroviral drugs to treat aids, recommending instead that south africans with aids follow a diet of beetroot, garlic, olive oil and african potato. in a recent south african population survey, a quarter of the population said they didn't believe in a link between hiv and aids (wilson 2007) . figure 7 .17 reveals the impact of misinformation on south africans perceptions regarding the sources of hiv transmission. of mozambique's population is hiv positive (bbc news 2007a). these lapses in political leadership highlight the need to incorporate political leadership into aids programs that are funded by multilateral, bilateral and private sector donors. multilateral institutions, such as the world bank, the imf and the global fund, and bilateral donors, including ngos and national governments, need to coordinate and harmonize their policies and financing conditions further in order to reduce were also infected "in order to finish quickly the african people". the catholic assigned to the private sector in delivering aid, in order to avoid the unnecessary diversion of funds away from the core goal of health care. in this regard, the experience to date suggests that social marketing is less effective and less equitable than the free provision of goods and services. particularly in the case of fastmoving infectious diseases, equitable and effective distribution should be paramount, since such diseases are unlikely to respect socio-economic boundaries, as the experience with hiv/aids and malaria has demonstrated. in particular, donors should not impose requirements to use patented medicines in place of cheaper, generic alternatives. the global business community has an important role to play in addressing global diseases. there are several key lessons that arise from the gbc's experience with hiv/aids. first, it is important to integrate health issues into overall business strategy, particularly since it takes about 3 years to fully implement such strategies. second, there is a need to standardize best practices on a more systematic the administrative burden on recipients and in order to avoid imposing conflicting conditions on recipients. donors also need to be careful with respect to the role basis and to create incentives for those best practices to be adopted by suppliers and business partners of global companies. in this regard, the creation of iso standards for company health policies would be extremely useful, particularly for fastmoving global infectious diseases that have the potential to cause widespread economic damage and human suffering, such as avian influenza and sars. the global response to the hiv/aids pandemic has highlighted the need to strengthen health care infrastructure in developing countries. there is a concern that the high profile given to the hiv/aids pandemic may have diverted funds away from other important health issues (england et al., 2007) . in chap. 4 we concluded that the focus on hiv/aids is justified, but it is important to address the hiv/aids pandemic in a way that strengthens the ability of national governments and multilateral organizations to address health concerns more generally. the following chapter examines the role of global health organizations in the hiv/aids pandemic, with a particular focus on the world health organization, which is the multilateral institution that is charged with addressing global health issues. the effect of population health on foreign direct investment private sector responses to hiv/aids: the case of debswana, botswana's diamond company funding the response to aids: why are donors not working together? are we spending too much on hiv? global fund to fight aids, tuberculosis and malaria: pdf global fund to fight aids, tb and malaria has improved its documenta-d07627 the framework document of the global fund to fight aids bringing hiv prevention to scale: an urgent global priority funding the response to aids: why are donors not working together? pound of flesh: joseph hanlon reports on how local people have wised up to a huge international hoax donor concern over imf cap on aid increases tion of funding decisions but needs standardized oversight expectations and assessments evaluation of the local fund agent system international assistance for hiv/aids in the developing world: taking stock of the g8, other donor governments, and the european commission. kaiser family foundation, center for strategic and international studies/unaids, data distribution of nets splits malaria fighters. www. nytimes.com. accessed 9 the new international aid architecture: new players, new challenges strengthening partnerships in hiv monitoring and evaluation: how joint missions build and strengthen partnerships to support the realization of national m&e systems global fund responsiveness to faith-based organizations imf must adapt and downsize or die socioeconomic obstacles to hiv prevention and treatment in developing countries: the roles of the international monetary fund and the world bank funding the response to aids: why are donors not working together? funding the response to aids: why are donors not working together? access to healthcare, mortality and violence in reports funding the response to aids: why are donors not working together? kenya: clinton foundation provides $38m for children living with hiv/aids the paris declaration following the funding for hiv/aids: a comparative analysis of the funding practices of pepfar, the global fund and the world bank map in mozambique, uganda and zambia the public interest: health, education, and water and sanitation for all funding the response to aids: why are donors not working together? a response to joseph hanlon's recent article, donor concern over imf cap on aid increases aids: free access to treatment, a public health necessity or an economic heresy? picard a (2007) quarantine sought for harsh tb strain: variant poses 'extreme risk hiv/aids: what is business doing? a survey of the business community's response to hiv/aids in kenya challenges and opportunities for the new executive director of the global fund: seven essential tasks global fund grant programmes: an analysis of evaluation scores hiv/aids and the private sector in africa: evidence from the investment climate survey data world bank chief calls for new direction for lender summary paper on the evaluation of the studies/specific_evaluations/terg_summary_paper the evolving hiv epidemic: what have we learned since the map began? powerpoint presentation hiv/aids in central america: the epidemic and priorities for its prevention an oed evaluation of the world bank's assistance for hiv our commitment: the world bank's africa region hiv/aids agenda resources/wb_hiv-aids-afa_2007-2011_advance_copy external /default/ wdscontentserver/ wdsp/ib/2007/09/07/000310607_20070907114625/ aids situation and response to the epidemic key: cord-005833-fizh495d authors: baumschlager, d.; haas-krammer, a.; rothenhäusler, h.-b. title: emotionale befindlichkeit, kognitive leistungsfähigkeit und lebensqualität bei hiv-patienten: ergebnisse einer explorativen untersuchung date: 2010-09-22 journal: nervenarzt doi: 10.1007/s00115-010-3124-3 sha: doc_id: 5833 cord_uid: fizh495d background: due to the change of hiv disease from an acute life-threatening disease to a chronic infection, it is more psychosocial rather than therapeutic aspects that have become of interest in scientific investigations. the purpose of this exploratory study was to evaluate emotional distress, health-related quality of life (hrqol) and cognitive performance. the diagnosis of hiv was considered a life event that may lead to post-traumatic stress syndrome. method: we recruited 37 hiv-positive outpatients and assessed the frequency of depressive (bdi) and post-traumatic stress symptoms (ptss) due to the diagnosis of hiv (ies), hrqol (sf-36) and cognitive performance (skt). further, the new diagnostic concept of adjustment disorder as a stress response syndrome according to maercker was considered. results: of the 37 patients, 67.6% (n=25) of the sample had a post-traumatic stress syndrome. the hiv-related ptss was considered adjustment disorder using the concept proposed by maercker. fourteen patients (37.8%) suffered from a depressive syndrome, and 27% (n=10) showed cognitive deficits (minimal: n=8; mild: n=1; moderate: n =1). hiv-positive patients with ptss had significantly unfavourable values in the sf-36 domains general health (p=0.003), vitality (p=0.007), social functioning (p=0.000), role-emotional (p=0.016) and mental health (p=0.000). conclusion: hiv-infected patients may face a major risk of hiv-related ptss in the sense of adjustment disorder according to maercker, depression and cognitive dysfunction. the presence of emotional distress is associated with impairments in quality of life. we therefore suggest an early and comprehensive bio-psycho-social assessment and therapy of hiv-infected patients. infolge der lebenszeitverlängerung durch haart treten zunehmend psychosoziale aspekte in den fokus wissenschaftlicher untersuchungen, um eine bessere versorgung und eine steigerung der medikamentenadhärenz erzielen zu können. psychosoziale faktoren sind für den erfolg der haart entscheidend, da eine exakte einhaltung des therapieregimes die wirksamkeit wesentlich beeinflusst und sich psychische probleme hier negativ auswirken [3, 4] . verschiedene autoren rezenter arbeiten [5, 6, 7] betrachten hiv bereits als chronischen, psychischen stressor. im bereich der posttraumatischen belastungsstörungen (ptbs) respektive stressreaktionssyndrome jedoch wurde die hiv-diagnosestellung kaum als mögliche ursache berücksichtigt. diese problematik spiegelt sich auch in den diagnostischen manualen wider, da "schwere erkrankung" erst in der version iv des dsm und der icd ab version 10 als potenzielle ursache für ptbs angeführt ist. mithilfe einer explorativen querschnittsuntersuchung evaluierten wir gezielt emotionale befindlichkeitsstörungen im gefolge von hiv-diagnosestellungen. hierbei beachteten wir für möglicherweise auftretende hiv-assoziierte, posttraumatische belastungssymptome (ptss; posttraumatic stress symptoms) die forschungsergebnisse von maercker et al. [9] durch berücksichtigung eines innovativen konzepts der anpassungsstörungen (ad, "adjustment disorder") als stressreaktionssyndrome. die autoren gehen davon aus, dass ad, analog zu ptbs, durch "intrusion", "vermeidung" und "fehlanpassungssymptome" gekennzeichnet sind. die ad werden hier als syndrome verstanden, die sich aus den genannten symptomgruppen zusammensetzen. auf die notwendigkeit einer revision der diagnosegruppe wurde aufgrund unklarer abgrenzung zu anderen entitäten und häufiger verwendung im klinischen alltag auch an anderer stelle hingewiesen [10] . in . tab. 1 sind die vorgeschlagenen diagnostischen kriterien der ad systema-tisch dargestellt. der hauptunterschied zwischen ptbs und ad bestehe in der schwere des traumas: die autoren gehen davon aus, dass ein leichteres trauma eher die entwicklung einer ad begünstige. erste empirische befunde zeigen, dass das neue konzept nach maercker eine fundierte basis für ein revidiertes ad-konzept sein könnte [11] . unserer einschätzung nach erfüllt die hiv-diagnosestellung eher die kriterien der lifeevent-forschung und nicht die kriterien von extremer, akuter bedrohung, wie sie für die diagnose der ptbs gefordert sind. wie auch in einer studie an 832 personen gezeigt wurde, hatten patienten mit einem "life event" durchschnittlich sogar mehr posttraumatische belastungssymptome (ptss) als personen mit einem trauma, wobei ptss infolge eines traumas persistenter sein dürften [12] . unter berücksichtigung dieser aktuellen forschungsergebnisse zielten wir in unserer explorativen studie auf eine realistische belastungseinschätzung ab. weiters evaluierten wir die gesundheitsbezogene lebensqualität, die lebenszufriedenheit und die kognitive leistungsfähigkeit von hiv-infizierten patienten, um mögliche zusammenhänge zwischen emotionalen befindlichkeitsstörungen, kognitiven beeinträchtigungen und lebensqualität zu finden. ein umfassenderes verständnis von möglichen psychiatrischen komplikationen im gefolge der hiv-erkrankung könnte die basis für eine verbesserte biopsychosoziale versorgung von hiv-patienten darstellen. background. due to the change of hiv disease from an acute life-threatening disease to a chronic infection, it is more psychosocial rather than therapeutic aspects that have become of interest in scientific investigations. the purpose of this exploratory study was to evaluate emotional distress, health-related quality of life (hrqol) and cognitive performance. the diagnosis of hiv was considered a life event that may lead to post-traumatic stress syndrome. method. we recruited 37 hiv-positive outpatients and assessed the frequency of depressive (bdi) and post-traumatic stress symptoms (ptss) due to the diagnosis of hiv (ies), hrqol (sf-36) and cognitive per-formance (skt). further, the new diagnostic concept of adjustment disorder as a stress response syndrome according to maercker was considered. results. of the 37 patients, 67.6% (n=25) of the sample had a post-traumatic stress syndrome. the hiv-related ptss was considered adjustment disorder using the concept proposed by maercker. fourteen patients (37.8%) suffered from a depressive syndrome, and 27% (n=10) showed cognitive deficits (minimal: n=8; mild: n=1; moderate: n =1). hiv-positive patients with ptss had significantly unfavourable values in the sf-36 domains general health (p=0.003), vitality (p=0.007), social functioning (p=0.000), role-emotional (p=0.016) and mental health (p=0.000). conclusion. hiv-infected patients may face a major risk of hiv-related ptss in the sense of adjustment disorder according to maercker, depression and cognitive dysfunction. the presence of emotional distress is associated with impairments in quality of life. we therefore suggest an early and comprehensive biopsycho-social assessment and therapy of hivinfected patients. mittels sf-36 (medical outcomes study short form survey) nach stewart et al. [13] in der deutschen übersetzung von bullinger und kirchberger [14] wurde die gesundheitsbezogene lebensqualität gemessen. der selbstbeurteilungsfragebogen umfasst folgende 8 gesundheitskategorien: die reliabilität (cronbach's α) liegt zwischen α=0,57 und α=0,94. depressive symptomatologie wurde mithilfe des bdi (beck's depression inventory) in der deutschen version von hautzinger et al. [15] , basierend auf dem bdi von beck et al. [16] erfasst. die interne konsistenz (cronbach's α) liegt bei α=0,88. die von uns angewandte einteilung der testergebnisse in unauffällig (0-10 punkte), mäßige depressive symptomatik (11-18 punkte) und klinisch relevante depressive symptomatik (19 und mehr punkte) stammt ebenfalls von den angeführten autoren. die ies (impact of event scale) nach horowitz et al. [17] unsere patienten wurden während unserer systematischen untersuchung dazu angehalten, die ies-aussagen auf die überbringung ihres positiven hiv-testergebnisses zu beziehen ("trauma") und auf der 4-stufigen skala anzugeben, wie oft das im jeweiligen item beschriebene symptom innerhalb der letzten woche aufgetreten war. die 9 subtests des skt (syndromkurztest) nach erzigkeit [19] dienen zur evaluierung von gedächtnis-und aufmerksamkeitsstörungen. er wird insbesondere bei verdacht auf demenz, kognitive leis tungsdefizite, hirnorganischen psychosyndrome, durchgangssyndrome und andere organisch bedingte psychische störungen eingesetzt. seine hauptanwendungsgebiete umfassen unter anderem auch klinische studien und grundlagenstudien. für die interne konsistenz (cronbach's α) konnten werte zwischen α=0,86 und α=0,88 ermittelt werden. diese ergebnisse wurden in anderen studien bestätigt [20] . die test-retest-reliabilität des skt-gesamtwertes lag bei 0,90 [21] . zwischen skt und anderen neuropsychologischen testverfahren konnten signifikante korrelationen gefunden werden [22, 23] . außerdem weisen ergebnisse darauf hin, dass der skt insbesondere zwischen leichten und mäßigen demenzformen gut differenzieren dürfte, [24, 25] , polytraumapatienten [26] , hepatitis-c (hcv)-patienten [27] und lebertransplantationspatienten [28] . im bereich der berufsausbildung konnten 3 personen (8,1%) keinen abschluss angeben, 14 (37,8%) hatten eine lehre absolviert, 9 patienten (24,3%) hatten eine berufsbildende schule abgeschlossen und 2 personen (5,4%) hatten eine meisterprüfung abgelegt. an höheren ausbildungen konnten 3 patienten (8,1%) einen fachhochschulabschluss angeben, 2 personen (5,8%) hatten ein universitätsstudium absolviert. weitere 2 patienten (5,8%) hatten eine andere berufsausbildung abgeschlossen und 2 (5,8%) hatten keine angabe zu ihrer berufsausbildung gemacht. zum untersuchungszeitpunkt waren 22 patienten (59,5%) vollzeitbeschäftigt, 4 (10,8%) teilzeitbeschäftigt und 2 patienten (5,4%) arbeitslos. während 4 personen (10,8%) regulär in ruhestand gegangen waren, waren 5 (13,5%) krankheitsbedingt frühpensioniert worden. von den untersuchten patienten lebten 12 (32,3%) allein. 19 personen (51,35%) lebten mit partnern oder familienangehörigen zusammen und 6 (16,2%) lebten in anderen wohnverhältnissen oder hatten keine angabe über ihre wohnsituation gemacht. bei 20 patienten (54,1%) fand die hiv-infektion höchstwahrscheinlich durch homo-/bisexuellen geschlechtsverkehr statt. 12 patienten (32,4%) dürften sich über heterosexuellen intimkontakt infiziert haben. bei 2 patienten (5,4%) gelangte das virus durch eine infizierte blutkonserve in den körper. ein patient (2,7%) infizierte sich auf dem mutter-kind-weg. von einem patienten (2,7%) ist der übertragungsweg nicht bekannt. nach ies konnten bei 12 patienten (32,4%) keine relevanten posttraumatischen belastungssymptome (ptss) nachgewiesen werden. 8 teilnehmer (21,6%) wiesen eine leichte ptss-symptomatik auf, 8 (21,6%) zeigten eine mäßige ausprägung und immerhin 9 patienten (24,3%) zeigten schwere ptss-symptomatik. der mittelwert des ies-scores betrug 25,41±21,3 (range: 0-69). dreiundzwanzig patienten (62,2%) zeigten keine relevante depressive symptomatik gemäß bdi. 7 personen (18,9%) hingegen wiesen eine leichte bis mäßige depressive symptomatik auf. bei weiteren 7 patienten (18,9%) deuteten die bdi-ergebnisse auf eine mäßige bis schwere ausprägung depressiver symptomatik hin. wir haben patienten mit auffälliger ptb-symptomatik mit unauffälligen hinsichtlich ihrer bdi-werte verglichen und konnten einen signifikanten unterschied (p<0,01) zwischen beiden gruppen feststellen. 13 von 14 patienten mit relevanter depressiver symptomatik nach bdi wiesen gleichzeitig auch relevante ptss-symptomatik nach ies auf. in . tab. 2 sind die sf-36-ergebnisse des mann-whitney-u-tests, den wir durchführten, um unterschiede in der gesundheitsbezogenen lebensqualität (hrqol; health-related quality of life) zwischen ptss-auffälligen und -unauffälligen patienten zu eruieren, aufgeführt. einen sig nifikanten unterschied zwischen beiden gruppen konnten wir für die kategorien allgemeine gesundheit, vitalität, so-ziale funktionsfähigkeit, emotionale rollenfunktion und psychisches wohlbefinden nachweisen. hier waren die werte bei relevanter ptss-symptomatik signifikant ungünstiger. beim vergleich der gemäß bdi als depressiv eingestuften patienten mit den als unauffällig evaluierten patienten hinsichtlich der sf-36-ergebnisse konnten wir signifikante unterschiede in allen 8 hrqol-kategorien nachweisen. . tab. 3 gibt die sf-36-ergebnisse des mann-whitney-u-tests für die beiden subgruppen wieder. bei 27 patienten (73%) wiesen die skt-ergebnisse nicht auf das vorhandensein kognitiver defizite hin. 8 patienten (21,6%) hatten minimale kognitive defizite, ein patient (2,7%) wies leichte zerebrale leistungsdefizite auf und bei einem patienten (2,7%) konnten wir eine mäßige ein-schränkung der kognitiven leistungsfähigkeit feststellen. durchschnittlich erreichten die teilnehmer unserer studie einen skt-gesamtwert von 2±2,83 (range: 0-11). die durchsicht unserer studienergebnisse veranschaulicht deutlich erhöhte punktprävalenzwerte von ptss-und depressionssymptomatik in unserer studien population hiv-positiver patienten im ambulanten setting. gleichzeitig konnten wir den einfluss der emotionalen befindlichkeitsstörungen auf kennwerte gesundheitsbezogener lebensqualität (hrqol) und lebenszufriedenheit nachweisen. beachtenswert ist der hohe anteil von patienten mit relevanten posttraumatischen belastungssymptomen (ptss; 67,7%) an unserer studienpopulation. aufgrund methodischer unterschiede bei der diagnostik der posttraumatischen belastungsstörung (z. b. selbstbeurteilungsskalen, fremdbeurteilungsinstrumente, strukturierte klinische interviews) sind häufigkeitsangaben unterschiedlicher populationen schwer vergleichbar. trotzdem kann man davon ausgehen, dass die werte bei unseren studienpatienten um ein vielfaches höher waren als jene der allgemeinbevölkerung (6%) [29] bzw. von patienten, die sich in allgemeinmedizinischer behandlung befinden (10%) [30] . bisher wurden wenige studien zur hiv-assoziierten posttraumatischen belastungsstörung (ptbs) publiziert. die angaben zur prävalenz der ptbs reichen von 13,3-64% [3, 31] . hervorgehoben muss an dieser stelle werden, dass selbst patienten mit vergleichbaren erkrankungen deutlich niedrigere ptbs-werte aufwiesen. für hcv konnte eine ptbs-prävalenz von 8,8% ermittelt werden [27] . diese erkrankung ist als chronische, virale infektion mit neurotropie mit der hiv-infektion vergleichbar. deutliche unterschiede sind hingegen im gesellschaftlichen bild und in den damit verbundenen psychischen belastungen beider krank in der literatur wird die prävalenz der depression bei hiv-patienten zwischen 5 und 49% angegeben [33, 34, 35, 36, 37, 38] . die von uns erhobene prävalenz depressiver zustandsbilder von ca. 38% ist mit diesen studienergebnissen konsistent. unsere untersuchungen wiesen einen deutlichen zusammenhang von ptss-symptomatik und depressivität gemäß bdi nach. in diesem kontext ist zu erwähnen, dass bei hiv-patienten depressive anpassungsstörungen im sinne von stressfolgeerkrankungen häufiger als depressive episoden im rahmen von depressiven störungen diagnostiziert werden [39] . je nach ies-wert unterschieden sich unsere patienten hinsichtlich der bdi-werte signifikant voneinander (u=60,00, p=0,003). lediglich ein studienpatient aus der depressiven subgruppe (n=14) wies kein relevantes ptss-syndrom auf. die hohe komorbidität zwischen posttraumatischen belastungsstörungen und de-pressiven erkrankungen wurde in mehreren studien bestätigt [4, 40, 41, 42, 43] [45] hingegen führten die hohen korrelationen zwischen ptbs, major-depression (md) und generalisierter angststörung (gas) an, definierten diese aber als unterscheidbar. ein dysphoriefaktor könnte sich auf ptbs, md und gas auswirken. ein 2-faktoren-modell der ptbs wurde von maes et al. [46] vorgeschlagen, das "depression-vermeidung" als ersten faktor und "angst-erregtheit" als zweiten umfasst. in der zusammenschau können wir feststellen, dass in der derzeitigen wissenschaftlichen literatur noch keine einigkeit darüber besteht, welche faktoren ein empirisch gesichertes modell der ptbs umfasst und ob depression einer dieser faktoren sein könnte oder lediglich mit ptbs assoziiert ist. zahlreiche studien konnten den einfluss von ptbs auf die hrqol und qol belegen [47, 48, 49, 50, 51, 52] . wenige studien hingegen haben sich mit diesem einfluss bei hiv auseinandergesetzt. zwei studien [53, 54] konnten nachweisen, dass symptomatische hiv-positive patienten ohne aids die besten hrqol-werte außer in den domänen körperliche rollenfunktion und allgemeine gesundheit hatten. sowohl asymptomatische hiv-positive patienten als auch aids-patienten hatten schlechtere werte. eine asymptomatische infektion könnte deshalb psychisch belastender sein, weil sie eine diffuse bedrohung darstellt, die kaum anhaltspunkte in der auseinandersetzung mit der krankheit bietet. diese problematik dürfte in unserer studienpopulation besonders relevant sein, da die cd4 + -werte unserer studienteilnehmer ein aids-stadium bzw. opportunistische infektionen infolge schlechter immunologischer abwehr ausschlossen. in unserer wie auch in anderen studien war vor allem die psychische hrqol erniedrigt [55, 56] . im gegensatz zur ptss-symptomatik wirkte sich depressivität in unserer explorativen studie signifikant auf alle sf-36-kategorien aus. der zusammenhang von depression und hrqol bei hiv konnte auch in anderen studien belegt werden [57, 58, 59] . die erste durchsicht der messergebnisse von gedächtnis-und aufmerksamkeitsstörungen unserer studienpopulation erweckte den eindruck einer hohen prävalenz neurokognitiver einschränkungen. es muss allerdings berücksichtigt werden, dass bei einem großteil (21,6%) unserer auffälligen studienteilnehmer (27%) die testergebnisse lediglich einen verdacht auf kognitive störungen zuließen. so blieb nur ein bruchteil übrig, bei dem von einer deutlichen kognitiven einschränkung ausgegangen werden konnte. selbst neuere untersuchungen, die nach flächendeckender einführung der haart durchgeführt wurden, geben deutlich höhere prävalenzen (33,3-84,3%) kognitiver leistungseinbußen an [60, 61, 62, 63, 64] . untersuchungen sprechen dafür, dass bestimmte aufgabentypen neurokognitiver tests besonders sensitiv für hiv-induzierte leistungseinbußen sind. diese könnten einen zu geringen anteil innerhalb der skt-subtests haben, weshalb wir eine niedrigere prävalenz fanden [65, 66, 67] . so sind die für die diagnostik früher stadien hiv-induzierter kognitiv-motorischer defizite sensiblen feinmotorikuntersuchungen wie der finger-tapping-test (tap) und der wiener reaktionstest (rt) beispielsweise im skt nicht repräsentiert [68] . unklar ist bis dato, ob sich hiv bei älteren patienten stärker auf die kognitive leistungsfähigkeit auswirkt. es gibt jedoch studien, welche diesen zusammenhang nahelegen. mit einem durchschnittlichen alter von mitte 40 besteht unsere studienpopulation aus eher jüngeren teilnehmern, die im vergleich zu andexvii international aids conference: from evidence to action-epidemiology alternative projections of mortality and disability by cause 1990-2020: global burden of disease study posttraumatic stress and trauma history in adolescents and young adults with hiv the differential impact of ptsd and depression on hiv disease markers and adherence to haart in people living with hiv lifetime and hiv-related ptsd among persons recently diagnosed with hiv symptoms of posttraumatic stress and death anxiety in persons with hiv and medication adherence difficulties common mental disorders among hiv-infected individuals in south africa: prevalence, predictors and validation of brief psychiatric rating scales hrsg) international handbook of traumatic stress syndromes, the plenum series on stress and coping adjustment disorders as stress response syndromes: a new diagnostic concept and its exploration in a medical sample anpassungsstörungen nosologische stellung und therapieoptionen anpassungsstörungen die erprobung eines neuen diagnostischen konzepts in einem ambulanten psychosomatischen setting symptoms of post-traumatic stress disorder after nontraumatic events: evidence from an open population study the mos short-form general health survey: reliability and validity in a patient population der sf-36-fragebogen zum gesundheitszustand. hogreve, göttingen das beck-depressions-inventar bdi an inventory for measuring depression impacts of events scale: a measure of subjective stress psychometric evaluation of horowitz's impact of event scale: a review skt ein kurztest zur erfassung von gedächtnisund aufmerksamkeitsstörungen the skt neuropsychological test battery factor structure and scoring of the skt test battery correlation of mmse, skt and clock test scores in patients with mild and moderate dementia differential validity of psychometric tests in dementia of the alzheimer type psychiatric and psychosocial outcome of cardiac surgery with cardiopulmonary bypass: a prospective 12-month follow-up study the effects of coronary artery bypass graft surgery on health-related quality of life, cognitive performance and emotional status outcomes: a prospective 6-month follow-up consultation-liaison psychiatry study relationship between posttraumatic stress disorder, quality of life, social support and affective and dissociative status in severely injured accident victims 12 months after trauma der zusammenhang zwischen emotionalen befindlichkeitsstörungen, kognitiver leistungsfähigkeit und gesundheitsbezogener lebensqualität bei mit dem hepatitis-c-virus infizierten patienten vor einer antivirealen therapie psychiatric and psychosocial outcome of orthotopic liver transplantation prevalence, co-morbidity and correlates of mental disorders in the general population: results from the german health interview and examination survey (ghs) prevalence, recognition and management of depression in primary care in germany: the depression 2000 study posttraumatic stress disorder in women attending human immunodeficiency virus outpatient clinics ptsd in somatic disease anxiety and depression among hiv-infected heterosexuals a report from india depression in hiv-infected patients. allopathic, complementary and alternative treatments mortality, cd4 cell count decline and depressive symptoms among hiv-seropositive women: longitudinal analysis from the hiv epidemiology research study depression in people living with hiv/aids attending primary care and outpatient clinics prevalence of axis i disorders in an aids cohort: a cross-sectional, controlled study social support, substance use and denial in relationship to antiretroviral treatment adherence among hiv-infected persons ten have t (2002) depressive and anxiety disorders in women with hiv infection incidence and impact of posttraumatic stress disorder and comorbid depression on adherence to haart and cd4+ counts in people living with hiv the relationship of post-traumatic stress disorder and depression to antiretroviral medication adherence in persons with hiv post-traumatic stress disorder among recently diagnosed patients with hiv/aids in south africa posttraumatic stress disorder in response to hiv infection testing alternative factor models of ptsd and the robustness of the dysphoria factor the structure of distress following trauma: posttraumatic stress disorder, major depressive disorder and generalized anxiety disorder the two-factorial symptom structure of post-traumatic stress disorder: depression-avoidance and arousalanxiety prevalence of post-traumatic stress disorder in patients with previous myocardial infarction consulting in general practice the prevalence of posttraumatic stress disorder in patients undergoing pulmonary rehabilitation and changes in ptsd symptoms following rehabilitation impact of comorbid anxiety disorders on health-related quality of life among patients with major depressive disorder ptsd diagnoses, subsyndromal symptoms and comorbidities contribute to impairments for breast cancer survivors the relationship between quality of life and posttraumatic stress disorder or major depression for firefighters in kaohsiung posttraumatic stress disorder and health-related quality of life among a sample of treatmentand pension-seeking deployed canadian forces peacekeeping veterans the role of psychological and behavioral variables in quality of life and the experience of bodily pain among persons living with hiv the economic costs and health-related quality of life of people with hiv/aids in the canary islands hiv symptoms and health-related quality of life prior to initiation of haart in a sample of hiv-positive south africans health-related quality of life in bereaved hiv-positive adults: relationships between hiv symptoms, grief, social support and axis ii indication a further investigation of health-related quality of life over time among men with hiv infection in the haart era neurocognitive impairment influences quality of life in hivinfected patients receiving haart the incidence of and risk factors for hiv-associated cognitive-motor complex among patients on haart neuropsychological impairment and the natural history of hiv-1 infection in spanish subjects prevalence of human immunodeficiency virus-associated cognitive impairment in a group of hispanic women at risk for neurological impairment motor function and human immunodeficiency virus-associated cognitive impairment in a highly active antiretroviral therapy-era cohort neurocognitive impairment in early hiv-positive individuals neuropsychological test profile differences between young and old human immunodeficiency virus-positive individuals cognitive impairment in asymptomatic stages of hiv infection. a longitudinal study the relationship between age and cognitive impairment in hiv-1 infection: findings from the multicenter aids cohort study and a clinical cohort clinical features, diagnosis and treatment of hiv-induced neuropsychiatric disorders age differences and neurocognitive performance in hiv-infected adults ren studienpopulationen aufgrund einer unterschiedlichen altersstruktur besser abgeschnitten haben könnten [66, 67, 69] . unsere studie weist ein querschnittsdesign auf. die studienteilnehmer wurden nicht in einem randomisierten verfahren gewonnen. aufgrund der gruppengröße und der unterrepräsentation von frauen könnte die aussagekraft vermindert sein. es wäre wünschenswert, dass die thematik in weiterführenden, prospektiven studien unter einbeziehung von vergleichsgruppen untersucht wird, um den in unserer studie nahegelegten zusammenhang von emotionalen befindlichkeitsstörungen und verminderter hrqol bei hiv-infizierten patienten zu untersuchen. key: cord-021326-yx0eb885 authors: croser, david title: infection control since hiv date: 2020-04-06 journal: nan doi: 10.1038/s41404-020-0359-y sha: doc_id: 21326 cord_uid: yx0eb885 nan h iv is the virus that can lead to acquired immunodeficiency syndrome or aids if not treated. there is no cure as yet but with proper medical care, hiv can be controlled. hiv is treated with antiretroviral therapy or art. if taken as prescribed, art can reduce the viral load so that it becomes undetectable. if it stays undetectable, patients can live long, healthy lives and have effectively no risk of transmitting hiv to an hiv-negative partner through sex, and more relevantly whilst being treated by a dental healthcare professional. today, someone diagnosed with hiv can live nearly as long as someone who does not have hiv. this is significant because there is an increasing number of hiv positive patients in the community whose bloodborne disease is controlled with art-many of whom will also need dental treatment. as a dentist, you have an obligation to provide dental care to those in need, including those who are or might be infected, and to offer the same high standard of care for all patients. it is unethical to refuse dental care to patients who disclose a positive diagnosis for a blood-borne virus (bbv) because you feel there could be an increased level of personal risk. it is also illogical to think you are safer by refusing to treat patients who disclose they have a bbv; just as many undiagnosed carriers of bbvs attend for dental treatment and, because they have not received any treatment, they may actually present a higher risk of infection. people living with hiv and the hepatitis viruses who are otherwise well, may be treated in general practice without the need for any restrictions or modification to their dental treatment. letting your patients know that you follow national guidelines on infection control -for example, by having a statement to this effect on your website or in your practice information leaflet or displaying a notice in the practiceand encouraging them to ask questions will reassure them that you are confident about your infection control procedures. patients trust the dental team to use effective infection control measures to keep them safe. you can enhance the patient's trust by occasionally pointing out the precautions used in the surgery -e.g. the use of disposable items, sealed single use items like anaesthetic cartridges and newly sterilised handpieces). it is a good use of your time and tells the patient that you care about them and reinforces their trust in you, at a time when a new strain of corona virus has emerged. the decontamination protocols required within the dental setting are defined in htm 01-05 (england and northern ireland). 1 it is not the intention of this article to describe those universal precautions. suffice it to say that, maintaining a constant high standard is dependent on a dental team that understands the importance of this aspect of patient safety; routinely applying the latest protocol and checking their compliance by using a regular audit. following the identification of the retrovirus causing hiv, the dental profession and patients have been exposed to information about blood-borne diseases and the benefits of testing. the internet and social media have helped to normalise the fact that some people do have a bbv; the fearful emotional reactions to hiv witnessed in the 1980s in the uk have now been replaced by an educated response based on reason. the pendulum has now swung the other way so that any healthcare provider who exposes their patients to the risk of acquiring a bbv through a breach in the htm 01-05 protocol, is likely to be shunned by society and exposed by the media. this is exactly what happened in leicester in 2014 when dentist desmond d'mello was suspended by the gdc over serious hygiene concerns and 22,000 patients from the practice were recalled and offered screening for hiv, hbv and hcv. since then, other cases have occasionally hit the headlines which inevitably challenges the trust placed in the dental profession. patients may not understand what good infection control should look like, so take a moment to make them aware of what you do to keep them safe from catching an unwanted disease in your surgery. covid-19 is currently having the same impact that aids generated when it first emerged, even though we are told that, in most cases, the effect of the disease will be mild. the fear of rapid spread and the absence of a cure makes it all the more important that healthcare workers set an example and respond to the challenge of limiting the spread of the disease by engaging with the science rather than emotions. the standard use of infection control protocols is now augmented by the need to consider delaying treatment for those patients who are identified as being at risk of having been in contact with covid-19. proposing a period of quarantine for a patient may be an unfamiliar step, but by following the doh guidance 2 primary care providers can protect themselves, their staff and the population at large. the guidance will evolve as more is learnt about covid-19 and the prospect of a vaccine becomes a reality. indeed, if required, dental surgeries could provide a convenient setting from which to support a programme of mass vaccination. ◆ health technical memorandum 01-05: decontamination in primary care dental practices covid-19: interim guidance for primary care advice bda advice to dentists regarding covid-19 is available at: www.bda.org/ news-centre/latest-news-articles/pages/ wuhan-novel-coronavirus-advice-fordentists.aspx https://doi.org/10.1038/s41404-020-0359-y © pbombaert/getty images plus david croser bda indemnity dental advisor, describes how time has normalised the way we care for patients with a once frightening disease as another one hits the headlines key: cord-004002-b35wm2db authors: gaborit, benjamin jean; tessoulin, benoit; lavergne, rose-anne; morio, florent; sagan, christine; canet, emmanuel; lecomte, raphael; leturnier, paul; deschanvres, colin; khatchatourian, lydie; asseray, nathalie; garret, charlotte; vourch, michael; marest, delphine; raffi, françois; boutoille, david; reignier, jean title: outcome and prognostic factors of pneumocystis jirovecii pneumonia in immunocompromised adults: a prospective observational study date: 2019-11-27 journal: ann intensive care doi: 10.1186/s13613-019-0604-x sha: doc_id: 4002 cord_uid: b35wm2db background: pneumocystis jirovecii pneumonia (pjp) remains a severe disease associated with high rates of invasive mechanical ventilation (mv) and mortality. the objectives of this study were to assess early risk factors for severe pjp and 90-day mortality, including the broncho-alveolar lavage fluid cytology profiles at diagnosis. methods: we prospectively enrolled all patients meeting pre-defined diagnostic criteria for pjp admitted at nantes university hospital, france, from january 2012 to january 2017. diagnostic criteria for pjp were typical clinical features with microbiological confirmation of p. jirovecii cysts by direct examination or a positive specific quantitative real-time polymerase chain reaction (pcr) assay. severe pjp was defined as hypoxemic acute respiratory failure requiring high-flow nasal oxygen with at least 50% fio(2), non-invasive ventilation, or mv. results: of 2446 respiratory samples investigated during the study period, 514 from 430 patients were positive for p. jirovecii. of these 430 patients, 107 met criteria for pjp and were included in the study, 53 (49.5%) patients had severe pjp, including 30 who required mv. all patients were immunocompromised with haematological malignancy ranking first (n = 37, 35%), followed by solid organ transplantation (n = 27, 25%), hiv-infection (n = 21, 20%), systemic diseases (n = 13, 12%), solid tumors (n = 12, 11%) and primary immunodeficiency (n = 6, 8%). by multivariate analysis, factors independently associated with severity were older age (or, 3.36; 95% ci 1.4–8.5; p < 0.05), a p. jirovecii microscopy-positive result from bronchoalveolar lavage (bal) (or, 1.3; 95% ci 1.54–9.3; p < 0.05); and absence of a bal fluid alveolitis profile (or, 3.2; 95% ci 1.27–8.8; p < 0.04). the 90-day mortality rate was 27%, increasing to 50% in the severe pjp group. factors independently associated with 90-day mortality were worse sofa score on day 1 (or, 1.05; 95% ci 1.02–1.09; p < 0.001) whereas alveolitis at bal was protective (or, 0.79; 95% ci 0.65–0.96; p < 0.05). in the subgroup of hiv-negative patients, similar findings were obtained, then viral co-infection were independently associated with higher 90-day mortality (or, 1.25; 95% ci 1.02–1.55; p < 0.05). conclusions: older age and p. jirovecii oocysts at microscopic examination of bal were independently associated with severe pjp. both initial pjp severity as evaluated by the sofa score and viral co-infection predicted 90-day mortality. alveolitis at bal examination was associated with less severe pjp. the pathophysiological mechanism underlying this observation deserves further investigation. over the last 10 years, survival benefits provided by steady advances in antitumor chemotherapy and immunosuppressant regimens for patients with autoimmune diseases, haematological malignancies, and solid organ transplants have substantially increased the number of adults living with immunodeficiencies [1, 2] . among opportunistic infections in immunocompromised adults, pneumocystis jirovecii pneumonia (pjp) was associated with high rates of intubation and mortality [3] . consequently, an early identification and optimal treatment of patients with pjp remains a key priority [4, 5] . since the advent of antiretroviral therapy, the incidence and mortality rates of pjp among patients positive for the human immunodeficiency virus (hiv) have decreased steadily [6] . however, pjp is being increasingly diagnosed in hiv-negative patients, in whom it carries a poorer prognosis [7, 8] . a higher proportion of neutrophils in broncho-alveolar lavage (bal) fluid during pjp was associated with higher risks of respiratory complications and mortality [9, 10] . in the same way, a low lymphocyte count in bal fluid was a risk factor for the failure of trimethoprim/sulfamethoxazole (tmp/smz) therapy [11] . early adjunctive steroid therapy for severe pjp dramatically decreased mortality rates in hiv-positive patients [12, 13] but had variable effects in their hivnegative counterparts [14] [15] [16] . these findings suggest that the immunological status and underlying diagnosis may influence the pathophysiology of pjp and the risk of mortality [17, 18] . however, bal fluid cytology profiles have not been adequately evaluated as potential prognostic factor and predictors of treatment responses. the identification of early predictors of pjp outcomes, including bal fluid findings, may help to determine which patients are most likely to benefit from intensive care and could justify adjunctive steroid therapy. the aim of this prospective study of patients with pjp was to identify early risk factors for severe pjp and 90-day mortality. this is a retrospective analysis of prospective cohort. from january 2012 to january 2017, all patients presenting an invasive fungal infection that has been diagnosed in our center have been included in a prospective registry (the french prospective surveillance programme, ressif network), thus pjp patients have been included in a subcohort. for each patient with a positive sample, the following clinical findings were prospectively investigated: dyspnea and/or cough in immunocompromised patients with interstitial syndrome by radiography or ct scan. among all respiratory samples investigated for 6 years (n = 2446), all positive tests for pneumocystis jirovecii samples (n = 430) were assessed by a biologist and a clinician to investigate criteria for pjp and include them in this prospective cohort. the following data were prospectively collected: age; sex; underlying disease; pjp prophylaxis; other medications including glucocorticoids taken during the past month; type of symptoms and symptom duration at pjp diagnosis and time from symptom onset to hospital admission. secondary, the clinical data were collected for all patients from medical records: laboratory findings (white blood cell count; absolute neutrophil count; c-reactive protein [crp] level; bal fluid findings including the cell profile assessed by an independent cytologist on centrifuged bal fluid samples prepared with the wright-giemsa and perls stains to allow the determination of macrophage, lymphocyte, neutrophil, eosinophil, and basophil counts); presence of p. jirovecii and/or other fungi and/or bacteria and/or viruses (influenza viruses, respiratory syncytial virus, adenovirus, cytomegalovirus) were recorded at pjp diagnosis. the sofa score on day 1 and the ratio of the arterial partial pressure of oxygen over the fraction of inspired oxygen (pao 2 /fio 2 ) on day 1 [19] were recorded for each patient at pjp diagnosis. anti-pjp medications, adjuvant glucocorticoid therapy, oxygen supplementation, and ventilatory support provided at admission were documented. finally, patient outcomes including 90-day mortality were recorded. the collection of follow-up data ended in december 2017. severe pjp was defined as hypoxemic acute respiratory failure requiring high-flow nasal oxygen with at least 50% fio 2 , non-invasive ventilation, or mv. because not all patients were hospitalized in intensive care units, we chose this pragmatic and reproducible severity criterion. according to the berlin definition [20] , severe acute respiratory distress syndrome (ards) was defined as the presence of the following criteria within 3 days after icu admission: new respiratory symptoms, bilateral opacities mortality. alveolitis at bal examination was associated with less severe pjp. the pathophysiological mechanism underlying this observation deserves further investigation. keywords: pneumocystis jirovecii pneumonia, early prognostic score, high flow oxygen, haematological malignancies, alveolitis on chest radiographs or by ct, absence of suspected hydrostatic/cardiogenic pulmonary oedema, and pao 2 / fio 2 ≤ 300. lymphocytic alveolitis [21] was defined as a bal fluid cell population containing more than 10% of lymphocytes and more than 5% of neutrophils combined with an activated macrophage phenotype, based on the diagnostic criteria for hypersensitivity pneumonitis, a condition characterised by alveolitis and migration to the alveoli of multiple cell types including activated t cells, monocytes, and natural killer cells [22] . finally, patients with other pathogens associated with p. jirovecii in respiratory or blood samples were classified as having coinfection at icu admission. patient characteristics were described using mean ± sd for continuous variables (or 95% confidence interval [95% ci] when appropriate) and proportions for qualitative variables. continuous variables were compared using the wilcoxon rank-sum and kruskal-wallis tests and qualitative variables using fisher's exact test with computation of the odds ratios (ors) and their 95% cis. overall survival was assessed using kaplan-meier curves and log-rank tests. factors associated with severity were identified by logistic regression analysis. factors associated with all-cause 90-day mortality were identified by logistic regression analysis of patients alive after day 90 versus patients who died before day 90. only factors reaching statistical significance (p < 0.05), with no more than 10% missing data, were included in the multivariate model. when collinearity between co-variates was detected, only the variable with the highest or was kept into the multivariate regression model. results are reported as log-transformed coefficients with their 95% cis. missing data were not interpolated. analyses were performed on the whole population, then excluding hiv+ patients. of the 2446 respiratory samples tested during the study period, 514 from 430 patients were positive for p. jirovecii. of these 430 patients, 107 met our pjp criteria and were included in the study; table 1 reports their main characteristics. the remaining 323 patients were classified as having bronchial p. jirovecii colonisation (fig. 1 ). the mean number of patients included per year was 21 and the number of patients per year increased gradually over time to reach a peak of 28 patients in 2015 (additional file 1: figure s1 ). of the 107 patients with pjp, 97 had positive bal fluid, by a direct examination of bal fluid in 49 patients and only detected by pcr in 48 patients. the induced sputum test was positive in 16 patients (by pcr, n = 15; time from onset to bal, days, mean ± sd 3 ± 5.5 pj visible in smears, n (%) 49 (45.8) alveolitis profile, n (%) 31 (29) co-infection at pjp diagnosis, n (%) viral infection 37 (35) bacterial infection 29 (18) invasive fungal infection 5 (5) and/or grocott-gomori stain, n = 3). both the bal fluid and the induced sputum test were positive in 6 patients. all patients were immunocompromised with haematological malignancy ranking first (n = 37, 35%), followed by solid organ transplantation (n = 27, 25%), hiv-infection (n = 21, 20%), systemic diseases (n = 13, 12%), solid tumors (n = 12, 11%) and primary immunodeficiency (n = 6, 8%). the mean hiv viral load at pjp diagnosis was 317,240 copies for hiv-positive patient. only 21 (19.6%) patients were given pjp prophylaxis. of these, 7 were compliant with the prescription during the last 2 months before diagnosis, which never consisted in tmp/smz. the first-line pjp therapy was tmp/smz for 100 (93.5%) patients, 6 (5.6%) patients being treated with atovaquone; no pjp therapy was given to the remaining patient, who died within 24 h after icu admission (pjp diagnosis was made post-mortem). adjunctive glucocorticoid therapy was given to 61 (57%) patients based on severity criteria. sixteen patients were switched from tmp-smz to atovaquone (n = 14) or pentamidine (n = 2), due to acute kidney injury (n = 9), myelotoxicity (n = 5) allergy (n = 1), or hepatic cytolysis (n = 1); 2 of these 16 patients had both acute kidney injury and myelotoxicity. of 97 patients with available bal fluid cytology results, 31 (32%) had evident alveolitis profile. mean bal fluid percentages in the 97 patients were 21.7 ± 21.3 for neutrophils, 46 ± 25.7 for macrophages, and 32 ± 24.7 for lymphocytes. bacterial co-infection was diagnosed in 19 (18%) patients, viral co-infection in 37 (35%) patients, and fungal co-infection in 5 (5%) patients (additional file 1: table s1 ). lymphocytes and neutrophils mean ratio were, respectively, 22% and 23% in hiv patients, 34% and 21% in non-hiv patients, 22% and 30% in dead patients, 35% and 19% in survivors patients, 36% and 21% with only pjp patients, 29% and 22% during coinfection. we did not observe in our study eosinophilic and neutrophilic alveolitis. of the 107 patients, 53 (49.5%) were classified as severe pjp (table 2) . icu admission was required in 50 patients, including 30 who received mv. the hiv serology was positive in 6/53 (11%) patients with severe pjp and 15/54 (28%) patients with non-severe pjp. early risk factors associated with severity in the univariate analyses were age > 55 years (or, 2.6; 95% ci 1.12-6.3; p < 0.02), albuminemia < 27 g/l (or, 3.3; 95% ci 1.25-9; p < 0.001), blood neutrophil count > 6.5 g/l (or, 6.5; 95% ci 2.4-20; p < 0.001), bal fluid neutrophils > 12% (or, 5.7; 95% ci 2-18; p < 0.001), p. jirovecii oocysts observed at direct examination of bal fluid (or, 2.8; 95% ci 1.2-6.9; p < 0.01), higher baseline lactic dehydrogenase value (or, 1.35; 95% ci 1.13-1.68; p < 0.002), and higher crp (or, 1.01; 95% ci 1.01-1.02; p < 0.001). a bal alveolitis profile was protective (or, 0.3; 95% ci 0.1-0.8; p < 0.01). by multivariate analysis, factors independently associated with severe pjp were older age (or, 3.36; 95% ci 1.4-8.5; p < 0.05), p. jirovecii oocysts observed at direct examination of bal fluid (or, 1.3; 95% ci 1.54-9.3; p < 0.05) and the absence of a bal fluid alveolitis profile (or, 3.2; 95% ci 1.27-8.8; p < 0.04). bmi, body mass index; icu, intensive care unit; ards, acute respiratory distress syndrome; saps2, simplified acute physiology score version 2; sofa score, sequential organ failure assessment score; hiv, human immunodeficiency virus; pjp, pneumocystis jirovecii pneumonia; crp, c-reactive protein; ldh, lactate dehydrogenase; pj, pneumocystis jirovecii a the total exceeds 100% because some patients had more than one cause of immunodeficiency b of these 21 patients, 7 followed their prescribed prophylactic regimen (aerosolised pentamidine, n = 6; and atovaquone, n = 1) and 14 did not (trimethoprim/sulfamethoxazole, n = 11; and aerosolised pentamidine, n = 3) two factors were independently associated with 90-day mortality by multivariate analysis, a worse sofa score was associated with higher 90-day mortality (or, 1.05; 95% ci 1.02-1.09; p < 0.001), whereas bal fluid alveolitis profile was associated with lower 90-day mortality (or, 0.79; 95% ci 0.65-0.96; p < 0.05) ( table 3) . hiv serology was a protective factor in univariate analysis but was not statistically associated with protective factor in the multivariate 90-day mortality analysis. in survival analysis hiv patients presenting with pjp was associated with statistically better prognostic than that of patients with hematologic diseases or solid cancer (additional file 1: figures s2, s3) . in the subgroup of hiv-negative patients, similar findings were obtained, then viral co-infection were independently associated with higher 90-day mortality (or, 1.25; 95% ci 1.02-1.55; p < 0.05) (additional file 1: tables s2, s3 ). factors associated with 90-day mortality in icu patients were sofa score and non-hiv patients (additional file 1: table s4 ). in this prospective study, over four-fifths of pjp patients were hiv-negative, and half met our criteria for severe disease. a worse sofa score on admission and viral co-infection were independently associated with higher 90-day mortality in both whole patients and hiv-negative patients. importantly, a bal fluid cytological profile consistent with alveolitis was associated with lower 90-day mortality. severe pjp on admission as defined for our study was associated with a 56% risk of receiving mv, in keeping with recent results from large cohort studies [3, 23] . given the prognostic significance of severity, an improved knowledge of early risk factors for severity is helpful to identify patients requiring more intensive monitoring and treatment. as illustrated here, hiv patients less often experienced severe pjp compared to their hiv-negative counterparts, in agreement with earlier data [5, 8, [24] [25] [26] [27] . the reduced severity of pjp in hiv-positive patients is more table 3 risk factors for 90-day mortality in the overall population (patients where bal was performed, n = 85) italics characters correspond to the analysis parameters with a statistically significant difference bmi, body mass index; saps2, simplified acute physiology score version 2, sofa score, sequential organ failure assessment score; hiv, human immunodeficiency virus likely to be associated with the particularity of hivinduced immunosuppression, of which pneumocystis is a hallmark of a severe adaptive cellular impairment. the co-morbidities in non-hiv patients (onco-hematology, solid organ transplantation and system diseases) may also contribute to their greater vulnerability. the immune recovery allowed by the initiation of antiretroviral therapy is probably correlated with better long-term outcomes in hiv-positive patients than in non-hiv patients whose profound immunosuppression is more frequently extended. hiv-positive patients also have lower neutrophil counts in bal fluid samples [28] , are less likely to develop severe pjp, and have lower mortality rates compared to hiv-negative patients [11, [26] [27] [28] [29] . the sofa score, viral co-infection and absence of alveolitis remained independently associated with a 90-day higher mortality in hiv-negative patients, suggesting that it is a strong independent marker in the whole pjp population. by univariate analysis, early risk factors for severity in hiv-negative patients were markers for vulnerability (older age and lower serum albumin) and for inflammation (systemic inflammatory syndrome characterized by blood and alveolar polynucleosis serum crp level). the patient subgroup at the most severe end of the spectrum had the highest crp levels and neutrophil counts, suggesting that anti-inflammatory treatments might improve patient outcomes. our study suggests that future prospective studies on adjunctive treatments for pjp should focus on hiv-negative patients, notably solid organ and haematological stem cell transplant recipients, meeting criteria for severe pjp (e.g., sofa score > 2 with a low pao 2 /fio 2 ratio). the potential relevance of a bal fluid cytology profile consistent with alveolitis should probably also be taken into account when assessing the efficacy of new treatment regimens. as expected, mortality within the first 90 days was significantly higher in patients with severe versus nonsevere pjp. the independent association between a worse sofa score at admission and 90-day mortality confirms the appropriateness of an evaluation with an intensivist to consider icu admission of patients with oxygen-dependent pjp [30] . the quicksofa, which is a simplified score based on three criteria, is easy to determine by non-intensivists and may be useful for determining when advice from an intensivist should be sought [31] . in addition to a worse sofa score, viral co-infection at the time of pjp diagnosis was associated with higher 90-day mortality. viral infections consisted mostly (67%) in reactivation of latent viruses (cytomegalovirus, herpes simplex virus, or epstein-barr virus), suggesting that this subgroup may have been characterised by a more profound immune deficiency. taken together, this finding supports the hypothesis that the outcome of pjp is also closely related to the underlying diagnosis and immune response. an important finding from this study is the clear association between a bal fluid cytology profile consistent with alveolitis (> 10% lymphocytes, > 5% neutrophils, and presence of activated macrophages) and less severe pjp and lower 90-day mortality. the presence of neutrophils in bal fluid has often been noted in clinical and experimental studies of acute respiratory distress syndrome (ards) [32, 33] . three patient subgroups can be identified in our population, one defined by alveolitis, the other one pjp occuring in hiv-positive individuals, both having a better prognosis and the last one defined by a sofa score above 2 on admission who have a more severe prognosis. taken together, these results suggest that specific immunological characteristics might allow the identification of patient subgroups with different treatment needs and outcomes. cd4 + t cell levels were non-significantly higher in patients with alveolitis, whereas glucocorticoid exposure was comparable in the two groups. to our knowledge, alveolitis during pjp has not been described as a good prognostic factor yet [21] . in murine models of cd4 + t-cell depleted mice, increased alveolar recruitment of cd8 + t cells induced by interleukin-7 therapy improved p. jirovecii clearance [34] . lymphocyte count in bal fluid is increased in patients with alveolitis, which was associated with a better prognosis in our study. thus, alveolitis during pjp might reflect maintenance of an effective pulmonary immune response including cd8 + t-cell recruitment. glucocorticoid therapy might, therefore, be unnecessary in patients with pjp and alveolitis, although their specific response to glucocorticoids has not been investigated to date. four-fifths of our patients had not been prescribed pjp prophylaxis, and among those with a prescription only one-third were compliant. these findings confirm earlier results [16, 35] and further identify the absence of tmp/smz prophylaxis as a major risk factor for pjp in high-risk patients [29] . providing appropriate prophylactic anti-microbial treatments to patients with immunosuppression, notably related to haematological diseases and transplantation, is crucial to improve patient outcomes. adherence to prophylactic treatment must be supported at each follow-up visit. pjp usually develops in patients with cd4 + counts below 300/mm 3 [36] [37] [38] , although the depth of lymphopenia does not correlate with pjp severity [39, 40] . a history of glucocorticoid exposure is an often reported risk factor for pjp in hiv-negative patients [41, 42] . among our patients, over half were receiving glucocorticoid therapy at the diagnosis of pjp, in a mean prednisone-equivalent dosage of 20 mg/day. adjuvant glucocorticoid therapy was given to most severe patients who failed antibiotic treatment alone. given the history of glucocorticoid exposure, the effects of adjuvant glucocorticoid therapy were difficult to assess. the role for adjuvant glucocorticoid therapy in patients with pjp is debated [26, [43] [44] [45] [46] [47] and is currently being assessed in a prospective study (clinicaltrials.gov identifier: nct02944045). serum ldh level at pjp diagnosis was associated with poor outcomes in hiv-positive and hiv-negative patients in several studies [48] . in our population, ldh > 7 µkat/l (420 u/l) levels were associated with severe pjp by univariate analysis but not with higher 90-day mortality by multivariate analysis. missing data or a role for unidentified confounders may explain this result. a limitation of our study is the use of non-validated criteria for defining severe pjp. the european conference on infections in leukaemia-5 group has defined severe pjp, like those defined for hiv-positive patients [49] such as pao 2 less than 8 kpa, oxygen saturation less than 91%, and radiological impairment. these criteria were only used in retrospective study of pjp patients [45] . however, these criteria have not been prospectively validated in non-hiv patients. hiv-patients less often experienced severe pjp compared to their hiv-negative counterparts, enhancing the need to propose an early severity scale adapted to this specific population. we, therefore, relied on criteria indicating hypoxemic arf, namely, fio 2 ≥ 50% or niv or mv. the strong association of severe pjp with higher 90-day mortality indicates that our definition successfully identified severe pjp. in a large prospective cohort of patients admitted for pjp, the sofa score on admission and presence of viral co-infection were independently associated with higher 90-day mortality. importantly, a bal fluid cytology profile suggesting alveolitis was associated with less severe pjp and lower 90-day mortality. our findings, notably the associations linking the sofa score and alveolitis to 90-day mortality, deserve further evaluation in a multicentre prospective study. future studies of adjunctive treatments for pjp therapy should differentiate between patients with and without alveolitis. clinical immune-monitoring strategies for predicting infection risk in solid organ transplantation opportunistic infections and biologic therapies in immune-mediated inflammatory diseases: consensus recommendations for infection reporting during clinical trials and postmarketing surveillance acute hypoxemic respiratory failure in immunocompromised patients: the efraim multinational prospective cohort study beyond tumor necrosis factor inhibition: the expanding pipeline of biologic therapies for inflammatory diseases and their associated infectious sequelae outcomes of pneumocystis pneumonia with respiratory failure in hiv-negative patients opportunistic infections in patients with and patients without acquired immunodeficiency syndrome acute respiratory failure due to pneumocystis pneumonia in patients without human immunodeficiency virus infection: outcome and associated features critical care management and outcome of severe pneumocystis pneumonia in patients with and without hiv infection cellular profiles in bronchoalveolar lavage fluid of hiv-infected patients with pulmonary symptoms: relation to diagnosis and prognosis cellular profiles of bronchoalveolar lavage fluid and their prognostic significance for non-hiv-infected patients with pneumocystis jirovecii pneumonia low lymphocyte proportion in bronchoalveolar lavage fluid as a risk factor associated with the change from trimethoprim/sulfamethoxazole used as first-line treatment for pneumocystis jirovecii pneumonia la voie l. corticosteroids as adjunctive therapy for severe pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. a double-blind, placebocontrolled trial a controlled trial of early adjunctive treatment with corticosteroids for pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. california collaborative treatment group use of adjunctive corticosteroids in severe adult non-hiv pneumocystis carinii pneumonia corticosteroids as adjunctive therapy for severe pneumocystis carinii pneumonia in non-human immunodeficiency virus-infected patients: retrospective study of 31 patients pneumocystis carinii pneumonia in critically ill patients with malignancy: a descriptive study pneumocystis carinii pneumonia. differences in lung parasite number and inflammation in patients with and without aids aids-related pneumocystis carinii pneumonia in the era of adjunctive steroids: implication of bal neutrophilia delivered oxygen concentrations using low-flow and high-flow nasal cannulas acute respiratory distress syndrome: the berlin definition cellular and cytokine changes in the alveolar environment among immunocompromised patients during pneumocystis jirovecii infection hypersensitivity pneumonitis and alpha-chemokines risk factors for the development of acute lung injury in patients with infectious pneumonia acute respiratory failure due to pneumocystis pneumonia: outcome and prognostic factors prognostic factors of pneumocystis jirovecii pneumonia in patients without hiv infection adjunctive steroid in hiv-negative patients with severe pneumocystis pneumonia pneumocystis carinii pneumonia in patients with hematologic malignancies: a descriptive study alveolar and blood t lymphocyte profiles in pneumocystis jirovecii-positive patients: effects of hiv status a multivariable prediction model for pneumocystis jirovecii pneumonia in hematology patients with acute respiratory failure changes in severity and organ failure scores as prognostic factors in onco-hematological malignancy patients admitted to the icu the prognostic performance of qsofa for community-acquired pneumonia molecular and immune biomarkers in acute respiratory distress syndrome: a perspective from members of the pulmonary pathology society mechanical ventilation induces inflammation, lung injury, and extra-pulmonary organ dysfunction in experimental pneumonia treatment with interleukin-7 restores host defense against pneumocystis in cd4+ t-lymphocyte-depleted mice pneumocystis polymerase chain reaction and blood (1 → 3)-β-dglucan assays to predict survival with suspected pneumocystis jirovecii pneumonia pneumocystis jirovecii pneumonia in hiv-negative patients: a prospective study with focus on immunosuppressive drugs and markers of immune impairment risk factors analysis for pneumocystis jirovecii pneumonia (pcp) in patients with haematological malignancies and pneumonia pneumocystis jirovecii pneumonia in patients with or without aids influence of type of cancer and hematopoietic stem cell transplantation on clinical presentation of pneumocystis jirovecii pneumonia in cancer patients peripheral blood cd4+ t-lymphocyte counts during pneumocystis carinii pneumonia in immunocompromised patients without hiv infection pneumocystis pneumonia in patients treated with rituximab pneumocystis carinii pneumonia among patients without aids at a cancer hospital adjunctive corticosteroids for pneumocystis jirovecii pneumonia in patients with hiv infection an official american thoracic society statement: treatment of fungal infections in adult pulmonary and critical care patients predisposing factors, clinical characteristics and outcome of pneumonocystis jirovecii pneumonia in hiv-negative patients clinical picture of pneumocystis jirovecii pneumonia in cancer patients analysis of underlying diseases and prognosis factors associated with pneumocystis carinii pneumonia in immunocompromised hiv-negative patients clinical course, treatment and outcome of pneumocystis pneumonia in immunocompromised adults: a retrospective analysis over 17 years pneumocystis carinii infection: current treatment and prevention publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the clinicians and microbiologists who contributed to the management of the study patients for their commitment to providing optimal patient care. supplementary information accompanies this paper at https ://doi. org/10.1186/s1361 3-019-0604-x. abbreviations ards: severe acute respiratory distress syndrome; bal: broncho-alveolar lavage; bmi: body mass index; ci: confidence interval; cmv: cytomegalovirus; crp: c-reactive protein; ebv: epstein-barr virus; fio 2 : fraction of inspired oxygen; hiv: human immunodeficiency virus; hsv1: herpes simplex virus; icu: intensive care unit; ldh: lactate dehydrogenase; mv: invasive mechanical ventilation; niv: non-invasive ventilation; or: odds ratio; pao 2 : arterial partial pressure of oxygen; pcr: real-time polymerase chain reaction; pjp: pneumocystis jirovecii pneumonia; rsv: respiratory syncytial virus; saps2: simplified acute physiology score version 2; sd: standard deviation; sofa score: sequential organ failure assessment score; tmp-smx: trimethoprim-sulfamethoxazole.authors' contributions bjg and bt analysed and interpreted the data. cs performed the cytological examinations of broncho-alveolar fluid samples. fm, ral, and bjg were members of the independent committee that adjudicated the pjp cases. bjg and jr wrote the manuscript. all authors read and approved the final manuscript no funding was received for this study. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. all authors read the final version of the manuscript and approved its submission to annals of intensive care. the authors declare that they have no competing interests. key: cord-030368-6z99rm0a authors: schwegler, ute; fritze, eugen; fritze, jürgen; pox, christian; schmiegel, wolff; may, burkard; merget, rolf title: infektionskrankheiten date: 2012 journal: die &#x000e4;rztliche begutachtung doi: 10.1007/978-3-642-21081-5_27 sha: doc_id: 30368 cord_uid: 6z99rm0a virale infektionen zählen immer noch weltweit, besonders in den entwicklungsländern, zu den häufigsten todesursachen. durch internationale systematische impfkampagnen der who ist die welt pocken-frei geworden. die inzidenz der infektionen, die durch eine immunprophylaxe verhindert werden können, wurde drastisch gesenkt (poliomyelitis, tollwut). in den 1980er jahren trat eine neue tierseuche beim rind auf, die bovine spongyforme enzephalopathie (bse), die mit der neuen variante der creutzfeldt-jakob-krankheit (vcjk) in zusammenhang steht (nahrungskette). im selben zeitraum begann die explosionsartige weltweite verbreitung eines neuen retrovirus, des hiv. trotz intensiver aufklärungskampagnen und neuen therapeutischen möglichkeiten (haart, hoch aktive antiretrovirale therapie) ist es bis zur jahrtausendwende nicht gelungen, die weitere ausbreitung der hiv-infektion zu verhindern. seit 2007 ist eine allmähliche eindämmung der infektion zu erkennen, die neuinfektionen sind weltweit rückläufig. durch den breiteren einsatz der haart sind lebensqualität und lebenserwartung enorm gestiegen, die sterberate der aids-infizierten ist seit 2005 um mehr als 50 % zurückgegangen. und meerkatzen in zentralafrika liegt. es ist nicht geklärt, ob diese tiere selbst an einem aids-ähnlichen syndrom erkranken oder nur virusträger sind. wahrscheinlich haben menschliche hi-viren und die von affen einen gemeinsamen vorfahren, aus dem sie über mehrere jahrhunderte entstanden sind. erkrankungen an aids sind in der bevölkerung zentralafrikas endemisch (hiv 2), die häufigkeit scheint dort bei männern und frauen gleich zu sein. homosexualität ist bei der afrikanischen bevölkerung fast unbekannt. man vermutet, dass das hiv aus zentralafrika nach haiti eingeschleppt und dort endemisch wurde. von dort ist es offenbar durch intensiven tourismus von homosexuellen in die großstädte der usa eingeschleppt worden und hat seit 1981 zu einem explosionsartigen ausbruch von aids geführt. etwa drei jahre später griff die krankheit auf die westeuropäischen großstädte über. der anstieg der erkrankungshäufigkeit folgte einer exponentialfunktion. indessen scheint der häufigkeitsanstieg durch weltweite aufklärungskampagnen von jahr zu jahr geringer zu werden. weltweit sind bisher seit ausbruch der pandemie 70-80 millionen menschen mit dem hi-virus infiziert worden (. tab. virale infektionen zählen immer noch weltweit, besonders in den entwicklungsländern, zu den häufigsten todesursachen. durch internationale systematische impfkampagnen der who ist die welt pocken-frei geworden. die inzidenz der infektionen, die durch eine immunprophylaxe verhindert werden können, wurde drastisch gesenkt (poliomyelitis, tollwut). in den 1980iger jahren trat eine neue tierseuche beim rind auf, die bovine spongyforme enzephalopathie (bse), die mit der neuen variante der creutzfeldt-jakob-krankheit (vcjk) in zusammenhang steht (nahrungskette). im selben zeitraum begann die explosionsartige weltweite verbreitung eines neuen retrovirus, des hiv. trotz intensiver aufklärungskampagnen und neuen therapeutischen möglichkeiten (haart, hoch aktive antiretrovirale therapie) ist es bis zur jahrtausendwende nicht gelungen, die weitere ausbreitung der hiv-infektion zu verhindern. seit 2007 ist eine allmähliche eindämmung der infektion zu erkennen, die neuinfektionen sind weltweit rückläufig. durch den breiteren einsatz der haart sind lebensqualität und lebenserwartung enorm gestiegen, die sterberate der aids-infizierten ist seit 2005 um mehr als 50 % zurückgegangen. nach der zahl der noch vorhandenen t-helferzellen wird dem jeweiligen klinischen stadium eine zahl zwischen 1 und 3 zugewiesen, sodass eine klassifizierung von a1 bis c3 möglich ist. die stadien a3, b3 und c1-3 gelten als aids (. tab. 27.2) z diagnostik nach infektion mit dem hiv können direkte verfahren zum virusnachweis, nach einsetzen der antikörperantwort indirekte immunologische methoden angewendet werden. in der zeit der diagnostischen lücke (zeitintervall von der infektion bis zur antikörperbildung), die bis zu 6 monaten dauern kann, kommen als direkte verfahren die pcr und der hiv-p24-antigentest zur anwendung. die direkten verfahren stellen keine routineuntersuchungen dar, sondern sind speziellen fragestellungen vorbehalten (neugeborene hiv-infizierter mütter, berufsbedingte infektionen). standardsuchtest ist der hiv-elisa, der regelhaft durch den western blot bestätigt werden muss. afrika südlich der sahara der indexpatient darf nur mit seiner zustimmung auf hiv untersucht werden. die ärztliche informationspflicht (d-arztbericht, ärztliche anzeige einer berufskrankheit) muss erfüllt werden. das robert koch-institut bittet darum, hiv-expositionen und ihre behandlung auf entsprechenden erhebungsbögen (▶ www.rki.de) anonymisiert zu dokumentieren und zu melden. die hiv-infektion ist eine tödliche viruserkrankung. durch die entwicklung neuer antiretroviraler chemotherapeutika mit dem einsatz der hochaktiven antiretroviralen therapie (haart) ist es gelungen, sowohl die lebenserwartung wie auch die lebensqualität der infizierten signifikant zu verbessern. die befürchtete explosionsartige verbreitung von hiv konnte weltweit, besonders in den subsahara-ländern, eingedämmt werden. z gutachterliche bewertung in der gesetzlichen unfallversicherung gelten grundsätzlich die gleichen kriterien zur anerkennung als berufskrankheit wie bei anderen infektionskrankheiten, die durch blut übertragen werden. da hiv ganz überwiegend in hochrisikogruppen verbreitet ist, muss bei meldung einer beruflichen infektion im einzelfall der bloße verdacht ausgeschlossen werden, ob sich der infizierte nicht als angehöriger einer hochrisikogruppe unfallunabhängig infiziert hat. bei der späteren begutachtung geschlossen werden. wenn der chirurg hiv-infiziert ist, liegt das risiko bei ca. 1/83.000 operationsstunden. valide daten existieren allerdings nicht. z therapie in der behandlung der hiv-infektion stehen medikamente aus verschiedenen wirkstoffklassen zur verfügung: nukleosidische reverse transkriptaseinhibitoren (nrti), nichtnukleosidische reverse transkriptaseinhibitoren (nnrti), proteaseinhibitoren, fusionshemmer, entryinhibitoren und integraseinhibitoren (ini). die zahl der zugelassenen einzelsubstanzen und kombinationspräparate ist auf weit über 20 angewachsen. seit der einführung der hochaktiven antiretroviralen therapie (haart), die lebenslang durchgeführt werden muss, -wann bei asymptomatischen hiv-infizierten behandelt werden sollte, ist unklar. keine der bisherigen therapiestudien hat diese frage eindeutig beantwortet. aus einer reihe von kohortenstudien lässt sich jedoch ableiten, dass bei einem behandlungsbeginn erst unterhalb einer cd4-zellzahl von 200/µl mit einer deutlich erhöhten morbidität und mortalität zu rechnen ist. ein abfall unter diesen wert sollte daher vermieden werden. asymptomatische hiv-infizierte mit einer cd4-zellzahl <200/ µl haben unabhängig vom ausmaß der virusreplikation ein deutlich erhöhtes risiko für eine immunologische und klinische progression, das durch eine antiretrovirale therapie vermieden werden kann. eine behandlung ist daher klar indiziert (evidenzgrad ai). zum serologischen nachweis sind nur für einen teil der erreger kommerzielle tests erhältlich (fsme, dengue-virus, wnf). die pcr-diagnostik darf nur in hochsicherheitslaboratorien durchgeführt werden. eine impfprophylaxe ist gegen gelbfieber, japan-enzephalitis und fsme (frühsommer-menigoenzephalitis) möglich. für laborpersonal steht ein inaktivierter impfstoff gegen die ost-und west-pferdeenzephalitis zur verfügung. z gutachterliche bewertung bei personen, die sich beruflich in endemiegebieten aufhalten müssen, ist wahrscheinlich, dass eine arbovirusinfektion als berufskrankheit anzuerkennen ist. virus übertragenden zecken finden sich nicht nur in wäldern und am waldesrand, sondern auch in parkanlagen und gärten. z symptomatik 60-70 % der infektionen verlaufen asymptomatisch. sie sind nur anhand der serokonversion zu erkennen. auch die klinisch inapparente infektion hinterlässt eine lebenslange immunität. ein drittel der infizierten entwickelt nach einer inkubationszeit von 1-2 wochen symptome. der krankheitsverlauf ist biphasisch. in der 1. phase treten grippeähnliche beschwerden auf mit kopf-, hals-und gliederschmerzen, fieber, erbrechen, schwindel. diese symptome verschwinden innerhalb einer woche spontan. nach einem fieberfreien intervall von 7-20 tagen treten bei 10 % der infizierten mit einem zweiten fieberanstieg neurologische symptome auf, zur hälfte eine benigne selbstlimitierte seröse meningitis, in einem drittel zusätzlich eine enzephalitis mit epileptischen anfällen, bewusstseinsstörungen und psychotischen symptomen. sehr selten kommt es zu einem meningomyeloenzephalitischen verlauf mit beteiligung des zentralnervensystems und des peripheren nervensystems mit schlaffen paresen. nach 2 wochen haben sich die neurologischen symptome zurückgebildet. restsymptome (paresen, anfallsleiden, kopfschmerzen) verbleiben bei weniger als 10 %. bei 1-2 % der erkrankten mit zns-beteiligung verläuft die infektion letal. z diagnostik diagnostische methode der wahl ist der fsme-virus-spezifische igm-und igg-nachweis im serum oder liquor mittels elisa. diese antikörper können mit beginn der 2. krankheitsphase nachgewiesen werden. bei atypischen verläufen gilt der alleinige igm-nachweis im blut als unzureichend spezifisch, weshalb empfohlen wird, einen 4-fachen titeranstieg in 2 serumproben nachzuweisen. zu beachten ist, dass nach fsme-impfung über längere zeit fsme-spezifische igm-antikörper vorhanden sein können. zu beginn der erkrankung ist eine virusisolierung aus blut und liquor möglich (spezielle pcr, westernblot). z therapie die therapie beschränkt sich auf symptomatische maßnahmen. z verlauf bei meningomyeloenzephalitischem verlauf kommt es bei ca. 10 % zu bleibenden neurologischen defiziten (bei kindern sehr selten, bei älteren erwachsenen in bis zu 40 %). die letalität beträgt 1-2 %. nach § 7 abs. 1 nr. 14 ifsg besteht namentliche meldepflicht bei erkrankung und tod. z prophylaxe durch die aktive fsme-schutzimpfung wird ein über 98 %iger impfschutz erzielt. schon nach der 2. impfung liegt der impfschutz bei 90 %. zur grundimmunisierung sind 3 impfungen erforderlich: die 2. impfung 21 tage bis 3 monate nach der 1. impfung, die 3. impfung 9-12 monate nach der 1. impfung. auffrischungsimpfung alle 3 jahre (stiko). indiziert ist die impfung bei allen personen, die sich ständig oder vorübergehend in der zeit von märz bis dezember in endemiegebieten aufhalten. eine postexpositionelle immunprophylaxe ist nicht möglich. in deutschland sind in den letzten 10 jahren keine mks-fälle aufgetreten. die pathogenität des mks-virus für den menschen ist sehr gering. infektionen wurden bei früheren ausbrüchen nur vereinzelt bei direktem kontakt zu infizierten tieren in der tierhaltung und beim schlachten beobachtet. begünstigend wirken mangelhafte arbeitshygiene, eine sehr massive exposition sowie hautverletzungen, die als eintrittspforte dienen können. verlauf nach einer inkubationszeit von 2-8 tagen entwickelt sich an der eintrittspforte eine primäraphthe. danach können eine diskrete fieberhafte allgemeinreaktion sowie aphthen an der mund-rachen-schleimhaut sowie an den fingern und zehen auftreten. der krankheitsverlauf ist unkompliziert, organmanifestationen an zns oder herz, wie sie vom tier bekannt sind, sind bei menschen bisher noch nie aufgetreten. die therapie ist symptomatisch. die erkrankung heilt folgenlos aus. impfung veterinärimpfstoffe stehen zur verfügung, sollen jedoch nur im notfall bei einem seuchenausbruch zum schutz der tiere in der weiteren umgebung eingesetzt werden. eine flächendeckende impfprophylaxe gibt es in der eu nicht. z gutachterliche bewertung gefährdet sind tierärzte, tierpfleger, melker, milchhändler, fleischer, aber auch verbraucher von unbehandelten tierprodukten. das masernvirus ist ein ausschließlich humanpathogenes rna-virus des genus morbillivirus in der familie der paramyxoviren. prognose und verlauf die prognose der subakuten sklerosierenden panenzephalitis (sspe) ist stets infaust. die masernerkrankung hinterlässt eine lebenslange immunität. die riesenzellpneumonie und die maserneinschlusskörperenzephalitis gehen mit einer letalität von ca. 30 % einher. auf etwa 10.000-20.000 masernerkrankungen entfällt eine erkrankung mit letalem ausgang. nach angaben des statistischen bundesamtes gab es seit 1998 pro jahr 1-2 masernsterbefälle (4 sterbefälle 1999). polioviren sind kleine sphärische unbehüllte rna-viren, die dem genus enterovirus und der familie der picornaviridae angehören. epidemiologie polioviren waren weltweit verbreitet. vor der einführung der oralen impfung war die verbreitung auch in mitteleuropa so ausgeprägt, dass der kontakt mit dem erreger meist schon im kindesalter erfolgte ("kinderlähmung" -paralytische poliomyelitis: rasche entwicklung asymmetrischer, schlaffer lähmungen der bein-(am häufigsten), arm-, bauch-, thorax-oder augenmuskeln. eine spezifische therapie gibt es nicht. nach der akuten behandlung ist eine längere physiotherapeutische, orthopädische nachsorge erforderlich. verlauf die letalität liegt unter 1 %. infektionen beim erwachsenen verlaufen schwerer als bei kindern. nach paralytischer poliomyelitis kann es noch nach jahrzehnten zu einer zunahme der paralysen mit muskelschwund kommen (post-polio-syndrom). präventivmaßnahmen grundimmunisierung aller säuglinge nach den empfehlungen der stiko mit der inaktivierten poliovakzine, die sicher wirksam ist, keine vakzine-assoziierte paralytische poliomyelitis hervorruft und auch bei immunschwäche risikolos ist. bei einer poliomyelitiserkrankung sollten alle kontaktpersonen unabhängig vom impfstatus ohne zeitverzug mit der inaktivierten polio-vakzine geimpft werden. polioverdachtsfälle müssen isoliert stationär behandelt werden. die bovine spongyforme enzephalopathie (bse) gehört zur gruppe der übertragbaren, chronisch degenerativen, spongyunverträglichkeiten ab und müssen individuell festgelegt werden. wichtig ist, dass die prophylaktischen medikamenteneinnahme bereits vor der reise begonnen und eine bestimmte zeit nach der rückkehr fortgeführt wird. in gebieten mit niedrigem oder mittlerem malariarisiko kann ggf. auf eine chemoprophylaxe verzichtet und stattdessen ein medikament als notfallmedikation mitgenommen werden. detaillierte aktuelle angaben zur chemoprophylaxe sind den empfehlungen der gesellschaft für tropenmedizin und internationale gesundheit zu entnehmen (▶ www.dtg.mwn.de). eine impfung steht derzeit nicht zur verfügung, der impfstoff rts,s/a02a, mit dem das risiko einer schweren malariaerkrankung um 58 % reduziert werden konnte, befindet sich noch im experimentellen stadium (agnandji et al. 2011). unter der schlafkrankheit werden die im tropischen afrika vorkommenden infektionen des menschen durch trypanosoma brucei gambiense und trypanosoma brucei rhodesiense verstanden. z epidemiologie, übertragung beide infektionen werden durch tse-tse-fliegen der gattung glossina übertragen und kommen in einem gürtel vor, der südlich der sahara bis nördlich der kalahrai-wüste reicht. z diagnose, therapie die erreger sind nur während der akuten infektion im blut nachweisbar, in der regel erfolgt die diagnose serologisch. in der therapie werden benznidazol und nifurtimox eingesetzt. die toxoplasmose wird durch eine infektion mit toxoplasma gondii bedingt. z diagnostik ein amöbenleberabszess kann in zusammenhang mit einer amöbenkolitis durch eine hämatogene verschleppung auftreten. in der regel geben die patienten bei diagnose aber keine durchfälle an und können sich häufig nicht an eine durchfallepisode erinnern. klinisch stehen fieber und rechtsseitige oberbauchschmerzen im vordergrund. der abszessnachweis erfolgt in der regel mittels bildgebender verfahren wie ultraschall oder computertomographie. in der diagnostik ist es wichtig, zwischen e. histolytica und apathogenen amöbenformen wie e. dispar zu unterscheiden. dieses wird durch fehlende morphologische unterschiede der zystenformen der beiden erreger erschwert. beweisend für eine infektion mit e. histolytica ist der nachweis hämatophager trophozoiten im frischen stuhl. die sensitivität der untersuchung beträgt jedoch bestenfalls 70 %. es werden daher vermehrt antigennachweise mit deutlich höherer sensitivität eingesetzt. bei einem amöbenabszess lassen sich häufig keine erreger im stuhl nachweisen, jedoch ist die serologie fast immer positiv. z therapie jede infektion durch e. histolytica ist behandlungsbedürftig. in der therapie werden bei asymptomatischen trägern paramomycin, bei invasiven erkrankungen metronidazol, gefolgt von paramomycin eingesetzt. der flagellat giardia lamblia ist weltweit verbreitet. eine infektion erfolgt in der regel über die aufnahme von zysten in verunreinigtem wasser oder lebensmitteln, eine fäkal-orale übertragung zum beispiel in kindertagesstätten ist ebenfalls möglich. nach aufnahme besiedelt der erreger den dünndarm. die die diagnose erfolgt in der regel anhand des trophozoiten-oder zystennachweises im stuhl, ggf. können duodenalasin seltenen fällen können frei lebende amöben wie naegleria oder acanthamoeba in das zentrale nervensystem eindringen und eine meningoenzephalitis hervorrufen. acanthamöben können zusätzlich insbesondere bei kontaktlinsenträgern eine keratitis hervorrufen. die erreger kommen in süßwassergewässern vor, die infektion erfolgt beim baden. 1997 wurden weltweit 179 fälle von durch frei lebende amöben bedingte meningoenzephalitiden registriert, die hälfte davon in den usa. der vor allem in schweinen nachweisbare erreger balantidium coli kann selten beim menschen eine akute durchfallerkrankung hervorrufen, die der amöbenruhr ähneln kann. häufig scheint die infektion aber symptomlos zu verlaufen. der erreger kommt weltweit vor, eine infektion erfolgt in der regel über verunreinigtes wasser oder lebensmittel. in manchen geographischen bereichen besteht ein so massiver wurmbefall der bevölkerung, dass für dort zum beispiel aus beruflichen gründen lebende europäer eine besondere infektionsgefährdung gegeben ist. die übertragung von askariseiern auf den menschen kommt gewöhnlich durch mit kot gedüngte, roh genossene gemüse oder salate oder auch durch verunreinigte nahrungsmittel zustande. die oral aufgenommenen eier entlassen im dünndarm die larven, welche die darmwand durchdringen und für ihre weitere entwicklung eines gewebsaufenthaltes bedürfen. in der lunge führen sie zum so genannten eosinophilen lungenfiltrat, welches meist um den 10. tag nach dem eindringen der eier auftritt und etwa nach einer woche, also etwa 3 wochen nach der infektion, sein maximum erreicht, um danach abzuklingen. die diagnostische erkennung der natur eines solchen lungeninfiltrates kann äußerst schwierig sein, weil der nachweis junger askariden im stuhl erst 5-6 wochen später möglich ist. auch askariseier treten erstmalig etwa 8-10 wochen nach einem solchen lungeninfiltrat auf (. abb. 27.9). symptomatik askariden können besonders bei ihrer vielzahl im darm zu ileusartigen krankheitsbildern und mit dem eindringen in den appendix, in den ductus choledochus oder pancreaticus zu entsprechender schwerer symptomatik führen. die infektion mit trichocephalus dispar ist besonders in feuchtwarmen ländern sehr häufig, aber auch in europa und besonders bei kindern nicht selten. nur gelegentlich kommt es zu katarrhalischen erscheinungen vonseiten des darmes, sehr selten auch zu heftigeren symptomen und unter umständen zu allergischen reaktionen. der befall durch ancylostoma duodenale in europäischen ländern, durch necator americanus und seltener durch ancylostoma brasiliense in nord-und südamerika ist besonders in tropischen und subtropischen gebieten weit verbreitet, weil die entwicklung der hakenwurmlarven entsprechende klimatische bedingungen voraussetzt. die mit galle und stuhl ausgeschiedenen eier infizieren schnecken, die sich darin entwickelnden zerkarien befallen fische, deren roher oder unzureichend gekochter genuss die infektion auf den menschen überträgt. der erreger fasciolopsis buski kommt in china, indonesien, vietnam, malaysia und indien bei schweinen und hunden, aber auch beim menschen vor, der sich nach einem ähnlichen entwicklungszyklus des erregers wie bei den schon erwähnten trematodeninfektionen durch den genuss bestimmter wasserpflanzen infiziert, an denen die zysten der zerkarien haften. bei massiven infektionen kommt es zu durchfällen und darmblutungen aus abszessen der darmschleimhaut. der lungenegel paragonimus westermani kommt in china, japan, korea und auf den philippinen, aber auch in westafrika bei hunden, schweinen und wildtieren vor. die eier gelangen mit dem sputum der tiere ins wasser, die daraus freigesetzten mirazidien infizieren schnecken, die sich entwickelnden zerkarien befallen krebse und fische, deren roher genuss den menschen infiziert. aus dem magen-darm-kanal des menschen wandern die larven in die lungen. chronischer husten, blutiger auswurf, gelegentlich auch durchfälle und lebervergrößerung sind die folgen. z gutachterliche bewertung wegen der weiten verbreitung und des großen infektionsrisikos spielt die bilharziose für den ärztlichen gutachter keine geringe rolle. kontakt mit infiziertem wasser bei der beruflichen tätigkeit oder beim baden ist in entsprechenden gebieten häufig gegeben. fasciola hepatica kommt als parasit in den gallenwegen von rindern und schafen überall in der welt vor. mit dem kot gelangen die eier in wasser. die sich daraus bei günstiger temperatur innerhalb einiger wochen entwickelnden mirazidien befallen wasserschnecken, in denen sie einen entwicklungsgang durchmachen. die die schnecke verlassenden zerkarien bilden an wasserpflanzen zysten, durch deren genuss sich der mensch infizieren kann. nach einer inkubationszeit von 1-2 monaten entsteht ein fieberhaftes syndrom, oft mit schmerzen im leberbereich, eosinophilie, leukozytose, leber-und milzvergrößerung. eitrig-septische entzündungen der gallenwege mit cholestase führen zur gelbsucht, zu ausgeprägter anämie und gelegentlich zu einer schweren septischen cholangitis mit tödlichem verlauf. auch eine biliäre zirrhose kann sich entwickeln. z gutachterliche bewertung neben dem krankheitsbild bei bestehender beruflicher exposition erlaubt der nachweis der eier im duodenalsaft und im stuhl die diagnose. eine positive komplementbindungsreaktion vermag sie zu erhärten. infektionsgefährdet sind landarbeiter und andere personen in gebieten mit infiziertem viehbestand. auch durch genuss von sauerampfer oder fallobst von feuchten wiesen, auf denen infiziertes vieh weidete, kann die infektion übertragen werden. wie bei der infektion durch fasciola hepatica, so führt auch die durch den ostasiatischen leberegel, clonorchis sinensis, zu einer leber-und gallenwegssymptomatik. in ostasien sind hunde und katzen, nicht selten auch der mensch infiziert. infektionen mit dibothriocephalus latus (syn. diphyllobothrium latum), dem sogenannten fischbandwurm, werden in den ländern des ostseeraumes, aber auch in den schweizer und norditalienischen seengebieten, im bereich des donaudeltas, in sibirien, japan, nordmandschurei und in nordamerika beobachtet. das ins wasser gelangte ei entwickelt sich zu einer larve, die von niederen krebsen gefressen wird, in denen sich ein weiteres larvenstadium entwickelt. wenn diese krebse von fischen gefressen werden, so wandern die larven in organe und muskulatur des fisches. der genuss rohen fischfleisches führt beim menschen zur infektion, die symptomlos sein, aber auch mit durchfällen und allgemeinsymptomen einhergehen kann. oft kommt es zu einer megalozytären anämie, weil der parasit das vitamin b 12 aufnimmt, ehe es zur resorption gelangen kann. die diagnose ist durch den nachweis der eier im stuhl zu stellen. infektionsgefährdet sind in entsprechenden gebieten menschen, die infizierte fische roh essen. bandwurmarten, die vorzugsweise in warmen ländern vorkommen und den menschen befallen, sind der zwergbandwurm, hymenolepis nana, und sparganum, der zur gattung diphyllobothrium gehört und im dünndarm von hunden und anderen karnivoren zum bandwurm ausreift. der zwergbandwurm bedarf keines tierischen zwischenwirtes, der mensch ist zwischenwirt und endwirt. die eier gelangen durch kotverschmutzung oder selbstinfektion in den magen-darm-kanal, die im dünndarm ausschlüpfenden larven entwickeln sich in die meisten bakteriellen infektionen können hier unberücksichtigt bleiben, weil ihre krankheitsbilder als heimische infektionskrankheiten gut bekannt sind und sie keine besonderen versicherungsrechtlichen oder gutachtlichen probleme aufwerfen. einige besonderheiten sind aber den hier im folgenden dargestellten bakteriellen infektionskrankheiten zu berücksichtigen. bakterielle infektionen im zusammenhang mit berufs-oder urlaubsreisen werden zunehmend häufiger. an erster stelle stehen infektionen des gastrointestinaltraktes, gefolgt von hautinfektionen und infektionen, die in tropischen oder subtropischen regionen durch insekten übertragen werden. der antikörpernachweis (agglutination nach widal) ist von untergeordneter bedeutung. für die antibiotische therapie werden vor allem chinolone und cephalosporine der dritten generation (ceftriaxon, cefotaxim) eingesetzt, wobei weltweit eine zunehmende resistenzentwicklung zu verzeichnen ist. der klinische verlauf ist bei typhus und paratyphus ähnlich, jedoch bei paratyphus meist leichter ausgeprägt. z gutachterliche bewertung für die gutachterliche beurteilung ist es für medizinisches personal und für personen, die im lebensmittelgewerbe tätig sind, von großer bedeutung, ob die erreger nach ausheilung der erkrankung im stuhl nicht mehr nachweisbar sind. bei etwa 2-5 % der infektionen mit salmonella typhi kommt es zu bakteriendauerausscheidung, die ein hohes infektionsrisiko darstellt. bei diesen in der regel beschwerdefreien personen persistieren die erreger häufig in der gallenblase. eine antibiotische sanierung von dauerausscheidern kann versucht (z. b. ciprofloxacin für 4 wochen) und ggf. eine cholezystektomie unter antibiotikaprophylaxe vorgenommen werden. dauerausscheidern kann die ausübung bestimmter beruflicher tätigkeiten ganz oder teilweise untersagt werden ( § 31 infektionsschutzgesetz, ifsg). insbesondere der einsatz in lebensmittelverarbeitenden bereichen ist nach § 42 ifsg verboten. betroffene beschäftigte erhalten eine entschädigung für verdienstausfall ( § 56 ifsg) oder berufswechsel, die kosten einer behandlung zur beseitigung dieses dauerausscheiderzustandes werden in der regel von der zuständigen berufsgenossenschaft, für medizinisches personal von der berufsgenossenschaft für gesundheitsdienst und wohlfahrtspflege, übernommen, wenn die infektion als berufskrankheit anerkannt wurde. die meisten infektionen des rachenraumes sind durch viren verursacht. bei etwa 15 % der fälle, bei kindern bis 50 %, werden β-hämolysierende streptokokken der gruppe a als erreger gefunden. das rheumatische fieber und die akute glomerulonephritis sind seltene spätfolgen von streptokokken-racheninfektionen bestimmter serotypen und können mit einer durchschnittlichen latenz von 18 bzw. 10 tage auftreten. eine genetische disposition scheint das auftreten der spätfolgen zu begünstigen. z gutachterliche bewertung epidemiologische studien haben gezeigt, dass bei engem kontakt von mensch zu mensch wie in kasernen und anderen gemeinschaftseinrichtungen, aber auch bei der beruflichen tätigkeit von medizinischem personal, erheblich größere infektionsgefährdung als bei der übrigen bevölkerung besteht. bei solchen und ähnlichen personengruppen mit erhöhter gefährdung wird daher das rheumatische fieber und seine komplikationen bzw. die glomerulonephritis als berufskrankheit oder bei soldaten als versorgungsleiden anerkannt. listeria monocytogenes, der erreger der listeriose, kommt ubiquitär in der umwelt vor und ist im landschaftlichen bereich weit verbreitet. der erreger wird im kot vieler tierarten gefunden, kann aber auch im stuhl von bis zu 5 % der asymptomatischen bevölkerung nachgewiesen werden. eine infektion die übertragung geschieht durch kontaminiertes trinkwasser oder nahrung. rund 80 % der infektionen verlaufen asymptomatisch. die inkubationszeit ist kurz (18 h bis 5 tage), die erreger werden noch 2-3 wochen nach überstehen der erkrankung im stuhl ausgeschieden. ursächlich für die diarrhöen ist ein von v. cholerae gebildetes enterotoxin. die letalität liegt unbehandelt bei 60 %. die therapie besteht aus oraler oder parenteraler flüssigkeits-, salz-und glucosesubstitution. hierdurch kann die letalität auf unter 1 % gesenkt werden. eine antibiose, in der regel doxycyclin, kann den krankheitsverlauf verkürzen. minierter lebensmittel (u. a. rohmilch und seine produkte, hackfleisch oder salate). bei immunkompetenten personen kommt es selten zu einer klinisch nachweisbaren infektion. bei abwehrgeschwächten personen können schwere verläufe mit meningoenzephalitis und septischem krankheitsbild auftreten. eine infektion während der schwangerschaft kann diaplazentar oder unter der geburt auf das kind übertragen werden und zu einer früh-oder totgeburt führen oder eine schwere erkrankung des neugeborenen hervorrufen. das erysipel durch β-hämolysierende streptokokken der gruppe a (streptococcus pyogenes) hat keine besondere versicherungsrechtliche bedeutung als berufskrankheit. der schweinerotlauf ist eine anthropozoonose. die krankheit kommt vor allem bei schweinen vor, der erreger erysipelothrix rhusiopathiae wird aber auch u. a. bei geflügel, fischen und schalentieren nachgewiesen. eine übertragung auf den menschen erfolgt durch direkten kontakt mit infizierten tieren oder tierprodukten über kleine verletzungen und hautläsionen. in der regel kommt es nach wenigen tagen am infektionsort zu einer lokalen schmerzhaften schwellung, systemische verlaufsformen sind selten. der verfügbare orale impfstoff ist mäßig wirksam, wird aber seit 2010 von der who für regionen mit endemischem auftreten empfohlen. von der stiko wird eine impfung für aufenthalte in infektionsgebiete empfohlen. milzbrand ist eine durch bacillus antracis verursachte zoonose, die vorwiegend bei pflanzenfressenden tieren auftritt. vom erreger gebildete sporen sind sehr widerstandsfähig. infektionen beim menschen sind selten und vorwiegend durch kontakt mit sporenhaltigem material bedingt: unbehandelt beträgt die letalität der beulenpest 50 %, die lungenpest oder pestsepsis ist in fast allen fällen tödlich. therapie der wahl sind streptomycin oder gentamicin. zur oralen therapie wird tetrazyklin eingesetzt. bis zu 60 % der reisenden erkranken während eines aufenthaltes in einem gebiet mit niedrigem hygienestandard an einer akuten durchfallerkrankung. diese treten in der regel in den ersten 2 reisewochen auf und dauern unbehandelt durchschnittlich 4 tage. ursache sind fast immer fäkal kontaminierte speisen oder getränke. das erregerspektrum umfasst am häufigsten enterotoxigene escherichiacoli-stämme sowie salmonellen, campylobacter jejuni und shigellen. daneben spielen auch viren (rotaviren, noroviren) und seltener protozoen (lamblien, amöben) und wurminfektionen eine rolle. die therapie beinhaltet insbesondere eine ausreichende flüssigkeitszufuhr. eine selbsttherapie mit motilitätshemmenden substanzen wie loperamid und antibiotika (am häufigsten gyrasehemmer) kann in den meisten fällen die durchfallintensität und -dauer verkürzen. eine prophylaxe besteht in der beachtung entsprechender hygienischer voraussetzungen ("boil it, cook it, peel it or forget it"). eine antibiotische prophylaxe sollte nur in ausnahmefällen erfolgen. z gutachterliche bewertung die tuberkulose lässt sich mit tuberkulostatika so effektiv und in so kurzer zeit behandeln, dass selbst ausgedehnte und kavernöse prozesse in der mehrzahl rasch ausheilen. meistens besteht bereits wenige wochen nach behandlungsbeginn keine infektiosität mehr. nach spätestens 6 monaten hat sich der röntgenologische befund in den meisten fällen residuenlos oder durch ausbildung produktiv-fibröser narben zurückgebildet. zu beachten sind resistenzen, eine entsprechende testung der resistenzlage ist obligat. nach wirksamer behandlung besteht keine notwendigkeit oder berechtigung mehr, die berufswahl einzuschränken, die aufnahme oder wiederaufnahme der beruflichen tätigkeit oder der ausbildung hinauszuschieben. nach effektiver chemotherapie, rückbildung des radiologischen lungenbefundes und bei konstant negativem bakterienbefund im sputum sollte der kranke als geheilt und arbeitsfähig betrachtet werden. für eine berentung oder für weitere rehabilitationsmaßnahmen besteht dann kein anlass mehr, abgesehen von fällen, in denen noch eine auf die krankheitsfolgen zurückzuführende leistungseinschränkung vorliegt. dies trifft für solche kranke zu, bei denen es zu einer funktionellen und anatomischen defektheilung gekommen ist. aufgrund des häufig gleichzeitigen auftretens von hiv-und tbc-infektion sollte gerade bei jüngeren patienten immer auf hiv getestet werden. in der regel ist eine rentenberechtigende mde nach komplikationslos abgelaufener vernarbter tuberkulose nicht mehr anzunehmen. in seltenen anderen fällen ergibt sich eine mde oder behinderung (gdb) aus dem ausmaß der bleibenden funktionsstörungen vonseiten der atmung, aus begleit-und folgekrankheiten und der auswirkung auf das herz und den kreislauf. das ausmaß der funktionellen einschränkung muss durch eine lungenfunktionsprüfung bzw. durch zeichen der rechtsherzbelastung objektiviert werden. die aktive, ansteckungsfähige lungentuberkulose bedingt arbeitsunfähigkeit. nach behandlungsbeginn ist z. b. im versorgungsrecht und im unfallversicherungsrecht eine mde von 100 % (bzw. ein gdb von 100) nicht mehr bzw. nur noch kurzfristig zum beispiel bis zum ende einer stationären therapie gerechtfertigt bzw. nur so lange anzunehmen, wie die gefahr eines wiederaufflackerns besteht. danach ist der kranke wieder arbeitsfähig. für die zeit weitergeführter chemotherapie, die das allgemeinbefinden beeinträchtigen kann, beträgt die mde für die dauer der medikation etwa 20 % und richtet sich weiterhin nach der funktionellen beeinträchtigung der atmung und des herz-kreislauf-systems. längere inaktivität des krankheitsprozesses rechtfertigt die reduzierung der mde bzw. des behinderungsgrades. der verbesserten prognose der tuberkulose, der kürzeren krankheitsdauer und der fast regelhaften heilung mit geringeren residuen muss in der höhe der mde und in der zeitlichen dauer der minderung der erwerbsfähigkeit rechnung getragen werden. eine impfung gegen tuberkulose (bcg, bacille calmette-guérin) wird nicht mehr empfohlen. z gutachterliche bewertung versorgungsansprüche aufgrund eines impfschadens (ifsg § 60) bestehen nur bei von den ländern öffentlich empfohleschüler und studenten sowie kinder in kindergärten sind unter den schutz der gesetzlichen unfallversicherung gestellt. dieser versicherungsschutz erstreckt sich z. b. auf den zeitraum des studiums, aber auch auf damit im zusammenhang stehende veranstaltungen. bei wehrdienstleistenden soldaten gilt für die anerkennung einer primärtuberkulose als wehrdienstbeschädigung, dass der nachweis der infektionsquelle nicht unbedingt erforderlich ist. es genügt, dass die tuberkulose während der zeit der ableistung des wehrdienstes erworben wurde. schutzimpfungen und medikamentöse behandlungsmaßnahmen haben den sinn, infektionskrankheiten vorzubeugen, ihren verlauf abzuschwächen oder zu unterbrechen. werden solche maßnahmen im zusammenhang mit versicherten tätigkeiten durchgeführt, gelten ihre unerwünschten nebenwirkungen als unfälle oder mittelbare unfallfolgen. impfungen stellen zweifelsohne eine der wichtigsten und effektivsten präventiven maßnahmen in der medizin dar. so ist es der pockenimpfung zu verdanken, dass pocken weltweit eliminiert werden konnten. seit aufhebung der pockenschutzimpfung 1976 gibt es in deutschland anders als in anderen ländern keine impfpflicht mehr. die ständige impfkommission (stiko ) am robert koch-institut (▶ www.rki.de) in berlin gibt jährlich aktualisierte impfempfehlungen heraus. impfungen von besonderer bedeutung für die gesundheit der bevölkerung werden von den obersten gesundheitsbehörden der länder auf der grundlage der stiko entsprechend § 20 abs. 3 des infektionsschutzgesetzes (ifsg) empfohlen. folgende standardimpfungen werden derzeit öffentlich empfohlen: -tetanus: impfung mit 2, 3, 4 und 12 monaten, auffrischung alle 10 jahre, 27.6 • infektionsprävention nen impfungen. impfschäden durch schutzimpfungen, die im zusammenhang mit einer beruflichen tätigkeit erforderlich wurden, werden jedoch durch die zuständige berufsgenossenschaft wie ein unfall entschädigt. in der regel sind die von der stiko empfohlenen impfungen gut verträglich. der impfende arzt hat die pflicht, über mögliche nebenwirkungen der jeweiligen impfungen aufzuklären. generell gilt, dass nach impfungen selbstlimitierende lokal-und allgemeinreaktionen auftreten können. folgende zusätzliche komplikationen nach impfungen können vorkommen: in . tab. 27.17 sind die meldepflichtigen krankheiten zusammengestellt. bestimmte bestehende krankheiten oder befindlichkeitsstörungen können unter tropischen klimatischen bedingungen probleme oder gefährdungen bedeuten. es gibt aber nur wenige offensichtliche gründe gegen einen beruflichen oder privaten aufenthalt in tropischen ländern. in der bundesrepublik deutschland sind gewerbliche unternehmen nach dem arbeitsmedizinischen grundsatz g 35 verpflichtet, ihre mitarbeiter vor und nach einem beruflichen auslandsaufenthalt durch einen von den berufsgenossenschaften hierzu ermächtigten arzt untersuchen zu lassen. eine tropentauglichkeitsuntersuchung unterscheidet sich nicht von einer eingehenden internistischen untersuchung einschließlich der erforderlichen anamnese. wichtig ist jedoch eine ausführliche beratung über lebensweise und krankheitsverhütung in tropischen ländern sowie erforderliche impfungen und ggf. eine malariaprophylaxe. bei physisch und psychisch gesunden und körperlich leistungsfähigen personen gibt es keinen zweifel an der tropentauglichkeit. die beurteilung bestehender gesundheitsstörungen kann allerdings eine schwierige aufgabe sein, weil der arzt eine prognose hinsichtlich der zu erwartenden anforderungen und risiken zu stellen hat. nicht geeignet für den aufenthalt in tropischen oder subtropischen ländern sind patienten mit krankheiten, die ständige ärztliche überwachung und behandlung erfordern, insbesondere wenn mit ihrem fortschreiten oder mit plötzlichen komplikationen zu rechnen ist. akute krankheiten müssen ausgeheilt sein. eine genaue auflistung der in frage kommenden krankheiten ist an dieser stelle nicht möglich. das urteil gegen einen aufenthalt in den tropen kann grundsätzlich und andauernd oder befristet sein. aufgabe der nach g 35 erforderlichen tropenrückkehruntersuchung ist es, erworbene infektionen oder andere krankheiten festzustellen. die untersuchung hat spätestens 8 wochen nach beendigung eines auslandsaufenthaltes von mehr als einem jahr dauer (u. u. auch bei kürzerer dauer) zu erfolgen. bei fortdauernden auslandsaufenthalten sind nachuntersuchungen im abstand von 2-3 jahren vorgeschrieben. die nachuntersuchung beinhaltet neben einer ausführlichen internistischen untersuchung parasitologische und bakteriologische stuhluntersuchungen. weitere untersuchungen können sich aus der vorgeschichte oder aus bestehenden beschwerden ergeben. -varizellen-impfstoff: sehr selten allergische reaktionen, einzelfälle von anaphylaktischem schock, herpes zoster und pneumonie. ein zusammenhang gewisser impfungen wie hepatitis b oder mmr mit bestimmten erkrankungen wie multipler sklerose oder autismus konnte wissenschaftlich nicht bewiesen werden. ein nachgewiesener, wenn auch seltener zusammenhang besteht jedoch z. b. zwischen der oralen poliomyelitisimpfung und einer polioerkrankung entweder des geimpften oder seiner unmittelbaren umgebung. hier besteht für den betroffenen ein anspruch auf entschädigung. 1998 wurde der orale lebendimpfstoff in deutschland aufgrund dieser möglichen nebenwirkung durch einen inaktivierten impfstoff ersetzt, bei dem durch die impfung keine poliomyelitis ausgelöst werden kann. die stiko gibt ebenfalls empfehlungen zur indikation und durchführung einer postexpositionsprophylaxe. diese kann in der gabe von immunglobulinen oder antibiotika bestehen: durch die postexpositionsprophylaxe sind in seltenen fällen gesundheitsschäden möglich. hier gelten dieselben versorgungsansprüche wie für impfungen. für weitere einzelheiten zu impfschäden und deren zuständigkeiten siehe ifsg § 60§ 68. das bundesseuchengesetz wurde am 1. country-wide hiv incidence study complementing hiv surveillance in germany two drugs or three? balancing efficacy, toxicity and resistance in postexposure prophylaxis for occupational exposure to hiv hiv-infizierte ärzte: ein ethisches dilemma hiv-1 infection transmitted by serum droplets into the eye: a case report use of laboratory tests and clinical symptoms for identification of primary hiv infection berufsbedingte hiv-infektion durch blutspritzer ins auge berufsbedingte hiv-infektion bei einer krankenschwester und ihrem kind operationen bei hiv-patienten: gefahren und indikationen. wie hoch ist das infektionsrisiko für den chirurgen? plasmaviral load and cd4*lymphocytes as prognostic markers of hiv-1 infection hiv-infektion als berufskrankheit bei einer krankenhausärztin zur problematik der nosokomialen übertragung von hiv berufsbedingte hiv-infektionen bei medizinischem personal zum verlauf der hiv-epidemie in deutschland bis ende postexpositionelle prophylaxe der hiv-infektion. deutsch-österreichische empfehlungen deutsch-österreichische leitlinien zur antiretroviralen therapie der hiv-1-infektion stand 04 hiv-infektion durch nadelstichverletzung (letter) report on the global aids epidemic 2010 failure of therapeutic coma and ketamine for therapy of human rabies tollwutschutzimpfung. in: spiess h, heininger u (hrsg) impfkompendium. thieme epidemiology of bat rabies in germany fledermaustollwut -infektionsgefahr auch in deutschland informationen zu den tollwutübertragungen durch spendeorgane rki (stand 07/2010) empfehlungen der ständigen impfkommission (stiko) am robert koch-institut tollwut in deutschland: gelöstes problem oder versteckte gefahr? konsensuspapier zur tollwutimpfung für reisende rabies and other lyssavirus diseases survival after treatment of rabies with induction of coma and investigations of west nile virus infections in recipients of blood transfusion and organ transplantation transmission of west nile virus from an organ donor to four transplant recipients österreichische gesellschaft für antimikrobielle chemotherapie (hrsg) infektionsnetz österreich ongoing outbreak of west nile virus infections in humans in greece west-nil-fieber: erster importierter erkrankungsfall in deutschland zu einem ausbruch von chikungunya-fieber in italien arboviren -durch arthropoden übertragbare viren: stellungnahme des arbeitskreises blut des bundesministeriums für gesundheit und soziale sicherung control of communicable diseases manual frühsommer-meningoencephalitis. prognose für kinder und jugendliche günstiger als für erwachsene rki (2010) fsme: risikogebiete in deutschland kapitel 27 • infektionskrankheiten 686 world/bse-specific-data/number-of-reported-cases-worldwideexcluding-the-united-kingdom paul-ehrlich-institut (pei) und bundesinstitut für arneimittel und medizinprodukte (bfarm) (2001) die bovine spongyforme enzephalopathie (bse) des rindes und deren übertragbarkeit auf den menschen creutzfeldt-jakob-erkrankungen in den jahren influenza a (h1n1)v in germany: the first 10,000 cases neuerungen in den aktuellen impfempfehlungen der stiko komplikationen von vzv-infektionen und einschätzung des robert koch-instituts zur aktuellen situation der vogelgrippe (aviäre influenza) impfung gegen die neue influenza a (h1n1) rückblick: epidemiologie und infektionsschutz im zeitlichen verlauf der influenzapandemie (h1n1) 2009 who (2006) information on infectious diseases pandemic influenza a (h1n1) 2009 breakthrough infections and estimates of vaccine effectiveness in germany british society for medical mycology proposed standards of care for patients with invasive fungal infections epidemiology of nosocomial fungal infections, with emphasis on candida species changing epidemiology of systemic fungal infections pathogenetische bedeutung der intestinalen candidabesiedelung. mitteilung der kommission "methoden und qualitätssicherung in der umweltmedizin fungal brain infections updates on the epidemiology of dermatophyte infections endemic mycoses in aids: a clinical review first results of phase 3 trial of rts,s/as01 malaria vaccine in african children the trypanosomiases rift-valley-fieber: ausbruch in den nordöstlichen und den küstenprovinzen kenias travelers' health: yellow book. health information for international travel tropenmedizin in klinik und praxis. thieme gelbfieber: übersicht, bericht über eine importierte erkrankung falldefinitionen des robert koch-instituts zur übermittlung von erkrankungs-oder todesfällen und nachweisen von krankheitserregern impfempfehlungen der ständigen impfkommission (stiko) am robert koch-institut, stand juli durchimpfungsgrad bei der schuleingangsuntersuchung in deutschland masern-eliminierung in deutschland -weitere verstärkte anstrengungen erforderlich zum impfschutz bei aufnahme in den kindergarten in schleswig-holstein im jahr zu 27 principles and practice of infectious diseases letzte vakzine-assoziierte poliomyelitis in deutschland globale polioeradikation -zwischen bangen und zuversicht zum welt-poliotag 2010: wieder polofälle in der who-region europa variant creutzfedt-jakob disease, current data possible transmission of variant creutzfeldt-jakob disease by blood transfusion oie: number of cases of bovine spongiform encephalopathy (bse) in the united kingdom oie: number of cases of bovine spongiform encephalopathy (bse) in farmed cattle worldwide: www.oie.int/animal-health-in-the prophylaxe und therapie der malaria in der merkblätter für ärzte: malaria 95 infektionsepidemiologisches jahrbuch meldepflichtiger krankheiten für reiseassoziierte infektionskrankheiten im jahr world malaria report. rbm.who.int/wmr2005/ html/ map1 zur diagnose von wurmbefall sleisenger and fordtrans' gastrointestinal and liver disease, 7 die lyme-arthritis clostridium difficile -more difficult than ever principles and practice of infectious diseases ratgeber: tularämie, hasenpest (francisella tularensis) merkblätter für ärzte: typhus ratgeber infektionskrankheiten: scharlach und andere infektionen durch streptococcus pyogenes merkblätter für ärzte: clostridium difficile reiseassoziierte infektionskrankheiten im jahr illness after international travel who (2010) cholera principles and practice of infectious diseases ratgeber infektionskrankheiten reiseassoziierte infektionskrankheiten im jahr infektionsepidemiologisches jahrbuch für lyme borreliosis principles and practice of infectious diseases ticks and tickborne bacterial disease in humans: an emerging infectious threat rickettsioses as paradigms of new or emerging infectious diseases neu und vermehrt auftretende infektionskrankheiten merkblätter für ärzte: q-fieber infektionsepidemiologisches jahrbuch meldepflichtiger krankheiten für klinische infektiologie chlamydia-psittaci-infektionen/ornithose ausgehend von einer geflügelschlachterei aktuelle statistik meldepflichtiger infektionkrankheiten tuberkulose als berufskrankheit, 2. aufl. ecomed bericht zur epidemiologie der tuberkulose in deutschland für hinweise für ärzte zum aufklärungsbedarf bei schutzimpfungen empfehlungen der ständigen impfkommission (stiko) am robert koch-institut kapitel 27 • infektionskrankheiten 688 key: cord-016041-427mbaqc authors: hengge, ulrich r. title: gentherapie date: 2008 journal: grundlagen der molekularen medizin doi: 10.1007/978-3-540-69414-4_16 sha: doc_id: 16041 cord_uid: 427mbaqc die gentherapie ist eine junge wissenschaft, die nukleinsäuren zur therapie einsetzt (hengge u. bardenheuer 2004). die somatische gentherapie befasst sich mit der behandlung von somatischen (körper-)zellen (⧁ tab. 4.1.1), wobei das therapeutische gen ein im organismus benötigtes protein kodiert. die gentherapie ist eine junge wissenschaft, die nukleinsäuren zur therapie einsetzt (hengge u. bardenheuer 2004) . die somatische gentherapie befasst sich mit der behandlung von somatischen (körper-)zellen (>tab. 4.1.1), wobei das therapeutische gen ein im organismus benötigtes protein kodiert. die erste klinische gentherapie wurde 1989 von rosenberg et al. (1990) durchgeführt, bei der ein retrovirus zur transduktion tumorinfiltrierender lymphozyten verwendet wurde, der für ein neomycin-markergen kodierte. hierdurch konnte das t-zell-infiltrat in melanomen über die zeit verfolgt werden. die erste therapeutische studie wurde an kindern mit adenosindeaminase-immundefektsyndrom (ada-scid) anfang der 1990er jahre erfolgreich durchgeführt (blaese et al. 1995; muul et al. 2003) . mittlerweile wurden weltweit mehr als 1.000 klinische gentherapiestudien mit über 10.000 patienten durchgeführt. eine aktuelle internationale internetdatenbank findet sich unter http://www.wiley.co.uk/ wileychi/genmed/clinical. grundsätzlich sind verschiedene aspekte zur strategie bzw. zur wahl des gentaxis (vektors) zu bedenken ( > tab. 4.1.2). bezüglich der strategie lassen sich in-vivo-von ex-vivo-methoden unterscheiden ( > abb. 4.1.1). bei der in-vivo-strategie wird genetisches material direkt in körperzellen transferiert. die therapeutische dna oder rna kann entweder direkt z. b. durch injektion oder elektroporation oder indirekt mittels liposomaler bzw. viraler vektoren in die zielzellen eingebracht werden. die hier in zielzellen bzw. gewebe eingebrachten nichtviralen vektoren werden durch die anwesenheit von nukleasen innerhalb weniger tage degradiert (hengge et al. 2001) . bei der ex-vivo-methode werden körperzellen entnommen, und der gentransfer erfolgt in kultur. nach entsprechender proliferation des gewünschten genkorrigierten gewebes lässt sich das transplantat dem wirt zurückgeben (z. b. modifizierte fibroblasten oder endothelzellen) ( > abb. 4.1.1). generell kann die erforderliche erbsubstanz (gen) mittels chemischer, physikalischer oder biologischer methoden ( > tab. 4.1.3) in die verschiedenen zelltypen bzw. organe eingebracht werden. je nach der beabsichtigten anwendung ist eine kurzzeitige herstellung des genprodukts (z. b. bei der dna-vakzinierung oder zur abheilung chronischer wunden) bzw. eine langzeit-oder dauersynthese (z. b. bei angeborenen stoffwechselkrankheiten) wünschenswert. nichtvirale vektoren nichtvirale vektoren führen zur transienten expression des kodierten genprodukts. neben liposomal, d. h. durch kationische lipide komplexierter plasmid-dna kann diese auch durch elektroporation (mittels elektrischer pulse) bzw. durch ultraschallbehandlung (transiente ruptur der zellmembranen) sowie auch durch direkte injektion sog. nackter plasmid-dna appliziert werden. während nahezu alle gewebe mittlerweile mit-tels nichtviraler vektoren transfiziert wurden, bietet die haut die möglichkeit der leichten zugänglichkeit und stellt ein potentes immunorgan dar. von unserer arbeitsgruppe wurde daher die methode der direkten in-vivo-injektion nackter dna, d. h. ohne virale vektoren, mit einer insulinnadel und tuberkulinspritze entwickelt (> abb. 4.1.2) (hengge et al. 1995 (hengge et al. , 1996 . hierbei wird plasmid-dna in die obere dermis unter aufbau eines hydrostatischen drucks injiziert und nach aufnahme transient für einige tage exprimiert (> abb. 4.1.3a-d) (hengge et al. 1995 (hengge et al. , 1996 . schon nach wenigen stunden kann das hergestellte protein (z. b. -galaktosidase) vorwiegend in den keratinozyten der epidermis nachgewiesen werden ( > abb. 4.1.3a-d). das entsprechende protein wird für 3 bis maximal 7 tage nach injektion der korrespondierenden dna synthetisiert. eine integration der zugeführten dna in das chromosomale erbgut findet nicht statt, was die sicherheit der methode . (hengge et al. 2001) . seit kurzem können wir die topische applikation nackter dna in form von sprayliposomen erfolgreich anwenden (meykadeh et al. 2005) . der intrakutane transport-und aufnahmemechanismus von dna in keratinozyten scheint an eine aktive proteinsynthese sowie an die existenz von dnabindenden proteinen (z. b. ezrin und moesin) gekoppelt zu sein (basner-tschakarjan et al. 2004) . im gegensatz zu den häufig in der gentherapie eingesetzten viralen vektoren lässt sich die "nackte" dna beliebig oft erneut in der haut exprimieren; es kommt hier nicht zum auftreten unerwünschter immunreaktionen, die bei wiederholter applikation die genexpression verhindern könnten. sog. lipoplexe oder molekulare konjugate führen zur komprimierung der dna und machen sie somit leichter aufnahmefähig (chen u. huang 2005) . diese polyplexe genannten kombinationen z. b. aus polyethylenimin und dna können auch systemisch verabreicht werden (erbacher et al. 2004) . gegebenenfalls werden die kleinen partikel mit spezifischen liganden, z. b. transferrin, ausgestattet, was ein selektiveres targeting an zielzellen ermöglicht (kloeckner et al. 2004 ). um die dna-aufnahme in den gewünschten zielzellen zu steigern, lassen sich polyplexe mit transferrin oder epidermalem wachstumsfaktor koppeln, was zur rezeptormediierten endozytose in die entsprechenden rezeptortragenden zellen führt (ogris et al. 2003) . diese auch als nanokomplexe bezeichneten molekularen konjugate besitzen eine geringe toxizität und eine gegenüber der klassischen lipofektion vergleichbare transfektionseffizienz, die sich sowohl in vitro als auch in vivo nutzen lässt. die selektivität der genexpression lässt sich durch den einsatz von promotoren erhöhen, die vorzugsweise im zielgewebe aktiv sind. für das maligne melanom zum beispiel sind dies melanozytenspezifische promotoren (z. b. tyrosinase). der melanozytenspezifische promotor (melanozyteninhibitorische aktivität, mia) in adeno-assoziierten virus-(aav-)vektoren zusammen mit dem hsv-thymidinkinase-(tk-)gen zeigte eine spezifische expression in melanomzellen (schoensiegel et al. 2004) . in tierexperimenten induzierte das in diesem konstrukt vorhandene hsv-tk-gen bei nachfolgender therapie mit ganciclovir einen rückgang der melanome (schoensiegel et al. 2004) . konditional replizierende adenovirusvektoren wurden ebenfalls in der therapie des melanoms eingesetzt (liu et al. 2004b) . ein mit einem targeting-peptid (rgd) gekoppelter adenovirusvektor, der das adenovirale e1a-protein selektiv in tyrosinasepositiven melanomzelllinien exprimierte, führte zu einer höheren transduktion von melanomzel-len. nach intratumoraler injektion zeigte sich in einem melanom-xenotransplantationsmodell darüber hinaus eine tumorregression (liu et al. 2004b) . auch in eigenen untersuchungen des menschlichen blm-melanoms führten trunkierte adenovirale e1a-proteine unter der kontrolle des humanen telomerase-promotors zur tumorgerichteten expression und regression von melanommetastasen im tiermodell (kirch et al. 2002) . in ähnlicher weise lassen sich leberspezifische, muskelspezifische oder neuronenspezifische promotoren einsetzen, um eine präferenzielle expression im erwünschten zielgewebe zu erreichen. neue verfahren des vektor-targetings sowie interessante techniken wie elektroporation und hydrodynamische injektion konnten die transgenexpression in vivo verbessern, indem eine verbesserte verteilung der plasmid-dna im zielorgan erreicht wurde (wolff u. budker 2005) . darüber hinaus wurden verschiedene attenuierte stämme von salmonellen, shigellen, listerien, yersinien und apathogenen e. coli-stämmen als vektoren zum transport von dna in säugetierzellen erfolgreich eingesetzt (vassaux et al. 2006) . viren haben während der evolution die fähigkeit entwickelt, dna oder rna in zellen eines organismus einzuschleusen und der transkriptionsmaschinerie zuzuführen. sie eignen sich daher als hervorragende vehikel für den gentransfer (>tab. 4.1.1). hierbei lassen sich nichtintegrierende [adenoviren (ad) und adeno-assoziierte viren (aav)] von integrierenden viren (z. b. retroviren) unterscheiden. zur einführung in den aufbau und die klonierung wird auf hervorragende übersichtsarbeiten verwiesen (adenoviren: volpers u. kochanek 2004; alba et al. 2005 ; aav: büning et al. 2004; ding et al. 2005; flotte 2005; muzyczka et al. 2005; vasileva u. jessberger 2005; retroviren: naldini u. verma 2000; mangeat u. trono 2005; herpes-virus-vektoren: glorioso u. fink 2004; epstein et al. 2005) . onkolytische viren replizieren in krebszellen mit höherer selektivität als in normalen zellen. indem sie in den zielzellen replizieren, lysieren sie diese. gerade für tumorzellen, bei denen der antivirale interferonsignalweg inaktiviert ist bzw. tumorsuppressorgene mutiert sind, ist in diesen zellen eine ungehinderte virale replikation möglich. vor allem onkolytische adenoviren oder herpes simplex-viren werden zu diesem zweck auch klinisch eingesetzt (galanis et al. 2005 (lin u. nemunaitis 2004; lorence et al. 2003) . eine ungeklärte frage onkolytischer viren mit hoher mutationsfrequenz besteht darin, ob das nach replikation freigesetzte virus mit dem ursprünglich injizierten jeweils identisch ist oder sich weiterentwickelt hat. darüber hinaus sind onkolytische viren hoch immunogen. sie sind deshalb auf eine lokale applikation und auf wenige verabreichungen beschränkt. eine weiterentwicklung der onkolytischen viren ist die ausstattung mit zytokinen, antiangiogenetischen faktoren bzw. immunologisch aktiven molekülen, auch "arming" genannt. ein problem dieser vektoren besteht in der möglichkeit des hervorrufens von autoimmunerkrankungen, z. b. der vitiligo (depigmentierung), die nach vakzinierung von melanompatienten mit einem vacciniavirus-konstrukt aufgetreten ist (kaufman et al. 2005) . darüber hinaus muss die fähigkeit onkolytischer viren zur rekombination zweifelsfrei untersucht und geklärt werden. auch die verschiedenen strategien der immunevasion, z. b. durch expression immunsuppressiver zytokine im zusammenspiel mit dem effektiven transgen, bedarf einer sorgfältigen präklinischen testung. ebenfalls ist das überschreiten der speziesrestriktion eine wesentliche, zu klärende frage, was durch das aviäre influenzavirus (vogelgrippe; h5n1) deutlich wird (chernajovsky et al. 2006) . lentiviren besitzen die wichtige eigenschaft, sowohl proliferierende zellen als auch ruhende zellen effizient zu infizieren und transduzieren. diese eigenschaft zeichnet sie gegenüber klassischen retroviren aus. beiden viren ist die insertion in das genom gemeinsam, was eine weitergabe des genetischen materials an die tochterzellen in sich birgt. um die retrovirale transduktion sicherer zu machen, wurde untersucht, ob ekotrope murine moloney leukämieviren (emmlv) zur transduktion menschlicher hämatopoetischer progenitorzellen eingesetzt werden könnten (yang et al. 2006) . die emmlv waren mit polylysin gekoppelt, um die normalerweise in humanen zellen nicht permissiven viren aufzunehmen. die transduktionsrate der emmlv-pl war gleich derjenigen amphotroper emmlv (mit oder ohne polylysinkopplung). auch lentivirale vektoren werden zur gentherapie eingesetzt. so konnte z. b. mit einem lentivirusvektor (nach pseudotypisierung mit einem sindbis-virus-oberflächenprotein) in einem melanom-mausmodell nach intravenöser injektion eine hohe transduktionsrate im tumor erzielt werden (morizono et al. 2005) . ein wesentlicher fortschritt in der lentiviralen vektortechnologie war die herstellung integrationsdefizienter lentiviraler vektoren, die eine stabile langzeittransduktion ermöglichen (vargas et al. 2004) . ein solcher vektor führte in postmitotischen geweben wie augen und gehirn von nagetieren zur langzeitexpression über mehrere monate (yanez-munoz et al. 2006) . alphaviren besitzen einen breiten tropismus und führen zu hohen konzentrationen an transgen (lundstrom 2005) . aufgrund einer präferenz für neuronale zellen haben alphavirusvektoren eine große beliebtheit in den neurowissenschaften erfahren. darüber hinaus wurden semliki-forest-virusvektoren zur aktivierung gegen viren und tumoren eingesetzt. aufgrund der hohen dichte an lamininrezeptoren auf krebszellen wurden konventionelle sindbis-vektoren zum tumorspezifischen targeting in tiermodellen eingesetzt (lundstrom 2005) . durch die anwendung von nanotechnologie wurden virusartige funktionen auf nanopartikel transferiert, was zur herstellung artifizieller viren führen wird (mastrobattista et al. 2006) . hierbei werden wenige nanometer große komplexe aus nukleinsäuren in kompakter form verwendet, die durch entsprechende virale liganden mit der fähigkeit zur bindung an zelluläre rezeptoren versehen werden. bei der dna-vakzinierung soll ein immunogenes antigen (eines pathogens oder eines tumors) im zielgewebe exprimiert werden und zu einer verbesserung der immunantwort führen. als die gewünschten transgene exprimierende zellen wurden anfangs myozyten oder keratinozyten gewählt. in den letzten jahren hat sich jedoch die transfektion von dendritischen zellen und epidermalen langerhans-zellen (ggf. mit aktivierung der toll-like-rezeptoren, tlr) etabliert, um eine potente antigenpräsentation zu erreichen. neben verschiedenen tumoren sind auch infektionskrankheiten einer therapeutischen vakzinierung zugänglich. in einer proof-of-concept-studie konnten wir eine dna-vakzinierung gegen leishmania major im modell der suszeptiblen balb/c-mäuse durchführen (walker et al. 1998) . durch wiederholte applikation des gp63-enthaltenden expressionsvektors konnten spezifische cd8-positive lymphozyten induziert werden, die zur protektion in 40% der vakzinierten tiere führten (> abb. 4.1.4) (walker et al. 1998 ). vorwiegend die problematischen infektionen wie hiv, ebola(nabel 2003) , dengue-virus (costa et al. 2006 ) oder sars (kong et al. 2005; wang et al. 2005; see et al. 2006 ) stellen eine große herausforderung dar. die meisten experten sehen eine beherrschung der hiv-infektion nur dann, wenn es gelingt, eine prophy-laktische vakzinierung zu entwickeln. eine solche funktionsfähige prophylaktische hiv-vakzinierung ist gegenwärtig nicht in sicht. neben der hochaktiven antiretroviralen therapie (haart) wurden fortschritte auf dem gebiet der therapeutischen hiv-vakzinierung gemacht. diese verfolgt das ziel, durch induktion hiv-1spezifischer t-zellen die hiv-replikation einzudämmen. hierzu sind verschiedene prime-boost-ansätze im sinne einer dna-vakzinierung und einer vacciniavirus-ankara-boosterung verfolgt worden goonetilleke et al. 2006) . neben der therapeutischen vakzinierung wurden verschiedene gentherapeutische strategien erprobt, um dem fortschreiten der hiv-infektion einhalt zu gebieten. hierzu zählen verschiedene strategien rna-basierter technologien, dominant-negative virale proteine oder intrazelluläre antikörper. rna-basierte gentherapeutische strategien gegen hiv-1, z. b. ribozyme, rna-decoys und sirna, wurden erfolgreich gegen hiv eingesetzt (michienzi et al. 2003) . katalytische rna, auch ribozyme genannt, greifen in die regulation der transkription ein und führen zur hemmung der hiv-vermehrung (puerta-fernandez et al. 2002) . mittels multimerer ribozyme gegen env-rna konnten humane, cd4-positive t-zell-linien durch ribozymexpression für mindestens 60 tage gegenüber einer hiv-infektion resistent gemacht werden (ramezani et al. 2002) . wie die sirna-technologie (7 kap. 4.3), so sind auch ribozyme immer dann nicht mehr wirksam, wenn das zielgen im hiv-genom mutiert. eine induzierbare anti-hiv-hairpin-rna gegen rev (unter der kontrolle des polymerase-ii-promotors) war nach induktion durch hiv-tat imstande, nach intrazellulärer prozessierung zu si-rna die hiv-vermehrung in infizierten zellen selektiv zu vermindern (unwalla et al. 2004) . weitere in-vitro-sirna-studien gegen rev und tat führten ebenfalls zum "gene silcencing" und werden experimentell klinisch gegen hiv eingesetzt (coburn u. cullen 2002; jacque et al. 2002) . lentiviren vom typ hiv wurden gegen die hiv-infektion eingesetzt, um sirna in humanen zellen dauerhaft zu exprimieren (trono 2000; mautino u. morgan 2002) . hierbei waren hiv-1-vektoren besonders gut in der lage, hämatopoietische stammzellen effizient zu transduzieren. auf diese weise exprimieren die nachfolgenden zellpopulationen das therapeutische genprodukt und lassen sich so ggf. vor einer hiv-infektion schützen morris u. rossi 2006) . effizienterweise sollten möglichst mehrere sirna-gene verwendet werden, um eine gewisse anzahl von genen simultan zu reprimieren. auch können lentiviren eingesetzt werden, um andere lentiviren zu verpacken (sog. "cross-packaging") (browning et al. 2001) . eine solche strategie, bei der hiv-1-vektoren durch felines immundefizienz-virus (fiv) verpackt wurden, war imstande, cd4-zellen vor einer hiv-infektion zu schützen (morris et al. 2004 ). das cross-packaging lentiviraler vektoren, wie z. b. hiv-1 mit einem fiv-packaging-system, stellt eine sichere methode dar, um lentivirale vektoren an zielzellen hiv-infizierter individuen zu targetieren. zusammen mit einer entsprechenden pseudotypisierung lassen sich auf diese weise zelltypen spezifisch und effektiv mittels lentiviraler vektoren transduzieren (kobinger et al. 2001) . auch das konzept der transkriptionellen repression durch blockade von transkriptionsfaktoren, welche an den hiv-promotor binden, stellt ein interessantes konzept dar. solche transkriptionsrepressoren betreffen die zinkfinger-dna-bindungsdomäne innerhalb des hiv-1 "long-terminal repeat" (ltr). diese strategie war bereits in vitro erfolgreich (reynolds et al. 2003) . auf diese weise ließ sich eine bis zu 100-fache reduktion der hiv-vermehrung durch inhibition des hiv-1-promotors erzielen (segal et al. 2004) . auch die intrazelluläre expression rekombinanter antikörper, sog. "intrabodies", führt zur intrazellulären immunisierung gegen hiv, z. b. gegen das vif-protein, das für die virusproduktion und die reverse transkription notwendig ist (goncalves et al. 2002) . lymphozyten, die einen intrabody gegen vif exprimierten, zeigten eine vermehrte resistenz gegenüber der hiv-infektion, und zwar sowohl gegenüber adaptierten als auch primären hiv-stämmen. eine deutliche wirkung ist vor allem dann zu erwarten, wenn sich diese intrabodies an proteine binden, die wirtsseitige interaktionspartner für hiv-proteine darstellen, da das virus keine strategie besitzt, nichtviral kodierte proteine zu synthetisieren. ein beispiel eines kritischen, wirtsseitig synthetisierten proteins stellt der chemokin-co-rezeptor cxcr-4 dar, dessen blockade zu reduzierter infizierbarkeit führt (bouhamdan et al. 2001). die meisten gentherapiestudien werden gegen krebs durchgeführt (mehr als 66% der indikationen) (prud'homme 2005; edelstein et al. 2004) . eine übersicht über die in deutschland laufenden gentherapiestudien findet sich auf der webseite des deutschen gentransferregisters (http://www.dereg.de), die gemeinsam von der deutschen gesellschaft für gentherapie und dem zentrum für klinische studien der universität freiburg etabliert wurde. mit blick auf das maligne melanom wurden verschiedene gentherapeutische strategien zur therapie des malignen melanoms entwickelt (>tab. 4.1.2). das konzept der immuntherapie gegen krebs basiert auf der annahme, dass tumoren zur generierung schwacher humoraler und/oder zellulärer immunreaktionen führen (sahin et al. 1995) . durch immunisierungsstrategien soll die immunologische toleranz durchbrochen und eine breite immunologische antwort auf die tumorzellen generiert werden, die die individuellen tumorzellen abtötet (hengge u. schadendorf 2000) . entsprechend lassen sich immunstimulatorische zytokine als adjuvanzien (z. b. il-2, il-12, il-15, gm-csf und interferon-) einsetzen, um eine systemische antitumorantwort zu forcieren. die transfektion von tumorzellen mit zytokin-genen soll zu einer aktivierung von natürlichen killerzellen, makrophagen und dendritischen zellen (dc) führen, die direkt oder indirekt an der bekämpfung der tumorzellen beteiligt sind. hierdurch lassen sich cd8-positive effektor-t-zellen und cd4-positive helfer-t-zellen induzieren. kürzlich wurde über eine dna-vakzinierung mit dem il-12-gen an patienten mit malignem melanom der stadien iii und iv berichtet. diese studie basiert auf dem b16-f10 mausmelanommodell, wo 12 von 15 mäusen mit subkutanen melanomen für bis zu 100 tage metastasenfrei blieben. bei den ersten 3 behandelten patienten fand sich ein klinischer rückgang der lymphknotenmetastasen nach vakzinierung mit 200 µg il-12-expressionsplasmid (heller et al. 2006) . in einer ähnlichen studie wurde ein il-12-expressionsvektor in einer dosiseskalationsstudie 3-mal pro zyklus für maximal 7 zyklen intratumoral appliziert (heinzerling et al. 2005) . hierbei zeigte sich bei 2 von 9 patienten je eine stabile erkrankung bzw. komplette remission. bei allen patienten fand sich eine antigenspezifische immunantwort gegen mage-1 und mart-1. bei einer weiteren zytokin-gentherapie wurde melanompatienten intratumoral ein canarypox-virus-vektor injiziert, der das il-12-gen exprimierte, und zur t-zell-akkumulation in injizierten melanomen führte (triozzi et al. 2005) . bei 3 von 9 patienten fand sich im serum ein anstieg von il-12 und interferon-. bei einem patienten fand sich eine komplette klinische remission. unerwünschte wirkungen waren grippale symptome, myalgie und müdigkeit. das "melanoma-differentiation-associated"-(mda-) 7/il-24-gen, das zu irreversibler wachstumshemmung und terminaler differenzierung führt, wurde mittels eines vermehrungsinkompetenten adenovirus in melanomzellen exprimiert (fisher et al. 2003) . bei patienten, die unter adenoviraler mda-7/il-24-gentherapie eine klinische teilremission und deutliche apoptose zeigten, fanden sich signifikant höhere il-6-und tnf-α-serumkonzentrationen . in der klinischen anwendung dieses vektors zeigte sich eine komplette und eine partielle remission . eine andere interessante anwendung ist die adaptive immuntherapie nach t-zell-rezeptor-transfer. hierbei wurde der rezeptor von αβ-t-lymphozyten, die cd8-positiv waren und zytotoxische eigenschaften besaßen, eingesetzt (willemsen et al. 2005) . um eine anhaltende immunität zu erzielen, wurden mhc-klasse-ii-restringierte cd4-t-lymphozyten mit einem hla-a1/mage.a1-spezifischen αβ-t-zell-rezeptor transduziert, was nach spezifischer stimulation zu einer starken interferon--, tnf-α-sowie il-2-produktion führte (willemsen et al. 2005) . bei der dc-vakzinierung werden antigenpräsentierende zellen mit definierten immunogenen peptiden (z. b. eines tumors) beladen, die t-zellen mit dem entsprechenden rezeptor aktivieren und zur expansion bringen sollen. so wurden melanom-antigen-gepulste dc seit einigen jahren in der therapie des metastasierten melanoms eingesetzt (nestle et al. 1998; thurner et al. 1999) . eine neuere studie mit dc und autologen lysaten (n=19) bzw. den melanomassoziierten antigenen mage-3.a2, tyrosinase, gp100 und mart-1 -zusammen mit dem adjuvans klh -, die wöchentlich in die inguinalen lymphknoten von 32 patienten injiziert wurden, bestätigt das immunologische wirkprinzip (hersey et al. 2004) . wie in früheren studien fanden sich auch hier drei partielle remissionen (3/19) in der gruppe von patienten, die mit lysat-gepulsten dc vakziniert wurden. in dieser studie zeigte sich, dass die "delayedtype hypersensitivity"-(dth-)reaktion auf autologe tumorlysate eine voraussetzung für das klinische ansprechen war (hersey et al. 2004) . jedoch fand sich keine interferon--produktion in der elispot-technik. es ist jedoch schwierig, einen zusammenhang zwischen tumorregression und der existenz einer durch die vakzinierung induzierten zytotoxischen t-zell-antwort unzweifelhaft festzustellen, da nicht alle patienten mit zellulären immunantworten auf den tumor eine regression desselben zeigen. zytotoxische effektor-t-zellen (ctl) sind zur tumorzelllyse befähigte t-zellen, die nach andocken an die zielzelle diese durch sekretion von granzym und perforin permeabilisieren und damit lysieren. nach der vakzinierung mit einem rekombinanten canarypox-vektor, der mage-3.a1-peptide exprimierte, zeigte sich, dass patienten mit zeichen einer klinischen tumorregression in 3/4 der fälle eine ctl-antwort aufwiesen, während dies nur bei 1/11 patienten ohne zeichen der tumorregression der fall war (karanikas et al. 2003) . kürzlich wurden verschiedene klonotypen der ctl-antworten gegenüber dem mage-3.a1 identifiziert (lonchay et al. 2004 ). hierdurch wurde es möglich, nicht nur die ctl-vorläuferfrequenz im blut als marker für die zytotoxische immunantwort zu untersuchen, sondern auch vorherrschende klonotypen der anti-mage-3 a1-immunantworten zu identifizieren. in jüngster zeit wird verbreitet auch rna in dc transfiziert und zur induktion tumorspezifischer t-zell-antworten eingesetzt (gilboa u. vieweg 2004; grunebach et al. 2005) . ein vorteil gegenüber der (konventionellen) peptidbeladung von dc besteht darin, dass eine vakzinierung gegen multiple, durch die rna kodierte epitope möglich ist. entsprechende klinische studien wurden sowohl gegen das prostatakarzinom (rini 2004) als auch gegen das melanom (kyte et al. 2006 ) durchgeführt. verschiedene fragestellungen wie die intradermale oder intranodale administration der vakzine sowie die optimale generierung und reifung von dc werden gegenwärtig untersucht und können noch nicht abschließend beurteilt werden. eine wichtige rolle für die resultierenden immunantworten scheint auch der tumorphänotyp und weniger die vakzinierungsplattform zu spielen (leitch et al. 2004) . die transduktionsraten von dc und die expressionsdauer wurden in abhängigkeit vom verwendeten vektor untersucht. je nach ziel des gentransfers ist diese bei langzeitexpression im gegensatz zur genetischen vakzinierung unerwünscht. nach intravenöser injektion von hochkapazitäts-adenovirus-vektoren sowie lentiviraler vektoren fand sich eine transduktion antigenpräsentierender zellen und b-zellen trotz verwendung leberspezifischer promotoren in bis zu bis zu 30%, während t-zellen refraktär waren. im gegensatz hierzu zeigte die in-vivo-applikation von aav8 und 9 nur eine expression in dc von unter 0,1% (riviere et al. 2006) . bei den meisten tumoren findet sich ein verlust der funktion von tumorsuppressorgenen. tumorsuppressorgene können zum wachstumsstopp von tumorzellen führen und diese der apoptose (programmierter zelltod) zuführen. ein paradebeispiel hierfür ist das p53-protein, das erst nach einem aufgetretenen dna-schaden den zellzyklus blockiert und die zellen der apoptose unterwirft (roth 2006) . die effektive expression von wildtyp p53 konnte tumorregressionen bereits etablierter, menschlicher tumoren induzieren und das wachstum humaner melanomzellen in kultur oder nacktmäusen blockieren (sauter et al. 2002) . die kombination von chemotherapie und adenoviral vermit-teltem wildtyp p53 war imstande, die amifostin-induzierte apoptoserate menschlicher bronchialkarzinomzellen zu erhöhen (pataer et al. 2006) . ein interessanter ansatz ist auch die wachstumsund invasionshemmung von melanomen durch inaktivierung des braf-onkogens mittels lentiviral vermittelter rna-interferenz (sumimoto et al. 2004) . nach lentiviralem transfer von sirna gegen die häufigste mutation des braf-antigens (v599e) zeigte sich, dass die meisten melanomzelllinien nach transduktion ein gehemmtes wachstum sowie eine reduzierte invasivität aufwiesen. die reduzierte invasivität zeigte sich auch in einer verminderung der matrixmetalloproteinase-aktivität und der β1-integrin-expression (sumimoto et al. 2004 ). daneben findet sich die gentherapie, bei der vorstufen zytotoxischer medikamente durch gentransfer metabolisierender enzyme möglichst selektiv in tumorzellen in toxische produkte umgewandelt werden (van dillen et al. 2002; fillat et al. 2003) . nach umwandlung der prodroge in einen toxischen metaboliten stirbt die transduzierte zelle und meist auch einige der nachbarzellen (sog. "bystander-effekt") (niculescu-duvaz u. springer 2005). so wurde zum beispiel die herpes-simplex-virus-thymidinkinase (hsv-tk) transferiert, um das nichttoxische ganciclovir selektiv in tumorzellen zum toxischen ganciclovir-triphosphat umzusetzen. als paradebeispiel der gentherapeutischen behandlung von stoffwechselkrankheiten dient die chronik der gentherapie der hämophilie (humaner gerinnungsfaktor viii bzw. ix) (lillicrap et al. 2006) . während bei den zurückliegenden versuchen an hämophilen mäusen und hunden (durch expression im muskel) mittels aav-gentransfer eine anhaltende faktor-ix-aktivität von 4-23% erzielt wurde (arruda et al. 2004; liu et al. 2004a) , war bei den initialen versuchen an patienten der muskel zwar zur langzeitexpression befähigt, jedoch ließen sich nur subtherapeutische serumkonzentrationen erzielen. daraufhin wurde der gentransfer in die leber als dem natürlichen bildungsort der gerinnungsfaktoren gewählt, wo zwar therapeutische spiegel erzielt wurden, jedoch die expression auf wenige wochen beschränkt war (manno et al. 2006) . insgesamt wurden bei den klinischen hämophiliestudien patienten mit missense-mutationen eingeschlossen, obwohl die immunreaktion bzw. die inhibitorformation bei vorliegen eines prämaturen stoppkodons oder frameshift-mutationen im faktor-ix-gen stärker ausgeprägt waren. auffällig war bei den bislang 7 behandelten patienten, dass die faktor-ix-aktivität innerhalb von 4 wochen auf unter 1% abfiel. zu diesem zeitpunkt traten bei den patienten hepatitiden mit erhöhten leberwerten (alt 600 u/l, ast 200 u/l) auf, weshalb zunächst keine weiteren patienten eingeschlossen wurden. diese unerwünschte wirkung wurde bei den vorangegangenen tierstudien nicht beobachtet. die systematische analyse der immunreaktionen mittels aav-kapsid-und faktor-ix-peptid-bibliotheken führte zur identifizierung hoher ctl-precursor-frequenzen für 2 hochkonservierte peptide (peptid 74 und peptid 82) der serotypen aav1-8. interessant war, dass bei patienten, die vor therapie hohe antikörpertiter gegen aav aufwiesen, die hepatitis und die entsprechende zelluläre immunantwort ausblieb und sie nur bei patienten mit geringen oder fehlenden aav-titern auftrat (manno et al. 2006) . dementsprechend werden patienten ab sofort vor einschluss in klinische studien hinsichtlich bestehender humoraler und zellulärer immunantworten gegen aav-kapside genau untersucht. eine immunantwort gegen das faktor-ix-protein war bei keinem der behandelten patienten feststellbar. als neue strategie wird bei diesen patienten die transiente immunsuppression für einen zeitraum von etwa 4 monaten erprobt, bis das aav-kapsid von den transfizierten zellen eliminiert ist. die expression des transgens im muskelgewebe wurde nach aav-gentransfer bis zu 2 jahre beobachtet (riviere et al. 2006) . in der haut findet sich ein tropismus von aav für den haarfollikel ( > abb. 4.1.3c,d) (hengge u. mirmohammadsadegh 2002) . bei einer anderen seltenen stoffwechselkrankheit stellt die gentherapie eine echte alternative zur knochenmarktransplantation dar, wenn entsprechende allogene hla-gematchte spender nicht zur verfügung stehen. so zeigte eine kürzlich erschienene deutsche studie die korrektur der x-gebundenen chronischen granulomatose (cgd) durch retroviralen transfer des gp91 phox , einem wichtigen protein in der oxidativen antimikrobiellen abwehr (ott et al. 2006) . diese arbeit ist in zweierlei hinsicht bemerkenswert: zum einen wurde hier die erste myeloide immundefizienz behandelt, indem die neutrophilen granulozyten von zwei betroffenen individuen phänotypisch und funktionell korrigiert wurden, die sich beide klinisch signifikant verbesserten. zum anderen zeigte diese studie nach nichtmyeloablativer knochenmarkkonditionierung die insertionelle aktivierung von 3 wenig erforschten genen mds1-evi1, prdm16 bzw. setbp1, die bezeichnenderweise die langzeithämatopoiese stimulierten und somit zur expansion der transduzierten zellpopulation beitrugen. nach dem gentransfer zeigten beide patienten zwischen 15% und 60% funktionell rekonstituierte neutrophile granulozyten mit guter lytischer aktivität gegenüber opsonisierten e. coli-bakterien. im gegensatz zu den gentherapiestudien der schweren kombinierten immundefizienz (scid-x1 und ada-scid) stellt die rekonstitution des gendefekts des gp91 phox keinen wachstumsvorteil für die myeloiden vorläuferzellen dar. außerdem bedürfen phagozyten als kurzlebige zellen einer konstanten erneuerung. diese ausgangsbedingungen lassen eine kurative gentherapie unter diesen gesichtspunkten schwierig erscheinen. offensichtlich war jedoch die hohe rate der vektorintegration in die o. g. drei loci von determinierten myeloiden progenitorzellen so effizient, dass dadurch das myeloide kompartiment unabhängig von wachstumsfaktoren eine rekonstitution erfuhr. derzeit werden verschiedene strategien erprobt, die klinische wirksamkeit der tumorvakzinierung durch kostimulation und amplifikation entsprechender immunzellen zu verstärken. generell haben die studien pilotcharakter, weshalb nur wenige zentren diese therapien anbieten können bzw. nur wenige patienten eingeschlossen werden konnten. außerdem handelt es sich durchweg um seltene entitäten bzw. in einigen fällen um individualisierte therapeutika, was die geringen patientenzahlen erklären hilft. dennoch lässt sich aus kleinen, aber sehr gut analysierten patientenkollektiven eine hohes maß an information gewinnen. beim neuroblastom war nach einer il-2-und lymphotactin-enthaltenden vakzinierung in 4 von 21 fällen eine komplette remission für mehr als 48 monate zu verzeichnen (brenner et al. 2000) . weiterhin fand sich bei 5 der 21 patienten eine stabilisierung der erkrankung für mehr als 400 tage. bei der untersuchung des zellulären immunsystems zeigte sich, dass nicht alle patienten auf die vakzine in der gewünschten weise antworten und dass nicht alle patienten mit nachweisbaren zellulären immunantworten wirklich imstande sind, den tumor zu kontrollieren. die vakzinierung gegen tumoren mit sog. potenten antigenen stellt kein großes immunologisches problem dar. jedoch stellt die vakzinierung gegen die häufigeren schwächeren tumorantigene (z. b. "latency membraneprotein"-1 und -2 von epstein-barr-virus bei nasopharyngealem karzinom und hodgkin-erkrankung) ein großes problem dar. hier ist es wichtig, entsprechende kombinationen (z. b. ablative chemotherapie oder spezifische antikörpertherapien) begleitend einzusetzen, um regulatorische t-zellen zu eliminieren, die inhibierende immunwirkungen bei der vakzinierung entfalten können. dies kann z. b. in form der zeitlich begrenzten applikation eines cd45-antikörpers oder mit ablativer chemotherapie im sinne einer lymphozytendepletion durchgeführt werden. so sind vakzinierungsanstrengungen in der zukunft ggf. mit temporärer lymphodepletion zu kombinieren, um synergistische effekte zu erhalten. im jahr 1999 ereignete sich der erste tödliche zwischenfall in der gentherapie bei einem jungen mit vererbtem ornithin-transcarbamylase-defekt nach adenoviralem gentransfer (raper et al. 2003) . bei hoher virusdosis und vorgeschädigter leber kam es nach intrahepatischer injektion des virus zum akuten leberzerfall durch massive immunaktivierung und einen zytokin-boost. drei jahre später fanden sich bei der sonst sehr erfolgreichen therapie der x-chromosomalen kombinierten schweren immundefizienz (scid) drei formen von leukämie (hacein-bey-abina et al. 2003a,b) . bei diesen patienten ist es zur insertionellen mutagenese mit nachfolgender inaktivierung von tumorsuppressorgenen gekommen. im jahr 2004 sind zwar keine neuen fälle insertionell bedingter tumoren oder leukämien in klinischen studien der retroviralen gentherapie aufgetreten; allerdings sind zwei der drei betroffenen kinder der scid-x1-studie an den folgen der insertionell bedingten malignen lymphoproliferation gestorben. zugleich zeigen jüngste daten aus mittlerweile mindestens 5 laufenden studien zur retroviralen korrektur von immundefizienzerkrankungen (mailand: ada-defizienz; paris: scid-x1; london: scid-x1 und ada-defizienz; nih: scid-x1; muul et al. 2003 ; frankfurt: cgd) positiven therapeutischen nutzen ohne das auftreten von sicherheitsproblemen. da das therapeutische potenzial des retroviralen gentransfers kaum mehr infrage steht, ist es nun das höchstrangige ziel, die früher beobachteten komplikationen durch ein besseres mechanistisches verständnis zu vermeiden. während nichtvirale vektoren keine integration in das genom zeigen und deshalb nur transient exprimieren, ist dies bei retroviralen und lentiviralen vektoren die regel. wie aus den zwischenfällen der retroviralen gentherapie bekannt wurde, hat jede integration in das genom ein gewisses risiko der insertionellen mutagenese. diese wurde sowohl in mausmodellen als auch in der klinischen scid-x1-gentherapie beobachtet (li et al. 2002; baum et al. 2003; hacein-bey-abina et al. 2003a,b) . im rahmen der aufklärung der drei lymphoproliferativen erkrankungen im rahmen der scid-x1-gen-therapie wurde in einem x-scid-mausmodell gefunden, dass das korrektive therapeutische gen die il-2-rezeptor-gammakette selbst in 1/3 der tiere zur lymphomgenese beitragen kann (woods et al. 2006) . bei der entwicklung der lymphoproliferativen erkrankungen nach scid-korrektur der il-2-rezeptor-gammakette ist es zur ungebremsten expression dieses lmo2-onkogens gekommen (hacein-bey-abina et al. 2003a,b) . im rahmen der experimente im x-scid-modell der maus zeigten sich in 33% der fälle t-zell-lymphome, die jedoch frühestens erst 6 monate nach transplantation auftraten. hierbei stellt sich die frage nach dem beitrag des transgens neben der insertionsstelle im lmo2-onkogen. in vorstudien zur klinischen gentherapie wurden weltweit 88 mäuse mit demselben transgen in retroviralen vektoren behandelt, ohne dass innerhalb einer experimentalphase von 6 monaten lymphome aufgetreten waren. auch die langfristige beobachtung von hunden, rhesusaffen oder schafen, die das humane gen für bis zu 1 jahr nach der transplantation exprimierten, zeigten keine lymphome. auch in der klinischen gentherapie traten die beobachteten, leukämieähnlichen krankheitsfälle 2-3 jahre nach transplantation auf. die lange latenz bis zum auftreten von lymphomen kann möglicherweise durch die akquisition weiterer komplementärer mutationen bedingt sein. um das risiko der insertionellen mutagenese zu reduzieren, wurden integrationsdefiziente lentivirale vektoren konstruiert, die eine stabile transduktion ermöglichen (saenz et al. 2004; vargas et al. 2004) . diese untersuchungen wurden in vivo in nagermodellen durchgeführt, bei denen in augen und gehirn injiziert wurde (yanez-munoz et al. 2006) . die hohe effizienz des gentransfers und der expression eines therapeutischen proteins führte zur dauerhaften klinischen besserung im modell der retinadegeneration (yanez-munoz et al. 2006 ). an diesem beispiel konnte gezeigt werden, dass für postmitotische gewebe eine vektorintegration keine unabdingbare voraussetzung für eine langzeitexpression darstellt. nachdem es in den vergangenen jahren gelungen ist, eine adäquat hohe expression des erwünschten transgens zu gewährleisten, stellen sich zunehmend probleme der immunogenität der häufig verwendete virusvektoren. so wurden beispielsweise die immunreaktionen auf die aav-serotypen 2, 5 und 8 systematisch untersucht, um zelluläre immunantworten gegen das viruskapsid bzw. gegen das therapeutische transgen zu definieren. insgesamt ging es um die frage, wie sich eine cd8-positive mhc-klasse-i-immunantwort gegen kapsidproteine entwickelt, wo bei diesem reaktionsweg doch die aufnahme der viruspartikel erfolgt und somit normalerweise eine mhc-klasse-ii-präsentation stattfinden müsste. am modell der c57bl/6-mäuse konnte mittels elispot und intrazytoplasmatischer zytokinfärbung in abhängigkeit von den zur immunisierung bzw. zur boosterung eingesetzten aav-serotypen die induktion interferon--positiver, cd8-positiver lymphozyten in der abstufung aav2/8 > aav2/5 > aav2/2 (serotypen des aav bei der prime-bzw. bei der boost-reaktion) nachgewiesen werden . bei dem versuch, die b-zell-antworten in analoger weise zu untersuchen (modellgen: hunde-gerinnungsfaktor ix), zeigte sich ein komplexes bild. interessant war, dass neutralisierende in-vitro-aktivitäten nicht automatisch mit einer in-vivo-neutralisationswirkung verbunden waren. die konsolidierungsphase der gentherapie scheint überwunden und berechtigter optimismus dahingehend angebracht, dass man in zukunft molekulare therapien für eine wachsende zahl klinischer erkrankungen entwickeln wird. trotz aller fortschritte dieses -noch nicht einmal 15 jahre alten -forschungsgebiets gilt es, lösungen für die gegenwärtigen probleme der gentherapie zu finden ( > tab. 4.1.5). die regulatorischen auflagen im umgang mit genetisch markierten organismen (gmo) und den voraussetzungen zur durchführung klinischer studien sind in den verschiedenen ländern unterschiedlich. auch die ethischen gesichtspunkte international durchgeführter klinischer studien sind von unterschiedlichen auflagen geprägt. es wäre für die vereinheitlichung und vergleichbarkeit der verschiedenen studienaktivitäten notwendig, eine internationale standardisierung zu erreichen. dies ist gerade deshalb so wichtig, weil z. b. in china eine adenovirale gentherapie zur expression des wildtyps p53 zugelassen ist. die dortigen sicherheitsbestimmungen sowie die regulatorischen auflagen entsprechen jedoch nicht den europäischen bzw. amerikanischen standards. dennoch wird eine solche hochbrisante therapie aus china in das europäische ausland und nach amerika exportiert. die positronenemissionstomographie-(pet-)technologie hat im molekularen real-time-imaging in den letzten 5 jahren große beliebtheit erfahren (serganova u. blasberg 2005) . neben dem klassischen reportergen-design wird "gene imaging" in absehbarer zeit zum überwachen der viral und zellbasierten gentherapie von tumoren eingesetzt werden (serganova u. blasberg 2005) . hierdurch lassen sich mittels nichtinvasiver invivo-darstellung quantitative aussagen zu vektorkonzentrationen und zur transduktionseffizienz in klinischen studien treffen, indem die lokalisation, das ausmaß und die dauer der transgenexpression verfolgt werden. weitere anwendungen stellen das zelluläre trafficking, das targeting und die replikation von vektoren dar. eine kürzlich veröffentliche studie zeigte bei systemischer anwendung von sirna nach hepatischer applikation der korrespondierenden hairpin-rna in einem aav-vektor schwere toxische reaktionen mit todesfolge in mäusen (grimm et al. 2006) . die hairpin-rna-therapie führte zur übersaturierung des mikror-na-stoffwechselwegs in der leber mit kompetition um das nukleäre karyopherin exportin-5. diese ergebnisse sollten die gentherapeuten zur umsicht und weiterem studium zum verständnis der sirna mahnen, bevor in klinischen studien möglicherweise schwere unerwünschte ereignisse auftreten. gutless adenovirus: last-generation adenovirus for gene therapy efficient lentiviral vectors for short hairpin rna delivery into human cells safety and efficacy of factor ix gene transfer to skeletal muscle in murine and canine hemophilia b models by adeno-associated viral vector serotype 1 uptake and trafficking of dna in keratinocytes: evidence for dnabinding proteins side effects of retroviral gene transfer into hematopoietic stem cells t lymphocyte-directed gene therapy for ada-scid: initial trial results after 4 years inhibition of hiv-1 infection by down-regulation of the cxcr4 co-receptor using an intracellular single chain variable fragment against cxcr4 phase i study of chemokine and cytokine gene-modified autologous neuroblastoma cells for treatment of relapsed/refractory neuroblastoma using an adenoviral vector primate and feline lentivirus vector rna packaging and propagation by heterologous lentivirus virions progress in the use of adenoassociated viral vectors for gene therapy phase i clinical trial safety of dna-and modified virus ankara-vectored human immunodeficiency virus type 1 (hiv-1) vaccines administered alone and in a prime-boost regime to healthy hiv-1-uninfected volunteers non-viral vector as vaccine carrier fighting cancer with oncolytic viruses potent and specific inhibition of human immunodeficiency virus type 1 replication by rna interference dna vaccine against the nonstructural 1 protein (ns1) of dengue 2 virus clinical and local biological effects of an intratumoral injection of mda-7 (il24; ingn 241) in patients with advanced carcinoma: a phase i study intracellular trafficking of adeno-associated viral vectors gene therapy clinical trials worldwide 1989-2004-an overview hsv-1-derived recombinant and amplicon vectors for gene transfer and gene therapy genuine dna/polyethylenimine (pei) complexes improve transfection properties and cell survival suicide gene therapy mediated by the herpes simplex virus thymidine kinase gene/ganciclovir system: fifteen years of application mda-7/il-24, a novel cancer selective apoptosis inducing cytokine gene: from the laboratory into the clinic adeno-associated virus-based gene therapy for inherited disorders phase i-ii trial of onyx-015 in combination with map chemotherapy in patients with advanced sarcomas cancer immunotherapy with mrna-transfected dendritic cells herpes vector-mediated gene transfer in treatment of diseases of the nervous system vif protein by intracellular immunization inhibits reverse transcription and viral replication induction of multifunctional human immunodeficiency virus type 1 (hiv-1)-specific t cells capable of proliferation in healthy subjects by using a prime-boost regimen of dna-and modified vaccinia virus ankara-vectored vaccines expressing hiv-1 gag coupled to cd8+ t-cell epitopes fatality in mice due to oversaturation of cellular microrna/short hairpin rna pathways p537 preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy new developments in dendritic cell-based vaccinations: rna translated into clinics a serious adverse event after successful gene therapy for x-linked severe combined immunodeficiency lmo2-associated clonal t cell proliferation in two patients after gene therapy for scid-x1 intratumoral injection of dna encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy evaluation of toxicity following electrically mediated interleukin-12 gene delivery in a b16 mouse melanoma model gene therapy and the skin cytokine gene expression in epidermis with biological effects following injection of naked dna safety and pharmacokinetics of naked plasmid dna in the skin: studies on dissemination and ectopic expression adeno-associated virus (aav) expresses transgenes long-term in epidermis and hair follicles modification of melanoma cells via ballistic gene delivery for vaccination expression of naked dna in human, pig, and mouse skin phase i/ii study of treatment with dendritic cell vaccines in patients with disseminated melanoma modulation of hiv-1 replication by rna interference monoclonal anti-mage-3 ctl responses in melanoma patients displaying tumor regression after vaccination with a recombinant canarypox virus targeting the local tumor microenvironment with vaccinia virus expressing b7.1 for the treatment of melanoma tumor-specific activation of htert-derived promoters by tumor suppressive e1a-mutants involves recruitment of p300/cbp/hat and suppression of hdac-1 and defines a combined tumor targeting and suppression system photochemically enhanced gene delivery of egf receptor-targeted dna polyplexes filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo modulation of the immune response to the severe acute respiratory syndrome spike glycoprotein by gene-based and inactivated virus immunization phase i/ii trial of melanoma therapy with dendritic cells transfected with autologous tumor-mrna ctl-dependent and -independent antitumor immunity is determined by the tumor not the vaccine murine leukemia induced by retroviral gene marking cellular and genetic therapies for haemophilia oncolytic viral therapies therapeutic levels of factor ix expression using a muscle-specific promoter and adeno-associated virus serotype 1 vector conditionally replicationcompetent adenoviral vectors with enhanced infectivity for use in gene therapy of melanoma correlation between tumor regression and t cell responses in melanoma patients vaccinated with a mage antigen overview of phase i studies of intravenous administration of pv701, an oncolytic virus biology and application of alphaviruses in gene therapy lentiviral vectors and antiretroviral intrinsic immunity successful transduction of liver in hemophilia by aav-factor ix and limitations imposed by the host immune response artificial viruses: a nanotechnological approach to gene delivery gene therapy of hiv-1 infection using lentiviral vectors expressing anti-hiv-1 genes topical application of plasmid dna to mouse and human skin rna-mediated inhibition of hiv in a gene therapy setting lentiviral vector retargeting to p-glycoprotein on metastatic melanoma through intravenous injection transduction of cell lines and primary cells by fiv-packaged hiv vectors lentiviral-mediated delivery of sirnas for antiviral therapy persistence and expression of the adenosine deaminase gene for 12 years and immune reaction to gene transfer components: long-term results of the first clinical gene therapy trial custom adeno-associated virus capsids: the next generation of recombinant vectors with novel tropism vaccine for aids and ebola virus infection lentiviral vectors vaccination of melanoma patients with peptide-or tumor lysate-pulsed dendritic cells introduction to the background, principles, and state of the art in suicide gene therapy tumor-targeted gene therapy: strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/dna complexes correction of x-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of mds1-evi1, prdm16 or setbp1 induction of apoptosis in human lung cancer cells following treatment with amifostine and an adenoviral vector containing wild-type p53 dna vaccination against tumors anchoring hairpin ribozymes to long target rnas by loop-loop rna interactions development and testing of retroviral vectors expressing multimeric hammerhead ribozymes targeted against all major clades of hiv-1 fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer repression of the hiv-1 5' ltr promoter and inhibition of hiv-1 replication by using engineered zincfinger transcription factors recent clinical development of dendritic cell-based immunotherapy for prostate cancer long-term expression and repeated administration of aav type 1, 2 and 5 vectors in skeletal muscle of immunocompetent adult mice gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction adenovirus p53 gene therapy unintegrated lentivirus dna persistence and accessibility to expression in nondividing cells: analysis with class i integrase mutants human neoplasms elicit multiple specific immune responses in the autologous host p53 alone or in combination with antisense cyclin d1 induces apoptosis and reduces tumor size in human melanoma mia (melanoma inhibitory activity) promoter mediated tissue-specific suicide gene therapy of malignant melanoma comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus attenuation of hiv-1 replication in primary human cells with a designed zinc finger transcription factor reporter gene imaging: potential impact on therapy inhibition of growth and invasive ability of melanoma by inactivation of mutated braf with lentivirusmediated rna interference vaccination with mage-3a1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic t cells and induces regression of some metastases in advanced stage iv melanoma intratumoral injection of ingn 241, a nonreplicating adenovector expressing the melanoma-differentiation associated gene-7 (mda-7/il24): biologic outcome in advanced cancer patients intratumoral administration of a recombinant canarypox virus expressing interleukin 12 in patients with metastatic melanoma lentiviral vectors: turning a deadly foe into a therapeutic agent negative feedback inhibition of hiv-1 by tat-inducible expression of sirna influence of the bystander effect on hsv-tk/gcv gene therapy. a review novel integrase-defective lentiviral episomal vectors for gene transfer precise hit: adeno-associated virus in gene targeting bacterial gene therapy strategies adenoviral vectors for gene transfer and therapy genetic immunization with glycoprotein 63 cdna results in a helper t cell type 1 immune response and protection in a murine model of leishmaniasis induction of antigen-specific cytotoxic t lymphocytes in humans by a malaria dna vaccine immune responses with dna vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in balb/c mice redirecting human cd4+ t lymphocytes to the mhc class i-restricted melanoma antigen mage-a1 by tcr alphabeta gene transfer requires cd8alpha the mechanism of naked dna uptake and expression gene therapy: therapeutic gene causing lymphoma effective gene therapy with nonintegrating lentiviral vectors retrovirus molecular conjugates: a versatile and efficient gene transfer vector system for primitive human hematopoietic progenitor cells die angegebenen zitate sind in den literaturteil integriert. ereignis referenz erste klinische markergen-therapie (tumorinfiltrierende lymphozyten) rosenberg et al. 1990 1990erste therapeutische gentherapie adenosindeaminase-immundefekt-syndrom (ada-scid) blaese et al. 1995; muul et al. 2003 1995 key: cord-023143-fcno330z authors: nan title: molecular aspects of viral immunity date: 2004-02-19 journal: j cell biochem doi: 10.1002/jcb.240591009 sha: doc_id: 23143 cord_uid: fcno330z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv60). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between 5 and 11 days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd4+ and cd8+ t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd4+ and cd8+ cells were detected by immunohistochemistry at 6 days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il-2, il-3, il-4, il-5, il-6, tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il-6, tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca 92037 although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd8+ cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within 2 hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp47 was both necessary and s f i c i e n t to cause retention of class i and icp47 expression in fibroblasts caused the cells to resist lysis by cd8+ t lymphocytes. icp47 is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp47 inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp47 have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp47 associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp47 are being assessed in proteosome and tap transporter assays. gst-icp47 fusion proteins tightly bind a 8.5 kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp47 binds to a single cellular protein of =55 kda on ligand blots. these proteins are good candidates as cellular targets of icp47 and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp47 is very effective i n blocking cd8+ t lymphocyte responses in vivo, perhaps explaining the predominance of cd4+ vs. cd8+ anti-hsv ctl i n vivo. we expect that icp47 may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma2, john p. carroll2, sara r. abromson-leeman2, joseph c. laning2, martin e. dorf2, and thomas l. benjamin2. idepartment of pathology, emory university school of medicine, atlanta, ga 30322, and 2department of pathology, harvard medical school, boston, ma 02115. susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h-2 haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c3wbida mouse and the highly resistant but mhc-identical (h-2k) c57bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs7bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c3wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c3wbida x c57bwcd.i) x c57bwcdj backcross mice cosegregated with mtv-7. inheritance of mtv-7 showed perfect concordance with absence of peripheral vp6+ t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv-7 showed no evidence of recombination between pyvs and mtv-7. strongly biased usage of vp6 by (a) polyoma-specific cd8+ ctl from virus-infected c57bwcdj mice and by @) cd8+ t cells infiltrating a polyoma tumor in a virus-immune c57bwcd.i host provide further evidence that t cells bearing this mtv-7 sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv-7 sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h-2, non h-2, level of cd4+ t cells, of cd8+ t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd8+ t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd8+ t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd8+ t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd8+ t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type 5 (ad 5) and human papillomavirus type 16 (hpv16)) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad 5e1a protein to which ctl clones were directed that could eradicate ad 5e1 induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad 5e1b protein. these ad 5eib specific ctl clones, however, were as effective as the ad 5e1a specific ctl clones in the eradication of ad 5e1 induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad 5e1 induced tumor cells with the activated ras oncogen the possibility of ad 5e1b specific ctl to recognize the ad 5e1 induced tumors was eliminated whereas the ad 5e1a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv16 induced mouse tumor model an immunosubdominant ctl epitope was identified in the e7 protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv16 induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv16 induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv16, that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a *0101, a *0201, a"0301, a*1101 and a *2401). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv16 induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of 1994. class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment3.4, biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd4 + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type 1 glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 5'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph8 environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al 18083 to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about 1 pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual 5' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are 106-10'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the 2-month old infant, an rsv vaccine will need to be effective when given to 1-month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: 1) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: 2) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd8+ cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd4' and cd8+ t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd4' t cells were more pathogenic than cd8+ t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m 2 (22k) protein were predominantly cd8' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd4+ t cells, and ctl. mice primed to g protein gave rise to predominantly cd4' t cells producing th2 cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type 2 cytokines, notably il-10, appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd4 t cells and protective effects by cd8' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d 4 ' t cells and b cells (immune complexes) in maintaining cd8+ t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn 38105. the analysis of the development and memory phases of the cd4+ "helper" n h ) and cd8+ cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type 1) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose (20 mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd4+ th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd44-high, l-selectin-low, cd49d (vla-4) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd49d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd49d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus 68 which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il-2 production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b7.1 and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il-4 vs il-i2/ifny are present at the initiation of the response of either cd8 or cd4 cells they dictate that the effectors generated will be polarized either towards il-4 and il-5 secretion or il-2 and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd4 effector cells not re-exposed to ag, produce no cytokines and they die within 3-4 days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il-2 produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il-2 and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around 450 trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to 7.0, the s ikes re ain their neutral configuration (1). probably as a consequence, the viral infectivity is totally preserved after an exposure of 2 hours at p 8 6.4 an cf 37t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein (2). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position 198, which is part of antigenic site 11, is important, although not essential, for the viral virulence. similarly, the arginine 333, which belongs to antigenic site 111, is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in 3). viral strains mutated at arginine 333 have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody (4). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus (5). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il 6061 1 theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd4 antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg2a antibody; (c) activated (le., ll-2rc) t cells infiltrating the cns are exclusively of the cd4+ phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids 74-86 of the vp2 capsid protein. a thl line specific for vp274-86 exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein 0. prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie9 bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by 9 amino acids between residues 96 and 167. au of the tg(mhu2m) mice developed neurologic drsease -200 days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu2m) mice with cjd prims produced mhu2mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu2m) mice wen differenl about 10% of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane >500 days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu2m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu2m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene -8-fold was not deleterious to themiw but it did shorten scrapie incubation times from -145 d to -45 d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon 101 (corresponding to codon 102 in hurp) pmdnced spontaneous, fatal neurcdegeneration between 150 and 300 d of age in two lines of tg(mohp-pio1l) mice designated 2866 and 2247. genetic crosses of tg(moprp-p101l)2866 mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between 150 and 160 days of age. the t g~o p r p -p l o l l ) 2 8 6~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast 10 the tg2866 and 2247 mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg2862 overexpress the mutant transgene -32-fold and develop fatal neurodegeneration behveen 200 and 400 d of age. tg2862 mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu2m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md 20892-0440. cd8' lymphocytes (tcd8+) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of 8 to 10 residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors (17 residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh2-terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the 17 residue precursor than from the nh2-terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv-1 envelope protein that contain overlapping determinants each presented by different class i1 mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv-1 envelope to provide help for neutralizing antibody (ab) and cd8+ cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p18 peptide from the v3 loop of hiv-1 iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p18 segment of both iiib and mn strains of hiv-1 in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all 3 responses, neutralizing ab, ctl, and th1 helper cells. the ctl specific for the mn strain p18 peptide crossreacted with strains sc, sf2,2321, and cdc4. the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp160 that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it 10 to 100-fold more potent in binding to the class i1 mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv-1 sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a2, with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/93. cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m1) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/89 and a/hawaii/91. this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/89 virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp120 generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il-4, months after immunization were also observed . immunization of rabbits with dna encoding l1, the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type 2 (hsv-2), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv-2. dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, 1993 2593745-1749 , 2dna cell biol, 1993 the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd4 t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd8 t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd8 cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e3) codes for more than 7 polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e3 gpl9k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by 4 distinct viral polypeptides, 3 of which (the ad e3 14.7k or the complex of the 10.4k and 14.5k proteins) are coded in the e3 region. the e3 polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e3 polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e3 polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e3 14.7k or ad e3 gpl9k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e3 genes in viral constructs, several transgenic mouse lines containing the ad e3 genes have been constructed for these experiments. the e3 genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e3 transgenic animals (h-2b'd) have been transplanted allogeneically to h-2d recipients and remained viable, secreting insulin until the end of the experiment at 94 days; in contrast, control nontransgenic islets of the same genotype were rejected by 21-28 days. the e3 genes behind the native e3 promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e3 promoter of the transgene is responsive to stimulation by the ad e1a following infection with an e3 minus ad 7001 and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e3 genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a 1993 outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr03121.) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g2 encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than 200,000 cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in 1993, a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus 7.5 k promoter and the s segment under control of the 11 k promoter. the m segment, which encodes the g1 and g2 envelope proteins, was included because of our findings that: (1) immunization with vaccinia or baculovirus-expressed g1 and g2 induced a neutralizing and protective immune response in hamsters; and, (2) neutralizing antibodies to g1 or g2 could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo7 pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd8 t cells and in the induction of virus-specific ctl activity. thereafter, the cd8 t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd8 cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl-2 in the t and b cells and in c57bl/6 ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd3, thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma 01772 thfectious disease unit, massachusetts general hospital, boston, ma 021 14 â§tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied 12 or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo0 for gag and 1/8500 for envelope. cd8+ lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd4+ cells. suppression mediated by unstimulated cd8 autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd8+ cells activated by an anti-cd3-specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv-1 molecular clone (nl43) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p24 and gp160 were observed in chimpanzees infected with n u 3 and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv-1, and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to 15 years with cd4 cells > 500/mm3. methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn-1 infected autologous cd4 cells, using target cells infected with recombinant vaccinia viruses expressing hn-1 proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr3 region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for 15 years, who by qc-pcr had <i67 viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv-1 variants, and both the p17 and p24 epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr3 region of the tcr in an individual with a vigorous response to gp41 suggested that approximately 6% of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv-1 infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf-1) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il-6 and il-7 receptor genes. to investigate the role of irf-1 related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o 5 pfu of coxsackie virus 83 (cvb3). homozygous irf-1 (+) and heterozygous irf-1 (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf-1 -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf-1 regulated gene products such as inos and ifn are essential for the early defense against cvb3-induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd4 and b-cell populations and a higher proportion of macrophages and cd8 cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il2, il4 and il5, but higher levels of il6 and ifng in the elderly as compared to the young mice. the addition of mf59 to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il2, il4 and il5 levels for both young and old, while il6 and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus 8 3 (cvb3), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb3 which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb3 is able to infect normal c57bl/6 mice and immunocompromised (cd4 ko and 62m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld5os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers 3-5 days pi.. large pathological changes were detectable only in heart tissue of cd4 ko mice, and were maximal at 1-2 weeks after infection. these results suggest the possibility that cd4' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd4+ and cd8' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p56lck are resistant to coxsackievirus b3 induced heart disease. tamara martino, karen aitken, joseph penninger, jan0 nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus 83 (cvb3) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p56kk are resistant to cvb3 induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb3 replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb3induced heart disease, cytokine expression in the hearts of cvb3 infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb3 infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd4+ t cells from 62m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd4+ t cells seen in normal mice 3 days after lcmv infection, previously presumed to be due to the activity of cd8+ ctl, still occurs in 62m-/-mice. however, 621114mice show an earlier rise in percentage and absolute numbers of cd4+ t cells by 7 days after infection. cd4+ t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd8+ t cells in normal mice after lcmv infection. cd4+ ciz activity is lower in 62m-/mice and have a later peak than cd8+ ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in 621114-mice. mice can assume some of the cytotoxic functions of the cd8+ cell population from normal mice, including immune-mediated pathology. further, cd4+ t cells from 62m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd8+ t cells from normal mice. mice genetically engineered to be deficient in 62-microglobulin in conclusion, these data suggest that cd4+ t cells from am-/australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd40 and its ligand, in the presence of b cell acting cytokines, such as interleukin 4 (il-4), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd40l and il-4, did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd4ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w2 lpg, u. k. and *nationallnstirute formedica1 research, london, nw7 1aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il-3 has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il-3 on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il-3 gene was disrupted in 129/sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il-3. mice were infected intranasally with a 2 strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il-3 deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd8+ cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd4+t helper (th) cells of the -1 subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within 7 days pi. c t l s were detected by h-2 specific lysis of ldv-specific target cells. these target cells were generated by transfection of 3t3-nm cells with a retrovirus vector carrying the gene (ow 7) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected 3t3-nih cells and cultured for 5 days in the presence of il-2. the ctls had largely disappeared in mice by 30 days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about 3 days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v3000) or an isogenic single-site attenuated vee mutant (v3032) (single amino acid change at e2 glycoprotein position 209 from glu-lys) and at 1,6,24,48,72, and 96 hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il-12, ifn-y, il-2, il-4, il-6, and il-10 gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il-12, ifn-y, il-10, and il-6 were detected in the lymph node, with no changes in either il-2 or il-4; however, whereas cytokine elevations were detected by 6 hours after injection of v3000, elevations were delayed until 24 hours following infection with v3032. cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il-10, and il-12 were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il-10 and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il-12 andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il-10. to confirm the importance of the immune system in clearing rotavirus infection we infected rag 2 knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. 1990, 121:149-160) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement 4b binding protein (bc4-bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature 1988, 355:176-179) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc4bbp ( kotwal, am. biotech. lab., 1994) and has also been shown to bind to two important complement components, c3 and c4, and block the complement cascade at multiple sites in vitro (kotwal et al., science 1990, 2502327-830; mckenzie, kotwal et al., j. inf. dis. 1992 , 1661245-1250 . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas 1992, 89:628-672) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature 1993,366:748-751) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn2 mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, 1993 mol. immunol. 30:20) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h-2 haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c5deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, 1981) . various immune regulatory molecules were investigated in inbred susceptible 129/j (129) mice and resistant c57bl/6j (b6) mice which share the same mhc haplotype. when they were given 200 eid, of sendai virus intranasally, 129 mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in 86 mice. virus was eliminated from the infected lung 3 days earlier in b6 mice than in 129 mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il-2, ifn-y, il-10, and il-4 producing cells were comparable for each strains of mice, while more il-6 producing cells were present in the lung of 129 mice. however, the numbers of these cytokine producing cells peaked in 129 mice 3 days later than in b6 mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il-10, and il-6 were present in 129 mice than in 86 mice. recall cytokine production in the draining mln showed only the presence of il-2, ifn-y, il-10, and il-6, while no il-4 was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in 129 mice. therefore, the susceptibility of 129 mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th2 pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il-2, ifn-y, tnf-a or il-7 in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il-4 by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il-4 by rvv was shown to inhibit the host il-2 response required for the development of ctl's. no production was also significantly suppressed by il-4. rvvs expressing il-5 or il-6, although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i2 (il-12) doses of >i00 ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd8+ t cell expansion and ctl activation, and up to 2 log increases in viral replication [orange, wolf, and biron, j. immunol. 152:1253, 19941 . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il-12 induced tnf-cx expression and that lcmv infection synergized with il-12 for this induction. administration of antibodies neutralizing tnf reversed the il-12-induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd8+ t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il-12-treated uninfected mice and were dramatically elevated in il-12-treated virus-infected mice, including: 1) decreases in body weights; 2) elevation of circulating glucocorticoid levels: and 3) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il-12 for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a 50% decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c57bu6 mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-or pkh26-gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately 10% of the transferred bone marrow cells accumulated in spleens after 20 hrs and 30% of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn-7 mrna by in sifu hybridization. treatment with anti-agmi or anti-nk1 .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box 334, canberra, act, 2601 australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c57bu6 (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd3 antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c57bu6 mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd3 antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn-7 and il-2. in contrast spleen cells from infected c57bu6 mice produced ifn-y and l-2 to levels that were only slightly less than uninfected controls. a-6 production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd3 stimulated lymph node cells from infected mice produced elevated ifn-1 suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il-4 or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl-2 proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl-2 does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il-4 produced a fully productive vsv infection at all times analyzed, up to 3 weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells (50-1 00 fold by week 1 and 1,000 fold by week 2). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il-5 and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va 22908 we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or 8-galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed 5 or 14 days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th2 type cytokines when stimulated with rsv infected spleen cells . however, the levels of 11-5 and 1fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il-2 were higher in the bulk culture from vac-f primed animals. the il-4 and il-5 production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed 14 days after intranasal inoculation produced much lower levels of il-4 and 11-5 while the levels of il-2 and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day 3 and subside as cd8+ t cell responses are activated at day 7 post-infection. in contrast, 02m-/-mice, lacking cd8+ t cells, have dramatically elevated nk cell responses on day 7 postinfection. the 02m-/-response is evidenced by increased nk cell activity, as well as up to 5-fold increases in blast and total nki.i+cd3-cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)-2independent, whereas day 7 nk cell responses in 02m-/-mice are csa-sensitive and il-2-dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il-4 and transforming growth factor4 (tgf-0) were examined. induction of il-4 mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days 0, 3, 5, 7, 9, and 14 post-infection demonstrated delayed induction of il-4 protein as compared to ctl activation. tgf-0, evaluated in biological and elisa assays, was induced maximally at days 7 to 9 post-infection. the kinetics of tgf-0 production by cells from infected 82m-/mice was similar to that of normal mice. however, cells from 02m-/-mice produced il-4 at early but not at late times postinfection. together, these results suggest that either il-4 is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the 02m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il-1p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days 1-6 after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after 6 days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with 103 pfu of either a wild type strain of mv(chicago-1,chi-1) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over 28 days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi-1 on d4 (105.7 pfu/ third of implant), and moron d21 (103.2 pfu/ third of implant). hematoxylideosin stained sections of chi-1-infected thymuses showed marked distortion of the cortex and medulla by d4 with thymocyte poilolosis and decreased cellularity. by d14, these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd3,cd4 and cd8 were analyzed by flow cytomehy. there were significant decreases in the cd4+cd8+ cell pop-ulation by d10 with complete loss of all such cells by d28 with chi-1, and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae3) or cd15 co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d4 in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d21. only rare cells stained both with mv ha and cd2 or cd4. mv ha was not expressed over background on any cd4+ cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl-5) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd4 were found to drop dramatically. other cell surface antigens such as cd18, cd43, and cd45 were unaffected during infection with myxoma virus. further more, the downregulation of cd4 by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd4 levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p56lck associates with the cytoplasmic domain of cd4 we have also examined the association of p56lck with cd4 as well as steady state levels of p56lck during viral infection. the modulation of surface cd4 has also been described in hiv infected t cells suggesting that the loss of cell-surface cd4 may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s 1 1 o r us2-6 gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before 18 months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd8+. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p2a(paucisymptomatic) group than in the pl(asymptomatic) and the p2b-f groups (symptomatic group). the cohort of children has now been followed during 4 years, and 46 children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c 10 weeks of age) showed no deletion of their cognate vp14* t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp14+ t cells characteristic of c3h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv-1 endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i1 (pol 11) template. the promoter contains a tata box 30 bp upstream of the transcription initiation site, binding sites for several pol i1 transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol 11. in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt-2, but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i1 promoter (6 pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used (60 pglml), in the range of a typical polymerase in (pol 111) promoter (va-i). purified tax, transactivates this promoter 5-to 10-fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the 0.5 m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i1 promoter and a unique pol i11 promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i1 template containing the 21 base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i1 expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as 16 hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within 48 hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and 11 mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd54 by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within 2 h of virus infection, while an indirect mechanism, regulated by type 1 interferon (ifn), operates within 24 h of virus infection. cd54 expression increased by 4-5 fold within 2h of wnv infection on hef, and by 6-7-fold within 24h. wnv-inactivated, conditioned supematants removed from infected hef cultures after 4 h incubation did not alter cd54 expression on unqimulated hef. whereas conditioned supernatants from 24 h-infected cultures increased cd54 expression by about 1.5-2-fold after incubation for 24 h, but not after 4 h, similar to cd54 induction by 200ulml of ifn-p. increased cd54 expression on hef by wnv was also cell-cycle dependent. cd54 increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd54 by types 1 and 2 ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus 2 and 5, and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd54 expression on hef after 24 h. another alphavirus, ross river, was able to induce cd54 but only by the indirect mechanism of type 1 ifn-dependent release. poly i.c, also, increased cd54 expression to the same extent as ifn-p after 24 h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd54 expression in a manner similar to wnv. the ability of flavivhses to induce increased cd54 expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b8 patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position 3 of the pi7 gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla 68 with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position 7, not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p2microglobulin in the er results in stable cell surface expression of the trimeric mhc-1 molecule. in previous studies we showed that in hpv-16 positive cervical carcinomas there was a loss of mhc-1 protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc-1 is mediated by an hpv-16 encoded protein. human keratinocytes were transfected withvarious hpv-16 constructs including pat16, the full length genome, pat16esx the full length genome with a premature stop codon in e5, puc.et16, the e6 and e7 oncogenes only, and pkve5, expressing e5 from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for 48 hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap-1 and mhc-1 heavy chains in the cells with pat16 as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat16esx and puc.et16 lines showed comparable tap-1 and mhc-1 protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap-1 and mhc-1 mrna between the different cell lines. the data indicate that expression ofhf'v-16 e5 leads to post-transcriptional loss of mhc-1, presumably by interfering with tap. to map and characterize functional differences between e1a of ad5 and adl2, we previously constructed a series of hybrid ad5/12 e1a genes and used them with ad12 e1b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad12 e1a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd8+ as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin-10 from a variety of organisms. many of the numerous immunological properties ascribed to interleukin-10 are shared by the product of bcrfl and this has led to it being termed viral interleukin-10. in order to investigate the activity of viral interleukin-i0 (vil-10) and its interactions with the human interleukin-10 receptor we have expressed the protein in a bacterial and the eukaryotic cos-7 expression systems. the bacterially expressed vil-i0 was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt4.l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp-1. a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil-10 protein that interact with the hil-10 receptor and confer its biological activity. a number of these mutants have been expressed in the cos-7 expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil-10 that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i0 within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the 10.4k or 14.7k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the 15 ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e3l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k3l protein shows sequence similarity to the eukaryotic initiation factor 2a (eif2a) that is phosphorylated and inactivated by pkr. the k3l protein competitively binds the kinase and blocks host eif2a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin-18, tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of 125i-ifna to u937 cells and binding of 125i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for 1fnc1fp. direct binding of 1251-ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd4+ t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a20 cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase 11, wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a118083 t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa 19107. antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p2-microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids 50-57 (h2-kk), 147-155 (h2-kd) and 366-374 (h2-db). the frameshift was introduced 26 amino acids upstream of the h2-kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h2-i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h2-kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h2-dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr8 the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np 383-391 srywairtr was studied using hla b27-restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b'2705 in vitro, and for presentation to ctl clones by hla 827positive targets. even conservative amino acid substitutions of the peptide residues p 4 , 7, and 8 profoundly influenced ctl recognition, without affecting binding to hla 8'2705. these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l 4 gene segment (but not those using tcr va12) were also sensitive to p1 substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class 1. these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla 827, p i , p4 and p8 are "flag" residues with tcr accessible side chains. the e3/19k protein of human adenovirus type 2 (ad2) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e3/19k protein mediates binding to mhc molecules. apart from a stretch of approximately 20 conserved amino acids in front of the transmembrane segment, e3/19k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad2 e3/19k protein. we show that e3/19k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in 293 cells. based on the differential binding of monoclonal antibody tw1.3 and cyanogen bromide cleavage experiments, a structural model of e3/19k is proposed, in which cys 11 and cys 28 as well as cys 22 and cys 83 are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e3/19k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e3/19k molecules. mutation of the three other cysteines at position 101, 109 and 122 had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e3/19k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd8' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent -75% of the u.s. population are being tested to determine which of 5 abundant cmv proteins they recognize by a cd8' ctl response: p28, p65, p150, ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd8' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class 1 epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a2.1 developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p65 and gb together with either of two lipid-based adjuvants, commercially available d0tapm (bcehringer-mannheim) or mf5gth (chiron, emeryville, ca). our preliminary studies with hsv-2 gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa2 as a restriction element in the transgenic model. the ctl response to sendai virus in c57by6 mice is directed almost exclusively to a single h-2kb-restricted epitope derived from the virus nucleoprotein, npj24-332 (sev-9). analysis of 18 independent t cell hybridomas generated from c57by6 mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev-9 bound to kb has shown that the side chaiis of peptide residues phpi, gl484, a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi484 and a d 5 protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev-9. to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev-9 analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp4 and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep1 and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev-9. these data are consistent with a critical role for the gi94 and a d 5 in governing tcr-sev-9eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla42.1 restricted ctl from hla-a2.1 uansgenic mice was the m1 peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/8/34-specific murine ctl that recognized an hla-a2.1 restricted epitope distinct from m1. using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/8 nsl protein. because these ctl did not recognize targets infected with the a/alaska/6/77 saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a2.1 specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common 8 amino acid sequence and two distinct sets of bmding mtif residues. however, the n0name.r peptide was able to sensitize ctl for half maximal lysis at 80-2500 fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions 4 and 8 and was not recognized at any tested concentration, although it bound with higher &ity to hla-a2.1 than the peptide from a/pw8. the a/pr/8 nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c57bv6 mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd8+ immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd8' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h-2b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd8+ ctl response directed against three wellcharacterized epitopes presented by h-2db molecules: "396-404 (fqpq-ngqfi), gp33-43 (kavynfatcgi) and gp276-286 (sgven-pggycl). the h-2db motif is characterized by a sequence of 9 to 11 a.a. with two anchor residues: asn at position 5 and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the 34 lcmv peptides and 11 known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides (28/34) did not bind to db. the other 6 (including the 3 epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb2 4at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt1.aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt1.aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently2~3. rt1.a alleles are naturally linked with the tapa or the tapb allelic group4. we have set out to characterise various alleles for the rt1.a molecule, and find that, for the majority of tapaassociated rt1.a molecules, 3 acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt1.a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b8. seventeen variant peptides were examined for their binding to hla b8; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b8 positive hiv-1 infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, 16132 genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd4+ human t cell lines and clones specific for different hiv antigens (gp120, p66, p24), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct 06520 listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p60 is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p60 is processed by the host cell into the nonamer peptide p60 217-225 and is presented to cytotoxic t lymphocytes by the h-2kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p60 to show that the rate of p60 217-225 production is proportional to the amount of antigen secreted into the host cell cytosol. p60 is degraded in the host cell cytosol with a half life of 90 minutes. the appearance of p60 217-225 is coupled to the degradation of newly synthesized p60. we have determined the rate of intracellular p60 secretion and by accounting for the rate of p60 degradation we estimate that approximately 35 p60 molecules are degraded to produce one p60 217-225 epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd8+ and class i hlarestricted by the b7 molecule. all three clones recognized lllb and rf but not mn strains of hiv-1. using vaccinia vectors expressing truncated versions of the hiv-1 envelope, the clones were found to recognize an epitope within amino acids 287-364, but not including 312-328 of gp120. further mapping of the epitope with synthetic 20-mer peptides overlapping by 10, or 25-mers overlapping by 8, was unsuccessful. the sequence of the region of gp120 recognized by these clones was compared to the predicted hla-87 peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the 10 aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc 835 gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of 835 allelic variants of ebv-lcl. by 835 restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described 8'3501 peptide epitope from pp65 was not able to prime the autologous 835 ebv-lcl for killing by the pp65-specific ctl, whereas a recombinant vaccinia virus expressing whole pp65 could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp41-specific cd8' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of 835 ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b'3502 allele are recognized and killed by the pp65-specific t cell clone, and cell lines carrying 8'3501 alleles are recognized by the hiv gp41-t cell clone. we conclude that the reported cmv pp65 b"3501 restricted epitope is not correct, since the ctl in question will only recognize 6'3502 alleles in combination with the correct pp65 epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus "147-155, ha204-212 and ha210-219 was found to be similar. an 11 amino acid peptide corresponding to ha204-214 which contains the 204-212 epitope was transported at a similar efficiency as the 9 amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue 215, this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at 260.8 fmolelminute (+/-30.5). the km for this peptide was found to be 231.9nm(+/-31.8). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., 19107. previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids 147-155) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct (147-158/r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus (75 < n < 100 amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs (50-158/r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed 6r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the 6r construct was presented at a level equivalent to that seen with the wild-type 50-158/rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den-2 was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den-2 will interact with mim and lymphocytes in different manners, we used two isolates --plo46 strain (obtained from a df patient during taiwan 1981 outbreaks) and 16681 strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after 24 hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd3icdi9 mab and complement prior to adherence procedure and the purity of mim culture was checked by cd14 surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den-2 strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den2 strain. in addition, this dhf-den2 strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv-1) and lymphocytic (h9) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den2 strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den2 strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that (1) different strains of dengue virus could orchestrate quite differently with immune cells, (2) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and (3) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. (2) when viral yields were enhanced early than day5 post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv-1 using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny 10016. primary isolates of hiv-1 have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd4-based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg12 is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd4 binding site (cd4-bs) on gp120. cd4-lgg2 is a recombinant molecule in which the variable domains of both heavy and light chains of lgg2 were replaced with the first and second immunoglobulin-like domains of human cd4. both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg12 and cd4-lgg2 were added at 25 pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p24 production was measured over 14 days of culture and an end-point titer of hiv-1 in the presence and absence of added antibody was determined. both igg12 and cd4-lgg2 were found to reduce the original hiv titer from seven plasma samples with high virus titer (>250 tcid50/ml) by up to 625-fold. this is in comparison to soluble cd4 which only reduced viral infectivity by 55-fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd4-bs of hiv-1 gp120 are able to effectively neutralize primary isolates of hiv-1 and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv 16 the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of 66 overlapping synthetic peptides corresponding to the entire hpv16 capsid proteins was used to generate hyperimmune sera. several antisera against 3 different peptides were reactive with intact hpv16 capsids at titers up to 1:150.000. hiv-1 serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md 20892. previously, we showed that hiv-1 proteins conjugated to 8. abortus (ba) could generate anti-hiv-1 neutralizing antibodies in mice even after depletion of cd4* t cells. in this study a 14-mer peptide from the v3 loop of hiv-1 (mn) was synthesized 013) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v3-klh induced mainly igg1, whereas v3-ba induced all igg isotypes but lgg2a predominated. fecal extracts from mice immunized with v3-ba were shown by elsa to contain iga antibodies. sera from these mice bound gp120, expressed on the surface of infected cells. sera from mice immunized with v3-ba inhibited syncytia formed between cd4' t cells and chronically infected [hiv-i (mn)] h9 cells. inhibition of syncytia, formed by other hiv-1 lab. strains correlated with the degree of their homology with the v3 region of hiv-i (mn). to mimic the efffect of hiv-1, mice were depleted of cd4' cells using anti-l3t4 at the time of primary or secondary immunization. following primary immunization, cd4+ t cell depletion abrogated v3-klh antibody responses, whereas responses to v3-ba were retained and sera from these mice were able to inhibit gp-120mediated syncytia. in secondary responses, cd4' t cell-depletion prevented boosting to v3-klh, but v3-ba increased anti43 and syncytia-inhibiting antibodies. these results suggest that: 1. 8. abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and 2. that infection with hiv-1 with subsequent impairment of cd4' t cell function would not abrogate anti-hiv-1 antibody responses if 8. abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh61-1p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn 38 10 i, 2departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c57bl/6j(b6) mice treated with the mab mel-14 to l-selectin have been analysed following i.n. infection with sendai virus. mel-14 treatment caused a 70-90% decrease in the lymphocyte recruitment to the mediastinal (h4ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd8+ ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day 10 after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel-14 antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel-14-treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd4+ th precursor cells into lymph nodes. facs sorting for l-selectin+, 8220+, and l-selectin-, b220+ cell populations from the mln and the cln of normal b6 mice 9 days post sendai virus infection, showed that the afcs were from the l-selectin-, b220+ cell population, a population which comprised 6-10% ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv-1 infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position 582 in gp41, is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd4 binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position 281, upstream from the v3 loop, does not involve the same site and does not involve v3. substitution of thr or ile also confers resistance. replacement of the v3 loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v3 peptide of mn but is affected by regions outside v3. the other appears to be conformational and outside v3, but its functional recognition is influenced by the v3 loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids 579-613 of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus 10 to 100 fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids 603-622 of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p<0.05). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv 16 proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e6 a n d e7 genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e7 either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e7-specific and cd4+ t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e7 protein of hpv 16 was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in 4 groups of mice (1) those not challenged with e7 (2) mice not grafted but challenged with e7 protein in the ear (3) mice primed by grafting with 107 hpv e7 expressing cells and challenged with e7 protein (4) mice primed by grafting with 5 x 105 hpv 16 e7 cells on day 7, grafted again with lo7 hpv 16 e7 cells on day 14 and challenged with e7 protein in the ear. mice optimally grafted and challenged (group 3) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group 4) exhibited igg antibody levels comparable to the control group (1). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp70) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during 18 months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr1 antibodies was also investigated in 100 hepatitis c viremic individuals and 25 non-viremic patients. a high frequency of positive reaction (90%) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp120 v3 loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., 1992) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of 18 reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd3cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = ,005) and with serum rotavirus iga titers stimulated by the different reassortants (p < ,001). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, 55 individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v3 profiles were determined using synthetic proteins derived from the amino acid sequences of the v3 region of 5 laboratory strains of hiv-1 in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v3 regions of mn, sf2, ny5 and han/sc. less than 50% of individual in each group recognized the v3 peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< .001). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p24 ag inhibition assays. although most patient serum was capable of inhibiting p24 ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v3 profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at 8 days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by 15 days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at 8 days post-infection, timepoints from 30 days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about 2-fold by 15 days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd4+ lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd29 were evaluated in 49 uninfected controls (group l), 84 hiv-1 positive patients with 220% cd4+ t cells (group 2), and 47 hiv-i-infected patients with ~2 0 % cd4+ t cells (group 3). most of these subjects also had 3-color staining for cd4\cd45ro\cd45ra. the appearance of positive cd29 and cd45ro on hivinfected and uninfected cells correlated well (r=.82 p<.ool). the percentage of cells staining cd4+\cd29+.(bright plus dim) was 43.3 (95%cl 37.3-49.4) in group 1, 28.9 (27.5-30.4 ) in group 2, and 10.2(8.6-11.9) in group 3. the respective values for these groups that were cd4+\cd2gbwm was 30.6 (26.9-34.3), 20.7 (1 9.3-22.2), and 7.4(6.3-8.6). values for cd4+\cd45ro+ were 33.7 (31.8-35.5), 21.8 (20.5-23.1), and 9.9 (8.5-11.3), respectively. in single factor discriminate function tests, the %cd4+\cd29+ cells best predicted subject group (87% correct), proving to be a better discriminator than %cd4+\cd29b'h' (77% ~orrect),cd4+\cd29~"" (5 l%), cd4+\cd45ro+ (75%) and cd4+\cd45ro+\cd45ra-(63%). overall, no advantage was seen to splitting the cd4+\cd29+ cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd4+\cd45ro+ compartment into cd45ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv-1 infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl60 recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp160 were performed at month 0 and 1 and two boosts of recombinant gpl60mn/lai at month 3 and 6 in alum or incomplete freund adjuvant(1fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from 18 out of 20 donors : seven out of eighteen (39%) were found to present envelope specific cytotoxic activity at months 2, 4, 7 or 12 post immunization ; this activity was characterized as a cd3+,cd8+, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd8+ cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al 35294. mhc class i restricted cd8' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il-2 and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd8' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd4+ and cd8' t cells by their expression on the surface of distinct d45r isoforms. cd45ra is expressed on naive or virgin t cells, while cd45ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd8+ t cells. the cd8' cells were stained for cd45ro' (pe) and cd45ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a 4 hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd45ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd45ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd8+/cd45ro+ t cells. similarly, cd8+/cd45rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd8+/cd45rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd8+ t cell response in lcmv infection we have observed a profound activation and proliferation of cd8+ t cells with a 10-40 fold increase in total number peaking at day 8-9 post infection. in c57bw6 mice, most of the viral antigen is cleared by day seven, and after day 9 the total cd8+ number per spleen drops about 10-fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd8+ cell remains remarkably stable between day 7-8 of the acute infection and for many months thereafter. thus, the decline in the cd8' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd4+ cells from c57bl6 female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd4+ cells either by magnetic bead depletion of non-cd4+ cells, or by facs after staining with anti-cd4 monoclonal antibody these enriched (>90% cd4+) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + 3x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at 111600 cd4+ cells, compared to a frequency greater than 1/1ooooo in naive controls. the memory cd4+ cells were further characterized as cd45rb-low (1/472) , cd44-high (1/294), lselectin-low (1/364), and cd49d-high (vla-4-high) (v102). this is close agreement with other phenotyping studies on cd4+ memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn 38105. the immune response of influenza a and sendai-virusspecific, memory cd8' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c57bu6 mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated 5 or 6 days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd8' t cells from the ln and spleen resulting in dna fragmentation of 12-18% ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent 9 days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb41 a3 cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while 1oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb41a3 cells were treated with 500pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by 500pm of nmda was reversed by 300pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c6 on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a118083 to carol s. reiss. pediatrics, university of iowa, iowa city, ia. 52242 mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. 40.90% of suckling c57bu6 (kbdb) mice inoculated intranasally with mhv-jhm at 10 days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at 3-8 weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c57bu6 mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c57bv6 mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids 510-518 (cslwngphl, db), 598-605 (rcqifani, kb), and 1143-1151 (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c57bu6 mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of 3-4 week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of 3-4 week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after 14 days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd8+ t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases3 research units, national animal disease center, usoa, agricultural research service, ames, ia 5001 0 a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a 3.0 kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel80 gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in 4-week-old or 4-day-old pigs, lltba2 replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba2-infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba2, 4-day-old pigs were infected intranasally with lltpa2 or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about 20-fold for lltpa2. abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba2-infected pigs. while replication of lltbres in the brain progressed until death at 7 days post-infection, replication of lltpa2 in the brain ceased by 9 days post-infection and the pigs exhibited only mild clinical signs. since lltba2 is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable 07 invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type 1 (hsv-1) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv-1 in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected 293 cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb2 iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of 3-4 week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day 60 post-infection. in contrast, in a large group of cba mice studied up until 393 days post-infwtion, viral rna could be detected by both techniques in 50% of mice until as late as 268 days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca 92037 our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within 4 months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p27 production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p27 production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v1 region of the siv env gene. the majority (>95%) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v1 region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class 1 positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, 10 fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd4+ t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd4+ t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd4+ t cells with monoclonal antibody gk1.5. both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd4+ t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md 20850; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn-1 epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn-1 subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv-1 infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv-1 of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp27. because of its early expression during the hsv replication cycle, lcp27 represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein4 (nsi) and the lcp27 sequence of hsv-2. the nsi-icp27 protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing 5 pg of nsi-icp27. responder cells obtained from draining lymphnodes were re-stimulated in vitro with p815 cells lransfected with icp27 and then lesled for cytolytic activity on icp27-p815 and control p815. the induction of icp27 specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h-2d haplotype: a dd restricted epitope from the gp160 protein of hiv-1 and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc 27599 a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately 50?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd-1 mice were inoculated subcutaneously with 2 x 10' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo5 ed, of influenza h s . au 24 corn1 mice suffered severe disease and 50% died. only one of 12 ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was 246, while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, 150. in a parallel experiment, no influenza infectivity was detected in the lungs of 12 ha vector immunized mice at 4 days postchallenge. in contrast, 8/12 pbs-inoculated mice and 5/12 inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of 3.04 and 1.93 x lo6 pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h1 hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from 100 to 0.1 ug of dna was inoculated by intramuscular and intradermal routes. from 0.4 ug to 0.0004 ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a 20 week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required 250 to 2,500 times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations (4 ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the 20 weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv40 promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg5rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l 4 caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl6 e7 hpv associated cervical cancer cells express hpv16e7 protein and antibody to hpv16 e7 can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e7 protein of hpv16, and expressing e7 protein in the skin, has recently been described (1) and these mice develop spontaneous humoral immunity to e7 protein similar to patients with cervical cancer(2). to determine whether immunisation could induce immunity to e7 sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv16e7 protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv16 e7 expressing tumour cells in nrro. we then used similar immunisation with e7iquil a to induce e7 specific immunity in fvb (h-29) mice. h-2qskin @s expressing e7 were not rejected by e7 immunised h-zq mice, though immunisation induced antibody to e7, and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl6 e7 lack a tc epitope in the context of h-zq, or that expression ofe7 in the skin from the e7 transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types 16 or 18, and continued expression of the e6 and e7 gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e6 and e7 from hpv16 and 18 with the aim of inducing e6 and e7 specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e6/e7 reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv16 â�¬7 specific ctl from c57bu6 mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi 6 e7 alone, with similar recognition of the defined immunodominant h-2db restricted epitope, e7 residues 49-57. ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa 19486 mice infected with the laboratory strains of a/pr/8/34 (hln1) or the mouse adapted a/hk/68 (h3n2) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h1 and h3 strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr8 and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/92 were susceptible to subsequent infection with the a/hk/68 strain although they were resistant to weight loss and death. other strains such as a/beijing/89 or a/georgia/93 provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/68 challenge than mice previously infected with a/bei/89 and a/ga/93, and were similar to mice that had been previously infected with a/bei/92. thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h-21-ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd8+ ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day 10 after infection. there was little effect on virus clearance. as found previously with cd4-depleted h-2b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd8+ t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd8+ cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd8+ ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd8+, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd8+ responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type 1 (hsv-1) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv-1 to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day 0 and day 21 resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv-1 vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv-1. viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl47-158 and plasmodium berghei cs protein 252-260 were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam3cyj) and dissolved in a 1% dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and 10% soya oil in water. 1 wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of 3 mice intra-peritoneally or subcutaneously at 1.10 and 20 days. 7 days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard 4 hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of 1w:l the plasmodium berghei peptide encapsulated in micmpartides gave 47% iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p3ccs252-260 gave a level of lysis of 82% at an e:t ralioof1w1. these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type 3 poliovirus (shah sauketf) together with the regular type 1 and type 2 canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i1 clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp1. as previously shown for hypsin-mated type 3 poliovirus @vm alone. trypsin used to modify the type 3 component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with 10-fold concentrated type 3 component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase 1 aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type 3 poliovirus. no unexpected sideeffects were recorded phase i1 trials comprised 50 adult volunteers with at least 5 years since the last dose of poliovirus vaccine and 50 children who were due to receive the third dose of the regular immunization schedule at about 2 years. in both groups. 25 individuals received tryipv and 25 were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type 3 poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with 0.4 ug of an h1 expressing plasmid dna (pcmvri1). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at 10 days post boost protection against the lethal challenge was complete at 10 days, 1 month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv-1 t-cell epitopes: generation of hla-b*3501-restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle6 and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv-1 patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys)4 and synthetic htlv-1 peptides which seem suitable for vaccine design. htlv-1 amino acid sequences were searched for eight to 14mer patterns carrying the anchor residues of the hla-b*3501 peptide motif at positions two and eight to fourteen. 65 candidate peptides were synthesized according to the matched sequence patterns. their hla-b*3501 affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b*3501 cells. the fourth group (controls) were inoculated with h3n2 (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i1 restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h3n2 group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i1 ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp120 la1 sequence. seventeen patients participating in a phase i gp160 protocol and 13 patients participating in a phase i gp120 protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into 96 well plates containing peptides overlapping the la1 sequence of gp120, pulsed on day 7 with tritiated thymidine and harvested and counted on day 8. results: the percentage of patient's pbmcs from each trial with an lsi 2 5 to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp160 or gp120 proliferate to multiple peptides within the gp120 molecule. reactivity from the end of c1 through early c2 (lai #i 12-21 1) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v3 loop peptide (la1 #300). although the percent reactivity to the entire gp120 molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c3 (lai #319-348). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il-12 production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] 1 and 2a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna 3 proteins (3a, 3b, 3c). and ebna 3c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna 1, 2, lp or lmp 1. the ebv-specific ctl lines expressed cd3/cd8 and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class-11. these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna 3 family of proteins and lmp 2a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology,2 genetic therapy, inc., gaithersburg, maryland 20878 replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. 1 :192-200; 1994) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb274nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of 100, 50 and 10. in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: 80.3 f 1.3% vs 6.2* 0.5%. 49.6*1.6% v s 5.7*0.4%. and 22.8*3.5% vs4.8*0.5% (meanrts.e.m., n=3; p<o.ol, all comparisons) a t e:t ratios of 100, 50 and 10, respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping 20-mer peptides derived from p24. two individuals (008 and 617) had ctl against p24.14 (a(mino) a(cids) 263-282, krwiilglnknrmysftsi), two others (038 and 157) recognized p24.17 (aa293-312, frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p26.14 (8653) and p26.17 (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of 9 aa overlapping 10-mers and to test for hiv-1isiv crossreactivity. both two p24.14 epitopes (possibly restricted by hla-a2) and the p26.14 epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p24.17 epitopes is in progress. the sn p26.17 epitope was also non-crossreactive, despite only one aa difference (position 6, s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p24 in humans (johnson ji 147,1512 ,1991 , buseyne jv 67,694,1993 and to p26.17 in cynomolgus macaques (gotch jmprim 22.1 19,1993) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd-4 and cd-8 1-cells, b-cells and adherent cells, while an average of 5% of virus was associated with lps-stimulated 8-cells and adherent cells. in the second experiment the combined function of antigen presenting cells, 1-helper cells and 8-cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day 0, and decreased at days 2, 3 and 4 post-cvb3, infection relative to unlnlected animals. conclusions: these data indicate that cvb3, associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb3, modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb3,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i11 j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih 3t3 fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of 2.8 kb in hind i11 j and of 7.7 kb in hind i11 j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i11 j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the 7.7 kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv5-4 progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v 5 -4 cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv5-4 was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a 5kb transcript in northern blot analysis. a 2 kb cdna was isolated and codes f o r a novel polypeptide o f 800 amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/305/57 in in uitro assays of viral infectivity of the mouse b-lymphoma a20-1.11. we have found that although infection sensitizes the a20 cells for recognition by both class i and class i1 restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a20 cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/305/57 from a different passage history, we find that n o t only are the infected a20 cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a 10-2 the therapeutic and prophylactic antiviral efficacy of interleukin-12 (il-12) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il-12 commenced 6 hours after mice were infected w i t h hsv, and was continued daily for a total of 5 days. il-12 therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. 1 .0x106we~ells/well of both adherent and nonadherent cell types were infected with two different den-2 virus strains : (1) thiland strain isolated from the dengue hemorrhagic fever(dhf1 patient and (2) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den2 and dhf virus strain replicated much more efficiently (2.3x104pfu/ml on day 3 p.i.1 than df strain; (b) nonadherent cells were 10 times less efficient to produce viral yields than adherent cells (dhf and2 df strains peaked at 6.3~10' pfu/ml and 1.5~10 pfu/ml on day 3 p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi-2/crmn38k gene trigger an inflammatory response by 36-48 hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at 48-72 hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo 63110 murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice 2-8 months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for 14 days. by this method, 1 pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted 1:lo. scid mice were used as a second detection assay for mcmv. using 46 scid mice and different doses of virus, the lds, of mcmv in scid mice was 2-3 pfu. mcmv infections were detected when 10-25 pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of 34 spleens, 34 kidneys, and 37 salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after 10-40 days. in addition, lytic mcmv was detected in scid mice 28 days after injection with 2(10)' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the 72kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp72 mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp72 expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp72, but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp72-negative cell line showed no difference from control viruses, and the presence of hsp72 did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp70 proteins act as molecular chaperones and that hsp72 can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp70 for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw2 in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp72 does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp72 during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs7/bl6 and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days 7 and 10 postinfection, mln or spleen cells from both strains of mice produced il-2, il-6, il-10 and ifn-y, but little or no il-4 or il-5. however, following in vivo treatment with anti-ifn-y antibody, production of 1l-4 and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs7/bl6 mice showed little change in cytokine profile. an increase in il-6 and 1l-10 production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of 1l-2 and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c57/bl6 mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa 19104 rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a 505 amino acid type i membrane glycoprotein with 3 potential n-glycosylation sites at asn37, asn247, and asn319. due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble 434 amino acid form of rgp lacking a transmembrane domain (rgpt434). rgpt434 was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt434 secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt434 was secreted only when asn3l 9 was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt434. although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of 6 his residues was added to the cooh-terminus of rgpt434. this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+2-agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev-21 locus is reduced 85 to 95% compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci (1,6,10 and 11 ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a9 -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad 65. one of the major isletcell antigens, shows a high degree of homology with the 2c protein of cbv-4. studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp60 and gad65, are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p53 alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p53 expression in 31 of 52 (60%). p53 detection by ipox is usually associated with mutated p53. sscp for exons 5-9 revealed p53 alterations in 2 of 25 specimens, and loh at exon 4 was seen in only 2 of 12 evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p53. we recently reported that sv40 induces mesotheliomas in rodents, and that sv40-like sequences are present and expressed in mpm. of note, sv40 large t antigen (tag) binds and inactivates wt p53 i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p53 may result from its physicdl binding with tag. tag was detected by ipox in 32 (62%) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p53 (p2 = 0.008, fisher's exact text), and preliminary experiments suggest co-precipitation of p53 and tag. finally, we demonstrate that waf-1 is not detectable in mpm expressing wt p53 suggesting that p53 is inactive. we speculate that tag expression in mpm binds to and inactivates p53 contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in 90% of cervical carcinomas and possibly 95% of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a2 expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type 16' and hla-a2'. culture media consisted of rpmi-10% human serum supplemented in three cases with 15 u/ml il-7, in another three cases with 3 u/ml il-2 and 15 u/ml il-7, and in the final three cases with 10 u/ml il-2 and 15 u/ml il-7. the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c33a (hpv, hla-a2') transfected with either the hpv type 16 e6 or e7 genes. one patient's cells maintained with 10 uiml il-2 and 15 ulml il-7 showed hpv e7 protein specific cytotoxicity in a hla-a2 restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k-562. the ctl efficiently lysed a c33a-e7 clone but in contrast a vector only transfectant was not lysed. the lysis of c33a-e7 was blocked with a-cd3 and a-cd8 antibodies at 66% and 41% respectively. facs analysis determined that this ctl population was 92.8 % cd3'cds' and 7.3% cd3'cd4'. limited e6 recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e7 responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv-1 infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv-1 infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b8 restricted epitopes: p17-3 gag and amino acids 18-26 of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv-1 infection may be influenced by an effective host immune response against hiv-1 and/or by viral properties. to investigate the cellular immune response to hn-1 we analyzed ctl activity against different hiv-1 proteins in long-term asymptomatic hiv-1 infection as well as during rapid progression to aids. methods: 10 longterm asymptomatic individuals with cd4 counts >500 celllpl after more than 8 years of infection were selected from the amsterdam cohort study on aids versus 10 subjects who progressed to aids < 5 years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv-1 ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus 70 which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus 11 isolates from an outbreak of disease in southem india. sequence analysis within the 5' untranslated region reveals that these isolates fall into two groups that differ by -20% (equivalent diversity to that seen between between published sequences of poliovirus 1 and coxsackie a 9 virus). these two groups of viruses also differ in their cell tropism. isolates defined as group 1 by their 5'utr sequence grow equally well on ht29 cells (a human colon carcinoma cell line) and vero cells. isolates of group 2, with one exception, grow only on ht29 cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the 5' untranslated region sequence of a group 1 virus but the protein profile and cellular tropism of a group 2 virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma 021 15 polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c3-i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py4 is the mouse mammary tumor virus 7 superantigen (mtv 7 sad gene, which deletes t cells required for py tumor immunosurveillance in h-2' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i1 and pedatteck (peru). both strains are susceptible to py as 100% of infected animals develop a full profile of tumors. crosses between cs7br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech 11, yield f1 progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs7br) x c57br) and ((czech i1 x cs7br) x c57br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i1 and peru mice by pcr using primers specific for mtv 7 sag indicates that both strains are negative for proviral mtv 7 sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i1 genomic dna. these results indicate that, like the c3hibi, the pedatteck and czech i1 contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c57br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i1 mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis-2 is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis-2 suppresses primary antibody response more efficiently in infected mice. some of the fts-2 infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis-2 show a high degee of homology between this variant and the prototype f-mulv clone 57. in this study we have attempted to localize the genomic determinant of fis-2 which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis-2 and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd4) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th2 cd4 t cell subset. interestingly, when cd4 responses were observed, cd8 tcell responses were also detectable. 2 . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex-3xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp1 was strain specific, and the polymerase (3d) was the most immunogenic and cross reactive. d) a construct comprising 3d and the immunodominant vp1 epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus 1 (ehv-1; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv-1. to generate useful amounts of individual ehv-1 glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv-1 infection. au three glycoproteins induced serum (elisa) antihodies to ehv-1, and ehv-1 gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv-1, protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv-1 antigen were observed for each of the ehv-1 glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv-1. molecular pathogenesis of ural infeetiom 52-331 enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb3, infection in adolescent c3h/hesnj mice. antigen presenting cell, 1-helper cell and 8-cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo5 plaque forming units of cvb3, at day 0 and with lo7 srbc's at days 0, 2, 3 and 4 post-cvbb, infection. splenocytes were harvested 4 days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb3, was associated with 12.9% to 17.4% of cd-8 positive t-cells and w a 11 % to 26% of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a 248% and 738% under non-stimulated conditions. an average of 1 % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. 1994, in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by 24% and 15% of residues, and the 1896 nt s genome is shorter by 163 nt. we surveyed 174 reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; 27 (15.6%) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california (4), arizona (3), new mexico (l), and mexico (2). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to 15.5%. but amino acid distances were less than 2%. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd8+ ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a2+ patients, htlv i-specific cd8+ ctl restricted by hla-a2 recognize a peptide derived from the htlv i tax protein (tax 11-19 llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd8' tax 11-19 specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag 556-564 vl&sdfri) presented by hla-a2. these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c3hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd4+ t helper cells in this autoimmune response by treating mice with the cw-specific gk1.5 monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c3h mice by showing that depletion of cd4+ cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b 1o.br mice than in c3h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl57 (h2n2) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues 204-212 and 210.219. when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl57-immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha 204-219 subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha 210-219 epitope of influenza strain a i m 5 7 in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha 210-219 specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv-1 gp41 and in the n-terminus of hla class i1 beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i1 molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = 0.027 by fisher two tail analysis)in a second, 7 year-longitudinal study of 12 progre.ssors and 12 stable patients we found: (1) the production of crab was seen in 70 -80% of rapid progresson, while the true stables produce only infrequent low-titers crab. (2) in rapid pmgressors, production of crab preceded by 2-3 years the marked drop in cd4 counts. (3) crab production did not correlate with the degree of hyperglobulinemia in these patients. (4) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd4 counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn-7 receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn-1 and 11-2 production, without detectable 11-4, and with decreased 11-10 secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i1-4 producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th-2-type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord-009891-gqrhbhbn authors: rassool, g. hussein title: current issues and forthcoming events date: 2003-09-03 journal: j adv nurs doi: 10.1046/j.1365-2648.2003.02701.x-i2 sha: doc_id: 9891 cord_uid: gqrhbhbn nan severe acute respiratory syndrome (sars) is a respiratory illness that has recently been reported in asia, north america, and europe. sars, an atypical pneumonia of unknown aetiology, has killed more than 200 people worldwide, and infected almost 4000. sars was first recognized on 26 february 2003 in hanoi, vietnam. a man (index case) was admitted to hospital in hanoi with a high fever, dry cough, myalgia and mild sore throat. over the next four days he developed increasing breathing difficulties, severe thrombocytopenia, and signs of adult respiratory distress syndrome and required ventilator support. despite intensive therapy he died on the 13 march after being transferred to hong kong special administrative region of china. on 5 march, seven health care workers who had cared for the index case also became ill (high fever, myalgia, headache and less often sore throat). the onset of illness ranged from 4 to 7 days after admission of the index case. sars is currently believed to be caused by a new coronavirus strain that is spread by close person-to-person contact. symptoms of sars typically begin with a fever greater than 100ae4°f [> 38ae0°c], headache, an overall feeling of discomfort, body aches, and mild respiratory symptoms. after 3-7 days, a lower respiratory phase begins with the onset of a dry, non-productive cough or dyspnoea, which might be accompanied by or progress to hypoxaemia. healthcare workers have been identified as a primary 'at risk' group for exposure to sars. caring for and treating persons with sars exposes individuals to infectious droplets. possible ways sars can be transmitted include touching people's skin or objects contaminated with infectious droplets, then touching your own eyes, nose, or mouth, or through airborne inhalation of aerosolized droplets. the centers for disease control and prevention (cdc), usa, reports that 'transmission to health care workers appears to have occurred after close contact with symptomatic individuals (e.g. persons with fever or respiratory symptoms) before recommended infection control precautions for sars were implemented (i.e. unprotected exposures).' there is also a possibility that the causative agent can remain viable for extended periods of time after drying on environmental surfaces. the risk of transmission of sars increases when caring for patients in the absence of appropriate infection control precautions. instituting proper infection control measures can significantly reduce the risk of exposure and prevent the development of sars among health care workers. as a primary step in infection control, screening and early identification of suspected sars cases is essential. prompt and appropriate isolation of patients suspected of having sars should also be done. at a minimum, health care workers should use the following precautions: • standard precautions • hand hygiene • eye protection for all patient contact. preliminary results of a large-scale trial of a candidate aids vaccine announced by the us-based biotechnology company vaxgen suggest that it is possible to protect some individuals from hiv infection. the trial of the company's aidsvax vaccine appears to show a protective effect among non-caucasian populations, especially african americans, although sample sizes were small. however, for the majority of the participants, who were caucasians, the effect of the vaccine was minimal. the company stressed, however, that the results only represent findings from an initial analysis. additional studies will be conducted to further clarify the data. 'these results are promising. the trial provides clear evidence that a vaccine can work', said dr peter piot, executive director of unaids. 'however, there is an urgent need for more targeted research to find out why the candidate vaccine only seems to work in certain population subgroups. in the meantime, we must continue to expand existing prevention efforts, which have proved their effectiveness when they are implemented at full scale'. this trial vaccine is a promising first step, but an effective vaccine providing widespread protection is still not on the horizon. the aidsvax phase iii trial was the first large-scale human trial of an hiv vaccine. the trial was made possible because of the involvement of over 5400 volunteers from the united states, canada, and the netherlands, the majority of whom were men who have sex with men. phase iii trials, which involve thousands of volunteers, focus on whether a candidate vaccine is effective and can safely protect people. these are only conducted once phase i and ii trials have ensured that the vaccine is safe and produces an immune response against the virus. the vaccine used in this trial was designed to reduce susceptibility to infection with hiv subtype b, which is prevalent in the americas, western europe, australia, and new zealand. to date, 11 subtypes of hiv-1 have been identified. one of the major challenges in hiv vaccine development is to develop one or multiple vaccines effective against all major subtypes of hiv. 'continued hiv vaccine research remains an urgent global need', said dr gro harlem brundtland, director-general of who. 'we will need many more trials to develop effective hiv vaccines, particularly against the most prevalent hiv subtypes, which are having a devastating impact on populations in sub-saharan africa'. dr brundtland added that national governments, the private sector and the international community must ensure that a robust programme of vaccine research is continued and that effective prevention, care and treatment programmes are available. vaxgen is also conducting another phase iii trial in thailand, involving a vaccine candidate based on hiv subtypes b and e. that trial, which involves more than 2500 volunteers, mostly injecting drug users, is expected to provide additional valuable information about the potential efficacy of this type of candidate vaccine. results are expected by late 2003. vaxgen is also currently conducting preclinical research to develop a vaccine against the most 'we are pleased that congress recognizes the nursing shortage and the need to invest in recruitment and retention', said ana president barbara a. blakeney. 'these funds will go a long way toward assisting nursing as a profession and will especially help implement the important programmes authorized by the nurse reinvestment act.' president blakeney noted that ana is particularly pleased with the increases to the nurse corps provision of the nurse reinvestment act, which will support scholarships and loan repayments for nursing students. also encouraging, said president blakeney, are the increases in funding for the magnet, career ladders, geriatric-training grant and facultydevelopment provisions of the act. 'these funding increases, combined with the increases for already existing diversity and nurse education and practice programmes, will go far in advancing the nursing profession', president blakeney noted. the nurse reinvestment act (pl 107-205), which was signed into law in august 2002, expands authority for existing nursing programmes and creates a number of new ones. for example, the new law authorizes scholarships and loan repayments for nursing students who agree to work in shortage areas after they graduate. in addition, the act authorizes public service announcements to promote nursing as a career, loan cancellations for nursing faculty, grants for geriatric nurse education, and grants to encourage nursing best practices, such as those in the american nurses credentialing center magnet recognition program, for excellence in nursing services. 'we thank senator mikulski for her diligence and commitment to securing the necessary funding included in this bill, as well as the other members of congress who supported it', president blakeney said. 'these new nurse education funds will be instrumental in stemming the nation's growing nursing shortage and in boosting the image of this most worthy and rewarding profession'. ana is the only full-service professional organization representing the nation's 2ae7 million registered nurses through its 54 constituent associations. ana advances the nursing profession by fostering high standards of nursing practice, promoting the economic and general welfare of nurses in the workplace, projecting a positive and realistic view of nursing, and by lobbying the congress and regulatory agencies on health care issues affecting nurses and the public. for more information visit http://www.ana.org council of nurses, the world confederation for physical therapy, the world dental federation, and the world medical association have joined in saying civilians pay the terrible price for war in death, injury and disease. speaking with one voice, the international organizations representing a broad spectrum of the world's health professions have spoken out against all armed conflict. the terrible health consequences of even 'conventional' war are borne overwhelmingly by civilians, with especially catastrophic effects on women and children's physical and mental health. death and debilitating injury are coupled with threatened access to safe water, sanitation and food, undermining the health of a population and creating circumstances favourable to epidemic diseases. as infrastructure, homes, and communities are destroyed and people seek safety, more families are added to the already huge population of 12 million refugees and 6 million displaced persons worldwide. precious resources, financial and human, are diverted to war -inevitably at the expense of investment in health, health systems, and education. according to the world health organization, about 35 people are killed every hour as a direct result of armed conflict. in the twentieth century, an estimated 191 million people lost their lives directly or indirectly as a result of armed conflict, the majority civilians. because of the overwhelmingly negative effects on health resulting from war, these health professionals oppose any armed conflict and have chosen to speak out at this time. we strongly encourage governments and ruling parties to find non-violent and democratic means to resolve conflicts and bring about peace. for the 1991 uk census showed that there were 10 times as many white elders as those from all minority groups, although this figure may change with the 2001 census. the diversity, wide geographical distribution and lack of knowledge about access to services among ethnic elders have led to an underrepresentation of their psychiatric needs. this situation is compounded by the fact that the younger ethnic minority population frequently does not share the traditional beliefs and expectation of support from the extended family held by their elders. information available in translated leaflets and posters is not as effective as it could be, for example, by dissemination though general practitioners and other stakeholders. the report recommends that: • acute psychiatric services involving assessment and treatment should remain within mainstream psychiatric services, with ethnic awareness and sensitivity emphasized by training staff in culturally sensitive issues. • services providing continuing care in the community should be developed specifically for the appropriate user group. • efforts could be made to recruit a racial mix of multidisciplinary staff members reflecting the population served. ethnic elders need appropriate and accessible mental health services. • a means to share information would be to set up a website on, or linked to, the royal college of psychiatrists' site. • there should be increased involvement and commitment by all interested stakeholders to involve general practitioners and other key players in establishing good practice for this group. the report comments that a reliable and informative database of good practice available on a website will also offer training opportunities to interested specialist registrars, and could lead to research that would define appropriate services for ethnic elders. for further information contact deborah hart or thomas kennedy in the external affairs department, tel. þ44 (0)20 7235 2351, e-mail: dhart@ rcpsych.ac.uk or tkennedy@rcpsych. ac.uk, or visit http://www.rcpsych.ac. uk/publications/cr/council/cr103.pdf for the full report. a new report finds that despite the wide availability of pain drugs, many patients diagnosed with cancer, are under treated for their pain and suffer unnecessarily. under treatment is the result of poor diagnosis, assessment, and treatment by physicians; coupled with an unfounded fear of the use of controlled drugs such as opioids. the report indicates that the key to better management of cancer pain is improved educational programmes and the development of dedicated pain management teams. cancer pain affects over 3 million people in the us and over half a million in the uk alone. this type of pain can have multiple causes but the most commonly seen are those associated with the tumour itself, and/or with conventional cancer treatments such as chemotherapy and surgery. the severity of cancer pain is often dependent on the type of cancer and stage of the disease. diagnosis of the pain type and severity are major determinants and the key to success of any treatment for cancer pain. failure to accurately assess pain can lead to under treatment. one reason for this failure is that assessment of cancer pain is often complicated by the presence of a number of different types of pain, in addition to a variety of painful conditions that can exist alongside the cancer (such as arthritis). the result is that poor diagnosis of pain in cancer patients remains a significant problem, with many physicians finding it difficult to differentiate between the various pain types; and, many underestimating its severity. cancer pain experts interviewed for the report believe that in many cases there is a failure by medical schools to sufficiently educate students in the basics of cancer pain management and palliative care. this contributes to a poor understanding of available diagnosis and assessment techniques. this is compounded by a lack of continuing education for medical professionals who are often unaware of newer diagnostic measures and the benefits of using more than one assessment measure prior, during and as follow-up to treatment. at present there are numerous best practice guidelines that provide an overview of the most appropriate methods of managing and treating cancer pain. despite the fact that such approaches have been found to be effective in relieving pain in almost 90% of patients, up to half of physicians do not make use of the guidelines. the most appropriate treatment of cancer pain is the use of non-opioid analgesics such as paracetamol for mild pain, a weak opioid such as tramadol for moderate pain, and a strong opioid such as morphine for severe pain. at each step, it may be appropriate to add an adjuvant-a drug that has a primary indication other than pain but is used to enhance analgesia in specific circumstances or to treat pain that is unresponsive to conventional analgesicssuch as an antidepressant. at present, pain physicians use a huge range of drugs; however, in comparison to the us, fewer patients in the uk are treated with opioids suggesting that physicians are still cautious about their use. it was found that 52% of patients in the us compared to 35% in the uk are treated with opioids for moderate pain; and 68% compared to 50% for severe pain. this is a significant difference and highlights that physicians are often using inappropriate treatments for more severe pain including low dosages for insufficient time periods. this is often a result of unfounded fears over addiction and side-effects of opioids -both by physicians and patients. low treatment rates and inappropriate use of available drugs highlight the need for increased education within the medical community. this is a great opportunity for pharmaceutical companies to become involved in awareness programmes, promoting more appropriate treatments, and thereby influencing physician prescribing. this would result in a win-win situation for both patients and pharmaceutical companies. experts believe an additional problem is poor communication between cancer physicians (oncologists) and their patients. physicians do not always inform patients of the likelihood of pain and many patients are still reluctant to report pain due to fears of its significance and attitudes towards treatment, in particular opioids. poor levels of communication not only exist between patients and physicians but among healthcare professionals, where there is still insufficient communication between physicians, in particular consultants to primary care physicians and this contributes to poor continuity of care. the research for this report has found that there are still few professionals involved in dedicated cancer pain management teams. governments and health authorities have a major part to play in making cancer pain management a priority and promoting the development of multidisciplinary cancer teams. the benefits of such a team is evident where they have been rolled out for terminally ill patientswith the patient referred to one palliative care specialist who takes responsibility for stabilizing the pain, setting up a treatment regimen, and informing the primary care physician about appropriate treatment in the outpatient setting. poor diagnosis, poor assessment, the choice of less appropriate treatments, plus patients and physicians fears about controlled drugs such as morphine all contribute to under treatment of cancer pain. education of patients is key to changing attitudes and improving communication between patients and physicians. awareness of the prevalence of pain as a symptom of cancer and the range of possibilities for treatment would aid patient presentation, compliance and ultimately reduce symptoms. physician education is key to better assessment and treatment. dedicated pain management teams have a role to play by trickling down ideas to the level of pcp and thus improving prescribing methods and alleviating unfounded fears over the use of controlled substances. further details on this are available from yasmeen khan at datamonitor, tel.: þ44(0)20 7675 7487, e-mail: ykhan@datamonitor.com type 2 diabetes is a known risk factor for stroke as well as for cardiovascular disease. but a british cohort study published in the february 2003 issue of stroke is the first to determine that type 1 diabetes is also a risk factor (laing et al. 2003) . the effect is similar in all ages, and the magnitude of risk is at least as great as with type 2 diabetes. 'the results from this group of patients with type 1 diabetes show that at all ages death from cerebrovascular disease is higher in the patients with diabetes than in the general population', says lead author susan p. laing from the institute of cancer research in surrey, uk. from 1972 to 1993, the diabetes uk cohort study identified 23 751 patients diagnosed with type 1 diabetes before the age of 30, and it followed them until december 2000, for an average of 17 years. of 1437 deaths, 80 were caused by cerebrovascular disease, which accounted for 4% of all deaths younger than 40 years and 8% of all deaths older than 40 years. standardized mortality ratios (smr) showed that these rates were significantly higher than expected when compared with the general population. smr was 3ae1 (95% confidence interval [ci], 2ae2-4ae3) for men and 4ae4 (95% ci, 3ae1-6ae0) for women. in subjects aged 20-39 years, the risk of cerebrovascular death was increased more than five-fold in men and seven-fold in women compared with the general population. separate analysis of haemorrhagic and non-haemorrhagic stroke still revealed a significant increase in mortality from non-haemorrhagic stroke. the authors conclude that 'at younger ages, the relative risks of cerebrovascular mortality in patients with type 1 diabetes are very high', and that at all ages they are 'still comparable to those of similarly aged patients with type 2 diabetes. 'these observations emphasize the vital need to identify and treat known cardiovascular risk factors in young people with diabetes', said dr laing. source: http://www.medscape.com mortality from cerebrovascular disease in a cohort of 23 000 patients with insulin-treated diabetes investigators have developed a rapid bedside test for plague, according to a report in the january issue of the lancet (chanteau et al. 2003) . the commentator suggests that in addition to being useful in endemic countries, this test will also enhance our preparedness for bioterrorism.'with this test, the rapid and costeffective diagnosis of bubonic and pneumonic plague could easily be achieved by health workers in remote sites', said lead author suzanne chanteau from the pasteur institute and the ministry of health in madagascar. 'use of the test could help to reduce mortality (through rapid and efficient treatment of patients), morbidity (by rapid implementation of preventive measures), and insecticide resistance of fleas (through rational use of expensive insecticides).' the diagnostic test, which takes up to 15 minutes, uses monoclonal antibodies to the f1 antigen of yersinia pestis. while detecting f1 antigen at concentrations as low as 0ae5 ng/ml, this test also detected 41ae66% more infections than did bacteriological methods, and 31% more infections than did elisa. sensitivity and specificity were both 100%, positive predictive value was 90ae6%, and negative predictive value was 86ae7%.in an accompanying commentary, david dennis and may chu from the centers for disease control and prevention in atlanta, georgia, praise this study for its comprehensive evaluation in health centres in madagascar where plague is endemic.'it is anticipated that it will soon be available in plague-endemic areas world-wide, providing opportunities for validations under various conditions', they write. ''in addition, the test…is expected to fill an important need in bioterrorism preparedness and response. key: cord-010689-d2qn1doq authors: chehardoli, gholamabbas; bahmani, asrin title: synthetic strategies, sar studies, and computer modeling of indole 2 and 3-carboxamides as the strong enzyme inhibitors: a review date: 2020-05-12 journal: mol divers doi: 10.1007/s11030-020-10061-x sha: doc_id: 10689 cord_uid: d2qn1doq abstract: indole derivatives have been the focus of many researchers in the study of pharmaceutical compounds for many years. researchers have investigated the effect of carboxamide moiety at positions 2 and 3, giving unique inhibitory properties to these compounds. the presence of carboxamide moiety in indole derivatives causes hydrogen bonds with a variety of enzymes and proteins, which in many cases, inhibits their activity. in this review, synthetic strategies of indole 2 and 3-carboxamide derivatives, the type, and mode of interaction of these derivatives against hlgp, hiv-1, renin enzyme, and structure–activity studies of these compounds were investigated. it is hoped that indole scaffolds will be tested in the future for maximum activity in pharmacological compounds. graphic abstract: [image: see text] the indole compound is classified in the family of heterocyclic organic compounds. the indole skeleton has a benzene ring fused to a pyrrole ring. from a chemical point of view, the nitrogen lone electron pair participates in the aromatic ring, and nitrogen does not have alkaline properties [1] . the numbering order of the indole molecule is presented in fig. 1 . medicinal chemistry, the study of heterocyclic compounds, especially indoles, is essential [2] . the presence of the indole scaffold in the amino acid tryptophan, neurotransmitter serotonin, and plant-based alkaloids has caused many medicinal properties. the chemistry and therapeutic study of heterocyclic compounds have been considered a powerful approach to treat a wide range of diseases [3] . in recent years, researchers have done many studies on the synthesis and biological evaluation of indole derivatives due to their biological properties and their potential to be the target [4, 5] . according to fig. 1 , the indole molecule has seven positions to accommodate different substitutions. thus, the new derivatives of the indole can be synthesized according to these seven positions. studies have shown that sites 1, 2, and 3 are of particular importance and are known as reactive sites for indole derivatives. the presence of the carboxamide moiety at positions 2 and 3 has led to the activity of these compounds tend to inhibit various enzymes and proteins. many studies have been done in this regard, and various properties such as anticancer [6] , antimalarial [7] , anti-inflammatory [8] , anti-diabetic [9] [10] [11] , antimicrobial [12] , antitubercular [13] , antibacterial [14] and cytotoxic [7] have been reported, for indole derivatives. based on our experiences on synthesis of various heterocycle compounds, such as: quinoxalines [15] , epoxides [16] , urazoles [17, 18] , pyrazolone [19] , benzoxazine [20] and especially the synthesis of 3h-indoles [21] , in this paper, we wish to present a comprehensive review about the synthesis, structure-activity relationship studies and computer modeling of indole 2 and 3-carboxamides as potent inhibitors of various enzymes. the synthesis of indole derivatives has long been of interest to researchers in medicinal chemistry and organic chemistry. the methods for synthesizing the derivatives of the indole are very diverse. the main focus of this review is on the methods for synthesizing derivatives of indole 2 and 3 carboxamide and does not refer to the synthesis of indole itself. according to fig. 2 , the generalization of synthetic methods for derivatives of indole 2 and 3 carboxamide has been shown. regarding the derivatives of indole 2-carboxamide, references are made to methods related to the years 2003-2016. to convert the derivatives of indole to indole 2-carboxamide, several reactants are used. however, in most cases, indole 2-carboxylic acid is used as a primer compound. in 2003, silvestri et al. synthesized indole 2-carboxamide derivatives, given the presence of arylsulfonyl and arylthio groups in the final products. they used arylsulfonyl chlorides, arylthiodisulfides, and indoles or ethyl indole-2-carboxylate to synthesize starting compounds. they used koh, etoh, and thf to synthesize carboxylic acid derivatives (route a). the critical step in the synthesis of indole 2-carboxamide derivatives is the transformation of carboxylic acid to amide derivative. in the route a, cdi and ammonia or hydrazine hydrate were used to obtain indole 2-carboxamide. cdi is commonly used to convert amines to amides, carbamates and urea. of course, the route a is very straightforward and useful, and many researchers have used cdi in the synthesis of indole 2-carboxamides [22] . interestingly, the reaction of route b uses the ester derivative as the main intermediate. trimethylsilyl diazomethane is used as a ch 2 source to convert indole 2-carboxylic acid to indole 2-carboxylate. in the next step, ammonium hydroxide or hydrazine hydrate is used for the synthesis of indole 2-carboxamide [22] . in the route c, silvestri et al. used bop, triethylamine, dmf solvent, and corresponding amines in their research on the synthesis and evaluation of sulfonyl indole carboxamide derivatives. bop reagent is commonly used to create peptide bonds. in the reaction of route c, bop activates carbonyl of indole 2-carboxylic acid and converts it into carboxamide using corresponding amines [23] . in 2006, ragno et al. provided suitable methods for the synthesis of indolyl aryl sulfone 2-carboxamide derivatives. they used oxazolidinone derivatives as amine agents, edci, dmap, and thf solvent for the conversion of indole 2-carboxylic acid to indole 2-carboxamide. edci is typically used to activate the carboxyl group and to connect it to amines for conversion to amides. in route d, dmap plays a nucleophilic catalytic role. they also used bop to synthesize their derivatives and gained high yields [24] . in innovative research, onda et al. synthesized new compounds with the title of n-bicyclo-5-chloro-1h-indole-2-carboxamide derivatives according to the route e. in their synthetic method, wsc.hcl is the same as edci used as hydrochloric salt. hobt is used for the synthesis of peptides and the conversion of carboxylic acid to the amide. their goal was to synthesize optically active derivative concerning the indole 2-carboxamide scaffold [25, 26] . in 2012, sindac et al. used ethyl isonipecotate as an amine reagent to synthesize indole 2-carboxamide derivatives. in route f, the synthesis steps are such that first, the ester derivative is converted to carboxylic acid and then to the carboxamide derivative. dipea in the reaction of route f does not have a nucleophilic role and only participates in the reaction as the base [27] . according to the pharmaceutical studies, shonberg et al. proposed route g for the synthesis of indole 2-carboxamide derivatives. their main goal was to connect a drug fragment to indole 2-carboxamide. they made several synthetic steps to synthesize this drug fragment. they used hctu as the ammonium coupling agent. hctu is a derivative of the hbtu compound. of course, considering that hctu has a chlorine atom at position 6, it improves reaction rate, and coupling reactions make fast and accessible [28] . in 2016, sweidan et al. provided a simple and common method to synthesize the new indole 2-carboxamide derivatives. according to route h, from the reaction of indole 2-carboxylic acid with excess thionyl chloride in dry chloroform, is obtained indole-2-acyl chloride. in the next step, the reaction of acid chloride derivative with aminoacetophenone/benzophenone in the presence of pyridine and triethylamine produced the final products [29] . liu et al. used tbtu to activate the carboxyl group and convert it into amide to synthesize the derivatives of indole 2-carboxamide (route i). tbtu was used as a catalytic amount. this compound is used as a coupling agent in the synthesis of peptides [30] . the synthesis of indole 3-carboxamide derivatives is very similar to the synthesis of indole 2-carboxamide derivatives. further, the synthesis of indole 3-carboxamide from 2001 to 2017 is presented. in 2001, duflos et al. proposed route j for the synthesis of indole 3-carboxamide derivatives. in this method, phosphoric anhydride, acyloxypyridinium salt, imidazolide, isourea ester, and acyloxyphosphonium salt were used as carboxylic acid activators. these compounds increase the conversion rate of carboxylic acid to carboxamide [31] . in a relatively different route, scheiper et al. used route k for the synthesis of indole 3-carboxamides. in route k, naclo 2 salt and nah 2 po 4 buffer cause the oxidation of the carbaldehyde derivative to the carboxylic acid derivative. thus, during the synthesis steps, the intermediate carbaldehyde is produced as the main intermediate. in the next step, n-boc-piperazine is used as an amine source. in route k, the mentioned reagents act as previous reactions, and the nmm is used as a catalytic base. hoat is used in biological reactions as a peptide coupler [32] . in 2014, boldron et al., using previous methods and relatively simple reagents (route l), succeeded in synthesizing n-[6-(4-butanoyl-5-methyl-1h-pyrazol-1-yl)pyridazin-3-yl]-5-chloro-1-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-1h-indole-3-carboxamide (sar216471) derivative as p2y12 antagonist [33] . nemoto et al. used me 2 alcl to perform various types of synthetic reactions such as carbamoylation of indoles (route m). an interesting point in their proposed method was the use of isocyanate derivatives in the synthesis of indole carboxamides. they synthesized indole 3-carboxamide derivatives directly from indole, isocyanate, and me 2 alcl. the highest yields of their reactions were obtained in a mixture of ch 2 clch 2 cl-hexane as solvents [34] . in 2017, shi et al. synthesized the indole 3-carboxamide derivatives, including amantadine ring, using oxalyl chloride, dmf, and et 3 n as the base (route n). they used (cocl) 2 as a source of chloride to convert carboxylic acid derivatives to chloride acid derivatives. eventually, the chloride acid derivatives convert to carboxamide derivatives with and high yield [35] . extensive research over many years by researchers has shown that indole compounds exhibit numerous biological activities. as the subject of this review is the study of the indole 2 and 3 carboxamide derivatives and these compounds in particular, have inhibitory activity against hlgp, hiv-1, and renin, the structure-activity studies of these compounds are discussed below. human liver glycogen phosphorylase is classified as the phosphorylase enzyme. in general, phosphorylase enzymes are a group of enzymes that add the phosphate function to a receptor. glycogen phosphorylase is the crucial enzyme in glycogen decomposition that causes the breakdown of this molecule by adding phosphate groups and generates glucose 1-phosphate. according to the crystallographic studies, glycogen phosphorylase molecule is a homodimeric enzyme and it has four ligand-binding sites: an allosteric site, a catalytic site, a caffeine-binding site, and a dimer interface site [36] [37] [38] . the level of phosphorylase activity is directly related to blood glucose control, so the study of the inhibitory activity of this enzyme plays an essential role in reducing the harmful effects of diabetes [39] [40] [41] . therefore, it is vital to discover and investigate the drugs that control the activity of hlgp (fig. 3) . researchers have studied indole 2-carboxamide derivatives for many years as suitable candidates for the inhibition of the hlgp enzyme. it is essential to understand the mechanism of interaction of hlgp and indole derivatives for the design of compounds that have the role of enzyme inhibitor. according to studies conducted in 1986 [42] and 1989 [43], it is possible to allosterically regulate the hlgp conformation by the binding of small molecule effectors and by phosphorylation of ser14. in 2000, rath et al. [44] studied the mechanism of interaction of the indole 2-carboxamide derivatives with hlgp, given the extensive research done in previous years [45, 46] on the design, synthesis, and crystallographic calculation of indole-2-carboxamide derivatives. eventually, they introduced a new allosteric site for binding of indole-2-carboxamide derivatives and hlgp that would serve as the basis for the design of new inhibitors. according to their crystallographic results, the presence of carboxamide and chloroindole moiety has a direct effect on the inhibition of hlgp activity. so, they introduced molecules 2-4 as candidates. due to the molecules 2-4, four ligand binding sites of hlgp, and experimental studies (table 1) , molecule 2 was identified as the most active compound. the results of the complexation of molecule 2 with hlgp are shown in fig. 4 . according to fig. 4 , molecule 2 spans the two types of inhibitor sites (nh carboxamide and cl), thereby increasing the inhibitory activity of this compound. by synthesizing compound 2 and examining its inhibitory activity, ic 50 of 6 nm was reported to be decreased compared to the previous samples (table 1 with hlgp [47] . their binding models showed important aspects of the inhibitor's conformation, subsite interaction, and hydrogen bonding. they used the results of the study by hoover et al. [45] and incorporated compound 5 as the basis for docking and 3d-qsar calculations. according to their experimental results (table 2) , compound 6 was identified as the most active compound and entered into molecular docking and 3d-qsar calculations. according to table 2 and the data obtained by liu et al., there is a significant relationship between the binding free energies (∆g) and the inhibitory activities (ic 50 ) of the derivatives studied. in other words, the more favorable the interaction energy of the indole 2-carboxamide derivatives with hlgp, the inhibitory potency increases. by comparing the ic 50 values of derivatives 5-14, it is found that the role of the x, r substitutions, and the chiral centers in the activity of these compounds are crucial. in fig. 5 , graphical results of the interaction of compound 6 and hlgp are shown. according to the results of docking calculations, the presences of indole ring and carboxamide moiety have a decisive role in the inhibitory activity of these compounds. the carboxamide moiety is very flexible and has both hydrophobic and polar properties. there are three hydrogen bonds between the indole derivatives and hlgp. according to fig. 5 , the first hydrogen bond is related to the interaction of indole nitrogen with the backbone carbonyl of glu190. the second hydrogen bond is the result of the interaction of the carboxamide nitrogen with the backbone carbonyl of thr380, and the third hydrogen bond is the interaction of the carbonyl in r moiety and the nitrogen atom of the lys191 side chain. in 3d-qsar calculations, they used comfa and comsia methods that had similar results and confirmed the results of molecular docking calculations. according to the research conducted up to 2008, the presence of carboxamide moiety in the indole derivatives is essential for their inhibitory activity against hlgp. in 2008, onda et al. devoted their research to adding different groups to the carboxamide moiety [26] . they put compound 15 as the basis for the design of new molecules, according to a study by martin et al. in 1998 [46] . their main purpose was to replace the phenylalanine group of compound 15 with substituents having the hydroxy group. accordingly, they synthesized indole 2-carboxamide derivatives and examined their inhibitory activity against hlgp (synthesized according to the route e). the results of their study in table 3 show that the presence of hydroxy group(s) and their appropriate position, a fluoro group, and nitrogen atom in the aromatic ring increases the inhibitory activity of the compounds studied. it can be concluded that steric hindrance is more important than electronic character in determining the inhibitory activity of indole 2-carboxamide compounds. according to their studies and the evaluation of table 3 , compound 19 was identified as the most active derivative, so was examined for inhibition of glucagoninduced glucose output in cultured primary hepatocytes and for oral hypoglycemic activity in diabetic db/db mice. their results showed that compound 19 inhibited glucose output dose-dependently with an ic 50 = 0.62 µm. interestingly, with the administration of 50 mg/kg dose of compound 19, the blood glucose level significantly decreased at 2 h postdose. the calculations of the binding interaction of compound 16 with hlgp are shown in fig. 7 . according to fig. 7 , in addition to the amide moiety interaction with the backbone of thr380, two hydroxyl groups have direct electrostatic interactions with the imidazole ring of his57 and the backbone of tyr185. this is important the presence of the carboxamide group in two respects: 1) the interaction of the carboxamide group with the backbone of thr380, 2) the polar groups attach to it, and the interaction of these groups with his57 and tyr185 (fig. 6 ). onda et al. continued their research to design and synthesize n-bicyclo-5-chloro-1h-indole-2-carboxamide derivatives as hlgp inhibitors (synthesized according to the route e) [25] . their strategy was to create fused rings to benzene carboxamide. during the biological evaluation, they found that the presence of large rings, such as 7-membered rings, caused steric hindrance, which interfered with the interaction of his57 with hydroxy groups and decreased inhibitory activity. it should be noted that adding methyl and hydroxy groups to the fused ring reduces the activity of these compounds, which can be attributed to the steric hindrance and intramolecular hydrogen bonding. the presence of fluorine atoms in the fused ring strengthens the inhibitory activity of the indole derivatives. according to fig. 7 , the steric hindrance around the central benzene ring is low and small substitutions such as fluorine can be added to the benzene ring [26] . according to their biological evaluation, compound 21 was identified as the most potent inhibitor with ic 50 = 0.02 µm (table 4 ). considering ic 50 = 0.02 µm for compound 21, further research in diabetic model mice has revealed some interesting points about this compound. compound 21 showed an inhibition glycogenolysis value equal to 0.69 µm. the important thing about this compound is that the dose used to reduce plasma glucose level at 2 h postdose is 10 mg/kg. in summary, the data obtained is not sufficient to nominate a drug for more advanced research and requires more complete data. therefore, they measured other properties of compound 21, such as pharmacokinetic profile, oral bioavailability, plasma half-life which were acceptable in male sd rats. the question that arises is why does the r-enantiomer have higher inhibitory activity than the s-enantiomer? to answer this question, they performed docking calculations using compound 21 (r-isomer) and hlgp. according to fig. 9 , the cause of the high activity of compound 21 is due to the lipophilic interactions of aliphatic fluorine atoms and the hydrophobic residues, such as phe53, pro188, and gly186. the difference in the inhibitory activity of r and s enantiomers, according to onda et al., is related to the inappropriate interaction of fluorine at position 1 and the methylene group at position 8. but the important thing about reducing inhibitory activity is associated with the inappropriate interaction of aliphatic fluorine groups and hydrophobic residues of hlgp. the fluorine and hydroxy groups in s-isomer have steric hindrance and thus do not interact with phe53, pro188, and gly186 (fig. 8 ). in controlling the inhibition of the hiv-1 virus, it is important to pay attention to reverse transcription (rt), protease, integrase enzyme, and viral entry/fusion. rt is a key step in the life cycle of retroviruses that is responsible for the synthesis of double-stranded (ds) dna from a viral singlestranded (ss) rna genome. therefore, drugs designed to inhibit hiv-1 virus are divided into several categories: nucleoside reverse transcriptase inhibitors (nrtis), nonnucleoside reverse transcriptase inhibitors (nnrtis), protease inhibitors (pi), and integrase inhibitors (ini) [48] . the main purpose of the design of hiv-1 inhibitors is to suppress the replication of hiv-1 in the long-term therapy and to maintain the function of the immune function [49] . given that delavirdine [50] with indole 2-carboxamide skeletal is used to treat hiv-1, other indole 2-carboxamide derivatives have the potential to inhibit hiv-1 virus activity. it should be noted that many researchers have used nevirapine and efavirenz as the control to compare with indole 2-carboxamide derivatives (hiv-1 inhibition). in the following, the mechanism of action of indole 2-carboxamide derivatives and their structure-activity relationships are discussed. their study showed that this compound is one of the most potent and selective nnrtis inhibitors of the hiv-1 reverse transcriptase [51] . of course, the main problem of compound 22 is its low solubility in water. in 2003, silvestri et al., according to the previous research [51] , designed and synthesized novel indolyl aryl sulfones and tested them against hiv-1 in acutely infected mt-4 cells [22] . the purpose of their study was to investigate the structural changes in the compounds studied. they used the strategy of replacing the carboxamide group to carboxy hydrazide to increase the solubility of compound studied, which reduced their activity. they modified the carboxamide side chain and examined its shift from position 2 to position 3. the results of their study showed that shifting the carboxamide moiety from 2 to 3 positions or switching it to 2-carboxy hydrazide reduced activity. finally, the results of their research clearly showed that the anti-hiv-1 activity of the investigated compounds was dependent on the presence of benzene sulfonyl and carboxamide moiety. as a general result, sulfone derivatives have lower cytotoxicity and higher activity potential than sulfur derivatives. they found that the best results were obtained when 3-benzene sulfonyl indoles contained 2-carboxamide moiety. the ester and hydrazide residues were less active, and carboxylic acid inactivated. given the results of their cell-based assays and the lack of inhibition of the rrt carrying the k103 mutation, it can be said that the compounds studied target the hiv-1 reverse transcriptase (fig. 10) . silvestri et al. in their subsequent studies used interesting strategies for the structural changes of compound 22 to test new sulfonyl indole carboxamide derivatives activity for cytotoxicity and against hiv-1 wt [23] . methyl groups were added to the benzenesulfonyl moiety of compound 22 and one to three glycinamide/alaninamide units to its carboxamide function. nevirapine and efavirenz were considered as reference compounds. the results of table 5 show that the addition of 2 methyl groups and d,l-alanine unit to molecule 22 reduces its cytotoxicity (molecule 23). compared to control drugs, molecules 22 and 23 have higher activity and less cytotoxicity. the results of their study show that the addition of methyl groups at positions 3 and 5 (phenyl ring) is most effective. the d,l-alanine unit establishes strong interactions with the target by increasing the number of hydrogen bonds (fig. 11) . in 2005, ragno et al. studied the binding mode and binding site of indolyl aryl sulfones using docking and 3d-qsar calculations. in their study, they used compound 22 as the reference compound in the interaction with 14 rts [52] . they used the structural data of 14 rts (pdb codes: 1dtq, 1dtt, 1eet, 1fk9, 1hni, 1hnv, 1jlq, 1rt1, 1rt3, 1rt4, 1rt5, 1rt7, 1vrt, and 1vru) to explore the binding mode of the compound 22. these codes represent different non-nucleoside binding sites related to reverse transcriptase. in docking calculations, in all cases, the docked conformations were in agreement with each other, the graphical result of which is shown in fig. 12 . the interesting point about the carboxamide function in position 2 of the indole ring is that the amide carbonyl group is in some instances with indole nh in cis-state and some cases in the trans-state. so they used 3d-qsar calculations to design new molecules that would have the proper hydrogen interactions at the carboxamide position. they used the data of silvestri et al. [22] in their calculations. according to the calculations, 2-hydroxyethylaminocarbonyl and 2-hydroxyethylhydrazinocarbonyl substitutions were attached to carboxamide. the selected derivatives were synthesized, and their activity against wt hiv-1 and mutant strains evaluated (table 6 ). according to table 6 , molecules 22, 24, and 25 have relatively good activity against wtiiib compared to nvp and efv. the important point about double mutant k103n-y101c is that molecule 25 has higher activity than molecules 22 and 24. in general, the number of factors must be considered to nominate a molecule as a drug (fig. 13) . in the years 2011 and 2012, regina et al. [53, 54] came up with new ideas for the design of indolyl aryl sulfones. their main purpose was to bind cycloalkylamino, aryl, or heteroaryl nucleus through methylene, ethylene, or ethoxy groups to the 2-carboxamide moiety and to investigate their activity against wt hiv-1 replication and hiv-1 clades in pbmc cells ( table 7) . the results of their study showed that compounds 26-29 had relatively good activity against wt hiv-1. in their study, they used hiv-1 clades in pbmc cells to measure the inhibitory activity of molecules 26-29 in primary t-lymphocyte cells. table 7 shows that these compounds sodium concentration activates the renin system. this pathway is first activated by the aspartyl protease enzyme, which eventually produces angiotensin i by hydrolysis of the angiotensinogen protein [55, 56] . since renin is the rate-limiting enzyme in this pathway, and since angiotensin has been recognized as the sole agent, a very effective anti-hypertensive have high inhibitory activity in primary t-lymphocyte cells. molecular docking calculations of the synthesized compounds showed that the hydrogen bonding between the nitrogen atom in the carboxamide chain and glu138:b of nnbs-hiv-1-wt rt plays a key role in controlling the activity of these compounds. considering the structure of compounds 26-29, it can be said that the presence of 5 and 6-membered rings without steric hindrance enhances the activity of these compounds. the presence of a nitrogen atom in these rings that cause hydrogen bonding is one reason for the increased activity of these compounds. for further studies on compounds 26-29, parameters such as cytotoxicity, selectivity index, and relative factor were measured that were acceptable (fig. 14) . renin or angiotensinogenase system is known as the blood pressure regulator as well as its electrolytes. lowering blood pressure, reducing circulatory volume, or lowering plasma strategy is to prevent angiotensin i production through renin inhibition [57] . if the renin-angiotensin system is abnormally activated, blood pressure will rise too. based on decades of efforts to discover renin inhibitors, several research groups have reported different renin inhibitors [58] . renin inhibitors are one of the main ways to lower blood pressure, treat heart failure and have adverse effects on diabetes. indole 3-carboxamide derivatives are very useful in inhibiting renin enzyme activity, which will be discussed further. from 2010 to 2011, scheiper et al. conducted extensive research on the discovery and optimization of new and non-chiral indole-3-carboxamide compounds as scaffolds for renin inhibition [32] . they reported the results of their study as an optimal compound, and in the next study used the reported compound as the basis of molecular design. using high-throughput screening, they selected compound 30 as the base compound for future research. due to the structure of compound 30, the other two derivatives were synthesized and evaluated. the crystallographic results of compounds 31 and 32 are shown in fig. 15 . in fact, with the blocking of nh-carboxamide, the interactions of carbonyl-carboxamide and nh-piperazine become essential. according to fig. 15 , nh-piperazine with residues asp32 and asp215 appeared to form ionic hydrogen bonding interactions. another interaction is related to the hydrogen-bonding between carboxamide oxygen and thr77-oγh. due to the structural properties of compounds 31 and 32, other indole 3-carboxamide derivatives were synthesized, and their inhibitory activity was investigated. finally, compound 33 was selected as the most optimal compound in interaction with the renin enzyme (table 8) . compound 33, with the 5-hydroxy group, establishes a favorable interaction with the imidazole ring of his287 (fig. 16 ). according to the results of the crystallographic studies, the presence of polar moiety in the indole core has a positive effect on the inhibition activity of the renin enzyme. therefore, in their next study, they used azaindole as the base compound. nitrogen atoms were positioned at the positions of 4, 6, or 5, 7 indole molecules. according to the results of their study, the activity of azaindole and indole compounds against the renin enzyme is similar. they used their candidate compounds in further study to measure factors such as caco-2 permeability, physicochemical data, and ic 50 values in human or mouse plasma. the results of their studies showed that some azaindole derivatives have the potential of intestinal absorption. therefore, the results of their study can serve as the basis for the initiation of advanced research [59, 60] . in 2012, jing et al. used the assp method to quantitatively characterizing the nonbonding interaction profile between renin and indole 3-carboxamide derivatives [61] . [61] in this method, the space containing the entire active site of renin is divided into thousands of small areas, and then the nonbonding potentials between renin and indoles are calculated. the advantage of the assp method is the use of graphical diagrams that illustrate the interactions. their calculations were performed using the data of the paper by scheiper et al. [32] . according to the results of their calculations, the interaction of indole 3-carboxamide derivatives with renin enzyme is primarily electrostatic, secondly hydrophobic, and thirdly steric (fig. 17) . the presence of polar and charged groups in the interaction with the renin enzyme causes hydrogen bond and salt-bridge networks. the presence of aromatic and aliphatic rings causes steric and hydrophobic interactions that are weaker than electrostatic interactions. according to their calculations, compound 33 had the highest activity against renin (fig. 17) . the activity of compound 33 depends on the presence of nh in the piperazine ring, c=o/-oh groups, f, and ch 3 groups, which enhance the electrostatic force and thus increase its activity. removal of any of the above-mentioned groups results in a decrease in the activity of this compound. these results confirm the research of scheiper et al. however, many computational and theoretical studies have been conducted by researchers to study and design indole 3-carboxamide derivatives, the results of which confirm previous studies [62, 63] . in recent years, indole derivatives are shown to be active against many enzymes renin [56] and proteins, indicating that these compounds are still in research [64] [65] [66] [67] . indole derivatives play essential roles in pharmaceutical studies. by reviewing the synthetic methods, it was found that bop, edci, hobt, and tbtu activate the carbonyl group and convert the indole carboxylic acid to indole carboxamide. the studies have shown that indole 2-carboxamide derivatives have inhibitory activity against hlgp and hiv-1, and indole 3-carboxamide derivatives have inhibitory activity against renin. considering the studies of investigating the inhibitory activity of indole derivatives, the presence of carboxamide moiety at positions 2 and 3 is significant from two aspects. 1) nh and co-carboxamide form hydrogen bonds with enzymes or proteins. 2) polar groups can bond to the carboxamide moiety and form new hydrogen bonds with enzymes or proteins. in fact, strengthening the electrostatic forces increases the inhibitory activity of indole 2 and 3-carboxamide derivatives. research on indole compounds is not limited to academic articles and studies. many of these derivatives are in the final stages of approval and entry into the pharmaceutical industry. table 9 shows the indole 2 and 3-carboxamide derivatives, which are in the late stage of discovery. the result of years of research by scientists has shown that these compounds have the potential to inhibit the activity of various enzymes such as hlgp, hiv-1, and renin. research on indole compounds is expected to continue and more drugs from these derivatives will reach final approval. chem of indoles gene-inspired mycosynthesis of skeletally new indole alkaloids the natural and synthetic indole weaponry against bacteria indole alkaloid sulfonic acids from an aqueous extract of isatis indigotica roots and their antiviral activity biosynthesis of fungal indole alkaloids structural optimization of indole based compounds for highly promising anti-cancer activities: structure activity relationship studies and identification of lead molecules synthesis of 1-indolyl substituted β-carboline natural products and discovery of antimalarial and cytotoxic activities indole-3-ethylsulfamoylphenylacrylamides: potent histone deacetylase inhibitors with anti-inflammatory activity synthesis of alpha amylase inhibitors based on privileged indole scaffold synthesis of 2-acylated and sulfonated 4-hydroxycoumarins: in vitro urease inhibition and molecular docking studies synthesis and biological evaluation of novel n-arylidenequinoline-3-carbohydrazides as potent β-glucuronidase inhibitors synthesis, antimicrobial, anticancer evaluation and qsar studies of 6-methyl-4-[1-(2-substituted-phenylamino-acetyl)-1h-indol-3-yl]-2-oxo/thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl esters synthesis, antimicrobial, antimycobacterial and structure-activity relationship of substituted pyrazolo-, isoxazolo-, pyrimidoand mercaptopyrimidocyclohepta[b]indoles cytotoxic and antibacterial polyketide-indole hybrids synthesized from indole-3-carbinol by daldinia eschscholzii qinoxaline ii. a practical efficient and rapid synthesis of new quinoxalines catalyzed by citric acid as a trifunctional bronsted acid at room temperature under green condition epoxidation of aromatic α, β-unsaturated ketones using pvp-h2o2 under mild and heterogeneous conditions 5-triazine-2,4,6-triyltrisulfamic acid (ttsa): a new organic solid acid for the nitrosation of secondary amines and oxidation of urazoles in the presence of nano 2 under mild and heterogeneous conditions oxidation of urazoles via in situ generation of cl + by using n, n,2,3,4,5,6-heptachloroaniline or a uhp/ mcln system under mild conditions new pyrazolone derivatives synthesis: comparison of the catalytic effect of three typically different brønsted acid catalysts on the reaction progression a simple and expedient method for the stepwise synthesis of 2-ethoxy-(4h)-3, 1-benzoxazine-4-ones citric acid as an ecient and trifunctional organo catalyst for onepot synthesis of new indolenines by fischer's method at reflux condition in ethanol novel indolyl aryl sulfones active against hiv-1 carrying nnrti resistance mutations: synthesis and sar studies simple, short peptide derivatives of a sulfonylindolecarboxamide (l-737,126) active in vitro against hiv-1 wild type and variants carrying non-nucleoside reverse transcriptase inhibitor resistance mutations design, molecular modeling, synthesis, and anti-hiv-1 activity of new indolyl aryl sulfones. novel derivatives of the indole-2-carboxamide design, synthesis, and pharmacological evaluation of n-bicyclo-5-chloro-1h-indole-2-carboxamide derivatives as potent glycogen phosphorylase inhibitors synthesis of 5-chloro-n-aryl-1h-indole-2-carboxamide derivatives as inhibitors of human liver glycogen phosphorylase a novel inhibitors of neurotropic alphavirus replication that improve host survival in a mouse model of acute viral encephalitis -cyano-3,4-dihydroisoquinolin-2(1h)-yl)ethyl)cyclohexyl)-1h-indole-2-carboxamide (sb269652), a bitopic ligand that acts as a negative allosteric modulator of the dopamine d2 receptor computer-aided design, synthesis, and biological evaluation of new indole-2-carboxamide derivatives as pi3ka/egfr inhibitors design, synthesis and structure-activity relationship study of novel indole-2-carboxamide derivatives as anti-inflammatory agents for the treatment of sepsis n-pyridinyl-indole-3-(alkyl)carboxamides and derivatives as potential systemic and topical inflammation inhibitors discovery and optimization of a new class of potent and non-chiral indole-3-carboxamide-based renin inhibitors sar216471), a novel intravenous and oral, reversible, and directly acting p2y12 antagonist me 2 alcl-mediated carboxylation, ethoxycarbonylation, and carbamoylation of indoles amidoalkylindoles as potent and selective cannabinoid type 2 receptor agonists with in vivo efficacy in a mouse model of multiple sclerosis acyl ureas as human liver glycogen phosphorylase inhibitors for the treatment of type 2 diabetes identification, synthesis, and characterization of new glycogen phosphorylase inhibitors binding to the allosteric amp site a new allosteric site in glycogen phosphorylase b as a target for drug interactions integrated effects of multiple modulators on human liver glycogen phosphorylase a recent advances in the allosteric inhibition of glycogen phosphorylase activation of human liver glycogen phosphorylase by alteration of the secondary structure and packing of the catalytic core the family of glycogen phosphorylases: structure and function human liver glycogen phosphorylase inhibitors bind at a new allosteric site indole-2-carboxamide inhibitors of human liver glycogen phosphorylase discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo inhibitory mode of indole-2-carboxamide derivatives against hlgpa: molecular docking and 3d-qsar analyses nucleoside and nucleotide analogue reverse transcriptase inhibitors: a clinical review of antiretroviral resistance declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. hiv outpatient study investigators challenges and approaches in the discovery of human immunodeficiency virus type-1 non-nucleoside reverse transcriptase inhibitors ) 5-chloro-3-(phenylsulfonyl)indole-2-carboxyamide: a novel nonnucleoside inhibitor of the hiv-1 reverse transcriptase docking and 3-d qsar studies on indolyl aryl sulfones. binding mode exploration at the hiv-1 reverse transcriptase non-nucleoside binding site and design of highly active n-(2-hydroxyethyl)carboxamide and n-(2-hydroxyethyl)carbohydrazide derivatives new nitrogen containing substituents at the indole-2-carboxamide yield high potent and broad spectrum indolylarylsulfone hiv-1 non-nucleoside reverse transcriptase inhibitors indolylarylsulfones as hiv-1 non-nucleoside reverse transcriptase inhibitors: new cyclic substituents at indole-2-carboxamide renin inhibition with aliskiren recent progress on the discovery of nonpeptidic direct renin inhibitors for the clinical management of hypertension the renin-angiotensin-aldosterone system and its suppression renin inhibitors -mechanisms of action structure-based design and optimization of potent renin inhibitors on 5-or 7-azaindole-scaffolds structure-based optimization of potent 4-and 6-azaindole-3-carboxamides as renin inhibitors exploring the substructural space of indole-3-carboxamide derivatives binding to renin: a novel active-site spatial partitioning approach non-chiral indole-3-carboxamide-based renin inhibitors theoretical design of new indole-3-carboxamide derivatives as renin inhibitors a novel harmine derivative, n-(4-(hydroxycarbamoyl)benzyl)-1-(4-methoxyphenyl)-9h-pyrido[3,4-b]indole-3-carboxamide (hbc), as histone deacetylase inhibitor: in vitro antiproliferation, apoptosis induction, cell cycle arrest, and antimetastatic effects subtle modifications to the indole-2-carboxamide motif of the negative allosteric modulator n-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1h)-yl)ethyl)cyclohexyl)-1h-indole-2-carboxamide (sb269652) yield dramatic changes in pharmacological activity at the dopamine d 2 receptor indole-2-carboxamidebased mmpl3 inhibitors show exceptional antitubercular activity in an animal model of tuberculosis infection antibacterial study of 3-(2-amino-6-phenylpyrimidin-4-yl)-n-cyclopropyl-1-methyl-1hindole-2-carboxamide derivatives: comfa, comsia analyses, molecular docking and admet properties prediction the authors gratefully acknowledge partial support for this work by the deputy of research, hamadan university of medical sciences (grant no: 9712147804). conflict of interest the authors declare that they have no conflict of interest.ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. key: cord-017227-66dx2dkv authors: humphreys, hilary; winter, bob; paul, mical title: immunocompromised patients date: 2012-08-21 journal: infections in the adult intensive care unit doi: 10.1007/978-1-4471-4318-5_10 sha: doc_id: 17227 cord_uid: 66dx2dkv the ominous prognosis of cancer patients with or without neutropenia in need of critical care has led to reservations with regard to admission of cancer patients to the icu. however, significant improvements in icu and in-hospital survival of cancer patients in icu have been demonstrated in studies in recent years [1–4]. risk factors for mortality have shifted from those related to the underlying condition to those related to the severity of acute illness similar to other critically-ill patients. neutropenia per se and the underlying malignancy (solid and hematological) do not have an impact on the outcome of patients in icu. recent chemotherapy is associated rather with improved survival [3, 5–7], while organ dysfunction, severity of disease scores, need for vasopressor treatment, need for mechanical ventilation immediately or after noninvasive ventilation, no definite diagnosis and a non-infectious diagnosis are associated with mortality [1–3, 8]. invasive aspergillosis is also associated with very high mortality rates in icu (see below). in several studies, admission to icu in the early stages of sepsis or other acute event was associated with better survival than admission later, after development of organ dysfunction. performance status is perhaps the most important and only variable relating to the underlying condition that is correlated with icu death. the prognosis remains guarded for certain cancer patients, including patients after allogeneic hematopoietic stem cell transplantation (hsct) with active uncontrolled graft versus host disease, those with relapse of the primary disease after allogeneic hsct and special cases of solid cancer including pulmonary carcinomatous lymphangitis and carcinomatous meningitis with coma [9]. associated rather with improved survival [ 3, [5] [6] [7] , while organ dysfunction, severity of disease scores, need for vasopressor treatment, need for mechanical ventilation immediately or after noninvasive ventilation, no de fi nite diagnosis and a noninfectious diagnosis are associated with mortality [1] [2] [3] 8 ] . invasive aspergillosis is also associated with very high mortality rates in icu (see below). in several studies, admission to icu in the early stages of sepsis or other acute event was associated with better survival than admission later, after development of organ dysfunction. performance status is perhaps the most important and only variable relating to the underlying condition that is correlated with icu death. the prognosis remains guarded for certain cancer patients, including patients after allogeneic hematopoietic stem cell transplantation (hsct) with active uncontrolled graft versus host disease, those with relapse of the primary disease after allogeneic hsct and special cases of solid cancer including pulmonary carcinomatous lymphangitis and carcinomatous meningitis with coma [ 9 ] . an "icu trial" consisting of patient admission and re-assessment after 3-5 days has been suggested for cancer patients [ 9 ] . outcomes were better associated with the hemodynamic and respiratory status after the fi rst stabilization phase than at the time of admission. another study supporting this concept showed that organ failure scores predicted survival more accurate on day six than at admission [ 7 ] . all patients who required initiation of mechanical ventilation, vasopressors, or dialysis after 3 days in the icu died. an early invasive diagnostic strategy should be pursued in immune compromised patients, since the differential diagnosis is broad including infectious and noninfectious etiologies and the spectrum of infectious agents is large. this includes bronchoalveolar lavage (bal) with or without lung biopsy for pulmonary disease, functional endoscopic sinus surgery (fess) for sinusitis/ rhinocerebral disease, endoscopy for colitis, biopsies from liver nodules, etc. some infectious conditions by organ system to be considered in immune compromised patients are provided in table 10 .1 . in additions, patients presenting with respiratory insuf fi ciency should be evaluated for community-acquired respiratory viruses using pcr, direct antigen tests and cultures of respiratory samples. these include in fl uenza, parain fl uenza, adenovirus, respiratory syncytial virus (rsv), and human metapneumovirus. empirical antibiotic treatment is recommended for neutropenic cancer patients (neutrophil count <500/mm 3 or <1,000/mm 3 and expected to decline to <500/mm 3 ) with fever, diarrhoea or suspected infection [ 10 ] . intravenous empirical treatment for high-risk patients should provide broad coverage against gram-negative (including pseudomonas aeruginosa ) and gram-positive bacteria. vancomycin should not be administered routinely, but is reserved for patients with hypotension or other hemodynamic compromise, those with a source of infection likely to be caused by staphylococci (skin/ soft tissue, catheter-related and pneumonia). antibiotic treatment should not be automatically discontinued with neutrophil recovery, even if infection has not been con fi rmed during neutropenia. rather repeated patient examination, imaging and microbiological evaluation for suspected sources of infection should be performed after neutrophil recovery to exclude new or exacerbations of pre-existing infections. among patients with documented infections during neutropenia, neutrophil recovery may be associated with "deterioration" in the status of the patient. local signs and symptoms of infection are frequently exacerbated. thus, pulmonary in fi ltrates may increase with new onset respiratory compromise (see figs. 10.1 and 10.2 ), an abscess may appear or enlarge or local signs of catheter-related infection may become manifest. in this case, treatment should not necessarily be changed or expanded. this is the normal response to neutrophil recovery. an immune reconstitution in fl ammatory response (iris), originally described among patients with hiv following treatment initiation (see below), has also been described among cancer patients following neutrophil recovery [ 11 ] . this syndrome represents an overly robust and dysregulated in fl ammatory response resulting in re-appearance or deterioration in clinical signs and symptoms of infection. it occurs usually later than the initial worsening following neutrophil recovery, days to weeks after immune reconstitution. diagnosis is dif fi cult and is based mostly on negative cultures and biomarkers for the initial infection and treatment is with corticosteroids. a speci fi c syndrome of adult respiratory distress syndrome (ards) has been described following neutrophil recovery in hematological cancer patients [ 12 ] . the only independent risk factor for ards is pneumonia during neutropenia. the importance of administering chemotherapy, if needed, cannot be overemphasized, even during an acute infection. while inducing immune suppression during an acute infection is counter intuitive, it is the underlying malignancy that is most commonly responsible for infection among cancer patients. without control of the underlying malignancy the long term outcomes of most or all infections remain ominous. close liaison with hematologists/ oncologists is recommended for oncological patients admitted to the icu [ 9 ] . cancer patients, especially hematological cancer patients are frequently thrombocytopenic as part of their underlying illness or as a result of chemotherapy. while randomized controlled trials have not shown an advantage to a threshold higher than 10 × 10 9 /l for thrombocyte transfusions, these studies did not include critically ill patients [ 13 ] . a higher threshold should probably be used (20-30 × 10 9 /l) in critically ill hematological cancer patients, especially with sepsis or with pulmonary involvement. pulmonary and intracerebral hemorrhage are frequent terminal events in these patients and prophylactic transfusions may prevent mortality. neutrophil transfusions have not been shown to improve survival for patients with severe neutropenia as part of the management of acute infections. however, when sub-grouped according to the dose of neutrophils transfused, survival was improved with average neutrophil doses of 1 × 10 10 . this dose can be obtained by pre-treating donors with granulocyte growth stimulating factors (g-csf) [ 14 ] . neutrophil transfusions should be reserved for severely neutropenic patients (<100/ ml 3 ) for whom the neutrophil count is expected to increase in a few days, as a bridge until bone marrow reconstitution. neutrophil transfusion is probably futile when there is no expectation that the natural neutrophil count will increase. hematological cancer patients may be hypoglobulinemic as part of the underlying hematological malignancy or following chemotherapy. there is no evidence from high-quality studies that intravenous immuneglobulins (ivig) reduces mortality in sepsis in general and in cancer patients speci fi cally [ 15 ] . in one randomized controlled trial speci fi cally assessing patients with hematological malignancies, there was no survival advantage with ivig [ 16 ] . ivig is used in some centers for infection prevention among patients with multiple myeloma or chronic lymphocytic leukemia, known hypogammaglobulinemia and recurrent respiratory infections [ 17 ] . patients with hiv may be seen in the icu as the fi rst presentation of their disease or following an infectious complication after diagnosis. highly active antiretroviral therapy (haart) has changed the epidemiology of hiv such that the latter group of patients is rare nowadays in locations where treatment is available (and depending on patient's compliance). clearly, the change in prognosis of hiv with haart has led to a shift in management such that hiv patients are offered maximal treatment, including full icu support, organ transplantation in the appropriate circumstances, or chemotherapy if needed. the impact of haart availability on mortality has been shown also in icu, where predictors of mortality in the haart era are no longer hiv-related [ 18 ] . a thorough discussion of the management of hiv patients is beyond the scope of this book. however, we will address a few critical decisions in the management of infections with suspected or known hiv. administering antiretroviral therapy in the icu is dif fi cult [ 19 ] . all antiretrovirals except zidovudine are available only as oral preparations, most only as tablets. beyond the poor bioavailability of orally administered drugs in the critically-ill patient, absorption and side effects of speci fi c antiretrovirals frequently depend on the provision of concurrent oral feeding. drug interactions are common; for example proton-pump inhibitors and histamine-2 blockers are contraindicated with protease inhibitors (pis). all nucleoside reverse-transcriptase inhibitors (nrtis), except for abacavir, require dose adjustment for renal failure. several pis require dose adjustment for hepatic impairment [ 19 ] . thus, fi xed drug combination usually cannot be used in patients with renal and hepatic impairment; individual drugs must be dose-adjusted and administered separately. drug-related adverse effects are common, although few may be relevant in the critical care setting. nrtis (mainly stavudine, didanosine, and zidovudine) may induce lactic acidosis. abacavir-related hypersensitivity is a serious adverse event and this drug should be administered only after genetic testing for hla-b*5701. the most common infectious scenario encountered in the icu will be the recently diagnosed patient presenting with an opportunistic infection. the question in this scenario is whether to initiate antiretroviral therapy early while treating the acute infection or after its successful management. aside from the practical dif fi culties in the administration of haart in icu, there is the fear of iris with worsening of the underlying infectious process during immune reconstitution. iris can occur between days to weeks after initiation of haart [ 19 ] . although logically predicted by rising cd4 counts, it can occur before signi fi cant cd4 cell count increase or viral load suppression [ 20 ] . there are few randomized trials to guide the strategic decision of early vs. deferred haart initiation during infection (table 10 .2 ). three trials have shown a clinical bene fi t for early, but not immediate antiretroviral drug initiation (e.g. within 2 weeks of starting anti-infective treatment), for patients mostly with pulmonary tuberculosis and pcp [21] [22] [23] . one study showed no difference in outcomes for patients with pulmonary and extrapulmonary tuberculoisis [ 24 ] . in contrast two trials assessing hiv patients with meningitis showed no bene fi t or increased harm with early initiation of haart initiation [ 25, 26 ] (table 10 .2 ) . there is no direct evidence on timing of haart initiation in the critical care setting. summarizing the evidence from existing trials, it seems that the initiation haart about 2 weeks after anti-infective therapy directed at the opportunistic infection is reasonable for patients with pulmonary infections, including tuberculosis. this allows the time for patient stabilization, hiv drug resistance testing and involving an hiv specialist in the management and all treatment decisions. with tuberculosis or cryptococcal meningitis, the start of haart should probably be deferred for longer. in previously treated hiv patients, the question is whether to continue or stop haart in icu. discontinuation could result in the selection of anti-viral resistance because of the different half-lives of the drugs included in the combination and functional monotherapy with the long-acting antiretrovirals. an expert recommendation is to try and continue haart for patients with virological suppression (plasma hiv rna below the limit of detection) [ 19 ] . in other patients, haart can probably be discontinued after consultation with an hiv expert. hiv/ hepatitis b co-infected patients require special consideration. antiretroviral treatment active against hepatitis b (lamivudine, emtricitabine, and tenofovir) should not be discontinued for fear of exacerabations of hepatitis b after discontinuation. adrenal insuf fi ciency is common among hiv patients and should be evaluated in all patients admitted to the icu. testing of stress cortisol concentration and lowdose adrenocorticotropic hormone (acth) corticotropin stimulation test (1-microg of acth) is recommended [ 27 ] . solid organ transplant (sot) recipients require life long immune suppression to prevent rejection. this immune suppression affects mainly the t-cell lymphocyte function. consequently, opportunist infections seen most frequently among sot recipients include herpesvirus infections, mainly cmv, pneumocystis pneumonia and more rarely fungal infections. ebv-associated post-transplantation lymphoproliferative disease (ptld) is a special entity. in the fi rst month after transplantation, most infections will be healthcareassociated, related to the surgical site, catheter or post-operative mechanical ventilation. lung transplant recipients are frequently colonized before transplantation by bacteria and pneumonia is very common in the fi rst year after transplantation (mostly in the fi rst month) and is associated with a high relative risk of death [ 28 ] . donors may also be colonized with antibiotic resistant bacteria [ 29 ] . prophylaxis for a more prolonged period (i.e. days) compared with non-transplant surgery (i.e. 1-2 doses of antibiotics) should be guided by the presence of pre-existing antibiotic-resistant bacteria in the recipient or the donor. all antibiotics continued after transplantation should be reviewed early with a view to discontinuation if the recipient is clinically well and there is no other evidence to suggest infection. antibiotics can be discontinued 48-72 h after transplantation if culture of donor and recipient samples is negative and the patient is stable. antibiotic prophylaxis in liver transplantation should provide a therapeutic concentration in the wound and within the biliary tract. in a european survey, all liver transplant centres administered antibiotic prophylaxis for liver transplantation, using a variety of different antibiotic regimens for a median of 3 days after transplantation [ 30 ] . given the shortage of organs, transplant centers are increasingly using marginal donors, sometimes with documented infections at the time of death, which might have been previously treated or not. studies describe non-inferior outcomes for recipients receiving organs from donors with clinical infections, including bloodstream infection and meningitis [31] [32] [33] [34] . treatment directed against the donor's isolate/s was given to recipients. procurement of organs from previously untreated patients with meningitis has not been described. pneumocystis pneumonia (pcp) caused by pneumocystis jirovecii most commonly affects patients with cellular immune de fi ciency, including lymphopenia or qualitative defects in lymphocyte activity, rather than patients with neutropenia or neutrophil dysfunction. susceptible patients therefore include patients with: multiple myeloma • chronic lymphocytic leukemia • following hsct with graft versus host disease (gvhd hematological cancer • patients receiving anti-lymphocyte antibodies such as rituximab and alemtuzumab (mainly lymphoma) sot recipients mainly during periods of high-corticosteroid therapy or anti-• lymphocyte antibody treatment for rejection non-immune-reconstituted hiv patients. • haart has changed the epidemiology of pcp such that most patients are now not hiv-positive. pcp is more severe and is associated with higher mortality in non-hiv patients. prophylaxis with trimethoprim-suphamethoxazole (tmp-smx) given daily or thrice weekly is highly effective in pcp prevention and patients who are receiving tmp-smx prophylaxis presenting with a clinical picture suspected of pcp probably do not have pcp. compliance with prophylaxis in the period before admission should be ensured by history taking, since discontinuation of pcp prophylaxis for adverse events is common and protection from pcp is reliable only while this drug is taken. less is known about the ef fi cacy of other prophylaxis agents, including dapsone or pentamidine, and their administration should not rule out the possibility of pcp in the appropriate clinical setting. pcp presents with dyspnea, tachypnea and hypoxia. lung imaging shows bilateral interstitial or ground glass in fi ltrates. initially the chest x ray may appear near normal but a high-resolution ct scan will show these opacities better. with more severe disease bilateral diffuse in fi ltrates can be seen also on the chest x-ray. the radiological picture is similar to than seen with cmv pneumonitis and actually coinfection of pcp and cmv is not uncommon. a diagnosis of one does not rule out the existence of the other and a search for cmv infection should be performed when pcp is diagnosed, especially in hematological cancer and sot patients. the interval from symptom onset to diagnosis of pcp was 3-14 days in one study [ 35 ] . diagnosis is established most commonly by examination of bal fl uid, but it is possible also with induced sputum (table 10 .1 ). giemza stain or immuno fl uorescence stain with human monoclonal anti-pneumocystis cyst antibodies will demonstrate p. jirovecii trophozoites or cysts. pcr is more sensitive but less speci fi c; p. jirovecii was identi fi ed by nested pcr in 68 % of people dying suddenly of non-infectious reasons, representing low-level colonization [ 36 ] . in the appropriate clinical scenario a positive pcr probably mandates treatment, but in other cases pcr results may represent colonization. tmp-smx is considered the most effective therapy for pcp [ 37 ] . it is administered using high doses of 15-20 mg/kg/day of the trimethoprim component and 75-100 mg/kg/day of the sulphamethoxazole component, divided in four daily doses. clinical response may be delayed until day 7 or later and treatment should be continued for 21 days. adverse effects are common and include mainly rash and tmp-smx-induced leucopenia. hematologists or oncologists may be reluctant to use tmp-smx in patients with neutropenia for fear of delaying neutrophil recovery. leucovorin (folinic acid) can attenuate the hematologic adverse effects of tmp-smx, but should probably not be used during active pcp infection since it has been shown to increase death and failure rates in hiv patients [ 38 ] . alternative agents include the combination of primaquine 30 mg/ day and clindamycin 600 mg thrice daily, a combination of dapsone with trimethoprim, atovaquone alone or intravenous pentamidine alone. each medication has its own adverse effect pro fi le which should be considered on a patient-by-patient basis before treatment and monitored for during treatment. adjunctive corticosteroid treatment for patients with hypoxemia (room air pao 2 <70 mmhg) is based on evidence of improved survival in hiv patients, but it is recommended in other immune compromised patients with severe pcp [ 37 ] . the dose recommended is 40 mg prednisone twice daily for 5 days, 40 mg/day for the next 5 days and 20 mg/day until day 21. the classical risk factors for invasive aspergillosis include severe prolonged neutropenia, the immune de fi ciency state following allogeneic hematopoietic stem cell transplantation, particularly in the fi rst year after transplantation and with gvhd and more rarely following sot, mainly lung transplantation. however, currently, invasive aspergillosis is also reported among other patient populations in the icu not "classically" considered as immune compromised. these include patients with chronic lung disease, cirrhosis, burns and others [ 39 ] . post-mortem studies show that invasive aspergillosis is under-diagnosed in the icu. therefore, in this setting, positive respiratory cultures for aspergillus sp . should not be automatically disregarded. aspergillus sp . may cause infectious, saprophytic and allergic syndromes. herein, we will address three of the more important infectious syndromes seen in icu: invasive aspergillosis, aspergillus tracheobronchitis and chronic necrotizing aspergillosis. invasive aspergillosis, the classical condition described in immunocompromised patients, involves the lungs, sinuses with or without cerebral extension and skin mostly. both contiguous extension disregarding normal anatomical barriers (as in the extension from the respiratory sinuses to the brain) and hematogenous dissemination (causing lung infarction) may occur. chest and sinus x-rays are notoriously insensitive for the diagnosis of invasive aspergillosis and ct is the imaging of choice. during neutropenia fi ndings are usually lacking or minimal, but after neutrophil recovery lesions frequently increase in size even with adequate treatment and control of systemic infection (fig. 10.3 ) . respiratory deterioration should also be expected at the time of neutrophil recovery. the classical signs of cavitation and crescent formation are usually observed at later stages of the disease (fig. 10.4 ) . lung hemorrhage is a feared complication, especially after biopsy, and thus attention should be given to correcting thrombocytopenia during invasive aspergillosis (see above). criteria for the diagnosis of invasive aspergillosis have been suggested [ 40 ] . these consist of at least one host risk factor and one clinical fi nding (table 10. 3 ). to diagnose probable invasive aspergillosis laboratory con fi rmation is needed in addition to host and clinical criteria (table 10 .4 ) and for proven disease histological con fi rmation is necessary. aspergillus spp . appear as narrow, non-septate, acutebranching hyphae. given the dif fi culties in obtaining histological specimens in cancer patients that are severely thrombocytopenic, diagnosis usually relies on culture, direct stains, pcr and galactommanan (gm, a cell wall component of aspergillus spp. and penicillium spp ). pcr and gm can be tested in serum or in bal fl uid. as can be seen in table 10 .5 , the sensitivity and speci fi city are slightly higher in bal, favoring the performance of bal for patients with suspected invasive aspergillosis. aspergillus tracheobronchitis represents infection of the major airways, with erythema, ulceration, nodules and pseudomembrane formation [ 39 ] . it has been described mostly among lung transplant recipients, but also among patients with copd and chronic necrotizing aspergillosis (or "semi-invasive aspergillosis") consists of a more chronic and diffuse form of lung infection, resembling pulmonary coccidioidomycosis or histoplasmosis [ 37 ] . it has been described among patients with chronic lung disease, diabetes mellitus, aids and with chronic corticosteroid therapy. the existence of the syndrome is important to recognize for patients presenting severe respiratory diseases and positive respiratory culture of aspergillus sp ., without the classical features of invasive aspergillosis. the primary recommended therapy for the infectious syndromes described herein is voriconazole [ 37, 45 ] . voriconazole is available both in oral and intravenous formulations. blood level concentrations should be monitored, since currently recommended dosing frequently results in subtherapeutic concentrations [ 46 ] . many other antifungals are active and recommended for the treatment of invasive aspergillus infections; [ 37, 45 ] fl uconazole is the only azole inactive against aspergillus sp . (see chap. 4 on antifungals). treatment failure and mortality rates are very high, especially with ongoing immune suppression. because of the ominous prognosis combinations of antifungals have been suggested as primary or salvage therapy. in one small randomized controlled trial, the combination of liposomal amphotericin b with caspofungin resulted in a higher rate of favorable response than liposomal amphotericin b, with no deaths in the combination arm (0/15 vs. 3/15 with monotherapy [ 47 ] . the most common combination reported in observational studies was voriconazole and caspofungin, but at this time no conclusions can be drawn on the effects of this combination over monotherapy [ 48 ] . 1. the patient in the case vignette underwent bronchoalveolar lavage (bal) with lung biopsy. cmv was isolated in cultures of the bal fl uid. cmv antigenemia was tested after neutropil recovery (the pp65 antigen is present in neutrophils which are required for cmv antigenemia assessment) and was negative. direct immuno fl uorescence and pcr for p. jirovecii in bal fl uid were negative. lung biopsy demonstrated diffuse alveolar damage with eosinophlic alveolar foam compatible with pcp. a methamine-silver and immunohistochemical stains for pcp were negative, as was the immunohistochemical stain for cmv. the patient was diagnosed with pcp based on the clinical presentation and histological sensitivity and speci fi city values refer to the diagnosis of probable or proven ia gm galactomannan, ci con fi dence interval a values refer to a gm cut-off of 0.5 optical density index fi ndings. cmv co-infection could not be ruled out. the positive cmv culture in bal fl uid could re fl ect cmv reactivation and infection in the presence of immune suppression, without disease. 2. the patient was empirically treated with high-dose trimethoprim-sulfamethoxazole and intravenous gancyclovir with no adverse events and these were continued after the results of the tests discussed above. 3. the dose of prednisone was increased and tapered down following the recommendations for severe pcp. 4. respiratory insuf fi ciency improved gradually until the patient was discharged with normal saturation on room air. notable was the appearance of pulmonary in fi ltrates and respiratory distress only after neutrophil recovery, although fever started during neutropenia and probably re fl ected the same infection. this exacerbation after neutrophil recovery is similar to the immune reconstitution syndrome seen with hiv patients after start of haart. the prognosis of acute respiratory failure in critically ill cancer patients survival in neutropenic patients with severe sepsis or septic shock icu and 6-month outcome of oncology patients in the intensive care unit prognosis of patients with acute myeloid leukaemia admitted to intensive care impact of neutropenia on the outcomes of critically ill patients with cancer: a matched case-control study impact of recent intravenous chemotherapy on outcome in severe sepsis and septic shock patients with hematological malignancies the icu trial: a new admission policy for cancer patients requiring mechanical ventilation characteristics and outcomes of patients with cancer requiring admission to intensive care units: a prospective multicenter study intensive care of the cancer patient: recent achievements and remaining challenges clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america the impact of the host on fungal infections risk factors for acute respiratory distress syndrome during neutropenia recovery in patients with hematologic malignancies prophylactic platelet transfusion for haemorrhage after chemotherapy and stem cell transplantation granulocyte transfusions for preventing infections in patients with neutropenia or neutrophil dysfunction intravenous immunoglobulin for treating sepsis and septic shock igma-enriched immunoglobulin in neutropenic patients with sepsis syndrome and septic shock: a randomized, controlled, multiple-center trial immunoglobulin prophylaxis in hematopoietic stem cell transplantation: systematic review and meta-analysis short-and long-term outcome of hiv-infected patients admitted to the intensive care unit intensive care of patients with hiv infection clinical features and serum biomarkers in hiv immune reconstitution in fl ammatory syndrome after cryptococcal meningitis: a prospective cohort study early antiretroviral therapy reduces aids progression/death in individuals with acute opportunistic infections: a multicenter randomized strategy trial timing of antiretroviral therapy for hiv-1 infection and tuberculosis timing of initiation of antiretroviral drugs during tuberculosis therapy time to initiation of antiretroviral therapy between 4 weeks and 12 weeks of tuberculosis treatment in hiv-1 infected patients. results from the time study. in: paper presented at the 22nd european congress of clinical microbiology and infectious diseases timing of initiation of antiretroviral therapy in human immunode fi ciency virus (hiv)-associated tuberculous meningitis early versus delayed initiation of antiretroviral therapy for concurrent hiv infection and cryptococcal meningitis in sub-saharan africa adrenal insuf fi ciency in critically ill patients with human immunode fi ciency virus pneumonia after lung transplantation in the resitra cohort: a multicenter prospective study impact of bacterial and fungal donor organ contamination in lung, heart-lung, heart and liver transplantation antimicrobial prophylaxis in liver transplant patients-a multicenter survey endorsed by the european liver and intestine transplant association the use of liver grafts from donors with bacterial meningitis intrathoracic organ transplantation from donors with meningitis: a single-center 20-year experience donors with positive blood culture: could they transmit infections to the recipients? clinical signi fi cance of donor-unrecognized bacteremia in the outcome of solid-organ transplant recipients clinical picture of p. jirovecii pneumonia in cancer patients pneumocystis colonization is highly prevalent in the autopsied lungs of the general population an of fi cial american thoracic society statement: treatment of fungal infections in adult pulmonary and critical care patients adjunctive folinic acid with trimethoprim-sulfamethoxazole for pneumocystis carinii pneumonia in aids patients is associated with an increased risk of therapeutic failure and death aspergillus infections in the critically ill revised de fi nitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group use of pcr for diagnosis of invasive aspergillosis: systematic review and meta-analysis evaluation of pcr on bronchoalveolar lavage fl uid for diagnosis of invasive aspergillosis: a bivariate metaanalysis and systematic review galactomannan detection for invasive aspergillosis in immunocompromized patients accuracy of bal galactomannan in diagnosing invasive aspergillosis: a bivariate metaanalysis and systematic review treatment of aspergillosis: clinical practice guidelines of the infectious diseases society of america monitoring plasma voriconazole levels may be necessary to avoid subtherapeutic levels in hematopoietic stem cell transplant recipients liposomal amphotericin b in combination with caspofungin for invasive aspergillosis in patients with hematologic malignancies: a randomized pilot study (combistrat trial) the role of combination antifungal therapy in the treatment of invasive aspergillosis: a systematic review key: cord-007188-tcq8lnwg authors: cunningham, anthony l.; grohman, gerhard s.; harkness, john; law, carmela; marriott, deborah; tindall, brett; cooper, david a. title: gastrointestinal viral infections in homosexual men who were symptomatic and seropositive for human immunodeficiency virus date: 1988-08-17 journal: j infect dis doi: 10.1093/infdis/158.2.386 sha: doc_id: 7188 cord_uid: tcq8lnwg gastrointestinal viruses, predominantly rotaviruses and adenoviruses, were detected by enzyme-linked immunosorbent assay, electron microscopy, or cell culture in >50% of two groups of homosexual men with symptomatic human immunodeficiency virus (hiv) infection, who did (54%) or did not (50%) have diarrhea. lower detection rates were observed in hiv-seronegative (15%) and asymptomatic hiv-seropositive (16%) men. in the patients with diarrhea, 95% of the isolates of virus were found in the most immuno suppressed patients, those patients with aids-related complex or opportunistic infections associated with aids. high excretion rates of these viruses are probably associated with both anal-oral transmission and immunosuppression. these viruses apparently cause acute episodes or relapses of diarrhea in some patients but may be co-pathogens or noncontributory to chronic diarrhea in others. or parasitic etiology. chronic diarrhea in the absence of cryptosporidium or other pathogens has been described as a common presenting feature of aids in africa [2] . hiv has been shown to infect colonic cell lines and has been suggested as the main cause of most idiopathic cases [3] . viruses may be significant gastrointestinal pathogens in other groups of patients with severe t lymphocyte deficiencies, such as bone marrow transplant recipients, although this group has the additional problem of graft-versus-host disease and chemoradiation damage that may involve the gut. yolken et al. [4] and we (0. m., j. h., g. s. g., k. atkinson, and j. c. biggs, unpublished observations) have observed that adenoviruses, coxsackieviruses, rotaviruses, and clostridium difficile toxin are detectable in the feces of marrow transplant recipients and hematology patients receiving chemotherapy. in yolken's study, patients infected with these organisms had significantly increased rates of diarrhea and abdominal cramps. the infections with rotaviruses and adenoviruses occurred at the same time that these viruses were prevalent in the normal pediatric population. the presence of these pathogens was associated with a marked increase in mortality [4] . adenoviruses have also been associated with diarrhea or colitis in patients with aids [5] . we have recently detected sporadic, as well as epi-demic, cases of norwalk-like viruses in adults [6] . immunosuppressed patients (including the marrow transplant recipients) have not been previously examined for these pathogens because of a lack of available antibody specific for norwalk virus. we report here the detection of viruses from the stools of a large proportion of patients with symptomatic hiv infection (aids, arc, and pol) and acute or chronic diarrhea when no other microbial pathogen could be identified. hiv-seronegative and -seropositive homosexual men presenting for hiv antibody screening provided control groups to examine the effects of transmission via anal-oral contact and infection with hiv, with or without significant depression of concentration of circulating cd4 lymphocytes. we studied stool samples from two groups of patients for the presence of viral and other microbial pathogens. group 1. all patients with symptomatic hiv infection (aids, arc, or pol) who presented with acute or chronic diarrhea to the st. vincent's hospital aids service between january 1986and january 1987were entered in the study. diarrhea was defined as more than three fluid stools (i.e., assuming the shape of the container) per day and as chronic if it persisted for more than three weeks. exacerbations of chronic diarrhea were defined as a transient marked increase in stool frequency and volume. fecal specimens were collected early in the course of acute diarrhea, during exacerbations of chronic diarrhea, or on several occasions during persistent chronic diarrhea. the specimens were examined (microbiology laboratory, st. vincent's hospital) by microscopy and culture, using standard methods, for the full range of potentially pathogenic bacteria and parasites occurring in these patients (including salmonella spp., shigella spp., campylobacter jejuni, yersiniaenterocolitica, aeromonas hydrophila, plesiomonas shigelloides, vibrio parahaemolyticus, atypical mycobacteria, cryptosporidium spp., isosopora belli, giardia lamblia, amoebae, strongyloides stercoralis, and c. difficile; toxigenic and enteropathogenic escherichia coli were excluded). c. difficile toxin was detected by a standard cytotoxic assay. group 2 (controls). homosexual men presenting to a sexually transmitted diseases (stds) center 387 for routine examinations for stds and for hiv antibody screening during the same period provided a comparison group. they were all examined by one of us (c. l.). on examination, 7010 of these men had acute diarrhea, and 18% had proctitis. stool specimens were obtained on three separate days over a one-to two-week period from all men in the group. all three specimens were screened for protozoa and helminths at the school of public health and tropical medicine (sydney university). two specimens were cultured for the same range of bacterial pathogens, and the first specimen was examined for viruses. all specimens for virological examination were transferred immediately to the virology department (institute of clinical pathology and medical research, westmead hospital), where they were examined for viruses. fecal specimens were processed for viral diagnosis as follows: a 10% solution was prepared in pbs (ph 7.2), held overnight at 4 c, and clarified by low-speedcentrifugation. aliquots of supernatant were inoculated into cell cultures (primary monkey kidney, hep-2, and human diploid fibroblasts) and also tested for rotavirus antigen by elisa (enzygnost'"; calbiochem-behring, sydney). the elisa-positive samples were later examined by electron microscopy (em) and tested in a blocking elisa (kallestad, shaska, minn) for rotavirus by using goat antibody to rotavirus (kallestad). the remainder of the supernatant was treated with arklone (1 ,1,2-trichlorotrifluoroethane; sigma, st. louis), held for i h at 4 c, and then ultracentrifuged. the pellet was resuspended, negatively stained with phosphotungstic acid, and examined by em. to detect norwalk virus by immune em, we incubated the resuspended pellet, before ultracentrifugation, with antiserum specific for norwalk virus. in the group of 137 homosexual men presenting to the std clinic for hiv antibody screening, the proportions of hi v-seronegative (12of 82) and asymptomatic seropositive men (seven of 43) with virus in their stools was not significantly different (15% vs. lamblia) did not significantly alter these proportions (12.5% vs. 14.3%; 'y} = 0.08). in the hivseropositive group, rotaviruses and adenoviruses were the predominant viruses detected (table 1) , and none of these viruses were associated with acute diarrhea. in the hlv-seronegative group, one of three rotaviruses identified and one of five enteroviruses was associated with acute diarrhea. in the group of 68 hospital patients with diarrhea, 107 stool specimens were examined for viral and other pathogens. twenty-eight patients had one episode of acute diarrhea, six had acute exacerbations of chronic diarrhea, and 34 had persistent chronic diarrhea. as shown in table 2, pathogens were isolated from 75% (51 of 68) of the patients, most commonly from those with arc (cdc class iva) and aids-associated opportunistic infections (aids-oi; t two patients progressed from arc to aids-oi and are included in both classes. (table 3) . almost all of these isolates were obtained from patients with arc or aids-o!. fifty percent of the adenoviruses detected were noncultivable and probably pathogenic. isolation of these strains in graham 293 cells, and subsequent typing, is in progress. none of the cultivable adenoviruses were type 35 [7] . in the hospital group (patients with diarrhea), 11 of the 37 patients with gastrointestinal viruses isolated had dual infections (five with adenovirus and five with rotavirus), whereas these viruses were found much less commonly in the randomly screened group (one of 25; ' 1. 2 = 4.79, p < .05). no seasonal clustering of rotavirus or adenovirus was observed. fifteen of the 26 rotavirus and 8 of the 18 adenovirus (3 noncultivable) isolates were associated with acute diarrheal episodes or exacerbations of chronic diarrhea. the remaining isolates of virus weredetected in patients with persistent chronic diarrhea, often in association with likely primary pathogens such as cryptosporidium or mycobacterium avium-mycobacterium intracellulare (table 2 ). in seven patients from whom three or more serial stool specimens were examined, rotaviruses or adenoviruses were found in two consecutive specimens in three patients. rotavirus was detected in specimens that were obtained 14and 33 d apart, and adenovirus was detected in specimens obtained nine days apart. all the rotavirus-positive stool specimens reported in this study were initially detected by elisa (enzygnost) and later examined by em. the elisa and em results correlated well, with all but three elisapositive stool specimens being confirmed by em. one specimen was em-positive for rotavirus but elisa negative. twenty-threeof the elisa-positive (7) note. for an explanation of cdc classes see table 2. * data are reported as the mean ± sd cd4 + cell concentration/mm" (normal concentration~400/mmj); n = no. of patients. stool specimens were available for retesting in a blocking elisa assay for rotavirus (kallestad). all were confirmed positive. no reoviruses were isolated in cell culture. other common enteric viruses were, however, isolated, as shown in tables i and 3. as reported above, the prevalence of viruses in stools showed a marked increase from asymptomatic to symptomatic hiv-infected patients, and 950/0 of the diarrheal patients with virus in their stools were found in the two most immunosuppressed classes (aids-oi and arc). the mean cd4 + lymphocyte concentrations for patients who did or did not have virus in their stools were not significantly different in any of the groups of patients (table 4). as expected there was a significant difference between the mean cd4 cell concentrations of asymptomatic and symptomatic hiv-seropositive men (t = 2.66; dj = 53, p < .02). persistent, profuse, watery diarrhea; malabsorption; or colitis are the most dramatic gastrointestinal manifestations of aids. most studies of gastrointestinal pathogens in patients with aids have focused on the microbial causes of these syndromes, including cryptosporidium spp., i. belli, m. avium-m. intraeellu/are, and cmv. in addition there are the usual pathogens that also cause diarrhea in hiv-seronegative homosexual men: campy/obaeter, salmonella, shigella, yersinia, giardia, and e. histo/ytiea. the common occurrence of acute diarrhea or exacerbations of chronic diarrhea in symptomatic hiv-seropositive patients and the possible association of this diarrhea with gastrointestinal viral pathogens other than cmv has often been overlooked in the literature, although a recent association between colitis and adenovirus has been reported [5] . in this study we showed that patients with aids or arc may present with acute diarrhea or exacerbations of chronic diarrhea and that in patients with symptomatic hiv infection and diarrhea, >50% excreted gastrointestinal viruses. the majority of these were rotaviruses and adenoviruses. these high detection rates for rotavirus and adenovirus in patients with arc or aids-oi are similar to those observed in marrow transplant recipients who also have a t cell immunodeficiency and often have gastrointestinal mucosal damage from graft-versus-host disease [4] . prolonged diarrhea and fecal excretion of rotavirus for more than six weeks have also been observed in children with t cell immunodeficiency. comparisons of stool isolation rates in hiv-seronegative homosexual men and asymptomatic or symptomatic hiv-seropositive men strongly suggested there were two factors responsible for the high detection rate: (1) life style factors involving transmission by direct or indirect anal-oral contact, a possibility supported by the lack of seasonal variation, and (2) immunosuppression. the latter factor was further supported by the predominance of gastrointestinal viruses (particularly adenoviruses and rotaviruses) in patients with arc or aids-o!. the demonstration of a high detection rate of gastrointestinal viruses (especially rotavirus) in patients with symptomatic hiv infection but no diarrhea and the transient association of rotaviruses and cultivable adenoviruses with other more-certain microbial pathogens in patients with aids-oi or arc and diarrhea suggest that these viruses may often be "passengers." however, the presence of rotaviruses and the usually pathogenic, noncultivable adenoviruses as the only significant microbes in the stools of patients with acute diarrhea (a finding that could not be readily attributed to other causes such as drug toxicity) strongly suggests a pathogenic role. this observation needs to be confirmed in future studies by testing serial stool samples during the course of acute episodes of diarrhea and by excluding other pathogens, including hiv itself, by using intestinal biopsies. the serial studies should also assist in determining the role of dual viral infection (especiallywith rotaviruses and adenoviruses) in the etiology of diarrhea in these patients. malabsorption and mucosal abnormalities of the small intestine in the acquired immunodeficiency syndrome slim disease: a new disease in uganda and its association with htlv-iii infection productive persistent infection of human colorectal cell lines with human immunodeficiency virus infectious gastroenteritis in bonemarrow-transplant recipients cytomegalovirus" colitis: can it be caused by adenovirus [abstract no. th8. 3j? in: program and abstracts of the iii international conferece on aids viral diarrhoea in children in australia molecular epidemiology of adenovirus type 35 infections in immunocompromised hosts chronic rotavirus infection in immunodeficiency intestinal infections in patients with the acquired immunodeficiency syndrome (aids) note added in proof in a recent study of 20 homosexual men with aids and diarrhea and 10 homosexual men with aids but without diarrhea, smith et at. [9] were unable to detect rotavirus antigen in any patients' stools by using elisa (abbott laboratories, north chicago, ill).in contrast, in the year after our study ended (1987), rotavirus continued to be the predominant virus detected in the stools of homosexual men with aids and diarrhea in sydney (21 [18%] of 116 patients). such disparate results suggest marked geographic variations in gastrointestinal viral infections in these patients. key: cord-018721-othar2uv authors: schwab, stefan; schellinger, peter; werner, christian; unterberg, andreas; hacke, werner title: infektionen date: 2012-03-17 journal: neurointensiv doi: 10.1007/978-3-642-16911-3_32 sha: doc_id: 18721 cord_uid: othar2uv trotz weiterentwicklung moderner antibiotika in den letzten jahren sind die letalitätszahlen der bakteriellen (eitrigen) meningitis weiterhin hoch; überlebende haben häufig neurologische residuen. die ungünstigen klinischen verläufe der bakteriellen meningitis sind meist folge intrakranieller komplikationen, wie z. b. eines generalisierten hirnödems, einer zerebrovaskulären arteriellen oder venösen beteiligung oder eines hydrozephalus. z ätiologie die häufi gsten erreger einer bakteriellen meningitis im erwachsenenalter sind streptococcus pneumoniae und neisseria meningitidis. ferner wird die bakterielle meningitis verursacht durch: listerien (<5 % der fälle), staphylokokken (je nach literaturangabe 1−9 % der fälle), gramnegative enterobakterien inkl. pseudomonas aeruginosa (<10 % der fälle) und haemophilus infl uenzae (1−3 %). die häufi gsten keime der eitrigen meningoenzephalitis im kindesalter sind pneumokokken und meningokokken und in der neugeborenenperiode streptococcus agalactiae (gruppe-b-streptokokken), gramnegative enterobakterien und listerien. meningokokkenmeningitisepidemien werden überwiegend durch serogruppe-a-meningokokken verursacht und kommen in entwicklungsländern vor, z. b. im "meningitisgürtel" afrikas (südlich der sahara und nördlich des äquators von der ost-bis zur westküste) sowie in südamerika und asien. meningokokken werden durch tröpfcheninfektion übertragen, die inkubationszeit beträgt in der regel 3−4 tage, kann aber in einem bereich zwischen 2 und 10 tagen liegen. die häufi gsten erreger der bakteriellen meningitis bei immunsupprimierten patienten sind gramnegative enterobakterien inkl. pseudomonas aeruginosa, ferner streptococcus pneumoniae und listeria monocytogenes [3] . otitis, sinusitis 50 (57, 5) chronische abwehrschwächende krankheiten -chronischer alkoholismus -malignome -diabetes mellitus -immunsuppressive medikamentöse therapie -terminales nierenversagen -chronische hepatitis (leberzirrhose) zunächst können bakterien, die durch einen parameningealen fokus, hämatogen oder iatrogen in das zns gelangt sind, weitgehend ungehindert proliferieren, da opsonierende substanzen wie antikörper oder komplement im zns nur in sehr geringen mengen vorhanden sind. freigewordene bakterielle zellwandkomponenten (z. b. lipopolysaccharide, teichonsäuren oder peptidoglykane), aber auch mikrobielle toxine (z. b. pneumolysin) werden schließlich über pathogenerkennungsrezeptoren (wie z. b. toll-like-rezeptoren) durch immunkompetente zellen erkannt [6] , es kommt zu einer aktivierung von transkriptionsfaktoren (z. b. nf-kappa-b bei der pneumokokkenmeningitis) und einer produktion von zytokinen und chemokinen, die die entzündungsantwort dirigieren [7] . in das zns eingewanderte entzündungszellen (vor allem granulozyten) werden aktiviert und produzieren toxische substanzen wie reaktive sauerstoff -und stickstoff moleküle, auf die das zns nicht vorbereitet ist [8] . die eigentlich gegen die eindringenden bakterien gerichtete immunantwort schädigt das körpereigene zns. infolge einer endothelialen funktionsstörung kommt es zu einer beeinträchtigung der zerebrovaskulären autoregulation, einer störung der kohlendioxidreaktivität zerebraler gefäße und einer störung der blut-hirn-schranke. die entstehung eines vasogenen hirnödems gehört zu den wichtigsten ursachen eines erhöhten intrakraniellen drucks im verlauf der meningitis. ein erhöhter intrakranieller druck kann durch entstehung einer zerebralen einklemmungssymptomatik und durch reduktion des zerebralen perfusionsdrucks mit der gefahr zerebraler ischämien gefährlich werden. 487 1-2 wochen nach bereits erfolgter guter klinischer besserung der patienten auft reten ("delayed stroke") [11] , [12] . wichtige ursachen eines erhöhten intrakraniellen drucks sind eine zunahme des intrakraniellen blutvolumens durch eine gestörte zerebrovaskuläre autoregulation oder eine septische sinus-oder venenthrombose sowie eine liquorzirkulationsstörung mit entstehung eines hydrozephalus. neben den zerebralen komplikationen können sich folgende extrakranielle komplikationen in der akutphase der bakteriellen meningitis entwickeln: septischer schock, verbrauchskoagulopathie, adult respiratory distress syndrome (ards), arthritis (septisch und reaktiv), elektrolytstörungen wie hyponatriämie, syndrom der inadäquaten adh-sekretion (siadh), zerebrales salzverlustsyndrom oder zentraler diabetes insipidus, rhabdomyolyse, pankreatitis, septische einseitige (selten beidseitige) endophthalmitis oder panophthalmitis, blindheit als folge einer vaskulitis und spinale komplikationen (z. b. myelitis oder spinale vaskulitis; [4] , [13] nierenversagen (hämofi ltration) 10 (11, 5) adult respiratory distress syndrome (ards) 6 (6, 9) meningitis-assoziierte intrakranielle komplikationen entwickelten sich bei 65 (74,7 %) und systemische komplikationen bei 33 (37,9 %) patienten. a bezogen auf alle patienten (bzw. 25,8 % der überlebenden). ein positiver befund allerdings nicht sehr hilfreich ist [15] . ferner wird die bestimmung des serumprocalcitonins für die unterscheidung einer bakteriellen von einer nichtbakteriellen meningitis herangezogen: procalcitonin ist bei der bakteriellen meningitis mit hoher sensitivität (bis 99 %) erhöht, die spezifi tät liegt jedoch unter 85 % [16] , [17] , [18] . in einer frühen krankheitsphase kann das procalcitonin (bei parameningealem entzündungsfokus und nicht primärer hämatogener entstehung der meningitis) jedoch noch normal sein, sodass ein negativer procalcitoninwert eine bakterielle meningitis nicht ausschließt. bei jedem patienten mit bakterieller meningoenzephalitis muss noch am aufnahmetag eine bildgebende untersuchung durchgeführt werden, in der regel ein schädel-ct mit knochenfenster [3] . mögliche befunde, die in der schädel-ct oder -mrt bei einem patienten mit bakterieller meningoenzephalitis nachgewiesen werden können, sind in der folgenden übersicht zusammengefasst. cosekonzentrationen (<5 mg/dl) fi ndet sich in der regel eine sehr große zahl von bakterien im liquor (bakterienrasen im gram-präparat). an einzelnen zentren wird die bestimmung von liquorlaktat (werte meist >3,5 mmol/l) der glucosebestimmung vorgezogen. liquorzellzahlen <1000 zellen/μl können bei der bakteriellen meningitis sehr früh im krankheitsverlauf, bei antibiotisch anbehandelten patienten, bei fulminanten krankheitsverläufen und bei abwehrgeschwächten (z. b. leukopenischen) patienten beobachtet werden. der erregernachweis im liquor ist mit verschiedenen methoden möglich: 4 mikroskopisch mittels gram-färbung (oder methylenblau-färbung), 4 bakteriologisch mittels kultur und 4 molekularbiologisch mittels polymerasekettenreaktion (pcr). der nachweis von bakterien im liquor ist mit den genannten methoden bei 70−90 % der patienten mit eitriger meningitis möglich. bei etwa 50 % der patienten mit bakterieller meningitis sind die blutkulturen positiv; blutkulturen müssen deshalb vor beginn der antibiotikatherapie angelegt werden. zudem stehen antigennachweise für n. meningitidis, s. pneumoniae, h. infl uenzae und streptococcus agalactiae zur verfügung [14] . im blut fi nden sich eine leukozytose sowie eine erhöhung des c-reaktiven proteins (mögliche ausnahme: immunsupprimierte patienten). eine metaanalyse ergab, dass der negative crp-befund bei patienten mit dem klinischen bild einer meningitis mit einer vorhersagewahrscheinlichkeit von größer 97 % für eine nichtbakterielle ursache spricht, [3] , [14] , [19] . c einheitliche empfehlungen liegen in der literatur nicht vor. eine antibiotikatherapie muss bei patienten mit verdacht auf bakterielle meningitis schnell begonnen werden, möglichst eine stunde nach aufnahme [14] . eine verzögerung der antibiotikatherapie um mehr als 3 h nach krankenhausaufnahme muss unbedingt vermieden werden [24] ; in einer prospektiven multicenterstudie bei 156 erwachsenen patienten mit pneumokokkenmeningitis konnte nachgewiesen werden, dass eine verzögerung der antibiotikatherapie um mehr als 3 h mit einer ungünstigen prognose vergesellschaftet ist. ferner wurde in einer retrospektiven datenanalyse (119 patienten mit einem alter ≥16 jahren und einer bakteriellen meningitis, 56 % hatten eine pneumokokkenmeningitis) gezeigt, dass patienten, die später als 6 h nach krankenhausaufnahme mit antibiotika behandelt wurden, ein 8,4-mal höheres risiko hatten, an der meningitis zu versterben [25] . liegt das antibiogramm vor, muss die intravenöse antibiotikatherapie entsprechend angepasst werden (. tab. 32.6 und . tab. 32.7, [3] ). die empfohlene behandlungsdauer mit antibiotika liegt bei unkompliziertem verlauf einer h.-infl uenzae-meningitis und meningokokkenmeningitis bei 7−10 tagen, bei einer pneumokokkenmeningitis bei (10−) 14 tagen. in der behandlung der listerienmeningitis und der durch gramnegative enterobakterien verursachten meningitis wird meist über 3 wochen (oder länger) therapiert. eine routinemäßige liquorkontrollpunktion ist nicht erforderlich. bei unbekanntem erreger und fehlender klinischer besserung kann -wenn keine kontraindikationen bestehen -eine erneute liquorpunktion erwogen werden. rasch (wenn möglich am aufnahmetag) die operative fokussanierung erfolgen. in abhängigkeit von der anamnese und vom klinischen befund soll nach anderen infektiösen foci gesucht werden (z. b. th oraxröntgenaufnahme, abdomensonographie/ct, echokardiographie). z therapie kantibiotikatherapie der bakteriellen meningitis ist der erreger nicht bekannt, wird empirisch unter berücksichtigung des alters des patienten, der prädisponierenden faktoren und der damit wahrscheinlichsten bakterien behandelt (. tab. 32.4 und . tab. 32.5, [3] ). bei erwachsenen mit ambulant erworbener bakterieller meningitis sind die häufi gsten erreger streptococcus pneumoniae und neisseria meningitidis, bei erwachsenen ab 50 jahren spielen listerien zudem eine wichtige rolle. infolgedessen wird bei erwachsenen mit ambulant erworbener meningitis eine empirische antibiotikatherapie mit ceft riaxon und ampicillin empfohlen. in einigen ländern wie frankreich, belgien, spanien oder den usa kam es in den letzten jahren zu einem starken anstieg penicillin-und cefuroxim-resistenter pneumokokken [14] , [21] , sodass bei entsprechender anamnese zusätzlich vancomycin in der initialtherapie verabreicht werden muss. in deutschland wurde zwar ein anstieg von penicillin-resistenten pneumokokken im nordosten des landes verzeichnet, cefuroxim-resistente pneumokokken fanden sich allerdings bisher als erreger einer bakteriellen meningitis nicht [21] . riellen zerebralen gefäßkomplikationen (arteriitis, vasospasmus) gibt es bislang keine gesicherten th erapieformen. in analogie zu vasospasmen nach subarachnoidalblutung können die gabe von nimodipin sowie eine hypervolämie mit hämodilution bei leicht hypertonen blutdruckwerten angestrebt werden. sollte nimodipin gegeben werden, ist eine intraarterielle blutdruckmessung aufgrund der gefahr einer induzierten hypotonie obligat. der wissenschaft liche beleg für die wirksamkeit einer antikoagulation septischer sinus-/venenthrombosen bei der bakteriellen meningitis ist nicht gegeben. prospektive kon trollierte studien liegen bisher nicht vor. in einer retrospektiven studie zeigte sich allerdings ein günstiger eff ekt der heparintherapie bei patienten mit septischer sinus-cavernosus-th rombose [27] . bei patienten mit meningitisassoziierter th rombose des sinus transversus wurde eine erhöhte blutungsgefahr berichtet [27] . derzeit wird deshalb die antikoagulation mit intravenösem heparin (ptt-wirksam) bei kernspintomographisch (oder in der dsa) nachgewiesenen septischen sinus-/venenthrombosen infolge einer bakteriellen meningitis -mit ausnahme bei beteiligung des sinus transversus (hier gefahr von intrazerebralen blutungen)empfohlen (7 kap. 30.1) . eine antiepileptikatherapie (z. b. schnelle intravenöse aufsättigung mit phenytoin, valproat oder levetiracetam) wenn es innerhalb von 2 tagen nach beginn der antibiotikatherapie zu keiner klinischen besserung kommt, müssen folgende ursachen bedacht werden: 4 auft reten von intrakraniellen komplikationen, 4 persistierender infektiöser fokus (insbesondere ein nichtsanierter oder unzureichend operierter parameningealer fokus wie z. b. eine mastoiditis, sinusitis oder otitis media), 4 inadäquates antibiotikaregime (z. b. unwirksames antibiotikum oder zu niedrige dosis). es müssen dann entsprechende diagnostische maßnahmen (z. b. bildgebung, hno-konsiliaruntersuchung) in die wege geleitet werden. wenn der erreger der eitrigen meningitis nicht isoliert werden konnte, sollte bei fehlendem ansprechen auf die antibiotikatherapie eine erweiterung bzw. ein umsetzen der antibiotika erwogen werden. ktherapie wichtiger intrakranieller komplikationen finden sich zeichen eines erhöhten intrakraniellen druckes, müssen hirndruck-senkende maßnahmen erfolgen (z. b. oberkörperhochlagerung auf 30°, osmotherapie mit mannit, bei beatmeten patienten normoventilation, bei sonst nicht beherrschbarem intrakraniellen druck möglichst kurzzeitige hyperventilation mit einem zielwert des pco 2 um 32 mmhg, tiefe sedierung, bei hydrozephalus externe liquordrainage) [26] . stuporöse oder komatöse patienten können von einem icp-monitoring profi tieren ( [20] ; 7 kap. 11). für die arte-. tabelle 32.5 antibiotikatherapie der bakteriellen meningitis (bei bekanntem erreger) zusammenfassend kann aufgrund der zur verfügung stehenden daten die gabe von dexamethason bei erwachsenen patienten mit verdacht auf eine bakterielle meningitis (d. h. klinischer verdacht plus trüber liquor, nachweis von bakterien im liquor in der gram-färbung oder eine liquorleukozytenzahl von >1000/μl) in deutschland empfohlen werden; dexamethason (fortecortin) wird in einer dosis von 10 mg i.v. unmittelbar vor gabe des antibiotikums verabreicht [3] . daraufh in wird mit 10 die wirksamkeit von dexamethason wurde in einer europäischen prospektiven, placebokontrollierten, randomisierten multicenterstudie bei 301 erwachsenen mit bakterieller meningitis untersucht [28] . dexamethason (10 mg) oder placebo wurden in dieser studie 15−20 min vor der ersten antibiotikagabe appliziert und dann alle 6 h für insgesamt 4 tage. in der studie konnte ein günstiger eff ekt der dexamethasonbehandlung gezeigt werden: dexamethason führte zu einer signifi kanten reduktion der letalität und der häufi gkeit ungünstiger klinischer verläufe. eine subgruppenanalyse zeigte, dass dexamethason nur bei den patienten mit pneumokokkenmeningitis wirksam war, nicht bei meningitiden anderer ätiologie, wie z. b. der meningokokkenmeningitis. der günstige eff ekt von corticosteroiden auf die letalität konnte in mehreren metaanalysen für länder mit einem hohen grad medizinischer versorgung bestätigt werden [29] , [30] , [31] . für entwicklungsländer mit eingeschränkter medizinischer versorgung und einem hohen anteil hiv-positiver patienten konnte keine wirksamkeit für dexamethason bei der bakteriellen meningitis nachgewiesen werden [32] , [33] , [34] . im falle eines impfpräventablen meningokokkenstamms als ursache der meningokokkenerkrankung beim indexpatienten (serogruppe a, c, w oder y) wird seit kurzem zusätzlich eine impfung mit einem entsprechenden impfstoff für enge kontaktpersonen (haushaltsmitglieder) empfohlen [36] . z prognose über 20 % der patienten mit einer pneumokokkenmeningitis und listerienmeningitis versterben [30] . die letalitätszahlen der meningokokkenmeningitis liegen bei 3−10 % (. tab. 32.8, [9] , [37] ungefähr 65 % der überlebenden patienten sind nach durchschnittlich 5 jahren zumindest grob neurologisch weitgehend rehabilitiert. jüngste neuropsychologische untersuchungen weisen jedoch darauf hin, dass ein breites spektrum von neuropsychologischen defi ziten -insbesondere einem subkortikalen muster entsprechend -auch noch nach >10 jahren bei der überwiegenden zahl der patienten mit einem hirnabszess bestehen, und zwar teilweise unabhängig von größe und lokalisation. der vor einiger zeit publizierte "imaging severity index" (isi) unterstützt eine frühzeitige potenzielle prognoseeinschätzung. die überwiegende zahl der abszesse im spinalkanal sind epidural lokalisiert, typischerweise thorakal und/oder lumbal sowie häufi g dorsal dem rückenmark anliegend. am häufi gsten werden sie im höheren lebensalter (7. lebensjahrzehnt) gesehen. sie erstrecken sich meist über nur wenige wirbelsegmente, können jedoch in einzelfällen auch deutlich ausgedehnter sein. in sehr seltenen fällen werden auch ein spinales subdurales empyem sowie ein intramedullärer abszess gesehen. alle eine transkranielle dopplersonographie erlaubt das frühzeitige erkennen einer arteriitis sowie deren monitoring. ein tuberkulintest ist nicht notwendig, da häufi g falsch positiv oder falsch negativ. in seltenen fällen kann eine meningeale biopsie indiziert sein, vor allem zur abgrenzung eines tuberkuloms oder einer granulomatösen lokalen meningitis von einem malignen tumor (z. b. lymphom). in der bildgebung wurden bei kindern und jugendlichen bestimmte computertomographische kriterien defi niert, die in kombination eine spezifi tät von nahezu 100 % und eine sensitivität von ca. 80−90 % zeigen, bei älteren und alten patienten mit tuberkulöser meningitis sind diese radiologischen parameter häufi g deutlich weniger ausgeprägt. eine hyponatriämie, am ehesten im sinne eines zerebralen "salt wasting syndromes" (csw), bedarf engmaschigsten monitorings der elektrolyte und resultiert nicht selten in intensivpfl ichtigkeit. gerinnungsuntersuchungen zeigen nicht selten einen zustand der hyperkoagulabilität, möglicherweise mit einem erhöhten risiko für zerebrale infarkte assoziiert. hiv-positive patienten mit intrakraniellen tuberkulomen können im rahmen des "immune reconstitution syndrome" (iris) eine durchaus dramatische klinisch neurologische verschlechterung erfahren, mit zunahme der neurologi-z diagnostik die untersuchung des liquor cerebrospinalis ist für die diagnose einer chronischen meningitis unverzichtbar, der liquor ist typischerweise klar, bei deutlich erhöhtem eiweiß auch xanthochrom wirkend. es fi ndet sich eine geringe bis mäßige gemischtzellige, gelegentlich überwiegend lymphozytäre pleozytose (bis zu 500 zellen/μl), bei akuten verläufen kann auch initial eine granulozytäre pleozytose bestehen. das liquoreiweiß ist auf bis zu 500 mg/dl erhöht, exzessive eiweißwerte (>1000 mg/dl) werden bei liquorzirkulationsstörungen gesehen. die liquorglucose (bzw. liquor-/ serum-glucoseratio) ist bei protrahiertem verlauf weitgehend normal, bei eher subakuten (akuten) verläufen gering bis mäßiggradig erniedrigt, sie korreliert mit der nachweisbarkeit von erregern im liquor cerebrospinalis. mittels ziehl-neelsen-färbung gelingt der nachweis von mycobacterium tuberculosis bei 10−25 % der patienten mit chronischer tuberkulöser meningitis, bei 30−50 % der patienten ist eine liquorkultur positiv. seriell angelegte liquorkulturen erhöhen die ausbeute auf >50 %. wenngleich die ergebnisquote des nachweises von mykobakterieller dna (mittels pcr) nicht höher liegt als die der liquorkultur, ist eine pcr trotzdem indiziert, da die ergebnisse schon nach 24 h vorliegen. die "nested-pcr", insbesondere die mpb-64-pcr, erhöht die sensitivität auf 90 % -dies bei vergleichbarer spezifi tät. weitere diagnostische methoden, die bereits erfolgreich zum nachweis von mykobakterien im sputum eingesetzt wurden, müssen noch auf ihre tauglichkeit bei einer zns-tuberkulose überprüft werden, die liquoradenosindeaminase kann als eine solche komplementäre diagnostische methode mit einer spezifi tät von >90 % und einer sensitivität von ca. 70 % gewertet werden. bei patienten mit bewusstseinsstörung und/oder neurologischer herdsymptomatik muss jeder lumbalpunktion die dreifachkombination (vierfach-/fünff ach-kombination) wird für mindestens 3−6 monate gegeben, anschließend eine zweifachkombinationstherapie für weitere 6−9 monate. regelmäßige klinisch neurologische kontrollen, neuroimaging-und liquorkontrollen sind essenziell. intrakranielle tuberkulome sind ebenfalls primär konservativ zu therapieren, in einzelfällen nehmen sie unter der spezifi schen chemotherapie an größe zu, in solchen fällen ist eine vierbis fünff achkombinationstherapie bis zum bildgebenden nachweis einer größenreduktion durchzuführen. eine frühzeitige externe liquordrainage bzw. die implantation eines ventrikuloperitonealen oder ventrikuloatrialen shunts verhindert bzw. behandelt die hydrozephalusbedingte icp-erhöhung. die endoskopische ventrikulostomie (3. ventrikel) ist im management eines obstruktiven hydrozephalus bei patienten mit tuberkulöser meningitis meist nicht zielführend. daneben ist auf ausreichende ernährung, engmaschigste elektrolytkontrollen (cave: siadh-/csw-syndrom) und entsprechenden elektrolytausgleich wert zu legen. im fortgeschrittenen stadium einer tuberkulösen meningitis bzw. bei drohender oder tatsächlicher spinaler symptomatik ist eine steroidtherapie (prednison 1 mg/kgkg) indiziert, wenngleich ein kürzlich vorgelegter cochrane-review [105] zur defi nitiven diagnose bedarf es des positiven ausfalls spezifi scher tests. der liquor cerebrospinalis zeigt in der überwiegenden zahl der fälle eine intrathekale igm-produktion, gelegentlich auch eine intrathekale igg-und iga-produktion. bei progressiver paralyse fi ndet sich fast immer eine liquorpleozytose, während dies bei tabes dorsalis nur in 50−75 % der fälle gesehen wird. eine meningovaskulitis zeigt ebenfalls in den meisten fällen eine pleozytose. das gesamtprotein sowie eine intrathekale igg-(häufi g auch igm-und iga-)produktion ergänzen den liquorbefund. die pleozytose ist häufi g lymphozytär, aber auch ein lymphoplasmazelluläres bild wird gesehen. bildgebende befunde (zerebrale ct-oder mr-untersuchung) zeigen unspezifi sche veränderungen im sinne einer arteriitis oder zerebraler ischämie oder auch unspezifi sche läsionen in der weißen substanz. z ätiologie und pathogenese zu den häufi gsten erregern akuter meningoenzephalitiden zählen in europa enteroviren, gefolgt von arboviren (diverse alpha-, flavi-, und bunyaviren). darüber hinaus kommen auch masern-, mumps-, epstein-barr-viren (ebv), humane immunodefi ziente viren (hiv) und "lymphocytic choriomeningitis"-viren (lcmv) in diesem zusammenhang vor [159] . die infektion erfolgt meist im rahmen eines systemischen virusinfekts. beim direkten erregerbefall gelangen die viren am häufi gsten auf hämatogenem weg ins zns. im gegensatz zu früheren annahmen scheinen die viren die blut-hirn-schranke relativ leicht überwinden zu können. der zns-befall hängt wohl vom ausmaß der virämie und die virämie von der verfassung des immunsystems ab. man vermutet, dass die viren die gefäßendothelzellen direkt befallen oder durch pinozytose/exozytose durch die zellen hindurchtransportiert werden. einige viren (rabies, hsv) können durch retrograden axonalen transport peripherer nerven in das zns gelangen. sicher müssen mehrere ungünstige faktoren zusammenwirken, damit sich aus einer der häufi gen virusinfektionen eine enzephalitis entwickelt. in der regel gehen die infi zierten nervenzellen zugrunde. dadurch werden z. b. entzündliche reaktionen ausgelöst, die weiteren schaden anrichten können. [148] , [170] . der klassische liquorbefund ist von der einer viralen meningitis nicht zu unterscheiden und folgendermaßen charakterisiert: 4 geringe bis mäßige zellzahlerhöhung: 20−1500/μl (selten bis 3000). cave: auch normale zellzahlen können vorkommen. bei sicherung einer enzephalitis sollte des weiteren eine infektiöse virale enzephalitis von einer akut disseminierten enzephalomyelitis (adem) unterschieden werden, die anamnestisch häufi g eine kürzlich erfolgte impfung bei kindern ergibt, visuelle störungen eines oder beider augen sowie zeichen der spinalen beteiligung und darüber hinaus multifokale entmarkungsherde in der mrt in beiden hemisphären [159] . die klinische untersuchung sollte auch das aufsuchen möglicher hautveränderungen (vzv) beinhalten. > gibt die klinische konstellation hinweise auf eine enzephalitis, muss sofort eine empirische antivirale therapie begonnen werden. kneuroradiologie die kranielle computertomographie (ct) und die magnetresonanztomographie (mrt) können charakteristische befunde zeigen und erlauben oft die abgrenzung anderer krankheitsbilder [145] , allerdings zeigt sich bei bis zu 10 % liquorchemisch nachgewiesener hsv-enzephalitisfälle ein unauff älliger kranieller computertomographie-oder magnetresonanztomographiebefund. [176] . in der kraniellen mrt können morphologische veränderungen bereits deutlich früher und sensitiver als in der cct nachgewiesen werden, wobei durch diff usionsgewichtete und sog. flair-sequenzen ein erheblicher informationsgewinn durch frühzeitige charakterisierung enzephalitischer läsionen herbeigeführt werden kann. die frontomesiotemporalen anteile, die insuläre region, der gyrus cinguli, der th alamus sowie der frontobasale kortex sind mit fokalen ödemen -manchmal sogar mit vereinzelter kontrastmittelaufnahme -häufi g betroff en. es gibt jedoch bildmorphologische hinweise darauf, dass bei säuglingen und kindern im gegensatz zu erwachsenen vermehrt extratemporale läsionen entdeckt werden [174] . eeg-untersuchungen zeigen bei liquor-pcr-bestätigten hsve-fällen in bis zu 90 % fokal auf den temporallappen bezogene spike-und slow-wave-aktivität, die jedoch häufi g unspezifi sch ist. charakteristischer liquorbefund ist eine lymphozytäre pleozytose von 15−200 (selten bis 700) zellen/μl. oft fi nden sich an tag 3−6 auch plasmazellen und eine mononukleäre pleozytose oder auch eine hämorrhagische komponente (erythrozyten, xanthochromie, siderophagen). der liquoreiweißgehalt ist in über 80 % der fälle erhöht. mittels pcr kann frühzeitig (tag 1 oder 2) virusspezifi sche dna im liquor nachgewiesen werden. allerdings korreliert die schwere der erkrankung nicht mit der zahl der viruskopien [178] . falsch-negative liquor-hsv-pcr-befunde sind am häufi gsten innerhalb der ersten 24−48 h sowie nach 10−14 tagen nach krankheitsausbruch. die hsv-pcr kann in seltenen fällen auch erst bei der 2. oder gar 3. liquoruntersuchung positiv werden, was auf eine zu frühe untersuchung oder zu geringe viruslast zurückzuführen ist. die therapie erfolgt bereits bei klinischem verdacht mit aciclovir mit einer dosis von 10 eine akut aszendierende parese, die einem guillain-barré-syndrom (gbs) ähnelt, allerdings mit einer pleozytose einhergeht, kann durch eine fsme-, hiv-infektion, rabies oder wnv-infektion bedingt sein [155] . z verlauf und spezifi ka einiger enzephalitiden aufgrund der verschiedenen verläufe wird auf die wichtigsten enzephalitiden speziell eingegangen. das hsv ist in westeuropa bei kindern, die älter als 6 monate sind, und bei erwachsenen die häufi gste ursache einer sporadischen enzephalitis. bei immunkompetenten erwachsenen wird die hsve in 90 % der fälle durch das hsv-1 ausgelöst, während hsv-2 meist nur eine benigne lymphozytäre meningoenzephalitis hervorruft , welche auch mehrfach remittieren kann (früher: mollaret-meningitis). bei neugeborenen und immuninkompetenten ruft hsv-2 eine diff use enzephalitis im rahmen einer systemischen, hämatogen fortgeleiteten infektion hervor. die durch hsv-1 erzeugte enzephalitis ist mit einer inzidenz von 2-4/1.000.000 die häufi gste sporadische enzephalitis in westeuropa [167] , und zwar ohne saisonale bevorzugung. ein drittel aller hsve-fälle tritt als primärinfektion auf. die mehrzahl aller patienten hat jedoch bereits antikörper gegen hsv, wenngleich nur 10 % aller hsve-patienten klinische zeichen einer rekurrierenden hsv-infektion aufweisen (z. b. herpes labialis). das virus gelangt vermutlich über die mund-und nasenschleimhaut zum bulbus olfactorius oder ganglion gasseri (n. trigeminus) und über durale nervenäste zur vorderen und mittleren schädelgrube. jedoch wurde bei proben aus ehemals gesundem hirngewebe hsv-gensequenzen auch außerhalb des ganglion gasseri gefunden [142] . hsv verursacht eine fokale enzephalitis, die vorwiegend temporo-und frontobasal gelegen und durch hämorrhagische nekrosen und eine erhebliche hirnschwellung charakterisiert ist. in einzelstudien gibt es hinweise auf virusunabhängige chronisch-progrediente gewebsuntergänge im langzeitverlauf der hsve [162] . die therapie unterscheidet sich nicht von der der hsve. alternativ kann auch brivudin eingesetzt werden. die mit windpocken assoziierte enzephalitis hat eine letalität von 30 %, meist bedingt durch die oft vorbestehende immuninkompetenz. in einem vor kurzem publizierten fallbericht wurde die kasuistik einer älteren patientin aufgearbeitet, welche nach erhalt einer vzv-lebendimpfung eine varizellen-assoziierte meninigits entwickelte [152] . zerebrale beteiligungen bei ebv-infektionen sind meist gutartig und kommen primär bei immunsupprimierten menschen vor. das ebv ist ein herpesvirus, welches verschiedene neurologische manifestationen verursachen kann (meningitis, enzephalitis, aids-assoziiertes zns-lymphom, myeloradikulitis und enzephalomyeloradikulitis). die neurologischen erscheinungen der ebv-infektion treten meistens als komplikationen der infektiösen mononukleose (in ca. 5−7 % der fälle) auf. die inzidenz der infektiösen mononukleose selbst liegt bei ca. 8/1000. die klassischen symptome einer infektiösen mononukleose sind fieber (76 %), pharyngitis (82 %) sowie lymphknotenschwellungen (94 %) und splenomegalie (52 %). neurologische symptome können sich vor, während und nach den klassischen symptomen manifestieren [149] . die ebv-enzephalitis kann als meningoenzephalitis, als zerebellitis (insbesondere bei kleinkindern) und in form von hirnnervenausfällen in erscheinung treten. es wurden auch polio-ähnliche krankheitsbilder beschrieben [179] . schwere krankheitsverläufe kommen insbesondere bei kleinkindern und immunsupprimierten patienten vor. diagnostiziert wird die erkrankung über die liquor-pcr. kontrollierte studien zur behandlung der ebv-enzephalitis fehlen. neben aciclovir kann auch ganciclovir über 3 wochen gegeben werden. das fsme-virus gehört zu der gruppe der arboviren, wobei das erregerreservoir aus kleinen wildnagern und vektorzecken besteht. die entwicklungszyklen der ixodes-ricinus-zecken führen zu einem saisonalen auft reten der erkrankung von märz bis oktober mit erkrankungsgipfel von april bis juli. durchseuchte zeckenpopulationen fi nden sich vornehmlich in süddeutschland, österreich, tschechien, ungarn und der slowakei [157] . im jahre 2009 sind zwar keine neuen risikogebiete hinzugekommen, jedoch trat erstmalig in schleswig-holstein ein fsme-fall auf, wobei frühere aufenthalte in risikogebieten nicht ausgeschlossen werden konnten. darüber hinaus wird derzeit durch die sog. gache-studie (german trial of acyclovir and corticosteroids in herpes-simplex-virus-encephalitis) der eff ekt von adjuvantem dexamethason auf folgeschäden bei patienten mit herpesenzephalitis untersucht. da bei der pathogenese dieser enzephalitis auch autoimmunmechanismen eine wichtige rolle spielen, scheint unter einer kombinierten th erapie mit aciclovir und dexamethason die rate von patienten mit schlechtem outcome geringer zu sein als unter alleiniger th erapie mit aciclovir [158] . die antivirale th erapie reduziert die zahl der viruskopien im liquor. in den meisten fällen führt die aciclovir-gabe somit zu einer raschen reduzierung des antigennachweises im liquor, sodass in den meisten fällen innerhalb von 15 tagen nach beginn der th erapie die liquor-pcr negativ ausfällt [174] . bei persistierend positiver liquor-pcr sollte an eine zusätzliche oder alternative antivirale th erapie gedacht werden. darüber hinaus ist die th erapie symptomatisch ausgerichtet, was in der regel ein monitoring bei intensivmedizinischer betreuung mit einbezieht. im einzelfall kann eine osteoklastische trepanation bei schweren verläufen aufgrund von fokaler hirnschwellung ein gutes ergebnis bringen [168] . eine antikonvulsive th erapie ist bei anfällen oder beim klinischen verdacht nichtkonvulsiver anfälle indiziert. die tatsächliche inzidenz der herpes-zoster-enzephalitis ist nicht bekannt. gefährdet durch schwere verläufe sind immunsupprimierte patienten, cmv-seronegative transplantatempfänger und malignompatienten während einer chemotherapie. ein besonders hohes risiko besteht für aids-patienten im stadium iv (chorioretinitis). die vzv-enzephalitis tritt in 1−2 von 10.000 fällen einer vzv-infektion auf, meist 1−2 wochen nach dem typischen exanthem. gelegentlich kann sie dem exanthem auch um bis zu 3 wochen vorausgehen. klinisch kommt es entweder zu einer meningoenzephalitis oder zerebellitis im anschluss an eine windpockeninfektion oder zu einer zosterneuritis-(gürtelrose-)assoziierten enzephalitis, die häufi ger bei abwehrgeschwächten vorkommt und als polioenzephalitis oder seltener als multifokale leukenzephalopathie verlaufen kann. meist beginnt sie 1−2 wochen nach dem exanthem, doch gelegentlich kann sie den windpocken auch um bis zu 3 wochen vorausgehen. neuropathologisch fi nden sich entzündliche läsionen, hämorrhagische nekrosen, vaskulitiden und infarkte durch gefäßstenosen und -verschlüsse. die kranielle mrt zeigt neben multiplen läsionen in der weißen substanz ischämische und hämorrhagische läsionen mit kontrastmittelenhancement. ein normales eeg im akutstadium spricht gegen die diagnose. die eeg-veränderungen können bis zu einem jahr persistieren. im liquor fi ndet sich eine lymphozytäre pleozytose, anfänglich mit einer granulozytose. derzeit sind 3 erregersubtypen bekannt (europäisch, östlich, fernöstlich). die letalität einer manifesten erkrankung beträgt beim westlichen erregersubtyp 1−2 % (bei der myelitischen form 20 %), beim östlichen subtyp 20 %. bei 27 % der patienten fi nden sich lang anhaltende neuropsychologische oder neurologische defi zite. eine virusisolierung gelingt in der akutphase des katarrhalischen infektes aus rachenspülwasser und liquor, aus blut nur selten. anfang 2004 wurde die falldefi nition des robert-koch-instituts für die fsme geändert: als fsme-fall gelten nur noch fsme-virusinfektionen, bei denen ein positiver befund vorliegt, welcher mit mindestens einer der 4 folgenden methoden erhoben wurde: eine spezifi sche therapie existiert nicht. es wird lediglich symptomatisch behandelt. menschen, die ein erhöhtes risiko durch vermehrten kontakt mit rabiesinfi zierten tieren haben, sollten eine präexpositionsprophylaxe erhalten. dabei handelt es sich um einen aktivrabiesimpfstoff , der intradermal oder intramuskulär am tag 0, 7, 21 oder 28 appliziert wird. es wird eine fäkal-orale und aerogene übertragung angenommen. zur vermeidung einer infektion mit enterovirus-typ-71 werden vor allem hygienische maßnahmen empfohlen. die diagnose erfolgt mittels nachweis von virus-rna im liquor-pcr in kombination mit pathologischen veränderungen in der mrt, insbesondere in den vorderhornzellen des rückenmarks, der dorsalen pons und der medulla oblongata. bei potenziell lebensbedrohlichen verläufen kann das präparat pleconaril verabreicht werden [171] . pleconaril ist ein oral applizierbares virostatikum, das die replikation von viren durch einen kapsidbindenden mechanismus hemmen kann. eine große post-hoc-analyse zeigte, dass pleconaril den verlauf der infektion bei milden erkrankungsformen lediglich minimal beeinfl ussen kann, wohingegen patienten mit einem schweren verlauf sowohl von dem präparat als auch von intravenösen immunglobulinen [163] profi tieren können. nisierungschemata. nach 3−5 jahren ist eine erneute booster-impfung erforderlich. der empfohlene zeitpunkt zum beginn der impfung ist im winter, da die zecken zu diesem zeitpunkt inaktiv sind. eine spezifi sche antivirale th erapie für die fsme gibt es nicht. rabies ist eine der ältesten bekannten zoonosen. erreger ist ein rhabdovirus der gattung lyssavirus, welcher alle säugetiere infi zieren kann. schätzungen zufolge versterben jährlich ca. 100.000 menschen an tollwut. in deutschland konnte die tollwut durch systemische bekämpfungsmaßnahmen nahezu eliminiert werden und ist daher extrem selten. so gab es 1 gemeldeten fall in 1996, 1 in 2004 und 4 in 2005. das reservoir des rabies-virus umfasst viele tierarten, darunter füchse, nager und fledermäuse, wobei die übertragung auf den menschen zu über 90 % durch hundebisse erfolgt. nach replikation im muskelgewebe bindet das virus an den acetylcholinrezeptor und gelangt über die neuromuskuläre endplatte und den peripheren nerven bis zum vorderhorn, wo es erneut zu einer virusvermehrung kommt. danach erfolgt die ausbreitung zu den speicheldrüsen über das sympathische nervensystem. hierbei ist das limbische system besonders vulnerabel, wobei im verlauf neben perivaskulären lymphozyteninfi ltraten als typisches merkmal sog. "negri-körperchen" auftreten. die inkubationszeit liegt zwischen 10 und 20 tagen (in einzelberichten bis 6 jahre). die größe der verletzung steht in umgekehrter korrelation mit der länge der inkubationszeit. die klassische klinische präsentation einer enzephalitischen rabies umfasst fieber und eine autonome hyperaktivität mit fl uktuierend mentalem status. in der akuten phase kommen krämpfe des larynx und des pharynx bei konsekutiver hydrophobie oder sogar aerophobie vor. der tod tritt in der regel im koma und unter den zeichen einer atemlähmung ein, zwischen auft reten der ersten symptome und dem tod liegen maximal 10 tage. bei klinisch manifester tollwut sterben die patienten praktisch immer, intensivmedizinische verfahren können lediglich den verlauf etwas aufh alten. bis zum derzeitigen zeitpunkt wurden in der literatur nur 5 patienten beschrieben, die trotz klinisch manifester erkrankung überlebten, wobei 4 von diesen personen eine postexpositionsprophylaxe (pep) vor ausbruch der erkrankung erhalten haben und ein erkrankter geimpft worden war. im jahre 2004 wurde in den usa von einem fall eines 15-jährigen mädchens berichtet, das an tollwut erkrankt war und ohne pep oder impfung überlebte. die patientin wurde über eine woche in ein künstliches koma versetzt sowie mit ribavirin-infusionen therapiert. die ursache für das überleben bleibt letztlich unklar [144] . die diagnose wird durch das klinische bild und den erregernachweis gestellt. im liquor fi nden sich oft eine schran-kapitel 32 · infektionen symptomatisch und supportiv, wobei die hälft e der patienten einer assistierten mechanischen beatmung bedarf. eine kürzlich publizierte arbeit [143] beschrieb den erfolgreichen einsatz eines spezifi schen humanen monoklonalen antikörpers m102.4, ein vom nipah-virus infi ziertes frettchen vor einer tödlichen erkrankung zu bewahren. dabei erfolgte die behandlung innerhalb von 10 h nach infektion. dies könnte ein wirksamer th erapieansatz sein, muss aber noch durch klinische studien untersucht werden. eine weitere vor kurzem erschienene arbeit [164] zeigt in einem in-vivo-tiermodell die inhibition einer nipah-virusinfektion durch eine künstlich angehängte cholesteringruppe an ein für die fusion des virus mit der zellmembran notwendiges protein. hierdurch konnte eine tödliche nipah-virusenzephalitis verhindert werden. es besteht die berechtigte hoff nung, dass dies in zukunft ein ansatz für eine prävention oder th erapie gegen nipah-infektionen sein könnte. auf der suche nach einem eff ektiven wirkstoff sowohl gegen das nipah-als auch gegen das hendra-virus wurden kürzlich mäuse mit partikeln des venezuela-equine-enzephalitis-virus beimpft , die glykoproteine entweder vom hendra-oder vom nipah-virus enthielten. daraufh in wurden hochpotente kreuzreagierende, neutralisierende antikörper gegen beide genannten viren produziert [150] . eine abgeheilte nipah-virusinfektion kann dennoch mit zunächst latenten residuen von später auft retenden persönlichkeitsänderungen oder persistierendem anfallsleiden einhergehen [145] . ein schubförmiger verlauf einer zns-entzündung mit dem nipah-virus im sinne einer "late-onset"-enzephalitis wurde bei ca. 8 % der initial mit dieser infektion überlebenden patienten beschrieben [172] . dies ist sicherlich als ein sehr ungewöhnlicher verlauf einzustufen. nach durchführung von mehreren autopsien (32 fälle) von durch nipah-enzephalitis verstorbenen patienten konnte nachgewiesen werden, dass diese häufi g eine systemische vaskulitis aufwiesen, die mit th rombosen und parenchymalen nekrosen insbesondere im zns assoziiert war. virale antigene konnten zudem in den zerebralen vaskulären endothelzellen nachgewiesen werden [180] . daher wird nun angenommen, dass das nipah-virus auf hämatogenem wege ins zns gelangt und dass die initialen neurologischen symptome ausdruck einer multifokalen vaskulitis sind mit daraus resultierenden multizentrischen th rombosen. auch direkte virusinfektion ist möglich. in den aktuelleren ausbrüchen von 2001 bis 2004 in bangladesh und in indien konnten in vielen fällen jedoch keine tierexpositionen als mögliche infektionsquelle nachgewiesen werden, sodass eine mensch-zu-mensch-transmission in betracht gezogen werden muss [146] , [153] . vorläufi ge ergebnisse legen die vermutung nahe, dass fledermäuse der gattung chiroptera das natürliche reservoir für das nipah-virus bilden. nipah selbst wird am ehesten durch den urin der fledermäuse verbreitet, indem beispielsweise schweine eine infektion durch direkte exposition dieser exkremente akquirieren [175] . die klinik bei schweinen verläuft in der regel relativ mild. bei menschen können verläufe von einer asymptomatischen infektion bis hin zu einer letalen enzephalitis vorkommen. zu beginn wird über grippeähnliche beschwerden mit fieber, halsschmerzen, kopfschmerzen, erbrechen und myalgien geklagt. nach etwa 3-14 tagen können schwindel, bewusstseinsstörung bis hin zum koma, fokal-neurologische symptome wie krampfanfälle, vegetative entgleisungen und atemregulationsstörungen folgen -allesamt anzeichen einer enzephalitis [160] . die inkubationszeit beträgt 4 bis 45 tage. die mortalität ist mit 73 % als sehr hoch anzusehen. diagnostiziert wird diese erkrankung durch den nachweis von serum-ak, liquor-pcr und anzüchtung in zellkulturen aus serum, liquor, rachenfl üssigkeit oder urin. in der kraniellen mrt der betroff enen patienten konnten multiple hyperintense läsionen subkortikal und im marklager in den t2-gewichteten und flair-sequenzen nachgewiesen werden. eeg-untersuchungen zeigten in der regel entweder schwere allgemeinveränderungen oder periodische slowwave-komplexe auf. eine spezifi sche antivirale therapie existiert derzeit nicht. die behandlung erfolgt in erster linie intensivmedizinisch auch systemische erkrankungen können die ursache einer chronischen meningitis darstellen, wobei dann mit zusätzlichen symptomen zu rechnen ist, auf die gesondert geachtet werden muss. im allgemeinen ist der verlauf chronisch fortschreitend mit wiederholten exazerbationen. vaskulitiden haben meist chronisch progrediente verschlechterungen mit krisenhaft en zuspitzungen, hingegen sind rezidivierende krisen mit intermittierender beschwerdefreiheit typisch für die mollaret-meningitis und auch für abszessrupturen. neben einem entzündlich veränderten liquor mit lymphozytärer pleozytose von einigen 100 zellen, eiweißvermehrung und glucosereduktion fi nden sich bei der diagnostik chronischer meningitiden häufi g allgemeinveränderungen in der eeg-untersuchung. kernspintomographische kontrastmitteluntersuchungen des gehirns oder des rückenmarks weisen häufi g ein meningeales enhancement auf und helfen darüber hinaus, eine geeignete meningeale lokalisation vor potenziell geplanter biopsie zu identifi zieren. sollte eine ursachenspezifi sche th erapie aufgrund fehlenden erregernachweises nicht zur verfügung stehen, wird längerfristig mit corticosteroiden behandelt. wie eine engmaschige kontrolle der atmungsparameter. im einzelfall kann eine osteoklastische trepanation bei schweren verläufen aufgrund von fokaler hirnschwellung ein gutes ergebnis bringen [168] . eine antikonvulsive th erapie ist bei anfällen oder beim klinischen verdacht nichtkonvulsiver anfälle indiziert. die immunisierung gegen bestimmte viren (fsme) ist daher umso bedeutender. bei ausgewählten viruserkrankungen (z. b. rabies, pocken) nach bereits stattgehabter infektion ist auch die gabe von hyperimmunglobulinen (passive immunisierung) notwendig. eine komatöse aufnahme ist für den patienten ebenso als prognostisch ungünstig zu werten wie ein im verlauf einsetzendes koma, ein erhöhtes lebensalter bzw. säuglingsalter sowie der nachweis einer intrathekalen igg-synthese. einführung chronische entzündungen der meningen können schwerwiegende neurologische störungen hervorrufen und sogar tödlich enden, falls nicht erfolgreich behandelt wird. der zustand kann dann diagnostiziert werden, wenn eine entzündung der hirnhäute über 4 wochen anhält, was sich in einem infl ammatorischen liquorprofi l widerspiegelt. [182] . es wurden th erapieversuche mit isoprinosin allein oder in kombination mit intrathekaler oder intraventrikulärer gabe von interferon berichtet, die die überlebensrate verlängert und bei manchen patienten eine gewisse klinische besserung gebracht hätten. allerdings gab es hierzu nie eine kontrollierte klinische studie [181] . ebensowenig gab es bisher eine klinische studie, die die hypothese bestätigte, dass das anti-apoptotische präparat flupirtin in kombination mit antiviraler th erapie den progredienten verlauf der krankheit aufzuhalten vermag [187] . die erkrankung endete früher in 80 % der fälle innerhalb von 3 jahren nach diagnosestellung letal. inzwischen sind durch gezielte behandlung von myoklonien, spastik, anfällen und weiterer komplikationen verläufe über 10 jahre möglich. diff erenzialdiagnostisch muss an eine progressive rötelnpanenzephalitis oder auch an leukenzephalopathien gedacht werden, die einen ähnlichen verlauf haben können. es wurden fälle beschrieben, in denen mrt-läsionen bei sspe-aff ektierten kindern auch im hirnstamm detektierbar waren [190] . in einer vor kurzem erschienenen fallberichtpublikation wurde von einem erstmaligen auft reten von sspe bei einem erwachsenen patienten im zervikalen myelon berichtet [186] . dies ist insofern interessant, als dass neben der ungewöhnlich späten erstmanifestation das rückenmark untypischerweise befallen wurde. der liquorbefund ist der einzige auff ällige laborparameter, klinik und infektparameter geben keinen hinweis auf eine infektion. im liquor und serum fi nden sich sehr hohe igg-titer gegen masern, der asi zeigt eine intrathekale synthese an. neueste daten zeigen, dass im liquor von sspe-patienten vermehrte plasmazellklone (cd-138+-zellen) krankheitsrelevante antikörper produzieren, die durch humane igg rekombinante antikörper (mabs) bilden und somit identifi ziert werden können [185] . das eeg ist stets pathologisch, und es fi nden sich alle 5−8 s gruppen hoher δ-wellen, die von rhythmischen hyperkinesien begleitet sind (rademecker-komplexe: charakteristisch, aber nicht pathognomonisch). eine gesicherte th erapie existiert nicht. z inzidenz, ätiologie, pathogenese die pml ist eine seltene demyelinisierende erkrankung des zentralnervensystems und wurde initial bei malignompatienten und bei iatrogen immunkompromittierten patienten beobachtet. sie zählt zu den opportunistischen infektionen. vor dem ausbruch von aids war sie eine extrem seltene erkrankung. es wird geschätzt, dass etwa 1 % der aids-patienten eine pml entwickeln werden, wohingegen mehr als 60 % der heute diagnostizierten pml-fälle aids-patienten sind. erreger ist das jc-virus, ein dna-virus, welches häufi g in der bevölkerung vorkommt, ohne eine infektion zu verursachen. allgemein gilt die hypothese, dass das jc-virus eine primärinfektion in der kindheit auslöst und anschließend in einer ruhenden phase verweilt, bis eine immunsuppression zu einer viralen reaktivierung führt [189] . nach der erstzulassung des ersten monoklonalen antikörpers natalizumab (tysabri) für die th erapie der schubförmigen multiplen sklerose wurden 3 die prognose ist infaust mit einem krankheitsverlauf von in der regel weniger als 1 jahr bei der scjd und von ca. 14 monaten bei der vcjd. eine aktuelle publikation [184] weist allerdings auf regionale unterschiede hin: so konnte gezeigt werden, dass bei scjd-patienten in japan die krankheitsdauer bis zum tod im schnitt 16 monate betrug, wohingegen sie in europa nur bei ca. 5 monaten lag. [201] ). die wahrscheinlichkeit, 3 jahre nach der hiv-infektion aids zu entwickeln, hängt mit der hi-viruslast und der t-helferzellanzahl eng zusammen. die statistische wahrscheinlichkeit einer berufl ichen hiv-infektion nach perkutaner exposition mit blut von hiv-infizierten (z. b. nadelstich-oder schnittverletzungen) liegt bei ca. 0,3 %. diese wahrscheinlichkeit steigt unter den folgenden bedingungen: tiefe verletzungen, frische und sichtbare blutspuren am penetrierenden instrument, verletzung durch eine kanüle, die früher in einer vene oder arterie lag, und hohe viruslast des quellenpatienten [193] . eine medikamentöse hiv-postexpositionsprophylaxe (hiv-pep) wird deshalb nach perkutanen verletzungen mit injektionsnadeln oder mit anderen hohlraumnadeln und nach schnittverletzungen unter beteiligung von körperfl üssigkeiten mit potenziell hoher hi-viruskonzentration empfohlen [197] . bei oberfl ächlichen verletzungen und bei zeit" (zwischen 4 und 12 wochen nach der primärinfektion) mittels polymerasekettenreaktion (pcr) der hiv-genomsequenzen festgestellt werden, bevor die hiv-antikörper nachweisbar sind. entscheidende parameter für die prognose der progression der hiv-infektion, die in korrelation mit den klinischen symptomen mitzurechnen sind, sind die virusbelastung (anzahl der hiv-rna-kopien in plasma) und die anzahl der cd4 + -t-helferzellen [195] . z therapie die behandlung von hiv-aids besteht aus 1. einer spezifi sch antiretroviralen th erapie, die eine mehrfachkombinationstherapie ist, die sog. hochaktive antiretrovirale therapie oder haart, 2. einer symptomatischen behandlung der assoziierten erkrankungen und 3. der prophylaxe opportunistischer infektionen. indikationen für den anfang einer spezifi schen antiretroviralen th erapie sind der klinische und/oder laborchemische nachweis des immundefekts (. tab. 32.24). ziele der th erapie der hiv-infektion sind: 1. die hi-viruslast maximal und dauerhaft zu reduzieren, 2. die immunfunktion wiederherzustellen, 3. die lebensqualität zu verbessern, 4. die entwicklung von resistenzen zu verhindern und 5. die hiv-assoziierte morbidität und mortalität zu reduzieren [200] . hiv verursacht -oder sekundär -als opportunistische infektionen und neoplasien -auf [205] . zerebrovaskuläre komplikationen unterschiedlicher genese sind überdurchschnittlich häufi g bei hiv-infi zierten patienten. die häufi gsten hivassoziierten neurologischen erkrankungen und deren pathogenese, der klinische verlauf, die diagnostik und die th erapie werden in den folgenden abschnitten beschrieben. die respiratorische insuffi zienz wegen pulmonaler komplikationen ist die häufi gste indikation für eine intensivmedizinische behandlung von hiv-/aids-patienten. eine solche kann aufgrund neurologischer komplikationen erforderlich sein, obwohl viele dieser komplikationen ambulant gut therapierbar sind. typische neurologische komplikationen im frühstadium sind insbesondere die hiv-meningoenzephalitis sowie eine der gbs analoge polyneuroradikulitis -häufi g stehen diese im zeitlichen zusammenhang mit der serokonversion und machen gelegentlich die aufnahme in eine intensivstation erforderlich. die spät auft retende und langsam progrediente aids-enzephalopathie bzw. -myelopathie und die oft sehr schmerzhaft e polyneuropathie sind in der regel kein grund zur intensivmedizinischen behandlung, treten aber als begleitkrankheitsbilder auf. die lebensbedrohliche erhöhung des intrakraniellen drucks wegen neuroinfektionen oder neoplasien sind indi-kontakt mit schleimhaut oder verletzter haut mit flüssigkeiten mit hoher hi-viruskonzentration kann eine hiv-pep angeboten werden. neurologische komplikationen entwickeln sich vor oder nach dem auft reten von hiv-antikörpern. hiv-assoziierte neurologische komplikationen treten entweder primär -von [192] . die gabe von cortison sollte nur im einzelfall durchgeführt werden (z. b. drohende einklemmung ), da die histologische abgrenzung zum lymphom dadurch erschwert wird. bei epileptischen anfällen sollten nur clonazepam oder gabapentin verwendet werden, weil alle anderen antikonvulsiva eine negative interaktion mit haart zeigen; über levetiracetam gibt es wenig daten bei hiv-patienten. die zytomegalievirusenzephalitis wird durch die reaktivierung einer latenten cmv-infektion verursacht und lässt sich immunhistologisch bei ca. 40 % der verstorbenen aids-patienten nachweisen. überwiegend kommt sie bei einer t-helferzellanzahl <100/μl vor . z symptomatik und diagnostik klinisch ist sie durch eine rasch progrediente enzephalopathie (mit demenz, psychischen veränderungen, gedächtnisund konzentrationsstörungen) charakterisiert. der nachweis des cm-virus-genoms im liquor mittels pcr sichert die diagnose [207] , dennoch wird die diagnose häufi g erst post mortem durch den nachweis der typischen riesenzellen mit einschlusskörperchen (eulenaugenzellen) im gewebe gesichert. z therapie die akuttherapie besteht in erster linie aus ganciclovir (2×5 mg/kgkg/24 h i.v.) und bei unverträglichkeit foscarnet (2×90 mg/kgkg/24 h i.v.). ganciclovir ist myelotoxisch, deshalb sind regelmäßige blutbildkontrollen erforderlich. foscarnet ist nephrotoxisch, aus diesem grund werden eine ausgeglichene flüssigkeitsbilanz und eine bestimmung der kreatininclearance empfohlen. fakultativ kann eine kombination beider substanzen indiziert werden [181] . nicht hiv-abhängige erkrankungen und unfälle sind ebenfalls gründe für eine intensivmedizinische behandlung von hiv-infi zierten. in solchen fällen sollte die einstellung oder umstellung der antiretroviralen th erapie in zusammenarbeit mit einem in der hiv-th erapie erfahrenen arzt erfolgen. zu beachten ist die gefahr der entwicklung einer th erapieresistenz. die zerebrale toxoplasmose ist die häufi gste opportunistische infektion des zns bei hiv-infi zierten patienten in westeuropa. diese erkrankung tritt bei 30 % der nichttherapierten patienten auf und ist in 10 % der fälle die erstmanifestation von aids. die zerebrale toxoplasmose wird von einer reaktivierung einer infektion mit dem protozoon toxoplasma gondii verursacht, dies wird in erster linie durch katzenkot oder unzureichend gebratenes fleisch übertragen. toxoplasma gondii bleibt nach einer primärinfektion als pseudozyste im gehirngewebe. z symptomatik und diagnostik klinisch kommt es bei 80 % der patienten zu fokalneurologischen symptomen. fieber und kopfschmerz treten bei ungefähr 50 % der patienten auf. in der kraniellen computertomographie (cct) und der kraniellen magnetresonanztomographie (mrt) werden bei 1/3 der patienten eine solitäre läsion, bei ca. 2/3 der patienten mehrere läsionen mit perifokalem ödem im marklager mit ringförmiger oder nodulärer kontrastmittelanreicherung gefunden. entscheidend für die diagnose ist das ansprechen der symptomatik und der nachweisbaren läsionen nach th erapie. wenn es nach 2−4 wochen th erapie zu keiner verbesserung kommt oder gar eine verschlechterung der klinischen z therapie die th erapie der wahl ist haart [196] , in einzelfallberichten sind längere überlebenszeiten als nur wenige monate möglich. das primäre zns-lymphom ist der häufi gste im zusammenhang mit aids auft retende tumor des zns. primäre zns-lymphome sind in der regel hochmaligne b-zelltyp-non-eine sekundärprophylaxe mit ganciclovir (5−6 mg/kgkg i.v. an 5 tagen der woche) oder foscarnet (1×90 mg/kgkg i.v. an 7 tagen oder 120 mg/kgkg i.v. an 5 tagen der woche) wird in der regel nach ca. 3 wochen akuttherapie lebenslang in reduzierter dosis eingesetzt. die kryptokokkenmeningoenzephalitis ist eine opportunistische infektion mit dem pilz cryptococcus neoformans, deren ausbreitung nach einer asymptomatischen besiedelung infolge inhalation von vogelkot hämatogen aus dem respirationstrakt erfolgt. sie tritt bei einer t-helferzellzahl <100/ μl auf. z symptomatik und diagnostik charakteristisch ist ein progredienter verlauf über tage oder wochen mit kopfschmerzen, fieber, übelkeit und somnolenz. meningitische zeichen treten bei nur 30 % der patienten auf. selten kommt es zu epileptischen anfällen und fokalneurologischen zeichen. der erregernachweis mittels tuschepräparat des liquors gelingt in 75 %, der antigennachweis in serum und liquor in >99 % der fälle. z therapie die akuttherapie besteht aus amphotericin b + flucytosin + fluconazol und dauert in der regel zwischen 4 und 6 wochen. danach wird eine konsolidierungstherapie mit fluconazol oder itraconazol eingesetzt, bis der kryptokokkenantigentiter im liquor um 2 stufen gefallen ist [204] . bei der akuten myelitis kann in über 50 % der fälle keine ursache gefunden werden. häufi ge ursachen sind vor allem die multiple sklerose [346] und virale entzündungen. die extramedullären entzündungen werden insbesondere durch hämatogene und lokale (per continuitatem) bakterienaussaat bedingt -z. b. nach bandscheiben-oder wirbelsäulenoperation, lumbaler drainage -und imponieren als abszesse, osteomyelitiden bzw. spondylitiden und bei beteiligung des bandscheibenfachs als spondylodiszitiden. häufi gste erreger sind staphylokokken, mycobacterium tuberculosis, e. coli spp., klebsiella spp., streptokokken und pseudomonaden. risikofaktoren für eine spinale infektion sind neben einer immunsuppression (hiv, immunsuppressive medikamentöse th erapie), patienten mit diabetes mellitus, alkohol-und drogenabusus, traumata und chronischen hepatischen und renalen erkrankungen [342] , [343] , [344] . auch im rahmen einer systemischen infektion (sepsis, endokarditis) kann es, vor allem bei den genannten risikogruppen, zu einer zusätzlichen spinalen manifestation der infektion kommen. myelitis (para-oder tetraparese mit symmetrischen sensiblen defi ziten und sphinkterfunktionsstörungen) und einer -vorwiegend jüngere frauen betreff ende -optikusneuritis ( sehstörungen, die oft mals schwerwiegender sind als bei der multiplen sklerose und bis zur blindheit führen können) [365] . z diagnostik die verdachtsdiagnose einer spinalen entzündung sollte zunächst durch das klinische bild erfolgen. die lokalisation der schädigung ist über die untersuchung der sensiblen dermatome, der myotome und der muskeldehnungsrefl exe möglich. hilfreich in der zuordnung der höhenlokalisation ist die untersuchung des vibrationsempfi ndens einschließlich der dornfortsätze. autonome störungen können beispielsweise über den analen sphinktertonus und blasenentleerungsstörungen mit restharnbildung (bestimmung mittels ultraschall) oder inkontinenz erfasst werden. umschriebene entzündungen der wirbelsäule und angrenzender strukturen gehen häufi g mit einem lokalen klopf-und stauchungsschmerz einher. (übersichtarbeiten zur diagnostischen herangehensweise und zu diff erenzialdiagnosen der akuten transversen myelitis bzw. der akuten und subakuten myelopathie fi nden sich in [354] und [364] die poliomyelitis verläuft klassischerweise in mehreren stadien und beginnt zunächst mit fieber, gefolgt von einem meningitischen stadium, bis sich dann das paralytische stadium anschließt. die mittlerweile seltene lues spinalis mit der tabes dorsalis (hinterseitenstrangmyelitis) als spätstadium der neurolues geht mit einer progressiven lähmung, sensibilitätsstörungen, lanzierenden schmerzen, refl exverlust und blasenstörungen einher. eine fsme-myelitis ist häufi g mit einer "hohen querschnittssymptomatik" mit beteiligung der arme, der hirnnerven und des zwerchfells verbunden und weist eine schlechte prognose auf [ 362] . die neuromyelitis optica (devic-syndrom) stellt eine demyelinisierende autoimmune erkrankung dar. charakterisiert ist sie durch das klinische bild einer akuten (transversen) -und meist ausgedehnten (>3 wirbelkörpersegmente) -falls kontraindikationen für die mrt-untersuchung vorliegen, kann bei extramedullären entzündlichen prozessen alternativ eine computertomographie mit kontrastmittel erfolgen. um die strahlendosis zu minimieren, ist eine vorherige höhenlokalisation anhand des klinischen bildes sinnvoll. eine erweiterte bildgebung (röntgen-th oraxaufnahme, computertomographie von z. b. th orax und/oder abdomen) ist bei verdacht auf infektiöse und tumoröse bzw. paraneoplastische prozesse erforderlich. neben dem klinischen bild ist die zeitnahe neuroradiologische bildgebung von besonderer bedeutung. sie dient einerseits zum ausschluss einer chirurgisch zu behandelnden ursache des spinalen syndroms und andererseits zur bestätigung der klinischen verdachtsdiagnose einer spinalen entzündung. aufgrund der hohen ortsaufl ösung, der guten diff erenzierbarkeit der verschiedenen gewebe und der sensitiven darstellung entzündlicher läsionen stellt die kernspintomographie (mrt; . abb. 32.5und . abb. 32.6) die untersuchungsmethode der wahl dar. entzündliche läsionen werden besonders gut in t2-gewichteten und t1-gewichteten aufnahmen nach kontrastmittelgabe dargestellt. um die räumliche ausdehnung zuverlässig beurteilen zu können, müssen bilder in mindestens 2 schnittebenen (bevorzugt axiale und sagittale schnittführung) angefertigt werden (. abb. 32.7). da pathologische veränderungen nicht immer auf dem klinisch vermuteten rückenmarksniveau liegen, sollte immer die gesamte wirbelsäule bzw. der gesamte spinalkanal untersucht werden. zum ausschluss einer zerebralen beteiligung (v. a. hirnstamm) ist, auch unter diff erenzialdiagnostischen aspekten (bspw. fi nden sich bei multipler sklerose meist auch zerebrale herde), bei zervikalen prozessen eine ergänzende zerebrale mrt sinnvoll. > ein initiales mrt der wirbelsäule kann bei bis zu 20 % der patienten mit einem spinalen syndrom ohne wegweisenden befund sein [368] . gründe dafür können sein: . bei verdacht auf eine bakterielle infektion muss immer eine erregerisolierung mittels liquorkultur oder pcr-diagnostik angestrebt werden. wenn der entzündliche prozess den subarachnoidalraum noch nicht erreicht hat, ist die liquordiagnostik in der regel nicht richtungweisend. in diesem fall gelingt -vor allem bei systemischen entzündungszeichenein keimnachweis mittels blutkultur. bei klar abgrenzbaren entzündlichen prozessen (spinaler abszess, diszitis) kann zur weiteren einordnung des entzündlichen prozesses ist neben der bildgebung die zytologische, chemische, bakteriologische und immunologische analyse des liquors essenziell. auch wichtige diff erenzialdiagnosen zur spinalen entzündung (z. b. spinale ischämie) können dadurch abgegrenzt werden (. tab. 32.35). bakterielle entzündungen gehen typischerweise mit einer deutlichen erhöhung von zellzahl (>1000 zellen/μl) und gesamtprotein einher. z diff erenzialdiagnose aus klinischer sicht muss bei akuten sensomotorischen ausfällen eine akute polyradikulitis (guillain-barré-syndrom) in betracht gezogen werden. die abgrenzung zur myelitis gelingt meist durch den typischen liquorbefund einer "zytalbuminären dissoziation" -mit erhöhung des liquorgesamteiweißgehaltes bei normaler zellzahl (7 kap. 37.1). auch medulläre tumoren (gliome, ependymome, sarkome, lipome, lymphome, abtropfmetastasen) müssen in die diff erenzialdiagnostischen überlegungen einbezogen werden. aber auch paraneoplastische myelopathien (z. b. beim bronchialkarzinom und m. hodgkin) sind beschrieben [352] . eine strahlenmyelopathie kann bei bestrahlungsdosen ab 20 gy mit einer latenz von mehreren wochen bis monaten und jahren als akute inkomplette bis komplette querschnittssymptomatik auft reten. zu den vaskulären spinalen syndromen zählen vor allem die spinale ischämie (z. b. nach aortenoperationen oder aortendissektion) und spinale arteriovenöse malformationen, angiome, kavernome und durale fisteln. letztere gehen häufi g mit einer venösen stauung und blutungen einher. metabolische myelopathien, die akut bis subakut verlaufen, sind insbesondere die funikuläre myelose bei vitamin-b 12 -mangel und die hepatische myelopathie bei leberinsuffizienz [359] . schwierigkeiten können bei der unterscheidung einer erregerbedingten von einer parainfektiösen myelitis auft reten. bei letztgenannten wird häufi g ein symptomfreies intervall zwischen der vorausgegangenen infektion und der myelitis beschrieben. die extramedullären entzündungen müssen von chronisch-entzündlichen rheumatischen wirbelsäulenerkrankungen abgegrenzt werden [363] . gedacht werden muss an die rheumatoide arthritis und die seronegative spondylarthropathie. zu den letztgenannten zählt man die ankylosierende spondylitis (m. bechterew), die psoriasisarthropathie, die enteropathische arthropathie, die reaktive spondylarthropathie und als sonderform der m. reiter. diagnostisch hilfreich ist bei den chronisch-entzündlichen rheumatischen auch eine ct-gesteuerte punktion zum keimnachweis hilfreich sein und sollte rechtzeitig erfolgen. virale entzündungen weisen neben einer leichten bis moderaten zellzahlerhöhung (meist 500 bis max. 1000 zellen/μl) üblicherweise nur eine leichte eiweißerhöhung auf. der nachweis spezifi scher antikörper (igg und igm) im liquor kann auf eine mögliche virale infektion hinweisen. eine intrathekale antikörperbildung kann zuverlässig durch ermittlung des antikörperspezifi schen index (ai) nachgewiesen werden. ein wert >1,5 ist verdächtig, werte >2 sprechen für eine antikörperbildung innerhalb des zentralen nervensystems. der antigennachweis mittels pcr ist eine schnelle und zuverlässige methode. sie kann insbesondere in der frühphase einer infektion, wenn die humorale antikörperantwort noch unzureichend ist, wichtige informationen liefern. autoimmune entzündungen weisen meist nur eine leichte pleozytose (<100 zellen/μl), aber auch schrankenstörungen und eiweißerhöhungen auf. bei der multiplen sklerose fi nden sich bei über 80 % der erkrankten oligoklonale banden im liquor. die neuromyelitis optica ist bei über 70 % der patienten mit spezifi schen antikörpern gegen aquaporin 4 (nmo-igg) im serum assoziiert [367] . die routine-labordiagnostik mit kleinem blutbild und c-reaktivem protein (crp) ist bei isolierten spinalen prozessen teilweise nicht richtungweisend und zeigt oft mals in der initialphase keine oder nur geringe entzündungszeichen. dennoch kann die crp-erhöhung bei bakteriellen spinalen entzündungen ein unspezifi scher hinweis sein, der dann eine detaillierte diagnostik nach sich ziehen sollte. besteht der verdacht einer systemischen entzündung aus dem kreis der kollagenosen, der rheumatischen erkrankungen und der vaskulitiden, ist der nachweis spezieller serologischer antikörper häufi g hilfreich. vaskulitiden können häufi g erst durch die histologische aufarbeitung von gefäß-und/oder nerven-bzw. muskelbiopsaten und immunhistochemischer färbung diagnostiziert werden. die diagnostik der funktionellen schädigung des nervensystems kann durch elektrophysiologische untersuchungen (v. a. somatosensibel und motorisch evozierte potenzikspezielle therapie auch wenn es für die th erapie der idiopathischen akuten transversen myelitis (iatm) keine randomisierten, placebokontrollierten untersuchungen gibt, die den einsatz einer cortisontherapie sicher positiv bewerten [357] , [358] , wird in analogie zur behandlung anderer entzündlicher erkrankungen und der klinischen erfahrung häufi g eine 3-bis 5-tägige intravenöse cortisonstoßtherapie mit 500-1000 mg methylprednisolon durchgeführt. klinisch schwer betroff ene patienten können evtl. auch von einer aggressiveren th erapie mit cyclophosphamid und plasmapherese profi tieren [351] . herpes-simplex-und varizella-zoster-assoziierte myelitiden werden mit aciclovir behandelt (3×10 mg/kgkg/24 h wirbelsäulenerkrankungen der nachweis von rheumafaktoren und bei den "seronegativen" spondylarthropathien die häufi ge assoziation mit hla-b27. neben den chronisch-entzündlichen wirbelsäulenerkrankungen müssen auch extramedulläre tumoren (neurinome, meningeome, angiome, sarkome) und metastasen (z. b. bronchial-, mamma-, prostatakarzinom, plasmozytom) in die diff erenzialdiagnostische aufarbeitung einbezogen werden. selten können spinale epidurale blutungen bei gerinnungsstörungen (antikoagulation!), zustand nach trauma, lumbalpunktion, periduralkatheter und vaskuläre malformationen eine querschnittsymptomatik verursachen. auch an degenerative erkrankungen mit wirbelkörperfrakturen, spinalkanalstenosen und bandscheibenvorfällen muss bei extramedullären prozessen gedacht werden. diese können durch kompression zu medullären schädigungen führen (kompressive myelopathie, t2-signalsteigerungen im mrt) und mit entsprechender klinischer symptomatik einhergehen [349] . z therapie > neben der (erreger)spezifi schen therapie sollten allgemeine maßnahmen wie anlage eines blasenkatheters bei blasenentleerungsstörungen, thromboseprophylaxe, lagerung, frühzeitige mobilisierung, physiotherapie und schmerztherapie sowie medikamentöse therapie einer spastik von anfang an durchgeführt werden. kallgemeine therapieprinzipien die medikamentöse th erapie hängt wesentlich von der zugrundeliegenden ätiopathogenese bzw. dem erreger ab. oft mals gelingt in der initialen phase keine eindeutige ätiologische zuordnung oder erregerisolation, sodass je nach dringlichkeit bei akuten erkrankungen die wahl der medikamente empirisch, entsprechend dem klinischen verlauf, den ergebnissen der initialen labor-und liquordiagnostik und dem zu erwartenden erregerspektrum erfolgt. bei unsicheren extramedullären befunden ohne erregerisolation sollte eine breite antibiotische kombinationstherapie mit einem zns-gängigen antibiotikum erfolgen. im vordergrund der medikamentösen th erapie steht immer der gezielte einsatz der antibiotika bzw. virustatika. die auswahl der präparate erfolgt entsprechend den ergebnissen der blut-und liquorkulturen bzw. punktatergebnissen (antibiogramm anfordern!) und den serologischen bzw. immunologischen resultaten. bei subakut oder chronisch verlaufenden erkrankungen sollte, wenn es die klinische situation zulässt, zunächst eine gezielte diagnostik -möglichst mit erregerisolation und ggf. diff erenzialdiagnostischer aufarbeitung -angestrebt werden. bei bakteriellen abszessen muss immer (soweit unter anatomischen und funktionellen gesichtspunkten möglich) zusätzlich zur antibiotischen th erapie eine (neuro)chirurgische herdsanierung diskutiert und individuell entschieden werden. . abb. 32.7 herpes-myelitis in höhe bwk 5 delt werden. gut zns-gängige antibiotika bei grampositiven erregern sind z. b. fosfomycin, ceft riaxon, cefotaxim, meropenem und linezolid. alternativ kann aber auch eine operative sanierung mit ausräumung der bandscheibe und anschließender stabilisierung notwendig sein. insbesondere bei kompression neuraler strukturen oder zeichen der instabilität sollte rechtzeitig ein chirurgisches vorgehen diskutiert werden. die neurosarkoidose, der neuro-behçet und lupus erythematodes werden immunsuppressiv behandelt. je nach schwere der erkrankung werden cortison und -vor allem in der langzeittherapie -auch methotrexat, azathioprin, ciclosporin und cyclophosphamid eingesetzt. die neuromyelitis optica wird in der akuten phase mit einer hochdosis-steroidtherapie (methylprednisolon täglich 1000 mg über 3-5 tage) behandelt. bei fehlendem ansprechen auf die steroide sollte über eine plasmapherese nachgedacht werden. im weiteren wird eine immunsuppressive th erapie mit azathioprin (2-3 mg/kgkg/tag, initial in kombination mit oralem prednisolon, bis die wirkung von azathioprin nach 2-3 monaten zu erwarten ist) oder rituximab i.v. für 10-14 tage). mittel der wahl bei cmv-infektionen ist ganciclovir (2×5 mg/kgkg/24 h i.v.). bei der seltenen aciclovir-unverträglichkeit kann bei hsv, vzv und cmv-infektion auch foscarnet (2×90 mg/kgkg/24 h) eingesetzt werden. die th erapie der neuroborreliose besteht in einer 2-bis 4-wöchigen antibiose mit ceft riaxon (1×2 g/24 h i.v.) oder cefotaxim (3×2 g/24 h i.v. a. lsp. lsp. lc. übersehen einer spinalen av-malformation, eines epiduralen abszesses oder eines intramedullären tumors schlechte bildqualität/aufl ösung (z. b. zu großes "fi eld of view" gewählt) zeitpunkt der untersuchung (z. b. häufi g fehlende kontrastmittelaufnahme nach beginn einer cortisontherapie, untersuchung mehrere tage nach einer transienten symptomatik die mit einem spinalen syndrom, jedoch ohne (wesentliche) mr-veränderungen einhergehen können (auswahl): metabolische störungen (z. b. vitamin-b 12 -mangel, kupfermangel, hepatische myelopathie), systemischer lupus erythematodes, sjögren-syndrom, sarkoidose, motoneuronerkrankungen, spinale ischämie, zns-vaskulitis, syphilis, hiv-myelopathie, paraneoplastische syndrome zerebrale läsionen imitieren ein spinales syndrom (z. b. falx-nahe tumoren in der zentralregion, infarkt im stromgebiet der a. cerebri anterior) > besonders die kombination mehrerer punkte kann zu falsch-negativen befunden führen! n. tibialis links / cz'-fz 10 tibialis rechts / cz'-fz 10.0 ms/div, 1.00 uv/div n. tibialis rechts / cz'-fz 10 motorischer cortex / rechtes bein 10.0 ms/div, 1000.0 uv/div, 2/0, 0ma re abb. 32.8 35-jähriger patient mit langjähriger multipler sklerose: a das mrt (t2w) zeigt myelitische herde in höhe hwk 4−6 und hwk 7 (pfeile). b tibialis-sep beidseits schlecht ausgeprägt mit deutlicher latenzverzögerung (li. 59 ms, re. 57 ms). c unterschenkel-mep mit deutlich verlängerter kortikaler latenz (li. 47 ms, re. 46 ms) bei normaler spinaler latenz und daraus resultierender erhöhter zentralmotorischer leitungszeit delayed cerebral thrombosis after initial good recovery from pneumococcal meningitis delayed cerebral thrombosis after initial good recovery from pneumococcal meningitis: past as prologue: delayed stroke as a parainfectious process of bacterial meningitis clinical features, complications, and outcome in adults with pneumococcal meningitis: a prospective case series therapy of communityacquired acute bacterial meningitis: the clock is running the potential roles of c-reactive protein and procalcitonin concentrations in the serum and cerebrospinal fl uid in the diagnosis of bacterial meningitis a new marker for bacterial infection serum procalcitonin level and other biological markers to distinguish between bacterial and aseptic meningitis in children: a european multicenter case cohort study serum procalcitonin levels aid in distinguishing bacterial from aseptic meningitis in children efns guideline on the management of community-acquired bacterial meningitis: report of an efns task force on acute bacterial meningitis in older children and adults pfi ster hw. discrepancies between brain ct imaging and severely raised intracranial pressure proven by ventriculostomy in adults with pneumococcal meningitis serotype distribution of invasive pneumococcal disease during the fi rst 60 days of life linezolid for the treatment of patients with central nervous system infection clinical experience with linezolid for the treatment of central nervous system infections detrimental role of delayed antibiotic administration and penicillin-nonsusceptible strains in adult intensive care unit patients with pneumococcal meningitis: the pneumorea prospective multicenter study delays in the administration of antibiotics are associated with mortality from adult acute bacterial meningitis neurocritical care of patients with central nervous system infections septic thrombophlebitis of major dural venous sinuses dexamethasone in adults with bacterial meningitis corticosteroids for acute bacterial meningitis. cochrane database syst rev empfohlen. weitere empfohlene substanzen, die bei versagen oder unverträglichkeit von azathioprin oder rituximab eingesetzt werden können, sind cyclophosphamid, mitoxantron aktuelle statistik meldepfl ichtiger infektionskrankheiten progress toward elimination of haemophilus infl uenzae type b invasive disease among infants and children -united states bakterielle (eitrige) meningoenzephalitis. leitlinien für diagnostik und therapie in der neurologie der dgn pneumococcal meningitis in adults: spectrum of complications and prognostic factors in a series of 87 cases new understandings on the pathophysiology of bacterial meningitis innate immunity to pneumococcal infection of the central nervous system depends on toll-like receptor (tlr) 2 and tlr4 protein expression pattern in experimental pneumococcal meningitis oxidative stress in pneumococcal meningitis: a future target for adjunctive therapy? clinical features, outcome, and meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study aetiological diagnosis of brain abscesses and spinal infections: application of broad range bacterial polymerase chain reaction analysis stereotactic aspiration and antibiotic treatment combined with hyperbaric oxygen therapy in the management of bacterial brain abscess characteristics of brain abscess with isolation of anaerobic bacteria diff usionweighted mr imaging in the preoperative assessment of brain abscesses the management of brain abscesses strategies for the management of bacterial brain abscess antibiotics for brain abscesses in people with cyanotic congenital heart disease postoperative central nervous system infection: incidence and associated factors in 2111 nerosurgical procedures fungal versus bacterial brain abscesses: is diff usion-weighted mr imaging a useful tool in the diff erential diagnosis the spectrum of complications during bacterial meningitis in adults: results of a prospective clinical study brain abscess in children managment of brain abscesses with sequential intravenous/oral antibiotic therapy multilocated pyogenic brain abscess: experience in 25 patients management of an extensive spinal epidural abscess from c-1 to the sacrum bacterial brain abscesses: a retrospective study of 94 patients admitted to an intensive care unit (1980-1999) the rational use of antibiotics in the treatment of brain abscess intracranial suppuration: a clinical comparison of subdural empyemas and epidural abscesses brain abscess in 142 patients: factors infl uencing outcome and mortality diff usion-weighted imaging of cerebritis subcortical defi cit pattern after brain abscess: a neuropsychological study spine and spinal cord emergencies: vascular and infectious causes tuberculous, brucellar and pyogenic spondylitis: comparison of magnetic nationwide implementation of adjunctive dexamethasone therapy for pneumococcal meningitis corticosteroids for acute bacterial meningitis dexamethasone treatment in childhood bacterial meningitis in malawi: a randomised controlled trial adjunctive dexamethasone in bacterial meningitis: a meta-analysis of individual patient data dexamethasone in vietnamese adolescents and adults with bacterial meningitis meningokokkenerkrankungen. rki ratgeber infektionskrankheiten -merkblätter für ärzte. www rki de empfehlung und begründung einer postexpositionellen meningokokken-impfung epidemic meningitis, meningococcaemia, and neisseria meningitidis acute bacterial meningitis in adults. a review of 493 episodes fungal brain abscess in transplant recipients: epidemiologic microbiologic, and clinical features multidrug-resistant spinal tuberculosis and giant paraspinal abscess ct-guided stereotactic biopsies of brain stem lesions: personal experience and literature review tuberculous spondylitis and pyogenic spondylitis:comparative magnetic resonance imaging features bacterial brain abscesses: prognostic value of an imaging severity index central nervous system infections in the intensive care unit central nervous system infections: meningitis and brain abscess effi cacy and safety of cefotaxime in combination with metronidazole for empirical treatment of brain abscess in clinical practice: a retrospective study of 66 consecutive cases positron emission tomographic fi ndings in a tuberculous brain abscess brain abscess: clinical analysis of 53 cases ehrlichia infection of the central nervous system factors aff ecting the outcome of neuroendoscopy in patients with tuberculous meningitis hydrocephalus: a preliminary study neurobrucellosis with transient ischemic attack, vasculopathic changes, intracerebral granulomas and basal ganglia infarction: a case report predictors of long-term neurological sequelae of tuberculous meningitis: a multivariate analysis coxiella burnetii infection cns manifestations associated with mycoplasma pneumoniae infections: summary of cases at the university of helsinki and review central nervous system tuberculosis immune reconstitution syndrome in a patient with aids with paradoxically deteriorating brain tuberculoma rapid diagnosis of tuberculous meningitis by polymerase chain reaction assay of cerebrospinal fl uid scrub typhus involving central nervous system acute disseminated encephalomyelitis as the fi rst presentation of cns tuberculosis: report of a case with brief review recurrent encephalopathy in cat-scratch disease the new global map of human brucellosis corticosteroids for managing tuberculous meningitis objective ct criteria to determine the presence of abnormal basal enhancement in children with suspected tuberculous meningitis rapid diagnosis of tuberculous meningitis: a comparative evaluation of in-house pcr assays involving three mycobacterial dna sequences, is6110-mpb-64 and 65 kda antigen cultivation of the bacillus of whipple's disease tuberculous meningovasculitis tuberulous meningitis presenting with unusually severe hyponatremia. the mount sinai timing of antiretroviral therapy initiation in tuberculosis patients with aids: a decision analysis atypische erregerbedingte meningoencephalitis intracranial complications of sinusitis: a 15-year review of 39 cases whipple's disease of the central nervous system neurology of whipple's disease defi nitive neuroradiological diagnostic features of tuberculous meningitis in children central nervous system disorders after starting antiretroviral therapy in south africa psychosis following mycoplasma pneumonia diagnosis of tuberculous meningitis: clinical and laboratory parameters mr imaging of central nervous system whipple disease: a 15-year review rickettsiosis: a clinical approach rare infections mimicking ms nocardial meningitis: case reports and review tick-borne neurological diseases cerebrospinal fl uid adenosine deaminase activity for the diagnosis of tuberculous meningitis in adults c. pneumoniae communityacquired pneumonia (cap) in mimicking mycoplasma pneumonia meningoencephalitis complicated by asthma ehrlichioses in humans: epidemiology, clinical presentations whipple's disease tuberculosis and other mycobacterial infections cerebral saltwasting syndrome due to infectious diseases of the central nervous system from cat scratch disease to endocarditis, the possible natural history of bartonella henselae infection matrix metalloproteinase-9 and intercellular adhesion molecule 1 are powerful staging markers for human african trypanosomiasis kapitel 32 · infektionen multicenter study of immunoblotting in serodiagnosis of lyme borreliosis neurosyphilis. in: noseworthy jh (ed) neurological therapeutics-principles and practice verlauf und langzeitfolgen der chronischen neuroborreliose severe course of lyme neuroborreliosis in an hiv-1 positive patient; case report and review of the literature epidemiology and diagnosis of lyme borreliosis microbiological and serological diagnosis of lyme borreliosis sexually transmitted diseases treatment practice guidelines for the treatment of lyme disease. the infectious diseases society of america double blind placebo-controlled trial of pleconaril in infants with enterovirus meningitis herpes simplex virus genomes in human nervous system tissue analyzed by polymerase chain reaction a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection recovery of a patient from clinical rabies update: outbreak of nipah virus -malaysia and singapore nipah virusassociated encephalitis outbreak diagnosis and treatment of viral encephalitis molecular analysis of cerebrospinal fl uid in viral diseases of the central nervous system severe neurological complications in association with epstein-barr virus infection induction of neutralizing antibodies to hendra and nipah glycoproteins using a venezuelan equine encephalitis virus in vivo expression system encephalitis, cerebritis, and brain abscess: pathophysiology and imaging fi ndings meningitis following varicella vaccine diagnostik und therapie in der neurologie, 4. aufl . thieme seltene bakterielle infektionen des nervensystems coagulant and fi brinolytic status in tuberculous meningitis central nervous system manifestations of q fever responsive to steroids chlamydia pneumoniae and meningoencephalitis clinicoradiological features of tuberculous meningitis in patients over 50 years of age mycoplasma pneumonia-associated transverse myelitis with unexpected rapid response to macrolide therapy: a case report tuberculous cranial pachymeningitis spine and spinal cord emergencies: vascular and infectious causes mycoplasma pneumonia: nervous system complications in childhood and review of the literature diagnosis of lyme borreliosis treatment of syphilis in hiv-infected subjects: a systematic review of the literature review: neurosyphilis: a historical perspective and review status epilepticus revealing syphilitic meningoencephalitis diagnosis and treatment of the neuromuscular manifestations of lyme disease is neuroborreliosis a medical emergency practice parameter: treatment of nervous system lyme disease (an evidence based review): report of the quality of standards subcommittee of the american academy of neurology facial palsy: etiology, outcome and management in children deterioration of mri fi ndings related to jarisch-herxheimer reaction in a patient with neurosyphilis a twist on lyme: the challenge of diagnosing european lyme neuroborreliosis delineation of borrelia burgdorferi sensu lato species by multilocus sequence analysis and confi rmation of the delineation of borrelia spielmanii sp. nov update on herpes simplex encephalitis emerging viral infections of the central nervous system, part 2 the long-term neuropsychological outcome of herpes simplex encephalitis in a series of unselected survivors viral encephalitis: familiar infections and emerging pathogens quantitation of herpes simplex virus type 1 dna in cells of cerebrospinal fl uid of patients with herpes simplex virus encephalitis poliomyelitis-like syndrome associated with epstein-barr virus infection nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis harrison's neurology in clinical medicine subacute sclerosing panencephalitis in papua new guinean children: the cost of continuing inadequate measles vaccine coverage progressive multifocal leukoencephalopathy in patients with rheumatic diseases: are patients with systemic lupus erythematosus at particular risk? duration of prion disease is longer in japan than in other countries measles virus-specifi c plasma cells are prominent in subacute sclerosing panencephalitis csf case report: cervical spinal cord signal changes in a case of adult-onset subacute sclerosing panencephalitis flurpirtine may stopp the progressive course of subacute sclerosing panencephalitis natalizumab and progressive multifocal leukencephalopathy: what are the causal factors and can it be avoided? pathogenesis of progressive multifocal leukoencephalopathy-revisited two cases of subacute sclerosing panencephalitis associated with brainstem involvement a 10-year follow-up study of tick-borne encephalitis in the stockholm area and a review of the literature: need for a vaccination strategy harrison's neurology in clinical medicine prospective analysis of 61 cases of enteroviral meningitis: interest of systematic genome detection in cerebrospinal fl uid irrespective of cytologic examination results tick-borne encephalitis (tbe) in germany and clinical course of the disease trends in the management of herpes simplex encephalitis viral encephalitis the neurological manifestations of nipah virus encephalitis, a novel paramyxovirus herpes simplex virus encephalitis: chronic progressive cerebral magnetic resonance imaging abnormalities in patients despite good clinical recovery clinical features, diagnosis, and management of enterovirus 71 moscona a inhibition of nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entr protein transfer at the blood cerebrospinal fl uid barrier and the quantitation of the humoral immune response within the central nervous system viral meningitis and encephalitis: traditional and emerging viral agents viral infections of the cns with special emphasis on herpes simplex infections craniectomy: an aggressive treatment approach in severe encephalitis diff usion mri in rasmussen's encephalitis, herpes simplex encephalitis, and bacterial meningoencephalitis acute encephalitis viral meningoencephalitis: a review of diagnostic methods and guidelines for management relapsed and late-onset nipah encephalitis laboratory diagnosis of central nervous system infections leitlinien zur antiretroviralen therapie der hiv-infektion leitlinien der deutschen gesellschaft für neurologie zur diagnostik und therapie hiv-1-assoziierter erkrankungen melarsoprol versus efl ornithine for treating late-stage gambian trypanosomiasis in the republic of the congo identifi cation of malaria retinopathy improves the specifi city of the clinical diagnosis of cerebral malaria: fi ndings from a prospective cohort study blantyre malaria project epilepsy study (bmpes) of neurological outcomes in retinopathy-positive paediatric cerebral malaria survivors: a prospective cohort study lethal african trypanosomiasis in a traveler: mri and neuropathology molecular identifi cation of trypanosoma cruzi i tropism for central nervous system in chagas reactivation due to aids tropical causes of epilepsy parasitic diseases of the central nervous system neurological manifestations of chagas' disease role of quinine in life-threatening babesia divergens infection successfully treated with clindamycin toxocara canis meningomyelitis quinine pharmacokinetics in cerebral malaria: predicted plasma concentrations after rapid intravenous loading using a two compartment mode meta-analysis: cysticidal drugs for neurocysticercosis: albendazole and praziquantel encephalitis due to a free-living amoeba (balamuthia mandrillaris), case report with literature review artesunate versus quinine in the treatment of severe falciparum malaria in african children (aquamat): an open-label, randomized trial artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial multifocal central nervous system lesions in three patients with trichinosis induction and maintenance therapy of cytomegalovirus central nervous system infection in hiv-infected patients meta-analysis of prophylactic treatments againts pneumocystis carinii and toxoplasma encephalitis a case-control study of hivseroconversion in health care workers after percutaneous exposure. centers for disease control and prevention needlestick surveillance group revised classifi cation system for hiv infection and expanded surveillance case defi nition for aids among adolescents and adults association of initial cd4 cell count and viral load with response to highly active antiretroviral therapy progressive multifocal leucoencephalopathy: improved survival of human immunodefi ciency virus-infected patients in the protease inhibitor era postexposure chemoprophylaxis for occupational exposures to the human immunodefi ciency virus kamps bs (hrsg) (2007) hiv.net 2007. www.hiv.net. steinhäuser, wuppertal-beyenburg goals and milestones during treatment of hiv-1 infection with antiretroviral therapy: a pathogenesis-based perspective prognosis in hiv-1 infection predicted by the quantity of virus in plasma guidelines for the use of antiretroviral agents in hivinfected adults and adolescents. department of health and human services guidelines for the diagnosis and management of neurological complications of hiv infection practice guidelines for the management of cryptococcal disease. infectious diseases society of america prospects for therapy of hivassociated neurologic diseases neue entwicklungen in diagnostik und therapie primärer non-hodgkin-lymphome des zentralen nervensystems diagnosis of cytomegalovirus encephalitis in patients with aids by quantitation of cytomegalovirus genomes in cells of cerebrospinal fl uid empfohlene websites 208 report of a case with arteritis human neurocysticercosis: comparision of diff erent diagnostic tests in cerebrospinal fl uid clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose malawian children falciparum malaria in children-a brief report of 305 patients from rourkela, eastern india serological studies of neurologic helminthic infections in rural areas of southwest cameroon: toxocariasis, cysticercosis and paragonimiasis effi cacy of combination of dfmo and diminazene aceturate in the treatment of latestage trypanosoma brucei infection in rats ischemic cerebral changes in the chronic chagasic cardiopathy pathology of human african trypanosomiasis with reference to experimental african trypanosomiasis and infections of the central nervous system neuroimaging fi ndings in children with retinopathy-confi rmed cerebral malaria spinal toxocara abscess treatment of severe malaria by exchange transfusion primary amoebic meningoencephalitis: two new cases from pakistan neurological fi ndings and outcome in adult cerebral malaria parasitic diseases of the central nervous system protozoal infections. in: noseworthy jh (ed) neurological therapeutics -principles and practice secondary cerebral amebiasis due to infection with entamoeba histolytica eosinophilic meningitis and radiculomyelitis in thailand, caused by cns invasion of gnathostoma spingerum and angiostrongylus cantonensis overwhelming strongyloidiasis: an unappreciated opportunistic infection trypanosoma cruzitriggered meningoencephalitis is a ccr1/ccr5-independent infl ammatory process entamoeba histolytica encephalitis diagnosed by pcr of cerebral spinal fl uid protozoa traversal of the bloodbrain barrier to invade the central nervous system cerebral sparganosis strongyloides stercoralis hyperinfection: diffi culties in diagnosis and treatment a trial of antiparasitic treatment to reduce the rate of seizures due to cerebral cysticercosis conventional and experimental treatment of cerebral malaria matrix metalloproteinase-9 and intercellulara adhesion molecule 1 are powerful staging markers for human african trypanosomiasis a rare case of toxocara canis cerebral vasculitis gnathostomiasis, another emerging imported disease first case of human babesiosis in germany -clinical presentation and molecular characterisation of the pathogen immunoblot diagnostic test for neurognathostomiasis helminthic invasion of the central nervous system: many roads lead to rome human african trypanosomiasis of the cns: current issues and challenges severe falciparum malaria: an important cause of multiple organ failure in indian intensive care unit patients novel in vitro and in vivo models to study central nervous system infections due to acanthamoeba spp african trypanosome infections of the nervous system: parasite entry and eff ects on sleep and synaptic functions acute granulomatous acanthamoeba encephalitis in an immunocompetent patient posterior reversible encephalopathy syndrome in neuro-malaria everyday and exotic foodborne parasites helminth infections of the central nervous system occurring in southeast asia and the far east central nervous system infections as a cause of an altered mental status? what is the pathogen growing in your central nervous system chemotherapy for chagas' disease: a perspective of current therapy and considerations for future research fungal infections of the cns: treatment strategies for the immunocompromised patient antifungal therapy in infants and children with proven, probable or suspected invasive fungal infections magnetic resonance imaging and angiography in cerebral fungal vasculitis combined activity in vitro of caspofungin, amphothericin b, and azole agents against itraconazloe-resistant clinical isolates of aspergillus fumigatus activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin b against 448 recent clinical isolates of fi lamentous fungi overview of the lipid formulations of amphotericin b central nervous system infectious complications early after liver transplantation unsuccessful treatment with voriconazole of a brain abscess due to cladophialophora bantiana meningoencephalitis due to blastomyces dermatitidis: case report and literature review invasive aspergillosis with central nervous system dissemination in a presumably immunocompetent, non-neutropenic patient: case report and review mri fi ndings in an immunocompromised boy with cns fungal infection fungal meningitis new antifungal drugs and new clinical trials: interpreting results may be diffi cult voriconazole versus amphotericin b for primary therapy of invasive aspergillosis central nervous system aspergillosis in an immunocompetent patient infection of the cns by scedosporium apiospermum after near drowning survival improvement in coccidioidal meningitis by high-dose intrathecal amphotericin b craniofacial mucormycosis: computed tomographic and angiographic fi ndings in two cases rhinocerebral phycomycosis and internal carotid artery thrombosis intraventricular cryptococcal amebas pathogenic for man difl uoromethylornithine, an eff ective new treatment of gambian trypanosomiasis dihydroartemisininpiperaquine against multidrug-resistant plasmodium falciparum malaria in vietnam: randomised clinical trial specifi c chemotherapy of chagas disease: controversies and advances important helminth infections in southeast asia diversity, potential for control and prospects for elimination amebic meningoencephalitides and keratitis: challenges in diagnosis and treatment an outbreak of angiostrongyliasis cantonensis in beijing research development of the pathogenesis pathways for neuroschistosomiasis dexamethasone proves deleterious in cerebral malaria. a double blind trial in 100 comatose patients praziquantel in treatment of cerebral schistosomiasis new treatment option for second-stage african sleeping sickness: in vitro and in vivo effi cacy of aza analogs of db289 epilepsy and neurocysticercosis in sub-saharan africa successful chemotherapy of transfusion babesiosis managment of servere malaria: a practical handbook, 2 nd ed. world health organization persistent cognitive impairment after cerebral malaria: models, mechanisms and adjunctive therapies fatal cerebral mycoses caused by the ascomycete chaetomium strumarium scedosporium cerebral abscesses after extra-corporeal membrane oxygenation rapid diagnosis of candidiasis and aspergillosis isolated central nervous system histoplasmosis in an immunocompetent patient: 53-month hiatus to diagnosis and treatment craniocerebral aspergillosis: a review of advances in diagnosis and management cerebral mucormycosis: proton mr spectroscopy and mr imaging rhino-orbitalcerebral zygomycosis in solid organ transplant recipients emergence of invasive cerebral aspergillosis in an hiv-positive patient on voriconazole therapy novel eff ect of voriconazole on conidiation of aspergillus species cerebellar and medullar histoplasmosis a retrospective study of catastrophic invasive fungal infections in patients with systemic lupus erythematosus from southern taiwan a new era of antifungal therapy neuroimaging and neurologic complications after organ transplantation spondylitis/spondylodiszitis vertebral osteomyelitis in northern spain. report of 62 cases vertebral osteomyelitis in göteborg, sweden: a retrospective study of patients during 1990-95 vertebral osteomyelitis at a norwegian university hospital 1987-97: clinical features, laboratory fi ndings and outcome prospective study of patients presenting with acute partial transverse myelopathy long-term outcome of acute and subacute myelopathies acute myelopathies. clinical. laboratory and outcome profi les in 79 patients compressive myelopathy mimicking transverse myelitis clinical practice. transverse myelitis idiopathic transverse myelitis. corticosteroids, plasma exchange, or cyclophosphamid paraneoplastic neurological syndromes 14-3-3 protein in the cerebrospinal fl uid of patients with acute transverse myelitis an approach to the diagnosis of acute tranverse myelitis prognostic predictors of acute transverse myelitis neurophysiological studies in acute cysts masquerading as racemose neurocysticercosis cryptococcal meningitis after neurosurgery liposomal amphotericin b: a review of its use as empirical therapy in febrile neutropenia and in the treatment of invasive fungal infections infectious and non-infectious neurologic complications in heart transplant recipients central nervous system aspergillosis in patients with human iummunodeffi ciency virus infection caspofungin: a new therapeutic option for fungal endocarditis oral versus intravneous therapy in the treatment of systemic mycosis nosocomial fungal infections: epidemiology, diagnosis, and treatment syndrome of the anterior spinal artery as the primary manifestation of aspergillosis coadministration of voriconazole and phenytoin: pharmacokinetic interaction, safety, and toleration fungal infections of the nervous system: current perspective and controversies in management fungal infections of the central nervous system in hiv-negative patients: experience from a tertiary referral center of south india cerebral mycosis: 7-year retrospective series in a tertiary center primary central nervous system phaeohyphomycocis: a review of 101 cases potential fungicidal eff ect of voriconazole against candida spp cerebral pseudallescheria mycosis after near-drowning central nervous system complications in renal transplant recipients in a tropical environment zygomycotic invasion of the central nervous system fungal infections. in: noseworthy jh (ed) neurological therapeutics-principles and practice. informa healthcare fungal brain infections neurologic complications after solid organ transplantation kapitel 32 · infektionen transverse myelitis is methyl prednisolone useful in acute transverse myelitis? transverse myelitis: pathogenesis, diagnosis and treatment the neurology of liver failure viral meningitis poliomyelitic-like illness in central european encephalitis entzündliche wirbelsäulenerkrankungen als ursache für rückenschmerzen approach to acute or subacute myelopathy efns guidelines on diagnosis and management of neuromyelitis optica proposed diagnostic citeria and nosology of acute tranverse myelitis the spectrum of neuromyelitis optica myelopathy but normal mri: where next? key: cord-016283-b6yywn9f authors: hasan, ashfaq; praveen, sai haranath; tarke, chandrakant; abdullah, fahad title: clinical aspects and principles of management of tuberculosis date: 2019-08-07 journal: mycobacterium tuberculosis: molecular infection biology, pathogenesis, diagnostics and new interventions doi: 10.1007/978-981-32-9413-4_20 sha: doc_id: 16283 cord_uid: b6yywn9f tuberculosis over the ages, has killed more people than any other infection has. notwithstanding the advances in modern science, clinical diagnosis sometimes remains elusive, owing principally to the frequent paucibacillary occurrence of the disease and the slow doubling time of the organism; empiric treatment is often fraught with risks in the era of increasing drug resistance. this chapter attempts to provide an overview of the disease, beginning with the pathogenesis and its protean clinical presentations. it also discusses the recent evolution of molecular methods that have lately provided an impetus to early diagnosis with a clear opportunity to unmask drug resistance before initiating “blind”, potentially ineffective, and sometimes harmful treatment with standard therapy. the chapter also provides insight into tuberculosis in special situations, and discusses briefly the treatments in uncomplicated cases as well as in special situations, and in instances of drug resistance. preventive methods including current and upcoming vaccines are mentioned. finally, a short discussion of the sequelae of tuberculosis—which have the potential to be confused with active disease—is presented. tb can involve all organs except the nails, hair and teeth [ekaterina kulchavenya. ther adv infect dis. 2014 apr;2(2): 61-70]. pulmonary tb is the most common form of tb. the symptomatology of tb has to do with its pathogenesis, the specific organ system involved and the duration of the disease. primary tb, which occurs in the mycobacterium-naive individual, usually presents with a frequently occult primary focus in the peripheral lung parenchyma (stead's focus) accompanied by a pleural effusion; or by a primary focus-frequently inapparent-in the lower part of the upper lobe or the upper part of the lower lobe (ghon's focus) accompanied by lymphadenopathy. other manifestations of primary tb such as erythema nodosum or phlyctenular conjunctivitis may be present. in young children with immature cellular immunity, or in immunocompromised individuals, the process may evolve to progressive primary disease. symptoms and signs relate to the compressive effect of enlarged lymph nodes on adjacent structures (collapse or hyperinflation of a lobe or segment), or rupture of caseating lymph nodes into a bronchus, with bronchiectasis later supervening in such a lobe. post-primary disease, the typical adult form of tb, usually occurs by reactivation of a dormant focus-or less frequently, by reinfection; the better aerated lung apices are preferentially involved. the classical triad of the constitutional symptoms of tb (fever, night sweats, weight loss)-most usually of several weeks' duration-is present in a high proportion of patients (heemskerk et al. 2015) . the extent of disease at the time of first diagnosis may be variable, from patchy consolidation to a large cavity. this not only reflects the protean pulmonary forms of the process but also has to do with host immunity, the virulence of the strain and, most of all, with the time elapsed between disease onset and diagnosis. cavitation is an important milestone in the timeline of the post-primary pulmonary tuberculosis. with ingress of o2-rich air into a fresh cavity (caused by the coughing out of a caseous collection), the aerobic mycobacterium is presented with an opportunity to multiply manifold in an aerobic environment: the infectiousness of the subject (and the possibility of a positive diagnosis on sputum testing) commensurately increases (golub et al. 2006) . cavitation also increases the propensity to haemoptysis (which may sometimes be massive) and that of cavitary colonization with a fungal ball (mycetoma) later on in healed disease. one third of all individuals affected with post-primary tb die within a few months ("galloping consumption") (reported tuberculosis in the united states, 2013 2014). the remainder either go on to develop a chronic pattern with a more gradual progressive decline or undergo spontaneous remission of the process and restoration to health. with chronicity, fibrosis is common. disseminated tb, which often casts "miliary" shadows in the lungs, occurs due to haematogenous dissemination following either primary or post-primary tb. the name derives from the resemblance of the widespread granulomatous lesions to millet seeds (seen in pathological specimens as 1-2 mm yellow granules). symptoms may be nonspecific and depend on the site and degree of organ involvement. hepatosplenomegaly, lymphadenopathy and pleural effusions may be present. fundus examination, often neglected, will frequently show choroid tubercles, which are virtually diagnostic of the condition in a likely setting. diagnosis often hinges on microbiology from broncho-alveolar lavage (bal) or on biopsy of involved organs (lewinsohn et al. 2017) though it may occasionally be made on sputum (sponaneously expectorated or induced). disseminated tb disseminated tb should always raise suspicion for an accompanying immune deficiency such as hiv disease. sometimes an occult disseminated form of tb ("cryptic miliary tb") presents as a "fever of unknown origin", usually in the elderly or immunocompromised patients. in difficult cases (as with the classic miliary form), bone marrow or liver biopsy with mycobacterial cultures may be diagnostic. unlike the post-primary form of disease, untreated miliary tb is predictably fatal. extra-pulmonary tuberculosis has been increasingly seen since the advent of the hiv epidemic. tb can involve any organ of the body (other than nails, hair and teeth). organ systems typically involved include pleura (pleural effusions), lymph nodes (generally cervical or mediastinal), airway (endobronchial tuberculosis), skeletal (joint or vertebral involvement, sometimes with a psoas abscess), gastrointestinal, genitourinary, meningeal and pericardial involvement. symptoms pertain to the system involved, but diagnosis may be difficult unless a high degree of suspicion is maintained. tissue or fluid samples from the involved organs with staining, mycobacterial cultures or molecular probe assays are often diagnostic although radiological patterns in specific cases may be very suggestive of the process (e.g. vertebral involvement with a paraspinal abscess) (lewinsohn et al. 2017) . the diagnosis of tuberculosis, especially in the developed nations, rests on the keystone of suspicion. physical examination is generally unrewarding. auscultation of the lung typically underestimates the lung problem relative to that seen on imaging. with a pleural effusion, breath entry is typically decreased, with a flat note over the effusion. cavernous bronchial breathing may be detected over a cavity, and tubular bronchial breathing over an area of consolidation or dense fibrosis. the radiologic features in the various forms of pulmonary tb have already been discussed above under the relevant heads. microbiological isolation of the mycobacterium tuberculosis on culture has been the only definitive test for tb thus far, though it could be argued that molecular probes for specific mycobacterial genes and other technologies (discussed below and also elsewhere in this book) offer viable and more attractive options for diagnosis. presumptive diagnosis is typically achieved by microscopy (usually of sputum, but also of fluid obtained by bal and other means) (pai et al. 2016 ). mycobacteria, not being stainable by "conventional" stains, require a modified acid stain (the ziehl-neelsen method) in order to be visualized; led microscopy though relatively expensive, by making the bacilli easy to see under low power, enables more fields to be scanned in a shorter period of time, and thus increases the sensitivity of microscopy several-fold (bhalla et al. 2013) . conventional culture methods are slow (4-6 weeks, less by radiometric assays), and frequently there is a compelling need to start appropriate anti-tubercular therapy as early as possible. rapid culture techniques are now in universal use. they use liquid rather than solid media. the mycobacteria growth indicator tube (mgit) method takes days rather than weeks and facilitates drug sensitivity testing. since relapses (see below) occur in approximately 5% of patients after 6 months of standard therapy, it is a moot point whether establishment of minimum inhibitory concentrations (mics) in the presence of a "critical concentration" of drug is relevant in selected cases (colangeli et al. 2018) . automated nucleic acid amplification technology (naat) has the potential of amplifying even a fragment of mycobacterial dna rapidly (in 2 h) and has revolutionized tb diagnosis. in its currently available form, the genexpert test is intended specifically to diagnose mycobacterium tuberculosis (and thereby differentiate it from other mycobacteria such as environmental mycobacteria), and also presumptively diagnose rifampicin resistance. though not invariable, rifampicin resistance also implies inh resistance. together, rifampicin and inh resistance fulfil the diagnosis of multiple drugresistant tb (mdr-tb). thus a positive genexpert test makes a presumptive diagnosis of mdr-tb (steingart et al. 2009 ). the test is more sensitive than direct microscopy and extremely specific (steingart et al. 2014 ). the test is not infallible, however, and any result that is discordant with the clinical picture should either be repeated or further information sought using another modality. false-positive naat results can occur due to laboratory contamination. importantly, naat can detect nucleic acid from dead non-viable bacilli, and so a positive test after completion of the course of treatment has no relevance in that situation (boyles et al. 2014) nor is it of relevance in monitoring response to treatment (friedrich et al. 2013) . it is important to realize that naat is not a substitute for afb smear and culture testing; culture is essential for species identification and for drug susceptibility testing (cheng et al. 2005) . a more sensitive version of xpert has been developed. the line probe assays (lpa) can permit rapid identification of specific gene markers associated with rifampicin resistance alone or in combination with isoniazid, and provide clinically relevant information about the level of inh resistance (low level associated with the inh-a gene; versus high level associated with the kat-g gene) (who treatment guidance for drug resistant tuberculosis 2016). definitive diagnosis has traditionally hinged on the culture characteristics of mtb: species discrimination can be achieved on the basis of colony morphology as well as on the basis of biochemical tests performed on the culture. conventional culture (lowenstein-jensen medium and its variants) is capable of detecting as few as ten bacteria per millilitre with the sensitivity and specificity of sputum culture approximating 80 and 98%, respectively (morgan et al. 1983) . following initial culture, species identification is done by any of the following techniques: biochemical methods (testing, nucleic acid hybridization probes, high-pressure liquid chromatography or mass spectrophotometry). drug sensitivity testing is subsequently carried out, usually at a reference laboratory (shinnick and good 1995) to first-line anti-tubercular agents, but sometimes to second-line agents as well when resistance to components of first-line anti-tubercular agents has been documented. in individuals living with hiv who have cd4 counts <100 cells/mm 3 , or those who are immunocompromised, mycobacterial cultures of blood and urine should complement conventional testing. point-of-care urinary detection of lipoarabinomannan, a component of the mycobacterial cell wall (urine lam test) may be useful for hiv-infected individuals, and its use may possibly confer a mortality benefit (reported tuberculosis in the united states, 2013 2014). the future of point-of-care devices is next-generation sequencing, and, perhaps, innovations like detection of volatile organic compounds in exhaled breath (garcia-basteiro et al. 2018) serological tests (tests for antibodies against tb) have poor sensitivity and specificity; false-positive tests are ubiquitous, and serologic testing for the diagnosis of tb is discouraged by the who. a pleural fluid adenosine deaminase (ada) >35 u/l has been reported to be 93% sensitive and 90% specific for the diagnosis of pleural tb in lymphocytic exudates (garcia-zamalloa and taboada-gomez 2012). tb infection can rapidly progress to disease in infants and young children (progressive primary tb). mortality is high in early childhood because of the severe forms of tb (such as miliary tb and meningeal tb) that occur more frequently in young children. progressive primary tb is a serious consequence of primary tuberculosis. consolidation or bronchopneumonia is more common than cavitary disease in children. haematogenous dissemination leading to miliary tb is also more common in infants and young children. tubercular pleural effusion, however, is less common in children below 5 years of age. central nervous tb is the most serious form of childhood tb (prevention 2014). the presentation is usually with nonspecific symptoms such as fever, headache, drowsiness and irritability, but in advanced cases, vomiting, neck rigidity, seizures, focal neurologic deficit and coma may supervene. since sputum samples for diagnosis are difficult to obtain in young children (children tend to swallow sputum rather than expectorate), gastric washings can rather be sent for microbiological examination. a ppd test that is greater than 10 mm in bcg-unvaccinated children and > 15 mm in bcg-vaccinated children is considered positive. (targeted tuberculin testing and treatment of latent tuberculosis infection. this official statement of the american thoracic society was adopted by the ats board of directors, july 1999. this is a joint statement of the american thoracic society (ats) and the centers for disease control and prevention (cdc). this statement was endorsed by the council of the infectious diseases society of america. (idsa), september 1999, and the sections of this statement 2000.) the principles of treatment of tuberculosis in children are similar to that of adults. paediatric tb patients should be monitored carefully for ethambutol-induced ocular toxicity, owing to the fact that children are less likely to report visual impairment than adults. in a prospective study of children on multidrug-resistant tuberculosis, children had lower serum concentrations in spite of higher dosing of moxifloxacin, a fact that was ascribed to increased drug elimination in children. quinolones have also been known to rarely cause arthropathy and tendon rupture. more recent studies have shown that most quinolones are quite well tolerated but very specific and narrow indications are present for quinolone use in children (patel and goldman 2016) . tuberculosis in pregnancy has important implications for the mother and child (loto and awowole 2012) . the diagnosis of tuberculosis in pregnancy can be challenging, since many of the symptoms of early tb may be confounded by pregnancy; for instance, the normal weight gain in pregnancy may temporarily mask the associated weight loss. chest radiograph is not an absolute contraindication and when necessary may be carried out with "abdominal shielding" of the fetus. the complications of tb in pregnancy include early spontaneous abortion, intrauterine growth retardation, preterm delivery, low birth weight and increased neonatal mortality. congenital tb is rare, but can be associated with high perinatal mortality. first-line oral drugs, i.e. isoniazid, rifampicin, ethambutol and pyrazinamide, can all be used during pregnancy safely. streptomycin and other aminoglycosides have been proven to be potentially teratogenic in pregnancy. they are also capable of causing eighth nerve paralysis, with deficits ranging from mild hearing loss to bilateral deafness (loto and awowole 2012) . concerns have been raised about the use of fluoroquinolones in pregnancy even though the weight of the evidence seems to point toward safety (acar et al. 2019) tb is considered an aids-defining disease [centers for disease control and prevention. appendix a: aids-defining conditions. mmwr. 2008;57(rr10):9]. the risk of developing tb is estimated to be between 16 and 27 times greater in people living with hiv than among those without hiv infection. hiv and tb have a salutary impact upon each other and expedite each other's progression. the risk of progressing from latent to active tb is estimated to be between 12 and 20 times greater in people living with hiv than among those without hiv infection (global tb report 2017). against the backdrop of hiv infection, the rate of progression of tb from the acquisition of infection to full-blown disease may take weeks, rather than years-as is the case in hiv-negative individuals (mayer and dukes hamilton 2010) . it is important to realize that tb can occur at all "stages" of hiv disease. the clinical presentation of hiv patients who have relatively high cd4 counts is no different to that which occurs in individuals without hiv. such individuals manifest with the classical manifestations of post-primary tuberculosis such as upper lobe infiltrates. on the other hand, lower lobe involvement, pleural effusions and intrathoracic lymphadenopathy are common in individuals with low cd4 counts (typically <200/μl). indeed the chest x-ray may sometimes only show vague interstitial infiltrates or even be normal (mayer and dukes hamilton 2010) . taken together, the fact that in advanced hiv disease (a) the sputum is less likely to be positive for afb (b) the ppd is usually non-reactive (c) the x-ray abnormalities are likely to represent other opportunistic infections or even non-infective conditions associated with hiv disease and (d) histopathology of involved tissues is characterized by lack of typical granulomata; these considerations make the diagnosis extremely challenging in this setting. in addition, there is increased frequency of extra-pulmonary tb among hiv-positive individuals. frequently, a positive naat (genexpert) test is the justification for starting treatment. however it must be cautioned that even a negative naat test cannot convincingly rule out disease, and, given that delays in treatment may be fatal, the decision to initiate treatment is sometimes taken on clinical grounds. the treatment of tuberculosis differs from the treatment of most other infections in certain respects. multiple drugs are required in a regimen since spontaneous mutations can lead to resistance to the action of one or more drugs. also, because the bacterium is relatively slow growing, it takes longer to achieve bacteriologic sterility. multidrug therapy, the norm for tb, has been very effective, with a modest rate of relapse (colangeli et al. 2018) . the standard four-drug regime ("short course") comprises a 2-month intensive phase with the four front-line drugs (rifampicin, isoniazid, ethambutol and pyrazinamide), followed by a continuation phase with the drugs (minus pyrazinamide and possibly ethambutol) for a further 4 months. the four front-line drugs have been chosen based on their efficacy in rapidly reducing the number of organisms initially (during the initial phase), thus reducing infectivity; and on their sterilizing potential (the ability to eradicate viable bacteria completely and so prevent relapses); and their relative lack of side effects (see table 20 .1). these drugs are combined into regimens. the six classes of second-line agents (fluoroquinolones, the aminoglycosides, capreomycin, ethionamide and prothionamide, para-amino salicylic acid (pas), cycloserine and terizidone) have a rather lower efficacy and a higher propensity for side effect and drug intolerance. several other antibiotics of uncertain efficacy have also been categorized as anti-tubercular agents and have a role in treating multidrug-resistant tb (mdr-tb) (see table 20 .2). two new agents, bedaquiline (a diaryl-quinolone) and delamanid (a nitroimidazole), are now approved for the treatment of mdr-tb. the who recommends using a daily regimen and fixed-dose combinations (guidelines for treatment of drug-susceptible tuberculosis and patient care 2017). administration of the drugs on a daily basis both during the intensive and during the continuation phase is preferable although daily administration during the initial phase and intermittent (thrice weekly) administration during the continuation phase is also acceptable (guidelines for treatment of drug-susceptible tuberculosis and patient care 2017). intermittent administration of drugs (especially in the initial phase) is not offered in the presence of hiv disease. if at the end of the intensive phase the bacteria are sensitive to the three most important agents (isoniazid, rifampicin and pyrazinamide), ethambutol may be discontinued (nahid et al. 2016 ). however, drug testing for three of the four frontline drugs for each patient at the end of the intensive phase is impractical, and limited testing (genexpert) is usually performed initially in those patients whose sputum remains positive. there is evidence to show that extending the continuation phase by 7 months (making up a total duration of treatment of 9 months) helps in preventing relapse in those patients who have cavitation on initial chest x-rays; and also in those who show positive cultures at the end of the initial phase of treatment (benator et al. 2002) ; as well as in those whose medication given during the initial phase did not include pyrazinamide. anti-tubercular drugs require to be supplemented with carefully monitored steroid therapy in two circumstances: tubercular meningitis (a short course of dexamethasone or prednisolone is typically given, tapered over 6 to 8 weeks) and tuberculous pericarditis (guidelines for treatment of drug-susceptible tuberculosis and patient care 2017). since many patients cannot be relied upon to regularly take their drugs, and irregular intake of drugs has the potential to result in drug resistance (see treatment failure and relapse; and drug resistant tb, below), it is imperative to ensure adherence to the regimen. directly observed therapy, short-course (dots) is the direct observation by another person, of the patient taking the medication (chaudhuri 2017) . the use of dots has improved adherence in subgroups of patients such as tb with hiv or if dots was at the site of dots centre or provider location. self-administered therapy (sat) modified with some form of monitoring incorporated into the process shows some promise. although dots could be administered by a family member, the preferred person is a trained lay provider or healthcare worker (guidelines for treatment of drug-susceptible tuberculosis and patient care 2017). the treatment of tb in hiv disease merits special consideration. antiretroviral therapy (art) should be started irrespective of the clinical stage of hiv disease and cd4 count. anti-tubercular therapy (att) should precede the commencement of art. art should then be commenced as soon as it is evident that att is being tolerated (typically between 2 weeks and 2 months). the immune reconstitution inflammatory syndrome (iris) is sometimes seen with the returning immune competence that follows art; an increased immune response to tubercle bacilli or antigens occurs. iris usually occurs between 1 and 3 months of the commencement of art and is associated with a decrease in viral load and an increase in cd4+ t cell count. there is a worsening in the clinical picture weeks or months into treatment, with an increase in the size of lymph nodes, development of pleural effusions and features of noncommunicating hydrocephalus in a patient with tb meningitis. although most patients with iris have mild to moderate symptoms, iris, especially when related to tb meningitis, can be life-threatening. an "unmasking iris" may occur in patients with unsuspected tb (raviglione 2017) . treatment of iris includes addition of steroids (which probably work by decreasing the levels of pro-inflammatory cytokines) and the continuation of att and art. with both att and art on board, drug-drug interactions especially involving rifampicin (by induction of the cytochrome p450 system) may occur. rifamycins induce the metabolism of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. rifampicin is a more potent inducer of the cytochrome p450 system than rifapentine, which in turn is more potent than rifabutin. despite potential drug interactions, rifamycins should nevertheless be included in tb regimens, with dosage adjustment if necessary. rifabutin is the preferred rifamycin in hiv-positive individuals. host-directed therapy to modulate the immune response to tb is an active area of research (kaufmann et al. 2014) . the use of an anti-diabetic drug-metformin, for example-is being investigated. also, studies of autologous mesenchymal stromal cell infusions is being investigated in mdr and xdr-tb (skrahin et al. 2014 ). an approach using high-altitude sanatoria was also explored (murray 2014) in a throwback to the pre-anti-tubercular chemotherapy era. study using atypical mycobacterial injection in tuberculous did not show any major outcome differences from placebo although steroids did decrease the subsequent development of constrictive pericarditis (mayosi et al. 2014) . given the role that vitamin d plays at the level of the antigen-presenting cell, the role of vitamin d deficiency as a possible player in tb predisposition has recently come into focus; indeed a recent meta-analysis has revealed that vitamin d supplementation in fact did not affect time to sputum culture conversion overall, though it did accelerate sputum culture conversion in patients with multidrug-resistant pulmonary tuberculosis (jolliffe et al. 2019). patients with tb may be admitted to an icu for a variety of reasons, including respiratory failure, multiorgan failure, decreased consciousness associated with tubercular meningitis, pneumothorax, cardiogenic shock (due to massive pericardial effusion) and liver or kidney failure due to a drug reaction induced by att. confluent tuberculous bronchopneumonia and tb ards are less common causes of respiratory failure (hagan and nathani 2013) . tuberculosis treatment is especially challenging in critically ill patients due to potential for poor gastric absorption and the high rates of organ dysfunction and drug toxicity in this subset of patients. deciding the optimal regimen and dosing becomes challenging in the presence of concomitant hepatic and renal failure. in a recent study done on tb patients requiring icu admission, mortality was 72.5% (tatar et al. 2018 ). treatment failure is suspected when the sputum remains positive for afb at the end of the first 3 months of treatment. it occurs due to factors that may involve incorrect regimens or dosing or indeed due to malabsorption or poor-quality drugs (lambregts-van weezenbeek and veen 1995) . patients are regarded as treatment failures when a microbiologic indicator like sputum smear/culture for tb remains positive after 3-4 months of starting treatment or comes back positive after a period of remaining negative (fall and rise phenomenon) (prasad et al. 2018) . patients may respond for a short time after starting treatment or do not respond to treatment from the inception. in any case the implication of treatment failure is drug-resistant tb (see below), and strenuous efforts must be made to look for microbiological resistance to first-and, in the proper context, to second-line agents-by the available tools. relapse, on the other hand, is the recurrence of tb after completion of treatment that has been successful, with documented culture negativity. relapse may be due either to a reinfection or reactivation of the dormant bacillus. drugresistant tb is to be considered in these settings. the implications of relapse are not as grave, the isolates generally being drug sensitive. in either case, the exact regimen chosen should be based upon the drug sensitivity pattern. specific recommendations upon the construction of such a regimen are available at who treatment guidelines for drug-resistant tuberculosis 2016 update (http://www.who.int). drug-resistant tb is a major global problem and is a threat to the success of the end tb strategy. mdr-tb or multidrug-resistant tb (mdr-tb) is defined by resistance of m. tuberculosis against to at least rifampicin and isoniazid. since these two drugs are the most effective drugs available against tb, resistance to both considerably shortens the odds of complete cure. xdr-tb or extensively drug-resistant tb is defined as resistance to rifampicin and inh plus resistance to at least one fluoroquinolone and one second-line injectable agent (amikacin, kanamycin or capreomycin). some of the reasons that lead to drug resistance have been mentioned in the section above. however the most important of these are failure to comply with the treatment regimen on the part of the patient and the addition of a single drug ("monotherapy") to a failing regimen; or a situation of unrecognized primary (that which is present at the start of therapy) or secondary (that which develops during treatment) drug resistance. inh mono-resistance of itself probably does not impact the outcome of therapy provided that ethambutol and possibly pyrazinamide are used for the entire duration of therapy. consideration should be given in this case to extending the duration of treatment to 9 months from the standard 6 (sadanand 2011). for reasons already discussed, rifampicin-resistant individuals should be managed as mdr-tb patients. in such patients it is not unusual to encounter resistance to additional drugs (e.g. ethambutol). it has been shown that clinical cure is possible in patients with mdr and even xdr-tb (who drug resistant tb 2016). however, owing to a variety of reasons not the least of which is drug intolerance, only about half of mdr-tb and a third of xdr-tb patients ever complete therapy. the who estimates there are over 600,000 mdr-tb or rifampicin-resistant cases annually (global tb report, who update 2017; wwww.who.int). the treatment of mdr-tb and xdr-tb is complex and requires specialized knowledge and close monitoring to ensure rational and safe therapy (lange et al. 2018) . the choice of drugs for treatment of mdr-tb is frequently difficult. formulation of drugs into regimens is facilitated by the organization of drugs into groups: group a ¼ levofloxacin/moxifloxacin, bedaquiline, linezolid. group b ¼ clofazimine, cycloserine/terizidone. group c ¼ ethambutol, delamanid, pyrazinamide, imipenem-cilastatin, meropenem, amikacin (streptomycin) , ethionamide/prothionamide, p-aminosalicylic acid. the who makes specific recommendations for short and long mdr-tb regimens. in the who's 2018 update (who treatment guidelines for multidrug-and rifampicin-resistant tuberculosis 2018), for those mdr-tb patients "who have not been previously treated for more than one month with second-line medicines used in the shorter mdr regimen or in whom resistance to fluoroquinolones and second-line injectable agents has been excluded," the who permits a shorter mdr-tb regimen of 9-12 months. for mdr/rr (rr¼ rifampicin resistant) tb patients on longer regimens, the who recommends that "all three group-a agents and at least one group-b agent (should) be included to ensure that treatment starts with at least 4 tb agents likely to be effective, and that at least three agents (are) included for the rest of treatment after bedaquiline is stopped. if only one or two group a agents are used, both group b agents are to be included. if the regimen cannot be composed with agents from groups a and b alone, group c agents are added to complete it. kanamycin and capreomycin are not to be included in the treatment of mdr/rr-tb patients on longer regimens" (who treatment guidelines for multidrug-and rifampicin-resistant tuberculosis 2018). a detailed discussion about the treatment of mdr-tb is available at the foregoing resource. novel anti-tubercular agents (e.g. bedaquiline and delamanid) have been discussed elsewhere in this text. the remodelling of the lung that follows tb can significantly contribute to morbidity and mortality. haemoptysis is frequently one of the remote sequelae and can on occasion be life-threatening. an unresolved cavity is frequently the cause, and bleeding from an unsupported blood vessel in the cavity wall or in a post tubercular bronchiectatic segment can be a source of troublesome bleed. cavities colonized by fungi (aspergillomas), broncholiths eroding into the bronchial wall and rarely expansion and subsequent rupture of an unsupported blood vessel in the cavity wall (rasmussen's aneurysm) can cause massive haemoptysis (van den heuvel and van rensburg 2006) . a scar carcinoma sometimes develops in an area of fibrosis and may sometimes be difficult to distinguish from a dense fibrotic infiltrate (gao et al. 2015) . post-tubercular bronchiectasis is predisposed to when caseation necrosis and inflammation with retention of secretions leads to destruction of bronchial walls with ensuing permanent dilatation. sometimes compression of bronchial lumen by enlarged lymph node can produce bronchiectasis by a different mechanism. postprimary tb involves the upper lobes of the lung, and so the gravitationally advantaged upper lobes do not usually undergo a process of chronic and recurrent infection unlike the lower lobes that are involved by bronchiectasis of other aetiologies. mycetoma (fungal ball) is a mass of fungal hyphal material that grows within a cavity. aspergillus fumigatus is the most common aetiological agent, but other fungi such as mucor or fusarium can also cause a fungal ball to develop (rippon 1988) . such a mycetoma is usually asymptomatic but can sometimes lead to haemoptysis. no treatment required in asymptomatic cases, but surgical treatment needs to be considered in patients with massive or recurrent haemoptysis. a list of adverse effects associated with anti-tubercular agents appears in table 20 .2. side effects are possible with any of the anti-tubercular agents, and on rare occasions, they may be serious and even fatal. the risk of liver injury with anti-tb therapy is always a concern. among the four first-line agents inh, rifampicin and pyrazinamide are all capable of causing hepatotoxicity. isoniazid is capable of causing asymptomatic mild elevation in transaminases in 15-20% as well as fatal severe acute hepatitis in 0.05-1%. jaundice with liver injury can occur in 0.5-1%. peripheral neuropathy due to pyridoxine deficiency can occur with inh therapy in up to 6.5% of elderly individuals (ruan et al. 2018 ); pyridoxine supplementation is required for all patients taking inh (john 2019). factors increasing the chances of inh hepatotoxicity include age, female gender, alcohol use, prior hepatitis, concurrent use of cytochrome-inducing agents and slow acetylator status (sharma et al. 2016) . acute inh toxicity can present as seizures due to decreased gaba levels. inh is metabolized to several metabolites including the hepatotoxic metabolite hydrazine, toxic-free radicals and mono-methylhydrazine. degradation by acetylation occurs via n-acetyltransferase-2 gene (nat2); a deficiency of the nat enzyme can lead to accumulation of hepatotoxic metabolite. ten percent of patients with anti-tubercular drug-induced hepatotoxicity show progression to acute liver failure; the rest are self-limited (badrinath and john 2018) . if the serum bilirubin rises to !3 mg/dl or serum transaminases exceed more than five times the upper limit of normal (or exceed three times the upper limit of normal in a symptomatic patient), all drugs should be stopped (nahid et al. 2016 ). the decision on what regimen to use on restarting should be individualized. the regimen, even if retailed, might then still include one or more potentially hepatotoxic drugs, and these should be restarted one at a time, only when liver function tests return to their baseline (e.g. the transaminases fall to less than twice normal). in general, a cholestatic type of derangement would prompt addition of inh first; with a non-cholestatic picture, rifampicin could first be added (sterling) . needless to say, careful monitoring with frequent testing of serum liver function is required. most physicians would omit pyrazinamide when restarting att except in the mildest of cases or with other compelling reasons (sterling) . patients receiving ethambutol (emb) should be questioned regarding their vision at each monthly visit. monthly visual acuity and colour vision discrimination testing is recommended in those patients in whom emb doses of greater than 15-20 mg/kg are being used, those who have been taking ethambutol for longer than 2 months and those with renal insufficiency in whom the levels of drug may be expected to rise (blumberg et al. 2003) . for patients who require a regimen with no hepatotoxic agents, potential agents include ethambutol, levofloxacin or moxifloxacin, an injectable agent and other second-line oral drugs. the optimal choice of agents and duration of treatment (at least 18-24 months) is uncertain. (see "antituberculous drugs: an overview", section on "second-line agents".) in the end, efforts to finally eradicate tb will need to hinge upon preventative aspects as much as upon its treatment. these strategies would include currently established practices, as well as novel strategies. the former include the following. tuberculosis is now the primary cause of death from infectious diseases exceeding hiv, aids and malaria. the who estimates 10.4 million new cases and 1.7 million deaths every year (who global tb report 2017). of these, 0.4 million are hiv patients who die mostly in lmic (low-and middle-income countries) (who global tb report 2017; www.who.int). within the next 16 years, the goal is to reduce tb incidence by 90% but the progress has been painfully slow. the most effective way to reduce the burden of tb in the community, to date, has been to identify patients affected with tb as early as possible and to treat them effectively with antitubercular therapy. the role of vaccines in this regard, though potentially vital, has been disappointing. of all vaccine tested to date, the bacillus calmette-guerin (bcg)-a live attenuated vaccine that was made in 1919 after an incredible 11 years and 231 sub-cultures (hasan 2014 )-is still the most effective vaccine we have today. yet its effect has been inconsistent, ranging from 80% to zero protection; several factors including geographical differences appear to play a role (british adolescents had better responses than south indian participants perhaps due to background exposure to mtb or atypical mycobacteria). the efficacy of bcg has been most manifested in its protection of infants and young children from the dangerous forms of tb (such as disseminated "miliary" tb and tb meningitis). also, its effects appear to wane with time (hasan 2014) . the bcg is not a single vaccine. several strains-all derived from the original 1921 bgc strain-are in use, which also partly explains its variable efficacy. in 1998, the sanger centre in cambridge, britain and paris's pasteur institute in france cracked the genetic code for the old h37rv strain of tubercle bacillus, and then, the genome of highly virulent cdc1551 (the "oshkosh" strain) was mapped at the tigr (fine 1989 ). these developments have led to the possibility of exciting new candidate vaccines, several of which are in fact in the pipeline. it has been estimated that one-third of the world has been infected with tb bacteria. a relatively small number of immunocompetent individuals affected with ltbi will ever progress to active disease. reactivation of tb occurs when the immune system is compromised by external factors such as steroids or other immunosuppressants or by immune deficiency syndromes, which enables the latent (dormant) bacteria to "awaken" causing active disease. there is unfortunately no test that can currently predict which of these individuals will progress to active disease immunocompromised individuals have a high possibility of doing so. given that perspective, treatment for latent tb infection is most relevant for children under 5 years, the elderly and individuals with hiv infection. to eliminate tb from the planet, a multipronged strategy incorporating early and effective treatment, vaccination and elimination of dormant reservoir of latent tb will be essential. ltbi treatment with inh ("chemoprophylaxis") is still the mainstay for treating latent tb infection but given that treatment extends to 6 months, and that there is one death from hepatotoxicity for every 25,000-40,000 cases with such treatment, alternate strategies including rifamycin-based regimens are being explored (rangaka et al. 2015) . pregnancy outcomes following quinolone and fluoroquinolone exposure during pregnancy: a systematic review and meta-analysis rifapentine and isoniazid once a week versus rifampicin and isoniazid twice a week for treatment of drug-susceptible pulmonary tuberculosis in hiv-negative patients: a randomised clinical trial performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis infectious diseases society of america: treatment of tuberculosis false-positive xpert (r) mtb/rif assays in previously treated patients: need for caution in interpreting results recent changes in technical and operational guidelines for tuberculosis control programme in india -2016: a paradigm shift in tuberculosis control molecular diagnostics in tuberculosis bacterial factors that predict relapse after tuberculosis therapy the bcg story: lessons from the past and implications for the future five-year follow-up of a controlled trial of five 6-month regimens of chemotherapy for pulmonary tuberculosis assessment of the sensitivity and specificity of xpert mtb/rif assay as an early sputum biomarker of response to tuberculosis treatment ct features of lung scar cancer point of care diagnostics for tuberculosis diagnostic accuracy of adenosine deaminase and lymphocyte proportion in pleural fluid for tuberculous pleurisy in different prevalence scenarios delayed tuberculosis diagnosis and tuberculosis transmission guidelines for treatment of drug-susceptible tuberculosis and patient care (2017) world health organization clinical review: tuberculosis on the intensive care unit adjunctive vitamin d in tuberculosis treatment: meta-analysis of individual participant data progress in tuberculosis vaccine development and host-directed therapies -a state of the art review control of drug-resistant tuberculosis drug-resistant tuberculosis: an update on disease burden, diagnosis and treatment infectious diseases society of america/centers for disease control and prevention clinical practice guidelines: diagnosis of tuberculosis in adults and children tuberculosis in pregnancy: a review synergistic pandemics: confronting the global hiv and tuberculosis epidemics prednisolone and mycobacterium indicus pranii in tuberculous pericarditis comparison of a radiometric method (bactec) and conventional culture media for recovery of mycobacteria from smearnegative specimens worth a try in extensively drug-resistant tuberculosis? official american thoracic society/ centers for disease control and prevention/infectious diseases society of america clinical practice guidelines: treatment of drug-susceptible tuberculosis tuberculosis diagnostics: state of the art and future directions safety concerns surrounding quinolone use in children multidrug-resistant tuberculosis/rifampicin-resistant tuberculosis: principles of management reported tuberculosis in the united states controlling the seedbeds of tuberculosis: diagnosis and treatment of tuberculosis infection (ed) harrison's infectious diseases, 9th edn. mcgraw-hill education medical mycology: the pathogenic fungi and the pathogenic actinomycetes isoniazid-induced hepatotoxicity and neurotoxicity in rats investigated by (1)h nmr based metabolomics approach harrison's infectious diseases miliary tuberculosis: a new look at an old foe diagnostic mycobacteriology laboratory practices autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase 1 safety trial xpert(r) mtb/rif assay for pulmonary tuberculosis and rifampicin resistance in adults treatment of drug-susceptible pulmonary tuberculosis in hiv-uninfected adults this official statement of the american thoracic society was adopted by the ats board of directors contributing factors to mortality rates of pulmonary tuberculosis in intensive care units who treatment guidance for drug resistant tuberculosis (2016) who treatment guidelines for multidrug-and rifampicin-resistant tuberculosis fluoroquinolones for treating tuberculosis key: cord-010499-yefxrj30 authors: yelverton, elizabeth; lindsley, dale; yamauchi, phil; gallant, jonathan a. title: the function of a ribosomal frameshifting signal from human immunodeficiency virus‐1 in escherichia coli date: 2006-10-27 journal: mol microbiol doi: 10.1111/j.1365-2958.1994.tb00310.x sha: doc_id: 10499 cord_uid: yefxrj30 a 15‐17 nucleotide sequence from the gag‐pol ribosome frameshift site of hiv‐1 directs analogous ribosomal frameshifting in escherichia coli. limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting. limitation for phenylaianine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting. protein sequence analysis demonstrated the occurrence of two closeiy related frameshift mechanisms. in the first, ribosomes appear to bind leucyl‐trna at the frameshift site and then slip leftward. this is the 'simultaneous slippage’mechanism. in the second, ribosomes appear to slip before binding amlnoacyl‐trna, and then bind phenylaianyl‐trna, which is encoded in the left‐shifted reading frame. this mechanism is identicai to the‘overlapping reading’we have demonstrated at other bacterial frameshift sites. the hiv‐1 sequence is prone to frame‐shifting by both mechanisms in e. coli. ribosomes normaiiy maintain a constant reading frame from aug to the finish, but they are capabie of slipping into an alternative reading frame at an average frequency of the order of 10 " (atkins etai, 1972; j. a. gallant etai, unpubiished) . in certain special cases, much higher frequencies of ribosome frameshifting occur. these cases include production of polypeptide release factor 2 of escherichia coli, which depends upon a rightward frameshift within the coding sequence (craigen et at.. 1985; craigen and caskey, 1987; weiss et ai, 1987; curran and yarus, 1988) ; translation of the reverse transcriptase of the yeast ty element, which also depends upon a rightward frameshift (clare ef ai, 1988) ; and translation of the rna of severai retroviruses, which express gag-pol and gag-pro-pol polyproteins by means of leftward frameshifts (reviewed by hatfield and oroszlan, 1990; cattaneo, 1989) . ribosomal frameshifting in both rightward and leftward directions has also been shown to occur at certain 'hungry' codons whose cognate aminoacyi-trnas are in short supply (gallant and foley, 1980; weiss and gailant, 1983; 1986; gallant et ai, 1985; kurland and gallant, 1986) . not all hungry codons are equally prone to shift: in a survey of 21 frameshift mutations of the rllb gene of phage t4, weiss and gallant (1986) found that oniy a minority were phenotypicaily suppressible when challenged by limitation for any of several aminoacyl-trnas. the context njies governing ribosome frameshifting at hungry sites are under investigation, and have been defined in a few cases (weiss et al., 1988; gallant and lindsiey, 1992; peter et ai. 1992; koior ef a/., 1993; lindsiey and gallant, 1993) . so far these sequences do not resembie any of the naturally occurring shifty sites summarized in the first paragraph above, in order to find out whether these two categories of ribosome frameshifting are mechanisticaliy reiated, we have tested the susceptibility of a well-studied retroviral frameshift site to manipulation by aminoacyl-trna limitation in e. coli we have directed our analysis to the shifty site at the gag-pol junction of hiv-1 both because of its clinical interest, and because certain features render it convenient for analysis. in some viral systems, baroque secondary structures in the mrna downstream of the frameshift site are required to augment frameshifting levels (jacks et ai, 1988b; brierley et ai, 1989) . in the case of hiv-1, however, although a stem-loop structure might exist downstream of the frameshift site (jacks et ai, 1988a) , direct modification or elimination of the stem-loop sequence has little effect on the rate of frameshifting (madhani et ai, 1988; weiss etai. 1989) . moreover, wilson etal. (1988) demonstrated that a short (21 nucleotide) sequence of hiv-1 without the stem-loop was sufficient to direct a high level of frameshifting in heterologous in vitro systems. the site of ribosomal frameshifting at the siippery sequence u-uuu-uua has been directly established by amino acid sequencing of frameshifted proteins (jacks et ai., 1988b) , and the participation of certain aminoacyl-trnas has been clearly implicated by mutagenesis of the monotonous tract of uridines (jacks et ai, 1988b; wiison et ai, 1988) . our purpose was to discover whether the ribosomal frameshifting directed by a very short sequence in hiv-1 could be reproduced by e coli ribosomes in vivo, and, if so, whether we could alter the rate of frameshifting by regimens that change the relative abundance of key aminoacyl trnas encoded at or near the frameshift site. weiss etal. (1989) have also reported that a 52 nucleotide fragment from hiv-1 is sufficient to direct ribosomal frameshifting in an e. coli system. in this report we present evidence that a much shorter 15-17 nucleotide sequence derived from hiv-1 is sufficient to direct the same ribosomal frameshift event in e. coli as in eukaryotes. we also show that in e. coli the rate of ribosomal frameshifting on that sequence can be increased by limitation for leucine, the amino acid encoded at the frameshift site. protein sequence analysis of the product indicates the occurrence of two siightiy different mechanisms of shifting. the strategy behind the construction of our assay system for ribosomai frameshifting may be understood with reference to fig. 1 . when eukaryotic ribosomes decode the hiv mrna sequence . . . uuuuuuaggg . . ., shown as nucleotides 1-10 in fig. 1a , the adenine at position 7 appears to be read twice: first, as the third position of a leucine codon (uua) and then again as the first position in the overlapping arginine codon (agg ratner et al. (1985) . in a heterologous mammalian in w/ro translation system, most of the frameshift product has the amino acid sequence . . . asn-phe-leu-arg (jacks et al., 1988b) , where leu is encoded by positions 5-7 and arg is encoded by positions 7-9. some mutations that result in increased or decreased expression of frameshift products in a heterologous test system are shown above and below the nucleotide sequence, respectively (wilson ef a/., 1988) . 'n' signifies a mutation to any non-u base. double underlines at positions -10 and +16 mark the boundaries of a fragment that directs the synthesis of a frameshift protein product in a heterologous yeast system (wilson et at.. 1988) . the singly underlined g at position -6 is the 5' boundary of a fragment that directs the synthesis of a frameshift protein product in a mammalian iri vitro system (jacks e( al., 1988b) . b. and c. a portion of &ie mrna sequences expressed from teczframeshift alleles hiv13, hiv13-a3, hiv201, and hiv201-u7 are depicted. numbers above the nucleotide sequence correspond to analogous positions of the hiv-1 gag-po/junction. doubly underlined nucieotides mark the twundaries of sequence thai is consen/ed with respect to hiv-1. synthesis of p-gaiactosidase from the alleles requires a leftward frameshift. amino acids are shown for the mature protein, afler in vivo cleavage of the initiating w-terminal fomiyl-met residue. constructs phiv13 and phiv201. and their variants phiv13-a3 and phiv201-u7, are described in fig. 1 . (the sequence of the critical heptanucleotide at the frameshift site is shown in parenthesis after each construct's designation.) all constructs were transformed into a derivative of cp79 {rela2 thr' leu his ~ arg~ thi~) carrying a complete deletion of lacz. methods of cultivation, and enzyme and protein assay were as described previously peter etal., 1992) . cells were grown into exponential phase in m63-glucose medium supplemenled with all required amino acids plus he and val. the lac promoter was induced (2mm iptg and 2.5mm camp) for about one doubling. data are reported ± standard error of the mean, with the number of replicate induced cultures in parentheses. these values include all the unstarved control cultures from the various starvation experiments. to take place in about 10% of ribosomal transits (jacks etai. 1988b) . in hiv-1, the outcome of the leftward ribosomal frameshift is the successful production of the gag-pol fusion protein, in the assay system we have devised, the outcome of an analogous ieftward frameshift by e coli ribosomes wiii be the successful production of the enzyme p-galactosidase from genetically frameshifted alleles of the /acz gene. we have previousiy used an assay system of similar design to demonstrate that lysyl-trna starvation can amplify ribosomai frameshifting in either direction at iysine codons, given certain context ruies (gailant and peter etai. 1992; lindsiey and gailant, 1993 ). aileles to be tested were constructed by the ligatlon of paired complementary oligonucleotides into the h/ndlll-bamh\ site of pbwhoo, as described in galiant and lindsiey (1992) . figure 1 shows the sequence of the translated strand from the region of our constructs that reproduces the gag-pol frameshift signal from hiv-1. the /acz frameshift alieles carried on plasmids pi^iv13 and phiv201 are constructed so that a shift to the left by one base, as in the expression of the gag-po/fusion of hiv, is required to generate active enzyme. the two constructs both carry a short sequence identical to the region around the frameshift site in the gag-po/overlap of hiv-1 for 15 nucleotides in phiv13 and 17 nucleotides in phiv201 (see fig. 1 ); they differ slightly from one another severai bases downstream of the frameshift site. host ceils carrying either of these piasmids produce active enzyme at about 1% of the efficiency of cells carrying a control lacz* plasmid (table 1) . this basal value is close to the value (1.8%) observed by weiss et al. (1989) for frameshifting on a much longer hiv-derived sequence in a similar p-galactosidase reporter. it is also much higher than the frequency of leftward frameshifting (0.03-0.2%) we observed previously at sequences unrelated to hiv (gailant and lindsiey, 1992) . the presence of the hiv sequence in our reporter thus leads to an unusually high frequency of leftward frameshifting. modification of the critical heptanucieotide sequence from u uuu uua to u uau uua in plasmid phiv13-a3 decreased frameshifting about fivefold, while modification of the heptanucleotide to u uuu uuu in phiv201-u7 increased frameshifting by two-to threefold (tabie 1). these genetic resuits are analagous to earlier findings in other reporter systems (jaci<s et ai. 1988b; wilson et ai. 1988; weiss et ai, 1989) and suggest that the heptanucleotide is the predominant site of leftward frameshifting in our reporter system as well. chemical evidence that this is indeed the case was obtained from protein sequence analysis. the protein encoded by phiv201 was purified and subjected to automated edman sequence analysis, as previously described peter et al., 1992) . the results ( fig. 2 ) are readily interpreted with reference to the nucleotide sequence and coding potential shown in fig. 1 . for the first 10 cycles, the amino acid sequence corresponds to normal transiation in the (0) reading frame: t-m-i-t-p-s-s-n-f-l. after this position, the signal (r-e-g-l-p) clearly corresponds to the leftward reading frame. thus, a leftward shift occurred after incorporation of the leucine at position 10, exactly the frameshift cfiaracteristic of hiv translation in eukaryotic systems (jacks et ai, 1988b) . similar resuits were obtained with plasmid phiv13 (data not shown). thus, our short cloned sequence reproduces the specificity of the hiv frameshift signal when translated by e. coli ribosomes. we asked whether the hiv sequence's rather high level of shiftiness could be modulated by stalling the ribosome at hungry codons within the frameshift region. phe and ieu are the last two amino acids encoded in the initial reading frame before the shift, and arg is the first encoded in the new reading frame after the shift. we imposed iimitation for phe-thna by inhibiting the synthetase with the analogue phenylaianine-hydroxamate (phx). graded limitations for arginine or ieucine were imposed by growing auxotrophic host cells on a range of concentrations of slowiy utiiized methyl esters of these amino acids (buston and bishop, 1955) . tables 2 and 3 present the results of ali these limitation regimens. considering first arg and phe (table 2) , we observe that both limitations substantiaily reduced the differential rate of enzyme synthesis expressed from both the frameshift construct and the lacz* control. the reduction was about the same in both genotypes, indicating that neither arginine limitation nor phe-trna limitation had a specific effect on the frequency of frameshifting within the hiv sequence. in order to ensure leucine sufficiency, the host was a ieucine prototroph isogenic with our normal host, and it was grown nevertheless in the presence of 100 jig ml" ^ leucine. the /ac promoter was induced for one doubling and two litres of cells were harvested. beta-galactosidase was isolated as described previously peter ef ai. 1992 ) and subjected to automated edman sequencing. the raw data for picomole amounts of pth amino acids in each cycle were corrected for background and lag using applied biosystems software, as described . the figure presents the sum of three independent sequencing runs, one performed at illinois and two at riverside. in every cyle, the most abundant pthamino acid released predominates over others by at least a factor of two, affording a clear signal of the primary sequence. secondary signals reflect a variety of artefacts. one is minor peaks which anticipate the next cycle (for example, f in cycle 8, r in cycle 10, e in cycle 11, and g in cycle 12}, presumably owing to a minority of the protein that is one amino acid shorter on the /v-terminus than the bulk of the material. exponential cells carrying plasmid phiv201 or the control lacz' plasmid pbwiloo were subjected to limilation for phenylalanyt-wna by addition of phenylalanine-i^ydtoxamate (phx) al the indicated concentrations; and for arginine by centrifuging and washing the cells, and resuspending them in growth medium containing the indicated concentrations of arginine-methyl-ester (ame), with a control culture containing arginine at our standard concentration of 100 jig ml v the /ac promoter was induced as in table 1 , and cells harvested for assay after about one doubling. cultures are listed in order of decreasing growth rate. growth rate varies directly with the concentration of ame, which serves as a source of required arginine, and inversely with the concentration of phx, which inhibits phenylaianine activation. in the case of leucine limitation, a different outcome was suggested in preliminary screening by a crude, qualitative petri plate assay (kuriand and gallant, 1986) . quantitative data on log-phase cells are shown in table 3 . in the lacz*ĉ ontroi, leucine limitation progressiveiy restricted enzyme synthesis, reducing it very severeiy at iow growth rates, in the hiv construct, on the other hand, there was no such restriction, but ratlner a smali stimuiation of enzyme synthesis. if this effect is caused by frameshifting at the ieucine codon within the hiv sequence, then it should be eliminated by eliminating that ieucine codon. as an additional control, therefore, we tested 201-u7, a construct that is identicai to 201 except for the conversion of the leucine uua codon to uuu (see fig. 1 ). in this construct, ieucine limitation did not stimulate enzyme synthesis as in 201, but ratfier reduced it, as it did in the wiid type (table 4) . the reduction was a good deal less than in the wiid type, suggesting that some frameshifting at various positions still occurs in 201-u7, so as to offset partly the effect of reduced lacz expression. however, the difference between tine response of 201 and that of 201-u7 demonstrates that the leucine codon eliminated in the latter must be a major determinant of frameshifting induced by leucine limitation. decisive identification of the frameshift site during leucine iimitation was obtained from protein sequence anaiysis, as described earlier. the resuits are shown in table 3 . relative growth rates and differential rates of (3-galactosidase synthesis during leucine limitation. exponential cells carrying plasmid phiv201 were spun, washed, and resuspended in medium supplemented with the indicated concentrations of leucine-methyl-ester (lme). these were grown in parallel with a control aliquot retumed to growth in leucine at lootigml''. the entries show growth rates and differential rates of enzyme synthesis relative to the control leucine culture, expressed as mean values i^ standard error (number replicate experiments). the mean control differential rates in these experiments were 2.3 ± 0.16 (6} for phiv201 and 191 ± 44 (4) forpbwiioo. fig. 2 and fig. 3 . in order to normalize to the same number of picamoles, all the values for the leucine-starved protein were multiplied by the ratio (unstarved/ starved) of the sums of the predominant peaks (n in cycle 8, f in 9, r in 11, and e in 12). the ratios of leucine to phenylaianine in cycle 10 of the two sets of data were as follows. for the unstarved case, the ratios in three runs were 2.55, 1.9, and 3.5, yielding an average of 2,65 with a standard error of 0.46. for the ieucine-starved case, the ratios in four runs were 0.52, 0.58, 0.68, and 0.51, yielding an average of 0.57 with a standard error ot 0.039. the difference is highly significant (p less than 0.005) by the la test (crow et ai.. 1969) . the nucleotide sequence in the region of interest is shown below the cycle bar plot, with the expected amino acids read in the initial (0) reading frame shown above the nucleotide sequence, and the expected amino acids read in the leftward reading frame shown below. solid bars = hiv-201 unstarved (+leu); open bars = hiv--201 stan/ed (+lme) normalized to unstarved values. fig. 3 . the protein sequence is very similar to that of the protein from unstarved cells (fig. 2) , with one significant exception. for the first nine cycles, the initial t-m-i-t-p-s-s-n-f sequence of the noimal (0) reading frame is clear, as it was in the protein from unstarved ceils. from cycle 11 onwards the signature of the leftward reading frame, r-e-g-i-p-g, is equaily clear. the one difference between the starved and unstarved proteins is at cycle 10, the position of the leucine codon. the predominant amino acid in the starved protein is f (phenyialanine) rather than l at this position. three considerations argue that carryover of f from cycle 9, the preceding position, does not contribute significantly to the peak of f in cycle 10 of the leucine-starved protein. first, the data in every cycle have been corrected for carryover by means of the applied biosystems lagcorrection aigorithm (huni^apiller, 1986) . the effectiveness (3) exponential cells carrying plasmid phiv201-u7 were treated as in table 3 . the mean control differential rate was 4.7 ± 0.20 (3). of this correction can be seen in the very low levels of carryover throughout the cycle bar plots of figs 2 and 3, and of our earlier publications (peter et ai, 1992; gallant and lindsiey, 1992) . second, amino acids differ in their susceptibility to carryover from one cycle to the next (proline, for example, being notoriously bad), and it is carryover of f that we need to assess in considering these data. in fact. figs 6 and 7 of gallant and lindsiey (1992) present exactly the needed controi: f was the amino acid present in cycle 7 of both sequences, and cycle 8 corresponded to a 'hungry' codon, just as in the present case. in those two sets of data, which represent the average of four independent sequencing runs in each case, carryover of f from cycle 7 to the next cycle averaged 8.3% (standard error = 2.8%), far less than the relative ievel of f found in cycle 10 of the present cycle bar plots. finally, the starved and unstarved proteins are virtually identical at every position except cycle 10, in which the difference in regard to phenylaianine and leucine was replicated in several independent sequencing runs on protein of each type. a summary of this comparison of the starved and unstarved proteins in and around cycle 10, and the statistics for this cycle, are presented in fig. 4 . phenylaianine corresponds to the uuu triplet overlapping the hungry ieucine codon in the leftward reading frame. we conclude that most ribosomes which produced p-galactosidase slipped ieftward when stalled at the leucine codon before aminoacyl-trna binding, and then bound phe-trna cognate to the overlapping phenylaianine codon. however, cycle 10 contains a significant secondary peak of leucine as well, suggesting that a minority of ribosomes bound leucyl-trna and then shifted left, just as in unstarved cells. our wild type lacz* controi shows a marked decrease in p-galactosidase synthesis during limitation for each of tiie amino acids tested. this response is typical of rela ~ cells, and is primarily caused by a dependence of /acztranscription on ppgpp during amino acid limitation (primakoff and artz, 1979; yangefa/., 1979; primakoff, 1981; foieyefa/., 1982) , compounded by errors in transiation at some hungry codons (reviewed by gallant, 1979; cashe! and rudd, 1987; parker, 1989) . expression of the /acz gene carrying the hiv frameshift signal should be subject to the same rela~ defects and is indeed reduced to the same extent as wild-type during limitation for arginine or for phenyialanyl-trna (table 2) . during (eucine limitation, in contrast, enzyme synthesis is increased rather than decreased in cells carrying the frameshift allele phiv201 (table 3 ). in 201-u7, which is identical to 201 except for the absence of the leucine codon at the frameshift site, leucine limitation provokes a decrease in enzyme synthesis (table 4) , as it does in the iacz* control. the fact that enzyme synthesis increases with leucine limitation in 201 means that an increase in frameshifting more than compensates for the sharp decrease in lacz expression found in tine control experiments with the wild type and with 201-u7. the increased frameshifting attributable to this one leucine codon can be estimated as the ratio of the effect of ieucine limitation on 201 to that on 201-u7. in the former case, enzyme synthesis was two times greater in 3)igmi"' of leucinemethyl-ester than in leucine, whereas in the �tter case it was three times less. by this estimate, therefore, the frequency of frameshifting at the critical leucine codon was increased about sixfold at 3|igmr^ of the leucine analogue, a limitation regimen which reduced growth about sevenfold. the protein sequence of the frameshifted material (fig. 3) demonstrates that the ribosomes read the sequence in a normal fashion for the first nine codons, up to the hungry codon calling for leucine at position 10. at this position, the predominant amino acid was f (phenylaianine) rather than l (leucine), implying that the ribosomes slipped leftward to decode the ttt triplet for phenyialanine, which overiaps the hungry codon from the left. this overlapping mode of reading is entirely analogous to the case of leftward frameshifting at a different hungry codon we have described earlier . however, there is also a significant secondary peak of leucine at position 10, amounting to one third of the total signal (fig. 3) . this is the amino acid encoded in (he initial or (0) reading frame. the presence of leucine here strongly suggests that a minority of ribosomes bound leucyl-trna and then slipped leftward, just as a majority of ribosomes did under conditions of (eucine sufficiency. thus, the hiv sequence is subject to starvationpromoted frameshifting on e. coli ribosomes, and by two seemingly different mechanisms. one is overlapping reading at the hungry codon. like the cases we have analysed elsewhere (weiss and gallant, 1986; gailant and linsdiey, 1992) . the second is zero-frame reading and subsequent siippage-inserting leucine at position 10 in this caseanaiagous to other studies of the translation of hiv (jacks etai, 1988b; weiss etal.. 1989) or rous sarcoma virus (rsv) (jacks etai. 1988a) in heteroiogous systems. in fact, a close reading of the data of jacks e/a/. (1988b) reveais that the hiv sequence also displayed both modes of frameshifting in the eukaryotic cell-free translation system they analysed. the predominant amino acid at the frameshift site in their study was leucine. but there was aiso about 20-25% phenyialanine (see fig. 2 of jacks et ai, 198bb) . indicative of overiapping reading. our results in unstarved cells (our fig. 2) indicate about 30% phenyialanine. these two modes of frameshifting share fundamentai similarities, most strikingly in regard to their sequence requirements in the four positions to the left of the frameshift site. in the case of the mechanism of (0) frame trna binding followed by slippage, mutation studies in this region strongiy suggest that the frameshift is facilitated by a leftward slip of peptidyl-trna in the p-site (jacks et al.. 1988a.b; wilson ef al., 1988; weiss et ai, 1989) . in the case of tiie overlapping reading mechanism, our mutation studies suggest a similar involvement of a peptidyl shiix {ko\ot et ai. 1993) . thus, in both mechanisms the leftward slippage at the ribosome's a-site is facilitated if the adjacent peptidyl-trna finds complementary pairing with a left-shifted triplet at the p-site. the distinction between the two meciianisms seems, at first thought, to reside in the order in which aminoacyl-trna binding and slippage of the message occur. however, this distinction may be more apparent than real. it could be that the aminoacyl-trna that reads tiie overlapping codon first enters the a-site in the (0) frame by way of a mismatch. in the present case, this would mean phe-trna pairing initialiy with the uua leucine codon, an interaction which demands one mismatch in the third position. this non-cognate interaction would of course be favoured by a shortage of leucyl-trna, particularly in our rela~ host cells which suffer increased recognition errors at hungry codons (gallant, 1979; cashel and rudd, 1987; parker. 1989 ). subsequently, leftward slippage of the phe-trna in the a-site to the overiapping uuu triplet would provide complementarity at all three positions, and thus improve stabiiity. in this way, an initial recognition error in the (0) reading frame could be resolved by way of a reading frame error, an example of what has elsewhere been termed 'error coupiing' (kuriand and gaiiant. 1986 ). the poi gene product expressed from intact hiv in human host cells has yet to be characterized at the amino acid sequence level. consequently, it is unknown whether the amino acid at the frameshift junction is ieucine or phenylaianine (or a mixed population), and it is therefore unknown which mode(s) of frameshifting operates under natural conditions. the overlapping reading mechanism is worth bearing in mind because of the obvious possibilities it presents for physiological controi. in prokaryotic systems, frameshifting by overlapping reading is a natural outcome of imbalances in the aminoacyl-trna pools (atkins et ai. 1979; gallant and foley. 1980; weiss and gallant, 1983; 1986; kuriand and gallant, 1986; bruce et ai. 1986; weiss and gallant. 1986 ). our present resuits demonstrate that a translational frameshift signal from hiv-1 can be reguiated in this fashion in response to changing ieveis of leucyl-trna. similar regulation might be a simpie but effective metliod for establishing physioiogical control of the expression of any frameshifted gene. many such genes function to replicate and spread viruses, transposable elements, and retroviruses (reviewed by hatfield and oroszlan, 1990; cattaneo. 1989) . in these cases, ribosome frameshifting during aminoacyl-trna imbalance might represent a mechanism for iinking element movement to the state of the celiuiar economy. the degree of aminoacylation of trnas provides a sensitive difference signal of ceilular activity. anabolism generates the amino acids which charge the trnas, while translation drains these amino acids into growing proteins. hence, the trna charging levei reflects the balance of these two flows, in bacterial celis, trna aminoacylation levels operate both specific attenuation control circuits (landick and yanofsky, 1987) and the highly pleiotropic stringent controi system (gallant, 1979; cashel and rudd. 1987) . and may regulate the expression of individual translation factor genes (grunberg-manago, 1987) . this speculation might provide a rationale for the various different shifty sequences found in the gag-pol aj\6 gagpro-pol overiaps of different retroviruses, and the comparable regions of different coronaviruses. perhaps each unique shifty sequence has been evoiutionariiy optimized to respond to a trna deficiency that is the most sensitive difference signal for its particular host ceils. likewise, evoiutionary pressure to maintain a response to a cell-specific aminoacyl-trna signal might also explain why retroviruses maintain non-optimal translational frameshift sequences which must in some cases be augmented by baroque mrna secondary structures downstream (weiss et ai. 1989) . finally, we might note that our data do not distinguish between a simultaneous slippage mechanism at the leucine codon (as proposed by jacks and varmus) and an overlapping reading mechanism at the next codon. if ribosomes in unstarved cells paused at the ggg codon following the leucine codon, then arg-trna could enter by overlapping reading and shift the reading frame leftward (see fig. 4 ). the amino acid sequence in this case would be exactly that ascribed to simultaneous slippage, and which we have demonstrated in unstan/ed e. coli cells. we have no independent reason to expect ribosome stalling at the ggg codon in growing celis, except that it is a rare codon and caiis for a rare trna. experiments designed to investigate this possibiiity are under way. frameshift alleles were constructed by modification of the plasmid vector pbwiioo. pbwiloo, a kind of gift of bob weiss, is a modified pbr322 in which nucleotides 1-1417 were replaced by nucleotides 1-4625 (nih genbank co-ordinates) of the e coli lac operon. the lac operon fragment has been modified by the deletion of nucleotides 641-1070, the removal of the natural ecori site within the /acz gene, and by the addition of the puc9 polylinker (vieira and messing, 1982) between codons 4 and 5. the resultant pbwiloo is lad z*y"a , and confers resistance to ampicillin. in typical constructions, about 1 jig of pbwiloo was cut to completion with restriction endonucleases h/ndlll and samhi (bethesda research labs) at their single sites within the puc9 polylinker. about loong of the prepared vector was used without purification in a 20 nl ligation containing a 10-fold molar excess of paired complementary oligonucleotide primers, kindly prepared by yim foon lee (howard hughes institute, university of washington) on an applied biosystems 380b synthesizer. ligations were carried out at room temperature for 3-12 h. then transformed into a lac~ strain for screening on xgal indicator plates. plasmid dna from (leaky) lac candidates was examined by restriction analysis and by dna sequencing of the salient region of the lacz gene. sequences of the oligonucleotide pairs were: hiv13/hiv13-a3, 5'-agctctaatt^tttagg-ga-3; and 5'-gatctccctaaaiaattag-3'; h1v201, 5-agctctaai 1 i i i iagggaagg-3' and 5-gatcccttcc-ctaaaaaattag-3'; hiv201-u7, 5'-agctctaal 1 i i i i i-gggaagg-3' and 5'-gatcccttcccaaaaaaattag-3'. bacterial strains and culture conditions were as described previously (weiss etai., 1988) , except that minimal glucose-m63 medium was supplemented only with isoleucine and valine in addition to the strains' growth requirements. the lacz plasmids were kept under selection through the presence of 1mgml ^ carbenicillin. tn most experiments. the host cells also carried ppyioii, a compatible plasmid (spectinomycin resistant, streptomycin resistant) carrying a complete lacl'^ gene. selection for this plasmid was maintained through the presence of 50|igmr'' spectinomycin in the overnight cultures used to inoculate our experimental cultures, but spectinomycin was not present during induction of lacz. cell-free extracts were made by resuspending cells at a final protein concentration of 1 -2 mg ml" \ and sonicating for 20 s at ice-water temperature in a braunsonic 1510 instrument; cell debris was centriiuged out at 26000 x g in a sorvaii refrigerated centrifuge. beta-galactosidase activity was measured by incubating up to 50 mi of extract with 250 iil onpg (0.8mgml ' in 50 mm sodium phosphate buffer. ph 7.0) at room temperature, and monitoring the accumulation of o-nitrophenol at a420 using a continuously recording gilford 2400-2 spectrophotomer. one enzyme unit is defined as the amount of enzyme required to produce a change in absorbance of one per minute. protein concentrations were determined by the method of lowry et al. (1951) using egg albumin as a protein standard. differential rates in enzyme units per mg of protein were computed for the period of induction (approximately one doubling in 2mm iptg, 2.5 mm camp) with or without growth limitation (as indicated). purification of 0-galactosidase by immunoprecipitation with specific antiserum was as described in gallant and lindsiey (1992) . n-terminal amino acid analysis of the purified protein was performed by automated edman degradation on applied biosystems 477 a pulsed-liquid phase sequencer and 470 a gas phase sequencer at university of illinois biotechnology center and university of california, riverside. data were analysed using applied biosystems software for background and lag correction. low activity of p-galactosidase in frameshift mutants of escherichia coii normal trnas promote ribosomal frameshifting characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot trna anticodon replacement experiments show that ribosomal frameshifting can be caused by doublet decoding synthetic a-amino-phydroxycaproic acids the stringent response how 'hidden' reading frames are expressed efficient translational frameshifting occurs within a conserved sequence of the overlap between two genes of a yeast tyl transposon translational frameshifting: where will it stop? bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2 use of trna suppressors to probe regulation of escherichia coli release factor 2 mechanism of the rel defect in beta-galactosidase synthesis stringent control in e. coli on the causes and prevention of mistranslation leftward ribosome frameshifting at a hungry codon some puzzle of translational accuracy regulation of the expression of aminoacyl-trna synthetases and translation factors the where, what, and how of ribosomal frameshifting in retroviral protein synthesis automated amino acid sequence assignment: development of a fully automated protein sequencer using edman degradation signals for ribosomal frameshifting in the rous sarcoma virus gag-po/region characterization of ribosomal frameshifting in hiv-1 gag-poi expression on the role of the p-site in leftward ribosome frameshifting at a hungry codon the secret life of the ribosome transcription attenuation on the directional specificity of ribosome frameshifting at a hungry codon protein measurement with the folin phenol reagent signals for expression of hiv pol gene by ribosomal frameshifting. in the control of human retroviral gene expression errors and alternatives in reading the universal genetic code context rules of rightward overiapping reading in vivo role of the rela* gene in the regulation of the /ac operon positive control of lac operon expression in vitro by guanosine 5'-diphosphate 3'-diphosphate complete nucleotide sequence of the aids virus, htlv-iii the puc plasmids, a m13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers mechanism of ribosome frameshifting during translation of the genetic code frameshifting suppression in aminoacyl-trna limited cells slippery runs, shifty stops, backward steps, and forward hops: -2, -1, +1, +2, -i-5. and +6 ribosomal frameshifting on the mechanism of ribosomal frameshifting at hungry codons e. coli ribosomes re-phase on retroviral frameshift signals at rates ranging from 2 to 50 percent hiv expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems differential sensitivity of gene expression in vitroio inhibitation of dna gyrase this work was supported by grant gm13626 and from grant gm07735 from the national institutes of health. key: cord-011855-0vetk6jd authors: shayo, elizabeth; van hout, marie claire; birungi, josephine; garrib, anupam; kivuyo, sokoine; mfinanga, sayoki; nyrienda, moffat j; namakoola, ivan; okebe, joseph; ramaiya, kaushik; bachmann, max oscar; cullen, walter; lazarus, jeffrey; gill, geoff; shiri, tinevimbo; bukenya, dominic; snell, hazel; nanfuka, mastula; cuevas, luis e; shimwela, meshack; mutungi, gerald; musinguzi, joshua; mghamba, janneth; mugisha, kenneth; jaffar, shabbar; smith, peter g; sewankambo, nelson kaulukusi title: ethical issues in intervention studies on the prevention and management of diabetes and hypertension in sub-saharan africa date: 2020-07-06 journal: bmj glob health doi: 10.1136/bmjgh-2019-002193 sha: doc_id: 11855 cord_uid: 0vetk6jd nan the incidence of diabetes and hypertension has risen sharply in sub-saharan africa alongside a continuing high burden of hiv infection. 1 in many settings, the prevalence figures among adults are 4%-5% for diabetes, above 25% for hypertension and 5%-20% for hiv infection. [2] [3] [4] all these conditions require lifelong treatment, and they have increased substantially the demand for chronic care services in africa, where health systems have, until recently, focused on tackling acute infectious diseases. 5 there is considerable inequity in service provision for chronic diseases. hiv services, including antiretroviral therapy, are available widely for free and are organised typically in stand-alone clinics. over 65% of people estimated to be living with hiv infection are in regular care. 6 in contrast, this figure is only about 5%-20% for people living with diabetes or hypertension. 3 7 a major challenge is that medicines for diabetes and hypertension are generally not provided free of charge and have to be purchased by patients. even in those countries that do provide free medicines for hypertension and diabetes, shortages are common and patients then have to purchase the medicines from private suppliers. our research collaboration is evaluating a biomedical diabetes preventive intervention in people living with hiv infection in a placebocontrolled randomised trial and, separately, evaluating integrated healthcare provision compared with standard care for people living with hiv, diabetes or hypertension in a clusterrandomised controlled trial. 8 there are no data on the effectiveness of these approaches from africa. therefore, these trials have clinical and health economic endpoints and the research is underpinned by an implementation research approach, which, for example, requires strong engagement with health policy makers . [9] [10] [11] [12] we discuss the implications of a limited supply of medicines and potential solutions to track the equity of medicine supply. in particular, we consider what should the ethical approach be for a research programme in terms of provision of a steady and sustainable supply of medicines for patients with diabetes and hypertension when alternative affordable and accessible supplies are unavailable? possible solutions and lessons from other contexts should the research be conducted in realworld conditions where medicines supply for hypertension and diabetes is patchy? if we conduct the research within the context of real-world conditions, then, under the ► conducting intervention studies in africa, where medicines supply for chronic conditions is inequitable and patchy, raises major ethical issues. ► here we discuss what should the ethical approach be for a research programme in terms of provision of a steady and sustainable supply of medicines for patients with diabetes and hypertension. integrated care model that we are testing, patients with different conditions would sit together in the same clinic and have consultations with the same healthcare providers. it will be morally challenging for clinical staff and researchers to turn away one group of patients because of a lack of medicines while for others, with hiv infection, treatment is available freely. in the past, in the vertical stand-alone models of care, the ethical dilemma was less stark because hiv and diabetes/hypertension clinics operated at different locations, sometimes on different days and involved different clinicians. as well as the issue of inequity, observing people living with diabetes and hypertension unable to access medicines, which are both low-cost and effective, could break the ethical principle of beneficence, which states that researchers should have the welfare of the participants as a goal. 12 also, if the research is conducted to real-world conditions, it may be of limited relevance by the time it is completed. this is because the provision of medicines for chronic disease management in africa is likely to increase in the next few years with the increased pressure that is now on donors and governments to support these treatment programmes. if drug shortages decrease, then the findings of our research programme, which would be available in a few years' time, would be of very limited relevance when they are published. thus, in our view, there are both moral and scientific reasons for ensuring patients entering such intervention studies have access to uninterrupted supplies of medicines for the duration of the research. should the research programme purchase the medicines for participants to enable the research to run smoothly? if the study identifies a model of care that is costeffective, it could give impetus to government health services to strengthen their medicine supply chains. on the contrary, by carrying the cost that should be met by governments and donors, it could potentially reduce the pressure on health authorities to find solutions, weaken the advocacy for patients' rights and inhibit the public from demanding their rights to access treatments. advocacy for the right to access antiretroviral therapy was crucial in hiv control in africa 13 and will likely play a major role in enhancing access to medicines for diabetes and hypertension. if the research programme provides the medicines for study participants, it would not be sustainable beyond the duration of the research programme and would mean that patients who access treatment services today may have to stop taking their medicines when the study finishes. while the study is running, provision of free drugs would be a strong incentive for participants to join the study. patients will have the right to decline and to receive the care they would otherwise have received, but if this means a less reliable supply of medicines (than in the research programme), then patients are very likely to join the research. the issue is whether or not this is undue coercion. in our view, the ideal situation here is that access to medicines is strengthened for all, ideally by ministries of health. for this to happen, researchers must work in partnership with policy makers and disease control managers, that is, policy makers and disease control managers must have ownership of the research. researchers should be prepared to purchase medicines for short-term use to cover any gaps that might occur. where ministries of health cannot achieve a reliable supply, even with the support of research programmes, then research in those settings may not be feasible. is there an obligation to provide medicines to non-trial participants? another ethical dilemma arises because the research programme will include only a fraction of all the patients with the target conditions attending the clinics and patients not in the research studies will not have access to any enhanced treatment. although the costs of treatment for diabetes and hypertension are relatively low, it is most unlikely that a research programme could bear the costs of treating large numbers of non-study participants and a requirement to do so would make the research nonviable. not providing medicines to non-study participants will cause inequity between patients in the trial and those who are not. it could compromise outcomes if participants share their medicines to spread the benefits, for example, with relatives with chronic conditions not in the study. it may also endanger community support for the study if this sends the message that we do not care about family members. there is no precedence with provision of drugs to large numbers of non-study participants. when combination antiretroviral therapy for hiv was introduced in high-income countries, it was not available in public health clinics in africa because of its high cost. research in africa at that time will have faced similar dilemmas but wide-scale provision only occurred more recently. at that time, there were also some calls that the standards in clinical trials around treatment and access to medicines should be the same in africa as in high-income countries. 14 however, the standardisation would have inhibited hiv research in africa and was opposed by global health researchers. 15 this enabled the research to be conducted quickly and at relatively low cost, and research on the prevention and management of diabetes and hypertension may need similar considerations. thus, although not ideal, priority of medicines for research subjects will be essential in some settings where the supply of medicines cannot be strengthened for all. should the health facilities be encouraged to procure a greater supply of medicines to facilitate the research? in some circumstances, health facilities might be able to procure a greater supply of medicines to facilitate the bmj global health research. in countries such as tanzania and uganda, under district fiscal decentralised systems, health facilities have flexibility in how they spend their resources. however, if the supply of medicines for diabetes and hypertension was augmented in this way, this could be at the expense of service provision for other conditions, raising further ethical concerns. moreover, there are clear ethical issues if health facilities procure medicines to support a research programme without ensuring that this supply will be maintained after the study. the ideal solution here is that health facilities are supported to strengthen all medicines supply, not just for diabetes and hypertension. research to inform strategies for the prevention and management of diabetes and hypertension is vital in africa. however, such research raises complex ethical issues relating to the limited supply of medicines and a pragmatic approach specific to the african context is needed. it is clear that the research would likely produce meaningless results if the supply of medicines was erratic, but equally, the research programme cannot just purchase the necessary drugs for its trial participants. a solution to this conundrum has to be through discussion and working in partnership with the key stakeholders: the policy makers, disease control managers, healthcare providers, patient groups and community representatives. indeed, a fundamental ethical requirement is meaningful engagement with the key stakeholders. 10 11 similar issues arose in the early years of research on hiv treatment in africa when antiretroviral therapy was prohibitively expensive and not available widely. the research that was conducted in these situations precipitated later pressure on the international community to ensure that life-saving medicines were made freely available to people living with hiv. hiv care and prevention would not have reached its current level without overcoming the initial obstacles to research on treatment. there is a pressing need to take on board the lessons from the progress made with hiv control to develop and expand research on diabetes and hypertension control. we have used our studies on three specific diseases-hiv infection, diabetes and hypertension-to highlight the ethical dilemmas, but the ethical challenges are likely to be common to other diseases. global, regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: a systematic analysis for the global burden of disease study 2016 unaids. global hiv and aids statistics diabetes in sub-saharan africa: from clinical care to health policy world health organisation. global health observatory data: raised blood pressure redesigning primary care to tackle the global epidemic of noncommunicable disease world health organisation. global health observatory data. antiretroviral therapy coverage among all age groups hypertension in sub-saharan africa: a systematic review integrated care for human immunodeficiency virus, diabetes and hypertension in africa developing the ethics of implementation research in health implementation research: what it is and how to do it implementation research: new imperatives and opportunities in global health world health organisation. ethical considerations for health policy and systems research. geneva: world health organization the history of aids exceptionalism the ethics of clinical research in the third world ethics of hiv trials provenance and peer review not commissioned; externally peer reviewed. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ 4. 0/. marie claire van hout http:// orcid. org/ 0000-0002-0018-4060 max oscar bachmann http:// orcid. org/ 0000-0003-1770-3506 jeffrey lazarus http:// orcid. org/ 0000-0001-9618-2299 luis e cuevas http:// orcid. org/ 0000-0002-6581-0587 shabbar jaffar http:// orcid. org/ 0000-0002-9615-1588 contributors es, ag, jb and sj wrote the first draft. mcvh made substantial contributions following the review by the journal. all the authors contributed to many iterations.disclaimer the views expressed in this publication are those of the author(s) and not necessarily those of the nihr or the uk department of health and social care or the european union.competing interests none declared.patient consent for publication not required. key: cord-018017-c8myq6bi authors: iversen, patrick l. title: the threat from viruses date: 2018-09-30 journal: molecular basis of resilience doi: 10.1007/978-3-319-98164-2_3 sha: doc_id: 18017 cord_uid: c8myq6bi infectious disease represent the most significant threat to human health. significant geologic cataclysmic events have caused the extinction of countless species, but these “wrath of god” events predate the emergence of homo sapiens. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic and are here to stay marking their presence with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but are never prepared. the long-term consequences often remain unrecognized and are always inconvenient including cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. viral success is based on rapid replication resulting in large numbers. single-stranded rna viruses with their high replication error rate represent a paradigm for resilience. contemplating recent career paths, i reviewed a broad range of scientific questions. most prominent was, why study an area of science that has no significant impact? in fact, why study anything that is not the most highly impactful area? this question demands a definition, what constitutes high impact? i decided to create a definition that impact and threat to life are related. further, threat to human life is the highest impact and it is likely that things threatening human life may also threaten all life. what presents the greatest threat to human life today? history should provide critical insights to answer this question. a comprehensive look would seek causes of mass extinctions over the past 3.5 billion years of life on earth. this perspective has been a trending subject in science with focus on five mass extinctions. there are inherent biases in focusing on mass extinctions such as these events fail to appreciate small living things such as single celled organisms or bacteria. another bias is in order to appreciate life in the past, the life form must occasionally produce a fossil when it dies. those concerns aside-geologic events have threatened the survival of living things (table 3 .1). the actual events causing these extinctions are frequently debated, for example, the asteroid impact 66 million years ago off the coast of mexico was accompanied by a massive tsunami that was responsible for mass extinctions in montana, the impact may have also triggered an enormous volcano in india resulting in ash and lava flow responsible for regional species extinctions, and the collective dust resulted in prolonged climate change which was probably responsible for even more species extinctions. the lesson remains the same, volcanic eruptions; climate change and asteroid impact threaten life in a highly significant way and are responsible for extensive selection pressure. unfortunately, these events are unpredictable and so enormous little can be done about them. a mass extinction may not represent a legitimate threat or selection pressure because large numbers of species are completely eradicated leaving few survivors to drive evolution. finally, outside of removing dinosaurs so that mammals could thrive, what impact did these events have on human life; humans were not yet on the earth when these events took place. refining the search to dramatic changes in human populations moves the period up to the last 100,000 years. mass migrations took place about every 23,000 years, which is in line with the precession of the earth leading to a possible linkage between earth's precession and human migration. this may have been due to regional climate change like ice ages in the northern hemisphere. however, human population changes on this timescale are not easily documented so estimates of climate and human population dynamics will be limited. time to refine the search. consider the exponential growth, outbreak, of tent caterpillars, malacosoma disstria, in montana reported by david quammen in his book spillover (quammen 2012) . the extensive caterpillar population consumed all of the leaves from the local elm and cottonwood trees in the summer of 1993. the caterpillar activity produced a crackle sound, "like a distant brushfire." the city attacked these insects with a broad arsenal of modern countermeasures but to no effect. however, a nuclear polyhedrosis virus (npv), uses the caterpillar host density like a critical mass in a nuclear weapon to explode destroying the caterpillars in an epic battle. the caterpillar population retreated to undetectable levels in a single season as a result of npv. human populations changed over the past 20,000 years with particular emphasis on the most recent 7000 years. a human population curve over this segment of time shows an exponential growth period interrupted in the middle ages by the plague. hypothesis: the plague and other pandemic infectious disease events appear to be the greatest threat to human life. infectious disease kills 15 million people each year, 26 percent of the total 57 million annual deaths in the global population (fauci and morens 2012) . a brief review of pandemic infections over the past 3000 years illuminates several events that reduced human populations. the plague of athens 430 bce killed 25 percent of the human population (table 3 .2). both bacterial and viral pandemic yersinia pestis is a bacterium causing plague. fleas can be infected with y pestis which transmit the bacterium to rodents, the primary hosts. changes in the environment may lead to the movement of rats into populated areas where humans become infected. homer points to such an infection in the iliad in his description of the trojan war in 1190 bce. plague has returned several times since the trojan war imposing enormous loss of human life (table 3 .2). the most recent plague epidemic killed over ten million people in india in the early 20th century. y pestis is still out there ready for favorable conditions to pounce on human populations but outcomes are likely to be less dramatic due to understanding of sanitation practices, quarantine, and availability of antibiotics. if infections are the greatest threat to human life, they should be critical drivers of evolution? clearly infections pose selection pressure on the human populations. origins of evolutionary thought did not include infection as darwin established key evolution concepts on the galapagos islands. these islands are isolated and an unlikely place for the spread of infections. the concepts speciation point to geographical separation of populations so infections would most likely be restricted to isolated populations. in many cases the survival selection pressure is not identified or ascribed to insufficient sources of food. unfortunately, common single-stranded rna viruses are so unstable that there are limited data for a viral fossil record. pandemic infections remain a threat to human survival in the presence of the information revolution, daily medical breakthroughs, and global travel. the human retrovirus hiv currently a global infection that infects up to 25% of the population in southern and eastern africa with a projected death toll of up to 100 million by 2025. measles killed 200 million people in the last 150 years and the development of an effective vaccine in 1963 reduced concerns for this infection but there were 777,000 deaths in the year 2000. vaccination programs are frequently disrupted due to complacence resulting from vaccine success, conflicts that shift healthcare focus, and social crisis such as the recent ebola outbreak in west africa. smallpox is also an ancient infection causing fever, skin lesions, and at times death. king ramses v of egypt is thought to have died from smallpox around 1200 bce. introduced into mexico in 1520, smallpox killed 3.5 million aztec indians or about half of the population in a period of 2 years and then proceeded to decimate the population of south america. variola is a highly infectious virus killing 300-500 million people during the 20th century inspiring the eradication campaign in 1967. variola was eradicated by december of 1979 (de cock 2001 , a rare triumph of public health. the who deserves acknowledgment for this unprecedented accomplishment and proof of concept that human suffering is not inevita-ble. however, variola is a dna virus with limited rate of mutation and a narrow host range so that animal reservoirs do not exist. the eradication of other viral infections will be more challenging. the world continues to confront a broad array of microbial threats. progress and preparedness make our engagement a likely success for those microbes that resurface and infections for which we have experience. medical and epidemiological uncertainties surround emerging infectious disease, those that challenge us with their novelty. pandemics dominate the infectious disease "fear factor" but each pandemic began as a much more frequent occurrence, an epidemic. most but not all epidemics come from emerging infectious agents, the most significant problem facing life on earth today. the concept of emerging is a human centric term as most of these infections are endemic in an animal host that serves as a viral reservoir. a 2005 report from the university of edinburgh identified 1407 human pathogens and 177 are emerging or re-emerging of which 75% are zoonotic, that is jump from an animal host to human. numerous emerging infections caused by viral agents have imposed high impact on human survival (table 3 .3). all the viral agents in table 3 .3 have genomes based on single-strands of rna except hbv which should focus scientific attention on rna. there are numerous questions that strike investigators as they ponder a collection of viral agents like those in table 3 .3. the viral polymerase errors in replicating single-stranded rna genomes are not corrected so the species are constantly changing. the apparent success of these viruses is that as they move from reservoir hosts to humans and as humans become immune to the initial infection, the population of diverse genomes offers multiple chances to adapt by finding a "fit" genome version which can propagate until the next transition requiring adaption. acquired immunodeficiency syndrome (aids) is caused by human immunodeficiency virus 1 (hiv-1), a retrovirus. these viruses have a single-stranded rna genome that is converted into dna, a paradigm shift in the flow of genetic information from dna to rna. the 36,000,000 human deaths caused by hiv-1 (table 3.3) is accompanied by a spectrum of clinical signs; eg. fever, diarrhea, peripheral neuropathy, pelvic inflammatory disease, cervical cancer, cytomegalovirus retinitis, kaposi's sarcoma, lymphoma, mycobacterium avium infection, recurrent pneumonia, and wasting syndrome. hiv-1 genome diversity includes base substitution, insertion, deletion, recombination, and gain or loss of glycosylation sites which all arises from the limited fidelity of the viral reverse transcriptase. these mutations are found in clusters or hypervariable regions indicating the fit virus is selected for from vast numbers of less-fit genome sequences. hiv-1 emphasizes key observations: (1) eid is a significant contemporary concern, (2) we are not prepared for the novel characteristics introduced by eid, (3) clinical signs can be diverse and often mimic symptoms of other diseases, and (4) the replication mechanisms are often error prone resulting in an array of fit viruses. dengue is a flavivirus with a positive sense single-stranded rna (+ssrna) genome carried by mosquitos to man. dengue has been a tropical disease for hundreds of years, typically a disease of young children but in 1953 an emerging severity was recognized in manila (table 3 .3), hemorrhagic fever (dhf) and dengue shock syndrome (dss). more than 2 billion people are at risk of dengue infection but only a small fraction of those infected will develop dhf or dhs. infected people develop antibodies that can lead to antibody-dependent enhancement (ade) in subsequent infections and more severe dhf or dss outcomes. it appears ade events occur in people that produce immunoglobins (iggs) with enhanced affinity to the activating fc receptor due to the igg1 subclass and lack of a fucose glycan modification of the igg (wang et al. 2017) aedes aegypti is the primary vector transmitting dengue but a new vector, aedes albopictus, now carries dengue to the southern united states. emergence of dengue in the southern united states is likely due to used tires imported from japan which provided a place for the asian tiger mosquito to live. outbreaks of dengue fever have become more numerous and more severe over the past three decades. viral "fitness" is constrained by the requirements imposed by the natural host so that it is a low probability event for a virus to move from the natural host to a human. while an insect frequently plays the role of vector carrying a virus from an animal reservoir to humans, several zoonotic viruses are transmitted by placing humans near rodents. several arenaviruses, minus sense single-stranded rna viruses, jump from their rodent natural hosts directly to humans through contact with rodent urine or saliva. notable arenaviruses are named for the hemorrhagic fever (hf) region of their zoonosis; bolivian hf is caused by machupo (macv), argentine hf is caused by junin (junv) and venezuelan hf is caused by guanarito (gotv). these south american outbreaks are caused by new world arenaviruses in contrast to lassa hf (lasv), an african or old world arenavirus. lasv is an endemic disease of west africa (table 3 .3) and can be confused with dhf or ebola infections. viruses that are transmitted by arthropod vectors are called arboviruses. in 1930, only yellow fever of six known arboviruses caused disease in humans. the discovery of arbovirus caused human disease expanded beginning in the late 1930s with western and eastern equine encephalomyelitis (weev, eeev) and st. louis encephalitis. both chickungunya and zika (table 3 .4) are arboviruses that have emerged to become global infections in the twenty-first century. zika not only became infamous for causing microcephaly in newborns of infected mothers and guillain-barre syndrome but is now sexually transmitted between humans. i worked on a therapeutic for the treatment of ebola infections from 2007 to 2013. the genome sequence recovered from each outbreak from 1976 to 2013 has been different. studies were conducted at usamriid in their bsl4 facility by expert ebola investigators. rhesus monkeys (macaca mulatta) were injected with 1000 plaque forming units (pfu) of zaire ebolavirus (zebov) kikwit into their thigh muscle. all of the untreated control monkeys died between days 8 and 10 following viral injection. we measured viral burden by quantitative polymerase chain reaction (qrt pcr) a measure of genome copies per milliliter (copies/ml) of plasma and plaque formation a measure of infectious viruses per ml (pfu/ml) plasma. we found an average of 1.7x10 8 genome copies per ml on day 8 post infection and 1.2x10 6 pfu/ml on day 8. nearly 100 genomes present for each successful virus in a nonhuvwas not observed, the dominant population of viral genome sequence did not change from one individual to another or from one time to another within the same individual. the existence of defective viral genomes may offer potential for rapid change but in a stable host, change was not rapid. when one considers the magnitude of differences in conditions as a virus jumps from a reservoir host to a human, rapid adaptability is a great advantage. a virus that kills all the hosts is not a successful virus. perhaps defective viral particles facilitate host immune responses giving them a chance to catch up to the rapidly proliferating virus. the ebola outbreak of 2014 illuminated the collateral damage that accompanies eid outbreaks. first, the first responders are local physicians and care givers are killed leaving a population lacking doctors and nurses. even physicians trained to exercise appropriate caution when interacting with patients such as dr. sheik humarr khan died of the ebola infection (bausch et al. 2014) . second, women are the main caregivers in their families and 75 percent of ebola deaths in the 2014 outbreak were women. this leaves families lacking traditional structure. third, the outbreak disrupts production, labor markets and trade resulting in scarcity and inflation of food prices. food security and nutrition are diminished which preferentially affects poor people as they spend 50 to 70% of their income on food. finally, public health measures are discontinued. before the 2014 outbreak, 97 percent of children in west africa were receiving routine vaccinations but that figure fell below 27 percent. the west africa loss of measles vaccinations was followed by measles out coronaviruses are unique in their large single-stranded rna genome (20,000 bases) and are often associated with mild disease, bronchitis and gastroenteritis. a 2003 outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in china rapidly spread to 8098 cases in 37 countries (table 3 .4). retrospective analysis of this outbreak linked zoonosis to a culinary trend in which people sought out exotic animals to eat. these nontraditional food animals appeared in street markets in urban areas bringing people close to the reservoir host triggering the outbreak. the civet cat was identified as the source of the first human cases but this is not the natural host. the natural host is the fruit bat, which infects a variety of small mammals including the civet cat. a point of intervention has been removal of the civet cat from markets but the natural bat host remains in the environment maintaining the virus for the next outbreak. a second coronavirus outbreak in 2014 of middle east respiratory syndrome coronavirus (mers cov) rapidly involved 23 countries including africa, western europe, and southeast asia. the number of cases is relatively low but the case fatality ratio is of great concern (table 3.4). the camel is reservoir host in this case but it may not be the natural host since camels often get sick as well. transition from animal-to-human transmission to efficient human-to-human transmission was observed quickly in the case of both sars-cov and mers-cov. this rapid adaptability of a highly infectious virus group should raise concern over endemic coronaviruses in companion animals since these viruses can clearly become pathogenic, can jump from animal to human and then become transmitted from human-to-human. an ongoing outbreak of yellow fever in africa (table 3 .4) is a striking reminder that eid can re-emerge into virtually naïve populations. some people recover from the acute symptoms but liver damage causes yellow skin, the reason for the name for the disease. the liver damage leads to bleeding and kidney damage and occasionally death. yellow fever is a flavivirus that originated in africa but was distributed to the world on barges and sailing ships to tropical ports. the virus arrived in the americas on slave ships. this virus interrupted the building of the panama canal which attracted the attention of walter reed, the military physician responsible for demonstrating transmission by mosquitos. an effective vaccine is used worldwide with immunity lasting over 10 years. the limitation of this vaccine like most is that populations need to be vaccinated regularly which in a world constantly changing due to political and economic drivers leads to vaccination gaps. cancer is one of the leading causes of human death but estimates are that 20 percent of all human cancers are directly caused by viruses (table 3 .5) (morales-sanchez and fuentes-panana 2014). the first tumor virus was identified by peyton rous in 1911. he took cell lysates from a chicken sarcoma which he passed through a filter known to hold back bacteria and then injected the filtered lysate into chickens. a tumor developed at the injection site. the virus is now known as rous sarcoma virus (rsv), a single-stranded rna virus. rous won a nobel prize in physiology or medicine for the discovery in 1966. rsv is a retrovirus that captured a tyrosine kinase, src gene, that triggers uncontrolled growth in infected host cells. the avian leukemia virus (alv) was discovered in denmark early in the twentieth century capable of causing disease in blood forming tissues of chickens. a strain of alv, avian myeloblastosis virus (amv) was described in 1952 that provided a convenient source of tissue for biochemical studies. during the 1930s a strain of inbred mice was found to develop leukemia between 6 and 18 months of (burkitt's lymphoma 2014) . after that he saw a second case at the jinja district hospital on the shores of lake victoria this became more than a curiosity. a review of hospital case notes confirmed the prevalence of these tumors of the jaw and they were accompanied by swellings in kidneys, ovaries, adrenal glands and liver. he assembled data from 38 patients and histological examination led to description of "lymphoma syndrome" or the "african lymphoma." burkitt received a grant in 1961 for ₤250 to visit 57 hospitals in eight african countries; uganda, kenya, tanzania, malawi, mozambique, zimbabwe, zambia, and republic of south africa to investigate african lymphoma. the tumor was found everywhere at the equator in areas where year-round temperature was above 60 °c but not in areas over 5000 feet in elevation. burkitt further determined the tumors only occurred where the annual rainfall was above 20 inches and not seen in the dry savannah of nigeria. burkitt contacted doctors in papua new guinea to discover these tumors were the most common childhood tumor in that country but only in wet coastal regions and not in the dry highland areas. burkitt's working hypothesis was that an insect transmitted virus infection was responsible for the lymphoma syndrome. he then observed adults with the lymphoma but only in individuals that moved into the "african lymphoma belt." these tumors were then observed in the united states and europe at a rate of 1-3 per million or about 100x less frequently than in africa. biopsy samples did produce viruses but none that came from insects so the working hypothesis was abandoned. a new hypothesis emerged between 1963 and 1966 in which p falciparum (a parasite causing malaria) was the causative agent for the lymphoma. it is believed that severe malaria does play a role in the development of burkitt's lymphoma. at the advice of peter clifford, burkitt administered methotrexate to treat patients which produced encouraging results. in 1961 burkitt gave a lecture at middlesex hospital, "the commonest children's cancer in tropical africa: a hitherto unrecognized syndrome." anthony epstein attended the lecture which changed his life and provides a punctuation mark in the history of science. epstein introduced himself after the lecture and the two sat down for tea. burkitt agreed to send tumor specimens to epstein and epstein received a grant from the us national institutes of health in 1963 for $45,000 which allowed him to employ two research assistants. yvonne barr was one of the research assistants that was able to propagate a virus after 26 tries with tumor specimens. the other research assistant was bert achong, an electron microscopist, who was first to identify the virus as a member of the herpes virus family. the virus bears the name epstein barr virus or ebv and is a causative agent of burkitt's lymphoma. an interesting historical note involves werner and gertrude henle at the children's hospital of philadelphia. they created antibodies to ebv infected cells and their survey of blood samples from american adults revealed over 90% to be ebv positive in 1966. they also showed that normal human lymphocytes could be made immortal by infecting them with ebv in 1967. we now know ebv silently infects children in the western world but in those infected a bit later in life become susceptible to infectious mononucleosis (the kissing disease) as adolescents. in 1972, a collaboration between george klein and the manolovs led to a discovery of a chromosome change that was specific to burkitt's lymphoma. this chromosome change was a translocation of 8:14 and rarely 8:2 or 8:22 (zech et al. 1976 ). the break points in chromosomes 2, 14, and 22 contained immunoglobin genes that become active in b lymphocytes as they produce antibodies during infection. a nobel prize in 1989 to michael bishop and harold varmus provided the significance to the break point in chromosome 8, bringing active immunoglobin genes near the oncogene c-myc. in 1970, a second cancer common in north and east africa as well as southern china was found to be ebv positive, carcinoma of the post-nasal space (nasal pharyngeal carcinoma or npc). the tumor is observed in epithelial cells not b-cells and is not geographically linked to endemic malaria regions. in this case, it appears that aerosol exposure to environmental carcinogens (possibly in preserved fish) may introduce mutations in nasal epithelial cells. the npc may be the result of silent ebv infection, carcinogen induced cellular mutations, and people carrying hla a*0207 and b*46 antigens. in 1975, several reports describing "fatal infectious mononucleosis" appeared. david purtilo described x-linked proliferative syndrome (xlp) based on an international registry of boys with fatal ebv infections (purtilo et al. 1977) . using genetic marker analysis from the international registry they narrowed xlp to a three million base pair segment on the x-chromosome. in 1998, a 384-base pair segment encoding the xlp protein of 128 amino acids was found to be lost or damaged in al xlp patients (nichols et al. 1998) . curiously, the xlp protein loss leads to a defect in nk and t lymphocytes which are necessary for recognition of ebv infected b lymphocytes. these individuals cannot make antibodies to ebv because their b cells require help from t lymphocytes. the development of potent immune suppressing drugs like cyclosporine a was linked to lymphoma after organ transplantation. two girls with acute lymphoblastic leukemia received bone marrow transplants from their hla-match brothers. the grafts were successful but they developed lymphoblastic leukemia from the transplant (male cells). these lymphomas do not present chromosomal translocation like burkitt lymphomas revealing a new path from ebv to lymphoma. the immune suppression upsets the ebv host homeostasis crippling t-cells that are required for keeping ebv under control. another situation where the virus can replicate without control is in hiv/aids patients and aids-lymphoma is yet another ebv induced casualty. ebv provides key insights into tumors associated with viral infections. first, the same virus is linked to multiple tumors in different populations including burkitt's lymphoma, hodgkin lymphoma, and nasopharyngeal carcinoma. second, tumorigenesis involves more than one mechanistic pathway but interest has focused on the latent membrane protein-1 (lmp1), ebv nuclear antigen 1 (ebna1), and bamh1-a reading frame-1 (barf1) genes. third, dissecting the association between infection with ebv and cancer has unfolded over a period of 30 years and led to a greater understanding of cancer, the immune system, and methods for immortalizing cells to study specific cell clonal populations. ebv has been a boon to scientific research while imposing a challenge to human survival. approximately 2 billion people have been infected with hbv and 360 million suffer from chronic infection. acute hbv infections are usually self-limiting associated with 5.2 million cases a year. chronic hbv infections lead to complications in 15-40 percent of cases resulting in 0.5 to 1.2 million deaths each year. hb v causes 60-80 percent of the world hcc cases which represents 5 percent of all cancers in the world. the hbx gene is considered key to oncogenesis. vaccination programs are effective in reducing mortality in infants and have reduced emerging prevalence of the disease. dietary exposure to aflatoxin enhances hbvrelated hcc (sun et al. 1999 ) as well as co-infection with hcv and excessive alcohol consumption. hepatitis b virus (hbv) was named due to differences in transmission: hepatitis a type virus follows fecal-oral transmission while b type virus is parenteral. the genome is circular dna that is not fully double-stranded with the end of the fulllength strand linked to dna polymerase. the genome is 3020-3320 nucleotides long (full length strand) and 1700-2800 nucleotides for the short strand. four genes are encoded including: (1) c the core protein (hbcag) synthesized by uorf aug to make pre-core protein, (2) p is the dna polymerase, (3) s is the surface antigen (hbsag) which has three aug start sites that divide the gene into pre-s1, pre-s2, and s, and (4) x is a gene that is not fully understood but may be a transcriptional transactivator. non-coding rna include hbv prealpha, hbv prebeta, and hbv rna encapsidation signal epsilon. there are 10 known genotypes which differ by 8 percent in sequence with distinct geographical distributions and are labeled a to j and at least 24 subtypes. type f is the most divergent form and found in central and south america, predominantly brazil. human hbv has a narrow host range infecting humans and higher primates, eg. chimpanzees. in the 1970s a woodchuck hepatitis virus (whv) was discovered which will not infect rodents. a ground squirrel form was identified (gshv) that is a distant relative of the marmot and woodchuck. indeed, dhbv infects ducks but not all species of duck, grey herons are infected with hhbv, the ross' goose with rghbv, the snow goose with sghbv, white storks with sthbv, and cranes with chbv. there are no hepadnaviruses in arthropods or insects. human t-cell lymphotropic virus (htlv-1) htlv-1 is a single-stranded rna retrovirus, defined by their use of reverse transcriptase, a polymerase, that makes a dna copy of the rna 7 kb viral genome. the dna viral genome is integrated into the host genome where it is referred to as a provirus and is replicated along with the host genome during cell division. only 1 percent of infected individuals will develop leukemia and this is observed 20 to 30 years after asymptomatic infection. the htlv-1 tax protein is likely to initiate cell transformation through interactions with transcription activators and cell cycle regulators. hepatitis c virus (hcv) hcv has infected approximately 3 percent of the world's population (~210 million) but screening of the blood supply has reduced prevalence. hcv is a flavivirus composed of a 9.4 kb single-stranded, positivesense rna. hcv is characterized by a single serotype but at least 6 major genotypes. genotype 1b is the most common genotype seen in the united states and taiwan. hcv becomes a chronic infection by evading host immune defenses through a combination of: (1) high replication rate (10 12 virions/day) and (2) lack of error proofreading by the viral polymerase leading to mutations in response to immune pressure. the genetic variability of hcv has limited efforts to design an effective vaccine. the polyomaviruses are small (3.4 kbp), double-stranded dna viruses. early studies with simian virus 40 (sv40) led to identification of the large tumor antigen (large t; lt). lt is also found in bk and jc viruses which are more suspect human tumor viruses and merkel cell polyomavirus (mcv) which is now well established as a human tumor virus (feng et al. 2008) . the n-terminus of lt contains an lxcxe motif that interacts with the retinoblastoma protein rb while the c-terminus contains an atpase/dna helicase domain that can inactivate p53. the ls-p53 complex activates insulin-like growth factor i (igf-i) which alone is capable of cell transformation. a recent report of morbidity and mortality reveals heart disease and cardiovascular events are the number one killer with neoplasia and infections following close behind. however, infections frequently cause neoplasia and cardiovascular disease leading to death. if we combine cardiovascular events and neoplasia caused by infection, then infectious disease is the most significant threat to human life and qualifies as the area of greatest impact. the picornavirus family are small (pico-), single-stranded, positive sense rna genome (7.4 kb) viruses that synthesize a single polyprotein that is cut into a small collection of functional proteins by virally encoded (2a and 3c) and cellular proteases. poliovirus is a picornavirus that has served as the prototype for the viral family. the enteroviruses are a group of picornaviruses that have been associated with cardiac disease. coxsackievirus b (cvb), coxackievirus a (cva), and echovirus infections lead to cardiac signs 3.2% of the time. about one-third of patients with acute cardiac disease (inflammation of the heart) are antibody positive for enteroviruses. while acute myocarditis is often self-limiting, chronic cardiac disease often leads to dilated cardiomyopathy (dcm) which is present with no heart inflammation. dcm is associated with heart failure which can be lethal (table 3 .6). these chronic infections lead to 50 percent of all cardiac transplants worldwide. the enteroviral 2a protease can also degrade dystrophin in the heart leading to cardiac necrosis, reduced ejection fraction, and then to dilated cardiomyopathy. transmission of these viruses is from contaminated food and water. the human herpesviruses are large double-stranded dna genome viruses. the human herpesvirus-5 (hhv5) or cytomegalovirus (cmv) has a 235 kbp genome encoding over two hundred genes. one problem in finding associations with cmv infections is that it is not an emerging disease, 50 to 99% of the population has been infected making comparisons to a control group challenging. most infections are observed in children and in newborns serious clinical findings can be observed. hence, most adults carry latent infections that are reactivated when individuals become immune suppressed following solid organ transplantation, malignant hematological disease, and aids. reactivation in immune suppressed individuals is associated with increased mortality. the association with cardiovascular disease has been demonstrated in two recent studies. in one, cmv infections were detected in 14.5 percent of coronary artery samples from bypass operations compared to 4 percent in of patients who needed cardiac surgery for reasons other than atherosclerosis hebar et al. 2015) . in the other, cmv reactivation (viremia) was detected in 16.5% of immunocompetent patients admitted for major heart surgery (roa et al. 2015) . the incidence of coronary artery disease is the major contributor to death from heart disease and if 14 to 16.5% of this disease is associated with cmv infections, this infection is a significant human health hazard. recognizing an emerging infectious disease involves well established strategies of surveillance; (1) identify unusual clusters of disease, (2) evaluate the spread of an outbreak, (3) estimate the magnitude of the problem, and (4) if possible identify the atheroscleotic plaque infectious agent. the strategy has proven valuable for known infectious and noninfectious diseases but has limited capacity to detect emerging infectious diseases. however, deviation from the traditional approach to surveillance is not likely to gain support. al smith, a veterinary virologist, approached me after a seminar i presented in 1997 in the college of veterinary medicine at oregon state university. i had just joined avi biopharma as the head of their research and development program. al was a veterinarian and professor and had devoted his career to the caliciviridae family of viruses dating back to his time in the naval research station in california. he had isolated the nonhuman vesivirus group of caliciviruses not only from suffering sea lions in the channel islands, but from reptiles in the san diego zoo and whales held in captivity. al and his capable technician, doug skilling successfully propagated the virus isolates in cell culture, an accomplishment not shared by other laboratories at the time. al developed nucleic acid probes and induced antibodies to these viral isolates. over decades of research he assembled an extensive collection of vesiviruses which were held in redundant −70° freezers. his singular vesivirus focus gave the appearance of a zealot but his ability to cultivate viral isolates, his extensive collection of isolates, and his one of a kind detection reagents made him a one of a kind virologist. al was well beyond retirement age and was an "old school" virologist ready at a moment's notice to take his bag into the real world and collect swabs from ailing animals. he maintained careful records of the condition of his patients, their location, and the setting of the animal in the community. every field trip led to work in the lab propagating virus from his swabs. al wanted to know if i would be interested in finding an antiviral agent for these viruses. my prime directive at avi biopharma was to explore the capabilities of our proprietary antisense technology and i had yet to investigate targeting a virus. the caliciviruses have positive sense single-stranded rna genomes which express three genes from a single polyprotein. we identified an active agent following an investigation with small collection of candidates (stein et al. 2001) . the vesivirus group of caliciviruses are considered animal only and are not believed to infect humans but al began exploring human samples with his collection of detection reagents. after several years making incremental progress, we found an association between human blood samples seropositive for vesivirus and markers of liver disease (smith et al. 2006) . we felt this evidence of an emerging infectious disease in humans and its potential to cause liver disease would be welcomed by the medical community. al and i made a trip to washington dc at our own expense to relate the findings in person. we meet with virologists at the national institutes of health but were met with judicious skepticism. harvey alter had been instrumental in the discovery of hepatitis c virus (hcv) and felt all viral liver disease is already accounted for by hepatitis viruses. hence, no interest in our findings. we met with the american red cross blood banking group in shady grove maryland and were met with concern. blood supplies are scrutinized by the nucleic acid test (nat) to eliminate viral contamination and any further elimination of blood samples would threaten an already limited inventory. our findings simply add complication to the blood supply finding an emerging infectious disease business. they asked for more compelling and more extensive data to add robustness to our claims. the only problem is that they were not interested in providing financial support for the recommended studies. the conundrum is all too common, you need more data to convince granting agencies to support the work but there is no support to add to the limited data. life on the cutting edge is frequently discouraging. our strategy took two paths: one to seek commercial support and the other to create proof of concept data for an antiviral solution. the most logical commercial solution was to meet with michael houghton at chiron. he was the driving scientific force in finding hcv and chiron might like adding to their reputation by finding another previously unrecognized hepatitis virus. indeed, dr. houghton invited us to bay area to discuss the project. he reviewed our data and the quality of al's detection reagents. chiron provided modest support and their in-house laboratory help to confirm our observations, a glimmer of hope. al created a company, calicitech, so that he could accept support and license his reagent patents from the university. testing an antiviral agent in humans would be expensive but in the absence of natural history of the infection would make human proof of concept impossible. however, al had been contacted by a cat rescue facility in atlanta, mommy cat, describing an outbreak of feline calicivirus (fecv) in their cattery. these cats had all been vaccinated with an approved fecv vaccine which failed to protect these cats. a veterinarian can decide to use alternative medicines if there are no alternatives and the owner oc the cats can sign the equivalent of informed consent. we provided our fecv specific antiviral to mommy cat along with a detailed protocol for treatment. the infected kittens we treated survived while those not treated died providing encouraging data. we then discovered a similar outbreak in the humane society facility in eugene oregon, our neighbors. again, we reached an agreement to treat infected kittens and were successful ). this was the first time we treated an infection based on symptoms in an "outbread" population of cats. with over 100 kitten patients and remarkable survival differences between treated and untreated, we had our proof of concept. there is no disease cluster to link to human vesivirus. norovirus is a calicivirus and is known to infect humans, in fact it is well known on cruise ships, day care centers, and extended care retirement facilities. our efforts to have human vesivirus recognized as an emerging infectious disease have failed. chiron was not impressed with our antiviral and no longer were interested in evaluating the detection reagents. if we are lucky, human vesivirus infections will remain mild clinical oddities. al smith has an interesting way of responding, "that virus is out there infecting humans. it won't go away." the existence of non-pathogenic viral infections led to the emergence of the study of the immune system, vaccination, gene therapy, and concern for future pandemics. we carry evidence of ancient retroviral infections in our genome from integration events that became vertically transmitted making up as much as 8 percent of the human genome (meyer et al. 2017) . these genomic fossils are called endogenous retroviral genomes (erv). most erv sequences accumulate sufficient mutations over evolutionary time that horizontal transmission is unlikely. these viral genome segments are trapped but can still be transcribed and encode some viral proteins. the significance of these integrated genomes is a current topic of investigation. adeno-associated virus (aav) is a single stranded dna virus that infects humans but are not known to cause disease. the lack of pathology has led to their use as viral vectors for human gene therapy. aav can infect dividing and quiescent cells and will persist extra chromosomally without integrating into the genome of the host cell. aav is a member of the parvoviridae family in the genus dependoparvovirus. is an arenavirus closely related to lassa virus and shares reservoir hosts. mv is naturally attenuated and nonpathogenic in humans. infection of humans with mv protects against lethal challenge with lv (wulff et al. 1977) . flaviviridae) named after g. barker, a surgeon, first identified in 1995. hgv infects one sixth of the world's population but does not cause human disease. a metaanalysis of 15 publications investigating gbv-c infections in hiv-positive individuals indicate coinfection with gbv-c slows the progression of hiv disease in individuals that have been seropositive for 2 years or more (zhang et al. 2006a) . two of the studies investigated gbv-c 5 years after documented hiv seroconversion estimate the hazard ratio of 0.36 (95% ci). is a retrovirus first identified in 1971 from a lymphoblastoid cell culture from a kenyan patient with nasopharyngeal carcinoma. hfv is homologous to primate foamy viruses and is most closely related to the chimpanzee foamy virus (sfvcpz). early studies raised alarm for association with autoimmune diseases but more extensive studies with more precise diagnostic reagents fail to find a disease associated with hfv. hfv is a rare human infection and concerns parallel sfv infections in humans. simian foamy virus (sfv; spumavirus-retroviridae) is a retrovirus infecting most primates born in captivity and people making contact with infected primates can become infected. human infections frequently occur in males probably requiring a bite from infected nonhuman primates but are harmless. the infected cells often fuse to form syncytia of giant foamy cells, which gives the virus its name. the error rate in sfv genomes is exceptionally low, 1.7 x 10 −8 substitutions per site per year, compared to hiv 10 −3 substitutions per site per year. since so-called crossspecies, infections have only been observed for a little over a decade the long term consequences are not known. these infections are watch and wait for everything from a zoonotic epidemic to identified disease clusters. perhaps this is exactly the sort of infection that will emerge as a significant human concern in the future. torque teno virus (ttv; alphatorquevirus) is a single-stranded, positive sense dna genome virus about 3.8 kb in size in the anelloviridae family. nearly 100 percent of even healthy individuals are infected in some countries. the virus was discovered in 1997 as the "transfusion transmitted virus (ttv)" in a japanese patient. it is often found in patients with liver disease but does not cause hepatitis on its own. closely related torque teno mini virus (ttmv) were isolated in 2007 and found to have smaller genomes of 2.8-2.9 kb. ttmv infections are also common but do not cause any described human disease. human adenovirus type 5 (rad5) is used to create an ebolavirus (ebov) vaccine encoding zaire ebolavirus glycoprotein failed to protect animals immune to ad5. a replication defective chimpanzee-derived adenovirus (chad3-ebo-z) provided protection against lethal ebov challenge in macaques but protection wane over several months. they boosted with a modified vaccinia ankara (mva-bn-filo) that led to durable protection (stanley et al. 2014 ). this vaccine progressed through phase i, single-blind, randomized human trials in mali between 2014 and 2015. a single dose of the chad3-evov-z is efficient as a prime vaccine strategy followed by mva-bn-filo as a boost was well tolerated in humans tapia et al. 2016) . is a 5229 base double-stranded dna virus infecting less than 5 percent of the human population. wu, named after washington university, is found as a co-infection in various respiratory infections but wu does not cause disease on its own. wu is closely related to ki virus that also is not known to cause clinical disease. however, related polyomaviruses that are clinically relevant include bk virus associated with nephropathy, jc virus associated with progressive multifocal leukoencephalopathy, sv40 virus associated with mesothelioma, and merkel cell polyomavirus associated with cancer. vaccinia virus (orthopoxvirus) is a large double stranded dna virus closely related to smallpox. edward jenner, the father of immunology, found the milkmaids exposed to cowpox (vaccinia) were immune to smallpox in 1798. this was the first vaccine (named after vaccinia) leading to the modern vaccine that has allowed for the eradication of smallpox. viral sequences are constantly mutating with no purpose other than seeking a survival/infectivity benefit. this means viruses with no current pathology represent a pre-mutation reservoir for the next catastrophic human pandemic. the popularity of rnaseq is likely to expand our catalog of nonpathogenic viral infections. however, the management of such information is in question. technology used to counter viral infections has resulted in over 90 approved drugs for the treatment of nine different human viral infections in just 50 years (de clercq and li 2016) . several different antiviral drug groupings have been reported, but the following arise from review of the mechanisms of action of antiviral drugs assembled in table 3 .7: (1) inhibition of viral attachment and entry, (2) inhibition of viral uncoating, (3) viral polymerase inhibitors, nucleotide analogues (ntrti) and non-nucleotide reverse transcriptase inhibitors (nnrti) and dna polymerase inhibitors, nucleic acid synthesis inhibitors and nucleotide pool size agents, (4) latency reversal agents, (5) integrase inhibitors, (6) protease inhibitors for both hiv and hcv, (7) neuraminidase inhibitors, (8) immune response modifiers, and (9) antisense inhibitors. administration of hyperimmune sera from immunized animals or human donors was the first effective treatment for infectious diseases. the practice has limitations but is still used to treat bacterial toxins and viral infections caused by cmv, rsv, hav, hbv, rabv, vzv and mev (keller and stiehm 2000) . the development of human or humanized monoclonal antibodies (humabs) has created a feasible way to rapidly generate novel antiviral therapeutics. humabs have advantages over serum therapy in that they are chemically defined reagents with minimal variability, greater activity per mass of protein, and they have no immunological consequences related to serum sickness. several mabs have been approved for treatment of infectious diseases including viral and bacterial pathogens (table 3 .8). the antiviral mab discovery field is exploding with activity particularly for hiv and hcv infections. a humanized mab targeting lymphocyte ccr5 receptors called pro140 has demonstrated potent and prolonged anti-hiv-1 activity and a large margin of safety (jacobson et al. 2010a) . administration of pro 140 by the subcutaneous route offers patients a way to self-administer the mab but importantly the mab is transported in the lymphatics providing enhanced access to binding to the cellular target (jacobson et al. 2010b) . the next generation of antiviral therapeutics are likely to be dominated by mabs. perhaps the only way to clear a viral infection involves a host immune response (table 3 .9). the innate immune response is particularly effective centered on a type 1-interferon pathway. unfortunately, many viruses carry mechanisms to evade host innate responses and innate immune effectors do not have the capacity for memory. the adaptive immune response can mitigate infection with antibodies, generally to surface antigens, which prevent the spread of the virus and t-lymphocytes, which can clear virus-infected cells. the first strategy will be to use an existing drug designed for another virus offlabel. this seems likely for antiviral drugs like cidofovir, foscarnet, and ganciclovir particularly for double-stranded dna viruses like ebv, hpv, and hhv6. ribaviran has been used for a number of single-stranded rna viruses including polio, junin, and lassa fever. secondary strategies will require investment of time and effort beginning with vaccine development and creation of monoclonal antibodies. platform technology rna-based therapeutics offer rational design for an expansive number of new antiviral strategies. this advantage is superimposed on the theoretical advantages of selectivity, specificity and affinity provided by watson-crick base pairing. rna-based therapeutics are expected to provide a substantially more narrow range of pharmacokinetic properties and toxicities thus are easier to compare to each other and ultimately combine into multi-agent cocktails. however, the mechanism of action may vary from rnase h or risc mediated degradation of the targeted rna or steric inhibition of rna function. the objectives of our antiviral program have been to exploit the broad understanding of rna-based therapeutics for antiviral activity with a common chemical type, the phosphorodiamidate morpholino oliogmer (pmo) and their enhanced derivatives. in this way the mechanism of action is common to all agents, which is steric blockade of rna function. studies reported by zamecnik and stevenson introduced the first approach to identification of an antisense antiviral agent stephenson and zamecnik 1978) . they used a 13-mer targeted to rous sarcoma virus. since the rous sarcoma virus pioneering efforts, antiviral rna based therapeutics have involved multiple oligomer chemistries with a variety of different mechanisms of action. chemical approaches to oligomers directed to hiv have been plentiful. a nonionic methylphosphonate oligonucleotide targeted to the splice acceptor site of hiv tat inhibited splicing of viral rna inhibiting syncytia formation and p24 synthesis at 3um concentration. poor aqueous solubility limited the utility of the methylphosphonate chemistry. phosphoramidate chemistry was investigated for inhibition of the splice-donor and splice-acceptor of hiv tat agrawal et al. 1988 ) and were more potent but these agents were cytotoxic and poorly water soluble. phosphorothioate chemistry targeting hiv-rev (matsukura et al. 1987 ) and hiv-tat were shown to be effective in inhibiting hiv replication, were not cytotoxic and were very soluble. further, the hiv-rev phosphorothioate oligodeoxynucleotide was stable in vivo with an acceptable pharmacokinetic profile (iversen et al. 1994) . a 25-mer phoshorothioate called gem91 targeting the initiation site of hiv-gag was evaluated in clinical trials (agrawal 1998 ) but the trials were discontinued. i focused on phosphorodiamidate morpholino oligomer chemistry which is both stable and net-neutral in charge at physiological ph. single stranded rna viruses with positive sense (ssrna(+)) these viruses are the most simple in terms of genome size, number of potential translated viral proteins, their genomes are all linear and they enter the cell ready for translation. the design of steric blocking rna-based therapeutics involves preventing translation, disrupting rna secondary structure and masking recognition sites for rna dependent rna polymerase (rdrp). the targeting of either the 5′-terminus and the first orf-aug are active. further, efficacy in animal challenge studies is observed with high fidelity when the most effective agent identified in vitro is employed. the family astroviridae with six different human astroviruses (huastv) responsible for 2-17% of all gastroenteritis. we found the 5′-terminus 1 to be the most effective site to target. this was also the optimal site for caliciviridae including vesivirus (vev; martin-alonso et al. 2005; stein et al. 2001), norovirus (nov; bok et al. 2008) , and feline calicivirus (fcv; smith et al. 2008) ; the flaviviridae including dengue (den; kinney et al. 2005; holden et al. 2006) , and west nile virus (wnv; deas et al. 2007; zhang et al. 2008) , arteriviridae including agriculturally important equine arterivirus (eav; van den born et al. 2005) , and porcine respiratory and reproductive virus (prrsv; zhang et al. 2006a, b) ; and the togaviridae exemplified by venezuelan equine encephalitis virus (veev; paessler et al. 2008 ). the family coronaviridae revealed a new active target site in the transcription regulatory sequence (5'-cgaac-3′) in both mouse hepatitis virus (mhv; neuman et al. 2004 ) and the severe acute respiratory syndrome virus (sars; neuman et al. 2005) . the picornaviridae active target site involved a highly conserved sequence in the internal ribosomal entry site (ires) in polio virus (pv; stone et al. 2008) , foot and mouth disease virus (fmdv; vagnozzi et al. 2007) , and the coxsackievirus (cvb3; yuan et al. 2006) . single stranded rna viruses negative sense (ssrna(−)) these viruses are generally more complex with respect to genome size, number of potential translated viral proteins, and multiple genome segments. the genome must be replicated prior to translation of viral proteins. the design of steric blocking rna-based therapeutics is similar to the positive sense rna genomes in that targets involve preventing translation and masking recognition sites for rna dependent rna polymerase (rdrp). we investigated 11 different targets in measles virus (mev), a member of the paramyxoviridae, finding the translation initiation start site of n the optimal target (sleeman et al. 2009 ). studies with the human respiratory syncytial virus (hrsv) found the translation site for l to be most active (lai et al. 2008) . the orthomyxoviridae studies investigated influenza a virus probing each of the 8 viral segments finding translation of pb1 active as well as the 5'terminal of vnp for h3n2 (ge et al. 2006 ) and h3n8 (lupfer et al. 2008 ) but a combination of targets was required in animal studies with a high pathogenic viral h7n7 isolate (gabriel et al. 2008 ). more extensive influenza a studies revealed a new target, the m-segment splice donor site. this target was evaluated in phase i clinical trials. the antisense platform technology has limitations in targeting viral sequences. inhibiting virally encoded proteins or blocking viral replication by interfering with the polymerase does not always work. the arenaviridae family proved difficult. we experienced some success with junin and lymphochoriomeningitis virus (lcmv) in cell culture observing 4 log 10 reductions in viral titer with a pmo targeting the highly conserved viral genome terminus. however, we failed to provide survival benefit to guinea pigs challenged with either junin or lassa virus. we then tried the mouse challenged with lcmv and failed again but we observed hemorrhagic disease in the mouse, a severe consequence of infection that mimics the worst aspects of arenaviral infection. we decided to try targeting host genes to mitigate disease in the mouse and found targeting il-17 provided a survival benefit (schnell et al. 2012) . the success in the face of failure puts targeting host genes at the center of attention for dealing with emerging infectious diseases. the antisense platform technology represents an excellent approach to rapid drug discovery for emerging infectious disease. rapid discovery demonstrated repeatedly but of course, the cost of advanced development is daunting. the application is best for immediate treatment of index cases, close contacts, and healthcare workers. we have an 800-pound gorilla in the room and everyone in the room considers it someone else's problem. the current course is to ignore the majestic creature until it starts tearing limbs from people and then the consensus is to kill the gorilla. agencies like the world health organization (who), the centers for disease control (cdc) and the national institutes of health (nih) can assemble highly skilled personnel and can confer with some of the greatest minds in the world. unfortunately, they are all aware of the potential problem that an emerging pandemic is likely to take us by surprise. significant speculation that a single-stranded rna virus will emerge killing tens of million people, costing hundreds of billions of dollars, and changing the course of human history. there is a high probability that the virus will be influenza a (h5n1 or h7n9) that will jump from a reservoir population of birds and establish human-to-human transmission. the pandemic will be a global event by the time an effective vaccine is available. neuraminidase (na) inhibitors as therapeutic and prophylactic agents in the setting of pandemic influenza a (flua) were called in to doubt in the past decade (michiels et al. 2013; jefferson et al. 2014) . indeed, 100% of isolates in the 2008/2009 a/h1n1 pandemic were found to be resistant to adamantanes, and resistance to oseltamivir (tamiflu; osl) was observed in virus recovered from individuals taking osl therapeutically or prophylactically (dharan et al. 2008; cdc mmwr 2009) , while effect on duration of shedding was not impacted. a recent outbreak of an influenza a (h7n9) virus caused 137 cases and 45 deaths in china revealed a novel na mutation r292k resulting in high level resistance to osl (wang et al. 2014) . therapeutic options for treatment of individuals with complicated influenza a are severely limited, perhaps no options. pandemic strains such as a/h1n1pdm09 carried significant morbidity and mortality, particularly in those who had not experienced h1-strain influenza in their lifetime. we are also witnessing more rapidly emerging highly pathogenic avian influenza strains that are resulting in human infection; some such as a/h5n1 and a/h7n9 appear to becoming more efficient in person-to-person transmission, and reports suggest osl resistance develops during treatment (de jong et al. 2005; lam et al. 2015) . we are not ready for an outbreak of avian flu or any other emerging single-stranded rna virus. why not? an estimate for the time and cost to develop a new drug is 10 years and $1 billion. the commercial use of a drug for an emerging infection is hard to estimate since by definition when you start development the infection has not emerged. most of us would be unlikely to use our retirement savings to invest in a drug development project with no reliable way to expect a return on our investment. it is a poor business model. when you consider there may be hundreds of emerging infectious diseases each times 10 years and $1 billion each the task is daunting. on which disease should we focus? consider the ebola drug avi-7537. the ebola therapeutic project began in 2004 following a laboratory accident at usamriid. we identified three compounds each with activity and when combined we observed unprecedented efficacy in a lethal challenge primate animal model (warfield et al. 2006 ). hundreds of experiments over the next 3 years optimized these agents (swenson et al. 2009 ). we were able to obtain research grants from the transformational medical technologies (tmt) division of the defense threat reduction agency (dtra) within the department of defense (dod) and we completed proof of efficacy studies (warren et al. 2010) . this led to submission of an investigational new drug application (ind) to the food and drug agency (fda) and phase i safety and tolerability studies were conducted i healthy human volunteers (heald et al. 2014) . after 11 years of continuous effort, we completed key aspects related to the fda approval process under the "animal rule" and we streamlined our treatment to a single agent, avi-7537 (warren et al. 2015) . however, shortly before the ebola outbreak in western africa, the us government "budget sequester" cancelled our project and the most advanced therapeutic on the planet was not deployed to treat those infected during the outbreak. as the outbreak continued, we found no viral resistance of avi-7537 unlike the monoclonal antibody therapy in use (khiabanian et al. 2014) . avi-7537 sits on a shelf, a political casualty and an unfavorable business model. the global virosphere may contain up to 10 31 virus/virus-like particles (suttle 2005) , the greatest reservoir of genetic diversity. the earth's atmosphere transports viruses all over the planet. viruses are found in soil at 1.5 x 10 8 to 6.4 x 10 8 particles per gram of dry soil (kimura et al. 2008) . the surface oceans carry approximately ten million viral particles in each milliliter of seawater, most of which are bacteriophages (phage). the small viral particles are easily carried into the upper environmental viral reservoirs atmosphere by up drafting winds. bacteria are deposited from the atmosphere at a rate of 0.3 to 8 x 10 7 per meter each day and viral deposition rates are 9-461 times greater (reche et al. 2018) . these phages influence bacterial lifecycles and play a role in natural energy and nutrient cycles fundamental to life on earth. the dynamics of phage-bacterial evolution drive changes in photosynthesis, phosphate, and nitrogen balance (breitbart 2012) . human accidental release of radioactive waste (discussed in chap. 2) and disposal of chemicals including potent antiviral and antibacterial compounds (discussed in chap. 7) may alter the eco-evolutionary dynamics producing unanticipated environmental consequences. the objective of this chapter has been to provide convincing evidence that infectious disease is the most significant threat to human health. the focus has been on viral infections because they rely on host ribosomes to produce their proteins, recent emerging infections have been from single-stranded rna genome viruses, and replication of rna viruses is error prone. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but have never prepared for these inevitable catastrophies. the long-term consequences often remain unrecognized and are always inconvenient such as cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. infectious disease will certainly continue to pose the most significant threat to human health in the age to cell phones, artificial intelligence, and global commerce. rapid replication of viral genomes combined with low fidelity polymerases provide the foundation for an unending source of new emerging infectious agents. these traits also make viral genomes sensitive to environmental contaminants in a way that may expand probabilities for zoonosis. infectious disease as part of our environment is not appreciated. the study of infectious disease is not a part of the curricula of students in environmental science/management. textbooks in in environmental studies do not include chapters in infectious disease. the integration of research at superfund sites focused on chemical contamination with infection and zoonosis would result in valuable insights into threat analysis. viruses with rna genomes lack sequence proofreading quality control during replication. the cumulative mutations in their genomes limits the genome size to under 30,000 bases. essentially, a larger genome would evolve out of existence, so called catastrophe evolution. the limited genome size makes these viruses exceptionally resilient to a changing environment. the virus must economize by combining functions. this means evolution and resilience are the same thing in the rna genome viruses. a unique insight is that in human evolution is restricted to the dna genome and resilience limited to rna, as it is in the rna genome viruses. antisense oligonucleotide-based therapy for hiv-1 infection from laboratory to clinical trials oligodeoxynucleotide phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus a tribute to sheik humarr khan and all the healthcare workers in west africa who have sacrificed in the fight against ebola virus disease: mae we hush inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of the genome marine viruses: truth or dare cancer virus oseltamivir-resistant 2009 pandemic influenza a (h1n1) virus infection in two summer campers receiving prophylaxis-north carolina approved antiviral drugs of the past 50 years the eradicatio of smallpox: edward jenner and the first and only eradication of a human infectious disease oseltamivir resistance during treatment of influenza a (h5n1) infection in vitro resistance and in vivo efficacy of antisense oligomer against west nile virus outbreak of antiviral drug-resistant influenza a in long-term care facility the perpetual challenge of infectious disease clonal integration of a polyomavirus in human merkel cell carcinoma morpholino oligomers targeting the pb1 and np genes enhance survival of mice infected with highly pathogenic influenza a h7n7 virus inhibition of multiple subtypes of influenza a virus in cell cultures with morpholino oligomers safety and pharmacokinetic profiles of phosphorodiamidate morpholino oligomers with activity against ebola virus and marburg virus: results of two single ascending dose studies cytomegalovirus infection and atherosclerosis in candidate of coronary artery bypass graft inhibition of dengue virus translation and rna synthesis by a morpholino oligomer targeted to the terminal 3′ stem-loop structure pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in the adult male rat following single injections and continuous infusion phase 2a study of the ccr5 monoclonal antibody pro 140 administered intravenously to hiv-infected adults anti-hiv-1 activity of weekly or biweekly treatment with subcutaneous pro 140, a ccr5 monoclonal antibody oseltamivir for influenza in adults and children: systemic review of clinical study reports and summary of regulatory comments passive immunity in prevention and treatment of infectious diseases viral diversity and clonal evolution from unphased genomic data ecology of viruses in soils: past, present and future perspectives inhibition of dengue virus serotypes 1 to 4 in cell culture with morpholino oligomers inhibition of respiratory syncitial virus infections in cell cultures and in mice with morpholino oligomers dissemination, divergence and establishment of h7n9 influenza viruses in china health impact of globalization: towards global governance inhibition of influenza a h3n8 virus infections in mice by morpholino oligomers isolation and characterization of a new vesivirus from rabbits phosphorothioate analogs of oligodeoxyribonucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus endogenous retroviruses: with us and against us the value of neuraminidase inhibitors for the prevention and treatment of seasonal influenza: a systematic review of systematic reviews antisense morpholino oligomers directed against the 5'-end of the genome inhibit coronavirus proliferation and growth inhibition, escape and attenuation of sars coronavirus treated with antisense morpholino oligomers inactivating mutations in an sh2 domain-encoding gene in x-linked lymphoproliferative syndrome inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oliogmers immunological disorders and malignancies in five young brothers spillover animal infections and the next human pandemic deposition rates of viruses and bacteria above the atmospheric boundary layer a prospective monitoring study of cytomegalovirus infection in nonimmunosuppressed critical heart surgery patients inhibition of acquired immunodeficiency syndrome virus by oligonucleotide methylphosphonates lymphocytic choriomeningitis virus infection in fvb mouse produces hemorrhagic disease ancient athenian plague proves to be typhoid inhibition of measles virus infection in cell cultures by peptide-conjugated morpholino oligomers vesivirus viremia and seroprevalence in humans virus specific antiviral therapy for controlling severe and fatal outbreaks of feline calicivirus infection chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge inhibition of vesivirus infetions in mammalian tissue culture with antisense morpholino oligomers inhibition of rous sarcoma viral rna translation by a specific oligodeoxynucleotide inhibition of multiple species of picornavirus using a morpholino oligomer targeting highly conserved ires sequence increased risk of hepatocellular carcinoma in male hepatiis b surface antigen carriers with chronic hepatitis who have detectable aflatoxin metabolite m1 viruses in the sea chemical modifications to phosphorodiamidate morpholino oligomer antisense molecules targeting vp24 modify their efficacy against ebola virus infection use of chad3-ebo-z ebola virus vaccine in malian adults with mva-bn-filo: a phase i, single-blind, randomized trial, a phase 1b, open label and double blind, doseescallation trial, and a nested, randomized, double-blind, placebo-controlled trial inhibition of foot-and-mouth disease virus in cell cultures with antisense morpholino oligomers antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture pcr for detection of oseltamivir resistance mutation in influenza a(h7n9) virus igg antibodies to dengue enhanced for fcγriiia binding determine disease severity gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers advanced antisense therapies for postexposure protection against lethal filovirus infections single component avi-7537 antisense compound provides greater protection than double component avi-6002 against lethal ebola virus infection in rhesus monkeys isolation of an arenavirus closely related to lassa virus from mastomys natalensis in south-east inhibition of coxsackievirus b3 in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosomal entry site inhibition of rous sarcoma virus replication and transformation by a specific oligodeoxynucleotide characteristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with burkitt and non-burkitt lymphomas effect of early and late gb virus c viremia on survival of hiv-infected individuals: a meta-analysis suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers west nile virus genome cyclization and rna replication require two pairs of long-distance rna interactions key: cord-009269-6fs0f4b7 authors: youde, jeremy title: is universal access to antiretroviral drugs an emerging international norm? date: 2008-12-12 journal: j int relat dev (ljubl) doi: 10.1057/jird.2008.10 sha: doc_id: 9269 cord_uid: 6fs0f4b7 the international community appears to have embraced a new norm — that of universal access to antiretroviral drugs. the process by which this norm has found acceptance raises interesting questions about how norm entrepreneurs frame their arguments, the role of non-state actors in realizing a norm, and the importance of existent complementary norms. to understand the success of the norm of universal antiretroviral access, i examine the failure of an earlier health-related norm — that of universal primary health care. the campaign for universal antiretroviral access points to a need for a more nuanced understanding of norm evolution within the international community and a more holistic vision of which actors can facilitate the realization of a norm. most visible by the 3 â 5 (to provide 3 million hiv-positive persons in the developing world with arv access by the end of 2005) and all by 2010 (to provide universal access by the end of 2010) campaigns, promotes providing access to these drugs regardless of ability to pay or country of residence. according to the precepts of this new norm, those infected with hiv in developing countries will have access to similar arvs as are available to people in developed countries. in 2001, only 240,000 people in low-and middle-income countries had access to arvs. by 2005, the number had jumped fivefold to 1.3 million. in addition, 21 low-and middle-income countries offered arvs to at least half of their citizens in need (unaids 2006: 151) . all regions of the world have seen dramatic increases in arv availability. because they see it as the right thing to do, national governments, donor states, international organizations, nongovernmental organizations, private philanthropic organizations, and multinational corporations have come together in a coalition to further expand arv access to those individuals who do not have the ability to pay for these drugs. the emergence and apparent adoption of a new norm is, in and of itself, a remarkable event. even more remarkably, this new norm emerged less than 30 years after an earlier attempt to promote universal health care for all failed to take hold within the international community. universal arv access is perhaps the most ambitious global public health campaign undertaken since the successful quest to eradicate smallpox. achieving this target will require international cooperation, vastly increased levels of financial assistance from developed countries, the active participation of international pharmaceutical companies, and rethinking and reapplying international intellectual property rights. perhaps more importantly, this new programme will require the international community to embrace a new norm that places the right to health and health care above concerns about the ability to pay and the sovereign right of states to manage their national health programmes. in their ambition, promoters set explicit deadlines for realizing this new norm's behavioural expectation. even though the 3 â 5 campaign failed to meet its target, the international community has remained energized around this idea of universal arv access and continues to move towards it -though its strategies for doing so are different from those generally recognized by the literature on norm evolution and acceptance. how has this new norm emerged and why has it emerged now? these two questions raise provocative and important concerns about the nature of norm entrepreneurs, the life cycle of norms in the international arena, and how norms evolve over time. in this case, universal arv access' norm entrepreneurs framed their campaign as an issue of individual human rights (an already existent and resonant norm) instead of as a collective public good (as the earlier promoters of universal health care for all did). reframing the norm allowed it to achieve greater success. most research on norm adoption and evolution focuses largely on the role of state actors and nongovernmental organizations, often presenting norms in relatively discrete terms. the case of universal arv access demonstrates the importance of actors who fall outside both traditional state structures and mass social movements. it also shows how norms adapt to changing international contexts to resonate with existing ideas. examining the case of universal arv access, we gain a nuanced view of norm adoption and internalization, a better appreciation for the range of actors important for promoting a norm, and an understanding of the importance of complementary international norms for successful norm adoption. to explain why the norm of universal arv access has emerged when earlier health-related norms failed, i begin by reviewing the literature on how norms evolve and take root within the international community. i then turn my attention to a case of a failed norm: universal primary health care, most prominently embodied in the 1978 alma-ata declaration and its attendant health for all by 2000 campaign. the third section focuses explicitly on universal arv access: where it came from, how entrepreneurs promoted it, and where we see evidence of its acceptance. i next look at the factors that allowed universal arv access to take root within the international community despite previous health-related norm failures. finally, i tie the specifics of universal arv access' history to our broader understanding of norm evolution, showing how it illustrates the need to reconsider the factors that promote the acceptance of international norms. finnemore defines norms as 'shared expectations about appropriate behaviour held by a community of actors ' (1996: 22) . a norm spells out how members of a group believe each other should act. it may or may not be explicitly codified, but members of a community understand the standards expected by the norm and hold each other accountable for conducting themselves in a manner consistent with it. it both constrains and enables action by defining the boundaries of acceptable behaviour (klotz 1995: 25-7) . for example, the norm of sovereignty posits that one state does not have the right to interfere or intervene in the affairs of another state. states share an understanding that following the norm of sovereignty is appropriate behaviour for members of the international community, and those who violate the norm face possible sanctioning. such behavioural expectations are not always formalized by international law or treaties, though they may eventually be; rather, they operate most prominently as a shared social expectation. this social aspect is crucial, since norms can and will change as shared understandings change. to return to the sovereignty example, the behavioural expectations that go along with it today are radically different than those from previous eras (hall 1999) , and continue to evolve today (wheeler 2000; finnemore 2003) . norms go beyond simple behavioural modifications. states begin to reenvision their own identities as they embrace a norm. as states internalize new standards of behaviour, they come to new understandings of themselves. they answer the question 'who am i?' in a different manner. states are willing to forgo the costs associated with upholding normative precepts because these norms are constitutive of how the state sees itself. when a state fails to live up to these behavioural expectations, they justify their actions by referencing the norm itself. in an important sense, the state has violated its own understanding of who it is. instead of taking actions to abide by the rules, states take certain actions and engage in certain behaviours (and refrain from others) because 'good people do (or do not do) x in situations a, b, and c' (fearon 1999: 29) . they connect their preferences to policy choices and instruments in different ways as their self-understandings change (kowert and legro 1996: 463) . finnemore and sikkink (1998) offer a three-stage 'life cycle' for norms. in the first stage, a norm emerges and is championed by norm entrepreneurs. these entrepreneurs use their organizational platforms (such as a nongovernmental or intergovernmental organization) to promote the norm to members of the international community. they actively promote the norm as 'appropriate or desirable behavior in their community' (finnemore and sikkink 1998: 896) . they must persuade a critical mass of important actors to adopt and embrace the norm in order to reach the second stage -the norm cascade. during this second phase, an increasing number of states begin to adopt the norm, even in the absence of domestic pressures or economic self-interests to do so, because they increasingly see it as appropriate. if enough states do this, the norm becomes internalized in the third stage. it becomes 'common sense' and few would even question the behaviours expected by the norm. states abide by the norm and its behavioural expectations because that is just what members of the international community do. it becomes part of the state's sense of itself and its obligations to others. despite the efforts of norm entrepreneurs, not all norms find a home within the international community. scholars have identified three particular factors that appear to increase the likelihood of a norm's acceptance: if its precepts concern the protection of vulnerable populations (keck and sikkink 1998: 27) , if the norm contains clear, consistent rules with a previous history of observance (legro 1997: 34-5) , and if the norm is both coherent and prominent (florini 1996: 374-7) . if a norm is going to stick, states need to share an understanding of what a given norm means from both a behavioural and a constitutive perspective (van kersbergen and verbeek 2007) . norm entrepreneurs, according to most scholars, concentrate their attentions at the state level (finnemore and sikkink 1998; ingebritsen 2002) . they tailor their actions to encourage government policymakers to change their understanding of a particular issue, modify their behaviour, and incorporate the norm's idea into the state's overall identity. they try to get a critical mass of states to adopt, and eventually internalize, a norm in hopes of creating a norm cascade that leads to the norm becoming 'common sense'. while this focus on states is understandable, it ignores the plethora of actors whose actions can put a norm's ideas into practice. international organizations, nongovernmental organizations, private philanthropic organizations, and even multinational corporations play an ever-increasing role in providing services and taking on traditional governance roles. because of this, norm entrepreneurs have started to recognize the utility of targeting these groups as well. these nonstate actors may have financial resources beyond those available to states. they may also possess a greater level of legitimacy and lack much of the historical baggage of states. universal arv access is not the first health-related norm to be promoted to the international community. in the 1970s and 1980s, norm entrepreneurs sought to inculcate the norm of universal primary health care. despite strenuous efforts by some actors, the international community failed to embrace this norm. the reasons for universal primary health care's failure are highly instructive for understanding universal arv access' apparent success. 3,000 delegates from 134 countries and 67 international organizations met in alma-ata, ussr (now almaty, kazakhstan), from 6 to 12 september 1978 at the international conference on primary health care. the conference, organized by the world health organization and unicef, was the first international meeting devoted solely to primary health care. unanimously adopted, the alma-ata declaration listed eight crucial components of primary health care: education on health concerns and how to treat them, promoting proper nutrition, ensuring adequate supplies of clean drinking water and proper sanitation, providing maternal and child health care, including family planning, immunizing populations against major infectious diseases, preventing and controlling local endemic diseases, providing appropriate treatment for injuries and illnesses, and providing access to essential drugs (world health organization 1978) . in order to achieve these goals, the alma-ata declaration set specific targets for signatory states. these goals included: spending at least five per cent of gross national product on health, having 90 per cent of children at the appropriate weight for their age, providing clean water within a 15-min walk of all homes and adequate sanitation either in the home or the immediate vicinity, making available trained personnel to attend to pregnancy and childbirth, and offering child care for children at least through one year of age (world health organization 1978) . these programmes sought to make essential health care accessible to all at an affordable cost and in line with a country's sovereign right to selfdetermination (world health organization 1978) . they afforded the majority of the country's population access to basic health care in line with locally determined needs. if states attained these goals and ensured the provision of primary health care, then it was hoped that the international community could meet its new goal -health for all by 2000. the impetus for promoting this new norm grew out of changes in the international community. the 1960s and 1970s saw a great wave of decolonization and liberation throughout the third world, and new governments often came to power promising better health care for all their citizens. while initially many of these new governments took steps to improve health care, often with the support and aid of western states, services tended to be overly concentrated in urban areas and failed to reach rural areas. this meant that the majority of the population in many newly independent states still had limited access to health care facilities (hall and taylor 2003) . at the same time, an increasing number of studies criticized the idea that improved health in developing states was simply a matter of transferring western technologies and health care systems to new places. these studies called for a more holistic approach to health care that emphasized integrating health care into overall social development (cueto 2004 (cueto : 1864 . researchers and activists increasingly called for a 'bottom-up' approach to health care that focused on local needs and ensuring equitable access without an emphasis on large hospitals or expensive technologies (magnussen et al. 2004: 168) . china, tanzania, and venezuela successfully trained local personnel to provide essential basic health care programmes. these programmes offered basic yet comprehensive health care services to rural areas. for example, china's 'barefoot doctors' focused their energies on preventative care within the communities from which they were drawn and combined western and traditional cures for treatment (cueto 2004 (cueto : 1865 . inspired by their example, and drawing upon his own experiences with health care policies in developing countries, who director-general halfdan mahler of denmark called upon the international community to apply the lessons from these cases throughout the world. he urged who and unicef to ensure 'health for all' by changing both the provision of health care in developing countries and the role of developed states in ensuring this aim. the conference in alma-ata concentrated on spreading the message of health for all and devising strategies for putting this idea into practice. it is hard to underestimate how revolutionary the alma-ata declaration and its health for all by 2000 programme were. up to this point, health care had generally been considered the sovereign domain of states. previous cooperation on international public health issues, while it certainly existed, had been driven largely by specific disease outbreaks that threatened commercial interests. the international health regulations, adopted in 1951, best reflect this concern. the ihr sought to 'ensure the maximum protection against the international spread of disease with minimum interference with world traffic' (world health organization 1983). states were required to report outbreaks of and take measures to prevent the spread of yellow fever, cholera, and plague -three diseases whose spread had long been associated with trade and travel (fidler and gostin 2006: 86) . the ihr thus made public health concerns subservient to economic relations among states, obligating national governments (and only national governments) to act only when disease threatened trade. the delegates to the alma-ata conference in 1978 functioned as norm entrepreneurs. they sought to create a change in how states viewed their responsibilities to their own citizens and those in other countries. much like the campaigns against slavery and apartheid (klotz 2002) , the alma-ata delegates engaged in normative debates that crossed ideological and economic lines. in the midst of the cold war, they sought to bring together democratic and communist states, encouraging them to look beyond their economic and political self-interest to embrace a greater good for the international community. by promoting the alma-ata declaration and health for all by 2000, norm entrepreneurs sought to have states declare that public health was no longer simply a concern for national governments. they wanted national governments to set specific targets and adopt a normative framework that equated good governance with the provision of adequate health care standards. they encouraged states to move beyond reactive concerns with specific maladies and toward a more proactive holistic understanding of health and health care. universal primary health care's advocates framed their advocacy in relatively amorphous terms. as noted above, they spoke of decolonization, fairness, and development. they saw the provision of primary health care, especially in terms of having developed countries provide funding for such programmes, as an obligation owed to developing states by those who had already prospered. they also framed universal primary health care as a public good -one that required more generalized economic and social development. according to the norm entrepreneurs, the market could not adequately provide primary health care, so the state should do so (gostin 2000: 4) . the alma-ata declaration noted that health care inequalities were 'politically, socially, and economically unacceptable andytherefore, of common concern to all countries' (world health organization 1978) . interestingly, the norm entrepreneurs generally saw the push for universal primary health care as something that developed states should support largely on altruistic grounds. the declaration reads, 'all countries should cooperate in a spirit of partnership and service to ensure primary health care for all people' (world health organization 1978) . universal primary health care was a public good that developed states should provide in conjunction with developing states out of a sense of moral obligation and fairness and in the spirit of decolonization. this framing shared many affinities with calls for a new international economic order (nieo). indeed, the third clause of the alma-ata declaration specifically located universal primary health care within the broader calls for the nieo. working through the united nations conference on trade and development, developing countries put forward a number of proposals that sought to improve their terms of trade, increase economic assistance, reduce the north-south divide, and rewrite the international economic rules to favor developing states. these proposals all fell under the rubric of the nieo (murphy 1984) . representatives from developing states argued that the nieo would correct structural imbalances and allow developing states to receive the same benefits as developed states. critics countered that blaming developed states for the lack of development in poorer states was misplaced. they dismissed charges that western development was immoral or that developed states needed to sacrifice their wealth for the benefit of the less fortunate (johnson 1976). the acceptance of universal primary health care had the potential to be a major shift in the international community's normative framework, as it would fundamentally alter what it meant to be a 'good' state. it could alter the framework from one that emphasized individual responsibility for health care to one that gave governments primary responsibility for ensuring the public's health, both in their own countries and abroad (fidler 2003: 23) . despite the efforts of the alma-ata delegates, the goals of health for all by 2000 quickly ran into difficulties. it soon became obvious that the norm entrepreneurs were failing to attract a critical mass of supportive states who could further propel and promote the idea of universal primary health care within the international community. no norm cascade developed, and states did not alter their behavioural expectations of themselves or others. the very idea of primary health care itself came under attack as wildly unrealistic and inappropriate. government officials in many developed countries refused to believe that developing states could or should implement the wide-ranging programmes encompassed in health for all by 2000 (hall and taylor 2003) . instead, they proposed a new solution, selective primary health care (sphc), that would provide only those health care services that would have the greatest benefit to children under five (walsh and warren 1979: 968-70) . primary health care's supporters, the norm entrepreneurs from alma-ata, saw sphc as a betrayal of the incipient norm's core beliefs. wisner (1988) alleged the sphc assumed that poor people were too ignorant to make proper health decisions, ignored existing local infrastructures and cultural practices, overlooked the role of grassroots efforts, and reinforced urban biases. hall and taylor remark, 'in effect, sphc took the decision-making power and control central to phc away from the communities and delivered it to foreign consultants with technical expertiseythese technical experts, often employed by the funding agencies, were subject to the policies of their agencies, not the communities ' (2003: 18) . sphc undercut the basic goals and ideals of health for all by 2000 and the alma-ata declaration. instead of encouraging broadbased participation and the equitable provision of health care to all groups within a society, the move towards sphc encouraged states to think in terms of economic self-interest. it removed the ability of developing states to determine their own needs and the best solutions to address those needs. sphc denied states policy autonomy. in the end, the norm of universal primary health care failed to gain much traction in the international community. its behavioural precepts failed to make an appreciable difference in state actions, and the shifts in identity associated with internalizing a norm never occurred. improvements in health care happened largely on an ad hoc basis with little international coordination or overriding guiding principles. what prevented the norm of universal primary health care from being adopted and internalized by the international community? a cursory examination highlights three key deficiencies. first, universal primary health care's supporters framed the norm as a collective public good. they called on developed states to provide a large outlay of funds to developing states en masse to right a perceived wrong. these pleas arose as the international community was dealing with a global recession, higher oil prices, and great economic uncertainty. few, if any, developed states were inclined to increase their foreign aid budgets. if anything, they were less inclined to provide assistance for health care in developing countries (people's health movement et al., 2005: 59-60) . further, as part of providing this collective public good, developed states were being asked to allow developing states to independently determine their health care policies (hall and taylor 2003) . the frame for universal primary health care combined large financial outlays with little oversight, making it rather unpalatable to many developed states. second, the norm of universal primary health care failed to resonate with existing norms in the international community. norm entrepreneurs argued for universal primary health care as an element of a fundamental right to health at a time when the right to health was highly contested. their arguments that universal primary health care fit with a broader context of decolonization and fairness failed to find a perch. in the same way that the nieo largely failed to resonate and led to little but token actions, universal primary health care put forward an idealistic vision that did not resonate with broader trends in the international community at the time. additionally, conceptualizing health as a collective public good with a prominent role for national governments to provide services ran counter to the increasingly prominent rhetoric of neoliberalism and privatization that emerged in the late 1970s and early 1980s (thomas and weber 2004) . us state department officials also derided universal primary health care as 'too political' and feared how it could potentially alter the balance between themselves and the soviet union (werner 2001) . when director-general mahler called the soviet union 'a pioneer since the first days of its revolution more than 50 years ago in placing health in the forefront of social goals and in linking its attainment with social justice and economic development' in his opening remarks at alma-ata (heyward 1978) , government officials in some states feared the relationship between universal primary health care and communism. murphy notes, 'american policy makers held that on fundamentals northern and southern views were incompatibleythey did not want to debate until both north and south had a single view of their common interests' (murphy 1984: 126) . interestingly, while american officials worried about how the ideological content of universal primary health care could promote communism and soviet ideals, the soviet union and people's republic of china vigorously disagreed with each other about the nature of universal primary health care (cueto 2004) . the disagreements between the two leading communist states further undermined universal primary health care's ability to find state supporters. finally, the norm entrepreneurs themselves were poorly placed to influence state governments or promote behavioural changes. universal primary health care's supporters targeted their appeals toward state governments, believing them to be the key to this norm being embraced by the international community. many were delegates to the alma-ata conference. most were bureaucrats either within their national health ministries or the world health organization. they may have had the technical expertise to understand the important of universal primary health care and perhaps the experience to implement it, but they lacked the political sway within governments to get them to reassess their behaviours and identities. in other words, they may have been norm entrepreneurs, but they were poorly placed norm entrepreneurs who lacked the ability to persuade enough others to adopt the norm. health ministries unfortunately have a tendency to be political backwaters with little influence beyond technical matters (vaughan et al. 1985) . the world health organization also lacked the stature to significantly affect international debates over universal primary health care. it lacked significant financial resources and, despite a near-universal membership, its political clout among member-states was negligible. the world health organization's low status was largely a reflection of the relatively low priority afforded to health within the international community. most states considered health to be a national responsibility and envisioned a limited role for the international community. the world health organization also sought, for much of its history, to consciously avoid political battles so as to avoid antagonizing its members (godlee 1994) . when it did try to take a more assertive role with universal primary health care, it faced the very real threat of having states like the united states withdraw its funding (walt 1993) . with the failure of health for all by 2000, international health norms largely fell off the global agenda. the international community came together to combat various diseases, but no overarching normative ideas concerning the behavioural obligations of states to others within the international community received much attention. the 3 â 5 initiative changed things, bringing the idea of the norm of universal arv access to the forefront of the international community. on 22 september 2003, the who, unaids, and the global fund to fight aids, tuberculosis, and malaria announced a new initiative to combat the failure to deliver arvs to people with hiv in developing countries. that year, unaids estimated that six million hiv-positive people in the third world required arvs, but less than eight per cent actually received them. while 84 per cent of those in need in central and south america had access to arvs, only two per cent of those in africa, the continent hardest hit by the aids epidemic, did (world health organization/unaids 2003: 4-5) . this new programme sought to correct that. it pledged to provide a sustainable and reliable supply of arvs to three million people in the developing world, half the number who needed the drug, by the end of 2005. although the leaders of this effort acknowledged that it was an incredibly ambitious goal, they based their calculations on an article published in 2001 in science. the article's authors cautioned that reaching this target would require optimal levels of both financing and technical capabilities. still, they considered it doable (schwartla¨nder et al. 2001 ) -as did, apparently, who and unaids. who and unaids declared the lack of arv access to be a global health emergency and an issue that urgently needed to be addressed. the 3 â 5 initiative did not create calls for universal arv access by any means (see, e.g. farmer 1999 and headley and , but it focused them and gave them far greater prominence within the international community. instead of being a relatively amorphous call to help people with aids, this new norm framed its calls for action in relatively concrete terms, of providing something tangible to individuals who could not otherwise acquire it, as a human right. by declaring a health emergency, though, the 3 â 5 initiative's promoters hoped to 'propel action and upend ''business as usual'' attitudes'. this new programme would 'demand new commitment and a new way or working across the global health community' (world health organization/unaids 2003: 6). to achieve this commitment, they situated the call within a framework of country ownership, human rights, and equity. not only would success require high-level political commitment but also the attendant financial outlays would also be quite high. when announcing the new programme, who estimated that it would cost at least us$5.5 billion to achieve the target (world health organization/unaids 2003: 24). however, the focus was not on the cost, it was on the realization of the norms of respect for universal human rights. who also saw the initiative as promoting the un's human rights agenda in two ways. first, the universal declaration of human rights declares that all people have the right to the highest possible standard of health -a promise reaffirmed to explicitly include hiv/aids during the united nations special session on hiv/aids in 2001. second, the initiative pledged to pay special attention to vulnerable groups who may have limited access to treatment and prevention programmes. by emphasizing equity, the initiative sought to overcome economic barriers that had prevented most people in developing nations from being able to afford arvs. it utilized the ideas of access to essential medicines and non-discrimination in the provision of care evident in the alma-ata declaration. harris and siplon identified a growing recognition, by developed states, of a norm promoting international assistance to developing states as 'the right thing to do ' (2006: 263) . in their paper, schwa¨rtlander et al. referenced the movement for realizing the right to health in africa as emblematic of the international community's growing respect for this ideal (2001: 2436). the 3 â 5 initiative's own materials were even more explicit. on its website, the initiative proclaimed that its efforts were 'a step towards the goal [sic] of making universal access of hiv/aids prevention and treatment accessible for all who need them as a human right' (world health organization n.d. a; emphasis added). from the earliest days, activists and organizations connected the drive for universal arv access back to the earlier efforts to promote health as a human right. tactically, norm entrepreneurs for universal arv broadened their reach. they did not solely focus on states as the entities responsible for realizing this norm. instead, they called on international organizations, nongovernmental organizations, multinational corporations, and private philanthropic groupsin addition to national governments -to work together. the norm entrepreneurs explicitly recognized this connection, noting, '''3 by 5'' is a target that many organizations are working together to achieve, including national authorities, un agencies, multilateral agencies, foundations, nongovernmental, faith-based and community organizations, the private sector, labour unions and people living with hiv/aids. to succeed, full support and participation from all partners and governments are needed' (world health organization n.d. b). this shift moved the norm from being a collective public good whose realization depended solely on developed states to being targeted toward individuals with diffuse responsibility for protecting the human rights of those in need. such a frame resonated with the increasing international embrace, for better or worse, of public-private partnerships and more holistic interpretations of governance (see bovaird 2004; flinders 2005, therien and pouliot 2006 for detailed discussions on the evolution of public-private partnerships and their associated costs and benefits). spearheading this drive, lee and piot played particularly important roles in mobilizing commitment, attracting international attention and support, and offering guidance. they served as norm entrepreneurs in every sense of the term. they made it their mission to try to convince donors, both governmental and nongovernmental, that the 3 â 5 initiative was in fact achievable. they had to convince a diverse array of actors to work together to find ways to lower the cost of arvs while still allowing the pharmaceutical manufacturers to earn a profit. they needed to get states to re-envision who they were and how they interacted with the rest of the world. they also had to convince states, private organizations, and multinational corporations that this was an issue of individual human rights as well as one in which they could play a significant role. at the end of the 3 â 5 initiative's timeframe, only 1.3 million people in developing countries were receiving arv treatment. this was less than half of the initiative's publicly stated goal. in many ways, this was still a remarkable success. in the span of two years, over one million new people gained access to life-prolonging drugs. over 20 per cent of those who needed arvs in the developing world now had them -a significant improvement over the seven per cent who had them in 2003. eighteen countries announced that they had met or exceeded their arv treatment targets (world health organization/ unaids 2006: 7). these are stunning accomplishments over an incredibly short period of time. these stunning accomplishments cannot diminish the fact that who and unaids failed to meet their goals. they pledged to provide arvs to half of the people in developing countries who needed them (a number that continued to grow over the two-year period from 2003), and they failed to do so. even with greater access to arvs, the worldwide rates of hiv infection continued to increase -meaning that even more people now required arv therapy and did not have access to it. critics lambasted the programme for being overly optimistic, relying on unrealistic modelling, and failing to properly coordinate programmes among the myriad of actors involved (economist 2005) . others noted that national aids control programmes often fell prey to petty turf battles and corruption, making them ineffective (itpc 2005: 6-7) . despite this apparent failure, the basic norm of universal arv access continues to hold sway within the international community. state governments, international organizations, nongovernmental organizations, private philanthropic organizations, and multinational corporations have repeatedly reaffirmed their belief in the norm and pledged additional funds (though still short of what is necessary) toward its realization. given the apparent failure of the 3 â 5 initiative, it was realistic to assume that the norm of universal arv access was dead. its proponents had set an explicit target with a very explicit timeframe -and they failed to achieve this. remarkably, this was not the case. instead of walking away from failure, the international community has embarked on an even more ambitious goal -all by 2010. all by 2010 is the latest attempt to put the emerging norm of universal access to arvs into practice. like the 3 â 5 initiative, all by 2010 combines the efforts of state and non-state actors to provide universal arv access as a constituent element of individual human rights. the central goal of all by 2010 is universal access to arv treatment. this means, according to most definitions, '80 per cent of all people in urgent need of treatment are receiving it' (avert n.d.) . based on current projections, the best estimate is that the all by 2010 programme will need to get 10 million people worldwide on arvs by the end of 2010 to meet its goals (as a shorthand, some also call this program 10 â 10). as with the 3 â 5 initiative, the leaders of all by 2010 explicitly state that this effort is designed to mobilize stakeholders, maintain momentum, and encourage states to contribute. the norm entrepreneurs are using their organizational platforms within who and unaids to encourage the adoption and internalization of a new norm. while expressing regret at its inability to achieve its initial target, the who and unaids' final report on the initiative discussed ways to rectify the problems it faced. the report argued the end of the initiative was just the beginning toward ensuring universal arv access for all. this provides evidence for the internalization of the norm through rhetoric and changes in constitutive identities. failure to achieve and the behaviours associated with it were explained within the context of the norm itself. 'the ''3 by 5'' target needs to be seen as an interim step toward the ultimate goal of universal access to antiretroviral therapy for those in need of care, as a human right, and within the context of a comprehensive response to hiv/aids' (world health organization /unaids 2006: 49) . the g8 nations, the very nations that provided the vast majority of funding for the programmes that came under the 3 â 5 initiative's umbrella, pledged in july 2005 to work toward universal access to arvs worldwide by 2010. at the g8 summit in gleneagles in july 2005, the leaders of the world's largest economies pledged at least an extra us$50 billion in aid annually, part of which would be specifically pledged for universal arv access (office of the prime minister 2005). two months later, the united nations passed a resolution calling on member states to work toward this goal and to pledge the necessary resources (avert n.d.) . in 2006, the un high-level meeting on aids produced a resolution that stated in part, '[we commit] to pursue all necessary efforts to scale up nationally driven, sustainable and comprehensive responses to achieve broad multisectoral coverage for prevention, treatment, care and support, with full and active participation of people living with hiv, vulnerable groups, most affected communities, civil society and the private sector, towards the goal of universal access to comprehensive prevention programmes, treatment, care and support by 2010' (united nations 2006) . african heads of state made a similar pledge in may 2006 at a summit in abuja, nigeria (agence france-presse 2006) . the clinton foundation and the gates foundation have both continued their arv access efforts and have expanded them beyond their initial plans. the international community has clearly embraced the normative rhetoric of universal arv access, tying it to the realization of individual human rights and a broadened conceptualization of governance. the united nations' 2001 declaration of commitment on hiv/aids resolved that 'access to medication in the context of pandemics such as hiv/aids is one of the fundamental elements to achieve progressively the full realization of the right of everyone to the enjoyment of the highest attainable standard of physical and mental health' (united nations 2001) . within months of the unveiling of the 3 â 5 initiative, all 192 member states of the who publicly endorsed the program and the norm contained within it. they publicly pledged to aggressively work toward the realization of this goal and, in a broader sense, to ensure that all those who needed arvs could get them. the un economic and social commission for asia and the pacific passed a resolution that called on states in the region to scale up their public health programs specifically in response to the 3 â 5 initiative (unescap 2004) . in may 2005, over 120 delegates from around the world came together in geneva to coordinate efforts to rapidly scale up efforts to expand access to arvs across political, economic, and religious lines. the us president's emergency plan for aids relief (pepfar), its primary aids programming effort, strongly emphasizes antiretroviral therapy (and its attendant infrastructure), considering it an integral part of its aids programmes and part of the us' obligation as a leading member of international society (office of the global aids coordinator 2006). when announcing pepfar during his 2003 state of the union address, us president george w. bush noted, 'because the aids diagnosis is considered a death sentence, many do not seek treatment. almost all who do are turned away. a doctor in rural south africa describes his frustration. he says, ''we have no medicines. many hospitals tell people, you've got aids, we can't help you. go home and die.'' in an age of miraculous medicines, no person should have to hear those words' (bush 2003) . this statement received a tremendous amount of applause. making this proclamation during his most important speech of the year shows that the norm of universal arv access is at least fomenting rhetorical changes. four years later, when bush called on congress to reauthorize pepfar by providing us$30 billion over the next five years, he highlighted the normative aspects of the programme. acknowledging the costs and the number of people affected by the programme, he emphasized, 'the statistics and dollar amounts i've cited in the fight against hiv/aids are significant. but the scale of this effort is not measured in numbers. this is really a story of the human spirit and the goodness of human heartsyour citizens are offering comfort to millions who suffer, and restoring hope to those who feel forsaken' (bush 2007) . evidence also shows that recipient states internalized this new norm. within months of the initiative's debut, 56 countries approached who, asking for assistance through this new programme (world health organization 2004: 9). these states sought to make the changes in their policies and infrastructure that would allow them to expand the ability of their citizens to access these drugs. they publicly acknowledged that they did not have the resources to enact such a programme, yet by approaching who, they also publicly acknowledged their desire to work with the international community to implement the norm's programme. further, nearly every country has created a country coordinating mechanism (ccm) to receive funding from the global fund and coordinate aids activities. these ccms explicitly incorporate representatives from the public and private sectors to promote the incorporation of all relevant voices (global fund to fight aids, tuberculosis, and malaria n.d.). these efforts show a willingness to adapt state structures in order to facilitate the provision of arvs. non-state actors play an increasingly important role in realizing the behavioural expectations of this new norm. international organizations like the world health organization and the joint united nations program on aids (unaids) serve as conduits of information for the international community. they gather and disseminate data, provide technical resources to actors trying to implement arv access programmes, and sponsor international meetings to facilitate networking. while they also provide some direct funding, they largely focus their energies on supporting the technical and logistical resources needed to bring the norm's objectives to fruition. for funding, the global fund to fight aids, tuberculosis, and malaria emerged in 2001. the global fund is an independent organization, with representatives from donor and recipient governments, nongovernmental organizations and the private sector, with responsibility for funding aids-related programmes. it explicitly does not implement programmes on its own. instead, it provides a centralized source for donors to contribute money and recipients to receive grants to implement programmes (van kerkhoff and szleza 2006) . unique among most international bodies, the global fund relies upon funds from national governments, nongovernmental organizations, private philanthropies, and the sale of specially branded consumer products (dyer 2006) . programmes funded by the global fund may be implemented by governments or nongovernmental organizations, broadening the realm of actors who can help realize the behavioural expectations of this new norm. private philanthropies and multinational corporations have also played a significant role in working toward universal arv access' behavioural precepts. the clinton foundation, former us president bill clinton's organization, has focused its energies on transforming the economic incentives for pharmaceutical companies. recognizing that these companies will not produce arvs without an ability to make a profit, the clinton foundation has helped to aggregate demand for arvs. it has sought to 'transform the antiretroviral marketplace from a low-volume, high-margin market to a high-volume, lowmargin market that serves millions of hiv/aids patients' (clinton foundation n.d.) . this strategy significantly reduces the price for arvs while still allowing generic and branded pharmaceutical manufacturers to recoup their investment in developing arvs. the foundation has forcefully argued that it has not asked for charity, but rather sought to ensure supply at an affordable price in the face of large demand (rauch 2007) . the bill and melinda gates foundation, the world's wealthiest philanthropic organization, collaborated with the government of botswana and the pharmaceutical company merck to create the african comprehensive hiv/aids partnership. this arrangement brings together the financial resources of the gates foundation, the manufacturing and distribution capabilities of merck, and the infrastructure of botswana to deliver arvs to those in need (gates foundation 2006; ramiah and reich 2006) . these two efforts demonstrate the significant role that non-state actors play in actualizing universal arv access. it is indeed true that, even with the diversity of actors involved, international funding for universal arv access has remained far below what experts and norm entrepreneurs claim is necessary. six months before the initiative formally ended, 'unaids estimates that at least an additional us$18 billion above what is currently pledged is needed for global hiv/aids efforts over the next three years' (world health organization 2005: 9; emphasis added). african governments pledged to increase their own budgetary outlays for health programmes within their own borders. by 2005, they promised to devote 15 per cent of their national budgets to health (including hiv/aids programmes) -but none of them met this target by 2005's end (itpc 2005: 4) . funds from some donor states like the united states have come with conditionalities that have hampered their ability to be accessed in a timely and efficient manner. despite this reality, the commitment to realizing the norm of universal arv access appears to remain intact. stephen lewis, the un's special envoy for hiv/aids in africa, proclaimed, 'mind you, i can even now hear the curmudgeonly bleats of the detractors, whining that we will fall short of the target of three million in treatment by the end of this year. tell that to the million people who are now on treatment and who would otherwise be dead. the truth is that the 3 by 5 initiative -which, i predict, will be seen one day as one of the un's finest hours -has unleashed an irreversible momentum for treatment' (un news service 2005; emphasis added). it is highly significant that no state ever predicated its behaviour on a rejection of the norm. no state stated that universal arv access was undesirable or unworthy. questions did arise as to how best to provide these medications to people in challenging environments and ensuring compliance with the drug regimen's requirements. even these discussions, though, referenced back to the emerging norm of universal arv access. the issue was not one of the appropriateness of universal arv access; it was one of delivery. these actions do not mean that the debates over universal arv access are over. the battles over funding levels alone demonstrate the continued discussion. those debates, though, are not evidence of the lack of a norm. van kersbergen and verbeek (2007) remind us that the details over implementing a new norm's behavioural expectations can continue for a while and even be contentious. what we see with universal arv access is a debate over how to realize the norm, not over whether the norm is appropriate. whereas the attempts to promote a norm of universal primary health care got bogged down in debates over its very appropriateness, universal arv access' norm entrepreneurs appear to have successfully convinced a significant portion of the international community that the basic idea is sound. the norm for universal arv access obviously picks up on some of the same themes as the earlier push for universal primary health care, yet it appears to be having more success in establishing itself and being internalized by the international community. what explains the difference? i suggest two important differences: the norm entrepreneurs themselves and the international normative context. first, norm entrepreneurs themselves make a difference, and this appears particularly true for aids-related issues. in the 1980s, many national governments specifically cited the personal lobbying of jonathan mann, then the head of the united nations' global program on aids, as the reason they increased their contributions to aids control efforts (gordenker et al. 1995: 74) . in the case of universal arv access, the effort was really spearheaded by lee, piot, and feachem (mann died in a plane crash in 1998) . these three had the connections and experience that allowed them access to the highest levels of governments. they also took a very intense personal interest in the promotion of this newly developing norm. all three men had impressive resumes working with hiv/aids and ensuring access to health care in developing nations. lee, who himself died suddenly in may 2006, devoted one of his last speeches to building on the lessons from the 3 â 5 initiative to promote universal arv access (lee 2006) . in addition, these three key norm entrepreneurs had impressive organizational bases -the world health organization, unaids, and the global fund to fight aids, tuberculosis, and malaria, respectively -from which to promote their norm and encourage states to adopt it. successful norm entrepreneurs can benefit from such a platform. mahler, during the promotion of health for all by 2000, held the same position as lee, so what explains the difference? for one thing, his was a more solitary voice. mahler was essentially the only leader on the international stage promoting universal primary health care. this made his task more daunting. second, international health organizations have greater prominence today than they did in the 1970s and early 1980s. the international community came together to form the global fund, unaids builds on the strengths of multiple un-affiliated agencies, and the who has received greater attention and respect thanks to its successful handling of crises like sars, avian flu, and the asian tsunami recovery efforts. in addition to the efforts of lee, piot, and feachem, a wide range of nongovernmental organizations took an active role in promoting the norm. these groups put pressure on their governments to live up to their promises, provided research to demonstrate the benefits of greater arv access, and worked to forge seemingly odd political coalitions (witness the alliance of irish rock star bono and conservative former us senator jess helms) to promote their cause. groups like the treatment action campaign, healthgap, and the international treatment preparedness campaign have forced governments around the world to respond to the burgeoning movement for universal arv access. the gravitas of figures like bill clinton, nelson mandela, and bill gates adds even more momentum to the calls for universal arv access. in addition, these groups regularly interacted with one another, sharing strategies and collaborating on international efforts. with norm entrepreneurs working from above (at the international organization level) and below (at the nongovernmental organization level), national governments found it harder to resist. second, the international normative environment has changed in a way more favourable to the embrace of universal arv access. cold war tensions, which partially bedeviled debates over universal primary health care, disappeared but it would be a mistake to attribute too much to this change. more importantly, universal arv access has also benefited from the internalization of related complementary norms within the international community. there is increasing recognition of health as a human right (mann et al. 1999) , which itself builds upon the embrace of universal human rights. farmer has written extensively and eloquently on the connections between health and human rights. he sees medical workers as the new vanguard for promoting human rights, as their actions can actually put the notion of health as a human right into practice. addressing health concerns in an unbiased manner necessarily involves the recognition of social and economic rights when healthcare workers provide these services for their fellow human beings without regard for ability to pay (farmer 2005: 219) . in this way, health promotes human rights in a less overtly political manner. within the framework of health as a human right, a number of groups specifically cite hiv/aids as a human rights issue -and a number of states have internalized this framework (youde forthcoming). if states agree with the idea that health is itself a universal human right and that aids, in particular, is a human rights issue, then it is a small stretch to embrace the notion that providing access to the drugs that combat aids is itself an important normative issue. by avoiding frames that emphasize providing collective goods for developing countries and instead focusing on realizing individual rights through broadbased participation, universal arv access' supporters positioned their ideas to resonate with existing international norms. this new norm then became seen as a natural extension of already-existing ideas. it fit in with prevalent norms about individual human rights and public-private partnerships. the growing discussions around health as a human right allows proponents of the norm of universal arv access to frame their issue in a manner that resonates with government officials and the public. advocates can use frames that match with ideas or images already present in a culture to gain support. this can be particularly important when high costs are involved. the proper frame encourages policymakers to look past their financial concerns to understand how that frame matches with an underlying constitutive identity. busby (2007) uses frames to understand how jesse helms, a conservative us senator, and bono, the irish rock star, came together to support debt relief for poor countries when the issue was framed as one of biblical justice. making this appeal in the context of an existing belief -a shared christian faith, in this case -allowed the issue of debt relief to move forward in spite of the financial cost. in the same way, universal arv access' advocates could draw upon the growing recognition that developed states have an obligation to help those less fortunate and that health is a fundamental aspect of dignified human existence (mann et al. 1999; harris and siplon 2006) . the international normative environment was thus primed to be more receptive to a call for widespread drug access in the early 2000s that was not present at earlier junctures. in many ways, universal arv access' path to acceptance has so far followed a path similar to that previously trodden by the norm of human rights. like universal arv access, human rights norms not only prescribe certain behaviours but they also allow states who internalize these norms to define themselves as liberal (risse and sikkink 1998: 8) . human rights norms did not simply emerge on their own, and states did not adopt them thoughtlessly. instead, the diffusion of human rights norms depended upon a sustained network of domestic and international networks that could connect to policymakers and international regimes. these networks put pressure on states, helped redefine the international normative context in a manner amenable to the embrace of these norms, and empowered actors to appeal for recognition of the norms (risse and sikkink 1998: 5) . activists framed human rights norms in ways that would resonate with existing domestic political cultures in various countries (risse and ropp 1998: 271) . some states may have initially embraced human rights norms for instrumental reasons (risse and sikkink 1998: 10) , but this process itself encouraged states to redefine their identities. human rights norms went from being policy choices to constitutive elements of how states saw themselves and their respect for basic human morality (donnelly 1999: 73) . human rights norms depended upon a combination of norm entrepreneurs and a favorable international normative context to succeed -the same processes that have assisted with the internalization of the norm of universal arv access. the moves toward universal access to arv therapy as embodied by the 3 â 5 initiative and all by 2010 programme represent the emergence of a new international norm born out of the ashes of an earlier failed attempt to inculcate a norm of universal primary health care access. it demonstrates how norms can evolve within the international community and take on new life when international political situations and norm entrepreneurs change. states are working toward universal arv access despite the very high costs and the potentially negative consequences for western pharmaceutical companies. to uphold the norm, they engage in actions that may not be economically profitable and explain their failures to live up to the norm's obligations in terms of those obligations themselves. this is more than just a story about arvs, though. the emergence of universal arv access enriches our understanding of how and why the international community embraces certain norms. it has shown the importance of moving beyond a state-centric view of norm emergence and adoption. nonstate actors, such as nongovernmental organizations, philanthropic groups, and multinational corporations play an increasingly important role in international governance, and that role extends to their influence on international norms. universal arv access also makes clear the importance of complementary norms for successful adoption. universal primary health care failed, in part, because it did not resonate with dominant norms within the international community at the time. finally, universal arv access highlights just how important framing by norm entrepreneurs can be. universal arv access' supporters cast the issue as one of individual human rights being realized by a broad-based coalition of state and non-state actors. this worked far better than the broader collective public good supported by developed states frame employed in the discussions around universal primary health care. universal arv access is indeed an ambitious goal but it could have immense international benefits. by internalizing this norm, states are redefining their obligations to each other when it comes to providing health care. they are establishing new standards of behaviour, standards by which their actions will be judged by others. international ethical obligations are changing for the better of humanity. earlier efforts to inculcate progressive norms that ensure access to health care may have failed, but contemporary norm entrepreneurs demonstrate that it is indeed possible to foster international health-related norms. african leaders pledge more access to aids, malaria treatment aids treatment target and results: all by public-private partnerships: from contested concepts to prevalent practice bono made jesse helms cry: jubilee 2000, debt relief, and moral action state of the union address president bush announces five-year, $30 billion hiv/aids plan hiv/aids initiative: drug access the origins of primary health care and selective primary health care preface the social construction of international human rights new fund-raising scheme fuses profit with philanthropy spin doctors infections and inequalities: the modern plagues pathologies of power: health, human rights, and the new war on the poor what is identity (as we now use the word)?', unpublished manuscript disease and globalized anarchy: theoretical perspectives on the pursuit of global health the new international health regulations: an historic development for international law and public health national interests in international society the purpose of intervention international norm dynamics and political change the politics of public-private partnerships the evolution of international norms working with botswana to confront its devastating aids crisis global fund to fight aids, tuberculosis, and malaria (n.d.) 'country coordinating mechanisms who in retreat: is it losing its influence? health for all beyond 2000: the demise of the alma-ata declaration and primary health care in developing countries' international relations and global ethics of hiv/ aids roadblocks on the road to treatment: lessons from barbados and brazil report of the alma-ata conference scandinavia's role in world politics missing the target: a report of hiv/ aids treatment access from the frontlines the new international economic order', university of chicago graduate school of business selected papers no norms in international relations: the struggle against apartheid transnational activism and global transformations: the anti-apartheid and abolitionist experiences norms, identity, and their limits: a theoretical reprise opening remarks at the consultation on aids and human resources for health which norms matter? revisiting the ''failure'' of internationalism comprehensive versus selective primary health care: lessons for global health policy the emergence of the nieo ideology gleneagles 2005: chairman's summary people's health movement, medact and global equity gauge alliance (2005) global health watch building effective public-private partnerships: experiences and lessons from the african comprehensive hiv/aids partnerships (achap) this is not charity the socialization of international human rights norms into domestic practices: introduction international human rights norms and domestic change: conclusions resource needs for aids the global compact: shifting the politics of international development? the politics of global health governance: whatever happened to ''health for all by declaration of commitment on hiv/aids', general assembly resolution a/res/s-26/2 draft political declaration -2006 high-level meeting on aids regional call for action to enhance capacity-building in public health. resolution 60/2 un goal of treating 3 million hiv/aids victims by 2005 unlikely to be met linking local knowledge with global action: examining the global fund to fight aids, tuberculosis, and malaria through a knowledge system lens the politics of international norms: subsidiarity and the imperfect competence regime of the european union can ministries of health support primary health care? some suggestions for structural reorganization and planning selective primary health care: an interim strategy for disease control in developing countries who under stress: implications for health policy elusive promise' saving strangers: humanitarian intervention and international society gobi versus phc? some dangers of selective primary health care the 3 by 5 initiative world health organization (n.d. b) 'partnerships: working together for declaration of alma-ata world health organization (1983) international health regulations progress on global access to hiv antiretroviral therapy: an update on '3 â 5 treating 3 million by 2005: making it happen progress on global access to hiv antiretroviral therapy: a report on ''3 â 5'' and beyond hiv/aids as a human rights issue emily atkinson provided invaluable research assistance for this article. i also wish to thank brent steele, tracy slagter, and jird's anonymous reviewers and editors for their thoughtful comments. any errors, of course, are solely my responsibility. key: cord-012503-8rv2xof7 authors: levintow, sara n.; pence, brian w.; powers, kimberly a.; sripaipan, teerada; ha, tran viet; chu, viet anh; quan, vu minh; latkin, carl a.; go, vivian f. title: estimating the effect of depression on hiv transmission risk behaviors among people who inject drugs in vietnam: a causal approach date: 2020-08-24 journal: aids behav doi: 10.1007/s10461-020-03007-9 sha: doc_id: 12503 cord_uid: 8rv2xof7 the burden of depression and hiv is high among people who inject drugs (pwid), yet the effect of depression on transmission risk behaviors is not well understood in this population. using causal inference methods, we analyzed data from 455 pwid living with hiv in vietnam 2009–2013. study visits every 6 months over 2 years measured depressive symptoms in the past week and injecting and sexual behaviors in the prior 3 months. severe depressive symptoms (vs. mild/no symptoms) increased injection equipment sharing (risk difference [rd] = 3.9 percentage points, 95% ci −1.7, 9.6) but not condomless sex (rd = −1.8, 95% ci −6.4, 2.8) as reported 6 months later. the cross-sectional association with injection equipment sharing at the same visit (rd = 6.2, 95% ci 1.4, 11.0) was stronger than the longitudinal effect. interventions on depression among pwid may decrease sharing of injection equipment and the corresponding risk of hiv transmission. clinical trial registration clinicaltrials.gov nct01689545. electronic supplementary material: the online version of this article (10.1007/s10461-020-03007-9) contains supplementary material, which is available to authorized users. despite global progress in combating the hiv epidemic, people who inject drugs (pwid) remain disproportionately at risk of hiv infection in southeast and central asia and eastern europe [1] [2] [3] [4] [5] . sharing injection equipment is one of the most efficient means of hiv transmission [6, 7] , and in these regions, pwid have limited access to and suboptimal use of harm reduction services and antiretroviral therapy (art) [8, 9] . the persistence of injection drug use and viremia, without adequate preventive services, results in a high risk of hiv transmission to injecting or sexual partners [10] . the burden of depression is high among pwid and may further interfere with hiv prevention efforts. up to 50% of pwid suffer from severe depressive symptoms [11] [12] [13] [14] [15] , and the presence and severity of depressive symptoms are closely linked to frequency of injection and risk of relapse, suggesting a bidirectional relationship between depression and injection drug use [16] [17] [18] . comorbid depression consistently results in poor hiv treatment outcomes, such as lowering art use and viral suppression [19] [20] [21] [22] [23] [24] . the online version of this article (https ://doi.org/10.1007/s1046 1-020-03007 -9) contains supplementary material, which is available to authorized users. depression may be an important driver of continued hiv transmission among pwid if symptoms increase transmission risk behaviors (e.g., sharing injection drug use equipment, engaging in condomless sex) in the absence of viral suppression. however, while the deleterious effect of depression on hiv treatment outcomes is well established across populations, its effect on the injecting and sexual behaviors that can facilitate hiv transmission or acquisition is not well understood among pwid. although there is substantial evidence that depression increases sexual risk behaviors among men who have sex with men (msm) [25] [26] [27] [28] [29] , few studies have focused on pwid and assessed injecting behaviors. specifically, in vietnam, the focus of this study and a setting where the hiv epidemic is concentrated among men who inject drugs [30, 31] , there have been no prior studies of the relationship of depression with injecting and sexual behaviors. existing studies on depression and hiv transmission risk behaviors among pwid have suffered from several methodological limitations. to our knowledge, all previous studies that include pwid populations have assessed only correlations between depression and transmission risk behaviors, without inferring causality [32] [33] [34] [35] [36] [37] [38] [39] [40] . in these studies, depression and risk behaviors have typically been evaluated for the same time period (e.g., self-report covering the last month), without the ability to infer whether depression preceded risk behaviors or vice versa [35-37, 39, 40] . potential confounders of the relationship between depression and risk behaviors were also measured for the same retrospectively assessed time period. studies that used traditional statistical adjustment for these contemporaneous covariates [34, 38] may have induced bias if these variables acted as causal mediators rather than confounders [41] . in addition, although depression is known to be episodic [42] , prior analyses have primarily relied on a single assessment rather than accounting for changes in both depressive symptoms and time-varying confounders [33, 34, 38, 40] . possibly stemming from these methodological issues, existing evidence for an association between depression and transmission risk behaviors in pwid is inconsistent. while an early meta-analysis (that included studies among pwid) found little evidence for an association between depression and sexual risk behaviors [32] , more recent studies in pwid and msm populations have found higher sexual risk associated with depression [34, 35, 40] or a non-linear association [36] in which mild symptoms are most predictive of sexual risk. the few studies that have evaluated the association of depression with injecting risk behaviors among pwid have suggested that depressive symptoms were associated with greater injecting risk behaviors [35, [37] [38] [39] . we sought to overcome past methodological issues by using a causal approach to estimate the effect of depressive symptoms on hiv transmission risk behaviors among pwid. we used marginal structural models, a tool for causal inference that accounts for time-varying exposures and confounders [43] [44] [45] , with longitudinal data from male pwid living with hiv in vietnam. we hypothesized that depression would increase behaviors associated with risk of hiv transmission to injecting partners (sharing injection equipment) and sexual partners (condomless sex). by examining depression as a potential underlying cause of hiv transmission through these behavioral mechanisms, we sought to provide clearer evidence about the potential for interventions against depression to avert future hiv infections among pwid. we used longitudinal data from a randomized controlled trial of an hiv stigma and risk reduction intervention among pwid living with hiv in thai nguyen, vietnam from 2009 through 2013 [46] . thai nguyen is a province in northeastern vietnam with an estimated hiv prevalence of 34% among its approximately 6000 pwid [47] [48] [49] . participants were recruited via snowball sampling from the 32 thai nguyen sub-districts (of 180 total) with the most pwid. recruiters (former and current pwid) approached members of drug networks in private places to discuss study enrollment and then accompanied or referred interested participants to the study site for screening. at screening, all participants were tested for hiv using two rapid enzyme immunoassay tests run simultaneously (determine: abbot laboratories, abbott park, il and bioline: sd, toronto canada), with discordant results resolved with a third rapid assay (hiv rapid test: acon, san diego, ca). the trial enrolled 455 participants who met the following eligibility criteria: 1) hiv-positive according to study test results, 2) male (given that 97% of pwid in thai nguyen are male), 3) age ≥ 18 years, 4) had sex in the past 6 months, 5) injected drugs in the previous six months, and 6) planned to live in thai nguyen for the next 24 months (the duration of the trial). questionnaire and laboratory data were collected at study visits every 6 months during 24 months of follow-up. the questionnaire collected information on demographics, injection drug use and other substance use, sexual behavior, depressive symptoms, quality of life, pre-study hiv diagnoses (baseline only), and art use. blood specimens were collected to confirm hiv infection at baseline and measure cd4 cell count at baseline and over follow-up. the exposure of interest was depressive symptoms over the past week, as assessed by the 20-item center for epidemiologic studies depression scale (ces-d), which has been validated as a reliable measure of depressive symptoms in vietnam [50, 51] . consistent with past work, we defined severe depressive symptoms as ces-d scores ≥ 23, mild depressive symptoms as scores [16] [17] [18] [19] [20] [21] [22] , and no symptoms as scores < 16 [15, [50] [51] [52] . the transmission risk behavior outcomes were any sharing of injection equipment (needles, syringes, solutions, or distilled water) and any condomless sex with a female partner, reported for the prior 3 months. we also descriptively examined the numbers of injection equipment sharing and condomless sex acts in the prior 3 months reported at each visit. questionnaire and laboratory data included potential confounders of the depression-risk behavior relationship. time-fixed covariates, which were reported at baseline and assumed to be stable throughout the study period, were marital status, age, employment status, intervention arm, history of overdose, alcohol use, hiv diagnosis prior to enrollment, and previous art use. employment and alcohol use could, in theory, vary over time, but these variables remained fairly constant in our population, motivating our decision to treat them as time-fixed. time-varying covariates measured at one time point may affect subsequent depression and risk behaviors; they may also be influenced by depression and risk behaviors from a previous time point. thus, time-varying covariates may act as either confounders or mediators, depending on the time point assessed [41, 43] . for this analysis, time-varying covariates were cd4 cell count category (< 200, 200-499, ≥ 500 cells/μl), depressive symptoms at the visit prior to exposure measurement, and transmission risk behaviors at the visit prior to exposure measurement. in the main analysis, we used marginal structural models to estimate the average causal effect of severe depressive symptoms on the risks of any injection equipment sharing or any condomless sex (separately) in the period three to 6 months later, controlling for time-fixed and time-varying confounders. we evaluated each risk behavior outcome (reported with respect to the prior 3-month period) at the next 6-month visit in order to temporally separate it from the exposure of depressive symptoms (hereafter referred to as the "longitudinal effect"). in a second analysis, to facilitate comparison with prior research, we used marginal structural models to estimate the association between depressive symptoms and risk behaviors reported at the same visit, where temporal ordering could not be differentiated ("cross-sectional association"). we repeated both analyses using three levels of depressive symptoms (severe, mild, none) in addition to the binary categorization (severe, not severe). we used inverse probability weighted estimation of marginal structural models [43, 53] . weights were estimated from a propensity score model for the probability of severe depressive symptoms as a function of time-fixed and time-varying confounders. time-fixed confounders had a constant (baseline) value over all visits; time-varying confounders used the value from the visit immediately preceding the visit at which depressive symptoms were assessed. in the main analysis, the propensity score model was estimated using logistic regression to model the probability of severe (vs. mild or no) depressive symptoms. in a second set of analyses, we used ordinal logistic regression to separately model the three levels of depressive symptoms (severe vs. mild vs. none). propensity score model diagnostics assessed positivity for all confounderdefined subsets of the study population. the denominator of the weights was the predicted probability of depressive symptoms from the propensity score model, and weights were stabilized using the marginal probability of depressive symptoms in the numerator. application of the weights to the study population removes the association between depressive symptoms and potential confounding variables included in the propensity score model, permitting estimation of a causal effect under key assumptions [53, 54] (see discussion). in the weighted study population, we estimated the risk difference (rd) for the risk behavior outcomes using generalized estimating equations (binomial regression models with an identity link) to account for repeated observations on participants [55] [56] [57] . for the longitudinal analysis, this weighted rd can be interpreted as the causal effect of depressive symptoms on the risk behavior outcome: that is, the difference in risk of the behavior in the period three to 6 months later if all participants had depressive symptoms compared with the risk if they all did not have depressive symptoms. to account for missing data due to missed study visits, we used multiple imputation by chained equations (mice) [58, 59] , imputing and analyzing 50 datasets. we included participants who were incarcerated or died during the study period in the main analysis up until the start of the 6-month follow-up interval during which incarceration or death occurred, censoring them after their final visit preceding death or incarceration. in sensitivity analyses, we instead used the imputed risk behavior outcome for that 6-month interval (and censored them at the start of the following interval), given the possibility of engaging in unmeasured risk behaviors prior to incarceration or death. for all estimates, our interpretation focuses on the point estimate and confidence interval, rather than statistical significance [60] . analyses were conducted using r version 3.4.3 [61] . this study was approved by the ethical review committees at all participating institutions. written informed consent was obtained from all participants. as required by inclusion criteria, all 455 participants were male, hiv-positive, and reported being sexually active and using injection drugs at baseline. the median age of participants was 35 years (interquartile range [iqr]: 30, 39), and half were married or cohabitating (47%) ( table 1 ). onethird had a high school education (34%), and the majority were employed full-time (69%). most participants were newly hiv-diagnosed at baseline (74%), while 15% had been previously diagnosed and were not taking art, and 11% reported being previously diagnosed and currently using art. the median cd4 cell count was 241 cells/µl (iqr: 126, 370). general health was rated as poor by 30%. nearly half reported injecting heroin daily (45%), 18% had a history of overdose, and 67% reported current alcohol use. participants completed between zero and four follow-up study visits (median = 4, iqr: 2, 4) at 6-month intervals over 24 months, with 87% completing at least one follow-up visit. at baseline, 44% of participants reported severe depressive symptoms (ces-d ≥ 23), 25% had mild symptoms (16 ≤ ces-d ≤ 22), and 30% had no symptoms (ces-d < 16). one quarter of participants reported sex without a condom in the prior 3 months (24%), with a median of 10 condomless sex acts reported for that period (iqr: 5, 20) . most participants reported sharing injection drug use equipment with injecting partners over the past 3 months (73%); these participants reported a median of 21 sharing acts during that period (iqr: 7, 52). after the baseline visit-when all participants received risk reduction counseling and the majority became newly hiv-diagnosed-sharing injection equipment and condomless sex decreased across trial arms (previously reported in [46] ). however, among the 397 participants attending ≥ 1 follow-up visit, 21% reported sharing injection equipment at ≥ 1 visit, and 7% reported condomless sex at ≥ 1 visit. the severity of depressive symptoms varied over time (supplemental fig. 1 ) with 59% of those attending ≥ 1 follow-up visit reporting severe depressive symptoms at least once. the percentage of participants experiencing competing events increased over time, with 8% incarcerated and 23% deceased at 24 months. in our main analysis, we estimated that severe depressive symptoms (compared to no or mild symptoms) increased the risk of sharing injection equipment by 3.9 percentage points (rd = 3.9%, 95% ci −1.7%, 9.6%) and decreased the risk of condomless sex by 1.8 percentage points (rd = −1.8%, 95% ci −6.4%, 2.8%) in the period three to 6 months later (table 2, fig. 1 ). in the crosssectional analyses, the association between severe depressive symptoms and contemporaneous injection equipment sharing (rd = 6.2%, 95% ci 1.4%, 11.0%) was stronger than the estimated longitudinal effect, while the association with condomless sex was attenuated (rd = −0.7%, 95% ci −4.5%, 3.0%). in analyses using three levels of depressive symptoms, there were small decreases in the risk of condomless sex as depressive symptoms increased, although all confidence intervals overlapped substantially ( table 2 , fig. 2 ). for injection equipment sharing, patterns of risk corresponding to the three levels of depressive symptoms differed between the longitudinal effect and the cross-sectional association. in longitudinal analyses, we observed a u-shaped relationship in which the risk of injection equipment sharing in the period three to 6 months later was 12.8% (95% ci 8.1%, 17.6%) among those with no depressive symptoms, 9.2% (95% ci 5.3%, 13.2%) among those with mild symptoms, and 13.8% (95% ci 9.1%, 18.5%) among those with severe symptoms. in contrast, in the cross-sectional analysis, we observed a monotonic increasing relationship in which those with no depressive symptoms had the lowest risk of 8.5% (95% ci 5.3%, 11.8%) while those with mild symptoms had a risk of 15.5% (95% ci 11.1%, 20.0%) and those with severe symptoms had a risk of 17.4% (95% ci 13.1%, 21.8%). we did not find appreciable differences in sensitivity analyses that varied censoring time for participants who were incarcerated or deceased (supplemental fig. 2 ). using longitudinal data and methods for causal inference, we found that severe depressive symptoms increased the risk of sharing injection equipment but not the risk of condomless sex among pwid. to overcome past methodological issues, we used marginal structural models to capture the episodic nature of depression, enforce temporal ordering of depression and transmission risk behaviors, and control time-varying confounding in the analysis. by focusing on pwid living with hiv in vietnam, a population at high risk of ongoing hiv transmission, we aimed to better understand depression as an underlying cause of behaviors associated with transmission. in our main analysis of injection equipment sharing in the period three to 6 months after assessment of depression, we found a rd of 3.9% (95% ci −1.7%, 9.6%), comparing participants with severe depressive symptoms to those with mild or no depressive symptoms. this longitudinal effect was only slightly weaker than the corresponding cross-sectional association (rd = 6.2%, 95% ci 1.4%, 11.0%) found in the analysis that did not enforce temporality. the 95% ci of the longitudinal effect (−1.7%, 9.6%) shows that a risk difference ranging from a 1.7 percentage point decrease, a small negative association, to a 9.6 percentage point increase, a substantial positive association, is compatible with the data. given that the overall risk of injection equipment sharing was 10% across follow-up visits, the point estimate of a 3.9% point increase is substantively meaningful. previous research has suggested a possible non-linear relationship between the severity of depressive symptoms and occurrence of sexual risk behaviors, although this literature has focused on msm, not pwid, and findings have been mixed. some studies have found that mild depressive symptoms are associated with higher levels of sexual risk behavior but decreasing risk with severe depressive symptoms [25, 36] ; others have observed increasing risk with increasing severity of depressive symptoms [26] [27] [28] . in contrast, our analysis of condomless sex according to three levels of depressive symptoms suggested slight decreases in condomless sex with increasing severity of depressive symptoms, consistent with our main analysis. participants with depressive symptoms -regardless of severity -may be experiencing fatigue, social isolation, and loss of interest in sex, thereby reducing the risk of engaging in this behavior [62] . although all participants reported sex in the 6 months prior to baseline (due to trial eligibility criteria), a loss of interest in sex over 24 months of follow-up, particularly among participants with depressive symptoms, may have contributed to our findings. in contrast to condomless sex, we observed possible nonlinearities in the relationship between depressive symptom severity and risk of sharing injection equipment, which have not been observed previously. prior studies have found an increasing risk of injecting risk behavior with increasing depressive symptom severity [38] or have not differentiated between mild and severe symptoms [35, 37, 39] . we found monotonically increasing risk with increasing depressive symptoms in our cross-sectional analysis, and a u-shaped risk in our longitudinal analysis, where those with mild depressive symptoms had the lowest risk. interestingly, the u-shaped relationship we observed for longitudinal injecting risk is the inverse of some previous findings on sexual risk among msm (where those with mild depressive symptoms had the highest risk) [36] . this may be due to mild depressive symptoms manifesting differently for injecting behavior compared to sexual behavior and inherent differences between pwid and msm populations. depressive symptoms could lead to cognitive distortions, maladaptive coping, and loss of risk aversion [63] [64] [65] , and such symptoms may need to become severe in order to be expressed behaviorally as increased frequency of injection drug use (to treat severe symptoms) and consequently, greater sharing of equipment. although various relationships between depression and hiv transmission risk behaviors have been studied previously, the unique contributions of this study are its focus on pwid living with hiv, a population for whom there is limited data on depression and risk behaviors, and its methodological rigor in inferring causality rather than correlation. our modeling approach controlled time-varying confounding and incorporated the episodic nature of depressive symptoms by using longitudinal data from five study visits over 2 years. given that the longitudinal effect enforced temporal ordering of depressive symptoms prior to risk behaviors, we believe that it more closely reflects the causal effect than does the cross-sectional association. however, it is important to consider the trade-off between temporal ordering and etiologic relevance in the context of data limitations particular to this study. separating the measurement of depressive symptoms and risk behaviors by 6 months (with a 3-month "blackout period" in between) was necessitated by the parent trial's data structure. this incomplete interval coverage could have attenuated effect estimates relative to what they might have been if the entire interval were included (that is, if depressive symptoms were more likely to influence risk behaviors in the first 3 months of the follow-up interval). a shorter time interval with more complete data coverage may allow better capture of the effect of episodic depressive symptoms on subsequent risk behavior. inferring causality relies on several key assumptions, which must be evaluated carefully in light of the limitations of this observational study [53, 54] . the assumption of no unmeasured confounding holds that there are no systematic differences between participants with and without depression beyond any differences in variables controlled for in the analysis. although we controlled for a variety of confounders, it is possible that unmeasured confounding biased estimates of the effect of depression on risk behaviors. we also assumed positivity (i.e., participants with and without depressive symptoms were in all confounder-defined subsets of the population) and that models were correctly specified without measurement error for covariates. importantly, this study's ascertainment of depression relied on ces-d score categories, and the ces-d is not diagnostic of clinical depression. however, we used a conservative cut-point for severe depressive symptoms with high reliability and validity [50, 51] . there may also have been under-ascertainment of risk behaviors due to social desirability and recall bias. however, participants reported high levels of drug use and had been recruited by former drug users (aware of their injection drug use), indicating a willingness to disclose sensitive behaviors. finally, the consistency assumption holds that there is no meaningful variability in treatment relevant to its effect on the outcome. here, we did not model a specific treatment on depression, and results should only be interpreted as the hypothetical effect of eliminating severe depressive symptoms without specifying the treatment used for elimination. our conclusions are specific to this study population, which was not randomly sampled and may not be representative of all pwid living with hiv. while men who inject drugs drive the hiv epidemic in vietnam, our findings may not be applicable to other groups, such as women or pwid in other regions. however, our findings may be broadly generalizable to other asian and european countries where the hiv epidemic is concentrated among similar groups. we also note that the sample size of this hard-to-reach population was relatively small, which limited our statistical power to detect small differences in risk between depression groups. importantly, the risk behavior outcome in our study does not allow direct prediction of forward hiv transmission risk, as we did not take into account viral suppression status, the frequency of risk acts, or partner susceptibility to hiv. these determinants of transmission will be incorporated into a future mathematical modeling analysis that will explicitly estimate forward transmission events from this study population. we found that severe depressive symptoms may perpetuate the risk of sharing injection equipment among pwid living with hiv in vietnam. during the study period (2009) (2010) (2011) (2012) (2013) , there was very limited access to mental health services for people living with depression in vietnam [66] . however, in recent years, mental health services have become a national health priority, and there is growing attention and funding for increasing local services and availability of depression treatment [66, 67] . screening and treating depressive symptoms among pwid presents an opportunity not only to improve mental health and drug abuse outcomes but also to reduce behaviors associated with hiv transmission risk. funding doctoral training support for sara n. levintow was provided by nida (r36 da045569), niaid (t32 ai070114-10), and viiv healthcare (pre-doctoral fellowship). the parent trial for this study was funded by nida (r01 da022962-01). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. this content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. conflicts of interest none. ethical approval this research was approved by the ethical review committees at the thai nguyen center for preventive medicine, the johns hopkins bloomberg school of public health, and the university of north carolina at chapel hill gillings school of global public health. all procedures performed were in accordance with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent written informed consent was obtained from all participants. global epidemiology of injecting drug use and hiv among people who inject drugs: a systematic review hiv prevention, treatment, and care services for people who inject drugs: a systematic review of global, regional, and national coverage the global hiv epidemics among people who inject drugs the hiv epidemic in eastern europe and central asia drug use as a driver of hiv risks estimating per-act hiv transmission risk a probability model for estimating the force of transmission of hiv infection and its application scaling up hiv prevention efforts targeting people who inject drugs in central asia: a review of key challenges and ways forward global, regional, and country-level coverage of interventions to prevent and manage hiv and hepatitis c among people who inject drugs: a systematic review the perfect storm: incarceration and the high-risk environment perpetuating transmission of hiv, hepatitis c virus, and tuberculosis in eastern europe and central asia prevalence of depressive symptoms and associated factors among people who inject drugs in china factors associated with symptoms of depression among injection drug users receiving antiretroviral treatment in indonesia depression and clinical progression in hiv-infected drug users treated with highly active antiretroviral therapy frequency of and risk factors for depression among participants in the swiss hiv cohort study (shcs) prevalence and predictors of depressive symptoms among hiv-positive men who inject drugs in vietnam longitudinal predictors of depressive symptoms among low income injection drug users depression as an antecedent of frequency of intravenous drug use in an urban, nontreatment sample depression severity and drug injection hiv risk behaviors interrelation between psychiatric disorders and the prevention and treatment of hiv infection psychiatric disorders and drug use among human immunodeficiency virus-infected adults in the united states role of depression, stress, and trauma in hiv disease progression depression in hiv infected patients: a review psychiatric illness and virologic response in patients initiating highly active antiretroviral therapy mortality under plausible interventions on antiretroviral treatment and depression in hivinfected women: an application of the parametric g-formula risk factors for hiv infection among men who have sex with men depression, compulsive sexual behavior, and sexual risk-taking among urban young gay and bisexual men: the p18 cohort study depression and oral ftc/tdf pre-exposure prophylaxis (prep) among men and transgender women who have sex with men (msm/tgw) depression, substance use and hiv risk in a probability sample of men who have sex with men a pilot study examining depressive symptoms, internet use, and sexual risk behaviour among asian men who have sex with men mortality and hiv transmission among male vietnamese injection drug users regional differences between people who inject drugs in an hiv prevention trial integrating treatment and prevention (hptn 074): a baseline analysis are negative affective states associated with hiv sexual risk behaviors? a meta-analytic review health psychol correlates of depression among hiv-positive women and men who inject drugs depression and sexual risk behaviours among people who inject drugs: a gender-based analysis people who inject drugs and have mood disorders-a brief assessment of health risk behaviors moderate levels of depression predict sexual transmission risk in hiv-infected msm: a longitudinal analysis of data from six sites involved in a prevention for positives study psychiatric correlates of injection risk behavior among young people who inject drugs association of depression, anxiety, and suicidal ideation with high-risk behaviors among men who inject drugs in delhi intimate relationships and patterns of drug and sexual risk behaviors among people who inject drugs in kazakhstan: a latent class analysis associations of depression and anxiety symptoms with sexual behaviour in women and heterosexual men attending sexual health clinics: a cross-sectional study the control of confounding by intermediate variables measuring depression over time or not? lack of unidimensionality and longitudinal measurement invariance in four common rating scales of depression marginal structural models and causal inference in epidemiology effect of highly active antiretroviral therapy on time to acquired immunodeficiency syndrome or death using marginal structural models marginal structural models for analyzing causal effects of time-dependent treatments: an application in perinatal epidemiology efficacy of a multi-level intervention to reduce injecting and sexual risk behaviors among hiv-infected people who inject drugs in vietnam: a four-arm randomized controlled trial ministry of health of vietnam. results from the hiv/sti integrated biological and behavioral surveillance (ibbs) in vietnam, round ii socialist republic of viet nam. vietnam aids response progress report 2014, following up the 2011 political declaration on hiv/ aids, reporting period thai nguyen provincial aids center and the division of social evils control and prevention, department of labor a self-report depression scale for research in the general population screening value of the center for epidemiologic studies-depression scale among people living with hiv/aids in ho chi minh city, vietnam: a validation study changes in depressive symptoms and correlates in hiv people at an hoa clinic in ho chi minh city vietnam constructing inverse probability weights for marginal structural models estimating causal effects from epidemiological data the r package geepack for generalized estimating equations estimating equations for association structures yet another package for generalized estimating equations. r-news multiple imputation for nonresponse in surveys mice: multivariate imputation by chained equations in r scientists rise up against statistical significance r: a language and environment for statistical computing depressive symptoms, social support, and personal health behaviors in young men and women sex, drugs and escape: a psychological model of hiv-risk sexual behaviours co-occurrence of treatment nonadherence and continued hiv transmission risk behaviors: implications for positive prevention interventions testing a social-cognitive model of hiv transmission risk behaviors in hiv-infected msm with and without depression mental health in vietnam: burden of disease and availability of services barriers and facilitators to the integration of depression services in primary care in vietnam: a mixed methods study key: cord-016690-3gsq724l authors: li, hongjun title: hiv/aids related respiratory diseases date: 2013-09-30 journal: radiology of hiv/aids doi: 10.1007/978-94-007-7823-8_17 sha: doc_id: 16690 cord_uid: 3gsq724l lungs are the most commonly involved organ by hiv/aids related diseases, and pulmonary infections are the main reasons for the increasing death rate from aids. pathogens of hiv related pulmonary infections include parasites, fungi, mycobacteria, viruses, bacteria and toxoplasma gondii. according to international reports, pathogens have different geographical distribution, which is also closely related to the socioeconomic status of the region to produce varied aids related diseases spectra. for instance, in the united states, pneumocystis carnii pneumonia (pcp), tuberculosis and recurrent bacterial pneumonia (at least twice within 1 year) occur frequently in hiv infected patients. an international report published 10 years ago indicated that pcp is the most common and serious pulmonary opportunistic infections in hiv infected patients. now its incidence has dropped with the application of antiretroviral treatment and preventive measures. pcp will continue to occur initially in patients who are aware of their hiv infection. in addition, hiv related viral and parasitic infections have been reported both domestically and internationally. in this section, the clinical manifestations and imaging findings of hiv related pulmonary infections are analyzed and discussed, which provide effective diagnosis basis, so as to reduce the incidence of hiv-related pulmonary infections. lungs are the most commonly involved organ by hiv/aids related diseases, and pulmonary infections are the main reasons for the increasing death rate from aids. pathogens of hiv related pulmonary infections include parasites, fungi, mycobacteria, viruses, bacteria and toxoplasma gondii. according to international reports, pathogens have different geographical distribution, which is also closely related to the socioeconomic status of the region to produce varied aids related diseases spectra. for instance, in the united states, pneumocystis carnii pneumonia (pcp), tuberculosis and recurrent bacterial pneumonia (at least twice within 1 year) occur frequently in hiv infected patients. an international report published 10 years ago indicated that pcp is the most common and serious pulmonary opportunistic infections in hiv infected patients. now its incidence has dropped with the application of antiretroviral treatment and preventive measures. pcp will continue to occur initially in patients who are aware of their hiv infection. in addition, hiv related viral and parasitic infections have been reported both domestically and internationally. in this section, the clinical manifestations and imaging fi ndings of hiv related pulmonary infections are analyzed and discussed, which provide effective diagnosis basis, so as to reduce the incidence of hiv-related pulmonary infections. pneumocystis has been believed to be a kind of protozoon. recently, based on its ultrastructure and ribosomal rna phylogenetic analysis, pneumocystis is now believed to be a kind of fungus, with high affi nity to the lung tissues. due to the compromised immunity, 95 % aids patients sustain different types of pulmonary infections, of which pcp is the most common life-threatening opportunistic infection with an incidence rate of about 60-85 %. about 90-95 % patients suffering from aids complicated by pcp are adolescents and adults with their cd4 t cell counts being less than 200/ μl. clinical manifestations of typical pcp are fever, cough (dry cough without phlegm), dyspnea, chest distress and shortness of breath. dyspnea is shown as progressive difficulty in breathing, which initially occurs after physical activities and develops into diffi culty breathing even in resting state. pcp is commonly accompanied by weight loss, fatigue, anemia, general upset and lymphadenectasis. all these symptoms are non-specifi c, but patients often report subjective feelings of severe symptoms while physical signs are mild. by auscultation, the lungs are normal or with slightly dry, moist rales. these are the clinical fi ndings characteristic to aids complicated by pcp. in most patients with pcp, the serum ldh level increases but it is non-specifi c. in cases of aids complicated by pcp, the blood po2 reduces, commonly being lower than 70 mmhg in patients in the middle and advanced stages. the diagnostic imaging for pcp includes chest x-ray and ct scanning. due to the low resolution of chest x-ray, its demonstrations are negative for pcp patients in the early stage or only include thickened pulmonary markings and decreased pulmonary transparency. however, ct scanning demonstrates tiny lesions or more detailed changes in lungs. especially with the application of hrct, the detection rate of pcp lesions has been greatly improved. it has been internationally reported that nearly 10 % of pcp patients show negative fi ndings by the chest x-ray but with abnormal fi ndings by hrct. due to the rapid progression of pcp as well as its complex pathological changes, ct scanning demonstrations are diverse with specifi city. according to different pulmonary ct scanning demonstrations in different stages of the illness, pcp is divided into early stage (exudative and infi ltrative stage), middle stage (fusion and parenchymal stage) and advanced stage (absorption or fi brosis stage). the early typical manifestations include intrapulmonary multiple miliary nodules, mainly distributed in both middle and lower lung fi elds. it may be accompanied by enlarged hilar shadow, which should be differentiated from acute miliary tuberculosis. the middle stage is a period of infi ltration. as the disease progresses, miliary and patchy shadows fuse and expand into a dense infi ltrative shadow with even density, showing a diffuse ground glass liked change. the typical manifestations include bilaterally symmetric foci with the hilus as the center. the foci infi ltrates from the hilus to bilateral pulmonary interstitium, progressing from the both middle lungs to both lower lungs. hrct can more clearly demonstrate the foci, showing a map liked or gravel road liked appearance, with clearly demonstration of gas containing bronchus penetrating the foci. the pulmonary apex is involved later. the exterior stripe of the lung fi eld has increased transparency, showing typical willow leaf sign or moon bow sign which is the manifestation of compensatory pulmonary emphysema. during the late compensatory repair period, the intrapulmonary lesions are mainly parenchymal changes and fi brosis, with large fl aky high density shadows as well as cords liked and reticular changes. pneumothorax, mediastinal emphysema, pneumatocele, pleural effusion and other complications may occur, with an incidence of pneumatocele in about 10-20 % patients. the autopsy grossly demonstrates swelling of the lung tissue, and the alveoli are fi lled with large quantity foamy liquid. the pathological changes mainly manifested as interstitial pneumonia, with early manifestations of increased permeability of the capillary wall basement membrane in the alveolar walls, which leads to fl uid exudation. the pneumocystis carinii proliferate in large quantity and adhere to cause degeneration of the type i alveolar epithelial cells and shedding of the basement membrane. vascular congestion, edema as well as infi ltration of lymphocytes, plasma cells and mononuclear cells can be found in the pulmonary interstitium. due to the extensive existence of aspergillus in natural world, sputum smear positive often fails to defi ne its invasive infection. in the cases with aspergillus infection, hyphae can be found in the sputum. fungal infections often occur in patients with cd4 t cell count below 100/μl, of which the most common pulmonary infection is aspergillus infection, followed by penicillium marneffei infection. pulmonary infections caused by candida albicans and histoplasma are rarely found. the incidence of cryptococcal pulmonary infection is still in a disagreement, which is increasing recently. there are also some common endemic fungal infections, such as the most commonly found fungal infections of aids complicated by penicillium infections in guangxi zhuang autonomous region and hong kong, china. aspergillus has an extensive existence in the natural world. aids complicated by aspergillus infection is related to the application of corticosteroid hormone or broad-spectrum antibiotics, which occurs commonly in the advanced/critical stage of aids. the cases of pulmonary fungal infections, with fi ndings of hyphae (aspergillus or candida) or yeast in cytoplasm (histoplasma capsulatum) in tissue sections and simultaneous fi ndings of histiocytic reactions including the infi ltration of neutrophilic granulocyte and the necrosis of histocytes, can be diagnosed as having invasive fungal infection. candida albicans is yeast liked fungus, which is widespread in the natural world. it can parasitize in the mocous of skin, oral cavity, intestinal tract and vagina of the human being. candida albicans cannot cause disease in immunocompetent people but is pathogenic in immunocompromised population. after its invasion into the tissues, it turns into mycelia and multiplies in large quantity with great toxicity. it also has the ability to fi ght against phagocytosis. clinically, its infection is characterized by a chronic onset and clinical symptoms of low and moderate grade fever but rarely high fever, cough, shortness of breath, cyanosis, irritation or dysphoria. the pulmonary signs include weakened breathing sounds by auscultation and obvious moist rales of lungs. the serious cases may have symptoms of systemic poisoning. the illness is prolonged and repeated during its whole progression. by diagnostic imaging demonstrations, it can be divided into the following types: (1) bronchitis type, with chest x-ray demonstrations of increased pulmonary markings in lower fi elds of both lungs; (2) pneumonia type, commonly with accompanying extrapulmonary lesions. the lesions are mainly located in the middle and lower lung fi elds and lesions in the lower lung fi eld are more common. the apex is generally not involved. the lesions are recurring one after another. a small number of patients may sustain complications of exudative pleurisy. (3) disseminated type, with miliary shadows, diffuse nodular shadows or multiple small abscesses. the lesions often involve the middle and lower lungs. chest x-ray demonstrates thickened pulmonary markings and accompanying spots, small fl akes and large fl akes of parenchyma shadows, in manifestations of bronchial pneumonia. in some serious cases, the foci may fuse together and enlarge to involve the entire lobe. ct scanning demonstrates pulmonary nodules and few have ground glass liked changes of the lungs. pathological changes include acute infl ammatory lesions in the lungs, alveolar exudation and infi ltration of monocytes, lymphocytes and neutrophils. acute disseminated lesions often cause multiple small abscesses, central caseous necrosis, spores and hyphae in and around the lesions. histoplasma capsulatum belongs to moniliales family, deuteromycetes class and fungal kingdom, whose growth requires organic nitrogen. it is often isolated from the soil with abundant contents of birds or bats faeces and spreads along with chickens, birds, dogs, cats, and mice. when the conidia and mycelial fragments of histoplasmosis are inhaled, most can be expelled by the defense mechanism of the human body. granulomas may form, but in immunocompromised patients, it may cause disseminated histoplasmosis. when the cd4 t cell count in aids patients is less than 150/μl, histoplasma capsulatum infection of lungs may occur. histoplasma capsulatum pneumonia has a higher incidence in south america, africa and india. in the slight cases, the clinical manifestations are similar to symptoms of the cold, with low-grade fever, cough, and general upset. in the serious cases, there are symptoms of infl uenza, including chills, high fever, cough, chest pain, dyspnea, fatigue and poor appetite. in the cases of acute cavity, thin-walled cavity may form within a month. complications may be pericarditis, arthritis, skin nodules, rash fi brous mediastinitis and mediastinal granuloma. diagnostic imaging demonstrations are non-specifi c, with scattering pulmonary acinus exudation, multiple nodules in a diameter of about 3 mm with accompanying thickened septa, and formation of granulomas with accompanying calcifi cation. it should be differentiated from bacterial pneumonia, tuberculosis and other pulmonary fungal infections by laboratory tests to defi ne the diagnosis. the specifi city of the glycogen antigen detection of histoplasma capsulatum is up to 98 %. mucor spreads through the respiratory tract. it commonly invades the blood vessels, especially arteries. it reproduces locally or causes thrombosis and embolism. clinical manifestations are high fever, cough, sputum, shortness of breath, chest distress, chest pain and hemoptysis (pulmonary artery involvement). the diagnostic imaging demonstrates fl akes infl ammatory foci, with manifestations of pulmonary cavity and pulmonary infarction. the pathological changes are hemorrhagic infarction of local tissue, pneumonia and exudation of neutrophils. hemorrhagic infarction of local tissue may be related to hyphae induced minor arteries lesions. it is estimated that one third of the world population was/is infected with tubercle bacillus and 9 % of them are aids patients. the who reported that there are 88,000 newly infected patients of tb each year and 8.4 % of them are caused by aids. it is estimated that each year in 1,000 hiv infected patients, 35-162 sustain active tb, and there is a great risk of active tb progressed from the latent tuberculosis infection. hiv infected patients with tuberculosis are commonly young and middle aged adults, with more male patients than female patients. tuberculosis can occur at any stage of aids and at any level of cd4 t cell counts. it has been internationally reported that hiv infection complicated by tb has no specifi c imaging demonstrations. it has an acute onset, with an incidence of acute onset 2.5 times as high as that in non-hiv infected patients. the lesions are morphologically diverse, which are different from non-hiv infected patients with tb. hiv infection complicated by tb has commonly an acute onset, while tb in non-hiv infected patients is commonly secondary to other lesions, with cavities, fi brosis, pleural thickening and calcifi cation. a study conducted in china has demonstrated that for aids complicated by tb, the acute cases mainly have military and exudative lesions, with an incidence of 33 and 49 % respectively; while the incidence of chronic cases including cavity, fi brosis and calcifi cation is declining, being 11, 11 and 2 respectively. a later occurrence of tuberculosis in hiv infected patients indicates a more seriously immunocompromised immunity, with less typical clinical manifestations and imaging demonstrations. when the cd4 t cell count level is above 350-400/μl, the systemic symptoms are fever, chills, night sweating, fatigue, poor appetite and weight loss. respiratory symptoms are cough, expectoration, hemoptysis, chest pain and dyspnea. it manifests as primary tuberculosis, with its foci distributing in the middle and lower lungs, involving multiple lobes and segments. when the cd4 t cell count decrease, the impact of tb increase including the occurrence of extrapulmonary tuberculosis and disseminated disease. when the cd4 t cell count drops below 200/μl, pulmonary tuberculosis manifests as acute onset (such as miliary tuberculosis) or extrapulmonary tuberculosis (such as ileocecal tuberculosis) and peripheral lymph nodes tuberculosis. its difference from the clinical manifestations of non-hiv infected patients is as the following: (1) more common pulmonary infi ltration with multiple involvements and rare cavities; (2) higher incidence of dissemination (87-96 %) commonly along with blood fl ow and higher incidence of extrapulmonary tuberculosis (60-70 %); (3) more common lymph node tuberculosis, such as hilar, mediastinal and extrapleural lymphadenectasis; (4) lower positive rate of tuberculin test (ppd); (5) more patients with no expectoration, with sputum smear for acid-fast bacilli staining is negative; (6) higher incidence of resistant strains, high recurrence rate, and higher mortality (table 17 .1 ). foci in the cases with aids complicated by pulmonary tuberculosis are change quickly. after anti-tb treatment, the lesions are absorbed quickly. those receiving no anti-tb therapy, the foci tend to fuse together to form a mass or diffusely distribute. bacterial septicemia often occurs in aids patients. many opportunistic pathogens can cause respiratory infections, including bacterial bronchitis, pneumonia and pleuritis. the incidence rate of bacterial pneumonia (bp) is 3-5 %. bp has a larger range of impact on hiv infected patients than on non-hiv infected groups. repeated episode of bp is considered to be the fi rst manifestations of latent hiv infection. therefore, for those individuals who have recurrent pneumonia without other risk factors, they should be alert to hiv infection. the common pathogenic bacteria include streptococcus pneumoniae, staphylococcus aureus, rhodococcus equi, haemophilus and pseudomonas aeruginosa. as non-hiv infected patients, the most common pathogens of pneumonia are streptococcus pneumoniae and haemophilus infl uenzae. legionella and klebsiella are also common. many factors, such as the reduced t lymphocytes in hiv infected patients, manufacturing disorders of neutrophils, mononuclear cells and cytokines, and dysfunctional b lymphocytes, provide chances for opportunistic bacterial infections. in addition, the application of broad-spectrum antibiotics also increases the chance of opportunistic infections. bp can occur in any stage of hiv and at any level of cd4 t cell count. when the cd4 t cell count decreases, the incidence of bp also increases. the clinical manifestations of hiv infected patients are the same as non-hiv infected patients, with acute onset (3-5 days) , high fever (39-40 °c) , chills, chest pain, dyspnea, cough, purulent sputum (bloody or rusty). being different from non-hiv infection, pulmonary infection in hiv-infected patients is often recurrent. the imaging demonstrations of hiv/aids related bacterial pneumonia are similar to those in non-hiv infected patients. most cases of streptococcal pneumonia and haemophilus pneumonia have unilateral, confi ned and partially fused foci with accompanying pleural effusion. the imaging demonstrations include thickened and deranged pulmonary markings, alveolar fi lling of infl ammatory exudates with the progression of the illness, large fl aks infl ammatory infi ltration shadows or parenchymal shadows, bronchial gas fi lling phase in the parenchymal shadows. the lesions distribute along the pulmonary segments or lobes, rarely with accompanying pleural effusion. during the absorption period, the density of the parenchymal shadows gradually reduces and the scope narrows down. there may be cavities in some individual cases. but in most cases it is completely absent after poor effi cacy and more side effects favorable effi cacy and less side effects 3-4 weeks. lesions absorption are slow in elderly patients and recurrent patients, which is diffi cult to be completely absorbed and often develop into organic pneumonia. rhodococcus equi was initially discovered in 1923 and nominated as corynebacterium equi. after structure analysis of the cell wall, it was found to be different from corynebacterium, and therefore it is classifi ed into rhodococcus. rhodococcus equi is generally considered as the pathogens of horses, pigs and cattle. human rhodococcus equi infection is rare. but in recent years, due to an increase in patients with immunodefi ciency syndrome, reports of rhodococcus equi induced human respiratory infection and sepsis are increasing. rhodococcus equi is an intracellular facultative parasitic bacterium. its optimum temperature for growth is 30 °c, and suitable temperature for its growth is 10-40 °c. the acid-fast staining for rhodococcus equi shows uncertain results. due to its various morphology, it is commonly mistaken as diphtheroids bacilli, bacillus or micrococcus. on sheep blood agar plate, the bacterium can have synergistic hemolysis with staphylococcus aureus, mononuclear listeria and corynebacterium pseudotuberculosis. toxicity mechanisms of rhodococcus equi has been recently discovered the existence of toxic plasmid, which provides a new idea for the full understanding of its pathogenesis. clinical symptoms are usually cough, orange red sputum, high fever and other symptoms. e marchiori et al. in 2005 studied fi ve cases of aids complicated by rhodococcus equi pulmonary infection. all the patients had a case history of cough and fever history for 1-2 months with accompanying shortness of breath and chest pain. li et al. in 2009 [ 106 ] studied a group of 13 cases. all patients had fever, with a body temperature being 38-40 °c, cough, orange red sputum. the typical clinical manifestations of the disease are fever, dyspnea and chest pain. other symptoms such as weight loss, diarrhea and joint pain are not representative. based on the course of the disease, the diagnostic imaging demonstrations of rhodococcus equi pulmonary infection can be divided into early stage, showing round liked fl aky blurry shadows surrounding unilateral hilum that has blurry boundary; middle stage (parenchymal change), showing central sphere liked high density shadow surrounding unilateral hilum, in parenchymal changes and with clear boundary; advanced stage (necrosis) showing secondary cavity of the pulmonary mass, possibly with hydropneumothorax and pleurisy. the imaging demonstrations are characteristic, but lack of specifi city. and it should be differentiated from pulmonary tumors. the pathological changes include the most commonly chronic suppurative bronchopneumonia and extensive pulmonary abscesses. the histopathology demonstrates massive bleeding in alveolar space, large quantity erythrocytes, intact cellular wall and large quantity epithelial cells. the predominant pathological changes may also be fi broblasts, with parenchymal changes of lung tissue and thickened alveolar septa. accumulating piles of strip liked purple rhodococcus equi can be found by pas staining. common pathogenic viruses of the opportunistic pulmonary infections in hiv infected patients are cytomegalovirus (cmv) and infl uenza virus. cmv is the most common pathogen of hiv/aids related pulmonary infection. by autopsy, 49-82 % patients with hiv/aids have cmv infection, only second to pneumocystis carinii pneumonia. moskowitz et al. [ 32 ] reported that among the direct causes of death in aids patients, 19 % is due to pulmonary cytomegalovirus infection. because of the lack of typical clinical manifestations and sensitive examinations for its early diagnosis, the defi nitive diagnosis rate of cytomegalovirus pneumonia is only 13-24 % before autopsy. the clinical manifestations of cmv infection are non-specifi c. the systemic symptoms are fever, soreness of joints and muscles. respiratory symptoms are paroxysmal dry cough, progressive shortness of breath, diffi culty in breathing during activities. pulmonary cmv infection may develop secondary fungal infection or be complicated by bacterial, fungal, and pneumocystis carinii infections. the cytomegalovirus can widely spread in the organs and tissues of the infected patients, and the infections can directly lead to the damage of infected host cells. in addition, the virus can also cause pathogenic effects via immune pathological mechanism. some scholars classifi ed cmv pneumonia into diffuse, miliary necrosis and cytomegalic. diffuse and cytomegalic cmv pneumonia are often accompanied by diffuse alveolar damage (dad), which is more common in the diffuse type of cmv pneumonia but less common in cytomegalic type of cmv pneumonia. the pathological basis of diffuse small nodules in lungs is hemorrhagic necrosis. sometimes cmv infection is concurrent with other infections in the lungs, and even co-infects one cell. pulmonary parenchymal changes indicate bacterial and fungal infections, such as fi ndings of inclusion bodies in the cells, commonly known as eagle's eye sign. the imaging demonstrations of cytomegalovirus pneumonia are diverse. some studies summarize that the lungs commonly have manifestations of diffuse interstitial infi ltration or alveolar infi ltration, with ground glass liked changes, pulmonary parenchymal changes, grid liked changes, thickend bronchial wall, bronchiectasis, pulmonary nodules or masses. the principal changes include the early lesions of ground glass liked changes and advanced lesions of pulmonary masses. lymphoid interstitial pneumonia is the abnormal hyperplasia of the pulmonary lymphoid tissue. its occurrence is related to autoimmune diseases, and is believed to be a direct response of the lungs to hiv. the clinical manifestations are recurrent infections, poor appetite, hepatomegaly and splenomegaly, and arrested development. the diagnostic imaging demonstrates no characteristic changes by ct scanning, with thickened bronchial wall, diffuse central lobular nodules or bronchiectasis, grid liked and cords liked shadows in uneven thickness. the pathological changes include accumulating lymphocytes and plasma cells that are mixed to infi ltrate the pulmonary interstitium and expand to surrounding areas of the bronchi. toxoplasma pneumonia is caused by the infection of the intracellular parasite, toxoplasma gondii. ludlam et al. in 1963 fi rstly proposed the concept of pulmonary toxoplasmosis, which was believed to cause atypical pneumonia [ 107 ] . the clinical manifestations are cough and expectoration. in the serious cases, dyspnea and cyanosis can occur. in the chronic cases, there are long term low grade fever, cough, weight loss and enlarged lymph nodes. the diagnostic imaging demonstrates bronchopneumonia, interstitial pneumonia and pleurisy. (1) bronchial pneumonia is also known as lobular pneumonia, with scattered patchy and blurry density shadows. (2) interstitial pneumonia has typical manifestations of reticular and nodular shadows. (3) pleurisy is rare, showing pleural effusion, limited diaphragmatic activity. the imaging demonstrations are non-specifi c, which can be defi ned in combination with the etiologic examinations. the pathological changes are congestion and edema of the surrounding connective tissue of the alveolar wall and bronchial walls, widened pulmonary interstitium, small quantity serous fi brin exudation from alveoli and pulmonary interstitium, and infi ltration of macrophages and lymphocytes. toxoplasma cysts and tachyzoites may be found in pulmonary interstitium and macrophages as well as alveolar epithelium. kaposi's sarcoma, a vascular tumor, was discovered in 1872, and is also known as multiple hemorrhagic sarcomas, multiple vascular sarcomas, or multiple pigmented sarcomas. kaposi's sarcoma is believed to be the defi ning tumor of aids. outbreak of ks occurred in male homosexuals in europe and the united states. data show that in about 30 % caucasian homosexuals, kaposi's sarcoma is a major complication of in hiv/aids patients. it has been confi rmed that, though kaposi's sarcoma has strong invasion, the disease itself has little impact on the mortality of aids. the cause of death in majority of the patients is still opportunistic infections. the clinical manifestations include face and neck lesions in dark red to purple red plaques. the plaques do not fade away when pressed, with surrounding brown ecchymosis. it commonly involves multiple organs including lungs, spleen, oral cavity, lymph nodes, gastrointestinal tract and liver. the lungs are the major target of invasion. the diagnostic imaging demonstrates hilar lymphadenectasis and its surrounding nodular infi ltration, bilateral interstitial changes, and pleural effusion that are its typical x-ray demonstration. early pathological changes are similar to those of common angioma; with gathering of capillaries into groups containing histocytes engulfed hemosiderin and orderly arranged vascular endothelial cells. it further progression see active proliferation of endothelial cells and fi broblasts, increased nuclear mitosis with anaplasia, and scattered lymphocytes and histocytes between blood vessels. in the advanced stage, occlusion and necrosis of the vascular lumen can be found. irregular lumen and fi ssures of the new capillaries can be commonly found in the tumor, fi lled with blood and common hemorrhage. in china, ks is relatively rare. its defi nitive diagnosis can be made by pathological examination. other hiv/aids related malignancies include burkitt's lymphoma, non-hodgkin's lymphoma, hodgkin's lymphoma and lung cancer. in summary, hiv/aids related pulmonary infections are important diseases in the disease spectrum of hiv/aids imaging. the diagnostic imaging is irreplaceable examinations for pulmonary infections. early prevention and correct diagnosis are the keys to improve the quality of life and prolong the lives of patients. the complexity and multiplicity of hiv/aids related pulmonary diseases present challenges for the clinicians. firstly, hiv/aids related diseases should be optimally classifi ed. each type should has a disease spectrum, which can be used for exclusion in combination with immunological indices to make the diagnosis and differential diagnosis. the diagnosis of hiv/ aids related pulmonary infections should be made in combination with case history and laboratory tests for targeting individualized diagnosis to serve clinical practice. carnii pneumonia (pcp) the pathogen is the trophozoites and cysts produced by pneumocystis carinii, principally living in the lungs. pneumocystis carinii was used to being categorized as as protozoon, but recently, it is believed to be belonged to fungus according to its ultrastructure and pneumocystis ribosomal rna phylogenetic analysis. the main infection route of pcp is airborne transmission and reactivation of in vivo latent pneumocystis carinii. infl ammatory and immune responses of the host include phagocytosis of pneumocystis carinii by the alveolar macrophages, infi ltration of lymphocytes in peribronchial and vascular area, proliferation of type ii alveolar cells, local and systemic increase of antibody. by naked eyes observation, there are extensive and diffuse invasion of lungs, which is soft like waterlogged sponge and in milky white with black spots. the fi lled foamy substance in the alveoli and bronchioles is a mixture of necrotic fungus and immunoglobulin. the alveolar septum has infi ltration of plasma cells and lymphocytes, resulting in thickened alveolar septa up to 5-20 times as the normal thickness that occupy 3/4 of the entire lung volume. the cysts are fi rstly located in the macrophage cytoplasm of the alveolar septa. subsequently, the alveolar cells containing cysts sheds off into the alveolar space. after the rupture of the cystic wall, sporozoite is discharged to turn into free trophozoites, which gains its access into the alveolar space. the alveolar exudates include plasma cells, lymphocytes and histocytes ( fig. 17 .1a-c ). the clinical symptoms include dry cough, shortness of breath and an indoor hypoxia. about 95 % aids patients have multi-pathogens induced pulmonary infections. the most signifi cant laboratory abnormality in most pcp patients is hypoxemia. based on correlation between pcp and arterial oxygen partial difference, hypoxemia is divided into three degrees. the slight cases at indoor conditions have their pao 2 being above 70 mmhg, or alveolar-arterial oxygen pressure difference being less than 35 mmhg, or both. the moderate and severe cases have their pao 2 being usually less than 70 mmhg, or alveolar-arterial oxygen pressure difference being above 35 mmhg, or both. the most common manifestations of aids complicated by pcp are progressive subacute onset of dyspnea, fever, dry cough and chest distress, the symptoms aggravating in a few days or weeks. pulmonary examination is usually negative in slight cases. as the disease aggravates, the cases show shortness of breath, cyanosis, tachycardia, and diffuse dry rales. pneumocystis carinii infection accounts for 60-85 % of aids patients, which is one of the major causes of death in aids patients. the diagnostic imaging examinations include chest x-ray, ct scanning and nuclear medicine examination. (1) chest x-ray is the conventional examination for screening. early lesions tend to be missed for the diagnosis due to the limited resolution or atypical lung lesions. (2) ct scanning with high resolution is superior to chest x-ray. (3) nuclear medicine examination demonstrates increased uptake of the isotope-labeled monoclonal antibodies in the lung tissues of the pcp patients. (1) by tracheal suction or lung tissue biopsy, the detection rate of pneumocystis carinii is up to 90 %. by tissue section staining, abundant protozoa can be found in intra-alveolar foamy eosinophil substance mass (by methenamine silver nitrate staining, the dark brown round or oval shaped cysts can be found in a diameter of 6-8 μm out of the cells). (2) by elisa, pneumocystis igg antibody can be detected and by latex particle agglutination test, the protozoa antigen can be detected. (3) molecular biology techniques, such as pcr can be applied for early diagnosis. the following examinations are non-specifi c, but can be used to assess the severity of pcp and its progression. (1) by arterial blood gas analysis, the patients may show reduced blood oxygen saturation and respiratory alkalosis. (2) by serum enzyme spectrum analysis, the patients may show increased ldh. (3) it can be detected to have increased alveolar-arterial oxygen partial pressure difference. in the early stage (exudation period), alveolar fl uids exudate, with diffuse granular shadows in the bilateral lung fi elds extend from the hilum to the surrounding. in the middle stage (infi ltration and fusion period), the intrapulmonary lesions fuse, with ground glass liked or cloudy shadows that are bilaterally symmetric like butterfl y wings. in the middle and advanced stages (parenchymal changes period), the lung tissues show parenchymal changes, with high density shadows and accompanying air bronchogram. the lung periphery shows stripes of transparent shadows. in the advanced stage (pulmonary fi brosis period), the pulmonary interstitium is thickened in dense cords liked appearance, with interval irregular patchy shadows. the pulmonary ventilation improves and the lung periphery shows dense parenchymal shadows with emphysema, pneumomediastinum and pneumothorax. in the early stage (exudation period), the lesions radiatus develop from the hilum to lung fi eld. in the early stage, the diffuse exudative lesions distribute as pulmonary acinus, with changes similar to pulmonary interstitial changes. it was believed to be interstitial pneumonia. however, acute pcp is actually exudation of alveoli and spaces containing a male patient aged 31 years was confi rmatively diagnosed as having aids by the cdc. he complained of dyspnea, cyanosis and wheezing for 3 weeks, with obviously decreased oxygen saturation. his cd4 t cell count was 45/μl. a female patient aged 37 years was confi rmatively diagnosed as having aids by the cdc. she complained of dyspnea, cyanosis and wheezing for 8 weeks, with obviously decreased oxygen saturation. her cd4 t cell count was 45/ μl. patients with acquired immunodefi ciency. the early symptoms include fever, dry cough and shortness of breath. the advanced symptoms are serious dyspnea, cyanosis, progressive hypoxemia and respiratory failure. by pulmonary examinations, scattered dry and moist rales can be heard. trophozoites of pneumocystis cysts can be found by liquid giemsa staining. slight and moderate interstitial infl ammation responses mainly involve lymphocytes and alveolar macrophages. the detection of cysts containing sporozoites is the basis to defi ne the diagnosis. chest x-ray chest x-ray demonstrations of pcp can be classifi ed into four types. (1) early pulmonary interstitial infi ltration and diffuse miliary alveolar exudation; (2) in the middle stage, there are alveolar exudates, with fusion and parenchymal changes; (3) in the middle-advanced stage, diffuse parenchymal changes; (4) pulmonary interstitial fi brosis and lung cavity or lung bulla, as well as pneumothorax and emphysema. for the cases with negative or atypical fi ndings by chest x-ray, ct scanning with high resolution should be performed. ct scanning demonstrates early lesions of multiple symmetric diffuse miliary nodal shadows, which have clear boundaries. in the middle stage, there are thin cloudy shadows or ground glass liked density shadows. in the middleadvanced stage, the lung tissues show parenchymal shadows, with trachea-bronchial sign. in the outer strip of the lung, a transparent area in shape of willow leaf can be demonstrated. in the advanced stage, fi brous cords liked shadows are demonstrated some lung tissues with compensatory emphysema and even pulmonary pseudocysts. the intake of the isotope-labeled monoclonal antibody by lung tissues of pcp patients increases. hiv/aids related pcp should be differentiated from bacterial pneumonia, pulmonary tuberculosis, viral pneumonia, fungal pneumonia, ards, and lymphocytic interstitial pneumonia (lip). bacterial pneumonia has more focal lesions but less diffuse lesions. pulmonary mycobacterium tuberculosis infection has manifestations of military pulmonary tuberculosis by chest x-ray, which is diffi cult to be differentiated from early pcp. hiv/ aids related pcp shows miliary nodules, which further fuse into cloudy or ground glass liked shadows or parenchymal changes. the lesions are commonly symmetrical, with the hilus as the center. the clinical manifestations include fever, dry cough or accompanying diffi culty breathing, and even cyanosis. but in the cases of pulmonary mycobacterium tuberculosis infections, most show miliary nodules, which further fuse into large nodules or mass. after about 2 weeks treatment in the early stage, the military nodules in both lungs can be absent, with common clinical symptom of high fever. correlation studies of miliary tuberculosis and peripheral blood cd4 t cell count have demonstrated that the general incidence of miliary tuberculosis is low, only 6-9 %, but it is the main manifestation of hiv/aids related pulmonary miliary tuberculosis. generally, when cd4 t cell count is below 200/μl, the incidence of cavity lesions is 29 %, noncavity lesions 58 %, complicated by pleural effusion 11 % and lymphadenectasis 20 %. when cd4 t cell count is between 200 and 390/μl, the incidence of cavity lesions and non-cavity lesions each accounts for 44 %, complicated pleural effusion 11 % and lymphadenectasis 14 %. when cd4 t cell count above 400/μl, the manifestation is commonly pneumonia type, in fl aky shadows or parenchymal shadows in just one pulmonary segment. the incidence of cavity lesions is 63 %, non-cavity lesions 33 %, complicated by pleural effusion 3 % and no lymphadenectasis. chest x-ray demonstrates cytomegalovirus pneumonia negative in 1/3 patients. the foci are commonly bilateral, with reticular particles in 33 % patients, alveolar foci in 22 % patients, nodular foci in 11 % patients, complicated by cavity in 11 % patients, cysts in 6 % patients, pleural effusion in 33 % patients and lymphadenectasis in 11 % patients. the incidence of diffuse foci in the cases of cryptococcus neoformans pneumonia is 76 %, interstitial foci or mixed foci 76 %, alveolar foci 19 %, nodular foci 5 %, lymphadenectasis 11 % and pleural effusion 5 %. hiv/aids related pcp is more likely to occur in children with aids, which presents diffi culty for its differentiation form lymphoid interstitial pneumonia. however, lymphoid interstitial pneumonia commonly has a chronic onset, with commonly manifestations of cough and dry rales. systemic lymphadenectasis and enlargement of salivary glands can also be found. by lung tissues biopsy, ebv-dna1 can be detected, which provides basis for their differentiation. pneumocystis, a unicellular organism, is the pathogen of pneumocystis carinii pneumonia. pneumocystis carinii pneumonia is one of common opportunistic infections in aids patients, which is also the leading cause of death in aids patients. in the initial episode of pcp, most patients have a cd4 t cell count of less than 100/μl. diagnostic imaging demonstrates bilaterally symmetrical ground glass liked shadows, which can be diffusely distributed and tend to mainly involve the periphery of the hilus or the middle and lower lung fi elds. hrct scanning is commonly applied to assess early pcp that is demonstrated negative by chest x-ray. hrct scanning demonstrates bilaterally symmetric patchy or fused ground glass liked shadows. the pathological basis of ground glass liked shadows and parenchymal areas refl ect that the acinus is fi lled by the foamy exudates, which are composed of surface active substances, cellulose and cell debris. all of the ground glass liked shadows, overlapping septa and the intralobular linear shadows are in gravel road liked manifestation. the septa and intralobular linear shadows demonstrate pulmonary interstitial edema or cellular infi ltration. in the middle-advanced stage of pcp, there are manifestations of small pulmonary nodules, pulmonary parenchymal changes, thickened interlobular septa, intralobular linear shadows, mass like lesions, pleural effusion, and lymphadenectasis. the cysts tend to mainly involve the upper lobes, which can be unilateral or bilateral pulmonary cysts, pneumothorax, mild or severe interstitial fi brosis and traction bronchiectasis. hrct scanning demonstrations of pcp are non-specifi c. its diagnosis should be in combination with hivph13 and etiological examinations. bacterial infections mycobacterium tuberculosis is still an important pathogen for pulmonary infection in hiv positive patients. since the mid-1980s, the main cause of the increasing incidence of tuberculosis is the prevalence of hiv infection. the incidence of tuberculosis in aids patients is 200-500 times higher than the general population. hiv infection is the most dangerous factor for progression of latent tuberculosis into active tuberculosis. tubercle bacillus belongs to mycobacterium family of mycobacterium genus, which is divided into types of human, bovine and murine. the main cause of human tuberculosis is human mycobacterium tuberculosis, which is known as acid-fast bacilli. tubercle bacillus wall is the complex containing high molecular weight fatty acids, lipids, proteins and polysaccharides, which are related to its pathogenicity and immune responses. lipid can cause the infi ltration of human monocytes, epithelial cells and lymphocytes to form tuberculous nodules. its protein contents can cause allergic reactions, and infi ltration of neutrophils and mononuclear cells. polysaccharides participate in certain immune responses (such as agglutination). these pathogenic factors lay the foundation for the occurrence of tuberculosis in aids patients. human immunity, allergic responses as well as the number and pathogenicity of tubercle bacilli are closely related to the quality, range, spreading rate and the progression of tuberculosis. its pathological changes are characterized by exudation, infi ltration, proliferation and hyperplasia, degenerative necrosis (caseous necrosis) and cavity formation. the manifestations include congestion, edema and infi ltration of leukocytes. the exudative lesions occur in early stage of tuberculosis infl ammation or when the lesions deteriorate. it can also be found in the serosa tuberculosis. there is neutrophilic granulocytes in the exudative lesions, which are gradually substituted by monocytes (phagocytes). the engulfed tubercle bacilli can be found in the large mononuclear cells. the exudative lesions are absorbed and dissipated through the phagocytosis of the mononuclear-phagocyte system, even with no scar. when large mononuclear cells engulf and digest tubercle bacilli, the phospholipid of the bacteria render the large mononuclear cells to enlarge and be fl at, similar to epithelial cells, which is known as epithelioid cells. these epithelioid cells gather into groups, with central langhans giant cells that pass the messages of the bacteria antigens to lymphocytes. surrounding the langhans giant cells, there are often many lymphocytes to form typical tuberculous nodules, which are characteristic lesions of tuberculosis. this is why it is called tuberculosis. in the tuberculous nodules, tubercule bacilli are usually undetectable. proliferation based lesions often occur in the cases with less bacteria invasion and when human cells mediated immunity is predominant. degeneration often occurs on the basis of the exudative or proliferative lesions. tubercle bacilli overcome macrophages and then continually proliferate in large quantity. after the cells become cloudy and swelling, the foci show fatty degeneration, dissolved into fragments, until the occurrence of necrosis. after the death of infl ammatory cells, proteolytic enzymes are released to dissolve the tissues that results in necrosis, which is coagulative necrosis. by naked eyes observation, they are yellowish gray, with loose and brittle quality like caseous. therefore it is known as caseous necrosis. microscopic examination demonstrates an area of solid and eosin staining red necrotic tissues with no tuberculosis. tubercle bacilli in the foci of caseous necrosis proliferate in large quantity to cause liquefaction, which is related to infi ltration of neutrophile granulocytes and large monocytes. part of liquefi ed caseous necrotic substances can be absorbed and part can be discharged by the bronchus to form cavities. otherwise, it may cause intrapulmonary spreading along with bronchi. the small caseous necrosis or proliferative lesions can be shrunk and absorbed after treatment, with only residues of slight fi brous scars. due to the compromised immunity in aids patients, the lesions rarely show fi ber tissues proliferation, but form cords liked scar. calcifi cation rarely occurs. if the necrotic lesions erode the blood vessels, tubercle bacilli can cause systemic miliary tuberculosis along with blood fl ow, including brain, bones and kidneys. large quantity sputum containing tubercle bacilli gains its access into the gastrointestinal tract. it can also cause intestinal tuberculosis and peritoneal tuberculosis. pulmonary tuberculosis can cause tuberculosis pleurisy via direct spreading to the pleura ( fig. 17 .19a-c ). clinically, it is a chronic progression, with rare acute onset. the clinical symptoms are commonly systemic, with fever and fatigue. the respiratory symptoms include cough and hemoptysis. pulmonary tb can be divided into primary and secondary, with the initial episode commonly being primary (type i). the residual bacteria after primary infection can cause secondary infection (type ii-iv) when the immunity is compromised via spreading along blood fl ow or direct spreading. it is common in hiv positive children. most cases are asymptomatic, sometimes with symptoms of low grade fever, mild cough, sweating, rapid heartbeat, and poor appetite. hiv/aids related miliary tuberculosis is one of the major manifestations of pulmonary tuberculosis, which is more common. the onset of acute miliary tuberculosis is rapid, with symptoms of chills and high fever with a body temperature up to 40 °c, mostly remittent fever or continuous fever. there may be decreased leukocytes count and accelerated sedimentation rate. the progression of subacute and chronic hematogenous disseminated pulmonary tuberculosis is relatively slow. infi ltrative pulmonary tuberculosis in aids patients commonly occurs in both middle and lower lung fi elds, with fl aky and fl occulent foci or parenchymal changes in lobes or segments. caseous lesions are rare. the early stage of infi ltrative pulmonary tuberculosis is commonly asymptomatic, with later occurrence of fever, cough, night sweating, chest pain, weight loss, expectoration and hemoptysis. this type of pulmonary tb rarely occurs in aids patients. in non-aids patients, chest x-ray demonstrates three major changes, namely cavity, fi brosis, and bronchial dissemination. in the aids patients, the pulmonary manifestations include single or multiple nodular shadows with clear boundaries. tuberculous pleuritis is an exudative infl ammation caused by the direct invasion of tubercle bacillus from the primary lesion near the pleura into the pleura, or hematogenous dissemination via the lymphatic vessels to the pleura. the routes for occurrence of tuberculous pleurisy include: (1) the bacteria in the hilar lymph tuberculosis counterfl ow to the pleura along lymph vessels. (2) tb lesions adjacent to pleura rupture to cause direct access of the tubercle bacilli or products of tuberculosis infection into the pleural cavity. (3) acute or subacute hematogenous disseminated tuberculosis causes pleuritis. (4) due to the increased allergic responses, the pleura highly respond to tuberculosis toxins to cause exudation. (5) thoracic tuberculosis and rib tuberculosis rupture into the pleural cavity. clinically, pleuritis can be divided into three types, dry pleuritis, exudative pleuritis and tuberculous empyema (rare). the common clinical manifestations are fever, cough with accompanying chest pain of the affected side and shortness of breath. (1) sputum smear examination is simple to manipulate, with high accuracy rate. the fi ndings of the tubercle bacilli can defi ne the diagnosis. it still is the golden criteria for the diagnosis of pulmonary tuberculosis. (2) sputum tubercle bacilli culture has high reliability. tubercle bacilli drug sensitivity test can be performed but requires 6-8 weeks to obtain the results. therefore, its application is limited. (1) tuberculin purifi ed protein derivative (ppd) test is commonly used. its positive result is one of the evidence confi rming a past history of tb infection. (2) bactec test can be performed to detect the metabolites of mycobacterium tuberculosis. generally, mycobacterium can be detected in 2 weeks. the quantity of mycobacteria can affect the period required for test results. (3) pcr has poor specifi city but high sensitivity of up to 98-100 %. both can be applied to observe the enlarged lymph nodes in the chest and mediastinum. in addition, they can be applied to obtain specimens for biopsy, which facilitates the diagnosis and differential diagnosis. diagnostic imaging examinations include chest x-ray and ct scanning. chest x-ray can demonstrate the location, quality and range of the lesions. it can also help to assess the therapeutic effi cacy. ct scanning can demonstrate small or hidden lesions, with a high resolution. primary pulmonary tuberculosis, also known as primary syndrome, is rare in adult aids patients. chest x-ray demonstrates intrapulmonary patchy or large fl aky parenchymal changes, hilar and mediastinal lymphadenectasis in connection to irregular cords liked shadows (located between intrapulmonary lesion and the hilum). lymph node tuberculosis is demonstrated to have mediastinal lymphadenectasis that sometimes fuse into mass. in aids patients, simple mediastinal lymph node tuberculosis is more common than primary syndrome. tuberculosis (1) the acute cases are demonstrated to have diffused miliary nodules in both lungs with even distribution, even size and even density. (2) the subacute and chronic cases are demonstrated to have nodules in both lungs, with uneven distribution, uneven size and uneven density. sometimes calcifi cation occurs in the nodules, with fi brous cords and thickened pleura. infi ltrative pulmonary tuberculosis are demonstrated to have patchy parenchymal changes in the middle and lower lung fi elds as well as parenchymal changes, cavities and fi brous cords liked foci in the segments and lobes. it can also occur in the upper lung fi elds, commonly with accompanying mediastinal and hilar lymph node tuberculosis. it commonly occurs in the advanced stage of aids,, with manifestations of pulmonary interstitial fi brosis and formation of cavities. this type of pulmonary tuberculosis is less common. it rarely occurs, mostly in the early stage of aids. it is rare in the middle and advanced stages of aids. dry pleuritis has manifestations of blunt costophrenic angle and limited diaphragm mobility. exudative pleuritis is manifested as small quantity pleural effusion and thickened pleura, commonly with encapsulated effusion of the lateral pleura. calcifi cation is rare. a male patient aged 28 years was confi rmatively diagnosed as having aids by the cdc. he complained of dull chest pain, dyspnea, fever, night sweating, fatigue and anorexia. his cd4 t cell count was 65/μl. a female patient aged 36 years was confi rmatively diagnosed as having aids by the cdc. she complained of dull chest pain, dyspnea, fever, night sweating and fatigue. her cd4 t cell count was 85/μl. a male patient aged 41 years was confi rmatively diagnosed as having aids by the cdc. he was infected hiv via blood transfusion, with complaints of cervical lymph node tuberculosis, ascites and abdominal infection, fungal stomatitis, biliary stones with infection and severe anemia. his cd4 t cell count was 33/μl. a female patient aged 39 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and night sweating. her cd4 t cell count was 73/μl. a female patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. she was hospitalized due to complaints of chest distress, cough and expectoration for 2 months, with after noon low grade fever and weight loss. on admission, she was confi rmed hiv positive and a cd4 t cell count of 120/μl. a female patient aged 37 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and chest pain for 2 months, with acid-fast bacilli positive in the pleural fl uid culture. her cd4 t cell count was 71/μl. a female patient aged 40 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and chest pain for 2 months, with acid-fast bacilli positive in the pleural fl uid culture. her cd4 t cell count was 891/μl. a female patient aged 34 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and chest pain for 2 months, with acid-fast bacilli positive in the pleural fl uid culture. her cd4 t cell count was 51/μl. cough expectoration, chest pain, dyspnea, fever, night sweating, fatigue, anorexia, lymphadenectasis and rapid progression of the conditions. ppd skin test with a resulted diameter of more than 5 mm should be considered as tuberculosis infection. but its positive rate remains low. the culture of sputum and bronchoalveolar lavage fl uid can detect the pathogens. nucleic acid analysis or dna probe technique, pcr and chromatography can be applied to detect tubercle bacilli. the commonly used diagnostic imaging examinations include chest x-ray and ct scanning. the main demonstrations include (1) intrapulmonary and extrapulmonary lymphadenectasis; (2) miliary tuberculosis manifestations; (3) infi ltrative (pneumonia type) pulmonary tuberculosis; (4) pulmonary interstitial fi brosis, cavity, pulmonary emphysema, nodules, emphysema, and bronchiectasis with accompanying infections. in the cases with their cd4 t cell count being above 400/μl, the imaging demonstrations are similar to those of non-hiv/aids patients with pulmonary tuberculosis. in the cases with their cd4 t cell count being above 400/μl, the manifestations are intrapulmonary large fl aky parenchymal changes, surrounding satellite lesions as well as mediastinal and hilar lymphadenectasis. sometimes the manifestations may be only mediastinal lymphadenectasis and their fusion into mass. in the cases with cd4 t cell count being above 200/μl, there may be accompanying extrapulmonary tuberculosis, such as tuberculous peritonitis, bone tuberculosis, brain tuberculosis and splenic tuberculosis. in the cases with their cd4 t cell count being lower than 100/μl, the manifestations are mostly miliary tuberculosis. hiv/aids related tuberculosis should be principally differentiated from pneumocystis carinii pneumonia, fungal infections, other pneumonia and lung cancer. hiv/aids related tuberculosis should be differentiated from pcp. pcp is mainly manifested as multiple lesions with hilum as the center to extending symmetrically to outside of the lungs. in the advanced stage, pcp has main lesions of pulmonary interstitial fi brosis, with less accompanying mediastinal and hilar lymphadenectasis. the laboratory tests can facilitate to defi ne the diagnose. hiv/aids related tuberculosis should be differentiated from fungal infections. fungal infections are relatively less common than tuberculosis. the imaging fi ndings of hiv/aids related pulmonary fungal infections are diverse, with manifestations of miliary, fl aky fl occulent liked, parenchymal, mass, and interstitial changes. in general, the diffuse lesions are mainly interstitial changes, while confi rmed lesions, compared to tb lesions, are more likely to have thick walled cavities. satellite lesions of fungal infections are less than those of tuberculosis, with less accompanying mediastinal and hilar lymphadenectasis. sometimes laboratory tests are necessary to defi ne the diagnosis. hiv/aids related tuberculosis should be differentiated from non-tuberculosis mycobacteria pneumonia. their imaging fi ndings are similar to each other, which presents challenges for their differential diagnosis. molecular biology examinations play an important role in the differentiation. hiv/aids related tuberculosis should be differentiated from other pneumonia. non-bacterial pneumonia (mycoplasma, viral and allergic) often shows patchy shadows, which are similar to the manifestations of early infi ltrative pulmonary tuberculosis. when bacterial pneumonia shows lobar lesions, it may be confused with tuberculous caseous pneumonia, which should also be differentiated for the diagnosis. symptoms of mycoplasma pneumonia are mild, with imaging fi ndings being always inconsistency with the clinical symptoms, which usually subside within 2-3 weeks. in the cases of allergic pneumonia, eosinophils in the blood increase, with intrapulmonary mobile shadows, which are the basis for their differentiation. bacterial pneumonia can have acute onset, with chills, high fever, rust colored sputum, and streptococcus pneumoniae positive. recovery is rapid after antibiotic treatment and all these symptoms can subside within 1 month. hiv/aids related tuberculosis should be differentiated from pulmonary abscesses. in the cases of infi ltrative pulmonary tuberculosis with cavities, it should be differentiated from pulmonary abscess. especially, tuberculosis with cavities in the apical segment of inferior lobe should be differentiated from acute pulmonary abscess. chronic fi brous cavity tuberculosis should be differentiated from chronic pulmonary abscess. the key points for the differentiation are tubercle bacilli positive by sputum culture in the cases of tb, while tubercle bacilli negative by sputum culture in the cases of abscesses. pulmonary abscess has an acute onset, with increased leukocytes and neutrophils as well as favorable therapeutic effi cacy of antibiotics. but sometimes tuberculosis with cavity may develop into bacterial infection, with undetectable tubercle bacillus by sputum culture. hiv/aids related tuberculosis should be differentiated from lung cancer. the central type of lung cancer has nodular shadow in the hilum or hilar and mediastinal lymph node metastasis, which should be differentiated from lymphatic tuberculosis. the peripheral type of lung cancer has small fl aky infi ltration and nodules in the periphery of the lungs, which should be differentiated from tuberculoma or tuberculosis infi ltrative lesions. lung cancers occur commonly in people aged above 40 years. the central type mainly is squamous carcinoma and the cases often have a history of long term smoking, with symptoms of no fever but diffi culty breathing or chest distress as well as gradually increasing chest pain. there are also symptoms of irritated cough with blood phlegm and progressive weight loss. the cases with supraclavicular metastasis have palpable harden lymph nodes. the intrapulmonary nodules can lobulated with fi ne spikes, no satellite lesions, generally no calcifi cation and possible vacuole sign. the peripheral type of lung cancer shows pleura invagination sign. tuberculin test often shows negative in the cases of lung, positive or weakly positive in the cases with tb, and negative or weak positive in aids patients. hiv infection is known to be the main factor for the development of latent tuberculosis into active tuberculosis. immunosuppression are similar to those in the cases with primary tuberculosis, with characteristic abnormal manifestations of hilar and/or mediastinal lymphadenectasis and parenchymal changes of the air chambers. ct scanning demonstrates enlarged nodules with low density. enhanced scanning demonstrates marginal enhancement of the lymph nodes. the incidence of military tuberculosis in aids patients is increasing due to reduced thymic t lymphocytes in aids patients and the defects of delayed allergic responses, which result in the formation of granulomas and impaired functions to kill bacilli and confi ne the lesions. nontuberculous mycobacteria (ntm) refer to the mycobacteria except for mycobacterium tuberculosis complex (human, cattle, african and vole) and mycobacterium leprae. the most commonly known nomination is nontuberculous mycobacteria (ntm). more than 100 kinds of ntm have been found so far. according to berger manual of systematic bacteriology, ntm is divided into two categories, rapid growth type and slow growth type. ntm are widely spread in nature, such as soil, dust, fl owing water and raw milk. under a microscope, ntm is morphologically similar to tubercle bacilli, with red stained fi ndings by acid-fast staining. according to the growth of ntm in solid medium, the runyon classifi cation divides ntm into the following four groups, light chromogenic bacteria; dark chromogenic bacteria that can cause cervical lymphnoditis in children, intrapulmonary or extrapulmonary infections and abrasive abscess; non-chromogenic bacteria including mycobacterium avium complex, intracellular mycobacteria that can cause pulmonary infections, lymphnoditis, arthritis and meningitis; rapid growth bacteria including mycobacterium fortuitum, mycobacterium, mycobacterium abscessus that can cause pulmonary diseases and skin infections. immunocompromised populations, such as hiv infected patients, patients with neoplasms, patients with long-term use adrenocortical hormone or immunosuppressive agents, are more susceptible to disseminated ntm infection. immunocompetent people may have mycobacterium kansasii and mycobacterium avium infections. it was reported in the united states that the occurrence of mycobacterium avium complex infection in hiv positive patients is up to more than 95 %. the pathological changes of ntm infections are similar to those of tuberculosis. ntm lymphnoditis is pathologically characterized by granulomatous infl ammation. tuberculous nodules formed by epithelioid cells and langhans giant cells are rare, with no accompanying central caseous necrosis. due to the weak pathogenicity of ntm, the pathological changes are slight, but there is difference in the pathological changes of ntm infections in terms of location, type and host. cavities are common in the cases with pulmonary ntm infection, commonly being multiple or multilocular thin wall cavities. the pleuron is rarely involved, with non-specifi c pathological changes of infl ammation but with large quantity pathogens of ntm. patients often have a history of chronic obstructive pulmonary disease, tuberculosis, silicosis, pulmonary abscess, bronchiectasis, cystic fi brosis, diabetes, ulcer as well as use of hormone or immunosuppressive agents. its occurrence is more common in males than in females. the symptoms include cough, expectoration, hemoptysis, chest pain, diffi culty breathing, low grade fever, weight loss and fatigue. the symptoms are nonspecifi c and the conditions progress slowly. for patients with suspected diagnosis of pulmonary ntm infection, sputum smear for acid-fast staining, sputum culture and bronchial lavage specimen culture can be performed. the positive fi ndings should be identifi ed with two to three times repeated culture. the same fi nding of ntm can defi ne the diagnosis. pathological biopsy can be performed for the diagnosis of ntm lymphnoditis. using 16s-23 srdna gene spacer sequence (igs) of ntm for pcr-restriction fragment length polymorphism analysis (pcr-rflp), ntm species can be identifi ed, which is more accurate, faster and simpler than the conventional morphological and biochemical examinations. mycobacterium tuberculosis and ntm have common antigen. ppd skin test produces cross-reaction, but there are still differences between mycobacterium tuberculosis and ntm. ppd-t of the mycobacterium tuberculosis and ppd-ntm of ntm are simultaneously obtained for mantoux skin tests. the induration diameter of ppd-t in ntm patients is generally within 15 mm. for the cases with the induration diameter of ppd-ntm skin test being 5 mm larger or over 25 % larger than that of ppd-t skin test, ntm infection can be confi rmed. both are the most commonly used imaging examinations. a female patient aged 26 years was confi rmatively diagnosed as having aids by the cdc. she complained of cough and chest distress for half a month, fever for 10 days, 1 day after cesarean section and fi nding of hiv positive for 1 day. her cd4 t cell count was 54/μl, with treponema pallidum antibody negative and ppd test negative. imaging demonstrations include various lesions such as infi ltration, cavity, nodules, fi brous caseation and extensive fi ber contraction in unilateral or bilateral lungs. the incidence of cavity is up to 80 %, being singular or multiple. cavities caused by intracellular mycobacterium are mostly found in the pleura, with thin wall and less surrounding exudates. the incidence of non-tuberculous mycobacterial infections in aids patients is high. mac infection is usually caused by the initial exposure rather than the reactivation of latent pathogens. in patients with complications of mac related lung diseases, most of the imaging fi ndings are normal. the most common manifestation is mediastinal or hilar lymphadenectasis. and the pulmonary symptoms are similar to those of tuberculosis. in the cases with multiple patchy parenchymal changes, cavities can be found, as well as nodules with blurry boundaries, pleural effusion and rarely found miliary nodules. sputum or bronchoalveolar lavage fl uid culture positive, clinical symptoms, imaging fi ndings, and response to treatment can defi ne the diagnosis. staphylococcus aureus is a gram positive coccus and is coagulase positive staphylococcus. the pathogenic substances of staphylococcus aureus mainly are toxins and enzymes, such as hemolytic toxins, leukocidin and enterotoxin, which play a role in hemolysis, necrosis, killing leukocytes and vascular spasm. the staphylococcus aureus coagulase is the main reason for suppurative infection. pneumonia caused by inhaled staphylococcus aureus through the respiratory tract often shows lesions in the large lobes or extensive fusion of bronchopneumonia lesions. bronchial and alveolar rupture allows gas to enter the pulmonary interstitium, which is communicated with the bronchi. in the cases of bronchiolar blockage by necrotic tissues or pus, the one-way valve effect is formed to cause tension pulmonary emphysema. in the cases with superfi cial pulmonary emphysema with excessively high tension, it ruptures to form pneumothorax or pyopneumothorax, as well as bronchooleural fi stula ( fig. 17.45a, b ) . the symptoms include chills, persistent high fever, cough, expectoration, chest pain and other symptoms. there is no sign in the early period. symptoms are scattered moist rales in both lungs, being in consistency to severe toxic symptoms and respiratory symptoms. yellow purulent sputum is the typical characteristics of staphylococcus aureus pneumonia. in the cases with larger lesions or fusion of lesions, signs of parenchymal changes, pneumothorax or pyopneumothorax can be found. in the sputum or pleural fl uid smears examinations, the bacteria with a concentration being no less than 107 cfa/ml is the pathogen, the bacteria with a concentration being 105-107 cfa/ml is the suspected pathogen, and the bacteria with a concentration being less than 105 cfa/ml is the contaminated bacteria. there are increased wbc count and neutrophils, leftward migration of the nucleus and possibly no increase of wbc count in aids patients. immunofl uorescence, enzyme-linked immunosorbent assay and counter immunoelectrophoresis can be performed to detect serum antigen or antibody of the pathogenic bacteria, which can defi ne the diagnosis. polymerase chain reaction has certain signifi cance in pathogen detection. the protected bronchoscopic specimen (pbs) and bronchoalveolar lavage (bal) can be applied to collect the specimen, which has reduced chances of specimens contamination by oral bacteria. biphasic tv monitors guided pulmonary puncture and suction for pulmonary tissues examinations can be performed to detect the real pathogenic bacteria. both are the most commonly used imaging examinations. the diagnostic imaging demonstrates staphylococcus aureus pneumonia as lesions in the inner zone of both middle lower lungs. there are singular or multiple parenchymal changes in patchy or lobar distribution that may fuse into large fl akes. it may be complicated by cavity and pulmonary emphysema, with surrounding compensatory emphysema. a male patient aged 37 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever with a body temperature of about 39 °c and chest pain for 2 months. his cd4 t cell count was 31/μl. chills, fever, cough, expectoration, chest pain and other symptoms commonly; hemoptysis and dyspnea rarely the patients may be found with fever appearance, rarely shortness of breath and cyanosis. in the serious cases, the body temperature can be as high as 39-40 °c and blood pressure decreases, with signs of shock. by chest examinations, decreased ipsilateral respiratory motion; increased or decreased fremitus, dull sound in percussion; bronchial breathing sounds or moist rales by auscultation; rarely pleural friction and weakened breathing sounds. increased wbc count and neutrophils, possible the nucleus left shift; no increase or even decrease of wbc count in aids patients; staphylococcus aureus positive by blood culture. sputum or pleural fl uid smears examinations for pathogenic bacteria culture is positive, and antibiotic sensitivity test is positive. by chest x-ray and ct scanning, the most common demonstrations are lesions of bronchial pneumonia. the fi ndings of pulmonary emphysema and cavities can facilitate the diagnose. the lesions should be differentiated from infi ltrative parenchymal bronchioloalveolar carcinoma. the smaller lesions should be differentiated from pulmonary infarction. the large lesions should be differentiated from obstructive pneumonia. it is diffi cult to identify the types of common bacterial pneumonia simply by chest x-ray and ct scanning. in combination to the laboratory tests, the diagnosis can be defi ned. the incidence of pyogenic bacterial infection is increasing in aids patients, which is caused by their weakened cellular and humoral immunity. the manifestations of these most common bacterial infections are similar to those of non-hiv infected patients by chest x-ray. bacterial pneumonia and purulent bronchitis are the most common causes of pulmonary infections in aids patients. particularly, they are frequently found in patients with a history of intravenous drug abuse and smokers. they are histologically characterized by infl ammations of the bronchi and bronchioles as well as infl ammatory exudates and mucus in the airway lumens. ct scans facilitates the diagnosis of bronchiolitis and early bronchial pneumonia. the demonstrations are characterized by (1) small centrilobular nodular shadows, which is the cross sectional demonstration of bronchioles fi lled with infl ammatory substances and its surrounding infl ammations; rhodococcus equi infection is one of the zoonotic diseases, which commonly occurs in the grazing areas. patients with t lymphocyte immunodefi ciency caused by aids and other factors are especially susceptible to the infection. rhodococcus equi was fi rstly discovered in 1923 and was nominated as corynebacterium equi. after structure analysis of the cell wall, it was found that the bacterium is quite different from corynebacterium, and therefore classifi ed as rhodococcus. rhodococcus equi infection in human is rare. but in recent years, due to an increase of patients with immunodefi ciency syndrome, reports on rhodococcus equi caused human respiratory infections and sepsis are increasing. in the past, the toxicity mechanism of rhodococcus equi was mostly speculated. until recently, the damage process of toxic plasmid to human tissue is discovered, which presents a new way for the study of the pathogenesis of rhodococcus equi infections. rhodococcus equi is one of the facultative parasites in the cells and its optimum growth temperature is 30 °c, with a suitable growth temperature of 10-40 °c. acid-fast staining of rhodococcus equi shows uncertain results. due to its morphological diversity, it is often mistaken as diphtheroid bacillus, bacillus or micrococcus. in sheep blood agar, rhodococcus equi can have synergistic hemolysis with staphylococcus aureus, listeria monocytogenes and corynebacterium pseudotuberculosis, which is a characteristic manifestation of rhodococcus equi. the most common pathological changes in rhodococcus equi infection are chronic purulent bronchitis and extensive lung abscess. imaging often demonstrates subacute pneumonia, commonly with cavities. the clinical manifestations are poor appetite, drowsiness, fever and shortness of breath. studies by e marchiori et al. [ 30 ] in 2005 revealed that all the 5 cases of aids complicated by rhodococcus equi pulmonary infection have cough and fever lasting for 1-2 months, with accompanying shortness of breath and chest pain. all the 13 cases, studied by li et al. in 2011 [ 106 ] , have fever with a body temperature up to 38-40 °c and cough. in addition, there are also expectoration with orange red sputum in 10 cases, hemoptysis in 4 cases, dyspnea in 11 cases, moist rales of lungs in 13 cases, emaciation in 6 cases, poor appetite in 6 cases, diarrhea in 2 cases, joint pain in 1 case, oral candidiasis infections in 13 cases, oral herpes in 4 cases, chest pain in 4 cases, no obvious symptoms in 1 case and hepatitis b in 3 cases. typical clinical manifestations of this disease are fever, cough, dyspnea and chest pain, while others such as emaciation, diarrhea and joint pain are not representative symptoms. identifi cation of the bacteria various specimens were inoculated on blood plates at a temperature of 35 °c for 18-24 culture, with bacteria growth of 18 strains. they are biologically characterized by a diameter of about 0.5 mm, non-transparent and slight yellowish colonies. after 48-72 h, the colonies expand to 1-2 mm, which can be emulsifi ed in mucous fl uid liked state. most of the colonies produce orange and orange red pigments, which can be cultured in ordinary agar. histopathological fi ndings are typical for rhodococcus equi infection. h&e staining demonstrates mainly bleeding in the alveolar space, large quantity epithelial cells, possibly predominant fi broblasts, parenchymal changes of lung tissue and thickened alveolar septa. pas staining demonstrates scattered or clustered rhodococcus equi in pink or purplish red. both are the most commonly used imaging examinations. biphasic tv monitor guided lung puncture can be performed to suck lung tissues for biopsy, based on which the real pathogenic bacteria can be detected. the typical demonstrations include central hilar sphere liked shadow with increased density in unilateral lung, accounted for 70 %. there are also manifestations of exudative infi ltration and large fl aky or spherical mass shadows in the right or left hilar area. the lesions are in patchy or fl aky appearance, radiating from the hilum to the lung fi eld with blurry boundaries. a male patient aged 30 years was confi rmatively diagnosed as having aids by the cdc. he complained of recurrent fever, cough and chest pain for 13 days; and was found hiv positive for 8 days. he was also a carrier of hepatitis c virus, with symptoms of fever with no known causes, cough, chest distress and weak limbs. by examinations, he was found to have complexion of chronic conditions; many moist and dry rales by cardiopulmonary auscultation. he had a past history of hiv positive for 8 years, with drug abuse and extramarital affairs. the history of present illness includes fever, paroxysmal cough with a little whitish yellow thick sputum since may 8, 2008 . he also had subjective paroxysmal dull pain in the left chest, and hemoptysis once which was bright red with blood clot in a volume of about 80 ml. his cd4 t cell count was 10/μl. by sputum culture, rhodococcus equi was found positive. after receiving antibiotic treatment, his conditions improved and he was discharged from the hospital. catalase test of the strain is positive, which is confi rmed as rhodococcus equi by api coryne system. examinations h&e staining demonstrates mainly bleeding in the alveolar space, large quantity epithelial cells or mainly fi broblasts, parenchymal changes of lung tissue and thickened alveolar septa. pas staining and masson staining demonstrate scattered or clustered distribution of rod rhodococcus equi in pink or purplish red. aids complicated by pulmonary rhodococcus equi infection shows subacute infl ammation. firstly, there are exudates in the surrounding area of unilateral hilum as well as sphere shaped mass shadows which is centrally dense and peripherally blurry, with its apex pointing to the hilum. the lesions can be complicated by cavities and fl uid level, with thick wall of the cavities. as the disease progresses, the abscess cavities have increased tension, with gradually thinner abscesss cavity walls and uneven wall thickness, even showing pleural effusion. these are its characteristic imaging fi ndings. it often needs to be differentiated from pneumocystis carinii pneumonia, tuberculosis, staphylococcus aureus pneumonia, central type lung cancer and other diseases. imaging fi ndings of pneumocystis carinii pneumonia usually are ground glass liked changes in the lung fi eld, with parenchymal changes and centrilobular nodules. tuberculosis often shows miliary tuberculosis, with lymphadenectasis, large tubercles and parenchymal changes. these characteristic pathological and imaging fi ndings of staphylococcus aureus pneumonia are similar to those of bronchial pneumonia (lobular pneumonia). the lesions are nodules with blurry boundaries in a diameter of 4-10 mm. the disease progresses rapidly, while pulmonary rhodococcus equi infection has a chronic progression. hrct scanning demonstrates staphylococcus aureus pneumonia as centrilobular nodules and branch linear shadows (tree buds sign), which can be found in 40 % patients, with 15-30 % will develop into commonly singular pulmonary abscess. chest ct scanning demonstrates round liked abscess cavity with thick wall and liquid level in it. the inner wall of the abscess cavity is often irregular, with various changes within short period. the central type of lung cancer is commonly demonstrated as round liked shadow of unilateral hilum with rough boundary. lobulation or bronchial stenosis sometimes occurs. however, aids complicated by rhodococcus equi pneumonia is demonstrated as sphere liked mass in the hilum; mostly sphere liked increased density shadow with hilum as the center. the shadow is centrally dense and peripherally blurry, with no bronchial stenosis. rhodococcus equi was fi rstly discovered in 1923 and was nominated as corynebacterium equi. after structure analysis of the cell wall, it was found that the bacterium is quite different from corynebacterium, and therefore classifi ed as rhodococcus. rhodococcus equi is generally believed to be the pathogen for horses, pigs and cattle. [ 106 ] showed a ct4 t cell count of lower than 49/μl. all the results are in consistency. in conclusion, aids complicated by pulmonary rhodococcus equi infection is mainly subacute infl ammation. there are exudation around the unilateral hilum as well as centrally dense and peripherally blurry sphere shaped mass shadows, with secondary cavities and parenchyma changes and even pleural effusion. all of these are characteristic imaging demonstrations. most cases of hiv positive complicated by respiratory or pulmonary diseases are caused by aspergillus fumigatus. aspergillus fumigatus belongs to filamentous fungi, which is a common opportunistic fungus and has a wide distribution in the nature. as conditional pathogenic bacteria, it can parasitize in the human skin and upper respiratory tract. human has certain resistance to aspergillus so it commonly fails to cause diseases. in immunocompromised aids patients, the pathogenic bacteria can pass through the defects in the skin and mucous membrane into the blood flow to infect the tissues and organs. aspergillus commonly violates bronchus and lung, with involvements of rhinal sinuses, external auditory canal, eye and skin. otherwise, it disseminates to organs of the body along with blood fl ow. the early lesions are diffuse infi ltrative and exudative changes. and advanced lesions are necrosis, pyogenesis or granuloma. large quantity hyphae can be found in the lesions. the hyphae penetrate the blood vessels to cause vasculitis, perivascular infl ammation and thrombosis. and thrombosis can cause ischemia and necrosis of the tissue. according to the pathological changes and imaging fi ndings, it can be divided into three major types: vascular invasion type, bronchopneumonia type and allergic bronchopulmonary aspergillosis type. (1) the vascular invasion type is the result caused by toxins released in the process of aspergillus spreading extensively from the primary focus to the lungs. vascular infi ltration of the pulmonary parenchyma and coagulative necrosis are believed to be the cause of vascular occlusion and pulmonary infarction. (2) bronchopneumonia type is acute bronchitis caused by inhalation of aspergillus spores. in the cases of hyphae invasion into the lung tissues, extensive infi ltrative pneumonia or focal granuloma are resulted in. it can also cause necrosis, pyogenesis and multiple small abscesses. spherical pulmonary aspergillosis is often secondary to bronchiectasis, tuberculosis, carcinous cavity and other lung diseases. mycelia multiply and gather in the cavities of the lungs to form a spherical mass with fi brin and mucosal cells, which are called aspergillar glomera, which do not invade the lung tissue. (3) allergic bronchopulmonary aspergillosis type is the proliferation and germination of inhaled aspergillus spore in the airway, often showing obvious related mucosal lesions and eventually resulting in bronchiectasis ( fig. 17.64a-c ) . the cases with acute onset have symptoms of high fever or irregular fever, cough, shortness of breath and green purulent sputum. the cases with a chronic onset have symptoms of repeated cough and hemoptysis, which are similar to those of tuberculosis. the pulmonary signs are not obvious, with occasional fi ndings of moist rales. most cases are asymptomatic and sometimes there are fever, cough, shortness of breath, and mucous purulent sputum. the main symptoms are persistent fever, cough and chest pain. in the serious cases, there is dyspnea. by microscopic examination of sputum, aspergillus hyphae can be found. the culture for aspergillus fumigatus is positive. serum ige is commonly above 2,500 μg/l. skin test for aspergillus antigen is positive. serum anti-aspergillus antigen igg antibody precipitin is positive. puncture of lungs and pleura for biopsy facilitates the diagnosis of pulmonary fungal infections. both are the most commonly used imaging examinations. hiv/aids related aspergillus bronchopneumonia is commonly demonstrated to have increased pulmonary markings, diffuse patchy blurry shadows and mass shadows in both lungs. spherical pulmonary aspergillosis is commonly demonstrated to have sphere liked aspergillar glomera suspending in the cavities to form a crescent shaped transparent area, in characteristic meniscus sign, rolling ball sign and fi ngertip sign. meniscus sign is nominated due to a meniscus liked space between the aspergillar glomera growing in the cavity and the cavity wall. rolling ball sign means that the aspergillar glomera moves along with the changes of posture. fingertip sign indicates that the substance formed by aspergillar glomera in dilated bronchi is in a fi nger shape, sometimes in v shape sign and y shape sign. invasive lesions refer to lesions invading or destroying lung structures, such as pneumonia, parenchymal changes and necrosis. a female patient aged 28 years was confi rmatively diagnosed as having aids by the cdc. she complained of cough and chest distress, with increased eosinophilic granulocytes. her cd4 t cell count was 45/μl. a male patient aged 36 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever or irregular fever, cough and shortness of breath. his cd4 t cell count was 96/μl. a male patient aged 38 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever or irregular fever, cough and shortness of breath. his cd4 t cell count was 76/μl. a male patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. he was hospitalized due to complaints of fever for 1 week, chest distress and shortness of breath for 1 day. on admission, he was confi rmed as hiv positive, with a cd4 t cell count of 9/μl. by physical examinations, he was in poor physical conditions, respiratory rate 27/min, lips cyanotic, coarse breathing sounds of both lungs with small quantity dry rales. his conditions progressed rapidly and death occurred due to respiratory failure after 3 days. lung and pleura puncture for biopsy can detect the growth of aspergillus hyphae. sputum culture can detect aspergillus hyphae, with fi ndings of aspergillus fumigatus positive. in the cases with allergic bronchopulmonary aspergillosis, the serum ige is above 2,500 μg/l. skin test of aspergillus antigen is positive. the serum anti-aspergillus antigen igg antibody precipitin is positive. characteristic ct scanning demonstrations of hiv/aids related parasitic aspergillar glomera include pulmonary cavities or cavity lesions with spherical contents, smooth boundaries of the spherical contents with even density, lunate shaped or ring shaped transparent shadows between cavity or cavity walls and the contents, migration of the contents with the body postures. according to the pathological and imaging demonstrations, it can be divided into three major types: in the early stage, ct scanning demonstrates soft tissue density nodules or light ground glass liked halo sign around the mass, which is the evidence for the diagnosis of the invasion type pulmonary aspergillosis. air cresent sign refers to round pulmonary infi ltration with accompanying central necrosis and surrounding lunate or ring shaped cavity. other noncharacteristic ct scanning demonstrations include multiple lobular parenchyma lesion shadows or lobular fusion shadows, parenchyma lesion shadows in the lobes, segments and subsegments, nodular or mass shadows and thin/thick wall cavities or low density areas in the mass shadows. it is demonstrated to have parenchymal lesions around the airway or/and central small nodules in the lower lobes. the parenchymal lesions prove the occurrence of mycotic bronchopneumonia. the most common imaging fi nding is the thickened bronchial wall. central bronchiectasis is its characteristic demonstration. in the cases of dilated bronchi containing sputum bolt or mucus, it shows fi ngertip shaped or toothpaste shaped shadow, which should be considered as its characteristic demonstration. hiv/aids related pulmonary aspergillosis should be differentiated from congenital bronchial atresia. most cases of the congenital bronchial atresia are atresia at the proximal pulmonary segment of the bronchi, often with a clearly defi ned mass. in the typical cases, there are bronchial branches and more branches in fi ngertip shape, pointing to the pulmonary hilum. confi ned pulmonary air retention in the pulmonary lobe and segment of the atresic bronchi is the important evidence for the diagnosis of congenital bronchial atresia. hiv/aids related pulmonary aspergillosis should be differentiated from allergic bronchial-pulmonary aspergillosis. in the cases of allergic bronchial-pulmonary aspergillosis have no clearly defi ned mass, with demonstrations of v shaped, y shaped, grapes shaped or fi ngertip shaped shadows with clearly defi ned boundaries, which are characteristic in those patients with bronchial asthma or a case history of exposure to dusts containing fungi. there is also increased proportion of eosinophilic granulocytes in the peripheral blood. detection of aspergillus in phlegm can defi ne the diagnosis. hiv/aids related pulmonary aspergillosis should be differentiated from central lung cancer. central lung cancer also can cause mucus impaction of the distal bronchi, with manifestations of bronchial arctia and/or truncation, and the surrounding soft tissue mass shadows. hiv/aids related pulmonary aspergillosis should be differentiated from pulmonary cavities and abscesses induced by dissolved tuberculoma, secondary pulmonary tb, chronic lung abscess and peripheral lung cancer as well as cystic bronchiectasis. except aspergilloma, spheric morphology caused by other causes is commonly irregular. the cavity contents cannot migrate with body postures, which is the key point for the differential diagnosis. hiv/aids related pulmonary aspergillosis can be caused by many pathogenic bacteria and aspergillus fumigatus is the most common one. the infection is often caused by inhaled aspergillus fumigatus in the environment. vascular invasion type of pulmonary aspergillosis usually has multiple lesions and nodular changes. generally in pathology, the center of nodule presents typical pale color; commonly with fi brous ring surrounding the nodules resulted from hemorrhage and/or lung parenchymal changes. histologically, they are characterized by coagulative necrosis of the lung tissues, infi ltration of large quantity hyphae in the necrotic tissue, pulmonary vascular infi ltration, but usually without responses of vasculitis and thrombosis. the enzymes released by neutrophile granulocytes can cause the separation of necrotic tissue from its adjacent lung tissues to form necrotic mass in the cavities. airway invasion type of pulmonary aspergillosis, also called aspergillus bronchopneumonia, accounts for 15-30 % of invasive aspergillosis. the most common imaging fi ndings are unilateral/bilateral fl aky parenchymal changes, centrilobular small nodules and branches liked linear shadows (tree buds sign). histologically, it is characterized by necrosis and infi ltration of neutrophil granulocytes. the lesions surround the bronchiole and the bronchiole. the invasion of the pulmonary artery can cause bleeding of the adjacent pulmonary parenchyma. allergic bronchopulmonary aspergillosis rarely has lesions, with no unknown pathogenesis, which is generally believed to be related to type i and type ii allergic reactions. it usually shows obvious asthma related mucosal lesions. hyphae generated by aspergillus fumigatus can induce the production of mucus and additional mucosal lesions, eventually leading to bronchiectasis. dilatate bronchial lumen is fi lled with mucus or with absence of epithelium, which is replaced by a granulomatous infl ammatory infi ltration. the most common imaging manifestations are migratory fl occulent, branched y shaped and v shaped (fi ngertip sign) shadows in the pulmonary parenchyma, which are related to the infi ltration of eosinophils. pathologically, bronchial cystic dilatation in the pulmonary segment and sub-segment occurs, with large quantity eosinophils in the bronchial mucus and scattered broken aspergillus hyphae. in combination with the case history, the diagnosis can be defi ned. compromised immunity is an important cause of cryptococcosis, especially in patients with aids or abnormal lymphoproliferative diseases. cryptococcus neoformans, a single phase mould, exists widely in the natural world. the cryptococcus has a diameter of less than 10 μm, which can be inhaled into the human body via respiratory tract. under the impact of a high concentration carbon dioxide, it forms a clearly defi ned protective layer composed of polysaccharide capsule to antagonize the defense mechanisms of the host. thus, lung infection occurs after its inhalation in immunocompetent people, which is commonly asymptomatic. of cryptococcus, inhalation of cryptococcus by aids patients can lead to hilar lymphadenopathy, as well as singular or multiple subpleural small nodules, being similar to those in the cases of mycobacterium tuberculosis infection. in the early stage of cryptococcal infection, only a mild infl ammatory reaction or diffuse infi ltrative exudative changes occur. but in the advanced stage, necrosis, suppuration or granuloma is formed. large quantity hyphae can be found in the focus. in the cases with hyphae penetrating the blood vessels, vasculitis, perivascular infl ammation and thrombosis occur. and thrombosis leads to ischemia and necrosis of the tissue (fig. 17.77 ). pulmonary cryptococcus infection in aids patients often is extensively disseminating, with symptoms of fever, cough, diffi culty breathing, expectoration, chest pain caused by pleuritis, and even acute respiratory distress syndrome (ards). hiv/aids related pulmonary cryptococcus infection has no characteristic imaging demonstrations. chest x-ray and ct scanning show multiple morphology of the lesions. in the slight cases, there are thickened pulmonary markings in both lower lungs or isolated nodular shadows, and occasionally cavities. in the cases of acute interstitial infl ammation, there are diffuse infi ltrative or miliary foci, with infi ltration, nodules or exudation in any lobe which is more common in bilateral middle and lower lungs, in unilateral lung or confi ned to one lobe. the foci may be isolated huge spherical or multiple nodular, without obvious surrounding infl ammatory responses, similar to those of tubercles or tumors. otherwise, they are diffuse miliary shadows or fl aky infi ltrative shadows. a male patient aged 60 years was confi rmatively diagnosed as having aids by the cdc. he was hospitalized on 2009-2-19 due to headache and vomiting for more than 10 days. in the csf, cryptococcus was found. by blood and sputum culture, cryptoccocus was detected. the diagnosis was cryptococcal meningitis, cryptococcal pneumonia and cryptococcal sepsis. after receiving amphotericin b antifungal treatment and dehydration, headache and vomitting were relieved. but chest ct scanning reexamination demonstrated increased pulmonary lesions, which was considered as tuberculosis. on 2009-4-1, he was given herv anti-tuberculosis treatment. twenty days ago he sustained weakened lower limbs, which gradually aggravated and completely paralyzed in the recent 1 week, his cd4 t cell count was 34/μl. hiv/aids related pulmonary cryptococcus infections should be differentiated from tuberculosis, primary or metastatic lung cancer. tuberculosis mostly is secondary tuberculosis, which is caused by repeated infections of tubercule bacillus. lesions show fl aky or fl occulent shadows in the two upper lungs, with blurry boundaries. the wrapped necrotic foci by fi bers develop into nodules. it can also show miliary shadows, mostly with mediastinal lymphadenectasis. it should also be differentiated from primary or metastatic lung cancer. cryptococcus is a relatively common pathogen of pulmonary fungal infection, and mostly develops in aids patients. usually, it is a disseminated disease, with common involvement of the central nervous system and the lungs. in immunocompetent patients, the nodular granuloma caused by the pathogen is similar to those of other pulmonary fungal infections. in patients with serious immunosuppression, wide tissue infi ltration of the pathogens may occur in the lungs. liked shadows, parenchymal changes of air cavity and miliary nodules. the pathogenic fungi can be found mainly in the pulmonary interstitium. imaging fi ndings include singular or multiple nodules or masses, parenchymal changes of lung lobes and lung segments with clear or unclear boundaries in size of 1-10 cm. there may be also miliary lesions, lymphadenectasis and cavity shadows. candida is an opportunistic pathogen, which widely exists in nature. candida albicans parasitize in the oral cavity, laryngopharynx, upper respiratory tracts, vaginal and intestinal mucosa of human being. pulmonary and bronchial moniliasis is commonly caused by candida albicans which has the strongest pathogenicity. after its invasion into the tissues, candida transforms into hyphae and multiplies in a large quantity, with strong toxicity and ability to fi ght against phagocytosis. aids patients may have disseminated pathological changes. only when the immunity is compromised, the pathogen invades into the bronchus or lungs to cause diseases. therefore, pulmonary candida infection is commonly secondary. candidosis can cause acute infl ammation in bronchus and lungs, mainly exudation of neutrophils, which can be divided into two types: bronchitis type and pneumonia type. the pathological changes in the early stage are acute suppurative infl ammation, accompanied with the formation of abscesses. by the naked eyes observation, they are large fl aky parenchymal changes, with central grayish white coagulative necrosis. under a microscope, the lesions are large fl aky caseous necrosis, accompanied with the formation of abscesses, and surrounding infi ltration of hyphae and phagocytes. in the advanced, there are caseous necrosis, formation of cavities, fi brosis and granuloma. symptoms in aids patients are mild, with frequent cough, with a small amount of white mucous phlegm or thick phlegm, no fever or low grade fever; scattered spots of white membranes in the mucosa of oral cavity, throat and bronchus. dry rales can be heard occasionally in both lungs. in aids patients, the manifestations are mostly acute pneumonia or sepsis, with chills, fever, cough, expectoration of white mucous jelly liked phlegm or thick phlegm often with blood or necrotic tissue. the thick sputum, candidal hyphae and shedding cell debris can be condensed into small colloid clumps, with yeast smell. other symptoms include even haemoptysis and diffi culty breathing. dry and moist rales can be heard in lungs. symptoms may be diffi culty breathing, rhinocnesmus, runny nose and sneezing. wheezing rales can be heard in both lungs. chest x-ray chest x-ray demonstrates nodular shadows and fl aky parenchyma changes in unilateral or bilateral lungs. sometimes there is miliary infection. ct scanning demonstrates most lesions in the middle and lower lung fi elds, with rare involvement of the apex. there are thickened lung markings or diffuse small fl aky/patchy shadows, some of which can fuse into large fl aky dense shadows, with blurry boundaries. nodules, due to bleeding around it, may be surrounded by ground glass liked shadows, which is necrotic bronchopneumonia, usually accompanied with a large quantity neutrophils. cough expectoration with white mucous phlegm or thick phlegm, hemoptysis and shortness of breath. examinations of the oral cavity and the throat demonstrate spots liked white membrane covering the surface, and dry and moist rales in the lungs. successive cultures of phlegm, lung tissue, pleural fl uid or cerebrospinal fl uid repeatedly demonstrate the same strain of candida, or direct microscopic fi ndings of large quantity pseudohyphae or hyphae and groups of spores can defi ne the diagnosis. it is demonstrated to have thickened and deranged lung markings in double lung, diffuse small fl aky/patchy shadows, fusion of some small shadows into large fl aky dense shadows, with blurry boundaries, enlarged hilum and blurry structures. the conditions progress rapidly, with repeated lesions occurrence. hiv/aids related pulmonary candida infection should be differentiated from bacterial pneumonia. bacterial pneumonia often has symptoms such as high fever, cough, expectoration, chest pain and shortness of breath. ct scanning demonstrates fl occulent infi ltrative shadows or parenchyma changes and cavities. the pathogen can be detected in the sputum or chest liquid. hiv/aids related pulmonary candida infection should be differentiated from virus pneumonia. viral pneumonia fi rstly causes upper respiratory tract infection, which spread downward to cause pulmonary infl ammation. the demonstrations a male patient aged 40 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever, cough, expectoration, chest pain and shortness of breath, with pulmonary parenchymal changes sign and moist rales. his cd4 t cell count was 18/μl. include ground glass liked changes in the lung fi elds or mass shadows. the defi nitive diagnosis should be based on throat swabs, virus isolation from the sputum and serum specifi c antibodies test. hiv/aids related pulmonary candida infection should be differentiated from pulmonary tuberculosis. in the early stage, the symptoms and signs include irritative dry cough, expectoration, hemoptysis and cavities in lungs. (detailed manifestations of tuberculosis see the section about tuberculosis in this chapter) its diagnosis mainly should be based on chest x-ray and fi ndings of tubercule bacillus in sputum or other specimens, or tuberculosis specifi c pathological changes. hiv/aids related pulmonary candida albicans is a widespread dimorphism bacteria. the oval shaped budding yeasts and hyphae both can be found in the tissues. candidiasis is a common disease in aids patients. chest x-ray demonstrates unilateral or bilateral patchy parenchymal changes of the air cavity and nodules with unclear boundaries. miliary lesions are common. hrct demonstrates multiple nodular shadows in both lungs, often accompanied with parenchymal changes. its defi nitive diagnosis should be based on the fi ndings of candida albicans in the tissues. penicillium marneffei (pm) is a newly found penicillium in 1956, which is a special strain with a distribution in south east asia and southern china. rhizomys is its natural host. in 1973, disalvo et al. [ 12 ] reported the fi rst case of natural human pm infection. in 1984, the fi rst case of human pm infection in china was reported in guangxi zhuang autonomous zone. pm is an opportunistic pathogen and immunocompromised people are susceptible to its infection. its spreading is along with soil contaminated by rhizomys feces to invade human body via the respiratory tract, the digestive tract and skin defects. pm infection is believed to be one of the most common opportunistic infections in aids patients in southeast asia, which has an increasing incidence. pm is the only dimorphic fungus in hyphomycetes penicillium, which is a special strain of penicillium. at different culture temperatures, it shows conversion of biphasic forms: fungal phase at 25 °c and yeast phase at 37 °c. the fungal phase is the hyphae of many cells, with certain biological morphology, such as penicillus, conidiophore, chain liked conidiospore and chains between spores. the yeast phase shows unicellular or bicellular form. in the growth process of pm, large quantity bright rosy or dark rosy pigments are produced, which is characteristically pm. the pigment of yeast phase is secondary metabolites of cells with strong hydrophobicity, which can promote the adhesion of conidiums in fungal phase and cells in yeast phase to the alveolar macrophages and other cells surface in the human body. the pigment monoclonal antibody (mab) can interrupt the pathological process of adhesion. in addition, this pigment can determine the expression of cluster-encoding genes mbr through diffusion and penetration of drugs to the cell membrane, thus preventing the penetration of hydrophilic antifungal drugs, such as fl uconazole. that is to say, it improves the natural antifungal resistance level of pm. the soluble components of the pigment in fungal phase can trigger the generation of anti-conidium antibody (only igg) in animals to prevent its spreading in the body. the phenomenon proves that, in terms of tissue invasion, fungal phase is less powerful than yeast phase. conidium in fungal phase is the carrier of pathogen while the cells in yeast phase are the real pathogenic factors. when pm spreads to the target organ along with the blood fl ow, it is engulfed by mononuclear phagocytes. in the cases of replication itself and further spreading, reactive proliferation of phagocytes is caused. mononuclear phagocyte system has strong defense ability. in the cytoplasm of proliferated mononuclear phagocytes, various amounts of pm can be found. pm mainly invades into the body via the respiratory tract, digestive tract and skin defects. in immunocompetent people, local abscesses form in the invasion site, which is characterized by the thick mucus fl uid, with mainly necrosis and liquefaction. vascular reactions and exudation of neutrophil leukocytes and body fl uids is less than abscesses induced by common purulent bacteria. the clinical manifestation is confi ned suppurative infl ammation. when the immunity is compromised, due to the insuffi ciency of immunologic factors, it is diffi cult for the immune cells to restrict and digest the engulfed pathogens, which leads to confi ned suppurative reaction. therefore, it often presents with diffuse lesions. the pulmonary lesions are principally interstitial exudative infl ammation. the typical penicillium marneffei disease has acute or subacute onset, along with fever, chills and shivers, cough and expectoration, hemoptysis, shortness of breath, abdominal pain, diarrhea, bloody stool, fatigue, central necrotic papula mainly in the head and face and scattering in the trunk and extremities as well as hepatosplenomegaly. bone marrow smear and pas staining can be performed to detect the pathogen. blood, bone marrow, pleuroperitoneal fl uid, phlegm and skin defect tissue are collected for the culture at double temperatures with sabouraud'broth medium. at the temperature of 25 °c, the colony is in dark red with villous surface, with surrounding red wine liked pigments to gradually spreading into the medium. biopsy of lymph nodes and skin defects with pas staining and wright & gimsa staining can be performed. wbc count, hemochrome, platelets, ast and cd4 t cell count. ct scanning and routine chest x-ray are the diagnostic imaging examinations of choice, which can facilitate to understand the size, morphology, location, quantity and density of the lesions. it demonstrates multiple small nodular shadows in the lungs, multiple honeycomb liked cavities in both lungs and mediastinal lymphadenectasis. abdominal scanning demonstrates different degrees of hepatic, splenic and retroperitoneal lymphadenectasis, which can fuse into a huge mass. routine chest x-ray it demonstrates thickened, deranged and blurry pulmonary markings, small cavities, military nodular shadows, mass liked shadows, spots and patchy shadows, ground glass liked changes, pleuritis and pleural effusion. a female patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. her husband had a history of drug abuse and she complained of abdominal pain and fever for more than 2 months, with accompanying face rash and diarrhea. her cd4 t cell count was 5/μl. by examinations, she sustained skin palpula, abdominal tenderness, central concave skin rashes on the face, neck, and upper limbs. there was a palpable mass in the upper left abdomen, hard and tenderness, in a size of 12 × 12 cm. more than 2 months before her admission, she had persistent dull abdominal pain that is commonly in the upper left abdomen, with accompanying fever and face skin rashes that is gradually increasing and spreads to the neck and upper limbs. she also had hepatosplenomegaly, abdominal aortic lymphadenectasis and ascites. a female patient aged 51 years was confi rmatively diagnosed as having aids by the cdc. she had an unhealthy sexual life, with complaints of fever and cough for more than 1 month. her cd4 t cell count was 7/μl. a male patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. he had been found to be hiv positive for 5 years, with complaints of irregular fever, cough, fatigue and dizziness for 10 days to be hospitalized. by examinations, his cd4 t cell count was 40/μl, hiv positive, subcultivation of strains demonstrated typical biphasic penicillium. by fungus culture, typical penicillus was found. by bone marrow smear, the round corpuscles mainly located in the macrophages. a male patient aged 32 years was confi rmatively diagnosed as having aids by the cdc. he had a history of extramarital affair, with complaints of fever, cough, chest distress, shortness of breath, fatigue, poor appetite, poor sleep, weight loss and shortness of breath after activities for more than 3 months. his cd4 t cell count was 23/μl. chest x-ray and ct scanning both demonstrate multiple nodules in different sizes and cavity shadows. the cavities cluster into honeycomb liked changes with uneven thickness of the walls, clear boundaries and surrounding infl ammatory exudates. some of the nodules infuse into mass dense shadows. lesions in both lungs have a symmetrical or asymmetric distribution, with no characteristic leions. mediastinal lymph nodes are obviously enlarged. sputum smear for direct microscopy demonstrates candida. pms are found by fi brobronchoscopy lavage smear and biopsy. (1) candida skin test shows positive. (2) fluorescent antibody test for pms is performed in procedures of direct smear, fungal colony culture and histopathological examination of the tissue sections. metabolites test of pm and pcr can be performed to determine the gene sequences of pm for the early diagnosis. hiv/aids related penicillium marneffei pneumonia should be differentiated from bronchiectasis and blood borne staphylococcus aureus pulmonary abscess. although the cases of bronchiectasis are demonstrated to have clustering round shadows on the cross sections, the wall is thinner and even, accompanying to the spots liked vascular shadows. some lesions have typical railway liked bronchial dilation signs. it is demonstrated to have multiple small cavity lesions in both lungs in line with the evenly distributing blood borne lesions. the lesions are rarely clustering. the wall of the cavities is thick and even with surrounding marginal exudates and blurry boundaries. with the steady increasing of hiv infections, reports on the complication of pm disease have also been increasing recently. the disease can be localized but mostly disseminated, with the involvement of lungs, liver, skin, lymph nodes and other tissues and organs. therefore, it is known as penicilliosis marneffei or disseminated penicillium marneffei infection in literature reports. due to the insuffi cient knowledge about the disease, diagnosis is delayed or missed. in thailand, penicilliosis marneffei has been the indicator disease of aids. about 20 % aids patients are infected by pm and 70 % have necrotic papula, which is characteristically disseminated penicillium marneffei infection. ct scanning demonstrates hiv/aids complicated by disseminated penicillium marneffei infection as fl aky parenchyma shadows in the lungs, clustering of cavities and nodular shadows, mediastinal lymphadenectasis and pleural infl ammation responses, which can facilitate its clinical diagnosis. mucormycosis is a rare kind of conditional fungal disease, with its pathogen, mucor, distributing widely in the natural world. it generally fails to cause diseases, but can cause systematic infections in immunocompromised people. mucor often invades into the human body via the nose to cause paranasal sinuses and orbital infections, which can further invade into the brain to cause meningitis and frontal abscesses. pulmonary mucormycosis is only second to the nasal-cerebral infection. it can spread via the respiratory tract to cause pulmonary infections. in addition, there are also skin and gastrointestinal mucormycosis as well as disseminated mucormycosis. the pathological changes of hiv/aids related pulmonary mucormycosis are mainly hemorrhagic necrotic infl ammation. the defense mechanism of immunocompetent people is to kill the fungal spores with the phagocytosis of macrophages and oxidation killing mechanism. in immunocompromised or immunodefi cient patients, the macrophages are often too weak to restrain the engulfed spores from germinating. therefore, the disease occurs. vascular vessels are susceptible to the invasion of mucor, especially the arteries. mucor can locally multiply to cause the formation of blood clots and embolization, and disseminate to other organs along with the blood fl ow. the main lesions are hemorrhage and necrosis of local tissues and exudation of neutrophils. the lesions of hemorrhage and necrosis are possibly related to the arteriole lesions caused by hyphae. the clinical manifestation of hiv/aids related pulmonary mucormycosis is a nonspecifi c pneumonia. the most common symptoms reported in literatures are persistent high fever, cough, hemoptysis, chest pain and diffi culty breathing. it has a rapid progression, with a high mortality rate of 65-96 %. lung lesions are hemorrhagic infarction or pneumonia, which can cause high fever, cough, expectoration, shortness of breath, chest distress, chest pain, hemoptysis (pulmonary artery involved) and other symptoms. moist rales can be heard in both lungs and pleural rubs can be heard in the cases of pleura involvement. 1. assisted by bronchofi broscopy, lung biopsy can be performed. 2. histological examinations include bronchial lavage fl uid examination, exploratory thoracotomy and puncture of lung tissues for biopsy. 3. chest x-ray and ct scanning are conventional effective examinations. the lesions are frequently found in the dorsal and medial segments of both lungs. early exudation shows miliary shadows, cavity shadows with no wall or with thin wall and small bronchiectasis shadows. their further progression may cause fusion infi ltration, parenchymal changes, nodules, masses, thick-walled cavities and pleural effusion, with accompanying mediastinal lymphadenectasis. 1. the fi ndings of mucor or their hyphae by sputum and bronchofi broscopic biopsy. 2. the diagnostic imaging demonstrates lesions commonly in the dorsal and medial segments of both lungs. there are diffuse scattering miliary shadows, cavity shadows with no wall or with thin walls and small bronchiectasis shadows. they further progress into fusion infi ltration, parenchymal changes, nodules, masses, thick-walled cavities and pleural effusion. 3. often with accompanying mediastinal lymphadenectasis. chest x-ray of pulmonary mucormycosis demonstrates progressive infi ltrated parenchymal changes, or masses, nodules, cavities and pleural effusion. it needs to be differentiated from miliary tuberculosis. hiv/aids related miliary tuberculosis are commonly demonstrated as chronic blood borne disseminated tuberculosis, with lesions distributing symmetrically in both lungs. its long term progression causes fusion into masses. otherwise, it can be cured by anti-tb therapies. the early lesions are no-wall cavities or thin wall cavities and small bronchioectasis shadows, based on which the differential diagnosis can be made. almost all cases of hiv/aids related pulmonary mucormycosis are found in immunocompromised patients. chest x-ray demonstrates parenchyma changes and isolated or multiple nodules or masses. the parenchyma changes are patchy or fuse, with unilateral or bilateral distribution. about 20 % patients have pleural effusion and less than 10 % patients have hilar or mediastinal lymphadenosis. ct scanning demonstrates singular or multiple nodules or masses, commonly with clustering or honeycomb liked cavities. cytomegalovirus pneumonia has extensive pathological changes in the lungs. pathologically, it shows interstitial pneumonia, with the lesions randomly blood borne distributing in the lungs. the distribution can be diffuse, panlobular or focal. the target cells of pathological changes include alveolar cells and macrophages. diffused pulmonary interstitial edema and fi brosis as well as alveolar swelling, focal necrosis, bleeding and hyperplasia occur after cmv infections to cause hypoxemia. gross observation of fresh specimens demonstrates pulmonary surface edema and fl aky blooding spots. fixed specimens demonstrate brown hard lung tissues. under a microscope, pulmonary interstitial congestion as well as infi ltration of lymphocytes and mononuclear cells can be found, with the involved epithelial cells enlarged. in the pulmonary interstitium and alveoli, there are intranuclear inclusions, cytoplasmic inclusions and fl uid containing abundant proteins. the classical intranuclear inclusions can be found in the cells, purplish red or purplish blue, round or oval, with surrounding halos in eagle eyes sign. atypical cytomegalic inclusions in cells are slender, long and round liked with abundant cytoplasm and accentric nucleolus, which are blurry, unclear and atypical ( fig. 17 .97a-e ). immunohistochemitry demonstrates hiv p24 antigen positive. the systemic symptoms of cmv infection include fever, joint and muscle soreness and pain, abdominal distension and orthostatic hypotension. the respiratory symptoms include paroxysmal dry cough, diffi culty breathing, cyanosis and three depressions sign. according to the imaging fi ndings of cmvp, cmv pneumonia can be classifi ed as diffuse, miliary and mass types, among which the diffuse type is the most common. cytomegalovirus can be separated from respiratory secretions culture and urine culture by using human embryonic fi broblasts. by urine sediment smear, giant cell with inclusions can be found. by using immunofl uorescence, indirect hemagglutination inhibition and complement fi xing test, the antibody titer can be found increased. indirect immunofl uorescence test and immunoenzymic staining test can be applied to detect the anti-cmv-igg and igm antibody. in addition, enzyme-linked immuno sorbent assay (elisa) can also be performed to detect the anti-cmv-igg and igm antibody. cmv-igm antibody positive indicates a recent infection, which has diagnostic value. a single serum cmv-igg antibody positive indicates a previous infection. and during the acute and recovery phases, double serum cmv-igg antibody titer being no less than four times increase has diagnostic value, indicating a recent infection. pcr can be applied to quantitatively determine the viral load in the whole blood, blood plasma, leukocytes, urine, bronchoalveolar lavage fl uid (balf), cerebrospinal fl uid and the tissue specimens, which is believed to be the best way for the diagnosis of invasive cmv infection. chest x-ray is the most commonly used examination. chest ct scanning is superior to chest x-ray in terms of resolution and detection rate of the lesions. pulmonary demonstrations by cmvp include diffuse interstitial infi ltration and alveolar infi ltration to form reticular shadows, nodules and parenchymal changes. a baby boy aged 6 months was confi rmatively diagnosed as having aids by the cdc. he was infected via vertical transmission from mother to child, with recurrent cough after being born and the most recent cough for 4 days as well as wheezing cough in throat for 1 day before he was hospitalized. he had a past history of premature birth, with primary apnea and bronchial pneumonia and was hospitalized for treatment. later, he was admitted for three times due to cough, which was diagnosed as interstitial pneumonia. by examinations, wbc 16.3 × 10 9 /l, lym lymphocyte count 11.7 × 10 9 /l, cmv-ab weak positive, blood sedimentation 11 mm/h, and tuberculosis antibody negative. after treated by broad-spectrum antibiotic therapy, the therapeutic effi cacy is unfavorable. cytomegalovirus (cmv) infection is an important cause of pneumonia in patients with compromised immunity. imaging fi ndings include nodular shadows with blurry boundaries and bilateral fl aky parenchymal changes of the lungs. the nodules tend to be bilaterally symmetric or asymmetric, with centrilobular distribution. histopathological manifestations are nodular alveolar hemorrhage, necrosis and infl ammatory lesions, and diffused alveolar lesions. the nodules tend to have a centrilobular distribution, indicating occurrence of bronchiolitis. in aids patients, if the diameter of nodules is under 10 cm, it is most likely to be viral infection. the size of the nodules can facilitate the differential diagnosis of pulmonary infections. herpes simplex viral pneumonia (hsvp) often occurs in the upper respiratory tract, and rarely in the lower respiratory tract. human herpes simplex virus can be divided into two types, namely herpes simplex virus type i (hsv-i) and herpes simplex virus type ii (hsv-ii). herpes simplex viral pneumonia mostly occurs in patients with immunodefi ciency. herpes simplex viral pneumonia can be caused by hsv-i and hsv-ii, both of which have a nucleocapsid with 20 surfaces. the thickness of the nucleocapsid is about 100 nm, which is composed of 162 capsomeres. the nucleocapsid contains the core of the virus dna. the virion gains the phospholipid rich viral envelope when it passes through the nuclear envelope. the nucleocapsid gemmates after it passes through the nuclear membrane and is released to the cell surface. the nucleocapsid can also be released outside the cells or gains its access into the neighbour cells for further reproduction. herpes simplex virus replicates itself in the cell nucleus to produce histopathologic changes of herpes virus replication, with visible cowdry a type intranuclear inclusions. the pathogenesis process of herpes simplex virus infection in the body can be divided into fi ve stages: initial skin mucosa infection, acute ganglia infection, latent infections, re-activation, and recurrent infections in susceptible hosts. patients infected by herpes simplex virus can produce igm, igg and iga antibodies to fi ght directly against virus protein, which may play a role in changing the severity of the infection. interferon also participates in the control of herpes simplex infection by inhibiting the virus or regulating the defense mechanism. genetic factors may be also related to the herpes virus infection. cellular immunity can confi ne the infection. herpes virus cannot reproduce in the alveolar macrophages of human body, which is also the reason why herpes virus is less than cytomegalovirus in lungs. currently, it is believed that herpes simplex virus is an important pathogen of respiratory infections, especially in immunocompromised patients. localized herpes simplex viral pneumonia occurs due to the direct spreading of virus in the upper and lower respiratory tract. diffuse herpes simplex viral pneumonia is caused by the virus spread from the reproductive organs lesions or oral lesions (most possibly blood borne). viremia caused by hsv-i or hsv-ii has been reported, and both are related to diffused infections. but in patients without herpes simplex viral infection in skin mucosa, herpes simplex viral pneumonia can also occur. herpes simplex viral pneumonia is caused by the direct spreading of the virus from the upper and lower respiratory tract. diffuse herpes simplex viral pneumonia is cause by the spreading of the virus from the reproductive organs lesions or oral lesions (most possibly blood borne). viremia cause by either hsv-i and hsv-ii have been reported, both of which are related to diffuse infections. in such cases, the lung tissues may have infl ammatory infi ltration, lung parenchyma necrosis, bleeding, cellular swelling and round, diffuse interstitial pneumonia. and in most cases, there are accompanying cellular changes of herpes virus infection such as the intranuclear eosinophilic inclusions, necrotic herpes simplex viral trachitis. herpes simplex viral bronchitis has demonstrations of mucosa erythema, edema, exudation and ulcer, with coverage of the surface by fi brous purulent membranous secretions. the common initial clinical symptoms of herpes simplex viral pneumonia are shortness of breath, cough, and fever with a body temperature being higher than 38.5 °c, decreased wbc count, hypoxemia, respiratory dysfunctioning and azotemia. hsv pneumonia may be accompanied by mucocutaneous lesions by hsv, which show earlier than those of pneumonia. there may be concurrent fungus, cytomegalovirus or bacteria infection. herpes simplex viral tracheobronchitis may show tracheal or bronchial spasm or stenosis. etiological examinations hsv can be detected in tracheobronchial secretions, bronchoalveolar lavage fl uid and lung tissues. early sampling should be performed under the guidance of a bronchofi broscope. tissue culture is the most sensitive and specifi c method for the diagnosis, which can also be used for the classifi cation of the virus. papanicolaou (pap) or tzank test is a fast and cheap method for cellular diagnosis. elisa can be used to detect herpes simplex virus, with a sensitivity of up to 95 % and a high specifi city. chest x-ray demonstrations are less valuable for the differential diagnosis. pulmonary ct scanning can be applied for the differential diagnosis. herpes simplex viral pneumonia includes three types, namely local, multiple or diffuse interstitial infi ltration. in the early stage, typical hilar or diffuse interstitial shadows with increased density can be found, with thickened bronchial wall. as the disease progresses, cloudy or patchy alveolar tamponade and fusion can be found. chest x-ray may demonstrate negative for herpes simplex viral trachitis and bronchitis. herpes simplex viral pneumonia should be differentiated from bacterial pneumonia, cmv pneumonia, and infl uenza pneumonia. hiv/aids related herpes simplex viral pneumonia is mostly demonstrated by multiple signs, including small nodules, ground glass liked shadows and patchy parenchymal changes. the nodules are in centrilobular distribution, mostly with accompanying branches liked shadows (tree buds sign). chest x-ray demonstrates diffuse lung parenchymal changes. imaging fi nding are parenchymal change areas with bilaterally blurry boundaries. generally, the nodules have a diameter of 2-10 mm. ct scanning with high resolution demonstrates nodules with surrouding ground glass liked density lesions. lymphoid/lymphocytic interstitial pneumonia (lip) is more common in children with aids. the cdc in the united states has defi ned lip in children under the age of 13 years as the diagnostic indicator of aids. the predictive diagnostic criteria include chest x-ray demonstration reticular nodular changes in pulmonary interstitium of both lungs for no less than 2 months, undetectable pathogens and no responses to the antibiotic therapy. currently, hiv/aids related lymphocytic interstitial pneumonia is considered to be related to hiv and epstein-barr virus, human t cell leukemia-lymphoma type i virus (htlv-i) and hiv-i. the infection of the above viruses causes pulmonary lymphatic hyperplasia and other systemic diseases. about 22-75 % children with hiv infection sustain lip, and 3 % in adults. most cases of non-hiv infected patients with lymphoid interstitial pneumonia are women, at average age of 56 years old, more commonly in the age group of 40-50 years old and above 70 years old. the pathological manifestations are infi ltration of small and mature lymphocytes as well as plasma cells in alveolar septum and the alveolus, extensive interstitial fi brosis and non-caseous granuloma. it is characterized by diffuse infi ltration of lymphocytes, plasmocytes and histocytes in the pulmonary interstitium. the lymph follicle with germinal center is more common. hyperplasia occurs in type ii alveolar epithelium, and the macrophage increases in the alveolar cavity. there are rare or mild intraalveoli organization and macrophage aggregation. staining of the immune globulin light chain demonstrates poly-clone b cells. the clinical symptoms are in progressive development, with cough and suffocation, rare hemoptysis and sjogren syndrome commonly in mouth and eyes. by examinations, the signs have slight difference between adults and children. in children, there are lymphadenectasis, hepatosplenomegaly, enlargement of parotid gland, clubbing fi ngers and wheezing sound. in adults, there are lymphadenectasis, slight fi ne bubbling rales, as well as hepatosplenomegaly and enlargement of parotid gland in 1/3 patients. 1. peripheral hemogram demonstrates increased lymphocytes and eosinophilic granulocytes. 2. myelogram demonstrates increased lymphocytes, plasmocytes and eosinophils. 3. blood biochemical examination demonstrates increased immune globulin, predominantly lgm. 4. blood gas analysis demonstrates hyoxemia. 5. pathogenic examinations by bronchofi broscopy, bronchial alveolar lavage and biopsy can defi ne the diagnosis. 6. pulmonary function examinations demonstrate restrictive ventilatory disorder, lower lung compliance and impaired diffusion function. impaired diffusion function is a more sensitive indicator in monitoring the progress of the disease. 7. chest x-ray is the most commonly used imaging examination, while chest ct scanning is commonly applied for the differential diagnosis. chest x-ray demonstrates hiv/aids related lymphoid interstitial pneumonia as reticular or reticular nodular shadows of lung markings in both lungs. hrct demonstrates bilateral diffuse ground glass liked density shadows. perivascular thin-walled pneumatocele is common. pneumatocele induced by lip is commonly found in the middle lung fi eld, which probably is due to the valve effects caused by infi ltration of cells around bronchioles. manifestations of pneumatocele, together with ground glass liked density shadows, highly indicate lip. centrilobular and subpleural small nodules and thickened intralobular septa can be occasionally found. dysfunctional diffusion is a more sensitive indicator in monitoring the progress of the disease. hiv/aids related lymphoid interstitial pneumonia should be differentiated from allergic pneumonia, carcinomatous lymphangitis and pneumocystis carinii cysts. ct scanning with high resolution demonstrates characteristic lesions of hiv/aids related lymphoid interstitial pneumonia, including intralobular linear shadows and honeycomb liked changes, with common involvement of the subpleural area and the basal lung. its characteristic manifestations are clustering gas containing thin-walled cyst in a diameter of 2-10 mm, with clear cyst wall. shared wall between cysts is its characteristic demonstration. the surrounding ground glass liked density indicates infl ammation. intralobular linear shadows indicate interstitial fi brosis. pulmonary toxoplasmosis (pt) is caused by the toxoplasma parasitizing in cells. ludlam et al. fi rstly proposed the concept of pulmonary toxoplasmosis in 1963 [ 107 ] , arguing that toxoplasma can cause atypical pneumonia. later, there are some pathological reports about pulmonary toxoplasmosis or disseminated toxoplasmosis with lung involvement. in recent years, due to the global prevalence of aids, the incidence of pulmonary toxoplasmosis is increasing, with most cases being disseminated toxoplasmosis with lung involvement. pt has been one of the important opportunistic infections in patients with immunosuppression, especially aids patients. hiv/aids related pulmonary toxoplasmosis is a zoonotic disease, with cats as its main transmission source, followed by pigs and sheep. people are infected by intake the water or food contaminated by cats' feces or without cooked meat. immunocompromised aids patients are susceptible to this disease, and its occurrence is rare in immunocompetent people. after its invasion into the human body, the sporozoite in the cystozygote and intracystic cystozoite overfl ows to penetrate the intestinal wall mucosa and spread to the whole body tissues along with blood or lymph fl ow. the brain, heart, lymph nodes, and lung are the most vulnerable tissues and organs for the infection. any abnormality in the process of defense mechanism can cause impaired immune functions to eliminate the toxoplasma, which ultimately causes systemic and pulmonary infections. pulmonary toxoplasma infection may also be caused by the blood borne spreading of reactivated toxoplasma infection in other body parts, with no exclusion of reactivated pulmonary infection or primary pulmonary infection. ludlam et al. generally nominated toxoplasmosis as atypical pneumonia [ 107 ] . catterall et al. divided toxoplasmosis into three types: necrosis, infl ammatory infi ltration and toxoplasma invasion [ 109 ] . it can also be classifi ed as type a: subclinical or occult infection; type b: interstitial and atypical pneumonia; type c: necrotic pneumonia; type d: lobar pneumonia; and type e: granulomas pneumonia (toxoplasmoma). by naked eyes observation, the involved lungs are solid, with congestion and red brown section. the pleura have bleeding spots, with moderate peribronchial lymphadenectasis. under a light microscope, there is exudation of serous fl uids in alveolar cavities, occasional formation of transparent membrane or fi brin purulent exudation, infi ltration of small quantity neutrophils, proliferation and shedding of alveolar wall cells, and trophozoite and/or cysts of toxoplasma in epithelial cells and macrophages. the pulmonary interstitium may have infi ltration of lymphocytes and plasmocytes as well as visible fi broblasts and macrophages. the granuloma changes are also found in the lung tissues, with central stripes or localized necrosis and surrounding lymphocytes and small quantity multinucleated giant cells. it is diffi cult to fi nd toxoplasma in granuloma, but it can be found in the normal tissues around or near the granuloma. almost all cases of aids complicated by pulmonary toxoplasmosis are caused by disseminated toxoplasmosis with pulmonary involvement. it is commonly diffuse pulmonary infl ammation with serious symptoms, including high fever, cough, cyanosis, breathing diffi culty, possible occurrence of skin rashes, lymphadenectasis and meningitis. the chronic cases may have long-term low grade fever, cough, and weight loss. direct light microscopy of the specimen smear and enprint such as blood, cerebrospinal fl uid, bone marrow, anterior aqueous humor, phlegm, urine, saliva, and other osmotic solutions, as well as lymph nodes, muscle tissue or other living tissues can be performed for pathogen examinations. sabin proposed that the staining test have high sensitivity and specifi city according to the fi ndings that mixture of fresh toxoplasma with normal serum can be stained deep by alkaline methylene blue staining, while its mixture with immune serum can be stained light or blank by the same staining. other assays including indirect fl uorescent antibody, indirect blood coagulation, and complement fi xation test can provide valuable reference for the diagnosis. toxoplasm is tested in pathological eaminations that can provide valuable reference for the diagnosis. chest x-ray is the most commonly used diagnostic imaging examination. and chest ct scanning can be applied for the differential diagnosis. imaging fi ndings of hiv/aids related pulmonary toxoplasmosis can be divided into four types: bronchial pneumonia, interstitial pneumonia, pleuritis and complication of cardiovascular disease. the type of bronchial pneumonia is also known as lobular pneumonia, with thickened pulmonary markings that distribute along with the bronchi in the middle and lower lung fi elds, scattered patchy shadows with uneven density and blurry boundaries, fusion of some shadows into large fl aky shadow and widened hilar shadow. the type of interstitial pneumonia is demonstrated as reticular and nodular shadows. the interstitial lesions widen the space between the bronchiole and the alveolar wall, with stripes and fl occulent shadows. the type of pleuritis is rare, with signs of pleural effusion. the type of complication of cardiovascular disease is demonstrated as heart failure (acute pulmonary edema), with signs of pericardial effusion. a male patient aged 39 years was confi rmatively diagnosed as having aids by the cdc. he complained of cough and fever. his cd4 t cell count was 29/μl. by direct light microscopy, toxoplasma tachyzoites can be found in the specimens such as blood, cerebrospinal fl uid, bone marrow, anterior aqueous humor, phlegm, urine, saliva, and other osmotic solutions, as well as lymph nodes, muscle tissues or other living tissues. the fl uorescent antibody and complement fi xation test are positive. the biopsy tissue culture and inoculation test are positive. in the lesions and their surrounding tissues of interstitial e c d fig. 17 . 105 (continued) pneumonia, necrotic bronchitis or granuloma, toxoplasma can be found. the diagnostic imaging demonstrates any one type of pulmonary toxoplasmosis, including bronchial pneumonia, interstitial pneumonia, pleuritis and cardiovascular disease, can be used as the evidence for the diagnosis of pulmonary toxoplasmosis. the type of bronchial pneumonia is also known as lobular pneumonia, with thickened pulmonary markings with a distribution in both middle and lower lung fi elds along with the bronchi, scattered patchy shadows with uneven density and blurry boundaries, fusion of some patchy shadows into large fl aky shadow and widened hilum. the type of interstitial pneumonia has typical demonstrations of reticular and nodular shadows. the interstitial lesions widen the space between the bronchiole and the alveolar wall, with strip and fl occulent shadows. the type of pleuritis is rare, with signs of pleural effusion. the type of cardiovascular disease may have signs of heart failure (acute pulmonary edema), and signs of pericardial effusion. hiv/aids related pulmonary toxoplasmosis should be clinically differentiated from infectious mononucleosis and mycoplasma pneumonia. with primary pulmonary lesions. pulmonary low malignant b cell lymphoma is the most common primary pulmonary lymphoma which is derived from mucosa related lymphoid tissue. the manifestations include slowly decreased alveolar transparency. pulmonary high malignant b cell lymphoma is extremely rare, which often occurs with singular lesion and primary disease such as immunodefi ciency. hiv/aids related malignant lymphoma is mostly caused by compromised immunity. hiv/aids related hodgkin's lymphoma is relatively rare. there are also reports about hiv/aids related t cell lymphoma with pulmonary involvement. hiv/aids related malignant lymphomas are mostly highly malignant large cells lymphoma. cerebral lymphoma is one of the defi ning diseases of aids. it has been reported that the clinical incidence of pulmonary infi ltration by malignant lymphoma is 10-20 %, but 29-50 % by autopsy. in the early stage, it is commonly asymptomatic. with its progression, symptoms of dry cough, suffocation, and small quantity clear phlegm occur. mediastinal lymphadenosis includes lymphadenectasis to compress the trachea by, blood vessels and nerves and lead to breathing diffi culty, superior vena caval obstruction syndrome, and hoarse voice. the pulmonary parenchyma lesions include reticular structure in the lungs. the clinical symptoms are cough, expectoration, suffocation and breathing diffi culty. a male patient aged 43 years was confi rmatively diagnosed as having aids by the cdc. he complained of chest distress and cough for more than 1 month. his cd4 t cell count was 56/μl. in patients with hiv/aids related kaposi's sarcoma, its serologic positive rate is 100 %. kaposi's sarcoma cells can produce il-6, during which il-6 plays a role as an autocrine factor to maintain the cell growth, paracrine cytokines, stim-ulate proliferation of other interstitial cells and induct the vascular growth. therefore, kaposi's sarcoma is a kind of tumor with abundant blood vessels. before the application of harrt, the incidence of kaposi's sarcoma in male homosexuals is 21 %. after the clinical application of harrt treatment, the incidence is decreasing. in addition to hhv-8, some studies indicated that most patients with kaposi's sarcoma have hia-dr5 alleles, suggesting a possible relationship between kaposi's sarcoma and the heredity. there is no obvious difference between hiv/aids related kaposi's sarcoma and classic kaposi's sarcoma in pathological changes. early pathological manifestations are chronic infl ammation or granulomatous infl ammation, with formation of new vascular and lymphatic vessels and accompanying edema and bleeding. the fi ndings of large and protruding endothelial cells in granuloma tissue with accompanying erythrocytic exudation and hemosiderin particles have great signifi cance for the early diagnosis. the pathological changes in the advanced stage are signifi cant proliferation of the endothelial cells, and proliferation of fi broblasts around capillaries. in the advanced stage, the lesions are often accompanied by extensive connective tissue hyperplasia, which presents diffi culty for its differentiation from common sarcoma. when it is diffi cult to defi ne the diagnosis by light microscopy, immunohistochemical examinations can be used to defi ne the diagnosis. the pathological changes are characterized by lesions confi ned to the epithelial lamina propria, gathering of spindle cells with mild heteromorphism around many lacuna vasorum with irregular lumen, erythrocytic exudation and hemosiderin sedimentation. the atypical lacuna vasorum can be compressed by proliferative spindle cells to be absent. vascular endothelial cells and peripheral spindle cells may have mitotic phase in the advanced stage, with increased heteromorphism cells. the infl ammatory cells are mainly plasma cells, with acidophilic corpuscles and pas staining positive, which can assist the pathological diagnosis. pulmonary kaposi's sarcoma in aids patients rarely has symptoms. it is commonly concurrent with pulmonary opportunistic infections, with symptoms of cough, diffi culty breathing and fever. other symptoms are related with the location of the tumors. the involvement of trachea or bronchi can cause luminal stenosis. the mediastinal tumor can compress and obstruct lymph vessels to cause pulmonary edema or a large quantity pleural fl uids, which result in respiratory diffi culty, and even respiratory failure. (1) sampling by bronchoscopy or endoscopy to prepare pathological section. (2) chest x-ray demonstrates its typical manifestation of pleural effusion. dr demonstrates enlarged and deranged hilum in both lungs in bird nest liked appearance. there is light density fl aky shadows in the both lower lungs. ct scanning demonstrates multiple rounds liked nodular shadows in the middle and lower lung fi elds of both lungs with clear boundaries. there are also mediastinal and hilar lymphadenectasis, with common involvement of the pleura and bilateral pleural effusion in a small quantity. a male patient aged 33 years was confi rmatively diagnosed as having aids by the cdc. he had been detected as hiv positive for 5 months, with complaints of recurrent cough and nausea for 10 days and was hospitalized on jan. 7, 2004. the transmission route was unknown because he denied histories of intraveneous drug abuse, paid blood donation, blood transfusion and unhealthy sexual behaviors. five months ago, he was diagnosed as aids in the stage of aids in our hospital, and hospitalized to treat pcp, with a cd4 t cell count of 17/μl. his symptoms were quickly relieved after pcp treatment and he continued the antiviral therapy for almost 5 months after being discharged. by physical examinations, he was in poor spiritual condition, a light blue nodule in size of 0.5 × 0.5 cm in the left upper chest wall with medium hardness, palpable lymph nodes in size of 1.0 × 2.0 in the opisthotic area and inguen, no tenderness and being movable. by the digital rectal examination, a palpable prominent nodule with wide base at 4 cm 7 points away from the anus, with fl exible texture and smooth surface. by the auxiliary examinations, wbc 3.9 × 10 9 /l, neμt 48.3 %, lym 34.9 %, mon 7.4 %, eos 9.0 %, rbc 3.27 × 10 12 /l, hgb 126 g/l, plt 210 × 10 9 /l, routine urine test normal, blood sedimentation 16 mm/h. by hepatitis b examinations, hbsag, anti-hbe and anti-hbe positive. his cd4 t cell count was 91/μl. by abdominal b ultrasound, multiple low echo nodules in the abdominal cavity, the largest in size of 1.2 × 1.0 cm, which are suspected to be enlarged lymph nodes. on jan. 14, he received inguinal lymph node biopsy, with pathological report of kaposi's sarcoma. during the treatment and following up, the involvement of lungs, digestive tract, lymph nodes and skin is suspected, with the diagnosis of phase ii kaposi's sarcoma and chemotherapy was recommended. reexamination by chest x-ray demonstrated normal cardiopulmonary phrenic. abdominal b ultrasound failed to fi nd enlarged lymph nodes. ct scanning demonstrated shrinkage of lesions in both lungs and mediastinal lymph nodes, with only palpable soybean sized submandibular lymph node. by examinations after chemotherapy, cd4 t cell count 67/μl, viral load 63,000 copies/ml. the patients had multimorphological erythema drug eruptions, which was suspected as drug allergies of chemotherapy, which were absent after symptomatic treatment. the following ups so far show no recurrence of kaposi's sarcoma, with his cd4 t cell count fl uctuating around 400/μl. he can work as usual. a male patient aged 36 years was confi rmatively diagnosed as having aids by the cdc. he had a history of homosexual behaviors, with complaints of fever and cough for 2 months as well as chest distress for more than 20 days. since, july 2010, fever with a body temperature of about 37.5-37.8 °c occurs, with cough, yellowish bloody sputum, and dark purplish patchy skin rashes. by examinations, his anti-treponema pallidum antibody positive, multiple dark purplish patchy skin rashes on the face, eyelid, lower jaw, hairline, chest and abdomen with skin surface desquamation, palpable bilateral cervical lymphadenectasis and the largest in size of 10 × 19 mm. by laboratory tests, wbc 5.98 × 10 9 /l, n 78.74 %, rbc 2.22 × 10 12 /l, hgb 71 g/l, plt 204 × 10 9 /l, cd4 12/μl. a male patient aged 27 years was confi rmatively diagnosed as having aids by the cdc. he complained of cough for more than 2 months, chest distress for more than 1 month, and bloody sputum for half a month and was hospitalized. he had a history of homosexual behaviors. by examinations on admission, multiple round purplish blue skin rashes nodules on the limbs. his cd4 t cell count was 9/μl. demonstrations can also be fl aky fl occulent areas with blurry density or parenchymal density areas along with bronchi. 3. lung puncture for histopathological biopsy demonstrates irregular vascular lumen in the dermis, proliferation of endothelial cell with accompanying heteromorphism. in some cases, there are tumor masses composed of spindle cells and epithelial cells. other sarcoma and vascular tumor hiv/aids related kaposi's sarcoma should be differentiated from other sarcoma and vascular tumor. ks invasion of the digestive mucosa can cause bleeding and upper gastrointestinal symptoms. the pathological lesions can be diagnosed by upper gastrointestinal endoscopy or biopsy. in the cases of no fever and exclusion of infections, the typical imaging demonstrations and bronchoscopy fi ndings can defi ne the diagnosis of pulmonary ks. hiv/aids related kaposi's sarcoma should be differentiated from pneumocystis carinii pneumonia. the lesions of pcp are mostly symmetric ground glass liked density shadows extending outwards from the hilum in both lungs. in the middle and advanced stages, nodules, fi brosis and cavities occur, rarely with pleural effusion. lung cancer commonly refers to the cancer in lung parenchyma, usually does not include those mesodermal tumors originating from other pleura, or other malignancies like carcinoid, malignant lymphoma, or metastatic malignancies for other body parts. therefore, the following lung cancer we are discussing about refers to the malignancies originating from bronchial, or bronchiolar epithelial cells, accounting for 90-95 % of the lung parenchyma malignancies. the cause of lung cancer is still not completely known. data have indicated that the risk factors of lung cancer include smoking (including second-hand smoke), asbestos, radon, arsenic, ionizing radiation, halogen alkenes, polycyclic aromatic compounds and nickel. long-term smoking can cause proliferation of the bronchial mucosal epithelial cells and proliferation of squamous epithelium to induce squamous epithelium carcinoma or undifferentiated small cell carcinoma. non-smokers can also develop lung cancer, but adenocarcinoma is more common among them. long-term exposure to radioactive substances, like uranium and radium, and its derivatives; carcinogenic hydrocarbons, like arsenic, chromium, nickel, copper, tin, ferri, coal tar, bitumen oil, petroleum, asbestos and mustard gas, all can induce lung cancer, which is commonly squamous carcinoma and undifferentiated small cell carcinoma. some chronic pulmonary diseases, such as tuberculosis, silicosis and pneumoconiosis, can concurrent with lung cancer. in the cases with these chronic pulmonary diseases, the incidence of cancer is higher than the general population. in addition,bronchopulmonary chronic infl ammation and pulmonary fi brous scar lesions may cause metaplasia or hyperplasia of squamous epithelium during their healing processes, based on which some cases can develop into cancer. the internal factors of the human body include family heredity, compromised immunity, metabolism and endocrine dysfunction. the lung cancers distribute more in the right lung than in the left lung, more in the upper lobe than in the lower lobe. its locations range from the major bronchus to the bronchioles. the central type of lung cancer has its origination from the major bronchial lobes and locates adjacent to the pulmonary hilum. the peripheral type of lung cancer has its origination from the lower parts of pulmonary segment bronchi and locates in the peripheral areas in the lungs. in the growth process of the lung cancer, it causes the extension and dilation of the bronchial walls, and penetrates the bronchial walls to invade the adjacent lung tissues and form masses. meanwhile it intrudes into the bronchi to cause luminal stenosis or obstruction. with its further progression and dissemination, it spreads from the lungs and directly extends into the chest walls, mediastinum, heart, major vessels and other adjacent organs and tissues. lung cancer can also transfer to other parts of the body along with blood and lymph fl ows or disseminates to other pulmonary lobes via the respiratory tract. the growth rate and transferring paths of lung cancer depend on its histological types, differentiation degree and other biological characteristics. lung cancer has no special symptoms in the early period, only has the common symptoms with common respiratory diseases, including cough, bloody sputum, low grade fever, chest pain and chest distress. therefore, it is often misdiagnosed. (1) face and neck edema; (2) hoarse voice is the most common symptom; (3) shortness of breath. lung cancer tends to occur distant metastases in the early stage. in the cases with metastatic lesions to the brain, the patients sustain persistent headache and blurry vision. in the cases with metastatic lesions to the bone, bone destruction may occur to cause fracture. (1) restrictive wheezing sound, mostly occurring in the inspiratory phase and recurring after cough; (2) hoarse voice, caused by lymph nodes transferring to compress and invade the recurrent laryngeal nerve; (3) superior vena cava syndrome, caused by the compresses or invasion to the superior vena cava by the mass and venous obstruction, with edema in the head, face, neck, and upper limbs, varicose veins and edema in the upper chest, and accompanying dizziness, chest distress, shortness of breath and other symptoms; (4) horner's syndrome, with enophthalmos of the affected side, blepharoptosis, shrinkage of the pupils, eye fi ssure stenosis, increased skin temperature in the upper chest of the affected side and no sweating due to compression or invasion of the apical cancer to the cervical sympathetic ganglia; (5) should and arm pain, which is radial burning pain in the shoulder and upper limbs of the affected side due to compression or invasion of apical cancer to the brachial plexus nerve; (6) phrenic nerve paralysis, with symptoms of shortness of breath and chest distress due to invasion to the phrenic nerve; (7) dysphagia, caused by compressed esophagus by mediastinal lymphadenectasis; and diffi culty breathing caused by compressed trachea by mediastinal lymphadenectasis; (8) pericardial effusion, shortness of breath, arrhythmia and heart dysfunctions due to pericardial invasion; (9) pleural metastasis, with chest pain and cancerous pleural effusion; (10) lung cancer metastasis, spreading of lung cancer along with blood fl ow to the bone, liver, brain, kidney, adrenal gland and subcutaneous tissues. intrapulmonary metastasis is also common. metastasis to different locations shows different symptoms and signs. (11) extrapulmonary signs, commonly including joint pain or joint hypertrophy, clubbing fi ngers and mental disorders. the diagnostic imaging is the most commonly used and an important examination for the diagnosis of lung cancer. it can facilitate to fi nd some specifi c manifestations in the lesions, which provide clues for the diagnosis of lung cancer. it is also the main basis for the staging of lung cancer, but fails to defi ne the qualitative diagnosis. chest x-ray is the main examination for the diagnosis of lung cancer. anteroposterior and lateral chest x-ray are used for preliminary screening. chest ct is the diagnostic imaging examination of the choice for the diagnosis of lung cancer. for the central type of lung cancer in the early stage, there are direct signs to defi ne the diagnosis. in the early stage, thin layer scanning with a layer thickness of 1.5-4 mm can be performed to observe the bronchial changes. mr imaging can demonstrate intraluminal nodules, luminal thickness and luminal stenosis of the bronchi from the transverse, coronal, and sagittal perspectives. mr imaging demonstrates favorably cancer in the lesions of obstructive pneumonia, and masses covered by the hilum. pet/ct scanning can be used for the screening of lung cancer metastasis and assessing the therapeutic effi cacy after treatment. dsa is used for infusion chemotherapy of bronchial artery in the cases of primary lung cancer. bronchoscopy is an important examination for the diagnosis of lung cancer. the pathological changes of the endothelium and the lumen of bronchi can be directly observed by using bronchoscopy. for the cases with caner or cancerous infi ltration by bronchoscopy, sampling of the tissues under the guidance of bronchoscopy for biopsy can be performed. otherwise, bronchial secretions can be suctioned under the guidance of bronchoscopy for cytological examinations to defi ne the diagnosis and the histological classifi cation. in most cases of primary lung cancer, the shed cancer cells can be found in the sputum, which can also facilitate to defi ne its histological classifi cation. after several examinations and short-term exploratory therapies, the qualitative diagnosis cannot be defi ned and the possibility of lung cancer cannot be excluded. therefore, exploratory thoracotomy can be performed if the patient's physical conditions permit. chest x-ray early lesions are confi ned within the bronchi, causing valve ventilatory disorder and changes of obstructive emphysema. the manifestations include restrictive pulmonary gas increase and sparse lung markings. in the cases with certain degree of bronchostenosis due to unfavorable discharge of the secretions, obstructive pneumonia occurs, showing patchy blurry shadows. in the cases with complete blockage of the bronchi, obstructive atelectasis occurs, showing decreased pulmonary volume, increased density and migration of the mediastinum to the affected side. obstructive pulmonary bronchiectasis has demonstrations of intrapulmonary cords liked shadows. lung a male patient aged 41 years was confi rmatively diagnosed as having aids by the cdc. he complained of repeated cough for more than 1 month and reported to have a history of unhealthy sexual behaviors. his cd4 t cell count was 65/ μl. cancer in the middle and advanced stages are mainly manifested as hilar mass and atelectasis. the mass has a high density with clear boundary. however, the cancer cannot be observed due to its common immersion in the large fl aky obstructive pneumonia lesion or large quantity pleural effusion. atelectasis is commonly manifested as shrinkage of pulmonary segments or shrinkage of unilateral lung, with high density. the shadow of atelectasis widens at the hilum to show prominent mass. in the cases of central type lung cancer in the right upper lobe, a transverse s shape is at the hilum (commonly known as pancoast cancer). early diagnosis of central type lung cancer by plain chest x-ray only shows some indirect pulmonary manifestations caused by bronchial obstruction. and these indirect signs are not characteristically lung cancer. in the cases of local obstructive emphysema, these indirect signs can be caused by foreign substances in the bronchi or early infl ammation. obstructive pneumonia is diffi cult to be differentiated from common pneumonia. obstructive atelectasis needs to be differentiated from many other conditions. (1) pathological changes in the bronchial lumen including polypoid, nodular or fl at papula masses. benign tumor has smooth boundary and malignant tumor has unsmooth boundary, commonly with wider base and thickened lumen wall. even the slight bronchial changes caused by the central type of lung cancer can be demonstrated by thin layer ct scanning, including slightly thickened bronchial wall, intraluminal small nodules and lumen stenosis or obstruction. in the middle and advanced stages, the direct signs of the central type lung cancer include thickened bronchial wall, irregular or unsmooth lining of the bronchial lumen. bronchial obstruction is suddenly truncation or gradual thinning of the lumen to obstruction. (2) hilar mass locates adjacent or around the bronchi, with smooth or arch shaped boundary. the indirect signs of the central type lung cancer in the middle and advanced stages include secondary changes to bronchial stenosis. obstructive pneumonia is manifested as patchy blurry shadows or parenchymal changes of the pulmonary segments/lobes, and decreased lung volume. mr imaging demonstrates the tumor as long t1 and long t2 signals. in the cases of central type lung cancer with secondary obstructive atelectasis and obstructive pneumonia, enhanced t1 demonstrates the tumor in the lesion of pulmonary atelectasis and obstructive pneumonia. the signal of atelectasis is higher than that of the tumor. pet/ct scanning can demonstrates increased and thick stained metabolites of metastatic lesions or residual lesions, which has a diagnostic sensitivity of above 90 %, and a reported specifi city of 80-90 %. in addition, it can be applied for the clinical consideration of it hilar, mediastinal lymph node metastasis and extrathoracic distant metastasis, which is an important method to decide clinical stages before lung cancer therapy. but pet has false negative diagnosis in the diagnosis of lung cancer with decreased metabolites, especially the alveolar cell carcinoma. for the diagnosis of pneumonia and pulmonary tuberculosis, it also has false positive results. dsa can demonstrate the blood supply of the tumors. miliary tuberculosis is more common in young adults, with obvious symptoms of systemic toxicity. anti-tuberculosis drug therapy can relieve the symptoms, with gradually absorbed lesions. (3) chest x-ray demonstrates hilar lymph node tuberculosis as mass shadows in the hilum of lung, which may be misdiagnosed as the central type lung cancer. hilar lymph node tuberculosis is more common in teenagers, commonly with symptoms of tuberculosis infection but rarely hemoptysis. lung cancer can be concurrent with pulmonary tuberculosis. (1) bronchial pneumonia; obstructive pneumonia induced by early lung cancer can be misdiagnosed as bronchial pneumonia. bronchial pneumonia has an acute onset with more obvious symptoms of infection. chest x-ray demonstrates patchy or spots shadows, with blurry boundaries and uneven density. the lesions are not confi ned within one segment or one lobe. (2) pulmonary abscesses; central necrosis and liquefaction of the lung cancer results in cancerous cavities. by chest x-ray, the central type lung cancer can be misdiagnosed as pulmonary abscesses. in the acute period, a pulmonary abscess has obvious symptoms of infection, with large quantity purulent sputum. chest x-ray demonstrates thin cavity wall, smooth inner wall, liquid level and infl ammatory changes in the surrounding lung tissues or pleura. pulmonary benign tumors including hamartomas, fi broma and chondroma have slow growth. chest x-ray demonstrates round liked mass shadow, with homogeneous density without lobation. joint united nations programme on hiv/aids (unaids) surveillance for aidsdefi ning opportunistic illnesses, 1992-1997 imaging and pathology of hiv related cytomegalovirus pneumonia roentgenographic patterns of pneumocystis carinii pneumonia in 104 patients with radiology distinction of pyogenic pulmonary infection from pneumocystis carinii pneumonia in aids patients atlas of differential diagnosis in hiv/aids. beijing: pmph the national institutes of health (nih) the centers for disease control and prevention (cdc), and the hiv medicine association of the infectious diseases society of america (hivma/idsa) guidelines for prevention and treatment of opportunistic infections in hiv-infected adults and adolescents acute respiratory failure associated with cryptococcosis in patients with aids: analysis of predictive factors pulmonary cryptococcosis: localized and disseminated infections in 27 patients with aids penicillium marneffei agent d'une mycose du system reticuloendothelial infection caused by penicillium marneffei description of fi rst natural infection in man cd4+ t cell-mediated fatal hyperinfl ammatory reactions in mice infected with penicillium marneffei infections caused by penicillium marneffei in china and southeast asia: review of eighteen published case and report of our mo re chinese cases disseminated histoplasmosis in the acquired immune defi ciency syndrome: clinical fi ndings, diagnosis and treatment, and review of the literature imaging of thoracic pathology in patients with aids clinical and radiographic features of hiv-related pulmonary tuberculosis according to the level of immunosuppression global tuberculosis incidence and mortality during 1990-2000. bull world health organ tuberculosis in patients with human immunodefi ciency virus infection pulmonary tuberculosis in kigali, rwanda. impact of human immunodefi ciency virus infection on clinical and radiographic presentation relationship of the manifestations of tuberculosis to cd4 cell counts in patients with human immunodefi ciency virus infection radiology of pulmonary tuberculosis and human immunodefi ciency virus infection gulu, uganda bacterial pneumonia in persons infected with the human immunodefi ciency virus. pulmonary complications of hiv infection study group microbiology of community-acquired bacterial pneumonia in persons with and at risk for human immunodefi ciency virus type 1 infection. implications for rational empiric antibiotic therapy the intracellular bacterium rhodococcus equi requires mac21 to mammalian cells virulence of rhodococcus equi isolates from patients with and without aids association between large plasmid and 15 to 17 kilo dalton antigens in virulent rhodococcus equi rhodococcus equi plasmids:isolation and partial characterization rhodococcus equi pneumonia in aids: high-resolution ct fi ndings in fi ve patients cytomegalovirus pneumonitis in patients with aids: fi ndings in an autopsy series immediate causes of death in acquired immunodefi ciency syndrome the causes of death in patients with humanimmunodefi ciency virus infection: aclinical and pathologic study with emphasis on the role of pμlmonary diseases cytomegalovirus pneumonitis: spectrum of parenchymal ct fi ndings with pathologic correlation in 21 aids patients rhodococcus equi -an increasingly recognized opportunistic pathogen a highly representative two hybrid genomic library for the yeast yarrowia lipolvtica rhodococcus equi infection in hiv infected patients rhodococcus equi infection in patients with and without human immunodefi ciency virus infection radiological fi ndings in nine aids patients with rhodococcus equi pneumonia comparison of nucleic acid amplifi cation, serology, and microbiologic culture for diagnosis of rhodococcus equi pneumonia in foals cytokine modulation alters pulmonary clearance of rhodococcus equi and development of granulomatous pneumonia diseases of the lung: radiologic and pathologic correlations pneumonia: high-resolution ct fi ndings in 114 patients radiologic distinction of pyogenic pulmonary infection from pneumocystis carinii pneumonia in aids patients high-resolution ct in the evaluation of clinically suspected pneumocystis carinii pneumonia in aids patients with normal, equivocal, or nonspecifi c radiographic fi ndings pneumocystis carinii pneumonia: spectrum of parenchymal ct fi ndings pulmonia in patient with aids discontinuation of prophylaxis for pneumo-cystis carinii pneumonia in hiv-infected patients treated with highly active antiretroviral therapy carcinoma of the lung in hiv-positive patients: fi ndings on chest radiographs and ct scans pulmonary complications of hiv-associated malignancies pneumocystis carinii infection and aids comparison of histologic stains in the diagnosis of pnemocystis carinii tuberculosis in patients with human immunodefi ciency virus infection comparative histopathological study of pulmonary tuberculosis in human immunodeficiency virus-infected and non-infected patients pulmonary tuberculosis in aids patients: transient chest radiographic worsening after initiation of antiretroviral therapy active pulmonary tuberculosis in patients with aids: spectrum of radiographic fi ndings (including a normal appearance) effect of hiv status on chest radiographic and ct fi ndings in patients with tuberculosis pulmonary mycobacterium avium complex disease without dissemination in hiv-infected patients mycobacterium avium complex infection in the acquired immunodefi ciency syndrome pulmonary disease due to infection by mycobacterium avium complex in patients with aids cd4 t lymphocyte count and the radiographic presentation of pulmonary tuberculosis. a study of the relationship between these factors in patients with human immunodefi ciency virus infection tuberculosis in the aids era mycobacterial pseudotumors of lymph node mycobacterial spindle cell pseudotumor of lymph node managenent of the hiv-infected patient tuberculosis in the aids era cytomegalovirus pneumonia in aids patients atypical cytomegalic cells are diagnostic for cytomegaloviral infection in aids cytomegalovirus as a primary pulmonary pathogen in aids infections with cryptococcus neoformans in the acquired immunodefi ciency syndrome endemic fungal pneumonia in immunocompromised patients disseminated histoplasmosis in adis: fi ndings on chest radiographs opportunistic fungal pneumonia pulmonary aspergillosis in the acquired immunodeficiency syndrome coccidioidomycosis during human immunodefi ciency virus infection. a review of 77 patients cryptococcal pulmonary infection in patients with aids: radiographic appearance pulmonary aspergillosis in patients with aids. clinical and radiographic correlations cryptococcal pneumonia in aids: is cryptococcal meningitis preceded by clinically recognizable pneumonia pulmonary manifestations of disseminated cryptococcosis in patients with clinical and bacteriological aspects of nocardiasis lung abscess in patients with aids focal mycobacterial lymphadenitis following initiation of protease-inhibitor therapy in patients with advanced hiv-1 disease rhodococcus equi endobronchial mass with lung abscess in a patient with primary pulmonary aids-related lymphoma: radiographic and ct fi ndings the expanding challenge of hiv-associated malignancies the pulmonary manifestations of aids-related non-hodgkin's lymphoma varied appearance of aids-related lymphoma in the chest lymphoma versus aids intrathoracic kaposi's sarcoma in women with intrathoracic kaposi's sarcoma. ct fi ndings distribution of human herpesvirus 8 dna in tumorous and nontumorous tissue of patients with acquired immunodefi ciency syndrome with and without kaposi's sarcoma kaposi's sarcoma in lymphnodes concurrent with hodgkin's disease the natural history of kaposi's sarcoma in the acquired immunodefi ciency syndrome pulmonary toxoplasmosis in patients in-fected with human immunodefi ciency virus: a french national survey diagnostic imaging, preautopsy imaging and autopsy fi ndings of 8 aids cases analysis on the imaging features of aids with pulmonary fungal infection liver injury in hiv-1-infected patients receiving non-nucleosides reverse transcriptase inhibitors-based antiretroviral therapy mri demonstrations of aids complicated by toxoplasma gondii infection in cervical spinal cord: with 3 cases reports diagnostic imaging in aids in china: current status and clinical application imaging and pathological fi ndings of aids complicated by pulmonary rhodococcus equi infection magnetic resonance spectroscopy in the diagnosis of cognitive impairment in aids patients ct image demonstrations of hiv-seropositive tuberculosis and their relationship with cd4+ t-lymphocyte count positron emission tomography of 18fdg uptake in localized pulmonary infl ammation penicilliosisde rhizomys sinensis accumulation of nuclear mitochondrial dna in the frontal cortex cells of patients with hivassociated neurocognitive disorders imaging fi ndings of aids complicated with pulmonary rhodococcus equi infection and correlated with pathology pulmonary toxoplasmosis? pathogenesis and prevention of rhodococcus equi infection pulmonary toxoplasmosis a female patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. she complained of cough, fever and no sputum. her cd4 t cell count was 45/ μl. filariform larvae of strongyloides stercoralis invade into skin or mucosa and reach the lungs through lymphatic vessels or venous system and the right heart. they develop into schistosomula in 3-30 days. a few schistosomula develop mature in the lungs or bronchi. most schistosomula penetrate the pulmonary capillaries into alveoli to cause a series of respiratory symptoms. in the cases of serious infection and in patients with compromised immunity, disseminated lesions occur in lungs and other organs. hiv/aids related pulmonary strongyloidiasis can have manifestations of local small bleeding spots, pimples, migratory linear or strips urticaria on skin, as well as manifestations of allergic bronchitis, lobular pneumonia or asthma. aids patient may have severe diffuse infection and systemic infection, with symptoms of fever, severe cough, expectoration, hemoptysis, shortness of breath, breathing diffi culty, and asthma. 1. laboratory tests 2. the pathological examinations include biopsy tissue culture and inoculation test. 3. chest x-ray is the most commonly used examination. there are demonstrations of small spots and fl aky shadows, thickened hilar shadow and thickened pulmonary markings. mitchell reported in 1992 that interstitial or alveolar infi ltration in both lungs accounts for 62 %, nodular shadows in both lungs 15 %, hilar or mediastinal lymphadenectasis 26 %, pleural fl uids 42 %, septal line 25 %, mediastinal lymphadenectasis and ascites are the important clues for the diagnosis. 1. the clinical manifestations of hiv/aids related pulmonary strongyloidiasis are non-specifi c. its diagnosis depends on the etiological examinations. the fi ndings of strongyloides sterocralis in the patient's sputum and feces can defi ne the diagnosis. increases to 20 × 10 9 ⁄l; eosinophils granulocytes 25-30 %, or even as high as 70-80 %; the serum total lge level increases by 50 %; 90 % cases with blood serum lgg and lge of fi lariform larvae antigen positive. in the cases with femal strongyloides stercoralis parasitizing in the bronchial epithelium, rhabditiform larva, fi lariform larva, schistosomula, adult strongyloides stercoralis and the eggs can be found in fresh phlegm, which can defi ne the diagnosis. 3. pathological examinations including biopsy tissue culture and inoculation test are positive. 4. by chest x-ray, the lungs have small spots and fl akes of shadows, thickened hilar shadow and thickened pulmonary markings. hiv/aids related pulmonary strongyloidiasis should be differentiated from hiv/aids related pulmonary toxoplasmosis, infectious mononucleosis and mycoplasma pneumonia. pulmonary infi ltration of hiv/aids related malignant lymphoma commonly has three types: primary pulmonary lymphoma, which is rare and accounts for 0. 3. the diagnostic imaging examinations are the most commonly used examinations for the diagnosis. the imaging demonstrations of hiv/aids related malignant lymphomas include: 1. mediastinal lymphadenectasis is the most commonly found pulmonary manifestations of malignant lymphoma. the lesions are mainly located in anterior and middle mediastinum, in asymmetric wave liked or lobulated mass. it occurs unilaterally or bilaterally, isolated or fusion. 2. the incidence of pulmonary parenchymal lesions is 20-30 %. chest x-ray demonstrates mediastinal lymph nodes extending directly into the lungs, which is susceptible to confusion with pneumonia. they are demonstrated as round shadows in the lung fi elds or distribute in the whole lung fi elds. chest x-ray demonstrates the lymphatic spread of lesions as military nodules in different sizes or isolated intrapulmonary nodules or cavities, commonly accompanying with mediastinal hilar lymphadenectasis. in the cases with its occurrence secondary to endobronchial membrance, obstructive pneumonia or atelectasis can be caused. some patients may have diffuse pulmonary interstitial changes. pulmonary infi ltration by non-hodgkin's lymphoma can also be divided into four types: (1) nodular type; (2) pneumoniaalveolar type; (3) bronchial-vascular-lymphatic type, which can be further divided into the central bronchialvascular type, and diffuse lymphatic type; (4) diffuse lymphatic type can have lesions of reticular or reticular nodular infi ltration and its progression into patchy changes. 3. miliary-blood borne spreading type is rare. 4. the pleural lesions is mainly pleural effusion, with bloody or serous pleural fl uid. a male patient aged 41 years was confi rmatively diagnosed as having aids by the cdc. he complained of chest distress and chest pain for more than 2 months. his cd4 t cell count was 36/μl. (1) tuberculoma is diffi cult to be differentiated from the peripheral type of lung cancer. tuberculoma is more common in young adults, with a long term course of illness. the lesions are commonly found in the apical posterior segment of the upper lobe or dorsal segment of the lower lobe. chest x-ray demonstrates the lesions with uneven density and satellite lesions. (2) miliary tuberculosis is diffi cult to be differentiated from diffuse bronchioloalveolar carcinoma. key: cord-004986-en7taikk authors: nagy, nathalie; remmelink, myriam; van vooren, j. p.; salmon, isabelle title: infections gastro-intestinales chez le patient immunocompromis date: 2002 journal: acta endoscopica doi: 10.1007/bf03016656 sha: doc_id: 4986 cord_uid: en7taikk the gastrointestinal tract is frequently involved in immunocompromised hosts. the most common digestive manifestations are dysphagia, odynophagia and diarrhea. these diseases are more frequent in patients with acquired immunodeficiency virus (aids). these gi diseases are of several categories: hiv related inflammatory conditions (hiv related enteropathy, idiopathic esophageal ulceration), infections due to germs also commonly present in immunocompetent patients (salmonellosis, shigellosis,…), opportunistic infections (cmv, mucormycosis,cryptosporidium, mycobacterium, isospora belli,…). the prevalence, pathogenesis, clinical manifestation, gross pathological findings and microscopic features are discussed for each entity. muqueuse, l'absence quasi complete de lymphocytes cd4+ au niveau intra-6pith61ial et de mani~re concomitante une diminution des lymphocytes cdll+ intra-6pith61iaux du gr~le et du rectum. le rapport t4/t8 de la lamina propria est identique h celui observ6 dans le sang p6riph6rique. le nombre et la fonction rdduits des cd4 rdsultent en une diminution du nombre de plasmocytes ig a s~crdtant au sein de la muqueuse. ii existe dgalement une rdduction de la s~cr~tion acide gastrique, probablement lide gt la presence d'anticorps anti cellules paridtales, ainsi qu'une diminution de la motilitd intestinale rdsultant d'une ddnervation autonomique. la combinaison de tous ces facteurs permet une colonisation aisle de l'intestin grgle par une flore anadrobie, source de diarrhdes pr~cddant trds souvent des infections opportunistes [3] . dans les pays industrialis6s, 75 % des patients hiv ou sida d6veloppent des diarrh6es persistantes ; ce chiffre atteint 100 % dans les pays en voie de d6veloppement. dans 80 % des cas, ces diarrhdes sont imputables ~ des ent6ropathog~nes dont la coccioidose est la plus fr6quente [1] . une malabsorption, des anomalies de la muqueuse gr~le en l'absence d'agent pathog6ne a pour la premiere fois 6t6 d6crite en 1984. le virus hiv a 6t6 d6tect6 dans la muqueuse intestihale, suscitant l'hypothbse qu'il soit lui-m6me responsable, du moins en partie, des 16sions observdes. n6anmoins, cette hypoth6se n'a pas 6t6 formellement confirm6e et certains auteurs sceptiques sugg6rent que ces diarrh6es seraient dues ?a des agents opportunistes non d6tectables par les techniques usuelles ou inconnus. enfin, les facteurs alimentaires tels que le r6gime pauvre en prot6ines, le d6ficit en acide folique ou en vitamine a peuvent 6galement affecter l'int6grit6 de l'6pithflium intestinal. approximativement 30 ?a 50 % des patients hiv pr6senteront durant le d6cours de la maladie une atteinte cesophagienne. l'agent pathog6ne le plus fr6quemment isol6 est le candida albicans survenant dans plus ou moins 50 70 % des cas, suivi des infections herp6tiques et cmv, estimdes h 15-35 % [4, 5] . n6anmoins, lorsque toutes les causes 6tiologiques causant des ulc6rations oesophagiennes ont 6t6 61imi-n6es, 40 a 50 % des patients hiv pr6sentent des ulc6rations idiopathiques. au sein du tissu de granulation, la prot6ine hivp24core a 6t6 d6tect6e, sugg6rant une participation active dans le processus d'ulc6ration en l'absence d'autre agent pathog6ne. ces ulc6rations surviennent le plus souvent lorsque le taux de cd4 est bas. les sympt6mes en sont gdn6ralement de l'odynophagie, l'aspect endoscopique mimant les ulc~res her-p6tiques et ~ cmv. ces ulcbres sont souvent difficile h gu6rir ; une instillation intral6sionnelle de corticoides de m6me que le thalidomide semblent donner de bons r6sultats. dans les proctocolites li6es au virus hiv, les marqueurs de l'inflammation sont non sp6cifiques, tels que l'augmentation du nombre de granules lysosomiaux au sein des lymphocytes intra-6pith61iaux, la pr6sence de cellules 6pith61iales apoptotiques, la pr6sence de structures r6ticulaires au sein des cellules endoth61iales, des lymphocytes ou des monocytes. le degr6 d'inflammation semble corr61er avec le taux d'antig~ne p24 d6cel6 dans la muqueuse ainsi qu'avec l'importance des sympt6mes cliniques, sug-g6rant un rfle 6tiologique du virus hiv [1] . l'ent6rocolite neutrop6nique est une condition inflammatoire non li6e au virus hiv ou aux infections opportunistes, 6galement appel6e typhlite. celle-ci est caract6ris6e par une inflammation aigue affectant essentiellement le caecum, l'appendice et parfois l'il6on terminal. d'abord d6crite chez l'enfant leuc6mique et s6v~rement neutrop6nique, l'affection se rencontre actuellement chez les patients souffrant d'h6mopathies malignes ou de cancers. la neutrop6nie associ6e aux effets de la chimioth6rapie permettent aux baet6ries intraluminales d'envahir et d'endommager la muqueuse. une septic6mie, le plus souvent causee par des bacilles gram n6gatifs, survient chez 79 % des patients. au niveau histologique, des h6morragies, un ~ed~me marqu6, une inflammation focale pauvre en cellules inflammatoires sont observ6s. parfois, une zone ndcrotique r6sultant d'un ulc6re bien ddlimit6, provoque une perforation. focalement, des colonies bact6riennes intramurales et des kystes gazeux sousmuqueux peuvent 6tre vus. les infections opportunistes representent la majo-rit6 des infections rencontrees chez l'hete immunocompromis, la plupart d'entre elles se rencontrant au niveau du tractus gastro-intestinal. le spectre de celles-ci s'est rapidement elargi. lors d'etudes anterieures (1990) , 43 % des causes d'enteropathies associees ~ des diarrhees chroniques 6taient inconnues. une etude 4 ans plus tard n'en revelait plus que 19 %, r6sultant de l'identification de nouveaux patho-g~nes [1] . les affections opportunistes varient egalement en fonction du sexe, du groupe h risque, de la localisation et de la periode etudiee. par exemple, la candidose eesophagienne, atteinte gastro-intestinale la plus frequente chez le patient hiv aux etats-unis et en europe, affecte plus particulierement les femmes. de m~me, les infections ~ cmv ou les cryptosporidioses surviennent plus frequemment chez tes homosexuels que chez les patients sida ayant contracte la maladie par drogues intraveineuses. l'isosporiase affectant 30 % des patients sida haitiens est pratiquement absente aux usa. lorsque les traitements sont efficaces et que la survie des patients augmente, l'epidemiologie des infections opportunistes se modifie. par exemple, les pneumonies resultant du pneumocystis carinii ont largement diminue depuis 1987, alors que les infections herpetiques et ~ cmv ont particuli~rement progress6 surtout chez les hommes homosexuels. dans 44 h 68 % des patients sida presentant une enteropathie due ~un ou plusieurs agents pathogenes concomitant, des symptemes gastro-intestinaux sont retrouves. le diagnostic d'infections opportunistes est en general base sur une combinaison de culture de selles, examen direct des selles ~ la recherche d'ceufs ou de larves, et d'une biopsie endoscopique. les symptemes gastro-intestinaux sont plus frdquents chez les patients africains que chez les europeens et les nord americains. les deux plaintes majeures sont l'odynophagie et les diarrhees. l'atteinte ~esophagienne survient chez 30 ~ 40 % des patients ; l'incidence des diarrhees est plus elevee atteignant 90 % particulierement chez les patients sevbrement immunocompromis. les infections virales se rencontrent chez tous les groupes de patients immunocompromis. l'infection cmv est le pathog~ne gastro-intestinal le plus frequent. l'infection herpetique semble 6tre plus frequente chez le patient hiv que chez les autres patients immunodeprimes. dans une importante etude prospective r6alisee sur 100 patients hiv pr6sentant une cesophagite her-petique, le virus hsv n'a 6te identifi6 que darts 5 % des cas alors que la prevalence du virus cmv atteignait 50 % [4] . les infections herpetiques chez l'hete immunocompromis semblent representer une reactivation d'une infection latente contractee plus tet dans la vie, et son incidence augmente lorsque l'immunodepression gagne du terrain. des anticorps igg specifiques diriges eontre l'anti-g~ne herpetique ont 6te documentes chez plus de 80 % des hommes homosexuels. chez le patient hiv, la reactivation du virus hsv survient lorsque le taux de cd4 diminue au-dessous de 50/ram 3. l'infection herpetique infecte essentiellement la muqueuse malpighienne ; d~s lors la muqueuse peri-anale et l'cesophage sont les muqueuses les plus frequemment tou-chees. l'oesophagite herpetique peut parfois, dans des formes severes, se compliquer d'une necrose transmurale associee ~ une fistule tracheo-~esophagienne. les une atteinte gastrique s'accompagne en g6n6ral d'une atteinte diss6min6e dans le tube digestif. les colites ~ cmv prddominent dans le caecum et le c6lon droit ; la muqueuse peut paraitre normale mais en g6n6ral on y observe de profondes ulcdrations. dans certaines atteintes s6vhres, une n6crose de la paroi avec perforation peut s'observer comme dans les m6gac61ons toxiques. l'aspect histologique des cellules infectdes par le virus est caract6ristique ; on retrouve au sein du cytoplasme ou du noyau des inclusions virales ; les cellules semblent ballonis6es. les inclusions nucl6aires sont acidophiles et fr6quemment entour6es d'un halo ; les inclusions cytoplasmiques sont souvent multiples, granulaires et basophiles (fig. 1) . le cmv infecte plusieurs types cellulaires, essentiellement les cellules endoth61iales, mais aussi les cellules musculaires lisses, les fibroblastes, et les cellules ganglionnaires. les cellules glandulaires sont nettement moins infect~es que les cellules malpighiennes. le diagnostic est r6alis6 par endoscopie et biopsies [6] . les immunomarquages et l'hybridation in situ sont des m6thodes plus sensibles que i'he ; la pcr repr6sente la technique diagnostique la plus sensible. le r6sultat du traitement par gancyclovir peut ~tre monitor6 par un grading histologique sur les sp6cimens biopsiques [4] . certains auteurs ont d6velopp6 un syst~me de gradation simple ddfini en trois grades selon le hombre de cellules infectdes visualis6es : grade i = de 1 ~ 4 cellules infect6es, grade ii = de 5 ~ 9, grade lii= plus de 10. les patients sida prdsentent plus fr6quemment des sympt6mes li6s/t ces organismes, ainsi que des infections prolong6es par rapport fi la population g6n6rale. la salmonellose r6sultant d'une infection ~ partir de salmonella typhimurium est 20 fois plus frequente chez les patients sida. la shigellose est souvent isolde des coprocultures chez ces patients sida; dans cette population, l'infection est potentiellement fatale. les infections ~ campylobacter ont 6t6 identifi6es dans approximativement 11% des coprocultures des patients sida, qu'ils souffrent ou non de diarrh6es ; ces patients, pr6sentant une incidence d'infection, sont 39 fois plus importants que dans la population g6n6rale. les femmes sont plus fr6quemment infect6es que les hommes. le clostridium difficile affecte la plupart des types de patients immunocompromis et est li6 essentiellement h la prise d'antibiotiques et aux hospitalisations prolong6es ou r6p6t6es [6] . la tuberculose gastro-intestinale reste rare malgr6 une atteinte pulmonaire fr6quente atteignant 60 70 % des patients sida ; celle-ci ne survient que dans 3 ~ 5 % des cas, chiffre comparable fi ceux retrouv6s dans la population gdn6rale (2 %). le tractus gastro-intestinal semble ~tre la porte d'entr6e de la mycobact6rie et son atteinte est deux fois plus fr6quente que la forme pulmonaire. bien que tousles segments du tractus digestif puissent ~tre entrepris, c'est la portion intestinale qui est le site primaire le plus fr6quent, le foie et la rate 6rant les sites les plus fr6quents de dissdmination [6] . le diagnostic est r6alis6 ~i partir de coprocultures ou de biopsies. au niveau histologique, la lamina propria de la muqueuse intestinale est diffus6ment infiltrde d'histiocytes, comblant les villosit6s et s6parant les cryptes. lorsque cette infiltration est massive, celle-ci est aisdment reconnuc aux colorations de routine d'he ; cependant, la coloration de ziehl facilite le diagnostic. cette coloration permet en effet de visua-liser les nombreux baciues au sein des macrophages mais aussi en extra-cellulaire. le diagnostic diff6rentiel se pose avec la maladie de whipple au sein de laquelle les macrophages sont fortement color6s par le pas et n6gatifs pour la coloration de ziehl. une culture sanguine positive 6tablit le diagnostic de mac diss6min6e, mais pas celui d'une infection active au niveau digestif. une coproculture positive sugg~re mais ne prouve pas une infection digestive. les cultures sanguines peuvent ~tre n6gatives n6cessitant des cultures r6p6-t6es, des biopsies de moelle osseuse ou des traitements empiriques. r6cemment, un nouveau type de mycobact6rie a 6merg6, il s'agit de la mycobactorie genovense. mycobactdrie gordonae est 6galement une cause rare d'atteinte digestive. la spiroch6tose survient relativement frdquemment chez les mgles homosexuels ; l'affection touche 6galement environ 5 % de la population gdndrale, et 30 % des homosexuels ne pr6sentent pas de tableau d'immunod6ficience. la spiroch6tose ne s'accompagne d'aucune 16sion macroscopiquement ou endoscopiquement visible. au niveau histologique, les spirochbtes adh6rent h la surface de la muqueuse ot~ ils apparaissent comme un tapis chevelu bleut6. ils se r6v~lent aux colorations de pas et de warthin-starry [7] . une majorit6 d'6tudes indique que la pr6valence des infections ~ hp est plus faible chez les patients sida que clans la population g6n6rale pour des cohortes appari6es pour le sexe, l'fige et les symp-t6mes. la pr6valence est certainement plus faible lorsque le taux de cd4 est inf6rieur ?a 200. ceci sugg~re un r61e des cd4 et une fonction immune ndcessaire aux infections a hp et aux ulc~res peptiques r6sultant de l'hp. cependant, lorsque les patients sida sont infect6s, la maladie peut 6tre particuli~rement virulente et la charge d'organismes, importante [8] . les candidoses gastro-intestinales surviennent dans une grande cat6gorie des patients immunocompromis mais affecte particuli~rement les patients sida. la candidose est l'affection gastro-intestinale la plus fr6quente chez le patient sida (31%). l'~esophage est l'organe cible. une co-infection avec le virus cmv est frdquente. chez les patients neutrop6niques, la candidose intestinale est une importante source de diss6mination h6matog6ne. le candida albicans est l'espbce la plus fr6quemment isolde. chez les patients sida, la flore orale dif-f~re de celle de la population g6n6rale par une diver-sit6 appauvrie; la pathogen~se de la candidose oesophagienne d6bute done par un remplacement de la flore orale par une flore moins complexe progressant vers l'oropharynx lorsque le nombre de cd4 diminue sous 179/mm 3, r6sultant en une infection eesophagienne invasive lorsque le taux de cd4 est > 129/ram 3 [4] . le diagnostic final se fait g partir de l'identification des filaments myc61iens et des spores h partir de sp6cimens biopsiques ou des brossages cesophagiens. au niveau histologique, la candidose invasive implique la pr6sence de filaments myc61iens p6ndtrant au sein d'une muqueuse intacte, alors que la colonisation se caract6rise par la pr6sence de filaments et de spores au niveau de zones ulcdr6es ou n6crotiques. les filaments peuvent ~tre ddtectds aux colorations de routine d'he mais sont mieux identifiables aux colorations de pas ou de grocott. une histoplasmose colique a 6galement 6t6 d6crite en association avec le syndrome de job. l'identification de l'agent pathog~ne sur mat6riel biopsique est primordiale 6tant donn6 que les mises en culture peuvent prendre plusieurs semaines [1] . la cryptoccocose digestive a 6t6 identifi6e dans 33 % des autopsies or) celle-ci se prdsentait sous sa forme diss6min6e. on la rencontre chez les patients sida mais aussi chez les patients souffrant d'h6mopathies malignes ou b6n6ficiant de corticoth6rapie au long court [1] . au niveau digestif, ce sont l'eesophage et le c61on qui sont le plus souvent affect6s. l'aspergillus infecte rarement le tractus digestif, l'cesophage reste le site pr6f6rentiel. l'aspergillose invasive se rencontre surtout chez les patients sdv~rement immunod6primds (fig. 2) la mucormycose est une affection rare et souvent fatale rencontr6e chez les h6tes immunocompromis. les facteurs de risque principaux incluent le diabbte, l'acidoc6tose, les neutrop6nies s6v6res, les leuc6mies et les traitements immunosuppresseurs. seuls quelques cas ont 6t6 rapport6s chez les patients sida. le tractus gastro-intestinal est rarement affect6, repr6sentant seulement 7 % de tousles cas [7] . l'infection initiale du tractus digestif r6sulte probablement de l'ingestion de spores. 66 % des infections digestives surviennent au niveau gastrique ; les perforations sont fr6quentes. le c61on (21%), le grole (4 %) et l'cesophage (2 %) peuvent 6galement 6tre infect6s. la caraet6ristique histologique principale est l'invasion locale des vaisseaux sanguins par les filaments, induisant des vasculites aigu~s, la formation de thrombi et de n6crose isch6mique du tissu adjacent. les filaments sont peu sept6s, branch6s de manibre irr6gulibre ~ 90 ~ peu color6s par i'he. la plupart des filament sont 6pais et atteignent 10 ~ 15 microns de diam6tre. certains filaments pr6sentent 6galement un aspect torsad6. les diarrh6es sont le sympt6me gastro-intestinal le plus frdquemment rencontr6 chez les patients sida, affectant 50 h 90 % des individus pr6sentant un taux de cd4 rdduit. les protozoaires sont actuellement reconnus comme les principaux agents pathog6nes des diar-rh6es infectieuses, les deux organismes les plus fr6quemment identifi6s 6tant le cryptosporidium et le comme les virus et les mycoses, c'est principalemerit l'intestin gr~le qui est affect6 en premier. ii existe certaines variations g6ographiques, surtout chez les patients sida, l'ispora belli est par exemple frdquemment rencontr6 ~ haiti, alors que le cryptosporidium est le pathog6ne le plus souvent responsable de diarrh6es h washington. une pneumocystose extra-pulmonaire a 6t6 identi-fi6e chez 2,5 % des patients sida/~ new york, avec une atteinte digestive de 22 %. l'affection digestive r~sulte en g~n6ral soit d'une diss6mination h partir des ganglions ou de la voie h6matog~ne suivant une infection pulmonaire soit par r6activation d'une infection gastrique latente. approximativement 50 % des patients prdsentant une forme extra-pulmonaire ont bdn6fici6 d'un traitement ~ la pentamidine, r6duisant le risque de la forme pulmonaire mais ne pr6venant pas la forme diss6min6e. la forme diss6min6e est typiquement un 6v6nement survenant tardivement dans le ddcours de la maladie, lorsque le taux de cd4 est infdrieur 50/mm 3. l'atteinte par pneumocystis a tout d'abord 6t6 d6crite au niveau ~esophagien et duod6nal chez un patient vivant en 1987 ; depuis lors, des infections gastriques, coliques et gr61es ont 6galement 6t6 identi-fi6es. au niveau histologique, il existe de nombreux organismes et macrophages spumeux dans la lamina propria [1] . le cryptosporidium est devenu l'agent pathog~ne le plus fr6quemment responsable des diarrh6es ren-contr6es chez les patients sida. identifi6 en 1976 comme agent responsable d'ent6rites chez l'humain, l'affection reste limit6e chez les h6tes immunocom-p6tents mais peut ~tre s6vbre chez les sujets immunocompromis. la cryptosporidiose survient chez 3 /a 11% des patients sida, quel que soit le groupe ~ risque mais est plus fr6quente chez les homosexuels. l'infection se rencontre surtout chez les hiv pr6sentant des diarrh6es chroniques. lorsque le taux de cd4 est inf6rieur ~ 50/mm 3, l'infection est souvent dramatique. d'autres maladies li6es au sida pr6-c~dent le d6veloppement de la cryptosporidiose dans 85 % des cas [1] . l'organisme infecte le plus souvent le j6junum, mais se rencontre 6galement ailleurs dans le tube digestif ou darts les voies biliaires. le c61on est le deuxi~me site le plus fr6quemment infect6, suivi de l'estomac et de l'~esophage. le diagnostic repose sur l'identification de l'organisme soit dans les aspirations duod6nales, soit sur mat6riel biopsique. les protozoaires sont clairement visualis6s aux colorations de routine d'he, mais sont mieux observ6s 5 la coloration de giemsa. ils se caract6risent par des organismes sph6riques, basophiles, mesurant entre 2 et 4 microns de diambtre et sont attach6s aux microvillosit6s formant la bordure des cellules 6pith61iales (fig. 3) . le sommet ainsi que les bords lat6raux des villosit6s comptent le plus grand nombre d'organismes au nlveau de la muqueuse gr~le; alors qu'au niveau colique, les cryptes et l'6pith61ium de surface sont infect6s de mani~re 6gale. la variabilit6 de taille des organismes correspond aux diff6rentes ~tapes du cycle de vie [4, 6] . le diagnostic peut 6galement 6tre 6tabli par identification des oocystes dans les selles, en utilisant une coloration de ziehl ou une immunofluorescence. les formes tissulaires sont elles ndgatives pour la coloration de ziehl ou les colorations du mucus. les microsporidium sont un groupe h6t6rog~ne de protozoaires, pr6sentant un cycle obligatoirement intra-cellulaires. identifi6s pour la premibre fois chez l'humain en 1985, ils peuvent se rencontrer dans le tractus digestif et les voies biliaires, la v6sicule biliaire et l'arbre respiratoire. l'incidence des diarrh6es ~ microsporidium identitides chez les patients sida varie de 1,7 a 39 %, mais m0me les patients asymptomatiques peuvent ~tre porteurs de l'organisme dans l'intestin gr~le. les ces agents pathog~nes sont difficiles ~ identifier en microscopie optique, la microscopie 61ectronique est souvent n6cessaire. ils peuvent ~tre mis en 6vidence dans les aspirations j6junales et les selles en utilisant une coloration acid-fast afin de d6tecter les oocytes, qui sont autofluorescents bleu lorsqu'ils sont examin6s en microscopie, en 6pifluorescence ultra-violette. la pr6sentation clinique est similaire a celle obser-v6e dans les infections dues au cryptosporidium parvum [9] . entomoeba histolytica est consid6r6 comme un agent commensal non pathog~ne chez les patients homosexuels. l'infection peut survenir lors d'ingestion de kystes par la voie oro-faecale. les patients hiv ne semblent pas pr4senter de susceptibilit6 particulibre ~ amoebae. cependant une colonisation m6me par des agents non pathog6nes peut 8tre responsable d'affections s6vhres chez les patients immunocompromis [6] . giardia lamblia peut occasionnellement provoquer une diarrh6e chez les patients sida. comme c'est le cas pour les entomoeba, le patient hiv ne pr6sente pas de susceptibilit6 particuli6re. la microscopie optique d6tecte largement les giardia, et moins fr6quemment les trophozoites, et constitue la m6thode diagnostique de choix. bien avant les 6pid6mies de sida, le toxoplasme 6tait reconnu comme protozoaire responsable d'infections opportunistes chez les patients immunocompromis. l'atteinte digestive r6sulte en g6n6ral d'une toxoplasmose diss6min6e et s'observe darts 20 % des autopsies. chez les patients sida, l'atteinte digestive peut otre g4n6ralis6e ou n'affecter qu'un seul segment, et peut exceptionnellement 8tre diagnostiqu6e du vivant du patient [7] . c'est un protozoaire obligatoirement intra-cellulaire, qui peut se d6velopper darts n'importe quel type cellulaire, ~ l'exception des globules rouges. certaines 6tudes rapportent des strongyloidoses diss6min6es chez des patients immunocompromis. le d6nominateur commun semble 4tre une corticothdrapie. on ne remarque pas d'incidence plus 61ev6e chez les patients sida. la leishmaniose visc6rale est une maladie s6vhre, acquise en g6n6ral au niveau du bassin m6diterran6en, se ddveloppant le plus souvent chez les h6tes immunocompromis lors de voyages dans les zones end6miques. l'infection s'aequihre par morsure de la femelle de la mouche des sables, transfusions sanguines, infections g6nitales et rapports sexuels. les infections visc6rales sont major6es chez les patients hiv et les atteintes digestives sont fr6quentes; certaines 6tudes rapportent 100% d'atteinte gastro-intestinale [7] . le diagnostic est r6alis6 par identification des amastigotes de la leishmania, dans les frottis ou les sp6cimens biopsiques. les biopsies j6junales pr6sentent des alt6rations villositaires telles que des villosit6s massu6es, dues h l'infiltration massive de la lamina propria par des histiocytes gorg6s de leishmania. les ent6rocytes bordant les cryptes et les villositds ne comprennent pas d'organisme. ceux-ci apparaissent bleut6s h la coloration de giemsa. chez les patients immuno-comp6tents, des granulomes peuvent 8tre observ6s dans la lamina propria ; ils sont en g6ndral absents ou peu form6s chez les immunocompromis. dans une 6tude, le blastocystis homminis 6tait le troisihme organisme le plus fr6quemment identifi6 aprss le cryptosporidium et le cmv chez les patients hiv. ils se rencontrent toujours lorsque l'immunosuppression est sdv6re dans un contexte de diarrh6es chroniques. les biopsies en gdndral apparaissent normales; lorsqu'elles sont anormales, elles exhibent des alt6rations non sp6cifiques. although gastrointestinal infections occur in all groups of immunocompromised patients, the frequency is highest in patients with acquired immunodeficiency syndrome (aids). the reduced number and the reduced function of the cd4+ cells affect the terminal differentiation of ig a-bearing to ig a-secreting b cells, resulting in a decreased number of mucosal iga-bearing plasma cells. there is also a reduction in gastric acid production, probably related to parietal cell antibodies and a decrease in intestinal motility resulting from autonomic denervation. the combination of all these factors promotes colonisation of the small bowel by anaerobic bacteria, which commonly causes diarrhea preceding opportunistic infections [3] . 100 % in some developing countries. at least 80 % of cases of chronic diarrhoea can be attributed to specific enteropathogens among which coccidial parasites are the most frequent [1] . malabsorption and mucosal abnormalities of the small bowel in the absence of detectable pathogens were first described in 1984. hiv has been detected within the intestinal mucosa and it has been proposed that the virus itself might be responsible, at least in part, for the enteropathy. however, no direct evidence of this theory has been produced so far and sceptics favour the suggestion that diarrhoea is actually due to opportunistic infection which has failed to be detected or to unrecognised pathogens. approximately 30-50 % of patients with hiv will have oesophageal disease some time during their illness. candida albicans is the most frequently isolated pathogen, accounting for 50-70 % of cases, followed by cmv and hsv in that order, accounting for 15 to 35 % [4, 5] . however, after all known aetiologies of hiv-related oesophageal ulceration are excluded, 40 to 50 % of patients have idiopathic oesophageal ulceration. hiv p24 core protein has been detected in the granulation tissue. these findings suggest that hiv is capable of producing ulcers in the absence of other pathogens. these ulcers occur mostly when the patients have a low cd4 count. these ulcers can be very difficult to treat, intralesional injection of steroids has shown some therapeutic potential as well as thalidomide. in hiv-related proctocolitis non specific markers of inflammation are seen such as an increased number of lysosomal granules in intraepithelial lymphocytes, focal crypt epithelial cell apoptosis, and tubuloreticular structures in endothelial cells, lymphocytes and monocytes. the degree of inflammation appears to correlate with the mucosal level of p24 antigen and clinical symptoms, suggesting an aetiologic role of hiv [11. first described in children with leukaemia and severe neutropenia, this condition is now known in immunocompromised patients suffering from lymphoma, anaplastic anaemia and cancer. the combined effects of neutropenia and chemotherapy allows luminal bacteria to invade and injure the bowel wall. septicaemia, mostly caused by gram-negative bacilli, occurs in more than 79 % of patients. histologically, haemorrhage, marked edema, and patchy inflammation with a paucity of inflammatory cells are seen. sometimes necrosis results in well demarcated ulcers that may perforate. focally, intramural bacterial colonies and submucosal gas cysts may be seen. in a large prospective study of 100 hiv-infected patients with ulcerative oesophagitis, hsv was identified in only 5 % of cases, whereas the prevalence of cmv was almost 50 % [4] . [1, 4] . pneumocystis carinii. cmv infections typically occur when immunodeficiency is severe (cd4+ < lo0/mm 3) and also represents reactivation of a latent virus conversely, persistent cmv infection itself may promote the decline of immune function. the infection may involve any part of the gi tract, and the risk of involvement of a given segment varies with the type of immunosuppression. in patients with aids, the colon is mostly affected; and in the upper gi tract, oesophageal disease is more common. in immunocompromised patients without aids, the lower and upper gi tract is equally affected and gastroduodenal disease is more common than oesophageal. in the oesophagus cmv preferentially affects the distal segment. gastric involvement is usually associated with involvement elsewhere in the gi tract. in cmv colitis', predominantly seen in the caecum and the right colon, the mucosa may appear normal but usually deep ulcerations are seen. severe cases may manifest as necrotising colitis or toxic megacolon with perforation. nuclear inclusion are acidophilic and often surrounded by a halo, cytoplasmic inclusion bodies are multiple, granular and often basophilic (fig. 1) . the diagnosis is made by endoscopy and biopsy [6] . immunohistochemistry and in situ hybridisation are more sensitive than he; pcr represents the most sensitive technique. results of treatment with ganciclovir can be monitored by means of histological grading of cmv in biopsy specimen. some autors developed a simple grading system that defines grade i as one to four, grade h as five to nine, and grade iii as ten or more cmv-infected cells per biopsy specimen [4] . clostridium difficile affects individuals with variable types of immunosuppression and related to the prevalence of antibiotic use and frequent hospitalisation [6] . these infections include mycobacterium tuberculosis, mycobacterium avium complex or intracellulare and other atypical mycobacteria. tuberculosis, despite extrapulmonary tuberculosis being common in aids patients (occurring in 60 to 70 %), rarely involves the gi tract (3 % to 5 %), and not significantly more frequently than in general population (2 %). oesophageal, small bowel and colonic tuberculosis have been described. oesophageal involvement may develop after mediastinal lymph node tuberculosis and can result in a tracheo-oesophageal fistula. [1, 4] . the gi tract appears to be the most common portal entry and gi involvement is twice as common as the respiratory tract. although segments of the gi tract may be implicated, the intestine is the most common primary site and liver and spleen are the most common sites for dissemination [6] . [7] . the majority of studies indicate that the prevalence of hp in aids patients is significantly lower than in age, sex, and symptom-matched hiv patients. the prevalence is certainly lower when the cd4 count is lesser than 200. this suggests a role of cd4 cell and immune function in sustaining hp infections and hp-related peptic ulcer disease. nevertheless, when aids patients become infected, the d&ease may exhibit particularly aggressive lesions and large number of organisms [8] . candidiasis is the most frequent aids-defining gi disease (31%). the oesophagus is the prime target organ. co-infection with cmv is frequent. in neutropenic patients, gi candidiasis is an important source of haematogenous dissemination. candida albicans is the species most commonly isolated. in aids patients the oral flora differs from that of the general population by a low level of genetic diversity; the pathogenesis of oesophageal candidiasis begins thus with the replacement of the normal oral flora with a less complex flora and progresses to oropharyngeal candidiasis as the number of cd4+ decreases below 179/mm 3 and results in invasive oesophageal disease when the cd4+ are < 129/mm 3 [4] . the definitive diagnosis rests on the identification of typical yeast forms in endoscopic mucosal biopsies or oesophageal brushings. histopathologically, invasive candidiasis implies hypheal penetration into intact mucosa as opposed to colonisation, which implies the presence of yeasts on an intact mucosa surface or in necrotic tissue. candida can be seen on he stains but is better visualised by a pas stain or grocott methenamine silver (gms). other species than c. albicans have been found in aids patients in 7 % to 8 % including c. parapsilosis, c. krusei, c. tropicalis and torulopsis glabrata. disseminated histoplasmosis develops in approximately 5 % of patients with aids in the midwest of the usa where histoplasmosis is endemic. colonic histoplasmosis has also been described in association with job's syndrome. recognition of the organism in biopsy specimen is crucial because culture may take several weeks [1] . cryptococcal gi disease has been identified at autopsy in 33 % of patients with disseminated cryptoccoccosis, including patients with aids and haematological malignancies as well as those receiving corticosteroid therapy. the oesophagus and colon are mostly involved [1] . aspergillosis rarely affects the gi tract, but the target site is the oesophagus. invasive aspergillosis affects severely debilitated patients (fig. 2) [7] . [1] . the organism infects the jejunum most heavily, but it can be found throughout the gi tract including the biliary tract. the colon is the second more common location, followed by the stomach and finally the oesophagus. the diagnosis is made by identification of the organism in either duodenal aspirates, stool, or tissue samples. the protozoan can be clearly observed on he stain, but is best seen with giemsa stain as rows or clusters of basophilic spherical structures 2 to 4 mm in diameter attached to the microvillous border of the epithelial cells (fig. 3) . tips and lateral aspects of villi show the greatest number of organisms in the small bowel whereas in the colon crypt and surface epithelial involvement appears equal. the variation of the size of the organisms corresponds to different stages of the life cycle [4, 6] . the diagnosis can also be made by identification of the oocysts in stools using acid-fast or irnmunofluorescent stains. however, tissue forms do not stain with acid-fast stains, and are mucous stain negative. microsporidia are a heterogeneous group of obligate intracellular spore-forming (coccidian) protozoa. first described in the human in 1985, they can be found in the gi tract, in the biliary tract, the gallbladder and in the respiratory tract. the reported incidence of microsporidium in aids patients with diarrhoea ranges between 1.7 % and 39 %, but even patients without symptoms may harbour the organism in the small bowel. microsporidiosis resulting from infection with enterocytozoon bieneusi affects exclusively the small intestine and the enterocytes, while septata intestinalis, first described in 1992, may disseminate and infect other organs like kidney and gallbladder parasites are then localised in macrophages, fibroblasts and endothelial cells. although electron microscopy of small bowel biopsies is considered to be the best method, the examination of a biopsy specimen stained with he, giemsa or a modified trichrome method, has sensitivities of 77 %-83 % and specificities approaching 100 % [1, 9] . diagnosis can also be made by identification of microsporidial spores in stools and duodenal aspirates. microsporidium is mostly identified in the jejunum as an intracellular parasite seen in the villous enterocytes from just above the mouths of the crypts to the tips of the villi where they are more numerous. parasites are 4 to 5 mm in diameter, supranuclear, either paler or darker than the surrounding cytoplasm, and contain prominent clefts. spores are less frequent than the parasite, but are supranuclear, clustered, dark, and refractile. the cytoplasm of the enterocyte becomes progressively vacuolated, and the nucleus is hyperchromatic. taking into account that albendazole is highly effective for e. intestinalis but largely ineffective for e. bienusi, it is important to make a specific diagnosis [6] . enteritis resulting from isospora belli has been observed in a number of settings of immunosuppres-sion, including aids, alpha-chain disease, lymphoblastic leukaemia and t-cell lymphoma. isospora is common in haitian and african patients with aids (15 % to 60 %), but is rare in the usa (3 % to 7 %). the symptoms are similar to those of cryptosporidiosis, but eosinophilia may be present. the diagnosis is made by identification of the oocysts in stool specimens using acid-fast stain, or by biopsy; the diagnosis is of importance because specific therapy is available. isospora can be recognised on he stains of small bowel specimens, the intracellular protozoon is larger than microsporidium and measures between 10 and 30pro in diameter the common merozoite is bananashaped and found at all levels of the enterocyte cytoplasm. although poorly stained and pale, the central nucleus, large nucleolus, perinuclear halo, and location within a parasitophorous vacuole give it a characteristic appearance. the infection produces mucosal atrophy and tissue eosinophilia. cyclospora [1, 4] . these pathogens are difficult to appreciate on optical microscopy, although electron microscopy is often diagnostic. they can be identified in jejunal aspirates and in stool specimens using acid-fast stain to detect oocysts which are autofluorescent blue when examined with ultraviolet epifluorescence microscopy. the clinical presentation is quite similar to that of cryptosporidium parvum [9] . entamoeba histolytica is often a non-pathogenic commensal in homosexual men. infection can occur by ingesting cysts through oral-anal contact. hiv patients do not appear to have an increased susceptibility to amoebae. as expected, colonisation, even with non-pathogenic strains, may cause significant disease in immunocompromised patients [6] . giardia lamblia can occasionally cause diarrhoea in aids patients. as in the case of amoebae, hiv patients do not appear to have an increased susceptibility to giardia. light microscopic detection of giardia cysts and, less frequently, trophozoites, continues to be the mainstay of diagnosis. even before the aids epidemic toxoplasmosis was well known as a protozoal opportunistic infection of a variety of immunocompromised patients. disseminated toxoplasmosis resulting in gi involvement, was observed at autopsy in 20 % of cases. aids-related gi toxoplasmosis may involve the entire gi tract or only one segment and may exceptionally be diagnosed antemortem by endoscopic biopsy [7] . it is an obligatory intracellular protozoan, that lives in any type of cells with the exception of red blood cells. trophozoites appear round to slightly oval and 2 to 4 pm in diameter in he-stained section. a central or eccentric haematoxylinophylic dot, representing the nucleus surrounded by pale cytoplasm, is seen in trophozoites. pas stains confirm the presence of glycogen-containing bradyzoites and true cysts. some studies report the cases of disseminated strongyloidiasis in a variety of clinical settings of immunosuppression. the common denominator appears to be corticosteroid therapy. visceral leishmaniasis is a severe disease, usually acquired (mediterranean countries) and usually developing in immunocompromised patients, by travel to an endemic region. the infection is acquired through bites of infected female sand flies, blood transfusion, genital infections and sexual contact. visceral infection is increasingly reported in hiv patients, and gi tact involvement is very frequent, some studies reporting an involvement of lo0 % of cases [7] . in one study blastocystis hominis was the third most common organism isolated after cryptosporidiosis and cmv in hlv patients. it is always recovered from severely immunosuppressed patients with chronic diarrhea. biopsies often appear normal; when abnormal, they only exhibit mild non specific abnormalities. gastrointestinal pathology, an atlas and text gastrointestinal disease in the immunocompromised patient. human pathol -review article : the therapy of gastrointestinal infections associated with the acquired immunodeficiency syndrome -aids and the gut recently recognised microbial enteropathies and hiv infection -gastrointestinal manifestations of aids in children c --idiopathic esophageal ulceration in acquired immunodeficieney syndrome : successful treatment with misoprostol and viscous lidocain -lower helicobacter priori infection and peptic ulcer disease prevalence in patients with aids and suppressed cd4 counts e --aids and the gastrointestinal tract. postgraduate medicine key: cord-017506-t86v3zw3 authors: knox, tamsin a.; jerger, logan; tang, alice m. title: alcohol, hiv/aids, and liver disease date: 2012-04-27 journal: alcohol, nutrition, and health consequences doi: 10.1007/978-1-62703-047-2_23 sha: doc_id: 17506 cord_uid: t86v3zw3 globally, there are over 33 million persons living with hiv/aids resulting in 1.8 million deaths annually. while the rate of hiv transmission is slowing, it is estimated that 2.6 million new infections occur yearly [1]. in the united states, there are approximately 1.2 million living with hiv/aids, with 50,000 new hiv infections and 17,000 deaths from the disease annually [2]. for those who can obtain effective antiretroviral therapy (art), hiv/aids has become a chronic disease with life expectancies over 30 years [3]. research in the last 10 years has revealed the importance of alcohol in the hiv/aids epidemic. alcohol use, in moderate or hazardous amounts, has been associated with increased acquisition of hiv infection, progression of hiv infection, deleterious effects on hiv treatment, and acceleration in the comorbidities of hiv infection [4–9]. yet alcohol remains the “forgotten drug” of the hiv/aids epidemic [10]. globally, there are over 33 million persons living with hiv/aids resulting in 1.8 million deaths annually. while the rate of hiv transmission is slowing, it is estimated that 2.6 million new infections occur yearly [ 1 ] . in the united states, there are approximately 1.2 million living with hiv/aids, with 50,000 new hiv infections and 17,000 deaths from the disease annually [ 2 ] . for those who can obtain effective antiretroviral therapy (art), hiv/aids has become a chronic disease with life expectancies over 30 years [ 3 ] . research in the last 10 years has revealed the importance of alcohol in the hiv/ aids epidemic. alcohol use, in moderate or hazardous amounts, has been associated with increased acquisition of hiv infection, progression of hiv infection, deleterious effects on hiv treatment, and acceleration in the comorbidities of hiv infection [4] [5] [6] [7] [8] [9] . yet alcohol remains the "forgotten drug" of the hiv/aids epidemic [ 10 ] . alcohol has a complex relationship with hiv acquisition. risky sexual behaviors, among heterosexuals or among men who have sex with men (msm), that promote hiv transmission are increased in the setting of alcohol these include increased frequency of sexual encounters with new or anonymous partners and reduced condom use [ 11, 12 ] . attention to the locations and clientele where alcohol is served [ 13 ] has led to the development of an "ecological epidemiology" of the interplay of multiple risk factors around hiv transmission [ 14 ] . once infected with hiv, alcohol use is associated with progression of hiv infection from asymptomatic infection, to symptomatic aids with declining immune function measured by low cd4 t-cell counts (<200 cells/mm [ 3 ] ) in the blood, to death from wasting or an opportunistic infection. again, the relationship between alcohol use and progression of hiv infection is multifaceted. hazardous drinking has been associated with delayed testing and treatment for hiv infection [ 12, 15, 16 ] , poor adherence to art therapy [ 6, 17 ] , and increased hiv viral replication and shedding [18] [19] [20] . simian immunode fi ciency virus (siv) infection in monkey models has con fi rmed fi ndings that regular intake of alcohol leads to more rapid progression of disease, weight loss, and death [21] [22] [23] [24] . alcohol use also complicates the care of persons with hiv infection. not only is adherence to art decreased, but drug interactions between alcohol and speci fi c art medications may increase the toxicity of therapy [ 25 ] . hiv infection has numerous comorbidities including coexisting infections such as chronic viral hepatitis or tuberculosis as well as progressive organ dysfunction involving the liver, cardiovascular system, neurological dysfunction, or pulmonary disease. concurrent alcohol use may have a deleterious effect on any of these conditions [26] [27] [28] [29] [30] . thus, the management of alcohol misuse is central to control and treatment of hiv/aids. this chapter summarizes recent research on the effects of alcohol on hiv infection. epidemiologic studies of alcohol use in hiv infection inconsistently de fi ne alcohol intake and problem drinking. many studies categorize alcohol intake as "none," "moderate" drinking (ranging from any alcohol intake to daily intake over the period studied), and "hazardous" drinking (including regular daily intake or binge drinking and may or may not include a diagnosed alcohol disorder). in addition, the studies screening for alcohol disorders use different criteria including the cage questions, audit questionnaire, self-reported drinking, or a physician's report of an alcohol disorder [ 31 ] . thus, varying methodology and study population selection will greatly in fl uence the results from studies of alcohol use in hiv. prevalence of alcohol use among populations with hiv. there are wide apparent differences in rates of alcohol use and hazardous alcohol use due to the populations surveyed, the de fi nitions of "problem" alcohol use even in the same cohort, and the methods used to determine alcohol intake. in general, the prevalence of alcohol use disorders is several fold higher among populations with hiv infection compared to the general us population. some of the highest prevalence rates from problem drinking are among us veterans and homeless veterans [ 6, 58 ] . among the veterans administration (va) population, hazardous drinking patterns are found more frequently in african-americans (26%) than in whites (18%, p < 0.001) [ 59 ] . cook et al. determined that the prevalence of moderate and hazardous drinking among women with hiv infection was also higher than in the general us population [ 42, 56, 57 ] . other characteristics were associated with hazardous drinking patterns such as lower education, unemployment, nonwhite race, depression, and drug use. in both this cohort and in a va cohort, hazardous alcohol use was associated with hepatitis c virus (hcv) infection [ 42, 60 ] . among veterans with hcv infection, 35% were hazardous drinkers compared with 12% hazardous drinkers among matched controls without hcv infection [ 60 ] . the increased alcohol use among idu and the high correlation of idu and hcv infection likely explain this fi nding [ 46, 61 ] . alcohol intake appears to decline over time in persons with hiv infection as it does in noninfected persons with medical illness [ 62 ] . lefevre et al. examined alcohol intake in a group of 111 hivpositive patients of a university hospital clinic, mostly msm. in surveys repeated every 6 months for a mean follow-up of 30 months, the frequency of drinking decreased from 6.4 to 3.9 drinks/week ( p < 0.001) [ 63 ] . in the swiss hiv cohort study, lower alcohol use was found in those who had been on art for longer periods of time [ 64 ] . cook analyzed data from the women's interagency hiv study (wihs) on 2,770 hiv-positive women followed for 11 years [ 42 ] . there was a slight, approximately 5%, decrease in hazardous drinking over time but no change in the overall amount of drinking, possibly as some switched categories from hazardous to nonhazardous drinking. however, there was a signi fi cant decrease in alcohol consumption among women who were coinfected with hepatitis c and hiv from 31% with hazardous drinking patterns in 1995 to 10% in 2006. alcohol has been implicated in accelerating the progression of hiv disease through a number of mechanisms. persons drinking alcohol heavily delay testing for hiv and have less connection with and retention in the health-care system [ 12, 15, 16 ] , delaying the initiation of art. thus, heavy alcohol use predisposes persons to late presentation in the course of infection, with high hiv viral loads, low cd4 counts, and opportunistic infections, and promotes continued spread of hiv [ 45, 65 ] . one of the central ways alcohol intake adversely affects hiv disease is by decreasing adherence to art. adherence to art is key to suppression of hiv replication, prevention of developing drug resistance, and long-term survival [ 66 ] . this has been well documented among all subgroups with hiv infection [ 6, 64, 65, [67] [68] [69] [70] [71] . while there are few studies of adherence in developing countries, one study from india con fi rms the association of alcohol use and risk of nonadherence or discontinuation of art medications [ 72 ] . convincingly, there is a dose-response relationship between alcohol intake and adherence, with higher amounts of alcohol or more hazardous drinking being associated with poorer measures of adherence. samet et al. found that the amount of alcohol consumption was the strongest predictor of adherence with highest levels of adherence being found in those abstinent from alcohol compared to moderate use or at-risk use [ 70 ] . chander et al., studying nearly 2,000 hivinfected persons receiving care at johns hopkins hospital in baltimore, maryland, found that adherence was 22% lower in moderate alcohol users and 54% lower in hazardous alcohol users compared to no alcohol use. adherence was further decreased by 68% with concurrent drug use [ 65 ] . there may be several reasons for lower adherence in persons who use alcohol. drinking pattern affects the likelihood of noncompliance. braithwaite et al., studying 2,700 members of the veterans administration aging cohort study (vacs), found that abstainers missed art on 2% of days. nonbinge drinkers missed medication on 4% of drinking days and post-drinking days but only on 2% of nondrinking days. binge drinkers, in contrast, missed art on 11% of drinking days, 5.5% of postdrinking days, and 4% on nondrinking days [ 6 ] . therefore, while medication adherence was lower on drinking days for binge and non-binge drinkers, missing medications was increased twofold among binge drinkers on days they were either not drinking or post-drinking. this suggests that nonadherence was also due to factors not directly related to alcohol but related to characteristics common among binge drinkers [ 6 ] . sankar et al. studied beliefs about alcohol and art medication interactions in a group of african-american patients treated for hiv [ 71 ] . over three quarters of those surveyed felt that "alcohol and art do not mix"; one-third attributed this to alcohol making art ineffective and another third felt that alcohol made art more toxic. in this study, participants reported purposely skipping art doses when they drank, with light drinkers skipping 64% of the times when they drank and moderate drinkers 55% of the times. however, heavy drinkers skipped art only 29% of the time when they drank and reported that they felt no ill effects from drinking and taking art [ 71 ] . thus, medication adherence is determined by amount of alcohol intake, drinking pattern (binge or non-binge drinking), and beliefs about the safety of alcohol combined with art. issues of medication adherence and alcohol are further discussed in chap. 18 and in a meta-analysis by hendershot [ 17 ] . alcoholics have increased susceptibility to bacterial infections including tuberculosis, pneumonia, and sepsis [ 73 ] . in vitro studies have shown that alcohol impacts several areas of immune function, acting largely as an immunosuppressant. alcohol decreases t-cell proliferation reducing cd4, cd8, and natural killer (nk) cell numbers [ 7 ] and reduces cd8 cell responses to bacteria [ 74 ] . cell-mediated immune responses are decreased [ 75 ] , and myeloid dendritic cells, which are involved in antigen presentation to the immune system, are decreased in number and function with chronic alcohol ingestion [ 76, 77 ] . alcohol increases expression of pro-in fl ammatory cytokines such as tnf-alpha [ 78 ] which may enhance immune dysfunction. experiments by bagasra et al. on human peripheral blood mononuclear cells (pbmc) have shown that cells from healthy persons who are infected in vitro with hiv-1 have higher levels of hiv replication when harvested after alcohol consumption [ 19 ] . enhanced hiv replication was associated with a concurrent inhibition of cd8 cells by alcohol [ 18 ] . siv infection, a macaque model for hiv, has produced evidence of the effect of alcohol on immune function and hiv replication. in rhesus macaques inoculated with siv infection, siv replication was 31-to 85-fold higher in monkeys with chronic alcohol ingestion compared to controls [ 21 ] . siv replication persisted in the central nervous system of alcohol-fed monkeys but was undetectable in control monkeys. poonia et al. proposed that the mechanism of alcohol's effect on siv replication is through its effect on intestinal lymphocytes since the small intestine is one of the most lymphocyterich organs. alcohol-fed monkeys had lower numbers of cd8 cells (before and after siv infection) and higher numbers of cd4 cells in the small intestine after siv infection. they suggested that the 1-2 log 10 increase in siv replication in alcohol-fed monkeys occurs because of the increase in number of cd4 cells susceptible to siv infection in the small intestine and reduction in cd8 cells which may control siv replication [ 22 ] . chronic alcohol ingestion also altered the course of hiv infection with alcohol-fed monkeys having lower cd4 cell counts, lower caloric intake, higher tnf-alpha expression, and a more rapid progression to end-stage siv disease (mean 374 days compared to 900 days in controls) [ 23, 24 ] . alcohol use has been shown to affect hiv progression and survival. in the pre-haart era, alcohol use was not associated with progression to aids [79] [80] [81] . however, two well-controlled, longitudinal studies since the introduction of combination art have shown that alcohol is associated with hiv disease progression. samet et al. studied alcohol use in 595 participants in the macs cohort over 7 years [ 82 ] . heavy alcohol use was associated with a lower mean cd4 cell count (by ~50 cells/ml) but not a decline in cd4 percentage or hiv viral load when adjusted for adherence. baum et al. studied 231 hiv-positive persons followed for 2.5 years [ 5 ] . frequent alcohol users of ³ 2 drinks/day were almost 3 times more likely to develop a cd4 count £ 200 cells/ml, which is an aids-de fi ning event. this effect was particularly marked in alcohol users not on art whose risk of developing a cd4 count £ 200 cells/ml was nearly 8 times nondrinkers. in this study, alcohol use was associated with higher hiv viral load in those on art but not in those without art. these results suggest that the effect of alcohol on hiv viral load is mediated through adherence. however, the effect of alcohol in lowering absolute cd4 count rather than percentage could be in fl uenced by the splenomegaly and secondary lymphopenia seen with alcoholism and chronic viral hepatitis [ 83 ] . moderate to heavy alcohol use has also been associated with increased hiv viral shedding in the female genital tract after controlling for plasma viral load [ 20 ] suggesting that alcohol may affect hiv transmission by physiological as well as behavioral risk factors. the vacs study has provided models for estimating the effect of alcohol on survival in hiv infection. using data on art adherence in the vacs cohort, braithwaite et al. developed a model simulating survival based on levels of alcohol consumption (nondrinkers, hazardous drinkers consuming ³ 5 drinks on drinking days, and nonhazardous drinkers) [ 84 ] . the model predicted decreased survival by >1 year in nonhazardous drinkers drinking at least once a week, 3.3 years in nonhazardous drinkers drinking daily, and up to 6.4 years in hazardous drinkers drinking daily. however, the vacs index, subsequently developed to predict decreases in life expectancy based on hiv and non-hiv characteristics, does not include a separate variable for alcohol or drug abuse beyond adjusting for severity of liver disease and coexisting hcv infection [ 85 ] . in addition, a longitudinal study of changes in physical function with age in the same cohort did not show an effect of alcohol [ 86 ] . further longitudinal studies in this cohort and others should de fi ne the impact of alcohol use on survival in hiv. persons with hiv infection are particularly vulnerable to the effects of alcohol. the detrimental effects of alcohol on the immune system have been covered above, and the effects of alcohol on general health and nutrition are covered in other chapters in this book. persons with hiv infection are at risk of poor nutritional status, and even a 3% weight loss has been associated with increased mortality [87] [88] [89] [90] . thus, further changes in nutritional status due to alcohol use, particularly lower body weight or micronutrient de fi ciencies, would exacerbate the nutritional effects of hiv [ 91, 92 ] . approximately one-third of persons with hiv infection are coinfected with hcv, and approximately 10% have evidence of chronic hepatitis b virus (hbv) infection [ 93 ] . the prevalence of hcv coinfection increases to almost 90% in those who acquired hiv from idu. persons with coinfection with chronic hepatitis have accelerated liver fi brosis leading to cirrhosis [ 9, 94 ] . in a study of liver histology of idu who had acquired hcv infection, those with concurrent hiv infection developed cirrhosis in a mean of 6.9 years after infection compared to 23.2 years among hcv mono-infected persons ( p < 0.001) [ 95 ] . persons with coinfection also have an increased risk of death from end-stage liver disease [96] [97] [98] [99] . they are also at higher risk for drug-induced hepatotoxicity from art [ 100, 101 ] which may be related to altered cytochrome metabolism with progressive liver disease [ 102 ] . other metabolic abnormalities are more common in coinfected persons including hyperglycemia, diabetes, and bacterial translocation from the small intestine to the portal system, predisposing coinfected chronic in fl ammation and progressive liver disease [103] [104] [105] . hazardous alcohol use is increased in some populations with coinfection, particularly idus [ 64, 106 ] . alcohol use further exacerbates the effect of coinfection on liver disease. alcohol use of > 50 g/ day is associated with increased hcv replication [ 107, 108 ] and progressive liver fi brosis assessed by serum markers [ 109 ] , transient elastometry [ 110 ] or by liver biopsy [ 9, [111] [112] [113] . death from endstage liver disease is also more common in coinfected persons who use alcohol [ 29, 30, 114, 115 ] . the incidence of, as well as deaths related to, hepatocellular carcinoma is also increased in those with coinfection who drink alcohol [ 30, 116 ] . only one study did not fi nd an association of alcohol use and an hcv-related severe event (including decompensated cirrhosis, hepatocellular carcinoma, or death) [ 117 ] , but in this cohort, only 10% consumed >30 g of alcohol daily. alcohol use also contributes to metabolic abnormalities in coinfected persons. it is associated with higher rates of liver steatosis [ 110 ] and drug-induced liver disease [ 25, 118 ] . the association of alcohol use with hepatocellular carcinoma is also discussed in chap. 32. the adverse effects of alcohol in coinfection argue strongly for intervention. hazardous alcohol use is a common reason for coinfected persons not receiving treatment for hcv infection, where treatment rates may be as low as 7% [ 106, [119] [120] [121] [122] . fortunately, alcohol use seems to decrease with interventions after hcv diagnosis in some populations [ 123, 124 ] . treatment of chronic viral hepatitis whether due to hcv or hbv infection slows the progression of liver fi brosis [ 125, 126 ] and reduces the incidence of drug-induced liver disease [ 127 ] . treatment outcomes with pegylated interferon and ribavirin [ 128, 129 ] and with the new protease inhibitors for hcv infection should continue to improve as more coinfected persons are being enrolled in treatment [ 130 ] . persons with hiv infection have an increased risk of cardiovascular disease, particularly accelerated atherosclerosis and myocardial infarction [131] [132] [133] . cardiovascular disease is likely due to a combination of additional risk factors found in hiv infection [ 26 ] including (1) chronic in fl ammation from hiv viral replication and subsequent immunode fi ciency [ 134 ] , (2) the effect of chronic in fl ammation on serum lipid levels [ 133 ] , (3) the metabolic effects of certain classes of antiretroviral medications [ 131, 133 ] , (4) increased prevalence of insulin resistance [ 135 ] , and (5) increased translocation of bacteria across the small intestine into the bloodstream as a result of immunode fi ciency [ 136 ] . persons with hiv infection have been shown to have more rapid progression of atherosclerosis measured by intermediate markers such as carotid intima-media thickness, and this has correlated with mortality [ 134, 137, 138 ] . alcohol use further increases the risk of cardiovascular disease in hiv infection. freiberg et al., studying the vacs cohort, found that the risk of cardiovascular disease was increased (or 1.55, 95% ci 1.07-2.23) in hiv-infected men with alcohol abuse or dependence, when controlled for cardiac risk factors, art use, and cd4 count [ 8 ] . furthermore, hcv infection may have an independent effect in increasing the risk of cardiovascular disease (or 4.7, 95% ci 1.7-12.7) although alcohol use does not seem to affect this relationship [ 139 ] . chapters 24 and 25 explore further the relationship of alcohol and cardiovascular disease. alcohol and hiv infection are both risk factors for pulmonary diseases. alcoholics have increased prevalence of oropharyngeal colonization by pathogenic bacteria and an increased risk of aspiration [ 140 ] . in addition, they have impaired pulmonary immune function leading to a higher incidence of pneumonia [ 27, 140 ] . studies have shown that alcohol use is a risk factor for the development of pneumonia in the absence of hiv, as well as more severe, multilobar pneumonia and more virulent pathogens including candida , gram-negative bacteria, and staphylococcus aureus infections. this, in turn, leads to longer hospitalizations and increased mortality related to alcohol use [ 141 ] . the risk for adult respiratory distress syndrome (ards), which has a mortality of 40-60% [ 142 ] , is increased three-to fourfold in those with heavy alcohol intake [ 143, 144 ] . similarly, persons with hiv infection are at an increased risk of community-acquired pneumonias, including unusual pathogens such as pseudomonas aeruginosa [ 145 ] . hiv infection is also associated with pulmonary opportunistic infections, such as pneumocystis [ 146 ] . while both alcohol use and hiv infection have an increased risk of pneumonia and tuberculosis, there are no studies to date that demonstrate the interaction of these risk factors for acute pulmonary disease [ 27 ] . there is suggestive literature that depletions in zinc levels or pulmonary glutathione stores may mediate impaired host defense [ 27 ] . chronic lung disease in alcoholics is largely related to associated tobacco use [ 27 ] . however, persons with hiv infection have an increased risk of emphysema, lung cancer, and pulmonary hypertension, independent of smoking, and this is particularly evident in those with poorly controlled hiv infection [ 147 ] . the adverse effects of alcohol use on hiv are evident, and interventions to mitigate alcohol use among hiv-infected individuals are needed. to date, clinical studies and a few randomized controlled trials (rcts) assessing the effectiveness of interventions have shown mixed results. in this section, we will brie fl y review the types of interventions that have been evaluated and discuss results from a few published trials. interested readers can refer to recent review articles for more complete reviews of the literature [ 13, 148, 149 ] . many types of alcohol interventions have been tested among hazardous alcohol users with and without hiv infection. these include brief interventions as well as more intensive behavioral, social network, and medication interventions. brief interventions, also referred to as brief motivational interviews, are typically a single session discussing the patients' alcohol use. studies employing this type of intervention often involve exploration of the pros and cons of a patient's alcohol use, self-assessment of the patient's alcohol consumption severity, and a more formal assessment of the patient's alcohol consumption as compared to the general population [ 150 ] . more extensive behavioral interventions have also been investigated, including cognitive-behavioral therapy, motivation enhancement, or 12-step programs. each of these behavioral interventions is directly aimed at investigating personal motivation behind alcohol consumption and developing personal behavior modi fi cation strategies [ 151 ] . these interventions typically require multiple sessions. in addition to individualized plans and programs for those with increased alcohol consumption, social network and structural interventions which target larger populations and communities have also been evaluated. social network interventions have most commonly focused on employing in fl uential community leaders to change speci fi c behaviors or promote health-conscious decisions. these studies, often referred to as popular opinion leader (pol) or peer-based model interventions, may be particularly effective in communities that are dif fi cult for outside researchers to impact [ 152 ] . alternatively, structural interventions, which may include political and legal action, may also be effective in altering individuals' behavior and environment. lastly, medications, such as disul fi ram, naltrexone, and acamprosate, have been shown to decrease alcohol consumption via physiologic effects, including decreasing cravings or causing adverse reactions when alcohol is consumed [ 149 ] . in addition to the type of alcohol intervention, there are several other factors to consider when evaluating results from clinical trials of alcohol interventions. the fi rst is the setting in which the interventions are conducted. interventions have been conducted in various settings including primary care clinics [ 153, 154 ] , hospital inpatient settings [ 155 ] , emergent care settings [ 156 ] , and social settings or drinking venues (places where alcohol is served) [ 13 ] . a second important factor to consider is the population being targeted, which may vary depending on severity of alcohol use (dependent vs. nondependent drinkers), geographic region, and cultural practices around drinking. a third factor to consider is the outcome that is being targeted. for example, previous trials have examined the effects of alcohol interventions on decreasing alcohol consumption, improving adherence to antiretroviral medication and/or reducing sexual risk behaviors. the combination of the type of intervention, the setting in which the intervention is implemented, the population that is being targeted, and the expected outcomes of the trial will all contribute to the success or failure of an intervention. the published literature on rcts of alcohol interventions among populations affected by hiv re fl ects the various combinations of factors described above. for example, one study targeting msm in the usa with alcohol use disorders combined two types of interventions (motivational interviewing and peer-group education/support strategies) and examined the effects on reducing at-risk drinking and sexual risk behaviors [ 157 ] . in this study, individuals receiving the combined intervention reported signi fi cantly lower number of days of drinking and number of heavy drinking days per 30-day period compared to control participants. another study tested the effects of a brief theorybased behavioral hiv-alcohol risk-reduction intervention on sexual risk behaviors in men and women recruited from informal drinking establishments in a suburban township of capetown, south africa [ 50 ] . the authors reported signi fi cant reductions in unprotected intercourse, increased use of condoms, and less use of alcohol before sex in the intervention group compared to controls, with the largest impacts among lighter drinkers. these two studies illustrate the success of individual counseling interventions for reducing risk behaviors around alcohol consumption among persons at risk for or living with hiv. other interventions among individuals with hiv who consume alcohol have targeted the outcome of antiretroviral medication adherence. in two speci fi c studies [ 151, 158 ] , motivational interviews and cognitive-behavioral skills training were not effective in improving long-term medication adherence. given the importance of adherence to art to controlling hiv infection, more research is needed to develop novel interventions targeting this outcome. interventions directed at alcohol-serving establishments have had mixed results. studies have focused on popular opinion leader (pol) models, in which community-de fi ned opinion leaders are identi fi ed and trained to help shift social norms and behaviors toward safer sexual practices [ 152 ] . this type of intervention in gay bars in several us cities signi fi cantly reduced episodes of risky sexual behavior compared to control bars [ 152, 159, 160 ] ; however, when this intervention was adapted for testing in several international settings, the fi ndings were negative in that comparable reductions in risky sexual behaviors and incidence of sexually transmitted infections were seen in both intervention and control communities [ 161 ] . another study testing the effects of a peer-based intervention on reducing episodes of unprotected sex with non-wife partners in beer halls in zimbabwe found no difference compared to controls [ 162 ] . in summary, interventions involving varied counseling approaches directed at decreasing alcohol consumption and/or risky sexual behavior appear promising in speci fi c settings. other areas of investigation, such as interventions aimed at improving art adherence among alcohol users or use of medications for alcohol dependence (such as naltrexone) in hiv-infected populations, need further research. more intervention studies will help to generalize fi ndings across different contexts and help to improve health outcomes and minimize the effects of alcohol on persons living with hiv. in this chapter, we have examined the prevalence of hazardous alcohol use in hiv which is much higher than found in the general us population. alcohol use and frequenting venues where alcohol is consumed has been shown to be an important risk factor for the acquisition of hiv infection. understanding the complex interrelationships between individual characteristics and venues should improve our approach to prevention [ 12, 14 ] . the effect of alcohol on adherence to art is well documented. there are also good laboratory models, particularly with siv infection in macaques, to show that chronic alcohol use accelerates the progression of disease. finally, alcohol use has deleterious effects on health, particularly related to progression of liver disease in persons with hiv/hcv coinfection. health-care providers may underestimate the extent of hazardous drinking among their hiv patients. a study in the va population showed that the sensitivity for health-care providers' ability to diagnose hazardous drinking was only 22% [ 163 ] . thus far, trials of interventions to reduce hazardous drinking in populations affected by hiv have shown mixed results. the underdiagnosis of hazardous alcohol use and lack of proven, effective treatment strategies raise the question of whether there is any "safe" level of alcohol intake in hiv. justice et al. examined the relationship of medical illness related to alcohol use in veterans with hiv infection [ 164 ] . for diseases associated with alcohol use (hcv infection, hypertension, diabetes, chronic obstructive lung disease, and certain infections), there was a linear relationship between alcohol intake category (none, moderate, hazardous) and the disease. this suggests that there may be no "safe" level of alcohol intake for hiv-infected persons [ 165 ] . more aggressive screening and treatment of alcohol-related disorders is clearly warranted to prevent hiv transmission and to improve treatment and outcomes of persons with hiv infection [ 84 ] . racial and sex disparities in life expectancy losses among hivinfected persons in the united states: impact of risk behavior, late initiation, and early discontinuation of antiretroviral therapy med care (alcohol in hiv infection: insights from the veterans aging cohort study and the veterans affairs national health information system) alcohol use accelerates hiv disease progression a temporal and dose-response association between alcohol consumption and medication adherence among veterans in care alcohol's role in hiv transmission and disease progression the association between alcohol consumption and prevalent cardiovascular diseases among hiv-infected and hiv-uninfected men incidence and predictors of severe liver fi brosis in human immunode fi ciency virus-infected patients with chronic hepatitis c: a european collaborative study alcohol: the forgotten drug in hiv/aids causal links between binge drinking patterns, unsafe sex and hiv in south africa: its time to intervene integrating hiv/aids and alcohol research social and structural hiv prevention in alcohol-serving establishments: review of international interventions across populations hiv risk and the alcohol environment: advancing an ecological epidemiology for hiv/aids alcohol and hiv disease progression: weighing the evidence health services utilization for people with hiv infection: comparison of a population targeted for outreach with the us population in care alcohol use and antiretroviral adherence: review and meta-analysis increased human immunode fi ciency virus type 1 replication in human peripheral blood mononuclear cells induced by ethanol: potential immunopathogenic mechanisms alcohol intake increases human immunode fi ciency virus type 1 replication in human peripheral blood mononuclear cells alcohol consumption and hiv-1 vaginal rna shedding among women increased viral replication in simian immunode fi ciency virus/ simian-hiv-infected macaques with self-administering model of chronic alcohol consumption chronic alcohol consumption results in higher simian immunode fi ciency virus replication in mucosally inoculated rhesus macaques chronic binge ethanol consumption accelerates progression of simian immunode fi ciency virus disease chronic alcohol accentuates nutritional, metabolic, and immune alterations during asymptomatic simian immunode fi ciency virus infection risk factors for severe hepatic injury after introduction of highly active antiretroviral therapy focus on the heart: alcohol consumption, hiv infection, and cardiovascular disease modeling hiv and alcohol's effects: focus on the lung focus on the brain: hiv infection and alcoholism prevalence factors associated with signi fi cant liver fi brosis assessed by transient elastometry in hiv/hepatitis c virus-coinfected, patients liver-related deaths in, h. i. v. infected patients between in the french, germivic joint study group network detecting alcohol problems in hiv-infected patients: use of the cage questionnaire the association between hiv infection and alcohol use: a systematic review and meta-analysis of african studies alcohol as a correlate of unprotected sexual behavior among people living with hiv/aids: review and meta-analysis early alcohol initiation and subsequent sexual and alcohol risk behaviors among urban youths alcohol use, partner type, and risky sexual behavior among college students: fi ndings from an event-level study hiv and sexually transmitted diseases: lethal synergy alcohol and sexual hiv risk behavior among problem drinking men who have sex with men: an event level analysis of timeline followback data alcohol use and risk of hiv infection among men who have sex with men alcohol use and sexual risk behavior among human immunode fi ciency virus-positive persons depressive symptoms, utilization of mental health care, substance use and sexual risk among young men who have sex with men in explore: implications for age-speci fi c interventions risk factors for hiv infection among men who have sex with men longitudinal trends in hazardous alcohol consumption among women with human immunode fi ciency virus infection alcohol consumption, art usage and high-risk sex among women infected with hiv hiv, alcohol, and noninjection drug use alcohol abuse and stage of hiv disease in intravenous drug abusers the impact of illicit drug use and harmful drinking on quality of life among injection drug users at high risk for hepatitis c infection alcohol use and sexual risks for hiv/aids in sub-saharan africa: systematic review of empirical fi ndings multiple recent sexual partnerships and alcohol use among sexually transmitted infection clinic patients socioeconomic status and risk of hiv infection in an urban population in kenya randomized trial of a community-based alcohol-related hiv riskreduction intervention for men and women in cape town south africa hiv rates and risk behaviors are low in the general population of men in southern india but high in alcohol venues: results from 2 probability surveys male alcohol use and unprotected sex with non-regular partners: evidence from wine shops in chennai women and substance use in india and bangladesh the impact of hiv and high-risk behaviours on the wives of married men who have sex with men and injection drug users: implications for hiv prevention estimation of hiv-1 incidence among fi ve focal populations in dehong, yunnan: a hard hit area along a major drug traf fi cking route the 12-month prevalence and trends in dsm-iv alcohol abuse and dependence: united states binge drinking among us adults associations between alcohol use and homelessness with healthcare utilization among human immunode fi ciency virusiinfected veterans alcohol problems and health care services use in human immunode fi ciency virus (hiv)-infected and hiv-uninfected veterans co-morbid medical and psychiatric illness and substance abuse in hcv-infected and uninfected veterans high prevalence of alcohol use among hepatitis c virus antibody positive injection drug users in three us cities reduction in alcohol consumption and health status alcohol consumption among hiv-infected patients self-reported alcohol consumption and its association with adherence and outcome of antiretroviral therapy in the swiss hiv cohort study hazardous alcohol use: a risk factor for non-adherence and lack of suppression in hiv infection adherence to protease inhibitor therapy and outcomes in patients with hiv infection gender differences in factors associated with adherence to antiretroviral therapy problem drinking and medication adherence among persons with hiv infection alcohol use and incarceration adversely affect hiv-1 rna suppression among injection drug users starting antiretroviral therapy alcohol consumption and antiretroviral adherence among hiviinfected persons with alcohol problems sero-positive african americans' beliefs about alcohol and their impact on anti-retroviral adherence access, adherence, quality and impact of arv provision to current and ex-injecting drug users in manipur (india): an initial assessment alcohol abuse, alcoholism, and damage to the immune system-a review chronic ethanol induces inhibition of antigen-speci fi c cd8+ but not cd4+ immunodominant t cell responses following listeria monocytogenes inoculation consequences of alcohol consumption on host defence alcohol exposure impairs myeloid dendritic cell function in rhesus macaques alcohol suppresses the granulopoietic response to pulmonary streptococcus pneumoniae infection with enhancement of stat3 signaling regulation of macrophage activation in alcoholic liver disease no evidence for a role of alcohol or other psychoactive drugs in accelerating immunode fi ciency in hiv-1-positive individuals. a report from the multicenter aids cohort study cofactors of progression to acquired immunode fi ciency syndrome in a cohort of male sexual contacts of men with human immunode fi ciency virus disease determinants of hiv disease progression among homosexual men registered in the tricontinental seroconverter study alcohol consumption and hiv disease progression the impact of cirrhosis on cd4+ t cell counts in hiv-seronegative patients estimating the impact of alcohol consumption on survival for hiv + individuals towards a combined prognostic index for survival in hiv infection: the role of 'non-hiv' biomarkers association of age and comorbidity with physical function in hivinfected and uninfected patients: results from the veterans aging cohort study malnutrition in a population of hiv-positive and hiv-negative drug users living in chennai, south india. drug alcohol depend weight loss and survival in hivpositive patients in the era of highly active antiretroviral therapy increasing risk of 5% or greater unintentional weight loss in a cohort of hiv-infected patients weight loss and body-composition changes in men and women infected with hiv in fl uence of chronic alcohol abuse on body weight and energy metabolism: is excess ethanol consumption a risk factor for obesity or malnutrition? inadequate dietary intake and altered nutrition status in early hiv-1 infection hepatitis c virus infection as an opportunistic disease in persons infected with human immunode fi ciency virus in fl uence of human immunode fi ciency virus infection on the course of hepatitis c virus infection: a meta-analysis human immunode fi ciency virus infection modi fi es the natural history of chronic parenterally-acquired hepatitis c with an unusually rapid progression to cirrhosis increasing mortality due to end-stage liver disease in patients with human immunode fi ciency virus infection rates and risk factors of liver fi brosis progression in patients with chronic hepatitis c natural history of liver fi brosis progression in patients with chronic hepatitis c. the obsvirc, metavir, clinivir, and dosvirc groups little evidence that hepatitis, c. virus leads to a higher risk of mortality in the absence of cirrhosis excess alcohol intake: the swiss hepatitis c cohort, study. j viral hepatitis hepatotoxicity associated with antiretroviral therapy in adults infected with human immunode fi ciency virus and the role of hepatitis c or b virus infection highly active antiretroviral therapy-induced liver injury ritonavir greatly impairs cyp3a activity in hiv infection with chronic viral hepatitis hepatitis c virus antibody-positive patients with hiv infection have a high risk of insulin resistance: a cross-sectional study the effect of haart and hcv infection on the development of hyperglycemia among hiv-infected persons human immunode fi ciency virus-related microbial translocation and progression of hepatitis c relative prevalence of comorbidities and treatment contraindications in hiv-mono-infected and hiv/hcv-co-infected veterans effect of alcohol use and highly active antiretroviral therapy on plasma levels of hepatitis c virus (hcv) in patients coinfected with hiv and hcv an overview of hiv and chronic viral hepatitis co-infection hazardous drinking is associated with an elevated aspartate aminotransferase to platelet ratio index in an urban hiv-infected clinical cohort risk factors for advanced liver fi brosis in hiv-infected individuals: role of antiretroviral drugs and insulin resistance liver fi brosis progression in human immunode fi ciency virus and hepatitis c virus coinfected patients. the multivirc group factors affecting liver fi brosis in human immunode fi ciency virusand hepatitis c virus-coinfected patients: impact of protease inhibitor therapy a comparison of fi brosis progression in chronic liver diseases the in fl uence of human immunode fi ciency virus coinfection on chronic hepatitis c in injection drug users: a long-term retrospective cohort study mortality related to chronic hepatitis b and chronic hepatitis c in france: evidence for the role of hiv coinfection and alcohol consumption hepatocellular carcinoma and nonhodgkin's lymphoma: the roles of hiv, hepatitis c infection, and alcohol abuse the french national prospective cohort of patients co-infected with hiv and hcv antiretroviral drugs and liver injury rates and predictors of hepatitis c virus treatment in hcv-hiv-coinfected subjects rates of hcv treatment eligibility among hcv-monoinfected and hcv/ hiv-coinfected patients in tertiary care referral centers barriers to treatment of hepatitis c in hiv/hcv coinfected adults in brazil barriers to treatment of hepatitis c in hiv/hcvcoinfected adults with alcohol problems medical care and alcohol use after testing hepatitis c antibody positive at std clinic and hiv test site screening programs awareness of hepatitis c diagnosis is associated with less alcohol use among persons co-infected with hiv liver fi brosis changes in hiv-hbv-coinfected patients: clinical, biochemical and histological effect of long-term tenofovir disoproxil fumarate use slower fi brosis progression in hiv/hcv-coinfected patients with successful hiv suppression using antiretroviral therapy managing symptomatic drug-induced liver injury in hiv-hepatitis c virus-coinfected patients: a role for interferon peginterferon alfa-2a plus ribavirin versus interferon alfa-2a plus ribavirin for chronic hepatitis c in hiv-coinfected persons peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection in hiv-infected patients care of hepatitis c virus infection in human immunode fi ciency virusinfected patients: modi fi cations in three consecutive large surveys between trends in rates of myocardial infarction among patients with hiv epidemiological evidence for cardiovascular disease in hiv-infected patients and relationship to highly active antiretroviral therapy cardiovascular disease risk factors in hiv patients-association with antiretroviral therapy. results from the dad study markers of atherosclerosis and in fl ammation and mortality in patients with hiv infection combination antiretroviral therapy and the risk of myocardial infarction microbial translocation is a cause of systemic immune activation in chronic hiv infection framingham risk score and early markers of atherosclerosis in a cohort of adults infected with hiv progression of atherosclerosis as assessed by carotid intima-media thickness in patients with hiv infection the association between hepatitis c infection and prevalent cardiovascular disease among hiv-infected individuals alcohol, immunosuppression, and the lung high alcohol intake as a risk and prognostic factor for community-acquired pneumonia incidence and outcomes of acute lung injury chronic alcohol abuse is associated with an increased incidence of acute respiratory distress syndrome and severity of multiple organ dysfunction in patients with septic shock the alcoholic lung: epidemiology, pathophysiology, and potential therapies bacterial pneumonia in hospitalized patients with hiv infection: the pulmonary complications, icu support, and prognostic factors of hospitalized patients with hiv (pip) study hiv-associated opportunistic pneumonias hiv infection and risk for incident pulmonary diseases in the combination antiretroviral therapy era effectiveness of hiv prevention for youth in sub-saharan africa: systematic review and meta-analysis of randomized and nonrandomized trials interventions targeting hiv-infected risky drinkers: drops in the bottle brief alcohol intervention and alcohol assessment do not in fl uence alcohol use in injured patients treated in the emergency department: a randomized controlled clinical trial motivational interviewing and cognitive-behavioral intervention to improve hiv medication adherence among hazardous drinkers: a randomized controlled trial randomised, controlled, community-level hiv-prevention intervention for sexual-risk behaviour among homosexual men in us cities screening in brief intervention trials targeting excessive drinkers in general practice: systematic review and meta-analysis effectiveness of brief alcohol interventions in primary care populations effectiveness of opportunistic brief interventions for problem drinking in a general hospital setting: systematic review preventive care in the emergency department: screening and brief intervention for alcohol problems in the emergency department: a systematic review reducing sexual risk behaviors and alcohol use among hiv-positive men who have sex with men: a randomized clinical trial a randomized controlled trial to enhance antiretroviral therapy adherence in patients with a history of alcohol problems community aids/hiv risk reduction: the effects of endorsements by popular people in three cities hiv prevention with male prostitutes and patrons of hustler bars: replication of an hiv preventive intervention results of the nimh collaborative hiv/sexually transmitted disease prevention trial of a community popular opinion leader intervention evaluation of a peer network-based sexual risk reduction intervention for men in beer halls in zimbabwe: results from a randomized controlled trial veterans aging cohort 3-site s. how harmful is hazardous alcohol use and abuse in hiv infection: do health care providers know who is at risk? med care (alcohol in hiv infection: insights from the veterans aging cohort study and the veterans affairs national health information system) role of alcohol in determining human immunode fi ciency virus (hiv)-relevant outcomes: a conceptual model to guide the implementation of evidence-based interventions into practice. med care (alcohol in hiv infection: insights from the veterans aging cohort study and the veterans affairs national health information system) the prevalence of alcohol consumption and heavy drinking among people with hiv in the united states: results from the hiv cost and services utilization study longitudinal assessment of the effects of drug and alcohol abuse on hiv-1 treatment outcomes in an urban clinic determinants of heterogeneous adherence to hiv-antiretroviral therapies in the multicenter aids cohort study key: cord-013442-kjfk7hq6 authors: muñoz-laboy, miguel; bamford, laura; benitez, jose; zisman-ilani, yaara; ripkin, alexandra; del castillo, laura; esteves-camacho, tracy; de la cruz, mario; katumkeeryil, elby title: “en la lucha”: strategies to improve hiv care for puerto ricans with opioids use disorders date: 2020-10-30 journal: j immigr minor health doi: 10.1007/s10903-020-01091-6 sha: doc_id: 13442 cord_uid: kjfk7hq6 background: clínica bienestar is a comprehensive hiv primary care clinic for spanish-speaking latinx with opioids use disorders (oud). this article describes the barriers and trajectories to hiv viral suppression for puerto ricans with a transnational profile and dual diagnoses (hiv and oud), and the strategies applied to increase retention in care. methods: case study methodology was used to select two patient life histories that illustrate the most common pathways to success in reducing hiv viral load to undetectable and achieving oud long-term recovery. results and discussion: patients’ major challenges included: (1) persistent migrating while seeking substance use treatment services with limited or no support from their sending and hosting communities; (2) intersectional stigmas; (3) untreated trauma; (4) language and cultural barriers. clínica bienestar’s service model included ten strategies to retain patients in care (e.g., case management to identify cases with high social isolation), six emerged as central to addressing transnational challenges. fewer than half of those with an opioid use disorder (oud) receive treatment in the united states [1] . these unmet oud treatment needs are more pronounced in latinx communities [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . in cities like philadelphia, pennsylvania, the zip codes with the highest concentrations of latinx residents also have the highest opioid overdose-related deaths (an indicator of untreated oud) and the highest annual hiv incidence rates over the past two decades [12, 13] . latinx with untreated oud have lower levels of retention in hiv care; and lower hiv viral suppression, and substance use treatment completion rates than other racial/ethnic groups, nationwide and locally in philadelphia [14] [15] [16] . in order to decrease hiv viral loads at the population levels in latinx communities, research examining detection, linkage and retention in hiv care is urgently needed, particularly amongst the subgroups with the highest incidence rates, such as latinx with dual diagnoses of hiv and oud. when describing the service needs and challenges experienced by this subgroup, it is imperative to recognize the impact of transnationalism on their health seeking behaviors. transnationalism describes the duality observed in many immigrant communities where community members may exhibit both, an allegiance and association with their "receiving" or hosting country, where they are making a new home and, simultaneously, their sending country or place of birth/origin [17] . the challenges faced by recent immigrants, who may need to access health services, to acculturate and adjust to new social norms, learn new vocabulary, and navigate the economic demands of daily life in a new place, can all contribute to difficult experiences accessing care. cultural barriers and migration patterns such as the "air bridge" (i.e. the cyclical migratory patterns between the territory of puerto rico and the east coast of the contiguous usa), are challenges to cities like philadelphia in the provision of hiv and oud services for latinx communities [18] [19] [20] [21] [22] . philadelphia has experienced a 46% increase in its latinx population in the past decade [23] . in response to this critical need for dual treatment for a transnational population, prevention point philadelphia (ppp), philadelphia fight community health centers (fight), and temple university's school of social work established clínica bienestar (spanish for "wellness clinic") funded initially by the special projects of national significance (spns) in december 2013. clínica bienestar is an hiv primary care clinic targeting spanishspeaking or bilingual puerto rican adults (aged 18 + years) with previous or current injection drug use, or who meet criteria for oud, who are newly diagnosed with hiv or who are not currently retained in hiv care (defined as being out of hiv care for at least 6 consecutive months in the previous two years-prior to intake) [24] . clínica bienestar's model of service provision consisted of outpatient clinical services two days per week combined with hiv patient navigation, case management, oud treatment, and recovery services throughout the week, as tailored to individual patient needs. the list of specific services has been published elsewhere [25] . the model has been effective at achieving hiv viral suppression with a statistically significant hiv viral suppression rate of 82.9% (95% confidence interval: 72.9%-93.1%) [25] . the objective of this article is to describe both the patient-level barriers identified and the contributing factors that influenced successful trajectory to hiv viral suppression for puerto ricans with dual diagnoses (hiv and oud); including the strategies implemented in the clínica bienestar service model to improve retention in care. while this feature of persistent migration was not present for all patients enrolled in the larger spns demonstration project, all patients (prior to clínica bienestar) experienced language and cultural barriers to hiv and oud care associated with their transnational backgrounds. thus, the service model was guided by a transnational framework-one which identifies, acknowledges, and builds upon the connections that latinx migrant populations may use to maintain ties to their sending countries/places of origin while living in the continental u.s [26] . having a basic understanding of the extent that this demonstration project addressed the needs of a transnational sample of latinx living with hiv who were out-of-care and dealing with co-occurring oud and other substance use disorders could provide insights into other programs caring for similar populations. based on more than two decades of organizational experiences since the onset of the hiv epidemic in philadelphia, and the review of the scientific literature in hiv care continuum outcomes, the leadership of both organizations and the intervention designers decided to establish clínica bienestar as guided by three principles: (1) colocation of services for transnational groups to increase utilization of hiv services by minimizing unnecessary navigation through complex health care systems (primary care services, hiv services, substance use disorder treatment) while decreasing duplicative costs to achieve similar health outcomes [27] . (2) integrated dual treatment approach (iddt), a well-documented, effective approach to mental health and substance use treatments, expanded to include hiv primary care services to substance use and mental health services for latinx with dual hiv and oud diagnoses [28] [29] [30] . (3) harm-reduction, critical time (cti) approach for transnational populations living with hiv focusing on people's readiness for treatment and engagement in their own hiv and oud treatment and care, and not requiring abstinence from substance-using before hiv treatment [31] [32] [33] [34] [35] . these above principles are relevant to understand the design of the overall spns intervention, as well as the tailoring of service delivery methods and strategies to address the needs of the patients who were also experiencing transnational challenges [36] . to conceptualize the experience of participants of clínica bienestar, we have to draw on the literature on intersectional stigmas, acculturative and minority stress. before entering the doors of clínica bienestar, the population from where our participants emerged have experienced systemic forms of durable oppressions, welldocumented to decrease the likelihood of engagement and retention in any form of health care service [37] . because of the racial/class structure in the usa, latinx are labeled into multiple combinations of minority statuses (racial, ethnic, gender expression, sexual orientation, social class) [37] . latinx who use substances, living with hiv and oud, experience additional forms of stigmatization manifested as systemic and interpersonal prejudice, rejection, discrimination, and violence against people who use substances and/or because of hiv status [19, [38] [39] [40] [41] . taken together, the additive effects of intersectional forms of stigma, in the absence of proper social support systems, result in experiencing high levels of minority and acculturative stress [37] [38] [39] [40] [41] [42] . minority stress refers to the continuum from distal to proximal stressors that derive from the personal embodiment of both internally and externally derived exclusionary processes, such as societal prejudice against people who use drugs, racial discrimination, antiimmigrant discrimination, and/or hiv stigmatization [39, [43] [44] [45] [46] [47] [48] . acculturative stress refers to the pressures of learning a new language, the reactions to language-related micro-aggressions, balancing differing cultural values, and having to broker between american and the culture of origin ways of daily living [49] . minority and acculturative stress further delay hiv and oud care because these are demonstrated predictors of anxiety, depressive, and substance use disorders severity, suicidal ideation, and low utilization and retention in hiv and substance use treatment and care services [43, [49] [50] [51] [52] [53] [54] [55] [56] [57] . the following analysis will provide the strategies that were used to mitigate the additive effects of intersectional stigmas, minority stress and acculturative stress for a transnational latinx population with dual diagnosis of hiv and oud. from september 2013 to december 2017, 70 individuals consented and enrolled (average age = 43.2 years; 81.4% males; 0% transgender) into the clínica bienestar spns project. the majority were born in the us territory of puerto rico (77.1%). between 10 to 15% of the sample at any given month during the longitudinal study was incarcerated at the county jail. 41.8% reported being homeless or being in an unstable housing situation most of the last month. most participants (79.2%) were single (not cohabitating, not married). most (88.5%) participants were out of hiv care before linkage to clínica bienestar, and the remaining were newly diagnosed with hiv (11.4%). the top five untreated physical health issues identified at baseline clinical assessment were: (1) hepatitis c virus (64.4%), (2) obesity/overweight/underweight (42.2%); (3) hypertension, pulmonary hypertension (30.1%), (4) gastroesophageal reflux disease, gerd (26.7%), and, (5) asthma (15.6%). the top five untreated mental health issues identified at baseline clinical assessment were: (1) tobacco use disorder (82.2%), (2) cocaine, stimulant, polydrug use disorders (64.4%), (3) depressive disorders (60.0%), (4) generalized anxiety disorders (33.3%), and, panic attack disorders (11.1%). close to a quarter of participants had severe neuropathy at baseline (22.2%). each patient received a personalized treatment plan for each co-occurring physical health or mental health concerns/conditions. to present detailed descriptions of the experiences and trajectories in accessing and obstacles to hiv and oud care; and the effects of the strategies used by clínica bienestar, we used cresswell's and yin's approaches to case study methodology, specifically confirming/disconfirming case study methodology (described below) [58] [59] [60] [61] . drawing on the principle of salutogenesis (briefly defined as a medical approach focusing on factors that support human health, resilience, coping, and well-being, rather than on factors that cause disease), our analytical focus was on cases that successfully reduced their viral load to undetectable and were retained in hiv/oud care for 24 months [62] . the qualitative process evaluation design of this spns demonstration project consisted of using case study methodology of life history interviews with the first ten individuals who completed the 24 months of the longitudinal study. to be considered for the interviews potential participants had to be hiv virally suppressed for at least one year, and currently in treatment and/or recovery for their substance use disorders. using the aforementioned criteria to develop a list of potential participants, individuals were contacted in the order of when they had completed the required 24 month period. the first ten patients who completed the 24 months of study participation were interviewed. drawing from the literature on biography research, a life history on hiv and substance use in this study refers to the construction of a chronological narrative of the events related to hiv acquisition, living with hiv, substance use trajectory, and experiences in care in the life of an individual; and the perceptions, reactions, meanings, feelings, thoughts, and life events that surround those experiences [63, 64] . the life history consisted of one initial open-ended, in-depth interview of 45 to 90 min; followed by a second interview, within two weeks, to clarify themes or inconsistencies in the narrative. participants received monetary compensation of usd$25 for their time. the interviews were conducted in spanish, english, or both, depending on the participant's choice. the interviews were audio-recorded and transcribed within two weeks from the day of the interview by a subcontracted independent transcription company. the life history interviews were covered under the consent for the clínica bienestar spns project, however, consent procedures were reviewed with each participant before the interview. interviews were conducted by a highly trained life history interviewer, who had more than 5 years of interviewing experience, and their only role on the project was as an interviewer for this specific research activity. the life history interview guide consisted of five primary elicitation themes: (1) migration trajectory to philadelphia; (2) hiv diagnosis and trajectory to engagement in care at clínica bienestar philadelphia; (3) substance use trajectory into current treatment and recovery; (4) mapping of kinship and community support systems; (5) services received for the past 24 months at clínica bienestar. the guide was divided into primary questions and probing statements. theme #1, the migration trajectory, served as the core element to build the narrative of the individual by asking questions about their journey to and arrival in philadelphia. then, the other thematic questions (#2-5) were asked with additional probes in relationship to the individual's migration history. this process allowed for topics related to their migration journey to be revisited throughout the interview and for a fuller narrative exploring their migration trajectory to be developed. apparent initial inconsistencies in any participant's narrative were revisited during the interview with additional probes or in the follow-up interview if needed. this study was approved by the institutional review boards of philadelphia fight community health centers and temple university. the first author, along with an independent coder, coded the life histories according to the theoretical framework: (1) family composition; (2) migration trajectory; (2) transnational life in philadelphia; (3) hiv treatment and care trajectory; (4) substance use treatment and care trajectory; (5) acculturation processes; (6) acculturative stress experiences; (7) minority stress experiences; and, (8) stigma experiences. the interviews were coded using atlas.ti software. to begin analysis of the interview data, the team utilized cresswell and yin's case study methodology. this methodology is characterized by first identifying cases that illustrate recurrent themes across all the cases [58] [59] [60] [61] . the team sorted through the ten cases and selected two cases that best illustrated all the five theoretical thematic codes. upon completion of all coding and team discussions of the narrative data, it was clear that not one case study represented all the histories within the group. thus, this qualitative analysis focuses on two of the 10 life histories conducted as part of the evaluation of clínica bienestar and its service model for this spns initiative: (1) ms. gloria, pseudonym, selfidentified as a woman, uses the pronouns, she, her, hers; was 49 years old at the time of the interview. (2) mr. tito, pseudonym, self-identified as a man, uses the pronouns, he, him, his; was 47 years old at the time of the interview. ms. gloria and mr. tito were selected because: (a) they represented opposite poles in their level of social assets/resources available in the context of transnationalism and accessing care and treatment services; (b) each case represented gradients of success in managing their substance use disorders, and; (c) they offered differing gender and sexual orientation perspectives from cis-gender heterosexual man (mr. tito) to cis-gender lesbian woman (ms. gloria). these two cases do not represent the total number of trajectories or the full spectrum of experiences into hiv and substance use treatments among study participants but rather provide an example of the variety of experiences identified across the entire sample [59] [60] [61] . the first participant selected as a case study, ms. gloria, grew up traveling back and forth between puerto rico and philadelphia. after beginning to engage in substance use at age 14, gloria agreed with her family to go to philadelphia to seek treatment for what she called her "vicio" (spanish for vice). she was 23 years old. her primary or sending home, however, was puerto rico, until that year. in philadelphia, she stayed with her aunt. at the end of that year, after turning 24 years old, she became homeless. her aunt expelled her from her house, when gloria did not complete her outpatient substance use treatment program. she began to use heroin and cocaine again, her primary drugs of choice. her oldest daughter was born in puerto rico when gloria was 16 years old. by her mid-twenties, she gave custody of her three daughters to her mother, who was raised at first in puerto rico and over the past decade in philadelphia. gloria is now a grandmother of a 7-month-old baby. currently, almost all of her family lives in philadelphia. during the four years before the interview, gloria traveled with less frequency between philadelphia and puerto rico, as she used to do before engagement in clínica bienestar. with regards to her migration pattern, she said: yendo y vieniendo a la isla no me ayudo going back and forth to the island, didn't help me during the winter months, she expressed her desire to return for long-periods to puerto rico, particularly to celebrate christmas and new year's. her level of contact with her kinship network in puerto rico, however, varied throughout the past two years. she expressed feeling isolated from her family in philadelphia and excluded from her family in puerto rico, a dual sense of loss associated with each place she has called home in her transnational experience gloria prefers speaking spanish over english; watches news and shows in spanish; and rarely speaks english except when she has to. this discomfort communicating in english has at times presented a barrier for her when trying to articulate her service needs or access care in philadelphia. gloria has had no support from her family on her substance use treatment trajectory. gloria's family communicates with her via text only, not in person, or by phone. gloria has been using substances since she was an adolescent. she does not provide details of those early experiences. she rather focuses on her trajectory through substance use treatment programs. she tried multiple programs without completing them. the longest period that she has gone without using substances was three years in her early twenties before relocating to the united states mainland. she thought that moving to philadelphia would allow her to "regain control" over her substance use. seeking and buying heroin and cocaine for herself and her girlfriend, who later became her wife, consumed most of gloria's time. at the height of her use, they rarely had sex together except when they were doing it in exchange for money or drugs. she tried suboxone on multiple occasions. it did not work, she thinks, because she was not truly invested. gloria sees the emergence of these co-morbidities as alerts to address her dependence on substances. over the last year, she has not used substances. currently, gloria is on daily methadone, and receiving psycho-social services for her substance use disorder at clínica bienestar. she has not had cravings. gloria explained how she walks to places that sell heroin or cocaine and she feels nothing. people give her "gifts" of substances that she has passed along to other users. "this has shown me how serious i am about my recovery," she expressed. in 2002, when she was in her late 20 s, gloria was diagnosed with hiv while in a correctional facility in puerto rico. back then, she said, there were no treatments available. people with hiv were dying constantly. the only way she could get hiv medications was inside correctional facilities. she had been imprisoned on drug-related charges both in puerto rico and pennsylvania. outside prisons and jails, hiv medications were too expensive. she is proud of achieving hiv viral suppression. gloria's wife was her closest source of social support until a year ago when gloria moved out of her wife's place. she has not dated anyone else since then. isolated from her family because of stigma and with few other sources of social support due to her transnational migration, dating was a form of support for her, for not feeling lonely. yet, her dating often led for gloria to relapse into using substances again. for her, going for more than 12 months without using drugs has been a major accomplishment that to some extent she adjudicated to living separately from her wife, who is still actively using opioids and other substances. this commitment to remaining sober is further evidence of the behavior and attitude change that she self-reported earlier in the transcript. in her experience, most of the discrimination she has suffered is because of multiple combinations or permutations of discriminatory rejections because of her homelessness, hiv status, drug use and/or her sexual orientation as a lesbian. en los half-way, te tratan como una ignorante, hechándoselas, dejándote saber quién manda in the half-way houses, they treated you as if you were ignorant, condescending and often showing off their power housing discrimination has been a challenge for gloria. from 2014 to 2018, she was homeless or living in between half-way-houses, which often only allow people to stay for a maximum of between 60 and 90 days. in the instances when she had accumulated enough money to find herself a place to rent, potential renters expressed to her that they did not trust that she would have a stable source of income to pay rent consistently. it is in those instances that she would feel her depression worsening. her hiv status made her feel even more depressed and would lead her to instances of catastrophizing her health conditions. gloria has also experienced discrimination by health care providers and in social services agencies because of her hiv status and her sexual orientation. cuando uno va al hospital y uno dice, "mira, yo soy hiv, soy una adicta", ahí rápidamente te ponen como un red flag… estás sellada, tú sabes, estás -una estampa. mira, esta es una ____. esta tiene hiv. sí hay maneras de -y que -que también los doctores cuando te suben, que te admiten y todo, ellos se paran a hablar de tu condición cuando pacientes van por aquí y alla when i went to the hospital, and one says: "i am hiv, i am an addict," quickly they put a red flag on you. look, she is a blank. she has hiv. yes, there are ways in which also doctors when they get you up to see them, and admit you, and they stand to talk about your condition in front of other patients coming in and out yet, the most powerful experiences of hiv related discrimination came from her family. no respeto para nadie y es triste. porque hay personas que no son fuertes y se han quitado la vida por eso, por la discriminación. como yo cuando mi familia lo supo, ellos limpiaban el toilet con -que yo me metí a bañar, cuando yo salía, ellos se -mi hermana se ponía guantes, cogía cloro. le decía a mi sobrina que cuando yo entrara al baño, que no entraran detrás de mí, que esperara -fuera a donde ella, le dijera, mira, yo usé el baño, para ya limpiarlo. hasta la ducha y todo. yo tenía una cuchara y un vaso para mí gloria was living under the bridge in kensington, philadelphia, pa, when one of the outreach workers of clínica bienestar approached her. the outreach worker was trained in addressing cultural barriers and using trauma-informed care approaches. the young woman outreach worker spoke to gloria about the syringe service program. gloria expressed how the outreach worker's welcoming and respectful approach made her feel safe. the non-judgmental invitation was distinct from previous stigma filled and negative experiences gloria had when trying to access services in the past, this had a direct impact on her engagement level upon entry into care and her retention in care and treatment services. gloria began to attend the sterile syringes exchange program weekly. two weeks later, gloria progressed into seeking services beyond the syringe service program ranging from lunches, clothes, mcdonald cards, wound care, and enrolling in clínica bienestar philadelphia (see table 1 clínica bienestar strategies). gloria had a full hiv primary care visit within three days of accepting becoming part of clínica bienestar. she was connected to a near-peer patient navigator (briefly defined as a person living with hiv of similar demographics to the patient who had also been through the process of hiv and substance recovery treatments) to show her through the navigation of hiv, substance use and mental health care services offer within the space of clínica bienestar and through referrals. over the next two years, gloria participated in intensive case management given her high social isolation and severity of her substance use disorder. she participated, and became one of the leaders of the women's support group. born in the bronx, new york, mr. tito began his transnational life at three years old, moving to new jersey, then back and forth between pennsylvania and puerto rico. his parents would move to puerto rico and stay there for years. at 15 years of age, tito and his brother were sent from puerto rico to rural pennsylvania to live with their aunt. less than a year later, both were arrested for their first time on drug possession and trespassing charges. during the four years before the interview, tito traveled once a year or once every two years to puerto rico but did not stay for periods longer than a month. he maintains close contact by phone and through social media with some of his family members in puerto rico. he expressed concern for friends or other "usuarios" (his word in spanish for substance users) who recently arrived and "no conocen las calles" (his phrase in spanish for not knowing the streets or being aware of drug culture in new york city). unlike gloria, he feels supported by his family in philadelphia and puerto rico. similar to gloria, tito also prefers speaking and writing in spanish over english; he watches news and shows in spanish; and rarely speaks english. this discomfort communicating in english has at times presented a barrier for him when trying to articulate his service needs or access care in philadelphia. tito tried cocaine at 15 years of age, and at "al principio todo es color de rosa" (spanish idiom for everything is wonderful at first) suggesting that his substance use was not as wonderful as he aged. months after trying cocaine, he started smoking marijuana which he continued doing from age 15 until his late 30 s. at 17 years old, he experimented with heroin and described in detail that first high on heroin. in summary, the high was more intense and superior than cocaine. he began snorting heroin regularly. later that year, he was arrested and sent to a county jail in pennsylvania. contraband heroin was the available drug inside that correctional facility. pero la cosa es que la cantidad que te daban no era suficiente para tú metértela por la nariz y emboyarte, sentir el arrebato. in 2005, he went to jail again for 2 months for possession of paraphernalia. "no soy un criminal o una persona que hace daño," (spanish for i am not a criminal or a person that causes harm). "boberias" (tito's word for stupid, silly things) have gotten him imprisoned including trespassing, accumulated unpaid citations, charges for small amounts of drug and paraphernalia possession. it was in jail in 2005 that he was diagnosed with hiv. he thinks that he didn't acquire hiv from a contaminated needle or sharing cookers but rather through sex. he learned how to inject and use clean needles from his relatives. furthermore, he remembers rarely using condoms in his teens and twenties. in his family, and neighborhood environment he doesn't remember being discriminated against for having been incarcerated, using substances or being puerto rican. however, tito described frequently being discriminated against when trying to get a job because of his criminal record; and several instances of racial discrimination. having hiv added a new layer of discriminatory experiences for tito. his sister and two of his nephews know about his hiv status. two years before arriving at clínica bienestar, he needed a place to stay so a friend offered to take him in. tito disclosed his hiv status to him. his friend made tito use disposable plates, cups, and utensils. since then, he doesn't disclose his hiv status unless the other person "really needs to know." tito has also experienced hiv related discrimination from doctors. in one of the examples, tito entered the physician's table 1 transnational challenges and strategies in hiv and substance use continua of care, clinica bienestar, philadelphia, 2013 philadelphia, -2018 challenges associated with transnational experiences nuances and differences between cases regarding challenge strategies to meet clinic participants needs (1) migrating to philadelphia seeking substance use treatment services with limited or no support from their sending and hosting communities having close family network connections that did not necessarily translated into social support when substance use has been a multigenerational issue (mr. tito's case), it increases likelihood of early onset of oud, and low treatment program completion strong held homophobic family beliefs, family beliefs that substance use is a failure of character, and poor family understanding substance use treatment produced further internalized stigmatization (ms. cariega) case management to identify cases with high social isolation, limited social support networks or challenging familial relationships, and using motivational interviewing techniques to design patient/client-centered strategies to manage the lack of family-based social support linkage to social support groups within the spaces of ppp and fight mitigating effects of the above in addressing challenges from transnational experience: (a) increased social connectivity; (b) increased informational, emotional, and instrumental support for patients (2) stigmatizing experiences including rejections because of their accents, being people who use substances, being people living with hiv, having criminal records, and, not having steady sources of income both cases had limited knowledge of their civil and legal rights, leading to housing issues, recurrent arrests; and recurrent sentences for criminal charges both cases illustrate the perverse incentive of having access to hiv medications through imprisonment, and no access while not incarcerated ms. cariega experienced additional discrimination because of being openly lesbian participants were exposed to educational materials on their rights legal aid services were provided onsite to assist on healthharming legal needs and referral to legal aid representation for criminal defense staff conducted strategic meeting to reinforce case management strategies to assist participants with housing, food, and supportive services mitigating effects of the above in addressing challenges from transnational experience: for healing and recovering from untreated traumas office, who extended his hand to greet and receive him. at the moment of leaving, the doctor would not respond to tito's extended hand to say good-bye. tito's attribution is that this change in salutation tone had to do with hearing about his hiv status, medical, and, substance use history. tito did not return to this provider. over the 2005 to 2014 period, tito attended more than ten substance use treatment programs. tito began treatment for substance use for the 11th time while he was beginning to receive syringe exchange services at ppp. he woke up in his bedroom one morning in october of 2015, with a rope tied around his neck, of which the other end was just lying on the floor next to him. he had no recollection of how he or the rope got there. he called his sister scared. she brought him to the hospital for detoxing and treatment for substance use. shortly after completing his initial stage of treatment, tito's nephew died from a lethal overdose. he recounted this event as devastating. his nephew died with the wave of friends and "fulanos" (spanish for acquaintances) who began to die rapidly in philadelphia from fentanyl and heroin-related overdoses. mira, fulano de tal se murió look! so-and-so died it is in this moment in his life, tito expressed, that he began to understand his depression, his sadness, the emotional and the physical pains he was experiencing as a result of his substance use, and his recurrent suicidal ideations. he was not prescribed antidepressants. his depression has continued until today, yet, he has not been able to stay in psycho-social therapy for his depression. nor is he interested in taking anti-depressants. throughout tito's account of this period, he mentioned how god and the virgin mary helped him from dying or killing himself. from his perspective, his faith and their mercy kept him alive. it is through the syringe exchange program mobile outreach that tito was recruited into clínica bienestar in 2014. at that point, he had not seen a non-emergency medical provider for more than 10 years. he was not taking hiv treatment medications. when one is without taking a shower for two to three days, one stinks. but here no one ever made me feel bad for not having showered. the way they treated me on that first day to today, it has been the same. never, anyone has made me feel bad. they treat with "señor" blank the literal translation from spanish to english of the word "señor" is "sir" or "mister," but neither of those captures the level of respect that the words "señor" or "señora" carry. the non-judgmental invitation was distinct from previous stigma filled and negative experiences tito had encountered when trying to access services in the past. also, like gloria, this respect for the individual and the recognition of their personhood had a direct impact on his engagement level upon entry into care and his sustained retention in care and treatment services. señor tito "bracamonte" (pseudonymn) was using heroin and cocaine during his first year of comprehensive hiv treatment. "estar solo no es facil" (spanish for being alone is not easy), is how tito described one of the major stressors of having hiv. disclosing his hiv status to intimate partners was and is something that he finds more difficult than using condoms. here the stigma that was feared from previous experiences went beyond solely being about his phyical appearance and housing status, there was also the fear of stigma associated with one's hiv status. the bienestar model of service provision, including case management and trauma-informed care and clinical services had a positive impact on tito's level of engagement and retention in hiv care and substance use recovery treatment. he has had sexual partners throughout his life, although not at the time of the interview. tito has achieved hiv viral load suppression and maintained it for the past 18 months. table 1 lists the transnational challenges in-common between the above two case studies of gloria and tito, and also the nuances and differences between cases regarding each challenge. there were four in-common transnational challenges: (1) migrating to philadelphia seeking substance use treatment services with limited or no support from their sending and hosting communities; (2) rejections because of their accents, being people who use substances, being people living with hiv, having criminal records, and, not having steady sources of income; (3) trauma associated with the transnational experience of people with severe substance use disorders; (4) language and cultural barriers, both cases did not know how to navigate highly complex mental health and health care systems. ten strategies were used by clínica bienestar to meet clinic participants' needs (see table 1 ). six of these strategies emerged as most influential and important to consider when providing care and services for hiv and oud to the transnational latinx population that travels between puerto rico and the united states mainland. they are: (1) case management to identify cases with high social isolation, limited social support networks, or challenging familial relationships; (2) linkages to social support groups within the spaces of ppp and fight; (3) staff conducted strategic meetings to reinforce case management strategies to assist participants with housing, food, and supportive services; (4) showing respect and offering trauma-informed care, and timely services in one location; (5) staff was trained on trauma-informed care for issues affecting transnational populations to increase their service delivery capacity; and (6) near-peer patient navigation to show respect and support participants through the navigation of mental and clinical health care systems. along with case management, both gloria and tito identified that attending social support groups and being paired with near-peer patient navigators, who had also been through the process of substance recovery treatment themselves, had a positive influence on their engagement and retention in care and services. these services were also identified as positive contributors to successful engagement in care and services by the majority of participants interviewed in this sample. additional research may yield deeper understanding of the degree to which each of the above-named strategies influence engagement or retention in care and reveal causal relationships or interdependence between individual strategies as drivers for patients' uptake of behavior change. dealing with the stressors produced by intersectional stigmas, addressing traumas connected to transnational migration, and managing experiences of discrimination when accessing clinical services, are all project objectives interwoven throughout the strategies listed in table 1 . the organizational and staff capacity and history of dealing with substance-using populations of ppp and fight facilitated a flexible implementation of strategies tailored towards the needs of each patient. furthermore, three-quarters of the staff members had personal experiences as members of transnational communities themselves (i.e. puerto rican born, first-generation puerto rican, recent migrants, firstgeneration members of latinx or other migrant communities) which contributed to their ability to provide care and services for the participants in this sample of the first ten out of seventy who completed the 24 months spns longitudinal demonstration project. the life histories of ms. gloria and mr. tito illustrate the social profiles of transnational puerto ricans dealing with dual diagnoses of hiv and oud (and other substance use disorders). in their social profiles, we see persistent and ongoing movement before engagement in treatment at clínica bienestar between the territory of puerto rico and the states of new york, new jersey, and pennsylvania. clínica bienestar offered culturally-appropriate services tailored for a transnational puerto rican audience seeking safe spaces to receive services. in these cases, as in the rest of the life histories, they minimized their traveling, on their own volition during the first twelve months of receiving out-of-patient services in clínica bienestar as a way of prioritizing and demonstrating a commitment to their engagement in care and recovery treatment. thus, the case studies above suggest that providing a transnational-cultural space, that may satisfy some needs of cultural connectedness to address social isolation, together with a safe organizational space that directly supports the management of the multiple stigmas and the stressors associated with hiv and oud, might have had positive effects in increasing retention in hiv/oud treatment and care. the examination of the life histories of successful case studies from clínica bienestar reaffirms the point that epidemics driven by structural factors, such as the hiv and oud, require structural responses such as the strategies used in clínica bienestar. the trajectories into hiv services for ms. gloria and mr. tito illustrate the trajectory of most patients/clients of clínica bienestar who had been diagnosed several years ago but rarely or never accessed hiv medical services. for a population, such as the cases presented, that has been lost to care in the medical and health and social services systems in both puerto rico and pennsylvania, the co-location of services is critical. we titled this manuscript, "en la lucha," spanish for in the fight, a phrase that ms. gloria and other patients/ clients in the clinic used to refer to the daily fight to manage their substance use disorder and adhere to hiv medications. as health professionals serving transnational populations of plwh with co-occurring substance use disorders requires, joining the fight for effective use of resources, for the development of cost-effective, sustainable, multi-level interventions, such as clínica bienestar philadelphia, that can bring health and hope to individuals such as ms. gloria, mr. tito, their families, and transnational medically-underserved communities. funding this study was funded by brazilian coordination for the improvement of higher education personnel (capes). laura bamford (grant no. h97ha26504). open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. health disparities in the latino population discrimination and substance use disorders among latinos: the role of gender, nativity, and ethnicity efficacy of maintenance treatment with naltrexone for opioid dependence: a metaanalytical review medication-assisted therapies-tackling the opioid-overdose epidemic can cultural competency reduce racial and ethnic health disparities? a review and conceptual model barriers to health promotion and disease prevention in the latino population barriers to eliminating disparities in clinical practice unequal treatment: the institute of medicine report and its public health implications the mayor's taskforce to combat the opioids epidemic overdoses from opioids in philadelphia profile of general population and housing characteristics: 2010 demographic profile data. am factfinder hiv care outcomes among hispanics or latinos with diagnosed hiv infection-united states understanding the association between socioeconomic status and physical health: do negative emotions play a role? hiv-related mortality among adults (≥ 18 years) of various hispanic or latino subgroups-united states transnational lives of new immigrants the puerto rico-new york airbridge for drug users: description and relationship to hiv risk behaviors determinants of health care use among puerto rican drug users in puerto rico and new york city azucar y nervios: explanatory models and treatment experiences of hispanics with diabetes and depression adoption and implementation of medications in addiction treatment programs organizational implementation of evidence-based substance abuse treatment in racial and ethnic minority communities the state of engagement in hiv care in the united states: from cascade to continuum to control providing hiv comprehensive care to latino/as who inject drugs: philadelphia sexual culture, structure, and change: a transnational framework for studies of latino/a migration and hiv. hiv prev with latinos theory: res pract utility functions that depend on health status: estimates and economic implications dual diagnosis: 15 years of progress dual diagnosis: a review of etiological theories dual diagnosis of major mental illness and substance disorder: an overview critical time intervention: model description and implications for the significance of timing in social work interventions a critical time intervention with mentally iii homeless men: impact on psychiatric symptoms preventing recurrent homelessness among mentally ill men: a" critical time" intervention after discharge from a shelter substance use reduction in the context of outpatient psychiatric treatment: a collaborative, motivational, harm reduction approach risk environments and drug harms: a social science for harm reduction approach culturally appropriate interventions of outreach, access and retention among latino/a populations initiative: intervention monographs racism in the united states: implications for the helping professions hiv, gender, race, sexual orientation, and sex work a qualitative study of intersectional stigma experienced by hiv-positive women in ontario. canada stigma and racial/ethnic hiv disparities: moving toward resilience framing mechanisms linking hiv-related stigma, adherence to treatment, and health outcomes practical strategies to fight stigma in mental health social stigma concerns and hiv medication adherence prejudice and discrimination as social stressors. in: the health of sexual minorities stigma, discrimination and the health of illicit drug users driving while brown: a proposal for ending racial profiling in emerging latino communities locus of control and anti-immigrant sentiment in canada, the united states, and the united kingdom immigration policies and social determinants of health: is immigrants' health at risk? stigma towards plwha: the role of internalized homosexual stigma in latino gay/ bisexual male and transgender communities acculturative stress, social support, and coping: relations to psychological adjustment among mexican american college students. cult divers ethn minor psychol minority stress and physical health among sexual minority individuals social and psychological well-being in lesbians, gay men, and bisexuals: the effects of race, gender, age, and sexual identity alcohol use exacerbates acculturative stress among recently immigrated, young adult latinas acculturative stress and specific coping strategies among immigrant and later generation college students acculturative stress and personal adjustment among hispanic adolescent boys acculturation and drug use disorders among hispanics in the us discrimination, acculturation, acculturative stress, and latino psychological distress: a moderated mediational model. cult divers ethn minor psychol acculturative stress, depression, and suicidal ideation among immigrant and second-generation latino adolescents research design: choosing among five approaches research design: qualitative, quantitative, and mixed methods approaches case study research and applications: design and methods. sage publications enhancing the quality of case studies in health services research the meanings of salutogenesis documents of life 2: an invitation to a modernity and self-identity: self and society in the late modern age key: cord-021990-a8ku5rke authors: tyring, stephen k. title: syndromal tropical dermatology date: 2016-12-02 journal: tropical dermatology doi: 10.1016/b978-0-323-29634-2.00001-8 sha: doc_id: 21990 cord_uid: a8ku5rke nan with increasing numbers of persons from industrialized, temperate countries traveling and / or working in tropical lands, there is a marked need for physicians to be able to diagnose accurately and treat tropical diseases with mucocutaneous manifestations. while some studies demonstrate that approximately one-third to two-thirds of travelers returning from tropical countries experience some health problem, diarrhea is the most prevalent complaint. mucocutaneous problems, however, are among the top five health complaints of the returned traveler, and comprise 10-15% of health concerns of persons returning from the tropics. 1 during international conflicts, soldiers from north america, europe, and australia are often required to serve in tropical lands and sometimes develop diseases not familiar to physicians of their home countries. this was the case for french soldiers serving in vietnam in the 1950s and american soldiers serving there in the 1960s and 1970s. recently hundreds of american and allied troops serving in iraq and afghanistan have developed "baghdad boils" (i.e., leishmaniasis), transmitted by sand fly bites ( fig. 1-1) . likewise, millions of persons from tropical countries now live and work in temperate lands and may present with medical problems with which the physician is not familiar. whereas the cutaneous problems in the returned traveler are frequently the the person traveled. for frequent travelers, the history may become complex if patients report having visited many destinations within the past few months. because vectors differ with the climate, the season of travel is also noteworthy. even in a tropical country where the temperature is always hot or warm, there may be a dry season and a rainy season. because seasons are reversed north and south of the equator, it is important to know the season at the destination. the duration of the stay is significant, not only because a longer stay increases the chance of acquiring an infectious disease, but also because it tells the physician whether the person was in the tropics during the incubation period of the suspected disease. whether the visitor was only in an urban environment or also in a rural area is relevant. whereas a sexually transmitted disease (std) could be acquired in either location, an arbovirus or a zoonosis might be more likely in a rural situation. the altitude of the destination could provide a clue to the etiology of the skin condition, as could the type of sleeping condition. for example, a sexually transmitted disease could easily be acquired in a five-star hotel, but an infection transmitted by a flea, louse, or mite would be more likely in someone who had slept on the ground and / or in a tent. the type and preparation of food and drink consumed by the traveler would not only help explain gastrointestinal symptoms, but could also be a clue to cutaneous signs (i.e., unsafe drinking water or milk or raw or undercooked meat, fish, or shellfish). a list of the patient's current and recent medications can be very useful and should include prescription drugs, illicit drugs, and herbal remedies, because the source of the cutaneous problem may not be directly related to the travel destination, but rather may be due to medications taken to prevent travelrelated illnesses. for example, many antimalarials, such as chloroquine, mefloquine, proguanil, quinine, and halofantrine, can cause cutaneous reactions, and chloroquine, doxycycline, and quinine can cause photosensitivity. interestingly, chloroquine can worsen psoriasis. a number of agents taken to treat or prevent diarrhea can also cause cutaneous reactions, such as quinolones (ciprofloxacin, ofloxacin, sparfloxacin, levofloxacin), furazolidone, metronidazole, trimethoprim-sulfamethoxazole and bismuth sulfate; quinolones are particularly likely to produce photosensitivity. anthelmintic medications, such as on the other hand, contaminated food may have originated in a tropical or subtropical area, such as when oysters from the gulf of mexico are shipped to the midwest usa and are consumed raw. the resulting vibrio vulnificus or hepatitis a infection thus produces gastrointestinal and cutaneous manifestations in individuals who may not have visited the source of the shellfish. therefore, it is always important to ask about new pets, changes in diet, or any other change in persons with a suspected tropical disease. on the other hand, travelers may have purchased non-consumable items that are the source of their dermatoses. for example, animal skins used for rugs or blankets may be the source of anthrax. a non-infectious cause may include nickel-containing jewelry to which the patient has developed contact dermatitis. whereas travelers naturally fear large carnivores while on camera safari, or sharks and a variety of other aquatic animals while swimming or diving, it must be remembered that the animal (indirectly) responsible for most morbidity and mortality is the mosquito (i.e., malaria, dengue, etc.) ( fig. 1-2 ). an example of a mosquito-borne disease that was considered primarily "tropical" in the recent past but is now relatively common in much of north america is infection with the west nile virus (fig. 1-3) . sometimes the skin findings on physical examination are not the reason for the visit to a physician or even the patient's complaint. such skin findings may be cultural, such as tattoos or scarification, or the result of the use of kava or of chewing betel nuts. some cultural practices, however, would be considered abuse in industrialized countries, but are widely accepted religious / cultural practices in certain lands. an example of such practice is female circumcision, which is practiced in many countries in sub-saharan africa. on the other hand, the skin changes may be much more benign, transient, and may even be the result of previous therapies, such as cupping and coining, widely practiced by immigrants from southeast asia. considerations for deciding the differential diagnosis of cutaneous manifestations of tropical diseases and / or of diseases acquired while traveling must be based not only on the type of lesions and systemic symptoms but also on the patient's history of travel. because the incubation period of various infectious diseases differs widely, it is important to know when temperate countries, but in the 21st century they have become rare in industrialized countries, except for imported cases. due to non-compliance with recommended vaccinations, however, 644 measles cases were reported in the us in 2014, the highest number in the 21st century. 3 a single outbreak in early 2015, resulting from an infected person visiting disneyland, california, resulted in 125 cases. 4 worldwide, however, almost one million children die of measles annually ( fig. 1-5 ) and rubella still causes many congenital abnormalities. measles is still the number one vaccine-preventable killer of children in the world. morbidity and mortality are often the result of secondary bacterial infections developing in malnourished infants with measles ( fig. 1-6 ). in east asia, sub-saharan africa, and many other parts of the tropical world, hepatitis b is very common and a major source of morbidity and mortality. although measles, rubella, and hepatitis b should not be a problem in the immunized traveler, many travelers have not received the proper immunizations because they or their parents had unfounded concerns about the safety of the vaccines. this problem continues to grow as more people reach child-bearing age without ivermectin, albendazole, and diethylcarbamazine, can also produce pruritus and rash. even diethyltoluamide (deet), used to prevent arthropod bites, can cause an irritant dermatitis when used in high concentrations. because many medications in tropical countries are sold over the counter and / or have different trade names to those in industrialized lands, patients are not always certain what they have received if treated during their travel. likewise, an injection or transfusion given in a tropical country might also carry an increased risk of contamination. a similar risk might be taken by having acupuncture, tattoos, or body piercing in tropical lands, but these procedures can be hazardous even in industrialized countries because the first intervention is occasionally done by non-medical personnel and the other two are almost never done by medically trained persons. a history of pretravel vaccinations and / or immunoglobulins would be useful for possible exclusion of certain suspected etiologies. for example, if the yellow fever vaccine and the hepatitis a and / or b vaccine series were administered in sufficient time before the travel, it is less likely that these viruses were the source of the medical complaint. the traveler's occupational or recreational exposure to dirt, water, or animals can be an important component of the history. an animal bite or scratch should be easy to remember, but the bite of many arthropods may not even be noticed until after a cutaneous reaction has appeared and the fly, mite, or flea that is responsible has moved on to the next victim. exposure to some animals may be more indirect. for example, the spelunker (cave explorer) may inhale aerosolized bat guano and develop rabies without ever touching a bat. a history of swimming, boating, or surfing can be a clue to an aquatic / marine etiology. such fresh-or brackish-water activities may increase the risk of infection with schistosomiasis or with free-living ameba, whereas marine activities may be associated with jellyfish stings, contact with the venomous spines of certain fish, or irritant dermatitis from fire coral. a preexisting skin abrasion or laceration, or a puncture wound from a sea urchin or sting ray, may result in a secondary bacterial infection. thus, a complete medical and travel history and physical examination are imperative in helping to narrow the differential diagnoses in the returned traveler, the immigrant, or the adoptee with a tropical origin. the qualitative and quantitative nature of the skin lesions is very important and is discussed in detail later in this chapter. specific attention must be given to the age of the patient as well as to the person who is immunocompromised due to human immunodeficiency virus (hiv), internal malignancy, organ transplantation, or another iatrogenic source of immunocompromise. blood tests (e.g., liver / kidney function tests, complete blood counts [cbc] with differentials, urinalysis, skin scraping, biopsy, and / or culture) are often necessary to confirm the diagnosis. a recent example of the importance of knowing both the patient's national origin and their immune status was seen when an hiv-seropositive man from myanmar presented with the first case of penicillium marneffei, recently renamed talaromyces marneffei, 2 reported from houston, tx ( fig. 1-4) . many viral diseases that were not considered "tropical" 50 years ago are now much more frequently seen in immigrants from tropical countries, or in travelers who did not receive their recommended childhood vaccines. three common examples are measles, rubella, and hepatitis b. until the 1960s, measles and rubella were very common sources of infection in • if the traveler is strongly suspected of having an std, did he / she visit a destination where chancroid, granuloma inguinale (gi), or lymphogranuloma venereum (lgv) (l serovars of chlamydia trachomatis) is prevalent? if so, the diagnostic tests and therapy might need to be expanded beyond those under consideration for stds acquired in temperate lands. when one std is confirmed, there is an increased possibility of acquisition of additional stds. not only is this the case because the source partner(s) may have had multiple stds, but also because having certain stds makes a person more susceptible to other stds. the best example of this phenomenon is the two-to fivefold greater risk of acquiring hiv if the person with a genital ulcer disease (gud) has sex with an hiv-positive individual. the reasons for this increased risk include the reduced epithelial barrier in all guds, as well as the infiltrate of cd4+ cells in certain guds such as genital herpes. these cd4+ cells are the targets for hiv infection. genital herpes is the most prevalent gud in industrialized countries. in fact, the centers for disease control and prevention (cdc) estimate that there are 45 million herpes simplex virus type 2 (hsv-2)seropositive persons in the usa. in the tropics, chancroid has been the most frequently diagnosed gud, followed by syphilis and genital herpes, but the last two diseases are becoming more prevalent in certain tropical countries. depending on the travel destination, lgv and gi must also be considered. the dates and duration of travel are important components of the history because the primary clinical presentation of all these guds ranges between 2 and 3 days (genital herpes and chancroid) and 4 weeks (syphilis and gi). currently, the world health organization (who) estimates that there are 46 million hiv-seropositive persons in the world. many of these people have gud, which may be changed both qualitatively and quantitatively by hiv. therefore the traveler may have a "non-classical" presentation of gud. in addition, it should be remembered that the signs and symptoms of gud can also appear on the perianal area / buttock or in or around the mouth. other locations are possible, but less likely. in general, however, multiple painful, usually bilateral, vesicles that progress to ulcers on skin or start as ulcers on mucous membranes, then heal over after 3-4 weeks without therapy or within 2-3 weeks with antiviral therapy, are consistent with genital herpes. because most true primary cases of genital herpes recur, a history of multiple recurrences of the vesicles or ulcers is highly consistent with genital herpes. this diagnosis can be confirmed by viral culture or serology. in the absence of these tools, a useful test is the tzanck smear, which usually demonstrates multinucleated giant cells in herpetic lesions, but is of low sensitivity and specificity. genital herpes, however, can present many diagnostic dilemmas because the first recognized clinical occurrence is often not the result of a recent infection but rather represents a first-episode, non-primary outbreak. whereas a true primary outbreak of genital herpes is usually consistent with acquisition of the virus 2 days to 2 weeks previously, a first-episode, non-primary outbreak may be consistent with an infection at any time in the past. in this case, the patient's recent travel history may be of less importance than his / her sexual encounters of the more-distant past. although syphilis is much more common in many developing countries than in the usa, western europe, or australia, a lack of travel certainly does not exclude syphilis. this diagnosis should be suspected when the patient presents with a single, ever knowing anyone who has suffered from the childhood diseases common in the first half of the 20th century. therefore, they do not understand that the approved vaccines are a millionfold safer than the diseases they are designed to prevent. stds should be considered at the top of the differential diagnoses when a patient presents with genital lesions and / or urogenital discharge. [5] [6] [7] although many of the same considerations would be true whether or not the patient was a recent traveler, certain factors should be given attention in travelers: • was the person traveling without his / her spouse / family and therefore outside his / her usual social structure? • did the person travel to countries where sex workers are readily available? although sex workers are available in most parts of the world, legally or illegally, the traveler might be less likely to acquire an std in mecca during a haj than in amsterdam, bangkok, or nairobi, where sex workers are very prevalent. • did the person attend parties where large amounts of alcohol and / or drugs were consumed (e.g., "spring break" in the usa)? fortunately, human papillomavirus (hpv) vaccines are now widely available, which can prevent up to nine of these sexually transmitted viruses. although the pustules of disseminated gonococcemia are distinctive, the consequences of untreated neisseria gonorrhoeae (gonococcus) are usually pelvic inflammatory disease, epididymitis, proctitis, pharyngitis, or conjunctivitis. pelvic inflammatory disease can also be caused by chlamydia trachomatis (non-l serovars), mycoplasma hominis, or various anaerobic bacteria. the non-l serovars of c. trachomatis can also cause epididymitis, proctitis, and conjunctivitis. pharyngitis can also be due to hsv-2 or entamoeba histolytica. the initial presentation of gc, c. trachomatis (non-l serovars), ureaplasma urealyticum, mycoplasma genitalium, trichomonas vaginalis, or even hsv-2 may be urethritis. vaginal discharge can be caused by any of these organisms as well as by candida albicans, gardnerella vaginalis, peptostreptococci, bacteroides spp. or mobiluncus spp. these organisms can usually be diagnosed by smear, wet-mount, dna detection, serology, or culture. antimicrobial therapy is usually initiated based on the physical examination and smear or wet-mount and modified, as needed, when other laboratory studies are completed. infection with hpv is one of the most common stds in the world, but the clinical implications of the infection vary widely. there are over 20 hpv types that can cause genital lesions, but most infections do not result in any visible lesions. because the incubation period of hpvs can be months, or even years, if and when genital lesions do develop, it is often very difficult for the patient to determine the source partner. therefore, it is usually a challenge for the physician to relate hpv lesions to travel, especially recent travel. non-oncogenic genital hpv, such as types 6 and 11, result in condyloma acuminatum, which can be treated with cytodestructive therapy, surgery, or with the non-tender, genital, perianal, or lip ulcer associated with nontender lymphadenopathy. whereas chancroid is uncommonly reported in industrialized countries, it is very common in the tropics. it is usually characterized by one or more painful genital ulcers and painful lymphadenopathy. in lgv the primary lesion is usually very transient and is often not seen. the clinical presentation is usually that of tender inguinal lymphadenopathy, sometimes with a suppurating bubo. the diagnosis of gi is very rarely made outside the tropics. the presentation is usually that of one or more non-tender genital ulcers with inguinal swelling. if any of these bacterial guds is suspected, the appropriate diagnostic tests must be initiated (i.e., serology for syphilis and lgv, culture for chancroid and lgv, or tissue examination for gi) and the appropriate antibiotic started. whereas a history of multiple recurrences of genital vesicles or ulcers would be consistent with genital herpes, a more difficult scenario is represented by the patient who reports a single outbreak of non-specific genital signs and symptoms that are resolved by the clinic visit. a western blot or type-specific serologic test for hsv-2 would determine whether the person was infected with this virus, but it would not be definitive proof that that hsv-2 was responsible for the resolved outbreak. for example, a hsv-2 serologically positive person may acquire syphilis, but the genital ulcer may resolve without therapy, or with inadequate treatment, before the clinic visit at home. thus, a careful history may reveal the need for serology for hsv-2 as well as for syphilis. because hiv can be acquired concomitantly with or subsequently to these guds, but not produce genital manifestations, hiv testing should be conducted as well. although many patients may be hesitant to admit sexual activity that puts them at risk for stds, others will worry about these activities following travel (or any time) and ask to be tested for "everything." if the sexual encounter with a new partner has been very recent, the serologic test may be false negative because serology for syphilis, hiv, or hsv-2 may require weeks to become positive in the majority of persons after initial infection. patients who ignore their primary genital lesions because of denial or difficulty finding medical care during their travels may believe that the problem is gone because the lesion has resolved. if syphilis is the cause of the gud, it may reappear weeks or months later as non-genital cutaneous manifestations in the form of secondary (or tertiary) syphilis ( fig. 1-7) . a careful history regarding the primary lesion may lead to the appropriate diagnostic tests and therapy. some stds may not produce any genital signs or symptoms and the disease may be diagnosed long after the travel (or the non-travel acquisition), making it more difficult to find the source of the infection. although over 90% of hiv-seropositive persons eventually develop indirect mucocutaneous manifestations of infection, the primary rash of seroconversion (if present) is not noticed by most patients. therefore, the diagnosis is usually made when the patient develops systemic signs and symptoms (e.g., fever, chills, diarrhea, weight loss, lymphadenopathy) and / or develops one or more of the opportunistic infections, neoplasms, or inflammatory skin problems frequently seen in hiv patients. similar to hiv, primary infection with hepatitis b rarely produces genital lesions. diagnosis is usually made long after infection due to systemic symptoms or non-specific skin changes such as jaundice. hepatitis b was the first std for which a prophylactic vaccine was available. therefore, a history of successful hepatitis b vaccination makes this diagnosis less likely. toxoplasmosis, trichinosis, tularemia, typhoid fever, and yellow fever. if the period between travel and fever / rash is up to a month, the list should be expanded to include hepatitis viruses (a, c, and e), hiv, rubella, schistosomiasis, and trypanosomiasis. if at least 3 months separate travel from fever / rash, the following infections should be considered: bartonellosis, filariasis, gnathostomiasis, hepatitis viruses (b and c), histoplasmosis, hiv, leishmaniasis, lyme disease, melioidosis, penicilliosis, syphilis, trypanosomiasis, and tuberculosis. in each case, however, the nature of the fever, the type of rash, the destination of the travel, and any other symptoms must be considered. because many infections producing fever with rash can be rapidly fatal and / or easily spread, it is imperative to initiate immediately diagnostic tests and antimicrobial therapy for the presumed cause of the infection. such infections include anthrax, bartonellosis, candida (macronodules), diphtheria, disseminated gonorrhoeae (papules and pustules over joints), hepatitis viruses, leptospirosis, meningitis (asymmetrical, scattered, petechiae, and purpura), plague, pseudomonas (ecthyma gangrenosum), relapsing fevers, rickettsia (scattered petechiae and purpura), staphylococcus (osler's nodes, diffuse toxic erythema), streptococcus (janeway lesions, diffuse toxic erythema), strongyloides (migratory petechiae and purpura), syphilis, tuberculosis, typhoid fever (rose spots) ( fig. 1-9) , various gram-negative bacteria (i.e., peripheral gangrene), vibrio (especially v. vulnificus), and viral hemorrhagic fevers (petechiae, purpura, hemorrhage). eosinophilia may be due to diverse processes, such as allergic, neoplastic, and infectious diseases. 8 although an allergic reaction could easily result from an exposure during travel, eosinophilia in the returned traveler may have nothing directly to do with the travel. on the other hand, it may be due to an infectious process or to a drug taken for prophylaxis or therapy during travel. if an infection is the cause of the eosinophilia, it is usually a parasitic disease, especially that due to a helminth. only a few viral, bacterial, or fungal diseases are associated with both rash and eosinophilia, for example, streptococcal fever (i.e., scarlet fever), tuberculosis, hiv, and coccidioidomycosis. protozoa only rarely provoke eosinophilia. immune response modifier imiquimod. oncogenic genital hpv, such as types 16 and 18, can result in anogenital cancer, the most prevalent of which is cervical cancer. cervical cancer is the second most prevalent cancer killer of women in the world and over 99% of all cervical cancer is caused by hpv. most cervical cancer deaths are in tropical countries, making hpv one of the world's deadliest tropical diseases (although rarely listed with the other major tropical diseases). there are many reasons why more cervical cancer deaths occur in tropical countries. first, in industrialized countries most women receive regular pap smears, which result in early detection and subsequent therapy of cervical abnormalities, thus reducing progression to cervical cancer. if cancer is detected, surgery, radiation therapy, or chemotherapy is available. in addition hpv vaccines are now widely available in most industrialized countries. in most tropical countries, regular pap smears are not the standard of care. therefore, cervical cancer is often detected too late for successful intervention, even if this is available. second, there appears to be a genetic susceptibility that allows oncogenic hpv to progress to malignancy. this genetic susceptibility appears to be more prevalent in certain tropical countries. the rarity of male circumcision in many tropical countries appears to be a risk factor for the development of cervical cancer in these males' partners. third, most of the world's estimated 46 million hiv-seropositive individuals live in tropical countries where no antiretroviral therapy is available. not only is cervical cancer an acquired immunodeficiency syndrome (aids)-defining illness in hiv-seropositive women, but the same hpv can also cause anal cancer, which is a major problem in homosexual men with hiv. molluscum contagiosum (mc) is a poxvirus that can be sexually transmitted, resulting in wart-like lesions on the genitalia. in contrast to hpv, however, mc does not progress to malignancy. like condyloma acuminatum, however, mc can be treated with cytodestructive therapy, surgery, or imiquimod. ectoparasites such as scabies, sarcoptes scabiei, and pubic lice, phthirus pubis, can be sexually transmitted. in contrast to many stds, however, these ectoparasites can be easily treated with topical medications such as lindane or permethrin. like all stds, if the sexual partner is not treated concomitantly then reinfection is common. the most common cause of fever after tropical travel is malaria, which usually does not have specific cutaneous manifestations. dengue fever is the second most common cause of fever in the traveler and does have somewhat specific cutaneous manifestations, making dengue fever the leading cause of fever with rash in the traveler returning from a tropical destination ( fig. 1-8) . other common causes of fever and rash include hepatitis viruses, rickettsia, and some enteric fevers. it should always be kept in mind, however, that fever in the returned traveler may not be due to exposure during travel. for example, the fatigue of travel (i.e., jet lag) may make one more susceptible to influenza or other common infections in temperate lands. when both fever and rash are seen, the time between travel and onset of signs and symptoms becomes increasingly important. if travel preceded fever and rash by less than 1-2 weeks, considerations should include anthrax, dengue fever, diphtheria, ehrlichiosis, hemorrhagic fever viruses, leptospirosis, lyme disease, measles, meningococcal infections, plague, rickettsia, be considered. therefore, consideration should be given to trichinellosis, strongyloidiasis, schistosomiasis, onchocerciasis, loiasis, hookworms, gnathostomiasis, dracunculiasis, and cutaneous larva migrans. pinworms, as well as protozoan infections such as amebiasis, giardiasis, and trypanosomiasis, are less likely to produce eosinophilia. pruritus and urticaria are possible with spirochetes such as borrelia (e.g., relapsing fevers), spirillum (e.g., rat-bite fever) and treponema (i.e., syphilis and pinta). yersinia (e.g., plague) is another bacteria that produces pruritus and urticaria, which can be present before buboes form. the hepatitis viruses (e.g., a, b, and c) can produce pruritus and urticaria, as can a number of ectoparasites and biting arthropods (e.g., ticks, scabies, bedbugs, lice, fleas, mites, and flies). [9] [10] [11] [12] [13] jaundice although hepatitis viruses can produce pruritus and urticaria, jaundice is a more specific indication that the problem has a hepatitic etiology. not only can all the hepatitis viruses (a-e) produce jaundice, other tropical viruses also do so commonly, e.g., yellow fever and rift valley fever. less frequently, dengue and epstein-barr viruses can cause jaundice, as can bacteria such as leptospira (i.e., leptospirosis), coxiella (i.e., q fever) and treponema (i.e., syphilis). protozoa, such as malaria, and drug reactions can also be responsible. although vesicles and bullae can appear as a result of contact dermatitis or drug eruption, including photodermatitis and photo-exacerbated drug eruptions as well as toxic epidermal necrolysis, many cases represent the early stages of a viral or bacterial infection. the most common viral etiology in the traveler or non-traveler includes the herpesviruses, especially herpes simplex virus 1 and 2, as well as varicella-zoster virus, both primary varicella and herpes zoster. measles and many enteroviruses (e.g., hand, foot, and mouth disease) can present with vesicles, as can certain alphaviruses. a number of poxviruses, such as vaccinia, variola, orf, tanapox, and monkeypox, can produce vesicles. less commonly, vesicles comprise an early stage of certain bacterial diseases such as those caused by vibrio vulnificus, bacillus anthracis, brucella spp., mycobacteria tuberculosis, mycoplasma spp., rickettsia akaru, and staphylococcus (bullous impetigo). other organisms such as fungi that cause tinea pedis, protozoa (e.g., leishmania brasiliensis), and helminths (e.g., necator americanus) can occasionally cause vesicles. a wide variety of infectious and non-infectious etiologies are related to both macules and papules. almost any of the vesicular diseases listed above may initiate first as a macule, then as a papule, before becoming a vesicle. a number of drugs, arthropod bites (e.g., mosquito or flea) and infestations (e.g., scabies and other mites) commonly cause macules and / or papules. a variety of terrestrial, freshwater, and marine contactants can elicit these cutaneous reactions, as can a spectrum of drugs. viral etiologies include hiv, as in the hiv seroconversion syndrome, epstein-barr virus (infectious mononucleosis), human herpesvirus 6 (roseola), parvovirus b-19 (fifth disease), measles, the principal helminth that causes eosinophilia is strongyloides. when strongyloides is disseminated, such as in the hyperinfection syndrome, skin lesions such as urticaria, papules, vesicles, petechiae, and migratory serpiginous lesions become common, especially if the patient is given systemic corticosteroids (because strongyloides was not considered). pruritic, erythematous papules can be seen as a result of schistosomal cercariae, as in swimmer's itch. eosinophils may be seen in the skin biopsy as well as in the blood. pruritic lesions of the skin and subcutaneous tissues are commonly associated with eosinophilia in onchocerciasis. lymphangitis, orchitis, and epididymitis are also commonly observed. in loiasis, fever and eosinophilia are typically seen. migratory lesions, especially angioedema, are usually erythematous and pruritic. likewise, gnathostomiasis produces recurrent edema after ingestion of raw fish. the skin lesions are usually erythematous, pruritic, and / or painful. drug hypersensitivity is a relatively common cause of eosinophilia and may be associated with non-specific skin changes, such as urticaria and / or phototoxic reactions. although most drugs that cause eosinophilia may not be taken for purposes related to traveling, increased sun exposure during travel may make the problem clinically apparent. because antibiotics may be taken for prophylaxis or therapy more frequently during traveling, they should be given careful consideration when eosinophilia is detected. such antibiotics include penicillins, cephalosporins, quinolones, isoniazid, rifampin, and trimethoprim-sulfamethoxazole. non-specific cutaneous manifestations of tropical diseases may include pruritus and urticaria. frequently, more specific signs may accompany pruritus and urticaria, which are useful in narrowing the differential diagnoses. if eosinophilia is found with the pruritus and urticaria, helminthic infections should an eschar can be seen in both temperate and tropical lands due to pseudomonas aeruginosa (i.e., ecthyma gangrenosum), but the most common infectious causes of eschars are rickettsia. eschars due to anthrax can be seen in persons who work with animal skins, but anthrax has received much attention recently owing to its potential use in bioterrorism. the best-recognized etiology of a non-infectious eschar is a bite from a brown recluse spider. petechiae and purpura can result from adverse reactions to a number of drugs. the most important infectious cause of petechiae and / or ecchymoses with fever is meningococcemia, which has a high rate of morbidity and mortality, is widespread throughout the tropical world, and is found sporadically in industrialized countries. other bacterial causes include borrelia, burkholderia, enterococcus, haemophilus, leptospira, pseudomonas, rickettsia, streptobacillis, treponema, vibrio, and yersinia. a number of hemorrhagic fever viruses can cause petechial or purpuric lesions, but the most prevalent viral causes are enteroviruses, cytomegalovirus, dengue, and yellow fever. protozoal diseases (e.g., malaria and toxoplasmosis) and helminths (e.g., trichinellosis) can also induce this clinical presentation. changes in pigmentation can be seen after a variety of medications, many of which are taken for prophylaxis or therapy related to travel. these agents include a spectrum of drugs such as antibiotics, antidiarrheals, anthelmintics, and antimalarials, many of which can also elicit photosensitization. a number of infectious agents can also alter pigmentation. leishmaniasis, pinta, and tinea versicolor may be associated with hypopigmentation or hyperpigmentation. leprosy, onchocerciasis, syphilis, and yaws are more often associated with hypopigmentation. erythrasma, hiv, chikungunya and loiasis are more frequently causes of hyperpigmentation. with the exception of the movements of scabies and the larvae of myiasis, mucocutaneous migratory lesions are usually due to infections with helminths. the best-recognized example is cutaneous larval migrans, but migratory lesions can also be due to dracunculiasis, fascioliasis, gnathostomiasis ( fig. 1-10) , hookworms, loiasis, paragonamiasis, sparganosis, or strongyloidiasis. since the first publication of tropical dermatology in 2006, many tropical diseases previously unknown in temperate countries have been reported to have been transmitted outside the tropics (e.g., ebola, chikungunya, and zika viruses), or have markedly increased their endemic areas (e.g., chagas disease). in addition, certain non-infectious diseases such as podoconiosis have become more widely recognized. rubella, and various hemorrhagic fever viruses. many bacteria can be responsible, such as rickettsia, bacillus anthracis, spirochetes (spirillum, leptospira, borrelia, treponema), coxiella burnetii, yersinia pestis, salmonella typhi, bartonella bacilliformis, and brucella. histoplasmosis and coccidioidomycosis are fungal diseases commonly associated with macules and / or papules. certain protozoa such as toxoplasma gondii and leishmania can also induce these types of lesions. among helminth diseases, hookworm disease, strongyloidiasis, and onchocerciasis can be associated with macules and / or papules. although otherwise similar, papules are usually less than 0.5-1.0 cm in diameter, whereas nodules are larger than 0.5-1.0 cm. except for certain poxviruses that cause orf and milker's nodules, as well as warts and malignancies induced by hpv, viruses rarely form nodules. on the other hand, all subcutaneous and systemic mycoses can induce nodules. bacterial causes of nodules include bartonella (verruga peruana and cat-scratch disease), buckholderia mallei (glanders), calymmatobacterium granulomatis (gi), chlamydia trachomatis (lgv), klebsiella rhinoscleromatis (rhinoscleroma), leptospira autumnalis (leptospirosis), mycobacteria spp. (atypical mycobacteria, cutaneous tuberculosis, leprosy, etc.), nocardia brasiliensis (and other bacterial causes of mycetoma), and treponema pallidum (bejel, yaws). protozoan causes of nodules include amebiasis, leishmaniasis, and trypanosomiasis. almost all helminthic infections that have mucocutaneous manifestations can induce nodules (e.g., coenurosis, cysticercosis, dirofilariasis, dracunculiasis, echinococcosis, filariasis, gnathostomiasis, loiasis, onchocerciasis, paragonimiasis, schistosomiasis, sparganosis, and visceral larval migrans). if the helminthic nodule contains sufficient fluid, it will produce a cyst. cysts can be seen in helminthic infections such as coenurosis, echinococcosis, filariasis, gnathostomiasis, loiasis, and onchocerciasis. there are also arthropod causes of nodules such as myiasis, scabies, tick granulomas, and tungiasis. although ulcers can form as a result of breakdown of previously normal skin, they frequently develop from nodules after inflammation destroys the epidermis and papillary layer of the dermis. herpes simplex virus is a very common cause of ulcers in both tropical and temperate regions of the world. other causes of gud are bacterial (e.g., chancroid, gi, lgv, and primary syphilis). other bacterial diseases that commonly cause ulcers include anthrax, bacterial mycetomas, diphtheria, glanders, melioidosis, mycobacterial diseases (e.g., buruli ulcer, leprosy, tuberculosis), plague, rickettsia, tropical ulcers, tularemia, and yaws. a number of fungi can form nodules that break down into ulcers, or they can induce ulcers from systemic spread (e.g., blastomycosis, chromomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, lobomycosis, mycetomas, paracoccidioidomycosis, penicilliosis, and sporotrichosis). the most common helminthic cause of cutaneous ulcers is dracunculiasis, when the worm erupts from the skin. two protozoan diseases cause ulcers -amebiasis and leishmaniasis. arthropod causes of ulcers include myiasis and tungiasis. many bites (e.g., those of brown recluse spiders and various snakes), stings (e.g., insect, jellyfish, and scorpion), or venomous spines of various fish can also induce ulcers. control, globalization, and emergence of another vector, a. albopictus, in addition to the main vector, a. aegypti. 17 travelassociated cases have been reported in the usa since 2006, with a significant increase in 2014 following the first case in the western hemisphere in december 2013 and the start of domestic transmission in 2014. 18 zika virus is a member of the family flaviviridae related to west nile, dengue, and yellow fever viruses ( fig. 1-12 ). 19 from 1951 through 1981, serologic evidence of human infection was reported from african countries and in parts of asia. 20 zika virus is transmitted to humans primarily through aedes mosquitoes. 21 in 2007, an outbreak of 185 cases was reported in yap island, micronesia. 20 since then, outbreaks have been reported in french polynesia, 22 and most recently in brazil and other countries in tropical south america. 23 zika virus transmission has not yet been documented in the usa, but cases have been reported in returning travelers. these imported cases, and the fact that the vector is the same as for dengue and chikungunya, may result in local spread of the virus in some areas of the usa. 21, [24] [25] [26] american trypanosomiais, also known as chagas disease (cd), is caused by trypanosoma cruzi. the disease is transmitted to humans by triatomine insects (blood-sucking bugs of the reduviidae family). these insects deposit their feces, which are laden with t. cruzi, at the time of biting. however, cd can also be transmitted via mother-to-child transmission, 27 food-borne transmission, and blood transfusion. for this reason, screening of the us blood supply for cd began in early 2007. cd infects ebola is a hemorrhagic fever disease caused by ebola virus, a member of the filoviridae family. ebola was first discovered in 1976 near the ebola river in congo. since then, outbreaks have appeared sporadically in central africa. the largest epidemic in history occurred in 2014, affecting multiple countries in west africa. 14 five different ebola viruses are known, with zaire ebola virus being the most virulent and causative agent in the most recent epidemic. 15 with a fatality rate of up to 90%, the world health organization (who) declared ebola a 'public health emergency of international concern' . 16 in the united states (usa) two imported cases, including one death, and two locally acquired cases in health-care workers were reported. ebola can be transmitted via direct contact with body fluids. dermatologists should be cautious of the high risk of contamination through skin biopsies and dermatologic examination. chikungunya is an acute febrile illness caused by the chikungunya virus, of the togaviridae family, and transmitted by aedes mosquitoes (fig. 1-11) . the spread of chikungunya worldwide has been attributed to a multitude of factors including mutation of the virus, absence of herd immunity, lack of efficient vector furthermore, cd is a leading cause of heart disease among people living in extreme poverty in the western hemisphere, especially in latin america ( fig. 1-13) . podoconiosis, a non-communicable, non-infectious, tropical disease, is a common cause of lower-leg lymphedema in tropical volcanic highland areas above 1000 meters with high annual rainfall. 31 clinical characteristics include below-theknee bilateral lower-limb elephantiasis. it is found in barefoot subsistence farmers in fields with red soils formed from alkaline volcanic rock (see fig. 3-6) . although it was only recently designated a "neglected tropical disease" by who, it is responsible for a significant public health burden in ethiopia and nine other countries in the horn of africa, northern india, and latin america. the finding of an association of certain hla class ii loci with susceptibility to podoconiosis suggests that it is a genetically predisposed, t-cell-mediated inflammatory disease. 32 in conclusion, the differential diagnoses of mucocutaneous lesions in the returned traveler, immigrant, or adoptee should be based on the morphology of the lesions, but the patient's symptoms, general medical, and exposure history must all be considered. [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] the physical examination and laboratory results must be integrated with the patient's vaccination and medication record. the travel destination(s), travel duration, living, work / recreation conditions, food and drink ingestion, and activities while traveling must all be taken into consideration. it must not be forgotten, however, that many mucocutaneous problems in the returned traveler or the immigrant / adoptee are not related to the travel or the country of origin, but can be the same disorders seen daily in patients who have never left their local communities. approximately 12 million people and kills about 60 000 yearly. 28 in some preliminary estimates, mexico ranks number three, and the usa number seven, in terms of the number of infected individuals with cd in the world. [27] [28] in the usa, approximately 300 000 cases are believed to be present, 29 although one alternative estimate reports more than 250 000 cases in texas alone, [29] [30] with up to one million or more cases nationwide. health problems after travel to developing countries disseminated talaromyces marneffei and mycobacterium intracellulare coinfection in an hiv-infected patient for healthcare professionals. clinical features morbid mortal wkly rep (mmwr) an overview of sexually transmitted diseases. part i (bacterial stds) an overview of sexually transmitted diseases. part ii (viral stds) an overview of sexually transmitted diseases. part iii. sexually transmitted diseases in hiv infected patients eosinophilia in travelers dermatoses associated with travel to tropical countries: a prospective study of the diagnosis and management of 269 patients presenting to a tropical disease unit cutaneous manifestations of intestinal helminthic infections parasitic infections of the skin skin problems in returning travelers dermatologic manifestations of parasitic disease ebola (ebola virus disease) cutaneous manifestations of the ebola virus world health organization. ebola virus disease mucocutaneous manifestations of chikungunya fever centers for disease control and prevention. chikungunya virus in the united states zika virus outside africa zika virus outbreak on yap island, federated states of micronesia clinical evaluation & disease zika virus zika virus outbreak centers for disease control and prevention. zika virus. areas with zika zika: the new arbovirus threat for latin america detection of zika virus in urine recent developments in dermatological syndromes in returning travelers an unfolding tragedy of chagas disease in north america trypanosoma cruzi and chagas disease in the united states historical perspectives on the epidemiology of human chagas disease in texas and recommendations for enhanced understanding of clinical chagas disease in the southern united states podoconiosis: endemic nonfilarial elephantiasis hla class ii locus and susceptibility to podoconiosis the travel and tropical medicine manual kaposi's sarcoma and other manifestations of human herpesvirus 8 human papillomaviruses mucocutaneous manifestations of viral diseases. london: informa healthcare tropical dermatology: viral tropical diseases lyme borreliosis dermatologic infectious diseases in international travelers mucocutaneous manifestations of helminth infections: trematodes and cestodes mucocutaneous manifestations of helminth infections: nematodes tropical dermatology: venomous arthropods and human skin: part ii. diplopoda, chilopoda, and arachnida tropical dermatology: venomous arthropods and human skin. part i. insecta tropical dermatology: marine and aquatic dermatology tropical dermatology: tropical diseases caused by protozoa tropical dermatology: bacterial tropical diseases tropical dermatology: fungal tropical diseases key: cord-004395-erqmbi2b authors: bugembe, daniel lule; ekii, andrew obuku; ndembi, nicaise; serwanga, jennifer; kaleebu, pontiano; pala, pietro title: computational mhc-i epitope predictor identifies 95% of experimentally mapped hiv-1 clade a and d epitopes in a ugandan cohort date: 2020-02-22 journal: bmc infect dis doi: 10.1186/s12879-020-4876-4 sha: doc_id: 4395 cord_uid: erqmbi2b background: identifying immunogens that induce hiv-1-specific immune responses is a lengthy process that can benefit from computational methods, which predict t-cell epitopes for various hla types. methods: we tested the performance of the netmhcpan4.0 computational neural network in re-identifying 93 t-cell epitopes that had been previously independently mapped using the whole proteome ifn-γ elispot assays in 6 hla class i typed ugandan individuals infected with hiv-1 subtypes a1 and d. to provide a benchmark we compared the predictions for netmhcpan4.0 to mhcflurry1.2.0 and netctl1.2. results: netmhcpan4.0 performed best correctly predicting 88 of the 93 experimentally mapped epitopes for a set length of 9-mer and matched hla class i alleles. receiver operator characteristic (roc) analysis gave an area under the curve (auc) of 0.928. setting netmhcpan4.0 to predict 11-14mer length did not improve the prediction (37–79 of 93 peptides) with an inverse correlation between the number of predictions and length set. late time point peptides were significantly stronger binders than early peptides (wilcoxon signed rank test: p = 0.0000005). mhcflurry1.2.0 similarly predicted all but 2 of the peptides that netmhcpan4.0 predicted and netctl1.2 predicted only 14 of the 93 experimental peptides. conclusion: netmhcpan4.0 class i epitope predictions covered 95% of the epitope responses identified in six hiv-1 infected individuals, and would have reduced the number of experimental confirmatory tests by > 80%. algorithmic epitope prediction in conjunction with hla allele frequency information can cost-effectively assist immunogen design through minimizing the experimental effort. computational algorithms are increasingly utilised in biological modelling and offer the potential to reduce the time and expense of immunological assays. computational algorithms were initially demonstrated as useful tools for predicting potential epitopes that might elicit quality t-cell responses [1, 2] . computational algorithms that predict potential hla binding t-cell epitopes can facilitate the design of vaccines capable of inducing t-cell immunity against hiv-1. the high variability of hiv-1 and the extensive genetic polymorphism of hla molecules can be managed in silico, allowing immunogen optimisation to increase breadth and magnitude of t cell responses in respect of hla allele frequencies and circulating virus strains in different populations. bioinformatics approaches were previously applied as proof of concept for an hiv-1 peptide-based vaccine for the env and gag genes [3] in cynomolgus macaques for a broad spectrum of hiv-1 clades. computational optimisation of immunogens facilitates the development of the multivalent and mosaic vaccines [4] necessary to control recombinant hiv-1 strains, an increasingly common occurrence in the epidemic in uganda [5] . computational approaches aim to identify optimal epitopes relevant to vaccine development and are not isolated to hiv-1 only, but a wide range of pathogens, including ebola virus [6] , therefore various statistical validation approaches have been applied for evaluation of these methods [7] [8] [9] [10] . for hiv-1 vaccine design purposes an important consideration for the suitability of a computational algorithm is the breadth of discrete number of t-cell epitopes it generates that could reach particular levels of coverage [11] of circulating viruses. the higher the number of epitope variants the more the reduction in their requirements to attain optimum coverage levels for any epidemic. previous data has shown that breadth of tcell response is associated to viral set point in chronic hiv-1 infection [12] [13] [14] [15] [16] [17] . in order to translate the computational epitope prediction into vaccine design, the number of discrete epitopes computationally generated from particular hiv-1 proteins is an important metric for further investigation [11] . a reliable pan-hla-specific algorithm netmhcpan4.0 [18] [19] [20] that has been improved by advances in hla binding data, covers 172 mhc class i molecules from human (hla-a, b, c, e), mouse (h-2), cattle (bola), primates (patr, mamu, gogo) and swine (sla) [20, 21] , and can also predict binding to alleles devoid of experimental data basing on similarity to known binders and non-binders [22, 23] . this is an artificial neural network (ann) algorithm for predictions of 8-14aa and capable of predicting epitopes for other hla alleles using data for similar alleles by positional similarity of residues in their binding motifs. netmhcpan4.0 is considered to be the tool of choice for such predictions considering the benchmarking done against other related tools [24] . nevertheless to have a conclusive outcome of the computational performance we compared netmhcpan4.0 to both an older and recent tool, netctl1.2 [25] [26] [27] and mhcflurry1.2.0 [28] respectively. the binding of ctl epitopes to mhc class i molecules is linear, anchoring at residues 2 and 9; hence the interface between ligand and ctl can be determined computationally [29] . validation of such computational applications can be done by comparing their predictions with suitable experimental data. despite the paucity of data validating the performance of computational methods relative to wet laboratory experiments, a few have documented them to achieve an area under the curve auc of over 90% [18, 19, 30, 31] by isolated experimental data. we have not come across a wet experiment that evaluated computational predictors to achieve a robust auc using a single set of wet laboratory experimental data. the previously reported 90% auc is largely based on positional specific scoring algorithms (pssm) for the collective isolated experiments alongside probability models used to establish affinity or binding scores. one study that explored the reliability of in-silico approaches in epitope prediction and its application for vaccine design reported a meagre 22, 44%, and relatively higher 78% match for three computational tools namely yfpeithi, ctlpred and iedb respectively [32] . using experimental epitope mapping data generated from 757 peptides tested on cells of 6 early hiv-1 infected individuals at paired time points, we show that netmhcpan4.0 can be useful for markedly reducing pooled peptide experiments as demonstrated by the 95% experimental and computational concordance. the data used was from an independent study that did not include this analysis in its objectives. experimental data of peptides previously mapped for hiv-1 epitope recognition of 6 individuals for a separate study (table 1) at 2 time points each was used for comparison with the computationally predicted binders. these were from a ugandan early hiv-1 serodiscordant couple cohort approved by the uganda virus research institute (uvri), research and ethics review board and the uganda national council of science and technology (uncst). all participants provided informed consent. six (6) participants whose experimental epitope recognition profile we evaluated were early hiv-1 infections (table 1) , enrolled under the following criteria: (i) detection of hiv-1 p24 antigen with a simultaneous negative hiv-1 antibody [34] .. the experimentally tested peptides totalled 757 (fig. 1) , were 17aa long, overlapping by 11aa and spanning the hiv-1 proteome consensus for subtypes a1 and d. cultured eli-spot assays using 200,000 cells/well as previously documented by obuku ae. et.al [34] . and ex-vivo ifn-γ elispot assay using 100,000 cells/well were used for testing peptide pools and epitope mapping respectively. experimental positive pools were 3 times the background wells and at least 600 spot forming units per million cells. "deconvolute this" software [35] was used to identify possible responding individual peptides from the pools or where it was not possible all the peptides in a pool were tested as single peptides. high resolution reference strand conformation analysis hla class i tissue typing for the early infected subjects was done using methods described elsewhere [36] . hiv-1 subtyping determination was performed on the gag gene [37, 38] using sanger method generated sequences. the sequences were input into the rega hiv-1 automated subtyping tool to determine the hiv-1 clade [39, 40] . hiv-1 subtypes a1 and d consensus sequences were used as inputs for the computational epitope prediction. these peptide sequences were all for the year 2004 downloaded from the los alamos database (hiv.lanl.gov/content/sequence/newalign/align. html). the web version of netmhcpan4.0 [19] (http://www.cbs.dtu.dk/services/netmhcpan/) was configured to predict 9mer through 14mer epitopes for 22 hla class i alleles ( table 1 ) that were expressed by the 6 hiv-1 infected donors. linux version mhcflurry1.2.0 [28] was used to predict 9mer epitopes and an earlier tool netctl1.2 was also used to predict 9mer epitopes for the 22 hla class i alleles expressed by the 6 study individuals. perl version 5.26.2 was used to extract the binders from all the netmhcpan4.0 predictions and also to compare the computational binders to the 93 mapped experimental 17aa peptides for 9mer through 14mer hits using a sliding window. an experimental peptide was considered a hit if any of the computational 9mer through 14mer sequence was contained in the 17 amino acid experimental peptide sequence as well as any of the hla-a, b or c expressed by the individual matched the netmhcpan4.0 hla class i type(s). if multiple computational epitope predictions were contained in a single 17mer experimental peptide they were counted as a single hit. these were determined by a blast search of the computational binders against the derivative experimental peptides to determine computational predictions from the same test peptide. the accession numbers of the sequences used to determine the hiv-1 subtypes for 5 of the 6 study subjects are; kt825896, kt825897, kt825898, kt825899, kt825900, kt825901, kt825902, kt825903, kt825904, kt825905, kt825906, kt825907, kt825908, kt825909, kt825910, kt825911 and kt82512. statistics computations and plots were generated using spss version 24.0.0.0. the netmhcpan4.0 computational performance was evaluated using a confusion fig. 1 elispot peptide consort; the experimental peptide mapping data was generated by culture elispot of multiple peptide pools tested in duplicate wells per time point, followed by ex-vivo elispot of potential candidate epitopes. to experimentally map a single time point required at least 541 assay wells matrix to classify true positives, true negatives, false positives and false negatives that were used for the receiver operator characteristic (roc) plot. the hit rate (sensitivity) and false hit rate (specificity) of binder predictions as determined by the netmhcpan4.0 threshold of peptides within the top 2% (with a score of 2 or less) were calculated and the strength of the model was determined by calculating the area under the curve, auc of the roc plot [41] [42] [43] . pearson's correlation coefficient was used to evaluate the relationship between the number of epitopes with various hiv-1 genes. to evaluate if there were any differences in the early versus late time point peptides for the binding ranking of the experimentally mapped peptides as predicted by the computational score the wilcoxon signed rank test was used. to evaluate if hiv-1 subtypes a1 and d affected the number of computational predictions generated, fisher exact test was used. to determine whether multiple computationally predicted epitope sequences were derived from the same experimental peptide sequence, a local blast database was set up using geneious version 9.0.5. both hiv-1 clades a1 and d experimental consensus sequences were used separately each as a reference sequence for the blast. the computational peptide sequences were then aligned against the consensuses to evaluate those derived from a single 17 amino acid experimental peptide sequence. where an experimental peptide was predicted by multiple or overlapping computational peptides, the average netmhcpan4.0 score was assigned as the computational score for this peptide. this score was also used during the generation of the roc curve and the confusion matrix. to compare the association between elispot spot forming units and netmhcpan4.0 scores or mhcflurry1.2.0 affinities and also the association between the values for experimentally mapped peptides for all participants and their cognate computational core 9-mer and a single 14-mer epitope sequence with scores. peptides shown in italic text were not algorithmically predicted as binders. multiple computational predictions contained in a single experimental peptide were counted as a single hit. participant's identifiers (id) beginning with e or l represent early or late time sampling points respectively the 2 computational tools, pearson's correlation coefficient was used. number of experimental assays compared to computationally guided prediction assay projections to experimentally determine epitopes for 757 peptides spanning the whole hiv-1 proteome for clades a and d as well as both time points of the 6 individuals required a total of 4230 test assay wells. for each test subject these included 9 antigen proliferation wells, 384 culture elispot wells and an average of 164 epitope mapping elispot wells (range; 148-186 test wells). using the 22 hla alleles represented in the study subjects we were able to computationally predict 95% of the experimentally mapped epitopes. this approach could have reduced the test assays by eliminating all the t-cell antigen proliferation and culture elispot steps totalling to 3258 assay wells (77%) and leaving only 972 (23%) epitope mapping assays required. applying a pooling strategy to the computational predictions similar to that used in the experimental pooling where each pool contained approximately 20 peptides with a coverage of 3 per peptide pool, the 923 potential peptides (95% of experimental peptides for epitope mapping elispot derived from the 972 (23%) eligible epitope mapping peptides) would make at most 46 pools. consequently the computational prediction approach could have reduced the experimental assays by at least 80%. the core 9mer epitope sequence was similar across 9mer through 14mer set length except for one 14-mer peptide (hit 72 in table 2 ) magnitude of epitope predictions are variable across hla alleles, hiv-1 proteins and clades the input hiv-1 subtypes a1 and d consensus whole proteome sequences evaluated for potential 9, 10, 11, 12, 13 and 14-mer binders to the 22 hla alleles represented in the six patients, varied in the distribution of predicted binders across hiv-1 genes and hla alleles. all the peptide hits predicted for 10 through 14-mer were also all predicted in the 9-mer set except for two 14-mer peptides. an expected positive correlation for hiv-1 protein length with number of epitopes predicted was observed as illustrated by spearman's rank order correlation; r s = 0.88 (fig. 2, a and b) . netmhcpan4.0 predicted 95% (88/93) ( table 2 ) of the experimentally mapped peptides as binders and missed 5% (5 out of 93) ( table 3) for the 12time points of the 6 participants. mhcflurry predicted 91% (85/93) of the experimental peptides and had a lot of similarity to netmhcpan4.0 for the predicted hla. netctl was the least performing tool with only 15% (14/ 93) predicted experimental peptides ( table 2) . comparison of the various epitope prediction length set showed that the 9mer setting was ideal for netmhc-pan4.0. the number of predictions were 88, 79, 55, 39, 39 and 37 hits out of 93 for 9, 10, 11, 12, 13 and 14-mer epitopes respectively. increasing the prediction length from 9mer through 14mer resulted in a smaller number of predicted binders as illustrated in fig. 3 . since we held the assumption that our wet experimental data was the gold standard we evaluated the sensitivity and specificity of netmhcpan4.0.the computational predictor had more predicted binders than those determined by the experimental mapping as presented in the confusion matrix in table 4 . the experimental positive's count also shown in table 2 under column "hit no" shows the test peptide count (1through 88) that contained the computational 9mer sequence. multiple computational epitopes may be contained in a single experimental peptide, as shown in the column "netmhcpan4.0 9-mer epitope prediction" in table 2 . overall hiv-1 clade a 9-mer predictions were fewer in number than clade d (fig. 2, c) though the difference did not approach statistical significance. the experimental peptide mapping data was derived from a baseline time point corresponding to hiv-1 fiebig stages iv, v and vi (table 1 ) and a later time point. ninety-three (n = 93) epitopes were experimentally mapped of which 12 were recognized at both baseline and later time points, 34 only at baseline and 54 only at the later time point. comparison of the ranked computational score for netmhcpan4.0 binders of early (n = 34) versus later peptides showed that the later time point predictions were stronger binders reaching statistical significance (wilcoxon signed rank p-value = 0.0000005) (fig. 4) . netmhcpan4.0 ranked binders as those predicted to false positive (37) computational negative false negative (5) true negative (627) the total number of peptides experimentally tested were 757 and these are broken down to show the fractions from both the experimental testing and netmhcpan4.0 computational predictions fig. 4 early versus late peptides. experimentally mapped peptides at baseline (n = 34) and at least 12 months later (n = 34) were compared using the 9-mer computational netmhcpan4.0 scores of the hits. the lower the computational score the stronger the predicted binding. late peptides were significantly stronger binders than early peptides (wilcoxon signed rank test, p = 0.0000005) be in the top 2% and assigned a score of 0.2 or below. any binder within the top 0.5% and assigned a score of 0.05 or below was ranked as a strong binder. considering only the 9-mer computational predictions, peptides that were derived from the same 17mer experimental peptide were determined by a blast mapping to their derivative sequences. the 17-mer peptides were then classified into a confusion matrix (table 4 ) as true positives, false positives, true negatives or false negatives. from the classification the true positive rate (sensitivity) was plotted against the false positive rate (1-specificity) using an roc curve and the auc attained reached 0.928 (fig. 5) . only 9-mer length epitopes were considered in the roc analysis as increasing the length to 10-mer through 14mer netmhcpan4.0 predictions neither raised the number of predicted binders nor improved the hit rate as all their predictions contained the sequence already predicted in the 9-mer set except 1, 14-mer peptide (hit 72 in table 2 ). comparison of the elispot magnitude of response (spot forming units) did not show any association to either netmhcpan4.0 scores or mhcflurry1.2.0 affinity values. similarly a comparison of the latter 2 computational predictors did not show any association between their assigned "affinity" values. netmhcpan4.0 registered the highest concordance to the wet experiments followed by mhcflurry1.2.0. in this analysis we showed that the computational method netmhcpan4.0 predicted 95% of previously experimentally mapped hiv-1 epitopes in 6 hiv-1 infected individuals expressing a total of 22 different hla class i alleles. in our ifn-γ elispot assays we evaluated 757 17mer peptides overlapping by 11 amino acids and covering the whole hiv-1 subtype a1 and d consensus proteomes. out of the 5 experimentally determined epitopes missed by the algorithm (table 3 ), 4 were actually computationally predicted as binders but were not included for lack of concordance with the participant's hla alleles. about one third (37) of 125 total positive predictions were not experimentally supported in our tests. these do not necessarily represent false positives, as elispot detection depends on the frequency of specific t cells in the participant's repertoire, and we observed changes in dominant t cell specificities within a given participant between early and later time points after hiv-1 infection. a formal roc evaluation of the score generated by netmhcpan4.0 as a classifier for peptides recognised/not recognised by pbmc in ifng elispot assays, produced an auc of 0.928. thus experimental confirmatory tests cannot be dropped altogether, however the netmhcpan4.0 algorithm could provide a considerable saving of time and resources in verifying just the predicted epitopes. as the participants had been enrolled in the acute/ early phase of hiv-1 infection and we had observed intra-participant changes in epitope recognition between early and late time points after infection, we compared the binding scores of confirmed epitopes at these time points and found a statistically significant change towards recognition of higher binding peptides as the infection entered the chronic phase. this might represent better support of the t-cell response directed at more stable hla/peptide complexes as the infection progresses into chronicity. the netmhcpan4.0 algorithm, which is based on binding affinity and integrates data on eluted naturally processed ligands, reflected optimal hla class i binding for 9-mers, producing a decreasing number of predictions when the peptide size was increased from 9 to 11 amino acids. with a single exception, predicted binders between 11 and 14 amino acids included at least one 9mer predicted to bind on its own, suggesting a destabilizing effect of the extra amino acids beyond the canonical hla class i binding pockets at positions 2 and 9 could account for fewer predictions. important limitations are the lack of predictions of hla class ii restricted epitopes, which might have contributed to a fraction of ifn-γ elispot responses. approximately 5% of the computational predictions may be false positives that only increase the size of planned wet experiments and approximately 1% of true positives may also be missed. in this analysis, using netmhcpan4.0, mhcflurry and netctl to predict previously experimentally mapped epitopes, we demonstrate that the computational methods reliably predict an acceptable portion of binder epitopes. we recommend the use of such computational methods to reduce the size of experiments required cost associated. vaccine design for h5n1 based on b-and t-cell epitope predictions methods for prediction of peptide binding to mhc molecules: a comparative study induction of broad cross-subtype-specific hiv-1 immune responses by a novel multivalent hiv-1 peptide vaccine in cynomolgus macaques first-in-human randomized controlled trial of mosaic hiv-1 immunogens delivered via a modified vaccinia ankara vector analysis of the history and spread of hiv-1 in uganda using phylodynamics mapping hla-a2, −a3 and -b7 supertype-restricted t-cell epitopes in the ebolavirus proteome enhancing in silico protein-based vaccine discovery for eukaryotic pathogens using predicted peptide-mhc binding and peptide conservation scores discovering a vaccine against neosporosis using computers: is it feasible? vacceed: a high-throughput in silico vaccine candidate discovery pipeline for eukaryotic pathogens based on reverse vaccinology a combined prediction strategy increases identification of peptides bound with high affinity and stability to porcine mhc class i molecules sla-1*04: 01 defining epitope coverage requirements for t cell-based hiv vaccines: theoretical considerations and practical applications control of human immunodeficiency virus replication by cytotoxic t lymphocytes targeting subdominant epitopes consistent cytotoxic-t-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities cd8 t-cell recognition of multiple epitopes within specific gag regions is associated with maintenance of a low steady-state viremia in human immunodeficiency virus type 1-seropositive patients induction of multifunctional human immunodeficiency virus type 1 (hiv-1)-specific t cells capable of proliferation in healthy subjects by using a prime-boost regimen of dnaand modified vaccinia virus ankara-vectored vaccines expressing hiv-1 gag coupled to cd8+ t-cell epitopes dynamics of viral evolution and ctl responses in hiv-1 infection broad and gag-biased hiv-1 epitope repertoires are associated with lower viral loads netmhcpan, a method for mhc class i binding prediction beyond humans netmhcpan-4.0: improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data pan-specific mhc class i predictors: a benchmark of hla class i pan-specific prediction methods a community resource benchmarking predictions of peptide binding to mhc-i molecules efficient peptide-mhc-i binding prediction for alleles with few known binders multipred: a computational system for prediction of promiscuous hla binding peptides automated benchmarking of peptide-mhc class i binding predictions an integrative approach to ctl epitope prediction: a combined algorithm integrating mhc class i binding, tap transport efficiency, and proteasomal cleavage predictions largescale validation of methods for cytotoxic t-lymphocyte epitope prediction reliable prediction of t-cell epitopes using neural networks with novel sequence representations mhcflurry: open-source class i mhc binding affinity prediction t-cell antigenic sites tend to be amphipathic structures emerging vaccine informatics prediction of peptide-mhc binding using profiles comparison of experimental fine-mapping to in silico prediction results of hiv-1 epitopes reveals ongoing need for mapping experiments dynamics of hiv viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary hiv infection macrophage inflammatory protein-1 beta and interferon gamma responses in ugandans with hiv-1 acute/early infections optimized determination of t cell epitope responses high resolution hla class i typing by reference strand mediated conformation analysis (rsca) profile of t cell recognition of hiv type 1 consensus group m gag and nef peptides in a clade a1-and d-infected ugandan population frequencies of gag-restricted t-cell escape "footprints" differ across hiv-1 clades a1 and d chronically infected ugandans irrespective of host hla b alleles a standardized framework for accurate, high-throughput genotyping of recombinant and nonrecombinant viral sequences an automated genotyping system for analysis of hiv-1 and other microbial sequences the meaning and use of the area under a receiver operating characteristic (roc) curve receiver operating characteristic (roc) curve: practical review for radiologists smallsample precision of roc-related estimates publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the study participants who provided specimen for the wet laboratory experiments, the aids information centre clinic kampala, uganda that steered the rubicon discordant couple cohort study, the late anthony kebba who initiated the rubicon cohort and christine watera who coordinated the recruitment clinic activities. we thank ruhena sargeant for hla typing, the late harr f. njai for hiv-1 elisa and western blot assays and deogratius ssemwanga for help with genbank submissions. this research is jointly funded by the uk medical research council (mrc) and the uk department for international development (dfid) under the mrc/dfid concordat agreement, the wellcome trust (grant wt078927ma), and edctp (project code: ta_05_40200_40203). most of the relevant data to support the manuscript has been included in the write-up. if any addition data is required will be availed once requested. the rubicon study, from which we derived the experimental data, was reviewed and approved by the uganda virus research institute, research and ethics committee (uvri-rec) and the uganda national council of science and technology (unct). all participants provided informed signed consent accepting to freely participate in this study. not applicable. the authors declare that they have no competing interests. authors' contributions dbl performed the peptide mapping experiments. nn provided hiv-1 subtyping. dbl analysed the data together with pp. dbl wrote the manuscript with major contributions from pp, aeo, pk and js. all authors reviewed the manuscript and or provided useful contributions as well as approved the final manuscript. key: cord-023729-dipjubn7 authors: serlin, michael h.; dieterich, douglas title: gastrointestinal disorders in hiv date: 2009-05-15 journal: global hiv/aids medicine doi: 10.1016/b978-1-4160-2882-6.50027-7 sha: doc_id: 23729 cord_uid: dipjubn7 nan gastrointestinal disease in human immunodeficiency virus (hiv) spans the entire gi tract from the mouth to the rectum. the spectrum of gastrointestinal symptoms in hiv ranges from odynophagia and dysphagia, to nausea and vomiting, to abdominal pain and fi nally diarrhea and tenesmus. as with normal hosts, gastrointestinal disorders are very common in hiv patients, whether it be from opportunistic infections secondary to the patient's immunosuppressed status, medication induced, or through other etiologies. almost all hiv and aids patients have some gastrointestinal complaints throughout the course of their illness. with the dramatic changes in hiv care because of highly active antiretroviral therapy (art) in the mid-1990s, the incidence of opportunistic infections are decreasing, and as a result, the clinical picture of gastrointestinal illnesses in hiv is changing. the evaluation of the hiv patient with gastrointestinal complaints requires a thorough history and physical exam, in addition to selected studies, in order to diagnose the correct disease and treat accordingly. patients with hiv and aids typically can have upper gastrointestinal symptoms, which can range from dysphagia, or diffi culty swallowing, to odynophagia, or the feeling of pain upon swallowing. at least one-third of patients with hiv before the art era had esophageal complaints, 1 and the incidence increased with the progression of the disease. most of the symptoms in these patients are secondary to opportunistic infections caused by the patient's immunosuppressed state, and related to the degree of immunosuppression. the most common etiologies of esophageal pathology and esophagitis in aids patients (table 23 .1) are candida species, herpes simplex virus (hsv), and cytomegalovirus (cmv). 2 in addition, there is also an entity of idiopathic esophageal ulcers (ieu) that is also seen in hiv patients, which may be immunologically mediated, 3 or caused by hiv itself. 4 other etiologies of esophageal complaints include malignancy (especially lymphoma and kaposi's sarcoma) and other noninfectious causes. however, with the introduction of protease inhibitors (pis) in 1996 and art, and the decreased incidence of aids, more esophageal complaints in hiv these days are related to common etiologies like gastroesophageal refl ux disease (gerd) than opportunistic infections. 5 in addition to the most common symptoms of dysphagia and odynophagia, other symptoms can also suggest esophageal disease in hiv patients, like chest pain, nausea, vomiting, anorexia and weight loss. the symptoms can be acute or have a more chronic, progressive course. in addition, dysphagia is often associated with candidal esophagitis, whereas odynophagia is generally symptomatic of esophageal ulcerative disease. patients can have both dysphagia and odynophagia, and because they also may have more than one illness concurrently, it is imperative to pursue a thorough investigation as to the etiology of esophageal complaints in hiv patients. the evaluation of hiv patients with esophageal symptoms does not defi nitively need to include endoscopy with biopsy, but this is the gold standard, as it allows the physician to visualize the esophageal lumen, and to biopsy affected sites ( fig. 23.1) . the history and physical exam is obviously important, as it may lead to a discovery of gerd, or pill-induced esophagitis. in addition, patients with disseminated cmv (e.g. cmv retinitis) with esophageal symptoms (especially odynophagia) may respond to cmv antiviral therapy in the absence of diagnostic endoscopy and biopsy. the most common sign on physical exam that relates to esophageal complaints is oral thrush, which can be suggestive of esophageal candidiasis in patients with esophageal complaints. in these patients, especially those with only dysphagia (or dysphagia and odynophagia, but not those with solely odynophagia) it may be benefi cial to document the response from an empiric trial of oral fl uconazole, as opposed to endoscopy. 6 if there is a symptomatic response to the fl uconazole, then it can be presumed the patient had candidal esophagitis and proceed accordingly. in addition to history and physical exam, there are other ways to evaluate esophageal complaints. barium esophagography is relatively insensitive and non-specifi c and should not be used for diagnostic purposes, but the most characteristic fi nding in candidal esophagitis is diffuse mucosal irregularity resulting in a 'shaggy' appearance mimicking diffuse ulceration. 7 cmv and ieu may appear as well circumscribed ulcers that may be shallow or deep, and are indistinguishable on barium swallow. 8 of course, radiography can determine a neoplastic origin to dysphagia in patients with malignancies. another form of evaluation is brush cytology, where a cytology brush is passed through a nasogastric tube and obtains tissue for viral cultures and immunohistochemistry. unfortunately, this leads to large sampling error, because it is done blindly without visualization of the lumen, and misses diagnoses (as patients may have two infectious processes, and ieu cannot be diagnosed). viral culture and cytologic brushings add little in the evaluation of aids patients with esophagitis over endoscopy with biopsy. 9 endoscopy with biopsy generally yields a viral or fungal diagnosis based on culture and hematoxylin and eosin staining (which will exclude a viral etiology); only after several biopsy samples do not show any etiology of the ulcerations can a tentative, exclusionary diagnosis of ieu be made. candidal esophagitis is the most common cause of esophagitis in hiv patients, especially in patients with complaints of dysphagia, or odynophagia and dysphagia. 10 the fungal isolate is generally candida albicans, though other species of candida can also affect the esophagus. it should be suspected in patients with a cd4+ lymphocyte count of <100 cells/mm 3 (though can occur at any cd4+ count), and esophageal symptoms with or without thrush, which can be absent in 30% of patients. 11 in patients with hiv and new onset symptoms, an empiric trial of standard dosed fl uconazole is an effective strategy, as 82% of patients in a prospective study responded. if there is no response, endoscopy can be pursued. 6 in patients that fail empiric antifungal therapy, the most common etiology (in 77% of the patients) is ulcerative esophagitis as opposed to persistent candidiasis. 12 fluconazole is the drug of choice for candidiasis, with a loading dose of 200 mg orally followed by 100 mg daily from 10-14 days. clotrimazole troches are also successful, as topical treatment of esophageal candidiasis, 13 but nystatin is not. 14 itraconazole and ketoconazole are effi cacious systemic therapies, but not as effective as fl uconazole. 15, 16 amphotericin b is also a helpful therapy, but is generally used only in azole-resistant patients because of the toxicity of the medication. low-dose amphotericin (0.3-0.5 mg/kg per day for 7-14 days) is usually adequate. caspofungin can also be used in candidal esophagitis, and is felt to be as effi cacious as fl uconazole and well tolerated. 17 however, it is only available in intravenous form and is more expensive, but may be the drug of choice for azoleresistant mucosal candidiasis because of the relative lack of toxicity compared with amphotericin b. primary prophylaxis of candidal disease is not recommended because of the non-life-threatening nature of the disease, the effectiveness of acute therapy, and the risk of antifungal resistance. while candida is the most common esophageal pathogen, it can also occur in addition to ulcerative esophagitis in hiv patients. cmv esophagitis is the most common etiology of odynophagia in hiv patients and therefore esophageal ulcerations, up to 45% of patients in one prospective study. 18 fever and substernal chest pain can be reported in addition to odynophagia, and thrush can be concomitant, but dysphagia is very uncommon. the diagnosis of cmv is best made by endoscopy and biopsy, with the pathology showing viral cytopathic effect in the gastrointestinal mucosa via intranuclear inclusions. immunohistochemistry is also helpful for confi rmation, as viral cultures are less sensitive and specifi c. 19 cmv is the most common viral pathogen in the esophagus in hiv patients, and esophagitis is the most common extraocular manifestation of cmv. 20 it can appear as a diffuse esophagitis, single or multiple ulcers, or giant ulcers involving the whole esophagus, and generally occurs when the cd4+ lymphocyte count is <50. it may be discovered only after treatment for candida, as they may exist concurrently in 20% of patients. 21 the incidence of cmv esophagitis has declined dramatically in the art era. treatment for cmv esophagitis involves a wide array of antiviral medications, namely ganciclovir, valganciclovir, foscarnet, and cidofovir. ganciclovir was the fi rst agent used to combat cmv and has the most data behind it; the response rate is about 70-80%. it is given intravenously in a 2-4 week induction period, 5-10 mg/kg per day, but has dose limiting side-effects, mainly bone marrow suppression (and resultant neutropenia and thrombocytopenia). oral ganciclovir can also be used as maintenance therapy, but the data is limited. intravenous foscarnet (90 mg/kg b.i.d., 2-3 weeks) is seen as equally effective to intravenous ganciclovir in the treatment of cmv esophagitis, but comes with a nephrotoxic side-effect profi le; however, randomized studies have shown equal effi cacy and safety. 22 foscarnet is generally used in cases of clinical failure after ganciclovir induction. 23 oral valganciclovir (900 mg/ day) has not been tested in cmv esophagitis, but does have 60% of the bioavailability of intravenous ganciclovir. cidofovir also can treat cmv, but is not typically used because of nephrotoxicity. hsv esophagitis is a relatively uncommon cause of esophageal ulceration in hiv patients compared with cmv and ieu. the endoscopic appearance is described as being well circumscribed and having a 'volcano' like appearance, distinguishing them from the ulcers seen in cmv infection, which tend to be linear or longitudinal and are deeper. 24 treatment is with oral acyclovir (15-30 mg/kg per day), though if patients have diffi culty with swallowing, intravenous acyclovir can be used. valacyclovir and famci-clovir can also be used because of their effi cacy and convenient dosing schedules. foscarnet intravenously (40 mg/kg b.i.d.) is used in cases of acyclovir resistance. 25 secondary prophylaxis with valacyclovir (500-1000 mg/day) is recommended in patients with frequent relapses. idiopathic esophageal ulcers (ieu) are a diagnosis of exclusion if none of the pathologic, fungal, or viral studies return a diagnosis. the treatment of idiopathic esophageal ulcers is done with oral corticosteroids, generally starting at 40 mg of oral prednisone daily. 26 if the patient cannot take medications orally, then the corticosteroids can be given intravenously. in addition, thalidomide can also be used for ieu if corticosteroids are not effi cacious. 27 in addition to the infections and ulcerations discussed above, there are other esophageal issues in hiv, namely motility and neuropathic issues. there is a form of hiv neuropathy that could lead to gastrointestinal complaints, like gastroparesis. these would be treated symptomatically, with prokinetic agents like metoclopramide. additionally, in patients with both symptomatic esophageal complaints, as well as those that are asymptomatic, there are fi ndings of esophageal motility abnormalities. this is probably because of the neurotropic nature of hiv leading to autonomic dysfunction in the gastrointestinal neurologic plexus. 2 the problems associated with the stomach in hiv patients are similar to the stomach problems of non-hiv infected individuals. opportunistic infections that affect hiv patients typically do not affect the stomach. symptoms and presentation are often related to abdominal pain, nausea or vomiting. the most common manifestations of gastric illness in hiv patients is like the general population, namely gerd, peptic ulcer disease (pud) and gastritis. care needs to be used when prescribing histamine-2 (h 2 ) blockers or proton-pump inhibitors (ppis), however, as these medications can interact with art, especially ppis. work-up is the same as with the general population, and endoscopy with biopsy is the gold standard for diagnosis. helicobacter pylori, a common cause of peptic ulcer disease in non-hiv positive patients, seems to have a decreased incidence in hiv patients compared with the general population; cmv may actually be the leading cause of pud in hiv patients. 28 as far as actual hiv-related gastric pathology, gastric lymphoma, kaposi's sarcoma, and some of the opportunistic organisms (e.g. cmv, tuberculosis, toxoplasmosis, and cryptococcosis) can be seen. in addition, dyspepsia, nausea and vomiting can all be related to the side-effects of various antiretroviral medications. one of the most common complaints of hiv patients is diarrhea. the reasons for diarrhea are multifold, but most commonly relate to opportunistic infection and antiretroviral medications. a more in depth coverage of diarrheal illness in hiv patients is discussed later in ch. 65: etiology and management of diarrhea in hiv-infected patients and impact on antiretroviral therapy, but an overview will follow here. as with esophageal disease, the incidence of opportunistic infections has decreased in the art era, though the incidence of chronic diarrhea has remained steady even in the art era. 29 the evaluation of diarrhea includes a thorough history and physical, as much can be determined from just eliciting the patient's history of symptoms. if the diarrhea occurs with upper abdominal cramps, or bloating, this suggests an upper intestinal source, or enteritis. bloody diarrhea, tenesmus, and lower abdominal cramping imply colonic involvement. in addition, patients with a history of receptive anal intercourse have a higher involvement of colitis and sexually transmitted pathogens (e.g. gonorrhea, hsv, etc.) in the anorectal area. homosexual or bisexual men have a 3-fold higher incidence of diarrhea than patients in other risk groups. obviously, travel and diet history can also be important in the histories of hiv patients with diarrhea. as with other aspects of hiv, opportunistic infections tend to be more common in the setting of lower cd4+ lymphocyte counts (and therefore greater immunosuppression). the diagnostic studies involved in the work-up of diarrhea in the hiv patient are similar to those in the general population. in addition to history and physical exam (exam specifi cally adds little to diagnosis, other than evidence of malnutrition), the fi rst route of investigation is often the stool examination. the tests to order include bacterial culture, clostridium diffi cile toxin assay, as well as looking for ova and parasites, especially isospora, cryptosporidium, and microsporidia. these generally need to be specifi ed as potential pathogens when the sample is sent to microbiology when looking at ova and parasites. in addition, a gram stain or methylene blue stain for fecal leukocytes can also be helpful, as evidence of fecal leukocytes may point towards a picture of colitis. another form of non-invasive testing is blood cultures and serologies, which can be helpful for the diagnosis of systemic opportunistic infections like mycobacterium avium intracellulare (mai) or viral etiologies like cmv. especially in the patients who have diarrhea and fever, mai may be a possibility. however, with all of these etiologies, a diagnosis may be treated, and symptoms may still persist as secondary infections may also be present. for colitis, a barium enema or abdominal radiography may detail the presence of a toxic megacolon, a complication of clostridium diffi cile colitis. in addition to the non-invasive studies listed above, in the absence of a diagnosis, the workup for diarrhea should include some invasive studies as well. the fi rst step is generally a fl exible sigmoidoscopy, which can give visualization of the colon (to diagnose or rule out pseudomembranous colitis, among other etiologies) and provide tissue for biopsy. abnormal tissue should be biopsied, but if the mucosa is normal then random tissue can be sent. the fl exible sigmoidoscopy is better than the alternatives as it does not require sedation. if small intestinal etiology is suspected, an egd (going past the second portion of the duodenum) can help determine the cause of the diarrhea through biopsies of the small bowel, sent not only for pathology, but also electron microscopy and culture analysis. if the fl exible sigmoidoscopy is nondiagnostic, and there is still no diagnosis from other studies, a colonoscopy can be performed so more biopsies can be done to rule out opportunistic infections, especially in the ileum. this is rarely done, however, and in general, the absence of defi nitive diagnosis leads to treatment and evaluation (as will be discussed later). the symptoms associated with enteritis are typically associated with diarrhea and prolonged malabsorption leading to malnutrition. this is generally because of opportunistic infections, and, similar to other pathologies in hiv patients, the incidence of enteritis has decreased in the highly active antiretroviral therapy (art) era. the main symptoms of enteritis are copious voluminous diarrhea (>2 l/day) with dehydration and malabsorption, as opposed to colitis, which is a bloody, painful diarrhea. the work-up of enteritis is detailed above, but specifi cally should include stool studies, and, if non-diagnostic, esophagogastroduodenoscopy (egd) with biopsy. the etiologies of enteritis in hiv patients are multifold, and include bacterial, viral, infections of the small intestine (enteritis) fungal, and parasitic pathogens. these can be diagnosed by the stool studies detailed earlier. parasites may be the most common etiology for enteritis, especially in those patients not on art and greatly immunosuppressed. parasites are typically diagnosed through stool analysis for ova and parasites, including direct fl uorescent antibody (dfa), enzyme-linked immunosorbent assay (elisa) or polymerase chain reaction (pcr). light microscopy can be used, though pcr can be diagnostic at much lower levels of parasitic infection. cryptosporidium parvum is the most commonly identifi able pathogen in aids related persistent diarrhea, especially in patients with cd4+ lymphocyte counts <200. it is typically treated with paromomycin 1500-3000 mg every 6-8 h orally, or azithromycin 900-1200 mg four times a day, though albendazole 400 mg twice a day has also shown to be effective. nitazoxanide (1 g twice daily for at least 2 weeks) can also be used in the treatment of cryptosporidiosis, but a cure is generally not possible if the cd4+ lymphocyte count is <50. however, a study with children showed no benefi t to nitazoxanide at all in hiv+ children (just hiv seronegative ones). 30 hyperimmune bovine colostrums can also be used, but typically not to cure the parasitic infection. 31 microsporidiosis is the next most commonly identifi able refractory diarrhea in hiv patients. this can be treated with albendazole as well (400-1600 mg every 12 h orally) but most cases are poorly responsive to treatment and require indefi nite therapy. isospora belli is rare in the usa, but is more common in developing countries. trimethoprim-sulfamethoxazole, one double strength tablet every 6 h for 10 days is the treatment of choice, but pyrimethamine 50-75 mg four times daily (with folinic acid 5 mg orally four times daily) is acceptable for patients with allergies to sulfa medications. 32 lastly, giardia lamblia and amoebic dysentery can also occur in hiv patients, at the same incidence as the general population, and can be treated with metronidazole 750 mg thrice daily for 5-10 days, or tinidazole 2 g orally once for giardiasis, 3 days for amebic dysentery. 30 for strongyloidosis, thiabendazole 25 mg/kg twice daily orally is the drug of choice. albendazole seems to be active against all of the parasitic organisms associated with diarrhea in hiv patients, and could be the fi rst line of therapy when parasitic infection is suspected, pending microbiological study. with all of the aforementioned parasites, treatment cannot only be targeted at the pathogen, but also at the diarrheal symptoms, with the use of somatostatin analogs like octreotide to try to reduce the amount of diarrhea. in addition, since the para-sites are both more common and more chronic at lower cd4+ lymphocyte counts, art, which reduces the degree of immunosuppression, can also be curative. opioids such as tincture of opium or codeine can provide symptomatic relief in cases of severe diarrhea through its constipating actions, as well as pain relief. 33 bulking agents, lactose-free diets, and antidiarrheal medications like diphenoxylate with atropine or loperamide are also benefi cial in treating diarrheal symptoms. viral infection in hiv patients can cause diarrhea, typically through colitis, but also rarely through an enteritis. cmv, in addition to causing esophagitis, can also affect the gi tract through diarrheal illness, and runs the spectrum from asymptomatic carriage to severe diarrheal illness including appendicitis, bleeding and perforation. it typically occurs in the setting of severe immunosuppression with a cd4+ lymphocyte count <100. the diagnosis of cmv enterocolitis is best made through demonstrating a viral cytopathic effect in tissue specimens, but viral stool cultures can also signify disease (though are less sensitive). the treatment for cmv enterocolitis is mainly ganciclovir and foscarnet, as described earlier in the esophagitis section. valganciclovir may not achieve adequate bioavailability because of the enterocolitis. other viruses can affect the hiv patient gastrointestinal tract, including rotavirus, adenovirus, norwalk virus, or unusually, picornaviruses, and coronaviruses. these tend to be less common than the other pathologies previously described, and are diffi cult to diagnose as well. adenovirus can cause a hemorrhagic colitis; acute diarrhea is seen in patients with only adenovirus in stools, but patients with adenovirus on biopsy specimen generally have a chronic diarrhea. 34 hsv can cause diarrhea via systemic infection in end-stage hiv patients, or can cause a colitis and proctitis through hiv mucosal lesions. hsv can be treated with acyclovir, valacyclovir, famciclovir, or, if acyclovir resistant, foscarnet (as previously described in the esophagitis section). in addition, hiv itself may be a cause of hiv enteropathy and a diarrheal pathogen, and may be identifi ed in gut tissue in up to 40% of patients, but this is controversial. idiopathic aids enteropathy, on the other hand, is the term used for a chronic diarrhea in an aids patient that is without identifi able pathogen or diagnosis (despite intensive investigation). mucosal hyperproliferation is noted on biopsy. for these etiologies, in addition to art to increase cd4+ lymphocyte count and reduce immunosuppression, should be treated symptomatically with bulking agents, antidiarrheals, and opioids. the combination of antidiarrheal therapy with art has been shown to be more benefi cial than antidiarrheal therapy alone in hiv patients with chronic diarrhea. 35 other agents that can cause enteritis include mycobacterium avium intracellulare (mai) and pneumocystis jiroveci (pcp). small intestinal disease is the most common site of gastrointestinal luminal involvement by mai. 36 it is often seen with diffuse small bowel infi ltration (mimicking whipple's disease) and causes severe malabsorption in patients with cd4+ lymphocyte counts of <50. if a malabsorptive diarrhea occurs with fever and night sweats, in addition to weight loss, mai must be considered, and blood cultures or a bone marrow biopsy may be diagnostic for disseminated mai infection. diagnosing mai enteritis is more diffi cult, however, as a positive stool is not diagnostic for gastrointestinal disease (though can suggest subsequent disseminated disease). 37 an endoscopic biopsy and acidfast staining can show acid-fast bacilli and give an ideal diagnosis. treatment is with a multitude of options, using combinations of clarithromycin 500 mg twice daily, ethambutol 800-1200 mg orally daily, azithromycin 600 mg daily, rifampin 600 mg daily, rifabutin 300 mg daily, amikacin 15 mg/kg three times weekly, and ciprofl oxacin 750 mg twice daily. these can reduce, but not eradicate, mai. luminal tuberculosis can also occur as an example of extrapulmonary involvement, but is rare; in contrast to mai, it can generally be treated to cure with antituberculous therapy. pneumocystis jiroveci (pcp) can also be seen as the cause of diarrhea in hiv patients, but is very uncommon, especially in the setting of pcp prophylaxis for aids patients; treatment is with antipneumocystis therapy, 38 generally with trimethoprim-sulfamethoxazole. bacterial infections in hiv patients typically cause a picture of colitis instead of enteritis, with bloody diarrhea and tenesmus. the most common bacterial pathogens seen in hiv patients include salmonellae, shigella, e. coli, campylobacter jejuni, and clostridium diffi cile. salmonellosis is 100 times more common in hiv patients than in immunocompetent hosts, 39 and recurrent salmonella bacteremia establishes the diagnosis of aids in an hiv patient. the diagnosis is straightforward, as salmonella can normally be cultured in stool specimens in addition to blood culture results. salmonella gastroenteritis can present with either watery diarrhea or dysentery (mucopurulent diarrhea) with or without fever, abdominal pain, or nausea and vomiting. though the diarrhea may be self-limited, the treatment is generally ciprofl oxacin 500 mg orally twice a day, for 2-4 weeks, for eradication. shigella has a similar presentation to salmonellosis, with a similar wide spectrum of presentation of illness. it does come with a high rate of severe complications, including anemia, hypoglycemia, sepsis, hemolytic uremic syndrome, disseminated intravascular coagulopathy and renal failure, with which mortality obviously increases. it also can be treated with ciprofl oxacin 500-750 mg twice a day orally for 5-7 days, and is diagnosed by stool culture as well. campylobacter jejuni generally presents as a watery diarrhea, and its incidence is probably decreased due to widespread pcp prophylaxis with trimethoprimsulfamethoxazole. it is typically harder to culture from stool, and may be diagnosed by endoscopic biopsy. antimicrobial therapy is not essential, though erythromycin 250-500 mg orally four times daily, or ciprofl oxacin 500 mg orally twice a day for 5-7 days may reduce the duration of the illness. e. coli may be seen (in any of several strains), and, like the other enteric bacterial diarrheal illnesses, can be treated with a fl uoroquinolone like ciprofl oxacin. as illustrated, in cases of suspected bacterial diarrhea, ciprofl oxacin would cover most enteric pathogens and would be the empiric drug of choice. clostridium diffi cile is seen in hiv patients not only in the presence of antibiotic therapy, but also in the absence of recent antibiotic therapy. the most common antibiotics that cause c. diffi cile are clindamycin, ampicillin, cephalosporins and aminoglycosides. the clinical presentations and response to therapy are not different in hiv patients than in patients without hiv. diagnosis is made by detecting c. diffi cile toxin in stool assay, and treatment is with metronidazole 250-500 mg orally every 6-8 h for 10-14 days, or tinidazole, in addition to stopping the offending initial antibiotic therapy. in resistant cases, oral vancomycin 250 mg every 6 h can be used, as can rifaximin 200 mg three times daily, though it is not as effective as vancomycin. in cases of suspected c. diffi cile without diagnosis via stool toxin assay, a fl exible sigmoidoscopy can look for pseudomembranous colitis, which can be diagnostic for c. diffi cile. fungal etiologies of diarrhea in hiv patients are relatively rare, but can occur in patients with immunocompromised states and low cd4+ lymphocyte counts. gastrointestinal histoplasmosis appears to be the most commonly described fungal etiology of diarrhea in hiv patients, and typically occurs in the colitis setting of a systemic infection. diagnosis is made by fungal culture and smear of tissue or blood, 40 and treatment is with amphotericin b 0.5-1 mg/kg per day intravenously initially, with maintenance therapy with itraconazole 200 mg orally daily. coccidiomycosis and cryptococcosis are also rare, and occur in the presence of systemic infections as well. in addition, as candidal infections are the most common opportunistic infections of hiv patients, a dehydrating diarrhea can also occur as a manifestation of the infection. obviously, not all causes of diarrhea in hiv patients are secondary to opportunistic infections. several noninfectious etiologies of diarrhea in hiv patients can occur as well, including the most common, drug-induced diarrhea. the most common drugs that cause diarrhea in hiv patients are nucleoside reverse transcriptase inhibitors (nrtis) and protease inhibitors (pis). with protease inhibitors, diarrhea is the most common side-effect reported with nelfi navir and saquinavir, most commonly occurs at the initiation of treatment, and is a cause of cessation of therapy and lack of adherence to treatment. 41 newer agents like lopinavir-ritonavir seem to cause less diarrhea than older pis like nelfi navir. 42 treatment is generally guided towards treatment of symptoms, namely with bulking agents or antidiarrheals like loperamide. other causes of diarrhea in hiv patients include infl ammatory bowel disease, including crohn's disease and ulcerative colitis, neither of which have increased incidence in hiv patients. treatment is tailored according to the disease process itself, though an active disease process may decrease cd4+ lymphocyte count; this may be reversed by colectomy. 43 in addition, aids related illnesses that are noninfectious but can also cause diarrhea and gastrointestinal issues include lymphoma and kaposi's sarcoma, both of which are diagnosed by biopsy. infi ltration of the mucosal tract by the neoplasm can lead to diarrhea and weight loss. anorectal disease is a big component of hiv gastrointestinal care, especially among homosexual males. interestingly, the incidence of anorectal pathology in hiv patients has not been affected by art. 44 many anorectal pathologies are seen in hiv patients, including anal fi stulas and fi ssures, perirectal abscesses, ulcerations and proctitis (box 23.1). in addition, anal neoplasms as a result of human papillomavirus (hpv) and other etiologies can also occur. anorectal carcinoma has an increased incidence in both hiv patients and among homosexual males, and is the fourth most common malignancy seen in hiv; 45 hiv+ homosexual men have twice the incidence than hiv negative homosexual men. anal squamous cell carcinoma is frequently associated with squamous intraepithelial neoplasia and hpv, much like cervical carcinoma, and can be detected by anorectal cytology, similar to papanicolaou smears. 46 the gold standard for diagnosis of anorectal neoplastic disease is still anoscopy with biopsy, though anorectal cytology can be useful as a screening test. in addition to anal carcinoma, other anorectal symptoms are common in the hiv population, especially among homosexual males. anal condyloma is the most common hiv related anal pathology, and is associated with hpv infection; treatment options include a variety of surgical options, including cryotherapy. the four most common infectious causes of proctitis in men who have sex with men are gonorrhea, herpes simplex, chlamydia and syphilis. 47 gonorrhea and chlamydia are typically treated together with ceftriaxone 125 mg i.m. once and azithromycin 1 g orally once; fl uoroquinolones, oral cephalosporins and doxycycline (100 mg orally twice a day for 7 days) can also be used. primary syphilis is treated with benzathine penicillin g 2.4 million units i.m. once (or doxycycline 100 mg twice daily for 2 weeks if penicillin allergic). hsv infections, as described earlier with esophagitis and colitis, can also cause perianal and rectal ulcerations, with associated symptoms of tenesmus, pain, and bleeding. treatment is with antiherpetic medications like acyclovir, valacyclovir, or famciclovir, as explained earlier, with foscarnet in acyclovir-resistant cases. other etiologies of proctitis include lymphogranuloma venereum, as well as other causes of colitis detailed above; clinical overlap can also happen in hiv patients. in addition to a thorough physical examination, all patients with anorectal symptoms should have anoscopy and sigmoidoscopy with mucosal biopsy to look for fi ssures, perirectal abscesses, and fi stulas in addition to searching for opportunistic infections, with microbiological studies sent for viral, fungal and bacterial cultures. oesophageal symptoms, their causes, treatment and prognosis in patients with aids esophageal motility disorders in hiv patients odynophagia from aphthous ulcers of the pharynx and esophagus in aids chronic idiopathic esophageal ulceration in aids: characterization and treatment with corticosteroids declining prevalence of opportunistic gastrointestinal disease in the era of combination antiretroviral therapy fluconazole compared with endoscopy for human immunodefi ciency virus-infected patients with esophageal symptoms aids therapy prospective endoscopic characterization of cytomegalovirus esophagitis in patients with aids prospective comparison of brush cytology, viral culture, and histology for the diagnosis of ulcerative esophagitis in aids natural history of hivassociated esophageal disease in the era of protease inhibitor therapy prospective evaluation of oropharyngeal fi ndings in human immunodefi ciency virusinfected patients with esophageal ulcer etiology of esophageal disease in human immunodefi ciency virusinfected patients who fail antifungal therapy comparison of oral fl uconazole and clotrimazole troches as treatment of oral candidiasis in patients infected with human immunodefi ciency virus oropharyngeal candidiasis in patients with aids: randomized comparison of fl uconazole versus nystatin oral suspensions fluconazole versus itraconazole for candida esophagitis in aids fluconazole compared with ketoconazole for the treatment of candida esophagitis in aids: a randomized trial a randomized double-blind study of caspofungin versus fl uconazole for the treatment of esophageal candidiasis esophageal ulceration in human immunodefi ciency virus infection: causes, response to therapy, and long-term outcome frequency of positive tests for cytomegalovirus in aids patients: endoscopic lesions compared with normal mucosa cytomegalovirus infection in patients with hiv infection cytomegalovirus and candida esophagitis in patients with aids treatment of cytomegalovirus esophagitis in patients with acquired immune defi ciency syndrome: a randomized controlled study of foscarnet versus ganciclovir foscarnet treatment of cytomegalovirus gastrointestinal infections in acquired immunodefi ciency syndrome patients who have failed ganciclovir induction herpes esophagitis: clinical syndrome, endoscopic appearance, and diagnosis in 23 patients successful foscarnet therapy for acyclovir-resistant mucocutaneous infection with herpes simplex virus in a recipient of allogeneic bmt a pilot study of oral corticosteroid therapy for idiopathic esophageal ulcerations associated with human immunodefi ciency virus infection thalidomide for the treatment of esophageal aphthous ulcers in patients with human immunodefi ciency virus infection. national institute of allergy and infectious disease aids clinical trials group low prevalence of helicobacter pylori but high prevalence of cytomegalovirusassociated peptic ulcer disease in aids patients: comparative study of symptomatic subjects evaluated by endoscopy and cd4 counts the changing etiology of chronic diarrhea in hiv-infected patients with cd4 cell counts less than 200 cells/mm 3 effect of nitazoxanide on morbidity and mortality in zambian children with cryptosporidiosis: a randomised controlled trial management of protozoal diarrhoea in hiv disease management of diarrhea in hiv-infected patients palliative care and aids: 2 -gastrointestinal symptoms enteric viral infections as a cause of diarrhoea in the acquired immunodefi ciency syndrome impact of protease inhibitors on the outcome of human immunodefi ciency virus-infected patients with chronic diarrhea atypical mycobacterial infection of the gastrointestinal tract in aids patients mycobacterium avium complex in the respiratory or gastrointestinal tract and the risk of m. avium complex bacteremia in patients with human immunodefi ciency virus infection pneumocystis colitis in a patient with the acquired immunodefi ciency syndrome risk factors for primary bacteremia and endovascular infection in patients without acquired immunodefi ciency syndrome who have nontyphoid salmonellosis disseminated histoplasmosis in the acquired immune defi ciency syndrome: clinical fi ndings, diagnosis, and treatment, and review of the literature management of protease inhibitorassociated diarrhea differences in rates of diarrhea in patients with human immunodefi ciency virus receiving lopinavir-ritonavir or nelfi navir infl ammatory bowel disease in individuals seropositive for the human immunodefi ciency virus anorectal pathology in hiv/aids-infected patients has not been impacted by highly active antiretroviral therapy cancer incidence in a population with a high prevalence of infection with human immunodefi ciency virus type 1 anorectal cytology as a screening tool for anal squamous lesions: cytologic, anoscopic, and histologic correlation etiology of clinical proctitis among men who have sex with other men key: cord-009096-3c5t70an authors: frankish, helen title: new who chief promises greater commitment to hiv/aids date: 2003-07-26 journal: lancet doi: 10.1016/s0140-6736(03)14007-x sha: doc_id: 9096 cord_uid: 3c5t70an nan w ith a pledge to give greater priority to hiv/aids and achieving results in poor countries, south korea's jong-wook lee took office as the new director-general of who on july 21. "we must scale up an integrated global hiv/aids strategy linking prevention, care, and treatment, prioritising poor and underserved areas", said lee in his inaugural address to about 500 who staff at the organisation's geneva headquarters. "i am, therefore, constituting an hiv/aids leadership team to ensure that who, working with local, national, and international partners, will be at the forefront of this effort." to promote synergy in combating infectious diseases, the who department in charge of tackling hiv/aids will be brought into one group together with tuberculosis and malaria. taken together, the three diseases are responsible for about 25% of all deaths worldwide each year. lee named american jack chow, former special representative for hiv/aids to us secretary of state colin powell, as the new head of the hiv/aids, tuberculosis, and malaria cluster. in the long term, lee said, who's goal would be to provide antiretroviral drugs to 3 million people in developing countries by the end of 2005-the so-called "three-by-five" target. "by dec 1 this year, world aids day, who's hiv/aids department, working with partners, will produce a global plan for reaching the three-by-five target", lee said. "together with partners such as unaids, who will use all available tools of advocacy to mobilise the political will and the additional resources needed to put this plan into action." the three-by-five target will guide much of who's work on hiv/aids, he said, but patterns of resistance to antiretroviral drugs would also be closely monitored through a global network in collaboration with who's partners. lee, a communicable diseases expert and former head of who's stop tb programme, also vowed to improve notification and monitoring systems to help tackle contagious diseases such as severe acute respiratory syndrome (sars). "the sars crisis illustrated who's essential role in coordinating the international response to infectious disease outbreaks", he said. but he conceded that the sars epidemic also revealed weaknesses in global disease surveillance. "we will work with our partners in the global outbreak alert and response network, and with bilateral and multilateral donors, to reinforce national and regional surveillance systems." on his first day in office, the new director-general also reinforced who's commitment to achieving the millennium development goals, targets that world leaders agreed on at the millennium summit 3 years ago. "we see the millennium goals as milestones on the road to health for all", he asserted. in his address, lee admitted that in recent years, who's resources have become increasingly concentrated in geneva, and that there has been "a gradual drift" towards programmes driven by headquarters' priorities, rather than towards programmes based on countries' individual needs. to ensure that who's resources are aimed at producing results where they are needed most, lee pledged to increase funds at country level over the coming months and years, adding that the organisation would ensure that country offices have the resources and authority they need to work more effectively in responding to national and local health needs. lee asked the 11 newly appointed assistant directors-general (see panel, p 298) to analyse the work of their respective areas and to propose specific steps for moving resources from headquarters to the various regions. "i will begin by deploying additional resources to priority country offices for building up capacity in hiv/aids control and health systems", he said. rights were not granted to include this image in electronic media. please refer to the printed journal. le gales-camus, a former scientific adviser to the director-general of health in france, as head of non-communicable diseases. yach, meanwhile, has been appointed to design a plan to strengthen who's response to non-communicable diseases. david nabarro, from the uk, will be replaced by germany's kerstin leitner, undp's resident representative in china, as head of the sustainable development and healthy environments cluster. nabarro will now lead who's health action in crises programme. and botswana's minister of health, joy phumaphi, will take over from tomris türmen, from turkey, as assistant director-general for family and community health, while türmen will now lead a team charged with developing policy recommendations regarding the health implications of intellectual property rights structures. concluding his address, lee called for the continued commitment of who's staff, the member states, and its national and international partners in order to meet who's goals in the years ahead. "who's founding vision, its achievements, its partners and, above all, its people create a solid foundation. guided by our principles of loyalty, transparency, and commitment to excellence, we will move forward towards the goal of health for all. together, learning from the past, we can change the future of global health." lee also pledged to tackle the shortage of skilled health-care staff worldwide, which will slow progress towards goals such as the three-by-five target and has hindered the millennium development goals. "our cooperation with other countries on this issue must intensify. together, we must build the health workforce using innovative methods of training, development, and supervision of allied and community health workers. community mobilisation is a key to success", he said. lee also outlined ways in which who could directly contribute to strengthening human resources within the health sector. in early 2004, he announced, who would launch the health leadership programme, an initiative to recruit promising young health workers from around the world, and provide them with the opportunity to work within who-at country level, in regional offices, and at who's headquarters in geneva. "mentored by senior who staff, these young professionals will form part of the next generation of international health leaders", he said. notably, lee also marked his takeover from the outgoing director-general, gro harlem brundtland, by replacing many of her most senior staff with a new team of assistant directors-general (see panel). david heymann, executive director of communicable diseases, who led the who's efforts earlier this year to control the sars outbreak, will now take charge of the drive to eradicate polio worldwide. ghanaian anarfi asamoa-baah, a who official who has been working for the agency since 1998, will take over from heymann as assistant director-general of communicable diseases. and south african derek yach, who spearheaded who's framework convention on tobacco control -adopted in may by the world health assembly after a 4-year negotiation period-will be replaced by catherine gro harlem brundtland hands over to jong-wook lee ap lee's newly appointed team of assistant directors-general uk), formerly chef de cabinet, will become director of the director-general's office anarfi asamoa-baah (ghana), currently executive director for health technology and pharmaceuticals, will lead the communicable diseases cluster director of the eastern mediterranean liaison office, will head the external relations and governing bodies cluster special representative of the us secretary of state for hiv/aids, will take charge of the hiv/aids, tuberculosis, and malaria cluster director of health equity at the rockefeller foundation in new york, will lead the evidence and information for policy cluster catherine le gales-camus (france), most recently scientific adviser to france's director-general of health will take leadership of the non-communicable diseases and mental health cluster un resident coordinator and undp resident representative in china, will be responsible for the sustainable development and healthy environments cluster russia), most recently the head of the department of general and clinical pharmacology at the russian university of people's friendship, will take responsibility for the health technology and pharmaceuticals cluster head of the health division at the swedish international development cooperation agency, will take control of the general management cluster joy phumaphi (botswana), minister of health in botswana and hiv/aids commissioner appointed by the un secretary-general key: cord-016255-kkko1xne authors: van der meer, j.t.m.; nouwen, j.l. title: 14 intravasale infecties en sepsis date: 2011 journal: microbiologie en infectieziekten doi: 10.1007/978-90-313-7944-6_14 sha: doc_id: 16255 cord_uid: kkko1xne infecties in het hart en de bloedbaan worden intravasale of endovasculaire infecties genoemd. de circulatie van bloed door het hart is essentieel voor de aanvoer van zuurstof en voedingstoffen naar weefsel en organen en voor de afvoer van afvalstoffen. in dit hoofdstuk worden infecties besproken die zich vooral afspelen in lymfoïde weefsels en meerdere orgaansystemen kunnen aantasten. cellen van het immuunsysteem spelen een belangrijke rol in de pathogenese van deze vaak chronische infecties. dit uit zich onder andere in gegeneraliseerde lymfadenopathie (lymfekliervergroting), een gemeenschappelijk klinisch kenmerk van deze ziekten. in dit hoofdstuk worden de pathofysiologische processen die aan deze lymfekliervergroting ten grondslag liggen kort samengevat en worden de belangrijkste infectieuze ziektebeelden met lymfadenopathie en hun verwekkers besproken. de lymfeklier verzamelt eiwitrijke vloeistof (lymfe) uit de extracellulaire ruimte van perifere weefsels. lymfe bevat antigenen, micro-organismen en fagocytaire cellen en vloeit continu via afferente lymfevaten naar de regionale lymfeklieren. vanuit de subcapsulaire ruimte van de lymfeklier bereikt de lymfe via weefselspletende sinussen -de hilus, waar zij de lymfeklier via de efferente lymfvaten verlaat. na passage van een aantal lymfeklierstations komt de lymfe uiteindelijk via de ductus thoracicus in de v. cava superior. tijdens de passage door de lymfeklier komt de lymfe in contact met een zeer groot aantal fagocyterende cellen die zich op een skelet van reticulinevezels hebben vastgezet. door fagocytose wordt de lymfe vrijwel volledig gefilterd van antigenen en micro-organismen. in de lymfeklier komen t-en b-lymfocyten in contact met antigene fragmenten die worden gepresenteerd door dendritische cellen. de lymfeklier fungeert dus behalve als mechanisch filter ook als kraamkamer van het specifieke immuunsysteem, waar door intensief contact tussen antigeenpresenterende cellen en lymfocyten een antigeenspecifieke cellulaire (t-cel) en humorale (b-cel) respons wordt gegenereerd. bij infecties kan zowel het niet-specifieke als het specifieke immunologische proces aanleiding geven tot lymfekliervergroting (lymfadenopathie). bij acute bacteriële infecties (zoals door staphylococcus aureus of streptococcus pyogenes) wordt de vergroting van de lymfeklier veroorzaakt door een toevloed van macrofagen en granulocyten, die in de lymfeklier doorgedrongen bacteriën afkapselen en fagocyteren. de vergroting is pijnlijk en gaat ook klinisch gepaard met andere ontstekingsverschijnselen (lymfadenitis), waarbij ook ontsteking van de afferente lymfvaten (lymfangitis) kan optreden. in de lymfeklier kan abcedering optreden. bij chronische virale infecties wordt daarentegen de lymfadenopathie veroorzaakt door een sterke toename van het aantal geactiveerde lymfocyten en immunoblasten. lymfeklieren bij chronische virale infecties zijn niet of nauwelijks pijnlijk. aspecifieke ontsteking en immuunactivatie kunnen ook naast elkaar voorkomen: in subacuut verlopende lymfadenopathieën, zoals veroorzaakt door bartonella henselae en chlamydia trachomatis, kunnen door histiocyten omringde microabcesjes optreden. lymfadenopathie door mycobacterium tuberculosis wordt gekenmerkt door het optreden van granulomen: haarden van epitheloïde cellen in palissadestand met veelkernige reuscellen en in het midden verkazende necrose. een overzicht van de belangrijkste ziektebeelden waarbij lymfadenopathie optreedt, met hun verwekkers en belangrijkste differentiële criteria, wordt gegeven in tabel 13.1. naast deze infectieuze oorzaken bestaan er tal van niet-infectieuze ziektebeelden die gepaard gaan met lymfadenopathie. hieronder vallen zowel systeemziekten (zoals bepaalde auto-immuunziekten en sarcoïdose) als maligne aandoeningen (primaire (non-)hodgkinlymfomen en secundaire lymfekliermetastasen). ten slotte kan lymfadenopathie optreden als reactie op bepaalde farmaca of corpora aliena (bijv. siliconen). mononucleosis infectiosa is een ziektebeeld met als belangrijkste klinische kenmerken moeheid, koorts, zwelling van (vooral de cervicale) lymfeklieren en een meer of minder uitgesproken faryngitis. de ziekte verloopt in de meeste gevallen subacuut en komt vooral voor bij adolescenten. in verreweg de meeste gevallen kan een recente infectie met epstein-barr-virus (ebv) worden aangetoond. ook kan het worden veroorzaakt door een recente cytomegalovirusinfectie (cmv-infectie) (5-10%), een toxoplasmose ( 1%), of een (primaire) hivinfectie. ebv en cmv behoren tot de humane herpesviridae (zie hoofdstuk 1). omdat lymfadenopathie tijdens de primaire infectie van ebv, cmv en hiv zo'n prominente rol kan spelen, zullen deze virussen in dit hoofdstuk uitvoeriger worden besproken. epstein-barr-virus (ebv, officiële taxonomische benaming: humaan herpesvirus 4, hhv-4) werd in 1961 ontdekt door epstein, achong en barr. zij zagen het virus met behulp van een elektronenmicroscoop in cellen die gekweekt waren uit een tumor die veel voorkwam bij kinderen in afrika (burkitt-lymfoom). vervolgens bleek dat de meeste volwassenen antistoffen hadden tegen ebv, passend bij een vroegere infectie. de toevallige observatie van de onderzoekers gertrud en werner henle dat zich antistoffen tegen ebv vormden bij een laboratoriummedewerker die mononucleosis infectiosa doormaakte, leidde tot de identificatie van ebv als een van de verwekkers van dit ziektebeeld. speeksel bevat wisselende hoeveelheden van dit virus, geproduceerd in de tonsilcrypten. primaire infectie via speekselcontact leidt tot productie van virus in de initieel geïnfecteerde epitheelcellen, waarop vervolgens de aanwezige b-cellen worden geïnfecteerd. infectie van deze b-cellen leidt tot een transformatie, wat in het laboratorium kan worden aangetoond met spontane uitgroei van lymfoblastoïde cellijnen. de infectie van b-cellen leidt tot een uitgesproken t-celrespons, waardoor in het bloedbeeld van patiënten een bont beeld van afwijkend gevormde (atypische) lymfocyten (geactiveerde t-lymfoblasten) wordt gezien. het kenmerkende symptomencomplex van mononucleosis infectiosa wordt niet rechtsreeks door virusreplicatie veroorzaakt, maar is het resultaat van de sterke t-celrespons (lymfadenopathie) en de daarmee gepaard gaande cytokine-en interleukineproductie, wat zich uit door extreme moeheid en koorts. deze t-celrespons is verantwoordelijk voor de onderdrukking van de door ebv geïnduceerde b-celproliferatie in vivo. ten gevolge van de virusspecifieke t-celrespons kan ebv na de primaire infectie alleen in latent geïnfecteerde b-geheugencellen voortbestaan. bij asymptomatische dragers is ongeveer 1 op de 100.000 b-geheugencellen geïnfecteerd. in deze cellen is weliswaar het ebvgenoom in de kern aanwezig, maar wordt slechts een beperkt aantal genen (10 van de ongeveer 100) tot expressie gebracht. hierdoor worden slechts weinig virale peptiden op het celoppervlak in de context van moleculen van hla-klasse i gepresenteerd, waardoor deze cellen niet door cytotoxische cd8+-t-cellen kunnen worden herkend. reactivatie van virusreplicatie treedt mogelijk op als gevolg van stimulatie van de b-geheugencel door herkenning van zijn specifieke antigeen. als gevolg van deze reactivatie kan bij 5-10% van de dragers uit speeksel virus worden geïsoleerd. het vermogen van ebv om b-celproliferatie te induceren kan leiden tot het ontstaan van b-celtumoren. aanvankelijk oligoklonale, in latere stadia vaak monoklonale b-cellymfomen kunnen worden gezien bij patiënten met een verstoorde t-celfunctie, zoals hiv-geïnfecteerde personen en ontvangers van orgaantransplantaten (post-transplant lymphoproliferative disorder, ptld). verder is aangetoond dat ebv-infectie een cofactor is in de pathogenese van burkitt-lymfoom en nasofarynxcarcinoom (frequent in china) en bij agressievere vormen van de ziekte van hodgkin. ebv komt wereldwijd voor. er is geen verschil in prevalentie tussen de geslachten of tussen etnische groepen. de leeftijd waarop infectie optreedt, wordt vooral bepaald door sociaal-economische factoren. in niet-westerse landen treedt infectie vaak al op de vroege kinderleeftijd op vanwege intensieve contacten; bij 5-jarigen ligt de seroprevalentie al boven de 50%, op volwassen leeftijd is uiteindelijk wereldwijd 90-95% geïnfecteerd. in zeldzame gevallen kan ebv-infectie aanleiding geven tot neurologische complicaties, zoals myelitis transversa, nervusfacialisparese, neuritis optica of cerebellitis. de soms heftig verlopende faryngitis kan naast slikstoornissen aanleiding geven tot pseudomeningisme door een verhoogde tonus van de cervicale musculatuur. respiratoire obstructie door zwellingen in hypofarynx en larynx komt incidenteel ook voor en kan ernstig zijn. karakteristiek is de rash die kan optreden tijdens ebvinfectie na empirische therapie met amoxicilline. een belangrijke hematologische complicatie is een hemolytische anemie op auto-immuunbasis. er zijn geen aanwijzingen voor betrokkenheid van ebv bij het chronisch vermoeidheidssyndroom. de diagnose ebv-infectie wordt gesteld op grond van serologisch onderzoek. waarschijnlijk ten gevolge van de massale polyklonale activatie van b-cellen kunnen tijdens de primaire ebv-infectie autoantistoffen en heterofiele antistoffen ontstaan (dat zijn meestal igm-antistoffen, gericht tegen erytrocytaire antigenen bij andere species). deze heterofiele antilichamen zijn dus niet gericht tegen ebv maar zijn een direct gevolg van polyklonale b-celstimulatie. de paul-bunnell-test, waarbij deze heterofiele antistoffen worden aangetoond in serum, is een specifieke surrogaattest voor ebv-infectie. deze test is positief bij 90% van de volwassenen met een primaire infectie, maar veel minder gevoelig bij jonge kinderen met een acute ebv-infectie. heterofiele antistoffen zijn vooral aantoonbaar in de eerste drie tot zes maanden na het begin van symptomen. voor screening, ook in de huisartsenpraktijk, zijn sneltests beschikbaar. specifieke ebv-serodiagnostiek kan ook worden verricht. hiermee kunnen igm-en igg-antistoffen worden aangetoond tegen het virale capsideantigeen (vca), een aantal vroege antigenen (early antigens, ea) en antigeen uit de latente cyclus van het virus (epstein-barr virus-associated nuclear antigen, ebna). de kinetiek van de verschillende antistofresponsen is weergegeven in figuur 13.2. aan de hand van het antistofpatroon kan een inschatting worden gemaakt van het moment van ebvinfectie. anti-vca-igg-antistoffen ontstaan tijdens de acute fase en blijven ook na herstel aantoonbaar. hoge titers anti-ea-antistoffen wijzen op een recente infectie. reconvalescentie gaat gepaard met het dalen van de anti-ea-titers en stijgende titers van anti-ebna-antistoffen. de kweek van ebv wordt niet gebruikt in de routinediagnostiek. de ontwikkeling van kwantitatieve moleculaire (pcr) technieken maakt het mogelijk om de hoeveelheid ebv in plasma te kwantificeren. bij patiënten die immuunsuppressieve therapie ontvangen vanwege een transplantatie wordt de hoeveelheid ebv gevolgd om ebv-gemedieerde posttransplantatie-lymfoproliferatieve ziekte vroegtijdig op te sporen en te voorkomen. de behandeling van een acute ebv-infectie is voornamelijk ondersteunend. aciclovir remt ebv-replicatie in vitro, maar heeft klinisch geen effect. dit past bij de beschreven vooral immunopathologische basis van het ziektebeeld. incidenteel kunnen corticosteroïden aangewezen zijn bij dreigende luchtwegobstructie of hemolytische anemie. een vaccin is nog niet voorhanden. bij ebv-positieve lymfoproliferatieve ziektebeelden speelt therapie gericht tegen b-cellen (anti-cd20 monoklonale antistoffen) een belangrijke rol. humaan cytomegalovirus (cmv, humaan herpesvirus 5) dankt zijn naam aan de reuzen-(megalo)cellen, die werden aangetroffen in organen van zuigelingen met een letaal verlopende congenitale infectie. humaan cytomegalovirus kent wereldwijd een groot aantal stammen met ongeveer 95% homologie op sequentieniveau; bij een groot aantal diersoorten (primaten, knaagdieren enz.) komen eigen cmv-soorten voor. besmetting met cmv treedt frequent prenataal of perinataal op (tijdens passage door het baringskanaal of door lactatie), of door contact met besmet speeksel of urine. monocyten kunnen tijdens de primaire infectie worden geïnfecteerd en produceren cmv na hun differentiatie tot macrofagen. op deze wijze dragen zij vermoedelijk bij tot de hematogene verspreiding van het virus. cmv infecteert onder andere epitheelcellen van nieren (proximale tubuli) en speekselkliercellen (ductusepitheel), endotheel en fibroblasten. het virus kan de placenta passeren en een intra-uteriene infectie veroorzaken (zie hoofdstuk 15). bij het onder controle krijgen van de primaire infectie spelen waarschijnlijk zowel specifieke cytotoxische t-lymfocyten als neutraliserende antistoffen een rol. de humorale en cellulaire respons leiden tot partiële immuniteit tegen andere cmv-stammen. in de latente fase kan cmv-dna worden aangetoond in monocyten en hemopoëtische stamcellen, maar niet in granulocyten, de cellen waarin het virus juist gedetecteerd wordt tijdens viremische episoden. actieve replicatie van cmv in de monocyten-/macrofagenreeks is waarschijnlijk beperkt tot gedefinieerde fasen in de differentiatie. de beperkte replicatie in de tussenliggende fasen draagt mogelijk bij tot de persistentie van de infectie. mogelijk geldt hetzelfde voor andere celtypen. bij immunocompetente dragers treedt episodisch een asymptomatische reactivatie op, waarbij het virus in urine en speeksel wordt uitgescheiden. lymfadenopathieën en hiv epidemiologie prenatale, intra-uteriene infecties kunnen ernstig verlopen (congenitale cmv, zie hoofdstuk 15), maar peri-en postnatale cmv-infecties verlopen vrijwel altijd asymptomatisch. overdracht vindt vooral plaats via speeksel, maar ook via moedermelk, urine, feces, bloed en sperma. tot 10% van de pasgeborenen wordt besmet met cmv tijdens de baring of via lactatie, maar intensief contact met leeftijdgenootjes die virus uitscheiden (crèches) zorgt voor een snelle toename van het aantal geïnfecteerden met de leeftijd. een tweede snelle stijging van de seroprevalentie wordt gezien bij jonge volwassenen. ook dan verlopen de meeste (90%) van de primo-infecties asymptomatisch. afhankelijk van de hygiënische omstandigheden varieert de seroprevalentie wereldwijd op volwassen leeftijd tussen 50 en 100%. ongeveer 10% van de primo-infecties op de volwassen leeftijd gaat gepaard met symptomen. deze treden op na een incubatietijd van gemiddeld zes weken, vergelijkbaar met ebv. ongeveer 5-10% van de klinische beelden van mononucleosis infectiosa wordt veroorzaakt door cmv. cmv-mononucleosis treedt doorgaans op wat latere leeftijd op dan ebv-mononucleosis. meestal blijven de symptomen beperkt tot twee à drie weken koorts. faryngitis en cervicale lymfadenopathie zijn minder gebruikelijk dan bij primaire ebv-infectie. het perifere bloedbeeld vertoont meestal een lymfocytose met atypische lymfocyten, en de leverfuncties kunnen gestoord zijn. volledig herstel treedt op na ongeveer zes weken. de paul-bunnell-reactie is kenmerkend negatief. zeldzame complicaties zijn hepatitis, pneumonie, aseptische meningitis en het syndroom van guillain-barré. ditzelfde ziektebeeld van koorts, leukopenie, atypische lymfocytose en splenomegalie kan optreden bij een cmv-seronegatieve ontvanger drie tot zes weken na bloedtransfusie met vers cmv-seropositief bloed. de kans op cmv-transmissie via bloedtransfusie (geschat op ongeveer 2,5% per unit getransfundeerd bloed) kan worden gereduceerd door gebruik van bloed van seronegatieve donoren, leukocytenarm bloed of bevroren bloed-of bloedproducten. ernstige ziektebeelden veroorzaakt door cmv treden vooral op bij intra-uteriene infectie (zie hoofdstuk 15) en patiënten met afweerstoornissen (zie hoofdstuk 17). bij orgaantransplantaties is cmv-ziekte een van de meest voorkomende complicaties. cmv-ziekte kan het gevolg zijn van reactivatie van het virus van de (seropositieve) ontvanger of infectie vanuit het donororgaan of door gedoneerd bloed. vanwege het beperkte aanbod van donororganen is het niet altijd mogelijk naast een goede hla-match ook de cmv-serostatus van donor en ontvanger op elkaar af te stemmen. cmv-infectie bij transplantatiepatiënten kan gepaard gaan met langdurige koorts, trombo-en/of leukopenie en gestoorde leverenzymen (hepatitis), spierpijn en ge-wrichtsklachten. daarnaast kunnen ernstige gastro-intestinale infecties optreden (oesofagitis, gastritis, colitis), die gepaard kunnen gaan met perforatie. cmvpneumonitis is vooral een levensbedreigende ziekte bij patiënten met een allogene beenmergtransplantatie. diverse vormen van cmv-ziekte kunnen optreden bij aidspatiënten, vooral bij een sterk gestoorde immuniteit (cd4+-cellen < 50/mm 3 ). naast pneumonitis, encefalitis en gastro-intestinale infecties is vooral retinitis een beruchte complicatie. de laatste manifestatie kan zich bilateraal voordoen en leidt bijna altijd tot blindheid, tenzij antivirale therapie wordt gegeven. incidenteel doet zich een ernstige cmv-colitis voor bij jonge volwassenen met een op het oog normale of hooguit licht gestoorde immuniteit (zoals bij zwangeren). diagnostiek de diagnose primaire cmv-infectie bij immuuncompetente gastheren wordt meestal gesteld aan de hand van het antistofpatroon. igm-antistoffen (in de vroege fase) en igg-antistoffen (levenslang) zijn aantoonbaar. een reactivatie van de cmv-infectie kan gepaard gaan met de hernieuwde vorming van igm-antistoffen maar dit is weinig betrouwbaar. een foutpositieve cmv-igm-test kan soms optreden bij een primaire ebv-infectie. het betreft hier antistoffen gegenereerd als gevolg van de tijdens de ebv-infectie optredende polyklonale b-celactivatie, of mogelijk antistoffen die gemeenschappelijke antigene determinanten van ebv en cmv herkennen. heterofiele antistoffen komen bij cmv-infectie echter niet voor. naast serologisch onderzoek bestaat de mogelijkheid om cmv als virus aan te tonen, door middel van kweek, door antigeendetectie of tegenwoordig vooral door cmv-dna-detectie. dit is vooral van groot belang bij de infecties van immuungecompromitteerde gastheren. cmv is in die gevallen als typisch systemische infectie aantoonbaar op vele plaatsen, zoals in de keel, in urine, in bronchiaal spoelsel en in leukocyten. ook bij congenitaal geïnfecteerde kinderen vindt soms nog jarenlang sterke cmv-uitscheiding in de urine plaats. positieve kweekresultaten wijzen niet noodzakelijkerwijs op een symptomatische cmv-infectie. asymptomatische uitscheiding van cmv komt regelmatig voor, in het bijzonder bij patiënten met een verminderde afweer. een goede indruk van de relevantie van een cmv-infectie is in die gevallen te verkrijgen door het meten van de hoeveelheid viraal dna in bloed of plasma. de drie middelen met klinisch bewezen werkzaamheid, ganciclovir, foscarnet en cidofovir, worden gekenmerkt door een hoge frequentie van toxische neveneffecten. ganciclovir is nauw verwant aan aciclovir, maar blijkt een veel effectievere remmer van de cmv-replicatie. dit is het meest toegepaste middel tegen cmv-infectie; het is ook oraal toepasbaar door middel van de prodrug valganciclovir. door het ter beschikking komen van snelle moleculaire tests voor de kwantitatieve bepaling van de hoeveelheid cmv in plasma wordt bij transplantatiepatiënten therapie vaak toegepast op geleide van de hoeveelheid cmv in het bloed. met deze strategie blijkt ernstige cmv-ziekte bij deze patiënten grotendeels te kunnen worden voorkomen (zie hoofdstuk 17). onderzoek naar preventie van cmv-ziekte door vaccinatie is gaande. immunisatie van ontvangers van niertransplantaten acht weken voor transplantatie met een verzwakte cmv-stam (towne) leidde niet tot een verminderde cmv-uitscheiding na transplantatie. de incidentie van cmv-ziekte was wel lager ten opzichte van de placebogroep en ook verliep de ziekte in die gevallen minder ernstig. naast onderzoek met dit type vaccin is ook onderzoek gaande met vaccins bestaande uit gezuiverd of recombinant gb, het voornaamste envelopglycoproteïne van cmv-virus. ook het voorkómen van schade door congenitale cmv-infectie is een belangrijk doel van vaccinatie. naar schatting 1% van de mononucleosis infectiosa-achtige ziektebeelden wordt veroorzaakt door toxoplasma gondii, een intracellulair protozoön. in hoofdstuk 18 zal nader op deze infectie worden ingegaan. casus 13.2 een 45-jarige vrouw wordt opgenomen in het ziekenhuis vanwege toenemende kortademigheid. tien jaar eerder werkte zij als reisleidster ruim acht maanden in tanzania, kort waarop zij een griepachtig beeld met lymfadenopathie ontwikkelde, dat toen werd geduid als mononucleosis infectiosa. twee jaar voor presentatie begon ze langzamerhand steeds meer last te krijgen van seborroïsch eczeem, genitale ulcera en eenmaal een ernstige gordelroos in het gelaat. de laatste maanden is ze 8 kg afgevallen. bij onderzoek blijkt er sprake van een verlaagde zuurstofspanning door een dubbelzijdige, interstitiële pneumonie. bij een longspoeling wordt pneumocystis in de alveolaire macrofagen herkend en wordt een hiv-antistoftest ingezet. deze blijkt positief. haar cd4-aantal blijkt 30/mm 3 . in 1981 werd in de verenigde staten een epidemie van longontstekingen met pneumocystis jirovecii beschreven, vaak gepaard gaand met een zeldzame vorm van huidkanker, het kaposi-sarcoom. al snel werd duidelijk dat een ernstige immuunstoornis aan deze verschijnselen ten grondslag lag en dat de slachtoffers allen in korte tijd overleden. aanvankelijk waren de slachtoffers jonge homoseksuele mannen, maar al snel werden vergelijkbare casus waargenomen bij heteroseksuele mensen uit haïti en bij ontvangers van bloedtransfusies. de aandoening werd aids genoemd (acquired immune deficiency syndrome) en gezien de epidemiologie vermoedde men al snel een infectieuze oorzaak. in 1983 isoleerden de latere nobelprijswinnaars (2008) barré-sinoussi en montagnier als eersten uit de lymfeklier van een franse aidspatiënt een nieuw retrovirus, dat uiteindelijk humaan immunodeficiëntievirus type 1 (hiv-1) werd genoemd. in 1986 werd een verwant virus, hiv-2, aangetoond bij aidspatiënten in west-afrika. inmiddels is duidelijk geworden dat beide virussen voorkomen bij primaten in afrika (als vormen van simian immunodeficiency virus, siv). men heeft aannemelijk kunnen maken dat de voorlopers van beide humane virussen in de jaren dertig (hiv-1) en veertig (hiv-2) van de vorige eeuw vanuit primaten bij de mens geïntroduceerd zijn op het afrikaanse continent. het begin van de epidemie in afrika is niet herkend, wel heeft men met terugwerkende kracht een geval van aids kunnen vaststellen bij een zeeman uit manchester in 1959, die vermoedelijk in afrika was geïnfecteerd. het virusdeeltje heeft een sferische vorm en is ongeveer 100 nm groot (figuur 13.3a). het virus heeft een envelop die bestaat uit de virale glycoproteïnen gp120 en gp41, en bestanddelen die afkomstig zijn van de gastheercelmembraan. deze envelop omgeeft de capside, met als voornaamste bestanddeel het virale p24-eiwit. binnen de capside bevinden zich twee identieke enkelstrengs rna-moleculen (het virale genoom) en een aantal moleculen van de enzymen reverse transcriptase, integrase en protease. het genoom is relatief klein (9 kb) en bevat negen genen, die in totaal coderen voor vijftien verschillende eiwitten. het genoom wordt aan weerszijden geflankeerd door een long terminal repeat (ltr), een niet-coderend deel van het genoom betrokken bij de regulatie van de virale expressie (figuur 13.3b). de genproducten zijn onder te verdelen in: 1 structurele proteïnen (o.a. de gag-genproducten (p24 en p17) en envelopglycoproteïnen (gp41 en gp120); 2 door het virus gecodeerde enzymen die nodig zijn voor de virale replicatiecyclus (reverse transcriptase, integrase en protease); 3 eiwitten die betrokken zijn bij de regulatie van de genexpressie (o.a. tat, rev en nef). hiv infecteert humane gastheercellen die op hun buitenmembraan een cd4+-receptormolecuul bezitten (figuur 13.4). deze cd4+-receptoren zijn uiteraard aanwezig op cd4+-positieve t-lymfocyten, maar in mindere mate ook op macrofagen en dendritische cellen. hiv gebruikt dit cd4+-molecuul als eerste receptor om de gastheercel te kunnen binden. door binding van het virale gp120 aan dit cd4+-molecuul ontstaat in de virusenvelop een conformatieverandering, waardoor het virus zich kan binden aan een tweede (co)receptor. deze tweede receptor komt uit de familie van de chemokinereceptoren, waarvan er twee belangrijk zijn voor hiv: ccr-5 en cxcr-4. de ccr-5-receptor bevindt zich vooral op macrofagen, dendritische cellen en cd4+-positieve t-lymfocyten, de cxcr-4-receptor komt vooral tot expressie op geactiveerde cd4+-positieve t-lymfocyten. op basis van hun coreceptorgebruik kan hiv worden onderverdeeld in varianten die vooral de ccr-5receptor gebruiken (r5-virussen, ook wel macrofagotrope virussen) en varianten die vooral de cxcr-4-receptor gebruiken (x4-virussen, ook wel lymfocytotrope virussen). er zijn ook varianten die beide receptoren kunnen gebruiken (duotrope virussen). het overgrote deel van de patiënten wordt geïnfecteerd met r5-trope virussen. gedurende de infectie kunnen virussen muteren met als gevolg dat bij ruim 50% van de geïnfecteerde individuen in de loop van de infectie virussen ontstaan die ook de cxcr-4-coreceptor kunnen gebruiken. na binding aan de coreceptor treedt een tweede conformatieverandering op en kan het envelopeiwit gp41 binden aan de gastheercelmembraan. binding van de virus-en celmembraan leidt tot fusie van beide membranen, waarop de nucleocapside het cytoplasma van de cel kan binnengaan. vervolgens worden met behulp van het enzym reverse transcriptase de twee enkelstrengs virale rna-kopieën omgezet in proviraal dubbelstrengs dna (figuur 13.4). dit dna migreert naar de kern en integreert in het gastheercel-dna met behulp van het reeds aanwezige virale integrase. het geïntegreerde dna noemen we een provirus. in feite is er nu sprake van een irreversibele ('ongeneeslijke') situatie: zo lang de geïnfecteerde gastheercel(populatie) overleeft, zal ook het geïntegreerde hiv-dna overleven. wanneer een provirus bevattende cd4+-cel geactiveerd wordt, wordt de transcriptiefactor nfkb geproduceerd. deze bindt aan de promotor van het provirus, waarop transcriptie wordt gestart. de virale eiwitten tat en rev die daarbij gemaakt worden, zorgen vervolgens voor een efficiënter verlopende transcriptie, waarop virale mrna's en de precursors van de structurele (glyco)proteïnen worden geproduceerd. de virale glycoproteïnen groeperen zich bij de celmembraan. het virale protease klieft het gag-precursoreiwit, waarop de eindproducten worden geproduceerd, onder meer de virale capside-eiwitten. samen met het genomisch rna assembleren deze eiwitten aan de binnenkant van de celmembraan tot nieuwe virionen, die zich door lokale uitstulping en afsnoering van de celmembraan (budding) buiten de cel begeven (zie figuur 13.4). men schat dat er bij een hiv-1-geïnfecteerde patiënt gemiddeld 10 8 -10 10 virusdeeltjes per dag worden geproduceerd, waarbij dagelijks 10 8 -10 9 cd4+-cellen worden geïnfecteerd. doordat het reverse transcriptase een slordig enzym is (1:10 4 nucleotiden wordt foutief gekopieerd), treden er mutaties op die leiden tot virusvarianten. men kan daardoor niet spreken van één virus maar van een viruspopulatie. afhankelijk van suppressieve factoren, zoals de gastheerimmuniteit tegen hiv of de aanwezigheid van medicatie, zullen vooral die virussen expanderen, die door hun mutatie(s) aan deze suppressieve druk kunnen ontsnappen (survival of the fittest). het kenmerk van een hiv-infectie is een geleidelijke afname van het aantal cd4+-positieve lymfocyten in het perifere bloed (figuur 13.5), waardoor uiteindelijk een functionele immuundeficiëntie ontstaat. aanvankelijk nam men aan dat de cd4+-daling een direct gevolg was van de celdood door virusreplicatie, dan wel door toegenomen geprogrammeerde celdood (apoptose), of door immuungemedieerde destructie. tegenwoordig neemt men aan dat de hiv-infectie een hyperactivatie van het gehele cd4+-compartiment veroorzaakt, waardoor de cd4+-cellen veel korter overleven en uiteindelijk onvoldoende kunnen worden aangevuld. aangezien de cd4+-lymfocyt een centrale rol speelt in de regulering van de immuunrespons, leidt het verlies van deze celpopulatie uiteindelijk tot deficiënties in die immuunrespons. vooral de cellulaire immuniteit is sterk afhankelijk van de aansturende rol van de cd4+-cel en is daarom als eerste gestoord. de cellulaire immuniteit controleert onder meer micro-organismen die in of op het lichaam latent aanwezig zijn, zoals schimmels, mycobacteria en herpesvirussen. wanneer de cellulaire immuniteit verminderd functioneert, kunnen deze micro-organismen tot ziekteverschijnselen leiden. men spreekt dan van opportunistische infecties (zie ook hoofdstuk 17). de humorale immuniteit ontwikkelt zich vooral in de lymfadenopathieën en hiv eerste levensjaren. de cd4+-cel speelt in die fase wel een belangrijke rol in deze ontwikkeling, maar als het humorale immuunrepertoire eenmaal gevormd is, wordt de rol van de cd4+-cel hierin minder belangrijk. een volwassen persoon met een laag cd4+-aantal kan gekapselde micro-organismen, zoals pneumokokken en stafylokokken, die vooral afhankelijk zijn van een humorale immuunrespons, daarom nog redelijk effectief bestrijden. jonge kinderen met hiv hebben daar duidelijk meer moeite mee door een nog onvoldoende gerijpt humoraal repertoire. vaccinatie, waarbij humorale immuniteit moet worden opgebouwd onder invloed van cd4+-cellen, is om diezelfde reden minder effectief bij hiv-geïnfecteerde patiënten met een laag cd4+-aantal. een hiv-infectie onderscheidt zich in het begin niet van andere virusinfecties waartegen het lichaam zowel een humorale als een cellulaire immuunrespons opbouwt. nadat het virus in de eerste weken vrij spel heeft gehad, waarbij grote hoeveelheden virus in het bloed kunnen worden aangetroffen (figuur 13.5), zorgen tegen hiv gerichte neutraliserende antistoffen en cytotoxische lymfocyten (ctl) voor controle over het virus, waarop de hoeveelheid celvrij virus in het plasma daalt. afhankelijk van de kracht van deze gastheerrespons en de mogelijkheid van het virus om hieraan te ontsnappen, ontstaat na een aantal weken een soort balans (set-point) waarbij hiv en het immuunsysteem elkaar in evenwicht houden. doordat het virus echter continu muteert, ont-staan virusvarianten die aan deze immuuncontrole ontsnappen, waarop een nieuwe immuunrespons tegen deze varianten de balans weer moet proberen te herstellen. doordat de daarvoor benodigde cd4+-cellen echter langzaam verdwijnen (en daardoor ook de ctlrespons tegen hiv vermindert), raakt de balans steeds meer verstoord en zal het virus deze strijd uiteindelijk winnen: de hoeveelheid virus in het plasma stijgt weer. in het algemeen geldt dat stijging van de hoeveelheid in het plasma (meer dan 100.000 viruskopieën per ml) gepaard gaat met een snellere cd4+-daling en dus een snellere ziekteprogressie. door de mutaties in de envelop kan het virus ook zijn tropisme veranderen: naast macrofagotrope virussen (r5) ontstaan duotrope (r5/x4) virussen, met als gevolg dat het virus in meer cellen kan repliceren. hierdoor stijgt de hoeveelheid virus snel en zal het aantal cd4+-cellen in korte tijd sterker dalen, wat gepaard gaat met snelle ziekteprogressie. het beloop van de hiv-infectie kent grote interindividuele verschillen: de duur van de asymptomatische eerste fase kan variëren van twee tot meer dan vijftien jaar. de genetische achtergrond, zoals de hla-typering van de geïnfecteerde persoon en ook andere genetische polymorfismen binnen het immuunsysteem, draagt daar belangrijk aan bij. zo zijn er individuen die een homozygote deletie hebben voor de ccr-5-receptor, waardoor infectie met r5-virussen niet kan plaatsvinden. bij personen met de heterozygote ccr-5-deletie kan een r5-virus zich minder gemakkelijk verspreiden en verloopt de ziekteprogressie trager. de kliniek rondom hiv-infectie is grofweg in drie fasen te verdelen: de acute hiv-infectie in de eerste drie maanden, daarna een relatief asymptomatisch verlopende fase, die twee tot meer dan vijftien jaar kan duren, en tot slot een symptomatische fase, die snel progressief kan verlopen en eindigt in aids. nadat iemand met hiv geïnfecteerd is geraakt, ontstaan in de acute fase klinische symptomen bij 80-90% van de personen, waarbij 50-60% medische hulp zoekt. de symptomen beginnen meestal twee tot vier weken na de infectie en lijken sterk op een griepbeeld of op mononucleosis infectiosa, dat eerder werd besproken bij de acute ebv-infectie. koorts treedt vaak op (> 90%), gepaard gaand met faryngitis (75%), lymfadenopathie (50-75%), spier-en gewrichtspijn (50-90%) en moeheid (80-90%). regelmatig worden bovenstaande verschijnselen begeleid door een uitgebreide maculopapulaire huiduitslag (30-70%), gewichtsverlies (50-70%), hoofdpijn (40%), darmklachten met diarree en/of braken (30-50%) en ulcera in mond, rectum en genitalia. bij bloedonderzoek ziet men vaak atypische lymfocytose (> 80%), leuko-en trombopenie (35-45%) en milde hepatitis (20%). meestal houden de klachten en afwijkingen niet langer dan twee weken aan. in deze fase van de acute hiv-infectie komt de immuunrespons tegen hiv op gang: er ontstaan antistoffen tegen alle hiv-eiwitten (seroconversie) en er ontstaat ook een sterke ctl-respons. initieel is er veel virus in het plasma aanwezig (hiv-rna viral load) en zullen de hiv-tests (die berusten op het aantonen van anti-hiv-antistoftests) nog negatief zijn; men spreekt van de window-fase. seroconversie treedt bij de meeste patiënten binnen acht tot tien weken op, zodat deze window-fase slechts enkele weken duurt. tijdens de acute hiv-infectie daalt het aantal cd4+-cellen in het bloed, wat zich meestal grotendeels weer herstelt. de daling kan soms zo diep zijn, dat ernstige opportunistische infecties kunnen optreden. de grootste daling van cd4+-cellen in de acute fase vindt echter plaats in het darmgeassocieerde lymfoïde weefsel (galt, gut associated lympoïd tissue). in dit compartiment vindt nauwelijks herstel van het aantal cd4+-cellen plaats. met het op gang komen van een krachtige immuunrespons daalt de hoeveelheid hiv-rna in het plasma en herstelt het aantal cd4+-cellen in het perifere bloed zich weer redelijk (figuur 13.5). na de acute fase volgt de al genoemde asymptomatische fase, waarbij virus en gastheer elkaar in evenwicht lijken te houden rond een soort set-point. algemene klachten zoals moeheid, nachtzweten en lymfadenopathie worden in wisselende mate gezien, evenals huidklachten zoals folliculitis en seborroïsch eczeem. bepaalde opportunistische infecties zoals herpes zoster en herpes simplex treden vaker op, evenals sinusitis en pneumokokkenpneumonieën, maar zo lang het aantal cd4+-lymfocyten hoger is dan 200/mm 3 zijn deze beelden mild en van voorbijgaande aard. zodra het cd4+-aantal duidelijk onder deze grens komt, worden de opportunistische infecties ernstiger. infecties door candida albicans van mondholte (stomatitis) en slokdarm (oesofagitis), longontstekingen met pneumocystis jirovecii (vroeger pneumocystis carinii genoemd) en diarree met giardia lamblia, cryptosporidium, isospora en microsporidia nemen in frequentie toe, naarmate het cd4+-gehalte lager wordt. bij cd4+-waarden onder de 100/mm 3 zorgen toxoplasmose (cerebraal abces), cryptococcus neoformans (meningitis, maar ook gedissemineerd), cmv (colitis en retinitis) en atypische mycobacteria (bacteriëmie, darmwand en beenmerg) voor ernstig verlopende en dodelijke infecties. los van deze cd4+-grenzen worden als belangrijke opportunistische problemen gewone tuberculose maar ook kwaadaardige aandoeningen als non-hodgkinlymfomen en kaposisarcomen gezien. wanneer een hiv-geïnfecteerde zich presenteert met een van deze opportunistische problemen, spreekt men per definitie van aids. onbehandeld is de gemiddelde overleving van een aidspatiënt meestal niet langer dan 18 maanden, afhankelijk van de ernst van de ziekte waarop de diagnose aids wordt gesteld. de belangrijkste aids-indicatordiagnosen zijn: -pneumocystis jirovecii-pneumonie (voorheen: pneumocystis jirovecii); -oesofageale candidiasis; -cerebrale toxoplasmose; -cryptosporidiose met > 1 maand diarree; -extrapulmonaire cryptokokkose; -cmv-ziekte van tractus digestivus, retinitis, pneumonitis, encefalitis; -hsv mucocutane ulcera > 1 maand, pneumonitis, oesofagitis; -pulmonale of gedissemineerde tuberculose; -gedissemineerde infectie met mycobacterium avium intracellulare of m. kansasii; hiv-wasting: > 10% gewichtsverlies en diarree, of langdurige febris e.c.i. en chronische malaise; -hiv-geassocieerde dementie; -kaposi-sarcoom; -non-hodgkin-of b-cellymfoom. sinds de eerste hiv-patiënten werden beschreven, is er al direct veel ontwikkeling geweest in het zoeken naar behandelingen. in eerste instantie bestond dit uit het steeds beter behandelen en voorkomen van opportunistische infecties. zo werd al snel duidelijk dat een van de beruchtste opportunistische infecties, de pneumocystis jirovecii-pneumonie (zie ook hoofdstuk 17) kon worden voorkomen als patiënten een profylaxe met co-trimoxa-zol ontvingen zodra hun aantal cd4+-cellen lager werd dan 200/mm 3 . vanaf 1987 werd het mogelijk de hiv-infectie zelf te behandelen met een middel dat reverse transcriptase remde: azidothymidine (azt). dit middel is een gemodificeerd nucleoside (thymidine), dat wanneer het in de dna-keten wordt ingebouwd verdere dna-verlenging (en dus uiteindelijk virusreplicatie) onmogelijk maakt. in eerste trials met dit middel was een duidelijk klinische verbetering aantoonbaar, maar het overlevingsvoordeel bleek van korte duur. nieuwe, vergelijkbare medicijnen, alle nrti's (nucleoside-analogue reverse transcriptase inhibitors) genoemd, kwamen op de markt, maar ook zij bleken in monotherapie geen aanwinst. duotherapie was effectiever, maar de echte doorbraak kwam pas met het gebruik van tripelcombinatietherapie. al snel werd het arsenaal medicijnen uitgebreid met een groep medicijnen die reverse transcriptase op een andere manier remmen (nnrti's, non-nucleoside reverse transcriptase inhibitors), maar ook met nieuwe nrti's en proteaseremmers. ook werd toen duidelijk waarom monoen duo-nrti-therapie niet werkten: deze middelen konden de virusreplicatie niet geheel remmen, omdat een of twee mutaties in het enzym reverse transcriptase al tot antivirale resistentie konden leiden. virussen met een tot twee mutaties waren al aanwezig in de viruspopulatie van onbehandelde mensen, met als gevolg dat deze mutanten werden uitgeselecteerd en konden repliceren. tripelcombinatietherapie (een proteaseremmer of een nnrti gecombineerd met twee nrti's) bleek wel tot volledige onderdrukking van de virale replicatie te leiden. hiervoor wordt het acroniem haart gebruikt (highly active anti-retroviral therapy). in enkele weken tijd kan de hoeveelheid hiv-rna in plasma tot onmeetbaar lage waarden worden teruggebracht, gevolgd door een snelle stijging van het aantal cd4+-cellen in het perifere bloed, deels als gevolg van reallocatie van deze cellen vanuit de lymfeklier, deels als gevolg van remming van virusgeïnduceerde celdood. met het stijgen van het cd4+-celaantal verdwijnen de ziekteverschijnselen snel en blijken de cd4+-waarden vervolgens geleidelijk te kunnen terugkeren tot relatief normale waarden, mits de virussuppressie voortdurend wordt gehandhaafd. belangrijk hierbij is dat haart dagelijks en goed geslikt wordt. met de eerstegeneratie-tripeltherapieën viel dit niet mee, aangezien zij bestonden uit grote aantallen pillen die meerdere keren per dag genomen moesten worden en bovendien frequent gepaard gingen met bijwerkingen als diarree, bloedarmoede en neuropathie. desalniettemin daalde in korte tijd de sterfte aan aids. ook bleek de virusoverdracht van moeder naar kind te kunnen worden geblokkeerd, door de hiv-positieve moeder tijdens het laatste deel van de zwangerschap een combinatiebehandeling te geven. na 2003 werden nog nieuwe klassen hiv-remmers geïntroduceerd: een remmer van de fusie van gp41 met de celmembraan (2003), een middel dat bindt aan ccr5-coreceptor (2008 ccr5-coreceptor ( ) en een integraseremmer (2008 , zodat het totale aantal geregistreerde antiretrovirale middelen in 2011 al ruim twintig telt. een bespreking van de aangrijpingspunten van antivirale therapie is te vinden in hoofdstuk 1 (paragraaf 1.7.2). meerdere combinaties van vrijwel steeds drie middelen kunnen op die manier worden samengesteld, waarbij ook resistente virussen goed kunnen worden geremd. behalve dit betere antivirale effect, maakten de nieuwere medicijnen combinaties mogelijk met een aanmerkelijk minder aantal pillen en ook met veel minder bijwerkingen. vooral deze laatste twee eigenschappen maakten dat patiënten daadwerkelijk hun medicatie goed konden blijven innemen. dankzij de haart lijkt de levensverwachting van hiv-geïnfecteerde personen inmiddels vergelijkbaar aan die van niet hiv-geïnfecteerden. langetermijnbijwerkingen, bijkomende ziekten (zoals chronische hepatitis b en c), maar ook ouderdomsverschijnselen spelen daarom een steeds grotere rol in de hiv-zorg anno 2011. met haart wordt gestart als het risico op opportunistische infecties begint te stijgen. aanvankelijk stelde men die grens onder een cd4+-aantal van 300/mm 3 , mede in overweging nemend dat de eerste combinaties ook veel bijwerkingen hadden. met de nieuwere combinaties is een vroegere start mogelijk en schuift de startgrens daarom op naar een hoger cd4+-getal (400/mm 3 anno 2010). haart is kostbaar en het bovenbeschreven succes kon aanvankelijk vooral worden bereikt in de rijkere landen. na 2000 is haart echter met vereende krachten ook geïntroduceerd in landen met de grootste patiëntenconcentraties. eind 2009 ontvingen zo ruim 5 miljoen patiënten over de hele wereld haart, weliswaar tegen een geschat totaal patiëntenaantal van 33 miljoen (2008). de bovenstaande beschrijving van het beloop en de behandeling van hiv-infectie betreft vooral hiv-1. wereldwijd is bij ongeveer 5% van de hiv-geïnfecteerde patiënten echter sprake van infectie met hiv-2. de prevalentie van dit virus is het hoogst in west-afrika, waar dit virus vermoedelijk rond 1940 door een meerkattensoort (zwarte mangabey) bij de mens werd geïntroduceerd. het ziektebeeld van hiv-2 verschilt niet van hiv-1, maar het beloop van een hiv-2-infectie is doorgaans veel milder en verloopt in een trager tempo. de hoeveelheid hiv-2-rna in plasma is veel lager dan bij hiv-1. dit draagt er waarschijnlijk toe bij, dat hiv-2 minder gemakkelijk kan worden overgedragen via seksueel contact of van moeder op kind. de diagnostiek van hiv-1 en hiv-2 verschilt enigszins: in de standaard hiv-serologie (elisa) zullen hiv-1-en hiv-2-infecties niet van elkaar onderscheiden kunnen worden. dit is pas mogelijk bij de zogeheten bevestigingstest (western-blot), waarbij specifieke antistoffen te-gen hiv-2-eiwitten apart worden gemeten. de pcr-test, die gebruikt wordt voor het meten van hiv-1-rna in plasma, detecteert hiv-2-rna niet. hierop dient men bedacht te zijn omdat een specifieke hiv-2-rna-test moet worden aangevraagd. ook therapeutisch zijn er verschillen tussen hiv-1 en hiv-2: non-nucleoside reverse transcriptase inhibitors (nnrti's) en fusieremmers zijn niet werkzaam tegen hiv-2 vanwege een andere structuur van het reverse transcriptase en gp41. het aantal behandelingsmogelijkheden tegen hiv-2 is daardoor beperkter. hiv kan worden overgedragen via seksueel contact, bloed-bloedcontact (zoals transfusie van bloedproducten en het gebruik van verontreinigde naalden) en door verticale transmissie (van moeder op kind). het risico op seksuele overdracht wordt vergroot wanneer er sprake is van slijmvliesbeschadigingen, zoals bij geslachtsziekten of bij agressieve c.q. traumatische seks (verkrachting), maar is ook afhankelijk van de seksuele techniek. zo is de transmissiekans groter bij anale seks ten opzichte van vaginale seks en ook zal de ontvangende partner eerder een infectie oplopen (tabel 13.2). de kans op overdracht wordt mede bepaald door de hoeveelheid aanwezig infectieus virus. bij een hoge virale load in het plasma, zoals vooral het geval is tijdens de acute hiv-infectie, is de transmissiekans veel groter dan bij een lage virale load. effectieve haart reduceert het transmissierisico tot bijna nihil. transmissie via bloed-bloedcontact verloopt efficiënter dan seksuele overdracht, maar is afhankelijk van het inoculum en ook weer van de hoeveelheid aanwezig virus. mondiaal gaat het hierbij vooral om hergebruik van injectienaalden bij intraveneus drugsgebruik, maar er zijn ook diverse voorbeelden van hergebruik van naalden en infuussystemen in medische setting (libië, china, roemenië), dan wel infusie van met hiv besmette bloedproducten. ook in nederland zijn vóór 1985 tientallen hemofiliepatiënten op deze manier met hiv geïnfecteerd, doordat er nog geen hiv-test beschikbaar was. het risico op verticale transmissie van hiv van moeder naar kind is 15-25% wanneer geen borstvoeding wordt geven, maar loopt op tot 40% als dat laatste wel gebeurt. de meeste kinderen raken geïnfecteerd in de laatste fase van de zwangerschap, vooral tijdens de partus. ook hier speelt de hoogte van de virale load weer een belangrijke rol. door alle zwangere vrouwen vroeg op hiv te testen en ten minste in het derde trimester van de zwangerschap met haart te behandelen (zie verder), is in de westerse wereld het risico op verticale transmissie tot minder dan 0,1% teruggedrongen. de incidentie van hiv-1 in de verschillende risicogroepen is sterk afhankelijk van de geografische lokalisatie en transmissieroute. in centraal-afrika, waar het virus eind jaren zeventig al wijdverbreid was, is heteroseksuele overdracht het frequentst en wordt het virus veel aangetroffen bij jonge mannen en vooral vrouwen. in sommige afrikaanse landen blijkt meer dan 30% van de zwangere vrouwen geïnfecteerd. ook infectie van pasgeborenen door verticale transmissie komt in afrika veel voor. in de verenigde staten, europa en australië komt een hiv-1-infectie vooral voor bij mannen die seks hebben met mannen (msm), ook al neemt het aantal heteroseksuele overdrachten geleidelijk toe. in oost-europa en veel aziatische landen, waaronder china en indonesië, is een snelle uitbreiding van de epidemie gaande, vooral als gevolg van intraveneus drugsgebruik. eind 2008 bedroeg het geschatte aantal in leven zijnde hiv-geïnfecteerden wereldwijd ongeveer 33 miljoen, waarvan het overgrote deel woonachtig was in afrika bezuiden de sahara. in nederland was eind 2010 het aantal in leven zijnde hiv-geïnfecteerden in de landelijke registratie 14.000, maar wordt het werkelijke aantal geschat op ruim 24.000. de belangrijkste transmissieroute bij de nederlandse patiënten is msm-contact (57,2 %), gevolgd door heteroseksueel contact (32%), (ex-)intraveneus drugsgebruik (3,2 %) en bloedproducten (1,5%). hoewel er direct na de ontdekking van hiv optimisme ontstond over het ontwikkelen van een beschermend vaccin, is hier tot op heden nog steeds geen zicht op. kandidaatvaccins bleken wel in staat tot het opwekken van hiv-specifieke immuniteit, maar dit bleek geenszins beschermend. toen uit een veelbelovende studie in 2008 bleek dat er wellicht meer hiv-infecties voorkwamen in de groep met het beoogde vaccin dan in de placebogroep, werden de verwachtingen nog verder getemperd, tabel 13.2 gemiddeld risico op hiv-1-transmissie bij eenmalig contact met een hiv-1-positieve, onbehandelde bron. al bleek er bij een intensieve gecombineerde vaccinatietrial in 2009 voor het eerst sprake van een zwak beschermend effect. goede voorlichting en vermijding van risicogedrag (veilige seks, schone naalden voor intraveneuze drugsgebruikers) blijven daarom vooralsnog de belangrijkste wapens in de strijd tegen de verspreiding van de hivepidemie. seksuele transmissie kan vooral voorkomen worden door goed en consequent gebruik van mannencondooms en ook mannenbesnijdenis blijkt de kans op transmissie te verlagen. geen van deze methoden biedt echter honderd procent bescherming. nieuwe methoden, zoals virucide vaginale crèmes en vrouwencondooms, bleken nog niet effectief. effectieve haart verlaagt ook het risico op seksuele overdracht, al maakt het daarmee condoomgebruik niet overbodig. transmissie via bloed-bloedcontact kan vermeden worden door goede screening van bloedproducten op hiv en het verstrekken van schone naalden aan drugsverslaafden. verticale transmissie kan worden voorkomen door het toedienen van antiretrovirale middelen aan de zwangere vrouw, waarbij in westerse landen ook de pasgeborene nog vier weken antiretrovirale therapie krijgt toegediend als een soort postexpositieprofylaxe (pep). in westerse landen worden daardoor weinig hiv-positieve kinderen meer geboren. in derdewereldlanden is een dergelijke benadering vaak niet mogelijk, maar lukt het dikwijls toch de transmissie terug te dringen door kort voor de bevalling een eenmalige dosis nnrti (nevirapine) toe te dienen. het aantal hiv-geïnfecteerde kinderen loopt hierdoor wel terug, maar de keerzijde van deze korte monotherapie zijn resistentieontwikkeling bij de moeders en de toch hiv-positief geboren kinderen. de resistentieprevalentie tegen nevirapine kan daarbij oplopen tot 60%. de genoemde postexpositieprofylaxe kan ook worden gegeven aan personen die door een incident (prikaccident, onbeschermd seksueel contact) aan hiv zijn geëxposeerd en mogelijk geïnfecteerd. hoewel hiervoor vooral dierexperimenteel bewijs bestaat, lijkt pep effectief als het zo snel mogelijk na het incident wordt toegediend. kernpunten -infecties met ebv, cmv, hiv-1 en t. gondii behoren tot de frequentste oorzaken van gegeneraliseerde lymfadenopathie. -een primaire infectie met ebv of cmv verloopt vaak asymptomatisch (kinderleeftijd) maar leidt op oudere leeftijd vaak tot een klinisch beeld met faryngitis, koorts, lymfadenopathie en atypische lymfocytose (mononucleosis infectiosa). -mononucleosis infectiosa kan ook gezien worden bij primaire infecties met toxoplasma gondii en hiv. -ebv en cmv zijn humane herpesvirussen die persisteren in latente vorm. vooral bij immuungecompromitteerde patiënten kunnen herpesvirussen ernstige systemische infecties of lymfoproliferatieve syndromen (ebv) veroorzaken. -een acute of doorgemaakte infectie met ebv of cmv is aan te tonen met serologisch onderzoek. moleculaire diagnostiek kan worden gebruikt om de hoeveelheid virusreplicatie te monitoren. -hiv is een retrovirus dat via seksueel of bloed-bloedcontact wordt overgedragen en een persisterende infectie veroorzaakt. ook perinatale transmissie komt voor. na de primo-infectie ontstaat een klinische latentieperiode die kan variëren van maanden tot jaren. -hiv-infectie leidt op den duur tot cd4+-t-celdepletie en een ernstige cellulaire immunodeficiëntie met als gevolg levensbedreigende opportunistische infecties en maligniteiten. -hiv-infectie kan worden aangetoond met serologisch onderzoek. de activiteit van de infectie kan worden vervolgd door seriële bepaling van het aantal cd4+-cellen en de hoeveelheid hiv in plasma. -hiv-infectie kan worden behandeld met een combinatie van antiretrovirale middelen (haart). haart leidt tot volledige virusonderdrukking met daarbij uiteindelijk goed herstel van de cellulaire immuniteit. -met behulp van levenslange antiretrovirale therapie kunnen patiënten een normaal leven leiden, met waarschijnlijk een bijna normale levensverwachting. hiv immunopathogenesis and strategies for intervention infectious diseases 3rd infectious mononucleosis principles and practice of infectious diseases clinical virology key: cord-011485-15wtv6bt authors: yang, wenbo; yang, dianlong; gong, shisong; dong, xiaobing; liu, luyao; yu, shengda; zhang, xiaolei; ge, shengxiang; wang, dong; xia, ningshao; yu, duli; qiu, xianbo title: an immunoassay cassette with a handheld reader for hiv urine testing in point-of-care diagnostics date: 2020-05-21 journal: biomed microdevices doi: 10.1007/s10544-020-00494-4 sha: doc_id: 11485 cord_uid: 15wtv6bt currently, most hiv tests are performed with blood samples, or alternatively saliva samples are used for hiv testing. simple hiv tests need to be performed in hospitals or other medical agencies instead of more invasive hiv blood tests. to enable point-of-care (poc) hiv diagnostics, based on a recently developed lateral flow strip for hiv urine testing, a microfluidic immunoassay cassette with a handheld optical reader is developed. based on lateral flow strip with gold colloid reporter, the integrated immunoassay cassette can perform sample introduction, metering, discharging, applying and detection which simplifies hiv testing. an indicator is incorporated into the cassette to guide sample introduction based on color change, and further, the excess test sample is stored inside the sealed cassette to avoid any contamination. the low-cost handheld optical reader can provide a test result within a few seconds, which is useful for simple, sensitive and affordable hiv onsite detection. instead of using normal white leds, a customized back light module embedded with green leds is adopted to illuminate the lateral flow strip with an appropriate working current to achieve optimal performance. compared to the standard lateral flow strips using a benchtop reader, with the disposable immunoassay cassette assisted by the handheld optical reader, more convenient, easier-to-operate, and more affordable hiv urine testing can be achieved in poc diagnostics. acquired immune deficiency syndrome (aids) is still a serious global disease since it cannot be cured completely. for people infected with hiv, aids is a stage when the level of their cd4 + t lymphocytes falls below acritical, for example 200 cells/μl (cheng et al. 2009 ). by 2018, it was estimated that the global number of people living with hiv was~37.9 million and more than two-thirds of them reside in developing countries (world health organization 2019; vos et al. 2016 ). there are common methods for hiv detection based on different principles chen et al. 2016) . using an immunoassay, hiv can be diagnosed by detecting anti-hiv antibody or hiv viral antigen. based on molecular analysis, hiv can be diagnosed by detecting viral nucleic acids. because hiv molecular diagnostic viral testing needs complicated sample processing for nucleic acid extraction and elaborate nucleic acid amplification, it is difficult to perform molecular diagnosis in developing countries where medical infrastructural facilities are lacking (zhao et al. 2019) . among different detection methods based on hiv immunoassay, lateral flow strips are widely used for on-site screening tests, especially in resource-poor settings at poc because of their ease of use, low-cost, simplicity and rapidity (granade et al. 2010) . various types of lateral flow strips have been developed for hiv immunoassays (paredes et al. 2006) . for example, anti-hiv antibody is can be detected in serum samples using lateral flow strips (faulstich et al. 2007 ). in principle, the detection result can be observed by eyes when the gold colloid reporter lateral flow strips are used. to avoid the interference from red blood cells, the raw whole blood sample needs to be centrifuged to obtain the cell-free serum sample. alternatively, an additional filtration pad adjacent to the sample pad can be incorporated into the lateral flow strip to block the red blood cell in lateral flow assay. hiv saliva tests can also be performed with lateral flow strip (delaney et al. 2006 ). compared to hiv blood testing, hiv detection can be implemented much more conveniently with saliva samples. to improve detection sensitivity, up-converting phosphor (ucp) reporter particles have been used to label the hiv antibody and avoid interfering background ). in this case, a companion optical reader is required to perform hiv saliva test with lateral flow strip when fluorescence labeling is adopted, which may partly limit its application field. urine testing is a routine examination in vitro-diagnosis. in fact, because of the superiority with noninvasive collection of samples, urinalysis is becoming a competitive format in poc diagnosis as more diagnostic biomarkers are identified in urine (mahoney et al. 2020; hart et al. 2011 ). for example, as an emerging application of urine analysis, in situ monitoring of activities of daily living (adl) is able to provide systematical information of the body status with continuous multi-parameter measurements of urine in a home setting for poc testing (taramasco et al. 2018 ). traditionally, urinalysis is able to provide not only valuable clinical information for diagnosis of various urologic and renal diseases, but also evidence in asymptomatic patients, for example, with sexually transmitted infections (sti's) (fogazzi and perazella 2010) . in poc diagnosis, widely used urinalysis devices include dip strips or sticks for pregnancy testing, urinary tract infections (utis), and urine test strips for other types of analytes of urine samples, for example, specific gravity, creatinine, glucose, ions, ketones, lactate, nitrite, ph, protein, and uric acid (kingston et al. 2003; suresh et al. 2018) . for high throughput detection, dna microarrays can be used for simultaneous detection of different dna markers in urine (gaber et al. 2017) . to avoid the inconvenience with urine sample storage and transportation, poc diagnosis devices, which can be easily implemented at the clinic or even at home, are highly desired in urine testing. with simple, convenient and easyto-handle operation, poc urinalysis devices developed based on microfluidic or lab-on-chip technology are able to detect infectious diseases or monitor chronic diseases with only modest resources (lepowsky et al. 2017; jalal et al. 2017 ). compared to blood testing requiring comparatively large sample volumes, poc microfluidics-based urine testing is quite promising and attractive for home use on account of the noninvasive nature of urine sample collection (lin et al. 2011) . to effectively control hiv epidemics, sensitive, simple, low-cost and easy-to-use detection methods need to be developed for rapid hiv test in poc settings. in this article, we describe a microfluidic immunoassay cassette with a companion handheld optical reader for hiv test based on urine sample analysis. based on a recently developed gold colloid lateral flow strip available for hiv urine testing, a disposable immunoassay cassette is developed to perform self-assisted urine metering, discharging, applying and detection. a low-cost handheld optical reader is developed to detect the test result from the cassette. once the raw, undiluted urine sample is loaded into the cassette, lateral flow immunoassay can be easily performed by the untrained operators themselves. excess sample will be retained inside the cassette to prevent any environmental contamination. the affordable handheld optical reader with a total component cost of seventy dollars improves the detection sensitivity, and logs, displays and transmits the test result if necessary. the performance of both the cassette and the handheld optical reader is systematically evaluated. experimental results show that successful hiv urine testing can be conveniently performed in poc settings with the test reported here. based on the immune specificity of hiv viral surface antibodies, an easy-to-operate, one-step lateral flow immunoassay was developed for hiv-1 antibody test with urine sample. for each test, the applied volume of urine sample is~80 μl. similar to existing lateral flow strips (qian and bau 2004) , as shown in fig. 1a , the hiv urine test strip (60 mm × 4 mm) is comprised of a backing, sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. on the conjugate pad, staphylococcal protein a (spa) conjugated to gold colloid nanoparticles is stored in dry form after diluted in a blocking reagent with bovine serum albumin (bsa). witnessing the fact of low concentration of biomarkers with urine sample, instead of using the sandwiched immunoassay which normally adopted in hiv blood testing, indirect immunoassay is adopted in hiv urine testing to ensure the detection sensitivity by capturing hiv antibody from urine sample through spa. moreover, to improve the detection sensitivity, large sample volume, e.g., 80 μl is adopted in hiv urine testing. in the test line area of the nitrocellulose membrane, hiv antigens are immobilized to form the capture line. in the control line area, the second antibodies are immobilized to form the verification line. once the urine sample is in contact with the sample pad of the lateral flow strip, it is wicked along the lateral flow strip by capillary force. at most 15 min later, the result becomes readable by observing the test or control line with red color. as shown in fig. 1b , in a predefined sequence based on indirect immunoassay, first the hiv antibodies in the test urine sample bind to the gold colloid particles stored on the conjugate pad through spa, and then they move together along the strip until captured by the test line based on the specific immune bioreactivity between the immobilized hiv antigens and the detected antibodies. finally, the remaining unbound free gold colloid particles are captured by the control line area for assay verification through the binding reaction between spa and the second antibody. the lateral flow strip recently developed by beijing wantai biological pharmacy enterprise co (beijing, china) for hiv urine testing has been evaluated thoroughly and systematically using a large sample bank for clinical trials. it has demonstrated 98.4% of accuracy with the hiv positive subjects without antiretroviral therapy (art), and 100% specificity with hiv negative subjects (beijing wantai 2019). compared to hiv blood testing, as a noninvasive detection method, hiv urine testing can be performed more easily and conveniently, which is suitable for self-test in poc settings. it is well known that hiv saliva test is a commercialized noninvasive detection method. compared to hiv salvia testing, hiv urine testing can be performed with relatively simpler procedure for sample collection and pre-treatment. however, the unfriendly smell from the urine sample could be one of the disadvantages with hiv urine testing. nevertheless, similar to hiv saliva test, hiv urine testing provides another choice for noninvasive hiv poc test. in poc diagnosis, to allow on-site hiv urine tests with the lateral flow strip performed by laymen instead of trained operators for example for home use testing, an immunoassay cassette with the capability of sample metering, discharging and applying was developed. figure 2 depicts the immunoassay cassette equipped with a lateral flow strip. as shown in fig. 2a , the immunoassay cassette consists of three major modules, e.g., sample introduction module on the top, sample metering module in the middle, and detection module on the bottom. the sample introduction module consists of a 30 mm × 20 mm × 6 mm poly(methyl methacrylate) (pmma) loading chamber whose top is partly covered with a pmma layer. as shown in fig. 2a , the urine sample from a normal container can be directly added into the sample introduction module through the loading port. an accessory tape layer on top is used to seal the loading port after sample loading, which reduce the risk of environmental contamination. the sample metering module consists of a 30 mm × 40 mm × 8 mm pmma storage chamber with a vent hole on top. as shown in fig. 2a , in the center of the storage chamber, there has an independent metering chamber with a size of~80 μl. meanwhile, the space around the metering chamber in the storage chamber is used as the overflow chamber to hold the excess urine sample. an absorbent paper layer (nanjing huiyuechi electronic technology company ltd.) is deposited into the overflow chamber to not only absorb any excess urine sample, but also to work as an indicator for sample loading since its color will obviously change from white to red on contact with urine. opposite to the metering chamber, an extruded connection head is fabricated on the bottom of the sample loading chamber to allow urine sample to smoothly enter into the metering chamber. the metering chamber also has an extruded head to establish a flow path to the lateral flow strip once the device is fully assembled. initially, the bottom of the extruded head is sealed with a thin 4 mm × 4 mm paper layer (shenzhen siweituoda technology company ltd.). the specific thin paper layer, which is made from stretched polytetrafluoroethylene (ptfe) with average pore sizes from 0.1 to 1.5 μm, has an average pore density larger than 80%. the specific thin paper layer is able to hold the urine sample inside the metering chamber while allowing air to pass through. the detection module consists of a lateral flow strip, a strip holder, and a sharp needle partly penetrating into the strip from the sample pad. to simplify the process of fabrication, different parts of the cassette were all cut with a laser machine (htc-0906-w80, jinan hantong digital control device enterprise co. ltd., jinan), and then laminated with the double-sided tape (type #9731, 3 m). to reduce the complexity of cassette fabrication and binding, before laser cutting, the pmma sheet was first laminated with the double-sided tape from one side to allow both of them to be cut simultaneously, which was helpful to improve the binding performance. initially, the sample introduction module has been bond with the sample metering module with double-sided tape, and the combined two modules are partly assembled with the detection module through two guiding shafts from both sides (as shown in fig. 2a) . after sample loading, the two combined modules can be conveniently assembled with the bottom detection module by hand. for safety, two doublesided tape layers on both sides are used to combine and fix three modules together after assembling (as shown in fig. 2a ). during assembly, the bottom needle (as shown in fig. 2a ) sitting on the detection module will penetrate through the thin paper layer under the sample metering chamber, which allows the urine sample to be continually imbibed by the sample pad of the lateral flow strip for immunoassay. as shown in fig. 2b , the top window of the detection module is covered by a transparent pmma layer for optical detection. a handheld optical reader (66 mm × 66 mm × 66 mm) with low-cost was developed to read and analyze the test result from the lateral flow strip, which allows the test result to be saved, displayed and transmitted if necessary. in principle, with the handheld optical reader, more sensitive, accurate and consistent test result can be achieved compared to typical eye observation which can be affected by a couple of issues. as shown in fig. 3a , a small-size cmos camera module (openmv3 cam m7, openmv, llc) equipped with an optical lens was used to collect image from the lateral flow strip when it was illuminated by a back light module from the top. since the gold nanoparticles have a peak wavelength of absorbing light around 525 nm, green light from the back light module consisting of green leds (wavelength: 510-525 nm) was adopted to illuminate the lateral flow strip from top to improve the detection sensitivity. a circuit module consisting of a single-chip microcontroller (n76e003at20, nuvoton technology corporation) was used to control the camera module to collect and analyze image, and then the test result was sent to the lcd module for display. a bluetooth module was connected to the single-chip microcontroller for data transmission. to reduce system power-consumption, instead of using an advanced camera, a compact camera module relying on a micro-controller based on arm7 (stm32f407, stmicroelectronics) was adopted, which was able to perform image processing with a built-in function library based on micro python after image collection. especially, for the image collection and processing module, a micro-controller with significantly low power consumption was adopted, which was critical to reduce the system total power consumption. the system currents for idle and working states are 160 ma and 90 ma, respectively. powered by two 1.5 v batteries, the device is able to continually work up to 4 h. figure 3c , d, respectively, depict the optical reader with a partly-and fully-inserted immunoassay cassettes. for the hiv urine test, once the urine sample is applied onto the lateral flow strip, the test result becomes detectable after 15 min. with the companion hand-hold optical reader, rapid, on-site, hiv urine testing with lateral flow immunoassay can be conveniently performed at home or in other poc settings. the total cost of components of the hand-hold optical reader is around seventy dollars, which is beneficial to poc diagnosis. a custom algorithm was developed to analyze the test, control lines and the background of the lateral flow strip to achieve the detection result for hiv urine testing. as shown in fig. 4a , the original image was captured by the cmos camera in the handheld optical reader with the resolution of 640 × 480 pixels. as shown in fig. 4b , an interest area was extracted from the original image for further analysis, and then it was converted into a gay image (fig. 4c) . as shown in fig. 4d , to improve the detection sensitivity, laplacian operator was used to process the gray image to strengthen the boundary between different parts of the lateral flow strip (tian et al. 2020 ). there are three major steps for image processing of the lateral flow strip. similar to the test or control line, a background line is adopted here to represent the background area of the lateral flow strip. because of the limited resolution of the captured image, the test, control and background lines are all regarded as a short line (g (x, y)) with a fixed height of one pixel. first is to search the control line within the image processing domain (fig. 4e) . the location of the control line is identified by analyzing the difference of the mean gray (graymean ) between the control line and its adjacent area. to reduce the time required by image processing, the boundaries of the searching area are defined as y = y c ± n (1 < n ≤ 5). here, y c is the estimated location of the control line. as shown in fig. 4f , gray mean at different locations, for example, y = y c or in between y = y c ± n(1 < n ≤ 5) can be calculated by eq. (1): the control line can be identified by comparing the difference of the mean gray between (y = y c ) and (y = y c ± n(1 < n ≤ 5)) with a predefined threshold when the image processing domain is scanned with different y = y c . here, the threshold was set to 10. within the image processing domain (fig. 4e) , in the x direction, the searching range of image processing domain is within w ¼ w 2 þ n à á − w 2 −n à á , and in the y direction, it is between y ¼ y 0 þ h 2 and y = y 0 + h. in the y direction, the searching range is limited to the lower half part of the domain to reduce the time required for image processing. the second step is to identify the test line. based on the location of the control line, similar algorithm can be used to find the test line within a specific image processing domain. third, the middle area between the control and test lines can be identified as the background line. to compensate the test fluctuation coming from the device or the process of detection, the gray difference between the test and the background lines (relative gray) is adopted as the calibrated result. finally, the detection result for hiv urine testing with lateral flow strip can be achieved by comparing the relative gray of the test line with a predefined threshold. urine samples from healthy donors were collected by national institute of diagnostics and vaccine development in infectious diseases (nidvd) of xiamen university with relevant guidelines and regulations approved by the ethics committee of the nidvd and stored in 1.5 ml collection tubes before use. all fresh urine samples were used within 30 min after collection. hiv serum sample with a high concentration was provided by beijing wantai. the original hiv serum sample was diluted with serum to achieve multiple test samples with different concentrations. hiv antibodies from beijing wantai were spiked in healthy urine to simulate hiv positive urine samples. the original hiv urine sample was diluted with urine to achieve multiple test samples with different concentrations. 3.1 urine metering, discharging and applying with immunoassay cassette as described above, in each test, the urine sample was first loaded into the metering chamber, and then discharged and applied onto the lateral flow strip for immunoassay. first, water was used as the test sample for performance evaluation of metering. in each experiment, approximately 300 μl of water was loaded into the cassette, and after a couple of seconds, the stored water from the metering chamber was weighted with a precise balance (fa1004b, shanghai yoke instrument co. ltd.) to determine its volume based on its density (1 g/cm 3 ). parallel experiments with urine samples were performed. next, experiments for performance evaluation of discharging and applying were also performed. similarly, both water and urine were used as the test samples. in each experiment, approximately 300 μl of water or urine was loaded into the cassette. first, the detection module of the cassette was weighted with a precise balance before sample discharging. secondly, after the sample was discharged into the detection module within a couple of min, the detection module was weighted again with a precise balance. based on the sample density, the volume of the discharged sample can be estimated by comparing the two weights of the detection module before and after sample discharging. figure 5a depicts the measured sample volume respectively from metering or discharging in different experiments. as shown in fig. 5a , it can be seen that either with water or urine, the volume deviation of the metered or discharged sample is within 80 ± 8 μl, which is acceptable for hiv lateral flow immunoassay. as shown in fig. 5b , the indicative absorbent paper works well since after sample loading, its color changes from white to red, which is helpful to guide proper operation. in principle, the gold nanoparticles have a peak wavelength of absorbing light around 525 nm. therefore, to optimize the system performance, different light sources including white led and green led were used to illuminate the lateral flow strip for comparison. to uniformly illuminate the entire detection area of the lateral flow strip for consistent performance, instead of directly using two independent leds as point light sources, a customized 33 mm × 27 mm back light module (shenzhen baohui photoelectricity company ltd.) was adopted. in the back light module, the light from multiple embedded white or green leds enters into a planar light waveguide from the side wall, which is able to provide uniform illumination. in all experiments, the driving current of the back light module was appropriately set to 1.5 ma. standard gold colloid lateral flow strips, instead of the developed cassette, were used to evaluate the performance of the handheld reader itself. hiv serum samples with different concentrations were used in experiments. in each test, 80 μl of hiv serum sample was applied onto the standard lateral flow strip specific for hiv serum detection and incubated for 15 min. figure 6 depicts the experimental results from two back light modules with different colors. error bars come from multiple repeated experiments. the signal amplitude is defined as the signal intensity difference between the test line and the background. as shown in fig. 6 , it can be found that with green leds, optimal performance can be achieved compared to white leds. inserts show photographs of the standard lateral flow strips. as shown in the insert of fig. 6 , a specific number (#) is assigned to each lateral flow strip based on a customized rule to represent the signal intensity of the test line. from #2 to #10, they respectively correspond to low-positive to high-positive. for the described handheld optical reader, low-positive detection close to #3 is the acceptable limit of detection. in fact, it is difficult for a person to locate the test line from the background for #3 low-positive detection just by eyes. therefore, compared to traditional eye observation, the handheld optical reader is helpful to improve the detection sensitivity of hiv urine test with lateral flow strip. as shown in fig. 6 , for both cases, instead of #2, #3 lateral flow strip with low-positive can be successfully detected. compared to white leds, significantly higher signal reading with the test line can be achieved with green leds for different sample concentrations (#3, #5, and #10 in fig. 6 ), which demonstrate that higher detection sensitivity can be achieved with green leds. therefore, a customized back light module with green light is adopted in the handheld reader. since the detection is performed in an enclosed box where the lateral flow strip is illuminated with a green lighting source, appropriate lighting conditions with a properly set driving current is critical to the detection performance. for comparison, experiments with different driving currents were performed. standard gold colloid lateral flow strips, rather than the cassette, were used to evaluate the performance of the handheld reader itself. hiv serum samples with different concentrations were used in experiments. in each test, 80 μl of hiv serum sample was applied onto the standard lateral flow strip and incubated for 15 min. figure 7 depicts the experimental results for three cases respectively with low (0.5 ma), medium (1.7 ma) and high (10.2 ma) driving currents. error bars come from multiple repeated experiments. as shown in fig. 7 , it can be found that with a medium driving current (1.7 ma), optimal performance can be achieved compared to others. as shown in fig. 7 , when the driving current is too low or too high, #3 lateral flow strip with low-positive cannot be successfully detected with acceptable credit. inserts are the recovered photographs of the standard lateral flow strips with medium-positive (#5) respectively with different driving currents, which are taken by the handheld optical reader with green light illumination. it has been demonstrated that optimal performance can be achieved with a medium driving current when the lateral flow strip is properly illuminated without saturation. it was found that comparable performance can be achieved when the driving current was between 1.5 ma and 1.8 ma. therefore, a medium driving current (1.7 ma) was applied in the handheld reader. the performance of the hiv immunoassay cassette was evaluated with hiv urine sample. hiv urine samples with different concentrations were used in experiments. in each test, more than 120 μl urine sample was loaded into the described cassette (80 μl was used in the test), and the lateral flow strip was detected with the described handheld optical reader after 15-min incubation, once the cassette was fully assembled. for comparison, parallel tests were performed with standard lateral flow strips. a benchtop immunoassay gold colloid reader (rtr-g100, xiamen innovax biotech co., ltd) was used to detect the standard lateral flow strip after 15-min incubation once 80 μl of urine sample was applied. figure 8 depicts the fig. 5 experimental results with metering and discharging: (a) metering or discharging sample volume respectively with water or urine sample; (b) two photographs of the immunoassay cassette before and after sample loading fig. 6 experimental results of lateral flow strip illumined with green or white light. inset shows photographs of standard lateral flow strips taken by a smartphone camera fig. 7 experimental results detected by the handheld optical reader with different driving currents. inset shows recovered photographs of standard lateral flow strips taken by the handheld optical reader experimental results for both cases respectively with the described cassette and the standard lateral flow strip. error bars come from multiple repeated experiments. as shown in fig. 8 , for both methods, both low (#3.5) and medium (#5) positive samples can be successfully detected and differentiated from the negative one. as shown in fig. 8 , acceptable detection sensitive with the described method can be achieved. inserts are the photographs for both cases with detection to negative, the low (#3.5) and medium (#7) positive samples. for each insert (negative, #3.5, and #7), the left photograph is from the described cassette, while the right one is from the standard lateral flow strip. it has been demonstrated that comparable performance between the described method and the standard lateral flow strip with a benchtop reader can be achieved. however, both the cost and the size of the described handheld optical reader are significantly less than those of the benchtop reader, which is more suitable for poc diagnosis at resource-poor settings. we describe a disposable, easy-to-operate microfluidic immunoassay cassette for hiv urine tests with a handheld optical reader for use at the point of care including home use. the hiv immunoassay cassette consists of three different modules respectively for sample introduction, metering and detection. the sample introduction module provides a user-friendly interface for users to conveniently add urine sample into the cassette. the metering module can adequately control the test sample volume with acceptable deviation. the detection module is able to perform hiv urine detection with an integrated lateral flow strip that uses gold colloid reporter. once the cassette is fully assembled, the metering chamber will be fluidically connected from its bottom by a sharp needle from the detection module, which allows the urine sample to be continually aspirated by the sample absorbing pad of the lateral flow strip for immunoassay. the whole operation can be performed just by hand without any instrument or special equipment. after 15-min incubation, the cassette can be inserted into a handheld optical reader to detect the test result within 5 s. a custom image processing algorithm was developed to search and locate the test and control lines of the lateral flow strip. the total cost of components of the handheld optical reader is around seventy dollars, which is suitable for poc diagnosis or home test. compared to the traditional test by just adding the urine sample to a lateral flow strip, there have a couple of advantages with the developed method. first, with the microfluidic cassette, the allowable test sample volume could be potentially increased for higher sensitivity. second, there has no need for any sample metering tools since there has a metering chamber within the microfluidic cassette. third, the risk of environmental contamination (including hiv sample contamination and even spread of the unfriendly smell from urine sample) can be reduced since both the lateral flow strip and the excess sample are sealed inside the cassette. fourth, the detection sensitivity and accuracy can be improved by combining the cassette with the handheld optical reader compared to direct eye observation which could be unfavorably affected by the operator and the environment. the performance for both the hiv immunoassay cassette and the handheld optical reader has been systematically evaluated. consistent metering, discharging and applying ensure adequate and reproducible test sample volumes. to improve the system performance, green light instead of white light was adopted to illuminate the lateral flow strip. a proper working current for the green back light module was chosen to ensure the system performance. hiv antibody-spiked urine samples were successfully detected by the immunoassay cassette with a handheld optical reader. it has been demonstrated that comparable results can be achieved with the described method comparing to the standard lateral flow strip with a benchtop reader. however, simple, easy-to-operate, low-cost, and integrated hiv urine testing, which is highly desired for poc diagnosis or even home test, can be conveniently achieved with the described method based on a disposable cassette and a handheld, affordable optical reader. user's manual: diagnosis kit for hiv-1 antibody test with urine sample a rapid, self-confirming assay for hiv: simultaneous detection of anti-hiv antibodies and viral rna enhancing the performance of a point-of-care cd4+ t-cell counting microchip through monocyte depletion for hiv/aids diagnostics performance of an oral fluid rapid hiv-1/2 test: experience from four cdc studies developing rapid mobile poc systems. part 1: devices and applications for lateralflow immunodiagnostics the urinary sediment: an integrated view, 3 rd edition comparison between real-time polymerase chain reaction and dna-microarray in detection and identification of mycobacterium species rapid detection and differentiation of antibodies to hiv-1 and h i v -2 u point-of-care oral-based diagnostics paper-plastic hybrid microfluidic device for smartphone-based colorimetric analysis of urine shelf life'of trichomonas vaginalis based assays for urine analysis urine analysis in microfluidic devices a timer-actuated immunoassay cassette for detecting molecular markers in oral fluids point-of-care urinalysis with emerging sensing and imaging technologies rapid hiv testing: a review of the literature and implications for the clinician analysis of lateral flow bio-detectors: competitive format finger-actuated, self-contained immunoassay cassettes a portable, integrated analyzer for microfluidic -based molecular analysis non-invasive paper-based microfluidic device for ultra-low detection of urea through enzyme catalysis a novel low-cost sensor prototype for nocturia monitoring in older people medical imaging and diagnosis of subpatellar vertebrae based on improved laplacian image enhancement algorithm global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: a systematic analysis for the global burden of disease study nucleic acid testing and molecular characterization of hiv infections publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest the authors declare that they have no conflict of interest. key: cord-011457-hqxybv1k authors: kirui, james; freed, eric o. title: generation and validation of a highly sensitive bioluminescent hiv-1 reporter vector that simplifies measurement of virus release date: 2020-05-19 journal: retrovirology doi: 10.1186/s12977-020-00521-5 sha: doc_id: 11457 cord_uid: hqxybv1k background: the continued persistence of hiv-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. here, we used nanoluc, a small and extremely bright luciferase protein, to develop an hiv-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. results: the reporter virus encodes a gag protein containing nanoluc inserted between the matrix (ma) and capsid (ca) domains of gag, thereby generating virus particles that package high levels of the nanoluc reporter. we observe that inserting the nanoluc protein within hiv-1 gag has minimal impact on gag expression and virus particle release. we show that the reporter virus recapitulates inhibition of hiv-1 particle release by gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-b methyl ester. conclusion: these results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small molecules that promote or inhibit hiv-1 particle production. hiv-1 remains a major global public health challenge despite the advances made in our understanding of its replication and in the development of antiviral drugs. the hiv field continues to require innovative research tools and techniques to provide new insights on virushost interactions that can guide the development of novel therapies. reporter viruses are an example of innovative tools that have been used to study hiv-1 replication. these viruses are engineered to carry reporter genes that enable the detection and quantification of virus infection and replication. an early hiv-1 reporter virus encoded a firefly luciferase gene in place of the nef gene [1] . several other hiv-1 reporter viruses have been developed using this approach [2, 3] . with this strategy, the reporter virus infects target cells and expresses the reporter protein, allowing for detection and quantification of virus infection. however, because the reporter protein is not packaged into progeny virions, the reporter cannot be used to detect virus released from the cell. another strategy that has been used to generate hiv-1 reporter virus involves inserting the reporter gene into the gene encoding the structural protein gag, often between the matrix (ma) and capsid (ca) domains but in other regions of gag as well [4] [5] [6] [7] . fluorescent reporter viruses made using the latter strategy have been used to detect viral transfer to target cells. a dual reporter virus encoding both a fluorescent protein-tagged gag and a second fluorescent protein in place of nef was used to study dynamics of hiv-1 replication in single cell infections [8] . the hiv-1 reporter viruses available today are highly useful tools for studying the early stages of the hiv-1 replication cycle. the early phase of the virus replication cycle begins with virus attachment and entry and ends with integration of the newly synthesized viral dna into the host cell genome; the late phase begins with viral gene expression and culminates in virus release and maturation. viruses that express a reporter gene (e.g., luciferase or gfp) in place of nef are suitable for studying the early steps of hiv-1 infection leading up to viral gene expression but not later steps. conversely, viruses with the reporter protein embedded in the gag protein can be used to study the late stages of hiv-1 replication. gag-gfp virus vectors, for example, have been used to monitor gag intracellular trafficking [5] , and a gag-yfp vector has been used to quantify virus release from cells [7] . additional strategies have been devised to engineer reporter viruses that carry the reporter protein, allowing quantification of released virus. these include co-packaging a vpr-gfp fusion protein with hiv-1 gag during virus assembly, leading to the production of viruses containing vpr-gfp [9] . another is the expression of a membrane-anchored gaussia luciferase upstream of the hiv-1 nef gene resulting in the labeling of the virus envelope with the reporter protein [10] . to enable simple and highly sensitive measurement of virus release from transfected cells, we generated hiv-1 reporter viruses in which nanoluciferase (nanoluc) was inserted between the ma and ca domains of gag (gag-inanoluc). nanoluc is smaller (~ 19 kda) and brighter (~ 150-fold) than firefly (~ 61 kda) or renilla (~ 36 kda) luciferases [11] . we demonstrate that the hiv-1 gag-inanoluc vector releases virus particles at similar levels to the wt hiv-1 molecular clone, enabling facile and highly sensitive detection of virus particle production from transfected cells. we further demonstrate the utility of the hiv-1 gag-inanoluc vector as a functional tool to study hiv-1 release by using it to recapitulate disruption mediated by gag mutations, a small-molecule inhibitor, and expression of the restriction factor tetherin. we show that although the infectivity of the gag-inanoluc reporter virus is impaired, infectivity can be rescued by complementing with the wt hiv-1 molecular clone. these results highlight the potential of gag-inanoluc as a tool for high throughput screening of host factors and compounds that specifically target the late stages of the hiv-1 replication cycle. to generate an hiv-1 reporter virus that enables quantification of the reporter protein directly from virus supernatant, we employed the previously reported strategy [4, 5, 7] of introducing the reporter gene between the ma and ca domains of gag, a location shown to tolerate genetic insertions with minimal effects on gag protein expression and processing [4] . the nanoluc gene was introduced between ma and ca domains of hiv-1 gag flanked by pr cleavage sites at the n-terminus or c-terminus, or both termini of nanoluc or not flanked by any pr cleavage site (fig. 1a) . upon pr-mediated gag cleavage, the resulting nanoluc products generated from these vectors are the nanoluc protein and ma-nanoluc, nanoluc-ca and ma-nanoluc-ca fusion proteins, respectively (fig. 1a) . to assess the effect of the nanoluc insertion into hiv-1 gag on gag protein expression and pr-mediated gag cleavage, we transfected the hiv-1 gag-nanoluc vectors into hek293t cells and after 48 h, lysed the cells and pelleted virus from the supernatant. we performed western blot analysis to evaluate the expression of hiv-1 gag-nanoluc fusion proteins in the cells and the pelleted virus. we observed no adverse effect on gag expression and processing and, importantly, the expected gag pr cleavage products were generated, including some cleavage intermediates (fig. 1b) . we also measured nanoluc activity from the cell lysate and virus supernatant and observed that all the vectors yielded robust nanoluc activity; i.e., up to 2.0x10 7 relative light units (rlus) (fig. 1c) . finally, we calculated virus release efficiency (vre) from the same samples and found that release efficiency was modestly reduced (< twofold) compared to that of wt gag but similar to the gag-igfp construct (fig. 1d ). vre is calculated as the ratio of virion-associated p24 ca to total gag (i.e., cellular p24 ca and pr55gag and viral p24 ca), normalized to wt vre, which is set to 100. because all four gag-nanoluc constructs were similar in terms of gag expression and nanoluc activity and displayed only modest (< twofold) differences in vre, we chose to proceed with the pnl4-3 gag-inanoluc vector for further characterization. because pnl4-3 gag-inanoluc has pr cleavage sites on both sides of the nanoluc protein, which allows for complete processing of the gag-inanoluc protein into the individual gag domains (as is the case for wt pnl4-3), it is the most representative of the native gag. to assess the sensitivity in the detection of virus release using the hiv-1 gag-inanoluc vector, we transfected hek293t cells with either a nanoluc expression vector, the wt hiv-1 molecular clone pnl4-3, or decreasing amounts of the hiv-1 gag-inanoluc vector (i.e. 1.0, 0.5, 0.25, 0.125 and 0.0625μg). at 48 h posttransfection, we lysed the cells and purified virions from the supernatant, analyzed gag levels by western blot (fig. 2a) , and measured nanoluc activity from the cell lysates and supernatants (fig. 2b) . we were able to detect nanoluc signal under all conditions tested, including at the lowest dna input at which virion-associated gag was undetectable by western blot. pnl4-3 gag-inanoluc vector-transfected cells produced significantly higher levels of nanoluc activity in both the cell lysate (> tenfold) and supernatant (> 1000-fold) relative to the nanoluc expression vector control. this implies that the nanoluc activity in the supernatant is derived from the nanoluc protein released with the hiv-1 gag during virus release. we also measured rt activity (fig. 2c ) and p24 protein levels (fig. 2d) in the virus supernatant and correlated both with supernatant nanoluc activity (fig. 2e , f ). we observed that the supernatant nanoluc activity was positively correlated with rt activity and p24 abundance, further reinforcing the specificity of the assay. we generated virus using either wt pnl4-3, the hiv-1 gag-igfp or the hiv-1 gag-inanoluc vectors by transfecting them into hek293t cells and collecting the supernatants containing the progeny virions at 48 h posttransfection. we quantified the relative amounts of virus in the supernatant by rt activity (fig. 3a) . we observed that rt activity in the supernatants of cells transfected with the hiv-1 gag-igfp and hiv-1 gag-nanoluc vectors was about twofold less than that of supernatants from cells transfected with wt pnl4-3. to test the infectivity of the virions produced from the hiv-1 gag-nanoluc vectors, we infected tzm-bl cells with the rt-normalized virus supernatants and measured the infectivity by quantifying the hiv-1 tat-driven firefly luciferase activity (fig. 3b) . we observed that the hiv-1 gag-nanoluc viruses were approximately tenfold less infectious than the wt virus while the gag-igfp virus was only about twofold less infectious than wt virus. we also transfected the supt1 t cell line with the hiv-1 gag-nanoluc vectors and monitored virus replication kinetics over several days and observed that replication was significantly impaired compared to the wt hiv-1 (data not shown). we generated viruses using the pnl4-3 gag-inanoluc vector complemented with different ratios of the wt hiv-1 molecular clone pnl4-3 and tested their infectivity by measuring the hiv-1 tat-driven firefly luciferase activity in tzm-bl cells. we observed that infectivity of the viruses generated with the pnl4-3 gag-inanoluc vector was rescued when complemented with the wt pnl4-3 vector. the infectivity increased with increasing pnl4-3 to pnl4-3 gag-inanoluc ratio; at ratios above 2:1 the infectivity was at wt hiv-1 levels (fig. 3c) . we also measured nanoluc activity from the same infected tzm-bl cells and observed that the virus generated with the pnl4-3 gag-inanoluc vector alone was able to express nanoluc activity in the target cell despite being poorly infectious. the nanoluc expression increased with increasing ratio of wt pnl4-3 to pnl4-3 gag-inanoluc vector used in generating the gag-inano-luc viruses. however, we observed that with higher ratios of wt pnl4-3 to pnl4-3 gag-inanoluc i.e., 5:1 and 10:1, the nanoluc expression decreased (fig. 3d) , presumably due to lower amounts of gag-inanoluc genome transduced into target cells. we analyzed purified virus particles generated using the gag-inanoluc proviral vectors either alone or complemented with the wt proviral clone by electron microscopy and we observed that the morphology of gag-inanoluc virus particles was abnormal; specifically, the viral cores displayed a spherical shape as opposed to the canonical cone shape (data not shown). consistent with the rescue of particle infectivity, normal, conical cores were observed when virus was generated by co-expressing the gag-inanoluc vector with wt pnl4-3 (data not shown). the rescue of virion morphology by co-expressing gag fusion proteins with wt gag has also been observed previously [4] . to examine the utility of the hiv-1 gag-inanoluc vector in functional assays for virus release, we constructed versions of the vector that lacked the ptap motif in the p6 domain of the hiv-1 gag protein (hiv-1 gag-inanoluc-ptap-) and the vpu gene (hiv-1 gag-ina-noluc-delvpu) by cloning the inanoluc cassette into previously reported ptap-and delvpu hiv-1 molecular clones [12, 13] . the p6 domain of hiv-1 gag is required for virus release [13, 14] because of its interaction with the escrt machinery [15] [16] [17] [18] . vpu is also required for hiv-1 release in the presence of the restriction factor tetherin (also known as bst-2), which blocks release of virions by tethering them to the plasma membrane [19] . vpu counteracts tetherin by mechanisms involving both [20, 21] we transfected hek293t cells with the wt, ptap-and delvpu versions of the hiv-1 gag-inanoluc vector with or without varying amounts of tetherin expression vector. at 48 h post-transfection, we measured nanoluc activity in the cell lysates and supernatants. we observed a twofold decrease in the nanoluc activity in the supernatant of cells transfected with the ptap-vs. the wt vector, but, as expected, no decrease in nanoluc activity in the cell lysates. likewise, co-transfection with a tetherin expression vector, but not an empty vector control, caused a four to tenfold decrease in nanoluc activity in the supernatant of cells transfected with the delvpu vector. the decrease in supernatant nanoluc activity was proportional to the amount of tetherin vector transfected (fig. 4a, b) . we performed western blot analysis of the cell lysates and the pelleted virions to analyze hiv-1 gag expression. we observed that virus release measured by virion-associated p24 levels corresponded with the nanoluc activity. finally, we tested the utility of the hiv-1 gag-inanoluc vector to detect impaired virus release induced by treatment of virus-producer cells with amphotericin b methyl ester (ame), a compound that inhibits hiv-1 particle production [22] . we transfected hek293t cells with the hiv-1 gag-inanoluc vector and at 24 h post-transfection treated the cells with either vehicle or increasing amounts (5μm or 10μm) of ame. at 24 h post-treatment, we collected the supernatant and measured nanoluc activity. we observed a decrease in supernatant but not cell-associated nanoluc activity in the presence of ame but not vehicle control. again, the decrease in supernatant nanoluc activity corresponded with reduced virion-associated p24 measured by western blot analysis. these results demonstrate that the gag-inanoluc vector provides a highly sensitive and quantitative tool for measuring the effects of gag mutations, host cell restriction factors, and small-molecule inhibitors on hiv-1 particle assembly and release. almost four decades since the emergence of hiv-1 and the aids pandemic, the virus is still a global public health concern. numerous technical innovations have led to detailed insights into the mechanism of hiv-1 replication. however, there still remain a number of knowledge a b c d fig. 4 hiv-1 gag-inanoluc vector recapitulates inhibition of hiv-1 particle release. a nanoluc activity in lysates and supernatants from hek293t cells transfected in 12-well plates with 1.0μg of either pnl4-3 gag-inanoluc wt, pnl4-3 gag-inanoluc ptap-or pnl4-3 gag-inanoluc delvpu and either 0, 0.1μg or 0.2μg ha-tetherin (bst2). nanoluc activity in lysates and supernatants was measured at 48 h post-transfection and normalized to that of pnl4-3 gag-inanoluc wt. b western blot analysis of hiv-1 gag (upper) in lysates and supernatant from a and ha-tetherin (lower) in cell lysates. c nanoluc activity in lysates and supernatants from hek293t cells transfected with 1.0μg of pnl4-3 gag-inanoluc and treated with either 5μm or 10μm ame or vehicle at 48 h post-transfection. ame treatment was done at 24 h post-transfection following replacement of the growth media. d western blot analysis of hiv-1 gag in lysates and supernatant from c. error bars ± sd, n = 3 independent experiments for a and c. *, p < 0.05 by one-way anova in panels a and c compared to wt pnl4-3 nanoluc activity and the 0 ame control nanoluc activity, respectively gaps, particularly regarding the late steps in the virus replication cycle, which are less amenable to high throughput analyses than the early steps. in this study, we set out to develop an hiv-1 reporter virus that would simplify and facilitate highly sensitive and quantitative measurements of the late stages of the virus replication cycle. we describe the generation of the hiv-1 gag-inanoluc vector that expresses the nanoluc reporter protein inserted into the viral gag protein between the ma and ca domains. the gag-nanoluc fusion protein is expressed in the cell and released at similar levels to wt gag, thereby enabling simple yet highly sensitive quantification of viral gene expression and virus particle production by measurement of the nanoluc reporter protein bioluminescent activity in the cell lysates and supernatants. importantly, we demonstrated that detection of virus release using the gag-inanoluc system correlates with detection using current assays that measure supernatant rt activity or p24 abundance, and that it functionally recapitulates inhibition of virus release by gag ptap mutation, tetherin expression and the small molecule ame. because of the highly sensitive nature of nanoluc detection, we hypothesize that the gag-inano-luc virus will also be useful for measuring the binding of hiv-1 particles to target cells. the gag-inanoluc vector reported here joins a number of other hiv-1 reporter vectors that are available to the field; these include gag-fusion reporter vectors [4, 5, 7, 9] , nef-substituted reporter vectors [1] [2] [3] 9] and virus envelope/membrane-anchored reporter vectors [10] . the nef-substituted reporter vectors have the advantage of being replication competent, although with lesser efficiency than wt hiv-1. however, because the reporter protein is expressed only in target cells following infection and not packaged into virus particles, these vectors are suitable for detecting infection in target cells but not for measuring virus release directly from the supernatant. on the other hand, gag-fusion reporter vectors are able to generate viruses that package the reporter protein thus allowing the quantification of virus release from cells directly from the cell culture media. compared to reporter viruses generated by co-packaging vpr fusion proteins with hiv-1 gag, the gag-inano-luc vector provides a higher sensitivity of detection because the nanoluc reporter is packaged into virus particles at higher amounts than a vpr-fused reporter, i.e., ~ 2000-3000 gag molecules per particle vs. ~ 10-30 vpr molecules per particle [23] [24] [25] . the high number of nanoluc proteins that are packaged into each virus particle, coupled with the inherent brightness of the nano-luc substrate, results in a high sensitivity of detection of virus particles in the supernatant. this allows the study of virus release dynamics at very low concentrations of the vector and thus minimizes possible cytotoxicity caused by expressing exogenous proteins and makes the gag-inanoluc vector a suitable tool for functional and high-throughput assays measuring virus particle production. the high sentivity of the gag-inanoluc system may lead to the detection of non-particle-associated gag-inanoluc, potentially dampening the magnitude of inhibition of virus release in the presence of mutations or inhibitors relative to that calculated using less sensitive methods like p24 western blotting or rt assay (e.g., see fig. 4 ). this phenomenon can be mitigated by optimizing transfection methods and the duration of transfection experiments to minimize non-specific release of nonparticle-associated gag-inanoluc. several gag-reporter virus vectors have been reported, most of which use fluorescent reporter proteins [4, 5, 8] ; these have primarily been used to study intracellular gag trafficking dynamics and as markers of infection in target cells and not for quantification of virus particle release from producer cells. the reason for this is that fluorescent reporter proteins generally have low signal-to-background ratios due to inefficiency of the photodetector in discriminating between excitation and emission photons and also the presence of other weak fluorophores in biological samples and cell culture media [26] which renders them less suitable for quantification of released virus particles and studying virus release dynamics. nonetheless, a gag-reporter virus vector expressing enhanced yellow fluorescent protein (eyfp) was developed for use in quantification of virus release [7] . eyfp was found to be more suitable for this purpose than enhanced green fluorescent protein (egfp) and enhanced cyan fluorescent protein (ecfp) as it had higher signal-to-background ratio. because the gag-inanoluc reporter vector encodes the bioluminescent nanoluc protein, it is more suitable for quantification due to the high signal-to-background ratio of luminescence assays compared to fluorescence assays. because luminescence relies on chemical excitation, as opposed to excitation by photons from an input light source to generate the signal i.e. emission photons, luminescence assays have very low background and hence high signal-to-background ratio. this high signal-to-background ratio results in increased sensitivity that can be as high as 1000-fold more than fluorescence assays [26] . due to the higher signal-to-background ratio of luminescence assays, coupled with the increased brightness of the nanoluc reporter, the gag-inanoluc vector can be used at very low concentrations and exhibits a wider dynamic range. recently, an hiv-1 nanoluc reporter virus vector developed by inserting the nanoluc gene upstream of the nef gene was reported by ventura et al. [27] . this vector generates replication-competent hiv-1 nanoluc reporter virus and was used to study hiv-1 replication dynamics in humanized mice during anti-retroviral therapy (art). our nanoluc vector generates virus particles that package the nanoluc reporter, enabling the quantification of released particles directly from the supernatant, making it a suitable tool for studying virus release. gag-fusion reporter vectors are generally impaired in their infectivity [4] . we found that the gag-inanoluc vector was similarly poorly infectious. we also observed that the gag-igfp virus is more infectious than the gag-inanoluc virus, despite the larger molecular mass of gfp relative to nanoluc. we speculate that the effect of gag insertions at the ma-ca junction on virus particle infectivity is not dependent on size alone but possibly also on how the insertion affects gag conformation and processing. as is the case for other gag fusion reporters [4, 7] , we demonstrated that the infectivity of particles generated with the gag-inanoluc vector can be rescued by complementing it with the wt hiv-1 molecular clone. we also observed that the gag-inanoluc virus could express nanoluc reporter in the target cell at 48 h following infection despite being poorly infectious. we hypothesize that the nanoluc activity in the target cell infected with the gag-inanoluc virus could be a result of packaged nanoluc in the infecting virus particles as well as newly synthesized nanoluc following infection. hiv-1 particle release is commonly measured by rt activity, p24 elisa or p24 western blotting assays. these assays are relatively insensitive, laborious, expensive and time consuming, and they often involve multiple steps that can introduce errors that can compound at each step to significantly affect the final results. the hiv-1 gag-inanoluc vector allows for a single-step assay to measure virus particle production and is significantly more sensitive and cheaper compared to other assays. accordingly, this vector provides a valuable tool for studying the late stages of hiv replication, in particular in the context of high-throughput screening for late-acting cellular factors that facilitate or restrict virus release or for inhibitor screening. previous studies have performed high-throughput screening for host factors affecting hiv-1 particle production by harvesting virus particles released from the screening/producer cells to infect target cells and measuring particle infectivity [28] [29] [30] . while this approach has generated many significant findings, a limitation is that the assay readout is the infectivity of the released particles rather than the number of virus particles per se, even though the two can often be correlated. effects on particle assembly/release therefore cannot be distinguished from effects on infectivity. the hiv-1 gag-inanoluc reporter vector simplifies virus production screening by eliminating the infection step because measurement of particle production can be performed directly from the producer-cell supernatant. this reduces the number of steps in the screening protocol and minimizes opportunities for errors and experimental variability, therefore making the hiv-1 gag-inanoluc reporter vector a valuable tool for functional studies of virus release and high-throughput screening for cellular factors and small molecules that affect hiv-1 particle production. the full-length hiv-1 molecular clone pnl4-3, a gfpexpressing hiv-1 molecular clone (pnl4-3gag-igfp), an hiv-1 molecular clone lacking the ptap motif (pnl4-3ptap-), a vpu-deleted hiv-1 molecular clone (pnl4-3delvpu) and an ha epitope-tagged tetherin expression plasmid (ha-tetherin) were used in this study [5, 12, 13, 19, 31] . the nanoluc gene sequence was amplified by pcr from puas-nluc (addgene plasmid no. 87696) although the origin of the sequence is from pnl1.1 (promega, madison, wi) [32] . the hiv-1 gag-nanoluc vectors were constructed by inserting the nanoluc gene between the ma and ca domains of gag flanked or not flanked by protease (pr) cleavage sites by overlap pcr. the pcr product generated encompassed the 5'utr-ma-nanoluc-ca with or without the pr cleavage sites sqnypivq flanking the nanoluc sequence. where the sqnypivq sequence was duplicated, synonymous changes were made on the dna sequence to avoid duplicating the dna sequence. the pcr product was then cloned in the hiv-1 molecular clone pnl4-3 via the bsshii and spei restriction sites. hek293t, hela and tzm-bl cells were cultured in dulbecco's modified eagle's medium (dmem) with l-glutamine (gibco), supplemented with 10% (vol/vol) fetal bovine serum (fbs), 100u/ml penicillin and 100μg/ml streptomycin at 37 °c and 5% co 2 . tzm-bl are heladerived indicator cells that express luciferase upon infection by hiv-1 [33] . supt1 cd4 + t cells were cultured in roswell park memorial institute (rpmi) 1640 medium with l-glutamine (corning, corning, ny) supplemented with 10% (vol/vol) fbs, 100u/ml penicillin and 100μg/ml streptomycin. pooled hiv-1 patient serum (hiv-ig) was obtained from the nih aids reagent program. hek293t cells were plated in 12-well plates and transfected with proviral plasmids using lipofectamine 2000 (thermo fisher scientific, waltham, ma) according to the manufacturer's protocol. at 48 h post-transfection, virus particles were purified by filtering the supernatant through a 0.45μm filter and pelleted by ultracentrifugation. cells and virion-containing pellets were resuspended in lysis buffer [(30 mm nacl, 50 mm tris-hcl, ph 7.5, 0.5% triton x-100, 10 mm iodoacetamide, complete protease inhibitor cocktail (roche applied science)]. 20ul of the cell-and virion-associated proteins were separated on 12% polyacrylamide gels by sds-page and the gag proteins detected by western blotting using hiv-ig antibodies. the vre was calculated as the amount of virion-associated p24(ca) as a percentage of the total (cell-and virion-associated) p24(ca) and pr55gag. where the virus release inhibitor amphotericin-b methyl ester (ame) [22] was used, hek293t cells plated in a 12-well plate were transfected with the proviral plasmids as outlined above. the cell media were changed at 24 h post-transfection and replaced with growth media containing ame and virus particles purified at 48 h post-transfection. viruses were generated by transfecting 2.5μg of proviral hiv-1 vectors into 0.5 × 10 6 hek293t cells in 6-well plates. the virus-containing supernatants were collected at 48 h post-transfection and assayed for reverse transcriptase (rt) activity as described [34] . the supernatants were used to infect 1.0 × 10 4 tzm-bl cells per well in a 96-well plate, the volumes of the supernatants used for infection were normalized by rt activity. at 48 h post-infection, the cells were lysed in passive lysis buffer (promega, madison, wi), and the luciferase signal was measured using britelite plus (perkinelmer, waltham, ma). infectivity is defined as the amount of luciferase activity from the lysates of tzm-bl cells infected with rt-normalized virus supernatants [34] . 3.0 × 10 6 supt1 cells were transfected with 3.0μg of hiv-1 proviral vector dna. the cells and the dna were mixed in 300μl of 0.8 mg/ml deae-dextran solution and incubated for 15 min at 37 °c. the cells were washed in 4 ml of stbs solution (25 mm tris-hcl [ph 7.4], 0.6 mm na 2 hpo 4 , 5mmkcl, 140mmnacl, 0.7 mm cacl 2 , 0.5 mm mgcl 2 ), pelleted by centrifugation, resuspended in 3 ml of rpmi media (with l-glutamine and supplemented with 10% fbs, 100u/ml penicillin and 100μg/ml streptomycin) and placed in culture. the transfected cells were split every other day starting from day 3 post-transfection and aliquots of the supernatants were collected for rt activity and nano-luciferase activity assays. to measure nano-luciferase activity, 15μl of cell lysates and virus supernatant (passed through a 0.45μm filter) from cells transfected with the hiv-1 gag-inanoluc vectors were diluted 1:2 in cell lysis buffer. luciferase activity was measured using the nano-glo assay system (promega, madison, wi); the nano-glo luciferase assay substrate was mixed with the nano-glo luciferase assay buffer at a 1:50 ratio and equal volume of the mix was added to the samples. the samples were incubated at room temperature for 2-3 min and luminescence was detected in a plate reader (perkin elmer wallac microbeta 1450). the concentration of hiv-1 p24 in culture supernatant samples was determined by the aids and cancer virus program using their in-house hiv-1 p24 antigen capture immunoassay. distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferaseencoding viruses use of a novel human immunodeficiency virus type 1 reporter virus expressing human placental alkaline phosphatase to detect an alternative viral receptor vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative sequence of human immunodeficiency virus type 1 (hiv-1) gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged hiv-1 fluorescent protein inserts in between nc and sp2 are tolerated for assembly, release and maturation of hiv with limited infectivity a simple fluorescence based assay for quantification of human immunodeficiency virus particle release single-cell and single-cycle analysis of hiv-1 replication visualization of the intracellular behavior of hiv in living cells a novel hiv-1 reporter virus with a membrane-bound gaussia princeps luciferase engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate a novel gene of hiv-1, vpu, and its 16-kilodalton product freed eo. p6gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release overexpression of the n-terminal domain of tsg101 inhibits hiv-1 budding by blocking late domain function tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding hiv-1 and ebola virus encode small peptide motifs that recruit tsg101 to sites of particle assembly to facilitate egress tsg101, a homologue of ubiquitin-conjugating (e2) enzymes, binds the l domain in hiv type 1 pr55(gag) tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu modulation of hiv-1-host interaction: role of the vpu accessory protein hiv-1 vpu -an ion channel in search of a job inhibition of hiv-1 replication by amphotericin b methyl ester: selection for resistant variants epitope-tagging approach to determine the stoichiometry of the structural and nonstructural proteins in the virus particles: amount of vpr in relation to gag in hiv-1 cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines tightly bound zinc in human immunodeficiency virus type 1, human t-cell leukemia virus type i, and other retroviruses the bioluminescence advantage promega corporation website2007 longitudinal bioluminescent imaging of hiv-1 infection during antiretroviral therapy and treatment interruption in humanized mice identification of host proteins required for hiv infection through a functional genomic screen genomescale rnai screen for host factors required for hiv replication identification of interferon-stimulated genes with antiretroviral activity production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone optogenetic control with a photocleavable protein effects of ccr5 and cd4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1 in vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank terra schaden-ireland from the aids and cancer virus program at the frederick national laboratory for cancer research for performing the hiv-1 p24 capture immunoassay. we also thank members of the freed laboratory for helpful discussion and critical review of the manuscript. both authors conceptualized and designed the project. jk performed experiments and collected data. both authors analyzed data, wrote the manuscript. both authors read and approved the final manuscript. the freed laboratory is supported by the intramural research program of the center for cancer research, national cancer institute, nih. work at the aids and cancer virus program is supported by contract no. 75n91019d00024 (formerly hhsn261200800001e) from the national cancer institute, national institutes of health. jk is a recipient of the 2019 nci director's innovation award. the authors declare that they have no competing interests.received: 2 march 2020 accepted: 12 may 2020 key: cord-017070-05vlz5dn authors: dimitrov, dimiter s. title: human monoclonal antibodies against hiv and emerging viruses date: 2008 journal: national institute of allergy and infectious diseases, nih doi: 10.1007/978-1-59745-569-5_34 sha: doc_id: 17070 cord_uid: 05vlz5dn nan human monoclonal antibodies against hiv and emerging viruses dimiter s. dimitrov throughout the centuries, viral diseases have killed hundreds of millions of people and continue to do so. more than 100 million died in the past from smallpox caused by the variola virus. influenza killed about 50 million people during the 1918 pandemic. more than 60 million have been infected with hiv since the first cases of the epidemic were reported in 1981, and more than 20 million have died from aids. although vaccines remain the most cost-effective way to prevent viral infections, in many cases (e.g., hiv) vaccines are not available and, in other cases, available vaccines have to be modified frequently to counteract virus escape (e.g., influenza). prophylaxis and treatment in such cases are critically important to fight viruses. a number of studies have shown the importance of neutralizing antibodies in recovery and protection from viral infections (1, 2) . sera from humans or animals containing antibodies have been widely used for prophylaxis and therapy of viral and bacterial diseases since the late 1800s (3) (4) (5) (6) . serum therapy of most bacterial infections was abandoned in the 1940s after antibiotics became widely available (5) . however, polyclonal antibody preparations have continued to be used for some toxin-mediated infectious diseases and venomous bites (3) . serum immunoglobulin (ig) has continued to be also used for viral diseases where there are few treatments available, although mostly for prophylaxis either prior to an anticipated exposure or following an exposure to an infectious agent (7) (8) (9) . antibody products licensed in the united states for prevention or treatment of viral diseases include human ig for use against hepatitis a and measles; virus-specific polyclonal human ig against cytomegalovirus, hepatitis b, rabies, respiratory syncytial virus (rsv), vaccinia, and varicella-zoster virus; and the humanized monoclonal antibody (mab) synagis (7) . polyclonal ig has also been used with various success for diseases caused by other human viruses including parvovirus b19 (10) (11) (12) (13) , lassa virus (14, 15) , west nile virus (16, 17) , some enteroviruses (18, 19) , herpes simplex virus (20) , crimean-congo hemorrhagic fever virus (21) , junin virus (22) , sars-cov (23, 24) , and hiv (25) (26) (27) (28) (29) (30) . although serum polyclonal antibody preparations have been clinically effective in many cases, problems related to toxicity including a risk for allergic reactions, lot-to-lot variation, and uncertain dosing have limited their use (3) . mabs including chimeric animal-human, humanized, and fully human mabs (hmabs) possess lower or absent immunogenicity, toxicity, and lot-to-lot variation. the molecular mechanisms of therapeutic efficacy of such antibodies are easier to dissect, and they can be engineered to further improve their therapeutic properties. recently, some mabs have shown clinical success. the humanized mab synagis (palivizumab), which is still the only mab against a viral disease approved for clinical use by the u.s. food and drug administration, has been widely used for prevention of rsv infections in neonates and immune-compromised individuals, and very recently has been further improved (31) . however, it is not effective for treatment of an already established infection; for example, there were no significant differences in the clinical outcomes between the placebo and the palivizumab groups for children hospitalized with rsv infection (32) ; in addition, resistance can develop relatively quickly: a recent study found f gene-resistant mutations in an animal model of the rsv infection (cotton rat) 12 weeks after infection including a completely resistant virus (33) . the development of hmabs for prophylaxis and treatment of diseases caused by hiv and emerging viruses is still in an initial stage. this chapter reviews hmabs with potential for prophylaxis and treatment of diseases caused by hiv-1, sars-cov, and henipaviruses (hendra [hev]) and nipah [niv]). mostly igg 1 are discussed unless specifically noted otherwise; other isotypes also could be useful but are not so frequently used and other formats including fabs and single-chain variable region fragments (scfvs) are noted when described. finally, possible implications for development of effective vaccines are discussed. the demand for new treatment options against hiv is becoming increasingly important as the side effects and the expansion and spread of drug-resistant virus within the infected population limit the clinical benefits provided by available anti-hiv drugs. despite the promise presented by synagis and its improved variants (31) , developing effective antibodybased therapeutics against hiv presents an especially difficult challenge. therapies based on small molecule-based drugs eventually fail because of the expansion of a drug-resistant virus population in the infected individual. antibody-based therapies are not immune to this problem. further, hiv-1 replicates and spreads within the densely packed cellular environment (reaching about 10 8 cells per ml) of the lymphoid tissues of the gut, spleen, and lymph nodes. antibodies may have difficulty preventing the cell-to-cell spread of virus in this seemingly impenetrable lymphoid environment. in fact, passive administration of anti-hiv antibodies as human immune plasma or polyclonal antibody preparations conferred, at best, only modest clinical impact (8, 29) . unfortunately, these trials were complicated by the relative absence of highly effective concentrations of hiv-neutralizing activity in the polyclonal preparations used. preparations containing high concentrations of hmabs that exhibited potent hiv-1-neutralizing activity in vitro (nhmabs), on the other hand, resulted in measurable decrease of plasma virus concentration (34) . however, the in vivo potency of this preparation was insufficient to completely block virus replication, and resistant virus rapidly emerged. despite this discouraging result, these studies suggest that antibodies with enhanced in vivo potency could have a more profound clinical impact. a major question is whether antibody-based therapeutics can provide long-term clinical benefits for patients with established infections that are comparable to or better than drugs currently in clinical use. a specific feature of antibodies compared to other drugs is that hiv has evolved a number of strategies to escape neutralization. such evasion strategies of the virus against polyclonal antibodies elicited during infection and strategies used by the immune system to generate broadly neutralizing hmabs have been extensively reviewed (1, (35) (36) (37) (38) (39) (40) . thus, a short answer to this question is perhaps it is possible if we can outsmart the virus by engineering potent antibody-based therapeutics against which the virus has not yet evolved protective strategies. here, we discuss our ongoing efforts to improve the potential clinical utility of already known hmabs and identify novel, more potent antibodies against hiv. hiv entry into cells is initiated by attachment of the viral envelope glycoprotein (env) to a host cell receptor (cd4). conformational changes follow, which enable enhanced exposure of a co-receptor (typically ccr5 or cxcr4) binding site and binding of the viral glycoprotein gp120 to the co-receptor. subsequent conformational changes finally result in fusion of the viral and cell membranes. in some cases, cd4 is not required and the env directly interacts with a co-receptor. because the env mediates hiv entry and is the only viral surface protein exposed to the surrounding environment, it is a major target for neutralizing antibodies and a potent immunogen (36) . env-specific antibodies are generated as early as a few weeks after productive infection or immunization. they do not typically neutralize current virus isolates but rather neutralize earlier isolates (41) . such antibodies are isolate-specific and lack broad neutralizing activity because the virus has evolved to hide conserved epitopes and escape neutralization by a number of mechanisms. as a result, most of the antibodies generated in natural infection or immunization are non-neutralizing or neutralize few isolates. more than 100 mabs have been reported as recognizing epitopes on gp120 and gp41, but only a small number exhibit neutralizing activity against primary isolates from different clades, denoted as broadly cross-reactive neutralizing hmabs (bcnhmabs). using phage display or b cell immortalization, several bcnhmabs were identified from hiv-infected patients whose sera contained a high titer of such antibodies. six major classes of such antibodies relevant to the binding location and properties of their epitopes have been identified: (1) antibodies that bind to the region containing the cd4 binding site (cd4bs) on gp120; (2) antibodies binding better to gp120 complexed with cd4 than to gp120 alone (cd4i antibodies); (3) carbohydrate-binding antibodies; (4) gp120 v2 or v3-binding antibodies; (5) gp41 antibodies targeting the membrane-proximal external region (mper); and (6) antibodies binding to other epitopes on gp41. the best characterized and very potent cd4bs antibody is b12, a hmab selected from a phage-displayed antibody library constructed from the bone marrow of an hiv-1-infected donor (42, 43) . the cd4 binding site is masked by v1/v2 variable loops and further shielded by n -glycan in the region. the limited accessibility of these conserved epitopes in the context of the oligomeric env could explain why most of the antibodies that are specific for the cd4 binding site bind weakly to env oligomers and exhibit weak to moderate neutralizing activities against primary hiv-1 isolates. recently, two novel nmabs (m14, m18) were identified by sequential antigen panning (sap) against purified env ectodomains (gp140s) of another phage-displayed antibody library derived from the bone marrow of three long-term nonprogressors with high titers of bcnhabs (44, 45) . these antibodies cross-react with envs from primary isolates and exhibit differential neutralizing activity with b12 to isolates from different clades. the env undergoes significant conformational changes after binding to cd4 leading to exposure of structures that contain epitopes or portions of epitopes targeted by cd4i antibodies. the best characterized cd4i antibody, 17b, is only weakly neutralizing as igg1, but its neutralizing activity increases significantly after its size is reduced to a scfv (46) . the neutralizing activity of the most potent and broadly neutralizing cd4i antibody, x5 (47) , also increases in many cases with decreasing its size to scfv or fab. one should note however, that for some isolates and in some assays including assays based on spreading infection in peripheral blood mononuclear cells (pbmcs), igg1 x5 is more potent than fab x5 likely due to an increase in avidity. thus, an interplay between avidity and size could be important in determining the neutralizing activity of cd4i antibodies. many gp41-specific antibodies have been identified of which three with linear epitopes, 2f5, 4e10, and z13, exhibit broad neutralizing activity and have been extensively characterized (48) . they can bind peptides containing stretches of the mper of gp41, which is relatively conserved. using a competitive antigen panning methodology, several new crossclade reactive anti-gp41 mabs have been identified (m43, m44, m45, m46, m47, and m48) that neutralize a variety of primary isolates in pbmc/infectious virus-based assays and bind to conformational epitopes distinct from those of other anti-gp41 antibodies (49) . there are several lines of evidence that antibodies can inhibit hiv-1 infections. first, it has been demonstrated that antibodies can exert selective pressure in humans suggesting that they inhibit infection (41) . second, hiv-1 concentration in plasma of patents decreases following administration of hmabs (34) . third, a triple combination (2g12, 2f5, 4e10) of nmabs at higher doses can delay viral rebound after cessation of antiretroviral treatment (50) . in addition, a number of experiments have shown that antibodies can prevent infection in monkeys (see ref. 37 for a recent review). naturally occurring antibodies with potent and broad neutralizing activity could be further improved. recently, scfv x5 was further improved in potency and breadth of neutralization by using the sap methodology and a library of x5 mutants; two scfvs, m6 and m9, which exhibited on average two-to threefold lower ic 50 than scfv x5 were identified (44) . the scfv x5 mutant library was also screened by surface plasmon resonance technique on immobilized envs. one scfv was selected on the basis of its higher kinetic association rate, and its activity is being evaluated. because cd4i epitopes may be available for limited time after cd4 binding to gp120 and before their interaction with a co-receptor, cd4i antibodies with faster binding kinetics would bind efficiently and are likely to have better inhibitory activity against hiv-1. we have been developing various other constructs including fusion proteins that are being evaluated for neutralizing activity. hiv has evolved a number of strategies to escape host immune surveillance, prominently by modifications of its env. thus, naturally occurring whole antibodies against its env may have little chance of significantly affecting viral replication and disease progression, as also evidenced by the lack of sustained significant effect in the few clinical trials that have tested such antibodies. this is mostly due to the rapid generation of resistant mutants, which remains a fundamental problem not only for antibodies but also for other antiretroviral drugs. antibodies against components of the entry machinery, engineered antibody fragments and their derivatives, and other antibodybased inhibitors that do not occur naturally and against which the virus has not developed defense mechanisms may be better able to control virus replication, although mechanisms of inhibitory escape such as generation of resistant mutants and difficult access could still be operating. the challenge is to fight these mechanisms and simultaneously ensure a relatively long half-life and biological effector functions. several directions of research appear promising in this aspect. one direction involves engineering antibody with higher binding affinity to conserved epitopes and with smaller size than the currently known antibodies. another uses the unique features of some anti-env antibodies (e.g., the mimicry of receptors) to engineer novel binding entities with high-binding affinity to many isolates. a third consists of engineering novel fusion proteins containing antibody-binding fragments. a fourth relies on using antibody effector functions, including antibody-dependent cellular cytotoxicity (adcc) and complement, to increase the efficacy in vivo . yet another direction of research aims to develop antibody-nanoparticle conjugates, or nanoliposomes in particular, that are able to irreversibly inactivate virus and cells expressing viral proteins. the development of novel approaches may also be fruitful in the future, as well as combining existing ones to develop antibody-based hiv inhibitors using other, yet undiscovered principles. whether these or other research directions will lead to clinically useful antibody-based inhibitors of hiv infections remains unknown; however, even if new engineered antibodies fail to inhibit hiv infections in a clinically useful way, there is still a basis for optimism in this endeavor because the new approaches can prove useful elsewhere. finally, one should note that all bcnhmabs have undergone a long maturation process and differ from the germline sequences by tens of mutations. one could speculate that generation of such antibodies in vivo following immunization is very unlikely because of the lack of b cells expressing surface-associated ig that is close in function to those bcnhmabs. this may represent a challenge in developing effective aids vaccines, and further studies are required to find novel approaches for elicitation of bcnhmabs in vivo . the sars-cov (51-54) caused a worldwide epidemic in 2002 and 2003, and infected more than 8,000 humans with a fatality rate of about 10%. although there are no recent outbreaks, the need to develop potent therapeutics and vaccines against a re-emerging sars-cov or a related virus remains of high importance. sars-cov infection leads to generation of potent neutralizing antibodies that can affect the course of infection and help clear the virus; they can also protect an uninfected host exposed to the virus. antibodies that neutralize the virus in in vitro assays were detected in sars-covinfected patients (55) (56) (57) (58) (59) (60) , and in mice (61) , hamsters (62) , and monkeys (63) infected with the virus. these antibodies also protected uninfected animals from sars-cov infection, e.g., passive transfer of immune serum to naive mice prevented virus replication in the lower respiratory tract following intranasal challenge (61) . patients infected with sars-cov were also treated with convalescent patient plasma containing polyclonal antibodies (64, 65) , improvements of the antibody preparations were suggested (24) , and batches of virus-inactivated hyperimmune globulins containing five to six times higher titers of sars-cov-specific antibodies than convalescent plasma were produced (66) . in an amazing pace of research, several groups have recently developed hmabs to the sars-cov spike (s) glycoprotein that neutralize the virus and have potential for therapy and prophylaxis of sars (reviewed in ref. 67 ) . recently, an improved method for epstein-barr virus transformation of human b cells has been developed based on cpg oligonucleotide (cpg 2006) that increases the b cell immortalization efficiency from 1-2% to 30-100%, and used for selection of hmabs specific for sars-cov proteins (68) . one of the selected antibodies (s3.1), which was specific for the s glycoprotein on the viral spikes, was about 500-fold more efficient in neutralization than convalescent serum. s3.1 prevented the cytopathic effect of the sars-cov at 300 ng/ml (68) , and inhibited entry of pseudovirus with s glycoprotein from urbani isolate with about the same ic 50 . however, it did not affect to any significant extent pseudovirus entry mediated by the gd03 isolate s glycoprotein and even enhanced the entry of virus pseudotyped with the s glycoprotein from the palm civet isolate sz16 (69) . in a mouse model of sars-cov infection, this antibody prevented viral replication in the lower respiratory tract (at doses of 200 and 800 âµg), and reduced it in the upper respiratory tract at the highest dose (800 âµg) used. unfortunately, data for the in vivo neutralizing activity of other nhmabs selected in this study (68) , including the most potent antibody (s215.13), which has a neutralizing concentration (1 ng/ml) 300-fold lower than that of s3.1, have not been reported. the high neutralizing activities of these two hmabs in igg1 format indicates possibilities for their use alone or in combination for prophylaxis and treatment of sars. phage display technology has been increasingly used to produce high affinity hmabs from both naã¯ve and immune libraries. an advantage of using a naã¯ve library is that b lymphocytes from an infected or immunized host are not required. recently, two human nonimmune scfv libraries containing about 10 10 members were developed from b cells of unimmunized donors, and used for selection of antibodies against a purified s fragment containing residues 12 through 672 (70) . one of the selected antibodies, igg1 80r, can neutralize 50% of the virus in a microneutralization assay at a concentration as low as 0.37 nm. it also blocked formation of syncytia, which could contribute to the spread of the virus in vivo , although at significantly higher concentration (25 nm) . its epitope overlaps the binding site of the sars-cov receptor ace2 suggesting a possible mechanism of neutralization by preventing the virus attachment to its receptor (70) . when 80r igg1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced to below assay limits (71) . one should note that the conditions used for evaluation of the neutralizing activity of different antibodies in this study are not exactly the same as in the study described previously and later; thus comparing the activity of different antibodies should be done with caution unless they are tested side by side under exactly the same conditions. three neutralizing hmabs were also selected from another large naã¯ve antibody library (72, 73) . they bound a recombinant s1 fragment comprising amino acid residues 318 to 510 that also binds the receptor ace2-the receptor-binding domain (rbd; ref. 73 ). the most potent of these hnmab, igg1 cr3014, required the residue n479 for binding (73) . this antibody exhibited in vitro 50% neutralizing activity at about 1 âµg/ml. more importantly, this antibody showed neutralizing activity in ferrets. in one set of experiments, ferrets were inoculated either with virus at two doses (low: 10 3 tcid 50 ; high: 10 4 tcid 50 ) or with virus preincubated with the antibody at 0.13 mg/ml for the low dose and 1.3 mg/ml for the high dose. animals exposed to the virus-antibody mixture had almost undetectable sars-cov in the lung, showed no lung lesions on day 4 or 7, and did not shed virus in their throats unlike control animals treated with irrelevant antibody. in a second set of experiments, the antibody at 10 mg/kg was administered 24 hours before challenge with virus and reached 65-84 âµg/ml serum concentration in three of the animals (<5 âµg/ml in the fourth one). in the three ferrets with high antibody concentration virus shedding in the throat was completely abolished, while in the fourth one it was comparable to that of the control group. the cr3014-treated animals had 3.3 logs lower mean virus titer than the controls and were completely protected from macroscopic lung pathology. note that the antibody dose used (10 mg/kg) was less than the one (15 mg/kg) used for prevention of rsv infections in infants, which is administered once a month. these results suggest a potential use of cr3014 for prophylaxis of sars-cov infections in humans if it can reduce the virus replication to the same extent as in ferrets. however, one should note that currently there is no available animal model of the sars-cov infection that results in death as in humans. two other hmabs (201 and 68) were derived from transgenic mice with human ig genes and evaluated in a murine model of sars-cov infection (74) . one of these antibodies [201] bound within the rbd of the s protein at amino acid residues 490 through 510, and the other one (68) to a region including residues 130 through 150. in a microneutralization assay based on protection to cytopathic effects the ic 50 for 201 was about 0.2 âµg/ml (74) . mice that received 40 mg/kg of these antibodies prior to challenge with the sars-cov were completely protected from virus replication in the lungs, and doses as low as 1.6 mg/kg offered significant protection. these antibodies have potential as therapeutics and research tools, and further studies are planned to evaluate the nhmab 201 for potential clinical use (74) . we have recently identified a novel cross-reactive potent sars-cov-neutralizing hmab, m396, by using a fragment containing residues 317 through 518 as a selecting antigen for panning of a large human antibody library constructed from the b lymphocytes of healthy volunteers (75) . this fragment was previously identified to contain the rbd (76-78) , which is a major sars-cov neutralization determinant (67, (79) (80) (81) (82) (83) (84) . it potently inhibited s-mediated cell fusion (ic 50 = 0.6 âµg/ml), pseudovirus entry (ic 50 = 0.01 âµg/ml), and replication of infectious virus in mice (complete protection at 0.2 mg per mouse; zhu et al., unpublished) . interestingly, this antibody also inhibited entry mediated by the s glycoprotein from the 2003/2004 gd03 isolate, which has a 487thr/ser mutation compared to the middle/late phase 2002/2003 isolate tor2, and is not neutralizable by other known potent hmabs including 80r and s3.1. it bound with high (pm) avidity to the rbd in a biacore chip and competed with ace2 suggesting a mechanism of neutralization that involves competition with the receptor for binding to the s glycoprotein (zhu et al., unpublished) . the epitope of this antibody was identified by crystallography and proposed as a possible vaccine immunogen (a retrovaccinology approach for design of vaccine immunogens [ 85 ] ). neutralizing antibodies directed to s inhibit sars-cov entry either by interfering typically with the s rbd-receptor interactions (70) or by other mechanisms including binding to other portions of s. recently, a human scfv, b1, was identified, which recognizes an epitope on s2 protein located within amino acids 1023 to 1189 (86) . this antibody recognized sars pseudovirus in vivo and competed with sars sera for binding to sars-cov with an equilibrium dissociation constant, k d = 105 nm. the b1 also had potent neutralizing activities against infection by pseudovirus expressing sars-cov s protein in vitro . other mechanisms of sars-cov infection inhibition could include steric hindrance that indirectly prevents virus attachment to receptors and binding to entry intermediates. mechanisms that could operate in vivo (and for lack of data are not discussed here) are related to the antibody biological effector functions conferred by the antibody fc, e.g., adcc. niv and hev are closely related emerging paramyxoviruses that comprise the henipavirus genus (87) (88) (89) (90) (91) (92) (93) (94) (95) (96) . the broad species tropisms and the ability to cause fatal disease in both animals and humans distinguish hev and niv from all other known paramyxoviruses (reviewed in ref. 1 ). they are biological safety level 4 pathogens and are on the niaid biodefense research agenda as zoonotic emerging category c priority pathogens that could be used as bioterror agents. there are currently no therapeutic modalities for treating niv or hev infections, and a vaccine for prevention of disease in human or livestock populations does not exist. although antibody responses were detected in infections caused by these viruses, the development of hmabs specific for hev and niv have only just been realized. we recently reported the identification of potent neutralizing hmabs targeting the viral envelope glycoprotein g by using a highly purified, oligomeric, soluble hev g (sg) glycoprotein as the antigen for screening of a large naã¯ve human phage-display library (97) . the selected seven fabs, m101-7, inhibited, to various degrees, cell fusion mediated by the hev or niv envs and virus infection. the conversion of the most potent neutralizer of infectious hev, fab m101, to igg1 significantly increased its cell fusion inhibitory activity-the ic 50 was decreased more than 10-fold to approximately 1 âµg/ml. the igg1 m101 was also exceptionally potent in neutralizing infectious hev; complete (100%) neutralization was achieved with 12.5 âµg/ml and 98% neutralization required only 1.6 âµg/ml. the inhibition of fusion and infection correlated with binding of the fabs to full-length g as measured by immunoprecipitation, and less with binding to sg as measured by elisa and biacore. m101 and m102 competed with the ephrin-b2, which we and others recently identified as a functional receptor for both hev and niv (98, 99) , indicating a possible mechanism of neutralization by these antibodies. the m101, m102, and m103 antibodies competed with each other suggesting that they bind to overlapping epitopes that are distinct from the epitopes of m106 and m107. in an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 g alanine scanning mutants and identified two mutants, p185a and q191,k192a, which significantly decreased binding to m101, and one, g183a, which decreased binding of m102 to g. these results suggest that m101-7 are specific for hev or niv or both, and exhibit various neutralizing activities; they are the first hmabs identified against these viruses and could be used for treatment, prophylaxis, diagnosis, and as research reagents and aid in the development of vaccine. recently, we matured in vitro m102 to a very potent crossreactive antibody, m102.4, which neutralized both infectious niv and hev with an ic 50 of 100 ng/ml (zhu et al., unpublished) . we developed a cell line that produces large amounts of igg1 m102. 4 . this antibody will be tested in an animal model of henipavirus infection and if successful, which is very likely, it could be clinically useful for humans. interestingly, the antibodies against sars-cov and henipaviruses were identified from naã¯ve libraries, and in contrast to hiv have only few mutations compared to the respective germline sequences. this correlates with the ability of various vaccines to elicit potent cross-reactive neutralizing antibodies against these viruses in contrast to hiv. one could speculate that humans have in their native repertoire antibodies that can bind with relatively high affinity envelope glycoproteins from these emerging viruses but not to the hiv env. further experiments are required to test this hypothesis, which if true could lead to novel approaches for design of effective vaccines against hiv and other viruses. the hmabs directed to the sars-cov s glycoprotein and the henipavirus g glycoprotein are currently in an advanced stage of development and offer the best hope as potential therapeutics. these antibodies specific for sars-cov, hev, and niv have potential for further development into a clinically useful product for prophylaxis and perhaps treatment of the diseases caused by these infections. they are very potent, and the viral infections to which they are specific are acute, such that only control or dampening of virus replication for a relatively short period of time (few weeks) is likely to be required after which the host immune system could control virus replication. in addition, these antibodies could be cross-reactive. thus, the problem of neutralization resistant mutants able to evade their inhibitory activity and the immune response is not as significant as for chronic infections with a high level of virus replication, e.g. hiv infections. a note of caution is that careful examination of candidate antibody therapeutics is required because of the possibility for infection enhancing effects, as e.g., for ebola, and animal model-dependent effects as well as in some cases, although rare, toxicity. a recent study also reported the possibility that neutralizing antibodies can enhance entry of sars-cov by a mechanism that involves antibody interactions with conformational epitopes in the s rbd (69) . in addition, it is known that in some cases antibodies that do not neutralize in the assay currently used for evaluation of their in vitro activity could exhibit potent neutralizing activity in vivo ; thus new approaches should be developed and antibodies tested also for their effector functions mediated by the fc including antibody-dependent direct cytotoxicity and complement-mediated immune responses. however, only further exploration of these antibodies and their extensive evaluation in animal models, likely in com-bination with other antiviral drugs (antibodies or small molecules), would allow identification of the best candidates for potential therapeutics. the rapid progress made in the last few years toward the development of potent neutralizing hmabs against emerging viruses and viruses of biodefense importance is a basis for further more accelerated development of neutralizing antibodies in the next 5 years and their testing in animal models. it is likely to see novel and even more potent antibodies against the sars-cov than the currently existing ones. they could be used alone or in combination with the existing antibodies in animal models of viral diseases and for evaluation of toxicity in human clinical trials. the currently available hmabs against niv and hev are likely to be tested in animals. if successful, which is very likely, they can undergo evaluation in human clinical trials. although the interest of big-and medium-size companies to such antibodies appears to be relatively minor, small companies and start-ups could be interested in developing such antibodies provided there is a continued governmental support by programs like bioshield. five years from now, it is likely to have at least several hmabs of potential clinical use in case of outbreaks or terror attacks. these antibodies could be used in combination with other therapeutics to increase potency and cope with resistance. several key issues are listed below: â�¢ continuation of research and development funding at the same or accelerated pace is of critical importance for development of potent and clinically useful therapeutic antibodies. â�¢ phage display techniques as well as novel methodologies will be critical for the development of fully human antibodies. â�¢ cloning, expression, and purification of novel antigens for screening of human antibody libraries is of critical importance. â�¢ crystal structures of antibody complexes with virus envelope glycoproteins and their use for further improvement of the antibodies and understanding of their interactions is of critical importance. â�¢ development of appropriate novel animal models that would be of critical importance for the accelerated testing of the therapeutic antibodies is necessary. â�¢ understanding of the pathogenesis and the design of antibodies acting through multiple mechanisms with an increased efficacy in vivo is of critical importance. â�¢ evaluation of combinations of antibodies and other antiviral drugs is of critical importance. acknowledgments. this study was supported by the nih nci ccr intramural program, the gates foundation, the nih intramural aids program (iatap), and the nih intramural biodefense program. virus entry: molecular mechanisms and biomedical applications passive antibody therapy for infectious diseases passive antibody therapies: progress and continuing challenges return to the past: the case for antibody-based therapies in infectious diseases serum therapy revisited: animal models of infection and development of passive antibody therapy preventing infectious disease with passive immunization antibodies for the prevention and treatment of viral diseases intravenous immunoglobulin for infectious diseases: back to the pre-antibiotic and passive prophylaxis era antiviral immunotherapy-a review of current status parvovirus b19 infection-related complications in renal transplant recipients: treatment with intravenous immunoglobulin pure red-cell aplasia of 10 years' duration due to persistent parvovirus b19 infection and its cure with immunoglobulin therapy successful intravenous immunoglobulin therapy in 3 cases of parvovirus b19-associated chronic fatigue syndrome chronic pure red cell aplasia caused by parvovirus b19 in aids: use of intravenous immunoglobulin-a report of eight patients lassa immune serum treatment of west nile virus encephalitis with intravenous immunoglobulin possible benefit of intravenous immunoglobulin therapy in a lung transplant recipient with west nile virus encephalitis intravenous immunoglobulin prophylaxis in an echovirus 6 and echovirus 4 nursery outbreak treatment of human enterovirus infections intravenous immunoglobulins suppress the recurrences of genital herpes simplex virus: a clinical and immunological study specific intravenous immunoglobulin for crimean-congo haemorrhagic fever importance of dose of neutralising antibodies in treatment of argentine haemorrhagic fever with immune plasma treating severe acute respiratory syndrome with hyperimmune globulins treatment of severe acute respiratory syndrome with convalescent plasma passive immunotherapy in aids: a doubleblind randomized study based on transfusions of plasma rich in anti-human immunodeficiency virus 1 antibodies vs. transfusions of seronegative plasma the use of intravenous immunoglobulins in symptomatic hiv infection. results of a randomized study intravenous immunoglobulin in symptomatic and asymptomatic children with perinatal hiv infection phase i/ii trial of hiv-1 hyperimmune globulin for the prevention of hiv-1 vertical transmission in uganda a passive immunotherapy, (pe)hrg214, in patients infected with human immunodeficiency virus: a phase i study passive immunization for the prevention and treatment of hiv infection ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization safety and pharmacokinetics of palivizumab therapy in children hospitalized with respiratory syncytial virus infection in vivo selection of respiratory syncytial viruses resistant to palivizumab passive immunization with the anti-hiv-1 human monoclonal antibody (hmab) 4e10 and the hmab combination 4e10/2f5/2g12 neutralizing antibody responses to hiv: role in protective immunity and challenges for vaccine design identifying epitopes of hiv-1 that induce protective antibodies antibodies: can they protect against hiv infection? the prospects for vaccines against hiv-1: more than a field of long-term nonprogression? hiv vaccine design and the neutralizing antibody problem antibody-based inhibitors of hiv infection antibody neutralization and escape by hiv-1 efficient neutralization of primary isolates of hiv-1 by a recombinant human monoclonal antibody recognition properties of a panel of human recombinant fab fragments to the cd4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1 identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody broadly cross-reactive hiv neutralizing human monoclonal antibody fab selected by sequential antigen panning of a phage display library access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1 broadly crossreactive hiv-1-neutralizing human monoclonal fab selected for binding to gp120-cd4-ccr5 complexes identifying epitopes of hiv-1 that induce protective antibodies selection of a novel gp41-specific hiv-1 neutralizing human antibody by competitive antigen panning virus isolates during acute and chronic human immunodeficiency virus type 1 infection show distinct patterns of sensitivity to entry inhibitors a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome sars-associated coronavirus chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection antibody responses against sars-coronavirus and its nucleocaspid in sars patients development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein sars corona virus peptides recognized by antibodies in the sera of convalescent cases s protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice severe acute respiratory syndrome coronavirus infection of golden syrian hamsters replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys treating severe acute respiratory syndrome with hyperimmune globulins sars: what have we learned? treating severe acute respiratory syndrome with hyperimmune globulins human monoclonal antibodies to the s glycoprotein and related proteins as potential therapeutics for sars an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association evaluation of human monoclonal antibody 80r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody the sars-cov s glycoprotein: expression and functional characterization the sars coronavirus s glycoprotein receptor binding domain: fine mapping and functional characterization a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine sars vaccine development receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies identification of a critical neutralization determinant of severe acute respiratory syndrome (sars)-associated coronavirus: importance for designing sars vaccines recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region single amino acid substitutions in the severe acute respiratory syndrome coronavirus spike glycoprotein determine viral entry and immunogenicity of a major neutralizing domain antibodies, viruses and vaccines a human sars-cov neutralizing antibody against epitope on s2 protein a morbillivirus that caused fatal disease in horses and humans the neurological manifestations of nipah virus encephalitis, a novel paramyxovirus nipah viral encephalitis or japanese encephalitis? mr findings in a new zoonotic disease fatal encephalitis due to nipah virus among pig-farmers in malaysia risk factors for nipah virus infection among abattoir workers in singapore high mortality in nipah encephalitis is associated with presence of virus in cerebrospinal fluid clinical features of nipah virus encephalitis among pig farmers in malaysia update: outbreak of nipah virus-malaysia and singapore nipah virus: a recently emergent deadly paramyxovirus introduction to current focus on hendra and nipah viruses potent neutralization of hendra and nipah viruses by human monoclonal antibodies ephrin-b2 ligand is a functional receptor for hendra virus and nipah virus ephrinb2 is the entry receptor for nipah virus, an emergent deadly paramyxovirus key: cord-018545-fk17n2bx authors: dorofaeff, tavey; mohseni-bod, hadi; cox, peter n. title: infections in the picu date: 2012 journal: textbook of clinical pediatrics doi: 10.1007/978-3-642-02202-9_268 sha: doc_id: 18545 cord_uid: fk17n2bx nan effective control of infections starts at the community level, outside the hospital. there are a number of important initiatives that, although simple and not necessarily intensive care related, have the greatest impact on the outcomes of infections. these include provision of adequate and age appropriate foods, breast feeding, drinkable water, provision of mosquito nets and shelter, avoidance of overcrowding, sanitization, and prevention of disease by vaccination. these are basic needs and requirements of mankind as a basis of good health. they are attainable in the largest cities or the most remote areas. infections are one of the commonest causes of mortality in the pediatric intensive care unit (picu), with a mortality of up to 50%, depending on the origin of the infection. infections in the intensive care unit can be divided into those that occur outside the hospital (community acquired) and those that occur within the walls of the hospital and beyond 48 h of admission (nosocomial). preventive measures give the most benefit, both outside and inside the hospital. good hand washing, good respiratory care practice, and judicious use of antibiotics are examples of effective interventions that reduce the rate of nosocomial infections. sepsis comprises up to 25% of admissions to a typical pediatric intensive care unit. shock and management of septic shock are discussed elsewhere in this text. however basic principles of management are the same and are not, and should not be, limited to the intensive care unit. treatment should commence as soon as the recognition of any septic process is underway be it in the field, the clinics, emergency departments, or on the wards. in the community, on the hospital wards, and in the picu, timely identification of illness and access to skilled healthcare personnel are crucial steps limiting the development of organ dysfunction and failure. early identification means early resuscitation and early treatment. this may be hours or in some cases days prior to the admission to the picu. this early recognition and intervention gives the patient the greatest chance of surviving a significant infection. crucial to the management of any serious infection in the intensive care unit and elsewhere is the early use of appropriate antibiotics. early identification of most bacteria is almost universally by the way of a gram stain. this can be performed in any microbiology lab, in the field, or a clinic that is suitably equipped. though references such as the ''red book'' (american academy of pediatrics) give invaluable information on the appropriate antimicrobial therapy for a given microbe or infectious syndrome, there is no substitute for a well informed, up-to-date infectious diseases physician or microbiologist. they are able to provide information on local isolates, patterns of sensitivity, and best management practices for a large variety of infections. the majority of the bacteria listed below will be referenced elsewhere; they are highlighted to reflect their frequency of identification in the picu. additionally, the immunocompromised host will be at risk from a number of opportunistic infections that will be discussed later in this chapter. these bacteria have a tendency for multiple antibiotic resistances. there are a number of other medically significant bacteria that only periodically present in the pediatric intensive care unit. these bacteria are prevalent in some regions and not elsewhere. they will not be discussed further in this chapter. fungi (of importance in the picu) classification • yeasts (e.g., candida or cryptococcus sp.) • molds -filamentous fungi (e.g., aspergillus sp. and trichophyton sp.) • dimorphic fungi -yeasts in tissue but grow in vitro as molds (e.g., histoplasmosis) candida. c. albicans has the highest incidence in the critical care environment followed by c. parapsilosis, c. tropicalis, c. glabrata, and c. krusei. localized and systemic infections in neonates and immunocompromised children. any organ can be involved: mucous membranes, larynx, esophagus, brain, eyes, lungs, heart, kidneys, liver, and spleen. aspergillus. a. fumigatus is the most common species in invasive aspergillosis, followed by a. flavus, and a. nigra. localized and systemic infection in immunocompromised children (particularly post stem cell transplant and in children with aml); in the skin, subcutaneous tissues, nasopharynx, lungs, brain, and virtually any other organ. endemic mycosis (histoplasmosis, blastomycosis). pneumonitis, hepatosplenomegaly, fever in children with t-cell dysfunction and in those with hiv infection. coccidioides immitis. pneumonia, meningitis in children with t-cell dysfunction and in those with hiv infection. cryptococcus neoformans. pneumonia, meningitis in children with t-cell dysfunction and in those with hiv infection. human herpes viruses 1. herpes simplex virus (hsv, types 1 and 2) (a) systemic infection in the neonate with shock and coagulopathy and severe liver failure (b) encephalitis, hepatitis (c) local (mouth, esophagus, larynx, lungs, heart, liver, kidneys, cns) or systemic disease in organ and stem cell transplant and immunocompromised patients 2. cytomegalovirus (cmv) (a) congenital infection in the neonate with systemic involvement (b) localized (liver, lungs, heart, kidneys, gi, cns, eyes) or systemic infection in solid organ and stem cell transplant as well as immunocompromised patients 3. epstein-barr virus (ebv) (a) infectious mononucleosis (b) burkitt's lymphoma, x-linked lymphoproliferative disorder (xl-lpd), post transplant lymphoproliferative disorder (ptld) (c) localized (liver, heart, lungs, kidneys, gi, cns) it is likely that viral infections have been underestimated both in their frequency and the degree of morbidity they cause. indisputably, in the modern day, hiv (human immunodeficiency virus) is one of the most significant viral pathogens worldwide, particularly in africa and developing nations that do not have the available resources to prevent spread among the community and more importantly maternal infection of the newborn. this leads to a range of morbidities as discussed in the immunocompromised section of this chapter. viral infections are mostly diagnosed clinically on the basis of history and physical examination as well as the regional prevalence of viral diseases. there are a number of ways to test for the presence of a particular virus from either patient blood or other body fluids. these are: tissue culture, serology and seroconversion, immunoflouresence, and pcr (polymerase chain reaction). only two of these are of any use to the intensivist: pcr and immunoflouresence. the turnaround time for viral culture and serum serology is inefficient in the critical care context. respiratory viruses (respiratory syncytial virus, influenzae, adenovirus, parainfluenzae, and human metapneumovirus) are the main contributors to viral disease in the picu, as they are in the general pediatric population. in the picu the majority of patients that develop significant degrees of illness are those who have significant comorbidities. conditions such as ex-prematurity, chronic lung disease, neuromuscular diseases, and congenital heart disease are probably the most common of these. herpes virus family, particularly herpes simplex virus (hsv), is the next important contributor to the burden of viral disease. all members of this family (hsv, ebv, cmv, and vzv) can cause serious infections in the neonate and in immunocompromised children. all services that treat the acutely unwell child (and adult) are at risk of being overwhelmed in an epidemic. national and regional planning needs to be undertaken prior to the advent of any serious infection where ever possible. (examples are sars -severe acute respiratory syndrome, or h1n1 ''swine flu.'') this is encapsulated in the worldwide pandemic planning taking lessons from the sars epidemic and the last major (in terms of mortality) influenza epidemic, the ''spanish flu'' that was prevalent from 1918 to 1920. at a hospital or an organizational level the concept of ''surge strategy'' is used. this is an organization based contingency plan to deal with large numbers of patients admitted simultaneously (i.e., mass trauma casualties or epidemics). in the case of influenza (or sars) this is relevant to the intensive care in that there is a finite capacity of any unit to provide mechanical ventilation. in addition to this, the institution is responsible for the protection of health care workers who are at high risk to contract an infectious illness and become a patient themselves. this would further increase the burden of illness and has the potential to limit available human resources. the prevalence of hiv in the general population and, in particular, in children in many developing countries poses significant stress on limited resources. hopefully, with more effective preventive programs to control vertical transmission of the infection and with availability of affordable anti-hiv medications, the quality of care for hiv-infected children will improve and the need for intensive care will diminish. regional or local experience is crucial in the management of many infections. dengue fever and viral hemorrhagic fevers, which are of more global importance, will be reviewed in more detail. dengue infections, caused by the four antigenically distinct dengue virus serotypes (den1, den2, den3, den4) of the family flaviviridae, are the most important arbovirus diseases. dengue is the most widely distributed mosquito-borne viral infection of humans, affecting an estimated 100 million people worldwide annually. dengue hemorrhagic fever usually occurs in children, with peaks in incidence at 7 months of age (with dengue-immune mothers), and at 3-5 years of age (during a second infection with a new serotype). it is spread throughout the tropical and subtropical zones between 30 n and 40 s where environmental conditions are optimal for viral transmission by aedes mosquitoes, principally aedes aegypti. the disease is endemic in se asia, the pacific, west africa, the caribbean, and the americas. global warming, by increasing the range of aedes mosquito, has the potential to lead to more widespread disease. who has classified the severity of dengue infection on the basis of a combination of clinical and laboratory findings (presence of hypotension and shock, tourniquet test, lowest platelet count, plasma leakage represented by high hematocrit level) in to: • dengue fever • dengue hemorrhagic fever • dengue shock syndrome (dss) viral hemorrhagic fever is a loosely defined category that includes infections from a host of viruses leading to similar clinical syndromes and sharing a similar severity of illness. otherwise, these viruses are different from each other with regard to their reservoir hosts, geographic distribution, and taxonomy. risk factors for exposure also vary among these infections and hence the control methods are geared to specific infections and their causative agents and intermediate hosts. in endemic areas diagnosis is by and large clinical and is confirmed by serological tests and viral pcr or culture. there are vaccines developed for some of these viruses. as a group, the treatment for these infections in the picu is mainly supportive and includes measures to: • optimize hemodynamic state and treat shock • monitor and control brain edema and intracranial hypertension • support ventilation and gas exchange with noninvasive or invasive ventilation • treat coagulopathic state if symptomatic • provide renal replacement therapy if needed; monitor and optimize glucose and electrolyte levels exhaustive discussion of this topic is beyond the current chapter, so the most important ones are briefly mentioned here. yellow fever is endemic in tropical africa between 15 n to 10 s and in parts of central and south america between 10 n and 40 s. in the life-cycle of this virus, in different parts of the world, mosquitoes (a. aegypti, haemagogus, and sabethes), monkeys, and people are involved; however, epidemic mosquito-borne human-to-human transmission can occur. after an incubation period of 3-10 days, fever, headache, malaise, nausea and vomiting, and musculoskeletal pain occur suddenly. initially, the clinical signs may include conjunctivitis, flushing of the skin, and relative bradycardia. in about 10% of cases the illness deteriorates with development of shock, systemic toxicity, gi bleeding, renal dysfunction, liver failure and jaundice, encephalopathy, and systemic bleeding. this latter picture is associated with a high mortality rate (30-50%). differential diagnosis includes other viral hemorrhagic fevers, viral hepatitis, leptospirosis, malaria, typhus, typhoid fever, brucellosis, rickettsial disease, and some intoxications. therapy is supportive and these patients may need intensive care admission for hepatic, renal and circulatory failure. who has recommended routine childhood vaccination in endemic areas (for children >4 months of age). vector control is important in highly populated areas to reduce the risk of epidemic transmission. lassa fever causes as many as 300,000 cases and 5,000 deaths each year in west africa and is a leading cause of maternal and fetal deaths. the virus is carried by mastomys huberti and mastomys erythroleucus, the rodent reservoirs whose infectious excretions are the source of human infections in west africa. in adults and children, early illness includes fever, malaise, headache, and musculoskeletal pain. these nonspecific symptoms progress over 4-5 days to include pharyngitis, cough, chest pain, diarrhea, and vomiting. in endemic areas, a purulent pharyngitis, with conjunctivitis, head and neck edema, and mucosal bleeding are highly specific signs of lassa fever. in severe cases, the illness may be complicated by hypovolemic shock, encephalopathy, respiratory distress caused by laryngeal edema, pleural effusions, or pneumonitis. liver failure, systemic and gi/gu bleeding, and myocarditis can occur. mortality is between 15% and 30%. there are anecdotal reports of the use of intravenous ribavirin in critically ill children with lassa fever but treatment is mainly supportive. lassa fever has been transmitted from person to person during hospitalization. universal exposure precautions should be observed as well as contact and droplet precautions. cchf is caused by a nairovirus (family bunyaviridae), and is transmitted by hyalomma ticks and by contact with infectious body fluids. the geographical distribution of the hyalomma ticks covers africa, the middle east and mediterranean areas, eastern russia, and west asia. the incubation period is from 2 to 9 days. illness onset is abrupt and nonspecific, with fever, chills, rigors, intense headache, and generalized muscle pain. onset of bleeding in the skin, mucous membranes, and the gi tract usually occurs after 3-6 days of illness. hepatitis, liver failure, circulatory failure, shock, and ards can ensue with mortality in up to 30% of cases. treatment is mainly supportive. the virus is sensitive in vitro to ribavirin, and this agent has been used in management of cchf with variable success (who). the value of immune plasma from recovered patients for therapeutic purposes has not been demonstrated, although it has been employed on several occasions (who). patients with suspected or confirmed cchf should be cared for by staff using added droplet and contact precautions. hfrs is caused by old world hantaviruses (family bunyaviridae). the reservoirs are small rodents, and humans are infected percutaneously or by direct exposure. clinical illness has an abrupt onset with fever, severe musculoskeletal pain, renal failure, systemic and gi bleeding, circulatory failure, and shock. this form of the disease is more common in asia and eastern europe. in hantavirus pulmonary syndrome (hps) (mainly seen in the americas), within 12-24 h of onset of symptoms, most patients develop some degree of hemodynamic instability and pulmonary edema accompanied by hypoxemia to full blown ards. petechiae of the head and neck are common but overt hemorrhagic symptoms are not. treatment is supportive and in those who survive, recovery is usually rapid. when given early in the course of illness, intravenous ribavirin has improved survival rate in hfrs but not in hps. steroids reduce the severity of the symptoms but do not increase the survival rate. malaria is singled out here because it is the most significant parasitic disease in humans with an estimated 500 million infections annually that result in 1-3 million deaths. the majority of these deaths are in children younger than 5 years of age and most are in africa. in developed countries malaria is the most common cause of febrile illness with no localizing signs in travelers returning from developing countries. the most important aspects of severe malaria are reviewed, which, for the most part, is caused by plasmodium falciparum. indicators of severe and complicated falciparum malaria and prognostic signs (world health organization 2000) cerebral malaria unrousable coma (gcs < 11/15), with peripheral p. falciparum parasitemia after exclusion of other causes of encephalopathy severe anemia hgb < 5 g/dl in the presence of parasitemia >10,000 per ml impaired consciousness of any degree, prostration, jaundice, intractable vomiting, parasitemia >2% in nonimmune individuals. levels of parasitemia should be interpreted in the light of immunity. patients with complicated malaria should be managed as severe malaria, i.e., with parenteral antimalarials even though they do not necessarily meet the criteria of severe disease. for details of management, review > chap. 101, ''malaria''. the world health organization defines cerebral malaria as unrousable coma in the presence of p. falciparum parasitemia when other causes of encephalopathy have been excluded. the precise etiology of cerebral malaria is not certain. most likely it is caused by sequestration of infected erythrocytes. this condition has a high mortality that likely results from brain micro vascular ischemia, infarction, and secondary cerebral edema. cerebral malaria is a medical emergency that requires: 1. supportive care: (a) continuous monitoring of vital signs. for a detailed discussion on sepsis, and the diagnosis and management of shock, please review the appropriate chapters (> chap. 61, ''bacterial sepsis and shock''). toxic shock and necrotizing fasciitis are two particular sepsis syndromes that require a special reference. toxic shock syndrome (tss) is caused by two bacteria: staphylococcus and streptococcus. s. aureus is a gram-positive coccus that is grouped in clusters. it is responsible for a number of infections ranging from skin sepsis, pneumonia, and joint infections to endocarditis. phage transformed staphylococcus produces a toxin that initiates a syndrome known as toxic shock syndrome (tss). this came to light in the 1980s with the ''menstrual shock'' syndrome. a non menstrual form was also identified. this was associated with staphylococcus sepsis at surgical sites, skin or joint infections, and with staphylococcal pneumonia. this syndrome is said to be ''superantigen'' mediated. the toxin proteins produced by the staphylococcus are able to ''cross-link'' the t-cell receptor without being processed by an antigen presenting cell (apc). this leads to an uncontrolled cascade of cytokines and immune system up regulation. at the level of the capillary this leads to inflammation and increasing permeability with secondary organ dysfunction (renal impairment, cardiac, pulmonary, and liver dysfunction). clinically this is manifested by skin erythema, tachycardia, hypotension, hypoxia and other critical organ dysfunction. initially this is subtle but rapidly develops into multi organ dysfunction. see the table below for the criterion upon which a diagnosis of staphylococcal toxic shock is made. treatment consists of recognition of the process, draining any collections of pus, and debridement, if that is appropriate. at the same time initiation of large volume fluid resuscitation, inotropic support and support of failing lungs with oxygen and ventilation if needed. antistaphylococcal antibiotics should be administered (this includes an antibiotic to cover for methicillin resistant staphylococcus). clindamycin being an anti-ribosomal antibiotic (50s bacterial ribosome) has a theoretical advantage in reducing the amount of toxin produced prior to antibiotic induced death of the bacterium. intravenous immunoglobulin (ivig) is a treatment for severe toxic shock that is progressing to multi-systems dysfunction. it has a proven efficacy in toxic shock in reducing the mortality of severe disease. this is thought to be via two general mechanisms. the first is by binding directly to the toxin. the second is by its immuno-mediatory properties. major criteria (all required) 1. fever !38.8 c 2. hypotension (orthostatic or shock) 3. rash (erythematous early and desquamative later) minor criteria (any three required) 1. gastrointestinal: vomiting or diarrhea 2. muscular: severe myalgia or cpk !2x upper limit of normal 3. mucous membranes: vaginal, oropharyngeal, or conjunctival hyperemia 4. renal: urea or creatinine !2x upper limit of normal, or urinalysis with >5 wbc per high-power field 5. hepatic: total bilirubin, ast or alt !2x upper limit of normal 6. blood: platelet count <100,000/ml 7. cns: disorientation or change in level of consciousness without focality, noted when fever and hypotension are absent streptococcal toxic shock is a syndrome that is analogous to staphyloccal toxic shock syndrome in that it is a superantigen mediated toxin related dysfunction of the immune system. group a beta-hemolytic streptococcus is most commonly associated with streptococcal toxic shock syndrome. clinical presentation is very similar to staphylococcal toxic shock. see table below. treatment consists of appropriate antibiotics. clindamycin is used for antimicrobial and antitoxin producing properties as previously mentioned. ivig here too has a role in reducing the mortality of severe disease. intensive care therapy consists of fluid resuscitation (large volume) and support of organ dysfunction (inotropes, ventilation, renal replacement therapy). hypotension or shock, plus any two of the following: 1. scarlet fever rash 2. abnormal liver function tests 3. renal insufficiency 4. disseminated intravascular coagulopathy (dic) 5. acute respiratory distress syndrome (ards) 6. soft tissue necrosis definite: preceding requirements + isolation of group a streptococcus from a normally sterile body site probable: preceding requirements + isolation of group a streptococcus from a non sterile body site necrotizing soft tissue infections are aggressive soft tissue infections that cause extensive necrosis, and include necrotizing cellulitis, fasciitis, and myonecrosis. the following clinical findings may be present: • erythema or discolored skin also the following systemic signs may be present: • local pain and tenderness out of proportion to physical findings • pain or tenderness that extends past the margin of apparent affected skin area necrotizing fasciitis is a surgical emergency. it is caused by a number of organisms: group a beta-hemolytic streptococci and other streptococci, staphylococcus, clostridium, pseudomonas, klebsiella, serratia, neisseria, escherichia, morganella, proteus, shigella, vibrio, salmonella, pasturella, enterobacter, corynebacterium, cryptococcus, fusobacterium, peptococcus, eikenella, bacteroides. the most common causative agent is group a streptococcus. pathologically it is characterized by micro angiopathic thrombosis and necrosis along superficial and deep fascial planes. the illness is associated with a breach of the integument. this can be by superficial infection, surgery or trauma. non steroidal antiinflammatory drugs are implicated in the pathogenesis. in children there is an association with varicella (chickenpox) infection. clinically the lesions appear either pale or have violaceous discoloration, often edematous, and there may be crepitus from gas forming bacteria. pain and tenderness in excess of that expected is a feature. the concern for the intensivist is the physiological decompensation that can lead to rapid cardiovascular collapse. broad-spectrum (and appropriate) antibiotics are indicated, and mechanical ventilation and cardiovascular support may be needed. urgent and wide surgical debridment of the affected areas is indicated. in cases of streptococcal necrotizing fasciitis there may be additional benefit from human immunoglobulin (ivig) therapy. though this has not been subjected to clinical trials, given the high mortality rate of necrotizing fasciitis and the biologically plausible consideration that ivig could neutralize the effects of streptococcal superantigens, its use can be justified. other treatments that have been used are: • vacuum-assisted wound closure (particularly in patients who have had large wound debridement) • hyperbaric oxygen (anecdotal evidence) the child, especially the infant, presenting with upper airway obstruction (uao) demands immediate attention. acute inflammation of the upper airway is of greater importance in small children because of the smaller diameter of the airway, hence the greater degree of obstruction from a similar amount of inflammation (resistance changes inversely to the fourth power of the radius of the airway). the following signs and symptoms are particularly worrisome: • inspiratory and expiratory stridor • active expiration (use of the rectus abdominis muscle when exhaling) • apnea or irregular breathing • increasing tachycardia (if no intervention is done tachycardia may be followed by decreasing heart rate which is usually a pre-arrest sign) • hypoxemia (late sign) • change in neurological status (becoming increasingly inconsolable and restless, or a child who ''stops fighting'' and becomes fatigued and hypotonic) there are many scoring systems for severity of the uao in children. the following is one suggested by downes et al. in 1980 . of note there is no mention of the neurological status in this scoring system. level of alertness and consolability of a small child are very important indicators of the severity of the uao. immediate management of acute severe stridor outside the picu, independent of underlying cause: • keep the child and the parent as calm as possible. do not separate the child from parent. • give the parent an oxygen mask to hold near the child's face. • call for help urgently from someone with expertise in airway management (usually an anesthesiologist). • give nebulized epinephrine (im epinephrine if airway obstruction is due to anaphylactic reaction). (skip nebulized epinephrine if you suspect epiglottitis.) • do not send the child to the radiology department for a lateral x-ray of the neck. • do not administer any sedative medications to the patient. • do not do attempt to draw blood for investigations. • place ecg monitoring leads and pulse oximetry probe without disturbing the child. • do not attempt to place an iv line (obviously you would place an iv/io access if the child has already had a respiratory or cardiac arrest). • the airway expert will decide to take the child to the or for intubation, or transfer to the picu. in children, there are many causes of acute uao, including infections (viral, bacterial) such as infectious mononucleosis, croup, epiglottitis, tracheitis, peritonsillar abscess, retropharyngeal abscess, diphtheria. noninfectious causes include foreign body, severe allergic reactions, acute angioneurotic edema, airway burn, trauma, and post-extubation in the picu. there are many causes of chronic/recurrent uao. in the history there may be chronic/recurrent symptoms. these patients may become symptomatic acutely (often with a viral respiratory infection) mimicking acquired acute upper airway obstruction. examples are: choanal atresia, laryngotracheomalacia, vascular ring, laryngeal web, subglottic stenosis, subglottic haemangioma, vocal cord palsy, recurrent angioneurotic edema. in this section the infectious causes of uao are addressed to. the more common infectious etiologies that may present with severe uao in children are: • croup or viral laryngotracheobronchitis • bacterial tracheitis • epiglottitis viral croup is the most common form of uao in children 6 months to 6 years of age (mostly 6 months to 2 years) and is more common in the autumn and early winter. the site of obstruction is the subglottic area. obstruction is caused by inflammation and edema. the most common viral etiology is parainfluenza, but influenza, enterovirus (coxsakie and echovirus), rsv, adenovirus, paramixovirus, rhinovirus, and hsv can cause a similar clinical picture. human metapneumovirus has been implicated in a few reports. in immunocompromised children, candida sp. can cause a similar presentation. there is a prodrome of mild fever and uri symptoms for 1-2 days before the onset of stridor. the stridor is characteristically harsh, dry, high pitched, and inspiratory. a ''barking'' or ''seal-like'' cough is prominent and usually worse at night. these children do have a voice, though hoarse, and they do not have trismus, dyphagia, or significant drooling. children with stridor at rest should be admitted for observation, while those with severe uao should be admitted to a picu. up to 15% of children with croup require hospitalization. usually no investigations are needed. administration of steroids (oral route is as good as intramuscular) in the emergency room has decreased the rate of hospitalization. hospitalized children with croup should receive a short course of oral or intravenous steroids (an example of a regimen is: dexamethasone 0.6 mg/kg iv/ po as an initial dose followed by 0.15 mg/kg q 6 h iv/po). inhaled nebulized epinephrine 1:1,000 solution (0.5 ml/kg, up to 5 mg) reduces the severity of obstruction and stridor. this can be repeated as required. the child must be observed for at least 2 h after a dose of nebulized epinephrine as the effects are transient. the decision on when to intubate a child with croup is a clinical one. if, despite maximum medical treatment there is not a clinical improvement or perhaps deterioration, a decision to intubate should be made or at least considered. a gentle and smooth intubation, using a tube one size smaller than usual for the age of the child, should be performed by a skilled and experienced practitioner. these children are at risk of accidental extubation and need proper securing of the ett, skilled nursing care, and adequate sedation only once the airway has been secured. most clinicians extubate the child 2-6 days later, when an audible air leak has developed around the ett and fever has settled. epiglottitis, or acute bacterial supraglottitis, is a bacterial infection of the laryngeal inlet, and is usually caused by h. influenzae type b (hib). with ''classical'' hib epiglottitis, the peak age of involvement is 2-3 years of age. since the introduction of the hib vaccine, the incidence of this disease has fallen dramatically, but the vaccine does not offer 100% protection. also, other organisms like s. aureus, s. pneumoniae, group a + b streptococcus, and n. meningitidis have been implicated as causative agents. the incidence of these latter organisms is higher in adolescents and older children. noninfectious causes of epiglottitis have been described in the following conditions: kawasaki's disease, stevens-johnson syndrome, airway burn, caustic ingestion, post-radiotherapy, angioneurotic edema, trauma (including trauma from intubation), leukemia, and lymphohistiocytosis. granulomatous states can cause a more chronic picture (sarcoidosis, tb, or wegener's granulomatosis). as fewer and fewer physicians have seen even one case of epiglottitis, it is important to have a high index of suspicion in any febrile child with uao. the following signs are highly suspicious of epiglottitis: • usually there are no prodromal signs and symptoms. • a few hours of high fever and tachypnea. • pain with swallowing, hence drooling is common. • reluctance to speak. • the child looks ill, with circumoral pallor, and a ''toxic'' appearance. • there is minimal or no coughing. • stridor is low-pitched and muffled, more like a snore. • child prefers to sit forward in the tripod position with mouth open and is reluctant to move his head or neck. if you have suspicion (on clinical grounds) that a child may have epiglottitis: • do not make the child lie down. • do not separate the child from parent. • do not examine the throat. • do not place an iv cannula. • do not order a lateral x-ray of the neck. • do not order any blood work. • do not transport a child with epiglottitis between hospitals unintubated. • the child should be accompanied by an expert in difficult airway management to the operating room for examination under anesthesia and securing airway if needed. the technique for induction of anesthesia is beyond the scope of this chapter. generally the inhalational method is performed in the sitting position (position of comfort for the child), and once the child loses consciousness intravenous access is secured and the rest of the monitoring is applied. laryngoscopy and intubation is only attempted after adequate depth of anesthesia has been obtained. blood cultures and a swab from the inflamed epiglottis should be sent and a 3 rd generation cephalosporin should be given once an iv is in place. when back in picu, accidental extubation can have disastrous consequences. skilled taping of the ett, nursing care, and adequate anaelgesia/sedation cannot be over emphasized. usually after 12-48 h of intravenous antibiotics the patient can be safely extubated, once the fever has subsided and presence of a leak is documented and the child is able to swallow (the child is not drooling). some practitioners prefer to reevaluate by direct laryngoscopy with the patient deeply sedated or anesthetized. if the causative organism is proved to be hib, in families with siblings under 4 years of age or families with an immunocompromised child, prophylaxis with rifampicin should be provided. bacterial tracheitis is characterized by profuse purulent secretions or sometimes by pseudomembrane formation in the tracheal lumen. the median age of the patient is 5 years. s. aureus is the most common etiology, though other gram-positive and less commonly gram-negative microorganisms might be causative. in immunocompromised children candida and aspergillus can cause tracheitis. these children usually have a high fever, they look toxic, and the stridor characteristically is high pitched and composed of both inspiratory and expiratory components. cough is usually prominent and they may have dysphonia or aphonia. drooling can be seen with bacterial tracheitis. these patients are at risk of airway obstruction. they require appropriate antimicrobial therapy, observation, and intubation by experts if warranted. community-acquired pneumonia (as opposed to nosocomial or hospital acquired pneumonia) is a common pediatric diagnosis that leads to admission to hospital for intravenous antibiotics and supportive respiratory therapy. pneumonia means inflammation of the lung parenchyma caused by infection and the diagnosis is made clinically in a febrile child with respiratory signs and symptoms who has evidence of consolidation on cxr. blood cultures frequently fail to reveal the infecting organism in pneumonia. tracheal aspirate, or more reliably, bronchoalveolar lavage (bal) and on occasions lung biopsy are required. children with immunodeficiency or malignancy undergoing therapy are a common example of where bal and/or lung biopsy may be necessary. • mycobacterium tuberculosis s. pneumoniae and s. aureus are the most important bacterial pathogens in children with pneumonia who need intensive care admission. as a general rule, truly focal disease, confined to a single lobe, is more likely to be due to bacteria. an ill child with unilateral pleural effusion most likely has s. pneumoniae or s. aureus pneumonia. viral • rsv • influenza a, b, c • parainfluenza routes to acquire infection: • inhalation of infected particles (most common) • aspiration • hematogenous invasion of the lower respiratory tract with viruses and bacteria leads to inflammatory changes characterized by migration of neutrophils into the alveoli. together with alveolar macrophages they provoke the production of inflammatory exudates and cellular debris that lead to consolidation of the lung parenchyma. the surrounding areas can be affected by atelectasis. • spo 2 <90% in high concentrations of inspired oxygen (>60% fio 2 ) • excessive work of breathing which may lead to exhaustion • shock and hemodynamic instability • change in neurological status (agitation or alteration of the level of consciousness) reasons of failure to respond to treatment on the pediatric ward or as outpatient: • development of an empyema or less commonly a lung abscess • underlying lung disease such as: bronchopulmonary dysplasia (bpd, in ex-premies), cystic fibrosis, inhaled foreign body, tracheobronchomalacia or post tracheal surgery, or infected congenital lung cyst • diagnosed or undiagnosed immunodeficiency states (primary, hiv, leukemia) • children with neuromuscular diseases, weakness, or spasticity such as muscular dystrophies, myasthenia, spinal muscular atrophy, or cerebral palsy • inappropriate antibiotics, inappropriately low dose or resistant bacteria • non bacterial pneumonia (viral pneumonia or alternative pathogen such as tuberculosis) once the culture results (bal, blood culture, sputum culture) and sensitivities are known the therapy should be tailored to the antibiotic sensitivities of the causative organism(s). • empyema. more commonly seen with s. pneumoniae and s. aureus pneumonia. generally a chest drain is needed. the use of fibrinolytics and surgery are areas for debate and local advice from thoracic or general surgeons and physicians from the respiratory and infectious diseases services should be sought. bronchiolitis is a seasonal viral infection of the lower respiratory tract that mainly affects infants. the usual cause is respiratory syncytial virus (rsv), although influenza, parainfluenza, adenovirus, and human metapneumovirus can cause a similar syndrome. in young infants, chlamydia and b. pertussis can cause respiratory illness with a more prolonged course that initially may resemble bronchiolitis. 20-25% of infants with bronchiolitis may have a secondary bacterial infection. infants with bronchiolitis have fever, cough, difficulty in feeding, and, on occasion, audible wheezing. on examination bronchiolitis is a syndrome characterized by respiratory distress, hyperinflation of the chest, and wheezes with fine inspiratory crackles heard on auscultation. apnea may occur even before onset of clinically significant respiratory distress, especially in ex-premature infections in the picu and very young infants. though not common, neonates may present with hypothermia and a sepsis like syndrome. the following groups are at increased risk of severe infection: • ex-premature infants and neonates • infants with congenital heart disease • infants with immune deficiency • infants with neuromuscular disease the virus causes direct damage to the respiratory epithelium with resultant inflammation, increased secretions, small airway obstruction. areas of hyperinflation and atelectasis exist simultaneously throughout the lung. this leads to ventilation and perfusion (v/q) mismatch and hypoxia. hyperinflation flattens the diaphragm and makes breathing less efficient. should they require respiratory support, many of these infants can be managed with noninvasive continuous positive airway pressure (cpap) at 4-8 cm h 2 o. this reduces the work of breathing and improves oxygenation. suction to maintain patency of airways is of crucial importance. if the saturations remain low and/or the infant continues to have frequent apneas despite providing noninvasive ventilatory support intubation of the trachea is indicated. • intubation is usually required for several days. • inadequate humidification and inadequate tracheal suctioning cause endotracheal tube blockage or lung atelectasis followed by increasing pressure and fio 2 requirements. • as a general rule, the best ventilatory mode is one that assists spontaneous respiratory efforts; keep the child's own respiratory and coughing efforts by providing enough comfort (sedation) and pressure support. • peep or cpap (initially at 4-6 cm h 2 o) may reduce the work of breathing. • apply enough peak inspiratory pressure (pip) to achieve visible chest excursions and if higher pressures (>30 cm h 2 o) are needed, let the paco 2 gradually rise to 75-80 mmhg with arterial ph > 7.2 (permissive hypercapnia). particular issues in infants with congenital heart disease and bronchiolitis: • infants with left to right shunts have more frequent viral and bacterial respiratory infections, and have higher morbidity and more prolonged course with bronchiolitis. • infants with palliated single ventricle physiology and those with limited cardiac output (for example severe valvar aortic stenosis) have high morbidity and mortality with bronchiolitis. • bronchiolitis and other viral respiratory infections in infants with congenital heart disease lead to operative delays and increasing complications post cardiac bypass surgery (e.g., pulmonary hypertension). in any infant with rsv bronchiolitis and congenital heart disease awaiting surgery, it is suggested to wait for 2-3 weeks before proceeding with bypass and surgery. the american academy of pediatrics has specific recommendations on prophylactic monthly injection of rsv monoclonal antibody in ''at risk'' infants during the cold season. however, the use of this approach has only been shown to aid a small number of patients. this section reviews: • myocarditis • infective endocarditis • infectious pericarditis • wound infection after cardiac surgery myocarditis is an inflammatory disease of the heart muscle characterized in its active phase by cellular infiltrates and myocardial necrosis. however myocarditis can have cellular infiltrates with little or no myonecrosis. most cases of myocarditis are thought to have a viral etiology; however, viruses are infrequently isolated. the most common viral causes include the enterovirus family particularly coxsackievirus b and adenovirus. other viral causes are influenza, cmv, hsv, parvovirus, rubella, varicella, mumps, hiv, and ebv. myocarditis has a number of other non viral etiologies, some infective and some not. they include bacteria, rickettsia, fungi, protozoa, pharmaceuticals, toxins, and connective tissue/autoimmune disorders. typically viral myocarditis begins as a systemic viral illness with flu-like symptoms. as the virus infects the myocytes the immune system is up regulated and cd4 t-helper cells and cd8 cytotoxic t cells are stimulated along with proinflammatory cytokines. persistence of the viral rna and production of no by the myocytes have been linked to myocardial tissue damage. myocarditis can present in a number of ways: • out of hospital cardiac arrest/sudden death • cardiogenic shock (may mimic sepsis) • congestive heart failure (increasing dyspnea, lethargy) • dysrhythymias -bradycardia, tachycardia whilst sepsis and hypovolemic shock are more common than cardiogenic shock from myocarditis, it should always be in the differential. sometimes acute ''decompensation'' of these children is heralded by abdominal distension and vomiting. teenagers may complain of a feeling of ''impending doom'' or severe chest discomfort. clinically, signs of tachycardia/tachypnea, gallop rhythm, hyperdynamic precordium, and displaced apex are often present. hepatomegaly and in older children elevated jugular venous pressure (jvp) are usually present. crackles on auscultation are often present in the chests of older children. chest x-ray (cxr) may show an enlarged heart, pulmonary venous congestion, alveolar edema, kerley b lines, and in some cases pleural effusions. in acute myocarditis the heart may often look normal in size on the cxr. a 12-lead ecg is useful to assess underlying rhythm, assess for ischemia and for the subtle ecg changes that are sometimes evident with myocarditis; st-t changes, reduced qrs voltage, widened qrs. echocardiography is absolutely necessary to assess structure and function of the heart and to assess for a pericardial effusion. involvement of appropriate specialists is important -cardiologists, intensivists, and cardiothoracic surgeons work cooperatively to manage and stabilize these patients. treatment of myocarditis is largely supportive. immunomodulation using steroids, intravenous immunoglobulin, and immunosuppressive agents is controversial. identifying any modifiable contributors, i.e., toxins and drugs, is of crucial importance. supportive therapy for heart failure associated with myocarditis ranges from diuretics and afterload reduction, addition of inotropic support to placing the patient on mechanical circulatory support. dopamine and dobutamine increase contractility but also heart rate and myocardial oxygen consumption. milrinone is an intravenous phosphodiesterase inhibitor that improves contractility and at the same time, reduces the afterload. enoximone is an oral phosphodiesterase inhibitor that is available in europe, but not in north america. levosimendan is a calcium sensitizer and improves contractility. it has limited availability world wide. positive intrathoracic pressure, given noninvasively via a face mask (cpap), reduces lv afterload and may improve cardiac output in the setting of lv dysfunction. failed medical therapy or deteriorating function will usually indicate the need for extracorporeal support and ultimately heart transplantation. mechanical support (ecls, ''berlin'' heart) is frequently used as a ''bridge'' to recovery or transplant. for a complete review of endocarditis review the cardiology chapter in this book. the major reasons a child with endocarditis may need admission to picu are: infections in the picu 1. congestive heart failure due to worsening valvar regurgitation 2. congestive heart failure with abrupt onset due to valve apparatus rupture/perforation, or dehiscence of a prosthetic valve 3. systemic to pulmonary artery shunt obstruction 4. arrhythmia 5. renal failure 6. embolic events to (a) brain (b) heart (c) lungs (d) bowel (e) extremities for a complete review of pericarditis and tamponade, please review the cardiology chapter in this book. acute inflammation of the pericardium in a previously healthy child has usually been assumed to be viral. in most cases a causative agent is not detected (hence the term ''idiopathic'' pericarditis). an upper respiratory infection usually precedes the onset of symptoms by 10-14 days. the reported viral pathogens include coxsackievirus, adenovirus, rsv, varicella, hepatitis b, hiv, and post influenza vaccine. primary infectious pericarditis that may need picu care is usually purulent bacterial pericarditis. these patients are generally toxic looking. the infection in the pericardium rarely occurs in the absence of infection elsewhere (hematogenous spread). in comparison with the viral (idiopathic) pericarditis, the incidence of tamponade and hemodynamic instability is much higher with purulent pericarditis. s. aureus is the most common cause of purulent pericarditis. other bacteria include h. influenzae, n. meningitides, and s. pneumoniae. in developing countries, tubeculous pericarditis is a common cause of chronic constrictive pericarditis. therapy depends on the hemodynamic status of the patient. a toxic-looking child with physiological signs and symptoms of tamponade should be transferred urgently to the catheterization laboratory or to the intensive care unit for percutaneous drainage of the pericardial collection. sometimes the pus in pericardium is so thick or organized (esp. with h. influenzae) that percutaneous drainage may not be sufficient and the child will need open surgical drainage. with tamponade physiology, administration of fluid boluses can temporarily increase the intracardiac ''filling'' and stabilize the patient until the definitive treatment (percutaneous or surgical drainage) is performed. broad-spectrum intravenous antibiotics with good antistaphylocccal coverage should be commenced promptly if purulent pericarditis is suspected. surgical wound infections after cardiac surgery can be categorized as superficial (cellulitis) or deep (mediastinitis). the patient usually presents a few days after the procedure, but may occur up to 2 months after the initial operation. the important signs are erythema and induration at the surgical incision. the child may be irritable and have a mild fever. there may be a leukocytosis with ''left shift'' and elevated inflammatory markers such as c-reactive protein (crp) or erythrocyte sedimentation rate (esr). in addition to the wound erythema with or without purulent discharge there may be signs of sternal instability with ''crepitus'' on direct pressure over the sternum. diagnosis nevertheless is a clinical one and relies on a high index of suspicion. the risk factors are: neonates, long cardiopulmonary bypass time, delayed sternal closure, and reexploration of the chest for postoperative bleeding. the most common organisms associated with sternal wound infections are s. aureus, s. epidermidis, enterococcus species, and candida species. antibiotic treatment should begin as soon as a sternal wound infection is suspected and a wound swab has been sent. blood cultures should be sent from both peripheral and central venous sites whenever possible. the initial antibiotic regimen should consist of broad-spectrum gram-positive (anti-staphylococcal) coverage, with the addition of gram-negative coverage if the patient is septic or mediastinitis is suspected. if there is not a rapid improvement or the patient deteriorates the sternal wound may need to be surgically explored and debrided. antibiotics should be given for 10 days to 2 weeks for cellulitis and for 4-6 weeks for deep wound infections. meningitis is an inflammation of the leptomeninges of the brain. for a review of ''aseptic'' meningitis which also includes viral causes of meningitis, please look at the neurology and infectious disease chapters in this textbook. suffice to mention that patients with ''aseptic'' meningitis are usually not as sick as those with purulent meningitis, and the csf abnormalities are not as prominent. patients with bacterial meningitis have a number of reasons for requiring intensive care. the most common clinical scenarios are coma and seizures. the local inflammatory response to bacteria multiplying in the csf involves polymorphonuclear leukocytes, the endothelium, complement, and cytokines. this results in an alteration in the cerebral blood flow and venous drainage, vascular inflammation, and obstruction to csf flow and reabsorption. the infection within the meninges may extend to the surrounding brain parenchyma. the commonest bacteria are s. pneumoniae, n. meningitidis, and h. infuenzae. in the neonatal period, the likely causative organisms are different: group b streptococcus, l. monocytogenes, and gram-negative bacilli are the commonest. this profile will be modified depending on the local vaccination policy, socioeconomic status of the children in the area, and local/regional epidemics of disease. none the less the intensive care management is similar: 1. broad-spectrum cns penetrating antibiotics with narrowing of spectrum of antibiotic cover once results of cultures of the blood and csf are known. antibiotics with high csf/brain tissue penetrance must always be used. in areas with high incidence of s pneumonia penicillin resistance (including the united states), empiric therapy for community-onset bacterial meningitis is both vancomycin and a 3rd generation cephalosporin. acute complications of bacterial meningitis are: • hyponatremia (serum sodium <135 micromole/l): this is usually due to the syndrome of inappropriate secretion of antidiuretic hormone (siadh). there is hyponatremia and low serum osmolarity without signs of hypovolemia. hyponatremia can cause convulsions. cerebral salt wasting is a much rarer condition that gives hyponatremia with signs of volume contraction. siadh is treated by free water restriction and cerebral salt wasting is treated with sodium (either iv or po) supplementation. in a hyponatremic child with convulsions give 3% nacl 3-5 ml/kg intravenously. it is important to note that the change of serum sodium (and hence serum osmolarity) is of more importance in some cases than the absolute serum sodium. a sudden drop in serum sodium (greater than 0.5-1.0 micromole/h) should be treated with hypertonic saline. • seizures: convulsions that occur early in the course of purulent meningitis are usually generalized and have less prognostic significance than those occurring later. etiology of convulsions in meningitis can be any of the following: brain edema, diffuse ischemia, hyponatremia, subdural collection, sinus venous thrombosis, or focal infarction. • subdural effusion: this complication is seen more commonly in neonates and infants. a good practice is to measure the head circumference daily in any infant with meningitis. the diagnosis is made or confirmed by neuroimaging. if the effusion is large, or if it is associated with focal signs, convulsions, or signs of increased intracranial pressure, a neurosurgical consultation is necessary. • obstructive hydrocephalus: obstructive hydrocephalus occurs when the pus (often with high protein content) in the ventricles blocks the outflow of csf. this situation occurs more frequently in small infants and neonates. similar to meningitis, seizures, focal or generalized signs, and coma, are common presentations. the list of differential diagnosis is long and includes: aseptic meningitis, post infectious encephalitis and noninfectious encephalopathies (metabolic, vascular, demyelinating disease, tumor). the most frequent causes of acute encephalitis include: enteroviruses, hsv, vzv, ebv, adenovirus, influenza virus, and m. pneumoniae. mumps, measles, and rubella infections are rarely seen in developed countries. in many parts of the world arboviruses are major causes of endemic encephalitides. tuberculosis is always high on the list of differential diagnosis in developing countries. diagnosis: csf can be completely normal, but usually contains >10 leukocytes/mm 3 (mainly lymphocytes), mildly increased protein level and mildly reduced to normal glucose level. in children with hsv encephalitis, the csf may contain red blood cells. csf in addition to cell count, chemistry and culture should be sent for pcr for viral agents. csf pcr can be helpful in a number of the infectious encephalopathies. for example: m. pneumoniae, mycobacterium, cmv, ebv, vzv; where the organism may not be cultured from the csf. brain imaging (ct or mri) can be helpful in the diagnosis. in hsv encephalitis there may be focal edema and enhancement seen in the temporal area. this is relatively specific for hsv infection of the brain. the electroencephalogram may show focal periodic epileptiform activity in frontal and temporal parts of the brain. this is common in hsv disease. diffuse slow waves generalized over the cerebral cortex may also be seen. this may either represent encephalitis or be secondary to sedatives and anticonvulsants used in the picu. the primary use of eeg is in the management of seizures. finally, when the etiologic diagnosis is not clear, or the patient is deteriorating despite treatment, brain biopsy may be performed. treatment is largely supportive. as for meningitis this consists of appropriate antimicrobial (antiviral) therapy. there should be no delay in starting acyclovir if hsv is suspected or considered. the dose is 30 mg/kg/day for 10 days. when m. pneumoniae is suspected antibiotics with good penetration into the brain (ciprofloxacin, or azithromycin) should be used. additional management includes airway protection for coma and seizures. medical and surgical therapies for management of intracranial hypertension, as discussed above should be adhered to. adem is an acute or sub acute inflammatory demyelinating disease of the cns (brain and spinal cord). in contrast to multiple sclerosis it is a monophasic illness. adem is considered a parainfectious disease and the precipitants include infection with upper respiratory tract viruses, influenza, group a streptococcus, ebv, vzv, measles, mumps, rubella, and mycoplasma. clinical presentation may involve fevers, seizures and a constellation of neurological phenomena. commonly these are coma, focal neurological deficits or alterations in personality and behavior. often a recent ''viral infection'' is present in the history. the lumbar puncture is done to exclude infections. csf may have normal white cell count or mild pleocytosis (mainly lymphocytes), and mild to significant protein elevation. the cultures and pcr should be negative. neuroimaging is the main diagnostic tool for adem mri is the modality of choice, as the ct is normal in 40% of cases. the typical mri findings are multiple disseminated asymmetrical hyperintense lesions on t2wi and flair in the white matter and basal ganglia. the cerebrum is more involved than the cerebellum. treatment: (1) continue antibiotics and antivirals until final csf cultures and pcr results are confirmed to be negative. (2) once infection is ruled out methylprednisolone ''pulse'' dose at 30 mg/kg/day (maximum 1 g) for 3 days followed by oral prednisolone 2 mg/kg/day for 2 weeks. this is followed by a 4 weeks weaning regimen. (3) plasma exchange or ivig for relapsed or refractory adem. gbs is an immune-mediated polyneuropathy that is usually preceded by a viral or bacterial infection of the respiratory or gi tract 1-3 weeks prior to presentation. gbs is the most common cause of acute paralysis in developed countries and is characterized by progressive, ascending, symmetric motor weakness and loss of reflexes. the sensory symptoms (extremity pain, paresthesia) and autonomic irregularities (tachycardia, bradycardia, hypertension, hypotension, arrhythmia) can be prominent. there are usually no sensory deficits in physical examination. infections known to precede the onset of paralysis are: cmv, ebv, vzv, campylobacter jejuni, mycobacterium tuberculosis, hiv, and m. pneumoniae. the pathogenesis involves an immune response against the infectious agent and has components that cross-react with those of the peripheral nervous system. diagnosis is clinical but is aided by csf and electrophysiology testing. the csf shows a high protein content and low/normal white cell count. this may be missed if the lumbar puncture is done early in the first week of the illness. electrophysiology will show decreased conduction velocity in the peripheral nerves. treatment: ivig at 2 g/kg/day for 2-5 days or plasma exchange. (two courses for mild gbs and four to five courses for severe illness.) corticosteroids have not demonstrated effectiveness in gbs and are not recommended. indications for picu admission: • for respiratory support • for plasma exchange • autonomic instability (hypo-or hypertension, arrhythmia) botulism is a toxin mediated disease caused by c. botulinum. symptoms start a few hours and up to 6 days after exposure to the toxin. the cranial nerves are involved initially with difficulty swallowing, abnormal speech (abnormal cry in infants) and eye movements (ptosis). other symptoms may include nausea, vomiting, constipation, and abdominal distension. as the illness progresses it causes paralysis of the extremities and respiratory muscles to various degrees. in infants the disease can be mild with hypotonia and constipation as the main findings. also in infants there is sometimes a history of ingestion of honey before the onset of symptoms (honey may contain the spores of c. botulinum). diagnosis is made from a combination of clinical findings and electromyography. stool for botulinum toxin or serum serology is confirmatory but takes time. treatment is with antitoxin to remove circulating toxin but this will not affect the toxin already present at the neuromuscular junction. specific botulinum immunoglobulin is not readily available world wide. the cost is prohibitive for a lot of countries and the cost-benefit analysis is only favorable for those patients requiring mechanical ventilation. penicillin and metronidazole are given to eradicate the source of toxin production. aminoglycosides and steroids should not be given as they may worsen the neurosmuscular transmission defect and increase muscular weakness. if the source of the c. botulinum is a wound (i.e., wound botulism) then it will need surgical debridement. indications for admission to picu: • respiratory support • autonomic instability children are increasingly surviving diseases that until recently were considered untreatable. there are more potent, intense chemotherapy regimens being used and increasingly there are more patients who undergo solid organ and stem cell transplants. these treatments and interventions particularly with immunosuppressants, though frequently successful, leave patients at considerable risk for severe infections. neutropenia is defined as the absolute neutrophil count (anc) [anc = pmn + band count] <1,000/mm 3 , and is generally associated with cancer and its treatment. the risk of infection is particularly high with: • rapid drop in anc • anc < 100/mm 3 (profound neutropenia) • prolonged neutropenia fever in neutropenic patients is defined as an oral/ tympanic membrane temperature >38 c in two repeated measurements over a 4 h period or one measurement above 38.5 c. the portals of entry of infectious agents are usually: the oral mucosa, the gut, the upper/lower respiratory tract, and central vascular lines. the most common organisms are gram-positive cocci (s. aureus, s. epidermidis, and strep. viridans), gram-negative bacilli (e. coli, k. pneumoniae, p. aeroginosa), and fungi (candida, aspergillus). recently the spectrum of pathogens has begun to change, with the emergence of more gram-negative and fungal infections. this is likely due to an increase in resistant pathogens in the face of the use of very broadspectrum antibiotics, intensity of therapy (high-dose chemotherapy and stem cell transplant) and prolonged neutropenia. the single most important risk factor for fungal infection is the duration of neutropenia. it is standard of practice to start antibiotics for a child with anc < 500 who is febrile. initial empiric therapy for febrile neutropenia consists of a b-lactam antibiotic and an aminoglycoside, plus a glycopeptide if a coagulase negative staph or enterococcus is suspected or isolated, if the child is in shock, has an endoprosthesis or a vascular tunnel infection. if patient has a history of arabinoside-c administration and has severe mucositis strep viridans infection is highly suspected and vancomycin should be added. if perianal infections in the picu tenderness is present add anaerobic coverage (metronidazole or clindamycin). after 4-5 days of fever and neutropenia adding an antifungal is the usual practice of most oncologists. infants with a severe combined or t-cell immune deficiency usually present early in the first few months. defects in cell-mediated immunity can result from congenital disorders such as digeorge syndrome, severe combined immunodeficiency disease (scid) and wiskott-aldrich syndrome. they can be secondary to lymphomas, immunosuppressive medications or chronic illness. acute viral infections such as measles and pertussis are also known to decrease a patient's cellmediated immunity. typical infections are pneumocystis jiroveci pneumonia (formerly carinii), cmv pneumonitis, rsv pneumonitis, disseminated enteroviral infection, and invasive fungal infection. patients are highly susceptible to infections with intracellular organisms such as salmonella, listeria, mycobacteria, herpes family viruses (cmv, ebv, and hsv), as well as fungi and protozoa. in older children and those with secondary immunodeficiencies, these infections tend to be reactivated disease. children with chronic mucocutaneous candidiasis and chronic granulomatous disease typically present early in life with recurrent candida and staphylococcal infections. neonates with adhesion molecule deficiency usually present with delayed separation of the umbilical cord stump, increased polymorphonuclear count, and increased incidence of bacterial infections. children with primary humoral immune deficiency usually present between 6 months and 5 years of age. the onset is consistent with the time when the level of placentally transferred maternal antibodies (igg) has declined. these defects as well as complement deficiency and asplenia are more commonly associated with infections by encapsulated microorganism such as h. influenza, n. meningitides, and s. pneumoniae. although patients with these conditions are mainly susceptible to bacterial infections involving the upper and lower respiratory tract, they can have protracted diarrhea with giardia or echovirus. defects in the late complement component (the ''attack'' component, c5-9) are prone to recurrent neisseria infections. children with early complement defects usually have autoimmune and rheumatologic manifestations. fulminant meningococcemia is also associated with properdin deficiency (alternative complement pathway). hsct is now an established treatment for a host of immunologic, metabolic, hematological, and neoplastic disorders. the ''stem cells'' may be obtained from the patient (autologous). alternatively from an hlacompatible related or unrelated donor (allogeneic). there is little risk of acute or chronic graft versus host disease (gvhd) with autologous hsct. currently there are three sources for stem cells: bone marrow, peripheral blood, and umbilical cord blood. generally with the umbilical cord stem cell transplant the speed of engraftment is lower, but so is the risk of gvhd. with peripheral blood stem cell transplant engraftment occurs faster but the risk of gvhd is also higher. with bone marrow stem cell transplant the speed of engraftment and the risk of gvhd is somewhere between the other two sources. with unrelated umbilical cord blood and t-cell depleted bone marrow or blood stem cell transplant, the risk of graft failure is higher. there is a higher risk of infection with occurrence of gvhd, and with graft failure. the conditioning regimens used to prepare the patients generally consists of high-dose chemotherapy with or without regional or total body irradiation. post transplant, there is a period of pancytopenia and though the neutrophil count usually normalizes after 3-4 weeks it is not unusual for these patients to need red cell or platelet transfusions for much longer. the risk of infection is influenced by rapidity of myeloid recovery and the rate of lymphoid reconstitution. the speed of restoration of adequate immune function is highly variable. the stem cell source, hla compatibility, purging or t-cell depletion of the graft prior to transplant, and severity of gvhd are important factors. in the early post-transplant period (first 100 days), transplant centers employ prophylactic measures to reduce the risk of infection. these measures vary between different centers, and include: • prophylactic antibiotics (for pcp, candida, hsv, cmv) • administration of ivig • environmental precautions (isolation and barrier nursing) in the early post-transplant period, patients are most susceptible to infections caused by both gram-negative and gram-positive organisms and by fungi. there is a higher risk of cmv pneumonitis in patients with gvhd (largely due to the need for immunosuppressive treatments). cmv negative recipients who receive transplant from a cmv positive donor are at highest risk for cmv pneumonitis. children who have undergone hsct and are admitted to picu have a particularly poor prognosis. pneumonia, mechanical ventilation, and the need for renal replacement therapy are especially poor prognostic factors. those with septic shock and line sepsis have the best prognosis. in addition to bacteria, the most commonly isolated organisms are cmv, rsv, adenovirus, candida, aspergillus, and pcp. in a recent report from great ormond street hospital in london, uk, only 56% of these patients survived to discharge. similar to children who have received hsct, children receiving solid organ transplants are prone to infections before and after the transplant. the main differences are: 1. patients after solid organ transplants are usually less immunosuppressed than hsct patients and are not at risk of immune reconstitution syndrome and its associated inflammatory and infectious complications. however they are at risk of surgery-related complications and postsurgical infection issues (wound infection, bacteremia, atelectasis/pneumonia, urinary tract infection). 2. infection of the transplanted organ due to latent or colonizing organisms present in either the donor or recipient can lead to invasive widespread disease in the immune suppressed post-transplant recipient. an example of this is the child with cystic fibrosis colonized with pseudomonas species; those colonized with b. cepacia are particularly at risk of developing resistant infection post lung or combined heart/lung transplants. 3. children receiving solid organ transplants are at risk of reactivation of latent infections, such as cmv. but, unlike hsct patients who most commonly present with cmv pneumonitis in recipients of solid organ transplants the cmv disease depends on the sites where the virus is latent and on the organ that has been transplanted. lung, heart/lung, and liver transplant patients are most vulnerable to systemic disease. cmv infection can precipitate rejection and increase vulnerability to other infections such as fungal infections. 4. ebv-associated post transplant lymphoproliferative disorder (ptld) is a potentially fatal complication of solid organ transplant. the risk of ptld is higher in children who were ebv seronegative prior to the sot. ebvrelated infection in sot patients may present in several different ways: asymptomatic or nonspecific viral syndrome, mononucleosis syndrome, and ptld. the latter can have a spectrum from fever, lymphadenopathy and diarrhea to full blown lymphoma. tissue biopsy is necessary to establish the diagnosis of ptld. the mainstay of therapy consists of decreasing immunosuppression. chemotherapy and biological treatments (such as anti-cd20 monoclonal antibodies) have been used. the most common reason children with hiv/aids are admitted to the picu is respiratory distress. septic shock and cns involvement (encephalopathy, encephalitis, and meningitis) are other common conditions leading to admission. in addition to the bacteria, viruses and mycoplasma that cause infections of the lower respiratory system in the non-hiv patient, there are a number of other opportunistic infections and inflammatory conditions to consider in the hiv-infected patient. these are commonly: • pneumocystis jiroveci • cmv pneumonitis • tuberculosis • fungal infections • lymphoid interstitial pneumonitis (lip) • immune reconstitution inflammatory syndrome (iris) most infants with respiratory failure will not have a previous diagnosis of hiv infection when they present. in non-endemic areas, especially during the colder season, such infants would be diagnosed and treated initially as cases of bronchiolitis. the possibility of an underlying immune deficiency and or aids should be considered in any infant who responds poorly to treatment or who has risk factors. these include failure to thrive, history of recurrent chest infections, hepatosplenomegaly, adenopathty, severe persistent oral thrush, or abnormal neurological signs. in sub saharan africa, tb and other bacterial pneumonias were common in both hiv-infected and uninfected children who presented with respiratory failure. however pneumocystis and cmv pneumonitis occurred almost exclusively in infants who were hiv-infected. the majority of cases of p. jiroveci pneumonia present in the first 6 months of life. usually these children are quite hypoxemic. high fever is uncommon compared with bacterial pneumonia. there is a diffuse, bilateral air space or interstitial involvement on the chest x-ray. occasionally there is a ''ground glass'' appearance. in an hiv positive patient with bilateral diffuse parenchymal or interstitial infiltrates on cxr, the development of pneumothorax is suggestive of p. jiroveci infection. diagnosis is by bronchoalveolar lavage (bal). even if a child develops pneumocystis while on prophylactic therapy, high-dose intravenous trimethoprim/sulfamethoxazole should be started. prophylaxis may have failed because of poor compliance. if one suspects drug resistance then other agents should be used (pentamidine, dapsone). methylprednisolone at 2-4 mg/kg/day divided in four doses should be administered in moderate to severe cases (practically all children with pneumocystis who are admitted to picu) for 5 days and then tapered. untreated, it is universally fatal. with proper therapy the mortality is less than 10%. the risk factors for mortality are the severity of respiratory failure and the severity of immunosuppression. lip in children with aids is associated with increased risk of lower respiratory tract infections including bronchiectatsis. this condition can produce severe ventilation/perfusion mismatch and hypoxemia but may also be asymptomatic. the cxr shows diffuse infiltrates and hilar lymphadenopathy persisting for >2 months despite antibiotic therapy. usually cxr changes are worse than clinical symptoms. lip can be related to ebv infection or to an exaggerated immunological response to inhaled or circulating antigens or both. steroids have been used in the treatment of symptomatic lip. coexistence of hiv and tb accelerates the course of both of these infections. the risk of miliary and extra pulmonary tb is higher in children with aids and the course is more likely to be severe and rapid. a child with hiv infection is five to ten times more at risk of active tb. in countries with high prevalence of tuberculosis, the who suggests bcg vaccination of all neonates at birth but not in any child/infant with clinical aids. for treatment of tb, please refer to the chapter in this book. mac can produce a systemic illness in children infected with hiv that is characterized by fever, chronic diarrhea, abdominal pain, malabsorption, lymphadenopathy, and obstructive jaundice. mac usually would not present with significant lung disease in these children. iris occurring weeks after the initiation of specific anti-hiv treatment may on occasion be severe enough to cause respiratory failure warranting admission to the icu, though it can also indicate latent or incipient mycobacterial disease. this (iris) is a diagnosis of exclusion and requires a bal and possibly a transbronchial biopsy. management is usually with corticosteroids. other than sepsis and respiratory failure, the other conditions that may bring the child with aids to the intensive care unit are (to name the more common ones): • cns infections (bacterial, mycobacterium, fungi, cryptococcus, viruses, and rarely cns toxoplasmosis), acute hiv encephalopathy • hiv-related cardiomyopathy • severe diarrhea and shock due to cryptosporidium or other microorganisms • liver failure due to infections or drugs • complications of antiviral medications such as acute pancreatitis, acute liver failure, stevens-johnson syndrome an important aspect of care for these children in the picu for the staff is risk of exposure to body fluids and of needle stick injury. universal exposure precautions should be strictly adhered to. it is imperative that all staff be aware of the guidelines and procedures after exposure to biological fluids in their institutions and seek advice from the occupational health department immediately if exposed. as increasing numbers of immunocompromised children with fungal and viral infections are admitted to the pediatric intensive care units, common antifungal and antiviral medications that are used against these infections are briefly reviewed. for a complete review of these topics, the reader is referred to chapters on individual fungal and viral infections in this book. increasingly systemic fungal infections have become more significant in morbidity and mortality of immunocompromised patients in intensive care units. factors that have been associated with this increase are: • use of more potent and broad-spectrum antibacterial agents • prolonged and severe neutropenia • prolonged and severe immune dysfunction (primary or secondary) • having central venous lines and invasive devices • total parenteral nutrition (tpn) the most common fungal pathogens causing systemic illness in critically ill children are candida and aspergillus species. in recent years there has been an increasing importance of uncommon fungal pathogens such as non-albicans candida species, fusarium species, trichosporon species, and dematiaceous fungi. in an immunocompromised patient with a positive fungal culture from a central venous line, current guidelines strongly advocate removal of the line. traditionally with invasive candidiasis, amphotericin b (amb) has been the first-line drug to use. however, intravenous flucanozole and itraconazole could be considered. in non-neutropenic patients positive for c. albicans (but not other candida species) fluconazole is as effective as amb. in empirical treatment of prolonged febrile neutropenic patients (>4-5 days) amb should be started. clinical trials have shown that liposomal preparations of amphotericin b (l-amb) have similar, but not better, efficacy compared with conventional amb preparations. some authors recommend a liposomal preparation of amphotericin as a preferred first-line treatment. unfortunately, high cost can be prohibitive in many parts of the world. in general, the l-amb agents cause less fever, rigors, nausea, and vomiting. they are also less nephrotoxic. voriconazole, a second generation triazole, can be used for empirical treatment of febrile neutropenic patients in place of l-amb. in patients with invasive aspergillosis it has been shown that initial therapy with voriconazole leads to a better response and improved survival with fewer side effects. echinocandins such as caspofungin have been used in combination with amb or voriconazole in more resistant cases of invasive aspergillosis with persisting fevers. micafungin and anidulafungin are two other agents in this family. antifiungals (old and new) have a large potential for side effects and drug-drug interactions. clinicians need to be aware of the specific profiles of the drugs they use from this antimicrobial family. below are the common agents likely to be used in the intensive care unit. hiv drugs have not been discussed. please refer to the chapter on hiv for more detailed discussion of these agents. there are two main groups of antiviral agents: various types of nosocomial infections in pediatric intensive care units, blood stream infections had the highest incidence, followed by lower respiratory infections and urinary tract infections. a basic mandate of medicine is ''primum non nocere'' -first do no harm. while it is inevitable that some patients may acquire nosocomial infections, these infections cause significant morbitiy and mortality. the overall mortality attributable to the various nosocomial infections within the picu has been estimated to be between 10% and 15% and infections acquired in the picu are associated with an increased risk of death, with a relative risk of 3.4. it is widely appreciated that these infections can be minimized by a number of simple interventions, most important of which is hand washing; ''clean hands save lives.'' in a review of the related literature between 1990 and 2002, it was shown that between 11% and 48% of nosocomial infections could have been prevented. blood stream infections are common. not surprisingly they are most common in those patients who are the most debilitated, receiving mechanical ventilation and have central venous lines in situ for longest time, urinary catheters and other artificial surfaces. the spectrum of infections also has a predictable frequency. gram +ve infections are the most common bacteremias (whether or not associated with a central line) followed by gram àve and then fungi. typically fungi are found in those patients on tpn or long term, broad-spectrum antibiotics and immuosuppressed. central venous lines (cvl) are used to provide secure intravenous access for administration of medications such as vasopressors and inotropes, to monitor pressures, blood oxygen saturations, and for intravenous nutrition. in a survey of picu's in the united states the rate of cvl infection was 7.6 per 1,000 catheter-days. in neonates the corresponding figure was 11.3 per 1,000 catheter-days. in europe the cvl infections occurred at a rate of 10.9 infections per 1,000 catheter-days. measures taken at time of insertion of the cvl significantly reduce the incidence of infection. strict aseptic technique (gowns/gloves/mask and wide sterile field), use of chlorhexidine (as opposed to povodine/iodine), and minimal trauma (use of ultrasound and experienced operators) are all very important factors at the time of insertion. chlorhexidine disks topically placed upon the skin at the insertion site and antibiotic impregnated lines are used by some units but not proven to be of value. cuffed and tunneled lines such as hickman lines, port-a-cath lines and picc (peripherally inserted central catheter) have a significantly lower rate of infections than standard central lines that are inserted in the intensive care. where long term therapy >1 week is required consideration should be undertaken to the insertion of one of these types of lines. site of insertion is important. the femoral vein is easy to cannulate with fewest insertion complications. however, it is more likely to become infected and thrombosed. it is good as a temporary line but early consideration should be given to removing and/or repositioning access. to prevent central line infection minimization of the ''opening'' of the line on a daily basis is important. asepsis on line ports prior to use (with alcohol or chlorhexidene) is critically important. ultimately to reduce infection rates lines should be kept for the briefest time possible. difficulty of insertion and type of ongoing therapy come into this cost-benefit analysis. when there is a suspicion that a central venous line has become infected then blood cultures should be drawn from the line and from a peripheral puncture. broad-spectrum antibiotics that cover the bacteria above (vancomycin and gentamicin for example) should be commenced. the line should be removed if at all possible. an attempt to sterilize the line may be made in the circumstances where the line is ''precious'' and not easily replaced. this can involve alternate infusion of antibiotics through all lumens and the use of antibiotic ''locks.'' this is defined as a respiratory infection that occurs 48 h post admission for mechanical ventilation. the respiratory infection is defined by: fever/hypothermia, crackles on physical exam, new respiratory infiltrates on cxr, deteriorating ventilatory status (tachypnea), cough, deteriorating gas exchange, elevated or depressed white cell counts. this may or may not be in the presence of bacterial isolates from a sterile respiratory sample (i.e., bal). there are age specific criteria for the diagnosis of vap. the cdc has produced a document that lists the specific criteria. this can be found at: http://www.cdc.gov/ncidod/hip/nnis/members/pneumonia/final/pneumocriteriav1.pdf. the incidence of vap in the picu is 6-11.6 per 1,000 ventilator-days. the diagnosis of vap is challenging and controversial. there are a number of simple interventions to reduce the incidence of vap. they are: 1. elevate head of the bed to 30 2. ventilator tubing: (a) dependant positioning of ventilator tubing to avoid aspiration (b) removal of excess condensate (c) limit frequency of tubing change unless required 3. suctioning: (a) limiting amount of saline lavage when suctioning (b) sterile technique (gloves and sterile catheter) + (gowns/masks and eye wear for protection of staff) 4. mouth care: frequent mouth cares with chlorhexidene-based wash 5. feeding: (a) early institution of feeding (b) avoidance of gastric over distension (c) limiting use of antacid therapy to high risk patients (i.e., burns, head injury) 6. avoid/limit antibiotic therapy to minimize chance of colonization with antibiotic resistant flora urinary tract infection is directly proportional to the length of time that a foley catheter is in place. frequently, patients are on antibiotics that will suppress urinary infections. however, virtually all intensive care patients with urinary catheters will acquire urinary sepsis if their stay is prolonged. more than 90% of hospital acquired uti's occur in catheterized children. the best intervention (as with central lines) is early removal of the catheter. when strict measurement of urinary output is not needed and the likelihood of urinary retention (due to illness or drugs) is not an issue then catheters should be removed. intermittent catheterization can be considered as an intervention to avoid a permanent foley catheter where retention is an issue in a longer term patient. if an icu patient develops fever or unexplained sepsis then it is mandatory that a urine specimen be sent for microscopy and culture. this is especially important in the patient with a catheter. if urinary sepsis is proven then consideration for catheter removal should be given. broad-spectrum antibiotics that cover the spectrum of bacteria listed above should be commenced. antibiotics should be specifically weighted to cover the gramnegative bacteria as these are most common. surgical site infections are a less frequent infection but none the less important source of infectious morbidity. if a wound is ''dirty'' or contaminated such as a traumatic soiled wound or contaminated peritoneum from perforated appendicitis then broad-spectrum antibiotics should be commenced in high dose. at the same time, appropriate surgical management should be undertaken to deal with the contaminated wound. the surgical team will usually offer guidance on this issue. if a wound is ''clean,'' for example a surgical incision, the surgical team will generally have a preference for antibiotic prophylaxis. commonly a second generation cephalosporin will be used. this should be given at time of the operation and for a defined and limited time thereafter. prolonged prophylaxis has been shown not to prevent inevitable wound infections and promotes emergence of multiple antibiotic resistances. for wounds that become infected in the intensive care unit, swabs should be taken of any discharge. surgical review should be initiated and the wound dressed (with frequent changes). appropriate antibiotics should commence. opening of the wound and drainage/debridement of infected tissue is the responsibility of the managing surgical team. all intensive care units should have the ability to isolate for airborne and body fluid infectious organisms. simple hand washing is very important (before and after examining patients or attending the bed side). where this is not infections in the picu practical an alcohol-based hand gel can be used. from simple hand washing a graduated appropriate degree of isolation and infection control processes should be undertaken, i.e., gowns, gloves, respiratory protection -masks with increasing filtering ability to full respirators. negative pressure rooms (with antechambers) are usually reserved for respiratory isolation for the protection of staff and other patients. positive pressure rooms are for protective isolation of the patient who is immunocompromised. negative pressure isolation and strict barrier isolation is reserved for highly infectious pathogens. sars and ebola virus are examples where this may be necessary. all intensive care staff should strictly adhere to hand washing practices (with a chlorhexidine based product). unfortunately this is not the case and medical staff are often the worst offenders in this regard. active and repeated awareness campaigns should be carried out to reinforce this basic but very important healthcare related activity. severe infectious processes are common reasons for admission to the pediatric intensive care unit. children in the picu are at risk for developing severe infections. increasingly children with a dysfunctional immune system survive their primary illnesses and are admitted to the picu with severe infections. secondary immune deficiency is common in the course of prolonged critical illness. rapid sampling of body fluids and commencement of broad-spectrum antibiotic cover is of the utmost importance. it is shown that even 5 min of delay in starting appropriate antibiotics has been associated with increased mortality. if there are reasons to believe there is an anatomical source of infection (collection of pus, infected central venous line, infected prosthesis etc.) often the antibiotics would not achieve their effects until the source of infection is dealt with effectively (surgical evacuation/ removal, drainage). optimizing the hemodynamic status of the patient (oxygen delivery, addressing preload, after load, and contractility) should start from the moment one considers the diagnosis of sepsis or severe life threatening infection. diagnostic and therapeutic interventions all go hand in hand and start in parallel from the initial encounter with the patient. it is vital that every unit has an updated knowledge of the prevalence and sensitivity of the micro organisms prevalent in their community and in the hospital. colleagues in clinical microbiology or infectious disease departments are invaluable members of any picu team in dealing with these issues. prophylactic measures such as effective hand washing, observing strict sterility while placing central venous lines, measures to reduce incidence of the vap, discontinuing the invasive lines and catheters when not indicated anymore, and adherence to universal exposure precautions should be implemented and monitored and audited regularly. they save more lives and money than much more expensive interventions. effective antibiotic stewardship, tailoring the antibiotic coverage when sensitivities of the causative organisms are known, and discontinuing broad-spectrum antibiotics as soon as clinically prudent will decrease the burden of antimicrobial resistance in the intensive care unit. supportive care of children with cancer: current therapy and guidelines from the children's oncology group incidence of pediatric and neonatal intensive care unit-acquired infections. national nosocomial infections surveillance system; pediatric prevention network viral sepsis in the pediatric intensive care unit bts guidelines for management of pleural infection in children infection control and the prevention of nosocomial infections in the intensive care unit ventilator-associated pneumonia in the pediatric intensive care unit: characterizing the problem and implementing a sustainable solution necrotizing fasciitis in children: diagnostic and therapeutic aspects clinical practice parameters for hemodynamic support of pediatric and neonatal septic shock: 2007 update from the american college of critical care medicine goal-directed management of pediatric shock in the emergency department cdc guidelines on the diagnosis of ventilator associated pneumonia toxic shock syndrome in children epidemiology, pathogenesis, and management manson's tropical disease british thoracic society guidelines for the management of pleural infection prognostic factors in pediatric cancer patients admitted to the pediatric intensive care unit epidemiology and outcome of necrotizing fasciitis in children: an active surveillance study of the canadian paediatric surveillance program acute bronchiolitis and croup ventilator-associated pneumonia in neonatal and pediatric intensive care unit patients risk factors for healthcare-associated infection in a pediatric intensive care unit a national point-prevalence survey of pediatric intensive care unit-acquired infections in the united states; pediatric prevention network concept of operations for triage of mechanical ventilation in an epidemic outcome of children requiring admission to an intensive care unit after bone marrow transplantation infections of the airway the global neonatal and pediatric sepsis initiative infections in the intensive care unit urgences et soins intinsifs paediatriques severe malaria: lessons learned from the management of critical illness in children sepsis and septic shock: a global overview pediatric critical care surge capacity critical care outcomes in the hematologic transplant recipient clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the infectious diseases society of america management of severe dengue in children infectious diseases in the pediatric intensive care unit rogers' textbook of pediatric intensive care red book: 2009 report of the committee on infectious diseases nosocomial infections in pediatric patients: a european, multicenter prospective study; european study group hand washing in the intensive care unit: a big measure with modest effects evidence behind the who guidelines: hospital care for children: what treatments are effective for the management of shock in severe dengue? epiglottitis and croup a prospective study of ventilator-associated pneumonia in children predictors of mortality in patients undergoing autologous hematopoietic cell transplantation admitted to the intensive care unit london world health organisation (2005) pocket book of hospital care for children: guidelines for the management of common illness with limited resources. who, geneva zar hj, apolles p, argent a et al (2001) the etiology and outcome of pneumonia in human immunodeficiency virus-infected children admitted to intensive care in a developing country infections in the picu key: cord-020494-d5sreohg authors: schmutzhard, e.; enzensberger, w.; schwarz, m. title: entzündliche erkrankungen date: 2006 journal: klinische neurologie doi: 10.1007/3-540-31176-9_32 sha: doc_id: 20494 cord_uid: d5sreohg nan unter einer meningitis versteht man eine entzündung von pia mater und arachnoidea. das erregerspektrum ist weit und reicht von bakterien, die hämatogen-metastatisch, fortgeleitet oder durch offene hirnverletzung zur eitrigen meningitis führen, über viren zu pilzen und parasiten. insbesondere bei den unbehandelt häufig letal verlaufenden eitrigen meningitiden ist eine rasche diagnose mit erregernachweis notwendig. unverzüglich ist daraufhin eine spezifische, der regionalen resistenzentwicklung angepasste therapie einzuleiten. die meningeale affek-tion im rahmen einer listeriose oder tuberkulose verdient aufgrund des klinischen bildes, des verlaufs und der spezifischen therapie besondere beachtung. die fungalen infektionen werden, da klinisch häufig als meningoenzephalitis imponierend, in kap. 32.3 abgehandelt. die bakterielle meningitis zeigt in europa und den usa eine inzidenz von 1-3 erkrankten/100000/jahr, wobei regionale häufungen bis 5-10/100000/jahr vorkommen. in tropischen ländern, insbesondere unter schlechten sozioökonomischen umständen, wurden inzidenzen von 70 erkrankten/100000/jahr berichtet. die altersspezifische inzi-▼ 665 denz ist bei säuglingen und kleinkindern am höchsten. beim erwachsenen wird eine bakterielle meningitis hauptsächlich durch neisseria meningitidis (meningokokken), streptococcus pneumoniae (pneumokokken) und -gelegentlich -haemophilus influenzae verursacht. eine pneumokokkenmeningitis kommt am häufigsten bei über 60-jährigen menschen, eine meningokokkenmeningitis im vorschulalter sowie zwischen dem 14. und 24. lebensjahr vor. bei bis zu 30% der eitrigen meningitiden ist ein erregernachweis nicht möglich. neben dem patientenalter und der geographischen herkunft wird das erregerspektrum der akuten bakteriellen meningitis von begleiterkrankungen sowie anderen zugrundeliegenden prädisponierenden faktoren und vorerkrankungen geprägt. die bei 70-75% der patienten mit »community acquired« bakterieller meningitis gefundenen prädisponierenden faktoren sowie die eine entwicklung der nosokomialen meningitis begünstigenden risikofaktoren sind in ⊡ tabelle 32.1 gelistet. ein wesentlicher epidemiologischer faktor ist die beobachtung der resistenzentwicklung im jeweiligen einzugsbereich. während für meningokokken in mitteleuropa nur wenige penicillinresistenzen berichtet wurden, stellt sich die resistenzsituation der pneumokokken gegenüber penicillin regional überaus unterschiedlich dar. in ungarn sind mehr als 50% der bronchial gewonnenen pneumokokken penicillinresistent, zwischen 10 und 25% der eine pneumonie verursachenden pneumokokken sind in frankreich und spanien penicillinresistent. in deutschland und österreich liegen die resistenzzahlen der pneumokokken nach wie vor sehr niedrig. vor wenigen jahren wurden in den vereinigten staaten erstmals neben penicillinresistenten auch (insbesondere gegenüber cephalosporinen der drit-ten generation) multiresistente pneumokokken isoliert. eine genaue kenntnis der resistenzsituation der einzelnen er reger erlaubt die bestmögliche adäquate initiale therapie. die gesamtmortalität der eitrigen meningitis liegt bei 10%. die erregerspezifische mortalität differiert weit: die haemophilus-influenzae-typ-b-meningitis zeigt eine mortalität von 2-5%, die meningokokkenmeningitis eine etwa 5%ige mortalität; bei auftreten einer meningokokkensepsis steigt die mortalität auf bis zu 20%. eine pneumokokkenmeningitis trägt immer noch eine mortalität von 20-32%. in den meisten fällen führt eine bakteriämie zur invasion des subarachnoidalen raumes. bakterielle replikation oder lyse führt zur freisetzung von zellwandbestandteilen (z.b. pneumokokken: lipoteichoinsäure). diese zellwandbestandteile rufen die synthese und freisetzung von zytokinen und anderen inflammatorischen mediatoren (in den liquor cerebrospinalis) z.b. reaktive sauerstoffspezies oder stickstoffmonoxid hervor. diese mediatoren aktivieren polymorphkernige leukozyten und führen zur expression von adhäsionsmolekülen an endothelzellen und granulozyten. dadurch wird die transendotheliale passage von granulozyten in den subarachnoidalraum einerseits sowie die adhäsion der granulozyten an gefäßwände andererseits gefördert. die eingewanderten weißen blutkörperchen werden durch bakterielle zerfallsprodukte oder zytokine dazu angeregt, weitere inflammatorisch wirkende substanzen freizusetzen, z.b. prostaglandine, toxische sauerstoffmetaboliten, plättchenaktivierenden faktor (paf), stick-stoffmonoxyd. diese verursachen erhöhte vaskuläre permeabilität, direkte neurotoxizität, letztlich hirnödem, veränderung des zerebralen blutflusses und steigerung des hirndrucks. eine infektion der paranasalen sinus, insbesondere auch der mastoidzellen, kann durch direkte ausbreitung zu einer sog. durchwanderungsmeningitis führen; sie birgt ein großes risiko der zerebritis und abszessbildung. das erregerspektrum, in abhängigkeit vom lebensalter, ist in ⊡ tabelle 32.2 dargestellt. grundsätzlich muss im erregerspektrum zwischen »community acquired« und nosokomialer infektion unterschieden werden ( tabelle 32.2). die kenntnis der erregerwahrscheinlichkeit in abhängigkeit vom lebensalter wird zu einem großen prozentsatz eine richtige empirische therapieentscheidung in der akutsituation erlauben. insbesondere bei mit antibiotika anbehandelten bakteriellen meningitiden, bei denen ein erregernachweis nicht mehr gelingt, ist sie essenzielle voraussetzung. leitsymptom der bakteriellen meningitis ist die trias aus allgemeinem krankheitsgefühl, heftigen kopfschmerzen/nackenschmerzen/nackensteifigkeit und fieber. häufig geht als erstsymptom eine infektion des oberen respirationstraktes voraus, diese ist in vielen fällen von malaise, myalgien, arthralgien, rückenschmerzen und allgemeinem krankheits-gefühl begleitet. innerhalb von stunden bis wenigen tagen entwickelt sich dann das klinische vollbild einer bakteriellen meningitis mit der bekannten konstellation von kopfschmerzen, photophobie, nackensteifigkeit, üblicherweise mit hohem fieber einhergehend. ein weiteres wichtiges symptom ist -bereits häufig in der frühphase zu beobachten -eine beginnende einschränkung der höheren und höchsten hirnleistungen. das weitere fortschreiten der erkrankung ist schwer vorhersehbar oder vorhersagbar. bei bis zu 40% der patienten werden in den ersten tagen generalisierte und/oder fokale zerebrale krampfanfälle beobachtet. hirnödem mit oft rascher entwicklung eines lebensbedrohlichen hirndrucks, ophthalmoplegie, hyp-bis anakusis (10-30%) und fokale neurologische herdzeichen (10%) können zu jeder zeit, d.h. auch in der frühphase, beobachtet werden. eine stauungspapille sollte immer an einen infektiösen raumfordernden prozess, z.b. hydrozephalus, abszess, subdurales empyem oder septische sinusvenenthrombose, denken lassen. die neurologische untersuchung wird durch eine sorgfältige hautuntersuchung auf petechien, purpura oder einen hämorrhagischen ausschlag ergänzt. mehr als zwei drittel der patienten mit meningokokkenmeningitis zeigen zumindest diskrete petechiale haut-bzw. schleimhautveränderungen als ausdruck der begleitenden gramnegativen sepsis. eine fulminant auftretende purpura (⊡ abb. 32 .1) zeigt einen malignen verlauf der meningokokkenerkrankung an; in einzelfällen kann eine meningokokkensepsis ohne begleitmeningitis bestehen. eine endotoxinämie führt zum klinischen bild eines gramnegativen schockgeschehens mit disseminierter intravasaler koagulopathie. eine begleitmyokarditis kompliziert den verlauf. die nackensteifigkeit kann beim komatösen pa tienten verlorengehen, bei vorliegen eines meningismus sind typische dehnungszeichen (lasègue, kernig, brudzinski) positiv. das lasègue-zeichen wird durch passives beugen des gestreckten beines im hüftgelenk (anheben des beines beim auf dem rücken liegenden patienten) ausgelöst, und deutet -falls beidseitig positiv -auf eine meningeale reizung hin. die antwort beim lasègue-zeichen sind radiku-667 lär ausstrahlende schmerzen. das kernig-zeichen wird wie das lasègue-zeichen ausgelöst, als antwort ist jedoch eine beugung im kniegelenk zu beobachten, und deutet ebenfalls auf eine meningeale reizung hin. das brudzinski-zeichen wird durch passive beugung des kopfes ausgelöst, und als antwort ist eine beugung in den kniegelenken zu beobachten. die typische klinische symptomatik einer bakte riellen meningitis führt in den meisten fällen direkt zur verdachtsdiagnose. die wichtigsten differentialdiagnosen sind in der oben stehenden übersicht aufgeführt. das entscheidende diagnostische kriterium ist die liquoruntersuchung. bei einer neurologischen herdsymptomatik, beim klinischen verdacht eines erhöhten intrakraniellen druckes, i.e. bewusstseinstrübung, dekortikationsoder dezerebrationszeichen, zustand nach generalisiertem tonisch-klonischem krampfanfall etc., muss vor der durchführung der lumbalpunktion eine zerebrale computertomographie oder mrt durchgeführt werden. grundsätzlich ist zur sicherung der diagnose eine lumbalpunktion anzustreben. ⊡ tabelle 32.3 zeigt den typischen liquorbefund bei bakterieller/eitriger meningitis im zeitlichen verlauf. die untersuchung des liquors auf zellzahl, zell art, glukose und eiweiß muss unbedingt durch die am bett stattfindende gramfärbung eines liquorausstriches und das sofortige anlegen einer liquorkultur ergänzt werden. der liquor ist bei bakterieller meningitis typischerweise trüb bis eitrig. in den allerersten stunden kann er noch wasserklar sein, in der resorptionsphase wird er wiederum klar werden. eine deutliche anspeicherung der meningen nach kontrastmittelgabe bestätigt die klinische verdachtsdiagnose einer akuten meningitis (⊡ abb. 32.2) . zirka 15% der patienten zeigen zerebrosvaskuläre (ischämische) läsionen (⊡ abb. 32 .3), ein ähnlich großer prozentsatz ein diffuses hirnödem. ein hydrozephalus (evtl. pyozephalus [⊡ abb. 32.4] ) wird in etwa 10-12% der fälle beobachtet. in seltenen fällen (2-3%) zeigen sich intrakranielle blutungen als möglicher ausdruck einer durch eine septische sinusvenenthrombose bedingten venösen stauungsblutung. bei einem liquorleck kann sich intrakraniell freie luft finden. ein hirnabszess, ein subdurales empyem, parameningeale entzündliche herde, z.b. sinusitis, mastoiditis etc., können frühzeitig durch die ct und mrt erfasst werden. in den meisten fällen findet sich bei der bakteriellen meningitis eine deutliche leukozytose mit linksverschiebung im differentialblutbild. bsg und c-reaktives protein sind deutlich erhöht. bei bereits fortgeschrittener allgemeinerkrankung, insbesondere bei beginnendem oder bereits manifest gewordenem sepsissyndrom, finden sich die typischen laborparameter mit zeichen eines multiorganversagens (harnstoff, kreatinin, leberfunktionswerte deutlich erhöht); in dieser phase kann sich eine leukopenie entwickeln, es kommt zu gerinnungsstörungen mit thrombopenie und absinken der plasmatischen gerinnungsparameter (z. b. inr). in der initialphase ist fibrinogen im sinne eines akutphasenproteins erhöht, bei fortschreiten des sepsis geschehens wird eine hypofibrinogenämie gefunden. insbesondere bei einer pneumokokkenmeningitis, häufig aber auch bei bakteriellen meningitiden anderer genese, findet sich eine flussgeschwindigkeitserhöhung als ausdruck einer vaskulitis bzw. eines vasospasmus. der zeitliche verlauf der transkrani elldopplersonographisch erhobenen flussgeschwindigkeit entspricht dem bei der spontanen sub arachnoidalblutung gefundenen: typischerweise steigt am tag 4 -5 die flussgeschwindigkeit um mehr als das doppelte an und sinkt zwischen tag 10 und 15 auf normalwerte ab. angiographisch können sich zeichen einer vaskulitis zeigen, in sehr seltenen fällen eine septische sinusvenenthrombose. elektrophysiologische untersuchungen wie eeg und evozierte potentiale (sep, aep) sind entsprechend dem klinischen verlauf unterschiedlich pathologisch; sie haben v.a. bei perakutem sehr schwerem verlauf prognostischen wert. eine radiologische untersuchung der nasennebenhöhlen, der lungen, eine hals-nasen-ohren-fachärztliche untersuchung, ein echokardiogramm und eine kardiologische un ter suchung (zum sicheren ausschluss einer endo karditis mit septischer embolisierung) komplet tie ren die möglichst frühzeitige apparative abklärung bei einem patienten mit bakterieller me ningitis. die prognose einer bakteriellen meningitis wird hauptsächlich durch das auftreten von komplikationen bestimmt. hierzu zählen arterielle und/oder venöse ischämien, diffuses hirnödem bis zur nicht beherrschbaren hirndruckerhöhung, hydrozephalus, hirnabszess, subduralempyem und bei kleinkindern subdurale ergüsse. in seltenen fällen können ein diabetes insipidus oder das syndrom der inappropriaten sekretion von antidiuretischem hormon (siadh) sowie spinale symptome beobachtet werden. bei bis zu 40% der patienten komplizieren generalisierte und/oder fokale zerebrale krampfanfälle den verlauf, bei bis zu 30% (in abhängigkeit vom erreger) werden hörstörungen beobachtet. in die antibiotische therapie der bakteriellen meningitis wird von den epidemiologischen gegebenheiten, insbesondere dem alter des patienten, der regionalen resistenzsituation der jeweiligen erreger und von evtl. zugrundeliegenden grunderkrankungen (pneumonie, paranasale infektionen, otitis, mastoiditis, zustand nach neurochirurgischer intervention, zustand nach schädel-hirn-trauma mit oder ohne rhinoliquorrhoe, zustand nach lumbalpunktion etc.) bestimmt. es ist insbesondere das vermutete pathogene agens, das die frühentscheidung über das empirisch zu verabreichende antibiotikum beeinflusst. ⊡ tabelle 32.4 zeigt die wahrscheinlichsten erreger in den entsprechenden altersgruppen und gibt das antibiotische regime, das sofort nach der diagnose einer eitrigen meningitis verabreicht werden soll, an. natürlich muss nach vorliegen des ergebnisses der gramfärbung des liquors, der kulturergebnisse (liquor, blut, rachenspülflüssigkeit) und v.a. des antibiogramms eine adjustierung des antibiotischen regimes vorgenommen werden. in weiten teilen mitteleuropas kann nach wie vor der »durchschnittliche« erwachsene patient mit einer eitrigen meningitis mit penicillin g behandelt werden. in teilen ostmitteleuropas, aber auch westeuropas (frankreich, spanien), insbesondere aber in den usa und in tropischen ländern gibt es zunehmend penicillinre sistente pneumokokkeninfektionen. diese berichte stützen sich überwiegend auf befunde, die bei pa tienten mit pneumokokkenpneumonien erhoben wurden. wenig ist bekannt über die resistenzsitua tion von pneumokokken, die aus dem liquor cerebrospinalis kultiviert und untersucht wurden. cephalosporine der 3. generation sind wirksame alternativen bei meningokokkenund pneumokokkenverdacht. an der ostküste der usa gibt es seit einigen jahren allerdings auch gegen cephalosporine der 3. generation resistente pneumokokken. die genera naegleria und acanthamoeba sind die wichtigsten protozoen, die eine primäre amöbenmeningoenzephalitis beim menschen verursachen. der klinische verlauf entspricht dem einer schwersten eitrigen hirnhautentzündung und ist differentialdiagnostisch von einer bakteriellen meningitis oft schwierig abzugrenzen. die behandlung erfolgt mit amphotericin b, kombiniert mit rifampicin. im neugeborenenalter, beim immunsupprimierten/-kompromittierten erwachsenen, aber auch gelegentlich beim sonst gesunden menschen kann listeria monozytogenes eine meningitis, meningoenzephalitis, rhombenzephalitis (⊡ abb. 32.5) sowie eine zerebritis verursachen. die grampositiven stäbchen sind gelegentlich schwer in der gramfärbung zu sehen und können grampositiven kokken (insbesondere streptokokken der gruppe b) ähneln. der klinische verlauf ist überaus variabel, von einer geringen meningitischen erkrankung bis hin zur fulminanten rhombenzephalitis und zerebritis. auffällig ist der sehr variable liquorbefund: neben einer reinen granulozytären pleozytose (bis mehr als 10 000 zellen/mm 3 ) kann auch eine fast ausschließlich mononukleäre pleozytose bestehen. das eiweiß ist üblicherweise mäßig bis deutlich erhöht, die glukosewerte sind oft gering erniedrigt. bei patienten mit zerebritis und v.a. hirnstamminfektion ist nicht selten die liquorkultur negativ, die blutkultur allerdings positiv für listeria monocytogenes. da listerien auch beim nicht immunkompromittierten menschen zu einer meningitis führen können, sollte insbe-sondere beim älteren menschen dieser erreger in die differenzialdiagnostischen überlegungen grundsätzlich miteinbezogen werden. dies ist insbesondere wichtig in hinblick auf die empi rische antibiotische initialtherapie, da listeria mo nocytogenes ausgezeichnet empfindlich gegenüber ampicillin (z.b. 4-mal 4 g) ist, jedoch überhaupt nicht empfindlich gegenüber cephalosporinen der dritten generation. die dauer der therapie einer zns-listeriose hängt von der klinischen manifestation ab; eine meningitis wird mindestens 2 wochen, eine zerebritis bzw. beginnende hirnab szessbildung bis zu 6 wochen therapiert. bei immunsupprimierten patienten kann eine sepsis (mit der typischen klinischen symptomatik wie bei einer »gramnegativen sepsis«) bestehen. nicht selten resultiert eine bakteriämie in eine metastatische aussaat der listerien in endokard, meningen und/oder gehirn. die die inzidenz beträgt beträgt 10-12/100000 personen/jahr. eine erregersicherung ist bei bis zu 45% möglich. die abgrenzung zur enzephalitis ist häufig unscharf. die altersspezifische inzidenz variiert sehr weit, bei kindern unter 1 jahr werden zahlen von 219 fällen pro 100000/jahr berichtet, bei kindern zwischen 1 und 4 jahren bis zu 19/100000/jahr. auch regional und geographisch gibt es deutliche unterschiede. enteroviren und arboviren sind in den wärmeren jahreszeiten deutlich häufiger zu beobachten. eine symptomatische therapie mit antipyretika und analgetika sowie initialer bettruhe ist zu empfehlen. bei immundefizienten patienten mit vermu teter spezifischer oder gesicherter virusätiologie werden virustatika (herpes zoster: aciclovir, zy tomegalievirus: ganciclovir, hiv: zidovudine) ge geben. in der folgenden übersicht sind die ursachen einer eosinophilen meningitis aufgelistet. diese diagnose wird gestellt, wenn mehr als 10% der im liquor cerebrospinalis gefundenen zellen eosinophile sind. in vielen fällen ist eine eosinophile meningitis nicht mit einer eosinophilie im peripheren blut zeitlich vergesellschaftet. der nachweis einer systemischen infektion mit m. tuberculosis erhärtet die diagnose: magensaft, sputum und harn sollen auf m. tuberculosis mittels ziehl-neelsen-färbung sowie kultur untersucht werden. tine-test sowie andere intradermale hauttests sind nur bedingt verwertbar, sie können bei bis zu 40% der patienten negativ sein. thoraxröntgen und thorax-ct sind bei etwa 50% der patienten mit zns-tuberkulose negativ. in ct und v.a. mrt zeigen sich charakteristische, aber nicht pathognomonische veränderungen: obstruktiver hydrozephalus, kontrastmittelanreicherung im bereich der basalen meningen sowie isch ämische läsionen können bei einem großteil der patienten in unterschiedlicher häufigkeit gefunden werden. eine zerebrale angiographie kann unspezifische vaskuläre stenosen oder okklusionen als vaskulitische veränderungen zeigen. elektrophysiologische methoden helfen in der pathogenetischen dif ferenzierung nicht weiter. die tcd kann frühzeitig eine sich entwickelnde vaskulitis entdecken helfen. nur selten ist eine biopsie der meningen für histologische, mikrobiologische und immunologische untersuchungen indiziert. die spezifische chemotherapie einer zns-tuberkulose besteht mindestens aus einer dreifachkombination mit isoniazid, rifampicin und ethambutol. bei langem bestehen bzw. klinisch oder bildgebend sehr ausgedehnten befunden wird eine vierfach-(gelegentlich eine fünffach-)therapie empfohlen. in solchen fällen werden pyrazinamid und evtl. cycloserin zugefügt. die dauer der spezifischen chemotherapie der zns-tuberkulose mit der dreifachkombination ist für mindestens 3 -6 monate, eine zweifachkombinationstherapie für weitere 6-9 monate unter entsprechenden klinischen, neuroimaging-und liquorkontrollen notwendig. die spezifischen chemotherapeutika, dosierung, applikationsart, zns-penetrationsfähigkeit sowie nebenwirkungen sind in ⊡ tabelle 32.8 aufgeführt. bei auftreten eines hydrocephalus occlusus ist die frühestmögliche durchführung eines ventrikuloatrialen bzw. ventrikuloperitonealen shunts unter der spezifischen tuberkulostatischen therapie essenziell. raumfordernde tuberkulome können eine entsprechende neurochirurgische intervention notwendig machen, normalerweise werden allerdings tuberkulome konservativ unter engmaschigem ct-monitoring behandelt. in einzelfällen wurde eine initiale größenzunahme der tuberkulome nach beginn der spezifischen chemotherapie beobachtet. bei patienten mit massiven hirnnervenausfällen, klinischen zeichen einer begleitvaskulitis sowie drohendem hydrocephalus occlusus kann die beigabe von steroiden (z. b. prednisolon 60 mg) überlegt werden. die gesamtmortalität der zns-tuberkulose beträgt 15-30%. eine chemotherapie sollte bereits bei begründetem verdacht auf eine zns-tuberkulose erfolgen. trotz adäquater therapie verschlechtert sich bei manchen patienten initial die tuberkulöse meningitis, und die tuberkulome vergrößern sich. bis zu 50% der patienten werden zumindest leichte residualsymptome davontragen, 25% deutli-che. residualsymptome können multiple infarkte, hydrozephalus und permanente hirnnervenschädigungen sein. die prognose von patienten mit atypischen mykobakteriosen ist deutlich schlechter. der hirnabszess ist eine form der eitrigen reaktion mit gewebseinschmelzung und kapselbildung, die in der mehrzahl der fälle hämatogen-metastatisch oder fortgeleitet, am häufigsten bei otitis, sinusitis und mastoiditis, entsteht. nur 10 % der hirnabszesse werden durch offene schädel-hirn-traumen oder neurochirurgische operationen hervorgerufen. die therapie erfolgt antibiotisch und ggf. neurochirurgisch. eine weitere fokale intrakranielle infektion ist das subduralempyem, ein eitriger erguss in dem von harten und weichen hirnhäuten präformierten hohlraum. das als »dringlichster aller neurochirurgischen notfälle« beschriebene subduralempyem macht 10 -20 % der fokalen intrakraniellen bakteriellen infektionen aus. die inzidenz des hirnabszesses ist während der letzten jahrzehnte, trotz antibiotika, stabil geblieben. es wird geschätzt, dass ungefähr 1 patient von 10000 krankenhausaufnahmen an einem hirnabszess leidet. in den letzten 1-2 jahrzehnten änderte sich durch das auftreten von aids sowie die zunehmenden immunsuppressionsmaßnahmen (transplantationsmedizin, tumortherapien) sowohl die häufigkeit als auch das erregerspektrum. generell kommen mehr männer als frauen (verhältnis 2:1) wegen eines hirnabszesses zur stationären aufname. hiv-assoziierten erreger (insbesondere toxoplasma gondii) werden im abschn. 32.5.7 be handelt. solitäre hirnabszesse finden sich typischerweise frontal oder temporal, seltener parietal, sehr selten zerebellar oder okzipital, intrasellär, in den stammganglien oder im hirnstamm. zu multiplen abszedierungen kommt es fast ausschließlich bei hämatogen-metastatischer genese. ⊡ tabelle 32.10 zeigt die lokalisation der hirnabszesse in abhängigkeit von prädisponierenden grunderkrankungen sowie die wahrscheinlichsten erreger. die klinische symptomatik und der klinisch-neurologische verlauf bei einem patienten mit einem/mehreren hirnabszessen kann von mild über protrahiert bis zu fulminant sein. etwa 75% der patienten mit hirnabszess haben eine anamnesedauer von weniger als 2 wochen. nur weniger als 50% zeigen die klassische trias mit fieber, kopfschmerzen und fokaler neurologischer herdsymptomatik. die klinik wird hauptsächlich durch den raumfordernden prozess bedingt, nur in seltenen fällen stehen akute infektionszeichen im vordergrund. nur 45-50% der erwachsenen haben fieber, bei kindern ist der prozentsatz mit 80% deutlich höher. die neurologische herdsymptomatik wird durch die lokalisation des abszesses, die perifokale ödementwicklung sowie evtl. zusätzlich vorhandene komplikationen (meningitis, arteriitis, septische sinusvenenthrombose) bestimmt. kopfschmerzen werden von etwa 70% berichtet, meistens mäßig bis schwer und gelegentlich halbseitenakzentuiert. in ⊡ tabelle 32.11 ist die typische symptomatik eines hirnabszesses zusammengefasst. bei klinischem verdacht auf einen hirnabszess ist der wichtigste diagnostische schritt die kraniale/zerebrale bildgebung. ct oder mrt mit und ohne km sind die diagnostischen methoden der wahl. die frühphase (zerebritis) zeigt sich in der ct häufig als hypodenses areal mit nur geringer oder teilweiser kontrastmittelaufnahme. sobald sich eine abszessmembran gebildet hat, kommt es zum typischen ringförmigen kontrastmittelenhancement mit perifokalem ödem, welches oft ausgedehnter ist als bei tumoren. vor allem bei infektion mit gasbildenden erregern kann durch einschmelzung des abszesses eine spiegelbildung zur darstellung kommen. na tivröntgen und ct mit knochenfenster zeigen entzündliche und/oder destruierende herde, insbesondere eitrige entzündliche prozesse der paranasalen sinus, des innenohrs oder der schädelknochen. die ct hat eine sensitivität von nahe 100% und eine spezifität von über 90%. die mrt scheint in den frühphasen der infektion (zerebritis) der ct überlegen zu sein und eine höhere spezifität zu haben. bei begründetem verdacht auf einen hirnabszess ist eine lumbalpunktion kontraindiziert, da der diagnostische wert der liquoruntersuchung nur sehr gering ist und die lp die gefahr einer einklemmung mit sich bringt. der liquor ist unspezifisch verändert mit vorwiegend geringer lymphozytärer pleozytose, geringer eiweißerhöhung sowie in 25% der fälle glukoseverminderung. weniger als 10% der liquorkulturen sind positiv, meist dann, wenn der hirnabszess in das ventrikelsystem rupturiert ist. bei etwa zwei dritteln der patienten findet sich eine leukozytose im peripheren blut, die hälfte der pa tienten zeigt eine beschleunigte bsg (mehr als 40 mm in der ersten stunde). bei etwas mehr als der hälfte der patienten zeigt sich eine linksverschiebung im differentialblutbild. die übrigen labor werte hängen von evtl. bestehenden grundkrank heiten bzw. zugrundeliegenden prädisponierenden fak toren ab. thoraxröntgen, thorax-ct, echokardiographie, ggf. auch abdomenultraschall und ct sind, bei entsprechendem klinischem verdacht, angezeigt. eine parameningeale infektion zeigt sich auch in nativröntgenuntersuchungen der schädelknochen. elektrophysiologische untersuchungen werden ein der klinik entsprechendes ergebnis erbringen. bei verdacht auf epileptische anfälle ist eine eeg erforderlich; eeg und evozierte potentiale können zur verlaufskontrolle dienen. es sollte möglichst versucht werden, die erreger kulturell zu isolieren und damit ein antibiogramm zu erhalten. die stereotaktische aspiration von abszessen ist mittlerweile eine diagnostische standardprozedur. bei entsprechend günstiger lokalisation kann auch eine offene neurochirurgische evakuation des abszesses in erwägung gezogen werden. beim leisesten verdacht auf eine endokarditis sind mehrfache blutkulturen bakteriologisch zielführend. bronchiallavagen bei bronchektasien bzw. lungenabszessen und die punktion von perimeningealen eitrigen herden (otitis, sinusitis) oder von intraabdominalen eitrigen herden bzw. eines pleuraempyems erlauben ggf. eine kulturelle sicherung des erregers. unter allen umständen sollte die antibiotische therapie entsprechend dem erreger geplant und eingeleitet werden. bei unbekanntem erreger bzw. in der initialphase (bis zum eintreffen des kulturergebnisses und des antibiogramms) kann mit einer kombinationstherapie -cephalosporin der 3. generation und staphylokokkenwirksames antibiotikum (oxacillin, fosfomycin, rifampicin) und anaerob wirksames antibiotikum (metronidazol) -begonnen werden. eine konservative, rein antibiotische therapie erzielt in den frühphasen der abszessbildung bzw. im zerebritischen stadium die besten ergebnisse. eine initiale antibiotische kombinationstherapie wird empfohlen, weil hirnabszesse häufig polymikrobiell sind. nach vorliegen des kulturbefundes und des antibiogramms kann auf eine monotherapie mit dem bestmöglichen antibiotikum bezüglich der antimikrobiellen kompetenz und der penetrationsfähigkeit der blut-hirn-schranke gewechselt werden. es ist dabei zu berücksichtigen, dass auch die für den hirnabszess verantwortlichen foci konservativ-antibiotisch oder chirurgisch zu sanieren sind. die dauer der antibiotischen therapie wird vom klinischen verlauf und den in 2wöchentlichen abständen durchzuführenden ct-/mrt-kontrollen abhängen. bei klinischen zeichen einer intrakraniellen drucksteigerung muss ein entsprechendes intensivmedizinisches management mit artifizieller ventila tion, osmotherapie (z.b. mannit) oder über wenige tage anhaltender verabreichung von dexamethason initiiert werden. der routinemäßige einsatz einer kortikosteroidtherapie bei hirnabszessen ist nicht durch kontrollierte studien abgesichert. möglicherweise wird der kapselbildende prozess verzögert und die antibiotikapenetration in die abszesshöhle vermindert. totalexstirpation des abszesses oder stereotaktische punk tion mit aspiration sind die derzeit im einzelfall zu entscheidenden operativen verfahren. günstig gelegene supra-oder infratentorielle abszesse sollten durch trepanation und exgrundsätzlich sollen abszesse mit einem durchmesser von mehr als 1-2 cm operativ saniert werden. kleine abszesse, insbesondere multiple kleine ab szesse mit nur geringem neurologischem defizit oder sehr ungünstig lokalisierte abszesse mit relativ geringem neurologischem defizit werden zunächst nur konservativ antibiotisch behandelt. klinisch-neurologisches und computertomographisches monitoring ist bei jeder therapiemethode unverzichtbar. eine möglichst rasche sanierung der primären infektionsquelle ist unter allen umständen anzustreben. breite, interdisziplinäre kooperation (hno, kieferchirurgie, pulmologie, thoraxchirurgie, kardiologie, allgemeinchirurgie) ist essenziell. bei rasch fortschreitender neurologischer symptomatik hat jedoch die therapie des hirnabszesses vorrang. ein patient mit hirnabszess hat eine schlechte prognose, 1. wenn die diagnose verspätet gestellt wird, 2. wenn eine ungünstige lokalisation vorliegt, 3. bei multiplen oder gekammerten abszedierenden läsionen, 4. bei ventrikeleinbruch (90-100% mortalität), 5. wenn der patient bei therapiebeginn komatös ist (80-100% mortalität), 6. wenn die antibiotische therapie unzureichend ist (cave: fehlendes kulturergebnis!), 7. bei pilzätiologie, 8. bei sehr jungem oder sehr hohem lebensalter, 9. bei sehr großen, insbesondere metastatischen abszessen. eine frühestmögliche spezifische therapie des abszesses und der grunderkrankung trägt dazu bei, die mortalität zu senken. leichtgradige neurologische langzeitfolgen treten bei 30-55% auf, etwa ein sechstel der patienten bleibt jedoch permanent stark behindert. mehr als ein drittel der patienten behält eine residualepilepsie. zwischen 10 und 20 % der fokalen intrakraniellen bakteriellen infektionen sind ein subduralempyem. dieses wurde als »the most imperative of all neurosurgical emergencies« beschrieben. das subduralempyem kommt deutlich seltener als ein hirnabszess vor (1:4). in fast allen fällen entstehen subduralempyeme per continuitatem aus einer paranasalen sinusitis, einer otitis media, einer mastoiditis oder als folge einer bakteriellen meningitis, nur selten durch infektion eines subduralen hämatoms oder hygroms nach neu ro chirurgischen operationen und/ oder penetrierenden verletzungen. in einzelfällen fanden sich hämatogen-metastatisch bedingte subduralempyeme. eine fo kale osteomyelitis liegt etwa 50% der fälle zu grunde, eine septische sinusvenenthrombose mit hä mor rhagischer infarzierung und oberflächlicher hirnabszessbildung kann ebenfalls ursache eines subduralempyems sein. bei weniger als 15% besteht eine begleitende purulente meningitis. ein perifokales hirnödem entwickelt sich sehr rasch und trägt zur verschlechterung der klinisch-neurologischen symptomatik erheblich bei. aerobe streptokokken werden in einem drittel und staphylokokken in einem sechstel der berichteten fälle aus dem eiter eines subduralen empyems kultiviert. in einzelfällen wurden pneumokokken, h. influenzae und gramnegative bakterien gezüchtet. anaerobier (inklusive bacterioides spp. ) sind häufig gefundene erreger. in den meisten fällen ist eine polymikrobielle infektion zu finden. das typische alter für ein subduralempyem ist die 2. und 3. lebensdekade, männer sind 4mal häufiger als frauen betroffen. bei 60 -90 % der patienten besteht eine -nicht selten asymptomatische -sinusitis oder otitis. die fortleitung dieser infektionen in den subduralraum führt zu fieber, herdförmigen kopfschmerzen, die später diffus werden können, zeichen einer meningealen irritation und erbrechen. innerhalb von 1-2 tagen entwickeln sich neurologische herdzeichen, entsprechend der lokalisation, und können rasch zu hirndruckentwicklung und bewusstseinsstörung führen. bei mehr als 50% der patienten werden zerebrale krampfanfälle, häufig fokal, berichtet. der verlauf eines subduralen empyems ist so rasch, dass sich nur bei weniger als 50% der patienten ein papillenödem entwickeln kann. komplikationen. eine septische sinusvenenthrombose, zerebrale krampfanfälle und hirndruckentwicklung sind potenziell lebensbedrohliche komplikationen eines subduralempyems. ein subduralempyem sollte bei jedem patienten mit meningismus und fokalem neurologischem defizit vermutet werden, insbesondere, wenn sich die klinik sehr rasch verschlechtert und auf eine hirnhemi sphäre beschränkt ist. eine vorausgehende sinusitis oder otitis macht die entwicklung eines subduralempyems besonders wahrscheinlich. bildgebung. ct und mrt (mit kontrastmittel gabe) sind die wichtigsten diagnostischen schritte mit sehr hoher sensitivität und spezifität (⊡ abb. 32.10). eine osteomyelitis oder eine sinusitis (mastoiditis etc.) kann auch im nativröntgenbild visualisiert werden. mikrobiologie. blutkulturen (anaerobier und aerobier) und punktate von nasennebenhöhlen und innenohr sollten unter allen umständen gewonnen werden. aufgrund der enormen einklemmungsgefahr ist eine lumbalpunktion absolut kontraindiziert. die antibiotische therapie sollte so schnell wie möglich empirisch bis zum eintreffen der spezifischen kulturergebnisse und der antibiogramme initiiert werden. bei erhöhtem intrakraniellem druck ist ein intensivmedizinisches/intensivneurologisches management unerläßlich, eine intubation mit artifizieller ventilation, eine osmotherapie mit mannit sowie evtl. eine dexamethasontherapie sind angezeigt. die notfallmäßige neurochirurgische entleerung, am besten mittels kraniotomie (bohrloch trepanationen sollten mit spülung des subdural raumes möglichst nur in den frühphasen ange wendet werden) ist unausweichlich notwendig, v.a. da septenbildung keine ausreichende eiterentleerung mittels eines bohr loches erlaubt. die post operative antibiotische therapie richtet sich nach dem kulturergebnis und dem antibiogramm. die initiale antibiotische therapie ist in ⊡ tabelle 32.12 aufgelistet. bei frühzeitigem therapiebeginn ist eine gute restitution zu erwarten, während jede auch nur kurzfristige verzögerung der therapieeinleitung das risiko permanenter neurologischer langzeitfolgen erhöht. die gesamtmortalität beträgt 15% -beim zu therapiebeginn wachen patienten lediglich 4-8%, beim zu therapiebeginn komatösen pa tienten 75%. fast die hälfte der überlebenden patienten leiden an einer residualepilepsie, fokal oder generalisiert. in den meisten fällen treten die ersten anfälle nach ab heilung der akuten erkrankung innerhalb von 1-1 1 / 2 jahren auf. bei der durch das herpes-simplex-virus (hsv) hervorgerufenen enzephalitis handelt es sich um eine sporadisch auftretende erkrankung. ihre inzidenz liegt bei 0,2 -0,4/100000/jahr. die hsv-enzephalitis wird meist durch das hsv typ 1 hervorgerufen. es handelt sich um eine akute, hämorrhagischnekrotisierende virusenzephalitis, die sich bevorzugt in teilen des limbischen systems im temporo-und frontobasalen hirngewebe entwickelt (meist linkshemisphärisch früher als rechts). neben einer hämatogenen entstehung bei virämie werden pathogenetisch die reaktivierung persistierender hsv-partikel in nervenzellen und eine virusausbreitung nach intrakraniell von der schädelbasis über die riechschleimhaut und den n. olfactorius (fila olfactoria) diskutiert. die hsv-enzephalitis kommt sowohl bei primärinfektionen als auch im rahmen von rekurrierenden infektionen vor, mit oder ohne haut-/schleimhautbläschen. ein bestehender immundefekt ist nicht die obligate voraussetzung für eine hsv-enzephalitis. lumbalpunktion. der liquor cerebrospinalis ist bei der hsv-enzephalitis schon frühzeitig pathologisch verändert. es zeigen sich eine pleozytose (30-300/µl, meist lymphomonozytär, anfangs auch granulozytär), eine leichte bis mäßige gesamteiweißerhöhung (0,6-1,5 g/l) und zeichen der störung der blut-liquor-schranke (albuminquotient pathologisch therapie der ersten wahl. die virustatische therapie mit aciclovir sollte bei jedem verdacht auf eine hsv-enzephalitis so früh wie möglich einsetzen, um die prognostischen chancen zu verbessern. bei entsprechender diagnostischer konstellation (zeichen der enzephalitis mit fokalen neurologischen defiziten bei mononukleär-entzündlichem liquor und initial unauffälligem ct) sollten keine weiteren mikrobiologischen »beweise« abgewartet und mit der parenteralen aciclovirtherapie begonnen werden. die standarddosierung beträgt 10 mg/kg kg alle 8 h über einen zeitraum von 14(-21) tagen. die einzeldosis wird über einen zentralen zugang als kurzinfusion mit 100 ml physiologischer nacl-lösung verabreicht, wobei die infusionszeit um 1 h liegen sollte. auf eine ausreichende allgemeine flüssigkeitszufuhr muss zum schutz der nierenfunktion geachtet werden. falls initial noch kein zentraler zugang vorhanden ist, kann die erste aciclovirgabe, in einem größeren lösungsvolumen verdünnt, auch langsam über eine große periphere armvene gegeben werden, um die zeit der therapieeinleitung zu verkürzen. bei eingeschränkter nierenfunktion muss die dosis entsprechend angepasst werden (⊡ tabelle 32.13). die zentraleuropäische frühsommermeningoenzephalitis (fsme) oder zentraleuropäische enzephalitis (cee) ist eine endemisch vorkommende erkrankung mit einem häufigkeitsgipfel im juli und august (märz bis november). ist eine aktive immunisierung durch 3-malige impfung gerechtfertigt und bei bis zu 99% der geimpften wirksam (aktive grundimmunisierung mit 0,5 ml fsme-impfstoff i.m. zu den zeitpunkten 0, 1 und 12 monate, auffrischung nach 3 jahren). allerdings müssen risiko und nutzen einer aktiven immunisierung individuell abgewogen werden, da einer fsme-erkrankungswahrscheinlichkeit von 1:500-1 : 5000 pro zeckenstich in endemiegebieten nicht ganz seltene fsme-impfkomplikationen (kopfschmerzen, polyneuritis, krampfanfälle) gegenüberstehen. die atypische chronische enzephalitiden (»prionerkrankungen«) wie die creutzfeldt-jakob-krankheit, kuru oder das bei verschiedenen viruserkrankungen kann es zu einer parainfektiösen enzephalitis kommen (perivenöse, zellulär vermittelte, entzündliche immun reaktion ohne behandlung verläuft die septische herdenzephalitis durch progrediente hirndrucksteigerung meist tödlich. erhält der patient eine frühzeitige antibiotische behandlung und werden alle eventuellen komplikationen berücksichtigt, so ist die prognose besser, die letalität liegt jedoch trotzdem noch um 50%. auch bei ausheilung der enzephalitis muss mit bleibenden residuen, auch persistierenden anfallsleiden, gerechnet werden. die fleckfieberenzephalitis wird durch rickettsien (rickettsia prowazeki) verursacht und durch den biss infizierter kleiderläuse übertragen. die erkrankung hängt daher in ihrer häufigkeit stark von den hygienischen lebensbedingungen ab und ist in westeuropa selten. die inkubationszeit liegt bei 11-12 tagen. die klinische symptomatik unterscheidet sich nicht in charakteristischer weise von anderen enzephalitiden, es kommt aber zusätzlich zum namengebenden exanthem (meist ca. 5. bis 10. tag). die untersuchung der haut auf läusebisse ist diagnostisch sinnvoll. gehäuft können sich im krankheitsverlauf sinus-und hirnvenenthrombosen entwickeln. die diagnose kann mikrobiologisch ab dem 10. krankheitstag durch blut-und liquoruntersuchung gestellt werden (weil-felix-reaktion, elisa, ift). die therapie erfolgt antibiotisch mit doxycyclin, initial 2-mal 0,2 g i.v., dann oral, bis 6 tage nach entfieberung. alter nativ: gyrasehemmer, rifampicin oder chloramphe nicol. lueserkrankungen kommen in westeuropa auch heute noch mit einer gewissen regelmäßigkeit vor. die in vielen neurologischen kliniken übliche screeninguntersuchung auf lues (tpha) ist daher durchaus sinnvoll, zumal die klinische symptomatik variabel sein kann. die erkrankung ist anonym meldepflichtig; im falle der therapieverweigerung besteht namentliche meldepflicht. einen zusätzlichen wandel hat das klinische bild im falle der koinfek tion mit hiv erfahren (32.5.6). die syphilis wird durch das in der regel sexuell übertragene treponema pallidum verursacht und verläuft über jahre und jahrzehnte in 4 typischen stadien. 3 wochen nach der ansteckung kommt es an der üblicherweise genitalen infektionsstelle im 1. stadium der krankheit (hautstadium) zur bildung eines schmerzlosen schleimhautgeschwürs, das auch ohne behandlung nach einiger zeit spontan abheilt. 6 wochen nach der infektion werden die spezifischen serologischen luesreaktionen im blut positiv. frü hestens 9 wochen nach dem infektionszeitpunkt kommt es im 2. krankheitsstadium zu einer generalisierten ausbreitung des erregers im organismus. klinisch ist dieses stadium meist gekennzeichnet von einem begleitenden spezifischen exanthem am gesamten integument. bei einem teil der patienten kommt es indiesem stadium auch bereits zu einer klinisch fassbaren neurologischen beteiligung in form einer frühluischen meningitis. wird auch dieses generalisierungsstadium undiagnostiziert und unbehandelt durchlaufen, schließt sich in der regel ein jahrelanges klinisch stummes latenzstadium an (lues latens seropositiva). nach einigen jahren kann es dann im 3. stadium der krankheit zu mesenchymalen (lues cerebrospinalis) und nach 10-20 jah wichtigster diagnostischer marker ist die spezifische luesserologie in serum und liquor. neben den vergleichsweise unspezifischen screeningparametern (tpha, vdrl) gibt es auch hochspezifische, luesbeweisende bestätigungsreaktionen (fta-abs-test, 19s-igm-fta-abs-test). die allgemeinen entzündlichen liquorveränderungen sind bei der neurolues in stadium iii und iv weniger ausgeprägt als in den früheren krankheitsstadien (z.b. bei der frühluischen meningitis), können jedoch regelmäßig nachgewiesen werden. es finden sich eine intrathekale igg-und igm-synthese, jedoch keine erhöhten iga-titer. die bildgebenden verfahren (multiple zerebrale infarkte bei der lues cerebrospinalis oder progrediente regressive hirnveränderungen bei der progressiven paralyse) und das eeg (herd-und/oder allgemeinveränderungen) erbringen keine die spezielle diagnose beweisenden befunde. eine sichere behandlung der neurolues gelingt mit der intravenösen gabe von penicillin g (2-3-mal 10 mega ie oder 4-mal 5 mega ie/tag über 14-21 tage unter erfolgreicher penicillinbehandlung ist mit einer besserung der klinischen symptomatik (infarktsymptome, demenz, exogene psychose) zu rechnen. rezidive sind bis zu 5 jahre nach erstbehandlung möglich. umgekehrt kann sich der patient nach erfolgreicher sanierung einer (neuro-) lues grundsätzlich wieder neu mit treponema pallidum infizieren. die besonderheiten der koinfektion mit hiv sind unter 32.5.6 erläutert. die erkrankung findet sich weltweit in gemäßigten, feuchten und waldreichen klimazonen, bestimmt durch die lebensbedingungen des vektors. die meisten infektionen verlaufen subklinisch (mittlere asymptomatische durchseuchung der bevölkerung in deutschland um 10%). die borreliose (nach dem ort der erstbeschreibung in den usa auch »lyme disease« genannt) wird durch bestimmte spirochäten (borrelia burgdorferi) verursacht, die durch zeckenstich (ixodes ricinus: gemeiner holzbock) auf den menschen übertragen werden. die erkrankung verläuft in »3 stadien«, von denen die ersten beiden stadien an dieser stelle nicht besprochen werden sollen (haut-und frühes organstadium). ohne antibiotische behandlung kann es nach einer latenzperiode von vielen monaten bis einigen jahren im 3. krankheitsstadium zu einem chronisch-progredienten oder mehr schubartigen enzephalomyelitischen krankheitsbild kommen. das enzephalomyelitische spätbild der neuroborreliose führt schleichend zu einer progredienten tetraparese sowie zu einer demenz. die diagnose wird serologisch gestellt, indem in serum und liquor (z.b. im elisa) spezifische igg-antikörper (in frühen stadien auch igm) nachweisbar sind und im western-blot-verfahren spezifische banden auftreten. ein teil der patienten wird bei entsprechend sorgfältiger exploration in den jahren vor der erkrankung einen oder mehrere zeckenstiche angeben, evtl. auch ein erythema chronicum migrans. diese anamnestischen angaben sind aber keine obligatorische voraussetzung für die diagnose einer neuroborreliose, da das frühstadium klinisch stumm verlaufen kann und der zeckenstich vom patienten nicht immer bemerkt wird. der liquor ist bei der neuroborreliose entzündlich verändert, mit einer pleozytose (über 30 lymphozyten/µl; typischerweise igm-haltige aktivierte b-lymphozyten) sowie einer gesamteiweißerhöhung (über 1 g/l). in der mrt zeigen sich im t2-gewichteten bild hyperintense entzündungsherde im periventrikulären marklager. der befund kann bildmorphologisch der multiplen sklerose ähneln. die therapie der wahl ist antibiotisch, wobei dem grundsätzlich wirksamen penicillin g aufgrund von in-vitro-ergebnissen cephalosporine der 3. generation vorzuziehen sind. die behandlung wird intravenös durchgeführt und sollte 14-28 tage dauern (⊡ tabelle 32.17). mit einer abnormen reaktion zu beginn der behandlung (vergleichbar der jarisch-herxheimer-reaktion bei der behandlung der lues) ist bei der spätform der neuroborreliose nicht zu rechnen (wohl aber bei der garin-bujadoux-bannwarth-polyradikulitis [akute neuroborreliose]). sollte eine allergie gegen cephalosporine bestehen, kann auf tetrazycline (z.b. doxycyclin, 0,2 g/tag i.v.) ausgewichen werden. bei tetrazyclinen muss auf eine adäquate dosierung und evtl. längere behandlungszeit (bis zu 4 wochen) geachtet werden, um ausreichende therapeutische sicherheit zu erzielen. eine liquorkontrolle nach 3-6 monaten ist sinnvoll; bei nicht ausreichend saniertem liquor ist eine erneute behandlung erforderlich. unter der beschriebenen antibiotischen behandlung mit einem cephalosporin der 3. generation ist mit einer besserung der klinischen symptomatik im verlauf von einigen wochen zu rechnen. bei den meisten parasitosen handelt es sich um in unseren breiten seltene erkrankungen, die jedoch aufgrund der steigenden zahl der fernreisen wieder an differenzialdiagnostischer bedeutung gewinnen. aufgrund der klinischen wertigkeit werden hier die zystizerkose und die echinokokkose ausführlicher besprochen. die zerebrale toxoplasmose ist in ihrer wachsenden häufigkeit eng an die ständig zunehmende zahl der hiv-infektionen und -erkrankungen gekoppelt und wird daher unter 32.5.7 detaillierter behandelt. die durchseuchung der bevölkerung mit toxoplasma gondii ist in deutschland hoch. sie steigt parallel zum lebensalter und erreicht v.a. in ländlichen regionen bis zu 80%. die infektion erfolgt über tierkontakte (katzen) und genuss von rohem fleisch. die erkrankung verläuft in der regel inapparent. eine zerebrale toxoplasmose ist im erwachsenenalter unter normalen immunologischen bedingungen sehr selten zu erwarten. die besondere situation bei hiv-kranken ist im zusammenhang mit aids (32.5.7) beschrieben. bezüglich der konnatalen toxoplasmose (neurologische leitsymptome und -befunde: mikro-oder hydrozephalus, intrakranielle verkalkungen, epileptische anfälle, mentale retardierung und augenbeteiligung) wird auf die lehrbücher der kinder-und frauenheilkunde verwiesen. bei der zystizerkose handelt es sich um die zerebrale absiedlung der finnen (cysticercus cellulosae) des schweinebandwurms (taenia solium). die zystizerkose ist nicht an besondere immunologische bedingungen geknüpft und tritt viele jahre nach aufnahme der taenieneier auf. die erkrankung verläuft multifokal (basale meningen, 4. ventrikel, großhirn; auch spinal möglich). die zystizerkose ist v.a. in mittel-und südamerika, afrika und indien gehäuft anzutreffen (reiseanamnese!). die zystizerkenenzephalitis weist in ihrem klinischen bild keine chrakteristischen besonderheiten auf. es kommt zu einem variablen progredienten organischen psychosyndrom, fokalen und generalisierten krampfanfällen, hirn-nervenlähmungen sowie zeichen des zunehmenden hirndrucks, ggf. auch zu spinalen symptomen. eine eosinophilie im differentialblutbild sowie in der liquorzytologie kann neben den allgemeinen entzündlichen liquorveränderungen bereits einen wichtigen hinweis auf diese parasitäre erkrankung geben. dazu kommen positive komplementbindungsreaktionen (kbr) auf das antigen der zystizerkenkapsel in serum und liquor und die befunde spezifischer igm-und igg-elisa. in der bildgebenden diagnostik (ct oder mrt) stellen sich multifokale rindennahe herde dar, die eine variable kontrastmittelaufnahme zeigen und bei der chronischen verlaufsform verkalkte anteile haben können (⊡ abb. 32.14). eventuell kann eine weichteilröntgenaufnahme oder eine ct der oberschenkelmuskulatur ebenfalls kleine intramuskuläre verkalkungen sichtbar machen. in der folgenden übersicht findet sich eine pathogenetisch orientierte auflistung der bei hiv-kranken vorkommenden neuromanifestationen. primäre [cdc] in atlanta/usa), manifestiert sich erst spät im krankheitsverlauf (zahl der cd4-lymphozyten meist unter 100/µl). diese neurolo gischen vollbildkomplikationen führen in der regel unbehandelt zum tode. in der therapie der hiv-krankheit sind in den letzten jahren bemerkenswerte fortschritte erzielt worden, die eine längerfristige suppression der hiv-vermehrung und eine verlangsamung des damit zusammenhängenden progredienten immundefektes erlauben. substanzen mit unterschiedlichen wirkprinzipien und angriffspunkten werden in den modernen anti-hiv-therapieschemata kombiniert eingesetzt, im sinne von zweier-oder dreierkombinationen ( tabelle 32.18). durch ein solches vorgehen lässt sich gleichzeitig das risiko einer hiv-resi stenz entwicklung vermindern. als parameter für die überprüfung der wirksamkeit einer eingeschlage nen anti-hiv-therapie steht neuerdings die direkte bestimmung der sog. »hiv-viruslast« (hiv viral load) zur verfügung, indem durch eine quantitative pcr die menge zirkulierender hiv-partikel bestimmt werden kann. zusätzlich zu den direkten anti-hiv-mitteln haben die letzten jahre auch wei terent wicklungen und fortschritte bei den behandlungsmöglichkeiten opportunistischer infek tionen erbracht. nachstehend werden die wichtigsten hiv-neuromanifestationen im einzelnen besprochen. die akute hiv-meningoenzephalitis wird nur bei einem kleinen teil der hiv-kranken beobachtet (ca. 1%) und ist damit deutlich seltener als die mononukleoseartige akute hiv-krankheit (5-10% der hiv-kranken). die diagnose kann in der akuten phase der erkrankung schwierig sein, wenn die enzephalitis der hiv-serokonversion etwas vorauseilt. in diesen fällen kann die diagnose durch einen hiv-antigennachweis in blut und liquor sowie evtl. durch eine hiv-pcr gelingen. ansonsten ist durch hiv-antikörperverlaufsuntersuchungen eine klärung evtl. erst retrospektiv möglich. der liquorbefund ist lymphozytär entzündlich. das eeg ist unspezifisch allgemeinverändert, z.t. mit zusätzlichen zeichen einer erhöhten zerebralen krampfbereitschaft. die bildgebende diagnostik (ct, mrt) ist bei der akuten hiv-enzephalitis normal. eine gesicherte spezielle therapie der akuten hiv-enzephalitis ist nicht bekannt, so dass zunächst nur unspezifische allgemeinmaßnahmen ergriffen werden können. trotzdem ist eine sinnvolle behand lung mit anti-hiv-medikamenten denkbar (nukleosidanaloga und proteinaseinhibitoren, tabelle 32.18). über den erfolg einer solchen therapie liegen aber bisher keine systematischen studien vor. die akute hiv-enzephalitis verläuft spontan günstig. es ist mit einer klinischen restitutio ad integrum innerhalb von tagen bis wochen zu rechnen. die verläufe können in ihrer schwere variieren. die akute hiv-enzephalitis prädisponiert nach derzeitigem kenntnisstand nicht für einen besonderen späteren verlauf der hiv-krankheit, auch nicht hinsichtlich primärer neurologischer hiv-spätmanifestationen. ganz im gegensatz zur akuten hiv-enzephalitis gehört die aids-enzephalopathie in die »immunologische endphase« der hiv-krankheit und zählt daher auch zu den sog. aidsdefinierenden erkrankungen, also zu den klinischen krankheitsmanifestationen, die den eintritt in das endstadium der hiv-krankheit (aids-stadium) anzeigen. die aids-enzephalopathie war in den jahren, in denen es noch keine anti-hiv-medikamente gab, die häufigste neurologische komplikation der hiv-patienten. über ein drittel aller hiv-kranken entwickelte im späten krankheitsverlauf dieses krankheitsbild. unter moderner hochaktiver antiretroviraler therapie (haart) ist die aids-enzephalopathie deutlich seltener geworden. die aids-enzephalopathie ist keine gewöhnliche chronische enzephalitis, zumal die greifbaren entzündungszeichen (liquorbefund, histologisches bild) im rahmen des progredienten immundefektes sehr gering sein können. vielmehr handelt es sich um eine primär hiv-bedingte, aber multifaktorielle hirnfunktionsstörung, die auch neurotransmitterstörungen einschließen kann. ihr zustandekommen ist regelmäßig an einen fortgeschrittenen immundefekt geknüpft (cd-4-helferzellzahlen unter 100/µl blut). weiter zeigt die klinische beobachtung, dass eine aids-enzephalopathie sich auch im zusammenhang mit anderen entzündlichen aids-komplikationen (z.b. einer pneumocystis-carinii-pneumonie) manifestieren bzw. einen verschlechterungsschub erfahren kann, mit parzieller remission nach überstehen der infektiösen zweitkomplikation. klinische charakteristika der aids-enzephalopathie sind ihre schleichend progrediente entwicklung sowie ihre ganz überwiegend psychopathologische symptomatik, neben diskreten einbußen in der feinmotorik. die patienten entwickeln über wochen und monate allmähliche störungen des kurzzeitgedächtnisses und der konzentration. die feinmotorik stö rungen können z.b. durch »finger tapping« oder reaktionszeittests standardisiert erfasst werden. zu beobachten ist auch eine nivellierung in der affektivität und ein rückgang im antrieb und in der eigeninitiative. im weiteren verlauf kann es zu einer zunehmend deutlichen einbuße höherer kognitiver leistungen kommen, wodurch die patienten nicht mehr in der lage sind, ihrem bisherigen beruf nachzugehen. in schweren fällen kann es auch zu einer hilfsbedürftigkeit im privaten lebensbereich kommen. nur ein kleinerer teil der patienten mit aids-enzephalopathie (ca. 30 %) bietet auch extrapyramidale und ataktische symptome. die diagnose der aids-enzephalopathie ist schwierig. es gibt allerdings konsensuspapiere und die deutsche geellschaft für neurologie legt entsprechende leitlinien vor. aus klinischer sicht ist die langsame krankheitsentwicklung sowie das fehlen fokal-neurologischer störungen diagnostisch richtungweisend. neben dem serologischen nachweis der bestehenden hiv-infektion kommt der immunologischen stadienzuordnung in das spätbild der hiv-krankheit große diagnostische bedeutung zu. die liquorveränderungen sind meist diskreter und chronisch-entzündlicher natur (oft normale zellzahl, allenfalls geringe pleozytose, leichte gesamt eiweißerhöhung, erhöhung des igg-anteils). der nachweis einer autochthonen spezifischen igg-erhöhung im liquor gelingt üblicherweise, ist aber kein beweis für die aids-enzephalopathie, da sie auch bei patienten ohne klinische zeichen der aids-enzephalopathie sowie in früheren stadien der hiv-krankheit anzutreffen ist. einen wichtigen diagnostischen beitrag kann das eeg leisten, wenngleich auch die veränderungen der hirnelektrischen tätigkeit nicht pathognomonisch sind. üblicherweise verlangsamt sich die eeg-grundtätigkeit parallel zur klinischen verschlechterung allmählich in den langsamen α-bereich (8-9 hz) bzw. bei schwereren klinischen bildern in den bereich der leichten allgemeinveränderung (um 7 hz) (⊡ abb. 32.15 bei erwachsenen hiv-patienten stellt die zns-toxoplasmose die häufigste opportunistische infektion des gehirns dar, 20% aller aids-patienten entwickeln in ihrem krankheitsverlauf eine toxoplasmoseenzephalitis (seit haart seltener geworden!). wenn bei einem patienten mit zns-toxoplasmose initial deutliche hirndruckzeichen bestehen (starke kopfschmerzen, erbrechen, meningismus, ausgeprägtes hirnödem in der ct), kann neben der spezifischen toxoplasmosetherapie vorübergehend zusätzlich dexamethason gegeben werden (bis 4-mal 4 mg/tag, bei besserung ausschleichend). im falle des auftretens von zerebralen krampfanfällen bei zns-toxoplasmose ist zusätzlich eine konsequente behandlung mit antikonvulsiva erforderlich (z.b. gabapentin, 900-2400 mg/tag). anders als bei carbamazepin ist dabei mit keinen relevanten wechselwirkungen mit der haart zu rechnen. unbehandelt führt die zns-toxoplasmose innerhalb weniger wochen zum tode. unter konsequenter therapie in der oben genannten weise und anschließender rezidivprophylaxe sind hingegen überlebenszeiten von über 6 monaten möglich. die endgültige individuelle prognose hängt dann nicht mehr von der zns-toxoplasmose, sondern von der hiv-grundkrankheit und ihrer optimierten behandlung ab (haart). das neurotoxin wird von clostridium botulinum sezerniert und entsteht in unsachgemäß anaerob konservierten dosennahrungsmitteln (anamnese!). das botulinumtoxin, eine zinkabhängige protease, führt zu einer präsynaptischen blockierung der azetylcholinfreisetzung an motorischen und autonomen nervenendigungen. die klinischen symptome beginnen 12 -36 h (6 h bis 8 tage) nach toxinaufnahme: allgemeine muskelschwäche (schlaffe paresen), dysphagie, dysarthrie, mydriasis, ptosis, akkomodationsstörungen, doppelbilder, mundtrockenheit, übelkeit, erbrechen, blasenatonie, paralytischer ileus, fehlende sensible störungen und ungestörtes bewusstsein. die diagnose wird durch anamnese, klinik und toxinnachweis aus patientenserum und speiseresten im mäusetierversuch gestellt (mit neutralisationstest). der liquor ist meist normal. therapeutisch wird nach eventuellem entfernen noch nicht resorbierten toxins in schwereren fällen möglichst früh trivalentes (typ a, b, e) botulismus antitoxin vom pferd gegeben, initial 500 ml (nach konjunktivaler anaphylaxieprüfung), je nach klinischem verlauf weitere 250 ml nach 4-6 h. intensiv überwachung und -pflege sind erforderlich, ggf. künstliche beatmung. die letalität liegt auch unter therapie noch bei 20-30%. das peripher und zentral durch blockade der freisetzung inhibitorischer neurotransmitter (v. a. gaba) wirkende neurotoxin wird von clostridium tetani gebildet. die aufnahme des erregers erfolgt über gartenverletzungen mit erde, tierbisse und verbrennungen (anamnese!). die klinische symptomatik beginnt nach einigen stunden bis 30 tagen, wobei eine kurze inkubationszeit einen schweren krankheitsverlauf erwarten lässt. die patienten zeigen kopf-und kieferschmerzen, unruhe, tachykardie, schwitzen, schluckstörungen, erbrechen, nackensteife, trismus, »risus sardonicus«, opisthotonus, progrediente generalisierte muskelspasmen, die durch externe reize triggerbar sind, zunehmende atemlähmung, fehlende sensible störungen und ungestörtes bewusstsein. die diagnose stützt sich auf die anamnese und das klinische bild. der nachweis von tetanustoxin im tierversuch ist möglich. im emg zeigt sich ein verlust oder eine verkürzung der »silent period« nach muskelkontraktionen. der liquor ist normal. zur therapie gehören: intensivüberwachung und -pflege, abschirmung gegen äußere reize, sedierung (z.b. mit diazepam oder chlorpromazin), ggf. künstliche beatmung, evtl. wundexzision, möglichst frühe gabe von humanem tetanusantitoxin (tetagam), 5000-10 000 ie am 1. tag, je 3000 ie an den folgetagen, außerdem aktive tetanusimpfung und antibiotische behandlung (z. b. 4-mal 5 mega-ie penicillin i.v. für 10 tage oder bei penicillinallergie 0,2 g doxycyclin/tag). die letalität liegt auch unter therapie in schweren fällen bei 40-60%. üblicherweise verläuft die infektion mit corynebacterium diphtheriae als pseudomembranöse tonsillar-oder rachendiphtherie. sie ist seit durchführung der aktiven impfung selten geworden. durch toxinbildung kann eine kraniale polyneuritis mit kaudalen hirnnervenstörungen auftreten, es finden sich aber auch fazialis-und augenmuskelparesen. proximal betonte schlaffe paresen und distal akzentuierte sensibilitätsstörungen kommen ebenfalls vor. in der behandlung wird neben penicillin g (10000 ie/kg kg für 14 tage) möglichst früh für 3 tage diphtherieantitoxin vom pferd eingesetzt (30000-50000 ie als einstündige infusion, in schwereren fällen, nach konjunktivaler anaphylaxieprüfung, doppelte dosis). gen, zuweilen begleitet von abnormem verhalten, organischer wesens änderung, verwirrtheitszuständen und exogener psychose. dann entwickelt sich sehr rasch eine demenz mit myoklonien, die besonders durch aku stische oder taktile reize ausgelöst werden. da neben zeigen sich zerebellare funktionsstörungen mit gang, stand-und zeigeataxie, störungen der basalganglienfunk tion in form von hypo-oder hyperkinesen sowie pyramidenbahnzeichen. im endsta dium sind die patienten mutistisch, sie zeigen beuge-oder streckautomatismen. der tod tritt durch pneumonien oder autonome regulationsstö rungen ein. gesichert werden kann die diagnose nur durch biopsie oder post mortem durch autopsie. sehr typisch sind periodische entladungen im eeg, die aus meist triphasischen »sharp-wave-komplexen« bestehen (⊡ abb. 32.21). allerdings sind diese periodischen eeg-veränderungen nur in bestimmten krankheitsphasen nachweisbar. deshalb werden mindestens 4 eeg-untersuchungen im verlauf gefordert. die spezifität dieser eeg-veränderungen wird mit etwa 75% bei einer sensitivität von 66% angegeben. eine cjk gilt klinisch als wahrscheinlich, wenn bei einer progredienten demenz von weniger als 2 jahren dauer und periodischen triphasischen eeg-komplexen und/oder dem nachweis von 14-3-3-protein im liquor mindestens zwei der folgenden vier symptome vorliegen: ▬ myoklonien, ▬ visuelle und/oder zerebellare störungen, ▬ pyramidale und/oder extrapyramidale störungen und ▬ akinetischer mutismus. sind diese klinischen kriterien bis auf den fehlenden nachweis der typischen eeg-veränderungen erfüllt, geht man von einer möglichen cjk aus. in der folgenden übersicht sind die diagnostischen krite rien zusammenfassend aufgelistet. (1) sicher (neuropathologisch) -bioptischer oder autoptischer nachweis des pathologischen prionproteins prp sc im hirngewebe (2) wahrscheinlich (klinisch) -demenz von < 2 jahren dauer -plus typische eeg-veränderungen und/oder nachweis von 14-3-3-protein im liquor -plus 2 von 4 symptomen: -myoklonus visuelle und/oder zerebelläre symptome pyramidenbahnzeichen und/oder extrapyramidale symptome akinetischer mutismus (3) möglich (klinisch) -wie (2), jedoch keine typischen eeg-veränderungen und keine liquoruntersuchung oder negativer 14-3-3-befund obwohl noch nicht in die offiziellen diagnosekriterien aufgenommen, gibt es noch andere liquorparameter und auffälligkeiten in der bildgebung mit hoher spezifität und sensitivität. zwar sind die üblichen liquorparameter (zellzahl, proteingehalt, intrathekale immunglobulinsynthese) unauffällig, doch lassen sich im liquor neben dem 14-3-3-protein (sensitivität >95%, spezifität >90%) noch andere spe zielle proteine nachweisen, die nach ausschluss e ines hirninfarktes, einer intrazerebralen blutung oder eines hirntumors mit einer hohen sensitivität und spezifität eine cjk anzeigen: (1) neuronenspezifische enolase (>35 ng/ml), (2) s-100 (≥4,2 ng/ml), das in großbritannien und frankreich ist über mehr als 150 patienten mit einer neuen variante der cjk (nvcjk) berichtet worden, die in zusammenhang mit der bovinen spongiformen enzephalopathie (bse) für öffentliche diskussionen gesorgt haben. die patienten erkranken durchschnittlich 40 jahre früher als die von der »klassischen« cjk betroffenen (mittel 26 jahre) und bieten ein ungewöhnliches klinisches bild mit frühzeitigem auftreten von psychiatrischen symptomen (reizbarkeit, missstimmung, angst, sozialer rückzug, interessenverlust) sowie ataktischen und sensiblen defiziten. zwar zeigen auch diese patienten eine progrediente demenz, myoklonien und extrapyramidale störungen in form von hyperkinesen, jedoch fehlen die als charakteristisch für die cjk geltenden eeg-veränderungen. die erkrankungsdauer ist mit durchschnittlich 22 monaten deutlich länger als bei der »klassischen« cjk. nvcjk-patienten zeigen im t2-gewichteten mrt das sog. pulvinar-zeichen in form einer bilateralen hyperintensität im thalamus. auch histopathologisch findet sich ein wichtiger unterschied. neben den bei cjk üblichen spongiformen veränderungen sind bei der neuen variante prionproteinplaques nachweisbar, die an kuru oder scrapie erinnern und von neuropilvakuolen »gänseblumenartig« umgeben sind. derartige prionproteinplaques sind auch bei der bse beschrieben. eine kausale verknüpfung mit der bse ist aufgrund des zeitlichen zusammenhangs des auftretens von bse und dieser neuen cjk-variante sowie des nachweises der histopathologischen ähnlichkeiten wahrscheinlich, aber bisher nicht bewiesen. bei der neuen variante lässt sich pathologisches prionprotein nicht nur im zns und auge, sondern auch in peripheren geweben (z. b. tonsillen) nachweisen. da deshalb die möglichkeit, die neue variante über blut und blutprodukte zu übertragen, nicht ausgeschlossen werden kann, wurden die gesundheitsbehörden zu neuerungen im transfusionswesen veranlasst. eine wirksame therapie der prionerkrankungen ist nicht bekannt. es werden medikamente gesucht, die die konformationsänderung von prp zum prp sc verhindern könnten. weiterführende literatur berlit p, fedel ch, tornow k, schmiedeck p (1996) 32.20a, b. histologische veränderungen bei cjk (53jäh rige patientin). a ausgeprägte spongisierung mit zahlreichen vakuolen im neuropil und gliose im gyrus parahippocam palis pfeile: reaktiv transformierte astrozyten). b fokale prionproteinimmunreaktive ablagerungen im kleinhirn (paraffinschnitt, abc-methode mit 3f4-antikörper). (prof. m. schröder, institut für neuropathologie, rwth aachen) ( farbtafel) typische periodisch auftretende triphasische »sharpwave-komplexe« im eeg eines patienten mit cjk normal host prion protein necessary for scrapie-induced neurotoxicity levels of rifampin and ciprofloxacin in nasal secretions: correlation with mic90 and eradication of nasopharyngeal carriage of bacteria dexamethasone in adults with bacterial meningitis aktuelle diagnostik und therapie opportunistischer hirnerkrankungen bei hiv-1 associated encephalopathy and myelopathy neurological complications in aids treatment of drugresistant pneumococcal pneumonia diagnostic medical parasito logy (eds) infectious diseases pneumococcal meningitis in adults: spectrum of complications and prognostic factors in a series of 87 cases utility of transcranial doppler ultrasonography in the diagnosis and follow up of tuberculous meningitis-related vasculopathy the efficacy of mr imaging in subdural empyema epilepsy following brain abscess. the evaluation of possible risk factors with emphasis on a new concept of epileptic focus formation diffusionweighted mri in patients with creutzfeldt-jakob disease bacterial brain abscess: microbiological features, epidemiological trends and therapeutic outcomes principles and practice of infectious diseases, 4th edn. churchill living stone meningococcal disease and travel risks of developing an oto genic intracranial abscess epidemiology of infectious diseases treatment of staphylococcal ventriculitis associated with external cerebrospinal fluid drains: a prospective randomised trial of intravenous compared with intraventricular vancomycin therapy bacterial meningitis: pathogenesis, pathophysiology and progress treatment of bacterial meningitis dexamethasone therapy for bacterial meningitis in children entzündliche erkrankungen des nervensystems fungal infections. in: noseworthy jh (ed) neurological therapeutics -principles and practice. dunitz protozoal infections. in: noseworthy jh (ed) neurological therapeutics -principles and practice. dunitz a randomized comparison of meropenem with cefotaxime or ceftriaxone for the treatment of bacterial meningitis in adults chemoprophylaxis for bacterial infections: principles of and application to meningococcal infections approach to the diagnosis and management of tuberculous meningitis sinusitis-induced subdural empyema clinical presentation and outcome of listeriosis in patients with and without immunosuppressive therapy central nervous system infections in the immune-competent adult differenzial diagnosis of acute meningitis: an analysis of the predictive value of inizial observation first hundred cases of variant creutzfeldt-jakob disease: retrospective case note review of early psychiatric and neurological features comparison of cryptococcal and tuberculous meningitis chronic meningitis -many causes to consider other viral infections. in: hacke w (ed) neuro critical care acute bacterial meningitis (gamma)-isoform detection distinguishes sporadic creutzfeldt-jakob disease from other dementias subdural empyema of otorhinological origin human prion protein with valine 129 prevents expression of variant cjd phenotype erweitertes krankheitsspektrum humaner spongiformer enzephalopathien oder prionkrankheiten chronic meningitis (eds) infectious diseases analysis of eeg and csf 14-3-3 protein as aids to the diagnosis of creutzfeldt-jakob disease current clinical diagnosis is creutzfeldt-jakob disease: identification of uncommon variants listeria monocytogenes meningitis in previously healthy adults: long-term follow-up komplikationen können dadurch entstehen, dass beatmungspflichtigkeit eintritt oder sich eine myokarditis entwickelt. die creutzfeldt-jakob-krankheit (cjk) gehört zusammen mit ihrer neuen variante (nvcjk), dem gerstmann-sträussler-scheinker-syndrom (gss), der kuru und der fatalen familiären schlaflosigkeit (ffs) zu den humanen spongiformen enzephalopathien. als wahrscheinlicher erreger dieser und der bei tieren vorkommenden spongiformen enzephalopathien, wie z. b. scrapie und bovine spongiforme enzephalopathie (bse), wird ein endogenes, pa thologisch verändertes protein, das prion (proteinaceous infectious agent) diskutiert. klinisch ist eine cjd wahrscheinlich, wenn zusätzlich zu einer progressiven demenz von weniger als 2 jahren dauer und typischen triphasischen »sharp-wave-komplexen« im eeg oder dem nachweis von 14-3-3-protein im liquor mindestens zwei der folgenden symptome vorliegen: myoklonien, visuelle und/oder zerebellare symptome, pyramidale und/oder extrapyramidale störungen, akinetischer mutismus. ge sichert werden kann die diagnose nur durch biop tischen oder autoptischen nachweis der spongiformen enzephalopathie und des pathologischen prionproteins. die übertragung der bse auf den menschen ist bisher nicht bewiesen, aber sehr wahrscheinlich. mit einer inzidenz von 0,5-1/1 mio. einwohner, die sich in den letzten jahren nicht geändert hat, ist die creutzfeldt-jakob-erkrankung sehr selten. frauen sind etwas häufiger betroffen als männer (1,2-1,5 :1). es handelt sich um eine erkrankung des höheren lebensalters mit einem mittleren erkrankungsbeginn von 66 jahren. bei einer durchschnittlichen erkrankungsdauer von nur 4-6 monaten versterben 90% der patienten innerhalb eines jahres. nur durch direkte inokulation infizierten gewebes ist eine übertragung von mensch zu mensch möglich. trotzdem sollten blut, liquor, urin, stuhl und kanülen als potenziell infektiös angesehen und entsprechend entsorgt werden. eine isolierpflege ist nicht erforderlich, handschuhe sollten jedoch getragen werden. bei nadelverletzungen sollte die haut für 10 min mit naoh-lösung (1 mol/l) und anschließend gründlich mit wasser gespült werden. die cjk beginnt oft mit einem wenige wochen dauernden unspezifischen neurasthenischen beschwerdebild. die patienten klagen über eine ungewöhnliche ermüdbarkeit, schlafstörungen, gliederschmerzen, schwindel, optische halluzinationen und schwer zu spezifizierende sehstörunkey: cord-005585-lc3fqhb0 authors: barbier, françois; coquet, isaline; legriel, stéphane; pavie, juliette; darmon, michael; mayaux, julien; molina, jean-michel; schlemmer, benoît; azoulay, élie title: etiologies and outcome of acute respiratory failure in hiv-infected patients date: 2009-07-03 journal: intensive care med doi: 10.1007/s00134-009-1559-4 sha: doc_id: 5585 cord_uid: lc3fqhb0 objective: to assess the etiologies and outcome of acute respiratory failure (arf) in hiv-infected patients over the first decade of combination antiretroviral therapy (art) use. methods: retrospective study of all hiv-infected patients (n = 147) admitted to a single intensive care unit (icu) for arf between 1996 and 2006. results: arf revealed the diagnosis of hiv infection in 43 (29.2%) patients. causes of arf were bacterial pneumonia (n = 74), pneumocystis jirovecii pneumonia (pcp, n = 52), other opportunistic infections (n = 19), and noninfectious pulmonary disease (n = 33); the distribution of causes did not change over the 10-year study period. two or more causes were identified in 33 patients. the 43 patients on art more frequently had bacterial pneumonia and less frequently had opportunistic infections (p = 0.02). noninvasive ventilation was needed in 49 patients and endotracheal intubation in 42. hospital mortality was 19.7%. factors independently associated with mortality were mechanical ventilation [odds ratio (or) = 8.48, p < 0.0001], vasopressor use (or, 4.48; p = 0.03), time from hospital admission to icu admission (or, 1.05 per day; p = 0.01), and number of causes (or, 3.19; p = 0.02). hiv-related variables (cd4 count, viral load, and art) were not associated with mortality. conclusion: bacterial pneumonia and pcp remain the leading causes of arf in hiv-infected patients in the art era. hospital survival has improved, and depends on the extent of organ dysfunction rather than on hiv-related characteristics. acute respiratory failure (arf) is the leading reason for intensive care unit (icu) admission in hiv-infected patients, with bacterial pneumonia and pneumocystis jirovecii pneumonia (pcp) accounting for most cases [1] [2] [3] [4] [5] [6] [7] [8] . effects of hiv infection that predispose patients to lung infections include depletion of alveolar cd4 t cells, impairment of humoral immunity, and functional alterations of granulocytes and alveolar macrophages [9, 10] . changes in the causes of arf were reported shortly after the introduction of combination antiretroviral therapy (art) [3, 6, 11] . the drop in hiv rna levels and increase in cd4 t-cell counts induced by art were associated with a reduced incidence of opportunistic infections and an increase in life expectancy [12, 13] . as a result of both primary prophylaxis and art, the percentage of arf cases due to pcp dropped from 70% to 20-40% [3, 4, 14, 15] . however, most of the available studies of arf in hiv-infected patients were conducted before 2000, at a time when art was not yet widely used, and more recent data are scarce [15] [16] [17] . thus, the possible influence of art advent on the causes and outcomes of arf in hiv-infected patients is not fully understood. the objectives of this study were to determine whether the causes and outcome of arf in hiv-infected patients have changed in the first decade following the advent of art, and to assess the effect of art use on these variables. to this end, we reviewed the medical charts of all hiv-infected patients admitted to our icu for arf between 1996 and 2006. 1 this retrospective study was conducted in the medical icu of the saint-louis hospital, a 650-bed university hospital in paris, france. all hiv-infected patients admitted for arf between 1 january 1996 and 31 december 2006, were included, unless they were receiving treatment for hematological malignancies or solid tumor. in patients with multiple admissions for arf during the study period, only the first icu stay was evaluated. arf was defined as a respiratory rate of more than 30 breaths/min and respiratory distress symptoms, pao 2 on room air of less than 60 mm hg, or need for invasive or noninvasive mechanical ventilation (mv). of note, admission policies of hivinfected patients remained identical throughout the study period in our icu and consisted in broad admission for arf, regardless of hiv-related history. the ethics committee of the french society for critical medicine approved the study. eligible patients were screened by independent query of the entire icu database by 2 of the investigators. the medical charts of the included subjects were then reviewed by 2 intensivists including a pulmonologist and an infectious disease specialist. both the icu charts and the charts from the infectious diseases department supplying hiv follow-up were reviewed. the characteristics of the hiv infection shown in table 1 were collected. aids-defining illnesses before icu admission were defined according to the latest statement of the centers for disease control and prevention (cdc) [19] . art was defined as a combination of 3 or more antiretroviral drugs belonging to at least 2 classes among the following: protease inhibitors, nucleoside reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors [20] . we defined patients as receiving art if the medication was prescribed for more than 30 days. however, patients whose medical charts from the infectious disease department indicated poor compliance with the prescribed art regimen were classified as not receiving art. poor compliance was defined as stationary viral load despite fully active antiretroviral therapy and no evidence of viral resistance according to available genotyping data, or no drug taken for more than 6 months. the demographic data, co-morbidities, and icu stay characteristics reported in tables 1 and 2 were collected. life-sustaining treatments used in the icu were recorded. the sepsis-related organ failure assessment (sofa) score was calculated in each patient and used to define extrarespiratory failures within the first 24 h of the icu stay [21] . the cause of arf was determined by consensus among all icu clinicians. four nonmutually exclusive diagnostic categories were used: bacterial pneumonia documented clinically and/or microbiologically, pcp, opportunistic lung infections other than pcp and bacterial pneumonia, and noninfectious diseases. clinically documented bacterial pneumonia was defined as an appropriate history and response to empiric antimicrobial therapy with focal pneumonia on chest x-ray, and either septic shock or predominantly neutrophils on bronchoalveolar lavage (bal) fluid examination, but no bacterial pathogen isolated. the diagnosis of pcp required documentation of p. jirovecii on respiratory samples. the diagnostic strategy, which has been described elsewhere [22] , included blood cultures, saline-induced sputum (is) for p. jirovecii testing, expectorated sputum for bacteria and mycobacteria, bal, distal protected specimen (dps), pleural fluid culture, legionella pneumophila serogroup i urinary antigen, and cryptococcus neoformans antigenemia. results are reported as medians and quartiles (25th-75th percentiles) or numbers and percentages. to provide a global overview of survival trends over time, the study period was subdivided into 3 subperiods, i.e., 1996 to 2000, 2000 to 2003, and 2004 to 2006. we sought to assess potential changes over time in term of icu admissions, frequency of inaugural arf, respective incidences of the 4 causal groups, and survival. patient characteristics were compared according to the use of art and the causes of arf using the chi-square test or fisher's exact test, as appropriate, for categorical variables, and the nonparametric wilcoxon's rank-sum test or the kruskal-wallis test for continuous variables. to investigate association between patient characteristics, use of art, and hospital death, we first performed univariate analyses to look for a significant influence of each variable on hospital mortality by logistic regression, estimating the odds ratio (or) with a 95% confidence 1 this work was presented in part at the american thoracic society international conference, 16-21 may 2008, toronto, canada (abstract no. 2086) [18] . interval (ci). variables listed in table 4 were entered into a backward, stepwise multiple logistic regression model in which hospital mortality was the outcome variable of interest. all tests were 2-sided, and p values smaller than 0.05 were considered statistically significant. analyses were done using the statview software package version 5.0 (sas institute, cary, nc). 29 .3%, and 20.6% for the same subperiods, respectively, p = 0.06) (fig.1) . at icu admission, 43 patients were on art overall (29.2%), without significant changes over the study period. no significant differences were found in terms of demographic characteristics, hiv risk factors, and co-morbidities when patients receiving art were compared with those who did not (table 1) . admission through the emergency room occurred in 89 (60.5%) patients. severity of organs failures at icu admission, assessed by the sofa score, did not differ significantly table 2) . at least 1 cause of arf was found in 145 patients (98.6%). infection was far more common than noninfectious pulmonary disease. table 3 lists the definitive diagnoses. half the patients had bacterial pneumonia, streptococcus pneumoniae being the most common pathogen. septic shock occurred in 28 (37.8%) patients with bacterial pneumonia. the second most common cause of arf was pcp, which was identified in about one-third of patients overall and in two-thirds of patients with newly diagnosed hiv infection. pneumonia due to other opportunistic pathogens was diagnosed in 19 (12.2%) patients, with mycobacterium complex tuberculosis being the most common pathogen (n = 8). table 2 compares patient characteristics across the 4 etiological groups. patients on art were more likely to be admitted for bacterial pneumonia or noninfectious pulmonary disease than other patients (p = 0.02). aidsrelated causes were significantly more prevalent in the group with newly diagnosed hiv infection (p \ 0.0001). neither the distribution of causes nor the prevalence of aids-related diagnoses changed significantly over the study period. two or more causes were found in 33 patients (22.4%). bacterial pneumonia was present concomitantly with pcp in 9 patients, other opportunistic infections in 8 patients, and noninfectious pulmonary disease in 8 patients. among the 52 patients with pcp, 8 (15.4%) also had other opportunistic infections, including 5 who had cytomegalovirus (cmv) reactivation with cytological evidence of cmv pneumonia on bronchoalveolar lavage (bal) fluid. most of the noninfectious pulmonary diseases were related to co-morbidities; examples were pulmonary edema related to heart failure and hypercapnic arf related to chronic obstructive pulmonary disease. factors independently associated with a diagnosis of pcp were diagnosis of hiv infection at icu admission (or, 5.31; ci, 1.31-21.48; p = 0.02), time from respiratory symptom onset to icu admission (or, 1.06 per day; ci, 1.02-1.1; p = 0.003), and pcp prophylaxis (or, 0.11; ci, 0.020.62, p = 0.01). steroids were given to all patients with pcp and pao 2 less than 70 mm hg on room air and/or mv (n = 46). niv was used in 22 (42.3%) of the 52 patients with pcp, of whom only 3 subsequently required tracheal intubation. invasive mv was used in 7 (13.5%) patients with pcp. pneumothorax occurred in 4 patients with pcp, including 1 patient who was receiving mv. overall in-hospital mortality was 19.7% (n = 29, including 16 deaths in the icu), and remained consistent through the study period. survival rates were not statistically different between the 4 etiological groups, even if mv was required ( table 2 ). significant results hiv human immunodeficiency syndrome, icu intensive care unit, pcp pneumocystis jirovecii pneumonia, iris immune restorationinduced syndrome, arf acute respiratory failure, copd chronic obstructive pulmonary disease, aids acquired immunodeficiency syndrome a clinically documented bacterial pneumonia was defined as an appropriate history and response to empiric antimicrobial therapy with focal pneumonia on chest x-ray, and either septic shock or predominantly neutrophils on bal fluid examination, without documented bacterial pathogen b including co-infection with haemophilus influenzae (n = 1) and staphylococcus aureus (n = 1) c including co-infection with streptococcus pneumonia (n = 1) and s. aureus (n = 1) d including the two co-infections with s. pneumoniae and h. influenzae e including rhodococcus equi (n = 1), toxoplasma gondii (n = 1), cryptococcus neoformans (n = 1) and aspergillus fumigatus (n = 1) of univariate analysis are listed in table 4 . most of variables associated with short-term prognosis reflected severity of organs failures, at admission or during the icu stay. when etiological parameters were tested, only pseudomonas aeruginosa pneumonia (or, 6.13; 95% ci, 1.29-29.14; p = 0.02) and cmv pneumonia (or, 6.69; 95% ci, 1.0642.11; p = 0.04) were found to be univariately linked to in-hospital mortality. kaposi's sarcoma was also associated with a poor prognosis (or, 8.67; 95% ci, 0.76-99.12), but the analysis was underpowered (n = 3, p = 0.08). by multivariate analysis ( this study reports the causes and short-term outcome of arf in 147 hiv-infected patients admitted to the icu during the first 10 years after the advent of art. the 19.7% hospital mortality rate is consistent with earlier evidence of recent survival gains among critically ill hiv-infected patients [11, 17] . however, in the 2 latest series from the san francisco general hospital (1996-1999 and 2000-2004) , almost 40% of hiv-infected patients with arf died before hospital discharge, and arf was among the critical illnesses associated with the worst outcomes in hiv-infected patients [14, 15] . comparable findings were obtained in the early art era [4, 6, 7, 17] . the lower mortality rate in our patients cannot be ascribed to lesser disease severity. severity of organ dysfunction was comparable to earlier studies, with onethird of patients requiring invasive mv and one-fourth requiring vasopressor support. differences in terms of hiv-related characteristics, such as cd4 cell count and hiv viral load, should be taken into account for the comparison of short-term outcome between other recent series [4, [15] [16] [17] and ours. nevertheless, and as in these previous studies, we found that these variables were not independent predictors of in-hospital mortality. the introduction of art does not explain the improved survival in our study. fewer than one-third of our patients were on art at icu admission, in keeping with other studies [3, 11, 14, 15, 17] , and this percentage showed no significant changes over the study period. it has been shown that the use of art, together with the cd4 t-cell count, influences long-term outcomes of hivinfected patients after icu discharge [2, 3] . our data suggest, however, that art may have no influence on short-term survival of hiv-infected patients admitted to the icu for arf. similarly, no independent influence of art on short-term outcome was noted in studies of hiv-infected patients admitted to the icu for any reason [17, 23] . in our study, art use was associated with nonopportunistic causes of arf and cd4 t-cell counts were significantly lower in patients with opportunistic infections. however, it should be noted that among the 104 patients with newly diagnosed hiv infection, cd4 counts and viral load were available in only 67 and 64 patients, respectively. nevertheless, the severity of organ failure at admission, as reflected by the sofa score, was not different between patients receiving art and those who did not. moreover, survival was not significantly different across the 4 etiological groups, and hiv-related causes were not independently associated with death before hospital discharge. thus, improved outcomes in patients with opportunistic pulmonary infections, mainly pcp, may contribute to explaining the lack of a survival advantage in patients on art. pcp was an independent predictor of death in most of the previous studies of critically ill hiv-infected patients, particularly when mv was needed or pneumothorax occurred, with hospital mortality rates ranging from 45% to 80% [1, 7, 11, [14] [15] [16] [24] [25] [26] . thus, the fact that pcp was slightly more frequent in other recent series [15, 16] could have contributed to the better overall survival in our cohort. however, the mortality rate in our patients with pcp was the lowest reported to date, and this cause was associated with the best short-term outcome. this difference cannot be ascribed to differences in steroids use, as steroids have been extensively prescribed to treat severe pcp since the early 1990s [27] . recent improvements in the survival of hiv-infected patients with pcp are related to advances in respiratory support modalities, such as the routine use of pressure support ventilation [28] and protective ventilation for intubated patients with pcp [29, 30] , who consistently meet criteria for acute lung injury or acute respiratory distress syndrome (ards). by multivariate analysis, independent predictors of hospital mortality included a need for invasive mv, a need for vasopressors, a longer time from hospital admission to icu admission, and the presence of more than 1 cause of arf. transfer of hiv-infected patients from another ward to the icu has been associated with higher mortality rates than icu admission from the emergency room [8] . in another study, early intensive management improved the prognosis of patients with severe bacterial sepsis [31] . in our cohort, half the patients had arf due to bacterial pneumonia, including 37.8% with septic shock. in two-thirds of the patients with bacterial pneumonia, faster icu admission from the emergency room may have allowed earlier initiation of appropriate sepsis management. thus, the lower hospital mortality rate compared to earlier studies [6, 32, 33] , most notably in patients with bacterial pneumonia, may be partly ascribable to the policy of prompt icu admission from the emergency department at our hospital. furthermore, our aggressive diagnostic strategy supplied the etiology of arf in nearly all our patients. establishing the etiological diagnosis has been shown to improve survival [34] . thus, our results suggest that hospital mortality of hiv-infected patients with arf depends chiefly on the severity of organ failure at admission and during the icu stay and that improvement in-hospital survival results primarily from advances in intensive care practices, as opposed to improvement in immune status and use of art. our results support earlier evidence that bacterial pneumonia is now a major cause of arf among hivinfected patients [4, 6, 8] . art reduces the risk of bacterial pneumonia during aids [35] , although hivinfected patients with mild cd4 t-cell depletion remain at increased risk [36] . this may explain why, despite art advent, bacterial pneumonia was the most frequent cause of arf in our study. two studies found a decrease in the incidence of pcp-related arf early in the art era [3, 4] . in our cohort, pcp was diagnosed in one-third of patients overall and was even more common among patients with previously unknown hiv infection. that incidence of pcp remains high is in keeping with recent series from san francisco and london, where pcp was the most common etiology [15, 17] . the fact that more than half the cases of pcp occurred in patients who were aware of their hiv infection suggests inadequate compliance with pcp prophylaxis. poor compliance may explain why the frequency of pcp remained unchanged throughout the study period, although the frequency of inaugural arf tended to decrease. the proportion of noninfectious non-hiv-related causes of arf was considerably higher than in studies done before the advent of art [4, 6, 24 ]. the immune system reconstitution and increased life expectancy obtained with art expose the patients to exacerbations of co-morbid conditions unrelated to hiv infection, such as chronic obstructive pulmonary disease or chronic heart failure. this observation is the main change in clinical and etiological patterns induced by art. our study has several limitations. first, it has the limitations inherent in its retrospective design. second, we defined the use of art and pcp prophylaxis as adherence to these treatments, as opposed to their prescription. we cannot exclude that some patients were misclassified as adherent or nonadherent by the infectious diseases physicians. difficulties with the evaluation of adherence occur in all studies of hiv-infected patients, even those conducted prospectively. third, our study was done in a single icu, which may have led to patient selection bias. the population admitted to an icu depends on local policies of admission and influences both etiological and outcome patterns [37] . however, hiv infection is not considered a reason for refusing admission at our icu, and the characteristics of our population are consistent with those of patients in other studies [11, 17] . in conclusion, hospital mortality of hiv-infected patients with arf depends mainly on the number and severity of organ failure at admission and during the icu stay and is not influenced by the extent of immune deficiency. hospital survival has improved when compared with studies from the pre-art era, which is not ascribable to the advent of highly active antiretroviral therapy. we believe that this evolution results from global progresses in intensive care practices, such as early icu transfer from the emergency department or other hospital wards, niv for pcp, and aggressive management of bacterial sepsis, even if our study was not designed to measure the individual effects of these procedures. finally, we found that bacterial pneumonia was the most common cause of arf in hiv-infected patients, even if pcp remains a major cause of life-threatening respiratory failure, most notably in patients whose hiv infection was previously unknown. respiratory disease trends in the pulmonary complications of hiv infection study cohort. pulmonary complications of hiv infection study group predictors of shortand long-term survival in hiv-infected patients admitted to the icu impact of haart advent on admission patterns and survival in hivinfected patients admitted to an intensive care unit intensive care in patients with hiv infection in the era of highly active antiretroviral therapy intensive care of patients with the acquired immunodeficiency syndrome: outcome and changing patterns of utilization reappraisal of the aetiology and prognostic factors of severe acute respiratory failure in hiv patients outcomes of intensive care for patients with human immunodeficiency virus infection clinical course, prognostic factors, and outcome prediction for hiv patients in the icu. the pip (pulmonary complications, icu support, and prognostic factors in hospitalized patients with hiv) study pulmonary complications of hiv infection infectious lung complications in patients with hiv/ aids characteristics and outcomes of hiv-infected patients in the icu: impact of the highly active antiretroviral treatment era epidemiology of human immunodeficiency virus-associated opportunistic infections in the united states in the era of highly active antiretroviral therapy highly active antiretroviral therapy decreases mortality and morbidity in patients with advanced hiv disease intensive care of human immunodeficiency virus-infected patients during the era of highly active antiretroviral therapy survival for patients with hiv admitted to the icu continues to improve in the current era of combination antiretroviral therapy improved survival for hiv infected patients with severe pneumocystis jirovecii pneumonia is independent of highly active antiretroviral therapy survival of hiv-infected patients in the intensive care unit in the era of highly active antiretroviral therapy acute respiratory failure (arf) in hiv-infected patients: trends in etiologies and prognostic factors on the first decade of highly active antiretroviral therapy (haart) (abstract no. 2086) revised surveillance case definitions for hiv infection among adults, adolescents, and children aged \18 months and for hiv infection and aids among children aged 18 months to \13 years--united states guidelines for using antiretroviral agents among hivinfected adults and adolescents the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure aids-related pneumocystis carinii pneumonia in the era of adjunctive steroids: implication of bal neutrophilia critical care of immunocompromised patients: human immunodeficiency virus aetiology and prognostic factors of patients with aids presenting life-threatening acute respiratory failure mechanical ventilation for pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome: is the prognosis really improved improvements in outcomes of acute respiratory failure for patients with human immunodeficiency virus-related pneumocystis carinii pneumonia a controlled trial of early adjunctive treatment with corticosteroids for pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome california collaborative treatment group noninvasive ventilation for treating acute respiratory failure in aids patients with pneumocystis carinii pneumonia low tidal volume ventilation is associated with reduced mortality in hiv-infected patients with acute lung injury meta-analysis of acute lung injury and acute respiratory distress syndrome trials early goaldirected therapy in the treatment of severe sepsis and septic shock bacterial pneumonia in hospitalized patients with hiv infection. the pulmonary complications, icu support and prognostic factors of hospitalized patients with hiv (pip) study the importance of bacterial sepsis in intensive care unit patients with acquired immunodeficiency syndrome: implications for future care in the age of increasing antiretroviral resistance management of patients with hiv in the intensive care unit effect on antiretroviral therapy on the incidence of bacterial pneumonia in patients with advanced hiv infection pyogenic bacterial lower respiratory tract infection in human immunodeficiency virus-infected patients variations in intensive care unit utilization for patients with human immunodeficiency virus-related pneumocystis carinii pneumonia: importance of hospital characteristics and geographic location acc/aha key data elements and definitions for measuring the clinical management and outcomes of patients with chronic heart failure: a report of the american college of cardiology/american heart association task force on clinical data standards (writing committee to develop heart failure clinical data standards): developed in collaboration with the standards for the diagnosis and treatment of patients with copd: a summary of the ats/ers position paper staging of chronic kidney disease: time for a course correction acknowledgments we thank antoinette wolfe for english revision of the manuscript. this study was supported by a grant from the assistance-publique hôpitaux de paris (aom 04139), a nonprofit institution.conflict of interest statement all of the authors declare that they have no conflict of interest. key: cord-009669-bcdjwpd1 authors: tsegaye, theodros solomon; pöhlmann, stefan title: the multiple facets of hiv attachment to dendritic cell lectins date: 2010-09-20 journal: cell microbiol doi: 10.1111/j.1462-5822.2010.01519.x sha: doc_id: 9669 cord_uid: bcdjwpd1 entry of enveloped viruses into host cells depends on the interactions of viral surface proteins with cell surface receptors. many enveloped viruses maximize the efficiency of receptor engagement by first binding to attachment‐promoting factors, which concentrate virions on target cells and thus increase the likelihood of subsequent receptor engagement. cellular lectins can recognize glycans on viral surface proteins and mediate viral uptake into immune cells for subsequent antigen presentation. paradoxically, many viral and non‐viral pathogens target lectins to attach to immune cells and to subvert cellular functions to promote their spread. thus, it has been proposed that attachment of hiv to the dendritic cell lectin dc‐sign enables the virus to hijack cellular transport processes to ensure its transmission to adjacent t cells. however, recent studies show that the consequences of viral capture by immune cell lectins can be diverse, and can entail negative and positive regulation of viral spread. here, we will describe key concepts proposed for the role of lectins in hiv attachment to host cells, and we will discuss recent findings in this rapidly evolving area of research. viruses need to access host cells to propagate and spread. the plasma membrane constitutes a physical barrier to infection, and enveloped viruses evolved specialized surface proteins (also termed envelope-or glycoproteins) to overcome this obstacle. these proteins mediate binding of viruses to host cells and subsequent fusion of the viral and a limiting host cell membrane, which allows the delivery of viral nucleic acid and protein into the host cell cytoplasm (harrison, 2008) . the first essential step in the entry cascade is the cognate binding of viral envelope proteins to components of the host cell surface, termed viral receptors (harrison, 2008) . receptor binding can limit infection efficiency, for example when receptor expression levels and/or receptor affinity of the viral envelope protein are low (bannert et al., 2000) . to circumvent this limitation, viruses can also engage cellular attachment factors, which promote viral binding to the cell surface and thus increase the possibility of successful receptor engagement and infectious entry. for instance, hiv can incorporate cellular proteins into its envelope, which interact with binding partners on target cells and thereby augment viral attachment and entry (cantin et al., 2005) . dendritic cells are professional antigen presenting cells, which can initiate primary and stimulate memory immune responses. immature dendritic cells are particularly adept in antigen capture, while mature dendritic cells efficiently present antigen to t cells. the major dendritic cell subsets in human blood are myeloid and plasmacytoid dendritic cells, which can produce high amounts of il-12 and ifn-a respectively (wu and kewalramani, 2006) . dendritic cells of myeloid origin also line body surfaces and attachment of hiv to these cells in the anogenital mucosa might play a prominent role in the sexual transmission of hiv, the major route of viral spread, as discussed below. initial evidence for an important role of myeloid dendritic cells in hiv transmission came from cell culture studies demonstrating that mature, myeloid dendritic cells isolated from blood boost hiv infection of cocultured t cells without becoming infected (trans-infection) (cameron et al., 1992) . subsequent studies extended these findings to monocytederived immature and mature dendritic cells (reviewed by wu and kewalramani, 2006) , model systems for myeloid dendritic cells in blood and tissues. a molecular basis for hiv trans-infection by immature dendritic cells was provided by geijtenbeek and colleagues, who showed that these cells express the lectin dc-sign (for dendritic cellspecific intercellular adhesion molecule 3-grabbing nonintegrin), and that dc-sign binds to glycans on hiv env and facilitates trans-infection of adjacent t cells (geijtenbeek et al., 2000) . based on these findings, and taking into account the natural ability of dendritic cells to migrate from the periphery into lymphoid tissue, it was proposed that sexually transmitted hiv hijacks submucosal dendritic cells via dc-sign to promote its dissemination, a concept referred to as the trojan horse model (geijtenbeek et al., 2000) . reports that other viruses and nonviral pathogens exploit dc-sign on dendritic cells to augment their spread (table 1) suggested that the trojan horse model might be paradigmatic for the host invasion by a broad spectrum of pathogens. recent work challenged important features of this model, but also demonstrated new mechanisms how pathogens can target dc-sign to propagate. in the present review, we will introduce potential consequences of hiv interactions with dc-sign and other immune cell lectins and we will discuss recent developments in the field. our focus will be on hiv, since important mechanisms underlying dc-sign-dependent augmentation of pathogen spread have been established with this virus, and apply to other viruses as well. dc-sign is expressed by monocyte-derived dendritic cells (mddcs) and by dendritic cells in mucosal and lym-phoid tissues, although concerns have been raised that dc-sign-positive cells found in some tissues might indeed be macrophages (granelli-piperno et al., 2005; gurney et al., 2005) . in addition, expression of dc-sign outside the macrophage/dendritic cell lineage has been noted; for instance a subset of b cells expresses dc-sign (rappocciolo et al., 2006a) . dc-sign is a type ii transmembrane protein, in which the following domains have been identified: a cytoplasmic domain, a transmembrane domain, a neck-region and a lectin domain. the cytoplasmic domain contains motifs involved in receptor internalization and signalling, while the neck region facilitates dc-sign tetramerization, which is required for highaffinity ligand binding (serrano-gomez et al., 2008; tabarani et al., 2009) . finally, the lectin domain, which requires calcium for its structural integrity (c-type), binds to high-mannose and fucose residues on pathogens and cellular proteins (guo et al., 2004) . trans-infection was reported to depend on dc-signmediated binding and cellular uptake of hiv into dendritic cells (geijtenbeek et al., 2000; kwon et al., 2002) , followed by intracellular transport of virions to sites of dendritic cell-t cell contact, termed infectious synapses (mcdonald et al., 2003) (fig. 1) . at the t cell site of the infectious synapse cd4 and coreceptor are concentrated (mcdonald et al., 2003) , resulting in the establishment of a microenvironment ideally suited for hiv trans-infection. collectively, these results suggest that hiv exploits mechanisms normally used for antigen presentation by dendritic cells to increase its infectivity for adjacent t cells. in the following, we will review recent work examining key features of this concept. initial work suggested that dc-sign was required for efficient mddc-mediated hiv trans-infection (geijtenbeek et al., 2000) . albeit this finding is controversial (gummuluru et al., 2003; boggiano et al., 2007) , a number of reports indicate that blockade of dc-sign by either antibodies, carbohydrates (baribaud et al., 2002; wu et al., 2002; wang et al., 2007a) or rnai (arrighi et al., 2004a,b) indeed diminishes hiv trans-infection (gurney et al., 2005) . in addition, dc-sign promotes hiv dissemination by migratory cells from cervical explants (hu et al., 2004) . however, the indicated role of dc-sign in hiv trans-infection by dendritic cells is not universal. thus, different types of dendritic cells employ different c-type lectins, dc-sign, mannose receptor (mr) and langerin, as well as cd4 for binding to hiv env, and dendritic cell maturation shifts env capture from lectins to cd4 (turville et al., 2002) . accordingly, dc-sign is believed to contribute to hiv trans-infection mediated by immature but not by mature dendritic cells (izquierdo-useros et al., 2007; wang et al., 2007a) , although one study reached a different conclusion (baribaud et al., 2002) , and coexpression of cd4 was found to diminish dc-sign-mediated trans-infection, possibly by facilitating uptake and transport of hiv into late endosomal compartments (wang et al., 2007b) . the list of lectins involved in hiv attachment to dendritic cells was recently expanded to include the c-type lectin dcir (dendritic cell immunoreceptor), but the relative contributions of dc-sign and dcir to hiv trans-infection remain to be established (lambert et al., 2008) . finally, hiv attachment to dendritic cells can also proceed in a lectin-and cd4-independent fashion (de witte et al., 2007a; hatch et al., 2009; izquierdo-useros et al., 2009 ). thus, dendritic cells have multiple means to capture hiv, and the prevalent mode of viral binding depends on the origin and the maturation status of the cells. kwon and colleagues found that hiv transfer to t cells by dc-sign-positive cells was dependent on lectinmediated viral internalization into low ph compartments (kwon et al., 2002) , in which viral infectivity was preserved (geijtenbeek et al., 2000; kwon et al., 2002) ( fig. 1 ). an ll motif in the cytoplasmic tail of dc-sign facilitates ligand endocytosis by dc-sign (engering et al., 2002) . however, a contribution of the ll motif to hiv transmission by dc-sign expressing cell lines was not observed by a subsequent study (burleigh et al., 2006) , and the role of a low ph compartment in hiv trans-infection has been questioned (nobile et al., 2005; wang et al., 2007a) . the former observation is in agreement with work demonstrating that dc-sign contributes to hiv uptake into dendritic cells, but targets the majority of internalized virions for degradation and mhc presentation (moris et al., 2004; (fig. 1) . the identification of lsp1 as a cellular binding partner of the cytoplasmic tail of dc-sign further supports such a scenario (smith et al., 2007) . thus, normal expression of lsp1 diverts hiv into the proteasome of dendritic cells, while lsp1 knockdown increases trans-infection (smith et al., 2007) . regardless of the role of dc-sign, it is undisputed that hiv is efficiently taken up into dendritic cells and important differences between immature and mature dendritic cells have been noted (frank et al., 2002; wang et al., 2007a) . thus, virions taken up into mature dendritic cells accumulate in large endocytic compartments, which resemble structures seen in macrophages upon hiv uptake by macropinocytosis (wang et al., 2007a) . in comparison, virions associated with immature dendritic cells are mostly found close to the cell surface, with only a few virions being present in intracellular vesicles, which exhibit a clathrin-coat (wang et al., 2007a) . notably, the ability to traffic hiv into deep intracellular compartments was found to correlate with protection of virus from proteases, and inhibitors of intracellular trafficking and cytoskeleton integrity were shown to inhibit trans-infection (wang et al., 2007a; , in agreement with the proposal that hiv traffics intracellularly, via a tetraspaninsorting pathway, to infectious synapses (garcia et al., 2005) . in contrast, a separate study postulated that virions reach infectious synapses exclusively by transport on the cell surface instead of travelling along intracellular routes (cavrois et al., 2007) . in agreement with this scenario, it was reported that hiv is routed towards the infectious synapse in a surface accessible, intracellular compartment (yu et al., 2008) . irrespective of the route of hiv trafficking, there is ample evidence that hiv capture does not preserve viral infectivity (turville et al., 2004; nobile et al., 2005; burleigh et al., 2006; wang et al., 2007a) , with the initially postulated conservation of viral infectivity likely being due to productive infection of the transmitting cells (nobile et al., 2005; burleigh et al., 2006) . collectively, hiv trans-infection driven by dendritic cells is short lived (hours) and dc-sign on immature dendritic cells contributes to this process in at least two ways: dc-sign promotes capture and potentially uptake of virions subsequently transferred to t cells. in addition, dc-sign seems to stimulate the formation of infectious synapses by a so far incompletely understood mechanism (arrighi et al., 2004a) . recent studies revealed a novel facet of hiv interactions with dc-sign on dendritic cells, the modulation of the immune response. it was demonstrated that binding of hiv to mddcs induces erk-dependent signal transduction, which correlates with production of the immunosuppressive cytokine il-10 and compromised maturation of dendritic cells (shan et al., 2007) . these effects were dependent on appropriate env glycosylation and on c-type lectin expression, and a role of dc-sign was postulated (shan et al., 2007) . removal of mannose-rich glycans from env improved immunogenicity of the protein (banerjee et al., 2009) , further pointing towards a role of mannose-specific lectins in immune responses shaped by dendritic cells. in agreement with these findings, hiv binding to dc-sign on dendritic cells was demonstrated to induce signalling via the rho guanine nucleotide-exchange factor larg and the small gtpase rhoa, which results in aberrant dendritic cell maturation (hodges et al., 2007) . thus, the cells fail to upregulate cd86 and mhc ii but readily form infectious synapses with t cells (hodges et al., 2007) , indicating that hiv signalling via dc-sign compromises the immune function of dendritic cells and simultaneously primes the cells for trans-infection. a separate study showed that binding of hiv to dc-sign induces signals via a multi-protein complex, including the kinase raf-1, which modulates tlr-induced cytokine production by regulating acetylation of the nfkb subunit p65 (gringhuis et al., 2007) . interestingly, activation of raf-1 was dependent on larg and rhoa and on the carbohydrate profile of the pathogen bound to dc-sign. thus, hiv and mycobacterium tuberculosis, which bound to dc-sign via high-mannose residues, activated raf-1 signalling and induced production of a cytokine profile different from that triggered by helicobacter pylori, which was due to recognition of fucose containing structures and did not involve raf-1 activation (gringhuis et al., 2009) (figs 1 and 2) . finally, signalling via tlr8 and dc-sign was required for nfkb-dependent recruitment of the transcription factor ptef-b to the viral promoter, and thus for the generation of full-length hiv transcripts in dendritic cells -a prerequisite for productive infection (gringhuis et al., 2010) (fig. 2) . however, hiv infection of dendritic cells is inefficient compared with t cells and macrophages (reviewed by wu and kewalramani, 2006) , and a contribution of this mechanism to viral spread in vivo remains to be established. in sum, dc-sign has signalling capacity, which can be exploited by pathogens to modulate immune responses and to establish productive infection of dendritic cells and adjacent target cells. langerhans cells are located in the top layer of the mucosa and are most likely the first cell type to come in contact with sexually transmitted hiv. langerhans cells are dc-sign-negative but express cd4 and the c-type lectin langerin (soilleux and coleman, 2001) . pioneer work by de witte and colleagues provided evidence that langerin constitutes a defence mechanism against hiv invasion (de witte et al., 2007b) . thus, langerin binds to env and targets bound virions into birbeck granules, an intracellular compartment specific to langerhans cells, where the virus is degraded (de witte et al., 2007b) . counter-intuitively, however, c-type lectins were reported to play a minor role in hiv entry into vaginal langerhans cells (hladik et al., 2007) , and examination of skin explants inoculated with hiv indicated hiv infection of langerhans cells, but not other types of dendritic cells, and transfer of virus from langerhans cells to t cells (kawamura et al., 2008) . the latter observations suggest that infection of langerhans cells might be an important early event in hiv transmission. such a scenario can be reconciled with the findings of de witte and colleagues, when taking into account that the barrier imposed by fig. 2. hiv induces signalling via dc-sign, which promotes dendritic cell infection and release of immunosuppressive cytokines. triggering tlr-3 or tlr-7 induces nfkb activation. concomitant engagement of dc-sign by hiv triggers raf-1-dependent signalling, which results in phosphorylation of ser276 of the nfkb subunit p65. phosphorylation of ser276 in turn induces acetylation of lysines in p65, which increases and prolongs transcription of the il-10 gene. il-10 inhibits the th1-mediated response and incapacitates the antigen presentation capabilities of dendritic cells. induction of tlr-8 signalling by hiv activates nfkb and allows synthesis of short hiv transcripts. parallel triggering of dc-sign signalling by hiv induces phosphorylation of the p65 subunit of nfkb at ser276. this phosphorylation event allows recruitment of the transcription-elongation factor ptef-b to the hiv promoter, which phosphorylates rna polymerase ii at ser2. phosphorylation increases processivity of the enzyme and thus allows synthesis of full-length hiv transcripts. langerin can be overcome by high doses of virus (de witte et al., 2007b) and that tnf-a, produced upon genital coinfections, like candida albicans, might increase permissiveness of langerhans cells to hiv infection (de jong et al., 2008b) -hypotheses that should be tested in animal models. dc-sign is targeted by different viruses which all contain envelope proteins with an appropriate glycan signature. three major consequences of viral engagement of dc-sign have been described: first, dc-sign can function as a bona fide entry receptor. in this case, expression of dc-sign is sufficient to render cells susceptible to infection. such a scenario has been suggested, e.g. for dendritic cell infection by human herpesvirus 8 (hhv-8) (rappocciolo et al., 2008) , the causative agent of karposi sarcoma and for ebolavirus infection of t cells (alvarez et al., 2002) . however, it is technically challenging to discriminate between entry mediated solely by dc-sign (receptor function) and entry augmented by dc-sign but facilitated by a coexpressed receptor, a process termed cis-infection (lee et al., 2001) . in fact, dc-sign most likely functions as an attachment factor in the context of ebolavirus entry into lymphoid cells (marzi et al., 2007) . second, dc-sign might augment cis-infection, as described above. dc-sign-driven cis-infection has been established for dengue virus infection of dendritic cells, which are important early targets of this pathogen (lozach et al., 2005) . polymorphisms in the dc-sign gene were shown to impact disease development, suggesting that cis-infection might facilitate viral spread in patients (sakuntabhai et al., 2005) . third, dc-sign can promote trans-infection of target cells. while this route has first been established for hiv, trans-infection of several viruses by dc-sign has been demonstrated, including measles virus (de witte et al., 2008) and ebolavirus (alvarez et al., 2002) , and might promote transmission of these pathogens. whether dc-sign engagement by viruses other than hiv also modulates cytokine release and thus impacts the establishment of an immune response remains to be determined. detailed information on the processes ensuing exposure of anogenital mucosa to sexually transmitted hiv is indispensable to understand viral dissemination and pathogenesis and to develop effective antiviral strategies. there is continued and well founded belief that dendritic cells play an important role in the establishment of hiv infection. however, the idea of the role of dc-sign in hiv interactions with dendritic cells has changed considerably (fig. 1) . it has become clear that dendritic cells have several means to bind and transfer hiv to cells, with c-type lectins being one of them. in addition, an appreciable protection of hiv from antiviral agents upon attachment to dendritic cells is under discussion, and the concept of long-term storage and subsequent regurgitation of infectious hiv by dendritic cells had to be abandoned. on the other hand, dc-signmediated hiv trans-infection, although found to be a short-lived process, and cis-infection might promote viral amplification in the genital mucosal, which could be critical for the establishment of the primary infection. conversely, langerin on langerhans cells was discovered to target hiv for degradation, suggesting a barrier function of this lectin. finally, intriguing new findings revealed a signalling capacity of dc-sign (and other lectins), which is exploited by hiv to compromise immune defences and to promote its spread. vaccine development should therefore encompass the optimization of the glycan profiles of candidate substances, and new strategies for immunotherapy can be envisioned. ultimately, key concepts need to be evaluated in improved cell culture systems and animal models for sexual transmission of hiv. an important but often overlooked parameter determining outcome and significance of such experiments is the source of the virus. glycosylation of hiv depends on the producer cell type, and viruses generated in macrophages are unlikely to be detected efficiently by c-type lectins on mucosal dendritic cells . consequently, these viruses might overcome the mucosal barrier with different efficiency and potentially by employing different strategies compared with viruses generated in t cells -and similar considerations apply to most if not all other dc-sign ligands. c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans dc-signmediated infectious synapse formation enhances x4 hiv-1 transmission from dendritic cells to t cells lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells enzymatic removal of mannose moieties can increase the immune response to hiv-1 gp120 in vivo the level of cd4 expression limits infection of primary rhesus monkey macrophages by a t-tropic simian immunodeficiency virus and macrophagetropic human immunodeficiency viruses quantitative expression and virus transmission analysis of dc-sign on monocyte-derived dendritic cells dc-sign interacts with mycobacterium leprae but sequence variation in this lectin is not associated with leprosy in the pakistani population helicobacter pylori modulates the t helper cell 1/t helper cell 2 balance through phase-variable interaction between lipopolysaccharide and dc-sign dendritic cell-mediated trans-enhancement of human immunodeficiency virus type 1 infectivity is independent of dc-sign infection of dendritic cells (dcs), not dc-sign-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to t cells the c-type lectin dc-sign (cd209) is an antigen-uptake receptor for candida albicans on dendritic cells dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to cd4+ t cells plunder and stowaways: incorporation of cellular proteins by enveloped viruses in vitro derived dendritic cells trans-infect cd4 t cells primarily with surface-bound hiv-1 virions dc-sign facilitates fusion of dendritic cells with human t-cell leukemia virus type 1-infected cells dendritic cell (dc)-specific intercellular adhesion molecule 3 (icam-3)-grabbing nonintegrin (dc-sign, cd209), a c-type surface lectin in human dcs, is a receptor for leishmania amastigotes sequence and expression of a membrane-associated c-type lectin that exhibits cd4-independent binding of human immunodeficiency virus envelope glycoprotein gp120 the dendritic cellspecific c-type lectin dc-sign is a receptor for schistosoma mansoni egg antigens and recognizes the glycan antigen lewis x the dendritic cell-specific adhesion receptor dc-sign internalizes antigen for presentation to t cells infectious and whole inactivated simian immunodeficiency viruses interact similarly with primate dendritic cells (dcs): differential intracellular fate of virions in mature and immature dcs hiv-1 trafficking to the dendritic cell-t-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse l-sign (cd 209l) is a liver-specific capture receptor for hepatitis c virus dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances trans-infection of t cells mycobacteria target dc-sign to suppress dendritic cell function dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin/ cd209 is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction c-type lectin dc-sign modulates toll-like receptor signaling via raf-1 kinase-dependent acetylation of transcription factor nf-kappab carbohydrate-specific signaling through the dc-sign signalosome tailors immunity to mycobacterium tuberculosis, hiv-1 and helicobacter pylori hiv-1 exploits innate signaling by tlr8 and dc-sign for productive infection of dendritic cells binding of human immunodeficiency virus type 1 to immature dendritic cells can occur independently of dc-sign and mannose binding c-type lectin receptors via a cholesterol-dependent pathway structural basis for distinct ligandbinding and targeting properties of the receptors dc-sign and dc-signr binding and transfer of human immunodeficiency virus by dc-sign+ cells in human rectal mucosa human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection viral membrane fusion glycosphingolipid composition of human immunodeficiency virus type 1 (hiv-1) particles is a crucial determinant for dendritic cell-mediated hiv-1 trans-infection initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1 activation of the lectin dc-sign induces an immature dendritic cell phenotype triggering rho-gtpase activity required for hiv-1 replication blockade of attachment and fusion receptors inhibits hiv-1 infection of human cervical tissue maturation of blood-derived dendritic cells enhances human immunodeficiency virus type 1 capture and transmission capture and transfer of hiv-1 particles by mature dendritic cells converges with the exosome-dissemination pathway dendritic cells mediate herpes simplex virus infection and transmission through the c-type lectin dc-sign tnf-alpha and tlr agonists increase susceptibility to hiv-1 transmission by human langerhans cells ex vivo significant virus replication in langerhans cells following application of hiv to abraded skin: relevance to occupational transmission of hiv dc-sign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses dc-sign specifically recognizes streptococcus pneumoniae serotypes 3 and 14 dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection the c-type lectin surface receptor dcir acts as a new attachment factor for hiv-1 in dendritic cells and contributes to trans-and cis-infection pathways cis expression of dc-sign allows for more efficient entry of human and simian immunodeficiency viruses via cd4 and a coreceptor differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein e2 dendritic cellspecific intercellular adhesion molecule 3-grabbing nonintegrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals recruitment of hiv and its receptors to dendritic cell-t cell junctions dc-sign enhances infection of cells with glycosylated west nile virus in vitro and virus replication in human dendritic cells induces production of ifn-alpha and tnfalpha dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus analysis of the interaction of ebola virus glycoprotein with dc-sign (dendritic cellspecific intercellular adhesion molecule 3-grabbing nonintegrin) and its homologue dc-signr dc-sign promotes exogenous mhc-i-restricted hiv-1 antigen presentation dendritic cells and hiv-specific cd4+ t cells: hiv antigen presentation, t-cell activation, and viral transfer dendritic-cellspecific icam3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquitocell-derived dengue viruses covert human immunodeficiency virus replication in dendritic cells and in dc-sign-expressing cells promotes long-term transmission to lymphocytes hepatitis c virus glycoproteins interact with dc-sign and dc-signr dc-sign on b lymphocytes is required for transmission of hiv-1 to t lymphocytes dc-sign is a receptor for human herpesvirus 8 on dendritic cells and macrophages human herpesvirus 8 infects and replicates in primary cultures of activated b lymphocytes through dc-sign a variant in the cd209 promoter is associated with severity of dengue disease structural requirements for multimerization of the pathogen receptor dendritic cell-specific icam3-grabbing non-integrin (cd209) on the cell surface hiv-1 gp120 mannoses induce immunosuppressive responses from dendritic cells leukocyte-specific protein 1 interacts with dc-sign and mediates transport of hiv to the proteasome in dendritic cells langerhans cells and the cells of langerhans cell histiocytosis do not express dc-sign dc-sign neck domain is a ph-sensor controlling oligomerization: saxs and hydrodynamic studies of extracellular domain dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells diversity of receptors binding hiv on dendritic cell subsets immunodeficiency virus uptake, turnover, and 2-phase transfer in human dendritic cells functionally distinct transmission of human immunodeficiency virus type 1 mediated by immature and mature dendritic cells cd4 coexpression regulates dc-signmediated transmission of human immunodeficiency virus type 1 dc-sign mediates avian h5n1 influenza virus infection in cis and in trans macropinocytosis and cytoskeleton contribute to dendritic cell-mediated hiv-1 transmission to cd4+ t cells measles virus targets dc-sign to enhance dendritic cell infection syndecan-3 is a dendritic cell-specific attachment receptor for hiv-1 langerin is a natural barrier to hiv-1 transmission by langerhans cells dc-sign and cd150 have distinct roles in transmission of measles virus from dendritic cells to t-lymphocytes dendritic-cell interactions with hiv: infection and viral dissemination functional evaluation of dc-sign monoclonal antibodies reveals dc-sign interactions with icam-3 do not promote human immunodeficiency virus type 1 transmission ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign hiv traffics through a specialized, surface-accessible intracellular compartment during trans-infection of t cells by mature dendritic cells we thank tf schulz for support and the bmbf (grant 01ki 0703) and dfg (sfb 900) for funding. we apologize to all colleagues whose work could not be cited because of space constraints. key: cord-017719-8lfn6mih authors: böhles, hansjosef; qirshi, mayyada title: infestationen und infektionen bei migranten – die wichtigsten erkrankungen date: 2018-02-09 journal: transkulturelle medizin doi: 10.1007/978-3-662-56035-8_10 sha: doc_id: 17719 cord_uid: 8lfn6mih skabies (krätze) ist bei migranten sehr häufig. über den gängen der krätzmilbe ist die haut entzündlich verändert. an händen und füssen sind die veränderungen vor allem in den interdigitalen räumen erkennbar. kopfläuse nehmen als problem zu. sie sind nur am menschlichen kopf überlebensfähig. flohstiche entstehen auch an bedeckten körperstellen. an der stichstelle entwickelt sich eine stark juckende quaddel mit einer zentralen blutung. die bettwanze sticht in der nacht und saugt blut. stiche sind typischerweise longitudinal angeordnet. unter den parasiten haben würmer eine große bedeutung. bei kindern in deutschland kommen madenwürmer (oxyuren) am häufigsten vor. wurmeier werden anal, perianal und auch vaginal abgelegt und führen zu einem starken nächtlichen analen pruritus. unter den infektionen hat die tuberkulose bei migranten eine höhere prävalenz als in deutschland gewohnt. unter den migranten aus dem subsaharischen afrika ist auf das vorliegen von hiv-infektionen zu achten. milben sterben nach der begattung. mit einer größe von bis zu 0,5 mm sind sie mit bloßem auge gerade noch zu erkennen. typisch für die milben ist ihr als bräunliches dreieck erscheinender vorderleib. die übertragung erfolgt durch körperkontakt und weniger durch kleidung oder wäsche. für eine infestation reicht die übertragung eines einzigen begatteten weibchens. skabies ist eine sehr stark juckende hauterkrankung. neben den pathognomonischen gängen sind rotbraune, ekzematöse hautveränderungen, die eine zelluläre immunreaktion auf die milben darstellen, typisch. die hautveränderungen über den gängen sind u.u. leicht erhaben und zeigen eine "kommaförmige" rötung. am ende des ganges kann die milbe als brauner punkt erkennbar sein. zu den prädilektionsstellen gehören: 5 kopf 5 gesicht 5 palmoplantarregion, die im frühen kindesalter, mit vesikeln und pusteln, besonders typisch verändert ist. die auftretenden papulösen hautveränderungen können auch nach erfolgreicher behandlung noch über monate bestehen bleiben (nicht mehr kontagiöse postskabiöse granulome). die hautreaktion ist von der milbenlast abhängig. extreme sind dabei: 5 "gepflegte" skabies mit nur marginalen ekzematoiden hautveränderungen. sie kommt bei patienten mit sehr guter körperhygiene vor. die zahl der milben ist dabei geringer als bei der klassischen skabies. 5 skabies crustosa (früher scabies norvegica). diese form ist durch einen massiven milbenbefall gekennzeichnet. eine begünstigende rolle spielen dabei eine immunsuppression (z. b. hiv), schwere zusätzliche erkrankungen, eine medikamentöse immunsuppression oder ein hohes lebensalter. diese form ist durch eine psoriasiforme palmoplantare hyperkeratose und auch eine erythrodermie gekennzeichnet. bei kindern ist sie eher selten. differenzialdiagnostisch muss vor allem bei afrikanischen kindern im 1. und 2. lebensjahr die acropustolosis im säuglings-und kleinkindesalter einbezogen werden. es bilden sich dabei stark juckende pusteln an handflächen und fußsohlen aus, die als skabies verkannt werden können. die therapie der wahl ist in allen altersgruppen die topische anwendung von 5 %igem permethrin (infectoscab ® also weder ein larven-noch ein puppenstadium. die aus dem ei schlüpfende nymphe sieht bereits wie eine kleine erwachsene laus aus. nach dem schlüpfen der nymphen bleiben die leeren eihüllen (nissen) am haar kleben. nissen sind somit nicht infektiös. läuse sind vektoren für bakterielle pathogene. nachgewiesen ist die übertragung von rickettsia prowazekii (erreger des klassischen fleckfiebers), von borrelia recurrentis (erreger des läuserückfallfiebers) und von bartonella quintana (erreger des fünf-tage-fiebers) boutellis et al. 2013) . durch flüchtlinge aus ostafrika wurde borrelia recurrentis nach europa eingeschleppt. im zeitraum von januar bis november wurden 28 fälle von läuserückfallfieber bei jugendlichen flüchtlingen aus somalia, äthiopien und eritrea dokumentiert (antinori et al. 2016) . die übertragung erfolgt durch direkten haar-zu-haar-kontakt, z. b. wenn personen kopf an kopf im bett liegen. kopfbedeckungen, bettwäsche, handtücher, bürsten und kämme spielen für die übertragung keine rolle (canyon und speare 2010) . typische verbreitungsorte sind vor allem kindergärten und schulen. in allen kulturkreisen sind mädchen häufiger betroffen als jungen. das verhältnis mädchen zu jungen reicht von 12:1 in der türkei bis zu 2:1 in mitteleuropa; (feldmeier 2012) . die häufung der pediculosis capitis bei mädchen wird auf geschlechtsspezifisches verhalten und die längeren haare von mädchen zurückgeführt. besonders die von teenagern geübte praxis der "selfies" ist mit häufigem haarkontakt verbunden. komponenten des läusespeichels verursachen nach dem biss eine immunantwort vom verzögerten typ. filzläuse (pediculosis pubis) befallen vor allem regionen mit apokrinen schweißdrüsen, wie scham-und achselhaare. an den stark juckenden bissstellen entwickeln sich kleine blutungen, die als kleine blaue flecke (tâches bleues; maculae coeruleae) imponieren. die sich bewegenden filzläuse können mit dem bloßen auge entdeckt werden. nach der erstinfestation treten die symptome an der kopfhaut nach ca. 4 bis 6 wochen auf. bei einer reinfestation dagegen treten die symptome bereits nach 24 bis 48 stunden auf. symptome treten nur bei14 bis 36 % der infestierten auf. bei den anderen betroffenen ohne pruritus ist die diagnosestellung rein zufällig (burgess 1995) . die immunreaktion induziert juckende, erythematöse papeln und quaddeln. der juckreiz führt zu kratzen und damit zu exkoriationen der epidermis und der bildung von krusten. sekundär können bakterielle superinfektionen auftreten. reaktionen der lymphknoten der kopfhaut manifestieren sich als linsenförmige knötchen in der kopfhaut. die sensitivste diagnostische methode ist das auskämmen der angefeuchteten haare mit einem engzahnigen läusekamm. die weißen nissen lassen sich im gegensatz zu schuppen nicht von den haaren abstreifen. der nachweis von nissen lässt nicht notwendigerweise den schluss auf eine aktive infestation zu. dimeticone hat eine gute wirkung gegen adulte läuse und deren eier. produkte sind: nyda ® , etopril ® und jacutin ® (hexachlorcyklohexan). die aufgetragenen substanzen werden nach einigen stunden wieder ausgewaschen. die behandlung muss nach einigen tagen wiederholt werden. nissen werden nach durchtränkung der haare mit essig mit einem feinen "läusekamm" ausgekämmt. sofort nach der behandlung können kinder wieder gemeinschaftseinrichtungen besuchen. flohbisse finden sich an bedeckten körperstellen (. abb. 10.4). sie sind in asymmetrischen gruppen angeordnet. an der bissstelle entsteht eine quaddel und unter dem glasspatel zeigt sich eine kleine blutung. es besteht ein starker juckreiz. antihistaminika. der menschenfloh pulex irritans kann durch eine gute wohnungshygiene eliminiert werden. einsprühen mit insektiziden (jacutin-spray ® , ddt-spray ® ). z tungiasis (sandflohkrankheit) die tungiasis wird durch den sandfloh tunga penetrans verursacht. es penetrieren nur weibliche sandflöhe. im gegensatz zu allen anderen flohspezies, die nur temporäre ektoparasiten sind, dringt tunga penetrans permanent in die haut des wirts ein. die tungiasis ist in mittel-und südamerika, auf den karibischen inseln und in afrika unterhalb der sahara endemisch. sie ist eine in typischer weise mit armut assoziierte erkrankung, die in der gesamtbevölkerung eine prävalenz von ca. 50 % und bei kindern bis zu 80 % erreicht. hunde, katzen, schweine, ziegen und ratten sind die wichtigsten tierreservoire (pampiglione et al. 2009 . kleinepidemien treten gerne am ende eines besonders warmen und feuchten sommers auf (müller-stöver et al. 2010) . hakenwürmer dringen innerhalb von minuten in die menschliche haut ein (haas et al. 2005) . die kutane larva migrans kann auch mittels infizierter wäschestücke übertragen werden. da sich die tierhakenwurmlarven im falschen wirt befinden, können sie die basalmembran der epidermis nicht durchdringen und wandern ziellos über wochen und monate durch die haut. um die migrierende wurmlarve entwickelt sich eine starke entzündungsreaktion. leitsymptom der erkrankung ist der charakteristische mäandrierende, erythematöse und über die haut erhabene gang, der sich pro tag um einige millimeter verlängern kann. daraus ist die englische bezeichnung "creeping eruption" abgeleitet. aus der länge des ganges kann auf den expositionszeitpunkt rückgeschlossen werden. in ca. 10 % entwickeln sich auch vesikobullöse läsionen. die effloreszenzen jucken außerordentlich stark. der juckreiz verstärkt sich in der nacht. juckreiz-assoziierte schlafstörungen sind daher nahezu konstant. die aus dem juckreiz resultierenden kratzspuren können sich bakteriell infizieren. es erfolgt eine impetiginisierung, welche die lokale entzündungsreaktion verstärkt. nach wochen bis monaten stirbt die larve in situ ab ). die diagnose ergibt sich aus dem erkennen des larvenganges. eine eosinophilie kann bestehen; sie ist aber nicht obligatorisch. es gibt keinen immunologischen nachweis. mittel der wahl sind ivermectin und albendazol oral (sunderkötter et al. 2015) . ivermectin oral in einer einmaligen dosis von 200 μg/kg bei kindern > 5 jahre und > 15 kg. in deutschland ist ivermectin nur für die skabiesbehandlung, aber nicht für die kutane larva migrans-behandlung zugelassen. albendazol: 400 mg/tag für 5-7 tage oder 15 mg/kg/tag bis zu einem maximum von 800 mg für ältere kinder und jugendliche. albendazol ist in deutschland nur für die behandlung der strongylosidiasis und echinokokkose zugelassen. die behandlung der larva migrans ist somit eine "off-label"-therapie. die entzündungszeichen und der juckreiz bilden sich innerhalb einer woche zurück. bei persistenz der symptome ist eine zweite dosis notwendig. fadenwurmerkrankungen oder filariosen machen die größte gruppe unter den humanpathogenen helminthosen aus. sie sind vor allem im subsaharischen afrika verbreitet und werden durch blutsaugende arthropoden (anopheles-, aedes-, culicoides-arten) übertragen. sitz der filarien ist das lymphsystem oder das subkutane bzw. das peritoneale bindegewebe. ca. 2 wochen nach der infestation kann sich eine pulmonale askariasis manifestieren, die als löffler-syndrom bezeichnet wird. charakteristisch dafür sind: 5 trockener husten 5 subfebrile temperaturen 5 retrosternales brennen die symptome können sich jedoch auch steigern und bis zu asthmoiden hustenattacken und angioneurotischen ödemen steigern. im differentialblutbild ist meistens eine eosinophilie nachweisbar. nach ca. 1-2 wochen sind die patienten wieder symptomfrei. die intestinale askariasis kann bei schwerem befall mit uncharakteristischen gastrointestinalen beschwerden einhergehen. die eiablage in den gallenwegen und im leberparenchym kann zu einer granulomatösen hepatitis führen. obstruktionen. am häufigsten werden als erstes bandwurmglieder (proglottiden) im stuhl entdeckt. sie werden in wasser gesäubert und zur zählung der uterusäste unter dem mikroskop zwischen zwei objektträgern zerquetscht (hämatoxilinfärbung nach van cleave). die zahl der seitenäste beträgt bei taenia saginata 18-32 und bei taenia solium 7-12. die taenieneier sind rund und haben eine radiär strukturierte schale. gelegentlich tritt eine eosinophilie auf. praziquantel in einer einmaligen dosis von 5-10 mg/kg. vermeidung des genusses von rohem rind-und schweinefleisch. einfrieren bei -20 °c für mindestens 5 tage tötet die zystizerken ab. vermeidung der fäkaldüngung. es handelt sich um die wurmgattung mit der weltweit größten bedeutung. große verbreitung besteht in den kanälen des nil. viele geflüchtete kommen aus hochendemiegebieten zu uns (dennebaum 2017 die wichtigste diagnostik ist weiterhin die mikroskopische beurteilung eines blutausstrichs. für die malaria tropica steht ein sehr zuverlässiger, sensitiver schnelltest zur verfügung. antigene sind: "histidine-rich protein" als auch die "plasmodienspezifische laktatdehydrogenase" (pldh). erst ein dreimalig negativer ausstrich im abstand von jeweils 12-24 stunden, schließt bei einem febrilen patienten eine malariaerkrankung aus. die malariaserologie spielt in der akutdiagnostik keine rolle und kann nur zum retrospektiven nachweis einer stattgefundenen erkrankung benutzt werden. laktatdehydrogenase (ldh) und haptoglobin zeigen den grad einer typischerweise vorliegenden hämolyse an. therapie 5 um ein optimales medikament auswählen zu können, müssen die genaue malariaform und die geographische lage des erkrankungsgebietes bekannt sein. 5 die therapie einer unkomplizierten malaria erfolgt mit act wie z. b. artemether-lumefantrin oder dihydroartemesin-piperaquin. act: "artemesin based combination therapy". artemisia ist das chinesische quing-hao-su. 5 die verwendung von act-präparaten erfordert das erkennen einer qt-zeit-verlängerung. initial sind tägliche ekg-kontrollen nötig. begriff aus dem hindi mit der bedeutung "schwarze haut". leishmanien werden durch den stich der sandmücke übertragen. leishmanien sind intrazelluläre parasiten und befallen makrophagen. hauptsächlich befallen werden: 5 leber und milz bei der viszeralen leishmaniose. unbehandelt führt sie (kalar-azar) zu leberversagen. 5 haut bei der hautleishmaniose. die parasiten sind in den tropen und den subtropen verbreitet. die who geht davon aus, dass sich 90 % aller leishmaniasisfälle in folgenden 6 ländern ereignen: afghanistan, iran, syrien, algerien, saudi-arabien, ägypten. die viszerale leishmaniose wird vor allem durch l. donovani (asien) und l. infantum (europa) ausgelöst. es kommt zum befall der inneren organe (milz, leber). schwere krankheitsverläufe finden sich vor allem bei kindern und erwachsenen mit beeinträchtigter immunabwehr. die erkrankung beginnt mit fieber, durchfällen, bauchschmerzen und gewichtsverlust. es entwickelt sich eine hepatosplenomegalie mit gerinnungsstörungen, panzytopenie und aszites. typisch ist die dunkle, makulöse pigmentierung der haut. im rahmen einer sonderform, der sog. dermalen post-kala-azar-leishmaniose kann es nach abheilung der viszeralen erkrankung sowohl zu papulösen, wie auch makulösen, hypopigmentierten hautveränderungen kommen. histologie aus dem randbereich der läsion. es zeigt sich ein granulomatöses, gemischtzelliges entzündungsmuster (stebut et al. 2012) . im serum besteht häufig eine starke erhöhung der igg-konzentration. die medikamentenauswahl ist an der auslösenden subspezies orientiert. eine klassische systemische therapieform (i.m., i.v.) sind pentavalente antimonpräparate (pentostam ® , glucantime ® ) oder liposomales amphotericin b. komplexe läsionen sollten immer systemisch behandelt werden. die wahl der medikation ist abhängig von: 5 auslösende leishmaniensubspezies. 5 immunstatus des patienten. mehrere wochen bis zu 3 monate nach einem sandmückenstich bildet sich eine erythematöse papel an der einstichstelle, die an größe zunimmt. in abhängigkeit der leishmaniensubspezies kann eine ulzeration auftreten. der randwall ist typischerweise erhaben und hyperkeratotisch ("vulkanzeichen"). nach ca. 1 jahr kommt es zu einer spontanen abheilung. es bildet sich eine flache, atrophische narbe aus (. abb. 10.11). es besteht bisher keine kausale therapie. wichtig ist eine frühzeitige intravenöse volumengabe, um die schockproblematik zu vermeiden. wegen der blutungsneigung dürfen keine gerinnungshemmenden medikamente, wie z. b. aspirin verabreicht werden. im gegensatz zur malaria-prophylaxe ist auch auf einen mückenschutz am tag zu achten. wirksam ist die imprägnation der kleidung mit insektiziden, wie z. b. pyrethroiden. das gelbfiebervirus gehört zur familie der flaviviridae, nach dem bei gelbfieberinfektion auftretenden ikterus. gelbfieber gehört zur gruppe der viralen hämorrhagischen fieber. das virus kann auf zwei unterschiedlichen wegen übertragen werden: 5 von mensch zu mensch durch aedes-aegypti-mücken (urbanes gelbfieber). es wird jedoch nicht direkt von mensch zu mensch übertragen. charakteristisch ist eine im verhältnis zum fieber bestehende bradykardie ("faget-zeichen"). die diagnose wird durch den molekularbiologischen virusnachweis gestellt. in der akuten phase können igm-antikörper nachgewiesen werden. der nachweis eines vierfachen igg-titeranstiegs sichert ebenfalls die diagnose. nach der gelbfieberimpfung lassen sich im neutralisationstest antikörper nachweisen. es ist keine spezifische behandlung möglich. es gibt eine impfung mit einem "17d"-gelbfieber-lebendimpfstoff (stamaril ® ). der impfschutz hält mindestens 10 jahre an. das humane zytomegalievirus (hcmv), nach neuer nomenklatur auch humanes herpesvirus-5 (hhv-5) wird durch schmierinfektion, also direkten kontakt von schleimhäuten mit infektiösen körperflüssigkeiten wie nasensekret, speichel, tränenflüssigkeit, harn, genitalsekreten oder auch muttermilch übertragen (buxmann et al. 2017) . nach der infektion repliziert sich das virus zunächst in epithelzellen der eintrittspforte und breitet sich danach hämatogen in einer vielzahl von organen und zelltypen aus. in fast allen organen verwandeln sich infizierte zellen durch den zytopathogenen effekt des hcmv zu riesenzellen mit kerneinschlusskörpern ("eulenaugenzellen"). nach der akuten virämischen phase, die nur beim kleineren teil der immunkompetenten infizierten von unspezifischen grippeähnlichen allgemeinsymptomen begleitet ist, persistiert hcmv lebenslang in lymphozyten. vor allem gestillte und kongenital infizierte kleinkinder scheiden häufig jahrelang asymptomatisch viren über speichel, tränenflüssigkeit und urin aus. die inkubationszeit beträgt ca. ein bis zwei monate. die infektion bleibt bei immunkompetenten personen fast immer asymptomatisch. bei erkrankung erinnert die symptomatik an eine mononukleose mit fieber, lymphadenopathie und hepatosplenomegalie. neugeborene mit einer konnatalen zytomegalie sind in ca. 90 % klinisch unauffällig. es können jedoch spätschäden auftreten, die von einer chorioretinitis über eine mikrozephalie bis zu einer hepatopathie reichen können. isolierung des hcmv aus körperflüssigkeiten (urin, speichel, blut) und nachweis des antigens pp65. igm-und igg-antikörpernachweis. konnatale zytomegalie: erregernachweis in der möglichst 1. lebenswoche. ursächliche behandlung mit ganciclovir (10-15 mg/kg/tag i.v. über 6 wochen). valganciclovir bietet die möglichkeit der oralen therapie. bei resistenten stämmen kommt foscarnet zur anwendung. jede frau im gebärfähigen alter sollte ihren hcmv-antikörperstatus vor einer schwangerschaft kennen. seronegative frauen sollten "speichelkontakt" vermeiden (z. b. trinken aus derselben flasche). die tuberkulose ist in den meisten herkunftsländern von asylsuchenden weiter verbreitet als in deutschland. die tb-prävalenz schwankt zwischen 17/100.000 in syrien und 338/100.000 in nigeria. die durchschnittliche tb-rate in europa wird mit 39/100.000 angegeben (who 2017). auf der flucht ist die gefahr einer ansteckung groß. auf engem, schlecht belüftetem raum zusammenzuleben und die gleiche luft zu atmen stellt ein hohes risiko dar. in erstaufnahmeeinrichtungen, in denen kinder unter 15 jahren mit hilfe eines tuberkulin-hauttests untersucht wurden, konnte in 5-7 % der fälle eine positive reaktion (> 10 mm) nachgewiesen werden (auer und jablonka 2017) . aus einer untersuchung von geflüchteten in schleswig-holstein ergab sich eine häufung von zwei tuberkulosefällen unter 5.000. vergleichsweise ist die prävalenz in der deutschen bevölkerung ca. 10-mal niedriger (ca. 2 fälle pro 50.000; ehrenstein 2015). in den erstaufnahmeeinrichtungen gehört daher eine röntgenaufnahme der lunge zu einem teil der untersuchung. tuberkulose ist in deutschland eine meldepflichtige erkrankung. die meldung erfolgt durch den arzt oder das labor an das zuständige gesundheitsamt. merke 5 nach hiv ist die tuberkulose die zweithäufigste tödliche infektionserkrankung weltweit. 5 aktuelle zahlen zur tuberkuloseprävalenz werden über die who online unter 7 http://gamapserver.who.int/gho/interactive_charts/tb/cases/atlas.html zur verfügung gestellt. 5 bei schweren kachektischen ernährungsstörungen von afrikanern ist häufig zusätzlich eine tuberkulose beteiligt. ursache der tuberkulose ist die infektion mit mykobakterium tuberculosis, m. bovis oder m. africanum. es können im prinzip alle organsysteme betroffen sein. der erreger wird durch tröpfcheninfektion übertragen und führt zu einer primärinfektion. an der eintrittsstelle in der lunge bildet sich der sog. primärherd, aus dem die erreger lymphogen zu den regionalen lymphknoten gestreut werden. primärherd und infizierter lymphknoten bilden den primärkomplex. es bestehen meist keine klinischen symptome und die mykobakterien können für viele jahre intrazellulär überleben. aus dem primärkomplex kann sich in abhängigkeit der immunologischen abwehr des patienten eine klinisch manifeste progrediente tuberkulose entwickeln. aus dem primärkomplex erfolgt bei absenkung der immunologischen abwehr die streuung in unterschiedliche organsysteme (niere, gehirn, knochen usw.), die dann als postprimäre streuherde bezeichnet werden. vor allem bei besuchern in indien muss auch noch mit einer fütterungstuberkulose, also mit einem primärkomplex im darm gerechnet werden, da in indien gerne die nicht pasteurisierte milch heiliger kühe getrunken wird, die tuberkelbakterien enthalten kann. die klinische symptomatik ist durch den jeweiligen organbefall geprägt. sie kann vor allem anfangs sehr diskret sein: subfebrile temperaturen, nachtschweiß, reduzierter allgemeinzustand. aber grundsätzlich gilt: 5 lang andauernder husten 5 fieber; fälle von fieber unbekannter ursache (fuo)! 5 nachtschweiß 5 gewichtsverlust sind weiterhin die klassischen zeichen einer hochkontagiösen tuberkulose. einen orientierenden hinweis bietet die röntgenaufnahme des thorax. in den erstaufnahmerichtlinien für flüchtlinge und migranten ist eine röntgenthoraxaufnahme vorgeschrieben (s. unten). tuberkulosefälle wurden vor allem bei migranten aus eritrea, äthiopien und aus den staaten der ehemaligen sowjetunion gesehen. in der deutschen bevölkerung dagegen hat die zahl der neuerkrankungen abgenommen. merke 5 in der deutschstämmigen bevölkerung nimmt die zahl der tuberkuloseneuerkrankungen ab, wogegen sie aber bei migranten zunimmt. bei persistierendem husten sollte immer auch an eine tuberkulose gedacht werden (mulenga et al. 2015 ) 5 bei jüngeren kindern präsentiert sich die erkrankung häufig nicht mit der klassischen trias aus husten, gewichtsverlust und subfebrilen temperaturen (marais et al. 2006 prophylaxe z bcg-impfung 5 die bcg-impfung ist in deutschland keine "empfohlene impfung" mehr. 5 die bcg-impfung ist keine schützende, sondern nur eine den schweregrad abschwächende impfung. dieser einfluss der impfung wird jedes jahr schwächer und ist maximal 10 jahre nachweisbar (tuberkulinpositivität, die jedes jahr schwächer ausfällt!). es bestehen zwei impfstrategien: 3-6 wochen nach der infektion entsteht in 40-70 % der fälle eine akute hiv-infektion. diese wird entsprechend der sog. fiebig-phasen unterteilt. in den meisten fällen liegt eine uncharakteristische grippale symptomatik vor. symptome wie fieber, pharyngitis, lymphadenopathie oder hautausschlag sind hinweise auf eine akute hiv-infektion. es können jedoch auch schwerwiegende symptome, wie ein akutes guillain-barré-syndrom auftreten. die symptomatik bildet sich innerhalb von 1-4 wochen spontan zurück. die chronische phase der hiv-erkrankung geht mit einer kontinuierlichen abnahme der cd4-zellen und einem anstieg der hiv-rna-viruslast einher. der verlauf kann über jahre asymptomatisch sein. das fortschreiten der immunsuppression und das auftreten klinischer symptome werden durch die cdc-klassifikation charakterisiert. diese basiert auf klinischen manifestationen: 5 a: asymptomatisch. der hiv-antikörper-test ist frühestens 3, spätestens 6-12 wochen nach der infektion positiv. die akute hiv-infektion ist mit: 5 hoher viruslast, 5 passagerem abfall der cd4-zellen und 5 anstieg der cd8-zellen assoziiert. die klassischen aids-definierenden erkrankungen manifestieren sich in der regel erst ab einer cd 4-zellzahl < 200 / μl blut. die infektion mit pneumocystis carinii tritt bei fortgeschrittener immundefizienz mit cd 4-zellen < 200/μl auf. die routinetestung auf hiv erfolgt durch den nachweis von hiv-antikörpern mittels elisa. in diesen tests werden nicht nur hiv-antikörper, sondern auch virales p24-antigen nachgewiesen. als bestätigungstest dient der proteinnachweis im western-blot. der früheste nachweis (ab 7.-14. tag nach infektion) gelingt durch nachweis viraler rna im serum. auf grund der diaplazentar übertragenen maternalen antikörper ist eine aussagekräftige hiv-testung erst ab dem 18. lebensmonat möglich. im rahmen der frühkindlichen hiv-diagnostik wird der hiv-rna-direktnachweis bei kindern ab der 4.-6. lebenswoche auch in entwicklungsländern eingeführt. > akute schlaffe asymmetrische lähmungen ohne sensorische defizite sind immer polioverdächtig. diagnostik 5 1 bis 2 wochen nach der infektion sind spezifische igm-antikörper nachweisbar, die meist 3 -6 monate persistieren. neutralisierende igg-antikörper steigen ca. 2 wochen nach der infektion an und persistieren langfristig. 5 meist besteht eine lymphozytäre liquor-pleozytose bei normaler glukose-und evtl. leicht erhöhter eiweißkonzentration. 5 virusnachweis mittels pcr aus stuhl oder rachenabstrich. 5 die vorderhornläsionen sind in der mrt darstellbar. sie korrelieren mit der lokalisation der lähmungen. die behandlung ist symptomatisch. während der akuten erkrankung ist körperliche schonung angezeigt. bei atemstörungen kann die mechanische beatmung notwendig werden (historisch: → "eiserne lunge"). 5 bei kontaktpersonen mit grundimmunisierung ist kein ausschluss aus einer personengemeinschaft notwendig. 5 die orale lebendvakzine nach sabin (sog. "schluckimpfung") wird trotz hervorragender impferfolge, wegen der ausscheidung von impfviren und des seltenen auftretens einer vakzine-assoziierten paralytischen poliomyelitis (vapp; impfpolio) und der evtl. beeinträchtigung immungeschwächter personen nicht mehr durchgeführt. 5 es wird nur noch die inaktivierte poliovakzine empfohlen. 5 "polioimporte" sind weiterhin durch reisende und migranten möglich. das vibrio cholerae ist ein gramnegatives "kommaförmiges" stäbchen. nur vibrionen mit den oberflächenantigenen o1 und o139 verursachen eine erkrankung. aufgrund unterschiedlicher physiologischer charakteristika werden unterschieden: 5 vibrio cholerae: klassische choleravibrionen. 5 vibrio el-tor: der derzeit häufigste erreger. cholera ist eine ausgesprochene wanderseuche. sie begleitet karawanen, pilgerreisende, religiöse feste und auch migrationsbewegungen. derzeit ist cholera ein weltweites gesundheitsproblem geworden, das 2017 im jemen einen neuen höhepunkt erreicht hat. die erreger werden vor allem über kontaminiertes wasser aufgenommen. nach einer latenz von stunden bis tagen beginnen die organismen choleratoxin auszuscheiden. das toxin (hitzestabiles, lokal wirkendes exotoxin) bindet an gm1-gangliosidrezeptoren der dünndarmzellen, wodurch eine irreversible stimulation der adenylatzyklase mit der bildung von camp bewirkt wird. camp führt zu einer verminderten intestinalen absorption von natrium-und chloridionen durch die villi und zu einer vermehrten sekretion von chlorid und wasser in den krypten. daraus resultiert eine massive wässrige diarrhoe. klinische zeichen treten nach einer inkubationszeit von 1-5 tagen auf, wobei nur ein teil der infizierten klinisch auffällig wird. ein schweres krankheitsbild tritt nur in etwa 10 % auf. klinisch ist der verlauf der leichten form von leichten durchfällen anderer ätiologie nicht zu unterscheiden. die krankheit sistiert meist innerhalb von 48 stunden. das vollbild der cholera zeigt folgende merkmale: 5 abrupt beginnende wässrige darmentleerungen und erbrechen mit einem volumen von bis zu 1.000 ml pro stunde bei erwachsenen. bei adäquatem flüssigkeitsersatz hört der durchfall nach 1-6 tagen wieder auf. 5 es treten schwere elektrolyt-und flüssigkeitsdefizite mit einer metabolischen azidose auf. 5 dehydratationszeichen: verminderter hautturgor, blutdruckabfall, heiserkeit, kalte extremitäten, anurie, nierenversagen. 5 die diagnose beruht auf dem klinischen bild. 5 ein kultureller nachweis aus dem stuhlsekret ist ohne schwierigkeiten möglich. mikroskopisch zeigen sich typische, "fischzugartig" angeordnete vibrionen. 5 beim erregernachweis im stuhl oder dem erbrochenen macht man sich die eigenschaften der "halophilie" (vorliebe für hohe salzkonzentrationen) und der alkaliresistez (bis zu ph 9) zu nutze. 5 die serumparameter sind unspezifisch und passend zu einer schweren dehydratation. > cholerapatienten haben kein fieber und kein blut im stuhl. therapie 5 im akuten stadium ist die intravenöse flüssigkeitszufuhr (ringer-lösung) unverzichtbar. 5 orale flüssigkeitssubstitution mit einer oralen rehydrierungslösung: ca. 10-20 ml/ kg/stunde. 5 eine antibiotische therapie ist nicht unbedingt notwendig. gegebenenfalls antibiose mit doxycyclin oder trimethoprim-sulfamethoxazol oder auch erythromycin. durch die antibiose verkürzt sich die dauer der erregerausscheidung. die infektion erfolgt hauptsächlich fäkooral über kontaminierte nahrung oder wasser. nach der oralen aufnahme vermehren sich polioviren in den epithelzellen der gastrointestinalen mukosa. voraussetzung ist der nur bei primaten vorkommende poliovirusrezeptor cd155, der auch an der neuromuskulären endplatte zu finden ist. es besteht für die polio somit kein tierreservoir. nach der allgemeinen virämie erreichen die viren auch das zns, insbesondere das lumbalmark und befallen die motorischen vorderhornzellen nestschutz"). 1988 erkrankten weltweit jedes jahr 350.000 kinder an kinderlähmung, davon allein 200.000 in indien und pakistan. im jahr 2014 gab es weltweit noch 359 poliofälle. europa gilt seit 2002 als poliofrei. endemisch ist die erkrankung nur noch in afghanistan nur bei ca. 1 % aller infizierten kommt es zu zentralnervösen manifestationen im sinne von auftretenden schlaffen lähmungen. diese manifestieren sich meist plötzlich am morgen des noch am vorabend neurologisch unauffälligen kindes. die lähmungen sind charakteristischerweise asymmetrisch und betreffen am häufigsten die oberschenkelmuskulatur. die muskulatur ist schmerzhaft und die sehnenreflexe sind im bereich der schlaffen lähmungen erloschen. die sensorik ist nicht beeinträchtigt. die bulbäre erkrankungsform tritt gehäuft nach einer tonsillektomie auf. bei einem wesentlichen teil der patienten bilden sich die funktionsstörungen zurück. bei jedoch über 50 % persistieren lähmungen und führen zu einer nachfolgenden atrophie. daraus entwickeln sich z. b. ein spitzfuß, ein neurogener klumpfuß oder eine skoliose. jahre bis jahrzehnte nach der erkrankung kann sich ein "postpoliosyndrom" mit zunahme von schwäche und atrophie entwickeln. zusammenfassend kann man drei klinische verlaufsmöglichkeiten der poliomyelitis unterscheiden: prophylaxe es steht ein oraler impfstoff (2 dosen im abstand von 1-6 wochen) zur verfügung personen mit chronischen erkrankungen, wie z. b. diabetes, malignomen oder immunsuppression sind zu schweren krankheitsverläufen prädisponiert. die meisten fälle waren mit saudi-arabien oder benachbarten ländern verbunden. patienten hatten immer kontakt zu dromedaren, die als quelle der infektion gelten diagnostik bei schweren pneumonien und atemnotsyndrom sollte auch an die möglichkeit von mers-cov gedacht werden, insbesondere wenn sich patienten in den 14 tagen vor erkrankungsbeginn in einem land der arabischen halbinsel aufgehalten und dort kontakt zu dromedaren gehabt haben. für die diagnostik ist wichtig ländern der arabischen halbinsel sollte der besuch von kamelfarmen oder kamelmärkten und der genuss von unvollständig erhitzten kamelprodukten vermieden werden louse-borne relapseng fever among east african refugees in europe flüchtlingsmedizin -was ist relevant für die kinderärztliche borrelia recurrentis in head lice human lice and their management primary human-cytomegalovirus (hcmv) infection in pregnancy indirect transmission of head lice via inanimate objects wenn würmer krank machen -helminthosen in der pädiatrie flüchtlinge erst einmal ins einzelzimmer. welt n24 03 parasitäre hauterkrankungen in der kinderärztlichen praxis -teil 1 pediculosis capitis -aktueller kenntnisstand epidemiologie, diagnose und therapie hookworm-related cutaneous larva migrans kutane larva migrans und tungiasis behavioural strategies used by the hookworms necator americanus and ancylostoma duodenale to find, recognize and invade the human host multidrug-resistant organisms in refugees: prevalences and impact on infection control in hospitals fecal colonization with extended-spectrum betalactamase-producing enterobacteriaceae and risk factors among healthy individuals: a systematic review and metaanalysis male circumcision and hiv infection risk identification of malassezia species from pityriasis versicolor in indonesia and its relationship with clinical characteristics länder in afrika sollen impfung einführen a refined symptom-based approach to diagnose pulmonary tuberculosis in children the role of clinical symptoms in the diagnosis of intrathoracic tuberculosis in young children in deutschland erworbene larva migrans cutanea sand flea (tunga spp) infections in humans and domestic animals: state of the art high prevalence of infectious diseases and drugresistant microorganisms in asylum seekers admitted to hospital; no carbapenemase producing enterobacteriaceae until multidrug-resistant organisms detected in refugee patients admitted to a university hospital larva migrans cutanées ankylostomiennes en dermatologie à lomé updates on the epidemiology of dermatophyte infections s1 guideline diagnosis and therapy of cutaneous larva migrans (creeping disease) cutaneous leishmaniasis as traveler´s disease. clinical presentation, diagnostics and therapy stellungnahme des robert koch-instituts zur frage des screenings von asylsuchenden auf multiresistente erreger (mre) q & a on the malaria vaccine implementation programme (mvip). www.afro.who.int; zugegriffen: 24 world malaria report key: cord-003895-m1y76ee5 authors: parcesepe, angela m.; nash, denis; tymejczyk, olga; reidy, william; kulkarni, sarah gorrell; elul, batya title: gender, hiv-related stigma, and health-related quality of life among adults enrolling in hiv care in tanzania date: 2019-03-30 journal: aids behav doi: 10.1007/s10461-019-02480-1 sha: doc_id: 3895 cord_uid: m1y76ee5 hiv-related stigma has been associated with worse health-related quality of life (hrqol) among people living with hiv (plwh). little is known about how different types of hiv-related stigma (i.e., anticipatory, internalized, or enacted hiv-related stigma) influence hrqol and whether these relationships differ by gender. the sample included 912 plwh aged 18 years or older enrolling in hiv care at four health facilities in tanzania. hrqol was assessed with the life satisfaction and overall function subscales of the hiv/aids-targeted quality of life (hat-qol) instrument. sex-stratified multivariable logistic regression modeled the association of anticipatory, internalized, and enacted hiv-related stigma on poor hrqol. across all participants, the mean life satisfaction score was 63.4 (iqr: 43.8, 81.3) and the mean overall function score was 72.0 (iqr: 58.3, 91.7). mean hrqol scores were significantly higher for women compared to men for overall function (5.1 points higher) and life satisfaction (4.3 points higher). fourteen percent of respondents reported recent enacted hiv-related stigma and 13% reported recent medium or high levels of internalized stigma. in multivariable models, high internalized and high anticipatory stigma were significantly associated with higher odds of poor life satisfaction and poor overall function in both men and women. psychosocial interventions to prevent or reduce the impact of internalized and anticipatory stigma may improve hrqol among persons in hiv care. future research should longitudinally examine mechanisms between hiv-related stigma, poor hrqol, and hiv care outcomes. the world health organization recommends antiretroviral therapy (art) for all people living with hiv (plwh), regardless of cd4 cell count [1] . this policy, often referred to as treat all, is informed by evidence that earlier hiv treatment is associated with improved outcomes across the hiv care continuum and reduced hiv transmission [2] [3] [4] [5] . in recent years, as access to art has expanded, due in part to the implementation and scale-up of treat all policies, hiv-related morbidity and mortality have decreased and life expectancy among plwh has increased [6, 7] . in many settings, treatment of hiv has shifted to a chronic care model of disease management [8] . given these shifts in the treatment and clinical outcomes among plwh, greater attention is needed to their health-related quality of life (hrqol) and potentially modifiable factors that influence hrqol among plwh. though precise definitions vary, hrqol is a multidimensional construct that reflects one's quality of life through assessment of physical, mental, emotional, and social functioning [9] . poor hrqol has been associated with suboptimal hiv treatment outcomes including worse engagement in care, poor art adherence, and increased mortality, as well as poor mental health in both resource-rich and resourcepoor settings [10] [11] [12] [13] . greater understanding of factors that influence hrqol among plwh is critical to identifying effective strategies to improve the health and well-being of plwh. hiv-related stigma is common among plwh and associated with poor hrqol [14] [15] [16] . hiv-related stigma can take many forms including anticipatory stigma, internalized stigma, and enacted stigma. anticipatory stigma concerns individuals' expectations of experiencing enacted hivrelated stigma as a result of being hiv-positive. enacted hiv-related stigma involves experiencing stigmatizing behaviors or negative treatment due to one's hiv status. internalized hiv-related stigma occurs when an individual adopts stigmatizing beliefs about plwh and applies these stigmatizing beliefs to themselves. in the framework of the relationship between hiv-related stigma and the health and well-being of plwh [17] , earnshaw and chaudoir posit that different dimensions of hiv-related stigma may differentially impact health-related outcomes, including hrqol, and emphasize the importance of understanding the relationship between hiv-related stigma and quality of life disaggregated by type of stigma and type of well-being measure assessed [17] . despite consistent findings that hiv-related stigma is associated with worse hrqol, understanding of how different dimensions of stigma (e.g., internalized, anticipated, or enacted) are related to different components of hrqol, remains limited, particularly in sub-saharan africa, which continues to bear a disproportionate burden of the hiv epidemic. a more nuanced understanding of the relationship between hiv-related stigma and hrqol is warranted. previous research has found differences in how men and women living with hiv perceive and experience hivrelated stigma [13, 18, 19] . for example, among plwh in the united states, hiv disclosure concerns were associated with increased health-related worries among women, but not among men living with hiv [20] . among plwh in china, women living with hiv endorsed significantly higher internalized hiv-related stigma compared to men [21] . gendered experiences of hiv-related stigma may be informed by differences in the social and economic position of men and women across societies and by the social construction of both stigma and gender across settings. previous research has found that women living with hiv often indicate concerns about the real or anticipated social and relational impact of hiv-related stigma in their lives and the lives of their children or other family members, including social rejection, isolation, and violence [22] . research with women living with hiv in the u.s. found that women's decision-making around hiv disclosure prioritized protecting social relationships and minimizing potential negative social effects of disclosure [22] . given these gendered differences in experiences of hiv-related stigma, the relationship between hiv-related stigma and hrqol may be meaningfully different for men and women living with hiv. however, little is known regarding whether the relationship between types of hiv-related stigma and types of hrqol differs by gender. it is important to understand to what extent hiv-related stigma and hrqol function differently among men and women. the objectives of this analysis were (1) to examine the relationship between three dimensions of hiv-related stigma (anticipatory, internalized, and enacted stigma) and two components of hrqol (overall function and life satisfaction) and (2) to assess whether these relationships differ by gender. data were collected as part of a study conducted at four hiv treatment clinics in the kagera region of northwestern tanzania. all clinics were supported by the tanzanian ministry of health with technical assistance from icap at columbia university via funding from the president's emergency plan for aids relief (pepfar) at the time of data collection. individuals were eligible to participate in the study if they were: (1) aged 18 years and older, (2) patients potentially eligible for study participation were identified by providers within 90 days of their first clinic visit. interested patients were referred to study staff for eligibility screening and consent procedures. data collection consisted of a structured interview conducted by trained research assistants which included questions on sociodemographics, hiv-related stigma, and health-related quality of life. health-related quality of life was assessed using the hiv/ aids-targeted quality of life (hat-qol) instrument. the hat-qol has demonstrated reliability and validity among plwh [23, 24] in resource-poor settings, including sub-saharan africa [25, 26] . this analysis includes two subscales of the hat-qol: the overall function subscale (6 items) and the life satisfaction subscale (4 items). participants were asked to report on how often they were able to complete a given health-related task or felt a particular way in the previous 4 weeks. response options ranged from 1 (none of the time) to 5 (all of the time). scores for each subscale were transformed linearly to a possible range of 0-100, according to a prescribed algorithm, with higher values indicating better functioning and higher life satisfaction. internalized stigma in the past 3 months was measured with the 5-item negative self-perception subscale (e.g. you felt that you did not deserve to live, you felt that you were no longer a person) of the hiv/aids stigma instrument, plwha (hasi-p), developed in sub-saharan africa [27] . an additional item not from this scale (you thought someone had cursed you) also was included. response options ranged from never (4) to most of the time (1) and were recoded so that higher scores represented higher internalized stigma (cronbach's alpha = 0.86). responses were categorized into three categories: none, low, and medium or high levels of internalized stigma. study investigators knew of no scales validated in sub-saharan africa to measure anticipatory hiv-related stigma at the time of data collection. thus, anticipatory stigma was assessed with 12 yes/no items created in accordance with the concept described by earnshaw and chaudoir (e.g., if others know or suspect you are hiv positive, your partner might get violent; your children might be abused or discriminated against; family members might treat you differently). a total anticipatory stigma score was constructed as the proportion of endorsed items from among all questions the participant was eligible to answer (i.e., participants without children or without a partner were not eligible to answer those respective items) (cronbach's alpha = 0.65). anticipatory stigma scores were categorized into tertiles based on distribution in the study sample. enacted stigma in the past 3 months was measured with eight items selected from hiv/aids stigma instrument-plwa (hasi-p) subscales (verbal abuse, fear of contagion, and social isolation); items inquired about how often the participant had experienced rejection due to their hiv status (e.g., you were told that you have no future, you were told that god is punishing you, someone stopped being your friend), with response options ranging from never (4) to most of the time (1) (cronbach's alpha = 0.70) [27] . because the reported frequency of occurrence of all items was low, the summary measure was coded as any recent enacted hivrelated stigma versus no recent enacted hiv-related stigma. sociodemographic variables included age, sex, education, religion, relationship status, employment status, food insufficiency, and time away from home. univariate analyses were conducted to assess the mean overall function and life satisfaction scores for the entire sample population and separately by gender. sex-stratified analyses of differences in group mean hrqol scores by type of hiv-related stigma were conducted using t-tests or anova procedures, as appropriate. logistic regression was used to model the association of hiv-related stigma and poor hrqol separately by gender. in multivariable analyses, poor quality of life was defined as being in the lowest quality of life quartile as compared to the top three quartiles. adjusted analyses controlled for age, relationship status, and employment status. all analyses accounted for clustering by health facility using proc survey logistic in sas version 9.4. because internalized stigma was highly correlated with enacted stigma, multivariable regression models were run separately to examine the relationship between each type of hiv-related stigma and hrqol. of the 912 participants included in this analysis, 63.2% were female ( table 1 ). the median age was 35 years (interquartile range [irq]: 28, 42). three quarters (77.9%) of participants had ever attended school. the majority of participants (58.1%) were married or in a relationship. most participants (61.8%) were catholic. past-year food insufficiency was table 1 sociodemographic characteristics and hiv-related stigma by health-related quality of life among adults enrolling in hiv care and not known to be eligible for art in tanzania a missing by variable: relationship status n = 1, age n = 6, food insufficiency n = 10, away from home for > 1 month n = 4, enacted stigma n = 79, internalized stigma n = 6, anticipatory stigma n = 7 relatively uncommon with 84.9% reporting never or seldom experiencing food insufficiency in the past year. the majority of participants were currently working for pay (61.1%) and had not spent more than 1 month away from home in the past year (72.1%). most participants (78.1%) had disclosed their hiv status to someone. among those who disclosed their status to someone (n = 712), 51.8% had disclosed their status to a spouse or partner, 33.3% had disclosed their status to a parent, 21.8% had disclosed their status to a friend, and 13.5% had disclosed their status to a child. across all participants, the mean life satisfaction score was 63.4 (iqr: 43.8, 81.3) and the mean overall function score was 72.0 (iqr: 58.3, 91.7). in bivariate analyses, mean hrqol scores were significantly higher for women compared to men for overall function (5.1 points higher) and life satisfaction (4.3 points higher) (fig. 1) . as shown in table 1 , for both overall function and life satisfaction, mean scores were highest among participants who were married or in a relationship and lowest among participants who had never been married. similarly, food insufficiency was significantly associated with mean overall function and life satisfaction scores, with those reporting experiencing food insufficiency never or seldom in the past year reporting significantly higher mean overall function (p < 0.0001) and life satisfactions (p < 0.0001) scores compared to those who reported food insufficiency sometimes, often, or always in the past year. employment status was significantly associated with mean life satisfaction score (p = 0.0133), but not with mean overall function score, while having been away from home for more than 1 month in the past year was significantly associated with mean overall function score (p = 0.0309), but not with mean life satisfaction score. fourteen percent of respondents reported having experienced recent enacted hiv-related stigma and 12.7% reported recent medium or high levels of internalized stigma. in relation to anticipatory stigma, almost two-thirds (63.5%) of respondents indicated concern that people might gossip about them if they knew or suspected that they were hivpositive. almost half (46.4%) of respondents with children worried that their children might become upset or fearful if they knew or suspected that the respondent was hivpositive. in bivariate analyses, experience of each form of stigma (anticipatory, internalized, and enacted hiv-related stigma) was associated with lower mean hrqol scores ( table 2 ). for both anticipated and internalized stigma, higher levels of stigma were associated with significantly lower mean overall function and life satisfaction scores overall and when stratified by gender. mean hrqol scores were significantly higher for those who reported no internalized stigma compared to those who reported medium or high levels of internalized stigma for overall function (women: 12.3 points higher, p < 0.0001; men: 12.6 points higher, p = 0.04) and life satisfaction (women: 16.2 points higher, p < 0.0001; men: 19.9 points higher, p < 0.0001). a similar pattern was observed between anticipatory stigma and hrqol for both women and men. in bivariate analyses, mean life satisfaction scores were significantly higher among those who reported no enacted stigma compared to those who reported having experienced enacted stigma overall (9.8 points higher) and separately among women and men (women: 8.5 points higher, p = 0.005; men: 15.0 points higher, p = 0.0009). having experienced enacted stigma was associated with significantly lower overall function score among women, but not among men (7.8 points lower among women vs 3.5 points lower among men). in adjusted multivariable analyses (table 3) , experience of each form of stigma (anticipatory, internalized, and enacted) was associated with higher odds of poor hrqol. women who reported high levels of anticipatory stigma had 2.0 [95% confidence interval (ci) 1.2, 3.6] times the odds of poor overall function and 2.1 (95% ci 1.2, 3.7) times the odds of poor life satisfaction compared to women who reported low levels of anticipatory stigma. a similar relationship between high anticipatory stigma and poor overall function and poor life satisfaction was found among men (table 3) . women who reported high levels of internalized stigma had 2.2 (95% ci 1.2, 4.1) times the odds of poor overall function and 2.2 (95% ci 1.2, 4.0) times the odds of poor life satisfaction compared to women who reported low levels of internalized stigma. a similar relationship between high internalized stigma and both poor overall function and poor life satisfaction was found among men (table 3) . enacted stigma was associated with significantly increased odds of poor life satisfaction among women [adjusted odds ratio [aor] 2.0 (95% ci 1.2, 3.4) ]. however, the relationship between enacted stigma and poor life satisfaction did not reach statistical significance among men [aor 2.1 (95% ci 1.0, 4.7)]. having experienced enacted stigma was not significantly associated with poor overall function among women or men. in a large sample of plwh enrolling in hiv care in tanzania, health-related quality of life was significantly lower among men as compared to women in both overall function and life satisfaction. previous research examining the table 3 sex-stratified bivariate and multivariable models of hiv-related stigma and health-related quality of life among adults enrolling in hiv care and not known to be eligible for art in tanzania *p < 0.05 a adjusted for age, marital status, and employment b models include enacted stigma, but not anticipatory or internalized stigma c models include internalized stigma, but not anticipatory or enacted stigma d models includes anticipatory stigma, but not internalized or enacted stigma relationship between gender and hrqol has found gender to be consistently associated with hrqol among plwh. however, the directionality of this relationship has varied across studies. similar to current findings, studies with individuals initiating art in ethiopia and with individuals testing hiv-positive in kenya found that hrqol was higher among women than men [13, 28] . however, another study with art patients in ethiopia found that men reported significantly higher quality of life compared to women [29] . the mechanisms through which gender influences hrqol across settings and populations warrant further investigation. high levels of anticipatory stigma were significantly associated with poor overall function and poor life satisfaction among both women and men. the authors were unable to identify previous research on the relationship between anticipatory hiv-related stigma and hrqol from sub-saharan africa. however, among plwh in sweden, anticipatory stigma, measured by concerns about public attitudes, was significantly associated with poor physical hrqol [30] . in this same study, though, concern regarding disclosure of one's hiv status, another component of anticipatory hivrelated stigma, was not significantly associated with worse hrqol [30] . among plwh in the united states, disclosure concerns were significantly associated with poor hrqol among men and women [20] . future research should examine to what extent the relationship between anticipatory stigma and hrqol varies by the component of anticipatory stigma assessed (e.g., disclosure concerns, concerns about public attitudes). factors that mediate or moderate the relationship between anticipatory stigma and hrqol should also be examined, including whether such mediating or moderating factors differ by gender. for example, effective coping strategies or greater social support may reduce the impact of anticipatory stigma on poor hrqol while maladaptive coping mechanisms, limited social support, or social isolation may have a synergistic effect on the relationship between anticipatory stigma and hrqol. greater understanding of the relationship between anticipatory stigma and hrqol can lead to the identification of potential intervention targets to mitigate the impact of anticipatory stigma on hrqol and improve the quality of life of plwh. such research is particularly needed with plwh in sub-saharan africa where the prevalence and impact of hiv-related stigma remain high [31] [32] [33] . internalized hiv-related stigma was associated with poor overall functioning and life satisfaction among both men and women. while research into the relationship between internalized hiv-related stigma and hrqol in sub-saharan africa remains limited, a study of plwh in spain similarly found that internalized stigma was associated with worse hrqol [34] . this study also found that the relationship between internalized stigma and hrqol was mediated by group identity, suggesting that interventions to foster social cohesion and increase social support may be a promising strategy to mitigate the negative impact of internalized stigma on hrqol [34] . peer support interventions may be particularly effective at reducing the prevalence and impact of internalized hiv-related stigma, particularly as a longitudinal study with plwh in uganda found that internalized stigma was associated with subsequent low levels of social support [35] . however, findings related to the relationship of peer support and hiv treatment outcomes remain equivocal [36] [37] [38] . internalized stigma has been consistently associated with poor mental health, including depressive symptoms and poor emotional well-being. examination of the extent to which psychological distress mediates the relationship between internalized hiv-related stigma and poor hrqol and whether this relationship differs by gender is warranted. evidence-based mental health interventions for plwh should consider incorporating components to reduce and manage internalized hiv-related stigma and adapted interventions should be implemented and evaluated. in multivariable analyses, enacted stigma was not significantly associated with overall functioning or life satisfaction among men. however, enacted stigma was significantly associated with poor life satisfaction among women. previous research into the relationship between enacted hiv-related stigma and hrqol remains equivocal. among plwh in spain, enacted stigma was significantly associated with worse hrqol [34] . however, no significant relationship between enacted stigma and hrqol was found when examined among plwh in sweden [30] . neither study examined the relationship between enacted stigma and hrqol separately by gender. however, research with women living with hiv in the united states found that women were particularly concerned about the negative social consequences of hiv-related stigma [22] . similarly, research in south africa found that the level of hiv-related stigma reported by one's community, operationalized as the percent of people in a village endorsing hiv-related stigma, was negatively associated with hiv testing among women, but not men [39] . gender differences in the relationship between enacted stigma and hrqol may be influenced by differences in the social positioning and socialization of women compared to men. if women place greater emphasis on preserving social relationships, women may be more negatively impacted by stigmatizing views and behaviors of community members compared to men. additional research into the relationship between gender, enacted stigma, and hrqol is needed. this work has limitations worth noting. first, we assessed the relationship between hiv-related stigma and hrqol prior to art initiation. future work should assess this relationship across the care continuum and examine whether there are critical points (e.g., diagnosis, disclosure) at which plwh are particularly vulnerable to the negative effects of stigma and poor hrqol. additionally, given the cross-sectional nature of our study, temporality of the relationship between hiv-related stigma and hrqol could not be established. it is possible that people with poor hrqol are more sensitive to experiences of hiv-related stigma. longitudinal research is needed to better understand pathways between hiv-related stigma and hrqol. further, data were collected from individuals at four hiv clinics in tanzania. as such, results cannot be generalized to plwh in other settings or plwh not engaged in care. as both hiv-related stigma and hrqol may pose barriers to engagement in care, the relationship between hiv-related stigma and hrqol may be meaningfully different among plwh not engaged in care. in addition, anticipatory stigma items in this study did not assess anticipatory stigma specifically in a health care setting or from health care workers. future research should examine anticipatory stigma specifically in relation to health care settings. finally, this analysis examined two aspects of hrqol. future work should examine the relationship between hiv-related stigma and other components of hrqol. hiv-related stigma was associated with worse hrqol among men and women. more nuanced understanding of the longitudinal pathways between stigma and poor hrqol can inform intervention development and adaptation. interventions to enhance the hrqol of plwh should incorporate stigma reduction components. psychosocial interventions to prevent or reduce the impact of internalized and anticipatory stigma may improve hrqol for plwh and should be considered across a range of hiv services. more research is needed to understand to what extent the mechanisms between these types of stigma and hrqol differ by gender and whether gender-specific anticipatory and internalized stigma reduction intervention strategies are warranted. antistigma interventions at the community level are also needed and may particularly benefit women living with hiv. guideline on when to start antiretroviral therapy and on pre-exposure prophylaxis for hiv. geneva: world health organization progress report 2016: prevent hiv, test and treat all. geneva: world health organization initiation of antiretroviral therapy in early asymptomatic hiv infection a trial of early antiretrovirals and isoniazid preventive therapy in africa prevention of hiv-1 infection with early antiretroviral therapy life expectancy living with hiv: recent estimates and future implications life expectancy among hiv-positive patients in rwanda: a retrospective observational cohort study the end of aids: hiv infection as a chronic disease measuring healthy days: population assessment of health-related quality of life quality of life in hiv-infected individuals receiving antiretroviral therapy is related to adherence health-related quality of life and survival among hiv-infected patients receiving highly active antiretroviral therapy: a study of patients in the aids therapy evaluation in the netherlands (athena) cohort predicting health-related quality of life in people living with hiv in nepal: mental health disorders and substance use determinants gender differences and psychosocial factors associated with quality of life among art initiators in oromia examining the associations between hiv-related stigma and health outcomes in people living with hiv/aids: a series of meta-analyses the role of hiv stigma in art adherence and quality of life among rural women living with hiv in india perceived social support, coping, and stigma on the quality of life of people living with hiv in nepal: a moderated mediation analysis from conceptualizing to measuring hiv stigma: a review of hiv stigma mechanism measures hiv/aids national strategic plans of sub-saharan african countries: an analysis for gender equality and sex-disaggregated hiv targets gender and care: access to hiv testing, care, and treatment gender differences in disclosure concerns and hiv-related quality of life gendered aspects of perceived and internalized hiv-related stigma in china stigma in hiv-positive women performance of a new, hiv/aids-targeted quality of life (hat-qol) instrument in asymptomatic seropositive individuals hiv/ aids-specific quality of life and adherence to antiretroviral therapy over time reliability and validity of two hiv/aids-specific quality of life instruments adapted for use in hiv-positive zimbabweans quality of life and the concept of "living well" with hiv/aids in sub-saharan africa validation of the hiv/aids stigma instrument-plwa (hasi-p) gender differences in health-related quality of life at the time of a positive hiv test-a cross-sectional study in a resource-poor, high prevalence setting in gender differences in health related quality of life among people living with hiv on highly active antiretroviral therapy in mekelle town the relationship between stigma and health-related quality of life in people living with hiv who have full access to antiretroviral treatment: an assessment of earnshaw and chaudoir's hiv stigma framework using empirical data stigma in ethiopia: association with depressive symptoms in people with hiv stigma and discrimination against people living with hiv by healthcare providers, southwest ethiopia hiv-stigma in nigeria: review of research studies, policies, and programmes enacted and internalized stigma and quality of life among people with hiv: the role of group identity the social context of food insecurity among persons living with hiv/aids in rural uganda high levels of adherence and viral suppression in a nationally representative sample of hiv-infected adults on antiretroviral therapy for 6, 12 and 18 months in rwanda effect of peer health workers on randomized control trial of peer-delivered, modified directly observed therapy for haart in mozambique hiv testing and stigma: the association of hiv testing behaviors and community-level and individuallevel stigma in rural south africa differ for men and women acknowledgements this project was supported by a research grant from the national institute of mental health (supplement to grant number r01mh089831). the clinics included in this analysis received support from icap at columbia university through funding from the president's emergency plan for aids relief. the content is solely the responsibility of the authors and does not necessarily represent the official views of the funders. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. conflict of interest the authors declare they have no conflict of interest.ethical approval all procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards.informed consent informed consent was obtained from all participants included in the study. key: cord-016472-jj7fqcen authors: freudenberg, nicholas title: health research behind bars: a brief guide to research in jails and prisons date: 2007 journal: public health behind bars doi: 10.1007/978-0-387-71695-4_24 sha: doc_id: 16472 cord_uid: jj7fqcen while most people make staying out of jail and prison a priority, a growing number of researchers are eager to get into correctional facilities in order to study the criminal justice system, the causes and consequences of incarceration, and the role of corrections in our society. for health researchers and their collaborators, the audience for this chapter, correctional facilities offer several unique advantages: a population at high risk of many health problems including infectious and chronic diseases, substance abuse, and mental health problems; social and physical environments that can enhance or impede well-being; a setting that is a focal point for the class, racial/ethnic, and gender differences that divide the united states; a site where health and mental health services and prevention programs are offered and can be evaluated; a controlled environment for administration of treatments such as directly observed therapy for tuberculosis; and a stopping point in the cycle of incarceration and reentry that so profoundly affects community well-being. while most people make staying out of jail and prison a priority, a growing number of researchers are eager to get into correctional facilities in order to study the criminal justice system, the causes and consequences of incarceration, and the role of corrections in our society. for health researchers and their collaborators, the audience for this chapter, correctional facilities offer several unique advantages: a population at high risk of many health problems including infectious and chronic diseases, substance abuse, and mental health problems; social and physical environments that can enhance or impede well-being; a setting that is a focal point for the class, racial/ethnic, and gender differences that divide the united states; a site where health and mental health services and prevention programs are offered and can be evaluated; a controlled environment for administration of treatments such as directly observed therapy for tuberculosis; and a stopping point in the cycle of incarceration and reentry that so profoundly affects community well-being. in this chapter, i consider the benefits and perils of doing health research in jails and prisons. the chapter begins with a brief overview of the different types of health research conducted within correctional facilities and among those leaving jail or prison. i then describe some of the unique obstacles that correctional health researchers encounter and assess some of the methods they have used to overcome these obstacles. since researchers in correctional settings face significant ethical dilemmas, i next consider recent frameworks for making ethical decisions about this research. finally, i suggest an agenda for future health research in correctional settings. health research behind bars: a brief guide to research in jails and prisons in recent decades, researchers from a variety of disciplines including health services research, public health, medicine, criminal justice studies, sociology, psychology, anthropology, organizational studies, and others have initiated studies on health in the correctional system. a brief typology of the different categories of questions these investigators have asked will help to set the stage for our consideration of approaches to correctional research. 1. what are the health and social characteristics of people in jail and prison? numerous studies have examined the health and demographic profile of incarcerated populations. these vary from large studies based on national samples and using multiple health outcomes such as the reports of the national correctional health care commission on the health status of soonto-released inmates (2002a, 2002b) or of the health status of inmates in texas prisons (baillargeon, black, pulvino, & dunn, 2000) to studies of a single outcome such as hepatitis c among california inmates (fox et al., 2005) . the various reports of the bureau of justice statistics on mental health, substance use, and other health conditions (e.g., james & glaze, 2006 , karberg & james, 2005 summarize data across u.s. jurisdictions, providing an opportunity for correctional and health officials to identify incarcerated populations in higher need. other studies describe patterns of health care utilization among inmate populations (leukefeld et al., 2006) . investigators often compare the health status of different subpopulations, e.g., men to women (peters, strozier, murrin, & kearns, 1997) , or african-americans and latinos to whites (rounds-bryant, motivaus, & pelissier, 2003) . these descriptive studies are used to identify the needs of various segments of the incarcerated populations, to compare changing incidence or prevalence of conditions over time, or to serve as a baseline for the subsequent evaluation of interventions. research imperatives in these studies are consistent definitions of dependent and independent variables, uniformity in data collection methods in multisystem studies, and sampling strategies that enable generalizations to other settings. 2. how does the health of inmates differ from that of nonincarcerated populations? a second group of studies compare the health of incarcerated populations with the health of the general population or with samples of nonincarcerated people. for example, teplin and colleagues' studies of the prevalence of mental health conditions among women and juveniles in chicago jails found higher rates of some psychiatric conditions in incarcerated populations than in similar populations living in the same catchment area from which inmates had been arrested (teplin, 1990; teplin et al., 1996) . these studies set the stage for the next group of studies. methodological issues in this type of study include selecting an appropriate comparison group. 3. how does incarceration itself affect the health of incarcerated populastions? both correctional and public health authorities want to know whether observed differences between incarcerated and nonincarcerated populations are due to differences in the composition of the populations or to the experience of incarceration, a variant of the classic epidemiological task of distinguishing between compositional (i.e., characteristics of the population) and contextual effects (i.e., characteristics of an environment). for example, numerous investigators have sought to determine whether the higher prevalence of hiv infection among u.s. prison populations was due to intraprison transmission or to criminal justice policies that led to incarceration for people already hiv infected (hammett, 2006; krebs & simmons, 2002) . most studies suggest the latter route is more important, reassuring correctional authorities that within-prison transmission, while it does occur, is not a major factor in higher rates. on the other hand, studies in the early 1990s established that tb transmission did occur within the facility, leading to substantial efforts to prevent such transmission (bellin fletcher, & safyer, 1993) . others have investigated whether incarceration is associated with homelessness and mental illness (mcneil, binder, & robinson, 2005) . the main analytic task in these studies is to distinguish between causal and noncausal associations between incarceration and selected health outcomes. 4. what are the health effects of criminal justice policies and practices on the health of inmates? criminal justice policies often have unintended effects on incarcerated populations. documenting the positive and negative impact of these policies can serve as a starting point for policy change. for example, a study in a large public hospital in new york city found that many admissions for diabetic ketoacidosis were related to the court practice of denying inmates access to insulin medications in court pens (keller et al., 1993) . health impact assessment, an analytic method developed to assess the health effects of both health and non-health-related policies, offers a promising approach to consider the health consequences of various prison and criminal justice policies (davenport, mathers, & parry, 2006; kemm, 2001 , veerman, barendregt, & mackenbach, 2005 . to date, however, this approach does not seem to have been used to assess the impact of u.s. correctional policies on inmate or community health. 5. what is the impact of interventions designed to care for or improve the health of incarcerated populations? a key practical question for correctional, public health, and correctional health officials is the effect of the programs they run on the well-being of the populations in custody. evaluation studies seek to document the utilization of health services (lindquist & lindquist, 1999) ; assess their impact on health or health care utilization (e.g., chan, vilke, smith, sparrow, & dunford, 2003; edens, peters, & hills, 1997) ; analyze the cost-benefits of an intervention (ncchc, 2002a) ; or compare the cost-effectiveness of various approaches to a specified health problem, e.g., screening for hiv or other infectious diseases within correctional settings (resch, altice, & paltiel, 2005; kraut-becher, gift, haddix, irwin, & greifinger, 2004) . in these studies, methodological issues include the specification of clearly defined outcomes, the use of standard accepted measures for assessing costs and benefits of various interventions, and the design of evaluation studies that are both methodologically sound and operationally feasible. 6. how does reentry affect the health of incarcerated populations? in the last decade, correctional health researchers have begun to follow their research participants back into the community, examining their success in finding health services or drug treatment (jarrett, adeyemi, & huggins, 2006; lincoln et al., 2006) , maintaining control of a mental health condition (wilson & draine 2006) , or in improving hiv care or reducing hiv risk behavior (bauserman et al., 2003; rich et al., 2001; myers et al., 2005) . these studies can be part of an evaluation of a reentry program (e.g., needels, james-burdumy, & burghardt, 2005) or a descriptive study of the outcomes of the reentry process (e.g., freudenberg et al., 2005) . 7. what is the impact of incarceration rates on the well-being of communities and populations? finally, a growing number of researchers are studying the impact of incarceration and correctional policies on the health of families, communities, and populations. for example, some research looks at the impact of incarceration on children and other family members (murray & farrington, 2005; barreras, drucker, & rosenthal, 2005) . researchers have asked whether incarceration policies have contributed to the community transmission of hiv infection (leh, 1999; johnson & raphael, 2006) or other sexually transmitted infections (thomas & sampson, 2005) , community rates of violence (rose & clear, 2003) , or disparities in health between black and white u.s. populations (taxman, byrne, & pattav, 2005; johnson & raphael, 2006; iguchi, bell, ramchand, & fain, 2005) . these studies can help policy makers consider the impact of various incarceration policy choices. this brief summary of the types of questions that correctional health researchers have sought to answer illustrates the scope of the field. for neophyte investigators, becoming familiar with the findings and methodological challenges in the extant literature relevant to their question of interest can save years of trial and error in this difficult setting and avoid duplication of effort. for more experienced researchers, a familiarity with the scope of prior research can help them move from descriptive to analytic and intervention studies. several recent reviews provide a good starting place for becoming familiar with recent correctional health research (edens et al., 1997; freudenberg, 2001; magaletta, diamond, dietz, & jahnke, 2006 , morris, 2001 pollack, khoshnood, & altice, 1999) . successful health research in correctional settings requires familiarity with the existing literature described in the previous section, a knowledge of the research methods applicable to the correctional setting, discussed in the next section, and an understanding of the various stakeholders in correctional health, discussed here. without a map of this organizational landscape, even skilled researchers can lose their way. key participants in developing and implementing research studies in correctional settings include correctional officials, correctional health providers, public health authorities, other researchers and research institutions, elected officials, funders, prison and reentry advocacy groups and inmates and their families. each of these constituencies has the potential both to improve research and to stop studies before they get off the ground. thus, the practical researcher will want to understand how to enlist each of these groups in supporting the research process. correctional officials need to approve and at least not oppose any research study conducted in their facility. their main concerns are the extent to which research may pose a threat to safety and regular prison routines, fear of bad publicity, cost and liability concerns, or additional demands on their staff. researchers who can reassure correctional officials on these matters will have an easier time pursuing their studies. investigators who are unable (or un willing) to provide these assurances may need to consider other approaches to their research, such as interviewing participants after their release from jail or prison. in most situations, research studies will need the tacit support of at least three levels of correctional authorities: senior departmental managers (e.g., commissioners/directors or sheriffs); wardens of the facility(ies) where the study takes place; and frontline correctional staff. each level brings different concerns and requires different assurances in order to allow the research to proceed. for example, frontline correctional officers who may be required to bring participants to the researcher for interviews or medical examinations want to make sure these procedures do not interfere with their routines or increase staff workloads. wardens often need to be assured that no research procedure will jeopardize security. in another example, a jail security warden was concerned that a stylus for a handheld computer device used for interviews with inmates could be used as a weapon. it took several meetings between a warden and a research team to agree on a type of stylus and interview procedures. senior officials of corrections departments are sometimes ambivalent about studying illegal behavior such as drug use or voluntary or coercive sexual behavior. if they know that a problem exists, they may have an obligation to address it so that agreeing to research on these topics can have significant administrative, legal, and cost implications. researchers will need to be prepared to address these concerns. correctional health providers have a constitutional mandate to provide health care to people in custody, offering a theoretical rationale for research that helps to improve care or make it more efficient or economical. in practice, however, since the types and quality of these services are often the subject of litigation (nathan, 2004) , health providers often filter requests for participation in research projects through their potential impact on current or future litigation. in addition, similarly to corrections officials, correctional health authorities often believe that if they know about a problem they will be required to take action to address it. this has made some officials reluctant to support research on difficult-and expensive-conditions such as hepatitis c (spaulding et al., 2004) . researchers who want to study such topics will need to be able to address these concerns. correctional health providers operate under a variety of auspices, including public departments of corrections or health, universities, voluntary hospitals, or for-profit companies (mellow & greifinger, 2006) . these differing organizational sponsorships influence a unit's openness to research and their motivation to participate in research studies. as with other potential stakehold-ers, researchers need to initiate a straightforward discussion to identify areas of common interest and potential conflict before beginning a study. in some cases, correctional health providers have themselves initiated evaluation studies to guide practice. for example, the university of texas, which has a contract to provide health services for inmates in texas prisons, commissioned an independent evaluation of its services. the report generally lauded the texas program and made several suggestions for more systematic quality assessment (texas medical foundation, 2005) . public health authorities often have a legal mandate to provide oversight of correctional health services and always have responsibility for providing core public health services to people returning to their communities. these obligations provide an incentive for research that can identify unmet needs, improve the effectiveness or quality of care or reduce its costs, or demonstrate the impact of interventions. in practice, some state and municipal health departments have close and positive relationships with correctional health researchers and enlist their help in identifying and solving problems. others, either as a result of fears of litigation, new mandates for service, or unfavorable media attention, may be reluctant to establish partnerships with researchers. other researchers and research institutions can provide an important resource for both experienced and neophyte correctional health investigators. they can share their frontline experiences doing research in specific correctional systems or facilities, the study designs and instruments they have used, their solutions to issues of confidentiality and informed consent, or their findings from their previous research. in the last decade or so, a number of research centers focused on correctional health or reentry have been established, gaining valuable experience and producing a body of work that can inform future studies. some of these are listed in table 24 .1. since some federal funding agencies prefer multijurisdiction research projects in order to increase generalizability, establishing partnerships with experienced centers can help to design such studies and win funding for them. elected officials in both the executive and legislative branches are sometimes needed to approve funding for research studies (e.g., evaluation of publicly funded health or reentry interventions) or to pose questions that need study to correctional or health officials (e.g., how best to provide substance abuse treatment services to people in and returning from correctional facilities). in order to help these officials take on these roles, researchers can provide them with information documenting the problem, cost arguments on the potential savings from new approaches, and the public health benefits of correctional health services. many elected officials worry that supporting health services or even research on the health needs of people in jail or prison might lead to charges that they are "soft on crime" or coddling criminals. research evidence that can reframe the issues as public health, public safety, or economic concerns may help to provide a rationale for interest. funders provide the financial support for correctional and reentry health research and thus for this research to develop they must be willing to provide the level and continuity of funding needed to develop the field. given that both private and public funders always have more requests for support than resources, that prison health is always a less popular choice than, say, children's health or education, and that many funders change their priorities (2004) http://cira.med.yale.edu/ regularly, researchers face an uphill battle in winning the resources they need to pursue a comprehensive research agenda on correctional health. funders who have provided significant support to correctional health research include public agencies such as the national institutes of drug abuse, alcohol abuse and alcoholism, mental health, and allergy and infectious diseases, the centers for disease control and prevention, the national institute of justice, and some state and local governments. private funders include the robert wood johnson foundation, the kellogg foundation, the open society institute, and the jeht foundation, among others. to ensure long-term support, correctional health researchers will need to educate public and private funders about the connections between correctional health and public safety, public health, and social justice as well as to find ways to integrate correctional health issues into research on a variety of health and social problems. prison and reentry advocacy groups serve as important bridge between inmates and their families and the wider community. they also have the potential to influence policy makers, elected officials, and the media. their opposition to unsafe or unhealthy prison conditions, inadequate medical care, or violations of civil liberties have contributed to the development of standards for correctional health care and greater public attention to these issues (nathan, 2004) . the mission, scope, and activities of these groups vary widely, from national organizations such as the national prison project of the american civil liberties union, which brings legal action against correctional systems alleged to violate inmate rights, and critical resistance, an alliance of regional groups dedicated to radical reform of the criminal justice system, to local groups that seek to coordinate reentry programs or organize prison visiting programs. for researchers, these groups can provide detailed knowledge about prison conditions, inmate perceptions of problems, and the local political climate on correctional issues including health. establishing relationships of mutual trust and respect, even when the two parties may disagree on the causes or solution to a problem of interest, can deepen investigators' understanding of the context in which their research is carried out. finally, inmates and their families can provide the insider knowledge that can determine the success or failure of a research project. their understanding of the real-world intersection of policy and practice, the actual living conditions of inmates, and the problems that people leaving jail and prison face when they return home can help researchers to design their studies, develop their research instruments, and interpret their findings. many researchers have noted the benefits of participatory research-deeper knowledge of the problem under study, greater engagement of research participants in the process, and more meaningful interpretation of results (israel, schulz, parker, & becker, 1998; metzler et al., 2003) . in summary, correctional health researchers interact with a variety of stakeholders. at worst, these interactions can appear as a gauntlet of opponents, each with contradictory perceptions and demands that threaten the integrity of the research process and have the potential to disrupt or even halt any study. at best, however, each stakeholder can offer unique insights into the research problem, contribute distinct resources to the research process, and assist in making findings lead to improvements in practice, policy, and health. thus, developing skills in successfully negotiating these interactions is an essential prerequisite for the correctional health researcher. researchers in correctional facilities have used a wide variety of data sources to study inmate health. these include surveys of inmates or correctional authorities, clinical studies of inmate health, secondary analyses of national datasets, ethnographies, and reviews of existing prison health or criminal justice records. each of these sources of data has unique advantages and disadvantages. increasingly, researchers combine different types of data in order to gain deeper insights into the question of interest. for example, many correctional health studies will integrate survey data from participants, medical records from a correctional health service, and official criminal justice records in order to assess the impact of intervention programs. in general, the methodological questions in correctional health are similar to those in other settings: e.g., how to define variables of interest consistently, how to ensure that the data collected are reliable and valid, and how to select appropriate samples and comparison groups. a variety of standard research texts can help investigators to become familiar with these issues (e.g., boruch, 2005; datzker, 1999; noaks & wincup, 2004; patton, 2001) . research in correctional settings does pose some particular methodological challenges. for example, longitudinal studies that follow inmates into the postrelease period face the problem of locating participants after release. since people leaving jail or prison often lack residential stability and may not want further contact with those associated with the incarceration experience, achieving acceptable follow-up rates can be difficult. strategies that have been used to increase follow-up rates include collection of multiple contact names at study entry; frequent interim contacts in order to maintain updated locators, use of both service and financial incentives, and use of public records (e.g., "rap sheets" and criminal records) in lieu of face-to-face contacts. correctional health researchers, like other investigators, often struggle to design and implement multilevel studies that seek to understand the cumulative impact of more than one level of organization on inmate or community health. they may collect data on individuals, social networks such as family and peers, communities, correctional facilities, and jurisdictions, then seek to analyze the contribution each level makes to a specified outcome. for example, a study of women and male adolescents leaving new york city jails examined the impact of individual characteristics, the jail and reentry experience, conditions in the returning community, and changing municipal policies on crime, welfare, and housing on returning inmates' drug use, hiv risk behavior, and reincarceration (freudenberg et al., 2005) . multilevel analyses consider the contributions of variables at multiple levels to the variability in a particular individual-level dependent variable, e.g., drug use. in public health, multilevel research is increasingly used to assess the relative influence of neighborhood and individual-level variables on health (diez-roux, 2001) . by comparing these two influences within different jurisdictions, a third level of organization (i.e., city or state policies or services) can be studied. health research in correctional settings also faces organizational and logistical issues. these include finding space for confidential interviews (an extremely challenging task in overcrowded jails and prisons), negotiating use of technology such as computer-assisted interviewing devices with prison security officials, providing clearance and escorts for researchers, and gaining consistent and reliable access to research participants within the security confines of the facility. solving these logistical problems requires a close and collaborative relationship between researchers and correctional officials. defining common objectives at the inception of research, developing procedures for resolving conflicts before they emerge, and maintaining open communications with all levels of correctional authorities-from frontline correctional officers to wardens and commissioners-can help to reduce logistical problems. most importantly, researchers who choose to work in correctional settings must be willing to act as guests in someone else's house, rather than expect to develop their own rules of conduct. researchers who are unable or unwilling to accept this reality will face difficulty in working in prisons or jails. perhaps the most challenging aspect of health research in correctional settings is meeting the competing demands for ethical research practice as mandated by various bodies as well as the researcher's own ethical standards. prisoners pose ethical dilemmas for researchers not only because they lack the freedom to make the choices that most individuals in the free world take for granted, but also because so many prisoners experience other problems that make them vulnerable as research subjects: low levels of literacy, hiv infection, mental illness, victims or perpetrators of violence, as well as being adolescents. ethical questions correctional health researchers must address include: â�¢ what procedures ensure that all incarcerated people involved in studies have been given the opportunity to give informed and voluntary consent to participate in the research? â�¢ what research practices can guarantee that inmates have as much right to choose to participate in research as any other population? â�¢ how do correctional health researchers balance their ethical responsibilities to the correctional officials who commission their work or provide access to inmates with their responsibility to inmates? â�¢ what level of individual or population benefits in correctional health research balances potential risks? â�¢ how can researchers ensure that participation in correctional health research studies will not lead to harm through disclosure of confidential medical or criminal justice information to third parties? â�¢ what ethical responsibility do researchers have to bring the findings of their research in correctional settings to policy makers or others who can act on these finding? a brief review of the recent history of ethical issues in prison research helps to illustrate the competing forces and changing policy priorities. more in-depth discussion of this history can be found elsewhere (gostin, vanchieri, & pope, 2006; kalmbach & lyons, 2003; degroot, bick, thomas, & stubblefield, 2001; haney & zimbardo, 1998; hornblum, 1998) . in 1997, hornblum observed that "from the early years of this century, the use of prison inmates as raw materials became an increasingly valuable component of american scientific research" (hornblum, 1997) . for example, in the 1960s, major pharmaceutical companies, dow chemical, and the u.s. army tested 153 experimental drugs at the holmesburg prison in pennsylvania (hornblum, 1998) . in 1976, based in part on disclosures of research abuses in prisons, the national commission for the protection of human subjects of biomedical and behavioral research (1976) issued a report that set the framework for subsequent federal involvement in setting ethical standards for human experimentation. their report called for additional protection for certain "vulnerable" populations, including children, neonates, pregnant women, and prisoners. in 1978, the commission issued a report titled "additional protections pertaining to biomedical and behavioral research involving prisoners as subjects" (u.s. dhhs, 2005) . the main goal of these early guidelines was to protect incarcerated individuals from serving as involuntary or coerced "guinea pigs" in research that offered no direct benefits and had the potential for harm. in the 1980s and early 1990s, the aids epidemic raised new ethical concerns for correctional health researchers. in some cases, prisoners with hiv infection or aids were not permitted to join clinical trials for new aids medications, based on various beliefs including their inability to give truly voluntary consent and their perceived unwillingness to comply with prescribed regimens. some health researchers and prisoners rights advocates argued that such a ban violated ethical principles and that prisoners should have the same access to experimental treatments and clinical trials as other sectors of the population. from this perspective, ethical guidelines should place a priority on ensuring access to potential beneficial treatments (dubler and sidel, 1989 ) -a priority that may conflict with the previous emphasis on protecting inmates from researchers. in 2006, the institute of medicine commissioned another review of ethical issues involved in prisoner research (gostin, vanchieri, & pope, 2006) . based on several reviews of the more recent literature and testimony from dozens of witnesses including researchers, inmates, and correctional officials, the committee on ethical considerations for protection of prisoners involved in research made fourteen recommendations in five broad categories (table 24 .2). these recommendations strive to find an appropriate and updated balance between the protection and access imperatives embodied in previous ethical standards. whether these institute of medicine recommendations lead to changes in federal guidelines for prison research or in practice remains to be seen. in practice, among the vexing problems correctional health researchers face are obtaining voluntary consent in jails or prisons, informing research participants about the benefits and risks of research, getting consent for randomized trials in which some participants receive no potential benefit, protecting the privacy of research participants, and negotiating with irbs that may lack expertise in the realities of prison research. defining "voluntary" consent in the coerced environment of a correctional facility is sometimes difficult. among the practices that can compromise free choice are promises of services not ordinarily available to inmates (e.g., certain types of health services), the presence of correctional officers in the area where consent is being solicited, the unavailability of the independent advice on participation that is normally available to research participants in the free world, or the implied offer to use participation in research in exchange for a shorter sentence or favorable consideration by a judge or parole board. since no set of rules can govern all the situations that can jeopardize voluntary consent, for any particular study the ethical researcher ought to consult experienced correctional researchers, correctional officials at the study site, prisoners rights advocates, and current and former inmates in order to obtain a variety of perspectives on the best procedures to insure voluntary consent. similarly, the process of informing research participants in correctional settings of the risks and benefits of a study can be challenging. many inmates have low levels of literacy; many distrust correctional and health authorities, sometimes based on their own past experiences; and, unlike most research in medical settings, an added risk is disclosure of information that can cause harm to participants from other inmates, correctional staff, legal authorities, or the wider public. research on stigmatized conditions such as hiv infection, mental illness, and substance use almost always poses such risks. methods that researchers have used to overcome these obstacles are to engage current and former inmates in the design of informed consent materials and as members of source: gostin et al. (2006) . irbs, to hire independent advisors who are not part of the research team to help inmates make decisions about participation, and to obtain federal certificates of confidentiality to minimize risk of disclosure of confidential information. while some inmates and ethicists express concerns about the coercion implicit in any research in the correctional setting, the recent iom report (gostin et al., 2006) also noted that other inmates strongly oppose restrictions on inmate participation in research. some are concerned about lack of access to cuttingedge treatments for hiv or cancer; others object to the loss of opportunities for compensation or enhanced living situations. a specific problem facing researchers involved in clinical trails in which some forms of treatment are withheld from some participants is convincing both staff and participants of the rationale for a randomized trial. from a researcher's point of view, the lack of definitive evidence of the benefits of an intervention is sufficient rationale for such a trial but for staff and participants, withholding services perceived to be beneficial may seem unethical. when staff are not convinced of the morality of a research study, they may intentionally or unintentionally undermine the study, either by providing services to the "control" group or by communicating their discomfort to research participants, thus discouraging enrollment in a study. for this reason, it is important for researchers to address this issue forthrightly. strategies to minimize this problem include offering all research participants some level of services above the standard care in the correctional facility, comparing different interventions to each other rather than to no special services, educating research staff about the ethics of offering unevaluated services to all participants, and, as the institute of medicine report on correctional research suggests (gostin et al., 2006) , joining advocacy efforts to improve the basic standard of care in all correctional facilities. in my experience, many correctional health researchers complain about the extensive and lengthy process required to get irb approval for their research study and suggest that it can discourage them from pursuing worthy projects. in some cases, several different irbs need to approve a single study and occasionally offer conflicting guidance on how to proceed. these complaints have a variety of sources: some investigators prefer the old way of business where researchers alone decided on the conduct of their studies. but even researchers who support the importance of protecting prisoners note that irb members often lack expertise in the day-to-day realities of correctional institutions and the nonresearch risks inmates encounter daily. they also report that irb committees often reflect the wider tension between protecting participants from research harm and ensuring access to beneficial services and in their effort to maximize both of these aims impose unreasonable demands on researchers. a possible solution is to assist irbs to find a member who is experienced in correctional settings and correctional research-not only to meet the dhhs regulatory requirement to include such a person but also to obtain practical advice on devising realistic and ethical resolution of problems. for example, one state prison system irb included an attorney who specialized in inmate litigation. another solution, as recommended by the iom report (gostin et al., 2006) , is to develop universal national standards for review of prison research so that all research is reviewed using uniform criteria. at present, correctional health researchers respond to a variety of heterogeneous influences -other criminal justice, medical, public health, and public policy researchers; local, state, and federal correctional and health officials; correctional health providers; a variety of professional organizations; elected policy makers; and various criminal justice and health advocacy organizations, among others. it is therefore not surprising that in this anarchic and complex environment correctional health researchers have yet to develop a coherent and comprehensive research agenda driven by existing scientific knowledge and public policy imperatives. however, the fact that it may be difficult to envision and articulate such an agenda should not stop the effort. in fact, as health and correctional officials and researchers request additional support for correctional health research, it is inevitable that they will be asked to set priorities. and if researchers themselves fail to take the lead in this process, others will impose an agenda on them. while the development of a comprehensive research agenda for correctional health is beyond the scope of this chapter, i conclude by suggesting some steps that might move the field in this direction. first, we need to begin a national dialogue on research needs that include researchers, correctional and health officials, policy makers, and advocates. questions to discuss include: what are the most promising avenues of research to lead to short-and middle-term improvements in the health of incarcerated populations? what are potential stable funding streams for this research? how best can we develop consistent frameworks for research so that clinical, practice, and policy decisions can be more evidence-based? who are the constituencies that will support a national research agenda on correctional health and how can these constituencies be organized into a coherent force? what correctional research might be particularly beneficial both to the health of the incarcerated and to the larger health of the public? organizations that can play a role in this national discussion include the national institute of justice, nih institutes and the centers for disease control and prevention, the national commission on correctional health care, various health professional organizations, and the reentry policy council. second, researchers need to synthesize the existing and disparate literature on correctional health to identify common findings, gaps in the literature, and future priorities. this literature is dispersed in several different disciplines and among the peer-reviewed and "gray" literatures, i.e., public and voluntary organization reports and studies. one possible sponsor for such a critical review would be the institute of medicine. third, as recommended by the recent iom report on correctional health research (gostin et al., 2006) , the united states should establish more consistent and uniform guidelines for ethical health research among incarcerated populations. such guidelines will protect researchers and inmates and help to resolve the continuing debate between protection from researchers and full access to the benefits of research. fourth, any agenda should consider the range of settings in which correctional health plays out, including courts, jails, prisons, parole and probation services, alternatives to incarceration, and reentry programs. too often, each setting has been its own silo with a cadre of researchers and officials. the evidence of the past decades suggests that in fact these settings constitute a single if sometimes disorganized system in which changes in one component affect all others. thus, health research needs to examine these systemic interactions in order to avoid shifting problems for one sector to another. finally, correctional health research has to be considered a branch of population health research and therefore address the broadest questions that affect the health of the public. in the past, some correctional health researchers have limited their attention to those individuals served in correctional health settings-the patients who walked through their clinic doors. while these concerns will continue to be important and warrant focused investigation, they are not sufficient to realize the full opportunity for correctional health researchers to improve health. research questions that need to be addressed in the coming decade include: how does incarceration influence socioeconomic, racial, and gender disparities in health in the united states? how does incarceration affect the health of the families and communities of incarcerated individuals? what role can correctional health services play in reducing community incidence, prevalence, severity, or costs of conditions such as hiv infection, hepatitis c, diabetes, asthma, addiction, violence, depression, or lack of health insurance? by expanding their focus to these questions, correctional health researchers have the potential to contribute to solving our nation's most pressing health problems. correlates of hiv infection among incarcerated women: implications for improving detection of hiv infection at the intersection between poverty, race, and hiv infection: hiv-related services for incarcerated women the disease profile of texas prison inmates the concentration of substance use, criminal justice involvement, and hiv/aids in the families of drug offenders hiv prevention with jail and prison inmates: maryland's prevention case management program association of tuberculosis infection with increased time in or admission to the new york city jail system randomized experiments for planning and evaluation: a practical guide (applied social research methods) impact of an after-hours on-call emergency physician on ambulance transports from a county jail reducing hiv risk among women visiting their incarcerated male partners readings for research methods in criminology and criminal justice use of health impact assessment in incorporating health considerations in decision making clinical trials in correctional settings: right or regression? investigating neighborhood and area effects on health understanding community re-entry among former prisoners with mental illness: a conceptual model to move new research on research on hiv infection and aids in correctional institutions treating prison inmates with cooccurring disorders: an integrative review of existing programs hepatitis c virus infection among prisoners in the california state correctional system jails, prisons, and the health of urban populations: a review of the impact of the correctional system on community health coming home from jail: the social and health consequences of community reentry for women, male adolescents, and their families and communities coming home from jail: a review of health and social problems facing us jail populations and of opportunities for reentry interventions ethical considerations for research involving prisoners reducing postrelease risk behavior among hiv seropositive prison inmates: the health promotion program hiv/aids and other infectious diseases among correctional inmates: transmission, burden, and an appropriate response the burden of infectious disease among inmates of and releasees from us correctional facilities the past and future of u.s. prison policy: twentyfive years after the stanford prison experiment a framework for management of hepatitis c in prisons they were cheap and available: prisoners as research subjects in twentieth century america acres of skin: human experiments in holmesberg prison how criminal system racial disparities may translate into health disparities review of communitybased research: assessing partnership approaches to improve public health mental health problems of prison and jail inmates bridging the gap: providing health care to newly released men the effects of male incarceration dynamics on aids infection rates among african-american women and men from evidence to action on hiv/aids in prisons: a report from the xvi international aids conference ethical and legal standards for research in prison substance dependence, abuse and treatment of jail inmates diabetic ketoacidosis in prisoners without access to insulin health impact assessment: a tool for healthy public policy cost-effectiveness of universal screening for chlamydia and gonorrhea in us jails intraprison hiv transmission: an assessment of whether it occurs, how it occurs, and who is at risk a review of the legal and ethical issues for the conduct of hiv-related research in prisons hiv infection in u.s. correctional systems: its effect on the community a prospective examination of high-cost health services utilization among drug using prisoners reentering the community treating incarcerated women: gender matters facilitators and barriers to continuing healthcare after jail: a communityintegrated program health behind bars: utilization and evaluation of medical care among jail inmates the mental health of federal offenders: a summative review of the prevalence literature incarceration associated with homelessness, mental disorder, and co-occurring substance abuse successful reentry: the perspective of private correctional health care providers through community-based participatory research partnerships the health of youth in the juvenile justice systems parental imprisonment: effects on boys' antisocial behaviour and delinquency through the life-course get connected: an hiv prevention case management program for men and women leaving california prisons taking stock of the accomplishments and failures of prison reform litigation: have the courts made a difference in the quality of prison conditions? what have we accomplished to date? community case management for former jail inmates: its impacts on rearrest, drug use, and hiv risk criminological research: understanding qualitative methods qualitative research and evaluation methods treatment of substance-abusing jail inmates examination of gender differences health care delivery strategies for criminal offenders cost-effectiveness of hiv screening for incarcerated pregnant women successful linkage of medical care and community services for hiv-positive offenders being released from prison linkages between in-prison and community-based health services incarceration, reentry, and social capital: social networks in the balance comparison of background characteristics and behaviors of african-american, hispanic, and white substance abusers treated in federal prison: results from the triad study effectiveness of antiretroviral therapy among hiv-infected prisoners: reincarceration and the lack of sustained benefit after release to the community effectiveness of antiretroviral therapy among hiv-infected prisoners: reincarceration and the lack of sustained benefit after release to the community report on a national survey of correctional health facilities: a needs assessment of health issues sex offenders, sentencing laws and pharmaceutical treatment: a prescription for failure racial disparity and the legitimacy of the criminal justice system: exploring consequences for deterrence the prevalence of severe mental disorder among male urban jail detainees: comparison with the epidemiologic catchment area program prevalence of psychiatric disorders among incarcerated women. i. pretrial jail detainees an evaluation of correctional health care services provided by university of texas medical branch correctional managed care to the texas department of criminal justice: an assessment of managed care service delivery systems, adherence to correctional health care standards and clinical outcomes high rates of incarceration as a social force associated with community rates of sexually transmitted infection code of federal regulations: title 45 quantitative health impact assessment: current practice and future directions collaborations between criminal justice and mental health systems for prisoner reentry reentry planning for mentally disordered inmates: a social investment approach key: cord-005327-bt7o8yxk authors: ahn, insung; son, hyeon seok title: epidemiological comparisons of codon usage patterns among hiv-1 isolates from asia, europe, africa and the americas date: 2006-12-01 journal: exp mol med doi: 10.1038/emm.2006.76 sha: doc_id: 5327 cord_uid: bt7o8yxk to investigate the genomic properties of hiv-1, we collected 3,081 sequences from the hiv sequence database. the sequences were categorized according to sampling region, country, year, subtype, gene name, and sequence and were saved in a database constructed for this study. the relative synonymous codon usage (rscu) values of matrix, capsid, and gp120 and gp41 genes were calculated using correspondence analysis. the synonymous codon usage patterns based on the geographical regions of african countries showed broad distributions; when all the other regions, including asia, europe, and the americas, were taken into account, the asian countries tended to be divided into two groups. the sequences were clustered into nine non-crf subtypes. among these, subtype c showed the most distinct codon usage pattern. to determine why the codon usage patterns in asian countries were divided into two groups for four target genes, the sequences of the isolates from the asian countries were analyzed. as a result, the synonymous codon usage patterns among asian countries were divided into two groups, the southern asian countries and the other asian countries, with subtype 01_ae being the most dominant subtype in southern asia. in summary, the synonymous codon usage patterns among the individual hiv-1 subtypes reflect genetic variations, and this bioinformatics technique may be useful in conjunction with phylogenetic methods for predicting the evolutionary patterns of pandemic viruses. over the past 30 yr, human immunodeficiency virus type 1 (hiv-1) infection has increased and now threatens the health of people worldwide. hiv-1, which is the most common cause of aids, has already infected more than 50 million people. an the strains of hiv-1 can be classified into three groups, group m (for main), group n (for non-m/non-o or new), and group o (for outlier), which are the result of three cross-species transmissions of chimpanzee viruses into the human population (pierre et al., 1994; beatrice et al., 2000) . group o hiv-1 is usually found in west-central africa, and group n, which was discovered in 1998 in cameroon, rarely appears. most of the hiv-1 infections belong to hiv-1 group m, which is divided into nine distinct subtypes (a, b, c, d, f, g, h, j and k) and 14 intersubtypes of hiv-1 recombinants, which are known as circulating recombinant forms (crfs). each subtype can be classified according to the criteria listed below. the subtypes are approximately equidistant from one another in terms of the env gene, and the env phylogenetic tree is, for the most part, congruent with the gag phylogenetic tree. moreover, two or more samples are required to define a new sequence subtype. envelope-coding sequences were used as the basis for classification in the 1992 compendium but, as the database of gene sequences has expanded, this basis has been increasingly challenged. for example, although subtype e viruses are equidistant from viruses of subtypes a to d in terms of their env-coding sequences, they appear to be similar to subtype a viruses with respect to their gag-coding sequences. the gag-coding sequences of subtype e viruses have not yet been documented. the subtypes have been used successfully as molecular epidemiological markers to track the course of the hiv-1 pandemic. for instance, africa has all the known hiv-1 subtypes and groups, reflecting the african origin of the epidemic (wouter et al., 1997) . subtype b viruses are prevalent in the americas and western europe, and subtype c is the most prevalent hiv type worldwide (josé and natt, 2000) . synonymous codon usage pattern analysis is a common method for examining the origin of species in many fields (bulmer, 1978; shields and sharp, 1987; moriyama and hartl, 1993; stenico et al., 1994; mclnerney, 1998; kanaya et al., 2001; duret, 2002; lynn et al., 2002) . synonymous codons often encode common amino acids during protein synthesis. however, they are not used randomly, and some codons are used more frequently than others (moriyama and hartl, 1993; mclnerney, 1998; duret, 2002; lynn et al., 2002) . in prokaryotes, such as thermophilic bacteria, the codon usage in highly expressed genes shows a shift towards a more restricted set of preferred synonymous codons (lynn et al., 2002) . codon usage patterns tend to mirror the distribution of trna abundances (shields and sharp, 1987; stenico et al., 1994) . the relative synonymous codon usage (rscu) values may be virus-specific (gu et al., 2004) and may not be affected by translational selection or gene length (jenkins and holmes, 2003) . jenkins and holmes (2003) analyzed codon usage patterns by applying correspondence analysis, which is a type of statistical perceptual mapping that relates categories of a contingency table. correspondence analysis is a compositional technique in which the perceptual map is based on the association between objects and a set of specified descriptive characteristics or attributes (hair et al., 1998; johnson and wichern, 2002) . it should be noted that almost all of the codon usage studies using correspondence analysis have been performed in bacteria or higher species but not in viruses. this study investigated the characteristics of genomic patterns, particularly with respect to the codon usage patterns of hiv-1, on the basis of continental regions, such as asia, europe, africa, and the americas, as well as nine non-crf subtypes. the hiv-1 genome contains three major regions, gag, pol, and env. we chose to determine the subtypes of the gag and env genes, which encode the inner and outer structures of hiv-1, respectively. the protease, reverse transcriptase, and integrase enzymes derived from the pol gene as well as tat (= transcriptional activation via rna target) gene (lee et al., 2004) were excluded, as the focus was on genes that are related to the specific structure of hiv-1. the matrix, which is located under the viral plasma membrane, plays an important role in transporting the gag-pol precursor polyprotein to the plasma membrane, while the capsid, which is derived from the gag gene, like the matrix, constitutes the core shell of the viral particle. if a mutation occurs in the capsid protein, that virus is not coated during the assembling process. among the env-encoded proteins, the surface glycoprotein gp120 triggers the viral entry process, while gp41 mediates the fusion interactions between the viral and cellular membranes in the initial stage of infection. these four genes, which are encoded from the env and gag gene regions, were analyzed in the present study. hiv-1 sequence files in the fasta format were collected from the hiv sequence database (http:// hiv.lanl.gov), which is based on hiv sequences downloaded from genbank (http://www.ncbi.nih.gov/ genbank), and all of the sequences were divided into four gene groups: matrix (784 sequences), capsid (829 sequences), gp120 (715 sequences), and gp41 (753 sequences), using java and the mysql database (version 3.23) on the linux operating system. the accession number was a primary key, and other fields, such as sampling region, country, year, subtype, gene name, and sequence, were also created in the database, which was constructed for this study. the sampling years of the sequences were from 1991 to 2004, and these sequences were grouped into five geographical regions, which included asia, europe, africa, and north and south america, on the basis of the 'sampling region' field. any sequences that had ambiguous characters or length were pre-screened, and no redundant sequences were sampled from the same patient. the non-crf (circulating recombinant form) subtypes, which included subtypes a, b, c, d, f, g, h, j and k, were extracted from the database and used to analyze the codon usage patterns of different geographical regions. in correspondence analysis, the rscu value for each codon is usually used to prevent the amino acid composition from influencing the codon usage values for each gene (sharp and li, 1986) . the rscu value is the number of times a particular codon is observed relative to the number of times the codon would be observed in the absence of any codon usage bias. if codon usage bias is absent, the rscu value is 1.00. the rscu was calculated as: where xij is the frequency of occurrence of the jth codon for the ith amino acid, and n i is the number of codons for the ith amino acid. for the correspondence analysis, each gene was represented as a 59 dimensional vector, with the exclusion of start and stop codons and the ugg codon, which encodes tryptophan without synonymous codons. all 59 codons were then pooled and collated in a contingency table according to the species genome in which they were included. in this study, we used correspondence analysis to compare the differences in rscu values among hiv-1 isolates on the basis of geographical region, non-crf subtype or country of collection. correspondence analysis is a type of multivariate analysis that represents associations either as tabulated frequencies or graphically as counts. each gene is presented as a 59-dimensional vector, and each dimension corresponds to the rscu value of one sense codon, excluding aug, ugg, and three stop codons (gu et al., 2004) . for a contingency table with i rows and j columns, the plot produced by correspondence analysis contains two sets of points: one set of i points that corresponds to the rows, and one set of j points that corresponds to the columns. the positions of the points reflect the associations. the rscu values for 59 codons, as described above, were calculated, and correspondence analysis was performed using the sas statistical program version 9.1 (cary, 2004) . the usual output from a correspondence analysis includes the "best" two-dimensional representation of the data, along with the coordinates of the plotted points and a measure (called the inertia) of the amount of information retained in each dimension (johnson and wichern, 2002) . the results provide information similar to that produced by factor analysis techniques, and they allow exploration of the structure of the categorical variables included in the table. genes or species that are strongly associated, as measured by their chi-square distances, lie in a similar direction from the origin (johnson and wichern, 2002; perrière and thioulouse, 2002) . the chi-square distance between two coordinates with the same row or column value, called the euclidean distance (perrière and thioulouse, 2002; gu et al., 2004) , has important statistical meaning, whereas there is no significant statistical meaning attached to the relationship between the row coordinate and column coordinate. the sigmaplot 8.0 program was used to create graphical correspondence analysis plots using coordinates that consisted of the firstand second-dimensional factors that resulted from the correspondence analysis. to compare the overall synonymous codon usage patterns, we created an analytical database that included the 3,081 sequences, including the genes from the matrix, capsid, gp120, and gp41, and performed correspondence analysis with the rscu values for each sequence using java codes ( figure 1 ). of the four genes, two gag-originating genes, for the matrix and capsid, showed more biased synonymous codon usage patterns for the dim1 gene than for the two genes that originated from the env gene region. the unit ranges of dim1 for the matrix and capsid were 0.69 (-0.34～0.35) and 0.68 (-0.25～0.43), while those for gp120 and gp41 were 0.41 (0.21～0.20) and 0.56 (-0.28～0.28), respectively. isolates from african countries revealed broad codon usage distributions; taking all the other geographical regions into consideration, asian countries tended to divide the isolates into two groups on the basis of dim1. european isolates also showed relatively broad distributions but the differences in the ranges were narrower than those for the african isolates. isolates from north and south america were clustered together for the matrix, capsid, and gp41 genes, but were divided into two groups for gp120. in addition to the codon usage analysis using the sequence datasets that were divided according to geographical region, we performed correspondence analysis using the rscu values for each non-crf subtype, i.e., subtypes a, b, c, d, f, g, h, j, and k, from all the countries available ( figure 2) . in contrast to the result described above (figure 1) , the hiv-1 subtypes were readily classified on the basis of their synonymous codon usage patterns. among these subtypes, subtype c showed the most distinct distribution. with regard to dim1 in the correspondence analysis plots, subtype c viruses were located opposite the other subtypes, with the exception of the capsid gene. on the other hand, subtype a viruses showed distributions for the capsid genes that were distinct from those of the other subtypes, although they shared similar patterns with subtype b for the other genes. subtype d showed similar patterns to subtype b for both the matrix and capsid genes (figure 2a and b) , but revealed somewhat different distributions for gp120 and gp41 ( figure 2c, d) . to understand why the codon usage distributions of isolates from the asian region were divided into two distinct groups (figure 1) , we extracted the sequences that originated in the asian countries, and analyzed them on the basis of individual countries of origin ( figure 3 ). for the capsid, gp120, and gp41 genes, the hiv-1 sequences from thailand commonly showed synonymous codon usage patterns that were distinct from those of other countries ( figure 3b , c and d), and the sequences from vietnam, myanmar, singapore, japan, and thailand for the matrix genes were clustered as a single group in the corresponding analysis plots ( figure 3a ). some sequences from singapore and myanmar were located at the same position as those from korea and china. using the same sequences, we classified the plots on the basis of their major subtypes, which included non-crf and crf subtypes, to determine the relationships between hiv-1 subtypes and their distributions in asian countries (figure 4) . the five major hiv-1 subtypes identified in this database, but not in the populations, were 01_ae, 08_bc, bc, b, and c. when compared with the results shown in figure 3 , it appears that high levels of 01_ae subtype viruses occur in southern asian countries, which include vietnam, singapore, thailand, and myanmar. unlike the sequences from korea, the sequences from japan were colocalized with this southern asian group. some of the sequences from myanmar were also found in the other groups, which may reflect the geographical location of myanmar, which lies between the southern asian countries and the chinese continent. hiv-1 isolates from china and india tended to be grouped together; subtypes bc and c were dominantly distributed in these countries, while subtype 01_ae viruses were mainly distributed in southern asian countries. subtype b viruses were predominate in korea. large amounts of genomic data have been collected and distributed on the web by various organizations such as the u.s. national center for biotechnology information (ncbi) and the european bioinformatics institute (ebi). thanks to these public databases, not only the survey data but also the genomic data have become important resources for the field of genetic epidemiology. since the initial reports of hiv-1 virus in africa, it has spread worldwide and generated biological subtypes, such as the nine non-crf viruses and several other crfs. as a result of geographical constraints, the distributions of these subtypes are so uneven that the differences in hiv-1 subtypes between countries can be used as a marker of epidemiological diversification. in this study, we determined the codon usage differences between hiv-1 isolate from each geographical region, to elucidate their evolutionary patterns. to investigate the overall patterns of synonymous codon usages among the isolates from asia, europe, africa, and north and south america, we extracted 3,081 nucleotide sequences from the hiv sequence database (figure 1 ). isolates from african countries showed broad codon usage distributions, taking all the other geographical regions into consideration. this result appeared reasonable because almost all hiv-1 subtypes are still distributed in african countries, especially near the central african region where the first hiv-1 infection occurred. in concordance with the previous phylogenetic analyses of hiv-1 subtypes (wouter et al., 1997; nicole et al., 2000; feng et al., 2001) , our results show that all the synonymous codon usage patterns exist in african countries. unlike the african isolates, the isolates from asian countries were divided into two distinct groups, particularly for the matrix and capsid genes. this is an interesting result, as the different codon usage patterns for these two groups may provide some clues as to the differentiation of epidemiological patterns of hiv-1 subtypes. we conducted an additional analysis that focused on the asian isolates. on the other hand, the broader unit ranges observed for dim1 of the gag-origin genes, such as the matrix and capsid genes, as compared to those of gp120 and gp41, suggest that genes responsible for virus-host interactions are more likely to be less biased or varied in terms of synonymous codon usage patterns than other structural genes. as the envelope glycoproteins gp120 and gp41 perform important roles in receptor binding and in interacting with neutralizing antibodies, they appear to be more conserved than other structural genes (lee et al., 2006) . the differences in synonymous codon usage patterns among the subtypes were also analyzed using the nine non-crf subtypes. we excluded the crf subtypes from this study because the focus was on the unique, not mixed, codon usage patterns of each subtype ( figure 2 ). all the sequences of the nine non-crf subtypes were extracted from the database and analyzed. interestingly, hiv-1 subtypes revealed a clear classification with respect to their codon usage patterns, regardless of the sampling country. among these non-crf subtypes, subtype c showed the most distinct distribution pattern. subtype c viruses, initially a rarely reported subtype, now dominate the global pandemic and constitute the majority of the newly transmitted cases of hiv-1 in many developing countries. there is growing evidence to suggest that subtype c viruses display characteristics that distinguish them from other subtypes, and that these differences affect transmission and pathogenesis (hong et al., 2002) . in the present study, this subtype proved to have different synonymous codon usage patterns. hiv-1 subtype c, which is currently ranked as the third most dominant hiv-1 subtype in the world, appears to have been successfully transmitted from the original source in central africa to countries such as india and saudi arabia (hiv-1 geography maps, 2006) . in the analysis of the overall patterns of synonymous codon usage among isolates from asia, europe, africa, and north and south america, the asian isolates tended to separate into two groups on the basis of dim1. to understand this phenomenon, we extracted the sequences for the asian isolates and analyzed the rscu patterns using correspondence analysis (figures 3 and 4) . among the asian countries, all the hiv-1 subtypes were distributed unevenly, showing distinct synonymous codon usage patterns for different geographical regions. interestingly, the sequences from southern asian countries, which include vietnam, singapore, and thailand, were grouped together, and the crf subtype 01_ae viruses predominated. although the dataset from japan used in this study was small, the crf 01_ae subtype viruses were also found in japan, and they showed the same codon usage patterns as isolates from the other southern asian countries. when we compared the patterns of the nine non-crf subtypes (figure 2 ), subtype c showed the most distinct patterns. however, the crf subtype 01_ae isolates weakened the effect of subtype c, being located in a distinctly different region to the other subtypes, including subtype c. according to nicole et al. (2000) , the highest prevalence of crf 01_ae is in the north of the democratic republic of congo (dcr), which is known as the epicenter for hiv-1 group m viruses in africa; this recombinant subtype uses the synonymous codons in a different manner in protein synthesis than do the other subtypes in our study. for various reasons, the crf 01_ae subtype seems to have adapted successfully to the southern asian countries, including japan in the far east. in contrast, subtype 01_ae and subtypes bc and c predominate in china and india, and cluster separately in the correspondence plots. to date, subtype c viruses have been found to play an important role only in the epidemics of southern africa (wouter et al., 1997) . however, subtype c viruses are found with increasing frequency in india, which is the gateway to the asian continent from the african continent. according to population-based studies, subtype b is the most predominant subtype in the world (hiv-1 geography maps, 2006) , especially in europe and the united states (ras et al., 1983) . unlike subtypes 08_bc, bc, and c, subtype b was distinctly grouped (figure 4) , showing similar but not identical codon usage patterns to other subtypes, with the exception of subtype 01_ae. some sequences from singapore and myanmar colocalized with those from korea and china. since myanmar is located between the southern asian countries and china, it is reasonable to find intermediate patterns between those two regions. on the other hand, for isolated singapore, it is difficult to explain why the two subtypes of subtype b subtype 01_ae were dominant. subtype b is often found in europe and america, so tourists from these countries to singapore may be transmitters of subtype b. for many years it was believed that errors in reverse transcriptase and the presence of virion rna in dimer form were the driving forces behind the genetic variation of hiv. however, since 1995, many studies have suggested that recombination plays a more important role in the genetic diversification of hiv-1 than was previously thought (wouter et al., 1997) . in the present study, we show that each hiv-1 subtype uses synonymous codons in a specific way, and that these codon usage patterns may play a role in the distribution of hiv-1 subtypes. in summary, the synonymous codon usage patterns among the hiv-1 subtypes reflect genetic variability, and this bioinformatics technique in conjunction with phylogenetic methods may be useful in predicting the evolutionary patterns of pandemic viruses. this study extends our knowledge of hiv-1 subtypes in terms of genomic analysis. further genetic epidemiology studies using genomic patterning on a population basis are needed to substantiate these findings, and bioinformatics techniques may be valuable tools in this respect. available at: www.unaids.org aids epidemic update 2 from united nations programme on hiv/aids (unaids) aids as a zoonosis: scientific and public health implications statistical analysis of nucleotide sequences of introns and exons in human genes sas ® r9.1.2 qualification tools user's guide evolution of synonymous codon usage in metazoans evidence of two distinct subsubtypes within the hiv-1 subtype a radiation analysis of synonymous codon usage in sars coronavirus and other viruses in the nidovirales multivariate data analysis hiv-1 geography maps at los alamos hiv sequence database phylogenetic and phenotypic analysis of hiv type 1 env gp120 in cases of subtype c mother to child transmission the extent of codon usage bias in human rna viruses and its evolutionary origin applied multivariate statistical analysis accelerating the development and future availability of hiv-1 vaccines: why, when, where, and now codon usage and trna genes in eukaryotes: correlation of codon usage diversity with translation efficiency and with cg-dinucleotide usage as assessed by multivariate analysis transduction of yeast cytosine deaminase mediated by hiv-1 tat basic domain into tumor cells induces chemosensitivity to 5-fluorocytosine structure-activity relationships of anti-hiv-1 peptides with disulfide linkage between d-and l-cystein at positions i and i+3, respectively, derived from hiv-1 gp41 c-peptide synonymous codon usage in subject to selection in thermophilic bacteria codon replicational and transcriptional selection on codon usage in borrelia burgdorferi codon usage bias and base composition of nuclear genes in drosophila unprecedented degree of human immunodeficiency virus type 1 (hiv-1) group m genetic diversity in the democratic republic of congo suggests that the hiv-1 pandemic originated in central africa use and misuse of correspondence analysis in codon usage studies isolation and envelope sequence of a highly divergent hiv-1 isolate: definition of a new hiv-1 group aquired immunodeficiency syndrome. a report of 2 south african cases codon usage in regulatory genes in escherichia coli does not reflect for 'rare' codons synonymous codon usage in bacillus subtilis reflects both translational selection and mutational biases codon usage in caenorhabditis elegans: delineation of translational selection and mutational biases the puzzle of hiv-1 subtypes in africa we acknowledge the contribution of all those who have made their invaluable data publicly available. this work was supported by the brain korea 21 project (2006). key: cord-022535-08hqmwlg authors: schmiedel, stefan title: infektionen, impfungen, reisemedizin date: 2013-06-26 journal: praxisleitfaden allgemeinmedizin doi: 10.1016/b978-3-437-22443-0.10009-2 sha: doc_id: 22535 cord_uid: 08hqmwlg nan syndrom (sars) 518 9.4.11 vogelgrippe 518 9.4.12 "neue influenza" 519 9.5 mykosen 520 9.5.1 allgemeines 520 9.5.2 candidosen (soor) 520 9.5.3 systemische mykosen 521 9.5.4 dermatophytosen 522 9.6 protozoeninfektionen 522 9.6.1 toxoplasmose 522 9.6.2 lambliasis (giardiasis) 524 9.6.3 sonstige protozoeninfektionen 524 9.7 wurmerkrankungen 525 9.7.1 madenwurm-infektion 525 stefan schmiedel (ascariasis) 526 9.7.3 bandwurm-infektion (zestoden) 526 9.7.4 echinokokkus-infektion 527 9.7.5 trichinose 528 9.8 sexuell übertragbare krankheiten 528 9.8.1 gonorrhö 528 9.8.2 syphilis (lues) 529 9.8.3 trichomoniasis 531 9.8.4 ulcus molle 531 9.8.5 lymphogranuloma venereum 531 9.8.6 condylomata acuminata 531 9. 9 hiv und aids 532 9.9.1 epidemiologie und übertragungswege 532 9.9.2 labordiagnostik 533 9.9.3 stadieneinteilung und verlauf 535 9.9.4 diagnostik bei hiv-positiven 537 9.9.5 häufige krankheitsbilder bei aids 537 9.9.6 therapie 542 9.9.7 • rekurrierendes fieber: in regelmäßigen abständen auftretendes, mehrere tage anhaltendes hohes fieber (> 39 °c) . malaria möglich, andere bakt. erkr. (diagn.: reiseanamnese, edta-blut für plasmodiennachweis, ▶ 9.10.8 und ▶ 9.10.9). • subfebril: temperaturerhöhung dem physiologischen tagesablauf angepasst, nicht über 38 °c. tumorfieber (bes. maligne lymphome; ▶ 19.4.3) , tbc (▶ 12.3.5) , arzneimittelfieber (drug fever; toxische oder allergische reaktion, v.a. bei sulfonamiden), lungenembolie (▶ 12.9.2) , hyperthyreose (▶ 17.6.2), chron. tonsillitis (▶ 23.3.2) , endokarditis (▶ 10.7.1), ra (▶ 18.3.1), polymyalgia rheumatica (▶ 18.5.3) , unter antibiotischer ther. • remittierendes fieber: max. schwankungen ≤ 1,5 °c, temp. abends höher als morgens. vorkommen: pyelonephritis (▶ 13.3.3) , tbc (▶ 12.3.5), rheumat. fieber, sepsis. diagnostik fieber objektivieren (selbst messen). ganzkörperstatus; labor: je nach befund, fieberverlauf und az, großes bb, crp, bsg, urinstatus, transaminasen, blutkulturen (mehrmalig; am besten bei fieberanstieg). evtl. rf, ana, hiv-test. abdomen-sono, rö-thorax. abhängig von befunden bzw. längerem persistieren klinikeinweisung zur weiterführenden diagn. bei psychisch auffälligen pat. an mögliche manipulation des thermometers denken! • kinder < 1 j.: bei trinkschwäche bzw. verweigerung von mehr als 3 mahlzeiten ggf. facharztüberweisung zum kinderarzt, je nach az evtl. klinikeinweisung in die kinderklinik ("durstfieber"). • kinder > 1 j.: zuwarten bei fieber bis 3 d, so weit kein klarer lokalbefund vorliegt und der az es erlaubt, rein symptomatische ther. (▶ 16.14 (z.b. autoimmunerkr., malignes lymphom) zurückzuführen ist, behandlung der grunderkr., ggf. facharztüberweisung oder klinikeinweisung bei reduziertem az, bei hohem fieber und vorliegen eines immundefekts (hiv-inf., immunsuppressive ther., z.n. splenektomie), sowie bei fortbestehen des fiebers über 1 wo. hinaus. bei diab. mell. vermehrt bz-kontrollen durch den pat. selbst oder den ha wegen gefahr der bz-entgleisung. fieber unbekannter herkunft, das länger als 2 wo. besteht: 40 % infektionen, 25 % tumoren, 20 % immunologische erkrankungen, 5-10 % seltene ursachen. die immunisierung durch impfungen ist eine der wichtigsten und wirksamsten maßnahmen zum schutz vor infektionskrankheiten. mit einer hohen durchimpfungsrate können einzelne krankheitserreger regional oder sogar weltweit ausgerottet werden. immunität gegen erkr. lässt sich passiv durch immunglobulingabe, aktiv durch impfung oder kombination beider methoden erreichen. in deutschland besteht keine impfpflicht. impfempfehlungen werden durch die obersten gesundheitsbehörden der länder ausgesprochen. diese öffentliche empfehlung hat im fall von impfschäden wichtige bedeutung für die weitere versorgung ( § 20 ifsg). informationen zu impfungen hersteller-informationen: in packungsbeilagen, fachinformationen und wissenschaftlichen basis-broschüren. hersteller können auch jederzeit telefonisch kontaktiert werden → service-nummern der arzneimittelhersteller finden sich z.b. in der roten liste. behördliche informationen: das robert-koch-institut unterhält eine ständige impfkommission (stiko), die impfempfehlungen regelmäßig überarbeitet (letzte fassung vom juli 2008) . die empfehlungen sind im anhang der "roten liste" abgedruckt. bestellung der impfempfehlungen der stiko bei: robert-koch-institut, kennwort " stiko-empfehlungen", nordufer 20, 13353 berlin, unter fax-nr. 01888-754-2628 • grav.: alle nicht dringend indizierten impfungen (▶ 9.2.4 ). • bei immunsuppression bzw. immundefekt: es gelten die herstellerangaben, die z.t. sehr restriktiv sind. • allergie gegen bestandteile eines impfstoffs (z.b. hühnereiweiß, ▶ 9.2.4). • nach auftreten von ko bei einer impfung besteht bis zur klärung der ursachen eine ki für denselben impfstoff. • banale infektionen mit subfebriler temperatur. • möglicher kontakt des impflings zu personen mit infektionskrankheiten. • krampfanfälle in der familie; fieberkrämpfe (bei bekannter disposition gabe eines antipyretikums bei der impfung). • chron. erkrankungen, nichtprogrediente zns-erkrankungen. • ekzeme u.a. dermatosen, lokalisierte hautinfektionen. • behandlung mit antibiotika, niedrig dosierten kortikosteroiden (≤ 10 mg hydrocortison-äquivalent) oder lokal angewendeten steroidpräparaten. • bei totimpfstoffen: angeborene oder erworbene immundefekte; neugeborenenikterus; frühgeburtlichkeit (frühgeborene sollten unabhängig vom geburtsgewicht gemäß dem empfohlenen impfalter geimpft werden). • art, risiken und behandlungsmöglichkeiten der zu verhütenden krankheit. • nutzen der impfung. • art des impfstoffs, durchführung der impfung. • nw und risiken des impfstoffs. • info. über ko d. punktion (blutung, verletzung von nerven u. gefäßen, inf. • "öffentliche impfungen": schriftliche aufklärung wird empfohlen; zusätzlich muss möglichkeit zum gespräch gegeben sein. aufklärungsmerkblätter (inkl. fragebogen und einwilligungserklärung) für impfungen im kindesalter sind erhältlich bei: deutsches grünes kreuz, schuhmarkt 4, 35037 marburg. impfanamnese • allgemeiner gesundheitszustand? • immunschwäche, -defekt? • durchgemachte infektionskrankheiten? • frühere impfungen, auftreten von impfreaktionen? • gravidität. • allergien. • wichtig für einen lang dauernden impfschutz: der bei der grundimmunisierung erforderliche mindestzeitraum (s. einzelne impfungen) zwischen vorletzter und letzter impfung darf nicht unterschritten werden. • es gibt keine unzulässig großen abstände zwischen impfungen. jede impfung gilt. auch eine für viele jahre unterbrochene grundimmunisierung muss nicht neu begonnen werden. • ist nicht bekannt, wann zuletzt bzw. ob überhaupt eine impfung stattgefunden hat, ist dies kein grund, eine grundimmunisierung nicht zu beginnen oder impfungen aufzuschieben oder nicht durchzuführen. • serologische kontrollen zur überprüfung des impfschutzes sind i.d.r. nicht angezeigt. es genügt meist 1 auffrischimpfung. spezielles schema beachten bei tetanus (▶ 9.2.3) , tollwut (▶ 9.2.3) , hepatitis b (▶ 9.2.3). • durch zusätzliche impfungen bei bereits bestehendem impfschutz steigt das risiko für ko nur bei bcg-impfung, tetanustoxoid (verstärkte lokalreaktion) und pneumokokken-impfung (systemische, allergische reaktionen). • lebendimpfstoffe: können simultan verabreicht werden; werden sie nicht simultan verabreicht, ist ein mindestabstand von 4 wo. zu empfehlen. voraussetzung: vollständiges abklingen der impfreaktion, keine ko. • totimpfstoffe (inaktivierte krankheitserreger, d.h. deren antigenbestandteile/toxoide): keine mindestabstände zu anderen impfungen erforderlich, auch nicht zu solchen mit lebendimpfstoffen. • bei wiederholungsimpfungen im rahmen der grundimmunisierung sollte für die weiteren impfungen das gleiche präparat verwendet werden. bei schlechter verträglichkeit bzw. unzureichender immunantwort nach abschluss einer impfserie kann eine impfung mit dem präparat eines anderen herstellers u.u. den gewünschten impferfolg haben. • kombinationsimpfstoffe helfen, injektionen und impftermine einzusparen; manchmal sind sie auch preiswerter als die summe der jeweiligen monovakzinen. umgang mit impfstoffen vor allem lebendimpfstoffe sind temperaturlabil und müssen vor erwärmung und gefrieren geschützt werden. alle impfstoffe sind bei 2-8 °c zu lagern, wobei die temperatur permanent mit einem geeichten minimax-thermometer kontrolliert werden muss. impfstoffe, die falsch gelagert oder eingefroren wurden, sind zu verwerfen. angebrochene amp. sofort verbrauchen, v.a. wegen der gefahr der bakteriellen kontamination (cave: spritzenabszess). für die injektion eine neue kanüle verwenden. an der aufziehkanüle anhaftender impfstoff kann zu lokalreaktionen der haut führen. injektionsort (herstellerangaben beachten!) • subkutan: am dors. oberarm oder ventrolat. am oberschenkel. bei antikoagulation auch i.m. impfstoffe (herstellerangaben beachten). • intramuskulär: die meisten totimpfstoffe werden i.m. injiziert. impfstoffmengen bis 1 ml in den m. deltoideus oder m. triceps brachii. injektion in den m. deltoideus ergibt bessere immunität als intraglutäale injektion. so lange der m. deltoideus nicht genügend ausgebildet ist, wird die injektion in den m. vastus lateralis (anterolateraler oberschenkel) empfohlen. hier besteht kaum gefahr, nerven oder gefäße zu verletzen. impfreaktionen häufig auftretende, milde, selbstlimitierende symptome. • lokalreaktionen: rötung, schwellung, schmerzhaftigkeit im bereich der injektionsstelle; klingen innerhalb von 72 h ab. bei anhaltenden beschwerden an eine infektion denken. • an den ersten beiden tagen nach impfung können subfebrile temperaturen auftreten. bei disposition zu fieberkrämpfen: simultane gabe von antipyretika. • mmr-impfung: zwischen 7. und 12. d ist eine leichte masernähnliche symptomatik mit subfebrilen temperaturen möglich. impfkomplikationen bei regelrechter anwendung der amtlich zugelassenen impfstoffe extrem selten. • bei verdacht: immunstatus und ggf. interkurrente infektionen zum zeitpunkt der impfung abklären → serum und ggf. mikrobiologische proben (serum-stuhlproben) asservieren. • meldung an das örtliche gesundheitsamt und die arzneimittelkommission der deutschen ärzteschaft, herbert-levin-platz 1, 10623 berlin, tel. (030) 40 04 56-5 00, fax (030) 40 04 56-5 55. • impfling bzw. eltern/sorgeberechtigte auf gesetzliche regelungen zur versorgung nach impfschäden hinweisen 66) . der antrag ist beim zuständigen versorgungsamt zu stellen. impfdokumentation • immer doppelte dokumentation durchführen (in den impfunterlagen des impflings und in den aufzeichnungen des arztes). • möglichst vorhandene dokumente (impfbücher) weiterführen. falls diese nicht vorliegen, kann eine separate bescheinigung ausgestellt werden. zum späteren nachtrag in impfbücher ist jeder arzt berechtigt. • bei neuausgabe von impfbüchern who-gerechte formulare bevorzugen ("internationale bescheinigungen über impfungen und impfbuch", erhältlich beim deutschen grünen kreuz, s.o.). • dokumentiert werden: tag der impfung; art der impfung; bezeichnung des impfstoffs (handelsname); chargen-nummer (sehr wichtig!); impfender arzt. • kassenleistung sind alle öffentlich empfohlenen impfungen. die einstufung erfolgt durch die stiko und kann sich von bundesland zu bundesland unterscheiden. • kosten für indikationsimpfungen aufgrund beruflicher risiken werden von der gesetzlich benannten stelle (i.d.r. arbeitgeber) übernommen. • sonder-und reiseimpfungen müssen privat abgerechnet werden. um mit möglichst wenig injektionen auszukommen, sollten kombinationsimpfstoffe bevorzugt werden. wichtige kombinationsimpfstoffe sind: varizellen impfstoff nichtspezifisches gammaglobulin (igg). indikation wenn aktive immunisierung nicht möglich ist. pep bei engem kontakt zu erkrankten bzw. konsum ha-virus-kontaminierter nahrungsmittel. immunglobulingabe bis zu 10 d nach exposition sinnvoll. strenge indikationsstellung! dosierung herstellerinformationen beachten. impfmodus grundimmunisierung: bei den neueren impfstoffen (z.b. havrix 1440 ® , vaqta ® ) 2 impfungen in den m. deltoideus. schema 0/6-12 mon. virushepatitiden ▶ 8.7.1. indikation reiseimpfung: längerer aufenthalt (> 4 wo.) während der übertragungszeit (in den tropen während der regenzeit, in gemäßigten zonen v.a. von spätsommer bis frühherbst) in endemiegebieten (süd-und ostasien), bevölkerung in endemiegebieten. impfstoff totimpfstoff; abgetötete viren (korea), z.t. auch chinesische lebendvaccine. impfmodus 3 impfungen von 1,0 ml s.c. an den tagen 0/7/30. kinder < 3 j. mit ½ dosis. auffrischimpfung nach 1 j. bei nachgewiesener und glaubhafter kompletter grundimmunisierung: • letzte impfung vor ≤ 5 j.: keine impfung. • letzte impfung vor 5-10 j.: -bei sauberen geringfügigen wunden keine impfung. -bei allen anderen verletzungen 1 impfung. • letzte impfung vor > 10 j.: 1 impfung. keine, fragliche oder unvollständige grundimmunisierung: • bei geringfügigen, sauberen verletzungen: beginn oder vervollständigung der grundimmunisierung. • bei allen anderen verletzungen: impfung + tetanus-immunglobulin. • bei 2 tetanus-impfungen in der vorgeschichte und wenn die verletzung nicht länger als 24 h zurückliegt, nur impfung und kein tetanus-immunglobulin. simultan und kontralateral zur impfinjektion 250 ie antitoxin, z.b. tollwut ▶ 9.4.8. postexpositionell; präexpositionell öffentlich empfohlen für risikogruppen (z.b. förster, veterinäre). reiseimpfung bei reisen nach afrika, asien und südamerika. impfstoff totimpfstoff. (pep) sofortiger beginn der behandlung! wunddesinfektion, wenn vertretbar, mit 70 % ethanol, sonst pvp-jod, z.b. betaisodona ® ; bei richtiger durchführung reduktion des infektionsrisikos um 90 %. 1 dosis, z.b. rabipur ® , 1 ml i.m. an den tagen: 0-3-7-14-28. am tag 0 zusätzlich homologes tollwut-hyperimmunglobulin, z.b. berirab ® tollwut immunglobulin, exakt 20 ie/kg kg i.m., nicht überdosieren! so viel wie möglich vom immunglobulin um die verletzungen herum instillieren, den rest entfernt vom verletzungsort injizieren. • expositions-kategorie 1: berühren oder füttern von tieren; belecken der intakten haut, berühren eines impfstoffköders bei intakter haut: keine schutzimpfung, vorsichtsmaßnahme! bislang ist noch keine tollwutinf. durch ein solches ereignis ausgelöst worden. • expositions-kategorie 2: knabbern an der intakten haut; oberflächliche kratzer, die nicht zum bluten führten; belecken der nicht intakten haut: aktive schutzimpfung durchführen. • expositions-kategorie 3: jegliche bissverletzung oder kratzwunden, die die haut durchdringen; kontamination von schleimhäuten mit speichel (z.b. lecken, spritzer), kontakt von hautverletzungen oder schleimhäuten mit impfstoffködern: aktive plus passive immunisierung. impfmodus präexpositionell 4 impfungen mit 1 ml impfstoff zum zeitpunkt: 0/1/12 mon. i.m. oder nach schnellschema: 0/7/21 o. 28 d/2-5 j. nebenwirkungen gelegentlich am injektionsort druckgefühl. schutzwirkung präexpositionell sehr sicher, postexpositionell abhängig von der lokalisation der wunde. ungünstige prognose bei bisswunden im gesicht. wiederimpfung nach 3 j. 1 dosis. immunisierung bei bissverletzungen abhängig vom zeitpunkt der letzten impfung: 1-5 j. → 0-3 d; > 5 j. → entsprechend ungeimpften personen. kontraindikationen bei postexpositioneller impfung keine, da erkrankung immer tödlich verläuft. präexpositionelle impfung: allg. ki (▶ 9.2.1). passive immunisierung nach exposition simultanimpfung (pep). indikation reisen (mittelmeer, orient, fernreisen). kinder ab vollendetem 2. lj. nur aktive immunisierung möglich, passive nicht verfügbar. impfstoff lebendimpfstoff = oralimpfstoff, sehr sicherer totimpfstoff thyphim vi ® 1 × s.c., besser 1 × 0,5 ml thyphim vi ® s.c./i.m. impfmodus an tag 1, 3 und 5 je 1 kps., z.b. typhoral l ® , 1 h vor der mahlzeit. nebenwirkungen gelegentlich übelkeit, durchfall, temperaturerhöhung. schutzwirkung 1 wo. nach der 3. impfung; schützt nur gegen salmonella typhi, nicht jedoch gegen salmonella paratyphi oder enteritis-salmonellen. schutzrate: 40-60 %; dauer: 3 j. wiederimpfung nach 2 j. vor oder bei massiver exposition jährlich. kontraindikationen kinder < 3. lebensmon., allg. ki (▶ 9.2.1). besonderheiten antibiotikatherapie und laxanzieneinnahme gefährden den impferfolg. beginn einer malariaprophylaxe frühestens 3 d nach letzter einnahme von typhus-impfstoff. impfstoff gereinigtes vi-kapselpolysaccharid von salm. typhi. sehr gute verträglichkeit. impfmodus 1 dosis, z.b. typhimvi ® 0,5 ml i.m. oder tief s.c. nebenwirkungen gelegentlich verstärkte lokalreaktion. schutzwirkung < 70 %, beginn 3 wo. p.v. wiederimpfung nach 3 j. kontraindikationen keine. wiederimpfung erneute impfung, wenn varizellen-ak eia < 100 iu. bei immunsuppression nach 3 mon. serologische kontrolle. kontraindikationen schwere immundefizienz, laufende chemother. (leukos < 1200/μl), aids; grav. (versehentliche impfung in der grav. kein grund für schwangerschaftsabbruch). indikation prävention bei seroneg. schwangeren; neugeborene bei varizellen-exposition; pat. unter zytostatischer therapie bei varizellen-exposition. dosierung anwendung je nach präparat i.v. oder i.m., z.b. varicellon ® , varitect ® 0,2 ml/kg kg i.m. aufgrund der epidemiologischen situation verschiebt sich die erkrankung immer mehr in das erwachsenenalter. dann jedoch verlaufen die windpocken oft schwer und das risiko der schwangerschaftsinf. steigt. durch impfung bleiben später der stress und die kosten des varizellen-immunglobulins bei varizellen-exposition von seroneg. schwangeren erspart. bei gut verträglichem impfstoff findet deshalb die allg. varizellen-impfung immer mehr befürworter. seroneg. frauen mit kinderwunsch sollte die impfung angeraten werden. indikation immunstimulation bei rezid. harnwegs-bzw. atemwegsinf. sowie vaginalen inf. ein gesicherter nutzen dieser maßnahmen ist nicht erwiesen. impfstoff lyophilisierte extrakte eines spektrums der üblicherweise bei den jeweiligen inf. beteiligten bakterien. nach möglichkeit sollten impfungen außerhalb der grav. erfolgen. wegen einer theoretisch möglichen gefährdung des feten keine parenteralen lebendimpfstoffe anwenden. • unbedenkliche impfungen in der grav.: typhus, tollwut (vitale ind.), polio-salk, tetanus, hep. a, hep. b, diphtherie, influenza. • impfung nur bei sorgfältiger risikoabwägung wegen fehlender erfahrung bei schwangeren: fsme, meningokokken, masern (bei bedarf passive immunisierung), pneumokokken, mumps, japan-enzephalitis. • kontraindiziert in der grav.: varizellen, masern und röteln: bei bedarf passive immunisierung, versehentliche impfung während der grav. keine ind. für schwangerschaftsabbruch. grundsätzlich keine ki für standardimpfungen sind: asthma, mukoviszidose, zöliakie, chron. herz-/lungenerkr., down-syndrom, stabile neurologische erkr., unterernährung, hypotrophe kinder und frühgeborene, postnataler ikterus. impfungen induzieren die hi-virus-replikation um das 3-bis 5fache. die hiv-konz. erreicht 4-6 wo. p.v. wieder die ausgangswerte. bedeutung für die progression bisher unklar. impfungen unter antiretroviralem schutz durchzuführen, ist eine mögliche vorsichtsmaßnahme. • asymptomatische hiv-inf.: keine einschränkung. • aids: nur totimpfstoffe und passive immunisierung. • hiv-pos. kinder: wegen übertragener mütterlicher ak kann erst im alter von 1 j. endgültig festgestellt werden, ob das kind tatsächlich infiziert wurde. empfehlungen der stiko in dieser situation: für alle impfungen gelten die gleichen ind. und ki wie für nichtinfizierte. eine allergische reaktion auf eine impfung kann durch den impfstoff selbst oder durch hilfsstoffe im präparat verursacht sein. um die immunität abzuklären, zuerst eine titerbestimmung der entsprechenden ak durchführen. wenn kein erhöhter ak-titer vorliegt, allergologische abklärung auf hilfsstoffe. hühnereiweiß impfstoffe gegen folgende krankheiten enthalten hühnerprotein (nach abnehmender konz. geordnet): gelbfieber, influenza, masern, mumps, fs-me, tollwut. v.a. hühnereiweißallergie vor impfung allergologisch abklären. einteilung der allergischen reaktion in 3 schweregrade. • grad 1: allg. unverträglichkeit von hühnereiern ohne allergische oder anaphylaktische symptome, neg. hauttest: keine gegenanzeigen für impfungen, auch nicht gegen influenza oder gelbfieber. • grad 2: im hauttest nachgewiesene, jedoch klinisch nicht bedeutsame hühnereiweißallergie: gelbfieber-impfung ist relativ kontraindiziert und darf nur in dringlichen fällen gegeben werden. impfung gegen influenza in der klinik. • grad 3: pat. mit sofortreaktion nach verzehr von hühnereiweiß mit urtikaria, laryngo-/bronchospasmus, rr-abfall. impfung gegen influenza und gelbfieber kontraindiziert. impfung gegen masern, mumps, fsme und tollwut (präexpositionell) in der klinik. neomycin u.a. in folgenden impfstoffen enthalten: masern, mumps, röteln, polio (je nach hersteller unterschiedlich). im hinteren abschnitt der roten liste ® (rosa seiten), befindet sich ein verzeichnis von notfalldepots, in denen folgende seren und plasmaderivate aufbewahrt werden: botulismus-antitoxin vom pferd, hep.-b-immunglobulin, polyvalentes immunglobulin, schlangengift-immunserum polyvalent europa, tetanus-immunglobulin, tollwut-immunglobulin, tollwut-impfstoff, varicella-zoster-immunglobulin. c1-inhibitor, diphtherie-antitoxin vom pferd, obidoxim, prothrombin-konzentrat (ppsb), röteln-immunglobulin. zusätzlich in der roten liste ® : • verzeichnis von informations-und behandlungszentren für vergiftungen für deutschland und europa (▶ tab. 3.3). • impfempfehlungen; impfvorschriften für den internationalen reiseverkehr. • empfehlungen zur malariaprophylaxe. typische erreger therapie erreger borrelia burgdorferi (spirochäte); durch zecken (10-30 % infiziert) auf den menschen übertragen. übertragung durch stechfliegen unwahrscheinlich. krankheitsbilder trachom (serotypen a-c); unspezifische genitalinf., neugeborenen-pneumonie, einschlusskörperchen-konjunktivitis (serotypen d-k); lymphogranuloma venereum (serotypen l1-l13; ▶ 9.8.5). erreger uncharakteristisches sepsisbild bei immunschwäche (▶ 9.9.5, ▶ tab. 9.27), klinikeinweisung bei verdacht. malaria ▶ 9.10.8, pneumocystis carinii ▶ 9.9.5, trichomoniasis ▶ 9.8.3. • maligne lymphome. • expositionsverdacht (z.b. durch nadelstichverletzung). • präventiv bei bordellbesuchern, prostituierten, sextouristen (krankenkasse zahlt nicht); vor/bei neuen sexualpartnern, bei kinderwunsch. • hiv-angst: aids-phobie (nur einmalig). grundsätzlich und ausschließlich persönliche mitteilung des testergebnisses, egal, ob pos. oder neg. grundsätzlich muss der pos. suchtest durch immunoblot-test bestätigt sein. bei definitiv hiv-pos. diagn. stützt sich das weitere vorgehen auf ein ungestörtes vertrauensverhältnis zwischen ha und pat. es dürfen keine unberechtigten hoffnungen geweckt werden, aber es muss das gefühl vermittelt werden, dass der ha dem pat. zur seite steht, wenn symptome "kontrolliert" werden müssen. prophylaxe von "kinderkrankheiten" in der umgebung von hiv-positiven (▶ 9.9.6); impfungen bei hiv-pos. kindern (▶ 9.9.6). zusätzlich zur medizinischen betreuung der kinder darauf hinweisen, dass die eltern durch aids pflegebedürftig werden bzw. versterben. kein erhöhtes infektionsrisiko durch übliche soziale kontakte, daher keine informationspflicht gegenüber dritten. die bescheinigung über die freiheit von infektionskrankheiten nach ifsg kann ausgefüllt werden. kategorie einteilung nach klinischen gesichtspunkten und anzahl der cd4-zellen/μl. bei neugeborenen hiv-pos. mütter wegen diaplazentarer übertragung nachweis von igg-hiv-ak; persistenz dieser leihantikörper bis zum 18. lebensmon. erst nach diesem alter ist ein pos. hiv-ak-test beweisend für eine inf. frühnachweis einer inf. mittels pcr und p24-ag möglich. bei neg. ergebnis wiederholung der pcr nach 6 wo. prognose unbehandelt in 33 % rasche progression zum stadium aids innerhalb von 2 j.; bei den übrigen 66 % langer asymptomatischer verlauf, mittl. überlebenszeit 8 j. verlaufskontrolle anhand von altersbezogenen werten der cd4-zellen. im stadium aids treten die gleichen opportunistischen inf. wie bei erw. auf, neurologische erkr. sind häufiger, kaposi-sarkome sehr selten. betreuung von hiv-pos. kindern durch ein pädiatrisches zentrum mit entsprechender erfahrung. individuell variabel. unbehandelt bei älteren pat. schnellere progression zum stadium aids als bei jüngeren. nach 10 j. bei 50 % der pat. aids, 20 % symptomfrei. nach 15 j. bei 65 % der pat. aids, 8 % symptomfrei. verlangsamung der progression durch eine spezielle ernährung oder lebensweise ist nicht belegt. durch antiretrovirale ther. starker rückgang der inzidenz opportunistischer inf. • detaillierte anamnese, vollständige klinische untersuchung; dabei bes. beachten: inspektion der gesamten haut, des mund-rachen-raums sowie der anal-und genitalregion auf verdächtige effloreszenzen (▶ 9.9.2); generalisierte lk-schwellungen, neurostatus, visus. körpergewicht dokumentieren. • bei asymptomatischen pat. ohne antiretrovirale behandlung im ersten halben j. 3-monatlich, um einen trend zu erkennen und um rechtzeitig die ind. für antiretrovirale ther. (▶ 9.9.6) und prophylaxe opportunistischer inf. zu stellen (▶ 9.9.6). -wenn cd4 < 350 bzw. virusload > 30 000/ml im rahmen des monitorings bei antiretroviraler ther. -resistenzbestimmung vor aufnahme einer behandlung. nach wie vor häufigste aids-definierende erkr. in europa und nordamerika. err. kommt weltweit und ubiquitär vor. durch zunehmendes know-how und prophylaxe (▶ 9.9.6) heute meist frühzeitig diagnostizier-und behandelbar. die hochaktive antiretrovirale therapie (highly active antiretroviral therapy = haart) vermag virusload unter die nachweisgrenze der quantitativen pcr zu bringen und eine remission der immunschwäche mit anstieg der cd4-zellen zur erreichen. der pool an hiv-infizierten zellen wird auch bei lang dauernder ther. nicht eliminiert, da weiterhin eine hiv-replikation auf sehr niedrigem niveau • impfungen und chemoprophylaxe (▶ 9.10.7). mutterpass und impfbefreiungszeugnis(se) mitführen (▶ 9.10.7 und ▶ abb. 9.3). für den notfall kann eine liste wichtiger begriffe bezüglich grav./geburt in englisch oder der jeweiligen sprache des reiselandes nützlich sein. relativ (abhängig von reiseziel, -art, -dauer): • im ersten trimenon und 4 wo. vor der entbindung. • mehrlingsschwangerschaft. • dehydratation bei kinetosen und diarrhö. • absolute und relative ki für (fern-)reisen (▶ 9.10.1). • zusätzlich klinisch manifeste zerebralsklerose und schwere inkontinenz. • grundsätzlich müssen strengere maßstäbe bei der beurteilung der reisefähigkeit angelegt werden als bei jüngeren pat. impfungen und chemoprophylaxe ▶ 9.10.7. reisetransportmittel bei flugreisen sind sgl. und akute otitis media, paukenerguss, tubendysfunktion, akute erkr. der nnh (▶ 23.5.2). cave: barotrauma sinnvoll ist auch, kaugummi mitzuführen: ständiges kauen und schlucken hält die tube offen. bei ohrendruck valsalva-manöver: nase zuhalten und gleichzeitig luft schlucken. sgl. und kleinkindern sollte flüssigkeit zum trinken angeboten werden, um durch schlucken den druckausgleich herzustellen. abgeraten werden. durch jet-lag-sy. (veränderung der zirkadianen hormone) und stress der reise (evtl. flugangst, hektik → erhöhter noradrenalin-spiegel) kommt es zur einer zusätzlichen labilisierung des stoffwechsels (kontrainsulinäre hormone ↑) an reisetagen möglichst alle 3 h andere essgewohnheiten berücksichtigen: z.b. wird in den mittelmeerländern selten vor 21 uhr zu abend gegessen → außerdem an mögliche diätprobleme und mangelnde verfügbarkeit von (diabetiker-)nahrungsmitteln im zielland denken autofahrten nur bei tag und in begleitung, bzw. möglichst alle 2 h pause mit zwischenmahlzeit. auch im auto kohlenhydratvorrat von mind • bei schiffsreisen muss ein antiemetikum griffbereit sein bei zeitverlängerung muss evtl. zusätzlich insulin gespritzt werden, bei zeitverkürzung entsprechend weniger (bz-kontrolle!) faustregel: 1 ⁄12 der üblichen dosis des basalen verzögerungsinsulins plus oder minus pro stunde zeitverschiebung. ab ankunft in der neuen zeitzone: umstellung der uhr und übliches schema wie zu hause. beispiel: • stuttgart -los angeles = zeitverlängerung 9 h: plus 9 ⁄12. • los angeles -stuttgart = zeitverkürzung cave: größte hypoglykämiegefahr während der ersten nächte nach der zeitverschiebung; aufgrund der höheren blutzuckerschwankungen, häufiger bz messen, um hypoglykämien durch zusätzliche mahlzeiten und durch evtl. nachspritzen von normalinsulin gegenzusteuern nadeln in ausreichender menge sowie ärztliche bescheinigung für den zoll mitführen (verwechslung mit drogenabhängigen vermeiden) ersatzbatterien mitnehmen, netzspannung kontrollieren • reiseapotheke: ▶ tab. 9.36. bes. wichtig sind: breitbandantibiotikum, desinfektionsmittel und verbandmaterial sowie antidiarrhoikum und antiemetikum (cave: ketoazidose und dehydratation bei durchfallerkr. und erbrechen). empfehlenswert sind auch instantsuppen und -bouillon als "durchfallther • verletzungen strikt vermeiden, bes. pat. mit pnp: z.b. strandschuhe mitnehmen herzschrittmacherpatienten • vorsorglich die magnetdetektoren an den flughäfen meiden und sich persönlich/manuell untersuchen lassen • ärztliches attest mitführen, das das implantat bestätigt/herzschrittmacherausweis dialysepatienten pat. können über die deutsche dialysegesellschaft niedergelassener ärzte e.v. ein verzeichnis anfordern, das praxen und dialysezentren im in-und ausland enthält, die plätze für gast-und feriendialysen anbieten eindringlich darauf hinweisen, dass nur die strikte einhaltung der verhaltensregeln prophylaktisch wirkt cave: immer darauf achten, dass der verschluss in restaurants erst am tisch geöffnet wird; in anderen situationen immer selbst öffnen. keine eiswürfel in getränken, keine offenen fruchtsäfte • selbstgekauftes obst und gemüse sehr gut waschen bzw • nur gut gekochte oder gebratene speisen verzehren, die frisch und noch warm serviert werden besser ist der verzicht auf rohes gemüse und salate, rohe wurstwaren, unvollständig gekochte/gebratene oder rohe fische und meeresfrüchte; generell speisen meiden, die schon längere zeit vor dem verzehr vorbereitet wurden (z.b. pudding) und/oder durch direkten handkontakt zubereitet wurden (z.b persönliche hygiene • hände vor jedem essen mit seife waschen stuhl und urin vermeiden kleidung • an kopfbedeckung und sonnenbrille denken (cave: sonnenstich, hitzschlag, konjunktivitis) auch am strand nicht barfuß gehen (cave: giftige insekten 10.8): haut bedeckt halten, auch an unterschenkeln und armen; lange hosen oder strümpfe versicherungsschutz eine private reisekrankenversicherung mit rückhol-garantie ist zu empfehlen. für privat versicherte: klären, ob die versicherung diese rückhol-garantie beinhaltet (nicht bei allen privatkassen vertraglich gesichert!). verhalten im reiseland süßwasserseen oder -tümpeln, flüssen und kanälen, auch nicht hände oder füße darin waschen (cave: bilharziose, lambliasis bzw. reisediarrhoe durch kontamination mit fäkalien) gesundheitsspritzen", sonstige spritzen; keine ohrdurchstechungen oder tätowierungen (cave: hiv-, hep (ausreichend) qualitätskondome aus apotheken und drogerien des heimatlandes mitnehmen (cave: hiv-, hep.-b-und -c-inf. u.a. geschlechtskrankheiten) oder sexuelle abstinenz reiseapotheke • art und umfang der reiseapotheke wird bestimmt durch reiseart, reisedauer, zielland, zahl und alter der mitreisenden (kinder? senioren?) sowie deren vorerkr. und/oder spezielle dispositionen für bestimmte erkr. cave: verschreibung ist keine kassenleistung hinweise zur verwendung der einzelnen mittel dem reisenden möglichst schriftlich mitgeben; nicht auf die informationen der beipackzettel verlassen, da diese für laien oft nicht verständlich sind cave: auf impfdokumente oder titer verlassen tropeninstitute, www.gesundes-reisen.de, adressen ▶ 35.1.2) unter berücksichtigung der reiseroute; einige länder verlangen einen impfnachweis schon für eine zwischenlandung ohne eigentliche einreise in das land abhängig von gesundheitsstatus, zielland, reiseroute und reiseart sowie reisetransportmittel cave: ki für impfungen und impfabstände impfbefreiungszeugnis falls obligatorische impfungen bei reisenden kontraindiziert sind, muss ein impfbefreiungszeugnis mit unterschrift des arztes und einem beglaubigungsstempel mitgeführt werden; je nach reiseland in englischer oder französischer sprache und evtl sonst muss eine nutzen-risiko-abwägung durch die schwangere selbst erfolgen. proguanil, z.b. paludrine ® , und chloroquin, z.b. resochin ® , können in schwangerschaft und stillzeit eingesetzt werden. mefloquin, z.b. lariam ® , nicht im 1. trimenon, also erst ab 2. trimenon. außerdem wird bei nicht-schwangeren während und bis zu drei monaten nach lariam-einnahme zur kontrazeption geraten stand-by") die in ▶ tab wobei die medikation prinzipiell verschieden von der prophylaxe sein soll. die behandlung soll jedoch, wenn möglich, immer im krankenhaus erfolgen, insbesondere bei rasanter verschlechterung des krankheitsbildes und bei indikation zur therapie mit chinin medikament dosierung chloroquin, z.b. resochin ® ab 6 mon paludrine ® < 11 mon. 25 mg/d, ab 11 mon. 3 mg/kg kg/d lariam ® ab 3 mon. bzw. 5 kg kg 5 mg/kg kg/wo malarone ® ab 11 kg kg 1 junior tbl. = ges. 62,5/25 mg pro 10 kg kg/d, max doxy v. ct ® ab 8 j. 1,5 mg/kg kg/d auswahl der medikamente in abhängigkeit von der zuvor verwendeten chemo bei malaria tertiana (p. vivax) ist im anschluss an die initialther. mit einem der o.g. chemotherapeutika wegen rezidivgefahr eine behandlung mit primaquin (importmedikament) zur eradikation der extraerythrozytären stadien erforderlich ausfall eine entscheidungshilfe (auch bei hoher parasitämie falsch neg. ergebnisse möglich) und ersetzen nicht die untersuchung von blutausstrichen. schnelltests sollten von medizin • nicht lesen; nach vorn blicken und ein objekt fixieren apfel, kaugummi) soll ebenfalls präventiv wirken • durch zeitverschiebung bei interkontinentalflügen hervorgerufene störung des zirkadianen rhythmus. dadurch beeinträchtigung mentaler und physiologischer funktionen, z.b. schlaf-und wachzustand zeitverschiebung ab 2 h werden mind. 24 h benötigt, um den jet-lag auszugleichen (sog. resynchronisationszeit) 41 einzelne malaria-medikamente mit indikation verschiebung der schlafphase) sind weniger beeinträchtigend als flüge von west nach ost (zeitverkürzung, häufig überspringen der schlafphase) entsprechend der zeitverschiebung kann reisenden ein verändertes einnahmeschema für medikamente mitgegeben werden, z.b. diabetikern ▶ 9 prophylaxe und therapie anpassung des schlaf-wach-rhythmus schon vor der reise; aus praktischen gründen meist jedoch nur für 2-3 h möglich risiken durch natürliche umweltbedingungen sonnen-und uv-lichtexposition trotz zunehmender aufklärung über kurzund langfristige risiken intensiver sonnenlichtexposition (melanome ▶ 26.10.3, basaliome ▶ 26.10.4, vorzeitige alterung der haut) wird das risiko nach wie vor unterschätzt. zu den vermeidbaren kurzfristigen folgen intensiver sonnenstrahlung, die das allgemeinbefinden am urlaubsort beeinträchtigen, gehören: polymorphe lichtdermatose ("sonnenallergie") ätiol.: sonnenstrahlung in verbindung mit duftstoffen, konservierungsmitteln, fett und emulgatoren in kosmetika und sonnenschutzmitteln. klinik: juckende, evtl. nässende pusteln oder quaddeln, vorwiegend an lichtexponierten hautstellen (dekolleté, arme, oberschenkel). prophylaxe: (▶ 26.9.2) allergiegetestete, möglichst fettfreie sonnenschutzmittel. evtl. weitgehend auf direkte sonnenexposition verzichten sonnenbrand prophylaxe durch konsequenten sonnenschutz mit mind uhr) höheren lsf, "sun blocker" bzw. strikte expositionsvermeidung: langsame und hauttypgerechte adaptation: morgens oder nachmittags, nie über die mittagszeit, helle haut nie entblößt in die sonne, ▶ 26.9.1. phototoxizität cave: doxycyclin, johanniskrautpräparate. uv-konjunktivitis immer kopfbedeckung tragen, gilt v.a. für kinder und ältere menschen (cave: glatze) risiken durch sport und freizeitaktivitäten aufenthalt in großen höhen/hochgebirge reizschwelle für die höhenanpassung beim gesunden liegt bei ca. 2500 m ü.m. bei zu schnellem aufstieg über diese höhe hinaus gefahr der akklimatisierungsstörungen (höhenkrankheit/akute bergkrankheit). entscheidend für die reizschwelle ist die tägl. schlafhöhe (übernachtung) müdigkeit am tag und schlafstörungen in der nacht keine zu großen höhenunterschiede tägl. bewältigen, keine gewalttouren; auf ausreichende flüssigkeits-und salzzufuhr achten; größere touren nur mit erfahrenen bergführern, die mit einer höhengerechten aufstiegstaktik vertraut sind klinik: höhenlungenödem mit dyspnoe, rg der atemwege und rapidem leistungsabfall (lebensbedrohlich!). ther.: sofortiger abstieg bzw. abtransport des pat. in tiefere lagen, sauerstoffgabe; medikamentös: nifedipin p.o. 10-20 mg plus 20 mg in retardform sofort der tauchsport verlangt ein hohes maß an körperlicher und mentaler fitness; minimum-check-up: herz-kreislauf-funktion und lungenfunktion. bei älteren reisenden evtl der anamnese ist vor einem tauchurlaub ein fachärztliches attest durch hno-arzt voraussetzung aufklärung über dekompressionskrankheit und luftembolie. -psychische stabilität und mentale fitness: es kann unter wasser bei bestimmter disposition zu (lebensgefährlichen) panikreaktionen oder euphorischen überreaktionen kommen cave: zusammenhang wird von laien oft nicht verstanden und deshalb nicht befolgt! -vor und nach tauchgängen keinen alkohol trinken abhängig von dauer und tiefe der tauchgänge darf für mind. 24-48 h danach nicht geflogen werden (genaue zeit wird nach dem letzten tauchgang berechnet, diese berechnung zu lernen, ist bestandteil einer seriösen tauchausbildung) definition durch im menschlichen venensystem (endwirt) lebende pärchenegel (schistosomen) verursacht. in warmem süßwasser werden von wasserschnecken (= zwischenwirt) die wimpernlarven (mirazidien) aufgenommen und als zerkarien (infektionslarven) freigesetzt. ikz bis 3 mon. inf. perkutan beim baden therapie unterschiedlich je nach subtyp: schistosoma haematobium (afrika, mittl. osten): befällt venengeflecht des kleinen beckens. klinik: blasenbilharziose mit hämorrhagischer zystitis. ko: blasenpapillome befällt leber und darm. klinik: darmbilharziose mit ruhrähnlicher kolitis. ko: perirektale abszesse, polypen eier dringen durch die darmwand ins darmlumen. klinik und diagn. wie schistosoma mansoni prophylaxe in den entsprechenden gebieten nicht in süßwasserseen und -tümpeln baden, waten oder sich waschen entwicklungsländern, vorwiegend in armutsvierteln (slums) für touristen ist das erkrankungsrisiko niedrig, da kein oder selten kontakt mit slumbewohnern oder aufenthalt in slums aedes aegypti), die in der nähe menschlicher behausungen in wasseransammlungen brüten. nach stich und eintritt in die blutbahn vermehrung in regionalen lk injektion der konjunktiven typisch. diffuses, stammbetontes erythem am 2.-3. d, danach morbilliformes exanthem am rumpf mit zentripetaler ausbreitung auf kopf und extremitäten, verbunden mit einem zweiten fieberschub. evtl. petechiale blutungen und epistaxis. abklingen der symptome nach 5-6 d therapie klinikeinweisung zur diagnosestellung, ggf. intensivmedizinische maßnahmen. cave: ass wegen vermehrter blutungsneigung, ausweichmittel paracetamol prophylaxe expositionsprophylaxe; haut durch kleidung und repellentien schützen. schlafen in räumen mit klimaanlage. keine impfung möglich ikz 3-6 d. vorkommen: norden südamerikas und zentralafrika klinik initialstadium (3 d): plötzlicher beginn mit fieber bis 40°c, schüttelfrost, starke kopf-und gliederschmerzen, übelkeit, erbrechen, evtl. relative bradykardie. remissionsstadium: fieberabfall am 3. oder 4. d, evtl. ausheilung. bei schwerem verlauf stadium der organschädigung: hepatitis mit ikterus und erbrechen, nephritis mit proteinurie, hämorrhagische diathese mit profusen blutungen diagnostik tropenanamnese bei ungeimpften (oder zu spät geimpften) personen mit typischer klinik, evtl. igm-ak-nachweis, pcr. therapie klinikeinweisung (verdachtsdiagnose "gelbfieber" angeben) zur diagnosestellung prophylaxe impfung (▶ 9.2.3) für alle reisen in endemiegebiete. cave: viele staaten, in denen gelbfieber nicht vorkommt, verlangen eine impfbescheinigung bei einreise aus infektionsgebieten. aktuelle information bei gelbfieberimpfstel übertragung vorwiegend fäkal-oral, verunreinigte gewässer, meerbuchten, aber auch durch blutprodukte (selten). ikz 2-6 wo. vorkommen: ubiquitär in ländern mit schlechten hygienischen verhältnissen ) meist anikterisch, oft nur gastrointestinale symptome. bei inf. im erwachsenenalter lange und schwere verläufe möglich. cave: bei reisenden über 50 j. ohne immunität häufiger als in jungen jahren fulminante verläufe mit höherer letalität havrix ® ) für alle fernreisenden; aufgrund der guten verträglichkeit und der fast 100 %igen schutzwirkung sollte die aktive schutzimpfung einer passiven impfprophylaxe (globulin i.m.) vorgezogen werden unkomplizierte reisediarrhö betrifft 20-50 % aller fernreisenden erreger 85 % bakt. (meist pathogene e.-coli-stämme), 10 % viral, 5 % protozoen blähungen; plötzlicher beginn, milder bis mittelschwerer verlauf über durchschnittlich 3-4 d orale rehydratationssalze (z.b. elotrans ® -pulver); in den apotheken tropischer länder ist "oral rehydration salts" (ors) verfügbar, dieses im angegebenen volumen abgekochtem wasser auflösen mg initial, dann 2 mg nach jedem wässrigen stuhlgang, max. 16 mg tägl.; kinder 8-13 j. 2 mg initial, dann 2 mg nach jedem wässrigen stuhlgang, max. 8 mg tägl.; bei kindern < 8 j. loperamid-lösung verwenden. -ethacridinlactat 8: ab 14. lj. am 1. und 2. krankheitstag jeweils 3 × 1 drg. nach dem essen, ab dem 3. krankheitstag 1 drg.; in schweren fällen 3 × 1 drg. über mehrere tage keine impfung möglich! komplizierte reisediarrhoe klinik fieber > 3 d und evtl ciprofloxacin: 2 × 500 mg/d für 3 tage • bei fieber oder blutigem stuhl auf motilitätshemmer verzichten • keine besserung bei fieber oder blutigem stuhl nach 2 d → arzt aufsuchen • keine besserung bei wässriger diarrhoe nach 3 d → v.a. lambliasis (häufiger durchfallerreger in warmen ländern) → metronidazol 3 × 500 mg für screening nach reiseende indikationen ein screening des zurückgekehrten reisenden mit exakter reiseanamnese (route, reisemittel, charakter der reise, dauer u.a.) ist nach längerem tropenaufenthalt unter schwierigen bedingungen angezeigt, z.b. nach arbeitsaufenthalten oder abenteuerreisen (auch wenn der rückkehrende symptomlos ist) und immer, wenn eines oder mehrere der folgenden symptome auftritt: • unklares fieber • schmerzen beim wasserlassen/blut im urin, genitale affektionen immer auch an das versagen der malaria-prophylaxe denken. frühzeitig kontakt mit tropenmediziner aufnehmen auf eosinophilie achten: hinweis für helminthiasis. • leberfunktionsparameter je nach ergebnis: überweisung an kollegen mit speziellen kenntnissen oder an eine tropenmedizinische klinik/ambulanz malaria pat. zur blutabnahme in geeignetes labor schicken, wo ausreichende erfahrung in anfertigung und auswertung z.b. eines "dicken tropfens" vorliegt; vorher telefonisch rücksprache nehmen fieber: schistosoma (katayama-sy., filarien, trichinella, toxocara). -git-beschwerden: schistosoma, strongyloides, toxocara. -hautsymptomatik: strongyloides, loa, onchocerca (toxocara). -lungensymptomatik: schistosoma, paragonimus, strongyloides, toxocara • serum-ige-spiegel > 1000 iu/ml alle in §6 aufgeführten erkr. müssen durch den behandelnden arzt an das zuständige gesundheitsamt übermittelt werden. daneben ist nach §7 ifsg der nachweis einer reihe von infektionserregern meldepflichtig 1 hinaus mitzuteilen, wenn personen, die an einer behandlungsbedürftigen lungentuberkulose leiden, eine behandlung verweigern oder abbrechen dem gesundheitsamt ist unverzüglich das gehäufte auftreten nosokomialer infektionen, bei denen ein epidemischer zusammenhang wahrscheinlich ist oder vermutet wird, als ausbruch nichtnamentlich zu melden bei denen ein epidemischer zusammenhang wahrscheinlich ist oder vermutet wird, wenn dies auf eine schwerwiegende gefahr für die allgemeinheit hinweist und krankheitserreger als ursache in betracht kommen, muss dem gesundheitsamt unverzüglich gemeldet werden mrsa-nachweis wird nach § 7 ifsg vom labor namentlich an das zuständige gesundheitsamt gemeldet meldung wann und wohin? die namentliche meldung muss unverzüglich, spätestens innerhalb von 24 h an das für den aktuellen aufenthaltsort des betroffenen zuständige gesundheitsamt (ga), bei meldung durch diagnostische institute an das für den einsender zuständige ga erfolgen virales hämorrhagisches fieber diese erkr. sollen nur in den kompetenz-und behandlungszentren in berlin erkrankung an oder verdacht auf (a = regelung für ausscheider: besuch von gemeinschaftseinrichtungen nach rücksprache mit dem gesundheitsamt möglich): cholera (a) typ b-meningitis; impetigo contagiosa; keuchhusten; ansteckungsfähige lungentuberkulose; lausbefall; masern; meningokokken-inf.; mumps; paratyphus (a) typhus abdominalis; paratyphus; cholera; shigellenruhr bei denen die möglichkeit einer übertragung der krankheitserreger über lebensmittel besteht umfangreiche informationen über die ww der in der hiv-ther. eingesetzten medikamente: www.hiv.net -leitlinien zu diagnostik und ther. der hiv-inf. und zur antiretroviralen ther. im erwachsenenalter: www.awmf-online.de -leitlinien für diagnostik und ther. der hiv-inf. und zur antiretroviralen ther. im erwachsenenalter und bei kindern. stand erreger bartonella henselae, gramneg. bakterium, verursacht bei immunkompetenten die katzenkratzkrankheit. in ⅔ d.f. sind auch hier verletzungen durch katzen der auslöser (▶ 9.3.8) . klinik lokal aggregierte oder disseminiert stehende rötliche, gelegentlich blaulivide oder hautfarbene knoten oder papeln. bis ca. 5 cm groß, mitunter auch exanthematische ausbreitung hunderter kleinster läsionen ohne bevorzugte lokalisation. konsistenz gummiartig, prall-elastisch. effloreszenzen: beginn als winzige, blaurote papeln, die ab einer bestimmten größe ulzerieren können und sich dann mit einer kruste überziehen. abheilung meist als restitutio ad integrum. manchmal bleiben eine hyperpigmentierung oder induration. bei rückbildung größerer knoten können sich atrophische, unter das hautniveau eingesunkene narben bilden. häufig befall von schleimhäuten und inneren organen. allgemeinsymptome: abgeschlagenheit, appetitlosigkeit, erbrechen, diarrhö, nachtschweiß, fieber, schüttelfrost, krampfartige abdom. schmerzen. differenzialdiagnose kaposi-sarkom. diagnostik nachweis von bakterien durch biopsie von angiomatösen neubildungen der haut; histologisch in der he-und warthin-starry-färbung bzw. durch pcr oder serologie. therapie gutes ansprechen auf roxithromycin, azithromycin, doxycyclin und ciprofloxacin. prognose unbehandelt letaler verlauf möglich. übertragung durch sexualkontakte, muttermilch und schleimhautkontakt. meist inapparenter verlauf, reaktivierung der latenten inf. bei immunschwäche. erkrankungsrisiko hoch, wenn cd4 < 100/μl. key: cord-018137-rmtyrbg0 authors: saad, farouk tijjani; sanlidag, tamer; hincal, evren; sayan, murat; baba, isa abdullahi; kaymakamzade, bilgen title: global stability analysis of hiv+ model date: 2018-12-29 journal: 13th international conference on theory and application of fuzzy systems and soft computing &#x02014; icafs-2018 doi: 10.1007/978-3-030-04164-9_109 sha: doc_id: 18137 cord_uid: rmtyrbg0 we developed and studied a mathematical model of hiv+. two equilibriums points were found, disease free and endemic equilibrium, and basic reproduction ratio [formula: see text] was also calculated by the use of next generation matrix. global stability analysis of the equilibria was carried out by the use of lyapunov function, and it was shown that the stability of the equilibria depends on the magnitude of the basic reproduction ratio. when [formula: see text] , the disease free equilibrium is globally asymptotically stable, and disease dies out. on the other hand if [formula: see text] , the endemic equilibrium is globally asymptotically stable and epidemics occurs. reported cases of 13646 hiv-1 positive were obtained in the year 2016 from ministry of health, turkey (moh). this data is used to present the numerical simulations, which supports the analytic result. [formula: see text] was found to be 1.98998, which is bigger than 1, this shows the threat posed by hiv in turkey. historically, infectious diseases posed a real threat to the human population. although they have been in human population all the time to some extent, the effects of epidemics are the most obvious and noticeable. only in the 14th century europe, around 25 million out of about 100 million individuals died from the black death [1] . several diseases were discovered in america which included smallpox, measles, influenza, and typhus, whooping cough, the mumps, and diphtheria. infectious disease was the main reason for the demise of the indians [2] . in the early 1980's, some homosexual men in the united states were diagnosed with a type of fungal infection and a tumor called candidiasis and kaposi's sarcoma respectively. paris pasteur institute in 1984 detected a virus responsible for those diseases and called it the human immunodeficiency virus (hiv) [3] . hiv is the virus that causes acquired immunodeficiency syndrome (aids). the virus is responsible for attacking and destroying the immune systems mainly the cd4+ t-lymphocytes or t-cells [1] . on a normal basis, these cd4+ t-lymphocytes or t-cells detect foreign and infected cells, and attack, spread and kill them [1] [2] [3] . hiv is able to infect cd4+ t-lymphocytes and insert its genome to host genome. this integrated hiv genome may exist in 2 states. they can be either transcriptionally active generating new viruses that can infect other cd4+ t-lymphocytes or in latent state which may become activated later. in transcriptionally active stage, the infected cd4+ t-lymphocytes die due to cytopathic effect of the virus. as a result, the number of cd4+ t-lymphocytes, which are able to recognize foreign and infected cells, declines, and this decrement lead stoper manent and lasting damage to the immune system [3] . the immune system finally loses its ability to fight and kill infections due to the number of cd4+ t-lymphocytes count which is so small. when an individual reaches this stage, the person is said to have aids [4] . the time between getting infected with hiv and advancing to aids is, in general, five years but changes due to many factors. if the cd4+ t-lymphocytes cells count falls below 200 cells/mm 3 , then the person is considered to be in the "aids phase", otherwise the person is said to be hiv infected [5] . the mode of transmission of hiv includes heterosexual intercourse, homosexual/bisexual intercourse, intravenous drug use, vertical transmission, and unknown reasons [6] . since its discovery (hiv/aids), the extensive increase and epidemic continues around the globe. the greatest number in any one year was in 2003, where almost five million individuals became newly infected, with a total of 38 million hiv/aids patients, and almost three million people died from aids in the same year [7] . to continue its record of one of the most destructive epidemics in history, it killed at least 25 million people by 2005. efforts to improve the use of antiretroviral treatment in some part of the world were still not enough to reduce a significant number of deaths, the hiv/aids epidemic claimed 3.1 million lives in 2005, of which about 570000 were children (unaids/who [8] ). the region that suffered most is africa, with sub-saharan africa as the home of the epidemic. in 2010, 2.7 million people were newly infected, and 1.8 million patients died of aids-related causes across the globe. by the end of 2010, around 34 million individuals were victims of hiv/aids [9] . china stated that about 2.8 million died of aids-related causes in 2011, and there were about 7.8 million hiv-infected individuals by the end of 2011. in the year 1985, two patients were diagnosed with aids in turkey, since then, the importance of aids started and still continue to be on the forefront. the number of new cases increases every year, with 34, 91, and 119 cases in 1990, 1995, and 1999 respectively [10] . in 2004, the ministry of health (moh) published the total number of hiv patients 1802, of which 76% are between the ages of 15 to 49 years and sexually active. among these patients, about 800 were aids patients. this is an official data from moh, however, these numbers does not reflect the actual figures of hiv infected individual in turkey, due to insufficiency of the registration system, and the lack awareness and phobia of the patients to attend health centers or hospitals [11] . at the end of 2011, the ministry stated that the total number of hiv/aids infected people was 5224. it also published in 2013 that at least 6000 individuals were infected with hiv, and the numbers of newly reported cases in 2010, 2011, and first six months of 2013 were 589, 1068, and 587 respectively. according to a report, poor knowledge of sexually transmitted diseases, poor socio-economic conditions, increase in number of unregistered sex workers, increase in number of homosexuals, and intravenous drug use contributed to the spread of hiv infection in turkey. it was reported that, the main way of transmitting hiv in turkey is via heterosexual sex (53%), then men having sex with men (msm at 9%), and intravenous drug users (idu at 3%) among the recorded cases. according to positive living association istanbul, personal communication, hiv/aids will become a major public health issue in turkey in the coming years, as such; it must be regarded as a rising disease for turkey [12] . the main ways in which hiv/aids is transmitted between individuals are now well understood, but the factors that contribute to the disparities in its prevalence and trends among populations remain an area of interest to scientific researchers. to understand these disparities, it is essential to understand the system, its components and its dynamics. mathematical models of hiv/aids transmission dynamics are important research tool in this category [13, 14] . primary purpose of any mathematical model of hiv transmission lies in using individual level inputs to project population level outcomes. some of the important outcomes that can be examined with a model are; the incidence of infection, the prevalence of infection, or the doubling time of the epidemic. more important than these however, is simply the likelihood of an epidemic to occur that is whether there is sufficient transmission potential for a chain of infection to be sustained. this outcome is termed by a simple summary statistic: the reproduction number of the infectious process, r 0 . in a susceptible population, r 0 represents the expected number of secondary infections generated by the first infected individual. if r 0 is equal or greater than 1 an epidemic is expected. if r 0 is less than 1, the infection is expected to die out [15] . the magnitude of r 0 is used to measure the risk of an epidemic or pandemic in any emerging infectious disease. it was used for understanding the outbreak and danger of sars. r 0 was also used to characterize bovine spongiform encephalitis (bse), foot and mouth disease (fmd), strains of influenza, and west nile virus [16] [17] [18] [19] . the incidence and spread of dengue, ebola, and scrapie have also been assessed by r 0 [20] [21] [22] . tropical issues such as the risks of indoor airborne infection, bioterrorism, and computer viruses also depend on this important parameter [23] [24] [25] [26] . in this paper, we shall first introduce the model involving systems of ode, and then discuss the biological meaning of the parameters involved. we shall study the global stabilities of both disease free and endemic equilibria by the use of lyapunov function. by the use of real data obtained from turkey in 2016, we will conduct numerical simulations to support the analytic result. the organization of the paper is as follows: in sect. 2, the model is presented and the basic reproduction number is obtained. in sect. 3, stability of the equilibria are investigated. in sect. 4, results are obtained by numerical simulations of the real data obtained from turkey in 2016. finally sect. 5 is the discussion and conclusion of the research. the system of ordinary differential equations derived is for the whole turkish population. consider the birth k(t) to the susceptible population per unit time. susceptibles individuals are removed through infection or through natural death. let µ be the natural death rate for the whole population. the removal rate of susceptible individuals through infection is the number of new hiv infections per unit time. we use this rate in calculating hiv incidence which by definition is the number of new infected persons in a specified period of time divided by the number of uninfected persons that were exposed for this same time. let each susceptible have c contacts per unit time. assume that a proportion h/n of these contacts are with infectives and at each of these contacts with infectives, a susceptible has a probability b of becoming infected. let a the incidence rate, then the total probability of one susceptible getting hiv infected from any of their contacts per unit time is ah/n. this is the expression for the force of infection. the force of infection is the probability that a susceptible will get an hiv infection per unit time. therefore in a population of s susceptibles, the number of new hiv infections per unit time is given by ahs/n. infectives are recruited through new hiv infections described above and removed through death at rate v and through natural death at rate µ. hence, 1/v is the duration spent in the infective stage and 1/µ is the life expectancy of the population. all these rates are assumed constant in the model. removed cases are recruited either through natural death l or through deaths due to hiv at the rate v. with these assumptions, we arrive at the following system of ode. (1) can be reduced to it follows from (1) that, thus the feasible region for (1) is for the simulation we make further assumptions as follows; (i) s(0) is considered to be the whole population in 2016 (ii) we considered homogeneous mixing in the population (iii) v ¼ 0:01493 is considered (that is averagely 67 years is the life span of hiv+ people) equating the equations in (*) to zero and solving simultaneously we find two equilibrium points. disease free and endemic equilibrium points. this is the number of secondary infections caused by a single infective individual in a completely susceptible population. it is denoted by r 0 . using the next generation of matrix (ngm) method we have, the spectral radius, which is the dominant eigenvalue, is as 0 v þ l hence the basic reproduction ration is; in this section stability analysis of the two equilibrium points is obtained by the use of lyapunov function. the conditions for the global stability of the equilibria in each case depends on the magnitude of the basic reproduction ratio r 0 . hence we have the following theorems and their proofs. theorem 1: the disease free equilibrium is globally asymptotically stable when r 0 1. we construct the following lyapunov function by the relation between geometric and arithmetic means and if as 0 v þ l ð þ. this implies _ v 0 if r 0 1. the endemic equilibrium e 1 is globally asymptotically stable when r 0 [ 1. proof: we construct the following lyapunov function by the relation between arithmetic and geometric mean and if in this section, results are calculated by simulating the model using the real data obtained from turkey in 2016. here we use the real data obtained from moh, in which there were a total of 13646 hiv-1 positive reported cases in the year 2016, in the year 2016 to study and predict the dynamics of hiv in turkey using our model. table 1 presents the values of the parameters as calculated based on the data obtained. since r 0 [ 1, the disease free equilibrium is not stable and the endemic equilibrium is stable. hence, there is going to be epidemics. simulating the above result, fig. 1 shows the epidemic of hiv/aids in turkey. a mathematical model for hiv+ is constructed and analyzed. two equilibrium points (disease free and endemic) are found and stability of each of the equilibrium point was shown to depend on the magnitude of basic reproduction ratio, using lyapunov function. it was shown that if r 0 ¼ as 0 v þ l is less than one, the disease free equilibrium is globally asymptotically stable. also, if the value is greater than or equals to one the endemic equilibrium is globally asymptotically stable. the turkish population in the year 2016 is 79814871, and the hiv positive population is 13646. the value of the basic reproduction ratio is 1.98998, which is bigger than one. this implies one hiv positive individual in turkey can be able to transfer the disease to almost 2 individuals, hence there is going to be hiv epidemic in turkey. numerical simulations were carried out and the results support the analytic findings. the results in the simulation shows that if appropriate measures are not taken, in 50 years to come, there will be more hiv positive individuals in turkey as there are susceptible individuals. the main limitation to our analysis may be that we did not account for the effect of behavioral change arising both from number of hiv cases in the community as well as awareness by governmental and nongovernmental organizations. secondary, the possible effects of extensive use of antiretroviral drugs (arvs) in terms of method of distributing drugs through public or private health institution or a combination of both could determine whether patients on arvs revert back to the infective class. this together with reduced infectiousness due to lower viral loads for those on treatment was not accounted for. despite the limitations mentioned above, there are several implications of our findings to public health. first, the endemic equilibrium should be brought as low as possible especially during the first wave of the epidemic. this model suggests that this can be achieved by prolonging the lifetime of the hiv patients for as long as possible. second, hiv prevalence at low prevalence levels become less sensitive to changes in the dynamics of hiv epidemic because it is overpowered by demographic changes especially the recruitment of susceptibles. at low prevalence levels, there is hence need to track trends in number of persons infected with hiv than tracking hiv prevalence. a preliminary study of the transmission dynamics of the human immunodeficiency virus (hiv), the causative agent of aids stability analysis of an hiv/aids epidemic model with treatment mathematical epidemiology of infectious diseases: model building, analysis and interpretation stability analysis of an hiv/aids epidemic model with screening stability analysis of an hiv/aids dynamics model with drug resistance analysis of the treatment costs of hiv/aids in turkey global analysis of an hiv/aids epidemic model dynamics of hiv/aids in turkey from 1985 to 2016 aids knowledge and attitudes in a turkish population. an epidemiology study status hiv/aids epidemic in turkey molecular epidemiology of hiv in a cohort of men having sex with men from istanbul mathematical models for hiv transmission dynamics: tools for social and behavioral science research hiv dynamics and behaviour change as determinants of the impact of sexually transmitted disease treatment on hiv transmission in the context of the rakai trial can population differences explain the contrasting results of the mwanza, rakai, and masaka hiv/sexually transmitted disease intervention trials: a modeling study a simple approximate mathematical model to predict the number of severe acute respiratory syndrome cases and deaths understanding the epidemiology of bse the foot -and -mouth epidemic in great britain: pattern of spread and impact of interventions transmissibility of 1918 pandemic influenza an epidemiological model for west nile virus: invasion analysis and control applications uncertainties regarding dengue modeling in rio de janeiro malaria and its possible control on the island of principe the basic ratio number of ebola and the effects of public health measures: the cases of congo and uganda a scrapie epidemic in cyprus risk of indoor airborne infection transmission estimated from carbon dioxide concentration emergence response to small pox attack: the case for mass vaccination role of awareness in controlling hiv/aids: a mathematical model key: cord-002746-qn34eyul authors: antzin-anduetza, irati; mahiet, charlotte; granger, luke a.; odendall, charlotte; swanson, chad m. title: increasing the cpg dinucleotide abundance in the hiv-1 genomic rna inhibits viral replication date: 2017-11-09 journal: retrovirology doi: 10.1186/s12977-017-0374-1 sha: doc_id: 2746 cord_uid: qn34eyul background: the human immunodeficiency virus type 1 (hiv-1) structural protein gag is necessary and sufficient to form viral particles. in addition to encoding the amino acid sequence for gag, the underlying rna sequence could encode cis-acting elements or nucleotide biases that are necessary for viral replication. furthermore, rna sequences that inhibit viral replication could be suppressed in gag. however, the functional relevance of rna elements and nucleotide biases that promote or repress hiv-1 replication remain poorly understood. results: to characterize if the rna sequence in gag controls hiv-1 replication, the matrix (ma) region was codon modified, allowing the rna sequence to be altered without affecting the protein sequence. codon modification of nucleotides (nt) 22-261 or 22-378 in gag inhibited viral replication by decreasing genomic rna (grna) abundance, grna stability, gag expression, virion production and infectivity. comparing the effect of these point mutations to deletions of the same region revealed that the mutations inhibited infectious virus production while the deletions did not. this demonstrated that codon modification introduced inhibitory sequences. there is a much lower than expected frequency of cpg dinucleotides in hiv-1 and codon modification introduced a substantial increase in cpg abundance. to determine if they are necessary for inhibition of hiv-1 replication, codons introducing cpg dinucleotides were mutated back to the wild type codon, which restored efficient gag expression and infectious virion production. to determine if they are sufficient to inhibit viral replication, cpg dinucleotides were inserted into gag in the absence of other changes. the increased cpg dinucleotide content decreased hiv-1 infectivity and viral replication. conclusions: the hiv-1 rna sequence contains low abundance of cpg dinucleotides. increasing the abundance of cpg dinucleotides inhibits multiple steps of the viral life cycle, providing a functional explanation for why cpg dinucleotides are suppressed in hiv-1. electronic supplementary material: the online version of this article (10.1186/s12977-017-0374-1) contains supplementary material, which is available to authorized users. the hiv-1 genomic rna (grna) has three major functions in the viral life cycle [1] . first, it serves as the pre-mrna that is spliced into over 70 different transcripts [2] [3] [4] . second, it acts as the mrna for the gag and gag-pol polyproteins that comprise the structural and enzymatic proteins, respectively [5, 6] . third, it is the genome that is packaged into virions and is reverse-transcribed upon infection of a new target cell [7, 8] . the grna can be divided into three regions: a 336 nt 5′ untranslated region (utr), a 219 nt 3′ utr, and an 8618 nt region that is densely packed with multiple open reading frames (nt lengths reference the hiv-1 nl4-3 strain [9] ). the 5′ utr contains several cis-acting elements in complex stem-loop structures that regulate multiple stages of the viral life cycle including transcription, splicing, grna dimerization, encapsidation and reverse transcription [8, 10] . the central 8618 nt region encodes nine open reading frames: gag, pol, vif, vpr, tat, rev, vpu, env and nef. in addition to encoding the amino acids of the viral proteins, the rna sequence underlying the open reading frames could regulate multiple steps of the hiv-1 life cycle including splicing, rna stability, rna nuclear export, translation and reverse transcription. indeed, a large number of cis-acting rna elements within the protein coding regions have been reported to regulate hiv-1 replication, some of which are highly conserved and under purifying selection [11, 12] . these include the programmed ribosomal frameshift sequence in gag for pol translation [13] , splicing signals in pol, vif, vpr, tat, rev and env [2, 3] , the rev-response element (rre) in env [14] and the polypurine tracts in pol and nef that are necessary for reverse transcription [15] . there is also extensive secondary and tertiary rna structure throughout the grna that could regulate viral replication [16] [17] [18] [19] . determining the full complement of cis-acting elements in the grna that regulate viral replication is necessary for a complete understanding of the hiv-1 replication cycle and may aid in the development of novel antiviral therapies [20] . furthermore, identifying and characterizing evolutionarily conserved cis-acting elements and structures is essential for understanding hiv-1 purifying and positive selection as well as recombination events [11, 12, [21] [22] [23] [24] . gag consists of four protein domains and two spacer peptides that control virion assembly [25] . matrix (ma/p17) mediates gag trafficking to the plasma membrane, capsid (ca/p24) forms the structure of the virion core, nucleocapsid (nc/p7) binds the genomic rna to mediate encapsidation, and p6 recruits the escrt complexes necessary for membrane fission during budding. within the ma open reading frame, there are a large number of proposed cis-acting rna elements that could be necessary for viral replication. these include a hnrnpa1 binding site that may regulate grna nuclear export [26] , an intronic splice enhancer [27, 28] , an internal ribosome entry site [29] , instability sequences that lead to rna degradation in the absence of rev [30] , sequences that base pair with the 5′ and 3′ utrs [31] [32] [33] [34] [35] , and elements that regulate encapsidation [7, 8] . however, the functional relevance of most of these elements for viral replication is unclear. some nucleotide patterns may also regulate hiv-1 replication and be under evolutionary selection. for example, the base composition of hiv-1 deviates from that of the human genome. hiv-1 rna has a high percentage of adenine (a, 36%) and low percentage of cytosine (c, 18%) [36] [37] [38] [39] [40] [41] [42] [43] . this nucleotide bias is found in groups m, n and o and is a general property of lentiviruses, though not all retroviruses [36, 38, 42, 44] . even though hiv-1 has a very high nucleotide substitution rate and sequence diversity, the a-rich bias has been conserved during the hiv-1 pandemic [42] . there are two hypotheses for why this has been maintained in the virus. first, the mutational pattern of reverse transcriptase or antiviral apobec3 proteins could impose an a-rich nucleotide bias [45] [46] [47] [48] [49] [50] [51] [52] . second, this bias could be required for viral replication and be under purifying selection [38, 53, 54] . in addition to understanding the rna elements that are necessary for viral replication, it is important to characterize the motifs that are underrepresented and may be deleterious. hiv-1 has a much lower than expected frequency of the dinucleotide cpg [36, 40, 44, [55] [56] [57] . this has been proposed to be under negative selection and the cpg dinucleotide abundance in hiv-1 may be linked to disease progression [58] . however, the mechanism by which cpg dinucleotides affect viral replication is unknown. these nucleotide biases cause the hiv-1 open reading frames to have a codon usage pattern that differs substantially from that of human mrnas [36] [37] [38] [39] [40] [41] 43] . the genetic code is redundant in that there are 61 codons for 20 amino acids and all of the amino acids except methionine and tryptophan are encoded by at least two codons. the preferred codons in cellular mrnas are thought to correlate with the availability of the aminoacyl-trnas but hiv-1 contains many rare codons [36] [37] [38] [39] [40] [41] . in this study, we investigated whether rna elements in the ma region of gag positively or negatively regulate hiv-1 replication. we initially focused on this region because of its high content of potential rna regulatory elements (discussed above). to change the rna sequence without altering the amino acid sequence, we codon modified this region by introducing large numbers of synonymous mutations. these mutations strongly inhibited viral replication by decreasing grna abundance, grna stability, gag expression, virion production and infectivity. we found that cpg dinucleotides introduced during codon modification were necessary and sufficient to attenuate hiv-1 replication. this highlights the functional importance of the suppressed cpg abundance in hiv-1 [36, 40, 44, [55] [56] [57] and shows that increasing the number of cpg dinucleotides in the grna inhibits multiple steps of the viral life cycle. jurkat cells were cultured in rpmi 1640 glutamax medium (gibco) supplemented with 10% fetal bovine serum (fbs) and 1% penicillin-streptomycin. hela, tzm-bl and 293t cells were cultured in dulbecco's modified eagle medium (gibco) supplemented with 10% fbs and 1% penicillin-streptomycin. all cell lines were grown at 37 °c in a humidified atmosphere with 5% co 2 . the phiv-1 nl4-3 constructs used in this study contain the provirus sequence from phiv-1 nl4-3 [9] cloned into the kpni and sali sites of pgl4.10 (promega). phiv-1 cm22-261, phiv-1 cm22-165, phiv-1 cm166-261 and phiv-1 cm22-378 have the designated sequences from phdmhgpm2 [59] chemically synthesized by life technologies and cloned into phiv-1 nl4-3 . for phiv-1cm 22-261 lowcpg and phiv-1 cm22-378 lowcpg , phiv-1 cpg22-261 and phiv-1 cpg22-378, the sequences shown in fig. 6 were synthesized by life technologies and cloned into phiv-1 nl4-3 . phiv-1 ∆22-261 and phiv-1 ∆22-378 have the designated region in gag replaced with a xbai site as in reil et al. [60] . the modified sequences in these plasmids were verified by dna sequencing (eurofins). pgfp and pvsv-g have been previously described [61, 62] . 4 × 10 6 293t cells were seeded in 10 cm plates and transfected with 10 μg of phiv-1 and 1.25 μg of pgfp using poly(ethlyleneimine) solution (pei) at a ratio of 5 μl pei per 1 μg dna. approximately 48-h post-transfection, the media was harvested, filtered through a 0.45 μm filter and quantified using a p24 gag enzyme-linked immunosorbent assay (elisa) (perkin-elmer). a total of 2.5 × 10 5 jurkat cells were plated in 1 ml of medium per well in 48 well plates and infected with 25 ng of p24 gag of each virus. supt1 cells were infected with 10 ng of p24 gag for each virus. supernatants were first collected when syncytia were first observed in the culture infected with hiv-1 nl4-3 . the amount of infectious virus present at each time point was quantified by infecting the tzmbl indicator cell line [63] [64] [65] . infectivity was measured by the induction of β-galactosidase using the galacto-star ™ system (applied biosystems). six-well plates of hela cells were transfected using transit ® -lt1 (mirus) according to the manufacturer's instructions at the ratio of 3 μl transit ® -lt1 to 1 μg dna. for each transfection, 0.5 μg phiv-1 and 0.5 μg pgfp or pvsv-g was used. media was recovered approximately 48 h post-transfection and filtered through a 20% sucrose cushion for 2 h at 20,000×g. the amount of infectious virus was quantified by using the tzm-bl indicator cell line [63] [64] [65] . approximately 48-h post-transfection, hela cells were lysed in radioimmunoprecipitation assay (ripa) buffer (10 mm tris-hcl, ph 7.5, 150 mm nacl, 1 mm edta, 0.1% sds, 1% triton x-100, 1% sodium deoxycholate). the media was clarified using a 0.45 μm filter. virions were pelleted through a 20% sucrose cushion in phosphate-buffered saline (pbs) solution for 2 h at 20,000×g. the pellet was resuspended in 2× loading buffer (60 mm tris-hcl (ph 6.8), 10% β-mercaptoethanol, 10% glycerol, 2% sodium dodecyl sulfate (sds), 0.1% bromophenol blue). cell lysates and virions were resolved by sds-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. the primary antibodies used were specific to hiv-1 p24 gag [66] , hsp90 (sc7947: santa cruz biotechnology), phosphostat1 (612132: bd transduction), ifit1 (gtx118713-s: insight biotechnology) or β-actin (ac-15: sigma). dylight ™ 800-conjugated secondary antibodies (5151s and 5257s: cell signaling) were used to detect the bound primary antibodies with the li-cor infrared imaging (li-cor uk ltd). hela cells were washed with 1xpbs and the rna was extracted using the rneasy kit (qiagen) following the manufacturer's instructions. 1 μg of rna was reverse transcribed to cdna using the high capacity cdna archive kit (applied biosystems). rna from virions was isolated using qiaamp viral rna mini kit following the manufacturer's instructions. because carrier rna is added to the lysis buffer, the total rna isolated was quantified using a qubit 3.0 fluorometer (ther-mofisher) and normalized so that 20 ng of rna from each sample was reverse transcribed using the high capacity cdna archive kit (applied biosystems). pcr reactions were performed in triplicate with taqman universal pcr mix using the applied biosystems 7500 real-time pcr system. hiv-1 nl4-3 grna primers were ggccagggaattttcttcaga/ttgtctcttcc-ccaaacctga (forward/reverse) and the probe was fam-accagagccaacagccccaccaga-tamra. hiv-1 nl4-3 total rna primers were taactagg-gaacccactgc/gctagagattttccacactg (forward/reverse) and the probe was fam-acacaa-cagacgggcacacacta-tamra. to analyze grna stability, 1 µg/ml actinomycin d (sigma aldrich) was added to hela cells ~ 45 h post-transfection. rna was isolated at the designated timepoints and grna abundance was measured. hela cells were stimulated with synthetic tlr ligands for 5 h at the concentrations indicated. ligands supplied by invivogen were polyic: polyic (tlrl-pic), gardiquimod (tlrl-gdqs), cl075 (tlrl-c75), r848 (tlrl-r848), pam3csk4 (p3c. tlrl-pms), ultrapure flagellin (flic-tlrl-epstfla-5). lps was supplied by enzo (alx-581-012-l002). cpg dna was synthesised by idt and 23s ribosomal rna by sigma. sendai virus (sev) was obtained from charles river labs. ifn-β was purchased from peprotech and was added to the culture for 1 h to activate ifn signaling. the "analyze base composition" tool in macvector was used to calculate the mono-and di-nucleotide frequencies for the hiv-1 nl4-3 grna (ncbi accession number m19921). the dinucleotide frequencies are calculated using the following formula: number of dinucleotide occurrences/(frequency of base 1 in pair × frequency of base 2 in pair) where frequency of base is number of occurrences of base/total number of bases in sequence. weblogo [67] was used to generate conserved nucleotides surrounding the cpg dinucleotides. to analyze the functional relevance of rna elements and nucleotide bias underlying the ma domain in gag, we introduced 80 synonymous mutations into nt 22-261 of hiv-1 nl4-3 gag (fig. 1a ). this codon modified (cm) provirus, hiv-1 cm22-261, has 69/80 codons in this region altered without affecting the amino acid sequence. the mutations were derived from phdmhgpm2, a codon optimized gag-pol construct in which many of the hiv-1 codons are replaced with codons used in highly expressed human mrnas [59, 68] . in addition, nt 22-165 or 166-261 in gag were codon modified to produce hiv-1 cm22-165 and hiv-1 cm166-216, which have 49 and 31 synonymous mutations, respectively. virus stocks were prepared by transfecting 293t cells with each proviral dna construct and the concentration of viral ca/p24 gag for each stock was measured by elisa. hiv-1 cm22-261 and hiv-1 cm22-165 had an ~ 65% and ~ 40% decrease in p24 gag concentration, respectively (fig. 1b) . to analyze the fitness of each virus, the viral inoculum was normalized so that jurkat cd4 t cells were challenged with 25 ng of p24 gag for each virus (fig. 1c) . the amount of infectious virus in the culture supernatant was monitored over 2 weeks using tzm-bl indicator cells [63] [64] [65] . hiv-1 cm22-261 replicated at a very low but detectable level and at day 12 had > 99.9% less infectivity than wild type virus. hiv-1 cm22-165 replicated slightly better than hiv-1 cm22-261, but was still > 99% lower than wild type hiv-1 at day 12. hiv-1 cm166-261 plateaued at the same level as wild type hiv-1 but with a delay of ~ 3 days. similar results were observed when supt1 cd4 t cells were challenged with the wild type and mutated viruses (additional file 1). we then used a single cycle infectivity assay to determine if hela cells were non-permissive for hiv-1 cm22-261, hiv cm22-165 or hiv-1 cm166-261 replication as well as to characterize which steps in the viral life cycle are inhibited by the synonymous changes in gag. hela cells were transfected with phiv-1 nl4-3 , phiv-1 cm22-261, phiv-1 cm22-165 or phiv-1 cm166-261 and the media and cell lysates were harvested ~ 48 h later. hiv-1 infectivity in the media was determined using tzm-bl cells and the abundance of virions in the media and gag in the cell lysate was analyzed by quantitative immunoblotting. compared with the wild type virus, hiv-1 cm22-261 infectivity was decreased to the limit of detection of the assay (fig. 2a) , indicating that the virus is attenuated in hela cells. virion production and intracellular gag expression were decreased ~ 90% (fig. 2b, c) . for hiv-1 cm22-165, the amount of infectious virus in the media was decreased ~ 98% with a < 50% decrease in gag expression and viron production. hiv-1 cm166-261 consistently yielded similar amounts of infectivity, virions and intracellular gag expression as wild type hiv-1. overall, there is a substantial reduction in infectivity for hiv-1 cm22-261 and hiv-1 cm22-165. hiv-1 cm22-261 also has a substantial defect in gag expression and virion production. to determine if the decrease in infectious virus production was due to a decrease in grna abundance, we performed quantitative rt-pcr (qrt-pcr) using a primer-probe set in a region of gag that was not mutated (fig. 3a, b) . phiv-1 nl4-3 , phiv-1 cm22-261, phiv-1 cm22-165 and phiv-1 cm22166-261 were transfected into hela cells. rna was isolated from the cell lysate and media ~ 48 h post-transfection. hiv-1 cm22-261 grna was reduced > 90% in the cell lysate and media compared with the wild type virus. hiv-1 cm22-165 grna was decreased ~ 70% in the cell and ~ 65% in the media. hiv-1 cm166-261 grna abundance was equivalent to wild type hiv-1 in the cell lysate and media. we then determined the effect of the synonymous mutations on infectivity/viral genome by infecting tzm-bl cells with an equivalent amount of viral genomes for each virus. when the input number of genomes was normalized based on the results in fig. 3b , hiv-1 cm22-261 infectivity was at the limit of detection of the assay and hiv-1 cm22-165 infectivity was decreased ~ 98% (fig. 3c ). this indicates that the decreased abundance of viral genomes in the media is not fully responsible for the loss of infectivity for hiv-1 cm22-261 and hiv-1 cm22-165. the hiv-1 grna can be spliced into over 70 different transcripts [4] and the gag-pol intron can be spliced out through one 5′ splice site and six 3′ splice sites [2] . a potential explanation for the decrease in intracellular grna abundance is that the mutations in gag could affect intronic splicing silencer (iss) sequences. if this occurred, grna abundance would decrease due to oversplicing but the total amount of hiv-1 rna would stay the same. to test this, we determined the total intracellular hiv-1 rna abundance using a primerprobe set upstream of the major 5′ splice donor (sd1). hiv-1 cm22-261 and hiv-1 cm22-165 had an ~ 80 and ~ 60% decrease in total hiv-1 rna abundance (additional file 2). since ~ 50% of the grna remains unspliced [2] , this is consistent with a specific reduction in the grna and does not appear to be a consequence of oversplicing. the synonymous mutations introduced into gag could inhibit hiv-1 replication by inactivating essential cisacting rna elements or introducing inhibitory elements. the region mutated in hiv-1 cm22-261 was designed to match the codons previously deleted by reil et al. [60] in hiv-1 hxbh10 ∆8-87/∆ct. in this virus, amino acids 8-87 error bars represent standard deviation. a rna was extracted from cell lysates and grna abundance was quantified by qrt-pcr. b rna was extracted from the media and grna abundance was quantified by qrt-pcr. c equivalent amounts of hiv-1 genomes were used to infect tzm-bl reporter cells to measure infectivity. the bar charts show the average values of three independent experiments normalized to the value obtained for hiv-1 nl4-3 . the average rlu for hiv-1 nl4-3 is 1,146,196. the dashed line represents 1000 rlu, which is approximately the limit of the assay for reproducible differential results (nt 22-261) in gag were deleted and a stop codon in env removed the cytoplasmic tail domain. because deletions in the globular core domain of ma prevent incorporation of env with a full cytoplasmic domain [69] , the truncated env cytoplasmic tail is necessary for virion infectivity. however, pseudotyping with heterologous envelope glycoproteins, such as that from vesicular stomatitis virus (vsv-g), allow viral entry into a target cell. hiv-1 hxbh10 ∆8-87/∆ct replicates as well as hiv-1 hxbh10 ∆ct in the mt4 cell line [60] , indicating that neither the protein or rna sequences in this region are necessary for viral replication in these cells. to determine whether the synonymous mutations inserted into nt 22-261 of gag removed essential cisacting elements or inserted deleterious sequences, we generated a hiv-1 nl4-3 ∆22-261 provirus construct (fig. 4a ) and compared it with hiv-1 cm22-261 in the absence or presence of vsv-g. in the absence of vsv-g, hiv-1 ∆22-261 produced very low levels of infectious virus (fig. 4b) , which was expected due to the role of ma in recruiting env with a full-length cytoplasmic tail. gag expression and virion production were similar for wild type hiv-1 and hiv-1 ∆22-261 (fig. 4c) , indicating that rna or protein sequences in this region are not necessary for these steps of the viral life cycle. when the viruses were pseudotyped with vsv-g, hiv-1 ∆22-261 infectivity was similar to wild type virus, confirming that the only functional defect for this virus in hela cells is env incorporation. in contrast, hiv-1 cm22-261 was not rescued by vsv-g pseudotyping and had a > 99.9% reduction in infectivity (fig. 4d) . reil et al. [60] also deleted amino acids 8-126 (nt 22-378) in ma and found that hiv-1 hxbh10 ∆8-126/∆ct replicated with moderately delayed kinetics compared to hiv-1 hxbh10 ∆ct in mt4 cells. we produced hiv-1 nl4-3 provirus constructs in which this region was either deleted (hiv-1 ∆22-378) or codon modified (hiv-1 cm22-378). vsv-g pseudotyped hiv-1 ∆22-378 had a small decrease in infectious virus production compared to wild type hiv-1 (fig. 4d) , which correlated with virion production (fig. 4e) . however, vsv-g pseudotyped hiv-1 cm22-378 had a > 99.9% decrease in infectious virus production with gag expression and virion production near the limit of detection (fig. 4d, e) . therefore, we concluded that codon modification of nt 22-261 or nt 22-378 of gag introduced inhibitory sequences into the hiv-1 genome that reduce gag expression, virion production and infectivity. to determine whether the synonymous mutations in gag altered the stability of the viral rna, hela cells were transfected with phiv-1 nl4-3 , phiv-1 cm22-261 or phiv-1 cm22-378 and, ~ 45 h post-transfection, rna polymerase ii-dependent transcription was inhibited by adding actinomycin d. rna was isolated from the cells immediately before actinomycin d addition (0 h) and then 1, 2, 4 and 6 h thereafter. grna abundance at the 0 h timepoint was substantially decreased for hiv-1 cm22-261 and hiv-1 cm22-378 (fig. 5a) and correlated with the length of codon modified sequence. since myc mrna has a half-life of < 1 h [70] , we used it as a control for rna stability and analyzed its abundance at each timepoint. as expected, myc mrna was rapidly degraded (fig. 5b) . the grna abundance for hiv-1 nl4-3 , hiv-1 cm22-261 and hiv-1 cm22-378 decreased by ~ 20, ~ 35 and ~ 70%, respectively, at the 6 h timepoint relative to its abundance at 0 h (fig. 5c) . this indicates that hiv-1 cm22-261 and hiv-1 cm22-378 grna is less stable than hiv-1 nl4-3 grna. comparing hiv-1 cm22-378 grna abundance to that of hiv-1 nl4-3 at the 6 h timepoint, hiv-1 cm22-378 grna was ~ 60% lower than wild type virus grna. if the degradation rate is constant, a 50% decrease every 6 h in grna abundance for hiv-1 cm22-378 relative to hiv-1 nl4-3 would be compounded to yield a 98.4% decrease after 36 h. this is consistent with the ~ 98% decrease in steady state grna for hiv-1 cm22-378 that we observed ~ 45 h post-transfection (fig. 5a) . overall, the synonymous mutations introduced into gag appear to decrease the stability of the grna. two types of rna dinucleotide patterns have previously been implicated in restricting rna virus replication, upa and cpg [71] [72] [73] [74] . the observed/expected ratio for upa in the hiv-1 nl4-3 grna is 0.92 (table 1 ) and the total number of upa dinucleotides in nt 22-261 and 22-378 decreased substantially in the codon modified sequence compared with the wild type sequence ( table 2 ). this indicates that upa dinucleotide content is not causing the inhibitory phenotype. the cpg dinucleotide observed/expected ratio is 0.21 in hiv-1 nl4-3 and it is the only dinucleotide substantially suppressed (table 1) . within nt 22-261 of gag, wild type hiv-1 has 4 cpg dinucleotides and the codon modified sequence has 22 (table 2) . similarly, codon modification of nt 22-378 increased the number of cpgs from 4 to 30. because cpg dinucleotides are underrepresented in hiv-1 (table 1) [40, 44, [55] [56] [57] and previous reports have shown that increasing the cpg dinucleotide abundance inhibits picornavirus and influenza virus replication [71] [72] [73] [74] , we hypothesized that the increased number of cpg dinucleotides in hiv-1 cm22-261 and hiv-1 cm22-378 could cause the decrease in hiv-1 infectious virion production. to test this hypothesis, we synthesized a hiv-1 gag sequence containing all of the synonymous mutations present in phiv cm22-261 with the exception of the codon changes that introduced cpg dinucleotides table 2 ). phiv-1 nl4-3 , phiv-1 cm22-261 and phiv-1 cm22-261 lowcpg were transfected into hela cells and single round infectivity assays were performed. in contrast to hiv-1 cm22-261, hiv-1 cm22-261 lowcpg infectivity, gag expression and virion production was similar to hiv-1 nl4-3 (fig. 7a, b) . we also cloned phiv-1 cm22-378 lowcpg , which has four cpg dinucleotides and 79 mutations compared with the 30 cpg dinucleotides and 109 mutations in hiv-1 cm22-378 (fig. 6, table 2 ). in a single round infectivity assay, hiv-1 cm22-378 lowcpg also had similar levels of infectivity, intracellular gag expression and virion production as hiv-1 nl4-3 (fig. 7c, d) . while codon modification of nt 22-261 and 22-378 in gag substantially decreased the a-rich nucleotide bias of these regions, eliminating only the cpg dinucleotides in the codon modified sequence did not restore the a-rich bias for hiv-1 cm 22-261 lowcpg and hiv-1 cm22-378 lowcpg ( table 2 ). this supports the hypothesis that the decrease in infectivity for hiv-1 cm22-261 and hiv-1 cm22-378 is due to the introduced cpg dinucleotides and not changes in the a-rich nucleotide bias. in sum, changing the codons that introduced cpg dinucleotides in nt 22-261 or nt 22-378 back to the wild type hiv-1 codons while maintaining all of the other mutations in these regions restored infectious virus production. toll-like receptor 9 (tlr9) recognizes unmethylated cpg dna motifs in endolysosomes within plasmacytoid dendritic cells, macrophages, and b cells [75] . upon ligand binding, tlr9 signaling stimulates interferon alpha (ifn-α) production, which binds the interferon alpha and beta receptor (ifnar) and induces interferon stimulated gene (isg) expression via the jak-stat signaling pathway. to determine whether hela cells are responsive to cpg dna or other tlr ligands, we tested a panel of ligands for tlr2, tlr3, tlr4, tlr5, tlr7, tlr8, tlr9 and tlr13 (additional file 3). as positive controls, hela cells were infected with sendai virus, which activates the cytoplasmic rna sensors rig-i and mda5 (rig-i-like receptors, rlr), or were treated with interferon beta (ifn-β), which also binds ifnar. both sendai virus and ifn-β stimulated stat1 phosphorylation and ifit1 expression, which is an isg. cpg dna did not stimulate stat1 phosphorylation or ifit1 expression. the only tlr ligand that stimulated the cells was poly(i:c), which is structurally similar to double stranded rna and can also signal via rlrs. therefore, it is unlikely that the additional cpg dinucleotides in hiv-1 cm22-261 and hiv-1 cm22-378 inhibit viral replication via tlr9 or other tlrs in hela cells. to determine if increasing the abundance of cpg dinucleotides is sufficient to inhibit hiv-1 replication, we synthesized gag sequences in which only the codons that introduced cpg dinucleotides in the codon modified sequence were changed (fig. 6) . these were inserted into phiv nl4-3 to produce phiv-1 cpg22-165, phiv-1 cpg22-261 and phiv-1 cpg22-378. we then used a spreading infection assay in jurkat cells to analyze the effect of the increased cpg abundance. 293t cells were transfected with each proviral construct to produce stocks of each virus and the abundance of p24 gag was (fig. 8a) . the viral inoculum was normalized so that jurkat cells were infected with 25 ng of p24 gag for each virus and replication was monitored over ~ 2 weeks. there are an additional 11 cpg dinucleotides in hiv-1 cpg22-165 and its replication was substantially reduced, with > 90% less infectivity at day 13 compared to hiv-1 nl4-3 (fig. 8b) . hiv-1 cpg22-261 has an additional 18 cpg dinucleotides and hiv-1 cpg22-378 has an additional 26 cpg dinucleotides. neither of these viruses replicated in the jurkat cells (fig. 8b) . therefore, introducing cpg dinucleotides into hiv-1 gag is sufficient to strongly attenuate viral replication in jurkat cells. to analyze the steps of the hiv-1 life cycle that were inhibited by the cpg dinucleotides, hela cells were transfected with phiv nl4-3 , phiv-1 cpg22-165, phiv-1 cpg22-261 or phiv-1 cpg22-378. ~ 48 h later, the media and cell lysates were harvested for infectivity, protein or rna analysis. in this single cycle assay, hiv-1 cpg22-378 infectivity was decreased ~ 99% (fig. 9a) . while there was no change in gag expression or virion production (fig. 9b) , hiv-1 cpg22-378 grna was decreased ~ 40% in the cell lysate and ~ 80% in the media (fig. 9c, d) . hiv-1 cpg22-261 had a ~ 75% decrease in infectivity with no difference in gag expression, virion production or grna abundance (fig. 9a-d) . there was no decrease in infectious virus production for hiv-1 cpg 22-165. overall, introducing 26 cpg dinucleotides into hiv nl4-3 decreased infectious virus production by ~ 99%, though this is a smaller attenuation than viruses containing cpg dinucleotides in the context of the codon modified sequence (figs. 2, 4, 7) . one potential explanation for why hiv-1 containing cpg dinucleotides in a codon modified context produce less infectious virus in hela cells than hiv-1 in which only cpg dinucleotides have been added is that a nucleic acid binding protein could bind the cpg dinucleotide to mediate antiviral activity. if a protein does directly bind the cpg dinucleotide, its binding site may encompass more than just the cpg and the surrounding nucleotides may affect binding. therefore, the five nucleotides 5′ and 3′ of the introduced cpg in hiv-1 cm22-378 and hiv-1 cpg22-378 were used to generate a 12 nt sequence. these were aligned and conserved nucleotides were identified using weblogo [67] . interestingly, the nucleotides surrounding the cpg in the codon modified sequence are more g/c-rich than in the wild type hiv-1 sequence (additional file 4a and b). herein, we show that introducing cpg dinucleotides into the hiv-1 genome inhibits viral replication. when only 11 cpg dinucleotides are inserted into gag in the context of the codon modified sequence (hiv-1 cm22-165), there is a large decrease in infectivity without a substantial loss of grna abundance, gag expression or virion production (figs. 2, 3) . when 18 or 26 cpg dinucleotides are added (hiv-1 cm22-261 and hiv-1 cm22-378, respectively), the intracellular grna stability and abundance is decreased which leads to reduced gag expression and virion production (figs. 2, 3, 4, 5 ). in addition, there is a defect in the pre-integration steps of the viral life cycle that is apparent when equivalent numbers of genomes are used to infect target cells (fig. 3c) . thus, manipulating the cpg abundance in gag can impart both producer and target cell defects in replication. determining whether these deficiencies are underpinned by a common mechanism such as shared host factors, or whether they are distinct from each other, will be an important direction of our future work. remarkably, none of the proposed cis-acting elements in nt 22-378 of gag appear to be necessary for infectious virus production in hela cells. we demonstrated this by comparing viruses that have nt 22-261 or nt 22-378 codon modified or deleted (fig. 4) . while codon modification of these regions inhibited hiv-1 infectivity, deletion of the same region did not substantially impair infectivity in a single round assay. furthermore, removing the introduced cpg dinucleotides in hiv-1 cm22-261 and hiv-1 cm22-378 while maintaining either 59 or 79 other nucleotide changes, respectively, almost completely restored hiv-1 infectious virus production (fig. 7) . this supports the observation by reil et al. [60] that the globular head of ma can be deleted in the context of env with a truncated cytoplasmic domain without substantially impairing viral replication in the mt4 t cell line or virion production in hela cells. one of the cis-acting elements proposed to be in the gag region that we have mutated is the grna packaging signal. while a relatively short sequence sufficient for packaging heterologous transcripts into virions has been identified for rous sarcoma virus and murine leukemia virus, delineation of the hiv-1 rna sequence that is sufficient for packaging has been more controversial [7, 8] . the core packaging signal for hiv-1 is in the 5′ utr; however, approximately 300 nt of the 5′ end of gag has been proposed to improve viral titre [7, 8] . we did not codon modify the first 21 nt of gag because this region is under purifying selection [11, 12] and nmr structures have shown that it base pairs with the u5 region of the 5′ utr to form the dimer promoting conformation of the grna that is packaged into virions [10, 76] . however, the relative importance of the sequence in gag beyond the first 21 nt for packaging the full-length hiv-1 grna, as opposed to heterologous transcripts, is unclear. mapping gag binding sites on the grna in living cells showed that the rna elements most frequently crosslinked to gag were in the 5′ utr and the rre [77] . in the context of the full-length virus, our data indicates that nt 22-378 in gag appear to have only a small effect on viral infectivity when this region is deleted or mutated without adding cpg dinucleotides (figs. 4, 7) . the requirement for this region for grna packaging may be different in the context of lentiviral vector genomes, which contain only a small portion of the hiv-1 grna [78] . in principle, codon usage changes in gag could affect grna translation [79] . however, the translation efficiency of a codon optimized gag mrna is only ~ 1.6fold higher than wild type gag mrna that contains theoretically suboptimal codon usage [80] . our data indicate that grna translation efficiency is not substantially affected by the changes in codon usage for hiv-1 cm22-261 since gag expression (fig. 2c) and intracellular grna abundance (figs. 3c, 5a) were both decreased by ~ 90%. changing the rna sequence could also affect the secondary or tertiary structure of the grna. while the amount of rna structure in the ma region of gag is much lower than that of the 5′ utr [32] , we cannot exclude that the synonymous mutations in gag have not altered nearby structures. the known structures in the nucleotides that we have mutated are the ires [29, 53, 81, 82] and the region that base pairs with the 3′ end of the genome [33, 35] . in the context of single cycle infectivity assays in hela cells (fig. 4) and replication in mt4 cells [60] , neither the ires nor circularization of the hiv-1 grna appear to be necessary because the region containing these elements can be deleted. however, it is possible that the phenotype for mutating an rna structure may not be the same as deleting it [83] and these structures could be necessary under conditions not tested in this study, such as cellular stress or the innate immune response [6] . two previous reports have shown that introducing synonymous mutations into gag or pol attenuates hiv-1 replication [54, 84] . martrus et al. [84] introduced codon pairs into gag or pol that are underrepresented relative to human mrnas, which strongly inhibited viral replication. however, codon pair bias in rna viruses has recently been shown to be due cpg and upa dinucleotide suppression and codon pair deoptimization increases cpg abundance [74, 85] . while not discussed in their study, the synonymous mutations that martrus et al. introduced increased the number of cpg dinucleotides in gag from 15 to 118. therefore, we hypothesize that the decrease in viral replication observed in this study is at least partially a result of increasing the cpg frequency instead of codon pair deoptimization. to analyze the role of the a-rich rna sequence for hiv-1 replication, keating et al. [54] codon modified regions within gag and pol by increasing the number of c-and g-rich codons, which also inhibited hiv-1 replication. phgag-pol [86] was used as the source of codon modified sequence and this has a large increase in cpg dinucleotide abundance compared with the wild type sequence. these additional cpg dinucleotides could be responsible for viral attenuation instead of altering the a-rich codon bias. supporting this hypothesis, klaver et al. [87] used phylogeny-instructed mutagenesis to increase or decrease the a-rich codon bias in an ~ 500 nt region of pol. in this study, the cpg dinucleotide abundance was decreased by one in the a-max mutant virus and only increased by four in the a-min mutant virus. substantially increasing or decreasing the number of a nucleotides did not affect hiv-1 replication, demonstrating that it is important to avoid introducing suppressed dinucleotides such as cpg in mutagenesis studies analyzing the functional relevance of nucleotide or codon bias. while cpg dinucleotides appear to be under negative selection in hiv-1 [36, 40, 44, [55] [56] [57] , the specific selection pressure has been unclear. there are at least four potential causes for the suppressed abundance of cpg dinucleotides in hiv-1. first, this could be due to a mutational bias caused by cytosines in a cpg context in the proviral dna becoming methylated and then undergoing rapid spontaneous deamination. however, we have shown that increasing the abundance of cpg dinucleotides inhibits viral replication, even in a single round assay (figs. 1, 2, 8, 9 ). second, cpg methylation-induced transcriptional silencing could inhibit hiv-1 gene expression [36, 40, 44, [55] [56] [57] 88] . however, this cannot cause the inhibition in the single cycle infectivity assays because the transfected proviral plasmids were amplified in bacteria, which does not methylate cpg dinucleotides. importantly, when an unmethylated plasmid is transfected into mammalian cells, the cpg dinucleotides are not methylated [89] [90] [91] . third, unmethylated cpgs in the dna could be recognized by tlr9 [88, 92] . this pattern recognition receptor is expressed in plasmacytoid dendritic cells, macrophages, and b cells [75] and therefore is unlikely to be expressed in hela cells. we confirmed that hela cells do not induce stat1 phosphorylation or ifit1 expression in response to cpg dna (additional file 3). in addition, ifn-α does not inhibit wild type hiv-1 infectious virus production in hela cells at most concentrations (10-1000 u/ml) and only has moderate inhibition at very high concentrations (10,000 u/ml) [93] . therefore, the inhibition that we observe in response to introducing cpg dinucleotides into hiv-1 is unlikely to be due to the production of type i interferon. the fourth possibility is that cpg dinucleotides in the viral rna could induce an antiviral response that restricts hiv-1 replication. the frequency of cpg dinucleotides is suppressed in many rna viruses that do not have a dna intermediate [57, 92, [94] [95] [96] , indicating that cpg dna methylation or activation of tlr9 cannot be responsible for cpg suppression in all viruses. introduction of cpg dinucleotides into picornaviruses or influenza a virus inhibits viral replication [71] [72] [73] [74] . it is unclear how cpg dinucleotides restrict rna virus replication but, for the picornavirus echovirus 7, it is not due to stimulating the interferon pathway, pkr, conventional pattern recognition receptors or altering the translation efficiency of viral proteins [71, 74] . it has been hypothesized that there is an innate immune sensor that detects cpg dinucleotides in viral rna and leads to the inhibition of viral replication, though the molecular details are unknown [71, 73, 74, 85, 92, [97] [98] [99] . we favor the hypothesis that the proposed active restriction pathway targeting cpg dinucleotides in rna viruses inhibits hiv-1 with an increased cpg abundance. when 26 cpg dinucleotides were added within nt 22-378 of gag (hiv-1 cpg22-378), which is < 5% of the hiv-1 grna, viral replication was inhibited in jurkat cells (fig. 8 ) and infectivity was decreased by ~ 99% in a single round assay (fig. 9 ). this indicates that the cpg dinucleotides induce a potent restriction. while this manuscript was under review, takata et al. [100] reported that introducing cpg dinucleotides into env inhibited hiv-1 replication by decreasing the abundance of cytoplasmic grna, gag expression, env expression and infectious virus production. they also demonstrated that depleting the cellular rna binding protein zap rescues replication of hiv-1 with increased cpg abundance and zap directly binds hiv-1 rna regions containing cpg dinucleotides. this indicates that zap restricts replication of hiv-1 containing increased cpg abundance, though it is unclear how zap promotes viral rna degradation. interestingly, we and others have shown that gag is efficiently expressed from mammalian expression vectors that encode codon-optimized gag or gag-pol cdnas containing large numbers of cpg dinucleotides [61, 68, 86, 101, 102] . therefore, it appears that cpg dinucleotides in the context of full length hiv-1 is more deleterious for protein expression than cpgs in the context of mammalian expression vectors. how the specific context of cpg dinucleotides affects zap binding to rna or modulates its activity will be an exciting area of future research. the hiv-1 rna sequence contains specific nucleotide features such as a low abundance of cpg dinucleotides. our data shows that introducing cpg dinucleotides into hiv-1 inhibits viral replication by affecting multiple steps of the life cycle. this provides a functional explanation for why cpg dinucleotides are suppressed in hiv-1 and we speculate this dinucleotide is under negative selection to avoid an active restriction system that may require zap. understanding how this restriction system inhibits replication of hiv-1 with increased cpg abundance may provide insight into how other rna viruses, such as picornaviruses and influenza a virus, are attenuated when cpg dinucleotides are introduced. posttranscriptional regulation of retroviral gene expression: primary rna transcripts play three roles as pre-mrna, mrna, and genomic rna regulation of hiv-1 alternative rna splicing and its role in virus replication alternative splicing: regulation of hiv-1 multiplication as a target for therapeutic action gene activity in primary t cells infected with hiv89.6: intron retention and induction of genomic repeats translational control of the hiv unspliced genomic rna regulation of human immunodeficiency virus type 1 (hiv-1) mrna translation life of psi: how full-length hiv-1 rnas become packaged genomes in the viral particles structural determinants and mechanism of hiv-1 genome packaging production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone nmr studies of the structure and function of the hiv-1 5′-leader synonymous site conservation in the hiv-1 genome extensive purifying selection acting on synonymous sites in hiv-1 group m sequences programmed ribosomal frameshifting in hiv-1 and the sars-cov the hiv-1 rev protein human immunodeficiency virus reverse transcriptase: 25 years of research, drug discovery, and promise structure-based alignment and consensus secondary structures for three hiv-related rna genomes comparison of siv and hiv-1 genomic rna structures reveals impact of sequence evolution on conserved and non-conserved structural motifs rna motif discovery by shape and mutational profiling (shape-map) architecture and secondary structure of an entire hiv-1 rna genome targeting the hiv rna genome: high-hanging fruit only needs a longer ladder towards realistic codon models: among site variability and dependency of synonymous and non-synonymous rates interplay between rna structure and protein evolution in hiv-1 rna structures facilitate recombination-mediated gene swapping in hiv-1 mapping of positive selection sites in the hiv-1 genome in the context of rna and protein structural constraints hiv-1 assembly, release and maturation synergistic stimulation of hiv-1 rev-dependent export of unspliced mrna to the cytoplasm by hnrnp a1 members of the heterogeneous nuclear ribonucleoprotein h family activate splicing of an hiv-1 splicing substrate by promoting formation of atp-dependent spliceosomal complexes the hiv-1 major splice donor d1 is activated by splicing enhancer elements within the leader region and the p17-inhibitory sequence the human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site distinct rna sequences in the gag region of human immunodeficiency virus type 1 decrease rna stability and inhibit expression in the absence of rev protein in vitro evidence for a long range pseudoknot in the 5′-untranslated and matrix coding regions of hiv-1 genomic rna high-throughput shape analysis reveals structures in hiv-1 genomic rna strongly conserved across distinct biological states circularization of the hiv-1 rna genome rna interactions in the 5′ region of the hiv-1 genome circularization of the hiv-1 genome facilitates strand transfer during reverse transcription nucleotide composition as a driving force in the evolution of retroviruses what can aids virus codon usage tell us? the tendency of lentiviral open reading frames to become a-rich: constraints imposed by viral genome organization and cellular trna availability unusual codon usage of hiv nucleotide composition bias and cpg dinucleotide content in the genomes of hiv and htlv 1/2 aids virus and htlv-i differ in codon choices the biased nucleotide composition of the hiv genome: a constant factor in a highly variable virus the unusual nucleotide content of the hiv rna genome results in a biased amino acid composition of hiv proteins codon and amino acid usage in retroviral genomes is consistent with virus-specific nucleotide pressure hiv genetic variation is directed and restricted by dna precursor availability lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase hypermutagenesis of rna using human immunodeficiency virus type 1 reverse transcriptase and biased dntp concentrations >a hypermutation of the human immunodeficiency virus type 1 genome: evidence for dctp pool imbalance during reverse transcription dna deamination mediates innate immunity to retroviral infection the cytidine deaminase cem15 induces hypermutation in newly synthesized hiv-1 dna broad antiretroviral defence by human apobec3g through lethal editing of nascent reverse transcripts human apobec3 induced mutation of human immunodeficiency virus type-1 contributes to adaptation and evolution in natural infection two ribosome recruitment sites direct multiple translation events within hiv1 gag open reading frame the a-rich rna sequences of hiv-1 pol are important for the synthesis of viral cdna various regulatory sequences are deprived of their uniqueness by the universal rule of ta/cg deficiency and tg/ct excess selection against cpg dinucleotides in lentiviral genes: a possible role of methylation in regulation of viral expression why is cpg suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? the cpg dinucleotide content of the hiv-1 envelope gene may predict disease progression packaging cells comprising codon-optimized gagpol sequences and lacking lentiviral accessory proteins. google patents efficient hiv-1 replication can occur in the absence of the viral matrix protein srp40 and srp55 promote the translation of unspliced human immunodeficiency virus type 1 rna hiv-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for gag processing but not for post-entry nuclear import sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor t-20 is modulated by coreceptor specificity defined by the v3 loop of gp120 emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (t-20) monotherapy effects of ccr5 and cd4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1 macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual v3 envelope sequence homogeneity in comparison with t-cell-tropic isolates: definition of critical amino acids involved in cell tropism weblogo: a sequence logo generator efficiency of transduction of highly purified murine hematopoietic stem cells by lentiviral and oncoretroviral vectors under conditions of minimal in vitro manipulation rescue of human immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins with short cytoplasmic domains the protein-coding region of c-myc mrna contains a sequence that specifies rapid mrna turnover and induction by protein synthesis inhibitors the influence of cpg and upa dinucleotide frequencies on rna virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication genetic inactivation of poliovirus infectivity by increasing the frequencies of cpg and upa dinucleotides within and across synonymous capsid region codons elevation of cpg frequencies in influenza a genome attenuates pathogenicity but enhances host response to infection rna virus attenuation by codon pair deoptimisation is an artefact of increases in cpg/upa dinucleotide frequencies microbial sensing by toll-like receptors and intracellular nucleic acid sensors nmr detection of structures in the hiv-1 5′-leader rna that regulate genome packaging global changes in the rna binding specificity of hiv-1 gag regulate virion genesis efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector synonymous codons: choose wisely for expression quantitative effect of suboptimal codon usage on translational efficiency of mrna encoding hiv-1 gag in intact t cells a conserved structure within the hiv gag open reading frame that controls translation initiation directly recruits the 40s subunit and eif3 a new type of ires within gag coding region recruits three initiation complexes on hiv-2 genomic rna opening of the tar hairpin in the hiv-1 genome causes aberrant rna dimerization and packaging changes in codonpair bias of human immunodeficiency virus type 1 have profound effects on virus replication in cell culture codon pair bias is a direct consequence of dinucleotide bias human immunodeficiency virus type 1-specific immunity after genetic immunization is enhanced by modification of gag and pol expression hiv-1 tolerates changes in a-count in a small segment of the pol gene source of cpg depletion in the hiv-1 genome inactive chromatin spreads from a focus of methylation dependence of transcriptional repression on cpg methylation density stability of patch methylation and its impact in regions of transcriptional initiation and elongation patterns of evolution and host gene mimicry in influenza and other rna viruses an interferon-alphainduced tethering mechanism inhibits hiv-1 and ebola virus particle release but is counteracted by the hiv-1 vpu protein modelling mutational and selection pressures on dinucleotides in eukaryotic phyla-selection against cpg and upa in cytoplasmically expressed rna and in rna viruses dinucleotide and stop codon frequencies in single-stranded rna viruses composition bias and genome polarity of rna viruses sequence-specific sensing of nucleic acids cpg rna: identification of novel single-stranded rna that stimulates human cd14 + cd11c+ monocytes distinguishing the immunostimulatory properties of noncoding rnas expressed in cancer cells cg dinucleotide suppression enables antiviral defence targeting non-self rna a rev-independent human immunodeficiency virus type 1 (hiv-1)-based vector that exploits a codon-optimized hiv-1 gag-pol gene retroviral mrna nuclear export elements regulate protein function and virion assembly the following reagents were obtained through the nih aids research and reference reagent program, division of aids, niaid, nih: tzm-bl from dr. john c. kappes, dr. xiaoyun wu and tranzyme inc; hiv-1 p24 hybridoma (183-h12-5c) from dr. bruce chesebro. we thank jonathan sumner in dr. stuart neil's lab for assistance in setting up the spreading infection assay and professor michael malim for helpful discussions. we also thank professor juan martin serrano, professor michael malim and dr. hendrik huthoff for critically reading the manuscript. additional file 2. codon modification of nucleotides 22-261 in gag decreases total hiv-1 rna abundance. the rna that was extracted from cell lysates as described for fig. 3a was quantified for total hiv-1 rna by qrt-pcr.additional file 3. cpg dna does not stimulate stat1 phosphorylation or isg expression. hela cells were stimulated with tlr ligands for 5 h. tlr3 was targeted with 0.1, 1, 10 μg/ml polyic, tlr7 with 0.3, 3, 30 nm gardiquimod (gard), tlr8/7 with 0.3, 3, 30 nm cl075, tlr7/8 with 0.01, 0.1, 1 μg/ ml r848, tlr9 with 0.01, 0.1, 1 μg/ml cpg dna, tlr13 with 0.01, 0.1, 1 μg/ ml 23s ribosomal rna (rrna). tlr 4, 2 and 5 were targeted with 0.1 μg/ml lps, pam3cys (p3c) or flagellin (flic), respectively. as controls for pattern recognition receptor signaling and jak-stat signaling, cells were infected with 50 hau/ml sendai virus (sev) for 5 h or stimulated with 0.01 μg/ ml ifn-β for 1 h, respectively. activation of ifn signaling was monitored by western immunoblotting against phosphorylated stat1 (pstat1) or expression of the isg ifit1. actin was used as a loading control. (* denotes the molecular weight marker).additional file 4. the cpg dinucleotide in the codon modified sequence is in a g/c-rich context. the sequence of the five nucleotides 5′ and 3′ to the cpg dinucleotides introduced into hiv-1 cm22-387 and hiv-1 cpg22-387 were aligned and a graphical representation of the sequence conservation was generated by weblogo. the authors declare that they have no competing interests. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. not applicable. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-017439-0c6ohmmg authors: hughes-oliver, jacqueline m. title: pooling experiments for blood screening and drug discovery date: 2006 journal: screening doi: 10.1007/0-387-28014-6_3 sha: doc_id: 17439 cord_uid: 0c6ohmmg pooling experiments date as far back as 1915 and were initially used in dilution studies for estimating the density of organisms in some medium. these early uses of pooling were necessitated by scientific and technical limitations. today, pooling experiments are driven by the potential cost savings and precision gains that can result, and they are making a substantial impact on blood screening and drug discovery. a general review of pooling experiments is given here, with additional details and discussion of issues and methods for two important application areas, namely, blood testing and drug discovery. the blood testing application is very old, from 1943, yet is still used today, especially for hiv antibody screening. in contrast, the drug discovery application is relatively new, with early uses occurring in the period from the late 1980s to early 1990s. statistical methods for this latter application are still actively being investigated and developed through both the pharmaceutical industries and academic research. the ability of pooling to investigate synergism offers exciting prospects for the discovery of combination therapies. the use of pooling experiments began as early as 1915 and was, initially, used in dilution studies for estimating the density of organisms in some medium. examples quoted by halvorson and ziegler (1933) include investigations of densities of bacteria in milk and protozoa in soil. prior to 1915, most dilution methods were inadequate because they failed to account for chance or error in observation. in 1915, mccrady presented a method of estimation based on probability which was then expanded by halvorson and ziegler (1933) to provide an estimator of density based on pooled data. fisher (1921) also used a similar pooling-based estimator. these early uses of pooling experiments were born of necessity, as explained below for bacterial density estimation. in order to determine the absence or presence of bacteria in a fluid, cultures are made of a number of samples (small amounts) of the fluid. growth of a colony of bacteria within a fluid sample indicates the presence of bacteria and no growth indicates absence of bacteria. the act of culturing this fluid can be viewed as applying a test, the result of which is "good" or "bad". this test is applied simultaneously to every molecule present in that sample of fluid and the results for all the molecules are pooled. the combined test results (from all samples) are then used to estimate the density of bacteria present in the source fluid. although it is virtually impossible to perform this test on individual molecules, it is quite simple to ascertain whether a culture from the pooled molecules is free of colony growth. today, pooling studies are not typically used from necessity. rather, they are used because of the economic gains, savings in time, or precision gains that can result. a useful review of pooling experiments from the point of view of composite sampling methods is offered by lancaster and keller-mcnulty (1998) . this chapter focuses on current usage for populations in which individuals are labeled with respect to one or more traits and where pooling experiments are optional. more specifically, the discussion addresses applications in blood testing and drug discovery. this is not meant to be an exhaustive review, but rather a vehicle for highlighting some important aspects of pooled screening in these two areas. applications in drug discovery require the identification of "hit compounds," which are those compounds having activity greater than some prespecified threshold in one or more biological assays. good hit compounds need to be identified quickly to allow progression to other phases of drug discovery (see chapter 4). one application in blood screening requires the identification of individuals with sero-prevalence (detectability in blood) of one or more diseases. cost effectiveness is important here because a balance must be struck between the cost of testing, which can be high, and the large populations that must be screened. a second issue that arises in blood screening is the need to estimate prevalences, possibly as a function of covariates. in order to address the two areas of application simultaneously, the term individual is used to mean either a person (in the context of blood testing) or a compound (drug discovery); the term active means either positive for one or more diseases (blood testing) or exceeding an activity threshold (drug discovery); and the term population means either a group of people being screened (blood testing) or a compound library being screened (drug discovery). two fundamentally different problems arise from pooling experiments, namely, estimation and classification. estimation involves the use of pooled samples for decreasing the cost-per-unit information when estimating the prevalence of active individuals in a population. these estimation results may then be used as the endproduct of analysis or they may be incorporated into a classification scheme. the estimation results serve as the end-product of analysis when the goal of the study is to estimate the prevalence of active individuals but there is no interest expressed in actually identifying these active individuals. in a classification scheme, the ultimate goal is screening for the purpose of identifying active individuals. the performance of a classification scheme is typically assessed by considering the expected number of tests required to identify active individuals with particular attributes. drug discovery is considered to be a classification problem, but results from the estimation problem can also be used to inform classification decisions. for blood testing, the application may be either a classification or an estimation problem, depending on the context. the most common assumptions of pooling experiments are briefly critiqued in section 2. in section 3, general accomplishments and advances in pooling experiments are reviewed, irrespective of their particular applications. sections 4 and 5 provide details specific to blood testing and drug discovery, respectively. pooling experiments are of two basic types, simple or orthogonal. in simple pooling, each individual appears in exactly one pool; see figure 1 (a), where each circle in the box represents an individual, and individuals in the same column are in the same pool. an active pool response must be followed by individual testing to determine which specific individuals in the pool are active. in orthogonal pooling using d dimensions, individuals appear in exactly d pools; see figure 1 (b) for d = 2. the determination of active individuals from orthogonal pooling is easier than that from simple pooling. consider orthogonal pooling over d = 2 dimensions corresponding to rows and columns. if a compound lies simultaneously in an active row and an active column, then it is reasonable to believe that this compound is active and that it should thus be assigned a favorable rank for individual testing. despite its benefits, orthogonal pooling adds many complexities and is, consequently, not as popular as simple pooling. all further discussion is limited to simple pooling. pooling experiments are based, historically, on several assumptions that are often blatantly unjustified. the first assumption is that individuals have equal probabilities of being active. in blood testing, genetic characteristics, environmental exposures, and demographic identities are widely accepted as sources of variability for disease status, thus suggesting that probabilities of activity are not constant across the population (dorfman, 1943) . in drug discovery, it is well recognized that structure-activity relationships (sars, see chapter 4), where activity is related to chemical structural features of a compound, lead to nonconstant probabilities of activity; see mcfarland and gans (1986) . a second assumption generally used is that interactions do not occur within a pool; that is, activity is neither enhanced nor degenerated by testing multiple compounds using a single test on a pool. it is possible, however, that individually inactive compounds can give an active test result when pooled together (borisy et al., 2003) , thus providing a case of "activity enhancement" by pooling. this phenomenon is called synergism and its detection is crucial to the development of combination therapies in the pharmaceutical industry. the reverse situation can also occur in that pooled testing of individually diseased samples can result in disease-free pool results (phatarfod and sudbury, 1994) , thus providing a case of "activity degeneration" by pooling. this phenomenon is called antagonism or blocking and is considered an undesirable potential effect of pooling in the blood testing application. blocking relationships that occur in drug discovery applications can have a positive impact on screening outcomes in that they provide further implicit evidence of structure-activity relationships. a third assumption concerns absence of errors in testing. both blood testing and drug discovery have strong potential for false negatives and false positives. errors in testing are inherently linked to assumptions regarding interactions within a pool. both concepts are, in turn, related to the sometimes arbitrarily chosen threshold value used for categorizing a continuous assay response into only two classes of "active" or "inactive". 3 history of pooling experiments dorfman (1943) has been credited with the origin of pooling experiments in the statistical literature. his ideas were popularized through the books of feller (1957, page 225) and wilks (1962) and became known as "the blood testing problem". many efforts were then made to refine dorfman's proposal by extending the number of stages using various retesting schemes and by relaxing assumptions. this section provides a brief summary of some key results from these efforts; see also chapter 9 for related work in factorial experiments. suppose that, in a large population of f individuals, each individual has, independently, the same probability p of being active. in this context, p represents a latent propensity for an individual to be active; some individuals ultimately express this latent feature and are thus labeled as actives, whereas others never express the latent feature and thus are labeled as inactive. if individuals are pooled into groups of size k and if pooling does not alter the behavior of individuals, then the resulting g = f /k pools will, independently, have the same probability θ = 1 − (1 − p) k of being active. hence, the number of active pools, x , follows a binomial distribution with parameters g and θ. of course, activity of pools or individuals must be revealed by some testing system and for now this system is assumed to be perfect. in other words, sensitivity (the probability that a test will identify, by testing outcome, an individual as active given that the individual is truly active) and specificity (the probability that a test will identify, by testing outcome, an individual as inactive given that the individual is truly inactive) are both assumed to be 1.0. dorfman himself did not believe these assumptions strictly but was able to build from the strength of the overall approach to make worthwhile reductions in the required number of tests over one-at-a-time testing. aspects of sensitivity and specificity are also discussed in chapters 4 and 6. dorfman's application was the need to identify world war ii selective service inductees whose blood contained syphilitic antigens. in other words, his was a classification problem and he wanted to minimize the number of tests required to classify all inductees. all individuals in inactive pools were declared to be inactive, without further testing. all individuals in active pools were subjected to one-at-a-time testing, thus leading to a random total number of tests t = f /k + xk (where f, k, and x are defined above). dorfman then needed to determine a pool size to minimize the expected total number of tests. pooling would only be advantageous if, on average, the total number of tests is less than f, which is the number of tests required by one-at-a-time testing. dorfman minimized the expected relative cost, for given p, with respect to k, to determine the best possible improvements offered by pooling experiments over one-at-a-time testing. for example, by pooling, he obtained an 80% cost savings in tests over one-at-a-time testing when p = .01 and k = 11. the savings decrease as p increases but are still appreciable even for larger p with, for example, 28% savings when p = .15 and k = 3. in fact, pooling, based on dorfman retesting for classification, is better than one-at-a-time testing when 1/k < (1 − p) k . the approximation (1 − p) k ≈ e − pk is sometimes used to claim that pooling is better than one-at-a-time testing when p < (ln k)/k. when p and k are both large, many active pools are observed and, consequently, more individual retests are required and this reduces the desirability of pooling. despite its simplicity, dorfman's retesting strategy is still very widely used today, especially in blood testing and drug discovery applications. his rough guidelines for choosing k such that p < (ln k)/k, coupled with recommendations by thompson (1962) , kerr (1971) , loyer (1983) , and swallow (1987) to use an a priori upper bound on p, is also commonly used today. indeed, the attraction of the dorfman strategy is its simplicity. improved methods for classification, some of which are discussed in this article, add various levels of complications that users may not yet be ready to accept. dorfman (1943) did not really address the problem of estimating the prevalence p but, using his assumptions, others did. gibbs and gower (1960) and thompson (1962) investigated the maximum likelihood estimator of p: where g = f /k is the number of pools and x the number of active pools. this is a positively biased, but consistent, estimator for p. based on the asymptotic variance ofp, peto (1953) and kerr (1971) determined that the optimum group size k satisfies (1 − p) k = .203. based on asymptotic considerations, thompson (1962) suggested that the group size should be approximately k = (1.5936/ p) − 1. he also argued, however, that the asymptotic results can be very misleading and offered small-sample exact bias and variance formulae. gibbs and gower (1960) , griffiths (1972) , loyer (1983) , and swallow (1985 swallow ( , 1987 also gave small-sample results. when c is the nontesting cost associated with obtaining an individual sample (for example, personnel time for drawing blood from an individual) divided by the cost of a test, sobel and elashoff (1975) showed that pooling is advantageous when for extremely costly tests, pooling can be beneficial for p as large as 2/3. extensions of dorfman's procedure follow four main branches: (i) development of different retesting schemes; (ii) strategies when p is unknown, as is usually the case; (iii) departures from binomial assumptions; and (iv) errors in testing. the literature is quite extensive (see hughes-oliver, 1991) , so only key papers are referenced here. for brevity, no attempt has been made to separate extensions for the goal of classification from extensions for the goal of estimation. many different retesting schemes have been suggested in the literature, some of which require infinite testability of the units. for example, sterrett (1957) proposed retesting individuals in an active pool only until an active individual is found. the remaining untested individuals are then retested as a single pool and the process is repeated. sobel and groll (1959) proposed a retesting scheme based on nested halving procedures. active pools are subdivided into two pools of size approximately k/2, each of which is tested. individuals in an inactive subpool are declared inactive but an active subpool is again halved. halving terminates when pool size becomes one, that is, at individual testing. sobel and elashoff (1975) proposed a general nested retesting scheme for estimation, of which nested halving is a special case. they found that a certain class of nested halving procedures is highly efficient and the savings over one-at-a-time procedures is even greater for the estimation problem than for the classification problem. they also found that, when the cost of obtaining individuals relative to the cost of a test is negligible, the optimal testing scheme does not include retesting. chen and swallow (1990) confirmed the finding that retesting is not advantageous for estimation when testing costs far exceed costs of obtaining individuals, but they showed that data from retesting can provide useful information for testing model assumptions. in contrast to the work of sobel and elashoff (1975) and chen and swallow (1990) , where the stated goal was to reduce cost per unit information for estimation in the presence of perfect testing, retesting has been shown to be useful for classification, especially when test results may be inaccurate. litvak et al. (1994) argued that, even when testing is correctly executed, it can lead to incorrect conclusions and, in these cases, retesting provides significant improvements over no-retesting for reducing error rates associated with labeling samples when screening low-risk hiv populations. based on nested halving, litvak et al. (1994) also proposed a new retesting scheme where inactive pools are subjected to a repeat test; if they again test inactive then all individuals in those pools are declared inactive, otherwise the pool is halved and subjected to additional testing. gastwirth and johnson (1994) , who were also concerned with error rates for labeling individuals assuming imperfect testing, proposed a "back-end" retesting stage where pooled testing is used to rescreen a subset of individuals who were declared inactive from "first-stage" pooled testing. the success of a pooling experiment depends heavily on the choice of a good value for the pool size k. unfortunately, optimal pool size depends on the value of p. in the absence of a priori information on p, le (1981) and a number of other authors recommended that different pool sizes be used and the resulting data on the number of active pools for each pool size be combined to yield an estimator. thompson (1962) argued that an a priori upper bound on p should be used to determine a single pool size, and hughes-oliver and swallow (1994) and hughes-oliver and rosenberger (2000) proposed two-stage adaptation to allow a single update of the pool size. these last authors also addressed the issue of pool size when there are multivariate responses from pools, motivated by the need to monitor prevalence rates for several diseases simultaneously. on the issue of departures from binomial assumptions, finucan (1964) considered a case where stratification occurs and results in different probabilities of activity for different individuals. a good early reference for various approaches to dealing with such situations is that of hwang (1984) . chen and swallow (1990) noted that model assumptions can be tested if data on unequal pool sizes are available. many recent articles also consider the situation where probability of activity is dependent on covariates. for small numbers of covariates, hung and swallow (2000) , vansteelandt et al. (2000) , xie (2001) , and tebbs and swallow (2003a,b) obtained estimates of prevalences in the different strata. for large numbers of covariates, xie et al. (2001) , zhu et al. (2001) , and yi (2002) obtained estimates of prevalences in the different strata then ranked the estimated prevalences to define a testing order for the classification problem. thus, the estimation problem was an intermediate step, not the ultimate goal, of the drug discovery applications of these authors. on a related note, remlinger et al. (2005) considered the design problem of assigning individuals to pools based on their covariates; the goal was classification in the presence of covariate-dependent prevalences. the problem of errors in testing has been examined by a host of investigators. references to investigators from a clinical/laboratory science viewpoint are given in section 4.2. from a statistician's viewpoint, gastwirth and hammick (1989) and hammick and gastwirth (1994) used trinomial models in which either a confirmatory pool test or an independent pool test was used to reduce the number of false positives. they also incorporated sensitivities and specificities (section 3.1) of the testing scheme into their estimator while maintaining individual anonymity. tu et al. (1994 tu et al. ( , 1995 also incorporated sensitivities and specificities of the testing scheme and showed that this leads to improved estimation accuracy. vansteelandt et al. (2000) took the same approach but with the added complication of covariate-adjusted estimation of prevalence. hung and swallow (1999) investigated robustness properties of the pooling estimator with respect to dilution effects and serial correlation models. wein and zenios (1996) also investigated dilution effects. in the area of drug discovery, langfeldt et al. (1997) , xie et al. (2001) , zhu et al. (2001) , yi (2002) , and remlinger et al. (2005) all investigated procedures that model possible interactions occurring within pools; such interactions may be mislabeled as errors in testing. pooling is now considered to be a routine option in blood screening, especially for the human immunodeficiency virus (hiv). there are many reports espousing the benefits of pooled testing in countries across the world, using a variety of assay techniques. there are actually three blood screening applications for which pooling has been beneficial. the two most common applications are the context of classification, where the goal of blood screening is to identify individuals with seroprevalence of one or more diseases. one classification application arises from the need to screen donated blood and blood products and the other from the need to screen for individual diagnoses. cost effectiveness, as measured by the reduction in the expected total number of tests, is the most commonly used assessment of pooling methods. the third application is the need to monitor changes in sero-prevalence over time for (possibly) different sets of individuals, where demarcation of individuals may occur along demographic lines or spatial/regional clusters. motivated by a more than 90% transmission rate of hiv by transfusion of blood and blood products, the world health organization (who) argued for 100% screening of donated blood. recognizing that developing countries can ill-afford the cost of 100% one-at-a-time screening, who issued recommendations for testing for hiv antibody on serum pools (who, 1991) in areas where seroprevalence is less than 2%. in fact, this figure of 2% sero-prevalence is much too restrictive. many investigators have achieved success with much higher prevalences. for example, soroka et al. (2003) described the successful use of pooling where prevalence was 9%. it is important to note that, for screening blood supplies, complete identification of sero-positive individuals is not necessary. all that is needed is a method for tracking the complete donated sample, without personal identifiers. this makes pooled screening very attractive for screening blood supplies because donors can be assured that their anonymity will be maintained. another classification problem occurs when individual diagnosis is the required outcome of a screening campaign. in such a campaign, personal identifiers must be maintained for the purpose of reporting back to individuals about their seroprevalence. moreover, diagnostic testing requires that sero-positive pools be subjected to confirmatory gold-standard tests. gastwirth and hammick (1989) and hammick and gastwirth (1994) approached the blood testing problem with a keen eye towards preserving individual privacy rights. they proposed screening strategies designed for estimating prevalences. rather than focus on the cost-saving advantages of pooling, these authors selected pooling because of the anonymity it provides to individuals being screened. they also reduced false predictive values by employing confirmatory tests to verify sero-prevalence. the standard practice in developed countries for determining hiv sero-prevalence is first to apply the cost-effective, but suboptimal, enzyme-linked immunosorbent assay (elisa) test. for those individuals who are identified as sero-positive by the elisa test, follow-up testing is then performed using the gold standard western blot test. unfortunately, the western blot is very expensive, difficult to standardize and often results in no clear diagnosis for some individuals (tamashiro et al., 1993) . to relieve the cost burden, the who recommends a series of repeat testing that uses cheaper tests, namely elisa or simple or rapid tests, to avoid the western blot while still maintaining testing accuracy. in general, the western blot best is up to six times as expensive as rapid or simple tests and 18 times as expensive as elisa; see, for example, who (1992) . rapid and simple tests provide results in less than one hour (less than 30 minutes for rapid tests) and may be performed by personnel having little or no laboratory training. elisa must be performed in a laboratory (so results are not immediately available) by extensively trained laboratory professionals. the who (1992) recommendations are shown in figure 2 and supporting text is given in table 1 . strategy i is recommended for screening contributions to a blood supply. it says that a contribution should only be accepted if it is seronegative according to either the elisa test, or the rapid test, or the simple test. sero-positive samples are not considered further. strategy i is also recommended when prevalence is high and the goal is hiv surveillance. who's strategy iii is recommended for diagnosing symptom-free individuals living in areas of low prevalence. it is the strategy that allows the greatest number of retests. if the first test is sero-positive, it is followed up with a second test that is not simply a repeat measurement of the first test. specifically, the assay procedure should differ from the first assay procedure in some substantial way; for example, different antigen preparation, different test principle (such as indirect versus competitive) or both. the first test should be very sensitive but the other two tests should have higher specificity than the first. if this second test is again sero-positive, a third and last test is applied. strategy ii is similar but with only two stages. effective clinical pooling studies for hiv classification and surveillance have been reported by a large number of investigators. emmanuel et al. (1988) cahoon -young et al. (1989) , kline et al. (1989) , behets et al. (1990) , archbold et al. (1991) , ko et al. (1992) , babu et al. (1993) , and perriens et al. (1993) have all reported successes for several different countries, including countries in africa and asia. "success" here is defined as the appropriate management of the logistics of pooling and the reduction of the amount of testing required. moreover, successes have been achieved based on several different testing protocols, including elisa, western blot, and rapid testing techniques; see also davey et al. (1991) , seymour et al. (1992) , raboud et al. (1993) , mcmahon et al. (1995) , verstraeten et al. (1998), and soroka et al. (2003) . these studies reported up to 80% reductions in cost for pooling experiments compared with one-at-a-time testing. since the late 1980s, statistical contributions to pooling for blood testing have focused on the following aspects: assessing changes in sensitivity and specificity due to pooling, designing pooling strategies to accommodate both cheap initial screens and gold-standard confirmatory screens, and estimation of covariate-dependent prevalences. let us first consider approaches to assessing changes in sensitivity and specificity due to pooling. as defined in section 3.1, sensitivity is the probability that a test correctly detects antibodies in a serum sample, and specificity is the probability that a test correctly identifies an antibody-free serum sample. these probabilities have been a major area of concern in pooling studies for blood testing (who, 1991) . the over-arching issue when screening a blood supply is whether dilution effects will cause a single sero-positive individual to be missed when combined in a pool with several (perhaps as many as 14) sero-negative individuals. this issue relates to the false negative predictive value as follows. a predictive value is the probability of truth given an individual's testing outcome; a false negative predictive value is the probability that the individual is truly active but is labeled as inactive from testing; a false-positive predictive value is the probability that the individual is truly inactive but is labeled as active from testing. when screening for diagnostic purposes, the major concern is that sero-negative individuals will be labeled sero-positive; this relates to the false positive predictive value. repeatedly, however, studies have indicated that, under their worst performance, these possible pooling effects are negligible. in fact, cahoon-young et al. (1989) , behets et al. (1990) , archbold et al. (1991) , sanchez et al. (1991) all reported reductions in the number of misclassified sero-negative individuals; for example, cahoon-young et al. (1989) found that there were seven misclassified sero-negative individuals out of 5000 tested, but no misclassified sero-negative pools out of 500 tested. for understanding sensitivity, specificity, false negative predictive value, and false positive predictive value, consider the four cells and two column margins of table 2 , where individuals are cross-classified with respect to their true serostatus versus observed sero-status. sensitivity is represented by s e = p(testing outcome + | truth is +) and specificity is s p = p(testing outcome − | truth is −). with these definitions and with p denoting the probability of an individual having table 2 . cross-classification of individuals for "true" versus "observed" sero-status (+, −) in terms of sensitivity s e , specificity s p , and probability p of positive sero-status (1 − p)s p positive sero-status, the false negative predictive value is fnpv = p( truth is + | testing outcome −) = p(1 − s e ) p(1 − s e ) + (1 − p)s p and the false positive predictive value is large false negative predictive values are particularly troubling when screening a blood supply because they allow sero-positive samples to enter the blood supply system, thus leading to possible transmission of deadly diseases. minimizing the false negative predictive value is probably more important than increasing cost efficiency of pooling for this application. of course, large false negative predictive values can arise even when screening is accomplished using one-at-a-time testing. false positive predictive values are of greater concern in diagnostic testing because they can cause undue stress for the falsely identified individuals and increase testing costs. notice that if s e = s p = 1, then fnpv = fppv = 0 and no misclassifications will occur. litvak et al. (1994) compared three pooling strategies and one-at-a-time testing with respect to their abilities to reduce fnpv, fppv, the expected numbers of tests required, and the expected numbers of tests performed for each individual. the first pooling study considered was dorfman retesting with pool size k = 15; that is, all individuals in sero-positive pools were tested one-at-a-time but no retesting was applied to individuals in sero-negative pools. the pool size of 15 was selected because, at the time, it was the largest acceptable size from a laboratory perspective for maintaining high sensitivity and specificity after pooling. litvak et al. (1994) called this screening protocol t 0 . their second pooling protocol, t 2 , was essentially the retesting method proposed by sobel and groll (1959) whereby sero-positive pools are recursively halved and testing of the subpools continues until no further splits are possible. in this strategy with k = 15, a serum sample must be positive four or five times before being declared sero-positive. their third pooling protocol, t + 2 , is similar to t 2 except that each sero-negative pool is subjected to one confirmatory pool test before all its individuals are labeled as seronegative. it was found that t 2 and t + 2 were comparable and that both provided huge reductions in fppv compared with one-at-a-time testing but smaller reductions compared with t 0 . for fnpv, t + 2 was the best protocol. in short, pooling reduced both false negative and false positive predictive values. the result from estimating sero-prevalence of hiv in the presence of errors in testing is really quite startling. tu et al. (1994 tu et al. ( , 1995 found that pooling actually increases estimator efficiency by reducing the effect of measurement errors. vansteelandt et al. (2000) extended the procedure to account for covariate adjustments. these results, along with the large number of empirical findings from investigators such as emmanuel et al. (1988) , clear the way for heavy reliance on pooling strategies to eliminate the backlog and reduce the cost of screening large populations. this is of particular importance to developing countries that are often cash-strapped but might benefit the most from 100% screening. even developed countries might want to rethink their screening strategies to take advantage of fewer but more informative pooled test results. twenty percent of sales from the pharmaceutical industry for the year 2000 were reinvested into research and development activities. this percentage is higher than in most other industries, including the electronics industry. at the same time, it is getting increasingly difficult to introduce (that is, discover, demonstrate efficacy and safety, and receive approval for marketing of) new drugs in order to recoup investment costs. on average, one new drug requires investment of $880 million and 15 years development (giersiefen et al., 2003, pages 1-2) . the days of profitability of "runner-up" or "me-too" drugs have long passed and the simple current reality is that survival and financial security of a pharmaceutical company demands that they find the best drugs as fast as possible. this means that the five major phases of drug discovery, as illustrated in figure 3 , need to be traversed aggressively. details on the phases of drug discovery can be found in chapter 4. here, attention is directed to the third phase, lead identification, which is where pooling experiments for screening in drug discovery usually occur. figure 3 . phases of drug discovery. given a large collection of compounds, say f = 500,000, the goal of lead identification is to find about 100 compounds that are (i) active for the assay-this allows them to be called "hits"; (ii) patentable; that is, their structures are novel and not already under patent; (iii) have good chemical properties such as stability, can be synthesized, are not toxic, and so on; (iv) something is understood about what makes them active; that is, their structureactivity relationships have been, at least partially, identified; (v) each compound is fairly different from the other ninety-nine. compounds that satisfy all these requirements are called leads or lead compounds. the need for properties (i)-(iii) is clear, but additional comments are warranted for the other properties. knowledge of structure-activity relationships allows chemists to focus on the essential substructures of the compound without wasting time with the portions that do not affect activity. the drug discovery phase that follows lead identification is lead optimization. in this phase, chemists expend enormous energies "tweaking" the leads to increase the chances of compounds making it through the grueling stages of preclinical and clinical development. it is imperative that the lead optimization phase produces very strong lead compounds to be investigated during preclinical and clinical development. once a compound reaches the preclinical and clinical development phase, extensive additional financial and time investments are made, so that heavy losses would be incurred if the compound had to be abandoned further down the drug discovery channel because it possesses undesirable features (see also chapter 4). the goals of drug discovery, as stated above, seem to be very similar to those of the blood screening for classification problem, but this is not at all the case. as mentioned, in earlier sections of this chapter, approaches to solving the blood testing for classification problem do not routinely incorporate covariate information. for the hiv blood testing problem, relevant covariate information for an individual may include the following: number of blood transfusions received, number of sexual partners, number of sexual partners who are hiv-infected, syringe use, drug use, sexual preference, and hiv status of parents. recent investigations have allowed the estimation of prevalence in different covariate-defined strata, but the number of strata is never large and is quite typically less than 10. in screening for drug discovery, on the other hand, the number of covariates is quite often at least twice the number of pooled responses available. indeed, the significant challenges that arise from the high-dimensional-with-low-sample-size data sets that usually result from "high-throughput screening" in drug discovery present major obstacles to analysis, even for one-at-a-time testing results. these difficulties are magnified in the presence of pooled responses. more information is given by langfeldt et al. (1997) , xie et al. (2001) , zhu et al. (2001) , yi (2002) , and remlinger et al. (2005) . arguably, the biggest difference between the two application areas discussed in this chapter is the potential for synergistic relationships between compounds in pools for drug discovery, whereas no such concept has arisen for blood testing. synergism has recently become the major supporting argument for pursuing pooling experiments in drug discovery yi, 2002; remlinger et al., 2005) . synergistic relationships can only be discovered through pooling studies where compounds are forced together, and it is these synergistic relationships that form the basis of combination therapies. these therapies involve deliberate mixing of drugs and they are now the standard of care for life-threatening diseases such as cancer and hiv. current combination therapies were discovered by combining individually active compounds after they had been approved by the food and drug administration. by investigating synergistic relationships in vitro, it is expected that one could find a combination where, individually, the compounds are inactive but, when pooled, their activities exceed all other combinations. borisy et al. (2003) demonstrated this quite nicely using several real experiments. for example, chlorpromazine and pentamidin were more effective than paclitaxel (a clinically used anticancer drug), even though individually neither drug was effective at tolerable doses. similar ideas were discussed by tan et al. (2003) . so, are cost considerations no longer important for drug discovery? the answer is "not really," or at least not as much as they used to be. before the advent of high-throughput screening (hts, see chapter 4) and ultrahigh-throughput screening (uhts), pooling was necessary for processing the large compound libraries typically encountered. in those days, a large screening campaign might screen a total of 50,000 compounds, and it would take months to complete. today, uhts can screen 100,000 compounds in a single day; see banks (2000) and niles and coassin (2002) . hts and uhts systems are centralized, highly automated, and are under robotic control so they can work almost around the clock with very small percentages of down-time. the two applications of drug discovery and blood testing are similar in how they process screening outcomes. comparing strategy iii of figure 2 with the extended view of lead identification in figure 3 , it can be seen that both methods use three tests in labeling the final selected individuals. the selected individuals are the gems for drug discovery applications but, for the blood testing problem, they actually cause concern because they are blood samples that have been confirmed to be diseased. a commonly used technique for analyzing drug discovery screening data from individuals is recursive partitioning (rp), more commonly known as "trees" (see, for example, blower et al., 2002) . in very recent times, efforts based on multiple trees (svetnik et al., 2003) have become the method of choice, despite the additional difficulties associated with them, because of their good predictive abilities. the number of researchers working to develop methodology appropriate for pooled drug screening data and who are allowed to discuss these issues outside the big pharmaceutical companies is very small. papers from these researchers have been reviewed earlier in this chapter, but a few additional comments are warranted. the bulk of the work has been divided into two major paths. one path concerns the search for the efficient placement of individuals within pools; that is, the design of pooling studies. because of the very large number of covariates, this is a difficult problem that requires computer-intensive techniques. remlinger et al. (2005) obtained structure-based pooling designs to assign pool placement in response to covariate-adjusted prevalences. zhu (2000) developed model-based designs for the same problem. the second major path concerns analysis methods, including nonparametric, semi-parametric, fully parametric, and bayesian approaches. nonparameteric results are based on recursive partitioning on pooled data and require the formation of pooled summaries and decisions of whether and how to include the retested data in the analysis without violating independence assumptions. for the semi-parametric work, yi (2002) modeled data from pooling experiments as missing data scenarios where missingness occurs at random. this was a novel use of the semi-parametric methodology to an area that had never before been considered. another interesting finding is that random retesting of both active and inactive pools can lead to improved estimators. litvak et al. (1994) and gastwirth and johnson (1994) were able to improve their estimators in the blood testing problem by retesting inactive pools. zhu et al. (2001) described a trinomial modeling approach that incorporates the phenomenon of blocking and used this model to develop criteria for creating pooling designs. these fully parametric models were also extended by yi (2002) who considered pairwise blocking probabilities. xie et al. (2001) used a bayesian approach for modeling blockers and synergism. finally, remlinger et al. (2005) also considered design pooling strategies, but from a completely structure-based approach. when it comes to designing and analyzing pooling studies for drug discovery, many open questions remain. single-dose pooling studies, which is an area still in its infancy, have been the focus of this chapter. multiple-dose pooling studies, which constitute a more mature area of research and application, can bring yet another level of interesting questions and evidence of the utility of pooling; see, for example, berenbaum (1989) . many modern developments and applications point to a bright future for pooling experiments. first, blood testing is ready to support heavy-duty use of pooling studies all across the world. the evidence of success is overwhelming whereas the costs are minimal. secondly, the drug discovery application still has a long way to go before it is fully developed, but researchers are making great strides. the ability to uncover synergistic relationships for discovering combination therapies is very exciting and offers many new challenges and possibilities. serum-pooling strategies for hiv screening: experiences in trinidad, ecuador, and the phillippines. vii international conference on aids abstract book reduction of the cost of testing for antibody to human immunodeficiency virus, without losing sensitivity, by pooling sera automation and technology for hts in drug development successful use of pooled sera to determine hiv-1 seroprevalence in zaire with development of cost efficiency models what is synergy? on combining recursive partitioning and simulated annealing to detect groups of biologically active compounds systematic discovery of multicomponent therapies sensitivity and specificity of pooled versus individual sera in a human immunodeficiency virus antibody prevalence study using group testing to estimate a proportion, and to test the binomial model pooling of blood donor sera prior to testing with rapid/simple hiv test kits the detection of defective members of large populations pooling of sera for human immunodeficiency virus (hiv) testing: an economical method for use in developing countries an introduction to probability theory and its applications the blood testing problem on the mathematical foundations of theoretical statistics estimation of the prevalence of a rare disease, preserving the anonymity of the subjects by group testing: application to estimating the prevalence of aids antibodies in blood donors a two-stage adaptive group-tesing procedure for estimating small proportions the use of a multiple-transfer method in plant virus transmission studies: some statistical points arising in the analysis of results modern methods of drug discovery: an introduction a further note on the probability of disease transmission application of statistics to problems in bacteriology group testing for sensitive characteristics: extension to higher prevalence levels strategic pooling of compounds for high-throughput screening estimation using group-testing procedures: adaptive iteration efficient estimation of the prevalence of multiple rare traits a two-stage adaptive group-testing procedure for estimating small proportions robustness of group testing in the estimation of proportions use of binomial group testing in tests of hypotheses for classification or quantitative covariables robust group testing the probability of disease transmission evaluation of human immunodeficiency virus seroprevalence in population surveys using pooled sera successful use of pooled sera to estimate hiv antibody seroprevalence and eliminate all positive cases a review of composite sampling methods optimal group testing in the presence of blockers. institute of statistics mimeograph series 2297 a new estimator for infection rates using pools of variable size screening for presence of a disease by pooling sera samples bad probability, good statistics, and group testing for binomial estimation the numerical interpretation of fermentation-tube results on the significance of clusters in the graphical display of structure-activity data pooling blood donor samples to reduce the cost of hiv-1 antibody testing miniaturization technologies for high-throughput biology use of a rapid test and an elisa for hiv antibody screening of pooled serum samples in lubumbashi a dose response equation for the invasion of micro-organisms the use of a square array scheme in blood testing combining pooling and alternative algorithms in seroprevalence studies statistical design of pools using optimal coverage and minimal collision workload and cost-effectiveness analysis of a pooling method for hiv screening use of both pooled saliva and pooled serum samples for the determination of hiv-1/hiv-2 antibodies by both conventional and rapid eia techniques group testing with a new goal, estimation group testing to eliminate efficiently all defectives in a binomial sample the use of simple, rapid tests to detect antibodies to human immunodeficiency virus types 1 and 2 in pooled serum specimens on the detection of defective members of large populations random forest: a classification and regression tool for compound classification and qsar modeling group testing for estimating infection rates and probability of disease transmission relative mean squared error and cost considerations in choosing group size for group testing to estimate infection rates and probabilities of disease transmission reducing the cost of hiv antibody testing experimenal design and sample size determination for testing synergism in drug combination studies based on uniform measures estimating ordered binomial proportions with the use of group testing more powerful likelihood ratio tests for isotonic binomial proportions estimation of the proportion of vectors in a natural population of insects screening tests: can we get more by doing less? on the information and accuracy of pooled testing in estimating prevalence of a rare disease: application to hiv screening regression models for disease prevalence with diagnostic tests on pools of serum samples pooling sera to reduce the cost of hiv surveillance: a feasibility study in a rural kenyan district pooled testing for hiv screening: capturing the dilution effect recommendations for testing for hiv antibody on serum pool. world health organization weekly epidemiological record recommendations for the selection and use of hiv antibody tests. world health organization weekly epidemiological record mathematical statistics regression analysis of group testing samples group testing with blockers and synergism nonparametric, parametric and semiparametric models for screening and decoding pools of chemical compounds. unpublished phd dissertation statistical decoding and designing of pooling experiments based on chemical structure. unpublished phd dissertation statistical decoding of potent pools based on chemical structure key: cord-000065-6c3zb3g4 authors: nguyen, thu anh; oosterhoff, pauline; pham, yen ngoc; hardon, anita; wright, pamela title: health workers' views on quality of prevention of mother-to-child transmission and postnatal care for hiv-infected women and their children date: 2009-05-13 journal: hum resour health doi: 10.1186/1478-4491-7-39 sha: doc_id: 65 cord_uid: 6c3zb3g4 background: prevention of mother-to-child transmission has been considered as not a simple intervention but a comprehensive set of interventions requiring capable health workers. viet nam's extensive health care system reaches the village level, but still hiv-infected mothers and children have received inadequate health care services for prevention of mother-to-child transmission. we report here the health workers' perceptions on factors that lead to their failure to give good quality prevention of mother-to-child transmission services. methods: semistructured interviews with 53 health workers and unstructured observations in nine health facilities in hanoi were conducted. selection of respondents was based on their function, position and experience in the development or implementation of prevention of mother-to-child transmission policies/programmes. results: factors that lead to health workers' failure to give good quality services for prevention of mother-to-child transmission include their own fear of hiv infection; lack of knowledge on hiv and counselling skills; or high workloads and lack of staff; unavailability of hiv testing at commune level; shortage of antiretroviral drugs; and lack of operational guidelines. a negative attitude during counselling and provision of care, treating in a separate area and avoidance of providing service at all were seen by health workers as the result of fear of being infected, as well as distrust towards almost all hiv-infected patients because of the prevailing association with antisocial behaviours. additionally, the fragmentation of the health care system into specialized vertical pillars, including a vertical programme for hiv/aids, is a major obstacle to providing a continuum of care. conclusion: many hospital staff were not being able to provide good care or were even unwilling to provide appropriate care for hiv-positive pregnant women the study suggests that the quality of prevention of mother-to-child transmission service could be enhanced by improving communication and other skills of health workers, providing them with greater support and enhancing their motivation. reduction of workload would also be important. development of a practical strategy is needed to strengthen and adapt the referral system to meet the needs of patients. prevention of mother-to-child transmission (pmtct) has been considered a simple intervention: just giving medication to prevent viral transmission from mother to child. now, though, pmtct is recognized as a comprehensive set of interventions requiring capable health workers. it starts with testing pregnant women for hiv, preferably during their first antenatal visit. when giving the test result, health care workers should provide good counselling, including information about pmtct options. the health system should ensure that hiv-positive women receive the pmtct services that they choose and should provide postnatal care. all along the timeline from finding out their serostatus to getting treatment for hivrelated problems, women and their children should be followed closely. the need for comprehensive and longterm care for hiv-infected women has become a challenge for health systems, particularly where lack of coordination among different facilities is common [1, 2] . viet nam's hiv epidemic is still in a concentrated phase, with the highest seroprevalence among high-risk key populations, including injecting drug users (idu), female sex workers (fsw) and men who have sex with men (msm). hanoi is one of 10 provinces/cities reported to have the highest number of hiv infections per 100 000 inhabitants. after the first case of aids was identified, in 1992, the hiv epidemic in hanoi increased sharply by 1994. the capital has 12 628 persons living with hiv/aids (plhiv), mostly idu from poor families. currently, there are 3623 aids patients and 2081 who had died of aids in the city. although hiv is predominantly concentrated among idu and fsw, it is gradually spreading among the general population. in 2007, hiv prevalence among pregnant women attending antenatal care (anc) clinics in the hanoi was 0.34% [3] . hanoi was selected as the study site because comprehensive pmtct care is theoretically available there. hanoi had 45 hospitals, 290 surgeries and five international hospitals. the national hospitals in hanoi serve as referral centres for the northern half of viet nam. hiv testing and counselling for pregnant women are offered at health facilities at district or higher level, but often only after the 28 th week of gestation. hiv-positive women are referred to provincial or national hospitals for arv prophylaxis, delivery and postnatal care. hanoi health services have sufficient supplies of prophylactic arv to meet the demand. antiretroviral therapy for adults is available at district level or higher. hiv-exposed infants are offered polymerase chain reaction (pcr) testing and free infant feeding formula for at least six months, while free paediatric arv is available for children three years of age or older. the extensive health care system in hanoi reaches the commune level, but multisectoral and cross-programme collaboration to link the pillars of the world health organization's (who) comprehensive approach to pmtct are weak [4] . for example, there is little collaboration between the programmes for hiv/aids and family planning. our previous work suggested that a large number of hiv-infected pregnant women remain undetected by the health system [5] . in addition, a number of barriers result in failure to access pmtct during pregnancy and delivery [6] [7] [8] [9] [10] [11] . among the weak points identified were that hiv-infected women received inadequate information about postnatal care, but even when they had knowledge, many expressed fear of stigma and discrimination that reduced their access to care; hiv testing is not available via health services at commune level, where many pregnant women go for care and delivery; and women feared lack of confidentiality of hiv test results [4, 12] . our previous studies on the experiences and views of women about the provision of pmtct in hanoi included criticisms about the quality of services provided by health workers [4] . other studies in asia found that health workers were unwilling to provide appropriate care for hivpositive pregnant women, often because of their own fear or lack of knowledge, or because of high workloads and lack of staff [13, 14] . inadequate health care delivery may be caused by a variety of factors, but we need to identify the main issues before planning interventions to strengthen it. we report here the health workers' perceptions on the factors that lead to their failure to give good quality pmtct. the findings should inform the development of a more effective programme for the fourth prong of the who-recommended comprehensive pmtct programme. sampling of study sites was based on availability of services in hanoi and their level and function in the health care system. all hospitals in hanoi that provide arv, pmtct and opportunistic infections (oi) treatment were selected, including all health facilities providing hiv testing. additionally, all health facilities in one high-prevalence and well-resourced district were selected. in that district, interviews were carried out at all levels of the health system involved in pmtct: district health centre, district maternity ward, district committee for family planning and mother and child health, and preventive medicine centres, including hiv testing sites. the research team also visited commune health stations in the district. details are presented in table 1 . selection of respondents was based on their function, position and experience in the development or implementation of pmtct policies/programmes. interviewees were first screened to check that they had appropriate positions and at least one year of experience with pmtct, so that they could provide insightful information. they included doctors, nurses, midwives, counsellors, laboratory technicians and programme managers. detailed information on interviewees is presented in table 2 . we conducted semistructured interviews with these 53 health workers about their experience in implementation of services for pmtct, their point of view about users of their services and their perception about the challenges they faced in providing good pmtct services in their health facility. in addition, unstructured observations were made in nine health facilities, in waiting rooms, counselling rooms, anc examination wards, delivery wards, postnatal wards, outpatient and inpatient clinics for arv and oi facilities, and laboratories. the interviewers were four trained public health and social science researchers. institutional ethical approval was obtained from the scientific committee of hanoi medical university and written informed consent was obtained from all interviewees, who were invited to participate voluntarily. the interviews were conducted privately and anonymously. a code book was developed focusing on key findings and terminologies. the transcripts of the semistructured interviews were coded, entered and analysed by means of n-vivo software. many hospital staff explained their reasons for not being able to provide good care; the most frequently heard was high workload. observation confirmed that the national and provincial hospitals' anc caseload was high: the wards were always crowded. one of the main obstetrics hospitals provides anc to between 200 and 400 pregnant women daily. none of the health facilities offering pmtct services had recruited additional staff to provide counselling. however, the workload in anc facilities at district and commune level was not so high, according to the respondents. "there are only very few health staff with only basic information on counselling in this hospital. poor knowledge and skill is common problem here." counsellor, anc provincial hospital especially at district or lower level, knowledge is limited regarding arv prophylaxis and on follow-up care, such as continuing replacement feeding (rf) supplies, infant testing and services for hiv-infected mothers and exposed infants. consequently, health workers cannot provide adequate counselling on these issues before women are discharged. "what i can do is to provide information about her hiv test result. i know that there is a medication to prevent transmission of hiv from mother to child, but i don't know exactly. our common practice is to refer her to provincial hospital." midwife, anc commune level the staff in most health facilities reported having had limited training on pmtct in general and on counselling in particular; that they apply knowledge and skills gained from observing colleagues conducting counselling sessions; and that refresher courses are rare. the duration of the training varied from two days to two weeks. after the training, counselling is added to their regular anc or maternity work. "what we do now is only to inform. [counsellors] lack knowledge. if they will be trained, who will train them?" manager, anc provincial hospital "we counsel from our experience. to me, our counselling may be not complete. we don't even know what might be our shortcomings". nurse, anc commune level an important point of the comprehensive pmtct approach is that hiv-infected women should be provided with several different services -such as arv prophylaxis, formula, counselling and hiv testing for exposed children -provided by different facilities. however, there are no inter-or intra-hospital linkages to make the pmtct comprehensive. for example, family planning services at a national obstetric hospital are not linked to other departments, including the infectious disease department that provides anc and delivery services for hiv-infected women. women are seldom referred to arv sites for clinical staging or immunological assessment. referral to postnatal care and social support for both mothers and children is not available at the hospital exit point. "there is no linkage with obstetric hospitals; they never directly inform us. it is very difficult to know which hiv+ patient or child has been referred to this paediatric hospital for follow-up by what hospital. we don't treat the mothers here. we only provide counselling to them. support groups? there are some but we don't know where they are." doctor, paediatric arv national hospital many health workers stated that their task is to provide services available in their own facilities but not services provided by other departments or facilities. lack of medications was another reason given for not being able to provide services for hiv-infected women. in many health care facilities, the arv was not consistently available. often even single-dose nevirapine (nvp) was lacking, for women who were tested only at the time of labour. even in the two pmtct sites in the city, shortage of arv for adults was observed to happen every few weeks. one problem with the nvp for children was that it is provided as a large bottle (200 ml) of syrup. once it has been opened, it cannot be kept for long, but very few hivexposed children were identified each day. that means that each bottle was not fully used, and that later, drugs were lacking when supplies ran out. "in some periods, there is a shortage of sd-nvp for pmtct. we could not do anything in that situation. in practice, the nvp syrup for children is very inconvenient to use. on one day, we have no more than two children to treat; we have to open one bottle for them and the rest of the syrup is unused. the syrup quickly runs out, and then we don't have medication for another child." manager, anc provincial hospital "we also counsel them to use condoms. if someone asks me what they should do to avoid unwanted pregnancy then i tell them. but i do not have condoms to give them for free for family planning." counsellor, anc district level another issue is that there are no national guidelines on counselling and testing. observation showed that facilities at provincial and national levels had counselling and pmtct guidelines and protocols developed by the projects that support those facilities, but most facilities at district or lower level do not have guidelines or even access to them. moreover, health workers at all levels often complained about the lack of attention to the needs of health workers when they have to work in a high-risk environment. "among 1,000 health workers, how many want to provide care and treatment for hiv-infected patients? there is no good compensation regimen to support staff working with hiv-infected patients. there is no benefit to save the life of patients in the late period, so how could we be enthusiastic?" doctor, paediatric arv national hospital "we receive extra pay for providing treatment for hivinfected people. but it is just for one health staff while all [12] staff in my department provide service. we have to share among us." doctor, adult arv district hospital fear of infection many respondents admitted that they were afraid of hiv transmission from patients, either because they feared being injured by the patients or through an occupational accident, because they lack protective equipment. observation at adult and pediatric arv sites supported this finding. health workers confront their fear of infectionto reduce both fear and risk of infection, health workers often find ways to protect themselves, either by trying to identify which patients might be infected with hiv, by using protective equipment, or by avoid exposure to patients as much as possible. "it is easier for us to prevent transmission if we know who among the patients is infected with hiv." midwife, anc provincial hospital observation revealed good practice of precaution when health workers assisted deliveries for hiv-positive women, but not always for hiv-negative women. all health workers said they knew how to protect themselves against occupational exposure to hiv and did so very carefully if they knew who was infected with hiv. "staff wear protective uniforms, maintain all hygiene practices and disinfection procedure on all equipment used." nurse, adult arv district level however, even if health workers want to protect themselves by using protective equipment, not all health facilities can provide these means for them. "health workers do not have enough protective clothes. we have to use cloth coats and short gloves when assisting deliveries for hiv-infected pregnant women. so we often wear a raincoat on top of the cloth coat." nurse, anc provincial hospital although hospital managers reported that occupational exposure is rare, among the study population we found five health workers who claimed to have had an exposure to blood that they thought might have put them at risk for hiv infection, either because of needle-sticks or blood that went to their eyes. they all informed us that arv prophylaxis for prevention of occupational exposure is free of charge at an arv site at district hospital. but only two of them took medication because the others turned out not to need prophylaxis after closer assessment of their exposure. prevention of hiv transmission became a good excuse for health workers to avoid taking care of hiv-infected women, or if they had to provide care, to isolate the hivinfected women for easier control and management. "we offer counselling, family planning, nutrition, delivery and care after delivery at home, vaccinations for tetanus. we offer this for normal pregnancies including those with hepatitis b. women who have high risk with hiv are referred. it is not our business." doctor, anc site "not everyone understands thoroughly about stigma and dread. the more they know, the more they fear and they try to push responsibility to others." manager, pmtct site many hospital managers emphasized that although the number of hiv-infected women attending their hospitals has been increasing, the number may still account for quite a small number of women in community. observation at all the health facilities revealed that hivinfected women were often placed in a separate room or area. even at a high-level hospital, where two or three patients often had to share one bed, there was still an empty bed in the room for hiv-infected patients. the manager in an anc facility explained: "hiv is an infectious disease, more severe than hepatitis b. therefore patients should be controlled carefully to avoid transmission to other patients and staff." not only fear of hiv infection influenced the quality of care provided to hiv-positive women. some of the weaknesses in providing service were related to the views of health workers regarding hiv-infected people, who are often seen as drug users or sex workers, or as having a "strange appearance". the real or expected behaviour of such people also induces fear and other negative emotional responses in the health workers. "they often have tattoos and never dress well. there are spots on their arms. easy to recognize their bluish black lips." doctor, anc national hospital "they inject in our department. how can we have a good attitude toward them?" nurse, anc national hospital health workers did admit that not all hiv-infected people behaved badly towards them. however, a few bad experiences could give all staff a negative attitude about hivinfected people in general. "almost all hiv-infected patients cannot be trusted. for instance, when they know they have opportunity to have care and support, they often find ways to get as much as possible. or if they want to leave the hospital, they often lie to the nurse that the doctor already agreed. or when they have to pay the hospital fee, they often tell the lie that they will pay tomorrow but after that they disappear." nurse, paediatric arv national hospital "when you have to work with them [hiv-infected patients], you will see the difficulties. it's already hard to gain trust from normal patients. now we have to serve the very scoundrel social class and at the same time, we receive very low salary. we have to provide service because it's our responsibility but we are not happy because they [hiv-infected patients] are drug users, they are very rude. my experience shows that health workers should not be too good to hiv patients." doctor, arv district level some of these attitudes are based on real experiences, but many are also based on prejudicial expectations, and women wishing to access pmtct services will be victims of that stigma, too. most of the health workers agreed that the quality of care could be improved by several interventions addressing both individual and structural issues. reducing workload and providing better compensation for working with risks were mentioned by almost all health workers at provincial and national level as important solutions. in the interviews, hospital managers suggested that it is very difficult to hire new staff because of the limited budget allocated from the government. a better solution would be to rearrange the services in a more logical structure, for example, to replace individual pretest counselling (which is often not offered anyway) by group counselling, to use peer counsellors to provide counselling and follow-up of care (reducing the burden for health workers and improving the quality of counselling) and to improve the quality of services at a lower level to reduce the burden on the higher levels. "if we have more equipment, we can deal with our workload with even few staff. for instance, if we are provided a video, leaflets on hiv and pmtct, and a room with table and chairs, we can do group counselling for pregnant women." manager, anc national hospital "i have seen in a hospital in thailand that peer counsellors can work in hospital to help health workers doing some administration work, providing counselling, making appointments. we may need to think about how to apply their experience." manager, arv district level however, many doctors and nurses are still unconvinced about the involvement of peer counsellors in the health service, because they feel that peer counsellors do not have enough capacity and the appropriate attitude to do this work, or even bring the potential for crime and corruption into the hospital. "you can find good peer counsellors in other countries but not in viet nam. they have low education. they may become drug dealers or whatever, we don't know." doctor, paediatric arv national hospital another possible intervention is training and updating information for health workers. midwives and nurses said they needed to improve their basic knowledge on hiv/ aids and pmtct because they received less training than doctors, while doctors preferred to have advanced pmtct training. all of them asked to be updated routinely on pmtct. "although training has often been provided to doctors but not nurses, in fact training should be conducted more often for both of them." doctor, paediatric arv national hospital regarding the system, hospital managers admitted that much needs to be done to improve the quality of service -for example, improving arv procurement, developing detailed pmtct guidelines taking into consideration the staff's high workload, increasing availability of high technology equipment and providing sufficient protective equipment for health workers. "although arv is sufficient for all hiv-infected women in hanoi, in the provincial hospital, there are some periods when they lack medicines. the provincial hospital should make a plan on how much medicine they need. the procurement facility should make administrative procedure short and easy for hospitals to get the medicines." manager, anc national hospital "we have implemented pmtct programme for more than five years without a pmtct guideline to instruct us. i think a pmtct guideline should be developed with involvement of all planners and implementers." manager, anc provincial hospital improvement of the referral system is seen as an important task that needs to be addressed in the near future. hospital managers propose to have regular meetings of the health network that provides different services for hiv-infected people. "when there are not a lot of patients, i think it's appropriate to divide tasks among different hospitals. but we also need to link all hospitals. in practice, the linkage is loose: that makes difficulties for patient access to services. it is due to lack of information about services provided by other places, lack of active coordination to link between different services, people are too busy to think about other services besides the treatment that we can provide. some health workers are not aware of the need to refer patients, or if they do, patients don't want to go and disclose their positive status in other hospitals, or may not go even because of lack of referral forms." manager, arv district hospital some health workers also proposed that all services should be offered at one point for better coordination. "now we have global fund project providing arv at district health centre. why don't we provide pmtct and paediatric treatment at the same facility? i have also heard that there are some self-help groups working at district level that can provide further support for hiv-infected people. the ministry of health should think about how to make one facility able to provide all services. that could help to avoid loss to follow-up, which is common among hiv-infected people." manager, anc national hospital the health workers did perceive not only problems but also solutions and seemed to have some motivation to solve the problems and to provide better services for hivpositive women seeking pmtct services. studies in viet nam have demonstrated that both hivinfected and non-infected women had many criticisms of anc and delivery services, about provision of information about pmtct and counselling, and about stigma displayed by health care workers [3, 15] . at present, the health service has not yet addressed this gap. access to comprehensive pmtct is still very poor, even in such a well-resourced setting as hanoi [16] . because the health care workers are subjected to many accusations about their performance in this context, this study was undertaken to find out their opinion regarding these gaps and weaknesses. health care workers usually want to do a good job and provide good care for patients. however, they are often unable to provide as good care as they would like, particularly in facilities with an overload of clients [17, 18] . results of a survey among women who had been pregnant in hanoi revealed that they attended and paid for anc services at higher-level health facilities (provincial and district hospitals) rather than go to commune health stations where anc services are free of charge [4] . high workloads were observed at provincial hospitals, while district hospitals and commune health stations appeared to have more time and space for pregnant women. studies of anc services in vietnam have identified a number of weaknesses. staff shortages and staff motivation can significantly affect the quality of service, especially for counselling, which takes a long time to do well [12, [19] [20] [21] [22] . in the case of hiv and pmtct, additional reasons for the unsatisfying performance included inadequate knowledge and skills due to lack of training, medicines, protective equipment and practice guidelines. health care workers had poor knowledge about hiv and about prevention of occupational exposure to hiv, especially at district or lower levels. even in the expected sources of expertise, the medical schools around vietnam, 70% of medical students and 48% of lecturers recapped used needles by hand, while two thirds always cleaned their hands with antiseptic after contact with blood. sixty percent of medical students and 37% of lecturers had been exposed directly to blood or body fluids and were worried about hiv transmission. however, 15% of the respondents recommended antibiotics for post-exposure prophylaxis, while one third proposed arv prophylaxis [23] . these results reveal a disturbing lack of knowledge and awareness about hiv, even among the medical profession. lack of practical needs can become an excuse for health care workers to justify their fear of hiv infection and their reluctance to provide good services for hiv-infected people [7, 24] . as the hiv epidemic has evolved in viet nam, both governments and international donors have given priority to prevention and surveillance activities. the main reason is that viet nam has had success up to now using surveillance and containment to control infectious diseases such as polio, sars and, more recently, avian flu. hiv/aids policy and practice also aims foremost at controlling the spread of the virus and has paid less attention to providing care and treatment to individuals already affected. in keeping with past experience in other epidemics, health staff perceived hiv-infected persons as sources of contamination, who should be isolated. health care workers are the key providers of medical care. stigma from health care workers can reduce patients' ability to manage their infection and gain access to health care [7, 25] . persons infected with hiv are often grouped with drug users and sex workers as marginalized, discriminated-against and criminalized elements in society, also by health workers. stigma towards hiv-infected persons has been documented in health care settings all over the world [26, 27] . showing a negative attitude during counselling and provision of care, treating in a separate area and avoidance of providing service at all are perceived as enacted stigma by hiv-infected patients. on the other hand, from the health workers' point of view, these actions result from a combination of factors: high workload and personal priority-setting influenced by fear of being infected as well as distrust towards almost all hiv-infected patients because of the association with antisocial behaviors [28, 29] . when health care workers have fear and lack knowledge, they can find reasons not to focus their attention on the hiv-positive patients and give those reasons for not providing service as they think/ know they should. moreover, health care workers are not only a source of stigma from the perspective of hivinfected people, but can also become recipients of stigma from colleagues and family because of their exposure to hiv-infected patients. the best and most feasible solution is to provide training and reference materials for health workers, to inform them about hiv transmission routes, universal precautions and post-exposure prophylaxis. reduction of workload would also be important [24] . the results of this study also suggest that the quality of pmtct service could be enhanced by improving communication and other skills of health workers and providing them with greater support and motivation. a positive atmosphere in the health facilities should be promoted by normalizing hiv-related services, and undertaking behaviour-change communication campaigns aimed at staff of the health facilities. feedback from service users could be used as one way to evaluate the quality of service. in addition, health facilities should make arv continuously available. the health workers' fear could also be reduced by ensuring that they have and use the protective clothing they need to maintain good hospital hygiene. it will be more difficult to address the issues of fear and stigma towards drug users and sex workers. self-help groups of both drug-using and non-drug-using women in hanoi and other countries were able to improve the relationship and communication between health care staff and patients/clients; peer counsellors and a buddy system led to improvements in the health care provided to and accessed by the women [3, 30, 31] . successful examples of this intervention have been documented among clubs for tuberculosis patients [32] , alcoholics, cancer patients and patients with chronic illnesses and mental problems [30] . continuous care and support for hiv-infected mothers after delivery was often not seen as a need to be addressed [12, 33] . in practice, the fragmentation of the health care system into specialized vertical pillars including a vertical programme for hiv/aids is a major obstacle to providing a continuum of care. medical treatment, including arv provision and medications for oi, is increasingly available but is often not accessible to plhiv because of a weak referral system and social stigma [3, 7] . the vertical organization of the health care system and the contradictory mandates between sectors obstruct the effective collaboration and referral between different services that the women and their families need. a lack of multisectoral collaboration is a barrier to effective information exchange for patients between national staff in different facilities [34] . providing information about topics such as abortion, clean needle exchange programmes and condoms is also politically sensitive in voluntary counselling and testing sites. the study suggests that information on available services should be made known to health workers. frequent meetings between different service sites should be organized, with the involvement of high-level health staff that can make decisions, to update information on services available and provide feedback on the quality of the referral system. development of a practical strategy is needed to strengthen and adapt the referral system to meet the needs of patients. for example, comprehensive services for hivinfected people should be provided at one service site at district level [2]. as information was collected by means of qualitative methods, the identified factors that lead to their failure to give good quality pmtct could not be quantified and be representative for the health care worker population in hanoi. in conclusion, the results of this study show that health care workers also face a number of barriers in their efforts to provide good pmtct services at different levels of the health services in hanoi. these include a high workload, a lack of equipment and materials, lack of training and skills updating, the common fear of the type of patients who may present with hiv, and little support to improve their performance. these weak points can be addressed by a number of feasible interventions. results of the study contribute to the picture of the pmtct programme not only in low-prevalence settings, as in asian countries, but also in high-prevalence settings with weak health care systems, such as african countries, and may require different interventions to improve the quality of the service. world health organization: prevention of mother-to-child transmission (pmtct) vietnam administration for aids control: result of the hiv sentinel surveillance in 2007 medical committee of the netherlands vietnam: towards a continuum of care in prevention of mother-to-child transmission programs: mid-term findings of rapid assessment action-research in north vietnam amsterdam: university of amsterdam: amsterdam school of social science a hidden epidemic among women in vietnam strategic approaches to the prevention of hiv infection in infants: report of a who meeting the stigmatization of the pregnant hiv infected women is the major factor of mtct [abstract promoting the positive: responses to stigma and discrimination in southeast asia women's reasons for not participating in follow up visits before starting short course antiretroviral prophylaxis for prevention of mother to child transmission of hiv: qualitative interview study hiv-related knowledge, attitude, perceived benefits, and risks of hiv testing among pregnant women in rural southern india barriers and opportunities to increase women's choices in entering pmtct in hanoi prevention of mother-to-child transmission of hiv in asia: practical guidance for programs washington: the linkages project rapid assessment of the pmtct program hanoi: vietnam ministry of health prevention of mother-tochild transmission of hiv in thailand: physicians' attitudes on zidovudine use, pregnancy termination, and willingness to provide care understanding hiv and aids-related stigma and discrimination hanoi: isds mtct training programme for village health nurses (vhn) in namakkal district identifying factors for job motivation of rural health workers in north viet nam improving motivation among primary health care workers in tanzania: a health worker perspective predictors of retention among hiv/hemophilia health care professionals rural population and health: baseline survey report: provision and utilization of reproductive health care services in seven unfpa-supported provinces in the 7th country programme hanoi: moh improving health worker performance: in search of promising practices netherlands: royal tropical institute evaluation of united nations-supported pilot projects for the prevention of mother-to-child transmission of hiv life gap project, cdc vietnam: an assessment of training needs in hiv occupational exposure in eight medical universities in vietnam hanoi: hmu managing aids stigma critical delays in hiv testing and care: the potential role of stigma interventions to reduce hiv/aids stigma: what have we learned? new orleans: horizons project the consequences of a positive prenatal hiv antibody test for women health care workers and hiv/aids: a critical review of the literature mandel js: hiv/aids related attitudes and practices of hospitalbased health workers in kampala a review of research on the effectiveness of self-help mutual aids groups empowering the people: development of an hiv peer education model for low literacy rural communities in india community tuberculosis care through 'tb clubs' in rural north ethiopia high acceptability of voluntary counselling and hiv-testing but unacceptable loss to follow up in a prevention of mother-to-child hiv transmission programme in rural malawi: scaling-up requires a different way of acting provision of care following prevention of mother-to-child hiv transmission services in resource-limited settings the authors would like to thank the health care workers at the following hospitals in hanoi, who enthusiastically participated in our research: dong the authors declare that they have no competing interests. tan, ah, pw, po and ypn devised the concept protocol. tan, ypn, pw and po participated in the data collection. tan, pw and ah analysed the data. tan, po, ah, pw and ypn drafted the manuscript. all authors read and approved the final manuscript. key: cord-016075-ind62t53 authors: hwang, stephen w.; dunn, james r. title: homeless people date: 2005 journal: handbook of urban health doi: 10.1007/0-387-25822-1_2 sha: doc_id: 16075 cord_uid: ind62t53 nan environment that warrant scrutiny. for example, both questions will lead to a consideration of the availability of low-cost housing and the ability of the health care system to care for patients with severe mental illness. however, the distinction between these two questions is important as they distinguish factors associated with the likelihood of being homeless due to health reasons versus the likelihood of consequences given homelessness. at another level, it is also important to distinguish if outcomes and their associated factors vary between cities, both in terms of the structural factors that generate homelessness and (given homelessness) health of those who are homeless. these questions frame the issue of the impact of the urban environment on the health of disadvantaged populations. in the course of such discussions, disagreement often arises as to whether homelessness should be considered primarily the consequence of individual vulnerabilities and failings, or the result of structural inequities in the social, economic, housing, and health care systems. rather than creating an either/or distinction, we will approach homelessness as the result of a complex interaction between individual vulnerabilities and structural forces in the urban environment. in most cases, the relative importance of these factors in determining the health of homeless people and the prevalence of homelessness remains the subject of ongoing debate. the burden of illness and disease is extremely high among homeless people (levy and o'connell, 2004) . however, any consideration of the common health problems of homeless people must first recognize the large degree of heterogeneity among people who are homeless. among street youth, single men, single women, and mothers with children, the patterns of illness differ notably. adolescents suffer from high rates of suicide attempts, sexually transmitted diseases, and pregnancy (greene and ringwalt, 1996; greene and ringwalt, 1998; greene, et al., 1999; feldmann and middleman, 2003) . female heads of homeless families tend to have far fewer health problems than single homeless women, although their health is poorer than their counterparts in the housed general population (robertson and winkleby, 1996) . homeless single men have a higher prevalence of alcohol abuse and drug abuse, whereas single women have a higher prevalence of serious mental illness (fischer and breakey, 1991) . health status also tends to be correlated with a person's history of homelessness. individuals with severe mental illness, substance abuse, and medical conditions are overrepresented among the chronically homeless, whereas those who are homeless for a transient period lasting only a few weeks or months are more likely to be relatively healthy (kuhn and culhane, 1998) . although chronically homeless people make up only about 10% of all individuals who experience homelessness in a given year, they account for a disproportionately large share of the demand for shelter beds and health care services for homeless people (burt, 2001) . in addition, the public's perception of homeless people often reflects a stereotyped image of this highly visible subgroup. cross-national comparisons of disease patterns among homeless people reveal the strong effect of social factors within each country. among homeless men in tokyo, japan, morbidity due to alcohol dependence (but not drug use) is common, as are musculoskeletal injuries incurred doing construction work (takano, et al., 1999b) . in contrast, 60% of homeless people in amsterdam, the netherlands, suffer from drug abuse or dependence (primarily heroin), and most are chronically homeless (sleegers, 2000c ). the prevalence of serious mental illness and substance abuse is high among homeless persons. in a nationwide u.s. survey of homeless people, 39% had mental health problems, 50% had an alcohol and/or drug problem, and 23% had concurrent mental health and substance use problems (burt, 2001) . common psychiatric diagnoses among homeless people include major depression, bipolar disorder, schizophrenia, and personality disorders. a systematic review of the prevalence of schizophrenia in homeless persons found rates ranging from 4 to 16% and a weighted average of 11% in the ten methodologically strongest studies . characteristics associated with a higher prevalence of schizophrenia were younger age, female sex, and chronic homelessness. marked cross-national variation is seen in the prevalence of schizophrenia, with prevalence rates of 23-46% reported among homeless people in sydney, australia (teesson, et al., 2004) . the prevalence of substance abuse is extremely high among homeless single adults. in a study from st. louis, missouri, large increases were seen in the prevalence of drug use among homeless men and women between 1980 and 2000. in 2000, 84% of men and 58% of women had an alcohol or drug use disorder (north, et al., 2004) . in another study, about three-quarters of homeless adults met criteria for substance abuse or dependence (o'toole, et al., 2004) . homelessness increases the risk of adverse health outcomes among substance abusers: in five canadian cities, the risk of a non-fatal overdose was twice as high among illicit opiate users who were homeless compared to those who were housed (fischer, et al., 2004) . homeless adolescents also have very high rates of mental health problems and substance abuse. in a study from seattle, 83% of street youths had been physically and/or sexually victimized after leaving home, and 18% met criteria for posttraumatic stress disorder (stewart, et al., 2004) . across the u.s., 55% of street youth and 34% of shelter youth had used illicit drugs other than marijuana since leaving home, in comparison to 13% of youth who had never been runaway or homeless (greene, et al., 1997) . street youth use a wide range of drugs, including hallucinogens, amphetamines, sedative/tranquilizers, inhalants, cocaine, and opiates. unfortunately, the initiation of injection drug use is quite common, with an incidence rate of 8.2 per 100 person-years among street youth in montreal (roy, et al., 2003) . infectious diseases are a common cause of health problems in homeless people (raoult, et al., 2001) . the most serious of these infections include tuberculosis (tb), human immunodeficiency virus (hiv) infection, viral hepatitis, and other sexually transmitted infections. outbreaks of tb among homeless people have been reported frequently, especially in individuals co-infected with hiv (barnes, et al., 1999; mcelroy, et al., 2003; morrow, et al., 2003) . the incidence of active tb in a cohort of homeless people in san francisco between 1992 to 1996 was 270 per 100,000, or 40 times higher than that seen in the u.s. general population in 1998 (moss, et al., 2000) . homeless people with tb require more hospital-based care than non-homeless people with tb, resulting in average hospital costs that are higher by $2,000 per patient. (marks, et al., 2000) contact tracing in the homeless population is difficult, and in one study only 44% of identified contacts completed treatment for latent tb infection (yun, et al., 2003) . among street youth, latent tuberculosis is more common than in the general population, but probably less prevalent than among homeless adults. in a study conducted in sydney, australia, 9% of homeless young people aged 12-25 years had latent tb infection (kang, et al., 2000) . homeless people are at increased risk of hiv infection. data from an older u.s. survey conducted from 1989 to 1992 in 14 cities found median hiv seroprevalence rates of 4.0% in adult men, 1.8% in adult women, and 2.3% in youths (allen, et al., 1994) . in more recent studies, hiv seroprevalence was 10.5% among homeless and marginally housed adults in san francisco in 1996, a rate five times higher than in san francisco generally (robertson, et al., 2004) . hiv infection was present in 1.8% of homeless veterans admitted to residential programs from 1995-2000 (cheung, et al., 2002) . female street youth and young homeless women who are involved in prostitution are at increased risk of hiv infection, due to both injection drug use and risky sexual behaviors (weber, et al., 2002) . in one study of homeless adolescents, the hiv infection rate was alarmingly high at 16% (beech, et al., 2003) . among substance users, homelessness is associated with higher rates of hiv seroprevalence (surratt and inciardi, 2004; smereck and hockman, 1998) . among hivinfected persons, those who are unstably housed (homeless or temporarily staying with friends or family) are less likely to receive adequate health care than those who are stably housed (smith, et al., 2000) . homeless people are at increased risk of viral hepatitis, primarily due to high rates of injection drug use. infection with hepatitis c was found in 22% of homeless men in los angeles , 32% of individuals using a mobile medical van in new york city (rosenblum, et al., 2001) , and 27% of homeless persons in oxford, england (sherriff and mayon-white, 2003) . in a veterans affairs population, the prevalence of anti-hepatitis c virus antibody was 41.7% and the prevalence of hepatitis b surface antigen was 1.2% (cheung, et al., 2002) . among street youth, the prevalence of these markers of infection was also high: 12.6% and 1.6%, respectively, in montreal (roy, et al., 1999; and 5.0% and 3.6%, respectively, in a northwestern u.s. city (noell, et al., 2001b) . sexually transmitted diseases (stds) are a particularly serious problem among street youth. in a longitudinal study of homeless adolescents, the annual incidence of chlamydia trachomatis infection was 12.1% in females and 7.4% in males; the annual incidence of herpes simplex virus type 2 was 25.4% in females and 11.7% in males. (noell, et al., 2001) a study of street youth and sex workers in quebec city, canada found that 13% of women less than 20 years old were infected with chlamydia trachomatis and 1.7% had neisseria gonorrhoeae (poulin, et al., 2001) . newer urine-based screening tests make it easier to screen homeless youth for stds in outreach settings (van leeuwen, et al., 2002) . common chronic diseases, including hypertension, diabetes, chronic obstructive pulmonary disease (copd), seizures, and musculoskeletal disorders, are often undiagnosed or inadequately treated in homeless adults. relatively little research has focused on these medical conditions in the homeless population. the prevalence of hypertension was higher among homeless clinic patients than among nonhomeless patients at an inner-city primary care clinic (65% vs. 52%) (szerlip and szerlip, 2002) . the prevalence of diabetes is similar in homeless and non-homeless individuals, but homeless people with diabetes face a number of serious barriers to appropriate disease management, including lack of access to a suitable diet and dif-ficulties coordinating medication administration with meal times (hwang and bugeja, 2000b) . glycemic control was found to be inadequate in 44% of homeless diabetics in toronto (hwang and bugeja, 2000b) . smoking rates are extremely high (about 70%) among homeless people (connor, et al., 2002) . as a result, copd is a common health problem among older adults. in a study of shelter residents in san francisco, the prevalence of copd based on spirometry was 15%, or more than twice the prevalence in the general population (snyder and eisner, 2004) . smoking also contributes to the high risk of cancer, especially among homeless single men. in a study from scotland that adjusted for age and socioeconomic deprivation, the incidence of cancer of the oral cavity and pharynx, larynx, esophagus, and lung in homeless men was 139%, 87%, 61%, and 23% higher than expected, respectively (lamont, et al., 1997) . homeless people are also less likely to receive recommended cancer screening than the general population: among homeless women age 40 and over in los angeles county, only 55% had undergone a pap smear and only 32% had undergone a mammogram within the last year (chau, et al., 2002) . thus, interventions such as smoking cessation treatment and routine preventive health services may provide significant benefit. although it is not surprising that homeless people with mental illness often receive inadequate care for medical comorbidities, the adequacy of care differs according to type of mental illness. homeless people with schizophrenia receive less detailed physical examinations, fewer primary care visits, and less preventive health services than homeless people with major depression . while it is unknown if these differences are due to patient factors, provider factors, or both, careful attention clearly needs to be paid to the physical health needs of homeless people with psychoses. trauma and injuries are significant hazards associated with life on the street (staats, et al., 2002) . in a sample of homeless and marginally housed people in san francisco, 32% of the women and 27% of the men had been sexually or physically assaulted in the last year (kushel, et al., 2003) . among women, being homeless (compared to being marginally housed) was associated with a more than 3-fold increase in the risk of sexual assault. in sydney, australia, 58% of shelter residents reported experiencing a serious physical assault in their lifetime, and half of the women reported having been raped (buhrich, et al., 2000) . among homeless youth in los angeles, reported exposure to violence was found to be equally high among males and females (kipke, et al., 1997) . foot problems are very common among homeless adults due to prolonged standing, long-term exposure to cold and damp, ill-fitting footwear, and inadequate foot hygiene. problems can range in severity from mild blisters and fungal infections to debilitating chronic venous stasis ulcers, cellulitis, diabetic foot infections, and frostbite. other common skin problems include sunburn and bites due to infestations by head lice, body lice, scabies, or bedbugs (stratigos and katsambas, 2003) . the prevalence of serious dermatologic conditions, while probably quite high among street-dwellers, appears to be relatively low among homeless people living in shelters that provide adequate clothing, laundry facilities, bathing facilities, and medical care. in a study of men staying at such a shelter in boston, the majority of individuals had relatively normal findings on skin examinations (stratigos, et al., 1999) . dental problems are an extremely prevalent and troubling but often-neglected problem for many homeless people. common conditions include advanced caries, periodontal disease, and ill-fitting or missing dentures. these problems may be related to poverty, lack of access to dental care, and substance use, rather than homelessness per se. in a study comparing homeless and domiciled veterans in veterans affairs rehabilitation programs for substance abusers, the two groups had similarly poor oral health (gibson, et al., 2003) . given the high prevalence of illness among homeless people and the adverse health effects of homelessness itself, it is not surprising that homeless people have very high mortality rates. men using homeless shelters are 2 to 8 times more likely to die than age-matched men in the general population (barrow, et al., 1999; hwang, 2000a) . homeless women 18-44 years of age have mortality rates that are 5 to 31 times higher than in the general population (cheung and hwang, 2004) . common causes of death among homeless people under the age of 45 are unintentional injuries, drug overdoses, aids, suicide, and homicide (hwang, et al., 1997; . in a longitudinal cohort study of street youth in montreal, the standardized mortality ratio was 11.4; hiv infection, daily alcohol use in the last month, homelessness in the last 6 months, drug injection in the last 6 months, and male sex were independent predictors of mortality (roy, et al., 2004) . among homeless women, major barriers to contraception include cost, fear of side effects or potential health risks, and the partner's dislike of contraception . pregnancy is particularly common among homeless adolescents. in a u.s. sur vey of runaway females age 14-17 years, 12% of streetdwelling youths and 10% of those residing in shelters were currently pregnant (greene and ringwalt, 1998) . in a group of pregnant homeless women, the risk of low birth weight (less than 2,500 gm) was 17%, compared to the national average of 6% (stein, et al., 2000) . lack of prenatal care and severity of homelessness (homelessness in the first trimester of pregnancy, number of times homeless, and percentage of life spent homeless) were independent risk factors for low birth weight. the health of children in homeless families has been the focus of relatively little research. some but not all studies of these children have found an increased prevalence of behavioral and mental health problems compared to children in housed low-income families (bassuk, et al., 1997; vostanis, et al., 1998) . infectious diseases are a significant concern in these children (ligon, 2000) . up to 40% of children in homeless families in new york city suffer from asthma, a rate six times higher than the national rate in children (mclean, et al., 2004c) . the medical literature has usually examined health problems from the perspective of the individual homeless person, and has given relatively little attention to the urban environment within which these health problems arise and must be ameliorated. this section addresses this gap by highlighting dimensions of the urban environment that affect, through interaction with individual vulnerabilities, the prevalence of homelessness and/or the health of homeless people. the following is not intended to be a comprehensive listing, but rather a selection of important determinants about which at least some information is available. these determinants have been grouped into categories encompassing the demographic and physical characteristics of urban centers (population and climate), their socioeconomic and service-delivery structures (income and poverty, social welfare systems, and health care systems), and their spatial and political organization (urban geography and urban governance). although these dimensions may have differential effects on the health of various subgroups of homeless people (e.g., youths, single adults, and families), these differences are not discussed in depth here. homelessness is a problem in cities across the u.s., as demonstrated by the fact that federally-funded health care for the homeless programs exist in 161 cities in all 50 states, the district of columbia, and puerto rico (health care for the homeless information resource center). there is limited information on the relationship between population size and prevalence of homelessness in different urban centers. one reason for this paucity of data is the logistical difficulty of conducting an accurate count of homeless persons, particularly those living on the street. another reason is that point-prevalence counts of the homeless population cannot be used to determine how many individuals are homeless in a city over an entire year, especially given seasonal fluctuations in the homeless population and the fact that homelessness is a transient state. counts of shelter users are particularly informative when all shelters contribute to a common administrative database, because this makes it possible to determine the total number of individuals who use shelters in a particular city over the course of a year, rather than simply the number of shelter users at a single point in time (metraux, et al., 2001) . in 1992, an estimated 1.0% of the 1.5 million residents of philadelphia and 1.2% of the 7.2 million inhabitants of new york city stayed at a homeless shelter at least once (culhane, et al., 1994) . in toronto, canada, 1.3% of the city's total population of 2.5 million used a homeless shelter during 2002. these figures are remarkably similar and strikingly high. thus, homelessness is quite common in large urban centers, although for many individuals the duration of homelessness is quite brief. in a u.s. survey of homeless people, 28% had been homeless for only 3 months or less, 26% had been homeless for 4-12 months, and 46% had been homeless for more than one year (burt, 2001) . cross-sectional counts of the number of shelter residents provide an important but somewhat less accurate picture of the homeless population. the maximum size of a city's shelter population is obviously determined by the number of available shelter beds. in a city with few shelters, this can create the illusion of a smaller homeless population than is actually the case. in addition, shelter beds may be less widely available in cities that do not experience severe cold weather in the winter. in the nine largest metropolitan areas in canada, the number of shelter beds per capita ranges more than four-fold, from 21 to 97 per 100,000 population (hwang, 2001) . the number of shelter beds per capita is not significantly correlated with population size. interestingly, the lowest number of shelter beds per capita in canada was observed in vancouver, a city with a very mild climate, and the highest figure was seen in calgary, a city with extremely cold winters. overall, this evidence suggests that episodes of homelessness are quite common among residents of major urban centers, but there is significant variation in the prevalence of homelessness across cities that does not necessarily correlate with population size. a related question is the role of migration in determining the size of the homeless population in urban centers. whereas some homeless people are migrants who were homeless before or upon their arrival in the city, others are local residents who have become homeless. in a nationwide u.s. survey, 56% of homeless people reported living in the same city where they became homeless (burt, 2001) . among the 44% of individuals who had moved from one location to another during their current episode of homelessness, the most common pattern was a net flux from urban fringes and medium-sized cities into large central cities. the most commonly cited reasons for these moves were lack of available jobs, lack of affordable housing, and eviction (burt, 2001) . climate is an interesting example of a characteristic of the urban environment that affects both the prevalence of homelessness and the health of homeless people. certain cities in warm regions may become a preferred destination for people who are homeless or at high risk for homelessness. as noted above, in cities with warmer climates, a larger proportion of the homeless population is likely to be found on the street rather than in shelters. people living on the street are more likely to be disengaged from the health care and social service systems, and typically these individuals have poorer health than shelter-dwelling homeless people (cousineau, 1997) . in colder climates, exposure to the elements has an obvious adverse impact on the health of homeless people, who face serious risks from trench foot, frostbite, and injury or death from hypothermia (tanaka and tokudome, 1991) . conversely, in hot weather, homeless people may experience severe sunburn, heat exhaustion, or heat stroke. the prevalence of severe poverty among the residents of an urban area is certainly an important factor affecting the prevalence of homelessness. poverty alone, however, does not necessarily lead to homelessness. data from nine u.s. cities demonstrate wide variation in the proportion of a city's poor residents that stays at a homeless shelter over the course of one year, ranging from a low of 1.3% to a high of 10.2% (metraux, et al., 2001) . some have argued, based on historical data, that an increase in the number of unmarried men with very low income is a particularly important explanatory factor for adult homelessness (jencks, 1994) . during the latter half of the twentieth century, the earning potential of men with limited education was greatly diminished by the decline of manufacturing jobs in urban centers (wilson, 1987; . at the same time, the availability of open-market sources of low-cost housing such as single-room occupancy hotels and rooming houses shrank steadily due to gentrification and urban renewal (hasson and ley, 1994) . in this setting, the level of government support for subsidized rental housing plays a key role in determining the availability of units that a low-income individual or family can afford; the 1980's saw a decline in this support in both the u.s. and the united kingdom (cohen, 1994) . some have suggested that income distribution, specifically the ratio of middleincome to low-income households within a given city, is an important determinant of homelessness among both single adults and families (o'flaherty, 1996) . o'flaherty argues that because the construction of new rental housing for lowincome individuals is economically unattractive, the main source of housing for poor people is deteriorating housing stock that has been vacated by middle-income people. o'flaherty theorized that cities with fewer middle-class people relative to the number of poor people have higher rents at the bottom of the market (because middle-income housing is not being "handed down" to the poor), resulting in higher rates of homelessness. members of ethnic and racial minorities are disproportionately represented in the homeless population (e.g., blacks and latinos in the u.s., and aboriginal people in canada) (burt, 2001; hwang, 2001) . the higher prevalence of poverty in these disadvantaged groups may explain this observation. however, other race-related factors in the urban environment may contribute to the excess risk of homelessness among people of color, including discrimination in the housing market and segregation of low-income minorities in neighborhoods with fewer economic opportunities than neighborhoods in which low-income whites reside. any discussion of the role that urban poverty plays in causing homelessness also raises questions about nature of the causal relationship between homelessness and poor health. poverty is consistently and strongly associated with poor health (marmot, et al., 1997) . thus, the poor health observed among homeless people may be explained in large part by the fact that they experience extreme poverty and deprivation, rather than the fact that they happen to be homeless at the present time. this is particularly likely to be the case for individuals who have only recently become homeless, and less so for the chronically homeless, who have been subjected to the adverse health effects of lack of housing for a lengthy period. to extend this concept further, homelessness is a marker for severe poverty in the urban environment, and it may be this level of poverty, rather than the negative impact of homelessness itself, that has the greatest effect on population health in urban centers. this issue is discussed further in section 4 of this chapter. social welfare systems in urban centers have a major impact on both the prevalence of homelessness and the health of homeless people. however, these systems are usually governed at the state or national level, rather than at the municipal level. wide variation is seen in the scope of social welfare programs, with more generous benefits typically seen in countries or regions that have less tolerance for high levels of income inequality and place a higher value on social cohesion (sleegers, 2000b) . for example, eligibility criteria for welfare benefits in the u.s. vary significantly from state to state. some states allow single men to collect welfare, whereas others exclude them. these policies would likely affect the risk of homelessness among low-income single men living in any city within a given state. in addition, u.s. federal funds may not be used to provide temporary aid to needy families (tanf) if an adult in the family has received assistance for more than 60 months, but individual states may elect to continue providing assistance to these families using state funds (state policy documentation project). in coming years, as families that are unable to become self-supporting begin reaching the 60-month federal time limits on benefits, their risk of becoming homeless may be greatly affected by the policies of the state in which they live. in contrast, most european union countries have extensive social welfare and public housing systems that make family homelessness less common. one area of controversy is whether the provision of cash entitlements or disability benefits has significant effects on the health of homeless people. on one hand, the health of homeless people should improve if public benefits allow them to obtain food, housing, and other essentials of life. on the other hand, increased income could be detrimental to health if the money is used to purchase alcohol or drugs. one of the few studies on this issue examined 173 homeless mentally ill veterans who applied for social security disability insurance (ssdi) or supplemental security income (ssi). the 50 individuals who were eventually awarded benefits did not differ in their past history of substance use from the 123 individuals who were eventually denied benefits. three months after the decision to award or deny benefits, the group that was awarded benefits had significantly higher average total income (by $277 per month) and higher quality of life than the group that was denied benefits. there was no evidence of increased alcohol or drug use or deterioration in psychiatric status among those who received benefits (rosenheck, et al., 2000) . most homeless people depend on their city's shelter system for housing, food, and other social services, and these shelters can therefore have a significant impact on the health of homeless people. the availability and quality of homeless shelters vary greatly. as noted previously, homeless people in cities with few shelter beds are more likely to live on the street or other places not intended for human habitation, with potentially adverse health effects. in addition, the staff at homeless shelters can play an important role in connecting homeless people to social services, job training, housing applications, and substance abuse treatment. the quantity and quality of food provided at shelters determines to a large extent the nutritional value of homeless people's diets, with potential downstream health effects (dachner and tarasuk, 2002) . finally, the physical environments at shelters range from extremely crowded, poorly ventilated, and unsanitary facilities to modern, clean, and well-run establishments. adverse shelter conditions have an impact on the transmission of tuberculosis and viral respiratory infections and the prevalence of health conditions such as skin infestations and asthma exacerbations. shelter conditions could also plausibly have an effect on mental and emotional well-being among residents. to date, however, little research has examined the effects of the physical shelter environment on the health of homeless persons, with the exception of the relationship between crowding and poor ventilation in shelters and the transmission of tuberculosis (advisory council for the elimination of tuberculosis, 1992b). the organization and financing of the urban health care system has an enormous impact on the health of homeless people, and to some extent on the prevalence of homelessness as well. in the u.s., 55% of homeless people lack health insurance, creating a significant barrier to obtaining care (kushel, et al., 2001) . these individuals are dependent on state-or city-based systems designed to provide care for the indigent. in many large urban centers in the u.s., a designated public, county, or charity hospital provides the majority of hospital-based health care for homeless people. some cities have free-care clinics or community health centers that provide ambulatory services for homeless persons as well as other low-income residents. in 161 u.s. cities, federally-funded health care for the homeless programs have established multidisciplinary teams of physicians, nurses, social workers, and outreach workers that provide care to homeless people on the street and in shelters. this limited set of health care providers is typically the only source of care available to homeless people in urban areas in the u.s., and the local funding and staffing level of these organizations is a critical determinant of access to health care. for homeless veterans, the proximity and availability of veterans health administration services is also an important factor. in countries such as canada and the united kingdom that have systems of universal health insurance, homeless people still face non-financial barriers to care. many access problems stem from the fact that a health care system designed to meet the needs of the general population may not accommodate the unique requirements of homeless people (crane and warnes, 2001; bugeja, 2000a, 2000b) . for example, the provision of universal health insurance does not necessarily result in the establishment of outreach programs for homeless people, appropriate treatment programs for homeless persons with mental illness or substance abuse, or an adequate supply of health care providers who are willing, able, and trained to work with this challenging population (buchanan, et al., 2004) . in the united kingdom, individuals must register with a general practitioner to obtain primary care, and some physicians are reluctant to accept homeless people into their practice because of their complex needs and the extra workload entailed (wood, et al., 1997) . health insurance does not protect against the fragmentation and discontinuity of care that homeless people often experience, nor does it eliminate the daily struggle to meet basic survival needs that may cause homeless people to place a lower priority on seeking health care (gelberg, et al., 1997) . inadequate access to primary health care may result in uncontrolled disease progression and frequent emergency department visits and hospitalizations (han and wells, 2003) . emergency department visits by homeless people should be seen as an indicator of high levels of unmet health needs, rather than inappropriate health care utilization (kushel, et al., 2002) . about 50% of homeless children with severe persistent asthma have had at least one emergency department visit in the last year, a finding indicative of inadequate access to health care and undertreatment of their disease (mclean, et al., 2004) . because individuals with severe mental illness who do not receive appropriate health care are at high risk of becoming homeless, the health care system can have a direct impact on the prevalence of homelessness. the role of deinstitutionalization in contributing to the problem of homelessness has been discussed extensively. beginning in the 1960's and 1970's, the advent of effective anti-psychotic medications to treat schizophrenia and an understandable desire to move people out of chronic mental hospitals, where conditions were sometimes horrendous, led to the discharge of tens of thousands of long-term psychiatric patients (dear and wolch, 1987; jencks, 1994) . the number of beds at psychiatric institutions fell precipitously. in theory, these patients were supposed to receive mental health care and social support in the community. in reality, many of these patients received little if any services and ended up swelling the ranks of the homeless population in the 1970's and 1980's. today, many decades after these events took place, "deinstitutionalization" is no longer the major cause of homelessness among people with serious mental illness. it is now uncommon for people with psychiatric disorders to have ever been institutionalized for an extended period, and any admissions tend to be quite brief. not surprisingly, individuals with severe illness, few social supports, and/or inadequate access to appropriate outpatient psychiatric care often become homeless. in a sense, homeless shelters have assumed the role that was played by chronic psychiatric hospitals fifty years ago. for these homeless people with severe mental illness, the delivery of appropriate health care is challenging but essential to improving their health and housing status. the assertive community treatment (act) model attempts to address this problem through a team of psychiatrists, nurses, and social workers who follow a small caseload of homeless mentally ill clients, seeking them out in the community to provide high-intensity mental health treatment and case management. studies have found that mentally ill homeless people receiving act spend fewer days hospitalized as a psychiatric inpatient and have somewhat greater improvement in symptoms than those receiving usual care (lehman, et al., 1997) . however, act is labor-intensive and costly, and its availability is often quite limited. the availability and type of addictive substances in the urban environment have an important effect on the prevalence of homelessness and on the health of homeless people (munoz, et al., 1998) . the advent of crack cocaine has been clearly implicated in the rise of homelessness in the u.s. in the 1980's (jencks, 1994) . in japan, alcoholism is the predominant addiction contributing to homelessness and morbidity among homeless people, whereas in the netherlands, homelessness is closely linked to chronic heroin addiction (takano, et al., 1999a; sleegers, 2000a) . access to addiction treatment is therefore a vital issue for a large proportion of homeless people. a number of treatment modalities for adults have been shown to be effective in controlled studies: admission to a post-detoxification stabilization program results in longer periods of abstinence than direct release into the shelter system (kertesz, et al., 2003) , and abstinence-contingent work therapy in a longterm residential setting has been shown to improve outcomes (milby, et al., 2000) . studies have examined the effectiveness of case management for homeless people with addictions, with mixed results (morse, 1999) . the forces underlying the urban geography of homelessness are aptly described in the seminal work of dear and wolch (dear, et al., 1987) . they examined how deinstitutionalization, rollbacks in entitlements to social assistance, and changes in the global economy in the late 1970s and early 1980s combined to create complex problems of poverty, inequality, and homelessness in north american cities that persist to this day. dear and wolch (1987) argued that these problems manifested themselves in the specific urban form of the "service-dependent ghetto," which refers to the spatial concentration in the inner city of service-dependent populations (such as people with mental illness, physical handicaps, addictions, or recent incarceration) and the organizations that assist them. while on one hand these can be characterized as areas of "urban blight," dear and wolch (1987) argued that they serve as a supportive environment and adaptive coping mechanism that can have a positive effect on the health and well-being of residents who have few other options. servicedependent ghettos are often the object of antagonism from surrounding communities. paradoxically, however, these more affluent communities often perpetuate the forces that create the service-dependent ghetto and entrench processes of inner-city decay through citizen resistance to housing and services for low-income people and exclusionary land use policies and zoning practices (dear and taylor, 1982) . in some cities, the tendency has been to isolate high-poverty urban neighborhoods rather than attempt to destroy them. davis argues that in los angeles and other cities, a conscious effort has been made to create geographic and physical barriers (such as expressways) that circumscribe poor and minority neighborhoods and cut them off from the rest of the city (davis, 1990) . this spatial isolation can further heighten the marginalization of these communities and limit residents' access to goods, services, and economic opportunities that are vital to health. since homeless people spend a great deal of their time in public spaces, the nature of these spaces can have a significant impact on their quality of life. some cities have numerous well-tended public spaces such as parks and squares that are conducive to those who wish to linger or rest, including homeless people. these spaces can serve a socially cohesive function if urban dwellers of diverse backgrounds perceive them to be safe "neutral" spaces in which to gather and socialize. in contrast, other cities have built environments that lack such public spaces and are instead dominated by privatized quasi-public spaces such as shopping malls. nonpurposeful lingering, which would be generally acceptable in a public space such as a park, is perceived as "loitering" in such places. in a relatively trivial but very specific expression of hostility toward homeless people, some cities have installed "bum-proof" benches that are designed to prevent reclining or sleeping on the seat. while these elements of the urban environment seem relatively minor, they may reflect a city's prevailing sentiment towards homeless and poor people that sets the tone of their daily existence (davis, 1990 ). homelessness is often perceived as having a negative effect on the quality of life in urban centers. some consider the visible presence of homeless people in parks, street corners, and other public spaces to be a manifestation of "urban disorder" and a barrier to the successful promotion of commerce and tourism. in response to these concerns, a number of cities have enacted by-laws against panhandling, loitering or sleeping in public places, public intoxication, or possession of shopping carts. some cities have instituted aggressive policing strategies to remove homeless people from public spaces (graser, 2000) . efforts to displace street youth and homeless people rather than offer them any meaningful help might have negative effects on health and in fact increase high-risk behaviors such as survival sex and unsafe injection drug use practices (o'grady and greene, 2003; wood, et al., 2004) . homeless people frequently interact with both police and paramedics, but they have much lower levels of trust in police than in paramedics (zakrison, et al., 2004) . by inhibiting homeless people from calling for needed emergency assistance, this distrust could result in serious harms to health. in a study of injection drug users in san francisco, 56% of those who had been present with an unconscious heroin overdose victim did not call for emergency services due to fear of police involvement (davidson, et al., 2002) . police action can also have direct adverse effects on the health through the excessive use of force (cooper, et al., 2004) . in a study in toronto, 9% of homeless people reported having been assaulted by a police officer in the last 12 months (zakrison, et al., 2004) . on a larger scale, issues of urban governance such as fiscal disparities affect all urban dwellers, but have the potential to have a particularly severe impact on homeless and poor people. fiscal disparities typically occur when an older central city with a significant number of high-poverty neighborhoods is surrounded by a ring of higher-income municipalities. the central city's primary revenue stream from property taxes is limited by a weak tax base, but at the same time the city is confronted by a high and rising demand for social services, some of which is driven by the downloading of 'unfunded mandates' by states onto central city municipalities (drier, et al., 2001) . meanwhile, the nearby ring communities have a strong property tax base and face a lower demand for social services, while at the same time its residents work in the central city and benefit from its economic activities and services (the socalled "free-rider" effect) (orfield, 1998; drier, et al., 2001) . these fiscal disparities greatly exacerbate the adverse effects of racial and economic segregation on homeless people and others living in extreme poverty in the central city. in an example of an effort to redress this problem, state legislation in minnesota, the fiscal disparities act, mandates the sharing of commercial property tax between outlying, high tax base municipalities to central city municipalities to assist in the provision of social services. enacted in 1971 by the minnesota legislature, the plan pools 40% of the increase in all communities' commercial/industrial property valuation. all cities and townships keep their pre-1971 tax bases plus 60% of the annual growth. the pool is then taxed at a uniform rate and redistributed among all local government entities. although this redresses some of the intrametropolitan disparities, it does little to reduce the payoffs of "externalizing" social problems with tools like exclusionary zoning in typically more affluent communities. moreover, the minneapolis-st. paul example depends on the existence of a strong regional-metropolitan level of governance, the met council, which although heavily studied (orfield, 1998; rusk, 1999; katz and bradley, 1999) , is still a concept that is strongly resisted by homeowners' associations, gated communities, and affluent municipalities (mckenzie, 1994; boudreau and keil, 2001 ). an alternative solution is to create cities that encompass lower and higher income areas, rather allowing them to separate into different jurisdictions. in toronto, ontario, this was effectively accomplished through the amalgamation of five contiguous cities into a single urban entity, although the amalgamation was motivated by a desire to increase operating efficiency rather than concern regarding fiscal disparities (boudreau, 2000) . does homelessness have a sizeable effect on population health? this question raises a number of complex issues. homeless people, especially those who are chronically homeless, tend to have poor health. however, homelessness is a temporary state, not a permanent trait. as many as 8 millions americans experience homelessness over a five year period, but most of these episodes of homelessness are quite brief (link, et al., 1994) . thus, at any single point in time only a very small proportion of a city's population is without a home. homeless people would therefore be expected to have a minimal impact on indicators of overall population health, such as health status or mortality rates. of course, this assumption may be incorrect in urban centers in the developing world, where extremely large numbers of people often live on the street or in encampments. some have suggested that homelessness may have an adverse effect on public health through the spread of infectious diseases, such as tuberculosis. compared to the general population, homeless people are clearly at increased risk of developing latent tuberculosis, which is not infectious to others, as well as active tuberculosis, which can infect those who come in close contact with the individual. during tuberculosis outbreaks, shelter residents, shelter staff, and health care providers are at increased risk of becoming infected (advisory council for the elimination of tuberculosis, 1992a). to date, however, outbreaks of tuberculosis among homeless people have not spread widely within the general population. the threat of tuberculosis is therefore an important health problem for homeless people, but one that has demonstrated relatively limited potential to affect overall population health in urban areas. the outbreak of severe acute respiratory syndrome (sars) in 2003 has raised the specter of rapid and uncontrolled spread of acute respiratory infections through the homeless population. the 2003 sars outbreak in toronto was almost entirely confined to travelers returning from abroad, health care workers, and their household contacts (svoboda, et al., 2004) . no homeless person became infected with sars. if this had happened, the large, transient, and difficult-to-locate shelter population would have made it almost impossible for toronto public health officials to implement their core strategy of identifying and quarantining all "household contacts" of patients with sars. such a situation could have had devastating effects on efforts to prevent the outbreak from spreading into the city's general population. given the threat of a recurrence of sars or the possible emergence of other new and potentially deadly respiratory infections, infection control measures to deal with a severe acute infectious disease outbreak in the homeless population require serious consideration. although this scenario is currently hypothetical, the potential implications for population health are considerable. homelessness may have major implications for population health, for reasons other than those discussed above. emphasis on the direct impact of homelessness on population health may be misplaced. instead, homelessness may be viewed as a sentinel event, a marker for dysfunction in multiple sectors including the housing market, job market, health care system, and social welfare system. homelessness represents the extreme end of a larger distribution of socioeconomic status and housing status, and it attracts attention precisely because of its dire nature. this conceptualization has been well-described in work by rose (1985) . as shown in figure 1 , the curve shown with a solid line represents the distribution of housing quality within a hypothetical population. homelessness represents the extreme low point along the dimension of housing quality. this approach views one's housing situation as a continuum and avoids creating a simple dichotomy between being homeless and being housed. for the sake of this discussion, we assume that housing conditions have an impact on health, an assertion for which there is ample support (fuller-thomson, et al., 2000; krieger and higgins, 2002) . figure 1 also illustrates two different approaches to improving health through improving housing conditions. the greatest effect in terms of population health may be gained through approach a (shifting the entire population distribution for the factor upwards slightly, to the distribution curve indicated by a dotted line) rather than approach b (focusing on improving conditions for the highest-risk group at the worst extreme of the distribution). a similar argument could be applied to the relationship between poverty and health, where the x-axis on the diagram would represent income rather than housing quality. the case of asthma is an excellent example of this dilemma. about 40% of children staying at homeless shelters in new york city have asthma (mclean, et al., 2004a) . although this is a disturbingly high rate, the prevalence of asthma is also very high among inner-city children living in substandard housing (malveaux and fletcher-vincent, 1995) . because homeless children represent a relatively small proportion of all children living in poverty in a given city, the population health effect of asthma among homeless children is likely to be far smaller than the population health effect of asthma in the much larger number of children who are housed but living in decrepit buildings. while homeless children are a distressing manifestation of urban poverty, they represent "the tip of the iceberg" of the broader issue of poverty and poor housing. thus, the problem of asthma among homeless children may be regarded as an extreme example of a much larger population health concern that may be a more appropriate target for intervention. a consideration of strategies to improve the health of homeless people raises the question of whether our first concern should be to attempt to deal with the problem of homelessness itself, or to intervene to relieve illness among homeless youths, single adults, or families. of course, this is not an either/or proposition. nonetheless, an excessive emphasis on the latter approach might result in producing healthier homeless people, yet fail to recognize that homelessness is the result not only of individual vulnerabilities, but also of deeper structural problems within our society. on the other hand, a focus on the former approach may founder on the assumption that providing homeless people with stable housing will necessarily improve their health. an example of this tension is the emergence of two contrasting service delivery models to meet the needs of chronically homeless adults with concurrent mental illness and substance abuse (tsemberis, et al., 2004; hopper and barrow, 2003 first model, known as the "continuum of care," attempts to move homeless people from the street into transitional congregate housing, in conjunction with a requirement that the individual engage in treatment for their mental illness and addictions. under this model, the person is allowed to make the transition to permanent housing only after they achieve abstinence from alcohol and drugs and their clinical status has been stabilized. in contrast, the "housing first" model is based on the belief that homeless people should be afforded permanent housing as a basic human right, not as a reward contingent on participating in treatment. in this model, homeless people can obtain housing in individual apartments without any preconditions, and they are then offered an array of harm reduction and treatment services through an act team (see section 3.5. above). a recent randomized controlled trial assigned 225 homeless adults with concurrent severe mental illness and substance abuse to one of these two approaches. individuals treated under the "housing first" model spent significantly less time homeless over the follow-up period, and at the end of 24 months about 80% were in stable housing as compared to only 30% in the "continuum of care" model. however, there were no significant differences between the two groups in terms of alcohol use, drug use, or psychiatric symptoms. this study highlights the need to acknowledge that ending homelessness is a worthwhile goal in and of itself, but that it is not synonymous with improving the health of homeless people. other strategies include adapting the health care system to better meet the unique needs of homeless adolescents, single men and women, or families. as discussed in section 3.5 above, a cornerstone of this effort is the use of multidisciplinary teams providing coordinated care at outreach sites, in combination with more traditional clinic-based health care services. for homeless people with severe mental illness, the availability of act services is vital, but the effectiveness of less resource-intensive systems of mental health care for homeless people needs to be assessed. for those with addictions, the availability of detoxification beds, postdetoxification stabilization programs, and longer-term (6 to 12 month) residential addiction treatment programs are important issues. in designing these services, the heterogeneous needs of different subgroups of homeless people (e.g., street youth, single men, single women, and mothers with young children) must be taken into account. while improving conditions at shelters is by no means the preferred route to better health for homeless people, it is important that shelters not contribute to ill health. certainly, the availability of adequate capacity to accommodate everyone who seeks a shelter bed is a reasonable first step towards protecting homeless adults from the elements. adherence to basic standards of cleanliness, nutrition, and food hygiene within shelters and the avoidance of overcrowding and inadequate ventilation are mandatory. perhaps equally important is the creation of a safe and welcoming environment that encourages clients to engage with service providers. at a broader level, interventions are needed to decrease the prevalence of homelessness and address the systemic issues that contribute to homelessness. these efforts may at least in some cases have health benefits as well. for homeless families, there is compelling evidence that the provision of subsidized housing is both necessary and sufficient to end their homelessness (shinn, et al., 1998) . the "housing first" strategy appears to be more effective in moving homeless people with concurrent mental illness and substance abuse into stable housing; further research is needed to examine the effectiveness of this approach with other subgroups of homeless people. serious attention needs to be paid to the impact of the social welfare system on homelessness and health. restrictions in eligibility for temporary aid for needy families and state-run welfare programs threaten to contribute to a potential rise in homelessness among families and single adults in coming years. further research is needed in this area and on the impact of receipt of welfare or disability benefits on the health of homeless people. finally, upstream from the distinctive and visible issue of homelessness is the larger problem of urban poverty. the existence of entire communities and groups who are cut off from a decent education, employment opportunities, housing, and access to health care should raise extremely troubling questions for anyone who cares about the health of our urban centers. while the adverse health effects of homelessness are clearly severe, this phenomenon is only a specific and extreme example of the larger problem of the effects of poverty and inadequate housing on population health. prevention and control of tuberculosis among homeless persons. recommendations of the advisory council for the elimination of tuberculosis prevention and control of tuberculosis among homeless persons. recommendations of the advisory council for the elimination of tuberculosis hiv infection among homeless adults and runaway youth foci of tuberculosis transmission in central los angeles mortality among homeless shelter residents in new york city determinants of behavior in homeless and low-income housed preschool children human immunodeficiency syndrome and hepatitis b and c infections among homeless adolescents the megacity saga: democracy and citizenship in this global age seceding from responsibility? secession movements in los angeles changing attitudes toward homeless people lifetime prevalence of trauma among homeless people in sydney helping america's homeless cancer risk behaviors and screening rates among homeless adults in los angeles county risk of death among homeless women: a cohort study and review of the literature viral hepatitis and other infectious diseases in a homeless population down and out in new york and london: a cross-national comparison of homelessness smoking cessation in a homeless population: there is a will, but is there a way characterizing perceived police violence: implications for public health health status of and access to health services by residents of urban encampments in los angeles the responsibility to care for single homeless people public shelter admission rates in philadelphia and new york city: the implications of turnover for sheltered population counts homeless "squeegee kids": food insecurity and daily survival witnessing heroin-related overdoses: the experiences of young injectors in san francisco city of quartz not on our street: community attitudes to mental health care landscapes of despair: from deinstitutionalization to homelessness place matters: metropolitics for the twenty-first century determinants of overdose incidents among illicit opioid users in 5 canadian cities the epidemiology of alcohol, drug, and mental disorders among homeless persons schizophrenia in homeless persons: a systematic review of the literature medical comorbidity and receipt of medical care by older homeless people with schizophrenia or depression the housing/health relationship: what do we know? competing priorities as a barrier to medical care among homeless adults in los angeles chronically homeless women's perceived deterrents to contraception a national survey of the oral health status of homeless veterans panhandling for change in canadian law prevalence and correlates of survival sex among runaway and homeless youth substance use among runaway and homeless youth in three national samples youth and familial substance use's association with suicide attempts among runaway and homeless youth pregnancy among three national samples of runaway and homeless youth inappropriate emergency department visits and use of the health care for the homeless program services by homeless adults in the northeastern united states the limits of neighbourhood empowerment: gentrification, resistance, and burn-out in kitsilano health care for the homeless information resource center two genealogies of supported housing and their implications for outcome assessment mortality among men using homeless shelters in toronto barriers to appropriate diabetes management among homeless people in toronto homelessness and health causes of death in homeless adults in boston the homeless prevalence of tuberculosis infection among homeless young people in central and eastern sydney divided we sprawl slowing the revolving door: stabilization programs reduce homeless persons' substance use after detoxification homeless youth and their exposure to and involvement in violence while living on the streets housing and health: time again for public health action applying cluster analysis to test a typology of homelessness by pattern of shelter utilization: results from the analysis of administrative data no door to lock: victimization among homeless and marginally housed persons emergency department use among the homeless and marginally housed: results from a community-based study factors associated with the health care utilization of homeless persons socioeconomic deprivation and health in glasgow and the west of scotland-a study of cancer incidence among male residents of hostels for the single homeless a randomized trial of assertive community treatment for homeless persons with severe mental illness health care for homeless persons infectious diseases among homeless children and adolescents: a national concern lifetime and fiveyear prevalence of homelessness in the united states environmental risk factors of childhood asthma in urban centers hospitalization of homeless persons with tuberculosis in the united states social inequalities in health: next questions and converging evidence outbreak of tuberculosis among homeless persons coinfected with human immunodeficiency virus privatopia: homeowner associations and the rise of residential private government asthma among homeless children: undercounting and undertreating the underserved report on socio-economic differences in health indicators in europe: health inequalities in europe and the situation of disadvantaged groups assessing homeless population size through the use of emergency and transitional shelter services in 1998: results from the analysis of administrative data from nine us jurisdictions initiating abstinence in cocaine abusing dually diagnosed homeless persons outbreak of tuberculosis in a homeless men's shelter a review of case management for people who are homeless: implications for practice, policy, and research tuberculosis in the homeless. a prospective study differential patterns of mental disorders among the homeless in madrid (spain) and los angeles (usa) incidence and prevalence of chlamydia, herpes, and viral hepatitis in a homeless adolescent population are rates of psychiatric disorders in the homeless population changing? risk factors for hepatitis c virus infection among homeless adults making room: the economics of homelessness a social and economic impact study of the ontario safe streets act on toronto squeegee workers selfreported changes in drug and alcohol use after becoming homeless metropolitics: a regional agenda for community and stability prevalence of chlamydia trachomatis and neisseria gonorrhoeae among at-risk women, young sex workers, and street youth attending community organizations in quebec city infections in the homeless hiv seroprevalence among homeless and marginally housed adults in san francisco mental health problems of homeless women and differences across subgroups sick individuals and sick populations hepatitis c and substance use in a sample of homeless people in new york city outcomes after initial receipt of social security benefits among homeless veterans with mental illness risk factors for hepatitis c virus infection among street youths drug injection among street youths in montreal: predictors of initiation mortality in a cohort of street youth in montreal hepatitis b virus infection among street youths in montreal inside game/outside game:winning strategies for saving urban america a survey of hepatitis c prevalence amongst the homeless community of oxford predictors of homelessness among families in new york city: from shelter request to housing stability similarities and differences in homelessness in amsterdam similarities and differences in homelessness in amsterdam similarities and differences in homelessness in amsterdam prevalence of hiv infection and hiv risk behaviors associated with living place: on-the-street homeless drug users as a special target population for public health intervention housing status and health care service utilization among low-income persons with hiv obstructive lung disease among the urban homeless state policy documentation project. center for law and social policy and the center on budget and policy priorities death by compaction in a garbage truck severity of homelessness and adverse birth outcomes victimization and posttraumatic stress disorder among homeless adolescents medical and cutaneous disorders associated with homelessness prevalence of skin disease in a cohort of shelter-based homeless men hiv risk, seropositivity and predictors of infection among homeless and non-homeless women sex workers in public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in toronto identification of cardiovascular risk factors in homeless adults disease patterns of the homeless in tokyo disease patterns of the homeless in tokyo accidental hypothermia and death from cold in urban areas psychiatric disorders in homeless men and women in inner sydney housing first, consumer choice, and harm reduction for homeless individuals with a dual diagnosis reaching homeless youths for chlamydia trachomatis and neisseria gonorrhea screening in denver mental health problems of homeless children and families: longitudinal study hiv risk profile and prostitution among female street youths when work disappears: the world of the new urban poor. random house the truly disadvantaged : the inner city, the underclass, and public policy displacement of canada's largest public illicit drug market in response to a police crackdown do the homeless get a fair deal from general practitioners? outcomes of contact investigation among homeless persons with infectious tuberculosis homeless people's trust and interactions with police and paramedics key: cord-000008-3dgjv0x1 authors: vali, bahareh; yue, feng yun; jones, r. brad; sheth, prameet m.; kaul, rupert; betts, michael r.; wong, david; kovacs, colin; loutfy, mona; common, andrew; halpenny, roberta; ostrowski, mario a. title: hiv-specific t-cells accumulate in the liver in hcv/hiv co-infection date: 2008-10-20 journal: plos one doi: 10.1371/journal.pone.0003454 sha: doc_id: 8 cord_uid: 3dgjv0x1 background and aims: hepatitis c virus (hcv)-related liver disease progresses more rapidly in individuals co-infected with human immunodeficiency virus-1 (hiv), although the underlying immunologic mechanisms are unknown. we examined whether hiv-specific t-cells are identified in the liver of hcv/hiv co-infected individuals and promote liver inflammation through bystander immune responses. methods: ex-vivo intra-hepatic lymphocytes from hcv mono-infected and hcv/hiv co-infected individuals were assessed for immune responses to hiv and hcv antigens by polychromatic flow cytometry. results: hcv/hiv liver biopsies had similar frequencies of lymphocytes but lower percentages of cd4(+) t-cells compared to hcv biopsies. in co-infection, intra-hepatic hiv-specific cd8(+) and cd4(+) t-cells producing ifn-γ and tnf-α were detected and were comparable in frequency to those that were hcv-specific. in co-infected individuals, viral-specific cd8(+) t-cells produced more of the fibrogenic cytokine, tnf-α. in both monoand co-infected individuals, intra-hepatic hcv-specific t-cells were poorly functional compared to hiv-specific t-cells. in co-infection, haart was not associated with a reconstitution of intra-hepatic cd4(+) t-cells and was associated with reduction in both hiv and hcv-specific intra-hepatic cytokine responses. conclusion: the accumulation of functional hiv-specific t-cells in the liver during hcv/hiv co-infection may represent a bystander role for hiv in inducing faster progression of liver disease. approximately 25% of human immunodeficiency virus-1 (hiv) infected individuals are also infected with hepatitis c virus (hcv) [1] . hiv adversely affects each stage of the natural history of hcv infection. fewer individuals recover spontaneously from hcv infection when also infected with hiv [2] . among those with persistent hcv infection, hiv co-infection is associated with higher hcv viremia and more rapid progression to cirrhosis and hepatocellular carcinoma [3] . a recent meta-analysis showed that hiv co-infection increased the risk of histological hepatic cirrhosis by two-fold and clinically decompensated liver disease by six-fold [4] . in addition, hcv co-infection is associated with increased incidence of haart (highly active antiretroviral therapy) related liver injury [5] . the mechanisms for hepatic damage in hcv/hiv co-infection are poorly defined. although intra-hepatic t-cell immune responses are necessary for hcv clearance, they have also been shown to play a central role in mediating hepatocellular injury by direct cytotoxicity or indirectly by releasing cytokines. in this regard, ifn-c has been shown to be anti-fibrogenic, whereas, tnf-a activates hepatic stellate cells, which induce fibrosis, and likely contributes to progression to cirrhosis [6, 7] . potent and broad cd4 + and cd8 + t-cell immunity are important for virologic control in both hcv and hiv viral infections. ex-vivo hcv-specific cd8 + t-cell responses in peripheral blood mono-nuclear cells (pbmcs) from mono-infected individuals are generally weak [8] . although, peripheral hcvspecific cd4 + and cd8 + t-cell responses are somewhat weaker in hcv/hiv co-infected individuals [9] , similar frequencies of intra-hepatic hcv-specific responses appear to be obtained in hcv versus hcv/hiv co-infection [10, 11] . however, ex-vivo hiv-specific cd8 + t-cell responses in pbmcs from hiv monoinfected individuals are about one log higher than ex-vivo hcvspecific responses in hcv mono-infection. in addition, impairment in cellular immune responses to hcv compared to hiv has been shown in hcv/hiv co-infection [12] . hiv-specific cd8 + t-cells are easily detectable in blood of untreated hiv infected individuals [13] . such high frequencies of hiv-specific t-cells circulating in peripheral blood led us to question whether these cells could also migrate to the liver in hcv/hiv co-infection and through bystander responses add to the inflammation induced by hcv-specific t-cells. hcv mono-infected and hcv/hiv co-infected individuals who required liver biopsies for work up of liver disease were recruited for the study (see results and table 1 ). all study participants provided informed, written consent and the study protocol was approved by the research ethics board at the university of toronto and st. michael's hospital. both blood and liver biopsy samples were received from each participant. liver biopsy samples were washed in rpmi-1640 to remove contaminating blood lymphocytes, manually homogenized with a plastic plunger, and treated with dnase (0.002%, sigma) and collagenase iv (0.02%, sigma) for 30 minutes, stirring at 37uc. the digested cell suspension was filtered through a 70 mm strainer, washed and re-suspended in r-10 medium (10% fetal calf serum). in order to identify candidate epitope-specific responses to be detected in ex-vivo liver samples, we first mapped antigen-specific t-cell responses in blood against the entire hiv-1 clade-b and hcv-1a proteome using the matrix approach by ifn-c eli-spot assay as described previously [14] . mapped peptides were then pooled to evaluate hepatic responses. in order to address the possibility that differing epitopes were only targeted in the liver, we also used four peptide pools that previously were shown to target a majority of responses. these pools spanned hiv-gag and hcv-ns3, hcv-ns4 and hcv-core protein (2 mg/peptide/ml, from national institute of health reagent program). of the hcv pools, the pool that gave the strongest elispot response in pbmcs was used for hepatic cell stimulation (see below). all the extracted cells from each liver biopsy were split in three wells and stimulated on the same day as pbmcs. 1610 6 pbmcs and liver-isolated cells were stimulated with either dmso, hiv or hcv peptide pools as described previously [14] . hiv pools consisted of peptides that were screened by the matrix approach in that individual plus the hiv-gag pool. likewise, hcv pools consisted of mapped peptides plus an hcv pool that gave the strongest response in pbmcs. cd107a antibody (pe-cy5, bd pharmingen) was added at the time of stimulation. the following antibodies were used for staining: cd8-pe texas-red (beckman coulter), cd4-pacific blue (e-bioscience), cd3-apccy7, ifnc-fitc, tnfa-pecy7, il2-apc, mip-1b-pe (bd bioscience), pd-1 fitc (biolegend) and dead cell stain aqua (invitrogen, molecular probes). cells were analyzed on a multi-color facsaria flow cytometer (bd biosciences). for blood samples between 500,000 to 1,000,000 total events and for liver biopsy samples between 50,000 to 200,000 total events were collected. data analysis was performed using flowjo version 8.6 (treestar inc., san carlos, ca). polychromatic flowjo data were analyzed with pestle software, and pie-chart graphs were generated using spice software (obtained from m. roederer, national institutes of health, bethesda md). multi-parameter analysis of hiv-gag: 77-85 (slyntvatl: sl9) specific cd8 + t-cells was conducted in both blood and liver of co-infected individuals initially identified with a positive elispot response to the 15-mer hiv peptide including sl9 epitope, using the corresponding tetramer (itag mhc class-i tetramer, beckman coulter). tetramer staining was performed prior to peptide stimulation, at room temperature for 20 minutes. tetramer stained cells were then washed and stimulated with 10 mg/ml of sl9 peptide followed by ics staining as mentioned above. additional hiv, hcv and cmv-specific pentamer staining (pro5 mhc class i pentamers -proimmune) was conducted followed by pd-1 staining. data were analyzed by performing two-tailed non-parametric mann-whitney test using graphpad prism version 4.00. p-values#0.05 were considered significant. three groups of individuals were studied as depicted in table 1 ; hcv mono-infected (n = 6), hcv/hiv co-infected who were not receiving haart (n = 8) and hcv/hiv co-infected who were receiving haart for greater than one year at the time of evaluation (n = 12). all individuals never received prior treatment for hcv and underwent liver biopsies for staging and evaluation for pegylated-interferon/ribavirin treatment. hcv/hiv co-infected individuals had higher hcv viral loads. on average, cd4 t-cell counts of hiv infected individuals were .400/ml in both groups. of note, the mean hepatic fibrosis scores were higher in the haart treated and mono-infected groups in this cohort, indicating that individuals in these groups had more advanced disease at the time of biopsy in this study. although similar frequencies of intra-hepatic lymphocytes were obtained in dual versus mono-infection, haart-treated individuals showed a trend towards greater percentages of lymphocytes in their biopsies ( figure 1a ). the percentage of intra-hepatic cd4 + t-cells was significantly reduced in dual infection [31.6%613.8 for hcv vs 6.5%62.9, for hcv/hiv therapy naïve, p,0.01], and was not associated with any improvement in haart-treated individuals, as previously shown in the gut [15] (figure 1b) . however, compared to hcv mono-infected individuals the percentage of intra-hepatic cd8 + t-cells was higher in both co-infected groups [33.8%65.5% for hcv vs 67.3%615.5% for hcv/hiv therapy naïve vs 59.5615.1% for hcv/hiv on haart, p,0.01] (figure 1b ). to determine the presence of intra-hepatic viral specific t-cell responses, liver isolated cells were stimulated with hcv and hiv peptide pools. summary data of viral specific responses are depicted in figure 2 . in response to stimulation with hiv peptide pool, untreated co-infected individuals showed significantly higher frequencies of intra-hepatic cd4 + t-cells producing ifn-c, compared to hcv mono-infected [0.1660.05% vs 0.0260.01%, p,0.05], and haart-treated co-infected individuals [0.1660.05% vs 0.0360.05%, p,0.05] (figure 2a ). untreated co-infected individuals showed a trend towards lower frequencies of intra-hepatic ifn-c producing cd4 + t-cells in response to hcv peptides. surprisingly, haart-treated co-infected individuals had significantly reduced hcv-specific ifn-c producing cd4 + t-cells when compared to untreated co-infected individuals [0.0260.01% vs 0.4660.11%, respectively, p,0.01] (figure 2a). therapy naïve co-infected subjects had greater ifn-c producing cd8 + t-cells in response to hiv peptides compared to hcv mono-infected individuals [1.3960.37% vs 0.0260.0%, p,0.05], and haart was associated with a significant reduction in the frequencies of these cells [1.3960.37% vs 0.3060.26%, p,0.05] (figure 2b). although there was a trend for enhanced intra-hepatic cd8 + t-cells producing ifn-c in response to hcv peptides in therapy-naive co-infection compared to hcv mono-infection, this was not found to be statistically significant. haart on the other hand, was associated with a significant reduction in hcv-specific, intra-hepatic cd8 + t-cells producing ifn-c [1.360.37% vs 0.0360.01%, p,0.05] (figure 2b). similarly, co-infected individuals had significantly greater intrahepatic tnf-a expressing cd4 + t-cells after hiv peptide stimulation compared to hcv mono-infected [0.260.05 vs 0.0260.01, p,0.01], although haart had no significant effect on their frequencies (figure 2c ). hcv mono-infected individuals showed significantly higher frequencies of hcv-specific tnf-a producing cd4 + t-cells compared to haart-treated co-infected individuals [0.9160.25% for hcv vs 0.2360.20 for hcv/hiv on haart, p,0.01] (figure 2c), but did not show significant differences with the untreated co-infected group. the therapy-naïve co-infected group showed significantly higher frequencies of intra-hepatic tnf-a producing cd8 + tcells in response to both hiv-1 and hcv antigens. both types of to study the functional profile of virus-specific t-cells in hcv/ hiv co-infection, simultaneous expression of 5 distinct cd8 + t-cell markers were analyzed in 3 individuals within each cohort using a previously developed multicolor flow cytometry method [16] . expression levels of degranulation marker cd107a and cytokines ifn-c, tnf-a and il-2, as well as the chemokine mip-1b were simultaneously measured in response to hcv or hiv peptides in both blood and liver of each individual. figure 3 depicts a representative multi-parameter analysis of cd8 + t-cell responses in liver and blood of a therapy-naïve, co-infected subject in response to hiv and hcv peptide pools. these data indicate that both hcv and hiv specific cd8 + t-cells expressing one or more functions are detectable in the liver and blood. compared to blood, the frequency of hiv-specific t-cells producing cd107a and il-2 was shown to be significantly higher in the liver of therapy-naïve, co-infected individuals. consistent with previously reported data [10] , hcv-specific responses were compartmentalized to the liver and stronger than peripheral hcv-specific responses (figure 3c ). recognition of functional ctl, specific for the hla-a0201-restricted hiv-sl9 epitope in hcv/hiv co-infected liver to determine if t-cells specific for an hiv immuno-dominant epitope are present in hcv/hiv co-infected liver, we quantified cd8 + t-cells specific for hla-a*0201-restricted slyntvatl (sl9) epitope in the liver of individuals with positive sl9 responses in their blood. we identified 3 co-infected hla-a*0201 individuals, among them one showed a response to the sl9 epitope of hiv-gag antigen. figure 4 shows the multi-parameter analysis of tetramer positive cd8 + t-cells in blood and liver of this therapy-naïve, co-infected individual. the tetramer cytokine response pattern was shown to be different in the liver compared to blood of the same individual, with diminished intra-hepatic tetramer-specific ifn-c responses and an increase in both cd107a and tnf-a responses, with the majority of sl9 tetramer positive cells expressing these two markers. we also included cmv as a non-hepatotropic control virus in our liver analysis. using a pool of hla-a*0201-and hla-a*2402-restricted, cmv-specific pentamers, we did not detect any cmv-specific cd8 + t-cells in hcv/hiv co-infected liver, although we could readily detect them in blood (figure 4c ). using the panel of markers tnf-a, ifn-c, mip-1b, il-2, and cd107a, we characterized the ability of cd8 + t-cells to simultaneously exert these 'functions' in response to both hcv and hiv peptides. figure 5 depicts a representative functional profile of virus specific cd8 + t-cells in blood and liver during hcv/hiv co-infection (fig. 5a) and hcv mono-infection (fig. 5b) . analysis of co-infected subjects demonstrates a very limited functional hierarchy of hcv-specific t cells in the blood, with majority of t-cells producing one function. hiv-specific tcells from blood had a more expanded functional hierarchy. in accordance with previous reports on hiv mono-infected individuals [16] , cells expressing all 5 functions were absent in the blood of co-infected subjects, mainly due to lack of il-2 production. in co-infection, intra-hepatic cd8 + t-cells responding to hcv peptides were within the single-responding and 2+ populations. intra-hepatic cd8 + t-cell responses to hiv peptides produced a larger spectrum of responses. cd107a responding cells were represented in nearly all of the different hiv-specific populations in the liver of co-infected individuals. as expected, hcv-specific responses in the blood of hcv mono-infected subjects were mainly single functional. the profile of hcv-specific responses in the liver of hcv mono-infected individuals showed the appearance of a very small population of 4+ responding cells in the liver. we and others have considered a cutoff point of more than 2 simultaneously expressed markers to demonstrate poly-functional characteristic of responding t-cells [16, 17] . figure 5c shows a comparison between average frequency of intra-hepatic viralspecific responses within the pool of cd8 + t-cell populations expressing 2 markers or less, and those within the pool of populations expressing more than 2 markers simultaneously. for both hcv and hiv-specific cd8 + t-cells the majority of responses had two or less functions. however, intra-hepatic hiv-specific responses demonstrated more poly-functionality, compared to hcv-specific responses either within co-infected or mono-infected individuals [0.0560.01 vs 0.00760.00, p,0.05; hiv-specific responses vs hcv-specific responses in hcv/hiv co-infected group]; [0.0560.01 vs 0.0160.00, p,0.05; hivspecific responses in hcv/hiv co-infected group vs hcvspecific responses in hcv mono-infected group]. in summary, although viral-specific t-cells, simultaneously expressing all 5 measured markers were rarely found in the liver, intra-hepatic hiv-specific t-cells showed greater functional capacity when compared to those being hcv-specific. based on the recently highlighted role of pd-1 contributing to the dysfunction of t-cells in chronic viral infections, we also determined whether hiv and hcv-specific intra-hepatic t-cells differ in the degree of pd-1 expression. in an hcv/hiv coinfected liver, we found that 100% of intra-hepatic hcv-specific cd8 + t-cells were pd-1 positive, compared to 48.8% of those cells that were hiv-specific (figure 5d ). this is the first study to demonstrate the presence of hivspecific t-cells within the liver of hcv/hiv co-infected individuals. the finding of hiv-specific t-cells within liver of co-infected individuals may not altogether be surprising, given the high frequencies of hiv-specific cd8 + t-cells found in the peripheral blood in untreated hiv infection. nevertheless, it is surprising to find functional t-cells of such viral specificities to be accumulating in liver. in contrast, we could not detect cmvspecific t-cells in co-infected liver despite their abundance in blood indicating that different viruses target t-cells to the liver. recent studies have demonstrated that systemic viral infections may recruit viral-specific t-cells to the liver. the significance of non-hepatotropic viral-specific t-cells that are found in liver is unclear. it has been postulated that the liver can non-selectively trap activated t-cells during any infection, and thus act as a 'sink' or 'graveyard' [18] , however it is unclear whether these cells are rendered anergic while traveling in the liver or contribute to inflammation and damage as a result of bystander activation. of note, is that hepatitis has been observed in measles [19] , sars [20] and in 20% of individuals with acute hiv infection [21] . polakos et. al. [22] found that some individuals infected with influenza-a develop transaminitis and showed in a murine influenza model that influenza-specific cd8 + t-cells migrate to figure 3 . polychromatic facs analysis of viral-specific t cells in hcv/hiv co-infection. shown are representative facs data of the hiv and hcv specific multi-parameter cd8 + t-cell responses from (a) liver and (b) blood of subject om 405, a therapy-naïve hcv/hiv co-infected individual, after in vitro stimulation using pool of hiv and hcv peptides. initial gating on forward scatter area (fsc-a) versus height (fsc-h) was used to remove doublets. the events were further gated on forward scatter (fsc) versus the dead cell marker to remove dead cells. lymphocytes were gated on the remaining live cells on a fsc versus ssc plot. gates on cd3 + /cd8 + cells were then generated. all responses are gated on a cd3 + /cd8 + population and presented against tnf-a on the x-axis. figure (c) shows a comparison of the frequency of hiv and hcv-specific cd8 + t-cells in the liver and blood of therapy-naïve, co-infected individuals. all intra-hepatic hcv-specific responses are significantly stronger than peripheral hcv-specific responses. doi:10.1371/journal.pone.0003454.g003 non-hepatotropic viruses such as hiv, cmv and ebv, in general do not induce chronic hepatitis, thus, it is possible that the coexistence of hepatotropic viruses may alter the hepatic environment to allow recruitment of activated t-cells non-specifically. this could be due to an up-regulation of integrins such as icam-1 and vcam-1 in hepatic sinusoids as previously shown during hcv infection [23] that could enhance t-cell recruitment. in this regard, spangenberg et. al. [24] demonstrated the presence of influenza-specific t-cells in about 50% of liver biopsies from hcv mono-infected individuals. there are several lines of evidence demonstrating that the liver efficiently clears many foreign pathogens, including rna viruses. it is shown that liver is a major organ for clearing simian immunodeficiency virus in rhesus monkeys [25] . there is also evidence for the detection of hiv rna in the liver of hiv infected individuals [26] . these findings support the identification of hiv-specific t-cells in the liver. in hcv/hiv co-infection, it is possible that intra-hepatic hcv-specific cd4 + t-cells become infected with hiv and recruit hiv-specific immune responses to this site. evidence for these potential mechanisms will need further analysis on liver biopsies of co-infected individuals. our analysis of liver biopsies from hcv/hiv co-infected individuals not only demonstrate that hiv-specific t-cells producing ifn-c and tnf-a are detected in the liver, but also exhibit comparable frequencies of responses to those that are hcv-specific. this observation may explain the added contribution of hiv-specific immune responses to the ongoing intrahepatic damage induced by hcv-specific t-cell responses that are inefficient in clearing the virus. therapy naïve co-infected individuals demonstrated a higher frequency of intra-hepatic cd8 + t-cells that produce tnf-a in response to both hcv and hiv antigen stimulation compared to hcv mono-infected individuals. in addition, we identified cd8 + t-cells specific for an immunodominant hiv epitope in co-infected liver, demonstrating high frequency of tnf-a expression. intra-hepatic tnf-a has been previously associated with liver fibrosis, and the accumulation of cells expressing this marker may explain in part the faster rate of liver disease progression found in hcv/hiv coinfection. further comparisons of tnf-a responses between immunodominant hcv and other hiv epitopes in a larger cohort of individuals are warranted. contrary to our expectation, viral-specific, intra-hepatic levels of ifn-c were also higher in the therapy-naïve co-infected group, which would be against the expected protective role of ifn-c. however, we interpret this observation as a potential effect of the fibrogenic tnf-a to mask ifn-c protection. on the other hand, viral-specific t-cells are composed of several major populations with unique functional patterns. therefore, measurement of only one or two t-cell functions may not provide a comprehensive picture of the quality of t-cell responses. recent lines of evidence demonstrate the importance of the qualitative rather than quantitative characteristics of cd8 + t-cell responses to efficient viral control [13, 27] . the significance of tcell populations simultaneously representing 5 different functions has been discussed as a hierarchical functional model in viral infections such as cmv and ebv which are effectively controlled by respective cd8 + t-cells [16] . hcv-specific cd8 + t-cells were not poly-functional which is consistent with the notion that although hcv-specific t-cells are found in hepatic tissue, their loss of poly-functionality may be associated with inefficient control of hcv replication. hiv-specific t-cells in the liver of co-infected individuals however, simultaneously could express 4 and 5 of the measured markers. recently, t-cell exhaustion has been related to signaling pathways through pd-1 [28, 29] . our analysis of pd-1 levels of antigen-specific cd8 + t-cells from co-infected liver demonstrates higher expression of pd-1 on hcv-specific t-cells, compared to those specific for hiv, supporting the notion that the former are less functional. the observed poly-functionality of intra-hepatic hiv-specific t-cells, should have little effect on hcv replication but would further enhance the cytokine milieu induced from bystander activation, and contribute to liver damage during co-infection with hcv. in this regard, we found that the degranulation marker cd107a dominates the hiv-specific cd8 + t-cell responses in the liver, with the majority of the responding cells expressing cd107a, a surrogate marker for the cytotoxic function of cd8 + t-cells. activated hiv-specific cd8 + t-cells with the potential to degranulate could induce bystander damage. in addition, the release of chemokines such as mip-1b by the same cells could also attract further lymphocytes without hcv specificity to the liver. bystander function of these non-specific t-cells could expand the tissue damage triggered by hcv infection and ultimately activate fibrogenesis. we found that the frequency of cd4 + t-cells within livers of coinfected individuals was reduced compared to hcv monoinfection. surprisingly, haart did not appear to reconstitute the cd4 + t-cell population within liver. despite this defect of cd4 + t-cell help, comparable frequencies of hcv-specific-cd8 + t-cells were found in co-infected livers. haart-treated biopsies showed further reduced frequencies of hcv-specific responses. these data support previous findings that show haart induces cd4 + t-cell recovery but not any restoration of hcv-specific tcell responses peripherally [30] . further investigation is needed to clarify the role of cd4 + t-cell help in affecting the frequencies of hcv-specific cd8 + t-cells in hcv/hiv-1 co-infection. haart was also associated with a reduction in frequencies of hiv-specific t-cell responses within liver, indicating that removing the hiv antigenic load may also reduce the opportunity for such cells to accumulate within hepatic tissue. here, we propose a novel mechanism for enhanced hcvrelated liver disease progression in hiv co-infection; that of bystander activation and induced inflammation from hiv-specific t-cells accumulated in the liver. our data however are limited in the cross-sectional nature of our cohort, the low number of analyzed liver biopsies and the narrow range of cd4 + t-cell counts among the studied individuals. we should also acknowledge that ex-vivo functional t-cell capacity may not exactly reflect the situation in-vivo. further studies, particularly, those which are prospective are warranted in order to understand the role that hiv-specific t-cells play in contributing to fibrosis and in particular how haart modulates these responses. frequency of cd8 + t-cells specific for hcv, hiv and cmv in hcv/hiv co-infected liver (om 385). liver isolated mononuclear cells were stained with pools of hla-a*0201 and hla-a*2402-restricted pentamers (pro5 mhc class i pentamers, proimmune), followed by staining for cell surface markers cd3 and cd8. the following pentamers were used for each group: hcv pentamers: ns3-cingvcwtv and ns3-klvalginav; hiv pentamers: pol-ilkepvhgv and gag p24-tlnawvkvv; cmv pentamers: pp65-nlvpmvatv and pp65-qydpvaalf. no cd8 + t-cells specific for cmv were detected in this co-infected liver sample. similar findings were found in another individual (data not shown). doi:10.1371/journal.pone.0003454.g004 background is shown to become extremely low when examining combinations of functions, nearly reaching 0 events for multiple functions simultaneously. this permits a very low threshold for detection of positive responses from multiple combinations. consequently, for multi-parameter analysis, the results were thresholded based on a minimum criterion of positivity, as calculated by spice software and presented as the 90 th percentile of negative values for each analysis. each pie chart generated by spice software, represents the hierarchy of responses to either hcv or hiv antigen stimulation. for simplicity, responses are grouped by number of functions and matched to the colored bars, with black bars representing the percentage of responding cells to hiv peptides and gray bars representing the percentage of responses to hcv peptide stimulation. in all pie charts, color red represents the 5+ responding population and the colors blue, green, turquoise, and yellow representing the 4+, 3+, 2+, and 1+ populations respectively. color-coded arcs represent the dominant marker within each pie slice, with color blue representing tnf-a, red for cd107-a, green for ifn-c and black for mip-1b. although il-2 is included in the presentation and demonstrated by bar graphs, the software would not allow for arc colors for more than 4 responses. as a result there is no arc representative for il-2. figure (c) represents the average frequency of intra-hepatic viral specific responses within the pool of cd8 + t-cell populations simultaneously expressing 2 functions or less, compared with those within the pool of populations expressing more than 2 functions simultaneously; as analyzed in 3 subjects within each cohort of hcv mono and hcv/hiv co-infected individuals. the cutoff point of simultaneous expression of more than 2 measured markers is considered to show cd8 + t-cell poly-functionality. effect of human immunodeficiency virus on hepatitis c virus infection among injecting drug users impaired hepatitis c virus-specific t cell responses and recurrent hepatitis c virus in hiv coinfection retrospective analysis of the impact of hiv infection and alcohol use on chronic hepatitis c in a large cohort of drug users influence of human immunodeficiency virus infection on the course of hepatitis c virus infection: a meta-analysis hepatotoxicity associated with antiretroviral therapy in adults infected with human immunodeficiency virus and the role of hepatitis c or b virus infection pathogenic roles of tumor necrosis factor receptor p55-mediated signals in dimethylnitrosamine-induced murine liver fibrosis the role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride impaired effector function of hepatitis c virus-specific cd8+ t cells in chronic hepatitis c virus infection hiv longterm non-progressors maintain brisk cd8 t cell responses to other viral antigens cd8+ cell responses to hepatitis c virus (hcv) in the liver of persons with hcv-hiv coinfection versus hcv monoinfection comparison of hcv-specific intrahepatic cd4+ t cells in hiv/hcv versus hcv human immunodeficiency virus type 1-hepatitis c virus coinfection: intraindividual comparison of cellular immune responses against two persistent viruses analysis of total human immunodeficiency virus (hiv)-specific cd4(+) and cd8(+) t-cell responses: relationship to viral load in untreated hiv infection hiv-specific cd8+ lymphocytes in semen are not associated with reduced hiv shedding severe cd4+ t-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy hiv nonprogressors preferentially maintain highly functional hiv-specific cd8+ t cells polyfunctional analysis of human t cell responses: importance in vaccine immunogenicity and natural infection the liver as a site of t-cell apoptosis: graveyard, or killing field? measles associated hepatobiliary disease: an overview clinical significance of hepatic derangement in severe acute respiratory syndrome acute human immunodeficiency virus type 1 infection kupffer cell-dependent hepatitis occurs during influenza infection inflammatory markers in chronic hepatitis c intrahepatic cd8+ t-cell failure during chronic hepatitis c virus infection the liver is a major organ for clearing simian immunodeficiency virus in rhesus monkeys identification and quantitation of hiv-1 in the liver of patients with maintenance of large numbers of virus-specific cd8+ t cells in hivinfected progressors and long-term nonprogressors pd-1 expression in acute hepatitis c virus (hcv) infection is associated with hcvspecific cd8 exhaustion pd-1 expression on hiv-specific t cells is associated with t-cell exhaustion and disease progression differences in hcv-specific t cell responses between chronic hcv infection and hiv/hcv co-infection we are grateful to the patients who contributed their time and effort to this study. we would also like to thank ms. dionne white for her expertise and assistance in flow cytometry and dr. george makedonas for spice and pestle software training. we specially thank dr. mario roederer for providing us with the mentioned software. key: cord-023854-w8kx5n8k authors: schuster, v.; kreth, h. w. title: virusinfektionen date: 2019 journal: p&#x000e4;diatrie doi: 10.1007/978-3-662-57295-5_14 sha: doc_id: 23854 cord_uid: w8kx5n8k dieses kapitel behandelt klinik, komplikationen, diagnostik, prophylaxe und therapie der wesentlichen virusinfektionen im kindesund jugendlichenalter, u. a. virusinfektionen der herpes-gruppe (hsv1 und 2 [u. a. schwere neonatale infektionen, herpesenzephalitis]), vzv [u. a. neonatale vzv-infektionen, herpes-zoster], ebv, cmv, hhv6–7 [dreitagefieber], hhv8 [kaposi-sarkom]. unter den virushepatititiden spielen bei kindern die hepatitis a, b (chronische hepatitis b) sowie c (chronische hepatitis b) die größte rolle. weitere bedeutsame viren sind parvovirus-b19 (ursache der ringelröteln, einer aplastischen krise oder eines hydrops fetalis), humane papillomaviren (bedeutsam v. a. genitalwarzen, larynxpapillome und karzinome), rotaviren (bedeutsame gastroenteritiserreger), fsme, hiv sowie die „typischen kinderkrankheiten“ masern, mumps und röteln. häufige erreger des oberen und unteren respirationstrakts sind rhinoviren, das rs-virus, das humane metapneumovirus (hmpv), das humane bocavirus (hbov), das humane coronavirus (hcov) sowie influenzaund parainfluenzaviren. herpes-simplex-virus-infektionen j epidemiologie infektionen mit dem herpes-simplex-virus (hsv) treten ubiquitär auf. die ansteckung erfolgt bei kindern überwiegend durch virushaltige körperflüssigkeiten (speichel) und engen körperkontakt, seltener auch durch organtransplantation. die durchseuchung von hsv-1 schwankt zwischen 30% (länder mit höherem lebensstandard) und 90% (ärmere länder). die häufigkeit von hsv-2-infektionen korreliert mit der sexuellen aktivität der jeweils untersuchten bevölkerungsgruppe. die inkubationszeit beträgt 2-12 tage. j ätiopathogenese es existieren 2 herpes-simplex-viren, typ 1 (hsv-1) und typ 2 (hsv-2): 4 infektionen mit hsv-2 sind häufig mit erkrankungen im genitalbereich assoziiert. insbesondere sind infektionen des feten oder neugeborenen meist durch hsv-2 verursacht. 4 hsv-1-infektionen sind überwiegend im gesichtsbereich lokalisiert. hsv repliziert sich in mukosazellen (v. a. rachenraum, genitalschleimhaut) . anschließend dringt das virus in die nervenendigungen von peripheren sensorischen nerven ein und wandert in ihnen retrograd bis zu den spinalen hinterstrangganglien (bei hsv-1 meist ganglion des n. trigeminus, bei hsv-2 häufig sakralganglien; . abb. 14.1). an diesem ort liegt hsv in latenter form vor (keine produktion von infektiösem virus) und persistiert lebenslang im wirt. durch verschiedene faktoren (z. b. immunsuppression, stress) kann das virus jederzeit reaktiviert werden. nach einer solchen reaktivierung "wandert" hsv anterograd über die peripheren sensorischen nerven zur mukosaoberfläche des entsprechenden dermatoms und führt dort zur bläschenbildung mit aktiver virusreplikation (herpes labialis, rekurrierender herpes genitalis). entscheidend für die immunologische bewältigung einer hsv-infektion ist die zelluläre immunität. diaplazentar übertragene hsv-neutralisierende antikörper können bei exponierten neugeborenen eine hsv-infektion u. u. verhindern oder zumindest die schwere der erkrankung abmildern. dagegen können hsv-spezifische antikörper weder rekurrierende hsv-erkrankungen noch exogene hsv-infektionen verhindern. intrauterine herpes-simplex-virusinfektion in seltenen fällen kann es zu einer diaplazentaren, hämatogenen hsv-infektion des feten (überwiegend hsv-2) kommen. betroffene kinder sind meist hypotroph (85%), fast alle zeigen kurz nach geburt ein bläschenför-. abb. 14.1 pathogenese des herpes zoster und des rekurrierenden herpes simplex. oben: nach abklingen der primärinfektion mit vzv (varizellen) oder hsv (gingivostomatitis oder primärer herpes genitalis) wandern die viren retrograd entlang der sensorischen nerven zu den spinalganglien des rückenmarks, wo die viren lebenslang persistieren. unten: durch verschiedene faktoren (z. b. immunsuppression) kann vzv und hsv reaktiviert werden. beide viren wandern anschließend entlang der sensorischen nerven zu haut-bzw. schleimhaut (dermatom), wo es zur lokalen virusvermehrung und ausbildung von bläschen kommt: herpes zoster bei vzv, herpes labialis oder genitalis bei hsv die infektion beginnt mit unspezifischen symptomen (fieber, kopfschmerzen, krankheitsgefühl) . nach 1-7 tagen kommt es zu einer progressiven neurologischen symptomatik (fokale oder generalisierte krampfanfälle, verhaltensauffälligkeiten, vigilanzstörungen) bis hin zum koma. unbehandelt versterben 70% der patienten. verschiedene genetische faktoren prädisponieren für eine herpesenzephalitis (u. a. mutationen in den genen stat1, nemo, tlr3, traf3, . bildgebende verfahren (kraniales ct oder mr) und eeg zeigen im "typischen" fall fokale veränderungen uni-oder bilateral v. a. im bereich der temporallappen. im liquor findet sich meist eine pleozytose (überwiegend lymphozyten) und eine starke ei weißerhöhung. in bis zu 85% ist der liquor als folge der ausge dehnten nekrosen im zns hämorrhagisch. im frühstadium einer hsv-enzephalitis kann der liquor noch vollkommen unauffällig sein. die aciclovirtherapie hat die letalität auf ca. 29% gesenkt. eine vollständige ausheilung ohne residualfolgen findet sich in 38% der mit aciclovir behandelten enzephalitispatienten, bei kindern liegt der prozentsatz höher. bei unbehandelten patienten mit einer herpesenzephalitis dagegen kommt es nur in 2,5% der fälle zu einer restitutio ad integrum. chronisch rezidivierende verläufe, auch bei mit aciclovir behandelten patienten, kommen gelegentlich vor. ösophagus, der gastrointestinaltrakt, der respirationstrakt (pneumonie), das zns (enzephalitis) und andere organe (leber, nieren, milz, nebennieren) betroffen. j diagnose häufig kann die diagnose einer herpesinfektion der haut oder der schleimhäute aufgrund der typischen herpeseffloreszenzen klinisch gestellt werden. in zweifelsfällen wird hsv leicht aus bläscheninhalt, schleimhautabstrichen und bioptischem material isoliert. methode der wahl für die diagnose einer hsv-enzephalitis ist der hsv-genomnachweis im liquor mittels polymerasekettenreaktion. der serologische nachweis von spezifischen hsv-antikörpern im serum oder liquor spielt in der frühdiagnostik von hsv-infektionen nur eine sekundäre rolle. bei einer unklaren enzephalitis kann der nachweis von intrathekal produzierten hsv-antikörpern am tag 7-10 nach auftreten der symptome die ursache der erkrankung nachträglich beweisen. j therapie mittel der wahl bei hsv-infektionen im kindesalter ist das nukleosidanalogon aciclovir. zur behandlung von neonatalen hsv-infektionen sowie der herpesenzephalitis wird aciclovir in einer dosierung von 3-mal 15(-20) mg/kgkg/tag i.v. (frühgeborene nur 2-mal 10 mg/kgkg/ tag i.v.) für mindestens 14, besser 21 tage eingesetzt. bei zu kurzer aciclovirtherapie einer hsv-enzephalitis (<14 tage) kann es zu rezidiven kommen. neugeborene mit hsv-infektion und neurologischer beteiligung sollten im anschluss an die i.v.-aciclovirtherapie eine orale suppressionstherapie mit 3×300 mg/m 2 kö aciclovir über 6 monate erhalten. hierdurch wird das outcome hinsichtlich der neurologischen entwicklung verbessert. > bei klinischem verdacht auf herpesenzephalitis beginne man sofort mit einer ausreichend hoch dosierten intravenösen aciclovirtherapie, ohne die endgültige labordiagnostik abzuwarten. eine stomatitis aphthosa oder ein herpes labialis beim immunkompetenten kind wird im normalfall nur symptomatisch (z. b. mit bepanthenlösung oder -salbe) behandelt. bei allen komplizierten hsv-infektionen einschließlich dem herpes genitalis ist aciclovir derzeit das mittel der wahl (i.v., oral, topisch) . bei aciclovirresistenten hsv-stämmen (immunsupprimierte patienten) kann ein therapieversuch mit foscarnet unternommen werden. für die topische behandung einer hsv-keratokonjunktivitis stehen verschiedene wirksame medikamente zur verfügung wie acicloviraugensalbe und trifluridinaugentropfen. die therapie muss immer in enger zusammenarbeit mit einem diesbezüglich erfahrenen augenarzt erfolgen. bei patienten >18 jahren kann ein genitaler herpes (primärinfektion, rezidive) auch mit famciclovir oder valaciclovir behandelt werden. j prophylaxe bei schwangeren mit aktiver genitaler herpesinfektion (sowohl hsv-erstinfektion als auch -rezidiv) am geburtstermin sollte die geburt durch kaiserschnitt erfolgen, sofern der blasensprung nicht länger als 4-6 h zurückliegt. bei frauen mit rezidivierendem herpes genitalis in der spätschwangerschaft senkt eine orale aciclovir-oder valganciclovirtherapie die häufigkeit von hsv-2-rezidiven zum zeitpunkt der geburt. mütter mit florider hsv-1-infektion dürfen nur dann stillen, wenn die brust frei von frischen hsv-effloreszenzen ist und andere aktive läsionen abgedeckt sind. familienangehörige mit floridem herpes labialis müssen beim besuch eines neugeborenen immer einen mundschutz tragen und dürfen das kind nicht küssen. die labialen herpesläsionen müssen außerdem vorher mit aciclovirsalbe abgedeckt werden. eine langzeitchemoprophylaxe mit aciclovir kann bei immunsupprimierten und transplantierten patienten die häufigkeit (und schwere) von hsv-infektionen und -reaktivierungen signifikant senken. j epidemiologie varizella-zoster-virus (vzv) kommt ubiquitär vor und ist hochkontagiös. eine krankheitshäufung findet sich in den späten wintermonaten und im frühjahr. varizellen treten vorwiegend im kindesalter auf; bis zum 16. lebensjahr sind über 90% aller kinder infiziert. die ansteckung mit vzv erfolgt meist durch direkten kontakt von mensch zu mensch, seltener aerogen. die infektiosität bei varizellen beginnt 1-2 tage vor auftreten des exanthems und endet ca. 5 tage nach exanthemausbruch (immunkompetente kinder). der herpes zoster ist weniger kontagiös als varizellen. der kontakt mit einem zosterpatienten führt bei einer seronegativen person zu windpocken. die inkubationszeit bei varizellen beträgt meist 2 wochen (10-28 tage). j ätiopathogenese vzv gehört zur gruppe der herpesviren. eintrittspforte für vzv sind die schleimhäute der oberen atemwege. nach initialer virusvermehrung tritt nach 3-4 tagen eine erste virämie auf. hierbei wird vzv in t-zellen über den blutstrom im ganzen körper verteilt. in leber und milz findet anschließend eine massive virusvermehrung statt. am tag 6-7 post infectionem (p. i.) kommt es zur 2. virämie: hierbei wird vzv auch in die peripherie zur haut und zu den schleimhäuten transportiert. infizierte haut-und schleimhautzellen gehen bei der infektion zugrunde, es bilden sich die typischen bläschen mit virushaltigem inhalt. nach abklingen der varizellen wandert vzv retrograd entlang der peripheren sensorischen nerven zu den spinalganglien des rückenmarks (n. trigeminus, thorakale ganglien u. a.), wo das virus lebenslang persistiert (. abb. 14.1). in diesen spinalganglien liegt eine latente vzv-infektion vor, d. h. es wird kein komplettes virus produziert. vzv kann bei nachlassender zellulärer immunität sowie durch noch unbekannte mechanismen jederzeit reaktiviert werden: vzv wandert nun entlang der sensorischen peripheren nerven anterograd an die hautoberfläche, wo es im bereich der betroffenen dermatome zur virusvermehrung mit bläschenbildung (herpes zoster) kommt. . abb. 14.4 herpes labialis bei einem 8 jahre altem mädchen im gegensatz zu varizellen, bei denen es im rahmen der 2. virämie zu einem schubweisen auftreten von bläschen kommt (sternenhimmelbild mit verschiedenen stadien von effloreszenzen), befinden sich die bläschen beim herpes zoster im gleichen entwicklungsstadium: es liegt ein uniformes exanthem vor. für die immunologische kontrolle einer vzv-infektion ist das zelluläre immunsystem entscheidend. vzv-neutralisierende antikörper können die schwere des verlaufs von varizellen abmildern und u. u. auch eine vzv-infektion verhindern, insbesondere dann, wenn sie vor eintritt der primären virämie verabreicht werden. > windpocken treten nur einmal im leben auf. zweiterkrankungen sind sehr selten (ca. 1-2%). meist manifestieren sich windpocken als typisches bläschenförmiges exanthem (. abb. 14.5) mit nur leichtem fieber in den ersten 2-3 krankheitstagen. die effloreszenzen treten zunächst v. a. im gesicht, am behaarten kopf, und am stamm auf, weniger häufig kommt es zu einer zentrifugalen ausbreitung auf die extremitäten. die handinnenflächen sind meist ausgespart. frisch aufgetretene bläschen, die klare virushaltige flüssigkeit enthalten, trocken rasch ein und bilden häufig krusten. daneben treten immer wieder neue bläschen auf. diese hautveränderungen entwickeln sich schubweise mit einer dauer von bis zu 8 tagen und sind oft von einem starken juckreiz begleitet. durch kratzen kann es in betroffenen hautregionen zu exkoriationen und späterer narbenbildung kommen. bei der enzephalitis (häufigkeit ca. 1:10.000), die bereits früher im verlauf der varizellen auftritt und die im allgemeinen eine schlechtere prognose hat, kommt es zu schweren krampfanfällen und bewusstlosigkeit mit exitus letalis oder ausgeprägten defektheilungen. zerebrale insulte in form von akut auftretenden hemiplegien können erst nach monatelanger latenz nach einer vzv-infektion auftreten. andere seltene komplikationen sind peri-/parainfektiöse thrombozytopenische purpura (itp), purpura fulminans, myokarditis, arthritis, nephritis und reye-syndrom. bei zellulären immundefekten und immunsuppression (organtransplantation, hiv-infektion, immunsuppressive therapie, maligne grunderkrankungen) kommt es bei kindern häufig zu schweren progressiven varizellen mit viszeraler beteiligung wie pneumonie, meningoenzephalitis, hepatitis und pankreatitis. die letalität beträgt bis zu 20%. vari zellen im 1. und 2. schwangerschaftstrimenon (v. a. in der 13.-20. schwangerschaftswoche) führen in bis zu 2% zu einem konnatalen varizellensyndrom (cvs) mit hautnarben, gliedmaßenhypoplasien, dystrophie, katarakt sowie zns-schädigungen (. abb. 14.6). varizellen während der schwangerschaft (v. a. in der 16.-33. schwangerschaftswoche) können außerdem (in über 1%) zum auftreten eines herpes zoster im ersten lebensjahr führen. ein herpes zoster bei einer immunkompetenten schwangeren dagegen führt nur extrem selten zu einer konnatalen oder neonatalen vzv-infektion. j ätiopathogenese ebv gehört zur gruppe der herpesviren. eintrittspforte für ebv ist der rachenraum (waldeyer-rachenring, tonsillen), wo das virus zu einer sog. lytischen infektion des lymphoepithelialen gewebes (b-zellen, epithelzellen) mit anschließender produktion von infektiösem ebv führt (invasive phase). im weiteren verlauf (nach ca. 2 wochen) kommt es zur virämie oder mehreren virämischen phasen. hierbei werden ebv-infizierte b-zellen über den blutstrom in andere organe (leber, milz, knochenmark, lymphknoten, evtl. zns) transportiert, erst nach einer inkubationszeit von 10-50 tagen treten die klinische symptome auf. in den im blut zirkulierenden b-zellen kommt es zunächst noch zu einer lytischen ebv-infektion mit produktion von infektiösem virus, später liegt nur noch eine sog. latente infektion vor, d. h. es werden nur noch wenige virusantigene (kernantigene ebna1-6 und membranantigene lmp1 und 2) exprimiert. diese b-zellen werden hierdurch zu lymphoblastoiden zellen "transformiert" und erwerben die fähigkeit zur unbegrenzten teilung und vermehrung (immortalisation). beim immunkompetenten menschen werden nach einer ebv-infektion rasch aktivierte zytotoxische t-zellen vom cd8+-typ gebildet, die selektiv nur die ebv-infizierten b-zellen weitestgehendeliminieren. diese aktivierten t-zellen bilden einen großen anteil der typischen "pfeiffer-zellen" (syn. lymphatische reizformen, virozyten) und der teilweise extremen lymphozytose im blutbild von patienten mit akuter infektiöser mononukleose (. abb. 14.8). nach durchgemachter ebv-infektion persistiert ebv lebenslang in ruhenden b-zellen im knochenmark. ebv kann in diesen zellen jederzeit reaktiviert werden. bei eingeschränkter zellulärer immunität (z. b. nach medikamentöser immunsuppression, aids) können sich diese b-zellen -abhängig vom ausmaß der immunsuppression -expandieren und so zu schweren lymphoproliferativen krankheitsbildern und b-zelllymphomen führen. in 70-90% tritt initial eine ausgeprägte tonsillopharyngitis mit fibrinbelägen (. abb. 14.10) auf, die in der 2. krankheitswoche meist rasch abheilt. eine splenomegalie findet sich bei 50-60% der patienten in der 2. und 3. krankheitswoche. seltener (15-25%) ist eine hepatitis mit und ohne ikterus. in 5-10% treten meist flüchtige morbilliforme exantheme auf. lymphoproliferative krankheitsbilder kinder und jugendliche mit angeborenen zellulären immundefekten (x-chromosomale lymphoproliferative erkrankung, xlp u. a.), aber auch mit erwor bener immundefizienz (organtransplantation, immunsuppressiver therapie, hiv-infektion) zeigen eine eingeschränkte immunkompetenz gegenüber ebv. hierdurch kommt es zu einer verschiebung des virus-wirt-gleichgewichts zugunsten des virus. ebv-infizierte b-zellen können daher unkontrolliert auswachsen und zu polyoligoklonalen b-zell-lymphoproliferationen bis hin zu monoklonalen malignen lymphomen führen. die häufigkeit dieser komplikationen ist direkt abhängig von der schwere der immunsuppression. ebv-assoziierte maligne erkrankungen ebv findet sich zu 100% in tumorzellen des endemischen burkitt-lymphoms und des nasopharynxkarzinoms. darüber hinaus lässt sich das virus in geringerer häufigkeit auch in anderen malignomen (m. hodgkin, b-und t-zell-lymphome) nachweisen. die rolle von ebv in der tumor entstehung und/oder -progression ist noch weitgehend unbekannt. j diagnose die infektiöse mononukleose kann meist klinisch diagnostiziert werden. labor im blutausstrich lassen sich typischerweise zahlreiche aktivierte t-lymphozyten (pfeiffer-zellen) nachweisen (. abb. 14.8). in zweifelsfällen wird bei immunkompetenten patienten die diagnose serologisch gesichert (. abb. 14.11). der mononukleoseschnelltest zum nachweis von heterophilen antikörper ist im kindesalter sehr wenig sensitiv und spielt in der pädiatrie keine rolle. die bestimmung der viruslast im blut mittels polymerasekettenreaktion ist bei immunsupprimierten patienten mit ebv-assoziierten lymphoproliferativen krankheitsbildern sinnvoll. immunsupprimierte patienten bei angeborenen immundefekten (xlp etc.) kann eine frühzeitige stammzelltransplantation (nabelschnurblut, knochenmark) zu einer immunrekonstitution führen und so spätere komplikationen durch ebv verhindern. bei auftreten von ebv-lymphomen im rahmen einer immunsuppressiven therapie führt die rechtzeitige reduktion der medikamentendosis häufig noch zu einer rückbildung der tumoren. der einsatz von monoklonalen anti-b-zellantikörpern (anti-cd20-rituximab) führt bei ebv-assoziierten lymphoproliferativen krankheitsbildern in bis zu 60% zu einer kompletten remission. die präsymptomatische therapie mit ganciclovir oder valganciclovir kann bei kindern nach organtransplantationen (v. a. lebertransplantation) wahrscheinlich die häufigkeit von ebvassoziierten lymphoproliferativen komplikationen reduzieren. bei organtransplantierten patienten kann die infusion von ebv-spezifischen zytotoxischen t-zellen des organspenders ebv-positive lymphome zur rückbildung bringen oder die neuentstehung von lymphomen verhindern (sog. "adoptiver immuntransfer"). zytomegalievirusinfektionen j epidemiologie die durchseuchungsrate mit dem zytomegalievirus (cmv) in der bevölkerung ist v. a. abhängig vom alter und lebensstandard. in deutschland sind ungefähr 50% der erwachsenen gesamtbevölkerung seropositiv für cmv. cmv-infektionen als folge von organtransplantationen treten meist nach 4 wochen bis 4 monaten auf, als folge einer bluttransfusion bereits nach 3-12 wochen. cmv wird horizontal über infektiöse körperflüssigkeiten (speichel, urin, muttermilch), blut, blutprodukte oder transplantierte organe, sowie vertikal (konnatale infektion) übertragen. cmv-positive säuglinge und kleinkinder können über wochen infektiöses cmv ausscheiden. j ätiopathogenese cmv gehört zur gruppe der herpesviren. cmv repliziert sich in epithelialen zellen der speicheldrüsen und der nieren, bei schweren generalisierten infektionen auch in leber, genitaltrakt, lungen und anderen organen. die produktive cmv-infektion führt in diesen zellen zu typischen intranukleären einschlüssen ("eulenaugenzellen"; . abb. 14.12) und zu massiver vergrößerung der infizierten zellen ("zytomegalie"). während der virämischen phase(n) findet sich cmv überwiegend zellassoziiert in der fraktion der polymorphkernigen granulozyten. nach einer primärinfektion persistiert cmv lebenslang im blut in monozyten/makrophagen sowie in anderen infizierten organen (speicheldrüsen, nieren). das virus kann bei immunsuppression jederzeit reaktiviert werden. bei der immunologischen bewältigung einer cmv-infektion spielt die zelluläre immunität (u. a. cmv-spezifische cd8+-t-zellen) eine entscheidende rolle. neutralisierende cmv-spezifische antikörper können bei einer cmv-infek tion die schwere einer cmv-erkrankung abmildern. personen die meisten cmv-infektionen verlaufen bei immunkompetenten personen asymptomatisch bzw. subklinisch. in seltenen fällen (1:1.000) manifestiert sich eine cmv-infektion als mononukleoseähnliches krankheitsbild mit ähnlichen klinischen symptomen und blutbildveränderungen (lymphozytose, atypische lymphozyten). das risiko und die schwere einer cmv-erkrankung korreliert mit dem ausmaß der immunsuppression, der virusmenge im blut und anderen faktoren, die eine reaktivierung von cmv begünstigen (akute graft-versus-host-erkrankung, infektion mit anderen herpesviren, cmv-seronegativer transplantatempfänger eines cmvpositiven organs). . abb. 14.12 eulenaugenzellen in der niere eines verstorbenen kindes mit konnataler zytomegalie . abb. 14.13 cmv-chorioretinitis bei einem 14 jahre alten patienen unter immunsuppressiver therapie (mit freundl. genehmigung von prof. dr. handrick, frankfurt/oder) konnatale cmv-infektionen die zytomegalie ist die häufigste konnatale infektion (ca. 0,2-0,4% aller neugeborenen). eine cmv-primärinfektion in der schwangerschaft führt bei ca. 7-10% der infizierten kinder zu einer schweren generalisierten cmv-erkrankung. eine cmv-reaktivierung oder -zweitinfektion in der schwangerschaft führt dagegen nur sehr selten zu einer symptomatischen zytomegalie beim kind. das risiko für eine symptomatische konnatale zytomegalie scheint direkt mit der höhe von maternalen neutralisierenden cmv-spezifischen antikörpern sowie der cmv-virusmenge im blut zu korrelieren. etwa 90% aller neugeborenen mit konnataler cmv-infektion sind bei geburt klinisch symptomfrei. ein teil dieser kinder (7-15%) kann aber später eine bleibende hörstörung entwickeln. aus diesem grund sollten bei allen kindern mit konnataler cmv-infektion wiederholt hör-und sehprüfungen veranlasst werden. eine symptomatische konnatale zytomegalie (nach cmv-erstinfektion in der schwangerschaft) ist eine multisystemerkrankung mit hoher morbidität und letalität. erkrankte kinder zeigen eine ausgeprägte intrauterine wachstumsretardierung, ikterus, hepatosplenomegalie, thrombozytopenie mit petechien (77%), pneumonie sowie schwerste zns-schädigungen (bis zu 70%) mit mikrozephalus (53%), intrazerebralen verkalkungen, chorioretinitis, späterer taub-und blindheit und geistiger behinderung. die letalität liegt bei bis zu 30%. für die einschätzung von späteren neurologischen defiziten scheint ein schädel-ct oder mrt die derzeit sensitivste methode zu sein. der nachweis von cmv-dna sowie eine eiweißerhöhung im liquor sind wahrscheinlich mit einer schlechteren prognose hinsichtlich der neurologischen entwicklung assoziiert. die schlechteste prognose haben konnatale cmv-infektionen häufig dann, wenn sie im 1. trimenon auftreten. peri-und postnatale cmv-infektionen infektionen, die durch cmv im zervikal-und vaginalsekret bzw. durch infektiöse muttermilch übertragen werden, verlaufen bei reifgeborenen, immunkompetenten kindern meist asymptomatisch oder mild. bei sehr kleinen frühgeborenen kann eine perinatale cmv-infektion (v. a. via cmv-positive muttermilch) eine schwere interstitielle pneumonie, hepatosplenomegalie und ein sepsisähnliches krankheitsbild verursachen. die letalität liegt bei 24%. j diagnose sensitive und spezifische parameter für eine floride cmv-infektion sind der quantitative nachweis des cmv-antigens pp65 oder des cmv-genoms (pcr) im blut (oder anderen körperflüssigkeiten). beide methoden erlauben eine bestimmung der viruslast und somit auch das monitoring einer virostatischen therapie mit ganciclovir bzw. valganciclovir. der "klassische nachweis" von cmv besteht in der isolierung aus verschiedenen körperflüssigkeiten (urin, speichel etc.). herpesvirus-typ-6-infektionen zu den begleitsymptomen und komplikationen, die meist schon im frühstadium (tag 1-4) auftreten, gehören gastroenteritis (55-70%), lidödeme (bis 30%), nagayama-flecken (papeln auf dem weichen gaumen und der uvula 65%), husten (50%), zervikale lymphadenopathie (30-35%), vorgewölbte fontanelle (26-30%) sowie fieberkrämpfe (5-35%). die angaben über die häufigkeit eines exanthema subitum nach einer primärinfektion mit hhv-6 schwanken stark. primärinfektionen mit hhv-6 stellen insgesamt eine häufige ursache von hochfieberhaften infekten (mit und ohne exanthem) bei kleinkindern dar. fieberkrämpfe und andere klinische manifestationen während einer hhv-6-primärinfektion kommt es nicht selten zu einer invasion des virus in das zns. bei bis zu 40% von kindern mit florider hhv-6-infektion kann das virus im liquor nachgewiesen werden, wobei entzündliche liquorveränderungen (pleozytose, eiweißvermehrung) fehlen. fieberkrämpfe treten in bis zu 35% auf. zu den seltenen neurologischen komplikationen gehören die meningoenzephalitis und das guillain-barré-syndrom. in wenigen fällen kann eine hhv-6-infektion, v. a. bei älteren kindern, auch mit einer mononukleoseähnlichen symptomatik, einer fulminanten hepatitis, einem schwer verlaufenden hämophagozytosesyndrom sowie mit der verschlimmerung einer idiopathischen thrombozytopenie (itp) assoziiert sein. organtransplantation kommt es häufig (in bis zu 80%) zu einer reaktivierung von hhv-6, die möglicherweise zu einer vermehrten transplantatabstoßung führt. nach hhv-6-infektion bzw. -reaktivierung können folgende klinische komplikationen auftreten: interstitielle pneumonie, graft-versus-host-erkrankung (gvhd) mit und ohne exanthem, enzephalopathie und knochenmarksuppression (nach knochenmarktransplantation). inwieweit diese komplikationen tatsächlich ursächlich nur durch hhv-6, oder möglicherweise erst in verbindung mit zusätzlichen infektionen (hiv, cmv und andere herpesviren) hervorgerufen werden, ist derzeit nicht bekannt. j diagnose die diagnose eines exanthema subitum kann bei typischer ausprägung klinisch gestellt werden. labor am 3. und 4. fiebertag fällt im blutbild häufig eine leukopenie mit relativer lymphozytose (bis zu 90%) auf. eine vermutete hhv-6-primärinfektion wird durch den serologischen nachweis von hhv-6-spezifischen antikörpern (anti-hhv-6-igm und/oder anstieg von anti-hhv-6-igg) bestätigt. eine hhv-6-reaktivierung bei immunsupprimierten kindern kann bei akut ansteigenden anti-hhv-6-antikörpertitern (bei bekannten ausgangstitern) vermutet werden. hhv-6-dna kann mittels pcr nachgewiesen werden (speichel, blut, plasma, liquor, urin). j therapie, prophylaxe eine spezifische therapie existiert nicht. bei hohem fieber erfolgt eine adäquate symptomatische fiebersenkung. bei (immunsupprimierten) patienten mit schweren hhv-6-assoziierten komplikationen (pneumonie, enzephalitis) ist ein therapieversuch mit foscarnet und/oder ganciclovir, zu erwägen. herpesvirus-typ-7-infektionen j epidemiologie auch herpesvirus typ 7 (hhv-7) kommt ubiquitär vor. die durchseuchung in der bevölkerung liegt z. t. bei über 90%. in den ersten 6 lebensmonaten ist eine hhv-7-infektion sehr selten, am ende des 1. lebensjahr sind bis zu 30%, am ende des 6. lebensjahrs bis zu 86% der kinder seropositiv. die primärinfektion mit hhv-7 erfolgt i. a. deutlich später als die mit hhv-6. die übertragung erfolgt über infektiösen speichel, u. a. innerhalb der familie, und möglicherweise auch über infizierte muttermilch. j ätiopathogenese hhv-7 gehört zur gruppe der herpesviren. die pathogenese ist ähnlich wie bei der hhv-6-infektion. j klinik hhv-7 ist (neben hhv-6) der erreger des exanthema subitum (dreitagefieber, roseola infantum). im vergleich zu hhv-6 scheint hhv-7 insgesamt in einer höheren frequenz zu fieberkrämpfen zu führen. das mittlere alter bei symptomatischen hhv-7-infektionen liegt bei 26 monaten, bei hhv-6-infektionen bei ca. 9 monaten. gelegentlich führt eine hhv-7-infektion bei älteren kindern auch zu einem mononukleoseähnlichen krankheitsbild. in den meisten fällen verlaufen hhv-7-infektionen allerdings subklinisch oder gehen mit einem unspezifischen fieberhaften infekt einher. j diagnose, therapie die diagnose eines exanthema subitum erfolgt bei typischer symptomatik klinisch. nur in ausnahmefällen scheint eine weitere virologische abklärung gerechtfertigt. hhv-7 kann mittels polymerasekettenreaktion im speichel, im peripheren blut, in lymphatischem gewebe und teilweise auch in der muttermilch nachgewiesen werden. der nachweis von hhv-7-spezifischen serumantikörpern erfolgt mittels indirekter immunfluoreszenz oder elisa. zu berücksichtigen ist hierbei, dass antikörper gegen hhv-7 teilweise auch mit hhv-6 kreuzreagieren können. eine spezifische therapie oder eine impfung gegen hhv-7 gibt es nicht. bei schwerem klinischem verlauf erfolgt eine entsprechende symptomatische therapie. herpesvirus-typ-8-infektionen j epidemiologie das herpesvirus typ 8 (hhv-8) kommt ebenfalls ubiquitär vor. die seroprävalenz in der bevölkerung ist in afrikanischen ländern und in japan (bis zu 100%) deutlich höher als in europa und in den usa (20-30%). hhv-8 wird überwiegend (homo)sexuell übertragen. die ansteckung von kindern und jugendlichen erfolgt wahrscheinlich über infektiösen speichel. bei nierentransplantationen kann eine transmission von hhv-8 in bis zu 10% erfolgen. j ätiologie hhv-8 gehört zur gruppe der herpesviren. hhv-8 zeigt in vitro und teilweise auch in vivo einen tropismus zu cd19+-b-zellen, zu endothelialen zellen und ganglienzellen. gefährdet sind u. a. patienten mit gleichzeitig bestehender chronischer hepatitis c. das krankheitsbild ist gekennzeichnet durch ein schnell zunehmendes leberversagen. die letalität ist hoch. j diagnose die diagnose einer hav-infektion sollte immer erwogen werden, wenn bei mehreren familienangehörigen oder anderen kontaktpersonen ein ikterus und gastrointestinale begleitsymptome auftreten. labor die diagnose eine hav-infektion wird durch den nachweis von virusspezifischen antikörpern im serum (anti-hav-igm positiv und/oder anstieg von anti-hav-igg) gesichert (. abb. 14.14). mit ausbruch der erkrankung werden anti-hav-igm-antikörper gebildet, die für 3-12 monate im blut nachweisbar sein können. die anti-hav-igg-antikörper persistieren (wahrscheinlich) lebenslang und sind ausdruck von immunität. j therapie eine spezifische therapie gibt es nicht. bettruhe und spezielle diäten haben keinen einfluss auf den krankheitsverlauf und sind nicht indiziert. symptomatische maßnahmen richten sich nach den jeweiligen beschwerden. im seltenen fall einer fulminanten hepatitis ist eine lebertransplantation zu erwägen. j prophylaxe die hepatitis-a-impfung (2 injektionen im abstand von 6 monaten; für kinder zugelassen ab dem 2. lebensjahr) kann bei rechtzeitiger gabe eine symptomatische wildvirusinfektion wirksam verhindern. sie wird allen potenziell gefährdeten immungesunden personen (u. a. seronegatives personal in medizinischen einrichtungen und kindertagestätten, reisende in regionen mit hoher hav-prävalenz, patienten mit chronischer hepatitis c) empfohlen. bereits 2 wochen nach der 1. impfung sind über 95% der geimpften geschützt. diese impfung schützt z. t. auch dann noch, wenn sie in der frühen inkubationszeit gegeben wird (sog. inkubations-oder "riegelungsimpfung"). nach einer vorausgegangenen wildvirusexposition kann durch frühzeitige einmalige gabe von immunglobulinen der ausbruch einer hepatitis a verhindert oder der verlauf abgemildert werden (postexpositionsprophylaxe). werden die immunglobuline erst nach einem intervall von 10(-14) tagen gegeben, ist keine wirkung mehr zu erwarten. im durchschnitt gelten patienten in einem zeitraum von 2 wochen vor bis 2 wochen nach ausbruch der erkrankung als ansteckend. hospitalisierte kinder mit einer hepatitis a sollten, sofern dies aus medizinischer sicht vertretbar ist, nach diagnosestellung schnellstmöglich nach hause entlassen werden. ansonsten erfolgt eine isolierung (einzelzimmer, kohortierung) für max. 2 wochen. neugeborene hbsag-positiver mütter erhalten am besten noch im kreißsaal, ansonsten innerhalb der ersten 12 h post partum simultan 0,5 ml hepatitis-b-impfstoff i.m. sowie auf der kontralateralen seite 0,5 ml/kgkg hepatitis-b-immunglobulin i.m. nach 4 wochen und 6 monaten erfolgt eine weitere impfung. geimpfte neugeborene dürfen gestillt werden. mutter und kind müssen nach geburt nicht isoliert oder voneinander getrennt werden. bei nichtimmunen personen mit fehlenden oder erniedrigten anti-hbs-antikörpern wird nach kontakt mit virushaltigem blut oder sekret schnellstmöglich (spätestens innerhalb von 12 h) hepatitis-b-immunglobulin i.m. verabreicht (postexpositionsprophylaxe). gleichzeitig sollte möglichst auch aktiv geimpft werden. in den ersten 1-3 monaten einer akuten hepatitis c kann der antikörpernachweis noch negativ verlaufen. die hcv-serologie ist daher v. a. für die diagnose von bereits einige zeit zurückliegenden oder chronischen hcv-infektionen geeignet. das hcv-genom kann mittels pcr im blut oder in der leberbiopsie nachgewiesen werden. die bestimmung der viruslast im blut (und ggf. auch im leberbioptat) sowie eine genotypisierung der vorliegenden hcv-variante sind hinsichtlich der indikation für eine interferon-α-therapie sowie für das anschließende monitoring wichtig. j therapie für eine antivirale therapie der akuten hepatitis c liegen für kinder und jugendliche bisher keine daten vor. da von einer hohen chronifizierungsrate ausgegangen werden muss, sollte wie bisher bei erwachsenen vorgegangen werden. die kombinationstherapie mit normalem oder pegyliertem (= retardiertem) interferon-α (zugelassen bei kindern ab 3 jahren) plus ribavirin ist derzeit bei kindern noch die standardtherapie der chronischen hepatitis c. prädiktoren für einen therapieerfolg sind der hcv-genotyp (v. a. typ 2 und 3), die dauer der krankheit und möglicherweise die hcv-konzentration im serum. bei erwachsenen mit chronischer hepatitis c führen neue virustatika (z. b. epclusa in kombination mit sofosbuvir und velpatasvir) zu einer ausheilung von meist über 90%. es ist zu hoffen, dass diese therapieoptionenn in einigen jahren auch bei kindern zugelassen sind. diese gehen dabei zugrunde, wobei die erythropoese kurzzeitig unterbrochen wird. eintrittspforte für parvovirus b19 ist der obere respirationstrakt. nach 4-5 tagen kommt es zu einer virämie, begleitet von einer retikulozytopenie. etwa 1 woche nach der virämie tritt meist das typische exanthem auf, möglicherweise hervorgerufen durch antigen-antikörper-komplexe. die entstehung eines hydrops fetalis nach intrauteriner parvovirus-b19-infektion wird v. a. mit der infektion von fetalen erythroblasten und der daraus resultierenden anämie erklärt. in bestimmten fällen kann möglicherweise auch die infektion des fetalen myokards mit parvovirus b19 zu einer eingeschränkten herzfunktion mit der folge eines hydrops fetalis führen. j klinik ringelröteln diese typische "kinderkrankheit" (syn: erythema infectiosum, 5. krankheit, epidemisches megalerythem, großfleckfieber, kinderrotlauf) wird in 15-20% aller frisch infizierten personen beobachtet. nach einem 2-3 tage andauernden prodromalstadium mit unspezifischen symptomen wie fieber, kopfschmerzen und schüttelfrost (zeitgleich mit der virämie) und einem anschließenden beschwerdefreien intervall von ca. 1 woche tritt im bereich der wangen ein hochrotes, leicht erhabenes konfluierendes exanthem auf (. abb. 14.19a). gleichzeitig kann eine periorale blässe wie beim scharlach bestehen. an den folgenden tagen treten . abb. 14.19 ringelröteln. a typisches konfluierendes exanthem im bereich beider wangen ("slapped cheek") mit perioraler blässe, b girlandenförmige exantheme a b an den extremitäten und am rumpf makulopapulöse effloreszenzen auf, die konfluieren. durch zentrales abblassen entstehen die typischen girlanden-netzförmigen muster (. abb. 14.19b) . in den folgenden tagen und wochen können immer wieder neue pleomorphe exantheme auftreten, teilweise provoziert durch sonnenlicht oder hohe temperatur. das allgemeinbefinden der p atienten ist meist nur wenig beeinträchtigt. andere begleitsymptome (juckreiz, kopfschmerzen, fieber, gelenkbeschwerden, bauchschmerzen) sind bei kindern eher selten. bei adoleszenten und jungen erwachsenen kommen auch vaskulitische exantheme vor mit strenger begrenzung auf die hände und füße ("handschuh-socken-syndrom"). die parvovirus-b19-assoziierte polyarthritis, die bevorzugt knie-, fuß-und die proximalen interphalangealgelenke befällt und häufiger bei mädchen und jungen frauen auftritt, ist praktisch immer selbstlimitierend. hydrops fetalis eine parvovirus-b19-primärinfektion in der schwangerschaft führt in bis zu 35% auch zu einer infektion des feten. in den allermeisten fällen ist diese fetale infektion klinisch stumm, in bis zu 5% der fälle kommt es zum abort (meist innerhalb von 3-6 wochen nach der mütterlichen infektion). die entwicklung eines hydrops fetalis nach einer mütterlichen (und fetalen) parvovirus-b19-infektion ist insgesamt selten, sie liegt bei ca. 4%. trotzdem ist die parvovirus-b19-infektion wahrscheinlich die häufigste ursache des nicht immunologisch bedingten hydrops fetalis. die fetalen komplikationen sind am höchsten bei einer parvovirus-b19-infektion zwischen der 13. und 20. gestationswoche. j klinik eine rotavirusinfektion führt bei kleinen kindern praktisch immer zu fieber, erbrechen sowie enteritis mit häufigen wässrigen, nicht blutigen stühlen. die durchfälle können bis zu 5-7 tage andauern. in der mehrzahl der fälle kommt es hierbei zu einer unterschiedlich ausgeprägten, meist isotonen, selten einer hypertonen dehydratation. ein teil der kinder zeigt zusätzliche klinische symptome wie leichte vergrößerung der zervikalen lymphknoten, otitis und rhinitis. bei immunsupprimierten kindern kann eine rotavirusinfektion chronisch und teilweise sehr schwer verlaufen. in sehr seltenen fällen können rotavirusinfektionen auch zu einer enzephalitis und zu krampfanfällen mit und ohne fieber führen. j diagnose die praktikabelste und schnellste methode zum nachweis einer rotavirusinfektion ist der rotavirusantigennachweis im stuhl (eia). follikuläre konjunktivitis häufig, meist unilateral 1-7, 9-11, 15-17, 19, 20, 22, 37 haut exantheme (morbilli-, rubelli-und roseolaform) j ätiopathogenese rhinoviren sind rna-viren aus der familie der picornaviren (pico, klein). rhinoviren infizieren v. a. die schleimhäute der nase und des oberen respirationstrakts, seltener auch die unteren atemwege. hier vermehrt sich das virus im zytoplasma. anschließend werden die neu gebildeten rhinoviren in großer menge (bis zu 106 viruspartikel pro ml nasensekret), v. a. in den ersten 3 tagen der erkältung, sowie meist noch 2-3 wochen danach ausgeschieden. während einer rhinovirusinfektion kommt es meist nur zu minimalen epithelsschäden ohne zilienverlust sowie zu einer lokalen infiltration mit granulozyten und monozyten. j klinik in den meisten fällen führt eine rhinovirusinfektion zu einer "erkältung" der oberen luftwege ("common cold") mit einer nur geringen allgemeinen klinischen beeinträchtigung. hauptsymptome sind schnupfen, husten und halsschmerzen. die regionalen zervikalen lymphknoten sind in ca. 50% vergrößert. eine meist leichte otitis media tritt in ca. 20% der fälle auf. die klinischen beschwerden sind in den ersten 3 tagen am ausgeprägtesten, in den meisten fällen bilden sie sich innerhalb einer woche, seltener nach 2 wochen zurück. vor allem bei jüngeren kindern können rhinovirusinfektionen auch zu schweren verläufen mit bronchiolitis, bronchopneumonie und obstruktiver bronchitis führen. todesfälle sind beschrieben worden. in den meisten fällen entwickeln die betroffenen kinder einen zunehmenden keuchenden husten und dyspnoe bei häufig nur leicht erhöhten temperaturen. in schweren fällen finden sich eine tachypnoe mit nasenflügelatmen und thorakalen einziehungen, eine zyanose sowie eine tachykardie. das atemgeräusch kann (als indirektes zeichen der überblähung bei atemwegsobstruktion) abgeschwächt sein. die röntgenaufnahme der lunge zeigt als typischen befund bei einer bronchiolitis eine überblähung der lungen und ggf. einige infiltrate. die blutgasanalyse ergibt häufig eine leichte bis mäßige hypoxie, eine hyperkapnie ist ausdruck der zunehmenden ateminsuffizienz, die einer dringenden intensivmedizinischen betreuung bedarf. bei früh-und neugeborenen kann eine rsv-infektion mit plötzlich auftretenden teils massiven apnoen, irritabilität, trinkverweigerung und lethargie -ohne äußere zeichen eines atemwegsinfekts -einhergehen. rsv-reinfektionen verlaufen i. a. milder und manifestieren sich häufig als oberer atemwegsinfekt oder tracheobronchitis. bei kindern mit schweren grunderkrankungen (bronchopulmonale dysplasie, herzfehler, immundefizienz) kann jede rsv-infektion zu einem lebensbedrohlichen krankheitsbild führen. j diagnose die diagnose einer rsv-infektion basiert auf der klinischen symptomatik, auf dem zeitlichen hintergrund (winterhalbjahr? bekannte rsv-epidemie?), auf der altersverteilung (kind <2 jahre) und der positiven virusdiagnostik. für den klinischen alltag sind rsv-antigen-tests zum virusnachweis in nasopharyngealsekret von großer praktischer bedeutung. daneben ist der nachweis von rsv durch anzucht in der zellkultur oder mittels pcr möglich. die serologische diagnostik spielt in der praxis keine rolle. j therapie im vordergrund steht die intensivmedizinische betreuung (u. u. kontrollierte beatmung) der meist schwer kranken kinder. symptomatisch können β 2 -sympathomimetika, racemisches epinephrin oder adrenalin verabreicht werden. kortikosteroide, theophyllin und mukolytika sind unwirksam. ribavirintherapie als virostatikum steht ribavirin zur verfügung. aufgrund unterschiedlicher, z. t. enttäuschender studienergebnisse hinsichtlich der wirksamkeit, der umständlichen inhalativen applikation sowie potenzieller nebenwirkungen (teratogenität) ist die indikation für ribavirin seit 1996 stark eingeschränkt worden. in über 90% lässt sich in tumorgewebe hpv nachweisen. zu den kanzerogenen typen gehören v. a. hpv-16 und hpv-18. j diagnose die diagnose erfolgt in den allermeisten fällen klinisch anhand der typischen manifestation (warzen, papillome). in unklaren fällen sollte immer eine histologische abklärung erfolgen. der nachweis und die typisierung von papillomaviren im biopsat erfolgt -falls überhaupt erforderlich -mit molekularbiologischen techniken. bei genitaler krankheitslokalisation sind immer auch andere geschlechtskrankheiten auszuschließen. bei kleinen kindern sollte auch an einen sexuellen missbrauch gedacht werden. larynxpapillome verursachen bei kindern eine mehr oder weniger ausgeprägte dyspnoe, die diagnose erfolgt mittels laryngo-oder bronchoskopie. j therapie vor jeder therapieentscheidung ist die hohe spontanremissionrate von warzen im kindesalter zu bedenken! hautwarzen werden durch kontaktvereisung mit flüssigstickstoff oder durch eine keratolytische und antiproliferative lokalbehandlung mit salicylsäureund 5-fluorouracil-haltigen präparaten (z. b. verrumal) behandelt. die früher durchgeführte warzenentfernung mittels elektrokauter ist wegen der schmerzhaftigkeit und der möglichen narbenbildung obsolet. filiforme warzen können chirurgisch im hautniveau exzidiert werden. larynxpapillome werden, v. a. bei entsprechenden atembeschwerden, mittels laser-oder kryotherapie behandelt. unterstützend kann zur proliferationshemmung lokal oder systemisch interferon α appliziert werden. die behandlung von genitalen warzen kann sehr schwierig sein und sollte immer von einem erfahrenen gynäkologen durchgeführt werden. die lokalbehandlung erfolgt u. a. mit podophyllinlösung (5-25%), kryotherapie sowie mittels co 2 -laser. die prodromi mit fieber und katarrhalischen symptomen (8-12 tage p. i.) signalisieren den beginn der immunologischen abwehrreaktion. so ist auch das exanthem folge der explosiven auseinandersetzung zwischen virusspezifischen t-zellen und virusinfizierten epithel-und endothelzellen. spezifische antikörper scheinen bei der überwindung der akuten phase keine rolle zu spielen. > masern gehen immer mit einer immunschwäche einher, die wochen bis monate anhalten kann. hauttests vom verzögerten typ (tuberkulintest!) werden vorübergehend negativ. außerdem kommt es durch die immunschwäche zu bakteriellen zweiterkrankungen oder zur aktivierung chronischer krankheitsprozesse. die pathogenese der para-(post-)infektiösen masernenzephalitis ist bisher nicht geklärt. histopathologisch finden sich im gehirn perivaskuläre demyelinisierungen und perivaskuläre lymphozyteninfiltrate. virale antigene oder rna-sequenzen lassen sich aber nicht nachweisen. j klinik die erkrankung beginnt mit hohem fieber und uncharakteristischen katarrhalischen symptomen, wie schnupfen, halsschmerzen, heiserkeit und bellendem husten (prodromalstadium). die patienten sind aufgrund einer konjunktivitis und einer (milden) keratitis ausgesprochen lichtscheu. gleichzeitig oder 1-2 tage später treten feine, kalkspritzerartige stippchen, bevorzugt an der wangenschleimhaut gegenüber den molaren, auf (koplik-flecken; . abb. 14.25). außerdem entwickelt sich ein fleckiges, dunkelrotes enanthem am weichen gaumen. nach leichtem fieberabfall geht das prodromalstadium 3-4 tage später unter erneutem hohem fieberanstieg in das exanthemstadium über. das makulopapulöse exanthem beginnt hinter den ohren (. abb. 14.27) und im gesicht und breitet sich weiter zentrifugal über den ganzen körper bis zu den füßen aus (. abb. 14.28). nach dem 3. exanthemtag folgt bei unkomplizierten verläufen rasche entfieberung und abblassen des exanthems. meist besteht eine generalisierte lymphadenopathie, wobei auch die hilären, paratrachealen und mesenterialen lymphknoten betroffen sind. bei ca. 50% der infizierten treten pathologische eeg-veränderungen auf, die sich später in den allermeisten fällen zurückbilden. 4 mitigierte masern treten bei jungen säuglingen auf, die noch maternelle antikörper besitzen und auch bei kindern nach gabe von immunglobulinen. 4 bei patienten mit schweren t-zelldefekten kann das exanthem völlig fehlen ("weiße masern"). es entwickelt sich eine riesenzellpneumonie, die fast immer zum tode führt. j komplikationen am häufigsten sind bakterielle sekundärinfektionen; und zwar bronchopneumonien, otitis media und diarrhö. weitere komplikationen betreffen das zentrale nervensystem. im vordergrund steht die akute masernenzephalitis mit einer häufigkeit von 1:500-1:2000. die masernenzephalitis tritt bevorzugt am 3.-9. tag nach exanthembeginn auf. typisch sind bewusstseinsstörungen (somnolenz, koma), zerebrale krampfanfälle, neurologische herdsymptome (hemiplegien, hirnnervenparesen) und gelegentlich auch myelitische symptome. die masernenzephalitis hat auch heute noch eine letalität von 30% und eine defektheilungsrate von ca. 20%. eine weitere seltenere komplikation ist die subakute sklerosierende panenzephalitis (7 abschn. 14.23.1). masern sind immer eine ernste und gefährliche krankheit. todesfälle kommen besonders im säuglingsalter, bei älteren probanden und besonders bei immundefizienten patienten vor. die krankheit verläuft besonders schwer in entwicklungsländern bei unterernährten kindern. . abb. 14.25 koplik-flecken . abb. 14.26 masernexanthem bei einem 8-jährigen jungen (hinter den ohren beginnend) . abb. 14.27 generalisiertes masernexanthem bei einem älteren säugling j diagnose im rahmen einer epidemie wird die diagnose meistens klinisch gestellt. labor ein typischer laborbefund ist die leukopenie mit erniedrigung sowohl der granulozyten als auch der lymphozyten. bei einzelerkrankungen sollte die diagnose serologisch bestätigt werden. das masernspezifische igm ist meist nach den ersten 3 exanthemtagen mittels enzymimmunoassay (elisa) nachweisbar. bei trotz impfung an masern erkrankten ist nur ein 4-facher titeranstieg im igg-elisa oder im hämagglutinationshemmtest (hht) diagnosesichernd (bei geimpften findet sich oft keine igm-antwort). in fraglichen fällen, z. b. bei immunsupprimierten patienten oder bei verdacht auf riesenzellpneumonie ist zur diagnosestellung der virusdirektnachweis (pcr oder virusisolierung) erforderlich. die diagnose einer masernenzephalitis beruht allein auf dem zeitlichen zusammenhang der enzephalitis mit einer akuten maserninfektion (igm-nachweis!), da sich im liquor in der regel weder das virus noch eine intrathekale antikörpersynthese nachweisen lassen. j diagnose wegen der ähnlichkeit mit anderen viralen und nichtviralen exanthemen ist die klinische diagnose oft schwierig. charakteristische blutbildveränderungen (leukopenie mit relativer lymphozytose und auftreten von plasmazellen) können von diagnostischer bedeutung sein. ansonsten muss die infektion serologisch bestätigt werden. beweisend sind ein 4-facher titeranstieg im hämagglutinationshemmtest (aus 2 serumproben) oder der nachweis von rötelnspezifischem igm mittels enzymimmunassay (elisa). je nach empfindlichkeit der testmethode sind spezifische igm-antikörper mitunter lange (bis zu einem jahr) im serum nachweisbar. um z wischen einer primären infektion und der (seltenen) reinfektion bei schwangeren zu unterscheiden, stehen spezielle tests zur ver fügung. bei der akuten rötelnenzephalitis findet man im liquor eine leichte lymphozytäre pleozytose. das liquoreiweiß ist normal. virale rna und oligoklonale banden lassen sich in der regel nicht nachweisen. bei mumpsmeningitis zeigt der liquor eine mäßige lymphozytäre pleozytose (10-2.000 zellen/µl) bei normalem bis leicht erhöhtem eiweiß und normalem bis leicht erniedrigtem liquorzucker. im liquor treten 2-3 wochen später virusspezifische oligoklonale mumpsantikörper auf als ausdruck einer intrathekalen immunreaktion. j therapie, prophylaxe eine spezifische therapie existiert nicht. auch eine symptomatische behandlung ist selten erforderlich. bei schweren verläufen (mumpsenzephalitis, orchitis) sind u. u. kortikosteroide indiziert. alle kinder (und noch seronegative adoleszenten und erwachsene) sollten 2-mal gegen mumps geimpft werden (7 kap. 17). spezielle immunglobuline zur passiven immunisierung stehen nicht zur verfügung. gemeinschaftseinrichtungen dürfen 9 tage nach beginn der parotitis wieder besucht werden. seit 2012 ist mumps meldepflichtig. j diagnose die diagnose wird in der regel nach klinischen symptomen und dem eeg-befund gestellt. gestützt wird die diagnose durch den nachweis von neuronalen destruktions-und glialen aktivierungsmarkern im liquor (neuronenspezifische enolase, proteine 14-3-3, s100-β-protein). die genannten marker sind allerdings nicht spezifisch für cjk. ansonsten ist der liquor unauffällig. eine definitive diagnose kann nur durch die untersuchung von hirngewebe gestellt werden. j therapie und prophylaxe es gibt bisher keine wirksame therapie. iatrogene übertragungen durch chirurgische instrumente können durch adäquate dekontaminationsmaßnahmen vermieden werden (z. b. dampfautoklavieren bei 134°c für 1 h, behandlung mit 2,5-5%iger natriumhypochloritlösung oder 1-2 n natronlauge für 1 h. prionenverseuchte nahrungsmittel dürfen auf keinen fall in den verkehr gebracht werden. humane spongiforme enzephalopathien -außer familiärhereditären formen -sind meldepflichtig. j epidemiologie tollwut ist eine weltweit verbreitete zoonose. das tollwutvirus wird durch infektiösen speichel bei kratz-und bisswunden von infizierten tieren (füchse, hunde, fledermäuse, katzen u. a.) übertragen. die jährliche inzidenz von tollwut beim menschen wird weltweit auf 40.000-100.000 fälle geschätzt. deutschland gilt derzeit als "tollwutfrei". die inkubationszeit beträgt 5 tage bis mehrere jahre. sie ist abhängig von der lokalisation der bissstelle (cave: gesicht, augenregion!) und der inokulierten virusmenge. bei kindern ist die inkubationszeit kürzer. 14.29 frühsommermeningoenzephalitis j epidemiologie die frühsommermeningoenzephalitis (fsme, zeckenenzephalitis, zentraleuropäische enzephalitis) kommt europaweit, v. a. in russland, im balkan sowie in zentral-und nordeuropa vor. die endemiegebiete in deutschland liegen hauptsächlich in bayern, baden-württemberg und im saarland. fsme-erkrankungen treten v. a. in den monaten april bis november auf. das fsme-virus wird überwiegend durch zeckenstiche übertragen. wichtigster überträger ist ixodes ricinus, der "gemeine holzbock". > in endemiegebieten sind ca. 0,1-1% der zecken durchseucht. etwa 12-15% aller fsme-erkrankungen betreffen das kindesalter. die inkubationszeit beträgt ca. 10 tage (3-28 tage). nach einer fsme-infektion besteht lebenslange immunität. j ätiopathogenese das fsme-virus gehört zur familie der flaviviren. über die pathogenese ist relativ wenig bekannt. nach der infektion kommt es zu einer starken virusvermehrung im retikuloendothelialen system und in gefäßendothelien. während der virämie (3-14 tage nach zeckenstich) können bei dem patienten grippeähnliche symptome auftreten. nach zns-invasion des fsme-virus und anschließender lokaler virusreplikation kann es in einer 2. krankheitsphase zum auftreten neurologischer symptome kommen. j klinik in 70-90% verläuft eine fsme-infektion asymptomatisch. bei 10-30% der infizierten kommt es nach meist ca. 10 tagen zu einem grippeähnlichem krankheitsbild für ca. 3-7 tage ("sommergrippe"). bei ca. 10% dieser personen tritt nach einem kurzen beschwerdefreien intervall eine 2. krankheitsphase mit fieberanstieg und folgender neurologischer symptomatik auf: 4 meningitis (60%), 4 meningoenzephalitis (30%), 4 meningoenzephalomyelitis (10%). die restitutionsphase beginnt nach etwa 3 tagen mit kontinuierlicher klinischer besserung im verlauf von 1-3 wochen. eine defektheilung (kopfschmerzen, konzentrationsstörungen, motorische lähmungen, muskelschwäche) tritt v. a. bei älteren menschen in ca. 10% auf. die letalität beträgt ca. 1% (beim östlichen fsme-virussubtyp ca. 20%). kinder und jugendliche haben in der regel leichtere (meningitische) krankheitsverläufe. j diagnose bereits anhand der anamnese (zeckenstich, endemiegebiet, jahreszeit) und der klinischen symptomatik (biphasischer krankheitsverlauf) ergibt sich der verdacht auf eine fsme-erkrankung. die diagnose wird durch den serologischen nachweis von fsme-spezifischen serumantikörpern gesichert. die liquoruntersuchung in der 2. krankheitsphase ergibt typischerweise eine lymphozytäre pleozytose (<100-5.000 zellen/mm 3 ) sowie eine liquoreiweißer höhung. j therapie, prophylaxe eine antivirale therapie existiert nicht. die behandlung ist rein symptomatisch. zur aktiven immunisierung steht ein impfstoff auch für kinder zur verfügung. > zielgruppe für eine fsme-impfung sind alle personen, die sich in endemiegebieten vermehrt im freien aufhalten. meldepflichtig sind erkrankung und tod nach einer fsme-infektion. j ätiopathogenese das "human immunodeficiency virus" (hiv) gehört zur gruppe der retroviren. es existieren 2 haupttypen (hiv-1 und hiv-2). in europa und nordamerika kommen praktisch nur hiv-1-varianten vor, in teilen afrikas findet sich auch hiv-2 bei einem großen teil der bevölkerung. hiv benutzt zur infektion einer zelle das hüllprotein gp120, mit dem es sich an den cd4-rezeptor und korezeptor (ccr5 oder cxcr4) der zielzelle (t-helferzellen, makrophagen, monozyten, gliazellen u. a.) anheftet. anschließend fusioniert die hiv-hülle mit der zellmembran, 2 rna-stränge und mehrere virusenzyme werden in das zytoplasma freigesetzt. mittels des viralen enzyms reverse transkriptase (rt) wird die rna in doppelsträngige dna "übersetzt" (provirus). diese dna wird anschließend durch das viral kodierte enzym integrase in das wirtsgenom im zellkern eingebaut (viruslatenz). durch verschiedene aktivierende faktoren kommt es zu einer ausgeprägten virusreplikation mit freisetzung von neuen hiv-partikeln in die blutbahn. der verlust der t-zellfunktion führt zu einem zunehmenden zellulären und humoralen immundefekt mit der folge von schweren opportunistischen infektionen und dem auftreten von lymphomen. j klinik die klinische symptomatik bei horizontal hiv-infizierten jugendlichen (infektion durch geschlechtsverkehr oder durch kontaminierte blutprodukte) entspricht im wesentlichen dem bild bei erwachsenen. der natürliche verlauf bei vertikal hiv-infizierten kindern variiert sehr stark. ohne therapie erkranken bereits 25-30% dieser kinder sehr früh, meist schon innerhalb des j diagnostik virologische diagnostik als sicherer nachweis einer infektion gelten wiederholt positive hiv-kulturen, wiederholte nachweise des p24-antigens oder der hiv-rna (rt-pcr). die letztere me thode erlaubt die bestimmung der viruslast und hilft mit bei der indikationsstellung für eine antiretrovirale therapie und beim therapiemonitoring. erst der wiederholte nachweis von hiv-antikörpern (elisa, immunoblot) nach dem 18. lebensmonat beweist eine hiv-infektion. bereits vorher nachgewiesene hiv-antikörper können von der mutter diaplazentar übertragen worden sein. immunologische diagnostik im rahmen einer hiv-infektion kommt es im verlauf zu einem zunehmend schweren kombinierten (zellulären und humoralen) immundefekt. im rahmen der initialen statusdiagnostik und späterer verlaufskontrollen sollten bei allen hiv-infizierten oder -exponierten kindern u. a. folgende parameter im blut bestimmt werden: 4 quantitative bestimmung der cd4 + -zellen, 4 serumimmunglobuline (igg, iga und igm), 4 impfantikörper nach erfolgter grundimmunisierung (dtp, hib, hbv, polio-salk), 4 antikörper gegen verschiedene herpesviren (nach stattgehabter infektion)! diese untersuchungen sollten möglichst immer nur am selben immunologisch ausgewiesenen speziallabor durchgeführt werden. die viruslast eines patienten muss immer mit derselben bestimmungsmethode quantifiziert werden! aids-definierenden krankheiten (kategorie c) dagegen tritt bei einem großen teil der kinder die klinische verschlechterung erst im grundschuloder schulalter auf. prädiktive parameter für eine schlechte prognose sind u. a. eine hohe viruslast im blut und ein schneller bei fortschreitender infektion und störung im immunsystem treten klinische zeichen hinzu, die schon eher an einen immundefekt denken lassen (kategorie b) weitere atemwegsassoziierte viren das humane metapneumovirus (hmpv), das erst 2001 entdeckt wurde, ist bei kindern der zweithäufigste erreger der bronchiolitis. darüber hinaus verursacht hmpv auch bronchitiden und pneumonien. die hospitalisierungsrate ist wahrscheinlich ähnlich hoch wie bei rsv-und influenza-infektionen. das humane bocavirus (hbov) wurde im sommer infektionen mit humanen coronaviren (hcov) können v. a. bei kleinkindern zu erkrankungen der unteren und oberen luftwege führen. nicht selten finden sich koinfektionen mit anderen respirationstraktviren der nachweis aller drei erreger in infektiösen körperflüssigkeiten gelingt meist mittels polymerasekettenreaktion. die therapie ist in allen fällen symptomatisch helfen meist auch bei der hiv-assoziierten autoimmunthrombozytopenie.chemoprophylaxe die früher häufigste opportunistische infektion, die pneumocystis-jirovecii-pneumonie, lässt sich durch orale gabe von cotrimoxazol (trimethoprim 150 mg/m2 kof an 3 aneinander folgenden tagen pro woche) zu fast 100% vermeiden.allgemeine schutzimpfungen nur bei einer noch asymptomatischen hiv-infektion wird eine masern-oder mmr-impfung empfohlen. auch gegen varizellen kann geimpft werden, sofern die relative cd4-zellzahl ≥25% liegt. ansonsten sollten keine lebendimpfungen erfolgen. im übrigen sollten hiv-infizierte kinder entsprechend dem impfkalender mit inaktivierten impfstoffen/ toxoiden (polio-salk-impfstoff, hepatitis b, hib, dpt) und zusätzlich gegen pneumokokken, meningokokken und influenza geimpft werden. bei kindern, die regelmäßig mit immunglobulinen behandelt werden, sind aktivimpfungen dagegen nicht mehr sinnvoll. eine hiv-infektion ist namentlich nicht meldepflichtig. es besteht nur eine anonyme labormeldepflicht. j epidemiologie influenzainfektionen sind ubiquitär. alle 3-5 jahre treten influenzaepidemien durch antigendrift der zirkulierenden influenzasubtypen bei nachlassender immunität der exponierten bevölkerung auf. in größeren zeitabständen (10-20 jahre) treten durch rekombination zwischen humanen und animalen influenza-a-virusstämmen neue subtypen auf (antigenshift), die zu schweren pandemien führen können. in unseren breiten tritt die influenzagrippe üblicherweise in den monaten dezember bis april auf. die übertragung erfolgt überwiegend durch tröpfcheninfektion (niesen, husten), seltener durch kontaktinfektion. die kontagiosität ist kurz vor ausbruch der klinischen symptome am höchsten, sie dauert bis zu 1 woche an. die inkubationszeit beträgt in der regel 2-3 tage (1) (2) (3) (4) (5) (6) (7) .j ätiopathogenese influenzaviren gehören zur familie der orthomyxoviren. es existieren 3 typen (a, b, c). influenzaviren enthalten 8 rna-segmente, die von einer hülle von strukturproteinen umgeben sind. diese enthält spikes aus hämagglutinin (h) und neuraminidase (n). gegenwärtig zirkulieren die influenza-a-subtypen h3n2 und h1n1 sowie zwei verschiedene influenza-b-virusstämme. influenzaviren führen zu einer lytischen infektion des respiratorischen epithels. hierdurch kommt es zu einem verlust der zilienfunktion, zu einer verminderten schleimproduktion sowie zur nekrose der epithelschicht. es folgt eine entzündungsreaktion mit infiltration von lymphozyten, histiozyten und granulozyten. in den folgenden 2 wochen kommt es wieder zur regeneration und erholung der epithelzellschicht. die klinische symptomatik wie fieber, abgeschlagenheit, kopf-und gliederschmerzen ist immunologisch bedingt (u. a. durch freisetzung von zytokinen).j klinik bei älteren kindern und erwachsenen führt eine influenzainfektion typischerweise zur klassischen virusgrippe mit hohem fieber, kopf-und gliederschmerzen, abgeschlagenheit, trockenem husten, pharyngitis und konjunktivitis. nach einigen tagen tritt meist entfieberung ein, gefolgt von einer bis zu wochenlangen rekonvaleszenzphase. bei 10% der patienten finden sich klinische und radiologische zeichen einer pulmonalen beteiligung.bei kleinen kindern manifestiert sich eine influenzainfektion klinisch meist nicht unterscheidbar von infektionen durch andere respirationstraktviren (rsv, adenoviren, parainfluenzaviren u. a.), unter dem bild einer bronchiolitis, einer obstruktiven bronchitis, einer pneumonie, einer akuten subglottischen laryngotracheobronchitis (infektkrupp) oder einer unspezifischen atemweginfektion. darüber hinaus können bei kleinen kindern gastrointestinale symptome (durchfall, erbrechen) auftreten. häufig kommt es auch zu fieberkrämpfen. bei kindern mit chronischen grunderkrankungen (z. b. zystische fibrose, immunsuppression) können influenzainfektionen sehr schwer verlaufen. die meisten erkrankungen verlaufen insgesamt gutartig. bei immunsupprimierten kindern können erkrankungen mit parainfluenzaviren äußerst schwer und u. u. letal verlaufen (riesenzellpneumonie).j diagnose eine spezifische diagnostik ist meist nur bei schweren krankheitsverläufen erforderlich. parainfluenzaviren können im nasopharynxsekret nachgewiesen werden (immunfluoreszenztest, elisa, rt-pcr). der serologische nachweis von parainfluenzavirusspezifischen antikörpern spielt im klinischen alltag keine rolle.j therapie, prophylaxe eine spezifische antivirale therapie oder eine impfung existieren nicht. die behandlung der klinischen beschwerden ist symptomatisch. bei immunsupprimierten kindern mit schwerer parainfluenzavirusinfektion (pneumonie) kann ein therapieversuch mit ribavirin unternommen werden. bezüglich hygienemaßnahmen gelten die gleichen empfehlungen wie für rsv-infektionen. key: cord-004600-5lhnzzvg authors: dennin, reinhard h.; doese, d.; theobald, w.; lafrenz, m. title: hiv-infektion – grenzen der präventionskonzepte: überlegungen zur verantwortung der betroffenen, der politik und der gesellschaft* date: 2007-03-26 journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: 10.1007/s00103-007-0212-z sha: doc_id: 4600 cord_uid: 5lhnzzvg despite the introduction of campaigns to prevent the continued spread of hiv/aids in germany, the number of annual firsttime hiv-diagnoses is continuing steadily. the concepts behind the current campaigns are largely based on models of new public health, of which social learning strategies are an essential element. the established personal and individual rights should be unimpeachable but the right not to know the status of hiv infection should be questioned for those people who spread their hiv infection intentionally and wilfully. confronted with more than 10,000 people in germany unconscious of their hiv infection, easy access to hiv testing and access of opportune therapy should be offered with the goal of reducing the number of new infections. expanded strategies on the responsibility to one’s personal health and that of the partner, understandable and adapted to special groups of the society, should be established and maintained at a high level of awareness. all measures must be performed voluntarily. in deutschland leben mit der hiv-infektion etwa 56.000 menschen, die gesamtzahl an der hiv-infektion verstorbener wird mit etwa 26.000 angegeben (stand ende 2006) [1] . angesichts der seit jahren im wesentlichen gleich bleibenden jährlichen zunahmen der zahlen hiv-infizierter seit beginn der hiv-epidemie [1, 2] in deutschland ist zu fragen, ob die konzepte und strategien der in den 1980er-jahren eingeleiteten präventionskampagnen wirkungsvoll genug waren. die hiv-epidemie ist bestimmt durch ein komplexes zusammenwirken von personengebundener transmission, über eine dekade langer, klinisch asymptomatischer zeit bis zur manifestation der krankheit, bei permanenter infektiosität. die individuelle intime sphäre des sexualverhaltens ist durch unsere gesellschaftliche verhaltensnormen geschützt; somit kann die hiv-infektion als personengebundene transmission nicht wie andere infektionen, die den community acquired infections zugerechnet werden, z. b. die tuberkulose, behandelt werden. warum hält der aufwärtstrend der hiv-erstdiagnosen an -trotz der präventionsarbeit? mit dem folgenden beitrag wird versucht, aus sicht der angewandten ethik, der verhaltensforschung, auf basis einer analyse der gesellschaftlichen entwicklung unter berücksichtigung infektionsepidemiologischer mechanismen grenzen der alleinigen anwendung der aktuellen präventionskonzepte aufzuzeigen. die präventionskampagnen beruhen im wesentlichen auf modellen der "new public health" (nph) [3] . als deren leitgedanke gilt, eine primärprävention durch lernstrategien zu erreichen. die zielsetzung ist, über kommunikation und breitenwirksame information ins besondere bei bereits von der hiv-infektion betroffenen und noch hivnaiven, aber sexuell risikobereiten (at risk) personen -neben intravenös drogengebrauchern (ivdu) -zeitstabile und wirksame verhaltensänderungen herbeizuführen. eine unterbrechung von hiv-infektketten soll erreicht werden. repressive maßnahmen sind in diesem "modell der verhältnisgestützten verhaltensmodifikationen" mit gutem grund ausgenommen. die intentionen der nph sind unter anderem beeinflusst von etablierten liberalisierungsideen; andere vorgaben, wie z. b. das anmahnen von selbstdisziplin und verantwortung für den mitmenschen, werden zurückgestellt. für gesellschaftliche als auch individuelle lebensbereiche wird die kategorie toleranz als wesentliches element hervorgehoben. angewandt auf die präventionskonzepte gegen die ausbreitung von hiv war damit ein vertrauens-und erwartungsvorschuss für bereits von der hiv-infektion betroffene wie noch hiv-naive mitmenschen verbunden, sich den an die kognitive ebene richtenden rationalen präventionsbotschaften entsprechend zu verhalten, d. h. neue hiv-infektionen zu vermeiden. übergeordnet gründen die präventionskonzepte in einer ethik, die dem menschen mehr zutraut bzw. mehr von ihm erwartet, als er teilweise wahrscheinlich von seiner naturbedingten herkunft zu leisten vermag und aufgrund etablierter persönlichkeitsrechte zu leisten bereit ist. nach unserer einschätzung sollten die auf nph gründenden präventionskonzepte und -strategien nach 20 jahren anwendung modifiziert werden. abgelöst von übergeordneten wertfragen haben sich zahlreiche der anfänglichen entscheidungen, die dem schutz der von der hiv-infektion betroffenen menschen dienen sollten (z. b. anonymitätsgarantie)‚ als nur teilweise wirksam erwiesen; es waren damit bedingungen vorhanden, die weitere infektionsketten möglich machten. eine dafür wesentliche entscheidung war, der hiv-infektion eine ausnahmestellung innerhalb der sexuell übertragbaren infektionen (sti) zum individuellen schutz zugunsten der damals von der hiv-infektion hauptbetroffenen einzuräumen [4] [5] [6] . seitdem hat es veränderungen in der zusammensetzung der hiv-infizierten gegeben, da auch der anteil an frauen und die heterosexuelle übertragung prozentual zugenommen haben. die argumente von vertretern der wesentlich auf konzepten der nph basierenden hiv-präventionskampagnen, diese hätten wesentlich zu den "geringen" zuwachsraten an hiv-infizierten in deutschland und -wegen der europäischen abstimmung -auch in europa beigetragen, sind nach der derzeitigen datenlage weder zu widerlegen noch zu belegen, da ein anderes modell der prävention nicht eingeführt wurde. unsicherheit über die effizienz dieser strategien wird weiterhin bestehen, da die evaluierungen aus retrospektiven und prospektiven erhebungen/befragungen von bereits hiv-infizierten und noch hiv-negativen atrisk-populationen wegen der subjektiven aussagen eingeschränkt bleiben. trotz einer nicht möglichen biostatistisch begründeten beweisführung solcher empirisch gewonnenen ergebnisse sind auch wir der meinung, dass die konzepte der nph zumindest zeitweise wesentliche beiträge zu präventionskonformen verhaltensänderungen bei den gefährdeten personen bewirkt haben. selbst aus kreisen hiv-infizierter wurde die nachhaltigkeit der angewandten präventionskampagnen im hinblick auf den zeitweiligen relativen rückgang der jährlichen hiv-erstdiagnosen wiederholt zur diskussion gestellt. [8] die folgen der übertragung einer infektion mit todesfolge werden durch leistungen verschiedenster art bisher von der gesellschaft aufgefangen. bei der hiv-infektion ist bekannt, dass mit den derzeitigen antiretroviralen medikamenten keine heilung möglich ist, und bei den meisten hiv-infizierten unter dauerhafter behandlung eine unterschiedlich hohe infektiosität für jahre aufrechterhalten wird. auch werden bei neu-oder superinfektionen partielloder multi-resistente hiv übertragen. das angebot zur toleranz insbesondere für von der hiv-infektion betroffene menschen wurde hier missbraucht. auch das liberale individualrecht ist damit trotz der verpflichtung für die gesellschaft missbraucht worden. die von der nph geforderte eigenverantwortung fehlt bei denjenigen, die die hiv-infektion weitergeben. danzinger weist auf diese problematik hin: "... a growing recognition of a possible weakness inherent in a strictly voluntarist approach to hiv prevention may herald a new approach to aids control ... by shifting the terms of debate away from individual rights towards a better understanding of individual and social responsibilities" [9] . peter piot (executive director, unaids) mahnte die beachtung der menschenrechte (human rights, hr) an, ohne zielgruppen zu benennen: "... a recognition of the inherent dignity and equal rights of all people and an understanding that the protection of human health is indispensible for the protection of human rights and fundamental freedoms of everyone" [10] . für eine erfolgreiche hiv-prävention sind zum schutz und aufgrund zukünftiger belastungen für die gesamtgesellschaft folgende fragen zu stellen: ist angesichts einer stetig voranschreitenden hiv-ausbreitung das recht auf informationelle selbstbestimmung mit der an den biologisch geprägten sexualtrieb gekoppelten hiv-übertragung -neben drogensucht -vereinbar? ist ausgelebte sexualität bis hin zum triebhaft gesteigerten verhalten als rechtfertigungsgrund bzw. duldung zur körperverletzung anderer menschen mit schwersten gesundheitlichen folgen in derzeitiger auslegung akzeptabel? diese in den frühen 1980er-jahren diskutierte frage stellt sich erneut bei zunehmender hiv-infektionszahl. steht das übertragen von hiv im gegensatz zu den intentionen des grundgesetzes (gg) -hier als eine nichtbeachtung des art. 2, abs. 1 gg [11] ? ferner lässt sich unserer meinung nach umgekehrt aus dem recht auf körperliche unversehrtheit (art. 2, abs. 2 gg) nicht postulieren, dass jeder seinen körper vorsätzlich schädigen darf, um dann die hilfe der gesellschaft rückhaltlos für sich einzufordern. die abwägung der frage von hiv-infizierung und der vereinbarkeit des art. 1, abs. 1, gg: "die würde des menschen ist unantastbar. sie zu achten und zu schützen ist verpflichtung aller staatlichen gewalt", sollte durchaus wieder andiskutiert werden. denn die weiterführung dieses grundsatzes bedeutet, dass nicht nur die würde des hiv-infizierten menschen unantastbar ist, sondern auch die würde aller anderen, noch nicht infizierten menschen. muss die körperliche unversehrtheit von mitbürgern nicht höher (höheres rechtsgut) bewertet werden, als die besonderen sensiblen empfindungen der derzeit von der hiv-infektion betroffenen menschen? verantwortung wird auch in den intentionen der human rights gefordert: "the international bill of human rights recognizes individual's duty to the community, creates absolute (nonderogable) rights, and outlines criteria for the limitation of other rights." [12] die mit den präventionskonzepten festgelegten rahmenbedingungen und die besondere stellung der hiv-infektion scheinen die nichtanwendung des strafrechts -bis auf seltene, augenfällige situationen bei der hiv-übertragung -zu begünstigen [13] : die im mittel lange klinische latenzperiode der hiv-infektion verwischt die psychologisch nicht wahrnehmbare kausale zuordnung zu einer individuell begangenen körperverletzung, teils auch mit zustimmung des partners, durch personengebundene übertragung einer infektion mit todesfolge: die manifestation von krankheit als folge der hiv-infektion kann ohne antiretrovirale behandlung bereits nach wenigen jahren p.i. ausgeprägt sein, im mittel ist sie aber nach 9-10 jahren ausgebildet, abhängig von individueller disposition des hiv-infizierten und der virulenz des infizierenden hiv. diese umstände erschweren eine beweisführung für den einzelfall bzw. machen sie nahezu unmöglich. es ist aufschlussreich, die gesellschaftliche entwicklung, die zu dieser unterschiedlichen betrachtungsweise von infektionen geführt hat, näher zu analysieren. sowohl die erklärung der menschenrechte als auch das bekenntnis zur menschenwürde antworteten jeweils auf herausforderungen ihrer zeit [14] . zielten die klassischen menschenrechtserklärungen mit der idee der freiheit an ihrer spitze auf politische befreiung und die begrenzung absolutistischer macht, und reagiert(e) das moderne bekenntnis zur menschenwürde auf diktatorische, das humanum mit füßen tretende schreckensregime (allen voran dasjenige des nationalsozialismus), so fehlt derzeit das bewusstsein für diese werte in demokratischen liberalen gesellschaften, da kein essenzieller widerstand, gegen den sie sich errichteten, mehr vorhanden ist. ehemals als schutzrechte gegen absolutistische herrschaftswillkür und unterdrückung entstanden, neigen die liberalen menschenrechte heutzutage dazu, sich selbst zu verabsolutieren. im rahmen der fortschreitenden säkularisierung übernehmen sie auch die funktion einer ersatzreligion, die das individuum verherrlicht und seine selbstentfaltung zum höchsten wert verklärt: sie werden zur bedingung des "gelingens der selbstdarstellung eines menschen als individuelle persönlichkeit" [15] . war die würde des menschen über fast zweitausend jahre europäischer geistesgeschichte -trotz z. t. fundamental wechselnder paradigmen -im wesentlichen gleich definiert, nämlich durch eine transzendente, sei es religiöse oder metaphysische bindung, so hat sie heute die tendenz bindungslos, wie das individuum selbst, zu werden. es fehlt teils eine sachliche begründung (vgl. dazu ausführlich [16] ), und das fehlen einer solchen begründung zeigt, wie diese form der würde aussehen kann: der auf den begriff gekommene ausdruck der selbstliebe eines menschen, der getrieben ist von einem "schier hemmungslosen streben nach glück und ich-genuss" [17] . dabei gehen werte zur gemeinschaftsfähigkeit verloren. die damit verknüpften konsequenzen der ausnutzung staatlich geschützter individueller freiräume klingen auch aus anderer sicht in allgemein gehaltenen verhaltens-/evolutionsbiologischen betrachtungen an: "nach popper liegt der einzige wesentliche unterschied zwischen den übrigen tieren und dem menschen alleine darin, dass letzterer es durch seine besonderen geistigen begabungen wie denken, vorstellungsvermögen, vernunft und logik geschafft hat, sich der direkten einwirkung der natürlichen auslese zu entziehen, indem bloße gedanken und vermutungen anstelle ganzer lebewesen dem irrtum geopfert werden." [ die festlegung auf hiv-testungen nur nach medizinisch begründeter indikation war in den 1980er-jahren wesentlich mit rücksicht auf die nicht-behandelbarkeit der hiv-infektion und den schutz der betroffenen entschieden worden. es haben sich, verbunden mit dem recht auf nichtwissen, über die jahre reservoire 3 siehe aber ausnahmen: "bug chaser", s. auch "barebacking" partys! zusammenfassung · abstract cess to hiv testing and access of opportune therapy should be offered with the goal of reducing the number of new infections. expanded strategies on the responsibility to one's personal health and that of the partner, understandable and adapted to special groups of the society, should be established and maintained at a high level of awareness. all measures must be performed voluntarily. hiv-prevention · individual rights · human rights · natural behaviour · unaware hiv infected hiv-prävention · persönlichkeitsrechte · menschenrechte · naturbedingte verhaltensmuster · unwissentlich hiv-infizierte despite the introduction of campaigns to prevent the continued spread of hiv/aids in germany, the number of annual firsttime hiv-diagnoses is continuing steadily. the concepts behind the current campaigns are largely based on models of new public health, of which social learning strategies are an essential element. the established personal and individual rights should be unimpeachable but the right not to know the status of hiv infection should be questioned for those people who spread their hiv infection intentionally and wilfully. confronted with more than 10,000 people in germany unconscious of their hiv infection, easy ac-unwissentlich hiv-infizierter als unerkannte infektionsquellen für die hiv-ausbreitung aufbauen können. etwa ein drittel der neu erkannten hiv-infizierten hat symptome der immunschwäche. entgangen sind ihnen rechtzeitige psychologische, medizinische versorgung und individuelle beratung (face-to-face) zur vermeidung von superinfektionen und zu präventionskonformem verhalten. die diskussion um eine optimierte präventionsstrategie bei der hiv-infektion ist kein alleinig deutsches problem. die sorgen um die voranschreitende hiv-ausbreitung und die voraussetzungen und bedingungen zu änderungen für die testung auf hiv werden zunehmend thematisiert [3, 4, 22, 24] . wesentlich dafür sind überlegungen, wie z. b. der wachsenden zahl unwissentlich hivinfizierter menschen begegnet werden kann. deren zahl wird in den usa mit wenigstens 250.000 personen [24, 25] angenommen, während für deutschland zumindest von etwa 14.000 personen auszugehen ist -basierend auf einem anteil von 25-30 % der auf 56.000 geschätzten lebenden hiv-infizierten. auf gleicher basis ist für west-und zentral-europa von zumindest 180.000 unwissentlich hiv-positiven personen auszugehen, als anteil der für diesen teil der welt geschätzten 740.000 hiv-positiven. in globaler dimension gesehen, wird von einem sehr hohen anteil (regional ist von bis zu 90 % die rede) unwissentlich hiv-positiven an den geschätzten 39,5 millionen lebenden hiv-positiven ausgegangen [26] . für die usa wurde das beachtliche ausmaß für sexuell bedingte neuinfektionen durch personen belegt, die nichts von ihrer hiv-infektion wissen [27] . ein ein weiterer, bisher wenig beachteter zukunftsweisender aspekt wurde bereits von baltimore angemerkt "the key fact about hiv is that it is a nonequilibrium infectious agent"; dieser weist auf entsprechende, evolutionäre potenziale des hiv hin [34] . die koevolution von hiv mit seinem neuen wirt homo sapiens hat erst begonnen; gefördert wird sie allerdings nur von jenen menschen der gesellschaft, die die hiv-infektion verbreiten, die sich der hiv-infektion aussetzen oder ihr ausgesetzt werden -z. b. durch zwangsprostitution. in diesem zusammenhang sind auch sog. bridging people zu sehen -auch als basis für die weitere evolution von hiv, die längst im gange ist [35, 36] . jede möglichkeit zur einflussnahme, die weitere ausbreitung einer infektion mit todesfolge zu mindern, sollte für jede diskussion offen sein, und sie muss freigehalten werden von ideologischer konfrontation. und: jedes land muss seinen beitrag aufgrund länderspezifischer bedingungen dazu leisten. hiv/aids in deutschland -eckdaten hiv-infektionen und aids-erkrankungen in deutschland changing the paradigm for hiv testing -the end of exceptionalism a misinterpretation of hiv-exceptionalism: the implementation of hiv surveillance in france, a case study kritische wertung der bisherigen anti-hiv/aids-präventionskonzepte in deutschland -ein diskussionsbeitrag aids -wissen, einstellungen und verhalten 1987 bis 1999. ergebnisse der jährlichen repräsentativbefragung an epidemic like any other? rights and responsibilities in hiv prevention human rights and public health in the aids pandemic jeder hat das recht auf die freie entfaltung seiner persönlichkeit, soweit er nicht die rechte anderer verletzt und nicht gegen die verfassungsmäßige ordnung oder das sittengesetz verstößt the universal declaration of human rights strafbarkeit des ungeschützten geschlechtsverkehrs hiv-infizierter themen parlamentarischer beratung aids: fakten und konsequenzen, 13/90; deutscher bundestag (hrsg) grundrechte als institution analytische einführung in die ethik das intelligente genom gefahren von aids und wirksame wege zu ihrer eindämmung. zur sache, themen parlamentarischer beratung applying public health principles to the hiv epidemic screening for hiv: a review of the evidence for the u.s. preventive service task force revised recommendations for hiv testing for adults, adolescents, and pregnant women in health care settings deutsche aids hilfe notes and quotes. national hiv prevention conference estimating sexual transmission of hiv from persons aware and unaware that they are infected with the virus in the usa routine testing: are we ready to throw human rights out of hiv testing policy? abstract weae0101/ abs. 2335; xvi international aids conference guidance on provider-initiated hiv testing and counselling in health facilities die offene gesellschaft und ihre feinde erstes kapitel: über zwei negative utopien hier: klappentext; rowohlt the enigma of hiv infection human immunodeficiency virus superinfection and recombination: current state of knowledge and potential clinical consequences identification of a newly characterized hiv-1 bg intersubtype circulating recombinant form in galicia, spain, which exhibits a pseudotype-like virion structure key: cord-005033-voi9gu0l authors: xuan, huiyu; xu, lida; li, lu title: a ca-based epidemic model for hiv/aids transmission with heterogeneity date: 2008-06-07 journal: ann oper res doi: 10.1007/s10479-008-0369-3 sha: doc_id: 5033 cord_uid: voi9gu0l the complex dynamics of hiv transmission and subsequent progression to aids make the mathematical analysis untraceable and problematic. in this paper, we develop an extended ca simulation model to study the dynamical behaviors of hiv/aids transmission. the model incorporates heterogeneity into agents’ behaviors. agents have various attributes such as infectivity and susceptibility, varying degrees of influence on their neighbors and different mobilities. additional, we divide the post-infection process of aids disease into several sub-stages in order to facilitate the study of the dynamics in different development stages of epidemics. these features make the dynamics more complicated. we find that the epidemic in our model can generally end up in one of the two states: extinction and persistence, which is consistent with other researchers’ work. higher population density, higher mobility, higher number of infection source, and greater neighborhood are more likely to result in high levels of infections and in persistence. finally, we show in four-class agent scenario, variation in susceptibility (or infectivity) and various fractions of four classes also complicates the dynamics, and some of the results are contradictory and needed for further research. focus on hiv/aids transmission among human groups in order to better understand its dynamical behavior. in epidemic modeling (see, e.g., bailey 1975; anderson and may 1991; murray 2005) , there are two frequently used methodologies: mathematical and simulation methods. for mathematical approaches, a cohort of people is often classified into susceptibles, infectives, and recovereds with (without) immunity (see, e.g., kermack and mckendrick 1927) . systems of differential equations are used to describe the linear (nonlinear) dynamics of epidemics. macroscopically, mathematical models can reveal the relationship among primary factors and describe their effects to epidemic spreading under certain assumptions. as to hiv/aids epidemic, many models have been proposed (may and anderson 1987; hyman et al. 1999; brauer and driessche 2001; wu and tan 2000, etc.) . however, mathematical approaches have some serious drawbacks due to its intractability and the complexity of epidemics. moreover, the complicated nature of hiv/aids transmission makes it even harder to obtain analytical solutions and difficult to study them. in the early 1990s, some researchers started to apply simulation approaches to this field. there is a large literature that addresses the computer simulation of epidemic dynamics (see, e.g., leslie and brunham 1990; atkinson 1996; rhodes and anderson 1996; rhodes and anderson 1997; ahmed and agiza 1998; benyoussef et al. 2003; tarwater and martin 2001) . particularly, cellular automata (ca) method (some literature refers to this as a lattice-based method) has been widely used in modeling complex adaptive systems. despite of its simple structure, ca is well suited to describing the propagation phenomena, such as rumor spreading, particle percolation, innovation propagation, and disease spreading. for instance, in epidemic modeling, fuentes and kuperman (1999) propose two ca models corresponding to the classical mathematical sis model and sis model respectively. ahmed and agiza (1998) develop a ca model that takes into consideration the latency and incubation period of epidemics and allow each individual (agent) to have distinctive susceptibility. gao et al. (2006) put forward a ca model for sars spreading which takes account of social influence. more recently, other methods such as agent-based modeling and system dynamics are introduced to this field (see, e.g., gordan 2003; bagni et al. 2002) . our paper contributes to this filed by developing an extended ca simulation model. we then use the new ca model to investigate some issues in hiv/aids epidemics. most models, including the foregoing ca models, have some limitations that fail to consider the peculiarities of hiv/aids epidemics and are thereby incapable of describing the epidemic accurately and completely (see frauenthal 1980 for more discussion). first, most of the models assume that there is no latent (or incubation) period. however, for some epidemics, especially aids, there are variously lasting periods of latency and incubation as well as behavior-varying infectivity (or susceptibility) during these periods. in fact, the development of aids involves a few stages in which an infected individual can exhibit different behaviors. those diversified behaviors, in turn, have some ignorable effects on the dynamics of hiv/aids. in light of this, we extend the conventional division of epidemic process (i.e., susceptible, infection, and removed) by dividing the infection period into three sub-stages, each corresponding to the clinical stage occurring in the course of aids development. due to the inability of classical ca approaches to accommodate those newly added state transitions events, we also borrow some ideas from discrete-event simulation techniques and make one agent's stage transitions being time-triggered instead of using some state-based transition rules. secondly, it is commonly assumed that individuals in the population are homogenous in the sense that they have equal infectivity and susceptibility, or they can exert the same influence on each other, etc. this assumption may be satisfied in commonly observed epidemics but not consistent with the hiv/aids epidemic. as we know, susceptibility and infectivity heavily depends on individuals' behavior. for examples, safe sex practices such as the use of condom could dramatically reduce the chance of infection. also, the way of hiv/aids transmission for one to another is various, depending on the interactions between people, and thus the probability of getting infected is determined in part by transmission routes and can be quite different between infected-male/susceptible-female and susceptible-male/infectedfemale interactions. under this assumption, the models that are confined to a single high-risk human group are not suitable in overall population cases. new models are needed to explicitly consider the complexity. therefore, we make an extension to the traditional ca model by introducing the extended definition of neighborhood and attaching some attributes to each agent such as infectivity and resistibility. we also define four types of agents that are characterized by different infectivity (and susceptibility) and various forms of neighborhood to represent four types of people in real life. in doing so, we will be able to investigate the dynamics of hiv/aids with heterogeneous groups in a realistic way. thirdly, classical ca models assume that agents in the grid are spatially fixed, that is, once an agent is placed in a cell, it does not move into another cell. this assumption is problematical because people in the real world are migratory. for instance, in china, millions of rural people leave their hometowns and seek jobs in the cities. the migration of population is a driving force for the spread of hiv/aids. ignoring the mobility of agents in epidemic models would jeopardize the creditability of the results obtained. considering this point, we incorporate agents' mobility into their behaviors. in our model, each agent is allowed to move randomly into one of its adjoined and unoccupied cells at random time intervals. recently, agent-based modeling is used in various fields to solve plenty of problems (see, e.g. zhang and bhattacharyya 2007; luo et al. 2007) . some reader might notice that our improved ca model have features that usually found in agent-based methodology. as a matter of fact, our method borrows much from agent-based simulation modeling. to make things simple, we prefer to view this model as being a ca models. this paper is organized as follows. in the next section, we present our extended ca simulation model. section 3 gives a detailed description of simulation results and analyzes some influential factors that affect the dynamical behavior of the model. section 4 concludes and points out some possible extensions and directions for future research. cellular automata have been extensively used as tools for modeling complex adaptive systems such as traffic flow, financial markets, chemical systems, biological groups, and other social systems (see e.g. gerhard and schuster 1989; gerhardt et al. 1990; weimar et al. 1992; karafyllidis and thanailakis 1997; karafyllidis 1998) . usually, a typical ca model consists of a regular two-dimension grid with a certain boundary condition and a swarm of agents living in the grid. 1 the neighborhood of an agent is defined to some (or all) of the immediately adjacent cells and the agents who inhabit in the neighborhood are called neighbors. agents are restricted to local neighborhood interaction and hence are unable to communicate globally. there are several states agents can be in at each time and an agent's state at time t + 1 is determined based on its neighbors' states at time t . the rules used in the determination of next-time states can be written as a mapping: where s is the set of states and t denotes simulation time. the mathematical properties of cellular automata have been studied in martin et al. (1984) . in our model, we consider a population of size n(t) at time t randomly distributed in a two-dimension w × w lattice. population growth rate r is fixed throughout the simulation. at each time, new agents are added to the model, and the dead removed. simulation time advances in a discrete way. the time interval (t, t + 1) is specified to represent one week in real life. this assumption makes the simulations run reasonably fast (with respect to the whole progress of epidemics) without losing any time-specific clinical properties associated with hiv/aids. explicitly modeling the post-infection progression to aids is one feature of our model compared with conventional ca models. classical epidemic models divide the closed population into three subgroups: susceptible, infective, and recovered (removed). this simplified classification is not consistent with the epidemics in real life. particularly, it is well established that an individual, once infected with hiv, undergoes roughly three clinical phrases towards the full-blown aids: (1) infected, not yet infectious, (2) infectious, not yet asymptomatical, and, (3) symptomatical (may and anderson 1987; may et al. 1988) . the lifetime of an individual should cover not only the process from health to infection, but also the sub-stages after infection. thus, we assume that each agent can go through the following states: • s1: healthy state, initially, each agent is set to be in s1 state. healthy agents have no risk of being infected. when a healthy agent moves into the neighborhood of an infectious one or an infectious agent approaches him, the healthy agent's state will change from s1 to s2 because contacts with infectives incur the danger of infection. as for an agent in s2 state, it can transit in two directions: one direction is to change from s2 back to s1, after all its infectious neighbors move away (or its dead neighbors are removed from the grid) or he leaves the neighborhoods of its infectious neighbors; the other direction is to change from s2 to s3 if he unluckily get infected. note that we assume infection is instantaneous, i.e., instantaneous transmission from an infected individual to a susceptible. a newly infected agent is unable to transmit hiv virus until seroconversion. the s3 state corresponds to the early stages of hiv infection. let t 1 denote the duration of this period. empirical works have been done to estimate the parameter. , anderson and medley (1988) report t 1 to lie between 40 and 60 days in transfusion-induced aids cases. in our model we assume that t 1 is a random variable following a normal distribution with the mean μ 1 and the variance σ 2 1 . after t 1 , the infected agent enters s4 state: infectious state. medically, the duration during which an infected is infectious but not yet symptomatic is called incubation period. we let t 2 donate the period. empirical work suggests an average incubation period of around 4 to around 15 years (medley et al. 1987 . a weibull distribution are commonly used to describe this incubation period (see, e.g., anderson 1988; anderson and medley 1988) . furthermore, , anderson and medley (1988) estimated t 2 with a weibull distribution (with a mean of 7.7 years and a median of 7.4 year) based on 545 transfusioninduced aids cases. for simplicity, we take t 2 as a real number drawn from a normal distribution with the mean μ 2 and the variance σ 2 2 rather than a weibull distribution. it should be pointed out that the simulation results generated with the normal distribution here are proved to have little, if any, difference compared with those generated when a weibull distribution is employed. during the t 2 period, hiv viruses in the victim's body are constantly cloning themselves and eventually the immunity system collapses. at this point, the victim starts to show some symptoms and thus transit to s5 state: symptomatic stage. as usually, let t 3 denote the duration of this period. rothenberg et al. (1987) report 5-7 year survival rates among 1660 idus (intravenous drug user) in new york city and find a median time of survival of 282 days. chang et al. (1993) report a median survival time of 10.5 months. empirical work shows that almost all hiv infectives, excluding those who die from other causes, will inevitably develop aids and die of it (may and anderson 1987) . similarly, we assume t 3 follows a normal distribution with the mean μ 3 and the variance σ 2 3 . eventually, the ill agent enters s6 state after t 3 passes by agents in s6 state will be removed from the population at the beginning of the next time and all of their uninfected neighbors will be released from s2 state, back to s1 state. generally, these state transitions take place in the order of s1, s2, s3, s4, s5, and s6. it is impossible for an agent to return from s3 state to s1 or s2 state. this backward transition s2 to s1, demonstrated by a dashed line in fig. 1 , is due to the disappearance of threats posed by infectious agents. moreover, although the transitions among s3, s4, and s5 state are not relevant to the propagation process, this process is closely related to hiv/aids transmission. taking account of this procession is essential for a better understanding of hiv/aids transmission. in the model, all the events triggering transitions could be divided into two categories. one category is a rule-based, such as healthy-to-dangerous, dangerous-to-infected, and dangerous-to-healthy state-transition events. these events occur according to the ca transition rules: an agent's state at time t + 1 is based not only on its own state but also on the states of its neighbors at time t . the other category is time-based, meaning that these events are scheduled at pre-specified times. for instance, an agent entering s3 state will be assigned a time indicating when to change to s4 state. after that amount of time elapse, the transition occurs spontaneously. despite the distinction between these two categories, the subtlety of implementing the two event-triggering mechanisms is very trivial and leaves no further elaboration necessary. actually, these six states can be divided into three "super" classes in terms of the taxonomy used in the classical mathematical models: s1 and s2 states correspond to the susceptible state; s3, s4, and s5 states belongs to the infection state, and s6 state is the removed state. obviously, s1 and s2 state could be treated as one single state without changing any results. the reason why we divide this into two sub-states is that this makes our model easily implemented and our logic decent and legible. hiv/aids epidemic differs from other epidemics in that its dynamics is heavily affected by individual's behavioral patterns and the interactions between them. for example, careful sex practices and sanitization measures in drug taking will make individuals less likely to be infected. behavioral patterns and interactions are mostly determined by individual's life styles, personalities, social networks, etc. however, the majority of models fail to take account of the heterogeneity in agents' behaviors. to capture this, we extend classical ca models by allowing each agent to have its own attributes such as mobility, infectivity, resistibility (susceptibility) 2 and different extent of neighborhood. assume that each cell in the grid can be occupied by at most one agent at a time. at time t , agent i can move from one cell into one of its adjacent cells with probability p m i . here, p m i is a measurement of agent i's activity level. it is a fixed real number, drawn from a uniform distribution (p m min , p m max ) (0 ≤ p m min ≤ p m max ≤ 1). when p m min = p m max , the activity level across agents is equal and therefore agents have the same inclination to move around. one extreme case is p m min = p m max = 0, which corresponds to the situation in which agents stay in their initial places during simulation, whilst p m min = p m max = 1 means that each agent will move into one empty neighboring cell at almost each time (he could get stocked and not move anywhere if it's neighborhood is occupied). it is easy to induce that the average time per move is calculated as 1/p m i . intuitively, high level of activity leads to speedy spreading. our simulation results verify this. besides the heterogeneity in agents' activity, another kind of heterogeneity is introduced when we assign various levels of infectivity and susceptibility to agents. let f i denote the infectivity level of agent i. f i is a real number drawn uniformly from the interval (0, 1). it measures the possibility that agent i transmits hiv viruses to others when they meets. evidently, greater values of f i indicate higher infectiousness of agent i. suppose also that each agent has resistance to being infected. we denote this resistibility as r i for agent i. similarly, r i is also a real number drawn uniformly from the interval (0, 1) and has the property that the greater the resistibility, the less is the chance of getting infected. note that the infectivity of an agent need not be a constant. an agent can have different level of infectivity, depending both on its state as well as on its behavior. it is widely believed that infectives experience two periods of high infectivity (see e.g. may and anderson 1987; may et al. 1988 ), one shortly after being infected and the other at the late stage of his illness. another example is that a patient might have high infectivity during the incubation period and low infectivity owing to good health care during the symptomatic period. although our model allow for various infectivity at different stages for a single agent, we adopt the fixed infectivity for each agent. in doing so, we can focus our attention on some significant issues. we leave various infectivity scenarios for future work. conventional ca models define two types of neighborhoods: moore neighborhood and von neumann neighborhood. in this paper, we extend the concept of ca neighborhood in order to better describe various situations encountered in agent-based modeling. figure 2 illustrates the definition. as we can see in fig. 2b shows the classical moore neighborhood, and fig. 2d classical von neumann neighborhood. figure 2c represents an extended moore neighborhood with the order of 2 × 2, and fig. 2e an extended 2 × 2 von neumann neighborhood. specially, fig. 2a can be simply viewed as an extended 0 × 0 moore (or von neumann) neighborhood. note that fig. 2b , f-i have the neighborhoods with one direction. this directional structure is able to capture the biases or preferences embedded in individuals' behavioral patterns and we can use different directions to represent variable ways of interactions. it is easy to see that the greater is the neighborhood, the larger extent to which an agent can exert its influence to its neighbors. given the above neighborhood definition, a concept of distance is naturally induced. let a pair of integer numbers (x, y) represent an agent's coordinates in the grid. the distance between agent i and j is thus given as (2) next, we specify that the influence indicator m i,j of agent i and j satisfies the following condition: that is to say, the influence intensity is inversely proportional to the distance between them if the influence can be exerted, and zero otherwise. therefore, the infective impact i i,j of agent i on agent j can be expressed as and the probability of an agent infected by one of its neighbors is defined as: where p(·) is a function satisfying the conditions: (1) p(·) is a real-valued function with the value between zero and one; (2) p(·) is increased in i i,j and decreased in r i . in this model, we assume p(·) takes the form of the following equation: here, p(i i,j , r i ) is interpreted as the probability of agent i being infected by its neighbor j . denoted by b i the set of all its neighbors, agent i's overall probability of infection is thus given by it indicates that this overall probability is determined by the most influential neighbor. such specification makes sense in most cases. the model developed in sect. 2 is implemented using java programming language with the repast software package. 3 detailedly commented source code is available from the authors upon request. next, we begin our analysis by considering first a typical simulation run as a benchmark case. 3.1 benchmark case table 1 lists the input parameters chosen for the benchmark case. in this case, the grid consists of 100 × 100 sites (w = 100). the population size n is set to 2000 with the initial infected ratio α = 0.005. all agents are homogeneous in terms of having the same infectivity f i = 0.2, resistibility r i = 0.5, and 1 × 1 moore neighborhood. they are uniformly distributed in the grid. the total simulation time t for each run is set to 2000. figure 3 shows a snapshot of the spatial distribution of the population at some time in a typical simulation. it is commonly believed that as the epidemic develops, its spread ends up with two typical situations: extinction or prevalence. figure 4 depicts these two situations. in fig. 4a , the number of infections 4 climbs early in the process. after about time t = 470, the infection level starts to drop slowly until it reaches zero at time t = 2000, while in fig. 4b , the number of infections increases slowly and reaches an equilibrium level after t = 1600. the intuition . 3 the spatial distribution of the population at time t = 1275 behind is that in the first case, newly infected continuously enter the pool of infectives at a fairly low rate in early stages. after the lengthy incubation period, these infectives begin to develop aids and eventually die. the total number of them drops when the infection rate is very low with regard to the rate at which infectives leave the pool for some reason (dead in the model). while in the second case, healthy agents get infected at a relatively high rate in early stages. the infection level continues to increases because the number of removed agents is relatively small in later stages. thus high infection rate often leads to prevalence as demonstrated in fig. 4b . these two results can be found in real-world situations. notice that fig. 4 the infections vs. time result in two simulations in the parameters setting, the growth rate r is almost zero (r = 0.0001). in next subsection, we will explore the effects of various factors, such as population density, initial infection ratio, and infectivity, on the epidemic. now we keep other parameters constant as before and let the population density β vary to see how β (β = n/w 2 ) affects the dynamics of hiv/aids transmission. tarwater and martin (2001) investigate this issue when studying the outbreaks of measles or measles-like infectious diseases. as one would expect, many common infectious diseases spread more rapidly at a high population density than at a low population density. figure 5 illustrates the time series of the mean numbers 5 of infectives for different population sizes n = 1500, 2000, 2500, 3000, and 3500. we can see that when population density is relatively low (n = 1500, 2000) , the infection levels are relatively low during the entire simulation and decline slowly in the later stages. this suggests that the epidemic died out eventually. in the and 400 at last. for n = 3000 and 3500, the infection numbers reach to a very high level and then drop rapidly. the collapse is because that so many infectives are removed from the model that the pool of infectives shrinks. for clarity, we also plot the fractions of infectives in the population vs. time in fig. 6 . clearly, in late stages of the epidemic, the fractions are greater when β is great than when β is small. in summary, hiv/aids infection is more likely to persist at higher population densities. this is due to that with the population density increasing, the population contact rate rise, leading to increases in the probability of infection. early work (see, e.g., rhodes and anderson 1996) suggests that there is a threshold below which the epidemic would eventually dies out and above which it would persist. due to the limitation of ca methods, it is diffifig. 7 mean number of infections vs. time for different initial infected ratios cult to pin down its exact value. however, with many simulation runs, we still can give an approximate interval in which the threshold lies. an interesting question one may ask is how epidemic spreading is affected by initial configurations of susceptibles and infectives, or whether the multiple infection sources will make the disease more likely to become endemic. with other parameters fixed as before, we run the simulations with α = 0.002, 0.004, 0.006, and 0.008, respectively. figure 7 presents the result. clearly, as α increase, the infection level shifts upwards. in the case of α = 0.004, the infection level reach 300 at time t = 2000, higher than 200 in the case of α = 0.002. by contrast, the level in the case of α = 0.006 climb to around 380 at time t = 1000 and drops slightly to 340 at time t = 2000. the maximum infection is reached in the case of α = 0.008 at time t = 1000, which is more than 440. spatially, more sources of infection imply greater chance of being infected within a certain area, letting hiv/aids epidemics to be more likely to spread out and persist. statistically speaking, an individual's probability of infection is generally proportional to the number of infectious sources. intuitively, the more migratory the population, the more likely that an epidemic is to spread. suppose an agents' activity can be measured by the number of contacts it makes with others within a unit of period of time. as a result, our model assumes that individuals' activity is measured by mobility. later stages. this is, in the case of p m max = 0.005, the level is above 120 and in the case p m max = 0.007, the level is in the range (180, 210). in the last case of p m max = 0.009, the infection level fluctuates above 300, higher than those of other cases. so we conclude that mobility plays a significant role in the dynamics. it could explain why chinese government took rather strong measures to control the migratory people and quarantine the infectives or the suspects during the outbreak of sars in the spring of 2003. as to our model, if agents are configured with higher mobilities, it is more likely that the hiv/aids infection can persist in the population, whilst if configured with lower mobilities, the infection would gradually diminish and eventually die out. next, we are to examine how neighborhood forms affect hiv/aids epidemic dynamics. the parameter sets are kept the same as in benchmark case, except for the adoption of different neighborhood forms. figure 9 illustrates the simulation results generated in two cases: one with 1 × 1 von neumann neighborhood and the other with 1 × 1 moore neighborhood. in the case where the von neumann neighborhood is used, the level of infection goes up to about 140. it is clearly greater than that of the case with 1 × 1 moore neighborhood in which the level only reach about 100. such result suggests that with wider neighborhood, an agent is more likely to get influenced by its neighbors and therefore the likelihood of getting infected increases accordingly. it is easy to induce that the infection level rises with the order of neighborhood. this result also suggests that hiv/aids epidemic dynamics is significantly affected by strong interactions between agents. we now turn to examine heterogeneous mixing, i.e., different at-risk groups coexist. usually, heterogeneous mixing will make the dynamics more complicated and unpredictable. in the following, we assume the whole population is divided into four different groups as shown in table 2 . as shown in table 2 , class p0 has very low infectivity, low susceptibility, and 1 × 1 von neumann neighborhood. it can represents children and (or) elders in the population who hardly infect others and are easy to be infected. class pl refers to ordinary people who have relatively low infectivity and high resistibility (therefore low susceptibility). in our model, this class amounts to a large fraction of the whole population. class ph and class ph+ can represent the two high-risk groups observed in real life. agents of class ph have high infectivity and low resistibility duo to their high-risk behaviors like incautious sex without protection, needle sharing, unhygienic blood transfusion and so on. the biased 0 × 1 (or 1×0) von neumann neighborhood captures their potential oriented or biased behaviors. in contrast, agents of class ph+ with both higher infectivity and higher susceptibility represent those who are, although being in the minority, the most dangerous and malevolent group. such group does exist in real life. for instance, some crimes were reported in china in recent years that a few aids infectives intentionally have sex with innocent people or shot people with contaminated syringes in public places. they blame their infection on the society and the government for not being able to provide necessary health service and compensating too little. the 2 × 2 moore neighborhood indicates their intensive influence on others. we distinguish class ph+ from class ph in order to see whether such malevolent behaviors have significant impacts on the spread of hiv/aids and to what extent. while such rough classification may be incorrect or even erroneous, it surely is reasonable and well supported by our extended ca model. table 3 gives the values of f i and r i used in the following simulations. as we will see later, the results generated with this classification are fairly consistent with those obtained through empirical work. figure 10 gives a typical simulation result in the four-class scenario. the fractions of four classes here are: n p0 = 0.1, n pl = 0.7, n ph = 0.1, and n ph+ = 0.1. the infection curve in fig. 10 is quite similar to that obtained in the single-class scenarios except that its level is fairly higher. in this section, we will investigate the effect of agents' susceptibility on the dynamics of hiv/aids. given n p0 = 0.05, n pl = 0.8, n ph = 0.1, n ph+ = 0.05 and others as before, let r ph+ vary. figure 11 gives the infection levels when r ph+ is set to be 0.6, 0.7, 0.8, and 0.9, respectively. as we can see, these equilibrium infections are almost at the same level, which is inconsistent with our expectation. the differences are so small that we cannot assure with confidence whether changes in susceptibility have impact on the epidemic dynamics. the possible reasons, we believe, are twofold: first, the role played by susceptibility may be not as decisive as the above factors. second, we may describe susceptibility in the wrong way and make it an inessential factor in our model. future work will reconsider this issue and find the better way to describe susceptibility. at last, we will examine whether changes in the fractions of some classes can affect epidemic behaviors. given n p0 = 0.1, n pl = 0.7 and other parameters as before, we take n ph = 0.12 (n ph+ = 0.08), 0.14 (0.06), 0.16 (0.04), 0.18 (0.02), 0.20 (0.0), respectively. figure 12 shows the infection levels in these five combinations. as observed from fig. 12 , we obtained very similar results, compared with those in the foregoing analysis. with n ph+ decreasing, infection level declines. recall that ph+ has more influence on its neighbors than ph, thus leading to greater transmission to others and larger infection rate. this finding suggests the government should pay more attention to those who have high-risk life styles and revengeful behaviors. this makes it the essential issue of how highly infectious and malevolent individuals are restricted and controlled. the focus of the paper is on the modeling of the entire course of hiv/acid epidemics and heterogeneity in agents' behaviors. even though classical ca models are capable of describing the spread of common epidemics but fail to represent the complicated epidemics like hiv/aids disease. the ignorance of heterogeneity gives rise to unacceptable errors in the prediction of the development trends. in addition, components of a conventional ca system such as topological forms of grid, the definition of neighborhood, and state transition rules are simple and unchanged over time. this makes the modeling of complicated dynamics such as hiv/aids transmission difficult and uncontrollable. in this paper, we have developed an extended ca model to capture key epidemiological and clinical features of hiv/aids epidemic. first, we explicitly models and simulates the whole progression of hiv/aids disease (i.e., infected but not infectious, infectious but asymptomatic, symptomatic, and deceased). such improvement can give us a better understanding of the dynamics during the entire hiv/aids epidemics. in order to examine various degrees of influence between agents, we have introduced an extended definition of neighborhood to represent the intensity and bias of influence. this lets us gain insight into how various degrees of interactions affects the hiv/aids epidemics. another type of heterogeneity in disease-related attributes such as susceptibility, infectivity and durations of epidemic phrases is also taken into consideration. moreover, we also consider the effect of agents' mobility on epidemics dynamics. given all the improvements, we have obtained richer simulation results similar to those usually found in the mathematical models or other classical simulation models. we have identified some influential factors that greatly affect the hiv/aids epidemic dynamics. the main findings are that 1) hiv/aids epidemic can end up in the two regimes: extinction and persistence; 2) with these factors such as agents' mobility, population density, initial infection ratio, and the extent of neighborhood increasing, the infection level get higher. after crossing some critical point, the regime generated could change from dying-out to persistence at some point. this result is robust across many of the tested parameter combinations; 3) in four-class scenarios, the great fraction of 'super' infectives (the ph+ class in our model) can also produce higher level of infection. however, our simulation study above is still preliminary. there are some issues needed to be addressed. first, as said before, we should redefine susceptibility in a better way to check its role in the dynamics of hiv/aids epidemic. second, most models posit that a virus carrier's infectivity is constant during the progress of a disease. however, this is not the case for hiv/aids epidemic. various infectivity at different stages could have substantial impact on the dynamics of hiv/aids transmission. this problem needs special attention. additional, further developments of our model, e.g. by adding age-related structure (griffiths et al. 2000) , different subgroup classification, and other heterogeneity, would greatly add to the appeal of this model. with these additions, a better understanding of hiv/aids and thorough empirical work are required. finally, a natural extension of the model is to include the assessment of various control policies and managerial strategies, and this will be a firm support for the decision-making in prevention programs against hiv/aids. on modeling epidemics. including latency, incubation and variable susceptibility the epidemiology of hiv infection: variable incubation plus infectious periods and heterogeneity in sexual activity infectious diseases of humans: dynamics and control possible demographic consequences of aids in developing countries epidemiology of hiv infection and aids: incubation and infectious periods, survival and vertical transmission a simulation model of the dynamics of hiv transmission in intravenous drug users a comparison of simulation models applied to epidemics the mathematical theory of infectious diseases and its applications dynamics of hiv infection on 2d cellular automata models for transmission of disease with immigration of infectives survival and mortality patterns of an acquired immunodeficiency syndrome (aids) cohort in new york state mathematical modeling in epidemiology cellular automata and epidemiological models with spatial dependence a heterogeneous cellular automata model for sars transmission a cellular automaton describing the formation of spatially ordered structures in chemical systems a cellular automaton model of excitable media a simple agent model of an epidemic an age-structured model for the aids epidemic the differential infectivity and staged progression models for the transmission of hiv a model for the influence of the greenhouse effect on insect and microorganism geographical distribution and population dynamics a model for predicting forest fire using cellular automata. ecological modelling a contribution to the mathematical theory of epidemics the dynamics of hiv spread: a computer simulation model flood decision support system on agent grid: method and implementation algebraic properties of cellular automata transmission dynamics of hiv infection the transmission dynamics of human immunodeficiency virus (hiv) [and discussion incubation period of aids in patients infected via blood transfusion the distribution of the incubation period for the acquired immunodeficiency syndrome (aids) mathematical biology i experiences creating three implementations of the repast agent modeling toolkit persistence and dynamics in lattice models of epidemic spread epidemic thresholds and vaccination in a lattice model of disease spread survival with the acquired immunodeficiency syndrome. experience with 5833 cases in new york city effects of population density on the spread of disease diffusion and wave propagation in cellular automaton models of excitable media modelling the hiv epidemic: a state-space approach effectiveness of q-learning as a tool for calibrating agent-based supply network models we would like to express our gratitude to the many people in xi'an jiaotong university who have participated in planning, data collection, and presenting the results. without their efforts, this simulation modeling would not have been possible. this work is supported by nsfc under contract 70601023. we are grateful to ting zhang and jie huang for excellent research assistance and the referees for many helpful comments that greatly improved the presentation. key: cord-020010-q58x6xb0 authors: nan title: 19th icar abstracts: date: 2006-03-13 journal: antiviral res doi: 10.1016/j.antiviral.2006.02.001 sha: doc_id: 20010 cord_uid: q58x6xb0 nan the society was organized in 1987 as a non-profit scientific organization for the purpose of advancing and disseminating knowledge in all areas of antiviral research. to achieve this objective, the society organizes an annual meeting. the society is now in its 20 th year of existence, and has about 600 members representing 30 countries. for membership application forms or further information, please contact dr. amy patick, secretary, isar; pfizer global r&d, department of virology, 10777 science center drive, san diego, ca 92121; phone +1 858 622 3117; fax +1 858 678 8182; e-mail amy.patick@pfizer.com. membership application forms will also be available at the conference registration desk, or from our website www.isar-icar.com. enzymes of the pol gene of hiv have been identified as important viral targets for the discovery anti-hiv therapeutic agents. while the viral targets, hiv reverse transcriptase and hiv protease, have been successfully investigated for the development of clinically useful therapeutic agents, research efforts on drug discovery on the third enzyme of the pol gene, hiv integrase, have not resulted in a single fda-approved drug. nevertheless, as integrase is essential for hiv replication, it remains an attractive target for the discovery of new anti-hiv agents. in this presentation, we report the discovery of a conceptually new beta-diketo acid, constructed on a nucleobase scaffold, that is a potent inhibitor of both the 3 -processing and strand transfer steps of recombinant hiv integrase. this inhibitor and the positive control compound, azt, were tested in a pbmc cellbased, microtiter anti-hiv assay against the clinical isolate, hiv-1teki (nsi phenotype) and hiv-1nl4-3 (si phenotype), and in a magi-x4 assay against hiv-1nl4-3 with hela-cd4-ltr-beta-gal cells. our integrase inhibitor was found to have highly potent in vitro anti-hiv activity and efficacy. the discovery of this remarkably active molecule, representative of a unique set of diketo acids bearing nucleobase scaffolds, has uncovered a new chapter in the chemistry and biology of integrase inhibitors and their potential therapeutic applications. berta bosch 1 , imma clotet-codina 1 , julia blanco 1 , eduard pauls 1 , gemma coma 1 , samandhy cedeño 1 , francesc mitjans 2 , anuska llano 1 , margarita bofill 1 , bonaventura clotet 1 , jaume piulats 2 , jose este 1 1 retrovirology laboratory, fundacio irsicaixa, badalona, spain; 2 laboratorio de bioinvestigación, merck farma y química, barcelona, spain macrophages are key cells for hiv infection and spreading inside the organism. macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (m-csf). in the monocyte to macrophage differentiation process with m-csf, av-integrins are upregulated concomitantly to the capacity of hiv to generate a productive virus infection. in the present study we show that an anti-av antibody, 17e6, inhibited hiv-1 infection of primary macrophages. the effect of 17e6 on r5 or x4 hiv-1 replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (ec50) of 17 ± 2 g/ml in the absence of cytotoxicity. similarly, a monoclonal antibody targeting the avb6 integrin (14d9.f8) also inhibited hiv-1 infection in this cell type. 17e6 reduced the detection of hiv-1 bal proviral dna in acutely infected macrophages but was completely ineffective against hiv-1 bal production in chronically infected macrophages, suggesting that 17e6 inhibited hiv infection at an early stage of the virus cycle. finally, a small molecular weight antagonist of the avb6 integrin reduced hiv replication at subtoxic concentrations. therefore, our results suggest that av-containing integrins could play a role in hiv replication in macrophages and indicate that small molecular weight compounds may be developed to interfere with hiv replication in macrophages through the interaction with av integrins. andrew vaillant 1 , hong lu 2 , shuwen liu 2 , carol lackman-smith 3 , roger ptak 3 , jean-marc juteau 1 , shibo jiang 2 1 replicor inc., laval, que., canada; 2 f. lindsay kimball research institute, new york blood center, new york, ny, usa; 3 southern research institute, frederick, md, usa the sequence independent antiviral activity of phosphorothioate oligonucleotides in inhibiting hiv-1 by blocking interactions between the v3 loop and cd4 has been previously described. this activity was attributed to their polyanionic activity. here we show that ps-ons (and their fully 2 -o-methylated derivatives) are also potent inhibitors of hiv-1-mediated membrane fusion and hiv-1 replication in a sequence-independent, sizedependent (optimal size ∼50-60 bases) and phosphorothioation dependent manner (independent of stabilization). ps-ons interact with the heptad repeat regions of gp41 and the hiv-1 fusion inhibitory activity of ps-ons is closely correlated with their ability to bind to these heptad repeats and block gp41 six-helix bundle formation, a critical step during the process of hiv-1 fusion with the target cell. the requirement for ps-on interaction was also found to be dependent on phosphorothioation, suggesting that the v3 loop/ps-on interaction may also have a hydrophobic component. the increased hydrophobicity of longer (≥40 base) ps-ons may contribute to their inhibitory activity against hiv-1 fusion and entry because these longer ps-ons have a greater hydrophobicity and are more potent in blocking the hydrophobic interactions involved in the gp41 sixhelix bundle formation than shorter ps-ons (<30 bases). this novel antiviral mechanism of action of long ps-ons has important implications for therapy against infection by hiv-1 and other enveloped viruses with type i fusion proteins. chris meier 1 , soenke jessel 1 , bastian reichardt 1 , olaf ludek 1 , jan balzarini 2 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium carbocyclic nucleoside analogues like abacavir showed very interesting antiviral properties. therefore, we are interested in a convenient stereoselective access to this class of compounds as potential antiviral agents. by using a new convergent synthetic strategy, starting from a chiral cyclopentenol, enantiomerically pure carbocyclic thymidine (carba-dt) was obtained as a key intermediate for further variations at the 3 -position. this pathway allows an entry to d-and l-configurated nucleoside analogues. however, using this approach a mixture of side products avoids the formation of the product in very high yields. however, we will present that the side products can be recovered by a stereoselective hydroboration leading to one intermediate only that can be use as well for the synthesis of carbocyclic nucleosides. the condensation of the carbocyclic moiety and different pyrimidine and purine nucleobase was achieved by a mitsunobu reaction. various analogues have been prepared via this strategy, e.g. d-and l-carba-bvdu, nucleoside analogues known to be antivirally active against hsv-1. additionally, carbocyclic ␣nucleosides and carbocyclic iso-nucleosides are accessible by this reaction sequence. all new nucleoside analogues were tested for their antiviral activity. particularly carba-dt was found to be a potent anti-hiv active derivative showing no toxicity. however, it can not be excluded that a non-activity of a compound is related to a missing phosphorylation to the monophosphate. in order to prove that, all nucleosides were converted into their cyclosal-phosphate trimesters and transferred into the nucleotides. detailed chemistry, enzymatic and antiviral activity data will be presented. in some cases the nucleotide releasing system showed improved antiviral activity as compared to the parent nucleoside. michela pollicita 1 , candace pert 2 , maria-teresa polianova 2 , alessandro ranazzi 1 , michael ruff 2 , carlo-federico perno 1 , stefano aquaro 1 1 university of rome tor vergata, italy; 2 georgetown university, washington, dc, usa monocytes/macrophages (m/m) are a strategic reservoir of hiv-1 commonly infected by ccr5-using (r5) strains of hiv-1. ccr5 is an attractive target for inhibition of ccr5 mediated hiv-1 entry. thus, ccr5 antagonists are expected to be a power-ful new class of receptor-based therapeutic agents against hiv-1 infection. d-ala-peptide t-amide (dapta) is an octapeptide derived from the gp120 v2 region of hiv-1, able to bind ccr5. dapta acts as selective viral entry inhibitor, displacing the binding of gp120 with ccr5. dapta anti-hiv-1 activity was evaluated in m/m infected with two different hiv-1 r5 strains, bal and 81a, in presence of several doses of the compound. dapta inhibited hiv-1 replication in m/m (>80% compared to control), measured by the p24 gag ag released in the cell culture supernatants, at concentration of 10-3 nm. pcr analysis of integrated hiv-1 proviral dna on cultured m/m proved that dapta is able to block hiv entry and so, to prevent hiv infection in m/m. moreover, the capability of different hiv-1 r5 strains produced and released by infected m/m in affecting neuronal homeostasis was assessed in a neuroblastoma cell line, sk-n-sh, expressing ccr5. in sk-n-sh were evaluated cell morphology, propidium iodide binding and fluorescenceactivated cell sorting (facs) analysis. dapta, at concentration of 10-3 and 10-4 nm, strongly inhibited apoptosis in sk-n-sh of 58 and 56%, respectively, compared to control. unexpectedly, tak-779 (a nonpeptidic ccr5 antagonist with potent anti-hiv-1 activity) inhibited apoptosis only of 30% compared to control. our results suggest that the development of new anti-hiv-1 compounds, such as dapta, could be important in synergistic combination with other antiretroviral treatments in prevent both central nervous system hiv-infection and the consequent neural damage. the mechanisms of dapta inhibition may include both suppression of hiv-1 r5 strains in the brain as direct inhibition of hiv-1 replication in m/m and gp120 related damage by ccr5 binding. pradimicin a (prm-a) is an antifungal non-peptidic benzonaphtacenequinone antibiotic that specifically inhibits human immunodeficiency virus (hiv) in cell culture. it markedly suppresses a variety of different hiv-1 clades in pbmcs, in primary macrophages and several hiv-2 and siv strains in laboratory cell lines (range of 50% effective concentrations: 0.69-11 g/ml; 50% cytostatic concentration: >50 g/ml). prm-a also inhibits syncytium formation between persistently hiv-1-infected hut-78 cells and uninfected sup t1 cells. prm-a behaves as an artificial lectin that selectively binds mannose-containing glycans. consequently, biacore experiments revealed that it binds to gp120 of hiv-1/mn in the presence of ca 2+ . prm-a is endowed with a high genetic barrier with regard to drug resistance development against hiv-1. a variety of multiple mutations at n-glycosylation sites in hiv-1 gp120 are required before the virus looses marked sensitivity to the drug. there is no clustered pattern of hiv-1 gp120 glycan deletions that occur under prm-a drug pressure. the resistance spectrum and mode of action is unique among any of the existing anti-hiv drugs and warrant further (pre)clinical investigations. acknowledgement: this research was supported by the flemish "fonds voor wetenschappelijk onderzoek," the centers of excellence of the k.u. leuven (no. ef/05/15), and the european commission (empro). jan muench 1 , ludger ständker 2 , knut adermann 2 , axel schulz 2 , michael schindler 2 , raghavan chinnadurai 2 , wolf-georg forssmann 2 , frank kirchhoff 2 1 department of virology university of ulm, albert-einstein allee 11, 89081 ulm, germany; 2 ipf pharmaceuticals gmbh, feodor-lynen-strasse 31, 30625 hannover, germany a variety of components in human blood might influence hiv-1 replication in infected individuals. peptide libraries derived from hemofiltrate (hf), an aqueous blood solution, contain essentially all circulating blood peptides with a molecular mass below 30 kda, including chemokines, defensins, and cytokines. to identify the most potent natural occurring factors inhibiting hiv-1 replication, we screened a hf-derived peptide library for antiviral activities. the most active fraction contained a 20-residue peptide corresponding to a c-terminal fragment of ␣1-antitrypsin (␣1-at), a highly abundant serine proteinase inhibitor. further analysis of the corresponding chemically synthesized peptide, termed virus inhibitory peptide (virip), demonstrated that it inhibits infection by all hiv-1 variants tested, independently of their subtype and coreceptor usage. notably, virip also blocked multi-resistant hiv-1 variants and primary isolates. virip specifically inhibited hiv-1 env function, and did not affect infection by virions containing hiv-2, siv, mlv, hcv, ebola or vsv env proteins. the antiviral activity proved to be highly specific for the 20-residue virip sequence since structurally closely related peptides were inactive. we found that virip inhibits hemolysis of erythrocytes induced by the hiv-1 gp41 fusion-peptide (fp). nmr spectroscopy confirmed that virip interacts directly with synthetic gp41 fp. our observations are evidence that a naturally occurring human substance inhibits hiv-1 infection by a new mode of action, i.e. binding of the highly conserved fp. furthermore, we performed a structure-activity-relation study with more than 350 virip analogs and found that specific amino acid changes enhanced the antiviral potency of virip by up to two orders of magnitude. experiments in cell culture and animal models further demonstrated that virip exerted no cytotoxic effects. thus, virip derivatives might become a new class of hiv-1 entry inhibitors. stefano aquaro 1 , valentina svicher 1 , roberta d'arrigo 2 , ubaldo visco-comandini 2 , andrea antinori 2 , mario santoro 1 , giovanni di perri 3 , sergio lo caputo 4 , pasquale narciso 2 , carlo-federico perno 1,2 1 university of rome tor vergata, italy; 2 inmi l. spallanzani, italy; 3 university of turin, italy; 4 sm annunziata hospital, florence, italy to investigate gp41-variability and correlation with viroimmunological parameters in 54 hiv-infected patients (pts) receiving t20 added as a single active drug to a failing regimen. two hundred and ten hiv-gp41 sequences and clinical follow-up from 54 t20-treated patients were analyzed from baseline up to 48 weeks (weeks) of treatment. the association of mutations with viremia (vl)/cd4 count (c/ul) was assessed by mann-whitney test. the addition of t20 to the failing antiretroviral regimen induced at 4 weeks a significant vl decrease from 5.1 log (stable in the last 12 weeks prior t20) to 4.3 log (p = .0002) and a significant cd4 increase from 48 c/ul (decreasing in the last 12 weeks prior t20) to 106 c/ul (p = .008). while vl rebounded to 4.7-5 log at 12-48 weeks, respectively, cd4 increased to 129 c/ul at 48 weeks. t20 resistance mutations, absent at bl, occurred shortly after treatment and usually alone. v38a was the most common sign of t20 failure (27.8% of pts). the viroimmunological outcome of t20-treated pts varied according to gp41-mutations. v38a/e (38.5% of pts) was associated with a cd4 increase from bl (48 c/ul) of 4.5-fold (142 c/ul) at 24 weeks and 6.0-fold (196 c/ul) at 36 weeks (p = .004 and .02 compared without v38a/e, respectively). no significant correlation with vl was observed (from 4.9 log at bl to 4.4-4.8 at 24-36 weeks). by contrast, q40h + l45m (11.1% of pts) was associated with cd4 loss from 71 c/ul at bl to 26 c/ul at 36 weeks (p = .02), without significant changes in vl (from 5 log at bl to 5 log at 36 weeks). mutation n126k (observed in 6 pts, but never found at bl) abrogates the 4th gp41-glycosylation site and correlated with 1.7-fold cd4 increase at 24 weeks. conformational changes induced by v38a/e in the highly conserved giv motif of gp41-hr1, are tightly related with a loss of hiv-induced damage of immune system. this facilitates cd4-recovery through mechanisms that can be virus-(loss of fusion efficiency) and immune-mediated (exposure of new epitopes) not applicable to protease/rt-inhibitors, and thus important for innovative therapeutic strategies. the spread of highly pathogenic h5n1 influenza viruses in humans in asia, with high mortality rates among infected individuals is a major public health concern. in the absence of a vaccine antigenically matching the pandemic virus, antiviral drugs can play an important role. in the present study we reported the antiviral activity of neuraminidase inhibitor oseltamivir against lethal h5n1 influenza virus infection in ferrets, an appropriate animal model that closely resembles clinical signs of human influenza. inoculation of young adult ferrets with a viral dose as low as 10 2 eid 50 of a/vietnam/1203/04 (h5n1) influenza virus caused high fever (39.8-41.8 • c), weight loss (25.4% of initial), anorexia, extreme lethargy and death of animals on days 7-10 post-virus inoculation (p.i.). oral administration of oseltamivir at a dose of 5 mg/kg/day for 5 days twice daily initiated 4 h p.i. inhibited the febrile response, reduced weight changes (14.6% of initial) and, most importantly, completely protected ferrets from lethal h5n1 infection. in the treatment groups, virus replication in the upper respiratory tract of ferrets was prevented, whereas untreated animals shed virus at titers of 2.8-6.5 log 10 eid 50 /ml on days 3, 5 and 7 p.i. systemic spread of the h5n1 virus was observed in untreated ferrets: virus was detected in multiple internal organs, including the brain. treatment with oseltamivir resulted in complete inhibition of virus replication in the lungs and small intestine on day 5 p.i. in the brains of treated animals virus was detected in one of the two animals tested with >100-fold reduction of titer. sequence analysis showed no amino acid substitutions at conserved residues in na or ha1 subunit in viruses isolated from ferret's internal organs after treatment. these results suggest that oseltamivir earlier treatment can prevent h5n1 mortality in ferrets, however, further studies investigating optimal doses and treatment durations required to achieve protection against infection with highly pathogenic influenza viruses are much needed. natalia ilyushina, erich hoffmann, rachelle salomon, robert webster, elena govorkova st. jude children's research hospital, memphis, tn 38105, usa in the present study we tested in the mouse model the hypothesis that combination chemotherapy with drugs targeting dif-ferent virus proteins may lead to more potent and beneficial effects. we applied plasmid-based reverse genetics technique to generate two recombinant a/vietnam/1203/04-like (h5n1) viruses. one virus possessed asparagine at position 31 of the m2 protein that was found in the naturally circulating virus (rgvn-1203) and confers resistance to amantadine. the other recombinant virus possessed serine at that position and was sensitive to amantadine (rgvn-1203sens) . balb/c mice were administered oseltamivir (1 or 10 mg/kg/day) and amantadine (1.5 or 15 mg/kg/day) twice daily for 5 days by oral gavage; the first doses were given 24 h before inoculation with 10 mld50 of h5n1 virus. combination treatment with 10 mg/kg/day oseltamivir and 15 mg/kg/day amantadine was given on the same schedule. single-drug oseltamivir produced a dose-dependent antiviral effect against both recombinant h5n1 viruses (p < 0.01). treatment with oseltamivir at dosage of 10 mg/kg/day significantly inhibited virus replication in the lungs, brain, spleen, and blood of mice at days 3, 6, and 9 after inoculation (p < 0.05), but resulted in low survival rate (20%). single-drug amantadine showed dose-dependent effect only against rgvn-1203sens strain. notably, risk of death for mice that received 15 mg/kg/day of amantadine or 10 mg/kg/day of oseltamivir was similar (p < 0.05). in contrast, prophylactic treatment of mice with combinations of oseltamivir and amantadine completely inhibited virus replication in the animals infected with rgvn-1203sens (p < 0.05) compared to singledrug usage and protected 90% of animals. importantly combination chemotherapy completely protected h5n1 virus spread to the brain of the mice: virus was not detected in brain of treated animals on days 3, 6, and 9 after inoculation and neurological symptoms were not observed. our results suggest that combination chemotherapy provides an advantage over single-agent treatment. this strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored. françois jean 1 , reid asbury 2 , meera raj 1 , david lawrence 3 , martin petric 3 1 the university of british columbia, vancouver, bc, canada v6t 1z3; 2 ge healthcare bio-sciences, piscataway, nj 08855, usa; 3 british columbia center for disease control, vancouver, bc, canada v5z 4r4 in late 2002, severe acute respiratory syndrome (sars) became the first new severe and easily transmissible human disease to emerge in the 21st century. although it abated after six months, sars serves as a modern paradigm for human emerging infections with 772 deaths reported from 29 countries. the causative agent was found to be a new sars-associated coronavirus (sars-cov) . while the sequence of sars-cov genome was first reported by the bc genome sciences center, the full set of viral and cellular proteins that compose the sars-cov virion remains unknown. to approach this problem, we have utilized two-dimensional gel electrophoresis and liquid chromatography-tandem ms (lc-ms/ms) to identify the viral and cellular proteins in purified sars-cov virions obtained from human infected cells [huh7: human liver] and primate (veroe6: monkey kidney) infected cells. interestingly, analysis of the proteins from purified sars-cov preparations has revealed that the enveloped virions contain not only the predicted viral structural proteins (e.g. spike glycoprotein, nucleocapsid protein, and membrane glycoprotein) but also an important number of differentially incorporated host cellular proteins into or onto the newly formed viruses. we have unambiguously identified over 50 host cellular proteins in sars-cov virions by lc-ms/ms. these proteins include members of the annexin superfamily, cytoskeletal proteins, chaperones, vesicular transport proteins, uracyl-dna glycosylases, and aldehyde oxidoreductases. this study provides the first comprehensive and comparative analysis of the viral and cellular proteins that compose infectious particles of sars-cov obtained from human and primate infected cells. the functions of these newly identified hostspecific proteins are currently being investigated using rna interfering systems; their contributions to structure, viral productive replication, and pathogenicity will be discussed. acknowledgement: supported by an early career ubc operating grant (f. jean) and cihr (m. petric) . dale barnard 1 , craig day 1 , robert montgomery 1 , kevin bailey 1 , matt heiner 1 , larry lauridsen 1 , robert sidwell 1 , kurt berg 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 panum inst., immi, the ifn-lab, copenhagen, denmark severe acute respiratory syndrome (sars) is a life-threatening respiratory illness caused by sars-cov. there are no approved therapies for sars. some drugs inhibit sars-cov replication in vitro including human interferons and selected antiinflammatory agents (chihrin and loufty, 2005. 3, 251-262) . interferons are very promising because of their potent in vitro inhibition of sars-cov. although anti-inflammatory agents are not very active in vitro, it is thought that they might be efficacious in reducing any deleterious inflammatory response associated with virus infections such as sars infections in humans. for example, troxerutin, a flavenoid with anti-inflammatory properties, is in clinical trials for treating rhinovirus (rv) infections, ameliorating rv-induced inflammation (turner et al., 2004. apmis 112, 605-611) . therefore, troxerutin was tested for inhibition of sars-cov replication in the lungs of infected mice using a mix of four hydroxyethylrutosides that included troxuretin. in addition, mouse interferon-alpha, used as a model compound for human interferon-alpha, was evaluated for inhi-bition of virus lung titers. both mouse interferon-alpha administered i.p. daily beginning 12 h pre virus exposure at doses of 100,000 and 10,000 iu and the hydroxyethylrutoside mix (100 and 10 mg/kg) administered i.p using the same schedule reduced virus replication in the lungs of mice to below detectable limits. when a hydroxyethylrutoside mix was given to mice in the drinking water at 1.32 mg/ml (likely equivalent to an i.p. dose of 100 mg/kg, assuming that the mice drank freely), virus lung replication was also completely inhibited. all treatments appeared to be well tolerated, since all groups of mice gained weight. we also report on the efficacy of various combinations of two doses of these drugs administered i.p., using the same dosing regimen as described. these data support the supposition that interferon might be a useful therapy for treating human sars infections and that hydroxyethylrutosides should be investigated further as a potential therapy. acknowledgement: supported by contract no. n01-ai-15435 from the virology branch, niaid, nih. treatment options for human respiratory syncytial virus (rsv) are limited. an effective vaccine is not yet available. neutralizing polyclonal antibody (respigam tm , medimmune) and a humanized monoclonal antibody (synagis tm , medimmune), are licensed for prophylactic use. ribavirin is the only approved antiviral against rsv, but its efficacy is controversial and its use is limited to treatment of high-risk patients. there is a clear need for new anti-rsv therapeutics with improved efficacy and ease-of-use. many early efforts to identify anti-rsv compounds focused on blocking the process of fusion. we have developed a cell-based screening platform to identify antivirals that inhibit rsv transcription and replication. the assay does not require infection with wild-type virus. it is based on an rsv subgenomic replication system in baby hamster kidney (bhk-21) cells that express the essential viral replication proteins (n, p, l and m2-1). the readout is expression of the reporter gene lacz from a subgenomic rna. screening of the apath small molecule library yielded 596 compounds (hit rate = 0.75%) with ec50 values ≤10 m and with selectivity index (si) values ≥10. seventy-two compounds demonstrated antiviral activity against wild-type rsv (strain a2) in a cytopathic effects inhibition assay (ec50 < 10 m; si > 10). these anti-rsv compounds represent nine different chemical classes. two compounds, a 4-aminoquinoline (ec50 = 0.25 m) and a thienopyrimidine (ec50 = 0.82 m), were shown to have desirable pharmacokinetic profiles and were chosen for efficacy testing in the cotton-tail rat model of infection. sar studies to identify the pharmacophore of the compounds have been initiated. preliminary studies to characterize the mechanism-ofaction in virological assays will be discussed. acknowledgement: supported by nih r44 ai047552-02. we have previously reported bicyclic furano pyrimidine nucleoside analogues (bcnas) as exquisitely potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for p-alkylphenyl substituted analogues such as lead compound cf1743(cf-1743) (1) . these compounds have entered preclinical development with fermavir pharmaceuticals. we now report the first chromatography-free synthesis of these agents, their scale up to multi-gramme amounts, and their pre-clinical characterisation. in addition, we were keen to address potential solubility and bioavailability issues of these highly lipophilic agents by the synthesis of more polar analogues in two categories; side-chain ethers (2) as new analogues in their own right, and 5 -phosphates (3) as potential more soluble prodrugs. we report data on both of these new families at this meeting. finally, we note the application of our phosphoramidate pro-tide approach to this family, with a series of bcna protides (4) designed as intracellular phosphate delivery forms to bypass the essential vzv thymidine kinase-mediated first phosphorylation step. graciela andrei 1 , joos van den oord 2 , pierre fiten 1 , ghislain opdenakker 1 , erik de clercq 1 , robert snoeck 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 pathology department, u.z. leuven, leuven, belgium varicella (chickenpox) , the primary infection caused by vzv, is characterized by viremia and skin lesions. reactivation of the latent virus results in skin lesions characteristic of herpes zoster (shingles). as keratinocytes are one of the main target cells for productive infection in vivo for vzv, human epithelial cells represent a relevant model for the study of vzv pathogenesis and evaluation of antiviral compounds. organotypic epithelial raft cultures permit full differentiation of keratinocytes via culturing of the cells on collagen matrix at the air-liquid interface. we have previously shown that the susceptibility of cultures to infection with vzv depends on the stage of differentiation of the rafts. we have now quantified the activity of reference anti-vzv compounds by measuring viral dna load by realtime pcr. quantitative pcr for vzv dna was performed by using specific primers and a mgb-probe for the orf29 gene (single-stranded dna binding protein) by the taqman method. two series of raft cultures were infected with the wild-type oka strain after 4 days of differentiation and treated with serial dilutions of the test compounds. at 12 days post-differentiation one series of the cultures was processed for histology and the other one for viral dna quantification. acyclovir (acv), penciclovir (pcv) and brivudin (bvdu) at 4 and 0.4 g/ml, foscarnet (pfa) at 40 g/ml and cidofovir (cdv) at 4, 0.4 and 0.04 g/ml inhibited viral dna content by more than 95%. these results were in agreement with histological examination of the rafts, no cytopathic effect being observed at these concentrations. as expected, only cdv and pfa inhibited the replication of the thymidine-kinase deficient (tk-) 07-1 strain. a correlation between the degree of protection as determined by histological examination and viral quantification could also be demonstrated for cdv and pfa against the tk-07-1 strain. since no animal model is available for the in vivo evaluation of antiviral agents against vzv, the organotypic cultures may be considered as a valuable ex vivo model to evaluate the efficacy of new anti-vzv antivirals. jae-seon hwang 1 , oliver kregler 1 , john c. drach 2,3 , leroy b. townsend 3 , elke bogner 1 1 institut für klinische und molekulare virologie, erlangen, germany; 2 department of biologic and materials sciences, school of dentistry; 3 interdepartmental graduate program in medicinal chemistry, college of pharmacy, university of michigan, ann arbor, mi, usa dna packaging is the key step in viral maturation and involves binding and cleavage of viral dna containing specific dnapackaging motifs. this process is mediated by a group of specific enzymes called terminases. we have previously demonstrated that the hcmv terminase is composed of two subunits, the large one encoding pul56 and the small pul89, where each protein has a different function. while the large subunit mediates sequence specific dna binding and atp hydrolysis, pul89 is only required for duplex nicking. inhibitors targeting pul56 and/or pul89 are attractive alternatives as hcmv antivirals since mammalian cell dna replication does not involve cleavage of concatameric dna. we now have screened several members of the benzimidazole ribonucleoside class of replication inhibitors in order to determine if a compound has the capacity to block the atpase activity of the large terminase subunit pul56. analysis by bioluminometric atpase activity assays identified bdcrb and one more compound [2-bromo-4,5,6-trichloro-1-(2,3,5-tri-o-acetyl-␤-dribofuranosyl)benzimidazole (btcrb)] with inhibitory effects. although only btcrb and bdcrb were inhibitors of the atpase activity, two other compounds, dbdcrb and cl4rb, inhibited virus replication in a plaque-reduction assay, thus indicating that those have a different mode of action. in addition, by electron microscopy of thin sections we observed that in the presence of btcrb only b-capsids and dense bodies were formed. furthermore, spherical capsids accumulated in the perinuclear cisternae indicating a block in nuclear egress thereby providing additional evidence that closely-related benzimidazole d-ribonucleosides may have differences in their antiviral modes of action. human cytomegalovirus (hcmv) is the cause of significant morbidity and mortality in a variety of immunocompromised patients. currently available anti-hcmv drugs interfere with dna replication; however, these drugs are highly toxic, pre-cluding their long-term use in humans. interrupting hcmv viral entry is largely unexplored as an antiviral drug development strategy and is potentially an ideal and tractable goal. hcmv is believed to rely upon formation of ␣-helical coiled coils in the viral glycoproteins gb and gh to promote virus-host membrane fusion; peptides encompassing heptad repeat sequences in these two proteins inhibit viral infection. we have explored nonnatural oligomeric molecules ("foldamers") that are designed to mimic elements of the putative ␣-helical segment of gb. this effort has led to the discovery of oligomers of ␤-amino acids ("␤-peptides") that block hcmv infection. the ␤-peptide scaffold offers several advantages for the design of protein-protein interaction inhibitors, as ␤-peptides are amenable to modular synthesis, resist proteolytic degradation, and can display large and tailored molecular surfaces. the most potent ␤-peptide inhibitor blocks hcmv infection with a micromolar ic 50 in a cell-based assay. these compounds show specificity for hcmv relative to closely related viruses. mechanistic studies suggest that these inhibitors interfere with membrane fusion between hcmv particles and host cells. current efforts are focused on understanding in greater detail the origin of the observed biological activity, exploring other foldamer scaffolds as bases for inhibitor design, and developing specific fusion inhibitors for other herpesviruses. previous reports have indicated that herpes simplex virus (hsv) activates nuclear factor-kappab (nf-kb) during productive infections. nonsteroidal anti-inflammatory drugs (nsaids) have significant inhibitory effects on nf-kb. therefore, two nsaids, indomethacin and aspirin, were assayed for antiherpetic effects and utilized as tools to further study the role of nf-kb in hsv-1 infection. we report that indomethacin and aspirin inhibited hsv-1 replication at non-cytotoxic doses. in vero cells, 500 um indomethacin and 20 mm aspirin reduced hsv-1 titers 99.999 and 99.5%, respectively. electromobility shift assays revealed that hsv-1 activation of nf-kb is inhibited by the nsaids at doses that coincide with reduction of hsv-1 titers. to investigate a pathway for nf-kb inactivation, protein levels of ikb-alpha, a cytoplasmic nf-kb inhibitor, were examined. ikb-alpha protein was present in uninfected samples, but decreased over time in all hsv samples, regardless of chemical treatment, suggesting localization of nf-kb to the nucleus. immunohistochemistry studies verified that p65, a component of the dimeric nf-kb complex, translocated to the nucleus of hsv-1 infected cells in the presence or absence of the nsaids. finally, direct effects on viral gene activity were assayed by real-time rt-pcr analysis. indomethacin and aspirin reduced mrna for icp4, an essential hsv immediate-early gene, 2.9and 2.5-fold, respectively, resulting in significant decreases of icp4 protein. but transcriptional analysis revealed that synthesis of mrna for thymidine kinase, an hsv early gene, was unaffected by chemical treatment. however, mrna for glycoprotein c, an hsv late gene was undetectable in indomethacin and aspirin treated samples. cumulatively, these data indicate that: (i) indomethacin and aspirin block hsv-1 replication and (ii) the in vitro anti-herpetic effects of nsaids may reside in their ability to block nf-kb activity within the nucleus, impairing activation of essentials hsv genes. increasing species-specificity constraints preclude study of human cytomegalovirus (hcmv) in animals, necessitating the use of rodent cmvs to model human disease. however, the susceptibility of animal cmvs to clinically useful antivirals is unpredictable. for example, the guinea pig cmv (gpcmv), a uniquely valuable virus for modeling congenital cmv infection, is highly resistant to ganciclovir (gcv) at medically relevant doses. we used a molecular virological approach to test the hypothesis that gcv susceptibility could be conferred on gpcmv by insertion of the human ul97 phosphotransferase gene into the gpcmv genome. the gpcmv genome, cloned as a bacterial artificial chromosome in e. coli, was modified by site-specific recombination, using a shuttle plasmid targeting the gp97 locus, and carrying the ul97 gene from hcmv strain towne. the resultant chimeric virus was replication competent, and was found to contain the hcmv ul97 by southern-blot and sequence analyses. northern-blot revealed that a hcmv ul97-specific transcript was expressed with late gene kinetics. western-blot, using a hcmv ul97-specific polyclonal antibody, detected protein in virus-infected cells. the chimeric virus was gcv-susceptible, compared to wild-type gpcmv, with an ic 50 of 15 m. chimeric virus also exhibited increased sensitivity to maribavir (mbv), exhibiting a 3-log reduction (compared to wild-type virus) in the presence of 50 m mbv, and an ic 50 of 5 m. to study the in vivo pathogenesis of chimeric virus, cyclophosphamide-immunocompromised strain two guinea pigs were challenged intraperitoneally, resulting in evidence of disseminated infection, and mortality. ganciclovir treatment (25 mg/kg/day) resulted in reduced weight loss, and mortality, compared to placebo. these studies confirm the key role of ul97 in cmv antiviral therapy, and demonstrate that a 'humanized' gpcmv can be generated with altered antiviral susceptibilities. genital herpes infections are a global health problem and impact hiv/aids epidemic. strategies to prevent transmission include treatment of infected subjects to suppress shedding and prophylaxis with vaginally-applied microbicides. we examined the in vitro and vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against hsv-2 infection of human cervical cells and in a vaginal murine model. rep 9 has broad-spectrum anti-herpetic activity with potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). at a concentration of 100 m, rep 9 inhibited 6-logs of hsv-2 infection if present during the entire experiment. synchronized infectivity assays demonstrate that, unlike sulfonated polyanions in clinical trials, which primarily block hsv attachment, rep 9 acts at multiple steps and inhibits binding, entry and post-entry gene expression. in our in vivo studies, mice were treated once intravaginally with rep 9 or pbs control at various times prior to vaginal challenge with a lethal dose of hsv-2 strain 186 (10 log 4 pfu). rep 9 prophylaxis provided protection to mice from hsv-2 infection and disease. protection was significant when challenged 30 min after treatment (p < 0.01). additionally, treatment with an analog of rep 9, which cannot activate tlr-9 mediated immune stimulation, was at least as active as rep 9, suggesting that direct antiviral activity and not stimulation of innate immunity is the mechanism of action in vivo. utilizing this analog, protection was significant when challenged 30 min after treatment (p < 0.001) with a trend toward protection when administered 60 min prior to challenge (p = 0.07). in summary, treatment with the rep 9 analog which has superior resistance to low ph and nuclease degradation was more effective than rep 9, in some experiments protecting 100% of mice from viral infection and disease. the testing of this ph resistant rep 9 analog in a gel formulation is currently underway. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. a phosphorodiamidate morpholino oligomer (pmo) designed to hybridize to a highly conserved region including the aug translation start site of hcv, called avi-4065, has been evaluated for efficacy, toxicity, and pharmacokinetic properties. avi-4065 inhibits translation initiated at the aug start site with ec50 of 308 nm (2.1 ug/ml) and shows positive cooperativity. this pmo retains most of the activity in the presence of point mutations in the hcv genome. huh-7 cells were incubated with normal human serum (nhs) or hcv infected human serum (is) and hcv replication observed by rt-pcr. avi-4065 produced robust inhibition of hcv in a dose and sequence-specific manner. studies conducted in vivo with avi-4065 in the hcv infected trimera mouse (xtl) show reduction in viral titer which is dose dependent with approximately 90% of mice with undetectable viral titer and the remaining mice show 1 log reduction in viral titer with 0.1 mg/mouse/day for 7 consecutive days. the fractional bioavailability of avi-4065 from a sq dose is approximately 1. the apparent elimination half life in rat, nonhuman primate and humans was 2.3, 4.5 and 11.4 h, respectively. the volume of distribution ranged from 0.6 to 1.0 l/kg and the cmax is linearly related to the dose in mg/m 2 . a phase i study in healthy volunteers in which 14 daily sq doses of 50 and 100 mg has been completed. no serious adverse events have been observed. treatment of infected patients is currently planned. inhibition of hcv polyprotein synthesis is anticipated to contain therapeutic benefis of both protease inhibitors and polymerase inhibition. hcv infection can progress to fibrosis, reduced liver function, hepatocellular carcinoma, and death. currently, the standard treatment for hcv infection involves treatment with pegylated interferon in combination with the nucleoside analogue ribavirin. this treatment regimen effects a cure in approximately 40-60% of the genotype-1 (gt-1) population; therefore a significant unmet clinical need exists in hcv therapy. virus-encoded polymerases have proven to be excellent molecular targets for chemotherapeutic intervention in numerous viral mediated diseases. in the case of hiv, hbv and herpes virus infections, deoxy-nucleoside analogues, which act as chain terminating agents, have been shown to have invaluable clinical utility. by analogy, appropriate ribonucleoside analogues might be expected to inhibit the essential rna polymerase (ns5b) encoded by hcv. here we describe the preparation of nucleoside analogues as inhibitors of the hcv polymerase. in our design of nucleoside analogs as potential anti-hcv agents, we chose to investigate the effect of 4 -substituted ribonucleoside derivatives. we reasoned that after incorporation of a ribonucleoside containing a 4 -substituent, a disruption in elongation of the growing rna could be effected through either steric hindrance or via a conformational change of the carbohydrate moiety. our investigations on several such analogues will be presented. of particular interest is 4 -azido-cytidine, which shows good activity in the genotype 1b sub-genomic replicon (ic50 = 1.28 m) with no measurable cytotoxic or cytostatic behavior. in addition, we have shown that the triphosphate of 4 -azido-cytidine is a potent and highly selective inhibitor of ns5b (ic50 = 0.32 m). joanna e. boerner, sue ma, choilai tiongyip, michael p. cooreman, teresa compton, kai lin novartis institutes for biomedical research, 100 technology square, cambridge, ma 02139, usa current drug discovery efforts for hepatitis c virus (hcv) focus on developing specific inhibitors of two viral enzymes, ns5b polymerase and ns3-4a protease. however, resistant viral mutants are likely to emerge during therapy, compromising the effectiveness of these inhibitors. an alternative and complementary strategy is to target host factors that are also essential for viral replication. cyclophilins, a family of peptidyl-prolyl isomerases and the cellular targets of cyclosporin a (csa), present such an opportunity. it was reported recently that cyclophilin b bound to hcv ns5b polymerase and stimulated its rnabinding activity, and that these functions were blocked in the presence of csa (watashi k. et al., molecular cell 2005) . nim811, a csa derivative, is a more suitable candidate for hcv therapy because it binds to cyclophilins with higher affinity than csa while lacking the immunosuppressive activity associated with csa. using the hcv replicon system we demonstrated that nim811 exhibited potent anti-hcv activities in vitro. moreover, the combination of nim811 with a specific non-nucleoside inhibitor of hcv polymerase led to synergistic antiviral effects with no significant increase of cytotoxicity. resistant clones against both inhibitors were obtained in vitro, however, it was much more difficult to generate resistance against nim811 than the polymerase inhibitor. also, there was no cross-resistance between the two inhibitors. finally, addition of nim811 to the hcv polymerase inhibitor drastically reduced the emergence of resistance compared to polymerase inhibitor alone. taken together, nim811, with a novel mechanism of action and a favorable pharmacokinetics and safety profile, represents a promising clinical candidate for treating hepatitis c and provides a rationale for specific combination therapy. the nucleoside analog r1479 was identified as a specific inhibitor of hcv replication in subgenomic hcv replicon cells. r1479-tp is a competitive inhibitor of cmp incorporation by hcv polymerase ns5b. in a transient replicon system r1479 inhibited hcv rna replication driven by genotype 1b polymerase with similar potency as compared to that driven by genotype 1a polymerase. r1479-tp inhibited native hcv replicase and recombinant ns5b from genotype 1a and 1b with similar potency. in contrast, r1479-tp did not inhibit human dna polymerases alpha, beta or gamma, including reverse transcriptase activities of dna polymerases beta and gamma, which were highly sensitive to inhibition by azt-tp and 3tc-tp. no significant inhibition was observed with human rna polymerases i, ii and iii derived from hela cells. in addition, the functionally related native influenza virus rna dependent rna polymerase (rdrp) activity in vitro was not inhibited by r1479-tp at concentrations up to 1 mm, suggesting high selectivity for the hcv rdrp. thus, r1479 was identified as a potent and highly selective inhibitor of hcv polymerase mediated rna synthesis. guangxiang luo, zhaohui cai, chen zhang, kyung-soo chang, jieyun jiang microbiology, immunology, and molecular genetics, university of kentucky college of medicine, lexington, ky, usa the study of hepatitis c virus (hcv) replication and the search for specific antiviral agents against hcv infection have been hampered by the lack of an efficient stable cell culture system of hcv infection and propagation. we have successfully constructed stable human hepatoma cell lines that contain a chromosomally integrated-genotype 2a hcv cdna and constitutively produce and secrete high titers of infectious virus into the culture media. transcriptional expression of the full-length hcv rna genome is under the control of a cellular pol ii polymerase promoter at the 5 end and a hepatitis delta virus ribozyme at the 3 end. the resulting hcv rna was expressed and replicated efficiently, as shown by the presence of high levels of hcv proteins as well as hcv rna in the stable huh7 cell lines. hcv secreted from the stable cell lines was infectious, as determined by antibody neutralization, blockage of putative hcv receptors, and inhibition of hcv replication by interferon. our findings demonstrate the establishment of a stable cell culture system of infectious hcv production and propagation, which allows the study of the entire hcv infectious cycle. the stable hcv-secreting cell lines are now being pursued to develop high throughput screens for effective hcv inhibitors. additionally, we established a novel and powerful hcv replication system in the mouse hepatocyte and mouse embryo fibroblasts (mef). hcv rna was found to replicate efficiently in both pkr +/+ and pkr −/− mef cells, demonstrating that hcv rna replication in mef cells is a powerful system to study host-virus interaction by using diverse gene-knockout animals. interestingly, hcv rna replicates more efficiently in the pkr −/− cell than in the pkr +/+ cell, suggesting a role of pkr in the control of hcv rna replication. however, ifn inhibited hcv rna replication in the pkr −/− cell with an efficacy similar to that in the pkr +/+ cell, suggesting a pkr-independent antiviral mechanism. clearly both pkr-dependent and pkrindependent antiviral mechanisms are important for the control of hcv replication and the mediation of the ifn-induced anti-hcv response. our studies set a stage for the development of transgenic mouse models of hcv replication and open up new avenues to study hcv and host interactions in mefs derived from diverse gene-knockout animals. andrea cuconati 1 , haitao guo 2 , gael westby 1 , anand mehta 2 , timothy block 1,2 1 institute for hepatitis and virus research; 2 drexel institute for biotechnology and virology research, doylestown, pa, usa the high levels of hepatitis b surface antigen (hbsag)-bearing non-infectious particles in the serum of infected individuals is thought to play a role in suppressing hepatitis b virus (hbv)specific immune response by titering out hbv-specific antibodies and lymphocytes. current hbv therapeutics do not directly reduce this viral antigenemia. our group has focused on the enhancement of the immune response through the inhibition of viral antigen secretion in the infected hepatocytes, with the therapeutic goal being the use of hbv vaccination for the treatment of acute and chronic infection. the high-throughput screening of a small molecule library of 80,288 drug-like compounds was undertaken to discover novel inhibitors of hbsag secretion. using the stably hbv-transfected, human hepatoma cell line hepg2.2.15, we developed an hts-compatible elisa protocol for the detection of hbsag secreted in the culture media. the screen resulted in 1758 initially positive hits, a hit rate of 2.2%. subsequent retesting for activity and toxicity by mtt assay has narrowed the number of confirmed, non-toxic hits to 77, currently categorized in twelve chemical series. we have previously reported on a trio of related pyrazolo-pyridines with ec50 measurements below 5.0 m and cc50 measurements >50.0 m. nascent structure-activity relationship (sar) suggests that a central moiety of the molecules is essential to activity, with an aromatic side group contributing to potency. among recently confirmed inhibitors, two currently under investigation include: (1) an isobutyl-acetamide with an ec50 of 87.0 nanomolar, and a cc50 of >50 m, and (2) a carbothiamide with an ec50 of 1.6 micromolar and a cc50 of >50 m. measurement of secreted hbv l and m antigens and cellular markers indicated that the pyrazolo-pyridines are not specific inhibitors of viral antigens, while the isobutyl-acetamide and the carbothiamide are indeed specific. measurement of intracellular viral dna indicated that none of these molecules are inhibitors of replication. we will be reporting on our studies of the potency, specificity, and potential mechanisms of action of these novel anti-hbv compounds. background: entecavir (etv) is a potent competitive inhibitor of hepatitis b virus (hbv) polymerase with activity versus all three enzymatic functions including priming, minus, and plus strand dna synthesis. virologic rebound due to etv resistance (etvr) has only been observed in lamivudine resistant (lvdr) hbv (m204v/i ± l180m), and requires at least one additional change in the reverse transcriptase domain (rt) at residues t184, s202, or m250. these substitutions surround the dntp binding site or primer grip of rt. the objectives of this work were to further characterize etvr and its mechanism(s) using cell culture, in vitro enzyme, and molecular modeling studies. methods: hbv cell culture assays used transfected hepg2 cells and quantitation of released, immunocaptured hbv nucleocapsids. gradient-purified intracellular nucleocapsids were used for in vitro rt assays. a 3d homology model based on the hiv-1 rt structure was used to model resistance changes in hbv. results: reduced etv susceptibility of etvr hbv was observed both in culture and enzymatically in vitro. kinetic studies showed various etvr substitutions in lvdr hbv selectively reduced etv-triphosphate (etv-tp) binding (k i ) to rt without markedly changing the affinity for dgtp (k m ) or inhibition by ddgtp. etvr rts also displayed reduced enzymatic activity (k cat ) relative to wildtype and etvr hbv appeared growth impaired. modeling studies suggested a novel etv-tp binding pocket in hbv rt that became constrained with etvr changes. m250 changes in the primer grip region of rt were unique in that resistance was primarily seen during synthesis of minus strand dna. etvr changes in the absence of lvdr substitutions had greatly reduced impacts on etv susceptibility, confirming models suggesting etvr is imparted through lvdr changes. summary: etv provides a high genetic barrier to resistance, requiring additional changes at residues t184, s202 or m250 along with pre-existing lvdr substitutions m204v/i ± l180m. kinetic parameters and molecular modeling indicated that etvr substitutions selectively affected etv-tp binding and reduced the replication capacity of hbv. a nonhuman primate (nhp) model of classical, lesional smallpox has been used to test the efficacy of intravenous (iv) cidofovir treatment. cynomolgus macaques were infected with a high dose (10 8 pfu iv) of variola to produce an artificial primary viremia, and then treated with cidofovir at 0, 24, or 48 h postinfection (pi). later treatment times were not evaluated. treatment at 24 or 48 h pi halted increases in peak blood viral genome titers measured by quantitative taqman-mgb real-time pcr, which were more than 10-fold less in cdv-treated animals compared to placebo. historically, the number of pox lesions provided the best correlation with human smallpox clinical severity, and cdv treatment in our model significantly reduced maximum pox lesion counts by >90%; the number and size of skin lesions, and in untreated animals contributed significantly to the total viral burden with lesions containing 10 9 -10 10 genomes/g. to better understand the role of viral burden and disease progression in major organ systems, a serial sample study was undertaken. in untreated animals at 24 h pi, viral replication in spleen exceeded 10 9 genomes/g while liver and bone marrow yielded 10 8 genomes/ml. in comparison, titers in other tissues ranged between 10 5 and 10 6 genomes/g and blood yielded 10 4 genomes/ml at 24 h, suggesting that the liver, spleen, and marrow may be initial sites of replication. levels of virus in the bone marrow reached a peak of approximately 10 10 genomes/g at day 5, then decreased to quantities consistent with those in blood. viral load in the blood increased with time, peaking around days 7-9 at 10 8 genomes/ml. virus was also detected in intestine, skeletal muscle, and late in infection, testes. the ability to successfully treat with cdv 24 h pi despite early extensive organ infection in the accelerated nhp variola model suggests that this treatment could be effective in reducing viremia and mortality after onset of symptoms in human smallpox, which demonstrates a more protracted disease course. work involving variola virus conducted in who-sanctioned cdc, atlanta bsl-4 laboratory. earl kern 1 , kathy keith 2 , robert jordan 2 , dennis hruby 2 , debra quenelle 2 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 siga technologies, inc., corvallis, or, usa although cidofovir (cdv) has been approved as an investigational new drug for emergency treatment of smallpox, its lack of oral activity and dose limiting toxicity dictates a need for continued development of better therapeutic agents for this potential bioterror disease. it has been reported previously that st-246, a low-molecular weight compound, inhibits replication of all the orthopoxviruses in vitro and protects mice infected with vaccinia or ectomelia virus. in the present study, we have utilized cowpox virus (cv) and vaccinia virus (vv) infections in vitro and in vivo to evaluate the efficacy of st-246 for treatment of orthopoxvirus infections. in plaque reduction assays in human foreskin fibroblast cells, both cv and vv were inhibited by about 0.1-0.5 um of st-246. for in vivo studies, st-246 was administered once daily by oral gavage to mice using 100 mg/kg for 5, 7, 10, or 12 days beginning 4 or 24 h after intranasal inoculation with vv or cv. st-246 was highly effective (p < 0.001) in preventing mortality due to vv or cv even when treatment was delayed up to 24 h post-infection. a dosing duration of 5 days was adequate for vv infected mice, but duration of 7 days or longer was required for efficacy in cv infected mice. when st-246 was given once daily for 14 days at 100, 30, or 10 mg/kg daily at 24, 48, or 72 h post-cv inoculation, mortality was significantly altered at all dosage levels and time points. to determine the effect of treatment on virus replication in target tissues, mice were inoculated with cv or vv and treated once daily with 50 mg/kg of st-246. on various days post-infection tissues were harvested and assayed for virus. in cv or vv-infected mice, st-246 treatment successfully reduced virus titers from 3 to 5 logs 10 in liver, spleen, and kidney. little effect was noted in lung tissue. these results indicate that st-246 has significant activity against vv and cv infections in vitro and in vivo and may be a potential chemotherapeutic agent for treatment of human orthopoxvirus infections. cidofovir (hpmpc) is a broad-spectrum anti-viral agent that is used (vistide ® ) to treat aids-related cmv retinitis. currently, cidofovir is of particular interest as a potential therapy for orthopox virus infections, including smallpox. an important limitation of cidofovir and analogous nucleotide drugs in a therapeutic role is their low oral bioavailability and poor transport into cells. in principle, bioavailability of a drug can be improved by structural modification targeting transporters expressed in human intestine. to be effective, the transported prodrug must be cleaved by endogenous enzymes to its parent compound. we will present synthetic studies of novel cidofovir and cyclic cidofovir (chpmpc) prodrugs incorporating amino acids or small peptides, comparing different drug-amino acid linkage strategies. the compounds were evaluated for transporter-mediated uptake and cellular and plasma hydrolysis. the results will be compared with similar studies carried out on a series of peptidomimetic conjugates of foscarnet, the trisodium salt of phosphonoformic acid (pfa), an anti-viral agent that also has very low oral bioavailability and poor cell penetration. the question addressed in this study is if wnv-reactive antibody can improve disease signs in a hamster model after the virus is demonstrated to be in the brain. the hypothesis is based on the high activity of a humanized monoclonal antibody, he16, in a mouse model when administered later in infection (oliphant et al., 2005. nat. med. 11, 522) . in this study, virus was demonstrated to be in the brains of hamsters at 5 days post-viral injection (dpi) by cell culture assay, quantitative rt-pcr, and immunohistochemical staining of wnv in neurons. eighty percent of hamsters treated i.p. 5 dpi with 100 mg/kg of humanized monoclonal antibody, he16, survived wnv disease, whereas, 37% of placebo-treated hamsters survived ( *** p < 0.001). if administered at 2 dpi, 100% survived. we tested the hypothesis that he16 is effective if delivered directly into the brain instead of by peripheral administration. the antibody was delivered into the brain 5 dpi using convectionenhanced delivery through a cannula implanted into the brain. the he16 was detected in the cns, but none was detected in the kidney. the survival of he16-treated hamsters was 88% as compared to 22% of placebo-treated animals ( *** p < 0.001). for additional proof, the majority of hamsters having wnv in their cerebrospinal fluid, a marker for cns infection, were protected with he16 administered i.p. at 5 dpi. this humanized monoclonal antibody, therefore, is a possible treatment for the post-exposure, wnv-infected humans that develop signs of neuroinvasive disease. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih, and grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. hemorrhagic fever viruses are of serious worldwide health concern as well as potential biological weapons. lassa fever virus in particular annually infects several hundred thousand individuals in west africa, and the export of this pathogen outside of this region, either intentionally or unintentionally, presents a serious risk to the developed countries of the world. the cdc and niaid have identified lassa fever virus as a category a priority pathogen, indicating the highest degree of threat to public health. no arenavirus-specific antiviral drugs are currently approved for use in humans. the purpose of siga's biodefense program is to develop safe and effective drugs for preventing and treating diseases caused by category a viruses. to that end, a large and diverse library of small molecule compounds was screened using a viral pseudotype assay to identify inhibitors that target the essential lassa surface glycoprotein (gp) and thus block viral entry into the host cell. twenty-six compounds were identified as quality hits, as defined by potency, selectivity, and chemical tractability. antiviral activity against authentic lassa fever virus was assessed in cell culture through a collaboration with colleagues at usamriid. a number of these potent antiviral compounds and their related analogs have exhibited informative chemical structure-biological activity relationships (sar). two potential lead compound series have emerged from these studies, each with 50% effective concentrations (ec50s) of less than 100 nm against lassa fever virus and with ec50s of less than 2 nm against lassa gp-pseudotyped virus. characterization of the in vivo properties of these compounds is underway. the in vitro antiviral potency and selectivity, animal pharmacokinetics, and the development process will be presented. these inhibitors represent an important step toward the development of a small molecule antiviral drug for lassa fever virus. sven enterlein 1 , pramila walpita 1 , allison groseth 2 , heinz feldmann 2 , ramon flick 1 1 university of texas medical branch, department of pathology, galveston, tx, usa; 2 national microbiology laboratory, public health agency of canada, winnipeg, man., canada nipah (niv) virus (family paramyxoviridae) is a recently emerged human and animal pathogen that can cause severe encephalitis with fatality rates of up to 70%. since no treatment or vaccination is available, and cross-species spread was observed, the virus has been classified as biosafety level 4 (bsl-4) agent. to avoid bsl-4 containment for the study of cis-acting signals as target for antiviral strategies, we used an optimized plasmid-driven t7 minigenome rescue system (without the need for recombinant vaccinia virus mva-t7) as well as an newly established rna polymerase i-based approach. minigenome rescue is based on transfection of the minigenome niv-cat and the plasmids encoding for the three nucleocapsid proteins n (nucleoprotein), l (polymerase), and p (phosphoprotein) and measured by enzymatic cat assays. we used the established plasmid-based minigenome rescue systems to screen for potential antiviral compounds. in a first step we tried to determine the optimal strategy for the delivery of small hairpin (sh) interfering rna molecules. for this we compared three shrna delivery systems against another bsl4 agent-reston ebolavirus (family filoviridae); (i) plasmid-mediated pol i and (ii) pol iii-driven shrnas, and (iii) exogenously (t7) produced shrna, for their ability to induce gene silencing. interestingly, beside the in vitro-generated or pol iii-driven shrnas, pol i transcripts showed very efficient inhibition of minigenome rescue. however, the most efficient delivery method was transfection of in vitro transcribed shrnas. we will present the results of this comparison and, based on the most efficient approach, also first results of shrnas targeted either to niv n, p, and l genes or to the leader/trailer noncoding regions to interfere with minigenome replication. conformative data with live virus experiments under bsl4 conditions will be included. filoviruses, which include ebola virus and marburg virus, are among the most notorious human pathogens because they cause sporadic outbreaks of severe hemorrhagic fever. unfortunately, very few therapeutic agents are available to treat infections with these viruses. antiviral screening methods that determine the effect of compounds on viral replication involve working with infectious virus, which is obviously not practical for these biosafety level 4 (bsl-4) agents. we developed an antiviral screening method based on a cell-based, infection-independent, ebola subgenomic replication system in which the expression of an easily measurable enzyme is dependent on the rna replication and transcription factors of ebola virus. using this system we screened a synthetic compound library for antiviral activity against ebola virus and have identified a number of inhibitors. we also used it to identify a peptide inhibitor directed against vp30. anti-ebola virus activity for many of the inhibitors was confirmed in a viral replication assay using a gfp-expressing zaire '76 strain of ebola virus. fifty-two small molecule inhibitors from at least six classes of compounds had ec50 values in the low micromolar range and good selectivity. several of these compounds have promising chemical, biological, and pharmacological profiles to pursue as potential anti-filovirus drugs. we are currently preparing to test these compounds in a mouse model of ebola virus. we have also begun a lead optimization program to improve antiviral potency and selectivity of aryl sulfonamide and 4-aminoquinoline compounds. acknowledgement: supported by nih r44 ai052917-02 and r01 ai066502-01. human papillomavirus (hpv) has been a difficult virus to target by traditional antiviral methods due to its small size, its small number of obvious therapeutic targets, and its resistance to propagation in vitro. nevertheless, antiviral compounds that reduce hpv dna load have the potential to prevent carcino-genic progression in infected patients. to that end, we developed an approach that dramatically reduces the hpv episomal dna load of keratinocytes in vitro by targeting viral dna sequences. pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. all fluorescent compounds rapidly localized to the nucleus of cultured keratinocytes following addition to the culture media. the compounds were then tested for their ability to alter keratinocyte hpv31 episomal dna content. two of the 19 compounds caused a dose-dependent reduction in hpv31 episomes as measured by taqman tm realtime pcr. while control and vehicle-treated cells maintained ∼1000 copies of hpv31 per cell, compounds 2-ta and 4-ta both reduced hpv31 dna levels to below 50 copies per cell after 48 h incubation with 10 m compound. an alternative taqman tm amplicon within the hpv31 e7 gene produced identical results. a multiplexed taqman tm real-time pcr reaction that followed the ratio of hpv31 dna to the human apoe gene also demonstrated dramatic loss of hpv31 dna copies, further confirming our initial observations. finally, cells were treated with polyamides for 48 h, polyamide-containing media was removed, and episome levels were followed for 8 days. at day 6, 4 days after removal of polyamide and 3 days after sub-culturing of the cells, viral episome levels remained approximately 60% lower than control samples. by day 8, 6 days after removal of polyamide, viral dna levels were beginning to recover but still remained significantly lower than control samples. together our results demonstrate that targeting the hpv origin of replication with dna-binding compounds dramatically reduces episomal dna levels. small interfering rnas (sirnas) are potent tools for gene down-regulation but are minimally stable in cells. to improve the efficacy of sirna, we replaced non-bridging oxygens in the phosphodiester linkages of natural rnas with bh3 groups. the resulting boranophosphates have unique properties, including enhanced nuclease resistance, altered hydrogen bonding of the phosphate, different interactions with metal ions, and increased thermal stability of rna:rna and rna:dna duplexes. anti-egfp sirnas containing boranophosphate modifications were prepared by in vitro transcription with t7 rna polymerase from ribonucleoside 5 -(alpha-p-borano)triphosphates, as well as normal and phosphorothioate sirnas. after confirming the presence of the borane modifications with maldi-ms, several properties of borano-modified sirnas were investigated: (1) the double stranded rna with borane modifications maintained the a-form conformation characteristics according to the circular dichroism (cd) spectra; (2) the borane groups in the sirnas increased the thermal stability, with an enhancement of t m by 0.5-0.8 • c per modification; and (3) sirnas with borano-modifications were shown to be at least 10-fold more resistant to rnase a digestion than normal ones. when these modified sirnas were used to down-regulate egfp expression in hela cell cultures, it was found that: (1) borano-modified sirnas were consistently more effective than sirnas containing the corresponding phosphorothioate modifications; (2) borano-sirnas were more effective than normal sirnas provided that the center of the antisense strand was not heavily modified; (3) borano-sirnas were more potent than normal or phosphorothioate sirnas at lower concentrations; and (4) finally, the silencing activity of boranophosphate singlestranded sirna (ss-rna) was comparable to that of unmodified ds-sirna. the borano ss-rna had excellent maximum silencing activity and was highly effective at low concentrations, and silencing activity was durable up to one week after transfection. results with anti-hpv sirnas will be discussed. boranophosphate modification is a potential new class of anti-viral therapeutic agents. this report describes the antiviral structure activity relationships that led to the discovery of phosphonomethoxy-2 -fluoro-2 ,3dideoxydidehydroadenosine (fd4ap, gs9148), a novel ntrti, with an excellent resistance profile toward hiv-1 variants containing major n(t)rti resistance mutations. methods: phosphonomethoxy analogs on purine and pyrimidine dideoxydidehydro (d4) and dideoxy (dd) ribose scaffolds were prepared. antiviral activity was measured against wildtype and n(t)rti-resistant recombinant viruses using cytopathic assay in mt-2 cells. mitochondrial toxicity was assessed in hepg2 cells by measuring mitochondrial dna content. results: the d4 scaffolds displayed superior antiviral activity compared to the dd scaffold and adenine was superior to other nucleobases. phosphonomethoxy-2 ,3dideoxydidehydroadenosine (d4ap) inhibited hiv-1 replication with a mean ec50 of 2.1 m and an 0.8-, 2.9-, and 2.9-fold change in potency against viruses containing m184v, k65r, and 6 thymidine analog mutations (tams), respectively. further exploration of d4ap was limited by its mitochondrial toxicity, which was then addressed in 2 ways: (i) preparation of l-d4ap or (ii) 2 fluorine substitution. l-d4ap exhibited an ec50 of 5.9 m but had substantially reduced potency (14-fold) toward m184v mutant viruses. fd4ap exhibited an ec50 of 12.3 m, with 0.8-, 1.2-, and 3.5-fold change in potency against viruses containing m184v, k65r, and 6 tams, respectively. no cytotoxic effects were measured up to 1 mm in mt-2 cells and no effects on mitochondrial dna were detected up to 300 m in hepg2 cells for both fd4ap and l-d4ap. conclusion: fd4ap is a novel phosphonate ntrti with antiretroviral activity toward wild-type and resistant mutant hiv-1 strains. compared to d4ap, the 2 -fluorine atom significantly improved the in vitro toxicity profile while retaining the favorable resistance profile. in subsequent studies, the monoamidate prodrug strategy was applied to fd4ap to achieve optimal in vivo pharmacokinetic properties. entry inhibitors, and ccr-5 antagonists in particular, have become one of the most actively pursued treatments for hiv within the pharmaceutical industry. recently, multiple groups have disclosed piperidine-based ccr-5 antagonists that -to the medicinal chemist's eye -might appear to share a common three-point pharmacophore comprised of a tertiary amine, a phenyl ring, and a carboxamide or sulfonamide group. in several of these cases, these pharmacophoric elements are tethered together by a flexible, aliphatic chain. we sought to improve the potency of and introduce structural novelty into this class of compounds by rigidifying this tether. herein, we describe stereoselective syntheses and sar of a series of ccr-5 antagonists wherein the tether has been replaced with four stereochemical isomers of a rigidified cyclopropyl scaffold. the regulation of hiv transcription is a complex, multistage process that requires the concerted action of viral and cellular proteins. we discovered the n-aminoimidazoles (naims) as a unique class of hiv inhibitors targeted at the viral transcription level. a prototype naim, nr-818, prevents the reactivation of dormant virus by inhibiting both the hiv-1 p24 and viral mrna production from latently hiv-1-infected cell lines upon stimulation with tnf-␣, pma, or tsa. extensive research revealed that nr-818 was unable to inhibit the nf-b activation pathway or chromatin remodeling at the viral promoter, both known to be crucial for viral transcriptional activation. focusing on the viral transcription process, chromatin immunoprecipitation (chip) experiments revealed that nr-818 was able to inhibit the ser5 phosphorylation of the c-terminal domain (ctd) of rna polymerase ii. this step is mediated by the cdk9 subunit of p-tefb, which is recruited to the viral promoter by the hiv-1 tat protein. since we did not find an inhibition at the level of cdk9 activity or tat-mediated transcription in tat-expressing cell lines transiently transfected with a ltr-gfp construct, we infer that nr-818 must interfere with the transcription process by a unique mode of action. evidence points towards a kinase, not belonging to the cdk family, to be the target of the naims, resulting in an antiviral action at the level of retroviral transcription. clara e. cases-gonzález 1 , sandra franco 2 , miguel a. martínez 2 , luis menéndez-arias 1 1 centro de biología molecular "severo ochoa", csic-uam, madrid, spain; 2 fundació irsicaixa, hosp. university germans trias i pujol, badalona, spain a ser-ser insertion at codons 69-70 together with substitutions t69s and t215y in the reverse-transcriptase (rt)-coding region of hiv-1 are known to confer resistance to zidovudine (azt) and stavudine (d4t). phenotypic resistance correlates with increased atp-dependent phosphorolytic activity on inhibitor-terminated primers. we have previously shown that an rt derived from a clinical isolate (ss rt) that contained the insertion and 10 additional mutations related to drug resistance (including t215y) showed >10-fold increased unblocking activity on azt-and d4t-terminated primers, when compared with an rt containing the insertion together with mutations t69s and t215y, in an otherwise wild-type bh10 sequence. these results suggested that other mutations associated with the complex t69sss/t215y in clinically relevant rts contributed to increase atp-mediated excision activity and conferred high-level resistance to azt and d4t in phenotypic assays. to identify residues increasing the excision activity, we obtained recombinant enzymes bearing ss rt residues 1-135 and wild-type bh10 rt residues 136-560 (l1 rt), or residues 1-135 of the bh10 rt and 136-560 of the ss rt (l3 rt), as well as an l1 rt variant with the substitution t215y (l2 rt) and an l3 rt derivative with t69sss (l4 rt). additional rts containing mutations m41l, a62v, or k70r together with the combination t69sss/t215y in the bh10 background were also obtained. atp-mediated excision activities on azt-and d4tterminated primers were determined and the effects of mutations were tested in phenotypic assays using recombinant hiv-1. the l2 rt containing mutations t69sss/t215y and additional changes in the n-terminal region showed the highest atp-dependent phosphorolytic activity on blocked primers, giving values similar to those reported for the ss rt. results were consistent with phenotypic data. in contrast, l1, l3, and l4 rts displayed low-level activity. further experiments revealed that three amino acid changes at the n-terminal region of the polymerase (m41l, a62v and k70r) were responsible for the increased excision activity shown by rts bearing mutations t69sss and t215y. from a series of phenyl-substituted thiazolobenzimidazoles, several compounds were identified as selective inhibitors of coxsackie b virus replication in vero cells. a structure-activity relationship was established, from which the 6-trifluoromethyl substituted analogs emerged as the most potent congeners. the compounds were active against all six coxsackie b strains tested. the in vitro antiviral activity of one of the most selective compounds, i.e. chi-033, was assessed by (i) mts-based cytopathic effect assays, (ii) virus yield reduction assays, (iii) real-time quantitative pcr (rt-qpcr) and (iv) by monitoring viral antigen expression. in all assays a clear concentration-response effect was obtained. the 50% effective concentration (ec50) was 0.30 ± 0.18 g/ml, while the cc50 (50% cytotoxic concentration) of chi-033 for vero cells was more than 100 g/ml, thus resulting in a selectivity index of >500. detailed single cycle time-of-drug-addition studies (in which viral replication was monitored by means of rt-qpcr) revealed that the compound interacts with viral replication at a time that coincides with the onset of intracellular viral rna synthesis. chi-033resistant virus is being generated by culturing the virus in the presence of increasing drug concentrations. drug-resistant virus will be genotyped, which should allow us to identify the (putatively viral) molecular target of this class of compounds. retroviruses 42 hiromichi tanaka 1 , kazuhiro haraguchi 1 , hiroki kumamoto 1 , takao nitanda 2 , masanori baba 2 , ginger e. dutschman 3 , yung-chi cheng 3 1 school of pharmaceutical sciences, showa university, tokyo, japan; 2 center for chronic viral diseases, kagoshima university, kagoshima, japan; 3 school of medicine, yale university, new haven, ct, usa our recent research program on the development of synthetic methods for 4 -carbon-substituted nucleosides has led to a new strategy, ring opening of 4 ,5 -epoxy-nucleosides with organoaluminum and organosilicon reagents. this enabled us to introduce alkyl, alkenyl, and alkynyl groups to the 4 -position. as a result of this study, 4 -ethynylstavudine (4 -ed4t) was found to be more anti-hiv active than the parent compound stavudine (d4t). this compound (4 -ed4t) has several additional appeals as a promising anti-hiv agent: much less toxic to various cells and also to mitochondrial dna synthesis, better substrate for human thymidine kinase than d4t, very much resistant to catabolism by thymidine phosphorylase, its activity enhances in the presence of a major mutation k103n known for nnrti-resistant hiv. in this conference, we present the synthesis and sar studies of 4 -ed4t analogues modified mainly in the sugar portion. negatively charged polymers (np) possess a broad immunoadjuvant and antiviral activity topically useful for vaccine, drug, and microbicide development. but their efficiency is limited over a reversibility of electrostatic kind of interference with virusspecific nano-objects. to overcome this limitation the purposemade intra-molecular modifications of np were studied among non-toxic maleic acid co-polymers (npsa), dextran and chitin derivatives (npps) within varied alicyclic modifiers application. the configurationally flexible alkyls (i), as non-alicyclic control, are ineffective synergist for np antiviral potency. monocycles (ii) are moderate active too. on the contrary the hardconformation frame-structured spheroids (iii-vi) exhibit ability (at optimal macromolecular parameters) to be super-effective synergists for strength and diapason of np antiviral action. unlike small molecular iii/iv-containing prototypes (amantadin, rimantadin, deitiforin, etc.), narrowly-effective inhibitors mainly of influenza a viruses, the np-coupled modifications become effective also against many other viruses, including the drugs resistant strains [antivir. res. 46 (1), 44]. in focus of the anti-hiv potency the ivs provide a 10-100-fold elevation of np activity. the more available and less toxic iii species are similarly active, but iii* (with spatial-optimally contactable double bond due to the exo-configuration) turns out the best synergist 20-500-fold amplifying the anti-hiv-1 selectivity up to is∼10000. augmentation of the frame cycles from iii-iv toward v-vi results in no essential enhancement of antiviral activity, but stimulates toxicity. the recently involved in the investigation vii, cholesterol-like systems, as tools for novel raft-targeted strategy, demonstrate capacity for at least 10-fold amplification of anti-hiv-1 potency our earlier studies showed that esterification of cidofovir (hpmpc) with alkoxyalkanols increased antiviral activity by more that two logs and promoted oral bioavailability. to evaluate this approach with purine based nucleoside phosphonates, we synthesized several alkoxyalkyl esters of acyclic purine phosphonates such as 2,6,-diamino-(9-[2-phosphonomethoxyethyl]purine (pme-dap) and 2-amino-6-cyclopropylamino-(9-[2phosphonomethoxyethyl]-purine (pme-cpr-dap) these purine phosphonates have been reported to be active against a wide range of viruses such as human immunodeficiency virus (hiv-1), other retroviruses, herpesviruses, poxviruses and hepatitis b virus. for this study several alkoxyalkyl analogs of acyclic 2,6diaminopurine nucleoside phosphonates were synthesized and evaluated against hiv-1. the alkoxyalkyl esters were more inhibitory than the unmodified compounds in p24 reduction assays in mt-2 cells infected with hiv-1. for example, hexadecyloxypropyl (hdp) and oleyloxyethyl (ole) esters of pme-cpr-dap were >3 logs more active than unmodified pme-cpr-dap. in spite of increased cytotoxicity in mt-2 cells, the selectivity indexes are more than 10-fold higher then for unmodified compound. in conclusion, esterification of pme-dap and pme-cpr-dap with hexadecyloxypropyl-or oleyloxyethyl-residues greatly increased their antiviral activity and selectivity against hiv-1 in vitro. victor kuz'min 1 , eugene muratov 1 , anatoly artemenko 1 , ludmila koroleva 2 , vladimir silnikov 2 , v. lozitsky 3 , a. fedchuk 3 1 a.v. bogatsky physical-chemical institute, odessa, ukraine; 2 institute of chemical biology and fundamental medicine, novosibirsk, russian federation; 3 ukrainian mechnikov research anti-plague institute, odessa, ukraine "chemical" ribonucleases hold promise as tools for studying the structures of rnas and rna-protein complexes, as reactive groups in conjugates intended for cleavage of particular rnas, as therapeuticals inactivating virus genome rnas or certain mrnas, and as a promising antiviral agents. drug design and development of new medicines directed against hiv are permanently actual tasks. the usage of modern quantitative structure-activity relationship (qsar) methods could allow us to solve these problems more effectively. the objective of the present work is qsar analysis of antiviral activity of various tetrapeptides-artifical ribonucleases and consequent molecular design of new antiviral agents. qsar approach based on simplex representation of molecular structure (sirms) has been used for the solution of the formulated problem. usage of sirms allows us to develop the molecular design of the new effective antiviral agents. thorough researches of relationship between antiviral activity (hiv-1, % of rna p-o bond cleavage) and a structure of artifical ribonucleases have been carried out. statistic characteristics for pls (partial least squares model) are quite satisfactory (r 2 = 0.836, q 2 = 0.788). on the base of these models the molecular fragments with positive or negative influence on the explored property have been determined. thus, for example, guanidine and triethylenediamine fragments promote antiviral action. it gives a possibility to realize based on elucidated rules molecular design of compounds with the high level of antiviral activity. the results of prognosis are verifying by the experimental investigations. thus, quite adequate simplex qsar model "anti-hiv activity-artifical ribonucleases structure" was obtained and used for drug design. the cyclotriazadisulfonamide (cada) compound specifically down-modulates the cd4 receptor expression on the surface of lymphocytes and monocytes/macrophages, the primary receptors utilized by hiv for infection of its target cells. cada thus inhibits the entry of hiv and hhv-7 (vermeire et al., 2002. virology 302, 342-353) . cada chemotherapy may not be susceptible to the production of drug resistant strains of viruses, as its mechanism of action is completely different from those of any other anti-hiv drugs currently in clinical use. the cd4 down-modulating and antiviral potencies of more than 25 cada analogs have been described (vermeire et al., 2003. mol. pharmacol. 63, 203-210) . structural modifications of cada were made to increase potency, reduce cytotoxicity, and improve physical properties. several head group analogs were synthesized with polar groups and good leaving groups ( fig. 1) . the anti-hiv and cd4 down modulation activities of these compounds are being studied. some of these head groups may regenerate the double bond of cada by elimination reactions, potentially producing water-soluble pro-drugs. isocada (sa05), an isomer of cada, was synthesized by cyclization of 1,5,7-triazabicyclo-[4.4.0]dec-5-ene (tbd) (fig. 1 ). this structural modification may reveal a relationship between the symmetry of the molecule and its biological activity. two new fluorine-containing analogs were also synthesized by modifying the toluenesulfonamide side arms (fig. 1) . the anti-hiv and cd4 down modulation activities of these new cada analogs are summarized. the center for drug discovery, university of georgia, athens, ga 30602, usa drug discovery targeted at the elusive viral enzyme, hiv integrase, has not resulted in a single fda-approved drug. in this presentation we describe our molecular modeling studies with conceptually novel inhibitors of hiv integrase that also possess potent in vitro anti-hiv activity. docking was performed on the catalytic core of integrase represented by chain c of pdb structure code 1bl3. building of molecules and primary modeling was done with sybyl 7.1 on a silicon graphics onyx3 (r14000) workstation. the program gold 3.0 (genetic optimization for ligand docking) was used extensively in evaluating the docking poses of these compounds with the active site of hiv integrase and to give information on key residues involved in the recognition and binding of these ligands. the gold function consists of three basic components: protein-ligand h-bonding energy, protein-ligand van der waals energy, and ligand internal energy. post-processing gold output was done with the program silver 1.1, a utility program supplied with gold for evaluating hydrogen-bonding interactions, metal coordination and van der waals factors. for comparison purposes, additional docking was performed using other docking protocols, notably the sybyl module flexx. data obtained from these and related studies including binding poses, binding affinities, functional and conformational considerations, and gold function scores will be presented and explained. the center for drug discovery, university of georgia, athens, ga 30602, usa hiv integrase is essential for hiv replication and is an attractive target for drug discovery against aids. however, research efforts on drug discovery pertaining to hiv integrase have not resulted in a single fda-approved drug for which mechanism of action is inhibition of hiv integrase. recently, we have been exploring a novel class of diketo acids that are constructed on nucleobase scaffolds and that have a specific arrangement of the functional and hydrophobic group on the scaffold. these compounds are inhibitors both key steps of hiv integrase. one lead compound from this group has also been found to have remarkable in vitro anti-hiv activity. however, the syntheses of the inhibitors are quite challenging. this presentation will describe the synthetic methodologies specifically developed in our laboratory for the preparation of some representative examples of these integrase inhibitors. purification approaches to produce highly purified compounds for biological studies will be explained. structural, functional and conformational data obtained from extensive spectroscopic studies will be discussed. representative anti-hiv integrase data and in vitro anti-hiv screening results will be presented. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, with little or low toxicity to the host cells. in this part i of the presentation on this subject, we report our preliminary findings on the mechanism of anti-hiv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues. in view of the fact that a number of hiv patients also suffer from hcv as a major coinfection, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. marina burshtein 1 , alexander serbin 2 , alissa bukrinskaya 1 d.i. ivanovsky institute of virology, moscow, russia; 2 health research and development found, moscow, russia introduction: amantadine is a well-known effective antiinfluenza drug. it was modified to enhance its antiviral activity by chemically linkage with the water-soluble polyanionic matrix via different spacer groups. the other group of used compounds was norbornene derivatives, as norbornene is an adamantane analogue on anti-influenza activity. methods: the absence of cytotoxic effect was shown by mtt test for estimating cytotoxic dose (ctd50). the antiviral effect of the compounds was analyzed in lymphoblastoid mt-4 cells and in hela cd4+/b-galactosidase cells ("magi" cells). the effect of the compounds was registered by immunoblotting of cell lysates and by measuring of b-galactosidase activity. results: the strong inhibition of hiv-1 replication was observed when the compounds were added with the virus and was expressed even when the compounds added with the virus were removed 1 h after infection. the anti hiv-1 effect of the compounds was gradually decreased if they were added 1 and 2 h after infection, no inhibition was observed when the compounds were added 4 h after infection. the compounds did not impair the virion structure. adamantane and norbornene derivatives were shown also to inhibit azt resistant viral strains. conclusion: adamantane and norbornene were shown to be active hiv inhibitors with the high selectivity index. the compounds are promising candidates for further investigation including preclinical studies. less is known about the effect of their intracellular half-lives on the maintenance of antiviral activity. to investigate this question, we developed a novel in vitro antiviral persistence assay. measurement of the antiviral persistence of tenofovir (tfv) and abacavir (cbv) was coupled to measurement of the half-lives of their tfv-dp and cbv-tp anabolites. methods: mt-2 cells or stimulated primary cd4+ t-cells were incubated with graded concentrations of tfv or cbv for 14 h (h); then extracellular drug was removed by washing. cells were further incubated without drug for 0-24 h and then infected with hiv-1 (iiib or bal). p24 was quantified on day 2; inhibition of hiv-1 replication due to intracellular drug persistence (pc50) was determined relative to a standard ec50. decay of intracellular dp/tps in cd4+ t-cells was measured using lc/ms/ms. results: in mt-2 cells, the pc50 value for tfv 12 h after drug removal remained unchanged relative to the ec50 (<3-fold shift) whereas the pc50 for cbv shifted >65-fold, indicating less persistence of cbv. in cd4+ t-cells, the pc50 value for tfv also showed a minimal shift relative to the ec50 (2.4-fold) 24 h after drug removal. cbv showed a much larger relative shift (>243-fold). quantification by lc/ms/ms of intracellular tfv-dp and cbv-tp in cd4+ t-cells in vitro demonstrated that tfv-dp had the longest intracellular half-life of the two drugs (tfv-dp, 21 h versus cbv-tp, 5 h). conclusions: a novel antiviral persistence assay was developed to study the relationship between intracellular nrti halflives and antiviral activity. in both mt-2 cells and primary activated cd4+ t-cells, tfv had the longest persistence of antiviral activity. in cd4+ t-cells, tfv-dp also had the longest half-life of the two nrtis. cbv-tp had a much shorter half-life than tfv-dp and showed less antiviral persistence. although both drugs are approved for qd dosing, the half-life of intracellular tfv-dp maintains antiviral suppression in vitro over a timeframe most consistent with qd dosing. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa isis 5320 is a phosphorothioate oligonucleotide with a molecular structure of t2g4t2. the g-quartet possessing molecule has been shown to be a potent inhibitor of hiv attachment and cell-cell fusion and acts by specifically interacting at the v3 loop of gp120. mapping studies with monoclonal antibodies targeting epitopes in and around the v3 loop have been used to define the binding site of isis 5320. in vitro, isis 5320 inhibits all laboratory and clinical strains of hiv-1 and hiv-2 tested, including representative subtype viruses, drug resistant viruses (including mdr viruses) and viruses that utilize the cxcr4 and ccr5 chemokine receptors. serial passage of virus in the presence of increasing concentrations of the oligonucleotide did not result in the selection of drug resistant virus strains and combination assays resulted in additive to synergistic interactions with other approved hiv inhibitors. the antiviral and toxicity profiles of isis 5320 resulted in the performance of human clinical trials for the therapeutic use of the oligonucleotide to treat hiv infection. the antiviral properties and mechanism of action of isis 5320 suggest that it may be an excellent anti-hiv topical microbicide. isis 5320 was found to be highly active in a cervical explant model of hiv infection with highly significant inhibition of ccr5-tropic strains of virus. activity was also observed in cell-free and cell-associated virus transmission assays, as well as in cd4-dependent and cd4-independent acute infection inhibition assays. in microbicidal specific combination assays, significant efficacy has been observed with isis 5320 used in combination with other microbicidal compounds. the results of these studies suggest that isis 5320 may represent a new and novel anti-hiv topical microbicide. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa though a variety of compounds are being developed as anti-hiv topical microbicides, such as polyanionic molecules, surfactants, natural products, peptides, proteins, heterocycles, and virucidal agents, clinical efficacy studies that demonstrate the ability of these agents to impact virus transmission are still in progress. it has been estimated that a microbicide that is only 60% effective would have the capacity to prevent millions of new infections each year. thus, one of the challenges in hiv drug development is the discovery of compounds that will inhibit the sexual transmission of infectious organisms between sexual partners. the rapid mutability of hiv and the known presence of drug resistant viruses in wild type virus populations suggests that microbicide development will suffer from the same problems that exist for all hiv therapies, namely the selection of resistant virus strains that will bypass the microbicide barrier and infect target cells in the vaginal or rectal environment even in the presence of the microbicide. thus, it is likely that haart-like combination drug therapies will become the most effective means of inhibiting the sexual transmission of hiv. we have evaluated a wide variety of anti-hiv and anti-sti compounds in vitro alone and in combination with one another and have demonstrated that certain patterns of inhibition (additivity, synergy, antagonism) occur between the various classes of compounds. recently, we have compared the combination anti-hiv activity of microbicide compounds in fresh human pbmcs infected with clinical isolates of hiv to the combination activity of the same test agents in cem-ss-based cultures. in general, these two assay systems yield similar combination assay results. to provide a rationale for the combination use of the compounds in a microbicide setting, the same combination of compounds was evaluated in a microbicide-like virus transmission assay. these combination results suggest that higher levels of synergy between virus attachment and reverse transcriptase inhibitors might be expected in the microbicide environment compared to levels predicted for the systemic therapeutic environment. the results of the combination assays with various microbicides will be presented. during the onset of the hiv disease, hiv rna is continually produced in the face of treatment with haart in circulating reservoirs and rt inhibitors are almost ineffective in the postintegration events. among the classes of anti hiv-1 drugs, protease inhibitors (pi) are the unique to inhibit the hiv-1 production in chronically infected macrophages. in the progression of hiv infection, the role of the monocytes-derived macrophages (m/m) is further confirmed as they represent chronologically the first cytotype where the viral replication restarts as a consequence of failure or interruption of antiviral therapy. aim of the work was to evaluate the rebound of hiv-1 production when pi have been removed in hiv-1 chronically infected m/m and, moreover, to verify the effect of this removal on virus maturation, infectivity and ability to trigger apoptosis in uninfected peripheral blood lymphocytes (pbl). a rebound of p24 gag protein was measured starting from 12 h after drug removal yet virus infectivity remained 1 log lower than control up to 1 week. inhibition of hiv-1 replication was still 48% and 18% upon amprenavir 20 and 4 m, respectively. these data were confirmed by western blotting and electronic microscopy showing production and release of immature viral particles. moreover, pi (amprenavir and indinavir) treatment dramatically reduced apoptosis of pbl co-cultured with chronically infected m/m and kept cd4/cd8 ratio above the levels of untreated controls until the 5th day of co-culture. taken altogether, these findings suggest a wide clinical importance for amprenavir and indinavir for their relevant long-lasting antiviral effect in persistently-infected reservoirs of hiv even in case of drug interruption and/or when hiv infection can restart in districts where drugs find not sufficient concentration. moreover, these results strengthen the evidence for an unique positive utilize of pi against ongoing and productive hiv infection. weili jin, salvatore santino, michael wang gilead sciences, foster city, ca, usa background: effective inhibition of hiv reverse transcriptase (rt) currently represents a crucial objective of antiretroviral therapy. capravirine is a second-generation non-nucleoside rt inhibitor (nnrti) that is capable of blocking the replication of certain nnrti-resistant strains of hiv and was recently in clinical development. in this study, we report on the in vitro selection and characterization of viral resistance to capravirine. methods: viral resistance selection experiments were performed in mt-2 cells with the hiv iiib isolate and increasing concentrations of capravirine. viruses were analyzed genotypically by population sequencing and by single genome sequencing (sgs). recombinant viruses with nnrti mutations were generated from proviral dna clones. phenotypic analyses were performed in mt-2 cells. results: capravirine resistance selections were initiated at 1 nm (ec 50 of 1.5 nm for capravirine). following nine passages in the presence of increasing concentrations of capravirine, the l100i mutation emerged in rt and additional passaging led to v179d and f227c mutations at higher concentrations (100-300 nm). further increases in capravirine concentrations led to the emergence of a l100i + v179d + f227c triple mutant, which confers >1000-fold resistance to capravirine. sgs of mixed viral populations from different passages showed that l100i, v179d and f227c were present on the same genome, with l100i as the primary mutation, and f227c and v179d were acquired sequentially at later passages. through sgs analysis, a l100i + k166r + v179d + f227c quadruple mutation on the same genome was also observed at higher capravirine concentrations (>1000 nm). recombinant viruses carrying these mutations were produced to assess their susceptibilities to capravirine. conclusions: after extensive in vitro passaging of hivinfected cells in the presence of capravirine, neither k103n nor y181c mutations in rt were observed. instead, the l100i mutation was initially acquired, followed by mutations f227c and v179d. addition of the k166r mutation to the triple mutant genome, l100i + v179d + f227c, appears to further enhance hiv resistance to capravirine. oluwafemi olawuyi 1 , adeyemi falegan 2 1 medical microbiology, university college hospital, ibadan, nigeria; 2 dentistry, university college hospital, ibadan, nigeria issue: the percentage of aids/hiv is increasing every year in the third worlds, and this is reinforced by the factor that majority of youth in third worlds do not know his/her hiv status. description: a self developed validated and reliable questionnaire [r = 0.87] was used to collect the data and percentage was used to analyze the data. the population of the study was made up of youth [female and male] in higher institutions, working places, market places and community streets in nigeria, 50,000-sample size, selected through simple random sampling technique. the mean age is 25.5 years old. relative risk [rr] calculated is 3.1, i.e. rr >1, indicating that the factor is the risk factor, and the confidential interval [ci] for rr at 95% significant level is 2.61 < 3.1 < 3.90 from the formula, ci lower limit < rr < ci upper limit. lessons learned: seventy percent of the sample population did know his/her hiv status and had had sexual intercourse in the past before, out which 20% had the unprotected intercourse once or more, 25% had protected sex while 25% were not sure of using protection means. while, 20% have knowledge about own hiv status and had had sexual intercourse before. ten percent have no knowledge about own hiv status and had no sexual intercourse before. conclusion: aids/hiv still remains a killer disease in the third world. however, the lack of knowledge of individual's hiv status remains the only highest risk factor for the spread of the disease in the third worlds. yuichiro habu 2,3 , jacob barnor 1,6 , norio yamamoto 4 , kahoko hashimoto 1,2 , naoko miyano-kurosaki 1,2 , koichi ishikawa 5 , naoki yamamoto 5 , david ofori-adjei 6 , hiroshi takaku 1,2,7 1 department of life and environmental sciences, chiba institute of technology, chiba, japan; 2 high technology research center, chiba institute of technology, chiba, japan; 3 japan foundation of aids prevention; 4 department of molecular virology, bio-response, tokyo medical and dental university, tokyo, japan; 5 aids research center, national institute of infectious disease, tokyo, japan; 6 department of virology, noguchi memorial institute for medical research accra-ghana, accra, ghana; 7 bach tech corp. rna interference (rnai) is a potentially strong gene interference tool, which had been successfully used to silence many pathogenic viruses including hiv. however, many recent reports have shown that, in long-term assay cultures involving rna viruses such as hiv, escape mutants breakthrough the silencing effect. in the light of this conundrum, it had been proposed that, vector designed to target multiple genes in a synergistic manner, may address the problem. hence, we designed a chimeric rna expression vector which express vif shrna and decoy tar rna by combining vif shrna and decoy tar rna with linker to which dicer was able to recognize for cleavage, as a second generation rnai expression vector system. the synergistic effect of these molecules enhanced the inhibition of hiv-1 replication in a long-term transduced pbmcs, h9, and jurkat cell culture assays (9 weeks) and prevented virus breakthrough associated with sirna-mediated escape variants. notably, hiv-1 replication was similarly suppressed in the control cells expressing only vif shrna for about 3 weeks, but an increase in virus replication was observed afterwards. hiv viral rna extracted and sequenced at this point indicated escape mutants in the cells expressing the vif target in hiv. we confirmed substitution of bases in the vif shrna target sequence. on the other hand, the incidence of mutation was not observed in a sequence of viral rna from the culture expressing the vif shrna-decoy tar rna at the fourth week. interestingly, virus production was inhibited for a long-term by an effect of decoy tar rna, through the rna-protein interaction. combining shrna with decoy tar rna as second-generation anti-hiv shrna may provide practical basis for applying sirna-based gene therapy to the treatment of hiv/aids. introduction: this is a designed efficient gene therapy against aids/hiv. the novelty of this aids vaccine design/concept is seen in the fact that the 'pol' gene encoding for nonstructural proteins (polyproteins that generate three enzymes: reverse transcriptase, integrase and protease) is cloned in a suitable retroviral vector and adult stem cells are transfected by this and reinfused into the circulation to effectively counter hiv replication and antigenic variation. method: the mrna are isolated from adult stem cells and transcribed into cdna with reverse transcriptase. the cdna are then cloned in a suitable retroviral vector (vacinia) carrying 'pol' gene that confers resistance to a strong reverse transcriptase inhibitor drug. the adult stem cells are transfected by the recombinant mixture, and reinfused into the circulation of hiv infected person. result: the transfected stem cells are reinfused to provide renewable source of more and better empowered normal blood cell types that would disrupt and half hiv replication in the circulation. there would be efficient induction of both humeral and cellular mediated immunity with prolonged expression of antigens and protective immunologic memory generation against hiv antigenic variation. conclusions: this aids vaccine design would lead to both efficient prophylactic and therapeutic therapy against aids in that it would effectively take care of the problematic factor of hiv antigenic variation which has long been the main obstacle to potent aids vaccine development. because of the real risk of interspecies transmission and/or reassortment between avian, swine and human influenza a strains, drug susceptibility monitoring of circulating avian and porzine virus strains appears to be warranted for effective application of antiviral drugs like amantadine. this study was designed to gain insight into amantadine susceptibility of 6 avian and 12 porcine influenza a viruses isolated in germany between 1981 and 2002. virus strains were isolated in embryonated chicken eggs and passaged one time in mdck cells. plaque reduction assays were applied to examine virus susceptibility to amantadine. genotyping was used to confirm drug resistance. in the result of these antiviral studies, only 3 of the 12 porzine isolates but all 6 avian isolates were shown to be amantadine-susceptible. interestingly, the three amantadinesensitive porzine strains were isolated between 1981 and 1987. all porzine influenza a viruses isolated later on were drugresistant and contained the aa substitutions g16e, s31n, and r77q in the matrix protein 2 (m2). additionally, l27a was detected in two h1n1 strains. s31n and/or l27a are well known amino acid substitutions in m2 that confer amantadine resistance. the role of the pig as an intermediate host of avian and human influenza a viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadineresistant porcine influenza a viruses suggest a real risk of emergence of amantadine resistant human viruses. therefore, further studies are ongoing now to evaluate the circulation of the resistant phenotype in pigs, birds and human. recently much attention has been devoted to searching for effective chemotherapeutic agents and vaccines for eradication of this notorious disease. at present only chemotherapy is available to combat avian flu, for instance, tamiflu, approved for the treatment by the us-fda. development of a simple, novel molecule with potential antiviral activity against is essential to treat avian flu viral infection. isatin (2,3-dioxoindole), is a versatile lead molecule for designing of potential antiviral agents and its derivatives were reported to possess broad spectrum antiviral activity. methisazone (nmethylisatin-3-thiosemicarbazone) was first clinically approved for treatment of pox viral infections, and its derivatives were documented to have anti-influenza activity. based upon this evidence, the present work was initiated to determine the antiviral activity of novel isatin derivatives against avian flu (h5n1) in mdck cells. antiviral activity was studied by virus yield assay (ec90), and cytotoxicity by neutral red uptake assay by uninfected mdck cells. all five compounds of a series inhibited the replication of avian flu (h5n1) virus replication in mdck cells and compounds spiii-5h and spiii-5cl were most active (ec90 5.5 g/ml, cc50 >100 g/ml and si > 18). details of these studies and results of treatment of influenza-infected mice are discussed. acknowledgement: supported in part by contract noi-ai-15433 and noi-ai-30048 from virology branch, niaid, nih]. arginine-rich peptide conjugated phophorodiamidate morpholino oligomers (arp-pmo) are nuclease resistant antisense compounds that hybridize to target rna in a sequence-specific manner resulting in disrupted rna function. eight arp-pmo were designed to base-pair with various regions of a/pr/8/34 (h1n1) rna and were then evaluated by hemagglutination and plaque assays for their ability to inhibit fluav production in vero cell culture. arp-pmo targeting the aug translation start site of the np or pb1 segment mrnas, or the 3 -terminus of their respective vrnas, were highly effective, reducing influenza virus titer by 1-3 orders of magnitude in a dose-dependent and sequence-specific manner over a period of 2 days. two of the p-pmo, targeting the pb1 translation start site region (pb1-aug) and the 3 terminus of np vrna (np v3 ), were evaluated by endpoint dilution (tcid50) or elisa assays against another h1n1 strain (a/wsn/33), as well as a/memphis/8/88 (h3n2) and a/thailand/1(kan-1)/04 (h5n1). the pb1-aug arp-pmo generated over 85% specific reduction of virus level, regardless of viral subtype or methodology, at concentrations in the range of 10-20 m. the np v3 p-pmo yielded similar results, with the exception of considerably lower efficacy against the h3n2 strain, with which it has two base mispairings. studies are planned to further evaluate of at least two arp-pmos in animal models for h1n1 and h5n1 fluva subtypes. macroheterocyclic compounds containing crown fragments and nitrogen atoms show large-scale biological activity. we synthesized series of aza-crown ethers and their derivatives. we also studied anti-influenza and antiherpetic action of some of them. anti-hsv action of studied compounds was tested using cyto-morphological method. hep-2 cells were infected with hsv-1 strain us in dose 5 ifu/cell. the cells were incubated in eagle's medium that contained compounds in a dose of 10 −4 m in experimental samples, or without them in control samples. then cells were fixed with 96% ethanol and stained with 0.01% acridine orange solution. the amount of infected cells with dna-containing virus inclusion bodies was counted by fluorescent microscopy. anti-hsv activity of compounds was calculated as the difference between of the percentage of infected cells in treated cell cultures to the percentage of infected cells in untreated cell cultures. anti-influenza activity was studied on the model of replication of a/hong kong/1/68 (h3n2) strain in tissue culture of chorio-allantoic membranes of chicken embryos. compounds were used in a dose of 10 −3 m during the study of their anti-influenza action. diaza-18crown-6 and two of its derivatives have showmen neither anti-hsv nor anti-influenza activity. diaza-18crown-6 derivatives that contain 2-oxyethyl-or ethoxycarbonyl-fragments decreased amount of cells infected by hsv-1 by 13 and 29%, respectively. both of these compounds inhibited replication of influenza virus on 1.7 log 10 tid 50 aza-15crown-5 did not show antiviral activity, but both its derivatives proved to be active inhibitors of hsv and influenza virus reproduction. aza-15crown-5 derivatives that contain 2-amino-3-phenyl-propanoyl-or 5-benzyloxy-3-oxapentyl-fragments decreased amount of cells infected by hsv-1 with virus-specific intranuclear inclusions by 41 and 47%, respectively. first compound inhibited replication of influenza virus on 1.5 log 10 tid 50 and the second one decreased virus amount on 2.0 log 10 tid 50 . the results of this study show that aza-crown ethers are the perspective class of compounds for search of new antiviral agents. acknowledgement: this work was partially supported by stcu (grant # 3147) . robert w. sidwell 1 , kevin w. bailey 1 , min-hui wong 1 , donald f. smee 1 , dale l. barnard 1 , shanta bantia 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 biocryst pharmaceuticals, inc., birmingham, al, usa the cyclopentane neuraminidase inhibitor, peramivir (bcx-1812, rwj-270201) has striking inhibitory effects on a spectrum of influenza viruses in vitro, and has also demonstrated significant effects against influenza a (h1n1, h3n2) and b virus infections when administered orally to mice and ferrets. unfortunately, clinical trials with the drug administered orally were not successful, probably due to low blood levels obtained after oral administration. significant plasma drug levels of peramivir persist up to 6 h after intramuscular (i.m.) injection; more importantly, however, is the observation that peramivir remains tightly bound to influenza virus n9 neuraminidase for over 24 h, suggesting single i.m. or intravenous (i.v.) therapy with the drug may be highly effective against an influenza infection. experiments now in press have indicated that single i.m. peramivir therapy administered up to 48 h after virus exposure was protective to mice infected with influenza a (h1n1) virus. in the present study, peramivir was administered i.m. or i.v. in a single injection 1 h pre-virus exposure in separate experiments to mice infected with an influenza a (h5n1) virus; efficacy was compared to similar dosages of oseltamivir and oseltamivir carboxylate run in parallel. dosages of 20 and 10 mg/kg of peramivir administered by either route significantly prevented deaths, lessened arterial oxygen (sao 2 ) decline, inhibited development of lung consolidation, and inhibited lung virus titers. the lung assays were performed at varying times after virus exposure. oseltamivir and oseltamivir carboxylate, which do not have the same neuraminidase binding abilities seen with peramivir, were less efficacious in these experiments. delaying the single i.v. therapy up to 72 h after virus exposure also significantly inhibited the virus infection. peramivir appeared to be well tolerated in toxicity control animals run concomitantly with these studies. these data indicate parenterally administered peramivir may hold promise as a therapy for clinical influenza a (h5n1) virus infections. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hiroshi saitoh 1 , naoko miyano-kurosaki 1,2 , hiroshi takaku 1,2 1 department of life and environmental sciences, faculty of engineering, chiba institute of technology, chiba, japan; 2 department of life and environmental sciences, faculty of engineering and high technology research center, chiba institute of technology, chiba, japan background: influenza virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. recently, small interfering rnas (sirnas) are a powerful tool for sequence-specific, post-transcriptional gene silencing and have a potential therapeutic and prophylactic application against cancer, as well as infectious diseases. here we show that short interfering rnas (sirnas) specific for conserved regions of the viral genome can potently inhibit influenza virus production in cell lines. the influenza virus np gene is a potential target for rnai technology. on the other hand, the baculovirus (acmnpv) can infect a variety of mammalian cells, facilitating its use as a virus vector for gene delivery in viral entry into cells. in this study, we describe the inhibition of influenza virus production by baculovirus-mediated shrna expression vectors. methods: the psv2neo-u6 plasmid vectors and pvl1393based baculovirus vectors were used in this study. the influenza virus a and b np genes were made into the target and the shrna expression plasmid vectors were constructed under the control of the human u6 pol iii promoter. the shrna expression plasmids or shrna expression baculovirus vectors introduced into mdck cells, and 24 h later the cells were infected with either a/pr8 or b/ibaraki virus at a moi of 0.01. at 72 h postinfection, culture supernatants were harvested and assayed to determine the virus titer by plaque assay. conclusion: the findings reveal that newly synthesized np proteins are required for influenza virus replication and provide a basis for the development of shrnas expression plasmids as prophylaxis and therapy for influenza infection in humans. julia serkedjieva, ekaterina krumova, tsvetanka stefanova, nadja nikolova, maria angelova institute of microbiology, bulgarian academy of sciences, sofia, bulgaria a semi-standardized polyphenol-rich extract (pre), obtained from geranium sanguineum l., inhibited the reproduction of influenza viruses types a and b in vitro and in ovo and protected mice from mortality in the experimental influenza virus infection (serkedjieva and manolova, 1992) . the selective in vitro virus-inhibitory activity of pre was fairly modest and this was in contrast with the significant protection in vivo. thus, the therapeutic effect of pre needed explanation. it was presumed that it might be attributed to a combination of more than one biological activities known for natural polyphenols. we have demonstrated previously that pre manifested strong antioxidant and radical-scavenging activities in model systems (sokmen et al., 2004) . the current study was undertaken to investigate the effect of the plant extract on the levels of the antioxidant enzymes superoxide dismutase (sod), catalase (kt) and peroxidase (po) in mice lungs during influenza virus infection as well as the effect of pre on the production of reactive oxygen species (ros) and reactive nitrogen intermediates (rni) by alveolar macrophages in influenza virus infected mice. mice were challenged intranasally (i.n.) with 5-10 ld 50 of a/aichi/2/68 (h3n2) influenza virus. pre was administered by i.n. instillation 3 h before infection in the dose of 10 mg/kg. it was established that influenza infection induced an increase in sod, kt and po production and on days 6 and 9 after infection their levels reached 140-160% of placebo control. the application of pre brought enzymes values to control levels. influenza infection caused also a significant increase of h 2 o 2 , o 2 •− and no production by alveolar macrophages; the generation of ros and rni peaked on day 9. pre-treatment before viral challenge reduced this excessive production. in conclusion, the obtained results outlined the antioxidant and radical scavenging properties of the plant extract; pre beneficially modulated the oxidative stress response in influenza virus-induced pneumonia. this alternative mechanism of action might contribute to the overall protective effect in the lethal murine experimental influenza infection. the antiviral activity of s11, a natural herb extract, ji-sun kwon 1 , hyun-jeong lee 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea the antiviral activity of s11, one of the traditional korean medical herb extract, against influenza virus was investigated. the 50% effective concentration (ec50) using plaque reduction assay was 31.25 ug/ml and the mean 50% cytotoxic concentration (cc50) using wst-1 assay in the mdck cells was 334 ug/ml. oral gavage treatment of the s11 to balb/c mice infected with a/pr/8/34 (h1n1) influenza virus showed the therapeutic effects as delaying clinical signs, significant inhibition of death and reduction of lung virus titers. to identify the lead molecules, the s11 was subjected to further fractionation, purification, and isolation of active compounds. the antiviral activity of these natural herb compounds will be discussed. these results suggest that the s11 is a possible candidate for the development of new antiviral medicine for influenza therapy. hiroshi takaku 1,2,4 , takayuki abe 3 , hitoshi takahashi 1 , naoko miyano-kurosaki 1,2 1 department life environ. sci., chiba inst. tech., chiba, japan; 2 high tech. res. center, chiba inst. tech., chiba, japan; 3 res. inst. microbial dis., osaka university, osaka, japan; 4 bach tech corp background: the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has long been used as a biopesticide and as a tool for an efficient recombinant protein production in insect cells. in this study, we examined the immunization of a recombinant baculovirus expressing the influenza virus hemagglutinin (ha) against lethal influenza infection in mice. protection was observed in mice immunized intranasally with not only the recombinant baculovirus but also a wild-type baculovirus. baculovirus was also shown to induce secretion of inflammatory cytokines, such as tnf-␣ and il-6, in murine raw 264.7 macrophage cell line. results: a varied route of immunization with a recombinant baculovirus expressing the influenza virus hemagglutinin protein of a/pr/8/34 (h1n1) virus against lethal influenza infection was examined in mice. the recombinant baculovirus encoding the hemagglutinin gene under the control of chicken ␤ actin promoter was inoculated twice, 2 weeks apart, at a dose of 1.1 × 10 8 pfu per mouse by intramuscular, intradermal, intraperitoneal, and intranasal routes. mice intramuscularly and intraperitoneally immunized with the recombinant exhibited higher level of production of serum anti ha antibody than those immunized via the other routes, but protection was only achieved by the intranasal immunization. surprisingly, mice immunized with a wild-type baculovirus with intranasal route were also protected from the lethal influenza virus challenge. sufficient protection in mice was achieved by the intranasal immunizations with 10 8 pfu of either the recombinant or wild-type baculovirus, as evaluated by the reduction of virus titer, production of inflammatory cytokines, and pulmonary consolidations in the lung. these results indicate that infection with a baculovirus induces a strong innate immune response and protection of mice from lethal influenza virus infection. conclusion: baculovirus (cpg motifs) induces a strong innate immune response and protection of mice from lethal influenza virus a and b infection. andrew vaillant 1 , annie lebel 2 , nathalie goyette 2 , guy boivin 2 , jean-marc juteau 1 , phil wyde 3 1 replicor inc., laval, que., canada; 2 chuq-chul and laval university, st. foy, que., canada; 3 baylor college of medicine, university of texas, houston, tx, usa potent antiviral activity of phosphorothioate oligonucleotides (ps-ons) was observed against influenza viral infections. antiviral activity was sequence-independent, size dependent (optimally active ps-ons were ≥40 bases in length) and dependent on the presence of the phosphorothioate modification (hydrophobicity). binding studies showed that rep 9 (a 40 mer degenerate ps-on) interacts with both neuraminidase and hemagglutinin although the sialidase activity of neuraminidase was not affected, suggesting that the structural interactions of these proteins required for influenza activity are the target for this compound. the requirement for hydrophobicity further suggests that the alpha helical regions of hemagglutinin are one of the regions of interaction. the antiviral activity of rep 9 was conserved in many influenza a and b strains suggesting potential therapeutic activity against avian flu and other newly emerging influenza strains. rep 9 aerosol has excellent characteristics for lung deposition and aerosol treatment with rep 9 was well tolerated and highly effective against infections with influenza a both in prophylaxis and 24 h after infection. these results demonstrate the therapeutic potential of aerosolized ps-ons against influenza infection. acknowledgement: supported by nih contract no1-ai-15437. irina v. alymova 1 , y. sudhakara babu 2 , allen portner 1 1 virology division, department of infectious diseases, st. jude children's research hospital, memphis, tn 38105, usa; 2 biocryst pharmaceutical, inc., birmingham, al 35244, usa bcx 2798 is a novel selective inhibitor of human parainfluenza virus infections, which design was based on the threedimensional structure of the hemagglutinin-neuraminidase (hn) protein of newcastle disease virus. compound exhibited striking activity against parainfluenza viruses in vitro and in vivo, and was efficacious in prophylaxis of lethal synergism between parainfluenza virus and streptococcus pneumoniae in a mouse model. present study was conducted to determine if bcx 2798's resistant variants of the recombinant sendai virus whose hn gene was replaced with that of human parainfluenza virus type 1 (rsev(hpiv-1 hn) could be selected in tissue culture and animals. for this purpose virus was serially passaged in llc-mk 2 cells at moi 0.1 in the presence of increasing (from 100 to 3200 m) concentrations of compound; infected 129×1/svj mice were treated with 10 mg/kg/day of bcx 2798 twice for five days. treatment started 4 h before infection. individual clones of viruses were analyzed for the presence of mutations. one mutation, e527k, on the globular head region of the hn protein was selected in tissue culture after the fifth and eleventh passages of rsev (hpiv-1 hn) . several mutations in hn gene of rsev (hpiv-1 hn) were selected in an animal model after the second passage of virus from mice treated with bcx 2798. two mutations, n23s and p29q, were located in the cytoplasmic domain of hn protein; mutations n173s and t553a were found on the globular head region of the glycoprotein. only nonconserved amino-acid residues of hn protein were involved in substitutions. all isolated mutant viruses were stable after the five passages in llc-mk 2 cells without drug; did not develop other substitutions in the presence of drug and displayed no resistance to bcx 2798 both in vitro and in vivo. infectivity of all mutants was not altered to compare with the wild type of rsev (hpiv-1 hn) virus. taking together our results indicate that prophylaxis/treatment of human parainfluenza virus infections with bcx 2798 may not lead to appearance of clinically significant variant of viruses. kie-hoon jung 1 , michelle mendenhall 1 , lawrence m. blatt 2 , robert w. sidwell 1 , brian b. gowen 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa hantavirus pulmonary syndrome (hps) is an acute human respiratory disease with remarkably high case fatality rates (30-50%) for which the etiological agents are members of the bunyaviridae family, genus hantavirus. maporal (map) virus is a recently identified hantavirus isolated in western venezuela, which is most similar phylogenetically to hantaviruses known to cause hps in southern regions of south america. despite the lack of evidence that map can productively infect humans and cause hps, infection of hamsters closely resembles disease manifestations associated with human hps. hantaviruses, in general, are known to produce little to no cytopathic effect (cpe) in cultured cell lines. unexpectedly, we found that map produces remarkable cpe in several vero cell lines facilitating the evaluation of known antiviral agents, ribavirin and interferon alfacon-1. both drugs were highly effective at reducing cpe, as determined by visual examination and neutral red dye uptake, associated with map infection. since much of the observed cpe may be due to apoptosis of uninfected bystander cells, we also developed a quantitative (q)rt-pcr assay to detect copies of map genomic sequence to more directly assess the inhibition of viral replication. data obtained using the qrt-pcr-based assay were consistent with the visual cpe reduction and neutral red-uptake cytotoxicity findings. the development of in vitro antiviral testing methods for map are essential to the evolution of the in vivo hamster disease model of hps. the latter is of utmost importance considering the current need for effective antivirals for the treatment of hps and the lack of a suitable model that does not require biosafety level 4 containment facilities. acknowledgement: supported by contract no1-ai-30048 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. nucleoside analogues are widely used in antiviral and anticancer chemotherapy. for this class of drugs, intracellular conversion of the nucleoside analogue into the corresponding 5mono-, 5 -di-, and 5 -triphosphate after target cell penetration is a prerequisite for biological activity. because of the structural differences from natural nucleosides, this conversion is often inefficient and, as a consequence, therapeutic efficacy is sometimes limited. the free phosphates, or nucleotides, have limited utility in therapy on account of their poor membrane permeability and chemical stability. one approach to improve the therapeutic potential of nucleoside analogues is the delivery of the corresponding nucleotide entities via neutral, lipophilic prodrugs, or protides. the nucleoside aryl phosphoramidate approach, developed by mcguigan and co-workers (2004) has been successfully applied to a number of different nucleosides (azt, d4t, dda, d4a). the general structure of aryl phosphoramidates encompasses two masking groups, an amino acid ester and an aryl moiety bonded to the phosphate group. in order to apply this protide technology to nucleosides with the potential for anti-hepatitis c virus (hcv) activity, we have undertaken studies designed to probe the effect of varying the natural and unnatural amino acid esters and the aryl groups used as masking groups in the target phosphoramidates. these compounds have been synthesised and evaluated using genotipe 1b sub-genomic hcv replicon. we have prepared a variety of arylphosphoramidate derivatives from a range of 4 -substituted nucleosides, including 4azido-cytidine, 4 -azido-uridine, and 2 ,3 -protected variants. with certain nucleoside phophoramidates, we have observed dramatic enhancement (>1000-fold) of replicon activity relative to the parent nucleoside. the synthesis, biological activity and sar of these compounds will be presented. reference mcguigan, d. cahard, balzarini, j., 2004. mini-review. med. chem. 4, 371-382 . we have identified a series of novel anthranilic acid derivatives that are potent, reversible inhibitors of hepatitis c virus (hcv) ns5b polymerase, an essential enzyme for viral replication. the micromolar ns5b polymerase inhibitors belong to the n-phenoxyacetylanthranilic acid chemotype. x-ray crystallography determined that the inhibitors bound to ns5b between the thumb and palm regions adjacent to the active site. guided by crystallography, subsequent modifications to the hydrogen bonding and lipophilic regions of the inhibitors resulted in greatly improved activity against ns5b. further sar studies revealed a second, more potent sub-series where the phenoxy group was replaced by an anilino group. analogs in both subseries showed antiviral activity in a cell-based replicon model of hcv. andrea brancale 1 , dimitrios vlachakis 1 , maria chiara barbera 1 , romano silvestri 2 , colin berry 3 , johan neyts 4 1 cardiff university, the welsh school of pharmacy, cardiff cf10 3 xf, uk; 2 universita' degli studi "la sapienza", dipartimento di studi farmaceutici, 00185 roma, italy; 3 cardiff university, cardiff school of biosciences, cardiff cf10 3us, uk; 4 rega institute for medical research, k.u. leuven, b 300 leuven, belgium hepatitis c is a viral infection that affects 170 million people worldwide, including 4 million in the united states and 8 million in europe. the virus establishes a chronic infection in 55-85% of cases and 20% of affected individuals develop cirrhosis. at the moment there is neither a vaccine nor an effective antiviral therapy available and efforts to identify a specific anti-hcv inhibitor have dramatically intensified in the last few years. many research groups have focused their interest on the enzymes involved in the viral replication and, among these enzyme, the viral helicase/ntpase has proven to be a suitable target for developing novel anti-hcv compounds. compound 1 is a potent inhibitor of the hcv helicase and, although its mode of action is still uncertain, it has been proposed that it acts as competitive inhibitor of rna binding. starting from this hypothesis, we have prepared a series of novel compounds based on the structure of 1 where the benzimidazole moiety has been replaced by different chemical groups, including the negatively charged carboxylate moiety, which should mimic the phosphate backbone of the nucleic acid. the synthesis, the enzyme inhibition and the biological evaluation in replicon of these novel compounds will be presented and analyzed. dale r. cameron migenix inc., 3650 wesbrook mall, vancouver, bc, canada v6s 2l2 hepatitis c virus (hcv), a leading cause of liver disease, continues to be an attractive target for new drug development. among the more favourable approaches to developing new hcv drugs is to target the rna-dependant rna (rdr) polymerase (ns5b), which has been shown to be an essential enzyme for replication. there are several published non-nucleoside inhibitors of this polymerase (some in clinical development) and several published allosteric binding pockets on the protein they target. to be successful, traditional lead identification can be timeintensive, costly and have large infrastructure requirements. increasingly, a push towards effective computational-based screening has led to the development of virtual screening tools. such tools allow investigation of large quantities of compounds in silico for particular properties without the need for compound synthesis or high throughput screening. moreover, these techniques require only a modest infrastructure investment and are very efficient. we employed the openeye set of screening tools (omega, rocs and eon) in concert with publicly available hcv inhibitor information, and commercial databases to identify novel leads. the inhibitor coordinates from a protein-inhibitor complex crystal structure were utilized as the target. available compound databases (asinex and chembridge) were utilized as the testset of compounds. filtered compound conformers were generated using omega and compared with the template using rocs with post-analysis by eon. visual analysis to maximize particular desirable binding features while minimizing protein-inhibitor steric clash allowed the list of potential hits to be further narrowed. multiple classes of compound were identified from the above procedure and after sourcing a subset of the actual compounds or close analogs, they were tested for enzymatic inhibition activity and further characterized. iteration of the process resulted in the identification of a lead compound class containing multiple active compounds, one with reasonable replicon activity. in conclusion, readily available structural and database information and virtual screening tools can be successfully utilized to identify novel inhibitors of hcv rdr polymerase which, in turn, can serve as novel leads for developing new therapies for treating hcv. synthesis, antiviral activity, and cytotoxicity of some novel quinazolin-4(3h)-one derivatives a series of novel 6-bromo/6,8-dibromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)-benzenesulphonamides were synthesized by condensation of 2-substituted benzo[1,3]oxazine-4-ones and sulphonamide. their chemical structures were assigned by means of spectral analysis (ft-ir, pmr, ms). synthesized compounds were screened for in vitro antiviral activity against human pathogenic viruses (hiv, hcv, hsv, vv). 6-bromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)benzenesulphonamide (sps-ii) and 4-(4-oxo-2-phenyl-4hquinazolin-3y-l)-benzenesulphonamide (sps-i) inhibits the replication of hiv-1 in acutely infected mt-4 cells at a concentration of approximately 10 g/ml, while not being toxic to the host cell at a concentration of 60 or >125 g/ml (selectivity index: 8 and >12), respectively. in huh 5-2 cells sps-i inhibited hcv rna synthesis at ec50 of 8 g/ml, while at cc50 for cell growth 32 g/ml. sps-ii inhibited the virus-induced cytopathicity in human embryonic lung (hel) cell infection with hsv-1, hsv-2 or vaccinia (vv) at a concentration of 50 g/ml, while not being toxic to the cells up to a concentration of 400 g/ml. further molecular modification in this series of compounds may help in optimising their antiviral activity. wengang yang, yongnian sun, avinash phadke, milind deshpande, mingjun huang achillion pharmaceuticals, new haven, ct 06511, usa hcv nonstructural protein ns5b is the catalytic subunit of the replication complexes, possessing a motif characteristic of rna-dependent rna polymerases. biochemical assays using recombinant ns5b have been used to investigate ns5b nonnucleoside inhibitors. however, the inhibitory effect of compounds often varies with the forms of recombinant ns5b and the concentrations of the template and/or primer used in the assays. in addition, it does not always correlate to that obtained with replicon-containing cells. these observations have cast concerns about the validity of these cell-free assays. in the report, we explored replication complexes, isolated as crude membrane fractions from replicon-containing cells, for their competency to synthesize viral rna in vitro as well as their responsiveness to ns5b inhibitors. after optimizing the experimental conditions, two species of nascent viral rna, one double-stranded and the other single-stranded, were readily detected. the addition of ns5b nucleotide inhibitor blocked synthesis of both species. the presence of nonnucleoside inhibitors, however, inhibited mostly single-stranded rna (ssrna) synthesis. in addition, the replication complexes isolated from the cells containing a replicon that carried a resistant mutation in ns5b to the nonnucleoside inhibitor were able to synthesize the same amount of ssrna in vitro regardless of the presence or absence of the inhibitor, demonstrating that the phenomenon is due to the specific inhibitory effect of the compound on ns5b. combining with kinetic studies that ssrna synthesis was inhibited only when the nonnucleoside inhibitor was present during the pulse period, we conclude that ssrna synthesis catalyzed by the replication complexes in vitro is likely derived from the de novo initiation. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, and little or low toxicity to the host cells. in this part ii of the presentation on this subject, we report our preliminary results on mechanistic studies of anti-hcv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues in the series. in light of the fact that hcv is a major co-infection in patients infected with hiv, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. nigel bourne, ronald veselenak, richard pyles, minkyung yi, stanley lemon the university of texas medical branch, galveston, tx, usa more than 170 million people worldwide are estimated to be infected with hepatitis c virus (hcv). in the majority of these people a chronic infection is established which can result in serious long-term liver damage including progressive fibrosis, cirrhosis and hepatocellular carcinoma. in fact, hcv is believed to cause more than 100,000 cases of liver cancer annually worldwide and accounts for at least 40% of liver transplants in the us. current treatment options are limited and there is a high treatment failure rate. thus, there is a real need for new treatment options. amantadine has been evaluated as a treatment for chronic hcv infection in a number of clinical studies both as a monotherapy and in combination with other therapeutics. however, the results of these trials have been contradictory and at this time the clinical potential of amantadine as a therapy for chronic hcv infection remains unclear. recent studies have shown that the small hydrophobic hcv p7 protein forms an amantadine sensitive ion channel providing a possible basis for antiviral activity. we examined the ability of amantadine to reduce hcv replication in both subgenomic and full-length hcv replicons of genotypes 1a strain h77c and genotype 1b strain n. in these studies amantadine failed to reduce viral rna replication in any of the replicons tested. further, in infectious virus assays using hcv genotype 2a strain jhf-1 10 um amantadine failed to reduce viral rna levels under any of the conditions tested. however, in these infectious virus studies, when the viral inoculum was treated with amantadine prior to infection of cell monolayers, or when the amantadine was added to cells 1 h after virus adsorption there was a significant reduction in the number of infectious viral foci observed after 72 h incubation (p < 0.05 and <0.01, respectively). these results suggest that even in the absence of a direct impact on rna replication amantadine has antiviral activity. we are currently evaluating amantadine for activity in infectious hcv genotype 1a assays to further define its antiviral spectrum of activity. studies to more fully define its mechanism of action in the virus life cycle are also underway. dominique dugourd, raymond siu, jeremy fenn migenix inc., vancouver, bc, canada celgosivir is an alpha glucosidase inhibitor that is being developed for the treatment of hepatitis c virus (hcv) infections in humans. the purpose of this study was to evaluate the in vitro antiviral activity of celgosivir and its primary active metabolite, castanospermine, when combined with current approved therapies (ribavirin, interferon ␣-2b, or both) in a surrogate model of hcv (bovine viral diarrhea virus (bvdv)). compounds alone or in combination were tested against bvdv in infected madin-darby bovine kidney (mdbk) cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). the celgosivirinterferon ␣2b combination was significantly more synergistic than the celgosivir-ribavirin combination (∼3-fold), or the ribavirin-interferon ␣2b combination (∼2-fold). similarly, the castanospermine-interferon ␣2b double combination was more synergistic than the castanospermine-ribavirin combination (∼5-fold), or the ribavirin-interferon ␣2b combination (∼3.3-fold). the combinations of celgosivir-interferon ␣2b or castanospermine-interferon ␣2b led to significant decreases in the ec50s of celgosivir (up to >20-fold) and castanospermine (up to >50-fold). the effective ec50s of celgosivir or castanospermine were further reduced by the addition of ribavirin. the cytotoxicity of the double and triple combinations was additive or less than additive, indicating that combinations of celgosivir or castanospermine with ribavirin and/or interferon ␣2b were generally less toxic than expected. these results indicate that the combination of celgosivir with interferon ␣2b or with interferon ␣2b and ribavirin may be effective in the treatment of hcv. pegylated interferon ␣ plus ribavirin is the current standard of care for the treatment of chronic hepatitis c virus (hcv) infections. this regimen results in sustained virologic response in only about 50% of patients and is associated with significant treatment-associated toxicities. a number of approaches are being used to identify novel therapeutic combinations with better tolerability and/or efficacy. inhibitors of endoplasmic reticulum (er) ␣-glucosidase have been shown to inhibit viral replication and secretion and may have utility as part of new multi-drug treatment cocktails. the ␣-glucosidase inhibitor celgosivir is currently being evaluated in combination with pegylated interferon ␣ and ribavirin in humans. the purpose of this study was to evaluate the antiviral effects of combinations of celgosivir and castanospermine, the primary active metabolite of celgosivir, with other antiviral agents having diverse mechanisms of action. the effect of the combination of celgosivir or castanospermine with the nucleoside analogue nm-107, amantadine, and another iminosugar, n-butyl-deoxynojirimycin (nb-dnj) was determined in a cytopathic assay using the hcv surrogate virus bovine viral diarrhea virus in madin darby bovine kidney cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). volumes of synergy indicated that the castanospermine and nb-dnj combination was additive, while the celgosivir and nb-dnj combination was synergistic at high nb-dnj concentrations (>100 m). celgosivir and castanospermine were synergistic with both amantadine and nm-107, with volumes of synergy between 60 and 150 m%. isobologram analysis confirmed these synergistic interactions. these results indicate that celgosivir could be considered in combination regimens containing drugs that directly target viral replication like nm-107. mechanism(s) of synergy are under investigation. department of biotechnology, yonsei university, seoul 120-749, korea hepatitis c virus (hcv) is an enveloped virus with positivestranded rna genome of approximately 9.6 kilobases and a major cause of non-a and non-b hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. combination of interferon-␣ (ifn-␣) and ribavirin is the current standard therapy for the treatment of hcv infection, but there is no specific antiviral therapy available. the hcv viral genome encodes a single polyprotein of approximately 3010 amino acids, which is proteolytically processed by a combination of host and viral proteases into at least 10 distinct structural and nonstructural proteins. the structural proteins include c, e1, e2, and p7 and the nonstructural (ns) proteins include ns2, ns3, ns4a, ns4b, ns5a, and ns5b. as new hcv specific therapies, small-molecule inhibitors against hcv enzymes including ns5b protein, the viral rna-dependent rna polymerase (rdrp), and ns3 protease are in clinical tests. however, rapid emerging of drugresistant mutants has been hampering their practical clinical applications. recently, we have shown that phosphorylation of hcv rna polymerase by protein kinase c-like 2 (prk2) regulates virus rna replication. hcv rna replication was inhibited when prk2 expression level was down-regulated by using a prk2-specific sirna. in this study, we investigated the anti-hcv effect of prk2 inhibitors in an hcv subgenomic replicon system. treatment of the replicon cells with prk2 inhibitors suppressing the endogenous prk2 activity inhibited the phosphorylation of hcv rna polymerase and resulted in suppression of hcv rna replication in a dose-dependent manner. furthermore, the prk2 inhibitor in combination with ifn-␣ more effectively inhibited hcv rna replication than ifn-␣ alone. because the prk2 inhibitor did not show cytotoxicity in the cell-based drug inhibition studies and cellular proteins rarely get mutated, prk2 can serve as a cellular target for therapeutic intervention of hcv replication. specific inactivation of prk2 activity will provide an opportunity to interfere with hcv rna replication. haitao guo 1 , tianlun zhou 2 , ju-tao guo 1 , andrea cuconati 3 , anand mehta 1 , timothy block 1 1 drexel university college of medicine, doylestown, pa, usa; 2 nucleonic inc., irvine, ca, usa; 3 hepatitis b foundation, doylestown, pa, usa more than 400 million people worldwide are chronically infected with hepatitis b virus (hbv). the major complication of chronic hepatitis b is the development of primary hepatocellular carcinoma (hcc), which causes an estimated 500,000 deaths annually. currently clinical treatments (␣-interferon and nucleoside analogs) of chronic hepatitis b rarely cure the virus infection. this is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) dna from the nuclei of infected hepatocytes. hbv cccdna is essential to the virus life cycle by serving as the template for the transcription of the pregenomic rna and of the subviral rna species. its elimination during chronic infection is considered critical to long-term therapy. however, cccdna has not previously been targeted in high throughput screens of small molecule libraries. to screen compound libraries for antiviral drugs targeting cccdna, we set out to develop a cell-based assay suitable for high throughput screening. since cccdna is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccdna level. we predicted that the secretion of hbv e antigen (hbeag) by hepad38 cells, a hepg2-derived tetracycline inducible hbv expression cell line, would be cccdna-dependent. this is because a large portion of pre-core mrna leader sequence in the 5 terminus of integrated viral genome was deleted, preventing hbeag expression from transgene, but could be restored from the 3 terminal redundancy of pre-genomic rna during viral dna replication and subsequent cccdna formation. our experimental results showed that following induction, hepad38 produced and accumulated cccdna, which became detectable between 7 and 8 days. hbeag synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccdna detected. therefore, the secretion of hbeag by hepad38 cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting hbv cccdna. kathy aldern, james beadle, karl hostetler university of california, san diego and the veterans medical research foundation, san diego, ca, usa (s)-hpmpa is a broad spectrum antiviral active against orthopoxviruses, hbv, cmv, hsv, and other herpes group viruses. we have shown that hdp-(s)-hpmpa has greatly enhanced antiviral activity against these viruses. in addition, while hpmpa itself is nearly inactive against hiv, we showed that hdp-(s)-hpmpa exhibited an ec50 >3 logs less than unmodified hpmpa in mt-2 cells by p24 reduction assay. to evaluate the metabolic basis for the increased antiviral activity, we studied and compared the cellular uptake of radiolabeled cdv, (s)-hpmpa and their hdp-esters and conversion to hpmpa-diphosphate (hpmpapp) and cdv-diphosphate (cdvpp) in mrc-5 human lung fibroblasts using hplc partisil sax ion exchange chromatography. cellular uptake of hdp-cdv and hdp-(s)-hpmpa was similar. however, when cells were exposed to the respective drugs for 6, 24 and 48 h, hpmpapp appeared much earlier than cdvpp and reached levels several fold greater than observed with hdp-cdv. drug wash out experiments were carried out in mrc-5 cells exposed to radiolabeled hdp-cdv and hdp-(s)-hpmpa. after 24 h, the culture medium was removed and replaced with complete medium without drug and the levels of hpmpapp and cdvpp were measured by hplc every 2 days for 0-10 days. levels of the diphosphates declined slowly with a t 1/2 of 5-10 days. in conclusion, hdp-(s)-hpmpa is converted to its diphosphate more rapidly than hdp-cdv and reaches higher intracellular levels. this may explain, in part, its greater antiviral activity. the antiviral activity and oral bioavailability of cidofovir (cdv) is enhanced when the phosphonate is esterified with various straight chain alkoxyalkyl groups. the length of this chain is an important determinant of antiviral activity and selectivity. however, in some cases, rapid metabolism to an inactive short chain metabolite was observed. to enhance the metabolic stability of these esters, we synthesized cidofovir alkoxyalkyl esters bearing methyl groups on the penultimate carbon of the alkyl chain. enzymatic stability of 15-me-hdp-cdv (1) was tested in liver s9 fractions from various species. in mouse and human liver s9 fractions, compound 1 was completely stable for 90 min while 15-20% of the straight chain hdp-cdv was metabolized. the branched alkoxyalkyl esters were then evaluated in cells infected with vaccinia, cowpox and ectromelia viruses. the branched methyl analogs were substantially more active than cdv and equal to or slightly more active than the straight chain analogs. compound 1 retained full activity compared to hdp-cdv and compound 2 showed greater activity against orthopoxviruses compared to its unbranched analog. we believe that the structural modification of the alkyl chain slows the formation of inactive metabolites, possibly by interfering with oxidation and may result in better pharmacokinetics and more potent antiviral activity against orthopoxvirus infection in vivo. cidofovir ([1-(s)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, hpmpc) is a broad spectrum antiviral agent clinically used for treatment of aids-related cmv retinitis. cidofovir has limited oral bioavailability (<5%), attributed to ionization of its phosphonic diacid moiety under physiological conditions. we have shown that masking of this group by conjugation of the cyclic form of the drug (chpmpc) via a ser side chain p-o ester linkage with x-ser dipeptides, where x = a hydrophobic amino acid, can result in prodrugs 1 that afford significantly improved biological availability of parent drug in an animal model. here we describe the total synthesis of novel cyclic cidofovir prodrugs 2ab of l-val and l-phe using an alternative conjugation strategy, viz. via an ethylene glycol link utilizing p-o and c-o ester bonds. the preparation of the hpmpc synthon from r-glycidol used our modification of the literature procedure (brodfuehrer et al., 1994) , involving reaction of tritylated (r)-glycidol directly with unprotected cytosine to achieve regiospecific opening of the epoxide ring, followed by reaction with benzoic acid anhydride to obtain the desired n-benzoyl intermediate needed to continue the synthesis. pybop was used as condensing agent in a convenient, one-pot conversion of hpmpc to chpmpc and subsequent esterfication of the latter by the ethylene glycol-modified amino acids. the prodrugs 2 were converted to drug by cellular (caco-2, hff) and tissue (liver and intestinal) homogenates, but did not show enhanced oral bioavailability when evaluated in a rat model, suggesting that such compounds may be useful for understanding the effectiveness of 1 in drug delivery. cidofovir (cdv) is a broad-spectrum anti-viral agent that is used to treat aids-related cytomegalovirus (cmv) retinitis and other cmv infections. cdv has good in vitro activity against orthopox viruses, including smallpox; however, its use is limited because of the drug's low oral bioavailability and poor transport into cells. in order to improve its oral bioavailability, our group has synthesized a series of dipeptide and amino acid prodrugs of the cyclic analog of cidofovir (ccdv). in the current project, we examined the cytotoxicity and antiviral activity of the prodrugs, showing that the compounds are not cytotoxic and have diverse activity against hcmv and orthopox viruses (vaccinia and cow pox) with 50% inhibitory concentrations ranging from 0.1 to 0.5 and 10 m and greater, respectively for the two virus types. in vitro and in situ perfusion studies established that the permeability of the prodrugs is enhanced more than 30-fold and that the transport is mediated, at least in part, by the intestinal dipeptide transporter. we also have found that the bioavailability of the prodrugs is dependent upon the prodrug structure and that we can achieve up to an eight-fold increase in bioavailability over the parent compound in vivo. drug stability experiments showed that in gastrointestinal and liver homogenates, the ccdv prodrugs are enzymatically hydrolyzed to the parent compound. it is clear from this work that the biologically benign dipeptide moiety, strategically linked to the drug to mask its anionic properties, significantly enhances intestinal transport of ccdv, creating the possibility of an orally bioavailable form of ccdv with low toxicity. acknowledgement: supported by funds from tsrl inc, the university of michigan, and nih grants r43ai056864 and u01ai061457. lawrence trost 1 , bernhard lampert 1 , lloyd frick 2 , merrick almond 1 , george painter 1 1 chimerix, inc., durham, nc, usa; 2 dmpk advisor, chimerix, inc., durham, nc, usa foscarnet, a pyrophosphate analog approved for the treatment of cmv retinitis and acyclovir-resistant herpes infections in immunocompromised patients, is active against highly drug resistant strains of hiv-1. however, the clinical utility of foscarnet is limited because it requires controlled intravenous infusion and is associated with high risks of renal impairment and seizure caused by alterations in plasma minerals and electrolytes. lipid conjugation has been shown to increase the in vitro activity, improve oral bioavailability, and reduce the toxicity of several antiviral drugs requiring intravenous administration because of poor bioavailability. in the case of foscarnet, conjugation to methylbatyl alcohol (cmx012) decreases the apparent ec 50 value against hiv-1 by up to 40-fold. cmx012 was esterified to produce cmx052 in order to increase solubility and to protect against decarboxylation of the foscarnet moiety during passage through the stomach. here we present the results of a preliminary toxicology and toxicokinetic study of the methylbatyl alcohol conjugate of foscarnet methyl ester (cmx052). rats were given oral doses of 10, 30 and 100 mg/kg cmx052 daily for 7 days. there were no clinical signs of toxicity. body weight and food consumption were comparable to controls and serum biochemistry, hematology, coagulation parameters and urinalysis were normal. there were no gross findings at necropsy, no effects on organ weights and no findings by histopathological examination of a wide range of tissues. importantly, there were no changes in serum biochemistry parameters or histopathological examination that were indicative of the renal impairment or serum electrolyte changes that are associated with foscarnet. oral dosing resulted in significant plasma exposure to cmx012 (c max > 4 g/ml), the biologically active deesterified form of cmx052. in conclusion, cmx052 is absorbed after oral administration, converted to cmx012, and has a good preliminary toxicity profile. these results support the development of cmx052 for the treatment of drug resistant hiv infection. zhiqian wu 1 , julie breitenbach 2 , ulrika erickson 1 , john hilfinger 3 , john drach 2 , gordon amidon 1 1 department of pharmaceutical sciences, college of pharmacy, the university of michigan, ann arbor, mi, usa; 2 school of dentistry, the university of michigan, ann arbor, mi, usa; 3 tsrl, inc., ann arbor, mi, usa vaccinia virus is a surrogate model system for study of pox virology and development of antiviral therapeutics. the potent anti vaccinia virus activity and various shortcomings of vidarabine make it a good candidate for improvement by utilizing prodrug strategy. vidarabine is a polar nucleoside drug with low membrane permeability and rapid degradation by adonesine deaminase. 5 -monoester prodrugs of vidarabine with various amino acids promoieties (l-valine, l-isoleucine, l-phenylalanine. laspartic acid, l-proline) are synthesized and evaluated for their stability, permeability and activity against vaccinia virus. prodrugs exhibit different hydrolysis rate in caco-2 cell homogenate (t1/2: 2-40 min). 5 -l-isoleucyl and 5 -l-valyl monoester prodrugs exhibit comparable bio-conversion rate and hpept1mediated uptake as well as caco-2 permeability with valacyclovir, a commercially marketed oral amino acid ester prodrug. both prodrugs have potent activity against vaccinia virus and are resistant to ada1. preliminary animal study shows 5 -lisoleucyl vidarabine results in >10-fold increase in circulating vidarabine level. the results suggest that it may be possible to use amino acids prodrug strategy to improve vidarabine as anti vaccinia virus agent. vidarabine [9-␤-d-arabinofuranosyl)adenine or ara-a) was originally investigated as an anti-tumor agent and was later found to be active against herpes simplex virus (hsv) types 1 and 2. it was the first fda-approved drug for treatment of systemic hsv infections. although replaced by acyclovir and analogs for most applications, vidarabine remains an alternative therapy for acyclovir-resistant hsv and varicella-zoster virus infections. despite its proven efficacy, vidarabine suffers some limitations including: (i) metabolism by adenosine deaminase (ada) to its inactive hypoxanthine homolog (ara-h); (ii) low lipophilicity and membrane permeability and (iii) poor aqueous solubility, thus limiting options for parenteral and peroral delivery. our recent interest in vidarabine was triggered by our discovery that it was ∼5-fold more active against vaccinia (vv) and cow pox (cpv) viruses than was cidofovir in plaque reduction assays. its activity was enhanced about 10-fold by combination with 1 m 2 -deoxycoformycin (pentostatin, a potent inhibitor of ada) thereby providing significant superiority to cidofovir. from these results and our earlier studies on 5 -substituted vidarabine analogs (lipper et al., 1978. mol. pharmacol. 14, 366-369), we determined that minimizing metabolism of vidarabine by synthesizing 5 -amino acid substituted prodrugs gave a significantly more potent anti-pox virus agent. we found that amino acid ester prodrugs of vidarabine are active against vv at non-cytotoxic concentrations. further, using cell homogenates, purified enzyme and intact cell systems, we showed that the prodrugs are resistant to inactivation by ada. the prodrugs also had enhanced transport potential, most likely targeting the intestinal dipeptide transporter. finally, oral delivery of the prodrug to the small intestine resulted in a 10-fold increase in vidarabine plasma levels when compared to unsubstituted vidarabine. these properties make the prodrugs of vidarabine good candidates as orally bioavailable anti-pox virus agents that are stable in the presence of ada. acknowledgement: supported by funds from tsrl inc. and the university of michigan. ulf goerbig 1 , anne baum 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katolieke universiteit leuven, leuven, belgium the cyclosal pronucleotide system has been designed for an intracellular delivery of therapeutically active nucleoside monophosphates. as part of recent work on the cyclosal approach, the interaction of cyclosal nucleotides with cholinesterases has been investigated. it is known that organo-phosphates may act as irreversible inhibitors of cholinesterases (suicide mechanism). in the case of cyclosal nucleotides, cholinesterase inhibition could lead to unwanted side effects in a possible therapeutic application. there are two types of cholinesterase found in humans, the highly specific, physiologically important acetylcholinesterase (ache) and the much more unspecific butyrylcholinesterase (bche) of unknown physiological importance. fortunately, no inhibition of ache was observed for a variety of different cyclosal nucleotides. in contrast, bche inhibition was found in some cases. the anti-hiv-active 3,5-bis-tertbutyl-6-fluoro-cyclosal-d4t monophosphate is the first cyclosal derivative combining three desired properties: successful intracellular delivery of nucleotides, sufficient hydrolytic stability and strongly reduced inhibitor activity towards bche. because of the promising properties of this compound, we combined this mask developed for d4t with the antiviral active nucleoside analogues like d4a, dda, azt and acyclovir. in this contribution we present the synthesis, hydrolysis stability, inhibition behaviour towards bche and anti-hiv data of these new compounds. henning jessen 1 , wolfgang fendrich 1 , tilmann schulz 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany, 2 rega institute for medicinal research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides are used for the delivery of antivirally active nucleotides into cells via a ph triggered selective hydrolysis. to distinguish between intra-and extra-cellular environment enzyme-cleavable side chains were introduced in the aromatic moiety of the pronucleotide to enrich the compound inside cells. this behavior will further be described as "lock-in"effect. many other different cyclosal pronucleotides have been designed, all showing different hydrolysis properties and antiviral data, both originating from the nature of the cyclosal-moiety as well as of the nucleoside. to examine these differences, analytical tools of high accuracy and sensitivity were needed, being structurally as close as possible to the lead compounds. these requirements are met by intrinsically fluorescent nucleosides coupled to different cyclosal masking groups. for analysis of the purine-type nucleosides iso-da with high intrinsic fluorescence properties was chosen and converted into iso-a, iso-dda and iso-d4a. for the pyrimidine-type series the fluorescent nucleoside m 5 k was synthesized as well as the dideoxy-compound dm 5 k. these nucleosides were transformed into different cyclosalpronucleotides and tested for their suitability for fluorescence analysis. in fact, an improvement of sensitivity by a factor of 5000 compared to uv-detection was found for some of the compounds (pmol detection). for all compounds fluorescence and absorbance spectra were recorded to determine the absorption and emission maxima. the new compounds lacked activity against hiv-1 and hiv-2 strains. however, the compounds showed low cytotoxicity, which is important for their usability as fluorescent probes in cells. due to the analytical sensitivity, a simple model uptake study could be carried out, employing an u-tube with two aqueous phases, which were separated by an unpolar organic solvent simulating a diffusion barrier. the properties of the aqueous phases were varied and an enzyme-driven enrichment of a "lock-in"-modified intrinsically fluorescent cyclosal-pronucleotide passing the diffusion barrier could be simulated. nicolas gisch 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides efficiently deliver therapeutically active nucleoside monophosphates in human cells. "lock-in"-cyclosal-pronucleotides -the so-called second generation of cyclosal-compounds -have been designed to trap the compound by intracellular cleavage of esterase-cleavable moiety. one disadvantage of the "lock-in"-compounds is their high chemical stability, which leads to a delayed drug delivery. therefore, conceptually different, enzymatically activated cyclosalpronucleotides have been developed. in this concept lipophilic donor substituents attached to the aromatic ring are converted into a polar acceptor substituent by intracellular enzymatic cleavage. as a consequence the liberated acceptor group leads to a strong decrease in hydrolysis stability and a rapid formation of a charged intermediate is the result. from the phosphodiester intermediate the nucleotide is released subsequently. the concept, synthesis, characterization and in vitro antiviral evaluation of the third generation of cyclosal-pronucleotides will be presented. tomas cihlar 1 , richard mackman 1 , adrian ray 1 , dean boojamra 1 , lijun zhang 1 , deborah grant 1 , hon hui 1 , jennifer vela 1 , neil parkin 2 , yolanda lie 2 , kirsten white 1 , michael miller 1 , gerry rhodes 1 , manoj desai 1 1 gilead sciences, foster city, ca, usa; 2 monogram biosciences, so. san francisco, ca, usa background: n(t)rtis are currently used as a backbone of antiretroviral combination therapy. however, their long-term benefit can be limited by adverse effects, resistance development, drug-drug interactions, and sub-optimal efficacy in treatment-experienced patients. therefore, we searched for novel nucleotide analogs with improved pharmacological profiles. methods: phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine (gs9148) was selected from a broad range of nucleoside phosphonate analogs. phosphoramidate prodrug technology previously explored with tenofovir was applied to gs9148, resulting in the identification of gs9131 (ethylalaninyl phenyl ester of gs9148). results: gs9131 exhibits potent anti-hiv-1 activity in primary lymphocytes and t-cell lines (ec 50 < 150 nm). low cytotoxicity (cc 50 > 100 m) was observed in multiple cell types including renal cells. diphosphate metabolite of gs9148 was shown to act as an obligatory dna chain terminator and a competitive inhibitor of hiv-1 reverse transcriptase (rt) (k i = 0.8 m). unlike ddi, d4t, or d4fc, neither gs9148 nor its prodrugs inhibited mitochondrial dna replication in hepg2 cells. in a phenosense assay, gs9148 retained its full activity against hiv-1 variants with k65r, m184v or l74v mutations in rt. viruses with ≥4 thymidine analog mutations showed ≤2fold reduced susceptibility to gs9148, a shift that was smaller than that of any other tested nrti. following an oral dose of 3 mg/kg gs9131 in dogs, the bioavailability of prodrug exceeded 20%, resulting in high intracellular levels (9.0 ± 2.3 m) and prolonged retention (t 1/2 > 24 h) of gs9148 diphosphate in blood lymphocytes. conclusions: both gs9148 and its prodrug gs9131 exhibit favorable in vitro pharmacological profiles including less resistance due to rt mutations than approved nrtis. gs9131 possesses good in vivo pharmacokinetic properties and thus represents an attractive development candidate with potential for clinical efficacy in both treatment-naive and nrti-experienced patients. martin mcdermott, gabriel birkus, ruth wang, holly macarthur, xiaohong liu, nilima kutty, tomas cihlar, craig gibbs, swami swaminathan, arnold fridland, william lee gilead sciences, inc., foster city, ca, usa gs-7340 and gs-9131 are alkylalaninyl phenyl ester prodrugs of tenofovir (tfv; 9-[(2-phosphonomethoxy)propyl]adenine) and a novel nucleotide analog fd4ap (phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine), respectively. both gs-7340 and gs-9131 exhibit potent in vitro anti-hiv-1 activity, favorable resistance profile, and low cytotoxicity. compared to tenofovir disoproxil (viread), both prodrugs are significantly more stable in plasma and deliver >10-fold greater levels of active diphosphate metabolites into pbmcs in vitro and in vivo. the initial step in the intracellular activation of gs-7340 and gs-9131 is the hydrolysis of the alanine carboxyester by an unknown hydrolytic enzyme. the isolation and identification of this enzyme from human pbmcs is reported here. results: a major enzyme capable of cleaving gs-7340 and gs-9131 was purified from human pbmcs and was separable from esterases able to cleave alpha napthyl acetate (ana). the increase in specific activity of prodrug hydrolase achieved was 3500-fold. sds-page analysis showed the presence of a prominent protein band at 29 kda, which was identified by ingel tryptic digestion and ms/ms sequencing of the resultant peptides as lysosomal carboxypeptidase a (cathepsin a, ec 3.4.16.5, cata). the biochemical properties of purified prodrug hydrolase matched those of cata. recombinant cata and the isolated prodrug hydrolase displayed nearly identical susceptibility to hydrolase inhibitors and substrate preference against a panel of tenofovir phosphoramidate prodrugs. incubation of both enzymes with [ 14 c]gs-7340 and [ 3 h]difluorophosphonate resulted in the labeling of an identical 29 kda protein (catalytic subunit). both labeled bands reacted with polyclonal antibodies specific for human cathepsin a. finally, following incubation with gs-7340, approximately 6-9-fold lower intracellular concentrations of tfv metabolites were detected in fibroblasts from patients expressing non-functional cat a (cat a-cells) compared to normal control fibroblasts (cat a+ cells). center for drug delivery and nanomedicine, university of nebraska medical center, omaha, ne, usa nucleoside 5 -triphosphate (ntp) is the biologically active form of many antiviral nucleoside analogs capable of efficiently blocking the production of viral nucleic acids in infected cells. we describe novel microparticulate formulations for encapsulation of ntp, drug delivery and antiviral therapy of respiratory infections. polymer networks (poloxagels) consisted of crosslinked poloxamers and cationic polymer molecules were designed, synthesized and characterized by loading with ntp and interaction with cells. poloxagel-ntp formulations could be obtained by simple mixing of the aqueous solution of ntp with the aqueous dispersion of poloxagel and subsequent lyophilization. drug loading was equal up to 30% by weight. in this form, phosphates groups of ntp are complexed with amino groups of polycationic backbone of poloxagels, and the formulations could be stored at room temperature for many months without degradation of ntp. the particle size of aqueous poloxagel-ntp dispersions was low, with a hydrodynamic diameter of 0.1-0.2 m. the rate of passive drug release in physiological solution was from 33 to 50% of loaded drug during the 24-h period. these formulations were effectively consumed by many types of cells. significant amounts of drug and poloxagels were detected in the cellular interior after only 1-2 h of incubation. in the presence of cellular membranes drug release from poloxagel-ntp formulations was dramatically increased. we attribute this effect to the triggered release of the bound ntp as a result of competitive interaction of polycationic backbone of poloxagels with phospholipids of cellular membranes. mucoadhesive properties of poloxamers may additionally enhance binding of poloxagels with airways/lung epithelium. 5 -triphosphate of 1-␤-d-ribofuranosyl-1h-1,2,4-triazole 3-carboxamide (ribavirin) was synthesized using phosphorylation with a tris(imidazolyl) phosphate in a convenient one-pot approach. formulations of different poloxagels with the ribavirin 5 -triphosphate were prepared and characterized as prospective antiviral formulations for prophylactic and therapeutic treatments of respiratory infections including influenza a virus. aerosolic route of application of these antiviral formulations and associated problems are discussed. edwin gong 1 , ebrima gibbs 2 , joel oger 2 1 department of pharmacology and therapeutics, the university of british columbia, vancouver, bc, canada; 2 neuroimmunology lab, ubc hospital, department of medicine, the university of british columbia, vancouver, bc, canada ifn-alpha and ifn-beta are currently employed in the treatment of many viral diseases, especially chronic hepatitis. ifn-beta is also employed for the treatment of multiple sclerosis (ms), a chronic and often debilitating disease of the central nervous system. however, as with other protein therapeutics, long-term ifn therapy can lead to the development of binding and neutralizing antibodies to ifns and thus lead to deceased clinical effect of ifns. in order to measure the bioavailability of ifns and the level of neutralizing antibody, we have developed a realtime rt-pcr (taqman) assay by quantitating the expression of mxa (an ifn-induced protein) mrna. the nucleotide sequences of mxa deposited in the genebank were aligned, and a pair of primers and the hybridization probe were designed based on the conserved regions. a house keeping gene, gapdh, was used as a calibrator for relative quantitation. the rna standards were generated by in vitro transcription from cloned mxa gene in a plasmid vector. the reaction parameters were optimized. the assay was validated using pbmcs of ms patients that were treated with ifn-beta. for evaluation of the ifn bioavailability, the total rna was extracted from pbmcs and quantitatively detected by one-step rt-pcr for both mxa and gapdh. the results calculated by the 2 (− ct) method showed that the difference (signal-to-noise ratio) between samples with neutralizing antibodies and samples from untreated ms patients or healthy donors were approximately 60-70-folds. this indicates that our assay is a reliable method for determination of ifn bioavailability. v. lozitsky 1 , i. kravchenko 2 , v. larionov 2 1 research anti-plague institute, odessa, ukraine; 2 national university, odessa, ukraine transdermal delivery (td) of drugs is a novel method for treatment of diseases. td is carried out by the help of transdermal therapeutic systems (tts), which are multilayer plasters that contain active ingredients. td have a number of advantages, such as: (1) prolongation of the drug's action; (2) drug's concentration is maintained in therapeutic range; (3) there is no trauma to patient's skin while using td; (4) removing tts from the skin immediately stops drug's entering to the organism; (5) first-pass effect in the liver is reduced; and (6) many highly active drugs are irritating the gastro-intestinal tract if administered orally, others have a short half-life time-these drugs do not have downsides mentioned above if used as tts. in our previous research we elaborated tts containing rimantadine (ttscr) and studied its efficacy during experimental influenza in mice. we had established that transdermal delivery of rimantadine is more effective than oral administration. the aim of this work was to increase the efficacy of ttscr. to solve this task we studied the influence of some permeability enhancers on anti-influenza efficacy of ttscr during experimental infection. applied tts had adhesion hydrogel matrix (polyvinyl alcohol and 1.2-propylenglycol). they consisted of a base and a plastificator, which improves the administration of active substances through the skin and does not induce irritation. ttscr (1 mg/mouse) were applied on shaven backs of experimental animals. tts for other groups additionally contained one of such permeability enhancers as: 10 mg/mouse of dmso or 10 mg/mouse of octanol or 1 mg/mouse of papain. tts were applied on shaven backs of mice and stayed there from 1 day before infection to 10th day after challenge. mice of all groups were infected intranasally with influenza virus a/pr/8/34 (h1n1), which is highly pathogenic for them. challenge was carried out using four animals for each virus dilution within the range of 10 −1 to 10 −7 . deaths of animals were recorded for 14 days. the results showed that proteolytic enzyme papain increased the anti-influenza efficacy of ttscr on 1 log 10 tid 50 . dmso and octanol did not demonstrate such activity. mikhail dobrikov 1 , serguei vinogradov 2 , barbara ramsay shaw 1 1 department of chemistry, duke university, durham, nc, usa; 2 center of drug delivery and nanomedicine, and college of pharmacy, university of nebraska, omaha, ne, usa nucleoside reverse transcriptase inhibitors (nrti) are widely used in the antiviral chemotherapy. most nrtis require stepwise phosphorylation to the respective nucleoside triphosphates, which inhibit the viral dna synthesis. however, the emergence of hiv-1 reverse transcriptase-dependent drug resistance limits the effectiveness of treatment by nrtis. the ␣-p-borano-nucleotide analogues show several unique physico-chemical and biological properties: (i) enzymatic studies indicate that the rp-isomer of ␣-p-borano-2 ,3 -ddndps is a better substrate for cellular ndp kinase than the parent ddndp; (ii) neither isomer of the ␣-p-borano-ddndps is a substrate for mammalian pyruvate kinase and shows very poor inhibitory properties to this enzyme; (iii) the rp-(␣-p-borano)-ddntp isomers are better inhibitors of drug-and multidrug-resistant viral reverse transcriptases and are poor substrates for dnadependent dna polymerises; and (iv) after incorporation into viral dna the borano-ddnmp residues are more resistant to atp-dependent removal from viral dna than parent ddntps. to by-pass inefficient phosphorylation of the nrtis, several prodrugs of ␣-p-borano-nucleotide analogues have been previously synthesized. a more efficient delivery system for ␣-p-boranonucleotide analogues based on nanosized cationic polymeric gel (nanogel) is proposed. selective inhibition of drug-and multi-drug resistant viral rts, poorer inhibition of intracellular kinases and dna polymerases by the ␣-p-borano-nucleotide analogues, and their specific delivery into infected cells in the complex with nanogel particles suggest a new approach to the design of more powerful antiviral drugs. acknowledgement: this work was supported by the nih grant r01 al52061 to b.r.s. we have developed a high-throughput, cell-based assay to address the critical need for antiviral drugs for the treatment of influenza. in consideration of the demand to screen high volumes of compounds, we targeted the development of a 384 microtiter plate format for the assay. in this assay, the inhibition of the influenza-induced cytopathic effect (cpe) in mdck cells was assessed using the celltiter-glo luminescent cell viability assay by promega. this reagent measures the amount of atp present in cells, which is directly proportional to the number of metabolically active cells. validation studies were executed to establish optimal cell density, viral concentration, dmso tolerance for compound dilution, incubation time for virus-induced cpe and effective control drug concentration. additional parameters, such as assay variability, reagent and read stability, edge effects, and ic50 stability were also investigated during validation. we are currently initiating use of the assay to screen chemical libraries and will report our findings from library screens in addition to the aforementioned validation. we believe the approach will also provide a mechanism for discovery of new antiviral leads for influenza as well as avian flu. fundacio irsicaixa, hospital universitari germans trias i pujol, 08916 badalona, spain we have developed bacteriophage lambda based genetic screen that can be used to isolate and characterize site-specific proteases. this genetic screen system is based on the bacteriophage lambda ci-cro regulatory circuit, in which the encoded repressor ci is specifically cleaved to initiate the lysogenic-to-lytic switch. we have adapted this simple, safe and rapid genetic screening system to predict the activities and phenotypes of human immunodeficiency virus type 1 (hiv-1) proteases in the course of viral infection and antiretroviral therapy. a specific target for the hiv-1 protease, p17-p24, was inserted into the lambda phage ci repressor. the target specificity of the ci-hiv repressor was evaluated by coexpression of this repressor with an hiv-1 protease construct. upon infection of escherichia coli cells expressing the two constructs encoding the ci-hiv-1 repressor and hiv-1 protease, lambda phage replicated up to 1000-fold more efficiently than in cells that did not express the hiv-1 protease. this assay responds appropriately to well-known hiv-1 proteases inhibitors and can be used to search for new proteases inhibitors. the high level of specificity of this system, in which modest differences in catalytic efficiency can be quantified, should be also useful for the characterization of different mutant viral proteases. we further demonstrated the broad applicability of this protease assay using other viral proteases and their cognate cleavage sites, including hepatitis c virus (hcv) ns3 protease and severe acute respiratory syndrome (sars) coronavirus (cov) (scov) 3c-like protease. compared with other protease assay methods, this assay has the following advantages: safe, highly sensitive, highly specific, easy quantification, and rapid generation of different protease cleavage substrates using molecular cloning and expression. this system may be useful for the development of a screening method to identify viral protease inhibitors and should be also useful to characterize cellular, viral, or other infectious agent proteases with different activities and specificities. karen m. watson, todd b. parsley, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa a virus transmission and rapid resistance selection assay has been developed in order to quickly evaluate the biological properties of anti-infective test compounds and rationally prioritize them for further development. the transmission assay specifically evaluates the ability of test agents to suppress and clear virus replication from cultures during the serial passage of virus in the presence of fixed concentrations of the test compounds alone or in combination. the growth and expansion of virus in the infected cultures has been shown to occur through the replication of originally infected cells in the absence of virus spread, through direct virus to cell infection, and through cell to cell transmission. in order to sterilize a culture a test compound must possess the ability to specifically and potently interfere with virus replication by each of these three methods and must be able to inhibit the replication of resistant viruses which pre-exist in the viral inoculum and which rapidly grow in the presence of the fixed concentrations of the test agent. twelve pyrimidinediones being evaluated for potential use as both anti-hiv therapeutic agents and topical microbicides were evaluated for their ability to inhibit virus transmission and to define their ability to rapidly select for resistant virus strains. these compounds were evaluated in parallel with known anti-hiv agents that inhibit virus entry (t20) and reverse transcription (sustiva, uc781 and azt). the results of the transmission assays suggest that significant biological differences exist between antiviral compounds and even between highly related congeners of the same class of pyrimidinediones, suggesting that the transmission and rapid resistant selection assay measures important antiviral properties of anti-hiv agents. biological studies that evaluate the mechanisms of virus growth in the presence of high concentrations of test compounds will be described. laure deflubé 1 , kerstin angner 1 , anna overby 1 , david stein 2 , patrick iversen 2 , ramon flick 1 1 utmb, department of pathology, galveston, tx, usa; 2 avi biopharma, inc., corvallis, or, usa in the family bunyaviridae, several members on the genus phlebovirus have been reported to cause disease in humans or livestock. among these, rift valley fever (rvf) virus is an important human/animal pathogen. its widespread geographic distribution and its ability to produce severe human disease makes this virus a worldwide public health concern. the phosphorodiamidate morpholino-oligomers (pmo) are a class of dna-like antisense agents typically synthesized to a length of about 20 subunits and contain purine or pyrimidine bases attached to a backbone composed of six member morpholine rings joined by phosphorodiamidate intersubunit linkages. they have been shown to be effective antiviral compounds for different virus families, e.g. coronaviridae and flaviviridae. pmo bind to rna preventing translation of the viral rnas. we used our recently developed plasmid-based minigenome rescue systems for uukuniemi (phlebovirus model virus) and rvf viruses to screen antiviral compounds based on the morpholino antisense oligonucleotide approach. for this the antiviral compounds were appraised on the basis of reporter gene activity (fig. 1b) . the inhibitory effects of the same compounds were also tested by measuring reduction in virus titer (fig. 1a) , by monitoring changes in viral antigen production using an indirect immunofluorescence procedure and facs analysis, and analysis of genome transcription/replication by rt-pcr. indeed several pmos could be identified with interfering effect at a low ic50 on bunyaviral minigenome rescue as well as virus proliferation. based on these results, we plan to confirm antiviral activity of the most promising compounds in suitable animal models. we have shown that the fractal approach to the problem of viruscell interaction gives the unique possibility to process the data through the sequence of the direct and inverse fourier transforms. the studies were carried on the herpes simplex virus us-1 interacting with the hep-2 sensitive cell culture. the object was imaged as system of bright peaks formed as a result of laser diffraction on the structural elements of the virus-cell system. the whole virus-cell interaction information is inserted into computer in a fastest parallel way. the laser intensity peaks, which form the speckle image of the system under consideration, could be transformed into the hierarchical system of the circles (or squares) according to the choice of the researcher, but conserving the same d value, which depends only on the true intermolecular interaction potential. this potential, being characteristic for every stage of virus-cell interaction, is responsible for the given structure of the dynamic virus-cell system and the unique, but the typical form of the fractal cluster corresponding both to the system itself and its image as well, was processed by computer techniques. the hierarchical fractal design of the virus-cell system, proposed here for the first time, gives the universality, needed for the quantitative description of any possible combination of the virus and corresponding sensitive cell. it should be noted, as well, that the fractal microscope use for viruscell dynamic system imaging have all the properties, required from all other experimental tools of monitoring, including the reliability, reproducibility and preciseness. this device could be used in drug design biological test stages with the scope of time and efforts economy during the compounds libraries screening. the fractal microscope combined with the qsar drug design technique makes the antiherpetic drug design more competitive as compared to the regular approaches. acknowledgement: the authors are indebted to the partial support of the stcu grant #3147. we have investigated experimentally the fractal properties of diffraction images obtained by laser irradiation of virus-cell system. it was shown that the diffraction process is mathematically equivalent to the direct fourier transform of the said system's components modeled with simple geometrical figures (e.g. circles). each viral family could be coded and described quantitatively with the average size of the free viral particle and the type of its symmetry. we propose here to use the inverse fourier transform of the virus-cell system in order to get the real enlarged image of the viruses attacking the sensitive cell as well as the cell's structural transformation caused by the sequent stages of virus reproduction process. the set of bright and dark spots, which forms the virus-cell system's diffraction image, could be coded into set of numbers (matrix form of correlation vector-function) using the quantification procedure. the correlation function was used as presented in polar coordinates because the system has the axial symmetry (laser beam taken as main physical axis). the full information included into the image peaks' diameters and color index is transformed using inverse fourier technique into set of intersecting bright and dark circles. the full in vitro dynamics of the structural changes of the virus-cell system are described by the changes of circles' diameters and the area of their intersection. it was shown, also, that the magnification of the fractal microscope could achieve 10,000× to 100,000×, depending on the laser power used. proposed fractal microscope could be applied as well in vivo experiments until the required magnification will not make us to use projection laser with the output exceeding 25 mw. we have shown that the fractal microscope based on the inverse fourier transform could be applied successfully in pharmaceutical antiviral drug design, laboratory and clinical trials of new antiviral preparations, especially effectively in hierarchic qsar research. acknowledgement: authors are grateful to the support under the stcu grant #3147. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for palkylphenyl substituted analogues . these compounds however are highly lipophilic and poorly soluble in water. we then reported a series of p-alkyloxyphenyl compounds containing a phenolic ether aiming to enhance water solubility whilst retaining antiviral activity (mcguigan et al., 2002) . we will now report the synthesis, characterisation and antiviral evaluation of a novel series of p-alkyloxyphenyls where there is at least one methylene spacer between the phenyl and ether group to potentially boost the pharmacokinetic profile. the alkyl chain length was fixed to retain a high clogp value, a parameter that has previously been shown to correlate with high antiviral potency . the target structures were prepared by the pd-catalysed coupling of a series of para-substituted alkoxyphenyl acetylenes with 5-iodo-2 -deoxyuridine, to give intermediate 5-alkynyl nucleosides, which were subsequently cyclised in the presence of cui to give the desired bicyclic systems. the antiviral activity, cytotoxicity, and solubility of these compounds are to be reported. we have previously reported on some novel nucleoside analogues containing and unusual furano bicyclic pyrimidine base and long side chain , which were discovered to be both potent exquisitely and selective towards the varicella zoster virus. following this discovery, three main sites for modification were identified and explored: (1) the side chain; (2) the bicyclic base; and (3) the sugar moiety. modification to the side chain by insertion of a phenyl ring, led to the most potent anti-vzv nucleoside to date (ec50 1 nm) . the investigation into modifications at the three sites stated above has continued and we herein report further adjustments to these analogues. replacement of the furo oxygen with sulfur on the parent nucleosides bearing an alkyl side chain has been reported to retain antiviral activity. however, those bearing a phenyl alkyl side chain are here shown to give a slight reduction in anti-vzv activity. modifications to the phenyl ring of the side chain have included halogen substitutions, and the fluorine in particular has produced some intriguing results in that, while the ortho and meta substitutions show some anti-vzv activity, the para analogue is completely devoid of antiviral activity. we now report further studies which include the di and tri substituted phenyl analogues. finally, we have also investigated sugar modification that has included substitutions of the 3 hydroxyl group. previous modifications which have replacements of the hydroxyl groups, resulted in loss of activity against vzv . we now present some new 3 substituted analogues which have provided interesting biological results. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) with subnanomolar activity for palkylphenyl substituted analogues srinivasan et al., 2001) . the sar is now further explored via the substitution of phenyl derivatives with electron withdrawing and electron donating groups. we now report the synthesis, characterisation, and biological evaluation of a novel series of mono substituted phenyl derivatives in order to probe the structure activity relationships in this region. the target compounds were synthesised under sonogashira conditions where a series of substituted phenyl acetylenes were coupled with 5-iodo-2 -deoxyuridine, to give intermediate 5alkynyl nucleosides that were subsequently cyclised in the presence of cui to give the desired bicyclic systems. diseases caused by herpes simplex virus (hsv) are widely distributed. prophylaxis and treatment of these infections are important health care tasks that require also the search, design and development of new antiherpetic drugs overcome drug resistance and toxic side effects of existing drugs. drug selection simply based on results of empirical screening is not very effective. computer-based technologies may help to optimize the structure of antiviral compounds as well as to design and develop new drugs. the objective of the present work is the quantitative structureactivity relationship (qsar) analysis of antiviral activity of various n,n -(bis-5-nitropyrimidyl)dispirotripiperazines in connection with consequent drug design. the well-established simplex representation of molecular structure (sirms) qsar approach has been used to fulfill this objective. it allows the molecular design of new effective antiviral drugs. thorough investigation of the relationship between: (a) cytotoxic (hela cells and gmk cells, cc 50 , g/ml); (b) antiherpetic activity (hsv-1 strain kupka, ic 50 , g/ml); and (c) selectivity index (ratio of cc 50 to ic 50 ) and the structure of 48 n,n -bis-5-nitropyrimidyl derivatives of dispirotripiperazine have been conducted. statistic characteristics for pls (partial least squares) models are quite satisfactory (r 2 = 0.816-0.991, q 2 = 0.637-0.868). the results are confirmed by experimental data. based on the obtained models, molecular fragments that promote and interfere with antiviral activity were defined. additionally, these models provide the possibility to predict molecular fragments that will enhance antiherpetic activity and to design new well tolerated highly virus-specific drugs. in summary, the developed simplex approach is an effective instrument for prediction and design of novel effective antiherpetic agents. several representatives of a series of 5-arylethynyl-2deoxyuridines (1a) bearing bulky aryl groups were recently shown to possess unexpected activity towards hsv-1. unlike common anti-hsv drugs, these compounds retain activity towards kinase-deficient acyclovir-resistant strains. therefore, an unusual mechanism of antiviral action is assumed. in order to investigate the mechanism and to discover more potent analogues we synthesized several novel 2 -deoxy (1a) and 2 -arabino (1b) uridine derivatives possessing different 5-arylethynyl substituents. dinucleosides 2 containing two uridine moieties coupled to a single polycyclic aromatic hydrocarbon (e.g. pyrene) represent another type of structural variation of nucleosides 1a. these compounds as well as some of 1a and 1b possess bright fluorescence that can be used in biological evaluations. cytomegalovirus (cmv) is a wide spread opportunistic pathogen which belongs to the beta subfamily of the herpesviridae. primary infection is generally asymptomatic resulting in life long latency. however, morbidity and mortality rates post-transplantation are greatly increased following reactivation or recrudescence of cmv. ganciclovir (gcv) and cidofovir (cdv) have both been successful in suppressing cmv viral replication in immunocompromised patients. although sustained use of these drugs has resulted in the emergence of multi-drug resistant strains of virus. in this study we used plaque reduction assays to determine the antiviral efficacy of two ribonucleotide reductase inhibitors, didox (dx; 3,4dihydroxybenzohydroxamic acid) and trimidox (tx; 3,4,5trihydroxybenzamidoxime) in inhibiting both wild type and drug-resistant strains of murine cmv (smith strain). the results presented here demonstrate that both dx and tx inhibit viral plaque formation in a dose dependent manner in both wild type and the resistant strain. a 43-and 39-fold increase in drug dose was required for cdv and gcv respectfully to inhibit plaque formation by 50% in the resistant strain (cdv wt: 0.03 m, r: 1.28 m/gcv wt: 3.17 m, r: 122.85 m). this compared to only a moderate increase in drug dose required for dx and tx to achieve 50% inhibition in the resistant strain (dx wt: 20.71 m, r: 28.88 m/tx wt: 7.12 m, r: 11.59 m), corresponding to a 1.4-and 1.6-fold increase respectfully. further work is currently underway to determine the possible mechanism of antiviral actions and toxicity profiles of these novel virostatics. in patients with human immunodeficiency virus (hiv) infection, coinfection with herpesviruses continues to be a problem for patients receiving antiviral hiv therapy. since treatment can be affected by the large number of drugs required for multiple infections, it would be useful to have antivirals that are active against both hiv and the herpesviruses. we reported previously that alkoxyalkyl ester prodrugs of cidofovir (cdv) are several logs more active against herpesvirus replication than unmodified cdv. to determine if this strategy would be effective for other acyclic nucleoside phosphonates which are active against hiv infections, hexadecyloxypropyl (hdp) esters were synthesized from 1-(phosphonomethoxyethyl)-cytosine (pme-c), 1-(phosphonomethoxyethyl)-5-bromo-cytosine (pme-5brc), 1-(phosphonomethoxyethyl)-5-fluoro-cytosine (pme-5fc), 9-(phosphonomethoxyethyl)-2,4-diaminopurine (pme-dap) and 9-(phosphonomethoxyethyl)-2-amino-4cyclopropylaminopurine (pme-cprdap) and assayed for activity against herpesvirus replication. overall, the hdp esters were more active than the unmodified acyclic nucleoside phosphonates, indicating that this is a useful strategy for increasing the antiviral activity of acyclic nucleoside phosphonates. one of the most active compounds was hdp-pme-cprdap which had ec 50 values of 0.2, 0.3, and 0.03 m in hff cells infected with hsv-1, hsv-2 or hcmv, representing a 12-26-fold increase in efficacy over the parent pme-cprdap. another promising compound was hdp-pme-dap, which had ec 50 values of 0.7, 0.2, and 0.6 um in hff cells infected with hsv-1, hsv-2, and hcmv, representing a 16-43-fold increase over the parent pme-dap. the results presented here indicate that modified acyclic nucleotides with antiviral activity against hiv also inhibit the replication of some of the herpesviruses. further evaluation of their activity against other herpesviruses that are a problem in hiv-infected patients, such as human herpesviruses type 6 and 8, is warranted and may provide new therapeutic options for patients with coinfections. julie m. breitenbach 1 , katherine z. borysko 1 , jiri zemlicka 2 , john c. drach 1 1 biologic & materials sciences, school of dentistry, university of michigan, ann arbor, mi, usa; 2 karmanos cancer institute, wayne state university school of medicine, detroit, mi, usa we previously described first (qiu et al., 1998. j. med. chem.) and second generation (zhou et al., 2004. j. med. chem.) methylenecyclopropane purines that have potent and selective activity against hcmv. strains selected separately for resistance to first-generation analogs (synadenol, synguanol) were 10-20-fold resistant to several first-generation purine analogs. similar resistance was observed to the second-generation guanine analog cyclopropavir [ic 50 's in plaque assays = 0.35 and 21 m, respectively for wild-type (wt) and synguanol-resistant (1982r) virus]. likewise a ul97 deletion mutant (prichard et al., 1996. j. virol.) was resistant to both first and second-generation compounds (ic 50 's = 2.1 and 0.25 m in wt; 100 and 15 m in ul97 del , respectively for synguanol and cyclopropavir). ul97 from the hcmv strain selected for resistance to the synadenol was sequenced and two mutations were identified: m460i and c603y. because hcmv with either m460i or the related c607y mutation alone was sensitive to synadenol and synguanol (baldanti et al., 2002 . antiviral res.), we hypothesize that two mutations are required for resistance to first-and second-generation analogs. this hypothesis was tested by construction of three strains of hcmv from hcmv ad169 bac with one, the other, or both mutations in ul97. as expected, the two strains with the single mutations were 3-to 6-fold resistant to ganciclovir but had little resistance to the first generation compounds synadenol and synguanol (0.5-to 1-fold). both strains were somewhat more resistant to the second-generation compound cyclopropavir (6to 8-fold) but less so than observed in the 1982r virus with two mutations. study of the resistance of the constructed virus with two mutations is underway. we conclude that a functional ul97 is required for activity against hcmv and that is likely that two mutations in ul97 are required for significant resistance. acknowledgement: supported by grants p01-ai46390 and r44-ai054135 from nih and funds from the university of michigan. svitlana zagorodnya 1 , nadiya nesterova 1 , inna alexeeva 2 , larisa palchikovskaya 2 , galina baranova 1 , alexander kobko 2 , anna golovan 1 1 zabolotny institute of microbiology and virology of nas of ukraine, kiev, ukraine; 2 institute of molecular biology and genetics of nas of ukraine, kiev, ukraine search of new effective preparations capable to inhibit herpesviruses reproduction is stipulated by their certain resistance to different groups of chemical preparations. new triazine bearing tricyclic bases and their n-glycosidic derivatives structures are widely used as potential antiviral agents. the objective of the present investigation was to study the activity triazine bearing tricyclic bases nos. 1 and 2, as well as n-glycosidic derivatives no. 3 against epstein-barr virus-lymphotropic and oncogenic virus from herpesviridae family. as a model of ebv-infection in vitro we used the line of lymphoblastoid b-cells raji, which infected by ebv. an inhibition of reproduction of ebv in a cell culture by no 1, no 2, and no 3 was determined by reduction of a number of genome-equivalents of ebv dna on a cell, which were revealed by quantitative pcr with use of primers and reagents "amply-senc-100 r" (russia). the first stage of investigation of substances was the analysis of their cytotoxicity for cell line raji. they have been studied in concentrations of 1000, 500, 250, 125, 64, 32, 16, 4 , 1, 0.5 and 0.1 g/ml. the concentrations that inhibited the quantity of live cells on 50% (id50) were equal to substances no. 1-750 g/ml, no. 2-625 g/ml and no. 3-125. the minimal inhibiting concentration (mic) of nos. 1, 2, and 3 was equal to 1 g/ml, because the amount of genome-equivalents of dna ebv on a cell was reduced with 28.0 up to 14. hence, the index of selectivity (is) was equal to 750 and 625 for triazine bearing tricyclic bases nos. 1 and 2, for n-glycosidic derivatives-125. in addition these compounds were also tested in transcription and replication model systems in vitro. our results indicate that bases and their n-glycoside derivatives effect rna and dna synthesis in different manner. r. sgarbanti 1 , l. nencioni 1 , g. macrì 2 , c. nucci 2 , u. benatti 3 , m. magnani 4 , e. garaci 5 , a.t. palamara 1 1 department public health sciences, university rome "la sapienza," rome, italy; 2 department biopathology, physiopathological optics, university rome "tor vergata," rome, italy; 3 department exp. medicine biochemistry section, university genova, genoa, italy; 4 inst. biochemistry, university urbino, urbino, italy; 5 department exp. med. biochem. sciences, university rome "tor vergata," rome, italy several studies have demonstrated that different viruses induce an imbalance in the intracellular redox state through a depletion of glutathione (gsh), the main intracellular antioxidant. the imbalance in the intracellular redox state represents a key event in the development of viral infection. indeed, our previous data showed that treatment with gsh prevents a decrease in intracellular gsh and inhibits replication of different rna and dna viruses in vitro and in vivo. our recent data demonstrated that a butanoyl derivative of gsh (gsh-c4), with increased hydrophobic properties, inhibited in vitro parainfluenza-1 and hsv-1 replication more efficiently than gsh. for this reason we evaluated the effectiveness of topical gsh-c4 administration in hsv-1-induced keratitis in rabbits. for infection, the corneal epithelium, previously scratched, was inoculated with 2 × 10 5 pfu/ml of hsv-1. gsh-c4, dissolved in a saline solution (150 mm, 100 ul/eye), was administered as eyedrops four times daily for ten days. a saline solution was used for the control group. the clinical evaluation of conjunctival and corneal involvement, performed by using 0.5% fluorescein sodium eyedrops and a slit lamp fitted with a cobalt blue filter, demonstrated that gsh-c4 treatment was effective in reducing the severity and progression of keratitis and conjunctivitis. moreover, in gsh-c4 treated animals, conjunctival hsv-1 titre, assayed by tcid50 on day 4 post-infection, was significantly reduced as compared to that of control animals (mean = 1.4 × 10 3 units/ml versus 1.1 × 10 5 units/ml, n = 10 for group). accordingly, similar results were obtained by measuring virus titre from the corneas of gsh-c4-treated animals versus placebo animals (mean = 3.5 × 10 3 units/ml versus 8.5 × 10 5 units/ml, n = 5 per group). such results highlight the antiviral activity of gsh-c4 in vivo and suggest that topical gsh-c4 treatment could be considered as complementary therapy of hsv-1-induced keratitis. debra quenelle 1 , deborah collins 1 , latisha pettway 1 , caroll hartline 1 , james beadle 2 , w. wan 2 , karl hostetler 2 , earl kern 1 1 university of alabama school of medicine, birmingham, al, usa; 2 department of medicine, university of california, san diego and veterans medical research foundation, san diego, ca, usa cytomegalovirus (cmv) can cause a wide variety of clinical manifestations in immunocompromised hosts or transplant recipients. we have utilized severe combined immunodeficient (scid) mice implanted with human fetal tissue and subsequently infected with hcmv or balb/c mice infected with mcmv to evaluate new antiviral therapies against cmv infection. in the current studies we used these two models to determine the efficacy of (s)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine ((s)-hpmpa), hexadecyloxypropyl-(s)-hpmpa (hdp-(s)-hpmpa), or octadecyloxyethyl-(s)-hpmpa (ode-(s)-hpmpa). in the hcmv model, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule of mice and inoculated 16-20 weeks later with 4700 pfu of hcmv. tissue samples were obtained at various time points for quantitation of hcmv titers by plaque assay. in general, replication of the toledo strain of hcmv in the implant tissue increased through 21-28 days and then gradually decreased to undetectable levels by 8 weeks post-infection. to determine efficacy of these compounds, oral treatment with vehicle or 10 mg/kg of (s)-hpmpa, hdp-(s)-hpmpa or ode-(s)-hpmpa was initiated 24 h after infection and continued for 28 days. cidofovir (cdv) at 20 mg/kg was administered i.p. daily as a positive control. results indicated that (s)-hpmpa, hdp-(s)-hpmpa and ode-(s)-hpmpa were highly effective in significantly reducing replication when compared to the vehicle control. in mcmv infected mice, hdp-(s)-hpmpa was highly effective in preventing mortality when administered orally at 30 or 10 mg/kg beginning 24 h post-viral inoculation and 30 mg/kg when treatment was delayed until 48 h postviral inoculation. these data indicate that these compounds were highly efficacious in two animal models of cmv infection and should be evaluated for use in hcmv infections in humans. cytomegalovirus (cmv) is a ubiquitous ␤-herpesvirus that asymptomatically infects immunocompetent individuals but leads to serious illness in immunocompromised individuals, such as transplant recipients, neonates and aids patients. thus, the need for well-tolerated and potent antiviral compounds with activity against cmv is well recognized. in our current studies, we have evaluated the in vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against murine cytomegalovirus infection (mcmv) in mice. rep 9 has potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). in our initial studies, infected mice were treated with rep 9 and compared to saline-treated infected control mice. compound was administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting at 2 days prior to infection. mice were infected with 5 × 10 5 pfu mcmv on day 0, at 3 h post-treatment. sera were collected at −22 h, at 36 hpi, and at 3 dpi for elisa analysis of ifn␥ production. spleens and livers were collected at 3 dpi for determination of virus titers. at 3 dpi, virus titers in the spleens and livers were significantly reduced by rep 9 treatment as compared to control mice. splenomegaly was observed in infected mice treated with rep 9 but not in saline treated, infected mice or in rep 9 treated, uninfected mice. ifn␥ levels in mice treated with rep 9 peaked at 36 hpi compared to 72 hpi for saline-treated control mice. these data suggests that immune stimulation might contribute to the antiviral activity of ps-ons, perhaps through ifn␥ levels. a second study comparing the in vivo activity of rep 9 with two oligonucleotide analogs that do not activate tlr-9 mediated immune stimulation suggests that direct antiviral activity of rep 9 and the analogs was the predominant therapeutic mechanism in vivo. moreover, one rep 9 analog exhibited even greater antiviral activity than rep 9 while causing no splenomegaly. additional experiments are underway to provide insights into the mechanism of action against mcmv infection. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. kathy keith 1 , joseph maddry 2 , namita bansal 2 , kochurani jacob 2 , secrist john 2 , earl kern 1 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 southern research institute, birmingham, al, usa a series of novel antiviral agents was prepared based on lead compounds related to acyclic nucleoside phosphonates. these agents consist of a purine nucleus bearing a pendant phosphonic acid group. the design strategy was two-fold: (1) following the approach of the hostetler group, to mask or partially mask the anionic phosphonate as a lipophilic ester and/or as an amino acid phosphonamidate prodrug that could enhance cellular uptake and be cleaved intracellularly; and (2) to investigate new substituents at the purine 2-and 6-positions. for proof of concept, a phosphonomethoxyethyl adenine (pmea) scaffold was employed. over 100 analogs of pmea substituted at the purine 2-or at the adenine n-6 site have been synthesized and evaluated for activity against orthopoxvirus infections. many n-6 substituents other than the previously recognized n-cyclopropyl have shown antiviral activity, and these structure-activity relationships are being investigated. an exciting finding has been that introduction of several novel moieties at the purine 2-position particularly the hydrazino, hydroxylamino, or the cyclopropylamino groups resulted in several compounds with excellent in vitro activity. for example, octadecyloxyethyl (ode) 2-amino-n(6)-cyclopropyl pmea had ec 50 values of 0.03-0.07 m and ode 2-hydroxylamino-n(6)-cyclopropyl pmea had ec 50 values of 0.3-1.6 m against cowpox and vaccinia viruses, respectively, using a plaque reduction assay in hff cells. under these conditions the parent molecule, pmea, was completely inactive. these two compounds had cc 50 values of 30-70 m giving selective indices of 200-1000. these studies indicate that several modifications in the pmea scaffold can result in good antiviral activity against orthopoxvirus infections in vitro and the most active compounds are currently being scaled up for evaluation in animal models. isatin (2,3-dioxoindole), a versatile lead molecule for designing of potential bioactive agents, and its derivatives have been reported to possess inhibitory activity against a variety of pathogenic viruses. methisazone(n-methylisatin-3thiosemicarbazone) was one of the first synthetic antiviral agents used clinically for the treatment of orthopox virus infections. the presence of the thiosemicarbazone, however, can result in immunosuppresion and we have attempted to replace the thiosemicarbazone with a sulphonamide in order to modify the antiviral activity. the present work was performed to evaluate the antiviral activity and cytotoxicity of some novel 4-[(1,2dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2pyrimidiny)-benzene sulphonamides against pox viruses such as vaccinia and cowpox virus in human fibroblast cells and the activity was compared with cidofovir(cdv). among the compounds tested,4-[(5-methyl-1,2-dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2-pyrimidiny)-benzene sulphonamide(spiii-5me), was the most active compound with an ec50 value of 18 mol, compared with cdv, which had an ec50 of 20 mol against vaccinia virus. all the compounds were non-toxic (>300 lm)using a neutral red uptake assay. substitution of a halogen atom in 5th position of isatin was found to abolish the antiviral activity. this compound should be evaluated in orthopox infections in animal to determine its potential for development as an effective agents for treatment of these infections. acknowledgement: supported in part by contract no1-ai-30049 from virology branch, niaid, nih, usa. evgeny belanov 1 , svetlana kotovskaya 2 , nikolay bormotov 1 , sergey balakhnin 1 , olga serova 1 , nataliya perova 2 , zina baskakova 2 , galina dzhumbaeva 2 , valerii charushin 3 , oleg chupakhin 2 1 state research center of virology and biotechnology "vector", koltsovo, novosibirsk reg., russia; 2 ural state technical university, yekaterinburg, russia; 3 institute of organic syntheses, yekaterinburg, russia during this study, we synthesized a series of 1,2,4-benzotriazine ( fig. 1) derivatives in order to evaluate the structural features required for anti-orthopoxviruses activity. these derivatives were tested for cytotoxicity and activity against the vaccinia, cowpox, mousepox, monkeypox, and in some experiments with variola viruses in vero and mk-2 cells. the results from studies of structure-activity relationship revealed that only compounds containing phenyl group at c-3 and the alkoxy and fluoro substitutes in the benzene ring of benzotriazines showed anti-orthopoxviruses activity. the antiviral activity was reduced or lost after substitution with other substitutes. thus, we find a new class of heterocyclic compounds with antiviral activity. acknowledgment: this research was funded by istc project #1989. yali chen, guang yang, kady honeychurch, dennis hruby, robert jordan siga technologies, inc., 4575 sw research way-suite 230, corvallis, or 97333, usa we have recently discovered a highly specific and potent antiorthopoxvirus compound (st-246) via high throughput screening (yang et al., 2005. j. virol. 79, 13139-13149) . marker rescue of st-246 resistant variants localized compound resistance to the f13l gene that encodes a major orthopoxvirus envelope protein (p37), which is required for extracellular viral particle formation. p37 participates in wrapping of intracellular mature virus (imv) in membranes derived from the trans golgi or late endosomal compartment to produce intracellular enveloped virus (iev) that are transported to the cell surface to form extracellular virus particles. to gain insight into the mechanism of action of st-246, we examined the effects of st-246 on the production of the extracellular viral particles in bsc40 cells infected with recombinant vaccinia virus containing a gfp-tagged p37 protein. in the presence of st-246, iev particle formation was dramatically reduced, plaque formation was almost completely inhibited, and imv particles appeared to be retained in intracellular vesicles as revealed by electron microscopy. furthermore, st-246 prevented the intracellular localization of p37 to the late endosome compartment as measured by confocal microscopy. in contrast, st-246 did not affect localization of p37 expressed from a st-246 resistant virus variant. more intriguingly, the compound did not affect the intracellular localization of p37 in transfected cells. these results suggest that st-246 inhibits an unknown virusspecific activity that requires f13l. this work underscores the exquisite specificity of st-246 and supports continued development of st-246 as a potential anti-orthopoxvirus drug. guang yang, chris harver, dennis hruby, robert jordan siga technologies, inc. 4575 sw research way, suite 230 corvallis, or 97333, usa st-246 is a potent, orally bioavailable anti-orthopoxvirus compound that is active in vitro and in vivo. the frequency of naturally occurring st-246 resistant variants was measured by fluctuation analysis and found to be 3.58 × 10 −6 . marker rescue of drug resistant variants localized changes associated with reduced compound susceptibility to the vaccinia virus f13l gene. the spectrum of mutations that confer st-246 resistance was determined using an error-prone pcr procedure to increase the frequency of compound resistance by 100-fold relative to the frequency of naturally occurring resistance. using this procedure, random point mutations were introduced into the f13l coding sequence by error-prone pcr and the mutated f13l alleles were transferred into wild-type virus genome by marker rescue. sequence analysis of the input error-prone pcr products prior to marker rescue identified numerous nucleotide changes in the f13l coding sequence, some of which created nonsense mutations. virus recombinants were selected that formed plaques in the presence of drug selection. this powerful selection procedure enriched for viruses that produced functional, st-246 resistant, f13l proteins. sequence analysis of the compound resistant f13l alleles identified numerous silent mutations scattered throughout the f13l coding sequence and 27 point mutations leading to amino acid changes that clustered around amino acid positions 258-302 within the f13l gene. seven of these mutations resulted in single amino acid changes and could be correlated with reduced compound susceptibility. taken together, these results suggest that: (1) mutations in at least 7 positions within f13l can confer resistance to st-246 and (2) st-246 resistant mutations cluster to a 58 amino acid domain in a region of the protein of unknown function. several 5-substituted pyrimidine analogs were identified as having antiviral activity against cowpox virus (cv) and vaccinia virus (vv) in primary human foreskin fibroblast cells. molecules containing benzopyran, cyanovinyl, and pyrazolone moieties at this position exhibited significant antiviral activity against both these viruses. three compounds in this series had ec 50 values below 10 m in a plaque reduction assay against both cv and vv. the antiviral activity of these compounds was also determined against herpes simplex virus (hsv) in a plaque assay. two compounds with cyanovinyl derivatives at the 5 position had ec 50 values below 15 m against both hsv-1 and hsv-2, whereas other substituents at this position exhibited weaker activity against one or both of these viruses. analogs containing the benzopyran substituents were the most effective against varicella zoster virus (vzv) and yielded ec 50 values below 10 m in a plaque reduction assay. none of the compounds were active against human cytomegalovirus. interestingly, all of the compounds were much less effective in a thymidine kinase (tk) negative strain of cv suggesting that the activation by this enzyme was important in their mechanism of action. tk deficient strains of hsv were also comparatively resistant to some of the compounds. the tk dependence of these compounds in cv and hsv taken together with the lack of activity against cytomegalovirus replication suggests that activation by a viral tk is important in their mechanism of action. these results indicate that pyrimidine analogs with large substituents at the 5 position are substrates for the distinct tk homologs encoded by the herpesviruses and orthopoxviruses and suggest that they may be effective against infections with these viruses. synthesis and testing of additional analogs is warranted and should help identify the most potent analogs for in vivo testing. department of pediatrics, university of alabama school of medicine, birmingham, al 35233, usa n-methanocarbathymidine ((n)-mct) is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. this compound inhibits the replication of herpes simplex virus (hsv) with ec 50 values below 1 g/ml, and vaccinia virus (vv) and cowpox virus (cv) with ec 50 values of 0.55 and 1.5 g/ml, respectively. assays using a thymidine kinase (tk) negative strain of cv yielded ec 50 values 14-fold greater than a tk positive isolate. similarly, a tk negative strain of hsv-1 was 90-fold less sensitive to the drug than wild-type strains. thus, the antiviral activity of this molecule is dependent on the type i tk in hsv and the type ii tk expressed by vv and cv viruses, suggesting that it is a substrate for these divergent forms of the enzyme. the drug is also a good inhibitor of viral dna synthesis in both viruses and is consistent with inhibition of the viral dna polymerase once it is activated by the viral tk homologs. it is also possible that the phosphorylated forms of the drug may inhibit other enzymes such as thymidylate synthetase and inhibit viral dna synthesis indirectly. the interesting tk dependence of this molecule explains the rather unusual spectrum of activity that includes orthopoxviruses, alphaherpesviruses, epstein-barr virus (ebv), and human herpesvirus 8 (hhv-8), since these viruses all express molecules with tk activity that could phosphorylate and thus activate the drug. conversely, n-mct is ineffective against the betaherpesviruses because they do not encode tk homologs. the compound is also highly effective in reducing the mortality of mice infected with cv, vv, and hsv when treatment is initiated 24 h after infection and at doses as low as 17 mg/kg. these results indicate that (n)-mct is active in vitro and in vivo and its mechanism of action suggests that the molecule may be an effective and selective therapeutic for orthopoxvirus and certain herpesvirus infections and that it warrants further development. we have previously reported the isolation and characterization of drug-resistant mutants obtained following repeated passages of the vaccinia virus (vv, lederle strain) in the presence of increasing concentrations of cidofovir (cdv). cdvr mutants encoded two mutations (a314t and a684v) not related to genetic polymorphism. we have now introduced these mutations in the pathogenic strain w western reserve and characterized the drug-susceptibility profile of the recombinant viruses and their pathogenicity in mice. both the a314t and the a684v recombinant viruses proved to be resistant to cdv and related compounds, such as cyclic cdv and -propoxy]pyrimidine}. the virus bearing both substitutions proved to be more resistant to cdv than the single mutants. interestingly, the a314t and the a684v mutants differed in their sensitivity to phosphonoacetic acid (paa); the a314t and the a684v mutants being, respectively, hypersensitive and resistant to paa. in contrast, the double mutant showed no change in sensitivity to paa as compared to the wild-type strain. unlike the a684v mutant that showed only a two to three-fold decrease in susceptibility towards the 3-hydroxy-2-phosphonomethoxypropyl (hpmp) purine derivatives, the a314t mutant showed cross-resistance to the hpmp purine derivatives. it should be noted that in the process of selection of cdv-resistant mutants in the presence of increasing concentrations of the compound, the a314t mutation appeared before the a684v substitution, and the latter mutation only occurred in conjunction with a314t. when tested for virulence in a lethal intranasal infection model in mice, all cdvr recombinant viruses proved to be attenuated, suggesting that cdvr mutations are associated with reduced pathogenicity. furthermore, we found that cdv at a dose of 50 mg/kg/day for 5 days was still able to protect mice (in terms of body weight loss) against an intranasal challenge with the a314t + a684v recombinant virus. evaluating the use of cpg dna as an antiviral therapy amanda phelps, linda eastaugh, amanda gates, david ulaeto, arthur krieg 1 defence science and technology laboratories (dstl), salisbury, wiltshire, uk; 6 coley pharmaceuticals ltd., ottawa, ont., canada at present there are no licensed antivirals against orthopoxvirus infections such as variola or vaccinia virus (vacv). although a stockpile of smallpox vaccine exists and has utility as a post-exposure treatment to infection, it is a live viral vaccine and as such cannot be administered to those with contraindications. bacterial dna contains unmethylated motifs that, together with their flanking regions, can stimulate an innate immune response. synthetic cpg dna mimics the immunostimulatory activity of bacterial dna and is recognised by intracellular toll-like receptor 9. there are four classes of cpg dna all of which have different properties, eliciting distinct initial immune responses. previous studies using an established lethal respiratory model of poxvirus infection demonstrated that a class b cpg dna (7909) could provide protection from lethality against vacv in balb/c mice when administered up to 7 days prior to challenge. in order to evaluate efficacy balb/c mice were challenged intra-nasally with vacv and treated with doses of 7909 ranging between 15 and 100 ug/mouse. treatment was administered intra-nasally under light anaesthesia either on the day of challenge, 1, 2, 3, or 4 days post-challenge. efficacy was determined by percentage body weight loss post-challenge. the optimum survival rate observed was 60% when treated with 30 ug 1 day post-challenge (70 mld50 challenge). a survival rate of 80% was observed when treated with 50 ug 2 days post-challenge (40mld50 challenge). the delay of treatment to either 3 or 4 days post-challenge was ineffective, indicating that the window of opportunity for delivery of 7909 is within 2 days. multiple doses of 7909 were used to attempt to extend this window of opportunity, delivering 7909 twice within a 3-day period. interestingly, this had a considerable detrimental effect, actually accelerating the onset of disease and ultimately death. further work is required to optimise the use of cpg dna as a potential antiviral therapy, and there is evidence to suggest that they may have immense utility as part of a co-administration therapy with other antiviral compounds, an area of work currently under investigation. © crown copyright dstl 2005. department of virology, hebrew university, hadassah medical school, jerusalem, israel the pathogenicity and immunogenicity of the lister (elstree) strain of vaccinia virus, used for vaccination against smallpox, was studied in the mouse model. the virus did not reach the brain when inoculated intranasally, but when injected intracranially at a dose of 5 × 10 5 plaque forming units (pfu), was lethal for 50% of the mice. lower doses of virus caused the mice to initially loose some weight but they completely recovered thereafter. a significant level of protection against a lethal dose of the wr strain was achieved in mice following immunization with the lister strain, while higher doses and repeated vaccination procedure, were required with modified vaccinia virus ankara (mva). we found that the lister vaccine strain applied in israel is comprised of heterogeneous virus population. we isolated and plaque-purified three virus variants differing in their plaque size in bs-c-1 cell cultures. they were named: l-large plaque, m-medium plaque and s-small plaque variants. these isolates could be neutralized by rabbit antibodies prepared against the western reserve strain of vaccinia virus and their one-step growth curves in bs-c-1 cells were quite similar. however, they differ in their pathogenicity to mice following intranasal inoculation of 10 7 pfu, or an intracranial injection of 5 × 10 4 pfu; the s variant being more virulent than the other two variants and resembles the pathogenicity of the lister strain. activity was also determined against a thymidine kinase (tk) deficient vaccinia virus in mouse and monkey cells. the potency of (n)-mct was similar to that seen with wild-type virus, suggesting that a cellular enzyme may be more important than viral tk to phosphorylate the compound. mice were intranasally infected with cowpox and vaccinia viruses followed 24 h later by intraperitoneal treatment with (n)-mct (2x/day for 7 days) or cidofovir (1x/day for 2 days). (n)-mct treatment at 100 and 30 mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival (placebo). statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100 mg/kg/day treated mice. these doses did not spare mice from lethal vaccinia virus challenge, however, but the 30 and 100 mg/kg/day treatments significantly reduced day 5 virus titers and lung weights, and the 100 mg/kg/day treatment reduced lung consolidation. cidofovir (100 mg/kg/day) protected all animals from death in both models. the evaluation of (n)-mct may be limited to mice based upon its greatly reduced efficacy in the cells of higher animals. acknowledgement: supported in part by contract no-1-ai-15435 from the virology branch, niaid, nih. chelsea byrd 1 , elena sbrana 2 , shu-yuan xiao 2 , marina siirin 2 , robert tesh 2 , dennis hruby 1 , robert jordan 1 1 siga technologies, inc., corvallis, or, usa; 2 university of texas medical branch, galveston, tx, usa st-246 is a potent small molecule inhibitor of orthopoxvirus replication that has been shown to protect mice from lethal challenge with vaccinia and ectromelia viruses. here we report the results of preliminary trials that show efficacy of st-246 against severe monkeypox virus infection in the ground squirrel model. ground squirrels infected with less than 1 pfu of monkeypox virus develop a fulminant disease resembling human hemorrhagic smallpox: the most severe and lethal form of the disease. oral administration of st-246 at 100 mg/kg once per day for 14 days protected ground squirrels from a lethal challenge with 100 and 1000 pfu of monkeypox virus. compound treated animals showed no weight loss or evidence of disease, and blood chemistry values were similar to uninfected animals. in contrast, placebo-treated animals showed elevated liver enzyme (alt and ast) levels and all animals died by day 12 post-infection. when treatment with 48, 72, and 96 h, 100% protection was observed in the 24, 48, and 72 h groups, and 66% protection in the 96 h group. severe pathologic changes were observed in the organs of the animals receiving placebo, especially in the lungs, liver, and spleen. in contrast, the organs of the animals receiving st-246 at 0, 24, 48, and 72 h postinfection appeared grossly and microscopically normal. thus, st-246 appears to be a promising candidate for continued development as a therapeutic agent for severe orthopoxvirus infection. inge vliegen 1 , guang yang 2 , dennis hruby 2 , erik de clercq 1 , robert jordan 2 , johan neyts 1 1 rega institute for medical research, k.u. leuven, belgium; 2 siga technologies, inc. corvallis, or, usa st-246 is a potent inhibitor of the replication of various orthopoxviruses. resistance of cowpoxvirus to st-246 maps to a mutation in v061, which is homologous to vaccinia virus f13l (yang et al., 2005. j. virol.) . the latter encodes the envelope protein p37 required for production of extracellular virus. deleting f13l resulted in a virus ( f13l-vac) that is replicationcompetent in cell culture but that produces smaller plaques than the wild-type wr-vac. whereas intravenous (i.v.) inoculation of nmri mice with 2 × 10 5 pfu of wr-vac resulted in 59 ± 11 pox tail lesions per mouse, the same inoculum of f13l-vac caused no lesions (p < 0.001). athymic nude (nu/nu) or scid mice inoculated iv with 2 × 10 5 pfu f13l-vac did not develop tail lesions. the mean day of death in nu/nu mice inoculated with f13l-vac was 22 ± 1 days as compared to 9 ± 1 days for wr-vac-infected mice (p < 0.001); scid mice survived the infection. we next studied whether f13l-vac is able to protect mice against a subsequent infection with wr-vac. to mimic the human vaccination protocol, nmri mice were infected intracutaneously (i.c.) by means of scarification at the lumbosacral area with 5 × 10 5 pfu f13l-vac or placebo. none of the infected mice developed lesions at the inoculation site. one week later, animals were infected ic with 5 × 10 5 pfu of wr-vac. all placebo animals, but none of the f13l-vac animals developed poxvirus lesions. in a second set of experiments, mice were again inoculated ic with placebo or f13l-vac and were infected one week later with 2 × 10 4 pfu of wr-vac by the iv route. placebo animals developed an average of 14 ± 4 pox tail lesions; no lesions developed in the f13l-vac animals (p < 0.001). in a third set of experiments, nmri mice were inoculated iv with either 2 × 10 4 pfu of f13l-vac or placebo, and none of the mice developed lesions. one week later, animals were inoculated iv with 2 × 10 4 pfu wr-vac. the placebo group developed an average of 11 ± 8 lesions as compared to 1.2 ± 0.7 lesions in the f13l-vac mice (p < 0.05). f13l-vac may thus be considered as a severely attenuated virus that may have potential for use as a smallpox vaccine. ji yuan 1 , travis lim 1 , shuan coughlin 1 , dexin qiu 1 , zhen liu 1 , dave stein 2 , decheng yang 1 1 the james hogg icapture centre for cardiovascular and pulmonary research, university of british columbia, vancouver, bc, canada; 2 avi biopharma, inc., corvallis, or, usa background: coxsackievirus b3 (cvb3) is the most common cause of viral myocarditis, but existing drug therapy is of limited value. antisense oligonucleotides (asons) designed to pair with viral rna could inhibit viral replication. however, the effectiveness of traditional asons is limited due to poor cellular uptake and degradation by nucleases. phosphorodiamidate morpholino oligomers (pmos) contain backbone modifications, which make pmos more resistant to nucleases. in addition, an arginine rich peptide (p007) is conjugated to the 5 end of the oligomer to improve its delivery into cells. these features make p007-conjugated pmos (p-pmos) promising candidates for the inhibition of cvb3 infection. methods: total 8 p-pmos targeting distinct regions of viral genome and one scrambled sequence were designed and chemically synthesized. fitc labeled p-pmos were used to observe their distribution of by confocal microscopy. viral protein vp1, viral titre, and cell viability were measured by western-blot, plaque assay, and mts assay, respectively. results: p-pmos showed increased cellular uptake compared to non-conjugated pmos. among the 8 p-pmos, p-pmo-6, targeting the internal ribosomal entry site in the 5 utr, showed the most potent anti-cvb3 ability in a dose-dependent manner. both infected hela and cardiomyocytes hl-1 cells treated with p-pmo-6 showed drastically reduced vp1 production and 3.0 log decreases in viral titres as compared to the controls. cell viability assay revealed that 83 and 89% of treated hela and hl-1 cells were still alive as compared to 11 and 10% of control-treated cells and 47% antiviral activity still existed after 5 days treatment. in addition, cells treated post-infection showed similar inhibition of viral replication. furthermore, the specificity of the p-pmos was demonstrated by their inability to inhibit rsv infection in hela cells. we have showed that p-pmos can effectively inhibit viral replication in vitro, providing a new possibility for antiviral intervention. picornaviruses are responsible for various human viral diseases including common cold, encephalitis, meningitis, myocarditis, etc. up to now, there is no specific antiviral therapy to treat or prevent such viral disease. the usage of modern computer technologies may help to solve this problem more effectively. the objective of the present study was the quantitative structure-activity relationship (qsar) analysis of antiviral activity of a set of [(biphenyloxy)propyl]isoxazole derivatives that inhibit cvb3 replication in hela cells. based on results from qsar, the structure of new potential antiviral agents should be predicted by using consequent molecular design. the qsar approach applied is based on simplex representation of molecular structure (sirms). the relationship between: (a) antiviral activity against the pleconaril-sensitive clinical cvb3 isolate 97-927 (ic 50 , g/ml); (b) cytotoxicity in hela cells (cc 50 , g/ml); and (c) selectivity index (si = ratio of cc 50 to ic 50 ), and structure of 21 [(biphenyloxy)propyl]isoxazole derivatives has been studied systematically. quite adequate qsar models (r 2 = 0.831-0.931, q 2 = 0.674-0.896) have been obtained using pls (partial least squares) method for all parameters studied. the models are in close correlation with experimental data. structural fragments with positive or negative influence on cytotoxicity as well as antiviral activity have been determined on the base of these models. for example, qsar analysis of antiviral activity of [(biphenyloxy)propyl]isoxazole derivatives revealed that the presence of m-nitrophenyl or p-trifluorophenyl fragment has distinctly negative influence on antiviral action. compounds with strong antiviral activity have to contain an oxadiazole fragment. moreover, our data allow the virtual screening and molecular design of new well-tolerated compounds with strong anti-cvb3 activity. ivanka nikolova 1 , roumena petkova 2 , boris atanassov 3 , stoyan chakarov 2 , angel s. galabov 1 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 scientific technological service, ltd., sofia, bulgaria; 3 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria analysis of the rna sequence of the disoxaril-resistant mutants of the coxsackievirus b1 was carried out. the wild-type disoxaril-sensitive strain (connecticut 5) and two disoxarilresistant mutants (one of them produced in fl cells and the other one isolated from brains of newborn mice) infected with coxsackievirus b1 and treated with disoxaril and a disoxarildependent mutant strain obtained from the resistant strain by 9 passages in cell culture were included in the present study. a rt-pcr assay with primer sets selected from a region of the coxsackievirus b1 genome coding for the capsid protein vp1 was carried out. a parallel comparative analysis of the sequences of resulting fragments from the disoxaril mutants studied and the genbank sequence of origin of the vp1 gene of coxsackievirus b1 was performed with the blast alignment tool. distinct alterations in the vp1 locus of the disoxaril-resistant and the disoxaril-dependent mutants compared to the sequence of origin from the genbank (namely, a deletion of uug at ntt. 2749-2751 and an insertion of uuu at nt.2769) were observed. high-degree similarity (97%) between the resistant mutant produced in cell cultures and the dependent strain was observed, while the similarity to the wild strain was only 91%. the resistant mutant obtained in mice was found to be very similar to the strain, developed in cell cultures. a putative 3-d model of the spatial folding of the target protein in disoxaril mutants is proposed. ralitsa vassileva-pencheva, angel s. galabov in previous study of ours we presented a new approach to combined application of antivirals-consecutive administration of the partners. this schedule could be considered especially suitable for treatment of enteroviral infections, in which the development of resistance is very rapid due to the extremely high viral mutation rate. this approach aims to restrict the resistance development in experiments in vivo, using antivirals with proved high efficiency in experiments in cell cultures. the screening of various double, triple, and quadruple combinations that we carried out showed that two of the triple combinations, namely disoxaril (win compound)/oxoglaucin (a new antiviral drug, developed in our laboratory)/ptu-23 (a classic enteroviral inhibitor) and disoxaril/oxoglaucin/guanidinehydrochloride (a classic enteroviral inhibitor) manifest significant effect of protection in newborn mice with neurotropic coxsackievirus b1 infection. in the current study the role of the chronology of arrangement of the antivirals included in the combinations was investigated. in the experiments carried out with the triple combination disoxaril/oxoglaucin/guanidine-hydrochloride, it was found that the optimal treatment course should start with disoxaril. the treatment course is quite successful when disoxaril is followed by guanidine-hydrochloride. the effect of the triple combination starting with oxoglaucine, followed by guanidine-hydrochloride was moderate. the course starting with guanidine-hydrochloride proved to be ineffective. furthermore, we studied the virus sensitivity to the inhibitorspartners (ic50 values) and some other phenotypic characteristics of the brain isolates, e.g. the size of the plaques and the pathogenicity for mice. recently our contribution to the development of new antipicornavirus agents has led to the discovery of 5methylthio-3-aryl-isoxazole-4-carbonitrile derivatives whose in vitro anti-coxsackievirus b1 activity were dependent on the nature of the substituents on the para position of the phenyl ring. particularly, compounds 5-methylthio-3-[4-(3-phenyl-1-propoxy)phenyl]isoxazol-4-carbonitrile (on-7) and 5-methylthio-3-[4-(4-phenoxy-1-buthoxy)phenyl]isoxazol-4-carbonitrile (on-10) exhibited an interesting antiviral activity with high selectivity indexes. in the present study, we investigated on the mechanism of action of these compounds. studies on time of addition experiments suggested that these compounds exert a different interference with an early step of the viral replicative cycle. in fact, compound on-7 was effective when added within 1 h after the end of the adsorption period and no reduction was observed if it was added during the adsorption period. whereas a reduction of virus titer was observed for on-10 when was added during the adsorption period, while no reduction was observed if the compound was added after this period (time 0). the influence of the compounds on virus adsorption step, studied by the infective center assays, indicated that on-10 primarily interferes with coxsackie b1 cellular attachment. at a concentration 100 times the id 50 , inhibition of adsorption of coxsackievirus by on-10 was complete, while similar concentration of on-7 had no effect. our experiments on neutralization of viral infectivity and on thermal stabilization demonstrated that the compounds were able to directly inactivate coxsackievirus, and the infectious titer was restored to the original value after extraction of the compound with chloroform. however, the compounds did not protect the viral infectivity against heat inactivation at the different concentrations used. the blood-brain barrier (bbb) fulfills a vital protective function by limiting entry of potential pathogens, toxins, and inflammatory cells into the central nervous system (cns). disruption of the bbb is a common component of many cns diseases, including viral diseases such as that caused by west nile virus (wnv). transforming growth factor-␤1 (tgf-␤1) has previously been shown to improve the function of an in vitro model of the bbb. we evaluated the role of the bbb in wnv infection in mice by determining the ability of intraperitoneally (i.p.) administered sodium fluorescein to move from the circulating blood to the central nervous system. to demonstrate bbb permeability a mean and normal range of permeability values was determined in 30 non-infected c57/bl6 mice. in subsequent experiments, any animal expressing a permeability value greater than 2 sd above the mean was considered abnormally high. we determined that elevations in bbb permeability can be detected in mice 8 days after subcutaneous inoculation with wnv. wnv inoculated animals were treated with doses of 3000, 1000, or 300 ng/kg/day of tgf-␤1 or with drug vehicle once daily via the i.p. route on 7 and 8 days post-virus inoculation (dpi), and then assayed for bbb permeability on 8 dpi. sixty-two percent (8/13) of placebo-treated animals had abnormally high permeability values, while animals treated with 3000 and 1000 ng/kg/day of tgf-␤1 had 29% (2/7) and 57% (4/7) of animals with abnormally high permeability values, respectively. in contrast, none of the animals treated with 300 ng/kg of tgf-␤1 (0/8) expressed abnormally high permeability values, which was significantly lower (p < 0.01) than placebo-treated animals. these results suggest that tgf-␤1 may improve the function of the blood-brain barrier in wnv infected mice. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. and contract no1-ai-15435 from the virology branch, niaid, nih. people infected with west nile virus (wnv) usually see their physicians after showing symptoms suggestive of neurological infection. wnv infects the central nervous system (cns) of rodents 3-5 days after s.c. viral challenge. yet, most published animal studies begin therapeutic treatments before or soon after viral challenge. the question addressed in this study is if neuroprotective agents can be efficacious when administered early before brain infection or later after the virus is demonstrated to be in the brain. the drugs evaluated in wnvinfected rodents were nmda and ampa receptor antagonists, modulators of nitric oxide synthase (nos) and nitric oxide production, and riluzole for reducing glutamate excitotoxicity. serial doses of diethyldithiocarbamate (ddtc) and n(g)monomethyl-l-arginine (l-nmma), an inducer or inhibitor of nos, respectively, administered i.p. daily for 10 days beginning 4 h before viral challenge slightly improved survival of mice, but the difference was not statistically significant. tolerated doses of two nmda-receptor antagonists, flupertine (7 mg/kg) and mk-801 (1 mg/kg), and one ampa-receptor antagonist, gyki 52466 (10 mg/kg), were administered twice daily (b.i.d.) on 4 though 9 days post-virus inoculation (dpi). gyki 52466 slightly improved mouse survival and weight gain, but the difference was not statistically significant. talampanel, an ampa-receptor antagonist and a derivative of gyki 52466, slightly improved hamster survival (p ≤ 0.05) when treatment began on 5 dpi, but repeated experiments using different doses and slightly different protocols gave mixed results. riluzole, the only drug shown to improved survival of amyotrophic lateral sclerosis (als), presumably by reducing glutamate excitotoxicity, was not effective against wnv disease when administered b.i.d. beginning 5 dpi. overall, neuroprotective agents did not consistently improve wnv disease, although slight improvements in animal survival might be relevant to improvement of neurological sequelae in wnv-patients. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hamster and mouse models for west nile virus (wnv) disease were used in this study to identify infected cells of the central nervous system (cns) early in the course of infection. this information may be relevant to therapeutic strategies since most wnv-infected people visit their physicians after showing symptoms suggestive of neurological infection. we subcutaneously infected adult mice and hamsters using 105.3 tissue culture infectious doses of wnv. tissues of infected and control animals from 3 to 11 days post-viral injection (dpi) were fixed by cardiac perfusion using 4% paraformaldehyde. we localized wnv, neuronal and astroglial markers in the paraffin embedded tissue sections by immunofluorescence. the images were captured using the confocal microscope (bio-rad, mrc 1020). we observed the presence of wnv antigen in cns tissues of mice and hamsters as early as 3 and 5 dpi, respectively. a strong wnv-specific immunofluorescence staining was observed in the cytoplasm of neurons from the spinal cord, cerebellum, cerebral cortex, and midbrain of these rodents. the wnv-specific staining co-localized with neuron-specific markers; however, astroglial markers were not co-localized with wnv antigen in brain sections. the lack of tropism by wnv for astrocytes was also confirmed in primary murine astrocyte cultures. interestingly, infected neurons in the midbrain of 7-day infected hamsters co-localized with calbindin, which is a calcium-binding protein and mostly expressed in the interneurons of the cns. therapies were evaluated in hamsters or mice at a time-point when wnv-stained neurons were identified in the cns. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. laboratory of virology, department of biological chemistry, school of sciences, university of buenos aires, argentina dengue virus (denv) is an arthropod-borne flavivirus that has re-emerged in recent years as an increasingly important public health threat with nearly 50 million infections occurring each year. at present neither specific antiviral therapy nor vaccine exists for the treatment and prevention of denv infections. carrageenans are sulfated galactans that can be extracted from red seaweeds and comprise diverse structures with a wide range of biological properties useful in biomedicine. in a previous study we have demonstrated the antiviral activity of commercialand -carrageenans against denv type 2 and 3 in vero (monkey kidney cells) and hepg2 (human hepatoma cells), showing inhibitory concentration 50% (ic 50 ) values in the range 0.14-1.1 g/ml and selectivity indexes (cc 50 /ic 50 ) in the range 1000-10000. in the present work we studied the mode of action of these polysulfates against denv-2 in vero and hepg2 cells, first analyzing the influence of time of addition of compounds on anti-denv activity by an infectious centre assay. the highest inhibitory effect was observed when the compounds were added during adsorption or at 1 h p.i., being ineffective at later times. then, the effect of compounds on virus adsorption and internalization was studied separately by a virus yield inhibition assay. significant antiviral efficacy was attained if compounds were present either only during denv-2 adsorption or internalization. the possible effect of carrageenans on viral protein synthesis, the subsequent stage of the virus cycle occurring during the first hour of infection, was analyzed by pulse-labeling with ( 35 s)-methionine. no alterations in denv protein synthesis in carrageenan-treated cells were observed. when cells were transfected with purified denv-2 rna in the presence of -carrageenan no inhibition in fluorescent cell focus formation and virus production was detected. besides, no significant direct virucidal effect on denv-2 was shown by the compounds. these results indicate that both denv adsorption and internalization seem to be the main target for these compounds, lacking effect on the steps that occur once the viral genome is inside the cell during in vitro infection of human and monkey cells. multiple members of the flavivirus genus of the family flaviviridae cause lethal hemorrhagic fever or encephalitis. the public health significance of the hemorrhagic fever and encephalitis caused by such flaviviruses is enormous and global and there is a tremendous need for antivirals. imino sugar glucosidase inhibitors have been shown to have selective antiviral activity against viruses such as bovine viral diarrhea virus (bvdv) and west nile virus (wnv) that have common requirements for their glycoprotein processing during virus production. we are developing imino sugar deoxynojirycin (dnj) derivatives through chemical synthesis of compounds with various alkyl side chains and antiviral testing against bvdv and wnv as well as in wnv subgenomic replicon assays. briefly, using a single step growth (virus yield reduction) assay for bvdv and wnv, a series of dnj derivatives containing various conformational locking side chains were shown to have antiviral activity. pre-liminary structure-activity relationships (sar) were obtained for further modification of the alkyl side chain and improvement of these dnj derivatives. the activity and mechanisms of action of these compounds will be presented. several flaviviruses cause life-threatening diseases in man. currently, there is no therapy available for these infections. in recent years, several highly selective inhibitors of the replication of hepatitis c virus (hcv) were designed. most small molecule inhibitors of hcv that are in preclinical or clinical development are either protease or polymerase inhibitors. most of these compounds are highly selective for hcv and are unlikely to exhibit activity against flaviviruses. nucleoside polymerase inhibitors, however, may have the potential to inhibit the replication of flaviviruses as well. we evaluated in vitro whether the active component of the anti-hcv compound valopicitabine, i.e. 2 -c-methylcytidine inhibits the replication of flaviviruses in cell culture. the compound was found to exhibit specific antiviral activity against yellow fever virus 17d (ec 50 = 3.1 g/ml in cpe reduction assays and >98% reduction at 5 g/ml as assessed by qpcr) and dengue fever virus type 2 (ec 50 = 16.5 g/ml in cpe reduction assays). the compound also efficiently inhibited west nile virus replication (>98% at 5 g/ml as assessed by qpcr and >98% by plaque reduction neutralization test at 50 g/ml). in the absence of any drugs for the treatment of flavivirus infections, it may be envisaged that nucleoside polymerase inhibitors, when marketed for the treatment of hcv infections, could be used off-label for the treatment of lifethreatening flavivirus infections. even if such drug would not be able to completely inhibit flavivirus replication, a partial reduction of the viremia during the acute phase of the infection may be sufficient to prevent the development of a fulminate disease and thus protect against virus-induced mortality. yuri klimochkin 1 , andrew shiryaev 1 , igor moiseev 1 , v sabynin 2 , larisa rustamova 2 , alexandr petkevich 2 1 state technical university, samara, russia; 2 institute of epidemiology and microbiology, minsk, belaruss arenaviruses are one of the most dangerous tools of bioterrorism in the view of pathogenicity and epidemiological threat. for the purpose of searching new remedies for treatment are-naviruses infections the synthesis of new derivatives of cage compounds has been carried out. the prepared compounds are bridgehead derivatives of cage compounds bearing different functional groups such as hydroxy, acylamino, alkoxycarbonylamino, alkylthiocarbonylamino groups as well as iminoalkyl adamantane derivatives, some adamantylated heterocycles and compounds containing two adamantane moieties in a molecule. the antiviral activity of the cage compounds has been studied in respect to arenaviruses lassa (sierra-leone strain) and pichinde (an-3739 strain) on the vero cells culture. different level of antiviral activity was shown by 15 compounds. the most active compounds are monosubstituted adamantane derivatives having sufur and nitrogen-containing substituent in the bridgehead position. the data on the activity confirm the availability of searching inhibitors of arenaviruses reproduction in the cage compounds series. brian gowen 1 , donald smee 1 , min-hui wong 1 , anne pace 1 , kie-hoon jung 1 , kevin bailey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa several arenaviruses endemic to the south american (junin, machupo, and guanarito) and african (lassa) continents are known to cause frequently fatal hemorrhagic fever. with the exception of ribavirin, which has demonstrated efficacy in cases of lassa fever, there are no other effective therapeutics for the treatment of arenaviral hemorrhagic fever. the outcome of treatment is ultimately dependent upon early diagnosis and the tolerability of ribavirin by patients at the high doses required for effective antiviral activity. we have recently demonstrated that consensus interferon-alpha (ifn-alfacon 1) can protect hamsters from lethal pichinde virus (pcv) infection (gowen et al., 2005 . antimicrob. agents chemother.), which serves as a model for acute arenaviral disease in humans. here we demonstrate highly effective therapy through the combined use of ribavirin and ifn alfacon-1 for the treatment of pcv infection in hamsters. ribavirin was given orally, twice per day for 7 days, and ifn alfacon-1 was administered intraperitoneally, once per day for 10 days. treatments were initiated 1-5 days post-infection with various dose combinations, many which were less than optimal when the drugs were given independently. combining suboptimal doses of ribavirin (5-10 mg/kg/day) with ifn alfacon-1 (5-10 mg/kg/day), we were able to show increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. our data indicate that synergistic activity resulted from combination therapy and that this activity may slow down the progression of disease and decrease fatality rates seen with severe arenaviral infections. further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity. acknowledgement: supported by contract no1-ai-15435 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. slobodan paessler 1 , laure deflubé 1 , andrew vaillant 2 , jean-marc juteau 2 , ramon flick 1 1 department of pathology, university of texas medical branch, galveston, texas, usa; 2 replicor inc., laval, que., canada rift valley fever virus (rvfv; genus phlebovirus, family bunyaviridae) is an arbovirus transmitted by many species of mosquitoes. this virus is a major public health concern in sub-saharan africa and egypt, which spread to yemen and saudi arabia. in this area, rvfv is responsible for dramatic epidemics/epizootics underlining the need for efficient antiviral/prophylactic measures. rep 9 is a 40 mer phosphorothioate oligonucleotide, which has previously been shown to have broad-spectrum activity in several viruses (vaillant et al., submitted for publication) . we used a vaccine strain (mp12) as well as the wild-type rvfv (zh501), to test the ability of rep 9 to inhibit bunyavirus proliferation. in vitro data showed reduction of virus titer for both strains using rep 9 at nanomolar concentrations. moreover, the absence of the phosphorothioate modification in a stabilized rep 9 analog resulted in a loss of antiviral activity, suggesting that as in other viruses, the increased hydrophobicity of rep 9 is essential for its antiviral activity. based on the inhibitory activity observed in vitro, we started with in vivo efficacy studies by utilizing a validated mouse model used in our laboratory. more animal experiments are ongoing to confirm the in vitro results and to evaluate the antiviral effect of the rep 9. adriana garozzo 1 , rossella timpanaro 1 , aldo stivala 1 , gianna tempera 1 , christian c.c. cutrì 2 , angelo castro 1 1 department of microbiological sciences, university of catania, via androne 8, 95124 catania, italy; 2 department of pharmaceutical sciences, university of catania, viale a. doria 6, 95125 catania, italy our previous studies described the synthesis and the antiviral activity of 3,4,5-trisubstituted isothiazole derivatives that were found to be particularly effective against picornaviruses. compound 3-methylthio-5-phenyl-4-isothiazolecarbonitrile (is-2) exhibited an interesting anti-poliovirus activity with high selectivity index. in the present study, we investigated on the mechanism of action of this compound. studies on the time of is-2 addition to poliovirus type 1 infected cells suggested that the compound may inhibit some early processes of viral replication. in order to determine its mechanism of action, we evaluated the rate of attachment and internalization of purified [ 3 h]uridine-labeled poliovirus to hep-2 cells in the presence or absence of is-2. no effect on poliovirus adsorption and internalization to host cells was detected. we also investigated the influence of the compound on virus uncoating using labeled poliovirus and measuring the radioactivity of oligoribonucleotides formed from viral rna susceptible to ribonuclease. these experiments demonstrated that poliovirus uncoating is influenced by is-2 action. justin julander 1 , aaron olsen 1 , john morrey 1 , lawrence blatt 2 , kristiina shafer 1 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa alpha togaviruses are medically important arboviruses, with clinical cases occurring each year in north, south, and central america. the recent increase in the threat of the use of these viruses as bio-terrorism agents has led to increased efforts to develop therapeutic agents for treatment of these viruses. venezuelan (vee) and western equine encephalitis (wee) viruses have been listed as category b priority pathogens by the national institute of allergy and infectious disease (niaid). the goal of these studies was to characterize animal models for vee and wee for use in evaluation of antiviral therapies. c3h/hen mice were infected through the intranasal (i.n.) route with a vaccine strain of vee, tc-83. virus was detected in the brain 2 days post-viral injection (dpi). brain titers increased to a peak titer of 10 9.5 50% cell culture infectious doses per gram tissue (ccid 50 /g) on 4 dpi, maintained a titer of 10 9 ccid 50 /g through 7 dpi, and dropped slightly to 10 8.6 ccid 50 /g by 8 dpi. virus was also detected in spleen, liver, and kidney. treatment of vee-infected mice with interferon alpha b/d, a human consensus interferon, resulted in 100% survival, whereas all placebotreated animals died by 9 dpi. syrian golden hamsters were infected with 10 3 ccid 50 wee through intraperitoneal (i.p.) injection. morbidity, including hind limb paralysis, tremors, nasal bleeding, and hunching, and some mortality were seen as soon as 4 dpi. the majority of deaths occurred on 5 dpi. virus was detected in all organs assayed (brain, liver, and spleen) with peak titers occurring 4 dpi. interferon alfacon 1 (ifn alfacon), a human consensus interferon, active in hamsters, was effective in significantly reducing mortality (p < 0.001 as compared to placebo). there was a trend for reduction of brain titers in ifn alfacon-treated animals (mean titer 10 4.8 ccid 50 /g) as compared with placebo (mean titer 10 9.5 ccid 50 /g), although this difference was not statistically significant. these models will be useful in screening potential antiviral agents for efficacy against vee and wee. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. justin julander 1 , kristiina shafer 1 , john morrey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa yellow fever virus (yfv) has caused significant morbidity and mortality for centuries. primates were the only animal models for visceral yfv. recently, hamsters were found to have morbidity and mortality when injected with a hamster-adapted jimenez strain of yfv (tesh et al., j. infect. dis. 183, 1431 -1436 . the objective of this study was to characterize this model of yfv viscerotropic disease for the study of effects of antiviral compounds and to test compounds with known efficacy for use as a positive control. animals were challenged with a 10 −4 dilution (a dilution previously shown to cause high mortality) of a liver homogenate made from livers taken 3 days post-viral injection (dpi) from hamsters challenged with the jimenez strain. there was 66% mortality in animals challenged with the virus up to 9 dpi. virus titers in the liver peaked 6 dpi as determined by qrt-pcr. a significant increase in serum levels of alt (6 dpi), alkaline phosphotase (6 dpi) and bilirubin (6 dpi), and a significant decrease in amylase (8 dpi), albumin (6 dpi), and glucose (6 dpi) were observed. hepatic icterus was observed in hamsters that exhibited disease signs at the time of necropsy. hamsters were treated with ribavirin or interferon (ifn) alfacon 1, a consensus interferon. ribavirin and ifn alfacon 1 both significantly (p < 0.001) reduced mortality as compared with placebo-treatment. there was also significant reduction in weight loss with ribavirin (p < 0.05) and ifn alfacon 1 (p < 0.01) treatment as compared with placebo. disease signs, such as lethargy and lying prostrate, were also reduced with treatment of ribavirin and ifn alfacon 1. viral liver titers from treated animals were not significantly different from titers in placebotreated animals. the hamster model of yfv disease will serve as a suitable model for the evaluation of antiviral compounds for efficacy against the virus. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. polyomaviruses are small dna tumor viruses that depend on the host cellular dna polymerase for their replication. three polyomaviruses have been associated with tumor formation in humans: jc virus (jcv), bk virus (bkv) and simian vacuolating virus (sv40). in addition, some of them have been associated with viral diseases. jcv can cause progressive multifocal leukoencephalopathy in immunosuppressed patients, while bkv is considered to be the causative agent of polyomavirusassociated nephropathy, which leads to kidney transplant failure. sv40 has not been associated with a well-defined disease, but viral dna sequences and protein expression have been detected mostly in central nervous system (cns) tumors which strengthens the evidence for the association of this virus with human cancer. the activity of various acyclic nucleoside phosphonates (anps) such as cidofovir and adefovir against murine polyomavirus and primate sv40 in vitro has already been demonstrated (andrei et al., 1998. antimicrob. agents chemother. 41, 587-593) . here, the activity of a new class of anp's, namely 6-[2-(phosphonomethoxy)alkoxy]-2,4diaminopyrimidines, against polyomaviruses was assessed. confluent uc1-b cells were infected with either of the four murine polyomavirus strains mn/rde toronto, pta, 2pta2 or lid-1, while bsc-1 cells were infected with either the primate sv40 strain a2895, the sv40 pml-1 strain ek or the sv40 pml-2 strain dar. after removal of the residual virus, serial dilutions of the test compounds were added. the viral cytopathic effect was recorded microscopically after 4-6 days (murine polyoma virus) or 5-7 days (sv40). hpmpo-dapy (2,4-diamino-6-(r)-[3-hydroxy-2-(phosphonomethoxy)propoxy]pyrimidine) and pmeo-dapy (2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine) were less active/selective than cidofovir and adefovir against the three sv40 strains tested. hpmpo-dapy and pmeo-dapy proved to be equally active as cidofovir and adefovir against the murine polyomaviruses. naresh sunkara 1 , sylvester mosley 1 , brian bakke 1 , joshua sadler 1 , katherine seley(radtke) 1 , sunny zhou 2 1 university of maryland-baltimore county, baltimore, md, usa; 2 washington state university, pullman, wa, usa inhibition of biologically significant enzymes critical to nucleotide metabolism and viral replication is a well-established chemotherapeutic approach to the treatment of many diseases. transcriptional 5 -capping of viral mrna has been implicated as an "elongation checkpoint" critical to the replication cycle of many viruses. this capping process is accomplished by various methyltransferases, therefore disruption of methylation becomes an attractive target for therapy. this can be accomplished in several ways; in particular, by direct inhibition of methyltransferases (metase) and/or indirect inhibition of s-adenosyl-l-homocysteine hydrolase (sahase), both established cellular targets for antiviral, antiparasitic and anticancer agents. modified nucleosides, in particular carbocyclic nucleosides, have exhibited potent inhibitory activity against both sahase and metase. inspection of the recent literature has revealed a close correlation between sahase inhibition and potent biological activity against negative stranded (−)-rna viruses (i.e. arenaviridae, paramyxoviridae, rhabdoviridae), double stranded (ą)-rna viruses (reoviridae), poxviridae, as well as hiv-1, thus supporting the importance of sahase as a viable chemotherapeutic target. herein we report the design, synthesis, and preliminary biological activity of a new class of structurally novel carbocyclic nucleosides. phosphorylation of ␣-p-borano substituted nucleoside diphosphates charlotta wennefors, mikhail dobrikov, barbara ramsay shaw chemistry department, duke university, durham, nc 27708-0346, usa most nucleoside antiviral agents require stepwise phosphorylation to their respective triphosphates in order to be activated in the cell. ␣-p-borano substituted nucleoside triphosphates are of interest because they have proven to be good substrates for hiv-1 reverse transcriptase (rt) and may therefore be useful antiviral agents. studies in our laboratory have indicated that the ␣-p-borano substitution of 2 3 -dideoxycytidine triphosphate (ddctp) resulted in a 28-fold increase in efficiency of incorporation by mmlv rt compared to non-substituted ddctp. however, the potency of these ␣-p-borano substituted nucleoside analogs as anti-viral drugs highly depends on their ability to be activated to nucleoside triphosphate (ntp). the phosphorylation of nucleoside analog diphosphates to their respective triphosphates has remained largely unexplored. here, the roles of several phosphorylating enzymes are examined. in our laboratory, nucleoside diphosphate kinase, creatine kinase, and pyruvate kinase are being evaluated for their specificity towards nucleoside analog diphosphates. the effects of nucleobase, ribose, ␣-phosphate substitution and stereochemistry of the boranophosphate group are of interest. the binding affinities of the substrates for creatine kinase (ck) and pyruvate kinase (pk) were determined using a fluorescence-quenching assay, which allowed us to investigate the substrate affinity in the pre-steady state. rabbit muscle ck and pk were titrated with a wide range of ndps and ntps by monitoring a decrease in enzyme intrinsic fluorescence. the affinities of these substrates were determined to establish a structure-activity relationship for ck and pk and to evaluate the effect of a substrate ␣-p-borano modification. ck showed stereospecificity towards the sp isomer of adp␣b whereas pk showed stereospecificity towards the rp isomer of adp␣b. negative cooperativity was observed for all studied substrates. steady-state experiments are also being performed directly following the product formation using uv-visible spectroscopy and high performance liquid chromatography (hplc). these kinases were investigated because they may serve as a means for activation of antiviral ␣-p-borano substituted ndps. traditional antiviral targets encoded by the small human papillomavirus (hpv) genome are lacking. for this reason, we chose to target dna sequences within the hpv genome in an effort to identify compounds that would block viral dna replication in cells. we chose compounds known as polyamides, which are related to distamycin and other natural products, as our dna binding agents. unlike many literature studies where polyamides were designed to block formation of the transcription complex for a particular gene, we chose to target sequences within the origin of replication (ori). thus, pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. the principles used to design these compounds will be described. we used "traditional" hairpin polyamides and some more unusual structures related to very recent literature reports. from the focused library that we prepared, two highly active molecules were identified. the rest of the molecules had minimal or zero activity. no cellular toxicity was observed, either in this project or in a related program where polyamides were used to affect cox-2 transcription (and subsequent expression) in rheumatoid synovial fibroblasts. of particular interest is the difference between the active molecules and two closely related compounds that were inactive: the active species bind and recognize two more hpv dna base pairs than do the related but inactive structures. this presentation will provide detailed chemistry background and structural information to complement our cell work that is also being presented at the meeting. discovery of the chemokine receptor ccr5 as a co-receptor for hiv-1 infections revealed a novel approach to hiv-1 treatments and preventions. ccr5, a member from the family of 7tm g-protein coupled receptors, thus became an attractive target pursued in the pharmaceutical industry. with the recent successful developments of several small molecules in clinic, these ccr5 antagonists hold great promise to be the next generation of anti-hiv medicines. this poster will describe our efforts at the n-terminal piperidine ring of template a to improve pharmacological properties of derived molecules. according to current models, proteolytic processing of hiv-1 gag precursor occurs within the virions which detach from infected cells. meanwhile, the viral protease is activated much earlier, and gag p55 cleavage initiates in infected cells. we followed the fate of matrix protein cleaved in infected cells (cma) in comparison with ma cleaved in the virions (vma) and showed that both forms differ in their localization in the infected cells and in the virions, both forms are involved into virus pathogenesis and represent the targets for antiviral compounds. mt-4 cells were labeled with [3h]-leucine or myristic acid, and 2 h after labeling protease inhibitor was added to separate the cleavage of cma from vma. cma was found in the nuclear and membrane fractions of infected cells while cca resided in cytoplasm. 18-20 h after labeling cma was found in the virions localizing in the cores. vma was located under lipoprotein envelope of the virions. new membranotropic antiviral compounds based on adamantane-and norbornene-related derivatives not toxic for the host cells were added to mt-4 cells before infection or 1-2 h later and at concentration 2-6 ug/ml blocked reverse transcription, the transport of cma into the nuclei, and the production of infectious virus. the compounds inhibiting very early step of virus life cycle are optimal candidates for microbicides. to enhance their antiviral activity, we plan to associate polyanionic matrix with ma imitating peptides and cholesterol-like fragments. kurt vermeire 1 , thomas bell 2 , sreenivasa anugu 2 , noah duffy 2 , roger le grand 3 , erik de clercq 1 , dominique schols 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 department of chemistry, university of nevada, reno, usa; 3 service de neurovirologie, fontenay-aux-roses, france the cyclotriazadisulfonamide (cada) compounds were shown to be potent inhibitors of hiv replication in human t-cell lines, pha-stimulated pbmcs, and monocytes/macrophages (ec 50 : 0.3-3.2 m). the prototype compound, cada, had consistent activity against laboratory adapted and primary clinical isolates of hiv-1, irrespective of chemokine receptor preference (r5, x4, r5/x4). cada acted synergistically when evaluated in combination with various other hiv drugs, such as reverse transcriptase (rt), protease, and virus entry inhibitors. flow cytometric analysis revealed a significant decrease in the cell surface and intracellular expression of the cd4 receptor in human cells after cada-treatment. moreover, the anti-hiv activity of cada correlated with its ability to down-modulate the cd4 receptor in human t-cells. here, we report the consistent antiviral activity of cada against: (i) drug-resistant viruses (i.e. viruses resistant to rt inhibitors, protease inhibitors, and enfuvirtide); (ii) different hiv-1 subtypes (a, b, c, d, a/e, f, h, o); and (iii) various hiv-2 strains examined. in addition, cada potently inhibited sivmac251 infection of pbmcs isolated from macaques (ec 50 : 1.6 m). comparable results were obtained in human cells infected with sivmac251. flow cytometric analysis also demonstrated a significant and dose-dependent down-regulation of the cd4 receptor expression at the cell surface of simian pbmcs after treatment with cada. the combination of cada with cellulose acetate 1,2benzenedicarboxylate (cap), an enteric coating polymer for capsules and tablets, resulted in a synergistic antiviral activity. in summary, our data indicate that cada may qualify as a potential anti-hiv microbicide drug candidate for the prevention of the sexual transmission of hiv. the preparation of gel formulations of cada (as single drug and in combination with cap) for vaginal administration in non-human primates is currently under investigation. department of micro & immuno, suny upstate medical university, syracuse, ny, usa varicella zoster virus (vzv, human herpesvirus 3) infection causes chicken pox, latency is established in neurons, and reactivation leads to shingles. acyclovir and its derivatives are the treatment of choice for both manifestations of vzv. new therapeutics are needed because acyclovir-resistant strains exist, and treatment must begin within 48 h. we have studied the anti-vzv properties of roscovitine, a cyclin dependent kinase (cdk) inhibitor. here, we tested more compounds that block the cell cycle and determined that vzv is acutely sensitive to them. their effects on vzv replication were tested in human foreskin fibroblasts (hffs) because these primary cultures should have a normal cell cycle (unlike tumor cell lines). the cytotoxicity of the drugs was determined by neutral red dye uptake assays. hffs were inoculated with a low moi (0.01) of vzvinfected cells, which remains entirely cell-associated, and then treated with drugs or diluent for 48 h. vzv spread and replication were measured by infectious focus assay and quantitative real time pcr. all of the drugs tested (acyclovir [acv], phosphonoacetic acid [paa], aphidicolin, aloisine a, purvalanol a, roscovitine, r-roscovitine, s-roscovitine, indole-3-carbanol [i3c], l-mimosine, dichloro-␤-d-ribofurano-sylbenzimidazole [drb]) had some anti-vzv activity. the selective indices of aphidicolin (833), purvalanol a (570), and i3c (1000) were greater than the positive controls acv (250) and paa (60). aphidicolin inhibits mammalian dna polymerase and is in clinical use for cancer; purvalanol a, a 2,6,9-trisubstituted purine, primarily inhibits cdk1; and i3c is derived from cruciferous vegetables and inhibits cell proliferation. the concentrations of these compounds that inhibited vzv replication were much less than those needed to cause cell cycle arrest, suggesting that vzv depends on the enzyme activities targeted by these compounds and not on cell proliferation per se. these three drugs will be studied next in skin organ culture and in the scid-hu mouse model of vzv pathogenesis. the results presented here demonstrate that targeting cell functions can inhibit vzv replication and help us better understand virus-host interactions. the viruses could be identified as supra-biopolymeric nanoscale complexes, parasitic intervention in cells of which occurs on an inter-polymeric reactions level. so the antiviral safety can not be fully provided without adequate nano-responsible antivirals (nav). here we discuss a strategy and methodology for the multi-functional nav development by rational macromolecular sar-cooperation of: (1) polyelectrolyte-specific interferon induction and immunomodulation; (2) electrostatic-selective prevention of viruses absorption on plasma membranes; (3) membrane-targeted blocking of post-absorption steps (fusion); (4) macromolecular prevention of structure-specific interactions of viral and cellular receptors; as well as (5) polymericassociated disruption of the latest stage of viral replication (virions assembly and maturation). a cooperation of the (1) and (2) functions was explored by synthesis and sar-optimization of succinate and carbohydrate polymeric derivatives modified with controllable combinations of anionic (a1/a2) groups. the immune-mediated protectors against tick-born, rabies, and other viral infections in vivo, and hiv-1 absorption inhibitors in vitro, were developed. this pre-nav generation was used as a macromolecular platform to step-by-step targeted design and synthesis toward high effective multi-functional nav where virusresponsible nano-selectivity was achieved by rational intra-or inter-molecular cooperation of virus-specific membranotropic vectors (bi), raft-targeted anchors (ci), and peptide-kind mimickers of virus usable receptors of human cells (pi), particularly ccr5/cxcr4. as a result, the novel nano-sensitive systems possessed unique wide multi-synergistic antiviral potency on a high level selectivity up to si = 20,000 (against hiv-1 strains) were created and purposed for advancement of antiviral vaccines, drugs, and microbicides. marina kukhanova 1 , alexander ivanov 1,2 , georgii galegov 3 , valeria andronova 3 , maxim jasko 1 1 engelhardt institute of molecular biology, russian academy of sciences, moscow, russia; 2 centre for medical studies, university of oslo, moscow, russia; 3 ivanovsky institute of virology, russian academy of medical sciences, moscow, russia novel acyclic purine phosphonate derivatives bearing a double bond conjugated with the nucleic base, namely, (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines, were synthesized, and their efficacies against hiv-1 and hsv-1 were evaluated in cell cultures. the activity of (z)isomer was higher against hiv than that of the reference 9-[2-(phosphonomethoxy)ethyl]adenine (adefovir) and comparable in respect to the activity of adefovir against hsv. the (e)-isomer showed low antiviral activity against both viruses. the compounds were less toxic towards cell cultures if compared to adefovir. the diphosphates (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines were evaluated as substrates towards hiv-1 reverse transcriptase and hsv dna polymerase. (z)-isomer was shown to be a more efficient substrate for both enzymes than the (e)-isomer. human dna polymerase alpha could incorporate neither of the diphosphates into the 3 -end of the growing dna chain. available to this virus. jev genome is an approximately 11-kb single-stranded positive-sense rna that has a cap structure at its 5 terminus but lacks a poly(a) tail at its 3 -terminus. the coding region of the genome is flanked by 5 -and 3 -untranslated region (utr). the 3 -utrs on both plus-and minus-strand jev genome contain important cis-acting elements required for the replication of the viral rna genome. peptide nucleic acid (pna) is a synthetic oligonucleotide, in which the phosphodiester backbone of dna/rna is replaced with a polyamine-(2-aminoethyl) glycine skeleton. pna offers a potentially powerful approach for recognition of rna and silencing of gene expression. in this study, we investigated the antiviral effect of the pnas targeted to the 3 -utr region of jev genome. to evaluate the pnamediated inhibitory effect on rna synthesis in vitro, the rnadependent rna polymerase (rdrp) of jev, ns5 protein, which plays a major role in replication of the viral genomic rna, was expressed in escherichia coli and purified to near homogeneity by sequential column chromatographies. the recombinant jev ns5 protein exhibited a primer-dependent rdrp activity in vitro on a homopolymeric template, poly(a). in addition, it was able to accept both plus-and minus-strand 3 -utrs as templates for rna synthesis in the absence of an exogenous primer. it could utilize the 3 -end 83-nt of jev genome as a minimal template. in vitro rdrp assays using this functional recombinant jev rdrp in the presence of the pnas targeted to the jev 3 -utr 83-nt showed a dose-dependent rna synthesis inhibition. delivery of the inhibitory pnas to the jev-infected cells suppressed jev replication, as determined by western-blot analyses and plaque assays. our results showed a sequence specific inhibition of jev replication by antisense pnas, suggesting the possible application of pna as a novel anti-jev agent. julia serkedjieva 1 , reneta toshkova 2 , milena nikolova 3 , reneta tsvetkova 3 , stefka antonova 4 , ivana roeva 1 , munnever sokmen 5 , bektas tepe 5 , medine gulluce 6 , fikrettin sahin 6 , atalay sokmen 5 1 institute of microbiology; 2 institute of experimental pathology and parasitology; 3 institute of botany, bulgarian academy of sciences; 4 faculty of biology, department of microbiology, sofia university, sofia, bulgaria; 5 faculty of art and science, department of biology, cumhuriyet university, sivas, turkey; 6 faculty of art and science, department of biology, atatürk university, erzurum, turkey natural products can be an important source of new pharmaceuticals. research on antivirals of natural origin is mainly focused on plants, since, among other reasons, they can be selected on the basis of their ethnobotanical use. plant extracts and natural plant products exhibit also a variety of biological activities with pharmacophoric utility. the population of the balkan peninsula, like people from all continents, has long applied poultices and imbibed infusions of hundreds of indigenous plants. the present report summarizes the antiviral screening study of 134 plant products, obtained from 67 bulgarian and turkish medicinal plants. they were tested for inhibitory effect on the reproduction of selected influenza virus (flu) strains in mdck cells and herpes simplex virus (hsv) strains in mdbk cells. the reduction of virus-induced cpe and infectious virus yields were used as measures of viral inhibition. fifteen samples (11.2%) inhibited flu reproduction, and twelve samples (7.5%) were active against hsv. the anti-flu activity was confirmed in vivo for all tested samples. the most effective products were tested further for their antiproteolytic, antioxidant and immunogenic capacities and for potential antibacterial and antifungal effects. the following correlations among the variety of biological and pharmacological activities of the plant products were observed: the anti-flu effect was associated with anti-hsv effect and vice-versa in 55.5%; the antiviral effect was connected with antioxidant activity in 100%; the anti-flu effect was associated with immunogenic properties in 100%; there was found no correlation between the antiviral effect and the antiproteolytic capacity, the anti-viral properties and bacterial or fungal inhibition, the anti-viral activity and the polyphenol contents. our previous investigations have revealed antiviral activity of some proteolysis inhibitors such as e-aminocaproic acid (e-aca) and para-aminomethylbenzoic acid (pamba) in vitro, in vivo and in clinic. construction of qsar computer-assisted hierarchical system for the effective anti-herpetic (anti-hsv) and anti-influenza (anti-flu) agents' selection as well as the elaboration of new methods of their synthesis are permanently the object of keen interest of our team. the objective of this study was to investigate the efficacy 2,6-di-substituted pyridines and their analogs combined with the fragments of proteolysis inhibitors in the framework of the qsar approach. molecules of new compounds consisted of "nucleus" (py or ar) and two symmetrical fragments: e-aca-carbonyl or pambacarbonyl taken from the inhibitors' molecules. anti-flu activity in dose 10-3 m was studied in vitro on the model of a/hong kong/1/68 (h3n2) reproduction in tissue cultures of chorioallantoic membranes of 12 days old chick embryos. anti-hsv activity in doses 10-4 m was studied on models of reproduction of hsv-1 on cell culture hep-2. compounds with py-nucleus, contained pamba-carbonyl or e-aca-carbonyl fragments, demonstrated sufficient anti-hsv activity (39.4 and 61% of reduction of intra-nucleus virus-specific inclusions on infected cells account accordingly). 3,5-dihydrazine-carbonyl-2,6-dimethylpyridine showed high anti-hsv (52%) activity. the efficacy of the designed antiherpetic compounds obtained with the combined efforts of qsar computer-assisted design, properties prediction, synthesis, and biological testing as well as the correction introduced after the iteration circle passsage have proven to be the efficient modern way of drug design. acknowledgement: this research was supported in part by stcu grant # 3147 and all the authors are indebted to stcu foundation courtesy. lubomira nikolaeva-glomb 1 , angelina trifonova 1 , stephan filipov 2 , angel s. galabov 1* 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria a series of aporphinoid alkaloids isolated from glaucinum flavum l. or obtained synthetically, were tested in vitro for antiviral activity against viruses belonging to picorna-, orthomyxo-, paramyxo-and herpesviruses. one of them, oxoglaucine, manifested a well-pronounced inhibitory effect on poliovirus 1 replication in fl cells measured by the semi-quantitative agardiffusion plaque-inhibition test. in virucidal activity testing the compound did not show direct virucidal effect on the extracellular virus. oxoglaucine's 50% inhibitory concentration for poliovirus 1 (mahoney) was found to be 0.188 g/ml in the cpeinhibition test and 0.041 g/ml in the classical plaque-inhibition test. similar values were obtained for the vaccinal poliovirus type 1 strain, lsc-2ab. the antiviral effect of oxoglaucine on the replication of viruses belonging to another enterovirus species was tested, i.e. coxsackie and echoviruses (hev-b). cva-9, the six coxsackie b viruses and 6 echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine by the endpoint dilution method in the multi-cycle cpe inhibition set-up in fl cells. oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. the concentrations that reduced virus titer by 1 lg ranged from 0.01 to 1.0 g/ml. selectivity index was greater than 100 and even greater than 1000 for some of the viruses tested. time-of-addition study by the one-step virus growth cycle set-up showed strong virus inhibition during the early periods of virus replication. milka mileva 1 , angel s. galabov 2 1 department of medical physics and biophysics, medical university, sofia, bulgaria; 2 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria in this study an investigation and comparison of the effects of plant flavonoid polyphenols quercetin and its sugar-containing homologue (rutinoside) rutin on the "oxidative stress" in liver, isolated from influenza virus a/aichi/2/68 (h3n2) (2.0 of ld50) inoculated mice, is carried out. it was found that experimental influenza virus infection is accompanied with graduated oxidative disturbances in the liver of mice, despite the absence of virus and inflammation in this tissue. it was found that experimental influenza virus infection is accompanied with a significant increase of lipid peroxidation products, a decrease of natural antioxidants (vitamin e, glutathione) and cyp, an inhibition of cytochrome c-reductase and liver monooxygenases (analgin-ndemethylase and amidopyrine-n-demethylase) as compared to control (non-infected) animals. the preliminary (5 days) supplementation of mice with rutin, quercetin or its combination, and their subsequent virus inoculation influence significantly all analyzed parameters as compared to controls. the protective effect of rutin against influenza virus-induced lipid peroxidation and activities of cyp and liver monooxygenases in liver was better expressed than the effect of quercetin may be due to containing of rutinoside part or difference of its metabolism. hyun-jeong lee 1 , ji-sun kwon 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea one of the traditional korean medical herb extract named s22 was investigated to determine the anti-influenza virus activity in vitro and in vivo. the s22 showed potent antiviral activities against a/pr/8/34 (h1n1) influenza virus with the 50% effective concentration (ec50) values of 62.5 g/ml and the 50% cytotoxic concentration (cc50) values of 673.86 g/ml in mdck cells. treatment with the s22 appeared capable of significantly ameliorating the influenza virus infection in mice by oral gavage treatment. the s22 treated mice showed significantly higher survival rate and lower pathogenic index as well as lung virus titers than untreated control mice. further, the s22 was extracted with chcl3, etoac and n-buoh for isolation of active compounds. the anti-influenza effects of these active compounds will be discussed. the antiviral effect of aqeous and ethanol extracts of ocimum gratissimum (og), terminalia catappa (tc), gynostemma pentaphyllum (gp), newbouldia laevis (nl), aspilia africana (aa) and phyllantus amarus (pa) leaves was examined by cultivation of virus and extracts in embryonated chicken eggs. extracts were inoculated immediately after virus (zero time) or 2 h after virus inoculation. virus replication was compared with those of controls by haemagglutination assay. at zero time, aqeous extracts of og, tc, pa, and gp inhibited virus growth by 50, 61, 72, and 94%, respectively whereas those of nl and aa did not. ethanol extracts of og, tc, pa and gp at same time inhibited by 25, 100, 72, and 100%, respectively whereas nl and aa did not. at two h after virus inoculation aqeous extracts of og, tc, pa and gp inhibited virus growth by 92, 6, 67, and 100%, respectively whereas nl and aa had no effect. ethanol extracts of tc, pa and gp inhibited the virus by 94, 78, and 94%, respectively whereas those of og, nl, and aa did not. the herbs were studied because they were being used by some herbalists in the treatment of human infectious diseases. the 20th international conference on antiviral research will be held in the palm springs, california area. the conference will begin on sunday, april 29, 2007 and will end on thursday afternoon, may 3, 2007. all scientific sessions will be held at the westin mission hills resort, rancho mirage, ca. the purpose of the international conference on antiviral research is to provide an interdisciplinary forum at which investigators involved in basic, applied, and clinical research worldwide can meet to review recent developments in all areas of antiviral research. specific topics to be covered in the program include synthesis and chemistry, biochemistry and mechanism of action, molecular biology and drug targeting, in vitro evaluation, animal models, pharmacokinetics, toxicology, and clinical trials. within these areas of interest, there will be invited overview speakers, oral presentations, and poster presentations. the famous el paseo shopping district of palm desert and downtown palm springs offer not only a large variety of galleries, boutiques and shops too numerous to mention, but there are restaurants for virtually every palate. whether your tastes run to burgers, sushi, pizza, escargot, steak, mexican or continental you will find it here with a california flourish in every price range. we hope you will take advantage of this opportunity to combine an important learning experience with a magnificent travel experience and join us in palm springs, california for the 20 th international conference on antiviral research. isar conference committee future conferences acknowledgement: the study was supported by rfbs 05-04-49500 and russian ministry of sciences (lot 11). key: cord-006381-fsg9x8n7 authors: howard, o. m. zack; oppenheim, joost j.; wang, ji ming title: chemokines as molecular targets for therapeutic intervention date: 1999 journal: j clin immunol doi: 10.1023/a:1020587407535 sha: doc_id: 6381 cord_uid: fsg9x8n7 despite the youth of the chemokine field, many antagonists of chemokine function have already been identified and tested at the preclinical level. these include neutralizing antibodies, peptidyl and non-peptidyl antagonists and non-specific immunosuppressive agents. these early studies suggest that chemokine agonists have the potential to regulate many diseases, ranging from hiv-1 infection and tumor growth to acute and chronic inflammation. clinical application will depend on pharmaceutical development. great strides have been made in defining structural domains of the chemokines involved in receptor binding and activation. the identification of receptors is rapidly progressing, but with 50 potential ligands and 15 characterized receptors, it is obvious that additional molecular studies are needed. the intriguing observation that several pathogens either use chemokine receptors as entry portals or produce chemokine decoys to subvert the immune system suggests that there is much to be learned about the immune system from studies of “virokines.” future studies should lead to the discovery and design of more effective inhibitors and antagonists with therapeutic benefit. ing aa between the first two of four or six cysteines (1) (2) (3) (4) . in addition, members of the defensin subfamily, which, at 4 kda, are half the size of cc chemokines, and have six cysteines with variable numbers of intervening aa's, also interact with one of the cc chemokine receptors (5) . a given receptor can interact with up to eight chemokines, and a given chemokine can interact with up to four receptors. furthermore, cells express multiple receptors. consequently, as is true of other cytokines, chemokines are subject to considerable redundancy in their activities. this is not the case for fractalkine, the only member of the cx3c subfamily, which interacts with cx3cr1 and lymphotactin, the only chemokine with one pair of cysteines, which interacts with xcr1. despite the considerable overlap in receptor utilization, studies of mice with "knocked-out" chemokine genes often reveal unique selective defects indicative of specialized and nonredundant in vivo chemokine functions. deletion of receptor genes usually has more wideranging consequences than ligand knockouts because multiple chemokine-induced responses are interfered with. it is, therefore, simpler to focus on the biological effects of activating a given receptor than to consider each individual ligand (tables i and ii) . chemokines are multifunctional. they chemoattract a variety of leukocytes and nonleukocytic cells to sites of inflammation and injury. chemokines also activate cells engaged in host immune responses, exhibit angiogenic or angiostatic effects, modulate hematopoiesis, promote fetal development, and regulate trafficking and homing of cells to appropriate tissue sites (tables i and ii) . overall chemokines contribute to various aspects of host defense and many of the chemokines have been detected in a wide variety of disease states involving inflammation, angiogenesis, and tissue injury and in neoplastic tissues (6) . although a number of chemokine-induced activities are essential for survival, chemokines can also mediate deleterious self-destructive effects and inhibitors can prove therapeutic. for example, chemokines contribute to self-destructive inflammation in autoimmunity, 280 0271-9142/99/0900-0280$ 16 .00/c 1999 plenum publishing corporation key words: chemokines; chemokine receptors; antagonists. over 50 members of the chemokine family have been identified to date. they activate a family of g proteincoupled seven-transmembrane receptors (stm) expressed by a wide variety of cell types. five cxc receptors have been identified that interact with the cxc subfamily of chemokines, which are characterized by having one amino acid (aa) between the first two of their four cysteines. nine cc receptors interact with members of the cc chemokine subfamily, which lack an interven-arteriosclerosis, and reperfusion injury, and they may promote tumor growth through angiogenesis. some viruses have subverted host defense mechanisms by utilizing chemokines or their receptors to their own advantage either as sites of cell entry as in the case of hiv-1 (7), by producing chemokine receptor analogues as by hhv8, which produces an il-8-like receptor (8) , or by producing antagonists that interfere with chemokine actions as in the case of mcvi and ii and the pox viruses (9) . imitation is the highest form of flattery, and viruses as well as knockout mice are teaching us the potential therapeutic benefits of targeting chemokines or their receptors. in this article we review the utilization of neutralizing antibodies, peptidyl and nonpeptidyl antagonists, and general suppressive agents that can be used to inhibit undesirable chemokine effects. the first group of chemokine inhibitors to be evaluated was neutralizing antibodies. these have the benefit, especially in the case of monoclonal antibodies, of having very specific inhibitory effects. antibodies have the disadvantage of being orally inactive, however, they have a relatively prolonged half-life and weekly injection can have prolonged inhibitory effects. the development of recipient immune responses to administered xenogeneic antibodies can be circumvented by using fully humanized antibodies obtained from transgenic mice (10) . consequently, reports of beneficial antichemokine therapy represent more than proof of principle and can be used in man. the first dramatic demonstration of the beneficial effects of inhibiting chemokine mediated inflammatory activities involved rabbits undergoing temporary pulmonary artery occlusion to induce hypoxia followed by reperfusion injury. prior administration of antibody to il-8 reduced the adhesion of neutrophils to the injured rabbit lung tissues and consequent morbidity and mortality due to this reperfusion injury (11) . a similar approach using anti-il-8 was subsequently shown to reduce the degree of stroke induced cns damage due to occlusion of the cerebral arterial blood supply (12) . anti-mcp-1 can also reduce myocardial reperfusion injury (13), suggesting both il-8 and mcp-1 induced by hypoxia can exacerbate posthypoxic inflammation. anti-il-8 treatment has also been shown to reduce the degree of inflammation and mortality of acute respiratory disease syndrome (ards) (12) and of acute glomerulonephritis (14) . anti-il-8 can inhibit tumor growth and metastases by interfering with the angiogenic effects of tumor produced il-8 (15) . anti-mip-1 a administration journal of clinical immunology, vol. 19, no. 5, 1999 can reduce chronic relapsing eae, while anti-mcp-1 blocks the initiation of eae (16) . however, one has to be judicious in blocking chemokine functions because this can reduce needed host defense as shown by studies in which anti-mip-2 (gro analogue) treatment of mice exacerbated klebsiella pneumonia infections (17) . there are many more reports and an earlier review of the beneficial effects of antibodies to chemokines and their receptors that demonstrate their potential utility in man (4). peptide antagonists of chemokines/chemokine receptors (table iii) are mostly generated based on structurefunction analyses of chemokine ligands. several aa residues at or near the n termini of the chemokines are crucial for receptor binding and/or signaling. the receptor binding domain and signaling domain of a chemokine are often separated. thus, an ideal peptide antagonist should be able to compete with the wild-type chemokine for binding to a high-affinity receptor, yet not initiate cell activation but, rather, block cell activation by the wildtype chemokine. libraries of large synthetic and natural peptide repertoires have been used to identify specific chemokine receptor antagonists. generally, the affinity of a peptide antagonist for a given receptor correlates with its length. the shorter the peptide, the lower the affinity. therefore, higher concentrations of the shorter peptide antagonists are required for competitive inhibition of receptor activation by wild-type chemokines. another intrinsic weakness of peptide antagonists lies in their potential antigeniclty and susceptibility to proteolytic degradation. this can be partially compensated for by designing shorter peptide sequences at the expense of reduced receptor affinity or by modification of the sequence by adding additional chemical groups or by using d-aa's, which are resistant to mammalian enzyme degradation. despite these limitations, many investigators have generated a variety of chemokine receptor-specific peptide antagonists that exhibit promising inhibitory effects on chemokines in various in vitro and animal models. due to the prominent role of il-8 in a variety of inflammatory conditions, considerable efforts have been made to develop peptide antagonists which may specifically bind to the receptors for il-8, but do not elicit signal transduction. moser et al. designed and synthesized a number of il-8 analogues which do not contain an intact elr motif (18) . one of those analogues, termed il-8(6-72), exhibited specific il-8 antagonist activity. il-8(6-72) displayed weak chemotactic activity for neutrophils at 1-10 mm concentrations but did not induce enzyme release or a respiratory burst. in contrast, it displaced binding of iodinated il-8 to neutrophils and potently inhibited il-8-induced neutrophil chemotaxis, mediator release, and respiratory burst. these results suggest that il-8(6-72) is a partial agonist which interferes with the signaling of the parental molecule. this property of il-8(6-72) was thought to be determined by the degree of receptor occupancy and the threshold of activation of a given signaling cascade. of particular interest is the capability of il-8(6-72) also to inhibit elastase release induced in neutrophils by gro and nap-2, two cxc chemokines with an elr motif that share the receptor cxcr2 with il-8. since il-8 is often generated together with other elr+ cxc chemokines under various pathological conditions, it is highly desirable for an antagonist to diminish or prevent the activity of all chemokines interacting with cxcr2. an analogue of the il-8-related chemokine gro/ mgsa was recently described by baly et al. (19) . the mutant mgsa h19a showed 100-200-fold lower agonist activity on neutrophils than the wild type of gro/ mgsa, in eliciting neutrophil chemotaxis, elastase release, and up-regulation of cd18. however, in receptor binding competition experiments, h19a showed only a 13-fold decrease in its affinity for cxcr2. furthermore, preincubation of cxr2 expressing cells with h19a inhibited cell migration, enzyme release, and calcium mobilization in response to wild-type mgsa. it appears that h19a binds to the receptor with a relatively high efficacy but is defective in inducing receptor signaling. this dissociation between receptor binding and activation is the basis for the design of specific peptide antagonists. regrettably, a long peptide antagonist has very limited therapeutic promise, since it is often highly antigenic and subject to rapid proteolytic cleavage, resulting in a short half-life in vivo. it is therefore necessary to develop shorter peptide antagonists which are less antigenic and easily modified to increase their resistance to enzyme degradation. these considerations led to the development by hayashi et al. of a hexapeptide, rrwwcr, which does not bear significant homology to il-8 or related cxc chemokines (20) . the l-aa rrwwcr, with an acetylated amino terminus and amidated carboxyl terminus (ac-rrwwcr-nh2), and the d-aa, ac-rrwwcr-nh 2 , are potent inhibitors of il-8 binding to human neutrophils and consequently inhibited il-8-induced neutrophil chemotaxis and enzyme release. twenty-five-fold lower doses of ac-rrwwcr-nh 2 were required to inhibit degranulation and rabbit skin edema in response to human il-8 than are required for inhibition of in vitro chemotaxis (20) . the advantage of these peptides lies in their lack of any agonist activity for il-8 receptors and greater ease of synthesis. the d-aa peptide appears to be the more potent il-8 receptorspecific antagonist. this may improve its therapeutic application since mammalian proteases cannot degrade d-aa peptides, thus resulting in a longer half-life in vivo. the peptide ac-rrwwcr-nh 2 has been named antileukinate and has been shown to inhibit the neutrophil accumulation in rabbit lungs in response to staphylococcal enterotoxin (21) . furthermore, antileukinate also inhibited the autocrine growth stimulating activity of mgsa/groa for several melanoma cell lines, but not the growth of a control hepatocyte cell line which was not mgsa/groa dependent (22) . in a nude mouse model, antileukinate inhibited the growth and incidence of lung metastasis of mgsa/groa-dependent human melanoma cells (23) . surprisingly, fujisawa et al. reported an autocrine growth stimulating effect of mgsa/ groa on human umbilical cord vein endothelial cells (huvec), and this effect was markedly inhibited by antileukinate through the obstruction of a binding site on huvec for mgsa/groa (24) . these observations suggest that this specific antagonist of cxcr2 may also have angiostatic effects. antileukinate is a promising candidate for further modification to yield more potent antagonists. several mcp-1 peptides have been generated based on the structure of mcp-1. zhang et al. used mutagenesis studies to determine the secondary structure of mcp-1 and identified several residues which are potentially important for receptor binding and function (25) . several mutated mcp-1 analogues, termed 7nd, r24f, y28d, and d3a, were able to inhibit monocyte migration in response to wild-type mcp-1, either by receptor competition or by forming heterodimers, which interferes with the normal function of the wild-type mcp-1. however, more potent peptide antagonists were generated by truncation of the n terminus of mcp-1. gong et al. reported that the n-terminally truncated mcp-1 analogues aa 8-76, 9-76, and 10-76 desensitized calcium flux in monocytes induced by wild-type mcp-1, while these analogues were themselves biologically inactive (26) . the most efficacious antagonist, mcp-1(9-76), competed for binding with an affinity only threefold lower than that of the wild-type mcp-1. mcp-1(9-76) also desensitized calcium flux in monocytes induced by mcp-3, which shares the receptor with mcp-1, but did not inhibit the signaling by cc chemokines that do not use ccr2. thus the antagonist activity of mcp-1(9-76) is receptor specific. this peptide was evaluated as a treatment of a mpl-1 pr mouse model of arthritis characterized by a monocyte-mediated chronic inflammatory response similar to human arthritis (27) . daily injection of mcp-1 prevented the onset of arthritis as measured by the degree of joint swelling and histopathology. injection of the antagonist even after the onset of arthritis resulted in a marked reduction in symptoms and histopathology in a substantial number of the ani-mals. in contrast, injection of the wild-type mcp-1 enhanced arthritic symptoms in mice. these results suggest a beneficial effect can be achieved using an mcp-1 receptor antagonist peptide in the treatment of an autoimmune disease despite the involvement of multiple cytokines and chemokines. since several chemokines may be produced at a given site of inflammation, antagonists of multiple chemokines may be more effective than antagonists of a specific chemokine to control cell migration and activation. gong et al. developed such multichemokine antagonists in the form of a number of truncated analogs of rantes, mcp-1, and mcp-3 (28). on the basis of their ability to compete for binding with their parental chemokines, three analogues were selected including rantes(9-68), mcp-3( 10-76), and mcp-1(9-76). these analogues bound to monocytic cells with a four-to six-fold lower affinity than the wild-type chemokines, but they did not induce cell migration or mediator release. ran-tes(9-68) competed for the binding and biological activity of wild-type rantes, mcp-1, and mcp-3. in contrast, native rantes inhibited only its own binding to cells. in addition, mcp-3(10-76) also inhibited the activity of wild-type rantes, mcp-1, and mcp-3 on monocytic cells. these observations suggest that modification of the n-terminal residues results in the generation of chemokine analogues which exhibit a broader range of receptor specificity. however, whether these deletion mutants are more effective in the treatment of disease models involving multiple chemokines remains to be determined. cxcr4 is a receptor for sdf-1 and serves as a fusion coreceptor for the t lymphocyte tropic and laboratory adapted strains of hiv-1. thus, the development of antagonists to cxcr4 becomes potentially useful in inhibiting hiv-1 infection. murakami et al. reported a peptide termed t22 [tyr5, 12, ly7)polyphemusin ii] consisting of 18 aa residues and an analogue of polyphemusin ii isolated from the hemocyte debris of american horse shoe crabs (limulus polyphemus) (29) . t22 specifically inhibited the ability of t-tropic hiv-1 virus to fuse with and infect peripheral blood mononuclear cells and cell lines transfected with cd4 and cxcr4. although the primary sequence of peptide t22 is unrelated to the cxcr4 ligand sdf-1, mechanistic studies revealed that t22 attenuated cxcr4-mediated calcium flux in response to sdf-1, but not ccr2-mediated signaling induced by mcp-1. since cxc chemokines have been proposed to possess a core structure of three antiparallel b sheets, it was postulated that the anti-cxcr4 property of t22 may be based on its one antiparallel b-sheet structure maintained by two disulfide bonds in t22 (29) . the cxcr4 specific inhibitory activity of t22 was confirmed by tamamura et al. (30) . a number of studies have focused on generating peptides derived from the structural analysis of sdf-1. the three-dimensional structure of sdf-1 was determined by nmr spectroscopy, which revealed sdf-1 to be a monomer with a disordered n-terminal region (31) . the 1-8 aa residues in this region form an important receptor binding site. however, only lys-1 and pro-2 seemed to be directly involved in receptor binding. modification of lys-1 and or pro-2 resulted in the loss of agonist activity of sdf-1. such a mutated sdf-1 analogue acts as a potent antagonist of cxcr4 by competitively interfering with the binding of wild-type sdf-1. furthermore, a dimeric peptide derived from residues 1-9 (p2g) was also a cxcr4 antagonist (32). heveker et al. generated a series of peptide domains of sdf-1, each comprising 13 aa residues, spanning the whole sdf-la sequence (33) . the antiviral and signaling properties of sdf-1 were retained by a 13-aa peptide corresponding to its amino terminus. as reported by crumps et al., removal of the first two residues yielded an antiviral sdf-1 analogue that also antagonized sdf-1-induced cxcr4 signaling. the investigators also identified a single-mutation analogue of sdf-1 which exhibited anti-hiv-1 activity but did not activate cxcr4 or antagonized the cxcr4 signaling induced by wild-type sdf-1. however, this anti-hiv-1 analogue of sdf-1 competed with monoclonal anti-cxcr4 antibody 12g5 for binding to cxcr4. these results indicated that the amino terminus of the sdf-1 is sufficient for signaling through cxcr4 and for inhibition of hiv-1 entry. however, these activities could be dissociated in a peptide analogue. therefore, peptide analogues derived from different domains of sdf-1 can antagonize selected activities and dissociate different functions of cxcr4. ccr5 and cxcr4 are crucial coreceptors for hiv-1 fusion with the host cells by interacting with viral envelope proteins during infection. studies with nterminally modified cc chemokine rantes, aop-rantes, which is a potent antagonist of ccr5, suggest that receptor internalization and inhibition of receptor recycling may represent a promising approach to inhibition of hiv-1 infection (34, 35) . the mechanism of hiv-1 inhibition by aop-rantes is based on a rapid decrease in cell surface expression of ccr5 on monocytes, macrophages, and t lymphocytes. more importantly, aop-rantes inhibited the subsequent recycling of internalized ccr5 to the cell surface, while the native rantes did not. this endows aop-rantes with a greater than wild-type rantes capacity to inhibit hiv-1 infection. extension of recombinant human rantes by a single aa methionine at the n terminus also is sufficient to produce a potent and selective antagonist of binding and cell activation by chemokines that share the receptors with rantes, such as mip-la and mcp-3 (36). met-rantes was shown to inhibit actin polymerization, release of oxygen intermediates, and calcium flux by human eosinophils stimulated with native rantes, mcp-3, and eotaxin (37) . therefore met-rantes may be an effective inhibitor of the accumulation of potentially self-destructive eosinophils in allergic diseases. in contrast, the addition of a methionine to the cxcr4 ligand sdf-1b resulted in an analogue which was more potent than wild-type sdf-1b in inducing calcium flux by cxcr4 expressing cells (38) . met-sdf-lb also induced the internalization of cxcr4, but with a potency comparable to that of native sdf-1b. the differences in receptor interaction between met-rantes and met-sdf-lb suggests that the functional consequences of modifying the n-termini is dependent on individual chemokines. a very creative approach favoring the retention of chemokine receptors ccr5 and cxcr4 has been described (39, 40) . rantes, mip-la, or sdf-la genetically engineered to express an endoplasmic reticulum retrieval peptide, kdel, at the c termini can retain newly synthesized coreceptors ccr5 or cxcr4 intracellularly, possibly by forming complexes. lymphocytes transfected with these modified chemokines (termed intrakines) are viable and resistant to hiv-1 infection. these observations further support the feasibility of using a receptor internalization and degradation strategy to prevent hiv infection. this section focuses on small nonpeptidyl antagonists of chemokine function that target chemokine receptor(s) and their in vitro and in vivo effects and potential therapeutic uses. the interaction of the chemokines and their receptors offers a target for the identification of selective small molecular antagonist to further define biochemical functions and to treat pathophysiological conditions. small nonpeptidyl antagonists, unlike peptide-based antagonists, have the advantage of not inducing a neutralizing immune response. another advantage of this class of antagonists is that, by modifying the delivery methods, bioavailability and toxicity problems can be reduced. the biggest challenge to the development of small nonpeptidyl antagonists is the initial identification of the prototype compounds; fortunately several programs have been successful at identifying antagonists of chemokine function. (table iv) . sb225002. the first selective nonpeptide antagonist for cxcr2, sb225002, was reported in 1998 (41) . sb225002 was identified using a combination of high-throughput screening based on inhibition of ligand binding and targeted drug design. the chemical formula for sb225002 is 7v-(2-hydroxy-4-nitrophenyl)-n'-(2bromophenyl urea). sb225002 selectively inhibited il-8-and groa-induced calcium mobilization by cxcr2 transfectants with 50% inhibitory concentrations (ic50) of 20 and 40 nm, respectively. in contrast, sb225002 did not inhibit il-8-induced calcium mobilization by cxcr1 transfectants. sb225002 inhibited neutrophil migration to both il-8 and groa in vitro, with ic50 values in the 20-60 nm range and inhibited neutrophil migration to il-8 in a rabbit model following intravenous administration. the development of sb225002 will provide an opportunity to dissociate cxcr1 and cxcr2 signals and assess their respective contributions to leukocyte recruitment in inflammatory disease. sb225002 has potential clinical applications as a suppressor of neutrophil recruitment in inflammatory conditions such as adult respiratory distress syndrome, reperfusion injury, chronic bronchitis, and asthma. since journal of clinical immunology, vol. 19, no. 5, 1999 (42) . antiviral activity was recently shown to be due to interaction with the hiv-1 co-receptor, cxcr4 (43, 44) . the prototype compound amd3100 is the octahydrochloride dihydrate of 1,1'-[ 1,4-phenylene-bis(methylene)]-bis-1,4,8,11 -tetraazacyclotetradecane. the antiviral bicyclams target only t-cell trophic (x4) virus and induce the development of an amd3100-resistant viral strain (45, 46), which also abrogated the inhibitory effect of exogenously added sdf-1. resistance to both amd3100 and sdf-1 is due to modification of the gp120 v3 loop (47). recent tissue culture studies suggest that prolonged amd3100 treatment will cause a shift to the less virulent r5 virus, but further in vivo studies are needed (48). the ic 50 for amd3100 antiviral activity in vitro was 1-10 nm. inhibition of binding by anti-cxcr4 was also observed in this range. inhibition of sdf-1a-induced calcium flux and receptor binding required about 10-fold less amd3100. the in vivo efficacy of amd3100 has not been reported. the potential for amd3100 to be used as an anti-hiv-1 and hiv-2 therapeutic is obvious. because cxcr4 and its ligand, sdf-1, are essential for embryonic vascular development (49, 50), it is tempting to speculate that amd3100 may interfere with the angiogenic effect of sdf-1 in tumor neovascularization and endothelial cell migration (51). recently, hesselgesser et al. reported the identification of a 4-hydroxypiperidine compound that selectively inhibits ligand binding to ccr1 (52). several 4-hydroxypiperidine compounds with ic 50 values less than 5 um were identified by a high-throughput assay that measured inhibition of mlp-la binding to ccr1. the prototype molecule has the chemical formula of (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl) valeronitrite and is referred to as compound 1. the ic 50 values for compound 1 inhibition of mlp-la and rantes binding to ccr1 were 41 and 61.5 nm, respectively. the ic 50 values for mip-la or rantes binding competition to ccr1 were 2.1 and 21.5 nm, respectively. compound 1 also inhibits mip-la-and rantes-induced cellular acidification and migration in the same 41-61 nm concentration range. compound 1 did not inhibit ligand binding to 18 other surveyed stm g protein-coupled receptors including ccr5, cxcr2, and cxcr4. the in vivo efficacy of compound 1 has not been reported. thus, compound 1 is a potent, selective in vitro antagonist of ccr1. ccr1 is one of the most promiscuous cc chemokine receptors, having eight reported ligands; compound 1 can be used to demonstrate the domains of ccr1 commonly used by each of these ligands to transduce the chemotactic signal. compound 1 has the potential to address the role of mip-la and rantes binding to ccr1 versus ccr5 in several pathophysiological conditions such as rheumatoid arthritis and asthma (53-55). due to the role of mip-la in experimental autoimmune encephalomyelitis (eae), one can speculate that compound 1 will be an important therapeutic for the treatment of chronic inflammatory conditions in the central nervous system such as multiple sclerosis (56). mip-la also participated in the activation of t cells in acute graft-versus-host disease, suggesting that compound 1 may also be an effective inhibitor of graft rejection (57). tak-779. baba et al. have identified a small nonpeptidyl antagonist of rantes binding to ccr5, tak-779, for which the chemical formula is n,n-dimethyl-n[4-[[[2-(4methylphenyl)-6,7-dihydro-5h-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2hpyran-4-aminium chloride (58). tak-779 was identified by a high-throughput screening method based on the inhibition of rantes binding to ccr5-transfected chinese hamster ovary cells. tak-779, like compound 1, but unlike sb225002, bicyclams, and distamycin a derivatives, is an asymmetric compound. tak-779 has been shown to inhibit rantes, mip-la, and mip-1b binding to ccr5 with an ic 50 of 1.0-1.5 nm and to inhibit rantes-induced calcium mobilization at 10 nm. the in vitro selectivity of tak-779 for inhibition of hiv-1 r5 fusion was demonstrated by comparison to the anti-hiv x4 activity of amd3100. like amd3100, the antiviral activity of tak-770 is at least 10-fold greater than the chemokine antagonist activity. the in vivo efficacy of tak-779 is being evaluated. there are several chemokines shared by ccr1 and ccr5: ran-tes, mip-la, and mip-1b (2). tak-779 and compound 1 could be used in combination to demonstrate the importance of ccr1 versus ccr5 in pathophysiological conditions. in addition to having anti-r5 hiv-1 activity, tak-779 may be beneficial in the treatment of rheumatoid arthritis, asthma, hhv8-encoded chemokine pathology, uveitis, and allergy (53-55, 59-61). distamycin a derivative. ureido analogues of distamycin were shown to have antitumor activity several years before a connection to the chemokine receptors was made (62) (63) (64) . interaction with chemokine receptors was suggested by the discovery of anti-hiv activity for many of the analogues (65) . several of the analogues had anti-hiv-1 activity, with ic 50 values of 1-10 um, but due to synthesis and availability concerns, nsc 651016 became the prototype molecule. nsc 651016 is a symmetric molecule with the chemical formula 2,2'[4,4'-[[aminocarbony 1] amino]bis [n-4' -di [pyrrole-2carboxamide-l,l'-dimethyl]]-6,8 naphthalenedisulfonic acid]hexasodium salt. unlike amd3100 and tak-779, nsc 651016 inhibits both r5 and x4 hiv-1 infection with an ic 50 of 1-3 um. it also inhibited hiv-2 and siv infection, although at higher ic 50 levels, 13.9 and 26 um, respectively. when tested for anti-hiv activity in a mouse hollow fiber model, no organ or cellular toxicity was observed at doses of 100 mg/kg body weight given three times daily, a dose that resulted in a 99% reduction in hiv-1 p24 production (66) . in vitro studies indicated that nsc 651016 selectively blocked chemokine binding and calcium mobilization mediated by ccr1, ccr3, ccr5, and cxcr4, but not the fmlp receptor (fpr), ccr2 or cxcr1, at 200 um. a more physiological result was the inhibition of mip-la-induced chemotaxis, with an ic 50 of 0.1 nm (67) . further mechanistic studies showed that nsc 651016, unlike the other nonpeptidyl chemokine receptor antagonists, induces receptor internalization after binding (67) . the peptidyl-based r5 antagonists, aop-rantes and nyy-rantes, appear to drive hiv-1 rapidly to select x4 virus, suggesting the development of a more virulent hiv-1 infection (68) . recent studies suggest that nsc 651016 may be able to inhibit ligand-resistant hiv-1 strains because it uses different receptor domains (69) . although nsc 651016 must be injected, it has a long in vivo half-life and, therefore, has the potential to be a broad-spectrum anti-hiv-1 therapeutic. additionally, nsc 651016 may have therapeutic effects in many inflammatory conditions, such as graft-versus-host disease. it will be interesting to evaluate other distamycin a analogues with receptor targets distinct from that of nsc 651016. hamycin. hamycin is a complex of polyene antibiotic compounds produced by streptomyces pimprina that is sometimes used as an antifungal. manna et al. investigated the ability of hamycin to inhibit il-8-induced biological functions (70) . their work demonstrated that hamycin blocked il-8 association with its receptor, resulting in decreased il-8-induced calcium mobilization, chemotaxis, and superoxide production. at 5 ug/ml hamycin appears to inhibit il-8-induced neutrophil responses, with little or no effect on fmlp or paf-induced responses, thereby demonstrating the selective nature of the hamycin effect. however, at 10 ug/ml hamycin inhibited all of the tested chemoattractants, suggesting a narrow range of selective function. the mechanism by which hamycin blocks il-8-induced responses appears to be by distorting the cellular membrane, causing the il-8 receptors to be structurally modified resulting in reduced binding of il-8. hamycin could potentially be used as a general antiinflammatory if the organ toxicity and bioavailability problems associated with polyene antibiotics are addressed (71) (72) (73) (74) . many of the established antiinflammatory therapies also regulate some aspect of chemokine-induced leukocyte infiltration. sozzani et al. have recently reviewed the role of proinflammatory cytokines such as lps, tnf, and il-1 and anti-inflammatory agents such as glucocorticoids on cc chemokine production and receptor expression (75) . they suggest that there is a balance between increased receptor expression induced by proinflammatory cytokines and decreased receptor expression induced by glucocorticoids. the net result depends on the degree of each treatment. in fact, a single concentration of dexamethasone inhibits the expression of the murine gro homologues kc and mip-2 but increases the expression of the murine homologue for ena-78, lix (76) . standiford et al. showed that il-8 expression by peripheral blood monocytes could be inhibited by either pge2 or dexamethasone, but only dexamethasone inhibited il-8 production by macrophages (77) . physiological concentrations of estrogen inhibit mcp-1 expression and mcp-1-induced chemotaxis, however, other estrogen receptor ligands, tamoxifen, and clomiphene blocked these effects (78) (79) (80) . therefore, steroid or prostaglandin therapy can either increase or decrease inflammation, depending on the targeted system. antiinflammatory cytokines suppress the production of most chemokines. the two cytokines most widely associated with this suppressive effect are il-10 and tgfb (81) (82) (83) . however, th2 cytokines il-4 and il-13 have also been shown to reduce mcp-1 and mip-la expression by activated epithelial cells and macrophages (84, 85) . a notable exception to the il-10-induced reduction of chemokine expression is hcc-4, whose expression is increased by il-10 (86) . presumably the antiinflammatory cytokines suppress chemokine expression at the messenger rna level (87) . a rather unique interaction between chemokines and tgfb is that pf4 selectively binds to the tgfb type 1 receptor (88) . since, tgfb has been shown to induce cell migration, it is difficult to correlate this unique observation with the inhibitory effects of tgfb. the g protein-coupled receptors are preeminent pharmacological targets and many agonists and antagonists have been identified. recently, an agonist, ib-meca, for the a 3 adenosine receptor was shown to suppress mip-la production by activated monocytes (89) . further, in a murine collagen-induced arthritis model, ib-meca reduced the degree of joint inflammation. the mechanism of ib-meca action has not been explored, but several of the purine nucleotide receptor agonists regulate the interface between the immune system and the central nervous system (90) . molecules that activate intracellular second messengers through unrelated receptors also may cause deactivation or desensitization of chemokine receptors, a process defined as heterologous receptor desensitization (91, 92) . it has been shown that stimulation of a classical g protein-coupled stm receptor named fpr, by the bacterial chemotactic formyl peptide n-formylmethionyl-leucyl-phenylalanine (fmlp), elicits a cascade of intracellular signaling events including increased camp and activation of protein kinase c (pkc). a recent study of various camp modulating drugs indicated that most inhibited chemokine expression and function (93) . pkc activation by fmlp is associated with functional attenuation of heterologous stm receptor responses to other chemoattractants, including chemokines (92) . several synthetic peptide domains derived from the hiv-1 envelope protein gp120 or gp41 have similarly been shown to be potent chemotactic agonist for the fmlp receptor fpr and its variant fprl1 (94) (95) (96) . one of the synthetic peptide domain derived from the v4-c4 region of the hiv-1 gp120, termed f-peptide, was shown selectively to activate fprl1 in human phagocytic cells and to induce down-regulation of ccr5 and cxcr4 expression and function in monocytes (96) . an acutephase protein, serum amyloid a (saa) at the high concentrations seen during acute phase responses, has also been identified as an agonist of the receptor fprl1 and potently desensitizes monocyte or neutrophil re-sponses to chemokines (97, 98) . thus, down-regulation of the function and/or expression of chemokine receptors by heterologous desensitization may represent an additional approach to the design of antiinflammatory and hiv-1 agents. heterologous desensitization does not involve agonist occupancy of the receptor and does not lead to arrestin-mediated receptor internalization. since heterologous desensitization has been reported to occur between a number of stm receptors, it has been postulated to play an important role in orchestrating the host cell response to multiple stimulants. for example, activation of two stm opiate receptors, 8 and u, by morphine or its endogenous analogue met-enkephalin resulted in a decrease in phagocyte migration to a number of chemokines (91) . further, activation of 8 and u opiate receptors induced chemokine receptor phosphorylation without a change in the level of cell surface expression or agonist-induced calcium mobilization (91) . these results suggest that the suppression of chemokine receptor function could be achieved indirectly by agents that indirectly perturb the integrity of chemokine receptor signaling machinery. chemokines and leukocyte traffic recent advances in chemokines and chemokine receptors chemokines: progress toward identifying molecular targets for therapeutic agents human beta-defensins promote adaptive immunity, by activating dendritic and t cells expressing ccr6 the role of chemokines in transplantation chemokine receptors: keys to aids pathogenesis a broad-spectrum chemokine antagonist encoded by kaposi's sarcoma-associated herpesvirus secreted poxvirus chemokine binding proteins fully human anti il-8 monoclonal antibody: potential therapeutics for the treatment of inflammatory disease states essential involvement of interleukin-8 (il-8) in acute inflammation pivotal role of interleukin-8 in the acute respiratory distress syndrome and cerebral reperfusion injury prevention of myocardial reperfusion injury in rats by an antibody against monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 the role of chemokines in tissue inflammation and autoimmunity in renal diseases inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in scid mice mip-lalpha and mcp-1 differentially regulate acute and relapsing autoimmune encephalomyelitis as well as thl/th2 lymphocyte differentiation neutralization of macrophage inflammatory protein-2 attenuates neutrophil recruitment and bacterial clearance in murine klebsiella pneumonia interleukin-8 antagonists generated by n-terminal modification a hisl9 to ala mutant of melanoma growth-stimulating activity is a partial antagonist of the cxcr2 receptor synthetic hexa-and heptapeptides that inhibit il-8 from binding to and activating human blood neutrophils peptide inhibitor of interleukin-8 (il-8) reduces staphylococcal enterotoxin-a (sea) induced neutrophil trafficking to the lung a synthetic peptide inhibitor for alpha-chemokines inhibits the growth of melanoma cell lines a synthetic peptide inhibitor for alpha-chemokines inhibits the tumour growth and pulmonary metastasis of human melanoma cells in nude mice inhibition of groalpha-induced human endothelial cell proliferation by the alpha-chemokine inhibitor antileukinate structure/activity analysis of human monocyte chemoattractant protein-1 (mcp-1) by mutagenesis. identification of a mutated protein that inhibits mcp-imediated monocyte chemotaxis antagonists of monocyte chemoattractant protein 1 identified by modification of functionally critical nh2-terminal residues an antagonist of monocyte chemoattractant protein 1 (mcp-1) inhibits arthritis in the mrl-lpr mouse model rantes and mcp-3 antagonists bind multiple chemokine receptors a small molecule cxcr4 inhibitor that blocks t cell line-tropic hiv-1 infection pharmacophore identification of a chemokine receptor (cxcr4) antagonist, t22 ([tyr(5,12),lys7]-polyphemusin ii), which specifically blocks t cell-line-tropic hiv-1 infection solution structure and basis for functional activity of stromal cell-derived factor-1 dissociation of cxcr4 activation from binding and inhibition of hiv-1 n-terminal peptides of stromal cell-derived factor-1 with cxc chemokine receptor 4 agonist and antagonist activities dissociation of the signalling and antiviral properties of sdf-1-derived small peptides aminooxypentane-rantes induces ccr5 internalization but inhibits recycling: a novel inhibitory mechanism of hiv infectivity potent inhibition of hiv-1 infectivity in macrophages and lymphocytes by a novel ccr5 antagonist extension of recombinant human rantes by the retention of the initiating methionine produces a potent antagonist the cc chemokine receptor antagonist met-rantes inhibits eosinophil effector functions enhanced inhibition of human immunodeficiency virus type 1 by met-stromal-derived factor ibeta correlates with down-modulation of cxcr4 inactivation of hiv-1 chemokine co-receptor cxcr-4 by a novel intrakine strategy phenotypic knockout of hiv type 1 chemokine coreceptor ccr-5 by intrakines as potential therapeutic approach for hiv-1 infection identification of a potent, selective non-peptide cxcr2 antagonist that inhibits interleukin-8-induced neutrophil migration potent and selective inhibition of human immunodeficiency virus (hiv)-l and hiv-2 replication by a class of hbicyclams interacting with a viral uncoating event amd3100, a small molecule inhibitor of hiv-1 entry via the cxcr4 co-receptor inhibition of t-tropic hiv strains by selective antagonization of the chemokine receptor cxcr4 new sulfonated distamycin a derivatives with bfgf complexing activity fce 27266, a sulfonic distamycin derivative, inhibits experimental and spontaneous lung and liver metastasis antitumor activity of fce 26644 a new growth-factor complexing molecule novel sulfonated and phosphonated analogs of distamycin which inhibit the replication of hiv inhibition of in vitro and in vivo hiv replication by a distamycin analogue that interferes with chemokine receptor function: a candidate for chemotherapeutic and microbicidal application small molecule inhibitor of hiv-1 cell fusion blocks chemokine receptor-mediated function highly potent rantes analogues either prevent ccr5-using human immunodeficiency virus type 1 infection in vivo or rapidly select for cxcr4-using variants naturally occurring ccr5 extracellular and transmembrane domain variants affect hiv-1 co-receptor and ligand binding function hamycin inhibits il-8-induced biologic response by modulating its receptor in human polymorphonuclear neutrophils analysis and assay of polyene antifungal antibiotics. a review suggested mechanisms for the antimycotic activity of the polyene antibiotics and the n-substituted imidazoles liposomal hamycin in the control of experimental aspergillosis in mice: effect of phosphatidic acid with and without cholesterol liposomal hamycin: reduced toxicity and improved antifungal efficacy in vitro and in vivo old and new chemokines. pharmacological regulation of chemokine production and receptor expression: mini-review the murine neutrophilchemoattractant chemokines lix, kc, and mip-2 have distinct induction kinetics, tissue distributions, and tissue-specific sensitivities to glucocorticoid regulation in endotoxemia regulation of human alveolar macrophage-and blood monocyte-derived interleukin-8 by prostaglandin e2 and dexamethasone estrogen regulation of je/mcp-1 mrna expression in fibroblasts monocyte chemotactic protein-1 (mcp-1) mrna is down-regulated in human dermal howard, oppenhe1m, and wang fibroblasts by dexamethasone: differential regulation by tgf-beta physiological concentration of 17 beta-estradiol inhibits chemotaxis of human monocytes in response to monocyte chemotactic protein 1 interleukin-10 inhibits interleukin-8 production in human neutrophils chemokine synthesis in the hsv-1-infected cornea and its suppression by interleukin-10 transforming growth factor-beta 1 regulates chemokine and complement production by human proximal tubular epithelial cells il-4, il-10 and il-13 down-regulate monocyte-chemoattracting protein-1 (mcp-1) production in activated intestinal epithelial cells expression and release of interleukin-8 by human airway smooth muscle cells: inhibition by th-2 cytokines and corticosteroids characterization of a novel cc chemokine, hcc-4, whose expression is increased by interleukin-10 the interferon-stimulated response element and a kappa b site mediate synergistic induction of murine ip-10 gene transcription by ifn-gamma and tnf-alpha platelet factor 4 selectively inhibits binding of tgf-beta 1 to the type i tgf-beta 1 receptor suppression of macrophage inflammatory protein (mip)-l alpha production and collageninduced arthritis by adenosine receptor agonists p2u agonists induce chemotaxis and actin polymerization in human neutrophils and differentiated hl60 cells opiates transdeactivate chemokine receptors: delta and mu opiate receptor-mediated heterologous desensitization chemoattractant receptor cross-desensitization effect of pde4 inhibitors on zymosan-induced il-8 release from human neutrophils: synergism with prostanoids and salbutamol t2i/dpi07, a synthetic leucine zipper-like domain of the hiv-1 envelope gp4l, attracts and activates human phagocytes by using g-protein-coupled formyl peptide receptors t20/dpi78, an cctodomain peptide of human immunodeficiency virus type 1 gp41, is an activator of human phagocyte n-formyl peptide receptor a synthetic peptide derived from hiv-1 gp120 down-regulates the expression and function of chemokine receptors ccr5 and cxcr4 in monocytes by activating the seven-transmembrane g protein-coupled receptor fprl-1/ lxar a seven-transmembrane, g protein-coupled receptor, fprli, mediates the chemotactic activity of serum amyloid a for human phagocytic cells serum amyloid a induces calcium mobilization and chemotaxis of human monocytes by activating a pertussis toxin-sensitive signaling pathway this project was funded in whole or in part by federal funds from the national cancer institute, national institutes of health, under contract no1-co-56000. key: cord-004198-h8ch3x14 authors: ebuy, hiluf; bekele, alemayehu; redae, getachew title: hiv testing, test results and factors influencing among infants born to hiv positive mothers in public hospitals of mekelle city, north ethiopia: a cross-sectional study date: 2020-01-21 journal: bmc infect dis doi: 10.1186/s12879-020-4790-9 sha: doc_id: 4198 cord_uid: h8ch3x14 background: timely infant testing for hiv is critical to ensure optimal treatment outcomes among exposed infants. while world health organization recommends hiv exposed infants to be tested between 4 to 6 weeks of age, in developing countries like ethiopia, access to timely infant testing is still very limited. the study is intended to assess timely infant testing, testing for hiv at the 18th month, test results and factors influencing hiv positivity among infants born to hiv positive mothers in public hospitals of mekelle, ethiopia. methods: a cross-sectional study design was employed on 558 hiv exposed infants, using consecutive sampling technique. a checklist was used to extract 4 years (january 2014–december 2017) secondary data, collected from january–april 2018. data were analyzed using spss version 20, and binary logistic regression model was used to examine the association of independent variables with the outcome variables. results: timely infant testing for hiv accounted for 346(62.0%). mothers who attended antenatal care (aor: 2.77; 95% ci: 1.17, 6.55) and who were counselled on feeding options (aor: 2.01; 95% ci: 1.11, 3.65) were strongly associated with timely infant testing. poor maternal adherence status was associated with infants’ hiv positivity at the 18th month of antibody test (aor: 15.93; 95% ci: 2.21, 94.66). being rural resident (aor: 4.0; 95% ci: 1.23, 13.04), being low birth weight (aor: 5.64; 95% ci: 2.00, 16.71) and not receiving arv prophylaxis (aor: 4.70; 95% ci: 1.15, 19.11) were positively associated with the overall hiv positivity. conclusions: a considerable proportion of exposed infants did not undergo timely testing for hiv. antenatal care follow-up and counselling on feeding options were associated with timely infant testing. mother’s poor adherence status was associated with infant’s hiv positivity at the 18th month of antibody testing. being rural resident, being low birth weight, and not receiving arv prophylaxis were the factors that enhance the overall hiv positivity. timely infant testing, counselling on feeding options and adherence should be intensified, and prevention of mother-to-child transmission program in rural settings need to be strengthened. there are many ways of hiv transmission. one way of transmission is from an hiv positive woman to her child during pregnancy, labor, and breastfeeding. it is also called vertical transmission which accounts for 14% of all new hiv infections worldwide. in developing countries, despite the availability of proven interventions for the prevention of mother-to-child transmission (pmtct), pediatric hiv is still a largely uncontrolled epidemic [1] [2] [3] . timely infant testing for hiv infection is critical to ensure optimal treatment outcomes among exposed infants. without testing and effective treatment, one-third of hiv-positive infants will die before the age of one year, and almost half by their second year of life. while world health organization (who) recommends hiv exposed infants to be tested between four to six weeks of age, in developing countries access to timely infant testing is still very limited [4, 5] . globally, in 2015, only 43% of infants exposed to hiv during pregnancy were tested within the recommended period of time. majority of the non-timely tested infants were from developing countries, particular sub-saharan africa. in sub-saharan africa, delayed infant testing is emerging as one of the challenging complex issues facing children infected with and affected by hiv. similar to the other sub-saharan african coutries, studies from ethiopia also showed that timely infant testing for hiv is very low [6] [7] [8] [9] . the prevalence of hiv among exposed infants can reach up to 45% if left without pmtct interventions. nearly two-third of pregnant women living with hiv in the middle east and north africa passed the virus onto their infants in the year 2015 alone 10, 11] . in ethiopia, vertical transmission, which accounted for more than 90% of pediatric hiv, is a very critical issue. accordingly, hiv related estimates and projections showed that the national estimate of mtct rate was 25% in 2013 and 17% in 2015. this high magnitude makes hiv/aids one of the top priorities of the health sector transformation plan (hstp) of ethiopia [12] [13] [14] [15] . although timely hiv testing of infants is not yet optimal, some strategies and solutions have proven successful, including community-based interventions and support and education of mothers. the joint united nations program on hiv/aids (unaids) 2016-2021 strategy set about ten targets to end the aids epidemic by 2030; one of the targets is to make new hiv infections among children zero and improve the health and wellbeing of mothers. the global plan to eliminate new hiv infections among children and improve the health of mothers targets to reduce the mtct rate to less than 5% among breast feeding population and to less than 2% among non-breast feeding population [5, 16, 17] . pertaining to the factors for hiv positivity of exposed infants, different studies reported various determinants. accordingly, mothers with high viral load, symptomatic disease, failure to use arv drugs during pregnancy, vaginal delivery, rupture of membrane > 4 h, low birth weight babies, premature births, and infants' failure to use antiretroviral (arv) prophylaxis were found to be associated with hiv positivity among infants [18] [19] [20] . in most resource-limited countries, deoxyribonucleic acid polymerase chain reaction (dna/pcr) test is not available for the timely testing of exposed infants, which plays a big role in infants' late initiation of treatment which then leads to hiv related pediatric mortality. in ethiopia, previous studies tried to demonstrate exposed infants' hiv testing and determinants of pmtct. however, most of the studies were confined to a single health facility and only tried to find out factors associated with hiv positivity at 6 weeks of testing. there is paucity of information regarding the factors affecting timely infant testing and hiv positivity at the 18th month of antibody test, until the infant completes the program. thus, the present study is intended to assess timely infant testing, testing for hiv at the 18th month, test results and factors influencing overall hiv positivity and hiv positivity at 18 months among infants born to hiv positive mothers in public hospitals of mekelle city, north ethiopia. this study was conducted in mekelle city. mekelle is the capital city of the tigray region and is located around 783 km north of addis ababa, the capital city of ethiopia. there are three public hospitals in the city namely; ayder comprehensive specialized, quiha and mekelle general hospitals. the college of health sciences (chs), ayder comprehensive specialized hospital (acsh) is a public hospital with a capacity of 500 inpatient beds and has the responsibility of rendering patient care, training for medical & health science students and also carrying out research. the institution has a clinic dedicated for maternal, neonatal and child health services (mnch) including pmtct services. quiha general hospital, which was established in 1985 by italian cooperation, served as a health center until 2012 and then become a general hospital. currently, the hospital has a capacity of 50 beds and the hiv care and treatment clinic is one of the core parts of the hospital's activities. mekelle general hospital, which was established in 1962, is the oldest general hospital in the region. it has its own separate fully functional art clinic and pmtct services. since there was no access to the dna/pcr test in all the public hospitals, the test was done in the regional laboratory after dbs was collected in the public hospitals. on the other hand, antibody test was conducted in the study setting [21] [22] [23] . a cross-sectional study design was employed in the public hospitals of mekelle city from january 2014-december 2017. study population, sample size and sampling procedure the study participants consisted of all mother-infant pairs who were eligible to be enrolled to the pmtct program in the public hospitals of mekelle city during the study period. four years data were extracted retrospectively, from january 2014-december 2017. the reason why we started extracting data from 2014 was due to the fact that in ethiopia option b plus strategy for pmtct was adopted in 2013 [24] . however, in the study area consistent and full documentation of option b plus services were available starting 2014. in the four years period, a total of 558 mother-infant pairs who fulfilled the inclusion criteria were enrolled to the study, using consecutive sampling technique. we used consecutive sampling technique because it was a secondary data, 30% of the participants were excluded from the study due to either incomplete information, loss to follow up or transfer out. all mother-infant pairs who sought and completed the pmtct program were included in the study. we excluded 57 participants who had an incomplete medical records, 148 lost to follow up subjects and 34 participants who were transferred into other health facilities. loss to follow up, which makes identifying and managing hiv-infected children very difficult, could be a reflection of failure in pmtct. after reviewing different literatures [8, 9, 25, 26] , a checklist developed by the authors was used to collect data regarding socio-demographic and health-related factors. data were extracted from secondary data sources retrospectively by reviewing patient charts (both paper and electronic medical recording systems), pmtct, antenatal, delivery, and postnatal care registration books and partographs from january-april, 2018. there is a special hiv detection test done for hiv exposed infants after dried blood spot (dbs) is collected called dna/pcr test. this hiv testing is recommended to be done within 4-6 weeks of age and is referred to as timely infant testing. this is the aggregate of mtct of hiv at the 6th week of dna/pcr test and the 18th month of antibody test, until weaning. ratios of art medication adherence equal to or greater than 95% were defined as good dosage adherence. adherence status of the participants was measured from a self-report. the checklist was prepared in english and reviewed by senior researchers and feedback was incorporated accordingly. the secondary data were extracted by three midwives, one for each hospital and training was given concerning the checklist, data collection technique, purpose of the study, and keeping confidentiality. the research team had day to day on-site supervision to make sure data extraction from secondary sources was going smoothly. the research team discussed how to take corrective measures like checking and discarding data if an error, such as illegibly documented checklists, occurred during the data collection. the collected data were checked for completeness, consistency, and clarity. the collected data were coded and entered into an excel spreadsheet; it was then exported to statistical package for social sciences (spss) version 20 statistical software for analysis. tables and figures were used for descriptive statistics. frequency and percentage were used for categorical variables. odds ratio (or) with 95% confidence interval was used to measure strength of the association. binary logistic regression model, with bivariate and multivariable analysis, was used to examine the association of independent variables with the outcome variables and calculate their crude as well as adjusted odds ratios. those variables which happened to have a p-value < 0.25 with crude analysis of logistic regression model were fitted into the final multivariable logistic regression model and their adjusted odds ratios were calculated. statistical significance was declared using an odds ratio and 95% confidence interval. absence of multicollinearity was checked and found to be satisfied with the maximum variance inflation factor (vif) value of 1.8. goodness of fit was checked by hosmer-lemeshow test which yielded a chi-square value of 1.4 with 8 df and p-value of 0.99 for the final model of overall hiv positivity. ethical clearance was obtained from the institutional review board of the ethiopian public health association (epha). the collected data was kept anonymous; mothers and infants' names were not recorded but instead were written down in code and additional measures like keeping the completed checklist in a safe place were taken. this study did not impose any harm to the community or to the research team, and it was conducted in line with national and international ethical guidelines. a letter of collaboration was also obtained from the institutional review board of the college of health sciences, ayder comprehensive specialized, quiha and mekelle general hospitals. socio-demographic characteristics of the study participants among the total 797 mother-infant pairs who were enrolled in the pmtct cohort over the past four years, 558(70.0%) of them fulfilled the inclusion criteria and were included in the final analysis. two hundred thirtynine (30.0%) participants were excluded from the study, of which 57(23.85%) had an incomplete medical record, 148(61.95%) were lost to follow up and the remaining 34(14.2%) were transferred into other health facilities. the mothers' ages ranges from 15 to 49 years; with a mean age of 28.89 years and a standard deviation of 4.65 years. regarding residence, the majority of mothers, 459(82.3%), were urban dwellers. there were 291(52.2%) female hiv exposed infants (table 1) pmtct service and clinical characteristics among mothers the majority (95.0%) of the mothers had attended anc and 529(94.8%) mothers were on art on entry to the pmtct cohort. of the 513(91.4%) mothers whose adherence status was known, 312(60.8%) had good adherence status. who clinical staging was documented in the majority (95.7%) of mothers and 505(94.6%) of them were in clinical stage i or ii (table 2) . the mean weight of the infants was 3.1 kg, with a standard deviation of 0.36 kg. eighty-four (15.1%) infants' were low-birth-weights. the majority (94.3%) of the infants were given arv prophylaxis. regarding the feeding of exposed infants, no mixed feeding was practiced. timely infant testing for hiv accounted for 346(62.0%) ( table 3) . overall 558 study participants were tested for dna/pcr. of these, 13(2.3%) (95% ci: 1.3, 3.8%) tested positive for hiv. among the 545 hiv exposed infants for whom the rapid antibody test was done, 7(1.3%) (95% ci: 0.4, 2.4%) tested positive for hiv. therefore, the overall mtct rate was 3.6% (95% ci: 2.2, 5.4%). in the multivariable logistic regression analysis, factors found to be significantly associated with timely infant testing were anc follow up and feeding option counselling. the odds of practicing timely infant testing of mothers who attended anc follow up were 2.77 times higher than those who didn't have anc follow up (aor: 2.77; 95% ci: 1.17, 6.55). the odds of practicing timely infant testing of infants whose mothers were counselled on feeding options were 2-fold higher than their counterparts (aor: 2.01; 95% ci: 1.11, 3.65)( table 4) . factors associated with hiv positivity at the 18th month at the 18th month of antibody test the mother's adherence status was the only variable that had a significant association with hiv positivity. the odds of hiv positivity of infants whose mother's adherence status was poor were 15.93 times higher than those with good adherence status (aor: 15.93; 95% ci: 2.21, 94.66) ( table 5) . in the multivariable logistic regression, the variables; place of residence, infants' birth weight, and infants' arv prophylaxis had a significant association with overall hiv positivity among exposed infants. accordingly, the likelihood of hiv positivity among rural residents was 4 times higher than urban dwellers (aor: 4.00; 95% ci: 1.23, 13.04). the odds of hiv positivity of low birthweight infants were 5.64 times higher than normal birthweight infants (aor: 5.64; 95% ci: 2.00, 16.71). the likelihood of hiv positivity among infants who did not receive arv prophylaxis was 4.7-fold higher than their counterparts (aor: 4.70; 95% ci: 1.15, 19.11) ( table 6 ). the present study was intended to assess timely infant testing, testing for hiv at the 18th month, test results and factors influencing overall hiv positivity and hiv positivity at 18 months among infants born to hiv positive mothers in public hospitals of mekelle, tigray, ethiopia. the percentage of hiv exposed infants tested for hiv timely (62.0%) in our study were comparatively higher than studies conducted in south gondar, ethiopia in 2014(25.3%) and asella teaching and referral hospital, ethiopia from 2012 to 2015(19.2%) [8, 9] . this high percentage of timely infant testing in our study could be due to the fact that the regional laboratory, where the dna/pcr test is done, is located in the same city where the study is conducted, which makes it easier for the dna/pcr test to be done in a timely manner. in the first six weeks the mtct rate of hiv, as determined by a dna/pcr test, was 2.3%(95% ci: 1.3, 3.8%). this was one of the lowest mtct rates of reports compared to a cross-sectional household survey conducted [8, 9, [26] [27] [28] . this could be attributed to the cor crude odds ratio, aor adjusted odds ratio *significant association ci confidence interval reason that almost all of our study participants gave birth in a health institution, which allowed them good access to the first six weeks of a pmtct program. in our study, the overall mtct rate of hiv infection among exposed infants was 3.6% (95% ci: 2.2, 5.4%), which was much less than a study conducted in brazil. a possible explanation for this might be that the study conducted in brazil was a 21 years study, which started when pmtct program wasn't as efficient and modernized as recently. our finding was also less than ethiopia's national estimate of mtct in 2013 and 2015 and from studies done in arsi add dire dawa, ethiopia [9, 12, 13, 20, 25] . this may be explained by the reason that no mixed feeding was practiced in our study, which enhances the odds of hiv positivity. in addition, the majority of our participants were from urban areas where they can easily access pmtct services, while the national estimates account for the overall mtct of the country, including rural residents and home deliveries. mothers who attended anc follow up through the course of the pregnancy were 2.77 times more likely to practice timely infant testing of hiv compared to the mothers who didn't have anc follow up. the conceivable reason could be that one of the components of pmtct services during anc is health education and awareness creation about the advantages of timely infant testing. this was given to the mothers who had anc follow up when they visited health institutions. counselling on feeding options was another factor associated with timely infant testing. the odds of practicing timely infant testing of infants whose mothers were counselled on feeding options were 2-fold higher than mothers of infants who weren't counselled on feeding options. this could be attributed to the reason that when mothers were counselled on feeding options, at the same time, they were also being counselled on other pmtct cascades, such as advantages of timely infant testing. mother's adherence status had a significant association with hiv positivity among exposed infants at the 18th month rapid antibody test. infants born to hiv positive mothers whose adherence status was poor were 15.93 times at higher risk of acquiring hiv infection than infants born to hiv positive mothers whose adherence status was good. the possible risk difference could be due to the fact that poor maternal adherence status causes drug resistance, which then leads to the elevation of maternal viral load, putting exposed infants at high risk of hiv positivity. the odds of hiv positivity of infants born to hiv positive mothers from rural residences were 4 times higher than infants born to mothers of urban residents. this is consistent with the finding of studies conducted in dil chora hospital in dire dawa, ethiopia and gondar university hospital in northwest ethiopia [25, 29] . a likely explanation is that mothers from rural settings might have limited access to anc clinics and therefore poor access to pmtct services compared to mothers who are urban dwellers. the odds of overall hiv positivity of low birth-weight infants were 5.64 times higher than the normal birthweight infants. this is consistent with researches done in arsi and addis ababa, ethiopia [9, 26] . the attributed risk difference could be due to the reason that the immature gastrointestinal tract in low birth weight infants might facilitate mtct of hiv through mother's breast milk. arv prophylaxis for infants was another factor associated with exposed infants' overall hiv positivity. the odds of acquiring hiv among infants who did not receive arv prophylaxis was 4.7-fold higher than those infants who received arv prophylaxis. the observed risk difference could be due to the reason that arv prophylaxis for exposed infants decreases vertical transmission of the virus from hiv positive mothers. this finding is in tune with studies conducted in brazil and ethiopia [20, 25, 26, 30] . this study gives a clue about the factors associated with timely infant testing and potential factors associated with hiv positivity among exposed infants. furthermore, the findings of the study are meant to strengthen and enhance the implementation of the pmtct program at the hospital level in the public health system. this study has some limitations. the fact that it is a crosssectional study design is one of the study's weakness. since the study used secondary data, the information gathered was incomplete. as a result, some sociodemographic variables such as household income and educational status were not documented. additionally, this study didn't specify the time when infants' arv prophylaxis was initiated. in conclusion, considerable proportion of exposed infants did not undergo timely infant testing for hiv. there was relatively low mtct rate of hiv at six weeks, eighteen months and for overall hiv testing. antenatal care follow-up and counselling on feeding options were associated with timely infant testing. mother's poor adherence status was associated with infant's hiv positivity at the 18th month of antibody test. infants born to hiv positive mothers from rural residences, low birth-weights and who didn't receive arv prophylaxis after birth were the factors that enhance the risk of overall hiv positivity. health care providers should work on strengthening timely testing of hiv among exposed infants. counseling on feeding options, anc and adherence needs to be provided in a sustainable way. continuous follow up of exposed infants has to be intensified. in addition, policymakers and program managers should focus on scaling up pmtct program in rural settings. a prospective study need to be conducted to assess the effectiveness of pmtct programs and to address additional factors associated with hiv positivity among exposed infants. deoxyribonucleic acid polymerase chain reaction; ebf: exclusive breast feeding; eff: exclusive formula feeding; epha: ethiopian public health association; hiv: human immunodeficiency virus; hstp: health sector transformation plan lsi: leadership in strategic information; mnch: maternal, neonatal and child health; mtct: mother -to-child transmission; or: odds ratio pmtct: prevention of mother-to-child transmission; spss: statistical package for social science; unaids: joint united nations programme on hiv/ aids; who: world health organization references 1. unaids. unaids report on the global aids epidemic mother-to-child transmission of hiv in the era of highly active antiretroviral therapy early infant diagnosis of hiv: review of current and innovative practices towards universal access: scaling up priority hiv/aids interventions in the health sector. aids epidemic update pediatric hiv/aids in sub-saharan africa: emerging issues and way forward risk of hiv and associated factors among infants born to hiv positive women in amhara region assessment of effectiveness of prevention of mother to child transmission of human immunodeficiency virus in asella hospital, ethiopia prevention of mother-to-child transmission ( pmtct) of hiv joint united nations programme on hiv/aids. aids by the numbers fdre; federal ministry of health. country progress report on the hiv response hiv related estimates and projections for ethiopia-2015 analysis of the prevention of mother-to-child transmission (pmtct) service utilization in ethiopia federal minstry of health. hiv related estimates and projections for ethiopia 2016. health sector transformation plan (hstp) joint united nations programme on hiv/ aids the global plan towards the elimination of new hiv infections among children by 2015 and keeping their mothers alive mode of delivery in hiv-infected pregnant women and prevention of mother-to-child transmission: changing practices in western europe factors associated with viral load suppression in hiv-infected pregnant woman in rio de janeiro, brazil maternal risk factors for hiv infection in infants in northeastern brazil mekelle general hospital public relations office quiha general hospital public realtions office, quiha general hospital profile report federal ministry of health. national comprehensive pmtct/mnch/rh training package guideline reference manual mother-to-child transmission of hiv infection and its determinants among exposed infants on care and follow-up in dire dawa city review of prevention of mother to child transmission of hiv in addis ababa, ethiopia. univ s afr pretoria reconstructing the pmtct cascade using cross-sectional household survey data: the pearl study determinant and outcome of early diagnosis of hiv infection among hiv-exposed infants in southwest ethiopia mother-to-child transmission of hiv and its predictors among hiv-exposed infants at a pmtct clinic in northwest ethiopia an assessment of the outcomes of prevention of mother-to-child transmission of hiv services in addis ababa publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the ethiopian public health association. our gratitude also goes to ayder comprehensive specialized, quiha and mekelle general hospitals for providing us the necessary information. last but not least we would like to acknowledge the data collectors.ethical approval and consent to participate this study was approved by the institutional review board of the ethiopian public health association. collected secondary data were kept anonymous; mothers and infants' names were not being recorded instead was written down in code. 2 ethiopian public health association, addis ababa, ethiopia. 3 authors' contributions he had taken a principal role in the conception of ideas, writing the protocol, developing methodologies, analyses and write up of the article and drafted the manuscript. ab contributed to the proposal writing and design, developing methodologies and made a critical revision to the paper and manuscript for intellectual content. gr contributed to the write up of the study protocol and made revision to the paper. ab and gr supervised the study. all authors read and approved the final manuscript. this work was supported by the ethiopian public health association. the funding agency had no involvement in the design of the study, data collection and analysis, interpretation of data and writing the manuscript. the datasets generated and/or analyzed during the current study are not publicly available due to ethical and confidentiality reasons but are available from the corresponding author on reasonable request under the ethics committee's approval. not applicable. there is no competing interest. key: cord-001079-v01vwu00 authors: thoden, j.; potthoff, a.; bogner, j. r.; brockmeyer, n. h.; esser, s.; grabmeier-pfistershammer, k.; haas, b.; hahn, k.; härter, g.; hartmann, m.; herzmann, c.; hutterer, j.; jordan, a. r.; lange, c.; mauss, s.; meyer-olson, d.; mosthaf, f.; oette, m.; reuter, s.; rieger, a.; rosenkranz, t.; ruhnke, m.; schaaf, b.; schwarze, s.; stellbrink, h. j.; stocker, h.; stoehr, a.; stoll, m.; träder, c.; vogel, m.; wagner, d.; wyen, c.; hoffmann, c. title: therapy and prophylaxis of opportunistic infections in hiv-infected patients: a guideline by the german and austrian aids societies (daig/öag) (awmf 055/066) date: 2013-09-14 journal: infection doi: 10.1007/s15010-013-0504-1 sha: doc_id: 1079 cord_uid: v01vwu00 introduction: there was a growing need for practical guidelines for the most common ois in germany and austria under consideration of the local epidemiological conditions. materials and methods: the german and austrian aids societies developed these guidelines between march 2010 and november 2011. a structured medline research was performed for 12 diseases, namely immune reconstitution inflammatory syndrome, pneumocystis jiroveci pneumonia, cerebral toxoplasmosis, cytomegalovirus manifestations, candidiasis, herpes simplex virus infections, varizella zoster virus infections, progressive multifocal leucencephalopathy, cryptosporidiosis, cryptococcosis, nontuberculosis mycobacteria infections and tuberculosis. due to the lack of evidence by randomized controlled trials, part of the guidelines reflects expert opinions. the german version was accepted by the german and austrian aids societies and was previously published by the arbeitsgemeinschaft der wissenschaftlichen medizinischen fachgesellschaften (awmf; german association of the scientific medical societies). conclusion: the review presented here is a translation of a short version of the german–austrian guidelines of opportunistic infections in hiv patients. these guidelines are well-accepted in a clinical setting in both germany and austria. they lead to a similar treatment of a heterogeneous group of patients in these countries. although opportunistic infections (ois) in human immunodeficiency virus (hiv)-infected patients have become rare in industrialized countries [1] , patients continue to present with advanced hiv disease and hiv-related ois. patients (so-called ''late presenters'') are often unaware of their hiv infection or have not received antiretroviral treatment. they present at a late stage and when their overall health status is already poor [2] . diagnosis and therapy of these ois remain a challenge. the aim of the recommendations presented here is to develop general and practical guidelines for the treatment and prophylaxis of the most common ois in germany within the framework of local epidemiological conditions. the tables in the different sections of the guidelines represent a summary of the therapeutic guidelines. with regard to diagnosis, the authors refer to the appropriate literature. at the time the guidelines were approved some articles were only available as congress abstracts; if these were published as peer-reviewed article at a later date, the published articles were cited. the kaad (clinical aids working group germany) guidelines conform to the international guidelines of the u.s. centers for disease control and prevention (cdc) (http:// www.cdc.gov/mmwr) [3] and guidelines formulated by the awmf (association of the scientific medical societies in germany) in the overlapping fields dermatology and neurology (http://www.uni-duesseldorf.de/awmf/ll/). members of other medical societies and the austrian aids society have also participated and have been consulted (see appendix). some of the following recommendations go beyond the approved use of drugs. in many cases, data from randomized controlled trials (rcts) are missing, and evidence is based on practical and clinical experiences not presented in published studies (expert opinion). in addition, we advise always checking interactions and toxicities of the applied drugs as these factors cannot be described in detail within the scope of this guideline. for the treatment of bacterial pneumonia, which is similar in hiv-positive and hiv-negative patients, the appropriate guidelines should be referred to. the indication for antiretroviral therapy (art) in germany is based on the guidelines by the german and austrian aids societies (daig and ö ag, respectively). however, general recommendations regarding when to start art with mostly art-naïve patients in the setting of an (acute) oi cannot be given. in the case of candidiasis, herpes virus infections or, for example, cryptosporidiosis, the immediate start of art is uncomplicated; in the case of progressive multifocal leukoencephalopathy (pml) it is even necessary and recommended. the situation is more difficult in cases of pneumocystis jiroveci pneumonia (pcp), cerebral toxoplasmosis, cytomegalovirus (cmv)-retinitis, tuberculosis (tb), atypical mycobacteriosis, and cryptococcosis. we refer to the corresponding sections of these guidelines. the recommendations given here represent the consensus of the guideline consensus group. the recommendations referring to medical therapies might involve off-label therapies that have not been officially approved. this is due to the lack of data from rcts on hiv-infected patients with oi. in such cases, the recommendation often refers to data on hiv-negative persons or personal experience (expert opinion). it should also be noted that drug-drug interactions or toxicities need to be excluded in each single case. the kaad was given the task to develop guidelines for the treatment and prophylaxis of oi by the daig in march 2010. the members of the daig, ö ag, and other german medical societies (in total 24 societies represented; see appendix) were asked to participate in the consensus process. the members formed small interest groups (n = 3-10 members) covering the different chapters of these guidelines. a first version was sent out in march 2010 based on the corresponding chapters of the digital version (http://www.hivbook.com). the different groups were free to base their chapters on this proposal after review of the relevant literature or to create new chapters. via an email system these new chapters were put together until the groups reached a consensus on a final draft. four weeks before a consensus conference in cologne on 25 june 2010, these drafts for all 12 chapters were sent out to all members of all groups and to all daig members with the request for suggestions for changes. the submitted suggestions for changes which were received were then sent out to the members prior to the meeting. during the consensus conference all suggestions were discussed and voted on separately. finally, each single chapter and the whole guideline proposal were voted on separately. there was an agreement of 100 % on the whole proposal between all members of the guideline group. in a third step the cologne proposal was sent out via email to all members of the daig four weeks prior to a daig member assembly in munich (17 march 2011) for comment. only minor revisions were asked for. the guidelines were again put to vote during the meeting. during the final vote the guidelines received 36 positive unanimous votes and were agreed on in the current version as the daig/kaad oi guidelines. the german version (long version) of these guidelines was submitted to the awmf on 30 august 2011 and was published online on 8 november 2011 (http://www.awmf. org/leitlinien/detail/ll/055-006.html). ö ag approved these guidelines on 9 november 2011. immune reconstitution inflammatory syndrome the immune system is expected to recover following initiation of art. some patients, however, show a paradoxical reaction. with widely varying symptoms, this pattern of disease is defined as immune reconstitution disease, immune reconstitution syndrome, or immune reconstitution inflammatory syndrome (iris) [4] [5] [6] [7] . different clinical case definitions exist [8, 9] manifestations of iris are diverse and range from unspecific symptoms, ois to autoimmune diseases, and malignomas [10] . regarding ois, the physician must differentiate between symptomatic relapse of a prior infection (paradoxical iris) and infections first appearing on art (unmasking iris). data on the incidence of iris vary widely, ranging between 10 and 23 % of all patients at initiation of an art [10] [11] [12] [13] . a prospective study showed an incidence rate in germany of 24.8 % [14] . an international meta-analysis showed a total incident rate of 16.1 % for iris, with the highest rates for iris uveitis, followed by tb, cryptococcal meningitis, pml, and rarer cases of kaposi's sarcoma or varizella zoster virus (vzv) infections [13] . the greatest risk factor would appear to be a low cd4 t-cell count of \50 cells/ll [12, 15] . patients starting an art with a cd4 t-cell count of \200 cells/ll and especially those who have a high viral load require close monitoring. patients with\50 cd4 t-cells/ll should also be tested for a latent mycobacterial infection (by culture). a large prospective trial [16] showed no difference for the development of an iris when art was initiated immediately after patients had started an oi therapy (patients with tb were excluded from the trial). in this study, corticosteroids were often given on initiation of art in a high number of pcp cases, which possibly suppressed some iris cases. for tb and cryptococcosis, however, several studies showed an higher incidence of an iris when art was initiated early [17] [18] [19] . corticosteroids are useful in cases of tb-iris [20] . steroid therapy for 2-6 weeks is recommended for cryptococcal-iris (increase of intracerebral pressure). the use of non-steroidal anti-inflammatory drugs (nsaids) and thalidomide was recommended in some studies, but a general recommendation can not be given for these agents [21] . art should only be interrupted in very severe cases. results of the swiss hiv cohort study prove that consequent isoniazid (inh)-prophylaxis in hiv patients with latent tb significantly reduces the risk of a relapse [22] . in general, prognosis for an iris is good and the mortality rate is not higher than that for patients without an iris [23] . pneumocystis jiroveci pneumonia is the most frequent oi in germany and appears predominantly in hiv-infected patients with advanced immunodeficiency (cd4 t cells \200/ll). if there clinical-radiological findings suggest pcp, therapy should be initiated immediately without awaiting results of a bronchoalveolar lavage. a mild pcp [bga: partial pressure of oxgen (po 2 ) [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] can be treated in outpatient medical care. if ventilation becomes necessary, non-invasive methods (continuous positive airway pressure) are beneficial if applied at an early stage [24] . with respect to the treatment of artnaïve patients, several experts believe that the initiation of art can be delayed until acute treatment is completed. however, one rct has shown advantages of an early start [16] . acute therapy should be given at least for 21 days, if necessary longer. the treatment of choice is a combination of trimethoprim and sulfamethoxazole (tmp/smx, cotrimoxazole). oral application of tmp/smx is only recommended in mild cases, but this therapy can be also considered after initial improvement during intravenous therapy. positive effects with lower doses of tmp/smx have been observed in some case reports, but data from controlled trials are missing [25] . all severe cases should be treated intravenously in hospital. in cases of respiratory insufficiency [po 2 \ 70 mmhg or alveolar-arterial oxygen tension difference (aado 2 ) c 35 mmhg on room air], most experts recommend (5)-10 days of adjuvant administration of prednisolone [approx.1 mg/kg body weight as a single dose or split dose twice daily (bid)]. with prednisolone, mortality risk of severe pcp can be reduced by half and significantly fewer patients require mechanical ventilation [26] . compared to tmp/smx, all alternative therapies are less effective. in the event of intolerance or sulfonamide allergy, intravenous pentamidine (4 mg/kg once daily (qd) for 14-21 days is recommended as a second choice; this agent is however more toxic and the dose may therefore have to be reduced after 5 days (2 mg/kg). treatment with inhaled pentamidine can be attempted in mild cases of pcp [27, 28] ; however, reports on experience with this approach are conflicting [29] [30] [31] . instead of pentamidine, the administration of atovaquone suspension or a combination of trimethoprime and dapsone or clindamycin and primaquine is possible [test for glucose-6-phosphate dehydrogenase (g6pd) deficiency!]. data are only available for mild to moderate pcp [32] [33] [34] . primaquine is no longer approved for use in germany, but it is available through international pharmacies. it can only be applied if there are no other alternatives and requires increased efforts in educating patients. according to a meta-analysis, the combination of clindamycin plus primaquine is the most successful therapy if cotrimoxazole therapy fails [35] ; this combination appears to be more effective than pentamidine alone [36] . patients with \200 cd4 t-cells/ll (or \14 % of total lymphocytic count) or a previous pcp require prophylaxis. the therapy of choice is tmp/smx, which also has a protective effect against bacterial infections and cerebral toxoplasmosis [37, 38] . daily administration is possibly more effective than three doses a week [39] . in cases of moderate cutaneous allergic reactions, desensitization is possible [40] . monthly pentamidine inhalations are a welltolerated alternative [41, 42] . a suitable inhalation system should be chosen and an inhalative ß-sympathomimetic should be administered beforehand. other options are dapsone [41, 42] and atovaquone, both of which have proved to be similarly effective as tmp/smx, dapsone, and pentamidine in two multi-center trials [43] [44] [45] . atovaquone, however, proved inferior to tmp/smx in another study [32] . pcp prophylaxis can be discontinued after successful immune reconstitution on art to c200 cd4 t-cells/ll for at least 3 months [46] [47] [48] [49] . only a few cases of reoccurring pcp have been reported for discontinuation at [200 cd4 t-cells/ll [50, 51] . if the hiv rna is well suppressed, [100 cd4 t-cells/ll may be sufficient to discontinue prophylaxis [52] . however, larger trials would be needed to submit a general recommendation regarding discontinuation for these patients. the recommendations concerning therapy and prophylaxis of pcp are summarized in table 1 . the incidence of cerebral toxoplasmosis has decreased to less than a quarter of that during the earlier years of the hiv epidemic, [53] . nevertheless, it remains the most important neurological oi in hiv-infected patients in europe [54] . cerebral toxoplasmosis almost always results from a reactivation of a latent infection with toxoplasma gondii. extracerebral manifestations are rare. standard therapy is a combination of pyrimethamine and sulfadiazine, which is effective in 75-89 % of cases [55, 56] . an equivalent alternative is pyrimethamine and clindamycin [55, 57] . tmp/smx is also possible, with the same doses as used in pcp [58, 59] . tmp/smx proved to be as effective as sulfadiazine/pyrimethamine in two rcts on ocular and cerebral toxoplasmosis [60, 61] . a cochrane review showed no superiority of any one specific regimen [62] . for pyrimethamine, a ''loading dose'' within the first few days has been used since the first studies [56] . however, the efficacy of this approach has not been proven. due to the myelotoxicity of pyrimethamine, it is important to add folinic acid (not folic acid) from the start [63] . other alternatives are atovaquone/pyrimethamine [64] or azithromycin/pyrimethamine [65] ; however, data are limited. acute therapy lasts for a period of at least 4 (to 6) weeks-longer for alternative therapies. in most cases, empiric treatment of toxoplasmosis is initiated upon identification by radiographic testing. any improvement or clinical deterioration should be evaluated clinically and by magnetic resonance imaging (mri) scanning during therapy (after 14 days). in the case of progression, an alternative diagnosis (i.e., cerebral lymphoma, tuberculoma) and a brain biopsy should be considered. maintenance therapy with a reduced dosage should be initiated when lesions have resolved at least by 50 %, the clinical course has improved, and contrast enhancement has been reduced or eliminated. art should be initiated as soon as possible. in cases of increased intracranial pressure or extensive edema, steroids can be given (dexamethasone, 3-4 9 4-8 mg/day). the choice for steroid therapy must be considered carefully as steroids distort possible differential diagnoses. for example, primary cerebral lymphomas also respond to steroids, and in the case of therapeutic failure, the validity of a potential biopsy can be reduced with steroids. antiepileptic therapy is indicated if epileptic attacks occur. due to rare interactions with art, gabapentin, pregabalin, and levetiracetam are applied. levetiracetam is also available as infusion. a distinction must be made between exposure prophylaxis, primary prophylaxis, and secondary prophylaxis after cerebral toxoplasmosis. • exposure prophylaxis: immunoglobulin g (igg)-negative patients should avoid eating raw or undercooked meat. an increased risk due to proximity to cats has not been proven [66] . stricter measures of hygiene should be followed. however, the importance of this recommendation under effective art is questionable. • primary prophylaxis: igg-positive patients with \100 cd4 t-cells/ll require primary prophylaxis. the drug regimen of choice is tmp/smx. in cases of allergy, desensitization may be considered [40] . see above for alternatives. primary prophylaxis can be discontinued if cd4 t-cell count is[200 cells/ll for at least 3 months. • secondary prophylaxis: in the absence of immune reconstitution, patients require lifelong secondary prophylaxis, usually consisting of half the dose needed for acute therapy [67] . clindamycin is presumably less suitable as secondary prophylaxis as it cannot cross the intact blood-brain barrier [63] . tmp/smx also seems less effective for secondary prophylaxis. however, it may be considered because it is simple. a higher dose than that for pcp is definitely required [68, 69] . prophylaxis may be discontinued safely if initial therapy has led to radiological resolution and if there is an immune reconstitution of [200 cd4 t-cells/ll for at least 3-6 months [31, [70] [71] [72] . the recommendations on therapy and prophylaxis of cerebral toxoplasmosis are summarized in table 2 . in germany, seroprevalence of cmv infection in the adult population is 50-70 %. the risk of a reactivation of cmv infection increases when the cd4 t-cell count is \100 cells/ll. in addition to cmv retinitis, impairment of other end-organs may occur. due to the limited data on cmv manifestations, the same systemic therapy is recommended in these latter cases as for cmv retinitis [73] . international guidelines are also available for this approach [3] . all patients with manifest cmv infection should start art immediately. the cmv-specific immune response is restored [74] , leading to a reduction of cmv viremia [75] and delaying progression of an existing cmv retinitis or its recurrence [76, 77] . in addition to art, a cmv-specific therapy should be initiated at the time of diagnosis. therapy of cmv retinitis can be performed locally or systemically. a local therapy alone does not provide protection against dissemination of infection in the contralateral eye or other organs, but it can be considered if systemic drug toxicity is high. for systemic therapy, four substances are available: gancyclovir, foscarnet, cidofovir, and valgancyclovir (valgcv). the reader is referred to the product information on these substances for the respective side effects. valgancyclovir is the only drug that can be administered orally. it is almost completely hydrolyzed to gancyclovir after resorption in the gastrointestinal tract [78, 79] . gancyclovir and foscarnet are both recommended as first choices for treating cmv retinitis even though foscarnet proved to be superior in pre-art times [80, 81] . the side effects of both drugs differ, but the response rates to therapy are similar with both substances [81] [82] [83] . as foscarnet must be administered via a central catheter, the administration of gancyclovir is easier and often preferred. valgancyclovir has proven to be effective in a comparative study and has the advantage of being less complicated to administer than intravenous infusion. intravenous treatment, however, may be necessary if foveal infections occur with acute risk of impairing visual acuity. in these cases, gancyclovir and foscarnet are equally recommended for first-line therapy. treatment with both agents consists of an induction therapy followed by life-long maintenance therapy. induction therapy usually lasts for at least 2-3 weeks until lesions resolve. without sufficient art, selection of resistant cmv mutations is frequent and accumulates as the infection progresses [84, 85] . several authors recommend valgcv for first-line therapy based on the results of a prospective randomized trial with valgcv and parenteral gancyclovir [86] and on those of studies on the pharmacokinetics of ganciclovir, with both showing similar results after the administration of valgcv [78, 79, 86] . other studies on the pharmacokinetics of gancyclovir following the administration of valgancyclovir either lack a comparison with parenteral gancyclovir [78] , or the administered doses were too low to show bioequivalence of valgcv and gancyclovir [79] . in summary, a clear recommendation in favor of valgcv cannot be given at the present time. in the presence of sight-threatening lesions, the panel strongly recommends against treatment with valgancyclovir due to the lack of clear evidence. some experts recommend a combination therapy of gancyclovir and foscarnet in full doses for acute sightthreatening lesions. maintenance therapy with valgcv can be initiated after lesions have completely resolved [87] ; however, this recommendation also lacks data. without sufficient art, a relapse is likely to occur, even under maintenance therapy with valgancyclovir. if lesions (zone ii and iii) are more anterior, therapy with valgcv may be attempted with weekly monitoring of the fundus. cidofovir has not been tested in controlled trials against gancyclovir or foscarnet. compared to a delayed therapy, cidofovir significantly slows down the progression of the infection [88] ; however, cidofovir is not recommended as first-line therapy due to its side effects. it does remain an important agent in the treatment of progredient cmv retinitis under gancyclovir or foscarnet therapy. sufficient art is crucial for a successful therapy of cmv retinitis. patients with progredient cmv retinitis on a gancyclovir regimen can be treated successfully with foscarnet or a combination of foscarnet and gancyclovir [89] . a good response is obtained in many cases with treatment with cidofovir, and this drug can therefore be an alternative. if foscarnet should fail, gancyclovir or a combination of gancyclovir and foscarnet can be effective. here too, therapy with cidofovir can prevent further progression. gancyclovir implants can still be effective after therapy failure under systemic gancyclovir or foscarnet due to the significantly higher intraocular gancyclovir concentration produced by the implants [90] . however, there is no protection against further spread of the infection to other organs or to the contralateral eye [91] [92] [93] . extraocular manifestations are always treated in the same way as a cmv retinitis, although only a few studies support this recommendation. in the presence of a cmv encephalitis or ventriculitis, clinical experience and smaller case studies indicate that a combination therapy with gancyclovir and foscarnet is superior to monotherapy [94] [95] [96] [97] [98] [99] . due to the toxicity of this therapy, the diagnosis should be confirmed. • primary prophylaxis: gancyclovir prophylaxis for cmv retinitis with a cd4 t-cell count of \50 cells/ll is effective, but this is usually too toxic. fundoscopy every 3 months is recommended but not necessary in the opinion of most experts (especially at a cd4 t-cell count of [100 cells/ll). • a dose-reduced secondary prophylaxis should be initiated, preferably with oral valgcv after about 3 weeks of acute therapy and after lesions have formed scars [87] . discontinuation of secondary prophylaxis to avoid side effects as soon as possible is recommended and feasible [77, 100, 101]-however, not before at least 6 months of maintenance therapy and immune reconstitution at a cd4 t-cell count of[100-150 cells/ ll. a small study showed that discontinuation after 18 months of art/maintenance therapy is already safe at a cd4 t-cell count of [75/ll [101] . in the first stage after discontinuation, patients undergo an ophthalmology control at least once a month. the required duration of a recurrence prophylaxis is not clear, nor is it as yet known for how long recurrences with other organ manifestations should be monitored. duration should therefore be handled as for cmv retinitis. the recommendations on therapy and prophylaxis of cmv manifestations are summarized in table 3 . from the roughly 200 candida species only about 15 different species are encountered in clinical daily practice. the most frequent species by far is c. albicans. clinical response to fluconazole of infections caused by c. albicans and candida parapsilosis is mostly good, whereas that to infections caused by c. glabrata or c. krusei is poor or totally missing. primary in vitro resistance of c. albicans to azoles is rare [102] . secondary resistance development under long-term azole therapy (fluconazole) was frequently observed in the pre-highly active art (haart) era. for the treatment of oral and vulvovaginal candidiasis, the reader is referred to the respective awmf guidelines [103, 104] . esophageal candidiasis (thrush) does not require an endoscopy to confirm the diagnosis in the presence of a typical clinical course and a mouth sore. the imidazole antimycotics, such as clotrimazole and the hydroxypyridone ciclopirox olamine, are suitable for local therapy of cutaneous candidiasis. if the immune status of the patient is good and/or in the case of a first episode of an oral candidiasis (oc), topical antimycotics, such as suspensions or pastilles (nystatin, amphotericin b, miconazole), are more inexpensive therapy options, although inferior to a therapy with fluconazole [105] [106] [107] . however, adherence is restricted with topically effective suspensions/pastilles. alternatives are mucoadhesive applications, although these are clearly more expensive. oral therapy with systemical azole derivatives (fluconazole, itraconazole, posaconazole, voriconazole) show a more rapid response, provide longer protection against recurrences, and are tolerated better by patients [108] [109] [110] [111] . fluconazole can be considered the drug of choice for oc and esophageal candidiasis. a once-daily oral therapy (100 mg for 5-14 days) has been established as the standard for oc [112] . single doses of up to 750 mg fluconazole have been tested in a small patient group (mostly without art) and was considered to be equivalent to a 14-day therapy. this therapy, however, should be confined to patients with compliance problems, as data on late relapses are limited [113, 114] . esophageal candidiasis is usually treated for 10-14 days with doses of 200-400 mg fluconazole qd. patients presenting with severe dysphagia can initially be treated intravenously and switched to oral application as symptoms improve. if fluconazole resistance has been detected, treatment with other azole derivatives is usually still effective and should be attempted before parenteral therapy is initiated (e.g., with echinocandin). traconazole, voriconazole, and posaconazole have demonstrated clinical efficacy for cases of fluconazole refractory oropharyngeal and esophageal candidiasis [115] [116] [117] [118] . all azole derivatives require a double dose on the first day of the regimen (loading dose). therapy with a higher dose of fluconazole (b800 mg/day & 12 mg/kg/day) or an antimycotic combination therapy [119] can be considered, but data are insufficient. therapy failure and/or fast relapses occur most frequently in patients with poor immune status (\100 cd4 t-cells/ll). data from randomized studies have shown that echinocandins (caspofungin, micafungin or anidulafungin) are as effective and well tolerated as fluconazole for the treatment of candida esophagitis [120] [121] [122] . however, application should be restricted to azole refractory infections with clear fluconazole resistance [120, 123, 124] . art should be initiated immediately if chronic recurring oropharyngeal/esophageal candidiasis is present and at the latest if resistance problems occur. azole refractory candidiasis as well as azole-resistant strains can disappear with sufficient immune reconstitution as a consequence of art [125, 126] . regular change of toothbrush and thorough cleaning of dentures are a basic recurrence prophylaxis for oc. oc in hiv-infected children and adults can be treated and relapses prevented by applying disinfecting mouth rinses containing chlorhexidine 0.12 % 1-29 daily for a 90-day period [127, 128] . in the pre-haart era, secondary prophylaxis or life-long therapy with fluconazole led to significant reductions of chronic recurring oropharyngeal candidiasis-but it has also led to the development of secondary resistance [129, 130] . in a randomized study comparing secondary prophylaxis after oc with intermittent therapy on oc recurrence, relapses and infections of systemic candidiasis were reduced by the long-term prophylaxis. however, no survival benefit has been demonstrated for any candidiasis prophylaxis [131] . primary prophylaxis is not recommended, and indications for secondary prophylaxis should be restricted to individual case. the recommendations on therapy and prophylaxis of candidiasis are summarized in table 4 . herpes simplex virus (hsv) infections are frequent in hiv-infected patients. chronic and atypical courses are possible especially in the setting of severe immune deficiency (\100 cd4 t-cells/ll). organs such as the esophagus, central nervous system (cns), eyes, and respiratory tract may also be affected. in these cases and with persistence of lesions for a period of[4 weeks, hsv infection is an aids-defining illness. topical treatment with acyclovir is adequate for patients with a good immune status and only discrete oral lesions. pencyclovir cream is probably as effective [132] . genital herpes lesions do not respond as well to topical treatment. for systemic treatment against hsv-1 and hsv-2, the drug of choice is still acyclovir. resistance is rare [133] , and healing of lesions can be accelerated by the therapy [134] . severe cases with organ involvement should be treated intravenously. as hsv levels are lower in the cns than in plasma, the dose to treat encephalitis should be increased. valacyclovir (valacv) and famcyclovir are equally effective alternatives to acyclovir [135, 136] . however, they are not approved for patients with immune deficiency and should only be applied if response to acyclovir fails [137] . for uncomplicated genital herpes lesions, shorter regimens of 2 days of 500 mg famcyclovir may be as effective, provided there is no immune deficiency [138] . according to the opinion of some experts, brivudine is an alternative for hsv-1 and vzv, although contraindicated for immunosuppressed patients and only approved for the treatment of vzv. however, results from controlled studies with hiv-infected patients are not available. in cases of painful mucocutaneous lesions, a local anesthetic can also be applied. treatment with foscarnet for several weeks may be helpful in exceptional cases, especially if lesions remain refractory to standard treatment [139] . primary prophylaxis is not recommended. an earlier meta-analysis in which acyclovir was found to reduce the risk of both hsv and vzv disease by more than 70 % [140] must be viewed in the context of art today. nevertheless, long-term treatment with low-dose acyclovir or valacv can still be effective treatments for recurrent hsv [141, 142] . the risk of hiv transmission, which is increased threefold by genital hsv-infection [143] , is not reduced by treatment with acyclovir [144] [145] [146] . between 70 and 90 % of patients with symptomatic hsv-2 infection and at least 20-50 % of patients with symptomatic hsv-1 infection experience recurring episodes within the first year. possible causes are local trauma, uv exposure, fever, and immune suppression. a long-term prophylaxis for at least 6 months is recommended for frequent recurrences. this prophylaxis can prevent further episodes in 70-80 % of cases. the recommendations on therapy and prophylaxis of genital hsv infections are summarized in table 5 . patients infected with hiv are at increased risk for vzv infection. multisegmental zoster or zoster generalisatus are often observed with low cd4 t-cell counts. chronic courses with ulcerating forms and involvement of other organs are rare. pneumonia or cns involvement should be considered. a monosegmental zoster can be treated with oral acyclovir. famcyclovir and valacv are alternatives. each complicated, multisegmental or facial zoster should be treated intravenously for 10-14 days. after clinical improvement is evident, a switch to oral therapy is possible. zoster neuralgia occurs less frequently in hiv-negative patients treated with the alternative drugs valacv, famcyclovir, and brivudine than when treated with acyclovir table 5 therapy and prophylaxis of genital herpes simplex virus infections a therapy/prophylaxis drug therapeutic regimen acute therapy (duration: 7-10 days), daily doses first choice acyclovir (3-) 5 9 400 mg p.o. severe cases 3 9 5-10 mg/kg i.v. 5 9 800 mg p.o. alternatives valacyclovir 2-3 9 1,000 mg or 3 9 500-1,000 mg (expert opinion) therapy for recurrent herpes simplex infection virus episodes acyclovir 3 9 400 mg p.o. for 5-10 days valacyclovir 2 9 1,000 mg for 5-10 days famciclovir 2 9 500 mg for 5-10 days long-term prophylaxis (duration: at least 90 days) acyclovir 2-3 9 400-800 mg valacyclovir 2 9 500 mg famciclovir 2 9 500 mg a daily doses [147] . however, according to a cochrane analysis, the results of this study are not clear [148] . brivudine is not licensed for the treatment of immunocompromised patients. acyclovir resistance is rare and most frequently observed under long-term therapy [149, 150] ; in these cases, foscarnet (3 9 40 mg/kg) or cidofovir (5 mg/kg, maximum 375 mg 19/week) can be given. early concomitant and monitored pain management with nsaids and/or other opiates in combination with amitriptyline and/or pregabalin is important. for further information on zoster pain, the reader is referred to the awmf guidelines. varicella vaccination seems to be fairly safe and effective for patients with a cd4 t-cell count of [400/ll [149] . vaccination should be considered if vzv serology is negative. in individuals with negative serology and exposure to vzv, administration of hyperimmunoglobulin may be attempted. long-term primary prophylaxis is usually not effective; however, a long-term low-dose therapy can be considered in the presence of persistent recurring episodes. the recommendations on therapy and prophylaxis of vzv-infections are summarized in table 6 . progressive multifocal leukoencephalopathy (pml) is a severe demyelinating disease of the cns caused by the john cunningham virus (jcv). prognosis for pml was poor in the pre-haart era, with the median interval between the onset of the first symptoms and death being 3-6 months. with effective art, there are significantly fewer cases of disease progression, and even complete remission seems possible [151] . nevertheless, mortality of patients with pml remains high at 50 %, albeit art [152] . there is no specific pml treatment with proven efficacy; consequently, the mainstay of therapy is immune reconstitution. as such, priority remains on the initiation and optimization of an art. treating physicians are recommended to apply intracerebral penetrating agents. a successful immune reconstitution accounts for a significant reduction in mortality [151] [152] [153] [154] [155] [156] . after initiation of art, a paradoxical worsening of clinical symptoms in terms of an immune reconstitution inflammatory syndrome (iris) has been observed in approximately 16-23 % of pml cases. administration of corticosteroids for pml-iris has only been described in case studies [157] , and evidence of a benefit was not provided. given the slight difference in the 1-year survival rate for pml patients and pml-iris patients [158] , the use of corticosteroids or a temporary discontinuation of art must be weighed up against the risks of a possible decline of the jcv-specific immune response. several supportive immunomodulatory approaches have been tested, but to date there is no convincing evidence for the efficacy of treatments with immunoglobulin, interleukin-2 (il-2), or il-a [159] . therapeutic regimens aimed at inhibiting jcv replication have also been attempted, but as yet relevant evidence supporting the clinical use of drugs such as cytosine arabinoside is not available [153, 160] . antiviral treatment with acyclovir, cidofovir, ganciclovir, brivudin, ribavirin, foscarnet and the combination therapy foscarnet and zidovudine have also proven to be ineffective [161] . recently, 5-ht2a inhibitors and/or serotonin receptor antagonists have been discussed for pml treatment. in vitro data for the suppression of jcv replicates via 5ht(2a)r inhibitors are contradictory [162] [163] [164] [165] [166] [167] . results from controlled clinical studies are missing. based on promising in vitro data [168] a phase i/ii study of mefloquine was initiated-only be stopped due to a lack of efficacy. in summary, specific treatments for pml cannot be recommended outside clinical trials. there is no prophylaxis. exposure prophylaxis is also not possible. the recommendations for therapy and prophylaxis of pml are summarized in table 7 . cryptosporidiosis is a parasitic intestinal disease with fecal-oral transmission, mainly caused cryptosporidium parvum (two other frequent types: c. hominis and c. [169] . infection of the biliary tract leading to sclerosing cholangitis is frequent, particularly among patients with severe immunodeficiency, but may be reversible with immune reconstitution [170] [171] [172] (level of evidence c). other rare manifestations are infections of the pancreatic duct and pulmonary infections [173, 174] . successful immune reconstitution under art can lead to complete resolution of clinical crytosporidiosis [175, 176] . symptomatic treatment with loperamide and/or tincture of opium should be given. octreotide (off label) can also be applied. sufficient hydration is important and infusions may even be required. no specific treatment has been validated [177] . rifaximin is promising, as first studies with aids patients show [178] ; however, results from randomized studies are still missing. nitazoxanide was found to be effective in a small randomized study in immunocompetent patients [179] . however, this drug is not approved for aids patients and showed no effects in a double-blind randomized study in hiv-infected children with cryptosporidiosis [180] . paromomycin has been found to have favorable effects on diarrhea [181] . however, a double-blind randomized study showed no benefit compared to placebo [182] . in a cochrane review for the prevention and treatment of cryptosporidiosis, paromomycin did not reduce diarrheal frequency permanently [177] . there is no generally accepted prophylaxis, although a protective effect of rifabutin and clarithromycin has been reported from retrospective studies. azithromycin showed no effect [183] . the usual hygienic measures (gloves) are usually adequate. patients do not need to be isolated. however, accommodation with other immunosuppressed patients should be avoided. the recommendations on therapy and prophylaxis of cryptosporidiosis are summarized in table 8 . for further information, refer to guidelines by the cdc for cryptosporidiosis in hiv-infected patients (cdc 2009; http://www.cdc.gov/mmwr) [3] . cryptococcosis occurs much more frequently in africa, the usa, and southeast asia than in europe. bird droppings (especially of pigeons) are presumably a key reservoir, but a direct transmission between humans has not been observed. although transmission occurs via inhalation, pulmonary symptoms or lung infiltration are only seen in 30-40 % of cases of hiv-infected patients. cryptococcosis infection is often followed by disseminated disease in hiv patients with severe immunodeficiency (\100 cd4 t-cells/ ll) and often involves the cns ([75 %, meningitis) [21] . recommended treatment for a cryptococcal meningitis is the combination regimen of amphotericin b deoxycholate (amb-d; 0.7-1.0 mg/kg/day i.v.) and flucytosine (100 mg/kg/day i.v. or p.o. if available), divided into four doses a day. acute therapy should be given for at least 14 days. if clinical response is good, a switch to monotherapy with fluconazole (400 mg/day) for another 8 weeks is possible [21] . liposomal amphotericin is slightly more effective than conventional amb-d and provides an alternative, if amb-d is not well tolerated [184] . monotherapy with fluconazole as initial treatment in hiv-infected patients is not sufficient, even with higher daily doses of 800-2,000 mg. thus, it is only considered as an option in countries with limited resources [21, 185] . in the pre-haart era, a triple combination therapy with amb-d, flucytosine, and fluconazole was favored for the treatment of cryptococcal meningitis in germany [186] . however, in one randomized study, the triple combination was not more effective than a combination with amb-d and flucytosine or amb-d and fluconazole or a monotherapy with amb-d [187] . the combination with amb-d and fluconazole is an alternative in regions with limited resources where flucytosine is not available. in a small study in thailand, a higher dose of fluconazole (800 mg/day) combined with amb-d (0.7 mg/kg/day) was more effective than monotherapy with amb-d alone or a regimen of amb-d ? fluconazole (400 mg/day). other combination therapies (e.g. fluconazole ? flucytosine) are possible alternatives, but lack sufficient data [188] . itraconazole plays no role in primary therapy and is less effective than fluconazole in maintenance therapy [189] . monotherapy with posaconazole showed a response rate of up to 50 % in a small case study on refractory diseases and therefore provides an alternative for this indication [190] . efficacy of voriconazole in salvage therapy is still not clear [191] . echinocandines show no in vitro effect against c. neoformans. in the case of an iris when art is initiated during antimycotic treatment, additional treatment with corticosteroids (0.5-1.0 mg/kg/day prednisolone equivalent) is required [21] . in refractory treatment situations, additional administration of c-interferon might be useful in individual cases [192] . treatment success is monitored based on the clinical course and repeated lumbal punctures. patients should have their intracranial pressure measured at time of diagnosis. if the intracranial pressure is very high, several punctures should be made in the first week until it is reduced to b20 cm. in individual cases, cerebrospinal fluid (csf) drainage can be considered to reduce the intracranial pressure if there are no contraindications [21] . for mild, isolated cryptococcal pneumonia (negative csf diagnosis), monotherapy with fluconazole (400 mg/day) is possible. treatment should continue for 6-12 months. severe cases of pneumonia with or without acute respiratory distress syndrome (ards) should be treated the same way as meningitis (see above). art-naïve patients at the time of diagnosis should start an art after a 2wo-week induction therapy with antimycotics. however, an optimal time for initiation of art is not yet clearly defined. primary prophylaxis can not be recommended to hivinfected patients in germany due to lack of a clear survival benefit [193] . after acute therapy of cryptococcal meningitis, secondary prophylaxis should be introduced. fluconazole (200 mg/day) is the regimen of choice and is also more effective than itraconazole [21] . secondary prophylaxis can be discontinued after at least 6 months maintenance therapy with sufficient immune reconstitution ([100 cd4 t-cells/ll and hiv-rna below detection limit for over 6 months). the risk of a relapse is high if maintenance therapy is discontinued too early [194] . the recommendations on therapy and prophylaxis of cryptococcosis are summarized in table 9 . human immunodeficiency virus-associated infections of nontuberculous mycobacteria (ntm) have declined in countries where art is available [195] [196] [197] . in addition to disseminated ntm diseases, which develop almost exclusively in the setting of severe cd4 t-cell depletion (\50 cd4 t-cells/ll) and which are mainly ([90 %) caused by the mycobacterium avium complex or m. intracellulare (mycobacterium avium intracellure complex, mai), incidences of ntm-iris as well as pulmonary ntm diseases are also observed. pulmonary ntm is frequently caused by other species, such as m. kansasii, m. xenopi, m. malmoense, and m. abscessus. for further information on diagnosis, the reader is referred to the american thoracic society criteria [198] . due to the ubiquitous occurrence of ntm, pre-exposure prophylaxis is not possible and an isolation of infected patients is not necessary. some specialists, however, recommend a screening of generalized mai infections in patients with cd4 t-cell counts of\50/ll prior to initiation of an art. given here are only recommendations for the mai therapy. with respect to ntm species other than mai, the reader is referred to the appropriate literature [198] or advised to consult experts (ntm-net). a combination treatment of macrolide (clarithromycin or azithromycin) and ethambutol plus/minus rifabutin is recommended [198] . rifabutin is preferred to rifampicin due to its in vitro efficacy against mai and its lower interaction potential. following the publication of data showing that rifabutin could be omitted from the treatment regimen [199] , another randomized study demonstrated a survival benefit with the triple combination clarithromycin, rifabutin, and ethambutol compared to clarithromycin with either ethambutol or rifabutin-the mortality rate was halved in the treatment arm receiving this triple (clarithromycin-containing) combination [200] . the doses for rifabutin must occasionally be adjusted to the art regimen [201] . clarithromycin increases the rifabutin serum level, while rifabutin decreases the clarithromycin level. treatment duration with rifabutin has not yet been determined in studies; however, experts recommend discontinuing rifabutin after a few weeks and with clinical improvement. the daily doses for clarithromycin should not exceed 2 9 500 mg, as a higher mortality risk has been described for patients receiving higher dosages [202, 203] . azithromycin can be administered instead of clarithromycin, as these two drug are comparably effective in combination with ethambutol, with slightly more rapid sterilization of blood cultures with clarithromycin [196, 199, 204] . as macrolides are the cornerstone of therapy, the development of resitance to macrolides must be avoided, and monotherapy with macrolides should not be administered. in the case of intolerance, alternative substances, such as fluoroquinolone, amikacin, cycloserine, dapsone, linezolid, or mefloquine, are available. however, clinical evidence for the treatment of mai infections with these alternative substances is still insufficient. in the case of ntm-iris, the extent and duration of an antimycobacterial therapy are not clear. it is possible that partial virus suppression is enough for a ntm-specific immune reconstitution [205] . it is easier to evaluate the clinical response to localized ntm infections. in cases of localized lymphadenitis and skin manifestations, therapy duration of 6 months is recommended after patients are culture-negative. if the clinical response is good and cd4 t-cells continue to increase under a still effective art, the regimen can be reduced after 3 months to a recurrence prophylaxis with a macrolide for a further 3 months. patients with abdominal localization have a poorer response and require a more aggressive and longer therapy [206, 207] . additive corticoid therapy has symptomatic indications. the treatment of patients with pulmonal ntm diseases not deriving from an iris are based on the guidelines for non-hiv-infected patients [198] . in the usa, placebo controlled trials for clarithromycin, azithromycin and rifabutin showed that primary prophylaxis significantly reduced mai-morbidity and -mortality in severely immunocompromised patients [208] [209] [210] [211] . all these studies, however, were undertaken in the pre-ha-art era. in addition, mai-infections are less frequent in europe, so that only a few patients receive primary prophylaxis [212] . due to the declining incidences since the introduction of art, primary prophylaxis can only avoid a small number of diseases [197] . ntm-associated iris can also not be prevented by prophylactic drugs [206] . therefore primary prophylaxis is not recommended in germany. after treatment of a disseminated mai-infection, patients lacking other art options, should receive secondary prophylaxis with a macrolide, provided cd4 t-cell count is under 50 cells/ll. weekly doses of azithromycin are convenient and efficacy is comparable to daily rifabutin [208] . secondary prophylaxis or maintenance therapy can be discontinued under an art and if patients are without symptoms and cd4 t-cell count is [100/ll for 6 months. the recommendations concerning therapy and prophylaxis of disseminated mai-diseases are summarized in table 10 . globally, tb is the most prevalent hiv-associated opportunistic infection. in germany, tb is rare. hiv-infected patients are affected by tb independent of their cd4 t-cell count [213] , although incidences increase with advanced immunodeficiency [214] . uncomplicated cases of tb can successfully be treated with a standard therapy regimen over a period of 6 months, regardless of hiv status. first-line drugs are rifampicin, inh, ethambutol, pyrazinamide, and streptomycin, with inh and rifampicin being the most effective. tb should always be treated with a combination of four drugs in the initial phase to prevent drug resistance. standard initial phase therapy is a 2-month course of rifampicin, inh, ethambutol, and pyrazinamide, followed by a continuation phase therapy of 4 months rifampicin and inh. in individual cases, such as incompliance, it may be necessary to extend the standard treatment duration to c9 months, especially if sputum cultures are still positive after 2 months. recurrences after successful therapy appear more frequently in hiv-infected patients [215] . if standard therapy has not been initially applied, treatment should always last for at least 9 months. alternatively, ethambutol, streptomycin, and reserve drugs such as ofloxacin or moxifloxazin, cycloserine, and linezolid may be administered. since this treatment is no different from that for multiresistant tb, these patients should be treated in specialized centers. adverse effects occur frequently with anti-tb therapy (refer to individual drug information for side effects, necessary testing, and drug interactions). severe side effects are observed more often in hiv-infected patients than in hiv-negative patients [216] . antiretroviral therapy significantly reduces the morbidity and mortality rate in hiv-infected patients [217] . a 6-month tb standard therapy achieves similar success in both hiv-infected and hiv-negative patients [218] . although a large retrospective and a large open-label, randomized trial showed a survival benefit with simultaneous art and anti-tb treatment, this approach proves to be difficult in practice due to overlapping drug interactions and side effects [219] . for tb meningitis, side effects are more frequent during the first 2 months of therapy if art and anti-tb therapy are initiated simultaneously. in this case, a delay of art by 2 months is possible without risking a higher mortality [220] . with regard to other forms of tb, 25-60 % of patients develop an iris in the first 3 months of art treatment [221] . a consensus on a uniform case definition of tb-iris was reached in 2008 [9] , which we refer to in the chapter on iris of this guideline. adherence to simultaneous hiv and mycobacterium tb treatment is difficult to achieve due to the high pill burden and overlapping toxicities. both rifampicin and protease inhibitors (pis) are metabolized by cytochrome p450 3a. concomitant therapy is therefore not recommended [222, 223] (table 11 ). rifabutin can be combined with boosted unboosted protease inhibitors are no longer recommended due to insufficient plasma levels. consider tdm pis, however the dose must be adjusted (table 12 ). it may be useful to determine serum levels [201] ; however, this approach has not been tested in clinical research with clear endpoints. recommendations can be given for first-line art therapy with tenofovir (tdf), tdf ? emtricitabine (ftc), and ftc plus efavirenz in combination with rifampicinbased tb therapy. alternatives are other efavirenz-based regimens (without adjustment of dose) with rifabutin [222] . to date, clinical data on combinations of rifamycin with new drugs, such as darunavir, raltegravir, or maraviroc, are limited. due to the strong inducing potential of cytochrome p450 3a, pis should be avoided and maraviroc should only be given under close observation. rifampicin also induces the enzyme ugt1a1, leading to increased glucoronidation and reduced plasma levels of raltegravir [224] . no interactions have been reported with tenofovir and t-20 [225] . the recommendations on the adjustment for combination of art/rifampicin in tb therapy are summarized in table 12 . treatment of active tb has clinical priority over art. in patients with \100 cd4 t-cells/ll, simultaneous treatment of both infections is indicated [222, 226] . however, even in this situation it is recommended to start tb therapy first for 2 weeks before initiating art to prevent possible side effects. for patients with 100-350 cd4 t-cells/ll, art can be delayed for 2 months until the anti-tb drugs can be reduced for the continuation phase. there is no evidence for an optimal timing of art when the cd4 t-cell count is [350 cells/ll [222] . the results of a large randomized trial indicate that mortality rate in patients with 200-500 cd4 t-cells/ll is reduced when art is initiated during tb therapy [219] . for hiv patients with\50 cd4 t-cells/ll, the results of a recent study show a benefit of a delayed art. the decision should be made carefully under consideration of the situation of each single patient [17, 227] . hiv-infected patients already on a successful art should remain on art, although the regimen may need to be modified [226] . the recommendations for co-administering art with rifabutin are summarized in the statement that adherence is the most important factor for therapeutic success and to avoid resistant tb strains. the world health organization (who) recommends a directly observed therapy for these patients. latent tuberculosis infection (ltbi) is defined by a positive mycobacterium tb-specific immune response in the tuberculin skin test (tst) or an interferon gamma release assay (igra) in the absence of active tb. clear values for a positive mycobacterium tb-specific immune response in hiv-infected patients do not exist. patients are not infectious as the tb is not active. however, hiv-infected patients with ltbi carry a higher risk of developing active tb. according to guidelines for the treatment of ltbi by the cdc [228] , hivinfected patients with a tst of [5 mm should be given treatment with inh for 9 months. this probably also applies to patients with positive igra test results, but convincing data are still missing [229] . alternatively, a 4-month course of rifampicin can be given. a 2-month course of rifampicin and pyrazinamide is no longer recommended, as it has been associated with significantly higher toxicities in hiv-negative patients [230, 231] . in 2006, 2.2 % of all tb patients showed multidrug resistance (at least resistance against inh and rifampicin). among these, 5 % were hiv-infected [232] . in addition to incidences of multidrug resistance (mdr), incidences of extensive drug resistance (xdr) were reported in at least 58 countries in 2010 [233] . xdr tb is defined by the who as tb which is additionally resistant to fluoroquinolones and at least one of the injectable drugs amikacin, capreomycin, or kanamycin. due to the complex therapy and an overall poor prognosis, patients with mdr/xdrtb should be treated in specialized centers. conflict of interests conflict of interest statements of all authors are published online: http://daignet.de/site-content/hiv-therapie/ open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. final consent to this version by the steering committee of the ö ag given on 9 november 2011. hiv-associated opportunistic infections-going, going, but not gone: the continued need for prevention and treatment guidelines patients presenting with aids in the ha-art era: a collaborative cohort analysis guidelines for prevention and treatment of opportunistic infections in hiv-infected adults and adolescents: recommendations from cdc, the national institutes of health, and the hiv medicine association of the infectious diseases society of america cytomegalovirus retinitis after initiation of highly active antiretroviral therapy focal mycobacterial lymphadenitis following initiation of protease-inhibitor therapy in patients with advanced hiv-1 disease immune reconstitution inflammatory syndrome: more answers, more questions aids: immune reconstitution inflammatory syndrome: a reappraisal immune reconstitution syndrome in hiv: validating a case definition and identifying clinical predictors in persons initiating antiretroviral therapy tuberculosis-associated immune reconstitution inflammatory syndrome: case definitions for use in resource-limited settings defining immune reconstitution inflammatory syndrome: evaluation of expert opinion versus 2 case definitions in a south african cohort incidence and risk factors for immune reconstitution inflammatory syndrome in an ethnically diverse hiv type 1-infected cohort incidence and risk factors for the immune reconstitution inflammatory syndrome in hiv patients in south africa: a prospective study immune reconstitution inflammatory syndrome in patients starting antiretroviral therapy for hiv infection: a systematic review and meta-analysis immunereconstitution inflammatory syndrome in a prospective german cohort. abstract 149. kit tuberculosis-associated immune reconstitution disease: incidence, risk factors and impact in an antiretroviral treatment service in south africa early antiretroviral therapy reduces aids progression/death in individuals with acute opportunistic infections: a multicenter randomized strategy trial integration of antiretroviral therapy with tuberculosis treatment early versus delayed initiation of antiretroviral therapy for concurrent hiv infection and cryptococcal meningitis in sub-saharan africa cryptococcal immune reconstitution inflammatory syndrome after antiretroviral therapy in aids patients with cryptococcal meningitis: a prospective multicenter study randomized placebo-controlled trial of prednisone for paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america reducing tuberculosis incidence by tuberculin skin testing, preventive treatment, and antiretroviral therapy in an area of low tuberculosis transmission immune reconstitution inflammatory syndrome in the first year after haart: influence on long-term clinical outcome noninvasive ventilation for treating acute respiratory failure in aids patients with pneumocystis carinii pneumonia good outcome with trimethoprim 10 mg/kg/day-sulfamethoxazole 50 mg/kg/day for pneumocystis jirovecii pneumonia in hiv infected patients adjunctive corticosteroids for pneumocystis jiroveci pneumonia in patients with hiv-infection treatment of mild to moderately severe pneumocystis carinii pneumonia with cotrimoxazole versus pentamidine aerosol. preliminary results of a prospective randomized therapy study pentamidine aerosol versus trimethoprim-sulfamethoxazole for pneumocystis carinii in acquired immune deficiency syndrome intravenous or inhaled pentamidine for treating pneumocystis carinii pneumonia in aids. a randomized trial inhaled or intravenous pentamidine therapy for pneumocystis carinii pneumonia in aids. a randomized trial treating opportunistic infections among hiv-infected adults and adolescents: recommendations from cdc, the national institutes of health, and the hiv medicine association/infectious diseases society of america comparison of atovaquone (566c80) with trimethoprim-sulfamethoxazole to treat pneumocystis carinii pneumonia in patients with aids oral atovaquone compared with intravenous pentamidine for pneumocystis carinii pneumonia in patients with aids. atovaquone study group clindamycin with primaquine vs. trimethoprim-sulfamethoxazole therapy for mild and moderately severe pneumocystis carinii pneumonia in patients with aids: a multicenter, double-blind, randomized trial (ctn 004). ctn-pcp study group second-line salvage treatment of aids-associated pneumocystis jirovecii pneumonia: a case series and systematic review clinical efficacy of first-and second-line treatments for hiv-associated pneumocystis jirovecii pneumonia: a tri-centre cohort study a metaanalysis of the relative efficacy and toxicity of pneumocystis carinii prophylactic regimens efficacy of trimethoprim-sulfamethoxazole for the prevention of bacterial infections in a randomized prophylaxis trial of patients with advanced hiv infection a randomized trial of daily and thrice-weekly trimethoprim-sulfamethoxazole for the prevention of pcp in hivinfected persons trimethoprim-sulfamethoxazole (tmp-smz) dose escalation versus direct rechallenge for pneumocystis carinii pneumonia prophylaxis in human immunodeficiency virus-infected patients with previous adverse reaction to tmp-smz meta-analysis of prophylactic treatments against pneumocystis carinii pneumonia and toxoplasma encephalitis in hiv-infected patients a randomized trial of three antipneumocystis agents in patients with advanced human immunodeficiency virus infection. niaid aids clinical trials group atovaquone compared with dapsone for the prevention of pneumocystis carinii pneumonia in patients with hiv infection who cannot tolerate trimethoprim, sulfonamides, or both. community program for clinical research on aids and the aids clinical trials group atovaquone suspension compared with aerosolized pentamidine for prevention of pneumocystis carinii pneumonia in human immunodeficiency virus-infected subjects intolerant of trimethoprim or sulfonamides atovaquone suspension for treatment of pneumocystis carinii pneumonia in hiv-infected patients discontinuation of prophylaxis for pneumocystis carinii pneumonia in hiv-1-infected patients treated with highly active antiretroviral therapy discontinuation of pneumocystis carinii pneumonia prophylaxis after start of highly active antiretroviral therapy in hiv-1 infection. euros-ida study group a randomized trial of the discontinuation of primary and secondary prophylaxis against pneumocystis carinii pneumonia after highly active antiretroviral therapy in patients with hiv infection. grupo de estudio del sida 04/98 pneumocystis carinii pneumonia recurrence in hiv patients on highly active antiretroviral therapy: secondary prophylaxis pneumocystis carinii pneumonia after the discontinuation of secondary prophylaxis discontinuation of secondary prophylaxis for pneumocystis carinii pneumonia in human immunodeficiency virus-infected patients: a randomized trial by the ciop study group pneumocystis jiroveci pneumonia prophylaxis is not required with a cd4 ? t-cell count\200 cells/microl when viral replication is suppressed incidence and risk factors for toxoplasmic encephalitis in human immunodeficiency virusinfected patients before and during the highly active antiretroviral therapy era prevalence, associated factors, and prognostic determinants of aids-related toxoplasmic encephalitis in the era of advanced highly active antiretroviral therapy treatment of toxoplasmic encephalitis in patients with aids. a randomized trial comparing pyrimethamine plus clindamycin to pyrimethamine plus sulfadiazine. the california collaborative treatment group treatment of central nervous system toxoplasmosis with pyrimethamine/sulfadiazine combination in 35 patients with the acquired immunodeficiency syndrome. efficacy of long-term continuous therapy pyrimethamine-clindamycin vs. pyrimethamine-sulfadiazine as acute and long-term therapy for toxoplasmic encephalitis in patients with aids cotrimoxazole therapy of toxoplasma gondii encephalitis in aids patients cotrimoxazole for treatment of cerebral toxoplasmosis: an observational cohort study during 1994-2006 randomized trial of trimethoprim-sulfamethoxazole versus pyrimethamine-sulfadiazine for therapy of toxoplasmic encephalitis in patients with aids. italian collaborative study group prospective randomized trial of trimethoprim/sulfamethoxazole versus pyrimethamine and sulfadiazine in the treatment of ocular toxoplasmosis management of toxoplasmic encephalitis in hiv-infected adults (with an emphasis on resource-poor settings) central nervous system toxoplasmosis in hiv pathogenesis, diagnosis, and therapy randomized phase ii trial of atovaquone with pyrimethamine or sulfadiazine for treatment of toxoplasmic encephalitis in patients with acquired immunodeficiency syndrome: actg 237/anrs 039 study. aids clinical trials group 237/agence nationale de recherche sur le sida, essai 039 a prospective, randomized trial of pyrimethamine and azithromycin vs pyrimethamine and sulfadiazine for the treatment of ocular toxoplasmosis cats and toxoplasmosis risk in hiv-infected adults thrice-weekly sulfadiazine-pyrimethamine for maintenance therapy of toxoplasmic encephalitis in hiv-infected patients. spanish toxoplasmosis study group comparison of high and low doses of trimethoprim-sulfamethoxazole for primary prevention of toxoplasmic encephalitis in human immunodeficiency virusinfected patients maintenance therapy with cotrimoxazole for toxoplasmic encephalitis in the era of highly active antiretroviral therapy discontinuation of primary and secondary toxoplasma gondii prophylaxis is safe in hiv-infected patients after immunological restoration with highly active antiretroviral therapy: results of an open, randomized, multicenter clinical trial immune recovery under highly active antiretroviral therapy is associated with restoration of lymphocyte proliferation and interferon-gamma production in the presence of toxoplasma gondii antigens restoration of toxoplasma gondii-specific immune responses in patients with aids starting haart guidelines for the treatment of cytomegalovirus diseases in patients with aids in the era of potent antiretroviral therapy: recommendations of an international panel restoration of cytomegalovirusspecific cd4 ? t-lymphocyte responses after ganciclovir and highly active antiretroviral therapy in individuals infected with hiv-1 loss of cytomegalovirus (cmv) viraemia following highly active antiretroviral therapy in the absence of specific anti-cmv therapy cytomegalovirus retinitis in advanced hiv-infected patients treated with protease inhibitors: incidence and outcome over 2 years long-lasting remission of cytomegalovirus retinitis without maintenance therapy in human immunodeficiency virusinfected patients pharmacokinetics of valganciclovir and ganciclovir following multiple oral dosages of valganciclovir in hiv-and cmv-seropositive volunteers single-dose pharmacokinetics of valganciclovir in hiv-and cmv-seropositive subjects increased survival of a cohort of patients with acquired immunodeficiency syndrome and cytomegalovirus retinitis who received sodium phosphonoformate studies of ocular complications of aids research group in collaboration with the cytomegalovirus retinitis trial. 4. visual outcomes. studies of ocular complications of aids research group in collaboration with the foscarnet and ganciclovir in the treatment of cmv retinitis in aids patients: a randomised comparison cytomegalovirus retinitis and viral resistance: ganciclovir resistance. cmv retinitis and viral resistance study group incidence of foscarnet resistance and cidofovir resistance in patients treated for cytomegalovirus retinitis. the cytomegalovirus retinitis and viral resistance study group a controlled trial of valganciclovir as induction therapy for cytomegalovirus retinitis a safety study of oral valganciclovir maintenance treatment of cytomegalovirus retinitis intravenous cidofovir for peripheral cytomegalovirus retinitis in patients with aids. a randomized, controlled trial combination foscarnet and ganciclovir therapy vs monotherapy for the treatment of relapsed cytomegalovirus retinitis in patients with aids. the cytomegalovirus retreatment trial. the studies of ocular complications of aids research group in collaboration with the treatment of relapsed cytomegalovirus retinitis with the sustained-release ganciclovir implant cytomegalovirus (cmv) retinitis in aids. gancilovir implantation in comparison with systemic therapy local therapy for cytomegalovirus retinitis treatment of cytomegalovirus retinitis with an intraocular sustained-release ganciclovir implant the development of cytomegalovirus encephalitis in aids patients receiving ganciclovir cytomegalovirus ventriculoencephalitis in aids. a syndrome with distinct clinical and pathologic features progressive cytomegalovirus encephalitis in successful ganciclovir therapy of cytomegalovirus retinitis in an aids patient cmv encephalitis during ganciclovir therapy of cmv retinitis cytomegalovirus encephalitis in two patients with aids receiving ganciclovir for cytomegalovirus retinitis ganciclovir therapy for cytomegalovirus (cmv) infection of the central nervous system in aids patients: monitoring by cmv dna detection in cerebrospinal fluid lack of reactivation of cytomegalovirus (cmv) retinitis after stopping cmv maintenance therapy in aids patients with sustained elevations in cd4 t cells in response to highly active antiretroviral therapy discontinuation of maintenance therapy for cytomegalovirus retinitis in hiv-infected patients receiving highly active antiretroviral therapy resistance of candida species to antifungal agents: molecular mechanisms and clinical consequences effendy i, et al. oral candidiasis guideline vulvovaginal candidosis: guideline of the german dermatological society, the german speaking mycological society and the working group for infections and infectimmunology of the german society for gynecology and obstetrics oropharyngeal candidiasis in immunocompromised children: a randomized, multicenter study of orally administered fluconazole suspension versus nystatin. the multicenter fluconazole study group the treatment of oropharyngeal candidiasis in hiv-infected patients with oral amphotericin b suspension oropharyngeal candidiasis in patients with aids: randomized comparison of fluconazole versus nystatin oral suspensions a multicenter randomized trial evaluating posaconazole versus fluconazole for the treatment of oropharyngeal candidiasis in subjects with hiv/aids fluconazole compared with itraconazole in the treatment of esophageal candidiasis in aids patients: a double-blind, randomized, controlled clinical study a randomized, double-blind, double-dummy, multicenter trial of voriconazole and fluconazole in the treatment of esophageal candidiasis in immunocompromised patients a double-blind comparison of itraconazole oral solution and fluconazole capsules for the treatment of oropharyngeal candidiasis in patients with aids mucosal and systemic fungal infections in patients with aids: prophylaxis and treatment single-dose fluconazole versus standard 2-week therapy for oropharyngeal candidiasis in hiv-infected patients: a randomized, double-blind, double-dummy trial single-dose therapy for oral candidiasis with fluconazole in hiv-infected adults: a pilot study itraconazole cyclodextrin solution-effective treatment for hiv-related candidosis unresponsive to other azole therapy treatment of fluconazole-resistant candidiasis with voriconazole in patients with aids posaconazole for the treatment of azole-refractory oropharyngeal and esophageal candidiasis in subjects with hiv infection safety and efficacy of posaconazole in the longterm treatment of azole-refractory oropharyngeal and esophageal candidiasis in patients with hiv infection high-dose therapy with fluconazole [or =800 mg/day a randomized double-blind study of caspofungin versus fluconazole for the treatment of esophageal candidiasis a randomized, double blind, comparative trial of micafungin (fk463) vs. fluconazole for the treatment of oesophageal candidiasis a randomized, double-blind trial of anidulafungin versus fluconazole for the treatment of esophageal candidiasis a phase 2, open-label study of the safety and efficacy of intravenous anidulafungin as a treatment for azolerefractory mucosal candidiasis a randomized double-blind study of caspofungin versus amphotericin for the treatment of candidal esophagitis resolution of azole-resistant oropharyngeal candidiasis after initiation of potent combination antiretroviral therapy declining rates of oropharyngeal candidiasis and carriage of candida albicans associated with trends toward reduced rates of carriage of fluconazole-resistant c. albicans in human immunodeficiency virus-infected patients efficacy of chlorhexidine gluconate rinse for treatment and prevention of oral candidiasis in hiv-infected children: a pilot study. oral surg oral med oral pathol oral radiol endod a randomized clinical trial of chlorhexidine in the maintenance of oral candidiasis-free period in hiv infection detection and significance of fluconazole resistance in oropharyngeal candidiasis in human immunodeficiency virus-infected patients development of resistance in candida isolates from patients receiving prolonged antifungal therapy a randomized study of the use of fluconazole in continuous versus episodic therapy in patients with advanced hiv infection and a history of oropharyngeal candidiasis: aids clinical trials group study 323/mycoses study group study 40 a comparison of topical application of penciclovir 1% cream with acyclovir 3% cream for treatment of genital herpes: a randomized, double-blind, multicentre trial resistance of herpes simplex virus infections to nucleoside analogues in hiv-infected patients impact of aciclovir on ulcer healing, lesional, genital and plasma hiv-1 rna among patients with genital ulcer disease in malawi valaciclovir: a review of its long term utility in the management of genital herpes simplex virus and cytomegalovirus infections valaciclovir versus aciclovir for herpes simplex virus infection in hiv-infected individuals: two randomized trials famciclovir for the treatment of recurrent genital herpes: a clinical and pharmacological perspective 2-day versus 5-day famciclovir as treatment of recurrences of genital herpes: results of the fast study treatment of acyclovir-resistant herpes simplex virus infections in patients with aids clinical efficacy of high-dose acyclovir in patients with human immunodeficiency virus infection: a metaanalysis of randomized individual patient data valacyclovir for the suppression of recurrent genital herpes in human immunodeficiency virusinfected subjects efficacy and safety of valacyclovir for the suppression and episodic treatment of herpes simplex virus in patients with hiv herpes simplex virus 2 infection increases hiv acquisition in men and women: systematic review and metaanalysis of longitudinal studies effect of aciclovir on hiv-1 acquisition in herpes simplex virus 2 seropositive women and men who have sex with men: a randomised, double-blind, placebo-controlled trial acyclovir and transmission of hiv-1 from persons infected with hiv-1 and hsv-2 effect of herpes simplex suppression on incidence of hiv among women in tanzania clinical practice. herpes zoster antiviral treatment for preventing postherpetic neuralgia. cochrane database syst rev prevention and treatment of vzv infections in patients with hiv clinical and virologic characterization of acyclovirresistant varicella-zoster viruses isolated from 11 patients with acquired immunodeficiency syndrome highly active antiretroviral therapy significantly improves the prognosis of patients with hiv-associated progressive multifocal leukoencephalopathy treatment options for aids patients with progressive multifocal leukoencephalopathy haart improves prognosis in hivassociated progressive multifocal leukoencephalopathy progressive multifocal leukoencephalopathy: improved survival of human immunodeficiency virus-infected patients in the protease inhibitor era progressive multifocal leukoencephalopathy in patients with aids receiving highly active antiretroviral therapy clinical course and prognostic factors of progressive multifocal leukoencephalopathy in patients treated with highly active antiretroviral therapy pml-iris in patients with hiv infection: clinical manifestations and treatment with steroids determinants of survival in progressive multifocal leukoencephalopathy progressive multifocal leukoencephalopathy: what's new? failure of cytarabine in progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. aids clinical trials group 243 team activities of various compounds against murine and primate polyomaviruses inhibitory effect of serotonin antagonists on jc virus propagation in a carrier culture of human neuroblastoma cells serotonin receptor 2a blocker (risperidone) has no effect on human polyomavirus jc infection of primary s112 human fetal glial cells mirtazapine in progressive multifocal leukoencephalopathy associated with polycythemia vera risperidone-induced reduction in jc viruria as a surrogate marker for efficacy against progressive multifocal leukoencephalopathy and hemorrhagic cystitis 5-ht2a inhibitors for progressive multifocal leukoencephalopathy: old drugs for an old disease mirtazapine use in human immunodeficiency virus-infected patients with progressive multifocal leukoencephalopathy identification and characterization of mefloquine efficacy against jc virus in vitro cryptosporidiosis among patients infected with human immunodeficiency virus. factors related to symptomatic infection and survival the natural history of cryptosporidial diarrhoea in hiv-infected patients sclerosing cholangitis with papillary stenosis in an hiv-infected patients with cryptosporidium infection a report of 95 cases aids and multiple system involvement with cryptosporidium respiratory cryptosporidiosis in a patient with malignant lymphoma treatment of hiv-1-associated microsporidiosis and cryptosporidiosis with combination antiretroviral therapy eradication of cryptosporidia and microsporidia following successful antiretroviral therapy prevention and treatment of cryptosporidiosis in immunocompromised patients resolution of severe cryptosporidial diarrhea with rifaximin in patients with aids treatment of diarrhea caused by cryptosporidium parvum: a prospective randomized, double-blind, placebo-controlled study of nitazoxanide high dose prolonged treatment with nitazoxanide is not effective for cryptosporidiosis in hiv positive zambian children: a randomised controlled trial paromomycin in cryptosporidiosis paromomycin: no more effective than placebo for treatment of cryptosporidiosis in patients with advanced human immunodeficiency virus infection possible effectiveness of clarithromycin and rifabutin for cryptosporidiosis chemoprophylaxis in hiv disease. hiv outpatient study (hops) investigators liposomal amphotericin b (ambisome) compared with amphotericin b both followed by oral fluconazole in the treatment of aids-associated cryptococcal meningitis management of cryptoccocal meningitis in resource-limited settings: a systematic review therapy of candidiasis and cryptococcosis in aids combination antifungal therapies for hivassociated cryptococcal meningitis: a randomised trial treatment of cryptococcal meningitis associated with the acquired immunodeficiency syndrome. national institute of allergy and infectious diseases mycoses study group and aids clinical trials group activity of posaconazole in the treatment of central nervous system fungal infections voriconazole treatment for less-common, emerging, or refractory fungal infections recombinant interferon-gamma 1b as adjunctive therapy for aids-related acute cryptococcal meningitis a randomized trial comparing fluconazole with clotrimazole troches for the prevention of fungal infections in patients with advanced human immunodeficiency virus infection. niaid aids clinical trials group long-term outcome of aids-associated cryptococcosis in the era of combination antiretroviral therapy aids-defining opportunistic illnesses in us patients, 1994-2007: a cohort study mycobacterium avium complex in patients with hiv infection in the era of highly active antiretroviral therapy disseminated mycobacterium avium-intracellulare complex (mac) infection in the era of effective antiretroviral therapy: is prophylaxis still indicated? an official ats/idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases a randomized, double-blind trial comparing azithromycin and clarithromycin in the treatment of disseminated mycobacterium avium infection in patients with human immunodeficiency virus a prospective, randomized trial examining the efficacy and safety of clarithromycin in combination with ethambutol, rifabutin, or both for the treatment of disseminated mycobacterium avium complex disease in persons with acquired immunodeficiency syndrome pharmacokinetic evaluation of rifabutin in combination with lopinavir-ritonavir in patients with hiv infection and active tuberculosis clarithromycin therapy for bacteremic mycobacterium avium complex disease. a randomized, double-blind, dose-ranging study in patients with aids. aids clinical trials group protocol 157 study team a prospective randomized trial of four three-drug regimens in the treatment of disseminated mycobacterium avium complex disease in aids patients: excess mortality associated with highdose clarithromycin. terry beirn community programs for clinical research on aids randomized, open-label trial of azithromycin plus ethambutol vs. clarithromycin plus ethambutol as therapy for mycobacterium avium complex bacteremia in patients with human immunodeficiency virus infection. veterans affairs hiv research consortium effect of potent antiretroviral therapy on immune responses to mycobacterium avium in human immunodeficiency virus-infected subjects nontuberculous mycobacterial immune reconstitution syndrome in hiv-infected patients: spectrum of disease and long-term follow-up mycobacterium avium complex immune reconstitution inflammatory syndrome: long term outcomes prophylaxis against disseminated mycobacterium avium complex with weekly azithromycin, daily rifabutin, or both. california collaborative treatment group incidence of mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency virus-positive patients once weekly azithromycin therapy for prevention of mycobacterium avium complex infection in patients with aids: a randomized, double-blind, placebo-controlled multicenter trial a randomized trial of clarithromycin as prophylaxis against disseminated mycobacterium avium complex infection in patients with advanced acquired immunodeficiency syndrome regional differences in use of antiretroviral agents and primary prophylaxis in 3122 european hiv-infected patients. eurosida study group response to treatment, mortality, and cd4 lymphocyte counts in hiv-infected persons with tuberculosis in abidjan risk factors for developing tuberculosis in hiv-1-infected adults from communities with a low or very high incidence of tuberculosis hiv-1 and recurrence, relapse, and reinfection of tuberculosis after cure: a cohort study in south african mineworkers adverse events and treatment interruption in tuberculosis patients with and without hiv co-infection outcome of hivassociated tuberculosis in the era of highly active antiretroviral therapy treatment of hiv-related tuberculosis in the era of effective antiretroviral therapy timing of initiation of antiretroviral drugs during tuberculosis therapy when to start antiretroviral therapy in hiv-associated tuberculosis clinical characteristics of iris syndrome in patients with hiv and tuberculosis office of aids research advisory council (oarac). guidelines for the use of antiretroviral agents in hiv-1-infected adults and adolescents. department of health and effect of rifampin, a potent inducer of drugmetabolizing enzymes, on the pharmacokinetics of raltegravir lack of enzyme-inducing effect of rifampicin on the pharmacokinetics of enfuvirtide treatment of tuberculosis in hiv-infected persons in the era of highly active antiretroviral therapy timing of antiretroviral therapy for hiv-1 infection and tuberculosis detection and prediction of active tuberculosis disease by a whole-blood interferon-gamma release assay in hiv-1-infected individuals managing drug interactions in the treatment of hiv-related tuberculosis. division of tuberculosis elimination national center for hiv/ aids, viral hepatitis, std, and tb prevention treatment of latent tuberculosis infection in hiv infected persons multidrug-and extensively drug-resistant tuberculosis multidrug and extensively drug-resistant tb (m/xdr-tb): 2010 global report on surveillance and response. who/htm/tb/2010.3. 2010. who acknowledgments we are indepted to fiona diekhoff for translation and preparation of the manuscript. key: cord-005335-u04cxiej authors: podder, c. n.; sharomi, o.; gumel, a. b.; strawbridge, e. title: mathematical analysis of a model for assessing the impact of antiretroviral therapy, voluntary testing and condom use in curtailing the spread of hiv date: 2011-05-05 journal: differ equ dyn syst doi: 10.1007/s12591-011-0090-6 sha: doc_id: 5335 cord_uid: u04cxiej this paper presents a deterministic model for evaluating the impact of anti-retroviral drugs (arvs), voluntary testing (using standard antibody-based and a dna-based testing methods) and condom use on the transmission dynamics of hiv in a community. rigorous qualitative analysis of the model show that it has a globally-stable disease-free equilibrium whenever a certain epidemiological threshold, known as the effective reproduction number [formula: see text], is less than unity. the model has an endemic equilibrium whenever [formula: see text]. the endemic equilibrium is shown to be locally-asymptotically stable for a special case. numerical simulations of the model show that the use of the combined testing and treatment strategy is more effective than the use of the standard elisa testing method with arv treatment, even for the use of condoms as a singular strategy. furthermore, the universal strategy (which involves the use of condoms, the two testing methods and arv treatment) is always more effective than the combined use of the standard elisa testing method and arvs. from hiv-related illnesses during the last 20 years. aids is now the leading cause of death in sub-saharan africa and the fourth-leading cause of death globally [23] . in addition to being a serious public health menace, hiv/aids has far reaching consequences to all social and economic sectors of society. it exacerbates poverty, reduces educational opportunities, devastates the workforce, creates large numbers of orphans, and exerts tremendous pressure on already limited health and social services [2, 9, 14, 36, 39, 43, 44] . another more troubling aspect of hiv disease is the fact that a sizable proportion (about 25% in north america) of those infected with the virus are not aware of their hiv status [6, 25] . thus, these individuals continue to unknowingly transmit the virus to others (in addition to not being able to benefit from clinical care to reduce morbidity and mortality). control measures against the spread of hiv are wide and varied. they include preventive measures (such as the use of condoms, public health education and counselling about safer sex practices, sterilization of needles in health care delivery, voluntary hiv testing) and the use of therapeutic agents. the currently available anti-hiv therapeutic measure is based on using anti-retroviral therapy, especially the highly-active anti-retroviral therapy (haart), which is known to significantly reduce viral load in treated individuals [13, 25, 29, 30, 34] . unfortunately, haart is still not widely accessible in many resource-poor nations, where hiv prevalence is the highest (an estimated 5.2 million people in low-and middle-income countries were receiving life-saving hiv treatment at the end of 2009 [38] ). the purpose of this study is to use mathematical modelling to gain insight into the impact of voluntary testing, the use of condoms and the use of anti-retroviral drugs in curtailing the spread of hiv in a community. two different testing methods, namely a standard antibody test (such as the enzyme linked immuno absorbent assay (elisa)) and a dna-based test, known as the nucleic acid amplification testing (naat), will be considered. unlike the standard antibody test (which typically detect antibodies in the blood after a minimum of three to four weeks of infection), naat can detect early infection (within the first few days of occurrence) [1, 31, 37] . the model to be developed in this study incorporates some of the well-known properties of hiv disease. these notably include the staged-progression aspect, where a typical hiv-infected individual passes through several infection stages, being highly infectious during the pre-antibody phase (characterized by high viremia with over 10 million viral copies per ml), maintaining low infectivity during the asymptomatic phase and becoming highly infectious as s/he progresses toward aids (i.e., the aids stage of hiv infection) [10, 13, 15, 16, 18, 22, 26, 30] . these stages are sometimes categorized into four groups namely, acute sero-conversion stage (less than 28 days of infection), early infection stage (less than 170 days of infection but prior to development of symptoms), established infection stage (after 170 days of infection) and the aids stage (characterized by the presence of clinical symptoms of aids and very reduced level of cd4 count) [1] . another important aspect of hiv disease is the fact that hiv rna levels are positively correlated with infectiousness [28, 34] . in addition to developing a reasonably realistic model for hiv spread, this study also contributes by including numerous anti-hiv strategies and carrying out rigorous qualitative analysis of the resulting model. the paper is organized as follows. the model is formulated in "model formulation" section, and rigorously analysed in subsequent subsections. numerical simulations are reported in third section. a deterministic compartmental modelling approach is used to design the model as follows. the total population at time t, denoted by n (t), is sub-divided into 10 mutually-exclusive compartments of susceptible individuals (s(t)), newly-infected individuals unaware of their status in the acute sero-conversion stage (i u 1 (t); less than 28 days of infection), infected individuals unaware of their hiv status in the asymptomatic stage (i u 2 (t); 28-170 days of infection), infected individuals unaware of their hiv infection status in the established (pre-aids) stage (i u 3 (t)), infected individuals unaware of their status in the aids stage of infection (a u (t)), treated individuals (t (t)), and (corresponding) infected individuals aware of their infection status (due to positive hiv diagnosis) at the aforementioned infection stages, denoted by i k 1 , i k 2 , i k 3 and a k , respectively. thus, the susceptible population is increased by the recruitment of new sexually-active individuals (at a rate ). susceptible individuals acquire infection, following effective contact with untreated infected individuals, at a rate λ, where in (1), β is the effective contact rate and the modification parameters η i (i = 2, 3, 4) are estimated as follows. the average transmission rate per coital act during the primary infection stage (i.e, less than 28 days of infection) is estimated to be 0.0082 [42] . assuming that an infected individual in this stage of infection has an average of six sexual acts per month, it follows that the transmission rate in the primary infection stage is β = 0.0082 × 6 × 12 = 0.59 per year. for the second stage of infection (i.e., 28-170 days of infection), it is assumed that the average transmission rate per coital act is 0.0035. this gives a transmission rate of β = 0.252 per year (so that, η 2 = 0.43). for the third stage of hiv infection (i.e., from 170 days of infection to the onset of clinical symptoms of aids), the transmission rate per act is 0.007 [42] , so that (by using an average of 3 acts per month) β = 0.0252 per year (hence, η 3 = 0.043). finally, following [42] , the per coital transmission probability in the aids stage is assumed to be 0.0028. using an estimate of 3 acts per month, it follows that the transmission rate in this stage is β = 0.1 per year (thus, η 4 = 0.17). it should be mentioned that, in the above, it is assumed that individuals in the pre-aids and aids stages have reduced number of sexual acts in comparison to those in the primary and early infection stages (the justification for this assumption is that individuals in the pre-aids and aids stages are too sick to engage in active sexual activity). in summary, these estimates (for show that the rate of hiv transmission is the highest during the primary infection stage (β = 1). it decreases during the second infection stage to β = 0.252, and further decreases to β = 0.0252 in the pre-aids stage. the transmission rate then increases to β = 0.1 in the aids stage. these estimates are consistent with the conclusions drawn in numerous hiv modelling studies, such as those reported in [19, 42] (it should, however, be mentioned that some studies, such as that reported in [35] , show that hiv transmission rate is the highest during the aids stage of infection). once infected individuals are made aware of their infection status (by positive hiv diagnosis), it is assumed that these individuals (who are moved to the class of individuals who know their positive hiv infection status) will reduce their risky behaviour by a factor of r . marks et al. [24, 25] estimated that knowledge of hiv status resulted in 57% reduction in unprotected sex (so that, r 0.43). condom use is modelled in terms of reduction in the infection rate, λ, by a factor of (1 − c κ), where 0 < c < 1 is the condom efficacy and 0 < κ < 1 is the fraction of sexually-active individuals that use condoms consistently and correctly (condom compliance). it is assumed that newly-infected individuals are unaware of their infection status until they are detected either by naat or the standard antibody test. it is assumed that individuals unaware of their infection status progress from the acute sero-conversion stage (i u 1 ) to the asymptomatic stage (i u 2 ), at a rate σ 1 . progression from the asymptomatic stage (i u 2 ) to the pre-aids stage (i u 3 ) occurs at a rate σ 2 . finally, pre-aids individuals progress to the aids stage at a rate σ 3 . individuals that are unaware of their infection status in the i u 1 class are tested (using naat) at a rate τ n , with efficacy n (0 < n < 1). those that are positively diagnosed move to the corresponding i k 1 class. standard (elisa) testing is used for individuals in the i u 2 , i u 3 and a u classes at a rate τ , with efficacy (0 < < 1); and those positively identified move to their corresponding i k class. individuals aware of their infection status progress through the various infection stages at the same rate (σ i ; i = 1, 2, 3) as those who are not (in other words, it is assumed that knowledge of hiv infection status does not alter the progression rate among untreated infected individuals). individuals aware of their status in the infection stages i k 1 , i k 2 , i k 3 and a k are treated at rates ψ 1 , ψ 2 , ψ 3 and ψ k , respectively. it is assumed, for mathematical tractability, that these (treated) individuals do not transmit infection. further, natural mortality occurs in all epidemiological classes at a rate μ (i.e., 1/μ represents the average duration of acquisition of sexual partners), and individuals in the aids stage suffer an additional disease-induced death (at rates δ u and δ k , for individuals unaware or aware of their status, respectively). it is assumed that treated individuals eventually succumb to the disease (at a reduced disease-induced rate θ 1 δ k , with 0 < θ 1 < 1). putting the aforementioned formulations and assumptions together, it follows that the model for hiv transmission, in the presence of the two testing methods, condom use and arvs, is given by the following system of differential equations (a schematic description of the model is given in fig. 1 , and the variables and parameters of the model are described in table 1 ): infected individuals unaware of their status in acute sero-conversion stage i u 2 (t) infected individuals unaware of their status in the asymptomatic stage infected individuals unaware of their status in the pre-aids stage infected individuals unaware of their status in the aids stage infected individuals aware of their status in acute sero-conversion stage infected individuals aware of their status in the asymptomatic stage infected individuals aware of their status in the pre-aids stage infected individuals aware of their status in the aids stage total population the model (2) is a modification of the model presented in [1] , in that it accounts for four hiv infection stages (as against the three infection stages considered in [1] ). furthermore, this study provides a rigorous mathematical analysis of the model (this is not given in [1] ). to be epidemiologically meaningful, it is important to prove that the solutions of the basic model (2), with positive initial data, will remain positive for all time t > 0. thus, t 1 > 0. it follows from the first equation of the differential equation system (2) that which is equivalent to, thus, so that, similarly, it can be shown that i u thus, all solutions of the model, with non-negative initial data, remain non-negative for all t > 0. adding all the equations of the basic model (2) gives, thus, local stability of disease-free equilibrium (dfe) since the model (2) monitors human populations, it is assumed that the variables and associated parameters are non-negative for all t ≥ 0. the dfe of the model (2) is given by consider the biologically-feasible region the following steps are taken to establish the positive invariance of d (i.e., solutions in d remain in d for all t > 0). the rate of change of total population, obtained by adding all the equations of the model (2), is given by since the right hand-side of (5) is bounded by − μn , a standard comparison theorem can be used to show that thus, d is positively-invariant. hence, it is sufficient to consider the dynamics of the flow generated by (2) in d. in this region, the model can be considered as been epidemiologically and mathematically well-posed [16] . the linear stability of e 0 is studied using the next generation operator technique in [8, 41] . the associated non-negative matrix, h, for the new infection terms, and the non-singular m-matrix, v , for the remaining transfer terms, are, respectively, given by it follows that the effective reproduction number, denoted by r eff , is given by where ρ denotes the spectral radius, and the threshold quantity, r eff , measures the average number of new secondary cases generated by a single infected individual in a population where the aforementioned anti-hiv control measures are implemented. it is worth stating that, in (6) , β is the infection rate and 1 k i (i = 1, . . . , 8) represent the respective mean duration in the infection classes an associated epidemiological threshold is the basic reproduction number (r 0 ), obtained in a similar way by considering the model (2) in the absence of any anti-hiv intervention (i.e., κ = c = τ n = n = τ = = ψ 1 = ψ 2 = ψ 3 = ψ k = 0), is given by where a 3 is as defined earlier (but with the aforementioned intervention-related parameters set to zero). the threshold quantity r 0 measures the average number of new infections generated by a single infected individual in a completely susceptible population. using theorem 2 of [41], the following result is established. the dfe, e 0 , of the system (2), given by (4), is locally-asymptotically stable (las) if r eff < 1, and unstable if r eff > 1. the epidemiological implication of lemma 2 is that hiv can be eliminated from the community when r eff < 1, provided the initial sizes of the sub-populations of the model (2) are in the basin of attraction of e 0 . in other words, an influx of small number of infected individuals into the community will not generate large outbreaks if r eff < 1. to ensure that disease elimination is independent of the initial population sizes of the state variables of the model, a global asymptotic stability result (of the dfe) is established below. the dfe of the model (2), given by (4), is globally-asymptotically stable (gas) in d whenever r eff ≤ 1. proof consider the lyapunov function with lyapunov derivative given by (where a dot represents differentiation with respect to t) it follows, from the lasalle's invariance principle [21] , that i u in the first and last equations of the model (2) shows that s → μ and t → 0 as t → ∞. thus, an alternative proof for theorem 1, using a comparison theorem argument, is given in appendix a. the existence of possible endemic equilibria of the model (that is, equilibria where the disease is endemic) is explored as follows. let, represents an arbitrary endemic equilibrium of the model (2) . further, let solving the equations in the model (2) at steady-state, in terms of λ * * s * * , gives t * * = b 9 λ * * s * * , with, using (8) in (7), and simplifying, gives where, the positive endemic equilibrium of the model (2) can then be obtained by solving for λ * * in (9) and substituting the result into (8) . clearly, λ * * = 0 is a solution of (9), which corresponds to the dfe (e 0 ). for λ * * = 0, the quadratic (9) can be simplified to since all the model parameters are non-negative, it follows that b 10 > 0 and c 1 > 0 for r eff > 1. thus, the linear equation (10) has a unique positive solution, given by λ * * = c 1 b 10 , whenever r eff > 1. the components of this solution are obtained by substituting the unique value of λ * * into (8). since r eff < 1 implies that c 1 < 0, it follows that, for r eff < 1, λ * * < 0 (which is epidemiologically meaningless). similarly, if r eff = 1, the coefficient c 1 = 0, so that λ * * = 0 (which corresponds to the dfe, e 0 ). hence, the model has no positive solution whenever r eff ≤ 1. these results are summarized below. the model (2) has a unique positive endemic equilibrium, given by e 1 , whenever r eff > 1, and no positive equilibrium otherwise. the local stability of the unique eep, e 1 , will now be explored for the special case where the disease-induced mortality is negligible (i.e., δ u = δ k = 0). setting δ u = δ k = 0 in the model (2) gives hence, it follows from (11) that n (t) → μ = n * as t → ∞. further, using the substitution (2) gives the following reduced model: where k 41 = k 4 | δ u =0 , k 81 = k 8 | δ k =0 and k 91 = k 9 | δ k =0 . it can be shown, using the above approach, that the system (12) has a unique endemic equilibrium, given by further, the following result holds (see appendix b for the proof): the unique endemic equilibrium, e 2 , of the reduced model (12) , is las whenever r c > 1. the epidemiological implication of theorem 2 is that hiv will persist in the community if the reproduction threshold (r c ) exceeds unity. the model (2) is simulated using the parameter values given in table 1 (unless otherwise stated). some of the parameter values are obtained from the literature (such as in [6, 7, 15, 18, 24, 25, 31] ). in particular, the stage progression parameters are estimated as follows. since the period for the acute sero-conversion stage (i 1 ) is less than 28 days of infection [1] , the average duration in this stage is set at 1 σ 1 = 4 52 (so that, σ 1 = 13 per year). similarly, the average duration in the second infection stage (i 2 ) is approximately 20 weeks (28-170 days [1] ). thus, 1 σ 2 = 20 52 (so that, σ 2 = 2.6 per year). the duration in the third (i 3 ) stage is assumed to be 15 years [1] (thus, σ 3 = 1 15 per year). it should be stated that although the model is parameterized using data largely from resource-rich countries, it is robust enough to allow for the evaluation of scenarios for resource-poor nations (by using appropriate parametrization). first of all, the model (2) is simulated to evaluate the effect of condom use, as a single anti-hiv intervention. comparisons are made with the following three different effectiveness levels of the combined testing (standard elisa and naat) and treatment strategy: (i) low effectiveness level of the combined testing and treatment strategy: τ n = τ = ψ 1 = ψ 2 = ψ 3 = ψ k = 0.4; (ii) medium effectiveness level of the combined testing and treatment strategy: τ n = τ = ψ 1 = ψ 2 = ψ 3 = ψ k = 0.6; (iii) high effectiveness level of the combined testing and treatment strategy: the aforementioned effectiveness levels, chosen arbitrarily, are used to address the problem of the uncertainty in the estimate of the values of the parameters related to the testing and treatment rates. using the low effectiveness level of the combined testing and treatment strategy, it is shown that the combination of the two testing methods and treatment is more effective (saves more new cases) than the use of condoms as a singular anti-hiv strategy followed by the use of only the standard elisa testing method with arv treatment (fig. 2a ). using the medium effectiveness level of the combined testing and treatment strategy, it is also shown that the combination of the two testing methods and treatment is more effective than the use of the only the standard testing method with arvs followed by the condom use as a singular strategy (fig. 2b) . similar trends were observed for high effectiveness level of the combined testing and treatment strategy (fig. 2c) . it should, however, be mentioned that the use of condoms as a singular anti-hiv intervention is marginally more effective than the low effectiveness level of the standard testing and arv strategy (but this situation is reversed if the effectiveness of the standard testing and arv strategy is increased to medium or high levels). figure 3 shows simulation results comparing the effect of the combined use of standard testing and arvs against a universal strategy (that entails the use of condoms, the two testing methods and arvs). here, too, the aforementioned three effectiveness levels of the combined cumulative number of cases averted using condoms only and various effectiveness levels of the combined testing and arvs intervention strategy. a low effectiveness level of the combined testing and treatment strategy (τ n = τ = ψ 1 = ψ 2 = ψ 3 = ψ k = 0.4; so that, r eff = 0.64 and r 0 = 2.65), b moderate effectiveness level of the combined testing and treatment strategy (τ n = τ = ψ 1 = ψ 2 = ψ 3 = ψ k = 0.6; so that, r eff = 0.61), and c high effectiveness level of the combined testing and treatment strategy (τ n = τ = ψ 1 = ψ 2 = ψ 3 = ψ k = 1; so that, r eff = 0.58). all other parameters are as given in table 1 testing and treatment strategy are used. the universal strategy saves more cases than any of the three effectiveness levels of the combined testing and treatment strategy (see fig. 3a-c) . a deterministic model is designed and used to monitor the impact of voluntary hiv testing (based on the use of a standard antibody-based and a dna-based testing methods), condom use and the use of arvs in curtailing the spread of hiv in a population. rigorous qualitative analysis of the model reveals that it has a globally-asymptotically stable disease-free equilibrium whenever a certain threshold quantity is less than unity. furthermore, the model has a unique endemic equilibrium when the threshold quantity exceeds unity. the endemic equilibrium is shown to be locally-asymptotically stable for a special case. numerical simulations of the model, using a reasonable set of parameter values, show the following: (i) the combined testing and treatment strategy is more effective than the use of the standard elisa testing method with arv treatment. the use of condoms as a sole anti-hiv strategy is marginally more effective than the low effectiveness level of the standard testing and arv strategy (this situation is reversed if the effectiveness of the standard testing and arv strategy is increased to medium or high levels). table 1 (ii) the universal strategy is always more effective than the combined use of the two testing methods and arvs (regardless of the effectiveness level of the latter strategy). overall, this study shows that the prospects of effectively controlling the spread of hiv using the interventions considered in this study are bright (particularly if they are used in combination). it is worth stating, however, that the simulation results presented in this study are sensitive to the choices of parameter and initial values used in the simulations. the uncertainties associated with the parameters related to the testing and treatment rates are accounted for by considering three (arbitrarily chosen) effectiveness levels of the combined testing and treatment strategy (a more detailed uncertainty analysis, based on latin hypercube sampling [4, 5, 27, 32] for example, can be applied if more data (related to the testing and treatment rates) becomes available). proof it should be noted, first of all, that the equations for the infected components in the model (2) can be written in terms of and h and v are as defined in "local stability of disease-free equilibrium" section. using the fact that the eigenvalues of the matrix h − v all have negative real parts, it follows that the linearized differential inequality system (a.1) is stable whenever r eff < 1. consequently, (i u 1 , i u 2 , i u 3 , a u , i k 1 , i k 2 , i k 3 , a k , t ) → (0, 0, 0, 0, 0, 0, 0, 0, 0) as t → ∞. it follows, by comparison theorem [20] , that (i u 1 , i u 2 , i u 3 , a u , i k 1 , i k 2 , i k 3 , a k , t ) → (0, 0, 0, 0, 0, 0, 0, 0, 0). substituting i u 1 = i u 2 = i u 3 = a u = i k 1 = i k 2 = i k 3 = a k = t = 0 into the first equation of the model (2) gives s(t) → μ as t → ∞. thus, → μ , 0, 0, 0, 0, 0, 0, 0, 0, 0 as t → ∞ and r eff < 1. hence, e 0 is gas in d if r eff < 1. proof let r c > 1, so that the unique eep of the reduced model (12), given by e 2 , exists. the proof of theorem 2 is based on using the technique in [17] (see also [11, 12] ), which employs a krasnoselskii sub-linearity trick. assume, first of all, that the linearization of the system (12), around the equilibrium e 2 , has solution of the form: 10) . substituting a solution of the form (b.1) into the linearized system of (12), around the unique endemic equilibrium e 2 , gives the following system of linear equations where, system (b.2) is simplified as follows. firstly, the negative terms in the last nine equations of system (b.2) are moved to their respective left-hand sides. we then solve for z 2 from the second, z 3 from the third and so on. the results are then substituted into the first equation of the system (b.2). finally, all the negative terms of the first one equation are moved to the left-hand side. these algebraic manipulations result in the following general system: where, the notation m(z ) i (with i = 1, . . . , 9) denotes the ith coordinate of the vector m(z). it should further be noted that the matrix m has non-negative entries, and the equilibrium e 2 satisfies e 2 = me 2 . furthermore, since the coordinates of e 2 are all positive, it follows then that ifz is a solution of (b.3), then it is possible to find a minimal positive real number s such that where, ||z ||= (|| z 1 ||, . . . , || z 9 ||) with the lexicographic order, and || · || is a norm in c. the main goal is to show that re(τ ) < 0. assume the contrary (i.e., re(τ ) ≥ 0). we then need to consider two cases: τ = 0 and τ = 0. assume the first case, τ = 0. then, (b.2) is a homogeneous linear system in the variables z i (i = 1, . . . , 9) . the determinant of the system (b.2) corresponds to that of the jacobian of the system (12) evaluated at e 2 , which is given by = eλs * * p n * + k 1 k 2 k 3 k 41 k 5 k 6 k 7 k 81 k 91 1 − s * * n * r c , (b.5) where, e = m 2 k 5 σ 1 {σ 2 σ 3 k 41 [k 6 k 12 (q k + k 91 )] + σ 2 k 41 k 81 (k 3 + k 6 ) + k 3 k 4 k 7 k 8 k 12 (1 + k 11 ) + k 3 k 4 k 8 k 11 (k 7 a 2 + σ 2 k 12 + a 3 σ 2 )} + m 1 k 2 k 3 k 41 {σ 1 σ 2 [σ 3 (k 91 + 1) + k 8 (1 + k 91 )] + k 7 k 8 [k 6 + k 91 (σ 1 + k 6 ) + σ 1 ]} + k 5 k 6 k 7 k 81 k 91 [σ 1 σ 2 (σ 3 + k 41 ) + k 3 k 41 (k 2 + σ 1 )]. by solving the equations of the model (12), at the endemic steady-state e 2 , and using the first equation of (12) , it can be shown that thus, using (b.6) in (b.5) shows that > 0. consequently, the system (12) can only have the trivial solutionz =0 (which corresponds to the dfe, e 0 ). now we consider the case τ = 0. in this case, re(f i (τ )) ≥ 0(i = 1, . . . , 9) since, by assumption, re(τ ) ≥ 0. it is easy to see that this implies | 1 + f i (τ ) |> 1 for all i. now, define f(τ ) = min | 1 + f i (τ ) | (for i = 1, . . . , 9) . then, f(τ ) > 1. hence, s f(τ ) < s. the minimality of s implies that ||z ||> s f(τ ) e 2 . but, on the other hand, taking norms on both sides of the second equation of (b.2), and using the fact that m is non-negative, we obtain then, it follows from the above inequality that || z 2 ||≤ s f(τ ) i u * * 1 , which contradicts re(f i (τ )) ≥ 0. hence, re(τ ) < 0. thus, the equilibrium e 2 is las if r c > 1. proceedings of the 10th pims industrial problem solving workshop antiretroviral therapy coverage rate in low-and middle-income countries randomized, controlled intervention trial of male circumcision for reduction of hiv infection risk: the anrs 1265 trial sensitivity and uncertainty analysis of complex models of disease transmission: an hiv model, as an example predicting the unpredictable: transmission of drug-resistant hiv the effectiveness of condoms in reducing heterosexual transmission of hiv on the definition and the computation of the basic reproduction ratio r 0 in models for infectious diseases in heterogeneous populations the impact of hiv and aids on africa's economic development theoretical assessment of public health impact of imperfect prophylactic hiv-1 vaccines with therapeutic benefits influence of vertical and mechanical transmission on the dynamics of dengue disease qualitative study of transmission dynamics of drug-resistant malaria immunopathogenic mechanisms of hiv infection developing economies shrink as aids reduces workforce mathematical study of a staged-progression hiv model with imperfect vaccine the mathematics of infectious diseases stability of the endemic equilibrium in epidemic models with subpopulations the differential infectivity and staged progression models for the transmission of hiv role of the primary infection in epidemics of hiv infection in gay cohorts stability analysis of nonlinear systems the stability of dynamical systems. regional conference series in applied mathematics. siam statistical analysis of the stages of hiv infections using a markov model global burden of disease and risk factors. a co-publication of the world bank meta-analysis of high-risk sexual behaviour in persons aware and unaware they are infected with hiv in the united states: implications for hiv prevention programs estimating sexual transmission of hiv from persons aware and unaware that they are infected with the virus in the usa a model of hiv/aids with staged progression and amelioration sensitivity and uncertainty analysis for a sars model with time-varying inputs and outputs plasma viral load and cd4+ lymphocytes as prognostic markers of hiv-1 infection adherence to antiretroviral therapy in sub-saharan africa and north america: a meta analysis mathematical analysis of hiv-1 dynamics in vivo detection of acute infections during hiv testing in north carolina mathematical study of the impact of quarantine, isolation and vaccination in curtailing an epidemic designing hiv vaccination policies subtypes and cross-immunity for the rakai project study group: viral load and heterosexual transmission of human immunodeficiency virus hiv epidemics driven by late disease stage transmission report on the global aids epidemic: executive summary/unaids: a unaids 10th anniversary special edition cost effectiveness of screening for acute hiv-infection: the north carolina stat program unaids: more than five million people receiving hiv treatment united nations department of economic and social affairs/population division: the impact of aids global health policy: the global hiv/aids epidemic: fact sheet reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission rates of hiv-1 transmission per coital act, by stage of hiv-1 infection who: the world health report: changing history. world health organization confronting aids: public priorities in a global epidemic key: cord-004247-lagv3tp7 authors: hooft van huijsduijnen, rob; kojima, somei; carter, dee; okabe, hisafumi; sato, akihide; akahata, wataru; wells, timothy n. c.; katsuno, kei title: reassessing therapeutic antibodies for neglected and tropical diseases date: 2020-01-30 journal: plos negl trop dis doi: 10.1371/journal.pntd.0007860 sha: doc_id: 4247 cord_uid: lagv3tp7 in the past two decades there has been a significant expansion in the number of new therapeutic monoclonal antibodies (mabs) that are approved by regulators. the discovery of these new medicines has been driven primarily by new approaches in inflammatory diseases and oncology, especially in immuno-oncology. other recent successes have included new antibodies for use in viral diseases, including hiv. the perception of very high costs associated with mabs has led to the assumption that they play no role in prophylaxis for diseases of poverty. however, improvements in antibody-expression yields and manufacturing processes indicate this is a cost-effective option for providing protection from many types of infection that should be revisited. recent technology developments also indicate that several months of protection could be achieved with a single dose. moreover, new methods in b cell sorting now enable the systematic identification of high-quality antibodies from humanized mice, or patients. this review discusses the potential for passive immunization against schistosomiasis, fungal infections, dengue, and other neglected diseases. the last three decades have seen a dramatic rise in the use of monoclonal antibodies (mabs) as therapeutics. by 2017, a total of 78 antibodies had been approved by the us food and drug administration (fda) or the european medicines agency (ema) [1] , with a further 11 approved by the fda in 2018 [2, 3]. beyond this, over 570 antibodies are in clinical development [4] . the global mab market reached us$100 billion annually in 2017 [5], underscoring the considerable economic importance of these medicines. the success of mabs starts with the general applicability of the technology used to make them. antibodies can be developed that have not only high affinity for their targets but also high selectivity, meaning they are less likely to have unwanted side effects and unexpected safety problems. mabs are particularly good at targeting cell-surface proteins and circulating protein factors; this is in contrast to small molecules, in which cell surface protein-protein a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in infectious disease, the biggest success story has been the use of antibodies to provide prophylaxis against infection by respiratory syncytial virus (rsv). rsv is responsible for over 30 million episodes of new lower respiratory tract infections, particularly targeting children 5 years and younger, resulting in an estimated 48,000 to 74,500 deaths globally (2015 estimates) [14] . palivizumab, dosed at 15 mg/kg, is given as a monthly intramuscular injection throughout the rsv season, which is suitable for small babies but would not be appealing for adults. a follow-on antibody-medi8897, now in phase ii clinical studies [15]-is 100 times more potent than palivizumab in vitro. it is being developed as a single 50 mg injection to cover the typical 5-month season. taken together, these cases illustrate, that after initial proof of concept, it is possible to find second-generation antibodies with more simple routes of delivery and sufficient potency to support relatively infrequent dosing in the field. a major objection to the use of mabs in infectious diseases as a therapeutic class is their high price, which is presumed to be a consequence of a high cost of goods. manufacturing costs are a particularly important attribute for any medication that is developed for diseases of poverty in low-and middle-income countries (lmics). however, production efficiency of mabs has increased dramatically over recent decades, and cell-culture expression levels around 4 g/l or even higher are common. a published analysis from a decade ago already noted that the costs of mab production were dropping from us$300/g to us$20/g when being produced at the 10-tonne-per-year scale, potentially using large, 100,000 l reactors [16] . a more recent analysis has similar estimates [17] , which-depending on process and volume-range from us$20/g to us$80/g [18] . for a public health application against an infectious disease in an lmic, an intramuscular or subcutaneous delivery would be preferable (rather than the intravenous route). the intramuscular route effectively limits the dose; an injection volume of 1.0 ml is probably close to the maximum acceptable in small children, and antibodies are typically only soluble to around 100 mg/ml, which gives a limitation on the total dose of around 100 mg, corresponding to a range of 5 to 20 mg/kg [19] . taken together, the cost of goods of an injection based on current numbers would be in the range of us$1 to us$8. benchmarking such costs is difficult, but as a comparison, the recently launched malaria vaccine provides less than 50% protection for 3 injections at around us$5 each. the programmatic cost of protecting a child from malaria for a year in the sahel using existing low-cost oral medicines has been estimated at us$3.40 by unitaid [20] . an injectable therapeutic that could protect a child for a season for this price provides a cost-effective option. the other important factor to manage is the reduction of the frequency of administration, to make the prophylaxis useful in resource-poor settings. mabs used in therapy are generally iggs (immunoglobulin gs), which have plasma half-lives of 20 to 25 days in humans [21, 22] . such antibodies can be used to provide several months of protection, as in the case of the once-per-season anti-rsv antibody medi8897 (nirsevimab) discussed earlier or the anti-calcitonin gene-related peptide (anti-cgrp) fremanezumab, recently approved for prevention of migraines with a dose of 675 mg every 3 months. an alternative to simply increasing the dose would be increasing the half-life of the circulating mab. several strategies have been proposed here [23] : mutations can be made in the igg fc (fragment crystallizable) region, which interacts with the receptors responsible for the uptake of antibodies. such mutations, as for medi8897, can significantly extend an mab's plasma half-life. mutations derived from the neonatal fc receptor (fcrn) increase the strength of the interaction between the fc region and the fc receptors under the acidic ph conditions in lysosomes, and as a result, the receptor-fc complex is recycled back to the cell surface, escaping degradation [24, 25] . this approach was followed for bevacizumab and cetuximab, by mutating met 428 to leu and asn 434 to ser. discussion about even longer periods of protection has been stimulated by recent results from virally mediated expression of antibodies targeting hiv [26] . these technologies hold out the promise of sustained serum titers of antibodies for more than a year from a single injection, although the technological development is more complicated than for the direct injection of antibodies. one final objection to the goal of administering monoclonals is that vaccination would instead provide lifelong coverage. however, for some diseases, the development of long-lasting potent vaccines still remains elusive. many pathogens will not raise an adequate immune response, due to immunosuppression. finally, antibodies give immediate protection, important during rapidly evolving epidemics or pandemics and for individuals being deployed into zones of high transmission. antibodies would thus form part of the overall armamentarium, along with drugs, bed nets and insecticides, and vaccines. first and foremost for the development of mabs is the question of suitable antigen targets. viruses are the simplest pathogens, and their genomes allow limited means to evade host immunity; therefore, passive and active vaccinations have been most successful in this area. therapeutic antibodies have been developed and marketed for respiratory syncytial, varicella zoster, vaccinia, and hepatitis b viruses (hbvs) [27, 28] . historically, immune responses against viruses were considered purely cellular and those against other types of pathogens mostly humoral (antibody based) and less effective. however, nowadays the interplay between both systems is better understood, for instance, with therapeutic opportunities against viruses such as rabies that rely on passive immunization. mabs that target the initial stages of an infection may provide prophylaxis, and the repertoire of potential antigens for such approaches is greatly helped by the increased availability of genomic and transcriptional information. passive immunization is especially effective in infections, such as those with the rabies virus, which avoids apoptosis of infected neurons while killing protective t cells so that carriers do not mount an immune response themselves [29] . a recent review [30] lists 21 mabs (and a nanobody, a single-chain type of antibody produced by camelids) that are currently in clinical trials in the united states. in addition, polyclonal antibodies have been approved in the european union or the us for cytomegalovirus, hepatitis viruses a and c, and postexposure prophylaxis against measles, rabies, rubella, and tetanus [31]. these approaches appear most suitable for virus infections and for protection against the consequences of snake venom but might also be applied to other types of disease. there are fewer marketed antibodies that target bacteria, and these target toxins, namely anthrax and clostridium cytotoxin [27] . this paucity may be due to a lack of discovery efforts, a consequence of the historic availability of cheap and effective antibiotics. however, with the worrying rise of antibiotic resistance, the tide is turning [32] . immunization against protist parasites and fungi is generally ineffective. one problem is that organisms such as trypanosoma brucei (which causes sleeping sickness) and plasmodium (malarial parasites) encode dozens or even hundreds of different surface antigens that are sequentially exposed at their most abundant stage, outpacing the host's immune system. for some pathogens, clinical trials in infected patients are not possible or desirable. in the case of inhalational anthrax, raxibacumab has been approved using the fda "animal rule" [33]. this provides a route forward in disease areas in which trials with infected human patients are not possible or not ethical. recently, the case for discovering mabs for neglected tropical and other infectious diseases was reviewed [34] . this work took a broad, category-wide approach (viruses, bacteria, parasites) and also included venoms. snake bites, and the challenge of envenoming, represent a serious health problem, resulting in 81,000 to 138,000 deaths yearly [35] , and were recently included in the world health organization (who) list of neglected tropical diseases. the discovery of mabs in this area logically follows the long-standing therapeutic successes with passive immunization [36] , and the costs and feasibility of developing mabs were recently reviewed [34, 37] [38], including specific clinical development and regulatory challenges [39] . in this review, we selected diseases prioritized by the global health innovative technology (ghit) fund, an international collaboration between the public and private sectors, supporting collaborations between japanese and non-japanese entities to advance global health research and development [40, 41] . the present vaccine discovery statuses for these diseases are listed in table 1 . human schistosomiasis is caused by infection, mainly with one of 4 species of the blood fluke belonging to schistosomatoidea. it is estimated that there are over 200 million cases annually in africa, with two-thirds being schistosoma haematobium and the remainder s. mansoni. in southeast asia, other species such as s. japonicum and s. mekongi cause smaller numbers of infections [64] . the mortality associated with schistosomal infections is difficult to calculate. comorbidities linked to anemia are common, and infection with s. haematobium can lead to female genital schistosomiasis, which results in increased susceptibility to hiv infection [65] . the life cycle of the parasite is complex, involving both a snail and a human host. in the infective stage, cercariae migrate and penetrate the human host skin to become schistosomula, which then move with the venous circulation, initially to the lungs. after further maturation, the parasite migrates via the heart to the portal circulation and the intestines; from there, eggs are excreted with the feces or, passing through the bladder wall, in urine. prophylaxis could therefore either be causal-and prevent entry and survival of the cercariae-or suppressive, by killing the juvenile and adult worms. clearly, as with the malarial parasites, the more stages of the parasite life cycle that can be inhibited, the more effective the protection will be. the molecular targets best addressed by antibodies are still not clear. some life cycle differences can be characterized: schistosomula recovered from the lungs are elongated and more resistant to antibody-dependent, cell-mediated cytotoxicity than newly transformed schistosomula. praziquantel, the standard treatment for schistosomiasis, acts only against adult worms and has no effects on the juveniles, thus repeat treatment is required to eliminate all the worms in a patient. an ideal treatment would therefore kill both adult and juvenile worms at a submicromolar concentration. this is effectively the target candidate profile for a small molecule inhibitor [66] . no target-product profile (tpp) for prophylaxis against schistosomiasis has been published, but it is possible to start to build one, with the recent characterization of praziquantel for such use in murine models and in pediatrics. clinically, this treatment reduces infection by 70% to 80% using doses of 40 to 60 mg/kg [67] . in a murine model with infection by s. mansoni cercariae, the maximum dose of 400 mg/kg killed 96% of all adult worms [68]. however, this murine dose produced a peak exposure of 6 ng/ml, some 10-fold higher than the exposure observed in children at the recommended regimen of 40 to 60 mg/kg [69, 70] . it seems pragmatic to project that an antibody that can reduce the worm burden by 80% in the mouse model, at a dose that is clinically achievable in humans, could be acceptable. the maximally practicable injection of an antibody in children is 2 to 5 mg/kg, and given the relatively consistent allometry between humans and mice for mabs, this corresponds to a dose of 80 to 200 μg in mice [19] . target identification for schistosomiasis is not straightforward. unlike malaria, the vaccine field offers no clues as to useful antibody targets. two vaccine candidates are currently being tested in humans. the first is based on the fatty acid-binding protein (fabp) sm-14 (schistosoma mansoni-14), which is being tested in 2 small, open-label pilot studies in senegal (nct03041766 and nct03799510). a larger 300-participant trial in uganda, nct03910972, is being planned for a vaccine based on sm-tsp-2 (schistosoma mansoni-tetraspanin-2) but is not expected to report before 2023. clues about other possible ways forward, in terms of antigen selection, come from a variety of areas.first, schistosoma are helminths (or flatworms), and the initial immune response is a characteristically strong t helper 2 (th2) cytokine reaction, which is not seen in viral or bacterial infections and is less pronounced in protozoan infections. the cytokines that are released include interleukin (il)-5, which drives the production of eosinophils to attack the parasite, and il-4 and il-13, which drive the production of immunoglobulin e (ige). epidemiological studies in endemic areas suggest that an age-dependent immunity may develop against infection, or against reinfection after treatment [71-73]. there is a good correlation between this protection and the development of ige antibodies, resulting from th2 responses [73, 74] . a hybridoma that produces a monoclonal ige antibody to s. japonicum, sj18ε.1, was identified [57]. this mab was protective in an in vitro, antigen-dependent, cellular cytotoxicity assay with rat macrophages or eosinophils and also in vivo during the early phase of infection second, beyond the cell-surface proteins, schistosomes also express a large number of glycans as part of their glycoprotein and glycolipid repertoire, and an antibody response against those glycans is mounted by the infected host [80] . in 2 cases, the specific antibodies produced could be identified using b cell cloning [81] or after infection of mice genetically modified to express human antibody repertoires [82] . interestingly, there has been no systematic analysis of the antibodies produced that target the various stages of the parasite life cycle, although early work on s. mekongi suggests schistosomula and adult worm extracts would induce a better response [83] . in addition to stimulating a th2 response, schistosome infection can suppress t-cell activation [84-86]; s. mansoni uses 2 distinct mechanisms to suppress t-cell activation, resulting in the selective up-regulation of pd-l1 on the surface of splenic f4/80 + macrophages. the presence of fatigued or anergized t cells opens up the possibility for cotherapy of low doses of mabs against programmed cell death protein-1 (pd-1) or pd-l1 with antibodies that reverse the anergy. the treatment of invasive fungal infections (ifis) remains a major challenge worldwide. there are few broad-spectrum antifungal drugs; the most effective have substantial toxicity concerns, and well-tolerated drugs used prophylactically frequently induce resistance. even with the best current treatment, the risk for mortality due to an ifi can be higher than 40%. for low-income countries, this figure is substantially worse, as many invasive infections are uniformly fatal without treatment, and estimates of global mortality involving fungal infections are as high as 1.5 million annually [87-89]. in addition, fungal infections are the cause of significant comorbidity and mortality in hiv patients. modeling studies suggest that optimal therapy could save the lives of 1.6 million hiv patients over a 5-year period [90]. the discussion here will focus on mabs developed for the following significant fungal pathogens: cryptococcus, pneumocystis and paracoccidioides, and candida. in addition to antibodies that directly target and inhibit the fungal pathogen, mabs can be directed to checkpoints that control the host immune response. this approach may be particularly useful against fungal pathogens for which sustained infection is characterized by a shift from a protective th1 or th17 response to a noninflammatory th2 response. the clinical use of anti-il17 in rheumatoid arthritis clearly exacerbates fungal infection [91], although it remains to be seen whether the reverse-stimulating this pathway-has a clinically useful effect. cryptococcus neoformans predominantly causes opportunistic infection in patients with hiv/aids and is responsible for a large burden of aids-related disease and death in sub-saharan africa [92] . although the rate of infection has decreased in recent years through greatly improved access to antiretroviral therapy (art), mortality in infected patients has not declined, demonstrating the failure of antifungal development to keep pace with improved antiviral treatment. cryptococcosis first manifests as a pulmonary disease but spreads hematogenously to the cerebrospinal fluid and brain to cause meningitis and meningoencephalitis, adding the complexity of crossing the blood-brain barrier to drug development. the ability of mabs to protect against a lethal cryptococcal infection in mice was first demonstrated over three decades ago [93], with subsequent work demonstrating the efficacy of various mabs that target the polysaccharide capsule, an essential virulence factor and the primary host-pathogen interface of cryptococcus infection [94] [95] [96] . among these, mab 18b7 was evaluated in a phase i clinical trial [50] and was found to produce a modest reduction in circulating cryptococcal antigen. further development has been hampered, however, by difficulties in securing funding for a disease for which financial returns are likely to be low-a common problem for neglected infectious diseases. subsequent studies have examined mabs that target cryptococcal melanin [97] , β-glucan [98] , or glucosylceramide ( [99] [100] [101] ; see [102] for additional in vivo and in vitro examples). host cd40 has also been targeted to stimulate the immune response [103] . in addition to highlighting the potential of mabs as therapeutics, these studies have demonstrated the diversity of inhibitory actions that mabs can perform on cryptococcal cells, which can include opsonization and increased phagocytosis, inhibition of fungal growth, capsular polysaccharide release and biofilm formation, antibody-mediated target cleavage, and augmentation of the host response [104] [105] [106] [107] . pneumocystis, like cryptococcus, is an important opportunistic pathogen in hiv/aids and other immunosuppressed patient populations, with estimates of up to 500,000 cases per year [87]. however, unlike cryptococcus, which is acquired from the environment, pneumocystis spp. are commensals, with different species occurring in the lungs of many mammals. the capacity for endogenous infection and for acquisition from asymptomatic carriers makes pneumocystis an attractive target for immunoprophylaxis. to this end, a variety of programs are aimed at developing a pneumocystis vaccine [108, 109] , and passive immunization studies have been initiated using mabs raised to pneumocystis epitopes. intranasal administration of 4f11, an mab that recognizes the pneumocystis kexin-like protein kex1, was able to prevent transmission of pneumocystis pneumonia from infected to susceptible cohoused mice, demonstrating the feasibility of this approach [110] . emerging evidence of the importance of b cellmediated immunity to pneumocystis infection strongly supports further research in this area [111] . paracoccidioides brasiliensis, while less globally prevalent than cryptococcus or pneumocystis, is the most important cause of ifis in latin america. mabs have been raised against the major p. brasiliensis antigens glycoprotein 43 (gp43) and gp70. in an infected mouse model, anti-gp43 activity, mediated by mab e3-enhanced phagocytosis of p. brasiliensis cells, increased interferon-γ production and led to a reduction in the fungal burden [112] , while anti-gp70 mabs significantly reduced fungal colony-forming units and almost completely abolished granuloma formation in the lungs [113] . antibodies raised against mouse cd25 + regulatory t (t reg ) cells, which control immunity and excessive inflammation, depleted cd25 + cells, resulting in less severe tissue inflammation, with reduced mortality in susceptible mice [112] . again, this demonstrates the potential for mabs to exert control of fungal infection, either directly by incapacitating the fungal cells or indirectly via modulation of the host response. candida albicans is the most commonly isolated fungal pathogen globally and is associated with significant morbidity and mortality, particularly in patients with hiv or tuberculosis (tb) infection [114] . a recent paper demonstrated the cloning of antibody genes from b cell cultures derived from patients infected with c. albicans [115] . these antibodies were capable of stimulating opsonophagocytic macrophage activity and provided protection in a murine model of disseminated candidiasis. in summary, mabs offer tremendous potential to augment the antifungal arsenal: animal models have provided promising results, there is a wide range of potential targets, they have the capacity to both inhibit the pathogen and augment the host response, and it may be possible to target diverse fungal pathogens with an appropriate mab cocktail or by targeting panfungal antigens. use of mabs as adjuvants to existing antifungal drugs also shows promise [116] . however, it should be noted that not all mabs are inhibitory; indeed, some may worsen infection, species and strain specificity can be an issue, and the mechanistic basis of inhibition by different mabs can vary dramatically [117] . to date, only 2 mab treatments have advanced to clinical trials: the previously mentioned 18b7 in cryptococcus, and mycograb, a heat shock protein 90 (hsp90)-specific antibody fragment, which showed promise for treating candida infections but failed to make it to market due to production difficulties and unresolved safety issues. antifungal immunomodulation is a complex area, and the field is still emerging. these preliminary studies highlight the potential for exciting new advances in mab research and application, both for understanding fungal immunity and for manipulating it to tackle lifethreatening fungal infections. dengue fever is a mosquito-borne viral infection found in tropical and subtropical regions around the world. the dengue virus (denv) is transmitted by female mosquitoes, mainly of the species aedes aegypti and, to a lesser extent, a. albopictus. there are 4 distinct serotypes-denv-1 to denv-4-of the virus, and all serotypes are presently circulating in endemic areas. denv infects cells of the human immune system and other cell types, leading to symptoms that include high fever, severe headache, severe pain behind the eyes, joint pain, muscle and bone pain, rash, and mild bleeding. in severe cases, plasma leaks out of the circulatory system, which can be fatal. the global incidence of dengue has grown dramatically in recent decades. one recent study estimated that approximately 390 million people are infected, of which 96 million manifest clinically each year [118] . who estimates that, globally, 500,000 people with severe dengue require hospitalization each year and that 2.5% of these infections are lethal. antibody-dependent enhancement (ade) is problematic in dengue infection. the presence of subneutralizing levels of flavivirus cross-reactive serum antibodies (acting against one member of the virus family) may result in an increase in infectivity via ade of another virus member or serotype, which is observed particularly after secondary dengue infection [119, 120] . despite decades of effort, there is no effective treatment against dengue. currently, dengvaxia is approved by the fda and is the only licensed dengue vaccine in the world. this is also a live attenuated tetravalent dengue vaccine developed by sanofi pasteur [121] that has been approved in several countries. however, interim results from long-term safety follow-up studies demonstrated an increased risk for hospitalization of vaccine-sensitized individuals [122] , suggesting that ade-related concerns are relevant. it has been reported that non-neutralizing levels of anti-denv antibody can enhance viral entry into host cells by forming a denvantibody complex [123, 124] . there is concern that an incomplete antibody against denvs may cause ade-mediated severe dengue disease. hence, there is a need for a safe and highly efficacious dengue therapy or vaccine that provides immunity against all 4 serotypes simultaneously. mab therapy is an alternative to vaccines and other therapies against dengue. many mabs against dengue from mice and humans have been characterized, and the use of mabs has also been explored as a therapeutic option. antibody sign-3c, identified by the singapore immunology network, neutralized all 4 dengue serotypes and decreased viremia of all serotypes in mice when given 2 days after infection [43] . a humanized mab visterra 513 (vis513), a panserotype anti-denv developed by visterra [44, 45] , which binds e protein domain iii (ediii) and neutralizes all 4 serotypes of denv, also showed useful antiviral utility. vis513 (25 mg/kg or 50 mg/kg) was administered at 5 days post infection in nonhuman primates, and no infectious virus could be detected by either plaque assay or virus isolation after treatment, a finding that was, however, not mirrored by reverse transcription pcr (rt-pcr) findings. a human challenge model is now available for clinical research in dengue [125] . in this model, the efficacy and safety profile of therapeutic antibodies can be evaluated rapidly in small-scale clinical settings, prior to traditional large-scale field studies with naturally infected patients. to develop mabs for dengue therapy, it is important to consider an approach that prevents or reduces ade, and several studies that address this link have been undertaken. a neutralizing human mab, d23-1b3b9, that targets the fusion loop in domain ii showed strong neutralizing activity against all 4 denv serotypes [46] . however, at subneutralizing concentrations, it also elicited ade activity in vitro [47] . to reduce the ade, injampa and coworkers [48] modified the d23-1b3b9 antibody fc domain at position n297q. the modified antibody kept the same cross-neutralizing activity to all 4 serotypes as those of wild-type antibody but lacked ade activity against all 4 serotypes at subneutralizing concentrations. in another neutralizing mab, sign-3c, 2 leucine to alanine mutations were engineered in the fc part, abrogating binding to fc gamma receptors [43] . this mutant fc version (sign-3c-lala) protected mice, while ade was completely abrogated. similarly, mab11/mutated fragment crystallizable region (mutfc) (an mab that is unable to bind to cells with fcγ receptors [fcγr]) and potentiate ade have been used as a prophylactic therapy [49] . passive immunization with this mab (at 25 mg/kg) reduced viral load and disease progression in nonhuman primates. here again, therapeutic antibodies can also be used for prophylaxis, affording immediate and reliable protection. tb tb is a good example of the gap between a pathogen's prevalence and burden. mycobacterium tuberculosis spreads easily among human populations; presently, about one-third of all humans are infected, and new infections occur in 1% of the population each year. among these billions of carriers, there are, however, "only" 10 million active tb infections, with 1.3 million deaths in 2016. thus, the vast majority of carriers keep the pathogen in check. tb exerts 2 levels of immune evasion: one in which it is maintained in a latent state and one in which it breaks free and causes active disease [126] . several studies have tested antibody therapy in tb, with varying success (reviewed in [127] ). even if an mab treatment would not be curative, shortening the standard treatment of patients infected with multidrug-resistant (mdr) and extensively drug-resistant (xdr) strains would represent a major advance. in addition, "checkpoint blockade during chronic tb infection requires further consideration" [128] . the role of protective antibodies in malaria was demonstrated over 40 years ago with the finding that the passive transfer of sera from mice with radiation-attenuated sporozoites delayed the development of infection in other mice [129] . the tpps for malaria are well described [130, 131] . these include a profile for seasonal malaria chemoprevention, a treatment successfully launched in sub-saharan africa in the last 5 years, consisting of a full treatment course of 3 days of amodiaquine and 1 dose of sulfadoxine/pyrimethamine. it is given monthly to children during the rainy season. antibody therapeutics for malaria could (1) prevent the entry (initial infection) of the parasite, (2) block entry of the sporozoite into the liver cells, (3) block entry of the merozoites into the erythrocytes, or (4) block the uptake of the gametocytes into the mosquito (breaking the transmission cycle). one difficulty in targeting the merozoites in symptomatic malaria is that the extracellular phase of the pathogen is relatively short-lived and is only a small part of the life cycle. additionally, the number of merozoites invading erythrocytes is very large (up to 10 12 ), compared with the number of sporozoites invading the liver stages (dozens). as such, the most interesting place to intervene with an mab is at the initial infection of the liver. currently there are 3 mabs published with potent activity against the circumsporozoite protein, csp, and these can reduce the parasitaemia in sporozoite-infected frg (triple mutant fah/raγ2/il2rγ) mice that carry a human liver implant [132] . these mabs include mab317, cloned from b cells obtained from a patient in the recent rts,s vaccine trial [133] , cis43 (circumsporozoite protein 43 [56]), and a set that included mgu12 [134] , cloned from patients vaccinated with irradiated sporozoites. antibodies that block the invasion process of the red blood cells by merozoites represent another possible approach. in studies of the merozoite protein rh5 (reticulocytebinding protein homologue 5) as a potential vaccine, some antibodies were described that could block the cycle of erythrocyte infection [135] . the specific merozoite epitopes are now being characterized, and this could form the basis of a second-generation antibody [136, 137] . a general observation with plasmodium infections is that the human host is unable to mount a sterilizing immune response. one theory is that the parasite can control the t-cell response and induce a state of fatigue or anergy similar to that seen in tumor-invading lymphocytes in immuno-oncology. several studies in mice have demonstrated that blocking pd-l1 or ctla-4 (cytotoxic t-lymphocyte-associated protein 4) improves clearance in mice infected with plasmodium bergheii [138] , and from this and similar experiments, a strong argument can be made that reversal of this fatigue by mild checkpoint inhibitor blockade may be a way of facilitating the host response [11]. hiv mab therapies have also been proposed for hiv. the case for hiv was recently reviewed [139, 140] . two such mabs are in phase iii trials: pro 140 and cenicriviroc. pro 140 targets ccr5 (cysteine-cysteine chemokine receptor type 5) and recently entered phase iib/iii trials for weekly subcutaneous dosing for monotherapy maintenance [141] . cenicriviroc is a dual ccr2 and ccr5 antagonist investigated for a number of indications, including hiv infection [142] . as noted earlier, ibalizumab was recently approved as a second-line treatment for hiv treatment [13]. for hbv infection, the hypothesis is that high circulating hbsag levels prevent a proper immune response. a novel mab, e6f6, is being evaluated for reducing hbsag levels in patients [52] . in addition, the hbv s protein is being targeted for the discovery of therapeutic mabs [143] . in the case of visceral leishmaniasis (vl), il-10 and glucocorticoid-induced tnf-receptorrelated protein have been considered as mab targets [144] , but there has been no systematic analysis of antigens or reported cloning of b cells from infected patients. although mabs have made a massive impact in controlling autoimmune disease, inflammation, and cancer, the relative impact in the world of infectious disease has largely been confined to viral diseases. the use of mabs to protect against rsv infections and the profile of secondgeneration antibodies shows that it is possible to obtain mabs that are sufficiently potent to provide long-term protection with a single intramuscular or subcutaneous injection. with the development of new technologies for cloning antibodies from b cells or plasma cells taken from patients infected with bacteria, viruses, fungi, or even protozoal pathogens, it is possible to quickly obtain fully human antibody collections with potential activity against pathogens in vitro and in vivo. technologies for expressing antibodies are now at the stage in which it is not uncommon to see extremely high levels of expression in cell culture, and taken together with the progress in reducing costs of production, the cost of goods for an antibody injection is starting to enter the range of us$1 to us$10, reaching the edge of what is affordable for infectious diseases of neglected populations. studies of mutations in the fc region confirm that mab half-lives can be extended, and the goal of a single injection to cover an entire season for those infections with seasonality is now a possibility. new technologies with viral delivery offer the promise that a single injection could give protection for even longer periods. for many infectious diseases, we are now seeing the buildup of a portfolio of potential antibodies. in cases in which little progress has been made, a systematic attempt is needed to identify the antibodies resulting from successful control of an infection in patients. beyond these basic antibodies, the availability of new monoclonals with anti-infective activity in vivo would open up the door to even more creative options: bispecific antibodies could be used in key immune cells and could effectively support the natural response to infection. studies in animal models of chronic malaria infection led to the observation that this results in a reduction in the impact of cytotoxic t cells and modulates the t reg capacity, leading to an "exhausted" or ineffective t-cell response [145] . clinical trials are already underway that use immune checkpoint blockers for chronic hiv and hbv infection [11] . this has important implications for any infectious disease in which a single infection does not drive a sterilizing immune response. for such diseases, which include malaria, one question for the longer term is whether immune checkpoint inhibition, or interfering with interferon-α signaling, could be used [128] . two decades ago, one of the biggest challenges of working in anti-infective drug discovery was the need to have new medicines with activities against the widest range of pathogens. in more recent times, the tide has changed, and clinical diagnosis now is such that medicines with a high degree of selectivity and specificity are often preferred-as long as they show good clinical activity. this shift to highly specific medicines favors the use of mabs. given the overall rise in interest for new treatments in infectious diseases caused by concerns about antimicrobial resistance, there is a real opportunity now to progress the newly emerging families of mabs to target infectious diseases of neglected populations. because of outdated preconceptions about this class of therapeutics, few research funds are being allocated to their discovery, resulting in an egg-and-chicken problem (the absence of conspicuous success driving additional efforts). one of the goals of the analysis that we present here is to promote making additional funds available to pursue the initial discovery of mabs for neglected diseases. monoclonal antibodies approved by the ema and fda for therapeutic use protective murine monoclonal antibodies to cryptococcus neoformans antibody-mediated protection in mice with lethal intracerebral cryptococcus neoformans infection monoclonal antibodies to cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice passive immunization with melanin-binding monoclonal antibodies prolongs survival of mice with lethal cryptococcus neoformans infection protection by anti-betaglucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence human antibodies against a purified glucosylceramide from cryptococcus neoformans inhibit cell budding and fungal growth monoclonal antibody to fungal glucosylceramide protects mice against lethal cryptococcus neoformans infection fungal glucosylceramides: from structural components to biologically active targets of new antimicrobials immunotherapy of cryptococcus infections. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases immunomodulation with cd40 stimulation and interleukin-2 protects mice from disseminated cryptococcosis a monoclonal antibody to cryptococcus neoformans glucuronoxylomannan manifests hydrolytic activity for both peptides and polysaccharides antibody to cryptococcus neoformans glucuronoxylomannan inhibits the release of capsular antigen immunoglobulins in defense, pathogenesis, and therapy of fungal diseases the yin and yang of current antifungal therapeutic strategies: how can we harness our natural defenses? frontiers in pharmacology immunization with pneumocystis cross-reactive antigen 1 (pca1) protects mice against pneumocystis pneumonia and generates antibody to pneumocystis jirovecii vaccine-induced immunogenicity and protection against pneumocystis pneumonia in a nonhuman primate model of hiv and pneumocystis coinfection passive intranasal monoclonal antibody prophylaxis against murine pneumocystis carinii pneumonia an emerging role of b cell immunity in susceptibility to pneumocystis pneumonia. american journal of respiratory cell and molecular biology the monoclonal antibody against the major diagnostic antigen of paracoccidioides brasiliensis mediates immune protection in infected balb/c mice challenged intratracheally with the fungus characterization of gp70 and anti-gp70 monoclonal antibodies in paracoccidioides brasiliensis pathogenesis candida albicans, a major human fungal pathogen single human b cell-derived monoclonal anti-candida antibodies enhance phagocytosis and protect against disseminated candidiasis immune modulators as adjuncts for the prevention and treatment of invasive fungal infections immunotherapy of fungal infections the global distribution and burden of dengue antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications development of the sanofi pasteur tetravalent dengue vaccine: one more step forward the impact of the newly licensed dengue vaccine in endemic countries antibody-enhanced dengue virus infection in primate leukocytes long-term safety assessment of live attenuated tetravalent dengue vaccines: deliberations from a who technical consultation experimental dengue virus challenge of human subjects previously vaccinated with live attenuated tetravalent dengue vaccines mycobacterium tuberculosis: immune evasion, latency and reactivation the role of antibodies in the defense against tuberculosis. tuberculosis-current issues in diagnosis and malaria drives t cells to exhaustion. frontiers in microbiology protective immunity produced by the injection of xirradiated sporozoites of plasmodium berghei hit and lead criteria in drug discovery for infectious diseases of the developing world new developments in anti-malarial target candidate and product profiles assessing drug efficacy against plasmodium falciparum liver stages in vivo structural basis for antibody recognition of the nanp repeats in plasmodium falciparum circumsporozoite protein a public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein human vaccination against rh5 induces neutralizing antimalarial antibodies that inhibit rh5 invasion complex interactions kilchip v1.0: a novel plasmodium falciparum merozoite protein microarray to facilitate malaria vaccine candidate prioritization. frontiers in immunology human antibodies that slow erythrocyte invasion potentiate malaria-neutralizing antibodies programmed death-1 ligand 2-mediated regulation of the pd-l1 to pd-1 axis is essential for establishing cd4(+) t cell immunity antibody-mediated therapy against hiv/aids: where are we standing now? broadly neutralizing anti-hiv-1 monoclonal antibodies in the clinic the return of pro 140, a ccr5-directed mab apobec3g/3a expression in human immunodeficiency virus type 1-infected individuals following initiation of antiretroviral therapy containing cenicriviroc or efavirenz a human monoclonal antibody against hepatitis b surface antigen with potent neutralizing activity combined immune therapy for the treatment of visceral leishmaniasis immune checkpoints and their inhibition in cancer and infectious diseases we thank dr. kiyoshi kita, dean and professor at the school of tropical medicine and global health, nagasaki university; dr. yoshimasa tanaka, assistant professor at the center for therapeutic innovation, nagasaki university; and dr. simon croft, professor at the london school of hygiene and tropical medicine, all of whom provided insight and expertise that greatly assisted the research. key: cord-004827-bnf3mvaf authors: desselberger, u. title: report on an ictv-sponsored symposium on virus evolution date: 2005-01-13 journal: arch virol doi: 10.1007/s00705-004-0466-9 sha: doc_id: 4827 cord_uid: bnf3mvaf nan genes etc. however, for viruses it should be noted that not all genes encoding proteins of similar functions (e.g. polymerases) stem from one single root. viruses in the retroviridae seem to be of very ancient origin as retroelements have been found in eukarya, bacteria and now also in archaea; in the latter, there is evidence for at least four different lateral gene transfers [22] . regarding the order of genes in a genome, patterns have often been maintained, but also often been rearranged. phylogenetic relationships have been found useful for virus identification, work on origin, speed and mechanisms of evolution, taxonomy, and the elucidation of transmission pathways (e.g. the transmission of hiv from a source to a victim [16] ). a case is being made for the use of rankless taxonomy clades (these being monophyletic groups without grading) instead of hierarchical formal linnean taxa for classification (see phylocode; www.ohiou.edu/phylocode). such a tree-based, rankless system could be constructed independently of taxonomy. in the discussion, the positions of clades within accepted phylogenies and the role of the quasispecies concept in a phylogenetically based classification system were considered. alexander e gorbalenya (leiden university medical center) spoke about "using evolutionary models to learn about rna viruses". starting from the idea that evolutionary models lead to the generation of structure/function studies, which in turn may or may not verify the model, the concept was developed that biopolymer alignments represent evolutionary models. proof of concept was explored using sequence data for members of the flaviviridae, nidovirales and birnaviridae as examples. citing data from lindenbach and rice [15] and the group of tautz (e.g. [33] ), it was concluded that hepaciviruses (e.g. hepatitis c virus) and pestiviruses (e.g. bovine viral diarrhea virus) have more in common than was originally thought; however, the phylogenetic analysis of hepacivirus and pestivirus genomes is still a challenge to the taxonomy. the nidovirales were established as an order that comprises the coronaviridae, arteriviridae and roniviridae families. this conclusion was based on the finding that viruses in the nidovirales share the mechanism of discontinuous transcription [30] and that they have motifs of their replicase enzymes in common [6, 32] . however, the taxonomy of the coronaviridae is under further review [5] . for instance, it has recently been found that the cysteine proteinases of an invertebrate nidovirus and of members of the potyviridae share unusual motifs [35] . viruses in the birnaviridae (carrying dsrna genomes) and some (but not other) members of the tetraviridae (carrying ssrna genomes and infecting insects) share a unique arrangement of motifs in their replicases [6] . a particular folding model of the replicase of infectious bursal disease virus, a member of the birnaviridae, has recently been tested and verified by the group of e mundt [34] . in the discussion, the relationship between coronaviruses and influenza c viruses (sharing neuraminate-o-acetyl esterase motifs and functions) was noted. graham hatfull (university of pittsburgh, pennsylvania) spoke about "mycobacteriophage genomics and the origins of mosaicism". given that there are an estimated 10 31 bacteriophages on earth (most of them in the sea), they represent an enormous genomic diversity and are also an excellent tool box with which to probe evolutionary theories. approximately 250 tailed dsdna bacteriophages have been completely sequenced, and 30 of those represent mycobacteriophages (of a genome size of approximately 2 mbp). partial genomic sequences of 14 of these phages (approximately 1 mbp each) have been subjected to phylogenetic comparison and analyses [7, 8] . their genes are closely packed and code for replication, integration, assembly and regulation functions. in the genomes, there is pervasive mosaicism, implying that horizontal exchange of genes has been an important component of their evolution. over 80% of the genes are only seen in mycobacteriophages but there are also some non-phage genes (which probably were picked up from host genomes). in terms of evolution and classification, each phage genome is considered to be a unique assembly of individual modules (a module either being an individual gene or a set of genes). in order report on ictv 631 to arrive at its present resting place, each module has a different phylogenetic history. the models for the generation of mosaicism are targeted recombination and random illegitimate recombination, followed by selection ('recombination reassorts genetic modules'). in order to conceptualize evolutionary relationships, the model of a three-dimensional web-like (or 'sweb') reconstruction of events was proposed. this would allocate unique 'sequence space' to each phage without ranks or preconceptions. in the discussion, the issues of the stability of mosaic genomes, the speed of recombination during evolution, the lack of a species concept, and the integration and reactivation of mosaic genomes were considered. simon wain-hobson (institut pasteur, paris) described and analyzed "the enormous multiplicity of hiv infection in vivo and the end of clonality". in addition to a minimum point mutation rate of 0.25/genome (increasing to 700/genome when nearing 'error catastrophe', see below), each hiv genome has undergone 3 recombination events on average, i.e. recombination creates much more diversity than point mutations [12, 14, 17] . in vitro, a single round of replication of hiv-1 in t lymphocytes generated on average 9 recombination events per virus [14] . hiv recombinants are frequently produced within individuals, and are even more frequently observed at epidemiological levels. siv recombinants are discovered within 15 days of infection. a prerequisite of recombination is a multiply infected cell (either co-or superinfected); such cells have been found in hiv-infected patients [17] . proviral sequences are randomly distributed on chromosomes; one chromosome can harbour several of them. there are also recombinant proviruses. there can be 600-700 proviral dna copies per cell, and the amount of dna in a cell can be increased threefold. one t cell can produce 500-4000 hiv particles that are spread preferentially by cell-to-cell contact. within an individual, donor cells (mostly dendritic, i.e. antigen presenting cells) carry sequences different from those found in recipient cells (mostly t cells). upon multiple infections the recombination rate increases and can reach the level of self-destruction ('error catastrophe', see below). concomitantly, the ratio [number of virus particles (nvp)/pfu], already high for all members of the retroviridae, increases further. in these circumstances, an accurate phylogeny cannot be constructed. john coffin (tufts university school of medicine, boston ma, and national cancer institute, frederick md) spoke about "retrovirus evolution and drug resistance". for retroviruses, host-virus co-evolution has been known for some time. the formation of endogenous retroviruses (ervs) as integrated proviral sequences leads to indefinite vertical transmission in the host. ervs can be considered and analysed as representing fossil records of previous virus-host interactions [9] . in human germlines, herv-k sequences are ubiquitous [10] . the history of retrovirus evolution in humans is long. ervs represent 6-8% of the human genome. strong parallels can be found in the phylogeny of erv of primates and that of primates themselves to the extent that the time points of evolutionary events in ervs and primates can be mutually determined. herv-k sequences entered human hosts approximately 30 million years ago. every human individual carries 30-50 different ervs of which 13 are considered as 'old' and 24 as 'new'. the analysis of long terminal repeats (ltrs) of ervs has allowed distinct waves of infection to be identified. mutations have accumulated as the species evolved. however, the 5 and 3 ends of the ltrs have not always co-evolved (about 6/36 human proviruses have 'mismatched' ltrs). approximately 50% of sequence changes are consistent with evolution by point mutations; other changes are due to multiple recombination events. hervs are still active and can be reactivated. using examples of the development of resistance of hiv to the action of the antiviral drug 3 -thiacytidine (3tc), mutations, selection, drift and linkage were recognized as genetic factors affecting the evolution of drug resistance. by using an ultrasensitive detection assay [20] , direct sequencing of hiv rna from limiting dilutions and the application of mathematical methods, extensive recombination events and evidence for 632 u. desselberger compensatory mutations were recognized as the main factors in the development of drug resistance. esteban domingo (centro de investigación en sanidad animal and universidad autónoma de madrid) spoke about "quasispecies dynamics and extinction of rna viruses". after an introduction in which basic genetic terms were defined (mutation and mutation rate, hypermutation, recombination, reassortment, segmentation etc), the quasispecies concept was presented according to which any sample of an rna virus represents a 'swarm' of closely related mutants. this composition allows the virus to adapt in a flexible way to changing environmental conditions. parameters of adaptability are: the number of mutations per genome (1-100), the population size (up to 10 12 infectious particles/host organism), the genomic length (9.5 kb for hiv, 3-30 kb for other rna viruses, i.e. relatively small for all rna viruses), and the number of mutations needed to produce a phenotypic change (can be very small). mutant spectra matter for the quasispecies of many rna viruses (vesicular stomatitis virus, picornaviruses [poliovirus, foot-and-mouth disease virus (fmdv)], lymphocytic choriomeningitis virus (lcmv), bunyaviruses etc). hypermutated (pre-extinction) rna often interferes with the infectivity of clonal rnas. assignment of a quasispecies to a phenotype is indeterminate. quasispecies have both deterministic and stochastic features [21, 27] . under bottleneck conditions (e.g. plaque-to-plaque passage in cell culture), the quasispecies spectrum will become narrower, and the fitness of the quasispecies to survive will decrease, due to the operation of muller's ratchet [3] . the fidelity of the transcriptase/replicase will go in parallel with the viability of a quasispecies distribution; with decreasing fidelity of these enzymes, the viral sequences will transgress via an error threshold to become random sequences. for fmdv, a constant rate of 0.25 mutations/genome/plaque transfer has been found during plaque-to-plaque passage. in the presence of a mutagen, viral extinction was frequently observed in vitro [2] . ribavirin, a licensed antiviral drug, was shown to be a mutagen as well. chronic infection of mice with lcmv was prevented (cured) by treatment of the animals with fluorouracil, a mutagen [28] . in the discussion, the influence of the ratio [nvp/pfu] on viability was considered. marilyn roossinck (samuel roberts noble foundation, ardmore ok) asked "what determines the quasispecies population size? lessons from plant viruses". using examples from the tobamovirus genus and the bromoviridae family, it was shown that the mutation frequency depended on virus host interactions [25, 26] . for brome mosaic virus, the control of diversity was located in rna segment 2, encoding the polymerase protein, and rna segment 3, encoding the cell-to-cell movement and coat proteins. bottleneck conditions limited diversity: of the 15 (silent) mutants in a mixed inoculum, only 7 were found in the 8th leaf and only 5 in the 15th leaf from the site of inoculation. the transmission frequency differed for different mutants. viruses with large host ranges were found to have large quasispecies 'swarms' (or 'clouds' [31] ). for further details see www.noble.org/virus evolution. ann palmenberg spoke about "rna structure and comparative picornavirology". for rna viruses, every viral base is to be regarded as a compromise forged by the totality of different selective pressures. those are mainly: protein recognition, mrna transcription, mrna translation, protein structure, and rna structure. different nucleic acids occur in different structural forms: in vivo, dna is usually in the b form, containing a wide major groove and rising by 3.4 a • /bp. duplex regions of rna occur in the a form, rising by 2.6 a • /bp and being more stable than dnas. the base stacking of rnas contributes hugely to their stability, and rna folding is largely driven by base stacking [4] . evolutionary co-variance of nucleotides is sometimes observed; compensatory changes may stabilize a stem, and such observations may help to confirm an rna structure. the most stable forms of rna or dna contain a minimum of free energy [36, 37] . using computer programmes developed by zuker's group, the optimal folding of rnas of relatively small size (picornaviruses) and large size (sars coronavirus) has been accomplished [19, 24] . the question arose: how can one recognize if a calculated fold is real? after randomizing and refolding the rna sequence of encephalomyocarditis virus (in silico), a highly stable form (∆g of −1720 kcal/mol) was obtained that was indistinguishable in stability from, and in the fold of, the real rna. thus, in reality 'the optimal fold' should be considered a myth; there is no single optimal structure. by mathematical derivation the number of alternative partners with which each base of an rna can interact (= p-num) can be obtained [36] and plotted against the sequence; troughs of the curve indicate regions of few alternative partners. the p-num derivative is a 'quantitative measure of the propensity of that base to become involved with the same or alternative pairing partners in a collection of suboptimal folds' [11, 19] ; it is thus a powerful parameter for locating wriggles in an rna structure. 'to maintain rna structure, evolution selects against better alternatives elsewhere in the genome'. the internal ribosome entry site (ires) of picornaviral rnas is a structural motif with a low p-num value. ires structures are similar to those of trnas in that they exhibit no significant sequence similarity, yet fold into virtually identical structures. the picornaviral cis-acting replication elements (cres), which display a cacaaa sequence to 3d polymerases, also have low p-num values, and again very different sequences adopt very similar 3-dimensional structures. vice versa, nucleotide sequence similarity does not always conserve rna structures. the aim of the talk was to show the significance of rna structural considerations for the evolution of viruses. at the conclusion of the symposium, andy ball thanked all speakers and discussants. the symposium was attended by approximately 150 participants. comments and suggestions on drafts of this report by andrew ball, jean cohen, esteban domingo, mary estes, denis fargette, anne-lise haenni, mike mayo and ann palmenberg are gratefully acknowledged. classification of papillomaviruses quasispecies dynamics and rna virus extinction multiple molecular pathways for fitness recovery of an rna virus debilitated by operation of muller's ratchet improved free-energy parameters for predictions of rna duplex stability a comparative sequence analysis to revise the current taxonomy of the family coronaviridae the palm subdomain-based active site is internally permutated in viral rna dependent rna polymerases of an ancient lineage bacteriophages with tails: chasing their origins and evolution bacteriophage genomics a novel, endogenous retrovirus-related element in the human genome resembles a dna transposon: evidence for an evolutionary link human endogenous retrovirus k solo-ltr formation and insertional polymorphism: implications for human and viral evolution improved predictions of secondary structures of rna multiply infected spleen cells in hiv patients imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches dynamics of hiv-1 recombination in its natural target cells flaviviridae: the viruses and their replication molecular evidence of hiv-1 transmission in a criminal case the nonclonal and transitory nature of hiv in vivo don't forget about viruses topological organization of picornaviral genomes: statistical prediction of rna structural signals new real-time reverse transcriptase-initiated pcr assay with single-copy sensitivity for human immunodeficiency virus type 1 rna in plasma reproducible nonlinear population dynamics and critical points during replicative competitions of rna virus quasispecies retroids in archaea: phylogeny and lateral origins the structure of a thermophilic archaeal virus shows a double stranded dna viral capsid that spans all domains of life generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted rna secondary structure evolutionary history of cucumber mosaic virus deduced by phylogenetic analyses plant rna virus evolution synchronous loss of quasispecies memory in parallel viral lineages: a deterministic feature of viral quasispecies lethal mutagenesis of the prototypic arenavirus lymphocytic choriomeningitis virus (lcmv) new haven ct 30. sawicki sg, sawicki dl (1998) a new model for coronavirus transcription genetic diversity in rna virus quasispecies is controlled by host-virus interaction unique and conserved features of genome and proteome of sars-coronavirus, an early split off from the coronavirus group 2 lineage of statistics and genomes vp1 of infectious bursal disease virus is an rna dependent rna polymerase the 3c-like proteinase of an invertebrate nidovirus links coronavirus and potyvirus homologs on finding all suboptimal foldings of an rna molecule prediction of rna secondary structure by energy minimization parc d'innovation, boulevard sébastian brandt, 67400 illkirch, france. -redaktion: sachsenplatz 4-6 key: cord-006716-n371b91w authors: cone, a. m.; nadel, s.; sweeney, b. title: flumazenil reverses diazepam-induced neonatal apnoea and hypotonia date: 1993 journal: eur j pediatr doi: 10.1007/bf01955914 sha: doc_id: 6716 cord_uid: n371b91w nan screening in pregnancy is now in progress in the usa [3, 4] discussing strategies aimed at limiting hiv-1 spread in children. these strategies are required in some european countries where the large proportion of intravenous drug user (ivdu) women of child-bearing age leads to increasing numbers of perinatally infected children [2] . sociological and cultural peculiarities in different areas do not allow for generalized conclusions but we think that our experience can be helpful to start the discussion in an european perspective. up to august 1992 we have observed 98 children (including three twin pairs) born to 85 hiv-i+ mothers. women were or had been: ivdu or ivdu and sexual partner (sp) of an hiv-1+ man (n: 50 = 58.8%); sp of an hiv-i+ ivdu man (n: 24 = 28.2%); sp of an hiv-i+ not ivdu man (n: 11 = 12.9%). we identified three conditions: (1) mothers who undertake and carry pregnancy to term conscious of being hiv+ and of risk for the child; (2) mothers having knowledge of their at-risk condition, but without awareness of being hiv-i+; (3) mothers unaware of either condition. pregnancies undertaken and brought to term without awareness of risk were more and more frequent among ivdu, sp of hiv-i+ ivdu men, sp of hiv-i+ not ivdu men (table 1) . six women (not ivdu) were seronegative early in pregnancy but seroconverted before delivery (6.3% of all pregnancies and 15.3% of pregnancies in not ivdu women). they all knew they were at-risk for hiv-1 infection. our data included also nine multiparous women: six (five ivdu) undertook and brought to term a subsequent pregnancy in spite of counselling. all had a first seroreverted child. in three other cases (one ivdu), women who gave birth to a second child when the first-born was hiv-i+ discovered their own and their children's infection status only after the second child's birth. abortion of the second pregnancy after the birth of a first hiv-i+ child occurred in four (two ivdu) women but in no mother of an uninfected first child. we observed that: (1) hiv-i+ ivdu women usually undertake a pregnancy conscious of their infection status. two main mechanisms work: intense desire of motherhood and poor consideration of risk for the child. motherhood is felt important as if it appears to be a way to recover from past or present mistakes, to be like normal women and to gain somebody who will live afterwards. these mechanisms have been already identified [1] and, as in others' data [1] , a prior unharmed experience seems to be a factor influencing reproductive decision; (2) hiv-1 testing in pregnancy is mostly useful for sexually infected women often unconscious of their condition. in a significant proportion of them seroconversion occurs during the course of pregnancy. in order to limit hiv-1 spread to children, we think that effective counselling of reproductive decision of hiv-i+ ivdu women would be the cornerstone, but our data show that it [2] did never seem to be enough to discourage ivdu women from pregnancy. thus, counselling should also be focused on the life-quality, morbidity and mortality in hiv-i+ children. theoretically, hiv-1 testing in our data could be useful in one third of women (less and less important in sp of not ivdu hiv-i+ men, sp of ivdu hiv-i+ men, ivdu women) but effective counselling in two thirds. in addition, individuation of hiv-1 infection in pregnancy is important in the care of at-risk infants, but it does not necessarily prevent from perinatal infection. screening might include an early evaluation which could be followed by abortion, but this latter step again needs effective counselling. in pregnant seronegative sp of hiv-i+ men an additional evaluation should be carried out close to delivery not for prevention but to identify at-risk children, since it is evident that these women are at high risk because of unprotected contacts. sir: we describe a case in which flumazenil, a specific benzodiazepine antagonist, reversed diazepam-induced neonatal apnoea and hypotonia. this is the first report of the use of flumazenil during resuscitation of the newborn. a pre-eclamptic woman underwent caesarian section. in the hour preceding surgery, she received diazepam, 20 mg, intravenously, because of worsening headaches, photophobia and hyper-reflexia. five minutes after induction of anaesthesia a male infant was delivered. he was apnoeic, blue, hypotonic and had a heart rate of 100 beats/rain. with oropharyngeal suction and manual ventilation there was a rapid improvement in heart rate and colour, but at 3 min of age apnoea and hypotonia persisted, and he was intubated. when, 10 min after delivery, his condition was unchanged, flumazenil (10 ~tg/kg) was given intravenously. within lmin respiratory effort began, accompanied by vigourous movement. at 15 min of age he was extubated and transferred to neonatal intensive care, where a fiumazenil infusion was commenced at 10 ~tg/kg per hour. there were no further episodes of apnoea or desaturation, and the infusion was weaned after 6 h. recovery was uneventful, with discharge to the postnatal ward 48 h later. diazepam crosses the placenta rapidly, and significant levels were likely to be present in the neonatal circulation at birth. neonates are susceptible to the effects of benzodiazepines administered to the mother antenatally and diazepam can cause repiratory depression and apnoea in the newborn [2] . rapid, complete and sustained reversal of prolonged apnoea and hypotonia following treatment with flumazenil suggests the inhibition of diazepam-induced neonatal depression by this drug. flumazenil is an imidazo-benzodiazepine, a specific antagonist which competes for benzodiazepine receptors in the cns. it has a relatively short half-life of i h, and may need to be given as a continuous infusion to prevent the return of benzodiazepine effects [4] . because the neonate had remained well and there was concern over possible adverse effects of flumazenil therapy, the infusion was weaned after 6h. diazepam levels were not measured, but in view of the prolonged half-life of this drug, it was likely that the infant would remain susceptible to its effects after cessation of the flumazenil infusion. discharge was therefore delayed for 48 h to allow observation to continue during this "at risk" period. flumazenil has been used safely in children to treat respiratory depression, muscular hypotonia and coma resulting from benzodiazepine poisoning [3] . in adults, anxiety, vomiting, seizures and arrhythmias can follow its administration [1] . whether neonates are susceptible to similar side-effects is unknown. c3a-desarg, c3bbbp and terminal complement complex (tcc) have been found in adult respiratory distress syndrome (ards) patients. in polytrauma patients at risk of developing ards, a prognostic value was ascribed to elevated c3a and c3a/c3 levels in the first few hours after injury. this prognostic value was, however, limited to a very small period of time e.g. 6h, since the increase in c3a and c3a/c3 was transient [4] . hack et al. [1] showed that patients with septic shock had significantly higher c3a levels than normotensive patients and that high plasma levels of c3a were associated with a fatal outcome. they also found no significant difference between patients who developed ards and those who did not. whether c3a precedes or follows the clinical course in these patients is controversial. schrod et al. [3] speculated on the role of c3a in the development of ards in newborn infants. these authors did not define the criteria for ards. the control group and asphyxia group did not differ in gestational age, in the need for artificial ventilation due to respiratory distress syndrome or in the severity of respiratory disease documented by fio2 requirement. the authors "assumed" a diagnosis of ards in only four infants of the asphyxia group; they did not "assume" ards in the three term neonates in the non-asphyxia group. no explanation was given for the three term infants with respiratory distress syndrome in the control group. we therefore must conclude that both control and asphyxia groups have a similar incidence of severe respiratory disease, but differ only in extent of c3a generation. generation of c3a in birth asphyxia is well in accordance with previously published data [5] . the authors do not comment on extremely high levels of c3a found in newborns with infection. none of these infants developed ards (5) . values for c3bbbp appear extremely high within both groups (normal range for adults 0-5 units/ml). if determined with the method by zilow et al. [4] values within this range are found exclusively after in vitro activation, for example in case of inadequate sample storage temperature or repeated freezing and thawing. since a different duration of in vitro activation has to be assumed, differences between the study groups in concentrations of both c3bbbp and c3 a cannot be interpreted. reproductive health policy and hiv: where do women fit in? perinatal hiv-1 infection in italy: general remarks hiv screening in pregnancy hiv screening in pregnancy death after flumazenil the use of flumazenil in a neonate flunitrazepam intoxication in a child successfully treated with the benzodiazepine antagonist flumazenii flnmazenil, a useful antagonist in paediatrics elevated plasma levels of the anaphylatoxins c3a and c4a are associated with a fatai outcome in sepsis complement. clinical aspects and relevance to disease complement fragment c3a in plasma of asphyxiated neonates complement activation and the prognostic value of c3 a in patients at risk of the adult respiratory distress syndrome sir: complement activation, leading to formation of anaphylatoxins, can occur in a variety of diseases key: cord-001532-kz3b01wq authors: gantt, soren; gachelet, eliora; carlsson, jacquelyn; barcy, serge; casper, corey; lagunoff, michael title: nelfinavir impairs glycosylation of herpes simplex virus 1 envelope proteins and blocks virus maturation date: 2015-01-29 journal: adv virol doi: 10.1155/2015/687162 sha: doc_id: 1532 cord_uid: kz3b01wq nelfinavir (nfv) is an hiv-1 aspartyl protease inhibitor that has numerous effects on human cells, which impart attractive antitumor properties. nfv has also been shown to have in vitro inhibitory activity against human herpesviruses (hhvs). given the apparent absence of an aspartyl protease encoded by hhvs, we investigated the mechanism of action of nfv herpes simplex virus type 1 (hsv-1) in cultured cells. selection of hsv-1 resistance to nfv was not achieved despite multiple passages under drug pressure. nfv did not significantly affect the level of expression of late hsv-1 gene products. normal numbers of viral particles appeared to be produced in nfv-treated cells by electron microscopy but remain within the cytoplasm more often than controls. nfv did not inhibit the activity of the hsv-1 serine protease nor could its antiviral activity be attributed to inhibition of akt phosphorylation. nfv was found to decrease glycosylation of viral glycoproteins b and c and resulted in aberrant subcellular localization, consistent with induction of endoplasmic reticulum stress and the unfolded protein response by nfv. these results demonstrate that nfv causes alterations in hsv-1 glycoprotein maturation and egress and likely acts on one or more host cell functions that are important for hhv replication. human herpesvirus (hhv) infections are ubiquitous and are responsible for substantial morbidity and mortality worldwide, particularly among people infected with human immunodeficiency virus (hiv). herpes simplex virus (hsv) and cytomegalovirus (cmv) infections can be recurrent and difficult to treat in hiv coinfected individuals [1] . moreover, genital hsv infection has been associated with greater risks of hiv acquisition, transmission, and progression of disease [2] . hhv-8 and epstein-barr virus infections cause the most common aids-defining malignancies, kaposi sarcoma and non-hodgkin lymphoma, respectively [3] . although greatly reduced by effective antiretroviral therapy (art), complications of hhv infections remain among the most common medical problems in people infected with hiv worldwide [3] [4] [5] [6] [7] . currently available antiviral drugs to treat or prevent complications of hhv infections all directly or indirectly target the viral polymerase [8] . each of these drugs has one or more important limitations, including selection of drugresistant viral mutants, significant toxicities, and/or poor bioavailability requiring intravenous administration. for example, treatment of acyclovir-resistant hsv or ganciclovirresistant cmv infections requires the use of intravenous foscarnet or cidofovir, both of which are associated with nephrotoxicity. as such, new agents that are effective for hhv infections are needed that are safe, orally bioavailable and have a high barrier to resistance. nelfinavir (nfv) is a first-generation hiv aspartyl protease inhibitor recently found to block production of multiple hhvs [9] . furthermore, because it also has potent antitumor and antiangiogenic properties, clinical trials are ongoing to evaluate nfv for the treatment of several cancers [10] [11] [12] [13] [14] [15] . the mechanisms by which nfv acts on tumor cells are multifactorial and include inhibition of cellular proteases, akt activation, and nf -b signaling, as well as induction of the endoplasmic reticulum (er) stress, the unfolded protein response (upr), and autophagy [11, 16, 17] . in contrast to the aspartyl protease required for hiv maturation, hhvs utilize a serine protease, which for hsv-1 is the gene product of ul26 open reading frame (orf) [18] . as such, we hypothesized that nfv does not inhibit hhv replication by acting on the viral protease. furthermore, we speculated that nfv acting on a nonprotease viral target would be improbable and that its antiviral activity is more likely due to one or more of its effects on host cells, which could impair efficient viral replication. we therefore investigated mechanism of action of nfv on hsv-1, by attempting to identify host cell or viral targets of the drug in vitro. human fibroblasts (hf) were cultivated in dulbecco's modified eagle's medium (dmem; gibco) containing 10% fetal bovine serum (fbs) and 100 units per ml penicillin g and 100 g per ml streptomycin (pen-strep) and maintained at 37 ∘ c in a humidified 5% co 2 atmosphere, as previously described [9] . hek293-t cells were maintained in dmem supplemented with 10% fbs and pen-strep. hsv-1 (strain f) was a gift from keith jerome (fred hutchinson cancer research center). drugs. nfv and indinavir (idv) were obtained through the aids research and reference reagent program, division of aids, niaid, nih. nfv was solubilized in dimethyl sulfoxide (dmso). idv and acyclovir (sigma-aldrich) were solubilized in water. the primary antibodies used were as follows: mouse monoclonal anti-hsv gb (virusys), mouse monoclonal anti-hsv1 gc clone 3g9 (abcam), rabbit polyclonal anti-lc3b (cell signaling technology), mouse monoclonal anti--actin, anti-flag m2 monoclonal, and anti-ha monoclonal antibody, clone ha-7 (sigma-aldrich). for western blotting, the secondary antibodies used were peroxidaseconjugated affinipure f(ab )2 fragment of goat anti-mouse igg(h + l) or peroxidase-conjugated affinipure f(ab )2 fragment of goat anti-rabbit igg(h + l) (jackson immunoresearch). secondary antibodies for immunofluorescence were alexafluor 594 f(ab )2 fragment of goat anti-mouse igg(h + l) or alexafluor 488 f(ab )2 fragment of goat anti-rabbit igg(h + l) (life technologies). blotting. biotinylated lectins used in the eastern blots (vector laboratories, inc.) were as follows: peanut agglutinin (pna), ricinus communis agglutinin i (rca i), wheat germ agglutinin (wga), and concanavalin a (cona). total cellular proteins (0.75-1.0 g loaded per lane) were separated by sds-page and transferred to a pvdf membrane. the membranes were blocked with 5% bsa in tris-buffered saline with 0.3% tween-20 (tbs-t), incubated with 1-2 g lectin/ml in blocking buffer for 1-2 hours, washed three times in tbs-t, and then incubated 1 hour with horseradish-peroxidase-conjugated avidin d (vector labs) at 1-2 g/ml in tbs-t. the blots were washed three times in tbs-t following the avidin incubation. detection was performed using an enhanced chemiluminescence method (pierce ecl plus). hf were infected with hsv-1 at an moi of 0.1 for 1 hour at 37 ∘ c and then incubated at 37 ∘ c with either 5 m nfv, 1 m acv, or no drug for 3-5 days until most cells were lysed. four passages were made for each group. virus was harvested with 3 freeze-thaw cycles followed by the addition of nonfat milk. virus titers were determined by plaque assay previously described [9] . 2.6. hsv-1 protease activity assay. the effect of nfv on hsv-1 maturational protease activity was assayed by transfecting hek293-t cells with vectors expressing the protease (vp24; n-terminal 247 amino acids of the ul26 orf gene product), as well as two substrates: the hsv-1 capsid scaffold protein (amino acids 307-635 of the ul26 orf gene product; full-length product of ul26.5; icp35) and a catalytically inactive mutant (s129a) of the full-length protease-scaffold protein transcript (amino acids 1-635 of the ul26 orf gene product) [18] . the protease coding sequence contained an n-terminal ha tag and each substrate contained an nterminal flag tag; all vectors were synthesized by blue heron biotech, llc. each substrate was expressed alone or in combination with the protease, in the presence of nfv or a vehicle control. expression was under the control of the cmv6 promoter. the plasmid pegfp-n2, which expresses a gfp variant from the cmv immediate-early promoter, was used to monitor transfection efficiencies. transfection was performed using mirus transit-293 reagent (mir 2700) according to the manufacturer's instructions. nfv (10 m final concentration) or dmso (0.1% final concentration) was added to the cells concurrently with the dna and transfection reagent. cultures were incubated 24 hours with no change of medium, at which time the cells were harvested on ice by scraping into ripa buffer containing protease inhibitors (roche complete mini edta-free protease inhibitor cocktail tablets). protein concentrations were determined by bca assay (pierce). extracted proteins (10 g/lane) were separated by sds-page and transferred to pvdf membranes by tank transfer overnight. the blots were blocked with 5% nonfat dry milk and probed with anti-flag m2 mouse monoclonal antibody (sigma), recognizing the tag on the scaffold protein, anti-ha monoclonal antibody, clone ha-7 (sigma), and recognizing the tag on vp24. human foreskin fibroblasts (hffs) were mock-infected or infected with hsv-1 (strain f) at an moi of 10 or 50. after 1 hour, the inoculum was replaced with medium containing 0.1% dmso or 10 m nfv. at 16 or 20 hpi the medium was replaced with a 1 : 1 mixture of dmem and 1/2 karnovsky's fixative [19] and the culture was returned to the incubator for 10 minutes. the mixture was replaced by 1/2 karnovsky's, and the cells were incubated a further 30-60 minutes at room temperature. the cells were scraped, transferred to a microcentrifuge tube, and pelleted at 200 ×g. the pellet was resuspended in 1 ml of fixative and then dehydrated, embedded, sectioned, and affixed to grids according to standard methods. the grids were examined on a jeol 1230 or a jeol jem 1400 transmission electron microscope at the electron microscopy lab at the fred hutchinson cancer research center. hffs were seeded in 4well chamber slides to a confluence of approximately 70%. they were mock-infected or infected with hsv-1 at an moi of 3. after 1 h, the inoculum was replaced by medium with 0.1% dmso or 10 m nfv, and the cultures were returned to the incubator. sixteen hours after infection, the cells were fixed in 4% paraformaldehyde in phosphate-buffered saline. cells were then permeabilized with 0.2% tween-20. endogenous peroxidase activity was inhibited with 3% h 2 o 2 . the cells were then incubated with 10% nonfat dry milk containing 1% normal goat serum (blotto/ngs; jackson) to block nonspecific binding. primary antibody binding was revealed with anti-mouse igg-hrp (jackson) followed by a 10-minute tsa amplification with tsa-594 (life technologies). the nuclei were stained with to-pro 3. slides were mounted after addition of slowfade (life technologies) and analyzed by confocal microscopy. confocal images were generated on an lsm 5 pascal system (zeiss). we passaged hsv-1 in the presence of nfv to determine if resistant mutants could be selected in an attempt to elucidate the mechanism of action of nfv on hhv production. as expected, [20] multistep passage under drug pressure was readily selected for high-level resistance to acyclovir ( figure 1 ). in contrast, no significant change in susceptibility to nfv was observed despite parallel passaging in the presence of nfv. of note, the inhibitory activity of nfv was not different between acyclovir-resistant and wild type isolates, further suggesting a distinct antiviral mechanism. to determine if nfv, an aspartyl protease inhibitor, could act on the essential hsv-1 ul26 serine protease, we tested whether nfv could inhibit the activity of the hsv-1 protease on its scaffold protein substrates using a cotransfection assay. at nfv concentrations that potently block production of infectious hsv-1, there was no effect on the activity of the hsv-1 protease expressed in hek293t cells (figure 2 ). attributed to a decrease in akt activation. one of the prominent effects of nfv on human cells that has been described is inhibition of the akt signaling pathway by reducing the phosphorylation of akt by phosphatidylinositol-3 kinase [21] [22] [23] . hsv-1 infection results in an increase in the level of akt phosphorylation (figure 3 ), as has been previously described [24] . the akt inhibitor ly294002 completely suppressed akt phosphorylation in hsv-1 infected cells, but nfv did not reduce the levels of phosphorylated akt even at drug concentrations that potently block virus production ( figure 3 ). furthermore, ly294002 treatment of hsv-1 infected hf cells did not reduce the production of infectious virus by plaque assay (not shown), consistent with published data [24] . therefore, it is unlikely that the documented inhibition of nfv on akt activation plays a role in the drug's inhibition of hsv-1. as shown by kalu et al. [25] and supported by our preliminary experiments (data not shown), late hsv-1 gene expression in hf cells was not reduced by nfv treatment. we therefore explored the effects of nfv on hsv-1 infected hf cells by transmission electron microscopy. normal numbers of virus particles appeared to be produced in nfv-treated cells. similar numbers of capsids were observed in the nucleus of untreated and nfv-treated cells (approximately 39 and 44, resp., figures 4(a) and 4(b) ). however, compared with untreated cells, capsids in nfv-treated cells appeared to be disproportionately retained within the cytoplasm (∼14 versus protease: figure 2 : nfv does not inhibit activity of the hsv-1 protease. the coding sequence of the full-length protein product of ul26 orf (pul26) and the expression proteins employed are diagramed in panel (a). transfection vectors were constructed to express the vp24 protease with an n-terminal ha tag ("protease"), the pul26.5 with an n-terminal flag tag (substrate 1), or the s129a pul26 mutant inactive protease-scaffold protein with an n-terminal flag tag (substrate 2). tags are depicted in grey at the left end of each construct. the release (r) and maturation (m) cleavage sites are shown. after transfection into hek293-t cells, construct expression and substrate cleavage were evaluated by western blot using anti-ha and flag antibodies. each protein expressed alone was of the expected size (28.8, 37.9, and 70.6 kd, resp., including tag). similarly, coexpression of protease with each of the 2 substrates resulted in the n-terminal cleavage products of the expected sizes. the generation of substrate cleavage products was not affected by the addition of nfv at concentrations that inhibit hsv-1 replication in vitro. 79, resp.), in which virus particles were more often located outside the plasma membrane (∼51 versus 3), suggesting a block in virus maturation or egress. in addition, in contrast to those in the cytoplasm of untreated cells, cytoplasmic capsids in nfv-treated cells were rarely observed to be enveloped (∼9 versus 0 in the fields shown; figures 4(c) and 4(d)). interestingly, although endoplasmic reticulum (er) stress, the unfolded protein response (upr), and autophagy are well known effects of nfv [16, [26] [27] [28] [29] , neither er dilation nor the abundance of double-membrane bound vesicles consistent with autophagosomes appeared consistently different between nvf-treated and untreated hsv-1-infected cells. are impaired by nfv. during evaluation of hsv-1 glycoproteins gb and gc expression by western blotting, it was apparent that nfv treatment of infected cells resulted in increased electrophoretic mobility (figure 5(a) ) compared to untreated controls or idv-treated cells. the apparent change in molecular weight of these viral proteins was estimated to be consistent with the reduction in glycosylation [30] [31] [32] . to assess the effect of nfv on protein glycosylation in hsv-1infected cells, eastern blotting was performed using lectins pna, rca-i, wga, and cona ( figure 5(a) ). compared to untreated or idv-treated cells, nfv resulted in a marked reduction in staining by pna, rca-i, and wga indicating decreased addition of galactose, n-acetyl-d-galactosamine, and n-acetyl-d-glucosamine [33, 34] . in contrast, cona staining was not appreciably reduced, suggesting relatively normal levels of oligomannose-type n-glycans. nfv resulted in a clear alteration in the subcellular localization of hsv-1 gb ( figure 5(b) ) by immunofluorescent antibody staining. compared with control treatments in which viral envelope glycoprotein staining uniformly delineated the plasma membrane of hsv-1-infected hf cells, with nfv treatment staining appeared predominantly perinuclear, suggesting improper trafficking to the cell surface. consistent with the electron microscopy results, using immunofluorescence, no increase in lc3-ii staining, a marker of autophagy, was apparent (data not shown). nfv is an hiv aspartyl protease inhibitor that, in addition to its antiretroviral activity, has complex effects on numerous human cellular functions, including on akt signaling, inhibition of cellular proteases, and induction of er stress, many of which might contribute to its ability to broadly inhibit tumor cell growth as well as replication of several non-hiv viruses [9, 11, 16, 17, 35, 36] . in this study, we specifically explored the mechanism(s) by which nfv might inhibit production of infectious hsv-1 in vitro. we found that nfv acts on hsv-1 late in virus production, without a detectable effect on late viral gene expression. furthermore, abundant virus particles were observed within infected cells, though envelopment and release of virus appeared to be substantially diminished. this is consistent with a recently published study by kalu et al., which also reported that nfv blocked hsv-1 maturation and egress [25] . nfv showed no activity on the vp24 protease, which was expected given that it is a serine protease that lacks structural or functional similarity with the hiv (aspartyl) protease. this was shown using a transfection system to examine enzymatic activity in trans and supports the finding that scaffold protein cleavage appears unaffected in hsv-1-infected cells treated with nfv [25] . no resistance to nfv could be selected under conditions that readily resulted in acyclovir-resistant hsv-1, which is again consistent with findings by kalu et al. [25] . though nfv-resistant hsv-1 may well be isolated using other conditions, this finding suggests a relatively high barrier to resistance in vitro and suggests a mechanism of action on a host cell function required for virus production, rather than a direct effect on a viral target [37] [38] [39] [40] [41] . indeed, many of the cellular functions affected by nfv have similarly been described to play a role in hsv-1 replication. nfv inhibits cellular proteases and the proteasome, which leads to accumulation and inefficient removal of misfolded proteins in the er and golgi [16, 42, 43] . the finding that nfv resulted in impaired viral protein glycosylation and trafficking is consistent with these processes and again validates the recent findings by kalu et al. [25] . of note, based on cona staining, n-linkage of immature (high mannose) carbohydrates appeared relatively normal [33] . these mannose structures are largely assembled in the cytoplasm, whereas trimming and modification of more complex sugar residues occur in the er and golgi. we found that the impairment of viral glycoprotein processing is at least one mechanism by which nfv reduces infectious hsv-1 production. agents that induce er stress, such as thapsigargin, similarly interfere with hsv-1 glycoprotein posttranslational processing and production of infectious virus [31] . numerous studies have reported that tunicamycin, which blocks the synthesis of the n-acetylglucosamine-lipid intermediates, and other inhibitors of protein glycosylation decrease the infectious yield of hsv-1 in vitro [44] [45] [46] . furthermore, tunicamycin does not affect the level of late viral gene product expression, and normal appearing capsids were noted within the cytoplasm, similar to the effects we observed with nfv. it is unclear, however, whether impaired hsv-1 envelope protein glycosylation would block virus egress based on studies using cell lines deficient in n-acetylglucosaminyl transferase activity, in which virus yield was only mildly reduced [47] . this work has several important limitations. the effects of nfv are highly pleiotropic, and we stress that nfv might affect the production of infectious hsv-1 through multiple mechanisms. in addition, based on what is known about nfv's effects on tumor cells [11, 21] , the most relevant mechanism(s) of action may differ with respect to individual hhv, cell type, and drug concentration. by necessity, all of the possible mechanisms by which nfv might affect hsv-1 replication were not evaluated. autophagy, a catabolic process that maintains cellular homeostasis under conditions of stress, is a prominent effect of nfv [11, 16] . hsv-1 encodes genes to block autophagy in infected cells, including infected cell protein (icp) 34.5, which is required for neurovirulence [48] . increased levels of autophagy can reduce production of infectious hsv-1 similarly to nfv, with relatively normal viral gene expression and formation of viral particles that are retained within cells [49, 50] . we were not able to formally show that nfv increased autophagy in hsv-1 infected cells under the conditions used. nfv treatment appeared to result in retention of cytoplasmic virus particles within single membranebound vesicles that could be late autophagosomes as in other reports [49, 50] . however, pathognomonic doublemembrane structures were not convincingly observed in greater numbers in nfv-treated versus untreated hsv-1infected cells nor were we able to show that nfv increased lc3-ii staining during hsv-1 infection. autophagy can be difficult to demonstrate [51] and cannot currently be excluded as a potential contributor to the effect of nfv on hsv-1 or other hhvs [48] . nfv is an orally bioavailable, fda-approved treatment for hiv infection and is well tolerated during long-term use. since the advent of more potent and convenient options, nfv is no longer widely prescribed for art [4] . however, because of its activity against a broad range of tumor cell types, there is intense interest in repositioning nfv as a cancer chemotherapeutic agent, perhaps at higher doses than those for art [11, 42, 52] . nfv could also be beneficial in the treatment or suppression of infection with hsv and other hhvs, particularly in the setting of resistance to first-line antivirals, in patients with cancer and/or hiv. additional studies are indicated to further elucidate the mechanism(s) of action of nfv on hhv infections and to evaluate its efficacy in clinical trials. guidelines for the prevention and treatment of opportunistic infections in hiv-infected adults and adolescents: recommendations from the centers for disease control and prevention, the national institutes of health, and the hiv medicine association of the infectious diseases society of america probability of hiv-1 transmission per coital act in monogamous, heterosexual, hiv-1-discordant couples in rakai, uganda global cancer statistics adolescents poagfaa, guidelines for the use of antiretroviral agents in hiv-1-infected adults and adolescents, department of health and human services burden of hiv-related cytomegalovirus retinitis in resource-limited settings: a systematic review frequent reactivation of herpes simplex virus among hiv-1-infected patients treated with highly active antiretroviral therapy kaposi sarcoma incidence and survival among hivinfected homosexual men after hiv seroconversion a cutting-edge view on the current state of antiviral drug development the hiv protease inhibitor nelfinavir inhibits kaposi's sarcoma-associated herpesvirus replication in vitro anti-hiv drugs for cancer therapeutics: back to the future? insights into the broad cellular effects of nelfinavir and the hiv protease inhibitors supporting their role in cancer treatment and prevention phase i trial of the human immunodeficiency virus protease inhibitor nelfinavir and chemoradiation for locally advanced pancreatic cancer phase i trial of the combination of the akt inhibitor nelfinavir and chemoradiation for locally advanced rectal cancer a phase i trial of the hiv protease inhibitor nelfinavir with concurrent chemoradiotherapy for unresectable stage iiia/iiib non-small cell lung cancer: a report of toxicities and clinical response phase i study of nelfinavir in liposarcoma nelfinavir, a new anticancer drug with pleiotropic effects and many paths to autophagy drug discovery using chemical systems biology: weak inhibition of multiple kinases may contribute to the anti-cancer effect of nelfinavir the herpes simplex virus 1 gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate a formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy resistance of herpes simplex virus to acycloguanosine: role of viral thymidine kinase and dna polymerase loci nelfinavir, a lead hiv protease inhibitor, is a broad-spectrum, anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo akt inhibitors mk-2206 and nelfinavir overcome mtor inhibitor resistance in diffuse large b-cell lymphoma nelfinavir augments proteasome inhibition by bortezomib in myeloma cells and overcomes bortezomib and carfilzomib resistance pi3k/akt signaling mediated apoptosis blockage and viral gene expression in oral epithelial cells during herpes simplex virus infection nelfinavir inhibits maturation and export of herpes simplex virus 1 nelfinavir inhibits regulated intramembrane proteolysis of sterol regulatory element binding protein-1 and activating transcription factor 6 in castration-resistant prostate cancer identification of endoplasmic reticulum stress-inducing agents by antagonizing autophagy: a new potential strategy for identification of anticancer therapeutics in b-cell malignancies preferential killing of triple-negative breast cancer cells in vitro and in vivo when pharmacological aggravators of endoplasmic reticulum stress are combined with autophagy inhibitors nelfinavir induces the unfolded protein response in ovarian cancer cells, resulting in er vacuolization, cell cycle retardation and apoptosis o-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the golgi apparatus expression of hsv-1 glycoproteins in tunicamycin-treated monkey kidney cells retinoic acid reduces the yield of herpes simplex virus in vero cells and alters the n-glycosylation of viral envelope proteins introduction to glycobiology ncbi bookshelf hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus hiv-protease inhibitors block the replication of both vesicular stomatitis and influenza viruses at an early postentry replication step host cell factors as antiviral targets in arenavirus infection novel hiv-1 therapeutics through targeting altered host cell pathways targeting host factors: a novel rationale for the management of hepatitis c virus potent hostdirected small-molecule inhibitors of myxovirus rna-dependent rna-polymerases role of host cell factors in flavivirus infection: implications for pathogenesis and development of antiviral drugs new prospects for nelfinavir in non-hiv-related diseases hiv-1 protease inhibitors nelfinavir and atazanavir induce malignant glioma death by triggering endoplasmic reticulum stress effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production antiviral activity of tunicamycin on herpes simplex virus the effect of ammonium chloride and tunicamycin on the glycoprotein content and infectivity of herpes simplex virus type 1 infectivity and glycoprotein processing of herpes simplex virus type 1 grown in a ricin-resistant cell line deficient in n-acetylglucosaminyl transferase i autophagy in herpesvirus immune control and immune escape dysregulation of autophagy in murine fibroblasts resistant to hsv-1 infection autophagy is involved in antiviral activity of pentagalloylglucose (pgg) against herpes simplex virus type 1 infection in vitro guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes nelfinavir: a magic bullet to annihilate cancer cells? this work was supported by grants from the nih: kl2 rr025015-01, ul1 rr025014, and university of washington center for aids research p30 ai027757. the authors declare that they have no conflict of interests regarding the publication of this paper. key: cord-009446-8keu2uay authors: kreer, christoph; gruell, henning; mora, thierry; walczak, aleksandra m.; klein, florian title: exploiting b cell receptor analyses to inform on hiv-1 vaccination strategies date: 2020-01-01 journal: vaccines (basel) doi: 10.3390/vaccines8010013 sha: doc_id: 9446 cord_uid: 8keu2uay the human antibody repertoire is generated by the recombination of different gene segments as well as by processes of somatic mutation. together these mechanisms result in a tremendous diversity of antibodies that are able to combat various pathogens including viruses and bacteria, or malignant cells. in this review, we summarize the opportunities and challenges that are associated with the analyses of the b cell receptor repertoire and the antigen-specific b cell response. we will discuss how recent advances have increased our understanding of the antibody response and how repertoire analyses can be exploited to inform on vaccine strategies, particularly against hiv-1. in order to protect from a vast number of different pathogens, human b cells are able to generate a remarkable diversity of different b cell receptors (bcrs). during b cell development and maturation, these receptors are built by recombination and mutation processes resulting in a virtually unlimited number of different antibodies (i.e., soluble bcrs). however, certain pathogens, such as hiv-1, challenge the immune system by the ability to rapidly escape from immune pressure [1, 2] , resulting in an ongoing adaptation of the immune response against these pathogens. this evolutionary arms race between pathogens and the immune system leaves footprints in our immunological memory that can describe developmental pathways towards an adapted immune response. deciphering these pathways holds the potential to greatly improve our understanding of critical steps in lymphocyte receptor development and may inform on novel vaccination strategies. for a long time, experimental setups and bioinformatics pipelines were limited in assessing the diversity of lymphocyte receptor repertoires or in identifying subtle immunological imprints after infections or vaccinations. the advent of advanced single cell cloning and next generation sequencing (ngs) methods has revolutionized the field and opened the door to investigate adaptive immune receptor repertoires (airr) at an unprecedented depth. in this review, we summarize recent developments that have fostered our understanding of b cell biology and antibody responses. focusing on the development of highly potent broadly neutralizing antibodies against hiv-1, we discuss how a detailed knowledge of the human b cell repertoire may support the development of novel vaccination strategies. the initial diversity of the b cell repertoire results from the assembly of the b cell receptor during early b cell development in the bone marrow. the recombination-activating gene (rag) 1/2 enzymes recombine variable (v), diversity (d), and joining (j) gene segments of the immunoglobulin heavy (igh) chain locus to first assemble the heavy chain variable region, followed by v and j gene segment recombination within the ig kappa (igk) and ig lambda (igl) loci [4] . junctional diversity is further increased by rag1/2 and other enzymes through the generation of palindromic (p) nucleotides, as well as by the terminal deoxynuclotidyl transferase (tdt) through the addition of non-template (n) nucleotides [5] (figure 1b ). heavy and light chain v genes exclusively encode for two complementarity determining regions (cdr1 and cdr2) that are usually structurally exposed at the tip of the antibody and contribute to antigen recognition. a third cdr (cdr3) is generated by the v(d)j recombination process and is the most variable part within b cell receptors and antibodies. the cdrs are interspersed and flanked with framework regions (fwr) that mainly function as a scaffold for the overall immunoglobulin (ig) fold. however, mutations in fwrs have been shown to also influence binding affinity as well as neutralizing activity [6, 7] . there are currently at least 56 functional v, 23 d, and 6 j genes described for the igh locus [8] , which results in a theoretical combinatorial number of 7728 different heavy chain variable regions. the igk locus can recombine to 205 different kappa light chain variable regions from 41 v and 5 j genes, whereas the igl locus might recombine up to 165 different lambda light chains from 33 v and 5 j genes [8] . combinatorial pairing of heavy and light chains yields a theoretical diversity of about 2.9 × 10 6 different antibodies. including the p/n nucleotides, the theoretical number of different antibody sequences is vastly higher than the total number of estimated b cells in the human body (10 12 ) [9] . however, all those antibody sequences are not equally likely to be generated. their generation probability spans 30 orders of magnitude for igh alone [9, 10] , with additional diversity being generated by insertions and deletions [11] . of note, due to sampling issues, the number of b cell clones whose size falls below the detection threshold is unknown, rendering estimates of total b cell counts unreliable [12, 13] . naïve b cells circulate between secondary lymphoid tissues (e.g., lymph nodes and spleen) until they recognize their cognate antigen [14] . upon antigen contact, b cells can be recruited to lymphatic structures called germinal centers present in secondary lymphoid tissues. there, the recognition event is able to trigger a second step of diversification called affinity maturation. affinity maturation is mediated by the enzyme activation-induced deaminase (aid) as well as b cell expansion and selection [15] . aid introduces shm including substitutions, insertions, and deletions into the variable regions, generating possible progenies that express b cell receptors that get selected for higher antigen affinity ( figure 1b) [15, 16] . shm is favored but not limited to hot spot motifs and multiple shm hotspots have been identified [17, 18] . in addition to their context preference, shms tend to occur close to each other along the sequence [10] . they are typically more pronounced in the cdrs than in the framework regions due to positive selection as well as higher frequency of aid motifs [18] . including shm into the calculation of potential bcrs results in an almost infinite number of different receptors. finally, aid activity is able to mediate heavy chain class switch from igm/igd to igg, iga, or ige. different b cell subtypes and antibody classes are of critical importance and their functions have been reviewed elsewhere [3, 14, 19] . dissecting the humoral immune response is a challenging task. analyses of antibodies on the serum level, for example by elisa, affinity chromatography, or mass spectrometry [20] , are usually limited to characterizing the polyclonal antibody response. genetic b cell analyses, on the other side, facilitate single cell (i.e., single antibody) resolution. to this end, antibody-coding nucleic acids (dna or rna) are extracted from b cells, amplified, and sequenced ( figure 2 ). importantly, complete sequences of matched heavy and light chains allow for recombinant production of antibodies and thus allow studying antibody functions on a monoclonal level ( figure 2 ). in this section, we describe the challenges arising at the different steps of genetic bcr analyses and discuss advantages and disadvantages of individual strategies. section, we describe the challenges arising at the different steps of genetic bcr analyses and discuss b cells can be subdivided into different subsets. these comprise (i) b cells at different developmental stages (e.g., pro-b cells, immature b cells, mature b cells), (ii) antigen-naive and antigen-experienced b cells, (iii) functional subsets, such as regulatory, effector, or memory b cells, or iv.) b cells with defined specificity (e.g., hiv-1 env -reactive). depending on the scientific question, it is often required to analyze an individual b cell subset and identification of such subsets can take place at distinct steps of an experimental pipeline ( figure 3 , first row). different b cell subsets can be enriched or isolated by sorting techniques such as magnetic-activated cell sorting (macs) or fluorescence-activated cell sorting (facs) [21, 22] . importantly, facs allows collecting and further processing target cells either in bulk approaches or as single cells in multi-well plates. recently, novel microencapsulation systems have been used to encapsulate single b cells into picoliter droplets [23, 24] . the combination of single cell encapsulation with fluorescence-activated sorting (fluorescence-activated droplet sorting (fads), reviewed in [25] ) allows processing of single antigen-specific b cells in compartments that are a million times smaller than the wells of multi-well plates, which significantly increases the throughput capacity. in order to isolate antigen-specific b cells, one of the following approaches (reviewed in [26, 27] ) can be applied ( figure 3 , first row): (i) antigen-derived baits that are fluorescently labeled can be used to identify and sort antigen-reactive b cells. this can be achieved by fluorescently-labeled proteins [28] [29] [30] [31] [32] [33] [34] , antigens presented on virus-like particles [35, 36] or cells [37] , or by pathogens themselves [38] . (ii) antibody libraries that are expressed on phages or yeast cells can be selected for binding to antigen-derived proteins or whole pathogens [39, 40] . of note, however, the random pairing of heavy and light chains in combinatorial libraries does not allow to infer a representative picture of the underlying antibody response. (iii) single b or plasma cells or immortalized b cells can be expanded and stimulated to secrete antibodies that can be tested for antigen-binding or neutralizing activity [41] [42] [43] [44] . importantly, recombinant proteins and other baits that are used for selecting antigen-specific antibodies can critically differ in their structure and glycosylation pattern from their native counterpart. indeed, the generation of optimized bait proteins [30] or native-like envelope trimers [45] were critical steps to improve the isolation of potent hiv-1 broadly neutralizing antibodies (bnabs) by antigen-specific sorting strategies [31, [46] [47] [48] . very recently, a combination of single cell co-encapsulation and dna-tagged recombinant proteins has even been used to directly map antibody sequences to their antigen specificity [49] . direct heavy or light chain rt-pcr and sequence analyses from bulk-sorted b cells allow to infer b cell repertoire characteristics such as clonal distributions, v(d)j recombination, and somatic hypermutation [50] [51] [52] [53] [54] [55] . however, the native pairing information of heavy and light chains is essential to fully describe an individual antibody, e.g., for recombinant expression. although pairing information can be restored to some extent from bulk analyses (i.e., by bioinformatic approaches) [56] , the most robust way to achieve these information is either by single cell sorting into multi-well plates [57] [58] [59] [60] or by co-encapsulation of single cells and rna-capture or barcode beads (e.g., with the 10× genomics chromium system) in picoliter droplets [23, 24, [61] [62] [63] [64] [65] (figure 3 , second row). single cell sorting into multiwell plates is typically limited in throughput to tens of thousands of cells, whereas encapsulation systems allow throughputs of hundreds of thousands of cells. however, droplet occupancy follows a poisson distribution and requires limiting dilutions of the sample and beads [61] . as a consequence, the majority of all droplets remains empty or contains only unpaired cells or beads, which can lead to a high loss of input material. alternatives for heavy and light chain paring comprise combinatorial yeast or phage display libraries that can be screened for reactivity (reviewed in [66] ). however, due to their stochastic nature, such display approaches also contain artificial heavy and light chain pairs. all current approaches require an initial pcr-based amplification of the bcr-encoding dna or mrna/cdna. the diversity of the b cell repertoire poses distinct challenges to this amplification step: (i) all possible v gene segments need to be covered by the pcr and (ii) priming sites may have been somatically hypermutated and are therefore prone to decreased amplification efficiencies (figure 3 , third row). pcr amplification of antibody heavy and light chains has thus been performed with v gene-specific multiplex primer mixes [57, 58, [67] [68] [69] [70] [71] . the majority of these primers were designed against the 5 end of the coding region of the v gene, which is sufficient for amplifying most antibody sequences. however, hiv-1 bnabs, for instance, have been shown to accumulate high levels of shm as well as insertions and deletions [7, 30, 72, 73] . in order to increase priming efficiency, primer mixes have been designed against the 5 end of the less-mutated leader region that encodes the antibody secretion peptide. these mixes have been demonstrated to be superior for the isolation of highly mutated hiv-1-reactive antibodies [72, 74] . whereas primer sets perform well in single cell cloning approaches, they may introduce primer biases in bulk pcr amplification approaches [75] . this can pose a critical disadvantage. a method that is able to overcome this limitation is the rapid amplification of 5 cdna ends (5 race) [76] , which has been adapted to bulk [77] and single cell approaches [60, 78] . commonly applied protocols include template-switching (ts) reverse transcription, which introduces a ts-oligo during cdna synthesis. the ts-oligo bears a universal priming site that can be used together with a constant region reverse primer to amplify any antibody variable region independent of the incorporated v gene segment. sequence analysis of amplified heavy and light chains can be demanding in several aspects ( figure 3 , fourth row). first, depending on the b cell subset of interest, the required throughput can vary from a few hundred to millions of antibody sequences. second, the sequencing method needs to reliably cover the whole region of interest (variable region~500-600 bp). third, shm needs to be distinguishable from sequencing errors, thus requiring low sequencing error rates or error-correction techniques. classical sanger sequencing is frequently employed in the analyses of b cell receptor subsets such as antigen-specific memory b cells in the blood [34, 79, 80] . due to the inability to sequence bulk-amplified heavy and light chains, next generation sequencing (ngs) techniques such as pyro-, ion-semiconductor-, or illumina dye sequencing are typically preferred over sanger sequencing for high-throughput analyses [50] [51] [52] [53] [54] [55] . however, they often suffer from shorter read lengths and higher error rates. molecular barcoding techniques and bioinformatics pipelines have been developed to account for both pcr-and sequencing-induced errors [81] . to this end, unique molecular identifiers (umi) are introduced during cdna generation by template-switching reverse transcription. moreover, protocols for long read parallel sequencing (e.g., smrt and nanopore sequencing) have been recently applied to analyze bcr sequences [63, 82] . finally, high-throughput analyses of millions of different antibody sequences require advanced bioinformatics pipelines. several bioinformatics tools have been reported [83] and standardized protocols on reporting antibody sequences have been developed by the adaptive immune receptor repertoire (airr) community [84, 85] . a detailed description of the methods applied is beyond the scope of this review and they are covered in previous reviews [83, 86] . isolated highly potent bnabs can serve as templates for the development of such strategies. over the last decade, numerous bnabs have been identified by bait-specific single cell sorts or b cell 230 microcultures. most of these antibodies were obtained from the memory b cell pool as well as, occasionally, from plasma cells [72, 87, 88] . this suggests that both, memory and antigen-secreting b only a small fraction of hiv-1-infected individuals develop highly potent bnabs and detailed analyses of b cell receptors and antibodies at a single cell level have been limited to a few dozen subjects. nevertheless, these critical investigations have revealed sequence and structural characteristics of potent hiv-1 neutralizing antibodies that were repeatedly observed across different individuals. these features can include high levels of somatic hypermutation, the presence of unusual insertions or deletions, and/or long heavy chain cdr3 (cdrh3) regions. thus, vaccine-mediated hiv-1 bnab induction may require specifically tailored strategies for b cell activation and maturation. isolated highly potent bnabs can serve as templates for the development of such strategies. over the last decade, numerous bnabs have been identified by bait-specific single cell sorts or b cell microcultures. most of these antibodies were obtained from the memory b cell pool as well as, occasionally, from plasma cells [72, 87, 88] . this suggests that both, memory and antigen-secreting b cells, can in principle serve as a valuable source for bnab isolation and characterization. all hiv-1 bnabs target epitopes on the hiv-1 envelope protein (env) that include the cd4 binding site (cd4bs), glycan-dependent targets on the variable env loops (v1/v2, v3), the fusion peptide and the membrane-proximal external region (mper) of gp41, and sites spanning the gp120 and gp41 subunits [27, 89] . in clinical trials, bnabs have been shown to suppress viremia and delay the time to viral rebound after interruption of antiretroviral therapy (art) [90] [91] [92] [93] [94] [95] [96] . of most relevance for hiv-1 vaccine efforts, however, bnabs are highly effective in preventing infection in animal models [97] [98] [99] [100] . as proof-of-concept trials for passive immunization using the cd4bs bnab vrc01 in humans are ongoing (clinicaltrials.gov: nct02716675, nct02568215), it is widely believed that induction of potent bnabs by vaccination will confer protection from hiv-1. among the highly potent hiv-1 bnabs, antibodies of the vrc01-and 8anc131-classes target the cd4 binding site (cd4bs) on the hiv-1 env. they are particularly noteworthy for their restricted v gene usage [30, 72, 101] facilitating the vh1-2 or vh1-46 gene segments. importantly, members of the potent vrc01-class of bnabs have now been identified in at least 12 individuals, demonstrating their capacity to be reproducibly induced [30, 72, 88, [101] [102] [103] [104] [105] [106] . such a reproducible development of very similar antibodies in different individuals is often referred to as convergent or stereotypical antibody responses or described as "public antibodies". identifying convergent immune responses is informative for vaccine design because strategies that induce such a repeatedly observed type of immune reaction may be broadly applicable on a population-level. indeed, b cell analyses have revealed convergent v gene responses not only against hiv-1 but several pathogens after infection and/or vaccination (table 1) . vrc01-class cd4bs bnabs demonstrate the same mode of cd4bs recognition that is dominated by the cdr2 of the heavy chain (cdrh2) [101] . to avoid steric clashes, they share an additional restriction for use of an unusually short (five amino acids) light chain cdr3 (cdrl3), which is found in only~1% of antibodies [102, 128] . finally, they show extensive levels of somatic hypermutation of up to >30% on the nucleotide level (i.e., >100 mutations) from their inferred antibody germline sequences [30, 72, 102, 104] . members of hiv-1 bnab classes targeting other epitopes are generally less restricted in terms of their v gene usage but often share other sequence and structural characteristics. for example, bnabs binding to the v1/v2 apex region typically carry cdrh3s of extraordinary length that are required to penetrate the extensive env glycan shield [129, 130] . compared to the average cdrh3 lengths of approximately 15 amino acids in the naïve and memory b cell receptor repertoires [131] , v1/v2-targeting bnabs have been identified that have >2-fold longer cdrh3s (e.g., vrc26.25 and pgdm1400 with cdrh3 lengths of ≥34 aa [31, 48] ). similarly, relatively long cdrh3s are also found in hiv-1 bnabs targeting other glycan-related epitopes (e.g., v3 loop, gp120/gp41 interface) or the gp41 mper [89] . in addition, some bnabs display poly-and/or autoreactivity [132, 133] , features that are often associated with long cdrh3s and are generally counterselected during b cell maturation [57] . overall, the consistent observation of one or multiple rare features in potent hiv-1 bnabs highlights some of the difficulties for their induction through vaccination. however, several antibodies with considerable breadth and potency but lower levels of somatic hypermutation and more regular cdrh3 lengths have now been identified [36, 105, 134, 135] . these antibodies may be more readily inducible and serve as blueprints for facilitating vaccine strategies. due to the unusual sequence and structural characteristics of most highly potent hiv-1 bnabs, unconventional approaches to vaccination are likely to be required. strategies that have been proposed include epitope-based and antibody lineage-based vaccine designs [136] . epitope-based vaccination strategies use immunogens that mimic the general structure of vulnerable env sites, in principle allowing for the development of multiple bnab classes against the same target region. however, when reverted to their inferred germline sequence, many hiv-1 bnabs show considerably reduced or fully abrogated binding to hiv-1 env [72, [137] [138] [139] . to this end, antibody-lineage based vaccination strategies employ designed immunogens that interact with inferred unmutated bnab precursors to initiate the development of a particular bnab lineage [140] . although bnab precursor cell frequency in the repertoire is only one of a number of factors that will determine the potential success of lineage-based vaccine design, a comprehensive understanding of the composition of the b cell receptor repertoire in healthy individuals can provide critical information to guide the development of vaccination pathways [141] . as seen for the majority of antibodies, most potent hiv-1 bnabs are strongly dependent on interactions mediated by the cdr3 of the heavy chain. compared to the distribution in the overall memory b cell repertoire, many hiv-1 bnabs have relatively long cdrh3s which have been suggested to be largely generated during vdj recombination [142] . particularly long cdrh3s are required for many bnabs targeting the v1/v2 apex region of env. notably, among näive b cell repertoires of healthy individuals, cdrh3 lengths of 28 amino acids and more have been identified in less than 0.5% of sequences [142] , and a cdrh3 length of 30 amino acids as seen for bnab pg9 was found to be exceedingly rare (0.01%) [143] . although this suggests difficulties for cdrh3-based hiv-1 vaccination, the potential contribution of bcr repertoire analyses to vaccine design was recently demonstrated when precursor frequencies of the cdrh3-dominated bnab bg18 [144, 145] were determined to inform on the selection of an immunogen targeting bg18-like precursors [146] . among 1×10 9 cdrh3 sequences from a total of 14 healthy donors, bg18-like sequences were identified in all individuals [55, 146] . importantly, rare immunogen-reactive b cells could subsequently be isolated from additional healthy donors [146] . moreover, antibody lineage-based vaccination strategies that aim to engage precursors of the vrc01-class of cd4bs bnabs have entered the clinical stage with the germline-targeting immunogen eod-gt8 (clinicaltrials.gov: nct03547245) [147] . as mentioned before, antibodies of this class are particularly restricted for usage of the vh1-2*02 allele and a 5 amino acid cdrl3 [101] . bcr repertoire analyses revealed that potential vrc01-class precursor b cells are exceptionally rare [147] [148] [149] . in addition, allelic variation can result in the lack of naïve b cells derived from the key vh1-2*02 allele [149] . nevertheless, eod-gt8-reactive naïve b cells could be identified in a majority of hiv-1-negative donors (14/18) [147, 148] , providing repertoire analysis-based support for advancing the eod-gt8 immunogen to be evaluated in a clinical setting. while germline-targeting immunogens are designed to initiate a particular b cell lineage, subsequent immunizations with additional antigens will likely be required to induce the development of broad and potent mature antibodies through additional rounds of affinity maturation [140] . to this end, interrogations of the natural development of bnabs in hiv-1 elite neutralizers may be highly informative for immunogen design. high-throughput parallel sequencing methods of the b cell receptor repertoire combined with bioinformatical processing and phylogenetic analyses have facilitated to reconstruct the inferred development of antibody lineages [56, [102] [103] [104] [105] 135, 139, [150] [151] [152] [153] [154] [155] [156] [157] [158] . of note, when informed by template sequences of bnabs obtained through single cell approaches, high-throughput bcr sequencing methods can identify antibodies with higher breadth and potency [159] . of particular relevance for vaccine design, longitudinal studies that investigate the co-evolution of hiv-1 and the neutralizing antibody response in single individuals may provide guidance for the design of antigens driving bnab potency and breadth. for example, several studies revealed that development of broad neutralization was preceded by viral diversification and/or supported by antibody helper lineages that selected for viral variants that drove bnab development [135, 152, 154, [156] [157] [158] 160, 161] . while these observations support a stepwise immunization approach, "dead-end" limbs of antibody lineages appear during the affinity maturation process. therefore, sequential immunogens will need to be carefully selected [48, 135, 156, 157] . besides the lineage specific analyses, high-throughput ngs approaches have also been used to investigate the whole b cell receptor repertoire of hiv-1-infected individuals either from combinatorial libraries [162] [163] [164] or from pbmcs or purified b cells [75, 154, [165] [166] [167] [168] . a recent study by waltari et al. detected slight shifts in v gene family usage, higher degrees of somatic hypermutation, and longer cdrh3s for hiv-1-infected individuals [167] . however, other studies could not find any differences but report variation within healthy or hiv-1-infected individuals to be as large as between the different cohorts [75, 166] . the current sampling and sequencing depths might therefore still hamper the identification of hiv-1 infection-induced changes on the b cell receptor repertoire. only a small fraction of hiv-1-infected individuals is able to mount a broadly neutralizing serum activity against hiv-1. over the last decade, advances in screening methods and single cell cloning techniques enabled the isolation of numerous broadly neutralizing antibodies. these antibodies have been shown to be promising candidates for hiv-1 treatment and prevention. however, molecular analyses also revealed special characteristics such as v gene restriction, long cdrh3s, and/or high loads of shm, which may restrict the development of highly potent bnabs in natural infection and hamper their induction by current vaccination strategies. to overcome potential roadblocks for the induction of bnabs through vaccination, a number of strategies have been proposed. all of these, however, will require the interaction of one or multiple immunogens with b cell receptors to effectively drive bnab development. thus, a detailed understanding of the naïve b cell receptor repertoire and the constantly adapting antibody response in the context of hiv-1 infection can be highly informative for vaccine design. novel experimental and bioinformatics pipelines have the capacity to integrate neutralization, antibody sequence, and structural data. these methods hold great promise to identify common pathways of potent immune responses that will be critical for developing effective vaccination strategies. measurement of the mutation rates of animal viruses: influenza a virus and poliovirus type 1 identification and characterization of conserved and variable regions in the envelope gene of htlv-iii/lav, the retrovirus of aids igg subclasses and allotypes: from structure to effector functions mechanisms of programmed dna lesions and genomic instability in the immune system lack of n regions in antigen receptor variable region genes of tdt-deficient lymphocytes hierarchy and extremes in selections from pools of randomized proteins somatic mutations of the immunoglobulin framework are generally required for broad and potent hiv-1 neutralization imgt, the international immunogenetics database olga: fast computation of generation probabilities of b-and t-cell receptor amino acid sequences and motifs high-throughput immune repertoire analysis with igor inferring processes underlying b-cell repertoire diversity genetic measurement of memory b-cell recall using antibody repertoire sequencing how many different clonotypes do immune repertoires contain? structure and function of immunoglobulins germinal centers immunoglobulin somatic hypermutation models of somatic hypermutation targeting and substitution based on synonymous mutations from high-throughput immunoglobulin sequencing data beyond hot spots: biases in antibody somatic hypermutation and implications for vaccine design how they develop and function sequencing and quantifying igg fragments and antigen-binding regions by mass spectrometry fundamentals and application of magnetic particles in cell isolation and enrichment: a review flow cytometry and cell sorting single-cell deep phenotyping of igg-secreting cells for high-resolution immune monitoring single-cell droplet microfluidic screening for antibodies specifically binding to target cells active droplet sorting in microfluidics: a review tools to therapeutically harness the human antibody response passive immunotherapy of viral infections: 'super-antibodies' enter the fray a method for identification of hiv gp140 binding memory b cells in human blood broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1. science recombinant hiv envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex vaccine-induced antibodies that neutralize group 1 and group 2 influenza a viruses recurrent potent human neutralizing antibodies to zika virus in brazil and mexico polyclonal and convergent antibody response to ebola virus vaccine rvsv-zebov pseudovirion particles bearing native hiv envelope trimers facilitate a novel method for generating human neutralizing monoclonal antibodies against hiv virus-like particles identify an hiv v1v2 apex-binding neutralizing antibody that lacks a protruding loop broad neutralization by a combination of antibodies recognizing the cd4 binding site and a new conformational epitope on the hiv-1 envelope protein fluorescently labeled dengue viruses as probes to identify antigen-specific memory b cells by multiparametric flow cytometry a large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals efficient recovery of high-affinity antibodies from a single-chain fab yeast display library an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis a next-generation cleaved, soluble hiv-1 env trimer, bg505 sosip.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies an hiv-1 antibody from an elite neutralizer implicates the fusion peptide as a site of vulnerability fusion peptide of hiv-1 as a site of vulnerability to neutralizing antibody new member of the v1v2-directed cap256-vrc26 lineage that shows increased breadth and exceptional potency high-throughput mapping of b cell receptor sequences to antigen specificity measurement and clinical monitoring of human lymphocyte clonality by massively parallel vdj pyrosequencing high-throughput immunoglobulin repertoire analysis distinguishes between human igm memory and switched memory b-cell populations naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation high frequency of shared clonotypes in human b cell receptor repertoires shaping of infant b cell receptor repertoires by environmental factors and infectious disease commonality despite exceptional diversity in the baseline human antibody repertoire mining the antibodyome for hiv-1-neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains predominant autoantibody production by early human b cell precursors efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning single-cell based high-throughput sequencing of full-length immunoglobulin heavy and light chain genes high-throughput sequencing of natively paired antibody chains provides evidence for original antigenic sin shaping the antibody response to influenza vaccination single-cell analysis and sorting using droplet-based microfluidics ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes high-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes tumor-infiltrating immune repertoires captured by single-cell barcoding in emulsion massively parallel single-cell b-cell receptor sequencing enables rapid discovery of diverse antigen-reactive antibodies beyond natural antibodies: the power of in vitro display technologies rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction tracing b cell development in human germinal centres by molecular analysis of single cells picked from histological sections a definitive set of oligonucleotide primers for amplifying human v regions v-gene amplification revisited-an optimised procedure for amplification of rearranged human antibody genes of different isotypes antibody repertoires in humanized nod-scid-il2rgamma(null) mice and human b cells reveals human-like diversification and tolerance checkpoints in the mouse sequence and structural convergence of broad and potent hiv antibodies that mimic cd4 binding antibodies in hiv-1 vaccine development and therapy openprimer for multiplex amplification of highly diverse templates toward a more accurate view of human b-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding rapid production of full-length cdnas from rare transcripts: amplification using a single gene-specific oligonucleotide primer construction of representative immunoglobulin variable region cdna libraries from human peripheral blood lymphocytes without in vitro stimulation amplification and analysis of cdna generated from a single cell by 5 -race: application to isolation of antibody heavy and light chain variable gene sequences from single b cells cross-specificity of protective human antibodies against klebsiella pneumoniae lps o-antigen repertoire and neutralizing activity of antibodies against hepatitis c virus e2 peptide in patients with spontaneous resolution of hepatitis bioinformatic and statistical analysis of adaptive immune repertoires in-depth determination and analysis of the human paired heavy-and light-chain antibody repertoire ireceptor: a platform for querying and analyzing antibody/b-cell and t-cell receptor repertoire data across federated repositories computational strategies for dissecting the high-dimensional complexity of adaptive immune repertoires potent and broad hiv-neutralizing antibodies in memory b cells and plasma identification of near-pan-neutralizing antibodies against hiv-1 by deconvolution of plasma humoral responses recent progress in broadly neutralizing antibodies to hiv virologic effects of broadly neutralizing antibody vrc01 administration during chronic hiv-1 infection viraemia suppressed in hiv-1-infected humans by broadly neutralizing antibody 3bnc117 effect of hiv antibody vrc01 on viral rebound after treatment interruption hiv-1 antibody 3bnc117 suppresses viral rebound in humans during treatment interruption antibody 10-1074 suppresses viremia in hiv-1-infected individuals safety and antiviral activity of combination hiv-1 broadly neutralizing antibodies in viremic individuals combination therapy with anti-hiv-1 antibodies maintains viral suppression highly potent hiv-specific antibody neutralization in vitro translates into effective protection against mucosal shiv challenge in vivo passive transfer of modest titers of potent and broadly neutralizing anti-hiv monoclonal antibodies block shiv infection in macaques a single injection of anti-hiv-1 antibodies protects against repeated shiv challenges broadly neutralizing antibodies targeting the hiv-1 envelope v2 apex confer protection against a clade c shiv challenge structural repertoire of hiv-1-neutralizing antibodies targeting the cd4 supersite in 14 donors multidonor analysis reveals structural elements, genetic determinants, and maturation pathway for hiv-1 neutralization by vrc01-class antibodies focused evolution of hiv-1 neutralizing antibodies revealed by structures and deep sequencing identification of a cd4-binding-site antibody to hiv that evolved near-pan neutralization breadth rapid and focused maturation of a vrc01-class hiv broadly neutralizing antibody lineage involves both binding and accommodation of the n276-glycan delineating antibody recognition in polyclonal sera from patterns of hiv-1 isolate neutralization lower igg somatic hypermutation rates during acute dengue virus infection is compatible with a germinal center-independent b cell response b cell gene signature with massive intrahepatic production of antibodies to hepatitis b core antigen in hepatitis b virus-associated acute liver failure infant and adult human b cell responses to rotavirus share common immunodominant variable gene repertoires vh1-46 is the dominant immunoglobulin heavy chain gene segment in rotavirus-specific memory b cells expressing the intestinal homing receptor alpha4beta7 immunodominance of the vh1-46 antibody gene segment in the primary repertoire of human rotavirus-specific b cells is reduced in the memory compartment through somatic mutation of nondominant clones ability to develop broadly neutralizing hiv-1 antibodies is not restricted by the germline ig gene repertoire structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine rapid development of broadly influenza neutralizing antibodies through redundant mutations electrofusion and epstein-barr virus transformation for peripheral blood lymphocyte immortalization b-cell repertoire dynamics after sequential hepatitis b vaccination and evidence for cross-reactive b-cell activation human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements therapeutic monoclonal antibodies for ebola virus infection derived from vaccinated humans isolation of potent neutralizing antibodies from a survivor of the 2014 ebola virus outbreak molecular ontogeny of the human antibody repertoire to the haemophilus influenzae type b polysaccharide: expression of canonical variable regions and their variants in vaccinated infants germline v-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus structures of preferred human igv genes-based protective antibodies identify how conserved residues contact diverse antigens and assign source of specificity to cdr3 loop variation cross-reactivity, and function of antibodies elicited by zika virus infection neutralizing human antibodies prevent zika virus replication and fetal disease in mice neutralization of zika virus by germline-like human monoclonal antibodies targeting cryptic epitopes on envelope domain iii preferential use of the vh5-51 gene segment by the human immune response to code for antibodies against the v3 domain of hiv-1 structural basis for germ-line gene usage of a potent class of antibodies targeting the cd4-binding site of hiv-1 gp120 structure of hiv-1 gp120 v1/v2 domain with broadly neutralizing antibody pg9 a broadly neutralizing antibody targets the dynamic hiv envelope trimer apex via a long, rigidified, and anionic beta-hairpin structure large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv-1 antibodies hiv-1 neutralizing antibodies: understanding nature's pathways hiv-1 neutralizing antibodies with limited hypermutation from an infant early antibody lineage diversification and independent limb maturation lead to broad hiv-1 neutralization targeting the env high-mannose patch hiv-1 vaccines based on antibody identification, b cell ontogeny, and epitope structure recombinant hiv envelope proteins fail to engage germline versions of anti-cd4bs bnabs structural basis for broad and potent neutralization of hiv-1 by antibody vrc01. science the effects of somatic hypermutation on neutralization and binding in the pgt121 family of broadly neutralizing hiv antibodies b-cell-lineage immunogen design in vaccine development with hiv-1 as a case study when designing vaccines, consider the starting material: the human b cell repertoire human peripheral blood antibodies with long hcdr3s are established primarily at original recombination using a limited subset of germline genes long antibody hcdr3s from hiv-naive donors presented on a pg9 neutralizing antibody background mediate hiv neutralization coexistence of potent hiv-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller structural characterization of a highly-potent v3-glycan broadly neutralizing antibody bound to natively-glycosylated hiv-1 envelope a generalized hiv vaccine design strategy for priming of broadly neutralizing antibody responses hiv-1 broadly neutralizing antibody precursor b cells revealed by germline-targeting immunogen the human naive b cell repertoire contains distinct subclasses for a germline-targeting hiv-1 vaccine immunogen differences in allelic frequency and cdrh3 region limit the engagement of hiv env immunogens by putative vrc01 neutralizing antibody precursors somatic populations of pgt135-137 hiv-1-neutralizing antibodies identified by de novo identification of vrc01 class hiv-1-neutralizing antibodies by next-generation sequencing of b-cell transcripts developmental pathway for potent v1v2-directed hiv-neutralizing antibodies maturation and diversity of the vrc01-antibody lineage over 15 years of chronic hiv-1 infection maturation pathway from germline to broad hiv-1 neutralizer of a cd4-mimic antibody of the mper-directed hiv-1-neutralizing antibody 10e8 viral variants that initiate and drive maturation of v1v2-directed hiv-1 broadly neutralizing antibodies hiv envelope glycoform heterogeneity and localized diversity govern the initiation and maturation of a v2 apex broadly neutralizing antibody lineage staged induction of hiv-1 glycan-dependent broadly neutralizing antibodies enhanced potency of a broadly neutralizing hiv-1 antibody in vitro improves protection against lentiviral infection in vivo co-evolution of a broadly neutralizing hiv-1 antibody and founder virus cooperation of b cell lineages in induction of hiv-1-broadly neutralizing antibodies characterization of human igg repertoires in an acute hiv-1 infection deep sequencing and circos analyses of antibody libraries reveal antigen-driven selection of ig vh genes during hiv-1 infection the potential of the human immune system to develop broadly neutralizing hiv-1 antibodies: implications for vaccine development dynamics of immunoglobulin sequence diversity in hiv-1 infected individuals 5 rapid amplification of cdna ends and illumina miseq reveals b cell receptor features in healthy adults, adults with chronic hiv-1 infection, cord blood, and humanized mice multi-donor longitudinal antibody repertoire sequencing reveals the existence of public antibody clonotypes in hiv-1 infection we thank all members of the klein lab for helpful discussions. the authors declare no conflict of interest. key: cord-003715-deqiets2 authors: warren, cody j.; meyerson, nicholas r.; dirasantha, obaiah; feldman, emily r.; wilkerson, gregory k.; sawyer, sara l. title: selective use of primate cd4 receptors by hiv-1 date: 2019-06-10 journal: plos biol doi: 10.1371/journal.pbio.3000304 sha: doc_id: 3715 cord_uid: deqiets2 individuals chronically infected with hiv-1 harbor complex viral populations within their bloodstreams. recently, it has come to light that when these people infect others, the new infection is typically established by only one or a small number of virions from within this complex viral swarm. an important goal is to characterize the biological properties of hiv-1 virions that seed and exist early in new human infections because these are potentially the only viruses against which a prophylactic hiv-1 vaccine would need to elicit protection. this includes understanding how the envelope (env) protein of these virions interacts with the t-cell receptor cd4, which supports attachment and entry of hiv-1 into target cells. we examined early hiv-1 isolates for their ability to infect cells via the cd4 receptor of 15 different primate species. primates were the original source of hiv-1 and now serve as valuable animal models for studying hiv-1. we find that most primary isolates of hiv-1 from the blood, including early isolates, are highly selective and enter cells through some primate cd4 receptor orthologs but not others. this phenotype is remarkably consistent, regardless of route of transmission, viral subtype, or time of isolation post infection. we show that the weak cd4 binding affinity of blood-derived hiv-1 isolates is what makes them sensitive to the small sequence differences in cd4 from one primate species to the next. to substantiate this, we engineered an early hiv-1 env to have high, medium, or low binding affinity to cd4, and we show that it loses the ability to enter cells via the cd4 receptor of many primate species as the binding affinity gets weaker. based on the phenotype of selective use of primate cd4, we find that weak cd4 binding appears to be a nearly universal property of hiv-1 circulating in the bloodstream. therefore, weak binding to cd4 must be a selected and important property in the biology of hiv-1 in the body. we identify six primate species that encode cd4 receptors that fully support the entry of early hiv-1 isolates despite their low binding affinity for cd4. these findings will help inform long-standing efforts to model hiv-1 transmission and early disease in primates. introduction compatibility with primate cd4 receptors might be dynamic, given that cd4 is highly diverged in its protein sequence at the interaction interface with hiv-1 env (fig 1a) . we began by cloning the cd4 genes of 15 primate species, plus human cd4. using retroviral transduction, we stably introduced these genes into cf2th cells (dog thymocytes) that had also been engineered to express the human c-c motif chemokine receptor 5 (ccr5) coreceptor for hiv-1 entry (s1 fig). expression levels of these cd4 receptors on cf2th cells were similar to endogenous expression levels of cd4 seen on human immortalized t cells and on primate primary t cells that we isolated directly from blood (s1 fig). our goal was then to infect these cells with an early hiv-1 isolate. for the sake of this study, we refer to all viruses or env clones that were isolated <150 days post infection as "early" and those isolated >150 days post infection as "chronic" isolates. this nomenclature is consistent with the fiebig scale classification, for which fiebig stages i-v (typically lasting until 70 to 150 days post infection) capture early infection stages and fiebig stage vi indicates progression into chronic infection [34, 35] . this is also consistent with other studies on transmitted or early hiv-1, which have used viruses isolated from human blood weeks to months after infection [5, 9, 31, 36, 37] . the properties of all of the hiv-1 env isolates used in this study are summarized in s1 table. the hiv-1 envelope clone "bg505" has risen to prominence in the study of early hiv-1. the overbaugh group amplified bg505 env from an infant approximately 6 weeks after delivery and showed that this env sequence was nearly identical to the two other env sequences amplified from this baby at the same time point [9] . this indicated that the infection probably started from a single virion and that the captured env sequence closely resembles the env of this transmitted virion. a cocrystal of bg505 env has been solved in complex with cd4 ( fig 1a) [28, [38] [39] [40] , and the alternate folding conformations of bg505 env have been characterized in depth [41] . we prepared hiv-1 pseudotyped with the bg505 env by cotransfecting 293t cells with two plasmids: one expressing the bg505 env, and another expressing an hiv-1 green fluorescent protein (gfp) reporter virus genome (q23δenv-gfp, representing a blood-derived clade a isolate from 1 year after seroconversion [30, 42] ). cell lines expressing human ccr5 and different primate cd4 receptors were then infected with this bg505 virus (similar infection results were obtained in cell lines expressing matched cd4 and ccr5 from each species; s2 fig). forty-eight hours post infection, cells were harvested and analyzed by flow cytometry. samples were first gated for live cells and further gated such that all samples were narrowed to the same log decade of cd4 receptor expression (s1 fig). gfp-positive (i.e., infected) cells were then enumerated within this population. we identified three categories of cd4 receptors that support hiv-1 entry to varying degrees ( fig 1b) . the cd4 receptors of some species (shown with purple bars: white-cheeked gibbon, olive baboon, gelada, black snub-nosed monkey, colobus, and owl monkey) behave similarly to human cd4 when challenged with bg505 hiv-1. these species are not our closest relatives but rather are distributed throughout the primate phylogeny ( fig 1b) . a second class of species (shown with gray bars: chimpanzee, gorilla, orangutan, and sooty mangabey) encodes cd4 receptors that support entry of this virus but at a level that is approximately 2-fold lower than human cd4. a third class of species (macaques, mandrills, sabaeus monkeys, and squirrel monkeys) encodes cd4 receptors that support entry at a level 25-fold or more reduced compared to human cd4. with some of the cd4 receptors in the latter class, infection was reduced by more than 100-fold compared to human cd4. while the chimpanzee cd4 allele tested here has a semipermissive phenotype, we have recently shown that some chimpanzee cd4 alleles are highly resistant to hiv-1 entry [48] . as a control, we determined that infection did not change the surface expression level of human or any primate cd4 receptor (s1 fig) . this allows us to conclude that the observed differences in infection are due to defects in entry and that our gating strategy did not exclude infected cells because they no longer express cd4. from this data, we can conclude that cd4 receptors are functionally variable from one primate species to the next with regards to supporting entry of an early hiv-1 isolate. prior to this, there was only one primate species known to encode a cd4 receptor compatible with early hiv-1 isolates, owl monkeys [33] , but now we can add five additional species to that list: white-cheeked gibbon, olive baboon, gelada, black snub-nosed monkey, and colobus monkey. hiv-1 entry requires engagement of the amino-terminal "d1" domain of the cd4 receptor (residues . this is evidenced by numerous env-cd4 cocrystals [43] [44] [45] and mapping of d1-domain residues that affect interaction with hiv-1 env [43, [46] [47] [48] . for instance, the macaque cd4 receptor can be rendered functional for hiv-1 entry by introducing a single amino acid substitution in the d1 domain (isoleucine "39i" mutated to the human residue, asparagine "39n") [31] . likewise, owl monkey populations circulate cd4 alleles that are both non-functional (39i) and functional (39n) for hiv-1 entry [33] . from the panel of primate cd4s that we have tested, we can now build on this observation that residue 39 in cd4 is particularly important for hiv-1 engagement. in fig 1b, the residues encoded at cd4 position 39 in each species are listed at the tips of the phylogeny, along with the total number of amino acid differences in the d1 domain compared to human cd4. from this, it appears that cd4 must encode an asparagine ("n") at position 39 in order to have any appreciable function as a receptor for bg505 hiv-1. indeed, position 39 in cd4 has experienced positive selection during primate speciation [20, 21] , and this may be because mutations that replace the asparagine protect individuals against infection. however, there are clearly sequence determinants outside of position 39 that also matter because not all receptors with an asparagine at position 39 function equally well. we next sought to determine whether the selective use of only some primate cd4 receptors is a property specific to early hiv-1 isolates. from this point forward, we focused on cd4 receptors from a subset of primate species, in particular the african primate species involved in the zoonotic transmissions of hiv-1 (chimpanzees and gorillas) and hiv-2 (sooty mangabeys) [17] , and the macaque species that serve as the current laboratory model for hiv-1 (expression histograms for these cell lines are shown in s2 fig) . all of these receptors were partially or highly defective for entry mediated by bg505 env (fig 1b) . we next tested envs isolated from the blood of four individuals chronically infected with hiv-1 at time points more than 6 months after the original infection [49] . these envs were pseudotyped onto q23δenv-gfp, as described above. these viruses were then used to infect stable cell lines expressing human ccr5 and cd4 from different primate species. two of the four envs demonstrated the specific use of human cd4 that we have described (fig 2a, left two panels) . the other two demonstrated a weakening of this selective cd4 phenotype but still a preference for human cd4 (fig 2a, right two panels) . this suggests that the phenotype of selective cd4 usage may be retained during later stages of disease and may not be unique to early hiv-1 isolates. to address this further, we looked at how cd4 receptor usage may change over time as infection progresses from early-to late-stages of disease. to test this, we made use of sets of molecular clones that had been amplified by single-genome amplification from patient blood early after infection and then again from the same patient 6 months later [3, 14, 50, 51] . from four such sets of molecular clones, we cloned the rev-env cassettes into a mammalian expression vector, which was then used to pseudotype each env onto q23δenv-gfp, as described above. these viruses were tested for their ability to enter cell lines stably expressing human or primate cd4 receptors, along with human ccr5. we observed no significant difference in receptor usage between the early env (grey bars) and its matched 6-month counterpart (black bars) (fig 2b) . in fact, for each patient, the envs derived from the two different time points (6 months apart) behaved almost identically. infection mediated by all of these envs was reduced in cells expressing chimpanzee or macaque cd4 receptors when compared to human cd4. one exception was seen with virus from patient ch40, whose env did mediate entry somewhat better via chimpanzee and macaque cd4 compared to other envs, although still less well than with human cd4. therefore, selective use of primate cd4 receptors appears to describe many hiv-1 isolates from the blood, including those isolated just after infection (weeks), later at 6 months (fig 2b) , or even during chronic stages of infection (fig 2a) . we next examined the transmission bottleneck directly by testing matched donor-recipient hiv-1 env pairs. hiv-1 was pseudotyped with envs from mother-infant pairs, of which the mother was chronically infected and her baby was newly infected (<6 weeks) [9] . for seven such pairs, we found that mother hiv-1 envs (black bars) were not different from infant hiv-1 envs (grey bars), in that all demonstrated the selective use of human cd4 (fig 2c) . in fact, corresponding mother and baby envs behaved very similarly. when the seven pairs were grouped, no significant differences were noted between maternal (chronic) and infant (early) envs in terms of their ability to infect cells bearing chimpanzee or rhesus macaque cd4 receptors (mann-whitney test, p > 0.05). the tested pairs represented most major hiv-1 group m subtypes, suggesting that selective use of cd4 may describe all or most globally circulating forms of hiv-1. we next verified this by challenging cell lines expressing different cd4 cells stably expressing human ccr5 and human or primate cd4 (x-axis) were infected with q23δenv-gfp bearing (a) chronic envelopes (envs) [49], (b) early (newly infected; grey bars) and chronic (6 months post infection; black bars) env pairs derived from the same patient [3, 14, 50, 51] , shown for one patient in each panel, or (c) envs derived from maternal (chronic; black bars)/infant (newly infected; grey bars) transmission pairs [9] , with one mother-baby pair shown in each panel. the percent cells infected (gfp-positive) was measured by flow cytometry and normalized to the percent infected with human cd4. error bars represent the mean + sem from two independent experiments, each with two to three technical replicates. data associated with this figure can be found in the supplemental data file (s2 data). ccr5, c-c motif chemokine receptor 5; gfp, green fluorescent protein. receptors with early hiv-1 isolates representing each of the four major group m (pandemic) hiv-1 subtypes. these four envs, isolated from patient blood shortly after initial infection [36, 52, 53] , were pseudotyped onto the q23δenv-gfp virus, as described before. while human cd4 supported entry of all of these viruses, primate cd4 orthologs supported levels of hiv-1 entry that ranged from 2-to 58-fold lower ( fig 3a) . collectively, our study has explored a breadth of hiv-1 isolates taken from patient blood, representing all of the major subtypes found globally, and has found that they can all be phenotypically described by their selective use of only certain primate cd4 receptors. next, we wanted to understand more about why blood-derived hiv-1 env isolates demonstrate this selective use of only some primate cd4 receptors. thus far, all of the envs that we have tested are from ccr5-tropic viruses isolated from the blood. there are three types of hiv-1 in the body of an infected person: ccr5 t cell-tropic (the most abundant form in the blood, utilizing the ccr5 coreceptor), cxcr4 t cell-tropic (in the blood but using an alternate coreceptor, c-x-c chemokine receptor type 4 [cxcr4]), and ccr5 macrophage-tropic (rare in the blood, usually found in the central nervous system) [29,54-56]. only the first of these transmits to new individuals, whereas the latter two types arise in special evolutionary niches within the human body during the course of chronic infection and rarely transmit [57] [58] [59] [60] [61] [62] [63] . in this study, we did not consider or test cxcr4-utilizing viruses. we next tested hiv-1 pseudotyped with envs from four macrophage-tropic viruses [31, [64] [65] [66] [67] [68] [69] [70] [71] [72] for their ability to infect cells bearing primate cd4 receptors ( fig 3b) . in stark contrast to the blood-derived isolates tested above (figs 1, 2 and 3a), macrophage-tropic viruses were promiscuous in their use of all cd4 receptors tested. for each primate cd4 tested, the median relative infection for all early hiv-1 envs (fig 3a) was significantly lower than macrophage-tropic envs (fig 3b; p < 0.05; mann-whitney u test). macrophage-tropic hiv-1 env is known to bind cd4 with higher affinity compared to other forms of hiv-1 env [73] [74] [75] . this is a selected property that these virions possess because macrophages have lower densities of cd4 molecules on their surface [74] . based on this, we hypothesized that weak cd4 binding affinity is what makes most blood-derived hiv-1 envs sensitive to small sequence differences in cd4 receptors from different primates. to test this, we first purified a soluble version of the human cd4 receptor for use in neutralization assays (scd4, consisting of the d1 and d2 domains of cd4) (s3 fig) . when scd4 is preincubated with hiv-1, it is known to competitively inhibit virus interaction with the cd4 expressed on the cell surface and block virus entry [76] . our purified human scd4 behaved identically to a commercially available version (s3 fig). hiv-1 pseudotyped with early or macrophage-tropic envs was preincubated with increasing concentrations of scd4 and then used to infect tzm-bl indicator cells (hela cd4/ccr5 cells that express luciferase in response to hiv-1 tat expression). hiv-1 pseudotyped with either of two different early envs (bg505 or ac10.0.29) was neutralized in a dose-dependent manner by human scd4 (fig 3c) . as expected, hiv-1 pseudotyped with either of two macrophage-tropic envs (bal or sf162) was neutralized by human scd4 to an even greater extent, consistent with these viruses having a higher affinity for cd4 ( fig 3d) . the ic 50 values are shown within each panel and are approximately 4-fold higher for early envs than for macrophage-tropic envs. in conclusion, the broadened ability to enter cells through the cd4 receptors encoded by all primates tested ( fig 3a and 3b ) correlates with tighter cd4 binding (fig 3c and 3d) . macrophage-tropic hiv-1 isolates, which are known to bind human cd4 more tightly, are promiscuous in their use of primate cd4 receptors. (a, b) cells stably expressing various primate cd4 receptors (x-axis), along with human ccr5, were infected with q23δenv-gfp pseudotyped with (a) early hiv-1 envelopes (envs) or (b) envs from common macrophagetropic hiv-1 isolates. the percent cells infected (gfp-positive) was measured by flow cytometry and normalized to the percent infected with human cd4. error bars represent the mean + sem from two independent experiments, each with two to three technical replicates. values above error bars represent fold decrease in viral entry relative to human cd4, only indicated for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < 0.05). (c and d) infection of tzm-bl cells with q23δenv-gfp pseudotyped with early (c) or macrophage-tropic (d) envs. each virus was preincubated with increasing concentrations of human soluble cd4 (scd4) as a competitive inhibitor. tzm-bl cells are hela cd4/ccr5 cells that express luciferase in response to hiv-1 tat expression, read in relative light units (rlus). error bars represent the mean + sd from one biological replicate (n = 3 to 4 technical replicates), representative of two independent experiments. error bars are not plotted in instances in which the size of the error bar is less than the symbol size. ic 50 values from the soluble cd4 neutralization assays are shown within each panel, for which the mean and sd values reflect the variation of the ic 50 calculations from two independent experiments. (e and f) same as c and d, only neutralization assays were performed with chimpanzee and rhesus macaque scd4 instead of human scd4. data associated with this figure can be found in the supplemental data file (s3 data). ccr5, c-c motif chemokine receptor 5; gfp, green fluorescent protein. we next wanted to confirm that weak binding to human cd4 receptors results in a loss of binding to primate cd4 receptors. we purified chimpanzee and rhesus macaque scd4 (s3 fig) and tested the ability of these proteins to neutralize different classes of hiv-1. viruses bearing macrophage-tropic envs (bal or sf162) were neutralized by chimpanzee and macaque scd4 (fig 3f) , while viruses bearing envs from early hiv-1 isolates (bg505 or ac10.0.29) were not neutralized to an appreciable degree ( fig 3e) . collectively, these data suggest that most early hiv-1 isolates from the blood, which have lower affinity for human cd4 compared to macrophage-tropic viruses, do not bind well to chimpanzee and macaque cd4. this shows that our entry assay for selective versus promiscuous use of primate cd4s is essentially a readout of cd4 binding affinity. to further test this premise, we next created a panel of envs engineered to have high, medium, or low binding affinity for cd4. bg505 env, the prototypic early env discussed above, was engineered to have two different point mutations, one at position 281 and the other at position 375. these mutations in env have been previously shown to increase binding affinity to cd4, although only one of them has been characterized within the bg505 env background [77, 78] . consistent with these previous reports, the a281v mutation subtly increased binding to human scd4 relative to wild type (demonstrated by increased neutralization by scd4), while the s375y mutation resulted in a more substantial increase ( fig 4a) . while both of these mutations were originally characterized because they increase entry via macaque cd4 [77, 78] , we found that both mutations also improve entry via all of the primate cd4 receptors tested, with increasing improvement as the binding affinity for cd4 increases (left to right in fig 4b) . this pattern of increased binding affinity for human cd4 and increased ability to enter cells via primate cd4s also correlated to increased binding affinity for primate cd4 receptors, as read in a neutralization assay with purified chimpanzee and rhesus macaque scd4 proteins (left to right in fig 4c) . finally, we engineered two additional mutations into hiv-1 bg505 env that have been shown to increase cd4 binding and to increase entry via macaque cd4 [77] . these mutations also make env able to more efficiently mediate entry via all primate cd4s tested, not just that of macaques ( fig 4d) . these data support the general premise that promiscuous use of primate cd4s can be created by mutations in env that increase cd4 binding affinity. further, the promiscuous use of primate cd4 receptors is not just a property of macrophage-tropic viruses but instead may be a common property of viruses engineered or adapted to bind cd4 more tightly. the fact that virtually no blood-derived hiv-1 isolate that we have tested has this phenotype (promiscuous use of primate cd4 receptors) suggests that this phenotype is strongly selected against in the human body. the reasons for this are unknown. we then employed a virus-free approach to confirm that the mechanism of selective use of certain primate cd4 receptors stems from weak cd4 binding, as opposed to other interactions between hiv-1 and cd4 that may occur during the course of an infection. cells expressing hiv-1 env on their surface were mixed with cells expressing human ccr5 and various primate cd4 receptors ( fig 5a) . cellular fusion was assessed using a split luciferase reporter system in which, upon cell-cell fusion, the two halves of luciferase merge and produce a functional enzyme [79] . we used this assay to test the interactions between four different envs and six different cd4 receptors, as shown in fig 5b. in this system, the phenotypes seen in our previous infection assays were largely recapitulated despite the fact that this assay is virus-free and only requires three proteins (env and cd4/ccr5). the early envs mediated fusion most efficiently with cells expressing human cd4, while the macrophage-tropic envs broadly engaged and fused with diverse primate cd4 receptors. this assay, when taken together with the experiments employing purified cd4 described above, reveal that weak binding to cd4 is an early env engineered to have weak, medium, or tight binding to cd4 becomes progressively more promiscuous in its use of primate cd4 receptors. (a-c) amino acid substitutions were introduced into the bg505 envelope (env) at position 281 and 375. the headers at the top describing these mutations pertain to panels a, b, and c. in each column, the indicted env was pseudotyped onto q23δenv-gfp. (a) each of the three viruses was preincubated with increasing concentrations of human soluble cd4 (scd4) and then used to infect tzm-bl cells, which are hela cd4/ccr5 cells that express luciferase in response to hiv-1 infection and tat expression, read in relative light units (rlus). error bars represent the mean + sd from one biological replicate, representative of two independent experiments. error bars are not plotted in instances in which the size of the error bar is less than the symbol size. ic 50 values are listed on each panel, for which the mean and sd values reflect the variation of the ic 50 calculations from two independent experiments. (b) cells stably expressing the indicated cd4s (x-axis) and human ccr5 were infected with each virus. the percent cells infected (gfp-positive) was measured by flow cytometry and normalized to the percent infected with human cd4. error bars represent the mean + sem from two independent experiments, each with three technical replicates. values above error bars represent fold decrease relative to human cd4 for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < 0.05). (c) same as panel a but with chimpanzee and rhesus macaque scd4 instead of human scd4. (d) two additional single point mutations were introduced into the bg505 env, which have also been shown to increase binding affinity for cd4 [77] . these envs were pseudotyped onto q23δenv-gfp and used to infect cells stably expressing the indicated cd4 (top of each graph) and human ccr5. the percent cells infected (gfp-positive) was measured by flow cytometry and normalized to the percent infected with human cd4. error bars represent the mean + sem from two independent experiments, each with two to three technical replicates. values above error bars represent fold increase relative what makes most blood-derived hiv-1 isolates (i.e., those fueling the global pandemic) selective in their use of only certain primate cd4 receptors. interestingly, macaques (both rhesus and pig-tailed) encode a cd4 receptor that is not permissive for entry of most blood-derived hiv-1 isolates (figs 1, 2, 3 and 4) , consistent with previous reports [30, 77, 78] . since macaques (predominantly rhesus macaques) serve as the current animal model for hiv-1, we sought to determine whether some allelic variants of rhesus macaque cd4 might encode receptors that better support hiv-1 infection. we sequenced the cd4 gene from 52 captive indian-origin rhesus macaques and identified alleles encoding five unique cd4 protein variants ( fig 6a) . all of these allelic versions of rhesus macaque cd4 behaved identically and were nonfunctional for entry of virus pseudotyped with bg505 env to wildtype bg505 env for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < 0.01). data associated with this figure can be found in the supplemental data file (s4 data). ccr5, c-c motif chemokine receptor 5; gfp, green fluorescent protein. https://doi.org/10.1371/journal.pbio.3000304.g004 [79] . these two cell types were then cocultured to induce membrane fusion, which was detected by a luminescent product following the addition of the renilla luciferase substrate. (b) the percentage of fusion was calculated relative to what was observed with human cd4. error bars represent the mean + sem from three to four independent experiments, each with four technical replicates. values above error bars represent fold decrease relative to human cd4 for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < 0.05). data associated with this figure can be found in the supplemental data file (s5 data). ccr5, c-c motif chemokine receptor 5. and also with env from another early hiv-1 isolate, cap210.2.00.e8 ( fig 6b) . therefore, while owl monkey and chimpanzee populations circulate cd4 alleles with dramatically different functions as receptors for hiv/siv entry [33, 48, 80] , it appears that the function of macaque cd4 is identical from one allele to the next. in this study, we have shown that envs from over 30 early and chronic hiv-1 isolates from human blood (mother-infant pairs, etc.) all demonstrate selective entry via only some primate cd4 receptors. we next tested 29 additional envs (n = 33 total, four of which were previously tested in infection assays herein; s1 table) using the cell-cell fusion assay described above. these envs were universally poor at mediating fusion with cells expressing rhesus macaque cd4 and ccr5 proteins (fig 6c) . similar data have been generated in other large screens of early hiv-1 envs [31,32]. collectively, it seems that virtually no hiv-1 isolated from the bloodstream is compatible with the macaque version of cd4. if we now understand the inability of hiv-1 to use macaque cd4 to be a reflection of weak cd4 binding, this leads us to understand that virtually all early-and late-stage hiv-1 isolates in the human bloodstream experience continual selection to retain weak cd4 binding. total rna was isolated from the blood of 52 rhesus macaque individuals. cd4 was amplified from cdna, and pcr products were sequenced to yield genotype information for each individual (104 chromosomes). genotype information was used to phase snps using phase 2.1 [81, 82] in dnasp v5 [83] . the table summarizes the unique cd4 alleles identified. for each allele, the residue encoded at each variable amino acid positions is noted. unique alleles were cloned and further verified by sanger sequencing. (b) dog thymocytes (cf2th cells) stably expressing human ccr5 and the indicated rhesus macaque cd4 alleles (x-axis) were infected with hiv-1 (q23δenv-gfp) bearing a subtype a (bg505) or subtype c (cap210.2.00.e8) envelope (env). the percent cells infected (gfp-positive) was measured by flow cytometry 48 hours post infection. error bars represent the mean + sem from two independent experiments, each with three technical replicates. values above error bars represent fold decrease relative to human cd4 for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < 0.05). (c) 293t cells transiently expressing diverse hiv-1 envs (see s1 table) and cf2th cells stably expressing the indicated cd4/ccr5 receptors were each transfected with plasmids encoding half of a split renilla luciferase [79] . these two cell types were then cocultured to induce membrane fusion, which was detected by a luminescent product following the addition of the renilla luciferase substrate. a total of 33 envs were tested from the following major hiv-1 subtypes: subtype a (n = 12), subtype b (n = 7), subtype c (n = 6), subtype d (n = 6), and subtype a/d (n = 2). the percentage of fusion was calculated relative to what was observed with human cd4/ccr5. error bars represent the mean + sd, for which n = 2 (two technical replicates from one experiment). data associated with this figure can be found in the supplemental data file (s6 data). nt, nucleotide; aa, amino acid; ccr5, c-c motif chemokine receptor 5; gfp, green fluorescent protein. https://doi.org/10.1371/journal.pbio.3000304.g006 selective use of primate cd4 receptors by hiv-1 we find that many hiv-1 isolates from the blood, including early hiv-1 isolates, enter cells poorly through a subset of primate cd4 receptors. this finding is highly analogous to what has been observed with primate restriction factors that block hiv-1 infection, in that they too are highly species-specific in their interaction with hiv-1 [84, 85] . like cd4, most restriction factors are also evolving under intense positive selection in primates [86] [87] [88] [89] , explaining why these proteins vary in protein sequence on virus-binding surfaces from one host species to the next. it appears that sivs, and probably other viruses as well, have put intense selective pressure on these genes to evolve in a way that changes the protein sequence on virus-binding surfaces. the selection and retention of new cd4 alleles that block virus entry could protect a species from infection, at least until the virus population counter adapts. when this type of arms race is playing out in multiple host species independently, it drives divergence in protein sequence at host-virus binding interfaces and has the long-term effect of making it difficult for viruses to move between species [16] . indeed, a prevailing theme that has emerged in recent years is that receptor sequence divergence serves as a potent barrier to the movement of viruses between species [16, [90] [91] [92] [93] [94] . likewise, this study suggests that hiv-1 entry is blocked by the cd4 receptor of some primate species that it might encounter, whether that be in the wild or in the lab. on the other hand, we identify several primate species that encode cd4 receptors compatible with unmodified isolates of hiv-1 from the human bloodstream, including early isolates. these species could possibly be useful as animal models of hiv-1 transmission and infection, assuming that restriction factor blocks can be overcome in these species. we show that selective use of only certain primate cd4 receptors occurs when hiv-1 env has weak binding affinity for cd4. this weak affinity makes hiv-1 env sensitive to sequence differences in the cd4 molecule. the strongest evidence in favor of this model is that viruses that have been selected or engineered to have tight cd4 binding affinity become agnostic to sequence differences in primate cd4 and can more efficiently enter cells using all cd4 orthologs that we have tested. while neutralization assays to assess the cd4 binding affinity of a given virus are laborious, our assay for selective or promiscuous primate receptor usage allowed us to screen a large number of hiv-1 isolates from human blood. based on this, we show that weak cd4 binding is a nearly constant property of hiv-1 circulating in the human bloodstream, including but not specific to early-stage virions. this underscores the need to further understand the significance of cd4 binding affinity to hiv biology. while it has long been known that different hiv types have different affinities for cd4, the functional consequences of this are unclear. this is important to understand because weak cd4 binding seems to be selected for in the human bloodstream, yet some of our current primate models use envs that are engineered for tight cd4 binding affinity so that they more efficiently engage the nonpermissive macaque cd4 receptor. for example, mutations at hiv-1 env 375, when engineered into shivs, render virons compatible with macaque cd4 [77] . however, mutations at this position do not emerge in vivo when hiv-1 envs are adapting to macaque cd4, whereas mutations at env 281 do [78] . based on our data, we can now speculate that envs with high binding affinity to cd4 have a fitness penalty in vivo. given that nearly all hiv-1 in the human bloodstream seems to be selected to bind cd4 weakly, why would this be preferable? although the reasons for this are currently unclear, there have been numerous speculations put forward [29] . it is possible that more efficient use of cd4 requires changes in env conformation that would increase susceptibility to antibody neutralization. indeed, arrildt and colleagues have shown that when compared to patient matched t-cell tropic isolates from the blood, macrophage-tropic isolates appear to be more sensitive to certain cd4 binding site antibodies [95] . thus, any conformational change that increases antibody neutralization sensitivity would likely be purged from the viral population. alternatively, tighter cd4 binding is known to enhance hiv-1 tropism for cells of the monocyte-macrophage lineage [73, 95, 96] . however, replication of hiv-1 in these cells appears to be slowed due to cell type-specific blocks, including the restriction factors samhd1 and apobec3a [97, 98] , delayed reverse transcription and integration [99, 100] , and transcriptional repression [101] . these blocks to replication in macrophages may put high-affinity cd4 binders at an evolutionary disadvantage during early stages of infection. finally, highly efficient macrophage infection may directly promote disease via inflammatory processes. hiv-1 infection of central nervous system macrophages coincides with severe inflammation and neuronal impairment [102] [103] [104] [105] . additionally, respiratory and cardiac dysfunction during latestage hiv-1 infection has been associated with chronic inflammation, of which macrophages may be playing a central role [106, 107] . viral infection is a delicate balance between replicating efficiently enough to transmit, yet limiting disease to maintain the health of the host. altering cellular tropism via high-affinity cd4 binding may be disadvantageous to the virus, disproportionately skewing infection toward a more diseased state. herein, as in many other studies, comparative genetics between humans and primates has revealed to us new lessons about hiv's interaction with its host. this has been a tremendously powerful tool in the hiv field, having led to the original identification of, and mechanistic insights into, many of the host factors that control hiv-1 biology (for only a few examples, see [86, [108] [109] [110] [111] [112] ). as primate research becomes more tenuous [113] , we need to keep in mind how powerful these comparative approaches have been in the study of hiv-1 and in the study of other viruses as well (for only a few examples, see [114] [115] [116] [117] ). hek 293t (atcc crl-11268), tzm-bl (nih arp # 8129), and canine thymocytes (cf2th) (atcc crl-1430) were cultured in dulbecco's modified eagle medium (invitrogen) with 10% fbs, 2 mm l-glutamine, and 1% pen strep (complete medium). all cells were maintained at 37˚c and 5% co 2 . . then, diluted blood was separated in density gradient media (ficoll-pacque plus ge17-1440-02) by 400 x g centrifugation for 40 minutes. the peripheral blood mononuclear cell layer (i.e., pbmcs or buffy coat) was removed and washed twice in three volumes of balanced salt solution per sample. each donors' pbmcs were then immediately subjected to a cd4-positive selection kit (easysep human cd4 positive selection kit ii cat #17852) to isolate cd4+ t cells. rhesus macaque and owl monkey cd4+ t cells were then activated for three days in rpmi supplemented with 100 u il2/ml (recombinant human-il2; sigma #11011456001) and 5 μg/ml or 0.5 μg/ml pha-p (sigma l1668), respectively. cd4+ tcell cultures were subsequently maintained in rpmi + 100 u il2/ml media. cd4+ t cells isolated from rhesus macaque or owl monkey whole blood (as described above) or human t cells (hut78 cells) were analyzed for cd4 expression by flow cytometry (beckman coulter cyan adp high-performance flow cytometer). cells were first incubated in fc receptor (cd16 monoclonal antibody (3g8), ebioscience cat #16-0166-82) blocking buffer (pbs + 2% fcs + 1mm edta + 0.5% bsa) on ice for 1 hour. these cells were then fixed in 1% pfa and stained for cell surface expression of cd4 (bd cd4 mouse anti-human, apc, clone: l200). on the cytometer, only live cells were gated and histograms of cd4 expression determined by unstained and isotype (bd mouse igg 1 , , cat #555751) controls. the following env clones were obtained from the nih aids reagent program, division of aids, niaid, nih: env clones from early infection (qf495. 23m [3, 14, 50 ,51]) were gifts from dr. beatrice hahn. we amplified the rev-env cassettes from these by pcr, ta-cloned them into pcr8 (clontech), and then used gateway cloning to move them into a gateway converted pcdna3.1 (invitrogen) mammalian expression vector. further description of these env clones, and those used in the fusion assay screens, are described in s1 table. nucleotide changes producing s375y/w/h and a281v were introduced into bg505.w6m.b1 by site directed mutagenesis using overlapping pcr primers containing the mutation of interest, following standard methods. cd4 and/or ccr5 was amplified from rna isolated from the following sources: jurkat t cells (human cd4, genbank mk170450), immortalized b-lymphocytes (rhesus macaque ccr5, genbank nm_001309402.1), whole blood (rhesus macaque cd4 alleles 1-5, genbank mf632286-mf632290; owl monkey cd4, genbank mk205145), and fibroblasts (gorilla cd4, genbank mk170451). pig-tailed macaque cd4 and ccr5 were subcloned into the pcr8/gw/ topo ta plasmid from pbabe retroviral vectors provided by dr. julie overbaugh [30] . human ccr5 was obtained through the nih aids reagent program, division of aids, niaid, nih (human ccr5 expression vector pcccr5 #3325 from dr. nathaniel landau) and subcloned into the pcr8/gw/topo ta plasmid. the remaining constructs were purchased as gene block fragments (idt), pcr amplified (phusion hifi master mix; thermo fisher), and ta cloned into the pcr8/gw/topo ta plasmid (thermo fisher). the genbank sequences used for gene block synthesis are as follows: chimpanzee cd4 (nm_001009043.1) and ccr5 (u89797.1), sooty mangabey cd4 (nm_001319342.1) and ccr5 (xm_012033360.1), gorilla ccr5 (af177901.1), sabaeus cd4 (xm_007967415.1), gelada cd4 (xm_025401282.1), mandrill cd4 (xm_011982990.1), colobus cd4 (xm_011952099.1), baboon cd4 (xm_003905871.3), black snub-nosed monkey cd4 (xm_017891844.1), white-cheeked gibbon cd4 (xm_004092147.1), squirrel monkey cd4 (af452617.1), marmoset cd4 (af452616.1), and orangutan cd4 (xm_024256502.1; incomplete cds, missing 3 0 sequence (15 nucleotides) was substituted with human cd4 residues). following ta cloning, an lr clonase ii reaction (invitrogen) was used to shuttle inserts into gateway-converted plpcx (cd4) or plhcx (ccr5) retroviral packaging vectors (clontech). to produce retroviruses for transduction, hek293t cells plated in antibiotic free media (1 x 10 6 cells/well in a 6-well plate) were transfected with a 2 μg of transfer vector (plpcx [cd4] or plhcx [ccr5] retroviral vector), 1 μg pcs2-mgp (mlv gag/pol), and 0.2 μg pc-vsvg (vsv-g envelope) using a 3:1 ratio of transit 293 (mirus) transfection reagent to micrograms of dna, according to manufacturer's directions. forty-eight hours post transfection, retrovirus vlps were collected, filtered through 0.22-μm cellulose acetate filters, and stored at −80˚c in single-use aliquots. cf2th cells were plated at 3 x 10 4 cells/well of a 12-well dish (approximately 15% confluent) and 24 hours later, transduced with 500 μl of retroviral supernatant by spinoculation at 1,200 x g for 75 minutes in the presence of 5 μg/ml polybrene. the following day, the cells were placed in complete medium containing antibiotic (3 μg/ml puromycin for plpcx or 250 μg/ml hygromycin-b for plhcx) and cultured until stable outgrowth was noted (>1 week). stable cell lines were maintained indefinitely in selection media. to produce hiv-1 reporter viruses, 13 x 10 6 hek293t cells were seeded into a 15-cm dish in antibiotic free media and 24 hours later transfected with 5.3 μg of q23δenv-gfp and 2.7 μg of env plasmid. q23δenv-gfp, encoding an approximately 400-bp deletion in env, and with gfp inserted into the nef gene (therefore destroying the function of nef), was obtained from dr. julie overbaugh and described previously [30] . fourty-eight hours post transfection, the cell supernatant was harvested, concentrated (approximately 100-fold) using amicon ultracell 100k filters (millipore), and stored at −80˚c in single use aliquots. virus titers were determined by facs analysis for gfp-positive cells, using a range of virus dilutions on cf2th cells stably expressing human cd4 and ccr5. for infection assays, cf2th cells stably expressing cd4 and ccr5 were plated at 3 x 10 4 cells/well of a 48-well plate (approximately 60% confluent) 24 hours prior to infection. the cells were then infected with hiv-1 pseudoviruses at a multiplicity of infection (moi) of approximately 0.6 in the presence of 5 μg/ml of polybrene by spinoculation at 1,200 x g for 75 minutes. fourty-eight hours post infection, the cells were harvested from the plate and fixed in 2% paraformaldehyde for 10 minutes. the fixed cells were washed three times with pbs and resuspended in 50 μl facs buffer (1x pbs buffer containing 2% fbs and 1 mm edta) antibody cocktail. all antibody pairs were used at 1:200 dilutions, and incubated at 4˚c with cells for 30 minutes as follows: for primate cd4/primate ccr5 (s2 fig), pecy7 mouse α-human cd4 clone l200 (bd #560644) and apc mouse α-human cd195 (ccr5) clone 3a9 (bd #560748); for all other studies, the combination of apc mouse α-human cd4 clone l200 (bd #551980) and pecy7 mouse α-human cd195 (ccr5) 2d7 (bd #557752) was used. stained cells were analyzed on a cyan adp (beckman coulter) flow cytometer. following live-cell gating, cd4 and ccr5 expressing gates were drawn and then the percent gfp positive cells was enumerated within the double positive population. the data from approximately 1 x 10 4 live cells was analyzed using flowjo version 10. paxgene (bd #762165) preserved whole blood was obtained from animals housed at the kccmr per blood collection protocols approved by the university of texas, md anderson cancer center iacuc. total dna/rna was extracted using the allprep dna/rna mini kit (qiagen). extracted rna was used as a template for oligo (dt) primed reverse transcription (superscript iii rt; thermo fisher). pcr amplification of cd4 was performed using pcr supermix hifi (thermo fisher) containing 1-10 ng of cdna and 0.2 μm final concentration of each pcr primer cd4-forward 5 0 aagcagcgggcaagaaagacg 3 0 and cd4-reverse 5 0 caagttcctgccctctgtgg 3 0 in a final volume of 25 μl. pcr cleanup was performed using exonuclease-i and shrimp alkaline phosphatase treatment (affymetrix) for 15 minutes at 37˚c, and then the cleaned-up products were sanger sequenced using the following cd4-forward 5 0 ggagttcaaaatagacatcg 3 0 and cd4-reverse 5 0 cagacacttcct tgttcttc 3 0 sequencing primers. the resulting genotype information was used to phase snps using phase 2.1 [81, 82] in dnasp v5 [83] . five unique rhesus macaque cd4 alleles were identified, ta cloned into the pcr8 gateway topo ta cloning vector (thermo fisher), and then subcloned into the gateway converted plpcx retroviral vector (clontech). all constructs were further verified by sanger sequencing. allele 1 represented the major allele circulating in this population and was used in all experiments except where designated otherwise. human, chimpanzee, and rhesus macaque cd4 cloned into the pcr8 plasmid (described above) served as a template for soluble cd4 (scd4) pcr. the d1/d2 domain (nucleotide positions 75-603, minus signal peptide) was amplified by pcr and cloned into phl-sec [119] (addgene #99845), which has been optimized for protein production in mammalian cells. 13 x 10 6 293t cells (three 15-cm dishes) were transfected with 25 μg of scd4 expression plasmids using transit-293 (mirus), and cell supernatant containing secreted scd4 was harvested at days three and six post transfection. cell supernatant was spun down at 1,200 x g for 5 minutes to remove cell debris, filtered using a 0.45-μm cellulose acetate membrane, and mixed 1:1 with wash buffer (25 mm na 3 po 4 ph 7.4, 500 mm nacl, 20 mm imidazole). cleared cell supernatant was then incubated with 1-ml bed volume of ni-nta agarose beads (qiagen, #30210) equilibrated in wash buffer for 2 hours at 4˚c. the mixture was then added to a gravity flow chromatography column and the flow-through was passed through the column a second time. the ni-nta beads were washed with 50 ml of wash buffer. bound protein was eluted with wash buffer containing 250 mm imidazole in 1-ml fractions. fractions containing scd4 were pooled, washed three times with pbs, and concentrated to 500 μl using an amicon ultra-15 ml centrifugal filter with a 10 kda molecular weight cutoff (emd millipore, #ufc901008). the sample was purified to homogeneity on a superdex 75 increase 10/300 gl (ge healthcare, #29-1487-21) in pbs. purified samples were concentrated to 1 mg/ml using an amicon ultra 0.5-ml centrifugal filter with a 10-kda molecular weight cutoff, aliquoted for single use, and flash-frozen in liquid nitrogen. for the neutralization assays, each virus (normalized by equivalent tdu/ml) was incubated with serial 2-fold dilutions of scd4 (range 30 μm to approximately 1 μm final concentration) at 37˚c for 1 hour. human, chimpanzee, and rhesus scd4 were produced in this study (methods above), and commercially available human scd4 was obtained through the national institutes of health aids reagent program: scd4-183 #7356 (from pharmacia, inc.). following incubation, the scd4 treated and untreated viruses were spinoculated (1, 200 x g for 75 minutes) onto 1 x 10 4 tzm-bl cells plated in 96-well plates, in the presence of 5 μg/ml of polybrene. following spinoculation, the cells were washed three times with pbs and given fresh media. at 48 hours post infection, infectivity was measured by firefly luciferase assays following the manufacturers protocol (promega). luminescence was determined using the bmg clariostar plate reader. cf2th cells stably expressing human or primate cd4 and human ccr5, and 293t cells, were plated at 3 x 10 5 and 1 x 10 6 cells/well of a 6-well dish, respectively. the next day, 1) cf2th cells were transfected with 2.5 μg of dna (1.25 μg of ½ renilla luciferase [79] and 1.25 μg of pcdna3.1 filler dna) using lipofectamine 3000 (invitrogen) and 2) 293t cells were transfected with 2.5 μg of dna (1.25 μg of ½ renilla luciferase and 1.25 μg of the env expression plasmid) using transit 293 (mirus). twenty-four hours post transfection, the cells were removed from the plate using an enzyme free 1x citric saline solution (10x solution, 1.35m kcl, 0.15m sodium citrate) diluted in pbs, counted, and resuspended to a final volume of 1x10 5 cells/ml. 100 μl of transfected cells in quadrupilicate (1 x 10 4 293t and cf2th cells) were mixed 1:1 and incubated for 6 hours in 96-well flat bottom plates. following incubation, fusion was assessed using a renilla luciferase assay (promega). luminescence was determined using the bmg clariostar plate reader. all data were plotted, and one-way anova and mann-whitney u tests were performed where indicated, using prism version 7.0a for mac (graphpad). ic 50 values were determined by first normalizing the relative luminescence units as follows: 100% and 0% inhibition was defined by wells receiving the highest concentration of scd4 and absence of scd4, respectively. a nonlinear regression curve was fit to the normalized data, and ic 50 values were calculated using prism (graphpad) software. the blood collections performed on the animals in this study were approved as protocol number 00000451-rn02 by the university of texas, md anderson cancer center, institutional animal care and use committee (mdacc-iacuc). the protocol approved by the mdac-c-iacuc adhered to the recommendations provided in the guide for the care and use of laboratory animals [120] and also adhered to the blood collection volumes recommended by the guidance document of the association of primate veterinarians (https://www.primatevets. org/guidance-documents) entitled guidelines for blood sampling in nonhuman primates (also known as blood sampling guidelines for nonhuman primates in biomedical research). hiv-1 transmission biology: selection and characteristics of infecting viruses human immunodeficiency virus type 1 population genetics and adaptation in newly infected individuals identification and characterization of transmitted and early founder virus envelopes in primary hiv-1 infection quantitating the multiplicity of infection with human immunodeficiency virus type 1 subtype c reveals a non-poisson distribution of transmitted variants envelope-constrained neutralization-sensitive hiv-1 after heterosexual transmission differences in the selection bottleneck between modes of sexual transmission influence the genetic composition of the hiv-1 founder virus wide variation in the multiplicity of hiv-1 infection among injection drug users a substantial transmission bottleneck among newly and recently hiv-1-infected injection drug users in st petersburg, russia neutralization escape variants of human immunodeficiency virus type 1 are transmitted from mother to infant transmitted/founder and chronic hiv-1 envelope proteins are distinguished by differential utilization of ccr5 characterization of glycosylation profiles of hiv-1 transmitted/founder envelopes by mass spectrometry env length and n-linked glycosylation following transmission of human immunodeficiency virus type 1 subtype b viruses antigenicity and immunogenicity of transmitted/founder, consensus, and chronic envelope glycoproteins of human immunodeficiency virus type 1 resistance of transmitted founder hiv-1 to ifitm-mediated restriction relative resistance of hiv-1 founder viruses to control by interferon-alpha how host genetics dictates successful viral zoonosis origins of hiv and the aids pandemic animal models for hiv/aids research modern-day siv viral diversity generated by extensive recombination and crossspecies transmission rapid evolution by positive darwinian selection in t-cell antigen cd4 in primates positive selection of primate genes that promote hiv-1 replication dual host-virus arms races shape an essential housekeeping protein evidence for ace2-utilizing coronaviruses (covs) related to severe acute respiratory syndrome cov in bats evolutionary reconstructions of the transferrin receptor of caniforms supports canine parvovirus being a reemerged and not a novel pathogen in dogs computational and functional analysis of the virus-receptor interface reveals host range trade-offs in new world arenaviruses nonmacrophage-tropic human immunodeficiency virus type 1 r5 envelopes predominate in blood, lymph nodes, and semen: implications for transmission and pathogenesis macrophage tropism of human immunodeficiency virus type 1 and utilization of the cc-ckr5 coreceptor generation and structural analysis of soluble oligomeric gp140 envelope proteins derived from neutralization-resistant and neutralization-susceptible primary hiv type 1 isolates effect of major deletions in the v1 and v2 loops of a macrophage-tropic hiv type 1 isolate on viral envelope structure, cell entry, and replication distinct biological and serological properties of human immunodeficiency viruses from the brain complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation characterization of antibody responses elicited by human immunodeficiency virus type 1 primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants the role of mononuclear phagocytes in htlv-iii/lav infection hiv-1 infects multipotent progenitor cells causing cell death and establishing latent cellular reservoirs an infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1 macrophage-tropic hiv-1 variants from brain demonstrate alterations in the way gp120 engages both cd4 and ccr5 quantification of entry phenotypes of macrophage-tropic hiv-1 across a wide range of cd4 densities macrophage entry mediated by hiv envs from brain and lymphoid tissues is determined by the capacity to use low cd4 levels and overall efficiency of fusion blocking of hiv-1 infectivity by a soluble, secreted form of the cd4 antigen envelope residue 375 substitutions in simianhuman immunodeficiency viruses enhance cd4 binding and replication in rhesus macaques a single gp120 residue can affect hiv-1 tropism in macaques conformational changes of the hiv-1 envelope protein during membrane fusion are inhibited by the replacement of its membrane-spanning domain cd4 receptor diversity in chimpanzees protects against siv infection a new statistical method for haplotype reconstruction from population data a comparison of bayesian methods for haplotype reconstruction from population genotype data dnasp v5: a software for comprehensive analysis of dna polymorphism data hiv restriction factors and mechanisms of evasion. cold spring harb perspect med macaque-tropic human immunodeficiency virus type 1: breaking out of the host restriction factors positive selection of primate trim5alpha identifies a critical species-specific retroviral restriction domain ancient adaptive evolution of the primate antiviral dna-editing enzyme apobec3g species-specific activity of hiv-1 vpu and positive selection of tetherin transmembrane domain variants ancient adaptive evolution of tetherin shaped the functions of vpu and nef in human immunodeficiency virus and primate lentiviruses the evolution and genetics of virus host shifts human viruses: discovery and emergence animal origins of the severe acute respiratory syndrome coronavirus: insight from ace2-s-protein interactions novel insights into cell entry of emerging human pathogenic arenaviruses parvovirus family conundrum: what makes a killer? phenotypic correlates of hiv-1 macrophage tropism the hiv env variant n283 enhances macrophage tropism and is associated with brain infection and dementia samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx apobec3a is a specific inhibitor of the early phases of hiv-1 infection in myeloid cells kinetics of human immunodeficiency virus type 1 reverse transcription in blood mononuclear phagocytes are slowed by limitations of nucleotide precursors characterization of the early steps of infection of primary blood monocytes by human immunodeficiency virus type 1 regulation of hiv-1 transcription in cells of the monocyte-macrophage lineage hiv-1 target cells in the cns hiv-1 replication in the central nervous system occurs in two distinct cell types neurocognitive dysfunction predicts postmortem findings of hiv encephalitis updated research nosology for hiv-associated neurocognitive disorders preferential destruction of interstitial macrophages over alveolar macrophages as a cause of pulmonary disease in simian immunodeficiency virus-infected rhesus macaques the macrophage: the intersection between hiv infection and atherosclerosis the cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys species-specific vulnerability of ranbp2 shaped the evolution of siv as it transmitted in african apes non-human primate schlafen11 inhibits production of both host and viral proteins a trim5-cyclophilin a fusion protein found in owl monkey kidney cells can restrict hiv-1 cyclophilin a retrotransposition into trim5 explains owl monkey resistance to hiv-1 monkey kingdom nuclear trim25 specifically targets influenza virus ribonucleoproteins to block the onset of rna chain elongation an intrinsically disordered region of the dna repair protein nbs1 is a species-specific barrier to herpes simplex virus 1 in primates dengue viruses cleave sting in humans but not in nonhuman primates, their presumed natural reservoir. elife species-specific deamidation of cgas by herpes simplex virus ul37 protein facilitates viral replication hiv type 1 variants transmitted to women in kenya require the ccr5 coreceptor for entry, regardless of the genetic complexity of the infecting virus a time-and cost-efficient system for high-level protein production in mammalian cells committee for the update of the guide for the care and use of laboratory animals we thank julie overbaugh, soraya shehata and joe timpona for comments on the manuscript. key: cord-000736-6f8vyziv authors: pripuzova, natalia; wang, richard; tsai, shien; li, bingjie; hung, guo-chiuan; ptak, roger g.; lo, shyh-ching title: development of real-time pcr array for simultaneous detection of eight human blood-borne viral pathogens date: 2012-08-17 journal: plos one doi: 10.1371/journal.pone.0043246 sha: doc_id: 736 cord_uid: 6f8vyziv background: real-time pcr array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. findings: we developed a real-time pcr array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (hiv-1 and -2), hepatitis b virus (hbv), hepatitis c virus (hcv), human t-cell leukemia virus-1 and -2 (htlv-1 and -2), vaccinia virus (vacv) and west nile virus (wnv). one hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with sybr green chemistry. the specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. the array detected: 10 genome equivalents (geq)/ml of hiv-2 and hcv, 50 geq of hiv-1 (subtype b), hbv (genotype a) and wnv. it detected 100–1,000 geq/ml of plasma of hiv-1 subtypes (a – g), group n and crf (ae and ag) isolates. further evaluation with a panel consisting of 28 hiv-1 and hiv-2 clinical isolates revealed no cross-reactivity of hiv-1 or hiv-2 specific primers with another type of hiv. all 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. the pcr array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for hiv-1, hcv or hbv at as low as several geq per pcr reaction. conclusions: the viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. further improvement in its sensitivity for the broad spectrum of hiv-1 subtypes is under development. rapid progress and improvement in molecular technologies have allowed researchers to switch from the traditional approaches of virus detection in clinical samples to multiplexing for simultaneous detection of multiple pathogens in a single assay. a number of different pcr based assays for detection and discovery of multiple pathogens have been developed [1] [2] [3] [4] . detection microarrays are proven to be useful in the identification and discovery of viruses homologous to known species. they have been used to guide the selection of samples for further analysis by sequencing [1] [2] [3] 5] . however, microarrays based on nucleic acid hybridization are too complex in design and performance for the routine donors testing, and exhibit a comparatively low sensitivity of detection, usually around 10021,000 genome copies of target virus per analyzed sample [1] [2] . several pcr based assays coupled with oligonucleotide microarray technology (so called resequencing arrays) have been designed to allow simultaneous detection or genotyping of a target group of viruses, such as some critical blood-borne pathogens (3 viruses) [6] , respiratory viruses (16-21 viruses) [7] [8] , and respiratory adenoviruses (6 different serotypes) [9] . such pcr based approach allows increasing the sensitivity of detection down to 10-100 copies of the target rna or dna in a sample. pcr multiplexing should be highly useful when both the volume of the samples and the time of testing are critical, as in the donor eligibility (de) testing for tissue or organ transplantation [10] . current regulation requires that de testing be performed using assays approved and licensed by the u.s. food and drug administration (fda). however, the automated assay systems that are designed to screen large numbers of samples, without the strict limitation of sample volumes, may not be completely suitable or ideal for the needs of de testing for tissue or organ transplantation. the main goal of the study presented here was to evaluate the feasibility of developing a sensitive and specific assay for rapid detection and identification of a group of target viral pathogens. the following viral pathogens were included in our array: human immunodeficiency virus types 1 and 2 (hiv-1 and hiv-2), human t-cell leukemia virus-1 and -2 (htlv-1 and htlv-2), hepatitis c virus (hcv) and west nile virus (wnv), all with singlestranded rna genome; vaccinia virus (vacv) and hepatitis b virus (hbv), both with double-stranded dna genome, hbv also has single-stranded rna stage. some of the listed viruses are included to the required de testing for tissue transplantation. besides, historically, some of the targeted viruses have been found to be allograft-transmitted to recipients [11] [12] . in the present study, a real-time pcr array with sybr-green chemistry targeting these eight viral pathogens listed above was developed and evaluated with analytical and clinical panels. the array demonstrated acceptable performance in the testing with both analytical panels and donors' clinical samples. the research study conducted at fda using previously frozen blood samples was reviewed by department of health and human services, food and drug administration, research involving human subjects committee (rishc protocol #10-008b entitled ''detection of infectious agents in previously frozen blood samples from patients with various illnesses and healthy blood donors''). the 17 clinical plasma samples positive for hbv, hcv or hiv-1 used in this study were existing clinical diagnostic samples kept in nih blood bank. information of these left over samples had been recorded in such a manner that subjects can not be identified, directly or through identifiers. the written informed consent from the participants was waived under 45 cfr 46.101 (b) (4) . the six plasma samples positive for hiv-1 were estimated to contain 50 to ,90,000 genome copy numbers per ml; six plasma samples positive for hcv were estimated to contain 780 to ,123,000 genome copy number/ml and five plasma samples positive for hbv were estimated to contain ,150 to 16,000 copy number/ml. the copy numbers of viruses in these samples were provided by the nih blood bank. no information about the subtypes or genotypes of these viruses was available. the amount of each clinical sample was sufficient to be tested only once by the pcr array in the study. all positive pcr products obtained in the testing using the pcr array were confirmed for validity by sequencing in the facility for biotechnology resources of fda/ cber. we used the ''insignia'' program (http://insignia.cbcb.umd. edu/query.php), a bioinformatics on line tool developed in the center for bioinformatics and computational biology, university of maryland [13] to choose a specific dna or rna ''signature'' (a sequence, with customized length and g/c content) for targeted viruses. comparative sequence analysis of the complete genomes was performed using mvista (http://genome.lbl.gov/vista/ mvista/submit.shtml). multiple nucleotide sequence alignments (nsas) were then created to visualize the most conserved genome areas using mega4 (http://www.megasoftware.net). specific criteria for the primers and amplicon selection for the sybr green based pcr array were: 1) the same range of annealing temperature (t) -57-60uc -for all primers, 2) high g/c content for primers, allowing higher specificity of annealing, and 3) an amplicon size in the range of 100-200 b.p. in order to have a high pcr amplification efficiency and to sufficiently distinguish the products from primer dimers based on melting t peak (tm). all primers were checked for potential dimer formation using ''primer express'' software (version 3.0, applied biosystems). after design, all primers were again checked using the national center for biotechnology information (ncbi) basic local alignment search tool (blast) (http://blast.ncbi.nlm.nih.gov/blast.cgi) to avoid any cross-reactivity with other species. newly designed and previously published primer sets adapted for the final version of the real-time pcr array are listed in table 1 . in addition, primers specific for the human beta-globin gene were included in the array as an internal control for the quality of dna/rna preparation as well as for estimation of viral copy number per host cell if needed (the last row of table 1 ). total cellular dna of the chronically infected cell cultures (for hiv-1, hiv-2; htlv-1, htlv-2 and vacv), viral genome cdna copy spiked into human dna (for hcv and wnv), and dna isolated from human plasma of the infected individual (for hbv) were used as the positive templates in the initial testing and are listed in the last column of table s1 . dna or rna panels were created by cloning of specific synthetic templates for each virus into the pgem-t-easy vector (promega) by ta cloning, following by in vitro transcription to obtain rna standards for hcv and wnv. all created plasmids are listed in table s1 . nucleotide numbers in table s1 refer to the location of the partial viral genome cloned into pgem-t-easy vector according to the following complete genome sequences available in genbank: htlv-1 -l03562.2, htlv-2 -m10060.1, hiv-1 -k02083.1, hiv-2 -j03654.1, hbv -af462041.1, hcv -af271632.1, vacv -ay243312.1, wnv -hq596519.1. to establish the real-time pcr standard curve the copy number was calculated for each plasmid carrying one copy of the specific viral gene. the size of each plasmid (x b.p.) was used to determine the molecular weight in daltons (g/mol): w (g/mol) = x b.p. (330 da 6 2). the copy number of the target viral gene (molecules/ml) was determined from the plasmid concentration (c dna ) and the molecular weight of each plasmid molecule: copy number = c dna (ng/ml) 6 6.02 6 10 23 (avogadro's number)/w. knowing the number of plasmid molecules with the target viral gene in a ml, a series of dilutions was made to generate a pcr standard curve. the developed analytical standards were used to calculate the intra and inter-assay reproducibility of quantification for each virus-specific primer set. mean c(t) values, standard deviation (sd) and coefficient of variation (cv) were calculated from the data obtained in three replicates of each standard dilution for the intraassay reproducibility, and in three real-time pcr assays consisted of three replicates each (nine total) for the inter-assay reproducibility. cv was calculated as sd/mean c (t) * 100%. preparation of viral rna/dna for pcr array analysis 0.5-1 ml of human plasma was used for the total viral rna/ dna extraction using ''qiaamp viral rna mini-kit'' (qiagen) and trna (sigma) was used as a carrier rna during the preparation. after the final elution step rna/dna in 160 ml of buffer ave was precipitated with 100% ethanol and 3 mm nacl at 220uc overnight. an rna/dna pellet was washed with 70% ethanol, dissolved in 10 ml of depc-treated water and then immediately used for cdna synthesis with superscript ii rt (invitrogen) and random hexamers (invitrogen) in a total reaction volume of 20 ml. the volume of cdna/dna sample was then adjusted up to 30 ml with depc-treated water and the whole pcr was performed using ''bio-rad cfx96 real-time system'' with ''power sybr green pcr master mix'' (applied biosystems). one reaction (25 ml total) contained: 12.5 ml of pcr master mix, 0.5 mm of each primer and 1.25 ml of dna/cdna template. in the single virus testing (sections 3 and 4 of the ''results'') we used 2.5 ml of dna/cdna template from 20 ml of sample after cdna synthesis. ''universal'' pcr conditions for all primer sets included to the array were: 95uc for 8 min (one cycle), then 50 cycles of: denaturation at 95uc for 15 s, annealing and extension at 60uc for 1 min, followed by melting curve read from 65uc to 95uc with increment 0.2uc for 5 s. real-time pcr data were downloaded in 96-well plate format from ''bio-rad cfx manager 2.1'' to ms excel and analyzed manually. two types of samples served as the background control for determination of the c(t) cut off. the 1 st type of negative control was 50 ng of human cellular dna. the 2 nd type of negative control was negative donors' plasma. data were collected in separate experiments from 8 human cellular dna controls and 3 negative donors' plasma. to standardized the c(t) cut off for all primers the threshold was set at the pcr machine default setting. based on a false positive rate of less than 5% the following method [14] was used to estimate the c(t) cut off from the range of c(t) obtained with negative samples for all 24 primer sets in the array: 1. calculate the margin of error of the confidence interval (ci), w = t* 6 sd/!n, where: n -number of obtained c(t) values, sd-standard deviation, df = n21, and t* (for 95% confidence) is a ''critical value of the t distribution'' [14] . 2. one side ci covers this range: m-w, where m is sample mean. the c(t) cut off calculated from the range (n = 50) of the 1 st type of negative control data was c(t)#41.03. the c(t) cut off calculated from the range (n = 50) of the 2 nd type of negative control data was c(t)#42.7. even some overlap between the c(t) measurements of truly positive and truly negative samples was detected in pcr array data, the tm parameter was used to define if the obtained pcr product is specific by comparison to the expected tm peak range. the analytical sensitivity of each primer set was determined in the single virus testing using fda/cber panels (kindly provided by dr. stephen kerby, fda/cber) consisting of various amounts of the viruses (0-1,000 genome copies/ml) spiked into the ''normal'' human plasma. panels for the following viruses were used in testing: hiv-1 (three different panels), hiv-2, hbv (based on genotype a), hcv (genotype 1b), and wnv (based on strain hu2002). these panels have been used for testing of commercially licensed nat assays and their development was described previously [15] . three separate hiv-1 rna panels were used for testing. the first consisted of various amounts of an hiv-1 group m subtype b isolate: 0, 5, 10, 25, 50, 100, and 500 copies/ml. the second consisted of 25 samples representing various concentrations of hiv-1 groups o and n, and group m subtypes a, c, d, e, f and g: 10, 100 and 1,000 copies/ml for each virus. the third panel consisted of hiv-1 circulating recombinant forms (crfs) ae and ag: 100, 1,000, and 10,000 copies/ml for each crf. np24 caacttcatccacgtttcacc a -to simplify the process of evaluation we used our primer names with sequential numbers, however some of the primers have been designed previously with their original names in the articles listed in the last column of this [25] [26] . hiv-1 groups and subtypes are based on designations reported by the nih arrrp. low passage virus stocks were prepared and median tissue culture infective dose (tcid 50 )/ml of virus containing supernatant determined in fresh human pbmcs isolated as previously described [27] . infectious unit (iu) of the virus stocks in tcid 50 s were titrated for each isolate and the number of iu used for viral rna isolation and pcr was calculated based on the dilution factor (1:100) and ranged from ,10 to 2,500 or 1 to 3.4 log 10 tcid 50 per pcr reaction. pcr approach based on sybr green chemistry, allowing simultaneous detection of multiple targets, was chosen to be applied for the array performance. virus-specific primer sets targeting at least three different genomic sites for each viral pathogen were designed for the real-time pcr array. we used the ''insignia'' program, a bioinformatics tool that helps to choose a specific dna or rna ''signature'' for different bacteria and viral pathogens that are included in the pre-built ''insignia'' database [13] . sequences of the highly conserved regions, such as coding viral polymerase or structural proteins were selected to design the candidate primers. in addition, we performed nucleotide sequence alignments (nsa) of the complete viral genomes and of the most conserved genome areas for different subtypes/genotypes or different isolates of all targeted viruses. some previously published primers designed for pcr detection of the target viruses were also adapted and evaluated. overall, a total of 120 primers were initially designed using specific criteria for the current real-time pcr array (see materials and methods) to cover the eight targeted viruses: hiv-1, hiv-2, hbv, hcv, htlv-1, htlv -2, wnv, and vacv. the primers were designed and tested for their effectiveness and specificity of amplifying the respective target under uniform pcr conditions (using the same annealing temperature for all primers). each candidate primer pair was first tested for its specificity and sensitivity of pcr amplification. this was assessed in a real-time pcr cross-testing against human pbmc dna (50 ng/pcr reaction) from a healthy donor, dna from human cell cultures infected by various target viruses, or human dna spiked with a known amount of genome copies of various viruses. it was important to ensure that the selected primers targeting a specific virus would not non-specifically amplify any dna in a sample. the melting temperature (tm) peak of the product amplified from the target virus should be clearly differentiable from the tm peak of primer dimers or any non-specific products produced in pcr. dna or rna panels were created from the cloned synthetic templates (listed in table s1 ) by spiking of 2-10 4 genome copies of each virus into 50 ng of human background dna. the sensitivity of each candidate primer set for each target virus was assessed using these panels. example of the experimental testing of hiv-1 specific primer set targeting gag gene (np3/4) for its sensitivity with dna analytical standards is shown in figure s1 . serial dilutions of a plasmid dna corresponding to 5-10 4 hiv-1 genome copies spiked into 50 ng of normal human dna, hiv-1 infected cells (h9/iiib) (''positive'' control dna) and uninfected human pbmc (''negative'' control dna) are used in the experiment. only a single melting peak tm = 72.5uc corresponding to hiv-1 specific product was observed and no unspecific amplification was registered. figure s1b revealed a standard curve showing the correlation between copy number of the target gene and ct values with a slope = 23.77. the limit of sensitivity was determined in this assay to be 10 viral genome copies/pcr. after evaluation, a total of 24 primer pairs targeting the eight different viruses were chosen for the real-time pcr array based on their specificity and sensitivity (table 1) . among them, five of the primer sets were previously published and the other 19 primer sets were originally designed in the current study. table 1 shows the sequences of the previously published primer sets selected for the real-time pcr array with the reference to the original source. analytical sensitivity expressed in genome copy/pcr for each primer set (table 1 ) was estimated using dna/rna analytical panels, as described above. coverage of variants (i.e., different subtypes or genotypes) for each virus (table 1 ) was estimated using the nsas. degenerative nucleotides were introduced into some of the primer sets based on the nsas performed in-house to obtain a broader coverage. the tm peak range of the product stated in table 1 for each primer set was established during further testing of selected primers with analytical and clinical panels. in addition, the intra and inter-assay reproducibility of quantification for all primer sets was evaluated using three replicates of each standard dilution (of dna or rna analytical standards) in each of three real-time pcr assay runs. the coefficient of variation (cv) for the c (t) values was #3.3% and #6.7% for intra-and inter-assay, respectively. all the data depicting mean c (t), standard deviation (sd), and cv for each primer set selected for the real-time pcr array with each standard concentration are shown in table s2 . to further evaluate the sensitivity of the selected primers, we used fda/cber panels (kindly provided by dr. stephen kerby, fda), consisting of various amounts of viruses spiked into ''normal'' human plasma, that are specifically developed and used for the evaluation of commercially licensed nat assays. table 2 summarizes the testing results for the selected primers against the target viruses. one out of four primer sets targeting hiv-1, np3/4, could detect the subtype b hiv-1 rna at the concentration of 50 copies/ml of human plasma. two other hiv-1 specific primer sets (np51/52 and np170/171) detected the hiv-1 rna at 100 copies/ml of plasma. the 4th primer set, np175/174, (targeting the conserved pol region and containing degenerative nucleotides to support broader variant coverage) could detect the virus only at 500 viral genome copies/ml of plasma. hiv-2 rna was detected with primer set np86/87 at the concentration of 10 copies/ml and with the primer set np76/77 at the concentration of 50 copies/ml. another primer set (np84/ 85) detected hiv-2 at 100 copies/ml. hbv (genotype a) dna was detected with the primer set np11/97 at 50 copies/ml and with np94/100 at 100 copies/ml. the third hbv specific primer set (np11/97-mod) detected hbv at 500 copies/ml of plasma. both hcv specific primer sets targeting the viral 59ntr detected hcv rna at the concentration of 10 copies/ml of plasma. two of the wnv-specific primer sets (np21/22, targeting e protein gene, and np176/177, targeting ns5) detected wnv at 50 copies/ml and the third primer set (np178/179, also targeting ns5) gave a positive signal at 100 copies/ml of plasma. the four hiv-1 specific primer sets were further evaluated by testing them against another fda/cber analytical panel containing a broad spectrum of hiv-1 subtypes. as summarized in table 3 , the hiv-1 subtype f (group m) and group o isolates in table 2 . the results of sensitivity testing of the real-time pcr array primer sets specific for hiv-1, hiv-2, hbv, hcv, and wnv the with fda/cber analytical plasma panels. table 3 . sensitivity of four hiv-1 specific primer sets selected for the real-time pcr array in testing with fda/cber analytical hiv-1 broad spectrum panel. detection limit (copy number/ml of plasma) was evaluated using fda/cber analytical panel, containing pre-set copy number of hiv-1 spiked into 1 ml of ''normal'' human plasma. rna from 1 ml of plasma was converted to cdna and divided into eight pcr reactions; two pcr repeats were performed for each primer set. the estimated copy number per pcr reaction is shown in parentheses. the panel could not be successfully amplified by any of the four selected primer sets (detection limit is .1,000 rna copy/ml of plasma). all other subtypes and crfs of group m, and the group n isolate could be amplified with at least one out of the four primer sets at 50 to 1,000 rna copies/ml of plasma (table 3) . the tm peaks of the pcr products amplified from different subtypes of hiv-1 varied within 1.5uc ( table 3 ). all of the four selected hiv-1 specific primer sets amplified hiv-1 subtype b (group m) with the highest sensitivity (50-500 copies/ml of plasma). to examine the specificity and the ability of the array to detect different isolates within subtypes of hiv-1 and different isolates of hiv-2 by the array, we additionally tested our primers with another panel, developed by the southern research institute. this panel contained three different isolates from each subtype (a to g) of hiv-1 group m, three isolates from hiv-1 group o, one isolate from hiv-1 group n and three different isolates of hiv-2. the infectious dose of each hiv isolate in the panel was determined by tcid 50 (median tissue culture infective dose) titration and the dose of virus used in each pcr reaction was calculated. the results of testing of hiv-1 specific primers are shown in figure 1 . in this experiment we evaluated the coverage of hiv-1 variants and estimated the relative sensitivity of the four hiv-1 specific primer sets based on cycle threshold (c(t)) values obtained with each isolate tested. two primer sets targeting the conserved pol regions (np170/171 and np175/174) were able to detect most of the hiv-1 subtypes with a high sensitivity (c(t) = 15-25). only one primer set (np175/174) detected both the group n isolate and all three isolates of group o with ct = 20-35. we did not detect crossreactivity of hiv-1 or hiv-2 specific primers with the other type of hiv. all three hiv-2 isolates studied (1-3 log 10 tcid 50 /pcr input) were detected with all hiv-2 specific primer sets with a low ct value: 12-20 (data not shown). after completion of sensitivity testing of primer sets with analytical panels we finalized the expected tm peaks range for each amplicon and arranged a working array in 96-well plate format ( figure s2 ). to evaluate the specificity and possible crossreactivity of primers in the array, 20-100 genome copies of each virus were spiked into 50 ng of human dna to be used as positive templates and the same amount of human dna was used as a negative control in each experiment. the array was tested with all targeted viruses using dna standards (listed in table s1 ). each positive template was tested in duplicate; one human dna negative control and one no-template control (ntc) were included in every testing. in these experiments the definition of ''positive'' signal was set up as the threshold cycle cut-off of c(t)#41 (see materials and methods) and all tm peaks are within the expected range ( figure s2) . no cross-reactivity was detected for any primer sets in the array using dna standards with this setting (data not shown). seventeen (17) clinical plasma samples (obtained from nih blood bank) from donors who tested positive for hiv-1, hbv, or hcv were used to evaluate the array sensitivity and specificity. the genome copy of each virus in virus-positive plasma samples was previously determined by the nih blood bank using commercial assays approved for donor screening. representative results from the testing of three hbv-positive plasma samples (pt.#13-pt.#15) using our array are shown on figure 2 . the threshold cycle cut off of the assay performed with plasma samples was set up as c(t)#43 (see materials and methods). the wells with c(t) lower than the cut-off and the obtained tm peaks within the expected tm range indicate positive amplification of hbv target genes and are highlighted with blue circles (figure 2 , green color code for hbv). for samples #13 and #14 tm peaks of the products, obtained with only two hbv specific primer sets (wells g2, g3-pt.#13 and wells g5, g6-pt.#14), were within the expected range, with c(t) values of 36.7-42.9 and 36.5-36.6 respectively. for sample #15, all three hbv specific primer sets gave the tm peaks within the expected range (wells g7, g8 and g9), and the c(t) values range was 33.8-34.2. the genome copy number of hbv in plasma samples #13-15 was 151-518 copies/ ml, corresponding to 6-21 copies/pcr. the wells (white color code, red circles) with the human beta-globin gene specific primer set, serving as the internal control, produced positive signals from all three plasma samples tested, with c(t) values of 25.5-26.3 (wells h3, h6 and h9 figure 2 ), which shows that the quality of the rna/dna sample preparation was equally good for the plasma of these three patients. the final tm range for each primer set was adjusted after testing of this clinical panel. based on the results from the testing of clinical samples and according to nsa performed during the design, two primer sets targeting the s gene region of hbv (np11/97 and np11/97-mod) have two different tm ranges depending on the hbv genotype ( figure 2 -wells g1 and g3). the lower tm range (74.5-75.5uc) was detected for genotype a and the higher tm range (76.8-77.4uc) -for genotypes b, c, and d. the difference in tm ranges occurred due to single nucleotide polymorphism (snp) in the amplicon sequence leading to a difference in the g/c nucleotide content of the products. table 4 summarizes the results from testing the 17 virus-positive clinical samples using the developed array. at least two different specific primer sets could detect a target virus in all cases, with one exception: one hiv-1 positive sample (#4) with ,4 viral genome copies per pcr reaction was amplified with only one primer set (np175/174). hiv-1 positive clinical samples (#1-#3) with 51-80 viral genome copies/ml of plasma or 2-3 copies/pcr reaction were detected with at least two primer sets. all six hcv-positive samples (#7 to #12) with 16-2,570 viral genome copies/ml of plasma tested positive with both hcv-specific primer sets. three hbv-positive samples (#15, #16, #17) at 21-66 copies/pcr tested positive by the three hbv-specific primer sets and two hbv-positive samples (#13, #14) with 6-10 copies/pcr tested positive with two out of three hbv-specific primer sets. it is important to note that none of the primer sets showed any crossreactivity with other viruses in the panel of clinical samples tested. in the study presented here we applied a real-time pcr array approach in a 96-well plate platform for detection of a group of target viral pathogens. in contrast to taqman pcr, which is commonly used for viral diagnostics, this platform based on pcr with sybr green chemistry supports simultaneous detection and identification of 24 different targets corresponding to eight different viruses. pcr based on sybr green staining of the double stranded dna is economically affordable and allows for the detection of mutants with snps within the amplicon sequence [34] . the strategy of primer selection for the array included the sequential use of different bioinformatics programs to identify highly-specific primer sets with a maximal variants' coverage of the targeted viruses, while working under ''universal'' pcr conditions. experimental selection process using a panel of dna or rna analytical standards allowed choosing two to four most sensitive and specific primer sets for each targeted virus. the sensitivity (in copy number per pcr reaction) and the intra and inter-assay reproducibility of quantification were characterized for each primer set selected for the array. fda/cber analytical plasma panels were used to assess the detection sensitivity of the primer sets selected for the working array. the hiv-2 and hcv rna was detected at as low as 10 genome copies/ml of human plasma by one out of three and two out two primer sets, respectively. similarly, wnv rna and hbv dna were detected at 50 genome copies/ml of plasma by two out of three and one out of three selected primer sets respectively. all four of the selected hiv-1 specific primer sets detected group m, subtype b, the most common subtype of hiv-1 in the americas and western europe [35] , at 50-00 genome copies/ml of plasma. however, the array was able to amplify all other subtypes, excluding subtype f, of hiv-1 group m with only one primer set (np170/171), targeting the conserved pol region, at 100-1,000 genome copies/ml of plasma. in addition, the array amplified the group n isolate at 1,000 copies/ml with another primer set (np175/174) targeting the conserved pol region. all the selected hiv-1 specific primer sets of the array failed to amplify the group o isolate (detection limit is .1,000 copies/ml of plasma). in comparison, the limit of detection (lod) of recently improved commercial multiplex nat assays for the broad spectrum of hiv-1 group m isolates is 40 copies/ml, and the lod for group o is 200 copies/ml [36] [37] [38] . thus, specific primer sets targeting group o isolates of hiv-1, as well as subtype f (group m) will need to be included in the array to increase the coverage of all existing subtypes/groups of the hiv-1. no crossreactivity was shown for hiv-1 or hiv-2 specific primers with the other type of hiv in a testing with medically relevant levels of viruses in a diversity panel containing 25 different hiv-1 isolates and three natural hiv-2 isolates collected worldwide. there is presently no us food and drug administration (fda) approved pcr-based nat testing for blood donors' screening for htlv-1 and htlv-2. there is no official fda (or world health organization (who)) viral panel released for these two viruses. it is also difficult to obtain htlv-1 or htlv-2 positive blood donors' samples in the united states. in the absence of the analytical panels and clinical samples we tested htlv-1, htlv-2 and vacv specific primers only with cell culture derived dna and with dna standards. the minimum detection limit of htlv-1 and htlv-2 specific primer sets estimated with analytical dna standards was 5-10 genome copies/pcr reaction, which is in the same range as for other primers included to the array. the coverage of the viral variants for the primers targeting these viruses was estimated in silico using multiple sequence alignments performed with complete genome sequences available in gen bank. the working array arranged in 96-well plate format was subsequently tested for specificity and potential cross-reactivity with human dna and with each of the targeted viruses. none of the primer sets selected for a particular target virus in the array produced non-specific cross-reaction toward the other viruses. comparative performance of the array was also evaluated through the testing of 17 clinical specimens from the united states patient population. all 17 samples were correctly identified in our pcr array with a high sensitivity to contain hiv-1, hcv, or hbv. we found that a combination of several primer sets targeting each virus in the array allows for the detection of different variants of the virus; however, it makes the absolute quantification with uniform dna/rna standards a challenge. quantification by the assay is not always possible when the genetic group of the viral isolate being tested is different than the assay standards. this is one of the reasons why in certain cases the commercial assays underestimate viral loads by up to 1-10 log 10 copies per ml [37] [38] [39] . nevertheless, relative quantification can be done using all primer sets selected for the array, and the absolute quantification can be performed with dna/rna standards using the primers targeting the most conserved genome areas. there are several commercial qualitative multiplex nat assays now available on the market simultaneously targeting three most important blood-borne viral pathogens (hiv-1, hcv and hbv) [40] . one of them was recently approved by u.s. fda for screening of blood and organ donors (http://www.fda.gov/biologicsblood vaccines/bloodbloodproducts/approvedproducts/licensedproduc tsblas/blooddonorscreening/infectiousdisease/ucm306073.htm). we compared the lod of these multiplex nat assays [40] with the results of our working pcr array in sensitivity of testing against these important viruses. the lod for hbv are 38.1-195 geq/ml by ''procleix ultrio tigris'' and 9.2-37. other pcr-coupled techniques have been developed previously for highly-sensitive pathogens' detection that could reach the sensitivity of the assay up to 1 genome copy per pcr reaction. for example the bioactive amplification with probing (bap), utilizing a nested pcr and magnetic bead-based hybridization with the specific probe, has been developed for the detection of bovine and avian viruses [41] [42] [43] . in spite of the exceeding sensitivity of such assays targeting a single virus, it may be difficult to adapt the approach or method to meet the main objective of simultaneous detection of multiple target viral pathogens by an array using universal pcr conditions. it is important to note that this array was developed to be adapted by any laboratory. comparison of experimentally obtained tm peaks to the range of expected specific tm peaks allows rapid identification or exclusion of the viral pathogen in a sample. this is an initial study to examine the suitability of using pcr arrays for the detection of a group of target viruses. the current array was developed utilizing five previously published and table 4 . tm and c(t) values obtained with primer sets specific for hiv-1, hcv, or hbv in testing of 17 human clinical samples in the format of pcr array targeting eight different viruses. 19 originally designed primers sets. however, it can be expanded to a larger number of targets for the same virus. targeting of several genome areas increases the detection sensitivity of the target virus and provides an intra-assay confirmation of positive signals. additionally, any new virus of interest can be added to the list of targeted pathogens. efforts are underway to test the utility of this real-time pcr array using samples with more diverse biological origin and pathogen content. panmicrobial oligonucleotide array for diagnosis of infectious diseases testing and validation of high density resequencing microarray for broad range biothreat agents' detection crossspecies transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony identification of pathogenic vibrio species by multilocus pcr-electrospray ionization mass spectrometry and its application to aquatic environments of the former soviet republic of georgia pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity an oligonucleotide microarray for multiplex real-time pcr identification of hiv-1, hbv, and hcv a low density oligonucleotide microarray for the detection of viral and atypical bacterial respiratory pathogens application of taqman low density arrays for simultaneous detection of multiple respiratory pathogens use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses eligibility determination for donors of human cells, tissues, and cellular and tissue-based products; final rule and notice. 21 cfr part 1271 infections and human tissue transplants: review of fda medwatch reports reported infections after human tissue transplantation before and after new food and drug administration (fda) regulations, united states insignia: a dna signature search web server for diagnostic assay development intuitive biostatistics development and evaluation of hiv-1 subtype rna panels for the standardization of hiv-1 nat assays genetic variation of hiv type 1 in four world health organization-sponsored vaccine evaluation sites: generation of functional envelope (glycoprotein 160) clones representative of sequence subtypes a, b, c, and e. who network for hiv isolation and characterization human immunodeficiency virus type 1 t-cell tropism is determined by events prior to provirus formation dual infection of the central nervous system by aids viruses with distinct cellular tropisms biologic and genetic characterization of a panel of 60 human immunodeficiency virus type 1 isolates, representing clades a, b, c, d, crf01_ae, and crf02_ag, for the development and assessment of candidate vaccines subgroup g hiv type 1 isolates from nigeria variability of human immunodeficiency virus type 1 group o strains isolated from cameroonian patients living in france identification of a new human immunodeficiency virus type 1 distinct from group m and group o biological and molecular variability of human immunodeficiency virus type 2 isolates from the gambia genetically divergent strains of human immunodeficiency virus type 2 use multiple coreceptors for viral entry aids verursacht durch hiv-2 preliminary characterization of an hiv-2 isolate derived from a german virus carrier development of hexadecyloxypropyl tenofovir (cmx157) for treatment of infection caused by wild-type and nucleoside/nucleotide-resistant hiv dna amplification for direct detection of hiv-1 in dna of peripheral blood mononuclear cells quantitation of hepatitis b virus dna in plasma using a sensitive cost-effective ''in-house'' realtime pcr assay a genotypeindependent real-time pcr assay for quantification of hepatitis b virus dna rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a taqman reverse transcriptase-pcr assay hpv dna detection and typing in cervical scrapes complete coding sequences of the rabbit pox virus genome sybr green-based real-time quantitative pcr assay for detection of west nile virus circumvents falsenegative results due to strain variability human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications evaluation of the roche cobas taqman and abbott realtime extraction-quantification systems for hiv-1 subtypes comparative rna quantification of hiv-1 group m and non-m with the roche cobas ampli prep/cobas taqman hiv-1 v2.0 and abbott real-time hiv-1 pcr assays a new real-time quantitative pcr for the diagnosis and monitoring of hiv-1 group o infection performance characteristics and comparison of abbott and artus real-time systems for hepatitis b virus dna quantification comparison of the analytical and operational performance of two viral nucleic acid test blood screening systems: procleix tigris and cobas s 201 development of a reliable assay protocol for identification of diseases (rapid)-bioactive amplification with probing (bap) for detection of bovine ephemeral fever virus development of a reliable assay protocol for identification of diseases (rapid)-bioactive amplification with probing for detection of avian reovirus development of a reliable assay protocol for identification of diseases (rapid)-bioactive amplification with probing (bap) for detection of newcastle disease virus key: cord-015831-s78omm53 authors: kaufman, joan title: civil society involvement in national hiv/aids programs date: 2019-05-20 journal: hiv/aids in china doi: 10.1007/978-981-13-8518-6_22 sha: doc_id: 15831 cord_uid: s78omm53 globally, the aids response relies on active participation of nongovernmental organizations (ngos) and civil society. in china, the government is the main provider of health and social services, and the role of ngos is more limited than in other countries. despite this, china has opened the door for ngo participation in its aids response, initially because of donor pressure but increasingly due to official acknowledgment of the important role these groups play in controlling the epidemic. since the first aids ngos were established in china in the 1990s, chinese aids ngos have made unique contributions to china’s aids response in critical areas like access to drugs, support for treatment compliance, outreach to marginalized at-risk groups, and efforts to reduce stigma among marginalized populations. however, there has been a substantial drop-off in donor funding in recent years, and although the chinese government has filled the funding gap, demonstrating its commitment to the sector, recent policy moves toward greater control over the work and funding of ngos threatens their survival. thus far, china’s aids response has been noteworthy, but these new ngo funding and regulatory developments pose significant challenges to the next phase of outreach, prevention, treatment, and care. in the last 15 years, china's aids response has gone from denial to global example. the combination of a strong national authority with resources and political will and a determination to apply evidence-based approaches has demonstrated the importance of both. in the years following the sars epidemic in 2003, china's public health leaders have aggressively analyzed and addressed their hiv/aids epidemic, putting in place pragmatic new policies and programs like harm reduction for people who inject drugs (pwid) and test-and-treat programs for large numbers of previously unidentified people living with hiv (plwh). in china, where government is the main actor for policy formulation and service provision, the role of nongovernmental organizations (ngos) has historically been weak. nevertheless, china's hiv/aids response has provided an opportunity for its fledgling ngo community to demonstrate its importance as an essential partner in achieving national goals, in this case the control of china's hiv epidemic. globally the hiv/aids response relies on active participation of ngos and civil society, and important progress has been made in the response due to their advocacy in critical areas like access to medicines, treatment compliance support, and outreach to marginalized at-risk groups. china has opened the door for ngo participation in the response, partly due to donor pressure, but also because of official acknowledgment of the important role these groups must play in controlling the epidemic. there is now strong policy support for the role of ngos in china's hiv/aids response, but this role is under threat as the political space for ngos shrinks. this may threaten future efforts to control china's hiv epidemic. in this chapter, the unique contributions of china's hiv/aids ngos and the expansion of the political space for their role over the last 20 years are reviewed. additionally, recent government moves to more closely control ngos, and their work and funding through more restrictive legislation is discussed as this may undermine china's success in controlling the epidemic in the coming years. since the beginning of the global aids epidemic, ngos have played a decisive role in the response, both for advocacy and for services. groups representing plwh have a mandated role in most international and national aids policy, program, and funding bodies. advocacy groups representing gay men and aids patients in the "global south" have put pressure on governments and industry to increase funding and access for treatment and prevention programs. this led to new norms about ensuring access to essential medicines and lifesaving drugs in poor countries. services provided by aids ngos, for outreach to gay men and other men who have sex with men (msm), condom distribution and promotion for sex workers, syringe exchange for pwid, youth sex education programs, sex worker legal protection services, treatment and support to aids patients, aids orphans support programs, and many other critical parts of global and national aids programs, have been a key feature of successful aids responses in many countries. these groups have been able to reach and represent hidden and underrepresented groups with essential risk-reduction education, recruitment for hiv testing and treatment, and assistance in accessing care and support. these ngos have also played an important role in representing the needs and perspectives of marginalized groups in policy and program formulation. once effective aids treatment became available in the 1990s, community groups and local ngos became the backbone of aids treatment support in rural communities where trained medical personnel are scarce (farmer et al. 2001 ). many of the important advances in the aids response have come through ngo organizing and advocacy. for example, act up in the united states (us) pushed for fast tracking approvals for aids medications. the treatment access campaign in south africa spearheaded a global movement for affordable aids drugs (heywood 2009 ). and finally, sonagachi in india demonstrated that protecting sex workers' rights increased their condom use and found that in project cities, hiv prevalence among sex workers was 11% compared to other indian cities where it exceeded 50% (jana et al. 1999) . these examples and many others attest to the effectiveness of both peer group approaches and prevention services provided by ngos and their value for supporting treatment programs in their communities. the participation of ngos in the aids response is mandated in numerous international agreements from global agencies. the joint united nations programme on hiv/aids (unaids) and the united nations (un) general assembly (ga) both support the essential role of civil society in responding to the epidemic, and unaids includes ngo representatives on its board. the global fund to fight aids, tuberculosis, and malaria (hereafter the global fund) requires civil society participation in its governance board, the ccm (country coordinating mechanism), for proposal submission and execution. furthermore, the "gipa" principle ("greater involvement of people living with hiv/aids") is promoted by unaids and calls for the greater participation in the response of people infected and affected by hiv. this has become an important mechanism for patient groups to form and participate in policy and program decision-making as well as service delivery. these global norms have secured a novel and secure political space for ngos in the global aids response (sidibé et al. 2010 ). while ngos are fully part of the architecture for service provision, governance, and advocacy in many countries, they have a more restricted and different role in china. there has been rapid growth of the civil society sector in china in the last 20 years. as of 2016, there were approximately 7000 foreign ngos in china and many times more domestic ngos-roughly 675,000 that were registered and perhaps as many as three million unregistered (hsu et al. 2016) . in other countries ngos often deliver direct services, but in china, the government system is the main provider of health and other social services to its vast population. as the number of chinese ngos has expanded, some have assumed roles in service delivery in partnership with government, but most operate as research organizations and as advocacy groups on issues like the environment, energy policy, hiv/aids, consumer rights, legal reform, and other issues of social and political concern. many of these organizations are linked to global and transnational networks, which have provided important support both financially and on strategy development, often in collaboration with global agencies and bilateral and multilateral donors. unique to china is the gongo sector-government-sponsored ngos. these parastatal agencies operate under the leadership of respective technical ministries or party-led organizations, and they are often led by former government officials. many receive government funding to carry out community-based or donor-funded initiatives through their networks at province and local levels. the ngo sector in china is highly restricted and governed by laws and regulations aimed at preventing anti-government political actions. the registration process for independent chinese ngos has involved registration with the ministry of civil affairs, which has included identification of a sponsoring government agency and a minimum amount of operating capital. requirements for social organizations include having an office, professional employees, and funds of 30,000 rmb, while requirements for "people-run non-enterprise" organizations include having an office, professional employees, and funds of 30,000-50,000 rmb. many ngos do not bother to officially register with the ministry of civil affairs and operate either without any registration or register as a company, a much easier path to legal status. some service-providing ngos work closely with government agencies or with gongos either through direct contract work or as members of advisory and governance boards, like the global fund's ccm. but other ngos, especially those doing advocacy or research, can be viewed with suspicion by government leaders, especially at the local level, who see them as outside government supervision and authority structures. there are restrictions on establishing nationwide networks (except for gongos). a new law, which went into effect at the start of 2017, has shifted oversight of foreign ngos from the ministry of civil affairs to the public security bureau, a move that will likely increase local mistrust. under the new law, foreign ngos are subjected to close scrutiny by the government and by police who have been given the right to question workers, inspect and close offices, review documents, and seize assets. only a fraction of foreign ngos in china has yet re-registered under the new law, and it is expected that some will instead pull out of the country (gan 2017). in china as elsewhere, the highest-risk groups for hiv infection are often among the most highly stigmatized groups in society (e.g., gay men, drug users, sex workers). the necessity of reaching these groups with hiv prevention services has contributed to limited societal acceptance and to a more sympathetic attitude to the ngos representing them. the hiv/aids response has often been cited as an example of how the policy environment has expanded for ngos in china. the combination of internal recognition of the essential roles that such ngos have played in the global response and external pressures and advocacy through funding mandates and global norms promoted by international partners has pushed forward a more embracing policy environment (kaufman 2012 ). the first time a government program involving ngos became involved in china's aids response was in 1997 and happened somewhat accidentally. this represented a milestone event in the history of ngo participation in china's response to hiv/ aids. australia's foreign aid agency, ausaid, provided 2 million australian dollars as a supplemental grant to a 100 million us dollar world bank project in china on disease prevention, known as health 7. health 7 worked with the ministry of health and the chinese academy of preventive medicine, which was the predecessor of the chinese center for disease control and prevention (china cdc). the initiative targeted 17 provinces and cities. the ausaid funds were exclusively to support ngo participation in hiv/aids programs. at that time, china had limited funding in the health sector and government institutes had little funding for hiv/aids programs. even while government partners were surprised and a bit shocked by the ausaid mandate, china's government felt obliged to follow the donor's requirement. but china aids ngos at that time had limited capacity and were unable to write project proposals or draft work plans. thus, the government worked with the ngos, wrote work plans for them, and gradually helped them increase their capacity. following the example of health 7, the united kingdom's department for international development (dfid) provided support for ngos for the world bank's basic health services project (health 8), which began in 1998. in china, real action often only happens in response to political will demonstrated via public endorsement by political leaders. the importance of such endorsements cannot be overstated. beginning in 2003, chinese government leaders have increasingly endorsed the role of ngos in the hiv/aids response. at the end of 2003, wu yi, who served as the vice-premier of the state council from 2003 to 2008 and as the minister of health from 2003 to 2008, clearly announced her support for a greater role for ngos in china's aids response and endorsed efforts to build a framework for government and ngo cooperation to effectively control and prevent the spread of hiv (cctv international, 2004) . her statements were echoed by many other high-ranking government officials including the then-party secretary to the ministry of health, gao qiang. officials in china's ministry of health stated that they hoped to further promote cooperation between government and ngos in hiv/aids control and prevention by way of public bidding and purchase of services. a new policy entitled "regulation on the prevention and treatment of hiv/ aids," drafted by the ministry of health and issued by the state council in 2006, provided an official endorsement and framework for promoting ngo participation in hiv/aids prevention and control. this was further elaborated in the state council's "decree no. 48" in 2010 (state council 2010), which contained language endorsing the purchase and contracting of services from ngos, as follows: reflecting this change in attitude, top leaders now regularly express their support for hiv/aids ngos and include ngos in important relevant meetings. former premier wen jiabao invited ngo leaders to participate in a panel discussion and premier li keqiang has done so as well. in november 2012, ahead of december 1, international aids day, china's then newly designated prime minister li keqiang promised to offer greater support to ngos tackling hiv/aids in china and has kept this promise with a special fund to support their work. these changes indicate the large government change in attitude toward aids ngos and their important role in supporting the formal health system. as noted above, the initial inclusion of ngos in china's hiv/aids response was driven by external forces (bilateral donors), even with little confidence among chinese officials that they had any real role to play. now, government officials and health sectors, even at local levels, recognize the important roles that ngo and community organizations play in the hiv/aids response in china and that without their active participation and critical contributions, it would be impossible to achieve national goals. support for aids ngos was enshrined in china's 12th five-year plan (2011-2015) and again in its 13th five-year plan (2015-2020). the hiv/aids action plan portions of these policies and related monitoring and evaluation frameworks contain clear language about the roles of ngos and include specific indicators for measuring it. at the local government level, support for ngos can vastly differ. some local governments have had long-term cooperation with ngos through work in poverty alleviation and social welfare and have had good experiences. for those, cooperation with hiv/aids ngos has been easier. in many instances they have been able to establish effective models for shared service provision and have provided funding for ngo partners. for example, in sichuan province in 2008, the provincial government declared its intention "to promote social forces to participate in aids prevention and treatment" and passed china's first local legislation to this effect. item 14 of the legislation states that "non-profit organizations held by social forces (including enterprise, public unit, village/community committee, civil organization and other related organization and individuals) and aiming at hiv/aids prevention and control, after being examined and approved by their local health administration, civil affair departments should approve their registration in accordance with related regulations." this provided professional authority for ngo participation in local hiv/aids programs. but in most places, local officials prefer to work with gongos, such as the labor union, communist youth league, and women's federation, rather than independent ngos. they are concerned about the political risk of engaging with independent ngos, a concern that has grown in recent years. although hiv/aids prevention and control efforts by gongos may be valuable, they rarely represent local communities. china has two main gongos working on hiv/aids, the chinese association for hiv/aids and std prevention and control (caspac) and the china preventive medicine association (cmpa), both with provincial and local branches, as well as many independent ngos operating at the national and local levels. both gongos are led by former government officials from the china cdc or ministry of health. both have been active and valuable players in china's aids response, serving as implementing agencies for government-supported and donor programs, especially the global fund and the bill and melinda gates foundation. in both instances they have served as pass-through organizations for many independent ngos and provided capacity building training and assistance in operations and management of funds. the independent ngos, however, are more legitimate representatives of their communities and to some degree have suffered in their own growth and funding by the concentration of funding to gongos instead of to them directly. the number of independent chinese aids ngos has grown rapidly in the last 20 years and peaked during the years in which the global fund provided funding from 2003 to 2012. the earliest groups operated online newsletters, networking activities, and education and outreach efforts. as funding increased for hiv prevention and care, many of the volunteer groups working with marginalized populations and plwh evolved and professionalized. china only openly acknowledged the extent of its aids epidemic in 2003 following the sars crisis. prior to that, the epidemic was played down, the government's response was weak, and policy was poorly organized (kaufman and jing 2002) . the epidemic was represented as mainly limited to china's southwest border areas and mostly among pwid. there were few independent chinese ngos working on hiv/aids. the china-uk hiv/aids prevention and care project funded by the united kingdom's department for international development (dfid) was the first bilateral project to tackle china's growing aids epidemic, even before official acknowledgment of the seriousness of the epidemic in 2003. launched in 1999 and working in yunnan and sichuan provinces in china's southwest, the china-uk hiv/aids prevention and care project provided important early funding to ngo outreach activities among msm, commercial sex workers, and pwid and helped foster good working relations with local government. groups like the chengdu community cares group (working with gay men) and daytop in yunnan (working with drug users) received funding and technical assistance to work with marginalized groups at risk for hiv. the program replicated best practices from neighboring countries like thailand for hiv prevention among sex workers and aimed to promote 100% condom use (yip 2014) . from the early 1990s, the ford foundation's china office also provided support for independent aids ngos in yunnan, beijing, and elsewhere. this funding built a backbone of technically strong, independent ngos working on hiv prevention for msm, sex workers, and youth and representing plwh. groups like yiteng, based in hong kong, worked to educate and protect the legal rights of chinese sex workers in hong kong and on the guangdong-hong kong border. aizhi action, an online platform started by chinese aids activist yanhai wan, documented the emerging and unacknowledged epidemic among paid plasma donors in henan province and profiled the work of dr. yaojie gao, an outspoken doctor on the frontlines of the epidemic calling for government accountability and action. it was one of the earliest accurate sources of information on the aids epidemic among paid plasma donors in central china and has remained an important advocacy group for the villagers in henan province who were inadvertently infected. positive art, a beijing-based art collective, and mangrove group at ditan hospital were two of the earliest support groups for plwh. finally, the chinese alliance of people living with hiv/aids (cap+), launched in the early 2000s, formed a larger coalition for grassroots groups, bringing together diverse groups such as rural women and young men living with hiv and linking them to the global network of people living with aids (gnp+) and other global and regional networks of plwh (kaufman et al. 2014) . one important group, friends exchange (with modest support by ford foundation, then barry and martin trust, and later unaids), played a critical role in mobilizing hiv prevention among msm in china. hiv infection among msm continues to grow in china. while now an acknowledged problem, at that time it was still a hidden issue. as of the end of 2015, 28.3% of all newly diagnosed hiv cases in china were acquired via male-male sexual contact. it was up more than tenfold since 2006 from 2.5% (pisani and wu 2017) . in the words of china's top aids prevention official, dr. zunyou wu, "the expanding epidemic among msm is undoubtedly the gravest of these new challenges regarding transmission of hiv." in the late 1990s, it was nearly impossible to find an entry point for work with msm in china. homosexuality and homosexual behavior were a hidden reality, highly stigmatized, and not acknowledged by the government, and there were no obvious groups with which to engage. friend exchange, an underground magazine that was providing information on safe sexual practices for homosexual men in china, was the exception, with each issue passed hand to hand so that it reached a wide audience. the magazine played a unique role in providing aids education to msm and also gave voice to a stigmatized and isolated community. the magazine eventually became official and began formal publication. the magazine's editor, beichuan zhang, a professor at qingdao medical university, also gave voice to the community's perspectives. the increasing tolerance and understanding of homosexuality in china is in no small way due to the influence of friend exchange and beichuan zhang's tireless efforts to humanize individual stories of stigma, the power of family expectations and roles in chinese society, and the journey and progress of sexual rights in china. at the same time, the publication opened political space for engagement and work on sexual rights and aids prevention, in part by challenging social conventions and forcing the chinese psychiatry association to remove homosexuality from its list of mental diseases (zhang and kaufman 2005) . published for 11 years, friend exchange reached countless homosexual, bisexual, and transgender men and women in china with lifesaving information about aids prevention as well as psychological support and understanding for lifestyles and sexual orientations that are still highly stigmatized in china. it became the most respected and authoritative source of information on homosexuality and lgbt (lesbian, gay, bisexual, transgender) issues in china, conducting countless surveys among its extensive readership and thereby generating information from the community itself on critical issues (e.g., condom use, numbers of sexual partners, attitudes toward safe sexual practices) for the understanding of the hiv epidemic among this key, vulnerable population. after 2003, donor funding for hiv/aids programs in china increased substantially and more funds were directed to the ngo sector. the global fund contributed over 400 million us dollars, the bill and melinda gates foundation provided 50 million us dollars, and usaid supported work in china's southwest provinces, all three with funding going directly to ngos. in china, the global fund entered the scene in the early 2000s and between 2003 and 2012, with six rounds of funding for aids programs, worked closely with the chinese government on numerous projects aimed at supporting government and ngo efforts to prevent and treat hiv, with specific funding earmarked to support organizations within civil society (huang and ping 2014; kaufman 2012) . as part of the global fund mandate, a country coordinating mechanism (ccm) was established with ngo representatives. this normalization of ngo participation in governance opened the door for the chinese aids ngo community to really demonstrate their value and build effective partnerships with national and local government agencies. empowered by donor engagement and funding, many groups professionalized and moved from volunteer organizations to organizations with greater capacity both technically and managerially. government and donor funding supported this process with trainings and capacity building workshops. when the global fund was established in 2002, its own mandated governance mechanism required establishment of a "country coordinating mechanism" (ccm) with civil society representatives to review, approve, and submit applications. china established a ccm, but initially it worked more as a "rubber stamp" for applications developed and executed by china's ministry of health and the china cdc. domestic aids ngos had a limited voice in the process. in 2006, the first ngo election was held to elect an ngo representative to the ccm. however, it was disputed, which precipitated a thorough review by the global fund and unaids. the result was a new election that was uniquely transparent, participatory, and accountable. the election, facilitated by the international republican institute (iri), a us ngo that has worked around the world to promote democratic elections, provided an opportunity for internet discussions and networked disparate groups around the country. several widely attended local meetings brought groups together often for the first time, with iri, unaids, and donor representatives to teach them how to conduct the elections. the election resulted in two elected representatives and two ngo committees, each constituted with 11 elected representatives from groups representing people with hemophilia, msm, former blood plasma donors (fbpd), and migrant workers (zhang and kaufman 2012) . the global fund election controversy and resolution served as a door opener for ngo participation in the aids response in china and established a mechanism, albeit still limited, for input by ngos into china's aids response. the global fund's round 6 was seen by many as a further mechanism to institutionalize the role of aids ngos in china's aids response because all of the fund's contributions were to be dispersed by ngos rather than by the government. however, its intended implementing agency (principal recipient) was switched at the last moment from a gongo, the china association for std and aids prevention and control, to the government's aids agency, the national center for aids/std control and prevention (ncaids) of the china cdc. after a negative review of the china program, china's global fund program was suspended in 2011. conditions tied to lifting the suspension of global fund moneys included the creation of a new sub-recipient that truly represented ngos and a commitment to channel 25% of funding to ngo groups (wong 2011) . however, before that happened, the global fund's round 11 grant to china was cancelled amid pressure from some donor countries to deploy global fund resources to africa instead of middle-income countries like china. the chinese government then announced that it would use domestic resources to substitute for cancelled global fund moneys and accelerated a process to contract directly with local ngos for program implementation, indicating the government's recognition of the essential role that ngos play. the acknowledgment of the need for ngo implementation resulted partly from the initial pressure and requirement from the global fund and partly from networking and advocacy by the groups themselves aided by external partners like iri, the hiv/aids alliance, icaso, pact, and other international ngos. china global fund watch, established around 2004, was an ngo that monitored compliance with global fund rules and published a regular online newsletter, modelled on a similar global watchdog organization (aidspan, which publishes the "global fund observer" online newsletter). china global fund watch played a leading role in publicizing the suspension of aids funding to china by the global fund and in representing the position of china's grassroots ngos in calls for reform of the governance mechanism of the china global fund grants (wong 2011) . china global fund watch played a watchdog role in raising and publicizing many issues and developments with china's own substantial global fund grants. the organization formed close alliances with similar groups in the region and the world and served as a forum for raising other important issues related to access to essential aids medicines, laws compensating plwh who were accidentally infected through medical procedures, and other sensitive issues. an important group working on the frontlines of stigma against plwh has been aids care china. this group, started by a guangzhou man who became infected with hiv and was evicted from his apartment when his hiv status became known, has gone on to become one of china's most well-established aids ngos, receiving funding from both the chinese government and international donors. originally focused on addressing stigma and providing shelter for plwh who were evicted from their homes, the group received substantial funding from donors and developed and expanded into a professional organization working in four provinces with counseling and treatment education centers known as red ribbon centers. these centers focused on improving treatment outcomes and adherence to medication by working with healthcare providers and hospital officials. aids care china and its founder and leader, thomas cai, received the red ribbon award from the un for work supporting care and treatment of plwh. funding for civil society from the global fund was critically important for the growth and development of china's aids ngo sector. many volunteer msm and plwh groups in many cities developed during this period and played a major role in china, reaching out to their communities with information and support often through contracted work to local cdcs. ngos working with aids orphans in central china (e.g., chi heng, aids orphan salvation association, and china orchid) emerged and began working closely with government agencies. all had designated representatives of their communities on the global fund's civil society board, the ccm. the bill and melinda gates foundation also provided substantial funding for hiv prevention during this period, mainly focused on testing for msm and prevention and treatment for plwh (yip 2014) . while most funding was channeled through the ministry of health and china's two main aids gongos (china preventive medicine association and china association for std and aids prevention and control), money passed through to local branches of these organizations, which engaged with local msm ngos to reach their communities with hiv testing. the testing program was controversial because those who chose to test were paid. however, the effort was important for helping to demonstrate the effectiveness of new, internationally recognized "test-and-treat" strategies, which are now a major thrust of china's national hiv/aids program. the last several years have seen a substantial drop-off in international funding for hiv/aids programs in china as china's economy has grown. today, its own resources and technical capabilities are the primary driver of its hiv/aids response, and global donors have called for a re-direction of funding to countries in greater financial need (chow 2010) . the global fund cancelled round 11 and ended its new funding to china. the bill and melinda gates foundation, usaid, and other donors have shifted their focus from supporting china's own response to hiv/aids to a "china in the world" orientation, focused on how to work with china as a development partner or source for lower-cost drugs in africa and elsewhere. an unfortunate consequence of the drop-off in funding has been the drying up of support for china's ngo sector working on hiv/aids. after the global fund's round 11 program to china was cancelled, the chinese government announced that it would use domestic resources to substitute for cancelled global fund moneys and accelerated a process to contract directly with local ngos for program implementation. but while some organizations have continued to operate with chinese government support contracted locally by local cdcs who depend on them for outreach, many others, especially those working on advocacy rather than service delivery, have struggled to continue their missions. as an indication of how valued the ngo service contribution to the aids response now is in china, the chinese government set up a special fund in 2015, exclusively for the support of ngos to ensure their continued input. the ministry of finance allocated 30 million rmb, but premier li keqiang felt this was insufficient, so he used his premier funds to add another 20 million rmb, for a total of 50 million rmb in 2015. the fund supports ngos to do outreach work with the msm, sex worker, drug user, and plwh communities. while the space afforded civil society organizations and ngos has expanded greatly over the past two decades, it appears to again be contracting. the foreign ngo law, which was passed in april 2016 and took effect in january 2017, shifted responsibility for foreign ngo oversight to the ministry of public security from the ministry of civil affairs and significantly increased the bureaucratic and regulatory requirements for all not-for-profit foreign organizations that work in china or provide funding to china-based organizations. restricting foreign funding to chinese ngos has adversely affected the operations of the domestic advocacy ngos even further. and, in light of the reductions in foreign donor funding in recent years, this could have serious negative consequences for china's hiv/aids response. many of the organizations highlighted in this chapter that have played a critical role in advocacy and research had already felt the impact of the draft law, which was issued in 2015. in many cases, their foreign funders retreated for fear of being out of legal compliance. yirenping, an ngo that has worked on employment discrimination against hiv and hepatitis b virus patients, had already been targeted by those tasked with enforcing this new law, most likely because of the foreign donor that supported its efforts. this law is one of a suite of new state security-oriented legislation recently passed. the ngo law, while primarily aimed at preventing foreign-funded civil society groups from organizing political movements that might challenge the authority of the current regime, has spilled over into work in the social sectors, even in areas like hiv/aids which is fully supported by the government. the type of donor-supported efforts that led to the development of ngos and the expansion of their roles in china's aids response for stigmatized groups like msm and criminalized populations like commercial sex workers and pwid is threatened at a time when access to these marginalized populations is essential for achieving national goals. global experience demonstrates, that it is nearly impossible to achieve success in aids prevention without actively involving the communities themselves in efforts to reach their members. in china, government is the almost exclusive provider of public services, and since 2002, the china cdc has done an admirable job of tackling many sensitive aids prevention challenges and achieved many successes in controlling and responding to the epidemic. the government frequently contracts with ngos for service provision for marginalized groups, especially msm and plwh, and has helped to begin normalizing the sector and formalizing the partnerships. however, although government effort is necessary, it is not sufficient to achieve success in hiv prevention among msm and other marginalized and stigmatized groups. ngos representing these groups are becoming less able to play the roles required of them because of the tightening political situation for chinese ngos accompanying the new foreign ngo law and reductions in overseas funding. these organizations, which are critical for reaching communities throughout china, are plagued by the lack of core financing (as opposed to project funding) especially in recent years and often depend too much on volunteers rather than staff. many groups have struggled to grow beyond their original vision and leadership and often compete for the more limited donor and government funds that trickle down to groups on the frontline of the epidemic. this situation is likely to worsen and may lead to the demise of some important groups that are unable to secure funding. to some degree, financial competition contributes to a lack of cohesion in the sector, further constrained by a regulatory environment that prohibits establishment of branches in different places (except for the gongo sector) or transnational networking. however, competition and poor capacity among china's ngos should not be used as an excuse to dismiss their value and necessity. without genuine capacity, core funding, networking, and better governance, these organizations are unable to play their much-needed role. true partnership of these ngos with government, the main service provider, architect, and enabler of policies and programs in china, is essential. without active engagement of china's aids ngos in china's hiv/aids response, the chinese government's effort to control the aids epidemic will be only partially successful. china's hiv/aids epidemic response has been noteworthy, but these significant challenges to the next phase of prevention must be taken seriously. non-governmental organizations will play a greater role in the field of aids prevention and control. cctv international china's billion-dollar aid appetite communitybased approaches to hiv treatment in resource-poor settings why foreign ngos are struggling with new chinese law-thousands could be operating in a risky legal limbo south africa's treatment action campaign: combining law and social mobilization to realize the right to health the state of ngos in china today ny: council on foreign relations creating an enabling environment: lessons learned from the sonagachi project china's evolving aids policy: the influence of global norms and transnational nongovernmental organizations china and aids-the time to act is now gender and reproductive health in china: partnership with foundations and united nations singapore: people's medical publishing house people, passion and politics: looking back and moving forward in the governance of the aids response notice of the state council on further strengthening hiv/aids response. regulation on the prevention and treatment of hiv/aids global fund lifts china grant freeze international philanthropic engagement in three stages of china's response to hiv/ aids the rights of people with same sex sexual behavior: recent progress and continuing challenges in china new paradigm of aids governance. beijing: peking union medical college press key: cord-013481-3zwq67do authors: guo, kejun; shen, guannan; kibbie, jon; gonzalez, tania; dillon, stephanie m.; smith, harry a.; cooper, emily h.; lavender, kerry; hasenkrug, kim j.; sutter, kathrin; dittmer, ulf; kroehl, miranda; kechris, katerina; wilson, cara c.; santiago, mario l. title: qualitative differences between the ifnα subtypes and ifnβ influence chronic mucosal hiv-1 pathogenesis date: 2020-10-16 journal: plos pathog doi: 10.1371/journal.ppat.1008986 sha: doc_id: 13481 cord_uid: 3zwq67do the type i interferons (ifn-is) are innate antiviral cytokines that include 12 different ifnα subtypes and ifnβ that signal through the ifn-i receptor (ifnar), inducing hundreds of ifn-stimulated genes (isgs) that comprise the ‘interferome’. quantitative differences in ifnar binding correlate with antiviral activity, but whether ifn-is exhibit qualitative differences remains controversial. moreover, the ifn-i response is protective during acute hiv-1 infection, but likely pathogenic during the chronic stages. to gain a deeper understanding of the ifn-i response, we compared the interferomes of ifnα subtypes dominantly-expressed in hiv-1-exposed plasmacytoid dendritic cells (1, 2, 5, 8 and 14) and ifnβ in the earliest cellular targets of hiv-1 infection. primary gut cd4 t cells from 3 donors were treated for 18 hours ex vivo with individual ifn-is normalized for ifnar signaling strength. of 1,969 ifn-regulated genes, 246 ‘core isgs’ were induced by all ifn-is tested. however, many ifn-regulated genes were not shared between the ifnα subtypes despite similar induction of canonical antiviral isgs such as isg15, rsad2 and mx1, formally demonstrating qualitative differences between the ifnα subtypes. notably, ifnβ induced a broader interferome than the individual ifnα subtypes. since ifnβ, and not ifnα, is upregulated during chronic hiv-1 infection in the gut, we compared core isgs and ifnβ-specific isgs from colon pinch biopsies of hiv-1-uninfected (n = 13) versus ageand gender-matched, antiretroviral-therapy naïve persons with hiv-1 (pwh; n = 19). core isgs linked to inflammation, t cell activation and immune exhaustion were elevated in pwh, positively correlated with plasma lipopolysaccharide (lps) levels and gut ifnβ levels, and negatively correlated with gut cd4 t cell frequencies. in sharp contrast, ifnβ-specific isgs linked to protein translation and anti-inflammatory responses were significantly downregulated in pwh, negatively correlated with gut ifnβ and lps, and positively correlated with plasma il6 and gut cd4 t cell frequencies. our findings reveal qualitative differences in interferome induction by diverse ifn-is and suggest potential mechanisms for how ifnβ may drive hiv-1 pathogenesis in the gut. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the type i interferons (ifn-is) are innate antiviral cytokines that include ifnα (12 different subtypes) and ifnβ [1] . these cytokines significantly inhibited hiv-1 replication in vitro, but human clinical trials with ifnα2 showed only moderate or no inhibitory effects on hiv-1 infection [2, 3] . all ifn-is bind to the same ifn-i receptor that is composed of two subunits, ifnar1 and ifnar2, resulting in phosphorylation of jak1 and tyk2. this in turn results in the phosphorylation of stat1 and stat2 that associate with irf9 to form the transcriptional activator, isgf3 [4] . isgf3 translocates to the nucleus, where it binds to promoters of genes encoding ifn response elements (isres), resulting in the induction of hundreds of ifn stimulated genes (isgs), collectively referred to as the 'interferome' [5] . recent studies highlighted ifnα as a potential adjunct to an hiv-1 curative strategy [6-10]. however, almost all hiv-1 clinical trials with ifnα were performed with only one subtype, ifnα2, with mixed results (reviewed in [2, 3] ). more recently, ifnα2 treatment of siv rhesus macaques under antiretroviral therapy did not reduce the latent hiv-1 reservoir [11] . our group and others reported that ifnα2 only moderately inhibited hiv-1 in humanized mice and primary cd4+ t cells compared to the more potent subtypes ifnα6, ifnα14 and ifnα8 [12] [13] [14] [15] . interestingly, the anti-hiv-1 potency of ifnα subtypes correlated with their binding affinity to ifnar2, suggesting that the antiviral differences between the ifnα subtypes were due to quantitative differences in ifnar signaling strength [12, 15, 16] . however, several human ifnα subtypes exhibit strong signals of purifying selection [17] , suggesting that these ifnα subtypes may have evolved to have specific, nonredundant functions. some ifnα subtypes were better at inducing certain immune responses in vivo [13, 18] and may trigger distinct intracellular signaling pathways [19] . nevertheless, the notion of qualitative differences between ifnα subtypes remains controversial [20, 21] . one reason is that most comparative studies normalized ifn-is using protein amounts or units/ml based on inhibition of vesicular stomatitis virus or encephalomyocarditis virus [1] . to unravel qualitative differences between the ifn-is, it would be important to normalize ifn-is for quantitative differences in ifnar signaling strength. it is widely accepted that ifn-i signaling can prevent acute retrovirus infection. genetic ablation of ifnar resulted in higher friend retrovirus replication in mice co-infected with lactate-dehydrogenase elevating virus, a potent ifn-i inducer [22] . moreover, ifnar blockade increased siv replication in rhesus macaques [23, 24] . administration of ifnα2 decreased retrovirus replication in mice, monkeys and humans [3, 18, 23] . however, in persistent virus infections, chronic ifn-i stimulation was linked to pathogenic outcomes [25] [26] [27] [28] [29] . although clinical administration of ifnα2 increased the number of low-dose mucosal inoculations needed for breakthrough siv infection in rhesus macaques, once the infection was established, lower cd4 t cell counts were observed in ifnα2-treated monkeys relative to untreated controls [23] . these findings highlight that hiv-1 infection shares features with 'interferonopathies' such as aicardi-goutières syndrome [30] , which are currently being targeted through ifn-i blockade strategies (clinicaltrial.gov nct03921554). in fact, ifnar blockade during chronic hiv-1 infection in humanized mice restored immune function, leading to better hiv-1 control [31, 32] . neutralization of (most) ifnα subtypes in siv-infected rhesus macaques prior to infection increased viral loads as expected, but also decreased subsequent immune activation profiles [24] . by contrast, blockade of ifn-i signaling in chronic sivtreated and untreated rhesus macaques decreased inflammation profiles associated with isgs but did not reverse t cell exhaustion or activation [33] . the basis for the protective versus pathogenic effects of ifn-is remains unclear. one hypothesis is that the initial ifn-i response is protective due to the induction of antiviral factors, whereas chronic stimulation promotes inflammation through other isgs with sustained, elevated expression. distinct ifn-is present in the acute versus chronic stages of persistent viral infection may induce divergent cellular immune responses. tissue compartmentalization may also play a role. the gut is a critical site not only for early hiv-1 infection, but also in driving chronic immune activation [34] . epithelial barrier dysfunction, partly due to the loss of th17 cells, leads to the translocation of enteric bacteria from the gut lumen to the lamina propria and systemic circulation, resulting in chronic immune activation [35] [36] [37] . we recently reported increased ifnβ gene expression in the gut, but not the blood, in persons with hiv-1 (pwh) infection compared to age/gender-matched hiv-1 uninfected controls [38] . by contrast, ifnα subtypes were downregulated in pwh, and ifnλ, a type iii ifn linked to mucosal immunity in mouse models [39] , was undetectable in these samples [38] . these findings suggest that among the diverse ifn-is, ifnβ may play a dominant role in the gut during chronic hiv-1 infection. here, we utilized transcriptomic approaches to evaluate whether the ifnα subtypes and ifnβ exhibit qualitatively different effects on gene expression. we then tracked how interferon-regulated genes were altered during chronic hiv-1 infection in the gut. our analyses highlight potential mechanisms driven by ifnβ that may influence mucosal hiv-1 pathogenesis. the relative anti-hiv-1 activity of ifnβ in primary lpmcs remains unclear, though studies using pbmcs suggest that ifnβ is relatively potent [40] . we previously reported a spectrum of antiviral potencies of the 12 ifnα subtypes against hiv-1 in primary lpmcs [12] . these data were extended to show a 10-fold difference in 50% inhibitory concentrations (ic50) between ifnα14 and ifnα2 [13] . using the same 6 lpmc donors used to calculate the ic50s of ifnα14 and ifnα2, we titrated the anti-hiv-1 potencies of ifnα1 (weak), ifnα8 (potent) and ifnβ. the ifn-is were added into lpmc cultures at the time of infection with hiv-1 bal . at 4 d post-infection, the percentage of hiv-1 gag p24+ cells were evaluated by flow cytometry. sigmoidal curves were fitted into the average inhibition data for 6 donors, and used to calculate ic50 values (pg/ml protein concentration). we also calculated a metric known as 'vres', which corresponds to the level of residual virus replication at maximal doses of ifn-is [41] . for comparison, previously reported inhibition curves for ifnα14 and ifnα2 are also shown [13] . ifnα8 showed ic50 concentrations over 10-fold lower than that of ifnα1 (fig 1) . notably, ifnβ had a similar potency as ifnα8 and ifnα14. we also calculated ic50s for individual donors (s1a fig) and show significantly lower inhibition by ifnα2 compared to ifnα8 and ifnα14 (s1b fig). ifnα8 and ifnα14 reduced hiv-1 infection levels to~30% at the maximal doses tested (10 ng/ml), whereas ifnα1, ifnα2 and ifnβ had higher vres between 38-42%. however, these differences in vres were not significant (s1c fig) . these findings validate prior results on the relative anti-hiv-1 activity of these ifnα subtypes and highlight ifnβ as a potent anti-hiv-1 ifn. quantitative differences were evident, as increasing the dose of weaker ifnα subtypes should enable these cytokines to inhibit hiv-1 just as well as the potent ifnα subtypes. the stronger anti-hiv-1 potencies of ifnα14 and ifnα8 compared to ifnα2 and ifnα1 were associated with higher isg induction [12] , but it remained unknown how the other ifnα subtypes and ifnβ compare. to test the isre signaling activity of ifn-is, we used a commercially-available ilite assay (see methods). the ilite cells are human u937 monocytic cell lines transduced with a luciferase reporter downstream of an isre from isg15, a canonical isg. serial 10-fold dilutions of the 12 ifnα subtypes and ifnβ were added into the ilite cells and relative light units were measured after 18 h. 50% effective concentrations (ec50) were then calculated from best-fit sigmoidal plots. fig 2a highlights the 482-fold ec50 difference in isre-activity between ifnα14 and ifnα1. the ec50s of the other ifnα subtypes and ifnβ fell in-between ifnα14 and ifnα1 data were normalized to mock (untreated) as 100% for each donor. dose-response curves were generated using a one-phase decay equation in graphpad prism 5.0 to determine ic50 values. vres, the percentage of cells that remain infected relative to the mock at the maximum dose tested, corresponded to plateau values from the decay equation. � note that data on ifnα14 and ifnα2 were previously published [13] . https://doi.org/10.1371/journal.ppat.1008986.g001 type i interferons in mucosal hiv-1 infection ( fig 2b) . notably, the isre ec50 of the ifnα subtypes significantly correlated with anti-hiv-1 potency data from our previous study (fig 2c) [12] . a significant positive correlation was also observed between isre-activity and the ic50 values from the 5 ifn-is tested in fig 1 that includes ifnβ (r 2 = 0.87, p = 0.02). importantly, isre ec50 values correlated strongly with published ifnar2 binding affinity values when comparing the ifnα subtypes ( fig 2d) [16] . however, the inclusion of ifnβ, which was reported to have a higher ifnar2 binding affinity than multiple ifnα subtypes [42, 43] , abolished the correlation (fig 2e) . these data demonstrate that isre-activity as measured by the ilite assay can be used to evaluate quantitative differences between the ifn-is, particularly for the ifnα subtypes. our data in figs 1 and 2, as well as data from other studies [12] [13] [14] [15] , provide strong evidence for quantitative differences among the ifn-is. to uncouple quantitative versus qualitative differences, we normalized our ifn-i treatments for isre-activity ( fig 2b) for unbiased transcriptomics. purified gut cd4+ t cells from 4 different donors were treated with 100 pg/ml ifnα14, and the other ifn-is were added at higher concentrations that match the isre-activity of this ifnα14 dose (e.g., 48.2 ng/ml ifnα1). purified cd4+ t cells were evaluated instead ifnα subtypes that had potent activity against hiv-1 in a previous study are highlighted in black. ifnβ is highlighted in green. note that the ec50s are negative log values; thus the higher the bar, the less concentration is needed to achieve an ec50. (c) isre-activity versus hiv-1 inhibition. hiv-1 inhibition values were previously reported [12] showing % inhibition of hiv-1 p24+ cells relative to mock in lpmc cultures. isre-activity versus ifnar2 binding affinity (d) without or (e) with ifnβ. ifnar2 binding affinity data were previously reported using surface plasmon resonance. for panels c to e, linear regression curves were plotted using prism 5.0 and evaluated using pearson statistics. https://doi.org/10.1371/journal.ppat.1008986.g002 type i interferons in mucosal hiv-1 infection of total lpmcs to reduce potential confounders due to cell type heterogeneity in lpmcs when performing rnaseq. cd4+ t cells account for majority of cells in lpmcs (65%) [44] and are the main cell types for the interaction between hiv-1 and antiviral isgs. given that only limited numbers of primary lpmcs can be obtained per donor, we selected ifnα1, ifnα2, ifnα5, ifnα8 and ifnα14, as these were highly expressed in hiv-1-exposed primary pdcs in vitro and in pbmcs during chronic hiv-1 infection in vivo [12, 38] . we also selected ifnβ because it is significantly upregulated in the gut during chronic hiv-1 infection on average, we obtained 34.6 million (range: 10.4 to 167 million) sequence reads per sample (s1 table) . we first evaluated the transcripts per million (tpm) levels of isg15, from which the isre was genetically linked to luciferase in the ilite assay. all ifnα subtypes tested and ifnβ induced isg15 to similar levels ( fig 3a, s2b fig). in fact, the treatment dose used type i interferons in mucosal hiv-1 infection also induced the canonical isgs rsad2 (viperin) and mx1 (fig 3b) , as well as oasl, bst2 and apobec3g (s3 table) to similar extents. these data confirmed that the treatments were indeed normalized for isre-activity and ifnar signaling strength. overall, the 5 ifnα subtypes and ifnβ altered 1,969 genes. upregulated genes (isgs) were more prevalent (68-80% of irgs) than downregulated genes across all ifn-is ( fig 3c) . we next compared our irg set with irgs catalogued in the interferome database [5] . a majority (70.8%) of the observed irgs were found in the interferome database ( fig 3d) . we then partitioned the data into individual ifn-is. strikingly, there were, on average, 2.7-fold more ifnβ-regulated genes than those regulated by any of the individual ifnα subtypes tested (fig 3c) . on average, ifnβ upregulated 2.4-fold and downregulated 3.9-fold more genes than the individual ifnα subtypes (fig 3c) . there was a strong correlation between ifnar2 binding affinity and the number of irgs or isgs (r 2 >0.94, p<0.01), but these correlations were lost if ifnβ was removed (p<0.05). in addition, our current analysis revealed 578 novel irgs (s2 table) . many of these novel irgs may not encode protein products and/or have tentative gene designations, potentially explaining why these genes are not in the interferome database. however, some long non-coding rnas such as nrir (negative regulator of the interferon response) [47] and bispr (bst2 interferon stimulated positive regulator) [48] could have critical roles for modulating ifn-i responses. some repressed protein-coding genes such as ccr9 appeared specific to just one ifnα subtype (s2 table) . the ifnα subtypes are homologous genes that activate ifnar, raising questions on whether their differential effects were primarily quantitative. we postulated that if the differences between the ifnα subtypes are mainly quantitative, then we should observe a substantial overlap between the irgs of cells treated with different ifnα subtypes that were normalized for isre-activity. the number of irgs that overlapped between any two of the 5 tested ifnα subtypes ranged from 59% (ifnα2 vs ifnα5) to 82% (ifnα2 vs ifnα1) ( fig 4a) . this included a core set of 266 irgs altered by all 5 ifnα subtypes tested (fig 4b; s3 table) . interestingly, many additional genes appeared to be specific to an ifnα subtype, particularly for ifnα5 and ifnα14 ( fig 4b and s4 table) . to test if the irgs unique to each ifnα subtype could have been regulated by other ifnα subtypes but excluded due to a stringent fdr cut-off of 20%, we investigated the median fdr of these unique genes against the other 4 ifnα subtypes. this analysis is illustrated in s3 fig, where the fdr values of each of the 201 ifnα5-specific genes or 257 ifnα14-specific genes ( fig 4b) were plotted against the gene induction datasets for the other ifnα subtypes tested. as shown, majority of the ifnα5 or ifnα14-specific genes had fdr values that were >90% in the other ifnα subtypes. these analyses were expanded on s5 table, where ifnα-specific irgs had a median fdr of at least 75%, with most >90%, when tested against genes sets from other ifnα subtypes. thus, irgs that were differently expressed by a specific ifnα subtype were unlikely to be significantly altered by the other ifnα subtypes. we next evaluated trends as to whether irgs altered by at least one ifnα subtype (a total of 1,257 genes) were similarly upregulated or downregulated. these comparisons were based on mean fold-induction values that can be visualized in the heatmap ( fig 4c) . surprisingly, a large number of genes (n = 367) that trended to be upregulated by ifnα1, ifnα2, ifnα8 and ifnα14 appeared to be downregulated by ifnα5 (fig 4c, s6 table) . as the irgs were based on an arbitrary cut-off (�1.5× relative to mock, fdr �20%), we next evaluated whether increasing our fc criteria changed the differential expression patterns. at 2×, 2.5× and 3× cut-offs, we still observed genes that were differentially regulated by ifnα5 to determine whether ifnα subtypes induced molecular programs distinct from each other, we subjected the irgs to ingenuity pathway analyses (ipa) [49] . s7 table provides a ranked list of activated and inhibited pathways for each ifn-i. as expected, 'interferon signaling' and 'pattern recognition receptors' were the top induced pathways predicted for the 5 79.0% of ifnα2-regulated genes were found among ifnα1-rgs, whereas 59.7% of ifnα1-rgs were found among ifnα2-rgs. (b) euler diagram showing the interferome overlap between the 5 ifnα subtypes tested. a core set of 266 irgs altered by all 5 ifnα subtypes were detected. ifnα-subtype specific genes were highlighted (e.g., 243 for ifnα14, 201 for ifnα5). the total number of irgs (n = 1,257) include genes not shown that were shared between 2 to 4 ifnα subtypes. (c) heatmap of irgs from distinct ifnα subtypes. highlighted areas in red correspond to core isgs, whereas those in blue correspond to genes differentially regulated by ifnα5 relative to the four other ifnα subtypes tested. (d) ingenuity pathway analyses of ifnα subtypes, highlighting z-scores for shared pathways and those predicted to be specific to ifnα5 and ifnα14. https://doi.org/10.1371/journal.ppat.1008986.g004 ifnα subtypes tested ( fig 4d) . death receptor, nfκb and inflammasome signaling were also highly induced by all ifnα subtypes (s7 table) . interestingly, ipa also predicted distinct pathways induced by the ifnα subtypes. cd28 signaling, ctl and nk function, p38 mapk signaling and sumoylation were predicted for ifnα14 but not the other ifnα subtypes (s7 table and fig 4d) . only the ifnα5 interferome was associated with downregulated apoptosis, ppar and lxr/rxr activation, likely due to some downregulated genes such as casp9 (s6 table) . these differential predictions provide confirmatory evidence of qualitative biological differences in endpoint effector mechanisms induced by different ifnα subtypes. our data in fig 3c revealed~970 more irgs for ifnβ than the individual ifnα subtypes. we pooled ifnα regulated genes independently of the subtype and compared them to ifnβ-regulated genes. almost half (46.4%) of ifnβ interferome genes were not regulated by any of the 5 ifnα subtypes (fig 5a) . these included cytokines and cytokine receptor genes such as il2, ifngr1, il21r and tgfb1 ( fig 5a; s8 table) . we compared the ipa results for the ifnα subtypes versus ifnβ regulated genes. ifnβ induced more pathways (fig 5b) than the ifnα subtypes, such as 'th2 pathway', 'erk/mapk signaling' and 'regulation of actin motility'. these findings indicated that ifnβ induced a broader interferome than ifnα subtypes. moreover, compared to ifnα, ifnβ likely regulated more cellular pathways in primary gut cd4+ t cells. we recently reported that during chronic, untreated hiv-1 infection, ifn-i inducible antiretroviral genes apobec3g, bst2 and mx2, as well as ifnβ, but not ifnα, were expressed to significantly higher levels when compared to hiv-1 uninfected individuals [38] . to expand on these findings, we performed rnaseq on these gut biopsies to more broadly investigate the table 1 provides the demographic characteristics of this clinical cohort, which included 19 pwh and 13 hiv-1 uninfected individuals [50] . on average, we processed 41.2 million sequence reads from colon biopsies per subject (table 1) . filtered data were normalized using transcripts per million (tpm), trimmed mean of m values (tmm) [51, 52] and deseq2 [53] methods. based on relative log expression plots [54] , the tmm method was most efficient at removing unwanted variation (s6 fig) . we next determined the expression levels in the colon biopsies in vivo of the 266 'core irgs' that were similarly regulated by the 5 ifnα subtypes and ifnβ in gut cd4+ t cells in type i interferons in mucosal hiv-1 infection vitro. the majority (92%; n = 246) of these core irgs that passed the rnaseq filter criteria were isgs (e.g., these genes were induced by the ifn-is tested, not downregulated). of these 246 core isgs, 126 (51%) were significantly altered in hiv-1 infected versus uninfected individuals at 5% fdr ( fig 6a) . the majority (89%) of these altered core isgs were upregulated during chronic hiv-1 infection. these included the antiretroviral genes apobec3g, bst2 and mx2, consistent with our previous report [38] . genes linked to innate sensing, immunomodulation and cell death/proliferation, as well as negative feedback regulation of the ifn-i response, were also expressed at significantly higher levels in pwh (fig 6b and 6c , s9 table) . since ifnβ induced a broader interferome than all ifnα subtypes tested (fig 5a) , we evaluated whether isgs that were unique to ifnβ (ifnβ-specific isgs) were also induced in the gut during chronic hiv-1 infection. of the 712 ifnβ-specific irgs, 57% (n = 406) were isgs that passed the rnaseq filter criteria (fig 3c) . nearly a third of these ifnβ-specific isgs (28%; n = 112) were significantly altered in pwh compared to hiv-1 uninfected controls (fig 7a) . only a few of the ifnβ-specific isgs were significantly upregulated in pwh (fig 7a) . by contrast, the vast majority (>90% (n = 102) of the altered ifnβ-specific isgs were downregulated during chronic hiv-1 infection in the gut (fig 7a) . these repressed ifnβ-specific isgs are involved in intracellular vesicle trafficking, transcriptional/translational regulation, protein ubiquitination and transport (fig 7b and 7c ; s10 table) . moreover, several known hiv-1 type i interferons in mucosal hiv-1 infection cofactors such as tsg101, nup54, and cul5 were downregulated [55] [56] [57] . we again emphasize that all these genes were induced by ifnβ in gut cd4 t cells ex vivo (s8 table) . given that a great majority of these ifnβ-specific isgs were downregulated in pwh, we conclude that a significant inversion of ifnβ-specific isgs was observed during chronic hiv-1 infection in the gut. we next investigated potential mechanisms for how these ifnβ-specific isgs may have been downregulated in the gut in the presence of high ifnβ levels. two known negative feedback regulators, usp18 [58] and unc93b1 [59] , were induced by all ifn-is tested ex vivo (s3 table) . gut ifnβ mrna levels positively correlated with usp18 and unc93b1 transcripts (s12 table) . however, none of the ifnβ-specific isgs altered in pwh correlated with usp18 (s11 and s13 tables). by contrast, 89% of these ifnβ-specific isgs negatively correlated with unc93b1 (s11 and s13 tables). thus, one potential mechanism for the inversion ifnβ-specific isgs in the gut during chronic hiv-1 infection may be the ifnβ-mediated induction of unc93b1. the clinical study described in table 1 involved cohorts where paired blood (pbmcs/plasma) and colon pinch biopsies (up to 30 per donor) were obtained. colon pinch biopsies (~20) were type i interferons in mucosal hiv-1 infection pooled for same-day flow-based immunophenotyping [50, 60] and the rest were frozen for later histology and transcriptomics. the study obtained comprehensive data including gut and pbmc ifnα and ifnβ transcript levels, plasma and gut viral loads, gut cd4+ t cell percentages (of cd45+ cells), myeloid activation (cd40 mfi in cd1c+ myeloid dcs), blood cd4+ t cell counts, markers of microbial translocation (scd27, lps, lta), inflammation (crp, il6), and epithelial barrier dysfunction (ifabp) ( table 1 and s11 table) [38, 50, 60-63]. we investigated how individual altered core isgs (n = 126) and ifnβ-specific isgs (n = 112) correlated with these clinically relevant parameters using linear regression models, after adjusting the data for age and gender. we used a 5% fdr threshold for these associations. five clinically relevant parameters-gut ifnβ mrna, plasma lps, gut cd4 t cell frequencies, blood cd4 t cell counts and plasma il6 levels-correlated significantly with the altered core and ifnβ-specific isgs (fig 8 and s11 table) . the expression of core and ifnβ-specific isgs significantly correlated with transcript levels of ifnβ (>90%) rather than ifnα (<1%) (s11 table) . interestingly, the directionality of the correlations was discordant between these 2 gene sets: higher ifnβ levels correlated with higher expression of 82% of core isgs, whereas higher ifnβ levels were associated with lower expression of 88% of ifnβ-specific isgs ( fig 8a) . both core isgs and ifnβ-specific isgs were significantly associated with plasma lps levels, but again with discordant directionalities (fig 8b) . gut cd4 t cell counts (as a percentage of cd45+ cells) were more significantly associated with core-isgs (78% of genes negatively correlated) than ifnβ-specific isgs (50% of genes positively correlated) (fig 8c) . by contrast, blood cd4 t cell counts were more significantly associated with ifnβ-specific isgs (87% negatively correlated) than core isgs (fig 8d) . notably, the lower expression of a substantial fraction of ifnβ-specific isgs (58%) was associated with higher levels of plasma il6. none of the core isgs correlated significantly with plasma il6 at the 5% fdr cut-off. the complete list of genes that correlated with the 5 clinically relevant parameters (gut ifnβ mrna, plasma lps, gut cd4 t cell frequencies, blood cd4 t cell count and plasma il6 levels) are described in s12 table. we highlight several core isgs with the highest correlations in fig 9a, 68] . nlrc5 and lag3 also positively correlated with plasma lps levels and inversely correlated with the percentage of cd4+ t cells in the gut (fig 9c and 9d ). since t cell exhaustion in persistent lcmv infection has been linked to the suppressive cytokine il10 [26] , we evaluated if ifnβ mrna levels correlated with cytokine transcripts in the rnaseq dataset. notably, ifnβ mrna levels were associated with increased levels of il10 and il10ra, which were in turn correlated with lag3 ( s7a fig). ifnβ mrna levels also correlated with transcript levels of tnfα and ifnγ, but not il18 and tgfβ (s7b fig) . these data suggest that increased ifnβ in the gut of chronic pwh may drive genes associated with sustained isg expression, antigen processing, t cell activation, inflammation and immune exhaustion. among the ifnβ-specific isgs (fig 9; panels in the right half), higher ifnβ transcript levels negatively correlated with: (1) eif4h, a eukaryotic translational initiation factor (fig 9e) [69]; (2) smad4, a key regulator of tgfβ [70] , which in turn promotes mucosal barrier integrity ( fig 9f) ; (3) vimp and sep15, selenoproteins that regulate protein folding in the endoplasmic reticulum [71, 72] that were linked to anti-inflammatory processes [72] ; and (4) two co-factors of hiv-1 vpr, nup54 and mus81 [56, 73] . nup54 is a nuclear pore component and mus81 is an endonuclease; both are involved in maintaining genomic dna integrity [74, 75] . reduced expression of vimp and sep15 were associated with lower gut cd4 t cell type i interferons in mucosal hiv-1 infection percentages (figs 8 and 9g) . downregulated nup54 and mus81 were associated with higher plasma il6 levels (fig 9h and 9i) , lower gut cd4 t cell percentages (s12 table) and blood cd4 t cell counts (fig 9h and 9i) . thus, our analyses link elevated ifnβ levels in the gut of pwh to decreased protein translation and decreased protection against dna damage, protein misfolding and barrier dysfunction. fig 6a) and ifnβ-specific isgs (fig 7a) in gut biopsies from the clinical cohort were determined against (a) gut ifnβ transcripts, (b) plasma lps levels, (c) gut cd4+ t cell percentages, (d) blood cd4 t cell counts and (e) plasma il6 levels using linear regression models, controlling for age and gender. the clinical cohort included pwh (n = 19) and matched hiv uninfected controls (n = 13). correlations with fdr � 5% were considered significant; the proportion of those core and beta isgs are plotted as pie charts, with red, blue and gray depicting positive, negative and no significant correlations, respectively. the top genes in the relevant categories with �50% representation are highlighted; a full list is available in s12 table. https://doi.org/10.1371/journal.ppat.1008986.g008 two aspects of type i ifn biology that could influence its translational potential remain insufficiently addressed. first, there is an ongoing debate as to whether the biological effects of ifn-is are primarily due to quantitative differences in ifnar signaling capacity. for example, would administration of higher doses of weakly antiviral ifn-is (e.g., ifnα2 for hiv-1 infection) achieve the same in vivo effect as that of more potent ifn-is (e.g., ifnα14)? second, the mechanisms governing how and why ifn-is become pathogenic during chronic hiv-1 infection remains unclear. which isgs are responsible for the discordant effects of ifn-is at distinct phases of infection with persistent viruses? we have undertaken an unbiased transcriptomics approach to gain deeper insights on these questions. our group and others reported diverse antiviral potencies of the ifnα subtypes and ifnβ that correlated with their ifnar binding properties. these data emphasize the contribution of quantitative differences in ifnar signaling in controlling acute viral infection [12] [13] [14] [15] 76] . however, data from multiple in vivo studies also revealed that the ifnα subtypes may have expression levels were based on tmm-transformed counts. linear regression was performed in r statistical package, adjusting for age and gender. a full list of the proportion of core and ifnβ-specific genes that correlated with clinical parameters is presented in s11 table. https://doi.org/10.1371/journal.ppat.1008986.g009 type i interferons in mucosal hiv-1 infection qualitative differences in mediating antiviral immunity (reviewed in [2] ). in fact, ifnβ has a significantly higher binding affinity to ifnar2 than all ifnα subtypes [42, 43] but did not exhibit higher isre activity and inhibitory activity against hiv-1. recently, schaepler et al utilized saturating concentrations of various ifnα subtypes to inhibit hiv-1 infection in activated pbmcs in vitro. since 25 canonical isgs were induced to similar levels at saturating ifn-i doses, the authors concluded that there were no qualitative differences between the ifnα subtypes [15] . we argue that this conclusion is premature, as there are hundreds of isgs encompassing the interferome [5, 20] . we postulate that if there were no qualitative differences, the interferomes of diverse ifn-is should have substantial overlap following stimulation of cells with ifn-is normalized for ifnar signaling strength. we utilized a luciferase reporter cell line linked to the isre of a canonical isg, isg15, to normalize for ifnar signaling strength. the 6 normalized ifn-is tested (ifnα1, 2, 5, 8, 14 and ifnβ) induced isg15 and other antiviral isgs to similar extents in gut cd4+ t cells as expected. interestingly, despite normalizing for quantitative differences in ifnar signaling strength, the overlap between the ifnα subtype 'interferomes' were not complete, ranging from 59 to 82%. ifnα14 altered >200 genes not found in any of the other ifnα subtypes. ifnα5 altered >200 additional genes and weakly downregulated genes that were induced by other ifnα subtypes, raising the intriguing possibility that ifnα5 may modulate the overall ifn-i response. while it was possible that these ifnα-subtype specific genes were an artifact of the low numbers of donors used in this study, these genes were not close to statistical significance of being differentially expressed by the other ifnα subtypes, as majority had fdr values >90%. the sample size we used for this work was also counterbalanced by utilizing a homogenous cell subpopulation (purified cd4+ t cells) treated in a controlled fashion in vitro that should decrease overall variability. in fact, >240 canonical isgs were captured by all ifns tested including our positive control, isg15. interestingly, ifnβ altered nearly three times the number of genes compared to the individual ifnα subtypes we tested. we speculate that the higher binding affinity of ifnβ to the ifnar2 may contribute in part to the increased gene induction numbers, but it was notable that enhanced binding affinity did not track with isre activity. the broader interferome associated with ifnβ was also reported by other groups using pbmcs [77, 78] , although it was unclear whether the doses were normalized for ifnar signaling strength. altogether, the incomplete overlap between the interferomes strongly suggests that there are qualitative differences between diverse ifn-is. the underlying molecular mechanisms for differential interferome induction remain unclear. one possibility is that subtle differences in ifnar binding may have triggered differential phosphorylation of diverse stats, jaks and mapks. this possibility is currently under investigation. we speculate that differences in interferome regulation may have consequences for the therapeutic use of ifn-is in vivo. clinical administration of ifnα2 and ifnβ in vivo were associated with a multitude of side-effects [79] . one approach to potentially reduce toxicity issues is to utilize ifnα subtypes with a more 'restricted' gene regulation signature. ifnα8 is ten times more potent at inhibiting hiv-1 than ifnα2, but did not induce as many genes as ifnα14. thus, ifnα8 may be a potent anti-hiv-1 therapeutic with limited side-effects. however, ifnα8 did not seem to inhibit hiv-1 when administered in humanized mice as encoded plasmids via hydrodynamic injection [14] . ifn-is with desirable immunomodulatory properties may also be useful in hiv-1 curative strategies [13] , as reactivation of latent hiv-1 was insufficient to reduce the reservoir during antiretroviral therapy [80] . ifn-is could be potent additive drugs for hiv-1 cure by activating immune cells with latent hiv-1 while stimulating immune responses that can kill infected cells and prevent subsequent infection through intrinsic restriction. ifnα14 strongly induced trail+ nk cells and apobec3g-mediated hypermutation compared to ifnα2 in humanized mice [13] and was predicted to induce pathways associated with immune function in gut cd4+ t cells compared to ifnα1, 2, 5 and 8. thus, ifnα14 could be a viable candidate to pursue for hiv-1 curative strategies. the current data could serve as a useful template to design transcriptome-wide studies that extend to the 7 other ifnα subtypes not studied here, as well as other cell types (dcs, nk cells, cd8+ t cells, b cells) in various tissue compartments. such follow-up studies may aid in designing focused ifn-i biologicals for various clinical applications. our recent study suggested that ifnβ may play an important role during chronic hiv-1 immunopathogenesis in the gut [38] . we observed downregulated ifnα, but elevated ifnβ levels in colon biopsies from pwh, while ifnβ was rarely detected in the pbmcs and plasma of these patients [38] . since ifnβ induced a broader interferome than the individual ifnα subtypes, we determined how isgs induced by all ifn-is tested ('core isgs') versus isgs specifically induced by ifnβ ('ifnβ-specific isgs') were expressed in the gut biopsies following rnaseq. one limitation of our approach is that colon biopsies encompass other cell types that the interferomes based on gut cd4+ t cells may not capture. specifically, it is possible that the decreased expression of ifnβ-specific isgs in pwh relative to uninfected controls may be due to the significant loss cd4+ t cells in pwh. if this was the case, we should have also observed a decrease in core isg expression in pwh. however, over a hundred core isgs were upregulated in pwh, correlating significantly with gut ifnβ expression. alterations in ifnar expression in mucosal cd4+ t cells during hiv-1 infection may also contribute to our results, but our recent study have not detected significant changes in ifnar2 expression in mucosal immune cells in pwh [81] . expanding the interferome analyses to other mucosal cell types may enable deconvolution of the bulk rnaseq data to specific cell types. consistent with our previous study [38] , many canonical isgs that include antiviral genes remained upregulated in chronic hiv-1 infection in the gut. core isgs positively correlated with ifnβ rather than ifnα transcripts, suggesting that ifnβ drove these responses in the gut. interestingly, core isg expression positively correlated with plasma lps levels. previously, we showed that many isgs were upregulated in gut cd4+ t cells following co-incubation with prevotella stercorea, a gram-negative microbe present in colon tissue of pwh [82] . these findings strengthen a link between microbial translocation, ifnβ, and elevated isg signatures in the gut during chronic hiv-1 infection. interestingly, we did not observe correlations between core isgs and the monocyte activation marker scd14 or myeloid dendritic cell activation. other markers such as scd163 [83, 84] may need to be investigated in future work. irf9, stat1 and stat2 (the components of isgf3) remained elevated in pwh, suggesting a mechanism for constitutive isg expression during chronic infection. notably, ifnβ expression in the gut correlated with gene markers of immune activation and inflammation (cd38, psmb9, nlrc5, tnfa, ifng), and exhaustion (lag3). ifnβ-mediated induction of these immunomodulatory genes may account for how ifnβ contributes to pathogenesis during chronic infection. specifically, ifnβ levels in the gut were associated with il10 and il10ra expression, which in turn correlated with lag3 levels, suggesting that ifnβ may drive immune exhaustion through the il10 pathway. it remains to be determined whether these core isgs are similarly regulated in the systemic circulation, where ifnα subtypes, and not ifnβ, are more prominent [38] . further, the rationale for why the gut appears primed to express ifnβ remains unclear. a recent study noted that helix 4 of ifnβ may have direct antimicrobial properties [85] . efforts are ongoing to investigate links between ifnβ levels, ifnβspecific genes and the altered microbiome in pwh. could ifnβ have distinct biological effects that are likely not due to other ifn-is? in persistent lcmv infection of mice, neutralization of ifnβ, and not ifnα, accelerated virus clearance and improved t cell responses [86] . in the present study, we observed that a substantial fraction of ifnβ-specific isgs, but not core isgs, correlated with plasma levels of the inflammatory cytokine il6. a link between ifnβ and il6 has previously been reported [87] . surprisingly, the ifnβ-specific isgs that correlated with il6 were downregulated during chronic hiv-1 infection in the gut. the magnitude of downregulation correlated with higher ifnβ levels, suggesting negative feedback control. among these negative feedback control genes, we identified unc93b1 as a potential driver for the downregulation of ifnβ-specific isgs, possibly through the regulation of tlr7 signaling via endosomal trafficking pathways [59, 88] . it is thought that constitutive expression of antiviral isgs may protect from virus infection [89, 90] , but decreased protein translation (eif4h) in pwh may minimize the contribution of antiviral isgs that are constitutively expressed. furthermore, our data suggest a potential link between ifnβ, decreased tgfβ signaling (smad4), increased unfolded protein response (vimp and sep15) and increased dna damage (nup54 and mus81) in chronic hiv-1 infection. these results raise the possibility that genes downregulated by ifnβ could have important consequences for mucosal hiv-1 pathogenesis. one possibility is that these altered ifnβ-specific genes (such as those that decreased protection from dna damage) may directly contribute to cd4+ t cell death, in line with previous studies linking ifn-is and cd4+ t cell depletion [91, 92] . given the predicted pleiotropic effects of ifnβ, our data also raise concerns in utilizing ifnβ for treating chronic hepatitis b virus infections that became refractory to ifnα treatment [76, 93, 94] . notably, ifnβ administered <7 days post-symptom onset may help resolve infection with the novel pandemic coronavirus, sars-cov-2 [95] . by contrast, delayed ifnβ treatment in murine coronavirus models exacerbated immune pathology [96, 97] . based on our studies as well as others, understanding the role of distinct ifns during the course of infection with diverse pathogenic viruses may yield important therapeutic insights. the mechanisms for the differential regulation of core versus ifnβ-specific isgs during chronic hiv-1 infection remain to be determined. studies in cell lines reveal that unphosphorylated stat1, stat2 and irf9 can assemble into an unphosphorylated isgf3 (u-isgf3) complex that could sustain the expression of canonical isgs after a single stimulation with ifnβ [90, 98] . constitutively expressed isgs regulated by u-isgf3 included antiviral genes such as bst2, apobec3g, mx2 that remained elevated during chronic hiv-1 infection. by contrast, isgs specifically regulated by phosphorylated isgf3 are more sensitive to negative feedback regulation. the isgf3/u-isgf3 model would imply that core isgs are regulated by u-isgf3, whereas the ifnβ-specific isgs are regulated by isgf3. investigations on the phosphorylation status of stat1, stat2 and irf9 in chronic hiv-1 infection are underway. in conclusion, we demonstrate that diverse ifn-is trigger non-overlapping interferomes in a single cell type, providing strong evidence for qualitative differences between the ifn-is. furthermore, conserved and qualitative differences in interferome induction correlated with clinically relevant markers of immunopathogenesis during chronic hiv-1 infection. further studies on the differences between the ifn-is and how these cytokines regulate distinct gene expression profiles in various tissue compartments could inform strategies to harness these biologicals and/or block these responses for hiv-1 control. the in vitro studies utilized disaggregated cells from macroscopically normal jejunum tissue that would otherwise be discarded. these tissues were obtained from patients undergoing elective surgery at the university of colorado hospital. the patients signed a release form for the unrestricted use of tissues for research following de-identification to laboratory personnel. the colorado multiple institutional review board approved the procedures and have given exempt status. colon pinch biopsies from pwh and age/gender-matched hiv-uninfected controls were obtained from archived samples from a completed, comirb-approved clinical study. this clinical study included 24 pwh and 14 hiv-uninfected controls, but archived colon pinch biopsies were available only for 19 pwh and 13 controls, respectively. study participants signed an informed consent. clinical parameters that include blood cd4-t cell counts (cells/ μl), plasma viral load, tissue hiv rna (per cd4 t cell), tissue cd4 t cells (% viable cd45 + cells), il-6 (pg/ml), crp (μg/ml), ifabp (pg/ml), scd27 (u/ml), cd14 (ng/ml), lps (pg/ ml), lta (optical density), gut ifnα and ifnβ transcripts, were reported previously [38]. ifn-is (pbl assay science, piscataway nj) were frozen into small aliquots for single-use. ilite type i ifn assay ready cells, purchased from svar life science ab (malmõ, sweden, cat# bm3049) were seeded into a 96-well flat-bottomed cell culture plate and maintained in complete dmem (with 10% fbs and 1% penicillin-streptomycin and l-glutamine). various doses of recombinant ifn-is were added to corresponding wells. after 18 h, the cells were lysed and luciferase activities were developed using bright-glo luciferase assay system (promega, madison, wi, cat# e2610) according to manufacturer's instruction. luciferase activity was subsequently measured using a perkin-elmer victor x5 plate reader. 50% effective concentrations were computed using dose-response curve in prism 5.0. hiv-1 bal (nih aids reagent program cat# 510) was prepared in molt4-ccr5 cells and titrated using an hiv-1 p24 elisa (advanced bioscience laboratories, rockville, md). 10 ng p24 of hiv-1 bal / 10 6 lpmcs (n = 6 donors) was spinoculated in the presence or absence of titrated doses of ifnα subtypes and ifnβ (pbl assay science). the cells were harvested at 4 d and analyzed by intracellular p24 flow cytometry as previously described [12, 99] . 50% inhibitory concentrations and vres were calculated using a one-phase decay equation in prism 5.0 as previously described [12, 13] . cd4+ t cells were purified from lpmcs (n = 4 donors) by negative selection using easysep™ human cd4+ t cell isolation kit (stemcell, vancouver, bc, canada, cat# 17952) and followed by flow sorting to reach a purity greater than 99%. 1x10 6 of these purified cd4+ t cells were treated with mock, 100 pg/ml ifnα14, or an equivalent isre-activity of ifnα1, 2, 5, 8 and ifnβ. after 18 h, rna was extracted using rneasy mini kit (qiagen, germany, cat# 74104). rnaseq libraries were constructed from 500 ng rna using quantseq 3' mrnaseq library prep kit (lexogen, austria, cat# 016.96). the rnaseq library quality was verified using 2100 bioanalyzer (agilent, santa clara, ca) before loaded into a hiseq 2500 by the genomics and microarray sequencing core facility at the university of colorado anschutz medical campus. the 96-well array contained primers for target genes isg15, arhgef3, lat and ahnak (s1 fig) , a reverse transcription control, a genomic dna control and a positive pcr control as well as the housekeeping gene gapdh (pph00150f). ninety-six-well custom rt 2 profiler pcr arrays were performed according to the manufacturer's instructions (qiagen, valencia, type i interferons in mucosal hiv-1 infection california, usa) using the bio-rad cfx-96 real-time pcr instrument (bio-rad, hercules, california, usa) and bio-rad cfx manager software (ver. 3.1). cdna templates were mixed with ready-to-use rt 2 qpcr master mixes and 25 μl of the pcr component mix was aliquoted into each well containing predispensed gene-specific primer sets. each plate was loaded with cdna from 6 individual samples. gene expression was normalized according to the average expression level of gapdh in the same sample. rna (500 ng) from the colon pinch biopsies were used to prepare the rnaseq libraries. the rnaseq work flow of library construction, quality control, and the subsequent sequencing were similar to that of ifn-i treated gut cd4+ t cells as aforementioned. rnaseq data were downloaded as fastq files and their quality examined and visualized using fastqc (babraham institute, https://www.bioinformatics.babraham.ac.uk/projects/ fastqc). the removal of illumina sequencing adapters and the by-base quality screening was performed with cutadapt (https://cutadapt.readthedocs.io/en/stable). the quality screened fastq files were mapped to the current human genome assembly grch38.p12 using hisat2 (johns hopkins university, https://ccb.jhu.edu/software/hisat2/index.shtml), taking into account the ensembl id, gene symbol and gene length. raw gene expression counts were extracted using featurecounts from the subread package (walter+eliza hall institute of medical research, http://subread.sourceforge.net). rnaseq raw counts were obtained for gut cd4 t cells treated with 6 ifn-is (ifnα1, 2, 5, 8, 14 and ifnβ) and mock from 4 donors. gene counts were normalized using transcripts per million (tpm). principal component analyses necessitated removal of one donor as the transcriptome of the mock condition was extremely skewed relative to the other 3 donors (s1a fig). using edger differential expression analysis, ifn regulated genes (irgs) were defined based on a 1.5-fold change (fc) cutoff relative to mock and a false-discovery rate (fdr) �20%. the rnaseq raw counts data had gene-level read counts for 13 hiv-1-uninfected donors and 19 pwh. lowly expressed genes which have average read counts less than 5 per library were filtered out. as a result, 19890 out of 43297 genes were kept for further analysis and gene counts were normalized using the trimmed mean of m values (tmm) normalization method from edger (version 3.24.3) in r (version 3.5.1) with the "estimatedisp" function and its default settings. the tmm method calculates the scaling normalization factor for each sample by accounting for library size and observed counts, while under the assumption that the majority of genes are not de. in our case, the tmm method provided the best normalization results in terms of removing the unwanted variation, according to the relative log expression (rle) plots (s6 fig) . two-group differential expression (de) analysis was performed to test for differences between healthy controls and pwh, using normalized counts in edger with default settings and false discovery rate (fdr) of 5% to control for multiple testing. the de analysis was done in edger by the exact test using the "exacttest" function and its default settings for two-group design. the benjamini-hochberg procedure (bh step-up procedure) controlling the fdr was used as the multiple testing correction method in this study. the normalized rnaseq counts were further transformed by the variance stabilization and bias reduction method called "regularized log transformation" (rlog) from deseq2 (version 1.22.2) [53] . the transformed counts were then used as the outcome in a multivariate linear regression model. specifically, the linear regression models were fit in a gene by gene fashion to test the correlation between rna expression levels and immunopathogenic markers of chronic hiv-1 infection, with one marker included in the model at a time, while adjusting for age and gender. based on the results of de analysis, significantly altered genes from core-isgs and ifnβ-specific isgs were selected to test the associations with clinical parameters through the above model, separately. several immunopathogenic markers were used in this part, including gut ifnβ transcript levels, gut cd4 t cell percentages, blood cd4 t cell counts, plasma il6 levels and plasma lps levels. genes were considered as significantly associated with the corresponding clinical parameter with an fdr cutoff at 5%. in addition, the difference in proportions of significant genes in each gene list were tested with chi-squared test in r, as well as the difference in proportions of positive correlations of each gene list. all the plots were generated by ggplot2 (version 3.1.1) package in r (version 3.5.1). next generation sequencing data were deposited in the sequence read archive prjna558974 (interferome dataset) and prjna558500 (colon pinch biopsies). these were also deposited in the gene expression omnibus archive with accession numbers gse156844 (interferome dataset) and gse156861 (colon pinch biopsies). (left panels) ifnα5-specific genes (n = 201) were classified based on significant induction/suppression in ifnα5-treated cells relative to mock at a 20% fdr cut-off. however, when these genes were evaluated in transcriptome datasets for ifnα1, ifnα2, ifnα8 and ifnα14, most had fdr values >90%, suggesting that these ifnα5-specific genes were not even close to being statistically-significant in these other ifnα subtypes. (right panels) evaluation of ifnα14-specific genes against gene datasets for ifnα1, ifnα2, ifnα8 and ifnα5. bars correspond to the frequency of genes that fell within the fdr values noted in the x-axis. type i interferons in mucosal hiv-1 infection the interferons: characterization and application interferon alpha subtypes in hiv infection the significance of type-i interferons in the pathogenesis and therapy of human immunodeficiency virus 1 infection the jak-stat pathway at twenty interferome: the database of interferon regulated genes interferon-alpha subtypes in an ex vivo model of acute hiv-1 infection: expression, potency and effector mechanisms interferon alpha subtype-specific suppression of hiv-1 infection in vivo gene therapy with plasmids encoding ifn-beta or ifn-alpha14 confers long-term resistance to hiv-1 in humanized mice dose-dependent differences in hiv inhibition by different interferon alpha subtypes while having overall similar biologic effects. msphere binding and activity of all human alpha interferon subtypes evolutionary genetic dissection of human interferons. the journal of experimental medicine interferon-alpha subtype 11 activates nk cells and enables control of retroviral infection type i interferon differential therapy for erythroleukemia: specificity of stat activation anti-retroviral effects of type i ifn subtypes in vivo type i interferon responses in rhesus macaques prevent siv infection and slow disease progression reduced chronic lymphocyte activation following interferon alpha blockade during the acute phase of simian immunodeficiency virus infection in rhesus macaques blockade of chronic type i interferon signaling to control persistent lcmv infection persistent lcmv infection is controlled by blockade of type i interferon signaling interferons and interferon (ifn)-inducible protein 10 during highly active anti-retroviral therapy (haart)-possible immunosuppressive role of ifn-alpha in hiv infection chronic cd4+ t-cell activation and depletion in human immunodeficiency virus type 1 infection: type i interferon-mediated disruption of t-cell dynamics downregulation of robust acute type i interferon responses distinguishes nonpathogenic simian immunodeficiency virus (siv) infection of natural hosts from pathogenic siv infection of rhesus macaques an altered intestinal mucosal microbiome in hiv-1 infection is associated with mucosal and systemic immune activation and endotoxemia edger: a bioconductor package for differential expression analysis of digital gene expression data differential expression analysis of multifactor rna-seq experiments with respect to biological variation moderated estimation of fold change and dispersion for rna-seq data with deseq2 rle plots: visualizing unwanted variation in high dimensional data tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding viral protein r regulates docking of the hiv-1 preintegration complex to the nuclear pore complex induction of apobec3g ubiquitination and degradation by an hiv-1 vif-cul5-scf complex usp18-based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response unc93b1 restricts systemic lethal inflammation by orchestrating toll-like receptor 7 and 9 trafficking gut dendritic cell activation links an altered colonic microbiome to mucosal and systemic t-cell activation in untreated hiv-1 infection brief report: inflammatory colonic innate lymphoid cells are increased during untreated hiv-1 infection and associated with markers of gut dysbiosis and mucosal immune activation low abundance of colonic butyrateproducing bacteria in hiv infection is associated with microbial translocation and immune activation enhancement of hiv-1 infection and intestinal cd4+ t cell depletion ex vivo by gut microbes altered during chronic hiv-1 infection elevated cd38 antigen expression on cd8+ t cells is a stronger marker for the risk of chronic hiv disease progression to aids and death in the multicenter aids cohort study than cd4+ cell count, soluble immune activation markers, or combinations of hla-dr and cd38 expression nlr family member nlrc5 is a transcriptional regulator of mhc class i genes roles, function and relevance of lag3 in hiv infection modulation of the helicase activity of eif4a by eif4b, eif4h, and eif4f tgf-beta signaling from receptors to smads selenoproteins: molecular pathways and physiological roles premature activation of the slx4 complex by vpr promotes g2/m arrest and escape from innate immune sensing nucleoporin 54 contributes to homologous recombination repair and post-replicative dna integrity the dna repair endonuclease mus81 facilitates fast dna replication in the absence of exogenous damage functional comparison of ifn-alpha subtypes reveals potent hbv suppression by a concerted action of ifn-alpha and -gamma signaling identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays ifn-beta induces greater antiproliferative and proapoptotic effects and increased p53 signaling compared with ifn-alpha in pbmcs of adult t-cell leukemia/lymphoma patients the interferons: 50 years after their discovery, there is much more to learn stimulation of hiv-1-specific cytolytic t lymphocytes facilitates elimination of latent viral reservoir after virus reactivation hiv infection does not alter interferon alpha/beta receptor 2 expression on mucosal immune cells the transcriptome of hiv-1 infected intestinal cd4+ t cells exposed to enteric bacteria soluble cd163 made by monocyte/macrophages is a novel marker of hiv activity in early and chronic infection prior to and after anti-retroviral therapy. the journal of infectious diseases irf9 and unphosphorylated stat2 cooperate with nf-kappab to drive il6 expression the chaperone unc93b1 regulates toll-like receptor stability independently of endosomal tlr transport unphosphorylated isgf3 drives constitutive expression of interferon-stimulated genes to protect against viral infections ifnbeta-dependent increases in stat1, stat2, and irf9 mediate resistance to viruses and dna damage cd4+ t-cell death induced by infectious and noninfectious hiv-1: role of type 1 interferon-dependent, trail/dr5-mediated apoptosis type i interferon contributes to cd4+ t cell depletion induced by infection with hiv-1 in the human thymus development of a novel site-specific pegylated interferon beta for antiviral therapy of chronic hepatitis b virus interferon-beta and interferon-lambda signaling is not affected by interferon-induced refractoriness to interferon-alpha in vivo triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice unphosphorylated stat1 prolongs the expression of interferon-induced immune regulatory genes microbial exposure alters hiv-1-induced mucosal cd4+ t cell death pathways ex vivo we thank eric lee and christine purba for expert technical assistance with the lamina propria aggregate culture model, and cheyret wood for helpful statistical advice. we also thank the patients who agreed to use discarded tissue for research purposes and the clinical study participants. key: cord-023925-qrr7jcwe authors: verhoef, jan; van kessel, kok; snippe, harm title: a8 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites date: 2011-07-12 journal: principles of immunopharmacology doi: 10.1007/978-3-0346-0136-8_8 sha: doc_id: 23925 cord_uid: qrr7jcwe in the middle of the 19th century, it became clear that micro-organisms could cause disease. effective treatment, however, was not possible at that time; prevention and spread of infectious diseases depended solely on proper hygienic means. at the beginning of the 20th century, passive and active vaccination procedures were developed against a number of these pathogenic micro-organisms to prevent the diseases in question (rabies, diphtheria, tetanus, etc.). thanks to the discovery of antimicrobial chemicals (by paul ehrlich) and antibiotics (by sir alexander fleming), the threat of infectious diseases seemed to be minimised. large scale vaccination programmes against childhood diseases (diphtheria, whooping cough and polio), started in the early 1950s, raised hopes of finally being able to eradicate these diseases from the planet. in the middle of the 19th century, it became clear that micro-organisms could cause disease. effective treatment, however, was not possible at that time; prevention and spread of infectious diseases depended solely on proper hygienic means. at the beginning of the 20th century, passive and active vaccination procedures were developed against a number of these pathogenic micro-organisms to prevent the diseases in question (rabies, diphtheria, tetanus, etc.) . thanks to the discovery of antimicrobial chemicals (by paul ehrlich) and antibiotics (by sir alexander fleming), the threat of infectious diseases seemed to be minimised. large scale vaccination programmes against childhood diseases (diphtheria, whooping cough and polio), started in the early 1950s, raised hopes of finally being able to eradicate these diseases from the planet. this approach was successful for smallpox (1980) . however, new infectious diseases have emerged [e.g., legionella, human immunodeficiency virus (hiv), helicobacter, sars, etc.] and new vaccines and antibiotics are needed. furthermore, due to intensive medical treatment with antibiotics and immunosuppressive drugs, hospital infections are a growing problem. bacteria hitherto deemed harmless are causing opportunistic infections in immunocompromised patients. the pathogens have developed resistance to many antibiotics and sometimes no effective antibiotics are available to treat these patients. to make the story even more serious, man is surrounded and populated by a large number of different non-pathogenic micro-organisms. in the normal, healthy situation, there is a balance between the offensive capabilities of micro-organisms and the defences of the human body. the body's defences are based on vital non-specific and specific immunological defence mechanisms. an infection means that the micro-organism has succeeded in penetrating those lines of defence, signalling a partial or complete breakdown of the body's defence system. the body's first line of defence comprises the intact cell layers of skin and mucous membrane, which form a physical barrier. the skin's low ph level and bactericidal fatty acids enhance the protection provided by this physical barrier. the defences in the respiratory tract and the gastrointestinal tract are mucus, the 'ciliary elevator' of the epithelium, and the motility of the small intestine. the presence of normal microbial flora (colonisation resistance) in the intestine also plays a role in protection against colonisation by external bacteria. the most important humoral natural resistance factors are complement, antimicrobial peptides, lysozyme, interferon, and a number of cytokines (see chapters a5 and a6). antimicrobial peptides are widely expressed as part of the professional phagocyte antimicrobial arsenal and rapidly induced at epithelial surfaces. they are found in mammals, invertebrates, and plants. in general, they are small, amphipathic molecules, contain positive charge and can be structurally divided into several categories. the mode of action goes beyond their antimicrobial capacities and they elicit a complex array of responses in different cell types. the most extensively studied mammalian gene families are the cathelicidins and defensins. in general the antimicrobial peptides disrupt lipid membranes and thereby induce microbial killing. micro-organisms have developed several countermeasures against antimicrobial peptides but the many structurally different peptide classes still provide protection against infection. lysozyme, which is found in almost all body fluids, degrades sections of the cell wall of gram-positive and -in combination with complement -gramnegative bacteria. this causes the otherwise sturdy cell wall to leak and the bacterium to burst. interferons are glycoproteins and may inhibit the replication of viruses. within several hours after the onset of a virus infection, interferons are produced in the infected cell and help protect the neighbouring unaffected cells against infection. this protection is brief, but high concentrations of interferons are produced at a time when the primary immunological response is relatively ineffective. cytokines, such as interleukin-2 (il-2), granulocytemacrophage colony-stimulating factor (gm-csf), and tumour necrosis factor-α (tnf-α), stimulate non-specifically the proliferation, maturation, and function of the cells involved in defence (see chapter a6). innate immune cells recognise microbes by tolllike receptors (tlr) (see section pathogenesis of shock), giving rise to the above production of cytokines in the early phase of the response. 128 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites micro-organisms that succeed in penetrating the first line of defence are ingested, killed, and degraded by phagocytic cells [polymorphonuclear leukocytes (pmn) or neutrophils, monocytes, and macrophages], which are attracted to a microbial infection through chemotaxis. the ingestion by phagocytic cells of the micro-organism is enhanced by serum proteins (opsonins), such as antibodies and the c3b component of complement, which are recognised by specific receptors on the phagocytes. after ingestion, the particle is surrounded by the membrane of the phagocyte, forming a vacuole known as a phagosome. the phagosome then fuses with some of the countless granules in the phagocyte, thus allowing the lysosomal microbicidal agents and enzymes to do their work. the formation of toxic oxygen radicals greatly contributes to the killing and elimination of the ingested micro-organism ( fig. 1 ) (see chapter a7). a special role in cellular natural resistance is reserved for the natural killer cells (nk cells), which display considerable cytotoxic activity against virusinfected cells. this nk activity is stimulated by interferons and, at a very early stage in the infection, serves to reinforce the non-specific defence mechanism. in the specific immune response, elements of the natural defence mechanism are directed against a specific enemy. depending on the micro-organism, either the cellular defence mechanism (tuberculosis) or the humoral antibody-dependent defence mechanism (influenza) is of primary importance. in many cases, a joint cellular and humoral response is needed to provide an effective immune defence (typhus). both t lymphocytes and macrophages play a role in cellular defence. during the first contact with an antigen, macrophages process the antigen and present its protein fragments (t cell epitopes) to t cells, which then proliferate and remain present for years in the body as memory cells. when a second encounter occurs, t cells produce lymphokines, which activate the macrophages. these activated macrophages grow larger, produce more and better degrading enzymes, and are now able to eliminate micro-organisms, which otherwise would have survived intracellularly (tuberculosis, typhoid fever). macrophages from non-immune animals are not able to eliminate these micro-organisms. specific resistance schematic representation of the progressive steps of phagocytic endocytosis. five different classes of antibodies can be distinguished in man, namely, igg, iga, igm, igd, and ige. they differ from one another in size, charge, amino acid composition, and glycosylation (see chapters a3, c2). in principle, the structure of the antibodies is the same, i.e. two heavy and two light chains: it is the variable part of these chains that recognises the micro-organism. the biological function (see below) is determined by the constant part (fc) of the heavy chain. with the exception of igd, all these antibodies are important in antimicrobial activity. • iga, which is found in all external secretions, reacts with the surface of micro-organisms, preventing them from adhering to sensitive cells and mucous membranes. • igg neutralises microbial toxins. • igg, igm, and c3b serve as opsonins, which promote phagocytosis. • igg, igm, and to a lesser extent iga activate the complement system after binding to the micro-organism. activation products c3a and c5a ensure that the phagocytes are attracted to the inflammatory response. • igg and igm, in combination with complement and lysozyme, have a lytic effect on gram-negative bacteria and enveloped viruses. • igg and igm inhibit the mobility of micro-organisms by attaching specifically to the flagellum. thereby the chance of phagocytosis increases and the chance of spreading of disease decreases. • igg, together with the killer or k cells, can eliminate infected host cells which carry viral or other foreign antigens on their surface. • ige is of importance in parasite infections. at the site of the infection, mast cells, bearing specific ige, release large quantities of vasoactive amines, which cause the contraction of smooth muscle tissue and increase the permeability of the blood vessels. in the intestine, this results in worms being detached and eliminated. several non-invasive bacteria, i.e. those that do not invade the body, cause disease through the production of exotoxins (tetanus, diphtheria, cholera). the immune system neutralises the toxin with the aid of antibodies (igg, igm). if the individual has not been inoculated, the toxin will act on certain cells in the body directly through a receptor. this bond is very strong (i.e. has a high affinity), and is difficult to break by the administration of antibodies. in practice, if there are clinical symptoms of the disease, then large doses of antitoxins must be administered. if one is trying to prevent the development of the disease, then the presence of small quantities of specific antibodies (igg) is sufficient. the adherence of bacteria to cells is effectively blocked by iga. oral vaccination against cholera, for example, is aimed at obtaining sufficient specific iga in the intestine, so that no colonisation of this bacterium can take place, and the cholera toxin can no longer adhere to its receptor. in general, defence against invasive bacteria is provided by antibodies (igg, igm) that are directed against bacterial surface antigens. in many cases, these bacteria have a capsule, which interferes with effective phagocytosis. antibodies against the antigens of these capsules neutralise the interference, with subsequent elimination of the bacteria by phagocytes. antibodies (igm, igg, iga) in combined action with complement kill bacteria by producing holes in the cell wall of the bacterium. although intracellular bacteria (tuberculosis, leprosy, listeriosis, brucellosis, legionellosis, and salmonellosis) are ingested by macrophages, they are able to survive and multiply. in these cases, cellular immunity alone provides the defence, since antibodies are not effective. only activated macrophages are capable of killing and degrading these bacteria. antibodies neutralise viruses directly and/or indirectly by destroying infected cells that carry the virus antigen on their surface. the mechanisms of this defence resemble those of humoral defence against bacterial surfaces. the antibody-dependent cellular cytotoxicity reaction is specific for defence against viruses. cells that carry an antigen encoded by the virus on their surface are attacked by cytotoxic k cells, bearing antibodies that fit the antigen on the target cell (k cells have fc receptors for igg). (fig. 2) not only humoral, but also cellular immunity plays an important role in virus infections. people with a genetic t cell deficiency are highly susceptible to virus infections. in cellular defence, it is primarily the virus-infected cells that are attacked and eliminated. cytotoxic t cells recognise mhc class i-presented t cell epitopes on the surface of virusinfected cells and kill them. the fungi responsible for human diseases can be divided into two major groups on the basis of their growth forms or on the type of infection they cause. pathogens exist as branched filamentous forms or as yeasts, although some show both growth forms. the filamentous types (trichophyton) form a 'mycelium'. in asexual reproduction, the fungus is dispersed by means of spores; the spores are a common cause of infection after inhalation. in yeast-like types (cryptococcus), the characteristic form is the single cell, which reproduces by division or budding. dimorphic types (histoplasma) form a mycelium outside, but occur as yeast cells inside the body. candida shows the reverse condition and forms a mycelium within the body. in superficial mycoses, the fungus grows on the body surface, for example skin, hair, and nails (epidermophyton, trichophyton), the disease is mild, and the pathogen is spread by direct contact. in deep mycoses (aspergillus, candida, cryptococcus, histoplasma), internal organs are involved and the disease can be life-threatening and is often the result of opportunistic growth in individuals with impaired immunocompetence. many of the fungi that cause disease are freeliving organisms and are acquired by inhalation or by entry through wounds. some exist as part of the normal body flora (candida) and are innocuous unless the body's defences are compromised in some way. the filamentous forms grow extracellularly, while yeasts can survive and multiply within phagocytic cells. neutrophils kill yeasts by means of both intra-and extracellular factors. some yeasts (cryptococcus neoformans) form a thick polysac-charide capsule to prevent phagocytic uptake. in addition, many cell-wall components of yeasts cause suppression of cell-mediated immune responses. the role of humoral and cellular immunity in controlling infections caused by fungi is not yet well defined, but cellular immunity is the cornerstone of host defence against (some) fungal infections. as a consequence, hiv infection, which affects the cellular arm of the immune system, results in previously uncommon infections such as those caused by c. neoformans. the immunological defence systems against parasites are considerably more complex than those against bacteria and viruses. this is due to various factors. in the first place, each parasite has its own life cycle, consisting of various stages with specific antigen compositions. moreover, parasites are able to avoid the host defence system (mimicry), to combat it (immunosuppression), or to mislead it (antigenic variation). both humoral and cellular immunity are important for the defence against parasites growing intercellularly, as we have seen in the case of bacteria and viruses. antibody concentrations (igm, igg, ige) are often elevated. ige also plays a special role in the removal of parasites (especially worm infections) from the intestine (see above). defence against bacteria, viruses, fungi, and parasites sepsis is a systemic inflammatory response to presumed or known infection. the resulting inflammatory response becomes over amplified, leading to multiple organ failure and death. pathogen-associated molecular patterns (pamp) in bacteria, viruses, parasites, and fungi initiate the host response by triggering families of pattern recognition receptors (prr). in gram-negative (fig. 3) bacterial infections, the interaction between bacterial endotoxin or lipopolysaccharide (lps; a major structural component of the cell wall) and various host-cell systems has been implicated in the pathogenesis of septic shock. in particular, the release of tnf-α and interleukin-1 (il-1) after the activation of host cells by endotoxin induces haemodynamic shock. biochemical and genetic evidence has identified tlr4 as the receptor that mediates cellular activation in response to lps. this family of tlr proteins (fig. 4) , which resemble the antimicrobial toll proteins of drosophila (fruit fly), has been identified in humans and mice. tlr4 was identified as the missing link in lps-induced cell signal transduction and responsiveness that is associated with md-2 and cd14. it is known that c3h/hej mice are hyporesponsive to the biological effects of lps. this proved to be the result of tlr4 deficiency. the tlr family members are coupled to a signalling adapter protein (myd88) and form differential dimers that may explain the discrete responses to tlr ligands such as lipoproteins, heat shock proteins, unmethylated cpg dna, viral dsrna and bacterial flagellin. intracellular signalling involves several kinases depending on 132 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites genotypical characteristics: chromosomal dna fragment analysis, nucleic acid sequence analysis, probes phenotypical characteristics: morphology, biotyping, serotyping, antibiotic resistance analytical characteristics: cell-wall analysis, lipid and protein analysis, enzyme typing (catalase) gram staining positive or negative aerobic, anaerobic: fermentation of different sugars naming and classification of viruses according to: structure: size, morphology (naked, enveloped), nucleic acid (rna, dna) molecular aspects: mode of replication, assembly and budding disease: encephalitis, hepatitis means of transmission: droplets, water, blood, insects host range: animal, plant, bacteria classification of fungi according to: structure: macroscopic morphology of hyphae (mycelium); microscopic morphology of hyphae, conidophores and conidia (spores); and shape and size cell features: nucleus, cytosol, plasmalemma (cell membrane which contains cholesterol), physiology, staining properties sexual characteristics: sexual and /or asexual reproduction, extended dikaryotic phase, basidium formation genotypical characteristics: chromosomal dna fragment analysis, nucleic acid sequence analysis, probes schematic illustration of the cell envelope of a gram-negative and a gram-positive bacterium. schematic illustration of cell activation through toll-like receptors (tlrs). the tlr involved and includes the map kinase and nf-κb pathways leading to a cellular response. pamp for tlr2 include a variety of agonists derived from gram-positive organisms such as peptidoglycan and lipoteichoic acid. therefore, it seems that tlr2 and tlr4 are activated primarily by different pamp to initiate the host response to gram-positive and gram-negative bacterial infection, respectively. this is further illustrated by the fact that tlr2 (but not tlr4) knockout mice are highly susceptible to gram-positive bacteria, like staphylococcus aureus, whereas tlr4 (but not tlr2) knockout mice are highly susceptible to gram-negative bacteria such as salmonella typhimurium. several lines of evidence support the current hypothesis that the monocyte/macrophage is the principal cellular mediator of endotoxicity. lpshyporesponsive tlr4-deficient c3h/hej mice are made responsive by transfer of macrophages of a closely related lps-sensitive strain. when the host is challenged with lps, soluble factors are produced by macrophages that mediate fever and an acute-phase response. these factors include the proinflammatory cytokines, il-1, il-6, il-8, and tnf-α. together, tnf-α and il-1 stimulate endothelial cells to produce and express proteins on their membrane that have adhesive properties for leukocytes, promoting the margination and passage of neutrophils from blood vessels through the endothelial layer, leading to neutrophil influx into the tissue. adhesion molecules that mediate the binding of neutrophils appear on the endothelium after an inflammatory stimulus, followed by molecules that are specific for adhesion of monocytes or lymphocytes, which may be why neutrophils enter before mononuclear cells. molecules that are currently known to be involved in leukocyte-endothelium interactions belong to three structural groups: the immunoglobulin gene superfamily, the integrin family, and the selectin family. concomitant with cytokine release, lps induces the activation of neutrophils, macrophages, and many other cells, resulting in the release of toxic oxygen radicals, which lead to tissue damage. at the same time, membrane-associated phospholipases are activated and products of the arachidonic acid cascade are released through the cyclooxygenase and/or lipoxygenase pathways (see chapter a7). platelet-activating factor (paf) is also generated, partly in response to the same signals. all these products contribute to a generalised inflammatory state with influx of neutrophils, capillary-leak syndrome, disturbances in blood coagulation, and myocardial suppression. endotoxin and tnf-α also trigger multiple abnormalities in coagulation and fibrinolysis, leading to microvascular clotting and diffuse intravascular coagulation. they also induce endothelial cells to produce plasminogen activator and il-6, which is an important modulator of the production of acutephase proteins by the liver. interestingly, despite having important structural differences, tnf-α and il-1 have multiple overlapping and few distinct biological activities, act synergistically, and mimic the whole spectrum of toxicity caused by lps (see chapter a5). il-8 is an important chemoattractant and activator of neutrophils and is crucial in the early stages of inflammation. infusion of endotoxin in healthy humans leads to an early and transient increase in plasma levels of tnf-α (detectable after 30 minutes, peaking after 90-120 minutes, and undetectable after 4-6 hours), which coincides with the development of clinical symptoms and pathophysiological responses encountered in gram-negative septicaemia. tnf-α, il-1, il-6, and il-8 levels are also increased in patients with sepsis syndrome, with high levels of these cytokines being correlated with severity of disease. all these observations support the concept that endotoxin largely acts by initiating an inflammatory response through the activation of monocytes/macrophages and the subsequent release of cytokines. it also activates the complement system (leading to the generation of c5a, which induces aggregation of neutrophils and pulmonary vasoconstriction) and factor xii of the intrinsic coagulation pathway (hageman factor). finally, it induces the release of endorphins, which are also involved in the complex interactions of the inflammatory response in endotoxic septic shock. gram-positive bacteria are frequently and increasingly cultured from blood obtained from patients in shock. unlike the pathophysiology of shock caused by gram-negative bacteria, not much is known about the sequence of events that controls the signalling of monocytes and macrophages that leads to the release of cytokines. cell-wall components, such as peptidoglycan and teichoic acid, are clearly important in the activation of these cells. exotoxins, however, may also play a role in the pathogenesis of gram-positive bacterial shock. recently, another protein family was identified that also participates by sensoring microbial components derived from bacterial peptidoglycan. the nod (nucleotide-binding oligomerisation domain) proteins nod1 and nod2 have that have been implicated in intracellular recognition of the core structure, γ-d-glutamyl-meso-diaminopimelic acid, present in peptidoglycan. a number of circulating inflammatory mediators have been investigated as marker tools to facilitate the early recognition of sepsis. these include il-1, il-6, tnf, pro-calcitonin, and triggering receptor on myeloid cells (trem-1). trem-1 is expressed on leukocytes and trem family members have been implicated in mounting the inflammatory response. pro-calcitonin (pct) and il-6 have proved to be the most prominent biomarkers of early sepsis. pct is the pro-hormone of the hormone calcitonin, and can be produced by several cell types and many organs in response to pro-inflammatory stimuli, in particular by bacterial products. more recently high mobility group box-1 (hmgb-1) has been implicated as a lethal mediator of systemic inflammation. hmgb-1 is a nuclear and cytosolic protein widely studied as a transcription and growth factor that is released into the extracellular environment. it has a weak proinflammatory activity by itself and it may work in concert with other pro-inflammatory cytokines. this molecule may also be useful as a biomarker in the stratification of sepsis. susceptibility to sepsis can be influenced by factors that include ethnicity, gender, age, genetic defects and environmental factors. single-nucleotide polymorphisms (involving single base-pair alterations) have been described in genes controlling the host response to infection such as alterations in tnf receptors, il-1 receptors, coagulation factors and tlr. it is now clear that sepsis is a complex, dynamic syndrome with great heterogeneity, and not a distinct disease. therefore, neutralisation of a single key mediator as a cure for all patients with sepsis is erroneous. the hiv is a retrovirus that infects cells bearing the cd4 antigen, such as t helper cells (th), macrophages, and dendritic cells. the cd4 molecule, together with other receptor molecules, like chemokine receptor ccr5, acts as a binding site for the gp120 envelope glycoprotein of the virus. in an attempt to respond to hiv antigens and concomitant secondary microbial infections, these cells are activated, thus inducing the replication of hiv in the infected cd4 t cells, which are finally destroyed. in contrast, hiv-1 infection of macrophages is self-sustained and results in an inexorable growth of chronic active inflammatory processes in many tissue compartments including the central nervous system. infected cells bear the fusion protein gp41 and may therefore fuse with other infected cells. this helps the virus to spread and accounts for the multinucleated cells seen in lymph nodes and brain. as a result of the decreased numbers of cd4 + th cells and defects in antigen presentation, depressed immune responses in these patients are observed. during the progression of the disease, opportunistic infections by otherwise harmless micro-organisms can occur. these include can dida albicans oesophagitis, mucocutaneous herpes simplex, toxoplasma in the central nervous system, and pneumonia caused by toxoplasma and pneumo cystis carinii; kaposi's sarcoma also occurs frequently in these patients. this has been linked to the presence of a previously unknown type of herpes virus (hhv-8). this immune deficiency syndrome is called 'acquired immune deficiency syndrome' (aids). it has been suggested that infected monocytes/macrophages carry the hiv virus into the brain where it replicates in microglia and infiltrating macrophages. as a consequence, many aids patients develop cognitive and motor brain impairments. however, the picture is complicated by the various persistent infections already present in these patients, which give rise to their own pathology in the brain. these include toxoplasma gondii, cryptococcus neoformans and jc virus. so far, no cure for hiv infection has been achieved. the main effort in the prevention of hiv infection lies 135 human immunodeficiency virus infection in mass public education programmes. treatment of infected individuals is possible but expensive. highly active antiretroviral therapy (haart) reduces morbidity and mortality among patients infected with hiv (see fig. 5 ). success is limited by the emergence of drug-resistance viruses that can be transmitted to newly infected individuals. resistance, drug toxicity, and poor patient adherence lead to treatment failure and necessitate continuous development of alternative treatment strategies that intervene with the hiv replication cycle: i.e. on the level of virus entry, critical viral enzymes [reverse transcriptase (rt), integrase (in) and proteases (pr)] or viral nucleocapsid (nc) protein. at this moment a triple therapy is being prescribed in western countries (two rt inhibitors and one pr inhibitor, fig. 4) , each of which interfere with specific steps in the process of hiv replication. one major problem that has arisen is the increasing resistance to these drugs. agents with novel mechanisms of action provide options for patients with drug-resistant virus. blocking of the chemokine receptor ccr5, a co-receptor on cd4 cells for hiv, is an alternative treatment for persons infected with the r5 hiv type. this notion is supported by a recent finding that a homozygous defect in this chemokine receptor accounts for resistance of multiple-exposed individuals to hiv-1 infection. currently a commercially available drug is being used that specifically binds to ccr5 on the surface of the cd4 cell and selectively blocks hiv-1 binding. pasteur and koch triggered the stormy development of vaccines (anthrax, rabies, cholera) at the end of the 19th century. while pasteur remained faithful to the principle of attenuated micro-organisms in preparing his vaccines, koch employed killed germs (cholera) as a vaccine. since diphtheria and tetanus cause disease by means of toxins, the next logical step in the development of vaccines was the use of detoxified toxins to induce protection against these diseases [diphtheria (von behring) and tetanus (kitasato)]. von behring and kitasato were the first to demonstrate that the source of the protective activity induced by vaccines was present in blood serum. von behring was also the first to prove that protective immunity could be passed on via serum. the development of new vaccines had its ups (yellow fever) and downs (tuberculosis). with the arrival 136 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites the effect of single and triple therapy on viral load and cd4 cells over time in hiv-infected individuals. the immune system in health and disease pathogenesis, treatment, and prevention of pneumococcal pneumonia harmful molecular mechanisms in sepsis chemokine coreceptor signaling in hiv-1 infection and pathogenesis 25 years after hiv discovery: prospects for cure and vaccine monoclonal antibody-based therapies for microbial diseases antibiotic-resistant bugs in the 21st century -a clinical super-challenge co-evolutionary aspects of human colonisation and infection by staphylococcus aureus outbreaks of infectious diseases centers for disease control and prevention of antibiotics, all work on new bacterial vaccines was suspended or severely curtailed, although some researchers continued to work on viral vaccines, such as rubella, measles, polio, and mumps.since it has proved difficult to consistently develop new antibiotics to combat antibiotic-resistant bacteria, interest in vaccines has gradually increased over the last 15 years (see chapter c1). today, thanks to new insights into the immune system and modern molecular biological and chemical techniques of analysis and synthesis, it is possible to produce well-defined vaccines. these contain only those determinants of the pathogenic micro-organism that induce protection (epitopes). these epitopes are usually short peptide or oligosaccharide chains, which can be produced synthetically or by means of recombinant dna techniques. the immunogenicity of these products can be enhanced by coupling them to a carrier (tetanus toxoid, liposomes) and/or by adding an adjuvant (a substance that strengthens the immune response non-specifically). the recombinant dna technique can also be used to obtain attenuated strains of micro-organisms, which are fully immunogenic and thus provide protection, but which are no longer virulent. one example of this is the development of a new cholera vaccine based on a bacterium that has all the characteristics of a virulent strain, except the toxin. the bacterium has retained all its adherence factors, which allow it to adhere to the intestinal mucosa; the length of time it spends in the intestine is sufficient to stimulate the local immune system. the newest trend in vaccinology is immunisation by introducing plasmid dna into the host. success has been attained by this method for hepatitis b vaccination.not only are new vaccines being developed, but it is also possible to heighten natural resistance for longer or shorter periods. various interleukins (il-2, gm-csf) and interferons are being studied in order to use them to combat infectious diseases (see chapter c7). passive antibody therapy with polyclonal or monoclonal antibodies (mouse or human igg with single specificity) for infectious diseases is experiencing a renewed interest. targeting soluble factors (like neutralisation of bacterial toxins and viruses) or common structures (like bacterial adhesins or viral entry factors) in high-risk patient groups may be ben-eficial alone or may enhance the therapeutic efficacy of other drugs. targets for clinical development of monoclonal antibodies include multi-resistant staphylococci and enterococci, bacillus anthracis toxin, hiv, hepatitis c virus (hcv), and respiratory syncytial virus (rsv). as outlined above for a number of bacteria and viruses, effective vaccines have been developed and applied worldwide. the eradication of smallpox (variola major) virus in the 1970s was a milestone for the world health organisation. the next goal of the who is to eradicate poliovirus in the coming years. major problems to be dealt with are the distribution of these vaccines, the costs involved, the registration and the compliance of the vaccinees and molecular techniques to trace the final bug. meanwhile new unexpected microbiological threats become in focus. hospital infections caused by multi-resistant micro-organism due to the abundance use of antibiotics and exchange of genetic material between micro-organisms impose major problems on patients and healthcare workers. new antibiotics and/or vaccines should be developed and new strategies employed to contain these infections. due to crowding and the high mobility of the world population, old and new pathogens, e.g. influenza and sars, threaten our society. the recent influenza a h1n1 ("mexican") flu outbreak in 2009 demonstrated how rapidly a new strain of flu can emerge and spread around the world. the sudden outbreak of this novel flu virus has tested the world's public health preparedness. in the netherlands, q fever emerged with large epidemic outbreaks. q fever is a zoonotic disease, that is passed from infected (farm) animals (cattle, sheep, and goats are the primary carriers) to humans. q fever is a disease caused by the intracellular bacterium coxiella burnetii. organisms are excreted in milk, urine, and faeces of infected animals. most importantly, during birthing the organisms are shed in high numbers within the amniotic fluids and the placenta. c. burnetii is resistant to heat, drying, and many common disinfectants, allowing it 137 infections in the new millennium 138 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites to survive for a long time in the environment. people can become infected by inhalation of the bacteria, but the risk of infection is low. only about one-half of all people infected with c. burnetii show signs of clinical illness (e.g. flu-like illness, pneumonia, and hepatitis). q fever can be treated with antibiotics, and most people will recover fully. as a control measurement pregnant goats on dairy farms had to be killed and a mandatory vaccination campaign was started.on top of this, terrorists might intentionally use micro-organisms (smallpox, anthrax, plague etc.), or bacterial toxins (botulism) to cause death and disease in humans or animals in a civilian setting. the recognition that an event was caused by a biological weapon presents a severe challenge to be prepared for such an attack, especially for medical care providers, and public health officials. strategies to combat bioterrorism have to be worked out but with the experience of 100 years of combating micro-organisms with hygiene measures, vaccination, antibiotic and anti-viral treatment, there must be a way out. key: cord-018864-c1r2n17o authors: pöhlmann, stefan; tremblay, michel j. title: attachment of human immunodeficiency virus to cells and its inhibition date: 2007 journal: entry inhibitors in hiv therapy doi: 10.1007/978-3-7643-7783-0_3 sha: doc_id: 18864 cord_uid: c1r2n17o the entry of enveloped viruses involves virus adsorption followed by close apposition of the viral and plasma membranes. this multistep process is initiated by specific binding interactions between glycoproteins in the viral envelope and appropriate receptors on the cell surface. in the case of hiv-1, attachment of virions to the cell surface is attributed to a high affinity interaction between envelope spike glycoproteins (env, composed of the surface protein gp120 and the transmembrane protein gp41) and a complex made of the primary cd4 receptor and a seven-transmembrane co-receptor (e.g., cxcr4 or ccr5) (reviewed in [1]). then a chain of dynamic events take place that enable the viral nucleocapsid to penetrate within the target cell following the destabilization of membrane microenvironment and the formation of a fusion pore. the entry of enveloped viruses involves virus adsorption followed by close apposition of the viral and plasma membranes. this multistep process is initiated by specific binding interactions between glycoproteins in the viral envelope and appropriate receptors on the cell surface. in the case of hiv-1, attachment of virions to the cell surface is attributed to a high affinity interaction between envelope spike glycoproteins (env, composed of the surface protein gp120 and the transmembrane protein gp41) and a complex made of the primary cd4 receptor and a seven-transmembrane co-receptor (e.g., cxcr4 or ccr5) (reviewed in [1] ). then a chain of dynamic events take place that enable the viral nucleocapsid to penetrate within the target cell following the destabilization of membrane microenvironment and the formation of a fusion pore. although it is generally accepted that hiv-1 attachment to its major cellular reservoirs (i.e., t helper cells and macrophages) occurs through the twostage receptor-interaction pathway, there is accumulating evidence indicating that the initial attachment step is a more complex phenomenon than initially thought. indeed it seems that adsorption of hiv-1 to the cell surface is modulated by a large variety of interactions between the viral entity and the target cell surface (reviewed in [2] ). this retrovirus may also attach to some cell types by cd4-independent interactions involving highly glycosylated groups or basic residues found on gp120 and polyanionic sulfated chains or lectin-like domains on some specific cell surface receptors (reviewed in [1] ). for example, heparan sulfate proteoglycans, which are expressed at high levels on different cell types, such as epithelial and endothelial cells, can interact with the envelope spike glycoprotein and serve as docking structures for hiv-1 [3] . heparan sulfate proteoglycans such as syndecans serve as the main class of attachment receptors for hiv-1 on different cell types, e.g., macrophages and endothelial cells, and are thought to play a cardinal role in virus transmission [4, 5] . gp120 can bind also to galactosyl ceramide and its sulfated derivative (i.e., sulfatide) [6, 7] that are found on macrophages and neural, glial and colon epithelial cells [6] [7] [8] . it can also associate with the mannose-specific macrophage endocytosis receptor (mr) [9] and other cellular lectins. in fact, the determinant role played by dendritic cells (dcs) in hiv-1 transmission might rely on specific interactions between gp120 and c-type lectins, of which the dc-specific intercellular adhesion molecule-3 (icam-3) grabbing nonintegrin (dc-sign) and dc-signr (for dc-sign-related) are the best studied [10, 11]. these two lectins are expressed on certain dc populations and endothelial cells, respectively, and are described in more detail in this article. hiv-1, as an enveloped virus, is released by budding through the plasma membrane of the productively infected cell. in addition to its own virus-encoded envelope glycoproteins, the virus incorporates many different cellular proteins normally found on the cell surface (reviewed in [12] [13] [14] [15] the process of incorporation of host cell membrane proteins was found to be conserved among all tested hiv-1 subtypes and strains that were expanded in natural cellular reservoirs, such as mitogen-activated peripheral blood lymphocytes and human lymphoid tissue cultured ex vivo [27] [28] [29] [30] [31] [32] . the physiological significance of this phenomenon is provided by two previous reports showing that host-encoded cell surface constituents were incorporated in plasma-derived clinical hiv-1 isolates [33, 34] . although different host cell constituents can be found embedded within hiv-1, the incorporation process seems to be selective. for example, cd45 is the most abundant leukocyte cell surface glycoprotein [35] , but is not acquired by hiv-1 [18, 36] . the cxcr4, ccr5, and ccr3 co-receptors are also excluded from hiv-1 [37] . this ability to incorporate discriminatory host antigens into mature virions has allowed two groups to demonstrate that cell-type-specific antigens can serve as markers of the cellular origin of hiv-1 replication [33, 38] . it has been estimated that between 375 and 600 hla-dr molecules are found associated with hiv-1 iiib emerging from h9 cells [39] . this observation suggests that virally embedded host hla-dr outnumbered virus envelope (env) glycoprotein gp120 by a factor of 8.9 to 28.6 considering that hiv-1 possesses an average of between 21 and 42 gp120 molecules per virion [40] . the molecular basis governing the selective incorporation of cell surface proteins within emerging hiv-1 particles is only beginning to be exposed. it was established that the virus envelope spike glycoproteins (i.e., gp120 and gp41) are not essential to achieve insertion of icam-1 into hiv-1 [41] . interestingly, icam-1 incorporation is governed by an intimate association between the cytoplasmic domain of icam-1 and the viral gag precursor polyprotein pr55 gag [42] . it can be proposed that besides interactions between gp120 and multiple attachment receptors, interactions can also occur between host-derived cell surface components incorporated within emerging virions and their natural counter-ligands. this scenario has been confirmed in numerous studies where such host cell membrane molecules were found to retain their biological activity when located on the virus. for example, hla-dr can increase hiv-1 infectivity for cd4-expressing t cells by about twofold [43] , whereas icam-1 alone augments virus infectivity for lfa-1 + target cells by up to 100-fold depending of the lfa-1 conformational state [22, 44, 45] . activation of primary human cd4 + t lymphocytes was found to result in lfa-1 clustering, an event that promotes the early events of hiv-1 replication cycle through an interaction between virus-embedded host icam-1 and lfa-1 clusters [46] . confocal analyses showed that hiv-1 is concentrated in microdomains rich in lfa-1 clusters [46] . virus entry studies including subcellular fractionation experiments with primary human cd4 + t cells illustrated that the acquisition of icam-1 by nascent hiv-1 modified the entry route of the virus within such target cells [47] . it was established that the icam-1mediated increase in virus infectivity was linked with a more productive entry process into primary cd4 + t lymphocytes (i.e., cytosolic delivery of viral material) [47] . it has been reported that the higher susceptibility of memory cd4 + t cells (cd45ro + subset) to hiv-1 infection is due to secondary interactions between virus-associated icam-1 and cell surface lfa-1 [48] . the presence of host-encoded cd28 in newly formed hiv-1 particles resulted in a close to 20-fold augmentation in virus infectivity when using target cells that express high levels of cd80 and cd86, two natural ligands of cd28 [49] . in addition, an increase in virus infectivity was also seen following insertion of host-encoded costimulatory molecules cd80 and cd86 within progeny viruses [50] . given that attachment of hiv-1 to host cells can be modulated by the additional interactions provided by virus-anchored host cell membrane proteins, it is thus not surprising to discover that virus susceptibility to blocking agents is affected. for example, icam-1-bearing virions are more resistant to antibodymediated neutralization and this decreased sensitivity is even more dramatic when target cells expressed on their surface the activated form of lfa-1 [51, 52] . additionally, it was reported that virions carrying host icam-1 on their surface are more resistant to the fusion inhibitor t-20 than are isogenic viruses lacking host icam-1 [53] . although the physical presence of such host constituents on the exterior of virions might be detrimental for the infected individual, the propensity of hiv-1 to acquire numerous host cell surface components could be exploited to control viral load. indeed, it has been shown in numerous reports that hiv-1 infectivity can be efficiently neutralized, both in vitro and in vivo, with antibodies specific for such host membrane proteins [22, 23, 26, 39, 44, 45, 54, 55] . interestingly, it was demonstrated that hiv-1 replication is diminished upon treatment with statin compounds (e.g., lovastatin) [56] , the primary drugs used in the treatment of hypercholesterolemia. the antiviral potency of lovastatin seems to be linked with its capacity to inhibit interactions between virusassociated host icam-1 and cell surface lfa-1. this in vitro work was confirmed by a proof-of-concept small-scale clinical study [57] . in this provocative study, six a1 stage hiv-1 patients not receiving combined therapy were given lovastatin for a month as their only medication. this short-term statin treatment clearly reduced serum viral rna loads in all patients and in general increased their cd4 + t cell counts. discontinuation of treatment was followed by a rebound in viral load. the prevention of hiv-1 infection by microbicides, topically applied inhibitors that block access of sexually transmitted hiv-1 to the host system, is an attractive strategy [58] . understanding which cell types are first targeted by sexually transmitted hiv-1 and how these cells interact with hiv-1 is key to the generation of effective microbicides. several studies suggest that dcs, professional antigen-presenting cells, might be intimately involved in the early local and subsequent systemic spread of sexually transmitted hiv-1 [59] . langerhans dcs in the top layer of the anogenital mucosa are probably the first cells exposed to sexually transmitted hiv-1. mucosal macrophages and submucosal dcs might subsequently get into contact with virus crossing the mucosal barrier via local breaches or with progeny virions generated by infected langerhans cells. dcs and macrophages express cd4 and chemokine receptors, and are thus permissive to hiv-1 infection, albeit infection of dcs is often relatively inefficient and depends on the subpopulation analyzed [59] . it has been proposed, however, that mere attachment of hiv-1 to mucosa asso-ciated dcs might be sufficient to promote hiv-1 spread, since these motile cells might ferry bound virus into lymphoid tissue, the major compartment of hiv-1 replication, as part of their migratory and antigen-presenting functions within the immune system [10] . several cellular lectins have been implicated in virus attachment to dcs, macrophages and other cell types relevant to hiv-1 spread. here, we discuss the role of the lectins dc-sign, dc-signr, mr and langerin in hiv-1 infection and introduce strategies to inhibit hiv-1 interactions with these molecules. dc-sign has initially been identified as a gp120-binding calcium-dependent lectin expressed in placental tissue [60] . the lectin has been "rediscovered" in 2000 when geijtenbeek and colleagues [10] showed that dc-sign is expressed on dcs and is involved in hiv-1 binding and subsequent transfer of the virus to t cells, the latter process presumably involving dc-sign-dependent endocytosis and conservation of infectious hiv-1 in a low ph compartment [61] . dc-sign seemed to mainly account for the ability of dcs to promote hiv-1 infection of cocultured t cells, and it was proposed that dcs might function as trojan horses, which take up hiv-1 via dc-sign and transport the virus into lymphoid tissue [10, 62]. geijtenbeek and coworkers also provided evidence that dc-sign interacts with icam-2 on endothelial cells [63] and icam-3 on t cells [64] , and proposed that these interactions contribute to extravasation of dcs from blood vessels into tissues and to the close contact between dcs and t cells required for efficient antigen presentation, respectively. thus, a scenario emerged in which dc-sign was involved in dc functions critical for the establishment of an effective immune response and simultaneously allowed hiv-1 to misuse dcs to ensure its spread in the host. a critical contribution of dc-sign to dc interactions with t cells/endothelial cells or hiv-1 has subsequently been challenged. it was reported that dc-sign or the related protein dc-signr bind to icams with submicromolar affinities similar to that observed for nonspecific cellular proteins [65] , suggesting that icam recognition might not account for a potential role of dc-sign in cell-cell interactions. it was also documented that hiv-1 capture by dcs does either not dependent on dc-sign [66, 67] or that the contribution of dc-sign is relatively modest with other factors playing an important role [68] [69] [70] [71] . in fact, truville and colleagues [72] provided evidence that different dcs bind to hiv-1 gp120 via different lectins or via cd4, as discussed below. moreover, it has been demonstrated that transformed cells frequently used to assess dc-sign function were not thp-1 monocytes, as reported [10], but most likely raji b-cells [73] , and that these cells as well as monocyte-derived dcs were permissive to infection by hiv-1 [74] [75] [76] . the latter observation suggests that the ability of dc-sign-expressing cells to maintain hiv-1 infectious over prolonged time is most likely due to productive infection of these cells [74] [75] [76] . indeed, dc-sign-dependent hiv-1 transmission is probably a short-lived process (fig. 1) , which is only observed a few hours after the dc-sign-positive, hiv-1-exposed cells make contact with target cells. mainly, hiv-1 might be endocytosed and processed for mhc presentation ( [77, 78] , fig. 1 ). finally, two reports indicate that dc-sign might not be a good marker for dcs in vivo [68, 79] , with dc-sign-positive cells in lymphoid tissue being of macrophage origin [68] . how these results relate to a series of previous studies demonstrating dc-sign expression on tissue dcs [10, 80, 81] is currently unclear. 36 s. pöhlmann and m.j. tremblay can a significant contribution of dc-sign to dc interactions with hiv-1, and thus to sexual transmission of hiv-1, be disregarded in the light of these results? probably not, since several studies also provide evidence for a role of dc-sign in hiv-1 capture and transmission by dcs. for example, arrighi and colleagues [82] demonstrated that sirna-mediated down-modulation of dc-sign diminishes hiv-1 capture by dcs. the contribution of dc-sign to this process might be due to an involvement of this lectin in the formation of an infectious synapse [83] , a microenvironment established between hiv-1bearing cells and target cells, which promotes efficient transfer of infectious virions [84] . interestingly, dc-sign did not contribute to hiv-1 infection of target cells in cervical explants but, together with cd4, was mainly responsible for hiv-1 uptake by migratory cells present in these explants [85] , suggesting that in hiv-1-infected individuals dc-sign might indeed contribute to hiv-1 dissemination by motile cells expressing this lectin. in this regard, it is noteworthy that platelets have been shown to express dc-sign and to capture hiv-1 in a largely dc-sign-dependent manner [86, 87] . these cells might bind hiv-1 via dc-sign once the virus has reached the blood stream and might promote its dissemination in the host system. similarly, a recent report suggests that a subset of b cells expresses dc-sign and facilitates hiv-1 transmission to t cells in a dc-sign-dependent manner [88] . finally, two groups found that certain polymorphisms in the dc-sign gene are associated with decreased risk of hiv-1 infection [89, 90] , highlighting that dc-sign might modulate important events leading to the establishment of hiv-1 infection. thus, further research is needed to clarify the role of dc-sign in hiv-1 infection and to evaluate whether this protein is a potential target for microbicides. , also termed l-sign (for liver sign) [91] , shares 77% sequence identity with dc-sign and is expressed by sinusoidal endothelial cells in liver (lsecs) and in lymph nodes, alveolar macrophages [92] and enterocytes of the small intestine [93] . moreover, dc-signr transcripts have been detected at sites of mucosal hiv-1 transmission [94] . dc-signr, like dc-sign, binds to high-mannose carbohydrates and captures hiv-1, hiv-2 and simian immunodeficiency virus [11, 91] . binding to icam proteins has also been demonstrated [91] . however, the natural function of dc-signr is currently unclear. expression of dc-signr in lymph node sinusoids might concentrate hiv-1 in this compartment, while dc-signr on lsecs might promote infection of this cell type, which was shown to be permissive in vitro [95] and in vivo [96, 97] . lsecs might therefore constantly release progeny virus into the blood stream, thereby promoting hiv-1 spread. dc-sign and dc-signr are both organized into a n-terminal intracellular domain, a transmembrane domain, a neck region containing 7.5 repeats of a 23-amino acid-comprising sequence and a c-terminal lectin domain. in contrast to the neck domain of dc-sign, which is highly conserved among individuals, the neck domain of dc-signr is polymorphic. while 7.5 repeats are most often found and are considered wild type (wt), alleles with 5.5 and 6.5 repeats are also frequent (28.9% and 12.2%, respectively, in the caucasian population [91] ). the impact of polymorphisms in the dc-signr neck region on susceptibility to hiv-1 infection has been analyzed by two studies. lichterfeld and colleagues [98] found no significant differences in dc-signr allele distribution between hiv-1-infected individuals and healthy controls. also, no correlation between dc-signr allele frequency and course of hiv-1 disease was observed [98] . in contrast, liu and colleagues [99] found that the 7/7 genotype was significantly less frequent in high-risk hiv-1-seronegative individuals compared to hiv-1-seropositive individuals, while the 5/7 genotype was associated with some protection against hiv-1 infection. it is currently unclear, however, how such a protective effect can be explained on the molecular level. thus, dc-signr variants with 5 and 6 repeats were found to form stable homo-oligomers [100] and to augment hiv-1 infection [101] with similar efficiency as the wt protein. also, coexpression of dc-signr alleles with 5 and 7 repeats allowed formation of stable hetero-oligomers and did not result in decreased hiv-1 interactions when compared to controls expressing the 7/7 allele combination [101] . a linkage between dc-signr polymorphisms and alterations in unrelated genes determining susceptibility to hiv-1 infection can therefore at present not be excluded. the observation that dcs can bind to hiv-1 independently of dc-sign raised the question whether related lectins might be involved. a detailed analysis of gp120 interactions with different dc subsets revealed that mr on dermal dcs might contribute to gp120 capture by these cells [72] . mr is an endocytic receptor that harbors multiple lectin domains and recognizes ligands bearing mannose, fucose or n-acetylglucosamine (glcnac) [102] . the lectin is expressed on dcs, macrophages and some endothelial cells [102] and might contribute to capture of hiv-1 virions by these cells. in fact, it has been demonstrated that an mr-specific antibody can reduce hiv-1 attachment to macrophages [103] . langerin contains a single carbohydrate recognition domain specific for mannose, fucose and glcnac and is expressed exclusively by langerhans cells [104, 105] . expression of langerin triggers formation of birbeck granules, which are part of the endosomal recycling machinery of langerhans cells [106, 107] . the lectin might function as an antigen uptake receptor that releases ligands upon exposure to low ph in endosomal compartments [105] . while langerin recognizes hiv-1 gp120, it needs to be determined whether it contributes to infection of langerhans cells, which are sus-ceptible to hiv-1 in culture and in patients [108, 109] , or to transmission of hiv-1 from langerhans cells to adjacent target cells. lectin-dependent hiv-1 attachment to cells can be prevented by interfering with lectin expression or by targeting domains in the lectin required for efficient ligand recognition. alternatively, carbohydrate structures in hiv-1-gp120, which are recognized by relevant lectins, are targets for intervention. down-modulation of lectin expression can be achieved by specific sirna [82, 110] and by sanglifehrin a [111] , an immunosuppressant that diminishes ctype lectin expression on dcs. however, issues with delivery (sirna) and possible unwanted side effects (sanglifehrin a) need to be addressed. several inhibitors that impede the interaction of dc-sign with hiv-1 or other viruses have been described. a synthetic, branched molecule that presents 32 mannose residues on its surface has been shown to inhibit hiv-1-gp120 binding to dc-sign [112] and to block dc-sign interactions with reporter viruses bearing the ebola virus glycoprotein [113] , a well-established dc-sign ligand [114, 115] . the antiviral activity of comparable molecules bearing sialic acid, the structure recognized by influenza hemagglutinin, has also been demonstrated in a mouse model for influenza infection [116] , underlining the feasibility of this approach. the inhibitory substances used to target lectinmediated hiv-1 attachment must not necessarily be of synthetic origin, since bovine lactoferrin [117] and a substance in human milk which harbors lewis x carbohydrates [118] were shown to bind to dc-sign and to inhibit hiv-1 transmission by dcs. similarly, a dc-sign inhibitory activity was identified in human cervicovaginal lavage fluid [119] . these natural substances might modulate the risk of hiv-1 transmission and merit further investigation. finally, inhibition of ligand binding to lectins can be achieved by monoclonal antibodies, and a variety of dc-sign-or dc-signr-specific monoclonal antibodies that inhibit pathogen interactions with these lectins have been generated [68, 69, 71, 120] . while several lectins expressed at the cell surface can mediate hiv-1 attachment, soluble human-, plant-and bacteria-derived lectins can be employed to inhibit this process. thus, mannose-binding lectin (mbl), a soluble lectin that is involved in innate immunity and is known to bind to hiv-1-gp120 [121] , inhibits dc-sign-dependent hiv-1 transmission to target cells, probably by competing with dc-sign for binding sites in hiv-1-gp120 [122] . a similar observation was reported for ebola virus [123] , validating that lectins with overlapping carbohydrate specificity can compete for binding sites in gp120, which can result in reduction of viral attachment. in fact, soluble lectins were shown to be effective against hiv-1 transfer by dcs and direct infection of dcs [124] , highlighting that lectins applied within a microbicide formulation might help to block hiv-1 infection upon sexual transmission. a promising candidate microbicide is cyanovirin n, a mannose-specific lectin obtained from the cyanobacterium nostoc ellipsosporum [125] . cv-n binds to the hiv-1-gp120 protein and inhibits hiv-1 interactions with dcs in vitro [124] and, when applied topically, infection of macaques with a simian/human immunodeficiency hybrid virus upon vaginal and rectal challenge [126, 127] . a more complete understanding of the possible contribution of virus-associated host proteins to the hiv-1 life cycle is crucial because it might lead to the development of alternative approaches for the treatment of hiv-1 infection and/or the design of an efficient vaccine strategy. interestingly, a therapeutic or vaccine strategy targeted at virus-associated host cell surface proteins might circumvent problems due to the great genetic variability displayed by hiv-1. elucidation of the molecular mechanisms underlying hiv-1 capture by cellular lectins and assessment of the contribution of this process to hiv-1 dissemination in and between individuals might help to define novel strategies for preventive or therapeutic intervention. moreover, lectins on dcs can be used as tools to target hiv-1 antigens to these important antigen-presenting cells [128] [129] [130] [131] , which might facilitate the generation of an effective hiv-1 vaccine. cell surface receptors, virus entry and tropism of primate lentiviruses hiv-1 attachment: another look human immunodeficiency virus type 1 attachment to hela cd4 cells is cd4 independent and gp120 dependent and requires cell surface heparans syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages syndecan captures, protects, and transmits hiv to t lymphocytes infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor inhibition of entry of hiv-1 in neural cell lines by antibodies against galactosyl ceramide acquisition of host cell-surface-derived molecules by hiv-1 host protein incorporation is conserved among diverse hiv-1 subtypes presence of host icam-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and cd4(+)-t-cell depletion in human lymphoid tissue, a major site of replication in vivo host cell-dependent alterations in envelope components of human immunodeficiency virus type 1 virions cellular compartments of human immunodeficiency virus type 1 replication in vivo: determination by presence of virion-associated host proteins and impact of opportunistic infection detection of hla-dr associated with monocytotropic, primary, and plasma isolates of human immunodeficiency virus type 1 cd45: a prototype for transmembrane protein tyrosine phosphatases cd86 (b7-2), and major histocompatibility complex class i and ii molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation exclusion of hiv coreceptors cxcr4, ccr5, and ccr3 from the hiv envelope human immunodeficiency virus type 1 derived from cocultures of immature dendritic cells with autologous t cells carries t-cell-specific molecules on its surface and is highly infectious cellular proteins bound to immunodeficiency viruses: implication for pathogenesis and vaccines envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/su), is the primary determinant of su content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus envelope glycoproteins are not required for insertion of host icam-1 into human immunodeficiency virus type 1 and icam-1-bearing viruses are still infectious despite a suboptimal level of trimeric envelope proteins interaction between the cytoplasmic domain of icam-1 and pr55gag leads to acquisition of host icam-1 by human immunodeficiency virus type 1 the presence of host-derived hla-dr1 on human immunodeficiency virus type 1 increases viral infectivity host-derived icam-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhances viral infectivity role of the leukocyte function antigen-1 conformational state in the process of human immunodeficiency virus type 1-mediated syncytium formation and virus infection regulation of lfa-1 activity through cytoskeleton remodeling and signaling components modulates the efficiency of hiv type-1 entry in activated cd4 + t lymphocytes presence of host icam-1 in human immunodeficiency virus type 1 virions increases productive infection of cd4 + t lymphocytes by favoring cytosolic delivery of viral material lfa-1 is a key determinant for preferential infection of memory cd4 + t cells by human immunodeficiency virus type 1 new insights into the functionality of a virion-anchored host cell membrane protein: cd28 vs hiv type 1 insertion of host-derived costimulatory molecules cd80 (b7.1) and cd86 (b7.2) into human immunodeficiency virus type 1 affects the virus life cycle interaction between virion-bound host icam-1 and the high affinity state of lfa-1 on target cells renders r5 and x4 isolates of hiv-1 more refractory to neutralization virion-bound icam-1 and activated lfa-1: a combination of factors conferring resistance to neutralization by sera from human immunodeficiency virus type 1-infected individuals independently of the disease status and phase susceptibility of hiv type 1 to the fusion inhibitor t-20 is reduced on insertion of host intercellular adhesion molecule 1 in the virus membrane macaques immunized with hla-dr are protected from challenge with simian immunodeficiency virus immunization with class i histocompatibility leukocyte antigen can protect macaques against challenge infection with sivmac-32h statin compounds reduce human immunodeficiency virus type 1 replication by preventing the interaction between virion-associated host intercellular adhesion molecule 1 and its natural cell surface ligand lfa-1 statins inhibit hiv-1 infection by downregulating rho activity inhibiting sexual transmission of hiv-1 infection the interaction of immunodeficiency viruses with dendritic cells sequence and expression of a membrane-associated c-type lectin that exhibits cd4-independent binding of human immunodeficiency virus envelope glycoprotein gp120 dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection dc-sign, a c-type lectin on dendritic cells that unveils many aspects of dendritic cell biology dc-sign-icam-2 interaction mediates dendritic cell trafficking identification of dc-sign, a novel dendritic cell-specific icam-3 receptor that supports primary immune responses characterization of dc-sign/r interaction with human immunodeficiency virus type 1 gp120 and icam molecules favors the receptor's role as an antigen-capturing rather than an adhesion receptor rhesus macaque dendritic cells efficiently transmit primate lentiviruses independently of dc-sign binding of human immunodeficiency virus type 1 to immature dendritic cells can occur independently of dc-sign and mannose binding c-type lectin receptors via a cholesterol-dependent pathway dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin/cd209 is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction quantitative expression and virus transmission analysis of dc-sign on monocyte-derived dendritic cells cell type-dependent retention and transmission of hiv-1 by dc-sign functional evaluation of dc-sign monoclonal antibodies reveals dc-sign interactions with icam-3 do not promote human immunodeficiency virus type 1 transmission diversity of receptors binding hiv on dendritic cell subsets raji b cells, misidentified as thp-1 cells, stimulate dc-sign-mediated hiv transmission immunodeficiency virus uptake, turnover, and 2-phase transfer in human dendritic cells covert human immunodeficiency virus replication in dendritic cells and in dc-sign-expressing cells promotes long-term transmission to lymphocytes infection of dendritic cells (dcs), not dc-sign-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to t cells dc-sign promotes exogenous mhc-i-restricted hiv-1 antigen presentation dendritic cells and hiv-specific cd4 + t cells: hiv antigen presentation, t cell activation, viral transfer tlr activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells expression of dc-sign by dendritic cells of intestinal and genital mucosae in humans and rhesus macaques constitutive and induced expression of dc-sign on dendritic cell and macrophage subpopulations in situ and in vitro lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells dc-sign-mediated infectious synapse formation enhances x4 hiv-1 transmission from dendritic cells to t cells recruitment of hiv and its receptors to dendritic cell-t cell junctions blockade of attachment and fusion receptors inhibits hiv-1 infection of human cervical tissue lentivirus degradation and dc-sign expression by human platelets and megakaryocytes dc-sign and clec-2 mediate human immunodeficiency virus type 1 capture by platelets dc-sign on b lymphocytes is required for transmission of hiv-1 to t lymphocytes association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type 1 infection analysis of genetic polymorphisms in ccr5, ccr2, stromal cell-derived factor-1, rantes, and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin in seronegative individuals repeatedly exposed to hiv-1 a dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv-1 infection cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus homozygous l-sign (clec4m) plays a protective role in sars coronavirus infection most dc-signr transcripts at mucosal hiv transmission sites are alternatively spliced isoforms primary cultures of endothelial cells from the human liver sinusoid are permissive for human immunodeficiency virus type 1 detection of hiv1 rna and p24 antigen in hiv1-infected human liver presence of hiv-1 in human parenchymal and non-parenchymal liver cells in vivo the tandem-repeat polymorphism of the dc-signr gene does not affect the susceptibility to hiv infection and the progression to aids repeat-region polymorphisms in the gene for the dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-related molecule: effects on hiv-1 susceptibility all but the shortest polymorphic forms of the viral receptor dc-signr assemble into stable homo-and heterotetramers impact of polymorphisms in the dc-signr neck domain on the interaction with pathogens pattern recognition receptors: doubling up for the innate immune response involvement of macrophage mannose receptor in the binding and transmission of hiv by macrophages the monoclonal antibody dcgm4 recognizes langerin, a protein specific of langerhans cells, and is rapidly internalized from the cell surface characterization of carbohydrate recognition by langerin, a ctype lectin of langerhans cells reproduction of langerin/cd207 traffic and birbeck granule formation in a human cell line model langerin, a novel c-type lectin specific to langerhans cells, is an endocytic receptor that induces the formation of birbeck granules epidermal langerhans cells -a target for htlv-iii/lav infection langerhans' cells are an actual site of hiv-1 replication rnai-directed inhibition of dc-sign by dendritic cells: prospects for hiv-1 therapy the novel cyclophilinbinding drug sanglifehrin a specifically affects antigen uptake receptor expression and endocytic capacity of human dendritic cells mannose hyperbranched dendritic polymers interact with clustered organization of dc-sign and inhibit gp120 binding mannosyl glycodendritic structure inhibits dc-sign-mediated ebola virus infection in cis and in trans dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans prevention of influenza pneumonitis by sialic acid-conjugated dendritic polymers lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type 1 transmission by blocking the dc-sign-gp120 interaction lewis x component in human milk binds dc-sign and inhibits hiv-1 transfer to cd4 + t lymphocytes human cervicovaginal lavage fluid contains an inhibitor of hiv binding to dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin internalizing antibodies to the c-type lectins, l-sign and dc-sign, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of t cell responses mannose binding lectin (mbl) and hiv inhibition of dc-sign-mediated trans infection of t cells by mannose-binding lectin mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization sugar-binding proteins potently inhibit dendritic cell human immunodeficiency virus type 1 (hiv-1) infection and dendritic-cell-directed hiv-1 transfer the highly specific carbohydrate-binding protein cyanovirin-n: structure, anti-hiv/ebola activity and possibilities for therapy cyanovirin-n inhibits aids virus infections in vaginal transmission models cyanovirin-n gel as a topical microbicide prevents rectal transmission of shiv89.6p in macaques dc-sign-specific liposomal targeting and selective intracellular compound delivery to human myeloid dendritic cells: implications for hiv disease high efficiency transduction of dendritic cells by adenoviral vectors targeted to dc-sign effective induction of naive and recall t-cell responses by targeting antigen to human dendritic cells via a humanized anti-dc-sign antibody recombinant adenovirus type 5 vectors that target dc-sign, chemr23 and alpha(v)beta3 integrin efficiently transduce human dendritic cells and enhance presentation of vectored antigens we acknowledge numerous contributions from various laboratories whose references were not cited in this review due to space limitations. the work presented in this review is supported by grants to key: cord-004335-bw3tziup authors: perez-zsolt, daniel; martinez-picado, javier; izquierdo-useros, nuria title: when dendritic cells go viral: the role of siglec-1 in host defense and dissemination of enveloped viruses date: 2019-12-19 journal: viruses doi: 10.3390/v12010008 sha: doc_id: 4335 cord_uid: bw3tziup dendritic cells (dcs) are among the first cells that recognize incoming viruses at the mucosal portals of entry. initial interaction between dcs and viruses facilitates cell activation and migration to secondary lymphoid tissues, where these antigen presenting cells (apcs) prime specific adaptive immune responses. some viruses, however, have evolved strategies to subvert the migratory capacity of dcs as a way to disseminate infection systemically. here we focus on the role of siglec-1, a sialic acid-binding type i lectin receptor potently upregulated by type i interferons on dcs, that acts as a double edge sword, containing viral replication through the induction of antiviral immunity, but also favoring viral spread within tissues. such is the case for distant enveloped viruses like human immunodeficiency virus (hiv)-1 or ebola virus (ebov), which incorporate sialic acid-containing gangliosides on their viral membrane and are effectively recognized by siglec-1. here we review how siglec-1 is highly induced on the surface of human dcs upon viral infection, the way this impacts different antigen presentation pathways, and how enveloped viruses have evolved to exploit these apc functions as a potent dissemination strategy in different anatomical compartments. dendritic cells (dcs) are the most potent antigen presenting cells (apcs) found in humans [1, 2] and their immune function is key to initiate immunity against invading viruses [3] [4] [5] . these cellular sentinels patrol distinct mucosae and upon infection, viral sensing triggers rapid innate immune responses that might initially contain viral spread. dc activation also elicits cellular migration towards secondary lymphoid tissues, where dcs acquire a fully mature phenotype and become competent for antigen presentation, activation of naïve t cells, and expansion of antigen-specific adaptive t cell responses [1, 2] . despite the immune activity exerted by dcs after viral infection, it has been known for decades that viruses evolved different strategies to escape dc antiviral activity [6] [7] [8] . furthermore, certain viruses exploit the immune function of dcs as a way to colonize distant tissues and effectively disseminate systemically [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . the discovery of the role of the receptor siglec-1/cd169, a sialic acid-binding ig-like lectin-1 expressed by dcs, has greatly contributed to our understanding of how viruses subvert dc activity. the siglec-1 receptor acts as an immuno-surveillance molecule [19] but can also be effectively hijacked by distinct enveloped viruses, which either infect dcs directly or are effectively transferred to bystander target cells that become productively infected [20] [21] [22] . hence, siglec-1 function on dcs clearly illustrates how these apcs can trigger antiviral immunity but also enhance viral spread via this receptor. siglec-1 is a type i transmembrane lectin with an amino-terminal v-set domain that interacts with sialylated ligands, preferentially n-acetylneuraminic acid (neu5ac) in an α2-3 linkage [19, 23] . several enveloped viruses including human immunodeficiency virus (hiv)-1 [24] [25] [26] and ebola virus (ebov) [22, [27] [28] [29] incorporate such sialylated ligands within their membranes as an integral part of the gangliosides that are dragged from the plasma membrane when viruses bud from infected cells. although siglec-1 affinity for sialylated ligands is in the micromolar range, high-avidity binding can be achieved upon clustering of thousands of gangliosides in the viral membrane with their receptors on the cellular membrane [19, 30] . moreover, as siglec-1 contains 16 ig-like c2-type extracellular domains that separate the ligand-binding site from the cell surface, it is available for interaction with external ligands and not bound in cis to cell-surface molecules, which is what usually happens with shorter siglecs that are also expressed by dcs [19, 23, 31] . in vivo, the role of siglec-1 during viral infection has been mostly studied in murine models, focusing on resident tissue macrophages that express this lectin and play key immunomodulatory functions. siglec-1-expressing macrophages are located in the subcapsular sinus of the lymph nodes, and they protect mice against vesicular stomatitis virus (vsv) infection by containing incoming viruses. viral sensing triggers cytokine release and promotes antigen presentation to b cells [32, 33] . however, studies using different retroviruses to infect mice have shown that the protective function of these macrophages can be hijacked for efficient viral infection and dissemination within tissues. indeed, robust infection of a particular retrovirus in lymphoid tissues and spleen requires siglec-1-expressing macrophages [34] . the pathogenicity of the infecting retrovirus is key to tip the balance of these siglec-1-expressing macrophages in favor of the protective immune function. the antiviral response dominates when the replicating virus has an expanded tropism [35] , as it also happens in the case of the amphotropic vsv infection [32, 33] . under these pathogenic conditions, viral capture via siglec-1 macrophages is necessary to elicit an effective antiviral cd8 + t cell response via antigen cross-presentation by dcs [35] . overall, these murine studies explain how siglec-1 can contain viral replication and induce antiviral immunity against highly pathogenic viruses, but also favor viral spread within tissues when retroviruses have a limited tropism. yet, how these findings correlate with the pathogenesis of different siglec-1-interacting human viruses, such as hiv-1 or ebov, remains largely unexplored. hiv-1 is the causative agent of acquired immunodeficiency syndrome (aids), a pandemic that has affected more than 70 million people worldwide [36] , while ebov is responsible for the intermittent outbreaks that produce a filovirus-associated disease (fvd) with high fatality rates [37] . in this review, we discuss how siglec-1 is induced on human dcs upon viral infection, to what degree that impacts different viral antigen presentation routes, and in which ways distant enveloped viruses have evolved to exploit siglec-1 function as a dissemination strategy in distinct anatomical compartments. siglec-1 is a receptor codified by an interferon-stimulated gene and is therefore potently upregulated on distinct human dcs, monocytes, and macrophages when these cells sense type i interferons (ifns) such as ifnα [38] [39] [40] . thus, infection with viruses such as hiv-1 or ebov tightly upregulates siglec-1 expression on apcs, as they directly trigger or indirectly promote the release of type i ifns via immune activating factors (figure 1 ). hiv-1 induces secretion of type i interferons (ifns) by plasmacytoid dcs (pdcs) through toll-like receptor (tlr) -7 and -9 sensing, which upregulates siglec-1 on dcs in a paracrine manner. in addition, lipopolysaccharide (lps) from bacterial translocation upregulates siglec-1 on dcs via tlr4 sensing and autocrine type i ifn release. (b) during ebov infection, type i ifns might also play a central role in enhancing siglec-1 expression on dcs, although this needs further investigation. pdcs may produce type i ifns in response to ebov infection in vivo, while bacterial translocation was suspected during a case of gram-negative septicemia in an ebov-infected patient. in parallel, viral components such as secreted ebov glycoprotein may induce activation of myeloid cells through tlr4 signaling, providing an alternative stimulus of autocrine type i ifns during ebov infection. while solid arrows indicate established mechanisms, dotted arrows suggest processes that require further investigation. ifnar: ifnα/β receptor; sgp: secreted glycoprotein. in the case of hiv-1 infection, ifnα levels are potently boosted during acute infection, and sustained-although to a lower extent-throughout the chronic stage, which is characterized by a persistent immune activation [41] [42] [43] . several dc types have been identified as the sources of ifnα production during the course of hiv-1 infection, and therefore contribute to siglec-1 induction. plasmacytoid dcs (pdcs) are considered the most potent type i ifn producers in blood [44] , and their capacity to secrete ifnα in response to hiv-1 sensing has been demonstrated in vitro [45] [46] [47] [48] [49] and in vivo [50] [51] [52] , both during the acute and chronic phases of the disease [50, 51, 53] . of note, pdc activation in response to hiv-1 sensing induces ifnα secretion through toll-like receptor (tlr) -7 and -9 signaling [47, 53] and maturation of bystander myeloid dcs [54] , and this ifnα response is more potent on pdcs derived from females [55] . in turn, secretion of this cytokine can directly upregulate siglec-1 expression on dcs [56] ( figure 1a ). in addition to pdcs, myeloid dcs also secrete type i ifns, although this release is mostly and indirectly triggered by immune activating signals present during the course of hiv-1 infection, which can induce the expression of ifn-stimulated genes on dcs in an autocrine manner [57] [58] [59] [60] . one of those factors is bacterial lipopolysaccharide (lps), which is increased in the plasma of hiv-1-infected individuals due to the bacterial translocation that takes place in the gut-associated lymphoid tissue as a consequence of the gut epithelial barrier disruption occurring early after hiv-1 infection [43, 61, 62] ( figure 1a ). lps induces siglec-1 expression on dcs [63] . moreover, plasma from hiv-1-infected individuals also stimulates siglec-1 expression on dcs signaling via type i ifn receptor [40] . this explains why on circulating monocytes of hiv-1 infected individuals, siglec-1 expression correlates in vivo with the levels of plasma viremia, and why these levels only diminish after introduction of combined antiretroviral treatment [40] . moreover, both types of siglec-1-inducing factors are also present throughout the course of ebov infections ( figure 1b ). secretion of ifnα has been detected in humans and nonhuman primate models [64, 65] , especially in lethal cases [64] , while asymptomatic ebov infections are characterized by the absence of this cytokine [66] . although in vitro pdcs exposed to ebov do not secrete ifnα [67] , activated pdcs have been found in ebov-infected nonhuman primates, suggesting that these cells might produce ifnα in vivo [68] ( figure 1b ). aside from pdcs, myeloid cells could contribute to ifnα secretion during ebov infection, as ebov-like particles induce ifnα production by murine bone marrow-derived dcs through tlr signaling [69] . ebov glycoprotein interaction with human monocyte-derived macrophages induced tlr4-dependent ifnα secretion by these cells [70] . moreover, a cleaved and secreted form of ebov glycoprotein signals through tlr4 [71] , although ifnα secretion in response to these glycoproteins remains unexplored ( figure 1b ). noteworthy, lps was also found in a case of ebov infection complicated with septicemia, possibly due to bacterial translocation [72] , which might account for indirect ifnα secretion during ebov infection as described for hiv-1 ( figure 1b) . overall, the presence of type i ifns throughout the course of these viral infections is well-established, although both hiv-1 and ebov have evolved particular molecular mechanisms via viral antagonistic proteins that aid to evade cellular immune sensing [73] [74] [75] [76] . intriguingly, the protective role of type i ifn responses is controversial, since the apparent antiviral function during the earliest stages of infection may, in turn, fuel pathogenesis during the later stages of viral disease. that seems to be the case not only for hiv-1 [43] , but also for ebov [76] , where clinical data collected during human outbreaks have indicated that elevated levels of circulating ifnα, as well as upregulation of type i ifn-inducible genes, correlates with fatal disease outcome [64, [77] [78] [79] . thus, hiv-1 and ebov infections trigger an immune activation state that upregulates siglec-1 expression on dcs, a situation that might favor early viral dissemination events in an otherwise antiviral environment [80] . viral sensing enhances siglec-1 expression on apcs, and this facilitates hiv-1 infection of dcs and other myeloid cells (figure 2a , top), such as macrophages [81] . although dcs are generally resistant to hiv-1 infection, a recent study showed that siglec-1 mediated hiv-1 productive infection of a population of human dc precursors known as pre-dcs [82] . even though all dcs express the hiv-1-interacting cellular receptor cd4 and the viral coreceptors [83, 84] , being therefore susceptible to viral infection in vitro [85] [86] [87] , infectivity was less prominent than in activated cd4 + t cells [88] [89] [90] [91] . importantly, the activation process that enhances siglec-1 expression on dcs further restricts viral infection, as mature dcs are 10-fold to 100-fold less susceptible to hiv-1 than immature dcs [9, 88, [92] [93] [94] . infection of dcs with hiv-1 also appears to be uncommon in vivo, although it has been reported for both cutaneous and mucosal dcs, including vaginal epithelial dcs [91, 95, 96 ]. yet, the role of siglec-1 in promoting hiv-1 infection of these dcs remains largely unexplored. in most dc subtypes, however, the restriction factor samhd1 inhibits reverse transcription, thus precluding immune sensing and the synthesis of viral antigens. bottom: conversely, hiv-2 encodes vpx, which counteracts samhd1 activity allowing reverse transcription of the viral genome, that can be sensed via cgas and trigger cytokine release. newly synthesized proteins lead to the production of viral particles, but also to proteosomal cleavage the de-phosphorylated form of the host factor sterile alpha motif histidine-aspartate domain-containing protein 1 (samhd1) is the most potent restriction factor that limits hiv-1 infection in dcs [97] (figure 2a, top) . however, if this lack of infectivity is actually beneficial for an immune control remains controversial, as the absence of viral replication impairs viral sensing and limits presentation of hiv-1-specific antigens to prime adaptive immune responses [98] [99] [100] . in contrast to hiv-1, hiv-2 naturally replicates on dcs [98] , which depends on the counteraction of samhd1 by the viral antagonist vpx [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] (figure 2a, bottom) . hiv-2 genome replication in infected dcs is detected by the innate cytosolic dna sensor cyclic guanosine-adenosine monophosphate synthase (cgas), which triggers immune responses upon dna sensing [98, 99] (figure 2a, bottom) . in contrast, hiv-1 restriction by samhd1 prevents viral dna (vdna) retrotranscription in the cytoplasm, impairing the induction of antiviral type i ifn responses [98] (figure 2a, top) . thus, viral replication on dcs can provide an additional source of viral components to be detected via immune sensors or presented to t cells [98, 102] (figure 2a, bottom) , but also compromise cell viability and release inflammatory factors that fuel viral pathogenesis, as previously described for sustained or chronic type i ifn responses. while hiv-1 replication on dcs remains hard to identify in vivo, it has been known for more than a decade that dcs are among the first target cells encountering ebov [11] . dcs are highly susceptible to ebov infection [11, 103] , and this is a complex process that involves several host factors whose function is still being identified [104] . indeed, siglec-1 expressed on activated dcs has recently emerged as a new host factor implicated in ebov attachment, a mechanism that facilitates subsequent cytoplasmic viral entry [22] ( figure 2b ). initial ebov attachment to the dc surface is mediated by several receptors that recognize different elements on the viral membrane and often have a redundant activity [105] . c-type lectin receptors (clrs) such as the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (dc-sign) and the liver/lymph node sinusoidal endothelial c-type lectin (lsectin) mediate viral attachment through binding to viral glycoproteins [106, 107] , while receptors of the tim/tam families (comprising the t cell immunoglobulin and mucin domain receptor along with tyro-axl-mer receptors) recognize phosphatidylserine lipids present on the viral envelope [108] (figure 2b ). ebov incorporates sialylated gangliosides on their membrane [27] , and we have recently shown that these viruses are effectively recognized by the siglec-1 receptor [22] (figure 2b ). siglec-1 recognition of sialylated gangliosides on ebov modulates the binding, uptake, and trafficking of filoviral particles into a sac-like virus-containing compartment (vcc) continuous with the plasma membrane ( figure 2b ). viruses stored in this compartment can be redirected into the classical endosomal pathway and facilitate viral entry into the cytoplasm [22] . indeed, ebov macropinocytosis allows trafficking into late endosomes, where cleavage of viral glycoproteins by cathepsin b (ctsb) facilitates the interaction with the endosomal receptor niemann-pick c1 [109] [110] [111] (ncp1) that triggers cytoplasmic viral entry [112] [113] [114] (figure 2b ). thus, siglec-1-mediated attachment facilitates viral access to the cell cytoplasm [22] . while siglec-1 contributes to ebov entry into dcs, filoviral replication within these cells compromises immune function and prevents adaptive immune responses by limiting cytokine secretion, downregulating the expression of major histocompatibility complex (mhc) and costimulatory molecules and also by reducing the ability of dcs to stimulate t cell proliferation [103, [115] [116] [117] . these results suggest that ebov suppression of dc function prevents initiation of adaptive immune responses and facilitates uncontrolled systemic virus replication [116, 117] through a mechanism that is enhanced by siglec-1 activity [22] . however, clinical data gathered during the west african 2014-2016 outbreak showed strong and sustained t cell activation [118] , challenging the in vivo relevance of this viral dc-escape mechanism [76] . overall, both hiv-1 and ebov can exploit siglec-1 activity to boost dc infectivity, although ebov replication is more prominent in these cells. yet, viral infection poses a difficult balance for apcs. on the one hand, infectivity triggers antiviral immunity via viral sensing and antigen presentation, but on the other hand, it also promotes cell death and suppression of immune responses through the activity of particular viral antagonist proteins. recently, it has been suggested that this apparent paradox is overcome by a division of labor between distinct dc subsets [119] . there is therefore a dissociation between viral infection and antigen presentation, which occurs in distinct dc subpopulations. by these means, susceptible infected dcs transfer viral antigens to resistant dcs, which remain competent to launch adaptive immune responses against viral infections [119] . while several pathways allow for antigen transfer between dcs, secretion of extracellular vesicles bearing particular antigens is among the most effective ones. although the functional paradigm of dc biology states that the particular apc that interacts with incoming viruses in the mucosa would be the one processing these viruses and then traveling to the lymphoid tissue, these cells may not always be the only ones presenting the captured antigens. rather, these pathogen-interacting dcs may transfer captured antigens to other apcs by several mechanisms, including secretion of extracellular vesicles bearing antigen-loaded fragments, which can even be already processed and presented in mhc molecules (figure 3, top) . by these means, the number of dcs bearing viral-specific antigens can be increased very quickly upon infection, thus amplifying the initiation of primary adaptive immune responses [120] [121] [122] . importantly, to induce naïve t cell stimulation in vitro, these extracellular vesicles require a competent activated dc to deliver the co-stimulatory signals to t cells [121] (figure 3, top) . thus, antigen-containing extracellular vesicles do not overcome the need for a competent apc to activate naïve t cells. among the distinct cellular receptors expressed by dcs, siglec-1 is key to capture secreted extracellular vesicles through the same mechanism hijacked by enveloped viruses [123] (figure 3 ). siglec-1 interacts with extracellular vesicles via recognition of sialylated gangliosides packaged on the vesicle membrane [124] , which assemble and bud from cellular membranes as viruses do [125, 126] . this result has been confirmed not only in vitro [63, 127] with extracellular vesicles derived from cell lines or primary cells but also in murine models where siglec-1 expressed on lymphoid tissues was required to trap extracellular vesicles in vivo [128] . upon capture of extracellular vesicles on activated dcs via siglec-1, these vesicles are trafficked along with the receptor towards a sac-like compartment invagination that is continuous with the plasma membrane and allows for extracellular vesicle retention [63, 123] (figure 3, top) . the siglec-1 positive compartment formed within activated dcs may serve as an antigen depot, controlling and sustaining adaptive immunity even if the source of antigen is not directly in contact with the apc, that still can trigger antigen-specific immune responses. these antigens could maintain immunity for prolonged periods, as it happens when dcs control endosomal acidification to preserve antigen cross-presentation over time [129] . although mature or activated dcs markedly downregulate their macropinocytic capacity, these cells are still able to capture, process, and present antigens internalized via endocytic receptors [130] , and that may also be the case for siglec-1 via extracellular vesicle trapping. moreover, as dcs continue to capture and present antigens after maturation in vivo [131] , they could also initiate responses to newly encountered antigens during the course of viral infections, a process that would be boosted by siglec-1 expression. overall, siglec-1 retention of distinct viruses on extracellular vesicle-containing compartments highlight how these cells might act as "trojan horses", capturing filoviruses or retroviruses in the peripheral mucosae and carrying them to secondary lymphoid tissues, where viruses can be effectively transmitted to target cells and contribute to the systemic spread of infection [9] [10] [11] 94, 141] . recently, we have identified the presence of siglec-1-expressing cells in cervical mucosa. these dcs are capable of capturing hiv-1 and mediate viral transmission to target cd4 + cells via transinfection, even in a basal state where no apparent activation is detected [56] (figure 4) . indeed, dcs directly isolated from human ectocervix displayed a basal siglec-1 expression that was sufficient to mediate viral transfer. however, at the endocervix, where the expression of siglec-1 is lower than at the ectocervix, this capacity was enhanced upon ifnα stimulation (figure 4) . hiv-1 is mostly acquired by sexual transmission, and in the cervical mucosa, there are two major sources of antiviral type-i ifn responses after retroviral infection: resident myeloid cells [142] and pdcs, which are the most potent producers of ifnα [44] and are soon recruited to the cervix [143] ( figure 4) . although increased antiviral ifnα secretion could limit initial viral infection, it could promote viral capture on cervical myeloid cells via siglec-1 induction as well. of note, in the cervical the fate of trapped extracellular vesicles on dcs is diverse, as they provide a source not only for antigen cross-presentation to cd8 + t cells, but also to stimulate antigen-specific naïve cd4 + t cell responses in vivo [120, 121] . cd4 + t cell stimulation can take place either by reprocessing the antigens contained in the captured extracellular vesicles or by the direct presentation of previously processed functional epitope-mhc complexes exposed in the vesicle surface [120, 121] . direct extracellular vesicle antigen presentation in the absence of lytic degradation within dcs was initially described using dc populations devoid of particular mhc-ii molecules, that were still able to activate cd4 + t cells because the necessary mhc-ii molecules were already presenting the antigen on the extracellular vesicles trapped by those dcs [121] . thus, extracellular vesicles displaying previously processed functional epitope-mhc complexes on their surface can be recognized, retained, and directly transferred from dcs to antigen-specific cd4 + t cells [121] (figure 3, top) . in turn, siglec-1 upregulation on activated dcs, which are competent apcs, could boost extracellular vesicle uptake and magnify antiviral immune responses. intriguingly, hiv-1 and other retroviruses exploit this antigen dissemination pathway usually engaged by extracellular vesicles to reach cd4 + t cells [123] , which are the main cellular targets of this particular retrovirus (figure 3, bottom left) . siglec-1 directs captured hiv-1 particles to the same vcc where ebola viral particles are retained [22] , that is in addition the same compartment where extracellular vesicles are trapped in activated dcs [63, 123] (figure 3, bottom left) . however, in the case of hiv-1 recognition, viral entry via siglec-1 does not lead to the productive infection of dcs as it happens with ebov, but favors the transfer of trapped viruses to bystander cd4 + t cells. thus, trapped viruses are efficiently transmitted across infectious synapses to susceptible lymphocytes [9, 132, 133] ( figure 3 ). this mechanism of viral transmission is known as trans-infection [134] and is mediated by siglec-1 on activated monocyte-derived dcs, monocytes, blood conventional dcs, pre-dcs, and primary myeloid cells isolated from lymphoid and cervical tissues [38] [39] [40] 56, 63, 82] . filoviral trans-infection from dcs to cd4 + t cells is improbable as lymphocytes are largely resistant to ebov infection [135] . nonetheless, filoviruses display a broad cell tropism, infecting hepatocytes, adrenal cortical cells, and endothelial cells, among other cellular targets [136] [137] [138] [139] . thus, aside from lymphocytes, other cellular targets could be transinfected (figure 3 , bottom right), as it was previously shown for a human cell line binding ebov that transinfected hela cells [140] . however, further research will be required to determine in which anatomical context dcs trapping ebov via siglec-1 could transfer that infectivity to susceptible cellular targets in vivo. overall, siglec-1 retention of distinct viruses on extracellular vesicle-containing compartments highlight how these cells might act as "trojan horses", capturing filoviruses or retroviruses in the peripheral mucosae and carrying them to secondary lymphoid tissues, where viruses can be effectively transmitted to target cells and contribute to the systemic spread of infection [9] [10] [11] 94, 141] . recently, we have identified the presence of siglec-1-expressing cells in cervical mucosa. these dcs are capable of capturing hiv-1 and mediate viral transmission to target cd4 + cells via trans-infection, even in a basal state where no apparent activation is detected [56] (figure 4) . indeed, dcs directly isolated from human ectocervix displayed a basal siglec-1 expression that was sufficient to mediate viral transfer. however, at the endocervix, where the expression of siglec-1 is lower than at the ectocervix, this capacity was enhanced upon ifnα stimulation ( figure 4) . hiv-1 is mostly acquired by sexual transmission, and in the cervical mucosa, there are two major sources of antiviral type-i ifn responses after retroviral infection: resident myeloid cells [142] and pdcs, which are the most potent producers of ifnα [44] and are soon recruited to the cervix [143] (figure 4 ). although increased antiviral ifnα secretion could limit initial viral infection, it could promote viral capture on cervical myeloid cells via siglec-1 induction as well. of note, in the cervical biopsy of a viremic hiv-1 + patient, siglec-1 + cells harbored hiv-1-containing compartments, demonstrating that in vivo, these cells can trap viruses [56] . interestingly, similar vcc-like structures have been detected in urethral macrophages of hiv-1-infected individuals under suppressive combination antiretroviral therapy [144] , but if siglec-1 is implicated in the formation of these particular structures remains to be determined. siglec-1 allows transferring viruses to bystander cd4 + t cells in the mucosa, but also the systemic viral dissemination upon dc migration to lymphoid tissues ( figure 4) . indeed, dcs bearing retroviruses are found in the draining lymph nodes of distinct animal models as soon as 24 h after vaginal challenge [12, [145] [146] [147] and these findings originally led to formulation of the trojan horse hypothesis, which states that dcs can serve as vehicles transporting the virus from the entry sites to distant tissues [9, 10] . . proposed mechanism of hiv-1 dissemination from the female reproductive tract. in the vaginal/ectocervical mucosa, basal siglec-1 + dcs may mediate local hiv-1 trans-infection to target cd4 + t cells. at the endocervical mucosa, lower siglec-1 expression is boosted in response to ifnα released by recruited pdcs sensing hiv-1. siglec-1 + dcs may also contribute to systemic hiv-1 spread due to their ability to migrate to secondary lymphoid tissues, where cd4 + t cells accumulate. while a similar mechanism could be exploited by ebov, further work needs to address this possibility. we hypothesize that the outcome of early interactions between dcs and enveloped viruses may be key to mount effective antiviral responses, but these early encounters also foster viral dissemination to distant tissues. the emerging roles of siglec-1 receptor on dcs clearly exemplify how the immune-mediated activity of this lectin can be effectively hijacked by unrelated viruses such as hiv-1 or ebov. future work should address if other enveloped viruses causing relevant human infectious diseases may contain sialylated gangliosides on their membranes and be recognized by siglec-1, as it has been already shown for the henipavirus [159] . moreover, the precise contribution of siglec-1 to viral immune containment or to pathogenic viral dissemination should be carefully evaluated, as understanding both mechanisms may provide novel avenues to combat infectious agents. the identification of individuals which naturally lack siglec-1 expression due to the presence of an early stop codon in the siglec1 gene in homozygosis [160] indicates that the role of this protein is not essential and that it may be therefore a safe therapeutic target. developing such . proposed mechanism of hiv-1 dissemination from the female reproductive tract. in the vaginal/ectocervical mucosa, basal siglec-1 + dcs may mediate local hiv-1 trans-infection to target cd4 + t cells. at the endocervical mucosa, lower siglec-1 expression is boosted in response to ifnα released by recruited pdcs sensing hiv-1. siglec-1 + dcs may also contribute to systemic hiv-1 spread due to their ability to migrate to secondary lymphoid tissues, where cd4 + t cells accumulate. while a similar mechanism could be exploited by ebov, further work needs to address this possibility. sexual transmission is not considered a major route of ebov infection. however, a case of sexually transmitted ebov has been well documented [148] . moreover, a recent mathematical model is consistent with a significant contribution of sexual ebov transmission during the 2014-2016 outbreak in west africa [149] . importantly, infectious viral particles are found in semen of ebov convalescent individuals several months following symptoms onset [150] [151] [152] [153] , and seminal fluid amyloids may enhance ebov infection [154] . as the cytokine tgf-β1 is abundant in semen, and it also upregulates siglec-1 expression on dcs [155] , the role of this receptor should be further assessed in the context of ebov sexual transmission. moreover, dcs are early and sustained targets of ebov that can disseminate infection from the portals of viral entry to the regional lymph nodes, spleen, and liver [11] . since siglec-1 is expressed in all these ebov replicating-tissues [23, 156] , this receptor could also boost systemic viral spread as previously suggested for hiv-1 [11, 13, 139, 157, 158] . collectively, these data indicate that siglec-1 on dcs may not only participate in viral transmission at the mucosa, but also promote systemic viral dissemination to secondary lymphoid tissues. we hypothesize that the outcome of early interactions between dcs and enveloped viruses may be key to mount effective antiviral responses, but these early encounters also foster viral dissemination to distant tissues. the emerging roles of siglec-1 receptor on dcs clearly exemplify how the immune-mediated activity of this lectin can be effectively hijacked by unrelated viruses such as hiv-1 or ebov. future work should address if other enveloped viruses causing relevant human infectious diseases may contain sialylated gangliosides on their membranes and be recognized by siglec-1, as it has been already shown for the henipavirus [159] . moreover, the precise contribution of siglec-1 to viral immune containment or to pathogenic viral dissemination should be carefully evaluated, as understanding both mechanisms may provide novel avenues to combat infectious agents. the identification of individuals which naturally lack siglec-1 expression due to the presence of an early stop codon in the siglec1 gene in homozygosis [160] indicates that the role of this protein is not essential and that it may be therefore a safe therapeutic target. developing such pharmacological agents to block siglec-1 interaction with viruses could pave the way to ameliorate viral systemic dissemination. moreover, exploiting the immune-surveillance function of siglec-1 receptor to induce antiviral immune responses may also prove valuable. in turn, dissecting how distinct viruses exploit common molecular pathways will advance future antiviral strategies to generate broad spectrum treatments. the dendritic cell system and its role in immunogenicity dendritic cells and the control of immunity visualizing priming of virus-specific cd8 + t cells by infected dendritic cells in vivo endogenous mhc class ii processing of a viral nuclear antigen after autophagy systemic activation of dendritic cells by toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity dendritic cells in viral pathogenesis: protective or defective? anti-immunology: evasion of the host immune system by bacterial and viral pathogens dendritic cell functions: learning from microbial evasion strategies dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to cd4 + t cells bone marrow-derived dendritic cells, infection with human immunodeficiency virus, and immunopathology pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques the molecule of dc-sign captures enterovirus 71 and confers dendritic cell-mediated viral trans-infection dendritic cells mediate herpes simplex virus infection and transmission through the c-type lectin dc-sign dendritic cells as achilles' heel and trojan horse during varicella zoster virus infection murine cytomegalovirus spreads by dendritic cell recirculation dc-sign and cd150 have distinct roles in transmission of measles virus from dendritic cells to t-lymphocytes replication in cells of hematopoietic origin is necessary for dengue virus dissemination siglecs and their roles in the immune system hiv-1 capture and transmission by dendritic cells: the role of viral glycolipids and the cellular receptor siglec-1 cd169-dependent cell-associated hiv-1 transmission: a driver of virus dissemination anti-siglec-1 antibodies block ebola viral uptake and decrease cytoplasmic viral entry characterization of human sialoadhesin, a sialic acid binding receptor expressed by resident and inflammatory macrophage populations retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides role of lipids in virus replication sialyllactose in viral membrane gangliosides is a novel molecular recognition pattern for mature dendritic cell capture of hiv-1 lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses quantifying lipid contents in enveloped virus particles with plasmonic nanoparticles in vivo oligomerization and raft localization of ebola virus protein vp40 during vesicular budding siglec regulation of immune cell function in disease sialic acid binding receptors (siglecs) expressed by macrophages subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral b cells subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus retroviruses use cd169-mediated trans-infection of permissive lymphocytes to establish infection a protective role for the lectin cd169/siglec-1 against a pathogenic murine retrovirus available online the pathogenesis of ebola virus disease interferon-inducible mechanism of dendritic cell-mediated hiv-1 dissemination is dependent on siglec-1/cd169 sialoadhesin expressed on ifn-induced monocytes binds hiv-1 and enhances infectivity hiv-1 immune activation induces siglec-1 expression and enhances viral trans-infection in blood and tissue myeloid cells interferon-alpha and tumor necrosis factor-alpha in serum of patients in various stages of hiv-1 infection induction of a striking systemic cytokine cascade prior to peak viremia in acute human immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis b and c virus infections microbial translocation is a cause of systemic immune activation in chronic hiv infection the nature of the principal type 1 interferon-producing cells in human blood cd4 + blood dendritic cells are potent producers of ifn-alpha in response to in vitro hiv-1 infection natural alpha interferon-producing cells respond to human immunodeficiency virus type 1 with alpha interferon production and maturation into dendritic cells endocytosis of hiv-1 activates plasmacytoid dendritic cells via toll-like receptor-viral rna interactions myd88-dependent immune activation mediated by human immunodeficiency virus type 1-encoded toll-like receptor ligands innate sensing of hiv-infected cells increased interferon alpha expression in circulating plasmacytoid dendritic cells of hiv-1-infected patients plasmacytoid dendritic cells accumulate and secrete interferon alpha in lymph nodes of hiv-1 patients plasmacytoid dendritic cells suppress hiv-1 replication but contribute to hiv-1 induced immunopathogenesis in humanized mice divergent tlr7 and tlr9 signaling and type i interferon production distinguish pathogenic and nonpathogenic aids virus infections human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomitantly induces the bystander maturation of myeloid dendritic cells sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv-1 dendritic cells from the cervical mucosa capture and transfer hiv-1 via siglec-1 maturation, activation, and protection of dendritic cells induced by double-stranded rna type i interferons produced by dendritic cells promote their phenotypic and functional activation a type i interferon autocrine-paracrine loop is involved in toll-like receptor-induced interleukin-12p70 secretion by dendritic cells autocrine type i interferon amplifies dendritic cell responses to lipopolysaccharide via the nuclear factor-κb/p38 pathways damaged intestinal epithelial integrity linked to microbial translocation in pathogenic simian immunodeficiency virus infections intestinal damage precedes mucosal immune dysfunction in siv infection siglec-1 is a novel dendritic cell receptor that mediates hiv-1 trans-infection through recognition of viral membrane gangliosides markedly elevated levels of interferon (ifn)-γ, ifn-α, interleukin (il)-2, il-10, and tumor necrosis factor-α associated with fatal ebola virus infection in vivo ebola virus infection leads to a strong innate response in circulating immune cells human asymptomatic ebola infection and strong inflammatory response ebola virus failure to stimulate plasmacytoid dendritic cell interferon responses correlates with impaired cellular entry infection with the makona variant results in a delayed and distinct host immune response compared to previous ebola virus variants ebola virus-like particles stimulate type i interferons and proinflammatory cytokine expression through the toll-like receptor and interferon signaling pathways ebolaviruses associated with differential pathogenicity induce distinct host responses in human macrophages shed gp of ebola virus triggers immune activation and increased vascular permeability a case of severe ebola virus infection complicated by gram-negative septicemia innate antiviral immune signaling, viral evasion and modulation by hiv-1 impairment of the type i interferon response by hiv-1: potential targets for hiv eradication filovirus pathogenesis and immune evasion: insights from ebola virus and marburg virus role of type i interferons on filovirus pathogenesis transcriptomic signatures differentiate survival from fatal outcomes in humans infected with ebola virus multi-platform 'omics analysis of human ebola virus disease pathogenesis kinetics of soluble mediators of the host response in ebola virus disease interferon-inducible cd169/siglec1 attenuates anti-hiv-1 effects of alpha interferon siglecs facilitate hiv-1 infection of macrophages through adhesion with viral sialic acids constitutive siglec-1 expression confers susceptibility to hiv-1 infection of human dendritic cell precursors efficient interaction of hiv-1 with purified dendritic cells via multiple chemokine coreceptors diversity of receptors binding hiv on dendritic cell subsets susceptibility of human peripheral blood dendritic cells to infection by human immunodeficiency virus replication of human immunodeficieacy virus type 1 in primary dendritic cell cultures differential susceptibility to human immunodeficiency virus type 1 infection of myeloid and plasmacytoid dendritic cells immature dendritic cells selectively replicate macrophagetropic (m-tropic) human immunodeficiency virus type 1, while mature cells efficiently transmit both m-and t-tropic virus to t cells virus replication begins in dendritic cells during the transmission of hiv-1 from mature dendritic cells to t cells during hiv-1 infection most blood dendritic cells are not productively infected and can induce allogeneic cd4 + t cells clonal expansion low levels of hiv-1 infection in cutaneous dendritic cells promote extensive viral replication upon binding to memory cd4 + t cells the maturation of dendritic cells results in postintegration inhibition of hiv-1 replication human immunodeficiency virus fusion to dendritic cells declines as cells mature dendritic-cell interactions with hiv: infection and viral dissemination blockade of attachment and fusion receptors inhibits hiv-1 infection of human cervical tissue hiv-1 replicates and persists in vaginal epithelial dendritic cells samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx a cryptic sensor for hiv-1 activates antiviral innate immunity in dendritic cells the capsids of hiv-1 and hiv-2 determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses vpx relieves inhibition of hiv-1 infection of macrophages mediated by the samhd1 protein mhc-i-restricted presentation of hiv-1 virion antigens without viral replication ebola and marburg viruses replicate in monocyte-derived dendritic cells without inducing the production of cytokines and full maturation mechanisms of filovirus entry characterization of the filovirus-resistant cell line sh-sy5y reveals redundant role of cell surface entry factors c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus phosphatidylserine receptors: enhancers of enveloped virus entry and infection endosomal proteolysis of the ebola virus glycoprotein is necessary for infection ebola virus entry requires the cholesterol transporter niemann-pick c1 small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection ebola virus entry: a curious and complex series of events host factors in ebola infection filovirus entry: a novelty in the viral fusion world impairment of dendritic cells and adaptive immunity by ebola and lassa viruses ebola virus: the role of macrophages and dendritic cells in the pathogenesis of ebola hemorrhagic fever the role of antigen-presenting cells in filoviral hemorrhagic fever: gaps in current knowledge human ebola virus infection results in substantial immune activation constitutive resistance to viral infection in human cd141 + dendritic cells exosomes: composition, biogenesis and function indirect activation of naïve cd4+ t cells by dendritic cell-derived exosomes membrane vesicles as conveyors of immune responses capture and transfer of hiv-1 particles by mature dendritic cells converges with the exosome-dissemination pathway lipids in exosomes: current knowledge and the way forward exosomes and hiv gag bud from endosome-like domains of the t cell plasma membrane higher-order oligomerization targets plasma membrane proteins and hiv gag to exosomes proteomics study of human cord blood reticulocyte-derived exosomes cd169 mediates the capture of exosomes in spleen and lymph node amigorena, s. nox2 controls phagosomal ph to regulate antigen processing during crosspresentation by dendritic cells mature dendritic cells use endocytic receptors to capture and present antigens dendritic cells continue to capture and present antigens after maturation in vivo recruitment of hiv and its receptors to dendritic cell-t cell junctions the infectious synapse formed between mature dendritic cells and cd4 + t cells is independent of the presence of the hiv-1 envelope glycoprotein dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances trans-infection of t cells apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses ultrastructural pathology of experimental ebola haemorrhagic fever virus infection association of ebola-related reston virus particles and antigen with tissue lesions of monkeys imported to the united states pathology of experimental ebola virus infection in african green monkeys: involvement of fibroblastic reticular cells marburg hemorrhagic fever: report of a case studied by immunohistochemistry and electron microscopy mannosyl glycodendritic structure inhibits dc-sign-mediated ebola virus infection in cis and in trans dc-sign: escape mechanism for pathogens blocking tlr7-and tlr9-mediated ifn-α production by plasmacytoid dendritic cells does not diminish immune activation in early siv infection glycerol monolaurate prevents mucosal siv transmission hiv-1 reservoirs in urethral macrophages of patients under suppressive antiretroviral therapy dendritic cells route human immunodeficiency virus to lymph nodes after vaginal or intravenous administration to mice simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus molecular evidence of sexual transmission of ebola virus effect of sexual transmission on the west africa ebola outbreak in 2014: a mathematical modelling study a case of ebola virus infection assessment of the risk of ebola virus transmission from bodily fluids and fomites persistence and genetic stability of ebola virus during the outbreak in kikwit, democratic republic of the congo clinical, virologic, and immunologic follow-up of convalescent ebola hemorrhagic fever patients and their household contacts enhancement of ebola virus infection by seminal amyloid fibrils transforming growth factor beta 1 up-regulates cd169 (sialoadhesin) expression on monocytederived dendritic cells: role in hiv sexual transmission the species-specific structure of microanatomical compartments in the human spleen: strongly sialoadhesin-positive macrophages occur in the perifollicular zone, but not in the marginal zone an analysis of features of pathogenesis in two animal models of ebola virus infection molecular pathogenesis of viral hemorrhagic fever virus particle release from glycosphingolipid-enriched microdomains is essential for dendritic cell-mediated capture and transfer of hiv-1 and henipavirus identification of siglec-1 null individuals infected with hiv-1 this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-010001-u0d5jkp1 authors: kotwal, girish j.; kaczmarek, jennifer n.; leivers, steven; ghebremariam, yohannes t.; kulkarni, amod p.; bauer, gabriele; de beer, corena; preiser, wolfgang; mohamed, abdu rahman title: anti‐hiv, anti‐poxvirus, and anti‐sars activity of a nontoxic, acidic plant extract from the trifollium species secomet‐v/anti‐vac suggests that it contains a novel broad‐spectrum antiviral date: 2006-01-22 journal: ann n y acad sci doi: 10.1196/annals.1352.014 sha: doc_id: 10001 cord_uid: u0d5jkp1 enveloped animal viruses such as human immunodeficiency virus (hiv), hepatitis b virus, hepatitis c virus, human papillomavirus, marburg, and influenza are major public health concerns around the world. the prohibitive cost of antiretroviral (arv) drugs for most hiv‐infected patients in sub‐saharan africa and the serious side effects in those who have access to arv drugs make a compelling case for the study of complementary and alternative therapies. such therapies should have scientifically proved antiviral activity and minimal toxic effects. a plant extract, secomet‐v, with an anecdotal indication in humans for promise as an anti‐hiv treatment, was investigated. using a previously described attenuated vaccinia virus vgk5, we established the antiviral activity of secomet‐v. chemical analysis showed that it has an acidic ph, nontoxic traces of iron (<10 ppm), and almost undetectable levels of arsenic (<1.0 ppm). the color varies from colorless to pale yellow to dark brown. the active agent is heat stable at least up to sterilizing temperature of 121°c. the crude plant extract is a mixture of several small molecules separable by high‐pressure liquid chromatography. the hiv viral loads were significantly reduced over several months in a few patients monitored after treatment with secomet‐v. secomet‐v was also found to have antiviral activity against the sars virus but not against the west nile virus. secomet‐v, therefore, is a broad‐spectrum antiviral, which possibly works by neutralizing viral infectivity, resulting in the prevention of viral attachment. enveloped viruses such as the human immunodeficiency virus (hiv), hepatitis b virus, and influenza virus remain a major threat to human health worldwide, infecting over 1 billion people. together, these three viruses affect at least 5-10 million individuals, or roughly 25% of the south african population, causing considerable morbidity and mortality. in 2004, hiv resulted in more than 3 million total deaths worldwide, with an estimated 5.3 million people infected in south africa alone (data provided by unaids). to combat diseases caused by hiv and other envelopedviruses, a sustained multipronged approach is needed such as the one described previously. 1 one of the most powerful weapons against hiv has been the lifeprolonging drug cocktails for aids treatment. unfortunately, the availability of such drug cocktails is limited to a very small fraction of those infected because of the high cost of treatment. to make matters worse, there are also severe side effects seen in people taking the drugs. therefore, there is an urgent need to continue to search for new therapies, which may be just as effective as new antiviral drugs. other enveloped viruses such as variola virus, the causative agent of smallpox, are considered to be prime candidates for bioweapons in the aftermath of terrorist attacks. in addition, the emergence of monkeypox as a disease-causing agent in the democratic republic of congo (drc) in central africa once again emphasizes a need to continue to search for an antiviral agent against enveloped viruses. the promise of chemotherapy for poxviruses (summarized in fig. 1) and complications from vaccination against smallpox with vaccinia virus seem quite bleak because of the severe side effects. 2 in addition, the looming threat of an influenza pandemic and lack of reliable treatment leave the world vulnerable to the upcoming emergence of new virulent strains. the spread of the sars-causing virus was nipped in the bud by the end of 2004, but that does not mean we can be complacent and ready to dispense a repertoire of antiserum treatments. antiviral drugs would be the best ammunition to bolster the preparedness against this agent. for centuries, humans have looked at finding natural solutions to health problems. recently, plants have been a focus of several investigators around the world in search of antiviral agents. [3] [4] [5] based on interesting anecdotal evidence suggesting properties protective against viral infections, a potentially promising plant extract was developed. reliable scientific evidence was needed to indicate that the extract was truly worth considering as a potential treatment for viral infections and doing more research was justified. with a well-established infrastructure and the methodology to cultivate and to titer viruses accurately 6 to evaluate antiviral effects, it was possible to show that indeed a small volume of the plant extract termed secomet-v was able to inactivate approximately 1 million virus particles of the attenuated recombinant vaccinia virus vgk5 7 in 1 minute consistently and reproducibly. the recombinant attenuated virus vgk5 was chosen because it grows as well as the wild-type western reserve strain of vaccinia virus in cell culture. the use of the highly attenuated vgk5 strain is also relatively safe, because of the lack of a neurovirulence factor 8 attributed to its inability to replicate in the brain and cause complications. 9 anecdotal evidence in humans had indicated that the plant extract had beneficial effects against common colds, influenza virus, and hiv. because secomet-v is registered as herbal medicine, it was provided by the secomet company only to patients with no access to antiretroviral (arv) drugs and under the care of a certified physician. the viral loads of all patients decreased significantly and to nearly minimal levels among the compliant patients, indicating that the extract does indeed have a positive effect on viremia. the levels do not decrease as rapidly as with arv therapy or highly active antiretroviral therapy (haart); however, there were no significant side effects with secomet-v, and the disease symptoms were reduced considerably soon after treatment. it is clear that the amount of active ingredient in the plant extract needs to be identified and increased. besides increasing the efficacy of the active ingredient by identifying, purifying, and characterizing it, the hope is to reduce any other component that may cause toxicity. it has become evident that changing the process of plant extraction (and avoiding the addition of charcoal), as well as using ultrafiltration with a 3-kda cutoff filter, greatly reduces both the long-term and short-term toxicity. secomet-v is a broad-spectrum agent that has the potential to be a safe and highly efficacious antiviral therapy. identifying the active ingredient would characterize the extract to the fullest extent possible and have a major impact on the development of a new synthetic antiviral originating from a natural source. another advantage of identifying the bioactive ingredient would be an understanding of the mechanism of action of secomet-v. currently, all the evidences indicate that the mechanism of action is through rendering the virus noninfectious and subsequently blocking viral attachment. the attenuated recombinant vaccinia virus vgk5 was produced in bsc-1 cells or chicken eggs in required quantities and titered in the presence or absence of the plant extract or the fractions from high-pressure liquid chromatography (hplc) columns as described earlier. 6 essentially, approximately 1 million virus particles were mixed with 10-20 µl of secomet-v. two different cell lines, ccrf cem and cem 174, were used and infected with a subtype b hiv virus (r482). plant extracts were diluted with phosphate-buffered saline. for the experiment, 12-well plates were used and in each plate the following was added: 1 ml rpmi-1640 medium (with pen and strep), 100 µl infected ccrf cem (424.4 pg/ml), or 100 µl infected cem 174 (20.3 pg/ml) and varying amounts of plant extract. for negative controls, the uninfected cell lines were set up in parallel with the infected cell lines. the positive controls were the infected cell lines on the days that the supernatant was collected. to look at the direct effects of the bioactive molecules on hiv infectivity, a pure virus stock of a million virus particles with varying amounts of the purified bioactive molecules was treated and compared with untreated samples. the p24 assay of surviving virus with and without treatment was also compared. additional anti-hiv assays were performed directly with secomet-v with direct contact with hiv at virologic, inc. (south san francisco, ca). varying amounts of african green monkey kidney cell lines (bsc-1 cells) in a monolayer on 96-well confluent plates were treated with varying amounts of the purified fractions and then stained with crystal violet to determine cell cytoxicity as judged by the integrity of the monolayer. secomet-v varies in color from almost colorless, when filtered with a 500-da cutoff filter, to dark brown when unfiltered. the bioactive agent therefore is smaller than or close to 500 da in size. autoclaving does not eliminate the activity, suggesting that the bioactive agent is heat stable up to at least 121°c. the ph of the undiluted extract is approximately 2.0. the virus comes in contact with the extract at a buffered ph of the medium (rpmi-1640) of approximately 3.5. the dry weight varies from approximately 3 mg/ml to 40 mg/ml based on the ultrafiltration cutoff. the arsenic (undetectable) and iron (<10 ppm) content was found to be well below toxic levels, within limits prescribed in the national formulary, usa (analyzed by using perkin-elmer atomic absorption spectrophotometer at the forensic chemistry lab, western cape police department, cape town, south africa). subjects have not complained about its taste, suggesting that it has at least a tolerable taste. secomet-v, an extract of an african plant also found elsewhere in asia, has been found to have potent antiviral activity against a poxvirus (vaccinia virus), rendering about 1 million particles noninfectious in 1 min with a 50th of a milliliter in in vitro assays (fig. 2) . independent reports of anti-hiv testing done by virologic, inc. by mixing the virus with the extract have shown that the hiv particles are neutralized by secomet-v. the same extract failed to cause any reduction in virus titers if the cells were first infected and then directly followed by addition of the plant extract, indicating that secomet-v neutralized viral infectivity but did not inhibit any of the post-entry steps in the life cycle of the virus. hiv-infected cells treated with plant extract showed no significant effect on the viral levels (table 1) . therefore, apparently there is not any effect on the life cycle of hiv once it has entered cells. this was also observed with vgk5 infection. if secomet-v is added after the virus is allowed to attach and internalize for 2 h, there is no significant effect on the virus replication and plaque formation, once again confirming the hypothesis that one or more of the small molecules in the plant extract is, by some as yet unknown mechanism, rendering the virus noninfectious. this suggests that once the virus enters the cells, the extract is no longer effective. this key finding indicates that it is most likely secomet-v acting as a broad-spectrum inhibitor of viral entry. because of this novel mechanism of inhibition, it would not have the same problems of resistance that are common with arvs, as well as the problems of viral evasion due to changes in a surface glycoprotein amino acid sequence. to determine whether secomet-v was able to reduce viremia, four hiv patients infected with hiv-1 subtype c and who had full-blown aids were given the plant extract. these patients had no access to arvs, and their cd4 counts had decreased quite significantly prior to treatment with secomet-v. viral loads were found to have been significantly reduced within a few months as observed (fig. 3a, b) . the sars corona virus was also found to have been rendered inactive by secomet-v (data not shown) when tested along with other agents as a coded panel. the west nile virus was similarly tested, but secomet-v had no effect on the west nile virus at concentrations at which other viruses were inactivated. the dry weight of 1 ml of the crude plant extract is approximately 22 mg and that of the ultrafiltered (with a 5-kda cutoff) plant extract is 3 mg. there was no difference in the effectiveness of the plant extract in rendering vaccinia virus noninfectious whether it was autoclaved or not, suggesting that the bioactive agent is most likely but not necessarily a heat-stable compound and not a small peptide. a preliminary hplc separation of the ultrafiltered plant extract on a c18 column showed 3 major peaks, 3 minor peaks, and approximately 12 quite tiny peaks. previously, it has been shown that the trifollium species has flavanoids and salicylic acid. besides testing for efficacy, preliminary studies were completed to determine the short-term and long-term toxicity of the extract. in a simple assay in which varying amounts of the extract were added to 3 million african green monkey kidney cells (bsc-1 cells), it was found that the 10 µl of the extract rendered half of the million virus particles inactive, and when 100 µl of the plant extract was added, the monolayer became detached and up to 50 µl of the extract was tolerated with no cytotoxicity present. therefore, at 10% of the concentration of the toxic dose, 50% of the virus was rendered inactive. when the extract was filtered through a 3-kda filter, the toxicity diminished, but the antiviral activity remained intact. similar studies have been conducted on potential long-term effects of the crude extract (ballardin et al., this volume) that could be carcinogenic. secomet-v was tested for mutagenicity using the ames gene mutation test in salmonella and the chromosome damage (clastogenic) micronucleus test in human lymphocytes. the crude extract presents weak clastogenic activity but powerful mutagenic activity in the ames test with the addition of exogenous metabolic activation. the purification of the extract without the addition of charcoal results in a drastic reduction of the extract's mutagenicity, which has been virtually reduced by means of further ultrafiltration (cutoff <3,000 da). if ultrafiltration almost eliminates the short-term and long-term toxic molecules, then pure bioactive molecules are likely to be free of all toxicity associated with the crude extract. these findings open new possibilities of hiv therapy because secomet-v, devoid of mutagenic activity, should reduce the possibility of the induction of resistant viral strains by previous arv drugs. hiv treatment and eradication in south africa vaccinia virus inhibitors as a paradigm for the chemotherapy of poxvirus infections korean medicinal plant extracts exhibit antiviral potency against viral hepatitis concerted inhibitory activities of phyllanthus amarus on hiv replication in vitro and ex vivo punica granatum (pomegranate) juice provides an hiventry inhibitor and candidate topical microbicide growing poxviruses and determining virus titer a novel approach using an attenuated recombinant vaccinia virus to test the antiviral effects of handsoaps mapping and insertional mutagenesis of a vaccinia virus gene encoding a 13,800 da secretory protein lack of n1l gene expression results in significant decrease in vaccinia virus replication in mice brain we thank abubakar jacobs and andre baard, forensic chemistry laboratory, cape town police department, for their help in analyzing the samples for arsenic and iron. g.j.k. is currently senior international wellcome trust fellow for biomedical sciences in south africa. a.p.k. and y.t.g. are recipients of the poliomyelitis research foundation (prf) of south africa and senior entrance fellowship and international students' fellowship at the university of cape town, south africa. this work was not supported by funding from secomet pvt. ltd. and is an independent study that was in no way influenced by secomet. key: cord-008672-luoxomif authors: mwachari, c.; batchelor, b.i.f.; paul, j.; waiyaki, p.g.; gilks, c.f. title: chronic diarrhoea among hiv-infected adult patients in nairobi, kenya date: 2004-10-29 journal: j infect doi: 10.1016/s0163-4453(98)90561-8 sha: doc_id: 8672 cord_uid: luoxomif objectives: chronic diarrhoea and wasting are well recognized features of aids in africa. however, because of resource constraints few ocmprehensive aetiological studies have conducted in sub-saharan africa which have included a broad range of microbiological investigations. we undertook a prospective cross-sectional study of adult patients admitted to a government hospital in nairobi, kenya, to determine possible bacterial, mycobacterial, parasitic and viral causes of diarrhoca; to consider which may be treatable; and to relate microbiological findings to clinical outcome. methods: stool specimens from 75 consecutive hiv-seropositive patients with chronic diarrhoca admitted to a nairobi hospital were subjected to microbiological investigation and results were compared with clinical findings and outcome. stool samples were cultured for bacteria and mycobacteria and underwent light and electron microscopy; lawns of escherichica coli were probed for pathogenic types and aliquots were tested for the presence of clostridium difficile cytotoxin. blood cultures for mycobacteria and other bacterial pathogens were performed as clinically indicated. results: thirty-nine (52%) patients yielded putative pathogens, the most common being cryptosporidium sp. (17%), salmonella typhimurium (13%), and mycobacterium tuberculosis (13%). of 41 patients investigated for pathogenic escherichia coli, enteroaggregative e. coli and diffusely adherent e. coli were each found in four patients. thirty-one (41%) patients died. detection of cryptosporidium cysts was the single most significant predictor of death (x(2) = 5.2, p<0.05). many patients did not improve (21; 285) or self-discharged whilst still sick (5; 7%) but five (7%) were diagnosed ante mortem with tuberculosis and treated and a further 13 (17%) showed improvement by time of discharge. conclusions: hiv-infected patients with chronic diarrhoea in nairobi have a poor outcome overall, and even with extensive investigation a putative pathogen was identified in only just over half the patients. the most important step is to exclude tuberculosis: and the most useful investigation appears to be ziehl-neelsen staining. other potentially treatable gram-negative bacterial pathogens, s. typhimurium, shigella sp. and adherent e. coli were, however, common but require culture facilities which are not widely accessible for definitive identification. further studies focussing on simple ways to identify sub-groups of patients with treatable infections are warranted. chronic diarrhoea and wasting, slim disease is a well recognized feature of african aids. 1'2 in nairobi it is a common cause of hospital admission in hiv-infected individuals, ranking fourth in cause of admission (after tuberculosis, acute pneumonia and enteric fever-like illness) and third as cause of death. 3 community studies also suggest that it is also a common cause of death, particularly when associated with significant body wasting. in europe the aetiology of hiv-associated chronic diarrhoea has been comprehensively investigated 4 because * please address all correspondence to: c. f. gilks, liverpool school of tropical medicine, pembroke place, liverpool l3 5qa, u.k. accepted for publication 20 april 1998. of the routine use of intensive diagnostic investigations, access to well equipped microbiology laboratories and frequent post-mortem studies. such services are not often routinely available in most government hospitals in sub-saharan africa; and despite the frequency of hiv-related chronic diarrhoea, the aetiology is far less well defined. most investigations into the cause of chronic diarrhoea have limited themselves to small patient numbers, 5' 6 to an intensive search for an individual pathogen 7-9 or have been narrowly based due to limited culture facilities, either ante-mortem or post-mortem. 6' ~o in particular, it is not clear whether any treatable causes of chronic diarrhoea are frequently missed because of the lack of appropriate diagnostic facilities. we undertook a 4-month study in nairobi, kenya, to determine possible bacterial, viral and parasitological causes of chronic diarrhoea and wasting using a broad range of investigations and related findings to patient outcome. from april to july 1992, consecutive patients reporting chronic diarrhoea (loose or watery stool for at least 4 weeks) were enrolled into a prospective clinical and microbiological study. all were adults (>16 years) admitted directly to the acute medical wards of the kenyatta national hospital (knh), the main government hospital serving nairobi. patients referred from other hospitals were excluded. recruitment occurred within 5 days of admission. all patients were examined, a brief history was taken and details were recorded on a standard clinical entry and follow-up form by a single observer (cm). patients received hiv counselling and informed consent was obtained before recruitment and testing. samples of stool and blood for serology were collected from all study patients. blood culture for mycobacteria and other bacterial pathogens was performed on patients according to clinical assessment. because of resource constraints there was limited access to the general diagnostic microbiology service when the study was conducted, and few stool or blood samples were routinely cultured in the service laboratory. all culture work was therefore carried out in the kenya medical research institute (kemri) microbiology research laboratory. relevant results were given by the study team to the clinicians caring for the patients in knh as soon as they were available. patients were otherwise investigated and treated as was appropriate and in line with local policy, and followed daily or until death. the majority of patients (75%) were given cotrimoxazole with or without metronidazole on admission. cd4 counts are not routinely performed as a service investigation in knh, and few families or patients were prepared to pay for them. the study had insufficient funds to carry out cd4 counts on all patients recruited. at the time of the study, sigmoidoscopy was not routinely carried out because of the limited capacity of the service, and no biopsies were taken. post-mortems were occasionally requested, but usually only a few relatives consented. it was concluded that it would not be possible in this study to obtain biopsy or autopsy material in a consistent fashion, so this was not attempted. the study was approved by the kenya hospitals national ethical committee and the kemri research committee. unless otherwise stated, the investigations described were applied to all patients. all specimens were processed for culture on the day of collection using oxoid (unipath, basingstoke, u.k.) media and incubated in air at 37 °c for 18 h, unless specifically stated. identification of isolates was by standard bacteriological techniques and confirmation of identification, along with phage typing and serotyping, where appropriate, were carried out by the public health laboratory service (phls), u.k. culture of stool samples for salmonella spp. and shigella spp. was carried out by direct inoculation onto xylose lysine desoxycholate agar (xld), brilliant green agar (bg) and selenite p broth. after incubation the broth was subcultured onto both xld and bg agars. campylobacter blood-free medium, with cefoperazone selective supplement, was directly inoculated and incubated microaerophilically at 37 °c for 48 h before examination. cefsulodin-irgasan-novobiocin (cin) medium was used to culture yersinia spp. after direct inoculation and after cold enrichment in phosphate buffered saline at 4 °c for 14 days. cin plates were incubated at 30 °c for 24h before examination. direct inoculation onto thiosulphate citrate bile salt sucrose (tcbs) medium and enrichment in alkaline peptone water were used to culture vibrio spp. aeromonas spp. were cultured on ryan's selective medium with 5 mg/1 ampicillin added. blood culture was carried out, for 18 patients, in brain-heart infusion broth incubated in coa for up to 7 days. patients were investigated for mycobacterial infection not as a putative cause of diarrhoea per se, but because a role for tuberculosis has been implicated in slim disease. 9 mycobacterial culture from stool was in selective kirchner medium (loml), following decontamination with 5% sodium hydroxide for 15 min. blood samples (2 ml) from 17 patients for mycobacterial culture were inoculated directly into kirchner medium (loml) at collection. all samples were incubated at 37 °c for up to 6 weeks. phenol-auramine stained smears were examined directly by fluorescent microscopy for mycobacterium spp. and cl~ptosporidium sp. modified ziehl-neelsen (zn) staining was used to confirm equivocal results. specimens were examined by light microscopy for ova, cysts and parasites directly and following formalin-ether concentration as wet preparations. the uvitex 2b fluorescent method was applied to examine for microsporidia in faecal smears from 36 patients. in addition, aliquots of faeces were preserved in 50% (vol/vol) formalin solution and transported to oxford phl, u.k. grids were prepared and examined for enteric viruses under a phillips 301 electron microscope following negative staining with 1% methylamine tungstate. of clinical features and outcome of hospitalization are shown in table i . one observer (cm) recruited all patients and 'marked weight loss' reflected the subjective impression of whether the patient was clinically wasted or not. most patients were unable to state their pre-morbid weight. thirty-seven (49%) patients reported fever and 18 (24%) were febrile on recruitment or during hospitalization. from stool samples from a random selection of 41 patients, lawns of presumptive e. coli were harvested and shipped to the laboratory of enteric pathogens, central public health laboratories, u.k. on columbia agar slopes for detection of pathogenic e. coli using dna probes directed at the following groups: enteropathogenic e. coli aliquots of faeces were stored at -70 °c and then transferred to oxford phl, u.k. for detection of c. difficile cytotoxin using mrc 5 human fibroblast cell lines. hiv antibody testing was performed using wellcozyme hiv-1 (wellcome diagnostics, dartford, u.k.) and confirmed using enzynergost hiv 1 + 2, (behringwerk ag, marburg, germany). one hundred and sixteen adults fulfilled the entry criteria for the study. all patients gave their consent and were entered into the study. of these, 21 (18%) were found to be hiv-seronegative, three (3%) had equivocal hiv serology and 17 (15%) either had inadequate clinical information recorded or inadequate samples collected to enable any useful analysis to be performed; all were excluded. results are presented for the remaining 75 patients. the wide range of potential pathogens detected can be seen in table ii . clostridium difficile toxin, vibrio spp. and yersinia spp. were not detected. light microscopy failed to detect ova, cysts or parasites normally associated with diarrhoea, although the following were detected: chilomastix mesnili (one), ascaris iumbricoides (two), entamoeba coli (two) and hookwork (two). no cases of isospora belli were seen. no patient yielded etec, epec, eiec or vtec. one patient had both eaggec and daec. the only salmonella sp. isolated was s. typhimurium, of which the commonest phage type (pt) was pt 56 (40%). four of 18 standard blood cultures (22%) were positive. one of 17 mycobacterial blood cultures was positive, yielding mycobacterium avium-intracellulare. fifteen patients had multiple pathogens isolated: most had just two, the commonest combinations being s. typhimurium with cryptosporidium sp. (n=5). potential pathogens (apart from hiv) were not found during the investigation of 36 (48%) patients. there was no signifcant difference in mortality rate between overall groups of patients yielding pathogens (17/39, 44%) and not yielding pathogens (14/36, 39%) (table iii) . however, mortality was significantly associated with detection of cryptosporidium cysts. the mortality rate in patients with cryptosporidium cysts was 9/13 (69%) compared with 22/62 (36%) for cryptosporidium-negative patients (x2=5.2; p<o.05). of the 44 patients who survived, 13 (17%) improved with bed rest, broad spectrum antibiotics and symptomatic therapy: five patients (7%) were diagnosed ante-mortem with tuberculosis and started on treatment; 21 patients (28%) failed to show any significant benefit from the hospital admission and were discharged home, and five patients (7%), all still sick, took their own discharge. all patients were considered to have clinical ads. cd4 counts were not available for the study patients. details in africa, chronic diarrhoea is common in adult patients presenting to hospital and is often associated with hiv the spectrum of pathogens recovered was largely in accordance with previous reports from africa, but was in some respects at variance with the experience of hivrelated illness in europe and america. cryptosporidium was the most commonly detected pathogen, being found in 17% of patients, compared with reported rates of 13% in burundi, 13 30% in zaire s and 32% in zambia. 14 other protozoa associated with diarrhoeal illness were not detected in our study. we saw no cases of isospora belli, a pathogen with marked variation in geographical distribution. 15 nor, like other workers in zambia, did we identify any cases of giardia lamblia or entamoeba. 16 a single patient yielded enterocytozoon, the only microsporidian seen, and our low detection rate differed from other studies in zimbabwe and zambia. 9' 17 although simple staining methods on unconcentrated stool samples have been shown to be effective, 9'15'17 small numbers may be revealed only after prolonged examination of preparations from at least two stool samples, and it is possible that more meticulous analysis would have yielded greater numbers. when profound weight loss is a feature of hiv-related illness in africa it is often referred to as slim disease. 1 in our study chronic diarrhoea was the primary entry criterion, but as this is inextricably linked with wasting in many patients it was considered important to seek evidence of mycobacterial infection as a cause of wasting and m. tuberculosis was isolated from the faeces of 10 (13%) patients. although these patients presented complaining of chronic diarrhoea, subsequent clinical evaluation identified five of these patients as likely to be suffering from pulmonary tuberculosis and appropriate therapy was started. these five patients survived, providing further evidence that initially unrecognized tuberculosis may be a significant and treatable contributing factor to the wasting seen in slim disease. 1° nevertheless, in a further five patients who were stool culture positive for m. tuberculosis, tuberculosis was not diagnosed as a result of routine clinical procedures. wasting may be the only noticable feature in a significant number of patients with tuberculosis; or it may be an agonal infection. only one patient yielded mycobacterium avium-intracellulare, which was grown from a blood culture. unlike europe and america, atypical mycobacterial infection is rarely seen in nairobi. 12 salmonella typhimurium was the leading bacterial pathogen in this study and was isolated from 12 patients, two of them from blood culture but not from stool. it is unclear how such acute disseminated salmonellosis, and the single case of e. coli bacteraemia, relate to chronic diarrhoea. these may be secondary infections in debilitated patients with marked immunosuppression. in a previous study, non-typhi salmonellae were found to be the most common cause of hiv-related bacteraemia in hospital admissions in nairobi. tm detection of eaggec or daec from 9/41 (22%) patients was an interesting finding. one patient yielded both of these forms of pathogenic e. coli. a significant association between colonization with adherent e. coli and chronic diarrhoea and wasting in aids patients has been demonstrated by kotler et al., 19 who found such strains in 17% of study patients in the usa. adherent e. coli have also been described from hiv-positive patients with chronic diarrhoea in zambia. 2° despite use of a broad range of microbiological investigations, there was failure to detect a pathogen in almost half the patients. it is likely that analysis of multiple stool samples taken at separate times might have increased yields, as might invasive procedures such as duodenal aspiration and rectal biopsy. ~ it is possible that novel agents remain to be characterized and associated with chronic diarrhoea and wasting in hiv patients, and hiv itself may be the cause of chronic diarrhoea in some patients. 21 whether any such additional investigations would identify more treatable infections remains to be seen. how then may these results contribute to the care of hiv-infected patients with chronic diarrhoea in areas with limited diagnostic and microbiological facilities? the study results clearly show that seropositive patients presenting with chronic diarrhoea have a poor outcome irrespective of whether a potential pathogen is isolated or not. careful diagnostic evaluation, in particular looking for pulmonary tuberculosis, should nevertheless be undertaken. sputum (or stool) zn microscopy for mycobacteria should be feasible almost everywhere and some patients with active tuberculosis will respond well to therapy. stool microscopy using modified zn staining can also identify cryptosporidium cysts, the presence of which is associated with a very limited prognosis. for such patients in the absence of effective therapy, it may be better to aim for symptomatic therapy and early discharge home as soon as the cysts are identified. what of the potentially treatable gram-negative enterobacterial pathogens identified, which were cultured from over 20% of study participants.) furthermore, as many as 20% of patients may be colonized with adherent e. coil, which may also play a role in pathogenesis and may respond to antibiotic treatment. to identify such infections requires access to a microbiology laboratory routinely performing culture and sensitivity testing. unfortunately, few hospitals in sub-saharan africa can at present afford to provide such a service routinely because of severe resource constraints. clinical trials focussing on these patients may be able to define more clearly signs and symptoms which predict bacterial infection without necessarily needing access to culture facilities, and thus more effectively identify a sub-group of patients with chronic diarrhoea for whom hospital admission and intensive antimicrobial therapy may be justified. slim disease: a new disease in uganda and its association with htlv-iii infection persistent diarrhoea, strongly associated with hiv infection in kinshasa, zaire the presentation and outcome of hiv-related disease in nairobi gastrointestinal infections in aids persistent diarrhoea in zairian aids patients: an endoscopic and histological study enteropathic aids in uganda. an endoscopic, histological and microbiological study prevalence of enteric viruses among hospital patients with aids in kinshasa faecal mycobacteria and their relationship to hiv-related enteritis in lusaka, zambia high prevalence of enteroeytozoon bieneusf infections among hivpositive individuals with persistent diarrhoea in harare contribution of tuberculosis to slim disease in africa parasitological observations of chronic diarrhoea in suspected aids adult patients in kinshasa disseminated mycobacterium avinm infection among hiv-infected patients in kenya infectious diarrhoea in african acquired immunodepression syndrome (aids). a prospective survey of 100 patients studied in bujumbura (burundi) hiv-related enteropathy in zambia: a clinical, microbiological and histological study cryptosporidial and mi-crosporidiai infections in hiv-infected patients in northeastern brazil intestinal parasites in zambian patients with human microsporidiosis in african aids patients with chronic diarrhoea life-threatening bacteraemia in hiv seropositive adults admitted to hospital in chronic bacterial enteropathy in patients with aids hep-2 cell-adherent e. coli in patients with human immunodefiency virus-associated diarrhoea small intestinal structure and function in patients infected with human immunodefieiency virus (hiv): evidence for hiv-induced enteropathy we would like to thank the medical and nursing staff of the kenyatta national hospital who participated in these studies and the director, kemri for permission to publish these findings. we are indebted to dr b. rowe for help with pathogenic e. coli detection. this study was funded by the wellcome trust (uk). key: cord-001707-piyo00yg authors: murray, jillian; cohen, adam; walaza, sibongile; groome, michelle; madhi, shabir; variava, ebrahim; kahn, kathleen; dawood, halima; tempia, stefano; tshangela, akhona; venter, marietje; feikin, daniel; cohen, cheryl title: determining the provincial and national burden of influenza-associated severe acute respiratory illness in south africa using a rapid assessment methodology date: 2015-07-08 journal: plos one doi: 10.1371/journal.pone.0132078 sha: doc_id: 1707 cord_uid: piyo00yg local disease burden data are necessary to set national influenza vaccination policy. in 2010 the population of south africa was 50 million and the hiv prevalence was 11%. we used a previously developed methodology to determine severe influenza burden in south africa. hospitalized severe acute respiratory illness (sari) incidence was calculated, stratified by hiv status, for four age groups using data from population-based surveillance in one site situated in gauteng province for 2009–2011. these rates were adjusted for each of the remaining 8 provinces based on their prevalence of risk factors for pneumonia and healthcare-seeking behavior. we estimated non-hospitalized influenza-associated sari from healthcare utilization surveys at two sites and used the percent of sari cases positive for influenza from sentinel surveillance to derive the influenza-associated sari rate. we applied rates of hospitalized and non-hospitalized influenza-associated sari to census data to calculate the national number of cases. the percent of sari cases that tested positive for influenza ranged from 7–17% depending on age group, year, province and hiv status. in 2010, there were an estimated 21,555 total severe influenza cases in hiv-uninfected individuals and 13,876 in hiv-infected individuals. in 2011, there were an estimated 29,892 total severe influenza cases in hiv-uninfected individuals and 17,289 in hiv-infected individuals. the incidence of influenza-associated sari was highest in children <5 years and was higher in hiv-infected than hiv-uninfected persons in all age groups. influenza virus was associated with a substantial amount of severe disease, especially in young children and hiv-infected populations in south africa. influenza virus causes substantial disease in low-and middle-income countries each year, but data on the disease burden are limited [1] . data on influenza from country-level surveillance can show the distribution of disease among the population and identify groups at high risk of infection. this information can subsequently help policy makers make evidence-based decisions on how to target influenza treatment and prevention programs, such as vaccination, and can lead to more effective allocation of resources within a country [2] . south africa is a middle-income country, but there is great variation in socioeconomic status with some provinces that are more similar to low-income countries [3, 4] . this results in some populations within the country having a disproportionately higher level of exposure to risk factors for communicable disease. these include environmental risk factors, such as crowded living conditions and exposure to indoor air pollution, as well as biological risk factors, such as malnutrition and underlying infections [5] [6] [7] . these risk factors may drive the burden of influenza in south africa to be greater than other countries with similar income level [3, 8, 9] . in particular, the high hiv prevalence in south africa likely leads to higher numbers of severe influenza-associated illness and thus more hospitalized cases [9] [10] [11] [12] [13] [14] [15] . the influenza season in south africa occurs in the austral winter months of may to august [14, 16, 17] . influenza vaccine has been available in the public sector in south africa for many years but coverage is only approximately 5%. the recommended target groups for annual vaccination include pregnant women, persons with underlying medical conditions (including hiv), children less than 5 years of age and persons over 65 years of age [18] . the estimation of national influenza burden is challenging particularly in low resource settings where data on the national number of consultations, hospitalizations and deaths are lacking in most instances. we estimated influenza-associated severe acute respiratory illness (sari) by hiv status in south africa using a rapid assessment methodology [2] . the protocol for the sari surveillance system was approved by the research ethics committees of the universities of the witwatersrand and kwazulu-natal. the surveillance data used for the model was deemed non-research by the united states centers for disease control and prevention and did not need human subjects review by that institution. all data that was analyzed was de-identified and consent was obtained from all patients before they were enrolled into surveillance. information from the surveys used was publically available. we utilized a multiplier model to estimate numbers of individuals with hospitalized and nonhospitalized influenza-associated sari for each province in south africa in four age groups (<5, 5-24, 25-44 and 45 years) for 2009-2011, stratified by hiv serostatus (fig 1) . non-hospitalized sari includes cases from the population that do not reach the hospital due to health care access barriers. this method was previously used in kenya and guatemala but was modified to include stratification by hiv serostatus to reflect the impact of hiv on influenza burden in south africa [2] . [19, 20] . sentinel surveillance is conducted in four out of the nine south african provinces (gauteng, kwazulu-natal, mpumalanga and north west). hospitalized children aged <5 years are enrolled in surveillance with physician-diagnosed lower respiratory tract infection, and individuals 5 years are enrolled if they meet a modified who case definition for sari (sudden onset of fever with cough or sore throat and shortness of breath or difficulty breathing with 7 days from presentation to the hospital) [21] . population-based surveillance is implemented at the chris hani baragwanath academic hospital (chbah) in gauteng province. chbah is a large public academic hospital in the soweto township in gauteng, south africa. it serves approximately 1.4 million lower income south african individuals [12] . estimation of the rate of hospitalized sari cases in soweto (gauteng province) (fig 1, step 1). we estimated the total number of sari hospitalizations using the number of enrolled sari cases at chbah, adjusting for non-enrollment (refusal to participate and nonenrolment during weekends) by age groups and hiv status as previously described [12] . the rates of hospitalized sari cases in soweto were obtained by dividing the estimated number of sari cases by the mid-year study population estimates by age group and hiv status. the hiv prevalence in the study population was obtained from the projections of the actuarial society of south africa (assa) aids and demographic model. the population of soweto represents approximately 11% of the population of gauteng province (referred thereafter as the base province). we assumed that the incidence rates for soweto reflected the incidence rate for the province. at the time of this study, chbah was the only public hospital serving soweto, where approximately 10% have private medical insurance ( [22] . more than 80% of uninsured and 10% of insured persons seek care at public hospitals such as chbah. therefore, the majority of individuals in soweto that seek care do so at chbah [12] . estimation of the rate of non-hospitalized sari in soweto (gauteng province) (fig 1, step 2). we estimated the rate of non-hospitalized sari in the base province using the data from two healthcare utilization surveys for sari that were conducted in soweto and klerskdorp, north west province in 2013. non-hospitalized cases of sari refer to cases of sari identified in the population that had symptoms severe enough to be hospitalized but did not seek care at a hospital, for reasons such as limited access to healthcare. briefly, between 4,000 and 6,000 community members were surveyed in each of the two sites as part of a cross-sectional door-to-door household survey and asked if they had a severe pneumonia in the past year (as defined as fever and cough and difficult breathing for 2-30 days or a physician diagnosis of pneumonia) and whether they were admitted to a hospital for the event. the ratio of those that sought care to those that did not was applied to the rate of hospitalized sari to get the rate of non-hospitalized sari in the base province [2] . estimation of the rates of hospitalized and non-hospitalized sari in the other provinces (fig 1, step 3). estimates of hospitalized sari rates for the other eight provinces in south africa were derived by adjusting the soweto (gauteng province) sari incidence rates for the provincial-level prevalence of known risk factors for pneumonia (from the south africa demographic and health survey and the assa model). risk factors included exposure to indoor air pollution, crowding, malnutrition, low birth-weight and non-exclusive breastfeeding; the last three were only included as risk factors for children under five years of age [2, 8] . the relative risks of sari associated with each risk factor were determined from the published literature [8] . in addition, we adjusted the provincial rates by the proportion of ari seeking care in the given province to the proportion of ari cases seeking care in the base province (gauteng province) using health and demographic surveys (dhs) [4] . an upward adjustment factor (greater than one) resulted in a greater incidence of sari in the province relative to soweto (gauteng province). similarly, a downward adjustment factor (less than one) resulted in a decrease in sari incidence in the province relative to soweto (gauteng province). the equations used for the provincial adjustment are provided in the technical s1 appendix. the rates of non-hospitalized sari in the remaining provinces were estimated using the provincial prevalence of known risk factors for pneumonia as described for the hospitalized cases. in addition, we adjusted the provincial rates by the proportion of ari not seeking care in the given province to the proportion of ari cases not seeking care in the base province (gauteng province) using health and demographic surveys (dhs) [4] . estimation of the rates of influenza-associated hospitalized and non-hospitalized sari (fig 1, step 4) . the estimates of hospitalized and non-hospitalized sari rates were subsequently multiplied by the percent of sari associated with influenza virus from surveillance (i.e., the influenza detection rate) to give the incidence for influenza-associated sari. data on the percent of sari due to influenza by age group in separate hiv-infected and-uninfected populations were pooled from all sentinel surveillance sites to give an average percentage that was used in the calculations for each province. estimating the provincial and national numbers of hospitalized and non-hospitalized sari cases and influenza-associated sari cases (fig 1, step 5). the number of hospitalized and non-hospitalized sari cases and influenza-associated sari cases was obtained by multiplying the estimated rates by the mid-year population estimates. provincial population estimates stratified by age group and hiv status were obtained from the assa 2008 population model. in an alternate method, we utilized a non-hiv-stratified model. to account for hiv prevalence in this model, we included hiv as a risk factor for sari in calculating the provincial adjustment factors rather than estimating separate incidences for hiv-infected and-uninfected individuals. the relative risk for hiv was determined from the literature to be 7.3 for children under five years of age and 5.6 for children and adults over five years of age, and was used in previous applications of this methodology [2, 8, 23, 24] . the base sari incidence rate in this method was also obtained from the population-based surveillance at chbah and the same healthcare utilization adjustments were made. data on the proportion of sari cases that were influenza-associated was pooled across surveillance sites to give an average percentage that was applied to all provinces. confidence intervals were estimated by bootstrapping each parameter included in the calculations 1000 times, including the sari rates in the base province, the influenza detection rate among sari cases, the prevalence of the provincial risk factors, the proportion of sari cases seeking care from the hus in the base province and the proportion of ari cases seeking care in each province form the dhs. the upper and lower limits of the 95% confidence intervals were the 2.5 th and 97.5 th percentile of these estimates, respectively. the highest rates of sari in both hiv-infected and hiv-uninfected populations were among children less than five years of age, ranging from 2,253-5,507 per 100,000 in the hiv-infected population and 1,609-2,027 per 100,000 in the hiv-uninfected population over the three year study period [12] . the incidence rate of sari in the hiv-infected population was consistently greater than the incidence in the hiv-uninfected population for all age groups. the rate ratio between hiv-infected and hiv-uninfected persons was highest in the 5-24 year old age group (range [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] and lowest in the under-5 year olds (range 1-3). sari rates varied by year, with 2009 having the highest incidence rate for most age groups among both hiv-infected and hiv-uninfected populations. base hospitalized influenza-associated sari rates, soweto, gauteng similar to the base incidence of sari, the base influenza-associated sari incidence rate was greater among hiv-infected individuals for all three years and highest in children <5 year of age, ranging from 140-844 and 93-366 per 100,000 person-years in the hiv-infected anduninfected populations, respectively, over the three year time period (table 1) . among adults, influenza-associated sari rates were highest among the oldest age group (>45 years) for hivinfected (range 143-266 per 100,000 person-years) and hiv-uninfected populations (20-54 per 100,000 person-years) ( table 1) . as the base province, gauteng had a risk factor adjustment factor of 1. the eastern cape consistently had the largest downward adjustment factor (range 0.9-1.1), while mpumalanga consistently had the largest upward adjustment factor (range 1.5-1.9; s1 table) . across the three surveillance years studied, mpumalanga and the northern cape consistently had the highest sari incidence rate across both hiv-infected and uninfected groups and the four age groups studied (s2 table) . rates for hospitalized influenza-associated sari stratified by age and province are given in table 1 . the estimated incidence of influenza-associated sari was consistently highest among the hiv-infected population across all age groups in all of the nine provinces for the surveillance years 2009-2011. within both the hiv-infected and hiv-uninfected populations, the highest incidence of influenza-associated sari was found in the <5 year olds for all provinces and surveillance years. in 2009, the national number of hospitalized influenza-associated sari cases was estimated to be 22,525 in hiv-uninfected individuals and 8,715 in hiv-infected individuals ( table 2) . at two sites where healthcare utilization surveys were conducted, 61% of influenza-associated sari cases were not hospitalized. in 2009, the total number of hospitalized and non-hospitalized influenza-associated sari cases was estimated to be 47,711 in hiv-uninfected individuals and 17,993 in hiv-infected individuals ( table 2 ). in 2010 and 2011, there were fewer overall estimated influenza-associated sari cases compared to 2009. in 2010, there were an estimated 21,555 total severe influenza cases in hiv-uninfected individuals and 13,876 in hiv-infected individuals ( table 2 ). in 2011, there were an estimated 29,892 total severe influenza cases in hiv-uninfected individuals and 17,289 in hiv-infected individuals ( table 2) . in our alternate method in which hiv was adjusted for as a risk factor rather than a stratifying variable, the greatest impact on the adjustment factors was for the 25-44 year old age group where the hiv prevalence is highest. the eastern cape province still consistently had the largest downward adjustment factor, except in the 25-44 year old age group where limpopo had the largest downward adjustment. however, kwazulu-natal province consistently had the largest upward adjustment factor across all age groups, due to the high hiv prevalence in that province. the results from method 2 yielded similar incidence of influenza-associated sari and number of hospitalized cases across the age groups and years included in the analysis (s3 table) . the non-stratified alternate method estimated a total of 74,558 cases of hospitalized influenza-associated sari for 2009-2011, only 6% more than the 70,607 cases estimated by the stratified model for the same years. influenza-associated sari represents a substantial burden of disease in south africa. using a rapid assessment methodology, we found that the highest rate was seen in children <5 years of age irrespective of hiv status. among adults, the highest number was seen in hiv-infected adults aged 25-44 years. previous studies of influenza in south africa have not calculated a national burden [25] [26] [27] [28] and have not estimated the non-hospitalized sari burden. our methodology allowed estimation of the number of cases in each province by age group and hiv serostatus, which can support decisions on targeted interventions within the confines of limited resources. our study highlights the important role that hiv infection plays when estimating severe influenza burden in settings with high hiv prevalence. among the age group with the highest hiv prevalence (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) year olds), hiv-infected persons accounted for 80% of the national burden of influenza-associated sari, despite representing only 30% of the national population [29] . hiv-specific incidences for influenza-associated disease in south africa have been reported previously to be high in hiv-infected individuals [11, 12] . a study in children 2-60 months of age in 1997 reported influenza-associated severe lower respiratory tract infection incidence rates of 1,268 and 148 per 100,000 for hiv-infected and hiv-uninfected children, respectively [11] . more recently, an analysis of population-based data from chbah found influenza-associated lower respiratory tract infections (lrti) to be highest in the hiv-infected population [12] . some countries may not be able to calculate hiv-specific influenza incidence rates due to lack of reliable hiv-stratified influenza data in the population. in the non-hiv-stratified alternate method, we derived similar estimates of burden using a model incorporating adjustment for hiv prevalence by province. this suggests that hiv-specific influenza incidence rates are not necessary to derive valid overall burden estimates because hiv prevalence by province is available in most african countries through demographic and health surveys or multicluster indicator surveys [4] . there were several limitations to our methodology. our study relies on extrapolating incidence rates from one surveillance site in one province to the rest of the provinces of south africa. although a high percentage of the base province is under surveillance from this one large surveillance site and we adjusted this incidence based on province-level risk factor for sari, there are likely additional unmeasured factors that can lead to variation in rates regionally. we assumed that the relative risk of sari for the different risk factors determined for the populations in the literature are the same relative risks for the population of south africa, which might not be the case [8, 23, 24] . second, the denominator estimates also might have been inaccurate. we used the assa 2008 population model to estimate the population and hiv prevalence by province and age group. these population estimates are extrapolations from the 2001 census. third, the healthcare utilization surveys used to calculate non-hospitalized cases was undertaken in only two sites in two provinces. moreover, the surveys did not stratify health-seeking by hiv status or age, which is likely to influence health-seeking behavior. fourth, these estimates reflect the dsease burden in the public sector; however, a similar study using a different modelling approach in the private health sector of south africa found similar incidence rates of influenza-associated respiratory hospitalizations [30] . lastly, we assume that the percent of influenza-associated sari is the same across all age groups, provinces and hiv status. this rapid assessment methodology has been used in kenya, guatemala and now south africa [2] . other countries that have influenza surveillance in place can also implement this methodology to estimate their national burden of severe influenza disease. we found that provinces in south africa vary in the prevalence of important risk factors for sari and influenza and that these impact the burden of severe disease. current recommendations in south africa include the vaccination of hiv-infected individuals with the seasonal influenza vaccine; however, the vaccine tends to be under-utilized in this population and in south africa in general [31] . the results of this rapid assessment can be used to focus efforts targeting influenza vaccination by demonstrating the distribution of disease across south africa. supporting information s1 global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis estimation of the national disease burden of influenza-associated severe acute respiratory illness in kenya and guatemala: a novel methodology surveillance and management of influenza in africa: an urgent need still to be met undernutrition can affect the invading microorganism social mixing patterns within a south african township community: implications for respiratory disease transmission and control risk factors for poor immune response to influenza vaccination in elderly people epidemiology and etiology of childhood pneumonia influenza-related mortality among adults aged 25-54 years with aids in south africa and the united states of america excess mortality due to pneumonia or influenza during influenza seasons among persons with acquired immunodeficiency syndrome increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodeficiency virus type-1 severe influenza-associated respiratory infection in high hiv prevalence setting, south africa naturally-acquired influenza-specific cd4+ t-cell proliferative responses are impaired in hiv-infected african adults influenza surveillance in 15 countries in africa lower respiratory tract infections associated with influenza a and b viruses in an area with a high prevalence of pediatric human immunodeficiency type 1 infection seasonal influenza epidemiology in sub-saharan africa: a systematic review twenty-five years of outpatient influenza surveillance in south africa recommendations pertaining to the use of viral vaccines: influenza 2013 regional perspectives on influenza surveillance in africa strategy to enhance influenza surveillance worldwide world health organization. who global epidemiological surveillance standards for influenza health and related indicators. south african health review morbidity in hiv-1-infected individuals before and after the introduction of antiretroviral therapy: a longitudinal study of a population-based cohort in uganda estimating the vaccine-preventable burden of hospitalized pneumonia among young mozambican children laboratory studies of the 1984 influenza epidemic on the witwatersrand surveillance of respiratory viruses. a 10-year laboratorybased study impact of the introduction of a/sydney/5/97 h3n2 influenza virus into south africa antigenic and molecular analysis of influenza a (h3n2) virus strains isolated from a localised influenza outbreak in south africa in 2003 south african national hiv prevalence, incidence, behaviour and communication survey 2008: a turning tide among teenagers? cape town hospitalizations associated with influenza and respiratory syncytial virus among patients attending a network of private hospitals in south africa coverage of high risk groups for influenza vaccination in south africa, 2011-2013 influenza seasons. options for the control of influenza cape town key: cord-016704-99v4brjf authors: nicholson, felicity title: infectious diseases: the role of the forensic physician date: 2005 journal: clinical forensic medicine doi: 10.1385/1-59259-913-3:235 sha: doc_id: 16704 cord_uid: 99v4brjf infections have plagued doctors for centuries, in both the diagnosis of the specific diseases and the identification and subsequent management of the causative agents. there is a constant need for information as new organisms emerge, existing ones develop resistance to current drugs or vaccines, and changes in epidemiology and prevalence occur. in the 21st century, obtaining this information has never been more important. population migration and the relatively low cost of flying means that unfamiliar infectious diseases may be brought into industrialized countries. an example of this was an outbreak of severe acute respiratory syndrome (sars), which was first recognized in 2003. despite modern technology and a huge input of money, it took months for the agent to be identified, a diagnostic test to be produced, and a strategy for disease reporting and isolation to be established. there is no doubt that other new and fascinating diseases will continue to emerge. infections have plagued doctors for centuries, in both the diagnosis of the specific diseases and the identification and subsequent management of the causative agents. there is a constant need for information as new organisms emerge, existing ones develop resistance to current drugs or vaccines, and changes in epidemiology and prevalence occur. in the 21st century, obtaining this information has never been more important. population migration and the relatively low cost of flying means that unfamiliar infectious diseases may be brought into industrialized countries. an example of this was an outbreak of severe acute respiratory syndrome (sars), which was first recognized in 2003. despite modern technology and a huge input of money, it took months for the agent to be identified, a diagnostic test to be produced, and a strategy for disease reporting and isolation to be established. there is no doubt that other new and fascinating diseases will continue to emerge. for the forensic physician, dealing with infections presents two main problems. the first problem is managing detainees or police personnel who have contracted a disease and may be infectious or unwell. the second problem is handling assault victims, including police officers, who have potentially been exposed to an infectious disease. the latter can be distressing for those involved, compounded, in part, from an inconsistency of management guidelines, if indeed they exist. with the advent of human rights legislation, increasing pressure is being placed on doctors regarding consent and confidentiality of the detainee. therefore, it is prudent to preempt such situations before the consultation begins by obtaining either written or verbal consent from the detainee to allow certain pieces of information to be disclosed. if the detainee does not agree, then the doctor must decide whether withholding relevant details will endanger the lives or health of those working within custody or others with whom they may have had close contact (whether or not deliberate). consent and confidentiality issues are discussed in detail in chapter 2. adopting a universal approach with all detainees will decrease the risk to staff of acquiring such diseases and will help to stop unnecessary overreaction and unjustified disclosure of sensitive information. for violent or sexual assault victims, a more open-minded approach is needed (see also chapter 3) . if the assailant is known, then it may be possible to make an informed assessment of the risk of certain diseases by ascertaining his or her lifestyle. however, if the assailant is unknown, then it is wise to assume the worst. this chapter highlights the most common infections encountered by the forensic physician. it dispels "urban myths" and provides a sensible approach for achieving effective management. the risk of exposure to infections, particularly blood-borne viruses (bbvs), can be minimized by adopting measures that are considered good practice in the united kingdom, the united states, and australia (1) (2) (3) . forensic physicians or other health care professionals should wash their hands before and after contact with each detainee or victim. police officers should be encouraged to wash their hands after exposure to body fluids or excreta. all staff should wear gloves when exposure to body fluids, mucous membranes, or nonintact skin is likely. gloves should also be worn when cleaning up body fluids or handling clinical waste, including contaminated laundry. single-use gloves should only be used and must conform to the requirements of european standard 455 or equivalent (1) (2) (3) . a synthetic alternative conforming to the same standards should also be available for those who are allergic to latex. all staff should cover any fresh wounds (<24 hours old), open skin lesions, or breaks in exposed skin with a waterproof dressing. gloves cannot prevent percutaneous injury but may reduce the chance of acquiring a bloodborne viral infection by limiting the volume of blood inoculated. gloves should only be worn when taking blood, providing this does not reduce manual dexterity and therefore increase the risk of accidental percutaneous injury. ideally, a designated person should be allocated to ensure that the clinical room is kept clean and that sharps containers and clinical waste bags are removed regularly. clinical waste must be disposed of in hazard bags and should never be overfilled. after use, the clinical waste should be doublebagged and sealed with hazard tape. the bags should be placed in a designated waste disposal (preferably outside the building) and removed by a professional company. when cells are contaminated with body fluids, a professional cleaning company should be called to attend as soon as possible. until such time, the cell should be deemed "out of action." there is a legal requirement in the united kingdom under the environmental protection act (1990) and the control of substances hazardous to health regulations 1994 to dispose of sharps in an approved container. in the united states, the division of health care quality promotion on the centers for disease control and prevention (cdc) web site provides similar guidance. in custody, where sharps containers are transported off site, they must be of an approved type. in the united kingdom, such a requirement is contained within the carriage of dangerous goods (classification, packaging and labelling) and use of transportable pressure receptacles regulations 1996. these measures help to minimize the risk of accidental injury. further precautions include wearing gloves when handling sharps and never bending, breaking, or resheathing needles before disposal. sharps bins should never be overfilled, left on the floor, or placed above the eye level of the smallest member of staff. any bedding that is visibly stained with body fluids should be handled with gloves. there are only three acceptable ways of dealing with contaminated bedding: the bbvs that present the most cross-infection hazard to staff or victims are those associated with persistent viral replication and viremia. these include hbv, hcv, hepatitis d virus (hdv), and hiv. in general, risks of transmission of bbvs arise from the possible exposure to blood or other body fluids. the degree of risk varies with the virus concerned and is discussed under the relevant sections. figure 1 illustrates the immediate management after a percutaneous injury, mucocutaneous exposure, or exposure through contamination of fresh cuts or breaks in the skin. hbv is endemic throughout the world, with populations showing a varying degree of prevalence. approximately two thousand million people have been infected with hbv, with more than 350 million having chronic infection. worldwide, hbv kills about 1 million people each year. with the development of a safe and effective vaccine in 1982, the world health organization (who) recommended that hbv vaccine should be incorporated into national immunization programs by 1995 in those countries with a chronic infection rate of 8% or higher, and into all countries by 1997. although 135 countries had achieved this goal by the end of 2001, the poorest countries-often the ones with the highest prevalence-have been unable to afford it. in particular these include china, the indian subcontinent, and sub-saharan africa. people in the early stages of infection or with chronic carrier status (defined by persistence of hepatitis b surface antigen [hbsag] beyond 6 mo) can transmit infection. in the united kindgom, the overall prevalence of chronic hbv is approx 0.2-0.3% (6, 7) . a detailed breakdown is shown in table 1 . the incubation period is approx 6 weeks to 6 months. as the name suggests, the virus primarily affects the liver. typical symptoms include malaise, anorexia, nausea, mild fever, and abdominal discomfort and may last from 2 days to 3 weeks before the insidious onset of jaundice. joint pain and skin rashes may also occur as a result of immune complex formation. infections in the newborn are usually asymptomatic. * in the united kingdom, written consent from the contact must be sent with the sample, countersigned by the health care practitioner and, preferably, an independent police officer. the majority of patients with acute hbv make a full recovery and develop immunity. after acute infection, approx 1 in 300 patients develop liver failure, which may result in death. chronic infection develops in approx 90% of neonates, approx 50% of children, and between 5 and 10% of adults. neonates and children are usually asymptomatic. adults may have only mild symptoms or may also be asymptomatic. approximately 15-25% of chronically infected individuals (depending on age of acquisition) will develop cirrhosis over a number of years. this may also result in liver failure or other serious complications, including hepatocellular carcinoma, though the latter is rare. the overall mortality rate of hbv is estimated at less than 5%. a person is deemed infectious if hbsag is detected in the blood. in the acute phase of the illness, this can be as long as 6 months. by definition, if hbsag persists after this time, then the person is deemed a carrier. carriers are usually infectious for life. the degree of infectivity depends on the stage of disease and the markers present table 2 . the major routes include parenteral (e.g., needlestick injuries, bites, unscreened blood transfusions, tattooing, acupuncture, and dental procedures where equipment is inadequately sterilized), mucous membrane exposure (including mouth, eyes, and genital mucous membranes), and contamination of broken skin (especially when <24 hours old). hbv is an occupational hazard for anyone who may come into contact with blood or bloodstained body fluids through the routes described. saliva alone may transmit hbv. the saliva of some people infected with hbv contains hbv-dna concentrations 1/1000-1/10,000 of that found in their serum (8) . this is especially relevant for penetrating bite wounds. infection after exposure to other body fluids (e.g., bile, urine, feces, and cerebrospinal fluid) has never been demonstrated unless the fluids are contaminated with blood. intravenous drug users who share needles or other equipment are also at risk. hbv can also be transmitted through unprotected sexual contact, whether homosexual or heterosexual. the risk is increased if blood is involved. sexual assault victims should be included in this category. evidence has shown that the virus may also be spread among members of a family through close household contact, such as through kissing and sharing toothbrushes, razors, bath towels, etc. (9) (10) (11) . this route of transmission probably applies to institutionalized patients, but there are no available data. studies of prisoners in western countries have shown a higher prevalence of antibodies to hbv and other bbvs than the general population (12) (13) (14) ; the most commonly reported risk factor is intravenous drug use. however, the real frequency of transmission of bbvs in british prisons is unknown owing to the difficulty in compiling reliable data. hbv can be transmitted vertically from mother to baby during the perinatal period. approximately 80% of babies born to mothers who have either acute or chronic hbv become infected, and most will develop chronic hbv. this has been limited by the administration of hbv vaccine to the neonate. in industrialized countries, all prenatal mothers are screened for hbv. vaccine is given to the neonate ideally within the first 12 hours of birth and at least two more doses are given at designated intervals. the who recommends this as a matter of course for all women in countries where prevalence is high. however, the practicalities of administering a vaccine that has to be stored at the correct temperature in places with limited access to medical care means that there is a significant failure of vaccine uptake and response. in industrialized countries, hbv vaccination is recommended for those who are deemed at risk of acquiring the disease. they include the following: 1. through occupational exposure. 2. homosexual/bisexual men. 3. intravenous drug users. 4. sexual partners of people with acute or chronic hbv. 5. family members of people with acute or chronic hbv. 6. newborn babies whose mothers are infected with hbv. if the mother is hbsag positive, then hepatitis b-specific immunoglobulin (hbig) should be given at the same time as the first dose of vaccine. 7. institutionalized patients and prisoners. ideally, hbv vaccine should be administered before exposure to the virus. the routine schedule consists of three doses of the vaccine given at 0, 1, and 6 months. antibody levels should be checked 8-12 weeks after the last dose. if titers are greater than10 miu/ml, then an adequate response has been achieved. in the united kingdom, this is considered to provide protection for 5-10 years. in the united states, if an initial adequate response has been achieved, then no further doses of vaccine are considered necessary. vaccine administration after exposure varies according to the timing of the incident, the degree of risk involved, and whether the individual has already been partly or fully vaccinated. an accelerated schedule when the third dose is given 2 months after the first dose with a booster 1 year later is used to prevent postnatal transmission. where risks are greatest, it may be necessary to use a rapid schedule. the doses are given at 0, 7, and 21-28 days after presentation, again with a booster dose at 6-12 months. this schedule is currently only licensed with engerix b. hbig may also be used either alone or in conjunction with vaccine. the exact dose given is age dependent but must be administered by deep intramuscular injection in a different site from the vaccine. in an adult, this is usually into the gluteus muscle. hbig is given in conjunction with the first dose of vaccine to individuals who are deemed at high risk of acquiring disease and the incident occurred within 72 hours of presentation. it is also used for neonates born to mothers who are hbeag-positive. between 5 and 10% of adults fail to respond to the routine schedule of vaccine. a further full course of vaccine should be tried before deeming the patients as "nonresponders." such individuals involved in a high-risk exposure should be given two doses of hbig administered 1 mo apart. ideally, the first dose should be given within 48 hours after exposure and no later than 2 weeks after exposure. other measures include minimizing the risk of exposure by adopting the safe working practices outlined in subheading 2. any potential exposures should be dealt with as soon as possible. in industrialized countries blood, blood products, and organs are routinely screened for hbv. intravenous drug users should be encouraged to be vaccinated and to avoid sharing needles or any other drug paraphernalia (see subheading 6.9.2.). for staff or victims in contact with disease, it is wise to have a procedure in place for immediate management and risk evaluation. an example is shown in fig. 1 . although forensic physicians are not expected to administer treatment, it is often helpful to inform persons concerned what to expect. tables 3 and 4 outline treatment protocols as used in the united kingdom. detainees with disease can usually be managed in custody. if the detainee is bleeding, then the cell should be deemed out of action after the detainee has left until it can be professionally cleaned. contaminated bedding should be dealt with as described in subheading 2.2. if the detainee has chronic hbv and is on an antiviral agent (e.g., lamivudine), then the treatment course should be continued, if possible. hcv is endemic in most parts of the world. approximately 3% (200 million) of the world's population is infected with hcv (15) . for many countries, no reliable prevalence data exist. seroprevalence studies conducted among blood donors have shown that the highest prevalence exists in egypt (17-26%). this has been ascribed to contaminated needles used in the treatment of schistosomiasis conducted between the 1950s and the 1980s (16) . intermediate prevalence (1-5%) exists in eastern europe, the mediterranean, the middle east, the indian subcontinent, and parts of africa and asia. in western europe, most of central america, australia, and limited regions in africa, including south africa, the prevalence is low (0.2-0.5%). previously, america was included in the low prevalence group, but a report published in 2003 (17) indicated that almost 4 million americans (i.e., 1.8% of the population) have antibody to hcv, representing either ongoing or previous infection. it also states that hcv accounts for approx 15% of acute viral hepatitis in america. the lowest prevalence (0.01-0.1%) has been found in the united kingdom and scandinavia. however, within any country, there are certain groups that have a higher chance of carrying hcv. these united kingdom figures are given in table 5 . after an incubation period of 6-8 weeks, the acute phase of the disease lasts approx 2-3 years. unlike hepatitis a (hav) or hbv, the patient is usually asymptomatic; therefore, the disease is often missed unless the individual has reported a specific exposure and is being monitored. other cases are found by chance, when raised liver enzymes are found on a routine blood test. a "silent phase" follows the acute phase when the virus lies dormant and the liver enzymes are usually normal. this period lasts approx 10-15 years. reactivation may then occur. subsequent viral replication damages the hepatocytes, and liver enzymes rise to moderate or high levels. eighty percent of individuals who are hcv antibody-positive are infectious, regardless of the levels of their liver enzymes. approximately 80% of people develop chronic infection, one-fifth of whom progress to cirrhosis. there is a much stronger association with hepatocellular carcinoma than with hbv. an estimated 1.25-2.5% of patients with hcv-related cirrhosis develop liver cancer (18) . less than 2% of chronic cases resolve spontaneously. approximately 75% of cases are parenteral (e.g., needle-stick, etc.) (19) . transmission through the sexual route is not common and only appears to be significant if there is repeated exposure with one or more people infected with hcv. mother-to-baby transmission is considered to be uncommon but has been reported (20) . theoretically, household spread is also possible through sharing contaminated toothbrushes or razors. because the disease is often silent, there is a need to raise awareness among the general population on how to avoid infection and to encourage high-risk groups to be tested. health care professionals should also be educated to avoid occupationally acquired infection. an example of good practice blood or blood-stained body fluids need to be involved for a risk to occur. saliva alone is not deemed to be a risk. the risk from a single needlestick incident is 1.8% (range 0-7%). contact through a contaminated cut is estimated at 1%. for penetrating bite injuries, there are no data, but it is only considered a risk if blood is involved. blood or blood-stained body fluids have to be involved in transmission through mucous membrane exposure. this may account for the lower-than-expected prevalence among the gay population. follow the immediate management flow chart, making sure all available information is obtained. inform the designated hospital and/or specialist as soon as possible. if the contact is known and is believed to be immunocompromised and he or she has consented to provide a blood sample, it is important to tell the specialist, because the antibody tests may be spuriously negative. in this instance, a different test should be used (polymerase chain reaction [pcr] , which detects viral rna). the staff member/victim will be asked to provide a baseline sample of blood with further samples at 4-6 weeks and again at 12 weeks. if tests are negative at 12 weeks but the risk was deemed high, then follow-up may continue for up to 24 weeks. if any of the follow-up samples is positive, then the original baseline sample will be tested to ascertain whether the infection was acquired through the particular exposure. it is important to emphasize the need for prompt initial attendance and continued monitoring, because treatment is now available. a combination of ribavirin (antiviral agent and interferon a-2b) (18) or the newer pegylated interferons (15) may be used. this treatment is most effective when it is started early in the course of infection. unless they are severely ill, detainees can be managed in custody. special precautions are only required if they are bleeding. custody staff should wear gloves if contact with blood is likely. contaminated bedding should be handled appropriately, and the cell cleaned professionally after use. this defective transmissible virus was discovered in 1977 and requires hbv for its own replication. it has a worldwide distribution in association with hbv, with approx 15 million people infected. the prevalence of hdv is higher in southern italy, the middle east, and parts of africa and south america, occurring in more than 20% of hbv carriers who are asymptomatic and more than 60% of those with chronic hbv-related liver disease. despite the high prevalence of hbv in china and south east asia, hdv in these countries is rare. hdv is associated with acute (coinfection) and chronic hepatitis (superinfection) and can exacerbate pre-existing liver damage caused by hbv. the routes of transmission and at-risk groups are the same as for hbv. staff/victims in contact with a putative exposure and detainees with disease should be managed as for hbv. interferon-î± (e.g., roferon) can be used to treat patients with chronic hbv and hdv (21) , although it would not be practical to continue this treatment in the custodial setting. hiv was first identified in 1983, 2 years after the first reports were made to the cdc in atlanta, ga, of an increased incidence of two unusual diseases (kaposi's sarcoma and pneumocystis carinii pneumonia) occurring among the gay population in san francisco. the scale of the virus gradually emerged over the years and by the end of 2002, there were an estimated 42 million people throughout the world living with hiv or acquired immunodeficiency syndrome (aids). more than 80% of the world's population lives in africa and india. a report by the joint united nations programme on hiv/aids and the who in 2002 stated that one in five adults in lesotho, malawi, mozambique, swaziland, zambia, and zimbabwe has hiv or aids. there is also expected to be a sharp rise in cases of hiv in china, papua new guinea, and other countries in asia and the pacific during the next few years. in the united kingdom, by the end of 2002, the cumulative data reported that there were 54,261 individuals with hiv, aids (including deaths from aids) reported, though this is likely to be an underestimate (22) . from these data, the group still considered at greatest risk of acquiring hiv in the united kingdom is homosexual/bisexual men, with 28,835 of the cumulative total falling into this category. among intravenous drug users, the overall estimated prevalence is 1%, but in london the figure is higher at 3.7% (6, 23) . in the 1980s, up to 90% of users in edinburgh and dundee were reported to be hiv positive, but the majority have now died. individuals arriving from africa or the indian subcontinent must also be deemed a risk group because 80% of the world's total cases occur in these areas. the predominant mode of transmission is through unprotected heterosexual intercourse. the incidence of mother-to-baby transmission has been estimated at 15% in europe and approx 45% in africa. the transmission rates among african women are believed to be much higher owing to a combination of more women with end-stage disease with a higher viral load and concomitant placental infection, which renders it more permeable to the virus (24, 25) . the use of antiretroviral therapy during pregnancy, together with the advice to avoid breastfeeding, has proven efficacious in reducing both vertical and horizontal transmission among hiv-positive women in the western world. for those in third-world countries, the reality is stark. access to treatment is limited, and there is no realistic substitute for breast milk, which provides a valuable source of antibodies to other life-threatening infections. patients receiving blood transfusions, organs, or blood products where screening is not routinely carried out must also be included. the incubation is estimated at 2 weeks to 6 months after exposure. this depends, to some extent, on the ability of current laboratory tests to detect hiv antibodies or viral antigen. the development of pcr for viral rna has improved sensitivity. during the acute phase of the infection, approx 50% experience a seroconversion "flu-like" illness. the individual is infectious at this time, because viral antigen (p24) is present in the blood. as antibodies start to form, the viral antigen disappears and the individual enters the latent phase. he or she is noninfectious and remains well for a variable period of time (7-15 years). development of aids marks the terminal phase of disease. viral antigen reemerges, and the individual is once again infectious. the onset of aids has been considerably delayed with the use of antiretroviral treatment. parenteral transmission included needlestick injuries, bites, unscreened blood transfusions, tattooing, acupuncture, and dental procedures where equipment is inadequately sterilized. risk of transmission is increased with deep penetrating injuries with hollow bore needles that are visibly bloodstained, especially when the device has previously been in the source patient's (contact) artery or vein. other routes include mucous membrane exposure (eyes, mouth, and genital mucous membranes) and contamination of broken skin. the higher the viral load in the contact, the greater the risk of transmission. this is more likely at the terminal stage of infection. hiv is transmitted mainly through blood or other body fluids that are visibly blood stained, with the exception of semen, vaginal fluid, and breast milk. saliva alone is most unlikely to transmit infection. therefore, people who have sustained penetrating bite injuries can be reassured that they are not at risk, providing the contact was not bleeding from the mouth at the time. the risk from a single percutaneous exposure from a hollow bore needle is low, and a single mucocutaneous exposure is even less likely to result in infection. the risk from sexual exposure varies, although it appears that there is a greater risk with receptive anal intercourse compared with receptive vaginal intercourse (26). high-risk fluids include blood, semen, vaginal fluid, and breast milk. there is little or no risk from saliva, urine, vomit, or feces unless they are visibly bloodstained. other fluids that constitute a theoretical risk include cerebrospinal, peritoneal, pleural, synovial, or pericardial fluid. management in custody of staff/victims in contact with disease includes following the immediate management flow chart (fig. 1 ) and contacting the designated hospital/specialist with details of the exposure. where possible, obtain a blood sample from the contact. regarding hbv and hcv blood samples in the united kingdom, they can only be taken with informed consent. there is no need for the forensic physician to go into details about the meaning of the test, but the contact should be encouraged to attend the genitourinary department (or similar) of the designated hospital to discuss the test results. should the contact refuse to provide a blood sample, then any information about his or her lifestyle, ethnic origin, state of health, etc., may be useful for the specialist to decide whether postexposure prophylaxis (pep) should be given to the victim. where only saliva is involved in a penetrating bite injury, there is every justification to reassure the victim that he or she is not at risk. if in doubt, then always refer. in the united kingdom, the current recommended regime for pep is combivir (300 mg of zidovudine twice daily plus 150 mg of lamivudine twice daily) and a protease inhibitor (1250 mg of nelfanivir twice daily) given for 4 weeks (27) . it is only given after a significant exposure to a high-risk fluid or any that is visibly bloodstained and the contact is known or is highly likely to be hiv positive. ideally, treatment should be started within an hour after exposure, although it will be considered for up to 2 weeks. it is usually given for 4 weeks, unless the contact is subsequently identified as hiv negative or the "victim" develops tolerance or toxicity occurs. weekly examinations of the "victim" should occur during treatment to improve adherence, monitor drug toxicity, and deal with other concerns. other useful information that may influence the decision whether to treat with the standard regimen or use alternative drugs includes interaction with other medications that the "victim" may be taking (e.g., phenytoin or antibiotics) or if the contact has been on antiretroviral therapy or if the "victim" is pregnant. during the second or third trimester, only combivir would be used, because there is limited experience with protease inhibitors. no data exist regarding the efficacy of pep beyond occupational exposure (27) . pep is not considered for exposure to low-or no-risk fluids through any route or where the source is unknown (e.g., a discarded needle). despite the appropriate use and timing of pep, there have been reports of failure (28, 29) . unless they are severely ill, detainees can be kept in custody. every effort should be made to continue any treatment they may be receiving. apply universal precautions when dealing with the detainee, and ensure that contaminated cells and/or bedding are managed appropriately. cases of this highly infectious disease occur throughout the year but are more frequent in winter and early spring. this seasonal endemicity is blurring with global warming. in the united kingdom, the highest prevalence occurs in the 4-to 10-years age group. ninety percent of the population over the age of 40 is immune (30) . a similar prevalence has been reported in other parts of western europe and the united states. in south east asia, varicella is mainly a disease of adulthood (31) . therefore, people born in these countries who have moved to the united kingdom are more likely to be susceptible to chicken pox. there is a strong correlation between a history of chicken pox and serological immunity (97-99%). most adults born and living in industrialized countries with an uncertain or negative history of chicken pox are also seropositive (70-90%). in march 1995, a live-attenuated vaccine was licensed for use in the united states and a policy for vaccinating children and susceptible health care personnel was introduced. in summer 2002, in the united kingdom, glaxosmithkline launched a live-attenuated vaccine called varilrix. in december 2003, the uk department of health, following advice from the joint committee on vaccination and immunisation recommended that the vaccine be given for nonimmune health care workers who are likely to have direct contact with individuals with chicken pox. any health care worker with no previous history of chicken pox should be screened for immunity, and if no antibodies are found, then they should receive two doses of vaccine 4-8 weeks apart. the vaccine is not currently recommended for children and should not be given during pregnancy. following an incubation period of 10-21 days (this may be shorter in the immunocompromised), there is usually a prodromal "flu-like" illness before the onset of the rash. this coryzal phase is more likely in adults. the lesions typically appear in crops, rapidly progressing from red papules through vesicles to open sores that crust over and separate by 10 days. the distribution of the rash is centripetal (i.e., more over the trunk and face than on the limbs). this is the converse of small pox. in adults, the disease is often more severe, with lesions involving the scalp and mucous membranes of the oropharynx. in children, the disease is often mild, unless they are immunocompromised, so they are unlikely to experience complications. in adults (defined as 15 yr or older), the picture is rather different (32) . secondary bacterial infection is common but rarely serious. there is an increased likelihood of permanent scarring. hemorrhagic chicken pox typically occurs on the second or third day of the rash. usually, this is limited to bleeding into the skin, but lifethreatening melena, epistaxis, or hematuria can occur. varicella pneumonia ranges from patchy lung consolidation to overt pneumonitis and occurs in 1 in 400 cases (33) . it can occur in previously healthy individuals (particularly adults), but the risk is increased in those who smoke. immunocompromised people are at the greatest risk of developing this complication. it runs a fulminating course and is the most common cause of varicella-associated death. fibrosis and permanent respiratory impairment may occur in those who survive. any suspicion of lung involvement is an indication for immediate treatment, and any detainee or staff member should be sent to hospital. involvement of the central nervous system includes several conditions, including meningitis, guillain-barre, and encephalitis. the latter is more common in the immunocompromised and can be fatal. this is taken as 3 days before the first lesions appear to the end of new vesicle formation and the last vesicle has crusted over. this typically is 5-7 days after onset but may last up to 14 days. the primary route is through direct contact with open lesions of chicken pox. however, it is also spread through aerosol or droplets from the respiratory tract. chicken pox may also be acquired through contact with open lesions of shingles (varicella zoster), but this is less likely because shingles is less infectious than chicken pox. nonimmune individuals are at risk of acquiring disease. approximately 10% of the adult population born in the united kingdom and less than 5% of adults in the united states fall into this category. therefore, it is more likely that if chicken pox is encountered in the custodial setting, it will involve people born outside the united kingdom (particularly south east asia) or individuals who are immunocompromised and have lost immunity. nonimmune pregnant women are at risk of developing complications. pneumonia can occur in up to 10% of pregnant women with chicken pox, and the severity is increased in later gestation (34) . they can also transmit infection to the unborn baby (35) . if infection is acquired in the first 20 weeks, there is a less than 3% chance of it leading to congenital varicella syndrome. infection in the last trimester can lead to neonatal varicella, unless more than 7 days elapse between onset of maternal rash and delivery when antibodies have time to cross the placenta leading to either mild or inapparent infection in the newborn. in this situation, varicella immunoglobulin (vzig) should be administered to the baby as soon as possible after birth (36). staff with chicken pox should stay off work until the end of the infective period (approx 7-14 days). those in contact with disease who are known to be nonimmune or who have no history of disease should contact the designated occupational health physician. detainees with the disease should not be kept in custody if at all possible (especially pregnant women). if this is unavoidable, then nonimmune or immunocompromised staff should avoid entering the cell or having close contact with the detainee. nonimmune, immunocompromised, or pregnant individuals exposed to chickenpox should seek expert medical advice regarding the administration of vzig. aciclovir (or similar antiviral agent) should be given as soon as possible to people who are immunocompromised with chicken pox. it should also be considered for anyone over 15 years old because they are more likely to develop complications. anyone suspected of severe complications should be sent straight to the hospital. after chicken pox, the virus lies dormant in the dorsal root or cranial nerve ganglia but may re-emerge and typically involves one dermatome (37) . the site of involvement depends on the sensory ganglion initially involved. shingles is more common in individuals over the age of 50 years, except in the immunocompromised, when attacks can occur at an earlier age. the latter are also more susceptible to secondary attacks and involvement of more than one dermatome. bilateral zoster is even rarer but is not associated with a higher mortality. in the united kingdom, there is an estimated incidence of 1.2-3.4 per 1000-person years (38). there may be a prodromal period of paraesthesia and burning or shooting pains in the involved segment. this is usually followed by the appearance of a band of vesicles. rarely, the vesicles fail to appear and only pain is experienced. this is known as zoster sine herpete. in individuals who are immuno-compromised, disease may be prolonged and dissemination may occur but is rarely fatal. shingles in pregnancy is usually mild. the fetus is only affected if viremia occurs before maternal antibody has had time to cross the placenta. the most common complication of shingles is postherpetic neuralgia, occurring in approx 10% of cases. it is defined as pain lasting more than 120 days from rash onset (39) . it is more frequent in people over 50 years and can lead to depression. it is rare in children, including those who are immunocompromised. infection of the brain includes encephalitis, involvement of motor neurones leading to ptosis, paralysis of the hand, facial palsy, or contralateral hemiparesis. involvement of the oculomotor division of the trigeminal ganglion can cause serious eye problems, including corneal scarring. shingles is far less infectious than chicken pox and is only considered to be infectious up to 3 days after lesions appear. shingles is only infectious after prolonged contact with lesions. unlike chickenpox, airborne transmission is not a risk. individuals who are immunocompromised may reactivate the dormant virus and develop shingles. people who have not had primary varicella are at risk of developing chickenpox after prolonged direct contact with shingles. despite popular belief, it is untrue that people who are immunocompetent who have had chicken pox develop shingles when in contact with either chicken pox or shingles. such occurrences are merely coincidental, unless immunity is lowered. staff with shingles should stay off work until the lesions are healed, unless they can be covered. staff who have had chickenpox are immune (including pregnant women) and are therefore not at risk. if they are nonimmune (usually accepted as those without a history of chicken pox), they should avoid prolonged contact with detainees with shingles. pregnant nonimmune women should avoid contact altogether. detainees with the disease may be kept in custody, and any exposed lesions should be covered. it is well documented that prompt treatment attenuates the severity of the disease, reduces the duration of viral shedding, hastens lesion healing, and reduces the severity and duration of pain. it also reduces the likelihood of developing postherpetic neuralgia (40) . prompt treatment with famciclovir (e.g., 500 mg three times a day for 7 days) should be initiated if the onset is 3 d ays or less. it should also be considered after this time if the detainee is over age 50 years. pregnant detainees with shingles can be reassured that there is minimal risk for both the mother and the unborn child. expert advice should be given before initiating treatment for the mother. this tiny parasitic mite (sarcoptes scabiei) has infested humans for more than 2500 years. experts estimate that in excess of 300 million cases occur worldwide each year. the female mite burrows into the skin, especially around the hands, feet, and male genitalia, in approx 2.5 min. eggs are laid and hatch into larvae that travel to the skin surface as newly developed mites. the mite causes intense itching, which is often worse at night and is aggravated by heat and moisture. the irritation spreads outside the original point of infection resulting from an allergic reaction to mite feces. this irritation may persist for approx 2 weeks after treatment but can be alleviated by antihistamines. crusted scabies is a far more severe form of the disease. large areas of the body may be involved. the crusts hide thousands of live mites and eggs, making them difficult to treat. this so-called norwegian scabies is more common in the elderly or the immunocompromised, especially those with hiv. after a primary exposure, it takes approx 2-6 weeks before the onset of itching. however, further exposures reduce the incubation time to approx 1-4 days. without treatment, the period of infectivity is assumed to be indefinite. with treatment, the person should be considered infectious until the mites and eggs are destroyed, usually 7-10 days. crusted scabies is highly infectious. because transmission is through direct skin-to-skin contact with an infected individual, gloves should be worn when dealing with individuals suspected of infestation. usually prolonged contact is needed, unless the person has crusted scabies, where transmission occurs more easily. the risk of transmission is much greater in households were repeated or prolonged contact is likely. because mites can survive in bedding or clothing for up to 24 hour, gloves should also be worn when handling these items. bedding should be treated using one of the methods in subheading 2.2. professional cleaning of the cell is only warranted in cases of crusted scabies. the preferred treatment for scabies is either permethrin cream (5%) or aqueous malathion (0.5%) (41) . either treatment has to be applied to the whole body and should be left on for at least 8 hours in the case of permethrin and 24 hours for malathion before washing off. lindane is no longer considered the treatment of choice, because there may be complications in pregnancy (42) . treatment in custody may not be practical but should be considered when the detainee is believed to have norwegian scabies. like scabies, head lice occur worldwide and are found in the hair close to the scalp. the eggs, or nits, cling to the hair and are difficult to remove, but they are not harmful. if you see nits, then you can be sure that lice are also present. the latter are best seen when the hair is wet. the lice bite the scalp and suck blood, causing intense irritation and itching. head lice can only be passed from direct hair-to-hair contact. it is only necessary to wear gloves when examining the head for whatever reason. the cell does not need to be cleaned after use, because the lice live on or near skin. bedding may be contaminated with shed skin, so should be handled with gloves and laundered or incinerated. the presence of live lice is an indication for treatment by either physical removal with a comb or the application of an insecticide. the latter may be more practical in custody. treatment using 0.5% aqueous malathion should be applied to dry hair and washed off after 12 hours. the hair should then be shampooed as normal. crabs or body lice are more commonly found in the pubic, axillary, chest, and leg hair. however, eyelashes and eyebrows may also be involved. they are associated with people who do not bath or change clothes regularly. the person usually complains of intense itching or irritation. the main route is from person to person by direct contact, but eggs can stick to fibers, so clothing and bedding should be handled with care (see subheading 6.5.3.). staff should always wear gloves if they are likely to come into contact with any hirsute body part. clothing or bedding should be handled with gloves and either laundered or incinerated. treatment of a detainee in custody is good in theory but probably impractical because the whole body has to be treated. fleas lay eggs on floors, carpets, and bedding. in the united kingdom, most flea bites come from cats or dogs. the eggs and larvae fleas can survive for months and are reactivated in response to animal or human activity. because animal fleas jump off humans after biting, most detainees with flea bites will not have fleas, unless they are human fleas. treatment is only necessary if fleas are seen. after use, the cell should be vacuumed and cleaned with a proprietary insecticide. any bedding should be removed wearing gloves, bagged, and either laundered or incinerated. bedbugs live and lay eggs on walls, floors, furniture, and bedding. if you look carefully, fecal tracks may be seen on hard surfaces. if they are present for long enough, they emit a distinct odor. bedbugs are rarely found on the person but may be brought in on clothing or other personal effects. bedbugs bite at night and can cause sleep disturbance. the detainee does not need to be treated, but the cell should deemed out of use until it can be vacuumed and professionally cleaned with an insecticide solution. any bedding or clothing should be handled with gloves and disposed of as appropriate. staphylococcus aureus is commonly carried on the skin or in the nose of healthy people. approximately 25-30% of the population is colonized with the bacteria but remain well (43) . from time to time, the bacteria cause minor skin infections that usually do not require antibiotic treatment. however, more serious problems can occur (e.g., infection of surgical wounds, drug injection sites, osteomyelitis, pneumonia, or septicemia). during the last 50 years, the bacteria have become increasingly resistant to penicillin-based antibiotics (44) , and in the last 20 years, they have become resistant to an increasing number of alternative antibiotics. these multiresistant bacteria are known as methicillinresistant s. aureus (mrsa). mrsa is prevalent worldwide. like nonresistant staphylococci, it may remain undetected as a reservoir in colonized individuals but can also produce clinical disease. it is more common in individuals who are elderly, debilitated, or immunocompromised or those with open wounds. clusters of skin infections with mrsa have been reported among injecting drug users (idus) since 1981 in america (45, 46) , and more recently, similar strains have been found in the united kingdom in idus in the community (47) . this may have particular relevance for the forensic physician when dealing with idus sores. people who are immunocompetent rarely get mrsa and should not be considered at risk. the bacteria are usually spread via the hands of staff after contact with colonized or infected detainees or devices, items (e.g., bedding, towels, and soiled dressings), or environmental surfaces that have been contaminated with mrsa-containing body fluids. with either known or suspected cases (consider all abscesses/ulcers of idus as infectious), standard precautions should be applied. staff should wear gloves when touching mucous membranes, nonintact skin, blood or other body fluids, or any items that could be contaminated. they should also be encouraged to their wash hands with an antimicrobial agent regardless of whether gloves have been worn. after use, gloves should be disposed of in a yellow hazard bag and not allowed to touch surfaces. masks and gowns should only be worn when conducting procedures that generate aerosols of blood or other body fluids. because this is an unlikely scenario in the custodial setting, masks and gowns should not be necessary. gloves should be worn when handling bedding or clothing, and all items should be disposed of appropriately. any open wounds should be covered as soon as possible. the cell should be cleaned professionally after use if there is any risk that it has been contaminated. during the last decade, there has been an increasing awareness of the bacterial flora colonizing injection sites that may potentially lead to life-threatening infection (48) . in 1997, a sudden increase in needle abscesses caused by a clonal strain of group a streptococcus was reported among hospitalized idus in berne, switzerland (49) . a recent uk study showed that the predominant isolate is s. aureus, with streptococcus species forming just under one-fifth (50% î²-hemolytic streptococci) (50) . there have also been reports of both nonsporing and sporing anerobes (e.g., bacteroides and clostridia species, including clostridia botulinum) (51, 52) . in particular, in 2000, laboratories in glasgow were reporting isolates of clostridium novyi among idus with serious unexplained illness. by june 12, 2000, a total of 42 cases (18 definite and 24 probable) had been reported. a definite case was defined as an idu with both severe local and systemic inflammatory reactions. a probable case was defined as an idu who presented to the hospital with an abscess or other significant inflammation at an injecting site and had either a severe inflammatory process at or around an injection site or a severe systemic reaction with multiorgan failure and a high white cell count (53) . in the united kingdom, the presence of c. botulinum in infected injection sites is a relatively new phenomenon. until the end of 1999, there were no cases reported to the public health leadership society. since then, the number has increased, with a total of 13 cases in the united kingdom and ireland being reported since the beginning of 2002. it is believed that these cases are associated with contaminated batches of heroin. simultaneous injection of cocaine increases the risk by encouraging anerobic conditions. anerobic flora in wounds may have serious consequences for the detainee, but the risk of transmission to staff is virtually nonexistent. staff should be reminded to wear gloves when coming into contact with detainees with infected skin sites exuding pus or serum and that any old dressings found in the cell should be disposed of into the yellow bag marked "clinical waste" in the medical room. likewise, any bedding should be bagged and laundered or incinerated after use. the cell should be deemed out of use and professionally cleaned after the detainee has gone. the health care professional managing the detainee should clean and dress open wounds as soon as possible to prevent the spread of infection. it may also be appropriate to start a course of antibiotics if there is abscess formation or signs of cellulites and/or the detainee is systemically unwell. however, infections can often be low grade because the skin, venous, and lymphatic systems have been damaged by repeated penetration of the skin. in these cases, signs include lymphedema, swollen lymph glands, and darkly pigmented skin over the area. fever may or may not be present, but septicemia is uncommon unless the individual is immunocompromised (e.g., hiv positive). co-amoxiclav is the preferred treatment of choice because it covers the majority of staphylococci, streptococci, and anerobes (the dose depends on the degree of infection). necrotizing fasciitis and septic thrombophlebitis are rare but life-threatening complications of intravenous drug use. any detainee suspected of either of these needs hospital treatment. advice about harm reduction should also be given. this includes encouraging drug users to smoke rather than inject or at least to advise them to avoid injecting into muscle or skin. although most idus are aware of the risk of sharing needles, they may not realize that sharing any drug paraphernalia could be hazardous. advice should be given to use the minimum amount of citric acid to dissolve the heroin because the acid can damage the tissue under the skin, allowing bacteria to flourish. drugs should be injected at different sites using fresh works for each injection. this is particularly important when "speedballing" because crack cocaine creates an anerobic environment. medical help should be requested if any injection site become painful and swollen or shows signs of pus collecting under the skin. because intravenous drug users are at increased risk of acquiring hbv and hav, they should be informed that vaccination against both diseases is advisable. another serious but relatively rare problem is the risk from broken needles in veins. embolization can take anywhere from hours to days or even longer if it is not removed. complications may include endocarditis, pericarditis, or pulmonary abscesses (54, 55) . idus should be advised to seek medical help as soon as possible, and should such a case present in custody, then send the detainee straight to the hospital. the forensic physician may encounter bites in the following four circumstances: a detailed forensic examination of bites is given in chapter 4. with any bite that has penetrated the skin, the goals of therapy are to minimize soft tissue deformity and to prevent or treat infection. in the united kingdom and the united states, dog bites represent approximately three-quarters of all bites presenting to accident and emergency departments (56) . a single dog bite can produce up to 220 psi of crush force in addition to the torsional forces as the dog shakes its head. this can result in massive tissue damage. human bites may cause classical bites or puncture wounds (e.g., impact of fists on teeth) resulting in crush injuries. an estimated 10-30% of dog bites and 9-50% of human bites lead to infection. compare this with an estimated 1-12% of nonbite wounds managed in accident and emergency departments. the risk of infection is increased with puncture wounds, hand injuries, full-thickness wounds, wounds requiring debridement, and those involving joints, tendons, ligaments or fractures. comorbid medical conditions, such as diabetes, asplenia, chronic edema of the area, liver dysfunction, the presence of a prosthetic valve or joint, and an immunocompromised state may also increase the risk of infection. infection may spread beyond the initial site, leading to septic arthritis, osteomyelitis, endocarditis, peritonitis, septicemia, and meningitis. inflammation of the tendons or synovial lining of joints may also occur. if enough force is used, bones may be fractured or the wounds may be permanently disfiguring. assessment regarding whether hospital treatment is necessary should be made as soon as possible. always refer if the wound is bleeding heavily or fails to stop when pressure is applied. penetrating bites involving arteries, nerves, muscles, tendons, the hands, or feet, resulting in a moderate to serious facial wound, or crush injuries, also require immediate referral. if management within custody is appropriate, ask about current tetanus vaccine status, hbv vaccination status, and known allergies to antibiotics. wounds that have breached the skin should be irrigated with 0.9% (isotonic) sodium chloride or ringer's lactate solution instead of antiseptics, because the latter may delay wound healing. a full forensic documentation of the bite should be made as detailed in chapter 4. note if there are clinical signs of infection, such as erythema, edema, cellulitis, purulent discharge, or regional lymphadenopathy. cover the wound with a sterile, nonadhesive dressing. wound closure is not generally recommended because data suggest that it may increase the risk of infection. this is particularly relevant for nonfacial wounds, deep puncture wounds, bites to the hand, clinically infected wounds, and wounds occurring more than 6-12 hours before presentation. head and neck wounds in cosmetically important areas may be closed if less than 12 hours old and not obviously infected. â�¢ dog bites-pasteurella canis, pasteurella multocida, s. aureus, other staphylococci, streptococcus species, eikenella corrodens, corynebacterium species, and anerobes, including bacteroides fragilis and clostridium tetani â�¢ human bites-streptococcus species, s. aureus, e. corrodens, and anerobes, including bacteroides (often penicillin resistant), peptostreptococci species, and c. tetani. tuberculosis (tb) and syphilis may also be transmitted. â�¢ dog bites-outside of the united kingdom, australia, and new zealand, rabies should be considered. in the united states, domestic dogs are mostly vaccinated against rabies (57) , and police dogs have to be vaccinated, so the most common source is from racoons, skunks, and bats. â�¢ human bites-hbv, hbc, hiv, and herpes simplex. antibiotics are not generally needed if the wound is more than 2 days old and there is no sign of infection or in superficial noninfected wounds evaluated early that can be left open to heal by secondary intention in compliant people with no significant comorbidity (58) . antibiotics should be considered with high-risk wounds that involve the hands, feet, face, tendons, ligaments, joints, or suspected fractures or for any penetrating bite injury in a person with diabetes, asplenia, or cirrhosis or who is immunosuppressed. coamoxiclav (amoxycillin and clavulanic acid) is the first-line treatment for mild-moderate dog or human bites resulting in infections managed in primary care. for adults, the recommended dose is 500/125 mg three times daily and for children the recommended does is 40 mg/kg three times daily (based on amoxycillin component). treatment should be continued for 10-14 days. it is also the first-line drug for prophylaxis when the same dose regimen should be prescribed for 5-7 days. if the individual is known or suspected to be allergic to penicillin, a tetracycline (e.g., doxycycline 100 mg twice daily) and metronidazole (500 mg three times daily) or an aminoglycoside (e.g., erythromycin) and metronidazole can be used. in the united kingdom, doxycycline use is restricted to those older than 12 years and in the united states to those older than 8 years old. specialist advice should be sought for pregnant women. anyone with severe infection or who is clinically unwell should be referred to the hospital. tetanus vaccine should be given if the primary course or last booster was more than 10 years ago. human tetanus immunoglobulin should be considered for tetanus-prone wounds (e.g., soil contamination, puncture wounds, or signs of devitalized tissue) or for wounds sustained more than 6 hours old. if the person has never been immunized or is unsure of his or her tetanus status, a full three-dose course, spaced at least 1 month apart, should be given. penetrating bite wounds that involve only saliva may present a risk of hbv if the perpetrator belongs to a high-risk group. for management, see subheadings 5.1.6. and 5.1.7. hcv and hiv are only a risk if blood is involved. the relevant management is dealt with in subheadings 5.2.5. and 5.4.6. respiratory tract infections are common, usually mild, and self-limiting, although they may require symptomatic treatment with paracetamol or a nonsteroidal antiinflammatory. these include the common cold (80% rhinoviruses and 20% coronaviruses), adenoviruses, influenza, parainfluenza, and, during the summer and early autumn, enteroviruses. special attention should be given to detainees with asthma or the who are immunocompromised, because infection in these people may be more serious particularly if the lower respiratory tract is involved. the following section includes respiratory pathogens of special note because they may pose a risk to both the detainee and/or staff who come into close contact. there are five serogroups of neisseria meningitidis: a, b, c, w135, and y. the prevalence of the different types varies from country to country. there is currently no available vaccine against type b, but three other vaccines (a+c, c, and acwy) are available. overall, 10% of the uk population carry n. meningitidis (25% in the 15-19 age group) (59) . in the united kingdom, most cases of meningitis are sporadic, with less than 5% occurring as clusters (outbreaks) amongst school children. between 1996 and 2000, 59% of cases were group b, 36% were group c, and w135 and a accounted for 5%. there is a seasonal variation, with a high level of cases in winter and a low level in the summer. the greatest risk group are the under 5 year olds, with a peak incidence under 1 year old. a secondary peak occurs in the 15-to 19-year-old age group. in sub-saharan africa, the disease is more prevalent in the dry season, but in many countries, there is background endemicity year-round. the most prevalent serogroup is a. routine vaccination against group c was introduced in the united kingdom november 1999 for everybody up to the age of 18 years old and to all firstyear university students. this has since been extended to include everyone under the age of 25 years old. as a result of the introduction of the vaccination program, there has been a 90% reduction of group c cases in those younger than under 18 years and an 82% reduction in those under 1 year old (60, 61) . an outbreak of serogroup w135 meningitis occurred among pilgrims on the hajj in 2000. cases were reported from many countries, including the united kingdom. in the united kingdom, there is now an official requirement to be vaccinated with the quadrivalent vaccine (acwy vax) before going on a pilgrimage (hajj or umra), but illegal immigrants who have not been vaccinated may enter the country (62). after an incubation period of 3-5 days (63,64) , disease onset may be either insidious with mild prodromal symptoms or florid. early symptoms and signs include malaise, fever, and vomiting. sever headache, neck stiffness, photophobia, drowsiness, and a rash may develop. the rash may be petechial or purpuric and characteristically does not blanche under pressure. meningitis in infants is more likely to be insidious in onset and lack the classical signs. in approx 15-20% of cases, septicemia is the predominant feature. even with prompt antibiotic treatment, the case fatality rate is 3-5% in meningitis and 15-20% in those with septicemia. (65). a person should be considered infectious until the bacteria are no longer present in nasal discharge. with treatment, this is usually approx 24 hour. the disease is spread through infected droplets or direct contact from carriers or those who are clinically ill. it requires prolonged and close contact, so it is a greater risk for people who share accommodation and utensils and kiss. it must also be remembered that unprotected mouth-to-mouth resuscitation can also transmit disease. it is not possible to tell if a detainee is a carrier. nevertheless, the risk of acquiring infection even from an infected and sick individual is low, unless the individual has carried out mouth-to-mouth resuscitation. any staff member who believes he or she has been placed at risk should report to the occupational health department (or equivalent) or the nearest emergency department at the earliest opportunity for vaccination. if the detainee has performed mouth-to-mouth resuscitation, prophylactic antibiotics should be given before receiving vaccination. rifampicin, ciprofloxacin, and ceftriaxone can be used; however, ciprofloxacin has numerous advantages (66) . only a single dose of 500 mg (adults and children older than 12 years) is needed and has fewer side effects and contraindications than rifampicin. ceftriaxone has to be given by injection and is therefore best avoided in the custodial setting. if the staff member is pregnant, advice should be sought from a consultant obstetrician, because ciprofloxacin is not recommended (67) . for anyone dealing regularly with illegal immigrants (especially from the middle east or sub-saharan africa) (e.g., immigration services, custody staff at designated stations, medical personnel, and interpreters), should consider being vaccinated with acwy vax. a single injection provides protection for 3 years. detainees suspected of disease should be sent directly to the hospital. human tb is caused by infection with mycobacterium tuberculosis, mycobacterium bovis, or mycobacterium africanum. it is a notifiable disease under legislation specific to individual countries; for example, in the united kingdom, this comes under the public health (control of disease) act of 1984. in 1993, the who declared tb to be a global emergency, with an estimated 7-8 million new cases and 3 million deaths occurring each year, the majority of which were in asia and africa. however, these statistics are likely to be an underestimate because they depend on the accuracy of reporting, and in poorer countries, the surveillance systems are often inadequate because of lack of funds. even in the united kingdom, there has been an inconsistency of reporting particularly where an individual has concomitant infection with hiv. some physicians found themselves caught in a dilemma of confidentiality until 1997, when the codes of practice were updated to encourage reporting with patient consent (68) . with the advent of rapid identification tests and treatment and the use of bacillus calmette-guã©rin (bcg) vaccination for prevention, tb declined during the first half of the 20th century in the united kingdom. however, since the early 1990s, numbers have slowly increased, with some 6800 cases reported in 2002 (69) . in 1998, 56% of reported cases were from people born outside the united kingdom and 3% were associated with hiv infection (70, 71) . london has been identified as an area with a significant problem. this has been attributed to its highly mobile population, the variety of ethnic groups, a high prevalence of hiv, and the emergence of drug-resistant strains (1.3% in 1998 ) (phls, unpublished data-mycobnet). a similar picture was initially found in the united states, when there was a reversal of a long-standing downward trend in 1985. however, between 1986 and 1992, the number of cases increased from 22,201 to 26,673 (72) . there were also serious outbreaks of multidrug-resistant tb (mdr-tb) in hospitals in new york city and miami (73) . factors pertinent to the overall upswing included the emergence of hiv, the increasing numbers of immigrants from countries with a high prevalence of tb, and perhaps more significantly, stopping categorical federal funding for control activities in 1972. the latter led to a failure of the public health infrastructure for tb control. since 1992, the trend has reversed as the cdc transferred most of its funds to tb surveillance and treatment program in states and large cities. from 1992 to 2001, the annual decline averaged by 7.3% (74) , but the following year this was reduced to 2%, indicating that there was no room for complacency. the who has been proactive and is redirecting funding to those countries most in need. in october 1998, a global partnership called stop tb was launched to coordinate every aspect of tb control, and by 2002, the partnership had more than 150 member states. a target was set to detect at least 70% of infectious cases by 2005. the acquisition of tb infection is not necessarily followed by disease because the infection may heal spontaneously. it may take weeks or months before disease becomes apparent, or infection may remain dormant for years before reactivation in later life especially if the person becomes debilitated or immunocompromised. contrary to popular belief, the majority of cases of tb in people who are immunocompetent pass unnoticed. of the reported cases, 75% involve the lung, whereas nonrespiratory (e.g., bone, heart, kidney, and brain) or dissemination (miliary tb) are more common in immigrant ethnic groups and individuals who are immunocompromised (75) . they are also more likely to develop resistant strains. in the general population, there is an estimated 10% lifetime risk of tb infection progressing to disease (76) . there has been an increase in the number of cases of tb associated with hiv owing to either new infection or reactivation. tb infection is more likely to progress to active tb in hiv-positive individuals, with a greater than50% lifetime risk (77) . tb can also lead to a worsening of hiv with an increase in viral load (78) . therefore, the need for early diagnosis is paramount, but it can be more difficult because pulmonary tb may present with nonspecific features (e.g., bilateral, unilateral, or lower lobe shadowing) (79). after an incubation period of 4-12 weeks, symptoms may develop (see table 6 ). the main route is airborne through infected droplets, but prolonged or close contact is needed. nonrespiratory disease is not considered a risk unless the mycobacterium is aerosolized under exceptional circumstances (e.g., during surgery) or there are open abscesses. a person is considered infectious as long as viable bacilli are found in induced sputum. untreated or incompletely treated people may be intermittently sputum positive for years. after 2 weeks of appropriate treatment, the individual is usually considered as noninfectious. this period is often extended for treatment of mdr-tb or for those with concomitant hiv. patient compliance also plays an important factor. the risk of infection is directly proportional to the degree of exposure. more severe disease occurs in individuals who are malnourished, immunocompromised (e.g., hiv), and substance misusers. people who are immunocompromised are at special risk of mdr-tb or mycobacterium avium intracellulare (mai). staff with disease should stay off work until the treatment course is complete and serial sputum samples no longer contain bacilli. staff in contact with disease who have been vaccinated with bcg are at low risk of acquiring disease but should minimize their time spent in the cell. those who have not received bcg or who are immunocompromised should avoid contact with the detainee wherever possible. detainees with mai do not pose a risk to a staff member, unless the latter is immunocompromised. any staff member who is pregnant, regardless of bcg status or type of tb, should avoid contact. anyone performing mouth-to-mouth resuscitation with a person with untreated or suspected pulmonary tb should be regarded as a household contact and should report to occupational health or their physician if no other route exists. they should also be educated regarding the symptoms of tb. anyone who is likely to come into repeated contact with individuals at risk of tb should receive bcg (if he or she has not already done so), regardless of age, even though there is evidence to suggest that bcg administered in adult life is less effective. this does not apply to individuals who are immunocompromised or pregnant women. in the latter case, vaccination should preferably be deferred until after delivery. detainees with disease (whether suspected or diagnosed) who have not been treated or treatment is incomplete should be kept in custody for the minimum time possible. individuals with tb who are immunocompromised are usually too ill to be detained; if they are, they should be considered at greater risk of transmitting disease to staff. any detainee with disease should be encouraged to cover his or her mouth and nose when coughing and sneezing. staff should wear gloves when in contact with the detainee and when handling clothing and bedding. any bedding should be bagged after use and laundered or incinerated. the cell should be deemed out of action until it has been ventilated and professionally decontaminated, although there is no hard evidence to support that there is a risk of transmission from this route (70). on march 14, 2003 , the who issued a global warning to health authorities about a new atypical pneumonia called sars. the earliest case was believed to have originated in the guandong province of china on november 16, 2002. the causative agent was identified as a new corona virus-sars-cov (80, 81) . by the end of june 2003, 8422 cases had been reported from 31 different countries, with a total of 916 deaths. approximately 92% of cases occurred in china (including hong kong, taiwan, and macao). the case fatality rate varied from less than 1% in people younger than 24 years, 6% in persons aged 25-44 years, 15% in those aged 44-64 years, and more than 50% in persons 65 years or older. on july 5, 2003, the who reported that the last human chain of transmission of sars had been broken and lifted the ban from all countries. however, it warned that everyone should remain vigilant, because a resurgence of sars is possible. their warning was well given because in december 2003, a new case of sars was detected in china. at the time of this writing, three more cases have been identified. knowledge about the epidemiology and ecology of sars-cov and the disease remains limited; however, the experience gained from the previous outbreak enabled the disease to be contained rapidly, which is reflected in the few cases reported since december 2003. there is still no specific treatment or preventative vaccine that has been developed. the incubation period is short, approx 3-6 days (maximum 10 days), and, despite the media frenzy surrounding the initial outbreak, sars is less infectious than influenza. the following clinical case definition of sars has been developed for public health purposes (82) . a person with a history of any combination of the following should be examined for sars: â�¢ fever (at least 38â°c); and â�¢ one of more symptoms of lower respiratory tract illness (cough, difficulty in breathing, or dyspnea); and â�¢ radiographic evidence of lung infiltrates consistent with pneumonia or respiratory distress syndrome or postmortem findings of these with no identifiable cause; and â�¢ no alternative diagnosis can fully explain the illness. laboratory tests have been developed that include detection of viral rna by pcr from nasopharyngeal secretions or stool samples, detection of antibodies by enzyme-linked immunosorbent assay or immunofluorescent antibody in the blood, and viral culture from clinical specimens. available information suggests that close contact via aerosol or infected droplets from an infected individual provide the highest risk of acquiring the disease. most cases occurred in hospital workers caring for an index case or his or her close family members. despite the re-emergence of sars, it is highly unlikely that a case will be encountered in the custodial setting in the near future. however, forensic physicians must remain alert for the sars symptoms and keep up-to-date with recent outbreaks. information can be obtained from the who on a daily basis from its web site. if sars is suspected, medical staff should wear gloves and a surgical mask when examining a suspected case; however, masks are not usually available in custody. anyone suspected of sars must be sent immediately to the hospital, and staff who have had prolonged close contact should be alerted as to the potential symptoms. the most consistent feature of diseases transmitted through the fecaloral route is diarrhea (see table 7 ). infective agents include bacteria, viruses, and protozoa. because the causes are numerous, it is beyond the remit of this chapter to cover them all. it is safest to treat all diarrhea as infectious, unless the detainee has a proven noninfectious cause (e.g., crohn's disease or ulcerative colitis). all staff should wear gloves when in contact with the detainee or when handling clothing and bedding, and contaminated articles should be laundered or incinerated. the cell should be professionally cleaned after use, paying particular attention to the toilet area. this viral hepatitis occurs worldwide, with variable prevalence. it is highest in countries where hygiene is poor and infection occurs year-round. in temperate climates, the peak incidence is in autumn and winter, but the trend is becoming less marked. all age groups are susceptible if they are nonimmune or have not been vaccinated. in developing countries, the disease occurs in early childhood, whereas the reverse is true in countries where the standard of living is higher. in the united kingdom, there has been a gradual decrease in the number of reported cases from 1990 to 2000 (83, 84) . this results from, in part, improved standards of living and the introduction of an effective vaccine. the highest incidence occurs in the 15-to 34-year-old age group. approximately 25% of people older than 40 years have natural immunity, leaving the remainder susceptible to infection (85) . small clusters occur from time to time, associated with a breakdown in hygiene. there is also an increasing incidence of hav in gay or bisexual men and their partners (86) . an unpublished study in london in 1996 showed a seroprevalence of 23% among gay men (young y et al., unpublished). the clinical picture ranges from asymptomatic infection through a spectrum to fulminant hepatitis. unlike hbv and hcv, hav does not persist or progress to chronic liver damage. infection in childhood is often mild or asymptomatic but in adults tends to be more severe. after an incubation period of 15-50 days (mean 28 days) symptomatic infection starts with the abrupt onset of jaundice anything from 2 days to 3 weeks after the anicteric phase. it lasts for approximately the same length of time and is often accompanied by a sudden onset of fever. hav infection can lead to hospital admission in all age groups but is more likely with increasing age as is the duration of stay. the overall mortality is less than 1%, but 15% of people will have a prolonged or relapsing illness within 6-9 months (cdc fact sheet). fulminant hepatitis occurs in less than 1% of people but is more likely to occur in individuals older than 65 years or in those with pre-existing liver disease. in patients who are hospitalized, case fatality ranges from 2% in 50-59 years olds to nearly 13% in those older than 70 years (84). the individual is most infectious in the 2 weeks before the onset of jaundice, when he or she is asymptomatic. this can make control of infection difficult because the disease is not recognized. the main route is fecal-oral through the ingestion of contaminated water and food. it can also be transmitted by personal contact, including homosexuals practicing anal intercourse and fellatio. there is a slight risk from blood transfusions if the donor is in the acute phase of infection. it should not be considered a risk from needlestick injuries unless clinical suspicion of hav is high. risk groups include homeless individuals, homosexuals, idus, travellers abroad who have not been vaccinated, patients with chronic liver disease and chronic infection with hbv and hcv, employees and residents in daycare centers and hostels, sewage workers, laboratory technicians, and those handling nonhuman primates. several large outbreaks have occurred among idus, some with an epidemiological link to prisons (87, 88) . transmission occurs during the viremic phase of the illness through sharing injecting equipment and via fecal-oral routes because of poor living conditions (89) . there have also been reports of hav being transmitted through drugs that have been carried in the rectum. a study in vancouver showed that 40% of idus had past infection of hav, and they also showed an increased prevalence among homosexual/bisuexual men (90). staff with disease should report to occupational health and stay off work until the end of the infective period. those in contact with disease (either through exposure at home or from an infected detainee) should receive prophylactic treatment as soon as possible (see subheading 8.3.7.). to minimize the risk of acquiring disease in custody, staff should wear gloves when dealing with the detainee and then wash their hands thoroughly. gloves should be disposed of only in the clinical waste bags. detainees with disease should be kept in custody for the minimum time possible. they should only be sent to the hospital if fulminant hepatitis is suspected. the cell should be quarantined after use and professionally cleaned. any bedding or clothing should be handled with gloves and laundered or incinerated according to local policy. detainees reporting contact with disease should be given prophylactic treatment as soon as possible (see subheading 8.3.7.). contacts of hav should receive hav vaccine (e.g., havrix monodose or avaxim) if they have not been previously immunized or had disease. human normal immunoglobulin (hnig), 500 mg, deep intramuscular in gluteal muscle should be used in the following circumstances: â�¢ has the detainee traveled to africa, south east asia, the indian subcontinent, central/south america, or the far east in the last 6-12 months? â�¢ ascertain whether he or she received any vaccinations before travel and, if so, which ones. â�¢ ask if he or she took malaria prophylaxis, what type, and whether he or she completed the course. â�¢ ask if he or she swam in any stagnant lakes during the trip. â�¢ if the answer to any of the above is yes, ask if he or she has experienced any of the following symptoms: a fever/hot or cold flushes/shivering. diarrhea â± abdominal cramps â± blood or slime in the stool. a rash. persistent headaches â± light sensitivity. nausea or vomiting. aching muscles/joints. a persistent cough (dry or productive) lasting at least 3 weeks. â�¢ take temperature. â�¢ check skin for signs of a rash and note nature and distribution. â�¢ check throat. â�¢ listen carefully to the lungs for signs of infection/consolidation. staff at higher risk of coming in to contact with hav should consider being vaccinated before exposure. two doses of vaccine given 6-12 months apart give at least 10 years of protection. there is no specific treatment for hav, except supportive measures and symptomatic treatment. although the chance of encountering a tropical disease in custo1dy is small, it is worth bearing in mind. it is not necessary for a forensic physician to be able to diagnose the specific disease but simply to recognize that the detainee/staff member is ill and whether he or she needs to be sent to the hospital (see tables 8-10) . this is best achieved by knowing the right questions to ask and carrying out the appropriate examination. tables 8-10 should be used as an aide to not missing some more unusual diseases. guidance for clinical health care workers: protection against infection with blood-borne viruses; recommendations of the expert advisory group on aids and the advisory group on hepatitis guidelines for hand hygiene in health care settings. recommendations of the healthcare infection control practices advisory committee and the hicpac/ shea/apic/idsa hand hygiene task force national model regulations for the control of workplace hazardous substances. commonwealth of australia, national occupational health and safety committee good practice guidelines for forensic medical examiners and the hospital infection control practices advisory committee. guideline for infection control in health care personnel report from the unlinked anonymous surveys steering group. department of health a strategy for infectious diseases-progress report. blood-borne and sexually transmitted viruses: hepatitis. department of health universal precautions for prevention of transmission of human immuno-deficiency virus, hepatitis b virus and other bloodborne pathogens in health-care settings risk factors for horizontal transmission of hepatitis b in a rural district in ghana familial clustering of hepatitis b infection: study of a family intrafamilial transmission of hepatitis b in the eastern anatolian region of turkey hepatitis b outbreak at glenochil prison during european network for hiv/aids and hepatitis prevention in prisons. second annual report. the network prevalence of hiv, hepatitis b and hepatitis c antibodies in prisoners in england and wales; a national survey the epidemiology of acute and chronic hepatitis c the role of the parenteral antischistosomal therapy in the spread of hepatitis c virus in egypt chronic hepatitis c: disease management. nih publication no. 03-4230. february department of health hepatitis c virus: eight years old laboratory surveillance of hepatitis c virus in england and wales: 1992-1996 last update aids/hiv quarterly surveillance tables provided by the phls aids centre (cdsc) and the scottish centre for infection and environmental health hiv and aids in the uk in 2001. communicable disease surveillance centre. an update mode of vertical transmission of hiv-1. a metanalysis of fifteen prospective cohort studies vertical transmission rate for hiv in the british isles estimated on surveillance data hiv post-exposure prophylaxis after sexual assault: the experience of a sexual assault service in london guidance from the uk chief medical officer's expert advisory group on aids. uk health department failures of zidovudine post exposure prophylaxis seroconversion to hiv-1 following a needlestick injury despite combination post-exposure prophylaxis department of health, immunisation against infectious disease. united kingdom: her majesty's stationery office chickenpox-disease predominantly affecting adults in rural west bengal prevention of varicella: recommendations of the advisory committee on immunization practices varicella-zoster virus epidemiology. a changing scene? use of acyclovir for varicella pneumonia during pregnancy outcome after maternal varicella infection in the first 20 weeks of pregnancy outcome in newborn babies given anti-varicella zoster immunoglobulin after perinatal maternal infection with varicella zoster virus varicella-zoster virus dna in human sensory ganglia epidemiology and natural history of herpes zoster and post herpetic neuralgia clinical applications for changepoint analysis of herpes zoster pain treatment of scabies with permethrin versus lindane and benzoyl benzoate treatment of ectoparasitic infections; review of the english-language literature nasal carriage of staphylococcus aureus: epidemiology and control measures centers for disease control and prevention. community-acquired methicillinresistant staphylococcus aureus infections-michigan methicillinresistant staphylococcus aureus, epidmiologic observations during a community acquired outbreak emergence of pvl-producing strains of staphylococcus aureus bacteriology of skin and soft tissue infections: comparison of infections in intravenous drug users and individuals with no history of intravenous drug use outbreak among drug users caused by a clonal strain of group a streptococcus. dispatchesemerging infectious diseases bacteriological skin and subcutaneous infections in injecting drug users-relevance for custody wound botulism associated with black tar heroin among injecting drug users isolation and identification of clostridium spp from infections associated with injection of drugs: experiences of a microbiological investigation team greater glasgow health board, scifh. unexplained illness among drug injectors in glasgow embolization of illicit needle fragments right ventricular needle embolus in an injecting drug user: the need for early removal departments of emergency medicine and pediatrics, lutheran general hospital of oak brook, advocate health system. emedicine-human bites prevention and treatment of dog bites human bites. department of plastic surgery guidelines for public health management of meningococcal diseases in the uk planning, registration and implementation of an immunisation campaign against meningococcal serogroup c disease in the uk: a success story efficacy of meningococcal serogroup c conjugate vaccine in teenagers and toddlers in england quadrivalent meningoimmunisation required for pilgrims to saudi arabia risk of laboratory-acquired meningococcal disease cluster of meningococcal disease in rugby match spectators immunisation against infectious disease. her majesty's stationery office ciprofloxacin as a chemoprophylactic agent for meningococcal diseaselow risk of anaphylactoid reactions joint formulary committee 2002-03. british national formulary notification of tuberculosis an updated code of practice for england and wales statutory notifications to the communicable disease surveillance centre. preliminary annual report on tuberculosis cases reported in england, wales, and the prevention and control of tuberculosis in the united kingdom: uk guidance on the prevention and control of transmission of 1. hiv-related tuberculosis 2. drug-resistant, including multiple drug-resistant, tuberculosis. department of health, scottish office control and prevention of tuberculosis in the united kingdom: code of practice epidemiology of tuberculosis in the united states nosocomial transmission of multi-drug resistant tuberculosis among hiv-infected persons-florida the continued threat of tuberculosis tuberculosis-a clinical handbook the white plague: down and out, or up and coming? a prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection influence of tuberculosis on human immunodeficiency virus (hiv-1): enhanced cytokine expression and elevated b 2 -microglobulin in hiv-1 associated tuberculosis the chest roenterogram in pulmonary tuberculosis patients seropositive for human immunodeficiency virus type 1 coronavirus as a possible cause of severe acute respiratory syndrome epidemiological determinants of spread of causal agents of severe acute respiratory syndrome in hong kong alert, verification and public health management of sars in post-outbreak period age-specific antibody prevalence to hepatitis a in england: implications for disease control phls advisory committee on vaccination and immunisation. guidelines for the control of hepatitis a infection control of a community hepatitis a outbreak using hepatitis a vaccine seroprevalence of and risk factors for hepatitis a infection among young homosexual and bisexual men outbreaks of hepatitis a among illicit drug users identifying target groups for a potential vaccination program during a hepatitis a community outbreak multiple modes of hepatitis a transmission among metamphetamine users past infection with hepatitis a among vancouver street youth, injection drug users and men who have sex with men implications for vaccination programmes key: cord-000130-dqqcajjd authors: smith?, robert j; gordon, richard title: the optaids project: towards global halting of hiv/aids date: 2009-11-18 journal: bmc public health doi: 10.1186/1471-2458-9-s1-s1 sha: doc_id: 130 cord_uid: dqqcajjd nan we face a unique, transitory opportunity in the history of the hiv/aids epidemic, because we have collectively pooled money faster than the epidemic has grown [1] . can we then seize the moment and halt this epidemic now? most scenarios for the future of hiv/aids project modest reductions spread out over decades [2] . the very timescale of such projections, beyond the persistence time of all models, makes them unreliable [3] . can we do better, quicker? the optaids project was conceived as a means to address this issue. its implementation thus far has been twofold: a workshop held in july 2008 and this supplement on the eradication of aids. the aims of the project are to address two questions: 1. can we optimally spend our way out of the hiv/aids epidemic? 2. can we work together to build a world halting aids model (wham) that would permit us to estimate the quickest way to halt hiv/aids, monitor our success, and adjust our strategy as we go? the optaids project grew out of a frustration with existing attempts to tackle the disease. aids exceptionalism means that hiv/aids is handled differently from other public-health epidemics, which has likely been detrimental [4, 5] . consequently, much of the funding of hiv/aids efforts has been for qualitative observations of the expanding epidemic rather than quantitatively effective intervention. although fund accumulation has recently outpaced the epidemic, we argue that plans to spend donor money are too long range in the face of a growing epidemic [6] . longrange scenarios have no reality to them, so that only shortterm solutions -those that fall within the persistence time of their models -have any possibility of being realistic [3] . furthermore, disease is a global problem that is only tackled locally [7] ; epidemics cross borders, whereas we fund mostly local or national "solutions". the optaids project was an outgrowth of the stop afghan aids project [8] . this project was led by mathematical modellers planning to continuously adapt their models to new data and predicting what data should be collected. the stop afghan aids project showed how it should be possible to intervene quantitatively in an epidemic. the usefulness of modelling in complex systems is not new. mathematical models of the economy tell us whether a decrease in income tax will result in an increase in investment or an increase in imported consumer goods. mathematical models of the atmosphere tell us what the effects of carbon dioxide emissions or of nuclear wars may be. mathematical modelling is used routinely in such things as aircraft design and the design of traffic systems [9] . so too, epidemics are quantitative creatures with predictable thresholds. models that can be adapted to new results and to changes in control policy have been identified as an integral part of disease-control programs [10] . modelling-led interventions were instrumental in halting the 2001 foot and mouth outbreak in the uk [11] . a mathematical model of the dynamics of measles in new zealand developed in 1996 successfully predicted an epidemic in 1997 and was instrumental in the decision to carry out an intensive immunisation campaign in that year. while the epidemic began some months earlier than anticipated, it was rapidly brought under control, and its impact on the population was much reduced [12] . the west african onchocerciasis (river blindness) control program successfully used modeling to supplement intervention programs [13] . by using clearly delineated endpoints, these models helped convince donors and the scientific community that the aims of the program were achievable [14] . as a result, mathematical models have retained a role in subsequent policy discussions [15] . insights from mathematical models during the sars epidemic helped determine how serious the epidemic might become, as well as the impact of proposed control measures. these models provided important guidance to public-health authorities at a critical time when little other information was available. insights from the models showed that, if unchecked, the virus could cause a significant epidemic, but that basic epidemiological control measures -patient isolation, contact tracing, etc -could have a substantial impact on the extent of the epidemic. subsequently, these control measures played a major role in limiting the spread of the 2003 epidemic [16] . weather prediction models provide a workable analogy. such models consist of continually updatable inputs, that must adapt to an enormous array of incoming data [17] . short-term predictions, especially those associated with discrete, extreme weather events such as floods and hurricanes, have proven useful in supporting emergency management strategies, unlike events such as earthquakes or acid rain, which have longer lead times [18] . complex mediating models which themselves have explanatory power and which embody techniques of modeling can be refined and passed down to successor models [9] . the virtue of mathematics in such a context is that it forces clarity and precision upon the conjecture, thus enabling meaningful comparison between the consequences of basic assumptions and the empirical facts [19] . existing scenarios for hiv control have typically been spread out over two or more decades [20] , which means that the reliability of their predictions is low. the basic concept of optaids is to spend more money up front, effectively, based on the best models and their parameters we can formulate, with the goal being a rapid halt to the epidemic with the fewest additional cases. this means that models can be shorter term and therefore more relia-ble, because we stay within the models' persistence time. optaids emphasises continuous monitoring to check the accuracy and adjust the parameters of the global model. mathematically, this is an optimal halting problem. the optaids workshop was the first of its kind: a scientific meeting held simultaneously in both a real world location and also second life ® http://secondlife.com, a virtual landscape that allows real-time communication. the broad topic was the eradication of aids using optimal spending models, but this encompasses an enormous number of issues surrounding the aids epidemic. topics covered included the impact of circumcision, the effect of traditional medicine, prevention strategies for countries with nascent epidemics and the difficulties of developing an hiv vaccine. mitacs http://www.mitacs.ca gave us a can$10,000 workshop grant for this meeting, which was held on july 29, 2008. given that this amount would only cover a few airfares, we decided to allow people to participate via second life ® . second life ® allows the creation of avatars [21] , so that users can participate in the world. within the virtual world, you can talk to other people's avatars, upload powerpoint slides and manipulate objects within the environment. twenty-two people convened over the day in toronto (figure 1) , including ten presenters. the traffic count during the day indicated 500 avatar arrivals in second life ® , four of whom were presenters. the workshop was advertised to pertinent groups in second life ® and open to the avatar public. the second life ® building includes a location where the original slide presentations can be viewed ( figure 2 ). two screens were used in toronto, showing the slides and the audience (represented by avatars), while second life ® participants could lecture by having their avatar stand near a virtual screen in second life ® . everyone could hear everyone else. a second virtual screen showed a live camera view of the audience in toronto (figure 3) . the all-day meeting had only a few short interruptions for technical reasons. a summary and follow-up discussion was presented at the annual meeting of the society for mathematical biology shortly afterwards. the four speakers presenting via second life ® were located in poland, seattle, denmark and los angeles. the technology allowed for interactive discussion, so speakers in toronto faced questions from second life ® participants all around the world, while second life ® speakers had their powerpoint presentations shown on a screen in toronto (operated by their avatars in second life ® and simultaneously by the organisers in toronto), and faced questions from toronto and other second life ® participants. the size of the turnout in second life ® demonstrated the effectiveness of virtual conferencing; many more people were able to attend the conference than would have been feasible otherwise. the event was covered by the national post, which reported on the innovative use of second life ® in an academic setting. all the presentations remain in second life ® http://slurl.com/secondlife/silver bog/21/27/22 as posters that can be clicked on by anyone interested. speakers can be asked to show up personally as avatars to go over the slides. the aim of this supplement is to discuss aids as a global phenomenon and address issues surrounding its eradica-tion. due to the scale of the epidemic, a great number of sub-issues arise. in thinking of aids as a global pandemic, we need to tackle the disease from as many directions as possible. some of the articles involve mathematical models, others involve a thorough examination of the state of resources, or an understanding of the effect of the disease on society. this supplement comprises fifteen articles (including this introduction), divided into six themes: 1. history 2. resources 3. demographics 4. in-host models 5. computation 6. spending our way out of the epidemic theme 1 comprises an introduction and overview of mathematical modeling [22] , as well as a history of aids in africa and its effects on human development [23] . theme 2 is concerned with the various resources that comprise our intervention arsenal: the allocation of resources [24] , cost-effectiveness of prevention [25] , antiretroviral pricing [26] , the effects of migration upon availability of health professionals [27] , and the relationship between mathematical models and resource allocation [28] . theme 3 looks at the effects of demographic changes in china on hiv [29] and the spread of hiv among men who have sex with men [30] . theme 4 examines in-host modeling -a crucial element in tackling the disease, often overlooked by epidemiologists -by proposing new methods for evaluating the efficacy of antiretroviral treatment [31] and examining antioxidant supplementation as hiv therapy, with a focus on injecting drug users [32] . theme 5 looks at using virtual epidemics to understand real ones [33] and develops an epidemic simulator of an agent-based, data-driven disease model [34] . finally, theme 6 examines the question at the core of the optaids project: spending our way out of the aids epidemic [6] . the collection of articles in this supplement run the gamut of topics related to hiv/aids. they examine the disease from a global perspective, in an attempt to untangle many of the problems associated with the epidemic. however, we view this as a starting point: the next step is for policymakers and the donor community to embrace the idea of global eradication. only by working together can we combat this disease. aids is the fourth worst infectious disease of all time, resulting in more deaths per day over the past 25 years than occurred on 9/11/2001 [35] . over 33 million adults are now infected with hiv, many in the developing world, where resources are scarce and infrastructure is struggling under the weight of this burgeoning epidemic. the hiv/ aids epidemic is often spoken of in terms of "reducing the spread" [36] , or achieving "sustainable financing" [37] however, this special issue demonstrates that, despite the immensity of the epidemic, eradication is not only possible, it is feasible. the time has come to stop thinking locally and to start acting globally. global health--the gates-buffett effect the case for expanding access to highly active antiretroviral therapy to curb the growth of the hiv epidemic useless arithmetic: why environmental scientists can't predict the future aids and the arrows of pestilence hiv testing, human rights, and global aids policy: exceptionalism and its discontents can we spend our way out of the aids epidemic? a world halting aids model why rich countries should care about the world's least healthy people aids 2006, xvi international aids conference 13-18 mathematical models: questions of trustworthiness epidemiological modeling for onchocerciasis control. parasitology today the uk foot-and-mouth disease outbreak -the aftermath predicting and preventing measles epidemics in new zealand: application of a mathematical model the role of mathematical modeling in evidence-based malaria control neglected tropical diseases: infection, modelling and control final report of the conference on the eradicability of onchocerciasis the geographic spread of infectious diseases: models and applications princeton university press representing model uncertainty in weather and climate prediction prediction in science and policy uses and abuses of mathematics in biology aids in africa: three scenarios to 2025 avatars: exploring and building virtual worlds on the internet berkeley mathematical epidemiology is not an oxymoron the impact of hiv/aids on human development in african countries the past, present and future of hiv, aids and resource allocation hiv prevention cost effectiveness: a review of the literature factors influencing global antiretroviral procurement prices addressing the migration of health professionals: the role of working conditions and educational placements recommendations for increasing the use of hiv/aids resource allocation models population profiling in china by gender and age: implication for hiv incidences a sex-role preference model for hiv transmission among msm population quantifying the treatment efficacy of reverse transcriptase inhibitors: new analyses of clinical data based on within-host modeling reconciling conflicting clinical studies of antioxidant supplementation as hiv therapy: a mathematical approach halting hiv/aids with avatars and havatars: a virtual world approach to modelling epidemics epidemic modeling with discrete-space scheduled walkers: extensions and research opportunities reducing the spread of hiv infection in sub-saharan africa: some demographic and economic implications sixtieth session, agenda item 45. follow-up to the outcome of the twenty-sixth special session: implementation of the declaration of commitment on hiv/aids. scaling up hiv prevention, treatment, care and support we would like to thank natalie k. björklundgordon the authors declare that they have no competing interests. rjs wrote the introduction, overview of the supplement and the conclusion. rg set the theme in the first draft and wrote the start of the introduction and the section on the workshop. both authors proofread and approved the final manuscript. key: cord-001385-rb5vwolt authors: reuven, eliran moshe; ali, mohammad; rotem, etai; schwarzter, roland; gramatica, andrea; futerman, anthony h.; shai, yechiel title: the hiv-1 envelope transmembrane domain binds tlr2 through a distinct dimerization motif and inhibits tlr2-mediated responses date: 2014-08-14 journal: plos pathog doi: 10.1371/journal.ppat.1004248 sha: doc_id: 1385 cord_uid: rb5vwolt hiv-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. hiv-1 infected cells show limited toll-like receptor (tlr) responsiveness via as yet unknown mechanisms. using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (tmd) of the hiv-1 envelope (env) directly interacts with tlr2 tmd within the membrane milieu. this interaction attenuates tnfα, il-6 and mcp-1 secretion in macrophages, induced by natural ligands of tlr2 both in in vitro and in vivo models. this was associated with decreased levels of erk phosphorylation. furthermore, mutagenesis demonstrated the importance of a conserved gxxxg motif in driving this interaction within the membrane milieu. the administration of the env tmd in vivo to lipotechoic acid (lta)/galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. our findings suggest that the tmd of env is involved in modulation of the innate immune response during hiv infection. furthermore, due to the high functional homology of viral env proteins this function may be a general character of viral-induced immune modulation. the ongoing race between pathogens and their hosts' responses to eliminate them led to the development of many mechanisms driven by the invading pathogen to impair immune responses. the cell populations that are mainly targeted by the virus include mononuclear phagocytes (e.g. macrophages and monocytes) and t cells [1] . several mechanisms of immune evasion and suppression have been described for the pathology of the human immunodeficiency virus type 1 (hiv-1) [2, 3] . regarding mononuclear phagocytes, studies implicated the importance of early genes, expressed by hiv-1 in advanced phases of infection, for immune manipulation [4, 5] . however, as these cells are hallmarks of innate immunity, there is also a requirement for immune manipulation at stages of viral entry and latency. little is known about the ability of hiv-1 to modulate innate immune responses of these cells during its entry and latent stages, particularly against members of the toll-like receptor (tlr) family. tlrs are critical in the immediate innate immune response against bacterial and viral pathogens [6, 7] . tlrs are conserved membrane receptors that recognize a wide variety of pathogenassociated molecular patterns (pamps), such as lipopolysaccharide (lps) from gram-negative bacteria, lipoteichoic acid (lta) from gram-positive bacteria, flagellin, in addition to intracellular molecules such as single-stranded dna and rna [8, 9] . to induce ligand recognition and subsequent signaling, the heterodimerization of tlr2 with tlr6 or tlr1 is required. this is coordinated through ligand binding to the extracellular regions of the proteins and conformational changes throughout the proteins [10, 11, 12] . the significance of the tlr2 and tlr6 tmds in the regulation and activation of formation of the receptor complex and in downstream signaling has been recently described [13] , revealing that activation of tlr2 increases resistance of macrophages to hiv-1 infection [14] . interestingly, dendritic cells (dcs) infected with hiv-1 were reported to be less responsive via tlr upon expression of env on the membrane [15] . these emerging studies link the manipulation of tlr2 responses and hiv-1 pathogenesis through as yet unknown mechanisms. hiv-1 infects cells via the hiv-1 env protein which mediates viral entry to host cells that express cd4 together with an additional co-receptor such as monocytes and dendritic cells, through membrane fusion. in addition to its fusogenic activity, the env protein binds proteins localized to membrane microdomains on macrophages and dcs. in addition, tlrs expressed on the cell membrane are recruited to cholesterol-enriched membrane microdomains upon their ligand recognition, initiating signal transduction [16, 17] . env is also targeted to cholesterol-enriched membrane microdomains through a well-defined localization signal located adjacent to its tmd. the tmd of env and its adjacent regions are extremely conserved among all clades currently reported of hiv-1, and are similar to the tmd of hiv-2 gp41 [18] . interestingly, recent studies showed that tlr2 recognition of several viral related glycoproteins (gp) induces activation of an antiviral response [19] . taken together, we hypothesized that the env plays a role in impairment of tlrinduced responses, contributing both to its evasion from recognition during the fusion process and to expropriate native tlr responses at latent stages, thus assisting in viral replication upon its initiation. here, we report that the gp41 tmd associates with tlr2 tmd in the membrane. as a result, cultured macrophages treated with peptides derived from the gp41 tmd displayed reduced tlr2-mediated signaling and decreased pro-inflammatory cytokine secretion. moreover, the ectopic expression of the intact gp41 and gp160 inhibited lta mediated cytokine secretion with gp41 having the highest potency. to demonstrate the tlr2 inhibitory effect in vivo, we show that the gp41 tmd protected mice against lta and d-galactosamine (lta/gln)-mediated acute sepsis, concomitant with a decrease in tissue damage. we synthesized a peptide derived from the n9 terminus of the env tmd region (gp41 tmd) previously reported to be involved in cellular responses to viral infection [20] . since this region harbors the well-defined gxxxg motif known to drive the assembly of tmds [21] , we also synthesized a peptide lacking this motif (gp41 mutant) (table s1 in text s1). we then utilized the macrophage cell line raw264.7 to study the functionality of tlr2. we activated tlr2 by purified lta that was recently shown to be regulated, at least in part, through interactions between the tmds of tlr2 and tlr6 [13] . we measured the level of phosphorylated erk1,2 upon treatment with lta, as these are key regulators of the tlr signaling pathway. figure 1a and b show that 20 minutes after addition of lta to raw cells that were pre-incubated with the gp41 tmd peptide, there was a significant decrease in erk1,2 phosphorylation compared to nontreated cells, indicating less receptor activation. as a negative control, pre-incubation with the gp41 mutant peptide did not change the levels of phosphorylated erk1,2. noteworthy, since the cells in culture that respond through the tlrs also respond to endogenous signals, the basal level of phosphorylated erk1,2 was also decreased by the gp41 tmd peptide but not by the mutant peptide ( figure 1a ). we next measured the expression levels of nfkb downstream genes; tumor necrosis factor a (tnfa), a hallmark cytokine of tlr activation, and monocyte chemotactic protein 1 (mcp-1), a major chemokine mainly secreted by macrophages [22] , in order to ensure that the inhibitive effect on erk1,2 phosphorylation is a result of inhibition of tlr2 signaling. treatment of the cells with gp41 tmd peptide resulted in a 45% decrease in mcp1 mrna expression levels compared to non-treated cells ( figure 1c ). the treatment also resulted in a 72% decrease in the mrna expression levels of tnfa ( figure 1d ). when cells were treated with the mutant peptide, only a 34% decrease in mrna levels was detected for tnfa ( figure 1c ), and no significant change was detected for the expression levels of mcp-1 ( figure 1d ). altogether, it is clear that the association between the tmds of gp41 and tlr2 functionally impacts receptor activity. in order to confirm that the effects observed at the transcription levels influence the protein expression levels we measured tnfa, mcp-1 and il-6 secretion levels. il-6 is an additional target gene of tlr2 but it is transcribed via a different transcription factor complex than tnfa. cells were pre-incubated with env tmd peptides for two hours prior to lta activation. cytokine expression was measured in accordance to the expected time of expression of the various targets. the results show that the envtmd peptide attenuates the secretion of tnfa (figure 2a ), il-6 ( figure 2b ) and mcp-1 ( figure 2c ). however, and in line with the mrna expression levels and phosphorylation inhibition, the gp41 mutant peptide did not decrease cytokine secretion. in order to validate that the effect observed is not limited to murine cells we evaluated the effect of the peptides on human thp-1 cells. we followed the secretion of tnfa under the same conditions as for the mice cell line. the data show that the wt peptide had similar effect on human cells as in mice cells and that the mutant peptide did not affect the secretion levels of tnfa ( figure 2d ). these results suggest that incubation of gp41 tmd peptides with cells leads to inhibition of tlr2/6 signaling. together, these results suggest a potential potent immunosuppressive effect of gp41 hiv-1 env protein via inhibition of tlr2 mediated signaling. in order to confirm this hypothesis we utilized the ectopic expression of hiv-1 env-yfp chimeric protein in raw 264.7 cells, which mimics the hiv-1 infection of macrophages, and tested their responsiveness to lta by measuring tnfa secretion. we expressed two forms of the env protein, the full length gp160 and the transmembrane protein gp41. lta responsiveness was compared to mock transfected cells (see materials and methods). lta treatment resulted in elevated tnfa secretion in mock cells, whereas gp160 expressing cells showed a reduction in tnfa secretion. furthermore, gp41 expressing cells showed even more reduction of tnfa secretion almost to the basal level of the nontreated cells (figure 3 ). the better efficiency of gp41 in inhibiting tnfa secretion compared to gp160 can be in part due to the smaller size of its ectodomain compared to that of gp160. this should result in less hindrance upon its interaction with tlr2. we confirmed similar expression levels of hiv-1 gp160 and gp41, as they showed similar extracellular expression levels (see example in to understand viral pathology and the tools needed to eliminate infection, it is important to understand how viral immune evasion occurs. one such mode of inhibition is the decreased responsiveness of toll-like receptors (tlrs). to date, the exact mechanism inducing this inhibition is not clear. in this study, we utilized a multidisciplinary approach and report on direct modulation of tlr2 activity by the envelope trans-membrane protein of hiv-1 through trans-membrane domain interactions. this interaction resulted in a decreased response in vitro of tlr2 to its natural ligand lta. through mutagenesis analysis we show that the gxxxg motif is the driving force of this interaction. interestingly, the inhibitory effect was also highly effective in protecting mice from lethal effects in a sepsis-like model. our findings implicate that env participates in innate immune impairment, which may occur during viral entry and at latent stages. furthermore, due to the high functional homology between viral env proteins, this function may exhibit a general character of viral-induced immune modulation. supplementary figure s5 in text s1). these data reveal that the fully processed env protein, once expressed on the cellular membrane, results in inhibition of tlr2 activation. we studied the ability of the env tmd to target the tlr2 tmd as it naturally resides in the membrane, and the tmds of tlr2/6 were shown to be important for their regulation and function. the gallex system [23, 24] was utilized to evaluate the hetero-association of gp41 and tlr2 tmds in biological membranes. b-gal activity was measured after the expression of lexa-tmd-mbp chimera proteins in which the tmds are those of env (wild type and mutant) and of tlr2. the gpa tmd sequence and its non-assembling mutant g83i (table s1 in text s1) served as positive and negative controls, respectively. strong dimerization results in low b-gal activity. the level of association between the tmds of gp41 wt and tlr2 was significantly higher than that of the tmds of the gp41 mutant and tlr2. moreover, there was no detectable association between the tmds of tlr2 and gpa (containing the gxxxg motif) ( figure 4a ). therefore, we conclude that the gxxxg motif contributes but is not sufficient for the assembly of the tmds of gp41 and tlr2. fret analysis was used to explore the direct interaction between the tmd peptides within phospholipid membranes (pc:chol 9:1). the decrease in the emission of nbd conjugated peptides (gp41 tmd/mutant), after the addition of successive amounts of rho-labeled tlr2 tmd, served as an indication of the interaction between a pair of peptides. the gp41 tmd peptide showed strong interaction with the tlr2 tmd peptide ( figure 4b ). this is evident from the large decrease in the emission signal at 530 nm after the first addition of the rho-tlr2 tmd peptide and by the dose dependent extent of association resulting in a decrease of over 60% at a 1:1 ratio. in comparison, the interaction between the gp41 mutant and tlr2 tmds peptides was significantly lower (less than 25%) (figure 4c ). to determine the degree of interaction between the tmds of env and tlr2 within macrophages we performed immunoprecipitation assays using the gp41 tmd and mutant peptides, and the antimicrobial peptide ll37 as a negative control. ll37 was used due to its strong membrane affinity properties and lack of specific protein targets in macrophages. figure 4d shows the strong precipitation of gp41 tmd with tlr2 compared to almost no precipitation of the gp41 mutant peptide. in order to validate that the inhibitory effect of the gp41 env peptide is due to inhibition of the interaction between the tmds of tlr2 and tlr6, we performed a fret competition assay between tlr2 and 6 in the absence ( figure s3 a in text s1) or presence ( figure s3 b in text s1) of the gp41 wt peptide. the data show that when the env peptide is present, the degree of association between the tlr peptides decreased from 40% fret to 16%. this inhibition was further corroborated by immunoprecipitation of tlr6-tmd peptides with the tlr2 protein in the absence ( figure s1 c, left lane in text s1) or the presence (figure s1 c, right lane in text s1) of the gp41 wt peptide. in order to verify that the tmd peptides preserved helical structures, we used circular dichorism (cd) spectroscopy in a membrane mimicking solution (1% lpc). all three peptides exhibited a-helical secondary structures, typical of a tmd peptide ( figure s2 in text s1). it is clear from the cd results that the gp41 mutant peptide (loss of the gxxxg motif) preserves a helical structure suggesting that any functional discrepancies are not as a result of major structural deformation. in order to corroborate the effect of the env tmd on tlr2 activation in vivo we utilized a murine model for acute sepsis caused by hyper-activation of tlr2 [13] . mice were injected intraperitoneally (i.p) with 100 mg of lta and 700 mg/kg dgalactosamine (gln), and were either treated intravenous (i.v.) with gp41 tmd peptide (5 mg/kg), mutated peptide (5 mg/kg) or pbs. upon injection of lta/gln, 7/9 pbs-treated mice and 6/ 9 of the gp41 mutant treated mice died while only 2/9 of the gp41 tmd treated mice died ( figure 5a ). histological analysis of the liver indicated that lta/gln treatment resulted in massive parenchyma damage associated with hemorrhage in both pbs and gp41 mutant treated mice ( figure 5b ). however in the gp41 tmd treated samples, the tissue resembled that of control mice ( figure 5b ) with no observable tissue damage or hemorrhage. this was also evident from the external feature of the tissues themselves, showing that mice not treated with the env tmd peptides had enlarged and inflamed livers and spleens ( figure s3 in text s1). we analyzed serum levels of tnfa and il-6 as these cytokines are secreted by resident macrophages and blood monocytes in the lta/gln model [13] . in accordance with the survival rates, our results indicate that the blood serum levels of tnfa and il-6 were significantly lower for the gp41 tmd peptide-treated group compared to the untreated group (figure 6a and b, respectively). we therefore examined spleen, in order to test activation of the immune system. the effect of an uncontrolled inflammatory response may lead to secretion of high levels of steroids, leading to massive cell death in the lymphocyte zones [25] . lta/gln injection resulted in a large number of apoptotic cell foci in the white pulp and congestion throughout the tissue in comparison to control mice spleens ( figure 6c middle and top panels, respectively). comparably, gp41 tmd treatment decreased apoptosis to levels similar to the naïve samples ( figure 6c lower panel) . cell death was also detected in the red pulp of untreated mice and was absent from gp41 tmd treated mice. this result indicates that gp41 tmd decreased monocyte activation, resulting in less apoptosis in the spleen. in order to test the specific attenuating effect of gp41 tmd on lta/gln mediated sepsis, we tested whether the treatment resulted in an inhibitive effect on macrophages, as macrophages are the main immune cells that orchestrate this type of inflammation. we investigated whether the inhibitory effect of gp41 tmd peptide was tissue specific or systemic by staining both liver (tissue specific) and spleen (systemic) for the monocyte activation marker, mac2 [26] . liver staining showed that the administration of lta/gln resulted in increase in mac2 positive cells, in addition to the appearance of round-shaped mac2 positive cells that could be either activated residential macrophages (kupffer cells) that phagocysed dead hepatocytes, or infiltrated activated monocytes ( figure 7a, white arrows) . the mutant gp41 showed minor amount of mac2 positive cells that resembled the non-treated control mice ( figure 7b ). these results suggest that the attenuating effect of gp41 tmd was systemic rather than tissue specific, with high specific effects on monocytes and macrophages. in this study, we report that the responsiveness of tlr2 is modulated by the env tmd. this inhibitory effect is driven through the direct association of the env tmd with the tlr2 tmd. functionally, the env tmd inhibits tlr2 signaling in response to lta and inhibits secretion of pro-inflammatory cytokines both in vitro and in an animal model of lta/gln sepsis. recent studies show that peptides derived from tmds of several membrane proteins interfere with their assembly and function [23, 27, 28] . we utilized this strategy to assess the ability of env to affect tlr2 activation, and to identify the important amino acids within the tmd that contribute to this interaction. it has been shown that the tmd of tlr2 regulates its function in response to natural and synthetic ligands. the env tmd possesses a gxxxg motif, which is well defined as a membrane dimerization motif. interestingly, the tlr2 tmd also possesses such a sequence ( figure s3 , boxed in text s1). the precise conformational changes of the tlr2 tmd that induce its dimerization with its partners leading to signal transduction are not known. nevertheless, it is conceivable that the gxxxg motif of the tlr2 and tlr6 tmds move in a piston mechanism (for an explanation on the piston motion, see ref. [24] ). this motion could explain how these motifs are important for the tlr function although they are not located parallel to each other within the membrane. mutating the gxxxg motif significantly decreased the ability of the env tmd to inhibit macrophage responses to tlr2 ligands. this is expected because the peptides' mode of action is by inhibiting dimerization of tlr2 and tlr6 through interactions within the membrane milieu. interestingly, the interaction of tmd sequences is not driven solely by the gxxxg motif, since the gpa tmd did not show any association with the tlr2 tmd. the specific nature of the interaction is demonstrated by the co-immunoprecipitation of tlr2 with the env tmd, while no interaction was detected with the mutated tmd. overall, the data suggest that the env tmd interferes with the dimerization of tlr2/6 tmds partially through their gxxxg motif. this subsequently leads to impaired complex signaling and to inhibition of cytokine secretion. the general effect of this inhibition is demonstrated by inhibition of the initial steps of signal transduction (erk phosphorylation), as well as, by inhibition of an array of end point targets of tlr signaling (tnfa, il-6 and mcp-1) each transcribed in a different complex of nfkb. note that it has been shown that the gxxxg is important, but not sufficient, for helix-helix interactions within the membrane [21] . as cd4 and tlrs may reside in similar membrane environments [29, 30] , and membrane-bound tlrs recognize viral glycoproteins, it is reasonable to assume that this leads to initiation of a response towards hiv-1 during fusion. furthermore, activation of tlr signaling prior to hiv infection causes dendritic cells to be less permissive to infection [31] . therefore, inhibiting the initial response towards the invading pathogen is a powerful tool to allow virus entry into the cell. an additional possible need for this inhibitory activity is at latent stages of viral infection. hiv-1 env and tlr2 both possess a membrane localization signal motif adjacent to their tmd ( figure s3 in text s1). this signal motif determines the precise environment within the membrane in which the proteins reside. although for t cells, env is expressed at low levels during latency, env expression levels on mononuclear phagocytes are high [32] . several studies indicated the impairment of tlr signaling after viral infection and incorporation [5] which may assist in the establishment of infection by opportunistic bacteria and fungi, in addition to the loss of cd4 + t cells [33] . together with our findings about the modulatory effect of env on tlr responses, this impairment may occur as a result of the interactions of the tmds of env and tlrs, leading to the prevention of tlr activation upon ligand recognition. noteworthy, several studies have linked the activation of tlr cascades to viral replication and burst. these studies focus on cells with established infection. this means that the virus successfully transmitted its genome into the cells and initiated the expression of its early genes [34] . in view of the small number of proteins expressed by the hiv genome, this immunosuppressive activity might exist with its fusogenic activity to help the virus to escape immune recognition and response towards it, prior to the activity of the viral early genes. in summary, we demonstrate that the env tmd inhibits the activation of tlr2 signaling by interacting with its tmd. this property could be of great importance during fusion and serves as an additional mean of regulation of the activity of tlr at stages of env expression. our findings further demonstrate the complex strategies used by hiv-1 to manipulate the immune system. interestingly, the env tmd is also involved in the late steps of the membrane fusion reaction. such dual activity further demonstrates how viruses although expressing a limited number of proteins have evolved to alter so many cellular processes. considering the strong ability of other gp41-derived segments to inhibit t-cell activation by various mechanisms, our study may indicate that these effects are also applicable to mononuclear phagocytes and additional targets should be further investigated. apart from hiv pathogenesis, specific gp41 derived peptides may provide potential tools for treating uncontrolled immune responses, manifested here by sepsis. all in vitro assays were performed on raw264.7 murine macrophages (atcc-tib71). cells were grown at 37uc in the presence of 5% co 2 in dmem supplemented with 10% fbs, lglutamine, sodium pyruvate, non-essential amino acids, and antibiotics (biological industries, beit-haemek, israel). for cytokine secretion measurements 2610 5 cells per well were cultured overnight in a 96-well plate. at the following day, media were replaced by fresh dmem, including all supplements. gp41 tmd peptides were dissolved in dmso and added to the cells in different concentrations. the final concentration of dmso was 1% for all groups. cells were incubated with the peptide for 2 hours, washed and incubated with fresh media containing lta. cells were incubated with lta for 5 hrs (tnfa) or 12 hours (il-6, mcp-1) at 37uc, after which samples of the media were collected and stored at 220uc. cytokine levels were evaluated using a mouse tnfa/il-6 enzyme-linked immunosorbent assay kit (elisamax, biolegend) according to the manufacturer's protocol. all experiments were done in triplicates. raw264.7 macrophages were transfected twice, 48 h and again 24 h prior to the experiment, either with p96zm651gp160-cd5-opt plasmid for the expression of hiv envelope protein gp160, or with gp41-yfp expressing plasmid for the expression of gp41 hiv fusion protein [35] . to increase the expression rate we utilized turbofect reagent (thermo scientific, waltham, ma, usa) according to the manufacturer protocol. surface expression of untagged gp160 was analyzed using immunofluorescence. briefly, cells were washed twice with pbs and fixed with 4% paraformaldehyde for 15 min at room temperature. antibody staining was conducted using a goat polyclonal anti-hiv-1 gp120-biotin conjugated antibody and a atto 488 conjugated, goat igg (h&l) antibody. fluorescence intensities were obtained using an inverted fluoview 1000 confocal microscope (olympus, tokyo, japan) with a 606 oil immersion objective (numerical aperture 1.35) at 25uc with a frame size of 5126512 pixels. atto 488 was excited with a laser diode at 488 nm and detected between 500 and 600 nm. large unilamellar vesicles (luvs) composed of phosphatidylcholine (pc) and cholesterol (chol) (9:1, w/w) were prepared using the extrusion method as described previously [36] . the fluorescence experiments were performed with pairs of peptides using 4-fluoro-7-nitrobenzofurazan (nbd, biochemika) -labeled peptides as donors and rhodamine-labeled peptides as acceptors and as described previously [13] . maximum fret values were obtained by calculating the changes in the emission of the donor peptide at 530 nm. cd measurements were performed by using an applied photophysics spectropolarimeter. the spectra were scanned using a thermostatic quartz cuvette with a path length of 1 mm. wavelength scans were performed at 25uc; the average recording time was 15 s, in 1 nm steps, through the wavelength range of 195-260 nm. peptides were scanned at a concentration of 50 mm in buffer only (hepes 5 mm) or in a membrane mimetic environment of 1% lysophophatidilcholine (lpc) in buffer. the hetero-interactions of the tmds within a natural membrane environment were studied using the gallex assay [37] . the expression vectors and e. coli strains required for the assay were kindly provided by dr. dirk schneider. briefly, two plasmids are used in the assay containing either the wild-type lexa sequence (pblm, encoding for resistance to tetracycline) or a mutated form of lexa (palm, encoding for resistance to ampicillin). the desired tm sequences are inserted between these two protein sequences using standard cloning techniques. the resulting plasmids were both transformed into an su202 e. coli strain, which then expressed the lexa-tm-male fusion proteins (after induction with 1 mm iptg). hetero-association of tm domains leads to the formation of lexa wt /lexa mut heterodimers that bind to a hybrid lexa promoter/operator in the reporter strain su202 (that recognizes the mutant lexa domain), and repress the expression of b-gal. furthermore, as the hybrid promoter is designed to bind only the wt and mutant heterodi-mers, it will not recognize wild-type lexa homodimers. thus, tm homo-oligomer formation does not interfere with the measurement of hetero-association. the tmds of glycophorina (gpa) and its g83i mutant, used as positive and negative controls, respectively, are itliifgvmagvigtilli and itliifgvmai-vigtilli. all the constructs were confirmed by dna sequencing. the dimerization propensity of the two constructs was measured by transforming an e. coli su202 indicator strain with the relevant palm and pblm plasmids (each harboring a different tm sequence) simultaneously. the bacteria were grown overnight in the presence of 1 mm iptg in lb medium with ampicillin and tetracycline in a 24-well plate. 10 ml of bacteria were resuspended in 100 ml of z-buffer (60 mm na 2 hpo 4 , 40 mm n a h 2 po 4 , 10 mm kcl, 1 mm mgso 4 , ph = 7.0, 10% chloroform, 1% b-mercaptoethanol), and the absorbance (od 600 ) was measured. bacteria were lysed with 50 ml of 2% sds in zbuffer. o-nitrophenyl-galactopyranose (onpg) was added and a kinetic reading of absorbance at od 405 was performed for 20 min to measure the activity of b-gal. vmax was calculated and divided by the absorbance at od 600 to correct for bacterial growth. strong dimerization results in strong inhibition of b-gal expression and less color formation. cell lysates were prepared in radioimmunoprecipitation (ripa) assay buffer (50 mm tris hcl ph 7.5, 150 mm nacl, 1% nonidet p-40, 0.5% sodium deoxycholate, 0.1% sds) containing 50 mm naf, 2 mm na3vo4, protease and phosphatase inhibitors (sigma-aldrich). protein concentration was measured using the bca protein assay kit (pierce chemical co.). fifty mg of protein was loaded and separated on 8-15% sds-page and transferred to a nitrocellulose membrane. the membrane was blocked using 5% bovine serum albumin (bsa) in phosphate-buffered saline containing 0.1% tween-20 (pbst) for 1 h at room temperature. the primary antibody was diluted in pbst containing 1% bsa and incubated with the membrane at 4uc overnight. after 3 washes with pbst, membranes were incubated with the secondary antibody in pbst containing 1% bsa at room temperature for 1 h. the following sequences were used for quantitative mrna analysis: tnfa forward: cttgtggcaggggccaccac reverse: ccatgccgttggccaggagg mcp-1 forward: tcacctgctgctactcattcacca reverse: agcacagacctctctcttgagctt anti phospho-erk1, 2 and anti-total erk1, 2 were purchased from sigma-aldrich and anti gapdh was purchased from millipore. goat polyclonal anti-hiv-1 gp120-biotin conjugated antibody was purchased from abcam (cambridge, united kingdom). atto 488 conjugated goat igg (h&l) antibody was purchased from rockland (gilbertsville, pa, usa). s. aureus lta was purchased from sigma-aldrich and d-galactosamine was purchased from calbiochem. tnfa and il-6 elisa kits were purchased from biolegend. the plasmid p96zm651gp160-cd5-opt was obtained through the nih aids reagent program, division of aids, niaid, nih: from drs. yingying li, feng gao, and beatrice h. hahn. the plasmid gp41-yfp, provided by roland schwarzer encodes a hiv-1 gp41 fusion protein with the c-terminal external parts of the protein replaced by a yellow fluorescent protein. animal studies were carried out in strict accordance with the israeli law and the national research council guidelines. all animal experiments were conducted at the weizmann institute of science and approved by the weizmann institutional animal care and use committee (iacuc permit no. 01190107-4). 12week-old c57 black female mice were injected ip with 50 mg/kg of s.aureus lta and 800 mg/kgof d-galactosamine in pbs, as previously described [13] , in combination with 5 mg/kg of gp41-tmd or mutated gp41-tmd or pbs, and monitored for survival for 36 hours. blood samples were collected for tnfa and il-6 serum levels measurements. serum was extracted by blood coagulation in rt followed by spin down centrifugation. serum samples were analyzed by elisa assays. spleen and liver samples were collected either post mortem or from sacrificed mice 36 hours after the injection of lta/gln and kept in formaldehyde (4%) for 18 hours and embedded in paraffin blocks. 4 mm sections were then prepared and stained with hematoxylin and eosin by standard protocols. peptides were synthesized by a 9-fluorenylmethoxylcarbonyl (fmoc) solid-phase method on rink amide mbha resin (calbiochem-novabiochem, san diego, california) by using an abi 433a automatic peptide synthesizer (applied biosystems, foster city, ca). peptide synthesis was followed by cleavage from the resin by incubation for 3 h with 95% tfa, 2.5% h 2 o, and 2.5% triethylsilane, followed by purification by rp-hplc (.98%) on a vydac c4 column (grace discovery sciences, deerfield, il), and then identification by electro-spray mass spectroscopy. the following fluorophores were used for fluorescent labeling: 4-fluoro-7-nitrobenzofurazan (nbd, biochemika) and 5(6)-carboxytetramethylrhodamine n-succinimidyl ester (tamra, bio-chemika). peptides used for in vivo experiments were treated twice with 20% acetic acid in order to replace the trifluoroacetate anion added during hplc purification. text s1 this file contains table s1 and figures s1-s5. figure s1 . this figure shows that gp41 tmd disrupts the interaction between tlr2 and tlr6 as revealed by fluorescence resonance energy transfer (fret) measurements. in this experiment the nbd-labeled tlr2 tmd peptide was added first from a stock solution in dmso (final concentration 0.1 mm and a maximum of 0.25% (v/v) dmso) to a dispersion of pc:chol luvs (100 mm) in pbs. this was followed by the addition of the following: a. rhodamine labeled tlr 6 tmd or in addition with un labeled wt gp41 (b), in sequential doses ranging from 0.01 mm to 0.1 mm (stock in dmso), generating a ratio of 1:10, 1:5, 1:2 and 1:1 rhodamine:nbd presented from top to bottom. in both graphs, the upper spectrum represents the emission of the nbd-labeled peptide alone. (c) ip of tlr2 from raw264.7 cells in the presence of the indicated rho-labeled peptides. the result of the last lane from the marker shows that in the presence of wt gp41 there is a decrease in the binding of the tlr6-tmd peptide to the tlr2 protein. figure s2 . this figure shows the secondary structure of the peptides used in this study as measured by cd spectroscopy. spectra were measured at 10 mm in a 1% lpc solution. graphs are the mean of 3 measurements. figure s3 . this figure shows that treatment with gp41 wt peptide rescues mice organs form inflammatory related damage. in this experiment tissue samples of liver (a) and spleen (b) from mice taken at the experimental end point. in both tissues inflammation is evident by the tissue color. figure s4 . this figure shows sequence alignment of the tmds of gp41 (top) and tlr2 (bottom). arrows indicate the predicted tmd. underline indicates the localization motif to cholesterol enriched regions within the membrane. figure s5 . monocyte-derived macrophages and myeloid cell lines as targets of hiv-1 replication and persistence immune evasion and counteraction of restriction factors by hiv-1 and other primate lentiviruses hiding in plain sight: how hiv evades innate immune responses hiv impairment of immune responses in dendritic cells human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells pathogen recognition receptors: ligands and signaling pathways by toll-like receptors sensing of microbial molecular patterns by toll-like receptors tlr2: cellular sensor for microbial and endogenous molecular patterns toll-like receptors linking innate and adaptive immune response toll-like receptor signalling crystal structure of the tlr1-tlr2 heterodimer induced by binding of a tri-acylated lipopeptide activating immunity: lessons from the tlrs and nlrs assembly of the tlr2/6 transmembrane domains is essential for activation and is a target for prevention of sepsis activation of toll-like receptor 2 increases macrophage resistance to hiv-1 infection dendritic cells from hiv-1 infected individuals are less responsive to toll-like receptor (tlr) ligands mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling hiv-1 broadly neutralizing antibody extracts its epitope from a kinked gp41 ectodomain region on the viral membrane toll-like receptor 2 on inflammatory monocytes induces type i interferon in response to viral but not bacterial ligands hiv-1 gp41 and tcralpha trans-membrane domains share a motif exploited by the hiv virus to modulate t-cell proliferation motifs of two small residues can assist but are not sufficient to mediate transmembrane helix interactions tolllike receptor (tlr) 2-9 agonists-induced cytokines and chemokines: i. comparison with t cell receptor-induced responses transmembrane domains interactions within the membrane milieu: principles, advances and challenges analyzing oligomerization of individual transmembrane helices and of entire membrane proteins in e. coli: a hitchhiker's guide to gallex the spleen as a target in severe acute respiratory syndrome neuronal accumulation of glucosylceramide in a mouse model of neuronopathic gaucher disease leads to neurodegeneration specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain computational design of peptides that target transmembrane helices lipid raft distribution of cd4 depends on its palmitoylation and association with lck, and evidence for cd4-induced lipid raft aggregation as an additional mechanism to enhance cd3 signaling location, location, location: is membrane partitioning everything when it comes to innate immune activation? grampositive bacteria enhance hiv-1 susceptibility in langerhans cells, but not in dendritic cells, via toll-like receptor activation biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160 cd4(+) t-cell depletion in hiv infection: mechanisms of immunological failure signaling through toll-like receptors triggers hiv-1 replication in latently infected mast cells the cholesterol-binding motif of the hiv-1 glycoprotein gp41 regulates lateral sorting and oligomerization characterization of the interacting domain of the hiv-1 fusion peptide with the transmembrane domain of the t-cell receptor gallex, a measurement of heterologous association of transmembrane helices in a biological membrane we thank dr. ori brenner of the weizmann institute of science for his help in analysis of the histological specimens and their interpretation. we also thank dr. avraham ashkenazi and mr. yoel klug for their critical revision of the manuscript and fruitful inputs. key: cord-008530-yni0poh9 authors: asensio, valerie c.; campbell, lain l. title: chemokines and viral diseases of the central nervous system date: 2004-01-07 journal: adv virus res doi: 10.1016/s0065-3527(01)56006-6 sha: doc_id: 8530 cord_uid: yni0poh9 this chapter discusses chemokines and their receptors in the evolution of viral infectious diseases of the central nervous system (cns). infection of the human cns with many different viruses or infection of the rodent cns induces vigorous host-inflammatory responses with recruitment of large numbers of leukocytes, particularly t lymphocytes and macrophages. chemokines coordinate trafficking of peripheral blood leukocytes by stimulating their chemotaxis, adhesion, extravasation, and other effector functions. in view of these properties, research efforts have turned increasingly to the possible involvement of chemokines in regulating both peripheral tissue and cns leukocyte migration during viral infection. the biological effects of chemokines are mediated via their interaction with receptors belonging to the family of seven transmembrane (7tm)-spanning, g-protein coupled receptors (gpcrs). in the normal mammalian cns, the number of leukocytes present in the brain is scant. however, these cells are attracted to, and accumulate in, a variety of pathologic states, many involving viral infection. although leukocyte migration into local tissue compartments, such as the cns, is a multifactorial process, it has become clear that chemokines are pivotal components of this process, providing a necessary chemotactic signal for leukocyte recruitment. the central nervous system (cns) has been viewed as a relatively immune privileged organ. in physiologic states, the cns contains few, if any, leukocytes, and an absence of professional antigen-presenting cells (apc); it is barren for the expression of key immune accessory molecules such as major histocompatibility molecules (mhcs), and is fortified by an effective blood-brain barrier. nevertheless, the past decade has witnessed a revision of this concept, as evidenced by the fact that activated t lymphocytes, regardless of their antigen specificity, migrate effectively through the blood-brain barrier (bbb), but also by the fact that the cns contains numerous resident cells, such as microglia, cerebral endothelial cells, and astrocytes, that can, under the appropriate conditions, acquire the expression of immune accessory molecules and may function as apc (hickey, 1999; owens et al., 1994) . significantly, in numerous pathological conditions, immune cells are readily recruited to and accumulate in the cns. infection of the human cns with many different viruses (e.g., human t cell leukemia virus type i [htlv-i]; measles virus, human immunodeficiency virus [hiv] type 1; and herpes simplex virus [hsv] type 2), or of the rodent cns (e.g., theiler's murine encephalomyelitis virus [tmev] , mouse hepatitis virus [mhv] , lymphocytic choriomeningitis virus [lcmv] and vesicular stomatitis virus) induces vigorous host inflammatory responses with recruitment of large numbers of leukocytes, particularly t lymphocytes and macrophages. this host response can be a two-edged sword that, on the one hand, is needed to control and extinguish the invading virus but, on the other, can produce immune pathology and tissue injury resulting in mental and physical debilitation, and often death, of the host organism. therefore, increasing our knowledge of the mechanisms that control the trafficking of immune cells into the cns, and knowledge of the subsequent interactions between these cells that contribute to cns damage, is an important objective. our understanding of the factors that govern leukocyte trafficking has been advanced considerably with the discovery of a large superfamily of mostly small proteins (termed chemokines) that function as leukocyte chemoattractants in immunoinflammatory states (for reviews, see bacon and oppenheim, 1998; rollins, 1997; taub and oppenheim, 1994) . chemokines coordinate trafficking of peripheral blood leukocytes by stimulating their chemotaxis, adhesion, extravasation, and other effector functions. in view of these properties, research efforts have turned increasingly to a focus on the possible involvement of chemokines in regulating both peripheral tissue and cns leukocyte migration during viral infection. however, it is clear that the scope of chemokine involvement in the pathogenesis of host/viral interactions extends well beyond leukocyte migration. with studies on hiv-1 in the vanguard, new roles for chemokines and their receptors in viral infection became apparent with the demonstration that certain classes of chemokine receptors functioned as essential coreceptors for hiv-1 binding and entry into the cell. yet more recent work has indicated that the genomes of some large viruses, belonging to the herpesvirus and poxvirus families, contain homologs of the mammalian chemokines and their receptors. these homologs may function to modulate local inflammatory responses and favor viral survival and spread. from the host perspective, accumulating evidence indicates that chemokines are plurifunctional molecules that may have a significant impact on the cns, regulating cellular communication in the developing and the normal adult cns (asensio and campbell, 1999) . here, we review the subject of chemokines and their receptors in the evolution of viral infectious diseases of the cns. as a prelude to the chemokines. atac, activation-induced chemokine-related molecule exclusively expressed in cd8 ÷ t lymphocytes; bca-1/blc, b cell-attracting chemokine/b-lymphocyte chemoattractant; gro, growth-regulated oncogene; il-8, interleukin-8; ip-10, inflammatory protein 10 kd; i-tac, interferon-inducible t cell alpha-chemoattractant; lix, lps-induced cxc chemokine; mcp, monocyte chemotactic protein; mig, monokine induced by interferon-gamma; mip, macrophage inflammatory protein; mrp-1, miprelated protein 1; pbp, platelet basic protein; pf4, platelet factor 4; rantes, regulated on activation-normal t cell expressed and secreted; sdf-1, stromal cell-derived factor-1; slc, secondary lymphoid tissue chemokine; chr., chromosome. main subject of this chapter, our discussion initially will provide the reader with a general background to the rapidly expanding area of chemokines and their receptors and the expression of these molecules in the cns. the chemokines are currently divided into 4 families ( fig. 1) : the cxc or ~ family; the cc or ~ family; the c or 7 family; and the cx3c or 5 family (zlotnik et al., 1999) . in general, within each chemokine subfamily, the individual members show considerable homology in their 130 val]~rie c. asensio and iain l. campbell amino acid sequence and often possess overlapping chemoattractant specificity. chemokines share a common three-dimensional structure including an nh2-terminal loop and three antiparallel ~ sheets that are followed by a c-terminal a helix. the amino acid backbone of chemokines belonging to the cc, cxc, and cx3c families contains four conserved cysteines, while the c family chemokine, lymphotactin, contains only two cysteines, which correspond to the first and third cysteines in the other groups. chemokines can be produced by a variety of immune cells such as t lymphocytes, macrophages, and natural killer (nk) cells, but also by organ-specific cell types such as resident glial cells in the cns (see the discussion below). depending on the chemokine and its cellular source, the expression of chemokines may be constitutive or inducible, and a new classification of chemokines, defined according to their expression pattern, has been proposed (mantovani, 1999a; sallusto et al., 1999a) . the regulation of chemokine expression can be mediated by soluble factors such as inflammatory cytokines, e.g., interleukin (il)-i, tumor necrosis factor (tnf)-a, interferon (ifn)-7, as well as by microbial products, e.g., bacterial lipopolysaccharide (lps) or hiv coat protein gp120 (baggiolini, 1998; baggiolini et al., 1997; premack and schall, 1996; strieter et al., 1996) . in contrast, counterregulatory cytokines can downregulate the production of chemokines. for example, il-10 and transforming growth factor (tgf)-~ have been shown to inhibit lps-induced microglial cell rantes mrna expression and protein release (hu et al., 1999) . the glucocorticoid dexamethasone is also a particularly potent inhibitor for cc and cxc chemokine production (tobler et al., 1992; villiger et al., 1992) . the biological effects of chemokines are mediated via their interaction with receptors belonging to the family of seven transmembrane (7tm)-spanning, g-protein-coupled receptors (gpcrs). all chemokine receptors contain two conserved cysteines, one in the nh2-terminal domain and the other in the third extracellular loop; these are assumed to form disulfide bonds critical for the formation of the ligand binding pocket. in accordance with the chemokine classification, the receptors are divided into four major groups, cxcr, ccr, xcr, and cx3cr (table i) . individual chemokine receptors may often be promiscuous, meaning that they are capable of binding several different chemokines, and conversely, individual chemokines can often bind to several different receptors. interaction between a chemokine and its receptor leads to generation of a coordinated series of signaling events such as mobilization and influx of ca 2+ and also activation of various signal transduction pathways (premack and schall, 1996) . lymphotactin a chemokines in boldface type are cross-subfamily ligand-binding receptors. bca-1/blc, b cell-attracting chemokine/b-lymphocyte chemoattractant; elc, epstein-barr virus-induced receptor ligand chemokine; ena-78, epithelial cell-derived neutrophil-activating factor-78; gcp-2, granulocyte chemoattractant protein-2; gro, growth-regulated oncogene; il-8, interleukin-8; ip-10, inflammatory protein 10 kd; i-tac, interferoninducible t cell alpha-chemoattractant; larc, liver-and activation-related chemokine; lix, lps-induced cxc chemokine; mcp, monocyte chemotactic protein; mig, monokine induced by interferon-gamma; mip, macrophage inflammatory protein; mrp-1, miprelated protein 1; pbp, platelet basic protein; pf4, platelet factor 4; rantes, regulated on activation-normal t cell expressed and secreted; sdf-1, stromal cell-derived factor-l; slc, secondary lymphoid tissue chemokine; teck, thymus-expressed chemokine. protein 10 kda); and mig (monokine induced by interferon-y), (see fig. 1 ). the presence or absence of an elr motif (glutamic acid-leucinearginine sequence) preceding the first cysteine further subdivides the cxc chemokines into elr or non-elr groups. examples of some common elr chemokines include il-8, gro-a, and mip-2, while non-elr chemokines include ip-10, mig and sdf-1. this grouping not only reflects a structural compartmentalization, but also a functional one in that elr cxc chemokines are predominantly chemoattractant for polymorphonuclear leukocytes and bind to the cxcr1 and cxcr2 receptors, while non-elr chemokines are chemoattractant for t and b cells and bind to the cxcr3 or cxcr4 receptors. in addition, members of the cxc chemokine family can either promote or inhibit anglogenesis, depending on whether they are elr or non-elr cxc chemokines, respectively. the elr-chemokines are generally produced during inflammatory responses provoked by bacterial infections. on the other hand, production of the non-elr chemokines, ip-10/crg-2 and mig, is commonly associated with viral infections where they likely function to recruit t cells and macrophages that predominate at sites of infection (amichay et al., 1996; asensio and campbell, 1997; asensio et al., 1999a; carr et al., 1998; charles et al., 1999; fisher et al., 1995; lahrtz et al., 1997; nazar et al., 1997; vanguri and farber, 1994) . this scenario is borne out by the recent demonstration of pronounced antiviral activity of mig and ip-10 when it is expressed ectopically by recombinant vaccinia viruses (mahalingam et al., 1999) . the antiviral actions of mig and ip-10 in this setting are mediated indirectly and require nk cells and ifn-a, ifn-~, and ifn-y. sdf-1 was originally isolated from bone marrow stromal cells as a pre-b cell growth-stimulating factor. in contrast to ip-10 and mig, sdf-1 is produced constitutively in many organs (e.g., heart, lung, and cns) and binds to the cxcr4 receptor. animals lacking sdf-1 or its receptor invariably die at or soon after birth and have major abnormalities affecting the immune system (an impairment in b lymphocyte development) as well as various organs including the heart and cns nagasawa et al., 1996; tachibana et al., 1998; zou et al., 1998) . these studies revealed that sdf-1 is a crucial mediator of cellular migration and patterning during organogenesis. to date, 5 cxc chemokine receptors have been described and their ligands identified (see table i ) (baggiolini et al., 1997; mackay, 1997; moser et al., 1998) . most of the early characterization of the cxc chemokine receptors was done with the il-8 receptors cxcr1 (also known as il-8ra) and cxcr2 (also known as il-8rb). cxcr2 is expressed on a variety of cells including t cells, monocytes, melanoma cells, synovial cells, neutrophils, and myeloid precursor cells. expression of cxcr1 is restricted largely to myeloid cells and neutrophils. while cxcr2 binds all the elr-chemokines, cxcr1 binds only il-8. mice lacking cxcr2 show normal development of neutrophils but have decreased numbers of myeloid progenitors (broxmeyer et al., 1996; cacalano et al., 1994) . cxcr2-deficient mice do not exhibit any other overt pathologic abnormalities. cxcr3 is the receptor for several of the non-elr cxc chemokines, ip-10, mig and i-tac. surprisingly, some cc chemokines such as eotaxin, mcp-4 (weng et al., 1998) , and 6ckine (also called exodus 2 or slc) also bind to cxcr3. through this interaction, eotaxin does not activate the cxcr3 receptor but effectively blocks ip-10 binding and prevents receptor activation. thus, these cc chemokines may serve as natural cxcr3 antagonists highlighting complexity in the regulation of chemokine actions. human cxcr3 has been shown to be produced specifically by il-2-activated cd4 ÷ t cells, b cells, and nk cells. cd8 ÷ t cell and nk cell clones have also been shown to express the cxcr3 receptor . this suggests non-elr chemokines such as ip-10 may exert a selective influence on thl and cytotoxic lymphocyte and nk cell recruitment, which may be important in the development of innate and adaptive immune responses to viral infections. cxcr4 and cxcr5 bind the other non-elr chemokines sdf-1 and bca, respectively. cxcr4, also known as lestr or fusin, serves as a major coreceptor for lymphotropic strains of hiv-1 (deng et al., 1996; feng et al., 1996) and is expressed in a variety of tissues including brain, heart, liver, and colon and in nonhematopoietic cells (lavi et al., 1997; ohtani et al., 1998) . cxcr4 mrna expression is also found in murine lymphocytes, macrophages, neutrophils, and glial cells . as indicated above, disruption of the cxcr4 gene causes defects in vascular development, hematopoiesis, cardiogenesis, and neurogenesis of the cerebellum. cxcr5, originally described as blr-1 (burkitt's lymphoma receptor-l), is highly related to the il-8 receptors (dobner et al., 1992) . cxcr5 mrna is expressed by b cell lymphomas and by granule and purkinje cells of the cerebellum. therefore, in addition to regulation of recirculating mature b lymphocytes (forster et al., 1996; forster et al., 1994; kaiser et al., 1993) , this receptor may be involved in some cns functions. however, mice lacking cxcr5 show significant phenotypic changes in lymphoid organs but not in the brain, suggesting that this receptor, unlike cxcr4, does not play an obligatory role in cns development (forster et al., 1996) . the cc or ~-chemokines contain the largest number of members and are so named because the nh2-terminal cysteines are adjacent to each other. individual cc chemokines may exert their actions on multiple leukocyte subtypes, including monocytes, basophils, eosinophils, t cells, dendritic cells, and natural killer cells. well-known members of this family include mcp-1, mip-la, c10, eotaxin, and rantes. a more comprehensive list of the members of this family can be found in fig. 1 . one of the most studied chemokines of this family is mcp-1, which attracts t cells and monocytes but not neutrophils (carr et al., 1994; matsushima et al., 1989) . mice with targeted disruption of the mcp-1 gene have defects in monocyte/macrophage recruitment in a variety of inflammatory and immunological models. conversely, transgenic mice with expression of mcp-1 targeted to pancreatic islets (grewal et al., 1997) or oligodendrocytes in the brain (fuentes et al., 1995) show an accumulation of monocytes at these sites. interestingly, these accumulating monocytes showed little evidence of activation, indicating that mcp-1 is an effective monocyte chemoattractant but lacks the ability to further activate these cells. another well-studied cc chemokine is mip-la, which induces migration of monocytes, t lymphocytes, and eosinophils (baggiolini et al., 1994) . mice with a targeted disruption of mip-la show no obvious hematopoietic abnormalities; however, they are resistant to coxsackievirus-induced myocarditis and clear influenza at a delayed rate (cook et al., 1995) . to date, 9 cc receptors (ccr1-ccr9) have been identified (table i) . ccr1 was originally designated as an mip-la/rantes receptor (gao et al., 1993; gao and murphy, 1995; neote et al., 1993) and was later shown to also bind mcp-2 and mcp-3 (benbaruch et al., 1995) . the ccr1 receptor is mainly found on circulating mononuclear cells. mice lacking ccr1 (receptor for mip-la, mip-5, mcp-2, mcp-3, and rantes) have a phenotype similar to that of mip-la deficient mice with a defect in inflammatory responses to microbial challenge such as coxsackie b and influenza viruses (gao et al., 1997) . in addition, these animals have impaired trafficking of subsets of myeloid progenitor cells. the primary receptor for mcp-1, ccr2, is expressed on monocytes, myeloid precursor cells, activated t lymphocytes, and b lymphocytes but not on neutrophils (bonecchi et al., 1999; frade et al., 1997; myers et al., 1995) . mice with targeted disruption of the ccr2 receptor have defects in monocyte/macrophage recruitment in a variety of inflammatory and immunological models (boring et al., 1998; kurihara et al., 1997; kuziel et al., 1997) . ccr3, which is the eotaxin receptor, is prominently expressed by eosinophils and is involved in the recruitment of these cells during allergic reactions. ccr4 is the main receptor for rantes and mip-la, while ccr5, expressed on monocytes/macrophages, t cells, and granulocyte precursors (alkhatib et al., 1996; deng et al., 1996) , binds mip-la, mip-1~, and rantes. the receptors ccr6 and ccr7, originally identified as orphan receptors, bind mip-3a, and mip-3~ and 6ckine respectively yoshida et al., 1997) . ccr8 binds 1-309 (goya et al., 1998) , tarc, and mip-i~ and is expressed in the thymus (zingoni et al., 1998) . ccr9, also designated as gpr-9-6, was shown to specifically bind teck (zaballos et al., 1999) . compartmentalization of cc chemokine receptor expression is emerging as an important regulatory component during the adaptive immune response. ccr5, the receptor for rantes, mip-i~, and mip-1~ is expressed by memory and activated t cells but not by naive t cells (bleul et al., 1997) . moreover, ccr5 is preferentially expressed by human thl cells, in contrast to ccr4 and ccr3, which are found predominantly on th2 cells (bonecchi et al., 1998; qin et al., 1998) . concomitant with their differential chemokine receptor expression, thl and th2 cells selectively migrate in response to the corresponding chemokine ligand. more recently, sallusto et al. showed that memory t cells can be distinguished with respect to their effector function in peripheral organs by their expression of the chemokine receptor ccr7 (sallusto et al., 1999b) . ccr7 ÷ memory t cells retain lymph node homing receptors and lack immediate effector function, whereas ccr7memory t cells express receptors for migration to inflamed tissues and possess effector function. ccr8, like ccr4 (bonecchi et al., 1998) , is preferentially expressed by activated th2 cells. ccr8 may be important in lymphocyte development as well as in th2-immune responses. polarized th2 cells have been shown to differentially express the chemokine receptors, ccr8 (zingoni et al., 1998) , ccr4 (bonecchi et al., 1998; sallusto et al., 1999a) and ccr3 (sallusto et al., 1997) . in the murine system, a recent study showed that thl and th2 cells expressed comparable levels of ccr1, ccr2, and ccr4 mrna. similar to human t helper cells, murine thl cells preferentially expressed ccr7 and ccr5, whereas th2 cells expressed more ccr3 (randolph et al., 1999) . in conclusion, depending on their antigenic stimulation and/or polarization toward thl versus th2 subsets, chemokine receptors are differentially expressed, which, in addition to their characteristic cytokine profiles, may thus further distinguish these cells (mantovani, 1999b ). lymphotactin, the lone member of the c-chemokine family, is produced by activated mouse t cells (kelner et al., 1994) . lymphotactin is unique because (1) it has only two cysteines, the second and the fourth, of the four cysteines conserved in the cxc, cc, and cx3c chemokines; and (2) the c-terminal sequence is much longer than those of other chemokines. lymphotactin, and the subsequently reported single motif cysteine (smc) chemokine-1 and the activation-induced, t-cell-derived, and chemokine-related molecule (atac), are all identical (muller et al., 1995; yoshida et al., 1995) . lymphotactin expression on activation is extremely rapid, which suggests that it has a role in the early phases of an inflammatory response. when lymphotactin is injected into peritoneum, it causes an influx of t lymphocytes and nk cells by 24h (hedrick et al., 1997) . yoshida et al. (1998) identified an orphan receptor, gpr5 (heiber et al., 1995) , as being a high-affinity functional receptor for lymphotactin this receptor was renamed xcr1. the single member of the cx3c or 8 family, named fractalkine, is also unique with a novel arrangement of three amino acids separating the first two conserved cysteines. furthermore, fractalkine exists in both soluble and membrane-bound forms (bazan et al., 1997; pan et al., 1997) . this chemokine is found on activated endothelial cells and is tethered to the membrane by a mucinlike stalk that suggests a novel role for this chemokine in juxtacrine signaling. the interaction of endothelial-cell-expressed fractalkine with its receptor, cx3cr1, on leukocytes mediates the initial capture, firm adhesion, and activation of circulating leukocytes independently of integrin or adhesion molecule involvement (fong et al., 1998) . thus, fractalkine not only induces the migration of leukocytes but also facilitates their adhesion directly to the endothelium at sites of inflammation. fractalkine has been shown to foster the migration of monocytes, t cells, and nk cells (hedrick et al., 1997; kelner et al., 1994) . the receptor for fractalkine was originally cloned from a rat brainstem cdna library and named rbsll (harrison et al., 1994) . subsequently, the human receptor was identified as v28 (raport et al., 1995) , or cmkblr1, which is highly expressed in the brain and in neutrophils, monocytes/macrophages, and t lymphocytes (combadiere et al., 1995) . imai et al. (1997) identified this molecule as being the fractalkine receptor (cx3cr1) that is expressed mainly by nk cells and monocytes and to a lesser extent in cd8 ÷ t cells. many of the large dna viruses are infamous for their ability to undermine host immunity. two genres of viruses, the herpesviruses and poxviruses, were recently shown to contain open reading frames (orfs) that encode homologs of mammalian chemokines and chemokine receptors (for detailed reviews, see ahuja et al., 1994; dairaghi et al., 1998; lalani et al., 2000; murphy, 1994; smith et al., 1997) . kaposi's sarcoma-associated herpes virus (kshv) encodes three cc-like chemokines, vmip-i, vmip-ii, and vmip-iii. the vmip-i and vmip-ii show 60% sequence identity to each other and 43% and 52% identity to mip-la, while vmip-iii is more distantly related to its viral and mammalian counterparts (stine et al., 1999) . of the three viral chemokines, the function of vmip-ii is the best characterized. receptor binding studies suggest vmip-ii is a ligand for ccr3 (boshoff et al., 1997) . in addition to being a potent inhibitor of hiv infection mediated principally through this receptor (boshoffet al., 1997), vmip-ii is able to mobilize calcium and stimulate the chemotaxis of eosinophils where ccr3 is predominantly expressed (boshoffet al., 1997) . in contrast to this agonist action on ccr3, vmip-ii also binds with a high degree of affinity to a number of other cc (ccr1, 2, 5) and cxc chemokine receptors (cxcr4), where it acts as an antagonist (kledal et al., 1997) , as well as to cx3cr1 . human monocyte chemotaxis induced by rantes, mip-i(~, and mip-i[~ is inhibited by vmip-ii, suggesting that this viral mediator may be a broad-spectrum chemokine antagonist. recently, vmip-i was shown to selectively bind to ccr8 (a cc chemokine receptor associated with th2 lymphocytes), acting as an agonist and stimulating ca 2+ mobilization on human t cells . interestingly, vmip-ii also binds to ccr8; however, this is associated with antagonistic actions . kaposi's sarcoma is an angioproliferative disorder, so it is interesting that in addition to deviation of the host chemokine response, vmip-i and vmip-ii both induce angiogenesis in a chick chorioallantoic assay (boshoff et al., 1997) . thus these viral chemokines might contribute directly to the pathogenesis of the proliferative angiopathy in kaposi's sarcoma. in addition to these chemokine homologs, herpesviruses also contain open reading frames (orfs) that share significant sequence conservation with the chemokine receptors. kshv and herpesvirus saimiri (hvs) contain orf 74 in hsv, whose sequence is most similar to the human il-8 receptors cxcr1 and cxcr2 (ahuja and murphy, 1993) . like cxcr1 and cxcr2, orf 74 binds il-8 as well as the other elr cxc chemokines, gro-(z and pf-4 (ahuja and murphy, 1993) . however unlike cxcr1 and cxcr2, orf 74 can signal in a constitutive agonist-independent manner and stimulate the proliferation of kidney fibroblasts (arvanitakis et al., 1997) . in addition to the stimulation of proliferation of nontransformed cells, orf 74 signaling may also lead to cellular transformation and tumorigenicity (bais et al., 1998) . thus, it would appear that orf 74 may contribute to the development of the transformed cell phenotype found with kshv-infected cells in kaposi's sarcoma lesions. cytomegalovirus (cmv) is a common opportunistic pathogen of immunocompromised hosts in whom the virus exhibits tropism for leukocytes. human and murine cmv possess genes that encode a variety of chemokine homologs (macdonald et al., 1997) . the murine cmv chemokine 1 and 2 (mck-1 and mck-2) peptides induce calcium signaling and adherence in peritoneal macrophages (saederup et al., 1999) . the human cmv gene ul146 encodes a protein designated as vcxc-1 and provides the first example of a virus-encoded cxc chemokine (penfold et al., 1999) . the vcxc-1 glycoprotein shows high-affinity binding to the cxcr1 and cxcr2 chemokine receptors, inducing calcium mobilization and chemotaxis of neutrophils. thus, the common property of the cmv chemokine homologs is to function as chemokine agonists. this in turn may promote leukocyte migration to sites of infection and may be responsible for more efficient dissemination of cmv in the host. in support of this notion, mck-1/mck-2 mutant cmv displays markedly reduced peak levels of monocyte-associated viremia in experimentally infected mice (saederup et al., 1999) . a gene encoding a viral homolog of ccr1 was found within the genome of the human cmv. this gene, orf us28, of human cytomegalovirus (hcmv) was shown to bind mip-la,-~, mcp-1, rantes , and mcp-3 (bodaghi et al., 1998) with higher affinity than their cognate receptors. thus, it is attractive to speculate that hcmv-infected cells expressing us28 may be used for sequestration of chemokines from the extracellular milieu. us28 can also serve as a coreceptor for hiv infection of cd4 t cells and may facilitate an interplay between hiv and hcmv (pleskoffet al., 1997) . the poxvirus molluscum contagiosum virus (mcv) infects humans, causing papules of the skin associated with virtually no immune response to the virus in the skin lesion. the genome of mcv contains an orf that encodes a protein (mc148r) that is a cc chemokine homolog (senkevich et al., 1996) . mc148r has homology to mip-la/[3 but does not induce chemotaxis and antagonizes mip-la-induced chemotaxis (krathwohl et al., 1997) . furthermore, mc148r inhibits the leukocyte response to several cc and cxc chemokines (damon et al., 1998) , indicating that it can act as a broad-spectrum chemokine antagonist. therefore, a primary function of this virus-encoded chemokine homolog might be to provide an immune evasion strategy that acts to limit the recruitment of effector leukocytes to mcvinfected epidermal cells. in conclusion, unquestionably these large dna viruses encode functional homologs of chemokines and chemokine receptors. although the precise role of the viral chemokine and chemokine receptor homologs (discussed above) in infectious processes remains to be determined, they clearly could contribute to the strategies employed by these viruses to subvert host immunity. the examples discussed here indicate that these strategies are diverse and, depending on the virus, range from wholesale suppression of leukocyte trafficking--providing immune evasion--to promoting selective leukocyte chemotaxis fostering the more efficient infection and dissemination of virus via its preferred target host cell. a concerted research effort has been mounted recently to ascertain the role of chemokines in immunoinflammatory disorders of the cns, such as multiple sclerosis (ms). as a result, it has become clear that most cells resident in the cns, including neurons, astrocytes, and microglia, can synthesize and secrete various classes of chemokines. importantly, these same cells are endowed with a variety of different chemokine receptors and therefore have the potential to respond to chemokines in their local environment. as recently speculated by us (asensio and campbell, 1999) and by others (mennicken et al., 1999) , it is likely the cns has its own chemokine ligand/receptor network, the function of which could extend well beyond the regulation of leukocyte trafficking in antiviral and other neuroimmune responses (see fig. 3 ). at present, the genes for only three chemokines, mcp-1, sdf-1, and fractalkine, are known to be expressed constitutively in the cns. evidence suggests mcp-1 is expressed in the cortex and hippocampus of the rat brain during development as well as in the adult animal (pousset, 1994) . more recently, mcp-1 expression at the protein level was shown in the human cerebellum, medulla oblongata, and pons (meng et al., 1999) . however, the function of constitutively expressed mcp-1 in the brain is unknown. the sdf-1 gene is found under physiological conditions in many organs including murine (tashiro et al., 1993) and human (shirozu et al., 1995) brain. sdf-1 has two isoforms, sdf-la and sdf-i~, that are produced by alternative splicing from a single gene (tashiro et al., 1993) . sdf-la rna is present at high levels in cultured rat astrocytes, at low levels in neurons, and is not detectable in microglia, while sdf-1~ rnais found at low levels in all these cell types . tanabe et al. (1997b) showed that sdf-la was able to induce migration of microglial cells but not astrocytes, even though the sdf-1 receptor, cxcr4, was expressed on both cell types. furthermore, human sdfla was able to induce calcium flux in cultured astrocytes (bajetto et al., 1999) . it was hypothesized that astrocyte-secreted sdf-1 could facilitate interneural cell communication . this is certainly the case during cns development, since, as noted above, knocking out cxcr4 results in a major defect in granule cell migration and cerebellar development zou et al., 1998) . the cx3c chemokine fractalkine is also found constitutively in the brain (bazan et al., 1997; pan et al., 1997) . fractalkine mrna is expressed at high levels by neurons in the olfactory bulb, cerebral cortex hippocampus, caudate putamen, and nucleus accumbens nishiyori et al., 1998; schwaeble et al., 1998) . in vitro studies have confirmed that fractalkine is constitutively expressed by neurons and, further, can be induced in astrocytes by tnf-a and il-i~ (maciejewski-lenoir et al., 1999) . in a model of peripheral nerve injury, harrison et al. have shown that fractalkine and its receptor were highly upregulated in the brain, with motor neurons being the primary source of the chemokine. fractalkine treatment of primary cultured rat microglial cells induces vigorous increases in intracellular ca 2+ levels and chemotaxis, which are inhibited by an antibody against the fractalkine (cx3cr1) receptor . although the precise function of fractalkine in the cns awaits clarification, the localization of the fractalkine receptor on microglia and the expression of its ligand by neurons clearly establish a spatial framework that could facilitate juxtacrine communication and migration between these cells (see fig. 3 ) nishiyori et al., 1998) . a majority of chemokines are not detectable in the cns under physiological conditions, but are present during diverse pathologic states including, as discussed below, viral diseases. it is beyond the scope of this chapter to detail all of the findings in these different pathologic states and the reader is encouraged to consult many excellent recent reviews dealing with this subject (asensio and campbell, 1999; glabinski and ransohoff, 1999; karpus and ransohoff, 1998; mennicken et al., 1999; ransohoff, 1997; ransohoff and tani, 1998) . it is clear from most, if not all, cases examined that glial cells represent an important source for the localized production of specific chemokines. for example, in ms, expression of ip-10 (balashov et al., 1999; sorensen et al., 1999) and mcp-1 (simpson et al., 1998; van der voorn et al., 1999) is prominent in astrocytes while mip-i(~ expression is strongly associated with macrophage/microglia (balashov et al., 1999) . in support of these clinicopathologic findings, studies in vitro demonstrate that expression of various cc and cxc chemokines can be induced in astrocytes and microglia by proinflammatory cytokines and lps. although differential expression of mcp-1 by astrocytes, and of mip-i(z by microglia, was reported for murine glial cells (hayashi et al., 1995) , this was not the case with human microglia, with expression of both these chemokines being induced in these cells by lps (mcmanus et al., 1998) . similarly, expression of ip-10 by murine astrocytes and microglia can be induced by ifn-~/or lps (luo et al., 1998; majumder et al., 1998; ren et al., 1998; vanguri, 1995) . in contrast to the glial cells, few inducible chemokines have been reported to be expressed by neuronal cells either in vitro or in vivo. how does cns chemokine expression contribute to the pathogenesis of neuroinflammatory disease states? clearly, one key role involves recruitment of leukocytes to the brain. differences in the chemokine gene expression patterns can be found in different neuroinflammatory diseases and correlate with a bias toward the involvement of particular leukocytes. in experimental and clinical bacterial meningoencephalitis, where cns lesions consist predominantly of neutrophils and monocytes, there is dominant cerebral production of the neutrophil and monocyte attractant chemokines il-8, mip-2, and groa, and mcp-1, mip-i~, and mip-i~, respectively (lahrtz et al., 1998; spanaus et al., 1997; sprenger et al., 1996) . in contrast, as discussed below, in viral meningoencephalitis, mostly lymphocytes and monocytes are found in the cns, in parallel with the cerebral expression of the chemokine genes encoding ip-10, mcp-1, and rantes, which are effective lymphocyte and monocyte chemoattractants (asensio and campbell, 1997; lahrtz et al., 1997) . transgenic studies show that individual chemokines can have a specific chemoattractant "signature." thus, cns expression of either the groa genes (tani et al., 1996) or the mcp-1 genes (fuentes et al., 1995) under the control of the myelin basic protein (mbp) promoter induces robust leukocyte infiltration of the cns that is composed predominantly of either neutrophils or monocytes, respectively. studies with intracerebral microinjection also demonstrate that the acute presence of chemokines in the cns leads to robust and cell-specific leukocyte recruitment. for example, mip-2 is more potent than il-8, ip-10, or mcp-1 in stimulating polymorphonuval]~rie c. asensio and iain l. campbell clear leukocyte recruitment to the brain (bell et al., 1996) , while the cc chemokine c10, when injected into the lateral ventricle, is able to induce a recruitment of macrophages, but not t cells (asensio et al., 1999b) . finally, neutralization of the chemokine mip-la, which is expressed in eae, is associated with a reduction in disease severity and in cns inflammation (karpus et al., 1995) . in all, these studies indicate that the qualitative makeup of chemokine production in the cns during disease can dictate the recruitment of selected leukocytes to the cns, and that targeting these molecules may provide an effective therapeutic approach to suppress cns inflammation. the cc and cxc receptors are expressed on a number of different cell types and their expression is often regulated by exogenous and endogenous stimuli. numerous in vitro and in vivo studies show that different neural cells express a variety of chemokine receptors under normal conditions and could therefore facilitate a direct interaction of these cells with chemokines. neurons have prominent expression of many chemokine receptors including cxcr2, cxcr4, ccr1, ccr4, ccr5, ccr9/10, cx3cr1, and duffy antigen-related chemokine (darc) (horuk et al., 1997; klein et al., 1999; lavi et al., 1998; meucci et al., 1998; sanders et al., 1998; vallat et al., 1998) . immunohistochemical staining of the human brain revealed cxcr1 is not detectable, whereas cxcr2 is expressed at high levels by subsets of projection neurons in different regions of the brain including the hippocampus, dentate nucleus, pontine nuclei, locus coeruleus, and paraventricular nucleus, and also in the spinal cord (horuk et al., 1996; horuk et al., 1997) . darc is expressed by subsets of endothelial cells and purkinje cells in the cerebellum, suggesting that this enigmatic receptor that lacks signaling function may have multiple roles in normal and pathological physiology, perhaps as a chemokine clearance receptor (horuk et al., 1996; horuk et al., 1997) . functional ccr3, ccr5, and cxcr4 receptors, with different patterns of distribution, were demonstrated on cultured fetal human and macaque neurons (klein et al., 1999) . expression of cxcr4 has also been detected by immunohistochemical staining on subpopulations of neurons in the normal adult brain (lavi et al., 1997; sanders et al., 1998; vallat et al., 1998) , while in the rat, levels of the cxcr4 receptor rna are higher on specific neurons, which include cerebellar purkinje cells, hippocampal hilar neurons, and cerebral cortical neurons (wong et al., 1996) . a clear theme emerging from all these studies is that neurons express a variety of chemokine receptors and that this expression in the cns exhibits marked regional and cellular diversity, suggesting that chemokines could have differential effects on different neurons. other than neurons, glial cells also express a variety of chemokine receptors. the fractalkine receptor cx3cr1 is found at high levels in rodent and human brain and shows strong localization to microglia (combadiere et al., 1998; harrison et al., 1998; imai et al., 1997) . the fact that fractalkine is expressed by neurons (as noted above), and its receptor by microglia, suggests a possible important role for this chemokine and its receptor in communication between neurons and microglia. a similar scenario may follow with sdf-1, whose receptor, cxcr4, in addition to expression by neurons, is found on astrocytes (heesen et al., 1996) and on microglial cells (tanabe et al., 1997b) . finally, the cc chemokine receptors ccr1, ccr5, and ccr3 have been shown to be expressed by astrocytes and/or microglial cells both in vitro and in vivo (boddeke et al., 1999; he et al., 1997; lavi et al., 1998; tanabe et al., 1997a; vallat et al., 1998; westmoreland et al., 1998; xia et al., 1998) . the expression of many of these chemokine receptors in the cns is not static and can be dynamically regulated by various stimuli. for example, in a model of excitotoxic brain injury induced by intracerebral injection of nmda in the rat, marked upregulation of ccr5 expression was observed by microglia and neurons (galasso et al., 1998) . in brain from alzheimer's cases, plaque-associated reactive microglia also show increased ccr5 expression . expression of the fractalkine receptor cx3cr1 is increased on perineural microglia following facial nerve axotomy in the rat . boddeke et al., have shown, by rt-pcr (reverse transcription-polymerase chain rear) analysis, that ccr1, ccr2, and ccr5 expression in cultured rat microglial cells is significantly upregulated after lps treatment (boddeke et al., 1999; spleiss et al., 1998) . the pathophysiological significance of altered cns chemokine receptor expression in disease states remains to be determined. however, it might be speculated that this provides yet another level of regulation in the already complex cellular communication processes mediated by the chemokines. cns infection, with a number of different classes of viruses, can provoke vigorous inflammatory responses with subsequent recruitment of large numbers of leukocytes (for a detailed discussion of this topic, see chapter 6 in this volume). in view of their functional properties, increasing interest has focused on the possible involvement of chemokines in regulating cns leukocyte migration during viral infection. however, as discussed below, studies of hiv-1 have revealed that chemokine involvement in the pathogenesis of host-virus interactions extends well beyond leukocyte migration. the lentivirus hiv-1 and its simian counterpart, simian immunodeficiency virus (siv), both infect the cns and are responsible for causing neurologic disease. in the case of hiv, a vast amount of research effort focused on this virus led to the discovery of new chemokine receptors that proved to be critical for understanding the process of hiv-1 entry into cells (bleul et al., 1996; feng et al., 1996; oberlin et al., 1996) . the interactions between this virus and the host cns have also been studied extensively, and more recently the focus has turned to the chemokines and their receptors. as discussed below, the findings point to a significant role for the chemokines and the chemokine receptors in hiv-1 neuropathogenesis. although cd4 was identified initially as a primary receptor for hiv-1 binding and entry, it was clear this could not account for many of the characteristics of hiv-1 infectivity, such as the existence of different strains of hiv-1 with selective tropism for either t lymphocytes (ttropic) or monocytes (m-tropic). a clue to the identity of other possible cofactors for hiv-1 entry into cells came with the discovery that the chemokines mip-la, mip-i~, and rantes produced by cd8 ÷ t cells could suppress hiv-1 infection of cultured cells (cocchi et al., 1995) . a seminal advance then came when it was shown that in addition to cd4, cxcr4 was required for t-tropic hiv-1 strains to infect host cells (bleul et al., 1996; feng et al., 1996; oberlin et al., 1996) . the identification of ccr5 as a receptor for mip-la, mip-i~, and rantes soon led to the demonstration that ccr5 served as a prominent coreceptor for cell entry by m-tropic strains ofhiv-1 (alkhatib et al., 1996; choe et al., 1996; deng et al., 1996; doranz et al., 1996; dragic et al., 1996) . the factors that govern hiv-1 entry into cells are clearly very complex, and while cxcr4 and ccr5 are the major coreceptors for ttropic and m-tropic hiv-1 strains, a large number of other chemokine receptors including ccr2b, ccr3, and ccr8, the us28 chemokine receptor homolog encoded by cytomegalovirus, as well as the orphan chemokine receptors gprl, gpr15/bob, and strl33/bonzo, can function as coreceptors for hiv-1 infection (deng et al., 1997; heiber et al., 1996; liao et al., 1997; littman, 1998; marchese et al., 1994; pleskoffet al., 1997) . the importance of the chemokine receptors for hiv-1 infection in vivo is illustrated in the case of individuals that are homozygous for a 32 base pair mutation in the ccr5 gene and who have marked resistance toward hiv infection (biti et al., 1997; o'brien et al., 1997; theodorou et al., 1997) . in addition, it has been reported that individuals with mutations in the cxcr4 and ccr2 genes also exhibit a retarded progression of hiv-l-associated disease . a significant number of patients with hiv-1 infection suffer from a variety of neurological problems known collectively as hiv-l-associated cognitive and motor disorder, or "neuroaids" (see chapter 18 in this volume). in its severest form, neuroaids can be responsible for subcortical dementia, memory impairment, and motor disorder. although the neuropathology of neuroaids is often variable, typical features include formation of multinucleated giant cells, diffuse gliosis, myelin pallor, and increased blood-brain barrier permeability. neurodegeneration, with disrupted synaptodendritic organization and loss of neurons, is observed in many but not all cases (everall et al., 1993) . the cause of neuroaids and the mechanisms that lead to brain injury are not well understood, although a combination of both host-and hiv-derived toxic factors is likely to be involved (gendelman et al., 1997) . it is clear, however, that productive hiv-1 infection of the cns is largely restricted to macrophage/microglia and infrequently to neurons or astrocytes. infection of the cns is thought to result from the entry of hiv-l-infected lymphocytes or macrophages--the subsequent presence of hiv-1 proteins in microglia and multinucleated giant cells suggests spread of the virus from the infiltrating infected leukocytes. as discussed above, resident cells of the cns are well endowed with a variety of different chemokine receptors. the levels and type of coreceptot expression are likely to be pivotal determinants of hiv-1 tropism in the brain. the coreceptors for m-tropic variants of hiv-1, ccr5, ccr3, and cxcr4 are expressed by microglia (he et al., 1997; lavi et al., 1997; vallat et al., 1998) . studies with primary cultures of human microglia have shown that ccr5 and ccr3 can serve as coreceptors with cd4 for hiv-1 infection, while cxcr4 is relatively ineffective in this capacity. microglia have very low surface-expressed cd4 (buttini et al., 1998) and this may thus preclude the efficient use of cxcr4 as a coreceptor. hiv-1 infection of microglial cultures can be inhibited by the cognate ligands mip-i~ (ccr5) and eotaxin (ccr3), further verifying the key role of the microglial-expressed cc-chemokine receptors for hiv-1 entry into cells. interestingly, the use of both ccr3 and ccr5 for hiv-1 infection of microglia contrasts with blood-derived macrophages in which ccr5, but not ccr3, functions as a coreceptor (alkhatib et al., 1996; deng et al., 1996; wu et al., 1997) . this suggests that m-tropic hiv-1 variants may arise in the brain that have distinct coreceptor requirements. in support of this, hiv-1 in the brain is typically m-tropic (korber et al., 1994; power et al., 1994) . moreover, he and colleagues (1997) demonstrated that yu2 and jrl env proteins that are cloned directly from hiv-l-infected brain use ccr5 or ccr3, together with cd4, for virus entry into transfected cells. a further determinant of hiv-1 entry and infection in the cns may be the chemokines themselves. increased levels of mip-la, mip-i~, rantes, mcp-1, ip-10, and sdf-1 have been reported in the brain or the csf of individuals with neuroaids (conant et al., 1998; kelder et al., 1998; kolb et al., 1999; letendre et al., 1999; sanders et al., 1998; schmidtmayerova et al., 1996; zheng et al., 1999b) . hiv-1 may directly regulate the expression of these chemokines. thus, infection of cultured microglia and/or astrocytes markedly increases the expression of mip-la and mip-i~ (schmidtmayerova et al., 1996) , while expression of sdf-1 rna decreases in hiv-l-infected microglia but increases in similarly infected astrocytes (zheng et al., 1999b) . in recent unpublished studies, we have observed induction of ip-10 gene expression in astrocytes in the brain of transgenic mice with astrocyte-targeted expression of hivgpl20. these transgenic mice exhibit similar neurodegenerative and other pathologic changes (e.g., astrocytosis) to neu-roaids (toggas et al., 1994) --our findings suggest that a further action of hivgpl20 is the induction of ip-10. the cns expression of ip-10 and other chemokines, such as mcp-1, correlates with viral load and increases with the progression of brain injury, indicating a possible role for these chemokines in the pathogenesis of neuroaids (kelder et al., 1998; kolb et al., 1999) . other than their potential contribution to brain injury (discussed in more detail below), the increased presence of these chemokines in the cns during hiv-1 infection may promote, via their chemoattractant activity, the further recruitment and accumulation of t lymphocytes and macrophages in the brain. in support of this idea, hiv-tat, protein-induced mcp-1 expression by astrocytes can stimulate the transmigration of monocytes across an in vitro model of the human bbb (weiss et al., 1999) . finally, since mip-la and rantes can effectively inhibit ccr5 coreceptor-mediated cell entry by hiv-1 (cocchi et al., 1996; dragic et al., 1996) , expression of these and other chemokines in the cns in neu-roaids might limit the spread of hiv-1 by antagonizing infection of target microglia. the wide distribution of chemokine receptors in the normal and hiv-infected human brain, as well as the increased expression of their cognate ligands, not only provides an avenue for hiv entry into the brain via the microglia, but might also contribute to the neuronal injury and loss that ensue. as indicated above, current evidence supports the idea that neuronal injury and death in neuroaids are consequences of indirect mechanisms involving a variety of host-derived molecules for example, cytokines and excitotoxic amino acids or hivencoded factors such as gpl20; it is probable that the host and viral products work in combination. loss of neurons, astrocytes, and microglia through apoptosis appears to be prominent in neuroaids (adle-biassette et al., 1995; gelbard et al., 1995; shiet al., 1996) and can be induced by hiv-1 infection of these cells in vitro (shi et al., 1996) . analysis of a panel of diverse hiv-1 primary isolates or strains revealed that, compared with blood-derived or t-tropic hiv-1 isolates, brain-derived m-tropic hiv-1 isolates are ineffective at inducing neuronal and astrocyte apoptosis (ohagen et al., 1999; zheng et al., 1999b) . thus, the primary determinant of neurodegeneration may not be the m-tropic hiv forms constituting the major cns reservoir of hiv-1, but rather, it resides with blood-derived viruses that are prominent during later stages of disease. furthermore, the env v3 region is the major determinant of neuronal apoptosis and also determines chemokine coreceptor usage for infection (ohagen et al., 1999) , implicating these viral coreceptors in the effector pathways that mediate neuronal damage during neuroaids. as mentioned above, the blood-derived, t-tropic hiv-1 isolates rely primarily on cxcr4 as a coreceptor, whereas the brain-derived, mtropic isolates use ccr5 and ccr3. in view of the congruity between the induction of neuronal apoptosis and chemokine coreceptor usage, and the fact that neurons express cxcr4 (as noted above), many recent studies have focused on the possible role of cxcr4 in mediating brain injury in neuroaids. hesselgesser and colleagues showed that signaling by cxcr4, following the binding of t-tropic hivgpl20, activates apoptotic cell death in human hnt neuronal cells (hesselgesser et al., 1998) . this does not appear to reflect an unusual property of hnt cells, which have a transformed phenotype, as induction of apoptotic cell death is also induced in rat hippocampal neurons exposed to hivgpl20 (meucci et al., 1998) . in these latter experiments, pretreatment of the neurons with the cognate ligand for cxcr4, sdf-1, partially protected against gpl20-induced neuronal apoptosis, suggesting that the effects of the viral product are mediated, in part, by cxcr4 binding. more recently, purified virions from t-tropic, but not m-tropic, hiv isolates were shown to induce cd4 independent neuronal signaling and apopto-sis that could be blocked by antibodies to cxcr4 (zheng et al., 1999a) . in these studies, the purified t-tropic hivgpl20 protein proved to be far less effective in affecting neuronal signaling and cell death than the whole virions. since the effects of the virions could be blocked by antibodies to gpl20 and gp41, the findings indicate that the envelope proteins in their native state are more potent mediators than their recombinant or purified derivatives. in all, the implication from these studies, which utilized either human neuronal cell lines or highly purified rodent or human primary neuronal cultures, is that direct binding of hiv gpl20 to the cxcr4 found on neurons activates apoptotic cell death in these cells. contrary to this notion, in mixed brain-cell cultures derived from embryonic rat cerebrocortex, gpl20-induced neuronal apoptosis was completely blocked by the tripeptide tkp, which blocks macrophages microglial activation (kaul and lipton, 1999) . this blockade by tkp was specific for gpl20 as sdf-l-induced neuronal apoptosis (as noted below) was unaffected by the peptide. interestingly, gpl20induced neuronal apoptosis in this model system was also blocked by the cc chemokines rantes and mip-i~, which bind to ccr5, suggesting there may be regulatory cross talk between the ccr5 and cxcr4 receptors. however, the conclusion from this study is that gpl20-induced neuronal apoptosis depends predominantly on an indirect pathway via activation of chemokine receptors on macrophages microglia. many possible explanations exist as to why there are differences between these studies with respect to the observed effects of gpl20's being conveyed by direct or indirect pathways. these include differences in the species and types of neuronal or brain-cell cultures used and the types, form, and levels ofgpl20 added to the cultures. while the question of whether the induction of neuronal apoptosis by gpl20 is consequent to interaction directly with the cxcr4 receptor on neurons or occurs indirectly, following activation of the macrophage/microglia, is unresolved, there is general agreement that hivgpl20 binding to cxcr4 can regulate a number of cellular signaling pathways (kaul and lipton, 1999; meucci et al., 1998; zheng et al., 1999a; zheng et al., 1999b) . these include inhibition of cyclic amp; activation of phosphoinositol (pi) hydrolysis, which is partially prevented by antibody to cxcr4 (zheng et al., 1999a; zheng et al., 1999b) ; activation of p38 mitogen-activated kinase (p38 mapk) (kaul and lipton, 1999) ; and increased ca 2+ mobilization (meucci et al., 1998) . hiv gpl20 neuronal apoptosis can be inhibited by inhibitors of p38 mapk (kaul and lipton, 1999) and by calcium/calmodulin-dependent protein kinase ii, protein kinase a, and protein kinase c (zheng et al., 1999a) , linking neuronal apoptosis, in part, to these signal transduction pathways. the preceding discussion indicates that hiv gpl20 neurotoxicity likely involves, in part, the chemokine receptor cxcr4, raising the possibility that hiv-1 subverts a key physiological cell death pathway. as we noted above, sdf-1/cxcr4 signaling is crucial for normal cerebellar neuronal migration and patterning during cns development. sdf-1 is expressed constitutively in the adult cns predominantly by astrocytes and to a lesser degree by neurons , raising the likelihood that this chemokine may regulate physiological processes in the adult brain. in support of this, sdf-la regulates a number of signal transduction pathways in neurons, causing decreased cyclic amp, increased pi hydrolysis, activation of p38mapk, and increased ca 2+ mobilization (kaul and lipton, 1999; meucci et al., 1998; zheng et al., 1999a) . functionally, exposure of hippocampal neurons to sdf-1 results in increased synaptic transmission, which is blocked by antibodies to cxcr4 (zheng et al., 1999a) . the similar effects of sdf-1 and hiv gpl20 in regulating cxcr4 signaling raise the question as to whether sdf-1 might also induce neuronal apoptosis. here, the findings have been conflicting. four studies revealed that sdf-1 promoted increased neuronal apoptosis (hesselgesser et al., 1998; kaul and lipton, 1999; zheng et al., 1999b) and activation of a key cell death gene, caspase 3 (zheng et al., 1999a) . in contrast to these findings, meucci and colleagues reported that, following treatment with sdf-1, there was marked protection of neurons from culture-and hiv gpl20-induced apoptosis (meucci et al., 1998) . the reason for these discrepancies is not known but again likely reflect differences in cell preparations and culture conditions. similar to hiv, siv infection of macaques can lead to the development of an aids-associated encephalitis that is virtually indistinguishable from neuroaids (raghavan et al., 1999; sasseville and lackner, 1997) . both t-tropic and m-tropic siv variants have been isolated and, like neuroaids, macrophage/microglia constitute the primary infected target cell population in the brain of siv-infected monkeys. the host immune system plays a major role in the infection of the cns with siv, where perivascular macrophages are continuously replaced via recruitment from the circulating monocyte pool through the blood-brain barrier (lane et al., 1996) . thus, infected t cells and monocytes likely enter the cns parenchyma during the asymptomatic phase of infection. in addition to leukocyte and endothelial adhesion molecules, chemokines could be critical components involved in siv infection of the brain. using immunohistochemical staining to investigate chemokine expression in the encephalitic brain of siv-infected macaques, sasseville and coworkers reported upregulation in the expression of a number of chemokines, including mip-la, mip-i~, rantes, mcp-3, and ip-10, by vascular endothelium and/or perivascular mononuclear cells . in contrast to neuro aids, mcp-1 was not elevated in the siv-encephalitic monkey brain. with the exception of mcp-1, expression of these proinflammatory chemokines in the siv-infected brain is similar to that found in the human brain in neuroaids (as noted above). however, like neu-roaids, the in vivo function of these chemokines in siv-encephalitis remains unknown; it is reasonable speculation however, to suggest that leukocyte recruitment to the siv-infected cns may be directed by these chemokines. in a follow-up study by this group, examining chemokine receptor expression in siv-aids encephalitis, the chemokine receptors ccr3, ccr5, cxcr3, and cxcr4 were found to be expressed by inflammatory cells within perivascular lesions (westmoreland et al., 1998) . in addition, expression of ccr3, ccr5, and cxcr4 was detected on subpopulations of large hippocampal and neocortical pyramidal neurons and on glial cells in both the normal and the siv-encephalitic brain. again, these findings show an overlap with those reported for the normal and the hiv-infected brain in humans (as noted above). m-tropic strains of siv utilize ccr5 as a coreceptor with cd4 for infection of macrophages deng et al., 1997) . expression of ccr5 is enriched on microglial cells in the macaque brain and may therefore provide the molecular framework for infection by siv in the monkey cns. overall, the preceding discussion highlights the overlapping expression patterns and potentially similar roles for the chemokines and their receptors in the pathogenesis of siv-encephalitis and neu-roaids. this idea is further underscored by a recent study in which chemokine receptor expression and signaling were examined in cultured neurons derived from either macaque or human brains (klein et al., 1999) . thus, both the qualitative pattern of expression and the functional behavior of the chemokine receptors, ccr3, ccr5, and cxcr4, in the two species are remarkably alike. in humans, multiple sclerosis is the most common and the bestknown cns demyelinating disorder. ms is thought to be an autoim-mune t-cell-mediated disease that is targeted at the myelin sheath. the etiology of ms is unknown (hailer, 1999) , although at one time or another, different viruses have been implicated in its pathogenesis, including epstein-barr virus (ebv), measles virus, and recently, hhv-6. the human t lymphotropic virus type i (htlv-1) infection of the cns can cause a progressive inflammatory demyelinating disease and shares many similarities with ms. in view of a possible viral role in ms, research has focused on several experimental viral models associated with the development of neuroimmune responses and primary demyelination that show many clinicopathologic similarities with ms. among the best-known and most widely studied of these experimental models are theiler's murine encephalomyelitis virus (tmev)-and murine hepatitis virus (mhv)-induced demyelinating disease. htlv-1 is the etiologic agent for adult t cell leukemia (atl) and for ham/tsp, which shows similar features to primary progressive ms (calabresi et al., 1999) . ham-tsp is a chronic progressive neurological disorder characterized by a perivascular demyelination and axonal degeneration, along with the presence of inflammatory infiltrating cells such as macrophages and cd8 ÷ t cells in the damaged areas (moore et al., 1989; umehara et al., 1993; wu et al., 1993) . these infiltrating cells produce several inflammatory mediators including the chemokine mcp-1 (umehara et al., 1993) . in addition, htlv-1infected t cell lines produce a number of chemokines including sdf-1, rantes, mip-la, and mip-i~ (arai et al., 1998; baba et al., 1996; mendez et al., 1997) . ham/tsp patients have a high frequency of cd8 ÷ ctl in their peripheral blood and cerebrospinal fluid (csf). biddison and coworkers have shown that htlv-i-specific cd8 ÷ ctl clones secrete mip-la, mip-i~, and il-16 (biddison et al., 1997) . thus, these htlv-1 ctls may be an important source of chemokines in ham/tsp, where they might contribute to the pathogenesis of this disorder. consistent with this, elevated mip-la protein was found in the csf in two of three patients with ham/tsp (miyagishi et al., 1995) . a further clinical study showed that mcp-1 was detectable on perivascular inflammatory cells and the vascular endothelium in active-chronic lesions of spinal cords of ham/tsp patients (umehara et al., 1996) . mhv belongs to the coronaviruses, a ubiquitous group of positivestranded rna viral pathogens of man and animals associated with a variety of respiratory, gastrointestinal, and neurological disorders. val]~rie c. asensio and iain l. campbell cns infection of susceptible murine hosts with neuro-adapted strains of mhv leads to a robust initial recruitment of leukocytes to the brain that follows an early peak of viral replication. this acute encephalomyelitis phase, with its resultant tissue injury, often leads to death of the host. however, in surviving animals, further viral replication is controlled by the host response and a chronic mononuclear cell inflammation, which occurs in the brain and spinal cord, is linked to a progressive demyelinating disease (buchmeier and lane, 1999; lane and buchmeier, 1997; weiner, 1973) . the mechanisms of demyelination in this model are not clear, although there is a requirement for a competent immune response. in a study of the kinetics and histological localization of chemokine gene expression in the brain and spinal cord of mhv-infected mice, lane and colleagues showed that a number of a-and ~-chemokines were expressed during acute or chronic stages of mhv-infection (lane et al., 1998) . expression of the transcripts for ip-10, mip-2, mcp-1, mcp-3, mip-1~, and rantes overlapped with the occurrence of acute viral encephalomyelitis, being present by day 3 postinfection and peaking at day 7 postinfection. during the chronic demyelinating phase of the disease, both ip-10 and rantes transcripts remained elevated. ip-10 rna colocalized with viral rna and was present in astrocytes and microglia associated with demyelinating lesions (lane et al., 1998) . with the exception of rantes, a similar pattern of chemokine expression was observed with cultured astrocytes infected with active, but not uv-inactive, mhv, indicating that viral replication in these cells can directly stimulate chemokine gene expression. therefore, in mhv infection of the brain, early viral-induced chemokine gene expression by resident cns cells such as astrocytes might promote the recruitment of t lymphocytes and macrophages and contribute to the maintenance of the chronic inflammatory response leading to demyelination. further studies in the mhv model using cd4 ko and cd8 ko mice show an important role for cd4 ÷ t-cells in the pathogenesis of inflammatory disease and demyelination (lane et al., 2000) . thus, mhvinfected cd4 ko mice have a marked reduction in the number of activated macrophage/microglia within their brains and spinal cords and significantly less demyelination. concomitant with the reduction in cns inflammatory disease in the mhv-infected cd4 ko mice, lower levels of rantes, but not other chemokine transcripts and protein were found indicating that cd4 ÷ t-cells represent one major source of rantes in the cns during mhv encephalomyelitis. administration of rantes neutralizing antisera to mhv-infected mice is associated with a significant reduction in macrophage infiltration and demyelination compared to control mice (lane et al., 2000) . these data clearly indicate that cd4 + t-cells have a pivotal role in accelerating cns inflammation and demyelination within infected mice possibly by regulating rantes expression. tmev-induced demyelinating disease is characterized by cns mononuclear cell infiltration resulting in a chronic cd4 + t-cell-mediated demyelinating disease (for more discussion, see chapter 7 in this volume). a study of the expression of cc and cxc chemokines in the cns throughout the disease course showed that expression of c10, ip-10, mcp-1, mip-la, mip-i~, and rantes mrna transcripts overlapped with the development of disease (hoffman et al., 1999) . expression of mip-i~ and mip-i~ protein was also detectable, being present in the spinal cord before the onset of disease and persisting throughout disease progression. these findings are somewhat reminiscent of those reported for mhv encephalomyelitis (discussed above) and reiterate the theme that chemokine expression in these viralinduced immune-mediated demyelinating diseases is a complex process that is clearly associated with disease progression. identifying the key chemokine protagonists in tmev-induced demyelinating disease awaits clarification. meningoencephalitis represents one of the most devastating consequences of viral infection of the cns, in which a concerted immunoinflammatory response by the host produces severe injury to the brain, leading to debilitating neurological disease and often death. in the viral meningoencephalitides, the specific mechanisms underlying the localization, extravasation, and activation of immune cells in the cns and the subsequent interactions between these cells are not well understood. studies from our own laboratory examined chemokine gene expression in lymphocytic choriomeningitis (lcm) that was induced by intracranial inoculation of mice with lcmv (asensio and campbell, 1997; asensio et al., 1999a) . following inoculation, lcmv infects and rapidly replicates in the meninges, the choroid plexus, and the ependymal membranes lining the ventricles. in this very well characterized model, immunocompetent adult mice infected with lcmv develop an acute monophasic disease characterized by the presence of infiltrating mononuclear cells in these same regions of the brain, and this leads to convulsive seizures and death by 6-8 days later (buchmeier et al., 1980; doherty et al., 1990) . infiltrating cells are predominantly t lymphocytes as well as macrophages. mhc class-i-restricted anti-lcmv cd8 ÷ cytotoxic lymphocytes (ctls), in addition to clearance of the virus, are also the primary effectors of lcm (buchmeier et al., 1980; doherty et al., 1990) . immune-compromised mice that are unable, or fail, to mount anti-lcmv cd8 ÷ ctl responses do not develop immune pathology in the brain and survive. we hypothesized that chemokines may be important regulatory signals for the cerebral recruitment and extravasation ofleukocytes in lcm (asensio and campbell, 1997; asensio et al., 1999a) . in examining this, we observed that the pattern of chemokine gene expression in lcm is dynamic and complex, with often overlapping expression of a number of different subclasses of chemokine genes. thus, by day 3 postinfection the expression of a number of chemokine genes is evident, including c10, mcp-3, mip-i~, mcp-1, crg-2/ip-10, and rantes. by day 6 postinfection the expression of all these chemokine genes increases markedly and the expression of the lymphotactin gene is also evident in the brain. a qualitatively similar but markedly decreased level of chemokine gene expression is observed in the brain of lcmv-infected athymic mice. a similar pattern of cerebral chemokine gene expression is also found in lcmv-infected ifn-y ko mice, although the levels of expression in the absence of ifn-y are about 50% of those in lcmv-infected wild-type controls. in both euthymic and athymic mice, expression of ip-10 was predominant at both early and late time points after infection and preceded detectable increases in proinflammatory and interferon cytokine gene expression and cns leukocyte recruitment in euthymic mice. in all, fig 2. the cxc chemokine ip-10 is expressed at high levels in the brain, following infection by many viruses. in this example mice were infected intracranially with lcmv, and the viral nucleoprotein (np) and ip-10 rna expression in the brain were analyzed by in situ hybridization. high expression of ip-10 rna was apparent by day 3 (a) postinfection and increased further by day 6 (b) postinfection. regional expression of ip-10 rna, on the whole, overlapped with sites of viral infection, as indicated by the expression of the lcmv-np gene. however, parenchymal expression of the ip-10 gene was also found in the absence of local lcmv-np expression. by using dual-label analysis to identify the cellular localization of ip-10 rna expression, these parenchymal cells were identified as gfap-positive astrocytes (c; arrows). from asensio, v. c., kincaid, c., and campbell, i. l. chemokines and the inflammatory response to viral infection in the 156 vall~rie c. asensio and iain l. campbell these observations, together with the finding that crg-2/ip-10, a prominently expressed chemokine gene in many different cns viral infections, is expressed by resident cns cells including astrocytes (see fig. 2 ), suggest that activation ofchemokine gene expression may be a direct, early, and localized host response to lcmv infection. these findings are consistent with the proposed involvement of chemokines as key signaling molecules for the subsequent migration of leukocytes to the cns, following lcmv infection. however, formal demonstration of the precise chemotactic, and possibly of other functions of chemokines in lcm, presently awaits verification. herpes simplex virus (hsv) is a common etiologic agent of acute focal encephalitis. pathologically, in hsv encephalitis, leukocytes infiltrate the subarachnoid space as a hallmark of meningitis and encephalitis, while csf analysis typically reveals a predominantly lymphocytic pleocytosis. to evaluate the possible contribution of chemokines to hsv encephalitis, rosler and coworkers examined the spectrum, quantity, and time course of csf chemokines in three patients with proven hsv type 1 encephalitis (hse-1) (rosler et al., 1998) . high chemokine levels were present in the csf of all hse-1 patients. peak levels occurred at the time of admission, with mcp-1 being greater than either mip-la or rantes. il-8 levels were elevated at 4 to 8 hours after admission. by contrast, plasma chemokine levels were considerably lower than csf levels, pointing to the localized nature of the cns chemokine response in these hse-1 patients. a comparison of mcp-1 levels with clinical status revealed a high reciprocal correlation. this single clinical study implicates chemokines, particularly mcp-1, in the pathogenesis of hse-1 and suggests these mediators may be useful indicators to determine the stage and severity of rise-1. experimental studies of hsv-1 in mice, following ocular infection, highlight the rapid and sustained upregulation of a number of chemokine genes including gro-a, mip-i~, mip-2, mcp-1, ip-10, and rantes (carr et al., 1998; thomas et al., 1998) . hsv stromal keratitis, associated with productive infection in the eye, results in significant accumulation of pmn, which may be driven by the presence in particular of the cxc chemokines gro-a and mip-2 (thomas et al., 1998) . a clinical survey of chemokine expression in csf in patients with viral meningitis, due to infection with paramyxoviruses or enteroviruses, implicated ip-10 and mcp-1 as important contributory factors in the accumulation of activated t cells and monocytes in the cns (lahrtz et al., 1997) . importantly, this study formally demonstrated that the ip-10 and mcp-1 levels in csf correlated with leukocyte chemotactic activity. mcp-1 was identified as an important chemoattractant for pbmcs while the combination of mcp-1 and ip-10 was required for migration of activated t cells. this study clearly establishes a direct link between the cns expression of the chemokines ip-10 and mcp-1 and cns leukocytosis in viral meningitis. in experimental studies, the paramyxovirus newcastle disease virus (ndv) induces ip-10 gene expression in infected astrocytes and microglia detectable by 3 hours postinfection (vanguri and farber, 1994) . uv-inactivated ndv proved to be as effective as live virus in inducing ip-10 gene expression and was not blocked by the protein synthesis inhibitor cycloheximide, indicating that the ip-10 gene functions as an immediate early response gene, following ndv infection. similar to ndv, measles virus (mv) is a paramyxovirus that induces ip-10 gene expression in human glioblastoma cells (nazar et al., 1997) . the promoter requirements for the induction of ip-10 gene expression are similar for ifn-~ and mv; however, these two stimuli use different combinations of dna binding factors, with stat 1 or nf~b being more important in the direct induction of ip-10 by ifn-~ or mv, respectively. while it is presently unknown whether these paramyxoviruses induce the expression of other chemokine genes, their potent ability to directly induce ip-10 expression by glial cells may constitute a significant cns source of production of this chemokine during viral meningitis. mouse adenovirus-type 1 (mav-1) is a double-stranded dna virus that causes a fatal hemorrhagic encephalopathy in c57b1/6 mice within 4-6 days (guida et al., 1995) , along with infection of cerebrovascular endothelial cells. chemokine gene expression was investigated and prominent induction of ip-10 was observed in the spleen and cns of susceptible mice, whereas mcp-1 and mip-2, respectively, were found in the spleen and brain of resistant mice (charles et al., 1999) . vascular endothelium and cns glia were identified as sites of ip-10 mrna expression in susceptible animals. in the normal mammalian cns, the number of leukocytes present in the brain is scant. however, these cells are attracted to, and accu-158 vali~rie c. asensio and iain l. campbell mulate in, a variety of pathologic states, many involving viral infection. although leukocyte migration into local tissue compartments such as the cns is a multifactorial process, it has become clear that chemokines are pivotal components of this process, providing a necessary chemotactic signal for leukocyte recruitment. generation of this signal in cns viral infection may involve localized production of proinflammatory chemokines by cells intrinsic to the brain, including the astrocytes and microglia. the activated glia have the potential to produce a spectrum of cc and cxc chemokines that may vary, depending on the nature of the invading pathogen. this in turn will determine the qualitative makeup of the leukocytes recruited to the brain. it is important to note, however, that how a chemokine that is secreted by a parenchymally located glial cell is "sensed" by circulating peripheral leukocytes is unclear at this time. roles beyond leukocyte chemoattraction are implied for the chemokines and their receptors. studies of hiv highlight the dynamic relationship between chemokine receptor usage and tropism for different isolates of hiv. on the other hand, the fact that chemokine receptor ligands are effective inhibitors of hiv entry into target cells suggests their local production may serve as a host response to limit further spread of the infectious agent. some chemokines such as ip-10 are documented as having direct antiviral functions. whether this is important in cns defense against viral infection where ip-10 is highly expressed, and whether any other chemokines have an antiviral function, remain open questions. a further consequence of the localized production of chemokines in the brain is likely the direct modulation of cns cell function. various classes of chemokine receptors are expressed by cns cells including neurons and the glia. in many respects the cns possesses its own chemokine network permitting autocrine and paracrine regulation of cellular activity. chemokines are implicated in many physiological functions including development, cell growth, angiogenesis, and cellular migration. perturbation of the cns chemokine network, resulting from viral infection, could therefore either be beneficial by promoting reparative processes or detrimental by contributing to tissue injury and loss. in conclusion, it is now apparent that, like their proinflammatory cytokine counterparts, the chemokines are important plurifunctional mediators in the host response to viral infection of the cns (see fig. 3 and color section). as such, a greater understanding of the neurocellular and viral targets and functions of the chemokines holds the promise of revealing new molecular focal points for therapeutic inter[for color reproduction, see color section.] chemokines are potentially multifunctional effectors in cns viral infections. in this schema, initial infection of the cns with a virus results in the localized production of chemokines by reactive astrocytes and microglia. some chemokines such as sdf-1 and fractalkine are produced constitutively in the cns and their production may also be altered by viral infection. increased chemokine production may then promote the further recruitment and extravasation of antiviral immune effector cells from the periphery. chemokine receptors are widely expressed by neuronal and glial cells and additional functions of the chemokines are likely. these may include direct antiviral actions (for example, by ip-10), and intercellular communication (for example, neuronally derived fractalkine can stimulate microglia). in addition, chemokines may exert detrimental actions; for example, sdf-1 can impair neuronal activity and stimulate apoptosis of these cells. m, macrophage; tc, t lymphocyte; n, neutrophil. vention that could more effectively control cns viral infections and prevent tissue injury. acknowledgments val6rie asensio is a postdoctoral fellow of the national multiple sclerosis society. the authors' studies were supported by nih grants mh 50426; mh 47680 and ns 36979 to ilc. this chapter is publication no. 13255-np, from the scripps research institute. neuronal apoptosis in hiv infection in adults chemokine receptors and molecular mimicry molecular piracy of mammalian interleukin-8 receptor type b by herpesvirus saimiri cc ckr5: a rantes, mip-la, mip-i~ receptor as a fusion cofactor for macrophage-tropic hiv-1 genes for chemokines mumig and crg-2 induced in protozoan and viral infections in response to ifn-7 with patterns of tissue expression that suggest nonredundant roles in vivo human t-cell leukemia virus type 1 tax protein induces the expression of lymphocyte chemoattractant sdf-1/pbsf human herpesvirus kshv encodes a constitutively active g-protein coupled receptor linked to cell proliferation chemokine gene expression in the brains of mice with lymphocytic choriomeningitis chemokines and their receptors in the cns: directing cellular communication chemokines and the inflammatory response to viral infection in the central nervous system with a focus on lymphocytic choriomeningitis virus c10 is a novel chemokine expressed in experimental inflammatory demyelinating disorders that promotes the recruitment of macrophages to the central nervous system constitutive expression of various chemokine genes in human t-cell lines infected with human t-cell leukemia virus type 1: role of the viral transactivator tax chemokines in disease models and pathogenesis chemokines and leukocyte traffic interleukin-8 and related chemotactic cytokines-cxc and cc chemokines human chemokines: an update g-protein-coupled receptor of kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator expression of chemokine receptors in the rat brain ccr5(+) and cxcr3(+) t cells are increased in multiple sclerosis and their ligands mip-lalpha and ip-10 are expressed in demyelinating brain lesions a new class of membrane-bound chemokine with a cx3c motif overriding the brain's intrinsic resistance to leukocyte recruitment with intraparenchymal injections of recombinant chemokines monocyte chemotactic protein-3 (mcp3) interacts with multiple leukocyte receptors. c-c ckr1, a receptor for macrophage inflammatory protein-1 alpha/rantes, is also a functional receptor for mcp3 identification of the cc chemokines tarc and macrophage inflammatory protein-1 beta as novel functional ligands for the ccr8 receptor human t cell leukemia virus type i (htlv-i)-specific cd8+ ctl clones from patients with htlv-i-associated neurologic disease secrete proinflammatory cytokines, chemokines, and matrix metalloproteinase hiv-1 infection in an individual homozygous for the ccr5 deletion allele the lymphocyte chemoattractant sdf-1 is a ligand tbr lestr/fusin and blocks hiv-1 entry the hiv coreceptors cxcr4 and ccr5 are differentially expressed and regulated on human t lymphocytes chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells cultured rat microglia express functional beta-chemokine receptors differential expression of chemokine receptors and chemotactic responsiveness of type 1 t helper cells (thls) and th2s upregulation of ccr1 and ccr3 and induction of chemotaxis to cc chemokines by ifngamma in human neutrophils decreased lesion formation in ccr2-/-mice reveals a role for chemokines in the initiation of atherosclerosis angiogenic and hiv-inhibitory functions of kshv-encoded chemokines involvement of interleukin (il) 8 receptor in negative regulation of myeloid progenitor cells in vivo: evidence from mice lacking the murine il-8 receptor homologue viral-induced neurodegenerative disease the virology and immunobiology of lymphocytic choriomeningitis virus infection novel role of human cd4 molecule identified in neurodegeneration neutrophil and b cell expansion in mice that lack the murine il-8 receptor homolog chemokines in chronic progressive neurological diseases: htlv-1 associated myelopathy and multiple sclerosis cytokine and chemokine production in hsv-1 latently infected trigeminal ganglion cell cultures: effects of hyperthermic stress monocyte chemoattractant protein 1 acts as a t-lymphocyte chemoattractant differential chemokine induction by the mouse adenovirus type-1 in the central nervous system susceptible and resistant strains of mice inactivation of hiv-1 chemokine co-receptor cxcr-4 by a novel intrakine stategy in vivo inhibition of cc and cx3c chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in wistar-kyoto (wky) rats by vmip-ii the beta-chemokine receptors ccr3 and ccr5 facilitate infection by primary hiv-1 isolates identification of rantes, mip-1 alpha, and mip-1 beta as the major hiv-suppressive factors produced by cd8+ t cells the v3 domain of the hiv-1 gpl20 envelope glycoprotein is critical for chemokinemediated blockade of infection cloning, chromosomal localization, and rna expression of a human ~ chemokine receptor-like gene gene cloning, rna distribution, and functional expression of mcx3cr1, a mouse chemotactic receptor for the cx3c chemokine fractalldne induction of monocyte chemoattractant protein-1 in hiv-1 tat-stimulated astrocytes and elevation in aids dementia requirement of mip-la for an inflammatory response to viral infection hhv8-encoded vmip-i selectively engages chemokine receptor ccr8. agonist and antagonist profiles of viral chemokines abduction of chemokine elements by herpesviruses broad spectrum chemokine antagonistic activity of a human poxvirus chemokine homolog identification of a major co-receptor for primary isolates of hiv-1 expression cloning of new receptors used by simian and human immunodeficiency viruses differentiation-specific expression of a novel g protein-coupled receptor from burkitt's lymphoma dissection of an inflammatory process induced by cd8+ t-cells a dual-tropic primary hiv-1 isolate that uses fusin and the beta-chemokine receptors ckr-5, ckr-3, and ckr-2b as fusion cofactors hiv-1 entry into cd4+ cells is mediated by the chemokine receptor cc-ckr-5 a review of neuronal damage in human immunodeficiency virus infection: its assessment, possible mechanism and relationship to dementia two orphan seven-transmembrane segment receptors which are expressed in cd4-positive cells support simian immunodeficiency virus infection hiv-1 entry cofactor: functional cdna cloning of a seven-transmembrane, g protein-coupled receptor regulatory mechanisms of murantes and crg-2 chemokine gene induction in central nervous system glial cells by virus fractalkine and cx3cr1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow a putative chemokine receptor, blr1, directs b cell migration to defined lymphoid organs and specific anatomic compartments of the spleen selective expression of the murine homologue of the g-protein-coupled receptor blr1 in b cell differentiation, b cell neoplasia and defined areas of the cerebellum characterization of the ccr2 chemokine receptor: functional ccr2 receptor expression in b cells controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1 excitotoxic brain injury stimulates expression of the chemokine receptor ccr5 in neonatal rats impaired host defense, hematopoiesis, granulomateus inflammation and type 1-type 2 cytokine balance in mice lacking cc chemokine receptor 1 structure and functional expression of the human macrophage inflammatory protein 1 alpha/rantes receptor human cytomegalovirus open reading frame us28 encodes a functional ~ chemokine receptor cloning and differential tissue-specific expression of three mouse ~ chemokine receptor-like genes, including the gene for a functional macrophage inflammatory protein-la receptor apoptotic neurons in brains from paediatric patients with hiv-1 encephalitis and progressive encephalopathy the neuropathogenesis of the aids dementia complex chemokines and chemokine receptors in cns pathology identification of ccr8 as the specific receptor for the human ~-chemokine 1-309: cloning and molecular characterization of murine ccr8 as the receptor for tca-3 transgeuic monocyte chemoattractant protein-1 (mcp-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic mcp-1 mouse adenovirus type i causes a fatal hemorrhagic encephalomyelitis in adult c57bl/6 but not balb/c mice the distinction blurs between an autoimmune versus microbial hypothesis in multiple sclerosis cdna cloning of a g-protein-coupled receptor expressed in rat spinal cord and brain related to ehemokine receptors role for neuronally derived i~actalkine in mediating interactions between neurons and cx3crl-expressing microglia production and function of monocyte chemoattraetant protein-1 and other ~-chemokines in murine glial cells ccr3 and ccr5 are co-receptors for hiv-1 infection of microglia lymphotactin is produced by nk cells and attracts both nk cells and t cells in vivo cloning of the mouse fusin gene, homologue to a human hiv-1 co-factor isolation of three novel human genes encoding g protein-coupled receptors a novel human gene encoding a g-protein-coupled receptor (gpr15) is located on chromosome 3 neuronal apoptosis induced by hiv-1 gpl20 and the chemokine sdf-1 alpha is mediated by the chemokine receptor cxcr4 leukocyte traffic in the central nervous system: the participants and their roles central nervous system chemokine expression during theiler's virus-induced demyelinating disease the duffy antigen receptor for chemokines: structural analysis and expression in the brain expression of chemokine receptors by subsets of neurons in the central nervous system inhibition of microglial cell rantes production by il-10 and tgf-beta identification and molecular characterization of fractalkine receptor cx3cr1, which mediates both leukocyte migration and adhesion the g protein-coupled receptor blr1 is involved in murine b cell differentiation and is also expressed in neuronal tissues an important role for the chemokine macrophage inflammatory protein-lalpha in the pathogenesis of the t cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis chemokine regulation of experimental autoimmune encephalomyelitis: temporal and spatial expression patterns govern disease patbogenesis chemokines and activated macrophages in hiv gpl20-induced neuronal apoptosis beta-chemokines mcp-1 and rantes are selectively increased in cerebrospinal fluid of patients with human immunodeficiency virus-associated dementia lymphotactin: a cytokine that represents a new class of chemokine a broad-spectrum chemokine antagonist encoded by kaposi's sarcoma-associated herpesvirus chemokine receptor expression and signaling in macaque and human fetal neurons and astrocytes: implications for the neuropathogenesis of aids identification of a t cell chemotactic factor in the cerebmspinal fluid of hiv-l-infected individuals as interferon-7 inducible protein 10 genetic differences between blood-and brain-derived viral sequences from human immnnodeficiency virus type 1-infected patients: evidence of conserved elements in the v3 region of the envelope protein of brain-derived sequences functional characterization of the c-c chemokine-like molecules encoded by molluscum contagiosum virus types 1 and 2 defects in macrophage recruitment and host defense in mice lacking the ccr2 chemokine receptor severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in cc chemokine receptor 2 chemotactic activity on mononuclear cells in the cerebrospinal fluid of patients with viral meningitis is mediated by interferon-gamma inducible protein-10 and monocyte chemotactic protein-1 chemokines and chemotaxis of leukocytes in infectious meningitis modulating chemokines: more lessons from viruses neuroinvasion of simian immunodeficiency virus coincides with increased numbers of perivascular macrophages/microglia and intrathecal immune activation dynamic regulation of a-and ~-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease murine coronavirus infection: a paradigm for virus-induced demyelinating disease a central role for cd4+ t-cells and rantes in virus-induced central nervous system inflammation and demyelination chemokine receptors in the human brain and their relationship to hiv infection cxcr-4 (fusin), a co-receptor for the type 1 human immunodeficiency virus (hiv-1), is expressed in the human brain in a variety of cell types, including microglia and neurons cerebrospinal fluid beta chemokine concentrations in neurocognitively impaired individuals infected with human immunodeficiency virus type 1 a novel chemokine receptor-like protein, functions as a fusion cofactor for both macrophage-tropic and t cell line-tropic hiv-1 chemokine receptors: keys to aids pathogenesis? chemokine receptor specific for ip10 and mig: structure, function, and expression in activated t-lymphocytes abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice the cxc-chemokine, h174: expression in the central nervous system impaired b-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in cxcr4-and sdf-l-deficient mice late expression of a beta chemokine homolog by murine cytomegalovirus characterization of fractalkine in rat brain cells: migratory and activation signals for cx3cr-l-expressing microglia chemokines: what chemokine is that? the interferon-inducible chemokines mumig and crg-2 exhibit antiviral activity in vivo regulation of human ip-10 gene expression in astrocytoma cells by inflammatory cytokines the chemokine system: redundancy for robust outputs chemokines. introduction and overview cloning of human genes encoding novel g protein-coupled receptors purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line mcp-1, mcp-2 and mcp-3 expression in multiple sclerosis lesions: an immunohistochemical and in situ hybridization study astrocyte-specific expression of human t-cell lymphotropic virus type 1 (htlv-1) tax: induction of tumor necrosis factor alpha and susceptibility to lysis by cd8+ htlv-l-specific cytotoxic t cells developmental expression of monocyte chemoattractant protein-1 in the human cerebellum and brainstem chemokines and chemokine receptors in the cns: a possible role in neuroinflammation and patterning chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity the role of ccr5 and ccr2 polymorphisms in hiv-1 transmission and disease progression macrophage inflammatory protein-la in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological diseases tropical spastic paraparesis: a model of virus-induced, cytotexic t-cell-mediated demyelination? lymphocyte responses to chemokines cloning of atac, an activation-induced, chemokine-related molecule exclusively expressed in cd8+ t lymphocytes molecular piracy of chemokine receptors by herpesviruses. infectious agents dis signal transduction and ligand specificity of the human monocyte chemoattractant protein-1 receptor in transfected embryonic kidney cells defects of b-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the cxc chemokine pbsf/sdf-1 a novel cxc chemokine pbsf/sdf-1 and its receptor cxcr4: their functions in development, hematopoiesis and hiv infection induction of ip-10 chemokine promoter by measles virus: comparison with interferon-gamma shows the use of the same response element but with differential dna-protein binding profiles molecular cloning, functional expression, and signaling characteristics of a c-c chemokine receptor localization of fractalkine and cx3cr1 mrnas in rat brain: does fractalkine play a role in signaling from neuron to microglia? hiv-1 infection in a man homozygous for ccr5 delta 32 the cxc chemokine sdf-1 is the ligand for lestr/fusin and prevents infection by t-cell-line-adapted hiv-1 apoptesis induced by infection of primary brain cultures with diverse human immunodeficiency virus type i isolates: evidence for a role of the envelope expression of stromal cell-derived factor-1 and cxcr4 chemokine receptor mrnas in cultured rat glial and neuronal cells inflammatory cytokines in the brain: does the cns shape immune responses? neurotactin, a membrane-anchored chemokine upregulated in brain inflammation cytomegalovirus encodes a potent alpha chemokine identification of a chemokine receptor encoded by human cytomegalovirus as a cofacter for hiv-1 entry developmental expression of cytokine genes in the cortex and hippocampus of the rat central nervous system demented and non-demented patients with aids differ in brainderived human immunodeficiency virus type 1 envelope sequences chemokine receptors: gateways to inflammation and infection the chemokine receptors cxcr3 and ccr5 mark subsets of t cells associated with certain inflammatory reactions morphological correlates of neurological dyshmction in macaques infected with neurovirulent simian immunodeficiency virus the role of ccr7 in t(h)i and t(h)2 cell localization and delivery of b cell help in vivo chemokines in neurological disease models: correlation between chemokine expression patterns and inflammatory pathology do chemokines mediate leukocyte recruitment in post-traumatic cns inflammation? the orphan g-protein-coupled recepter-encoding gene v28 is closely related to genes for chemokine receptors and is expressed in lymphoid and neural tissues lipopolysaccharide-induced expression of ip-10 mrna in rat brain and in cultured rat astroeytes and mieroglia. molec time course of chemokines in the cerebrospinal fluid and serum during herpes simplex type i encephalitis cytomegalovirus-encoded beta chemokine promotes monocyte-associated viremia in the host chemokines and chemokine receptors in t-cell priming and thl/th2-mediated responses two subsets of memory t lymphocytes with distinct homing potentials and effecter functions selective expression of the eotaxin receptor ccr3 by human t helper 2 cells chemokines and receptors in hiv encephalitis neuropathogenesis of simian immunodeflcieney virus infection in macaque monkeys chemokine expression in simian immunodefieieney virus-induced aids encephalitis human immunodeflciency virus type i infection alters chemokine beta peptide expression in human monocytes--implications for recruitment of leukocytes into brain and lymph nodes neuronal expression of fractalkine in the presence and absence of inflammation genome sequence of a human tumorigenic poxvirus: prediction of specific host response-evasion genes apoptosis induced by hiv-1 infection of the central nervous system structure and chromosomal localization of the human stromal cell-derived factor 1 (sdf1) gene expression of monocyte chemoattractant protein-1 and other ~-chemokines by resident glia and inflammatory cells in multiple sclerosis lesions vaccinia virus immune evasion expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients the c-c chemokine 6ckine binds the cxc chemokine receptor cxcr3 the viral chemokine macrophage inflammatory protein-ii is a selective th2 chemoattractant c-x-c and c-c chemokines are expressed in the cerebrospinal fluid in bacterial meningitis and mediate chemotactic activity on peripheral blood-derived polymorphonuclear and mononuclear cells in vitro cloning of rat hiv-l-chemokine coreceptor ckr5 from microglia and upregulation of its mrna in ischemic and endotoxinemic rat brain chemokines in the cerebrospinal fluid patients with meningitis virally encoded chemokines and chemokine receptors: genetic embezzlement of host dna the role of chemokines in models of human disease the chemokine receptor cxcr4 is essential for vascularization of the gastrointestinal tract murine astrocytes express a fimctional chemokine receptor functional expression of the cxcchemokine receptor-4/fusin on mouse microglial cells and astrocytes neutrophil infiltration, glial reaction, and neurological disease in transgenic mice expressing the chemokine n51/kc in oligodendrocytes signal sequence trap: a cloning strategy for secreted proteins and type i membrane proteins chemokines, inflammation and the immune system hiv-1 infection in an individual homozygous for ccr5 delta 32 herpes simplex virus replicationinduced expression of chemokines and proinflammatory cytokines in the eye: implications in herpetic stromal keratitis glucocorticoids downregulate gene expression of gm-csf, nap-1/il-8, and il-6, but not of m-csf in human fibroblasts central nervous system damage produced by expression of the hiv-1 coat protein gpl20 in transgenic mice immunocytochemical analysis of the cellular infiltrate in the spinal cord lesions in htlv-i-associated myelopathy expression of adhesion molecules and monocyte chemoattractant protein-1 (mcp-1) in the spinal cord lesions in htlv-i-associated myelopathy localization of hiv-1 co-receptors ccr5 and cxcr4 in the brain of children with aids expression of mcp-1 by reactive astrocytes in demyelinating multiple sclerosis lesions interferon---inducible genes in primary glial cells of the central nervous system: comparisons of astrocytes with microglia and lewis with brown norway rats ifn and virus-inducible expression of an immediate early gene, crg-2/ip-lo, and a delayed gene, i-ao~ in astrocytes and microglia molecular cloning, functional characterization and mrna expression analysis of the murine chemokine receptor ccr6 and its specific ligand mip-3a monocyte chemoattractant protein-1 (mcp-1) expression in human articular cartilage. induction by peptide regulatory factors and differential effects of dexamethasone and retinoic acid pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) hiv-1 tat induces monocyte chemoattractant protein-l-mediated monocyte transmigration across a model of the human blood-brain barrier and up-regulates ccr5 expression on human monocytes binding and functional properties of recombinant and endogenous cxcr3 chemokine receptors chemokine receptor expression on resident and inflammatory cells in the brain of macaques with simian immunodeficiency virus encephalitis rat lcri: cloning and cellular distribution of a putative chemokine receptor in brain neuroaxonal dystrophy in htlv-l-associated myelopathy/tropical spastic paraparesis: neuropathologic and neuroimmunologic correlations ccr5 levels and expression pattern correlate with infectability by macrophage-tropic hiv-1, in vitro immunohistochemical study of the beta-chemokine receptors ccr3 and ccr5 and their ligands in normal and alzheimer's disease brains molecular cloning of a novel human cc chemokine ebil-ligand chemokine that is a specific functional ligand for ebi1, ccr7 identification of single c motif-lflymphotactin receptor xcr1 molecular cloning of a novel c-)~ or ~/-type chemokine, scm-1 cutting edge: identification of the orphan chemokine receptor gpr-9-6 as ccr9, the receptor for the chemokine teck lymphotropic virions affect chemokine receptor-mediated neural signaling and apoptosis: implications for human immunodeficiency virus type 1-associated dementia intracellular cxcr4 signaling, neuronal apoptosis and neuropathogenic mechanisms of hiv-l-associated dementia the chemokine receptor ccr8 is preferentially expressed in th2 but not thl cells recent advances in chemokines and chemokine receptors function of the chemokine receptor cxcr4 in haematopoiesis and in cerebellar development key: cord-000158-d08buwtu authors: corti, davide; langedijk, johannes p. m.; hinz, andreas; seaman, michael s.; vanzetta, fabrizia; fernandez-rodriguez, blanca m.; silacci, chiara; pinna, debora; jarrossay, david; balla-jhagjhoorsingh, sunita; willems, betty; zekveld, maria j.; dreja, hanna; o'sullivan, eithne; pade, corinna; orkin, chloe; jeffs, simon a.; montefiori, david c.; davis, david; weissenhorn, winfried; mcknight, áine; heeney, jonathan l.; sallusto, federica; sattentau, quentin j.; weiss, robin a.; lanzavecchia, antonio title: analysis of memory b cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from hiv-1-infected individuals date: 2010-01-20 journal: plos one doi: 10.1371/journal.pone.0008805 sha: doc_id: 158 cord_uid: d08buwtu background: the isolation of human monoclonal antibodies (mabs) that neutralize a broad spectrum of primary hiv-1 isolates and the characterization of the human neutralizing antibody b cell response to hiv-1 infection are important goals that are central to the design of an effective antibody-based vaccine. methods and findings: we immortalized igg(+) memory b cells from individuals infected with diverse clades of hiv-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. culture supernatants were screened using various recombinant forms of the envelope glycoproteins (env) in multiple parallel assays. we isolated 58 mabs that were mapped to different env surfaces, most of which showed neutralizing activity. one mab in particular (hj16) specific for a novel epitope proximal to the cd4 binding site on gp120 selectively neutralized a multi-clade panel of tier-2 hiv-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other cd4 binding site-specific neutralizing mab b12. a second mab (hgn194) bound a conserved epitope in the v3 crown and neutralized all tier-1 and a proportion of tier-2 pseudoviruses tested, irrespective of clade. a third mab (hk20) with broad neutralizing activity, particularly as a fab fragment, recognized a highly conserved epitope in the hr-1 region of gp41, but showed striking assay-dependent selectivity in its activity. conclusions: this study reveals that by using appropriate screening methods, a large proportion of memory b cells can be isolated that produce mabs with hiv-1 neutralizing activity. three of these mabs show unusual breadth of neutralization and therefore add to the current panel of hiv-1 neutralizing antibodies with potential for passive protection and template-based vaccine design. neutralizing antibodies provide one arm of the adaptive immune response against the human immunodeficiency virus type 1 (hiv-1). several reports demonstrated that the neutralizing antibody response exerts selective pressure during hiv-1 replication in vivo, which accounts in part for the extensive variation in the env gene observed soon after primary infection [1, 2] . furthermore, selective pressure imposed by neutralizing antibodies has been demonstrated in a human trial where three neutralizing monoclonal antibodies (mabs) administered during haart treatment-interruption led to a reduction in viremia followed by selection of escape mutants [3, 4] . passive transfer studies in macaques showed that the administration of hiv-1 neutralizing mabs protects against vaginal or intravenous challenge with siv-hiv-1 chimeric viruses (shiv) [5, 6, 7, 8, 9] . in some models protection depended not only on viral neutralization but also on fc-mediated antibody effector functions [10, 11] . given the predicted low-titer inoculum driving hiv-1 sexual transmission, a vaccine capable of eliciting antibodies that neutralize a broad spectrum of viral strains could potentially reduce or prevent infection. it has been anticipated that the identification of broadly neutralizing mabs from hiv-1 infected individuals, and the characterization of their cognate epitopes will be instrumental in the design of immunogens capable of eliciting such a broad neutralizing response [12] . this idea has led to a major international cooperative effort within consortia of laboratories with complementary expertise in human immunology, structural biology and vaccine design [13, 14] . hiv-1 is characterized by an extraordinary genetic diversity, reflected by the presence of several clades (subtypes), a fact that represents a significant impediment to vaccine development. env is the most variable hiv-1 gene, with up to 35% sequence diversity among clades, 20% diversity within clades, and 10% diversity in a single infected individual [15, 16, 17] . several conserved epitopes have been defined by a small panel of neutralizing mabs isolated using different experimental approaches. one epitope that appears to be relatively conserved and overlaps with the cd4 binding site (cd4bs) on the surface env glycoprotein gp120 is recognized by mab b12, which is the most potent and broadly-reactive mab of such specificity [18, 19, 20] . this site was recently shown to be a significant target of neutralizing antibodies present in the sera of selected patients [21, 22] . however, b12 was derived from a phage library in which heavy and light chains have been randomly reassorted, thus its relevance to naturally-occurring b cell responses in hiv-1 infection is unclear. a second epitope in a carbohydraterich region on the outer domain of gp120 is composed of glycans recognized by mab 2g12, which shows an unusual interlocked vh domain-swapped dimer generating an extended and monovalent binding surface [23, 24, 25, 26] . the 2g12 epitope is not present in the majority of clade c isolates [27] , but, of more concern, no 2g12-like activity has been detected in the sera of hiv-1 infected individuals [21, 22] , suggesting that this type of neutralizing antibody may not be generally amenable to elicitation by b cells. a third target of the broadly-neutralizing mab 4e10, and relatively broad mabs 2f5 and z13, is located on the membrane-proximal external region (mper) of the transmembrane glycoprotein gp41 [28, 29, 30, 31] . as with 2g12, mperspecific neutralizing antibodies are infrequently encountered in the sera of hiv-1-infected individuals [21, 32, 33] , potentially reducing their relevance to vaccine development strategies. moreover, mper-specific mabs have features that suggest they are products of autoreactive b cells, casting doubts over the possibility of inducing such antibodies by vaccination [34] . a fourth target of neutralizing mabs is the v3 loop, which is immunogenic but also a highly variable region involved in env-coreceptor binding. antibodies to the v3 loop, such as mabs 447-52d and f425-b4e8, are mostly effective against neutralization sensitive (tier-1) viruses and have generally a limited breadth of reactivity almost exclusively specific for clade b viruses, reflecting their provenance from clade b-infected individuals [11, 35, 36, 37] . a cluster of human v3 mabs raised from b cells derived from non-b cladeinfected individuals showed variable neutralizing activity for two ag and two c clade primary isolates, although these were particularly neutralization sensitive [38] . finally, the cd4-induced (cd4i) site has been described as a neutralization epitope primarily on tier-1 isolates with limited accessibility on tier-2 isolates [39] . very recently, the isolation and partial characteriza-tion of two related mabs from a clade a-infected donor that appear to have very broad and potent neutralization profiles was reported [40] . these mabs recognize a discontinuous epitope only expressed on the native env trimer, made up of segments of the cd4i, v2 and v3 loops. this publication is the first to provide new broadly neutralizing reagents from a non-clade b donor and provides proof-of-principle that new neutralization specificities remain to be discovered on hiv-1 env. since the number of existing neutralizing mabs with a relatively broad neutralization profile is very limited, and those described to date, with the exception of the walker et al mabs were isolated from clade b virus-infected donors, we undertook the task of isolating new human mabs from hiv-1-infected donors. our primary aims were: i) to isolate novel mabs with cross-clade neutralizing activity from non-b clade donors; ii) to analyze the memory b cell repertoire in a representative panel of predominantly non-b clade-infected individuals. by using an improved memory b cell immortalization method [41] , combined with highthroughput parallel screening with a panel of recombinant envbased antigens, we isolated a panel of 58 human mabs which we have characterized with regard to epitope specificity and breadth of neutralization. we have more fully characterized three mabs from this panel that demonstrated complementary and relatively broad cross-clade neutralizing profiles that target the gp120 cd4bs and the v3 crown, and the gp41 heptad repeat 1 (hr-1). the study was approved by the ethical committee at institute of tropical medicine and queen mary university. all participants gave written informed consent. tzm-bl and hos.cd4-r5 cells were obtained from the nih-aids research and reference reagent program (arrrp). 293t/17 cells were obtained from the atcc. mabs 2g12 [29] , 2f5, 4e10 [42] , b12 [18] , 3d6 [43] and 5f3 [29] were obtained from polymun scientific gmbh (austria), as part of the collaboration for aids vaccine discovery program. the clade b and c hiv-1 reference panels of env clones [44, 45] and the sf162 clone were obtained through the nih-arrrp. other non-clade b isolates were provided by the comprehensive antibody vaccine immune monitoring consortium (ca-vimc). hiv-1 subtype b clone jrfl was provided by dennis burton (scripps institute, la jolla, us). pseudoviruses were produced by co-transfecting hek293t/17 cells with the envexpressing plasmids and the complementing viral-genome reporter gene vector, pnl4-3.luc + .e 2 r + (kindly provided by john r. mascola, vrc, niaid, nih, us). recombinant gp140 from hiv-1 isolates 92ug37, ca18, cn54, k530, ug21 and br29 were provided by simon jeffs (imperial college london, uk). recombinant gp120 from hiv-1 isolates cm235, 93th975, bal and sf162 were obtained through the nih-arrrp, while gp160 from isolates mn and lai were purchased by prospec-tany technogene ltd (israel). recombinant yu2 gp120 and the two mutants d368r and i420r were provided by john r. mascola and richard wyatt (vrc, niaid, nih, us). recombinant soluble cd4 and recombinant gp120 from hiv-1 iiib and cn54 were obtained from the cfar, nibsc (uk). the recombinant ectodomain of gp41 from isolate hxb2 (amino acids 541-682) was purchased by vybion inc. (us). hr-1 is a modified version of gp41 [46] exposing residues hllqltvwgikqlqarilave (hxb2). 5hb was produced as described [47] . hr-1-fp comprises gp41-residues 512-594 (hxb2) including the fusion peptide and hr-1. hr-2 (hxb2) comprises gp41 residues 591-693 (hxb2) including the cys-loop region, hr-2 and mper flanked by two coiled coil regions. patients were selected for inclusion into the study on the basis of the clade of their infecting virus (predominantly non-b clade) and on the ability of their plasma to neutralize a panel of hiv-1 primary isolates using two differerent assays. at the institute for tropical medicine a panel of 4 primary a strains (vi191, 92ug37, vi820, vi 1031), 4 primary c strains (vi829, vi882, vi1144, vi1358) and 6 primary crf02 strains (vi1090, vi2680, ci20, ca18, vi1380, vi2727) was used to select patients with .80% neutralization was measured at a 1/20 dilution of plasma. the plasma dilution was mixed with virus for 24 hours and absorbed to pha/il-2 stimulated freshly isolated pbmc for 1 hour. virus-antibody mixture was then removed by extensive washing and p24 elisa was performed after 14 days of culture to determine neutralization. at queen mary university of london, the tzmbl-based recombinant pseudovirus assay was employed with a selected panel of tier 2-type clade a, b, c, crf02_ag, _ae, f and bf viruses to measure .50% neutralization at a 1/20 dilution. mabs were isolated using a previously described improved ebv immortalization method [41] . culture supernatants were harvested 14 days after immortalization and assayed in parallel for binding to trimeric gp140 proteins (ug37, clade a and cn54, clade crf07_bc) [48] , monomeric gp120 (cn54, crf07_bc and iiib, clade b) and gp41 recombinant ectodomain (hxb2, clade b). those that were positive were then subcloned and grown up on a large scale for supernatant purification. the purity of all mab preparations was assessed using a chromogenic lal endotoxin assay (genscript) and endotoxin levels were always ,0.05 eu/ml. a standard elisa was used to determine binding of mabs to the panel of hiv-1 envs. briefly, elisa plates were coated with each env antigen, blocked with 10% fcs in pbs, incubated with human mabs and washed. bound mabs were detected by incubation with ap-conjugated goat anti-human igg (southern biotech). plates were then washed, substrate (p-npp, sigma) was added and plates were read at 405 nm. the relative affinities of mabs binding to respective coated antigens were determined with elisa by measuring the concentration of each mab required to achieve 50% maximal binding at saturation (k 50 ). the ability of mabs to inhibit binding of scd4 to gp120 or gp140 was evaluated by elisa. serial dilutions of mabs were pre-incubated with gp120 (or gp140) and added to plates pre-coated with scd4. after 1 h plates were washed and incubated with sheep polyclonal antibody d7324 (aalto bio-reagents) followed by washing, incubation with ap-conjugated rabbit anti-sheep igg antibody (abcam, cambridge, uk), extensive washing and detection as above. mabs were purified on protein g columns (ge healthcare) and biotinylated using the ez-link nhs-peo solid phase biotinylation kit (pierce). the competition between unlabeled and biotinylated mabs for binding to immobilized env antigens was measured by elisa. briefly, unlabelled competitor mabs were added at different concentrations. after 1 h biotinylated mabs were added at a concentration corresponding to the 70-80% of the maximal od level. after incubation for 1 h, plates were washed and bound biotinylated mab was detected using aplabeled streptavidin (jackson immunoresearch). the percentage of inhibition was tested in triplicates and calculated as follow: (12[(odsample-odneg ctr)/(odpos ctr -odneg ctr)])6100. overlapping linear 15-mer and cyclized 15-mer peptides based on gp160 of hiv-1 ug037 and 93mw965 and the overlapping 15-mer peptides of gp41 strain sf162 were synthesized on polypropylene support (minicards), and were tested for reactivity with mabs as described [49, 50] . pepscan peptide binding analysis was carried out by an elisa-based format in which the colored substrate was quantified with a charge-coupled device (ccd)camera and an image processing system. the values mostly ranged from 0 to 3000, a log scale similar to 1 to 3 of a standard 96-well plate elisa-reader. a single-cycle infectivity assay was used to measure the neutralization of luciferase-encoding virions pseudotyped with the desired hiv-1 env proteins, as previously described [51] . briefly, appropriate dilutions of the virion-containing culture supernatants were pre-incubated at 37uc for 1 h with mabs at various concentrations. the virus-mab mixtures were added to hos-cd4-ccr5 cells and incubated for 3 days at 37uc. a similar protocol was used for supernatants screening using tzm-bl cells [45] . the cells were then lysed with britelite reagent (perkin-elmer) and the relative light units in the cell lysates were determined on a luminometer microplate reader (veritas, turner biosystems). the 50% inhibitory dose (ic50) was defined as the sample concentration at which relative luminescence units were reduced 50% compared to virus control wells. some of the neutralization data for the reference mabs b12, 2g12, 2f5 and 4e10 are taken from previous analyses [41, 42] carried out under identical standardized conditions to those used for the new mabs reported here. a multi-cycle virus replication assay was used to measure the neutralization of replication competent luciferaseencoding viruses in 5.25.egfp.luc.m7 cells [52] . this cell line is a genetically engineered clone of cemx174 that expresses multiple siv and hiv-1 entry receptors (cd4, ccr5, cxcr4, gpr15/bob) [53] . for the neutralization assay, 5000 tissue culture infectious dose 50 (tcid 50 ) of virus was incubated with multiple dilutions of test sample in triplicate for 1 hr at 37uc in a total volume of 150 ml in 96-well flat-bottom culture plates. a 100 ml suspension of cells (5610 5 cells/ml of growth medium containing 25 mg deae dextran/ml) was added to each well. plates were incubated until approximately 10% of cells in virus control wells were positive for gfp expression by fluorescence microscopy (approximately 3 days). at this time, 100 ml of cell suspension was transferred to a 96-well white solid plate (costar) for measurements of luminescence using the britelite luminescence reporter gene assay system (perkinelmer life sciences). plasma samples were obtained from patients entered into the antwerp and london cohorts who were infected with a variety of hiv-1 clades. patients were selected for mab preparation on the basis of the clade of their infecting virus (predominantly non-b clade), and on the ability of their plasma to neutralize, to .80% at a 1/20 dilution, members of a selected panel of tier 2-type clade a, b, c, crf02_ag, ae, f and bf viruses in both a pbmcbased infectious primary isolate virus assay [54] and in the tzmbl-based recombinant pseudovirus assay [55] . in some cases only a restricted plasma neutralization analysis was possible due to limiting amounts of patient plasma. twelve and nine infected donors from the london and antwerp cohorts respectively were selected for b cell immortalization. summarized clinical and virological data including hiv-1 clade, viremia, cd4 + t cell counts, therapy and breadth of neutralization are shown in table s1 . igg + memory b cells were isolated from the selected donors and immortalized with ebv in the presence of cpg as described [41] . the immortalization efficiency of memory b cells from hiv-1 patients was significantly lower than that of non-hiv-1-infected donors (3% versus 20%, n = 21, p,0.001). this finding may be explained by the recent report that hiv-specific b cells are present within a population of ''exhausted'' memory b cells, characterized by the expression of inhibitory receptors and low levels of cd21 [56, 57] . to maximize detection of antibodies recognizing conserved features, cell culture supernatants were screened by parallel highthroughput elisas using five recombinant hiv-1 env proteins, including trimeric gp140 [48] , monomeric gp120 and gp41 recombinant ectodomains. from the 21 donors interrogated we selected 58 b cell clones producing mabs that bound to at least one of the screening antigens. the mabs were purified and further characterized for binding specificity and neutralizing activity using an extended panel of recombinant env proteins and pseudoviruses representative of several hiv-1 clades with diverse coreceptor usage, geographic origin and conformation. the binding data for each mab are displayed in figure s1 and expressed as half-maximal binding concentrations at equilibrium (k 50 ), which approximate to the equilibrium dissociation constants [58] . of the 58 mabs, 37 bound to gp120 and 21 to gp41. several gp120-specific and gp41-specific mabs showed a broad pattern of reactivity with most recombinant proteins, although usually within a broad range of k 50 values. overall, there was no relationship between the donor's hiv-1 clade and the clade specificity of the isolated mabs. the neutralizing activity of the mabs was initially measured using 20 pseudotyped hiv-1 primary isolate envs characterized by different sensitivity to neutralization [44, 45] . the mabs were tested at a fixed concentration (100 mg/ml) in a luciferase-based neutralization assay using hos-cd4.r5 as target cells. out of the 46 mabs tested against the whole virus panel, 37 showed neutralizing activity on at least one isolate ( figure 1a) . three mabs, hj16, hgn194 and hk20, stood out for their breadth of neutralizing activity, neutralizing 10, 11 and 17 out of the 20 pseudoviruses, respectively ( figure 1a) . these mabs were further characterized in the same assay for their potency ( figure 1b) . hj16 showed high neutralizing activity, while hgn194 and hk20 were less potent. in addition, while most antibodies preferentially neutralized tier-1 isolates, hj16 preferentially neutralized tier-2 isolates ( figure 1a-b) . since it has been reported that hiv-1 neutralization is target cell type-sensitive [59, 60] , we next used tzm-bl as target cells and titrated the mabs against a larger panel (n = 46) of predominantly (38/46) tier-2 pseudoviruses (table s2) . generally the results obtained with the extended panel confirmed the previous results and showed that several mabs had potent neutralizing activity, with ic 50 values ,20 ng/ml. a striking exception was hk20, which neutralized 17/20 pseudoviruses in the hos-based assay, but only 3/46 pseudoviruses in the tzm-bl based assay. we established the breadth of neutralization of these three novel mabs by comparing them in the tzmbl assay with the five mabs currently considered to be the most broadly reactive, namely b12, 2g12, 2f5, 4e10 and 447-52d. we used a large multi-clade panel comprising 10 tier-1 and 82 tier-2 isolates including clade b early-transmitted viruses [61] . the three new mabs showed a pattern that was clearly distinct from that of previously described mabs (figure 2 ). in particular, hgn194 neutralized with high potency all tier-1 viruses from clade a, b and c and 11% of tier-2 viruses. by contrast hj16 neutralized only 1 out of 10 tier-1 viruses, but neutralized 39% of tier-2 viruses. thus hj16 is comparable to b12 and 2f5 in terms of percentage of tier-2 isolate neutralization and is superior to 2g12 and 447-52d. 4e10, as previously reported [27, 41] showed an extremely broad pattern of reactivity. taken together the above results indicate that novel neutralizing mabs can be isolated from memory b cells of nonclade b hiv-1-infected individuals, some of which display relatively broad neutralizing activity. since one of our primary aims was to isolate and characterize novel mabs with unusually broad and potent neutralization activity, we focused our attention on hj16, hk20 and hgn194. mab hj16, derived from a donor infected with clade c, has a unique neutralization profile with potent and selective neutralization of multiple tier-2 pseudoviruses ( figure 1b and figure 2 ). when compared with the cd4bs-specific mab b12 for gp120 binding, hj16 showed similar binding curves ( figure 3a ) and inhibited to a comparable extent the binding of gp120 to solid-phase scd4 ( figure 3b , ic 50 values of 1.57 and 1.16 mg/ml, respectively). however, cross-competition analysis of hj16 and b12 for binding to gp120 revealed incomplete heterologous inhibition, with plateau values of approximately 80% ( figure 3c-d) . these results suggest that hj16 and b12 recognize related but non-overlapping cd4bsproximal epitopes. to further characterize hj16 specificity we measured its binding to two yu2 gp120 mutants: the d368r mutant, which is not bound by cd4 or cd4bs-specific mabs [21] and the i420r mutant, which is not recognized by cd4i-specific mabs [62] . as already reported, b12 bound the i420r cd4i mutant, but failed to recognize the d368r cd4bs mutant. by contrast, hj16 bound both mutants and indeed bound better to the d368r cd4bs mutant compared to the wild-type molecule ( figure 3e-f) . attempts to map the epitope by pepscan analysis using overlapping linear peptides were unsuccessful (not shown), consistent with hj16 recognition of a discontinuous epitope. hj16 and b12 neutralized 1/10 and 8/10 tier-1 and 32/82 and 35/82 tier-2 pseudoviruses, respectively ( figure 2) . interestingly, 22/32 tier-2 isolates neutralized by hj16 were not neutralized by b12, and reciprocally 24/35 tier-2 isolates neutralized by b12 were not neutralized by hj16. another interesting finding is that hj16 does not discriminate between clades as much as b12, 2g12 and 2f5 (e.g. b12 and 2g12 rarely neutralize clade a isolates while 2f5 and 2g12 rarely neutralize clade c isolates, table s3 ). these results reveal a largely nonoverlapping pattern of reactivity of hj16 and b12 and suggest that the combination of these two mabs could be effective against a dominant fraction of hiv-1 isolates (57/82, i.e. 69%). taken together these results are consistent with hj16 recognition of a surface proximal to the cd4bs domain within gp120, but with an epitope completely distinct from that recognized by b12. mab hgn194, isolated from a donor infected with crf02_ag clade, neutralized 11/20 pseudoviruses in the hos-based assay and 19/92 pseudoviruses in the tzm-bl-based assay. of note, hgn194 neutralized all tier-1 isolates tested (10/10 neutralized) across clades a, b, c and recombinant ag and bc. using pepscan analysis with linear and cyclic peptide libraries of gp120 the epitope recognized by hgn194 was mapped to the sequence rrsvrigpgqtf in the crown of the v3-loop ( figure 4a) . similarly, the epitope of 7 additional gp120specific mabs was mapped to the same v3 region using different peptide libraries generated from the sequence of the isolate to which each mab was mostly reactive ( figure 4a) . the minimal sequence recognized by these mabs ranged from 7 to 17 amino acids and, with a single exception, comprised the g(a)pgr/q/k sequence, which is modeled to interact with ccr5 or cxcr4 during the viral entry process [63] . however, in contrast to hgn194 which neutralizes with high potency all tier-1 isolates tested, the other v3-specific mabs neutralized only a few tier-1 isolates ( figure 1a -b and figure 2 ), similar to the activity of other recently characterized v3 loop-specific mabs derived from non-b clade infected individuals [38] . we confirmed the neutralization activity of hgn194 in an m7 cell-based assay challenged with replication-competent hiv-1 viruses carrying a luc reporter [52] and we observed that 3/5 clade b viruses were neutralized (table s5) . to better characterize the epitope recognized by hgn194 we performed a replacement scanning of each position with the 18 complementary amino acids. this analysis revealed only 3 positions (rrsvrigpgqtf) where amino acid substitutions abrogated binding. remarkably, only one mutation out of the 21 found in viral isolates (i to m in position 6) affected binding of hgn194 ( figure 4b-c) . however, we observed that several hiv-1 isolates that were not neutralized by hgn194 encoded the same amino acid sequence shared by other hiv-1 isolates that were neutralized ( figure 4b ). for instance, the epitope rksvrigpgqtf on 93mw965.26, which is strongly neutralized by hgn194 (i.e. ic50 ,0.02 mg/ml), is shared with du422.1, zm197m.pb7 and du172.17, isolates not neutralized by hgn194. this implies that the hgn194 v3 epitope is unavailable for antibody recognition on the assembled trimer of these non-neutralized isolates. taken together these results indicate that hgn194 has unusual potency and breadth for a v3-specific monoclonal antibody. of note, hgn194 appears to have a broader reactivity than 447-52d since it neutralizes all tier-1 isolates and 11% of tier-2 isolates, while 447-52d neutralizes 88% of tier-1 and 4% of tier-2 isolates (figure 2 and table s3 ). another v3 mab with unusual breadth of activity is f425-b4e8 [37] . we have not compared hgn194 with f425 here, but f425-b4e8 appears somewhat broader than 447-52d, neutralizing 1 clade c and 2 clade d pseudoviruses [37] . further comparison between f425-b4e8 and hgn194 is difficult as different viruses and assays were used to determine neutralization breadth and potency. hk20, an hr-1 specific mab with target cell-specific neutralizing activity hk20 was initially characterized as a gp41-specific mab with broad neutralizing activity in the hos-based assay but lacking robust activity in the tzm-bl assay. to map the epitope, we tested hk20 against all overlapping 15-mer peptides of the extracellular region of hiv-1 gp41. hk20 bound peptide qqhllqltvwgikql that overlaps the hydrophobic pocket sequence of hr-1 ( figure 5a ). the specificity of hk20 was confirmed by immunoprecipitation of the 5-helix bundle (5hb) construct [47] and of a trimeric hr-1 construct that includes the gp41 fusion peptide, indicating that the fusion peptide does not interfere with hk20 binding ( figure 5b ). in elisa, hk20 bound to 5hb and hr-1 gp41 constructs with k 50 values of 210 and 95 ng/ml, respectively and also to the gp41 ectodomain, although with lower avidity (1.27 mg/ml) ( figure 5c ), a finding that may be due to the partial unfolding of solid phase-bound gp41. taken together, the above results indicate that hk20 recognizes a highly conserved site within the hr-1 region of gp41. since hr-1 occupies a restricted surface only transiently exposed during the hiv-1 entry process, we hypothesized that accessibility of the hk20 target epitope might be limited by the size of an igg molecule. we therefore compared intact igg and fab fragments of hk20 for their capacity to neutralize a panel of 22 pseudoviruses in the hos-based assay. the hk20 fab fragments showed markedly increased breadth and potency compared to igg, being able to neutralize 21/22 isolates with ic 50 values ranging from 0.01 to 5.7 mg/ml ( figure 5d -e and table s4 ). by contrast, hk20 igg neutralized 19/22 isolates with ic 50 values ranging from 1.46 to 84 mg/ml. in addition, when the hk20 fab fragment was tested in the tzm-bl assay it neutralized 10 out of 33 isolates with ic 50 values ranging between 0.4 and 8.8 mg/ml (table s2) , thus suggesting that the lack of activity observed with hk20 igg was possibly associated to a different susceptibility of this cell line to this type of entry inhibitors. overall, these findings suggest that the reduced size of the fab molecule allows increased access to the hr-1 region and imply that hr-1 accessibility is largely dependent on the target cell used. this is similar in concept to the enhanced neutralization profiles of fab and scfv specific for the cd4i surface [39] . further studies using m7 cells [52] challenged with replication-competent hiv-1 viruses carrying a luc reporter showed that hk20 exhibits broad neutralization consistent with the results from the hos cell assay but unlike the tzm-bl assay, being able to neutralize 4 out 5 viruses tested (table s5) . the epitopes recognized by the 55 remaining mabs were mapped using three experimental approaches: i) cross-competition against mabs of known specificity or soluble cd4; ii) binding to gp120 mutants or gp41 constructs representing different conformational intermediates; iii) pepscan analysis. the results of the analysis performed on the gp120-specific and gp41-specific mabs are presented in table 1 and table 2 , and the data are summarized in a pie chart in figure 6 . thirty-seven mabs bound to gp120 targeting primarily the cd4bs and the v3 loop and to a lesser extent the cd4i site ( table 1) . a first group of mabs was assigned to the v3 loop based on cross-competition with two v3 loop specific mabs (hr10 and hga9). this group comprised 8 mabs, which were already mapped to the v3 loop using the pepscan analysis approach, and 7 additional mabs that were not analyzed by peptide scanning (i.e. hgf9, hgt4, hgp21, hgp51, hgw48, hgd129 and hgp27). four mabs (hx44, hgp105, hgw7, hgy38) reacted with wt and cd4bs yu2 mutants but failed to bind the cd4i mutant, and therefore were assigned to the cd4i cluster. notably, one mab (hgp68) was mapped by pepscan analysis to a novel epitope in the v2 loop (tvyalfyrldivp) and neutralized 4 tier-1 isolates (table s2) . fifteen other mabs were assigned tentatively or conclusively to the cd4bs based on their capacity to inhibit gp120-scd4 binding. most of these mabs competed other cd4bs-specific mabs, such as b12 and hj16, while in some cases this assay could not be performed due to lack of epitope expression on the gp120 proteins used. furthermore, this group of mabs showed variable reactivities with the yu2 mutants. while most of them did not bind the d368r mutant similar to b12, others including hj16, bound this mutant avidly. in addition, most of these new cd4bs-specific mabs also showed decreased binding to the cd4i mutant, suggesting that they may span a broader, as yet undescribed region that includes elements of the cd4bs and cd4i sites. finally, for 2 mabs these analyses did not provide any relevant information. twenty-one mabs bound to gp41 by targeting primarily the immunodominant c-c region, the hr-1 and the region recognized by 5f3 mab [64] (table 2) . mabs hgk129, hgn146 and hgn35 were assigned to the c-c loop since they competed with 3d6 [43] , reacted with an hr2 construct and recognized synthetic peptides within this region. mabs hgw17 and hgy25 were provisionally assigned to the c-c region since they competed with 3d6. these data are consistent with the immunodominance of the c-c region and with the notion that the antibody response against this region overlaps with the 3d6 epitope. several mabs competed with 5f3 and hk20, and bound both the hr-1 and 5hb constructs, suggesting that they may bind between the fusion peptide and the hr-1 region. hgb33 was provisionally assigned to the hr-1 region according to competition with hk20 and binding to the 5hb construct. other mabs bound specifically the 5hb construct but did not compete with any of the mabs tested, thus indicating that the complete hr-1 coiled coil region exposed in 5hb harbors antibody epitopes available for b cell recognition in the gp41 pre-hairpin conformation [65, 66] . the concomitant binding to recombinant gp41 suggests that the latter may partially resemble the gp41 prefusion state. of note, three gp41 binding mabs (hgp40, hgp48 and hgy50) neither competed with any of the mabs tested, nor bound to any constructs representing pre-hairpin conformations or native gp140 ( figure s1 ), indicating that they recognize as yet uncharacterized regions that are only available in the recombinant gp41 protein. interestingly, mab hgf24 was assigned by pepscan analysis to an epitope in the c-terminal region of hr-2 (tnliytlieesqn), proximal to the 2f5 epitope, and neutralized 4 tier-2 isolates (table s2) . this mab competed with hk20 for gp41 binding, in spite of their distinct cognate specificities (hr-2 and hr-1, respectively). this finding would be consistent with the proximity of hr-1 and hr2 in the six-helix bundle structure. finally, although the gp41 protein used in the primary screening includes the 2f5 and 4e10 epitopes, no mper specific mabs were isolated, reinforcing the idea that this portion of gp41 is poorly immunogenic in humans [21, 22] in conclusion, regardless of their limited breadth of neutralization, this extended panel of human mabs may represent a useful tool for understanding the molecular basis of env recognition in humans. using an improved ebv immortalization method combined with a broad screening strategy we isolated from memory b cells of hiv-1 infected donors 58 mabs that cover an extensive antigenic surface of env. of these, 37 neutralized at least one of the isolates tested and 3 mabs in particular bound to the cd4bs, v3 crown and hr-1, showing considerable neutralizing breadth against a panel of hiv-1 pseudoviruses of different clades and spanning tier-1 and tier-2 isolates. several studies have questioned the existence of individual broadly neutralizing antibody specificities as part of a normal immune response to hiv-1 infection. in this respect, b12 was isolated from a phage library, while 2f5, 4e10 and 2g12, although isolated from memory b cells, do not appear to have a counterpart in human sera [22] . in a recent study, nussenzweig and coworkers used trimeric gp140 to isolate antigen-binding memory b cells from which 500 antibody sequences were retrieved by single-cell pcr [62] . surprisingly, although the b cell donors had broadly neutralizing serum activity, none of the mabs isolated had this property. this raised the possibility that broad serum neutralizing activity results from multiple clonal responses each of unique epitope specificity but restricted breadth of viral strain recognition. however, an alternative interpretation might be that since available recombinant trimeric antigens vary in structure, the 'bait' used for b cell isolation was not optimal for enriching those secreting neutralizing antibodies. our results indicate that monoclonal antibodies with a limited spectrum of neutralizing activity can be easily isolated, while those with a broad pattern of cross-clade reactivity are rare, in line with the study of walker et al [40] and consistent with inferences from serum neutralization specificity mapping analyses [21] . in this respect our study demonstrates the feasibility of the ebv immortalization method, which is limited only by the size of the blood samples obtained (20-50 ml of peripheral blood). the characterization of the specificity and neutralizing activity of this new panel of human mabs reveal some interesting features of the human antibody response to hiv-1 within a population of hiv-1-infected donors. first, most of the gp120-specific mabs showed neutralizing activity (37 out of 58), indicating that they bind to sites available on the functional env trimer. the selection of a highpercentage of trimer binding specificities may be a consequence our use of trimeric env antigens during the screening of the evb-b cell supernatants. the second aspect relates to gp41-specific antibodies, which, with a few remarkable exceptions, were not neutralizing. interestingly the non-neutralizing antibodies bound gp41 in the context of the recombinant trimer, whereas the most effective neutralizing mabs bound to gp41 fusion intermediates. the three new neutralizing mabs hj16, hgn194 and hk20 show some interesting features. hj16 showed a breadth of neutralizing activity comparable to, and generally complementary to b12, which aside from pg9 and pg16 [40] is currently the most potent of the relatively broad neutralizing antibodies available. hj16 also showed selective neutralization of multiple tier-2 isolates, making it particularly relevant as a template for vaccine design. hgn194 binds to an extremely conserved epitope in the v3 crown, and neutralizes all tier-1, but only 11% of tier-2 isolates tested. hgn194 showed broader activity than the well-characterized v3-specific mab 447-52d against a panel of 92 pseudoviruses. the preference for tier-1 isolates characteristic of v3-specific mabs is consistent with the idea that the v3 loop is displayed to varying degrees in the context of the native trimeric env protein of individual isolates [67, 68] and with the recent observation that scd4 broadens neutralization of v3 mabs [69] . the results obtained with hk20 are particularly intriguing, since this mab has a broad pattern of neutralization observed with hos but not tzmbl target cells. this mab recognizes a highly conserved epitope in the hr-1 trimer and it is more effective as an fab fragment as compared to intact igg, a fact that is may be explained by the limited accessibility of the epitope, which is likely to be exposed only transiently on the cell surface during the viral entry process [70] . intriguingly, a recent study indicates that in tzm-bl cells hiv-1 pseudoviruses penetrate the cytoplasm from within endosomes rather than from the cell surface [71] , a finding that might help explain the lack of activity of hk20 in the tzmbl assay if binding of this mab is affected by the endosomal environment. further studies on primary cells will be required to address the relative importance of these two entry pathways and to establish whether hk20 may be capable of exerting a broad and potent neutralizing activity in vivo. preliminary studies using m7 cells [52] challenged with replication-competent hiv-1 viruses cross competition with biotinylated mabs of known specificity: +, 90-100% inhibition, +/2, 50-90% inhibition, 2, ,50% inhibition. binding to different gp41 constructs by elisa. minimal linear epitopes using the pepscan analysis and specificity assignment. c-c, c-c loop; 5hb, 5-helix bundle; fp, fusion peptideshown is also the number of isolates neutralized in the hos-based and tzm-bl based assays. doi:10.1371/journal.pone.0008805.t002 carrying a luc reporter showed that hk20 exhibits reasonably broad neutralization consistent with the results from the hos cell assay, and similar to fab hk20 in the tzm-bl cell assay. whereas the tzm-bl assay is sensitive for certain antibodies such as cd4bs mabs, some mabs such as 2g12 to other epitopes are less sensitive to neutralization in this assay [72] , perhaps owing to the unnaturally high level of ccr5 expression on the tzm-bl cells [60] . in this respect, low coreceptor expression levels have been associated with delayed fusion kinetics and hence enhanced virus sensitivity to hr-1 binding inhibitors, such as t-20 [73] . therefore, despite the advantages of high throughput with the tzm-bl assay, alternative neutralization assays might have closer relevance to the in vivo situation. in conclusion, we have shown that in hiv-1-infected individuals a high proportion of hiv-specific b cells produce neutralizing antibodies but only a few possess neutralizing activity with relatively broad neutralization coverage. in the short term, some of these reagents will be tested for their ability, singly or in combination, to prevent hiv-1 or siv transmission in a macaque challenge model as done previously with other mabs [5, 6, 7, 8, 9] . in the longer term, and combined with an atomic-level analysis of epitope specificity, some of these mabs may facilitate the templatebased design of immunogens for the development of a vaccine able to induce neutralizing antibodies against the wide range of hiv-1 strains present in the global pandemic. figure s1 binding of gp120 and gp41-specific mabs to a panel of 15 recombinant env proteins from different clades. found at: doi:10.1371/journal.pone.0008805.s001 (0.47 mb pdf) figure 6 . epitope specificity and neutralizing activity of the mab panel. the pie chart shows the specificity of the 58 mabs isolated from the 21 interrogated individuals. the fraction of antibodies with neutralizing activity against at least one isolate is indicated in parentheses. partial cd4bs, incomplete inhibition of cd4 binding; hr-1/ fp, epitope located in between hr-1 and the fusion peptide as determined by 5f3 competition; hr-1/5hb, recognition of trimeric hr-1 and 5-helix bundle; 5hb, 5 helix bundle; c-c region, gp41 immunodominant region; hr-2, heptad-repeat 2; nd, not defined epitopes within gp120 or gp41. doi:10.1371/journal.pone.0008805.g006 antibody neutralization and escape by hiv-1 rapid evolution of the neutralizing antibody response to hiv type 1 infection in vivo and in vitro escape from neutralizing antibodies 2g12, 2f5, and 4e10 delay of hiv-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies protection of macaques against pathogenic simian/human immunodeficiency virus 89.6pd by passive transfer of neutralizing antibodies protection of macaques against vaginal transmission of a pathogenic hiv-1/siv chimeric virus by passive infusion of neutralizing antibodies antibody protects macaques against vaginal challenge with a pathogenic r5 simian/ human immunodeficiency virus at serum levels giving complete neutralization in vitro human neutralizing monoclonal antibodies of the igg1 subtype protect against mucosal simian-human immunodeficiency virus infection complete protection of neonatal rhesus macaques against oral exposure to pathogenic simian-human immunodeficiency virus by human anti-hiv monoclonal antibodies fc receptor but not complement binding is important in antibody protection against hiv neutralization of primary human immunodeficiency virus type 1 isolates by the broadly reactive anti-v3 monoclonal antibody antibodies, viruses and vaccines hiv vaccine design and the neutralizing antibody problem medicine. the need for a global hiv vaccine enterprise consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection regulation of human natural killer cell migration and proliferation by the exodus subfamily of cc chemokines human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications a large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals efficient neutralization of primary isolates of hiv-1 by a recombinant human monoclonal antibody recognition properties of a panel of human recombinant fab fragments to the cd4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1 broad hiv-1 neutralization mediated by cd4-binding site antibodies profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes b and c dissection of the carbohydrate specificity of the broadly neutralizing anti-hiv-1 antibody 2g12 antibody domain exchange is an immunological solution to carbohydrate cluster recognition molecular characterization of five neutralizing anti-hiv type 1 antibodies: identification of nonconventional d segments in the human monoclonal antibodies 2g12 and 2f5 the mannose-dependent epitope for neutralizing antibody 2g12 on human immunodeficiency virus type 1 glycoprotein gp120 comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2f5 generation of human monoclonal antibodies against hiv-1 proteins; electrofusion and epstein-barr virus transformation for peripheral blood lymphocyte immortalization anti-human immunodeficiency virus type 1 (hiv-1) antibodies 2f5 and 4e10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize hiv-1 broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41 analysis of neutralization specificities in polyclonal sera derived from human immunodeficiency virus type 1-infected individuals factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv-1 antibodies the v3 loop is accessible on the surface of most human immunodeficiency virus type 1 primary isolates and serves as a neutralization epitope crossclade neutralizing activity of human anti-v3 monoclonal antibodies derived from the cells of individuals infected with non-b clades of human immunodeficiency virus type 1 analysis of the neutralization breadth of the anti-v3 antibody f425-b4e8 and re-assessment of its epitope fine specificity by scanning mutagenesis cross-clade neutralizing activity of human anti-v3 monoclonal antibodies derived from the cells of individuals infected with non-b clades of human immunodeficiency virus type 1 access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1 broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1 characterization of monoclonal antibodies to human immunodeficiency virus type 1 gp41 by hiv-1 polypeptides expressed in escherichia coli human immunodeficiency virus type 1 env clones from acute and early subtype b infections for standardized assessments of vaccine-elicited neutralizing antibodies genetic and neutralization properties of subtype c human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in southern africa design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery protein design of an hiv-1 entry inhibitor expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of hiv-1 antigenic structure of the central conserved region of protein g of bovine respiratory syncytial virus rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces human monoclonal antibodies specific for conformation-sensitive epitopes of v3 neutralize human immunodeficiency virus type 1 primary isolates from various clades evaluating neutralizing antibodies against hiv, siv and shiv in luciferase reporter gene assays association of chemokine-mediated block to hiv entry with coreceptor internalization neutralization kinetics of sensitive and resistant subtype b primary human immunodeficiency virus type 1 isolates recommendations for the design and use of standard virus panels to assess neutralizing antibody responses elicited by candidate human immunodeficiency virus type 1 vaccines b cells in hiv infection and disease evidence for hiv-associated b cell exhaustion in a dysfunctional memory b cell compartment in hiv-infected viremic individuals identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody international network for comparison of hiv neutralization assays: the neutnet report increased efficacy of hiv-1 neutralization by antibodies at low ccr5 surface concentration identification and characterization of transmitted and early founder virus envelopes in primary hiv-1 infection broad diversity of neutralizing antibodies isolated from memory b cells in hivinfected individuals structure of a v3-containing hiv-1 gp120 core generation of human monoclonal antibodies against hiv-1 proteins; electrofusion and epstein-barr virus transformation for peripheral blood lymphocyte immortalization atomic structure of the ectodomain from hiv-1 gp41 core structure of gp41 from the hiv envelope glycoprotein cryptic nature of a conserved, cd4-inducible v3 loop neutralization epitope in the native envelope glycoprotein oligomer of ccr5-restricted, but not cxcr4-using, primary human immunodeficiency virus type 1 strains antibodies that are cross-reactive for human immunodeficiency virus type 1 clade a and clade b v3 domains are common in patient sera from cameroon, but their neutralization activity is usually restricted by epitope masking soluble cd4 broadens neutralization of v3-directed monoclonal antibodies and guinea pig vaccine sera against hiv-1 subtype b and c reference viruses hiv infection does not require endocytosis of its receptor hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes hiv sensitivity to neutralization is determined by target and virus producer cell properties sensitivity of hiv-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics we thank john mascola and richard wyatt for generously providing yu2 mutant molecules, marlen aasa-chapman for experimental support, jane anderson and werner leber who managed the follow up of patients and provided blood samples; we are also thankful to all patients who participated in this study. we would like to express our thanks to vasia dekou who coordinated reagent supply and the work within our consortium. key: cord-003307-snruk3j2 authors: schmidt, julius j.; lueck, catherina; ziesing, stefan; stoll, matthias; haller, hermann; gottlieb, jens; eder, matthias; welte, tobias; hoeper, marius m.; scherag, andré; david, sascha title: clinical course, treatment and outcome of pneumocystis pneumonia in immunocompromised adults: a retrospective analysis over 17 years date: 2018-11-19 journal: crit care doi: 10.1186/s13054-018-2221-8 sha: doc_id: 3307 cord_uid: snruk3j2 background: despite modern intensive care with standardized strategies against acute respiratory distress syndrome (ards), pneumocystis pneumonia (pcp) remains a life-threatening disease with a high mortality rate. here, we analyzed a large mixed cohort of immunocompromised patients with pcp, with regard to clinical course and treatment, and aimed at identifying predictors of outcome. methods: this was a single-center retrospective analysis in a tertiary care institution across 17 years. diagnosis of pcp required typical clinical features and microbiological confirmation of pneumocystis jirovecii. epidemiological, clinical, laboratory and outcome data were collected from patient records. results: a total of 52,364 specimens from 7504 patients were sent for microbiological assessment (3653 with clinical suspicion of pneumocystis pneumonia). pcp was confirmed in 240 patients, about half of them hiv positive (52%). the remaining subjects were either solid organ transplant recipients (16.3%) or suffered from malignancy (15.8%) or autoimmune diseases (11.7%). of note, 95% of patients with pcp were not receiving chemoprophylaxis. overall in-hospital mortality was 25.4%, increasing to 58% if icu admission was required. multivariable regression identified lactate dehydrogenase (ldh) as predictor of in-hospital mortality (adjusted or 1.17 (95% ci 1.09–1.27), p < 0.0001). mortality in ldh quartiles increased from 8% to 49%, and a cutoff value of 495 u/l predicted mortality with sensitivity and specificity of 70%. with regard to treatment, 40% of patients received trimethoprim-sulfamethoxazole at doses that were lower than recommended, and these patients had a higher mortality risk (hr 1.80 (95% ci 1.10–3.44), p = 0.02). conclusions: pcp remains a life-threatening disease among immunocompromised patients. about half of patients with pcp do not have hiv infection. initial ldh values might serve as a stratifying tool to identify those patients at high risk of death among patients with hiv and without hiv infection. electronic supplementary material: the online version of this article (10.1186/s13054-018-2221-8) contains supplementary material, which is available to authorized users. pneumocystis pneumonia (pcp) is a severe disease with high morbidity and mortality, which almost exclusively affects immunocompromised patients. pcp has long been known for its high prevalence among human immunodeficiency virus (hiv)-positive patients [1] . since the implementation of combination antiretroviral therapies (cart) and chemoprophylaxis its incidence has been continuously decreasing [2] . nowadays, most patients with hiv-associated pcp are treatment-naïve with very low cd4 cell counts; some of these patients do not know that they are hiv positive until they attend hospital [3] . on the other hand, pcp is also frequently diagnosed in non-hiv-positive patients as immunosuppressive regimens are being increasingly used in a wide range of patient populations. consistently, the incidence of pcp in non-hiv-positive patients is increasing [2] . pneumocystis infections have first been described in preterm infants following world war ii [4] and in patients with malignancies in the late 1960s [5] , more than a decade before the hiv epidemic emerged. defects in cell-mediated immunity and use of glucocorticoids are among the strongest risk factors for the development of pcp [6, 7] . the epidemiology of pcp has been described in several retrospective studies [2, 8] . a study from the mayo clinic in 116 non-hiv-positive patients found that most frequent underlying diseases were hematological malignancies (30%), organ transplantation (25%), autoimmune disease (22%), solid tumors (13%) and other reasons [6] . although, most of these reports were published more than a decade ago and might not reflect the current epidemiological situation, a report from france was published a few years ago [9] . roux et al. reported overall mortality of 17.4%, which was significantly higher in non-hiv-positive patients (27%) than in hiv-positive patients (4%) [9] . a few parameters such as age, prior episode of pcp, low cd4 cell count, lactate dehydrogenase (ldh), and coinfections have been reported to predict unfavorable outcome in patients with hiv infection [10] [11] [12] . reports on outcome predictors in non-hiv-positive patients are sparse. based on the high burden of pcp and the likelihood of unfavorable outcome particularly in non-hiv-positive patients, chemoprophylaxis with trimethoprim-sulfame thoxazole (tmp-smx) is recommended in high-risk populations [13] . tmp-smx is also the treatment of choice for pcp. adjunctive corticosteroid therapy is recommended in hiv-positive patients with severe respiratory failure [14, 15] . however, while beneficial outcomes of higher dosage of corticosteroids in hiv-positive patients during pcp are reported [16] , the outcome of using corticosteroids in non-hiv-positive patients is not clear [17] . we here report comprehensive epidemiological, clinical, laboratory, therapeutic and outcome data on 240 cases of pcp, including a high percentage of non-hiv-positive patients, in a tertiary care center over the last 17 years. additionally, we aimed at identifying predictors of outcome. we performed a retrospective, single-center cross-sectional analysis of all patients with a positive finding of pneumocystis jirovecii on direct immunofluorescence testing or detection by diff-quick staining in the bronchial washing fluid or broncho-alveolar lavage (bal) fluid, from january 2000 to june 2017. our hospital is a university tertiary care center with approximately 1500 beds. written informed consent was waived by the ethics committee due to the anonymized retrospective nature of the analysis. all bronchial washing fluids or bal samples from patients clinically suspected to have p. jirovecii pneumonia were evaluated within the study period. for every patient, clinical data on demographic characteristics, underlying disease, status of immune competence, treatment regimens of immunosuppression, pcp therapy regimen and mortality, were gathered in the study database. date of diagnosis was defined as the date of microbiological confirmation. p. jirovecii direct immunofluorescence was performed using the monofluo kit p. carinii (biorad laboratories, years 2000 to 2016) or the detect if pneumocystis carinii kit (axis-shield diagnostics ltd., year 2017). diff quick staining was performed using the stain sets provided by dade behring or siemens ag, respectively. standard descriptive statistics were used to summarize the data (e.g. continuous: mean ± standard deviation/ count: absolute and relative frequencies/time-to-event: kaplan-meier estimator). to identify predictors of in-hospital mortality or survival we applied logistic and cox proportional hazards models. consequently, both odds ratio (or) and hazard ratio (hr) estimates were reported. to obtain multivariable adjusted estimates, all main effects with univariate p values less than or equal to 0.15 were investigated simultaneously. in addition, confidence intervals (ci) were calculated with coverage of 95%. finally, we also created receiver operating characteristics (roc) curves for in-hospital death, which were summarized by area under the curve (auc) estimates. roc analyses were based on an increasingly complex (leave one out) logistic regression model with in-hospital mortality as the predicted outcome and the following predictors, which were all entered linearly: ldh alone, ldh + age, ldh + age + body mass index (bmi), and for ldh + age + bmi + estimated glomerular filtration rate (egfr). all reported p values are nominal and two-sided. in this explorative study, we applied a significance level of α = 0.05 (two-sided). all statistical analyses were done using graphpad prism 5.0 (la jolla, ca, usa) or r 3.4.2. during the observation period, 52,364 bal or bronchial washing fluid samples were investigated. a clinical suspicion of p. jirovecii infection was raised in 3652 of these specimens of which 252 (i.e. 6.9%) were microbiologically confirmed by immunofluorescence as positive for pcp. there were ultimately 240 patients enrolled into the study (12 were excluded due to incomplete data sets) (fig. 1 ). in the overall population, 67 patients were female (27.9%) and the average age was 45 ± 15 years. there were 125 patients (52%) with hiv infections; 39 (16.3%) had undergone solid organ transplant and 38 (15.8%) had undergone chemotherapy due to hematologic or oncologic malignancies: 28 patients (11.7%) received immunosuppressive therapy for rheumatoid autoimmune diseases (is/rd) and 10 patients (4.2%) had other underlying diseases (miscellaneous (misc)), e. g. common variable immune-deficiency (cvid) ( table 1) . five patients could be classified into multiple categories. after case reevaluation, patients were classified to the most recent active disease prior to the pcp event. the average cumulative incidence of pcp in our institution was 13 ± 5 cases per year, with a peak in the years 2005-2010 (additional file 1: figure s1a ). of the 240 patients with pcp, 41.7% were admitted to the intensive care unit (icu), with 36.6% in need of mechanical ventilation, 16.3% in need of renal replacement therapy (rrt), and 4.5% in need of extracorporeal membrane oxygenation (ecmo) support (fig. 2a) . the overall in-hospital mortality was 25.4%. the mortality was 58% in patients requiring icu treatment, 74.4% in patients requiring rrt and 81.8% in patients requiring ecmo support, and only 1.6% of patients died on regular (non-icu) wards (fig. 2b) . mortality during the observation time from 2000 to 2017 slightly fluctuated but did not trend toward an improvement in more recent years (additional file 1: figure s1b ). the underlying disease was associated with outcome ( table 1 ). the lowest mortality was observed in hiv-infected patients (12.8%); the respective mortality rates in the non-hiv group were 38.4% in solid organ transplant recipients, 30.0% in patients with rheumatic diseases and 44.7% in patients with hematologic -oncologic diseases, respectively (additional file 1: figure s1c ). figure 2c , d summarizes icu admission and icu mortality with regard to the etiology of the patients' immunosuppressive disease. of note, only 12 patients (5%) were receiving chemoprophylaxis with tmp-smx at the time when pcp was diagnosed ( table 1) . univariate regression analysis revealed that age, bmi, gfr and initial ldh were associated with death from pcp. both logistic and cox multivariable regression analyses identified ldh as a predictor of unfavorable outcome in pcp (table 2 , adjusted or 1.17 (95% ci 1.09-1.27) per 50 u/l, p < 0.0001, adjusted hr 1.07 (95% ci 1.04-1.10) per 50 u/l, p < 0.0001). the initial ldh levels were significantly higher in later non-survivors (443 ± 214 vs. 673 ± 373 iu/l, p < 0.0001, fig. 3a ) and in patients that required admission to the icu (411 ± 199 vs. 627 ± 328 iu/l, p < 0.0001, fig. 3b ). stratification of patients into ldh quartiles showed increasing (in-hospital) mortality rates among those. mortality ranged between 8% in the lowest and 49% in the highest ldh quartile (fig. 3c) . a roc curve for in-hospital death had an estimated auc of 0.724 (95% ci 0.65-0.80) (p < 0.0001 for ldh alone, additional file 1: figure s2 ). potential alternative cutoff values and their impact on usually reported statistics are summarized in additional file 2: table s1 for descriptive purposes. we used a cutoff of 496 iu/l for further analysis. patients with initial ldh below 496 iu/l had lower mortality than the overall population (13.1 vs. 25.4%) and lower mortality than those with initial ldh > 496 iu/l (13.1 vs. 43.9%, p < 0.001, fig. 3d, e) . the addition of other clinical quantitative variables to ldh in the auc model such as age, bmi and egfr did not improve the predictive value of ldh alone (additional file 1: figure s2 , additional file 2: table s2 ) in the overall cohort. however, when icu patients were analyzed separately, we observed that the addition of age as a variable increased the predictive performance of ldh alone from an auc of 0.61 (95% ci 0.49-0.72) to an auc of 0.71 (95% ci 0.60-0.81) (p = 0.024). of note, we did not detect any etiology subgroup that showed better performance in the roc analysis (additional file 1: figure s3 ), nor did we detect temporal trends when applying the ldh prediction models in three strata (2000) (2001) (2002) (2003) (2004) (2005) 2005 -2010, 2010-2017) (additional file 1: figure s4 ). fig. 3 influence of initial lactate dehydrogenase (ldh) as an outcome predictor. the scatter plot shows initial ldh levels at admission in later survivors (alive) and non-survivors (dead) (443 ± 214 vs. 673 ± 373 iu/l, p < 0.0001) (a) and in non-icu versus icu patients (411 ± 199 vs. 627 ± 328 iu/l, p < 0.0001) (b). c bar graph showing overall mortality (right y-axis) stratified for initial ldh quartiles (left y-axis) (q1, < 310; q2, 311-436; q3, 437-599; q4, > 600 iu/l). d mortality in all patients with pneumocystis pneumonia with ldh levels ≤ and > 496 iu/l (cutoff value was derived from roc curve of all patients, with sensitivity and specificity of 70%.) the gray area highlights the overall mortality of 24.8% without ldh risk stratification. e kaplan-meier analysis of survival in patients stratified for ldh ≤ and > 495 iu/l during 120 days (p log-rank test < 0.0001) there were 224/240 patients (93.3%) initially treated with the gold standard, tmp-smx. the remaining 16 patients had alternative regimens with chemotherapeutics from the second line, mostly due to side effects and intolerance of tmp/smx (e.g. atovaquone and/or pentamidine, fig. 1) . among the 224 patients treated with tmp-smx, 159 (71%) were dosed in the recommended range above 15 mg/kg bodyweight (bw) per day, while 65 patients (29%) received a dose lower than 15 mg/kg bw (additional file 1: figure s5a ). of note, these patients had a higher mortality risk (hr 1.80 (95% ci 1.10-3.44), p = 0.02). solid organ transplant (sot) recipients and patients with rheumatoid diseases were more likely to receive lower dosages (additional file 1: figure s5b ). on the other side, we observed that under-dosed patients also had significantly lower egfr (48.9 ± 43 ml/min vs. 104 ± 32 ml/min, p < 0.0001). interestingly, the absolute dose of tmp-smx was not different in survivors (16.9 ± 5.5 mg/kg) and non-survivors (15.6 ± 7.1 mg/kg, p = 0.93). still, kaplan-meier survival curves revealed a significant difference when patients were grouped into those treated with < 15 mg/kg and those treated with ≥ 15 mg/kg tmp-smx (additional file 1: figure s5c , p log-rang test = 0.02). however, tmp-smx dose did not independently predict outcome in the multivariable regression analyses. here we present the single-center experience with regard to epidemiology, treatment and outcome of pcp in a tertiary care center over a period of 17 years. a substantial group of our pcp cases (about 50%) were not related to hiv infections and these patients had a worse clinical course with higher icu admission and mortality rates than patients with hiv-associated pcp. three major non-hiv-positive groups were identified: (1) solid organ transplant recipients, (2) patients with malignancies and (3) patients with rheumatic diseases. pcp occurred almost exclusively in patients who did not receive chemoprophylaxis with tmp-smx. overall, about one quarter of all patients suffering from pcp did not survive the disease. in those who required intensive care (approximately 40% of all patients), the in-hospital mortality increased up to 58%. based on the -in principle -reversible nature of pcp, almost no patients were denied intensive care in the event of acute respiratory failure, as the in-hospital mortality rate in non-icu patients was 1.6%. moreover, we found that the extent of initial ldh elevation was a predictor of in-hospital mortality, and that in univariate comparisons, in-hospital mortality was higher in patients whose tmp-smx dose was below 15 mg/kg bw. non-hiv-positive populations at risk of pcp have been described before. the distribution across these risk groups varies widely (sot 3-31%, hematologic diseases 26-39%, rheumatic diseases 11-25%), which is likely due to the clinical emphasis in predominantly monocentric studies [9, [18] [19] [20] . several centers have reported a decrease in the percentage of hiv-infected patients among all cases of pcp in the era of cart [19, 21] . we did not confirm this decrease, nor did fillatre et al. [18] . our data showed that the clinical course and outcome of pcp differed between hiv-infected and non-hiv-infected patients. in general, hiv-infected patients require fewer icu admissions and have better overall survival than non-hiv-infected patients, which is in line with previous reports [9, 22] . the pcp mortality rate in hiv-positive patients was in the range of 1-15% [9, 18, 19] compared to 30-40% in hiv-negative patients [23] [24] [25] . the better outcome of hiv-positive patients might be the consequence of (1) lower age (− 3.8 years in our cohort), (2) fewer co-morbidities, (3) the reversible nature of the immune defect upon anti-viral treatment strategies and hypothetically (4) greater awareness of pcp in hiv-positive individuals, where it is the most common aids-defining disease. in mixed cohorts, icu admission and mechanical ventilation occurred in 22-40% and 13-22% of patients, respectively [9, 18, 22] . both events are frequently reported as negative predictors of survival. nevertheless, newer data on icu mortality in patients with pcp are scarce. only one recent study reported relatively high icu mortality in hiv-negative patients of 53% compared to 15% in hiv-positive patients [18] . mortality in patients with invasive mechanical ventilation was 60% and 28%, respectively, in these two groups [22] . compared to other reports [21, 26] , we had a relatively high icu admission rate of 25% in hiv-positive patients, which can in part be explained by the selective transfer of more patients with more progressive disease to our university hospital as a tertiary center. the literature is inconclusive on outcome predictors, but on multivariable analysis, a large prospective study from france demonstrated a significant survival benefit in younger patients [9] . although observed in hiv-positive patients before [27] , here we report for the first time that in a mixed population of hiv-positive and non-hiv-positive patients the initial levels of serum ldh at hospital admission are not only useful in the diagnostic evaluation [28] but may also be an independent predictor of survival. this might potentially help the clinician in early identification of the sickest patients at high risk of unfavorable outcome. with regard to the specificity/sensitivity, the ideal ldh cutoff value to predict outcome has yet to be defined. with regard to the actual treatment of pcp we found that a reduction in the recommended tmp-smx dose below 15 mg/kg bw (for whatever reason) might be associated with higher mortality (13.1 vs. 55.8%). given that this observation was not confirmed in the multivariable analysis, relevant confounders must be considered. our study has several limitations. this was a retrospective observational study based on the medical records of patients with pcp from a single institution. consequently, causal claims cannot be made. the observation that under-dosing with tmp-smx is associated with higher mortality might be confounded by a higher percentage of patients with renal impairment. more robust evidence from well-designed studies is needed to make firm conclusions or even to make implications about changes in standard pcp management. in summary, pcp is a rare but potentially fatal disease in immunocompromised patients with diseases of different etiology. about 50% of cases were non-hiv-associated. initial ldh levels -if validated by others -might be a useful predictor of in-hospital mortality, and tmp-smx treatment doses in patients at high risk of death (e.g. icu admission + ldh > 495 u/l) should probably not be reduced below 15 mg/kg bw. additional file 1: figure s1 . cumulative incidence of (a) and inhospital mortality in (b) pneumocystis pneumonia (pcp) at hannover medical school from 2000 to 2017. figure s2 . receiver operating characteristic (roc) curves for in-hospital mortality applied to the total sample. figure s3 . roc curves for in-hospital mortality applied to subgroups regarding underlying etiology of immunosuppression and for the icu cohort. figure s4 . roc curves for in-hospital mortality with the ldh prediction model applied in three strata (years 2000-2005, 2005-2010, 2010-2017) . figure s5 . association between trimethoprimsulfamethoxazole (tmp-smx) dose and mortality (docx 21 kb) additional file 2: table s1 . suggested, alternative data-derived ldh cutoff values and their impact on usually reported statistics (with 95% confidence interval in parenthesis) related to the prediction of in-hospital mortality in patients with pcp. table s2 . estimated aucs (with 95% confidence interval) of the ldh-related logistic regression models for the prediction of in-hospital mortality in patients with pcp. table s3 . additional descriptive information (with 95% confidence interval in parenthesis) on patient characteristics and their potential value as predictors of in-hospital mortality in patients with pcp. table s4 . pneumonia in the acquired immune deficiency syndrome opportunistic infections in patients with and patients without acquired immunodeficiency syndrome severity and outcomes of pneumocystis pneumonia in patients newly diagnosed with hiv infection: an observational cohort study parasitic pneumonia. interstitial plasma cell pneumonia of premature, caused by pneumocystis carinii pneumocystis carinii pneumonia in the united states. epidemiologic, diagnostic, and clinical features pneumocystis carinii pneumonia in patients without acquired immunodeficiency syndrome: associated illness and prior corticosteroid therapy pneumocystis carinii pneumonia among patients without aids at a cancer hospital pneumocystis: epidemiology and molecular approaches pneumocystis jirovecii pneumonia in patients with or without aids prognostic factors of early fatal outcome and long-term survival in patients with pneumocystis carinii pneumonia and acquired immunodeficiency syndrome prognostic factors influencing the outcome in pneumocystis carinii pneumonia in patients with aids survival of patients with aids, after diagnosis of pneumocystis carinii pneumonia, in the united states risk factors of pneumocystis pneumonia in solid organ recipients in the era of the common use of posttransplantation prophylaxis a multicenter randomized double-blind placebo-controlled trial of adjunctive corticosteroids in the treatment of pneumocystis carinii pneumonia complicating the acquired immune deficiency syndrome acute respiratory failure secondary to pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. a potential role for systemic corticosteroids adjunctive corticosteroids for pneumocystis jiroveci pneumonia in patients with hiv infection. cochrane hiv/aids group use of adjunctive corticosteroids in severe adult non-hiv pneumocystis carinii pneumonia incidence of pneumocystis jiroveci pneumonia among groups at risk in hiv-negative patients pneumocystis pneumonia suspected cases in 604 non-hiv and hiv patients infectious disease ward admission positively influences p. jiroveci pneumonia (pjp) outcome: a retrospective analysis of 116 hiv-positive and hiv-negative immunocompromised patients population-based analysis of invasive fungal infections pneumocystis pneumonia in hiv-infected and immunocompromised non-hiv infected patients: a retrospective study of two centers in china outcomes and prognostic factors of non-hiv patients with pneumocystis jirovecii pneumonia and pulmonary cmv co-infection: a retrospective cohort study predisposing factors, clinical characteristics and outcome of pneumonocystis jirovecii pneumonia in hiv-negative patients analysis of underlying diseases and prognosis factors associated with pneumocystis carinii pneumonia in immunocompromised hiv-negative patients intensive care of patients with hiv infection: utilization, critical illnesses, and outcomes. pulmonary complications of hiv infection study group plasma il-6/il-10 ratio and il-8, ldh, and hbdh level predict the severity and the risk of death in aids patients with pneumocystis pneumonia utility of lactate dehydrogenase vs radiographic severity in the differential diagnosis of pneumocystis carinii pneumonia no external funding was received to conduct this study. however, dr david's laboratory is supported by a grant from the german research foundation (da1209/4-3). the datasets used and analyzed during the current study are available from the corresponding author on reasonable request. carl-neuberg-strasse 1, 30625 hannover, germany. 2 authors' contributions jjs, cl, sd and sz collected the data. as performed the statistical analysis. experts in particular field were involved for specific data interpretation (ms for hiv; hh, jg, tb, mmh for transplantation; me for malignancies). jjs, cl, mmh, as and sd wrote the manuscript and all authors read, commented on and approved it.ethics approval and consent to participate written informed consent was waived by the local ethics committee at hannover medical school due to the anonymized retrospective nature of the analysis. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-006260-ux32zanj authors: sarkar, paul; gould, ian m. title: antimicrobial agents are societal drugs: how should this influence prescribing? date: 2012-09-17 journal: drugs doi: 10.2165/00003495-200666070-00001 sha: doc_id: 6260 cord_uid: ux32zanj this paper is concerned with how those who prescribe antimicrobials should consider the wider repercussions of their actions. it is accepted that in an ecological system, pressure will cause evolution; this is also the case with antimicrobials, the result being the development of resistance and the therapeutic failure of drugs. to an extent, this can be ameliorated through advances by the pharmaceutical industry, but that should not stop us from critically appraising our use and modifying our behavior to slow this process down. up to 50% of prescribing in human medicine and 80% in veterinary medicine and farming has been considered questionable. the alliance for the prudent use of antimicrobials (apua) was approached by the who to review the situation. their recommendations include decreasing the prescribing of antibacterials for nonbacterial infections. in the uk, there has been an initiative called ‘the path of least resistance’. this encourages general practitioners to avoid prescribing or reduce the duration of prescriptions for conditions such as upper respiratory tract infections and uncomplicated urinary tract infections; this approach has been successful. another recommendation is to reduce the prescribing of broad-spectrum antibacterials. in uk hospitals, the problems identified with the inappropriate use of antibacterials are insufficient training in infectious disease, difficulty in selecting empirical antibacterial therapy, poor use of available microbiological information, the fear of litigation and the fact that the majority of antibacterials are prescribed by the least experienced doctors. with close liaison between the laboratories and clinicians, and the development of local protocols, this can be addressed. another recommendation is to tighten the use of antibacterial prophylaxis and to improve patient compliance. through a combination of improved education for doctors and patients, and improved communication skills, these problems can be addressed. a further recommendation is to encourage teaching methods that modify prescribing habits. it has been shown that workshops have led to a significant reduction in the prescribing of broad-spectrum antibacterials in the community. auditing the prescribing of antibacterials has also been recommended. surveillance systems around the world monitor trends in resistance: the european antimicrobial resistance surveillance progamme (earss) monitors antibacterial resistance; the who and the international union against tuberculosis and lung disease collaborate to monitor tuberculosis; the who and the international aids society monitor hiv. in the third world, a bigger problem than resistance is whether drugs are even effective, as they are often spoiled by climactic conditions, and poor quality generics and counterfeit drugs are common. also, patients may not be able to complete a course for financial reasons. facts about antimicrobial resistance in animals (and agriculture) and impact on resistance (faair) was commissioned by apua. they conclude that the nonhuman use of antibacterials can lead to the development of antibacterial resistance in human pathogens. the european commission banned the use of antibacterials as growth promoters in 1999. in the western world, we should improve our diagnosis of sepsis, access local guidelines and consider withholding treatment pending investigations, decide if treatment can be stopped earlier and treat the patient not the result. many developing countries need improved access to more antimicrobials, preferably in the controlled environment of appropriate medical advice. up to 50% of prescribing in human medicine and 80% in veterinary medicine and farming has been considered questionable. the alliance for the prudent use of antimicrobials (apua) was approached by the who to review the situation. their recommendations include decreasing the prescribing of antibacterials for nonbacterial infections. in the uk, there has been an initiative called 'the path of least resistance'. this encourages general practitioners to avoid prescribing or reduce the duration of prescriptions for conditions such as upper respiratory tract infections and uncomplicated urinary tract infections; this approach has been successful. another recommendation is to reduce the prescribing of broad--spectrum antibacterials. in uk hospitals, the problems identified with the inappropriate use of antibacterials are insufficient training in infectious disease, difficulty in selecting empirical antibacterial therapy, poor use of available microbiological information, the fear of litigation and the fact that the majority of antibacterials are prescribed by the least experienced doctors. with close liaison between the laboratories and clinicians, and the development of local protocols, this can be addressed. another recommendation is to tighten the use of antibacterial prophylaxis and to improve patient compliance. through a combination of improved education for doctors and patients, and improved communication skills, these problems can be addressed. a further recommendation is to encourage teaching methods that modify prescribing habits. it has been shown that workshops have led to a significant reduction in the prescribing of broad-spectrum antibacterials in the community. auditing the prescribing of antibacterials has also been recommended. surveillance systems around the world monitor trends in resistance: the european antimicrobial resistance surveillance progamme (earss) monitors antibacterial resistance; the who and the international union against tuberculosis and lung disease collaborate to monitor tuberculosis; the who and the international aids society monitor hiv. in the third world, a bigger problem than resistance is whether drugs are even effective, as they are often spoiled by climactic conditions, and poor quality generics and counterfeit drugs are common. also, patients may not be able to complete a course for financial reasons. facts about antimicrobial resistance in animals (and agriculture) and impact on resistance (faair) was commissioned by apua. they conclude that the nonhuman use of antibacterials can lead to the development of antibacterial resistance in human pathogens. the european commission banned the use of antibacterials as growth promoters in 1999. in the western world, we should improve our diagnosis of sepsis, access local guidelines and consider withholding treatment pending investigations, decide if treatment can be stopped earlier and treat the patient not the result. many developing countries need improved access to more antimicrobials, preferably in the controlled environment of appropriate medical advice. "we need to relearn the use of antibiotics and the prudent use of antibiotics (apua), the europeadvocate for a more controlled application of anan society of clinical microbiology and infectious tibiotics for therapeutic use only so that strains disease (escmid) and other authorities have made susceptible to drugs will re-emerge." dr stuart recommendations, working in partnership with the levy, president of the alliance for the prudent use who. in this context, they are acting as stewards on of antibiotics (apua) an international level. there are also national and this paper is concerned with how we can change local stewards, such as stichting werkgroep antibithe attitudes of both prescribers and consumers with oticabeleid (swab) in the netherlands and the regards to the use of antimicrobials. the problem is swedish strategic programme for the rational use of resistance and poor quality use. [1] it is established of antimicrobial agents and surveillance of resisthat there is an association between antibacterial use tance (strama). in the us, the equivalents would and the prevalence of resistance. [2] whether antibacbe the infectious disease society of america (idterial control measures today can reduce levels of sa) and the center for communicable disease conresistance is not so clear. [3] trol and prevention (cdc). the european centre for disease prevention and control (ecdc) is a new european union agency that opened in 2004 to provide stewardship in europe against infections, the use of antimicrobials is wide-ranging and including influenza, severe acute respiratory syndiffers from country to country; [4] they are condrome (sars) and hiv. in the uk, most clinicians sumed in human medicine, veterinary medicine, will follow local guidelines or refer to established animal farming and agriculture. in the us, it has references, such as the monthly index of medical been quoted that 50% of antibacterial use is in specialties (mims) or the british national formuhuman medicine, of which 80% is used in the comlary (bnf). munity and 20% in hospitals; it has been suggested the dose is calculated by weight or age in chilthat 20-50% of prescribing is unnecessary. [5] veteridren, but a standard does is generally used for all nary and farming use accounts for the remaining otherwise healthy adults. prescribing schedules 50%, of which 20% is therapeutic and 80% is for have been established by population studies, but this prophylactic or growth promotion purposes; 40-80% of this usage has been questioned. [5] has tended to group variables together. it is evident that target organisms will have different minimum the key to reducing inappropriate use of aninhibitory concentrations and that the tibacterials is to develop guidelines for prescribers pharmacokinetics of drugs will be different in differthat generate a maximum health gain with a minimum development of resistance. the alliance for ent patients. the result in an unlucky individual (who is infected with a more resistant organism and 1. reduce the prescription of antibacterials for inin whom the peak concentration or the area under fections that are not bacterial, i.e. viral upper respirthe curve is reduced) may be treatment failure as a atory tract infections. result of sub-therapeutic antibacterial concentrations 2. reduce the overprescription of broad-spectrum at the site of infection. the extent of this problem antibacterials. and its contribution to the global problem of antimi-3. tighten the use of antibacterials in prophylactic crobial resistance is yet to be established, [6] but we situations, especially with respect to timing and feel it is likely to be highly significant in selecting duration. resistant strains. 4. encourage patient compliance to prescriptions, including addressing the hoarding of antibacterials the use of antibacterials outside standard regifor future use. mens, such as prescribing long-term low-dose treat-5. review the use of antimicrobial-containing cleanment, is also problematic. guillemot and carbon [7] ers and disinfectants. showed convincingly that this led to an increased likelihood of penicillin-resistant pneumococci 6. promote the use of non-antimicrobial intervenemerging in children. on the other hand, not finishtions, such as condoms, bed nets with or without ing a course of antibacterials may be no bad thing, insecticide impregnation, and vaccination. provided relapse of the infection is avoided. even 7. encourage forms of teaching that are likely to with adequate dosages, prolonged courses breed result in modification of prescribing behaviour, more resistance; therefore, the traditional advice to these include interactive and repeated teaching ses-"keep taking the tablets" is no longer appropriate. [8] sions, such as workshops and problem-solving sessions. the who wished to develop a global strategy for 8. use of audit to monitor prescribing behaviour. [9] the containment of antimicrobial resistance. they as in all medicine, any guidelines that are prosought a synthesis of recommendations from expert duced should be evidence based. apua has propolicy groups and approached the apua. their duced a comprehensive framework that can be folrecommendations are based on the following four lowed with good clinical governance. while the facets: evidence base behind these recommendations may • the establishment of effective surveillance sysnot be confirmed by randomised clinical trials, they tems to assess the shift of antibacterial resistance represent a synthesis of expert opinion and common patterns on a local, national and global scale, and sense. use of this information to guide prescribers on the there is the issue that antibacterials are drugs of appropriate use of antimicrobials. fear, and perceived, rather than real, health risks lead • the alteration of patient and provider behaviour clinicians to overprescribe broad-spectrum agents to reduce the inappropriate use of antimicrobial instead of observing the clinical development of agents. symptoms pending the result of bacteriological in-• to encourage the research and development of vestigations. the non-biomedical reasons for the new antimicrobial agents that can address the overprescription of antibacterials have been investiproblems of established resistant organisms, and gated by lam and lam, [10] who found that, in the to treat infections caused by those that surveilpublic sector, antibacterials were often prescribed as lance has indicated may cause future disease. a time-saving measure. in the private sector, they • to recognise the use of antibacterials outside were often prescribed for fear of losing clients. human medicine. a comparable quantity is used to summarise, the relationship between the use in the farming industry and veterinary practice. of antibacterials and the development of resistance the impact on this in human health is being in humans and in the ecosystem we live in is probassessed. lematic. [11] current prescribing patterns indicate the the recommendations of apua on how antimi-problem can be improved through good control and crobial prescribing should be influenced are as fol-stewardship. we need to consider how to change lows: these patterns and to establish that improving pre-reduce the overprescription of scribing patterns will improve quality of use and broad-spectrum antibacterials reduce resistance. broad-spectrum antibacterials, such as amoxicillin/clavulanic acid and cefotaxime, tend to be pre-2. examples of different situations on a scribed when the patient is deemed to be septic; this global scale is correct, as inadequate initial therapy will compromise patient outcome. in medical and surgical wards, however, only 70% of fever is due to infection, so an infection severity assessment is criti-countries, using the uk as an example cal. [14] if the patient has been in hospital for >2 days, the possibility of a more resistant bacterial infection will be considered, so an extended-spectrum an-of all antibacterials prescribed, 80% are in genertibacterial may be considered. if the patient had al practice; most of these are prescribed for respira-mrsa recently and appears to be septic, then an tory tract infections, and the second most common antibacterial active against mrsa, such as vancoreason for prescription is urinary tract infections. [12] mycin, teicoplanin or linezolid, will also be consid-the common hospital pathogens tend to show the ered. most resistant antibiogram and consist of meticillinresistant staphylococcus aureus (mrsa) and an to reduce the overprescription of broad-specassortment of gram-negative bacilli. they are most trum antibacterials in the hospital setting, there is problematic in the presence of a long-term prostheclose liaison between the microbiologist and clinisis, a central line or a catheter, when the patient has cian with regard to positive cultures and, when been exposed to broad-spectrum antibacterials. it is asked for, other advice. the aim is to stop or narrow generally accepted that someone has developed a the spectrum of the antibacterial used as soon as hospital-acquired infection if sepsis develops >48 investigations indicate what appropriate treatment hours after admission. may be. on the whole, the impact of this intervention is not as substantial as it could be because of poor or delayed communication of results. where the following paragraphs discuss what is being there are high prescribing rates of antibacterials in done in the uk to alter prescribing behaviour, using critically ill patients, such as an intensive care unit apua's eight recommendations as a framework (icu), there are combined ward rounds to facilitate (see section 1). appropriate prescribing, with the most up-to-date results that may not yet be available on the computer. the antibacterial committee, comprising a microbiologist, an infectious disease clinician and a to reduce prescription of antibacterials for infec-pharmacist, generates a list of restricted antibacteritions that are not bacterial (i.e. viral upper respirato-als that can only be prescribed after consultation ry tract infections in the community), there is an with them. they establish a local protocol of approinitiative named the path of least resistance. the priate first-line antibacterials for a given clinical advice is not to prescribe antibacterials for sore scenario that targets the likely pathogens, with the throats, coughs or colds, to limit prescribing for knowledge of local sensitivity patterns. the anuncomplicated urinary tract infections in healthy tibacterial formulary is reviewed periodically. anwomen to 3 days, and to limit the telephone pre-other approach often considered is a mandatory rescribing of antibacterials. it appears that this has had view of intravenous antibacterials prescribed after an effect. the antibacterial prescribing rates in the 24-48 hours. [15] this is mainly to avoid inappropricommunity have fallen in england from 49.4 mil-ately long courses of intravenous therapy but also lion scripts per year in 1995 to 36.9 million in 2000. serves as a prompt to review any positive microbio-the largest decrease has been seen in prescriptions logical results. once a likely pathogen has been for children. [13] isolated, it is the responsibility of the clinician to encourage forms of teaching that modify rationalise therapy. this is an area where there is prescribing behaviour room for improvement. it is recognised that didactic teaching does not problems identified in hospitals leading to the work as well as problem-solving workshops in modinappropriate use of antibacterials include, insuffiifying prescribing behaviour. a study has shown cient training in infectious diseases, difficulty in that antibacterial workshops have led to a 15.4% selecting the most appropriate antibacterial empirireduction in the prescribing of broad-spectrum cally, insufficient use of microbiological informaantibacterials in the community. [18] the hospital ention available and the fear of litigation. also, the vironment, especially if it is a teaching hospital, is majority of antibacterials are prescribed by the least conducive to ongoing learning. for the more isolatexperienced doctors. [14] ed doctors, perhaps in general practice, it might not tighten the use of prophylactic antibacterials be so easy to keep up to date. however, the advent of websites with guidelines should even the dispari-to tighten the use of antibacterials as prophylaxty. it is unrealistic to expect doctors to be up to date is, the re-education of junior surgeons on an annual in all things, but it is reasonable for them to keep basis goes some way towards addressing this, but abreast of the current guidelines in the drugs that has by no means controlled it. problems include they most commonly prescribe, which include inappropriate timing and duration of administration, antibacterials. the scottish executive and the britalong with confusion over what is the appropriate ish society of antimicrobial chemotherapy (bsac) agent or agents to use. the scottish intercollegiate have funded a website for medical undergraduates to guidelines network (sign) has guidelines on this. learn the prudent prescribing of antibacterials it allows for input of local data to assess the approthrough a framework of learning objectives and priateness of prophylaxis in the local situation. [16] reflective learning on a web-based format called 'appropriate antibiotic prescribing for tomorrow's to improve patient compliance with antibacteridoctor' (apt). [19] als in the community, patient education and the the use of audit to monitor prescribing habits has improved. communication skills is an area that has been successful. databases keep a record of antibacbeen highlighted at an undergraduate and postgraduterials prescribed and are retrospectively analysed. ate level in the uk in the last 10 years, with the in this way, individuals or departments can receive introduction of role-playing scenarios and playing feedback and modify prescribing habits where apback video recorded consultations. multimedia edupropriate. computerised prescribing will allow easicational campaigns are being carried out in the uk. er audit without interfering with the prescribing there was a two-phase campaign, with the first process. a recent european-wide audit of hospital phase in the autumn of 1999 to support health proantibacterial prescribing is accessible at wwfessionals in the management of acute upper respiraw.abdn.ac.uk/arpac. tory tract infections by reducing a patient's expectation for a prescription and encouraging the use of in an ideal world, everyone with a bacterial infecsymptomatic relief. the second was in 2002 pro-tion requiring an antibacterial would have a firm moting the message that antibacterials kill good microbiological diagnosis and the most narrowbacteria that keep us healthy. it used a combination spectrum antibacterial commenced for the minimum of the press, magazines and media relations, and duration required for a cure. this is not the current provided resource packs for schools and health au-situation and a degree of pragmatism is required to thorities, and non-prescription pads to general prac-result in the majority of patients receiving approprititioners and hospitals. [17] ate antibacterial therapy. apua's fifth and sixth recommendations, the to give an indication of the problem of clinically use of antimicrobial-containing cleaners and non-relevant resistant pathogens in europe, there is useantimicrobial interventions, are not about prescrib-ful information on the european antimicrobial reing so will not be addressed here. sistance surveillance programme (earss) website (www.earss.rivm.nl/) and the antibiotic resistance testing in conjunction with national or area anti-prevention and control (arpac) website (www. tuberculosis treatment-resistance surveillance. abdn.ac.uk/arpac). earss is an international net-the aims of this project are to estimate the magwork of national surveillance systems that collects nitude and pattern of anti-tuberculosis treatment recomparable and validated antimicrobial susceptibilisistance, monitor trends, evaluate effectiveness of ty data for public health action. it collects informatreatment programmes, plan and evaluate intervention on streptococcus pneumoniae, s. aureus, estion and prevention programmes, aid in developing cherichia coli, enterococcus faecalis and e. faecituberculosis policies at a country level, strengthen um, which have been identified as causing invasive laboratory services, advocate for increasing political disease. it collects data from 600 laboratories servcommitment and identify research needs. [21] ing 970 hospitals in 27 countries and has amassed 2.1.3 antivirals data from 65 000 isolates. it is co-ordinated by the the most commonly prescribed antiviral treat-dutch national institute for public health and the ment for hiv is highly active antiretroviral therapy environment (rivm). (haart). it consists of two nucleoside reverse the earss data on s. pneumoniae causing invatranscriptase inhibitors with either a non-nucleoside sive disease were analysed to assess trends in resisreverse transcriptase inhibitor or one or two protease tance to penicillin and erythromycin from 1999 to inhibitors. the british hiv association (bhiva) 2002. overall, the resistance to penicillin was 10% produces treatment guidelines. [22] it is possible to and to erythromycin, 17%. there was a decrease in carry out resistance testing. the patients with poor penicillin resistance of 5.3% per annum, an increase compliance will tend to select out the resistant viin erythromycin resistance of 5.9% per annum and ruses, which will result in an increasing viral load an increase of dual resistance of 7.6% per annum. and treatment failure along with increasing their earss predict that by 2006 there will be 20.4% infectivity with resistant virus. this is an increasingresistance to erythromycin and 8.9% dual resisly common problem. the global hiv treatmenttance. [20] arpac deals with a much broader selecresistance surveillance network is another who tion of resistant bacteria, specifically regarding hosstrategy for the containment of antimicrobial resispital infections and their control by antibacterial tance. [23] prescribing and infection control policies. the treatment of tuberculosis and hiv have in common a longer duration of therapy compared with other infections. this makes patient compliance and the incidence of tuberculosis in the uk is low. the control of adverse effects all the more important. this is thanks to a highly effective national control policy. where a patient has come from overseas, 2.2 human medicine in developing their country of origin is relevant, as it may be more countries, using sub-saharan africa as likely that they have multidrug-resistant tuberculoan example sis. in this situation, second-line agents should be used. directly observed therapy can be used to en-the majority of morbidity and mortality in develsure the treatment is taken. this is of greater releoping countries is in children <5 years old. they are vance in undeveloped countries where people may dying of lower respiratory tract infections, malaria, be less inclined to complete a treatment course when bacillary dysentery and measles, as well as malnutrithey start feeling better. tion. other significant infectious diseases include hiv and tuberculosis. the global project of anti-tuberculosis treatmentresistance surveillance has been in place since 1994. the who has devised an integrated manage-it is a collaboration between the who, the interna-ment of childhood illness (imci). this is a syntional union against tuberculosis and lung dis-dromic approach to clinical management. the anease, and other partners. it has formed a network of timicrobials used would depend on government polsupranational laboratories to aid national reference icy. the range of antimicrobials is limited and the laboratories in quality assurance and susceptibility laboratory services are somewhat lacking. it is a government's responsibility to ensure the knowl-safety; addressing threats to antibiotic sensitivity; edge of local antimicrobial sensitivity patterns is preventing and controlling zoonotic emerging infecavailable and disseminated through district medical tious diseases; protecting environments and ecosys-officers (dmos). if a non-governmental agency is tems; participating in bio-and agro-terrorism preworking in an area, they should liaise with dmos paredness and response; using their skills to conand adhere to national guidelines. [24] front non-zoonotic diseases (such as malaria, hiv/ aids, vaccine preventable diseases, chronic diseas-most importantly, it must be ensured that es and injuries); strengthening the public-health inmedicines are still efficacious after transportation, frastructure; and advancing medical science through given the climactic conditions. the temperature of a research." [26] container exposed to the sun may rise to >50°c and spoil antibacterials. the appearance of counterfeit veterinary practice uses an equivalent quantity of drugs, poor quality generics and the availability of antimicrobials to human medicine, but mainly for antibacterials bought over the counter (otc) are growth promotion and disease prevention. common adding to morbidity in these countries. the countertherapeutic uses include treating mastitis in dairy feit or generic drugs may be less efficacious, and the cattle and respiratory illnesses in battery-reared otc antibiotics may be taken for too short a time or chickens. non-therapeutic uses include growth proin too low a dosage because of expense. motion in chickens. in agriculture, streptomycin and a background document for the who global oxytetracycline are used to spray fruit in orchards to strategy for the containment of antimicrobial resismake them more visually appealing. to investigate tance called drug resistance in malaria concludes this situation further, the apua initiated a 2-year that people need more access to antimalarials. [25] it project called facts about antimicrobial resistance is accepted that this will lead to increasing levels of in animals (and agriculture) and the impact on resistance, so there must also be investment in con-resistance (faair). [27] it is concerned with the trol measures to increase the useful lifespan of a overall effects of antimicrobial use from an ecologitherapy, and to allow time for new treatments and cal point of view. there are two ways they consider controls to be developed. that resistant infections can be acquired from the the problem with prescribing for tuberculosis is environment; the first is from direct infections from compliance with therapy. the who feels so stronghuman pathogenic bacteria, such as salmonella from ly about this that they advise not to commence chickens, and the second is from the human acquisitreatment if supervision over the full duration of tion of resistant nonpathogenic bacteria from the therapy can not be assured (usually 6 months). this environment, which can act as a reservoir of resisis of particular relevance in the refugee setting. tance genes that maybe transferred to human patho-the use of non-antimicrobial interventions is genic bacteria. the findings and conclusions are that mainly targeting malaria (with bed nets) and hiv antimicrobial use selects resistant bacterial strains (with low-priced condoms), and a national vaccinaand genetic vectors specifying resistance genes. the tion programme (which is incorporated into the imauthors see the propagation of antimicrobial resis-ci). tance as an ecological problem and suggest that reducing it would require an understanding of the 2.3 non-human uses of antimicrobials commensal microbiota of mammals and genetic vectors of resistance. when one antimicrobial dr calvin schwabe talks of 'one medicine', the selects out resistance, because of the genetic linkage bringing together of human and veterinary of resistance genes, other antimicrobial resistances medicine. "human health provides the most-logical will emerge. the period between the use of an unifying or apical cause in veterinary medicine's antimicrobial and the emergence of resistance varhierarchy of values. veterinarians in all aspects of ies, but once the prevalence reaches a certain level, the profession have opportunity and responsibility reversal is difficult. the effect of this on the global to protect the health and well-being of people in all problem of antimicrobial resistance is unknown. that they do, including protecting food security and faair's conclusions are that any antimicrobial used anywhere for anything can affect anyone any-prescriber's attitudes cover a spectrum from conwhere in the world. this will cost us financially and servative to aggressive. the use of antimicrobials is our health may be affected, although at a population easily justified in life-threatening conditions, but level the cost of antibacterial resistance is not often they are prescribed to reduce morbidity and known. therefore, the elimination of the nonthera-prevent the occasional complication. the word peutic use of antimicrobials in food animals and 'doctor' is derived from the greek word for 'teachagriculture will benefit us all. the european com-er'. it is important to educate patients about the mission banned the use of antibacterials as growth condition they have as fear is usually a result of promoters in 1999. ignorance. reassurance and addressing why a problem is occurring or recurring should not be forgot-several groups are looking into ways to evaluate ten. for developed countries, we should influence the safety of ingested antimicrobial residues used in our prescribing in five ways: veterinary medicine on the human intestinal microflora. [28] one group has identified the presence 1. improve our diagnosis of sepsis. of multidrug-resistant enteric bacteria in dairy farm 2. access the latest local guidelines on the subject. topsoil and implicated this as a reservoir for bacteri-3. decide if antimicrobial treatment can be withheld al resistance against clinically relevant antibacteripending positive microbiology. [30] als. [29] the problem of resistance can be further 4. decide if treatment can be stopped or rationalised compounded by the handling of waste from food once results return and chase up results if they are animals which will often pollute a widespread area not received on time. by contaminating water supplies. food animals may 5. treat the patient, not the results. also develop antibacterial-resistant diseases but the advances in information technology and near scale of the problem has not been ascertained. the patient testing could also improve prescribing behamechanism of increased disease in humans from viour. antimicrobial resistance in animals is multifactorial many developing countries need improved acand includes an increase in colonisation rates, an cess to more antimicrobials preferably in the conincrease in pathogenicity and a decrease in the effitrolled environment of appropriate medical advice. cacy of treatment. [27] don't keep taking the tablets? lancet antibiotic resistance: 22. british hiv association. bhiva guidelines for the treatment of synthesis of recommendations by expert policy groups on hiv infected adults with antiretroviral therapy availbehalf of the world health organisation by the alliance accessed 23. world health organization, international aids society. the what are the non-biomedical reasons which make family doctors over-prescribe antibiotics for upper rehivaids/network/en/index.html [accessed use of antimicrobial agents and drug resistance. n www.who.int/imci-mce drug resistance in malaria veterinary medicine protecting and promoting antibiotic prescribing: are there lessons the public's health and well-being the effect of intrave-27. faair scientific advisory panel. select findings and conclunous-to-oral switch guidelines on the use of parenteral antimicrobials in medical wards approaches in the safety evaluations available from url: http:// effects on the human intestinal microflora. j vet pharmacol www.sign.ac.uk/guidelines/fulltext/45 available from url: http:// resistant enteric bacteria in dairy farm topsoil primary care workshops can reduce and rationalise antibiotic prescribing the apt project: appropriate prescribing for tomorrow's doctors trends of penicillin and erythromy cin resistance amongst invasive streptococcus pneumoniae in 7nb the authors received no funding for the preparation of this manuscript and have no conflicts of interest relevant to its antimicrobials are unique amongst all drugs in contents. that their use can result in reduced efficacy. in many areas, there is room for improvement in the way they references are used. how should this influence prescribing? key: cord-009388-k3exf8a4 authors: agarwal, yash; beatty, cole; biradar, shivkumar; castronova, isabella; ho, sara; melody, kevin; bility, moses turkle title: moving beyond the mousetrap: current and emerging humanized mouse and rat models for investigating prevention and cure strategies against hiv infection and associated pathologies date: 2020-04-10 journal: retrovirology doi: 10.1186/s12977-020-00515-3 sha: doc_id: 9388 cord_uid: k3exf8a4 the development of safe and effective combination antiretroviral therapies for human immunodeficiency virus (hiv) infection over the past several decades has significantly reduced hiv-associated morbidity and mortality. additionally, antiretroviral drugs have provided an effective means of protection against hiv transmission. despite these advances, significant limitations exist; namely, the inability to eliminate hiv reservoirs, the inability to reverse lymphoid tissues damage, and the lack of an effective vaccine for preventing hiv transmission. evaluation of the safety and efficacy of therapeutics and vaccines for eliminating hiv reservoirs and preventing hiv transmission requires robust in vivo models. since hiv is a human-specific pathogen, that targets hematopoietic lineage cells and lymphoid tissues, in vivo animal models for hiv-host interactions require incorporation of human hematopoietic lineage cells and lymphoid tissues. in this review, we will discuss the construction of mouse models with human lymphoid tissues and/or hematopoietic lineage cells, termed, human immune system (his)-humanized mice. these his-humanized mouse models can support the development of functional human innate and adaptive immune cells, along with primary (thymus) and secondary (spleen) lymphoid tissues. we will discuss applications of his-humanized mouse models in evaluating the safety and efficacy of therapeutics against hiv reservoirs and associated immunopathology, and delineate the human immune response elicited by candidate hiv vaccines. in addition to focusing on how these his-humanized mouse models have already furthered our understanding of hiv and contributed to hiv therapeutics development, we discuss how emerging his-humanized rat models could address the limitations of his-mouse models. despite combination antiretroviral therapy (cart)mediated suppression of human immunodeficiency virus (hiv) replication and promotion of immune reconstitution in patients, hiv-associated morbidity persists and is associated with the latent reservoir, unresolved immune abnormalities, and fibrosis in lymphoid organs [1] . additionally, hiv transmission remains endemic across the globe, and development of a functional cure and/or an effective vaccine will be required to end this epidemic [2] . hiv is a human specific pathogen; thus, animal models for evaluating the safety and efficacy of therapeutics and vaccines directly against hiv requires the incorporation of human lymphoid tissues and/or hematopoietic lineage cells. such mouse models exist, and are termed, human immune system (his)-humanized mice [3, 4] . to construct his-humanized mice, immunodeficient mice are myoablated to eradicate residual mouse bone marrow stem cells, and then engrafted with human peripheral blood mononuclear cells (pbmcs) or human hematopoietic stem cells (hsc) with or without transplantation of lymphoid tissues, such as thymus and/or spleen [5] . over a period of a few weeks to months, transplanted mice develop human immune cells, and reconstitution is confirmed by flow cytometry [5] (fig. 1) . his-humanized mice can then be employed in studies investigating hiv prevention or cure strategies [5] . in this review, we will discuss the myriad of approaches for developing hishumanized mouse models, and their applications in hiv therapeutics and vaccine development studies, along with their limitations. finally, we will discuss the potential of an emerging his-humanized rat model, which has a longer lifespan and greater physiological similarity to humans compared to mice, designed to enable longitudinal studies in evaluating therapeutics against hiv reservoirs and vaccine-induced immunity [6, 7] . human cd4 + t cells are the major target for hiv infection; thus, a mouse model with human cd4 + t cells provides a platform for modeling hiv/aids. various immunodeficient mouse models lacking mature t, b, [8] and nk cells, along with defects in macrophage phagocytic function [9] support robust reconstitution of human cd4+ t cells and other lymphocytes (e.g. cd8 + t cells) following transplantation of human pbmcs or cd4+ t cells. such cells can be transplanted via intravenous (iv) or intraperitoneal (ip) injection into myoablated, immunodeficient juvenile mice (6-8 weeks old) at a dose of 5-10 × 10^6 cell per mouse, to generate peripheral blood lymphocyte (pbl)-humanized mice. human cd4 + t cells are readily detectable in the blood fig. 1 construction of the human immune system-humanized mouse models. (i) immunodeficient mice are myoablated via irradiation or busulfan, followed by the administration of antibiotics and analgesics. general anesthesia is induced prior to surgery. (ii) to generate human lymphoid tissue xenografts along with autologous immune cell reconstitution, human fetal lymphoid tissue(s) and liver are processed into 1 mm 2 pieces, and autologous cd34 + hscs are isolated from the fetal liver via immunomagnetic selection. cd34 + hscs are then transplanted via retro-orbital injection following renal capsule transplantation of the lymphoid tissues. alternatively, to generate human immune cell only, pbmcs, cd4+ t cells, or cd34 + hscs are transplanted via iv or ip injection. (iii) transplanted mice are maintained under specific pathogen-free conditions and the human lymphoid tissue(s), and/or immune cell reconstitution in the peripheral blood and murine lymphoid tissues (humanized-murine tissues) are allowed to develop over a period of 2-10 weeks (or more), resulting in the his-humanized mouse model at 4 weeks post-transplantation [9, 10] , hence providing a humanized mouse model that can be generated in a relatively short period. the pbl-humanized mouse model supports hiv replication and provides a means of evaluating the efficacy of direct-acting therapeutics (e.g. antivirals drugs, antibodies) geared towards preventing hiv transmission [11] and controlling hiv replication [9] . additionally, pbl-humanized mouse models constructed using pbmcs from hiv-infected individuals with undetectable viral load can be employed as an in vivo assay (mouse-quantitative viral outgrowth assay) for evaluating the eradication of the hiv reservoir in said individuals [12, 13] . a major limitation of this model is the rapid development of graft-versus-host (gvhd) disease within 6-7 weeks following transplantation of lymphocytes, thus significantly restricting the experimental window [14] . additionally, the pbl-humanized mouse model does not incorporate human macrophages, which are a major hiv reservoir in various organs, including the brain [9, 15] . however, modification of the pbl-humanized mouse model via transplantation of hiv-infected monocyte-derived macrophages in the brain supports hiv infection and pathogenesis in the brain [15] . in order to generate a de novo human immune system and reconstitute a broader spectrum of human immune cells in his-humanized mice, myoablated, immunodeficient mice are transplanted with human cd34 + hscs via intrahepatic or intracardiac injection in neonatal mice [10, 16] or iv injection in juvenile/adult mice [16, 17] . these hscs can be obtained from a myriad of sources, including fetal liver tissue [17, 18] and neonatal cord blood cells [4, 16, 19] . human immune reconstitution in the hsc-humanized mouse model requires 10-12 weeks to develop [20] . various hematopoietic lineages, including t cells, monocytes/macrophages, b cells and dendritic cells are developed in the blood and other tissues (e.g., spleen, liver, brain) [20] . moreover, the hsc-humanized mouse model generates a naïve human immune system, which negates confounding factors associated with prior pathogen exposure [16, 21] . the hsc-humanized mouse model supports hiv infection, cd4+ t cell depletion, chronic immune activation and limited anti-hiv t and b cell immune responses [4] . a major advantage of the hsc-humanized mouse model over the pbl-humanized mouse model is the delayed and reduced incidence of gvhd, which provides the opportunity for long-term modeling of hiv infection and replication [9] . the hsc-humanized mouse model provides a means of evaluating the efficacy and safety of directacting therapeutics (e.g. antivirals drugs, antibodies) and immune-modulatory agents (e.g. pdc modulators [22] ) geared towards preventing hiv transmission, controlling hiv replication, and ameliorating cd4+ t cell depletion and chronic immune activation [4] . the hschumanized mouse model supports hiv transmission via the iv (along with ip) route [18] ; however, conflicting reports exist for the mucosal route of transmission [23, 24] . additionally, the reconstituted human t cells are educated in the mouse thymic epithelium, thus limiting antigen-specific responses [25] . this limitation of t cell education in the murine thymic epithelium has been partially overcome by the construction human leukocyte antigen (hla) class i transgenic-immunodeficient mice to support robust t cell development of hla-matched hsc transplants [26] . moreover, lymph nodes and spleen are poorly reconstituted, including a limited development of human b and myeloid cells in the white and red pulps of the spleen [27] . several modifications have been made to the hsc model to address these limitations. li et al. constructed an immunodeficient mouse model that incorporated a lymphoid tissue-stromal cytokine transgene (i.e. thymic-stromal-cell-derived lymphopoietin) and demonstrated improved lymph node development in hsc-humanized mice [27] . additionally, studies have demonstrated enhanced human b and myeloid cell development in murine secondary lymphoid tissues via transgenic expression of critical cytokines (i.e. il6; il3, gm-csf and scf) for b and myeloid cell maturation [28] [29] [30] . although incorporation of requisite human transgenes in his-humanized mice has been successful in demonstrating improved development of immune cells, often the resultant lineage is skewed, as the transgene expression is not synchronized for physiological expression and supporting stromal cells and other essential cytokines are absent [28] [29] [30] . another strategy for improving human immune cell development in his-humanized mouse models is to implant human lymphoid tissues containing the requisite microenvironment for supporting robust immune cell development. to facilitate human t cell education and associated function, human thymic tissues are incorporated in his-humanized mice, and termed, bone marrow-liver-thymus (blt)-humanized mice [31, 32] (fig. 2) . blt-humanized mice have served as a key animal model for hiv research for over a decade and are a cost-effective alternative to the surrogate, simian immunodeficiency virus (siv)-non-human primate (nhp) models. the blt-humanized mouse model is generated by surgically transplanting myoablated, immunodeficient mice with fetal human liver and thymus tissues, followed by iv injection of autologous cd34 + hscs [31, 33, 34] . transplanted mice require 10-12 weeks for systemic reconstitution of human cells post-transplantation [31, 33, 34] . the most widely utilized strain for constructing blt mice is the nod-prkdc scid il2rg tm1wjl (nsg) [21, 32] , which is readily available from jackson laboratory. blt-humanized mice can also be constructed using comparable immunodeficient mouse strains, such as, c57bl/6 rag2−/−γc−/−cd47−/− (tko) [21, 35] . the key benefit of the blt-humanized mouse model over pbl-and hsc-humanized mouse models is the presence of human thymic microenvironment, which facilitates t cell education in an autologous human tissue that contains the requisite stromal cells (as well as cytokines and factors, presumably at physiological levels) [21] . blt-humanized mice have systemic tissue reconstitution with human immune cells, including in mucosal tissues, which enables mucosal transmission [36] [37] [38] [39] [40] [41] [42] [43] and recapitulates the main route of hiv transmission in humans [36] [37] [38] [39] [40] [41] [42] [43] [44] . other hallmarks of hiv infection and replication in blt-humanized mice include robust t cell depletion [36, 42] , central nervous system infiltration [45, 46] , immune response [35, [47] [48] [49] [50] , and latency [51] [52] [53] . the blt-humanized mouse model is a robust platform for evaluating hiv prevention and cure strategies, including antiretroviral therapy, pre-exposure prophylaxis (prep), latency reversing agents (lra), vaccination, proviral excision, and t cell engineering (table 1) . despite significant advances gained from the blthumanized mouse model, the system does have some disadvantages. construction of blt-humanized mice requires advanced surgical expertise and extensive experience; therefore, these animals are constructed predominantly by specialized core facilities. additionally, blt-humanized mice are prone to gvhd, which limits the experimental window these animals can be utilized to approximately 6 months post-engraftment [54, 55] . however, blt-humanized mice constructed with a c57bl/6 immunodeficient background are resistant to gvhd [35, 53] . another disadvantage involves the use of human fetal tissues in constructing the model; these tissues are not readily available. furthermore, a typical human fetal thymus and autologous fetal liver-derived hscs can only support the construction of 15-25 blt-humanized mice. the limited availability of said human fetal tissues creates logistical and operational constraints. recently, a novel blt-like humanized mouse model has been developed using non-autologous human cord blood-derived hscs and human neonatal/pediatric thymus, which enable investigators to construct > 1000 blt-like humanized mice using cryopreserved thymus tissues and readily available cord blood-derived hscs [96] . recent studies demonstrate that these blt-like humanized mice develop human immune cells, support hiv infection and replication, and exhibit anti-hiv immune response (unpublished data from elie haddad, chloé colas, et al., at the cancure 5th annual general meeting-2019, poster session, in montreal, canada). despite systemic immune cell reconstitution and hiv-specific immune responses, both neonatal/pediatric tissue-and fetal tissue-derived blt-humanized mouse models blt mice do not develop a complete human immune system. the current widely used immunodeficient mouse models possess an il-2 receptor γ chain deletion [97] [98] [99] [100] . as a result, mouse lymphoid organs do not fully develop in such models, [21] and the loss of lymphoid tissue microenvironment impairs the ability of blt-humanized mice to develop a robust humoral immune response, as immunoglobulins are skewed towards igm or weak igg response [35, 49, 99, [101] [102] [103] . constructing blt-humanized mice using immunodeficient mouse models with requisite human transgenic factors/cytokines, may optimize human b cell development and overcome the limitations of humoral immune response in the model [28, 104, 105 ]. an alternative strategy, which is consistent with the blt-model to construct his-humanized mice and rats, immunodeficient mice and rats are myoablated, followed by engraftment of human lymphoid tissues (thymus with or without human spleen) under the kidney capsule, along with injection of autologous human cd34+ hematopoietic stem cells. in representative images, we show the engrafted human lymphoid tissues (human thymus xenograft-thymus, white tissue; human spleen xenograft-spleen, dark brown tissue and the reconstituted rodent-spleen (humanized spleen-hspleen). note: mouse and rat organs are not at the same scale pre-exposure prophylaxis (prep) c5a peptide [66] cc-griffithsin [67] cd4 asics [68] cd4-expressing lactobacillus acidophilus [69] cd4mc p-iii-48 [70] dtg-ultra la [71] efda [72] g2-s16 pcd [73] iga [74] mvc [75] ral-la [76] rpv-la [39, 77] siccr5 lfa-1 i-tsnp [78] tnv gel [79] [80] [81] vrc01 bnab [82] ftc, taf [83] ftc, tdf [36, 43, 84, 85] taf, evg [86] b12, vrc01, vrc07 g54w bnabs [87] latency-reversing agents (lras) azd5582 [88] panobinostat [89] suw133 (bryostatin analog) [90] vaccines plga-gag microparticles [49, 91] recombinant gp140∆683 [49, 91] proviral excision sacas9/sgrna [92] t cell engineering ccr5 shrna [93, 94] cd4 car [95] 3tc, lamivudine; asics, aptamer-sirna chimeras; azt, zidovudine; bnab, broadly neutralizing antibody; ccr5, c-c chemokine receptor type 5; car, chimeric antigen receptor; cd4, cluster of differentiation 4; cd4mc, cd4 mimetic compound; cc, caulobacter crescentus recombinant expressing; dca, didehydro-cortistatin a; ddi, didanosine; dtg, dolutegravir; dtg-ultra la, long acting dolutegravir; efda, 4′-ethynyl-2-fluoro-2′-deoxyadenosine; evg, elvitegravir; ftc, emtricitabine; idv, indinavir; ifnα14, interferon α suptype 14; iga, immunoglobulin a; lfa-1 i-tsnp, lymphocyte function-associated antigen-1 integrin-targeted and stabilized nanoparticle; mab, monoclonal antibody; mvc, maraviroc; pcd, polyanionic carbosilane dendrimers; pd-1, programmed cell death protein 1; plga, poly(lactic-co-glycolic) acid; ral, raltegravir; ral-la, long-acting raltegravir; rpv, rilpivirine; rpv-la, long acting rilpivirine; sacas9/sgrna, staphylococcus aureus crispr-associated protein 9/single-guide rna; shrna, short hairpin rna; siccr5, small interfering rna ccr5; taf, tenofovir alafenamide; tdf, tenofovir disoproxil fumarate; tnv; tenofovir strategy, is to incorporate the requisite human secondary lymphoid tissue (i.e. spleen) microenvironment for robust human immune cell (e.g. b cells, macrophages) development and response [5] . to address the limitations of the blt-humanized mouse model, namely, poor development of secondary lymphoid tissue and modest macrophage reconstitution, we incorporated human spleen into the blt-humanized mouse model, and termed these animals, bone marrow-liverthymus-spleen (blts)-humanized mice [5] (fig. 2) . blts-humanized mice exhibit significant improvement over the blt-humanized mice by addressing several limitations [5] . successful spleen growth dramatically lowers the incidence of gvhd in blts-humanized mice, thus allowing for experimental studies that extend up to 9 months post-transplantation [5] . we speculate that the decreased gvhd in blts-humanized mice results from appropriate modulation of t cell activation by the human antigen-presenting cells in the human spleen xenograft. the human spleen in the blts-humanized mouse model recapitulates human adult spleen architecture and facilitates better reconstitution of immune cells, including human red pulp macrophages, which are poorly reconstituted in the blt-humanized mouse model [5] . it is well established that macrophages can serve as a reservoir for hiv [106] [107] [108] ; thus, the blts-humanized mouse model provides a system for investigating human splenic macrophage-hiv interactions [5] . additionally, the spleen is a major lymphoid tissue reservoir, with b cell follicles in the white-pulp serving as an immune privilege site for anti-hiv t-cells [109] . the human spleen in bltshumanized mice provides a model for investigating anti-hiv immune response within the white-pulp and the role of b cell follicle in mediating hiv persistence. the blts-humanized mouse model supports cart-mediated hiv load suppression, and replication competent hiv reservoirs can be detected in human spleen tissues [5] . lymphoid tissue fibrosis is an immuno-pathogenic feature associated with hiv infection and plays a major role in mediating chronic inflammation and abrogating the development of a robust immune response [110] . a major advantage of the blts-humanized mouse model is that hiv infection results in lymphoid tissue fibrosis; this disease manifestation is absent in hiv-infected blt-humanized mice [5] . although the blts-humanized mouse model exhibits more robust immune reconstitution compared to its blt counterpart, the two models share some limitations. the transplantation of the human tissues under the renal capsule requires an individual with advanced surgical skills. the blts-humanized mouse model uses human fetal tissues, which introduces logistical and operational constraints. the use of frozen fetal tissues and hscs can alleviate some of those constraints (unpublished data). demonstrating robust anti-hiv immunity in his-humanized mouse models has been a long-term goal in the field because said system would allow robust evaluation of hiv vaccine candidates against circulating viral strains. the incorporation of human primary and secondary lymphoid tissues in hishumanized mice brings us closer to this goal. at present, we are actively investigating the anti-hiv human immunity in the blts-humanized mouse model to determine if this system provides a means for evaluating hiv vaccines. prior to the development of genetic engineering technologies for creating transgenic and knockout mice, rats were the predominant specie of rodents employed in biomedical research [6] . advantages of using rats include their longer lifespans (~ 3.5 years) and larger size (~ 350 grams), which facilitates longer experimental window and larger sampling volumes compared to the short lifespan (< 1 year) and small size (< 25 g) of mice [6] . rats provide a more ideal platform for in vivo imaging of diseases, as the larger size of rats provides better resolution [111] . additionally, rat models exhibit advanced cognitive skills and critical physiological parameters (e.g. heart rate, drug metabolism) that more closely mimic humans [112] [113] [114] [115] . recent advances in genetic engineering, such as the crispr/cas 9 technology has enabled the development of several immunodeficient rat models for transplanting and regenerating human tissues and cells [7, [116] [117] [118] . similar to currently used immunodeficient mouse models, immunodeficient rat models carry mutations in rag1/2 and il2rγ genes, with or without sirp1α transgene [7, [116] [117] [118] . a recent study demonstrated that these immunodeficient rats can be reconstituted with a myriad of human immune cells following transplantation with human-fetal liver-derived hscs and autologous thymus tissues [7] (fig. 2) . these his-humanized rat models could provide a means for robust longitudinal studies on the safety and efficacy of therapeutic agents targeting the hiv reservoir. additionally, his-humanized rats could provide a means for modeling hiv-associated end organ diseases, such as cardiovascular, neurocognitive and lung diseases. despite successful prevention of hiv transmission with antiviral drugs, it is likely that an effective vaccine provides the only means of ending the hiv epidemic [2] . over the past decades, several promising vaccine candidates have failed in large-scale safety and efficacy clinical trials [2] . the failure of those clinical trials suggests that improved "gate keeper" animal modeling systems are needed for better prediction of vaccine candidate outcomes in human clinical trials [119] . currently, the surrogate siv-nhp model is the sole "gate keeper" animal model for determining potential of vaccine candidates [119] . candidate hiv vaccines selected using this gatekeeper system have been unsuccessful in human clinical trials [119] ; suggesting, major improvements in animal modeling are needed. although significant advancements are still needed in improving his-humanized models, several recent advances, such as improved human-secondary lymphoid tissue development, along with the previously developed, robust primary lymphoid tissue development has made it possible to evaluate human immune responses to vaccines [5, 27] . ideally, these "improved" his-humanized mouse models will complement nhp models in addressing critical gaps, such as vaccine-induced immune responses against circulating hiv strains, vaccine safety in the context of hiv transmission, and human-correlates of immunity. although cart has significantly reduced the morbidity and mortality associated with chronic hiv infection, the hiv reservoir persists in people living with hiv (plhiv) and is associated with chronic inflammation, lymphoid tissue damage, and a myriad of end-organ diseases [1] . therefore, eradicating the hiv reservoir and associated chronic inflammation and end organ diseases remains a major challenge. the mechanisms of hiv persistence in plhiv, despite robust cart-mediated suppression of the virus, are thought to be multifactorial. these factors include persistence in transcriptionally quiescent resting memory cd4+ t cells in the peripheral blood and lymphoid tissues, infection of long-lived resident tissue macrophages in lymphoid tissues and immune privilege organs (e.g. brain, testes, b cell follicle, etc.), and dysregulation in anti-hiv immunity. by virtue of the multitude of factors that play a role in hiv persistence, in vivo models that recapitulate human host-hiv interactions are necessary for determining the safety and efficacy of therapeutic agents for eradicating hiv reservoirs. improved his-humanized mouse models with systemic reconstitution of human immune cells and robust lymphoid tissues development provide a means of evaluating both directacting and immune-modulatory hiv-cure therapeutics. further advances in improving the human-immune system in his-humanized mouse and rat models will provide better in vivo systems for evaluating the safety and efficacy of therapeutics and vaccines for hiv prevention and cure. not applicable. moses turkle bility is the senior author authors' contributions mtb conceived this review and contributed to the preparation of the manuscript. ya, cb, sb, ic, sh and km contributed equally to the preparation of the manuscript. all authors read and approved the final manuscript. this work was supported by the following nih grants (r21od024789, (national institute of allergy and infectious diseases) r21ai135412). not applicable. the human fetal liver, spleen and thymus (gestational age of 18-20 weeks) used in the transplantations were obtained from medically or elective indicated termination of pregnancy through magee-women' ). the use of human hematopoietic stem cells was reviewed and approved by the human stem cell research oversight (hscro) at the university of pittsburgh. the use of biological agents (e.g., hiv), recombinant dna, and transgenic animals was reviewed and approved by the institutional biosafety committee (ibc) at the university of pittsburgh. all animal studies were approved by the iacuc at the university of pittsburgh and were conducted following nih guidelines for housing and care of laboratory animals. why and where an hiv cure is needed and how it might be achieved the complex challenges of hiv vaccine development require renewed and expanded global commitment small animal models for human immunodeficiency virus (hiv), hepatitis b, and tuberculosis: proceedings of an niaid workshop humanized immune system mouse models: progress, challenges and opportunities human immunodeficiency virus infection induces lymphoid fibrosis in the bm-liver-thymus-spleen humanized mouse model rats! disease models & mechanisms an immune system-modified rat model for human stem cell transplantation research hu-pbl-scid mice: a model for human immune function, aids, and lymphomagenesis a simple mouse model for the study of human immunodeficiency virus chronic, acute, and reactivated hiv infection in humanized immunodeficient mouse models hu-pbl-scid mice can be protected from hiv-1 infection by passive transfer of monoclonal-antibody to the principal neutralizing determinant of envelope gp120 the mouse viral outgrowth assay: avatars for the detection of hiv-1 reservoirs high activation and skewed t cell differentiation are associated with low il-17a levels in a hu-pbl-nsg-sgm3 mouse model of hiv infection xenogeneic graft-versus-host-disease in nod-scid il-2rgammanull mice display a t-effector memory phenotype generation of cytotoxic t cells against virus-infected human brain macrophages in a murine model of hiv-1 encephalitis parameters for establishing humanized mouse models to study human immunity: analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the il2rgamma(null) mutation hiv-1 infection and pathogenesis in a novel humanized mouse model humanized mice engrafted with human hsc only or hsc and thymus support comparable hiv-1 replication, immunopathology, and responses to art and immune therapy comparison of human fetal liver, umbilical cord blood, and adult blood hematopoietic stem cell engraftment in nod-scid/gammac-/-, balb/c-rag1-/-gammac-/-, and cb-17-scid/bg immunodeficient mice generation of immunodeficient mice bearing human immune systems by the engraftment of hematopoietic stem cells humanized mice for immune system investigation: progress, promise and challenges flt3l-mediated expansion of plasmacytoid dendritic cells suppresses hiv infection in humanized mice rag2(-/-)gamma(-/-)(c) mice transplanted with cd34(+) cells from human cord blood show low levels of intestinal engraftment and are resistant to rectal transmission of human immunodeficiency virus mucosal transmission of r5 and x4 tropic hiv-1 via vaginal and rectal routes in humanized rag2-/-gammac -/-(rag-hu) mice the analysis of the functions of human b and t cells in humanized nod/shi-scid/gammac(null) (nog) mice (hu-hsc nog mice) generation of functional human t-cell subsets with hla-restricted immune responses in hla class i expressing nod/scid/il2r gamma(null) humanized mice a human immune system mouse model with robust lymph node development a novel humanized mouse model with significant improvement of class-switched, antigen-specific antibody production development and function of human innate immune cells in a humanized mouse model development of human cd4 + foxp3 + regulatory t cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing nod-scid il2rgamma(null) humanized mice reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and cd34(+) cell transplantation antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv-1 in humanized blt mice humanized mice mount specific adaptive and innate immune responses to ebv and tsst-1 immunodeficient mouse model for human hematopoietic stem cell engraftment and immune system development blt-humanized c57bl/6 rag2-/-gammac-/-cd47-/-mice are resistant to gvhd and develop b-and t-cell immunity to hiv infection antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv-1 in humanized blt mice art influences hiv persistence in the female reproductive tract and cervicovaginal secretions animal models for hiv/aids research long-acting rilpivirine (rpv) preexposure prophylaxis does not inhibit vaginal transmission of rpvresistant hiv-1 or select for high-frequency drug resistance in humanized mice immune reconstitution of the female reproductive tract of humanized blt mice and their susceptibility to human immunodeficiency virus infection superior human leukocyte reconstitution and susceptibility to vaginal hiv transmission in humanized nod-scid il-2rgamma(-/-) (nsg) blt mice intrarectal transmission, systemic infection, and cd4 + t cell depletion in humanized mice infected with hiv-1 human breast milk and antiretrovirals dramatically reduce oral hiv-1 transmission in blt humanized mice setting the stage: host invasion by hiv reduced antiretroviral drug efficacy and concentration in hiv-infected microglia contributes to viral persistence in brain t cells establish and maintain cns viral infection in hiv-infected humanized mice induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized blt mice alpha interferon and hiv infection cause activation of human t cells in nsg-blt mice humoral immune responses in humanized blt mice immunized with west nile virus and hiv-1 envelope proteins are largely mediated via human cd5 + b cells rapid evolution of hiv-1 to functional cd8 + t cell responses in humanized blt mice generation of hiv latency in humanized blt mice hiv latency in the humanized blt mouse an advanced blt-humanized mouse model for extended hiv-1 cure studies graft versus host disease in the bone marrow, liver and thymus humanized mouse model human immune system development and survival of nonobese diabetic (nod)-scid il2rgamma(null) (nsg) mice engrafted with human thymus and autologous haematopoietic stem cells in vivo suppression of hiv rebound by didehydro-cortistatin a, a "block-and-lock" strategy for hiv-1 treatment efficient inhibition of hiv replication in the gastrointestinal and female reproductive tracts of humanized blt mice by efda pd-1 blockade in chronically hiv-1-infected humanized mice suppresses viral loads efficacy of broadly neutralizing monoclonal antibody pg16 in hiv-infected humanized mice therapeutic efficacy of vectored pgt121 gene delivery in hiv-1-infected humanized mice early initiation of antiretroviral therapy can functionally control productive hiv-1 infection in humanized-blt mice long-acting parenteral combination antiretroviral loaded nano-drug delivery system to treat chronic hiv-1 infection: a humanized mouse model study elite suppressor and chronic progressor hiv-1 isolates replicate vigorously and cause cd4 + t cell depletion in humanized blt mice targeted cytotoxic therapy kills persisting hiv infected cells during art concurrent administration of ifnα14 and cart in tko-blt mice enhances suppression of hiv-1 viremia but does not eliminate the latent reservoir protection efficacy of c5a against vaginal and rectal hiv challenges in humanized mice a caulobacter crescentus microbicide protects from vaginal infection with hiv-1jr-csf in humanized bone marrow-liver-thymus mice inhibition of hiv transmission in human cervicovaginal explants and humanized mice using cd4 aptamer-sirna chimeras blocking hiv-1 infection by chromosomal integrative expression of human cd4 on the surface of lactobacillus acidophilus atcc 4356 a small-molecule cd4-mimetic compound protects bone marrow-liver-thymus humanized mice from hiv-1 infection ultra-longacting removable drug delivery system for hiv treatment and prevention hiv pre-exposure prophylaxis for women and infants prevents vaginal and oral hiv transmission in a preclinical model of hiv infection prevention vaginally of hiv-1 transmission in humanized blt mice and mode of antiviral action of polyanionic carbosilane dendrimer inhibitory effect of hiv-specific neutralizing iga on mucosal transmission of hiv in humanized mice role of semen on vaginal hiv-1 transmission and maraviroc protection a long-acting formulation of the integrase inhibitor raltegravir protects humanized blt mice from repeated high-dose vaginal hiv challenges nanoformulations of rilpivirine for topical pericoital and systemic coitus-independent administration efficiently prevent hiv transmission rnai-mediated ccr5 silencing by lfa-1-targeted nanoparticles prevents hiv infection in blt mice rectal transmission of transmitted/founder hiv-1 is efficiently prevented by topical 1% tenofovir in blt humanized mice one percent tenofovir applied topically to humanized blt mice and used according to the caprisa 004 experimental design demonstrates partial protection from vaginal hiv infection, validating the blt model for evaluation of new microbicide candidates topical tenofovir disoproxil fumarate nanoparticles prevent hiv-1 vaginal transmission in a humanized mouse model vrc01 antibody protects against vaginal and rectal transmission of human immunodeficiency virus 1 in hu-blt mice nanoencapsulation introduces long-acting phenomenon to tenofovir alafenamide and emtricitabine drug combination: a comparative preexposure prophylaxis efficacy study against hiv-1 vaginal transmission prevention of vaginal and rectal hiv transmission by antiretroviral combinations in humanized mice systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous hiv-1 transmission in humanized blt mice tenofovir alafenamide and elvitegravir loaded nanoparticles for long-acting prevention of hiv-1 vaginal transmission vectored immunoprophylaxis protects humanized mice from mucosal hiv transmission systemic hiv and siv latency reversal via non-canonical nf-kappab signalling in vivo in vivo analysis of the effect of panobinostat on cell-associated hiv rna and dna levels and latent hiv infection in vivo activation of latent hiv with a synthetic bryostatin analog effects both latent cell "kick" and "kill" in strategy for virus eradication immunization of blt humanized mice redirects t cell responses to gag and reduces acute hiv-1 viremia in vivo excision of hiv-1 provirus by sacas9 and multiplex single-guide rnas in animal models a highly efficient short hairpin rna potently down-regulates ccr5 expression in systemic lymphoid organs in the hu-blt mouse model engineering hiv-1-resistant t-cells from short-hairpin rna-expressing hematopoietic stem/progenitor cells in humanized blt mice stem-cell based engineered immunity against hiv infection in the humanized mouse model a humanized mouse model generated using surplus neonatal tissue cryptopatches are essential for the development of human galt generation of improved humanized mouse models for human infectious diseases human immune system mice: current potential and limitations for translational research on human antibody responses defective lymphoid development in mice lacking expression of the common cytokine receptor γ chain th1 and th17 immunocompetence in humanized nod/scid/il2rγnull mice the analysis of the functions of human b and t cells in humanized nod/shi-scid/ cnull (nog) mice (hu-hsc nog mice) hypogammaglobulinemia in blt humanized mice-an animal model of primary antibody deficiency improved b cell development in humanized nod-scid il2rgamma(null) mice transgenically expressing human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3 enhanced antibody responses in a novel nog transgenic mouse with restored lymph node organogenesis the hiv reservoir in monocytes and macrophages hiv-1 reservoirs in urethral macrophages of patients under suppressive antiretroviral therapy macrophages sustain hiv replication in vivo independently of t cells the b-cell follicle in hiv infection: barrier to a cure lymphoid tissue fibrosis is associated with impaired vaccine responses technical and conceptual considerations for performing and interpreting functional mri studies in awake rats rodent models in neuroscience research: is it a rat race? rat models of cardiovascular diseases autoimmunity to myelin oligodendrocyte glycoprotein in rats mimics the spectrum of multiple sclerosis pathology 2011:497841. research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year • at bmc a novel immunodeficient rat model supports human lung cancer xenografts severe-combined immunodeficient rats can be used to generate a model of perinatal hypoxic-ischemic brain injury to facilitate studies of engrafted human neural stem cells sprague dawley rag2-null rats created from engineered spermatogonial stem cells are immunodeficient and permissive to human xenografts monkeying around with hiv vaccines: using rhesus macaques to define 'gatekeepers' for clinical trials publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-021872-rhi7hi9m authors: wilkes, rebecca p.; hartmann, katrin title: update on antiviral therapies date: 2015-12-04 journal: august's consultations in feline internal medicine, volume 7 doi: 10.1016/b978-0-323-22652-3.00007-4 sha: doc_id: 21872 cord_uid: rhi7hi9m nan update on antiviral therapies rebecca p. wilkes antiviral chemotherapy use is still relatively uncommon in veterinary medicine. controlled studies evaluating the efficacy of antiviral drugs in cats are lacking, or, if studies have been done, in many cases, the data are insufficient to determine effective dosing for these drugs. with the exception of the recombinant feline interferon (rfeifn)-omega, so far no antiviral drugs are specifically licensed for veterinary medicine, which leaves the veterinary community with the option to use off-label antivirals made for humans to combat viral diseases in feline patients. the goal of research in antiviral chemotherapy is the discovery of antiviral agents that are specific for the inhibition of viral multiplication without affecting normal cell division; however, because viruses are dependent on host cell machinery for replication, drug targets are often nonspecific. this makes antivirals inherently more toxic than antimicrobials are because the antiviral drugs are damaging to not only the virus but also the host cells as well. in addition, agents considered safe for human use are not always safe when administered to cats. 1 antivirals made for systemic use often require host and/ or viral metabolism to be active. therefore, agents designed for use in humans are neither reliably nor predictably metabolized by cats or their viruses. thus antiviral agents should always be tested first in vitro for efficacy and safety, and then followed by pharmacokinetic studies in cats. 1 systemic antivirals often have a relatively narrow safety margin, and special considerations should always be given to patients with reduced hepatic or renal function. well-designed blinded, placebo-controlled studies in client-owned animals should follow studies in laboratory-bred, experimentally infected cats to confirm results in genetically diverse cats. 1 most of the human antivirals are specifically intended for treatment of human immunodeficiency virus (hiv) or human herpesvirus infections. therefore, feline immunodeficiency virus (fiv) and feline herpesvirus type 1 (fhv-1) infections have been the most important indications for antiviral chemotherapy in veterinary medicine. topical antiviral therapy has been mainly used for herpetic ocular disease, but studies have evaluated a systemic antiviral compound (famciclovir) for treatment of multiple clinical syndromes associated with fhv-1 infections. even though combination antiviral therapy has been successful in slowing disease progression in people with hiv, similar therapy has not been thoroughly evaluated in cats. 2 recent studies have focused on combination therapy and evaluation of additional hiv drugs that have not been previously evaluated in feline cells. it is hoped that expanding the number of drugs that are shown to be effective for fiv will lead to effective combination therapy for feline patients. some additional feline infections that have been the focus of current antiviral studies are feline leukemia virus (felv) infection and feline infectious peritonitis (fip). some of the hiv antivirals, such as raltegravir, are nonspecific, and they display activity against additional retroviruses, including felv, in in vitro studies. identification of the human coronavirus that causes severe acute respiratory syndrome (sars) has led to evaluation of antivirals for treatment of various coronaviruses, including the feline coronaviruses (fcovs) that cause fip, although testing is mainly in in vitro stages. several studies have also evaluated the use of rfeifn-omega for treatment of multiple feline viruses. a review of the literature for antiviral treatment in cats, including current recommendations for drug dosages and use, is given in table 7 -1. feline immunodeficiency virus infects lymphocytes, cells of the monocyte-macrophage lineage, and cells of the central nervous system causing a variety of clinical signs (figure 7-1) . the viral replication cycle of fiv is highly similar to hiv. feline immunodeficiency virus binds to host cells by an initial interaction of the fiv envelope (env) glycoprotein with the cd134 molecule on the host cell, resulting in subsequent interaction with the co-receptor cxcr4 on the host cell, followed by viral envelope fusion with the host cell membrane. this allows entry of the viral nucleocapsid into the cytoplasm. the viral rna is released into the cytoplasm and transcribed to complementary dna (cdna) by the reverse transcriptase (rt) enzyme, which is specific to retroviruses. the cdna is subsequently synthesized to double-stranded dna, transported to the nucleus, and integrated into the host genome by another virus-specific enzyme, the integrase. viral messenger ribonucleic acid (mrna) and genomic rna are then transcribed and transported to the cytoplasm. viral proteins are translated and processed by a third virus-specific enzyme, the protease. the immature virion moves to the cell membrane and acquires the viral envelope and glycoproteins and then is finally released from the cells. 2 antiretroviral drugs studied extensively in hiv infection have targeted the three virus-specific enzymes (protease, rt, and integrase), as well as some additional targets, interfering with different steps of the virus replication cycle. 3 as of 2014, approximately 30 compounds are approved by the u.s. food and drug administration (fda) for treatment of different stages of hiv infection. 2 some of these drugs can also be used for fiv, and steps that can be inhibited include: (1) virus entry into susceptible cells by blocking attachment to the host cell co-receptor cxcr4; (2) reverse transcription of viral genomic rna; (3) viral dna integration into host genomes; and (4) proteolytic processing of precursor viral proteins into mature viral proteins (figure 7 -2). 2,3 dose indication close similarities exist between the rt of hiv and fiv, and it has been shown that several rt-targeted antiviral compounds active against hiv are also effective in inhibiting fiv replication in vitro. 4 the rt of hiv is actually the target for three classes of inhibitors: nucleoside rt inhibitors (nrti), nucleotide rt inhibitors (ntrti), and nonnucleoside rt inhibitors (nnrti). nucleoside rt inhibitors and ntrti interact with the catalytic site (the substrate-binding site) of the rt enzyme, whereas nnrti interact with an allosteric site located at a short distance from the catalytic site. for the nrti and ntrti to interact with the substrate-binding site, they need to be phosphorylated. 3 all , and emtricitabine) can be considered as nucleoside analogues, and they act in a similar fashion. after they have been taken up by the cells, they are phosphorylated three times to the active triphosphate form, and they act as competitive inhibitors of the normal deoxynucleoside triphosphate (dntp) substrates, which are used by the cell to make dna. unlike dntp substrates, nrti lack a 3′-hydroxyl group on the deoxyribose moiety. once incorporated into the dna chain, the absence of a 3′-hydroxyl group, which normally forms the 5′-to 3′-phosphoester bond with the next nucleic acid, blocks further extension of the dna by rt, resulting in dna chain termination. the analogues cannot be cleaved from the active center and thus block the rt enzyme. 3 nucleoside analogues are not only accepted as false substrates by viral enzymes, but also by cellular enzymes, and infectious diseases mononuclear cells. 4 six of these drugs (abc, ddi, emtricitabine, 3tc, d4t, and azt) had been previously evaluated in feline cells, and three (amdoxovir, racivir, and dexelvucitabine) had not. significant differences among the drugs were not found, but based on the data obtained, amdoxovir, dexelvucitabine, and racivir appear to be options for future studies investigating their potential use in fiv-infected cats. though pharmacological data for cats are not available for these drugs, cytotoxic properties of these compounds suggest they could likely be used in vivo at dosages comparable to that for azt. 4 nucleotide rt inhibitors are distinguished from nrti as they are nucleotide analogues (not nucleoside analogues), which means that they only need two (not three) phosphorylation steps to be converted to their active form. most importantly, they contain a phosphonate group that cannot be cleaved by hydrolases (esterases), which would make it more difficult to cleave off these compounds, once incorporated at the 3′-terminal end, compared with their regular nucleotide counterparts. use of these compounds also results in dna chain termination. one of these drugs, cidofovir, is active against virtually all dna viruses, including polyoma-, papilloma-, adeno-, herpes-, and poxviruses. cidofovir has been used for treatment of fhv-1 (see feline herpesvirus type 1). adefovir (9-(2-phosphonylmethoxyethyl)adenine [pmea]) has a spectrum of activity that partially overlaps with cidofovir, in that both are active against herpesviruses, but adefovir is also active against hepadnaviruses (hepatitis b) and retroviruses, including fiv and felv. the antiviral activity spectrum of tenofovir (pmpa) is narrower than that of pmea, in that it no longer extends to herpesviruses but is confined to hepadna-and retroviruses. 6 this drug has been tested in vitro against felv (see feline leukemia virus). adefovir has been tested in fiv-infected cats in a 6-week placebo-controlled, double-blinded, clinical trial; 10 cats received adefovir (10 mg/kg subcutaneously [sc] twice weekly) and 10 cats received a placebo. 7 there was no decrease in the proviral or viral loads in treated cats, and the cats developed a progressive, life-threatening anemia. this is a common adverse effect of some nucleotide analogues. 7 adefovir was also tested in combination with the co-receptor inhibitor plerixafor (see co-receptor inhibitors) in the same study, producing the same outcome as seen with use of the adefovir alone. 7 a related drug, (r)-9-(2-phosphonylmethoxypropyl)-2,6diaminopurine (pmpdap), has been shown previously to be a potent inhibitor of fiv replication in cell culture and has reduced the viral load in three of four cats experimentally infected with fiv when treated at 20 mg/kg sc three times per week for 6 weeks. there were no changes in the red blood cell counts or hemoglobin values with treatment. 8 a recent study evaluated the efficacy of this drug in a placebocontrolled, double-blind study with a population of 20 cats naturally infected with fiv. 8 no significant differences were found between pmpdap-treated (25 mg/kg sc twice weekly for 6 weeks) and placebo-treated cats, although cats treated with pmpdap showed a tendency for improvement this is the major cause of their toxicity. zidovudine is the nrti most studied in cats, including in vivo studies evaluating the clinical response of experimentally and naturally fivinfected cats treated with the drug. zidovudine can increase the cd4+/cd8+ ratio and improve clinical condition scores in fiv-infected cats; however, it can result in adverse effects, such as dose-dependent nonregenerative anemia and neutropenia. 4, 5 in addition, mutations producing resistance against the drug can develop. 4,5 therefore, a study evaluated nine nrti to inhibit fiv replication in feline peripheral blood receptor and co-receptor proteins dna a significant decrease in the provirus load but did not lead to improvement of clinical or immunological variables. a statistical decrease in serum magnesium levels was observed in the treatment group, without clinical consequences. no development of resistance of fiv isolates to plerixafor was found during the treatment period, making it a potential treatment for fiv-infected cats. 7 limited oral bioavailability and short half-life preclude clinical use of plerixafor in hiv infection, 2,7 but additional cxcr4 antagonists are under development and should be tested for efficacy against fiv when available. integrase catalyzes strand transfer (3′-end joining), which inserts both viral dna ends into a host cell chromosome. 3 integrase inhibitors are used to treat hiv infection. one of the integrase inhibitors (raltegravir) has been shown to be effective for inhibition of felv (see feline leukemia virus). administration of a combination of drugs from different classes, termed highly active antiretroviral therapy (haart), to hiv-infected patients has turned an invariably fatal disease into a chronic but manageable condition. 2, 3 the goals associated with the use of combinations of three (or more) anti-hiv compounds are: (1) to obtain synergism among different compounds acting at different molecular targets; (2) to lower the individual drug dosages to reduce their adverse side effects; and (3) to diminish the likelihood of development of drug resistance. 3 combination therapy has not been thoroughly investigated for treatment of fiv infection in cats, 2 and use of multiple classes of drugs is more difficult in cats because some of the drug classes that are effective for hiv do not work for fiv. 2, 4 however, the need for combination antiretroviral therapy for feline patients has been the focus of recent studies. the goal of antiviral therapy should be improvement of the cat's clinical status. this is not always correlated with virus replication, as measured by a plasma viral load. 9 it has been suggested that antiretroviral therapy should be administered to fiv-infected cats in the later stages of the asymptomatic phase of infection, during which the cat does not show clinical signs and the immune system is relatively normal and more likely to respond to treatment. 5 after experimental infection, when the cd4+/cd8+ ratio decreases, viral load increases markedly, and clinical signs of immunosuppression begin to appear. however, the situation in naturally infected cats is different, and the quality of life is not associated with the viral load. 11 therefore, it is debated at which time point antiviral therapy should be started and whether it should be administered to asymptomatic cats. in a recent study, antiretroviral therapy was initiated during the later stages of the asymptomatic phase of infection in naturally infected cats. the cats were defined as being in the later stages of the in their clinical signs and cd4+/cd8+ ratios. mild hematological side effects (slight decline in packed cell volume and hemoglobin values) were seen in the treatment group. compared with other ntrti, pmpdap seems to be slightly less toxic. 8 unlike the nrti and ntrti, nnrti are an active form, with no dependence on intracellular metabolic pathways. nnrti inhibit the rt by binding to the enzyme in a hydrophobic pocket that is located away from its catalytic site. the interaction of the compounds with the rt induces conformational changes that affect the catalytic activities of the enzyme. 9 nonnucleoside rt inhibitors are considered highly specific inhibitors of hiv-1, and thus not active against other retroviruses, including fiv. 9 this is due to differences in the structure and/or flexibility of fiv rt that prevent nnrti from interacting with the fiv rt. 10 protease inhibitors are based on the "peptidomimetic" principle, that is, they contain a hydroxyethylene scaffold that mimics the normal peptide linkage (cleaved by the hiv protease) but which itself cannot be cleaved. they thus prevent the hiv protease from carrying out its normal function, which is the proteolytic processing of precursor viral proteins into mature viral proteins. 3 despite similarities between the hiv and fiv proteases, all but one of the currently available hiv protease inhibitors have failed to inhibit the protease of fiv. the one compound of interest, tipranavir, has only been tested against fiv in vitro so far. 4 however, studies have demonstrated that these compounds can be used to inhibit fcov replication (see feline coronavirus). co-receptor inhibitors block viral attachment by binding to receptors on the host cell membrane to obscure the site of interaction of env with the receptor. 2 most of the receptor homologues or antagonists are highly selective for hiv and not useful for veterinary medicine. one exception can be used in cats with fiv infection, the class of bicyclams (e.g., plerixafor). plerixafor (1,1′-[1,4-phenylenbismethylene]bis(1,4,8,11-tetraazacyclotetradecane)-octachloride dehydrate, [amd3100], [ jm3100]), is the prototype compound among the bicyclams. bicyclams are dimeric low-molecular weight nonpeptidic compounds that bind selectively to the chemokine receptor cxcr4. this is the cell surface co-receptor used by both hiv and fiv for attachment and infection of susceptible cd4+ lymphocytes, and the amino acid sequences of human and feline cxcr4 are highly similar. drug binding inhibits attachment of the viral envelope to the host cell. the efficacy of plerixafor against fiv was recently investigated in naturally fiv-infected cats that were treated in a placebo-controlled, double-blind clinical trial. 7 plerixafor was administered at 0.5 mg/kg sc every 12 hours. treatment of fiv-infected cats with plerixafor caused infectious diseases agriculture (usda) as a treatment aid for cats infected with fiv or felv. the primary therapeutic effect is activation of progenitor cd4 t-cells to mature cells, which then produce cytokines, including interleukin (il)-2 and interferon (ifn). a few studies performed by the manufacturer are highlighted in a review article. 13 the studies suggest reduced virus load, improved clinical signs, and improved hematological parameters with treatment. however, the data for placebocontrolled studies were not shown, and a field study with naturally infected cats lacked a control group. independent placebo-controlled, blinded studies are warranted. additional information about immunomodulators and immunostimulants is provided in the feline herpesvirus type 1 and feline coronavirus sections. feline leukemia virus, like fiv, is a member of the family retroviridae, but unlike fiv, felv is a gammaretrovirus and not a lentivirus. feline leukemia virus causes a wide variety of clinical signs in infected cats (figure 7 -3). structural differences affect the susceptibility of gammaretroviruses to anti-hiv drugs, but the similarities in mechanism of replication suggest that some of these drugs can also inhibit felv. this is true of most nrti. 14 zidovudine effectively inhibits felv replication in vitro, and in vivo in experimental infections. however, in naturally felv-infected cats, it did not reduce plasma virus load, improve immunological and clinical status, increase quality of life, nor prolong life expectancy. 15 its bone marrow toxicity can also cause adverse side effects (e.g., nonregenerative anemia) that are more pronounced in felv-infected cats than in fiv-infected cats. therefore, it is not recommended as a first line of therapy for felv infection. 16 asymptomatic phase of infection when the cd4+/cd8+ ratio reached 0.9, because at this stage of infection, the viral load increased markedly, and clinical signs of immunosuppression began to appear. the ratios were calculated every 4 months for 2 to 5 years prior to initiation of the antiviral therapy, and viral loads of all cats were quantified once a year. the cats were randomly assigned to treatment groups of eight cats each. treatment included combination therapy, but no placebo group was used, and the study was not blinded. 5 the follow-up was performed over 1 year, through clinical evaluation and the determination of viral loads and cd4+/cd8+ ratios. comparisons of pretreatment and post-treatment values from the cats were performed, as well as comparison of values between treatment groups. a combination of two nrti (azt + 3tc, 25 mg/kg every 12 hours orally [po]) was compared to treatment with azt alone (5 mg/kg every 12 hours po). the combination of azt and 3tc is often used in hiv-infected patients, given that both drugs show a synergistic effect. treatment with azt alone or in combination with 3tc induced a significant increase in the cd4+/ cd8+ ratio and a significant decrease in viral load within and among groups, with an even greater reduction with combination therapy than with azt alone. only mild side effects, including vomiting in one of eight cats, anorexia in two of eight cats, and anemia in one of eight cats, were seen with this treatment combination, but therapeutic interventions resolved the problems, and treatment did not have to be stopped. 5 however, the lack of a control group and lack of blinding make the results of the study very difficult to interpret. therefore, treatment of asymptomatic fiv-infected cats with antivirals cannot be generally recommended based on the currently available data. an earlier in vivo study was performed in experimentally fiv-infected cats that were treated with a high-dose azt and 3tc combination (100 or 150 mg/kg/day po for each drug). the combination had no anti-fiv activity in these chronically infected cats. severe side effects, which included fever, anorexia, and marked hematologic changes, were observed in some of the cats with such high-dose dual-drug treatment, but the toxic effects were reversed when the dose was lowered to 20 mg/kg every 24 hours. 12 ideally, combination therapy for feline patients will contain at least two to three drugs from at least two different classes, as recommended for human patients. 9 as previously mentioned, pmea (an ntrti) was tested in combination with the co-receptor inhibitor plerixafor; however, because of the toxicity associated with the pmea, this combination cannot be recommended. 7 therefore, use of plerixafor in combination with other ntrti that are less toxic than pmea or compounds of other drug classes are should be further investigated in the future. lymphocyte t-cell immunomodulator (ltci), a protein produced by a bovine-derived thymic stromal epithelial cell line, is conditionally licensed by the united states department of alphaherpesviruses, such as herpes simplex virus 1 (hsv-1). they have been investigated for treatment of fhv-1. members of this group of antiviral agents include acyclovir (and its prodrug, valacyclovir), ganciclovir, and penciclovir (and its prodrug, famciclovir). all require three phosphorylation steps for activation. the first of these steps must be catalyzed by the fhv-1 viral enzyme, thymidine kinase. this makes the drugs less toxic in vivo compared to many of the other antiviral drugs. however, the activity of the thymidine kinase in fhv-1 is not equivalent to the enzyme of human herpesviruses. the second and third phosphorylation steps must be performed by host enzymes, which are not as effective in cats as they are in humans. this knowledge helps explain why the acyclic nucleoside antiviral agents developed for humans infected with hsv-1 are not predictably effective when administered to cats infected with fhv-1 and why pharmacokinetic and efficacy studies are always needed to establish appropriate dosing in cats. 1 acyclovir has been adequately tested in cats for the treatment of fhv-1, but it has a relatively low antiviral potency and poor bioavailability. a very high dose is required for effective treatment, which is associated with unacceptable toxicity, with signs related to bone marrow suppression and nephrotoxicity. 1 a prodrug of acyclovir, valacyclovir, was developed for increased bioavailability in humans, but use for fhv-1 treatment in experimentally infected cats induced fatal renal and hepatic necrosis and bone marrow suppression, and did not reduce viral shedding or clinical disease severity. 1, 19 therefore, despite its superior pharmacokinetics, valacyclovir should not be used in cats. 1 ganciclovir appears to be at least 10-fold more effective against fhv-1 than acyclovir in vitro. ganciclovir is available for systemic as well as topical use in the form of a 0.15% ophthalmic gel formulation in humans. ganciclovir holds promise for feline fhv-1 infection and currently available formulations warrant safety and efficacy studies in cats. 1 tenofovir, an ntrti used for treatment of hiv, has been shown to be effective against felv in vitro. 14 the anti-felv mechanism of tenofovir is probably similar to what has been described for hiv-1. tenofovir is given in the form of a prodrug, which is converted to an acyclic nucleoside phosphate. once converted to the active diphosphate form, tenofovir is incorporated by rt into viral dna, where it acts as a chain terminator to inhibit further elongation of the viral dna. 14 however, in vivo studies in felv-infected cats are lacking. another compound currently used for human hiv therapy, raltegravir, could be considered for the treatment of felv-infected cats. 14 the high degree of conservation across lentiviruses, betaretroviruses, gammaretroviruses, and alpharetroviruses of integrase active sites suggests that felv might be highly sensitive to integrase inhibitors. 16 the mechanism of action against felv is the same as for fiv, inhibition of integration of the viral dsdna that is produced by reverse transcription of the viral rna genome. 14 an in vitro study evaluated the effective 50% inhibitory concentration (ec 50 ) for felv inhibition of raltegravir in several feline cell lines and found these values are in the range of that observed for hiv and a related gammaretrovirus, xenotropic murine leukemia virus, and are well below the minimal plasma concentrations found in humans. 16 the effective concentration of raltegravir had no appreciable effect on cell viability nor induced apoptosis, suggesting that this could be an effective and safe drug also in vivo. 16 however, raltegravir is partly eliminated as glucuronide, a metabolic pathway that is not very efficient in cats, and it would increase the risk of toxicity resulting from drug accumulation. 16 as of 2014, no in vivo studies have been published. feline herpesvirus type 1 is a member of the subfamily alphaherpesvirinae, order herpesvirales. herpes simplex viruses 1 and 2 and varicella zoster virus are also members of this subfamily, and antivirals developed for the treatment of these human viruses have been used for treatment of fhv-1 in cats. feline herpesvirus type 1 typically infects epithelial and mucosal surfaces and travels retrograde along sensory axons to establish latency in the trigeminal ganglia. reactivated virus travels down those same axons to infect similar tissues to those that were originally infected, potentially resulting in recurrent or chronic sequelae, including keratitis, conjunctivitis, rhinosinusitis, dermatitis (figure 7-4) , and potentially blindness. 17 whereas drug combinations have become standard procedure for the treatment of hiv infections, the treatment of other virus infections, including herpesviruses, is routinely based on the use of a single antiviral drug. 18 a group of antiviral drugs known as acyclic nucleoside analogues are used for the systemic treatment of human effective against fhv-1. 23 this is potentially an alternative therapy to the use of topical drugs, the majority of which require multiple daily applications. 23 however, an implantable silicone polymer device impregnated with penciclovir has been developed that holds promise for long-term, steadystate subconjunctival delivery of the drug for the treatment of ocular herpetic disease. 17 although herpetic ocular disease is commonly treated with topical antiviral ophthalmic solutions or ointments (including idoxuridine, vidarabine, or trifluridine), 1 these antivirals do not require a virus-specific phosphorylation step for activation. moreover, they damage host cells, specifically resulting in bone marrow suppression. therefore, they should not be used systemically. 1 for good reviews of these topical drugs, see the reports of maggs 1 and gould. 24 cidofovir, a member of the ntrti class of drugs, has been tested for topical treatment of fhv-1 ocular disease but not for systemic use. it appears to be efficacious topically and is a newer drug (therefore it is included in this section). cidofovir requires the typical two host-mediated phosphorylation steps without virally mediated phosphorylation. 1 its safety when given topically arises from its relatively high affinity for hsv dna polymerase compared with human dna polymerase. it is commercially available only in injectable form in the united states for treatment of a human betaherpesvirus. when applied topically as a 0.5% solution twice daily to cats experimentally infected with fhv-1, it led to reduced viral shedding and improvement of clinical disease compared to the placebo group. 25 its efficacy with only twice daily administration (despite being virostatic) is believed to be due to the long tissue half-lives of the metabolites of this drug. there are reports of its experimental topical use in humans and rabbits being associated with stenosis of the nasolacrimal duct, but this has not been shown in cats. the fact that a twice-daily topical treatment is sufficient, whereas all other topical antivirals require application every 3 to 4 hours, makes cidofovir a useful alternative for ocular topical treatment. 1, 24, 25 small interfering rna small interfering rnas (sirnas) designed to target the fhv-1 dna polymerase 26 and glycoprotein d 27 have been used in vitro to induce rna interference in an immortalized cell line and in primary feline corneal epithelial cells to inhibit fhv-1 replication. rna interference is a posttranscriptional, rna-guided gene-silencing mechanism present in eukaryotes. 28 interference of the fhv-1 essential genes resulted in reduction of virus replication up to 98 ± 1%. this type of therapy is intended for topical treatment of chronic herpetic disease. however, a preliminary in vivo study evaluating topical delivery of sirnas to feline corneas was unsuccessful. 29 the lack of delivery was likely the result of sirna dilution and rapid removal by tear film and blinking. studies are ongoing to identify a means of increasing the most promising systemic drug for the treatment of fhv-1 is famciclovir, a prodrug of the active compound penciclovir, which has been shown to be highly efficacious in inhibiting fhv-1 replication in vitro. penciclovir is absorbed poorly when given orally, so the oral form famciclovir was developed with increased bioavailability and uptake from the intestinal tract. 1 famciclovir requires di-deacetylation, mainly in the blood, and oxidation by a hepatic aldehyde oxidase for conversion to the active compound penciclovir. unfortunately, hepatic aldehyde oxidase activity is basically absent in cats, which makes the pharmacokinetics of this drug complex and results in lower than expected plasma penciclovir concentrations despite administration of relatively high doses of famciclovir. 20, 21 despite this, studies evaluating famciclovir in vivo have shown it to be safe and efficacious for use in feline patients. 20, 22 cats experimentally infected with fhv-1 and receiving famciclovir 90 mg/kg po three times daily for 21 days had significantly improved outcomes for systemic, ophthalmic, clinicopathologic, virologic, serologic, and histologic variables when compared with placebo-treated cats. treatment was initiated on day zero, the same day the cats were infected. 20 even though this study did not mimic how cats with natural infection would be treated, results from a clinical case study suggested this drug is likely effective for treatment of clinical cases, though it was not blinded and placebo controlled. 22 clinical cases with primary ocular disease, rhinosinusitis, and dermatitis each attributed to fhv-1 (though not definitively diagnosed), were treated with famciclovir at doses of 62.5 mg po once or twice daily for ocular herpetic disease or rhinosinusitis or up to 125 mg po three times daily for dermatitis. famciclovir was well tolerated with each dose and had a positive effect on each clinical condition. 22 a definitive dose rate has not been established for famciclovir. however, penciclovir has no appreciable in vitro effect if present for 24 hours prior to infection, suggesting that famciclovir should be administered more frequently than once every 24 hours to ensure exposure to penciclovir as additional epithelial cells become exposed to viral infection. 21 current pharmacokinetic data suggest that dosing three times daily is required, 21 and 40 mg/kg po three times daily has been suggested for treatment of cats infected with fhv-1, based on effective concentrations obtained in in vivo studies 20, 22 and determination of new in vitro 50%-inhibitory concentrations. 21 the most commonly reported adverse effects of famciclovir treatment in humans include urticaria, hallucinations, headaches, and confusion (especially in elderly humans), which would likely be more difficult to detect in animals. for these reasons, judicious use of this drug is recommended in client-owned cats, especially those with preexisting hepatic or renal insufficiency. 21 pharmacokinetic studies have also evaluated the concentration of penciclovir in tears, and treatment with an oral dose of 40 mg of famciclovir/kg three times daily achieves a penciclovir concentration at the ocular surface likely to be a bolus and not added to food. any benefit from lysine therapy is likely only possible with daily, lifelong treatment of cats with chronic herpetic disease, rather than use of lysine as a treatment during acute or recrudescent episodes. potentially, daily therapy would reduce episodes of viral recrudescence. however, clinical studies in pet cats are lacking. the cost of this therapy should be weighed against the potential benefits. owners should be made aware that this is only an adjunctive therapy and that administration of antiviral drugs might be necessary to gain better control of signs. polyprenyl immunostimulant (pi) is an immunomodulator that has a conditional license in the united states for treatment of fhv-1 infection. in blinded, placebo-controlled, experimental challenge studies, pi started on the day of virus exposure significantly reduced the severity and duration of rhinitis and conjunctivitis associated with acute fhv-1 disease (legendre and kuritz, manuscript in preparation). according to the manufacturer, pi upregulates the innate immune system and modulates the immune response toward a cellular response. this activity was attributed to positive effects associated with treatment of fhv-1, which requires a cell-mediated immune response for control. viral titers were not compared between treatment and control groups in the studies, but based on the reduced signs associated with treatment, clinical studies are warranted. feline infectious peritonitis is associated with clinical signs that can affect almost any body system (figure 7-5) . currently, there is no effective treatment for fip despite its importance as the leading infectious cause of death in young cats. 33 following the discovery that sars is caused by a coronavirus (sars-cov), efforts to find an antiviral drug for coronaviruses increased. a few antiviral agents that target different steps in the replication cycle have been tested against feline fcov. coronavirus spike proteins on the viral envelope initially bind to receptors on the host cell membrane. 33 the spike protein mediates fusion of the viral envelope with host cell membranes. during this process, heptad repeats 1 and 2 (hr1 and hr2) of the spike protein assemble to form a complex, resulting in a conformational change that is necessary for fusion. 34 peptides have been used as antivirals by inhibiting the hr1-hr2 interaction, thus preventing membrane fusion. 34 the spike protein must be cleaved for entry of the virus into the cytoplasm. feline coronavirus infection is dependent on cathepsin b, a host cysteine protease found within the cell, making this the likely protease responsible for spike protein cleavage. therefore, cathepsin b can serve as a potential target for the development of therapeutic drugs against fcov. following entry into the cell, fcov produce viral polyproteins that are processed into contact time between the corneal cells and sirnas to allow delivery. twice-daily oral l-lysine bolus administration, initiated prior to experimental infection, reduced the severity of conjunctivitis in cats undergoing primary infection. l-lysine bolus administration also reduced viral shedding in latently infected cats experimentally infected with fhv-1, following changes in husbandry and housing but not following corticosteroid administration. in vitro, lysine supplementation led to reduction of fhv-1 replication. 1 arginine exerts a substantial growth-promoting effect on fhv-1 and is an essential amino acid for viral protein synthesis, and lysine antagonizes this effect. lysine and arginine competitively inhibit transport of each other by using a common transport system, and lysine induces arginase, an enzyme that causes the degradation of arginine. arginine deficiency inhibits synthesis of infectious viral particles and downregulates synthesis of viral proteins. however, unlike the protocol for hsv-1-infected humans, owners of cats receiving lysine for fhv-1 should not be advised to restrict their cat's arginine intake 1 because feeding a diet lacking l-arginine is associated with a severe risk of hyperammonemia and encephalopathy. 30 it has been suggested that the ratio of l-lysine to l-arginine, rather than the concentration of each amino acid, is critical in achieving an inhibitory effect on viral replication. dietary supplementation increases mean plasma concentrations of l-lysine without reducing l-arginine concentrations and has been shown to be safe for use in cats, up to 86 g/kg of diet. supplementation with higher doses has been shown to result in reduced food intake. 30, 31 despite promising initial in vitro data and in vivo results from experimental studies, current studies question whether viral inhibition with increased lysine concentrations, in the absence of decreased arginine concentrations, can be biologically important. a new study evaluating the effect of various ratios of l-lysine and l-arginine on fhv-1 dna replication in vitro demonstrated only a modest reduction in viral dna (less than 1 log) at ratios considered difficult to obtain in vivo in healthy cats. 31 a lack of efficacy of l-lysine supplementation has also been demonstrated in vivo in shelter settings. 1, 32 dietary supplementation was unsuccessful, likely because the cats were anorexic during peak disease and were not ingesting the lysine when they needed it the most. bolus administration was also unsuccessful, likely because of stress associated with the lysine administration. 1, 32 the stress of bolus administration in shelter situations could negate its effects and even cause transfer of pathogens among cats by shelter workers administering the lysine. however, data do not support dietary supplementation. 1, 32 unfortunately, no studies to date have been conducted on client-owned cats; however, anecdotal evidence suggests that there is a benefit from administration of lysine in individuals. dosing is 500 mg po twice daily, which should be given as infectious diseases ment to the host cell. the antiviral effects were concentration dependent, and nelfinavir displayed cellular toxicity at higher doses. gna was a better inhibitor of fcov, and when the two agents were added together, a synergistic antiviral effect was produced. the results suggest that the combined use of gna and nelfinavir could have therapeutic potential in the treatment of cats with fip. 35 viral fusion has also been targeted effectively with a synthetic peptide based on the putative hr2 sequence of fcov. virus replication was significantly inhibited in vitro compared to controls, and the peptide was nontoxic. 34 this peptide was also used in combination with human ifn-alpha. the two displayed a synergistic effect, but the cells were pretreated with ifn prior to infection by the virus. 34 see the section on interferon for further information about interferon treatment for fip. immunomodulators have been considered because fip is an immune-mediated disease. 35 a drug that has shown promise for immunomodulation is pi. 36 this drug has a conditional license in the united states for treatment of fhv-1 infection. in a case series of three cats, pi was associated with prolonged survival in cats with noneffusive fip. 36 no placebo group was included for comparison, so definitive conclusions about the effectiveness of this drug for treatment of fip cannot be drawn. additional immunostimulants such as immunoregulin (propionibacterium acnes), an inactivated bacterin, and a t-cell receptor peptide (manufactured by imulan biotherapeutics), have been suggested for treatment of fip. these are not antiviral drugs; instead, each of these products is reported to stimulate the immune response toward a cell-mediated response or to reduce an overactive type 2 helper t-cell (th2) response. an imbalance in t-cell versus b-cell immune response has been suggested to contribute to the development of fip. it has been proposed that a strong cell-mediated immune response protects a cat from the development of fip, whereas the production of antibodies is counterproductive, enhancing the uptake and replication of feline infectious peritonitis virus (fipv) in macrophages and contributing to the pathology by producing a type iii hypersensitivity vasculitis. however, this hypothesis has been questioned. 37 therefore, even though the use of these types of drugs for stimulation of a cell-mediated response might seem a logical approach for the treatment of fip, there is currently no data to support their use. an additional non-antiviral drug that has been evaluated for treatment of fip is propentofylline. this drug appears to downregulate proinflammatory cytokines, which in turn can reduce vasculitis. vasculitis, as stated earlier, is responsible for pathology associated with fip. however, in a placebocontrolled, double-blind study in cats with late stage fip, mature proteins by viral-specific proteases, the main protease (3c-like [3cl] protease) and the papain-like protease. because the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is also an attractive target for therapeutic intervention. 33 in an in vitro experiment in crandell-rees feline kidney cells, 3cl protease inhibitors and cathepsin inhibitors were tested for their ability to inhibit fcov replication. 33 both types of drugs produced effective inhibition with ec 50 values in the nanomolar range and each drug tested was nontoxic to the cells at effective concentrations. the 3cl protease inhibitors were more effective than the cathepsin inhibitors and when used in combination, these drugs had strong synergic effects. there have not been any in vivo studies with these drugs in cats to date. in one in vitro study, 16 antiviral compounds, including nucleoside analogues used to treat herpesviruses, nrti used for hiv, and protease inhibitors also used for hiv, were tested for their ability to inhibit fcov in cell culture. 35 among the 16 drugs tested, two showed significant inhibition of fcov when compared to the untreated cells. these were nelfinavir and galanthus nivalis agglutinin (gna). nelfinavir is a hiv protease inhibitor that has been shown to target the 3cl protease of sars-cov by interacting with 18 residues of the protease. the drug was slightly less effective against fcov than against sars-cov, likely because only seven of the corresponding residues of the 3cl protease of fcov are identical to the sars-cov protease. 35 gna, a carbohydrate-binding agent, exhibits its antiviral effect by binding to coronavirus glycosylated envelope glycoproteins, thereby inhibiting viral attachineffective within a few weeks because of the development of neutralizing antibodies that limit its activity. 40, 41 rhuifn-α can be given orally for a longer period because no antibodies will develop during oral treatment. unlike rhuifn, rfeifnomega, being a feline recombinant product does not induce neutralizing antibodies when administered sc. this means that the high-dose parenteral protocol can be used safely and efficiently even if repeat administration is required. this is an important factor to consider in a condition where management needs to be lifelong. 42 given po, ifns are inactivated by gastric acid and destroyed by trypsin and other proteolytic enzymes in the duodenum and therefore are not absorbed and cannot be detected in the blood after oral administration. direct antiviral effects are unlikely after oral application; however, ifn still seem to have immunomodulatory activity. type i ifns likely bind to mucosal receptors in the oral cavity, stimulating the local lymphoid tissue, leading to cytokine release from lymphatic cells in the oropharyngeal lymphoid tissues, triggering a cascade of immunologic responses that act systemically. 43 interferons have been used for the treatment of feline retrovirus infections. treatment with ifn improved the clinical condition scores of cats infected with felv and fiv, but not because of a reduction in viral load. this suggests that the improved clinical condition seen with treatment is not specific to an antiviral effect, at least not for fiv and felv, but instead is a result of immunomodulation, potentially associated with the innate immune response. 40, 42, 44 some clinical signs in fiv-infected cats are caused by immunopathological reactions, such as gingivostomatitis and uveitis. immunomodulation might be the cause of improvement of some clinical signs associated with ifn treatment, probably the result of an effective control over inflammatory cytokines in diseased organs. 44 it has been suggested that a nonspecific stimulation of the immune system with ifn therapy might be contraindicated in fiv-infected cats because it could lead to a rise in viral replication produced by the activation of lymphocytes and macrophages harboring latent infections and therefore accelerate disease progression in these cats, and the use of ifn in hiv-infected humans is controversial. 5 however, use of low-dose oral huifn (natural, not recombinant in this study) in ill fiv-infected cats (50 iu per kg on the oral mucosa daily for 7 days on alternating weeks for 6 months, followed by a 2-month break, and then repetition of the 6-month treatment) resulted in improvement of clinical signs in a placebo-controlled, doubleblind study. 44 parenteral rfeifn-omega used according to the licensed protocol (table 7 -1) resulted in decreased mortality rates in felv-infected cats, compared with the control group in another placebo-controlled study. 40, 45 in another study evaluating fiv-and/or felv-infected cats housed in a shelter, hematologic values remained within reference ranges, and there were no biochemical abnormalities there was no statistically significant difference in the survival time, the quality of life, or any clinical or laboratory parameter in cats treated with this drug versus cats receiving a placebo. 38 of the cats in the study, 21 of 23 cats displayed effusion at the start of the study. the drug might be more useful in cats without effusion because it may have a chance to prevent vasculitis and therefore effusions, but studies are lacking. interferons are molecules produced by vertebrate cells in response to viral infections or certain inert substances, such as double-stranded rna, and other microbial agents. there are three types of ifns. type i ifns comprise the largest subfamily and include ifn-α, ifn-β, and ifn-omega. type i ifns are produced by various cell types, such as leukocytes and fibroblasts, in direct response to virus infection. 39 there is only one member of the type ii ifn subfamily, ifn-γ, that is an immunomodulatory cytokine, produced in response to recognition of infected cells by t lymphocytes and natural killer cells of the host's immune system. 39 type iii ifns, which contain three ils, (il-28a, il-28b, and il-29), are identified. this subfamily also has the ability to interfere with virus replication and has been suggested to be the ancestral antiviral system of vertebrates. 39 interferons are not virucidal; rather, they trigger expression of various antiviral proteins and thus induce an antiviral state within the host cell to limit replication and spread of viruses. further, type i ifns have been shown to potently enhance innate and adaptive immune responses in vivo, through various immunomodulatory effects, such as activation of dendritic cells (dcs), amplification of antibody response, and enhancement of t-cell and natural killer cell cytotoxicity. 40 viruses causing lysis of their target cell are most effectively inhibited by ifn through their antiviral activity in noninfected cells. therefore, ifns have their highest utility in the prophylaxis or early postexposure management for virus infections. given that ifns are not specific for a particular virus, they have been tested for the treatment of multiple feline viruses, including fhv-1, fiv, felv, feline calicivirus (fcv), and fcov. 40 two molecules of type i ifns are currently being used for therapy in cats: human recombinant interferon alpha (rhuifn-α), and rfeifn-omega, which is licensed for use in cats and dogs in europe, australia, and some asian countries. ifns are not strictly species-specific in their effects; however, their biologic activity and toleration are greater in cells of genetically related species. in vitro results suggest that rfeifn-omega would likely be more effective than rhuifn-α in vivo, although both ifns have been shown to have therapeutic value in cats. 40 there are two common treatment regimens for use of rhuifn-α in cats: injection of a high dose (10 4 to 10 6 international unit per kg sc every 24 hours) or oral application of a low dose (1 to 50 international unit [iu] per kg every 24 h). when given parenterally to cats, rhuifn-α becomes feline coronavirus shedding was reduced but not significantly, and there was not enough fpv detected in the population to draw any significant conclusions. 41 however, there was no placebo group used for this study, and without a placebo group, it is difficult to determine definitively if the results are due to antiviral effects of ifn or are just consistent with the natural resolution of viral shedding. in a separate study, 36 cats with naturally acquired upper respiratory tract disease housed in a humane society facility were treated with one drop of rfeifn-omega solution (10 6 unit/ml), rhuifn-α solution (10 6 iu/ml), or saline (0.9% nacl) solution (12 cats/group) in each eye twice daily for 14 days for the treatment of keratoconjunctivitis. 46 there was no statistical difference between the treatment groups and the placebo group with regard to clinical scores or viral shedding (fhv-1 and fcv), determined by real-time quantitative pcr from oropharyngeal and conjunctival swabs. feline herpesvirus type 1 shedding was lower, though not statistically significant, on day 14 compared with day 0 for all groups (including the placebo group), and clinical scores were significantly decreased on day 14 compared to day 0, again for all groups including the placebo group. 46 therefore, comparing results between days 0 and 14 in the treated cats without the inclusion of the placebo group would have resulted in a different conclusion for this study. these cats were not infected with felv, and even though the fiv status was unknown for all the cats, the ones that were tested were negative. however, this study highlights the need for a placebo group for accurate evaluation of the effect of ifn therapy on fhv-1 and fcv shedding and associated disease. oral and sc ifn therapy has been associated with an improvement in oral ulcers and gingivitis and gingivostomatitis in cats infected with fiv, 40,41 a condition that is common in cats with fcv infections. 41 feline calicivirus is also associated with chronic gingivostomatitis in cats not infected with fiv or felv, 43 and a study evaluated the efficacy of rfeifnomega (10 5 iu/day for 90 days by topical oromucosal administration) for the treatment of fcv-associated feline chronic gingivostomatitis (fcgs) and caudal stomatitis in fiv-/ felv-negative cats. 43 cats were included in the study if they continued to show persistence of clinical signs of fcgs at least 2 months after periodontal treatment (scaling, subgingival débridement, and polishing), tooth extraction, and 3 weeks of antibiotics with analgesic and anti-inflammatory drugs as needed. twenty-four cats were treated with the ifn, and the effect was compared with a positive control group that received a standard corticosteroid therapy. the results suggested that the ifn therapy was as effective as the corticosteroid treatment for this condition for improvement in clinical signs. 43 feline calicivirus viral loads were not evaluated in this study, and there was no placebo group used for comparison. however, assuming the ifn therapy was the cause of the improvement seen in these cats, the results add to the hypothesis that improvement in oral lesions in fivand/or felv-infected cats likely is associated with the effect of ifn on opportunistic viral infections. differences in the outcome of the different studies could be due to different associated with rfeifn-omega treatment used according to the licensed protocol. 41 these findings suggest that ifn treatment is safe for treatment of fiv-and felv-infected cats, 40, 41, 44, 45 but further studies are required to clearly demonstrate its efficacy against fiv and felv in vivo. a recent study evaluated the use of oral administration of rfeifn-omega for the treatment of symptomatic naturally infected, client-owned fiv-infected cats. 42 the treatment protocol was 10 5 iu/cat po every 24 hours for 90 consecutive days, administered by the cats' owners. a historical group that was treated sc with the licensed protocol 41 was used as a control for comparison, but no placebo group was included. treatment resulted in significant improvement of clinical scores between pretreatment and post-treatment values, and there was no significant difference between the sc historical control group and the po group, suggesting that po administration of rfeifn-omega could be used effectively as an alternative to the licensed protocol, at a significantly reduced cost. 42 an additional benefit of using ifn therapy for fiv and felv treatment could be the effect of ifn on opportunistic infections by other viruses, including fhv-1 and fcv. 41 in fact, the effect of ifn on these additional viral infections might be the cause of the improved clinical scores associated with ifn treatment. 40, 41 both fiv and felv replicate in lymphoid and monocytoid cell subsets and cause immunosuppression. in fiv-infected cats, most of the clinical signs are not directly caused by the fiv itself but are the result of secondary infections, as well as neoplasia. 40, 46 although felv causes more severe clinical syndromes than fiv does, diseases secondary to immunosuppression account for a large portion of the syndromes seen in felv-infected cats as well. 11 considering that ifn therapy seems to have no effect on fiv and felv virus load but it is immunomodulatory, it would seem advisable to treat retrovirus-infected cats with ifn when they have clinical signs, as they would benefit from its effects in improving their clinical condition. 40 a recent study attempted to evaluate the hypothesis that improvement in clinical scores with ifn treatment in fivand felv-infected cats might be a reflection of reduction in viral shedding of secondary viruses in these cats. 41 sixteen naturally infected fiv-and/or felv-infected cats (seven fiv, six felv, and three coinfected) were followed during rfeifn-omega therapy (used according to the licensed protocol) to monitor clinical signs and to correlate them with excretion of concomitant viruses (fcv, fhv-1, fcov, and feline parvovirus [fpv] ). shedding of these viruses was evaluated by real-time quantitative polymerase chain reaction (pcr) (fhv-1 and fcov) or conventional pcr (fcv and fpv). pretreatment and post-treatment samples were compared. feline calicivirus shedding was detected in 13 of 16 cats on day 0 and not detected on day 65. the amount of fhv-1 shedding was significantly reduced in the cats at the end of the study (day 65), compared with the beginning. before euthanasia with a mean survival time of 18 days. there was only one long-term survivor (>3 months) in the rfeifnomega group. interferon treatment might be more effective if started earlier, but this is not of relevance in the treatment of cats with fip in the field. 47 however, ifn therapy might be useful for treatment of cats with chronic fcov shedding, but further studies are required. as previously mentioned, treatment with rfeifn-omega (licensed sc protocol) was associated with a decrease in fcov shedding in fiv-and/ or felv-infected cats; however, the results were not compared with a placebo group. 41 in conclusion, antivirals are still in their infancy for the treatment of feline diseases. however, as new drugs are produced for human viral diseases that can be used for feline patients, and testing of currently available drugs continues, it is hoped that determination of effective protocols for treatment of feline viral diseases will be possible in the future. application methods (e.g., ocular versus oral); however, definitive conclusions cannot be drawn without additional studies that also evaluate viral load in naturally infected cats that are randomized, placebo-controlled, and double-blinded. ifn has also been evaluated for treatment of fip. in a randomized placebo-controlled, double-blinded treatment trial, 37 cats with fip were treated with rfeifn-omega or placebo. 47 in all cats, fip was confirmed by histology and/or immunohistochemical or immunofluorescence staining of fcov antigen in effusion or tissue macrophages. all cats received glucocorticoids, either as dexamethasone in case of effusion (1 mg/kg intrathoracic or intraperitoneal injection every 24 hours) or prednisolone (2 mg/kg po every 24 hours). cats also received either a placebo or rfeifn-omega at 10 6 iu/kg sc every 24 hours for 8 days and subsequently once a week. there was no statistically significant difference in the mean survival time of cats treated with rfeifn-omega versus the placebo. cats survived for a period of 3 to 200 days antiviral therapy for feline herpesvirus infections pharmacological inhibition of feline immunodeficiency virus (fiv) anti-hiv drugs: 25 compounds approved within 25 years after the discovery of hiv antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells evaluation of different antiretroviral drug protocols on naturally infected feline immunodeficiency virus (fiv) cats in the late phase of the asymptomatic stage of infection acyclic nucleoside phosphonates: past, present and future bridging chemistry to hiv, hbv, hcv, hpv, adeno-, herpes-, and poxvirus infections: the phosphonate bridge efficacy and adverse effects of the antiviral compound plerixafor in feline immunodeficiency virus-infected cats -phosphonylmethoxypropyl) -2,6-diaminopurine (pmpdap) in the treatment of feline immunodeficiency virusinfected cats their discovery, development, and use in the treatment of hiv-1 infection: a review of the last 20 years susceptibility of feline immunodeficiency virus/ human immunodeficiency virus type 1 reverse transcriptase chimeras to non-nucleoside rt inhibitors clinical aspects of feline immunodeficiency and feline leukemia virus infection is azt/3tc therapy effective against fiv infection or immunopathogenesis? lymphocyte-cell immunomodulator (ltci): review of the immunopharmacology of a new veterinary biologic discovery of drugs that possess activity against feline leukemia virus a trial with 3′-azido-2′,3′-dideoxythymidine and human interferon-a in cats naturally infected with feline leukaemia virus inhibition of feline leukemia virus replication by the integrase inhibitor raltegravir controlled release delivery of penciclovir via a silicone (med-4750) polymer: kinetics of drug delivery and efficacy in preventing primary feline herpesvirus infection in culture a 40-year journey in search of selective antiviral chemotherapy effects of valacyclovir in cats infected with feline herpesvirus 1 evaluation of orally administered famciclovir in cats experimentally infected with feline herpesvirus type-1 in vitro cytotoxicity and antiviral efficacy against feline herpesvirus type 1 of famciclovir and its metabolites treatment of feline herpesvirus-1 associated disease in cats with famciclovir and related drugs pharmacokinetics of famciclovir and penciclovir in tears following oral administration of famciclovir to cats: a pilot study feline herpesvirus-1: ocular manifestations, diagnosis and treatment options effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus-1 infection in cats evaluation of the effects of small interfering rnas on in vitro replication of feline herpesvirus-1 use of interfering rnas targeted against feline herpesvirus 1 glycoprotein d for inhibition of feline herpesvirus 1 infection of feline kidney cells therapeutic sirna: principles, challenges, and strategies evaluation of delivery agents used for introduction of small interfering rnas into feline corneal cells excess dietary lysine does not cause lysinearginine antagonism in adult cats effects of physiologic concentrations of l-lysine on in vitro replication of feline herpesvirus 1 oral supplementation with l-lysine did not prevent upper respiratory infection in a shelter population of cats potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3c-like protease peptides corresponding to the predicted heptad repeat 2 domain of the feline coronavirus spike protein are potent inhibitors of viral infection synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus effect of polyprenyl immunostimulant on the survival times of three cats with the dry form of feline infectious peritonitis an update on feline infectious peritonitis: virology and immunopathogenesis randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis interferons: signaling, antiviral and viral evasion use of recombinant interferon omega in feline retrovirosis: from theory to practice relevance of feline interferon omega for clinical improvement and reduction of concurrent viral excretion in retrovirus infected cats from a rescue shelter oral recombinant feline interferon-omega as an alternative immune modulation therapy in fiv positive cats: clinical and laboratory evaluation comparative efficacy of a recombinant feline interferon omega in refractory cases of calicivirus-positive cats with caudal stomatitis: a randomised, multi-centre, controlled, doubleblind study in 39 cats lowdose interferon-treatment for feline immunodeficiency virus infection therapeutic effects of recombinant feline interferon-omega on feline leukemia virus (felv)-infected and felv/feline immunodeficiency virus (fiv)-coinfected symptomatic cats effects of topical ocular administration of high doses of human recombinant interferon alpha-2b and feline recombinant interferon omega on naturally occurring viral keratoconjunctivitis in cats effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis key: cord-023364-ut56gczm authors: nan title: education day monday: plenary session 1 monday: parallel sessions date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00651.x sha: doc_id: 23364 cord_uid: ut56gczm nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-007237-8y7218oj authors: manning, ashleigh; willey, samantha j.; bell, jeanne e.; simmonds, peter title: comparison of tissue distribution, persistence, and molecular epidemiology of parvovirus b19 and novel human parvoviruses parv4 and human bocavirus date: 2007-05-01 journal: j infect dis doi: 10.1086/513280 sha: doc_id: 7237 cord_uid: 8y7218oj background. parv4 and human bocavirus (hbov) are newly discovered human parvoviruses with poorly understood epidemiologies and disease associations. we investigated the frequencies of persistence, tissue distribution, and influence of immunosuppression on replication of these viruses. methods. at autopsy, bone marrow, lymphoid tissue, and brain tissue from human immunodeficiency virus (hiv)—infected individuals with acquired immunodeficiency syndrome (aids) and those without aids and from hiv-uninfected individuals were screened for parvovirus b19, parv4, and hbov dna by means of quantitative polymerase chain reaction analyses. results. b19 dna was detected both in hiv-infected study subjects (13 of 24) and in hiv-uninfected study subjects (8 of 8), whereas parv4 dna was detected only in hiv-infected study subjects (17 of 24). hbov dna was not detected in any study subjects. the degree of immunosuppression with hiv infection did not influence b19 or parv4 viral loads. b19 or parv4 plasma viremia was not detected in any study subjects (n = 76; viral load <25 dna copies/ml). a significantly older age distribution was found for study subjects infected with b19 genotype 2, compared with those infected with b19 genotype 1. two genotypes of parv4 were detected; study subjects carrying prototype parv4 (genotype 1) were younger (all born after 1958) than those infected with genotype 2 (parv5; study subjects born between 1949 and 1956). conclusions. tight immune control of replication of b19 and parv4 was retained despite profound immunosuppression. recent genotype replacement of parv4, combined with absent sequence diversity among genotype 1 sequences, suggests a recent, epidemic spread in the united kingdom, potentially through transmission routes shared by hiv. ruses (aavs) in the dependovirus genus. recently, 2 additional human parvoviruses have been discovered [1, 2] . parv4 was detected in a large-scale screening of exogenous dna and rna sequences in a panel of blood samples from individuals with acute infections of undiagnosed etiology [1] . parv4 does not resemble any other known mammalian parvoviruses and probably will be classified as the sole member of a new parvovirus genus. at the same time, a second novel human parvovirus was discovered in pooled respiratory samples [2] . the virus has been named "human bocavirus" (hbov), since it is most closely related to 2 other parvoviruses, bovine parvovirus 1 and a minute canine virus in the bocavirus genus (i.e., bovine/canine) of parvoviridae. hbov infections have been found to be widely distributed among young children worldwide [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] , with growing evidence for an etiological role in severe lower respiratory tract infections. in contrast, detection of parv4 infections to date has been confined to the original infected study subject [1] and to occasional pooled plasma samples used for blood-product manufacturing [13] . thus, the prevalence, epidemiology, transmission routes, and clinical associations of parv4 remain undetermined. on the basis of the observation of relatively short-lived viremia and the development of a strong immune response that conferred protection from reinfection, b19 infections have been considered to be acute and spontaneously resolving. however, more recent evidence from tissue biopsy specimens and autopsy samples has indicated frequent persistent infection in previously exposed individuals [14] [15] [16] [17] [18] [19] , even though infection is controlled effectively by the immune system to the extent that persistent viremia is infrequent and has an invariably low titer among seropositive individuals [20] [21] [22] . the combination of long-term persistence and immunity from reinfection has been shown to create a so-called bioportfolio in which b19 and possibly other persistent dna viruses, such as aavs, that are encountered early in life may be preserved lifelong, thus constituting a record of virus variants that had circulated several decades previously [19] . in the current study, we examined a series of samples of different tissues, collected at autopsy, for the presence of hbov and parv4 dna sequences and compared the frequencies of detection, viral loads, and genetic variability with those for b19. samples from hiv-infected individuals who did not have aids (hereafter referred to as "pre-aids study subjects") and those with terminal aids at time of death were compared with corresponding samples from hiv-uninfected individuals, to investigate the relationship between viral load and immunosuppression and, thus, the role of the immune system in controlling virus replication. bone marrow, lymphoid tissue, and brain tissue were examined as part of an initial investigation of the cellular tropism of these different parvoviruses in vivo. the autopsy tissue samples used in this project were obtained from the edinburgh medical research council hiv brain and tissue bank at western general hospital, edinburgh. consent for use of postmortem tissue was obtained from the lothian research ethics committee. for the studies of viral load in autopsy tissue, lymphoid (lymph node or spleen), brain (frontal or occipital lobe), and bone marrow tissue samples were obtained from 13 hiv-infected individuals with aids at time of death, 11 hiv-infected individuals without aids, and 8 hiv-uninfected individuals (table 1). samples were stored at ϫ80њc for use in subsequent polymerase chain reaction (pcr) analyses. all but 2 of the study subjects with aids were untreated with antiretroviral drugs before death. all pre-aids study subjects had a cd4 count of у200 cells/ml at the time of death, except for 1 study subject with a cd4 count of 167 cells/ml. all presymptomatic study subjects died from aids-unrelated causes [23] . plasma samples were obtained from a total of 36 previously untreated hiv-1-infected individuals at varying stages of disease progression, which was reflected in their cd4 lymphocyte counts (table 1). plasma samples were obtained from 40 low-risk hiv-negative individuals who were approximately age matched to the hiv-positive individuals; samples were collected over the same period of time (2003) (2004) (2005) . dna extraction. tissue samples were extracted as described elsewhere [24] . extracted dna was quantified by spectrophotometry at 260/280 nm. a maximum of 0.5 mg dna was assayed by pcr. dna was extracted from 0.4-ml volumes of plasma by use of the qiaamp dna blood minikit (qiagen). extracted dna was resuspended in 50 ml elution buffer, and 5 ml was assayed by pcr. amplification of parvovirus b19, hbov, and parv4 dna sequences. nested-pcr methods were used for amplification of b19, hbov, and parv4 dna. b19 primers were based on a highly conserved region in the ns gene, with the following sequences (position of the 5 base in b19-au sequence m13178 in parentheses): outer sense, gtgtatcagcaatttgtrs-aatt (2478); inner sense, tytgtttgacttagttgctcg (2867); inner antisense, actggrtgataaggcatgggg (2984); and outer antisense, taagtcttcactagataatac (3038). for screening hbov and parv4 dna, we used previously described primer sequences from the ns gene [12] . a shared amplification protocol for each target was used [12] . nested pcrs for the 3 virus targets were sensitive for single copies of the target sequence [12] , enabling use of limiting dilution [24] for quantitation of positive samples. for the autopsy samples, initial screening was performed by use of 0.5 mg dna from each sample, and positive samples were titrated further in 5-fold dilution steps until limiting dilution was observed in 4 or 8 replicate reactions; viral loads then were calculated from the frequencies of positive reactions at limiting dilution [25] and were expressed as copies per million cells. plasma samples were screened for b19 and parv4 sequences by means of pcr using 5 ml of the extracted dna (40 ml of the original sample). based on single-copy detection [12] , the assay had a theoretical sensitivity of 25 dna copies/ml of plasma. to rule out the presence of impurities in extracted dna that could reduce pcr sensitivity, 1-mg quantities of extracted dna from 15 different autopsy tissues and 3-5-ml volumes of 8 different plasma samples were amplified in reactions spiked with 10 copies of b19 or parv4 dna. no inhibition of amplification was detected in any sample, when compared with the 10-copy amplification controls. nucleotide sequencing. parv4-positive samples were sequenced in the region amplified by the screening primers, by use of the big dye terminator kit (applied biosystems). because the b19 amplicon obtained from screening was short, sequencing used the following alternative set of primers in the ns gene: outer sense, gugguaaraaaaayacmcugugg (1638); inner sense, suguuccaguruauggcaugg (1716); inner antisense, aaguaaugucaccauugcugg (1943); and outer antisense, guaascccaugucagggcugcauc (2048). all samples positive with the screening primers were able to be amplified with the sequencing primers. accession numbers. the nucleotide sequences of b19 and parv4 obtained in this study have been submitted to gen-bank and have been assigned accession numbers ef371379-ef371417. detection of parvoviruses in autopsy tissue. dna samples extracted from lymphoid tissue (lymph node or spleen, depending on availability), bone marrow, and samples of brain tissue (cerebral cortex) were screened for parvovirus dna by means of nested pcr using sets of b19-, hbov-, and parv4specific primers (table 2) . b19 dna was frequently detected in study subjects from all 3 categories (hiv negative, pre-aids, and aids), whereas parv4 dna was detected only in those study subjects with hiv coinfection (7 of 11 pre-aids study subjects and 10 of 13 study subjects with aids). in contrast to the frequent detection of b19 and parv sequences, hbov dna was not detected in any study subjects. because of the previously described association of hbov with respiratory infections, we also assayed autopsy lung samples from 3 study subjects with aids, but pcr analysis showed that all samples were negative for hbov dna (data not shown). samples positive for b19 and parv4 dna were quantified by limiting-dilution pcr (figure 1). viral loads varied widely within each tissue-sample type and subject category, ranging from undetectable (!6 dna copies/10 6 cells) to several samples with 11000 copies of b19 or parv4/10 6 cells. when data from all 3 subject categories were combined, the highest b19 viral loads were found in bone marrow samples, with a median level of 582 dna copies/10 6 cells versus 92 dna copies/10 6 cells in lymphoid tissue ( , mann-whitney nonparametric u p p .07 test) and 69 dna copies/10 6 cells in brain tissue ( ) p p .006 ( figure 1a ). among the hiv-infected study subjects, a similar difference was found in parv4 viral loads in bone marrow versus lymphoid tissue (median values of 220 and 46 dna copies/10 6 cells, respectively; ) (figure 1b). p p .0005 neither b19 nor parv4 showed an association between increased viral load and immunosuppression (figure 1). for b19, median viral loads for hiv-uninfected individuals had a range of values similar to that for the pre-aids and aids subject categories (e.g., median viral load in lymphoid tissue from hivuninfected individuals was 203 dna copies/10 6 cells, compared with 120 and 46 dna copies/10 6 cells for pre-aids study subjects and those with aids, respectively [ ]). among hiv-p 1 .05 infected study subjects, a correlation between cd4 lymphocyte count and b19 viral loads was not found in any of the tissues examined, as determined by means of the nonparametric spearman's rank correlation test ( to .04). using previously r p ϫ.01 determined hiv viral loads for the subjects in this study group [23, 26] , we also found no association between the extent of b19 infection and the extent of hiv infection in any of the tissues examined ( to .20). r p ϫ. 22 although parv4 was found only in the lymphoid tissue and bone marrow of hiv-infected individuals, there was no correlation between immunosuppression and parv4 viral load. viral loads were similar in pre-aids study subjects and those with aids ( figure 1b) (table 2) . the individuals in these study groups were demographically similar to the hiv-positive study subjects from whom autopsy samples were obtained and also showed a range of cd4 lymphocyte counts that substantially overlapped with that of the latter group (table 1). given the likelihood that a substantial proportion of the tested individuals were persistently infected with b19 and parv4, negative findings from analysis of their plasma samples indicate that persistent infection with either virus is not associated with frequent viremia. b19 and parv4 genotypes. amplified dna from samples positive for both b19 and parv4 was sequenced to identify which genotypes infected the study subjects (figure 2). among hiv-infected and -uninfected study subjects, sequences of b19 genotypes 1 and 2 were detected (12 [60%] and 8 [40%] study subjects, respectively), with a significantly different age distribution between genotypes (figure 2a). the median year of birth for genotype 1-infected study subjects was significantly later than that for genotype 2-infected study subjects (1963-1964 vs. 1955, respectively; ). genotype 2 sequences differed p p .004 from genotype 1 sequences by 9.5% in the region sequenced (positions 1737-1922 in the ns region of the b19-au prototype sequence) and showed a highly constrained pattern of variability; all substitutions between and within genotypes were confined to synonymous sites (i.e., non-amino acid changing). the pattern of sequence variability among parv4 sequences was remarkably similar to that of the b19 sequences. two variants of parv4 were detected and differed from each other by 13% in the region sequenced (positions 1455-1569 in the complete parv genome sequence nc_007018); all substitutions again were confined to synonymous sites. fourteen of the sequences were identical to nc_007018 (referred to as "genotype 1"), and the remaining 4 (provisionally termed "genotype 2") showed greater intragroup variability (mean pairwise distance of 3.1%). genotype 2 infections were found only in those born before 1956 (median year of birth was 1953, compared with 1964 for those with genotype 1 infections [ ; figure 2b] ). p p .003 as indicated by their frequent detection in autopsy tissue, both b19 and parv4 showed evidence for the lifelong persistence of infection, which contrasted markedly with the findings for hbov. the high frequency of detection of b19 in this study is consistent with previous pcr-based evidence for frequent, potentially lifelong persistence of infection [19, 27] -that is, strong anti-b19 cytotoxic t cell reactivity among seropositive individuals [28] and the appearance of the igg4 subclass of antibody to b19, which usually is associated with chronic viral infection [29] . indeed, ongoing immunoreactivity to b19 at sites of replication has a potential etiological role in several chronic inflammatory diseases, such as arthritis [22, [30] [31] [32] [33] [34] [35] , although high frequencies of b19 detection also have been reported in a variety of tissues (synovium, liver, bone marrow, myocardium, and lymphoid tissue) in study subjects without inflammatory disease [14] [15] [16] [17] [18] [19] . as indicated by the results of the current study, persistence may be the usual outcome of b19 infection [14] , and the detection of b19 may be incidental to inflammatory diseases [15, 36] . since a high frequency of b19 infection was detected in lymphoid cells in this and a previous study (figure 1a) [19] , the higher viral loads in inflamed tissues, such as synovial tissue in subjects with arthritis, may have arisen through the migration of b19-infected inflammatory cells into affected tissues. although the highest b19 and parv4 viral loads were detected in bone marrow (which is consistent with the propensity of parvoviruses to target dividing cells) and both were found in lymphoid tissue, only b19 was detected in the brain autopsy samples. the spread of b19 into the central nervous system (cns) occurred in all study subjects and was not related to immunosuppression (figure 1a); these findings are consistent with the frequent detection of b19 dna sequences in prefrontal cortex samples in a recent study [37] . the detection of b19 dna sequences in autopsy brain samples is unlikely to be simply a spillover effect associated with the entry of lymphoid or stem cells into the cns; parv4 and b19 viral loads were similar in lymphoid and bone marrow-derived cells, but parv4 was consistently absent from the brain samples (table 2) . a surprising finding in the current study was the similarity in b19 viral loads between immunocompetent and immunosuppressed individuals ( figure 1a ). despite the destruction of the immune system in patients with terminal aids, b19 replication remained under tight control, with no evidence of increased viral loads in autopsy tissue from the study subjects with aids, compared with pre-aids and hiv-uninfected individuals; b19 or parv4 viremia was undetectable in a larger study population of 76 individuals that included several with low cd4 lymphocyte counts. the behavior of b19 and parv4 contrasted markedly with other persistent dna viruses, such as human cytomegalovirus, for which reactivation leading to clinically significant disease frequently develops during aids. as another example, the ubiquitously distributed and persistent small dna virus torquetenovirus (ttv) also shows evidence of increased viral replication in immunosuppressed individuals [38] [39] [40] . using the same autopsy samples that were analyzed in the current study, an ∼1000-fold increase in ttv viral load was found in the bone marrow, lymphoid tissue, and brain tissue samples from the study subjects with aids, compared with the samples from pre-aids and hiv-uninfected study subjects (s. j. willey, g. j. hughes, s. lucas, j. e. bell, and p. simmonds, unpublished data). while b19 dna sequences were detected in study subjects in all 3 categories, detection of parv4 dna was confined to hiv-infected individuals (table 2) . three hypotheses might explain this restricted distribution. it is possible that, in immunocompetent individuals, the immune system controls parv4 replication to such an extent that parv4 dna becomes undetectable in autopsy samples, whereas reactivation of parv4 occurs in immunosuppressed individuals through hiv infection. alternatively, parv4 infection may be nonpersistent in immunocompetent individuals, but the pre-aids study subjects and those with aids in the current study may have developed a persistent infection after reexposure to parv4 while infected with hiv. finally, it is possible that exposure and infection with parv4 is restricted to the hiv-infected group because of shared routes of transmission, such as sexual contact or parenteral exposure. the latter possibility is supported by the finding of a history of injection-drug use in each of the parv4-infected study subjects. without an antibody assay for parv4, it is currently difficult to distinguish between these hypotheses. however, if the replication of parv4 is controlled by the immune system in way that is similar way b19 replication, then several observations in the current study point toward the third hypothesis. first, evidence from the measurement of parv4 viral loads in pre-aids study subjects or those with aids does not indicate that the degree of immunosuppression had any effect on viral replication ( figure 1b) . if the lack of parv4 detection in hivuninfected individuals was the result of immune control, then viral loads would be expected to be much lower in the pre-aids group, since they retain a modestly functioning immune system, compared with the study subjects with aids. second, there was clear differentiation in parv4 infection status, with concordant results obtained from lymphoid and bone marrow samples from all but 1 individual. there was similarly a distinct distribution of parv4 viral loads among infected individuals from negative samples (figure 1b). if parv4 infection was under varying degrees of immune control at different stages of hiv disease progression, such a clear differentiation between hiv-infected and -uninfected individuals would be unlikely. future investigations of autopsy samples from hiv-infected individuals, non-injection-drug users (idus), and hiv-uninfected idus will help resolve the underlying reasons for the association of parv4 infection with hiv infection in the current study. understanding of the epidemiology and transmission patterns of b19 has advanced greatly, owing to the discovery of a temporal succession of infections with genotypes 1 and 2 over the past 60-70 years [19] , as well as recent evidence of much higher rates of sequence change in parvoviruses than had been thought previously [41, 42] . our finding of a significantly older age distribution among study subjects infected with b19 genotype 2 closely reproduced findings from a finnish study that demonstrated that b19 variants detected in biopsy samples corresponded to the original infecting strains [19] . the observed disappearance of genotype 2 infections in all but 1 individual born after 1965 closely matches data in the current study, in which the last genotype 2 infection was detected in an individual born in 1962 (figure 2a); this similarity provides some evidence for long-range b19 transmission networks between quite distantly separated countries in northern europe. evidence was obtained for an equivalent temporal succession of infection with 2 different parv4 genotypes in the study group. infection with divergent variants of parv4, provisionally referred to as genotype 2 in the current study and corresponding to the previously described parv5 variants [13] , was restricted to study subjects born in 1956 or earlier; this age range was even more sharply differentiated from that for genotype 1, compared with the difference observed between the b19 genotypes. if parv4 infection is associated with needle sharing or hiv infection (as discussed in the previous subsection), then the time scale for this succession would be substantially more recent than that for b19. thus, parv4 infection might have occurred only after 1982, when the first cases of hiv infection occurred in edinburgh. this hypothesis of a very recent source of parv4 infection in this risk group is supported by the nucleotide-sequence diversity of parv4. no sequence variation was observed in the 13 genotype 1 sequences of 114 bp. the recently described high rates of sequence change in b19 and canine parvovirus [41, 42] can be used to infer maximum time since divergence for these variants, based on the assumption that substitution rates at synonymous sites in the viral nonstructural regions are similar between different genera of parvoviridae. by application of the observed mean substitution rate of substitutions ϫ4 1.8 ϫ 10 per site per year (95th percentile range, to 3.0 ϫ 10 ϫ4 ϫ5 7.5 ϫ 10 substitutions) [41] , the parv4 variants that infected the study subjects, as well as the individual in whom parv4 was originally discovered, would have diverged within the last 14 years (range, 8-34 years) before death. this prediction indicates a very recent introduction and rapid dissemination of parv4 infection in the hiv/idu risk groups in edinburgh in the 1980s-1990s. in contrast, the same calculation applied to b19 genotype 1 variants from the study subjects (mean pairwise distance, 0.47%) predicted an earlier average divergence time of 52 years, which is remarkably consistent with the observed replacement of b19 genotype 2 by genotype 1 in the 1960s (figure 2) [25] . rapid turnover and changes in transmission dynamics of genetic variants of b19 and parv4 revealed by these new models of parvovirus evolution may apply more broadly to other members of parvoviridae. the combined set of 125 published sequences from the np1 gene of hbov sequences, including those from geographically disparate sources such as sweden [2] , canada [8] , japan [5] , and australia [3] , showed a mean pairwise distance of 0.003, which corresponds to a predicted time of origin of 34 years (range, 21-83 years). as an intriguing alternative to the hypothesis that hbov was not detected in autopsy samples because infections were nonpersistent (see above), the genetic data support the possibility that hbov also might have spread to scotland very recently and was not circulating during the childhood years of the study subjects (1930s-1970s) , when hbov infection usually seems to be acquired. further studies, including the development of serological assays for antibody to hbov, are clearly required in order to understand more fully the transmission dynamics and persistence of hbov, parv4, and b19 infections. new dna viruses identified in patients with acute viral infection syndrome cloning of a human parvovirus by molecular screening of respiratory tract samples evidence of human coronavirus hku1 and human bocavirus in australian children frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections detection of human bocavirus in japanese children with lower respiratory tract infections bocavirus infection in hospitalized children the association of newly identified respiratory viruses with lower respiratory tract infections in korean children human bocavirus infection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus in french children simmonds p. epidemiological profile and clinical associations of human bocavirus and other human parvoviruses novel parvovirus and related variant in human plasma persistence of parvovirus b19 dna in synovial membranes of young patients with and without chronic arthropathy a study of the role of parvovirus b19 in rheumatoid arthritis integrity and full coding sequence of b19 virus dna persisting in human synovial tissue evidence for persistence of parvovirus b19 dna in livers of adults high prevalence of viral genomes and multiple viral infections in the myocardium of adults with "idiopathic" left ventricular dysfunction bioportfolio: lifelong persistence of variant and prototypic erythrovirus dna genomes in human tissue persistent b19 infection in immunocompetent individuals: implications for transfusion safety persistent parvovirus b19 infection without the development of chronic anemia in hiv-infected and -uninfected children: the women and infants transmission study parvovirus b19 infection-persistence and genetic variation an immune control model for viral replication in the cns during presymptomatic hiv infection human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers limiting dilution assays for the determination of immunocompetent cell frequencies. i. data analysis identification of shared populations of human immunodeficiency virus type 1 infecting microglia and tissue macrophages outside the central nervous system hedman k. persistence of human parvovirus b19 in human tissues prolonged activation of virusspecific cd8 + t cells after acute b19 infection parvovirus b19-specific dna in bone marrow from b19 arthropathy patients: evidence for b19 virus persistence clinical and laboratory findings in immunocompetent patients with persistent parvovirus b19 dna in bone marrow presence and significance of human parvovirus b19 dna in synovial membranes and bone marrow from patients with arthritis of unknown origin persistence of b19 parvovirus in synovial membranes of patients with rheumatoid arthritis chronic human parvovirus b19 infection in rheumatic disease of childhood and adolescence persistence of parvovirus b19 dna in synovium of patients with haemophilic arthritis parvovirus b19 and chronic arthritis-causal or casual association? detection of adeno-associated virus 2 and parvovirus b19 in the human dorsolateral prefrontal cortex ttv viral load as a marker for immune reconstitution after initiation of haart in hiv-infected patients effect of immune modulation on tt virus (ttv) and ttv-like-mini-virus (tlmv) viremia tt virus infection: prevalence of elevated viraemia and arguments for the immune control of viral load phylogenetic evidence for the rapid evolution of human b19 erythrovirus high rate of viral evolution associated with the emergence of carnivore parvovirus we are grateful to frances carnie for technical assistance with the brain and other autopsy tissue samples and to gareth hughes and jill douglas for providing plasma samples from hiv-infected and -uninfected individuals. key: cord-000868-vnwpzsu8 authors: eissmann, kristin; mueller, sebastian; sticht, heinrich; jung, susan; zou, peng; jiang, shibo; gross, andrea; eichler, jutta; fleckenstein, bernhard; reil, heide title: hiv-1 fusion is blocked through binding of gb virus c e2d peptides to the hiv-1 gp41 disulfide loop date: 2013-01-22 journal: plos one doi: 10.1371/journal.pone.0054452 sha: doc_id: 868 cord_uid: vnwpzsu8 a strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. hiv-1 patients benefit from a coinfection with gb virus c (gbv-c), since hiv-positive individuals with long-term gbv-c viraemia show better survival rates than hiv-1 patients without persisting gbv-c. a direct influence of gbv-c on hiv-1 replication has been shown in coinfection experiments. gbv-c is a human non-pathogenic member of the flaviviridae family that can replicate in t and b cells. therefore, gbv-c shares partly the same ecological niche with hiv-1. in earlier work we have demonstrated that recombinant glycoprotein e2 of gbv-c and peptides derived from the e2 n-terminus interfere with hiv entry. in this study we investigated the underlying mechanism. performing a virus-cell fusion assay and temperature-arrested hiv-infection kinetics, we provide evidence that the hiv-inhibitory e2 peptides interfere with late hiv-1 entry steps after the engagement of gp120 with cd4 receptor and coreceptor. binding and competition experiments revealed that the n-terminal e2 peptides bind to the disulfide loop region of hiv-1 transmembrane protein gp41. in conjunction with computational analyses, we identified sequence similarities between the n-termini of gbv-c e2 and the hiv-1 glycoprotein gp120. this similarity appears to enable the gbv-c e2 n-terminus to interact with the hiv-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit hiv fusion and should inspire the development of this new class of hiv-1 entry inhibitors. introduction gb virus c (gbv-c) is a common human virus that can be transmitted sexually, parenterally and vertically from mother to child [1, 2] . infection of immunocompetent individuals usually leads to clearance of gbv-c viraemia within the first years; however, gbv-c can cause persistent infection in approximately 25% of cases [3] . as a member of the flaviviridae family, for which a fourth genus, termed pegivirus, was recently proposed, it is related to the hepatitis c virus (hcv) [4] . however, acute and persistent infection with gbv-c does not appear to be associated with hepatitis or any other diseases in humans (reviewed in [5, 6] ). the gbv-c genome is a positive-sense, single-stranded rna (9.4 kb) that contains one long open reading frame (orf) [7] [8] [9] . the polyprotein is post-translationally processed by cellular and viral proteases into the structural proteins such as e1 and e2, as well as the nonstructural proteins ns2, ns3, ns4a/b and ns5a/b [10] . in most industrialized countries 1% to 5% of healthy individuals are viraemic for gbv-c and 10% to 15% have developed anti-e2 antibodies as evidence of past gbv-c infection [11] . in contrast to hcv, in the majority of gbv-c infected individuals, the occurrence of anti-e2 antibodies is associated with clearance of the virus [12] . however, gbv-c elimination has been documented without the appearance of anti-e2 antibodies, indicating that a diagnosis based merely on the detection of anti-e2 antibodies in serum might lead to an underestimation of prevalence of prior infection. based on the common transmission routes, the prevalence of gbv-c in hcv and hiv-1-positive individuals is much higher than in the general population. gbv-c rna prevalence rates range from 20% to 30% in hcv coinfected individuals and from 15% to 40% in hiv-1 patients (reviewed in [3] ). several, but not all, epidemiological studies and a metaanalysis have reported that gbv-c viraemia has a beneficial effect on the course of hiv-1 disease progression and survival [13] [14] [15] [16] [17] [18] [19] [20] . furthermore, mother-to-child transmission of gbv-c reduces the vertical transmission of hiv-1 from gbv-c/hiv-1 coinfected mothers [21] , and recently it was reported that accidental gbv-c acquisition via transfusion is associated with a significant reduction in mortality in hiv-infected individuals [22] . gbv-c is a lymphotropic virus that replicates primarily in b and t cells [23] . therefore, it occupies, at least in part, the same ecological niche as hiv-1. several effects have been proposed to explain the interference with hiv-1, including induction of chemokines, cd4 receptor and coreceptor modulation, prevention of th2 cytokine profile, reduction of t cell activation, as well as downmodulation of fas-mediated apoptosis in gbv-c coinfected individuals [24] [25] [26] [27] [28] [29] [30] [31] . cell culture experiments revealed a direct influence on hiv-1 replication by at least two gbv-c proteins, i.e., the nonstructural protein ns5a and the e2 envelope protein [31, 32] . whereas ns5a leads to decreased hiv-1 receptor expression and stabilization of the th1 profile, the e2 protein prevents hiv-1 entry by a mechanism as yet unknown [31, [33] [34] [35] . using synthetic peptides presenting different regions of e2, we recently demonstrated that this interference with hiv-1 entry can be ascribed to the n-terminal part of the gbv-c e2 protein ranging from residue 29 to 72 (according to genbank accession no. af121950) [33] . two overlapping 20 mer peptides (p4-7 and p6-2) presenting amino acids 37 to 56 (wdrgnvtllcdcpngpwvwv) and 45 to 64 (lcdcpngpwvwvpafcqavg), respectively, of the gbv-c e2 protein, were shown to be most potent with ic 50 s between 2 and 0.2 mm in a tzm-bl-based hiv-1 replication assay. cell entry of hiv-1 is mediated by noncovalently associated trimers of gp120 and gp41 subunits assembled into functional spikes on the surface of virions. the noncovalent association between the two subunits appears to be maintained by interaction of the n-and c-termini of gp120 with the disulfide loop region of gp41 and parts of the flanking n-and c-terminal heptad repeat regions (nhr and chr) (reviewed in [36] ). the primary receptor for gp120 is cd4, which is expressed on the surface of monocytes, macrophages, and on subsets of dendritic and t cells [37] . upon engagement of cd4, gp120 undergoes a conformational shift, enabling binding to the chemokine coreceptors cxcr4 or ccr5, respectively [38, 39] . this interaction is thought to induce additional conformational changes in the envelope trimer that result in exposure of the hydrophobic fusion peptide of gp41 and its insertion into the host cell membrane. subsequently, the nhr and chr regions of gp41 fold back and interact with each other, in order to form a six-helix bundle (6-hb), that promotes the convergence of the viral and cellular membrane and the formation of the membrane pore (reviewed in [40] ). in this study, we aimed to elucidate the mechanism of action by which gbv-c e2 20 mer peptides p4-7 and 6-2, representing the e2 residues from 37 to 64, inhibit hiv-1 entry. we demonstrate that e2 peptides act late after cd4 and coreceptor engagement via binding the gp41 disulfide loop region, a new promising target for hiv-1 entry inhibition. peptides n-acetylated or fluorescein-conjugated peptides derived from the gbv-c glycoprotein e2 (p4-7, p4-7s, p6-2, p6-2s, p9, and p28) were obtained from emc microcollections (tübingen, germany). all other peptides (listed in table s1 ) were synthesized by a standard solid-phase fmoc method using a peptide synthesizer. the n-and c-termini of n36 and c34, as well as the gp120 n-terminus peptides, were acetylated and amidated, respectively. the gp41 disulfide peptides (loop36ox and loop36s) were n-terminally biotinylated for site-selective attachment to streptavidin-coated assay plates. loop36o6 was cyclized by formation of a disulfide bridge between the two cysteine residues through air oxidation at ph 8. the peptides were purified to homogeneity (.95% purity) by high-performance liquid chromatography and verified by laser desorption mass spectrometry (perseptive biosystems, framingham, ma) and by esi mass spectrometry, respectively. peptide stocks were dissolved in 75% dmso/h 2 o and diluted for experiments in respective buffers or medium. hiv-1 virions containing the blam-vpr chimera were produced as previously described [33] . briefly, 293t cells were cotransfected with pnl4-3 proviral dna, pcmv-blam-vpr, and padvantage vectors. after 2 days of cultivation, the viruscontaining supernatant was centrifuged for 10 min at 3006g to remove cellular debris. the hiv-1 virion-containing supernatant was overlaid onto a 20% sucrose cushion and ultracentrifuged at 350006g at 4uc for 90 min. the resulting pellet was resuspended in medium and aliquots were frozen at 280uc until usage. pha/il-2-stimulated pbmc or tzm-bl cells were incubated with e2 peptides (25 mm), phorbol 12-myristate 13-acetate (pma; 40 ng/ml), sdf-1a (1 mg/ml) or rantes (50 nm) for 6 hr. cd4, cxcr4 and ccr5 surface expression was analyzed by using standard facs staining protocols. cells were washed with pbs and incubated for 30 min at 4uc with pe-labeled antibodies (cd4 clone sk3, cxcr4 clone 12g5, and ccr5 clone 2d7, bd biosciences, germany). subsequently, washed cells were resuspended in pbs and analyzed with a facscalibur tm flow cytometer (bd biosciences, germany). the virion-based fusion assay was performed essentially as described elsewhere [41] . briefly, 5610 4 tzm-bl cells were infected with sucrose-purified hiv-1 nl4-3 virions containing blam-vpr (5 ng p24-gag per approach). under temperaturearrested state (tas) conditions, binding of the hi virions to target cells was allowed at low temperature via spinning inoculation, for which virions were added to cells and centrifuged at 20956g, 4uc for 30 min. to remove unbound virus particles, cells were washed with cold medium and incubated with 20 mm of each e2 peptide or respective controls (amd-3100 [10 mm], 2f5 [0.5 mg/ml], b12 [0.3 mg/ml], and b4 [0.3 mg/ml]) at 23uc for 1 h. virus-cell fusion was initiated by a temperature shift to 37uc for a total of 2 hr. under standard conditions, e2 peptides, or defined control agents, were added to cell culture shortly before hiv-1 inoculation, and cells were incubated at 37uc for 2 hr. subsequently, cells were washed with hbss, loaded with the ccf2-am substrate, as described by the manufacturer (invitrogen, germany), and incubated overnight at 20uc. blam activity was quantified using a fluorescence plate reader. the extent of viruscell fusion was determined from the ratio of blue (460 nm) and green (510 nm) emission upon exciting the cells at 405 nm. hiv-1 gp120 binding to cd4, ccr5, and cxcr4 hiv-1 gp120 binding studies using flow cytometry were performed as previously described [42] . briefly, supernatants containing soluble fc-gp120 jrcsf fusion protein or the fc control protein were obtained from transiently transfected 293t cells. for binding studies, 293t cells were transiently transfected with pcd4 expression plasmid or an empty plasmid as control, and preincubated with 40 mm of the e2 peptides, 1 mm scd4, 3 mg/ml vrc01, or 10 mm of the ccr5 inhibitor tak-779. similar amounts of soluble fc-gp120 or fc control, were added for 45 min on ice, and cells were washed and stained with anti-human igg, fitc-conjugated secondary antibody (dako, germany) for 30 min at 4uc. subsequently, washed cells were resuspended in pbs and analyzed with a facscalibur tm flow cytometer. data were collected with bd cellquest tm and analyzed with fcs express v3. in parallel, cd4 and ccr5 expression was determined by staining with the antibody clones sk3 and 2d7, respectively (bd biosciences, germany). hiv-1 gp120 binding studies by immunoblotting were performed as previously described [43] . briefly, 293t cells were transiently transfected with either pcd4 or pccr5 expression plasmids. verification of expression was performed 2 days after transfection via flow cytometry. after 2 days of cultivation, the cells were resuspended in binding buffer (50 mm hepes, 5 mm mgcl 2 , 1 mm cacl 2 , 5% bsa, 0.1 mm nan 3 , ph 7.5). in a 50 ml binding assay, either 40 mm tak-779 or maraviroc, 0.8 mm scd4, 40 mg/ml vrc01, 160 mg/ml 447-52d, 200 mg/ ml f425b4e8 (all: nih aids research and reference reagent program), 40 mg/ml 5f3 (polymun scientific), 0.6 mm e2 340 -fc or fc control, 0.2 mm p4-7, p6-2 or p28 and additionally 10 mg/ml hiv-1 bal or hiv-1 iiib gp120 protein (nih aids research and reference reagent program) were added. after incubation for 1 h at 37uc, cells were washed with cold pbs and lysed in triton x-100 and tween-20 containing buffer. lysates were separated by a 5% sds-page and verified with monoclonal gp120 antibody f425 b4a1 (nih aids research and reference reagent program) or with sheep a-hiv-1-gp120 (aalto bio reagents ltd.) in western blot analyses. hiv-1 gp120 (5 ng) was used as a standard. detection was achieved with hrp-conjugated antihuman antibody and chemiluminescence reagents. 293t cells were transiently transfected with an hiv-1 env expression vector, coding for the cxcr4-tropic nl4-3 gp160 precursor (x4env) for surface expression of gp120-gp41 env trimers or empty plasmid as control. after 2 days of cultivation, cells were incubated with 1 mg scd4 and 1 mm fitc-conjugated e2 peptides for 1 h at 37uc respectively. subsequently, cells were washed, resuspended in pbs, and analyzed by flow cytometry using a bd tm lsr ii flow cytometer (becton dickinson biosciences, germany). flow cytometer data were collected with bd facsdiva tm and analyzed with fcs express v3. in parallel, gp160 expression was determined by staining with hiv-1 gp120 mabs (2g12 and 17b) and an anti-human igg-fitc pab (dako, germany). flat-bottom, 96-well microtest elisa plates (sarstedt, germany) were coated overnight at 4uc with recombinant hiv-1 gp120 (0.5 mg/ml), gp41 (0.22 mg/ml) proteins, or with streptavidin (4 mg/ml) in coating buffer (0.1 m nahco 3 , 0.1 m na 2 co 3 , ph 9.87). nonspecific binding was blocked (blocking buffer: 1% bsa, 0.1% tween-20 in phosphate buffer, ph 7.2) for 1 h at room temperature, followed by washing steps (wash buffer: 0.1% tween in phosphate buffer, ph 7.2). streptavidin-coated plates were then incubated with gp41 disulfide loop peptides (loop36ox or loop36s; 2.5 mm). all plates were then incubated with recombinant e2 340 -fc or fc protein in serial dilutions in sample buffer (phosphate buffer, ph 7.2) for 3 hr at room temperature. to perform the competitive elisas, the recombinant e2 340 -fc protein was adjusted at 5 mg/ml and the monoclonal gp41 antibodies (246-d, f240, t32, d50, 2f5, 4e10, chessie 8 [all: nih aids research and reference reagent program], and 5f3 [polymun, austria]) at 100 mm. after washing procedure, the plates were incubated for 1 h with polyclonal rabbit anti-human igg peroxidase antibody (dako, germany) 1:6000 in sample buffer and washed. plates were developed with tmb peroxidase substrate solution (kpl, usa), or with opd/h 2 o 2 solution in the dark. absorbances (ods) were read at 450 or 492 nm, respectively, using a multi-channel photometer (elisa-reader axsym, abbott laboratory, usa). inhibitory activity of the peptides (p4-7, p6-2, and p28) on 6-hb formation was measured by a modified elisa-based method as previously described [44] . briefly, 96-well polystyrene plates were coated with the 6-hb-specific monoclonal antibody, nc-1 igg, (described by [45] ) (2 mg/ml in 0.1 m tris, ph 8.8), and then blocked with 2% non-fat milk. n36 (0.25 mm), the tested peptides and c34 as control were added at graded concentrations into the wells. after incubation for 30 min at 37uc, c34-biotin (0.25 mm) was added, followed by incubation for 45 min at 37uc and washing with wash buffer (pbs containing 0.05% tween-20) six times. streptavidin-labeled horseradish peroxidase (invitrogen, grand island, ny) and the substrate 3,39,5,59-tetramethylbenzidine (sigma, st. louis, mo) were added sequentially. absorbance at 450 nm (od 450 ) was measured. the percent inhibition of 6-hb formation by the peptides was calculated as described before [46] . globular and disordered regions in e2 were identified using globplot2.3 [47] with standard settings. secondary structure prediction was performed using the consensus prediction method provided by the nps@ server [48] . structural homologs were identified with the bioinfo.pl meta prediction server [49] , and modelling was performed with modeller6.2 [50] , using the crystal structure of a single-variable-domain antibody (pdb code: 2z8w) as template [51] . sequence comparison between different e2 proteins and between e2 and gp160 was performed with lalign (http://www.ch.embnet.org/software/lalign_form.html) using the local alignment option. the gp160 signal peptide comprising residues 1 to 30 was removed prior to the similarity searches because it is not present in the mature protein. all statistical analyses were performed using wilcoxon rank-sum test (two-tailed). it has been shown that the hiv-1 receptor/coreceptor density plays a critical role in the efficiency of hiv-1 fusion and infection [52] . thus, we addressed the question of whether or not the two overlapping gbv-c e2 20 mer peptides p4-7 and p6-2 have an impact on the cell surface presentation of the hiv-1 receptors cd4 and cxcr4 or ccr5, respectively. the cell surface presentation of these receptors was quantified via flow cytometry after incubation of primary peripheral blood mononuclear cells (pbmcs) and of hela-derived hiv indicator cells expressing cd4, ccr5 and cxcr4, designated as tzm-bl cells, with the e2 peptides p4-7 and p6-2. the non-hiv-1-inhibitory peptide p28 derived from the c-terminal part of e2 (residue 271 to 290), served as a negative control [33] . as shown in figure 1 , both cd4 and coreceptor expression on pbmc or tzm-bl cells remained largely unaffected by e2 peptides, thereby eliminating a change in receptor density as a mode of action of the e2 peptides. the effect of gbv-c e2 peptides arises after gp120/cd4 and gp120/coreceptor engagement to find out whether early or late steps of hiv-1 entry are affected by the e2 peptides, the e2 peptide activity was determined before and after gp120/cd4 engagement using the vpr-b-lactamase (vpr-blam) enzyme-based virus-cell fusion assay under standard (pre-cd4 binding) and temperature-arrested state (tas; post-cd4 binding) conditions, respectively. based on the temperature sensitivity of envelope-mediated membrane fusion, low temperature (,28uc) during and after cd4/gp120 engagefigure 3 . e2-derived peptides do not influence hiv-1 gp120 binding to cd4 and ccr5. (a) 293t cells expressing cd4 were preincubated with e2-derived peptides and specific controls that bind to gp120 (scd4; vrc01, directed against cd4 binding site [bs]) or ccr5 (tak-779). subsequently, cells were incubated with soluble fc-gp120 (hiv-1 jrcsf ) and binding to cd4 was analyzed by flow cytometry using a fitc-conjugated anti-human igg pab. binding to cells in the absence of inhibitor was set as 100%. columns show average values 6sd of three independent experiments each performed in duplicate. *: p,0.05 in (b) and (c) 293t cells expressing cd4, ccr5, or cxcr4 were preincubated with e2-derived peptides, e2 340 -fc and fc protein as well as specific controls that bind to gp120 (scd4, vrc01, 447-52d, f425 b4e8), gp41 (5f3), or ccr5 (tak-779, maraviroc). soluble hiv-1 gp120 was added to the cd4-, ccr5-, or cxcr4-expressing cells and binding was investigated by separating the cell lysates on sds-page and visualized by performing western blot analyses. no influence of e2 peptides or intact e2 protein on the binding efficiency between gp120 and cd4, ccr5, or cxcr4 respectively, was observed after normalization of band intensity with the gel loading control hsp90. doi:10.1371/journal.pone.0054452.g003 ment leads to an arrest of the fusion process at an intermediate state and the blockade of coreceptor binding, 6-hb formation, and final membrane fusion [53] [54] [55] . as shown in figure 2 , the effect of antibodies that interfere with the initial cd4/gp120 interaction (b12 and b4) was largely abrogated under tas conditions. in contrast, p4-7 and p6-2 showed full activity under both conditions, comparable to known fusion inhibitors like amd-3100 and the neutralizing mper antibody 2f5 that act after cd4 engagement. therefore, these data provide evidence that p4-7 and p6-2 implement their hiv-1 inhibitory activity post-cd4 binding of gp120. to substantiate this conclusion, next we assessed the impact of e2 peptides on gp120 binding to cd4. immunoblotting and flow cytometry data revealed that e2 peptides and the whole e2 ectodomain fused to the fc domain of human igg (e2 340 -fc) that showed hiv-1 inhibitory activity in earlier studies [32] do not interfere with the interaction of recombinant gp120 with cellular expressed cd4 ( figure 3a, b) . in a related approach, the effect of e2 peptides or intact e2 protein on the interaction between gp120 and the coreceptors ccr5 and cxcr4 expressed on cells was tested by immunoblotting. to ensure efficient binding of gp120 to the coreceptors, soluble cd4 (scd4) was added simultaneously. use of ccr5-antagonists maraviroc and tak-779 or anti-gp120 coreceptor binding site (v3-loop) antibodies 447-52d or f425b4e8, respectively, as positive controls revealed a clear reduction in the gp120/ccr5 and a less pronounced decline (densitometric analysis ,50%) in the gp120/cxcr4 interaction. however, after normalization of the band intensity (hsp90; data not shown) there was no indication for any e2-effect on the binding efficiency between gp120 and the hiv-1 coreceptors ccr5 or cxcr4, respectively ( figure 3c , d). recombinant gbv-c e2 protein interacts with gp41, while n-terminal gbv-c e2 peptides abrogate this interaction previously, we have shown that the target of the hiv-1inhibitory e2 peptides is most likely located on the hiv-1 particle, rather than on the cell [33] . the surface of the hiv-1 particle consists of a lipid bilayer with incorporated host cell proteins and a limited number of functional envelope (env) trimers (on average 1467) [56] . therefore, we tested whether the hiv-1-inhibitory e2 peptides interact with trimeric hiv-1 env proteins expressed on the surface of transfected cells. for this purpose, 293t cells were transiently transfected with an hiv-1 env expression vector, coding for the gp160 precursor of the cxcr4-tropic nl4-3 envelope (x4env). binding of fitc-labeled e2 peptides was monitored by flow cytometry. although we could observe a background attachment of p4-7 and p6-2 to mock-or vectortransfected 293t cells, there was a significant increase in the binding of p4-7 and p6-2 when cells expressed hiv-1 env and were pretreated with scd4 ( figure 4a ). in contrast, the increase of binding was not observed with the negative control peptide p28. these data suggest that the hiv-inhibitory e2 peptides bind to hiv-1 env and that this interaction seems to be cd4-dependent, since the induction of conformational changes in gp120 and/or the increase in availability of gp41 after cd4 engagement facilitates e2-binding. in order to prove the assumption that gbv-c e2 interacts with hiv env, subsequent binding assays were performed with plate immobilized subunits of hiv-1 envelope proteins (gp120 and gp41). to improve the significance of this experiment, here we used the whole e2-fc fusion protein (e2 340 -fc) as a ligand. the fc domain alone served as a negative control. ninety-six-well plates were either coated with recombinant full-length gp120 (gp120 iiib [immuno diagnostics, 1001-10], and gp120 mn [immune technology, it-001-002mnp]), expressed in eukaryotic cells, or with gp41, expressed in e. coli, consisting of the complete (gp41 iiib [abcam, ab68129]) or truncated gp41 ectodomain (gp41 mn [nih, 12027] ). in gp41 mn , the hydrophobic fusion peptide (fp), the transmembrane region (tm), and most of the c-terminal part of the cytoplasmic tail (cp) were omitted (illustrated in figure 5a ). whereas no binding of the recombinant e2 340 -fc fusion protein to gp120 was observed, e2 340 -fc protein was shown to interact with both gp41 variants in a dose-dependent manner ( figure 4b ). to test whether gbv-c e2 binds hiv gp41 with the nterminal domain, competition assays were performed with the nterminal e2 peptides p4-7 and p6-2. the results of this experiment revealed that both hiv-1-inhibitory e2 peptides abrogated the protein-protein interaction between hiv-1 gp41 and gbv-c e2 in a dose-dependent manner, whereas increasing amounts of the negative control peptides p9 and p28 had no effect ( figure 4c , left panel). the e2 peptide p9 (residue 81 to 100 of gbv-c e2, see table s1 ) was chosen as an additional control [33] , due to its more comparable content of cysteine and hydrophobic residues. noteworthy, p4-7 and p6-2 contain either two or three cysteines, respectively. to elucidate the characteristics of the e2-gp41 interaction, next we tested the competition ability of peptide variants p4-7s and p6-2s in which the cysteine residues were replaced with serine. the results revealed that the serine variants p4-7s and p6-2s had lost their ability to abrogate the e2-gp41 binding, suggesting that the cysteine residues within the hivinhibitory e2 peptides play a crucial role for gp41 recognition ( figure 4c, right panel) . in combination with these findings, we are able to show that the n-terminal region of gbv-c e2 (,residue 37 to 64), that is represented by the hiv-1-inhibitory peptides p4-7 and p6-2, interacts with hiv-1 gp41 and that the cysteine residues within this region appear to contribute substantially to the gp41 binding. we next sought to localize the e2 peptide binding site within gp41. the transmembrane protein gp41 consists of several functional regions ( figure 5a ). the fp, tm and cp regions of gp41 do not appear to be involved in the interaction with gbv-c e2 or e2 peptides, since the absence of these regions (gp41 mn ) did not abrogate or decrease the e2 binding to gp41 ( figure 4b ). to test whether the e2 peptides interact with either the nhr or chr regions of gp41, which would lead to a blockade of 6-hb formation, a competitive elisa was performed. in this assay, peptides n36 and biotinylated c34, which present the nhr and the chr region of gp41, respectively, interact with each other, forming stable 6-hb structures in vitro [57, 58] . the 6-hb-specific mab nc-1 was used as a capture antibody [45] and the gbv-c e2 peptides were tested for competition with n36 and c34, respectively. neither p4-7 nor p6-2 was able to interfere with the formation of 6-hb, suggesting that the binding site of the e2 peptides is neither located in the nhr (n36) nor in the chr (c34) regions of gp41 ( figure 5b ). to further dissect the e2 binding site within gp41, we tested whether the e2 peptides compete for gp41 binding with different monoclonal anti-gp41 antibodies with known specificities, including mabs 246-d, f240, t32, d50, 5f3, 2f5, 4e10, and chessie 8. of the eight antibodies, only the gp41 interaction of f240 and t32 was inhibited in a dose-dependent manner by p4-7 and p6-2, but not by the negative control p28 ( figure 5c ). no unspecific binding between the e2 peptides (p4-7 and p6-2) and the antibodies f240 or t32 was observed that could have account for the same competition results (data not shown). therefore, we conclude that p4-7 and p6-2 compete with f240 and t32 considerably for the same binding region. f240 and t32 represent two of three tested antibodies that recognize the immunodominant disulfide loop region of gp41. their epitopes overlap partially with each other, implying that the e2 peptides bind approximately between residues 596 and 612 of gp160. the epitopes of all tested anti-gp41-mabs are shown in figure 5a and table s2 . again, the serine variants of p4-7 and p6-2 lost their ability to compete for the binding target with the disulfide loop antibody f240 or were at least less efficient (t32). finally, we performed a direct binding assay with biotin-tagged 36mer gp41 disulfide loop peptides (residues from 588 to 623 according to hiv gp160 hxb2 ) and gbv-c e2 340 -fc. we immobilized via streptavidin an oxidized (closed loop) or a linear variant of the disulfide loop, in which the cysteine residues were replaced with serine (table s1 ) onto plates and the binding of graded concentrations of gbv-c e2 340 -fc was monitored with hrp-anti-human antibodies. indeed, a distinct binding of recombinant e2 340 -fc protein to the gp41 disulfide loop peptides was evident in a dose dependent manner, whereas for the fc control only a low background binding was observed. the binding of e2 340 -fc was clearly diminished when the linear serine variant of the gp41 disulfide loop was tested ( figure 5d ). specific intra-and intermolecular interactions within hiv-1 env are essential for the formation of a native and functional hiv-1 envelope trimer, as well as for concerted conformational changes during the entry process. therefore, peptides or small molecules that mimic env regions involved in these interactions are potential inhibitors of the formation of properly folded tertiary and quaternary env structures. examples of this strategy are potent peptide fusion inhibitors, which resemble the gp41 nhr or chr sequences, including enfuvirtide (t20) [59] , sj-2176 [60] , or n36 [61] . hence, we performed a similarity search between the gbv-c e2 ectodomain and hiv-1 gp160 and identified a local similarity of two sequence stretches of the gbv-c e2 n-terminus to the hiv-1 gp120 n-terminus ( figure 6a ). the sequence stretches 33-46 and 54-70 of e2 exhibit sequence similarities of 85.7% and 76.5% to the n-terminus of gp120. this region of similarity also agrees very well with the sequence region spanned by e2-derived peptides (residues 29-72) that proved to be potent in hiv-entry inhibition [33] . we have also put this sequence similarity in the structural context of gp120 and gp41. high-resolution structural information on gp120-gp41 interaction remains elusive (reviewed in [36] ). however, several mutagenesis studies and a recent crystal structure of gp120 suggest that the n-and c-termini of gp120 interact predominantly with the disulfide loop region of gp41 [62, 63] . interestingly, the n-terminal sequence stretch of gp120 that has been proposed to be involved in the gp120-gp41 interaction is located within the gp120 region that resembles the hiv-1-inhibitory n-terminal gbv-c e2 peptides ( figure 6b ). furthermore, residues within the gp41 trimer, which have been proposed to be involved in the gp120 interface, are located near or within the epitopes of the two antibodies t32 and f240 whose interaction with gp41 was disrupted by the e2 peptides p4-7 and p6-2 ( figure 6c ). to prove the assumption that a similarity between the gbv-c e2 and hiv gp120 n-termini enables e2 or e2 peptides to bind to gp41 in a gp120 manner, a corresponding n-terminal gp120 peptide should be able to disturb the e2-gp41 interaction. therefore, we assessed in e2-gp41 competition assays the effect of a gp120 peptide (n35, gp120 residue 31 to 65) that includes the respective e2-similarity region (figure 7) . we observed that n35 efficiently competed with e2 for gp41 binding, implying that e2 and gp120 share the same gp41 interaction site. not surprisingly, the gp41 affinity was more pronounced for n35 (ic 50 :0.7 mm) than for the e2 peptides (ic 50 s: 7.0 mm for p6-2 and 11.7 mm for p4-7). the c54s variant of this peptide (n35s) still abrogated the e2-gp41 interaction in a dose dependent manner, but less efficiently than the wild-type n35 peptide (ic 50 :4.6 mm). together, these results strongly support the hypothesis that a mimicry phenomenon between the n-termini of the non-related viral glycoproteins e2 gbv-c and gp120 hiv, enables the e2 n-terminus to approach the gp120 binding site on gp41 that is believed to be the disulfide loop region. since no structural information on the gbv-c e2 protein is currently available, we performed structure predictions and globularity analyses of the gbv-c e2 protein in order to get an idea whether the n-terminus of e2 is accessible to the interaction with hiv-1 env involved in membrane fusion. our analysis consistently suggests the presence of a folded region spanning mn) and peptides (n36, c34) used in elisa as well as the binding epitopes of different monoclonal gp41 antibodies. gp41 consists of the n-terminal fusion peptide (fp), the n-and c-heptad repeats (nhr, chr) that are connected by the disulfide loop region (c-c loop), the membrane proximal external region (mper), the a-helical transmembrane-spanning domain (tm), and the cytoplasmic tail (cp). (b) inhibitory activity on 6-helix-bundle (6-hb) formation of peptides p4-7, p6-2, and p28, as well as c34 as control was measured by elisa using the nc-1 monoclonal antibody that detects the 6-hb formation between c34-biotin and n36 peptides. each sample was tested in triplicate. this experiment was repeated twice and similar results were obtained. the statistical significance (p,0.01) was achieved for all measure points (c34 vs. p28). (c) competitive binding of e2 peptides and different gp41 targeting mabs to immobilized recombinant gp41 mn . simultaneously to mab incubation e2 peptides were added with increasing amounts. the graphs show average values of three independent experiments each performed in duplicate. *: p,0.05 (p4-7 or p6-2 vs. p28) (d) binding of recombinant e2 340 -fc protein and fc protein as control to cyclic (loop36ox) and linear (loop36s) peptides presenting the hiv-1 gp41 disulfide loop (residues 588-623). the graphs show average values of three independent experiments each performed in duplicate. the statistical significance (p,0.05) was achieved with more than 0.003 mm concentrations of each protein (loop36ox+e2 340 -fc vs. loop36ox+fc). doi:10.1371/journal.pone.0054452.g005 figure 6 . local similarities between the n-termini of gp120 and e2. (a) similarities of the two local sequences in the n-termini of hiv-1 gp120 and gbv-c e2. the detected e2 stretches (residues 33-46 and 54-70) are shown in the context of the entire e2 n-terminus and are color coded in green and orange, respectively. illustrated are the sequences of the peptides that proved to be potent in hiv-1 entry inhibition [33] . these peptides almost exclusively cover the e2 sequence stretch that exhibits similarities to the gp120 n-terminus. molecules are shown in space-filled presentation and the functionally important regions are colored. (b) structure of monomeric gp120. the n-terminal region of gp120 that exhibits local similarity with the active e2-derived peptides is shown in blue. residues that were deduced from mutational analyses to be relevant for the gp120-gp41 interaction (reviewed in [36] ) are additionally shown in green. (c) structure of trimeric gp41. the disulfide-bonded loops that are recognized by the t32 antibody (residues 596-612) are shown in red. residues 592-596 additionally present in the f240 epitope are shown in orange. residues that were deduced from mutational analyses to be relevant for the gp120-gp41 interaction (reviewed in [36] ) are additionally shown in green. the coordinates for structure presentations were taken from pdb entries 3jwd and 2ezo for gp120 and gp41, respectively. doi:10.1371/journal.pone.0054452.g006 residues 76 to 195 of gbv-c e2. the remaining extracellular part, in particular the n-terminal region including amino acids 1 to 75 that comprise the hiv-1-inhibitory domain, does not contain significant amounts of regular secondary structure and is therefore predicted to form no globular conformation ( figure 8) . consequently, the e2 n-terminus is likely to be rather flexible which could plausibly account for its interaction with gp41 during hiv-1 entry. in this study, we have explored the molecular mechanism underlying the previously reported interference with hiv-1 entry by two overlapping peptides derived from the n-terminal part of the gbv-c envelope protein e2. we are able to demonstrate that the e2 peptides interfere with late stages of hiv-1 entry. this notion is evidenced by the observations that neither the cell surface expression of hiv-1 receptors, nor the binding of gp120 to cd4 and coreceptors, respectively, appears to be affected. instead, hiv-inhibitory e2 peptides showed full activity on hiv entry at a post-binding stage. in agreement with these findings, binding experiments revealed an interaction between e2 or e2 peptides and the hiv-1 transmembrane protein gp41 responsible for late membrane fusion activity. in an earlier study, we demonstrated that the hiv-1 inhibitory region ranges from residue 29 to 72 of the gbv-c e2 n-terminus [33] . in this study we show that the e2 peptides p4-7 and p6-2 (representing e2 residues 37 to 64) compete with e2 protein for gp41 binding. therefore, we assume that the hiv-inhibitory region or at least the inner domain ranging from residue 37 to 64 reflects a gp41-binding site of gbv-c e2. interestingly, the sequence stretch covered by the hiv-inhibitory peptides includes the strongest conserved part of the nonglobular n-terminus of related flaviviridae e2 proteins ( figure s1 ). in particular, several cysteines (c46, c48, c60) and tryptophans (w55, w65) are highly conserved among gbv-c and gbv-a isolates, suggesting that the respective e2 proteins might also exhibit a similar anti-lentiviral activity. the alignment shown in figure s1 also reveals that the sequence conservation is less pronounced for the more distantly related e2 proteins of gbv-b and gbv-d. the n-termini of the e2 proteins from gbv-c and hcv show no detectable sequence homology at all rendering functional similarity rather unlikely. the results of several experiments aimed at dissecting the e2binding domain within gp41 suggest that e2 interacts with the gp41 disulfide loop region. the gp41 disulfide loop region structurally connects the nhr and chr domains and contains two conserved cysteines [64] . noteworthy, the n-terminal e2 region contains three cysteine residues (cys46, cys48 and cys60) as well. previously we could show that variants of the e2 peptides (p4-7s and p6-2s), in which cysteines were replaced with serine residues, lost their hiv-inhibitory capacity [33] . in agreement with these observations, in this study, p4-7s and p6-2s lost their ability to compete with e2 for gp41binding. in addition, the cysteine residues within the disulfide loop peptide appeared to be crucial for the interaction with recombinant e2 protein. this implies that the cysteine residues within the hiv-inhibitory e2 peptides may interfere with the oxidation state of the respective cysteines in the gp41 disulfide loop region. a variety of evidence suggests that a number of viral envelope glycoproteins depend on a dynamic thiol/disulfide balance to mediate virus-cell fusion (reviewed in [65] ). for hiv-1 it has been shown that after cd4 binding a cell surface-associated reductase activity leads to cleavage of disulfide bonds at least within gp120 and that this event is obligatory for triggering membrane fusion [66] . however, the insights into the mechanistic role of the disulfide loop cysteines for the fusion reaction are still limited and need further evaluation. future studies will show, whether reducing agents would change the interference effects of gbv-c e2-derived peptides. based on cryo-em structural information, the gp41 transmembrane protein is expected to be at least partially buried in the trimeric gp41-gp120 structure [67] . thus, the transient states of gp41 appears to be valid hiv-1 inhibitor targets, as evidenced by a number of known hiv-1 fusion inhibitors, including gp41targeted peptides and low-molecular-weight inhibitors. these inhibitors typically bind to the nhr or chr regions during the prehairpin stage in order to prevent the formation of the 6-hb. however, münch et al. [42] isolated the natural hiv-1 entry inhibitor virip from human hemofiltrates, targeting the gp41 fusion peptide, and broad neutralizing antibodies, like 2f5, 4e10, and z13e1, bind the mper, an epitope that is also transiently accessible at a late stage of hiv-1 entry. our results show that the interaction of a peptide with the gp41 disulfide loop region can block hiv-1 fusion, thus introducing the gp41 disulfide loop as a new and promising target for hiv-1 entry inhibition. the relevance of disulfide loop-targeting hiv-1 inhibitors is further supported by two reports that describe low-molecular-weight hiv-1 inhibitors causing resistance-conferring mutations within that disulfide loop region [68, 69] . the gp41 disulfide loop is an immunodominant region termed cluster i [70] , suggesting that this region is permanently or at least transiently accessible [71] . however, unlike the e2 peptides studied here, antibodies directed against cluster i appear to have no relevant hiv-1 neutralizing capacity. the difference of action between the cluster i antibodies and e2 peptides is an interesting question that needs further investigation. one possible explanation may be the fact that steric forces prevent globular antibodies to gain access to the gp41 disulfide loops at the right time, whereas peptides may have sufficient flexibility for this purpose. however, it is tempting to speculate whether in vivo the intact e2 ectodomain is able to reach the gp41 disulfide loop in a peptide-like manner? while no structural information on e2 is available, our computerbased structural analysis revealed that the n-terminal region of e2 (residues 1 to 75) appears to be largely unstructured, suggesting that this protein domain may be sufficiently flexible to interact with the disulfide loop of gp41 during hiv-1 entry. in this context, experiments addressing the amount of circulating e2 (virus-versus possibly exosome-associated or processed e2) in plasma of gbv-c coinfected hiv-positive individuals appear appropriate. the surface glycoprotein gp120 and the transmembrane gp41 subunits are noncovalently associated on the viral surface. however, little is known at the atomic level about the critical gp120-gp41 interface. several mutagenesis studies suggest that the n-and c-termini of gp120 interact with the disulfide loop of gp41 and flanking parts of the nhr and chr regions [62, 63, [72] [73] [74] . , for which a structural model could be generated, is shown in backbone presentation and colored according to the secondary structure type. the n-terminus (residues 1-75), which is not predicted to adopt a globular three-dimensional structure, is schematically depicted as circles indicating the identity of the respective amino acids. cysteines, which may form disulfide bonds, are shown as diamonds and their sequence position is indicated. residues belonging to the p4-7 and p6-2 peptides investigated in the present study are highlighted in red and blue, respectively. overlapping residues present in both peptides are shown in magenta. doi:10.1371/journal.pone.0054452.g008 however, our knowledge about the dynamic gp120-gp41 interplay during the different phases of hiv-1 entry is sparse. we do not know which parameters allow gp120 to stabilize gp41 in a metastable conformation in the unliganded trimer and after cd4 engagement but support gp41 rearrangements after coreceptor binding. a recent crystal structure of an hiv-1 gp120 core with intact gp120 n-and c-termini revealed three topologically separate and structurally plastic layers [75] . the conformational mobility of these layers together appears to be relevant for buffering movements of gp120 from gp41. it should be noted that the extended gp120 n-terminus appeared particularly flexible, such that interactions with gp41 in the viral spike could easily induce termini movements [75] . this plasticity of the termini might also offer an explanation for the fact that the interactions of the gp120 n-terminus with gp41 can be mimicked by flexible peptides. computational and experimental data from this study indicate that n-terminal gbv-c e2 peptides mimic the hiv-1 gp120 n-terminus and are therefore able to compete with gp120 for binding to the disulfide loop region of gp41. competition experiments confirmed that e2 peptides (p4-7 and p6-2) and a corresponding n-terminal gp120 peptide ranging from residue 31 to 65 of gp120 share the same binding region in gp41. therefore, we conclude that the respective e2 peptides may displace gp120 and disturb the interface between gp120 and gp41 after cd4 or coreceptor binding. this phenomenon may result in impeding the concerted interplay between stabilization (native, unbound state) and destabilization (receptor-bound and following states) of the gp120-gp41 complex, which is critical for hiv-1 entry to take its proper course. in particular, binding of e2 or e2-derived peptides to the gp41 disulfide loop may influence the proper disulfide formation of gp41, the flexibility of gp41 that is needed to promote the 6-hb formation, or the membrane accessibility of gp41 that is essential for lipid mixing. however, the elucidation of the precise mode of action of the disulfide loop targeting peptides will be an interesting subject for future evaluations. in this context, it would be interesting to consider chimeric peptides that incorporate sequences of gbv-c e2 and the hiv-1 n-terminus of gp120 as a new approach to enhance the antiretroviral activity of disulfide loop-targeted peptide inhibitors. previously, herrera et al. [76] reported that peptides derived from the n-terminus of gbv-c e2 (ranging from residues 31 to 78), as well as several more downstream regions of e2, interact with the hiv-1 fusion peptide, resulting in suppression of hiv-1 replication. our data do not confirm the association of the gbv-c e2 n-terminus with the hiv-1 fusion peptide during hiv-1 entry. however, it is very plausible that other e2 regions interact with the n-terminal gp41 fusion peptide, in addition to the interaction of the gbv-c e2 n-terminus with the gp41 disulfide loop proposed in this study. most recently, xiang et al. [35] reported that the gbv-c e2 protein or a peptide representing the e2 residues 276 to 292 interfered with hiv-1 entry, when expressed in jurkat cells. however, when added to cells the respective peptide did not inhibit hiv-1 entry unless it was fused to tat for cellular uptake. taken together, these observations support the relevance of gbv-c e2 for hiv-1 entry interference but suggest that additionally to the n-terminus at least the c-terminal part of the e2 ectodomain is involved in hiv-1 entry inhibition. this may also explain the modest effect on cxcr4 cell surface presentation upon expression of full length e2 that we do not see after cell incubation with n-terminal e2 peptides. however, it needs further evaluation to what extent e2-presenting gbv-c particles or gbv-c infected cells via a bystander effect, proposed by xiang et al. [35] , contribute to the e2-mediated hiv-1 entry impairment. in conclusion, we propose a new strategy of hiv-1 entry inhibition based on sequence resemblance between the n-termini of gbv-c e2 and hiv-1 gp120. our results demonstrate that peptides targeting the gp41 disulfide loop are able to inhibit hiv-1 fusion, introducing a novel design concept for hiv-1 fusion inhibitors. figure s1 multiple sequence alignment of related e2 proteins. multiple sequence alignment of e2 proteins from gbv-c (black), gbv-a (green), gbv-b (red), and gbv-d (blue). the gbv-c isolate used in the present study is shown in the first line and the sequence stretch covered by the active peptides is underlined. the second line shows a distantly related e2 protein from chimpanzee gbv-c to highlight the sequence divergence within the gbv-c isolates. conserved cysteines and aromatic residues are shown as bold letters. (tif) table s1 sequences of synthetic gbv-c e2 and hiv-1 peptides. *numbering follows gbv-c e2 genbank accession no. af121950 or hiv-1 hxb2 gp160 (hiv databases: http://www. hiv.lanl.gov), respectively x: e-aminohexanoic acid. (doc) gb virus c transmission by blood products prevalence of gb virus type c/hepatitis g virus rna and of anti-e2 in individuals at high or low risk for blood-borne or sexually transmitted viruses: evidence of sexual and parenteral transmission gb virus type c interactions with hiv: the role of envelope glycoproteins the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in genus pegivirus within the family flaviviridae g-pers creepers, where'd you get those papers? a reassessment of the literature on the hepatitis g virus gb virus type c/hepatitis g virus sequence and genomic organization of gbv-c: a novel member of the flaviviridae associated with human non-a-e hepatitis molecular cloning and disease association of hepatitis g virus: a transfusiontransmissible agent isolation of novel virus-like sequences associated with human hepatitis hepatitis g virus encodes protease activities which can effect processing of the virus putative nonstructural proteins humoral immune response to the e2 protein of hepatitis g virus is associated with longterm recovery from infection and reveals a high frequency of hepatitis g virus exposure among healthy blood donors age-dependent acquisition of hepatitis g virus/gb virus c in a nonrisk population: detection of the virus by antibodies gb virus c/hepatitis g virus infection: a favorable prognostic factor in human immunodeficiency virus-infected patients? carriage of gb virus c/hepatitis g virus rna is associated with a slower immunologic, virologic, and clinical progression of human immunodeficiency virus disease in coinfected persons infection with gb virus c and reduced mortality among hiv-infected patients effect of gb virus c/hepatitis g virus coinfection on the course of hiv infection in hemophilia patients in japan persistent gb virus c infection and survival in hiv-infected men effect of coinfection with gb virus c on survival among patients with hiv infection effect of hepatitis g virus infection on progression of hiv infection in patients with hemophilia. multicenter hemophilia cohort study effect of early and late gb virus c viraemia on survival of hiv-infected individuals: a meta-analysis reduced mother-to-child transmission of hiv associated with infant but not maternal gb virus c infection acquisition of gb virus type c and lower mortality in patients with advanced hiv disease gb virus c replicates in primary t and b lymphocytes inhibition of hiv strains by gb virus c in cell culture can be mediated by cd4 and cd8 t-lymphocyte derived soluble factors gb virus type c infection modulates t-cell activation independently of hiv-1 viral load gbv-c coinfection is negatively correlated to fas expression and fas-mediated apoptosis in hiv-1 infected patients regulation of cc chemokine receptor 5 in hepatitis g virus infection slower progression of hiv-1 infection in persons with gb virus c co-infection correlates with an intact t-helper 1 cytokine profile gb virus c coinfection in advanced hiv type-1 disease is associated with low ccr5 and cxcr4 surface expression on cd4(+) t-cells inhibition of hiv-1 replication by gb virus c infection through increases in rantes, mip-1alpha, mip-1beta, and sdf-1 an 85-aa segment of the gb virus type c ns5a phosphoprotein inhibits hiv-1 replication in cd4+ jurkat t cells hiv entry inhibition by the envelope 2 glycoprotein of gb virus c peptides derived from a distinct region of gb virus c glycoprotein e2 mediate strain-specific hiv-1 entry inhibition gb virus type c infection polarizes t-cell cytokine gene expression toward a th1 cytokine profile via ns5a protein expression characterization of a peptide domain within the gb virus c envelope glycoprotein (e2) that inhibits hiv replication hiv envelope: challenges and opportunities for development of entry inhibitors the t4 gene encodes the aids virus receptor and is expressed in the immune system and the brain cc ckr5: a rantes, mip-1alpha, mip-1beta receptor as a fusion cofactor for macrophage-tropic hiv-1 hiv-1 entry cofactor: functional cdna cloning of a seven-transmembrane, g protein-coupled receptor entry inhibitors in the treatment of hiv-1 infection a sensitive and specific enzyme-based assay detecting hiv-1 virion fusion in primary t lymphocytes discovery and optimization of a natural hiv-1 entry inhibitor targeting the gp41 fusion peptide the thetadefensin, retrocyclin, inhibits hiv-1 entry identification of a critical motif for the human immunodeficiency virus type 1 (hiv-1) gp41 core structure: implications for designing novel anti-hiv fusion inhibitors a conformation-specific monoclonal antibody reacting with fusion-active gp41 from the human immunodeficiency virus type 1 envelope glycoprotein n-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion globplot: exploring protein sequences for globularity and disorder protein structure prediction. implications for the biologist 3d-jury: a simple approach to improve protein structure predictions comparative protein structure modeling. introduction and practical examples with modeller structure of an ignar-ama1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition sensitivity of hiv-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics the temperature arrested intermediate of viruscell fusion is a functional step in hiv infection a covalent inhibitor targeting an intermediate conformation of the fusogenic subunit of the hiv-1 envelope complex evidence that the transition of hiv-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion distribution and three-dimensional structure of aids virus envelope spikes core structure of gp41 from the hiv envelope glycoprotein atomic structure of the ectodomain from hiv-1 gp41 peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection hiv-1 inhibition by a peptide a trimeric structural subdomain of the hiv-1 transmembrane glycoprotein alanine scanning mutants of the hiv gp41 loop peptide mimic of the hiv envelope gp120-gp41 interface model for the structure of the hiv gp41 ectodomain: insight into the intermolecular interactions of the gp41 loop cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus glycosaminoglycans and protein disulfide isomerase-mediated reduction of hiv env subunit organization of the membrane-bound hiv-1 envelope glycoprotein trimer sensitivity to a nonpeptidic compound (rpr103611) blocking human immunodeficiency virus type 1 env-mediated fusion depends on sequence and accessibility of the gp41 loop region a lowmolecular-weight entry inhibitor of both epitope map of human immunodeficiency virus type 1 gp41 derived from 47 monoclonal antibodies produced by immunization with oligomeric envelope protein epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies a recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein role of the hiv gp120 conserved domain 1 in processing and viral entry structure of hiv-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility effect of synthetic peptides belonging to e2 envelope protein of gb virus c on human immunodeficiency virus type 1 infection a small-molecule, nonpeptide ccr5 antagonist with highly potent and selective anti-hiv-1 activity we thank lu lu key: cord-001972-1zisomq5 authors: wang, xue; tan, jiying; biswas, santanu; zhao, jiangqin; devadas, krishnakumar; ye, zhiping; hewlett, indira title: pandemic influenza a (h1n1) virus infection increases apoptosis and hiv-1 replication in hiv-1 infected jurkat cells date: 2016-02-02 journal: viruses doi: 10.3390/v8020033 sha: doc_id: 1972 cord_uid: 1zisomq5 influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. it is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (hiv)-1 replication in hiv-1-infected patients. using a lymphoma cell line, jurkat, we examined the in vitro effects of pandemic influenza a (h1n1) virus (ph1n1) infection on cell death and hiv-1 rna production in infected cells. we found that ph1n1 infection increased apoptotic cell death through fas and bax-mediated pathways in hiv-1-infected jurkat cells. infection with ph1n1 virus could promote hiv-1 rna production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated b cells (nf-ĸb), nuclear factor of activated t-cells (nfat) and activator protein 1 (ap-1) through mitogen-activated protein kinases (mapk) pathways and t-cell antigen receptor (tcr)-related pathways. the replication of hiv-1 latent infection could be reactivated by ph1n1 infection through tcr and apoptotic pathways. these data indicate that hiv-1 replication can be activated by ph1n1 virus in hiv-1-infected cells resulting in induction of cell death through apoptotic pathways. it is estimated by the world health organization (who) that approximately 36.9 (34.3~41.9) million persons worldwide are infected with human immunodeficiency virus (hiv), which is the etiologic agent of acquired immunodeficiency syndrome (aids), including 1.7 million in the united states [1] . influenza a virus can cause acute respiratory infection in humans and animals throughout the world, and has continued to be a significant public health threat, leading to substantial global morbidity and mortality and an average of approximately 23,600 deaths annually in the united states alone [2] . the impact of influenza virus on hiv infection has not been well investigated. little is known about influenza virus infection in hiv-positive individual. hiv infection has been shown to be related to worse prognosis of influenza. hiv-infected patients in canada who also had pandemic 2009 influenza a (h1n1) virus (ph1n1) infection had more severe illness than those who did not have the co-infection [3] , and fatality was higher than for patients who were not co-infected in california (usa) [4] . recent reports indicate that adults with aids experience substantially elevated influenza-associated mortality [5, 6] . influenza virus infection has been associated with viremia in human and animal models. viral rna has been detected in blood in severe human ph1n1 infection [7] [8] [9] . encapsidated ph1n1 rna is stable in blood derived matrices [10] and influenza viruses can be transmitted by blood transfusion in ferrets [11] . normally, influenza a virus infection is confined to the airways where the virus replicates in respiratory epithelial cells. cumulated reports indicate that influenza viruses can infect and replicate in blood cells, such as dendritic cells [12] , primary monocytes/macrophages, and t cells [13, 14] . infection with hiv-1 can result in apoptotic cell death through activation of both death receptor-mediated and bax/mitochondrial-mediated apoptotic pathways [15] , which cause a progressive depletion of a select group of immune cells namely the cd4+ t helper cells leading to immunodeficiency. while hiv directly and selectively infects cd4+ t cells, the low levels of infected cells in patients is discordant with the rate of cd4+ t cell decline and argues against the role of direct infection in cd4 loss [15] . a viral protein, neuraminidase (na), derived from the human influenza virus was reported to enhance the level of hiv-1-mediated syncytium formation and hiv-1 replication [16, 17] . however, it is not known whether influenza a virus in blood affects hiv-1 replication, or reactivates hiv-1 replication in hiv-1-infected cells. here, we showed that pandemic influenza a (h1n1) virus infection increased apoptotic cell death and hiv-1 replication in hiv-1 infected jurkat cells. rabbit polyclonal antibodies against bax, bcl-x l (b-cell lymphoma-extra large), caspase-3, caspase-8, cavoelin-1, cd4, cd28, cytochrome c, fadd (fas-associated protein with death domain), fas, flip ((fadd-like il-1β-converting enzyme)-inhibitory protein), p53, zap-70 (zeta-chain-associated protein kinase 70), and gapdh (glyceraldehyde 3-phosphate dehydrogenase were from santa cruz biotechnology (santa cruz, ca, usa). ap-1, erk (extracellular-signal-regulated kinases), jnk (c-jun n-terminal kinases), p38, nfat, and nf-kb p65 were bought from cell signaling technology, inc. (danvers, ma, usa). all other chemicals were from sigma (st. louis, mo, usa). the human jurkat t cell line (clone je6.1) and j1.1, a latently hiv infected cell line cloned by limiting dilution from hiv-infected jurkat cells were obtained from national institutes of health (nih) aids reagent program (germantown, md, usa) and cultured at 37˝c in 5% co 2 in rpmi 1640 medium containing 10% fetal calf serum, 2 mm glutamine, 50 µg/ml penicillin, and 50 µg/ml streptomycin. for hiv-1 infection, jurkat cells (clone je6.1) were seeded at 2ˆ10 5 cells/ml for 24 h, and infected with known amounts of hiv-1 (hiv-1 mn strain, 10 9 copies per 10 6 cells) for 2 h, washed twice with pbs, and cultured for periods of time indicated. quantitative real-time reverse-transcriptase (rt) pcr was used for quantitation of viral rna. viral rna was isolated from 140 µl of culture supernatant by using the qiaamp viral rna mini kit (qiagen inc., valencia, ca, usa) according to the manufacturer's protocol. the primers and taqman probe were designed in the gag capsid (p24) region, which is the variable region among most of the hiv-1 subtype b isolate sequences according to genbank database. the forward primer was 5 1 -gacatcaagcagccatgcaa-3 1 , corresponding to nucleotides 1367-1386, and the reverse primer was 5 1 -ctatcccattctgcagcttcct-3 1 , corresponding to nucleotides 1430-1409. the taqman probe was oligonucleotide 5 1 -attgatggt ctcttttaaca-3 1 , corresponding to nucleotides 1488-1507, coupled with a reporter dye (6-carboxy fluorescein) (fam) at the 5 1 end and a non-fluorescent quencher and a minor groove binder (mgb), which is a tm enhancer, at the 3 1 end. the nucleic acids were amplified and detected in an automated taqman 7500 analyzer by using quantitect™ probe rt-pcr kit (qiagen inc.). the 25-µl pcr mixture consisted of 100 nm primers and 100 nm probe. following three thermal steps at 55˝c for 5 min, at 50˝c for 30 min, and at 95˝c for 10 min. 45 cycles of two-step pcr at 95˝c for 15 s and at 60˝c for 1 min were performed. the data are expressed as copy numbers/ml. known concentrations of hiv-1 (mn) viral rna (serially diluted: 10 8 to 100 copies) were used as templates and quantitative rt-pcr performed to generate a standard curve. each value represents the average concentration of six reactions in triple isolated repeats based on the standard curve. enzchek ® caspase-3 assay kit #1, z-devd-amc substrate from invitrogen (grand island, ny, usa) was used to test caspase-3 activity, which was performed according to the manufacturer's instruction. cell viability was determined by trypan blue exclusion analysis (life technologies, waltham, ma, usa). to generate membrane and cytoplasmic lysates, subcellular protein fractionation kit for cultured cells was used (thermo scientific, rockford, il, usa). lysis of cells generated membrane and cytoplasmic protein extracts. the protocol was performed according to the manufacturer's instructions. normalized portions of each extract (10 µg) were analyzed by western blotting using specific antibodies against proteins from various cellular compartments, including cytoplasmic (cytochrome c) and plasma membrane (caveolin-1) ( figure s1 ). proteins were isolated from the culture of jurkat cells with ripa buffer (1ˆpbs, 1% (v/v) np-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sds, 0.1 mg/ml pmsf, 30 µl/ml aprotinin, 1 mm sodium orthovanadate). equal amounts of protein were boiled in the loading buffer (100 mm tris-hcl, 200 mm dtt, 4% sds, 0.2% bromphenol blue, 20% glycerol) and separated on sds-page and blotted onto polyvinylidene difluoride membranes. for immunoprecipitation (ip), 1 µg of antibody was added to 500 µg of total protein in 500 µl, rotated for 2 h at 4˝c, then incubated with 20 µl of protein a-sucrose beads (santa cruz biotechnology) for another 2 h, spun down at 500ˆg, and washed three times with ripa buffer. the data represented are from three independent experiments. the unpaired student's t test was used for data analyses as indicated, and a value of p < 0.01 was considered very significant (**). it was reported that influenza virus infection could cause severe complications in hiv-1-infected individuals leading to an increased risk of complications and death compared with uninfected individuals [5, 6] . we found that ph1n1 could infect and replicate in jurkat cells and primary cd4+ t cells ( figure s2 ). to examine whether ph1n1 infection increased cell death due to hiv-1 infection, jurkat cells were infected with hiv-1 for three days, followed by incubation in the presence of ph1n1 for another three days. as shown in figure 1a , ph1n1 infection caused increased cell death in hiv-1 infected cells compared with uninfected cells (control). we also tested caspase-3 activities ( figure 1b ,c) and found that more caspase-3 was activated by ph1n1 in hiv-1 infected cells than uninfected cells (control). we also obtained the similar results with using primary cd4 t cells incubated with hiv-1 for seven days and then infected with ph1n1 for another three days ( figure s3a ,b). the total cell lysates were subjected to test caspase-3 activities with enzchek®caspase-3 assay kit #1-z-devd-amc substrate from invitrogen (grand island, ny, usa), which was performed according to the manufacturer's instruction; (c) the total cell lysates with ripa buffer were subjected to western blot analysis to detect caspase-3.the unpaired student's t test was used for data analyses and a value of p < 0.01 was considered very significant (**) relative to control. these data indicated that ph1n1 infection caused cell death through apoptotic signaling pathways in t cells. recently, we found that ph1n1 is able to induce apoptotic cell death in a549 cells [18] . to determine whether ph1n1 can promote death through fas-mediated apoptotic pathway in jurkat cells, total cell lysates from cells infected with hiv-1 followed by infection with ph1n1 for an additional three days were used for ip with anti-fas antibody and western blotting with anti-caspase-8 antibody to evaluate death-inducing signaling complex (disc) formation. as shown in figure 2a , more disc formation was found with the ph1n1/hiv-1 infection relative to hiv-1 infection or ph1n1 alone. figure 2b showed that high level of expression of p53 and bax proteins was detected in jurkat cells infected with ph1n1 infection and hiv-1-infected cells, compared with ph1n1 or hiv-1 alone. these data indicate that pandemic influenza a (h1n1) infection can induce both fas-mediated and bax-mediated apoptotic pathways. a higher level of activation of both apoptotic signaling pathways was found in ph1n1 infected jurkat cells after hiv-1 infection. recent reports indicate that ph1n1 infection was associated with increase in mortality in patients with late and advanced hiv disease [5, 6] and increased cell death was found in ph1n1-infected jurkat cells after hiv-1 infection ( figure 1a) . we determined whether ph1n1 infection could influence hiv-1 replication. as shown in figure 3a , jurkat cells infected with hiv-1 and incubated with ph1n1 for another three days, had higher levels of hiv-1 rna relative to cell with hiv-1 infection alone, suggesting that ph1n1 infection increases hiv-1 replication in co-infected cells in jurkat cells and in cd4+ t cells ( figure s3c ). it has been well-known that some host transcription factors are required for hiv replication, including nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), nfat, ap-1, which are up-regulated when t-cells become activated [19] [20] [21] . they stimulate viral gene expression through transcription with the long terminal repeat (ltr) of hiv-1 [22] . cells treated with ph1n1 had higher level of nf-kb phosphorylation and increased protein expression of nfat and ap-1 ( figure 3b ) relative to hiv-1 infection alone, suggesting ph1n1 infection can activate host transcription factors required for hiv-1 replication in jurkat cells. the mitogen-activated protein kinases (mapks) are central components of signal transduction pathways activated by diverse extracellular stimuli. several studies have shown that the mapk signal pathway can positively regulate replication of hiv-1, and pathway inhibitors can block hiv-1 replication [23, 24] . our results indicate that ph1n1 infection promotes mapk pathway molecules, such as p38, erk, and jnk phosphorylation ( figure 3c) . these data approve the conclusion that pandemic influenza a (h1n1) infection increases hiv-1 replication in infected cells through activation of nf-kb, nfat, ap-1, and mapk pathways that are necessary for hiv-1 replication. it is well known that cd4 is the receptor of hiv-1 [25, 26] . hiv-1 gp120 binds to the cd4 molecule on the surface of host cells [24] . since hiv-1 entry takes place in lipid rafts of the plasma membrane [27] , we examined whether ph1n1 infection affects cd4 expression on the plasma membrane in jurkat cells infected with hiv-1 and ph1n1. as shown in figure 4a the t cell activation and proliferation control hiv-1 replication and gene expression [28] ; and activation of t cells through involvement of tcr, cd3/cd8, or cd28, can trigger and activate downstream molecules, such as zap-70 and pkc8, and which activate transcription factors, ap-1 nfat and nf-κb [19, 29] , and increase hiv-1 gene expression and replication directed by the hiv-1 ltr [22] . activation events of t cells require association with lipid rafts of the plasma membrane [24, 27] . our results showed that ph1n1 infection increased more zap-70 and pkc8 expression on plasma membranes relative to hiv-1 infection alone ( figure 4b,c) . these data indicate that pandemic influenza a (h1n1) infection can increase accumulation of cd4 protein and induce t cell signaling and activate host transcription factors required for hiv-1 replication. hiv-1 infection is presently readily controlled with combination antiretroviral therapy. the persistence of hiv-1 in the face of potent antiretroviral drugs appears to be largely due to the ability of the virus to establish a state of latent infection in a long-lived population of resting memory cd4+ t cells [30] . this latent reservoir is widely considered to be the major barrier to curing infection and is currently the subject of intense research [30] . to determine whether ph1n1 infection can reactivate hiv-1 latent infection, j1.1 cells were infected with ph1n1 for three or seven days days. as shown in figure 5a , measure with real-time pcr analysis showed that significant high level of hiv-1 rna production was detected in ph1n1 infected cell relative to non-ph1n1 infected controls. cell fractionation showed that ph1n1 infection caused increased accumulation of cd28 and zap-70 proteins in plasma membrane in j1.1 cells ( figure 5b ). western blotting analysis showed that ph1n1 infection increased nfat expression and activated nf-kb signaling ( figure 5c ). ph1n1 infection could activate both apoptotic pathways with an increased expression of pre-apoptotic proteins (such as fas, disc formation, fadd, and bax) and a decreased expression of anti-apoptotic proteins (such as flip and bcl-2) (figure 6 ), suggesting that apoptotic pathways may be also involved in ph1n1-induced reactivation of hiv-1 replication. these data indicate that pandemic influenza a (h1n1) infection is able to reactivate latent hiv-1 infection in vitro. during the 2009 h1n1 influenza a virus pandemic, it was reported that some patients developed severe pneumonia leading to acute respiratory distress syndrome (ards) and multiple organ dysfunction, associated with high (17%-54%) mortality [31, 32] . however, there is little information available on the direct interaction between influenza virus and the host immune system that pertains to severe disease outcomes. it has been reported that pathogenesis due to influenza infections was associated with alterations in the lymphohematopoietic system, leading to the destruction of lymphocytes and histopathological necrosis of lymphoid tissues [33, 34] . there is little information available on whether and how influenza infection could damage the immune system directly and alter its ability to control other infectious pathogens that ultimately are detrimental to the host. our study showed that pandemic influenza a (h1n1) virus infection is able to enhance cell death in hiv-1-infected cells (figure 1) . hiv-1 infection may be associated with a worse prognosis of influenza and adults with aids experience substantially elevated influenza-associated mortality, which has declined with widespread introduction of haart (highly-active anti-retroviral therapy), but not completely eliminated [35] . in hiv patients, well controlled on haart, pandemic influenza a (h1n1) virus infection has resulted in a similar clinical outcome and prognosis to that of non-hiv patients [34] and oseltamivir treatment has been known to shorten the duration of influenza viral shedding in children [36] and adults [37, 38] . cd4 cell count and plasma hiv-1 rna did not differ before and 4-6 weeks after influenza a h1n1 diagnosis in the presence of oseltamivir treatment in the hiv-positive group [37] . however, hiv-1-infected subjects that had progressed to immune deficiency presented an increased mortality by ph1n1 [5] . this information suggests that hiv-1 activation, induced by influenza virus infection, can be controlled by oseltamivir in "normal" hiv-positive persons on haart treatment, and may not be controlled in hiv-infected patients under illness condition [5] . influenza a virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells [18, 33, 34, 39] , and apoptosis is a source of tissue damage during infection [33] . distal lung epithelial cell apoptosis and apoptosis-resulting in necrosis were described as a classical feature of h5n1-or pandemic h1n1-induced ards [40] . epithelial cell apoptosis is generally seen as a major underlying cause of diffuse alveolar damage in many forms of ards [40] . so far, it has been clear that influenza infection can affect both intrinsic and extrinsic apoptotic pathways [18] . it was also reported that after influenza virus infection, some cd4+ cells in the spleen and thymus may express viral proteins [41] , and a population of t cells is susceptible to influenza a virus in infected mice [42, 43] . influenza virus infection has been shown to activate tcr-signaling pathways in jurkat cells [16] and pandemic influenza a (h1n1) virus infection causes apoptotic death in hiv-1-infected jurkat cells (figures 4 and 5b,c) . previously, we reported that pre-apoptotic molecules from apoptotic pathways increase hiv-1 replication, while anti-apoptotic proteins inhibit viral rna yield [23] . consistent with our previous observation, pandemic influenza a (h1n1) virus infection can increase hiv-1 replication with up-regulation of pro-apoptotic molecules, down-regulation of anti-apoptotic proteins, and with an increased activation of t cells through tcr-related signaling pathways ( figures 1c, 2 and 6 ) required for t-cell activation and hiv-1 replication; however the detailed mechanisms by which pandemic influenza a (h1n1) virus infection induces hiv-1 replication needs further study. in conclusion, our results demonstrate that pandemic influenza a (h1n1) virus infection can induce cell death through apoptotic signaling pathways and promote hiv-1 replication through the mapk and tcr-related signaling pathways in hiv-1-infected jurkat cells. pandemic influenza a (h1n1) virus infection is also able to reactivate hiv-1 replication from its state of latent infection through activating apoptosis and tcr-signaling pathways. stimating seasonal influenza-associated deaths in the united states: cdc study confirms variability of flu. cdc. available online canadian critical care trials group h1n1 collaborative. critically ill patients with 2009 influenza a (h1n1) infection in canada california pandemic (h1n1) working group. factors associated with death or hospitalization due to pandemic 2009 influenza a (h1n1) infection in california severe 2009 pandemic influenza a (h1n1) infection and increased mortality in patients with late and advanced hiv disease influenza-related mortality among adults aged 25-54 years with aids in south africa and the united states of america clinical and virological factors associated with viremia in pandemic influenza a/h1n1/2009 virus infection evidence of viremia in 2 cases of severe pandemic influenza a h1n1/09 influenza viral rna detection in blood as a marker to predict disease severity in hematopoietic cell transplant recipients hewlett, i. stability and infectivity of novel pandemic influenza a (h1n1) virus in blood-derived matrices under different storage conditions highly pathogenic avian influenza a virus (h5n1) can be transmitted in ferrets by transfusion high susceptibility of human dendritic cells to avian influenza h5n1 virus infection and protection by ifn-alpha and tlr ligands efficient generation and expansion of antigen-specific cd4+ t cells by recombinant influenza viruses active nf-kappab signalling is a prerequisite for influenza virus infection hiv-1 induced bystander apoptosis syncytium formation and hiv-1 replication are both accentuated by purified influenza and virus-associated neuraminidase neuraminidase from a bacterial source enhances both hiv-1-mediated syncytium formation and the virus binding/entry process novel pandemic influenza a (h1n1) virus infection modulates apoptotic pathways that impact its replication in a549 cells regulation of nf-κb induction by tcr/cd28 nfat and irf proteins regulate transcription of the anti-hiv gene, apobec3g modulation of hiv-1 transcription by cytokines and chemokines akt/nox2/nf-κb signaling pathway is involved in tat-induced hiv-1 long terminal repeat (ltr) transactivation hewlett, i. molecules from apoptotic pathways modulate hiv-1 replication in jurkat cells some mechanisms of flip expression in inhibition of hiv-1 replication in jurkat cells, cd4+ t cells and pbmcs assembly and architecture of hiv the functional roles of lipid rafts in t cell activation, immune diseases and hiv infection and prevention viral entry, lipid rafts and caveosomes hostile takeovers: viral appropriation of the nf-kb pathway hiv reservoirs: pathogenesis and obstacles to viral eradication and cure pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico critically ill patients with 2009 influenza a(h1n1) in mexico apoptosis: a mechanism of cell killing by influenza a and b viruses depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h5n1) virus isolated from humans clinical presentation and prognosis of the 2009 h1n1 influenza a infection in hiv-1-infected patients: a spanish multicenter study pandemic influenza a (h1n1) outbreak among 15 school-aged hiv-1-infected children acute respiratory distress syndrome due to influenza virus a/h1n1v in a patient with hiv/hcv co-infection influenza a h1n1 in hiv-infected adults aryl hydrocarbon receptor activation during influenza virus infection unveils a novel pathway of ifn-gamma production by phagocytic cells early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection influenza h5n1 and h1n1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation role of itk signalling in the interaction between influenza a virus and t-cells antigen signal strength during priming determines effector cd4 t cell function and antigen sensitivity during influenza virus challenge the authors wish to acknowledge of mohan haleyurgirisetty and viswanath ragupathy for critical review of this manuscript, and acknowledge of the nih aids reagent repository for the reagents. this work was supported by an internal cber modernizing science grant. the findings and conclusions in this article have not been formally disseminated by the food and drug administration and should not be construed to represent any agency determination or policy.author contributions: x.w. and i.h. conceived and designed the experiments; x.w., j.t., s.b., j.z., and k.d. performed the experiments; and x.w. and z.y. analyzed the data, and x.w. and i.h. wrote the paper. all authors have read and approved the final manuscript. the authors declare no conflict of interest. key: cord-000849-rrezynbs authors: kumar, rajesh; andrabi, raiees; tiwari, ashutosh; prakash, somi sankaran; wig, naveet; dutta, durgashree; sankhyan, anurag; khan, lubina; sinha, subrata; luthra, kalpana title: a novel strategy for efficient production of anti-v3 human scfvs against hiv-1 clade c date: 2012-11-15 journal: bmc biotechnol doi: 10.1186/1472-6750-12-87 sha: doc_id: 849 cord_uid: rrezynbs background: production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing hiv-1 infection, however only a few such antibodies have been generated till date. isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. this has been avoided by the process described in this paper, wherein, by combining the strategy of ebv transformation and recombinant dna technology, we constructed human single chain variable fragments (scfvs) against the third variable region (v3) of the clade c hiv-1 envelope. results: an antigen specific phage library of 7000 clones was constructed from the enriched v3positive antibody secreting ebv transformed cells. by ligation of the digested scfv dna into phagemid vector and bio panning against the hiv-1 consensus c and b v3 peptides followed by random selection of 40 clones, we identified 15 clones that showed v3 reactivity in phage elisa. dna fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. expression of the positive clones was tested by sds-page and western blot. all the 13 anti-v3 scfvs showed cross-reactivity against both the clade c and b v3 peptides and did not show any reactivity against other unrelated peptides in elisa. preliminary neutralization assays indicated varying degrees of neutralization of clade c and b viruses. ebv transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scfv generation, thus avoiding the problems of hybridoma technology. moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. after selection, the phage clones were propagated in a clonal manner. conclusions: this strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. this is the first study to generate anti-v3 scfvs against hiv-1 clade c. there is a rapid increase in the number of human immunodeficiency virus (hiv-1) infected individuals worldwide and so far we have met with little success in slowing down or preventing the progression of this pandemic disease. in order to use broadly neutralizing antibodies as effective reagents for passive immunotherapy to slow or to halt the disease progression in hiv-1 infected individuals and for immunogen design for vaccination to prevent the infection, the generation of large numbers of human hiv-1 specific monoclonal antibodies is desirable. although a few human broadly neutralizing antibodies (bnabs) to hiv-1 exist [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] , these antibodies have limited reactivity against non-clade b viruses, which are responsible for more than 85% of the infections worldwide [4] . few bnabs exist so far, that are effective against the clade c viruses, which include the 4e10, antibodies from the caprisa cohort and the recently isolated monoclonal antibodies pg9, pg16 and vrc01 [9] [10] [11] [12] . in order to evaluate their utility in combating hiv-1 infection, and to tackle the problems posed by the extensive diversity of hiv-1, it is essential to generate a large panel of human anti-hiv-1 antibodies of different specificities. further, it may be necessary to evaluate several antibodies to find rare but highly effective molecules. the methods used for the generation of human monoclonal antibodies include the hybridoma technology, recombinant technology by phage display and the recently employed techniques such as single b cell sorting followed by amplification of heavy and light chain genes [8, 13, 14] . generation of antibodies by the conventional hybridoma technology is not adequate enough to meet the challenge of assessing large numbers of human monoclonal antibodies from hiv-1 infected individuals at various stages of their clinical course. our approach to the problem has been to combine the antigen specific pre-selection of ebv transformed b cells with the construction of a phage library. phage display is a scalable method for antibody production against a wide variety of antigens [15] [16] [17] . investigators are using this technology for the production of antibodies with the desired isotype specificities and affinity for research, clinical and industrial applications. antibody gene variable regions are amplified and expressed on the surface of filamentous bacteriophage as a fusion protein [14] and a number of antibodies can be produced from a single library and can be expressed and produced in a prokaryotic system. the major drawback in this technology is the requirement for the construction of large sized phage libraries (10 9 -10 10 ) with an optimum diversity because the diversity of these libraries primarily determines the successful isolation of the desired antibodies. construction of such large libraries requires a number of ligation and electroporation reactions. screening of positive clones from these libraries generally require at least three to seven rounds of biopanning to select specifically binding clones [18, 19] . it is also difficult to maintain such large sized libraries. keeping all these factors in mind we have described here a simple and efficient method for the production of human monoclonal antibodies using a combinational approach of ebv transformation, antigen preselection and phage display technology. a similar approach was previously used by kempf et al. to isolate fabs to the gp120 of hiv-1 [20] . here, we adopted a modified strategy and isolated for the first time, human anti-v3 scfvs from the ebv transformed lymphocytes of a clade c hiv-1 infected patient. this method can be used to produce large panels of viral specific phage libraries in less time and in a cost effective manner. a limited number of monoclonal antibodies are generated against the hiv-1 clade c which is the most predominant subtype worldwide and in india [21] . the v3 region of hiv-1 gp120 is an important target to induce neutralizing antibodies against different strains of hiv-1. it exhibits a high degree of sequence variability in the most structurally conserved region of the hiv-1 envelope [22, 23] and it interacts with the co-receptors ccr5 and cxcr4 [7, 24, 25] . in this study, we constructed a scfv library from the ebv transformed b cells of an hiv-1 infected patient #254, reactive to the third variable (v3) region of gp120 of hiv-1 clade c. the plasma sample of this patient was previously tested and found to exhibit cross neutralizing activity against a standard panel of pseudoviruses of different clades (table 1 ) and also had cross reactive binding antibodies to clade c and b v3 peptides [26, 27] . the modified strategy used here employing ebv transformation; antigen preselection followed by phage library construction enabled the successful selection of human monoclonal recombinant scfvs specific to the v3 antigen of hiv-1. construction of human scfv phage library from these antigen preselected b cells led to an efficient and cost effective approach for the production of anti-v3 scfvs. the plasma antibodies of the hiv-1 infected patient #254 displayed neutralizing activity against 8 out of 10 hiv-1 viruses from subtype-a, b and c ( table 1 ). the plasma sample also showed a cross subtype-b and c binding reactivity in context of the anti-v3 loop directed antibodies ( figure 1 ). a total of 4.8 million pbmcs were isolated from the hiv-infected patient (# 254) and ebv transformed in 48 wells of a 96 well culture plate ( figure 2 ). after 2 weeks, the cultures were screened (first screening) for the presence of anti-v3 antibodies and 7/48 wells showed high v3 binding reactivity with od> 2. cells from these 7 v3 positive wells were expanded to a 24 well plate and after a second screening, cells from 5 out of 7 wells showing v3 binding with od > 0.6 were expanded in a six-well plate, pooled and finally transferred to a t-25 flask. in the 3rd (final) screening of the cells in the flask, the cells showed high reactivity to v3 (od > 1) ( table 2 ). these v3 positive (enriched) cells were processed for recombinant anti-v3 scfv generation. total rna from the v3 enriched antibody producing b cells of the patient #254 was isolated. a total of 200ng of rna was reverse transcribed to cdna. use of random hexamer and oligo dt in the cdna synthesis process was shown to be more effective than either of them alone or the 3 0 primers [15, 20] . it also enhanced the chances of amplification of the antibody genes even when the amount of rna is low [15] . a total of 54 combinations were used to amplify the heavy and light chains (24 for vh and 30 for vl). among the 24 combinations of heavy chains, 18 were successfully amplified. the heavy chains vh1, vh4, vh5 and vh6 were preferentially more expressed while vh2 and vh3 were very less expressed ( figure 3a ). all the 30 kappa light chain combinations were successfully amplified and expressed almost at the same level, except for vlk2, which were relatively less expressed ( figure 3b ). a heavy chain and light chain pool was made by mixing equal concentration of each combination (50ng) irrespective of their expression level, so that each combination of the heavy chain has an equal probability to combine with the light chain and vice versa and to avoid the predominant expression of one antibody subclass and finally to have new combinations of antibodies for a particular antigen that are naturally not elicited during the natural course of infection. a final pull through pcr was performed using the forward and reverse primers. these scfvs were then ligated into the pcantab-5e vector that contains the sfi1 and noti sites ( figure 3 ). the ligated product was transformed into tg1 using calcium chloride mediated transformation. the incubation time after the heat shock treatment during transformation was maintained for a maximum of 40 min to avoid repetition of similar types of clones in the library. we obtained 200-300 transformed colonies per plate. a total of 7000 colonies were obtained with 400ng of digested scfv dna that were ligated into 1 μg of phagemid vector. the ligated dna was transformed into e.coli tg1. to check the diversity of antibodies in the library, we randomly selected 10 clones from the unselected library. amplification of these 10 scfvs by pcr, followed by digestion with bstn1 and comparing their dna fingerprint patterns showed that 9/10 clones were distinct from each other. clone 3 and clone 6 showed identical dna fingerprinting patterns ( figure 4 ). we further confirmed the diversity of the antibody fragments by dna sequence analysis of 29 randomly selected scfv clones from the unselected library which showed high diversity of clones i.e. 73% (21/29) in the library. the heavy chain (vh) sequences showed a limited diversity, igvh4 (13/29) and igvh5 (15/29) were the most preferentially represented vh sequences that combined with all the representatives of ighd genes except for ighd1. diversity of the vl paratope was also limited with igkv1, igkv2 and igkv3 being most represented, except for one occurrence of igkv7 ( table 3) . as the size of the library was very small, one round of biopanning was done against the v3 c and v3 b peptides. forty clones were randomly selected and checked for figure 2 schematic overview of the strategy used for construction of antigen specific phage library. the peripheral blood mononuclear cells (pbmcs) were isolated from an antiretroviral drug naïve patient (#254) whose plasma exhibited neutralizing activity against a panel of viruses and also displayed cross clade reactive anti-v3 antibody binding potential. pbmcs were subjected to ebv transformation in 96 well culture plate, wells showing high level of anti-v3 antibodies were selected and expanded to 24 well stage and then to six well stage . the wells showing high titre of anti-v3 abs were pooled together and cultured in t25 flask. total rna was isolated from these antigen specific enriched b cells and cdna was synthesised. heavy and light chains were amplified and scfvs were constructed and cloned into a pcantab-5e phagemid vector. scfv phage library of 7000 clones was constructed. their binding, of which 15 showed positivity in phage elisa with both the peptides ( figure 5 ). four clones were randomly selected for elisa with purified phage. the binding specificity of these anti-v3 scfvs was checked with phage from the 24 well plate and peg precipitated purified phage. the 24 well elisa and pure phage elisa binding assays showed similar results. three clones exhibited almost similar binding in both the formats of elisa while one clone had lower binding reactivity in pure phage elisa although it was not completely lost (data not shown). dna fingerprinting analysis using bstn1 followed by sequencing revealed that 13/15 clones were distinct (table 4 ). all the distinct 13 anti -v3 scfvs that were finally selected, showed cross-reactivity against both the v3 peptides and did not show any reactivity against other unrelated peptides. one round of biopanning was found to be sufficient to get v3 positive scfv clones with diversity. the antigen binding clones showing positivity/binding in the phage elisa were further processed for soluble scfv expression by induction with 1mm iptg [28] . after induction, the periplasmic lysate, inclusion bodies, culture supernatant and whole cell extract were prepared and analysed for scfv expression on a 12% reducing sds-page. the scfv fusion protein was found to be expressed in the cell lysate, culture supernatant, periplasmic extract, with highest expression in the inclusion bodies. scfv expression was also observed in the uninduced culture ( figure 6 ). the scfvs were expressed in e. coli hb2151 cells by inducing for 6 to 8 h with 1 mm iptg at 24°c. the a total of 4.8 million pbmcs were isolated. 2 number of cells plated per well was 100000. 3 forty eight wells were subjected to ebv transformation. 4 all the 48 wells were successfully transformed (transformation efficiency 100%). 5 7/48(approx 14.6%) wells showed positive binding (od>2) with v3 peptide and expanded to 24 well stage. 6 antibody fragments were purified from the periplasmic extract and the purified product was analysed by sds-page. the scfv protein of 32kda was expressed in different elute fractions e1 to e5 ( figure 7c ) and protein samples were concentrated using ultrafiltration columns (ambion) over a 10kda cut off. expression of the scfv fusion protein (32 kd) in the periplasmic extract was confirmed by western blotting. the cell lysate of hb2151 was used as a negative control ( figure 7d ). the functional activity of the newly generated clones 3e6b and 3e7b was assessed by their binding to the v3 peptides of clade c and b in elisa. both the scfvs showed specific binding to the v3 peptides ( figure 8 ). in addition, these scfvs did not bind to other unrelated peptides like mper and id loop of hiv, peptide pool (unrelated viruses) and bsa. the purified scfvs were tested for the viral neutralization potential against a panel of pseudoviruses from clades a, b and c hiv-1 viruses (table 5) we have demonstrated in this study a strategy that successfully yielded recombinant human scfvs against the v3 region of hiv-1 using a pool of antigen selected ebv transformed b lymphocytes and phage display technology. the use of preselected b cells producing anti-v3 antibodies for phage scfv library construction proved to be an efficient strategy for the isolation of v3 specific clones. moreover, this approach has prevented any loss of unstable or slowly growing clones which may have useful and unique binding specificities (that often occurs during cloning and propagation). stringency during the initial stages helps in elimination of most of the false positive clones. beginning with the 96 well stage and up to the final screening of the cells in the t25 flask, we selected only those wells with high v3 binding reactivity for further expansion and finally for the phage library construction. by this approach, we obtained a phage library with an adequate number of v3 binding clones. amplification of the heavy chain gene showed that the vh1, vh4, vh5 and vh6 were preferentially more expressed than the vh2 and vh3 and corroborated with the previous reports [29] that anti-v3 antibodies more preferentially exhibit the above gene usages. it is well established that in hiv-1 infection, vh3 genes are less preferentially used [30, 31] . from the sequencing data, we observed that twenty nine randomly selected clones from the ebv transformed cells library exhibited the vh4 and vh5 gene usage suggesting that our library may be biased towards antigen specificity. in healthy individuals, vh3 genes are most frequently used and vh5 is used only by a low percentage of antibodies (http:imgt.cines.fr). the light chain did not show any preferential gene usage and all the light chains were successfully amplified. as heavy chain amplification was biased towards the expression of a set of heavy chain genes, to avoid predominant expression of one subset of genes in the phage library, we took an equal concentration of all the heavy chain genes which in turn increased the chances of the new combinations that are not expressed in natural infection. successful isolation of positive clones with higher affinity and specificity is possible when a library is more diverse and has less number of clones with incomplete scfvs sequences [15, 32] . if phage carrying incomplete scfvs are more in number, they overwhelm the library after multi-step panning thereby the presence of the full size insert will decrease, this being a common problem with phage libraries. interestingly, our library exhibited 90% diversity of clones and 100% clones had complete scfvs ( figure 7a,b) , this might be because of the optimum incubation time (30-40 min) maintained during the transformation process that reduced the chances of repetition of clones. in addition, the combination of the sfi1-noti site containing pcantab-5e vector that we used in the library construction may have reduced or eliminated the chances of getting incomplete scfv sequences because these restriction enzyme sites are rarely present in the scfv dna sequence [15, 33] . furthermore, none of our clones had internal sfi1-noti sites. stringency in the biopanning also reduces the number of false positive clones. initially, phage were allowed to bind to the plastic plate, next, to coated milk, followed by bsa and finally onto a pool of coated unrelated antigens in the plates. this stringency reduced the number of nonspecific phage binders and made the screening process more convenient and specific. kempf et al., using a similar strategy of ebv transformation, constructed a fab library of 10 7 clones from which they isolated three fabs after one round of biopanning. their scoring of positive clones however was less, probably because they selected cells positive for gp120 binding from the 96 well stage and also included wells that were low binders ( od> 0.1) [20] . we therefore selected only those wells with high v3 binding reactivity (od>0.6) at all stages for the phage library construction. this reduced the number of false positive clones and enriched the v3 specific cells. there are several reports on the generation of scfvs from different sources like pbmcs [34, 35] , bone marrow [36] , tonsils [37] , hybridomas [38, 39] , but none of them employed ebv transformed antigen specific cells to generate a scfv phage library. generation of phage library from ebv transformed lines after preselection with antigen is very useful for laboratories with limited resources because the antibody production by traditional hybridoma technology costs ranging between $8000 to $12000 and 80% of the costs are incurred in the post ebv immortalization steps like fusions and cloning. our strategy does not require the expensive process of electroporation because the cost of cuvettes and instrument mainly limits the construction of phage libraries with such a large size and diversity; also it eliminates the large number of biopanning rounds followed by cumbersome screening processes. such small libraries are easy to maintain and can be produced by routinely used calcium chloride mediated transformations. we have for the first time, generated anti-v3 human scfvs against clade c hiv-1. by antigen pre-selection, the phage library served as an adequate source of v3 positive clones. the combination of ebv transformation and selection of immortalized lines that exhibit the desired antigen binding characteristics with phage display technology provides a useful strategy for specific recombinant antibody generation. our study validated the generation of recombinant libraries as a powerful tool for the generation of diverse recombinant antibodies. the hiv-1 seropositive patient (# 254) enrolled for the construction of the anti-v3 scfv phage library was recruited after obtaining written informed consent from the department of medicine, aiims, new delhi. the study was approved by the institute ethics committee. whole blood was collected in edta vaccutainers. the plasma was separated from whole blood, aliquoted and stored at −70°c until tested. the peripheral blood mononuclear cells (pbmcs) were separated by phycoll hypaque centrifugation and processed immediately for ebv transformation. the plasma sample of the patient # 254 was previously tested and found to exhibit good neutralization potential against a diverse panel of viruses (table 1 ). it was also tested for the presence of binding anti-v3 antibodies and the data is shown in figure 1 . the neutralization efficiency of the plasma #254was tested against a standard panel of pseudoviruses of clades a, b and c obtained from the nih aids research and reference reagent program, by tzm-bl assay [40] . the standard panel of pseudoviruses has been categorized from tier 1 to tier 3, based on the decreasing order of susceptibility to neutralization by the known monoclonal antibodies [41] . the neutralization assay was carried out in 96-well tissue culture plates. briefly, 50 μl of the heat inactivated plasma/purified scfv, at different dilutions in duplicate, was added to 200 tcid 50 of the virus and incubated for 1 h. a cell control well containing culture media only and a virus control well containing both the culture media and the virus were tested in parallel. the rest of the procedure is the same as described for calculating tcid 50. the scfv (hepb), an scfv against the hepatitis b surface antigen [42] and 1418, a human antibody to parvovirus b19 protein, were used as negative controls. for all dilutions of test plasma, the percent neutralization was calculated based on the relative luminescence units (rlu) in the presence of plasma divided by the virus control. the cell control value was subtracted from the plasma rlu value as the background cutoff. the 50% neutralization titer (id50 titer) was determined for the plasma sample #254 against each virus by plotting percentage neutralization against the dilution of the plasma tested. a non-linear regression straight line was drawn by the method of least squares, and the reciprocal id50 titers were extrapolated. the experiments were performed in duplicate and repeated at least twice and the mean id50 titers were calculated. the anti-v3 antibody content in the hiv-1 seropositive plasma sample #254 and in the supernatants of the ebv transformed pbmcs in culture at different stages was determined using v3 peptide elisa. thirty five mer peptides of v3c (ctrpnnntrksirigpgqtfyatgdi igdirqahc) and v3b (ctrpnnntrksihigpgrafy ttgeiigdirqahc) were synthesised (sigma aldrich, usa), based on the consensus v3 sequences. the v3 peptides (1 μg/ml) were coated onto 96 well nunc-immuno plates (nunc: cat# 439454) using antigen coating buffer (150 mm na 2 co 3 , 350 mm nahco 3 , 30 mm nan 3 , ph 9.6) at 4°c overnight. plates were washed using phosphate buffered saline with 0.1% tween-20 (0.1% pbst) thrice using a plate washer. plates were then blocked with 100 μl of 15% fetal calf serum and incubated at 37°c for 1.5 h. following blocking and washing, heat inactivated plasma (100 μl, dilution range=300-100000) or 100 μl of supernatants from ebv transformed pbmc cultures was added to each well and incubated for 1hour at 37°c. after 3 washings with pbst (0.1%), the bound v3 specific antibodies were detected by addition of 100 μl of alkaline-phosphatase conjugated anti-human igg fc (1:2000 in pbst). immune complexes were revealed with ap-substrate in dae buffer and the colorimetric reaction was stopped by the addition of 6n naoh. the optical density was read at 405 nm. id 50 titers were calculated for the plasma sample against each of the peptide by plotting the absorbance at 405 nm against the dilutions of the plasma sample tested. a non-linear regression straight line was drawn by the method of least squares and the id 50 titers were extrapolated. b95 cell line comprises of ebv transformed human lymphoblastoid cells which secrete ebv in the supernatant. ebv transforms human b cells [43] . we obtained the b95 cell line from american type cell culture (cat. no. vr-1492). b95 cells were grown in t-75 tissue culture flasks, at 100,000 cells/ml at 37°c, 5% co 2, in complete rpmi media. after 10 days, the viral supernatant was centrifuged at 300 × g and filtered with 0.45 μm filters. peripheral blood mononuclear cells (pbmc) (100,000 cells/well) from the patient #254 were ebv transformed by mixing with 100 μl of the viral supernatant in a 96-well plate and cultured with a polyclonal b cell activator, cpg (2 μg/ml), which enhanced ebv infection and b cell transformation [44] and csa (0.5 μg/ml). the plate was incubated at 37°c in a 5% co 2 incubator overnight. next day, cells were fed with 100 μl of complete medium containing csa and cpg. cultures were fed twice per week and half of the culture supernatant was replaced with fresh complete media, 200 μl/well (no csa and cpg). after two weeks of culturing the b cells in the 96 well plate, we screened for the presence of v3 antibodies by v3 peptide binding elisa as described above. the positive b cell clones were further expanded to 24 well plates and screened in the 3 rd and 4 th week; v3 positive clones were transferred to a six well plate, pooled and further expanded to the t-25 flask ( table 2) . total rna from the v3 specific antibody producing b cells (from flask stage) was isolated by trizol reagent (sigma, usa) and then reverse transcribed to cdna, using the reverse aid mmulv reverse transcriptase (fermentas, usa). a total of 200 ng of rna was reverse transcribed in a reaction volume of 50 μl containing, 10ng of random hexamer, 20μm oligo-dt, 1.5 μl of rnase inhibitor (40u/μl), all dissolved in 1x rt buffer. the rna was heated to 65°c for 5 min and then immediately chilled on ice for at least five minutes. following the addition of the reaction mixture, the tube was incubated at 42°c for 60 min, at 70°c for 5 min and quickly chilled on ice and stored in −20°c. heavy chain variable region genes were amplified using a total of 24 combinations (6 forward primers and 4 reverse primers representing all human immunoglobulin subfamilies) and for light chain kappa, a total of 30 combinations were used (6 forward primers and 5 reverse primers) as described previously [15] . a total of 54 independent reactions were performed to generate the variable regions of heavy and light chains. the heavy chain 5 0 primers included a sfii site and the light chain 3 0 primer included a noti site. light chain 5 0 primer included part of the linker region (gly 4 ser) 3 and this was compatible with the heavy chain 3 0 primer. each variable heavy region was amplified using hot start taq dna polymerase (fermentas) in a pcr reaction of 50 μl containing 2.5 μl cdna, primers 1 μl (10 pmole each) both forward and reverse. pcr reaction was performed for 34 cycles (94°c for 3 min initial denaturation, 94°c for 1 min, annealing at 63°c for 1 min, extension at 72°c for 2 min) using eppendorf master cycler. light chain variable region were amplified with the similar protocol except the annealing temperature used was 57°c. each variable region gene was purified from the agarose gel using gel extraction kit (qiagen, germany). an equimolar mixture of pooled heavy and light chain dna was used in the second round assembly pcr. the assembly pcr reaction was cycled 20 times (94°c for 1 min, 94°c for 45 sec, 62°c for 50 sec, 72°c for 2 min) the assembly reaction was performed using pfu dna polymerase and without primers. full length scfvs were amplified using a pull through pcr reaction using taq dna polymerase and the following primers ptfw 5 0 cct ttc tat gcg gcc cag ccg gcc atg gcc 3 0 and ptrv 5 0 cag tca ttc tcg act tgc ggc cgc acg 3 0 (94°c for 1 min, annealing at 62°c for 1min, extension at 72°c for 1 min and final extension at 72°c for 5 min). the scfvs were agarose gel purified using gel extraction kit (qiagen, germany). the scfv dna fragments and pcantab-5e vector were digested with noti /sfii (new england biolabs, usa) respectively. the digested scfv dna fragments and pcantab-5e vector were gel purified using gel extraction kit (qiagen, germany). the scfv dna was ligated into vector at a 3:1 molar ratio using t4 dna ligase (new england biolabs, usa). the ligated dna was transformed into chemically competent cells of e.coli tg1 and placed on ice for 1h followed by heat shock treatment for 90 seconds and chilled on ice for 5 min. next, 800 μl of 2xyt media was added and incubated in a rotating shaker at 200 rpm for 40 min. the transformed cells were plated on to 2xyt medium agar plates containing ampicillin (50 mg/ml) and 2% glucose, and incubated overnight at 37°c. the following day, colonies were scraped into 1ml of 2xyt medium with 20% glycerol and stored at −70°c. sequence of the assembled scfv was confirmed by an automated abi prism sequencer using gene specific primers. the phage were rescued by infection with helper phage (m13-ko7), followed by precipitation with peg/nacl, resuspension in pbs and titration for the determination of phage concentration. the phage were then subjected to a single round of enrichment by bio-panning. the panning procedure was carried out in immuno 96 microwell plates. plates were coated with 100 μl of v3 peptide 1 μg/ml in 0.1 m nahco 3 (ph 8.6) overnight at 4°c. a phage library of 10 12 phage was incubated for 1h in milk coated wells to remove the non-specific binders. after one hour, unbound phage were transferred to the peptide coated wells for 30 min at rt. the unbound phage was eliminated by washing 10-15 times with pbs containing 0.1% tween 20. the bound phage was eluted with 0.2 m glycine ph 2.2 for 10 min at rt. the eluted phage were neutralized with 1m tris hcl ph 9.2 and immediately infected onto tg1 (od 0 .4 to .5) for 30 min at 37°c and for 30 min with shaking at 37°c. cells were spun down and plated on 2xty agar containing ampicillin (50 mg/ml) and 2% glucose. individual colonies were picked and grown in 96 well sterile culture plates (corning) and a glycerol stock was made and stored at −70°c. individual colonies were grown in 1ml 2xyt broth containing ampicillin and 2% glucose overnight with shaking at 37°c at 160 rpm. a small inoculum was transferred to 1ml 2xyt broth containing ampicillin (50 mg/ml) and 2% glucose at 37°c with shaking at 200 rpm till the od reached 0.4 to 0.5. helper phage were added and the plate was incubated at 37°c without shaking and then for 30 min with shaking at 180 rpm at 37°c. the cells were spun down at 1500 × g, the supernatant was discarded and pellet was washed with 2xyt broth. the pellet was then resuspended in 2xyt broth containing ampicillin (50 mg/ml) and kanamycin (100 mg/ml) (no glucose) with shaking at160 rpm at 30°c for 16-18 h. it was then centrifuged at 6000 × g and the supernatant was collected and stored at 4°c and an aliquot was tested in the phage elisa. the elisa plates were coated with 100 μl of v3 peptide (1 μg/ml) in 0.1 m nahco 3 (ph 8.6) and incubated overnight at 4°c. the plates were washed once with 1x pbs and blocked with 4% non-fat milk (titan biotech, india) for 2 h at 37°c. the plates were then washed three times with 1x pbs. phage supernatant (100 μl) was added to each well and incubated for 1h at rt. phage supernatant was discarded and the plates were washed four times with pbst(0.1%). 100 μl of anti m13 antibody (diluted 1:2000) was added (sigma) and incubated for 1h at rt. the plates were washed four times with pbst (0.1%). 100 μl of anti rabbit hrp (jackson) diluted 1:3000 were added and incubated at rt for 1h. the plates were then washed four times with pbst (0.1%). 100 μl of tmb substrate was added, and the reaction was stopped by adding 8n h 2 so 4. absorbance was measured at 450nm. individual bacterial colonies were grown in 5ml 2xyt medium containing ampicillin (50 mg/ml) and 2% glucose with shaking at 200 rpm at 37°c till the o.d reached 0.4 to 0.5. next, 1 μl of ko7 helper phage (10 18 ) was added and incubated at 37°c for 30 min without shaking, followed by 30 min shaking at 200 rpm. the cells were centrifuged at 2500 × g for 10 min, supernatant was discarded and the pellet was washed again with 2xyt broth. the pellet was resuspended in 50ml 2xyt broth containing ampicillin (50 mg/ml) and kanamycin (100 mg/ml) (no glucose) at 30°c with shaking at 160 rpm for 12-16 h. it was then centrifuged at 10000 × g for 20 min at 4°c. the supernatant was transferred to glass bottles and ¼ volume of peg/nacl was added and kept on ice for 4 to 6 h followed by centrifugation at 20000 × g for 20 min at 4°c. the supernatant was discarded and the pellet was dissolved in sterile and autoclaved 1x pbs and stored at 4°c. the transformation unit (tu) was calculated. twenty nine scfvs clones were randomly selected from the unselected library and sequenced by macrogen (south korea). the sequences were analysed using immunoglobulin blast [45] and v base software [46] . the diversity of the scfv repertoire was analysed by comparing the restriction digestion pattern of scfvs. ten clones were randomly selected from the primary phage library and the plasmid was isolated. the scfv sequences were amplified using primers ptfw 5 0 cct ttc tat gcg gcc cag ccg gcc atg gcc 3 0 and ptrv 5 0 cag tca ttc tcg act tgc ggc cgc acg 3 0 (94°c for 1 min, annealing at 62°c for 1min, extension at 72°c for 1 min and final extension at 72°c for 5 min). the amplified pcr products were digested with a frequent cutter restriction enzyme bstn1 (neb) and analysed on 2% agarose gel. clones showing positivity/binding in phage elisa were selected for soluble scfv expression. these were than transformed into hb2151 using calcium chloride mediated transformation for soluble scfv expression. the hb2151 cells carrying pcanta-5e plasmid were grown in 10 ml 2xty medium overnight at 37°c with shaking at 200 rpm. the next day, 1/100 th volume of the overnight culture was inoculated in 1 litre of 2xty medium and grown at 37°c with shaking at 240 rpm till the od reached 0.6 and then the culture was induced by 1 mm iptg for 6 to 8 h at 24°c [33] . cells were harvested and different fractions were prepared. scfvs were purified from the periplasmic fraction. briefly, the cells were harvested by centrifugation at 4000 × g for 15 min at 4°c. the supernatant was discarded and pellet was resuspended in 30 mm tris-cl. 20% sucrose, ph 8.0 at 80 ml/ gram wet weight. the cells were placed on ice for 20 min and 500 mm edta was added to a final concentration of 1 mm edta. the cells were spun down at 8000 × g for 15 min at 4°c. the pellet was resuspended in 5 mm mgso 4 and the cells were placed on ice for 10 min with slowly stirring, pelleted down at 8000 × g for 15 min at 4°c and the supernatant collected for purification. purification of e-tagged scfv was carried out using the recombinant phage antibody system (rpas) purification module (amersham biosciences) as per the manufacturer's instructions. the periplasmic extract was filtered through a 0.45 μm filter to remove any remaining cell debris. the anti-e tag column was regenerated by washing the column with 15ml of elution buffer (0.1 m glycine, ph 3.0) and was then equilibrated with 25 ml binding buffer (0.02 m phosphate buffer, 0.005% nan 3 , ph 7.0). the e-tagged scfv in the periplasmic extract was then allowed to bind to the column by passing the extract through the column. the unbound excess e. coli proteins were removed from the column by washing it with 25 ml binding buffer. the flow rate at each step was maintained at 5 ml/min through the column. finally, the bound scfv was eluted by 15ml elution buffer. several fractions of the eluted scfv (900 μl each) were collected in tubes containing 100 μl neutralization buffer (1 m tris, 0.05% nan3, ph 8.2). the amount of protein in each fraction was estimated using bca method and the fractions containing considerable amount of scfv were pooled together and concentrated. soluble elisa was performed, as described in the phage elisa. hundred microliter of soluble scfv periplasmic extract/purified scfv was added and incubated at room temperature for 1h. elisa plates were washed three times with 0.1% pbst and incubated with 1:1000 dilution of primary antibody in 2% mpbs. the plates were washed three times with 0.1% pbst and incubated with 1:2000 diluted anti rabbit hrp conjugated secondary antibody in 2% mpbs. the elisa plates were washed again as described above. 100 μl of tmp substrate was added and incubated at rt till the colour developed. reaction was stopped by adding 8nh 2 so 4. absorbance was read at 450 nm. sds-page was done as described [33] . proteins were separated on a 12% running gel and 5% stacking gel and visualized by coomassie brilliant blue (cbb) staining. for western blotting, gel was blotted onto nitrocellulose membrane using electroblotting, (100 v for 1h) and probed with primary antibody. anti-rabbit hrp was used as secondary antibody and colour was developed with dab as the substrate. broadly cross-reactive hiv-1-neutralizing human monoclonal fab selected for binding to gp120-cd4-ccr5 complexes the cross-clade neutralizing activity of a human monoclonal antibody is determined by the gpgr v3 motif of hiv type 1 efficient neutralization of primary isolates of hiv-1 by a recombinant human monoclonal antibody cross-clade neutralizing activity of human anti-v3 monoclonal antibodies derived from the cells of individuals infected with non-b clades of human immunodeficiency virus type 1 fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the cd4 binding site of human immunodeficiency virus type 1 gp120 an igg human monoclonal antibody that reacts with hiv-1/gp120, inhibits virus binding to cells, and neutralizes infection cd4-dependent, antibody-sensitive interactions between hiv-1 and its co-receptor ccr-5 broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1. science insensitivity of paediatric hiv-1 subtype c viruses to broadly neutralising monoclonal antibodies raised against subtype b potent and broad neutralization of hiv-1 subtype c by plasma antibodies targeting a quaternary epitope including residues in the v2 loop generation of human monoclonal antibodies to human immunodeficiency virus filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface a compact phage display human scfv library for selection of antibodies to a wide variety of antigens making antibodies by phage display technology monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice generating recombinant antibodies for research, diagnostics and therapy using phage display isolation of potent neutralizing monoclonal antibodies from an siv-infected rhesus macaque by phage display the rescue by phage display of human fabs to gp120 hiv-1 glycoprotein using ebv transformed lymphocytes global and regional distribution of hiv-1 genetic subtypes and recombinants in 2004 structural basis for coreceptor selectivity by the hiv type 1 v3 loop alternative conformations of hiv-1 v3 loops mimic beta hairpins in chemokines, suggesting a mechanism for coreceptor selectivity envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human ccr5 as a coreceptor for viral entry and make direct cd4-dependent interactions with this chemokine receptor hypervariable region 3 residues of hiv type 1 gp120 involved in ccr5 coreceptor utilization: therapeutic and prophylactic implications relative reactivity of hiv-1 polyclonal plasma antibodies directed to v3 and mper regions suggests immunodominance of v3 over mper and dependence of high anti-v3 antibody titers on virus persistence neutralization of tier-2 viruses and epitope profiling of plasma antibodies from human immunodeficiency virus type 1 infected donors from india reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system human anti-v3 hiv-1 monoclonal antibodies encoded by the vh5-51/vl lambda genes define a conserved antigenic structure molecular characterization of human monoclonal antibodies specific for several hiv proteins: analysis of the vh3 family expression human antibody variable region gene usage in hiv-1 infection isolation of a nanomolar scfv inhibiting the endopeptidase activity of botulinum toxin a, by single-round panning of an immune phage-displayed library of macaque origin plückthun a: selection, characterization and x-ray structure of anti-ampicillin single-chain fv fragments from phage-displayed murine antibody libraries one-step detection of aflatoxin-b(1) using scfv-alkaline phosphatase-fusion selected from human phage display antibody library generation of a large complex antibody library from multiple donors optimal construction of non-immune scfv phage display libraries from mouse bone marrow and spleen established to select specific scfvs efficiently binding to antigen human antibodies with subnanomolar affinities isolated from a large non-immunized phage display library characterization and molecular modeling of a highly stable anti-hepatitis b surface antigen scfv improved breadth and potency of an hiv-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning measuring hiv neutralization in a luciferase reporter gene assay tiered categorization of a diverse panel of hiv-1 env pseudoviruses for assessment of neutralizing antibodies humanization of high affinity anti-hbs antibody by using human consensus sequence and modification of selected minimal positional template and packing residues an effective method for establishing human b lymphoblastic cell lines using epstein-barr virus an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the patient participant for providing blood sample. we profoundly thank dr susan zolla pazner from new york university for providing the antiparvovirus antibody 1418. we thank dbt (bt/pr 10511/med/29/66/2008) for the funds and for the junior research fellowship provided to rajesh kumar. we thank all india institute of medical sciences and national brain research centre for providing the facilities for conducting this study. the authors declare that they have no competing interests.authors' contributions kl and ss designed and conceptualized the study and finalized the manuscript. rk constructed the phage library, constructed the scfvs and wrote the manuscript. ra screened the plasma of patients for neutralization and performed the ebv transformation experiments. at provided valuable inputs in phage library construction. ssp, dd,as and lk helped in protein expression, purification and phage elisa. nw provided the patient samples. all authors have read and approved the final manuscript. key: cord-007890-bie1veti authors: nan title: ecc-4 abstracts date: 2002-04-16 journal: int j antimicrob agents doi: 10.1016/s0924-8579(02)00033-x sha: doc_id: 7890 cord_uid: bie1veti nan f spain introduction: the prevalence of erythromycin resistance (er-r) in group a streptococci (gas) has increased in spain since early 90s with current rates exceeding 40% in some regions. this study determined the emm -types associated to erythromycin resistance in spain. material and methods: isolates belonged to the sauce* surveillance collection. rapid sequence analysis of specific pcr products was used to deduce emm -types corresponding to the majority of the known gas m serotypes. pcr primers used: gasm1 (5?-tattgcgct-tagaaaattaa-3?) and gasm2 (5?-gcaagttctt-cagcttgttt-3?). sequencing was done with the big dye terminator mix and autosequenator (applied biosystems). dna sequences were subjected to homology searches against the bacterial dna database. results: overall, 670 gas isolates (345 er-r) were analysed. three m-types (m4, st1812 and m12) accounted for 66.4% of the er-r isolates, whereas they just represented a 16.7% of the ery-s. for er-r isolates the strongest association was seen with m4 (or0/12; 95% ci 6.7 á/22.1), and m75 was second after m4 only in the last temporal period of the study (1998 á/1999) . no homogeneous distribution of er-r m-types by centres was seen. conclusions: few m-types (leading by m4) are responsible for the er-r in spain. but for m4, the remaining er-r m types (st1815, m12 and m75) did not show a temporally nor geographically homogeneous distribution. *sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en espana' (susceptibility to the antimicrobials commonly used in the community in spain ) and is the spanish word for the willow tree. significant increase in the prevalence of erythromycin-resistant, clindamycin and miocamycin-susceptible (m-phenotype) streptococcus pyogenes in spain (1998 ( á/2001 purpose: a variety of methods is used for a molecular typing of enterococcus spp. and related gram-positive bacteria. these include dna-based methods such as macrorestriction analysis using pulsedfield gel electrophoresis (pfge), ribotyping, and amplification-based methods such as rapid amplification of polymorphic dna (rapd) and amplified fragment length polymorphism (aflp). we used a homogeneous strain collection of 24 transconjugants resulting from filter-matings with different antibiotic-resistant e. faecium and a recipient isolate from our lab. the influence of transferred antibiotic-resistance determinants on the outcome of different typing methods was investigated. results: fragment patterns resulting from pfge indicated minor differences between the transconjugants and the recipient. in respect to different primers used for rapd, none or only a single fragment shift was detected in the resulting fragment patterns. aflp clusters all transconjugants into a group of major relatedness, but the result was strongly dependent on the mathematical method used for cluster analysis. fragment patterns of digested plasmids showed the possession of different or only widely related plasmids in the transconjugants. conclusions: the results of this study clearly show that under certain situations typing methods commonly used for enterococci and related gram-positive bacteria come to their limits. the sasss network aims to set up a national surveillance study to obtain standardized information on antimicrobial susceptibility to various bacterial pathogens. currently, 25 hospitals are participating in the project from different geographical regions in saudi arabia. during the 1st year (2001), the sasss focused on setting up this network. overall, high frequencies of resistance to antibiotics to different bacterial pathogens in saudi arabia were seen. geographical variations of resistance were noticed, which could be related to different prescribing practices. approximately, 9 and 32% of escherichia coli and k. pneumoniae , respectively, were extended spectrum â-lactamases (ebls) producers. resistance of enterobacteriacae group to carbapenem and pipracillin/tazobactam is low. resistance of pseudomonas aeurginosa to various anti-pseudomonal antibiotics including carbapenem is high and alarming. methicillin resistant staphylococcus aureus (mrsa) comprised 36% of s. aureus isolates. no vancomycin intermediate s. aureus (visa) was detected. high level resistance to gentamicin in enterococcus were seen in 12% of the isolates and only 2% of enterococcus facieum were glycopeptide resistant. resistance of streptococcus pneumoniae to penicillin ranged between 2% to almost 52%. surveillance of antibiotic resistance on a national level is necessarily to give guidance to practicing physicians on the best agents to use. the world-wide problem of betalactam resistance (r) in streptococcus pneumoniae (sp) has been complicated by increasing r to macrolides and some older fluoroquinolones (fq) (ciprofloxacin cip). aim of our study was to evaluate rate of acquisition of resistance to different fq: cip, sparfloxacin (spx) and levofloxacin (lev) of sp strains with different levels of susceptibility to penicillin (p). fifteen strains were serially and daily passaged in subinhibitory concentrations of these four antibiotics by a gradient plate method until acquisition of resistance. clinical strains isolated from children in day-care centers were used. five strains were susceptible (s) to penicillin (p) (one reference strain, four clinical isolates: p micsb/0.064 mg/l): five were intermediate (i) to p (one reference strain: p mic 0.19 mg/l, four clinical strains p mics: 0.5 á/1 mg/l), five were resistant (r) to p (p mics 1.5 mg/l). mean of number of passages (n ) necessary to reach i or r level with each fq as selecting agent are in the following table: spx and lev induced resistance but more slowly than cip. our results show that rate of acquisition of resistance to fq is strongly related to alteration of susceptibility to p, probably by modification of cell wall. these results are concordant with clinical results. clinical relevance of phase variation in pneumococcal opacity: nasopharyngeal (np) colonization in children from day care centers (dcc) soa8.11 carsenti h, mancini g, bensoussan m, dunais b, pradier ch, dellamonica p. archet hospital, infectious disease, nice, france streptococcus pneumoniae (sp) adherence to nasopharyngeal (np) epithelium is a prerequisite for induction of otitis transparent sp (t) have been shown to colonize the np of infant rats better than opaque (o) sp. opaque sp has proven more virulent than the t form during systemic infection in a mouse model. aim of this study was to evaluate phase variation in the nasopharynx of children. sp strains were isolated during a winter epidemiology study of np samples in children from family dcc. mics determinations were performed by e -test for penicillin (p), amoxicilline (amx) and ceftriaxone (cro). serotypes were performed using the quellung reaction. upon oil immersion microscopic examination short chains of six to eight cocci were noted as 0, '/, '/'/, '/'/'/ for absence, 1, 2, !/3 chains by field, respectively. phase variation was detected on catalase trypticase soja plates, amx and cro mics and bactericidal activity was determined for 10 pairs of o and t variants with different serotypes and susceptibility to penicillin. seventy strains of sp were screened for phase variation. nine out of 42 with chain length 0, '/ had o variants while 23 out of 28 strains with chain length '/'/ or '/'/'/ showed o variants. proportion of o variants was predominant when chain length increased. serotype 23f was prevalent. bactericidal activity of o variants showed a four-to eightfold increase of mbc. o variants may be present in np of children while t are predominant form for colonization. these virulent variants with lower level of autolysis showed less susceptibility to killing by antibiotics. they may persist in np and explain the absence of eradication by active molecules. antimicrobial resistance among clinical strains of s. pneumoniae isolated from patients with community-acquired respiratory tract infections (carti) in russia soa8.12 kozlov rs a , bogdanovitch tm a , sivaya ov a , agapova ed b , ahmetova li b , furletova b , gudkova lv b , gugutsidge b , ilyina vn b , marusina b , multich ig b , ortenberg ea b , schetinin ev b , shturmina purpose: to determine the antimicrobial resistance of pneumococci causing carti in different russian cities. methods: a total of 142 non-duplicate strains isolated in 14 russian cities in 2001 were studied. antimicrobials tested included penicillin (pen), amoxicillin (amo), erythromycin (ery), azithromycin (azi), clarithromycin (cla), midecamycin (mid), spiramycin (spi), clindamycin (cli), levofloxacin (lev), vancomycin (van), rifampicin (rif), tetracycline (tet) and co-trimoxazole (sxt). susceptibility testing was performed by broth microdilution with interpretation of the results according to nccls guidelines (2001) a map of bacterial resistance in a hungarian region soa8.16 farkas a a , juhasz a b , orosi p a , miszti c c , balogh m d . a kenezy teaching hospital, hygienie, debrecen, hungary , b kenezy teaching hospital, laboratory, debrecen, hungary , c university of debrecen, microbiology lab, debrecen, hungary , d regional hospital berettyóújfalu, laboratory, debrecen, hungary background: regional trends of microbiological resistance pattern constitute basic data and qualifying criteria for effective infection control. purpose: the aim of our study was to establish an internationally compatible regional database in a hungarian county hajdú -bihar. methods: our model is the national nosocomial infections society publications' format from the u.s. published in 2001. it contains data regarding various icu types, ambulatory patients and hospitalised patients. the same format is used for antibiotic utilisation data and device related infections' rates as well. we collected cleaned data of years 2000 á/2001 from all the microbiological laboratories of our county. results: ciprofloxacin p e. coli 6.8 4.5 9.6 75 á/above 90 susceptibilities not significantly different from u.s. data were as follows: mr cns, streptococcus pneumoniae /penicillin and 3rd generation cephalosporin, pseudomonas aeruginosa /piperacillin and enterobacter spp. and escherichia coli /ceftriaxon. conclusions: this database proved to be a very useful tool for choosing primary wards of active surveillance including places for infectious disease physician's visit (icu, rehabilitation unit). additional analysis is needed at an individual institution's level for other heavily used (or useable) antibiotics and bacteria as well (aminoglycosides, beta-lactam á/beta lactamase inhibitor combinations, 2nd generation cephalosporins, corynebacteria . to compare epidemiological, clinical, and immunological features of add before and after haart introduction, between the 436 patients (p) diagnosed in 1985 á/1995, and the 103 p detected since 1997, in a case-control study. though the mean number of newly diagnosed aids p had a sharp drop in the haart era, from 65 p/year in 1991 á/ 1995 to 22 p/year since 1997 (p b/0.001), the distribution of add and underlying immunodeficiency showed limited changes. when excluding a greater frequency of tuberculosis (tb) (p b/0.001) and wasting syndrome (p b/0.04), all other add did not show a different frequency before and after 1997. a tendency towards a higher mean cd4 count at aids disease was noticed: 78 vs 61 cells/ml (p b/0.003), with a significant difference for candida esophagitis , toxoplasmosis, kaposi sarcoma and tb (p b/0.002 á/b/0.03). the limited variation of clinical and immunological presentation are attributable to the poor impact of haart before aids recognition: 88.4% of p detected since 1997 did not receive haart or had insufficient compliance to antiretrovirals, so that 58.2% of p were aids presenters. during the haart era, an increase of mean age and sexual transmission was found (p b/0.001). notwithstanding the effects of haart on the natural history of hiv disease, the consequences on add distribution and related immunodeficiency were negligible, since most p could not benefit from haart before aids onset. a high clinical suspicion for add should be maintained when facing p with missed or undertreated hiv disease. radata */communication internet platform management of resistance analysis guided haart switch for implementation in clinical practice of hiv-infected individuals soa9.3 paech v, lorenzen t, stoehr a, plettenberg a. ifi, interdisciplinary infectiology and immunology, hamburg, germany purpose: hiv-resistance analyses are indicated to prepare switch of haart in hiv-infected individuals with failure to ongoing haart regimen. specialists at several responsible sites often feel lack of complementary informations if interpretation of resistance analyses is done independent from each other. clinical benefits from resistance analysis assays are sigfnificantly higher for those physicians, who can access external advice from hiv-experts for possible treatment options. the database concept 'radata' (www.radata.de) was developed in germany to generate expert advice for implementation in haart switch. results: fifteen hiv-treatment centres, seven laboratories and 15 high ranked authorities in hiv-medicine contribute to radata database since it is started in january 2002 in germany. hiv-infected subjects are eligible to participate at the project after presentation of failure to haart (viral load !/1000 c/ml). expert advice is generated after all data are evaluated and based on recommendations of 2 á/4 external hiv-experts. observation after therapy switch is scheduled for a period of 12 months. conclusions: radata is a novel database concept with features for evaluation of data and availability of complementary information to participating sites. the project is designed to provide its proficiency to patients and centres from germany and foreign countries. further information will be provided after the number enclosed subjects have enlarged. in vitro effects of hiv infection on abc transporter expression and antiretroviral drug efficacy soa9.4 therefore, we evaluate in primary cultures of human monocytederived macrophages (mdm) and lymphocytes, effects: (1) of retroviral infection and haart on the expression and activity of p-gp and mrp; and (2) of specific inhibitors of these host proteins on antiretroviral activities of nrti, non-nrti and ip. results: on the one hand, we evidenced a transitory increase of p-gp mrna expression in lymphocytes and mdm in response to in vitro hiv infection. this was correlated to an increased p-gp cell surface expression and activity, and an increased tnf-alpha production and mrna. in contrast, no significant modulation of mrp was observed. on the other hand, psc833 and probenecid potentiated in vitro the anti-hiv activity of azt and indinavir. these effects were accentuated when psc833 and probenecid were combined. conclusion: these results showed that: (1) hiv infection by increasing abc transporter expression could favorise the efflux of antiretroviral drugs and decrease their pharmacological effects; and (2) specific inhibitors of these transporters could reverse these deleterious effects. effects of interferon alpha plus ribavirine therapy on frequencies of hcv, hiv and cmv specific cd4-t-cell responses in peripheral blood of hiv/hcv coinfected patients after 6 months of treatment soa9.5 methods: two groups of patients with chronic hcv infection were studied: 26 hiv coinfected progressors with antiretroviral therapy and 13 hiv-negative controls. twelve hcv/hiv and 9 hcv patients have already reached 6 months of ifn-alpha'/ribavirine therapy. virusspecific cd4-t-cells in peripheral blood were analyzed by ifngamma-elispot-assays using hiv-p24, one cmv and three hcv (core, ns3, ns4) antigens. results: (1) at baseline, hcv-specific cd4-th1-cells frequencies were significantly lower than hiv-and cmv-specific ones; (2) frequencies of cd4-th1-cells against hcv as well as against cmv were similar in the two groups; (3) in hcv'//hiv'/, hcv specific cd4-t-cell frequencies did not change between baseline and 6th month of anti-hcv treatment, decreased in three and increased in only one case. hiv-and cmv-specific frequencies were decreased in seven patients. similar results were observed in hiv-negative group. conclusion: (1) hcv-specific immune responses might be more prone to tissue compartmentalization than hiv-specific ones; (2) immune defects induced by hiv infection might not be responsible for the low level of hcv-specific responses observed in hiv-progressors; (3) ifn-alpha'/ribavirine therapy influence on hcv-and hivspecific cd4-t-cell frequencies after 6 months of treatment will be discussed. frequencies of hiv-1-p24 specific th1 cells (elispot) are correlated with plasma hiv-1 viral load in a cohort of lt-np and slow progressors soa9.6 martinez v a , alatrakchi n a , costagliola d b , bonduelle o a , agut h c , autran b a , alt study group a . a hopital pitié-salpêtrière, laboratoire d'immunologie cellulaire, paris, france , b faculté de medecine saint-antoine, inserm sc4, paris, france , c hopital pitié-salpêtrière, laboratoire de virologie, paris, france background: hiv-1-specific t helper-1 cell responses have been associated with long-term-non-progression (lt-np) in hiv infection but the correlation between frequencies of hiv-1-p24-specific th1 cells and viral load has not yet been studied. we prospectively quantified these frequencies by using an ifn-gamma elispot assay in a cohort of lt-np. methods: a cohort of 62 lt-np and slow progressors (infection !/ 8 years and cd4 counts !/600/mm 3 ) was analysable. hiv-1-p24specific t cells were analyzed using: proliferation, ifn-gamma eli-spot assays and ifn-gamma production in cell supernatants. results: wide ranges were observed in the frequencies of hiv-1-p24-specific cd4 th1 cells as assessed by elispot (0-3770 sfc/106 pbmc) with a median of 70 sfc/106 pbmc. these frequencies were negatively correlated with viral load (r0/(/0.304, p 0/0.026) but not with cd4 counts and associated with a low level of t cell activation assessed by cd71 on cd4 cells (r0/(/0.266, p 0/0.035). similar results were obtained with t cell proliferation and ifn-gamma production. conclusion: interestingly, the numbers of hiv-1-p24-specific th1 cells correlate with plasma viral load, independently of cd4 counts indicating that: (1) the defect in hiv-1-specific cd4 th1 cells does not reflect the global cd4 depletion; and (2) these responses are strongly correlated to the control of virus replication. impact of drug á/drug interactions on therapeutical management of active tuberculosis in hiv infected patients soa9.7 martinez v a , truffot c b , caumes e c , katlama c c , jarlier v b , bricaire f c , jouan m d . a department of infectious and tropical diseases, pitié salpêtrière hospital, institut pasteur, unité de génétique mycobactérienne, paris, france , b department of bacteriology, pitié salpêtrière hospital, paris, france , c department of infectious and tropical diseases, pitié salpêtrière hospital, paris, france , d institut pasteur, unité de génétique mycobactérienne, paris, france since 1996 and the use of haart, management of hiv patients with active tuberculosis raised the question of drug á/drug interactions and therapeutical management of both infections. retrospective cohort study: follow-up of 43 hiv patients with active tuberculosis diagnosed between 1996 and 2000. studied data included evolution of tuberculosis and hiv, cd4 cell counts, plasma hiv viral loads, antituberculosis and antiretroviral regimens. ninety-four percent of patients were treated by quadruple combination antituberculosis drug with rifabutin for five patients. fourteen patients were treated by double antiretroviral therapy of nucleoside reverse transcriptase inhibitors (nrtis) and 29 by triple or more drugs (nrtis and/or nonnucleoside reverse transcriptase inhibitors nnrtis and/or protease inhibitors pis). the median follow-up was 24 months. there was no difference on cd4 cell counts and viral loads in the two groups and between the patients treated by nnrtis and pis at the diagnosis of tuberculosis, at the time of antituberculosis drug discontinuation and concerning cure rates of tuberculosis. on the other hand, the plasma hiv viral load was significantly better controlled in patients with nnrtis than with pis (p b/0.005). in hiv patients with active tuberculosis receiving haart, antiretroviral combination including nnrtis allows a better control of viral replication than regimen including pis without impact of the use of rifampin or rifabutin. adherence is essential to the effectiveness of antiretroviral therapy. a pharmacy visit would improve the patient advisement. a survey was carried out over a period of 3 months in the u.m.i.t. a self-report was distributed to 150 patients. ninety were evaluated. for 66%, information's given by the clinician were sufficient and for 76% the treatment advice cards were useful. however, 72% of them would like to attend a pharmacy visit. the topics they would prefer to be tackled were drug interactions (51%), side effects (49%) and effect of forgetting (44%). the treatment was well accepted and tolerated for, respectively 91 and 65% of the patients. the viral load and the cd4 count were well known by, respectively 52 and 46%. however, inaccurate pattern of treatment was frequent ( !/50%) and bad adherence was observed: treatment forgotten occasionally (60%), regularly (30%) or inadequate attitude when the treatment was forgotten (76%). number of pills, dose frequency, length of the treatment would be risk factors of nonadherence. for the majority of patients, a pharmacy visit is necessary and beneficial. the first result shows a better understanding of the treatment, an improvement of the adherence and an enhancement of plasma concentration of antiretroviral drugs. in vivo activity of glycopeptides against s. aureus infection in a rabbit endocarditis model: is mic predictive for in vivo efficacy? soa10.3 asseray n, caillon j, lemabecque v, jacqueline c, batard e, potel g, bugnon d. laboratoire antibiologie, faculte de medecine, nantes, france we have studied the in vivo efficacy of vancomycin (v) and teicoplanin (t) against five staphylococcus aureus (sa) strains: two methicillin-susceptible (mssa 1 and 2), two methicillin-resistant (mrsa 3 and 4) and one glycopeptide-intermediate (gisa 5) strain, in a rabbit endocarditis model. mics of v and t (v/t) were 0.5/0.25, 1/0.5, 1/1, 0.5/0.5, and 4/8, for mssa 1, mssa 2, mrsa 3, mrsa 4, and gisa 5, respectively. the animals were randomly infected with one of these strains, then treated for 2 days by v or t. a continuous infusion of v, simulating a 30 mg/kg/24 h human dose was used. t was infused as a continuous infusion allowing simulating a 6 mg/kg human dose, following an initial bolus. these regimens achieved clinically relevant serum steady-state concentrations of glycopeptides ( !/20 mg/ l). results were as follows: expressed in log cfu/g of vegetations (mean9/sd, followed in parenthesis by the number of rabbits used). purpose: to determine the prevalence of the decreased glycopeptides susceptibility among 187 clinical isolates of staphylococcus aureus collected from patients hospitalized in strasbourg university hospital between 15/01/2000 and 01/03/2000. the susceptibility to glycopeptides of 38 s. aureus isolates collected from hospital environment during approximately the same period was also investigated. methods: the susceptibility to glycopeptides was studied among the mrsa isolates, using: á/ detection of the decreased susceptibility to glycopeptides using agar plates containing 6 mg/l teicoplanin, á/ detection of hetero-visa strains using agar plates containing 4 mg/ l vancomycin, á/ determination of the mics of vancomycin and teicoplanin using the agar dilution and the e -test strips methods. results: thirty-nine percent of s. aureus clinical isolates (74 out of 187 strains) are mrsa. no visa or hetero-visa strain was detected. six percent mrsa isolates are teicoplanin intermediate s. aureus strains. in the environment, 13% s. aureus isolates are methicillinresistant (five out of 38). the five strains are all susceptible to glycopeptides. conclusion: the results regarding vancomycin are reassuring. however, the high rate of mrsa and the presence of teicoplanin intermediate s. aureus isolates prove that prevention and control measures need to be improved. comparative investigation of polymerase chain reaction and a conventional methods for detection of methicilin resistant staphylococcus amont clinical isolates soa10.5 kantardjiev tv a , vacheva-dobrevski rs b , panajotov sv a , bachvarova am a , velinov ti a , levterova vs a . a national center of infectious and parasitic diseases, microbiology, sofia, bulgaria , b military medical academy, clinical microbiology, sofia, bulgaria purpose: identification on methicillin resistant staphylococci has a great clinical implication and significant impact of antibiotic therapy. the aim of this study is to compare the disc-diffusion test (ddt), oxacillin agar screen test (oast) and pcr for detection of mec a gene. fifty selective clinical isolates (41 staphylococcus aureus and nine s. epidermidis ) determined as methicillin resistant by ddt were enrolled in the study. ddt was performed with oxacillin disk (1 mkg/ml) on mueller-hinton agar (mha) without nacl (nccls, 2000) . oast was performed on mha with 4% nacl, oxacillin 6 mkg/ ml, t 35 8c. these strains was genotypically characterized for the mec a gene presence by pcr method using the mec a 1-5?-aaa atc cat ggt aaa ggt tgg c-3? and the mec a 2 á/5?-agt tct gca gta ccg gat ttg c-3? primers (gibco, brl) . results: in the group of 50 mrs isolates, detected by pcr, positive results were as follow: 23 s. aureus and six s. epidermidis . for six s. aureus isolates ddt and oast were positive; pcr-negative. for two s. aureus and two s. epidermidis isolates pcr was positive; phenotypic methods-negative. conclusions: accurate and rapid detection of mrs is a constant challenge for laboratories. the pcr assay (first time in our country) appears to be more reliable than routine susceptibility testing for the rapid diagnosis of mrsa infections at hospitals, particularly due to the heterogeneous resistance of many strains. 7.39/1.7 (6) 9.79/0.9 (5) 8.79/0.9 (5) 8.49/1.3 (8) 8.29/1.1 (4) v 3 . 0 9/1.3* (7) 8.39/1.5 (5) 3.09/0.9* (4) 6.69/1.6 (6) 6.79/1.9 (6) t 3 . 0 9/0.6* (4) 7.59/0.4 (4) 4.59/1.6* (6) 8.29/0.6 (4) 5.79/1.9 (6) distribution and antibiotic susceptibilities of bacteria isolated from suspected urinary tract infections of inpatients in hungary soa13.2 rozgonyi f, csukás z, kamotsay k, szabó d, ostorházi e, berek z, maródi c. institute of medical microbiology, semmelweis university, budapest, hungary between l january 1997 and 31 december 2000, a total of 12, 143 urine samples were cultured 40% as native urine (nu) and 60% as uricult-plus (up) (orion diagnostica, finland) . cultivations were negative in 39% of nu and 42% of up specimens, while contamination was revealed in 20% of nu and 30% of up. in the clinical bacteriologically evaluable positive 1726 nu and 1656 up cultures, the distribution of the gram-negatives was very similar with the predominance of escherichia coli (43 and 41%) followed by enterobacter spp. the distribution of gram-positives differed significantly according to the types of specimens. nu resulted in 13% group-d and 6% group-b streptococcus while up did 22 and 1.5%, respectively. third generation cephalosporins and fluoroquinolons were equally very effective against e. coli strains, while ampicillin inhibited growth of 42% only. carbapenems, cefepime and the fluoroquinolons were the most active against enterobacter strains. interestingly, trimethoprim'/ sulfarmetoxazole combination could inhibit more than 75% of enterobacteriaceae strains. piperacillin'/tazobactam (89%), imipenem (83%), and ciprofloxacin (82%) could be the drog of choice against pseudomonas aeruginosa . enterococcus strains were highly sensitive to glycopeptides (100%), nitrofurantoin (99%), imipenem (95%) and amoxicillin'/clavulanic acid (94%). antimicrobial susceptibility levels of escherichia coli isolates cultured from urine at a tertiary care teaching hospital. temporal trend and comparison between community-acquired and nosocomial urinary tract infection soa13.3 nanetti a, manfredi r, valentini r, calza l, chiodo f. uni versity of bologna, infectious diseases, bologna, italy in order to assess the local temporal trend of antibiotic sensitivity of the most common urinary tract bacterial pathogen, all urine-cultured escherichia coli isolates were reviewed as to susceptibility profile, and specimen source (community-versus hospital-acquired infection). when evaluating sensitivity levels of 2070 community-acquired pathogens (1999 á/2001), a significant resistance rise was limited to cotrimoxazole (p b/0.01) and nalidixic acid (p b/0.02), while a tendency towards increased resistance regarded norfloxacin (p 0/ 0.05) (fig. 1) . when 1570 community-acquired e. coli isolates were compared with 2687 nosocomial strains (tested in the years 2000 á/ 2001), a greater susceptibility of community-acquired e. coli isolates was limited to cotrimoxazole versus all other compounds in the year 2000 (p b/0.03), while it was extended to amoxicillin, cephalotin, nitrofurantoin and piperacillin in the year 2001 (p b/0.0001) (fig. 2) . on the whole, e. coli showed an elevated sensitivity rate ( !/90% of tested strains) to nitrofurantoin, gentamicin, amikacin, and 2nd-and 3rd-generation cephalosporins, while only amoxicillin and piperacillin had a mean resistance rate !/30%, regardless of the community or nosocomial origin. a permanent surveillance of sensitivity levels of the most common pathogens responsible for infectious diseases enables to identify local antimicrobial activity and its temporal variations, and plays a key role in starting empiric therapy, pending bacterial identification and in vitro assays. conclusion: in uti, the antimicrobial agents such as 1st cg combined with aminoglycosides are recommended as initial treatment as well as the 3rd cephalosporin generation at monotherapy. in addition, the fluoroquinolones and aminoglycosides are effective in uti. prevalence of resistance mutations to antirretrovirals and relation to virological failure s3.4 garcia f a , suarez s a , alvarez m a , martinez nm a , valera b b , pasquau j b , hernandez quero j a , maroto mc a . a hospital san cecilio, microbiology, granada, spain , b hospital virgen nieves, microbiology, granada, spain purpose: to investigate the prevalence of resistance mutations in the reverse transcriptase (rt) and protease (p) genes of hiv and to relate it with the type of virological failure (vf), we have studied 88 patients (11% naïve or pregnant women, 31% were first vf, 23% were second vf, 35% more than two vf. resistance mutations were investigated using trugene hiv-1 genotyping kit (visible genetics). results: global prevalence of resistance mutations for rt inhibitors (rti) has been !/20% for m41l, d67n, k103n, m184v, l210w, t215yf, and for l10i, m36i, l63p, a71vt, l90m for p inhibitors (pi). the prevalence of resistance mutations for the naïve patients studied was very low (a98g, v118i for rti and l10i, m36i, m46i */all n0/1 */and l63p n0/4 for pi). for patients on first vf only k103n, m184v, t215yf (rti) were !/20%, as well as l10i, d30n, l63p (pi); when patients on second vf were studied, then m41l, e44d, k103n, m184v, g190a, l210w, t215yf, k219qe (rti) and m36i, l63p, a71t (pi) were !/20% prevalence; finally, when patients with more than two vf were studied, the following resistance mutations were !/20%: m41l, d67n, k70r, k103n, v118i, y181c, m184v, g190a, l210w, t215yf (rti), and l10i, m36i, m46il, l63p, a71t, l90m (pi). conclusions: the prevalence of primary resistance in the population studied is very low; the prevalence of mutations in the reverse transcriptase and protease genes increase in parallel to the type of virological failure. genotypic resistance in hiv-1 rna from patient plasma compared with rapid virus isolation and phenotypic resistance in patient pbmcs s3.5 stuermer m, groeschel b, cinatl j, doerr hw. institute for medical virology, university clinic frankfurt, frankfurt, germany objective: to compare hiv-1 virus isolation in the presence of antiretroviral drugs with plasma hiv-1 genotyping. materials and methods: hiv-1 genotyping was performed using the viroseqtm vers. 2 from applied biosystems. interpretation of genotype was done according to international standards. cd4-cells were purified from patient plasma and cultivated in microtiter plates coated with anti-cd3 and anti-cd28 antibodies in the presence of different concentrations of antiretroviral drugs. virus production was measured using a p24 antigen assay. phenotypic activity was expressed as 50% reduction of p24 concentrations. results: seventeen samples were analyzed. for 11 samples results were obtained from both methods, two samples could not be analysed by phenotyping and four samples not by genotyping. only 3/11 samples showed total and 2/11 samples partial concordance, 6/11 samples showed discordance between the two assays. in discordant samples the genotype gave a definite interpretation. conclusion: hiv-1 virus isolation and phenotyping from pbmcs may overcome the problem of currently used resistance assays, which analyse only the reverse transcriptase and the protease gene of hiv-1. possible mutations in other regions may influence viral fitness and therefore contribute to the growth of the virus population present. the lack of concordance between the two assays is related to the different blood compartments used. the clinical value of resistance tests using pbmcs is under investigation. interleukin-6 co-operates with a new type i ifn, ifn-tau to inhibit early steps of hiv-i biological cycle s3.6 rogez c a , clayette p a , martin m a , dereuddre-bosquet n a , martal j b , dormont d c . a cea, drm, fontenay-aux-roses, france , b inra, physiologie animale, jouy-en-josas, france , c cea, crssa, ephe, drm, fontenay-aux-roses, france background: type i interferons (ifn) exhibit efficient antiviral activities notably against hiv, but severe side effects restrict their clinical uses. ifn-tau is an ovine or bovine non-cytotoxic type i ifn which displays higher inhibitory effects towards hiv replication than ifn-alpha, particularly in human monocyte-derived macrophages (mdm). the antiretroviral activity of ifn-tau seems to involve antiviral and immunomodulatory mechanisms: il-6 synthesis is increased in dose-dependent manner in mdm treated with ifn-tau and a specific inhibition of il-6 biological activity decreases its antiretroviral efficiency. results: after a 1-h infection, a significant decrease of intracellular hiv rna amount was found in mdm treated with ifn-tau. in parallel, no additive inhibition was observed with ifn-tau during the elongation of proviral dna. these results suggest either an inhibition of hiv nucleocapsid uptake or an immediate hiv rna degradation, and the expression of 2?,5?-oas, mxa protein and pkr was then measured. ifn-tau induced the expression of these three host cell factors. the role of il-6 on these different steps was evaluated and we showed that il-6 co-operates with ifn-tau during the very early step of hiv biological cycle. conclusion: altogether, these results evidence that ifn-tau uses the same antiretroviral pathway as others type i ifn in mdm, and that il-6 takes part to its inhibition of early steps of hiv biological cycle. actinomycin-d as a modulator of resistances due to cell-wall active agents like bacitracin (bc) and lysozyme (lz) s4.7 chakrabarty an a , dastidar sg b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india it was observed that development of lzr in the lzr mutants took placed at three different levels and was accompanied with unselected, distinctive and elevated levels of bcr. similarly, bcr in bcr-mutants were also detected at three different levels. although the levels of bcr (100/200 mg/ml) in the bcr mutants could be raised only by persistent efforts, an increase in the levels of lzr (as cross-resistance) in the same mutants could be easily achieved. a correlation of actinomycin-d resistance with lzr and bcr of the mutant bacteria and the effects of lipase treatment on the same showed a 4 á/20-fold rise in actinomycin-d resistance of the lzr and bcr mutants of gram-positive bacteria compared with their correspondence wild-types. these findings suggest that lzr and bcr are controlled by several genes accounting for reduced cell-wall and cell-membrane permeability and indirectly, by phenotypic alteration of the lipid content of the cell-wall. thus, the alteration of cell-walls and membranes and a phenotypic extra lipid layer can work in conjunction with the efflux pump mechanisms finally determining the levels of drug-resistance. experimental development of drug resistance to non-antibiotics: a role of alteration of membrane fluidity and efflux systems s4.8 dastidar sg a , mazumdar k a , asok kumar k a , chakrabarty an b . a jadavpur university, pharmaceutical technology, calcutta, india , b department of medical microbiology, calcutta university, calcutta, india drug resistance among clinical strains was studied by selecting mutants resistant to promazine (pr) and methdilazine (md). the results showed that successive step-up mutants of pr and md developed cross-resistance to several unrelated drugs, which in subsequent steps had broader resistance spectra with higher levels of resistance. experiments on the membrane fluidity or permeability of bacterial cells using diphenyl hexatrine (dph), a fluorescent probe on md-mutants showed that three was marked reduction in the membrane fluidity and permeability. when several analogues of the basic phenothiazine structure, e.g. 2-chlor-methyl-n -methyl-pyrrolidine (cmp), methyl-1-methyl-2-pyrrolidone-4-carboxylate (mmpc), 3 hydroxymethyl-n -methyl-pyrrolidine (hmp) and md with final substitution were tested for antibacterial function on different strains, highest activity was observed with respect to md. with anaerobic bacteria the resistance(s) dependent on efflux pumps showed higher levels of resistance even to md. we have found the non-antibiotic agents triflupromazine, trimeprazine and diclofenac sodium have high degree of activity against vibrios, staphylococci and pseudomonads. the explanation of such a phenomenon in terms of possible efflux pumps will be discussed. csf, plasma and urine pcr in lyme neuroborreliosis s7.3 pícha d a , moravcová l a , lásiková š a , marešová v a , ž ïárský e b . a charles university, 2nd medical school, 1st clinic for infectious diseases, prague, czech republic , b department of cellular and molecular biology, charles university, 3rd medical school, 1st clinic for infectious diseases, prague, czech republic the main reason for high diagnostic value of pcr in neuroborreliosis (nb) is the direct way of spirochete detection. two sets of primers in nested pcr were used: one for plasmide gene encoding ospc protein and second for chromosomal gene 16s rdna. so far 25 patients with clinically manifested involvement in nb were enrolled into the prospective designed study (being continued). the main including criterion was positive prove of intrathecal specific antibody secretion (in 22 patients) and pcr positivity in csf (in 3). all patients were repeatedly examined by neurologist and samples of csf, plasma and urine were taken: (1) before treatment; (2) after treatment; (3) after 3 months. before treatment were 12 patients pcr positive in csf, six in plasma, and 10 in urine. five were parallel positive in csf and plasma and four in all three body fluids. urine after treatment was positive in seven (28%) cases and completely negative after 3 months. the pcr has had relative high sensitivity (44 %), but does not rich the sensitivity of antibody index (88%). supported by grant mzcr 6244; 111300003. consumption of imipenem correlates with b-lactam resistance in pseudomonas aeruginosa s12.5 lepper pm a , hö gel j b , trautmann m a , grusa e c . a department of medical microbiology and hygiene, university of ulm, ulm, germany , b department of biostatistics, university of ulm, ulm, germany , c hospital memmingen, central pharmacy, memmingen, germany purpose: in the present study we investigated the monthly consume of three anti-pseudomonas-active antibiotics, namely imipenem, piperacillin/tazobactam (pt) and ceftazidime during a period of 3 years (1997 á/2000) . the use of these antibiotics was correlated to the rate of resistance in pseudomonas aeruginosa . results: inspection of the time series for use of imipenem, ceftazidime, and pt, and the corresponding time series for resistance (each available from july 1997 to july 2000) indicates a remarkable coincidence between use of imipenem and resistance against the three antibiotics mentioned. pearsons's coefficient of correlation for the use of imipenem and the resistance against imipenem was 0.62 (p b/0.001), between imipenem use and pt resistance was 0.57 (p b/0.005), and between imipenem use and ceftazidim resistance 0.56 (p b/0.005). we found positive regression coefficients quantifying an association with imipenem use in the same month (p b/0.01) and with the use during the preceding month (p b/0.05). the same was true when checking dependence of ceftadizime resistance (p b/0.05) and pt resistance (p b/0.01) on imipenem use observed during the same month. neither the use of ceftadizime nor of pt could be identified as factors creating resistance to one of the three antibiotics under consideration within a reasonable period of time. conclusion: there might be a strong pressure towards resistance created by carbapenems. this could limit the use of carbapenems for initial empiric therapy. treat hard and fast: short course antibiotic treatment and its relation with patient compliance and effectiveness s12.6 perez-gorricho bpg a , ripoll m b , pechere jc c . a niño jesus hospital, infectious diseases, madrid, spain , b insalud, outpatient consult, madrid, spain , c university of geneve, microbiology, geneve, switzerland 'treat hard and fast ': short course antibiotic treatment and its relation with patient compliance and effectiveness. finding the important implications for the way in which physicians manage patients with mild á/moderate respiratory tract infections, and the relation of this management with the perception of antibiotic effectiveness, and the compliance with the antibiotic regimen has been the main purpose of the research. in a pan-european market research study of more than 3000 patients, designed to determine behaviour to the antibiotic management of mild-moderate respiratory tract infections, patient expectations of antibiotic therapy were identified, particularly those aspects that relate to efficacy and compliance. the study identifies three key drivers of patients perceived antibiotic efficacy: length of antibiotic course, time to onset of symptom relief and time to complete resolution of symptoms. the results demonstrate that once daily treatment for short periods is perceived by patients to be significantly more effective than longer antibiotic courses and thus better meets patient expectations of therapy. in this study, a macrolide, azithromycin, was selected as the drug therapy of shortest course, being the antibiotic with the shortest dosage schedule for common outpatient infections. the perception of efficacy with short course therapy also correlates with overall satisfaction with management by the physician and with patient compliance with antibiotic therapy. purpose of the study: group b streptococci (gbs) remain a major cause of neonatal infections. consensus guidelines have recommended an intrapartum antibioprophylaxis by amoxicillin, which has reduced the incidence of early-onset neonatal gbs infections. however, an increased incidence of beta-lactam-resistant gram-negative neonatal sepsis has been reported. the aim of our study was to analyse the consequences of this antibioprophylaxis on the intestinal microbial colonization of newborns. a study of the fecal flora was carried out on 50 stools samples from 3 days-old newborns divided into groups: group a intrapartum treated mothers (n 0/25); and group b untreated mothers (n0/25). both groups were matched with regards to known factors affecting intestinal microbial colonization: gestational age, type of delivery and feeding. results: colonization by enterobacteria and enterococci was not significantly different and occurrence of amoxicillin-resistant enterobacteria was similar (11/13 and 13/16 in groups a and b, respectively). however, the colonization by clostridia was modified: the number of newborns colonized was significantly less important in group a than in group b (group a: 3/25 and group b: 10/25 p b/0.05). conclusion: in our study, intrapartum antibioprophylaxis did not affect intestinal colonization by aerobes but reduced significantly colonization by clostridia, potentially anaerobic pathogens. impact of an antibiotic policy restricting the use of b-lactams and macrolides on the incidence of clostridium difficile associated diarrhoea in general medical, renal and elderly patients s12.8 boswell tc a , pacey s b , broomfield s c , westmoreland d c , yates c c . a nottingham city hospital, microbiology, nottingham, uk , b nottingham city hospital, pharmacy, nottingham, uk , c nottingham city hospital, infection control, nottingham, uk the purpose of the study: to investigate the short-term impact of a new antibiotic policy for the treatment of urinary and respiratory infections on the incidence of clostridium difficile associated diarrhoea (cdad) in hospitalised medical, elderly care and renal patients. the results obtained: a policy restricting the use of b-lactams (except parenteral penicillin), and promoting alternative antibiotics including levofloxacin for pneumonia, and doxycycline for non-pneumonic respiratory infections, was launched in july 2000. as a result there was a significant and sustained reduction in use of aminopenicillins, cefuroxime and macrolides, with a corresponding increase in doxycycline and levofloxacin. the incidence of cdad was determined during the 1st 12 months of the new policy and compared to the last 12 months of the old policy. the incidence of cdad fell from 10.02 to 4.91 per 1000 patients, and from 1.2 to 0.59 per 1000 in-patient days (p b/0.00001). in contrast, there was no change in the incidence of cdad in other specialties (surgery, oncology etc.) that had not introduced the new policy. there was no change in the incidence of nosocomial bacteraemia with quinolone-resistant coliforms or mrsa, despite the increased use of levofloxacin. conclusions: hospital-wide reduction of b-lactam and macrolide use in medical patients can result in a significant and immediate reduction in cdad. longer follow-up will determine if this effect is sustained. use of imipenem/cilastatin i.v. (tienam i.v.) for the treatment of low respiratory tract infections in intensive care units s12.10 izzo l a , orsetti r b , boschetto a a , binda b a , della casa u a , caramanico l a , la mazza a a . a department of surgery, universitá degli studi di roma 'la sapienza','p. valdoni', rome, italy , b s. camillo-forlanini, intensive care unit, rome, italy ventilator associated pneumonia (vap) is considered the most frequent infection in the intensive care unit (icu), occurring in 9 á/24% of patients intubated for longer than 48 h besides nosocomial pneumonia is a common complication in the critically ill surgical or trauma patient. inadequate treatment can lead to the complications of acute respiratory distress syndrome (ards), empyema, and lung abscess. the most important aetiological agents both in vap and in pneumonia which arise as complication in surgical or trauma patients are bacteria, whit a marked predominance of staphylococcus aureus and pseudomonas aeruginosa . the authors present their experience (30 cases) on the employment of imipenem/cilastatin i.v. (tienam i. v.) as initial empirical monotherapy at the dose of 500 mg)/3/day or 1 g)/3/ day for the treatment of the serious lower respiratory tract infection in an icu. tienam is a well tolerated broad spectrum antibacterial agent that is effective against the majority of gram-positive and gram negative aerobic and anaerobic bacteria including most pseudomonas species. except one patient deceased for causes related to his very poor general conditions and three cases in which has been necessary the addition of an aminoglycoside, in all the other patients the imipenem/ cilastatin (tienam) monotherapy has shown satisfactory clinical and bacteriological responses. clinical auditing of the impact of recommendations on antibiotic treatment s12.11 kinoo j a , david-ouaknine f a , hacquard b a , echard y a , decazes jm b . a centre hospitalier lagny marne la vallée, lagny sur marne, france , b hospital saint louis, paris, france the aim of this study was to assess the impact of curative antibiotic recommendations on suitable prescriptions at lagny-marne la vallée hospital (general hospital, 714 beds). two prospective exhaustive audits were made (all complete hospitalizations, excluding psychiatry, february á/may 1997 and 2000) of the detailed curative antibiotic prescriptions, before and after distribution of internal recommendations. the same methodology, designed by a multidisciplinary team, was used for both periods. the same antibiotics were available at the pharmacy. the prescriptions were assessed by an infectious diseases specialist and a pharmacist using pre-established criteria: literature recommendations (1997 audit), internal recommendations (2000 audit). six hundred and fifty-six prescriptions for 498 patients were collected and analysed in 1997, 738 for 497 patients in 2000. exhaustivity of the recovered prescriptions was over 95%. patient characteristics, infection sites and microbiological findings were similar for both groups. suitable prescriptions were significantly increased (47 á/59%, p b/0.001). unsuitable prescriptions (economic reasons, too short or too long course, incorrect administration, or underdosage) were significantly reduced. prescriptions for incorrect indications were unchanged and necessary combined treatment not being prescribed, increased. local recommendations improved prescriptions, but efforts have to be done in order to go on the improvement of the practice behaviour. cost-effectiveness analysis of antibiotic therapy in hospitalized patients with copd exacerbations (ae-copd) s12.12 beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy antibiotic costs represent a high burden of total drug costs for hospital administrations. a scientific approach considering also the economic aspects of each therapeutic decision may gain optimal treatment objectives at pondered costs. in our study we retrospectively evaluated the clinical effectiveness and costs of antibiotic therapy in patients with ae-copd. from 1997 to 2001 , 1058 our retrospective study results support previous pharmaco-economic considerations according which in choosing an antibiotic regimen for ae-copd we must take into consideration the expected clinical and microbiological results without forgetting to consider the economic burden of our decisions. significant increase in fungaemia due to non-albicans candida species s20.4 shah pm. klinikum der j.w. goethe universitaet, schwerpunkt infektiologie, frankfurt am main, germany until 1996, predominant candida species in blood cultures was candida albicans . it accounted for 72.8% of all candida species cultured from blood. since then we have observed a gradual increase in number of non-albicans candida species. from 1999, onwards nonalbicans candida species out-number c. albicans . this observation is especially important as non-albicans candida species are generally non-susceptible to azole derivatives and empirical use of azoles in suspected candidaemia should not be recommended. amphotericin b is uniformally active against almost all candida species. echinocandin may be an alternative. see figure below. a search for newer antifungal chemotherapeutics s20.5 chakrabarty an a , dastidar sg b , saha b b , basu l b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india fungal infections due to the mucor-rhizopus (m-z) group present formidable problems due to lack of appropriate and effective drugs against them, as seen in increasing number of clinical situations; death due to mycoromycosis is nearly inevitable. we analysed the biological 'weak-spots' of the mucor-rhizopus group and attempted to devise suitable drugs using their weak-spots. we have noted that like many free-living fungi, the m-z fungi are facultatively chemoautotrophic (can grow on simple sources of carbon and nitrogen and a solution of mineral salts), like the human pathogenic chemoautotrophic nocardioform bacteria. we devised a minimal medium based on that of davis and mingioli, supplemented with simple chemical compounds as sole sources of carbon and nitrogen. the key chemical here was diphenylamine with trypan blue (dpa á/tb) and other similar sources of c and n. we found that while media free of these chemicals (controls) allowed good growth of different strains of m-z fungi, a mixture of dpa á/tb completely prevented their growth over a wide concentration range. experiments with immunocompromised mice showed that these drugs at the concentrations used are well tolerated; mice experimentally infected with several clinical isolates of m-z fungi and receiving these chemicals showed that these fungi could not grow in vivo. in vitro activity of newer fluoroquinolones against multi-drug resistant salmonella typhimurium s33.4 nolones resistance is being also reported. we have studied the in vitro activity of b-lactams and fluoroquinolones against multi-drug resistant s. typhimurium from human sources. material and methods: fifty multi-drug resistant s. typhimurium were tested against cefazolin, cefuroxime, cefotaxime, cefepime, ofloxacin, levofloxacin, and moxifloxacin, by the agar dilution method according nccls guidelines. results and conclusions: all the strains were resistant to four or more of the following antibiotics: ampicillin, tetracyclines, chloramphenicol, streptomycin, sulphonamides and nalidixic acid. a high proportion of strains were intermediate or resistant to amoxicillin/clavulanate. we found no resistance to cephalosporins. nevertheless, 26% were intermediate to first and/or second gen. cephalosporins. cefotaxime and cefepime were the most active cephalosporins (mic50: 0.1 mg/l). though increasing fluoroquinolones resistance has been described among this kind of strains, no resistance to fluoroquinolones was found here. levofloxacin was the most active fluoroquinolone (mic90: 0.06 mg/l), followed by ofloxacin (mic90: 0.1 mg/l) and moxifloxacin (mic90: 0.2 mg/l). high rates of resistance to antibiotics by salmonellae from diarrhoeic children in zliten-libya s33.5 ghenghesh ks a , ben ali m b , abuhelfaia a b , dufani ma a . a faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya , b faculty of arts and sciences, el-ghomes university, biology, el-ghomes, libyan arab jamahiriya salmonellae are major bacterial cause of diarrhoea in libya particularly in children. included in the present study 23 salmonella species isolated from 169 children with diarrhoea in zliten city-libya. the children aged between a few days to 10 years. the organisms were tested for their susceptibility to antibacterial agents using the disc diffusion method. of the isolates examined, 23 (100%) were resistant to ampicillin, 22 (95.7%) to amoxicillin á/clavulanic acid combination, 20 (87%) to cefoxitin, 22 (95.7%) to chloramphenicol, 21 (91.3%) to doxycycline, 18 (78.3%) to gentamicin, 1 (4.3%) to nalidixic acid, 1 (4.3%) to trimethoprim á/sulphamethoxazole and none (0.0%) were resistant to norfloxacin. a strong relationship was observed between the availability of antibiotics in the pharmacies of the city and resistance of the isolated salmonellae to these drugs. the misuse of the antibiotics by the community may be an important factor (among others) in the emergence of these high rates of resistance by the salmonellae examined. effect of ceftriaxone along with probiotics administration on intestinal ecosystem and betalactamase activity s34.6 bertazzoni minelli e a , benini a a , zoppi g b . a department of medicine and public health-pharmacology section, university of verona, verona, italy , b department of paediatrics, university of verona, verona, italy oral bacteriotherapy during antibiotic treatment is a much debated topic. aim: to study whether different probiotics can prevent imbalance of the intestinal ecosystem (dysbiosis) in children during therapy with ceftriaxone (cx). methods: fifty-one children (mean age 5.1 years) with febrile respiratory tract infections were treated with cx 50 mg/kg/day iv, alone (therapy 1) and along with the following preparations: saccharomyces boulardii (2); enterococcus spp. (3); lactulose (4); l. casei gg (5); l. rhamnosus , l. bifidus and l. acidophilus (6); b. bifidum and l. acidophilus (7); and a mixture of various lactobacilli and bifidobacteria at high concentrations (8). faecal samples, collected before and after treatment, were analysed for microflora composition, cx concentration, and beta-lactamase (bl) activity. results: cx causes intestinal dysbiosis. no c. difficile was found. faecal bl increased after therapy in all treated groups. cx alone increased bl activity in 60% (3/5) of children (no activity before treatment); a higher incidence (75 á/80%) was found in groups 2 and 5. after therapies 3, 4, 6, 7, and 8, bl activity was found in 1 or 2 more children. cx was detected in 36% of faecal samples. conclusions: the probiotics administration seems to protect against dysbiosis caused by cx and to contain the increase in faecal bl activity. the effects differ according to the probiotic administered and are peculiar to certain bacterial species. these preliminary data need further studies. comparative study of initial and acquired drug resistance in pulmonary tuberculosis in iran s37.4 mansoori d, arami s, mirabolhasani z. nritld, infectious disease, tehran, islamic republic of iran purpose: resistant to anti-tuberculosis agents particularly multiple drug resistant (mdr) is a major obstacle in treatment tuberculosis in the world. between september 1996 and march 2000 for 273 smear and culture positive pulmonary tuberculosis patients (old 0/86, new0/ 187) pretreatment susceptibility tests of isolated bacilli to inh, rif, emb and stm were performed by standard proportional method and the results were attributed to three groups: (i) newly diagnosed without any history of treatment; (ii) patients with history of treatment for one course; (iii) patients with history of treatment for two or more courses supposed to be mdr cases. the results were collected for each drug individually and different combinations of two, three and four medications. results: resistance to one, two, three and four drugs was significantly increased in group iii comparing to groups ii and i, also in group ii compared to group i. we observed a high rate of primary resistance to inh and stm in groups i and ii and a high rate of mdr (inh and rif resistance) in groups ii and iii. conclusion: the duration of bacilli exposure to antituberculosis agents in the past is a major factor in developing resistance. in contrast to who's guideline, due to high rate of primary resistance especially to stm in our area, we do not recommend addition of stm for treatment of patients whose initial four-drug regimens have been failed (group ii). donors, to understand host interactions with this bacteria, to develop new methods of diagnosis and define new vaccine candidates. nineteen tb patients and seven healthy donors were enrolled in french hospitals. cellular immune responses were evaluated by lymphoproliferation and ex-vivo quantification of specific th1 cells by elispot-ifn-gamma assays. four recombinant proteins of m. tuberculosis were tested: esat-6, 85b, erp and tb b1.3 and compared with tuberculin. we confirmed that 85b (but not esat-6 in our study) gives higher responses in tb patients compared to donors according to the results in proliferation assay (p 0/0.04). in addition, frequencies of th1 cd4 specific cells for esat-6 and tuberculin were statistical different between the two groups (p0/0.04 and 0.028, respectively). 41.1% for 85b and 70.5% for esat-6 of the patients tested were responders in elispot-assay versus 57.9% for both in proliferation assay. for the new antigens erp and tb b1.3, no difference was observed between the two groups. in conclusion, 85b and esat-6 are recognised by a large number of our patients. they seem to be promising antigens for the development of new methods of diagnosis or for the development of new vaccines. erp and tb b1.3 are not preferentially recognised by tb patients. other exported antigens will be tested. the incidence of tuberculosis (tb) is increasing worldwide. in recent some years, geographical differences in the incidence of tb in former yugoslavia have been observed. an important rise in tb cases was registered in the bordering region of bosnia. it is likely that poorer living conditions, influenced by war and emotional stress, may promote such rising incidence of tb. renal tuberculosis was diagnosed in 25 patients (female 21, male 4) from district brcko in bosnia, during the period of 2 years, 2000 á/2001. at the same time none patient had active pulmonary tb lesions, fibrous lesions were noticed in 18 patients, but we did not diagnose any signs of previous pulmonary tb in seven patients. seven patients developed relapse of renal tb after 1 á/5 years of previous treatment. guided by clinical parameters, precisely done renal echosonography enabled early suspicion and searching for renal tb, by radiological and other methods. bacteriological diagnosis was performed by detection mycobacterium tuberculosis on loewenstein á/jensen medium in 18 patients. pcr as simple, fast and highly sensitive method enabled diagnosis in incipient stadium of disease, so antituberculous therapy could be instituted some months earlier. prompt diagnosis of renal tuberculosis (using pcr, besides standard methods) is necessary, otherwise delayed diagnosis may be dangerous. a study on resistance to first generation anti-tuberculosis drugs in mycobacterium kansasii s37.7 mirsaeidi sm, farnia p, mohammadi f, mansoori sd, jabbari r, taghizadeh r, masjedi mr, velayati aa. national research inistitute of tuberculosis and lung diseases, infectious diseases and immunology (nritld ), tehran, islamic republic of iran purpose: this research has been performed to determine antibiotic resistance of atypical mycobacteria especially mycobacterium kansasi . results: twenty-three pigmented colonies which indicated atypical agents from nritld's mycobacterium culturebank were selected, they then underwent type identification and antibiogram for inh, rif, etb, stm. nine samples were m. kansasi and 14 were other non-mtb, 4 was m. gordonei , 2 m. xenopi , 3 mac, 1 m. bovis , 2 m. tererra , 1 m. asiatioum , 1 m. marinum and 1. mean age in m. kansasi cases was 34'/ 8.1 year and in non-kansasi cases 52'/ 15.6 year and in whole society ntm was 44.9'/16.4. frequency of resistance in kansasi group were 4 to inh (44%), 4 to rif (44%) 4 to etb (44%), 5 to stm (55%) and prevalence of mdr was 4 (44%) and in non-kansasi group frequency of resistance were 13 (92%) to inh, to rif 9 (64%), to etm 9 (64%) and to stm was 10 (71%), to mdr was 9 (64%). conclusion: a significant difference was seen between the age groups of patients who are affected with m. kansasi and non-kansasi (p b/ 0.01), also in frequency of resistance to first generation anti-tb drugs. m. kansasi is detected as the most common atypical mycobacterium agent in pulmonary infections and attention to antibiogram is recommended before treatment. buruli ulcer caused by mycobacterium ulcerans , is the third most common mycobacterial after tuberculosis and leprosy in west africa. nowadays, the only effective treatment is surgery. it consists in a large excision of the lesions, often followed by a skin transplant. in this study, the effectiveness of rifampin, amikacin and their combination were estimated in the treatment of mice, which were infected experimentally by m. ulcerans . after 15 weeks of treatment with rifampin, amikacin or their combination, no more viable bacilli were found in infected tissues. the animals were kept for 3 other months. among the mice treated with rifampin alone, two mice out of 30 relapsed. the minimal inhibitory concentration of these isolated strains went from 0.5 to 8 mg/ml. the dna sequence, obtained from a 93-pb of the rpob gene from these strains, showed a missense mutations, which affect a ser-415 replaced by a phenylalanine. this modification on the gene leads to an important inefficacy of treatment when rifampin was used alone. this study showed that rifampin and amikacin have a bactericidal action on m. ulcerans and that a combination of these antibiotics is necessary to avoid the selection of resistant mutants. histopathologic and electron microscopy studies of a severe isolated hiv enteropathy detected in an aids presenter. favorable response to haart introduction or1.1 manfredi r, calza l, chiodo f. university of bologna, infectious diseases, bologna, italy advanced hiv infection was detected in a heterosexual female with a 1-year history of chronic diarrhoea and severe wasting, as expressed by a body weight of 39 kg, a cd4'/ count of 64 cells/ml, and a plasma viraemia of 2.5 million copies/ml. a malabsorption syndrome was confirmed by d-xylose test, but repeated pathogen search tested negative at stool examination and light microscopy, scanning electron microscopy (em), and transmission em study of enteric mucosa. em assays detected an ultrastructural modification of duodenal mucosa never reported to date: an extensive thinning of enterocytic microvilli, disappearance of glycocalix, and large vacuolization of the enterocyte cytoplasm. two weeks after starting an indinavir-based haart, diarrhoea disappeared and our patient significantly gained body weight: 6 kg after 4 months,15 kg after 8, and 18 kg after 1 year, paralleling a cd4'/ increase to 299 cells/ml, and undetectable hiv viraemia. the subsequent 3-year follow-up confirmed absence of gut disturbances, a stable body weight, a cd4'/ count of 350 á/480 cells/ml, and hiv viraemia persistently b/50 copies/ml. repeated endoscopy and related histopathologic and em assays documented a notable improvement of mucosal damage, with complete cure reached after 2 years of haart. a direct intestinal localization of hiv may be responsible for severe diarrhoea, malabsorption, and wasting, though the morphological features of hiv enteropathy are still unclear. haart acts favourably also against isolated hiv-related enteropathy. kaposi's sarcoma in a non-hiv immunocompetent adult: relapsing due to the development of a squamus cell carcinoma or1.2 sioula e a , magira ee a , georgopoulou c a , rontogiani d b , gounaris t a . a evagelismos, internal medicine and infectious diseases, athens, greece , b evagelismos, pathology, athens, greece a 26-year-old heterosexual hiv negative girl was diagnosed with cutaneous kaposi's sarcoma. the disease was started 2 years earlier with the appearance of lesions on the left feet and on the right knee. absolute number of cd4 and cd8 were 1200 and 486 cells/dl, respectively with a decreased lymphocyte proliferation. human herpesvirus type 8 had been detected in biopsy specimens and she placed on recombinant interferon alpha-2b. follow up few months later the lesions decreased in size. two years after the onset of the disease the patient readmitted because of a mass on the left paratracheal region along with mediastinitis. her body temperature was increased. the patient underwent thoracic ct scan, which demonstrated mediastinal well defined soft tissue infiltration associated with mediastinitis and a well-defined mass in the left paratracheal region. the mass biopsy revealed squamous cell carcinoma. several violaceus lesions were observed on the arms, hands and face. severe bilateral lymphedema of the legs with a reddish papules nodules and tumors from 0.5 to 7 cm in diameter on the soles, toes and calves were present. this case illustrates the significant relapsing of the cutaneous kaposi's sarcoma soon after the appearance of and the carcinoma and the mediastinitis. a 58-year-old male patient with insulin-dependent diabetes underwent cardiac surgery for aortocoronary bypass 2 years ago. two weeks after surgery he developed mediastinitis and sternal osteomyelitis caused by methicillin-resistant staphylococcus aureus (mrsa). twice, revisions and plastic surgery for sternal osteomyelitis were performed. the patient received initially treatment with vancomycin. then the patient received intravenous outpatient treatment with teicoplanin for 6 weeks followed by by treatment with fusidic acid and then trimethoprim/sulfametrol. the fistula was closed. four months later he presented again with substernal pain and purulent discharge. the culture revealed the growth of staphylococci which were first mistaken for coagulase-negative staphylococci. after closer investigation these staphylococci were identified as small variant mrsa. computer tomography (ct) revealed multiple mediastinal abscesses. the patient was treated with intravenous linezolid 600 mg bid for 10 days and then switched to oral linezolid 600 mg bid. the oral therapy was pursued for 16 weeks under close surveillance. the patient improved substantially, the purulent discharge disappeared. the mediastinal abscesses were not detected any longer by ct at the end of treatment. the treatment with linezolid was well tolerated. platelets decreased initially but rose to normal values without treatment modification. nosocomial pneumonia due to stenotrophomonas maltophilia in a profound granulocytopenic patient hospitalized for community-acquired staphylococcus aureus severe sepsis or1.5 radulescu a a , sasca n b , lupse m c , tatulescu d c . a university of medicine and pharmac, epidemiology, cluj-napoca, romania , b the teaching hospital of infectious diseases, laboratory, cluj-napoca, romania , c university of medicine and pharmacy, infectious diseases, cluj-napoca, romania objective: to present the diverse opportunistic infections in immunocompromised patients and treatment difficulties. findings: a 35-year-old women was admitted to the teaching hospital of infectious diseases cluj with a 2-day history of fever, myalgia, lumbar pain, hemorrhage syndrome. severe sepsis was diagnosed and the conditions that evolved in it were paronychia in a patient with chronic leukemia having prolonged and profound granulocytopenia due to aggressive treatment with ifn. the condition at admission was critical due to trombocytopenia ( b/2000 platelets/ml) and hemorrhage syndrome. the evolution was favorable under antimicrobial treatment (imipenem), blood and platelet transfusion, intravenous immunoglobulins, granulocyte colony-stimulating factor, antifungal prophylaxis and supportive care. blood and pus cultures revealed mssa. in the 6th day of hospitalization she developed bronchopneumonia and respiratory failure. the sputum culture was positive for stenotrophomonas maltophilia susceptible to ceftazidime, fluoroquinolones. treatment was unsatisfactory until introducing ticarcilline á/clavulanate and ciprofloxacine. she had uneventful recovery despite remaining granulocyto-trombocytopenic. conclusions: treatment of infections with emerging agents in immunocompromised patients is difficult, guidance by results of susceptibility testing being misleading with a poor correlation between the tests and treatment outcome. early disseminated listeriosis in a liver transplant recipient (ltr): a rare case due to an in vitro multiresistant strain or1.6 manfredi r, de ruvo n, vivarelli m, bellusci r, montalti r, la barba g, abtueli aden a, cucchetti a, attard l, calza l, cavallari a. university of bologna, infectious diseases, bologna, italy a ltr receiving cyclosporin, azathioprine and steroids, developed an extraordinary episode of sepsis and pleural effusion due to a multiresistant listeria monocytogenes (lm) isolate. a lm strain serov. 4 showing the same, extensive resistance pattern (all penicillins and 1stand 2nd-generation cephalosporins), was isolated from multiple blood cultures and pleural fluid 2 weeks after surgery, while stool exam was negative. our p spent her life in countryside and bred some animals, but denied consumption of uncontrolled food. iv cotrimoxazole administration achieved a complete clinical and microbiological cure in 10 days. underlying immunodeficiency may prompt unusual/severe lm infection, but because of its usual community-acquired origin, lm disease remains infrequent in hospitalized p. only seven anecdotal reports of lm infection were described in ltr, 1986 á/2000, all but 1 occurring months á/years after surgery. an early respiratory and systemic infection caused by a community-acquired lm strain which proved resistant to first-choice antibiotics, but had a favorable response to cotrimoxazole (used only once in a ltr with lm sepsis), characterized our episode. an epidemiological survey retrieved the possible source of this usually community-acquired infection. lm should be regarded as an emerging opportunistic pathogen in ltr, and specific risk factors should be seeked. when immunodeficiency is of concern, the unpredictable sensitivity of lm should prompt in vitro assays to adjust antimicrobial therapy problems for discussion hbv á/hcv and liver carcinogenesis: where does the viral influence end? or2.2 pappas ga. university hospital, internal medicine, ioannina, greece hepatocellular carcinoma (hcc) is a major clinical problem worldwide, usually evolving over a long-standing liver pathology, in the latter stages in the form of cirrhosis. hbv and hcv chronic infection is a common etiology of cirrhosis, and hence, hcc. a number of studies have attempted to clarify the role of these viruses into the progression towards hcc. does their end in the stage of cirrhosis? is progression towards hcc independent of the etiology of cirrhosis (since alcoholic cirrhosis also proceeds to hcc)? do the trials with interferon alfa for patients with hbv or hcv cirrhosis exhibit a favorable result due to the antiviral properties of interferon, or is interferon exhibiting anti-oncogenic potential?, and is hcc cytokine and hormone sensitive (view ongoing trials with somatostatine analogues versus hcc)? (hence, if we treat alcoholic cirrhosis patients with interferon could we have a favorable response?). which patients with hbv or hcv cirrhosis are eligible for interferon treatment?: interferon treatment is a potential hazard for those with thrombocytopenia. how ethical is it to conduct a randomised trial where one leg of cirrhotic patients is left without antiviral therapy? and on the basis of which classification system should the two legs of such a trial be separated? moreover, do viral proteins with oncogenic potential exist (the controversy over the recently discovered hbv protein is still, unresolved)? a major topic awaiting for a major debate. to assess the role of hiv-associated campylobacteriosis (c) according to haart availability, 17 patients with positive culture were identified since 1991. compared with the â/1000 hiv-infected p followed in the last decade, no epidemiological differences were shown, save a greater sexual exposure to hiv (p b/0.005). the introduction of haart caused a drop of frequency of c (from 1.8 to 0.9 episodes per 1000 p-year; p b/0.0001), and modified clinical features, with disappearance of dissemination and mortality, reported in 7 and 2 patients before 1996 (p b/0.03). hiv-related immunodeficiency and disease stage were significantly related to c features before and after haart availability: p b/0.0001 for cd4 and neutrophil count, p b/0.007 for aids diagnosis. most cases (15) were community-acquired, but alimentary or environmental risk factors were never found. ten patients received cotrimoxazole prophylaxis (nine before 1996; p b/ 0.03), while no relationship occurred with steroid or antibiotic use, caused 14 cases out of 17. a 100% sensitivity was found to quinolones, followed by cephalosporins (82.3%), gentamicin (76.5%), macrolides (64.7%), and cotrimoxazole (47.1%). a 8 á/18-day antimicrobial therapy cured 16 p , but relapses caused by similar strains occurred in 6 patients within 2 á/8 weeks, all in the pre-haart era (p b/0.05). c still occurs in the haart era, probably due to its varied mode of transmission. the frequency of c is greater in hiv-infected patients, but less frequent visceralization, recurrences, and mortality characterized the haart era. objective: to determine the incidence and risk factors for nosocomial viral respiratory infections (nrvi) and involvement of human coronaviruses (hcov) in a neonatal and pediatric intensive care unit. methods: prospective observational study. nasal samples were obtained by cytological brush at admission and weekly thereafter for all hospitalized infants. nasal samples were taken monthly from staff. virological studies were performed, using immunofluorescence for respiratory syncitial virus (rsv), influenza viruses, paramyxoviruses, and adenoviruses; both immunofluorescence and rt-pcr were used for hcov detection. results: during 1998, 42 hcov related nrvi were detected in 152 nn and six in 92 children. three hcov-related outbreaks were observed (february, august and december), associated with a high prevalence of infection in staff. during august outbreak, 18 hcovinfected nrvi were detected over 23 hospitalized infants. seventy-five of hospitalized preterm nn with gestational age under 32 weeks and 52.4% of staff members were infected. risk factors for nrvi in nn were birth weight, gestational age, ventilation, oxygenation and hospitalization length. ninety-two percent of infected preterm nn were symptomatic, mainly with bradycardia and respiratory worsening. conclusions: these data provide additional evidence for a significant role of hcov in nrvi occurring in hospitalized preterm nn. strain typing and screening of 8 dna targets to assess echinococcus sp transmission in new and old geographic endemic foci or2.5 bart jm a , piarroux r a , dia l b , benchikh-elfegoun mc c , vuitton da a , bardonnet k a . a serf, parasitology, besancon, france , b national centre of veterinary study, parasitology, nouakchott, mauritania , c university of mentouri, parasitology, constantine, algeria purpose: cystic echinococcosis is due to echinococcus granulosus. parasite cycles depending on the main intermediate host species involved in different foci have been described promoting mixed infection in the same definitive host. strain typing is a tool to identify the main intermediate host involved via the dogs in the human infection route and to focus the control measures. many dna targets have been used to compare samples and to access the parasite cycle in different countries. but no study has compared the value of each of these targets. eight targets have been tested in mauritania where echinococcosis is an emergent disease, and in algeria where strain typing has never been done. thirty-five cyst samples from human, ovine, camel and bovine have been tested with six nuclear and two mitochondrial targets. results: the two mitochondrial targets and four out the six nuclear targets have allowed to discriminate the different foci. two strains have been found infectious to human : the 'sheep' strain in algeria and the 'camel' strain in mauritania. conclusion: although overlapping geographically sometimes, this raises the question of the respective genetic evolution of the different strains and of their involving in human infection. alveolar echinococcosis in france: an update or2.6 bardonnet k, bresson-hadni s, bartholomot b, gérard a, watelet j, beytout j, saurin jc, piarroux r, vuitton da, who centre collaborating for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the highest prevalence rate for alveolar echinococcosis (ae) in europe has been found in france. in 1997, a french observatory of human ae was done in order to get data that could be used to evaluate presentation, evolution and management of ae. material-methods: french cases were collected for the period 1982 á/ 2002. registration of every case was performed with the subject's agreement. a questionnaire was filled in by referring to the patients' medical files or to practitioners or to patients themselves. completeness of the collection of cases was ensured by multiplying the sources of information. results: two hundred and sixty nine french patients were registered. sex ratio averaged 1. mean age at diagnostic was 54.8 years. 12.5% of diagnosis was performed in 'echinococcosis free' french areas. symptoms, but not always specific liver symptoms, were present at diagnosis in 76.4% of cases. the liver was the main location of lesions in 93.6% of cases. a wide spectrum of management of the patients was observed, accounting for regional differences. conclusion: this french observatory of human ea will facilitate a better management of the disease at the national level. it shows new epidemiological trends, and especially an extension of the endemic area. can coins and paper currency transmit bacillus anthracis ? or2.7 ghenghesh ks. faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya anthrax is an often fatal bacterial infection caused by bacillus anthracis . recent events that began in september 2001 in us has gained the organism worldwide attention and heightened awareness of and concern about anthrax. many cases of anthrax with a number of deaths have been reported as a result of contact with envelopes, sent through postal mail, containing b. anthracis endospores. a number of studies have shown that currency is colonized with bacterial organisms, that include enteropathogens (e.g. shigella sp.), other enteric flora (e.g. escherichia coli ) and potential pathogens (e.g. staphylcoccus sp. , pseudomonas sp. and bacillus sp.). furthermore, methicillin-resistant s. aureus (mrsa) isolates that produced enterotoxin (seb) and toxic shock syndrome toxin-1 also been reported. all of these studies do agree on that currency may be considered as a method of spreading potentially pathogenic and pathogenic bacteria in the community. therefore, currency could also be a vehicle for spreading other highly pathogenic organisms that include b. anthracis . in addition, the introduction of the 'euro' could also allow such bacteria greater freedom to travel across the euro zone. the threat of using currency, particularly paper notes, in spreading lethal organisms should be investigated and proper measures to prevent the use of such a method by terrorists should be implemented. salvage of temporary femoral catheters for haemodialysis using antibiotics in ambulatory patients or2.8 gerasimovska v, oncevski a, dejanov p. department of nephrology, clinical centre, skopje, the former yugoslav republic, macedonia the stay of femoral catheters (fc) for haemodialysis is typically short-term for several days. we used fc as a temporary vascular access (va) for a longer period of time in outpatients going on regular ambulatory haemodialysis, who had a problem with their permanent access. we analysed 43 patients who were discharged from hosptal with fc. duration time of fc was between 13 and 183 days (average 44.2 days) with cummulative total of 1989 days. the incidence of bacteriaemia was 2.51 episodes/1000 catheter days. in six patients we had signs of infection, so according to our protocol we took blood cultures from peripheral vein, and from catheter (at same time) and started with antibiotic therapy (ab) systemically and locally (ab was 'locked' in catheter) with different duration of time. dominant microorganism was staphylococcus coagulasa negative, and much less staphylococcus aureus , and enterococcus.ab that were frequently used were: cefotaxim, vancomycin and ciprofloxacin. at one of six patients we removed catheter at once without trying to save the catheter. catheter tip was sent for microbiological analysis too. criteria for catheter-related bacteriemia (crb) was found in only one patient, and for possible crb in five patients after we removed the catheters. in the absence of clinical signs of infection, ab treatment was not provided for positive tip culture alone or for positive blood culture of the catheter with negative blood culture from peripheral vein. advances in meningitis education or2.9 holt de a , tait mi b , cavanna al b , worgan-brown s a , hart b a . a the meningitis trust, stroud, uk , b the computer-aided learning unit, school of health science, university of wales, swansea, uk background: meningitis remains an important cause of death worldwide despite improvements in diagnosis, treatment and prevention. clinical and lay awareness of the disease relies on education, however educational delivery has changed and the introduction of material suitable for computer and internet application is now necessary. we have developed educational material on cdrom and on the internet applicable both at tertiary university and secondary school level. application: a computer-aided learning program on cdrom, covering all aspects of meningitis has been produced. it is suitable for undergraduate teaching of healthcare professionals from student nurse and doctors to pharmacists. in order to reach school children in a form acceptable to both pupils and teachers, we have developed a curriculum-linked website. these applications are simple to use and can be incorporated into existing courses of study, so that issues raised can be discussed with tutors and group peers. comment: the introduction of new methods of teaching and learning mean that compatible educational material must be produced. we believe that these applications, focusing on meningitis, are the first of their kind and that they offer tutors the opportunity to progress their teaching of the disease both in methodology and content. brivudin compared to famciclovir for improved therapy of herpes zoster: effects on acute disease and postherpetic neuralgia or3. potentially treatment-related adverse events occurred in 11.8% of the brivudin recipients and in 10.1% of the famciclovir recipients (p 0/ 0.26). conclusions: in zoster patients ]/50 years, brivudin 1)/125 mg and famciclovir 3)/250 mg showed equivalent effects on prevalence and duration of phn. brivudin is as effective as famciclovir in stopping viral replication in acute herpes zoster. brivudin offers the advantage of a once daily dosage regimen while being as well tolerated as famciclovir. activity of complexes of pt(ii) and pd(ii) with pyridine-2-carbaldehyde thiosemicarbazone (hfotsc) (acta virol., 2001, 45, 87) with selectivity index (si) 1.5 times higher than that of acyclovir (acv). in order to evaluate virus specific response and structure á/activity relationships we continue our investigations with three pt(ii) and three pd(ii) complexes. the activity was evaluated against sensitive to acv hsv 2 (strain bja) and resistant strains r-100 (hsv 1) and pu (hsv 2) and compared to that obtained against strain victoria (hsv 1) infection. si was indicative for activity. the virus specific response was demonstrated by the fact that viruses sensitive to acv were also sensitive to pt(hfotsc) 2 ]cl 2 , while acv resistant viruses were sensitive to [ptcl(fotsc) ]. the structure á/activity relationship was proved by the fact that the less active against hsv infection was [pd(fotsc)]. influenza diagnosis, treatment, and the impact of new antivirals on current treatment behaviours during influenza outbreaks or3.3 schaetz l a , sessa a b , a hoffman-la roche f. basel, switzerland , b italian college of general practitioners, italy introduction: annual influenza epidemics severely affect individuals, families, health care systems and society. the availability of new and specific antivirals provides an opportunity for better management of influenza. methods: during the 1999/2000 and 2000/2001 influenza seasons, physicians ( â/100/country) and public ( â/1000/country) in the usa and europe were interviewed to determine perceptions of influenza and behaviours for its treatment. results: patients recognise influenza illness as severe and identify it by symptoms of fever, muscle aches/pains and cough. physicians use these symptoms to diagnose influenza clinically (93% fever, 78% muscle aches/pains, 47% cough); their main treatment objective being to reduce complications. antibiotics for influenza treatment are broadly recommended/prescribed by about 30% of european physicians, whereas currently available antivirals are only recommended by 10%. the recommendation of antivirals by us physicians increased from 47% (season 99/00) to 62% (00/01) and markedly decreased antibiotic use (from 25 to 11%). experience from the two influenza seasons shows that influenza antivirals are only used while the virus is circulating and that the volume of use is proportional to the size of the outbreaks. conclusions: experiences in the usa show that with prompt outbreak information antivirals can be used appropriately in times of influenza activity. influenza treatment with oseltamivir: costs and benefits for the individual as well as for society or3.4 objective: to evaluate the effects of treatment of influenza with antivirals (oseltamivir) on health outcomes and costs to patients and society. methods: based on clinical trial data and data from the literature a simulation model has been developed. the underlying clinical pathway covers morbidity and mortality due to influenza and its specified complications. health outcome data and costs were attached to events in the model. the model compares various scenarios, which are defined by treatment schemes within defined populations and other parameters. application of the model is shown using uk unit cost data simulating an otherwise healthy adult population comparing oseltamivir with usual care. results: early treatment results in reduced morbidity, which translates into faster recovery and return to normal activities (1.93 days). lower morbidity and mortality make this a cost-effective intervention from a societal perspective. the analysis covers more than 10 different scenarios and the incremental cost effectiveness ratios will be discussed. conclusion: antiviral treatment appears to be effective in terms of health outcome and cost for otherwise healthy adults from the perspectives of both the individual patient and society. however, this effect is very sensitive to time when treatment is started and the accuracy of the diagnosis of influenza. oseltamivir is well tolerated by all patient groups or3.5 thakar b a , dutkowski r b , froelich e c , gilbride j a , ward p a . a roche global development, welwyn, uk , b f hoffman-la roche, nutley, usa , c f hoffman-la roche, basel, switzerland introduction: oral oseltamivir, the ethyl ester pro-drug of a potent inhibitor of influenza virus neuraminidase, is licensed for the treatment and prophylaxis of influenza in the usa. patients and methods: safety data [adverse events, laboratory safety evaluations] derived from clinical trials involving !/12 000 subjects (including â/1000 children and â/1200 high-risk adults) and 400 healthy volunteers in a large study investigating ecg parameters. spontaneous event reports from medwatch or yellow-card reports following use by â/2 000 000 individuals worldwide. an observational case-control study of !/10 000 subjects with influenza-like illness treated with oseltamivir. results: oseltamivir was well tolerated in clinical trials; drug-related side-effects were limited to transient gi effects occurring in 5/1:10 exposed individuals. these resolved spontaneously and caused drop out in b/1% of treated subjects. no effects on ecg parameters were noted at doses ]/sixfold above the licensed regimen. oseltamivir had no adverse effects on pulmonary function. no additional effects were identified among high-risk adults or children, or following prolonged dosing for prophylaxis. occasional reports of liver dysfunction have been documented post-marketing but causal association has not been established. conclusions: oral oseltamivir is an effective and safe antiviral suitable for influenza management in all patient groups. the decision to stop the vaccination against smallpox and the loss of specific immunity of a high proportion of the population made apocalyptic the perspective of a natural or provoked re-emergence of smallpox. therefore, it is important to improve the current capacities to prevent or to treat the orthopoxvirus infections. uracil dna glycosylase (udg) is one viral enzyme indispensable to the replication of poxviruses. udg of the copenhagen strain of vaccinia virus (vv) was characterized with the aim of defining specific inhibitors susceptible to be used as a new class of active antiviral substances on the viruses of the orthopoxvirus genus. the activity of this enzyme was analysed in real time, in an original method, on a pcr quantitative instrument by digestion of amplified dna revealed by fluorescent intercaled molecules. this technique was used to screen and select several active antiviral substances on udg. moreover, the antiviral activity was estimated by the cytopathic effect of the vv on infected vero cells. the cytotoxicity was determined by inhibition of trypan blue exclusion. the specificity of action of each tested compound was estimated by the selective index (50% cytotoxic dose/50% effective dose). two antiviral compounds were selected for their inhibitory effect on udg activity and on vv replication in vero cell culture : ('/)-5-iodo-2?-deoxyuridine and 4-chlorouracil. these compounds are candidates for the chemotherapy of poxvirus infections. objective: to study the efficacy and tolerance of russian antiviral drugs produced from dna in a limited resources context. results: the drug derinat was produced from salmons' milt. mm of dna was 270 á/500 kda, hyperchrome effect !/37%, protein content b/0.5%. the conjugation of the dna with fe3'/ resulted in a new drug named ferrovir which influences dna and rna synthesis during early stages of hiv-1 replication by blocking the virus's action on cells' metabolism and reduces cytomegalovirus titre in fibroblast cells for 1.5 á/2.0 ig tcid50. a protective effect of ferrovir against fatal herpes encephalitis mice was found. the drugs are not toxic. ic50!/ 4000 mg/ml. ec90 of ferrovir against hiv-1 was 800 mg/ml. in limited clinical trials patients received 75 mg of drugs twice daily (7 á/14 days). administration was well tolerated and no side effects were observed. derinat in 88.1% cases of herpesvirus infection (42 patients) improved the healing and shortened duration of illness. hiv-infected patients (34) treated with ferrovir showed sustained, elevated cd4'/ counts and a significant reduction in hiv-1 viral load (median 1.8 ig). the apparent remission was found in patients with concomitant hiv and herpes virus infection. conclusions: antivirals show good antiviral potency against rnaand dna-viruses; are well tolerated by patients and are useful in case of mixed infections; low price makes them accessible to populations with low financial resources. ortho total hcv core antigen assay can aid early prediction of response in patients treated with interferon/ribavirin or3.8 lunel f a , veillon p b , payan c b . a ahu angers, laboratoire de bacterio-virologie, angers, france , b chu angers, laboratoire de bacterio-virologie, angers, france aim: evaluate the predictive value of total hcv core antigen assay and viral kinetics in patients with chronic hcv. methods: one hundred and twenty two patients infected by genotype 1, 4, 5 or pretreatment viral load (bdna 2.0, chiron) !/3 meq/ml, with no previous treatment, received 6 mu interferon (ifn) during 12 months (m). ribavirin was given with ifn after 3 months therapy, for 9 months in patients with detectable rna. viral load was expressed as log (ui/ml) and hcv ag as log (pg/ml)/10 000). results: pretreatment ag values were correlated with viral load (r 2 0/0.779). we observed a rapid decrease of ag (5.2 log pg/ml) and viral load (5.1 log ui/ml) after m1 in sustained responders (sr). in patients who relapsed (rr) after ifn alone, the fall was less important (2.6 log pg/ml, 3.6 log ui/ml) during m1. in sr and rr to combination therapy, the decrease of ag and viral load at m1 was, respectively, (ag: 1.2 and 1.4 log pg/ml; rna: 2.4 and 1.5 log ui/ml). we did not observed significant variation of ag and viral load in nonresponders. the negative predictive value of hcv rna and ag after m1 of treatment were 100%, and positive predictive values were 81 and 82%. after 1 month of ifn alone, the hcv ag decrease was highly predictive of sr, correlated with rna negativation and early reduction of hcv rna ( !/2 log). conclusion: early measurements of total hcv core antigen are useful to predict long-term response to treatment. lamivudine in the treatment of acute hepatitis b or3.9 vincenti a, meini m, luchi s, de gennaro m, ricciardi l, moneta s, scasso a. infectious diseases department, infectious diseases, lucca, italy acute hepatitis b is a self-limiting infection, but in some cases its course may be particularly severe. we report a case of a 77-years-old woman affected by acute hepatitis b treated with lamivudine. on admission in the hospital the alanino-aminotransferase was1060 u/l, the aspartate-aminotransferase 1017 u/l, bilirubin 10,2 mg/dl, hbsag, hbcigm and hbeag were positive, hbv dna was 300.000 copies/ml. during the following days, the levels of ast and alt gradually rose; on the 14th day prothrombine time was 39%, bilirubin 30 mg/dl and the patient developed signs of encephalopathy. four plasmapheresis were practiced without benefit, so the patient was treated with lamivudine, 100 mg/day. after 24 days of therapy, lamivudine was discontinued because of the appearance of diffuse maculopapular rash. at this time the results of liver function tests were normal; after four months hbsag and hbv dna were no longer detectable. in our patient lamivudine prevented an acute hepatic failure. our experience suggests a promising role of lamivudine in the treatment of acute hepatitis b, but how long such therapy have to be practiced and in which patients? prospective, controlled, clinical studies using lamivudine in patients with acute-hepatitis b are necessary. the cost-effectiveness of amantadine versus symptomatic care in the treatment of influenza or3.10 morris s a , carman wf b , barber j c . a city university, london, uk , b west of scotland specialist virology centre, glasgow, uk , c alliance pharmaceuticals, chippenham, uk aim: to assess the cost-effectiveness of amantadine versus best symptomatic care in the treatment of influenza in the uk. methods: we constructed an economic model populated with parameters from the published literature. the model structure is the same as that used in the economic evaluation of zanamivir published by the national institute for clinical excellence in the uk. we conducted a cost-utility analysis (incremental cost per qaly gained) of amantadine versus best symptomatic care. the analyses are conducted for all adults (average-risk group) and the at-risk population (high-risk group), based on the prevalence of influenza over an average season and when the virus is circulating. the perspective is that of the nhs. results: in the average-risk group the incremental cost per qaly gained of amantadine relative to best symptomatic care is uk£30,488 during an average influenza season and uk£12,538 when the virus is circulating. for high-risk individuals the figures are uk£35,278 and uk£14,526, respectively. the results are sensitive to the hospitalisation rate. conclusions : if the threshold for cost-effectiveness is £20,000 per qaly gained amantadine represents value for money in the treatment of influenza in a variety of scenarios, including the baseline for both average-risk and high-risk groups when the virus is circulating. background: surveillance studies all over the world have revealed an extraordinary increase in the prevalence of penicillin resistant streptococcus pneumoniae . the newer quinolones are believed to have broad activity against s. pneumoniae . methods: a total of 87 penicillin resistant clinical strains isolated from patients at hacettepe children's hospital, ankara, turkey between 1999 and 2001 were tested for their in vitro susceptibility to various antibiotics that are commonly used in the treatment of respiratory tract infections. the minimum inhibitory concentrations (mics) of the penicillin, amoxicillin/clavulanic acid, doxycycline, azithromycin, clarithromycin, ceftriaxone, ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin were determined using the nccls recommended procedure for e -test. results: the range of mics, mic50 and mic90 values for all agents tested against the strains are shown in the table. gemifloxacin and moxifloxacin had the highest in-vitro activity among the quinolones tested. all strains tested were susceptible to b/0.2 mg/ml gemifloxacin, b/1 mg/ml moxifloxacin and 2 mg/ml levofloxacin. conclusions: there is some degree of resistance to all the drugs except the newer quinolones which were active against all isolates studied. purpose: stenotrophomonas maltophilia prevalence is growing, mainly in some hospital areas. s. maltophilia is frequently multidrug resistant. fluoroquinolone (fq) resistance varies from one to another study, but in whole resistance is moderate to high. gyra and parc qrdr partial codes have been recently described. we have studied correlations between fq-resistance and mutations in these sequences in s. maltophilia clinical strains. material and methods: gyra and parc qrdr regions from six fqresistant and two fq-susceptible s. maltophilia clinical strains were amplified and sequenced. mics of ciprofloxacin (cfx), gatifloxacin (gfx) and clinafloxacin (cnfx) were determined by the agar dilution method, according guidelines defined by nccls for p. aeruginosa . results and conclusions: mics ranges of cfx, gfx and cnfx for resistant strains were 8 á/32, 1 á/32 and 0.5 á/8 mg/l. susceptible strains had mics of cfx, gfx and cnfx of 1, 0.2 and 0.06 á/0.1 mg/l, respectively. most susceptible and resistant strains had no significant mutations in the fragments sequenced. only one resistant strain (mic of cfx 16 mg/l) and one susceptible strain (mic of cfx1 mg/l) had a significant gyra mutation, the same in both strains (ile112 á/val). thus, fq resistance in s. maltophilia shall derive from changes in other areas in the topoisomerases or probably from other mechanisms of resistance, such as efflux pumps. purpose: corynebacterium urealyticum is the cause of encrusted cystitis and other inespecific utis and systemic infections. it is frequently multi-drug resistant, with a high rate of resistance to fluoroquinolones (fq). the mechanisms of resistance to fqs have not been described in c. urealyticum . we describe the c. urealyticum parc gene qrdr region and its relationship with quinolone resistance. materials and methods: the activity of ciprofloxacin (cfx), levofloxacin (lfx), gatifloxacin (gfx), clinafloxacin (cnfx) and moxifloxacin (mfx) against 30 c. urealyticum clinical strains was determined following nccls guidelines for enterococci. we amplified and sequenced their parc qrdr by standard methods. results and conclusions: five strains (16.6%) were cfx-susceptible (mic 0.1 á/0.5 mg/l), 5 had mics 1 á/2 mg/l and 20 (66.7%) were highlevel cfx-resistant (mic 32 á/128 mg/l). cnfx was 64-fold more active than cfx. mfx and gfx had mics90 of 4 and 8 mg/l. all the strains, including the type strain, showed a c to t change at the 2614 position referred to wild type s. aureus parc gene, leading to a ser-80-phe change, described as the main parc change in fq-resistant s. aureus . this finding suggest that this mutant sequence, as compared with parc sequences from other grampositives, might be the wild-type for this species, and might explain in part its high resistance rate, and its apparent lightness to develop high-level resistance. purpose: during routine surveillance, we identified 32 ciprofloxacinresistant (mic!/8 mg/l) pneumococcal isolates and compared clinical details and resistance patterns. results: they were isolated from sputa (29) and blood cultures (3) from adults, most with heart or lung disease. hospital admissions were common; half had been inpatients in the previous 3 months. nineteen patients received quinolones in the preceding 3 months, in part reflecting the local policy (introduced in 1997) of penicillin and ofloxacin for first line treatment of pneumonia. thirteen patients had radiological signs of pneumonia and 10 were pyrexial with raised inflammatory markers. agar dilution mics for quinolones, including norfloxacin with and without reserpine, penicillin and erythromycin were performed. an increase in norfloxacin mics was noted over the period 1997 (16 á/64 mg/l) to 2001 (256 mg/l). fluoroquinolone efflux was suggested in three isolates. resistance to moxifloxacin (mic 8 á/16 mg/l) was noted from 1998 onwards. all isolates were serotype 9v and resistant to penicillin (mic!/0.5 mg/l). thirty-one were resistant to erythromycin (mic!/1 mg/l). conclusion: the 1997 policy of using quinolones may have contributed to the development of quinolone resistance and this cluster of isolates. the increasing levels of quinolone resistance observed raise concerns about the future use of newer quinolones for the treatment of respiratory infections. s. maltophilia has emerged in the last years as an important nosocomial pathogen, inherently resistant to most of the antimicrobial agents. new quinolones has been proposed as a treatment of choice because their enhanced activity, but several parameters (t a , atmosphere, method) can affect the results of mics. methods: we have performed mics using two different methods (agar dilution and microdilution) and different conditions: 35 and 30 8c of temperature; atmosphere of o 2 and co 2 , and incubation times of 18, 24 and 48 h. a total of 78 strains were assayed with nine quinolones following standard nccls. comparisons were made between results with 18 á/24 and 24 á/48 h using the x 2 -test (a 0/0.05). results: no differences were found between 18 á/24 and 24 á/48 h results with agar dilution, except with 3 atbs in the case of mics at 30 8c co 2 . on the contrary, almost all the atbs showed significant differences in the results with 24 and 48 h using microdilution method, at any condition of ta or atmosphere. comparison of mics (p values, significance level 0/95%) with incubation times of 24 and 48 h at different procedure conditions. the incubation time is a parameter that seems to affect significantly the results of mics of quinolones when microdilution method is used, whereas only few differences can be encountered with the agar dilution method. results: breakpoints were used as proposed by nccls 1999. during the study period the pneumococci resistance was noted as follows: 65% to pc, 39% to em and 73% to sxt. the rank order of activity of the five fqs against multi-drug resistant pneumococci was: cip (mic 90:2 mg/l), ofx (mic 90:2 mg/l), lvx (mic 90:1 mg/l), grx (mic 90:0.25 mg/l), tvx (mic 90:0.25 mg/l). conclusions: in romania, fluoroquinolones represent alternative treatment to beta-lactams and macrolides for first-line empirical treatment for respiratory tract infections caused by pneumococci but, continued vigilance for emerging resistance to fqs is further indicated. introduction and material/methods: susceptibility testing (semiautomated broth microdilution method, sensititre, trek diagnostics, usa, following nccls recommendations) was performed with six different quinolones to 817 streptococcus pneumoniae isolates with a ciprofloxacin-cip */mic ]/2 mg/l collected in two consecutive sauce$ surveillances in spain (1996 á/97/1998 á/99). nccls resistance (r) breakpoints were used ( ]/8 for ofloxacin-ofl and levofloxacin-lev; ]/2 for sparfloxacin-spa; ]/4 for gatifloxacin-gat */and moxifloxacin-mox), but for gemifloxacin-gem */where ]/1 was used. results were as follows. conclusions: for cip-r isolates gem and mox were the most active agents. gem was the only agent not influenced by cip mic increase regarding prevalence of r , with 0% resistance for strains with cip mic ]/8 mg/l. $sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en españ a' (susceptibility to the antimicrobials commonly used in the community in spain) and is the spanish word for the willow tree. in vitro activity of gatifloxacin and seven other antibiotics against respiratory and urinary tract pathogens from the community. first results of the basic */study ps110 grimm h, on behalf of a european multicenter study group, institute for med. microbiology, 88250 weingarten, germany a total of 24 centers in austria, belgium, france, germany, italy, portugal, spain and switzerland are involved in the basic study (bacterial annual susceptibility information collection). the mics of gatifloxacin (gati), ciprofloxacin (cipro), clarithromycin (clari), benzylpenicillin g (pen), amoxicillin (amox), amoxicillin/clavulanic acid (augm), cefuroxime (cur) and cefixime (cix) were determined using the microdilution method. each center is requested to investigate 30 strains each of the following species: s. pneumoniae (spn), s. pyogenes (spy), s. aureus (sau), e. faecalis (efa), m. catarrhalis (mca), h. influenzae (hin), e. coli (eco), k. pneumoniae (kpn), p. mirabilis (pmi) and p. aeruginosa (pae). so far approximately 1400 strains are enrolled. some important mic90%/percentage resistance were as follows: from the oral antibiotics tested gatifloxacin has the highest activity and broadest spectrum against all relevant respiratory and urinary tract pathogens. gatifloxacin is a promising alternative for therapy of respiratory tract bacterial infections. in vitro activity of gatifloxacin against bordetella pertussis in comparison with erythromycin, ciprofloxacin and levofloxacin ps111 bourgeois n, pangon b, ghnassia jc, doucet-populaire f, de versailles ch. microbiologie, le chesnay, france purpose of the study: bordetella pertussis infections are far more common in adults and adolescents than is generally estimated. however, they are often not recognised. infected or colonised adults can act as a reservoir of infection, passing it to children. fluoroquinolones are currently recommended for the treatment of respiratory tract infection in adult patients, which is usually empirical. gatifloxacin is a novel 8-methoxyquinolone, with a potent activity against both gram-negative and -positive bacteria. the in vitro activity of gatifloxacin was compared with those of erythromycin, the drug of choice for both treatment and prophylaxis of pertussis, ciprofloxacin and levofloxacin, against 52 clinical isolates strains of b. pertussis including 12 erythromycin resistant strains. results: we used the agar dilution method on mueller á/hinton medium supplemented with 10% horse blood to determine the mic of each antibiotic. gatifloxacin (mic90, 0.125mg/l) was as active as ciprofloxacin and levofloxacin (mic90, 0.06mg/l) against both sensitive erythromycin (mic90, 0.015 mg/l) and resistant erythromycin (mic90, !/512 mg/l) strains. conclusion: gatifloxacin may be an effective drug in the treatment or prophylaxis of adults with suspected or confirmed pertussis. ex vivo serum activity (killing rates) after gemifloxacin 320 mg versus trovafloxacin 200 mg single doses against ciprofloxacin-susceptible andresistant streptococcus pneumoniae ps112 calvo a a , giménez mj b , alou l a , gó mez-lus ml a , aguilar l b , prieto j a . a microbiology department, universidad complutense, madrid, spain , b glaxosmithkline, medical department, tres cantos, madrid, spain serum bactericidal activity was measured ex vivo after single dose administration of gemifloxacin (gem) 320 mg and trovafloxacin (tro) 200 mg to 12 healthy volunteers in a randomized, cross-over phase i trial. blood samples were collected 1 h (cmax) after dosing and serum killing rates were determined against a serotype 3 penicillin (pen) á/ciprofloxacin (cip) susceptible strain (s3) (mics of 0.03, 1, 0.015 and 0.06 mg/l for pen, cip, gem and tro) and a serotype 9 pen á/cip resistant strain (s9) (mics of 2, 4, 0.03 and 0.25 mg/l for pen, cip, gem and tro). tubes with 1.6 ml of serum sample and 0.4 ml broth (50% todd á/hewitt'/50% hbss) were incubated over 3 h at 35 8c. final inocula was 10 7 cfu/ml. mean colony counts for samples and controls (k ) are shown in the figure: gem exhibited higher colony counting decrease of the initial inocula, versus tro, for both strains. after 3 h incubation, the initial inocula decrease obtained with tro and the cip susceptible strain was similar to that obtained with gem and the resistant strain, showing a lower influence of cip mic increase in the ex vivo bactericidal activity of gem versus tro. urine bactericidal activity after administration of gemifloxacin and trovafloxacin single doses in a phase i study ps113 garcía-calvo g a , parra a a , giménez mj b , ponte c a , aguilar l b , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain urine bactericidal activity after o.d. administration of gemifloxacin (gem) 320 mg and trovafloxacin (tro) 200 mg, was assessed in six adult males in a cross-over phase i trial. urine killing rates (ukr) against escherichia coli atcc 25922 (mic mg/l of 0.008 and 0.015 for gem and tro) and s. saprophyticus atcc 1970 (mic mg/l 0.015 and 0.06 for gem and tro) were performed with samples collected at 0, 2 á/4, 16 á/24 and 60 á/72 h. a 1.6 ml of iso-sensitest broth and 0.4 ml of bacterial logarithmic growth were added to 2 ml sample, giving a final inoculum of 107 cfu/ml. colony counting was performed after 1, 2, 3 and 4 h incubation. percentages of initial inocula reduction (iir) were calculated. mean urine concentrations measured by bioassay were (mg/l): 28.4, 14.7 and 0.5 for gem, and 3.6, 3.0 and 0.1 for tro. against e. coli , an iir of 99.9% was obtained after 2 h incubation with all samples except with tro at 60 á/72 h. against s. saprophyticus an iir of 90% was obtained after 3 h incubation with all samples except with tro at 60 á/72 h, where bacterial regrowth was found. the maintenance over 72 h of gem urine antibacterial activity suggests its efficacy in the treatment of uncomplicated cystitis. influence of the decreased susceptibility to ciprofloxacin on gemifloxacin versus levofloxacin efficacy in experimental pneumococcal pneumonia in guinea pigs ps114 garcia-olmos m a , parra a a , gimenez mj b , garcia-calvo g a , ponte c a , aguilar l b , soriano f a . a fundacion jimenez diaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain the efficacy of ciprofloxacin (cip), levofloxacin (lev) and gemifloxacin (gem) in the treatment of pneumococcal pneumonia was assessed in a guinea pig model using three strains (s) with mics (mg/l) of 2, 2 and 0.03 (s1), 32, 4 and 0.25 (s2) and 64, 32 and 1 (s3) for cip, lev and gem, respectively. intraperitoneal treatments started 1 h after s. pneumoniae intratracheal inoculation, and continued t.i.d up to four doses. ten animals were included in each group. doses (mg/kg) used were 20, 16 and 6 for cip, lev and gem, respectively, in order to mimic auc0-24 h and cmax obtained in humans after standard doses. animals that survived 48 h after inoculation were sacrificed and colony counts were performed in lungs ( the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of nosocomial pneumonia. our aim was to study the pharmacokinetic á/pharmacodynamic appropriateness of lfx 500mg iv bid in the treatment of six inpatients with ventilator-associated pneumonia (vap) (459/25 years; 4m á/2f; 749/9 kg). blood and urine samples were collected in steadystate conditions at appropriate intervals. lfx concentrations were analysed by hplc. the aetiological agent was identified in all the cases and its in vitro sensitivity to lfx was always assessed. the results obtained mean values (9/sd) of the major pharmacokinetic parameters were: cmax, 8.789/2.05 mg/ml; vdss, 1.259/0.36 l/kg; t1/2b, 4.699/0.87 h; cl, 3.659/1.01 ml/min/kg; auc0-t, 32.479/12.80 mg/ml h. cumulative urinary excretion was 82.899/10.23%, confirming that lfx clearance is mainly renal. clinical cure and microbiological eradication were obtained in all the patients after a 7 á/13 day therapy. a suprainfection due to acinetobacter anitratus insensitive to lfx occured in 1 case. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/10; auc/mic !/125) in all the cases. the conclusion reached the findings suggest that lfx 500 mg bid iv may be considered effective in the treatment of vap caused by sensitive bacteria. comparative pharmacokinetics of levofloxacin in patients with lower respiratory tract infections (lrti) being treated with sequential therapy ps116 pea f a , brollo l a , lugatti e b , di qual e a , dolcet f b , talmassons g b , furlanut m a . a institute of clinical pharmacology and toxicology, dpmsc, university of udine, udine, italy , b division of pneumology, sm misericordia hospital, udine, italy the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of lrti. our aim was to study the pharmacokinetic á/pharmacodynamic appropriateness of a standard switch lfx iv/os regimen (500 mg iv od for 5 á/7 days followed by 500 mg os od for 4 á/10 days) in the treatment of seven inpatients with lrti (709/17 years; 6m á/1f; 769/15 kg). blood samples were collected in steady-state conditions at appropriate intervals. lfx plasma concentrations were analysed by hplc. the aetiological agent was identified in 2/7 cases and its in vitro sensitivity to lfx was assessed. the results obtained absolute oral bioavailability was 1.079/0.19, with a cmax of 10.899/3.39 vs 8.369/3.90 mg/ml after iv and oral administration, respectively. no significant difference in the main pharmacokinetic parameters was observed between the two routes. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/10; auc/mic !/125) in the two assessable cases. all the patients were clinically cured after a 9 á/15 day therapy. the conclusion reached the ad interim findings show that lfx 500 mg od may guarantee per os an exposure similar to that achievable after iv administration, suggesting that sequential therapy may be considered effective in the treatment of lrti. levofloxacine in the exacerbations of copd due to pseudomonas ae ps117 micheletto c, tognella s, pomari c, dal negro r, ospedale orlandi, div.pneumologia, bussolengo, italy development of antibiotic resistance in bacteria is a problem of great concern. gram-negative bacteria, including multidrug-resistant (mdr) pseudomonas aeruginosa (ps), are responsible for a significant proportion of episodes of copd exacerbations, particularly in elderly (1). aim was to check the susceptibility to common antimicrobial treatments against ps strains isolated from bronchial secretions in patients with severe exacerbations of copd. methods: microbial investigations were conducted on 290 specimens: spontaneous purulent sputum (88.4%), and tracheobronchial aspirates (11.6%, collected with a protected specimen brush). results: fifty-seven ps pathogen strains (106 cfu) were identified (19.6%) and tested over a 6-month period: ps. aeruginosa 91.2%; ps. putida 3.5%; ps. fluorescens 3.5%, and burkholderia cepacia 1.8%. the assessed susceptibility to most common antibiotics was: levofloxacine (90%), ciprofloxacine (84%), ipenem cil. (88%), ceftazidime (84%); amikacin (84%), and piperacillin'/tazobactam (74%). a much lower susceptibility was found for ticarcillin á/clavulanic acid (58%), gentamicin (48%), and netilmicin (40%). (1) at present, levofloxacine proves the most effective antimicrobic option for treating copd exacerbations due to ps infection; (2) a much more efficient policy of antibiotic prescribing should be promoted in order to prevent the selection of resistant strains in these cases. these results confirm the excellent 'in vitro' activity of levofloxacin against nosocomial gram-negative pathogens, including the esbl producing strains (90% of escherichia coli , e. cloacae and k. pneumoniae were inhibited at 0.5 mg/l). levofloxacin was more rapid than ciprofloxacin to determine a bactericidal effect particularly against s. maltophilia . moreover, considering the favourable pk/pd profile, levofloxacin can represent a valid therapeutic option for the treatment of severe gram-negative nosocomial infections. moretti f, quiros-roldan e, casari s, chiodera a, viale p, carosi g. university of brescia, institute of infectious and tropical diseases, brescia, italy a 34-year-old man, ivdu, hiv positive was attended in aur hospital for fever and toracic pain. a x-chest radiography revealed a round lesion of 5 cm near the lingula with central hyper-diaphan area. lymphocytes cd4'/ count was 21 cells/mm 3 and viral load 91.900 cp/ ml. hospital stay rhodococcus equi was found in cultures of peripheral blood, faecal and sputum specimens. antibiotic treatment with oral rifampin (600 mg/qd) and with intravenous imipenem (500 mg tid) was started. due to the persisting fever, immodificated radiography and negativity for p. carinii , mycobacteria and bacteria in bal coltures, imipenem was substituted by parenteral vancomycin (400 mg bid). after 10 days, because of persisting fever and increase of the diameter of the lung lesion (6 cm) vancomycin was sustituted by oral levofloxacyn (500 mg bid), continuing rifampin. after a 4 days course of levofloxacyn therapy the fever remitted. the patient was discharged with levofloxacyn (500 mg bid) and rifampin and, after 2 months of follow-up, a radiological control pointed-out a remarkable resolution in the lung lesion. we may suppose that levofloxacyn can be effective for the treatment of r. equi infection, even if more studies (particularly controlled studies) are necessary. liberti a a , izzo b a , loiacono l b , calabria g a , patarino t a , izzo e a . a ii department, naples, d. cotugno hospital, italy , b iii department, naples, d. cotugno hospital, italy the increasing prevalence of salmonella typhi strains with reduced susceptibility of chloramphenicol had prompted the search for other antibiotics with the same efficacy.quinolones are a class of antibiotic with an activity in vitro and in vivo against enteropathogenes. we inwestigated the use of levoxacin in two regimens of treatment of typhoid and paratyphoid infection. patients and methods: thirty-two adult patients were incluted in this study from september 1999 to april 2001; 26 patients had positive culture for s. typhi and six had positive cultures for s. paratyphi . all isolated were fully susceptible to levoxacin. we compared treatment with levoxacin for 7 days, 500 mgr b.i.d. (group 1, 16 patients), with treatment for 10 days, 500 mgr b.i.d. (group 2, 16 patients). clinical cure was defined as defervescenze of fever by day 3 of treatment, with an absence of complications and no clinical relapse during the followup. results and conclusion: the clinical cure rate was 87% (14 patients) for group 1 and 94% (15 patients) for group 2; the difference in these rates was not statistically significant. the blood cultures of all patients were sterile by day 2 of treatment and remained so until the 6th month of follow-up, no subjects had clinical or microbiological relapse and all stool cultures remained negative, also. the two regimens of treatment was good tollerated and no adverse event was registered; it was concluded that levoxacin treatment for 10 days in enteric fever is not necessary the mulidrug-resistence of s. typhi led to the use of quinolones as the first-line drug in the treatment of enteric fever. pefloxacin in the treatment of patient with acute infectious diarrhoea ps121 troselj-vukiae tvb a , strahinja v b , poljak i a , stojanoviae d c , nikoliae n d . a department of infectious disease, university hospital center, rijeka, croatia , b glaxosmithkline, marketing, rijeka, croatia , c dept. rijeka, institute of public health, rijeka, croatia , d dept. rijeka, maritime academy, rijeka, croatia the purpose of the study was to investigate clinical and bacteriological efficiency in 5 and 7 days pefloxacin treatment and to compare it with symptomatic therapy. the results obtained in 47 patients treated with pefloxacin the therapy was clinically effective already in the third day while in the control group this happend in the 7th day. bacteriological eradication was noted in 18 patients (95%) of the first and 25 patients (93%) of second group in the 5th days of the treatment. they all had negative cultures 1 and 4 weeks after pefloxacin protocols were completed. only 22 patients (63%) in control group had negative stool cultures in the 7th day of the treatment and all of them 4 weeks after it ended. there was no statistically significant difference in clinical (p 0/0.232) and bacteriological (p 0/0.972) efficiency between 5 and 7 days pefloxacin treatment protocols. both protocols significantly differed in clinical (p b/0.001) and bacteriological (p0/0.017) eradication from the control group. the conclusion reached is that the efficiency of pefloxacin (quinolones) in the treatment of acute infectiuos diarrhoea and justifies their use in the more severe forms of the disease. dalhoff a a , ullmann u b , schubert s b . a bayer ag, wuppertal, germany , b university of kiel, institute of med. microbiology, kiel, germany background: the antibacterial activity of moxifloxacin (mxf) was compared to levofloxacin (lev), amoxicillin (amx), clarithromycin (cla) and erythromycin (ery) in an in vitro model. method: pharmacokinetics in bronchial mucosa (bm) and serum (s) following single oral doses of 400 mg mxf or cla and 500 mg lev, amx or ery were simulated using a one compartment model. bacteria tested staphylococcus aureus (nos. 133, 25895), streptococcus pneumoniae (nos. 4241, 672). aliquots were taken (0 á/8 h) and plated on to brain heart infusion agar for enumeration. results: s. pneumoniae 4241 was eliminated by all agents studied. significant differences were apparent with s. pneumoniae 672 and the s. aureus strains: objective: to compare the safety and efficacy of once-daily moxifloxacin with once-daily ceftriaxone in the treatment of cap in hiv-infected patients (pts). methods and results: in a retrospective survey, oral moxifloxacin (400 mg daily)/12 á/15 days) was compared to standard regimen of i.v. ceftriaxone (2 g daily)/10 á/12 days) for treatment of cap in hiv'/ pts. adults pts with clinical signs and symptoms of cap and consistent chest x-ray findings were included. pts had a median age of 39 years (range 29 á/529 and 70% were male). demographic characteristics were similar in both treatment groups; 15 pts received mxifloxacin and 25 pts ceftriaxone. clinical success rates were 94% for moxifloxacin and 96% for ceftriaxone. at a post-study evaluation approximately 8 weeks later, 2 moxifloxacin-treated pts and 3 ceftriaxone-treated pts had relapsed. the adverse events reported were comparable for both treatment groups. there were four-related adverse events (3 gi, 1 headache) for moxifloxacin-treated and 7 (4gi, 3 skin) for ceftriaxonetreated pts. conclusion: the results of this study show that moxifloxacin as oral therapy is as effective and well tolerated as i.v. ceftriaxone in the treatment of hiv'/ pts with cap. therapy with moxifloxacin was not associated with any significant clinical or laboratory abnormalities. these data suggest that once-daily oral administration of moxifloxacin is potentially convenient and cost-effective alternative therapy for cap in pts with hiv infection. moxifloxacin in the treatment of acute maxillary sinusitis after first-line therapy failure or acute sinusitis with high risk of complications ps124 gehanno p a , berche p b , perrin a b , arvis p c . a ent department, bichat claude bernard hospital, paris, france , b microbiology department, necker enfants malades hospital, paris, france , c bayer pharma, medical affairs department, paris, france the efficacy and safety of moxifloxacin (mxf) 400 mg once daily for 7 days was evaluated in the treatment of acute maxillary sinusitis after first-line therapy failure, or acute sinusitis with high risk of complications. in this prospective, multicenter study, a total of 216 patients with acute bacterial sinusitis confirmed by sinus x-ray were valid for efficacy analysis: one hundred and seventy five patients (81.0%) had an acute maxillary sinusitis which failed to respond to a previous antibiotic therapy given for a mean duration of 7.2 days, and 41 (19.0%) had an acute sinusitis with high risk of complications (frontal: 24, pan-sinusitis: 15 and sphenoid: 2). ninety two patients (42.6%) were microbiologically valid. clinical cure and continued clinical cure rates at 7 á/10 and 28 á/35 days post-therapy were 92.6 and 99.0%, respectively. clinical cure rates at 7 á/10 days post-therapy were 94.9 and 82.9% in sinusitis after first-line therapy failure and in sinusitis with high risk of complications, respectively. bacteriological eradication rate during therapy (day 3 á/4) was 95.7%. at 7 á/10 days post-therapy. bacteriological success rates were 97.1% in patients who failed to respond to a previous antibiotic and 95.4% of patients who had sinusitis with high risk of complications. 12.2% of patients experienced drug-related adverse events, abdominal pain (2.4%), nausea (2.4%) being the most frequently reported events. mxf was rapidly effective and a well-tolerated treatment for this kind of infection. neisseria gonorrhoeae with decreased susceptibility to penicillin and ciprofloxacin: novel mutation patterns in the gyr a and par c genes of ciprofloxacin resistant isolates and plasmid profile of penicillin resistant isolates of n. gonorrhoeae in india (delhi) ps125 chaudhry u, saluja d. dr. b. r. ambedkar center for biomedical research, university of delhi, delhi, india commercial sex workers (csws) serve as the most important reservoir of gonorrhoea. periodic monitoring of the antimicrobial susceptibility profile of neisseria gonorrhoeae in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. in india, such a surveillance of the in vitro antimicrobial susceptibility of n. gonorrhoeae was established in 1997. significant increasing trend of penicillin and ciprofloxacin resistance with high mic of 2 á/16 and 1 á/32 mg/ml, respectively were found over the years (1997 á/2001) . the molecular basis of ciprofloxacin resistance, i.e. mutations in the gyr a and the par c genes of 170 isolates, were analyzed. four isolates (with an mic of 32 mg/ml for ciprofloxacin) harbored triple mutations (ser-91 to phe, asp-95 to asn and val-120 to leu) in the gyr a gene. the third mutation of val-120 to leu, lies downstream of the quinolone resistance determining region of the gyr a and has not been described before in gonococcus. in addition, these isolates had a phe-100 to tyr substitution in the par c, a hitherto unknown mutation. the alterations in the par c gene were seen in these isolates only in the presence of changes in the gyr a gene and comprised amino acid changes at codons 91, 100, 104, 109, and 131. the presence of b-lactamase plasmid among the penicillinresistant isolates was determined by their plasmid profiles and further confirmation was carried out by a pcr based protocol. our findings suggest that emergence of penicillin and ciprofloxacin resistance in n. gonorrhoeae isolates from a major std center in india, indicates the need for the increased awareness and prudent use of antimicrobials. in vitro activity of newer antibiotics against methicillin-resistant staphylococcus aureus ps126 gutierrez zufiaurre mn, sanchez hernandez j, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: mrsa are frequently co-resistant to a number of structurally unrelated antibiotics. more than 70% mrsa are resistant to gentamycin, ciprofloxacin, macrolides and clindamycin. newer antibiotics active against multi-drug resistant grampositives have been developed. we have tested the in vitro activity of newer antibiotics against genetically-characterized, high level ciprofloxacin resistant mrsa. material and methods: thirty-six ciprofloxacin-resistant, gyra/grla mutant mrsa clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), gatifloxacin (gfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method, according nccls guidelines. results and conclusions: all the strains were resistant to cfx, 55,5% were lfx-susceptible and 63.9% were gfx-susceptible. nevertheless, mics of lfx and gfx for all susceptible strains was in the highest extreme of the susceptibility range. mfx was the most active quinolone. almost all the strains were high-level er-resistant with constitutive mlsb phenotype. tl did not improve significantly its behaviour, although it was 10-fold as active as er against the only er-susceptible strain. va, lin and syn had excellent activity against all the strains. they showed a very homogeneous behaviour against all the strains that were included in a range of 1 á/4 mg/l of lin and va and 0.5 á/1 mg/l. sanchez hernandez j, gutierrez zufiaurre mn, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: corynebacterium urealyticum is the etiologic agent of encrusted cystitis and inespecific utis, and can be also involved in systemic infections. c. urealyticum is frequently multi-drug resistant, so only glycopeptide antibiotics and tetracyclines have high susceptibility rates, while fluoroquinolones resistance rates vary significantly. we have tested the in vitro activity of linezolid, telithromycin, synercid and newer fluoroquinolones against multi-drug resistant c. urealyticum clinical strains. material and methods: sixty-four c. urealyticum clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method according guidelines defined by the nccls for enterococci. results and conclusions: results confirm the high resistance rate to older fluoroquinolones and macrolides, with !/50% cfx resistance and 100% er-resistance. lfx was more active (mic90 32 mg/ml). mfx was the most active fluoroquinolones (mic90 4 mg/ml). tl improve its behaviour in respect to er (range 0.5 á/1 mg/ml). va, lin and syn had excellent antimicrobial activity. no resistant strains were found. mic90 were 1, 0.5 and 1 mg/ml, respectively. mics were similar for all the strains independently of their resistance to other antibiotics plasma concentrations, urinary excretion and bactericidal activity of linezolid (600 mg) versus ciprofloxacin (500 mg) in healthy volunteers after a single oral dose ps128 wagenlehner fme a , wydra s a , onda h a , kinzig-schippers m b , sö rgel f b , naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, (ibmp), nürnberg-heroldsberg, germany purpose of the study: in a randomized cross-over study 12 volunteers received a single oral dose of 600 mg linezolid versus 500 mg ciprofloxacin to assess plasma concentrations (up to 24 h), urinary excretion (by hplc), and urinary bactericidal titers (ubt) up to 120 h. the mean maximum plasma concentration of linezolid was 12.1 mg/ ml and of ciprofloxacin 2.4 mg/ml. the cumulative renal excretion (mean) of parent drug was 40%/41% for linezolid/ciprofloxacin. ubts were determined for a reference strain and five gram-positive clinical uropathogens with the following mics (mg/ml) for linezolid/ciprofloxacin: s. aureus atcc 27278 (4/0.25), s. aureus (mssa) (2/32), s. aureus (mrsa) (2/128), s. saprophyticus (msse) (2/0.5), e. faecalis (2/1), e. faecium (2/2). results: median ubts measured within the first 6 h for linezolid were 1:96 for enterococcal strains and 1:128 to 1:256 for the four staphylococal strains. median ubts for ciprofloxacin were 1:64 for the two enterococcal strains, 1:384 to 1:512 for the two ciprofloxacin susceptible, 1:1.5 for the two resistant staphylococcal strains. areas under the ubt á/time-curve showed statistically significant differences only for the two ciprofloxacin resistant staphylococcal strains in favour of linezolid. conclusion: linezolid exhibited the same bactericidal activity against ciprofloxacin resistant as susceptible strains. linezolid should be tested for treatment of complicated uti due to gram-positive uropathogens in a clinical trial. whitehouse t a , cepeda ja a , tobin purpose: we performed pharmacokinetics within a double-blind, randomised trial comparing linezolid and teicoplanin in intensive care patients with known or suspected gram-positive infection. they received either 600 mg linezolid intravenously 12-hourly, or 400 mg teicoplanin 12-hourly for the first three doses and once daily thereafter (or every 3rd day if renally impaired). blood samples were collected to create serum pharmacokinetic profiles. linezolid was quantitated by hplc and teicoplanin by fluorescence polarization immunoassay. results: twenty two patients were studied in the linezolid group (m á/f 13:9, mean age 54 years [range 19 á/90 years]). median treatment duration was 8.5 days (range 4 á/18). eighteen patients were treated with teicoplanin (m á/f 13:5, mean age 57 years [range 17 á/86]) for median 9 days (range 4 á/17). steady state peak concentrations (mean9/ sd) for linezolid and teicoplanin were 12.89/5.0 and 10.59/4.7 mg/l, respectively. trough concentrations at day 4 were 4.79/4.3 mg/l for linezolid and 7.99/2.5 mg/l for teicoplanin. recommended breakpoints of staphylococcus aureus are 4 mg/l for linezolid and 8 mg/l for teicoplanin. accumulation occurred in one 90-year-old linezolidtreated patient with impaired renal function. conclusion: current recommended dosing regimens for linezolid and teicoplanin are generally appropriate in the critically ill, though a more detailed analysis is required. stamos g, lebessi e, ioannidou s, paleologou n, kallergi k, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of the study was to investigate the susceptibility of methicillin resistant staphylococcus aureus (mrsa) isolates from a 500-bed paediatric hospital to quinupristin/dalfopristin (q/d, streptogramin combination) and linezolid (lzd, oxazolidinone). material: we performed a retrospective analysis of 85 mrsa strains, isolated from patients hospitalized in miscellaneous medical departments [neonatal unit (35) , surgical wards (15), orthopaedic wards (9), oncology unit (7), other wards (8) and outpatient clinic (11)], during a 3-year period (1998 á/2000) . the sources of isolation were pus (23), throat (22), nasal (8), bronchial (5), skin (6), stool (6) ear (4) and other (11) specimens. all isolates were sensitive to glycopeptides, while 22.4% were resistant to gentamicin and 7.1% to erythromycin. methods: the sensitivity testing was performed by a disk diffusion method (bbl sensitivity disks, becton dickinson), according to nccls guidelines. the breakpoint zone diameters for lzd and q/ d were ]/21 and ]/19 mm for susceptibility and 5/17 and 5/15 mm for resistance, respectively. results: all isolates were proved to be susceptible to both antibiotics. the mean inhibition zones were 29.1 mm for lzd and 26 mm for q/d. conclusions: lzd and q/d are very promising antimicrobial agents, showing excellent activity against mrsa clinical isolates. the prudent therapeutic use is strongly recommended to avoid the emergence of resistance. in vitro activity of streptogramins and oxazolidinones against streptococcus pneumoniae clinical isolates ps131 stamos g, lebessi e, paleologou n, psatha m, sanida p, zaphiropoulou a, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of this study was to evaluate the in vitro activity of linezolid (lzd), a member of oxazolidinones and the streptogramin combination quinupristin/dalfopristin (q/d) against clinical isolates of streptococcus pneumoniae from a tertiary care paediatric hospital. material: a total of 53 pneumococcal isolates exhibiting reduced susceptibility to common antibiotics were included in the study. the strains were isolated from middle ear fluid (34), eye (4), nasal (5) blood (6) and other (4) cultures during the last 4 years. the percentages of the isolates that were resistant to penicillin, erythromycin, cotrimoxazole and clindamycin were 67.9, 67.9, 67.9 and 26.4%, respectively. methods: the susceptibility testing was performed by a standard disk diffusion method (bbl sensitivity disks, becton dickinson). in case of marginal results or intermediate sensitivity to quinupristin/ dalfopristin, the mic was determined using the e -test method (ab biodisk). results: all isolates were found to be sensitive to lzd. q/d was very active as well, except for two isolates that exhibited intermediate susceptibility, showing cross-resistance to macrolides and clindamycin, as well. conclusions: the new antimicrobial agents show excellent activity against resistant to common antimicrobials pneumococcal isolates, but the clinical use is not suggested, unless no other therapeutic solutions are available. linezolid is a new oxazolidinone with excellent activity against gram-positive organisms including glycopeptide-resistant strains of staphylococci and enterococci. in icu linezolid has been used for the treatment of severe gram-positive infections in a control trial. the susceptibility pattern of all gram-positive isolates from icu patients has been studied. methods: a total of 2427 specimens from icu patients were processed, 767 were from patients enrolled in the antibiotic trial. all methicillin-resistant staphylococcus aureus (mrsa), coagulase-negative staphylococci (cons), enterococcus sp and methicillin-sensitive staphylococcus aureus (mssa) were tested. the break point for linezolid was 4 mg/l and for teicoplanin 8 mg/l. isolates were tested for susceptibility by e -test. results: linezolid (1063 isolates) mic 50/90 (mg/l) were as follows: mrsa (n 0/541) 0.6/2, cons (n0/143) 0.9/1.4, enterococcus sp (n0/39) 0.7/0.8, mssa (n0/340) 0.8/1. teicoplanin (782 isolates) mic 50/90(mg/l) mrsa (n0/415) 1.9/3.5, cons (n 0/100) 4/7.4, enterococcus sp (n0/24) 0.9/1.4, mssa (n0/243) 2.2/2.6. all grampositive isolates were inhibited by concentrations of linezolid below the breakpoint including eight strains of staphylococci resistant to teicoplanin. conclusions: linezolid was highly active against grampositive isolates. resistance to teicoplanin was similar to other reported series. there was no emergence of resistance to linezolid. mrsa colonization is often a limiting factor for discharge from icu. clearance of mrsa is seldom achieved with conventional glycopeptide treatment. the oxazolidinone, linezolid, has excellent soft tissue and respiratory tract penetration and might be expected to eradicate carriage in some patients. we recently performed a doubleblind randomized trial in 204 icu patients with known/suspected gram positive infection. 100 received intravenous linezolid, 600 mg b.d., and 104 patients received teicoplanin 400 mg o.d. mrsa clearance was assessed at end of treatment (eot), at 7-and 21days follow-up. results: in the linezolid and teicoplanin groups, 45 and 43 were known to be colonized with mrsa at study entry, respectively, while 39 and 40 were not. detection of clearance of mrsa colonization at eot was 51% for linezolid vs 19% for teicoplanin group (x b p b/ 0.005), at 7 days it was 35% vs 14% (x b p b/0.1), and at 21 days 19% vs 33% (ns). conclusion: short-term mrsa clearance can be achieved in significantly more patients treated with linezolid however this was not maintained at 21 days, either because of incomplete initial eradication or recolonization. further analysis will follow when molecular typing of the isolates is completed. penetration of linezolid into bone, fat and muscle during hip arthroplasty ps134 lovering am, bannister gc, zhang j, macgowan ap, southmead hospital, bcare, bristol, uk there are limited data describing the concentrations and penetration of linezolid (lzd) into tissues, such as bone, that can be used to guide therapy for non-vascular infections. here we report the concentrations and penetration of lzd for bone, fat and muscle in comparison with cefamandole (cmd). twelve patients received 600 mg lzd as a 20 min infusion and 1000 mg cmd as a bolus injection immediately before hip arthroplasty. bone, fat, muscle and blood samples were collected at timed intervals after the infusion and assayed by a validated hplc method. for bone, peak levels of both agents occurred 10 min after administration with mean levels of lzd 9.1 mg/kg versus cmd 17.5 mg/kg, decreasing to lzd 5.7 mg/kg versus cmd 9.7 mg/kg at 30 min. correction for blood concentrations gave penetration of lzd 51% versus cmd 25% at 10 min and lzd 46% versus cmd 23% at 30 min. for fat and muscle, peak levels occurred 20 min after infusion. mean levels were lzd 5.2 mg/kg versus cmd 10.9 mg/kg (fat) and lzd 13.4 mg/kg versus cmd 10.9 mg/kg (muscle). correction for blood concentrations gave penetration of lzd 37% versus cmd 19% (fat) and lzd 95% versus cmd 31% (muscle). we conclude that linezolid exhibits rapid penetration into bone and associated soft tissues achieving levels in excess of the mic for sensitive organisms; with a similar distribution and penetration profile to agents currently used for treatment of infections in these tissues. bacqué e a , barrière jc a , berthaud n b , desmazeau p b , dutruc-rosset g b , dutka-malen s b , ronan b b . a aventis pharma, chemistry, paris, france , b aventis pharma, disease group, paris, france xrp2868 is a new oral streptogramin composed of 2 semi-synthetic synergistic components in a 30/70 (w/w) association: rpr202868 (5d-(1-morpholino)methyl-5d,5g-dehydro pristinamycin i e ) from pi a and rpr132552 [(16r )-16-deoxy-16-fluoro pristinamycin ii b ] from pii b by original synthetic routes. the association has antibacterial activity against staphylococci including methicillin */mls b -resistant strains (mic90 range: 0.12 */1 mg/ml), streptococci (mic90: 0.25 mg/ml), pneumococci including multidrug resistant strains (mic90: 0.50 mg/ ml), enterococci including vancomycin-resistant strains (mic90: 4 mg/ ml), m. catarrhalis and neisseria spp. (mic90: 0.12 mg/ml), h. influenzae (mic90: 1 mg/ml), legionella spp. (mic90: 0.06 mg/ml) and anaerobes (mic range: 0.06 á/4 mg/ml). xrp2868 is generally bactericidal at the concentration of 2 á/4)/the mic against s. aureus , s. pneumoniae , h. influenzae and demonstrates consequent pae (2.1 á/ !/5.7 h at 2 á/4)/mic, following an exposure of 0.25 á/2 h). mutants of s. aureus to xrp2868 were isolated at low frequencies (3.6)/ 10 (9 á/9.7)/10 (10 ) at 2)/ and 4)/mic while no mutant could be isolated at 8)/mic. these results suggest that xrp2868 (30/70 w/w) is a promising compound for the treatment of community-acquired infections. ex vivo evaluation of rpr202868/rpr132552 (xrp2868), a new oral streptogramin ps136 berthaud n, diallo n. aventis pharma sa, infectious disease group, paris, france the intra cellular activity of xrp2868, was assessed in j774 murine macrophages containing ingested staphylococcus aureus (three strains) or l. pneumophila (one strain). at the concentration of 8 )/ the mic, growth of s. aureus was strongly inhibited after a 3-h period of incubation (d log 10 cfu/ml vs t 0 controls range: (/2.16 á/(/1.24, according to the strain tested). at the concentration of 8)/ the mic, growth of intracellular l. pneumophila was inhibited after a 48 á/ 72-h period of incubation (d log 10 cfu/ml vs t controls about (/1.30 and (/1.48 at 48 and 72 h, respectively). rpr202868 and rpr132552 alone had also inhibiting effect on bacterial growth (d log 10 cfu/ml vs t 0 controls after 72 h of incubation about (/1.54 and (/0.87, respectively). the bactericidal activity of xrp2868 was also assessed against slowly growing s. aureus , under experimental conditions mimicking those observed in patients with an infected indwelling device (first step of infection: adherence to inert support; declared infection: biofilm model). under the experimental conditions, xrp2868 demonstrated a rapid and potent bactericidal effect against s. aureus adherent to an inert support or included in biofilm. this effect was observed at each of the three concentrations tested (5, 10, 50 and 8, 100 and 200)/ mic, respectively). in vivo evaluation of xrp2868 (rpr202868/rpr132552), a new oral streptogramin ps137 berthaud n, huet y, aventis pharma sa, infectious disease group, paris, france the oral efficacy of xrp2868, was assessed in staphylococcus and pneumococcal murine infections. mice were challenged ip ( )/10, and )/100 times ld 50 ). abscesses were established by intramuscular injection of about 10 7 bacteria into the right thigh of mice. pneumonia was established by intranasal injection of about 10 7 bacteria. mice were treated twice a day for 1 (staphylococcus aureus septicaemia and abscesses), 2 (s. pneunomoniae septicaemia) or 3 days (s. pneumoniae pneumonia). six to 15 days post infection, for septicaemia and abscesses the results were expressed as ed 50 , whereas for pneumonia they were expressed as the dose yielding an average survival time (ast) significantly longer than that of the untreated infected controls. xrp2868 was efficacious in the treatment of experimental infections in mice caused by mls b -sensitive and -constitutively resistant s. aureus (ed 50 range: 20 á/44 and 24 á/60 mg/kg/administration in septicaemia and abscess models, respectively). it was also efficacious in the treatment of infections caused by s. pneumoniae whatever the serotype and the resistance profile of the strains tested (ed 50 range: 28 á/55 mg/ kg/administration in septicaemia, ast: 50 mg/kg/administration). these results suggest that xrp2868 might be effective for the treatment of staphylococcal and pneumococcal community-acquired infections. xrp2868 (rpr202868/rpr132552), a new oral streptogramin: bactericidal activity and pharmacokinetics in a model of streptococcus pneumoniae mouse pneumonia ps138 berthaud n, huet y, diallo n. aventis pharma sa, infectious disease group, paris, france the bactericidal activity against streptococcus pneumoniae of xrp2868, a new oral streptogramin composed of two semi-synthetic synergistic components in a 30/70 (w/w) association (rpr202868, pristinamycin i derivative and rpr132552, pristinamycin ii derivative), was assessed in lungs of mice with pneumonia. mice were inoculated intranasally with about 10 7 cfu of strain 6254-01 (mls bresistant). eighteen hours later (t 0 ), animals received xrp2868 (120 mg/kg p.o). the administration was repeated 6, 24, 30, 48 and 54 h afterwards. to study the influence of varying the ratio of pi to pii component administered on activity and pk parameters, ratios of rpr202868 to rpr132552 ranging from 0/100 to 100/0 were also administered under the same conditions. after an oral unitary administration at 120 mg/kg, xrp2868, as well as ratios of rpr202868 to rpr132552 ranging from 50/50 to 90/10, demonstrated strong and quick bactericidal activity in lungs. lung levels of rpr202868 and rpr132552 were generally equal or two times higher than blood levels. resulting rpr202868/rpr132552 ratios in blood and lung, although not in accordance with the initial ratio administered, were synergistic for 3 á/4 h in blood and for 1 á/3 h in lungs explaining the activity observed. conclusion: based on in vitro data, telithromycin is a good candidate for the treatment of rti. in vitro evaluation of abt-773, telithromycin and azithromycin against streptococcus pneumoniae , moraxella catarrhalis , haemophilis influenzae and methicillin-resistant staphylococcus aureus ps140 steele-moore l, berg d, barnes i, couch k, klein j, holloway w. christiana care, infectious disease, wilmington, us macrolide resistant streptococcus pneumoniae (sp) is a worldwide concern predominantly because these isolates tend to be multiply drug resistant. new agents with increased activity against these pathogens are clinically important. the ketolide class of antimicrobial agents demonstrate excellent in vitro activity against sp, even those that are macrolide resistant. the in vitro activities of the ketolides abt-773 (a) and telithromycin (t) were compared to azithromycin (az) against clinical isolates of sp, moraxella catarrhalis (m.cat), haemophilis influenzae (h.flu) and mrsa. organisms tested: 51 strains of sp (including 14 az resistant), 25 h.flu, 25 mrsa and 10 m.cat. microdilution mic tests were performed following nccls recommendations using freshly prepared plates containing haemophilis test medium for h.flu, cation adjusted mueller-hinton broth (camhb) with laked horse blood for sp and camhb for m.cat and mrsa. the new ketolides, a and t had superior activity against sp including the az resistant strains (mic90s: a0/0.12 mcg/ml, t 0/0.25, az0/4). all compounds had excellent activity against m.cat while none demonstrated activity against mrsa. h.flu activity was comparable among a, t and az. these new ketolides are not currently approved by the fda; however t has been approved in europe. ló pez h, vidal gi, salomó n jm, scaglione m, zitto tr. centro de infectología, infectious diseases, buenos aires, argentina objective: we evaluated the impact of the initial treatment failure rate, hospitalisation and costs in outpatient treatment of adult cap in argentina comparing amoxicillin, clarithromycin and telithromycin. method: a probabilistic model was implemented in outpatient treatment of cap. we estimated an initial treatment with amoxicillin, clarithromycin or telithromycin. we assumed expected clinical cure at 90.1, 88.5 and 94.6%, respectively. for those patients with failure treatment we evaluated a second-line of antibiotics (amoxicillin followed by clarithromycin and clarithromycin followed by new fluorquinolone) or hospitalisation. patients with telithromycin and failure treatment must be hospitalised without a second line of outpatient treatment. costs of cap included drug's costs by 10 days, medical visits, chest radiographies, analysis and hospitalisation. results: we estimated treatments in 100 patients and first-line drug failure in 10, 11 and 5 patients with amoxicillin, clarithromycin and telithromycin, respectively. costs in outpatient treatment were: hospitalisation $12,224 and $13,489.4; second-line drug $1039 and $1045; second-line hospitalised $1320.4 and $1232 with amoxicillin and clarithromycin, respectively and hospitalisation with telithromycin $6137. conclusions: telithromycin showed lower clinical failure, hospitalisation and costs in cap. some studies suggest shortening cap telithromycin treatment to 7 days helping adherence to treatment and decreasing costs even more. the new macrolides */a good alternative of tetracycline in the treatment of mediterranean spotted fever ps142 popivanova nip a , petrov aip a , boykinova obb a , kazakova zkk a , baltadjiev agb b . a medical university, infectious disease, plovdiv, bulgaria , b department of anatomy, medical university, plovdiv, bulgaria mediterranean spotted fever (msf) caused by rickettsia conorii appears an endemic disease for some regions in bulgaria. frequently the disease has a severe course with multiple organ lesions. the early and adequate treatment is of extreme importance for the outcome of the disease. searching an alternative antibiotic treatment of this disease we considered macrolides for their good cell and tissue penetration and dose-dependent bacteriostatic and bactericide effect. we treated 27 msf patients with doxycycline 2)/100 mg/day, 25 patients with clarithromycin 2)/250 mg/day, as well as 25 patients with midecamycin 2)/400 mg/day and midecamycin acetate 30 mg/kg/day. as a surrogate marker for treatment evaluation the effect on the febrile syndrome was used. our findings showed that by the 5th day of treatment the fever normalized in 89.47, 88.24 and 76.47% of the patients treated with doxycycline, clarithromycin and midecamycin, respectively. for the same period the patient fever decreased below 38 8c in 100, 100 and 94.12%, respectively. the intoxication symptoms were influenced within the same period equally in all treated patients. conclusions: we suggest that the new macrolides appear a good alternative of tetracycline on patients with msf. erythromycin resistance of gram /'// cocci in bulgaria. benefits of the new macrolides in the treatment of respiratory infections ps143 popivanova ni a , yovtchev ip b , dobreva md c , argirova ta c . a medical university, infectious disease, plovdiv, bulgaria , b medical university, ear-nose-throat disease, plovdiv, bulgaria , c medical university, microbiology, plovdiv, bulgaria in the recent 2 years we tested erythromycin sensitivity of 474 species of gram /'// cocci isolated from throat and nose secretions, ear and eye effusions, sputa, cerebrospinal fluid and blood cultures, vaginal and urethral secretions, urine and fecal samples from patients with inflammatory diseases of the listed organs and systems. staphylococcus aureus , staphylococcus coagulase /(//, streptococcus â-haemolyticus , enterococcus were isolated. of these microorganisms s. aureus was the most abundant. our resistograms revealed sensitivity of the gram /'// cocci in 76.97 and 68.60% for 2000 and 2001, respectively, and resistance of 23.03 and 31.40%, respectively. in the tests with midecamycin and midecamycin acetat the same microorganisms showed sensitivity of 85.38% and resistance of 14.62%. the clinical findings showed excellent effect of the new macrolides including clarithromycin and azalides */azithromycin. we conclude the resistance of gram /'// cocci and especially of s. aureus to erythromycin increases very quickly and has reached dramatical extent. by now, the new 14, 15, and 16-membered ring macrolides and azalides show high antibacterial activity and good clinical effect. pappas ga, liberopoulos e, tsiara s, elisaf m, tsianos e. university hospital, internal medicine, ioannina, greece quinupristin/dalfopristin (q/d) is a novel injectable streptogramin antibiotic which initiation in therapeutics was hailed as an important step towards treatment of vancomycin-resistant enterococcus faecium (vref) species. initial reports concluded in an excellent response of vref to q/d. reports of q/d-resistant strains of e. faecium have emerged, both in usa and europe. we report two cases of e. faecium bacteremia in which the responsible isolate was not sensitive to q/d. the first patient was a woman with acute leukemia and septicemia. e. faecium was cultured from blood samples: the species was resistant to almost all antibiotics, exhibiting sensitivity only to tetracycline (!), while its sensitivity to q/d was indermediate. the second patient was a man with endocarditis, in whom blood cultures isolated e. faecium sensitive to a number of antibiotics, including ciprofloxacin and vancomycin; still the sensitivity to q/d was indermediate. q/d has not been officially introduced to the antibiotic arsenal of greek medicine. moreover, the drug has never been used in our hospital, not even experimentally. other e. faecium species isolated in our hospital have been sensitive to q/d. how much hope can be put on a drug that seems to be partly useless before its initiation? increasing reports of v/ q-resistant strains of e. faecium from all over europe raises fears that the v/d story might well end before it begins. varadinova t a , diakov t a , karagiozova d a , genova p a , pardon p b , baudry c b , quideau s b . a sofia university, virology, sofia, bulgaria , b et institut du pin, centre de recherche en chimie moleculaire, universite bordeaux 1, bordeaux, france vegetable tannins posses a wide range of biological activities. the aim of the present study was to evaluate the cytotoxicity of five purified vegetable tannins against mdbk cells. the maximal nontoxic concentrations (mnc) and the concentrations required to inhibit cell growth by 50% (cc 50 ) were evaluated after 24, 48 and 72 h periods of action. mnc values after 48 h indicated that compounds stimulated cell surveillance when applied in concentrations lower than 0.01 mm. cc 50 values indicated: (i) a decrease in cytotoxicity after 48 h as cc 50 were up to 70 times lower than those observed after the 24 h period, and (ii) a re-increase in cytotoxicity when the period of action was prolonged up to 72 h as cc 50 were 2500 times lower than those observed after the 48 h period. these data thus appear to reveal the capability of the investigated natural polyphenolic products to stimulate cell surveillance in a time-dependent manner. antibacterial effect survey of enoxolone on periodontopathogenic and capnophilic bacteria isolated from specimens of patients with periodontitis ps146 salari mh a , kadkhoda z b , sohrabi n a . a tehran university of medical sciences, pathobiology, tehran, islamic republic of iran , b tehran university of medical sciences, dentistry, tehran, islamic republic of iran objectives: most of the microorganisms associated with periodontitis are capnophilic and anaerobic bacteria. purpose of this study was to detection of antibacterial effect of enoxolone on periodontopathogenic and capnophilic bacteria. methods: in this study 136 periodontopathogenic and capnophilic bacteria were isolated from 400 specimens of patients with periodontitis by culture method. an anti-bacterial activity of enoxolone against these microorganisms was investigated by minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) methods. results: based on our findings the mic, mbc and lethal does 50 (ld50) of enoxolone for actinobacillus actinomycetemcomitans , eikenella corrodens and capnocytophaga were '8, 16, 64', '16, 16, 32', and '8, 16, 32' mg/ml, respectively. conclusion: our results show enoxolone has antibacterial effect on a. actinomycetemcomitans , e. corrodens and capnocytophaga spp. sakalova stn. medical university, microbiology, grodno, belarus we have synthesized diamides of dicarboxylic acids, with components such as 5-nitrothiazole benzolsulphamides and triazol. all the above compounds exhibited bacteriostatic activity towards some microorganisms. for further studies of bacteriostatic activity of amides and diamides of dicarboxylic acids, as well as for determination of 'structure á/activity' relationship, we have synthesized a range of monoamides. antibacterial activity of the synthesized compounds was studied in vitro by agar dilution methods. for this purpose, approximately 50 various gram-positive and -negative microorganisms including clinical strains of staphylococcus aureus , bacillus subtilis , serratia marcescens , escherichia coli , proteus morganii , micrococcus lisodeicticus , staphylococcus epidermidis , shigella sonnei , salmonella typhimurium , yersinia enterocolitica . minimum inhibitory concentration was expressed in mg/ml. nitazole was used as a comparison substance. analysis that new derivatives of 5-nitrotiasole have high antibacterial activity relative towards certain microorganisms included strains obtained from infection department's patients. results will be shown. microbial susceptibility to the essential oil of ziziphora clinopodiodes lam. ps148 purpose of the study: antimicrobial activities of essential oils vary from oil to oil and from one micro organism to another. the antimicrobial and chemical properties of essential oil from ziziphora clinopodiodes lam. has not been studied and hence the present study was planned to evaluate those properties against a series of micro organisms viz, escherichia coli , staphylococcus aureus , pseudomonas aeroginosa , klebsiella pneumonia , bacillus subtilis , bacillus licheniformis , streptococcus faecalis , candida albicans and saccharomyces cerevisiae . results: z. clinopodiodes lam. essential oil was found to have remarkable antimicrobial property against all the microorganisms but p. aeroginosa . the oil exhibited its best antimicrobial activity within a maximum of 45 min. seventeen components were identified by gas chromatography and mass spectrometry (gc and gc/ms) analysis of the oil, out of which pulegone (24.5%), neomenthol (12.8%), cyclohexene,5-methyl-3-(1-methenyl)trans (12.2%), 2,4-cycloheptadien-1-one,3,6,6-trimethyl (9.2%), piperitone (4.2%), and limonene'/ 1,8-cineole (4.1%) constitute major parts of the oil. conclusion: monoterpenes seem to have antimicrobial role. it seems necessary to explore antimicrobial properties of new harmless antimicrobial agents from natural sources as substitutes for common chemical drugs. methanol extract of carpobrotus edulis enhances killing of methicillin resistant staphylococcus aureus phagocytosed by thp-1 human monocyte derived macrophages and promotes the release of modulators of cellular immunity ps149 although alkaloids from the family mesembryanthemaceae have anti-cancer activity, species of this family have received little attention. because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, we have studied a crude methanol extract of carpabrotus edulis , a common plant found along the portuguese coast, for properties normally associated with plasma membrane active compounds. the results of this preliminary study show that the extract is non-toxic at concentrations that prime thp-1 human monocytederived macrophages to kill ingested methicillin resistant staphylococcus aureus and promote the release of lymphokines associated with cellular immune functions. the extract also induces the proliferation of thp-1 cells within 1 day of exposure and 2 days earlier than that induced by phytohemagglutinin. similar results were obtained with monocyte-derived macrophages isolated from human peripheral blood. the active component or components of the plant extract may be exploited as intracellular active anti-bacterials as well as modulators of cellular immunity. enhancing of erythromycin production by saccharopolyspora erythraea nur001 with common and uncommon oils ps150 hamedi j a , malekzadeh f a,b saghafi-nia ae b . a department of biology, faculty of science, university of tehran, tehran, islamic republic of iran , b shafe-e-sari co., antibiotic production co., teheran, iran enhancing effect of various oils on the erythromycin production by saccharopolyspora erythraea nur001 were evaluated in a complex medium consisting soybean flour and dextrin as the main substrates. the biomass, erythromycin, dextrin and oil concentrations, and ph value were measured on a daily basis. also, the kinds and frequencies of fatty acids of the oils used were determined. saturated fatty acids in the shark oil were higher than that of vegetable oils used. erythromycin concentration in the melon (cucumis melo var. inderus cultivar mashhad ) seed oil containing medium was 4.56 times higher than that of the control medium (without oil) and 1.18 times higher than that of rapeseed oil containing medium. erythromycin concentration in the other oil containing media, including rapeseed, soybean, shark (carcharhinus dussumieri ), and safflower oils was 3.88, 3.03, 2.59, 2.44 time higher than that of control medium, respectively. the melon seed oil had the least enhancing effect on the biomass production, and thus decreasing the cost of the biomass separation. can varicella be eliminated by universal childhood vaccination ? */ epidemiological and economic data from germany ps151 wutzler p a , banz k b , neiss a c , goertz a d , bisanz h d . a institute for antiviral chemotherapy, university of jena, jena, germany , b outcomes international, basle, switzerland , c institute of medical statistics and epidemiology, technical university, munich, germany , d glaxosmithkline, pharma, munich, germany purpose: universal varicella vaccination in childhood is expected to reduce substantially the number of uncomplicated cases of chickenpox and to decrease the number of complicated cases requiring hospitalization. to generate fundamental data for decisions of the health authorities epidemiological and health-economic data were collected in two large studies. using an age-structured decision analytic model the benefits, costs and cost-effectiveness of a varicella immunization program over a period of 30 years were assessed. results: it could be shown, that the vast majority of varicella cases occur in children aged less than 8 years. in 16.3% of the cases a severe course was assessed. overall incidence of complications was estimated to be 5.7%. a routine varicella vaccination program targeting healthy children could prevent 82.6% of varicella cases and over 4700 major complications per year provided the coverage rate would be 85%. under these conditions the elimination of varicella is predicted to be achievable within 15 á/20 years. a combined measles, mumps, rubella and varicella vaccine is expected to provide the required coverage. conclusions: routine childhood varicella vaccination appears to be a highly efficient strategy to significantly reduce the sizeable burden of varicella and leads to significant savings from both societal and payer's perspective. bulgakova va, balabolkin ii, sentsova tb. scientific center of child health, russian academy of medical sciences, scientific research institute of pediatrics, moscow, russian federation objective: to estimate efficacy of vaccine influvac at children with allergic diseases. methods: twenty children aged 3 á/18 years with allergic diseases received vaccine influvac (solvay pharma). for the control group of children, with allergic pathology did not receive this vaccine because of an intolerance of chicken protein (10 children). results: all vaccinated children for the observable season of 6 months did not get influenza. general and aboriginal reactions to a vaccine did not occur. in control group for the observable season two children were ill with influenza and four children with acute respiratory virus infection (60%). among vaccinated children there was an increase in titre to a protective level (1:40 and above) to all to three strains of influenza 30 days after injection. vaccine influvac can be recommended for an immunisation against influenza of children with an allergic pathology because of efficacy and absence of side effects. ló pez h, zitto tr, vidal gi, cánepa mc, salomó n jm, scaglione m. centro de infectología, infectious diseases, buenos aires, argentina objectives: our study examines the possible economic impact of the influenza on health working adults in argentina, and the intervention cost saving with immunization. methods: this is a theoretical study based on a mathematical model. population data was published in 2001s national statistics. the global incidence of influenza infection was estimated at 5%. we have estimated the direct cost on influenza infection (outpatient visits, drugs and hospitalization) and indirect cost (work absenteeism and productivity loss) and projected net saving for the 20 á/64 year-old vaccinated group. the vaccine effectiveness was estimated at 70 and 90%. the price of vaccine was $8 each. results conclusion: influenza vaccination is effective in diminishing cases of flu and reducing working-day loss. its a safe and cost-effective vaccine. ló pez h, zitto tr, cánepa mc. centro de infectología, infectious diseases, buenos aires, argentina flu infection is a major cause of illness and one of the most common cause of work absenteeism, increasing institution costs, healthcare provider visits, use of drugs, and decreasing work productivity. vaccine against flu has an effectiveness between 70 and 90%, in health adults. objective: evaluate the impact of flu-like respiratory tract infections in a health institution staff during 1 year, comparing vaccinated with not-vaccinated groups. methods: we evaluated all causes of absenteeism along 1 year (2001), based on the written note made by the professional who has evaluated ill people, selecting flu-like respiratory infection causes. we evaluated age, working days loss related to illness, and cost on vaccinated and not-vaccinated groups. results: one hundred and sixty eight of the total staff (479 people) were vaccinated, 21 of them had flu-like infection, resulting in 40 working days lost. for not-vaccinated group, 33 people had flu and 56 lost days. lost cost for vaccinated group was of $1267, and for notvaccinated group, $1874. conclusion: we observed a decrease in working days loss and money waste related to flu-like infections on the vaccinated group. because of safety and effectiveness of vaccine against flu, the implementation of vaccination will be cost-effective for all institution staff. we studied functioning of the interferon system in 108 children with atopic bronchial asthma (ba) at the age of 2 á/14 years. the control group included 10 healthy children. we investigated the interferon status (method of grigoryan s.) and serum concentrations of ifngamma (ifng) (elisa). there was a decrease in the ifn-producing ability of leukocytes to the synthesis of ifna at 50% and ifng at 84% of children with ba. serum level of ifn of children with ba during all period of illness is compared to the children without predisposition to atopy (94.99/10.3 and 192.39/7.8 pg/ml accordingly) was significantly decreased. production of ifna increased after using viferon (recombinant a2b ifn and antioxidants). decreased ability of gammainterferonogenesis in the most children was not affected by the action of immunomodulators. there was shown interferon system's dysfunction in the development of atopy and increasing predisposition to respiratory infections and to persistent of atypical infections in children with ba. harxhi a, pilaca a, pano k. university hospital center of tirana, infectious diseases, tirana, albania congo-crimean haemorrhagic fever is viral disease with a high rate of mortality that is caused by a nairovirus, bunyaviride species. this is a zoonotic disease, which affects sporadically humans and is geographically distributed even in eastern europe and balkan. during the months of may and june 2001, in northeast of albania were reported eight cases of haemorrhagic fever. serologic tests performed in the laboratory of reference in thesaloniki, greece confirmed the diagnosis of congo-crimean haemorrhagic fever. in the mean time, who reported the outbreak in southwest kosovo of 69 cases suspected for haemorrhagic fever from which 18 were confirmed laboratory as congo-crimean haemorrhagic fever. we are describing here the clinical history of one of eight cases with cchf in albania. from the epidemiological point of view this case was considered peculiar, as it was the only one hospitaly acquired, and due to the gravity of the haemorrhagic syndrome was admitted at the intensive care unit at infectious diseases service, university hospital center of tirana. results: in the study were included 150 patients ]/18 years of age with documented hbsag-carrier ]/6 months (average age */39.4 years, male */58%, female */42%). hbv dna in serum was tested by qualitative and quantitative pcr (commercial test-system ampli-sens hbv). hbeag, hbeab, hbsag were detected by elisa (hoffmann la roche). hbv dna by qualitative pcr was detected in 41% patients, by qualitative pcr was detected in 13% patients in the concentration ]/10 5 copies/ml, in 4.3% ]/10 6 copies/ml, in 4.3% ]/10 7 copies/ml, in 2.1% ]/10 8 copies/ml ( fig. 1 ). hbv dna level distribution among the hbsag carriers. elevated (1.5 the upper limit of normal) alt level was determined in 3.8% of the hbv dna negative and 25.4% of the hbv dna positive patients. hbeag was detected in 10.2% of the hbv dna positive patients and had not been determined in the hbv dna negative patient. eleven percent of patient had the combination of the biochemical, serological and virology criteria, which are typical for active chronic hepatitis b (hbsag-carrier !/6 months, hbv dna ]/ 10 )/5 copies/ml, elevated alt). conclusion: in smolensk 41% of the hbsag-carriers have viral replication confirmed by qualitative pcr. eleven percent of them have active chronic hepatitis b. kandemir o a , polat a b , kaya a a . a medicine of faculty, mersin university, clinical microbiology and infectious disease, mersin, turkey , b medicine of faculty, mersin university, pathology, mersin, turkey the exact potential of nitric oxide in the pathogenesis of chronic viral hepatitis is not known. the elevated nitric oxide production is assumed to be responsible for the pathological changes in many inflammatory conditions, mainly via peroxynitrite, a potential oxidant that is produced by the reduction of superoxyde anion with nitric oxide. the intensity and the distribution of the immunohistochemical staining of intrahepatic inducible nitric oxide synthase were studied in the biopsy specimens obtained from 63 patients with viral hepatitis and 13 patients with elevated transaminase levels from other etiology. hepatic inducible nitric oxide synthase staining was significantly more intense in the viral hepatitis group (p 0/0.000). inducible nitric oxide synthase staining levels correlated well with the severity of the viral hepatitis using the knodell's liver histological activity index (r0/0.393, p 0/0.002). among the viral hepatitis group, the pathological distribution of the inducible nitric oxide synthase staining favored the periportal regions whereas less staining was observed in the bile duct and parenchyma regions. as nitric oxide mediated nitration of hepatocellular proteins is found to be elevated in the inflamed hepatic tissues and it well correlated with the severity of the disease, we suggest that inducible nitric oxide synthase can possibly have a critical role in the pathogenesis of chronic viral hepatitis. there were 36 patients under observation who were divided into two groups, the first of 18 patients (eight chronic virus hepatitis; three chronic virus hepatitis'/steatosis; four steatohepatitis; three chronic cryptogenic hepatitis; there were 14 men and four women aged from 35 to 74. the second group consisted of 18 patients, eight with chronic virus hepatitis; four with steatohepatitis; six with chronic cryptogenic hepatitis. there were 15 men and three women aged from 40 to 80. diagnosis was confirmed with the help of clinical data, biochemical tests, serological markers, psr-diagnostics and ultrasound examination and computer tomography of the abdomen. in the first group of patients the treatment with ursofalk was administered at the dosage of 10 mg/kg of body mass from 1 month to 3 years with improvement in general condition of the patients: heaviness, pain under the right rib, nausea and skin itch have disappeared. in all the cases, improvement in the biochemical blood analysis took place during treatment. the average index of alt activity was before */105.88 u/l, after */62.06 u/l and ast */156.44 and 95.05 u/l; alp */196.39 and 171.87 u/l; ggt */163.2 and 82.86 u/l; chol */5.38 and 4.8 mmol/l; tg */2.15 and 1.75 mmol/l. in the second group of patients the treatment was carried out with various hepatoprotectors during the courses from 1 to 6 months. before the average index of alt activity was 181.3 u/l, after */165.5 u/l; ast */126.9 and 129.0 u/l; alp */229.8 and 213.5 u/l; ggt */204.2 and 238.0 u/l; chol */5.85 and 5.67 mmol/l; tg */1.72 and 1.8 mmol/l. treatment of patients suffering from hepatitis of viral and other aetiologies with ursofalk produces a positive effect on both clinical symptomatic and biochemical indices. remission was more stable during a long period of taking the preparation. the hepatoprotective effect of ursofalk during the 3 years was sustained for the whole of the period of the treatment. after stopping, an acute attack of cytolytic syndrome was observed. with other hepatoprotectors we did not get any improvement in clinical scene of the disease or in biochemical indices. shavlov nm, kletsky sk. minsk, belarus hsv-1 and -11 have possibility to damage different organs and systems. sometimes they cause damage of the liver, which resemble viral hepatitis. the etiology of such hepatitis may be confirmed only by results of liver biopsy. we have diagnosed 12 cases of herpetic hepatitis: eight children and four adults. clinical course was different. in five cases the acute beginning took place: high temperature, the jaundice at the 2 á/3 day (the level of bilirubin was 200 á/350 mkmol, especially direct), cholestasis, the pain in upper right part of abdomen. the ascites was found in three patients with acute hepatitis during 1st week from the beginning of disease. in seven cases the beginning was gradual. the temperature was subfebrile, prolonged; malaise and moderate pain in upper part of abdomen were constant complaint. the jaundice was moderate; bilirubin increased until 120 mkmol. the level of alt was moderately increased (5 á/7 times). the blood analysis showed moderate leukocytosis with neutrophilia, and increased sre. the serological markers of hepatitis a, b, c, d were negative in all cases. hsv-1 and -2 were found in the blood. the diagnosis was defined by the results of hystochemical investigations, when the viruses were found in liver bioptat, and confirmed with the results of specific treatment. specific damage of liver cells was found: protein dystrophy and specific inclusions in cell nucleus. in all cases the treatment with acyclovir were given. the results we have observed during 1st week: the temperature became normal, the jaundice decreased and bilirubin was normal during 5 á/10 days. in one case the recidive took place 2 weeks later after treatment. the second course of acyclovir with intron a gave good results. nesic z a , delic d b , prostran m a , vuckovic s a , stojanovic r a . a department of pharmacology, school of medicine, university of belgrade, belgrade, yugoslavia , b clinical center of serbia, institute for infection and tropical disease, belgrade, yugoslavia the large number of unsolved cases of acute and chronic hepatitis has most probably the viral etiology. in mid 1990s, two independent groups of authors reported a new human hepatotropic virus, with flavivirus like genomes, hepatitis g virus (hgv). the aim of this pilot study was to determine the prevalence of hepatitis g viral infection among patients at high risk of exposure to blood and blood products, as well as to evaluate if the risk of hgv infection was higher among them than in the general population. immunoenzyme test on microtitration plate for detection antibodies against hgv e2 antigen in plasma or sera (r&d systems, minneapolis, usa) was used for evidencing anti-hgv igg antibodies in sera. anti-hgv antibodies were detected in the control group (blood donors) in 8.3% (2/24) patients. prevalence of anti-hgv antibodies among i.v. drug users was evidenced in 42.8% (9//21), in hemophiliacs 41.2% (7/17), in patients acquiring multiple blood transfusion 42.8% (3/7), in hemodialyzed patients 33.3% (4/12) and in patients with transplanted organs 57.14% (4/7). our results suggest that patients exposed to blood or blood products have a higher risk of hgv infection than general population. evaluation of ortho total hcv core antigen assay in assessment and follow-up of patients treated for chronic hcv ps162 lunel f, veillon p, payan c. chu angers, laboratoire de bactério-virologie, angers, france an assay to quantitate 'total' hvc core antigen (hcv ag) in serum or plasma, may reflect viral load, has been developed by ortho-clinical diagnostics. methods: we evaluated hcv ag with two quantitative assays for hcv rna: bdna 3.0 (bayer) and monitor 2.0 (roche). we studied 191 samples from untreated patients and 237 from 144 patients with chronic hcv treated with ifn or ifn/ribavirin. results: correlation of ag and quantitative assays was high (r0/ 0.85 for bdna 3.0 and 0.83 for monitor 2.0). no difference between the levels of rna and ag among hcv genotypes (r0/0.82 á/0.96) was found. ag values, before treatment, were significantly lower in sustained responders (sr) than in other groups (5.4 log versus 6 log, p b/0.001). in patients treated with ifn or combination therapy, we found very good correlation between decrease and negativation of ag and viral load: 2 log iu/ml decline after m1 of interferon was significantly correlated with the negativation of hcv ag and sr. thirty-eight/41 of sr had a rna load decrease !/2 log iu/ml and 39/ 41 had a negativation of hcv ag after m1. conclusion: total hcv core ag appears to be a new tool for monitoring patients with hcv infection. hepatitis c virus rna and hcv core antigen kinetics predict the efficiency of interferon-alfa and ribavirin therapy in naive patients infected by hcv genotype 2 or 3 ps163 fifty-five patients infected by genotype 2 or 3 were treated with a primary dose of 3 (if hepatitis c virus (hcv) rna b/3 meq/ml) or 6 million units of interferon alfa-2b (ifn) thrice weekly for 12 months. ribavirin was added at month 3 (m), until m12 if hcv rna was found positive after m2 of ifn. the viral kinetic was assessed during the follow up by serial measurements of hcv rna (bdna 3.0 and monitor 2.0) and using a new assay from ortho-clinical diagnostics which is able to quantify total hcv core antigen. sustained virologic response was observed in 65% of the patients (36/55). after 1 month of ifn treatment, sustained responders had a fall of hcv rna and hcv core antigen higher than non-responders (4.339/1.84 log ui/ml versus 0.159/0.31 log ui/ml, p b/0.001, for hcv rna) and (1.689/ 0.89 log (pg/ml)/10 000) versus 0.179/0.18 log (pg/ml)/10 000) p b/ 0.001, for hcv core antigen). after 1 month of ifn, the positive and negative predictive values of sustained response were, respectively, 100 and 17.4% for hcv rna negativation and 97.5 and 37.5% for hcv antigenemia negativation. these results suggest that both kinetics of viral load and antigenemia are highly predictive of sustained response. theodorou m, petinelli i, pontikaki d, mela c, blana a, papanastasiou a, toliopoulos a, stavrakaki m, sagkana e. microbiology department, western attika general hospital, greece, egaleo, greece greece has accepted a big number of economic immigrants lately. we investigate the prevalence of hepatitis b/c as well as the epidemiological features that might influence the public health. 1.235 economic immigrants from: albania 630, eastern europe 411 and from asiatic-african countries 194, visited our hospital to be checked in order to get a health certificate to obtain the green card. they where tested for hepatitis b/c. the serological markers were determined by immunoenzymatic method. all hbsag('/) and anti-hcv were further tested for hbv dna and hcv rna by competitive rt pcr. hcv rna('/) were genotyped by strip hybridization immunoassay. in 630 albanians 17.5% were hbsag('/), 14.6% hbv dna('/), 1.1% anti-hcv('/) and 0.8% hcv rna('/). in 411 east europeans 5.1% were hbsag('/), 4.4% hbv dna('/), 2.43% anti-hcv('/) and 1.9% hcv rna. in 194 asians-africans, 2.6% were hbsag('/) and 2% hbv dna('/). in 101 pakistanis, 26.7% were anti-hcv('/) and 22.0% hcv rna('/). of the rest of asians-africans, 11.82% were anti-hcv('/) and 9.3% hcv rna('/). albanians: higher prevalence of hbv infection (14.6%). greek blood donors: 1% pakistanis: hcv infection is 22% (predominance of 3a type), general greek population: 2%. public health services in greece and europe must take appropriate measures. el zawawy la a , mohamed on b , ali sm a , eissa me a , allam sr a . a faculty of medicine, parasitology, alexandria, egypt , b high institute of public health, microbiology, alexandria, egypt the purpose of the study was to investigate the influence of schistosomal suppression on the antibody response to hepatitis-b vaccine (hbv) and to study if the vaccine has any protective effect on experimental () infection. the results obtained revealed that infection reduced the serum antibody level against hbv. parasitological and histopathological findings showed significant protection against infection. the conclusion reached was; in order to reduce the incidence of virus-b infection especially in schistosomiasis endemic areas, public health officials should evaluate a policy for regulation of hbv booster vaccination to enhance the population immunity against hepatitis-b infection. cooper e, fisher t, shingadia d. newham general hospital, family clinic, london, uk sustained anti-retroviral combination chemotherapy requires excellent adherence to the regimen so as to suppress viral replication sufficiently to delay the emergence of resistance. if chemotherapy were taken to scale, e.g. in africa, erratic adherence might soon lead to multi-resistant circulating virus. we reviewed our experience in a well established london family clinic with a team including community nurses. we reviewed the records of the 23 african immigrant children, aged 2 á/14, treated with anti-retrovirals exclusively at our centre throughout 2001. whereas 14 had undetectable hiv rna within the year, only four had undetectable rna throughout the year. four failed therapy through proven resistance mutations, but nine were considered through circumstantial evidence to have rising viral loads primarily because of poor adherence. three were known to have stopped taking drugs for extended periods. the three boys over 11 years were unreliable in adherence, but the one girl in this age-group was fully adherent. our preliminary assessment is that for the children in our families, despite a team approach and home visits, nonadherence to haart may be twice as common as selection of a dominant viral mutant as a primary cause of failure to sustain viral suppression. quiros-roldan e, moretti f, castelli f, el-hamad i, carosi g. the prevalence of hiv related lipodystrophy-syndrome depending on the definition and severity of lipodystrophy ranges from 5 to 80%. we have retrospectively reviewed the medical records of 58 african patients followed. the characteristics are shown in the 3.4% of africans had triglycerides !/200 mg/ml and 1.7% had cholesterol !/250 mg/ml, none had both metabolic alterations. glycemia !/120 mg/ml was observed in 8.6% patients. it is interesting highlight that in any the africans morphological changes were noted and all of them showed weight stable. although the low prevalence of metabolic alterations may be attributed to the different ethnic alimentary behavior if self-body perception by african is not as accurate as by caucasian on the estimation of the body changes have to be investigated. alabaz d a , alhan e a , yaman a b , evliyaoglu n a , kocabas e a , aksaray n a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey hepatitis a virus (hav) infection is usually asymptomatic in children. however, it may occasionally cause a severe disease with high morbidity and mortality, and loss of school or business days. in a previous study, we have shown that every one of two to three school children from upper social classes living in adana carries high risk of hav infection. it is well known that maternally transmitted anti-hav antibodies interfere with hav vaccination. in an effort to determine the optimal age for hav vaccination, 122 babies (52% girls and 48.5% boys) born in our hospital were prospectively followed up at least 24 months for the presence of maternal antibodies to hepatitis a (anti-hav igg). anti-hav igg titers were measured from the blood specimens obtained at birth from the mothers and from the offsprings at months, 0, 3, 6, 9, 12, 15, 18, 21 and 24. the prevalence of positive anti-hav at birth (95%) was similar to those of hav seroprevalance studies carried out in adults in our area. the disappearance of antibodies occurred between the 1st and 21st month of life. the prevalence of anti-hav igg among children aged 0, 9, 12, 15, 18 and 21 months were 95, 60, 32, 9, 4 and 0%, respectively. in light of these findings, we suggest that hepatitis a vaccination be given after 21 months of age. earlier vaccination may be ineffective due to interference with maternally transmitted anti-hav antibodies. ghaderi b, alaghebandan r, rastegar lari a. department of microbiology, iran university, tehran, islamic republic of iran diarrhoea is a major public health problem in developing countries. amoebiasis is one of the most common causes of diarrhoea in iran as an endemic area for amoebiasis. little, however, is known about the extent of the condition in our society. the aim of this study is to determine socio-demographic and clinical characteristics of patients with intestinal amoebiasis. during july and august 2001, we collected 90 patients with diarrhoea among 5000 patients who visited at a referral hospital in shahriar area (in countryside of tehran), iran. thirty out of 90 patients (33%) had intestinal amoebiasis and were followed up prospectively until the resolution of the illness. nineteen of 30 (63.3%) patients were male and the remaining of 36.7% was female. the patients were aged 1 á/76 with mean of 28.4 years. most of the patients (70%) were below 30 years of age and the peak of occurrence was between the age of 17 and 26 years. watery diarrhoea with abdominal cramps was the main clinical feature. seventy percent of patients were resident in urban area and the remaining (30%) in rural area. average family income was low and all patients were in low socioeconomic level. water supplying system for all patients was pipeline water. low socioeconomic level associated with poor personal hygiene was the most important factor for highly prevalence of this problem in our society. also it seems that food plays important role in transmission of protozoa then water. the new strategy for allele identification of the genes coding for pertactin and pertussis toxin subunit s1 in bordetella pertussis ps170 bordetella pertussis strains demonstrate a significant polymorphism in toxin s1 subunit and pertactin, which are major protective antigens of the organism. monitoring the changes in prevalence of particular alleles of genes coding for these proteins in local b. pertussis populations is an essential issue in cases of the observed decrease of vaccination effectiveness. we have developed a new method for allele identification of these genes, which eliminates the necessity of dna sequencing. the approach is based on the identification of the number of repeats or the presence of specific nucleotides in the polymorphic regions or residues, respectively, of the genes and utilises products of their full or partial pcr amplification. the nucleotide heterogeneity in each polymorphic site is analysed either by the differential digestion of the amplicons or by the arms (amplification-refractory mutation system) methodology. numbers of repeats in particular regions of the genes are revealed by the size analysis of the adequate pcr products or their restriction fragments. in all cases the presence, size or pattern of dna molecules obtained is visualised by the agarose gel electrophoresis. the preliminary analysis of the recent and archival b. pertussis strains identified in poland was performed using the described approach. the presented strategy provides a much easier, faster and more cost-effective than dna sequencing mean to study the polymorphism of the major b. pertussis antigens. vaccination coverage and history of vaccine preventable infectious diseases among students in second year of medicine and pharmacy of tours university ps171 borderon jc a , hamed a b , ragot s b . a centre hospitalier universitaire, tours, france , b médecine préventive universitaire, tours, france the purpose of this study was to determine the level of infectious risk in students who will be exposed to patients. information was obtained by a questionnaire for each student, and by checking medical records for immunization coverage and vaccine preventable infectious diseases. answers could be specified for 138 students, of whom 97 females (f) and 41 males (m). the number of non-immunized students was against diphtheria: two, tetanus: three, pertussis: four, poliomyelitis: two, hepatitis b: six, and hepatitis a: 127, respectively. among the 52 students non-vaccinated against measles, 14 (nine f and five m) had no history of that disease. among 56 (31 f and 25 m) non-vaccinated against rubella, 17 (10 f and seven m) had no history of that disease, uncertain in seven others (six f and one m). the date of vaccination was often late regarding recommendations. fifteen students had no history of varicella. one student had not received bcg vaccination. fifty-eight students had received two, and 14 three bcg vaccines. post-bcg tuberculin skin testing was missing after 28 first bcg, 14 second and 3 third bcg. the date of the first tuberculin test was often one or several years after bcg vaccination. adverse effects of vaccination were rarely reported: two cases of fever (dt polio, measles); three cases of local reaction (dt polio, dtp polio). one case of contraindication for influenza vaccine: egg allergy. the survey shows failure in immunization coverage actually recommended in health care students. objectives: to present the morbidity of rabies and evaluate the efficiency of our prophylaxis scheme in lasi county. material and method: we made a retrospective study of the rabies cases in the patients admitted in our unit in a 13th-year period. we have analysed all the clinical, epidemiological and biological aspects. results: in a 30-year period, 22 cases of rabies were admitted in the clinical infectious diseases hospital lasi. the highest incidence was for 1986 á/1990 */eight cases (36%); the highest yearly cases were three cases in 1974 and 1986. most of the patients were male (59%), came from suburban areas (21 patients). eight cases occurred in may á/june, wild animals were involved in half the cases (fox, wolf). for 18 patients, no prophylaxis was performed and an incomplete course in four cases. the period of time to the appearance of the first symptom was 18 á/120 days. the prophylaxis scheme led to a good protection. conclusions: in lasi county, rabies is a problem with a prevalence of 0.73%/year. trends in the use of antimicrobials in riyadh in 2000 were analyzed. data was obtained from a survey of 652 randomly selected families of school children aged 6 á/8 years in a 3-month period in 2000. one hundred and ninety-nine (33.8%) students were on antibiotics in the month preceding the study; 108 (56%) received antibiotics for the diagnosis of pharyngitis; 177 (90%) students antibiotics were prescribed by a physician; and in 161 (83%) the duration of antibiotics was less than 1 week. this study shows a major problem in antibiotics prescription in our community and also the need to establish effective antibiotics policy in general practice to limit the potential emergence of drug resistance bacteria in the community. mir s a , cura a a , erdogan h a , guler s a , sengul gn a , koyu a a , ozinel ma b . a department of pediatrics, ege university medical faculty, izmir, turkey , b department of microbiology, ege university medical faculty, izmir, turkey antibiotic susceptibility spectrum of childhood urinary tract infection agents are geographical variation. the current antibiotic regimens and the selection of antibiotics for prophylaxis should be re-evaluated periodically. the objective of our study was to determine the local resistance rates to antibiotics and to give a direction for the selection of antibiotics in uti treatment. we evaluated 4124 urinary culture assays retrospectively, sent from pediatrics and pediatric surgery inpatient and outpatient clinics of our hospital during the last 6 months, and investigated the isolated pathogens and the resistance rates to antibiotics. in addition, the data obtained were compared with of 5 years ago. with respect to the resistance rates to antibiotics of uti pathogens, the resistance rates of e. coli for carbapenems, aminoglycosides and third generation cephalosporins were 1, 10 and 10%, respectively as before, but the rates for ampicillin increased from 67 to 75% and for tmp-smx it increased from 49 to 61%. we concluded that the resistance profiles to antibiotics should be reviewed every 5 years at least and thus the selection of proper antibiotics would lessen the morbidity as well as the medical expenses. chanturidze tk, tsiklauri r. ministry of labor, health and social affairs, public health, tbilisi, georgia purpose: since 1991 infectious diseases (id) are increasing in georgia. this study is aimed to reveal economic barriers of effective id control by assessing financial contribution to id from public and private sources, household's total spending on health and their capacity to pay. sources: 1) national household expenditure and revenue survey. 2) who fair financial contribution methodology. 3) meta-analysis. results: 51.8% of population leaves under the poverty level; 70% out of total household expenditure (average 270 gel; us$ 130) 30% is spent on food */non-subsistence income covers expenditures on goods and services including health; 15% of population refuses health services because of inability to pay; public spending on health comprises 33% of total health expenditures; public spending on id control is below 1 gel per capita; almost all private spending goes to id treatment and equals to 28.6 gel per patient. conclusions: insufficient public spending on id control transfers the burden to the population with extremely low capacity to cover health expenses. refusal to utilize health services, and incomplete treatment and increases the threat of id spread and drug resistance. government should increase the allocations to id from public sources for effective id control in georgia. antimicrobial consumption trends in children's university hospital ps177 ratchina s a , averchenkova l b , jarkova l a . local surveillance of antimicrobial (am) consumption is essential to promote the rational use of this group of drugs. the purpose of this study was to analyze the trends of am use in the children university hospital in 1998 and 2000. data on am usage were obtained from the hospital drugstore requests in the 250-beds multi-ward children university hospital. consumption was expressed as the number of ddd per 100 bed-days (b á/d). the total am consumption figures were similar in 1998 and 2000 (7.7 and 8.2 ddds/100 b á/d, respectively) with notable differences in am prescribing patterns. penicillin consumption increased from 1.8 to 3.8 ddds/100 b á/d mostly due to amoxicillin. the overall aminoglycoside usage remained comparable (0.95 vs. 0.82 ddds/100 b á/d) though amikacin has considerably replaced gentamicin. there was a sevenfold increase of ciprofloxacin (0.03 vs. 0.2 ddds/100 b á/d) along with the evident decrease of tetracycline and co-trimoxazole consumption found (1.5 vs. 0.9 ddds/ 100 b á/d and 0.8 vs. 0.1 ddds/100 b á/d, respectively). the tendency to prescribe more effective in respect of the local resistance data and/or more safe am was detected in 2000 comparing with 1998 that can be explained by the introduction of the local guidelines for infectious diseases management in 1999. clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis */a pilot study ps178 the aim of this pilot study was to determine the efficacy and tolerability of clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis (ct). fifty-two patients older than 18 years of age with diagnosed chronic chlamydial prostatitis were enrolled. the presence of ct in expressed prostatic secretion or urine specimen voided immediately after prostatic massage was confirmed by isolation on mccoy cells and lugol staining. the majority of patients suffered from suprapubic pain and pain in the groin. twelve patients had no clinical symptoms. according to rectal palpation, prostate gland was normal in 35 patients, tender and soft in 12 and firm in five patients. clarithromycin was administered orally 500 mg twice daily for 14 days. simultaneously the patients' partners received 500 mg orally twice daily for 7 days. clinical efficacy and tolerability of administered clarithromycin were evaluated 1 á/7 days and 4 á/6 weeks after the end of treatment. bactericidal efficacy of administered drug was evaluated 4 á/6 weeks after the end of treatment. the eradication of ct was achieved in 30 out of 40 patients, while 28 patients were clinically cured. two patients had nausea and elevated serum transaminases. in asymptomatic patients, the eradication of ct was achieved in 10 of 12 patients who reported no side effects. this pilot study has shown an excellent efficacy and tolerability of clarithromycin in the treatment of patients with chronic chlamydial prostatitis. women with diagnosis of urinary tract infections (uti) often demonstrate vaginal colonisation with alpha-haemolytic escherichia coli strains. in the present study we decided to evaluate a distribution of virulence genes encoding for cytotoxic necrotizing factor type 1 (cnf1), p-fimbriae, s/f1c-fimbriae aerobactin (aer), and afa genes in alpha-haemolytic e. coli strains isolated from gynaecological material in our region and to compare the detected sequences in clinical isolates of other diagnostic groups. of 127 alpha-haemolytic e. coli strains, 41 were isolated from urine, 44 from gynaecological specimen, and 42 were faecal strains. e. coli strains were tested for the production of haemolytic phenotype on blood agar plates. the amplification of virulence factors was performed by pcr according to previously described protocols (le bouguenec et al., 1992; blanco et al., 1996; and yamamoto et al., 1995) . we found that all gynaecological alphahaemolytic strains were positive for cnf1, (p b/0.001 compared to 78% of urine strains, and p b/0.0001 compared to 61% of faecal strains). similarly, sfa/foc specific dna sequences were found in 100% of gynaecological isolates (p0/0.01 compared to 85% of urine strains and p 0/0.005 compared to 83% of faecal strains). from this point of view, the female genital tract seems to be a potential reservoir of these uropathogenic e. coli strains. azithromycin in the treatment of pelvic inflammatory disease caused by chlamydia trachomatis ps180 administered in 35 hospitalized patients with chlamydial pid. the diagnosis was made prior to hospitalization. microbiological analysis of urine, blood and swab specimens collected from endocervix, vagina and urethra confirmed c. trachomatis to be the single suspected causative pathogen of pid. the presence of c. trachomatis in swab specimens from endocervix was examined by dnk/rnk hybridization. azithromycin was administered 5 á/7 days after samples for microbiological analysis were collected in dose of 1)/500 mg iv for 5 days. clinical efficacy and tolerability of therapy were assessed 1 á/7 days after the end of therapy and clinical and microbiological analysis 3 á/4 weeks after completion of therapy. the eradication of c. trachomatis and normalization of gynecological findings were achieved in 33 and disappearance of subjective symptoms in 30 patients. no side effects and deviations from normal values in hematologic and biochemical blood parameters were recorded. this study showed high bactericidal efficacy, rapid clinical effect and good tolerability of once-daily administration of 500 mg azithromycin for 5 days in the treatment of patients with pid caused by c. trachomatis . altindis m a , cevrioglu s b , aktepe oc a , cetinkaya a . a kocatepe university school of medicine, microbiology, afyon, turkey , b kocatepe university school of medicine, obstetrics and gynecology, afyon, turkey diagnosis of the causative organism of the vaginitis is usually based on clinical criteria. a standardized, laboratory based and rapid diagnostic test for the identification of these organisms is desirable. to determine the laboratory method that best predicted the causative organism, we calculated the sensitivity, specificity and predictive value of positive and negative test for clinical criteria, an oligonucleotide probe test (affirm vpiii-bd usa) and compared them with the combination of positive vaginal culture and gram-stained vaginal smear. we evaluated 40 consecutive women aged 21 á/48 years, attending for vaginal discharge. vaginal swab specimens were used for culture of gardnerella vaginalis , trichomonas vaginalis and candida sp, preparation of a vaginal smear for gram-stain interpretation and wet mount evaluation and affirm test. affirm detected g. vaginalis in 10 (25%), candida sp in three (7.5%) women and no trichomoniasis case found by any methods. the sensitivity and negative predictive values of affirm test and clinical signs were same (100%) in identifying of bacterial vaginosis. however, affirm test was more specific (94 vs 72%) and also has higher positive predictive value (80 vs 53%) than clinical signs. we did not evaluate the results for patients with candidiasis because of less number. according to these results affirm-microbial identification tests are objective and specific for the rapid diagnosis of the bacterial vaginosis. comparison of efficacy of single dose of tinidazole with standard dose of metronidazole in giardia lamblia infection (preliminary report) ps182 fallah m a , moshtaghi aa b . a university of medical sciences, parasitology, hamadan, islamic republic of iran , b university of medical sciences, pediatrics, hamadan, islamic republic of iran objectives: giardia lamblia is the most common intestinal protozoa in developing countries. treatment of the infection with metronidazole, the drug of choice, requires a long course of therapy and produced some side effects. the object of this study is to determine efficacy and side effects of tinidazole in g. lamblia infection. this is a preliminary report of an ongoing trial. methods: a randomized controlled clinical trial was carried out and 47 subjects with g. lamblia infection were treated with tinidazole or metronidazole. tinidazole 50 mg/kg single dose and metronidazole 25 mg/kg three times a day for 7 days were given orally to 24 and 23 children, respectively. parasitological cure was documented when there were consecutive negative stool examinations at 1 á/2 weeks after therapy. results: twenty-one of 23 individuals treated with tinidazole and 20 of 24 children treated with metronidazole had parasitological cure. cure rates between two groups were not significant statistically. no major side effects were observed except one case in metronidazole group who had mild headache and abdominal pain for 2 days. conclusions: we concluded, tinidazole at the dose has efficacy equal of metronidazole in the treatment of g. lamblia infection. because of single dose prescription, short course of therapy and good compliance of patients, this preparation is preferred to metronidazole in giardial infection. ebeid fa, seif el-din sh. theodor bilharz research institute, pharmacology, cairo, egypt b-carotein was given in different doses starting from 2.5 to 10 mg/kg body weight (b.w.) for different groups of albino mice 4 days before infection with s. mansoni . infection of animals was done by body immersion using 80 egyptian strain of s. mansoni cercariae/mouse. forty-nine days after infection the animals were sacrificed and hepatic and mesenteric worms were extracted and determined. ova count in liver and intestinal tissue and the total number of worms/animals were also determined in experimental groups comparing with infected control animals. the results indicated marked decrease in number of worms and ova count in both liver and intestine comparing with control ones. this reduction increased significantly with increasing dose. it was concluded that b-carotein could be used as a prophylactic agent against s. mansoni infection. barisic z, babic-erceg a, borzic e, zoranic v, carev m, kaliterna v. department of microbiology, public health institute, split, croatia the aim of this study is to determine frequency of pseudomonas aeruginosa urinary tract infection (uti) in outpatient's population in south croatia and to suggest optimal antimicrobial treatment for these patients. during 3 months long observation period, from total number of 13 158 examined urine specimens, significant bacteriuria was found in 2341 specimens. p. aeruginosa was the sixth most common isolate, it was isolated from 94 specimens (4.02%). these 94 specimens were taken from 57 different patients. susceptibility testing was performed by disk diffusion method, and the following results were obtained: resistance to cefibuten occurred in 94.74% patients, to norfoxacin in 49.12%, to ciprofloxacin in 47.37%, to gentamicin in 42.11%, to netilmicin in 35.09%, to amikacin in 24.56%, to ceftazidime in 3.51% and to imipenem in 1.75% patients. p. aeruginosa strains showed better susceptibility to tested parenteral antibiotics than to antibiotics for oral use which complicated treatment in outpatients. the best susceptibility was shown to imipenem, but this drug is inappropriate for use in outpatients setting, so the best choice for treatment p. aeruginosa uti in our outpatients is treatment with ceftazidime, and the second choices are aminoglycoside drugs amikacin and netilmicin. silan l a , breza j b , krcmery v jr. c . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school, bratislava, slovakia , c department of pharmacology, st. elisabeth cancer institute, bratislava, slovakia we studied the clinical efficacy of oral treatment with ciprofloxacin/ cpf/ alone and combined with clarithromycin in patients with complicated urinary tract infection/cuti/ with or without an indwelling catherer. patients were randomly allocated to 600 mg cpf/cpf group/ or to 600 mg cpf plus 600 mg cam/combination group/ for 14 days. evaluation was done on day 14 according to the criteria advocated by the japanese urinary tract infection committee. in patients with a urinary catherer, the combination achieved a higher complete bacterial elimination rate /50%/ and clinical efficacy rate / 84%/ than cpf alone /30 and 61.5%, respectively/. while no significant difference was found in the bacterial elimination rate between the two groups, the clinical effect of the combination /40%/ was superior to that of cpf alone /23%/ in patients with an indwelling catherer. the better clinical efficacy of the combination may partly be attributed to the antibiofilm effect of cam in the clinical setting. the results also indicated that difficulties still remain in the treatment of cuti in patients with an indwelling catherer. in conclusion, clinical study suggested that cam might have an inhibitory action on biofilm formation in the clinical setting. combination of cam with other appropriate antimicrobial agents may have a favorable effect on the treatment of cuti. vesicoureteral reflux and urinary tract infections */the management of primary vesico-eureteral reflux in children ps186 the children studied presented with primary vesicoureteral reflux at derer s universitz hospital in bratislava between 1974 and 1994. seven hundred and sixty patients, 179 boys and 581 girls, suffering from primary vesicoureteral reflux in age from 6 months to 18 years were tested and systematically analyzed outcomes data for seven treatment alternatives. key outcomes identified were probability of reflux resolution, likelihood of developing pyelonephritis and scarring, and possibility of complications of medical and surgical treatment. available outcomes data on the various treatment alternatives were summarized and the relative probabilities of possible outcomes were compared for each alternative. conclusions: increased of urinary tract infection, vesicoureteral reflux nephropathy includes early diagnosis, appropriate evaluation, effective atb therapy, and surgery indicated. the main determinants of renal damage are bstruction, age, sex, predisposition on renal scarring, reflux grade and laterality, therapeutic delay, individual susceptibility, bacterial virulence and immunogenetic status. 3) the one and only absolute indication for surgical management is failure of medical therapy to prevent chronic recurrent pyelonephritis, renal injury or other reflux complications and eliminations of the reflux condition will minimize their likelihood. genetically conditioned immunopathogenic mechanisms are involved in the pathogenesis of the chronic recurrent pyelonephritis in patient suffering from vur. for most children we recommended continuous antibiotic prophylaxis as initial treatment-medical therapy is based on the principle that reflux often resolves with time, and antibiotics maintain urine sterility and prevent infections while the patients awaits spontaneous resolution. 6) vur predispose an individual to renal infection, the immunological and inflammatory reaction caused by a pyelonephritic infection may result in renal injury or scarring. silan l a , breza j b . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school of medicine, bratislava, slovakia elderly patients with uti are believed less likely to be cured by antimicrobial therapy than younger patients. the reasons for this poorer outcome have not yet been clarified. we have investigated the efficacy of antimicrobial therapy in elderly patients with complicated uti. five hundred patients, 260 men and 240 women, who had complicated uti/268 symptomatic and 240 symptomatic and were 21 á/80 years of age, were treated with one of three different drugs, one was a never quinolone and two were oral cephems. multivariate logistic regression analysis of treatment outcome revealed that the clinical response was significantly related to general underlying diseases and diseases of the urinary tract, but not to age, symptomatic or asymptomatic uti, or infection site such as the kidney or bladder. we concluded that the clinical effectiveness of an antimicrobial agent was not directly related to age, and that urological examination for underlying disease and control of them is quite important for effective treatment and control of complicated utis, especially in elderly patients. the study on the frequency and antimicrobial resistance of escherichia (e ) coli in urine isolates of patients admitted to maribor teaching hospital in 1998 and 2000 ps188 rebersek gorisek j a , baklan z a , unuk s a , novak d b . a department for infectious diseases, teaching hospital maribor, maribor, slovenia , b department for microbiology, regional institute of public health maribor, maribor, slovenia purpose: the aim of the study was to determine the frequency and antimicrobial resistance of escherichia coli isolated from urine samples of patients admitted to maribor teaching hospital in 1998 and 2000. the frequency and the antimicrobial resistance were compared between years 1998 and 2000. methods: in the prospective study going on between 1998 and 2000, all urine isolates from patients at maribor teaching hospital were collected and analysed. urine cultures were done using the modified sanford method. the susceptibility testing was performed by disk diffusion method according to nccls. results: in the year 1998, 2563 urine isolates and in the year 2000, 2325 urine isolates were analysed. e. coli represented 39.1% of urine isolates in 1998 and 39.4% of urine isolates in 2000. e. coli resistance rates (%) to amoxycillin was 39.5 in the year 1998 and 38.4 in the year 2000; to amoxycillin/clavulate was 15.4 and 10.5; to cefalotin was 7.8 and 5.7; to cefaclor 2.8 and 2.18; to trimethoprim sulfamethoxazole was 15.0 and 18.4; to ciprofloxacin was 4.9 and 6.9; to gentamicin was 2.3 and 2.2. conclusion: compared to 1998, the frequency of e. coli isolated from urine samples is similar to that in the year 2000. the resistance to amoxycillin, cefaclor and gentamicin is stable. the resistance to trimethoprim sulfamethoxazole and ciprofloxacin is increased and the resistance to amoxycillin/clavulate and cefalotin is decreased. prevalence of the resistance to metronidazole, furazolidone and nitrofurantoin in helicobacter pylori clinical strains ps189 de la obra sanz p a , roman jl a , lomas e a , villar h b , lopez-brea m a . a hospital de la princesa, microbiology, madrid, spain , b hospital de san agustin, microbiology, aviles, spain the objective of this study was to determine the prevalence of metronidazole, furazolidone and nitrofurantoin resistance in helicobacter pylori clinical isolates. methods: a total of 286 strains of h. pylori were included in this study. all these were tested against metronidazole, and 164 against furazolidone and nitrofurantoin by an agar dilution method. resistance was defined as: metronidazole, mic !/8 mg/l; and mic !/4 mg/l for furazolidone and nitrofurantoin. results: sixty-eight strains were resistant to metronidazole (23.8%). the mic50 and mic90 values were 1 and 32 mg/l, respectively. three of 164 strains (1.8%) were furazolidone resistant (mic 0/4 mg/l), two of these strains were metronidazole resistant (mic0/16 mg/l) and they had mic of 2 mg/l for nitrofurantoin. the mic50 and the mic90 were 0.125 and 0.5 mg/l, respectively for furazolidone. only one of the 164 strains (0.6%) was nitrofurantoin resistant (mic 4 mg/l), this strain was metronidazole resistant (mic 128 mg/l) and had a mic0/1 mg/l for furazolidone. the mic50 and the mic90 were 0.5 and 1 mg/l, respectively for nitrofurantoin. conclusion: the low frequency of furazolidone and nitrofurantoin resistance, compared to metronidazole suggests that the furazolidone and the nitrofurantoin may be good alternatives to metronidazole for treatment of h. pylori infections. antimicrobial resistance of campylobacter isolated from human origins ps190 zhukhovitsky vg, drabkina iv. department of bacteriology, botkin hospital, moscow, russian federation the purpose of the study was to determine the antimicrobial resistance of 50 thermophilic enteropathogenic campylobacter spp. (tec) isolated from human under acute diarrhoea in 2001 in moscow. among 50 tec strains 45 c. jejuni and five c. coli were identified. the antibiotic tested by disk diffusion test on mueller-hinton agar with sheep blood were ampicillin (a), amoxycillin/clavulanate (ac), imipenem (i), meropenem (m), erythromycin (e), clarithromycin (cl), tetracycline (t), doxycycline (d), gentamicin (g), azithromycin (az), chloramphenicol (ch), lincomycin (l), ciprofloxacin (c), nalidixic acid (na). the resistant rate of the tec isolates was highest for na (42%) followed by a (38%), t (36%), d (20%), n (20%) and cl (16%). a moderate resistance rate was obtained for a (8%), ch (6%), az (6%), ac (2%). none of the isolates demonstrated resistance to i, m and g and four of 50 isolates (8%) were sensitive for all the antibiotics tested. mic90 to na was estimated as 64 mg/l. among 21 na resistant tec strains 20 (40%) were identified as c. jejuni and one (2%) as c. coli . among c. jejuni and c. coli na resistant rate was 44 and 20%, respectively. one na resistant c. coli and nine na resistant c. jejuni were resistant to ciprofloxacin. ring c, atanassova v. department of food hygiene and microbiology, school of veterinary medicine, hannover, germany aim of the study: poultry meat is known to be often contaminated with salmonella and other foodborne pathogens and thus has to be considered as a possible source for human infections. the aim of the study was to monitor the resistance of salmonella isolates from poultry meat of different european countries to various antibiotics. material and methods: from september 2001 to december 2001 a total of 422 samples of frozen poultry meat from france, germany, italy, spain, the netherlands and portugal were examined for the prevalence of salmonella using classical cultural detection as well as rflp-pcr. all isolates were tested for their sensitivity towards ampicilline, kanamycine, ciprofloxacine, tetracycline, trimethoprim, sulfamethoxazole, nalidixic acid and erythromycine using standard procedures. results: from 18.01% of all examined samples salmonella spp. were isolated. of these isolates 32.9% were characterized as salmonella , 42.1% as s. hadar and 17.1% as s. typhimurium . nearly 100% of all isolates were resistant to erythromycin. resistance towards four or more isolates was observed in several cases. discussion: the consumption of poultry meat, if insufficiently prepared, has still to be considered as a major source for human infection with salmonella spp. the question arises whether the resistance of the isolates to various antibiotics is of clinical importance in the treatment of the patients. objective: to provide insight into the epidemiologic situation of salmonellosis for the nis area (the largest area in serbia, with inhabitants */648,000). methods: the material was processed at the institute for public health (epidemiology and microbiology divisions). isolation of microorganisms was performed on apparatus for rapid identification (vitek-biomerieux) and by applying elisa tests and classical microbiological methods. results: in the period 1992 á/2000, 2857 salmonella laboratory confirmed cases were reported. the greatest number of diseased in the 0 á/4 years group. the most frequent isolated salmonellae were: s. enteritidis (83.7%) and s. typhimurium (11.2%), s. hadar , s. agona , s. virchow , s. infantis , s. derby , s. enteritidis showed the greatest sensitivity to antibiotics with the infrequent resistance to ampicillin and trimethoprim-sulfamethoxazole. s. typhimurium showed the greater resistance to the wide spectrum of antibiotics and some isolates were resistant to all antibiotics tested. the less common types of salmonella were sensitive to all antibiotics except trimethoprimsulfamethoxazole and ampicillin. conclusion: specific resistance to some antibiotics was related to serotypes. typhoid fever */retrospective study of 52 cases in lebanon ps193 tohme a, abboud j, ghayad e. hôtel-dieu hospital, internal medicine, beirut, lebanon objectives: to present epidemiological and clinical features of typhoid fever in lebanon. methods: fifty-two patients were seen at hotel-dieu hospital of beirut between 1995 and 1999. diagnostic criteria were positive blood culture for s. typhi or paratyphi and/or a somatic o agglutinin titer ]/ 1/160 as determined by the widal test with symptoms suggestive of typhoid fever. we also present an epidemiological study of 3864 cases registered by the ministry of health during the same period. results: among the 3864 cases, 40% of the patients' ages were between 15 and 40 years and 33% were less than 14 years. the overall male to female ratio was 0.98 and 24% of cases were seen on january, february and 11% on august. among the 52 patients, young adults were the most affected. average duration of symptoms before the diagnosis was 109/8 days. the main presenting symptoms were: fever (96%), diarrhoea (37%), abdominal pain (31%) and headache (29%). complications were noted in 33% of cases and digestive complications were the most prevalent. leucopenia was not a helpful diagnostic marker. s. typhi was the most frequent (84%) serotype identified. resistance to ampicilline was 13%, to cotrimoxazole and chloramphenicol 10% for each. the mortality rate was 2%. conclusion: typhoid fever is still an endemic disease in our country and the occurrence of resistant strains of s. typhi will favor ceftriaxone or fluoroquinolones in the treatment. maaloul i a , hammami b a , zambaa f a , elleuch r a , hammami a b , -ben jemaa m a . a chu hedi chaker, service des maladies infectieuses, sfax, tunisia , b chu habib bourguiba, laboratoire de microbiologie, sfax, tunisia although its not very frequent, the psoas abscess is not an exceptional entity. in order to specify its clinical, biological, radiological and evolutionary features, a retrospective study has been led in our service, on a period of 14 years (january 1988 á/december 2001). on the whole, 25 cases have been listed. they were 16 men and 9 women. the age average was 40 years (extreme 14 á/74 years). the study did not find any underlying diseases, except diabetes mellitus for three patients. the clinical symptoms were dominated by fever with abdomino-lumbar aches (20 cases), and psoitis (eight cases). biology showed an inflammatory syndrome in all cases and a hyperleucocytosis in 17 cases. the diagnosis of psoas abscess, evoked on clinical data, has been confirmed by the imagery data: ultra-songraphy (16 cases), ct scanning (six cases), magnetic resonance imaging (three cases). the tubercular etiology has been confirmed in six cases, among which two were associated to escherichia coli (one case) and to brucella melitensis (one case). the other etiologic agents were dominated by staphylococcus aureus (eight cases), b. melitensis (two cases), e. coli (one case), bacteroides fragilis (one case), streptococcus anginosus (one case), fusobacterium nucleaticum (one case) and candida glabrata (one case). all patients received an anti-infectious treatment adapted to the micro organism in question. a drainage of the abscess has been realized for 15 patients (percutaneous: nine cases, surgical: six cases). the evolution was favourable for 23 patients. however, two patients had a relapse after stopping the treatment. conclusion: the diagnosis of the psoas abscess, difficult on the clinical data, is based on the imagery techniques (us, ct, rmi). the percutaneous drainage guided by the imagery is recommended (in an etiological and therapeutic aim). associated to an adapted antibiotherapy, it allows to defer a surgical drainage. zezoski mbz, nikolova o, gavriloski pmg. medical center, infectious diseases, prilep, the former yugoslav republic, macedonia purpose: to make a list of the most frequent abdominal changes in patients with human brucellosis. materials and methods: there were 769 new patients with human brucellosis, between 01 1991 and 12 2001. diagnosis was made using standard clinical, biochemical and serological investigations (bab, wright, coomb's, rvk, 2-mercaptoethanol, elisa igm and igg), and specially ultrasound examination of the abdomen and retro peritoneum. results: weight loss is the most frequent change, presented in 665 (86.5%) patients. follow atypical abdominal pain in 89 (5.3%), vomiting in 59 (7.7%), diarrhea in 44 (5.3%), enlarged liver in 594 (77.2%), enlarged spleen in 573 (74.5%) and hepatic lesion with increased ast and alt in 174 (22.6%). conclusion: although frequent, abdominal changes seldom could be missed in patients with human brucellosis. we recommend routine ultrasound examination with standard biochemical test for liver function, due to avoid unnecessary complications. osteoarticular complications are common in brucellosis. the most common site of involvement is the sacroiliac joint. the osteoarticular complications such as, sacroiliitis and spondylitis are diagnosed with radiologically. in the present study, we aimed to determine the severity (grade) of sacroiliitis by using some laboratory parameters such as esr, crp and tube agg test. seventy-two (34 male, 38 female) patients with brucellosis were included in the study. osteoarticular involvement was present in 44 patients. the most common osteoarticular finding was sacroiliitis in the patients (86%). twenty (20) healthy subjects were formed the control group. there was statistically significant difference between patients and controls regarding esr, crp, and tube agg test (p 0/0.000, 0.0017, 0.000, respectively). in addition, sacroiliitis has an effect on esr and crp. there was a positive correlation between the grade of sacroiliitis and the value of crp (p0/0.006, r 0/0.414). in conclusion, it has been suggested that, crp may be used as an auxiliary or a secondary parameter in grading sacroiliac joint involvement in brucellosis. magira ee a , papandreoy s a , gounaris t a , spirelis ma a , tasopoulos g a , anagnostopoulou m b , paniara o b , gounari p a , sioulal e a . a evagelismos, internal medicine, athens, greece , b evagelismos, microa 65-year-old greek farmer was admitted to the hospital because of painful scrotal swelling, hepatosplenomegaly, lumbar pain and lowgrade fever accompanied by profuse sweating. his life style included occupational animal exposure ingestion of raw milk and dairy products. the laboratory data were within the normal ranges. focal hypoechoic right testicular lesions, swelling of the concurrent epididimis along with an increase in the vascularity of the right testis were seen on an echo examination. these findings were consisting in unilateral epididimo-orchitis. a ct scan of the lumbar spine area showed a decrease of the signal intensity localized in the anterior aspect of l5 vertebral body at the diskovertebral junction involving the subchondrial parts of the l5 and s1 vertebrae standard tube agglutination test was positive for antibodies to brucella melitensis (titer !/1/1280). cultures of blood specimens were positive for b. melitensis . the patient had been given to a combination of antibiotics with doxycycline, streptomycin and rifampin. a remarkable improvement of his clinical condition was showed 2 weeks later. this case illustrates the following point: in areas in which brucellosis is endemic when scrotal abnormalities are seen the possibility of genitourinary tract complications of brucella should be considered. pappas ga a , akritidis nk b , mastora m b , tsianos e a . a university hospital, internal medicine, ioannina, greece , b 'g. hatzikosta ' hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and forms of complications associated with brucella infection. patients and methods: we studied the 100 most recent patients, in all larger series approaching 1000, diagnosed as suffering from brucellosis, and assessed the presence of signs and symptoms of arthritis and spondylitis, or other forms of bone involvement. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures or cultures from other media. results: osteoarticular complications were noted in 23 patients, 13 presenting with arthritis, and 10 presenting with spondylitis. eight patients presented with genitourinary complications, either orcheoepididymitis (four patients), or hematuria resolving with treatment (four patients). meningitis was present in two patients. gastrointestinal complications (vomit and diarrhea) were present in three patients, while one patient presented with ascites. respiratory tract complications, in the form of pneumonia (four patients) or bronchitis (three patients) were noted in seven patients, while one patient with pneumonia exhibited pleural fluid. skin rashes, of macular type, were present in three patients. no patient presented with complications from the heart. hematologic complications were frequent, in the form of severe (one patient) or moderate (two patients) pancytopenia, isolated thrombocytopenia (three patients), or lymphocytosis (eight patients). akritidis nk a , pappas ga b , mastora m a . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and modes of bone and joint involvement in the course of brucellosis. patients and methods: we studied the 100 most recent patients, in all larger series approaching 1000, diagnosed with brucellosis, and assessed the presence of arthritis and spondylitis. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures. results: twenty-three patients exhibited a form of osteoarticular involvement. arthritis was present in 13 patients, most often involving the knees, but also the hips, elbows, even smaller joints as intephalangeal joints of the hand. synovial fluid, when aspirated, was often characterised by an intense mononuclear infiltrate. spondylitis was present in 10 patients, most often involving the lumbar spine, but also the thoracic spine. the characteristic erosion on the upper anterior crest of the vertebral body was visible in plain x-rays in three patients, while mri and bone scan were helpful in other cases. discussion: osteoarticular involvement in the course of brucellosis is the most common focal presentation of the disease. acute brucellosis is often accompanied by bone and joint ache, especially of the lumbar spine, still frank involvement in the form of arthritis and spondylitis is not rare. arthritis usually presents in the acute form of the disease, while spondylitis tends to be characteristic of a chronic form of the disease, often necessitating prolonged use of antibiotics. bosilkovski m, krteva l, caparoska s, grozdanovski k, sajn b. clinic for infectious diseases and febrile conditions, medical faculty, skopje, the former yugoslav republic, macedonia one hundred and twenty-six patients with brucellosis were studied prospectively. seventy-eight (61%) of them had osteoarticular involvement. peripheral arthritis in 46 (59%) patients was the most frequent, followed by spondilitis in 27 (35%), sacroiliitis in 15 (19%), rarely bursitis, tendinitis and osteomyelitis. the overall male to female ratio was 2:1. their average age was 39 (sd 20) years. direct contact with animals was the reason for acquisition of the illness in 59% of patients, in 29% alimentary or aerogenous route was incriminated, and in 12% the route of aqisition was unknown. the average duration of the symptoms from the onset to establishing the diagnosis was 68 (sd 78) days. the main presenting symptoms were joint pain (100%), sweating (77%), fatigue (58%) and fever (57%). hepatomegaly was present in 58%. in 49% of patients, involvement of some other system was evident. comparison with patients, who did not have osteoarticular illness, showed that patients with osteoarticular involvement had significantly more often joint pain, fatigue, weight loss and more prolonged duration of symptoms before the diagnosis was established. doxycycline and chloroquine as combination therapy for chronic q fever endocarditis ps201 calza l, attard l, manfredi r, chiodo f. division of infectious diseases, university of bologna, s. orsola hospital, bologna, italy introduction: endocarditis is the main clinical manifestation of chronic q fever, occurring in about 60 á/70% of all reported cases, and it is diagnosed almost exclusively in patients with either cardiovascular abnormalities or an immunocompromised condition. case report: a 62-year-old caucasian male patient with biological prosthetic aortic valve was first hospitalized because of an interstitial pneumonia. six months later, our patient was re-admitted owing to intermittent fever, chills and weight loss. echocardiographic study showed a small vegetation of 12 mm in diameter on left cusp of aortic valve. serology for coxiella burnetii revealed a complement-fixing igg antibody titer to phase i antigen of more than 1:2048, consistent with chronic q fever endocarditis. antimicrobial therapy with i.v. doxycycline and oral chloroquine was started, leading to a clinical and echocardiographical recovery. therapy was continued by oral doxycycline and chloroquine, and the patient remained asymptomatic during a 2-year follow up. conclusion: the optimal treatment of q fever endocarditis has not been well established: the most effective antimicrobials are fluoroquinolones and rifampin, but chloramphenicol, doxycycline and trimethoprim are also useful. the role of chloroquine in combination with doxycycline seems to be promising, because chloroquine may increase the lysosomal ph, enhancing the doxycycline bactericidal activity. akritidis nk a , pappas ga b , mastora m a , liappis e a , tsianos e b . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scopes: to review the incidence and the forms of lower respiratory tract infection in patients suffering from q fever, and their clinical and radiological characteristics. patients and methods: twenty-seven patients diagnosed as suffering from q fever, were assessed for the presence of lower respiratory tract infection. the diagnosis was confirmed serologically. results: thirteen patients expressed lower respiratory tract pathology, as confirmed by clinical examination and chest x-ray. in 11 of these patients the main cause of admission was respiratory tract symptoms, ranging from dry cough to hemoptysis. chest x-ray was pathological in 13 patients: 10 patients had lobar pneumonia, two of them multiple nodular opacities, and one of them bronchopneumonia. hepatitis was a common finding. all patients were treated with tetracycline. discussion: although coxiella burnetii infection is acquired via the respiratory tract, it is paradoxical that symptoms attributed to the lung are not invariably positive. q fever pneumonia is an atypical pneumonia that usually follows a benign course. diangostic suspicion is usually raised by the epidemiologic pattern and the accompanying mild hepatitis. pleural effusion is not a common finding. the usual radiologic appearance of q fever pneumonia is that of a lobar or segmental pneumonia. one important aspect of q fever pneumonia is its common presentation in the form of multiple nodular opacities often necessitating the exclusion of malignancy. (16), culture (4), and serology'/culture (2). there were 0 á/4 cases per year, mainly in october (73%). a history of exposition to hare was present in 24/25 (96%) and to marmot in 1/25 (4%). skinning (18/25 72%), animal contact (5/25 20%), bite (1/25) and wound during bait preparation with frozen meat for hunting (1/25) were noted. the initial clinical presentation was ulceroglandular (77%), glandular (19%) and pneumonic (4%). the involved nodes distribution was axillary (12/24), cubital/axillary (10/24) and cubital (2/24). median incubation period was 3 days (range 1 á/6); time to consultation 3 days (range 0 á/7), and time for effective treatment 7 days (range 0 á/23). an initial diagnosis of tularemia was made presumptively in 42%. effective antibiotic regimen used was aminoglycosides in 73% (19/26), and tetracyclines in 27% (7/26). note that intravenous netilmicin was used in 6 cases. complication rate was 35% (9/26) with one death (4%), and was associated with delay in effective treatment ( !/8 days of illness) (p b/0.05). conclusion: in our area tularemia occured mainly in male population, during autumn, with a short incubation period and history of hare contact. delay before appropriate treatment increased the complication rate. bompolaki i, doukakis s, triantafillidou d, polimili g, kastanakis m, nikiforakis k, vittorakis e, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece a severe frontal and/or retroorbital headache represents the most common neurologic manifestation of murine typhus. other neurologic manifestations as confusion, stupor, nuchal rigidity and in severe cases delirium, extreme agitation or coma appear less commonly. eightyfour patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi, were studied from our team. seventy-four patients (88%) presented headache and nine patients (11%) presented confusion. one patient (1.1%) presented nuchal rigidity in combination with severe headache and confusion giving us the suspicion of meningitis. in this case a lumbar puncture was performed emergently and the cerebrospinal fluid (csf) was examined. the findings of csf were proteins: 9 mg/dl, wbc: 16/ml and glucose: 73 mg/dl and its culture was negative. all patients were treated with a specific anti-rickettsial treatment. the outcome of murine typhus was favorable for all 84 patients (100%). no one patient presented neurologic sequelae. conjunctivitis usually accompany rickettsial diseases such as rocky mountain spotted fever, epidemic typhus and murine typhus. eightythree patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january 1993 and the first semester of 1998. the clinical examination of these patients revealed the presence of conjunctivitis in 21/83 patients (25.6%). in the same time these patients referred retroocular pain and mild photophobia. this study showed that in murine typhus the injection of conjunctivae is rather common. almost a quarter of the patients presented conjunctivitis despite the fact, that this ocular manifestation is less severe than in other typhus and spotted fevers. eighty-three patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january 1993 and the first semester of 1998. three blood samples were obtained from each patient for the study of their hematological abnormalities. the first sample was obtained on admission, the second sample 2 weeks after the first, the third sample, 1 month after the second. on admission 26/83 patients (31%) presented anemia, 6/83 patients (7%) presented leukopenia and 42/83 patients (51%) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was 12.4 g/dl, 6.1)/10 3 and 147)/10 3 /ml, respectively. two weeks later anemia was presented in 45/83 patients (54%), 3/83 patients (4%) presented leukopenia, 3/83 patients (4%) presented leucocytosis and 15/83 patients (18%) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was 11.3 g/dl, 7.0)/10 3 and 248)/10 3 /ml, respectively. one month later 11/42 patients (26%) had anemia and 2/42 patients (5%) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was 12.4 g/dl, 6.4)/10 3 and 242)/10 3 /ml, respectively. our study showed that early thrombocytopenia and anemia are frequent in murine typhus and that white blood cells count is usually normal. renal function in murine typhus: a study of 83 cases ps207 doukakis s, polimili g, triantafillidou d, kastanakis m, vittorakis e, palla k, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece the clinical course of murine typhus is usually uncomplicated and the mortality rate is low ( b/1%). advanced age and prolonged interval before administration of a specific anti-rickettsial treatment are correlated with severity of the disease. renal function in murine typhus is usually unaltered except in elderly patients with prolonged hypotension. eighty-three patients with compatible clinical status of murine typhus and high serological titres of antibodies against rickettsia typhi, were studied from our team, during a period of time between january 1993 and the first semester of 1998. three blood samples were obtained from each patient for the study of their renal function. the first sample was obtained on admission, the second sample approximately 2 weeks after the first, the third sample, taken from the half of the patients, was obtained one month after the second. on admission 4/83 patients (5.0%) presented acute renal failure. the outcome of murine typhus was favourable for all 83 patients (100%). the four patients who presented acute renal failure reversed after the administration of anti-rickettsial treatment and careful administration of fluids. in murine typhus the induction of hypovolaemia insufficiently corrected by normal homeostatic mechanisms may lead to prerenal azotaemia. in these cases the immediate onset of an antirickettsial treatment and the correction of hypovolaemia are essential for the rapid clinical improvement of the patient. experimental ocular toxoplasmosis: clinical, histopathological, immunological and therapeutic studies ps208 el zawawy lae a , hammoda na a , allam sr a , ali sm a , galal as b . a faculty of medicine, parasitology, alexandria, egypt , b faculty of medicine, ophthalmology, alexandria, egypt the purpose of the study was to investigate clinical, histopathological, immunological and therapeutic features of an experimental model of ocular toxoplasmosis in sensitized and non sensitized rabbits and to assess the influence of treatment by interleukin2 (il-2) on ocular lesions. the results obtained was that 'toxoplasma' retnochoroiditis developed in both groups of rabbits with more pronounced effect in non sensitized animals. administration of il-2 improved ocular lesions in both groups with more evident effect in sensitized rabbits. immunological findings were consistent with clinical and histopathological observations. the conclusion reached was that; ocular lesions were manifested in non sensitized rabbits more than in sensitized ones. il-2 revealed a significant impact on improving the host defense against toxoplasm infection in eye. immunotherapy with il-2 would open the way for a new range of treatment based on immunomodulation. express-diagnostic of streptococcus antigen for the adequate antibiotic therapy in patients with pharyngitis ps209 pertseva to, konopkina li, kireeva tv. dsma, internal medicine , dniepropetrovsk, ukraine the purpose of the study: evaluation of effectivness of streptococcus express-diagnostic for the adequate antibiotic therapy in patients with pharyngitis. results: we deal with clinical and microbiological comparison in 53 patients with pharyngitis. using of streptococcus antigen express diagnostic in swabs from the backside of pharynx allowed to get positive results in the seven cases (13.2%). following cultural study has confirmed these results. positive test was more probable in patients with pronounced fever (more than 38 8c), headache, weakness and in cases associated with chronic tonsillitis. isolated streptococcus pyogenes was susceptible to ampicillin, claritromicin, erytromicin, azytromicin, , clindamicin, ceftriaxon, levafloxacin, oxacillin, cefuroxim, roxytromicin. conclusion: using of the express diagnostic of streptococcus antigen allows to restrict groundless prescription of antibiotic therapy in patients with other types pharyngitis (i.e. viral, candidal etc.). alabaz d a , turgut m a , kocabas e a , tumgor g a , yaman a b , alhan e a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey chickenpox is a common viral infection that usually follows a benign, self limited course in healthy children. the most common complication in children with varicella is superimposed cutaneous infections with pyogenic bacteria (streptococcus pyogenes and staphylococcus aureus ). varicella gangrenosum, a necrotising soft tissue infection complicating the vesicular eruption of chickenpox, is rare. here we present three cases with necrotising soft tissue infections following chicken pox. these children were admitted because of common crusted lesions and necrotising soft tissue infection over the neck, the back, and the inguinal area. they all had the contact history and ensuing vesiculopapular rush. these infections were caused by group a streptococci in two cases, and s. aureus in one case. after instituting of appropriate antibiotic therapy, each patient underwent a surgical exploration with fasciotomies and debridement. widespread use of varicella vaccine may decrease invasive infections in children, adolescents, and adults, thus decreasing the burden the disease with its complications impose up on the family and the society . cefprozil is a second generation cephalosporin. the aim of this open, multicentre, non-comparative study was to investigate the efficacy and safety of cefprozil in the treatment of streptococcal tonsillopharyngitis. fifty-eight patients were clinically assessed for signs and symptoms of streptococcal infection. laboratory confirma-tion was sought using three tests; culture, rapid strepto test and estimation of antistreptolysin (aso). one or more tests were done in 47 of the 58 patients. treatment was for 5 days, 25 mg/kg per day in children, 500 mg per day in adolescents. patients were again clinically assessed on the 5th á/6th day. the results showed clinical success in 56 patients (96.5%) and in 46 of the 47 who had laboratory tests (97.8%). two patients had treatment withdrawn because of nausea and abdominal pain (3.5%). of the 47 patients with laboratory tests at least one test was ositive. the most helpful was the strepto test giving the quickest positive result in 92% of those tested (38/41). culture was positive in 58% of those tested (24/41). the aso test was of limited value. in conclusion, cefprozil showed high clinical efficacy and safety in the treatment of streptococcal tonsillopharyngitis. tsiara s, militadou g, milionis c, elisaf m. internal medicine department, university of ioannina, ioannina, greece streptococcus group b agalactiae (gbs) is a rare pathogen for healthy male adults. we present an old man in whom (gbs) was isolated in blood cultures. case report: a 79-year-old man was admitted to the hospital in order to investigate osteolytic lesions in the lumbar spine. two weeks before, he experienced severe low back pain radiating to the right leg and fever arising to 39.50 8c with chills and rigors. a gbs was isolated from blood cultures and treatment with penicillin was initiated. a spine ct scan revealed osteolysis in the t12 and s1 vertebrae and the patient transferred to us. he was a previously healthy man. he received only antihypertensive therapy. on admission he was afebrile with arthralgias and myalgias. on clinical examination there was tenderness on the right pleurospondylic angle radiating to the right leg. laboratory data on admission: hb: 11.2 g/dl, wbc: 7.4)/10 9 /l, esr: 77 mm/h. biochemical values, serum protein electrophoresis, rectal examination and a prostatic specific antigen (psa) were normal. a bone marrow aspiration and biopsy were negative. a transoesophageal ultrasound revealed vegetation on the right cusp of the aortic valve, with low grade regurgitation. an mri of the lumbar spine revealed infectious myositis with concomitant osteomyelitis involving the l4, l5 vertebrae without any evidence of osteolytic lesions. a thorough investigation did not revealed any underlying immunosuppressive disease. treatment with vancomycin and gentamycin iv was initiated and the patient discharged from the hospital in excellent health after 6 weeks. discussion: gbs infections usually occur in neonates and pregnant, or in adults with underlying disease as diabetes mellitus or immunosuppression. the most common site of infection is soft tissue. we present this case because gbs infections are rare in the elderly in the absence of underlying disease. common sites of involvement are soft tissues, while bone, joints, and heart valves account to 1 á/2% of the involved organs. although our patient had more than one site of involvement he responded well to medical treatment without surgical debriment or heart valve replacement. objective: to evaluate the safety and efficacy of rhugm-csf in combination with broad-spectrum antibiotics for the treatment of ccnf. methods and results: a retrospective review of all patients (pts) with ccnf treated with antibiotics'/rhugm-csf at our hospital from january 1997 to december 2001 was performed. five patients were identified with the diagnosis of ccnf. ages ranged from 33 to 71 years; there were three women and two men. dental infection was the most common source of ccnf in 90%. one patient had acute tonsillitis leading to ccnf. all cases studied experienced infection of the neck with spread into the submandibular, submental, sublingual, retropharyngeal, and parapharyngeal spaces. all infections were polymicrobial. diabetes mellitus was a co-morbidity in one case. all pts were treated with dual antibiotic coverage (vancomycin'/meropenem), rhugm-csf (10 mcg/kg/daily given s.c.) and aggressive wound care. rhugm-csf was given for 7 á/10 days. in spite of the severity of the infection all pts recovered and do not experienced local or systemic complications. discussion: ccnf is a severe bacterial infection of the cervical fascia resulting in extensive fascial necrosis with widespread undermining of the surrounding tissues. prompt antibiotic therapy combined with rhugm-csf resulted in a 100% overall survival in our experience. to our knowledge, this is the first report describing the successful treatment of ccnf with use of broad-spectrum antibiotics combined with rhugm-csf. the following case adds to the clinical manifestations and course of meningococcal disease. a previously healthy 3-year-old girl presented acutely with high fever purpuric rash including conjuctival haemorrhages and hypotension. the child had also neck stiffness. a presumptive diagnosis of meningococcal septicaemia was made and treatment with penicilline, chloramphenicole, fluids and inotropes was initiated. laboratory investigations showed wbc: 4300/ml, hb: 12.2 g/dl, hct: 39.5%, esr: 1 mm in the first hour, pt: 26 s, aptt: 90 s. neisseria meningitidis group b was isolated from the blood cultures. csf obtained after antibiotics were started did not grow n. meningitidis . the patient had an adverse outcome. she died after 12 h of hospitalization. this patient developed a fulminating meningococcal septicaemia. shock is a clinical diagnosis arising from the failure of compensatory mechanisms that maintain perfusion of vital organs at the expense of non vital. septic shock results from loss of circulating plasma volume due to increased vascular permeability and maldistribution of intravascular volume. in young children there is a prevalence of serogroup b meningococcal disease which can be explained by the immaturity of the immune system and by the fact the group b capsule synthesis is known to inhibit alternative complement pathway activation. this case emphasizes the need for further protection against n. meningitidis group b. . results: forty-eight patients were included in this study. the etiological agents were: viral (n 0/26, 54.2%, mean ('x ) age0/28 years), streptococcus pneumoniae (n 0/8, 16.6%,'x age0/53), neisseria meningitidis (n0/7, 14.6%, 'x age0/28), s. viridans (n0/6, 12%, 'x age0/34), p. multocida (n0/1, age 75). the peak incidence of bacterial meningitis was in winter (pneumococcal 73%, meningococcal 86%, s. viridans 83%) .the cerebrospinal fluid (csf) findings in viral meningitis were 'x white cells 0/340/mm 3 , 'x pmn0/27%, 'x glucose csf/ serum 0.55, 'x protein 39 mg/dl and in bacterial meningitis were 'x white cells0/5800/mm 3 , 'x pmn0/80%, 'x glucose csf/ serum0/ 0.2, 'x protein0/350 mg/dl, gram stain was positive in 55%, culture was positive in 45%. all pneumococcal and meningococcal strains were susceptible to penicillin. the case fatality rates for pneumococcal and meningococcal meningitis were 25 and 14.3%, respectively. conclusions: the cases of bacterial meningitis were according to typical epidemiological features of age and season. the case fatality rate of pneumococcal meningitis appear to be high regardless of susceptibility to penicillin. none had received pneumococcal vaccine prior to becoming ill. diagnosis and therapy of meningococcal meningitis */trend and particularities of a 'romanian model' ps216 lintmaer i, moroti r, popescu a, popescu c. institute of infectious diseases matei bals , unit 5 , bucharest, romania background: newer diagnosis methods and antimicrobials are expected to change the management of menigococcal meningitis (mm) and to improve its prognosis. objectives: to determine the changes in the diagnosis methods and therapy of mm patients in a infectious diseases hospital. to compare mm management in bucharest with literature data. methods: retrospective rewiew of mm in adult patients hospitalized over a 6-year period. our results were compared with other studies made in the 90s, taken from medline. results: there were 97 episodes of mm during the study period (52 episodes in 1995 á/1997 and 45 episodes in 1998 á/2000) . we noticed a defined diagnosis increase and increased blood culture specificity. the antimicrobial monotherapy was maintained but penicilin was replaced by ceftriaxone. hhc was replaced by dexamethasone in pathogenic therapy. we noticed a shorter length of treatment and a reduced lethality. the most important differences between our results and other studies are: monotherapy regimens are less frequent and therapy lengths are longer; however, prognosis is similar. conclusions: the mm management has been modified in the last 3 á/ 4 years: prognosis is improved, but the changes do not bring clear cost/ effective benefits. tiouiri th, kilani b, amari l, zouiten f, kanoun kf, ghbontini a, ben chaabene t. rabta hospital, infectious diseases, tunis, tunisia objectives: in order to study epidemiological, clinical and therapeutical characteristics of the infective endocarditis (ie). methods: we reviewed all the cases of ie fulfilling the duke criteria. data were collected during a 19-year period (1980 á/1998) in the unit of infectious diseases. results: one hundred and eight cases were identified. the mean age was 37.5 years. sex ratio was 0.8. eighty-five ie (78.6%) occured in patients with native valve, and 23 ie (21.4%) with prosthetic valve. fever was the most common sign, 12% had a congestive heart failure, 26.9% had cutaneous signs. the most common primary focus of ie was orthodontic. blood cultures were positive in 63% of cases. in one case, serological test identified rickettsia conori . streptococci and staphylococci were isolated in 27.8 and 25.9%, respectively. echocardiography detected abnormalities in 63.4% of cases. rheumatic heart disease was the most predisposing condition. empirical therapy was based on combination of b lactam with aminoglycoside. recovery was obtained for 60 patients. cardiac surgery was performed in 26 cases. overall mortality rate was 19.4%. conclusion: ie affects young persons. prevention needs eradication of acute rheumatic arthritis. a major outbreak of legionnaires' disease in spain: diagnostics aspects ps218 guerrero c a , toldos cm a , yagü e g b , ramírez c a , rodríguez t a , -segovia m a . a hospital morales meseguer, servicio de microbiología, murcia, spain , b departamento de microbiología, facultad de medicina, universidad de murcia, murcia, spain objective: to evaluate the value of different methods (serological tests, culture of respiratory secretions, blood cultures and urinary antigen testing) for the diagnosis of legionella pneumophila pneumonia during an outbreak in spain. results: we have studied 542 patients from a recent outbreak of legionellosis in murcia (spain). the diagnosis was achieved in 305 patients. urinary antigens were positive in 187 patients. in the 355 patients with urinary antigen negative the serological response was demonstrated by indirect immunofluorescence (ifa) in 118 patients. all blood cultures processed were negative. sputum samples were obtained from 154 patients, of these l. pneumophila was isolated only in six patients. in all of them direct immunofluorescence test (dfa) was positive. conclusions: although the serological diagnosis was the most sensitive method the urinary antigen testing was of great value in the rapid diagnosis of the legionella's outbreak in murcia. the isolation of l. pneumophila by culture showed a poor sensitivity probably because of the low severity of the illness. purpose: to evaluate chloramphenicol for an initial empiric antibiotic treatment of purulent meningitis in adults. study group: one hundred and twenty patients hospitalized for the diagnosis purulent meningitis in the department in years 1997 á/2000, 67 males and 53 females, age range 15 á/81 years, mean age 52.6 years. children up to 15 years were not included. method: a retrospective analysis of the study group focused on antibiotic treatment both initial and changes during treatment. results: chloramphenicol was used as an initial antibiotic in 59 (49%), 3rd generation cephalosporin in 30 (25%), penicillin in 21 (17%), ampicillin in five (4%) and other antibiotic in five (4%), respectively. during treatment chloramphenicol was switched for 3rd gen cephalosporin in seven of 23 patients with streptococcus pneumoniae meningitis and in five of 28 patients with meningitis of unknown etiology. the reason for the change was non-improving csf formula in three, persisting csf culture positivity in two and persisting coma in seven patients. conclusion: because of repeated necessity to switch chloramphenicol for 3rd gen cephalosporin during treatment of purulent meningitis of pneumococcal and unknown etiology the initial treatment strategy was changed in 2001. third gen cephalosporin is now used as a first choice antibiotic, what is in consent with international recommendation of treatment. to evaluate and compare groups treated initially with chloramphenicol and with 3rd gen cephalosporin will need several more years. low prevalence of multi-drug resistant mycobacterium tuberculosis in jerez de la frontera-cadiz (spain) ps220 alados jc, aller ai, de miguel c, de francisco jl, calbo l. hospital del sas-jerez , microbiologia, jerez de la frontera, cadiz, spain introduction and aim: previous reports indicate that multi-drug resistance mycobacterium tuberculosis (mtb) is an worldwide problem. the aim of this study was to review the resistance of mtb to the first-line antimycobacterial agents in our area. material and methods: over a period of 4 years (1998 á/2001), 151 strains of mtb isolated from non-treated patients with tuberculosis (38 strain in 1998, 48 in 1999, 37 in 2000 and 28 in 2001) were studied. these isolates were tested for in vitro drugs susceptibility to isoniacid-i, rifampicin-r, streptomycin-s and ethambutol-e using the bact/ alert method (organon teknica) as described by the manufacturer. results: our results showed that 10.6% (16/151) strains were resistant to one or more drugs. single drug resistances were detected on nine strains to i (6.3%), one to r (0.66%), two to s (1.3%), one to e (0.66%). three mtb strains were resistant to more than one drug but only one was multi-drug resistant (i'/r).the incidence of i-resistant strains over the period fell from 13% in 1998 to 3.6% in 2001. conclusions: (1) multi-drug resistance is not an important problem in our area. (2) isoniacid resistance was declined to an admissible level. to investigate the anti-tuberculosis drug resistance pattern of pulmonary tuberculosis isolates in southern taiwan, an area with higher tuberculosis incidence and mortality than other regions of the island, we performed a hospital-based surveillance at a southern taiwan medical center from 1996 to 2000. the combined drug resistance rates to at least one of five first-line agents was 84.8%, and to both isoniazid and rifampin (multi-drug resistance, mdr) was 11.4%, indicating high resistance rates compared with those reported in the who/iuatld global project and in northern taiwan. the resistance rates to two second-line drugs, cycloserine, and kanamycin, were 75.7 and 23.7%, respectively. a significant decreasing trend in resistance rates to all drugs except streptomycin was observed during the 5-year period. though combined drug resistance rate may not be the most accurate tool as it includes previously treated cases which inflates the resistance rate, the observation of trends in the susceptibility of pulmonary tuberculosis in accompany with the increasing percentages of tuberculosis patients receiving complete treatment course and the decreasing percentages of cases lost of follow-up in kaohsiung after the institution of new governmental regulations for case management in 1997 suggest the usefulness of intervention programs. lipid profile in patients with multidrug resistant pulmonary tuberculosis ps223 extrapulmonary tuberculosis may sometimes present with confusing clinical manifestations. a 77-year-old female patient was admitted with a history of recurrent supra-sternal abscess for 1 year. mri confirmed the presence of sternal osteomyelitis and an anterior mediastinal mass. the diagnosis of tuberculosis was proved by histologic examination and acid-fast stain. the patient was treated with first-line agents, which isoniazid, rifampin, pyrazinamide, and ethambutol. tobacco smoking as a factor of the decrease of chemotherapy effectiveness and of the development of the drug resistance in patients with pulmonary tuberculosis ps225 shprykov as a , zhadnov vz a , shprykova on b . a medical academy, department of tuberculosis and lung diseases, nyzhny novgorod, russian federation , b medical clinic for infectious #2, laboratory of bacteriology, nyzhny novgorod, russian federation studies of the effect of smoking on the results of chemotherapy of 798 patients with tuberculosis of lungs. intensive tobacco smoking slowed down clearance of positive sputum and of lung tissue destruction (in smokers 92.1% and 70.1 vs. 98.0% and 90.6% in nonsmokers, p b/0.01). drug-resistant mtb strains have been found to be isolated more often in smokers */43.4 vs. 19.4% in non-smokers, p b/ 0.01. resistance to streptomycin and isoniazid prevailed, reaching in heavy smokers 50.0 and 30.5%, respectively. resistance to rifampicin increased 1.5 times. the concentration of rifampicin in the blood serum of heavy smokers decreased in 1.8 times. clinical data are in complete correlation with the findings of our experiments: 67% of experimental cultures developed resistance to streptomycin, isoniazid and less to rifampicin in the study of drug sensitivity under the effect of tobacco smoke condensate. thus, our findings show the development of drug resistance in mtb under the effect of components of tobacco smoke and also showed less effectiveness in therapy. kilani kb, ammari la, tiouiri ht , ben chaabène tbc. rabta hospital, infectious diseases, tunis, tunisia guerrero c a actinomycosis is a chronic disease characterized by abcess formation, tissue fibrosis and draining sinuses. it is caused by anaerobic bacteria belonging to the genus actinomyces. thoracic actinomycosis may involve the lungs, pleura, mediastinum or chest wall. the authors present a case of pulmonary actinomycosis complicating a cervicofacial site. a 32-year-old man with a history of cervicofacial actinomycosis treated by penicillin g 2 years ago was admitted because of right-sided chest pain for 2 months before presentation, cough and fever. physical examination shows a painless indurated mass in the neck with multiple fistula of the sternum. chest radiograph and ct scan revealed a mass in the upper lobe of the right lung with an infiltrate of the upper lobe of the left one. magnetic resonance imaging confirms the previous lesions, with extending process to the sternum and right collar bone. bronchoscopy was performed while patient was on antimicrobial therapy. culture of bronchoalveolar lavage fluid was negative. transbronchial biopsy was not conclusive. fungal serologies were negative for aspergillosis, histoplasmosis, blastomycosis. bacterial examination of purulent drainage from sternal wound shows inclusion bodies identified as actinomyces. he was treated then with penicillin iv for 2 months, than switched to doxycycline. after 8 months of treatment, he is asymptomatic with radiological improvement. kanellopoulou m a , skarmoutsou n a , iglezos i b , mylona e a , martsoukou m a , apostolopoulou f b , papafrangas e a . a laboratory of clinical microbiology, sismanoglio general district hospital of attica, athens, greece , b 2nd department of pneumology, sismanoglio general district hospital of attica, athens, greece introduction: achromobacter xylosoxidans is a rare human pathogen. it is an important cause of bacteremia in patients with cardiac diseases, malignancies and immunosuppression. it has been recently recognized as an emerging microorganism in cystic fibrosis (cf), whose its pathogenic role is unknown. aim: to investigate the sensitivity to eleven different antibiotics of 27 a. xylosoxidans strains isolated from adults with cf, during 2001. methods: the susceptibility was tested by kirby bauer and microdilution methods (wider i, fransisco soria melguizo, s.a.), according to nccls recomendations. results: the resistance to antibiotics was as follows : gentamicin, tobramycin, aztreonam 100%, amikacin 96%, ceftazidime 85%, ticarcillin 66%, carbapenems, cotrimoxazole 59%, colistin 51% and piperacillin 22%. conclusions: (1) a. xylosoxidans isolated from cf patients appeared resistant to the most usually tested antibacterial agents. (2) colistin which is used as aerolized antibiotic for cf patients seems to be effective in the half of the isolated strains. (3) piperacillin was the most active antibiotic against a. xylosoxidans . morris ka, perry jd, jain s, gould fk. microbiology department, freeman hospital, newcastle upon tyne, uk alafosfalin, l-alanyl-l-1-aminoethylphosphonic acid, is an antibacterial peptide mimetic which inhibits peptidoglycan biosynthesis. we report the in-vitro activity of this compound in combination with ceftazidime, cefsulodin, fosfomycin, piperacillin/tazobactam, aztreonam, ciprofloxacin and timentin. drug combinations were evaluated against 20 burkholderia cepacia strains, and 20 pseudomonas aeruginosa strains isolated from patients with cystic fibrosis. for this purpose a chequerboard technique was adopted using doubling dilutions of each antibiotic incorporated into a highly defined agar medium free of antagonists. the minimum inhibitory concentrations (mics) and fractional inhibitory concentrations (fics) of all the antibiotic combinations were determined which revealed the antibiotic interaction occurring. alafosfalin in combination with ceftazidime, meropenem, piperacillin/tazobactam and timentin demonstrated the highest percentages of synergy in both b. cepacia and p. aeruginosa . synergy was shown to occur in 15, 15, 10 and 10% of b. cepacia strains respectively, and in 10, 5, 5 and 5% of p. aeruginosa strains. these four combinations were re-tested with all 40 isolates and the results were shown to be reproducible. alafosfalin shows potential as a treatment for cystic fibrosis patients colonised with p. aeruginosa and/or b. cepacia , when applied in combination with these agents. community-acquired pneumonia */does its aetiology matter? ps229 lintmaer i, popescu a, popescu c. institute of infectious diseases matei bals, unit 5, bucharest, romania the aetiology of a pneumonia is not one of the criteria used to determine pneumonia's severity. however, it is accepted that identified based á/based therapy is less expensive (and possibly more effective). objectives: our study aims were to: (1) to evaluate the role of aetiology identification in pneumonia; (2) to evaluate the first-line therapy in pneumonia. methods: we conducted a retrospective study in an infectious diseases hospital on 1126 patients with pneumonia. we excluded all the cases with nosocomial pneumonia. primary end-point was the 28day clinical failure (deaths, icu admission), secondary end-points were the average time of fever and length of stay and the antimicrobial regimen changes. results: causative agent identification rate was 24.6%. the evolution was different for patients with identified aetiology compared with other patients in terms of: 28-day failures, length-of-stay and changes of the antimicrobial regimen. the 61 patients with inadequate first-line therapy had a more severe course of illness with a greater rate of 28day clinical failure, longer fever and length-of-stay. conclusions: pneumonia's treatment was better for the patients with identified causative agent. that is why we should include aetiology among the pneumonia severity criteria, especially at an 'after 3-day therapy' re-evaluation. results: pneumomococcal aom was detected in 94 children (41.0%) and s.pn. was the only pathogen in 80.9%. the resistance rates of the organism to antibiotics were as follows: penicillin 48.9% (micb/1 mg/ml; intermediately resistant 31.9%, mic 1.5 mg/ml 8.5%, and mic!/2 mg/ml; highly resistant 8.5%), erythromycin 55.3%, clindamycin 11.7%, cotrimoxazole 41.5% and chloramphenicol 6.4%. all isolates were uniformly susceptible to rifambicin and vancomycin. the large majority of pneumococcal isolates (80.8%) had the mphenotype and the remaining strains (19.2%) the constitutive mls phenotype. a various of serogroups were detected; the serogroup 19 was the most predominant one (50.0%), followed by serogroups 14 (14.9%), 23 (10.6%) and 6 (6.4%). the non-typable s.pn. strains compromised the 8.5% of the strains. conclusions: high prevalence of resistance to penicillin, macrolides and cotrimoxazole in pneumococcal aom of childhood was recognized. a strategy for preventing aom caused by drug-resistant pneumococci is mandatory to start. material and methods: a total number of 82 strains were examined. the sensitivity test was performed by kirby bauer, microdilution method (pasco, difco) according to nccls guidelines and by e -test. results: a percentage of 5.5 % of s. pneumoniae strains revealed high level resistance to penicillin (mic]/2 mg/ml), while the 28% showed intermediate resistance (mic 0.12 á/1 mg/ml). the resistance to erythromycin and cotrimoxazole was 8.3 % (mic ]/1 mg/ml) and 5.5 % (mic]/4/76 mg/ml), respectively. all strains were sensitive to cefotaxime (mic 0.05 mg/ml), vancomycin (mic5/0.5 mg/ml), meropenem (mic 5/0.25 mg/ml) and levofloxacin (mic5/2 mg/ml). conclusions: (1) the prevalence of high resistance s. pneumoniae to penicillin seems to be low in examined strains (5.5%). (2) intermediate resistance to penicillin of s. pneumoniae isolates was high as expected (28%). (3) most of the strains were sensitive to erythromycin (91.7%) and cotrimozaxole (94.5%). (4) s. pneumoniae isolates were completely (100%) sensitive to levofloxacin, vancomycin and meropenem. beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy aim: of this study was a retrospective (1997 á/2001) evaluation of the more effective and practical antibiotic treatment in 1058 ae-copd patients (pts) admitted to our unit. methods: before introducing any antimocrobial drug, sputum specimens were collected for microbiological purposes, while blood analysis, to monitor adverse systemic effects, were performed at the beginning and the end of treatment. antibiotic treatment ranged from 8 to 15 days according to four regimens: regimen a (832 pts)0/oral therapy only: 30.9% with amc 1 g b.i.d.; 24.4% with cip 500 mg b.i.d.; 17.4% with dox 100 mg u.i.d.; 10.2% with lev 750 mg u.i.d.; 8.5% with cla 500 mg b.i.d.; 7.8% miscellaneous. regimen b (94 pts)0/sequential therapy (e.v. for 3 days0/oral): 56.4% with amc 1 g b.i.d.; 37.2% with cla 500 mg b.i.d. regimen c (68 pts)0/e.v. therapy only: same drugs. regimen d (64 pts)0/an association of two antibiotics. results: of 1058 evaluated pts, only 199 (19.6%) required a second regimen of treatment because of failure of the previous one: 19.7% of regimen a; 12.7% of regimen b; 23.5% of regimen c, and 14% of regimen d. mild adverse effects were detected only in four pts. our results confirm that oral antibiotic treatment is practical, safe, and effective, and can be considered as the first line regimen also in hospitalized patients with ae-copd. becher g a , gillissen a a , rothe m b . a st. george medical center, robert-koch-hospital, leipzig, germany , b filt, lung and chest diagnostics ltd., berlin, germany patients with severe form of chronic obstructive pulmonary disease (copd) are prone by frequent exacerbations. bacterial infections are judged to cause at least half of exacerbations. haemophilus influenzae and streptococcus pneumoniae are the most frequent isolates, gramnegative bacilli account for the severe cases, aggravating the inflammatory process in the airways eventually leading to respiratory insufficiency. the aim of this ongoing placebo controlled, parallel group, mono center study trial is to evaluate beneficial effect of cefixim to reduce bacterial load and pulmonary inflammation in patients (n0/ 30) with acute bacterial exacerbation of severe copd. thus, 30 patients received in randomized fashion either cefixim (400 mg/day) or placebo (5 days). on days 1, 2, 5 and 8 breath condensate is collected using 'ecoscreen' (jaeger germany) for ltb4-, il-8-, nitrite-and ph-analysis. in parallel sputum gathered for detection of bacterial strains, and for ltb4-, and il-8 quantification purposes. these data are compared to clinical outcome parameters such as lung function tests, radiographic findings, serum inflammatory markers and length of hospital stay. the preliminary data obtained confirm successful antibiotic therapy with oral cefixim in bacterial related acute exacerbations of copd is a useful approach to reduce bacterial load, and concomitantly lower inflammatory indices of the central and peripheral airways leading to clinical improvement of the patients. soriano garcia f a , fenoll a b , fernandez-roblas r a , coronel p c , gimeno m c , rodenas e c , garcia m a , granizo jj d . a fundacion jimenez diaz, microbiology, madrid, spain , b instituto de salud carlos iii, centro nacional microbiologia, majadahonda, spain , c tedec meiji farma, scientific, alcala de henares, spain , d fundacion jimenez diaz, epidemiology, madrid, spain purpose: to describe the susceptibility of streptococcus pneumoniae against cefditoren and 10 other antimicrobials by serotype a multicenter study in south europe was carried out. a total of 877 strains were collected between september 2000 and march 2001 from adult patients (more than 16 y.o.) with respiratory tract infection (respiratory tract samples and blood cultures). all the isolates were sent to a central laboratory (fundació n jiménez díaz, madrid, spain) where susceptibility test was performed by broth microdilution (sensititre) following nccls recommendations. serotype was determined by quellung reaction and dot assay in carlos iii institute in 806 strains. results: a total of 25 strains (3.1%) were not typable. the most prevalent serotypes were 23 (15.4%), 6 (11.4%), 19 (10.8%), 3 (9.6%), 14 (8.9%) and 9 (8.1%). two hundred and sixty-four strains were grouped in 27 different serotypes. the proportion of susceptible strains by serotype to penicillin, erythromycin and levofloxacin were: serotype 3 (90.9, 93.5, 100%); 6 (50.0, 34.8, 96.7%); 9 (36.9, 86.2, 93.8%); 14 (38.9, 48.6, 94.4%); 19 (60.9, 44.8, 97.7%); 23 (58.9, 49.2, 99.2%). the mic 90 to cefditoren was 5/0.03 (serotype 3); 0.5 (serotype 6, 9 and 19) and 1 mg/l (serotype 14 and 23). conclusions: the most prevalent serotype was 23. the susceptibility was higher in serotype 3 than in serotypes 14 and 23. community acquired pneumonia */a study among closed military community of young people ps235 martynova av, turkutyukov vb, vostrikova aa, andryukov bg. vladivostok state medical university, epidemiology, vladivostok, russian federation purpose: the etiology of pneumonia is still partly unknown. we should like to clear up an etiological role of respiratory pathogens in community-acquired pneumonia among youth. and we had chosen for it a model of a closed community both investigation of etiology of disease and for further investigation of mechanisms of transmission drug-resistant mechanisms. methods: we studied 300 adults in age of 17 á/25 from closed military collectives who presented to two public hospitals (one urban and one rural) with acute radiologically confirmed pneumonia during winter 2000 á/2001. we did blood and lung-aspirate cultures, mycobacterial cultures, serotype-specific pneumococcal antigen detection, and serology for viral and atypical agents. results: streptococcus pneumoniae is recognized as an important cause of community-acquired pneumonia, it probably accounts for 65% of cases of community-acquired pneumonia among youth. chlamydia pneumoniae and mycoplasma pneumoniae responsible for approximately 20% of cases. haemophilus influenzae caused 7.5% sever cases of disease, 3% of all cases were due to moraxella catharralis . conclusion: pneumococcial infection accounted for 65% of the cases diagnosed. s. pneumoniae was the most common bacterial infective agent, with a low incidence of both m. pneumoniae and s. pneumoniae . other causative pathogens occurred only within groups of individuals with deficiency of immunological status. berezin en a , cardenuto md a , nobuko e b , guerra ml c , brandileone mc d . a santa casa, pediatrics, s. paulo, brazil , b santa casa, microbiology, s. paulo, brazil , c adolfo lutz, microbiology, s. paulo, brazil , d adolfo lutz, bacteriology, s. paulo, brazil to determine antimicrobial susceptibility of sp isolated from the upper respiratory tract, we collected np swab specimens from 80 children, between 3 months and 5 years old. those children attended the outpatient clinic in s. paulo city, with diagnosis of bacterial infection requiring antibiotic therapy between march 15, 2000 to may 20, 2001. penicillin susceptibility of isolates was determined by screening with oxacillin 1 mcg disk and performing the minimal inhibitory concentration by the e -test. we performed also susceptibility test for amoxicillin and cefaclor. results: sp was recovered from np of 42 children (52.5%). the carriage of sp was more prevalent in children attending day care centers, children with young siblings at home, and children with tobacco users at home. the prevalence of penicillin non-susceptible strains was 40.4% all of them with intermediate resistance. all the strains were susceptible to amoxicillin and 19.2% were resistant to cefaclor. serotypes 14.6b, 19f, 9n, 4, 6a and 5 were the most common. these findings suggest that nasopharyngeal isolates of streptococcus pneumoniae from children with upper respiratory infections can be used to conduct surveillance for antimicrobial resistance in a defined geographic area. we were able to conclude also that penicillin intermediate resistent strains can be susceptible to amoxicillin. hinojosa rm a , saenz a a , collazo m a , echaniz g b . a universidad autonoma de nuevo leon, infectologia, monterrey, mexico , b instituto nacional de salud publica, epidemiologia, cuernavaca, mexico the emergence of penicillin-and multidrug-resistant pneumococcal strains has become a global concern, necessitating the identification of the epidemiological spread of such strains. material: ninety streptococcus pneumoniae clinical isolates were collected from march 1997 through march 1999. typing was done by the capsular reaction with pooled, type, or group antisera. susceptibility testing to 11 antimicrobials was done by the e -test and the disk diffusion method. results: only 46 (51%) s. pneumoniae strains were classified by serotyping; the most frequent types were 6a/b, 23f, 9v, 19f and 4. the oxacillin screening test detected 37.2% penicillin-resistant s. pneumoniae strains isolated from children and 44.6% from adults. the susceptibility percentage of s. pneumoniae to ceftriaxone was 93% in both children and adults. s. pneumoniae isolates from children exhibit a susceptibility of 88% to azithromycin, while in adults 91% of the isolates were susceptible. for the rest of the antimicrobial agents, the susceptibility ranged from 69 to 89%. s. pneumoniae had a lower susceptibility to ceftazidime and ciprofloxacin. conclusions: ceftriaxone and azithromycin had a good in-vitro activity against s. pneumoniae strains. but the percentage of penicillinresistant s. pneumoniae detected in this study is alarming, therefore we conclude that a continuous surveillance system is necessary in mexico. vertkine al, prokhorovitch ea, alexanyan la. a retrospective analyses of 221 cases of ambulant pneumonia with fatal outcome was made. among the patients who died from ambulant pneumonia the prevailing age was over 60 (63.8%) and the prevailing sex was male (68.8%). 95.9% had pneumonia accompanied with some pathology: chronic lung disease (43.9%), alcoholism (26.2%), diabetes mellitus (10.4%). 62.5% of the patients had a big volume of lungs lesion */55.7% of the patients suffered from bilobular pneumonia and 6.8% */from trilobate pneumonia. in 57.0% of the cases pneumonia was complicated with abscess formation and/or exudative pleurisy. we studied the antibiotics therapy used for the patients treatment. the change of antibiotics was made only in 53 cases (24.0%) whereas in the other cases no change of preparations was made though the signs of the therapy non-effectiveness were obvious. thus, the rational antibiotics therapy with the timely change of non-effective antibacterial drug is significantly important. while choosing the antibiotics, the patient's age, the accompanying diseases, the volume of the lungs lesion and complications which define pneumonia seriousness are to be taken into consideration. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to describe the management of lower respiratory tract infections (lrti) in healthy adults, by general practitioners (gp). methods: a questionnaire was sent to a representative national sample of 4092 gps. this questionnaire assessed their perception and management of lrti, the indication for antibiotics (ab) in a case of lrti in a healthy adult with no focal signs and no signs of severity, knowledge of the micro-organisms responsible for acute bronchitis and knowledge of the afssaps (french agency for the safety of health products) recommendations. results: three thousand seven hundred and thirty-eight gps, who reported seeing an average of 131 patients per week, including 10.39/ 9.6 patients with lrti, returned the questionnaire. the main results are presented in the following for the majority of gps, the main objective of the visit is to determine the indication for antibiotics. according to gps, alp and whooping cough are rare, while atypical pneumonia is frequent. gps also declare that the diagnosis of alp is often easy right from the first visit, in contrast with that of atypical pneumonia. complementary investigations are not often requested. gps consider that they often delay prescription of antibiotics (41%) and declare that they tend to prescribe a macrolide as first-line treatment. finally, gps have a poor knowledge concerning the micro-organisms responsible for acute bronchitis and the majority of gps declare to be familiar with afssaps recommendations. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to study the management of one case of lower respiratory tract infection (lrti) in adults by general practitioners (gps). methods: prospective study conducted on a representative national sample of 4092 gps. each gp had to include the first healthy adult patient seen during the 3-week data collection period, either on a home visit or in the office for recent cough and acute fever !/37.8 8c. clinical data, the diagnostic perception and the therapeutic approach to the patient were collected by means of a standardised questionnaire, distributed by abbott laboratories. results: three thousand seven hundred and thirty-eight general practitioners included 3738 patients. conclusions: during lrti in adults, gps observe few focal signs, confirming the marked predominance of bronchitis compared to pneumonia. in view of the frequency of the signs, the diagnosis of pneumonia appears to be overestimated. one-third of clinical situations were diagnosed as 'bacterial superinfection of acute bronchitis', despite it is not a recognised diagnostic entity. antibiotic prescription was immediate in 87% of cases and delayed in 10% of cases. this last point shows that clinical practice differs from the gp's perception of their described prescribing practice shown in a simultaneous survey (56% of gps declared that they prescribed antibiotics immediately, while 41% delayed this prescription). results: thirty-three patients (20 men, mean age 49 b 19 years, mean apache ii score 15.3 &bdqup;b 7.3) during the years 1998 á/ 2001 were prospectively studied. thirteen patients (39%) had no identifiable risk factor for severe cap. an etiologic factor was revealed in 15 patients (45%). in 14 of them this was achieved with noninvasive methods. psb cultures were taken from eight patients and were positive in only 1. the offending organisms included: streptococcus pneumoniae in six cases, gnb as the sole pathogen in six cases, haemophilus influenzae (with s. pneumoniae or klebsialla pneumoniae ) in two cases, s. aureus in two and legionella pneumophila in one patient. initial antibiotic regimen a combination of marcodile '/3rd gen cephalosporin9/aminoglycoside was successful in 12 patients who all survived and had to be changed empirically or according to culture results in 17 patients who had a mortality of 65%. the overall mortality rate was 42%. the identification of the causative factor did not seem to have any impact on survival. conclusion: severe cap in our icu was most often caused by s. pneumoniae and gbn. the high mortality of this entity seems to be influenced by the immediate use of the appropriate antibiotic combination and not by the identification of the causative organism. this underscores the need for knowledge of topical microbiology which helps in designing an effective empirical initial antibiotic regimen. mily mn a , golubev sa b , lugovoy vy a , voronov gg b . a vitebsk emergency hospital, pharmacotherapy unit, vitebsk, belarus , b basic and clinical pharmacology department, vitebsk state medical university, vitebsk, belarus purpose: the study aims were to assess the spectrum and predictors of the antibiotic use during pneumonia management in a regional emergency hospital in belarus. patients, treatment and physicians characteristics of 195 cases (2000 á/2001) were collected and possible associations were examined with using defined daily doses methodology (ddd) and american thoracic society (ats) guidelines. results: the mean treatment duration was 13.99/7.4 days, the total antibiotic ddd/100 bed-days was 130.4. the ddd/100 bed-days of the most used antibiotics were: penicillins 54.0; aminoglycosides 29.5; macrolides 21.9; cephalosporins 9.8; tetracyclines 8.2. in manova certain ats categories were associated with ddd/day, but not with frequency of definite antibiotic use. ddd/day was higher in case of multilobar infiltrates ( conclusion: our study indicated the low rate of macrolides and cephalosporins using, and the high one of aminoglycosides. antibiotic prescriptions were associated with disease severity and physician personality rather then with empirical choice rules recommended. acute exacerbation of copd: most frequent infecting agents and their suspectability to the different types of penicillins. analysis of medical documentation ps243 pertseva to, bogatska ke, gashynova ky. dsma, internal medicine , dniepropetrovsk, ukraine number of copd cases has been increased in ukraine. treatment of acute exacerbation (ae) of copd is not always successful because of inadequate antibiotic therapy. the aim of study was to reveal most frequent infecting agents and their susceptibility to the different types of penicillins in patients with ae of copd. medical documentation of 48 patients with ae of copd (type i) was studied. data of sputum analysis and susceptibility of isolated agents to penicillin, ampicillin, oxacillin and amoxicillin/clavulone acid were evaluated. there were patients with haemophilus influencae/parainfluencae (43.8%), klebsiella pneumoniae (25%), staphylococcus aureus (12.5%), pseudomonas fluorescens , pseudomonas putida , serratia marcescens , serratia liquefaciens (6.8% each), streptococcus agalactiae , acinetobacter baumanii (4.1% each) in samples of sputum. in 31.3% cases there was mixt infection. 6.8% had no any bacterial agents. only 10% of agents were susceptible to penicillin, 35% */to ampicillin, 15% */to oxacillin. however, 70% of microorganisms were susceptible to amoxicillin combined with clavulone acid. this study has shown that most frequent infecting agents caused ae of copd were gram-negative microorganisms and s. aureus . according to antibiogram the prescription of amoxicillin/clavulone acid is most expedient in this case. pertseva to, bogatska ke, konopkina li, kireeva tv, gashynova ky. dsma, internal medicine department, dniepropetrovsk, ukraine we examined 32 patients (22 men, mean age 52.39/4.7 years) with acute exacerbation of cob (type i). the most frequently isolated agents were haemophilus influenzae and parainfluenzae */eight patients, gram-negative rods in 8, staphylococcus aureus in two and mixed in six and one patient had no bacterial agents isolated in their sputum. high susceptibility to azithromycin was found in all cases of gram-positive agents and in h. influenzae and parainfluenzae . other gram-negative agents were resistant to this drug in vitro. however, treatment with 500 mg/day during 3 days was clinically effective in 93.7% of cases. only 25.0% of patients had a further acute exacerbation of cob. there were peculiarities of the infecting agents causing acute exacerbation of cob in this study: klebsiella pneumoniae and s. aureus were found more frequently than in other studies. high efficacy of surnamed in the treatment of acute exacerbation of cob was established in 68.7%. 25.0% had partial positive clinical effect after this therapy. there were no patients with adverse events. efficacy and tolerance of amoxicillin 30 mg/kg bid versus amoxicillin 15 mg/kg tid in the treatment of acute otitis media (aom) in children 5/2 years ps245 borek m a , guggenbichler jp b . a biochemie gmbh, international medical department, kundl, austria , b department of pediatrics, university of erlangen, erlangen, germany five hundred and sixteen patients (mean age 49/2.6 years) with clinical and otoscopic diagnosis of aom were included in a randomized, double blind, multicentre study, and were treated 10 days either with amox 30 mg/kg bid or amox 15 mg/kg tid. assessments were made during therapy (day 3 á/5), after end of therapy (eot, day 12 á/14) and follow up (fu, day 38 á/46). the primary efficacy endpoint was the clinical response at eot defined as success (cure/improvement) or failure. both regimens were well tolerated; one or more drug-related adverse events (aes) were reported in 17.2% (11/64) of bid patients and in 21.7% (13/60) of tid patients. the most frequently reported drugrelated aes in each group were gastrointestinal symptoms (bid 11.5% vs. tid 13.5%), which were mainly of mild or moderate severity. both regimens were clinically equivalent. the higher cure rates in the bid group suggest a possible higher benefit from bid therapy in children 5/ 2 years. children attending family day-care (fdc) should be less exposed to upper respiratory tract infections than those in group day-care (gdc) and therefore to antibiotic treatment; fewer should thus carry resistant bacteria. to test this hypothesis, np carriage of sp and hi with reduced susceptibility to penicillin (pdsp and hi bl'/, respectively) was investigated among children in fdc (maximum three children) and in gdc (25 á/100 children) in the alpes maritimes (france) between november 1999 and march 2000. a two stage cluster sample of children attending gdc or fdc was selected. np samples were cultured for sp and hi. penicillin susceptibility was tested by disk diffusion and e -test, and b-lactamase production by api-nh † tests (biomerieux, lyon). two hundred and thirty-five children in fdc and 298 in gdc aged 6 á/36 months were sampled. age and sex distribution were similar in both groups. sp was isolated in 80 children in fdc (34%), and in 163 (54.7%) children in gdc (p b/10 á/6 ). proportions of pdsp were 52.5 and 55.8%, respectively (p0/0.6). hi was present in 37.6% of children in gdc vs. 23.8% in fdc (p b/ 0.001). proportions of hi bl'/ were 40.2% vs. 46.4%, respectively (p0/0.4). antibiotic exposure during the previous 3 months concerned 46.2% of children in gdc vs. 48.7% in fdc (p 0/0.6). there was no correlation between antibiotic use and carriage of pdsp or hi b'/ strains. sp and hi carriage rates are significantly lower among children in fdc than in gdc. advising alternative types of daycare for children attending gdc should reduce exposure and thus limit the spread of resistant bacteria. however, the proportion of pdsp and hi bl'/ is similar in both groups and comparable patterns of antibiotic use are observed. continued efforts must concentrate on parental education and enforcement of recommendations for management of pediatric upper respiratory tract infections. during a period between january 1999 and august 2001, 43 invasive haemophilus influenzae (hi) isolates had been collected at the children's hospital of tunis. we used haemophilus test medium to test antibiotic susceptibility. the mic of beta-lactams was measured by e -test. beta-lactamase production was determined by using the cefinase test and biotyping by apinh. presence of capsular antigen was determined by using hi typing anti sera. hi strains were isolated from meningitidis (35), bacteremia (5) and arthritis (3). all strains were serotype b and 62.2% of them belonged to biotypes i and ii. amoxicillin resistance with beta-lactamase producing mechanism occured in 34.8%. mic90 of beta-lactamase producing strains was 32 vs 0.5 mg/l in non-producing one. there is no betalactamasenegative amoxicillin resistant among these invasive isolates. antibiotic resistance concerned chloramphenicol: 9.3%, trimethoprim-sulfamethoxazole: 11.6%, tetracycline: 4.6% and kanamycin: 11.6%. invasive hi infections in tunisian children's were always associated with type b strains. introduction of a hib vaccine programme in tunisia is recommended. the aim of this study was to analyse the clinical picture and treatment of neurological manifestations of neuroborreliosis in children. the study included 22 children (2 á/16 years) with neuroborreliosis diagnosed on the basis of the clinical and serologic criteria. symptoms of facial palsy occurred in six children symptoms of iii á/vi cranial nerves palsy in three children, meningitis in four and paresthesias in three. symptoms of v or viii nerve palsy, mental disturbances, radiculoneuritis or cerebellitis were found in singular cases. all children received ceftriaxone intravenously 3 á/4 weeks. total recovery was obtained in 18 children following the first course of therapy. recovery following the second course of therapy (amoxicillin) occurred in one child with mental disorders and one with vi nerve palsy. improvement was achieved after the second course in the patient with radiculitis, however, muscular atrophy persisted. irreversible, unilateral deafness was found in a child with viii nerve palsy in spite of three courses of therapy applied. infection with borrelia burgdorferi in children causes a wide spectrum of neurological manifestations. facial palsy was the most common sign in our study. applying ceftriaxone in the treatment of neuroborreliosis is characterised by a good effectiveness. double infection by c. pneumoniae and m. pneumoniae as a cause of cystic changes in the lungs ps249 streharova a, moravcikova d. department of infectious diseases, university of trnava, trnava, slovakia chlamydia pneumoniae and mycoplasma pneumoniae are human respiratory pathogens manifested in early childhood. immunological disbalance could trigger autoaggressive diseases. the authors describe the case of a15-year-old girl with development of multiorgan failure and septic state, which followed multiple cystic changes in the lungs. the girl did not have a diagnosis of cystic fibrosis. the authors consider that cystic changes are a consequence of double infection by c. and m. pneumoniae. dzarlieva m, momeva l, temelkovska g, balevska p, pejkovska m. medical centre, neonatology, bitola, the former yugoslav republic, macedonia unicef skopje has supported a nationwide safe motherhood needs assessment in representative samples of hospitals. eighteen of 28 maternity wards and facilities renovated and certified as 'babyfriendly'. all mature newborns with successful adaptation to extra uterine life and satisfactory vital parameters are 24 h during the day with their mothers at rooming-in system. aim: with rooming-in system we reached decreasing of incidence of neonatal infections. material and methods: history records of newborns from our department. for the period of 9 months, 907 neonates have been borne. with suspection of infection there */49 babies (5.4%). newborns borne through meconium stained liquid */40 (4.4%). results: microbiological findings: from blood culture */staphylococcus coagulaza negative from swabs */staphylococcus aureus , escherichia coli , staphylococcus epidermidis. conclusion: in 1999, from all babies who had risk factors for infection in 56 newborns (5%) we had positive findings and in year 2000 (before rooming-in sistem), in 44 (3.9%). after that period (with rooming-in) 15 newborns (1.5%). with rooming-in system we reached decreasing in incidence of neonatal infections by breaking the chain of infection */only mothers take care of their babies with help of the staff. newborns are in their micro environment, the same they will have at their home. with this practice we have also reduction of nosocomial infections. a study on antibiotic susceptibility and resistance factor transmissibility among antibiotic resistant salmonellae isolated from children affected to diarrhea ps251 sharifzadeh a. azad university of shahrekord, microbiology, shahrekord, islamic republic of iran in spite of happened drug resistance, antibacterial therapy still is the best route of treatment of salmonellosis in man and animals. in order to detection of dominants serotypes of salmonellae in children and detection of antibiotic susceptibility and r-factor transmissibility among those isolated salmonellae. this study was conducted on 400 diarrheic stool samples were collected from children affected by diarrhea in ayatollah kashany hospital of shahrekord, during spring of 1999 to autumn of 2000. after isolation and identification of salmonellae, seven serotypes were detected. one of those was s. typhi and another six serotypes were s. paratyphi b . in order to detection of antibiotic different antibiotic disks were used in disk diffusion method. best results were taken from ceftizoxim, cephtriaxon, cephazolin and chloramphenichol. the r-factor were transferred from isolated salmonellae to escherichia coli k12 in all of cases of resistance to penicillin and ampicillin. pharyngeal colonization by streptococcus pneumoniae and group a bhs were evaluated in 652 randomly selected school children aged 6 á/ 8 years in riyadh, saudi arabia. fifty-six children (8.6%) had positive culture for either organisms of the 56 isolates from school children, 17 (30%) were s. pneumoniae , of them 14 (82%) were penicillin-sensitive, three (18%) were penicillin-resistant, and two (11.7%) were resistant to two antimicrobials. forty isolates of bhs (71%) were group a bhs. all isolates were penicillin and erythromycin sensitive. the carrier rate among school children for penicillin-resistance s. pneumoniae and resistance to two antimicrobials were (4.6%) and (3%), respectively. the carrier rate of group a bhs was (6.1%). riyadh has a low rate of antibiotic-resistant s. pneumoniae and a similar rate of group a bhs carriers among school children as that seen in temperate areas. boukadida j a , boukadida n b , hannechi n a , said h b , erraii s a , elmhabrech h a . a chu f. hached, laboratoire de microbiologie, sousse, tunisia , b c s base, sousse, tunisia the acute pharyngitis is a very frequent pathology in which group a streptococcus is the most incriminated bacteria. however, other non a b-haemolytic streptococcus (sbna) could be responsible. the aim of this work is to determine the part of each non a b hemolytic streptococci (sbna) in acute pharyngitis and the related antibiotics susceptibility pattern. the study was realized in sousse-tunisia (north africa) during 5 months from may 2001. the origin materials of isolates are throat swab (transystem venturi, copan, bovezzo). the mean age of patients is 23 years with extremes 3 á/72 years. the samples are cultured on blood agar plates in a delay of 3 h maximum. identification was done to samples that have over than 20 b-hemolytic colonies, groupage with pastorex strep. sanofi pasteur france, susceptibility pattern according to nccls norms, mic is determined by e -test. the control strain is s. pneumoniae atcc 49619. twentyone clinical isolates of sbna are distinguished from 80 clinical isolates of b-hemolytic streptococci recovered from 143 patients with acute pharyngitis without symptoms of viruses' infections (tearing, corysa, sneeze). all b-hemolytic streptococcus represents 55.94% of all collected samples. sbna were 26.25% of the isolates. sbna were 11 strains group g streptococci, seven strains group c streptococci and three strains group f streptococci. susceptibility pattern of each sbna to antimicrobial agents is as follow: group g streptococci: peni g 0/100%, amoxicillin 0/100%, pristinamycin 100%, clindamycin and erythromycin0/45.46%, tetracyclin 0/9.1%, telithromycin 63.63% and levofloxacin0/45.45%. group c streptococci: peni g0/ 100%, amoxicillin 0/100%, pristinamycin 100%, clindamycin0/ 57.15%, erythromycin0/42.86%, tetracyclin0/71.42%, tel-ithromycin0/85.72%, levofloxacin0/100%. group f streptococci-peni g0/100%, amoxicillin0/100%, clindamycin0/66.66%, erythro0/33.34%, tetracyclin 0/66.66%, telithromycin 0/100%, levofloxacin0/66.66%. all sbna have mic to penicillin g under 0.1 mg/l. according to available data, penicillin g and amoxicillin still the reference treatment of acute bacterial pharyngitis in spite of the new antibiotics introduction. berezin en a , quevedo sg b , nicolla l b , viegas d c , eizenberg b d , pedrosa f b , santos ag a . a santa casa, pediatrics, s. paulo, brazil , b elly lilly, scientific, s. paulo, brazil , c fac. abc, pediatrics, santo andre, brazil , d university hospital, pediatrics, s. paulo, brazil a multicenter, open label, prospective, randomized trial in which patients 2 á/16 years of age with proven gabhs pharyngitis were randomized to receive either 10-day course of the broad spectrum oral cephalosporin, cefaclor or a 10-day course of amoxicillin. patients were included if they have signs and symptoms of streptococcal tonsillopharyngitis and a rapid streptococcal rapid test positive . patients were evaluated at days 3 á/5, 13 á/16 and 24 á/35 posttherapy. pharyngeal cultures were conducted at baseline and at follow-up visit (13 á/16 days). we considered for bacteriologic eradication analysis only patients with positive culture to gabhs. there were 148 patients with a rapid streptococcal rapid test. clinical success were achieved in around 95% of the patients. for evaluate eradication of the initial pathogen we considered 42 patients of the amoxil arm and 40 patients of the cefaclor arm. thirty-four of 42 patients of the amoxil arm (80.9%) and 36 of 40 (90%) patients of the cefaclor arm were considered bacteriological cured at the second culture performed at day 13 á/16. ten days of a penicillin or amoxicillin therapy may not be the best therapeutic choice for all pediatric patients. in developing countries where rheumatic fever is still an important problem to evaluate the bacterial eradication achieved with the different antibiotics may be important. prophylactic affect of saccharomyces boulardii for antibiotic-associated diarrhea in a paediatric age group ps256 erdeve o, tiras u, camurdan mo, tanyer g, dallar y. ankara education and research hospital, pediatrics, ankara, turkey the process resulting in antibiotic associated diarrhea is the alteration of enteric flora due to bacteria. saccharomyces boulardii is a yeast, isolated from cover of a kind of hazelnut and its usage became widespread recently. there is no enough study about s. boulardii activity at pediatric ages. we aimed to define s. boulardii activity on azithromycin and sulbactam-ampiciline, and associated diarrhea at pediatric age group. the 18.9% of cases only with antibiotic usage developed diarrhea, whereas rate was 5.7% for probiotic using group (p b/0.05). all of the clostridium difficile toxin a defined cases were from sulbactam-ampiciline using group. the rate of diarrhea for sulbactam-ampiciline using group was 25.6%, while it was 5.9% for group used s. boulardii as probiotic beside sulbactam-ampiciline (p b/ 0.05). it was observed that probiotic usage decreases diarrhea rate four times (p b/0. 05). when age groups considered, the rate of sulbactamampiciline associated diarrhea increased at 1 á/5 ages and s. boulardii effect on preventing diarrhea was significant at 1 á/12 ages (p b/0.05). antibiotic associated diarrhea is a common clinical problem at pediatric age group. s. boulardii is a hope giving probiotic especially for sulbactam-ampiciline associated diarrhea. grey e a bilikova e a , hafed bm a , kovacicova g a , chovancova d b , huttova m b , krcmery v a . a school of health, trnava university, trnava, slovakia , b postgraduate academy of medicine, neonatal clinic, bratislava, slovakia the purpose of the study: the aim of the study was to find out, whether artificial abortion of a mother has impact on neonatal infection of her future baby. the results obtained: therefore, we compared 39 neonates with infection, who were born to mothers with past history of abortion (1 á/7 artificial abortions within 10 years), with the babies of mothers, who have not experienced abortion before. control group consisted of 207 neonates hospitalized at the same clinic, at the same time period. according to the analysis of risk factors for neonatal infections, it was found, that prematurity (28 á/32 weeks of gestation) (30.77 vs. 14.01%, p 0.019) and low birth weight-1500 á/2500 g (58.97 vs. 39.13%, p 0.03) were significantly more frequently observed in babies of mothers with past history of abortion. drug abuse (heroin) (12.82 vs. 1.45%, p 0.003) and nicotine use (15.38 vs. 2.42%, p 0.0027), were more significantly related to neonatal infections in babies of mothers with past history of abortion. etiological analysis showed that only candida albicans (5.13 vs. 0%, p 0.024) was significantly related to neonatal infections in mothers with past history of abortion. the conclusion reached: in conclusion, artificial abortion has not only direct impact to the health of the mother, but also on her next pregnancy. bacteraemia and patient mortality in a peadiatric intensive care unit ps258 armenian sh, singh j, arrieta ac. children's hospital of orange county, infectious disease, orange, usa purpose: this study will identify the factors that significantly contribute to mortality in patients with bloodstream infections (bsi) at a pediatric intensive care unit (picu). results: medical records of 63 patients who were admitted to the picu and had a documented bsi were reviewed. there were 74 separate episodes of bsi's, with nine patients having multiple bsi's during their hospital stay. a casecontrol model was used. cases were bsi's with eventual mortality (n0/27; 36.5%) and controls were those who survived bsi (n0/47; 63.5%). patients who died were older (5.7 vs. 2.4 years; pb/ 0.01), more likely to have a nosocomial bsi (42.9 vs. 16.7%; pb/ 0.05), longer hospitalization prior to bsi (30.5 vs. 9.8 days; pb/ 0.05), and have a polymicrobial bsi (66.7 vs. 30.6%; pb/ 0.05). infection related mortality (irm)-defined as death within 7 days of bsi */was significantly higher in those receiving inadequate antibiotic treatment at the time of diagnosis of bsi (54.5 vs. 12.7%; p b/ 0.01), as well as in those with gram negative bacteremia and/or fungemia (35 vs. 13%; p b/ 0.05). logistic regression was used to adjust for potential confounding variables. conclusions: we found that being older, multiple organisms, and a longer hospitalization prior to the bsi were significantly associated with overall patient mortality. irm was significantly higher for those with inadequate initial antibiotic coverage and in those with gram negative bacteremia and/or fungemia. somer a a , gü r d a , diri s a , yalcýn i a , salman n a , ongen b b , gü rler n b . a department of pediatric infectious diseases, istanbul university istanbul medical faculty, istanbul, turkey , b department of microbiology and clinical microbiology, istanbul university istanbul medical faculty, istanbul, turkey teicoplanin is a glycopeptide antibiotic active against a broad range of gram-positive pathogens including methicillin-resistant staphylococci and offers the advantages of once daily administration, choice of administration route, lack of requirement for routine therapeutic drug monitoring and lower propensity to cause nephrotoxiticy and anaphylactoid-like reactions. in this study the efficacy and safety of teicoplanin were evaluated retrospectively in children with serious bacterial infection. sixty-three children (18 girls, 45 boys) aged between 1 month and 15 years were treated with teicoplanin (three loading dosages of 10 mg/kg at 12 h intervals, followed by a maintenance dosage of 10 mg/kg/day). the infections treated were pleural empyema (n0/25), joint and bone infections (n 0/22), septicemia (n0/9), skin and soft tissue infections (n0/4), and lung abscess (n0/3). the pathogens isolated were staphylococcus aureus (n 0/30, 16 of which were methicillin resistant), coagulase-negative staphylococci (n0/4), s. pneumoniae (n0/4), and group a hemolytic streptococci (n 0/2). the duration of therapy ranged from 14 to 81 days (median 28 days). clinical success (cure plus improvement) was achieved in 96.8% of cases. no side effects attributable to teicoplanin therapy were encountered. teicoplanin appears to be an effective and well-tolerated treatment for serious gram-positive infections in children. the risk of infection is present in all children with acute leukemia. two hundred and sixty episodes of febrile neutropenia were analyzed in 149 children (aged 1.5 á/16 years) with aml */43 cases and all */ 106 cases over 10 years during cytostatic therapy (protocols: mbfm */ 87 aml and mbfm */90 all). a degree of fn occurred in 90% of cases in aml, in 53% */in all. the sites of infection were: blood'/ central venous catheter (32%) and respiratory tract (49%). pathogens isolated from blood were: gram-positive */cns */70%, streptococcus spp. */18.7%, gram-negative */enterobacteria */41%, pseudomonas aeruginosa */23%, candida spp. */23.5%, aspergillus spp. */5.9%, other */5.9%. for the treatment of fn we used empirical antibiotics regimens. clinical response was noticed: in 1st line of therapy */ cephalosporins 3 á/4 generation used */45%, carbapenems used */60% as 2nd á/3rd lines */vancomycin */70%, amphotericin b */80%. thirtytwo percent of children with aml and 5.2% children with all died because of sepsis. conclusion: carbapenems are more active in the 1st line of antibiotics therapy both for patients with aml and all. vancomycin is useful in the 2nd line for patients with all. amphotericin b and vancomycin are useful in combination in the 2nd line for patients with aml. pavlenishvili ivp. medical academy, pediatrics, tbilisi, georgia our drug of choice in cases of complicated neonatal sepsis i. pavlenishvili, g, iobashvili tbilisi, medical academy georgian society of pediatrics chemotherapy (gspc) with collaborations of drug and therapeutic committee (dtc) of childers hospital 'republic' finished the observation study about nosocomial infections of neonatal icu in above mentioned hospital. study begins from november 2000. during this period we observe 45 cases of neonatal sepsis, most of them were due gram-negative bacteria. thirty-four cases of neonatal sepsis were cause different stains of escherichia coli , proteus spp. and acinetobacter . we notice that during last 2 years the role of acinetobacter in etiology of neonatal sepsis and nosocomial complications of neonatal sepsis in our hospital is gradually increased. as our observation reveals most optimal in the case of complicated neonatal sepsis was (according criteria of dts) use of ceftasidime. almost in all cases we used ceftasidime with success. we have only one case of mortality. since the macrolide resistance (mr) in streptococcus pyogenes has been increasing in europe, we studied the incidence and genetic basis of mr in s. pyogenes in russia. s. pyogenes isolated at baseline and during follow-up visits from children with acute pharyngitis receiving penicillin or midecamycin (16-membered macrolide) were studied. mr was evaluated by agar dilution (nccls), resistance mechanisms */by pcr. a total of 149 s. pyogenes were obtained at the baseline, 21 (14%) of which were erythromycin-resistant: 19 strains (90%) were erm (a)-positive, one */mef (a)-positive, and one was negative for all primers used. all erm (a)-strains were inducibly resistant to clindamycin and represented seven pfge profiles with one profile found in 11/ 19 strains. in three midecamycin treated patients mr was selected during the therapy (one strain had erm (a), one */mef (a), one */ unknown resistance determinant). mef (a)-strain obtained during follow-up visit had different pfge pattern with mef (a) strain isolated at baseline, while both the baseline and follow-up strains with unknown mechanism of resistance had unique pfge pattern. these data showed moderate incidence of erythromycin-resistant s. pyogenes in smolensk. ribosomal methylation (erm (a)) was the most common mechanism and though the polyclonal nature of mr was established, most erm (a)-strains belonged to only a few clones. bacillus cereus infections in traumatology-orthopaedy department: retrospective study and re-evaluation of healthcare practices ps263 bacillus cereus was cultured for 30 patients from traumatology department who had developed postoperative wound infections between august 1997 and march 2000. all patients presented inferior members open fractures, frequently contaminated with telluric material and requiring external fixators. genomic study of clinical isolates by pulsed field gel electrophoresis and random analysis polymorphic dna, allowed us to eliminate an outbreak. furthermore, the reduced delay in which patients developed the infection (6 days'//-2) led us to re-evaluate the procotols used in our institution. indeed, all patients had received amoxycillin '/ clavulanate iv 2g for antibioprophylaxis during anesthetical induction then relayed per os for 48 hours. because of the production of a potent beta-lactamase by the bacteria, this association could not be efficient. furthermore, accordingly to the afnor en 1040 norm, we have tested clinical isolates' sensitivity to the principal antiseptics used for antisepsis and disinfection (iodophors, chloride derivated and biguanidines) and observed a major resistance of all strains tested. even if these postoperative wound infections are considered as nosocomial because of the delay in appearance, we actually think that bacillus cereus were initially present in telluric material. this fact led us to propose a systematic screening for bacillus at admission for this type of wound and to administrate quinolones such as ciprofloxacin for prophylaxis. internal thiols and reactive oxygen species in the candidacidal activity exerted by a n-terminal peptide of human lactoferrin ps 264 the purpose of the study: the emergence of candida albicans strains resistant to current antifungals points to the need for new antifungal agents, e. g. antimicrobial peptides. the results obtained : we report that hlf(1-11), a synthetic nterminal peptide of human lactoferrin, displays excellent killing effect against fluconazole-resistant c. albicans and that sub-optimal concentrations of this peptide combined with fluconazole act synergistically. previous investigations revealed that hlf(1-11) required an energized mitochondrion, atp release by candida , and ligation of atp receptors for its killing effect. we now report that reactive oxygen species (ros) are involved in the killing effect of hlf(1-11). since internal thiols protect cells from oxidative damage, our observation that hlf(1-11) caused a 20% reduction of internal thiols in candida is of interest. as expected, n-acetyl-l-cysteine (nac), which is a precursor of glutathione and a ros scavenger, inhibited the killing effect of hlf(1-11). diamide, which oxidizes internal thiols, was candidacidal and hlf(1-11) and diamide acted synergistically in killing c. albicans and ros production. moreover, the hlf(1-11)-induced activation of mitochondria was inhibited by nac, indicating that internal thiols/ros affect mitochondrial activity. the conclusion reached : the candidacidal activity of hlf(1-11) involves ros production and reduction of internal thiols. objective : an audit was conducted to explore the observation of increasing resistance to ciprofloxacin in enterobacteriacea isolated from specimens from such patients in the hematology unit. results : enterobacteriacea isolates in specimens from such patients processed over the years 1997 to 2001 were examined. number of specimens per year was */109, 152, 132, 160 and 170 respectively and the annual percent ciprofloxacin resistant enterobacteriacea from these was 2%, 9%, 12%, 14% and 21%. conclusion : conclusion drawn from the audit was that rapid rise in ciprofloxacin resistance may possibly be attributed to the use of this fluoroquinolone as a single agent in dose of 250/500 mg [cut off patient weight 40 kg] twice a day in neutropenic patients till neutrophils exceed 500/c.mm. subsequent to the audit, prophylaxis protocol was modified to use of oral colistin 3-mega units/twice a day in combination with ciprofloxacin 250/500 mg twice a day. a prospective audit is proposed to test the benefits of this combination. rhinocerebral zygomycosis: diagnostic dilemma for emergency physician: can the associated morbidity and mortality in this rare but deadly disease be reduced? ps 266 samavedam s, guleri as. western infirmary, clinical microbiology, glasgow, united kingdom rhinocerebral zygomycosis [rcz] is a rare, invasive, rapidly progressive opportunistic infection caused by ubiquitous fungi of the order mucorales. it usually occurs in diabetics or immunocompromised patients. the emergency physician will typically see patients with rcz in its earliest stages masquerading as a variety of other less serious diseases. the key markers like necrotic patch on hard palate, nasal septum or turbinate, marked facial pain, and cellulites with marked eye and neurological signs may present late in disease. we report a case of rcz caused by rhizopus arrhizus [oryzae] in an 84 year old woman with poorly controlled diabetes. she presented with a right-sided facial droop of short origin and being generally unwell. ct scan was non-conclusive and delayed presentation of key markers of rcz permitted disease to rapidly progress. despite an intensive antifungal therapy with ambisome and insulin sliding scale, patient rapidly succumbed within 4 days. fine needle aspiration cytology is less invasive, easier and equally effective alternative to pre op biopsy. the key to successful reduction in morbidity and mortality associated with this rapidly fatal disease is -increasing awareness of the disease, an early diagnosis, correction of underlying metabolic derangement, prompt intensive antifungal therapy with amphotericin b and radical surgical debridement of the necrotic tissue. an 'optimal dosage' of ambisome requires discussion. clinical audit in the haematology ward of a tertiary care hospital: study of degree of correlation between bacteraemia and oro-pharyngeal screens in immunocompromised patients over five years and role of antibiotic prophylaxis ps 267 guleri as, butcher i. western infirmary, clinical microbiology, glasgow, united kingdom introduction : a clinical audit was carried out over 5-years [1997] [1998] [1999] [2000] [2001] in the immunocompromised patients including neutropenic patients and bone marrow [autograft] transplant recipients in hematology ward of gartnaval general hospital, glasgow, a tertiary care center. objective : it was aimed to establish the degree of correlation between bacterial isolates in oro-pharyngeal screen during bacteraemia episodes and role of antibiotic prophylaxis. methods purpose : to assess whether antiretroviral therapy (art) intensification, gm-csf use and remune initiation before stopping art lead to viremia containment, and long periods off art. methods : ten adults with chronic hiv disease, hiv-1 rna levels (vl) b/1.7 log copies/ml and median cd4'/ -t cell count of 385/ml were enrolled. after art intensification with ddi (6 months) '/ hydroxyurea [hu](5 mo.) '/ gm-csf (3 mo.) and a remune dose, art was stopped but remune continued. art was resumed if rebound vl did not decrease to b/4.7 log in 3 months or if cd4'/ counts decreased to b/200. results : vl rebounded in all patients after stopping art, and 7 developed an acute retroviral syndrome (ars). cd4'/-t cells decreased, and cd8'/38'/ increased (5-fold). after a median stoppage of 16 weeks, art was resumed in 9 patients and vl decreased to b/ 1.7 log, cd4'/ counts were regained, il-2 and il-15 levels rose. at the 2nd interruption, 9/9 patients had a rebound, and 3/9 had a 2nd ars, but peak vl and loss of cd4'/ were lower (p 0/0.003). after 21.3 weeks off art, 8 patients resumed therapy. the breadth and magnitude of hiv-specific activity increased and thymus size grew. the patients were off art for a median of 49.5 out of 80 weeks. two of them are off art for 80 and 44 weeks respectively ( vlb/4 log). conclusions : this approach led to a high ars incidence, long periods off art, increases in hiv-specific responses, il-2 and il-15 levels, and thymus size. urinary tract infection in hospitalized population is a significant problem. this is a study of catherized patients urinary infection in corfu hospital. in 2000 á/2001, 3500 urine cultures were sent to our bacteriology laboratory. 650 of them were collected from the catheters. clinical data of age and type of disease were analysed. the samples were cultured on mc conkey agar-urotube and vitek cards. the organisms identified with vitek automatic system. the sensitivity was tested with vitek and kirby-bauer method. results: no growth of organisms in 46.5%. positive cultures 35%. 62.2% of the bacteria were gram((/) rods (45.6% e. coli , pseudomonas spp. 0%), 16% were gram('/) cocci (enterococcus spp.) and 22% was candida spp. the resistance of e. coli was: 37% to ampicillin , 17% to co-trimoxazol and 2% to quinolons. e. faecalis was resistant 10% to vancomycin . conclusions : (1) the possible interference of acetaminophen in the amoxicillin/ clavulanic acid (a/c) or erythromycin (ery) efficacy in the treatment of acute otitis media (aom), and its possible role in the evolution to otitis media with effusion (ome), were determined in a gerbil model. a 23f streptococcus pneumoniae strain exhibiting a mic of a/c and ery of 1/0.5 and 0.12 mg/l, respectively, was used. both antibiotics were tested at 2.5 and 10 mg/kg. acetaminophen at 50 mg/kg was administered 30 min before each antibiotic dose. antibiotic concentrations in serum and middle ear exudate were determined. both antibiotics significantly reduced the number of culture-positive ears and colony counts, with serum concentrations over the mic of the microorganism for ]/15% of the dosing interval. antibiotic concentrations in middle ear exudate were almost identical in animals receiving and not receiving acetaminophen. clinical and microbiological efficacy was correlated with antibiotic concentrations in middle ear exudate ]/1.7 times the mic of the microorganism, for both antibiotics. both antibiotics demonstrated efficacy in the treatment of pneumococcal aom, with the same rate of ome. acetaminophen, concomitantly administered, did not interfere the efficacy of the two antibiotics tested and did not prevent the evolution of aom to ome. parra a a , ponte c a , cenjor c b , garcía-olmos m a , giménez mj c , aguilar l c , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b fundación jiménez díaz, otorrinolaryngology, madrid, spain , c glaxosmithkline, medical department, madrid, spain a gerbil model of otitis media with effusion (ome) induced by haemophilus influenzae (amoxicillin/clavulanate-a/c-and erythromycin-ery-mics of 1/0.5 and 4 mg/l, respectively) was used to evaluate the efficacy of a/c (10/2 and 15/3 mg/kg) and ery (20 and 50 mg/kg). antibiotics were administered subcutaneously 2 h post-middle ear inoculation, and continued t.i.d for 24 h, with or without acetaminophen (ap), at 50 mg/kg, administered 30 min before each antibiotic dose. antibiotic concentrations in serum and middle ear (me) were measured by bioassay. me samples for colony counting were collected on day 2. a/c reduced (p 5/0.05) positive me samples and colony counts versus untreated controls or ery: me positive cultures of 90% for controls, 0% for a/c 15, 7% for a/c 10, 17% for a/c 10'/ap, 90% for ery 20, 57% for ery 50 and 87% for ery 50'/ap. this was due to a/c (but not ery) concentrations in me exceeding 1.8 times the mic despite the higher percentage of antibiotic penetration of ery versus a/c (43 versus 8/14%). animals receiving ap showed less polymorphonuclear cells and more bacteria in me than those receiving only antibiotics, suggesting that the anti-inflamatory drug diminish the phagocytes and therefore, the efficiency in bacterial clearance. amoxycillin treatment for acute otitis media caused by penicillinresistant streptococcus pneumoniae . a pharmacodynamic analysis pm103 parra a a , ponte c a , cenjor c b , garcía-calvo g a , giménez mj c , aguilar l c , soriano methods: a serotype 23f streptococcus pneumoniae strain exhibiting a mic of amoxycillin of 1 mg/l was used in an experimental model performed in gerbils (meriones unguiculatus ) following previously described procedures. amoxycillin was tested at the following doses: 0.2, 0.4, 0.8, 1.25, 2.5 and 5 mg/kg. amoxycillin concentrations in serum and middle ear exudate were determined after drug administration. results: doses of ]/2.5 mg/kg significantly reduced the number of culture-positive ears, colony counts and otorrhoea (p 5/0.05) as compared with untreated controls or animals treated with doses lower than 1.25 mg/kg. doses of ]/2.5 mg/kg achieved antibiotic concentrations in the middle ear 1.4 á/2.4 times higher than the mic of the infecting strain and serum concentrations over the mic for 14 á/19% of the dosing interval. conclusions: amoxycillin at doses achieving serum concentrations similar to those obtained in children after standard doses, obtained therapeutic and microbiological efficacy regardless the susceptibility of the infecting strain. better correlation was found between antibiotic efficacy and antibiotic concentrations in middle ear exudate than between efficacy and serum concentrations, which were suboptimal from the pharmacodynamic perspective. increasing prevalence of amoxycillin á/clavulanate-resistance among e. coli strains in a hungarian university hospital pm104 veréb i, vígh a, urbán e, hajdú e, nagy e. department of clinical microbiology, university of szeged, szeged, hungary background: amoxicillin á/clavulanate resistance (acr) is an emerging problem in escherichia coli as reported from different parts of europe. the aims of the present study were to evaluate statistically the prevalence of acr among e. coli isolates and to investigate the genetic background of the resistance. methods: all e. coli strains isolated between 2000 1 1 and 2001 9 1 were screened for acr by kirby á/bauer disc diffusion method. the resistance to other beta-lactam á/beta-lactamase inhibitor combinations and to different beta-lactam antibiotics were also tested. selected strains underwent determination of beta-lactamase activity. confirmatory tests for suspected extended spectrum beta-lactamase were performed. pcr testing for tem and shv genes were carried out on plasmids isolated from selected strains. results: in 2000 out of 1109 e. coli strains 102 (9.2%) were found to be resistant to amoxicillin clavulanate (amc). most of the resistant strains (95%) were obtained from the genitourinary tract and no acr isolate was found in blood cultures. in 2001 out of 2167 isolates 239 (11%) proved to be acr and 2.5% were isolated from blood cultures and 66.9% from the genitourinary tract. thirty-five selected strains were further analysed. thirty-two were also resistant to (sam) and six were further resistant to tzp. quantitative beta-lactamase determination showed increased activity in strains which were partially susceptible to amc. the presence of esbl could be proved only in three acr isolates. this open parallel-group study compared the efficacy and tolerability of cef with cfix 400 mg once daily in the treatment of community acquired uncomplicated uti. seventy-eight female patients were randomized to receive either oral cef or cfix for 5 or 10 days. the efficacy of treatment was evaluated by clinical response (by symptoms of uti dysuria frequency urgency suprapubian pain and by clinical signs) by bacteriologic response and health status measures at baseline and posttherapy. results: the clinical cure (complete resolution of symptoms and signs) rate for patients receiving cef was 95.12% of the 41 evaluable patients and 89.18% of the 37 patients receiving cfix. bacteriologic response (based on the results of urine cultures obtained posttherapy) the pathogen was eradicated in 82.3% for cef 73.3% for cfix. no drug related side effects have been reported in cef and side effects were experienced by 5.40% of the patients receiving cfix. improvement in health status comparing visual scale scores baseline and poststudy to have detected a higher change in average score from 17 to 94 in cef, from 20 to 82 in cfix. wilcoxon improvement value was significant on the 3rd day of therapy in case of cef and on the 5th day of therapy in cfix group. in conclusion, the results of this study indicate that cef course is more effective than cfix in producing a favourable clinical outcome and achieving higher bacteriologic eradication rate, furthermore cef was better tolerated. cefepime is a fourth generation cephalosporine that has a broader spectrum of antibacterial activity than the third generation cepfalosporines and is more active in vitro against gram-positive aerobic bacteria. the purpose of this study was to measure cefepime concentrations in plasma, bile fluid and gall bladder tissue in patients undergoing cholecystectomy. thirty patients 12 male, 18 female, mean age: 48 years had data acceptable for analysis and were included in this study. all patients received iv 2 g of cefepime. several hours after administration and at different time intervals, during surgery, samples were obtained from plasma, bile fluid and gall bladder tissue concomitantly. antibiotic levels were measured by an agar diffusion method. the mean delta time was 2849/255 min. the values for plasma, bile fluid and gall bladder tissue, were 33.69/28.5, 6.69/4.3 and 16.19/12.9 mg/ml, respectively. the plasma/bile fluid ratio was 2.69/ 2.4. there was a significant correlation between plasma and gall bladder tissue concentration (r0/0.771, p0/ b/0.0001). a correlation between bile fluid and plasma cefepime concentration was not observed. the minimum inhibitory concentration (mic) data from previous in vitro studies indicate that the cefepime concentration observed in plasma bile and tissue samples of this study would be adequate against typical biliary tract pathogens. furthermore, these cefepime concentrations correlated well with the favorable clinical outcome reported in previous clinical studies in biliary tract infections. there was also good correlation between delta time and plasma and tissue concentrations and if the dose were given closer to the time of surgery, cefepime concentration would be higher reducing the possibility of an infection. objectives: the use of antibiotics may lead to decreased colonization resistance and increased formation of resistant bacteria. present concept was developed to overcome these untoward effects. methods: b-lactamase of bacillus licheniformis was overproduced in bacillus subtilis . this targeted recombinant b-lactamase enzyme (trbl) was released in the small bowel from a controlled-release formulation. beagles (n0/6) were treated bid with either 20 mg/kg ampicillin (i.v.)'/placebo (p.o.), 20 mg/kg ampicillin (i.v.)'/trbl (p.o.) or only placebo (i.v.'/p.o.). stool was collected at days 4 and 10. samples were cultured for total and main groups of aerobic and anaerobic bacteria and yeast. temperature gradient gel electrophoresis (tgge) was used to separate the ribosomal rna genes. results: ampicillin'/placebo group had clearly decreased counts of both aerobic and anaerobic bacteria during the treatment, whereas those receiving trbl had only minor overall changes and some occasional changes by single species. intravenous ampicillin decreased the fecal similarity percentage to 60%. the similarity percentage during treatment with ampicillin'/trbl did not differ from that of placebo (86 vs. 81%). conclusions: according to our results the trbl can maintain the large intestinal microflora almost unchanged. these results indicate that trbl is a promising novel approach for overcoming the ecological adverse effects on gut flora caused by b-lactam antibiotic agents. adamis since broad-spectrum â-lactams combined with amikacin are often applied for nosocomial infections, their pharmacokinetic interactions might be interesting. one gram of aztreonam and 0.5 g of amikacin were administered intravenously single and in combination in six healthy volunteers. blood samples were collected at regular time intervals and concentrations of antimicrobials were determined by a microbiological assay applying a strain developing resistance to single agent after serial passages. mean concentrations of amikacin in serum when administered alone and in combination with aztreonam were 26.2 and 20.3, 11.8 and 12.3, 8.4 and 12.2, 6 .3 and 9.7, 1.8 and 4.3 and 0 and 1.3 mg/ml immediately after and 0.5, 1, 2, 4 and 8 h after infusion of antimicrobials. respective concentrations of aztreonam were 63.5 and 25.3, 27.5 and 21.6, 24.2 and 10.5, 19.5 and 13.6, 5.8 and 4.4 and 1.8 and 4 .0 mg/ml. aucs for amikacin when administered alone and in combination with aztreonam were 35.59/10.9 and 50.59/7.8 mg h/l, respectively. respective auc for aztreonam were 99.79/35.2 and 76.49/31.5 mg h/l. it is concluded that the co-administration of aztreonam and amikacin results in earlier clearance of aztreonam and in higher levels of amikacin compared to the administration of each single antimicrobial. molecular modelling of b-lactams reveals the structural basis for their inhibition of penicillin-binding proteins, susceptibility to b-lactamases and oral bioavailability pm110 grail bm, gupta s, payne jw. school of biological sciences, university of wales, bangor, uk b-lactam antibiotics are peptide mimetics that act as suicide substrates for transpeptidase enzymes that cross link bacterial cellwall peptides. for the first time, the structural and electronic features needed for their recognition by transpeptidase have been fully described, using innovative molecular modelling techniques to compare the conformational forms adopted by cell-wall peptides and blactams. comparison of features in the backbone and c-terminal regions of conformers of active b-lactam antibiotics and model cellwall peptides, has allowed definition of the molecular recognition template required for substrate recognition by transpeptidase. these shared structural features allow both to act as substrates and to acylate the active-site serine. however, a significant difference in a critical backbone torsion between the two substrates, provides an explanation for the inability of the enzyme á/antibiotic complex to undergo the deacylation step that causes inhibition of transpeptidase. on the other hand, b-lactamases appear to have evolved molecular mechanisms that facilitate the deacylation reaction through compensating for the altered structural orientations in b-lactams caused by the different backbone torsion. finally, analysis of the conformer repertoires of blactams for structural features required for substrate uptake by peptide transporters, provides insights into how their structures can be tailored for optimal oral absorption. antimicrobial susceptibility of proteus mirabilis clinical isolates producing extended spectrum beta-lactamases (esbls) pm111 objectives: the aim of the present study was to determine in vitro susceptibility to antimicrobials of proteus mirabilis isolated from urinary tract infections. methods: we studied the susceptibility profile of esbl positive p. mirabilis strains in three adopted children from india with age range from 20 months to 2 years. esbl was identified using the synergic effect of clavulanate with betalactams (ceftazidime and cefotaxime the in vivo efficacy of amoxicillin (amx) sub-therapeutic doses (3.12 mg/kg, t.i.d for 48 h, achieving serum levels over the mic of only 3% of the dosing interval) and concomitant specific serotherapy (single intraperitoneal dose of 1/4 diluted hyperimmune serum (hs) obtained from mice immunized with the heat-inactivated strain) was assessed in a pneumococcal sepsis balb/c mouse model. mice (five mice/ treatment group) were intraperitoneally infected with 1.5 )/10 8 cfu/ ml of a serotype 6b penicillin-resistant strain (mic of 2 and 4 mg/l for penicillin and amx, respectively). treatments started 1 h after bacterial inoculation. study groups were: control (k; receiving nonimmune serum (nhs)), amx'/nhs, hs, and amx'/hs. survival rates (%) over time were: purpose of the study: the study was performed to determine the consumption of imipenem and resistance of gram-negative pathogens (pseudomonas aeruginosa , acinetobacter sp., klebsiella sp., escherichia coli , proteus mirabilis , serratia marcescens , enterobacter sp. ) to imipenem. gram-negative pathogens were isolated at the sestre milosrdnice university hospital from zagreb, croatia, in 1999 and 2000. the imipenem sensitivity testing was performed by disk diffusion and e -test methods. the consumption of imipenem was expressed in ddd/100 hospital days in the same periods. results obtained: imipenem resistance of acinetobacter sp. decreased significantly in the year 2000 (p0/0.0052), especially in the first 6 months (p 0/0.021) when the lowest consumption of imipenem was recorded. imipenem resistance of other gram-negative pathogens did not decrease significantly. conclusion reached: comsumption of imipenem might lead to changes in resistance to imipenem among acinetobacter strains. vacheva-dobrevski rs, savov ez. military medical academy, clinical microbiology, sofia, bulgaria purpose: acinetobacter baumanii is becoming increasingly frequent nosocomial pathogen at our hospital, and beta-lactam resistant strains are on the increase, especially among icu isolates. to study the susceptibility of a. baumanii clinical isolates to beta-lactams and to determine the esbl-producing strains during 2001, year. a total 438 gram-negative nonfermenters (gnnf) isolates was investigated by semiautomated mini api system (bio merieux, france). eighty-four a. baumanii non-repeated isolates was studied for esbl-producing by double-disk synergy test (ddt) and atb-blse test (bio merieux, france). mics for beta-lactams were determined by e -test (ab biodisk, sweden). results: a. baumanii (n0/84) showed a multidrug resistance. the isolates were resistant to cefotaxime (79%), cefoxitin (96%), ceftazidime (49%), amoxicillin/clavulanate (76%), piperacillin (72%), aztreonam (84%), imipenem (12%). the 32 (38%) of investigated a. baumanii expressed esbl activity and originated more frequently from icu (78%). esbls producing strains were isolated from endotracheal aspirate (52%), surgery wounds (29%), blood culture (5.7%). conclusions: in general resistance levels were higher in clinical isolates a. baumanii to beta-lactams. the ddt seems to be a practical method for esbl-screening; atb-blse method is more sensitive. our study display to be the first report of esbl-producing a. baumanii strains from our country. carbapenems seems to be the most active agents against a. baumanii . salmonella infantis , strain 111, was isolated from a newborn baby at wassila bourguiba maternity in tunis. it exhibited high resistance to penicillins, extended-spectrum cephalosporines (cefotaxime, ceftriaxone, ceftazidime, cefpirome) and aztreonam but remained susceptible to cefoxitine and imipenem. involvement and characterization of enzymatic mechanism in b-lactam resistance were investigated in strain 111. isoelectricfocusing revealed that this strain produced a b-lactamase of pi 6. this enzyme had a broad-substrate profile, hydrolyzing amoxicillin, ampicillin, ticarcillin, cephaloridine, cefuroxime, cefotaxime, ceftriaxone, cefpirome and ceftazidime. the highest specific activity was observed with ampicillin. cefotaxime was hydrolyzed the most efficiently of the extended-spectum cephalosporines. the pi 6 extended-spectrum b-lactamase (esbl) was inhibited by clavulanic acid and sulbactam. no inhibition of the esbl was observed with 1 mm edta. thus, no metal ion is involved in hydrolysis for this b-lactamase. resistance due to the production of the pi 6 esbl was transferred with dna plasmid into escherichia coli . on the basis of substrate and inhibition profiles and isoelectric point, the pi 6 esbl was not previously described in s. infantis in tunisia. the presence of such a resistance on a plasmid raises concer for rapid dissemination among bacteria and loss of effectiveness of blactams. poizot-martin i a , enel p b , benhaïm s c , vion-dury f c , dinh t c , drogoul mp c , gastaut ja c . a assistance publique hôpitaux de marseille, cisih sud, pr ja gastaut, marseille, france , b assistance publique hôpitaux de marseille, cellule santé publique dmi2, marseille, france , c assistance publique hôpitaux de marseille, cisih sud, marseille, france objective: to assess liver biopsy (lb) practices in a cohort of 255 co-infected hcv and hiv patients followed up in an hiv specialized medical unit. method: transversal study with questionnaire among patients in pre-therapeutic's evaluation with pcr'/ and without lb at 6 months. results: among the 255 patients, 28 (11%) are lost of follow up, 159 (62.3%) have had lb, 68 (26.2%) have no lb. the characteristics of these 68 patients are: median age 0/38.59/6 years; sex ratio 0/2.14, cdc-stage a0/33.3% b 0/47.0% c 0/19.7%, undetectable viral load0/33.3%, median cd40/3269/204, anti-retroviral therapy0/ 95.5%, hcv-genotype 10/46.7%; 3a0/31.7%; 40/21.7%. causes of non-made lb are: (1) refusal from patients because of biopsy's fear0/ 35.3%; (2) contraindications because of hiv infection0/33.8% (clinical events0/16.2% which contraindicate anti-hcv treatment, grade iii thrombocytopenia0/8.8% which contraindicate biopsy, non-adherence to previous hiv follow up0/8.8%); (3) other0/30.9% (alcoholism0/13.2%, psychiatric/depressive disorders0/11.8%, decompensated cirrhosis0/5.9%). drug use or methadone/buprenorphine treatment are not considered as contraindication. conclusion: one-third of patients are afraid of lb. alcoholism and psychiatric/depressive disorders are the principal contraindications to anti-hcv treatment. it seems important to improve information of patients about lb and to focus on alcohol and psychiatric/depressive disorders management in such population. kashiwagi kk a , furusyo nf a , nakashima hn a , kashiwagi sk b , hayashi jh a . a department of environmental medicine and infectious disease, kyushu university, fukuoka, japan , b national kyushu medical center, fukuoka, japan the purpose of the study: the aim of this prospective study was to explore the effect of htlv-i co-infection on the development of hcc among patients with chronic hcv viremia. a total of 764 consecutive patients with chronic hcv viremia were studied and followed-up over a mean period of 5.1 years: 172 (22.5%) were infected with htlv-i infection and 592 (77.5%) were not. the results obtained the annual hcc development rate was 3.4% in patients co-infected with hcv and htlv-i and 1.4% in patients infected with hcv alone. hcc was significantly higher in 33 (19.2%) of the 172 patients co-infected patients than in 39 (6.6%) of the 592 patients infected with hcv alone (p b/ 0.001, logrank test). in patients under the age of 55 years, hcc development was significantly higher in seven (17.1%) of 19 patients co-infected with hcv and htlv-i than in eight (2.9%) of 220 patients with hcv alone (pb/ 0.05, logrank test), whereas there was no significant difference in hcc development between patients over age 55 with or without htlv-i infection (26 (2.0%) of 131 and 31 (9.9%) of 312, respectively). the conclusion reached htlv-i infection accelerates the development of hcc in chronic hcv patients, especially among patients under the age of 55 years. to analyze hbv genotype-related clinical differences among patients with chronic hbv infection, all 158 patients were serially tested for serum alanine aminotransferase (alt) and hepatitis b e antigen (hbeag) and followed up for a mean 10.8 (6.4) year period. genotypes b and c were found in 58 (36.7%) and 100 (63.3%) of the patients, respectively. hbeag positivity and alt abnormality rates at the start of the observation period were significantly higher in genotype c patients (66.0 and 84.0%) than in genotype b patients (34.5 and 22.4%). the annual rate of spontaneous hbeag disappearance in genotype b patients was much higher than in genotype c patients (8.38 versus 2.34%, respectively). patients with genotype c who were continuously hbeag negative from entry had significantly higher alt abnormality (58.8%) than those with genotype b (19.2%). interestingly, patients with genotype c who became hbeag negative by interferon treatment had high alt abnormality (58.8%). all patients with alt abnormality were serum hbv dna positive. these findings indicate that hbv genotype c patients are more severe liver deterioration because of the delay of hbeag disappearance and continued hbv replication after hbeag disappearance. nossik nn a , nebolsin ve b , zheltukhina ga c , yevstigneeva rp c . a the d.i. ivanovsky institute of virology, viral reproduction, moscow, russian federation , b pparminterprisis co., chemistry, mocow, russian federation , c moscow state academy of fine chemical technology, piptide chemistry, moscow, russian federation objective: to study the effects of 'gamma'-l-glutamylhistamine (glu-ha) derivates on non-specific immunity ('alfa'-ifn, 'gamma'-ifn and nk cell activity) and antiviral activity on the experimental influenza and herpes virus infections in mice. the glu-ya and its derivate glu-ii were synthesized by peptide chemistry techniques. the glu-ha and glu-ii was administered i.p. 0.05 and 0.5 mg/kg before and after influenza virus (type a/aichi) and showed a protective effect even at the high infective dose (100ld50) */the rate of protection0/ 42 á/51/60% in the positive/control group. they were not very effective in the protection of herpes simplex virus encephalitis in mice. the model of the physico-emotional stress in mice was used to investigate the ifn system and nk cell activity. the production of ifns and nk cell activity of splenocytes decreased in 2 h after the stress and back to normal level in 7 á/10 days. it was shown that glu-ha and glu-ii can protect or substantially prevent the decrease in nk cell activity and ifns synthesis in post-stress period (so normally did not induce the ifns' synthesis). conclusions: the glu-ha and glu-ii showed antiviral effect against influenza virus infection in mice. the immunomodulating activity and ability to normalize the ifn synthesis and nk cell activity depressed the post-stress period and probably play an essential role in the antiviral activity. no dose adjustment of an anti-influenza prodrug oseltamivir is required in patients with hepatic impairment pm120 oo c a , snell pr b , liu b a , martin d b , simkins t b , small i b , ward p b . a hoffmann-la roche inc., global development, nutley, usa , b roche products ltd., global development, welwyn garden city, uk background: oseltamivir (ose; ro 64-0796, tamiflu † ) is an oral ethyl ester prodrug of its active metabolite oseltamivir carboxylate (oc: ro 64-0802), a potent and selective neuraminidase inhibitor of the influenza virus. the purpose of the study is to evaluate the need for ose dosage adjustment in hepatic impaired patients (hi). method: healthy volunteers (hv) versus hi (child-pugh score 7 á/ 9) [matched on the basis of age (9/10 years), gender and weight (9/ 20%)] were compared. each subject received 75 mg ose. results: based on c max (ng/ml) and auc inf (ng h/ml) analysed using nominal times, ls mean ratios and 90% ci between hi and hv were similar. ose* values in hi were marginally elevated but not sufficiently to require dose adjustment. the aim of this study is to investigate the influence of molecular structure of macrocyclic pyridinophanes and their analogs on antiinfluenza and antiherpetic activity of these compounds. we used 4d-qsar approaches on the basis of simple representation of molecular structure. such representation for biologically active substances allows the description of the spatial structure of compounds with the complete stereochemical information. it determines spatial structures either promoting or interfering of the concrete biological activity. it is easy to realize the molecular design of compounds with the given level of activity with the help of the combinations of simplexes. statistic characteristics for qsar of partial least-squares models are satisfactory (r0/0.92 á/0.97; cvr0/0.76 á/0.86). the molecular fragments that increase the antiviral activity were defined and will be demonstrated. this information was used for design and directed synthesis of several novel antiviral agents with predicted high anti-influenza or antiherpetic activities. predicted activities were confirmed experimentally. 4d-qsar approaches are useful for development of antiviral compounds. this work was partially supported by intas foundation (grant intas 97-31528). lozitsky vp. ukrainian mechnikov research anti-plague institute, chemotherapy, odessa, ukraine the purpose of this study was to research the anti-influenza activity of proteolytic inhibitor e-aca. it prevents the enhancement of proteolysis during the interaction of virions with cell membranes and decreases penetration of virions into cells. e-aca brings down proteolytic cleavage of ha-precursor to ha-1 and ha-polypeptides and reduces the infectious virus harvest. it shows the prophylactic and therapeutic action during the experimental influenza reducing the enhancement of alkaline proteases activity in lungs after infection. e-aca promotes the intensification of specific antibodies production and cell immunity, prevents vessels' permeability and hemorrhagic phenomena, decreases the destruction of bronchi's epithelium. it reduces the duration of intoxication, catarrhal instances and hyperthermia in sick children. e-aca improves the indexes of immunity, non-specific resistance and decreases the rate of bacterial complications. application of e-aca for treatment influenza and other arvi in children is recommended in ukraine on the base of results of our researches. the higher effects demonstrated as a result of combine usage of e-aca with specific ig, or deitiforin, or unithyol, or ribavirin. in our opinion, the study of effectiveness of e-aca combine application with inhibitors of influenza na is the perspective direction of anti-influenza researches development. we should we treat immediately all varicella patients with acyclovir if the patient is older than 20 years pm123 during past 2 years in institute for infectious and tropical diseases in belgrade, 89 immunocompetent varicella patients were treated and cured. among them 32 were older than 20 years (35.95%). x-rays were performed in all patients. diagnosis of pneumonia was made in 32 patients (35.95%), but in 26 (81.25%) patients older than 20 years. varicella is a benign, self-limited disease, if it strikes early, i.e. preschool, school children and teenagers. at that time there is no need for specific therapy. but in neonates, immunocompetent adults and in all immunocompromised patients it can be difficult and life-threatening disease. in immunocompetent adult population pneumonia is a very serious, sometimes fatal complication. knowing the pathophysiology of primary varicella á/zoster infection, specific therapy with acyclovir should be started immediately after making the diagnosis in patients older than 20 years, without waiting for x-ray proof of pneumonia. brivudin compared to acyclovir for the treatment of herpes zoster: effects on acute disease and posttherapeutic pain pm124 objective: comparison of efficacy and safety of brivudin 1)/125 mg and acyclovir 5)/800 mg, both for 7 days, in the treatment of herpes zoster. methods: randomised, double-blind study on 1227 immunocompetent patients ]/18 years (brivudin: n 0/613, acyclovir: n 0/614). a subgroup of patients ]/50 years (brivudin: n0/309, acyclovir: n0/299) was examined for the occurrence of posttherapeutic pain in a poststudy survey. posttherapeutic pain was defined as any zoster-associated pain, regardless of intensity, after the end of acute zoster. results: brivudin was superior to acyclovir in reducing time to last occurrence of new vesicles (rr(itt): 1.13 [1.01 á/1.27], p 0/0.01). the advantage of brivudin was more pronounced in patients ]/50 years (rr(itt): 1.16, [1.01 á/1.34], p0/0.02). incidence of posttherapeutic pain was significantly lower with brivudin (32.7%) than with acyclovir (43.5%, p0/0.006). duration of pain was comparable in both treatment groups (rr: 1.11, [0.93 á/1.32], p 0/0.27). potentially treatment-related adverse events occurred in 7.7% of the brivudin recipients and in 10% of the acyclovir recipients. conclusions: brivudin 125 mg once daily for 7 days is superior to standard acyclovir in stopping viral replication in acute herpes zoster. in patients ]/50 years, brivudin is more effective than acyclovir in reducing the risk of developing posttherapeutic pain. brivudin is as well tolerated as acyclovir. varadinova t a , genova p a , garcia-raso a b , terron a b , fiol j b , badenas f b . a laboratory of virology, sofia university, sofia, bulgaria , b chemistry, universitat de les balears, palma de mallorca, spain we have published that cu(ii) complexes of acyclovir (acv) are active against hsv infection (mbd, 1996) . here we present data on the activity of acv complexes of ni(ii), cd(ii), co(ii) and ag(i) against resistant to acv hsv 1 strain r-100 in comparison with the effect against acv sensitive strain victoria. selectivity indexes (si) compared to that of acv were indicative for activity. the following data were obtained: (i) 1 was 10 times less selective inhibitor of strain r-100 than of strain victoria; (ii) under the action of [cd(acv)cl 2 ], [ni(acv) 2 (h 2 o) 4 ]cl 2 ×/2acv and [ni(acv)(no 3 )] 3 ×/5h 2 o was up to 90% higher than that in the control; (ii) 2 was 28 times less selective inhibitor than acv; (iv) si of 3 was two times higher for strain r-100 and five times lower for strain victoria then that of acv. these data show that the selectivity of acv against resistant hsv 1 strains can increase when acv is bond to a proper metal ion. methods: an antiviral activity against herpes simplex virus of the type i (hsv-i/leningrad/248/88) and variant hsv-1 (vvt/4/00r) resistant to acyclovir was determined using commonly accepted method. viruses were grown on a continuous culture. maximal toxic dose was determined by the administration of compounds orally (300 mg/kg) or intraabdominally (100 mg/kg) to white mice that had mass 18 á/20 g. condition of the animals was controlled during 72 h. mice pneumonia model was used for the testing activity in vivo. results: derivatives tested have activity against hsv-1 and hsv-1 resistant to acyclovir. maximum protection of the cells up to 80% was reached at concentration of compounds 100 á/10 mg/kg. tested compounds have low toxicity and animals did not die after intraabdominal and after per oral administration of the substances. using these compounds led to essential relief of diseases in animals. the number and square of virus specific areas of inflammation in lung was decreased to compare with control untreated group. tested compounds protected animals similar to acyclovir that was used as control. conclusion: derivatives of carboalkoxysulfanilic acids are active against hsv in vitro and in vivo and act on the acyclovir resistant variant viruses. markiewicz r a , szepietowski jc b . a jelfa s.a., medical department, jelenia gora, poland , b department of dermatology, university of medicine, wroclaw, poland background: denotivir is a 5-benzoamino-4?-chloro-3-methyl-4isothiazolecarboxanilide anti-inflammatory agent with antiviral and immunomodulatory activities. it possesses also mild antibacterial and antifungal action. the purpose of the study: the aim of this presentation is to give an overview of recent studies demonstrating denotivir efficacy in herpetic infections. the results obtained: in vitro studies revealed that denotivir in the doses below its cytotoxicity (about 25 um) significantly inhibited (by 90 á/99%). herpes simplex virus (hsv)-1 and hsv-2 replication in fibroblast and kidney cell cultures. moreover, it was showed that denotivir in the dose of 37 mg/ml markedly inactivated hsv-2 after 30 min incubation in 37 8c. in giunea pigs research, 2% denotivir in 90% dmso appeared to be superior to 90% dmso alone and untreated groups in the therapy of animal skin infected by hsv-2. there was no huge difference in eythema and oedema scorings between studied groups, however in the group treated with denotivir, in contrast to others, no vesicles developed. several clinical studies showed usefulness of denotivir in controlling herpetic infections in dermatology, ophthalmology and otolaryngology. in the majority of studies within few hours after the drug application itch and pain relief was noted and within 1 á/2 days the vesicular lesions were dried up. the conclusion reached: in conclusion, denotivir is an effective antiherpetic agent. this study was conducted on 12 cows affected with teat papillomatosis. in the first step, each cow was located on one of three groups. the first group contained four cows from 1 to 4 years that were treated with fig tree (ficus carica) latex. the second group contained four cows from 1.5 to 4 years that were treated with a 10% solution of salicylic acid, and the third group contained four cows as control. in group one and two following treatment with fig tree latex and salicylic acid, superficial necrosis begun from day 5 and all of the warts disappeared by day 30. in the control group, after day 20, there were no changes in number of lesions, but some of them were larger than first observation. on day 25, one of the marked warts disappeared and on day 35 another wart was disappeared but six were present until day 45. comparison of effects of salicylic acid and fig latex showed similar effects in treatment of udder papillomatosis in cow. laboratory markers of skeletal muscle toxicity in hiv-infected patients: a cross-sectional case-control survey of frequency, potential correlation with antiretroviral therapy, clinical significance, and outcome pm130 manfredi r, motta r, patrono d, calza l, chiodo f, boni p. to assess skeletal muscle toxicity among â/1000 hiv-infected outpatients (p), the 129 p who had ]/1 altered cpk assay ( !/195 u/l) between may and november 2001, were compared with 387 p randomly selected among those who had ]/2 laboratory exams in this 6-month interval, in a 1:3 case-control study. among the 129 p with altered cpk levels only six were females, and 110 received antiretrovirals. the overall frequency of altered cpk among all p who underwent ]/2 laboratory workouts in 6 months was 14.4%. cpk alteration was transient in 98 p, with values ranging from 196 to 3463 (mean 256.29/62.3) u/l, but was recognized ]/2 times in 2 á/6 months in 31 p (24%), 24 of them showing concomitant high aldolase levels (3.1 á/10.8 u/l). a myopathy or a rhabdomyolisis were recognized in four p only; a myositis was confirmed in one p by histopathology. in a multivariate logistic regression analysis, when excluding the unexpected prevalence of the male gender (p b/0.0001), no significant difference emerged between p and controls as to age, risk for hiv infection, iv drug use, duration of hiv infection, prior anti-hiv therapy and its length, selected drug combinations, administered nucleoside analogues,hiv disease stage, mean cd4'/ count and hiv viremia, signs and duration of lipodystrophy, increased glucose, triglyceride and cholesterol levels, and other therapies. muscle abnormalities, though frequently asymptomatic, are underestimated hiv disease complications, and the role of metabolic (i.e. mitochondrial) alterations, deserves investigation. poor efficacy of non-nucleoside reverse transcriptase inhibitor (nnrti)based salvage haart in hiv-infected patients heavily pre-treated with all other classes of antiretroviral compounds pm131 manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy poorly comparable literature series show conflicting results of nnrti-based rescue haart: 20 á/70% rate of virologic success. to assess the response to a 4 á/5-drug rescue haart including a nnrti, 72 patients (p) treated with nucleoside analogues (na) and protease inhibitors (pi) for !/18 and !/15 months, respectively but naïve to nnrti, with a viremia !/10 000 copies/ml, were prospectively followed during 1 á/2 years, provided that they ensured a !/90% adherence. efavirenz was used in 41 p, nevirapine in 29, and delavirdine in two. most p (88.9%) had an early laboratory improvement, but mean peak viral load decrease was 0.4 log, and a significant reduction vs baseline (p b/0.02) lasted 3 months only. a mean 17% increase of zenith cd4 count was obtained (p b/0.001), but only 18.1% of p remained !/200 cells/ml 1 year after switch to nnrti. in a multivariate analysis, the concurrent introduction of novel pi(s) (32 p) and/or different na(s) (19 p) acted favorably until the 9th month of follow-up (p b/0.04), while genotypic mutations conferring nnrti cross-resistance, usually associated with a broad resistance profile, predicted failure in all p (p b/0.001), and the response did not vary according to duration and type of prior therapy, and selected nnrti. a deep salvage nnrtibased haart has a poor and transient virologic outcome also in nnrti-naïve p, while a more evident and sustained immunologic response is expected. p who can introduce novel pi/na and have no mutations impairing nnrti activity are entitled to a better outcome. fatal lactic acidosis without elevation of liver-enzymes during the treatment with stavudine, didanosine and efavirenz: a case report pm132 winzer r, langmann p, väth t, zilly m, klinker h. medizinische poliklinik der universität würzburg, schwerpunkt hepatologie/infektiologie, ürzburg, germany nucleoside reverse transcriptase inhibitors (nrtis) cause various side effects, many of which are thought to be due to their effects on mitochondria. a 36-year-old hiv positive (hiv rna: 6000 copies/ml, cd4 cell count: 359/ml), obese (body-mass-index: 40.9), therapy-naïve female patient, who after 7 months of well tolerated and effective antiretroviral therapy (stavudine, didanosine, efavirenz), had slight gastrointestinal discomfort and suddenly developed a lactic acidosis (arterial-ph 7.03 [7.36 á/7.44 she died 4 days later despite intensive care (continuous venovenous haemodiafiltration, sodium-bicarbonate infusion, high doses of vitamins, respiration). the pathologic examination showed an enlarged liver (2370 g) with yellowish appearance and pasty consistency, which microscopically appeared as a massive macro-and microvesicular fatty degeneration, and only slight signs of terminal pancreatitis. this reported case gives evidence that a massive lactate acidosis may develop without previously disarranged laboratory parameters for liver or pancreatic function. a fatal outcome may evolve without further accompanying-illnesses. efficacy and tolerability of atorvastatin in the treatment of hypercholesterolemia in hiv-infected patients receiving haart pm133 calza l, manfredi r, chiodo f. division of infectious diseases, university of bologna, bologna, italy introduction: significant increases in plasma triglyceride and cholesterol levels have been reported in patients treated with haart, and prolonged metabolic imbalances could significantly act on the longterm prognosis and outcome of hiv-infected persons. patients and methods: fourteen hiv-infected patients on pi-based haart since at least 12 months and presenting hypercholesterolemia ( !/290 mg/dl) of at least 6-month duration and unresponsive to a hypolipidemic diet and physical exercise, have been treated with a single daily dose of atorvastatin (20 mg) for 18 months. results: one patient was ecluded from evaluation due to early dropout. ongoing antiretroviral treatment included ritonavir in four cases, indinavir in four, nelfinavir in three, and saquinavir hard-gel in two. at the close of 18-month follow-up of atorvastatin therapy, a decrease of total cholesterol level of 21.9% versus respective baseline value was observed; eight out of 13 patients reached normal values for cholesterol. mild gastroenteric symptoms were found in only one of the 13 treated patients, while no skeletal muscle and liver toxicity has been observed. discussion: in our study, pharmacological treatment with atorvastatin proved certainly effective in the management of diet-resistant hypercholesterolemia, and was associated with a favourable tolerability and adherence profile. the effect of combination antiretroviral therapy regimens on hiv-1 proviral dna level in peripheral blood mononuclear cells (pbmcs) was examined in 12 hiv-1-positive patients, using endpoint dilution pcr and serially cloning and sequencing of the gag region of hiv-1. the major clone was defined as the most numerous of 10 analyzed clones, and observation periods ranged from 8 to 32 months (mean, 19.89/10.2 months). in five patients (one with primary-stage hiv-1 infection) receiving three antiretroviral drugs, hiv-1 rna levels reduced to undetectable (i.e. b/100 copies/ml). hiv-1 proviral dna levels and the number of major clones reduced in four of these patients. hiv-1 rna levels reduced, but remained detectable, in five other patients. in the two remaining patients (both receiving two rather than three antiretroviral drugs) hiv-1 rna levels increased. these results suggested that the population of the major clones may be affected when hiv-1 rna levels reduce following combination regimens of antiretroviral therapy. saquinavir hard gel (shg) as a part of a spontaneous 12 á/18-month deintensification anti-hiv regimen following successful highly active antiretroviral therapy (haart) pm135 manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy the induction-maintenance concept was poorly studied in hiv'/ patients (p), and shg was never assessed after prolonged response to potent protease inhibitors (pi)-based haart. shg-naïve p who refused indinavir, ritonavir, or nelfinavir-based haart after achieving long-term viral suppression, and resorted to shg'/2 nucleoside analogues (na), were followed prospectively. in 60.7% of the 61 p assessed for 12 á/18 months, ]/1 na was changed. prior haart was interrupted after 8.49/2.9 months, due to adverse events (46 p), or p's request (15 p), while a viremia b/50 copies/ml was present since 4.79/ 1.5 months. a viremia of 50 á/1000 hiv-rna copies/ml was maintained in 49 p (80.3%), while a higher viral load occurred in 12 p after 5.99/0.6 months, and was related to a pre-haart viremia !/100 000/ ml, a more frequent !/100% recovery of cd4 count, mutations of codons 48 á/90, and failure to change na (p b/0.05 á/0.001). a cd4 drop !/20%/150 cells/ml was found after 7.19/0.5 months in only eight p, who also had virologic failure: immunologic deterioration was earlier and deeper when na were not changed (p b/0.05). all the 37 p who introduced shg'/novel na after a successful !/6-month induction with a potent pi-based haart had a stable 12 á/18-month outcome. a suboptimal haart including the less effective but better tolerated shg may be effective for !/1 year, especially when novel na are introduced, and specific mutations are absent. despite a lower potency, drugs with a good safety and compliance profile may be recovered for simplified regimens. objective: to evaluate efficacy of antiretroviral therapy (art) with two or three drugs in the nervous 'reservoir'. patients: thirteen acute neurological and art naive aids patients underwent a paired and simultaneous sample from plasma and cerebrospinal fluid (csf) for a quantitative detection of hiv-1 rna (amplicor roche) before art. all patients underwent a ct and/or mr of the brain to perform a diagnosis. all of them had an hiv related neurological acute inflammatory disease. after diagnosis all patients received art: 7/13 received two nrti and 6/13 received haart including two nrti and one protease inhibitor. all patients underwent a paired and simultaneous follow-up from plasma and csf during the 2nd month of treatment. results: in all patients baseline levels of hiv-rna were higher (p b/ 0.05) in the plasma (log 10 5.37'/0.93) than in the csf (log 10 4.33'/1.379). the 7/13 patients who received dual therapy had undetectable levels (cut-off 200 copies/ml) of viral rna at the follow-up in csf, but not in plasma: three of these seven patients had a detectable plasma hiv-1 rna. all 6/13 patients with haart had undetectable hiv-1 rna both in plasma and in csf at the follow up. conclusions: dual nrti therapy is rapidly effective in csf (because of an high penetration of drugs through a more permeable blood á/brain barrier and lower hiv rna baseline levels) but not in plasma. haart is rapidly and equally effective both in csf and plasma. there are no reports of fulminant and fatal hepatic failure after the start of highly active antiretroviral therapy (haart) in an hiv subject without chronic viral hepatitis. case report: a 30-year-old naive aids woman with clinical symptoms due by a pcp was observed. baseline alt was increased (0.5 m.n.v.) because of a mild hepatosteatosis and a silent cholelithiasis. serology for hbv, hdv and hcv was negative; igg anti-hav, anti-ebv, anti-cmv and anti-hsv were present. hiv-1 rna was 5.7 log 10, cd4'/ count was 26/ml. during the pcp treatment with cotrimoxazole alt values increased ( !/5 m.n.v.); nevertheless, she completed the treatment. liver enzymes returned to the pre-treatment values over several days. then she started haart with stavudine, lamivudine and efavirenz. after 10 days the patient showed an efavirenz-related skin rush that resolved within 5 days, without treatment discontinuation. fourteen days after the start of haart jaundice appeared. laboratory revealed severe alt increase ( !/8 m.n.v.) and hyperbilirubinemia (17 mg/dl) and she died because of an acute liver failure syndrome within few days. an haart efavirenzbased regimen can result highly hepatotoxic when given in presence of a hepatosteatosis, of a recent hepatotoxicity caused by a nonantiretroviral treatment and of a previous idiosyncratic reaction to efavirenz. our experience with bulgarian herbal extracts for improving the general condition of hiv-positive patients pm138 methods: we used a combination of 19 bulgarian herbal extracts and treated six patients, divided in two groups: three with symptomatic and three with asymptomatic hiv-infection. the all three patients with asymptomatic hiv-infection were treated only with herbal extracts, another three patients with symptomatic hiv-infection were treated with combination of herbal extracts and anti-retroviral therapy. the general status of patients has been evaluated by both subjective and objective surveillance. the immunologic monitoring has been performed by absolute count of cd4'/ lymphocytes. results: all patients have shown an obvious improvement in their general condition: high spirit and working capacity, good appetite and sleep, a restoration of body weight. the number of cd4'/ lymphocytes has been lightly increased or constant. conclusion: the combination of bulgarian herbal extracts has shown significant positive effect on the general condition and improve the quality of life. antifungal activity of in vitro and in vivo combinations of voriconazole with 5-fluorocytosine and amphotericin b against candida and cryptococcus spp pm139 hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: the present study was designed to determine whether the activity of voriconazole (vor), a novel triazole, was reduced against candidal and cryptococcal infections by the addition of standard antifungal agents, amphotericin b (amb) and 5-fluorocytosine (5-fc). vor was tested in combination with standard antifungal agents both in vitro, using a checkerboard mic determination test, and in vivo in immune normal guinea pig models of fungal infections. the results indicate that the efficacy of vor against candida albicans and c. neoformans was not antagonised by amb or 5-fc in vitro. furthermore, in guinea pig models of systemic candidiasis and intracranial cryptococcosis, no antagonism was observed between the lower doses of vor and either amb or 5-fc on the basis of reductions in tissue fungal loads. at the highest doses of vor, both amb and 5-fc showed some antagonism, but the combinations were still effective in significantly reducing fungal tissue loads compared with vehicle-treated control animals. conclusion: these results suggest that vor may be used in combination with standard antifungal agents, and future studies to elucidate the clinical potential of vor combination therapies in the management of candida and cryptococcus infections are warranted. itraconazole in the treatment of pityriasis versicolor pm140 tiodorovic j, jovanovic d, binic i, nikolic lj. faculty of medicine, clinic of dermatovenerology, nis, serbia, yugoslavia a comparison of two short-term dose schedules with itraconazole was carried out in 50 patients with pityriasis versicolor. the patients were divided in two groups. each group consisted of 25 patients who completed the therapy and controls. the clinical diagnosis was confirmed mycologically, by direct microscopic examination. the first group received 400 mg of itraconazole daily for 3 days. the second group received 200 mg daily for 5 days. the patients were controlled clinically and mycologically 15 and 30 days after the initiation of treatment. erythema, scaling and pruritus was evaluated clinically. clinically and mycologically cured patients accepted as cured. the cure rate were 76% in the first group and 72% in the second group at day 30. the effects of these two groups are similar. none of the patients reported side-effects. fungal urinary infections: emerging species, antifungal susceptibility trends and antibody response pm141 badawi he a , kamel ai b , fam ns a , el-sayed me a , elian sae c . a theodor bilharz medical research institute (tbmri ), microbiology, giza, egypt , b theodor bilharz medical research institute (tbmri ), urosurgery, giza, egypt , c faculty of medicine, medical microbiology and immunology, cairo university, cairo, egypt objectives: to assess the role of candida species in 120 patients with urinary tract infections (utis) with or without schistosomiasis and/or cancer bladder, to compare chromogenic; chromagar (cma), biggy agar; morphologic (corn meal, rice agar-tween 80) media and biochemical candifast test for identification of candida species. susceptibility to antifungal agents using e -test and candifast and the performance of elisa test for detection of anticandida antibodies (igm and igg) in serum were evaluated. results: c. albicans was the most frequent (43.4%) species responsible for fungal utis. however, non-albicans species, c. glabrata (23.3%), c. tropicalis (20%) and c. krusei (13.3%) were also isolated. rice agar-tween 80 was found to be cheap, available and sufficient to make a final identification (100%). cma could not identify c. glabrata . biggy agar could not adequately differentiate candida species. candifast biochemical identification showed low sensitivity of 83.3%. e -test on sabouraud dextrose agar (sda) is simple method for mics determination and could detect s-dd strains in case of azoles. conclusion: the emergence of non-albicans species such as c. glabrata , c. tropicalis and c. krusei have contributed to complicated utis. this necessitates accurate isolation and identification of candida to the species level. morphology on rice agar-tween 80 and antifungal susceptibility using e -test on sda is a simle rapid scheme for routine identification of clinically important yeasts. purpose: classification of allergic fungal rhinosinusitis (afr) is based on the immunologic relationship of the host to the fungus. afr must be differentiated from other fungal rhinosinusitis infections, which include acute invasive, chronic invasive, fungal balls and saprophytic colonization. although many cases of fungal rhinosinusitis is caused by species of aspergillis , dermatiaceous moulds have become an emerging pathogen in immunocompetent individuals. results: our case study involved a 36 year male suffering from facial pain, headache, postnasal drip and loss of smell. he was hiv negative and a nonsmoker. the following laboratory tests were performed: ige-2331.40 (0.00 á/25.00) iu/ml iga-2.16 (0.70 á/4.00) g/l igm-0.61 (0.40 á/ 2.30) g/l allergen specific ige for alternaria */20.00 (0.00 á/0.5) ku/l fbc-normal except eosinophils slightly raised 0.60 (0.00 á/0.50))/10 9 / l. ct scans indicated fungus proliferation, bone erosion and extension of disease into adjacent anatomic area. sinus tissue following debridement was sent for microscopy and culture. hyphae was microscopically observed and cultures yielded two dermatiaceous fungi, bipolaris spp and alternaria spp . conclusion: it is important to differentiate these two species from curvularia , helminthosporum , drechelria and exserohilum . knowledge of these dermatiaceous fungi is important in directing appropriate antifungal therapy and selecting the correct antigens for postsurgical immunotherapy after initial debridement and irrigation. antifungal activity of in vitro and in vivo combinations of voriconazole with 5-fluorocytosine and amphotericin b against aspergillus fumigatus pm143 hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: a key requisite for a new antifungal drug is to demonstrate that it is devoid of significant antagonism in combination with other agents. combinations of the new triazole, voriconazole (vor), and standard antifungal agents (5-fluorocytosine or amphotericin b; 5-fc or amb) were tested against aspergillus fumigatus in vitro and in guinea pig models of infections to confirm that antifungal activity was not antagonised by using combination therapies. vor was studied in combination with amb or 5-fc in vitro, using a checkerboard mic determination test, and in vivo, using immune normal and immunocompromised guinea pig models of systemic aspergillosis. results: the results indicate that the potency of vor was not antagonised by amb or 5-fc in vivo; indeed, at lower concentrations of vor, significant improvements in reducing fungal burden in both in vivo models were achieved by the addition of amb. in vitro, no antagonism was found between vor and amb, although 5-fc had a significant antagonistic effect on vor activity. conclusion: these results from in vitro and in vivo models of aspergillosis suggest that vor may be used in combination with standard antifungal agents and, therefore, justify further examinations of vor combination therapies in a clinical setting. in vitro activity of caspofungin compared to that of amphotericin b, fluconazole, and itraconazole against candida species pm144 arikan s, sancak b, hascelik g. department of microbiology and clinical microbiology, hacettepe university medical school, ankara, turkey purpose: to evaluate the in vitro activity of caspofungin against various candida spp. and particularly against isolates with decreased amphotericin b, fluconazole, and itraconazole susceptibilities. methods: susceptibility tests were done by nccls m27a microdilution guidelines for 239 clinical candida strains. the mics (mg/ml) were read at 24 and 48 h. results: caspofungin mics at 24 h are shown in the table. mics at 48 h were similar to 24 h readings. expectedly, no evidence of crossresistance was detected between caspofungin and other drugs tested. caspofungin was similarly active against fluconazole-or itraconazolesusceptible and resistant isolates. conclusions: (1) caspofungin is active in vitro against all candida spp. tested. (2) caspofungin mics are slightly higher for c. parapsilosis compared to other species. (3) its activity against fluconazole-and itraconazole-resistant isolates is noteworthy. (4) validation of these data require clinical investigations. oropharyngeal microbiological samples of 80 bmt patients were evaluated. weekly cultures (days (/7, 0 and '/7) revealed presence of fungi in 27 patients (33.7%): in four (5%) patients before bmt only, in 12 (15%) after bmt only, and in 11 (13.8%) both before and after bmt. in one patient candida norvegensis was isolated from the throat, buccal and palatal surfaces. three c. albicans , two c. krusei , and three c. norvegensis from four patients were chosen to comper their antifungal sensitivities and extracellular virulence factors. using fungitest method we determined the sensitivities of these isolates to flucytosine, amphotericin b, miconazole, ketoconazole and fluconazole. the fluconazole sensitivities were also determined by the e -test. on the basis of the fungitest data the three c. norvegensis isolates were sensitive to flucytosine, amphotericin b, and also to ketoconazole. in case of fluconazole and miconazole they proved to be susceptible dependent upon dose. the mic fluconazole values determined by the e -test for the three c. norvegensis isolates were 96, ]/256 and ]/256 mg/ml, respectively. the oropharyngeal isolates of c. norvegensis produced high amounts of extracellular aspartic protease and phospholipase similarly to c. albicans strains. these enzymes may contribute to the pathogenesis of this new emerging candida species. in vitro activities of antifungal and antiseptic agents against rhodotorula sp pm148 preney l, théraud m, guiguen c, gangneux jp. laboratory parasitologie-mycologie, chu de rennes, france purpose of the study: rhodotorula species are common saprophyte yeasts widespread in nature. since the last 10 years, they have been implicated in several severe infections, especially in immunocompromised patients, and various antifungal therapies were used. however, only limited data are available on the susceptibility of rhodotorula sp. to antifungal and antiseptic agents. material and methods: in this work, we evaluated the in vitro activities of eight antifungal agents against 30 strains of rhodotorula (21 strains of r. rubra and nine strains of r. glutinis using atb fungus system (biomerieux) and etest strips (ab-biodisk). beside, the effect of eight antiseptic agents was assessed on a suspension of r. rubra . the quantification of yeasts after exposure to antiseptic agents was performed by subculturings using a microtitration method in 96well plates. results and discussion: all strains tested were susceptible to amphotericin b, 5fc, and nystatin. twenty-nine out of 30 strains were susceptible to ketoconazole, 21 out of 30 were intermediate to econazole. all strains were resistant to fluconazole (cmi!/64 mg/ml) and itraconazole (cmi!/1 mg/ml), and 29 out of 30 were resistant to miconazole, suggesting that antifungal therapy must be adapted when rhodotorula yeasts are implicated in invasive infection. beside, 5 min exposure to sodium hypochlorite 128, chlorhexidine 0.5% or ecodiol (isopropyl alcohol'/alkylamin) showed fungicidal activities. susceptibility testing of aspergillus fumigatus and emerging aspergillus pathogens by a modification of the nccls m38-p method pm149 logotheti m a , kapsanaki-gotsi e b , velegraki a a , zagoura d b . a department of microbiology, mycology reference laboratory, medical school, university of athens, athens, greece , b biology department, section ecology and systematics, university of athens, athens, greece aspergillosis in high risk groups of patients is still associated with high mortality rate (30 á/90%). aspergillus fumigatus is the primary pathogen, while other opportunistic aspergillus species are emerging. amphotericin b (ab), itraconazole (it), voriconazole (vo) and terbinafine (te) minimum inhibitory concentrations (mic) were determined by modifying the nccls m38-p microdilution method. stock drug solutions were prepared in rpmi 1640, dimethyl sulfoxide (dmso), and polyethylene glycol (peg 400). inocula, of the a. fumigatus group (7), a. flavus group (6) (2) and the m38-p quality control strains were prepared according to, and by modifying, the nccls guidelines. plates were incubated at 30 and 35 8c and read at 24 and 48 h. peg 400 effectively dissolved it and vo, while either dmso or peg dissolved te. low 30 and 35 8c á/48 h ab, it, vo and te mics (0.062 á/0.5 mg/l) were recorded. a. terreus (1) and a. parasiticus (2) were resistant to ab. certain clinical isolates demonstrate clinical and in vitro resistance. standardization of susceptibility testing would offer reliable assistance in selecting and monitoring antifungal therapy. otag f, aslan, g, ozturk c. microbiology department, faculty of medicine, mersin university, mersin, turkey rates of opportunistic fungal infections have risen markedly. because some of these species have potential resistance to antifungal agents, rapid presumptive species level identification is crucial in allowing for directed antifungal therapy. in this study, 55 isolated yeasts from the clinical specimens were identified by atb id 32 c (biomerieux, france). the number of identified yeasts were, respectively; 33 (60%) candida albicans , six (10%) c. glabrata , five (9.1%) c. tropicalis , three (5.4%) c. parapsilosis , two (3.6%) c. krusei , two (3.6%) c. kefyr , one (1.8%) c. guillermondii , one (1.8%) c. dubliniensis . twenty-one of 55 strains were investigated for antifungal sensitivities by atb fungus kit (biomerieux, france). the results are as follows: 100% sensitivity was detected to myconasol, 94% to flusitozin, nystatin and econasol, 95% to amphtericin b and ketokonazol. it is important to achieve empirik treatment of the opportunistic candida infections and the following of resistance to antifungals. shakhmatov da, strelchenco, ov. novosibirsk state medical academy, dermatovenerology, novosibirsk, russian federation at the present stage in russia with a background of a high case rate of syphilis, it becomes necessary to exclude biological false positive serological tests. because the serodiagnosis of syphilis has significant limitations, the direct detection of t. pallidum in suspect blood may serve as an alternate diagnostic strategy. polymerase chain reaction (pcr) has been the most widely used amplification method. the study of 56 patients receiving examination related and treatment for syphilis in std clinic and persons directed from other hospitals where routine serologic examination revealed doubtful results. pcr reaction was carried out with nested primer pairs based on the dna sequence of the 47 á/1 and 47 á/2 kda gene of t. pallidum . pcr was utilized with whole blood. a complex of serological tests: fta-abs and tit was used as the &rdqup; gold standard''. as a result the sensitivity of pcr was 91.2% and specificity was 90.9%. selective comparison of pcr results with vdrl, the fta-abs and treponemal immobilisation test (tit) has shown concurrence 96.8%. in conclusion, the preliminary results of pcr in whole blood in syphilis detection revealed its high sensitivity and specificity; possibility to obtain rapid results in unclear cases. chlamydia pneumoniae (cp) is an atypical pathogen whit intracellular location, whose eradication is very difficult. in the past years it has been objects of many studies that lead to the demonstration of a relationship between its presence and the development of widespread multifactorial pathologies such as atherosclerosis and asthma. the lack of its eradication can become an important clinical and social problem. the study objective is the comprehension of pathogen á/host interaction mechanism, to characterize therapeutics protocols that cold lead to complete eradication of cp from organism. the research had been principally made on the studying the molecular mechanisms that are at the root of pathogen permanence inside host cell. using proliferation and apoptosis tests we underlined a different behaviour of infected cells towards control cells. in presence of p1 (20 mg/ml), i.e. a peptide that can inhibit the proliferation and induce apoptosis in vitro inhibiting nf-kb, uninfected cells proliferation decreased of 35% in comparison whit the controls, while the one of infected decreased only of about 15%. moreover, using various apoptosis-inducers, the infected cells showing apoptosis were about 10% while the uninfected were about 40%. the caspace iii activity increased significantly in uninfected cells. in conclusion, cp could delay its elimination from the host inhibiting the apoptosis via nf-kb activation. hryniewiecki t a , gzyl a b , rawczynska-englert i a , a department of acquired valvular heart disease, national institute of cardiology, warsaw, poland , b department of sera and vaccines, national institute of hygiene, warsaw, poland infective endocarditis (ie) frequently causes problems in diagnosis, especially where blood cultures are negative and with fungal etiology (also as a fungal superinfection in bacterial ie). the purpose of the study: the purpose of the study was to evaluate the usefulness of broad-range fungal pcr in diagnosis of fungal superinfection of bacterial ie. twenty-five blood samples were taken for analysis from patients with infective endocarditis. ie was diagnosed according to duke criteria including positive blood cultures. suspicion of fungal superinfection was established on serological investigation in five patients, confirmed by blood culture in two patients. control group consisted of 15 patients without infection. dna was isolated using the commercially available s.n.a.p. kit. amplification products were analyzed by gel electrophoresis stained with ethidium bromide. the results obtained: fungal dna was found in two patients with fungal superinfection of bacterial ie confirmed by culture. in the remaining patients with ie and controls no fungal dna was found. the conclusion reached: broad-range fungal pcr is a fast and inexpensive tool for the detection of fungal dna, but it is more prone to contamination than species-specific pcr. the method may be valuable in the identification of fungal superinfection of bacterial ie or diagnosis of fungal ie. rivanera d, lilli d, lozzi ma, piunno m, mancini c. microbiology, science and public health, rome, italy aim: the aim of this study was to evaluate the eia method for detection of antibody to ttv virus (ttv) and to investigate the anti-tt virus prevalence in patients with hepatitis b (hbv) virus, hepatitis c (hcv) virus, in group of 'high risk'subjects to hepatitis and in healthy subjects. the elisa methods (nuclear laser vienna lab) using ttv s and ns antigens: orf1 (770 aa) and orf2 (202 aa) was applied to detect anti-ttv; the serological screening was performed from 250 samples to italian subjects. results: the positive rates of anti-ttv antibodies were 11.29% in 62 patients with hepatitis b á/c and 14.06% in 119 'high risk' hepatitis patients. the anti-ttv was also found in 7.56% in 69 healthy people. conclusions: the anti-ttv were detected in all groups studied, however, its positive rate was similar in patients with hepatitis b á/c and in 'high risk' hepatitis respect to heathly people. our results shown that tt virus is frequent in italy both in patients infected by others transmitted viruses and in general population. the positivity found in healthy adults included in our studies suggests that the virus might be transmitted non-parenterally. the study of pattern of antibody to ttv may be an infectious marker of ttv similar to that of anti-hcv. a stress test on a miniaturized identification system designed for neisseria and haemophilus pm155 rich m a , bannatyne rm a , memish za b . a king fahad national guard hospital, division of microbiology, riyadh, saudi arabia , b king fahad national guard hospital, infection prevention and control, riyadh, saudi arabia we report an incident that occurred in our laboratory when the bbl crystal identification system for neisseria and haemophilus was used to identify a haemophilus-like-organism. the numerical profile generated was not in the system database. conventional biochemical tests subsequently revealed an identification of brucella melitensis , a common isolate in our area. as a result of this revelation we subjected this system to a mini 'stress-test' with a collection of 20 isolates of b. melitensis . two numerical profiles were obtained, 1740616606 and 1740616607, neither of which are listed in the system database. brucella species have been misidentified as moraxella species, moraxella phenylpyruvica , and as haemophilus influenzae biotype iv in various identification systems. two cases of laboratory-acquired brucellosis have been attributed to misidentification. to its credit the bbl crystal identification system for neisseria and haemophilus neither generates a profile number with a misidentified organism nor assigns a confidence level. instead it properly directs the user to resort to conventional methods to secure an identification. if further studies on additional brucella isolates and strains from different geographical sources confirm the unique biochemical profiles identified here, it may be worthwhile to incorporate these into the database where they would be of considerable assistance in areas where brucellosis is widespread. cloning and characterization of aflmp1 in aspergillus flavus pm156 chong tk, woo pcy, leung asp, yuen ky. the university of hong kong, microbiology, hong kong, hong kong purpose of the study: to clone and characterize an antigenic protein for serodiagnosis of infection caused by aspergillus flavus which is the commonest aspergillus species causing aspergilloma (ao) and invasive aspergillosis (ia) in asia. result obtained: we cloned the aflmp1 gene, which encodes the first antigenic cell wall protein in a. flavus . aflmp1 codes for a protein, aflmp1p, of 273 amino acid residues, with sequence features that are present in mp1p and afmp1p, the antigenic cell wall mannoprotein in penicillium marneffei and aspergillus fumigatus that we described previously. it contains a serine-and threonine-rich region for o glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. specific anti-aflmp1p antibody was generated with recombinant aflmp1p protein purified from escherichia coli to allow further characterization of aflmp1p. indirect immunofluorescent staining indicated that aflmp1p is present in the cell walls of the hyphae and conidia of a. flavus . furthermore, it was observed that patients with ao and ia due to a. flavus develop a specific antibody response against aflmp1p. conclusion reached: this suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with ao or ia, and the protein may represent a good cell surface target for host humoral immunitiy. grape purpose: to investigate the basis for increasing resistance to trimethoprim and sulphamethoxazole. methods: pcr screening for integrons of 105 clinical urinary tract isolates was performed. isolates were tested for resistance to 12 antibiotics. integrons in 14 isolates were sequenced. results: integrons of class 1 were found in 43 isolates and class 2 integrons were found in 10. eight isolates in the study were resistant to five antibiotics or more and not shown to carry any integron. nineteen of 69 isolates resistant to trimethoprim did not carry integrons. only one of these isolates was shown to carry sul1 and is thus probably also carrying an integron. none of the 19 isolates were shown to carry dfr8 , one of five trimethoprim resistance genes known to exist outside integrons. three isolates were resistant to sulphonamides but were not shown to carry neither sul1 nor sul2 . only dfr and aad gene cassettes were found in the sequenced integrons. conclusions: resistance to trimethoprim in 19 of 69 trimethoprim resistant isolates is mediated by genes not detectable, as in the case with three sulphonamide resistant isolates. sequenced integrons that contain dfr genes do not carry any gene cassettes mediating resistance to modern antibiotics. unusual diagnosis tool for an unusual presentation of alveolar echinococcosis: report of two cases of local progression after an animal bite pm158 bardonnet k, bart jm, loiseau j, gérard a, estavoyer jm, heyd b, badet jm, dubiez a, piarroux r, bresson-hadni s. who collaborating centre for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the classical human contamination route for alveolar echinococcosis (ae) is ingestion of eggs. two exceptional human cases are reported with extensive local evolution of ae after a bite. case no. 1: between 1954 and 1972, a patient underwent surgery seven times for a muscle growing tumour which developed after a bite. the diagnosis of muscle ae was assessed on histopathological examination. in 1980, serological tests were in accordance with echinococcus sp infection. case no. 2: in 1985, a man presented 'cat-scratch fever' with a right supraclavicular tumefaction following a cat bite. between 1986 and 2000, five recurrences occurred. different surgical explorations indicated multiple abscesses of the cervical muscles. in 2000, serological tests were in favour of echinococcus sp infection and the pathologist described a parasitic wall suggesting hydatidosis, but specific pcr from histological samples prompted the diagnosis of ae. conclusion: in these exceptional observations, the liver which is the most usual location of ae was lesion-free. the chronic inflammatory ae lesions have developed in the local lymphatic chain area of the bite site. to perform diagnosis in these very unusual forms of ae, it is necessary to add unusual tests such as specific pcr to classical tests. antibiotic resistance in foodborne salmonella is an emerging public health concern. integrons are now recognized as the main genetic vehicles of antibiotic resistance in gram-negative bacteria, including in salmonella . the purpose of the present study was to investigate the presence of class i integrons in resistant isolates of several serotypes of salmonella isolated from poultry products and to determine their association with multidrug-resistance phenotypes. a total of 20 isolates of salmonella belonging to seven different serotypes were tested. the most frequent multiresistant phenotype, found alone or together with other resistances, was to streptomycin and tetracycline. all but seven were resistant to three or more antimicrobial agents, including quinolones and amoxicillin. pcr analysis with the 5?cs and 3?cs primers detected the presence of class i integrons of 1.5 kb in one isolate, with the multiresistant phenotype: amoxicillin, chloramphenicol, streptomycin, trimethoprim-sulphametoxazol and tetracycline. our findings suggest that the uncontrolled use of the antimicrobial agents in food animals may have contributed to the development of the pattern of resistance observed in salmonella isolates. also the presence of integrons in low prevalent human salmonella serotypes but associated with food animals underscores the public health problem of antibiotic resistance acquisition and spread. prevalence and antimicrobial resistance of campylobacter jejuni and c. coli isolated from broilers and pigs in france pm161 avrain l a , humbert f b , sanders p c , kempf i a . a afssa, umb, ploufragan, france , b afssa, hqpap, ploufragan, france , c afssa, lermvd, fougères, france in 1999, 620 caeca from standard, export or free-range broilers and in 2000, 600 fecal samples from pigs, were collected in french slaughterhouses. prevalence of campylobacter jejuni and c. coli strains was 56.6% in standard, 51.3% in export and 80% in free-range broilers. in standard and export productions, the most often isolated species was c. jejuni , whereas c. coli was predominant in free-range production. 53.8% samples collected from pigs contained c. coli . the sensitivity of strains to ampicillin, nalidixic acid, enrofloxacin (broilers) or ciprofloxacin (pigs), tetracycline, erythromycin and gentamicin was tested by an agar dilution method. in broilers, the percentages of resistant strains were, respectively 22, 25, 17, 57, 0.3 and 0% for c. jejuni and 29, 43, 40, 70, 31 and 0% for c. coli . in pigs the percentages of resistant c. coli were, respectively 12, 21, 12, 83, 65 and 0%. in broiler production, significant differences between distributions of species or percentages of resistant strains were observed according to type of production or administrated antimicrobials. the enzyme dhps (dihydropteroate synthase) participates in the folate synthesis pathway, and is well recognized as the target for sulphonamides. the enzyme preceding dhps in this pathway, pppk (dihydropterin pyrophosphokinase), is another interesting candidate drug target. the metabolic role of pppk is to provide one of the substrates for dhps. earlier studies have suggested that pppk and dhps enzymes need to have physical contact with each other for full enzyme activity. studies of potential interactions between the enzymes have been initiated. so far, indication of a weak interaction has been detected in gelfiltration experiments and the two-hybrid system. to confirm these results, we are currently developing a method to study substrate channeling, as interfering with such interactions could lead to impaired growth and thus be used as inhibitory drugs. we have also cloned and sequenced the operons coding for the enzymes in the folate biosynthesis from different clinical isolates of streptococcus pyogenes . comparisons revealed some isolates with a mosaic structure in the operon, suggesting that horizontal transfer of genetic material has occurred. multi-resistance gene cluster on a plasmid in a clinical isolate of e. faecium pm163 werner g, hildebrandt b, klare i, witte w. department of nosocomial infections, robert koch institute, wernigerode branch, germany purpose: strain uw786 was isolated from an urine sample of a patient with a permanent catheter. the purpose of our study was to identify and localize the resistance determinants in this isolate. results: isolate uw786 was resistant to the following antibiotics: penicillin, ampicillin, gentamicin (high-level), streptomycin (highlevel), erythromycin, clindamycin, vancomycin, teicoplanin, ciprofloxacin, moxifloxacin, nourseothricin, rifampicin, and fusidic acid (lowlevel, mic0/4 mg/l); but showed susceptibilities to oxytetracycline, phosphomycin, chloramphenicol, trimethoprim/sulfamethoxazol, linezolid, and quinupristin/dalfopristin. hybridization, pcr and sequencing experiments localized a cluster consisting of several resistance genes in a composite element on a plasmid. the cluster included genes and transposons tn1546 (vana) á/tn917 (ermb) á/tn1505 (aade á/ sat4 á/apha-3). the plasmid itself was not transferable in filter-matings into a fusidic acid high-level resistant enterococcus faecium recipient while selecting either for erythromycin or vancomycin resistances. however, after transposing a tn916-related determinant into uw786, determinants became mobilizable with the help of the conjugative transposon. transconjugants were, besides others, high-level resistant to fusidic acid, but susceptible to penicillin and ampicillin. pfge of transconjugants demonstrated a pattern almost identical to the recipient but clearly different from the donor. conclusion: resistance genes in e. faecium could be arranged in a cluster and are mobile via mobilizable/transferable plasmids. lilli d, rivanera d, barbacini ig, lozzi ma, mancini c. department of science and public health, university la sapienza, microbiology, rome, italy aim: hepatitis g virus (hgv), a new rna virus that is parenterally trasmitted has frequentley been found in patients with chronic hepatitis c infection but its role in chronic liver desease is unknown. the aim of this study was to determine the prevalence of hgv infection in patients infected with hcv. ninety-eight patients infected with hcv were evaluated for the presence of hgv rna. the hcv genotypes distribution was 30 genotype 1b, 10 genotype 1a, 54 genotype 3a and four genotype 4c/4d. hcv rna and hgv rna were detected by rt-nested pcr. results: infection with hepatitis g virus was detected in 21 (21.4%) patients and 77 (78.6%) were hgv rna negative. none of our patients with genotypes 1a and 4c/4d results hgv rna positive. prevalence of hgv infection was 10% in patients infected with hcv genotype 1b and 33.3% with genotype 3a. conclusions: infection with hgv occurred frequently (21.4%) in this sample of patients with chronic hepatitis c. we observed a height prevalence of hcv/hgv coinfection in patients infected with hcv genotype 3a. this association with hcv genotype 3a was indipendent of the source of infection, infact some of our patients have not history of intravenous drug use. characterization of extended-spectrum beta-lactamase (esbl)mediated resistance in salmonella spp. from durban, south africa pm165 moodley p a , essack s b , gajee k a , sturm w a . a department of medical microbiology, nelson r. mandela school of medicine, school of infection, university of natal, durban, south africa , b school of pharmacy and pharmacology, university of durban westville, durban, south africa background: gastroenteritis is a common condition among the paediatric population presenting to king edward viii hospital in durban, south africa. from july 2001, we noticed that the susceptibility of the salmonella spp. isolated from stool samples among these children were resistant to multiple antibiotics. aim: to characterize the phenotype of the resistance mechanisms involved. methods: minimum inhibitory concentrations (mics) of ampicillin, azithromycin, ciprofloxacin, cefepime, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, cefoxitin, chloramphenicol, cotrimoxazole and gentamicin were determined by means of the agar dilution method. isolates were subjected to the e -test for extended-spectrum betalactamase (esbl) production. isoelectric focusing was performed as a preliminary step in enzyme characterization. results and conclusion: thirty isolates of multiresistant salmonella spp. were obtained. antibiogram typing revealed six different resistance phenotypes. all isolates depicted ceftazidime/ceftazidime á/clavulanate ratio of !/8 and were considered putative esbl-producers. isolates expressed 1 á/3 beta-lactamases each with pi values ranging between 5 and 8.2 indicative of tem-, shv-and/or ctx-m-related esbls. nine isolates expressed two beta-lactamases each and two isolates expressed three beta-lactamases each. there was evidence of the simultaneous expression of both tem-and shv-derived esbls as well as the simultaneous expression of multiple tem-or shv-derived esbls in single isolates, a phenomenon reported in esbl-positive klebsiella pneumoniae isolated at the same hospital. neutrophils exhibit reduced chemiluminescence response to serum opsonized klebsiella pneumoniae producing extended spectrum b-lactamases (esbl) pm166 objective: to investigate the ability of esbl and non-esblproducing klebsiella pneumoniae isolates treated with human serum to induce a chemiluminescence response in neutrophils. methods: oxidative burst induced by the interaction of esbl (n0/ 81) and non-esbl-producing (n0/152) klebsiella pneumoniae isolates with neutrophils from healthy individuals was monitored by measuring the chemiluminescence response (cl). pooled sera from healthy individuals served as source of complement for pretreatment of the bacteria. cl responses triggered by serum treated zymosan served as positive control. the serum opsonized klebsiella strains were arbitrarily graded as high (h) and low (l) inducers of cl when the cl response induced by the bacteria was cl b/60 and !/60%, respectively, of that induced by opsonized zymosan. results: out of 152 non-esbl-producing klebsiella isolates, 61.8% induced high cl response in neutrophils whereas only 42% of 81 esbl-producing klebsiella isolates did so (pb/ 0.02). conclusions: strains harboring the esbl plasmid were more virulent than non-esbl-producing strains by virtue of their higher tendency to escape serum-dependent recognition by neutrophils. osiris */an automated system for susceptibility testing in agar diffusion technique pm167 chegrani f, kolbert m, shah pm. universitätsklinik frankfurt, zentrum der inneren medizin med iii schwerpunkt infektiologie, frankfurt am main, germany objective: osiris measured zone sizes were compared to manually measured inhibition zones using round 90 (rp) and 120 mm mueller hinton square agarplates (sqp). variations of 9/3 mm in zone size measurements were defined as tolerable. 'very major errors' (vme) were defined as classification of a resistant organism as sensitive by osiris. thirty thousand two hundred and ninety-eight single measurements testing 42 antibiotics on 352 staphylococci and 113 enterobacteriaceae were done according to the din 58940 recommendations. results: vancomycin, rifampicin, gentamicin gave the best results on rp with a concordance of 96, 88 and 86%. vancomycin, rifampicin, teicoplanin performed best with 97, 94, 93% on sqp testing staphylococci . worst results on rp gave cefuroxim (43.8%) and fosfomycin (55.1%), on sqp fosfomycin (36.4%), ofloxacin (88.1%). for enterobacteriaceae amikacin (100%), gentamicin (100%), ciprofloxacin (100%) performed best on rp; worst nalidixinacid (32.5%), piperacillin (82.5%). concordance on sqp amikacin (100%), cefotaxim (100%), gentamicin (100%), nitrofurantoin (72.5%), cotrimoxazol (90%). very major errors were seen in b/1% of all test performed. interpretation: osiris is a rapid and reliable system for susceptibility testing with round and square agarplates and has an excellent expert system. altindis m a , aktepe oc a , kocagoz t b . a kocatepe university school of medicine, microbiology, afyon, turkey , b diomed inc. tr, istanbul, turkey dio-bacit, in a two section plate, that contains 5% sheep blood agar on one side and sheep blood agar with bacitracin (2 mg/ml) was compared for its efficiency in identification of group a beta hemolytic streptococcus (gabs) with other two different growth plates, one containing 5% sheep blood agar with bacitracin (b) and the other containing b-sxt. we used latex-agglutination for this comparision. throat specimens obtained from 490 cases were inoculated to dio-bacit plates, first to one side with 5% sheep blood agar and to the other side with b. after an overnight inoculation at 37 8c, colonies with beta hemolysis an 5% sheep blood agar but no growth with b, were inoculated to 5% sheep blood agar again and antibiogram identification discs containing 0.04 u b and 1.25 and 23.75 mg sxt (oxoid, uk) were placed onto the plate and incubated overnight at 37 8c. after that, colonies with beta hemolysis were defined as b-sensitive while colonies resistant to sxt were defined to be gabs. all colonies are serologically classified by latex-agglutination (oxoid, uk). seventy-one (14.5%) inoculations revealed growth of gabs at dio-bacit plates. after inoculating these colonies to 5% sheep blood agar, 72 of them were found to be sensitive to b, while 67 were found to be sensitive to b but resistant to sxt and 58 of them were defined as gabs by latex test. when compared with latex-agglutination test, we found dio-bacit method's sensitivity and spesificity to be 92 and 96.9%, respectively. method: agar diffusion technique as recommended by din 58940 was used to determine izs (read using aura and manually) for 178 staphylococci and 22 enterobacteriaceae . variations in automated measured zone sizes of 9/3 mm to the manual readings were considered to be within acceptable range. results: six thousand and fifty-two zone sizes were determined for staphylococci and 748 for enterobacteriaceae . mha displayed tendency to smaller zone sizes in automated readings than isa, as well in staphylococci and enterobacteriaceae . on the other side automated readings presented on isa more precise results than mha. overall less major discrepancies ( b/3 mm) were found on isa. izs were generally smaller on mha. the tables below show differences in manually and automated measured zone sizes on different media and species. we discovered seven more cases of resistance in the case of metronidazole. we did not have such experience with clarithromycin. conclusion: our results show that e -test is comparable to ad for clarithromycin, but for metronidazole our findings confirm nccls recommendantion. classical ad is time consuming for every day use in the laboratory. the use of screening agar plate with 8 mg/ml of metronidazole to detect possible resistance could be the solution. rokosz a a , sawicka-grzelak a a , meszaros j b , luczak m a . a department of medical microbiology, the university medical school, warsaw, poland , b department of bacteriology, state institute of hygiene, warsaw, poland purpose: to identify esbl-positive strains and to compare two methods applied for the detection of extended-spectrum beta-lactamases (esbls). methods: two hundred and sixty strains of gram-negative rods were cultured from clinical specimens from hospitalized patients. identification of strains was performed in the automatic atb system (biomerieux, france). these strains were identified as esbl-positive on the basis of the double-disc synergy test (ddst according to jarlier et al., 1988) results. all strains were also determined using a novel method of esbl detection (dd, diagnostic disc) according to appleton (1999) . two discs were applied in this test: cpd (cefpodoxime) and cd 01 (cefpodoxime/clavulanic acid) (oxoid, england). results: consistent results of two methods (ddst and dd) were obtained in the case of 166 from among 260 of examined strains (60.4%). consistent results concerned 161 out of 222 strains of enteric rods (72.5%) and only five from among 38 other strains (mostly nonfermenting rods). conclusions: the novel method of esbl-producers detection (dd) is more objective and easier for interpretation than the double-disc synergy test (ddst). diagnostic disc test should be used as the basic one or to confirm the results of ddst in difficult cases. assessment of e -test for determining penicillin resistance in pneumococci pm172 sener b, yeniþehirli g, ercis s, hasçelik g. department of clinical microbiology, hacettepe university medical faculty, ankara, turkey there is a greater need for susceptibility testing methods that distinguish between susceptible and resistant pneumococci. an alternative method could be the e -test, which is compared with the reference agar dilution method in this study. penicillin susceptibility of a total of 149 pneumococci was determined by e -test and agar dilution methods. streptococcus pneumoniae atcc 49619 and enterococcus faecalis atcc 29212 were used as controls. the results were given in the effect of anoxic conditions on the minimum inhibitory concentration of metronidazole in helicobacter pylori pm173 de la obra sanz p, lomas e, roman jl, alarcon t, lopez-brea m. the objective of this study was to determine the effect of incubation under anoxic conditions on the metronidazole resistance of helicobacter pylori . methods: a total of 35 clinical isolates were used in this study. mics were determined by an agar dilution method using mueller-hinton agar plus 7% lysed horse blood. three plates series contained twofold dilutions of metronidazole from 256 to 0.008 mg/l were prepared. the first one was incubated under microaerophilic conditions (oxoid) for 3 days; second and third series were incubated anaerobically (anaerobic system, oxoid) for 8 and 18 h, respectively, and were then transferred to the microaerophilic enviroment up to complete 3 days of incubation. results: with microaerophilic incubation, 12 of 35 strains were resistant (mic50 and mic90 were 1 and 32, respectively). with 8 h anaerobic preincubation, four of 35 strains were resistant (mic50 and mic90 were 0.5 and 4, respectively). with 18 h anaerobic preincubation, 1 of 35 strains was resistant (mic50 and mic90 were 0.25 and 1, respectively). conclusions: anaerobic preincubations causes an increase in sensitivity to metronidazole, the extent of which was dependent on the length of the anaerobic period. methods: the susceptibility to antibiotics was performed by microdilution method according to nccls guidelines. the production of b-lactamase was tested by nitrocefin sticks (oxoid). results: the mics50/mics90 (mg/ml) appeared, respectively: ampicillin 1/4, amoxicillin/clavulanic 0.06/0.12, cefaclor 1/1, ceftriazone 0.06/0.5, erythromycin 0.12/0.25, azithromycin 5/0.03/0.06, clarithromycin 0.12/0.12, ciprofloxacin 0.03/0.06, imipenem 5/0.015/0.06, tetracycline 5/0.25/5/0.25, trimethoprim/sulfamethoxazole 0.25/0.25. b-lactamase was detected in 98.5% of the strains. conclusions: (1) m. catarrhalis isolates were uniformly susceptible to all tested antimicrobials except ampicillin. (2) the production of blactamase was responsible for ampicillin resistance (98.5%). (3) m. catarrhalis strains had almost the same behavior 'in vitro' to the tested microlides (erythromycin, azithromycin, clarythromycin). rokosz a, sawicka-grzelak a, luczak m. department of medical microbiology, the university medical school, warsaw, poland purpose: to isolate, identify and determine the drug-susceptibility of fungal strains cultured from fecal samples routinely submitted for detection of clostridium difficile and its toxins in cases of antibioticassociated diarrhea (aad). methods: one hundred fecal samples from hospitalized patients were examined (may á/october 2001). c. difficile toxins a/b were detected directly in stools with c. difficile tox a/b ii test (techlab † , usa). fecal specimens were inoculated on ccca and candida id (biomerieux, france) media. c. difficile and fungi were identified with standard microbiological procedures. susceptibility of fungal strains to anti-fungal agents was determined (atb fungus, biomerieux, france). results: c. difficile toxins were detected in 38 and c. difficile strains were isolated from 23 of examined specimens. sixty-two fungal strains of 8 genera were cultured from 50 stool samples (24 c. albicans isolates). massive fungal growths were observed on primary plates in all cases. fifty-five fungal strains were susceptible to nystatin, 53-to 5fluorocytosine, 52-to amphotericin b, 45-to ketoconazole, 43-to miconazole and 39-to econazole. conclusions: in some cases of antibiotic-associated diarrhea fungal strains are responsible for symptoms of this disease. certain persons having aad should be treated with anti-fungal agents. results: a total of 291 isolates (33.2%) were penicillin nonsusceptible (intermediate and resistant) and 308 (35.1%) were erythromycin non-susceptible. non-susceptibility to both antibiotics was found in 183 (20.9%). conclusions: if penicillin administration eliminates all penicillin susceptible strains, the prevalence of penicillin non-susceptible strains will increase as well as the erythromycin non-susceptible ones. this means that the proportion of erythromycin non-susceptible strains should increase from 35.1 to 62.9%. at the same time, if erythromycin eliminate all susceptible strains to this antibiotic, the prevalence of penicillin non-susceptible strains would increase from the initial 33.2 to 59.4%. these data can explain the co-selection results observed in different surveillance studies. antimicrobial susceptibility and capsular types/groups of streptococcus pneumoniae isolates causing pneumococcal diseases in bulgaria pm178 setchanova l a , gergova r a , ioneva m b , sredkova v c . a department of microbiology, medical university, sofia, bulgaria , b department of microbiology, ii city hospital-sofia, sofia, bulgaria , c department of microbiology, faculty of medicine, pleven, bulgaria a prospective study of pneumococcal infections was performed in cooperation with five clinical microbiology laboratories in bulgaria. mics values to 12 antimicrobials and serotype/serogroup distribution were determined for 242 strains of streptococcus pneumoniae . pneumococci were isolated from patients with systemic or respiratory infections. the incidence of penicillin g-intermediate and penicillin gresistant isolate was 27.3 and 10.3%, respectively. the rates of resistance to other antimicrobials were: cefotaxime/ceftriaxone */ 6.2%; erythromycin */21.5%; clindamycin */7.8%; tetracycline */ 24%; chloramphenicol */26.4%; trimethoprim/sulfamothoxazole */ 38%; ciprofloxacin */10.7%; rifampin */3.7%. the s. pneumoniae isolates belonged to 19 capsular types/groups. the most common serotypes/serogroups in bulgaria are 1, 3, 5, 6, 9, 14, 19 we aimed to determine the pneumococcal antibiotic resistance rates and the serotypes of those resistant isolates in our hospital. the mic values of 212 isolates (year 1996 á/2001) were determined by agar dilution method. serotyping was performed by using 12 pooled antisera of the pneumo-test. the results were as follows (n 0/212). objectives: to asses the antimicrobial susceptibility of clinical isolates of pseudomonas aeruginosa obtained from 1997 to 2000 and to monitor trends in antimicrobial resistance. methods: mics were determined by microdilution testing according to nccls. the antibiotics tested were: ceftazidime (caz), aztreonam (atm), imipenem (imp), gentamicin (cn), tobramycin (tb), amikacin (ak) and ciprofloxacin (cip). results: a total of 1293 isolates were included. urine was the most common site of isolation for outpatients isolates (61.2%) while for hospitalized patients respiratory samples were the most frequent (40.2%). susceptibility to antibiotics was: 90% caz, 87.5% atm, 93% imp, 76.8% cn, 95.5% tb, 96.7% ak and 81.9% cip. comparison of susceptibility data through 1997 á/2000 showed that the increase in the resistance rate was significative for caz (4.6 vs 12.9%), atm (9.9 vs 18%), cn (18 vs 26%), tb (1.85 vs 6.9%), ak (1.6 vs 5.7%) and cip (15.17 vs 21%). significant differences were found under the following circumstances: isolates from intensive care units and from inpatients were significatively more resistant to caz, atm and imp. isolates from respiratory samples were more resistant to caz and atm and isolates from urine samples were more resistant to cip. conclusions: although the antimicrobial susceptibility level has been decreasing p. aeruginosa isolates still show good susceptibility percentages for all antibiotics tested. antimicrobial susceptibility testing of clinical isolates of bordetella pertussis : report on 27 isolates from rouen, france pm181 lemee l a , nouvellon m a , caron f b , lemeland jf a . a chu rouen, bacteriologie, rouen, france , b chu rouen, maladies infectieuses et tropicales, rouen, france reports of an increased clinical incidence of pertussis and the development of resistance by bordetella pertussis to erythromycin prompted the collection and antimicrobial susceptibility testing of recent clinical isolates from patients, who were hospitalized in rouen between 1991 and 1999. mics of nine antimicrobial agents (erythromycin, josamycin, spiramycin, roxithromycin, ketolide hmr 3647, cotrimoxazole, ciprofloxacin, rifampicin and amoxicillin) were measured by agar dilution method on mueller-hinton agar containing 5% horse blood. mbcs of erythromycin and rifampicin were also determined against four isolates of b. pertussis . all isolates were fully susceptible to the nine antimicrobial agents tested. mics 90 (mcg/ml) were 0.03 for erythromycin, ketolide hmr 3647 and ciprofloxacin, 0.06 for josamycin, 0.25 for spiramycin, roxithromycin and rifampicin, 0.5/2.5 for cotrimoxazole, and 1 for amoxicillin. mbcs (mcg/ml) were 0.125 á/0.5 for eyrthromycin and 0.5 á/1 for rifampicin. in conclusion, our isolates of b. pertussis remain extremely susceptible to all antimicrobial agents tested, especially macrolides. no resistance was detected. finally, if erythromycin remains the molecule of choice, other macrolides (c14 and c16) also confirm their good in-vitro activity. in addition, the good in-vitro potency of rifampicin, together with its great diffusion within the respiratory tract, suggests that rifampicin has potential clinical efficacy in pertussis too. the emergence of streptococcus pneumoniae (sp) with diminished susceptibility to penicillin g (psdp) suggests the use of other antibiotics such as newer fluoroquinolones (fq). the resistance phenotypes of 252 consecutive pneumococcal strains isolated from patients of four hospitals (observatoire régional des pneumocoques du nord-pas de calais) were studied: 39 strains were susceptible to penicillin g and 213 were psdp. reference strains provided from the centre national de réfeacute;rence des pneumocoques were added to the study. the activity of pefloxacin, ciprofloxacin, norfloxacin, sparfloxacin, levofloxacin and moxifloxacin was studied. reserpine was used to detect the efflux phenotype. methods used were performed according to the recommendations of the comité de l'antibiogramme de la société française de microbiologie. for each strain, the resistance phenotype to fq was deduced by comparison of mics or diameters obtained with those obtained with the reference strains of known phenotypes. fq resistance phenotypes were not correlated to blactam agent susceptibilities. wild type phenotype was observed among 76.9 and 77.5% of the susceptible and psdp strains, respectively. a 'wild efflux' mechanism, deduced by addition of reserpine to norfloxacin, represented the predominant phenotype. it was detected among sp susceptible to penicillin g (23.1%) as well as among psdp (17.4%). the aim of this study was to determine macrolide resistance phenotypes of sp isolated in three french departments (alpes maritimes, doubs, nord) from nasopharyngeal aspirates of children aged 3 months to 3 years attending a dcc. a random sample of children attending randomly selected dccs was obtained during three periods (spring, autumn and winter 1999) in each department (10 494 children attending dccs and 2565 children sampled). analysis of macrolide susceptibility of sp strains was performed using the ca-sfm method. out of 1262 strains, 64.7% had decreased susceptibility to penicillin (spdp) and 77.5% were resistant to erythromycin. the triple disk diffusion method (erythromycin (e), clindamycin (cl) and spiramycin) was used to determine resistance phenotypes. macrolide resistance is a well known phenomenon in france and is confirmed by our study. these results show that the constitutive phenotype is predominant as in other parts of europe and the frequency of efflux mechanism is lower than that observed in the usa and canada. developing antibiotic resistance surveillance of helicobacter pylori in england and wales pm184 elviss nc, owen rj. central public health laboratory, laboratory of enteric pathogens, london, uk purpose: helicobacter pylori antibiotic resistance is a key contributing factor in â/10% of infected patients failing drug treatment. our aim was to survey rates of primary in-vitro resistance at different locations, and links to disease severity. antral gastric biopsies/cultures were received from phls in chelmsford, mid-essex (1115 isolates 1995 á/2001); london (103 isolates 1999 á/2000); and bangor, north wales (165 isolates1999 á/2001). susceptibilities to metronidazole (mtz), clarithromycin (cla), tetracycline (tet) and amoxicillin (amx) were tested by disc diffusion and also by e -test for cla and mtz. results: overall resistance rates (1383 isolates) were 38% for mtz and 8% for cla. all were susceptible to amx and tet. dual resistance rate was 5%. breakdown by location showed some marked differences. mtz resistance was highest in london (63%) compared to 37% in chelmsford and 20% in bangor. by contrast cla rates were 11% for london, and about 5% for bangor and chelmsford. in london, the majority of mtz resistant isolates were from non-uk borne individuals (75% non-uk vs 25% uk). comparison of duodenal ulcer-associated isolates with those from non-ulcer patients indicated similar rates of mtz resistance (28%). conclusion: resistance rates may vary significantly between locations depending on the local population with non-uk birth being a key risk factor for primary resistance with a mtz resistant strain. local resistance rates should be taken into account in test and treat strategies. potrykus j, benetkiewicz m, wegrzyn g. department of molecular biology, university of gdansk, gdansk, poland purpose of the study : because of their ability to extrude a wide range of compounds, multidrug efflux pumps have recently become an important issue in combating bacterial infections. acrab-tolc is the major efflux system of escherichia coli . we investigated the effect of acra on plasmid-borne and intrinsic chloramphenicol, tetracycline and ampicillin resistance. results and conclusions: recently, we reported a chloramphenicol sensitivity of e. coli mutant expressing cat , the chloramphenicol resistance gene. the strain was shown to bear a nonsense mutation in the acra gene. our studies indicate that this mutation is, at least in part, responsible for the observed chloramphenicol sensitivity phenotype. the mutation seems also to influence the strain's susceptibility to ampicillin and teta (c )-mediated (plasmid-borne) tetracycline resistance. although the teta(c) protein retained its biological function, there was a considerable growth impairment of the mutant strain when cultured in tetracycline containing medium. deletion of the acrab locus prevented any growth in the presence of tetracycline. upon the addition of ampicillin, the mutant underwent lysis more rapidly than the control strain. such was also observed in acrab deletion derivatives of other e. coli strains. we are trying to elucidate the role of the acra gene product in the phenomena described above. existence of efflux pumps in wild type isolates of drug-resistance bacteria pm186 raja ray rr. medical microbiology and parasitology, calcutta university, kolkata, india efflux pumps possessed by the bacterial cells of different kinds of bacteria had presented as a newer mode of drug resistance in many organisms. the capacity of bacterial cells to cause outward flow of noxious agents was known, however, for a considerable time with respect to tetracycline. recently, interest in the efflux pump system has brought to light some previously ill-understood mechanisms of drug resistance, involving noxious agents, toxins or poisons. we have found high level of resistance in pseudomonads towards cetrimide and other germicides for which no definite chromosomal/plasmid-mediated genes/mechanisms could be identified. likewise, occurrence of nonantibiotic sensitive vibrios, staphylococci and pseudomonads in the background of their high level of resistance to most of the common antibiotics suggest a mechanism of interference with the efflux pump, which accounts for such sensitivity in such cases. involvement of multiple resistance of marine isolates of v. parahaemolyticus to numerous clinically used antibiotics to which they have never been exposed also suggests a possible role of efflux pumps in determining such resistance */that these can simultaneously develop against multiple marine toxins/poisons and other noxious agents. interaction between oxacillin and glycopeptides in a teicoplanin-resistant mutant of staphylococcus epidermidis with reduced susceptibility to vancomycin pm187 greco aa, ben hassen a. laboratory service of national bone-marrow transplant center, tunis, tunisia we selected a laboratory-generated mutant of staphylococcus epidermidis capable of growing in the presence of 256 mg/l of teicoplanin (353b tm256), from a methicillin-resistant (mic!/256 mg/l), teicoplanin-sensitive (mic 4 mg/l) and vancomycin-sensitive (mic 2 mg/l) clinical isolate of s. epidermidis (353b so). in a previous work, we studied the different phenotypic characteristics acquired by the teicoplanin-resistant mutant 353b tm256 (20th interdisciplinary meeting on anti-infectious chemotherapy, december 2000, poster sessions, 82/p1). in this work, we examined the interaction between oxacillin and glycopeptides against this teicoplanin-resistant mutant of s. epidermidis with reduced susceptibility to vancomycin. to study the combined antibiotic activity of oxacillin and glycopeptides, we used different methods: a modified disk diffusion test, the e -test, time-kill assays and population analysis profiles. the synergistic activity of glycopeptides in combination with oxacillin against the teicoplaninresistant mutant 353b tm256 was demonstrated with a bactericidal effect. no synergy was seen against the parental strain 353b so. moreover, the synergy between glycopeptides and oxacillin occurred with suppression of the subpopulation with the highest level of glycopeptides resistance. we concluded that combination of glycopeptides and oxacillin may be a possible alternative in the treatment of infections caused by methicillin-resistant, teicoplanin-resistant s. epidermidis . compositional changes in microcosm biofilms induced by application of minocycline: a preliminary study pm188 the aim of the study was to observe the effect of application of minocycline upon microcosm dental plaques. the plaques were cultivated in a constant departmenth film fermentor (cdff), which produces biofilms under conditions mimicking those present in vivo. the composition of the biofilms was determined by viable counting on selective and non-selective media. the proportion of antibiotic resistant genera within the biofilm was determined by viable counts utilising media containing minocycline (16 mg/ml). before commencing antibiotic pulsing, the biofilms had a total viable anaerobic count of 1.26)/10 10 cfu per biofilm, with negligible (6 cfu/biofilm) minocycline-resistant bacteria. however, 24 h after introduction of the antibiotic, the total count had been reduced to 1.02)/10 9 cfu/biofilm whilst the number of minocycline-resistant bacteria had risen to 1.23)/10 6 cfu/biofilm. at the final sampling time point (192 h) the total viable anaerobic count was 5.82)/10 8 cfu/biofilm whilst the number of minocycline-resistant bacteria was 1.12)/10 7 cfu/biofilm. hence, there is a very low basal level of inherent resistance to minocycline within microcosm dental plaques, but this increases considerably once the biofilms are exposed to minocycline. mechanism of resistance to aminoglycosides (amg) e. coli isolated from children with community-acquired urinary tract infections (cau-tis) pm189 methods: during the 2000 á/01 years nine centers took part in the study. the mics of antimicrobials were determined by the agar dilution method as described in the nccls guidelines. results: a total of 710 consecutive urine isolates from 692 children aged 1 month to 18 years with cauti were collected. the most frequently isolated species from children with cauti was e. coli (52.3%), followed by klebsiella spp. (8.0%) and proteus spp. (7.6%). results of the in vitro susceptibility testing of e. coli to amg are shown in table below. resistance of the strains was conditioned on production of amg-modifying enzymes. there has been found following phenotypes among resistance strains: gentamicin á/ tobramycin á/netilmicin (77.8% */aac(3)-v and 13.9% */aac(3)-iv enzymes) and gentamicin á/tobramycin (8.3%, due to ant(2ƒ) enzyme). conclusion: amikacin is most active amg against e. coli . resistance to gentamicin and netilmicin was mainly determined by production of aac(3)-v enzyme. effect of b-lactamase inhibitors (b-l-i) on the evolution of resistance (r) to b-lactams (b-l) in gram the b-lactams antimicrobials still are the most frequently used. among the bacteria responsible of high resistance to b-lactams are gramnegative rods; its most frequent mechanism is the production of b-lactamase. the use of b-l-i has reversed partially this mechanism of resistance. we expect changes in the frequencies of r using b-lƒci after more than 10 years. since 1988, the venezuelan group of bacterial resistance, with 29 health institution in the country; analyse and publish data on bacterial resistance of isolates from patients with bacterial infection coming from hospitals. it was used diffusion disk, according nccls. the software program whonet (world health organization net) was used. we follow the trends of r of gram-negative rods to b-l alone and with b-l-i during the decade 1988 á/1998. statistical analyses were made by evaluating the differences among percentages of resistance between the two series (p 5/0.05). results and discussion: the difference in r between b-l and b-l/b-l-i are: (1) piperacillin, piperacillin/tazobactam: between 10 and 30% of r for most isolated, except for escherichia coli (45%) and serratia spp. (60%); (2) ampicilin, ampicilin/sulbactam: between 10 and 30%; (3) cefoperazone, cefoperazone/sulbactam: between 5 and 25%. how is expected gramnegative rods resistance to b-lactams with a b-l-i is lower than the b-lactam alone; furthermore the difference between both series, grows higher with time. these results are relevant and they were not expected, since b-l-i have been shown to be b-lactamase inductors. trends in the resistance (r) to b-lactams and others antimicrobials in p. aeruginosa in venezuelan medical centres. nosocomial (nos) and communitarian resistance pm191 in order to approach the infection produced by resistant bacteria, it is convenient to consider the hospital and the community as two separate ecosystems. the hospital ecosystem has special relevance in the infection and r of gramnegative aerobic bacilli. today, they are the main responsible of nos infection, with special reference to pseudomonas aeruginosa . infection by resistant bacteria is a world wide problem, specially related to nos. since 1988, the venezuelan group of bacterial resistance, with 29 health institution in the country; identify, analyse and publish data on bacterial r to antimicrobials: b-lactams, quinolones and aminoglicosides of isolates from patients with bacterial infection coming from hospitals and the community. it was used diffusion disk, according nccls. the software program whonet (world health organiza-tion net) was used. statistical significance (p 5/0.05) was determined by application of the x 2 technique. we show significant differences in the r of p. aeruginosa nos and communitarian (highest differences: piperacilina 40/7%, tobra 40/4%). we also established significant differences between the r arising in public hospitals and private hospitals (highest differences: ceftazidime 50/5%, amika 25/ 5%). we show the tendency in decreasing of frequency of r since 1998; this is more evident in private hospitals (b-lactam and aminoglicosides). new antimicrobials and new mechanism of action, and in the future the new technology will solve today's problem. however, the most important tools we have today are prevention and antimicrobials, and we must make them suitable. susceptibility to antibiotics of enterobacter cloacae and citrobacter freundii from drinking water pm192 quintera sm, sousa jc, peixe l. department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal the increased use of antimicrobials in farming, together with the practice of raw sewage discharge into receiving waters, has resulted in a significant increase in the number of antibiotic resistant bacteria present in aquatic environment. our objective was to determine the antimicrobial susceptibility, with focus on b-lactam resistance, among enterobacteriaceae strains isolated from raw drinking water samples. several isolates (n0/107) of enterobacter cloacae and citrobacter freundii obtained from drinking waters were screened for antibiotic susceptibility patterns, using the agar diffusion technique, according to nccls's procedures. only 55% of e. cloacae strains, as well as 37% of c. freundii strains show resistance to amoxicillin and amoxicillin/ clavulanic acid. a reduced incidence of resistance to several others antibiotics was also observed. the obtained results suggest that strains isolated from raw drinking water have greater susceptibility to antimicrobial agents than pathogenic strains from hospital or outpatients infections. the 'natural' antimicrobial resistance phenotypes, usually described for c. freundii and e. cloacae , only seem to apply to strains isolated from human infections. notwithstanding the high susceptibility of the tested isolates to b-lactams, the role of environmental bacteria as a reservoir of resistance genes justify its periodical monitoring as a valid index for resistance spreading. a snapshot of the soil. using bacterial communities for tracing the evolution of metal-resistance pm193 quintera sm a , sousa jc a , peixe l a , monteiro nm b . a department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal , b department of zoology and anthropology, faculty of sciences, university of oporto, oporto, portugal it is well known that pathogenic bacteria, specially those resistant to antimicrobial agents and heavy metals poses public health risks of great concern, and its detection, namely in soils is generally related to pollution. in this study, the heavy metal resistance patterns of the microflora isolated from polluted (dump area) and unpolluted soil environments were examined. the plate growth covering percentage in the soil samples was determined using mueller-hinton plates supplemented with different heavy metal (al3'/, cd2'/, cu2'/, pb2'/, hg2'/ and zn2'/) concentrations. parallelly, using icp-aes, it was possible to ascertain the real heavy metal concentration for each soil sample. we found that the percentage of plate growth covering from the used samples was closely linked to the level of chemical pollution measured for each location. moreover, using anova, we found significant differences between locations. the dump site showed the highest tolerance to all the tested metals (newman á/keuls test). this pattern of results was consistent when using the data from the icp-aes. furthermore, it was possible to observe that pseudomonas spp., with a relatively high mic for the studied metals, might become a relevant model for both public health issues and eco-toxicological studies. biochemical characteristics of environmental isolates of listeria monocytogenes pm194 moshtaghi h a , garg sr b , mandokhot uv b . a shahrekord university, food hygiene, shahrekord, islamic republic of iran , b haryana agricultural university, food hygiene, hisar, india purpose: the investigations were carried out to study the biochemical reactions of listeria monocytogenes isolated from different sources in the environment. results: a total of 12 isolates of l. monocytogenes were obtained from 230 samples of agricultural soil, faecal matter of animals and sewage. all the isolates were gram-positive, small rods, catalase positive, oxidase negative, motile with tumbling motility in hanging drop at 22 á/25 8c, aerobic, facultative anaerobic, fermentative and produced acid from glucose. all the isolates of l. monocytogenes were beta haemolytic and positive for camp reaction with staphylococcus aureus . all the isolates were negative for phenyl alanine deaminase, ornithine decarboxylase, lysine decarboxylase, malonate utilization and beta galactosidase tests. these were also negative for acid production from arabinose, d-xylose, mannitol, soluble starch and sucrose but acid was produced in rhamnose, salicin, and trehalose. hydrogen sulfide production was recorded in tripticase soy broth with lead acetate paper strips but negative with triple sugar iron agar. all the isolates were found to hydrolyse aesculin. out of 12 isolates of l. monocytogenes only two produced acid from lactose. in serotyping all the isolates were serotype 4b. conclusion: we can conclude that l. monocytogenes serotype 4b at least in fermentation of lactose shows different reactions. methods: the samples were pre-enriched in bhi broth with and without vancomycin (6 mg/l) and then plated onto m-enterococcus agar with and without antibiotics: vancomycin (6 mg/l), gentamicin (125 mg/l), kanamycin (500 mg/l), and streptomycin (1000 mg/l). representative colonies of each morphology were isolated and identified as enterococcus sp as previous described. pcr was used to identify e. faecium and e. faecalis and to characterise vancomycin resistant genotype. api20strep was also used in the identification. susceptibility testing to 11 antibiotics was performed by an agar dilution method (nccls). results: three hundred and fifty-three enterococci were isolated from 92 of a total of 99 faecal samples (93%, n 0/92/99). the majority of enterococci were identified as e. faecium , e. faecalis and enterococcus sp. resistance to almost all antibiotics studied was observed: vancomycin */3.0%; teicoplanin */3.0; ampicillin */21.2%; tetracyclin */81.8; erythromycin */86.9%; ciprofloxacin */78.8%; chloramphenicol */54.5%; gentamicin */17.2%; streptomycin */ 78.8%; kanamycin */77.7%; linezolid */0%. the vancomycin resistant enterococci presented a vana genotype. conclusion: resistance to several common antibiotics used in therapy was observed among enterococci isolated from healthy human from community. many of these isolates presented multi-resistance. of concern is the presence of vana genotype among these populations that may constitute a reservoir of vancomycin resistant genes. antimicrobial resistance in tetracycline-resistant oral bacteria pm196 mercury release from dental amalgam may select for mercuryresistant oral bacteria. mercury resistance is often associated with multiple antibiotic resistances. the aims of this study were to determine whether tetracycline-resistant oral bacteria from children with and without amalgam fillings were also resistant to: (a) mercury; and (b) multiple antibiotics. tetracycline-resistant organisms were isolated on iso-sensitest/blood agar containing tetracycline (8 mg/ml). the mic of hgcl 2 and several antibiotics were determined using agar dilution (bsac). one hundred and three organisms were isolated from patients without amalgam. ninety-one were streptococcus species, seven neisseria species, three veillonella dispar and two rothia species. fifty-seven percent exhibited resistance to at least one antibiotic, 12% were mercury-resistant, 42% were penicillin-resistant, 11% were ampicillin-resistant and 23% erythromycin-resistant. fiftytwo organisms were isolated from patients with amalgam. forty-five were streptococcus species, five neisseria species, one v. dispar and one staphylococcus aureus . sixty-three percent exhibited resistance to at least one antibiotic, 23% were mercury-resistant, 38% penicillinresistant, 11% were ampicillin-resistant and 31% showed erythromycin-resistance. statistically, the results showed that in tetracyclineresistant organisms, the presence of dental amalgam did not affect the level of resistance to mercury or to the antibiotics tested. conway-wallace hl a , mullany p a , bedi r b , wilson m a . a eastman dental institute, university college london, microbiology, london, uk , b eastman dental institute, university college london, transcultural oral health, london, uk the purpose of this study: to determine the prevalence of antibioticresistant oral bacteria in children who had not received antibiotics during the 3 months prior to sampling. plaque samples were obtained from 16 children aged 4 á/6 years and plated onto media containing: penicillin, ampicillin, tetracycline, erythromycin and vancomycin. resistant isolates were enumerated, sub-cultured and frozen for subsequent identification. the process was repeated 6 and 12 months later. the results obtained: bacteria resistant to each of the antibiotics were present in all of the children at each sampling time (except in the case of ampicillin and penicillin at 0 months). the proportion of antibiotic-resistant bacteria in the oral microflora ranged from ]/13.7 (erythromycin) to 5/0.5% (ampicillin). the proportions of bacteria resistant to a particular antibiotic remained reasonably constant over the 12-month sampling period. in only two cases (penicillin and ampicillin) was there a statistically significant change in the proportions of resistant bacteria at different time periods. the conclusion reached: the results of the study have revealed that bacteria resistant to a wide range of antibiotics may be isolated from children who have not been administered these agents during the 3 months prior to sampling. furthermore, in many cases the proportion of bacteria resistant to a particular antibiotic remains constant over a 12-month period. antimicrobial use in the intensive care unit: results of a pharmacoepidemiological study in italy pm198 periti p. e.i.f.t. srl, firenze, italy a retrospective survey of antimicrobial chemotherapy use in 560 intensive care units in italy was carried out in 1999 using a computerized questionnaire under the auspices of the journal of chemotherapy. of the icus contacted, 43.4% replied, being mainly general or post-surgical and pediatric units having a mean of 10 beds, nine doctors and 20 nurses. the antimicrobial agents used in these wards were almost always polychemotherapy with prevalent use of beta-lactams, aminoglycosides and glycopeptides or as empirical treatment during the first 72 h after hospital admission. the continual use of medium-high dose combinational antimicrobial chemotherapy was justified by microbiological testing, which revealed that more than one-third of bacterial pathogens were resistant. approximately, 60% of gram-positive bacteria were methicillin-resistant, whereas about 13% of gram-negative strains were resistant to at least one of the tested antibiotics. forty percent of the responding icus furnished microbiological testing data, of which three quarters indicated the incidence of chemoresistance of the isolated strains. fungal infections were less frequent than bacterial, the most commonly isolated agent being candida spp. in conclusion, the sample of icus examined showed adequate and reasonable use of antimicrobial agents, with heavy reliance on medium-high dose combination therapy due to the elevated incidence of resistant isolates found. plasma concentrations (p), urinary excretion (u) and bactericidal activity of gatifloxacin (gat) 400 mg versus ciprofloxacin (cip) 500 mg in 12 healthy volunteers after a single oral dose pm199 boy d a , kinzig-schippers m b , sö rgel f b , well naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, ibmp, nürnberg-heroldsberg, germany twelve volunteers received a single oral dose of 400 mg gat versus 500 mg cip to assess p up to 36 h, u (by hplc), and urinary bactericidal titers (ubt) in eight intervals up to 120 h. the mean pmax of gat/cip was 3.35/2.12 mg. the ucum (mean) for gat/cip was 81.0/43.2%. the ubts, i.e. the highest twofold dilution of urine still bactericidal, were determined for nine uropathogens and one reference strain */mics (mg/ml) (microdilution) for gat/cip: escherichia coli atcc 25922 (0.008/0.008); e. coli (0.06/0.06); klebsiella pneumoniae (0.03/0.016); proteus mirabilis (0.25/0.06); pseudomonas aeruginosa (1/ 0.125); s. saprophyticus (0.125/0.25); two strains of s. aureus (0.06/ 0.125); two strains of e. faecalis (0.5/1 and 8/32). the median ubts measured within the first 6 h for gatifloxacin were between 1:16 and 1:1024 for the five gram-negative strains (incl. p. aeruginosa ) and between 1:8 and 1:1024 for the five gram-positive strains. the median ubts for ciprofloxacin were between 1:64 and 1:1024 for the gramnegative strains (incl. p. aeruginosa ) and between 1:1.5 and 1:768 for the five gram-positive strains. for the ubts up to 12 h, gat was significantly superior to ciprofloxacin in all gram-positive strains, not different in the two e. coli strains, and inferior in the klebsiella , proteus and pseudomonas strains. for the ubts at 12 á/24 h, gat was generally superior to cip, but showed no difference in the proteus and pseudomonas strains. gat showed overall comparable urinary bactericidal activity as cip. this is in agreement with a clinical study performed previously. malaria is one of the most prevalent endemic infectious disease affecting humans. in bichat hospital 111 cases of malaria acute illness were reported during 2001. among them, 96 patients were hospitalised and intravenously treated by quinine. this retrospective study consisted of comparing the therapeutic drug monitoring (tdm) of quinine distinguishing, respectively 36 and 60 patients cured in infectious medical department (imd) and intensive care unit (icu) where a standardised quinine regimen was established (62 and 47% malaria attacks, respectively). in icu, the treatment consisted of an infused loading dose 16 mg/kg/4 h of quinine diluted in 5% glucose followed by 24 mg/kg/day. plasma quinine maximal concentrations were assessed after selective liquid á/liquid extraction and spectrofluorometry detection. statistical analysis was performed using t -test. results showed that patients had comparable weight (71.49/19.7 and 70.89/17.9 kg) but quinine doses and plasma concentrations were significantly different in icu and imd, respectively (20.29/6.5 versus 22.79/3.7 mg/kg/day, pb/ 0.001 and 11.49/3.3 versus 10.39/3.2 mg/l, p b/ 0.01). in icu and imd, respectively: 57 and 49% were in the therapeutic range (10 á/15 mg/l) with 29 and 43% below the requested therapeutic concentration (10 mg/l) and 13 and 8% above the limit of toxicity (15 mg/l) conveying the importance of tdm in intravenous quinine treatment to avoid infra-therapeutic or toxic concentrations. simultaneous central nervous system distribution using microdialysis and pharmacokinetic á/pharmacodynamic modelling of the electroencephalogram effect of norfloxacin in rats pm201 chenel m, marchand s, dupuis a, bouquet s, couet w. university of pharmacy, pharmacology, poitiers, france purpose: to investigate the epileptogenic activity of norfloxacin by a pharmacokinetic á/pharmacodynamic (pk á/pd) modelling approach and to assess the contribution of distributional processes across the blood á/brain barrier (bbb) to the delayed effect. methods: rats (n0/10) received an iv bolus dose of norfloxacin (150 mg/kg). convulsant effect was quantified by electroencephalogram (eeg) recording during 9 h post-dose. arterial blood samples were collected for drug assays in plasma. unbound norfloxacin concentrations were monitored in brain extracellular fluid (ecf) using microdialysis with in vivo calibration of the probes by retrodialysis with ciprofloxacin. results: the eeg effect reached its maximum between 70 and 190 min post-dose. a pk/pd effect compartment model was successfully fitted to these data. the relationship between effect and concentration at the effect site was best described by a spline function. norfloxacin concentrations in brain ecf were relatively low compared to plasma levels (ecf/plasma areas under curve (auc) ratio equal to 9.79/ 2.8%), but central distribution was rapid. therefore, the effect versus brain ecf concentrations curves still exhibited a marked hysterisis. conclusion: the delay observed between plasma concentrations and norfloxacin convulsant effect cannot be explained by a slow distribution of norfloxacin across the bbb. pagoulatou a a , kanellakopoulou k b , vafiadou m a , kostakopoulos th c , kastriotis i b , giamarellou h c . a department of anesthesia, sismanoglio general hospital, greece , b 4th department of internal medicine, athens medical school, athens, greece , c department of urology, athens medical school, athens, greece csf kinetics of van and fu were studied in 57 patients who underwent short urological surgery under spinal anesthesia. patients were excluded if they were already receiving an antibiotic or were suffering from renal and hepatic dysfunction. van was administered at 1 g over 1 h infusion. serum and csf samples were collected post-dose and the mean serum levels were as follows: 30 min á/1 h: 21.1 mg/ml (five patients), 1 á/2 h: 10.1 mg/ml (five patients), 2 á/4 h: 8.5 mg/ml (six patients), 4 á/6 h: 6.7 mg/ml (six patients) and 6 á/8 h: 5.15 mg/ml (seven patients). fu was administered at 500 mg dose over 1 h infusion. serum and csf samples were taken post-dose and the mean serum concentrations were found as follows: 0 á/30 min: 43.08 mg/ml (six patients), 30 min á/1 h: 41.7 mg/ml (six patients), 1 á/2 h: 33.8 mg/ml (five patients), 2 á/4 h: 27.8 mg/ml (six patients), 4 á/6 h: 25.6 mg/ml (five patients). in csf, both van and fu were undetectable. it is concluded that in the absence of meningeal inflammation van and fu do not penetrate (with the applied microbiological assay) the csf barrier. comparison of the pharmacology of intravenous and orally given moxifloxacin in an in-vitro model pm203 wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: the intravenous form (iv) of 400 mg moxifloxacin (mox), one of the newer fluoroquinolones, has been recently approved by the fda. during the iv treatment higher peak serum concentrations are achieved in comparison to the oral administration (po) of the same dose. the antibacterial activity of fluoroquinolones is concentration dependent. we therefore simulated human pharmacokinetics of single po and iv dosages of 400 mg mox in an in-vitro model using six different gram-negative and -positive pathogens to elucidate the different effect of these two dosing schedules. results: the comparison of the pharmacological parameter auc/ mic shows an increase ( table 1 ) that could predict an enhanced antibacterial effect. however, the analysis of the killing curves with the following parameters, ka.max (maximal killing activity) and aac (area above the killing curve between 0 and 24 h), reveals no major difference between the po and iv dosage. conclusion: the serum concentration after oral administration is already sufficiently high to show the optimal bactericidal effect of mox that can only be slightly increased by higher peak concentrations and higher auc/mic ratios. thus the concentration dependence is not linear but ends already at concentrations achievable by oral dosing and documents that auc/mic calculations cannot easily be translated into dosing schedules. background : bacteria growing in vivo multiply much more slowly than in vitro. whether the bactericidal activity of quinolones may be affected by an increase in generation time (g) was studied in batch cultures. methods: by limiting the nutrient supply, generation times were lengthened from approximately 0.45 to 1.5 h up to 3.9 h. alternatively, the quinolones were added to the bacterial cultures during the lag-, exponential-and stationary phase. recent clinical isolates of escherichia coli were exposed to multiples of the mics of ciprofloxacin or norfloxacin. the 'killing rates' were calculated in analogy to the growth rate. results: the bactericidal activity of the quinolones tested against e. coli was minimally influenced by the reduced generation time. ciprofloxacin concentrations of ]/2)/mic eliminated the test strains within 5/2 h from the test system if added during the lag or exponential growth phase; four times higher concentrations were needed to reduce cfus by 99% within 2 h, if added during the stationary phase. norfloxacin was significantly less active. conclusion: in contrast to norfloxacin, the bactericidal activity of ciprofloxacin is minimally affected by the generation time or growth phase of the bacteria. wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: moxifloxacin (mox) is one of the newer fluoroquinolones, now available for parenteral application. the pharmacology of an intravenous once-daily dose (od) of 400 mg mox was determined with five gram-negative and -positive pathogens (streptococcus pyogenes , streptococcus pneumoniae , moraxella catarrhalis , escherichia coli , and klebsiella pneumoniae ). a twice-daily dose (bid) of 400 mg mox was simulated with the gram-positive species in order to increase the bactericidal effect. results: to determine the efficacy, killing curves were analyzed, and following parameters were calculated: ka.max: maximal killing activity [log cfu]; ka. conclusion: an intravenous once-daily dose of mox is active against all tested pathogens. the gram-negative species are rapidly killed (ka.1 h similar to ka.max). there is no pronounced initial effect on the two gram-positive species but a general slow reduction in the viable cell count (ka.max is reached after 24 h). the efficacy of mox (measured as aac and ka.max) on s. pyogenes and s. pneumoniae is to some extent increased after the second dose. however, the analysis of the killing curves reveals no major difference between od and bid. even the od nearly gives the maximal bacterical activity of mox against gram-positive pathogens. objectives: to evaluate the dose proportionality of amoxicillin and to compare the respective pk/pd parameters of two dosage regimens. methods: the dose proportionality of amoxicillin was evaluated using linear regression of mean auc 0-inf and c max data of 13 different bioequivalence studies (n0/477 volunteers) performed with formulations containing various amounts of amoxicillin alone or in the combination with clavulanic acid. the volunteers received a single oral dose in the range of 250 á/1000 mg. amoxicillin plasma concentrations were determined by hplc/uv or lc/ms/ms methods. time above mic (tmic) expressed in% of dosing interval was calculated with three target mic values (0.5, 1.0 and 2.0 mg/l) for 500 mg 8hourly and 1 g 12-hourly dosage regimens. results: the absorption of amoxicillin (auc 0-inf ) showed a linear dependence with a correlation coefficient of 0.975. the correlation coefficient of the linear regression for the cmax dependence on the actual dose was 0.909. the respective tmic for both dosage regimens were very similar, with largely overlapping confidence intervals, supporting a pd breakpoint of 2 mg/l for the 1 g 12-hourly regimen (tmic ]/2 mg/l: 42.8%, 95% ci 38.6, 46.9%). conclusion: this analysis shows the dose proportionality of amoxicillin over the dosage range of 250 á/1000 mg and supports the pharmacodynamic rationale for a 1 g bid dosage regimen. piperacillin/tazobactam concentration profile after high dose administration pattern in nosocomial pneumonias due to mecanical ventilation pm207 pedeboscq s a , gruson d b , bassoua v a , hilbert g b , pometan jp a . a st. andré hospital, pharmacy, bordeaux, france , b pellegrin hospital, reanimation, bordeaux, france the piperacillin (p)/tazobactam (t) antibacterial spectrum covers the largest part of bacteria responsible for pneumonias due to mechanical ventilation. but, due to important bacterial inoculum and pharmacokinetic parameter modifications in intensive care patients, high doses of beta-lactamines seem to be necessary to obtain antibiotic concentrations above suspected bacteria's mic (minimal inhibitory concentration) . this led us to compare, in patients with pneumonia due to mechanical ventilation, two intermittent administration patterns: 4 g three times a day (usual pattern) versus 4 g four times a day (high dose pattern). this study is carried out in collaboration with intensive care unit, bacteriological department and pharmacy where antibiotic concentrations are determined. twenty-three takings of blood are executed within a 48 h period, in addition to two bronchial secretion samples. concerning p seric concentrations, the high dose pattern seems to be more adapted because of relatively high residual concentrations ( !/20 mg/ml). three hours after each injection, t seric concentrations are lower than the 4 mg/ml activity threshold. first and second day residual bronchial concentrations of p seem to be sufficient although t concentrations are below activity threshold. these results are to be correlated with the mic determined by the bacteriological department, and only this correlation will make us able to conclude the better efficacy of the high dose pattern in intensive care patients. anti-inflammatory drugs interference in absorption and tissue penetration of amoxycillin pm208 del fiol fs a , menon sz b , caramez th b , celotto tf b , lopes ras b . a university of sorocaba, pharmacology, sorocaba, brazil , b school of pharmacy, university of sorocaba, sorocaba, brazil antibiotics and anti-inflammatories are frequently associated in clinical practice. there is some concern about the quantity of antibiotic that reaches the infection sites, which may be reduced in the presence of an anti-inflammatory drug. the purpose of the present study was to analyse how steroids (dexamethasone (dexa)) and aines (celecoxib (cele)) influence on the penetration of amoxicillin to inflamed tissues. thirty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs to form granulomatous tissue. one week later the animals were divided into three groups. one group received only amox (40 mg/kg), another received amox (40 mg/kg) plus cele (6.0 mg/kg) and the last received amox (40 mg/kg) plus dexa (0.3 mg/kg). one hour later the animals were sacrificed and the concentration of amoxicillin in the serum and tissue investigated. there was no difference among the groups in the quantity absorbed (amox0/18.879/2.05 mg/ml; amox'/cele0/18.209/1.89 mg/ml and amox'/dexa0/18.139/2.28 mg/ml). there was a reduction in the tissue concentration of amoxicillin (p b/ 0.01 tukey-kramer) for the group that received the drug with dexamethasone. for the other groups, there was no difference in the tissue concentration of amoxicillin. the results indicated that in inflamed tissue, a significant reduction of antibiotic penetration was induced by sinultaneous dexamethasone therapy. prediction of the optimal amoxicillin dose regimen based on coupling of pharmacokinetic data and bactericidal activity pm209 background: given its short half-life, amoxicillin (amx) should be administered at least three times a day to patients with acute exacerbations of chronic bronchitis, in order to achieve serum concentrations well above the mic of the responsible pathogen. however, several authors have recommended twice-daily administration of a higher dose for a shorter period. we assessed the relationship between amx sputum concentrations and antibacterial activity following two treatment schedules in healthy volunteers. subjects and methods: twelve healthy volunteers were randomized to receive amx for 4 days at a dose of either 1 g bd or 500 mg bd. serum and sputum were collected every day, 3 h after the morning administration, and again 2 days after the last dose. amx concentrations were determined by hplc with fluorometric detection. sputum killing activity was determined against haemophilus influenzae , streptococcus pneumoniae and moraxella catarrhalis . results: mean serum concentrations measured 3 h after the morning administration were 1.5 (500 mg bd) and 1.95 mg/l (1 g bd), and were above the mics of the three microrganisms. in contrast, sputum concentrations were always below 0.5 mg/l. in terms of sputum killing activity, 1 g bd was more effective than 500 mg bd against s. pneumoniae and m. catarrhalis , whereas no sputum samples were active on h. influenzae . conclusion: the optimal amoxicillin treatment schedule cannot be established on the basis of serum pharmacokinetics only. galmiche h, louchahi k, tod m, drugeon h, giroud jp, rouveix b. service de pharmacologie clinique, hopital cochin, paris, crepit 93, hopital avicenne, bobigny, service de microbiologie, hopital laennec, nantes, france background: cysteine-based mucolytics are commonly used in combination with antibiotics to treat patients with acute exacerbations of chronic bronchitis (aecb). they are also used to allow in vitro mic determination in sputum specimens. we conducted an in vitro and ex-vivo compatibility study designed to detect a possible interaction between mucolytics and antibiotics. methods: serial samples of bronchial secretions were collected from aecb patients and from healthy volunteers who received 1 g of amoxicillin twice a day for 4 days. two mucolytics were used to fluidify sputum specimens: 2,3-dihydroxy-1,4-dithiolbutan (digest-eur † ) and acetylcysteine (10% solution). amoxicillin was assayed using a chromatographic system with fluorometric detection. each sample was also tested in a microbiological assay. results: amoxicillin could not be detected in the presence of the mucolytic agents. conclusions: this mucolytic á/amoxicillin interaction may be explained by amoxicillin fixation to fluidified mucoproteins, and should be taken into account when assessing antibiotic efficacy in vivo. del fiol fs a , ferro c b , albuquerques et b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil physicians frequently recommend that macrolides should be administered with milk to decrease the discomfort they cause. thus the objective of this study was to verify the interference of milk in the absorption and distribution of erythromycin (eryt); clarithromycin (clar); roxithromycin (roxi) and azithromycin (azit). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs eryt, clar, roxi and azit with and without milk (3.5 ml/kg [ca'/'/]0/1.1 mg/ml). the animals were sacrificed and the serum and tissue concentration of the drugs was investigated. there was no reduction (pb/ 0.05 tukey-kramer) in the serum and tissue concentration in the presence of milk for azit and clar. there was a 27% reduction for roxi in the serum concentration in the presence of milk (11.84 9/1.35 and 8.639/1.34 mg/ml), but no alteration in the tissue concentration. there was a 33% reduction for eryt (p b/ 0.05), in the serum concentration in the presence of milk (10.209/ 1.06 and 6.839/0.88 mg/ml) and a 40% reduction in the tissue concentration. the milk decreased the effectiveness of treatments with erythromycin and roxithromycin and the bioavailabilities of this macrolides were affected by co-administration with milk. del fiol fs a , souza gp b , duzzi mr b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil the degree to which tetracyclines are absorbed differs greatly. this absorption is impaired by the concurrent ingestion of divalent and trivalent cations. thus the objective of this study was to investigate the interference of milk in the absorption and distribution of tetracycline (tetr), oxytetracycline (oxyt), minocycline (mino) and doxycycline (doxy). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their back for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs: tetr; oxyt; mino; and doxy with and without milk (3.5 ml/kg [ca'/'/]0/1.1 mg/ml). the animals were sacrificed and the drug concentrations in the serum and tissue were determined. there was no reduction (pb/ 0.05 tukey-kramer) in the serum and tissue concentrations in the presence of milk for mino. there was a 25% reduction (p b/ 0.05) for doxy in the serum concentration in the presence of milk (11.249/0.61 and 8.409/0.41 mg/ml) and 32% in the tissue concentration. for oxyt, there was a reduction of 34% (pb/ 0.05) in the serum concentration in the presence of milk (14.579/1.51 and 9.549/1.51 mg/ml) and 16% in the tissue concentration. the tetr results show a 35.4% reduction (p b/ 0.05) in the serum concentration in the presence of milk (13.469/3.39 and 8.699/3.03 mg/ml) and 40% in the tissue concentration. milk decreased tetracycline bioavailability and effectiveness. isotopic studies with oxine labelled platelet. platelet kinetics in thrombocytopenic malaria patients pm213 introduction : thrombocytopenia is a common feature in human malaria (1). excessive splenic platelet pooling has been suggested to play a role in uncomplicated cases of malaria, but a moderately shortened platelet life span during the period with decreasing parasitaemia seems the most plausible cause of the frequently observed thrombocytopenia (2, 3). consumption coagulopathy, eventually manifested as disseminated intravascular coagulation, has been described in malaria (4). in uncomplicated malaria, however disseminated intravascular coagulation is rarely found (3). results: in malaria patients the sequestration was not different to normal. platelet half-life was reduced in patients with p. falciparum malaria to 10 á/17 h (normal â/8 days). in one patient with p. vivax malaria platelet half live was 59.5 h. conclusion: no significant differences in the sequestration of platelets when compared to healthy individuals could be detected by 111 in-labelled platelet scintigraphy. especially, no enhanced splenic sequestration, as previously expected, was the cause of the thrombocytopenia. therefore, other mechanisms than sequestration are responsible for the dramatically reduced life span of the platelets during acute malaria. zaharanka ag, rozhdestvensky da. vitebsk state medical university, vitebsk, belarus aim: the studying of chromatingeterogenic test (ct) results in sperm of subjects taking doxycycline (d) and some macrolides (erythromycine (e), jozamycine (j), and azytromycine (a)) in moderate therapeutic doses. methods: forty healthy volunteers (20 á/23 years) were studied. daily dose of d was 0.2; e was administered in dose 0.25 four times per day 10 days; j */0.5 before meals twice daily 10 days; a */0.25 before meals once daily 5 days. ct for evaluation of dna condition in human spermatozoids was performed before treatment (twice), on the 5th and 10th days of treatment, as well as after 1 and 2 months after treatment course completing. results: ct data analysis revealed that the mean amount of defective spermato-zoids before treatment was 18.5'/3.5%. by the 5th day of d treatment the index of de-natured dna was 63.4'/6.5% (pb/ 0.001), by the 10th day */80.4'/7.2% (pb/ 0.001). one and 2 months after the d treatment course the amount of generative cells with denatured dna was 54.5'/4.6 and 42.5'/3.8%, respectively (p b/ 0.001 in both case). under e treatment the amount of defective spermatozoids changed as 38.2'/4.2 (5th day), 42.4'/2.7 (10th day), 35.6'/2.5 (after 1 month), and 30.5'/4.2% (after 2 months) (pb/ 0.05 in any case). under a using the ct results at the same control points were 39.5'/4.7, 46.5'/3.2, 40.2'/3.5, 25.6'/3.2% (pb/ 0.05 in any case); and under j treatment */18.2'/2.5, 20.3'/2.5, 17.2'/3.2, 15.2'/ 4.2%, respectively (p !/0.05 in any case). conclusions: the data obtained permit to conclude that d demonstrates the high level of toxicity to male generative cells. this effect preserves during 2 months after the course of d treatment. objective: to study the effect of aggressive isolation and decontamination measures to control an outbreak of multi-resistant acinetobacter baumanii (mr-ab) in an icu. the outbreak: the index case was transferred from a mediterranean hospital, directly into an open-plan 10-bedded icu, with severe injuries to his head and thorax. he died shortly after admission. sputum, bronchoalveolar lavage fluid, blood cultures and a chest drain swab grew mr-ab, resistant to ampicillin, co-amoxiclav, aztreonam, amikacin, ceftazidime, cefotaxime, cefuroxime, ciprofloxacin, gentamicin, meropenem, piptazobactam, tobramycin and sensitive only to colistin. within 10 days, mr-ab was isolated from two further icu patients. all isolates demonstrated identical antimicrobial susceptibility profiles. the icu was closed to admissions and thoroughly cleaned. all patients were isolated and their contacts screened. the icu was reopened, however, mr-ab was isolated from a fourth patient. this patient was isolated, the icu closed, for a second time, thoroughly cleaned, and all contacts isolated until discharge. all subsequent patients screened were negative for mr-ab conclusion: this illustrates the importance of aggressive isolation measures and thorough supervised cleaning in control of an outbreak, and the need to screen patients for resistant bacteria before admission to the intensive care unit in a general hospital. extended spectrum beta-lactamase-positive bacteria isolated in neonatal intensive care unit pm216 sandorcinova z a , siegfried l a , kmetova m a , viragova s b . a institute of medical microbiology, faculty of medicine, university of p.j. safarik, kosice, slovakia , b hospital, kosice-saca, neonatal intensive care unit, kosice, slovakia extended-spectrum beta-lactamases hydrolyse all penicilins, cephalosporins, including third-generation cephalosporins and aztreonam. esbl are predominantly produced by klebsiella spp. but may be presented in other enterobacteriaceae, too. the aim of present study was to investigate the occurrence of esbl-producing bacteria isolated from patients hospitalized at the neonatal intensive care units (icu). fifty escherichia coli and 35 klebsiella spp. were isolated from rectum of patients hospitalized at the neonatal icu. the mics of antimicrobial agents were determined by the standard agar plate dilution method according to the nccls guidelines. for screening of esbl production we investigated strains showing reduced susceptibility (mic equal and/or more than 2 ml/l) to at least one of third generation cephalosporins. esbl production was detected by double disk synergy test (ddst), e -test for esbl, and pcr employing specific primers for the presence of blashv and blatem genes. by using ddst and e -test, among e. coli isolates, an expression of esbl was detected in the three by the former method, while among klebsiella spp. isolates, a production of esbl was found in the two by the latter method. in esbl-positive e. coli strains the presence of blatem genes fragments was detected while in esbl-positive klebsiella spp. genes encoding for shv-type beta-lactamases were found. isolation of staphylococci from wound swabs and their susceptibility to antibiotics pm217 markov mij, shopovski e, despotovski v, angelevski a, nikolovski b. military hospital, microbiology, skopje, the former yugoslav republic, macedonia purpose: to determine percent of staphylococci from wound swabs and to establish their susceptibility to antibiotics. material and method: the wound swabs have been evaluated with standards microbiological techniques. bacteria have been identified with strips from the 'atb expression' system. the susceptibility testing has been performed with strips with dilution technique, read by the same system. results: during the last 5 years (01 01 1997 á/25 02 2002), a total of 1978 wound swabs have been evaluated in the military hospital in skopje. positive bacterial finding have been determined in 1345 (68%) swabs with 1575 isolated bacterial species, from which 587 (37.3) were staphylococci: staphylococcus aureus 460 (29.2%) isolations (173 methycillin resistant s. aureus ); s. epidermidis 79 (5.0%); s. haemolyticus 21 (1.3%); s. hominis 11 (0.7%); s. chromogenes and s. lugdunensis nine (0.6%); s. intermedius , s. lentus , s. sciuri and s. warneri all with seven (0.4%) isolations. the susceptibility of s. aureus was to: penicillin 2%, ampicillin 15%, amoksicillin/clavulonic acid 67%, ceftazidime 66%, gentamicin 29%, tetracycline's 28%, erythromycin 61%, lincomycin 44%, ciprofloksacin 95%, cotrimoxazole 82%, vancomycin 100%, fusidic acid 96%, cefixime 57%, and azitromicin 78%. conclusion: in our study most frequently isolated bacteria from the wound swabs were staphylococci, especially s. aureus . susceptibility, except for the penicillin (2%), was high to other antibiotics. the study went on from january 1999 to december 2000. at maribor teaching hospital, 2288 staphylococcus aureus isolates were collected. in 1999, 114 (10%) and in 2000, 73 (6.4%) were mrsa. mrsa were recovered from routine clinical material and from surveillance swabs (nose, throat, skin). for isolation, conventional culture media were used and for surveillance swabs mrsa-screening plate (manitol salt agar with 6% oxacillin) and trypticase soy broth with 6.5% nacl were added. s. aureus was identified by catalase, dna-se and tube-coagulase test. antibiotic susceptibility was determined by the disk-diffusion method according to ncci guidelines. all 187 mrsa isolates were tested for sensitivity to the following antimicrobials: gentamicin, netilmicin, ciprofloxacin, erythromycin, chloramphenicol, tetracycline, cotrimoxazol, vancomycin and clindamycin. it was found that all mrsa isolates were sensitive to vancomycin and partially or totally resistant to the rest. there were no important differences between the years 1999 and 2000. our mrsa isolates were completely (100%) susceptible to vancomycin, but resistant to the other antimicrobials in use to some extent. although the monitoring of mrsa susceptibility to antimicrobials once a year did not show any important change in antimicrobial resistance, the periodical monitoring of mrsa susceptibility to antimicrobials and revaluation of current treatment regimens of mrsa infections is necessary. staphylococcus aureus strains with reduced susceptibility to vancomycin among clinical isolates in university hospital in warsaw pm219 the visa and especially h-visa are very difficult to be found in the routine laboratory. in our investigations we examined 1011 of s. aureus strains isolated and stored in our laboratory for several last years (1997 á/2001) . most strains were isolated in 2000, and some in 2001. for all staphylococcal strains mrsa as well as mssa the mics of vancomycin were performed by the standard dilution method. among strains isolated in the last year three strains were recognized as visa (mic values were 8 mg/l). the frequency of visa was 0.3%. in the aim of founding the h-visa strains the population analysis was used. for this analysis all strains growing on the concentration 4 mg/l of vancomycin from the inoculum 10 6 were chosen. it was 54 strains, but only 32 of them were recognized as h-visa. the frequency of h-visa among investigated strains was about 3%. most but not all of the h-visa and all visa strains were methicillin resistant. in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci isolated from patients of surgical intensive care unit pm220 kucukates e, karayel n, kansiz e. institute of cardiology, university of istanbul, istanbul, turkey objectives: oxacillin-resistant staphylococci have emerged as a major infection control problem in our hospital. the aim of this study was to evaluate the in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci. material and methods: this study was performed between january 2000 and december 2001, at university of istanbul, institute of cardiology. the antimicrobial susceptibilities of 179 staphylococci isolates for vancomycin, teicoplanin and oxacillin have been investigated by e -test according to nccls guidelines. results: fifty-five (30.73%) of 179 clinical isolates were staphylococcus aureus . one hundred and twenty-four (69.27%) of 179 clinical isolates were coagulase negative staphylococci (cns). none of 179 staphylococci isolates were resistant to vancomycin. but three of cns isolates were intermediate and six of cns isolates were resistant to teicoplanin. twenty-eight (50.9%) of 55 s. aureus were resistant to oxacillin. ninety (72.60%) of 124 cns isolates were also resistant to methicillin. conclusions: nosocomial staphylococcal infections, especially in intensive care units increase day by day. staphylococcal infections are a major problem in many hospitals. according to our experiences the rate of oxacillin resistant staphylococci isolates in our hospital has also increased. methicillin-resistant staphylococcus aureus (mrsa) is a clinically significant pathogen because mrsa is resistant to many kinds of antibiotics and causes nosocomial infections around the world. the antiseptics are used for prevention of infections by mrsa. antisepticresistant mrsa strains have been isolated from clinical specimens. antiseptic resistance genes confer resistance to many kinds of drugs structurally and the resistance mechanism is the energy-dependent drug efflux system. in addition, the fluoroquinolone (fq)-resistance gene, nora , confers also resistance to many kinds of antiseptics. we studied the relation of the susceptibility to antiseptics and fqs of mrsa strains isolated in japan. a total of 420 strains of mrsa were isolated from 14 hospitals in japan from 1998 to 1999. acriflavin (af), acrinol, benzethonium chloride, benzalkonium chloride and chlorhexidine digluconate were used as the antiseptics. norfloxacin and sparfloxacin were also used in this experiment. the mic was determined by agar double-dilution method as recommended by the nccls. about 65% of mrsa showed resistance to af (mic: !/64 ug/ml). no strain was resistant to a specific antiseptic. fq-susceptible strains were susceptible to all antiseptics. this study showed that antiseptic-resistant mrsa are widely spread at hospitals in japan. the drug of choice in treatment of serious infections caused by mrsa was still vancomycin, however sometimes failures were observed, especially in monotherapy. some conflicting are present in literature about an effect of combined action of vancomycin and betalactams. in the present work, the common effect of vancomycin and methicillin against chosen staphylococcus aureus strains was examined. the investigated strains were characterized as visa, h-visa and clones obtained from h-visa in population analysis. two methods were performed: e -tests with methicillin and vancomycin placed on the media supplemented with the second antibiotic and the chessboard micro-analysis with increasing concentrations of both antibiotics. the fic indexes were calculated for different combinations of concentrations. on the basis of the fic indexes it was shown that the simultaneous action of vancomycin and methicillin was synergistic in all examined strains visa and h-visa, but only in appropriate concentrations. in different combinations the observed effect was addition or indifference. antagonism was never observed. the synergistic effect was not observed in the case of standard s. aureus strain sensitive to methicillin. supplementation of media with 4% of nacl substantially decreased the observed effect. incidence of antibiotic resistance in staphylococcus aureus strains in hungary with special reference to mrsa pm224 ghidán á , maró di c, csukás z, kamotsay k, szabó d, ostorházi e, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary between january 1997 and december 2000, a total of 3109 staphylococcus aureus strains isolated from patients admitted to the clinics of the semmelweis university were examined for antibiotic sensitivity with the disc diffusion test. resistance to individual antimicrobials were as follows: penicillin 84%, oxacillin 25%, erythromycin 31%, ciprofloxacin 15%, amikacin 12%, netilmicin 8%, tobramycin 10%, gentamicin 20%, clindamycin 22%, mupirocin 4%, tetracyclines 34%, chloramphenicol 9%, teicoplanin 2% and vancomycin 0%. all mrsa were b-lactamase producer. they showed coresistance to erythromycin (53%), ciproflxacin (36%), amikacin (29%), netilmicin (23%) and mupirocin (10%). multiple resistant mrsa strains to mupirocin'/tetracyclines'/chloramphenicol amounted to 0.9%. triple resistance to oxacillin'/ciprofloxacin'/netilmicin was 14%. the detection of meca gene by pcr in randomly chosen mrsa qualified with 1 mg oxacillin disc resulted in only 15% meca positivity indicating that the traditional disc diffusion test overestimates the frequency of mrsa strains particularly in such an environment where the usage of penicillins and cephalosporins is so liberal as in hungary. consequently, the selective pressure for blactam-resistance and b-lactamase induction exists everywhere. this conclusion is coherent with the relatively low frequency of multiple resistant mrsa strains and urge the need of a routinely available genetic method to apply for mrsa detection. objectives: the main objectives of this study were to monitor antibiotic resistance, identify new/emerging resistance mechanisms at an early stage, prevent their dissemination, early detection and prevent the outbreaks. methods: our laboratory used antibiotic disc sensitivity testing methodology (nccls 1993) . zone sizes were measured objectively using a biomic automated radius zone reader. results: throughout 2 years (january 1999 till december 2000) we have surveyed 3190 organisms collected from outpatient departments (1145, 35.8%), radio-oncology department (992, 31.1%), medical department (765, 23.9%), obg department (217, 6.8%), surgical-oncology non icu department (207, 6.5%), icu department (175, 5.5%). from 1543 (100%) strains of enterobacteriaceae 68 (4.4%) were resistant to 4th generation fluoroquinolone and 44 (2.9%) strains were esbl positive. from 416 (100%) strains of staphylococcus aureus were only five (1.2%) strains resistant to methicilin (mrsa). we collected 370 (100%) strains of enterococci, whereabout only two (0.5%) were resistant to glycopeptides (vre). from 235 (100%) strains of pesudomonas aeruginosa , 97 (41.2%) were resistant to aminoglycosides. conclusions: national restrictive antibiotic policy hand in hand with local hospital antibiotic policy and regular rotation of antibiotics used in prevention and treatment on all departments is leading in our case in positive situation in antibiotic resistance in comparing with other slovakian and european centers. antimicrobial resistance of nosocomial strains of staphylococcus aureus pm226 dekhnich av, stratchounski ls, edelstain ia, narezkina ad. institute of antimicrobial chemotherapy, smolensk, russian federation purpose: to determine the antimicrobial resistance of staphylococcus aureus causing nosocomial infections in smolensk regional hospital. results: a total of 140 s. aureus strains isolated during 2000 á/2001 were studied. antimicrobials tested included oxacillin (oxa), erythromycin (ery), clindamycin (cli), gentamicin (gen), vancomycin (van), linezolid (lnz), tetracycline (tet), chloramphenicol (chl), rifampicin (rif), fusidic acid (fus), trimethoprim/sulfamethoxazole (ts), ciprofloxacin (cip), mupirocin (mup), quinupristin/dalfopristin (qd). susceptibility testing and its interpretation were performed by agar dilution according to nccls guidelines where applicable. results are presented in the conclusions: the most active antimicrobials were vancomycin, linezolid, quinupristin/dalfopristin, fusidic acid, mupirocin, followed by co-trimoxazole, rifampicin. beta-lactams, macrolides, lincosamydes, tetracyclines and chloramphenicol should not be used for the treatment of nosocomial s. aureus infections. we investigated all staphylococcal infections within 2 years among 246 neonates hospitalized for infection in the neonatal icu in a tertiary neonatal referral center. univariate analysis, to assess risk factors for neonates infected with staphylococcus aureus (121) vs. without s. aureus (125) was performed. from the total number of 246 cases, in 121 cases s. aureus was isolated from various samples; in 16 cases from blood cultures, in 36 cases from urine, in 18 cases from eye swabs and in 29 cases from gastric content (no significant differences in comparison with control group). colonization with s. aureus , was a predictor of infection: nasal swabs, throat swabs, ear swabs, skin swabs and umbilical swabs were significantly more commonly observed in neonates infected with s. aureus , than with other infections. etiological analysis showed that co-pathogens escherichia coli and viridans streptococci were significantly more frequently associated with neonatal infection caused by s. aureus , in comparison to other organisms. according to localization of infection site, conjunctivitis and thrush stomatitis was the commonest s. aureus neonatal infections. outcome was similar to other infections and without any significant differences between both groups. mortality was similar to other infections, probably because: initial therapy in our centre contains an antistaphylococcaly active agent (cefuroxime or cefotaxime plus aminoglycosides). morozova ot, semina na. laboratory of hospital infections, central research institute of epidemiology, moscow, russian federation purpose is to study the role of enterococcus spp. in the aetiology of nosocomial infections among the patients of the childrens clinical hospital and susceptibility of these strains to antibiotics. methods: the strains of enterococci were isolated from 66 patients with hospital infections in 2000 á/2001. results: the aetiological structure of enterococcus -infections showed the predominance of skin and soft tissue infections (39.4%), urinary tract infections (33.3%), bloodstream infection (10.6%), pneumoniae (4.5%), infection of central nervous system, gastrointestinal tract, eye, surgical wound infections were of rare incidence (3 á/ 1.5%). various nosological forms of infections were caused more often by e. faecalis than e. faecium (72.7, 27.3%). the antibiotic resistance to ampicillin and other beta-lactams occured in 100% of e. faecium isolates, but all strains of e. faecalis were susceptible to these drugs. high-level gentamicin resistance demonstrated e. faecalis isolates */ 29.0%, e. faecium */100%; and high-level streptomycin resistance showed e. faecalis */67.7%, e. faecium */88.8%. all the e. faecalis were active against fluoroquinolones, but e. faecium were resistant in 22.0%. there were no vancomycin resistant enterococci. conclusion: e. faecalis predominated in the aetiological structure of nosocomial infections due to enterococcus spp. antibiotic resistance patterns for two species of enterococci were different, all the strans were susceptible to vancomycin. evaluation of antimicrobial resistance of enterococcus spp. experience of 6 years (1996 á/2001) pm229 lopez-barba j, jesus de la calle i, solino-ocañ a i, rodríguez-iglesias m, perez-ramos s. microbiology laboratory, puerto real university hospital, cádiz, spain objective: determination of quantitative changes in antimicrobial resistance of enterococcus isolated from clinically significant not urinary samples of patients remitted to the laboratory of microbiology during a 6 years period (1996 á/2001) . material: the period of the study was comprised between 1996 and 2001. the samples has been processed for the isolation of enterococcus following conventional methods. were isolated 3424 enterococci strains. the identification and susceptibility to antibiotics have been performed in automated system microscan(c) dade-behring(c) through panels combo cgp. the data were processed by the statistical system statgraphics plus 4.1. results: of the 3424 enterococcus , have been identified 3298 e. faecalis and 126 e. faecium . the resistance is shown in table 1 . conclusions: the resistance to va and tei of e. faecalis remains through last 4 years (2 á/3%). the high resistance to erythromycin and tetracilin ( !/70%) and the resistance (30 á/31%) to quinolones, antibiotics all of them used in community-acquired infections justify the susceptibility testing to the clinical strains isolated of this group of microorganism. in e. faecium the antimicrobial resistances was high and increasingly to imipenem, meropenem, erythromycin and quinolones. characteristics of strains e. faecium colonizing the neutropenic patients pm230 abbassi ms, achour w, gréco a, ben hassen a. laboratory of bone marrow transplant center, tunis, tunisia digestive colonization by enterococcus faecium in the neutropenic patients under gut decontamination is important. seventeen multiresistant strains of e. faecium isolated from stools of seven neutropenic patients were the target of an epidemiological analysis through the determination of the mics of amoxicillin, gentamicin, vancomycin, the transferability gentamicin resistance to the recipient strain e. faecalis jh2-2 by filter-mating assay, analysis of plasmid profiles of e. faecium -strain and of transconjugants and the amplification by pcr of the gene aac(6?)-aph(2??) coding for the bifunctional enzyme by using primer m13771 who gives a fragment of 174 kb. among the seventeen strains, eleven had the same antibiotype a1, 12 had a gentamicin mic!/4096 mg/l. the mic90 of the amoxicillin was of 128 mg/l. all the strains were sensitive to vancomycin. ten strains harbored a plasmid of 70 kb transfered at a frequency of 6.10 á/5, also found in gentamicin-resistant transconjugants. however, strains belong to nine distinguished plasmids profiles. all high-level gentamicin resistant-strains had a positive pcr amplification of the aac (6?)-aph (2ƒ) gene. the features of the studied strains establish their endogen origin, specific for every patient, sharing only high-level resistance to gentamicin. gut decontamination treatment with gentamicin enhance either the spread and the preservation of easy-transferable plasmid carrying genetic transposable element. frequency and antibiotic resistance of bacteria isolated from patients suffering infectious complications following the implantation of prosthetic devices pm231 kristó f k, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary for patients with indwelling joint prosthesis, early recognition and prompt therapy for infection in any location may be critical to reduce the risk of seeding the joint implant heamatogenously. a year period (2001) a total of 2440 swabs of aspiration from patients with infectious complications following the implantation of prosthetic devices were cultured. cultivation and identification of the strains were performed by conventional methods and by vitek system (biomȇrieux) and susceptibility testing by disc diffusion. potentional pathogens were recovered in 324 cases (13.27%). gram positive cocci, in particular staphylococcus spp. proved to be the most commonly isolated bacteria. coagulase-negative staphylococcus (cns) was isolated more frequently (48%), followed by s. aureus (28%), enterococcus faecalis and e. faecium (11%), gram-negatives (3%), anaerob isolates (7.5%). resistance to individual antimicrobials of s. aureus and cns were as follows: methicillin 8 and 47%, clindamycin 4.5 and 21.7%, fluoroquinolones 0 and 18%. mupirocin resistant strains of s. aureus were not found, while 7.7% were among the cns strains. our results could be essential for the rational selection of treatment at our orthopedic wards. results: in the analysed period the percentage of isolated enterococcus sp. strains among all non-repetitive clinical isolates in 1999, 2000 and 2001 was 3.9, 3.1 and 51%, respectively. cultured strains were identified as e. faecalis, e. faecium, e. gallinarum and e. avium . the most prevalent was e. faecium strains, isolated with a frequency of 75, 62 and 75%, respectively. vancomycin-resistant strains were all identified as e. faecium and in 2001 they comprised 17% of all isolates of this species. conclusions: (1) the frequency of isolation of enterococcus sp. in blood cultures of haematological patients remained relatively stable in 1999 á/2001. (2) the predominant enterococcal species isolated from these patients was e. faecium . (3) in 2001 we recorded for the first time an emergence of vancomycin-resistant e. faecium , which comprised 17% of all isolates of this species. kalai s, ben hassen a, achour w, greco a. laboratory of microbiology, national bone marrow transplantation center, tunis, tunisia from april 1998 to june 2000, 47 non-repeated strains of pseudomonas aeruginosa were isolated from 42 immunocompromised patients. thirty-six percent of strains were isolated from abscess, 27% from blood culture and 11% from urine. susceptibility to antibiotic was studied by the routine disk diffusion method (ca-sfm). mics were determined using agar dilution to 11 antibiotics (5b-lactams, four aminosides and two fluoroquinolones). serotyping of the different trains was performed using antisera to the international antigenic typing systems serotypes. the study showed 80% of resistance to c cochin port royal hospital, service de gynecologie-obstetrique site st vincent-de-paul, paris, france , d cochin port royal hospital, cclin, paris, france aims: to determinate risk markers of an outbreak of postpartum endometritis due to group a streptococcus . design: a case-control study using data collected with a structured form. setting: the cases of postpartum endometritis were diagnosed in the department of obstetric of the paris hospital network during 5 days (december 1999 á/january 2000). the group of controls consisted of women delivered in the same department during the same period. participants: cases (n0/3) and controls (n0/59). findings: cases had smoked more often during pregnancy (67 vs. 16%; p 0/0.089) and received more often immunosuppressive treatment than controls (33 vs. 0%; p 0/0.060). instrumental delivery has been needed more often for cases than controls (67 vs. 13%; p 0/0.062). cases had been hospitalized after delivery in a ward z of the department more often than controls (100 vs. 23%; p 0/0.017). they had been examined after delivery more often by a midwife x (100 vs. 18%; p0/0.009) and a nurse y has provided care to cases and not to controls (33 vs. 0%; p 0/0.051). conclusion: smoking, receiving immunosuppressive treatment during pregnancy, and instrumental delivery were significantly associated with postpartum endometritis (pb/ 10%). a midwife and a nurse might be involved in the transmission of the infection. petoukhova i a , sokolova e a , dmitrieva n a , nummaev b b . a laboratory microbiology, cancer research center of russia, moscow, russian federation , b department of oncogynecology, cancer research centre, moscow, russian federation the aim of the study was to assess efficacy of perioperative ap. total 241 pts were included. two hundred and one pts with cervical cancer (cc) undergone extensive hysterectomy, 22 pts with cancer of vulva (cv)-extensive vulvoectomy, 18 pts with ovarian cancer (oc) á/ extensive/combined operations. fifty-one pts (group 1) received ap with amoxicillin/clavulanate (am/cl) 1.8 g iv 30 min prior to operation, then 1.2 g iv thrice per day for 3 á/5 days. fifty pts (group 2) received cefotaxime (ctx) 2 g iv 30 min prior to operation, then 1 g four times per day for 3 á/5 days'/metronidazole (mtz) 500 mg two times per day for the same period. one hundred and forty pts were retrospective control (they received ii á/iii generation cephalosporins or linkosamides only after operation). the rate of swi in pts with cc, cv and oc was 13.5, 73, 29%, respectively; dwi */16.2, 0 and 21%, respectively; uti */72, 80, 42.8%, respectively. the ap with am/cl was more effective compared to ctx'/mtz (swi */6 vs 18%, respectively, pb/ 0.05, dwi */9.8 vs 14%, respectively, p b/ 0.05). the rate of postoperative uti was equivalent in two groups (62 vs 74%, p 0/n.s.). thus, ap with am/cl is preferrable option in extensive operations in og pts. fifty-eight patients were randomized into two groups. group 1, a treatment consisting of 31 patients and group 2, consisting of 27 patients as a control. the patients were at the age of 679/1.2 and had had different surgical interventions with general anaesthesia from 1 to 3 h and accompanying copd (89%). artificial pulmonary ventilation was used in 45 cases. in the early post-surgery period the patients of group 1 were administered inhalation therapy, including ipratopium of bromide in the combination with fenoterol (atrovent, berodual) and ambrocsol (lazolvan) through a nebulizer. the inhalation therapy was not administered to the patients of group 2. under the influence of the inhalation therapy pulmonary ventilation and respiratory metabolism was restored faster in all the resuscitation patients (in group 1 */by the end of the first 24 h, in group 2 */on the 3rd á/4th day). the percent rate of pef1 was 76.7'/4.1 and 56.4'/4.5, respectively. artificial pulmonary ventilation ended in 9.2 and 7.3 h, respectively. the time of the patients' stay in the resuscitation department was 3.2 days in group 1 and 4.6 days in group 2. by the end of the 1st week pneumonia developed in one patient from group 1 and in eight patients in group 2. aerosol therapy application accelerates medication delivery to the respiratory tracts, increases the local activity and effects good prophylaxis for surgical hospital pneumonia. variation in ethiology of early and late onset ventilator associated pneumonia pm250 nikolopoulos j, daganou m, michailidou m, karabela e, kavada k, retsou s, antoniadou a, rasidakis a. department of respiratory abstracts s100 failure and icu, sotiria general and chest disease hospital, athens, greece purpose: to compare the distribution of causative microorganisms, their susceptibility to antibiotics and outcome of 'early' and 'late' vap in a greek icu. methods and results: retrospective study of mechanical ventilated (mv) patients (pts) with early and late vaps during a 6-month period. diagnosis of vap was made by clinical, radiographic criteria and quantitative cultures of bronchial secretions. vap was diagnosed in 17 pts (26%) out of 67 consecutive admissions in icu. all pts before the development of vap received antibiotics. three episodes of vap (17.6%) were developed before the 7th day of mv (early vap) and were caused: (1) by multi-resistant acinetobacter; and (2) by antibiotic-susceptible pseudomonas aeroginosa . in this group one pt died from septic shock related to vap and two pts survived. fourteen pts (82.4%) developed vap after the 7th day of mv (late vap). five cases were caused by multi-resistant p. aeroginosa , two cases by mrsa, two cases by multi-resistant acinetobacter, two cases by susceptible to antibiotics klebsiella pneumoniae , and three were polymicrobial and caused by multi-resistant microorganisms (mrsa and gnb). four pts died (28.6%) from septic shock related to vap, five pts (36%) died because of another cause and five pts (36%) survived. conclusions: early and late onset episodes of vap were caused by 'potentially drug-resistant bacteria'. p. aeroginosa as a cause of early vap was susceptible. mortality attributed to early and late vap was similar. antibiotic prophylaxis in oncological and major reconstructive orthopaedic surgery pm251 de biase p, ciampalini l, astone a, capanna r. azienda ospedaliera careggi, oncological and reconstructive centre, aoc, florence, italy during last year patients scheduled for oncological surgery or major reconstructive procedures were randomised to either vancomycin or teicoplanin prophylaxis. prophylaxis was performed with either vancomycin 1 g i.v. twice daily or teicoplanin once daily 400 i.v. two hundred patients were included. four patients did not agree the study protocol and were excluded. we treated 98 patients with teicoplanin and 98 patients with vancomycin. out of the 196 patients 117 were operated for oncological disease, while the remaining 79 underwent major orthopaedic procedures. we experienced 10 cases of red man syndrome, and five cases of moderate hypotension. five patients had postoperative complications: two deep venous thrombosis, one pulmonary embolism, two postoperative haematoma. in five patients we observed a wound dehiscence; two of these patients showed clinical sign of ssi and microbiological examinations were positive for mrsa. one patient recovered from infection with medical therapy, while the other patient showed a local tumour recurrence and was amputated at the thigh. at last surgery infection was still present clinical and at microbiological examination. in conclusion we had an infection rate of 1.02% which is comparable to the infection rate of a 'clean' surgery in patients with normal risk of infection. teicoplanin showed lower toxicity, has a longer half-life and has a simpler way of infusion and it is our current choice in high risk surgery. injuries with contaminated sharp articles in health care workers in general hospital celje, slovenia pm252 lesnicar g, sibanc b. department of infectious diseases, medical center celje, celje, slovenia in a prospective study carried out from january 1997 till june 2001, we registered 133 subcutaneous injuries with sharp objects, mostly in nurses and cleaning service workers. in 19% of cases the incident occurred outside the hospital, in persons who were not medical workers. in 69 cases the injury causing object was a needle that had been used in known patients, 14 of which were hepatitis b positive. fifty-five (48.2%) of the injured health workers had been previously vaccinated against hepatitis b; the protective antibodies to hepatitis b in the blood were found in 35/114 (30.7%) health workers only, while the tests for antibodies to hepatitis c and hiv were negative in all cases. following the incident, the majority of the injured persons, i.e. 102, were vaccinated against hepatitis b, while 46 persons (34.6%) also received passive prophylaxis with human immunoglobulin against hepatitis b. none of the injured persons have developed the disease or showed evidence of sero-conversion. in the year 2000 the general as well as specific preventive measures practised in our hospital became more rigorous. thus, approximately 80% of our health workers at risk have already been vaccinated against hepatitis b. to improve measures preventing dissemination of multidrug-resistant bacteria (mrb), a cross-sectional survey (2000) was conducted to analyse healthcare workers' (hcws) isolation precaution knowledge for mrb infection at 21 investigation and outpatient departments excluding four declaring not to be involved in care to mrb patients (emergency, obstetrics, nuclear medicine, and bacteriology). two hundred and eight hcws answered (67% of the paramedical staff, 28% of the physicians). thirty-three percent of them reported they do not know frequently or always the patient mrb status. they (82%) wish mrb status to be mentioned on the test form or on the advice request letter. mrb patient visit or test was appropriately timed in 62% answers. gowns (61%) or masks (58%) use were not systematically reported. other hcws (74%) reported better isolation precaution knowledge than physicians (58%) and nurses (81%) than investigation assistants (58%). physicians declared lower compliance with use of gowns, gloves or draw-sheets than other hcws. they had also lower education in isolation precautions and were less interested in education programs. this study suggests the necessity to improve mbr infection information. physicians and investigation assistants seem to be insufficiently aware of hospital infection control. therefore, education strategies targeted at physicians and investigation assistants working at outpatient and investigation departments should be developed. outbreak of clostridium difficile -associated diarrhoea in infectious disease department: risk factors and hygiene measures assessment pm254 henoun loukili n, martin m, remy v, hansmann y, christmann d. hôpitaux universitaires de strasbourg, maladies infectieuses et tropicales, strasbourg, france (shock)'/4* (bedridden status)'/4* (age !/65 years)'/3* (previous antibiotic treatment) and points (women)0/6* (shock)'/4* (bedridden status)'/4* (age !/65 years)'/3* (immunosuppression). the vast majority of patients (89 and 87% of males and females, respectively) could be classified in the subgroups with lower scores (six points or less) which had a very limited risk of death (0 á/2.5 and 0 á/2.3% for men and women, respectively), whereas for patients in the highest score subgroup (11 points or more), the risk was 100% for men and 91% for women. conclusion: risk stratification of patients with ap is possible from simple clinical variables available on admission. procalcitonin (pct) and c-reactive protein (crp) for differentiation of systemic inflammatory response syndrome (sirs), sepsis and severe sepsis pm259 lupse m a , ursu l b , slavcovici a a , carstina d a . a clinical department, teaching hospital of infectious diseases, cluj-napoca, romania , b laboratory department, teaching hospital of infectious diseases, cluj-napoca, romania objectives: to evaluate the value of pct in the differentiation of patients with sirs, sepsis, severe sepsis and bacteremia in comparison to crp. design: prospective study including patients who meet criteria for sirs, sepsis or severe sepsis (consensus conference of the accp/ sccm) admitted over 18-month period. patients and method: a total of 67 patients were included: eight with sirs, 35 with sepsis and 24 with severe sepsis. sixteen from 59 patients had bacteremia. pct and crp were evaluated in the first 24 h after admission: pct by brahms pct-q test and crp by turbidimetric assay. the sensitivity, specificity, predictive value of different cutoff points for crp and pct were determined. results: with a cut off point of 0.5 ng/ml for pct and 0.6 mg/dl for crp sensitivity and specificity for sepsis were 73%, respectively 75% (ppv 95.6, npv 27.3) and 96%, respectively 17% (ppv 87.2, npv 75). a cutoff point of 2 ng/ml for pct accurately predict severe sepsis (sensitivity 80%, specificity 100%, ppv 100). a pct level of at least 10 ng/ml was a good predictor for bacteremia (sensitivity 86%, specificity 90%, ppv 73.7 and npv 95). conclusion: pct is a good discriminating marker to characterize the level of inflammation caused by infection and can predict bacteremia . giamarellou h a , aoun a b , klastersky j b , anagnostopoulos n c , galani l c , grecka p c , panaretou e c , papageorgiou e c , repoussis p c , syrseloudis pct has been considered as a useful diagnostic marker in neutropenic patients with bacteremia and/or severe sepsis (giamarellos-bourboulis ej et al. clin infect dis 2001; 32:1718) . in an attempt to define its value in the diagnosis of localized infections in neutropenic hosts, daily determinations of pct and of c-reactive protein (crp) were performed before and after the onset of fever in 50 subjects 27 male and 22 female aged 48.69/18.7 years with various haematologic malignancies (aml 26, nhl 17, mds 5, all 2) developing neutropenia ( b/500 pmns/mm 3 ) after chemotherapy. thirty-three patients were presented with fever of unknown origin (fuo) and 17 with localized bacterial infections (lbi; pneumonia 7, acute pyelonephritis 4, soft tissue infections 3, acute pharyngitis 3). pct was determined by an immunoluminometric assay and crp by nephelometry. it is concluded that febrile neutropenia followed by a localized bacterial infections is accompanied by significantly higher levels of pct than in case of fuo (47.99/3.64 vs 0.519/0.16 ng/ml). similar differences are not observed with crp, which lacks the appropriate specificity. our study included 35 patients who were categorized as having proven (9), probable (11), or possible (5) systemic fungosis according to eortc criteria; 10 showed no sign of infection, and were used as controls. blood samples were received on the 1st, 3rd, and 5th day from the onset of signs of a fungal infection, and then twice a week. pct levels were determined by an immunochemioluminent assay, and candida and aspergillus antigen levels by elisa. in only five patients pct indicated early signs of infection, albeit at barely detectable limits. six patients, however, showed significantly increasing titres preceding time of death. positive antigens titres were observed only in 10 patients who had proven or probable systemic fungosis. only half of the control group had negative antigen titres; a high rate of false negatives was also observed. both pct and antigens titres increased in parallel in 4/6 patients with unfavorable outcome. pct and antigens titres cannot reliably indicate early diagnosis of systemic fungal infections although may be used as a prognostic tool of severity. lactulose, a factor that decreases endotoxaemia, in obstructive jaundice? pm262 koutelidakis im a , papaziogas v a , makris i a , giamarellos-bourboulis ej b , giamarellou h b , papaziogas t a . a thessaloniki med school, 2nd surgical clinic, thessaloniki, greece , b 4th department internal medicine, athens medical school, athens, greece bacterial translocation is a process implicated in the pathogenesis of spontaneous peritonitis. in order to evaluate the impact of lactulose administration on systemic endotoxaemia, obstructive jaundice was induced in 11 rabbits by common bile duct ligation. animals were divided into two groups, group a of five rabbits not receiving lactulose and group b of six rabbits, which received 1.5 ml/kg of lactulose orally by an oral catheter. blood was collected daily, before and after operation for a total duration of four days. samples were applied for culture and for determination of endotoxins (lps) by the lal qcl-1000 assay. concentrations of lps (mean9/sd) of group a were 1.159/0.74, 2.249/0.74, 1.609/0.81 and 2.349/0.70 eu/ml on the 1st, 2nd, 3rd and 4th day, respectively. respective concentrations of lps (mean9/sd) of group b were 0.849/0.28, 0.659/0.12, 0.819/0.20 and 0.609/0.42. all blood cultures were sterile in both groups. differences activity of linezolid against nosocomial strains of staphylococcus aureus in russia: results of multicentre study pm221 were included in the study. antimicrobial susceptibility testing was performed by agar dilution method in accordance with the nccls recommendations. all tested strains including 295 mrsa strains (33.6% of all strains) were found to be susceptible to linezolid with the mic ranged from 0.5 to 4 mg/l. both mic 50 and mic 90 were 2 mg/l. conclusions: linezolid had excellent in vitro activity that was not affected by resistance to other classes of antimicrobials susceptibility to antiseptics of mrsa isolated in japan during 1998 á 60% to piperacillin, 55% to ceftazidime, 71% to cefepime, 13% to imipenem, 51% to amikacin and 55% to ciprofloxacin. fiftythree percent of p. aeruginosa strains were multiresistant (25 strains) and were isolated in 12 patients. wild phenotype to b-lactams was observed in 30% of strains. the most frequent b-lactams resistance phenotypes were: cephalosporinase over production (34%) and penicillinase (11%). imipenem, ceftazidime and piperacillin-tazobactam were the most active b-lactams (mic 50 of 4.16 and 32 mg/l, respectively) these results showed high rates of antibiotic resistance and predominance of o11 serotype in multiresistant strains compared to the o12 serotype in europe. infectious complications sustained by stenotrophomonas (xanthomonas ) maltophilia in hiv at present, very limited informations are available about s. maltophilia infections in the setting of hiv disease. patients and methods: a retrospective survey of clinical and microbiological records of 1374 hiv-infected patients referring to out tertiary care centre between 1991 and 2000 was performed, in order to identify all episodes of s. maltophilia infections, and analyze its epidemiological, clinical, and microbiological variables. results: sixty-one episodes of s. maltophilia infection were observed in 59 patients: sepsis/bacteraemia in 48 cases (78.7%), lower airways infection in five, urinary tract infection in four, pharyngitis in two, lymphadenitis and liver abscess in one case each. forty-seven out of 61 episodes of s. maltophilia infections (77%) occurred as nosocomial disease, generally in association with advanced immunodeficiency, neutropenia, instrumentation, and prior antimicrobial therapy. bacterial isolates showed an elevated resistance profile against many betalactam compounds, aztreonam, imipenem, and aminoglycosides. conclusion: s. maltophilia represents an emerging opportunistic pathogen in hiv-infected patients extended spectrum beta-lactamases producing germs in intensive care units pm235 university of medicine and pharmacy 'victor babes ', microbiology, timisoara, romania injury and complications lasted 22 months, demanded for 11 surgical procedures and total cost was 1 comparison of different methods for detection of extended spectrum beta lactamases (esbls) and their genetic relatedness among enterobacteriaceae clinical isolates in a research medical institute pm242 methods: one hundred out of 178 isolates that were screened positive for esbls were tested with double disk synergy test (ddst), three dimensional test (tdt), e -test-esbl and vitek-esbl test. pulsed field gel electrophoresis (pfge) analysis was applied to 13 esbls; five klebsiella pneumoniae and eight escherichia coli . results: revealed the prevalence of esbls in 25.8% of clinical isolates. the sensitivities of the ddst, tdt, e -test and vitek were 100, 73, 86.9 and 47.8%, respectively. in the ddst, aztreonam was the most sensitive indicator (93.4%). pfge demonstrated that 80% of k. pneumoniae were derived from a single clone whereas 62.5% of e. coli isolates were derived from two different clones. non-clonal origin was demonstrated in 20% of k. pneumoniae and 37.5% of e. coli . conclusion: there is an increased prevalence of esbls. the ddst is the most sensitive, practical and cost effective diagnostic method reliable for routine use in our laboratory. both clonal spread and plasmid dissemination contributed to the concurrent nosocomial outbreaks caused by esbl-producing k severe nosocomial infections due to stenotrophomonas maltophilia pm243 the teaching hospital of infectious diseases total 241 og pts after extensive hysterectomy (201 pts), extensive vulvoectomy (22 pts) and extensive/combined operations for ovarian cancer (18 pts) were analysed. ic developed in 74 pts. one hundred and sixtyseven pts had no ic. twenty-eight of 50 rf analysed were independent rf of ic. most important included: age !/50 years (p0/0.0002), grade 2 á/3 obesity (p 0/0.0002), diabetes mellitus (p 0/ 0.0002), diagnosis of cervical cancer (p 0/0.0001), history of pre-cancer of vulva postpartum endometritis due to group a streptococcus : a case-control study pm247 unite operationnelle d 'hygiene all isolates were sensitive to vancomycin. among gram-negative bacteria klebsiella spp. was isolated in 21.6% of cases, acinetobacter baumanii in 13.5%, enterobacter spp. in 8.1%, providencia spp. in 2.7%. of the klebsiella spp. isolates 25% were resistant to amikacin, 85% to cephalosporins, 87% to piperacillin/ tazobactam. all were sensitive to imipenem. of the a. baumanii isolates 100% were resistant to amikacin, aztreonam, cefoperazone, cefotaxime, ceftriaxone, piperacillin; 60% to ampicillin-sulbactam, 80% to ceftazidime, and 40% sensitivity to imipenem antimicrobial activity of selected pharmacopoeial antiseptics analysed according to european standards pm270 european committee for standardisation approved several european standards (en), describing test methods establishing, whether an antiseptic has or does not have a bactericidal or fungicidal activity under the laboratory conditions defined by en. the aim of the study was to investigate, if some chemical compounds in concentrations recommended by polish pharmacopoeia for skin disinfection, comply european standards requirements. methods: basic bactericidal (en 1040) and fungicidal (en 1275) activity were investigated as well as bactericidal activity of products for hygienic and surgical handrub and hand wash used in human medicine (pren 12054). all methods and used neutralizers were validated. standard strains: staphylococcus aureus , pseudomonas aeruginosa , escherichia coli , e. hirae , candida albicans and a. niger were used, when en standards were evaluated. results: ethanol, izopropanol and n -propanol caused viable microbial count reduction required by ens in pharmacopoeial concentrations the purpose of the study: we collected 226 bacteriological samples from adult and neonate patients who were admitted in intensive care units (icu) . the aim was to observe the colonization status with microbes that may have a nosocomial potential and to establish circulating phenotypes in icus. the results obtained from a total of 226 samples 61 strains of gram negative bacteria (enterobacteriaceae family) were isolated. fourteen strains showed extended spectrum beta-lactamases (esbl) phenotype (eight strains of klebsiella pneumoniae , three of escherichia coli , two of klebsiella ornithynolitica , one of klebsiella oxytoca ). we used both disc diffusion test (extended antibiotic susceptibility test and synergy test to visualize 'champagne stopper' pattern) and mini api † system.the conclusion reached: we put in evidence a massive colonization with germs that may have a nosocomial potential especially microbes that produce esbl (22.9% from all enterobacteriaceae isolated) which implies a rational policy in prescribing antibiotics in hospitals from western part of romania.carbapenem activity against nosocomial gram-negative rods pm236sawicka-grzelak a a , rokosz a a , meszaros j b , luczak m a . a department of medical microbiology, university medical school, warsaw, poland , b department of general and transplantation surgery, university medical school, warsaw, poland purpose: to determine a susceptibility of nosocomial gramnegative rods to carbapenems.methods: two hundred strains of gram-negative rods were cultured from clinical specimens from hospitalized patients (july á/november 2001). identification of strains was performed in the automatic atb system (biomerieux, france). susceptibility of strains to carbapenems: imipenem and meropenem was determined with disc diffusion method according to nccls recommendations. esbl-producing strains were detected with double-disc synergy test (ddst according to jarlier et al., 1988) or a novel method of esbl detection (dd, diagnostic disc) according to appleton (1999) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd 01) (oxoid, england) .results: one hundred and ten strains of enteric rods and 90 strains of non-fermenting rods were cultured. twenty eight (14%) esblpositive strains were detected. carbapenems were active against 96% of enteric rods. the percentage of non-fermenting rods susceptible to imipenem was 70 and to meropenem */60.conclusions: carbapenems: imipenem and meropenem demonstrated high activity against clinical strains of enteric rods. however, the antibiotics were less active against nosocomial strains of nonfermenting rods.inhaled antibiotics against multiresistant bacteria in bronchial secretions of icu patients: a preliminary report pm237horianopoulou m a , kanellopoulou m b , paraskevopoulos i a , valakis k a , kyriakidis a a , lambropoulos s a . a intensive care unit, sismanoglio general hospital, athens, greece , b department of microbiology, sismanoglio general hospital, athens, greece purpose: the aim of this study was to assess the effectiveness of aerosolized ampicillin/sulbactam, ceftazidime and colistin, in icu patients with multiresistant acinetobacter baumannii or pseudomonas aeruginosa colonization of the respiratory tract.methods: fifty-three intubated, mechanically ventilated patients participated in the study. multiresistant a. baumannii , sensitive only to ampicillin/sulbactam, or p. aeruginosa , sensitive to ceftazidime or colistin, were isolated from the bronchial secretions (10 5 á/10 8 cfu/ ml). all 53 patients were subsequently treated with intravenous ampicillin/sulbactam, ceftazidime or colistin, whereas 27 of them were also given the same antibiotic in aerosolized form.results: a decrease in the number of colonies by 10 3 á/10 6 cfu/ml was observed, following 2 á/4 days of combined treatment with both intravenous and inhaled antibiotic. none of the 27 patients developed vap. in the 26 patients who only received the antibiotic intravenously, the decrease ranged from zero to 10 4 cfu/ml, after 7 days of treatment. two of 26 patients developed vap.conclusions: our results suggest that the administration of aerosolized antibiotics represents an effective means of preventing ventilator-associated pneumonia caused by a. baumanni and p. aeruginosa . introduction: pseudomonas aeruginosa is an important nosocomial pathogen. resistance to certain beta-lactam antimicrobial agents among p. aeruginosa is increasing. despite the development of new antibiotics multiresistant strains of p. aeruginosa represent an important therapeutic problem. the aim of this study was to investigate the activity of imipenem, amikacin, piperacillin, ciprofloxacin, ceftazidime, against clinical isolates of p. aeruginosa . methods: a total of 54 isolates by tracheal aspiration from hospitalized patients, admitted to intensive care units were identified as p. aeruginosa using an algorithm that included: gram stain, pigment, oxidaze ('/0,) and gram negative identifications microscan walkaway-96 (dade behring) were used according to the manufactures instructions. minium inhibitory concentrations were determined using walkaway, interpretation based on ncclsm 100-s9, january '99.results: the respiratory tract was the single site of isolation for this study. the best activity was showed by imipenem 25%, followed by amikacin 22%, piperacillin, pip/tazobactam, ciprofloxacin had the same sensitivity 11%.conclusion: a high level resistance to antibiotics was observed to p. aeruginosa isolated from tracheal aspiration. carbapenems seem to be the most active against p. aeruginosa in this study. materials and methods: clinical samples were collected from patients admitted to this hospital. only one isolate per patient was included. antimicrobial susceptibility testing was performed as recommended by the nccls. all bacterial isolates were tested by dd and ad to provide a comparison of both test results. very major error was considered when the strains were resistant (r) by ad and susceptible (s) by dd and major error when s by ad and r by dd. categories of s and r were stablised using the breakpoints suggested by mensura (2000) . colistin r strains was typed by rep-pcr.results: among the 35 strains included in this study 30 (85.7%) were s to colistin and five (14.2%) were colstin r by ad, of this five colistin r strains four (11.4%) were s to colistin by dd and one was r by both methods. all r isolates were similar by rep-pcr.conclusions: most of the ad colistin resistant strains were s when tested by dd indicating that this method is not useful to determine the resistance to colistin. rep-pcr patterns show that the spread of a colistin r clone seems to be involved. methods: gnb were isolated from per-operative biopsies (pob) and/ or from articular punction (ap). patients (pts) received cfp, 2 g bid'/ ofl, 200 mg tid or cip, 400 mg bid intravenously for 28 days, followed by a prolonged oral fq monotherapy. cure was defined as: resolution of all clinical signs of infection, normalization of the biological inflammatory profile at the end of treatment (eot) and absence of infection at the same site during the post-treatment followup period (ptfu).results: all of the 20 studied patients [mean age 0/48 years] had hospital acquired bji. seventeen/20 had an infected orthopedic device (prosthetic joints0/6, other orthopedic prosthetic devices0/11). culture of 17 pob and 5 ap yielded to pseudomonas sp. (11), enterobacter cloacae (6), others (4). vancomycin was added for six pts co-infected by gnb-mrsa. nineteen/20 pts underwent a surgical intervention (debridement0/11, removal-replacement0/7, amputation0/1). after ptfu period of 17 months (range 3 á/29), the overall success rate was 12/17 (88.2%) without serious adverse events.conclusion: cfp á/fq combination was safe and efficient in the treatment of hypercase gnb, bji.treatment of posttraumatic mrsa osteomyelitis of the femur with longterm cotrimoxazole */a case report pm241 the authors report a case of posttraumatic osteomyelitis of the femur caused by methicillin-resistant staphylococcus aureus (mrsa), following the shot injury. relapses of the infection occured in 3 months interval and were treated by revision, debridement, lavage and vancomycin. because of laboratory signs of renal insufficiency vancomycin became contraindicated for treatment of the third relapse of infection and the different approach was employed: classic open treatment of bone infection sec. orr was combined with a long-term administration of high-dose cotrimoxazole. the patient was given cotrimoxazole 9600 mg daily divided in four doses (120 mg/kg/24 h) for 2 months, then for gastrointestinal complaints with lowered dose of 5760 g daily for next 6 months. the wound completely healed. during 18 months after the final surgery there was no relaps of infection, but the atrophic pseudoarthrosis of the femur resulted. the patient can walk with a rigid orthesis and two crutches. the whole treatment of the objective: to present a variety of severe nosocomial infections due to stenotrophomonas maltophilia in patients hospitalized in tertiary medical units from cluj.results: during the last year nine strains of s. maltophilia obtained from patients with severe infections and hospitalized in different wards were isolated. all but one were considered nosocomial infections: four cases of pneumonia, one urinary tract infection, three cases of surgical wound infections and one case of endocarditis under surgical treatment. the cases of pneumonia were either primary occurring in a granulocytopenic patient with leukemia or secondary in patients that underwent surgical treatment. in the case of endocarditis the ethiology was established after surgery from the damaged valve in a negative hemoculture patient with a poor outcome under medical treatment. in all cases of surgical wound infection bacteremia occurred diagnosed on clinical basis in the presence of severe sepsis or hematogenous dissemination in the lung. the urinary tract infection occurred in a patient after urinary surgery and having a catheter in place. the immediate evolution was favorable in all cases but treatment was difficult due to the highly resistant strains and to underling diseases.conclusions: s. maltophilia should be considered in nosocomial severe infections and prophylaxis by interrupting environmental transmission has to be promoted. sawicka-grzelak a, rokosz a, luczak m. department of medical microbiology, university medical school, warsaw, poland purpose: to identify and determine the drug-susceptibility of esblpositive strains isolated from urine samples.methods: seven hundred and twelve strains of gram-negative rods were cultured from 4000 urine samples from hospitalized patients during 5 months (july á/november 2001). identification and susceptibility were performed in the automatic atb system (biomerieux, france) using id 32 e, id 32 gn and atb ur 5 strips. esbl-activity was detected with double-disc synergy test (ddst according to jarlier et al., 1988) or using a novel method of esbl detection (dd, diagnostic disc) according to appleton (1999) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd 01) (oxoid, england).results: five hundred and ninety-five strains (83.6%) belonging to enterobacteriaceae family, 115 strains (16.1%) of non-fermenting rods and two strains (0.3%) of other gram-negative rods were isolated. eighty-two esbl-producing strains (11.5% of all strains) were detected. fifty-nine esbl-positive strains were susceptible to nitrofurantoin, 58-to norfloxacin and ciprofloxacin and 54-to fosfomycin.conclusions: esbl-positive strains were detected most frequently among enteric rods (80 strains). nitrofurantoin and quinolones were the most active in vitro antibacterial agents against examined esblpositive uropathogens. results: the mechanisms of resistance were evaluated phenotypically using 12 different aminoglycosides. a total of 257 aminoglycoside resistant gram-negative strains were studied. one hundred fifty-eight strains were collected in 1995 á/1996, 52 in 1998 and 47 in 2000. the resistant profiles were determined: enterobacteriaceae */152; pseudomonas aeruginosa */62; acinetobacter spp. */43. the most frequently phenotypes were gt (gentamicin, tobramycin) */48% and gtnet (gentamicin, tobramycin, netilmicin) */35%. the gt phenotype due to production ant(2ƒ)-i enzyme, the gtnet á/aac(3)-v (87 strains), aac(3)-iv */one strain and aac(2ƒ)-i */one strain. the resistance to amikacin in 7% strains was due to production aac(6?)-i (3%) and aph(3?)-vi (4%). the most of examined strains were simultaneously resistant to kanamycin and neomycin caused by production of aph(3?)-i (69%). only seven strains were resistant to all aminoglycosides due to impermeability of outer membrane. no substantial differences were observed between years.conclusions: the main mechanism of aminoglycoside resistance is fermentative modification. the high rate to gentamicin and tobramycin was due to production of ant(2ƒ)-i and aac(3)-v. amikacin and isepamicin were the most active aminoglycosides against gramnegative nosocomial isolates.the incidence of clostridium difficile associated diarrhoea (cdad) increased in our department from january to june 2001.objective: to confirm the out break of cdad, to identify the risk factors and assess the effectiveness of the measures implemented for controlling this outbreak.methods: cdc definitions were used to identify the cases. the scope of the outbreak was defined. cdad incidences during the outbreak period and during the same period in 2000 were compared. risk factors (reduced mobility, antibiotic treatments . . .) were studied for patients whom length of hospital stay (lhs) was more than 6 days. contact precautions and environmental cleaning with clona implemented were assessed.results: seventeen episodes of cdad were identified. sex ratio: 1.41, mean age0/58.5, mean lhs0/36 days, mean delay for cdad occurring0/21 days. one hundred and fifty-two patients involved in the study of risk factors. relative risk (r.r.) evaluated were: blactams (r.r.0/7.62, ic95%: 1.03 á/56.56), reduced mobility (r.r.0/ 7.61, ic95%: 1.76 á/32.85). incidence of cdad was less than two cases per 1000 days of hospitalisation after june 2001.conclusion: we confirmed the outbreak of cdad in our department b-lactams and reduced mobility were identified as risk factors for cdad. measures implemented to control the outbreak were effectiveness.positive heart transport fluid cultures associated with severe infections in heart transplant recipients pm255 at the mount sinai hospital in new york city 256 heart transplants were performed between 1986 and june 2000. cultures were routinely performed on all heart transplant transport fluids. culture data was available for 204 of these patients. in total 24/204 (11.8%) were positive for bacteria, fungi or both. the organisms isolated included coagulase negative staphylococci (13), pseudomonas aeruginosa (2), staphylococcus aureus (1), acinetobacter baumanii (1), serratia mercescens (1), enterobacter cloacae (1), escherichia coli (1), proteus mirabilus (1), enterococcus faecalis (1), viridans streptococci (1), and fungi (aspergillus fumigatus (1), penicillium species (1), and rhodotorula rubra (1). two heart transplant recipients had two organisms isolated from the transport fluid. isolation of resistant gram-negative bacilli in the transport fluid was associated with significant infection in 3/5 patients (60%) with the same organism. the observed infections were pneumonia secondary to e. cloacae , sternal wound infection secondary to p. aeruginosa , and bacteremia secondary to p. aeruginosa . it appears prudent to provide prophylaxis against resistant gram negative bacilli to prevent infections. zacharof ak, flevaris c, petrogianopoulos c, karachalios g, vroulis j, chartzoulakis g, drakogiorgos g, loizidou a, svoukas g. 2nd department of internal medicine, hellenic red cross hospital, athens, greece objective: we studied the trends of nosocomial bloodstream infection and calculated the population-attributable risk for death among hospitalized patients. methods: we perform a 15-year retrospective study for all patients (n 0/79 160), admitted to our department between 1986 and 2000.results: between 1986 and 2000, a total of 1577 patients developed 1763 episodes of nosocomial bloodstream infection. the crude infection rates increased linearly from 7.2 to 25.4 per 1000 discharges (1.11 á/1.93 episodes per 1000 patient-days) during the 15-year study period. increases in the infection rates were due to gram-positive cocci, yeasts and essentially explained by infections caused by coagulasenegative staphylococci, staphylococcus aureus , enterococci, and candida species, respectively. although the crude mortality in patients with nosocomial bloodstream infections decreased from 45% in 1986 to 28% in 2000, the in-hospital population-attributable mortality among infected patients increased from 5.5 deaths per 1000 discharges in 1986 to 8.2 per 1000 discharges in 2000. the etiologic fraction or the proportion of deaths in patients with bloodstream infection to all deaths occurring in the hospital increased from 15.4% in 1986 to 26.4% in 2000.conclusions: the incidence and the population-attributable risk for death among patients experiencing nosocomial bloodstream infections increased progressively during the last 15 years in our department.ventilator-associated pneumonia before and after intensive care unit temporary closure pm257 results: we compared the incidence, causative organisms and mortality of vap in two different time periods. period 1 june to december 1999. period 2 june to december 2000. between those two periods the icu remained closed for 4 months because of reconstruction works.period 1: sixty-seven consecutive patients (pts) were studied with bronchial secretions cultures at least 2 days after mechanical ventilation (mv) initiation. the vap was diagnosed by clinical, radiological and microbiological criteria in 17 pts (26%). causative organisms included: pseudomonas aeruginosa 8, acinetobacter 5, staphylococcus aureus 4, klebsiella pneum. 3, enterobacter 1. in three cases vap was proved polymicrobial. fourteen (14) episodes of vap (82%) were developed after 7 days mv (late vap) and were attributed to multiresistant microorganisms. mortality of vap was 29%.period 2: among 66 consecutive pts, vap was diagnosed in 21 (32%). causative organisms included: acinetobacter 10, p. aeruginosa 8, s. aureus 4, k. pneumoniae 3, escherichia coli 1. five (5) cases were polymicrobial and 14 cases were 'late vap' (67%). causative microorganisms had similar patterns of sensitivity to antibiotics (compared to period one). mortality of vap was 38%.conclusion: temporary icu closure had no significant influence on the incidence, distribution of causative organisms, their sensitivities to antibiotics and mortality of the vap. efstathiou sp a , pefanis av a , tsioulos di a , tsiakou ag a , zacharos id a , kanavaki s b , mountokalakis td a . a third university department of medicine, sotiria general hospital, athens, greece , b microbiology laboratory, sotiria general hospital, athens, greecepurpose: the aim of this study was to derive a scoring system for the prediction of outcome in adult patients with acute pyelonephritis (ap) severe enough to need hospitalization. therefore, the charts of 225 patients (102 men, median age 67 years) were reviewed.results: logistic regression analysis identified in both sexes four independent correlates of in-hospital mortality, the coefficients of which divided by 0.5 and rounded to the nearest interval, resulted in the following integer-based scoring system: points (men)0/6* between concentrations of lps of the two groups were statistically significant on the 2nd and the 4th day (p b/ 0.05). it is concluded that the administration of lactulose may decrease systemic endotoxaemia in the field of obstructive jaundice.nosocomial infections: a prevalence study in the island of crete pm263 doukakis s a , tzimis l b , perogambrakis g a , kalloniatou m a , christodoulakis n a , evaggelopoulos a a , koutsoumba d a , kastanakis s a . a first medical department, 'saint george ' general hospital, chania, greece , b pharmacy department, 'saint george ' general hospital, chania, greeceprevalence surveillance is a rapid and inexpensive mode to estimate the problem of hospital-acquired infections (hais). to study the problem of nosocomial infections in our hospital, a prevalence study was made from our team in 1999. the study included 265 patients (the total number of hospitalized patients at the time of the study). from these patients 129 were males (49.0%) and 136 females (51.0%). one hundred and ninety-one patients (72.0%) belonged in the groups of age between 50 and 89 years. fifty-seven patients had a urine catheter (22.3%). one hundred and fifty-five/265 patients (58%) received antibiotics and from these 99 patients received one antibiotic and the remaining 56 patients two or more. a nosocomial infection was found in 13 patients and consequently the prevalence of hais was 5.0%. among these, urinary tract infections were six (46.1%), lower respiratory tract infections were three (23.0%), surgical site infections were three (23.0%), and bloodstream infections was one (7.7%). the incidence of multiresistant bacteria was primarily enterococcus spp and secondary, pseudomonas aeruginosa , enterobacter spp, klebsiella pneumoniae , escherirchia coli , staphylococcus aureus , enterobacter cloacae . unfortunately prophylactic chemotherapy of long duration was found despite the suggestions of the infection control committee. regarding age the highest incidence of hais occurred in the third age group. bagirova ns, dmitrieva n. laboratory of microbiology, cancer research center of russia, moscow, russian federation objectives: to determine the pathogens and susceptibility to antimicrobials.methods: blood samples were collected from 430 adult pts (1997 á/ 2001). the bacteraemic episodes were classified according to the definitions of the cdc. laboratory detection of bacteraemia and fungaemia was performed according to cumitech 1b (blood cultures iii, 1997). susceptibility testing was performed by disk diffusion method (nccls).results: the total number of blood samples */1371, 249-positive (18.2% episodes of significant bacteraemias). bsi was confirmed microbiologically in 137 of 430 febrile pts (31.9%). the most frequent pathogens were gram('/) cocci (62.7%) (p b/ 0.0001), gram((/) bacilli */22.7%, fungi */11.4%. coagulase-negative staphylococci (cns) represented 38.9%, staphylococcus aureus 12.4%, streptococcus spp. 4.9%, enterococcus spp. 5.9%, enterobacteriaceae 15.1%, pseudomonas aeruginosa 3.2%, other non-fermenting */5.9%, yeast 8.1%, mould 3.2%, anaerobes 2.7%. one hundred percent cns were resistant to penicillin, 68.4% to oxacillin, 36.6% to clindamycin, 39.5% to cefazolin, 80.3% to ceftazidime, 48.7% to ciprofloxacin, 51.3% to gentamicin, and no isolate was resistant to vancomycin. the predominant pathogens in all types of hm were gram('/) cocci (mainly cns). all gram('/) microorganisms were sensitive to vancomycin. katashinsky o a , opriatova t b , tchuev p c . a state clinical hospital, anaesteziology, odessa, ukraine , b state clinical hospital, bacteriology, odessa, ukraine , c medical university, anaesteziology, odessa, ukrainethis investigation was carried out in odessa state clinical hospital during 2000 á/2001. of the patients with post-operation complications, 60% of 518 gram-negative cultures were sensitive to ceftazidime and 72% to amikacin. sixty-nine percent of 143 gram-positive cultures were resistant to penicillin g, but they were sensitive to vancomycin and nitrofurantoin in 100 and 92% of cases, respectively. sixty percent of 366 isolates of pseudomonas aeruginosa were sensitive to ceftazidime and 71% to amikacin. rates of resistance to carbenicillin and gentamicin were 60 and 66%, respectively. one hundred and fortythree isolates of escherichia coli were studied and 65% of them were resistant to ampicillin 67% to cephalothin and 70% to tetracycline. of the isolates tested against ciprofloxacin, all were sensitive. one hundred and eight isolates of staphylococcus aureus were studied and they were resistant only to penicillin g (69%). staphylococcus aureus was sensitive to erythromycin (65%), tetracycline (65%), oxacillin (83%) and vancomycin (100%). all 34 isolates enterococcus faecalis were sensitive to nitrofurantoin and 70% to ciprofloxacin. the majority of s. aureus and e. faecalis isolates were susceptible to most other antibiotics, but the majority of e. coli isolates were resistant to the studied antibiotics expect ciprofloxacin.campylobacter foetus bacteraemia in an immunocompromised patient: a case report pm266monno r a , ierardi e b , rendina m b , ceci g a , luzzi i c , de vito d a , rizzo g a , francavilla a b . a department of internal medicine and public health, university of bari, bari, italy , b department of emergency and organ transplantation, university of bari, bari, italy , c istituto superiore di sanità, rome, italy a 33-year-old woman was admitted for recurrent fever. the patient underwent a liver transplantation and splenectomy in 1985. she had followed immunosuppressive therapy until 1995 when tacrolimus was added for chronic rejection. in 1999 non-hodgkin lymphoma was diagnosed and chemotherapy was started. six months later because of the presence of two metastatic encephalic foci affecting the optic chiasm, a new chemotherapy course was started with the regression of lesions. in january 2000 she was treated with steroid recycle and cyclosporine á/azathioprine á/prednisone reintroduction. fever occurred after 2 months and cytomegalovirus (cmv) infection was diagnosed. treatment with ganciclovir was started with clinical remission. in november 2000 cmv infection recurred and blood cultures were positive for a bacterium that was identified as campylobacter fetus . the patient was successfully treated with intravenous ciprofloxacin. bacteremia frequently occurs in cancer patients. bacteremia due to c. fetus are rare, occurring mainly in immunocompromised patients. c. fetus expresses a proteinaceous surface layer that confers serum resistance. in our patients steroid and immunosuppression may have contributed to the development of lymphoma. all of these factors and chemotherapy have contributed to cmv infection and all have made the patient susceptible to bacteremia with this infrequently found bacterium. the clinical microbiologist should be aware of this infection in immunocompromised host. minenko sv, dmitrieva nv, sokolova en, ptushkin vv. bone marrow transplantation department, n.n. blokhin cancer research center, moscow, russian federation c'/a is the standard regimen as empirical therapy for febrile neutropenia (fn). activity of c against g'/ bacteria and g(/ bacteria, producing chromosomally-mediated b-lactamases (e.g. ampc) is suboptimal. cep is active against a broad range of g'/ and g(/, including ampc producing bacteria. the purpose of the study was to compare the efficacy of two regimens in the treatment of fn.methods: patients with fn received either cep (2 g/8 h) or c (2 g/8 h) plus amikacin (15 mg/kg/day) or netilmicin (5 mg/kg/day). data were collected prospectively.results: a total of 35 pts with 41 episodes (35/41) of fn were included. fifteen/19 in cep group and 20/22 in c'/a group. the median duration of neutropenia grade 4, distribution of age, sex and underlying disease were comparable in both arms. mdi was in 37 and 24%, cdi in 21 and 18% fuo in 57 and 63% in fep and c'/a groups correspondingly. response to the initial empirical regimen according to ihs criteria was in 58% of cep and 27.3% of c'/a groups (p 0/ 0.05). modification of therapy with a change of cep or c to carbapenem took place in 31 and 40% of cep and c'/a groups (p0/0.5). no patient in either treatment group died due to the presenting infection. tolerability of cep was good and no laboratory abnormalities took place. transient elevation of serum creatinin level was observed in two patients c'/a group.conclusions: cep monotherapy is as effective as c'/a combination in the treatment of patients with fever and granulocytopenia purpose of the study: retrospectively, we analysed 60 patients with intraabdominal infection for aetiology, risk factors, and outcome from 11 hospitals in slovak republic within 1 year (march 00 á/march 01). results: in this group significantly more frequent were older patients ( !/65 years) with cancer (65 vs. 16%, p b/ 0.0007). acinetobacter spp. (50 vs. 7%, p b/ 0.0001) and enterobacteriaceae (83 vs. 39%, pb/ 0.04) were predictive for monoinfection. pre-treated patients with other antibiotics had inferior prognosis and more risk factors: permanent urinary catheter (95 vs. 67%, pb/ 0.05), ventilation or intubation (91 vs. 17%, pb/ 0.0001) and polymicrobial infection (86 vs. 33%, pb/ 0.0002). the risk factors with poor prognosis were enterobacteriaceae (30 vs. 14%, pb/ 0.04), diabetes mellitus as underlying disease (18 vs. 54%, p b/ 0.2) and uraemia (4 vs. 40%, p b/ 0.002). surprisingly, negative prognostic factor was also non-effective previous antibiotic therapy. failed patients died significantly more frequently patients due to underlying disease. cefoperazone/sulbactam was shown to be useful, effective and well tolerated also in one group of patients with 75% efficacy of treatment, and it belongs to a group of antibiotics suitable for treatment of nosocomial infections. key: cord-015372-76xvzvdg authors: nan title: national scientific medical meeting 1996 abstracts date: 1996 journal: ir j med sci doi: 10.1007/bf02945204 sha: doc_id: 15372 cord_uid: 76xvzvdg nan zero months months months months group a 5.53 5.09* 5.66** 6.11"* 6.26** 4 zero months group b 6.06 6.4** values are means; *p<0.05 and **p<0.0l~ most other studies show neutral effects on insulin sensitivity, with minimal incidence of glucose intolerance. this may be partly explained by the diversity of age, diagnosis (whether insufficiency or deficiency state), other pituitary deficiencies and replacement therapy, and possibly by dosage ofgh utilised in these studies. clostridium difficile-associated disease (cdad) is primarily a nosocomial condition. community-acquired disease has been described but the incidence is low "). during a recent outbreak, we prospectively reviewed all new cases of cdad to determine what proportion of cytotoxin positive cases were community or hospital acquired. during a 4 month study period, 73 cases were identified. history of diarrhoeal episodes were recorded for each case. selected isolates were typed using pyrolysis mass spectrometry (pms). community-acquired cdad was defined as diarrhoea, on or within 72 hours of admission, in association with a positive stool cytotoxin test for c.difficile and in the absence of hospitalisation within 60 days. sixty-five cases (89%) were hospital acquired. fifteen patients (20.6%) had cdad on admission; 8 (11%) were community acquired, 7 (9.6%) had been recently hospitalised (4 at st. james's hospital, 3 at other hospitals). pms typing of faecal isolates revealed that 2 predominant strains were responsible for the hospital outbreak; one of these strains was also isolated in community-acquired cases. this study suggests that the incidence of communityacquired cdad may be higher than previously reported. we suggest that all newly admitted or transferred patients with diarrhoea should be screened for this organism. several studies have confirmed that activated protein c resistance (apc-r) occurs in 20-60% of thrombosis patients and is therefore more common than congenital deficiencies in the inhibitors of coagulation such as atii1, proteins c and s. homozygosity for the factor v (fv) gene mutation is associated with a 50-100 fold increased risk of venous thrombosis while heterozygosity is associated with a 5-10 fold increased risk. the mutation, however, is highly prevalent in the general population, a prevalence of 5% has been reported in several european countries. its frequency in the population of northern ireland (ni) has not yet been reported. we screened a group of 72 generally healthy elderly individuals (av. age 81.3; range 76-97 yr on several occasions for apc-r using an assay based on the prolongation of activated partial thromboplastin time by the addition of apc. a mean ratio of 2.64 (range 1.72-3.46) was measured. seven individuals (9.8%) had ratios <2.3 (av. 2.11; range 1.72-2.28). these subjects were then analysed for the fv mutation by pcr amplification and restriction analysis. the 4 individuals with the lowest ratios (av. 2.0; range 1.72-2.15) were found to be heterozygous for the mutation. none of these 4 individuals were deficient for protein c, protein s or atiii. the frequency of apc-r (5.8%) within this ni elderly group is similar to that reported by others in the uk whose studies would have included a generally younger population. the successful ageing of these individuals perhaps underlines the low risk associated with heterozygosity. alternatively a higher prevalence of the mutation may exist in the general population of ni, where the incidence of heart disease is one of the highest worldwide. the insulin-like growth factor ii gene (igf2) is imprinted. thus, in contrast to most genes where both maternal and paternal copies (alleles) are transcribed into rna and expressed, 1gf2 is normally only expressed from the paternal copy (monoallelic expression). alterations causing biallelic expression of igf2 may lead to excess growth factor production, and thus, to tumourigenesis. this study evaluated igf2 expression in a series of childhood cancers. to date 41 tumours have been evaluated using pcr based methodology (26 wilm's tumours, 12 rhabdomyosarcomas, 3 miscellaneous). dna was extracted and a polymorphic site apal within the igf2 gene was amplified and digested. this identified 10 samples as heterozygous for igf2, meaning that separate maternal and paternal alleles were distinguishable. rna from these informative samples was extracted, pcr amplified and restriction digested, to identify monoallelic versus biallelic profiles at the expression level. samples with normal imprinting (monoallelic) displayed allele a (236bp) or allele bl/b2 (176/63 doublet). biallelic samples displayed both alleles. using this approach 3/5 informative wilm's tumours and 2/ 4 rhabdomyosarcomas demonstrated biallelic expression. in conclusion, biallelic expression of igf2 was detected in a significant number of wilm's tumours and rhabdomyosarcomas, and should be considered; with other genetic alterations, as a candidate mechanism in tumourigenesis. the proinflammatory cytokines, tnf~, il8 and il-6, may mediate host metabolic and immune responses to cancer possibly leading to paraneoplastic phenomena such as cachexia. the cellular origin of these cytokines in the cancer patient, in many cases, remains unknown. we examined proinflammatory cytokine levels intracellularly, using flow cytometry, in pbmcs from oesophageal cancer patients (n=14) and age and sex matched controls (n=10). tnf~ and il-6 levels were significantly increased (p<0.05) in pma stimulated t cells and monocytes from the cancer patients when compared to the healthy controls. these results were confirmed using standard elisa assays. following cotlagenase digestion, increased levels of tnfa and il-6,were detected in oesophageal tumour infiltrating t cells when compared to cells from normal mucosa. there was also increased production of tnf~ and il-6, but not il-ib, in malignant epithelial cells when compared to normal and halothane and maintained by 50% nitrous oxide-oxygen, 0.75-1.5% halothane, with spontaneous ventilation. tc rose marginally in group 1 and fell in group 2 (not significant, ns). tp in groups 1 and 2 at induction and 20 and 30 minutes were, respectively, (mean + sem, celsius) 31.4+0.33 vs 31.6+0.3, ns; 33.9+0.3 vs 33.4+0.3, ns and 34.5+0.4 vs 33.2+0.3, (p<0.05). overall incidence of shivering-was 14.%, but not significantly different between the groups. the data suggests that preemptive application of the space blanket increases tp in paediatric patients during general anaesthesia and tends to conserve tc. chronic actinic dermatitis (cad) an uncommon, eczematous, photosensitive eruption is diagnosed on history, clinical examination, skin biopsy and abnormal light tests. drug induced photosensitivity may look identical clinically, have a similar history and patients with cad may be treated with potentially photosensitising drugs. we therefore reviewed all patients with cad and compared their monochrumator light tests with patients who had drug induced photosensitivity. phototesting was performed on unaffected skin of the back with an irradiation monochromator; the minimal erythema dose (med) determined for a series of wavelengths between 300 and 400 nm, in 14 patients with drug induced photosensitivity and 7 patients with cad. of ten females, four males with drug induced photosensitivity, age range 40-77 (mean 61 yrs), ten (71%), were photosensitive in the uva range (320-370nm), the implicated drugs including, quinine, sparfloxacin, amiodarone, doxycycline, mefenamic acid, nalidixic acid, fenbrufen, diclofenac, enalpril and prochlorperazine maleate. three patients with rosacea were photosensitive to doxycycline. the re/nainder (29%), were tested after discontinuation of the drug and their light tests were normal. in the cad group, (four males and three females), age range 38-86 (mean 71.5 yrs), three patients (43%), were sensitive to uva, uvb and visible light, four (57%) to uva and uvb. in conclusion, uva dissociated from uvb photosensitivity seems a relative but not absolute sign of drug induced photosensitivity. this pattern of light tests should prompt a detailed drug history to elucidate the causative agent. phototesting should be performed while on the offending drug as testing days or weeks after discontinuation will give normal results. patients at risk of bone fractures by measuring bone mineral density (bmd) and markers of bone turnover and to assess the correlation of these with the severity of cld. twenty three patients with cld had bmd measured by dual-energy x-ray absorptiometry scanning of hip and lumbar spine. bone formation was assessed using serum levels of procollagen type 1 peptide, osteocalcin and bone alkaline phosphatase, and bone resorption was assessed using 2 hour urinary excretion of hydroxyproline, pyridinoline and deoxypyridinoline. 48% and 39% of patients had evidence of osteoporosis at the lumbar spine and femoral neck respectively. biochemical results showed that 74% of patients had an increase in all 3 bone resorption markers and 42% had an increase in markers for bone formation. bmd at the lumbar spine was lower in patients with cholestatic liver disease compared to patients with other types of'liver disease (p=0.004). no correlation was found between bmd and patient age, bilirubin, albumin, inr or duration of liver disease. conclusions: osteopenia occurs in up to 62% of patients with cld due to a high bone turnover state where bone resorption exceeds formation. osteoporosis is most severe in those patients with cholestasis. a detailed profile is presented of all 632 leukaemia and multiple myeloma (icd-o code 169) patients registered by the southern tumour registry during the 8-year period 1983/1990 "). annual age-adjusted rates of 13.9 and 8.7 per 100,000 were seen for males and females respectively. these levels indicate a lifetime (up to 75 yr) risk of 1 in 68 for males and 1 in 116 for females. the main morphological sub-types registered were multiple myeloma (31%), cll (25%), aml (18%) cml (9%) and all (8%). one, two and five-year survival rates were examined; age at diagnosis and lesion type were extremely significant factors in relation to patient outcome. the overall incidence levels indicate that irish rates were relatively high by internatiomil standards r we assessed effects of reducing the volume of hyperbaric bupivacaine by giving half the volume as isobaric bupivacaine. when using 0.5% hyperbaric bupivacaine for spinal blockade, the segmental spread and cardiovascular effects of the block have been shown to be dependent on the volume of local anaesthetic injected. 40 patients undergoing elective surgery were studied in a prospective, randomised, double-blind trial: group 1 (20 patients) received their local anaesthetic as two equal aliquots of 0.5% hyperbaric bupivacaine and 0.5% isobaric bupivacaine respectively; group 2 (20 patients) received their local anaesthetic as two equal aliquots of 0.5% hyperbaric bupivacaine. there was no significant difference found between the two groups with regard to maximal height of block (group 1, mean (range), t7 (t4-ti 1); group 2, t8 (ts-t11)), rate of onset of blockade, or time to maximal blockade (group 1, mean (sem), 23.55 (2.41) rain; group 2:20.89 (2.95) min). there was no difference found between each group in either cardiovascular stability or vasopregsor usage. the administration of a mixture of 0.5% isobaric bupivacaine and 0.5% hyperbaric bupiv/~caine confers no advantages over administration of the same volume of 0.5% hyperbaric bupivacaine alone. propofolis used as a sedative during regional anaesthesia. providing titrateable sedation, it can compromise haemodynamic stability. a propofol ketamine combination provides stable haemodynamics during total intravenous anaesthesia, avoiding emergence phenomena m. we compared two sedative regimes in patients having spinal anaesthesia. following informed consent, 40 patients, asa i-ii, undergoing spinal anaesthesia were randomized to one of two groups (n=20). group i (propofol-ketamine) received loading doses of 0.4mg/kg propofol, 0.1mg/kg ketamine followed by an infusion of 1.2mg/kg/h and 0.3mg/kg/h respectively. group ii (propofol) received bolus 0.5mg/kg and infusion 1.5mg/kg/h. subsequent infusion rates were titrated to effect using a sedation score. heart rate, blood pressure, oxygen saturation, end tidal c02 and oxygen requirements were recorded. observation continued for the recovery period and patients visited the following day. data were analysed using t-test, chi 2 test and anova. groups were demographically comparable. sedative and respiratory indices were similar for both groups. there was no difference in total propofol requirements between the groups; group i -146_+194mg, group ii -137_+1:52mg (mean _+ sd). there was a large difference in mean arterial pressure, being much lower in the propofol only group. both groups had an uneventful recovery without emergence phenomena. our results do not confirm the described additive effect of andketammc . ketamine.with propofol for sedation propofol ' =~2) confers haemodynamic stability during spinal anaesthesia. we designed a controlled study to investigate whether there is a direct relationship between the degree of postoperative pain and the development of negative middle ear pressure in adults following tonsillectomy. middle ear pressure was measured by tympanometry. pressures were classified as type a (o to -99 mmh20), type b (flat) or type c (-100 to -350 mmh20) tympanograms. 30 patients with type a tympanograms, undergoing tonsillectomy were enrolled in the study. patients had daily tympanometry whilst in hospital and then weekly until amrmalisatign. a questionnaire incorporating visual analogue pain scores was filled in at the same time. a control group of 26 patients with type a tympanograms, undergoing appendicectomy and endotracheal intubation was used. follo~v up was available on 26 patients. 13 patients (50%) developed type c tympanograms, 5 patients 09%) type b and 8 (31%) patients remained unchanged. no member of the control group developed any change in middle ear pressure (chi squared = 27.5, p < vol. 165, 1996 irish journal of supplement no. 2 medical science 0.001). there was no relationship between pain scores for throat pain or otalgia and the development of negative middle ear pressure. 8 patients recorded higher pain scores for throat pain at day 7 then day 1, only 3 of this group had negative middle ear pressure. middle ear pressure reverted to normal at day 7 in 15/18 patients and in the remaining 3/18 it was normal at day 14. this study demonstrates the development of transient negative middle ear pressure following tonsillectomy in 69% of patients. this change is unrelated to the degree of postoperative pain nor is it associated with otalgia. postoperative ward analgesia remains suboptimal. this may be partially related to inadequate early use of opioids to attain minimum effective analgesic concentration (meac). we examined the incidence and predictors of severe postoperative pain on admission and discharge from our postoperative recovery room (rr). verbal pain scores were obtained in a pilot study of 202 patients on rr admission and discharge. procedures were classed as open cavitary, laparoscopic, orthopaedic, ent or body surface surgery. intraoperative use and dosage of narcotics and nonsteroidal (nsaid) analgesics, anaesthetists' experience (prefellowship or post-fellowship nchd, consultant) were noted, and rr opioid usage recorded. pain scores and analgesic use were examined using mann-whitney, ~2 analysis and logistic regression. moderate or severe pain was experienced by 25% of patients on either arrival or discharge. median intraoperative morphine dosage was 5 mg. opioid use was slightly (median morphine dosage 8 mg, p < 0.05) higher in patients undergoing cavitary surgery; these patients had the highest pain scores on rr arrival and departure. 21 patients (10%) received > 10 mg morphine intraoperatively. discharge scores of 6/10 or higher occurred in 24 patients (12%). opioid usage and pain scores were unrelated to level of training. nsaid use/nonuse was unassociated with differences in opioid use or rr pain scores. no morbidity attributable to analgesic use (desaturation, slow respiratory rate) occurred. nonattainment of meac is frequent after open cavitary surgery. conservative opioid dosages continue to be employed despite inadequate early postoperative pain relief; this does not change with increasing experience. reporting such findings in departmental audit may help to alter perioperative management; such data may serve as a baseline for future interventional studies. diclofenac is frequently used for analgesia after tonsillectomy. recently concern has been expressed about the effect of diclofenac on prolonging bleeding time. one recent retrospective study found its use in tonsillectomy was associated with an increase in reactionary haemorrhage. we designed a randomised controlled study to compare the effects of rectal diclofenac and im pethidine given at induction with pethidine alone, in children undergoing tonsillectomy. fifty nine patients were entered into the study. there were 26 males and 33 females, mean age 6 years, range (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) . patients were randomised according to chart number. thirty five patients received rectal diclofenac after induction. twenty four patients acted as controls. there were no significant differences in operating time or operative blood loss between the two groups. in the recovery room the diclofenac group was significantly less restless than tl~e control group (p < 0.05, chi squared test), with less crying, movement and agitation. there was no difference in postoperative recovery and no primary or reactionary haemorrhage. one patient in the diclofenac group developed a secondary haemorrhage. this study demonstrates a significant reduction in restlessness in the recovery room in children receiving rectal diclofenac. no increase in reactionary haemorrhage was demonstrated. diclofenac remains a safe and effective analgesic agent in children undergoing tonsillectomy. elderly patients have decreased dose requirements for many drugs compared to the young. few studies have examined dose requirements of opioids in the elderly when administered via patient controlled analgesia (pca) 1. we compared the pca morphine requirements between young and elderly patients. records were retrospectively analysed from 1,000 consecutive patients receiving pca for post-operative pain. inclusion criteria (i) age less than 10 years or greater than 60 years, (ii) upper abdominal surgery, and (iii) morphine pca usage. 238 patients fulfilled the inclusion criteria. 89 patients were young and 149 were elderly. the mean age in the young was 29 years and in the elderly was 69 years. 51% were female in the younger group, 40% were female in the older group. pain scores at rest and on movement were similar in both groups 3.8 and 6.7 respectively in the young, 3.2 and 6.2 in the elderly. (p > 0.05, students t-test). morphine usage over 48 hours was 58 + 32.1mg in the young, and 37 + 20.3 in the elderly. (mean + s.d.) (p.< 0.05, students t-test). elderly patients required significantly less morphine via pca to achieve the same pain scores as the young. these findings are consistent with studies showing decreased requirements of other drugs in elderly. the erythrocyte sodium-lithium countertransport (slc) is abnormal in essential hypertension and some other forms of cardiovascular disease (cvd) but the considerable overlap in its activity in patients with these conditions and in normotensive healthy subjects remains a strong point against its possible utility as a marker for cvd. we sought to address this issue in greater detail. twenty-nine hypertensive patients (19 with family history of cvd) aged 56.4+1.9 years (mean + se) and 32 normotensive subjects (12 with family history of cvd) aged 45.9 + 1.9 years, participated after informed consent. slc were determined in 35, 50, 70, 100 and 140mm sodium chloride; and the vmax and km of the transporter determined. hypertensive and normotensive individuals with family history of cvd (n = 31) had higher slc activity (0.331 + 0.011 vs 0.238 + 0.011, p < 0.0005), greater vmax (0.473 + 0.019 vs 0.397 + 0.018 mmol/lcell.h, p < 0.006) and lower km 56.0mm (median) vs 95.5 (p < 0.001) than hypertensive and normotensive subjects without such a history (n = 30). however, none of these parameters was sufficiently discriminatory as evidenced by the considerable overlap in the scattergrams for the two groups. on the other hand not only was the median quotient vmax/km significantly different 7.89 vs 4.20 (p < 0.001), but also the scattergram separated the two groups. this may reflect an effect of hereditary factors on the identified rate-limiting step in the transport system. capsaicinadministration results in depletion of substance-p sensitive nerves. this study was carried out to observe the impact on the morphology of mitral valve endothelium. the experimental group received capsaicin i.p. on day four of life; control animals received drug vehicle only. animals were anaesthetised by chloral hydrate and hearts were removed following perfusion of 4% glutaraldehyde, and were routinely processed for scanning electron microscopy. normal endothelial morphology showed an ordered and structured pattern, with large raised nuclei covered in discrete microappendages: no zoning was observed over the valve surface. following capsaicin administration, valves were seen to be torn and possessed a denuded endothelium. nuclear bulges changed in both apparent height and area, with the surface partially denuded of microappendages. one month following systemic administration of capsaicin to neonatal rats, a serious alteration of mitral valve endothelium morphology and integrity had occurred. depletion of substance-p may have resulted in mechanical insufficiency of the mitral valve. this study was funded by the health research board. with expanding applications and increasingly aggressive stress protocols, concerns about the safety of dobutamine stress echocardiography (dse) have arisen. the purpose of this study was to analyse prospectively the safety, adverse event profile, and complication rate of dse. prospective data was recorded in a consecutive series of 309 patients undergoing dse for diagnostic evaluation of chest pain, for risk assessment following myocardial infarction or for detection of hibernating myocardium. the maximum dose of dobutamine used was 50 mcg/kg/min in 24.6% of patients and 40 mcg/kg/min in 73.1%. atropine was used in 6. 8% long-term outcome following coronary artery bypass grafting may be related to the prevalence of major risk factors and their treatment following surgery. we aimed to establish the prevalence and current management of coronary risk factors in a group of consecutive patients attending our hospital. this is a report of the first 100 patients. data was collected by a structured patient interview, chart review, physical measurements and blood sampling. there were 74 male and 26 female patients, average age 61.5 years. the mean length of time since surgery was 2.2 years. thirty-nine patients had a recurrence of angina and this occurred on average ll months after surgery. as regards risk factors, 22 were active smokers, 56 were ex-smokers and only 22 had never smoked. two thirds of the patients were taking regular exercise; only 6 took no exercise at all. seventy-two percent of patients had a cholesterol greater than 5.5mmol/l, yet only 10 of the patients were on lipid lowering drug therapy and a further 16 were on a lipid towering diet. twenty-nine patients had a systolic bp > 160 or a diastolic bp > 90 and 15 of these were on antihypertensive therapy. seventy-seven patients were overweight but most of these had received specific advice regarding weight reduction in the preceding year. our results show a high prevalence of treatable risk factors in this high-risk group with inadequate treatment in many cases. new combined primary and hospital care strategies for cardiac rehabilitation and long-term secondary prevention of coronary heart disease are required. if the goal of modern therapy of acute myocardial infarction (ami) is preservation of myocardium, the occurrence of cardiac failure could be regarded as a treatment failure. in recent studies of iv thrombolysis and primary ptca the frequency of left ventricular failure (lvf) following ami has been as low as 3.5%. this very low rate might be explained by selection bias in patients recruited to randomized trials. the purpose of this study was to examine the frequency of lvf in a consecutive alterations in nitric oxide (no) synthesis have been implicated in cardiomyopathy, ischaemic heart disease and septic shock. recent work has suggested a possible role for nitric oxide in cardiac arrhythmogenesis. the effects of inhibiting no synthesis with l-name (n c -nitro -l -arginine methyl ester) on cardiac electrophysiology have not been fully determined. the dominant frequency of electrically induced ventricular fibrillation was determined in l0 anaesthetised pigs (30-42 kg) using a fourier transform. the dominant frequency (7.8 _+ 0.5 hz in lead ii) was not altered by treatment (8.5 _+ 0.5 hz) with l-name in a group of 16 pigs (45-58 kg) the effect of l-name (10 mg/kg) was assessed in relation to energy required to defibrillate. there was no significant difference in the energies required to achieve successful defibrillation on 80% of attempts between the l-name group (104.9 _+ 8.0 j) and the control group (111.1 _ 11.9 j), and on 100% of occasions, l-name (122.5 + 11.5 j) and control (137.5 _+ 13.1j). the results show that inhibition of no synthesis has no significant effect on the dominant frequency of ventricular fibrillation or on the efficacy of defibrillation in the pig heart. received either 100mg (7pts) or 200mg (19 pts) oral flecainide followed by a maintenance dose of 100mg bd. all pts were euthyroid. none had significant hypertensive, valvular or ischaemic heart disease. 23 pts had normal echocardiograms, 3 had mildly dilated atria. of the 26 pts 17 (65%) converted to sinus rhythm, 15 within 72 hrs and 2 at 12 and 21 days respectively. 2 subsequently, he was found to have paralysis of all the muscle groups of his right upper limb apart from some flexor movements in his fingers with areflexia and sensory loss over the c5/c6 dermatomes. the findings were thought to be in keeping with a brachial plexus.lesion. an mri scan showed a "haematoma" or "fibrosis" around the brachial plexus~ emg studies revealed a complete lesion from c5/c7 with evidence of partial function at c8 and a good function at c4. despite physiotherapy, at follow up 2 months later there was no improvement in the emg findings. though brachia~ plexus injury has always been considered a complication of central venous line insertion <l), there have been no cases described, in the literature in the last 10 years. central lines are mandatory irt major surgical cases but one must always be aware of the possible complications caused by them. body weight (bw) in icu patients fluctuates due to alterations in body mass (1) and water (2) and is usually estimated rather than measured. we hypothesised that (i) estimated weights are inaccurate and (ii) changes in true body mass (tbm) are masked by changes in water content. on admission, icu patients whose expected length of stay (los) was > 5 days were placed on a bed incorporating 4 load cells (accuracy of _ 0.1 kg). bw was estimated by icu staff. mbw was then recorded 12 hourly under standard conditions. changes in mbw due to pack insertion/dressing changes were excluded. we calculated tbm as mbw minus cumulative fluid excess, corrected for insensible losses c2~. patients received standardized nutritional support from day 3 and urinary nitrogen on day 6 was calculated we studied 20 patients whose mean (sd) age, mbw, los and apache ii score were 44.3 yr (18.54), 70.5kg (14.39), 10.0 days"(6.78) and 18 (3.8) respectively. mean error in estimated weight on admission was 10.3% (nurses) and 12.2% (doctors). mean protein and calorie intake was 64g and 1670 kilocals/day. mean decrease in mbw during icu stay 0.22 kg/day (range 0.21 kg (gain) to 0.49 kg). mean reduction in tbm was 0.56 kg/day (range 0.28-0.86 kg/ day) and this correlated with urinary nitrogen loss (r=0.7). in icu, (i) estimated weight is significantly inaccurate and should not be used in physiological calculations and (ii) rapid and significant decreases in body mass occur which may be underestimated due to fluid accumulation. dopamine appears to influence pituitary function and is associated with decreased circulating human growth hormone and insulin-like growth factor i m which could exacerbate catabolism, and therefore wasting, during critical illness. at 12 hrly intervals from admission, patients whose expected length of stay exceeded 5 days, were weighed (measured body weight, mbw) under standardized conditions,-using a bed incorporating 4 electronic load cells (accuracy + o.1 kg). standard demographics, apache ii score and use of dot~amine by infusion was recorded. changes in weight due to removal./ insertion of prostheses, packs/dressings were excluded mbw was converted to true body mass (tbm) using measurements of cumulative fluid balance (1000 ml = 1 kg) and insensible lossr patients received nutritional support, starting on day 3, based on their admission mbw. there were no significant differences in mean age, weight, length of icu stay, admission apache ii scores and mean daily protein/calorie intake between 14 patients who had received dopamine by infusion (group d) and 18 who had not (group nd). mean (sd) decreases in mbw during icu stay were 0.42 (0.08) kg/day (group d) and 0.20 (0.05) kg/day (group nd) (p<0.0i). mean decrease in tbm was 0.55 (0.10) kg/day and 0.35 (0.07) kg/day respectively (p<0.05). thus group d were losing an additional 1.4 kg body mass per week relative to group nd. the use of dopamine by infusion is associated with an accelerated loss of lean body mass during critical illness. adhesion of polymorphonuclear leucocytes (pmns) to pulmonary endothelial cells is an initial step in the inflammatory process characterising the adult respiratory distress syndrome. previous studies using human umbilical vein endothelial cells (huvecs) have suggested that lipopolysaccharide (lps) is a potent stimulus for pmn adhesion to endothelial cells. the aim of this study was to investigate the effect of lps on pmn adhesion to human pulmonary artery endothelial cells (hpaecs). human pmns were coincubated with hpaecs _+ lps (0.001-10mg/ml) with 2% serum for 1 hour. the effect of phorbol myristate acetate (pma) (125 ng/ml) was also examined. percentage adhesion stimulated by pma was 66+10sd. lps did not significantly increase adhesion at any of the concentrations used. to confirm the activity of lps pmns were incubated at 37~ with 0.01-10mg/ml lps + 2% serum for 1 hour and labelled with flourescent antibodies to the mac1 adhesion molecule complex (cd 18/cd 1 lb). facs analysis indicated upregulation of both cd18 and cdllb. thus in the system used, lps did stimulate pmn adhesion molecule expression, indicating that the lack of adhesion reflects a difference in hpaec response to lps compared to that reported for huvecs. this work was supported by the health research board ireland. although inhaled corticosteroid therapy is of undoubted benefit in the management of asthma, dysphonia is a recognised sequela. this study was designed to examine longitudinally the effect of inhaled steroids on the voice and vocal cords of newly diagnosed and previously untreated asthmatics. twenty subjects were recruited and underwent voice and vocal cord assessment prior to and 3 months after starting inhaled steroid treatment. the assessment consisted of 1) rating dysphonia using a visual analogue scale, 2) acoustical analysis of the voice and 3) videostroboscopic examination of vocal cord activity. prior to commencing inhaled steroid therapy for their asthma 14 subjects had normal voices, 5 subjects were mildly hoarse and one was moderately hoarse. vocal cord pathology was noted in 12 subjects, 2 patients had vocal cord nodules and the remainder were noted to have mildly oedematous cords together with a glottic chink. at 3 month follow up, improvement in voice was noted in 2 subjects, one patient felt more dysphonic but there was no change in vocal cord appearance. one subject was noted to have developed a mid glottic chink with no associated change in voice. one subject had clearing of mild vocal cord oedema and improvement in voice. this study demonstrates that 60% of subjects commencing inhaled steroid therapy for asthma have mild vocal cord pathology. voice is more likely to be improved following use of inhaled steroids for 3 months then made worse. although the relationship between elastin degradation and emphysema is well known, recent evidence suggests that a more complex process of pulmonary remodelling occurs within the emphysematous lung. the aim of this study was to assess the extent of extracellular matrix remodelling by ultrastructural examination of its two major components, elastin and collagen. emphysema was induced in rats by the intratracheal administration of porcine pancreatic elastase (1.2u/g body weigh0 and human lungs were obtained at surgical resection for lung carcinoma. emphysema was confirmed histologically in both animal and human samples by measurement of the mean. linear intercept. matching sections were immersed in 2.5m naoh and 88% formic acid to digest elastin and collagen respectively. scanning electron microscopy with stereo-pair imaging allowed 3-d visualisation of elastin and collagen frameworks. the distribution of emphysema was primarily panacinar in rat lungs a'nd centriacinar in human lungs. as expected in both types of emphysema, elastic lamellae were disrupted and perforated with multiple fenestrations. accompanying this disintegration was a marked increase in thickness of collagen fibrils which in some cases coalesced irish journal of medical science imparting a sheet-like appearance to the airspace walls. unique to human centriacinar emphysema, collagen formed helices which spiralled around alveolar septae to form bulky walls between adjacent airspaces. in conclusion, these findings lend support to the novel concept of aberrant collagen remodelling in the pathogenesis of emphysema. small cell lung carcinoma (sclc) is the most aggressive of the four common cell types of lung carcinoma. less than 10% of patients with sclc are alive two years after diagnosis. staging procedures, treatment regimens and survival results were reviewed in a small regional centre to make a comparison with larger treatment centres. thirty-one cases of sclc seen by one physician from 1985 to 1993 were reviewed. staging was clinical. treatment was undertaken in conjunction with the local oncology and radiotherapy services. 42% of patients had limited disease where as 58% had extensive disease at diagnosis. in patients with limited disease, 20% were alive at 18 months and there was a 10% long term survival rate i.e. greater than three years. average length of survival in limited disease was 456 days. survival results were comparable with those treated with chemotherapy alone and combination chemotherapy and radiotherapy. in patients with extensive disease the best results were from those treated with a combination of chemotherapy and radiotherapy with art average survival of 353 days. these figures compare favourably with those from larger multidisciplinary centres. the factors contributing to our relative success may relate to continuity of care achieved in a smaller centre. nasal cpap is a very effective therapy for osa, but is cumbersome, and compliance varies. we prospectively evaluated 91 consecutive osa patients treated with ncpap, who were asked to complete questionnaires before, and 2 to 12 months after starting therapy. this stttdy intended to examine both the patient's subjective response to ncpap and their bed-partner's impressions also. replies were received in 84 (92%) patients. patients were divided i~to 4 groups depending on whether they had a bed-partner, and according to their response to an initiat question assessing overall improvement in sleep quality and daytime al'ertness with ncpap, ranging from 0 (minimal/none) to +4 (excellent). patients were called responders if they scored > 2. group a (45 pts, 54%) were responders with bed-partners; group b (10 pts, 12%) non-responders with bed-partners; group c (15 pts, 18%), were responders (12 pts, 14%) and nonresponders (3 pts, 4%) without bed-partners; and group d (14 pts, 17%) had stopped ncpap. nine questions were directed at the bed-partner, and assessed their perception of changes in both the patient's and their own sleep quality, daytime alertness, mood and quality of life, and also to changes in the relationship between patient and bed-partner following institution of ncpap. these questions scored from -1 (worse) to +3 (marked improvement). significant improvement in all parameters for the patient (mean + sd = 2.4 + 0.7) were noted in group a. in addition, group a bed-partners reported ~ubjective improvement in the same parameters (1.7 + 1.1). group b improvements were less, (1.8 + 1.0 in patients, and 0.6 + 1.1 in partners). overall, the data indicate a subjective success of therapy in 68% of patients, but the bed-partner's replies indicate this figure underestimates the true response rate. furthermore, the results show significant improvements in the bed-partner's sleep quality, daytime alertness, mood and quality of life, indicating that successful treatment of osa patients with ncpap also gives significant benefits to their bed-partners. vincent's hospital, dublin. neutrophil collagenase (mmp 8) is a member of the matrixmetalioproteinases (mmps), a family of highly homologous zinc endopeptidases which play a crucial role in many physiological processes. the aim of this project was to develop a purification system for mmp 8 from purulent sputum and raise polyclonal antibodies. after initial extraction, contaminating proteins were removed with a zinc chelate affinity column. mmp 8 was then separated, from another closely related mmp, gelatinase b, on a q sepharose ion exchange column using a nacl gradient. the final purification step was carried out with an orange sepharose affinity column. sds-page analysis indicated the presence of purified protein with bands corresponding to latent neutrophil collagenase (85kd) and products of coll~igenase autodegradation at lower molecular weights (55kd & 57kd). a 10fold increase in specific activity was observed, with a 20% final yield, which provided mg quantities of pure enzyme. this work is funded by forbairt and the. irish american partnership. cd30 is a protein first described as a surface marker on hodgkin's lymphoma cells. recently cd30 has been demonstrated on th2-type t lymphocytes (produce il-4, 5, 6, l0 and 13), which have a pro-inflammatory cytokine profile. but is not found on thl-type t lymphocytes (produce il-2 and ifn-gamma). its ligand, cd30l, has also been described, cd30-cd30l interaction has been shown to aid the development oft lymphocyte clones into a th2 rather than a th 1 phenotype. th2-type cytokines are inextricably linked to the aetiology of inflammatory airway disease. firstly we investigated serum cd30 levels in various patient groups. we have demonstrated significant differences in serum cd30 levels in the following groups, atopic asthmatics (mean = 149 iu/l, n = 62), non-atopic asthmatics (mean = 107 iu/l n = 13) and atopic rhinitis/dermatitis (mean = 90 iu/l n = 36) and normal controls (mean = 14 iu/l, n = 9). secondly we cultured peripheral blood mononuclear cells from allergic individuals and normals. when these cultures were stimulated with house dust mite antigen (der p 1) and il-2 or der p 1 with both il-2 and i1-4, surface expression of cd30 on t lymphocytes could be demonstrated using fluorescent staining and flow cytometric analysis, after 9 days culture in the allergic individuals but not in normals. the presence of i1-4 in the culture increased the degree of surface cd30 expression. these results are important as they show that allergic individuals have an expandable population of memory t lymphocytes which respond to allergen by expressing cd30 and developing th-2 phenotype. most work on cd30 and th-2 cytokines has hitherto been carried out an t cell clones. we have developed a relatively simple in vitro system of looking at t lymphocyte response to allergen which will allow the testing of novel therapeutic interventions with a view to modulating the immune response in allergic disease. our work also suggests that even non-allergic patients with inflammatory airway disease may have increased th2 activity, which has not been shown previously. scimitar syndrome is a rare congenital disorder consisting of a spectrum of abnormalities including hypoplasia of the right pulmonary artery, dextroposition of the heart, anomalous pulmonary venous drainage of the right lung into the inferior vena cava and anomalities of the right diaphragm. bronchiectasis and respiratory tract infections on the right side are the usual clinical presenting features. a 70 year old male patient was referred to the outpatient clinic with a history of recurrent chest infections which were slow to resolve following antibiotic therapy. physical examination revealed decreased air entry, coarse crepitations and a prolonged expiratory wheeze in the right lower lobe. the only abnormalities on routine biochemical and haematological screening were an elevated esr of 69 and a slightly raised white cell count of 15. a chest x-ray revealed hypoplasia of the right lung when compared to the left. in addition to this there was a vascular shadow present in the right lower robe representing an anomalous pulmonary vein which appeared to drain to below the diaphragm on the night side. bronchoscopy showed a normal left bronchial tree. on the right, no apical segment was detected in the right lower lobe. otherwise no endobronchial lesion was seen. a dynamic computerised axial tomographic scan of the thorax was performed. this showed a dilated anomalous right lower pulmonary vein which was clearly seen to enter the inferior vena cava below the diaphragm. in addition the right lung was again noted to be hypoplastic when compared to the left. these findings were pathognomonic of the scimitar syndrome. "the patient was treated symptomatically and is presently stable. conclusion: scimitar syndrome, with its wide spectrum of abnormalities should be considered when reviewing plain chest x-ray in patients with recurrent right lower lobe respiratory tract infection. recent studies indicate that the ability of circulating neutrophils to regulate surface levels of adhesion molecules may be altered in disease situations. the aim of this study was to determine if changes in neutrophil responsiveness accompanies chronic inflammation in cf. neutrophils in blood samples from 17 cf patients and 13 age-matched control subjects were analysed by flow cytometry for expression of l-selectin and mac-1 (cd1 lb) following stimulation by interleukin-8 (il-8) and fmlp. as expected, both il-8 and fmlp provoked a decrease in surface levels of l-selectin and an increase in cdi lb levels. however, the magnitude of these changes was significantly lower in cf patients than in control subjects (table) . these results suggest that chronic exposure to inflammatory stimulii in vivo may alter neutrophil responsiveness in cf. given the emphasis on rational prescribing, we reviewed drug use in a 208 bedded long-stay unit. prescribing patterns were analysed on an appointed day thereby obtaining a "snapshot" of prescribing practices. one hundred and ninety four long-stay residents, with a mean age of 78, were on 1188 drugs, the maximum number of drugs per patient was 12, the minimum 1 and the average 6.1. sixty percent of prescriptions fell within one of the following therapeutic categories:-central nervous system (cns) preparations (321 prescriptions), analgesics (145), gastrointestinal preparations (139) and cardiovascular preparations (i 25). there were 19 ,prescriptions for respiratory drugs and only 36 prescriptions were for antibiotics. the most commonly prescribed cns preparations were anti-psychotics (127), benzodiazepincs (1 t 3), anti-depressants (30). 43% of all analgesics prescribed (63) were nsaids. the most commonly prescribed h2 blocker was cimetidine (33). nuseals aspirin (43), digoxin (35) and captopril (24) were the most commonly prescribed cardiovascular drugs. 25% of drugs were issued on an as required basis, i.e. "prn". the most commonly prescribed prn therapeutic classes were analgesics (100 prescriptions) followed by gastrointestinal (74) and cns preparations (68). these results contrast with prescribing patterns in hospitals and general practice and may provide an insight into the challenges and realities of management in long-stay units. supported by the health research board. evaluation of physician requests to hospital based clinical pharmacist for (1) drug information, (2) possible adverse drug reaction (adr) was undertaken over a two year period from jan '94 to dec '95 (admissions -1,667, opd attendances -5,908). overall 55 requests were made. (1) drug information: advice/information on new drugs, formulation, dosage, safety consideration prior to drug prescribing was given in 23 cases. (2) suspected adr: a total of 32 suspected adverse drug reactions were investigated. in 4 cases, no adr link was established, after extensive literature/data base search. adr's were confirmed in 28 cases of which 11 were reported to n.d.a.b. regular on-going interaction between physicians and clinical pharmacy allowed critical analysis of new drugs and heightened awareness of, potential adverse drug reactions in current clinical practice. we previously demonstrated that commonly prescribed medications are not easily identified by patients, doctors or nurses in the hospital setting o,2~. we then investigated the ability of hospital pharmacists, 33 in all, to identify the same 12 commonly prescribed branded and generic drugs. correct identification as follows:-bendrofluazide k (32/33), cimetidine (33/33), diazepam 5mg (31/33), diazepam 5mg generic (30/33), digoxin (24/33), ferrous sulphate (14/33), frusemide (16/33), mefenamic acid (33/33), paracetamol (27/33), prednisolone (1 l/ 33), temezepam (33/33), theoph3/lline (25/33). pharmacists had 78% correct answers compared with 62% for nurses and 42% correct for doctors. the pharmacists had no difficulty recognising drugs with brand names written on them e.g. cimetidine, but like nurses and doctors had difficulty identifying the plain white tablets e.g. prednisolone. generic drugs were tess well recognised. a number stated that they were unwilling to definitively identify medication with no clear marking. pharmacists were also asked to list the top 5 prescribed drugs, l0 got 1/5 correct and 23 got 0/5 correct. in contrast 36 out of 80 doctors got 1/5 correct, 25 got 2 right and only 4 got 3 right. we conclude that hospital pharmacists are generally better than doctors or nurses at identifying commonly prescribed drugs but their knowledge of the top 5 prescribed drugs is not as good at that of doctors. all professionals need assistant in this important task. suggesting reduced activity or more iranians with inherited variants of cholinesterase. one iranian subject with very low activity (dibucaine number below 20, atypical) had a history of apnea. these data indicate that the frequency of atypical and heterozygote genes for cholinesterase activity leading to prolonged apnea with succinylcholine (suxamethonium) is much higher in iranian than irish populations. this study emphasises the importance of ethnic pharmacology. it is has been advocated that funding for the prescibing of methadone in general practice should be provided separate from the indicative drugs budgeting scheme on the assumption that this may act as a disincentive to g.p.s to take on care of drug addicts. the objective was to analyse the current level and cost of methadone prescribing in 550 general practices in the eastern health board over a six month period. there was a review of methadone prescriptions for gms patients from jan. to jun. 1995. 1,087 persons received prescriptions. 3,945 scripts were issued. the age-specific prescribing rate for the total population was 6/10,000 (males 10/i 0,000, females 3/10,000). males aged 25-34 years had the highest age specific rates (44/100,000). the cost of methadone prescriptions amounted to s for the six months there was a trend towards an increase in the number of g.p.s who prescribed methadone over the period. only four of the 70 g.p.s (5.7%) who prescribed methadone issued in excess of 50 scripts for the period studied. for a small number of g.p.s methadone prescribing is a significant cost item on their budget. in the light of this, government policy should be reviewed with a view to excluding methadone from the indicative drug budgeting scheme. sciences, mashhed, lran. there is increasing evidence that some of the wide variation in the response to medicines has a genetic origin which may be expressed on a racial basis. to further study inter-ethnic differences in pharmacology we compared the activity of an enzyme responsible for the breakdown of endogenous substances and drugs -serum cholinesterase (pseudocholinesterase), dibucaine and fluoride numbers -in 200 irish and 200 iranian healthy subjects. irish subjects had significantly higher serum cholinesterase activity (7.28 + 0.14 vs 5.22 + 0.09 u/ml, mean + sem, p < 0.01 drug prescribing data may reflect changes in therapy and disease pattern. we reviewed current drug use among patients (n = 578) in a dublin teaching hospital in "snapshot" fashion on a designated day, and compared it with that obtained in 1985. in 1985, patients received an average of 7.8 different drugs each, with 41% on 5 or more and 3% on none. by 1995, the average was 6.8 (range i -19), with 62% on 5 or more. the percentage of patients receiving heparin fell from 42% to 13%, due mainly to a reduction in use on the medical side. the proportion of patients prescribed hypnotics fell from 55% to 37%, while ssri's are now the most used anti-depressants. antibiotic choice changed from amp/amoxicillin to coamoxiclav and the cephalosporins. diuretics remained the most frequently used cardiovascular agents, accounting for 4% of all drugs used and prescribed for around one quarter of patients. digoxin use remained constant, and by 1995, 20% of patients were on anti-platelet doses of aspirin. at least four different agents were in use in each of calcium antagonist, beta blocker and ace inhibitor classes. some of these changes in therapy reflect therapeutic advances, changes in disease management, greater choice of therapy and amendment of less than desirable therapeutic practices. on the other hand, some may reflect fashion or pharmaceutical promotion, rather than change as a consequence of evidence-based practice. acknowledgements: pharmacy staff, st. james's hospital and the health research board. studies of in vivo endothelial function in humans have usually involved intraarterial cannulation and the subsequent administration of substances that stimulate the endothelium to produce nitric oxide (no). such techniques are invasive and potentially hazardous. an alternative non-invasive method would be of benefit. animal studies have indicated that reactive dilation of vascular beds may be at least partially endothelium dependent. this study aimed to determine whether reactive hyperaemia in the human forearm was an endothelial dependent process with the potential to be used as a non-invasive method of stimulating the endothelium. ten volunteers underwent brachial artery cannulation and randomly received either placebo or n-monomethyl-l-arginine (l-nmma) (8~mol/min), an no synthase inhibitor, for 10 minutes. following this reactive hyperaemia was induced by the inflation of an arm cuff to 200 mmhg for 5 minutes and the response to this was measured by strain-gauge plethysmography. when flows had returned to baseline the process was repeated with the remaining substance. results were analysed by repeated me~isures anova. l-nmma resulted in significant reduction of basal forearm blood flow (p<0.01). there was no significant difference in reactive hyperaemia with either l-nmma or placebo. in conclusion, no does not contribute to reactive hyperaemia in the human forearm. dublin 8. home-based infusion therapy has been widely recognised as the optimum for treatment of disease states that require daily intravenous therapy from a patient-care aspect. conditions necessitating intravenous therapies in hiv disease include: cmv retinitis, intractable cryptosporidial diarrhoea, azole-resistent candidosis, nutritional support with total parenteral nutrition, chemotherapy for aids-related malignancies and palliative care in the terminal phase of the disease. the need for such therapies is increasing as patient survival improves. in 1993, the home-infusion service was set up in recognition of the need to treat patients, requiring intravenous therapy, in the home environment. this has been brought about by the development of small, light-weight pumps suitable for ambulatory use, the development of a service for aseptic compounding and the availability of permanent in-dwelling venous catheters. we describe the impact of this service on our patient cohort. to date, sixty-five hiv positive patients have received parenteral therapy at home. patients' age, sex, risk group, cdc stage, cd4 count, indication for therapy, complication rate and response to treatment are described. the provision of this service has reduced the number and length of patient admissions with associated improvement of quality of life. in addition, it recognises that patients prefer to be treated in the home environment aided by a co-ordinated multidisciplinary approach. since blood alcohol levels over 80 mg/100 ml are now illegal for vehicle drivers we have investigated if the commonly held "safe" limit of two drinks will bring the young adult over the legal limit and if this amount of alcohol will affect their psychomotor skills. following informed consent 33 healthy volunteers, nonhabitual drinkers on no medication (16 male, 17 female), with a median age of 24 (range 20-27) years participated and refrained from alcohol for at least 2 days. each drank within 15 minutes two standard drinks (35.5 ml each ) of 37.5% vodka (2.7 units of alcohol) plus 60 ml of orange juice at about 90 minutes after a standard mid-day meal. their psychomotor performance was estimated by the number connecting technique at 45 minutes after alcohol consumption and they were also asked to rate their feelings (which included alertness, clear-headedness, competence and attentiveness) using a visual analogue scale of 1 to 10. blood samples at one and two hours later were collected from the antecubital vein and analysed on the same day for alcohol content using enzymatic methods. mean (+ sem) blood concentrations of alcohol at 1 and 2 hours respectively were 25.39 + 6.19 and 21.62 + 4.58 mg/100 ml. in males and 36.43 + 11.89 and 38.66 + 3.43 mg/l-00 ml in females. values were significantly higher in females. blood concentrations in females were also higher (p < 0.01) than in males when expressed per kg body weight. while the blood alcohol in both the genders was considerably lower than the current legal limit in ireland their psychomotor skills as estimated from their task completion time and their answers to questionnaires were indicative of an impaired cns function. thus while 2 drinks may keep many subjects below the legal limit, there is considerable inter-individual variation with females showing higher concentrations and both genders have evidence of impaired performance at these lower levels. a non-linear approach was used to develop an hrv parameter, robust to both data non'-st~itionary and missing data points. unlike the standard chaos approach, using higher dimensional embeddings and time-delays, we employed a onedimensional correlation integral plot. the parameter thus obtained, allows an estimate of the spatial spreading of the attractor (ssa) or spatial variation of rr intervals along a straight line. heart rate data from 18 volunteers (22:00 to 06:00 hr), ~ifter oral placebo or propranolol 80 mg, investigated the ability to detect drug effect. vitamin e (~-tocopherol) is the most important dietary antioxidant in lipid and cell membranes and its intake reversely relates to the incidence of coronary heart disease and certain cancers. estrogen regenerates oxidized tocopherol radical in vitro m but such interaction has not been investigated in postmenopausal women receiving estrogen containing hormone replacement therapy (hrt) although estrogen containing oral contraceptive may reduce plasma vitamin e levep. we studied 21 healthy post-menopausal women (aged 46 -56) ammenorrheic for at least one year. fifteen subjects took a combination of harmogen provera therapy and 6 acted as a control group. blood samples were taken from all subject at baseline and after 4 weeks. in the hrt group, serum fsh levels were greatly reduced (86.46 + 25.27 vs 68.67 + 11.59 iu/1, p < 0.04, mean + sd, after hrt) with an increased serum oestradiol level (< 20 -28.68 vs 603.67 + 343.56 umol/1, p < 0.0001). no change occurred in the control group. vitamin e status, measured either as plasma or red cell ~-tocopherol respectively showed no change in both groups (hrt group 26.48 + 5.42 vs 27.17 + 3.39, 23.79 + 5.19 vs 24.93 + 4.37 gmol/1, p > 0.05). we conclude that in post-menopausal women, 4 weeks estrogen containing hrt did not alter vftamin e concentrations in vivo. we assessed the clinical benefit of the newer markers of bone formation: osteocalcin (oc), procollagen 1 carboxyterminal peptide (picp), bone alkaline phosphatase (balp), and bone resorption: carboxyterminal telopeptide of type 1 collagen (ictp) and urinary deoxypyridinoline crosslinks (dpd) over traditional assays such as total alkaline phosphatase (talp) and urinary hydroxyproline (oh/pr) in 23 patients with primary hyperparathyroidism (phpt). patients were sampled basally, then at 2, 6, 24, 48 and 72 hours post surgery and again at 1.5, 3, 6 and 12 months post op. the mean basal p1cp level was 89+26 ug/l (normal: 38-202) this increased to a peak at 48 h (168+_74 ug/l), then declined to normal at 6 weeks (116+56 ug/l). mean basal urinary dpd levels were raised at 8.6+1.2 nm/mm cr. (normal 2.0-6,0), they had normalised by 6 months to 5.9-+1.4 nm/mm cr. mean balp levels were always normal, although normal the yearly mean oc level was significantly lower than the basal value. mean 1ctp, oh/pr and talp levels were always normal. therefore bone turnover in phpt is best assessed by the newer markers picp and dpd. we have previously described seasonal variation in fibrinogen with higher levels in winter. as fibrinogen is an acute phase reactant, the winter rise may be a response to seasonal infections. the present study investigates this hypothesis by examining seasonality infibrinogen and markers associated with infection: white cell count (wcc), interleukin-6 (1l-6), human herpes virus 6 (hhv6) and herpes simplex virus (hsv) antibodies. monthly blood samples from healthy volunteers age 75 and over were measured for fibrinogen, wcc, il-6, hsv and hhv6 reactivation over a 1 year time period. a rhythmometric method was used to examine the data for seasonality. statistical significance was measured using the fstatistic. a highly significant seasonal variation (sv), peaking in mid-february, was found for fibrinogen (n=24; sv=0.629 g/ 1; f=76.148; p<0.01). no significant seasonal variation was present for measures of wcc (n=24; sv=0.209 e9/l; f= 1.940; p>0.05), hhv6 (n=24; sv=0.065 au; f=0.653; p>0.05), hsv (n=7; sv=6.055 au; f=2.048; p>0.05) or il-6 (n=7; sv=1.235 pg/ml; f=0.558; p>0.05). the present investigation does not support the hypothesis that seasonal variation in fibrinogen is a direct effect of the acute phase response, initiated by a seasonal variation in level of infection. the explanation for the seasonal changes in fibrinogen remains unknown. increased plasma homocysteine and reduced plasma antioxidants are risk factors in the development of vascular disease. design: 131 subjects drawn from elderly people living in the community (median age 85 yr, range 60-102 yr; 81 female). total plasma homocysteine, vitamin c, gamma tocopherol, retinol and beta carotene were measured by high pressure liquid chromatography. homocysteine levels in elderly males [median (range) = 12.35 um (4-18.1), n=241 were significantly higher than in vol. 165, 1996 supplement no. 2 irish journal of medical science elderly females [8.45 um (4.8-24.8), n=30]. these values were also higher than in a younger (30-49 years) male cohort [mean = 7.85 um, n= 610]. no correlations to vitamin concentrations were found, nor was there a correlation to age within the elderly cohort. within the elderly females, a significant negative correlation with age was found in vitamin c, gamma tocopherol and beta carotene (p<0.05). however a significant increase in retinol was noted. a very strong correlation between vitamin c and gamma tocopherol levels was noted in the elderly population sample (p<0.001 after multiple regression). conclusion. homocysteine levels in the elderly are higher than in samples of a younger population. a gender difference is maintained in the elderly. the provision of extended care forms one part of a spectrum of health care for older people. in the eastern health board area all patients over the age of 65 must be assessed by the multidisciplinary geriatric team prior to placement. we report on the experience of the total number of referrals for assessment for extended care to one department of geriatric medicine in a 268 bed teaching hospital. ninety-eight patients listed for extended care in 1994. the mean number of days between listing for long term care and placement was 49 _+ 45 days (range 3 to 206). almost one quarter of patients died while in hospital awaiting long term care: this underlines the frailty of patients who are admitted to hospital and request long term care. two patients were transferred to other institutions and 10 patients were able to get home. of the remaining patients 35 (63%) were placed in statutory or voluntary long term care accommodation and only 37% were eligible (usually financially but in some cases due to significant disability) for nursing home care using the terms of the 1993 nursing home act. patients who are listed for long term care through a general hospital are in general very frail, they tend to have a very extended length of stay and the provisions of the nursing home act only apply to a minority. these findings underline the need for provision of adequate statutory and voluntary extended-care places within the eastern health board area. there are over 40 screening assessments for cognitive function and choosing the most appropriate may be difficult. increasingly the importance of behavioural dysfunction is recognised. can any of the cognitive assessments help to predict behavioural dysfunction.'? we compared and contrasted the folstein mini mental state examination (mmse) and the cognitive assessment schedule (cas) of the clifton assessment procedures for the elderly (cape) and compared them with the behavioural rating scale (brs) of the cape. the study was carried out on a total of 21 referrals to the occupational therapy departments by geriatricians in the meath hospital and st. james's hospital. all subjects were over 65 and medically stable. the time scale involved was may-july 1995. the mmse and the cas were administered within the one sitting and each was timed. brs was rated the same day by either a staff or family, member. the average time to complete the mmse (416 + 124 s) was longer than the cas (342 + 171 s) but this was not statistically significant. the mmse and cas were significantly correlated (r = 8208, p < 0.0001). the cas was significantly correlated with the behaviour scale (r = 0,551, p < 0.01) whereas the mmse was not. these results suggest that equivalent assessments of cognitive function may be made with the mmse or cas, but a low cas score will be a better prediction of behavioural dysfunction. a spectrum of neurological and myopathological changes are associated with patients in intensive care units. we observed several patients post discharge from icu who presented with unexplained dysphagia which we suspected may be associated with the neurological complications of sepsis. the particular complication of dysphagia as a neurological manifestation of sepsis has not been documented. our descriptive study presents a series of three patients with persistent dysphagia which may represent a similar phenomenon. we selected patients for the study ranging from 75-87 years of age and on the basis of medical history including icu stay, sepsis, and intubation. all patients presented with dysphagia as observed on videofluoroscopy. we studied the video findings in-depth in order to ascertain if similar swailow patterns were present in these patients and if this could be correlated with their medical history. each of the three patients presented with similar dysphagia signs. the oral phase of the swallow was moderately atypical but the pharyngeal phase was significantly atypical. it was felt that intubation alone was not the sole causative factor of this dysphagia. the polyneuropathy associated with sepsis in icu may explain the atypical swallow patterns observed in these patients. the severity of the persistent dysphagia can cause serious respiratory and medical consequences. there is a need for further investigation of this phenomenon to identify patients who are at risk. little attention has been paid to the prevaience and phenomenology of behavioural disturbances among medical patients despite awareness of the high prevalence of cognitive vol. 165, 1996 supplement no. 2 impairment in this patient population. we screened 79 consecutive admissions to a department of acute geriatric medicine. patients were evaluated over a 2 week period using a modified version of the brief agitation rating scale. medication use, cognitive function and impact on nursing time were also measured. the prevalence of behavioural disturbance in this population was 19/79 (24%). the most frequent behavioural abnormalities were restlessness (12), complaining (12) and screaming (6). the most common underlying disorders were dementia, stroke disease, personality disorder and paranoid psychosis. the behavioural disturbance was only documented in the medical notes in 7 patients (37%) and in only 5 cases was a psychiatric consultation sought. these findings demonstrate that behavioural disturbances are not only common but also under-documented in elderly medical patients and there is a need for training in the detection and management of behavioural symptoms in this patient group. in lower limb trauma where there is severe compound fracture, the successful treatment of this depends on adequate bone and soft tissue debridement. as a result, subsequent bone defects can lead to instability and often require large amounts of bone grafts, and major soft tissue reconstruction is reaquired to obtain skin cover. large soft defects can by reduced bv primary bone resection and shortening of the limb. this will improve the chance of bone healing if performed in the presence of an external fixator, then lengthening at a site away from the traumatised area can gradually restore limb length. two cases are presented to demonstrate .the effect of compression / distraction techniques on soft tissue and bone injuries in these difficult situations. wegener's granulomatosis -wg (15), churg strauss syndrome -css (4), polyarteritis nodosa -pan (2) and unclassified (5). using the chc definitions, the diagnoses were wg (5), microscopic polyangiitis -mpa (10), pan (1) and undefined (10). there was concordance in only 5 patients (all wg). there is significant discordance between these two criteria sets. since the acr criteria does not recognise mpa, they tend to overdiagnose wg. in addition, the chc criteria cannot be applied without a biopsy and therefore surrogate features which predict the underlying histology are required to allow more practical application of the chc definitions. the objective was to determine the value of examination of dried freshly produced saliva, under light microscopy, in patients with xerostomia related to secondary sjogrens syndrome. ten patients with known connective tissue disease or rheumatoid arthritis attending rheumatology clinic were enrolled into the study, all with symptomatic xerostomia and dry eyes. all had an abnormal schirmer's test. five normal patients were enrolled, all of whom were without clinical evidence of rheumatological disease. control patients were enrolled who had no clinical evidence of rheumatological disease, a salivary sample was collected and examined by light microscopy. serum was also examined for the presence of anti-ro/la, rheumatoid factor, and anti-nuclear factor. all ten patients demonstrated 'reindeer horn' type ferning of saliva, a pattern of shorter thicker clubbed branches of crystallised mucus, in contrast to the normal ferning pattern of the healthy subjects. conclusion: we have shown in this preliminary report that light salivary microscopy is a simple test easily performed in an outpatient setting which could be a useful diagnostic procedure in sjogrens syndrome. recently, two sets of criteria have been proposed for the nomeclature of primary vasculitides, the 1990 american college of rheumatology (acr) classification criteria and the 1992 chapel hill consensus conference (chc) definitions. the aim of this study was to determine the concordance of these two systems in a cohort of patients with primary systemic vasculitis. patients with systemic vasculitis were recruited who had a biopsy proven diagnosis or, who had typical clinical features associated with a postive antineutrophil cytoplasmic antibody (anca). the case notes were reviewed and patients were classified according to both sets of criteria. twenty-six patients were recruited, 21 of whom had a positive biopsy. applying the 1990 acr criteria, the diagnoses were, primary pulmonary hypertension (pph) typically affects young individuals, and has a high morbidity and mortality. secondary pulmonary hypertension complicating connective tissue diseases likewise carries a poor prognosis. we evaluated the acute and chronic effects of ketanserin, a selective serotonin type-2 receptor antagonist in 2 patients with pulmonary hypertension in the acute study ketanserin was administered as a peripheral venous infusion during right heart catheterisation. following encouraging results during catheterisation oral administration of ketanserin 160mg daily in divided doses was instituted. in patient 1, a 30 year old female with probable pph, serial cardiac catheterisations over a 1 year period showed a significant, sustained reduction in both mean pulmonary artery pressure from 49 mmhg at baseline to 24 mmhg at 1 year (normal 10-20 mmhg) and pulmonary vascular resistance 1118 units at baseline to 288 units at 1 year (normal <200 units). in patient 2, a 61 year old female with limited scleroderma (crest) echocardiography after 1 month's oral ketanserin showed a reduction in estimated peak right ventricular systolic pressure from 60 mmhg at baseline to 13 mmhg (normal range 15-30 mmhg). the acute and long term response to ketanserin with improyement in pulmonary haemodynamics in these patients suggests that if a beneficial effect is detected during catheterisation long term oral therapy may be worthwhile. levels were low (< 7.9 iu/1); normal though above average (>-8 -<10 iu/l) and moderate high (>10 -<15 iu/l) respectively. gonadotrophins for ovarian stimulation were commencing initially at 225 iu for group a & b and at 450 iu for group c. ivf performance was poor in most aspects (total follicles, oocytes & embryos transferred) in group b comparing with group a or c; the cumulative ongoing pregnancy rate (pr) over 2 ivf cycles in group b; was 15% comparing with 27.6% in group a (p < 0.05) however there was no significant difference in pr in group c (20%) comparing with other two groups. cycle day 3 fsh screening is predictive of follicular development in ivf. high initial dose of gonadotropins help to improve the pregnancy rate in the presence of moderate high level of fsh. the purpose of this study was to evaluate patient satisfaction with antenatal care provided in the perinatal day centre (pndc). a self administered questionnaire was administered to 61 consecutive patients. the main indications for referral were suspected small-fordates (34%), non-proteinuric hypertension (23%), glucose tolerance testing (15%), reduced fetal movements (10%) and post-term evaluation (7.5%); 51% were nulliparae. thirty-two percent of patients were reviewed in the pndc on the day of referral; the rest within 7 days. twenty eight percent of patients lived more than 10 miles from the hospital and 48% spent more than 30 minutes in travelling there. eighty five percent of patients scored their level of satisfaction with the service provided in the pndc as > 7 out of 10; only 6.5% would have preferred admission; 42% said that they would prefer to visit the pndc 5 times per week to avoid admission. the main area of dissatisfaction related to the waiting time for review prior to discharge, with 69.5% of patients waiting over 3 hours. patients attending the pndc report a high level of satisfaction; changes to reduce the visit duration have been introduced. to examine the change of taking-up the essential preconceptual measurements; rubella immune status, cervical cytology and prophylactic folic acid intake; following specific advice and publicity through 3 general public meetings with new patients prior to in vitro fertilization (ivf) programme. in 1994 we studied 281 new couples for ivf for the presence of some specific pre-conceptual data (group a). in this study we follow-up the same intake in another 229 new women interviewed to commence ivf programme from january 1995 till september 1995 (group b). in group (b) the taking-up measurements were dramatically improved. however, 30% and 18% stilldid not have rubella immunity test and cervical cytology performed; compared to 54% and 36% in group (ai respectively (p<0.001). while folic acid intake was sustained at >90% in both groups. following specific advice the rate of taking-up of preconceptual measurements prior commencing ivf programme was improved. there is a future need for continuous enhancement of the publicity and advice regarding the importance of preconceptual measurements. the aim of this study was to introduce icsi to ireland for treatment of specific cases of male factor infertility. following an introductory proving period using the bovine model, thirtyeight couples with infertility attributed to the male were selected for an icsi attempt. ovulation induction, oocyte retrieval and luteal management were as described for conventional ivf tm. the average age of patients selected for icsi were 34.5 + 3.7 years and 36.4 + 4.6 years for the female and male respectively, with an average duration of infertility of 4.8 + 2.6 years. a 64 year old woman presented with a three day history of parasthesia in her lower limbs and difficulty walking. neurological examination revealed sensory loss in her limbs and truncal ataxia. rombergs sign was positive. pelvic examination revealed a large pelvic mass that was distinct from the uterus. routine blood investigations were normal. csf culture, ct brain and serum electrophoresis were negative. anti-purkinjie cell antibodies were not present. ca-125 levels were elevated at 159 micrograms/litre. laparotomy revealed a 20 cm left ovarian tumour. a total abdominal hysterectomy, bilateral salpingo-oophorectomy and omentectomy was performed. histology revealed a poorly differentiated clear cell adeno-carcinoma of the left ovary. the capsule was intact and peritoneal washings were negative. she made a good postoperative recovery. she received six courses of carboplatin without ill effect. her neurological symptoms resolved. subacute cerebellar degeneration can occur as a paraneoplastic disorder in ovarian carcinoma. the mechanism by which cancer can cause neurological disorders is not fully understood. paraneoplastic cerebellar degeneration occurs with or without the presence of purkinjie cell antibodies. the aim was to review all red cell transfusions in gynaecological surgery in 1994. a retrospective review of blood bank records and individual charts was carried out. 4611 patients underwent gynaecological surgery; 97 (21%) were cross matched and 60 (1.3%) were transfused. 184 units were transfused. there were no single unit transfusions. the mean number of units transfused per patient was 3. this accounted for 33% of all units transfused this year. 92% of patients were undergoing elective surgery. the overall cross match/transfusion ratio was 2. intraoperative difficulty was recorded in 56% of cases. 64% of patients were transfused perioperatively and 36% postoperatively. the percentage of patients requiring blood transfusions in the the main individual operation categories was as follows: radical surgery: 57%; total abdominal hysterectomy and salpingo-oopherectomy, 17%; vaginal hysterectomy and repair, 16%; subtotal abdominal hysterectomy, 30%; vaginal hysterectomy alone 12.5%; and total abdominal hysterectomy alone 5%. adverse reactions to transfusions were seen in 5% of patients. conclusion: the majority of patients transfused were undergoing elective surgery. vaginal hysterectomy was associated with greater blood loss than abdominal hysterectomy. only half of all units cross matched were transfused. dilatation and curettage (d+c) is the most common operation performed in the u.k. the liberal use of d+c has been criticised. the objective of this study was to evaluate the use of outpatient endometrial pipelle biopsy and determine its safety in terms of detecting abnormalities. complications and financial costs were also evaluated. data was reviewed from an active gynaecological unit from february 1993 to january 1995, using theatre and outpatient records. a total of 303 d+cs and 104 endometrial pipelle biopsies were performed in this period. 9 malignancies were detected by d+c and 1 by pipelle biopsy. a total of 24 and 3 benign abnormalities were detected by each method respectively. there was a higher complication rate in the d+c group but the failure rate was higher in the endometrial pipelle biopsy group. the monetary savings over this period is estimated at s there were no missed malignancies to our knowledge over the 8 year period since endometrial pipelle bioposy was introduced to this hospital. our study indicates that outpatient endometrial pipelle biopsy appears to be safe, efficacious and economical. while ultrasound findings may sometimes be in conflict with clinical examination, it is the case that there are instances when ultrasound findings have, following subsequent laparotomy, been found to be wholly incorrect. it is therefore not surprising that there remain some gynaecologists who view ultrasound with scepticism, preferring to rely solely of their clinical findings. there have been few studies that directly compare clinical, ultrasound and surgical findings in the detection of pelvic masses. the objective of the study was firstly to directly compare the reliability of clinical and ultrasound examination findings in the detection of pelvic masses proven by subsequent laparotomy and secondly to determine the accuracy of ultrasound in detecting malignancy. this was as a retrospective review of 86 women who underwent a laparotomy because of a pelvic mass between january 1993 to february 1995. information was obtained from theatre and patient records. real time abdominal ultrasound was used. findings at laparotomy were correlated with clinical and ultrasound findings. the sensitivity and specificity of ultrasound in detecting a uterine mass was 94% and 99% respectively. this contrasts sharply with clinical examination (sensitivity = 74% and specificity = 94%). similar findings were obtained when ultrasound was compared to clinical examination in detecting ovarian masses. ultrasouud is capable of predicting benign disease with reasonable confidence but the prediction of malignancy is less reliable. in conclusion, ultrasound is more sensitive and specific in detecting pelvic masses compared to clinical examination. vincent's hospital, dublin. osteoporosis occurring during pregnancy or lactation is a rare event despite the homeostatic demands of the foetus for calcium. we investigated the case of a 24 year old woman who, immediately following vaginal delivery of her first child, developed severe back pain due to a vertebral compression deformity of the second lumbar vertebrae. bone mineral density (bmd) was measured by dual-energy x-ray absorptiometry. calcium metabolism and bone turnover were studied. there was a severe reduction in bmd in the spine (z-score = -4.8) and f e m o r a l neck ( z -s c o r e = -3.6); but, serial measurements showed no further reduction in bmd. indices of calcium metabolism and bone turnover were normal. pregnancy-induced osteoporosis is a severe but self-limited disorder in calcium homeostasis of unknown aetiology. women with low bmd prior to pregnancy may be at increased risk. in view of increased demand, supplemental calcium and vitamin d should be considered during pregnancy and lactation. crumlin, dublin 12. the aim of this study was to assess the clinical status on admission and the critical care m a n a g e m e n t of children p r e s e n t i n g with m e n i n g o c o c c a l i n f e c t i o n . t h i s was a retrospective study of the charts of 46 consecutive admissions. mean age was 3.43 years (+3.46). the average duration of symptoms prior to admission was 20.4 hours (+11.09). on admission 17.4% were hypotensive, 45.6% had clinical signs of haemodynamic instability and 54.8% of cases that had a wood gas analysis on admission had a metabolic acidosis (bases excess < -5.0). the mortality rate was 10.9%. 80% of deaths were hypotensive on admission and all had a metabolic acidosis. of the 41 survivors 9.7% were hypotensive on admission, 39% had clinical signs of cardiovascular compromise, 78% were admitted to the high dependency unit, 25% required invasive pressure monitoring and 7.3% were ventilated and received inotropic support. in this study children presenting with m e n i n g o c o c c a l infection have a high incidence of cardiovascular instability. successful management is dependent on early presentation and initiation of therapy and on aggressive intensive care monitoring and support of the cardiovascular i11 and vital organ systems. the normal crying curve and incidence of colic for term infants are well known. we studied prospectively the crying pattern and the incidence of colic in preterm'infants to determine if prematurity influenced these behaviours. the subjects were consecutive preterm infants admitted to the cork neonatal units for two and a half months from july 1995. a continuous 24 hour diary was completed on each infant by the neonatal nurses, when the babies were on full oral feeding and no longer required intensive care. the parents completed the diaries 'after discharge. colic was defined according to wessel's rule of threes. two unwell babies were excluded. the duration of follow up was from 2 to 16 weeks. fifty infants were recruited and 45 completed the study (4 lost to follow up and one withdi'awn due to sepsis). their mean (range) gestational age was 31 (26-36) weeks and birthweight was 1.52 (0.64-2.4) kg. the mean (range) age of crying onset was x(y-z) weeks; crying, peak 'was x(y-z) weeks and crying offset was x(y-z) weeks. one baby developed colic in the period of follow up. conclusions: the incidence of wessel's colic was less than expected in these preterm babies. the crying pattern according to chronological age was different from that clescribed in term babies. in general preterm babies had a delayed onset of crying, but the pattern became similar to term babies when allowance was made for gestational age. the findings'suggest that the crying patterns of early infancy have a developmental basis. we re-evaluated 18 children who had had rs (1979-86), with their closest-age siblings using the wechsler scales, coopersmith self esteem inventory and achenback child behaviour checklist (acbc) (duffy j. et al 1991). the rs patients' means were consistently lower than that of their sibs. however, comparison of mean raw data, using "t-tests", yielded significant differences only in the acbc scores, in that rs children exhibited significantly more problem behaviours than their sibs (p=0.033). after categorisation of iq data, further comparisons between the groups (using x2), found rs patients were significantly more likely to score "below average" in tests of verbal iq compared to their sibs (p=0.04). age of onset and clinical stage were also found to be more important predictors of outcome. children less than 1 year of age at onset of rs had significantly lower iq scores on all measures of cognitive ability (p=0.003) and more problem behaviours (p=0.01) than children over 1 year of age. no significant differences were found in comparison with sibs. clinical stage to which rs progressed affected only verbal iq scores. children in whom consciousness had been impaired had significantly lower iq scores than both their sibs and rs children in whom consciousness was less impaired (p=0.02). in conclusion outcome remains cautiously positive, with 13/18 rs children attending mainstream schools or in employment without apparent difficulties. a national breastfeeding policy was introduced by the department of health in 1994. factors identified for promotion of breastfeeding were based on the who/unicef "ten steps for successful breastfeeding". we present clinical cases which suggest that one step may need to be modified. the charts of breastfed babies admitted to the special care baby unit were reviewed for one year following the introduction of the national breastfeeding policy to this hospital. thirteen term breastfed babies were admitted because of fever and dehydration. none of the babies had water or bottle feed supplements. ten of the thirteen mothers were primigravida. eleven babies were admitted in the six months following the introduction of an exclusive breastfeeding policy. the nursing staff were then alerted to the risk of dehydration, but two further babies of mums committed to exclusive breastfeeding were admitted in the subsequent six months. routine biochemistry, haematology and a limited septic screen was performed in all babies. three of the thirteen babies had lumbar punctures. the mean (range) weight loss on admission was 9.5 (4.1-16)%. the mean (range) plasma sodium level was 147.6 (138-156)meq/1 and the mean (range) urea was 6.5(2.1-13.4)meq/t. there was no growth from the cultures of the blood, urine, csf and swabs. all the babies were given intravenous fluids and parenteral antibiotics for 48 hours. the outcome was satisfactory in all babies and breastfeeding was reestablished in eleven of the thirteen babies. conclusions: the common factor to these babies was inadequate fluid intake prior to admission associated with stricl~ adherence to the policy, and avoidance of all supplements including water. we conclude that the who/unicef step 6 "not to give food or drink other than breastmilk unless medically indicated" is too restrictive in the immediate postpartum period. 42.5% of children reported headache in the previous 12 months 44.8% of girls and 40.5% of boys reported headache (p < 0.0001). 0.6% of children reported daily headache 5.9% of children reported weekly headache 9.4% of children reported monthly headache 22.5% of children reported headache less often than monthly the percentage of children with headache at each frequency, other than daily, increased with increasing age. in girls headache showed a marked rise at ages 14, 15 and 16 years. reported prevalence of headache in the past year in 5 to 15 year old aberdeen children was 66% and 48% was recorded for swedish children aged 8, 11, 13 and 17 years old. this study is the first community based prevalence study of headache in irish schoolchildren. our aim was to test the hypothesis that there is no correlation between the type of feeding & swallowing disorder the child has with the neurological diagnosis or the radiological findings. a further purpose was to develop a classification of the feeding & swallowing disorders which would guide us towards a management plan. a retrospective analysis of the data collected between the years 1988-94 from the feeding & swallowing clinic at booth hall children's hospital was done. 73 children were included in our study 9 ages ranged from 4 months to 17 yrs. all the children were assessed by the members of the feeding & swallowing team and had videofluroscopic assessment by the same radiologist. neurological signs, speech therapy assessments & videofluroscopy findings were compared between children with spastic quadriparesis & those without. significant differences were noted. a clinical classification was devised using cluster analysis. we conclude that there is no causal relationship between the neurological diagnosis & the type of dysphagia. there are three distinct groups of children who require different strategies of clinical management. surveillance commenced in january 1995 to continuously monitor the incidence of surgical site infections (ssi). employing modern optical scanning technology (formic for windows version 2.0, formic limited, london) a questionnaire was designed, which required minimal completion time. the questionnaire includes relevant data based on the american national nosocomial infections surveillance system for ssi including the ssi risk index. surveillance commences at the time of surgery and continues until the patient's discharge. optical scanning technology allows rapid reading of surveillance questionnaires thereby bypassing the bottleneck of manual data entry. by october 1995, details of 2,156 procedures had been recorded. the crude ssi rate for these patients was 2.7%. the patient risk index used demonstrated that there were increased chances of developing ssi in certain patient groups. seventy-nine per cent of ssi had presented by the 10th post-operative day. the length of stay increased by an average of 4 days in patients developing ssi. regular feedback to individual surgeons, theatre and ward staff maintains awareness and highlights possible problems. we recommend optical scanning technology to all those engaged in surveillance work. this system would be especially useful were data collected is transported from outlying hospitals to a central receiving centre for collation and analysis. in 1872 francis crumpe published a paper ~ in which he described the therapeutic effect of poisonous mussels (psp) on a case of tetanus. he obtained the mussels from tralee ship canal on the occasion of its infrequent emptying, and entertained the idea of using them in tetanus after treating a young girl with psp who recovered after 24 hours. prior to the use of psp he described it's paralytic effect in two cockrels who both recovered. in concluding his successful use of psp he speculated as to the clinical nature and role of the toxin tralee ship canal was opened in 1846. the water was relatively stagnant and would have contained plenty of nutrients. as such it would have been an ideal habitat for toxic algae which may have been brought across the atlantic as spores in bilge water 2. the emptying of the canal may well have been done at times of algal blooming. this is the first irish report of psp and a most remarkable use of saxitoxin (?) in the treatment of tetanus, antedating the current management by seventy years. this study was carried out to quantify the published research on smoking in irish medical journals; to ascertain the type of research carried out; and to identify the authors of that research. during the 35 years under study, 50 papers explicitly dealing with smoking were published. only 2 papers appeared in forum. there was a decline in published papers in the eighties with a resurgence in the early nineties. of the 50 papers, a majority were observational and ten were editorials. only one paper dealt with smoking cessation, and one with preventive work. general practitioners were poorly represented as authors. one doctor (prof. r. mulcahy) published at least one paper on smoking in each quinquennium since 1960. this study underlines the relative insignificance of smoking as a topic for research in ireland. a major sea change in attitude will be required if the government's targets for smoking cessation are to be realised, particularly if they are to be achieved by relying on the medical profession. radiology represents a major cost centre within a hospital. lack of awareness of cost amongst doctors may result in the inappropriate requesting of radiology services. this study assesses doctors knowledge of the cost to a tertiary referral hospital of commonly performed radioj'ogical procedures and investigations. doctors were asked to estimate the cost of 9 items namely -chest,x-ray, arch aortogram, ultrasound abdo., lumbosacral spine, barium enema, ct brain, ct abdo., ultrasound abdomen, i.v.p. and percutaneous gastrostomy tube insertion under radiological screening. 60 doctors in st. vincent's hospital were surveyed. doctors as a group overestimated the cost of all 9 individual tests, by margins ranging from 10% (i.v.p. & gastrostomy insertion) to 100% (ct brain/ct abdo.) the total cost to the hospital of all 9 items was s consultants'overestimated this total cost by 107%, followed by registrars, interns and s.h.o's who overestimated the total cost by margins of 51%, 43% and 12% respectively . conclusion: doctors tend to overestimate what radiological procedures and investigations cost a public hospital, often by quite wide margins. thus, any excessive requesting of radiology services by docto.rs is not due to a lack of awareness of their true cost to the hospital. (ded) in dublin. secondly, to identify the major cancers contributing to years of potential life lost (ypll) for the ehb, each cca and ded in males and females. in ireland little work has been done to date on disease specific premature mortality. crude death rates weight all deaths equally; in comparison, ypll emphasise deaths among younger persons and provide a measure of the burden of premature mortality. premature mortality for the 322 deds in dublin for the years 19988 and 1989 was estimated using ypll, which was calculated by subtracting the date of death form 65. ypll due to each of the major disease groups were ranked for each ded. seventy-one thousand four hundred and sixty-eight &471468) years of potential life were lost in dublin in the 1988 and 1989. 21.9% of ypll was due to injury and poisoning, 20.64% to cancer, 15.6% to circulatory disease. however, when the major cause of ypll was established for each ded, injury and poisoning was the number one cause of death in 25.5% of the deds; cancers 24%, congenital and perinatal conditions 21.5% and circulatory disease 14%. by emphasising deaths in younger individuals ypll is a valuable tool for planning and monitoring local health promotion initiatives. serum total homocysteine (thcy) levels are inversely associated with dietary intake of folic acid and b vitamins and raised levels have been linked with chd. we have examined the association between thcy concentration and the risk of chd in middle-aged men in 18 british towns. we used a nested case control study design, within an ongoing prospective study, thcy concentration was measured in serum samples, stored at entry to the study, from 110 incident cases of myocardial infarction and 118 controls. cases and controls were frequency matched by town and age group. levels of homocysteine [geometric mean (95%c1) were significantly higher in cases than'controls: homocysteine 13.5 (12.6 -14.3)gmol/l vs 11.9 (11.3 -12.6)lamol/l; p = 0,005. there was a graded increase in the relative risk (odds ratio; or) of chd in the 2nd, 3rd and 4th quartile of thcy (or 1.4, 1.9, 2.2; trend p = 0.006) relative to the first quartile. adjustment for age, town, social class, body mass index, smoking, physical activity, alcohol intake, hypertensive status, serum cholesterol and serum creatinine did not attenuate this association, (or 2.1,2.3, 2.7; trend p = 0.04). the findings suggest that thcy is an independent risk factor for chd with no threshold level. in summer 1995, a diagnosis of cryptosporidiosis was made 25 in a child who had visited a pet farm. this child had participated in a summer project involving 161 children and nine adults. reports of a similar illness among other project members, prompted an outbreak investigation. a cohort study consisting of two phases was initiated. 95% (162/170) of project participants responded to a self-administered questionnaire in the first phase. thirteen children met the case definition, of whom seven had cryptosporidium detected in their stools. illness was significantly associated with having visited a pet farm. (p<0.000005). 75% of those ill sought medical attention, of whom two were hospitalised the second phase of the cohort study was conducted among those who had visited the pet farm. 95% (52155) were interviewed. illness was significantly associated with play in sand, to which animals had access, at a stream's edge beside a picnic area (p<0.005). contact with various animals was not statistically significantly associated with illness. however the small numbers involved may have obscured such an association. this outbreak highlights a potential hazard for children visiting pet farms and also that cryptosporidiosis is a significant but often overlooked cause of morbidity in healthy children. managers of pet farms need to be aware of the potential for transmission of disease to visiting children. strict implementation of hygiene measures is essential to minimise risk. the mrc vitamin trial highlighted the importance of folic acid in the prevention of neural tube defects(l). since 1993, the department of health has recommended periconceptional folic acid supplements. the objective of this study was to document the knowledge and behaviour of women in child bearing years to periconceptional folic acid. a cross sectional community survey was conducted using an interviewer administered questionnaire in dublin. three hundred and thirty five women took part in the study. approximately two thirds 213/335 (63.6%) had heard of folic acid. knowledge was significantly associated with higher social class and higher education (p<0.o5). few 18/335 (5.4%) had been advised to take folic acid before pregnancy. only 9/335 (2.7%) of the women in the study were currently taking folic acid supplements. three quarters of the group (75.9%) would be willing to take periconceptional folic acid supplements if they knew it would reduce the risk of malformations. the majority (77.4%) would prefer to take folic acid in tablet form. this study clearly shows that few women in childbearing years have been advised on folic acid. however, if advised appropriately the majority would be willing to take periconceptional folic acid in tablet form. future publicity campaigns involving all health professionals should address these issues. unstable intra-articular fractures with or without dislocation of the phalangeal joints often lead to joint stiffness ana loss of function. nine patients with comminuted intra-articular phalangeal fractures were treated in our unit by dynamic external fixator using "pins and rubber bands traction system". the mean age was 32.3 years, and the follow-up average was 4.6 months. five patients had full and good range of motion in the involved joints. three patients had poor results, and one patient underwent open reduction one week following the original procedure. the technique and our results are discussed. this dynamic frame is compact, comfortable for the patient, easy to apply and allows early mobilisation. careful selection of patients and close follow-up in the first few weeks are needed. this study examines how gp's store and handle vaccines. all 144 gp's in a health board region were invited to take part. gp's were interviewed in their practice premises about how they dealt with vaccines fridges were examined and temperature recorded post interview. oral polio was taken from 20 randomly selected fridges for potency testing. cold chain monitors and freeze watch indicators were used to monitor batches of vaccine stored. of the 144 gp's, 142(98.6%) agreed to participate, 140 used 111 fridges to store vaccine, 2 store vaccine at room temperature. of the 111 fridges, 6 (5.4%) had the power supply safeguarded, 9 (8.1%) had thermometers, 36 (32.4%) had vaccine only stored therein. during defrosting, vaccine was inadequately protected in 22 (19.8%). of the 138 gp's who use multi-dose vaccine vials, 133 (97.2%) keep them for further use at the end of a day/session, 3 store them at room temperature. 42 (37.8%) fridges had temperatures outside the recommended range. 13 (28.2%) coldchain monitors indicated vaccine exposed to more than 10~ 18 (90%) of the oral polio samples showed a reduction in total titre of live virus, however, none were below the minimum acceptable. this study indicates that vaccine potency could be seriously compromised due to breaks in the cold-chain and suggests the need for guidelines to be drawn up, implemented and monitored to ensure the integrity of immunisation schemes. comparison was made with a study carried out in 1983. in addition the range of antimicrobial agents tested included new oral cephalsporins and quinolones that were not then available. three hundred microorganisms isolated from mid stream urine (msu) samples were examined by standard microbiological techniques. antimicrobial susceptibility testing to 12 antimicrobial agents was performed on significant pathogens (>105 organisms per ml) by disc diffusion test, and minimum inhibitory concentrations of antibiotics was carried out by e test on organisms found resistant by disc testing. by comparison with 1983 resistance amongst e.coli, the most commonly isolated pathogen, had increased for the following: ampicillin by 6% to 61%, co amoxyclav by 4.4% from 0%, trimethoprim by 18% to 28% and nitrofuradantin by 5% from 0%. no increase in resistance occurred to cephradine (4%), or nalidixic acid (5.4%). resistance to cefixime, ofloxacin and ciprofloxacin, was 3%, no resistance was encountered to cefotaxime. for proteus species resistance to ofloxacin, ciprofloxacin and cefotaxime was 4%, and for enterobacter sp 20%. enterococci were sensitive to ampicillin and augmentin but the numbers were small. pathogens isolated from patients domiciled in the inner city were significantly more resistant to nalidixic acid (20%), cefotaxime (6%), cefixime (14%), ofloxacin and ciprofloxacin (12%) than those isolated from patients in rural areas. the purpose of this study is to examine the relative importance of obstetric complications (0c%) in the aetiology of schizophrenia and mania. using the dublin psychiatric case register, birth records of 635 patients with an icd-9 diagnosis of schizophreniaor mania were obtained. these records were evaluated, for obstetric complications using two scales, the lewis, owen and murray scale (lom) it~ and the parnas scale 121. the mothers of those going on to develop schizophrenia did not differ from those going on to mania as regards maternal age, parity, social class, or period of pregnancy. however, males who developed schizophrenia when compared to males developing mania, experienced significantly more oc's when rated by the lom scale (p=0.007) and.more frequent oc's on the parnas scale (p<0.001) of greater severity (p=0.018). no significant differences were found between females with schizophrenia and those with mania. dublin. the aim was to evaluate the diagnosis, symptomatology and level of functioning of patients presenting with a first episode of psychosis to a catchment area service and a private psychiatric hospital. all patients presenting with a first episode of psychosis were assessed using the scid-p,,the positive and negative syndrome scale (panss) and the global assessment of functioning scale (gaf). fifty-eight patients (34 male, 24 female) ranging in age from 15 to 65 years (mean + sd = 28.4 + 10.8) were included in the study. the mean total panss score was 84.8 (sd + 21.6) and was strongly correlated with the gaf score (p < 0.001) but independent of age (p = 0.16). males had a significantly lower gaf score compared to females (p = 0.04) but there was no gender difference in the total panss score (p = 0.20), twenty-five patients (43%) had a lifetime prevalence of drug abuse or dependence but only 12 patients (21%) had signs of drug abuse or dependence in the month prior to presentation. level of functioning was strongly influenced by the severity of psychopathology. substance abuse is common in individuals presenting with a first episode of psychosis. the aim was to evaluate the presence of involuntary movements in patients presenting with first episode psychosis to a catchment area service and a private psychiatric hospital. patients presenting with first episode schizophrenia and schizophreniform psychosis were assessed for involuntary movements using the involuntary movements scale (a.i.m.s.). 28 patients (17m., llf.), age range 15-67 years (mean=30.5 years.) were included in the study. one patient (3.5%) satisfied the strict criteria of schooter and kane for spontaneous dyskinesia. 7 patients (6m., if.), had minimal involuntary movements in at least 2 body areas, predominantly orofacial. the total a.i.m.s. score was positively correlated with the number of days spent in hospital per year of follow up (p=0.01). the group with involuntary movements were found to have spent more days in hospital per year of follow up, 94 v. 55 days (p=0.047). for patients with first episode schizophrenia or schizophreniform disorder spontaneous dyskinesia is not common. however involuntary movements at presentation may be a predictor of poorer outcome. psychiatry has moved from custodialcare towards care in the community. adequate reprovision will have to be made in order to discharge the remaining continuously hospitalized patients. the objectives of this study were to describe a.n entire long-stay hospital population, to examine the differences between the old and new long stay groups within this populatian and to evaluate the needs for community residential and day care facilities in order for hospital closure to take place. the study group consisted of the total long-stay population of st. davnet's hospital, monaghan. the patients were assessed using the community placement questionnaire (cpq) one hundred and twenty four patients were included in the study. fifty-six were female and 89% were single. the mean age of the total group was 68.7 years. the majority suffered from schizophrenia. the assessment revealed a globally disabled group with multiple handicaps. the new long-stay group were disabled as the old long-stay group. the patients were characterised into four groups with regard to placement recommendations. these were a specialist unit for chronically disturbed geriatrics, a geriatric unit, a high support hostel and a medium support hostel. the remaining population of this hospital were highly dependent with multiple handicaps but would live in community with adequate support. there is little difference between the needs of the old long-stay and those of the new long-stay. failure of the immune system to identify self peptides is likely to lead to the development of an autoimmune reaction. susceptibility to autoimmunity is strongly influenced by genes clustered in the hla region (chromosome 6p) particularly class i (a, b and c) and class ii (d, q and p). it has been suggested that there is an autoimmune component in the aetiology of schizophrenia. of many conflicting reports from case/control studies using hla antigens the most consistent finding has been an increased frequency of hla-a9 (now split into a23/a24). additionally, a susceptibility locus for schizophrenia has been reported near the hla locus. to attempt to confirm the hla a9 hypothesis, we have genotyped a preliminary sample of 63 familial schizophrenic probands and 77 unrelated controls at the hla-a region, using a pcr-ssop technique. the frequency of hla-a24 (the major component of a9) in patients and controls respectively was 12.5% vs 15.5%. these findings do not support the hypothesis. some of the discrepancy may be due to unspecific cross-reactions produced by commercial antisera used in the microlymphocytotoxicity method of previous studies. however it is also possible that the hla associatton with schizophrenia may reflect linkage disequilibrium with unidentified gene(s) within the hla region which is less strong in the irish population. schizophrenia is a common mental disorder affecting about 1% of the general population with a devastating disturbance of mind and personality. family, twin and adoption studies have demonstrated that the disease is largely genetic with a polygenic mode of: transmission. dopamine receptors have been implicated in the aetiology of the disease. as yet 5 dopamine genes have been identified (d1-d5). in particular the d3 receptor is expressed in the limbic regions of the brain, implicated in the control of emotions. association studies of a d3 polymorphism (glycine to serine substitution at position 9,) with schizophrenia have produced conflicting findings, many of which, however, have demonstrated a significant excess of homozygosity, or excess of the 1 -l genotype at this polymorphism. in this study, 200 familial schizophrenics and 239 irish unrelated controls were genotyped. the result show a small increase in the frequency of the 1-1 genotype which did not attain statistical significance (patients, 41.5% vs. controls, 38.5%). homozygosity (alleles 1-1 and 2-2) was also slightly increased in the patients (patients, 55.5% vs. controls, 48.9%). the small increase in the frequency of the 1-1 genotype and of homozygosity in the patients is in keeping with earlier findings but suggests that the effect, if any, of d3 sequence variation in the development of the disease is small. lack of information about general practitioners' (gp's) ability to prescribe psychotropic medication may affect patients' compliance. in this study, 73 out of 75 patients attending a psychiatry out-patient clinic completed a questionnaire which documented how many had run out of medication, the steps taken if they had and the role each patient thought their gp played in their treatment. 82% indicated that their gp knew what their current medication was but only 46% thought that their gp could provide them with a prescription if they did not have one from the clinic. this figure was similar in those who had (21%) and had not (79%) run out of medication in the past. on running out of medication, 42% of patients waited until their next appointment, 29% attended their gp and the remainder either contacted the department or went to a chemist. in conclusion, many patients do not appreciate the entitlement of their gp's to prescribe psychotropics for them. literature regarding whether or not the social class distribution of patients with psychiatric illness may differ from the general population remains controversial. we sought to clarify this by examining social class at the time of birth, to see whether patients with serious psychiatric illnesses (schizophrenia and mania) differ from the general population. paternal occupation of 450 schizophrenic patients, and 77 manic patients, from the dublin psychiatric case register, were obtained from birth registration details and categorised according to central statistics office criteria, the same-sex previous live birth was used as a matched control. there was no difference between the social class of patients with schizophrenia or mania (p=0.32). neither patients with schizophrenia (p=b.31) nor mania (p=0.153) differed from controls in social class distribution. paternal social class was found to be related to amount of time spenr in hospital (p<0.001', mean=439.62), and educational age (p<0.001, mean=15.01) and "age at onset of the illness" (p=0.03, mean=31.59). these results suggest that social class of origin may not be related to the development of either schizophrenia or mania. however, social class of origin may be relevant in terms of presentation of schizophrenia for treatment. cognitive function is widely recognised to be impaired in schizophrenia but there is an ongoing debate as to whether this impairment is generalised or localised, progressive or static, similar in both sexes, or related to symptoms. using the positive and negative syndrome scale (panss)'we measured psychopathology in 48 chronic in-patients (27m, 21 f; mean age 68.0+12.7) who satisfied feighner criteria for schizophrenia. subsequent to this, we assessed their global cognitive function using the mini-mental state examination (mmse) and their frontal cognitive function using a new instrument, the executive interview (exit). poor performance on the exit was associated strongly with increasing severity of negative (r=-0.74, p<0.001) but not positive (r=--0.09, ns) symptoms,'in both males (r=--0.73, p<0.001) and females (r=--0.76, p<0.001). overall exit performance declined modestly with increasing age (r=--0.32, p<0.05) but this phenomenon was co'nfined to females (r=--0.64, p<0.01; males: r=--0.13, ns). mmse performance was also associated with negative symptoms (r=--0.67, p<0.001) but decreased mm:e prominently with age (r=--0.59, p<0.001) and showed no gender difference. frontal dysfunction in schizophrenia appears to be intimately related to negative symptoms over the course of severe chronic illness, and may reflect among males a more static' trait deficit than is accessed by the mmse. this study was supported by the stanley foundation. while determinants of the course of schizophrenia are unclear, emerging evidence suggests that the longer psychosis proceeds unchecked before initiation of anti-psychotic therapy, the poorer may be long-term outcome. we have reported that, among older in-patients, increasing duration of initially untreated psychosis in the pre-neuroleptic era was associated with a deterioration to a state of muteness (after controlling for intervening variables). the current survivors of this population have now been examined more extensively using the positive and negative syndrome scale (panss), the mini-mental state examination (mmse) and the executive interview (exit). among these 48 patients (mean age 68.0+12.7), after controlling for age and for the duration and continuity of subsequent antipsychotic treatment, increasing duration of initially untreated psychosis was associated with greater severity of negative symptoms (p<0.005) and with lower scores on the mmse (p<0.05) but not with executive dysfunction on the exit (p=0.3). increasing duration of initially untreated psychosis appears to be associated with the evolution of more prominent negative symptoms and cognitive impairment in a manner consistent with an active, morbid process in schizophrenia that can be ameliorated by anti-psychotic drugs. this study was supported by the stanley foundation and the health research board. patients who are selected and who agree to participate in the royal college examinations play an important role. as psychiatrists and exam organisers, we should be aware of the potentially stressful experience which this might present. the purpose of our study was to elicit attitudes to the exam, and also knowledge of the examination procedure. a questionnaire comprising 10 questions was circulated to 30 patients who had participated in the royal college examinations. responses were received from 28 (93%) of the 30 patients. there were 14 males and 10 females in the responding group. none of the patients had previously participated in the examinations. all of the respondents (n=28) felt that the candidate had been polite towards them during the interview. 38% (n=9) of the patients were nervous prior to the examination, and this group was predominantly female (n=6). 21% of the patients (n=5) did not know that they would receive payment for their participation. 67% (n=l 6) did not know that they might be physically examined as part of the examination procedure. 17% (n=4) of the patients described experiences which had been upsetting for them the results of our study suggest that on the whole patients tolerate the exam procedure quite well. one of the central issues concerning physiotherapists in stroke rehabilitation is the emergence of abnormal tone. rehabilitation involves re-establishing a normal postural control mechanism (ncpm)"~. abnormal tone may develop in the presence of severe sensory and proprioceptive loss. the patients' attempts to move and find a stable base can lead to compensatory movement patterns and asymmetrical postures. positioning is used by physiotherapists to influence the distribution of muscle tone and facilitate symmetrical postures. it is essential that the patient is made aware of his 'position in space' as failure to do so presents no feedback regarding movement resulting in inertia ~2~. standardised positioning charts have been used in hospitals. the physiotherapist liaises with nursing staff regarding correct use on a 24 hour basis. this study looked at the role of a more individualised approach to positioning in the form of a photograph. patients were randomised to two positioning groups group a standard vs group b photograph, and their positioning was scored by a 'blinded' research physiotherapist over an eight week period. the results of a pilot study on a small number of patients revealed that nursing staff preferred a positioning chart individually tailored to the patient's problems. from a physiotherapy perspective, improved postural awareness correlated with better positioning scores in group b. the prevention of compensatory movements, posture is critical in stroke rehabilitation, the use of an individualised positioning chart requires further evaluation. we discuss the case of a fourteen year old boy who presented with bilateral ptosis present since birth, microcephaly and pigmentory retinopathy"!. he was found to have mild facial and proximal limb weakness. creatinine kinase and ldh were raised. muscle biopsy showed ragged red fibres consistent with a mitochondrial myopathy ~2~. electron microscopy showed abnormal mitochondria. the term mitochondrial myopathy describes a diverse range of clinical disease ~3~ and this is discussed. she developed a vasculitic skin rash with pruritis and oedema associated adenopathy, low grade fever and mouth ulcers. lab tests showed leucocytosis, eosinophilia and abnormal liver function. skin biopsy indicated an inflammatory picture without vasculitis. ct thorax confirmed axillary and para-aortic adenopathy. lymph node biopsy confirmed a reactive lymphadenopathy. the aim of. this study was to assess the characteristics of patients referred for pudendal nerve studies over a one year period. 31 consecutive patients were asked a standard questionnaire and nerve studies were performed as described by kiff and swash m and swash and snook~2k of the 31 patients, only 3 were male. the age range was from 21 to 79 (mean 50). 10 presented with constipation and 19 with faecal incontinence. two had both symptoms. bladder incontinence was presented in 10 of 31 patients. of these, faecal incontinence was the cardinal symptom in 8 patients, constipation in 2. 12 patients were nulliparous. of the remaining 17, 14 had a history of complicated births involving forceps (7), caesarean section (4), post partum haemorrhage (1), breech without forceps (2) . 14 patients had pelvic surgery and one had major trauma. 22 of 31 patients had bilaterally delayed pudental nerve terminal motor latency (pntml). 7 of 31 people had unilaterally delayed pntml, 2 were right sided, 5 were left sided. 2 had normal studi~s. the range of measurements was 1.8 -9.2 ms with a mean of 3.65 ms. in conclusionl delayed pntml was seen in 11 of 12 patients with constipation and 19 of 20 with faecal incontinence. pelvic surgery and a complicated obstetric history were significant. urological symptoms were also a common association. the p50 component of the middle latency-auditory evoked potential is attenuated in response to the second of paired clicks in a normal population. in schizophrenia, this attenuation is minimal. in alzheimer's disease (ad), the results have varied between centres depending on the frequency of the stimuli and the interclick interval. we studied 10 ad patients, 10 elderly controls (ec) and 10 young controls (yc) using a paradigm of 32 sets of paired clicks. in contrast to previous studies, our study demonstrates significantly larger absolute p50 generation and recovery amplitudes in ad patients compared to elderly controls and young controls. the purpose of study was to establish a simple screening vol. 165, 1996 irish journal of supplement no. 2 medical science test to identify asymptomatic intracranial aneurysms (icas). an association between atherosclerosis and icas is recognised. elevated serum lipoprotein (a) [lp(a)] is an independent risk factor for atherogenesis. we aimed to assess the degree of correlation between serum lp(a) and the occurrence of sporadic ruptured aneurysms and familial asymptomatic aneurysms. lp(a) levels were measured in (a) 50 patients with icas and 42 normal controls, (b) 25 first degree relatives of patients with familial subarachnoid haemorrhage (sah). icas were detected by cerebral angiography. patients with sporadic icas had significantly elevated lp(a) levels when compared with matched controls. mean level was 20.1 mg/dl in patients and 10.8mg/dl in controls. in the familial studies, 10 out of i1 subjects with asymptomatic icas had elevated lp(a) levels. one young female with elevated lp(a) had a pre-aneurysmal dilatation at operation. six out of 14 subjects without icas had elevated lp(a) levels; four of these were in the second or third decade of life and may yet develop aneurysms. conclusions: lp(a) has potential as a biological marker for icas. follow-up studies are required on angiographically negative subjects. we have begun a genetic case-control study to establish if particular apoprotein (a) gene polymorphisms can be correlated with the occurrence of icas. post mastectomy breast reconstruction has undergone several changes in the recent years. attitudes have changed towards the problem from both the patient and the reconstructive surgeon, as aesthetic outcome receives a greater emphasis than previously. there is a shift towards using autologous tissue as a means of reconstruction; these new technically difficult procedures entail a longer learning. centralization of this type of reconstruction in highly specialized centres only will serve the patient better. we share our experience of post mastectomy breast reconstruction spread out over the past five years. seventy-eight consecutive cases of breast reconstruction are included in the study. different techniques of breast reconstruction were used with a recent switch to transverse rectus abdominis myocutaneous (tram) flap; we feet that tram flap is the gold standard of breast reconstruction as far as the ultimate cosmetic result is concerned. ours is only a moderate sized study compared to some published, yet it is representative of the experience of most of the plastic surgery units in the british isles. clinically significant paraneoplastic neurologicaldisorders are rare, most are associated with small cell lung, female genital tract and breast carcinoma. the malignancy is often silent and the neurological manifestations vary from encephalomyelitis, cerebellar degeneration, sensory neuropathy to neuromuscular block. prognosis is usually poor. pathogenesis is thought to be related to cross reaction with neurons of antibody produced to tumour antigens. detection of these antineuronal antibodies in serum has assisted diagnosis of paraneoplastic encephalomyelitis in which anti-hu antibodies are present and cerebellar degeneration, in which anti-yo are found. in our lab, we used avidin-biotin-complex immunocytochemistry to detect anti-hu (cortical neuron antibodies) and anti-yo (purkinje cell antibodies) in patients' sera. tests were performed on human frontal cortex and cerebellum, at 1 in 500 and 1 in 4,000 dilutions, with positive and negative controls. of 68 sera, 6 were positive. two patients had repeat positives; in one, antibody titre rose in the second sample. subsequent patient review showed 3 positives (2 patients) had identifiable carcinoma with paraneoplastic cns signs, 2 had no identifiable malignancy but had no other cause of their cns disorder and are being followed up; details of one patient were unavailable. these results are similar to other centres. the proliferation of tumour cells despite the presence of tumouricidal mediators could be due induction of a heat shock response, a universal cellular defence mechanism in host cells and possibly tumour cells. protection may be mediated either by increasing intracellular levels or surface expression of heat shock proteins (hsp). the aim was to assess the effect of heat shock induction on tumour cell protection against host effector cells. the heat shock response was induced in sw707 colorectal cells by either sodium arsenite (0-320~tm for 6hr) or by hyperthermia (42~ for 20 rain). monocyte (m0)-mediated cytotoxicity or flow cytometry to evaluate surface expression of hsp60 and hsp70 were assessed. cytotoxicity showed a significant decrease in all treated groups (p<0.002) when compared to the control value. there was also a significant decrease in all groups (p<0.001) when compared to the 42~ value. no significant alteration in surface expression of either hsp60 or hsp70 was seen. conclusion: heat shocking tumour cells significantly protects them from m0-mediated tumour cell lysis. since the flow cytometric data indicate that there is no concomitant increase in surface expression of hsp60 and hsp70 on the tumour cell following heat shock, it can be inferred that induction of intracellular hsp levels are responsible for the protective effect on the tumour cells. a 4 week qol study in 157 consecutive advanced cancer patients was undertaken to compare the subjective 28 question fact-g with simple subjective global tools (visual analog, categorical scales: vas, cas), objective tools (spitzer qli and ecog performance status) and verbatim patient description. we anticipated the high drop out rate enrolling 157 to achieve 87 complete study patients for statistical purposes. the study sample appeared representative of the advanced cancer population in the usa. generally qol was satisfactory despite the severity of illness. there were significant differences in all measures between those who described qol in verbatim responses as positive and negative, particularly cas, vas, and qli (p<0.0001). there were significant intercorrelations between qli and ps (observer rated), vas and cas (subject rated) respectively (p<0.0001). taking patient description as the gold standard, simple, global qol measures e.g. vas or cas are as effective as multidimensional ones (fact-g and qli). irish journal of medical science males had more dysphagia. survival from diagnosis was greater for females </=65 years (female median 18 months, male median 13 months, p<0.0016). anorexia was associated with reduced survival since diagnosis in the total population and in males. anorexia anddysphagia were associated with reduced survival from referral. advanced cancer patients were polysymptomatic. symptom prevalence differed with age, gender and cancer site. the pattern of g1 symptoms was related to gender and severe weight loss. specific symptoms were associated with reduced survival. there was a gender difference in survival favoring females. the administration of recombinant interleukin-2 (rll-2) is limited by the induction of increased microvascular permeability. the in vivo antineoplastic effects of taurine in combination with rll-2 were investigated and its impact on the associated vascular leak was examined. lung metastases were established in mice via tail vein injection. ten days after injection mice were randomized into treatment groups. on day 18 the mice were sacrificed; lungs were removed, weighed and metastases counted. treatment of tumour-bearing mice with rll-2 alone resulted in a significant reduction in tumour nodule incidence compared to a control group, while the~group recei'~ing rll-2 + taurine showed an even fuither reduction in the incidence of lung nodules. animals receiving rll-2 showed a significant increase in mean wet lung weight compared to control lung weight, while mean wet lung weight of the rll-2 + taurine group was significantly less than that of the rll-2 group.and comparable to control values. animals receiving ril-2 + taurine in vivo demonstrated significantly enhanced splenocyte-mediated antimelanoma activity ex vivo compared to animals receiving rll-2 alone. thus taurine may have an important role in modulating both metastatic growth and the associated toxicity of rll-2 immunotherapy. a prospective analysis of symptoms in 1,000 patients on initial referral to the palliative care service of the cleveland clinic was made using the paradox relational database. the median number of symptoms per patient was 11 (range 1-27). the 10 most frequent symptoms were pain 82%, easy fatigue 67%, weakness 64%. anorexia 64%, >10% weight loss 60%, lack of energy 59%, dry mouth 55%,'eonstipation 51%, dyspnea 51% and early satiety 50%. patients 65 years and under had more pain, sleep problems, depression, anxiety, vomiting and headache (all p<0.01 ).'the prevalence of early satiety, nausea, vomiting and anxiety were greater in females; dysphagia 3nd hoarseness in males. patients with >/= 10% weight loss had more gi symptoms; of these females had more nausea, early satiety; the progn.o'stic significance of abnormalities in the p53 tumour suppressor gene and in the expression of its protein in colorectal carcinoma may be influenced by the method of analysis used. we studied p53 abnormalities in 66 patients with colorectal cancer followed for more than 10 years. single-strand conformation polymorphism analysis (sscp) was used to detect alterations in exons 5-8 of the p53 gene. paraffin sections were examined immunohistochemically for p53 overe,xpression with the monoclonal antibody do-7 (dako) both with and without microwave antigen retrieval. abnormalities of the p53 gene were found in 41% of cases by sscp analysis but were unrelated to age, sex, tumour size or differentiation. outcome was unrelated to sscp abnormalities (p=0.19). overexpression of p53 protein was seen in 47% of cases by immunohistochemistry without microwave antigen retrieval and in 52% of cases with microwaving. poor long-term survival was related to immunohistochemical expression of p53 protein either with (p=0.03) or without (p=0.02) microwave antigen retrieval. these results suggest that immunohistochemical detection of the p53 protein product may be more useful than sscp analysis of the encoding p53 gene in identifying those at high risk of colorectal cancer recurrence and death. dublin 9. the anti-tumour activity of tumour infiltrating lymphocytes (tils) is known to be poor and therapeutic manipulation of these cells has met with little success. suppressor macrophages (smo) influence t cell cytotoxicity and proliferation. we hypothesized that smo are a component of the lymphoreticular infiltrate and that these cells may be related to lymphocyte numbers within the tumour. tgf-b may influence macrophage phenotype. colorectal and breast tumours were obtained within an hour of resection. tumours were dissaggregated with collagenase and dnase for three hours. antibodies was used to identify smo (rfd 1 and rfd7) and t cell subsets (cd4 and cd8) by flow cytometry on the resulting cell suspension. pre-op blood was collected from patients and tgf-b levelsdetermined by elisa. conclusions: we have shown for the first time that smo, defined by the antibodies rfd1 and rfd7, are present within breast tumours. we have also shown that the balance of t cell subsets is different in these tumours and may be related to smo content. circulating tgf-b levels are increased in breast cancer and associated with greater smo numbers. this was not found to be the case in colorectal cancers. these results imply the existence of a fundamental difference in the make-up of the lymphoreticular infiltrate between these cancers. swelling of the upper limb is an uncommon but well irish journal of medical science recognised complication of breast cancer treatment. in severe cases, patients have limited arm function and feel disfigured. in a pilot study, the incidence of arm swelling following complete axillary clearance in the immediate post-operative period and at long term follow-up was investigated. arm volume measurements were performed using an opto-electronic volometer (bosl medizintechnick, hamburg). both ipsilateral and contralateral arm volumes were assessed. the expected volume of the ipsilateral arm volume was calculated using the formula vr = v1 = 31 mls for right handed people and v1 = vr = 25 mls for left handed people (vr and v1 = volume of the right and left arms respectively). the difference between the expected and actual volume of the ipsilateral arm was expressed as a percentage of the expected volume. twelve patients undergoing axillary clearance for breast cancer were prospectively evaluated pre-operatively, 48 hours and 5 days post-operatively. a second group of 48 patients who had had axillary clearance at least 3 months previously (range 3 to 116 months) were also evaluated. there was no significant change in arm volume in the immediate post operative period. clinically detectable arm welling was found on 3 patients who had undergone axillary clearance at least 3 months previously but none had any impairment of arm function. we conclude that axillary clearance can be performed safely and that arm swelling is an uncommon complication. a larger study is planned to investigate factors such as the influence of pectoralis minor division, duration of the operation and the number of axillary nodes retrieved on upper limb volume. epidermal growth factor (egf) is a potent mitogen and has been shown to accelerate healing of epithelial damage both in the skin an.d the gut. in the skin egf is not produced locally as the requisite mrna is not present but egf receptors are present on the surface of basal keratinocytes. egf is produced in various sites in the gi tract including the submandibular salivary glands. we have hypothesised that as there is upregulation of salivary egf production in some enteropathies a similar situation may occur in disorders of the skin with an associated enteropathy. using a sensitive radio-immunoassay, egf activity was estimated in stimulated saliva from 87 patients with various skin disorders, 49 patients with gastrointestinal disease, 8 patients with mixed dermatological and gut disease and 41 normal healthy volunteer controls. elevated egf activity was found in the following groups of patients : skin cancers, psoriasis, acne, oesophagitis and ulcerative colitis. the hypothesis of up-regulation of salivary egf production in skin associated enteropathy was rejected but the discovery of elevated egf activity in skin cancers and psoriasis may have aetiological and therapeutic implications. the malignant fibrous histiocytoma (mfh) is considered an uncommon malignancy. its potential for invasion, metastasis and death of patient has been reported in literature. it can be confused with other tumours including fibrosarcoma. salient histologic features include cells of both the fibrocytic and histiocytic series. mfh with its high recurrence rate and lethal potential merits an aggressive evaluation and treatment. we present unusual case of recurrent mfh treated in our unit with an open question as to what qualifies to be adequate primary surgical excision. the recommended management of localised merkel cell carcinoma has been wide surgical excision, combined with adjuvant radiotherapy in selected cases. the risk of recurrent regional disease is reported to be between 30% and 45%. a 70 year old woman with merkel cell tumour on the cheek is presented; this patient was treated exclusively with radiotherapy to a total dose of 50 gy over 18 days. the tumour regressed rapidly during the treatment, and there were no signs of local or regional recurrence. the patient is still alive and free of disease for 45 months. immune in origin with a heightened cutaneous immune response to ultraviolet light. the coexistence of cad and pbc is a new association which has not previously been documented and may not be fortuitous given the similar pathogenesis of both diseases. chronic actinic dermatitis (cad) is a rare photosensitive disorder which primarily affects elderly men resulting in an eczematous reaction to ultraviolet-radiation and sometimes visible light. the pathogenesis has been attributed to an autoimmune process, possibly in response to a photoallergen which has yet not been identified. we report a 71 year old female patient who developed cad four years after being diagnosed with primary biliary cirrhosis (pbc). abnormal monochromator irradiation tests were detected with narrow band ubv, uva and in addition visible light wavelengths. phot0provocation tests induced florid vesicular eczema and multiple patch and photo-patch tests were positive, findings typical of cad. immunoglobulin g was elevated at 2290 mg/dl and liver histology was typical of pbc with an elevated anti-mitochondrial antibody. routine biochemical and immunological tests were ~ormal and porphyrin screen was negative. azathioprine 50 mg/day induced remission of cad. pbc is an auto-immune disorder where cell-mediated immunity is impaired, suggesting that sensitized t lymphocytes may cause damage to bile ducts. the pathogenesis of cad may be autowe present two cases of cutaneous polyarteritis nodosa (pan) associated with seronegative arthritis: the first patient, a 50 year old male presented in 1993 complaining of a7 year history of pain, stiffness and swelling affecting his right ankle. he also noted intermittent tender nodules on the dorsum of his foot and over his ankle over the preceding three years. the second patient, a 51 year old male, presented .in 1994 complaining of a 5 month history of tender nodules on his shins, and pain and swelling of his right ankle. skin biopsies of the nodules in both cases showed medium vessel vasculitis consistent with polyarteritis nodosa. neither patient had any symptoms or signs to suggest systemic involvement. the only abnormality.0n laboratory investigations for vasculitis was elevated esr. x-rays showed periosteal elevation and new bone formation in case 1, and were normal in case.2. bone scan demonstrated increased uptake at the talo-navicular joint in case 1 and at the fight ankle in case 2. synovial biopsy and mri confirmed the presence of an inflammatory arthropathy in patient 1. joint involvement has been a prominent feature throughout the course in both cases requiring aggressive treatment with vol. 165, 1996 irish journal of supplement no. 2 medical science cyclophosphamide and systemic corticosteroids in case 1. cutaneous pan is a localised cutaneous vascular disorder with a benign chronic relapsing course. in one reviewl, 12 of 23 patients had arthralgias but an association with arthritis has not been emphasized in the literature to date. we conclude that this condition may present as a seronegative arthropathy in which the joint symptoms may be the most prominent feature and aggressive immunosuppresive therapy may be required for control. cardiac transplantation patients have an increased risk of skin disease. in our centre, 119 heart transplants were performed with a 5 year survival of 75%. eighty three patients are now alive and 39 have required dermatological assessment. the mean age of patient was 48.3 years, (range 11-64 years); 99 males, 20 females. skin infections were diagnosed in 13 of 39 patients. drug side effects, including sebaceous hyperplasia and steroid acne, were common. in the patients who developed skin cancer, mean time from transplant to development of lesions was 3.1 years. eleven of 36 patients had 32 non melanoma skin cancers (nmsc), 28 squamous cell carcinomas (scc), 4 basal cell carcinomas (bcc), giving scc/bcc ratio of 7:1. three of 11 patients had multiple skin cancers, one had 12 tumours. nine of 39 patients had actinic keratoses, two thirds of whom had sccs. nineteen of 39 patients had viral warts, two of whom had sccs. viral warts, premalignant and malignant lesions were located on sun exposed sites. skin complications of cardiac transplantation though mild were very common. the observed incidence of nmsc in age matched cardiac transplant recipients, appears much higher than the expected incidence of 171.38 per 100,000 population (national tumour registry 1994). regular dermatological assessment of cardiac transplant patients is necessary to detect skin disease and early skin cancer. the increased incidence of warts and skin cancer in renal transplant recipients {rtr} is well known. the oncogenic potential of unusual human papilloma virus {hpv} types has been postulated from warts and in both premalignant and nonmelanoma skin cancer (nmsc). the possible etiological role of sun-exposure in facilitating the development of hpv associated skin disorders is also suggested. a clinical study to assess the risk factors for development of these lesions in 50 rtr attending the dermatology servic& age and sex matched haemodialysis patients were similarly examined as controls. 40 male and 10 female patients with a mean duration of transplant of 10.6 years, range 1 to 25 years. a total of 191 nmsc (range 1 to 57),175 scc and 16 bcc, ratio 10.9:1, were excised from 26 rtr of which over 95% had viral warts, mosle commonly occurring on sun exposed sites and always predated the development of neoplastic lesions. both were associated with mean duration from transplantation, 4 years for warts and 10.8 for skin cancer and not the type of immunosuppressive treatment. none of the control patients had similar findings. conclusions: the close clinical association of viral wart lesions and development of skin cancer in these patients suggests a close relationship to immunosuppression, in addition to exposure to ultraviolet radiation. this study highlights the high rate of nmsc in rtr. these patients justify early and regular skin assessments soon after transplantation with advice on sun protection. sensitivity to ultraviolet (uv) light may be established by exposure to broad band uva and uvb radiation. the minimal erythema dose (med) can be determined at individual wavelengths using a monochromator. uv action spectra of photosensitive disorders may thus be constructed. we examine the value of this process in distinguishing two clinically similar photosensitive disorders. the radiation from a xenon arc is separated into component wavelengths using the monochromator. each wavelength is focused on unaffected skin, on the patient's back. the patient is exposed to a range of doses of w radiation. the med is determined for a series of wavelengths from 300 to 400 nm. chronic actinic dermatitis (cad) and drug induced photosensitivity are photosensitive disorders which may have similar clinical history and presentation. ten cad and 14 drug induced photosensitivity patients were tested. uva photosensitivity was seen in 71% of the latter group. the remaining 29% had normal mlts as the implicated drug had been discontinued prior to testing. cad patients were sensitive to both wa and wb radiation. forty-three percent of these patients were also sensitive to visible light (400 to 800 nm). monochromator light test (mlt) results show that uva photosensitivity dissociated from wb photosensitivity is indicative of a drug induced light sensitive disorder. sensitivity to both wa and wb however indicates a diagnosis of cad. mlts can therefore distinguish between clinically similar photosensitive disorders. bartholomew's hospitals, london. patients with mpd have an increased incidence of both thrombosis and haemorrhage suggesting a pivotal role for; platelets in these conditions. this study aimed to examine platelet activation antigen expression in stable patients with mpd and to examine the predictive value of these antigens prospectively. 52 patients with mpd had p selectin and gp53 measured using a refined minimally manipulative flow cytometric technique. expression of p selectin -median 5.1% (inter quartile range 2.1-10.9), control 2.0% (1.3.-4.0) and gp53 -median 4.0% (1.8-5.7), control-2.1% (1.0-2.9), were significantly elevated p<0.01. patients were followed for a median of 26 months. 15% experienced thrombosis and 6% bleeding during follow up. at entry to the study 48% of patients had previously experienced thrombosis, median disease duration 8 deaths, 3 of which were caused by thrombotic events in which the mpd was a major risk factor. increased expression of p selectin or gp53 expression failed to predict thrombosis or bleeding in this study. nor was any significant retrospective relationship demonstrated. however, previous thrombotic events were strongly associated with future events (p<0.05). this association was independent of disease, duration, age and medication. not surprisingly disease duration was also correlated with thrombotic/bleeding events. taurine levels fall in gut mucosal cells during critical illness. however, taurine transport into human intestinal cells is poorly understood. the aim was to establish the efficiency of taurine uptake by enterocytes, and to examine uptake under stressful conditions. to investigate efficiency of taurine uptake, confluent caco-2 cells were incubated for time points up to 10h. in a second study, cells were incubated for 9h with medium containing dexamethasone and / or cytokines. media for both studies was supplemented with [3h]-taurine. radioactivity was related to mg/ml protein to calculate rate of taurine uptake for each time point. study 1: uptake exhibited a steady linear response which approached saturation at 7h. maximal uptake occurred at 9h after which the rate levelled off. study 2: dexamethasone alone reduced taurine uptake by 75.4% (p<0.001) and in combination with tnf-c~ and ifn ~/ it decreased transport by 66.3%. (p<0.001). lps alone impaired uptake by 56% (p<0.001). conclusion: we have established the time course over which taurine transport reaches its maximum rate in caco-2 cells, and that corticosteroids and cytokines significantly impair uptake of taurine in these cells. elderly individuals have an increased risk of infection suggesting that immune responsiveness is altered with age. changes in the level of proinflammatory cytokine production may be an important indication of any such age related change. using flow cytometry we examined intracellular tnfet, il-lf~ and il-6 in pbmcs from normal healthy volunteers of different ages ranging from 22 up to 85 yr (n=28). tnf and il6 levels from pma stimulated cd3 positive cells (t cells) were shown, using this technique, to increase in an age dependent manner (p<0.05). no il-113 was detected in any t cell sample. no significant differences were observed between the different age groups for tnfa, il-1 g or il-6 in cd 14+ cells (monocytes). the age related changes detected by flow cytometry have been confirmed using conventional elisas. this novel method of proinflammatory cytokine detection has detected increased tnf and il6 levels in t cells from elderly healthy volunteers which may help explain some of the exaggerated inflammatory responses seen in elderly patients. detection of proinflammatory cytokines by conventional elisa or bioassay is problematic due to the presence of naturally occurring biological inhibitors. flow cytometry allows the simultaneous detection of both intra and extracellular antigens thus intracellular cytokine levels can be quantified while cell surface markers allow cell type identification. a range of monoclonal antibodies were examined for tnfcx, il-lg and il-6 using saponin permeabilisation oft cells (cd3), monocytes (cd14) and epithelial cells (ber-ep4). t cells and monocytes were grown in ~culture, t~p :to 72 hr with or without pma activation, and intracellular cytokine levels were shown to increase with time, with the stimulated samples producing more cytol<ine than the spontaneous.samples (p<0.05). elisas performed on these same samples (n--:24) correlated well with the flow cytometric results (r=0.52). tnf~x, il-113 and il-6 are detectable !n monocytes while only tnfa and il-6 have been detected in tcells and epithelial cells using this methodology. this novel technique will allow us to identify the cytokine producing cell while also avoiding the influence of the extracellular millieu, thus being useful for a number of research and diagnostic applications. all the viral hepatitides are said to be common in the middle east. hepatitis a viru s (hav) seroprevalence is said to be 100% in the adult population of gulf countries. hepatitis b, c and d seroprevalence are taken from blood banks data which are biased by selection criteria and the background of blood donors. very little is known about hev in the uae. vol. 165, 1996 supplement no. 2 irish journal of medical science the aim was to investigate the seroprevalence of hav, hbv, hcv, hdv & hev in the uae. we prospectively recruited volunteers drawn from all the uae, socioeconomic classes and from different age groups in both sexes. each donated 10ml. of venous blood and using an indirect enzyme immunoassay were tested for total ig to hav; hbsag using monoclonal antibody t.o hbsag; for hcv antibodies using recombinant antigens hc-34, hc-43, c-100 and ns-5, (3rd generations); for total ig to hdv and for total ig to hev. positive samples were retested in duplicate for confirmation. this large sero-epidemiological study clearly shows that hav is decreasing in the uae. hbv is low but significant, hcv is similar to southern european countries, hev is lower than expected and hdv does not exist in hbsag carriers in the uae. reactive oxygen and nitrogen species produced by inflammatory cells plays a role in tissue injury and inflammation. taurine, a bamino acid present in high concentrations in leukocytes functions as an anti-oxidant by scavenging the mpo derived oxidant, hypochlorous acid, to form taurinechloramine. experimental colitis was induced in male sprague-dawley rats and randomised into two groups. one group received tap water whilst the second group was supplemented with taurine (4%). these groups were sub-divided into those receiving tnbs/ ethanol (n=8) or saline (n=8). an increase in wcc count, particularly neutrophils was found in colitis. tat~rine was found to reduce the severity of the disease (ns) usin.g a colon macroscopic score. in the first group, respiratory burst activity was reduced in animals that had developed colitis (p<0.05) although mpo activity was augmented (p<0.05). nitric oxide production was 2.2 and 1.8 fold respectively higher in inflamed and non-inflamed areas of the colon than that by normal colonic mucosa. animals supplemented with taurine demonstrated attenuated m po activity (p=0.001). administration of taurine did not lead to a reduction in nitric oxide production. thus, taurine appears to have a beneficial role in reducing the severity of disease. a reduction in mpo and respiratory burst may be beneficial in reducing tissue damage. to women subsequently identified by a national screening programme which revealed 438 to be positive for rna of hepatitis c, type lb. 94 were referred to this centre for evaluation. 10 had moderate activity by modified knodell index, mean 8 (_+1.4), bridging fibrosis and a mean alt of 77 (42-176). six months dual therapy was administered. two patients withdrew due to depression (2) and hyperthyroidism (1). one patient di d not respond. seven responded with normalisation of alt and negative pcr. eight patients showed improved histology (kai 4 +_ 1.5), fibrosis was unchanged. adverse reactions in the treated group included marrow suppression (1) and thyrotoxicosis (1) . at six months follow up four remain in remission with normal alt negative pcr, three have relapsed, (pcr positive, mean alt 87(50-150). we conclude that despite adverse prognostic indicators in this homogenous group with chronic hepatitis c, lb and long disease duration, dual therapy with interferon and ribavirin achieved results comparable to prolonged high dose interferon in patients with more favourable initial prognoses. the aim of the study is to evaluate a 5 day course of azithromycin in combination with omeprazole in eradication of h.pylori. twenty-four patients with proved h.pylori infection and peptic ulcer disease were treated with omeprazole 40 mg daily for 2 months plus azithromycin 500 mg daily for 5 days. h.pylori was diagnosed on the basis of clo test and histology. this was done at the beginning and 3 months after the treatment. eradication was defined as negative clo test as well as negative histology. out of 24 patients, 2 patients were lost to follow-up. only 21 patients were analysed. sixteen patients responded to treatment and had successful eradication as well as healing of their ulcers. these included 8 patients with gastric ulcers (79.95%), 4 patients with duodenal ulcers, 2 patients with gastric and duodenal ulcers and 3 patients with non-ulcer dyspepsia. five patients did not respond including 2 with non-ulcerative dyspepsia. 2 duodenal ulcers and 1 gastric ulcer. however their ulcers have healed and symptoms relieved and they were treated with other h.pylori eradication regimes. conclusion. in this small study we have shown that azithromycin 5 mg oncedaily for 5 days is an alternative safe effective and well tolerated monotherapy for eradication of h.pylori. it has been suggested that hepatitis c virus (hcv) infection is associated with a high prevalence of autoimmune disease, but that this risk may be diminished in patients who received a low viral load at initial infection. the aim was to determine the prevalence of symptoms, signs and markers of autoimmune disease in a group of women vol. 165, 1996 irish journal of supplement no, 2 medical science infected with hcv contaminated anti-d immunoglobulin. 50 patients, mean age 44.3 years (range 32 -65 yrs) were examined. 7 patients (14%) were rheumatoid factor positive. ana was positive in 6 patients (12%) but titres were 1/10 in 4/6. thyroid globulin antibodies were found in 3 patients (6%) with 1 also being tm positive. 2 further patients were positive for thyroid microsomal ab. prevalence of apa, ara, sm were 4%, 2% and 2% respectively. one patient had cryoglobulins detected while antibodies against mitochondria and lkm-i were not found only one patient of 50 was symptomatic. these levels of antibodies are no greater than levels in the general population and similar to other groups infected with hcv contaminated anti-d. these results further support the hypothesis that either low viral innoculum or long duration of infection may in some way result in a low level of autoimmunity. a previous study (ilan et al, arch int. med., 1993; 3: 1588-1592) suggested that lga deficiency disposes towards hepatitis c virus (hcv) infection in some patients. it has also been suggested.that iga deficiency may occur as a result of the viral infection as a secondary event. the aim was to determine the prevalence of selective and partial lga deficiency in patients with hcv infection using a radio-immunodiffusion technique. serum lgg and igm levels were also examined. immunoglobulin levels were measured in 67 patients, 59 women and 8 men, mean age 45.3 years (range 29 -72 years) infected with blood between 1977 and 1990. none of the patients were found to have selective or partial iga deficiency. mean iga = 2.59 g/l (range 0.86 g.1 -5.19 g/l). furthermore no patient exhibited deficiency of igg or igm. conclusion : no evidence of iga or other immunoglobulin deficiency exists among this group of patients with hcv infection. while it may be possible that lga levels decrease following infection, no evidence exists that this phenomenon persists in the long term. hereditary non-polyposis colorectal cancer (hnpcc) is the most common form of hereditary colon cancer and accounts for up to 10% of the overall cancer incidence. the disease is inherited in an autosomal dominant manner andis characterised by predominantly right-sided tumours. hnpcc is caused by mutations in one of four mismatch repair genes. these genes are homologues of the bacterial mismatch repair systerfi and their function is to detect and correct defects in dna. the hmsh2 gene is reported to account for up to 60% of cases, hmlhi, hpms1 and hpms2 account for 30%, 5% and 5% of cases respectively. we have collected eighteen irish hnpcc families which satisfy the amsterdam criteria for the disease. we have screened the entire hmsh2 gene in eight of these families. a combination of single strand conformational analysis (sscp) followed by dideoxy sequencing was used to detect defects in the dna. sequence analysis revealed the presence of a novel polymorphismin exon 5 in one family and a possible splice site mutation in the remaining ten families, using a novel mutation detection technique. departments of medicine and immunology, university college hospital, galway. serial iga gliadin antibody (igaga) studies are used as a surrogate for repeated small intestinal biopsy to monitor dietary compliance in treated coeliac patients. we have correlated igaga levels with indices of hyposplenism (platelets and erythrocyte "pits") to determine if either index can be used to monitor adherence to a gluten free diet. the study population of 129 biopsy proven coeliacs was stratified according to dietary status into 114 gluten free diet (groups a) and 15 normal diet (group b). the iga gliadin antibody titres were significantly lower in group a than group b (44.5 : 126 units p = 0.007). in group a gliadin antibody titres showed a statistically significant correlation with both platelet (p = 0.039) and pits count (p --0.04) ; in group b gliadin antibody titres correlated with platelet counts only (p -0.003). those ona gluten free diet were further subdivided according t ~ reported degree of adherence to the diet (strict diet -sgfd, lax'diet -lgfd). significantly higher gliadin antibody titres were found in lgfd than sgfd (103.6; 27.9 p = 0.003), but the ptatelet and.pit counts were similar in the two groups. igaga is superior to platelet or pit coums in monitoring dietary'compliance in coeliac disease. hospital, galway. iga endomysial antibodies (igaea) are said to be more specific and sensitive indicators of coeliac disease than iga gliadin antibodies (igaga). initially the igaea substrate was monkey oesophagus and more recently umbilical cord. a new commercial product has becomeavailable using primate smooth muscle (accu track -diagnostic slides). we have undertaken a preliminary study to compare the performance of this lgaea assay and igaga elisa in treated and untreated coeliacs, coeliac relatives and controls. the study population consisted of 127 biopsy-proven coeliacs who were stratified according to "reported" dietary status into 89 good gluten free diet (group a), 24 poor gluten free diet (group b) 14 normal diet (group c) and 10 unaffected coeliac relatives (group d) and 5 controls (group e). elevated igaga (>25 units) were found in the following groups : a 37%, b46%, c 50%, d 20%. igaea were positive in a 27%, b 33%, c 50%. with respect to igaga positivity, there were 11 false positive igaeaa 18%, b 12.5%, c 14% and 23 false negative -a 8%, b 8%, c 14%, d 20%. in this preliminary study in untreated coeliac patients the performance of the igaea test was on a par with the igaga assay. alt and fibrosis may be associated with a non-alcohol steatohepatitis and these processes may be synergistic. finally, number and type of riba bands is not a predictor of inflammatory activity. ~stepping hill hospital, stockport, uk sk2 7je. 2royal oldham hospital, rochdale rd., oldham ol1 2jh. we investigated upper gut bleeding in patients aged 75 years and over. a proforma addressing demography, drug therapy, clinical status, timing of endoscopy / surgery, and outcome was used. 109 consecutive patients (median age 80 years, range 75-92) were studied over 6 months. 106 (97%) underwent gastroscopy with a 97% diagnostic yield. 32 patients had severe oesophagitis, 2 had oesophageal malignancy, 29 had gastric ulcers -5 of which are malignant and 23 had duodenal ulcers. ulcerogenic drugs were implicated in 52 patients. 38 patients were referred for surgery, 8 operated upon with one postoperative death. 51 had haemoglobins of 10g/dl or less. all malignant lesions were inoperable. the overall mortality was 6% reducing to 2% if neoplasla were excluded. co-morbidity influenced mortality. 92 patients were discharged with a median hospital stay of 15 days. information on cause of bleeding greatly influenced management. the prognosis of gut haemorrhage in the very old need not be so poor. a few require surgery but the majority respond to active medical resuscitation which is a key factor in determining outcome. we advocate low threshold for endoscopy, judicious use of ulcerogens and adherence to guidelines on management of upper gut haemorrhage. haemochromatosis (hh), a common recessively inherited disorder of iron metabolism is closely linked to the hla-a locus on chromosome 6. linkage studies have demonstrated a close association between (hh) and the hla alleles, a3 and b7 ("ancestral haplotype"). heterogeneity at the molecular level may account for the variance in clinical phenotypic expression. the aim was to evaluate phenotypic expression of hh in the presence/absence of the a3-b7 ancestral haplotype. 32 probands (26m:6f) from unrelated irish families were investigated. phenotypic variability was assessed with regard to l) age; 2) % trans.sat.; 3) serum ferritin; 4) liver bx iron grade; 5) body iron stores and 6)symptomatology. three males were homozygous for a3b7, 15 were heterozygous for a3b7 and 2 were non-a3b7. symptomatology, trans, sat., serum ferritin and liver bx grade were not influenced by homozygousity or heterozygousity for a3b7. conclusion: there were no significant differences in phenotypic expression on comparison of the three haplotype groups. no predominant genotype appears to be responsible for phenotypic severity in irish families indicating the possibility of multiple mutagenicity of the hh gene. of 1013 riba positive anti-d associated chronic hepatitis c patients, 438 were pcr positive but had surprisingly mild disease. the disease status of the pcr negative patients was hitherto uncertain and is the subject of this study. 20/72 riba positive patients referred to this centre were biopsied because of elevated alt (4) or florid symptoms which dated from inoculation (16). 1 2 3 4 histological activity index * 5,1,0,1 3,2,5(f), 2(f), 0,4(f), 2,1,2 0,2,1,2 2,2,3 no bile duct damage, lymphoid follicles or aggregates was observed. 3/20 had mild periportal fibrosis (f), 2 of these had steatohepatitis with obesity (1) and impaired glucose tolerance (1) suggesting dual pathology. we conclude that riba positive, pcr negative patients have minimal disease activity. elevated the association between the hla locus and haemochromatosis (hh) has allowed early identification of affected siblings. it is unclear what proportion of subjects who are predicted to be homozygous or heterozygous for the disease by hla typing develop the disease. studies correlating clinical features with hla type in families from ireland -a putative source of this celtic trait have not been described. the aim was to correlate clinical, biochemical and pathologic features of hh with hla typing in 67 first degree relatives of 12 probands. initial analyses identified 12 homozygous (hh), 40 heterozygous (hn) and 15 normal (nn) individuals. however, 11/40 hn individuals had stainable iron on liver biopsy, confirming hh. further hla analysis revealed 7 homozygous x heterozygous matings and identification of all disease haplotypes within each pedigree allowed final classification of 30 hh, 25 hn and 12 nn individuals. vol. 165. 1996 supplement no. 2 conclusion: this study demonstrates the importance of hla typing in the clinical management of families with hh, furthermore, in multiply affected families the incidertce of homozygous x heterozygous matings is high indicating the high degree of "pseudodominance" in the irish population. the degree of acute hepatic failure after severe trauma and sepsis is related to the extent of hepatocyte (hc) damage and cell death resulting from either necrosis or apoptosis. we have previously demonstrated that tnf-ct and lps can directly lead to hc necrosis, but not apoptosis. recent, studies have shown that reactive oxygen intermediates (roi) and nitric oxide (no) are capable of inducing apoptosis in eukariotic cells. however, it is unclear whether roi or no are involved in hc cell death. the aims of this study were to evaluate the role of no and roi in hc cell death (apoptosis vs necrosis). hcs were isolated from sprague-dawley rats, and cultured with the no donor, sodium nitroprusside (snp) or the roi generation system, hypoxanthine-xanthine oxidase (hx-xod) and h202. the effect of lps, tnf-t~, and ifn-y alone or in combitmtion with different antioxidants and the no synthase inhibitor, n-methyl arginine (nma) on hcs was also assessed. snp caused a dose-dependent increase in hc apoptosis. roi generated by hx-xod and h202 did not induce hc apoptosis, but were responsible for hc necrosis. tnf-ct alone failed to induce hc apoptosis, but when ~combined with antioxidants resulted in increased hc no production and apoptosis. this effect was attenuated by nma. snp also induced hc damage and hc necrosis. moreover, tnf-ct-mediated hc damage and necrosis could be further reduced by the combination of antioxidants and nma. these results indicate that roi preferentially induce hc necrosis, but not apoptosis. induction of no resulted in both hc apoptosis as well as hc necrosis, which suggest that overproduction of no may be detrimental during the sirs. irish journal of medical science intervention was not uniform. mean albumin was 34.3g/1. mean weight was 57.8kg. poor cognitive status greatly increased the requirement for dietetic consultation time. lack of dietetic resources results in inadequate monitoring of these patients following discharge. this study highlights the need for a dedicated clinical nutrition service, for medical services for older people. periconceptual consumption of folic acid has been shown to decrease the incidence of neural tube defects. the preventative strategy of universal food fortification with folic acid presents the possible risk of masking the diagnosis of cobalamin deficiency in pernicious anaemia. in addition, the ultimate longterm effect of universal exposure of adult or foetal cells to a synthetic substance, ie. folic acid, is unknown. in this study, the threshold oral dose of folic acid in a number of foods above which metabolically-unaltered vitamin appeared in serum postprandially was determined in a young and elderly population by microbiological assay of serum pre-fractionated by hplc. subjects on a five-day regime of fortified cereal and bread along with their normal unfortified diet. were shown to have a threshold level of 400~g/d, abovewhich unaltered folic acid appeared in the serum. individuals given folic acid in either isotonic saline, milkor white bread exhibited a threshold level of 200~tg per serving. from patterns of food consumption in ireland, even moderate levels of fortification are likely to lead to some population groups being exposedto excessive amounts of un-altered folic acid in serum. many older people are nutritionally compromised. there is clear evidence that: nutrition intervention reduces morbidity m and mortality in older patients. to identify the spectrum of nutritional abnormalities referred for dietetic intervention and the problems associated with nutritional assessment, 50 elderly patients were alphanumerically selected from files of the department. of nutrition and dietetics. the most common dietetj.c interventions were: use of supplements 60%; high protein high calorie diet 46%; nasogastric feeding 26%; reduction fat 8%; iron/thiamine assessment 6%; high fibre diet 6%; diabetic diet 4%; nutrition swall6w programme 2%; lipid lowering diet 2%. some 60% referred required nutritional supplements, but the profile of an increase in oxidative stress in cystic fibrosis patients has been suggested. activated neutrophjls in the presence of chronic lung inflammation in addition to increased activity of the electron transport chain in cfmay. increase free radical generation. antioxidant protection against free radical attack is likely to be compromised as a i'esult of deficiencies in fat soluble antioxidants vitamins. in the present study stimulated thiobarbituric aoid reacting substances (tbars) were measured to determine the ability of plasma to withstand lipid peroxidation. copper was used to in!tiate the breakdown of lipids to lipid hydroperoxides and eventually to aldehydes, mainly malonyldialdehyde (mda). pooled cf plasm a and pooled control plasma were incubated for 0, 30, 60, 90, 140 and 200 min. mda complexes with thiobarbituric acid which absorbs at approximately 535 rim. there is a lag phase where antioxidartts vol. 165, 1996 irish journal of supplement no. 2 medical science in the plasma or tissue protect against lipid peroxidation, then a log phase where the protective effect is overcome and finally the reaction reaches a plateau when lipid peroxidation is complete. absorbance at 532 nm was measured in all samples and zero order and first derivative spectra were obtained. the lag phase appears to be longer in the pooled cf plasma compared with controls. plasma t~-tocopherol levels were within the normal range in both groups, indicatin~ an alternative protective effect in cf. mild hyperhomocysteinaemia is an established risk factor for heart disease. a source of homocysteine in humans is the essential amino acid methionine found in protein of animal origin. in an 8-week study weekly fasting plasma homocysteine levels were examined in a group of healthy male subjects (n=6) under normal dietary conditions (weeks 1 to 4) and in response to graded increased methionine intakes (weeks 5, 6, 7). nutrient intakes, including methionine, were calculated from 4x3-day food records. under normal dietary conditions weekly mean plasma homocysteine levels were not significantly different (anova) from each other ranging from 6.82+1.77 to 9.42+2.73~tm/1. doubling daily methionine intakes (supplementing with 25mg/ kg/d) did not result in a significant increase in plasma homocysteine (8.56+3.68~m/1), however, significant increases were achieved when diets were supplemented with methionine at levels of 50 and 75mg/kg/d resulting in mean plasma homocysteine levels of 13.37+5 9 and 18.051am/1+11.08, respectively. mean plasma homocysteine levels returned to baseline (8.76 + 3.42 ~tm/l) 10 days post supplementation. we conclude that supplementary methionine results in a significant increase in plasma homocysteine only when levels of five times the normal dietary intake are reached. this study is evaluating the use of a synthetic construct which encompasses primer binding sites for lpl and a variety of cytokine and other transcripts, to quantify lpl expression in cultured human monocyte-derived macrophages. following isolation of total rna at various times during cell culture, its reverse transcription (rt) using random hexamer primers generating first strand cdna and specific amplification of lpl cdna targets by polymerase chain reaction (pcr), generates products identifiable on gel electrophoresis. quantitation of message is obtained by incorporating the pawl08 construct in the rt assay in varying quantities as an internal standard with known amounts of monocyte-macrophage rna (l~tg). pcr amplification of this construct yields size distinguishable products from that produced by the monocyte-macrophage lpl cdna transcript. pcr conditions for the assay have been optimised at 29 cycles of denaturation (tmin @94~ annealing (1.5min@55~ and extension (lmin@72~ lpl mrna has been detected in cultured monocytes and macrophages throughout their differentiation. also increased expression of monocyte lpl mrna has been observed following 15 hr incubations with chylomicrons (30t~g/6x 106 mononuclear cells/ ml)-when compared with controls. interestingly, little or no lipase mrna was detectable in circulating monocytes using identical pcr conditions to preparations of mrna from the day 4 and day 8 cultured cells. this methodology will now permit investigation of the factors controlling lpl expression in cultured human monocytic cells. replacement growth hormone (gh) therapy in adult hypopituitarism is attracting increasing interest. in 1992 markussis (l) detected premature atherosclerosis by ultrasonography in the untreated patient 9 we have shown plaque regression with patients on replacement gh (norditropin) in a 4-month trial. 11 females and 9 males were recruited, mean age 49.6 years. at each timepoint plaque characteristics were measured by duplex ultrasound. 6 patients showed a large reduction in plaque size (mean 21%) after four months treatment (p value < 0~00l). similarly, highly significant values in cholesterol, hdl and ldl and apo a1 are achieved. chol hdl ldl apo a 1 pretreatment 6.54 1.23 5.04 119 posttreatment 5.64 w 1.24 w 3.79 w 109 ~ w achieve a high degree of statistical significance (p< 0.001). the significant reductions achieved in plaque characteristics in six patients studied who showed plaque formation correlates with other parameters traditionally accepted as reducing cardiovascular risk. hypertension is found in approximately 70% of patients with cushing's syndrome, but the mechanism is poorly understood. previous studies in our unit have examined levels of exchangeable sodium, plasma renin and angiotensin ii and cardiac sensitivity to phenylephrine. one previous study has demonstrated enhanced pressor responsiveness to noradrenaline in a group of patients with cushing's syndrome due to adrenal adenoma. we have investigated the blood pressure response to noradrenaline in 7 patients with pituitary dependent cushing's syndrome and in 7 controls matched for age, sex and bmi. noradrenaline was infused for 10 minute intervals at five different concentrations between 0.01 and 0.18 mcg.kg.min -~ multiple systolic and diastolic readings were recorded and the infusion was stopped if the systolic pressure became > 200 mmhg, diastolic _> 1 l0 mmhg or the systolic pressure rose > 35 mmhg. basehne blood pressure in the patients with cushing's disease (cd) was 140/88 + 6/3 compared with 122/83 + 6/6 mmhg in the normal controls (nc). in 6 of the 7 patients with cushing's disease, the test had to be stopped before completion of the protocol, whereas this was necessary in only one control subject the change in blood pressure from basehne to the blood pressure value recorded either at the time the test was stopped or at the peak blood pressure reading during equivalent noradrenaline infusions was compared between the matched pairs. the mean change in diastolic pressure was 22 + 5 mmhg in cd compared with 7 + 2 in nc (p<0.05). there was no statistically slgmficant difference in either systolic pressure (28 + 5 vs 26 + 5 mmhg) or mean arterial pressure (23 + 5 vs 14 + 3 mmhg). these results demonstrate an increased diastolic pressor response to noradrenahne tn cushing's disease. increased pressor sensitiwty to uoradrenaline may contribute to the elevated blood pressure seen in cushing's disease. increased plasma homocysteine (thcy) is an independent risk factor for premature vascular disease. patients with insulindependent diabetes have an increased prevalence of cardiovascular disease. accordingly, we measured plasma thcy concentration in 119 such patients (15-59y), randomly selected, and in 51 control subjects. in controls, thcy was higher in males than in females (supine: geometric mean (95% ci): ,8.3 (7.2,9 6) v 5.9 (5.1,7.0) i.tmol/l, p<0.001), as previously described, but there was no gender difference in patients. male patients, without microvascular complications, had lower thcy than controls (supine: 6.5 (5.4, 7.8) v 8.3 (7.2,9 6) ~mol/l, p<0.05), but values in female patients without complications were similar to those of female controls, thcy significantly correlated with age in diabetics but not in controls, thcy increased in patients with increased severity of microvascular complications, partly due to the effect of age. thcy was higher when standing.than when supine in both controls (8 0 (7.0,9.1) v 7 1 (6.4,8.0)lalmol/l, p<0.05) and patients (6.9 (6.5,7.4) v 6.4 (6.0,6.9)l.tmol/l, p<0.02). the absence of gender difference, the association between thcy and age, and higher levels with increasing microvascular complications suggest thcy could be of pathogemc significance in iddm patients, despite unexpectedly low levels in male patients without complications. differences.between supine and erect samples may be due to haemodilution of albumin-hound thcy in the latter a review of the treatment outcome of thyrotoxicosis with standard dose/doses of radio-active iodine (sdrai) in 67 consecutive patients presented to the endocrinology department, uchg, from december 1992-december 1993 was analysed. the mean pre-treatment levels of free thyoxine (ft4) was correlated with the treatment outcome. there was statistically significant difference in the pre-treatment ft4 between responders and nonresponders to the first dose rai (p = 0.001). response with hypothyroidism and/or euthyroidlsm was considered successful treatment 43/67 (64 2%) responded to a single dose rai; 26/ 67 (38.8%) and 17/67 (25 4%) responded with euthyroldism and hypothyroidism respectively 19/67 (28.4%) and 3/67 (4.5%) responded to the second and third doses of rai respectively giving a total response rate of 92.5% and 97% respectively. interestingly, 2/67 (2.99%) patients failed to respond even to the fourth dose rai 4/67 (5.97%) patients with t3 toxicosis (3 females and 1 male). two responded to the first dose (one with hypothyroidism and the other with euthyroidism), the remaining 2 required a second dose, which produced the same results. no statistically significant difference in the response rate between t3 and t4 toxicosis (p = 0.9) was observed. inherent in the st. vincent declaration targets is the need for continuous data collection and audit. we present preliminary information from the mater database, the first prospective audit of patients from a homogenous irish population. 1,051 iddms (527m:524f) were identified with the following characteristics (mean + sd), age 37.3 + 14.4 years, duration of dm 185 + 11.2 years, bmi 24.4 + 3.0 (males) and 24.2 + 4.0 kg/m2 (females), hbalc 8.6 + 1.97% (n<6.2%). no male:females differences existed in the above nor in macrovascular complication rate 10.1% (predominantly peripheral vascular &sease and lschaemic heart disease). however, males were more likely to be current smokers (34% vs 27%, p = 0 01). hypertension rates (17.2m vs 15.4%f), cholesterol > 6.5 mmol/1 (6.2m vs 9.8%f) were similar but more males had cholesterol < 5.2 mmol/l (64.8m vs 53.8%f, p < 0.01). clinical nephropathy was present in 11.4% of males vs 8.7% in females (p<0 05) 11.9% had clinical peripheral neuropathy. retinopathy will be described elsewhere. 10.4% of females and 2.3% of males had a history of hyperthyroidism and 2.5% of females vs 1.4% of males of hypothyroidism. 7.8% had history of psychiatric disease. conclusion although not a population based study, care of iddm in ireland is almost totally hospital clinic based cigarette smoking is identified as the major problem to be addressed patients with diabetes meltitus (dm) are at a higher risk of developing vascular complications, including coronary artery disease (cad). we performed a detailed analysis of predictors of cad and its seventy in patients with dm and chest pain 19 patients in total single vessel cad (svd) in 4, double vessel (dvd) in 5, and triple vessel (tyd) in 5 on cine contrast angiography clinical, biochemical and dobutamme stress echocardiographic findings are tabulated below for patients with angiographically proven coronary artery disease. patients with tvd had a longer duration of dm (19 4 years) and were more likely to have retinopathy (100%) the sensitivity of dse was excellent for severe disease. conclusion' duration of dm, retinoapthy, and a positive dse were the best predictors of severe cad in a diabetic population with chest pain the haemodynamic hypothesis for the pathogenesis of diabetic microangiopathy argues that an initial increase in microvascular flow leads to sclerosis and disturbed microvascular autoregulation. we have recently demonstrated impairment of vasoconstrictor responses to endothelin-1, a potent endothelium-derived constrictor substance, in niddm and have suggested that this could contribute to the initiation of microangiopathy the purpose of this study was to determine whether responsiveness to endothelin-i is also impaired in iddm. non-specific vascular smooth muscle contraction was assessed using high dose serotonin eleven patients with iddm and 11 control subjects underwent forearm blood flow (fbf) measurement by venous occlusion plethysmography in response to local infusions of endothelin-1 (5 pmol/min for 60 minutes) and serotonln (30 la g/min for 2 minutes) control subjects showed slow onset vasoconstriction in response to endothehn-1 reaching maximum at 35 minutes (p<0.01) the diabetic group did not respond to endothelin-i group differences were significant (p=0.02). the two groups showed similar vasoconstriction in response to serotonln. in conclusion, vasoconstriction in response to endothelin-t is impaired m iddm non-specific vascular smooth muscle contraction is preserved. impaired vascular responsiveness to endothehn-i is a possible common mechanism for the pathogenesls of microanglopathy in 1ddm and niddm. we measured total corrected (tca) and standardised ionised calcium (lca) in a population of 76 intensive care (1cu) patients (with a mean age of 57+22 years, 59% male) to determine the prevalence of abnormalities in circulating calcium and its possible determinants severity of illness was measured by the apache ii score (acute physiological and chronic health evaluation). for comparison of ica we examined 20 subjects undergoing arterial gases which proved to be normal and 40 non-critically 111 hypoxlc subjects ica was measured on arterial gas samples and corrected for ph 84% of icu patients had a total ca (unadjusted) of < 2 2mmol/l. after adjustment for serum albumin, 55% of icu patients had an tca <2 2 mmol/1 30% of icu patients had a serum phosphate of <0.8 mmol/1 ica in controls was 1.44 + 0.017 mmol/1 and 1.38 + 0.015 mmol/1 in hypoxlc non icu patments (ns) ica in icu was lower: 1.34 +0.016 (<0.003). tca and lca were not slgmficantly related. tca and ica did not sigmficantly differ between patients who died and who survived in the icu, and they were not related to apache ii score. belfield, dublin 4. from july 1987 to june 1995 there was a 16-fold increase m the annual number of specimens submitted to the virus reference laboratory because of a perceived risk of contracting hiv through a needlestick injury, blood splash, human bite, or through occupational exposure. needlestick-associated specimens also comprise an increasing proportion of 'at-risk' specimens, rising from 0.9% m the year july 1987-june 1988 to 9.4% in the year july 1994-june 1995. between july 1992 and june 1995 a total of 1787 patients had specimens submitted for hiv antibody testing after a perceived'exposure to hiv of these only 209 patients had more than 1 specimen taken. although the time of putative exposure is rarely avadable, the median interval between 1st and 2nd postexposure specimens for these 209 patients is 3 months with 136/ 209 (65%) lying between 1 to 6 months. if the risk of hiv refection from a needlestlck injury is assessed as sufficient to warrant serological investigation, the timing and number of blood samples are important. a negative report from a single early specimen may not indicate an absence of infection a basehne specimen and follow-up specimens at 6 weeks, 3 months and at a minimum of 6 months post-exposure are recommended appropriate serology for other viral mfections (hepatitis b and hepatitis c) should also be considered. since the beginning of 1995 a significant sustained increase in the numbers of hepatitis a cases has occurred. the number of cases m 1995 was 229 against 121 in 1994. this reverses the continuous fall observed over previous years. the reason for this increase remains unidentified at present this increase has occurred following a dramatic increase in the total number of hepatitis a igm tests carried out by the vrl since february/ march 1994 the increase in the number of tests carried out since this time is primarily attributable to increased hepatitis testing following receipt of hepatitis c-contaminated rhesus anti-d immunoglobuhn. analysis of the age profiles of the positive patients and of all the referrals indicates that 95.5% of positive results were found m patients aged 50 yr or less whdst 31% of all hav igm tests were performed on individuals aged 51 yr or greater. this raises the question whether greater selectivity should be employed when requesting hepatitis a tests studies report compliance rates ranging from 30 to 50% in hiv negative patients there has been no comprehensive study of compliance in hiv positive patients. accurate measurements of compliance are not easy; easy measurements of comphance are not accurate "). to determine the compliance rate in hiv posmve patients attending st. james hospital, dublin, one hundred consecutive patients attending the service were interviewed (homosexual 46, ivdu 45, heterosexual 9). the questionnaire was divided into three sections. firstly, a medical review was completed by the clinicmn which included demographic data, cd4 count, cdc staging, karnofsky index and prescribed medication. secondly, the pharmacy detailed the medication dispensed to each patient. the third section comprised a patient interwew to determine adherence to, and understanding of prescribed drug therapy. we report an overall comphance rate of 61%. this was unevenly distrtbuted between the two main patient groups (89% in homosexuals, 24% in ivdu) the following factors were found to mfluence compliance: number of medications, cdc stage, karnofsky index, dysphagia, educational and socio economic factors. we also found that poor patient understanding of the prescribed therapy significantly affected compliance. the aim was to determine the incidence of stds in patients presenting for hiv testing at the department of genito-urinary medicine, saint james's hospital. a retrospective analysis of all patient notes who presented for hiv testing between july '94 and december '94 was undertaken. according to clinic policy all patients had been screened for the following stds; neisseria gonorrhoea, chlamydia trachomatls trlchomonas vagmalis, candida, human papilloma virus, herpes simplex virus syphilis and hepatitis b in addition intravenous drug users (ivdus) were also screened for hepatttls c all patients underwent pre test counselhng. sex, age, risk groups and diagnoses were noted. 504 patients presented for hiv testing, of whom 66% were male, 34% female, wtth an average age of 28.5 years. of the total, 8% were, or had previously been ivdus. of the total 46 ivdus, 37 were heterosexual males 2 were bisexual and 4 were females 10.2% of the male patients were homosexual and 4 4% btsexual there were 15 posmve hiv tests (3% of total); 14 males and 1 female. in this group there were 5 patients with hepatitis c, all of whom were ivdus. no other stds were detected in the hiv negative group hepatitis c was diagnosed in 20, hepatttis b in 4, anogenital warts in 58, herpes gemtalis in 10, syphilis in 3, n. gonorrhoea in 9, t. vagmalis in i, c. trachomatls m 12, g vaginalis in 5 and candldlasis in 47. this study confirms the importance of std screening in all patients requesting a hiv test. of the total testing for hiv, 30% had a concurrent std diagnosis although no stds were identified in the hiv positive group this may be more a reflection on the makeup of the irish hiv positive population, the majority being ivdus, rather than a difference m the mode of sexual toxoplasmosis is the most common opportumstlc infection of the central nervous system in aids patients. in clinical practice the diagnosis depends on clinical, radiographic and serological findings coupled to chnical response to therapy brain biopsy is not routinely performed. in this retrospective review, we describe our experience with diagnosing toxoplasmosis we examined (a) the clinical demographics and presentation, radiographic findings, response to therapy and patient outcome (b) role of polymerase chain reaction (pcr) in detecting toxoplasma gondii from blood and csf samples (c) the usefulness of serology in diagnosing acute infection. all cases diagnosed as toxoplasmosls based on the above criteria were reviewed. pcr to detect toxoplasma gondn dna used primers to the b 1 gene giving a 223bp amphficatton product serological tests used were sabln-feldmen dye test and latex agglutination. there were 25 cases diagnosed (17 m, 8f, cd4 0-300; cdc iv 19). 3 panents unknown to be hiv posmve presented with cerebral toxoplasmosis 22 patients were not receiving continuous systemic prophylaxis against pce diagnostic value oft gondii pcr in blood and csf showed a sensmvlty of 20%, specificity of 100%, ppv was 100% and npv was 40% determination of lg subtype was of limited value 25% (6) of patients were seronegatlve of whom 50% (3) had histologically proven disease 2 of these latter 3 cases were pcr negative the dye test was of poor predictive value this review confirms the need to combine all parameters in making a diagnosis of toxoplasmosis in lmmunocompromlsed hosts. the performance of the newly developed, rapid and fully automated m~croparticle enzyme immunoassay abbott imx hiv-1/hiv-2 3rd generation plus assay for the detection of antibody to hiv-1 and hiv-2 including subtype o m human serum or plasma was assessed the assay was evaluated by testmg specimens from blood donors, diagnostic populations and hospitalized patients, hiv seroconverslon panels confirmed hiv-positive specimens, and potentially interfering specimens. the abbott imx hiv-1/hiv-2 3rd generation plus assay showed an overall apparent specificity of 99.97% (lower limit of 95% ci 99.84%) in the tested blood donor populations (n=347t). this comparable to the specificity found for the abbott imx hiv-i/hiv-2 3rd generation plus eia (99 93%) and the axsym h1v-1/hiv-2 assay (99 90%). the apparent sensitivity of the abbott imx hiv-i/hiv-2 3rd. generation plus assay is at least equivalent to that of the abbott imx hiv-i/hiv-2 3rd generation plus eia and the axsym hiv-i/hiv-2 assay. of 21 hiv-i seroconversion panels tested, the abbott imx hiv-1/hiv-2 3rd generation plus assay detected seroconverslon earlier on up to 6 panels, depending on the comparison assay. among 785 specimens from asymptomatic and symptomatic hiv patients, the abbott imx hiv-1/hiv-2 3rd generation plus assay detected 100 (785) including 7 specimens characterized as hiv-1 subtype o. the abbott imx hiv-1/hiv-2 3rd generation plus assay is an extremely sensltlve and highly specific assay for the early detection of antibody to hiv-1/hiv-2 and shows at least an equivalent performance to the abbott imx hiv-i/hiv-2 3rd generation plus eia and the axsym hiv-1/hiv-2 assay. the fully automated imx instrument system offers ease of use and rapid results on a widely accepted and reliable platform. streptokinase (sk), a 47kd protein produced by group c b hemolytic streptococci, is a widely used thrombolytic agent. anti-sk antibodies arise either as a result of therapeutic administration of sk or following natural mfection with streptococci although the clinical significance of antl-sk antibodies is not clear, there is evidence that some anti-sk antibodies arising from natural infections can interfere with sk activity tn vtvo, resulting in thrombolytlc failure. to facilitate further investigations of these antibodies, we have developed and validated a highly sensitive functional assay, which measures sk neutralisatlon activity of serum independently of other circulating inhibitory factors in the sample, and a rapid and convenient enzymeimmunoassay for the detection of anti-sk antibodies. analysis of over 200 random serum samples from the local blood bank with the enzymeimmunoassay showed the prevalence of antl-sk antibodies to be approximately 13%. all the positive samples and an equal number of the negative samples randomly selected were analysed by the functional assay the agreement between the results of the two assays was excellent indicating that our enzymelmmunoassay was a convement method for detection of anti-sk antibodies which could neutrahse sk activity m vitro irish journal of medical science tuberculosis drug therapy, isolation precautions and prophylaxis. conventional methods of detection such as microscopy and culture either lack sensitivity and specificity or are timeconsuming. in this study we investigated the use of a pcr based diagnostic assay for the detection of m. tuberculosis in sputum samples this assay has been developed by bioresearch ireland (bri) and raggio italgene. sputum samples were lysed and pcr amplified using an m. tuberculosis complex-specific primers. the results obtained using the bri/c-trak tm technology were initially compared to the amplicor system (hoffman la roche). both probe detection methods represent fast and reliable methods for the detection of m. tuberculosts in clinical samples. this test is designed to eliminate the possibility of obtaining false negatives. strongyloldes stercoralis infection in humans ts endemic in the tropics. as travel is becoming more common, it will be seen more frequently. two cases of this infection in irish people are described. case i. a 21 year old women had travelled and worked in poor rural areas of mexico for one month, three years before presentation. two and a half years later she developed abdominal discomfort, anorexia and sore throat. myalgia, arthralgta and a transient skin rash began to appear in the next month. eosinophilia, mild anaemia and raised liver blood tests were noted. elisa test for strongyloides was positive but parasites were not seeri in the faeces. ivermectin was given and the patient feels better. case ii. a 31 year old nurse had arthralgia, fatigue and some weight loss for 18 months'. on two occasions in the last four years, she had been travelling extensively in s.e. asia for a total of four months. she was admitted to hospital because of acute fever and loin pain. a urinary tract infection was diagnosed. absolute eosinophil count was i'aised 0.63x10^9/1. esr was 17mm/hr. strongyloides elisa was positive and treatment administered as above. strongylotdes is the most important nematode in the returned tropical traveller. it can multiply and persist within the body for long periods of time and it can cause hypertnfection syndrome, a protean fulminating infection of bowel, lungs, blood stream and brain, in those who are lmmunocompromised. diagnosis can be difficult by stool microscopy. thlabendazole has side effects but ivermectin is safe and effective. june 1995, there was an outbreak of c.difficde-associated diarrhoea (cdad) at st. james's hospital. the aims of this study were to determine the incidence and outcome of cdad in hiv positive and negative patients we prospectively reviewed all patients with diarrhoea, a positive c.difftcile cytotoxin assay, and in whom no other infectious cause for diarrhoea was identified demographic data, history of diarrhoeal episodes, risk factors and outcome were recorded. the incidence of cdad in hiv negative patients was 1.2 per 100 hospital admissions, compared to 1 per 100 admissions m hiv positive patients. the average number of courses of antibtotlcs received, in hiv negative patients prior to the onset of symptoms was 2.3, and 76% of this group were exposed to third generation cephalosporins. hiv positive patients received an average of 3.4 courses of antibiotics and no patients received third generation cephalosporins. there were no deaths due to cdad in hiv positive patients however 4 hiv negative patients died from severe pseudomembranous colitis in conclusion we documented a unexpectedly low incidence and complication rate of cdad in hiv positive patients this is surprising considering their multiple hospital admissions and exposure to ant~microbial and chemotherapeutic agents. the number of new positive hiv specimens detected at the virus reference laboratory has risen from a cumulative total of 638 m july of 1987 to 1597 in september of 1995. we examined our data to determine the proportional make up of these positives by major risk group. in august 1987 64.7% of positive specimens were from intra venous drug abusers (ivda) by september 1995 ivda made up 49% of the total positive hiv specimens. positive specimens from homosexual individuals rose fro.m l0 9% of total positives in august 1987 to 20.6% in september 1995 there were no recorded positive specimens from heterosexual exposure in august 1987 but in september 1995 8.9% of positive specimens recorded heterosexual exposure. a further category which includes blood donors, haemophiliacs, transplant patients and organ donors made up 25% of total positives in august 1987 and in september 1995 made up 22.4% of total positives. we further examined our data in order to show when these changes occurred by ascertaining how many new positive patients have been discovered per year in each of the main risk groups. see in the united kingdom echovlrus type 22 (echo-22) is regularly isolated, with nearly 200 reports annually to the central public health laboratory. reports increased during 1986-1988 and overall it is the second most commonly reported echovlrus in the u.k. echo-22 epldemiology is different to that of other enteroviruses; over 90% of patients with echo-22 tsolated are less than 2 years of age echo-22 shows distinctive and unique cytopathogenic features in tissue culture, and based on sequence analyses, it seems to belong to a separate subgroup of picornaviruses. echo-22 has been associated with respiratory symptoms in premature infants, myocarditis and severe encephalitis. in 1989 an outbreak of acute flaccid paralysis associated with echo-22 was described in jamaica in six patients, four of whom died. we describe three cases of sudden death in infants associated with echovirus type 22 infection case i: s d. born 20/7/94; birth asphyxia and death at two days of age; echo-22 isolated on 2217194 from spleen. this study assessed the antibiotic sensitivity of organisms causing urinary tract infections (uti) among genito-urinary medicine (gum) clinic attenders in order to determine whether it is worthwhile giving tetracycline for dipstick (nitrite) positivity, even in the absence of clinical features of uti. we looked retrospectively at 100 laboratory confirmed uti's diagnosed among gum clinic attenders over a period of eight months. we assessed antibiotic sensitivities of the organisms involved, and determined how many dipstick positive urines which were left untreated turned out to be real uti's. 86% of uti's were due to coliforms and 66% of these were sensitive to tetracycline. 4% of uti's were due to staphylococcus saprophyticus, 3% due to beta haemolytic streptococcus group b, 2% due to enterococcus, 2% due to proteus species and 2% due to coagulase negative staphylococci. 25% of nephur positive urines were left untreated. 32% of these were nitrite positive. failure to treat a positive urine dipstick which turned out to be a uti necessitated a further clinic visit for adequate treatment. nitrite positive urines should be treated as a uti, even in the absence of clinical features of uti, either with trimethoprim or tetracycline. the number of untreated uti's and unnecessary extra visits to gum clinics would have been reduced with the use of judicious antibiotic therapy for nitrite positive urines. strains of enterococci resistant vancomycin have been reported with increasing frequency. in 1995, we investigated an increase in the frequency of vancomycin-resistant enterococcus faecium (vref) among patients in the haematology/oncology unit using pulse-field gel electrophoresis (pfge) to genotype these isolates and to assist in establishing the source of these vref. eighteen clinical isolates of vref from blood, urine sputum and-faeces and two environmental isolates were collected from separate patients between march and july 1995. minimum inhibitory concentrations (mics) to several antibiotics including teicoplanin and vancomycin were determined by agar dilution. pfge were performed following smal restriction endonuclease digestion. antimicrobial susceptibility testing revealed high level resistance to vancomycin and teicoplanin; mics >128 mg/ l and >32 mg/l respectively. this antibiogram is consistent with the van a phenotype. pfge of all 20 isolates revealed identical patterns indicating clonal spread of vree subsequent implementation or infection control measures reduced the frequency of vref isolation. pfge proved useful in demonstrating clonal spread of vref and.in emphasising the need for infection control measures. a prospective audit of baeteraemia in our 600 bed teaching hospital was carried out from february 1991 to march 1993. clinical and microbiological data were collected on 520 episodes of bacteraemia in 78 patients. of these 230 (62%) were hospital acquired and 200 (38%) community acquired. urinary tract and respiratory tract sources were implicated in 30% and 14% of community acquired episodes, making e. coli and s. pneumoniae the commonest community acquired isolates (41% and 16% respectively). other gram negative bacilli accounted for 13% and s. aureus for 11%. coagulase negative staphylococci were the commonest hospital acquired isolate (24%) followed by s. aureus (22%), e. coli (i5%) and enterococcus spp. (10%). enterobacter spp. were the second commonest gram negative isolate (5%). central venous cannulae were implicated in 43% of hospital acquired cases. urinary tract infections accounted for 20%. 63% of which were catheter related. invasive diagnostic procedures (angiography, prostate and liver biopsies, sinography) were implicated in t0 episodes. gentamicin resistance was found in 9% of hospital acquired aerobic gram negative bacilli and mrsa accounted for 13% of hospital acquired s. aureus. these figures are higher than expected but may be explained by outbreak of mrsa and gentamicin resistant entercobacter spp. which occurred during the study period. the past severalyears have seen a significant increase in the recognition of moraxella (branhamella) catarrhalis as a respiratory pathogen (~). the pathogenic mechanisms employed by the organism are largely unknown, but adherence may play a role ~2~. in our investigation the haemagglutinating ' activity of 40 isolates of m. catarrhalis was determined by a microtitre method. no isolate agglutinated horse, chick or sheep red blood cells (rbc). seventeen isolates agglutinated human rbc, x~hile 7 of these 17 isolates also agglutinated rabbit red. blood cells. haemagglutination of human and rabbit red blood cells was inhibited by porcine mucin. galactose inhibited the haemagglutiriating activity of the 7 isolates which agglutinate both human and rabbit rbc and yet bad no effect on the haemagglutinating activity of the isolates which haemagglutinate human rbc alone. .electron microscopy studies of the bacteria demonstrated a diffuse outer fibrillar layer on the surface of haemagglutinating positive isolates, thislayer was subsequently removed following trypsin treatment, as was the haemagglutinating activity. a 200 kda trypsin sensitive protein appears to be associated wfth haemagglutinating properties. mrsa is an increasingly important cause of morbidity, and is spreading from large hospitals to smaller community-based facilities and nursing homes. the objective of this survey was to obtain an indication of the size of the mrsa problem in ireland prior to introducing national mrsa control guidelines. a survey of all microbiology laboratories in ireland was carried out over two weeks in spring 1995. for patients from whom mrsa was isolated during the study period standard demographic and clinical data were requested and period prevalence/1000 discharges was calculated. all 45 microbiology laboratories surveyed responded. mrsa was isolated from 448 patients during the 2 week period. the period prevalence of mrsa/1,000 discharges was 16.5. males aged 65+ had the highest rate of infection (50/1000 discharges). half of all isolates were from patients in surgical or medical wards, but 4% were from community-based sources e.g. gps, nursing homes, hospices. thirty-two percent of mrsa patients were infected rather than colonised. mrsa is clearly a substantial problem in ireland. while it is largely a hospital problem at present, the increasing trend for day procedures and shorter stays means that infection will increase in the community. a survey in a university hospital in the usa revealed 41% of mrsa cases to be communityacquired. tonsil core specimens were cultured for bacteria including mycoplasma, chlamydia and ureaplasma urealyticum in 70 children undergoing tonsillectomy for recurrent acute tonsillitis. serology for chlamydia and mycoplasma pneumoniae was obtained in 55 of the children. the polymerase chain reaction (pcr) was used to investigate the presence of chlamydia pneumoniae in core tonsil tissue. ureaplasma urealyticum was cultured in three children (4.3%) and mycoplasma salivarium in two children (2.8%). culture was negative for chlamydia pneumoniae and mycoplasma pneumoniae. the complement fixation test for chlamydia species was positive in 16/55 children (29%) indicating previous infection. specific immunofluorescence testing for c. pneumoniae was positive for lgg (titre> 16) in 10/55 (18%). igm antibody to c. pneumoniae and antibodies to c. trachomatis and c. psittaci were not detected. ninechildren (16%) had titres > 32 to m. pneumoniae. pcr failed to demonstrate c. pneumoniae. aerobic and anaerobic bacteria were cultured from all specimens. the culture of ureaplasma urealyticum in 4.3% of our patients indicates a higher rate of colonisation then previously thought. this study 49 irish journal of medical science demonstrates past infection with c. pneumoniae in 18% and with m. pneumoniae in 16% of children with recurrent tonsillitis. however c. pneumoniae and m. hominis do not play a significant role in childhood recurrent tonsillitis. multiply resistant enterococci are increasingly common causes of serious infection in hospitalized patients. high level gentamicin resistance (mic > 1000 mga) in enterococci further compromises the therapy of such infections. we have identified seven clinical isolates of enterococcus hirae demonstrating high-level gentamicin resistance (hlgr: mic > i000 mg/1). to our knowledge this is the first report of hlgr for this enterococcus species. plasmid analysis has demonstrated the presence of a single, large plasmid in all seven isolates, as well as several smaller plasmids in some of the isolates. filter mating experiments have revealed that in all seven cases, hlgr was transferred to a laboratory recipient e. faecalis jh-22 by conjugation. plasmid analysis of transconjugant strains confirmed transfer of the large plasmid in all cases. based on restriction enzyme profiles, two distinct conjugative plasmids were identified for the e. hirae isolates investigated. at present we are using southern blot techniques with oligonucleotide probes designed to hybridise to the hlgr determinant found in other species of enterococcus. the results will confirm whether or not the same resistance determinant is responsible for the dissemination of hlgr in the genus enterococcus. dublin 8. aminoglycosides remain commonly used in the treatment of severe gram negative infection and have conventionally been given on a twice or thrice daily basis. single daily dosing offers advantages with respect to less nephrotoxicity, better bactericidal activity, convenience, nursing time, cost and should avoid subtherapeutic dosing which has a significant impact on outcome. we reviewed serum gentamicin assays from january to december 1995 to assess potential toxicity and subtherapeutic dosing in patients who received once daily gentamicin and those who received multiple daily dosing. 4346 assays were performed in the study period. 520 of those were random assays and not included. there was a trend towards significantly less potentially toxic levels in the once daily group compared to the multiple daily group (p<0.1). once daily dosing produced significantly less subtherapeutic dosing (p<0.001). over 98% of peak assays in the once daily group were in the recommended range. we conclude that current practice of multiple daily dosing of gentamicin leads to significant underdosing and more potentially toxic trough levels. measurement of trough assays only in patients who are treated with once daily aminoglycosides is sufficient and will have considerable cost savings. respiratory, syncytial virus (rs virus) is a major respiratory pathogen of infants less than 1 year old. it occurs in annual epidemics during the winter and early spring in temperate climates. during rs virus epidemics a significant number of infants less than 6 months old are hospitalised with symptoms of bronchiolitis and pneumonia. rs virus exists in two antigenically distinct subgroups, a and b which are known to cocirculate in the same community during the same rs virus season. there is much debate regarding the virulence of one strain over the other. using a panel of monoclonal antibodies specifically directed against the two rs virus strains, 167 rs virus isolates from specimens sent to the virus reference laboratory, university college dublin, over seven consecutive rs virus seasons (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) were typed and the rs virus subgroup predominance monitored. subgroup a was the most predominant of the rs virus isolates accounting for 67.7% of the total and was found to be the predominant rs virus strain in six out of the seven rs virus seasons studied. subgroup b predominated in a season in which the number of rs virus detections peaked much later than normal. treatment of fungal infections in patients in intensive care unit (icu.) is usually empiric. the aim of this study was to identify candida species isolated from i.c.u. patients and to test their susceptibility to antifungal agents to enable more directed therapy. forty candida sp. from 23 patients in i.c.u. were isolated from the following sites, blood culture (5), central venous catheter (7), chest drain fluid (5), wounds (8), catheter urine (8), bronchial lavage (3), sputum (4). strains were identified by standard procedures. minimum inhibitory concentrations of amphotoeracin b, 5-flucytosine and fluconazole were obtained by agar dilution test and e test. the isolates were identified as c.albicans (22), c.glabrata (8), c.krusei (5), c.tropicalis (2), c.parapsilosis (2), c.kefyr (i). all the candida species were sensitive to amphoteracin b (mic = 1-<025mg/l), and 5 flucytosine (mi c= <025mg/l). c.albicans and c.parapsilosis were fluconazole sensitive (mic = 16-15mg/l). four of eight c.glabrata were fluconazole resistant (mic = >256mg/l). c.krusei, c.tropicalis and c.kefyr were also resistant (mic = >256 mg/l). wbilst there were no major discrepancies between the agar dilution test and the e test, the agar dilution test was laborious and required a high degree of skill. the e test was easier to read and more reliable results were obtained provided the inoculum was carefully standardised. this study shows that azole antifungals should not be ganglion is probably the commonest tumour encountered in the hand and the wrist. it often arises from tendon sheath or lining of a joint capsul. the treatment can be surgical or nonsurgical, the latter includes aspiration with or without injection of steroids. surgical treatment of ganglion can pose a difficult situation to deal with. it requires hand surgeon to deal with one such problem, we present a 46 year old man with tender mass in the hypothenar eminence. during surgical exploration it was obvious that the ganglion was infiltrating the wall of the ulnar artery, and the histology proved this later. the clinical features, management arid the outcome of this unusual case are discussed. ischaemic preconditioning (ipc) of the myocardium with repeated brief periods of ischaemia and reperfusion (i-r) prior to prolonged ischaemia significantly reduces subsequent infarction. following ipc two "windows of opportunity" (early and late) exist during which prolonged ischaemia can occur with reduced myocardial infarction, we investigated if ipc of skeletal muscle prior to flap creation improved subsequent flap survival and perfusion in either early or late windows. the latissimus dorsi muscles (ldm) of sprague-dawley rats were used. group 1: (control, n=12). the ldm was elevated as a thoracodorsatly based island flap group 2: (early ipc, n=8). the ldm was preconditioned with two 30 minute episodes of normothermic global ischaemia with intervening 10 minute episodes of reperfusion prior to elevation. group 3: (late ipc, n=8). the ldm was elevated 24 hours after ipc ischaemia was created by occlusion of the thoracodorsal artery and vein and the intercostal perforators having previously isolated the muscle on these vessels. muscle perfusion was assessed by a laser doppler perfusion imager. one week after flap elevation the percentage of muscle necrosis was measured by computer-assisted planimetry ipc significantly reduced muscle flap necrosis (table) in both early and late windows. muscle flap perfusion was similar in all groups. this study compares the biochemical, serological and histopathological findings in 20 women with chronic hepatitis c virus (hcv) infection with 20 age-matched women with established autoimmune hepatitis (aih). there is increasing evidence of autoimmunity in hcv liver pathology. because of different treatment regimens for hcv (d-interferon) and aih (steroids, immunosuppression), clear distinction between the two diseases is desirable. liver enzymes (alt, ast, alkaline phosphatase), antinuclear factor (anf), anti-smooth muscle (asm) and antimitochondrial (ama) antibodies are compared for both groups of patients. liver biopsies from all women were compared using the grading and staging system of ishak et al (1995) . the results show that while some women in both groups show elevated liver enzymes, positive anf and asm autoantibodies, the values are much higher in aih. similarly aih patients show overall more severe disease on liver histology. both groups demonstrate poor correlation between histological features and autoantibody titre. we conclude that distinction between hcv and a!h is usually possible with liver histology and serology. some women with chronic hcv and positive autoantibodies however," demonstrate a histological and serological picture suggesting that chronic hcv may be mediated by an immunopathogenic mechanism. irish journal of medical science terminal changes only, 5 showed lymphocytic meningitis and 4 of these also had perivascular lymphocytic inflammation (cd3 positive) in the subependymal regions, brainstem and choroid plexus. two brains showed purulent meningitis and one case had central pontine myelinolysis probably related to profound metabolic disturbance. basal ganglia mineralization, hiv encephalitis (hive) or hiv leucoencephalopathy (hivl) were not present. these findings differ considerably from those described in us cases, in whom the majority have evidence of hiv within the cns. relatively early death from systemic infection may account for the lack of hive/hivl in these cases. the lymphocytic meningitis and perivascular inflammation may represent an immuno-allergic reaction, previously reported as "early" changes, regarded as important in inducing vascular damage which allows subsequent entry of h1v into the brain. the aim of this study is to assess apoptosis in areas of interface hepatitis and spotty necrosis in hepatitis c virus (hcv) infected liver biopsies, and to correlate the degree of apoptosis with severity of histological activity. 25 liver biopsies were randomly selected from a group of type lb hcv positive women. these patients were diagnosed by recombinant immunoblot assay (riba) test and the presence of hcv rna was confirmed using the polymerase chain reaction (pcr). apoptosis was demonstrated in the biopsies by the oncor apoptag in-situ hybridisation technique. the average number of apoptotic hepatocytes per portal tract, and within the parenchyma per 10x objective, was determined. the modified histological activity index (h.a.i.) was used to score each biopsy. comparison of the results shows that increasing numbers of apoptotic hepatocytes are consistently associated with increasing scores for interface hepatitis and spotty necrosis. it is concluded that apoptosis occurs in hcv infected livers and that it correlates with increasing histological activity .indicating a significant role for apoptosis in the pathogenesis of hcv liver disease. and university of edinburgh, scotland. paediatric aids represents only 5% of cases worldwide, in most published series parental iv drug use was the main risk factor, cns pathology was a late feature of the disease and antiretroviral treatment had been given. we studied eleven brains from romanian children with probable postnatal hiv infection using standard neuropathological stains and immunostains for lymphocyte markers and hiv p24 antigen. death was due to systemic infection, mostly pneumonia or gastroenteritis; antiretroviral treatment had not been given. three cases showed we studied the role of the p53 and bcl-2 genes in the pathogenesis of post-transplant lymphoproliferative disease (ptl). ten cases were examined by immunohistochemical and molecular methods. immunohistochemistry was performed using standard and antigen retrieval methods, with the p53 do-7 and the bcl-2 oncoprotein clone 124 antibodies. dna was extracted from paraffin blocks and subjected to pcr, and single-strand conformation polymorphism (sscp) analysis searching for mutated p53 genes. samples showing any evidence of aberrant migrations were further analysed by direct sequencing. pcr was also used to detect bcl-2 gene rearrangements. we employed a technique called representational difference analysis to search for previously undescribed translocations or deletions which may be involved in breast carcinogenesis. dna was isolated from both invasive ductal carcinoma of the breast and normal tissue from the same patient. following restriction enzyme digestion and tigation of oligonucleotide linkers, pcr was carried out on both tumour and normal dna using oligonucleotide specific primers to obtain a representation of each genome (amplicons). digestion with the same restriction enzyme removed the original linkers, and a second set of oligonucleotides were ligated to normal amplicons only. the tumour derived amplicons were subtractively hybridised to the normal and subsequent pcr was used to isolate fragments unique to the normal dna (difference products). this was possible since oligonucleotides were ligated to normal dna only. following a series of further subtractive hybridisations and subsequent amplifications, purified difference product was obtained. difference products in the size range 500-1500 bp were obtained. following further rounds of subtractive hybridisation and amplification, purified difference product will be sequenced and characterised by comparison with known gene sequences. the chromosomal location of the affected gene will be established by in-situ hybridisation and somatic ceil hybridisation, using the difference product as probe. protein s is secreted by osteoblasts and case reports of reduced bone mineral density in patients with total protein s deficiency have lead to the hypothesis that this inherited disorder is associated with generalised osteoblast dysfunction predisposing to osteoporosis ~. we have assessed bone formation in 8 patients (2 male and 6 female) with total protein s deficiency and 4 controls (2 male and 2 female) using two recently available sensitive markers; serum osteocalcin (oc) and serum procollagen 1 carboxyterminal peptide (p1cp), both secreted by the osteoblast. the mean total protein s level amongst the patients was 40 _+ 12% (ref range 62-135%), mean oc was 14.2 ng/ml (ref ~'ange 5.2-59 ng/ml) and the mean picp was 81.5 + 15 uga (ref range 38-02 ug/1). in the control group, mean oc was 12.6 _+ 5 ng/ml and the picp was 124 + 37 ug/1. there was no statistical difference between both groups using either marker. in conclusion bone formation as assessed by serum osteocalcin and p1cp appears to be normal in patients with total protein s deficiency. hereditary spastic paraparesis (hsp) is a variably expressed neurodegenerative disorder which exhibits clinical and genetic heterogeneity. hsp can be inherited in an autosomal dominant (ad), autosomal recessive (ar) or x-linked manner. ad-hsp has been linked to a number of loci. we have ruled out linkage to these loci in a large irish family affected with ad-hsp. the aim of this study was to determine whether ad-hsp is linked to spinocerebellar ataxia loci (sca), sca-i & sca-ii. ad-hsp can be clinically similar to sca. dna was extracted from blood taken from 45 co-operating family members. microsatellite markers spanning the sca-i and sca-ii loci were amplified by pcr. individuals were genotyped and linkage analysis was carried out using the linkage set of programs. significantly negative lod scores were obtained for both sca-i and sca-ii loci. d6s274 gave maximum exclusion of 18cm on either side of the sca-i locus with a lod of -2.11 at a recombination fraction of 0.18. d12s105 gave maximum exclusion of 13cm on either side of the sca-ii locus with a lod of -2.08 at a recombination fraction of0.13. other markers examined also outruled linkage to these loci. we conclude that the gene for ad-hsp is unlinked to the major sca loci. serum vitamin b12 is frequently measured in the investigation of anaemia, and in screening neurological and other disorders. frequently, patients are found with low serum vitamin b12 level with a normal hb and without clinical abnormalities relevant to vitamin b 12 deficiency. this study was carried out to determine the significance of a low serum vitamin b 12 level. 959 vitamin b12 measurements were carried out over an 8 month period using a chemiluminescence method (abbott imx). clinical data was obtained retrospectively. of the 959 samples 81 (8.4%) representing 65 patients had a low serum vitamin b12 level (>180 pg/ml) with a mean of 152 pg/ml (39-179). data was available on 44 patients. 20 (45%) had a hb below the normal range with median serum vitamin b12 level of 158 pg/ ml (75-184). 19 (45%) had a normal hb, mcv and mchc with a median serum vitamin b12 of 152 pg/ml (52-210), and 5 had a normal hb with an abnormal mcv or mch. lft's, autoantibodies, schillings test and bone marrow examination data will be presented. in conclusion in patients with a low serum vitamin b 12 level, there was no significant difference in the b12 levels in those patients with a normal or a low hb concentration. it would appear that serum vitamin b12 is a poor discriminatory test but that changing the normal range may not help in screening. low serum vitamin b12 on its own may not appear to provide adequate grounds for lifelong replacement therapy. of 1 in 133 for males and 1 in 172 for females. as expected the overall incidence of hodgkin's was lower with one third of male and one quarter of female lymphoma cases affected by the disease. a distinct age specific pattern is evident depending on lesion type. marked variation in incidence levels were noted throughout the study region. an extremely varied pattern is evident in the survival rates for lymphoma patients. the cork and kerry rates for malignant lymphoma are relatively low when compared with international levelstzl obstetric complications and schizophrenia: methodology and mechanisms perinatal complications and clinical outcome within the schizophrenia spectrum 96) negative symptoms, cognitive impairment and duration of initially untreated psychosis in schizophrenia davnet's hospital, monaghan, and royal college of surgeons in ireland retinal pathology in kearns sayre syndrome mitochondrial dna and disease mitochondrial myopathies: clinical features, investigation, treatment and genetic counselling phenytoin induced pseudolymphoma. a report of a case and review of the literature cutaneous reactions in head injured patients receiving phenytoin for seizure prophylazis hydantoin induced pseudopseudolymphoma branhamella catarrhalis: an organism gaining respect as a pathogen correlation between branhamella catarrhalis adherence to oropharyngeal cells and seasonal incidence of lower respiratory tract infections 206) epidem1ology and survival rates for all 324 lymphoma patients registered in cork and kerry over the eight year period an indepth review of all 324 lymphoma (icd -o code 196) patients registered by the sourthern tumour registry during the eight year period 1983/1990~l annual age adjusted rates of 7.6. and 5.4 per 100,000 were seen for males and females respectively. these levels indicate a lifetime (up to 75 yr) risk references 1. cancer, the irish experience cancer incidence in five continents volume v immunohistochemistry for bcl-2 oncoprotein without antigen retrieval gave negative results, but with antigen retrieval, showed positive staining in 6 out of 8 cases. no bcl-2 rearrangements were detected by pcr. the combination of sscp and sequencing confirmed only wild type dna in all cases, p53 immunohistochemistry by standard methods revealed positive staining in only one out of nine samples analysed. when the antigen retrieval method was employed for this antibody, positive staining was seen in > 10% of tumour cells in four further cases.our results suggest that p53 does not play major role in ptld. bcl-2 overexpression but not rearrangement may contribute to the development of ptld. transplant arteriopathy (ta) is the major cause of death in cardiac allograft recipients. the pathogenesis is unclear. we have previously shown a plasma cell predominance in the infiltrate of ta, leading us to hypothesise a role for epstein-barr virus (ebv) infection in its pathogenesis. an association between cytomegalovirus (cmv) and ta has previously been suggested. the aim of the study was to investigate the role of epstein-barr virus (ebv) and cytomegalovirus (cmv) in the pathogenesis of ta.we performed pcr for cmv and ebv dna and protein (lmp) in seven cases of ta, involving cardiac allografts. restriction mapping was used to confirm that pcr products were either cmv or ebv dna respectively.cmv dna was found in four cases. ebv dna was found in six of the seven cases and ebv lmp staining was present in six cases. ebv was detected in all cases by either pcr or ihc.our results suggest that ebv infection may play a pathogenic role in transplant arteriopathy. the evidence for a similar role for cmv ~s less strong. st. vincent's hospital. hereditary spastic paraplegia (hsp) is a neurodegenerative disorder characterised by progressive spasticity, primarily of the lower limbs. it can be inherited in an autosomal dominant (ad), autosomal recessive or x-linked manner~ we have identified a large irish family (family a) affected with ad hsp that cosegregates with dementia. three. loci have previously been identified that are linked to ad hsp in families of different ethnic origin. the locus on chromosome 2 is reported to be the major hsp locus. the aim of the present study was to examine family a for linkage to the chromosome 2 hsp locus.dna has been extracted from blood taken from all 45 co-operating family members for genotyping. polymorphic microsatellite markers from chromosomal region 2p24-21 have been amplified by pcr, electrophoresed on a denaturing polyacrylamide gel and detected by silver staining. linkage analysis was carried out using the linkage series of programs. linkage analysis excluded the hsp gene from the chromosome 2p locusl the most significant marker was d2s367, with a lod score of-2.12 for recombination fraction 0.19, thereby excluding approximately 19cm either side of this marker. negative lod scores were also obtained for the other markers chosen (d2s391, d2s392, d2s352, d2s 174) excluding 20cm, 8cm, 4cm and 8cm respectively.the current study has therefore successfully excluded linkage of ad hsp in family a to the major locus on chromosome 2p. further studies are underway to exclude linkage of hsp in this family from other candidate loci, prior to carrying out a genome wide search. the presence of dementia in this family in association with hsp suggests that a new and as yet unidentified gene is responsible. vincent's hospital. eye and ear hospital, dublin. ched is a corneal endothelial dystrophy characterised by diffuse bilateral corneal opacities resulting in impaired vision. both autosomal dominant and autosomal recessive modes of inheritance have been described. another endothelial dystrophy, posterior polymorphous dystrophy (ppmd) has been linked to 20qll.we have used homozygosity mapping to analyse a pedigree with autosomal recessive ched for linkage to 20ql.1. all affected individuals are offspring of consanguinous matings. homozygosity mapping is based on the principle that these offspring would be homozygous for genetic markers near the disease gene. homozygous regions would be random between different offspring of these matings, except at the di-sease locus shared by affected offspring.dna was extracted from blood taken from 22 family members, 14 of which have ched. allele frequencies were determined in pooled dna from affected individuals. pooled dna from unaffected individuals was used as a control. at the disease locus, a shift in allele frequencies towards a single homozygous allele would be observed in the affected dna pool. pooled dna was genotyped by pcr for 3 polymorphic microsatellite markers in the region of20qll. pcr products were separated on a polyacrylamide gel and visualised by silver staining. similar allele frequencies were observed at these loci in both dna pools demonstrating independent assortment of alleles. in addition, affected and unaffected family members were individually genotyped at these loci and no significant loss of heterogeneity in the affected individuals at these loci was observed. these data indicate exclusion of linkage of the ched gene to 20qll. key: cord-000077-d441jam3 authors: zhang, hao-jie; wang, yong-xiang; wu, hao; jin, dong-yan; wen, yu-mei; zheng, bo-jian title: the y271 and i274 amino acids in reverse transcriptase of human immunodeficiency virus-1 are critical to protein stability date: 2009-07-03 journal: plos one doi: 10.1371/journal.pone.0006108 sha: doc_id: 77 cord_uid: d441jam3 reverse transcriptase (rt) of human immunodeficiency virus (hiv)-1 plays a key role in initiating viral replication and is an important target for developing anti-hiv drugs. our previous study showed that two mutations (y271a and i274a) in the turn rt (gln(269)-arg(277)) abrogated viral replication, but the replication capacity and rt activity was discordant. in this study, we further investigated why alanine substitutions at these two sites would affect viral replication. we found that both rt activity and rt protein were almost undetectable in viral particles of these two mutants, although the pr160(gag-pol) mutants were properly expressed, transported and incorporated. using protease inhibition assay, we demonstrated a correlation between the degradation of the rt mutants and the activity of viral protease. our native gel analysis indicated that the mutations at 271 and 274 amino acids might cause conformational changes, leading to the formation of higher order oligomers instead of dimers, resulting in increased protein instability and susceptibility to viral protease. thus, residues 271 and 274 are critical to rt stability and resistance to viral protease. the conservation of the two amino acid residues among different strains of hiv-1 lent further support to this conclusion. the knowledge gained here may prove useful in drug design. reverse transcriptase (rt) of human immunodeficiency virus (hiv)-1, encoded by pol gene, is a multifunctional enzyme that possesses rna-and dna-dependent polymerase activities as well as rnase h activity [1] . rt is indispensable for hiv-1 and it converts the single-stranded viral rna into double-stranded dna upon viral entry into host cells. due to its important role in viral life cycle, rt is one attractive target for antiviral drug design [2] . the biologically active form of hiv-1 rt is a heterodimer consisting of two subunits, p66 (66 kda) and p51 (51 kda). the p51 subunit is derived from p66 by proteolytic cleavage of its cterminal domain [3] . the polymerase domain of p66 and p51, resembling a right hand configuration, consists of four subdomains, which are known as fingers, palm, thumb and connection. the fingers, palm and thumb subdomains of p66 form a nucleic acid binding cleft and the connection subdomains of the two subunits form the floor of the nucleic acid binding site [4] [5] [6] [7] . the thumb subdomain has four a helices. two antiparallel ahelices of them, a-h (asn 255 to ser 268 ) and a-i (gln 278 to thr 286 ), are important for holding the primer/template in position during the translocation in polymerization. the primary sequence (val 254 to ala 288 ) in the vicinity of these two a helices has been found to share homology with several other nucleic acid polymerases and has been termed the ''helix clamp'' [5, 8] . extensive studies have been carried out to shed light on the relationship between the ''helix clamp'' and function of rt. the effects of alanine-scanning mutations in a-h and a-i on polymerase activity, primer/template (p/t) binding, fidelity and enzyme kinetics have been determined. while mutations in a-i do not affect p/t binding or fidelity significantly, several a-h mutants exhibit lower binding affinity, processivity and frameshift fidelity [9] [10] [11] [12] . previous studies have demonstrated that mutations in these two helices can have significant effect on rnase h activity, minus-strand dna transfer activity and removal of polypurine track primer [13, 14] . although alanine substitutions at sites 269, 270, 271 and 277 have been investigated in two studies [9, 10] , detailed studies on the functional structure of the ''turn'' (gln 269 -arg 277 ) between a-h and a-i were limited. in our previous study on hepatitis b virus rt, conserved residues located at the ''turn'' of helix clamp motif were found important for pregenomic rna encapsidation during the assembly of nucleocapsids [15, 16] . since this homologous helix clamp motif is also present in hiv-1 rt, we hypothesized that residues in the turn may play important roles in viral life cycle. our recent study showed that alanine substitutions at 271 and 274 of hiv-1 rt drastically affected viral replication, but discordance between viral replication and rt activity was observed [17] . in this study, we confirmed our previous observations and further investigated why the two mutations abrogated viral replication. our study demonstrated that these two mutations lead to rapid degradation of rt in viral particles, indicating that the residues of 271 and 274 are critical for maintaining the stability of hiv rt. the parental hiv-1 proviral plasmids, plai.2, pnl4-3-de-egfp and phef-vsv-g, were obtained through the nih aids research and reference reagent program, division of aids, niaid, nih [18] [19] [20] . the pnl4-3-de-egfp based mutants (y271a, g273a, i274a, k275a, v276a, r277a) and plai2 based mutants (y271a and i274a) were constructed by sitedirected mutagenesis using quikchange ii xl site-directed mutagenesis kit (stratagene, usa) according to manufacturer's instruction. the open reading frame of rt p66 subunit was amplified from wild type or mutant pnl4-3-de-egfp using a pair of primers and cloned into pet-28b vector (novagen, shanghai) to obtain expression plasmid pet-p66 with the 66 his tag at 39 terminus. the paired primers used for site-directed mutagenesis and construction of plasmids are listed in table 1 . cell lines 293ft, hela and u373-magi-cxcr4 cem [21] were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum and antibiotics (invitrogen, usa). mt2 cells [22, 23] were maintained in rpmi 1640 supplemented with 10% fetal bovine serum and antibiotics. to prepare pseudoviruses and live viruses, 293ft cells were cotransfected with 7.5 mg wild-type or mutant pnl4-3-de-egfp, and 2.5 mg phef-vsv-g, or 10 mg wild-type or mutant plai.2 using lipofectamine 2000 (invitrogen, usa) according to the manufacturer's instructions. supernatants were collected 48 h after transfection and stored at 280uc after spinning down and filtering to remove cell debris. the cells were rinsed with ice-cold phosphate-buffered saline (pbs), scraped from each plate and lyzed in cell lysis buffer (boehringer, germany) and 16 protease inhibitor cocktail (roche, usa) on ice for 30 min. cell lysates were stored at 220uc after centrifugation at 13,0006g at 4uc for 15 min to remove cell debris. one cycle infection assay was carried out using normalized pseudoviruses as described previously [20] . briefly, jurkat cells (0.5610 6 ) were infected with viral supernatants containing 250 ng p24, which was measured by vironostika hiv-1 antigen microelisa kit (biomerieux bv boxtel, netherlands). the virus and cells mixture was spun at 1,800 g at 30uc for 2 h. after the 2h spin infection, jurkat cells were washed with 2 ml culture medium twice, then cultured in 24-well plates at 37uc for 48 h. the cells were collected and washed twice with pbs. after the cells were fixed with 1% paraformaldehyde in pbs for 30 min on ice, the infected cells, as determined by the expression of gfp, were measured using a facscalibur instrument (becton dickinson, usa) and analyzed with cell quest software (becton dickinson, usa) as described previously [20] . infection with live viruses was conducted in u373-magi-cxcr4 cem and mt-2 cells. u373-magi-cxcr4 cem cells were infected with normalized wild type or mutant live viruses in 24-well plate as described previously [21] . briefly, triplicate wells (6610 4 cells/well) were infected with live viruses (60 pg p24/well) which were diluted in dmem containing 20 mg/ml of deaedextran (amersham biosciences). after cultured at 37uc in 5% co 2 incubator for 48 h, the cells were fixed in 1% formaldehyde-0.2% glutaraldehyde in pbs for 5 min. after washing twice with pbs, the cells were stained with 400 mg/ml of x-gal (5-bromo-4chloro-3-indolyl-b-d-galactopyranoside), 4 mm mgcl 2 , 4 mm potassium ferrocyanide, and 4 mm potassium ferricyanide in pbs for 2 h at 37uc. the plate was washed twice with pbs and blue foci were observed under microscope. infection with live viruses was also carried out in mt-2 cells as described previously [22, 23] . in brief, mt-2 cells (1610 4 cells/well) in 96-well plate were infected with live viruses (20 pg p24/well). after cultured for 6 days, the virus-specific cytopathic effect (cpe) was observed under microscope. the rt activity in pseudoviruses was measured by a rt assay using colorimetric kit (roche, usa). briefly, viral supernatants containing 2 mg p24 were centrifuged at 4uc for 2 h at 40,0006g and the viral pellets were resuspended in 50 ml lysis buffer. lyzed viral pellets were 10 fold serially diluted and the subsequent procedures were carried out according to the manufacturer's instructions. rt activities of mutants were calculated and compared to that of wild type pseudovirus. expression and subcellular localization of precursor gal-pol polyprotein were detected by immunofluorescence microscopy as described previously with some modifications [24] . briefly, hela cells cultured on coverslip in 24-well culture plate were transfected with 600 ng pnl4-3-de-egfp and 200 ng phef-vsv-g. two days post-transfection, the cells were fixed with 500 ml 4% paraformaldehyde (pfa) at room temperature for 15 min. after washing 3 times with pbs, 500 ml of 50 mm ammonium chloride was incubated with the cells for 10 min to neutralize residual pfa. the cells were washed 3 times with pbs and treated with 0.05% triton x-100 for 3 min. after washing 3 times with pbs, 500 ml 10% normal rabbit serum (nrs) in pbs was added to block the slip overnight at 4uc. primary antibody (mouse monoclonal antiintegrase, 1:100, santa cruz) and secondary antibody (texas red dye-conjugated rabbit anti-mouse igg, 1:100, jackson immu-noresearch) were incubated with samples in dark at room temperature. following each incubation, samples were subjected to 3 washes with 1% nrs in pbs. with 3 additional pbs washes, the coverslip was mounted using fluorescence mounting medium (dako) and observed under lsm510 meta confocal microscope (carl zeiss). gal-pol polyprotein and its products were tested by western blotting as described previously with some modifications [25] . briefly, viral proteins were separated by 10% sds-polyacrylamide gel electrophoresis (sds-page) and electro-blotted onto hybond-p pvdf membrane (ge healthcare, bio-sciences). after blocking with 5% skim milk for 1 h at room temperature, the membrane was incubated with primary antibodies (rabbit polyclonal anti-rt, 1:3000; mouse monoclonal anti-rt, antiintergrase, anti-protease or anti-capsid, 1:500) for 1 h. membrane was washed three times with pbs containing 0.1% tween 20 (pbs-t) and then incubated with horseradish peroxidase (hrp)-conjugated secondary antibody (goat anti-rabbit igg or goat anti-mouse igg, 1:4000) for 1 h. after the blots were rewashed three times in 0.1% pbst, signals were visualized using ecl western blotting substrate reagents (amersham biosciences, usa) and kodak biomax scientific imaging film (eastman kodak). wild type and mutant pet-p66 expression vectors were respectively transformed into e. coli bl21(de3) and the expression was induced with 0.3 mm isopropyl b-d-1-thiogalactopyranoside (iptg) when e. coli grew up to an od600 of 0.7,1.0. e. coli expressing p66 was spun down and re-suspended in the binding buffer (50 mm nah 2 po4, 300 mm nacl, 10 mm imidazole). after the bacteria were disrupted by ultrasonication and centrifuged at 15 000 g for 30 min at 4uc, the wild type and mutant p66 in supernatants were purified using ni-nta magnetic agarose beads (qiagen, germany). the purified p66 proteins were subjected to nativepage novex bis-tris gel analysis and western blotting according to the protocol recommended by invitrogen, usa. rt sequences of 1083 hiv-1 strains obtained from the hiv complete sequence database (http://www.hiv.lanl.gov/content/ sequence/newalign/align.html) were aligned and compared. based on the x-ray crystal structures of hiv-1 rt (1rth) from the research collaboratory for structural bioinformatics protein data bank (rcsb pdb), structural models of wild type and mutant rts were analyzed using swiss-pdbviewer software (http://spdbv.vital-it.ch/). statistical analysis of rt activities was performed by student's t test using stata statistical software. results were considered significant at p#0.05. the effect of six rt mutations at the helix clamp turn in hiv-1 rt on viral replication was tested with pseudoviruses. when jurkat cells were infected with the wild type and mutant pseudoviruses (250 ng p24 virus/5610 5 cells), viral replication was almost completely inhibited in the mutants y271a and i274a, while replication of other mutants did not show significant difference as compared to that of the wild type (fig. 1a) . a similar result was also obtained when the cells were infected with lower amount (150 ng p24) of pseudoviruses (data not shown). to confirm the above results, replication of wild type, y271a and i274a live viruses was further monitored in u373-magi-cxcr4 cem and mt2 cells. mutants y271a and i274a were undetectable in u373-magi-cxcr4 cem cells (fig. 1b) and no virus-specific cpe was found in mt-2 cells (fig. 1c) . since reverse transcription is the first step for hiv replication after viral entry into host cells, our results suggested that the two mutations might have affected the rt activity. rt activity and rt proteins were undetectable in pseudoviral particles of mutants y271a and i274a we thus interrogated the pseudovirus mutants for rt activity. the rt activity recovered from mutants y271a and i274a were almost unnoticeable, as compared with that of wild type ( fig. 2a) . this result was further confirmed using wild type, y271a and i274a live viruses (data not shown). next, we asked if the viral particles contain dysfunctional rt or the rt was not incorporated into the viral particles. by western blotting, rt protein was basically undetectable in viral particles of these two mutants (fig. 2b) . the results suggested that the mutations could affect the incorporation of rt into the viral particles, leading to a defect in reverse transcription which initiates viral replication. mutations y271a and i274a did not affect pr160 gag-pol expression and transportation we next investigated whether the loss of rt in viral particles of the mutants was attributed to altered expression and transportation of the precursor protein, polyprotein pr160 gag-pol , during virus assembly. pr160 gag-pol in lysates of cells transfected with the wild type and mutants was detected by western blotting. as shown in fig. 3a , similar levels of pr160 gag-pol , gag protein (pr55 gag ) and capsid protein p24 (ca p24) were found in cells transfected with the wild type or mutant constructs, indicating that the expression and stability of rt precursor protein were not affected by the mutations. immunofluorescence staining further demonstrated a normal subcellular localization of mutant gag-pol polyproteins as compared to that of the wild type, suggesting that the transportation of gag-pol was unlikely to be affected by alanine substitutions (fig. 3b) . the gag-pol polyprotein was incorporated into mutant virions of y271a and i274a, but the rt was degraded by viral protease to investigate whether the precursor gag-pol polyprotein was indeed incorporated into pseudoviral particles of mutants y271a and i274a, products of the gag-pol polyprotein, integrase (in), protease (pr) and p24, in wild type and mutant pseudoviral particles were examined by western blotting. the results showed that, except for rt (fig. 2b) , all products of the gag-pol polyprotein, in p32, pr p11 and ca p24, were detected in the mutants y271a and i274a at a level similar to that of the wild type pseudoviral particles (fig. 4a) . thus, pr160 gag-pol was indeed incorporated into the virions and processed properly. to study if the rts in the viral particles of y271a and i274a mutants were degraded by proteolysis that made them undetectable, pseudoviruses of wild type and mutants were generated in the presence or absence of indinavir, a highly specific inhibitor of hiv-1 protease. indinavir treatment was effective, because the treatment resulted in a dose-dependent inhibition of pr55 gag processing into p24 for wild type and mutant viruses (fig. 4b ). as shown in fig. 4c , in the absence of indinavir, both rt p66 and p51 were readily detected in the wild type pseudovirus, but not in the mutant viruses. in the presence of indinavir, however, both rt p66 and p55 were markedly reduced in the wild type virus but became detectable in mutant y271a viral particles, while rt p66 was also detectable in mutant i274a virus. we also detected the rt subunits in the viral supernatants collected at earlier time points (12, 24 and 36 hours post-transfection), but the mutant rts were still undetectable (data not shown). these results suggested that the y271a and i274a rts were degraded after incorporation of gag-pol polyprotein into the virions, which might be attributed to the activity of viral protease. since rt dimer is the stable form which remains resistant to the proteolysis, we tested whether treatment of dimerization enhancer could reduce the mutant rt proteolysis. wild type and mutant pseudoviruses were generated in the presence or absence of efavirenz (efv), the most potent dimerization enhancer [26] . the results showed that the rt mutants were still undetectable in the presence of efv (fig. 5a) . the conformation of the wild type and mutant rt p66 was further analyzed by native gel electrophoresis followed by western blotting (fig. 5b) . under native conditions, wild type p66 subunit formed homodimer. however, the formation of homodimer in mutant y271a was markedly reduced and a higher order oligomer appeared, which might be tetramer according to its molecular weight, while mutant i274a existed as at least two higher order oligomers, which were likely tetramer and octamer based on their molecular weights. however, the exact nature of the oligomers formed by mutant p66 subunits remained to be clarified. furthermore, the treatment with b-mercaptoethanol could partially disrupt the higher order oligomers and improve dimer formation. this result implicated that dimer formation might be necessary for the stability of rt and its resistance to proteolysis after incorporation of gag-pol polyprotein into the viral particles. amino acids at 271 and 274 are relatively conserved and big side chains may be important in maintaining rt stability and resistance to proteolysis by comparing 1083 complete sequences of hiv-1, it was found that the amino acids at 271 and 274 were relatively conserved. as shown in table 2 , tyrosine (y) is the predominant naturally existent amino acid at 271 residue (99.19%), while phenylalanine (f), histidine (h) and cysteine (c) occur rarely (,1%). similarly, isoleucine (i) is the predominant naturally existent amino acid at 274, which accounts for 98.43%, while in less than 2% of all cases, valine (v) and leucine (l) are found at this site. this finding suggested that these amino acids might probably play important role in maintaining the active conformation of rt. consistently, structural analysis suggested that amino acids at these two sites are buried in the thumb region of rt (fig. 6a) . it was further revealed that all wild type rts have relatively big side chains, but they are absent from the mutants 271a and 274a (fig. 6b & 6c) . thus, the loss of the side chains in the rt mutants plausibly leads to conformational change of rt, leading to aberrance in dimer formation and susceptibility to proteolysis by hiv-1 protease. hiv rt plays a key role in viral replication and is an important target for development of anti-hiv drugs. however, the turn between helices h and i of hiv-1 rt thumb region (amino acid residues 269 to 277) has not been well characterized yet. to understand the structure-function relationship of this turn in details, we investigated whether alanine substitutions in this region would affect rt activity. except for residues 269 and 270, which have been reported to have no significant influence on rt activity [9, 10] , and for residue 272, which is originally an alanine, six mutant pseudoviruses (residues 271 and 273 to 277) were constructed and viral replication was compared with that of the wild type virus. the replication of two mutants, y271a and i271a, were found to be almost completely abolished (fig. 1a) . this result was further confirmed in live virus system (fig. 1b & 1c) . since it has been reported that bacterially expressed y271a mutant has only 1% activity of wild type enzyme [9] , we asked whether the mutants would similarly affect rt activity in the viral particles. the results showed that the rt activity was basically undetectable in viral particles of these two mutants ( fig. 2a) . it was also found that the loss of rt activity in the viral particles might be attributed to the absence of rt in the viral particles rather than the incorporation of dysfunctional rt into the virions (fig. 2b) . after viral entry into the cells, the first step of hiv-1 replication is reverse transcription. our results thus suggested that replication of mutant viruses was abrogated, because the mutant rt did not exist in the virion. since hiv-1 rt is incorporated into the virion in the form of pr160 gag-pol , which is transported to cell membrane where the virus is packaged, through interaction with pr55 gag [27] [28] [29] [30] , we thus investigated whether the mutations would affect pr160 gag-pol expression and transportation. our results showed that pr160 gagpol was properly expressed and transported in cells transfected with mutant constructs, y271a and i271a (fig. 3) , indicating that these mutations did not affect either the production of the precursor or the interaction between pr160 gag-pol and pr55 gag . it was further found that the pr160 gag-pol was indeed incorporated into the virions of these two mutants, because except for rt, all other products of pr160 gag-pol including p24, protease and integrase [31, 32] , could be detected in the viral particles of mutants y271a and i271a (fig. 4a) . although it has been reported that the domain between residues 183 and 305 of rt is likely responsible for rt incorporation [33] , our study ruled out the involvement of rt was detected in wild type and mutant pseudoviruses generated in the presence and absence of efv, the most potent dimerization enhancer, by western blotting using mouse monoclonal anti-rt and anti-ca p24 antibodies, respectively. (b) purified wild type and mutant rt subunit p66 were analyzed by native gel electrophoresis followed by western blotting using mouse monoclonal anti-rt antibody in the presence or absence of b-mercaptoethanol (b-me). compared with wild type p66, which basically existed as homodimer, p66 subunit of the 271a and 274a mutants formed higher order oligomers, suggestive of conformational change. doi:10.1371/journal.pone.0006108.g005 the turn (gln 269 -arg 277 ). another report has suggested that two mutations in rt, l234d and w239a, led to premature cleavage of the gag-pol precursor and reduced levels of viral enzymes in the virions [34] . our results also excluded that the mutations of y271a and i271a affected the cleavage of the gag-pol precursor. by a viral protease inhibition assay using indinavir, we finally demonstrated that the rt mutants were degraded after incorporation of the precursor polyprotein into the virions and the degradation was associated with the activity of viral protease (fig. 4c) . our results indicated that the mutations in residues 271 and 274 would reduce the stability of rt inside the viral particles, rendering it susceptible to viral protease. post-incorporation degradation of rt has been reported previously. wapling et al. [35] have reported that mutations of w401l and w401a in rt can inhibit rt dimerization, resulting fig. 6a . a big side chain (blue) is found in naturally occurring residues but not in 271a. (c) structural models of naturally existing 274i, 274v and 274l residues as well as the lethal 274a mutation of p51 subunit. the same region was highlighted above in the right panel of fig. 6a . a big side chain (pink) is found in naturally occurring residues i, v and l, but not in 274a. doi:10.1371/journal.pone.0006108.g006 [36] . another mutation, l289k in p66 subunit, has also been reported to be able to abrogate dimerization [37] . another group, when they tried to generate infectious molecular clones of simian immunodeficiency virus, found that glutamic acid replacement at position 287 affected the stability of rt [38] . the biologically active and stable form of hiv-1 rt is a heterodimer consisting of p66 and p51, while the immediate precursor of p66/p51 heterodimer is the p66 homodimer. proteolytic removal of the rnase h domain in one of the p66 homodimer subunit by hiv-1 protease leads to the formation of stable heterodimer p66/p51 [39] . rt exists as an equilibrium mixture of monomers and dimers that include the p66/p51 heterodimer and the p66/p66 and p51/p51 homodimers, among which the heterodimer is the most stable and the p51/p51 homodimer is the most unstable [40] [41] [42] [43] . plausibly, the degradation of rt as reported in previous studies may be ascribed to the inhibition of rt p66 dimerization by the mutations. the mechanism of rt degradation in the virions observed in this study, however, may be different. our results showed that rt mutants were still undetectable in the viruses generated in the presence of a potent dimerization enhancer (fig. 5a ). our native gel analysis showed that the dimer form of rt mutant 271a was detected, although it was markedly reduced as compared to the wild type, while mutant 274a could not form dimer. instead, higher order oligomers of rt were detected in both mutants (fig. 5b ). it has been reported that higher order oligomerization may occur in hiv-1 rt [41, 44, 45] . our results have also indicated that y271a and i274a substitutions may change the conformation of rt, leading to oligomerization. the higher order oligomers formed in these 2 mutants, perhaps tetramer and octamer, may be associated with instability of rt mutants and their susceptibility to viral protease. furthermore, according to the locations of residues 271 and 274 (fig. 6a) , it is highly probable that alanine substitutions at 271 and/or 274 can affect the conformation of p51 thumb region and subsequently its interaction with rnase h domain of p66, resulting in the exposure of the 7-amino-acid p51-rnas h cleavage sequence in the p66 subunit [7, 46] and consequent proteolytic degradation by hiv-1 protease. the relative conservation of rt 271 and 274 residues and the loss of big side chains at these two sites in the mutants as shown in the structural models (fig. 6b & 6c ) also support their important roles. we tried to demonstrate this in vitro, by treating wild type and mutant rts with recombinant protease, but wild type rt, as well as mutant rts, could not be digested in vitro (data not shown). this result is consistent with that reported by abram and praniak [36] . this could be explained by different proteolytic stability of rt in vitro and in virions. taken together, our study showed that alanine substitutions at residue 271 or 274 of hiv-1 rt could cause conformational changes, rendering rt unstable and susceptible to viral protease. thus, it is demonstrated that the ''turn'' (gln 269 -arg 277 ) between two helices may be important in maintaining protein stability and formation of bioactive dimer. mutations in residues 271 and 274 of rt may inactivate the virus, which may enter cells but can not replicate. these findings may prove useful in anti-hiv drug design and vaccine development. considering the emergence of resistance to the current rt inhibitors, this turn, especially amino acid residues 271 and 274, may be an ideal new target for antiviral drug design. the agents targeting this region may be able to affect the stability or dimerization of hiv-1 rt and subsequently inhibit viral replication. moreover, the potential drug resistant mutations in this region, especially at residues 271 and 274, may probably inactivate the virus, which prevents the appearance of drugresistant virus. hiv reverse transcriptase structure-function relationships molecular targets for aids therapy expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in escherichia coli and analysis of mutants structure of unliganded hiv-1 reverse transcriptase at 2.7 a resolution: implications of conformational changes for polymerization and inhibition mechanisms crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded dna at 3.0 a resolution shows bent dna the structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1 crystal structure at 3.5 a resolution of hiv-1 reverse transcriptase complexed with an inhibitor the 'helix clamp' in hiv-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases structure/ function studies of human immunodeficiency virus type 1 reverse transcriptase. alanine scanning mutagenesis of an alpha-helix in the thumb subdomain role of the ''helix clamp'' in hiv-1 reverse transcriptase catalytic cycling as revealed by alanine-scanning mutagenesis reduced frameshift fidelity and processivity of hiv-1 reverse transcriptase mutants containing alanine substitutions in helix h of the thumb subdomain a minor groove binding track in reverse transcriptase effects of mutations in the polymerase domain on the polymerase, rnase h and strand transfer activities of human immunodeficiency virus type 1 reverse transcriptase residues in the alphah and alphai helices of the hiv-1 reverse transcriptase thumb subdomain required for the specificity of rnase h-catalyzed removal of the polypurine tract primer a putative new domain target for anti-hepatitis b virus: residues flanking hepatitis b virus reverse transcriptase residue 306 (rtp306) mutational analysis revealed that conservation of hepatitis b virus reverse transcriptase residue 306 (rtp306) is crucial for encapsidation of pregenomic rna mutational analysis of the ''turn'' of helix clamp motif of hiv-1 reverse transcriptase efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of hiv-1lai, hiv-1mal, and hiv-1eli novel single-celllevel phenotypic assay for residual drug susceptibility and reduced replication capacity of drug-resistant human immunodeficiency virus type 1 indicator cell lines for detection of primary strains of human and simian immunodeficiency viruses metabolism and anti-human immunodeficiency virus-1 activity of 2-halo-29,39-dideoxyadenosine derivatives infection of htlv-iii/lav in htlv-i-carrying cells mt-2 and mt-4 and application in a plaque assay severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release immunogenicity in mice of tandem repeats of an epitope from herpes simplex gd protein when expressed by recombinant adenovirus vectors nonnucleoside reverse transcriptase inhibitors are chemical enhancers of dimerization of the hiv type 1 reverse transcriptase rapid localization of gag/ gagpol complexes to detergent-resistant membrane during the assembly of human immunodeficiency virus type 1 the nonmyristylated pr160gag-pol polyprotein of human immunodeficiency virus type 1 interacts with pr55gag and is incorporated into viruslike particles requirements for incorporation of pr160gag-pol from human immunodeficiency virus type 1 into virus-like particles characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and gag-pol precursor incorporation characterization of ribosomal frameshifting in hiv-1 gag-pol expression hiv expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems incorporation of human immunodeficiency virus type 1 reverse transcriptase into virus-like particles mutations in the primer grip of human immunodeficiency virus type 1 reverse transcriptase impair proviral dna synthesis and virion maturation mutations that abrogate human immunodeficiency virus type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer virion instability of human immunodeficiency virus type 1 reverse transcriptase (rt) mutated in the protease cleavage site between rt p51 and the rt rnase h domain structure/function studies of hiv-1(1) reverse transcriptase: dimerizationdefective mutant l289k generation of infectious molecular clones of simian immunodeficiency virus from fecal consensus sequences of wild chimpanzees the packaging and maturation of the hiv-1 pol proteins conformational stability of dimeric hiv-1 and hiv-2 reverse transcriptases human immunodeficiency virus-1 reverse transcriptase heterodimer stability dimerization of human immunodeficiency virus type 1 reverse transcriptase. a target for chemotherapeutic intervention effects of efavirenz binding on the subunit equilibria of hiv-1 reverse transcriptase a model for reverse transcription by a dimeric enzyme chemical crosslinking of the subunits of hiv-1 reverse transcriptase structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer the following reagents were obtained through the aids research and reference reagent program, division of aids, niaid, nih: plai. conceived and designed the experiments: hjz yxw ymw bjz. performed the experiments: hjz yxw hw. analyzed the data: hjz yxw hw dyj ymw bjz. wrote the paper: hjz dyj ymw bjz. key: cord-002757-upwe0cpj authors: sullivan, kathleen e.; bassiri, hamid; bousfiha, ahmed a.; costa-carvalho, beatriz t.; freeman, alexandra f.; hagin, david; lau, yu l.; lionakis, michail s.; moreira, ileana; pinto, jorge a.; de moraes-pinto, m. isabel; rawat, amit; reda, shereen m.; reyes, saul oswaldo lugo; seppänen, mikko; tang, mimi l. k. title: emerging infections and pertinent infections related to travel for patients with primary immunodeficiencies date: 2017-08-07 journal: j clin immunol doi: 10.1007/s10875-017-0426-2 sha: doc_id: 2757 cord_uid: upwe0cpj in today’s global economy and affordable vacation travel, it is increasingly important that visitors to another country and their physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population thwaites and day (n engl j med 376:548-560, 2017). this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population [1] . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. emerging infectious diseases are a result of a convergence of numerous factors and comprise complex interactions among multiple variables. some of those factors are human movement, land use change, encroachment and wildlife translocation, rapid transport, and climate change. several studies demonstrate that for a pathogen to persist in a population, a minimal host population size that is specific for the type of pathogen and host population. of particular relevance to emerging infections is the pattern of rapid population growth. in the tropics, before wwii, most regional ecosystems consisted of few large cities and scattered human settlements separated by large areas of cropland, pastureland, or undisturbed forest. today, the pattern is the opposite: many large cities have developed with only patches of undisturbed forest or grassland. domestic vectors have therefore expanded their population with increasing urbanization and this markedly impacts the interactions between vectors and pathogens [2] . human activities such as deforestation, use of pesticides, pollution, etc. lead to the loss of predators that naturally regulate rodent and insect populations. this contributes to emerging zoonotic diseases and explains why they occur more frequently in areas recently settled. in today's global economy and affordable vacation travel, it is increasingly important that visitors to another country and their the southern, with a reduction in the number of cold days per year, changes in rainfall (more winter precipitation and summer droughts) [4] , and together these changes increase the risk of several vector-borne diseases in new areas. climate change involves not only global warming but also changes in precipitation, wind, humidity, and the location and frequency of extreme weather events like floods, droughts, or heat waves. changes in climate produce changes in pathogens, vectors, hosts, and their living environment. increases in precipitation can increase mosquitoes for example, but heavy rainfalls may cause flooding that temporarily eliminates larval habitats and decreases mosquitoes. flooding may force rodents to look for new habitats in houses and increase the opportunities of vector-human interactions, as occurs for example in the case of epidemic leptospirosis. humidity is another very important factor of climate change in the development of vector-borne diseases. mosquitoes and ticks do not survive well in dry conditions. therefore, weather impacts infectious pathogen distribution in complex ways that are not predictable by the forecast. extreme weather events can precipitate outbreaks of infection. an increase in the frequency and intensity of natural disasters like hurricanes and tsunamis, in relation to the el niño/southern oscillation phenomenon, may result in more flooded grasslands, which favor the breeding of aedes and culex mosquitoes [5] , and impact water sanitation fostering outbreaks such as cholera. flooded areas can displace rodents leading to plague. tornados and other severe weather can stir up soil leading to infections with soil fungi leading to episodic outbreaks of invasive fungal disease such as mucormycosis such as apophysomyces trapeziformis [6] . malaria is a common disease that can vary dramatically depending on weather, and extreme weather can alter the very landscape, providing new bodies of water to support larval development. if the melting of glaciers and the polar ice caps bring coastal cities underwater, or if overpopulation and waste cause drinkable water shortages in certain regions of the world, we can expect mass migrations. these could change the patterns of infection and drive outbreaks. migrants traversing tropical forests, or feeding with meat from game or carcasses, are but two scenarios that could be envisioned for the emergence of zoonotic infectious diseases [7] . several predictive models have been developed to evaluate the impact of climate change on the emerging infectious diseases: climex, dymex, miasma models. nevertheless, it remains difficult to predict when and where pathogen behavior will deviate from its typical pattern. climate change primarily affects vector-borne diseases by increasing rates of reproduction and biting and by shortening the incubation period of the pathogen they carry. ticks have gained spread from the mediterranean basin to northern and eastern europe, as well as appearing at higher altitudes. increased survival, density, and activity have also been reported following shorter, milder winters. climate change has also resulted in more days of activity per year for mosquitoes. as temperatures rise, more parasites are viable within regions ranging from the mediterranean and tropical zones, up to the balkans, russia, scandinavia, and the uk. for some ticks and fleas, temperatures over 25°c with relative humidity of over 85% are optimal for their proliferation and activity throughout the year [4, 8, 9] . for example, dengue fever is usually limited to a tropical latitude and a low altitude, since mosquito eggs and larvae lose viability with sustained low temperatures. during unusually warm summers, however, dengue has been reported as high up as 1700 m above sea level. warmer temperatures also result in smaller adult mosquitoes, which need to bite more frequently to feed themselves and be able to lay eggs, thus increasing the rate of transmission [8] . in contrast, the incidence of malaria has followed mixed patterns of increase and decrease along recent decades, and computer models have failed to predict the spread. the explanation for this is, in part, that climate change also results in diminished survival of the vectors (warming over 34°c affects the survival of both parasites and vectors), and in part, that the effect of climate change is non-linear and complex [10] . the frequency of emerging vector-borne infections varies per changes in land use, human activity, intervention maneuvers to eradicate the vector or prevent transmission to humans, drug treatment, and vaccines. both ecologic and economic changes may bring together rodents and humans. hunting activities may change vector distribution and large-scale animal movements can impact disease distribution. impoverishment of cities and overcrowding in slums, but also reforestation, golf club development, and the urbanization of rural suburbs facilitate exposure to ticks and rodents [11] . many patients with pidd require immunoglobulin replacement. immunoglobulin products have been demonstrated to improve outcomes in hepatitis a and measles [12, 13] . at least some neutralizing antibody is present directed to rsv and group b streptococci [14, 15] . this raises three distinct issues for patients: (1) difficulty in the diagnosis of infections in travelers because locally produced immunoglobulin may have antibody titers to local infections that are not typical for other countries, (2) safety concerns about locally produced immunoglobulin, if the patient resides in the location long enough to require immunoglobulin from a local provider, (3) the decision to use locally produced immunoglobulin products to provide superior prevention of infection compared to the patient's usual product. there are limited data on each of these subjects. serologic diagnostic testing in patients on immunoglobulin therapy will be addressed below in terms of issues related to lack of specific antibody produced by the patient (potentially) after infection. the converse can also be an issue. patients arriving from countries with significant occurrences of infections unusual in their current country may have igg antibodies to those infections simply through their immunoglobulin product and not reflecting a true infectious event in the patient. this can lead to diagnostic confusion when serologic testing demonstrates the presence of antibodies due to the infusion product. patients will often ask if immunoglobulin products from other countries are held to the same rigorous standards as their home country. today, commercially produced immunoglobulin is safe and tightly regulated. all commercially produced immunoglobulin around the world has one of the time-tested viral inactivation procedures such as nanofiltration, caprylate absorption, pasteurization, solvent/detergent, vapor heating, and low ph treatment. these procedures uniformly inactivate enveloped viruses. many emerging viruses are specifically tested for their ability to withstand the purification process. much has been learned since the transmission of hepatitis c viruses through immunoglobulin products in the early 1990s [16] . nevertheless, vigilance is important. in 2009, counterfeit immunoglobulin was identified. therefore, patients should ensure that they receive only brand name products while traveling. the subject of whether a patient's interests would be best served by using a local immunoglobulin product, with antibodies to pathogens that are prevalent in the community, or have their home physician ship their usual product, for which the patient has a known tolerance is hotly debated. patients with a history of intolerance to immunoglobulin products should not switch unless necessary. however, there are compelling reasons to consider a locally produced product when patients are in a foreign country for an extended period. it is known that antibodies to west nile virus have tracked with the distribution of the virus as it has emerged in several areas [17, 18] . titers in products using donors from the usa have higher neutralizing titers to west nile virus than those using donors from europe, although there is a 400-fold difference in titers between lots from the usa [18] . similarly, protective antibodies to concerning pathogens may be optimal in locally produced immunoglobulin. it is critical to inquire where the plasma source is derived. in most countries, the utilized immunoglobulin is produced in europe or the usa. having a different name does not ensure that the plasma pool comes from a different country. most lots of immunoglobulin are produced from 3000 to 60,000 plasma donors. the infrastructure to support such an endeavor is not easy to establish in each geographic area. serologic testing is commonly used for the diagnosis of infection. this approach relies upon detection or quantitation of antibodies made by the host against specific pathogens. the presence of igm antibodies against a specific pathogen indicates recent infection, while igg antibodies against a specific pathogen indicate past infection. importantly, serologic testing can only be applied for the diagnosis of infection if the host can mount a specific antibody response to the pathogen. conversely, serology cannot be relied upon to diagnose infection in the setting of immune deficiency where there is impairment of the specific antibody response, such as in the case of primary antibody deficiencies, cellular immune deficiencies, combined immune deficiencies, and other secondary immune deficiencies affecting t and/or b cell function. in these situations, the causative pathogen must be identified by alternate means such as culture of the organism, antigen detection, or molecular approaches (nucleic acid hybridization, nucleic acid sequencing, or oligonucleotide probe arrays). molecular approaches are particularly relevant for the diagnosis of infection in patients with pidd. signal and target amplification techniques can be coupled with nucleic acid hybridization or probe assays to allow detection of pathogen dna or rna that is present in very small amounts in clinical samples. in patients with pidd, vaccines could play an important role in preventing infections with vaccine-preventable diseases. even pidd patients may generate some protective responses [19, 20] . the decision to immunize such patients or not depends on the type and severity of pidd as well as the type of vaccine to be administered (live or inactivated) ( table 1 ). in general, inactivated vaccines are safe for pidd patients while immunization with live attenuated vaccines is a known hazard to patients with serious immunodeficiencies of t cell, b cell, and phagocytic cell origin (table 1 ). in less severe pidd, the vaccine can induce adequate protection as in healthy individuals or the efficacy may be reduced [20] [21] [22] . of note, immunoglobulin replacement therapy induces passive immune protection to some vaccine-preventable infections, such as measles, mumps, rubella (mmr), and varicella. in addition, live viral vaccines have greatly reduced efficacy while on immunoglobulin replacement. therefore, vaccine administration in patients receiving regular immunoglobulin replacement treatment should be withheld until at least 3 to 11 months (depending on dose) after cessation of such treatment, if cessation and vaccination are safe. in addition, pidd patients who have received hematopoietic stem cell transplantation but have incomplete immune reconstitution or are under immunosuppression should not receive live attenuated vaccines. in general, the decision of administering live viral vaccines should be made by clinical immunology experts [23] . in developing countries where polio is still endemic and oral polio vaccine is essential for eradicating the disease, it is of utmost importance that all pidd patients and family members should not receive live oral polio (opv) because of the reported prolonged excretion of the virus for months and even years [24] . in addition, vaccine-associated paralytic polio is a real risk for some with pidd. these patients and family members should receive inactivated polio vaccine (ipv) instead of opv. similarly, the hazards of administering bacillus calmette-guerin (bcg) vaccine to pidd patients have been documented. in a series of 349 bcg, vaccinated severe combined immunodeficiency (scid) patients from 28 centers in 17 countries, 34% of scid patients developed disseminated bcg infection and had the worst outcome [25] . patients with chronic granulomatous disease vaccinated with bcg also developed local and disseminated bcg infection. recently, vaccine strains of rubella virus were found to be associated with skin granulomas in pidd patients [26] [27] [28] . siblings and household contacts of patients with suspected or diagnosed pidds should receive all the national immunization scheduled vaccines. ipv should be substituted for op in families where an antibody-deficient patient exists. yearly influenza vaccination of family members is recommended in order to reduce the risk of household-social transmission [20, 22, 29] . diseases where routine vaccination has reduced incidence can occasionally experience a resurgence in times of economic hardship with reduced attention to vaccination. diphtheria is currently seen in venezuela for this reason. war and disruption of health infrastructure are other common reasons for resurgence in vaccine-preventable diseases. in other settings, antivaccination sentiment has led to outbreaks of diphtheria, measles, and mumps. an additional consideration is that not all countries provide the same level of vaccination, and therefore, vaccine-preventable illness can still be seen regionally. these outbreaks represent a significant risk to patients with pidd. a universal consideration for patients with pidd is the concern about antibiotic resistance, which varies dramatically around the world. for certain high impact infections, the emerging antibiotic resistance patterns will be discussed below. antimicrobial resistance occurs naturally, but misuse and overuse of antimicrobials are accelerating this process. in nearly every country, antibiotics are overused and misused in people and animals leading to antibiotic resistance in every country. key resistance patterns to common bacteria include emergence of carbapenem-resistant klebsiella pneumoniae around the world with high frequency of resistance (due to different mechanisms) in the mediterranean, with greece, italy, and turkey having endemic spread of this pathogen [17] . carbapenem-resistant strains among other genera of enterobacteriaceae have also been recognized. they are particularly common in greece, but have been found widely distributed [30] . the new delhi metallo-beta-lactamasemediated resistance, which is endemic in the indian subcontinent but becoming increasingly spread worldwide, is a growing concern [30, 31] . as a common cause of urinary tract infections, colistin is the only recourse when carbapenemresistant enterobacteriaceae, and colistin resistance has recently emerged in small outbreaks throughout the world [32] . in these cases, the infection is essentially untreatable. fluoroquinolone-resistant escherichia coli, a common cause of urinary tract infections, now accounts for over half of the isolates in some asian countries [33, 34] . t he emergence of resistance to antibiotics in grampositive pathogens has become a major international problem as there are fewer, or even sometimes no effective, antimicrobial agents available for infections caused by these bacteria. methicillin-resistant staphylococcus aureus is common in many countries and in fact has spawned a nomenclature recognized by the general public: mrsa. several studies have reported resistance to the newer antimicrobial agents like linezolid, vancomycin, teicoplanin, and daptomycin [35] . thus far, these isolates appear to be uncommon and have been found in <1% of isolates in brazil, china, ireland, and italy, with nearly undetectable rates elsewhere. vancomycinresistant enterococcus (vre) is growing in frequency and can now be a cause of primary bacteremia in immunocompromised individuals [36] . a key message is that antibiotic resistance is increasing (generally) and travelers should be alerted to resistance to commonly encountered organisms, and if antibiotic prophylaxis is required, their prophylaxis is adjusted. neisseria gonorrhoeae is a specific organism for which resistance has become especially problematic. it has progressively developed resistance to virtually all antimicrobial agents since introduction of sulfonamides in mid-1930s. the current treatment guidelines recommend dual antimicrobial therapy (ceftriaxone 250-500 mg × 1 plus azithromycin 1-2 g × 1) as first-line regimen [37, 38] . although dual therapy is very effective, development of concomitant ceftriaxone and azithromycin resistance is likely to emerge [39] . the risk of untreatable n. gonorrhoeae demands better global antimicrobial surveillance system, clinical trials on combined therapy of existing drugs as well as novel agents in monotherapy, and development of a gonococcal vaccine. for pidd patients, guidance on barrier methods for the prevention of sexually transmitted diseases should be a part of any pre-travel counseling. mycobacteria tuberculosis (mtb) is an age-old pathogen with emerging resistance. drug-resistant tb, fueled by the hiv epidemic, is a global threat. in 2015, who estimated 480,000 new cases of multidrug-resistant tb (mdr-tb) and an additional 100,000 new cases of rifampin-resistant tb (rr-tb) who would also require mdr-tb treatment. treatment of mdr-tb and mycobacterium bovis disease is beyond the scope of this text and reader is referred to recent who mdr treatment guidelines [40] . regions of the world with >6% mdr tb include regions of azerbaijan, kazakhstan, russia, uzbekistan, china, georgia, and eastern europe. extensively drug-resistant tb (xdr tb) refers to mtb resistant to isoniazid, rifampin, any fluoroquinolone and at least one second-line drug. xdr tb has been reported in 105 countries. on average, 10% of patients with mdr tb have xdr tb. as is true for all types of mtb, xdr tb is contagious and small outbreaks related to person-person transmission have been reported. non-tuberculous mycobacteria (ntm) cause significant systemic infections in patients with defects of the il-12/ ifnγ/stat1 axis as well as in gata2 deficiency and can cause significant pulmonary infections in pidd patients with bronchiectasis. compared to tb, ntm is acquired from the environment and not from person-to-person transmission; therefore, acquisition of antibiotic-resistant strains is less common. however, in these individuals with pidd, ntm disease is often chronic and can be difficult to eradicate, and resistance can then easily develop during therapy. using combination of antibiotics is essential, and consultation with those familiar with treatment of treatment refractory ntm disease is recommended. aspergillus species are ubiquitous inhaled molds with worldwide distribution that cause opportunistic infections in immunocompromised patients [41] . aspergillosis also occurs in pidds associated with quantitative and/or qualitative phagocyte defects; it develops most frequently in chronic granulomatous disease (cgd) patients (prevalence,~40%), while it is seen less often in patients with gata2 deficiency, card9 deficiency, and congenital neutropenia syndromes [42, 43] . upon inhalation, aspergillus species cause invasive pulmonary disease in susceptible hosts with the exception of card9 deficiency, where aspergillosis has a predilection for extrapulmonary tissues with sparing of the lungs [44] . diagnosis is established by fungal culture and/or histopathology showing acute-angle septate hyphae and/or detection of galactomannan, an aspergillus cell wall component released during invasive infection, in serum or bronchoalveolar lavage fluid [41] . while azole-susceptible aspergillus fumigatus is still the most common infecting species in pidd patients, the emergence of azole-resistant a. fumigatus and nonfumigatus aspergillus species underscores the importance of a high index of suspicion in patients who do not respond to front-line voriconazole treatment [45] . the advent of fungal molecular diagnostics has demonstrated that patients with pidds are more prone to infections by uncommon low-virulence aspergillus species with intrinsic resistance to azole antifungal agents that do not infect patients with iatrogenic immunosuppression. these primarily include aspergillus viridinutans, aspergillus tanneri, and neosartorya udagawae, which pose major diagnostic and therapeutic challenges due to their impaired sporulation and propensity for contiguous and distant tissue spread, respectively. in addition, acquired azole resistance in a. fumigatus can be seen in patients on long-term exposure to azole drugs used as treatment and/or prophylaxis [46] . azole resistance in these strains is predominantly caused by point mutations in the lanosterol 14α-demethylase gene that encodes the cyp51a protein, the primary target of azole drugs. importantly, infection by azole-resistant a. fumigatus strains without prior exposure of patients to azole antifungals has recently emerged as an important global health concern due to the widespread use of sterol demethylase inhibitor fungicides in agriculture that results in cross-resistance with the triazole antifungals used in clinical practice [47] [48] [49] . fungicide-driven azole-resistant environmental aspergillus strains were first observed in the netherlands in 2007 and have since then been documented in other parts of europe, south and north america, the middle east, australia, africa, and asia. the prevalence of these azole-resistant aspergillus strains among clinical aspergillus strains recovered from patients in 19 european countries was reported to be 3.2%, while alarmingly in some areas >20% of recovered strains exhibited azole resistance [50] . the emergence of such aspergillus environmental strains poses serious threats to the treatment of immunosuppressed patients. mortality rates as high as 88% have been seen due to delays in diagnosis and suboptimal treatment with azole antifungals [51] . although no prospective data exist for the treatment of patients with such resistant fungi, the use of amphotericin b-and echinocandin-based regimens are preferred over azoles [52] . in the absence of azoles, the lack of alternative oral antifungal agents is particularly challenging for pidd patients such as those with cgd who require long-term suppressive antifungal treatment. candida species are commensal yeast fungi that colonize the mucosal surfaces of~60% of healthy individuals [53] . when perturbations in immunity and/or microbiota occur, candida causes opportunistic mucosal or systemic infections that depend on clearly segregated immune responses for host defense. specifically, t cells of the th17 program are critical for mucosal and phagocytes for systemic immunity [54] . indeed, a proportion of patients with cgd and complete myeloperoxidase deficiency develop systemic, but not mucosal candidiasis [42] , whereas patients with monogenic syndromes of chronic mucocutaneous candidiasis due to mutations in the il-17 signaling pathway (il17ra, il17rc, il17f, act1) or in other genes that adversely affect th17 differentiation (rorc, stat3, stat1, aire, dock8, stk4, irf8) do not develop systemic candidiasis. the only known pidd to date that results in combined mucosal and systemic candida infection susceptibility is card9 deficiency. systemic candidiasis in card9-deficient patients has a predilection for the central nervous system, associated with brain-specific impaired recruitment and effector function of neutrophils [55] [56] [57] . diagnosis of candida infections is established by culture. azole-susceptible candida albicans is still the most common infecting species in pidd patients; however, emergence of azole-resistant c. albicans is not uncommon during chronic azole use, making long-term therapy challenging due to lack of alternative oral antifungal treatment options [58] . beyond c. albicans, non-albicans candida species can rarely infect pidd patients, some of which have intrinsic resistance to azole antifungals, including candida glabrata and candida krusei [59] . most recently, candida auris has emerged as a global public health concern due to its resistance to antifungal drugs, high virulence potential, propensity for health careassociated horizontal transmission and outbreaks in health care settings, persistence in the human skin and hospital environment, inherent resilience to antiseptics, and misidentification by routine biochemical methods as other yeasts (most often candida haemulonii, but also candida famata, rhodotorula glutinis, or saccharomyces cerevisiae). c. auris was first recovered from the ear canal of a patient in japan in 2009 and has since then been reported to cause life-threatening infections and hospital outbreaks in europe, asia, africa, the middle east, and south and north america [60] [61] [62] [63] . most of the reported strains of c. auris have intrinsic resistance to fluconazole and other triazole antifungal agents, while a significant proportion of strains has elevated minimum inhibitory concentrations to amphotericin b and echinocandins, with some strains reportedly resistant to all three classes of azoles, polyenes, and echinocandins [64] . avoidance of azole antifungals is important in c. auris-infected patients, and echinocandinor amphotericin b-based regimens are preferred, guided by strain-specific in vitro susceptibility patterns. this section on vector-borne infections is a major focus of this review because the infections are often more severe in immunocompromised individuals and because there are mitigation strategies that should be considered even in the absence of defined medical treatments for infection. prevention of mosquito bites depends somewhat on the endemic species but there are generalizations. the use of a mosquito repellant such as deet, oil of lemon eucalyptus, ir3535, or picaridin is as important as using long sleeves and long pants while in an affected area. deet and picaridin are safe in pregnancy, and some data support their greater efficacy [65] . air conditioning and fans tend to keep mosquitoes away but netting at night is essential in mosquito-prone areas. light-colored clothing is less attractive to mosquitoes than dark clothing, and scented detergents and use of dryer sheets tend to attract mosquitoes, hence should be avoided. aedes species prefer to bite indoors and thrive in urban areas with small puddles of water. they bite most frequently around dawn and dusk. anopheles species have very similar behaviors. culex mosquitoes, in contrast, bite primarily at night. tick and fly bite prevention is focused on physical and chemical prevention. for ticks, physical inspection for biting ticks should also be incorporated. arthropod-borne viruses (arboviruses) are transmitted to humans through the bites of infected insects: mosquitoes, ticks, sand flies, or midges. some arboviruses can be transmitted through blood transfusion, organ transplantation, perinatal transmission, consumption of unpasteurized dairy products, or breastfeeding. there are >100 arboviruses causing human disease. most arboviral infections are asymptomatic. infectious manifestations range from mild febrile illness to severe encephalitis. arboviral infections are often categorized into two primary groups: neuroinvasive disease and non-neuroinvasive disease. tables 2 and 3 list the encephalitigenic viruses and the febrile/hemorrhagic disease causing viruses. in this section, we will highlight west nile virus, the most common of the encephalitogenic arboviruses. west nile virus is a single-stranded mosquito-borne rna virus of the family flaviviridae. the natural transmission cycle of the virus occurs in culex mosquitoes and birds. humans and horses are dead-end hosts for the virus. the most common mode of transmission to humans is through the bite of infected mosquitoes. other less common modes of transmission include blood transfusions, organ transplantations, occupational exposure in laboratories and mother-to-child transmission during pregnancy, childbirth, and breastfeeding. west nile virus has been diagnosed in >2000 people in the usa with slightly more than half having neuroinvasive disease. since 1999, >40,000 people in the usa have been infected. it is also common in africa, europe, and asia [66] . infection with west nile virus is asymptomatic in most individuals [67, 68] . the incubation period lasts for 2 to 6 days. however, it can be significantly longer in immunocompromised hosts. clinical manifestations following infection develop in 20-40% of those infected and include fever, headache, myalgia, arthralgia, vomiting, and rash. severe neuroinvasive disease leading to meningitis, encephalitis, and flaccid paralysis develops in less than 1% of infected individuals. the overall case fatality is approximately 10% which is a disproportionately high mortality in patients with encephalitis and myelitis. diagnosis of west nile virus rests on demonstration of specific antibody responses especially specific igm antibodies in the serum or csf of infected individuals by enzyme immunoassays. plaque reduction neutralization tests can differentiate cross-reactive antibodies. detection of virus in csf, blood, or tissue specimens by culture or pcr is particularly useful in immunosuppressed individuals who may have impaired serological responses. west nile virus has been reported in the context of both primary and secondary immunodeficiency. severe neurological manifestations have been reported in hiv-positive individuals, individuals receiving immunosuppressive therapy including rituximab, and individuals with pidd. infection with wnv has been reported in individuals with common variable immunodeficiency, idiopathic cd4 lymphopenia, gata2 deficiency, and a case of probable good's syndrome [69] [70] [71] . individuals with antibody defects, neutropenias, and impaired t cell responses are potentially at an increased risk of severe manifestations of wnv disease including severe neurological involvement. this section highlights the four important non-neuroinvasive arboviruses based on current geographical distribution: dengue, yellow fever, zika, and chikungunya (table 3) . approximately 100 countries/territories have reported local transmission for both chikungunya and dengue viruses [72] . dengue is due to infection with one of four dengue virus serotypes, transmitted by a mosquito (typically aedes aegypti). this febrile illness affects all ages with symptoms appearing 3-14 days after the infective bite. symptoms range from mild to high fever, with severe headache, musculoskeletal pain, and rash. severe dengue (also known as dengue hemorrhagic fever) occurs in 0.5-5% of cases and is characterized by fever, abdominal pain, persistent vomiting, bleeding, and breathing difficulty and is a potentially lethal complication [73] . paradoxically, the main risk factor for dengue hemorrhagic fever is pre-existing antibodies. early clinical diagnosis and comprehensive management by experienced clinicians increase survival. dengue is ubiquitous throughout the tropics with the highest infection rates in the americas and asia. dengue is now endemic in 100 countries, causing up to 50 million infections a year and 22,000 deaths, mainly among children. over half of the world's population inhabits areas at risk for dengue infection [74] . the presence of a. aegypti in over 125 countries potentially puts almost the whole world at risk of becoming infected with this virus. pcr is widely used as serologic methods to diagnose dengue. immunity to one serotype does not confer protection against the other three serotypes, and heterologous antibody may be a risk factor for hemorrhagic dengue [73] . the natural history of dengue has been studied in hiv patients where hiv did not appear to increase severity. there have been no reports of patients with pidd having dengue; however, dengue infection after solid transplantation has been reported [75] [76] [77] [78] with some patients having severe complications suggesting that t cell compromise in pidd could be a risk for severe disease. there are no antiviral medications utilized for dengue virus. care of patients with hemorrhagic disease requires meticulous approach to fluids and coagulation status. one dengue vaccine has been registered in several countries (cyd-tdv) for individuals from 9 to 45 (or 60) years old. it is a live attenuated recombinant tetravalent vaccine with backbone of the attenuated yellow fever 17d virus genome with the prm and e genes that encode the proteins from the four wild-type dengue viruses. the who has suggested its use in regions where seroprevalence of dengue virus of any serotype is 70% or greater, but has not recommended it to hiv-infected, immunocompromised individuals, nor pregnant or lactating women [79] . most people infected with the yellow fever virus have no illness. symptoms of yellow fever include sudden onset of fever, chills, headache, musculoskeletal pain, nausea, vomiting, fatigue, and weakness. the incubation period is typically 3-6 days, and symptoms may appear after return from travel. most people improve after the initial presentation, but 15% of cases progress to develop a more severe form of the disease, usually after a day of presumed recovery. the severe form is characterized by high fever, jaundice, bleeding, and eventually shock and failure of multiple organs [80] . yellow fever virus is an rna virus that belongs to the genus flavivirus. it is transmitted from mosquitoes after biting an infected primate. it is widely distributed in the equatorial tropics [80] . aedes species of mosquitoes are primarily responsible for transmission. large epidemics of yellow fever occur when the infection enters heavily populated areas with a high mosquito density and where most people have little or no immunity. west africa has undergone a large-scale vaccination campaign with impressive results and yellow fever is now uncommon in west africa [81] . serologic testing for yellow fever is the diagnostic standard. pcr can be performed on tissue samples. there are no published studies of yellow fever in immunocompromised people, but the elderly, very young, people with autoimmune disease, or who are post-thymectomy are at risk from the attenuated vaccine strain. thus, it seems likely that any immunodeficiency would be associated with more severe wildtype disease. currently, no specific antiviral drug for yellow fever exists. treatment of dehydration, liver and kidney failure, and fever improves outcomes. the yellow fever vaccine is highly effective; however, immunodeficient patients should not receive it. infection with zika virus is often asymptomatic. it represents a mild infection for those who have any symptoms [82] . the zika virus has been detected in urine, semen, and saliva of infected individuals, and transmission from transfusion and sexual relations has been reported. it is also detectable in breast milk, but breastfeeding-associated transmission has not been reported so far [83] [84] [85] . contact with highly infectious body fluids from patients with severe zika infection has also been suggested as a possible mode of transmission [86] . of tremendous importance is the presence of prolonged shedding of zika virus in a congenitally infected newborn [87] . the main public health risk of zika virus is microcephaly in newborns from infected mothers [88] . zika virus is capable of infecting human neural progenitor cells in vitro. infection results in disruption of cell cycle, increased cell death, and attenuated neuron growth [89] . zika is not thought to be a major risk for people with pidd (based on the experience with hiv patients, but our recognition of zika is very recent. there is no known specific treatment for zika; however, there is an important effort to develop a vaccine. chikungunya fever is an acute febrile illness caused by the alphavirus, chikungunya virus. the incubation period is usually 3-7 days after the bite of an infected aedes mosquito. there is abrupt onset of high fever, and the fevers can be biphasic [90, 91] . severe polyarthraligias develop after the onset of fever. the joint pains can affect any joint, but the pattern is usually symmetric and a true acute arthritis is not uncommon. the proportion of infected people with rash has varied across studies. when seen, the rash appears after the fever as a truncal maculopapular type of rash [92] . cervical adenopathy is another common feature of infection. death is uncommon in chikungunya, but serious complications such as myocarditis have been seen. over half of the patients report continued joint symptoms 1 year after acute illness and 12% have long-term joint symptoms [93] . chikungunya originated in central/east africa. in forests, the virus circulates in aedes mosquitoes and non-human primates. in urban centers, the virus circulates between humans and mosquitoes similar to the pattern of dengue. there have been periodic urban outbreaks in asia and africa since 1960 with an acceleration in spread since 2004 [94] . an important consideration is the periodic outbreaks with high attack rates in naïve populations. areas at risk currently are east africa, central africa, la reunion, india, and southeast asia. diagnostic testing utilizes pcr or serology. the threat to immunodeficient patients is not entirely clear. there are a few provocative cases where the immunocompromised appears to have been associated with fewer joint symptoms, but there were two patients, medically immune compromised, who had very severe disease [95, 96] . this suggests that the presentation may be atypical and the course may be severe in immunodeficient people. treatment is supportive, although chloroquine, acyclovir, ribavirin, interferon-ɑ, and steroids have limited preclinical data to support clinical trials. babesia microti (the main species in the usa) infection can be asymptomatic, but many people develop fever, chills, headache, myalgias, anorexia, nausea, or fatigue [97] . babesiosis often causes hemolytic anemia. b. microti is spread by ixodes scapularis ticks in the usa and babesia divergens (the main species in europe) is spread by ixodes ricinus. symptoms begin 1-3 weeks after a bite from an infected tick with b. divergens having a higher mortality rate and greater symptomatology compared to b. microti. the main geographic areas involved are the coastal eastern usa and cattle breeding areas throughout europe. the diagnosis is usually by inspection of a blood smear or through serology. a pcr test has just been developed. immunodeficiency, asplenia, and older age are recognized risk factors for severe disease and even death [98] [99] [100] . thus, congenital asplenia would be considered a major risk for severe disease. a combination of atovaquone and azithromycin is generally used for therapy, although clindamycin and quinine have been used with success. patients with severe illness have been treated with exchange transfusions. five different types of plasmodium (plasmodium falciparum, plasmodium vivax, plasmodium ovale, plasmodium malariae, and plasmodium knowelsi) infect humans. malaria is transmitted primarily by female anopheles mosquitoes. symptoms vary depending on the type of plasmodium involved but usually include high fever, chills, and headache. in some cases, the illness can progress to severe anemia, kidney and respiratory failure, and death. the incubation period typically ranges from 9 to 14 days for p. falciparum, 12 to 18 days for p. vivax and p. ovale, and 18 to 40 days for p. malariae. in p. vivax and p. ovale infections, relapses can occur months or even years without symptoms. p. vivax and p. ovale have dormant liver stage parasites that must be specifically eradicated through medical therapy. malaria has been a global health concern throughout history and is a leading cause of death and disease across many tropical and subtropical countries. over the last 15 years, new control measures have reduced malaria by over half [101] . the democratic republic of the congo and nigeria account for over 40% of the estimated total of malaria deaths globally. high rates of malaria are seen in india as well. nevertheless, malaria exists in most tropical regions of the americas, africa, and asia [101] . the diagnosis of malaria depends on the demonstration of parasites in the blood, usually by microscopy. the threat to immunodeficient patients is not entirely clear, but patients with hiv seem to have no additional burden of disease other than an increase in placental malaria, suggesting that t cells are not central to the defense of malaria [102, 103] . asplenia is a known risk factor for severe malaria [104] . antibodies appear to be both protective and pathologic [105, 106] . treatment and prophylaxis depend on the region of the world because the parasites and resistance are highly variable and highly dynamic. therefore, it is best to consult an infectious disease specialist familiar with the prophylaxis before travel and for treatment of acute cases. leishmaniasis is due to infection with an obligate macrophage intracellular protozoa of the genus leishmania. it causes a spectrum of disease ranging from a cutaneous ulcer to mucosal disease and the most severe form, visceral leishmaniasis (vl). the liver, spleen, and bone marrow are major sites of parasite growth and disease pathology in vl [107] . purely cutaneous leishmaniasis is most often caused by leishmania major, leishmania. tropica, leishmania aethiopica, leishmania infantum, and parasites belonging to the leishmania mexicana complex, the leishmania braziliensis complex, and the leishmania guyanensis complex. mucocutaneous disease is most often due to l. braziliensis complex, leishmania panamensis, leishmania amazonensis, and rarely by leishmania guyanensis. vl is most often caused by leishmania donovani and leishmania infantum (previously l. chagasi) [108] . cutaneous leishmaniasis can have many variations but is most often an ulcer that develops after an indolent papule. the incubation period ranges from weeks to months. the ulcer usually heals within months to years, and there can be mild adenopathy. mucocutaneous leishmaniasis follows a cutaneous ulcer and is only caused by l. braziliensis parasites. oral and respiratory mucosa are most often involved with granulomatous lesions that may be extremely destructive. vl is associated with fever, lymphadenopathy, hepatosplenomegaly, wasting, hypoalbuminemia, and pancytopenia. this picture evolves over months to years. there can be secondary immune deficiency due to the pancytopenia. the epidemiology has changed dramatically and has been impacted by climate change [109] . the sand flies that spread the parasite are affected by temperature and rainfall. in most endemic regions, leishmania has a patchy distribution due to micro-ecologic factors. poverty has been demonstrated to be a major risk factor for leishmaniasis [110] . it has been estimated that up to half a million new cases of vl occur every year, but the majority are in resource-poor countries such as bangladesh, nepal, india, sudan, ethiopia, and brazil. emergence of resistance to antimony-based drugs has also led to a major resurgence of disease. the primary reservoir for leishmania is forest rodents, but dogs are increasingly important. the growing spread of leishmania is due to a combination of factors, and now 88 countries have reported cases. immunodeficient patients are more susceptible to infection, and relapse occurs more frequently [111] . the risk of developing vl is estimated to be between 100 and 2300 times higher in hiv-infected than in non-hiv-infected individuals [112] , and these patients have higher rates of treatment failure with the illness often taking a prolonged chronic course and higher mortality rates [113] . a similar picture has been seen in patients with vl-infected post-kidney transplantation [114] . dendritic cells, t cells, and the generation of reactive oxygen species have been shown to be essential for parasite control [115] [116] [117] . pidd with impaired il-12 production have been associated with severe disease [118] . a patient with cd40l deficiency, associated with poor il-12 production, had chronic leishmania and died in spite of aggressive treatment. vl has been reported in cgd patients [119] . six cgd patients were infected by leishmania, and they developed hemophagocytic syndrome with a poor outcome for one of them [120] . the diagnosis of leishmaniasis is usually by visual inspection for parasites. immunofluorescence microscopy, direct agglutination, skin test, and pcr have been used. treatment is long-term and difficult. emerging resistance to first-line treatment is increasingly problematic. pentavalent antimonials are the mainstay of treatment in most countries, but liposomal amphotericin is widely used where resistance occurs. newer drugs with more favorable side effect profiles have been used in certain geographic settings: miltefosine, paromycin, and sitamaquine. rickettsiae are small gram-negative bacteria. they are obligate intracellular parasites, and the primary target in humans appears to be endothelial cells with subsequent thrombosis and clinical presentation of vasculitis [121] . the rickettsiaceae family, originally defined by non-specific phenotypic characteristics, was reclassified into different strains and subspecies based on gene sequencing and genetic phylogeny ( table 4 ). the clinical presentation of rickettsial disease can vary, but the classic triad of fever, rash, and headache still provides major clues for the diagnosis [122] [123] [124] . however, rash is not an obligatory sign, and the incidence of rash can range between 100% for rickettsia conorii infection,~90% for rickettsia rickettsii, 30% for rickettsia africae, and less than 10% in the case of anaplasma phagocytophilum infection. therefore, fever in patients with exposure to a potential vector should raise a concern for a rickettsial disease, especially if there is also evidence of rash, inoculation eschar, or localized lymphadenopathy. additional supporting laboratory findings can include neutropenia, thrombocytopenia, and increase in liver transaminases. the geographic distributions of rickettsioses and ehrlichioses are mostly dependent on their vector distribution [125] . as such, louse-borne and flea-borne are worldwide, reflecting the worldwide distribution of lice and fleas, with a tendency to parasite poor people in cold places and, characteristically, during wars. ticks, on the other hand, depend on their environment and most do not have a worldwide distribution. with the exception of the dog tick, vector for r. conorii in asia and north africa, r. rickettsii in the usa, rickettsia massiiae and erhlichia canis worldwide, most other tick-borne diseases are restricted to areas of the world correlating with the distribution of their vector [126] . for that reason, it should be anticipated that climate and environmental changes will affect vector distribution and its reservoir host and, hence, the geography and epidemiology of tick-borne diseases [10, 127, 128] . diagnosis presents a challenge, as it is extremely difficult to grow these organisms in culture. immunohistochemistry and pcr can be helpful. the severity of rickettsial disease varies with the causative agent and the host. r. rickettsii, rickettsia prowazekii, and orienta tsutsugamushi are considered most pathogenic. as for host factors, although severe and fatal cases have been described in healthy immunocompetent hosts [129, 130] , there is evidence to suggest that children under the age of 10 [130] and immunocompromised hosts either secondary to hematologic malignancies, immunosuppressant treatment for organ transplantation, or hiv infection are at a greater risk to develop more severe disease with higher case fatality rates [131, 132] . all rickettsiaceae are intracellular pathogens, and one could expect an increased risk for severe disease in pidds with abnormal t cell function. five to 7 days of doxycycline is the preferred treatment for non-pregnant adults and children. treatment should not be delayed while awaiting diagnostic testing [133] and can be given to children despite a minimal risk for dental staining. alternative treatments include azithromycin for mild disease [134] and chloramphenicol for pregnant women. anaplasma is an intracellular bacterium that infects wild and domestic mammals, including man. a. phagocytophilum was formerly known as human granulocytotropic ehrlighiosis but is now known as human granulocytotropic anaplasmosis [135] . e. chaffeensis infects monocytes and causes human monocytic ehrlichiosis [136] . anaplasma and ehrlichia have historically cycled within non-human enzootic hosts, and man has become infected through increasing interactions with the environment. ehrlichia and anaplasma are transmitted by ixodes species of ticks, and their ranges include the eastern usa, south central usa, and scattered regions of europe, as far north as sweden. these infections have not been seen in humans in the southern hemisphere, but there are reports of organisms being identified [137] . a less common mode of transmission is through transfusions. the symptoms of ehrlichia and anaplasma infections are similar [136] . abrupt onset of an influenza-like illness occurs about 12 days after a tick bite. ehrlichia can cause a mild rash (30% of adults and 60% of children), but rash is uncommon in anaplasma infections. highly suggestive laboratory features are leukopenia and thrombocytopenia. mortality in the general populations appears to be <5%, but icu admission is not uncommon. hemophagocytosis has been described with anaplasma [138] and ehrlichia [139] . both infections are more severe in any setting of immune compromised, including asplenia [129, 135] . the diagnosis is typically made by pcr, and doxycycline is the recommended treatment. intracellular inclusions can be seen on cbc smears (more often in anaplasma than ehrlichia). an uncommon but well described facet of these infections is that the tick vector can also transmit borrelia burgdorferi and babesia microti, and simultaneous infection with multiple organisms can occur. c. burnetii is a highly pleomorphic gram-negative coccobacillus and the causative agent of q fever. q fever is a zoonosis, and the most common reservoirs are cattle, sheep, and goats but many other animals can be infected by c. burnetii [140, 141] . when infected, these domestic animals can shed the organism in urine, feces, milk, and especially birth products. the pathogen survives within the phagolysosome of host cells, and a spore stage has been described. this spore stage explains the ability of c. burnetii to survive in unfavorable environmental conditions, and it can be an environmental risk for months to years after shedding from an infected animal. q fever is considered an occupational disease affecting people with direct contact with infected animals; however, indirect contact through exposure to contaminated animal products has also been described to cause disease outbreaks. humans are infected by inhalation of contaminated aerosols. following an average incubation period of 20 days, infected patients can present with severe headache, fever, chills, fatigue, and myalgia. other signs and symptoms depend on the organs involved. in contrast to rickettsial diseases described above, rash rarely occurs in the early stages of the disease. c. burnetii can cause a range of clinical symptoms. a self-limited febrile illness is probably the most common form of q fever. pneumonia, either atypical or severe, is also common and can be a part of acute q fever syndrome. in contrast, a variety of manifestations can be recognized in chronic q fever, including endocarditis, endovascular infection, osteomyelitis, hepatitis, interstitial pulmonary fibrosis, prolonged fever, and purpuric vasculitic rash. q fever diagnosis is based on serologic testing with indirect immunofluorescence being the best for differentiating between acute and chronic q fever (high antiphase i antigen titer). the treatment of choice for acute q fever is doxycycline, with co-trimoxazole, chloramphenicol, or rifampin being an accepted alternative. there is no agreement on the treatment for q fever endocarditis, and a combination of doxycycline with either fluoroquinolone or hydroxychloroquine is recommended. there is also controversy regarding the duration of treatment, ranging from 2 years to indefinite treatment. old evidence suggests that q fever is more common in immunocompromised patients. a french study showed higher incidence of antibodies to c. burnetii in hiv positive compared to hiv-negative patients (10.4 vs 4.1%). in addition, 5 out of 68 hospitalized patients were hiv positive (7.3%), suggesting a more frequent symptomatic disease [142] . a smaller similar study performed in central africa failed to show increased incidence of seropositivity in hivpositive patients [143] . two case reports describe severe disease in immunocompromised patients. the first was a case of fatal q fever disease in an 11-year-old male with cgd [144] . the patient was treated with broad spectrum antibiotics, but without coverage for q fever. the second case was a 53-yearold asplenic male who presented with fever, jaundice, and encephalopathy and was diagnosed with acute q fever [145] . the patient was successfully treated, but the two case reports could suggest susceptibility in cases of phagocytic disorders. the bartonellaceae are fastidious, facultative intracellular gram-negative bacilli (table 5 ). most species infect primarily non-human animals, and in most cases, human are considered incidental hosts. the documented common human pathogens include bartonella bacilliformis, bartonella henselae, and bartonella quintana, and it is believed that humans are the primary mammalian reservoir of b. quintana. infection occurs through inoculation of bartonella-infected arthropod feces into breaks in the skin. the epidemiology of bartonella infection in humans follows the distribution of the vector. as such, infection with b. bacilliformis follows the distribution of the sand fly vector (lutzomya) and is confined to the andes mountain in peru, ecuador, and colombia at heights of between 500 and 3200 m. the human body louse pediculus humanus is the vector of b. quintana, which explains the global distribution of this pathogen and worldwide outbreaks of trench fever, mostly in conditions of poor sanitation and upon exposure to body lice. trench fever was responsible for over a million infections during world war i. fever, either abrupt or indolent in onset, with a maculopapular rash, conjunctivitis, headache, myalgias (most often affecting legs), and splenomegaly was described. urban b. quintana infections occur most often among homeless and have a distinct clinical picture with fever as the most common manifestation. endocarditis occurs in many [146] . bartonella henselae is globally endemic, and domestic cats are a major reservoir. the major arthropod vector of b. henselae is the cat flea, which is responsible for cat-to-cat transmission. human infection, called cat scratch disease, is assumed to involve inoculation of bartonella-infected flea feces into the skin during a cat scratch. b. henselae causes primarily adenopathy and neurologic symptoms [147] . b. bacilliformis causes a condition with two phases: the acute phase with fever, anemia, and transient immunosuppression followed by nodular dermal eruption [148] . recently appreciated are the ongoing systemic features during the eruptive phase such as arthralgias, adenopathy, and anorexia [149] . diagnosis of bartonella-associated diseases can be achieved by direct examination of clinical material, bacteriologic culture methods, serologic and immunocytochemical studies, pcr-based assay, or combination of these methods. bartonella infection can present differently in immunocompromised hosts [150] . in addition to higher prevalence of bartonella infection in hiv patients [151] , both b. quintana and b. henselae can induce neovascular proliferation which might involve the skin, lymph nodes, and a variety of internal organs including the liver, spleen, bone, brain, lung, bowels, etc. these neovascular lesions, known as bacillary angiomatosis/peliosis (ba/bp), were initially described in hiv-infected patients with advanced disease and was later described in other immunocompromised hosts secondary to immunosuppressant treatment for solid organ transplantation or hematologic malignancy [152] [153] [154] [155] . cutaneous ba lesions can vary in presentation and can be subcutaneous, dermal nodules, single or multiple papule that can be flesh colored, red, or purple. lesions may ulcerate and bleed. they can change in number and size (millimeters to centimeters; few to hundreds) and can involve mucosal surfaces or deeper soft tissues. similar variation can be seen with visceral involvement. histologically, lesions consist of lobular proliferation of small blood vessels and neutrophilic predominant cell infiltration. the term bacilliary peliosis is used to describe bloodfilled cystic spaces mostly involving the liver, spleen, and lymph node. pathogenic bacteria can be isolated from vascular lesions. while both pathogens were associated with cutaneous lesions, only b. henselae was associated with visceral bp [156] . based on hiv literature, it is reasonable to expect an unusual presentation of bartonella infection especially in pidds involving t cell dysfunction. bartonella infection was described as a cause for hepatitis in a single cd40l deficiency patient [157] . in addition, since cases of granulomatous disease due to bartonella infection [158] [159] [160] have been described, it should be considered in the differential diagnosis of pidd with granulomatous inflammation. borrelia spp. the genus borrelia belongs to the spirochaetaceae family. it includes b. burgdorferi which causes lyme disease and species that cause relapsing fever. the latter are further divided into tick-borne species and louse-borne species. louse-borne relapsing fever (lbrf) is caused only by borrelia recurrentis and is spread by human body louse. the disease was epidemic in the early twentieth century, and it is estimated that more than 50,000 died of lbrf during world war ii. with sanitation improvement lbrf is now found only in the horn of africa and among homeless people in europe. more recently, cases of lbrf in refugees and migrants were described [161, 162] . tick-borne relapsing fever (tbrf) is caused by a group of pathogens which are maintained by and survive in softticks (orinthodoros genus). each tbrf borrelia species depends on one specific species of soft-body tick. except for australia and antarctica, tbrf species can be found in all continents. the animal reservoir includes small animals and rodents. since the spirochetes persist in the tick salivary gland, disease transmission occurs when humans intrude the tick's environment. tick bites are painless, and history of a tick bite is often missing. therefore, a history of potential exposure can be valuable. the major clinical symptom is relapsing fever. after a median incubation period of 7 days, patients present with febrile episode that can last 2-7 days, followed by afebrile period of 4-14 days. patients with tbrf can have up to 30 febrile relapses, while lbrf is usually associated with only one relapse. other symptoms include myalgia, arthralgia headaches, and vomiting, and physical findings include lymphadenopathy and splenomegaly with rash occurring only in third of the patients. a range of neurologic complications as well as systemic inflammatory response syndrome also have been described [163] . diagnosis is based on identifying the spirochetes on blood smear. sensitivity of blood smear is higher during febrile period (about 70%), and a negative blood smear does not exclude rf. in lbrf, the spirochete load can be low and specific stains can be helpful. other diagnostic methods include serologic testing, pcr, and mouse inoculation. doxycycline, tetracycline, and penicillin are the preferred treatment, with most patients treated with 7-10 days of doxycycline. jarisch-herxheimer reactions with high fever and leukopenia occur in half of the patients following the first antibiotic dose and can develop into a severe reaction with hypotension, respiratory distress, and death [163] . without treatment, tbrf carries a mortality rate of up to 10% with even higher 40% mortality rate for untreated lbrf. two cases of meningoencephalitis with borrelia miyamotoi in heavily treated immunocompromised patients have been described [164, 165] . lyme borreliosis is the most common vector-borne disease in the northern hemisphere caused by a group of at least 13 genospecies. lyme disease is a multisystem illness affecting the skin, joints, nervous system, and heart. human infection is caused mainly by three species: b. burgdorferi is the most common cause in north america but also found in europe, and borrelia afzelii and borrelia garinii which cause the disease in europe and asia. emerging infections in the mid-western usa with borrelia mayonii cause a condition similar to lyme disease. most tick species do not carry borrelia species. the vectors of all borrelia species are the ixodid tick species; this includes the deer tick, i. scapularis, in the northeast and midwest of the usa, ixodes pacificus in the west, the sheep tick, ixodes ricinus, in europe and the taiga tick, ixodes persulcatus, in asia. the ixodid tick demonstrates a complex vector ecology with preferences for different hosts in different geographical regions and at different stages of its development. more than 300 different species, including deer, rodents, birds, and reptiles, have been described. infection rates also show seasonal variation with highest rates during lyme disease follows several stages starting with localized disease at the site of inoculation, followed by dissemination stage and, later, persistent infection [166] . however, an individual patient can show highly variable disease progression with different patterns of organ involvement and disease severity. erythema migrans (em) is often seen at the site of the tick bite after 3-30 days of incubation. regional lymphadenopathy can be seen. secondary skin lesions represent hematogenous dissemination. at this stage, constitutional symptoms of general fatigue, fever and headaches, migratory musculoskeletal pain, conjunctivitis, and cardiac involvement occur. in total, 15% of untreated patients can develop frank neurologic manifestations of meningitis, encephalitis, and variable forms of neuritis with fluctuating symptoms. persistence can occur in untreated (on inadequately) patients. antibiotic refractory arthritis is well described. however, even without treatment, intermittent or persistent attacks usually resolve completely within several years. co-infection with a. phagocytophilum and b. microti can cause diagnostic confusion [167, 168] . the diagnosis of lyme disease is established based on clinical symptoms, history of potential exposure, and serologic studies. although positive culture can confirm the diagnosis, it can usually be obtained only from early em lesions. pcr testing is superior to cultures and can be performed on joint fluid samples [169] . cdc recommendations for the diagnosis of lyme disease are based on serology which might be impossible in pidd patients with abnormal humoral response. cdc guidelines require both an elisa (or comparable test) to be positive and a western blot (2 out of 3 bands (23, 39, or 41 kd) on the igm or 5 out of 10 bands on the igg (18, 23, 28, 30, 39, 41, 45, 58, 66, 93 kd) . most lyme manifestations can be treated with oral antibiotics, while patients with neurologic abnormalities and some patient with lyme arthritis require intravenous therapy [170] . doxycycline is the treatment of choice for early and disseminated disease, with amoxicillin as the second-line choice. jarisch-herxheimer-like reactions can appear in the first 24 h in about 15% of the patients. for patients with clear neurologic symptoms, 2-4 weeks of iv ceftriaxone is the most commonly used therapy. few cases of neuroborreliosis and hiv have been described with a good response to treatment. descriptions of lyme disease in pidd patients are lacking. zoonoses are infectious diseases that pass between animals and humans and span the spectrum of pathogens including viruses, bacteria, fungi, and parasites. zoonoses are very common and range from mild such as certain forms of tinea to lifethreatening infections such as rabies. some of the zoonoses that are vector-borne will be covered in other sections. risk mitigation strategies for zoonoses include patient education, proactive advice about risk scenarios, and avoidance of infected animals. several zoonoses are associated with contact with mammals such as rodents or domestic farm animals through direct contact or through contact with their feces. for instance, hantavirus infections are often associated with exposures to mouse droppings when staying in cabins in the western usa. occupational exposures can occur with buffalopox or parapoxvirus (causing orf infection) through direct contact with buffalo and goats/sheep, respectively [171] [172] [173] [174] . in general, there are very few cases of pidd with zoonotic infections acquired from mammals. however, there are a few special considerations. for instance, lymphocytic choriomeningitis virus is acquired through exposure to house mice primarily, with hamsters being a less common source of infection. both domestic and wild mice can carry the infection without exhibiting symptoms. although infection is rare, there have been severe cases in patients with t/ nk cell dysfunction, such as a case in xlp1 and cases in solid organ transplant recipients [175] . therefore, in patients with severe t/nk defects, consideration should be given to whether small rodents are appropriate household pets. tularemia is a disease of animals and humans caused by the bacterium francisella tularensis. rabbits, hares, and rodents are the main reservoirs. humans become infected through direct contact, ingestion of contaminated water, or inhalation of organisms. ticks and deer flies can also transmit the disease through bites. fever is universal, but other features depend on the mode of transmission. a patient with cgd had a complex course suggesting myeloid defects are a risk for more severe disease [176] . rabies is an almost universally fatal infection caused by contact with oral secretions from infected mammals, typically raccoons, bats, or foxes, and there is no suggestion that pidd or immune compromised modifies the prognosis. for individuals with high-risk exposures, such as those working with wildlife or traveling in endemic areas, pre-exposure prophylaxis is given with vaccination, and if an exposure occurs, rabiesspecific immunoglobulin is provided as well as vaccination. however, for those with humoral immunodeficiencies who cannot respond to the typical pre-exposure vaccination, there needs to be counsel on the additional risk without vaccination. in table 6 , several of the bacterial and viral zoonoses are summarized with their typical endemic areas, which is somewhat limited by diagnostic abilities and reporting, as well as the typical clinical scenarios, known cases in immunodeficiency and an approach to diagnosis and therapy. nipah virus causes a range of infectious phenotypes ranging from asymptomatic infection to acute respiratory distress and encephalitis. nipah virus was identified in 1999 on pig farms in malaysia, leading to identification of 257 human cases, including 105 human deaths and the culling of one million pigs [177] . the natural host is the fruit bat: pteropodidae pteropus. symptoms of infection from the malaysian outbreak were primarily encephalitic in humans, but later, outbreaks have caused respiratory illness, increasing the likelihood of human-to-human transmission. fever, headache, cough, abdominal pain, nausea, vomiting, weakness, problems with swallowing, and blurred vision were common. seizures were seen in 25% and coma in 60%. relapses of encephalitis have been described [178] . increasing infections due to nipah virus is thought to be due to an increasing overlap between bat habitats and pig sties in malaysia. all outbreaks thus far have been in india, bangladesh, or malaysia. the diagnosis of nipah virus relies on pcr of fluid samples, serology in convalescent samples, and immunofluorescence of tissue. there have been no infections of immune compromised patients reported. therapy is largely supportive, although preliminary reports of ribavirin use have been encouraging. a vaccine is under development. severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) are two zoonotic coronaviruses. the sars pandemic in 2002-2003 resulted in 8096 reported cases in 27 countries. no further sars cases were reported after the pandemic except isolated cases linked to laboratory accidents. patients usually presented with fever and respiratory symptoms, but occasionally had diarrhea and vomiting. about 20-30% of sars patients required mechanical ventilation, with a case fatality rate of about 9% [179] [180] [181] . mers was first noted in saudi arabia in 2012, and countries around the arabian peninsula are now endemic for mers-cov. patients usually present with fever, cough, chills, sore throat, myalgia, and arthralgia rapidly progressing to pneumonia with over 50% of patients requiring intensive care. about one-third of patients present with diarrhea and vomiting, and acute renal impairment is a striking feature of mers. risk factors for poor outcome include diabetes, hypertension, and renal and lung disease. cases have been exported to at least 26 countries with travel occasionally causing cluster of secondary outbreaks. one such example is the mers-cov outbreak involving 82 patients in south korea, and the median incubation period was estimated to be 7 days with a range of 2 to 17 days [182] . at the end of 2015, there were 1621 confirmed mers with a 36% mortality rate [179] [180] [181] . bats are the natural reservoirs of both sars-cov and mers-cov. sars-cov crossed the species barrier into palm civets and other animals in live animal markets in china, which then infected human, while a mers-cov ancestral virus crossed species barrier into dromedary camels. abundant circulation of mers-cov in camels results in continuous zoonotic transmission of this virus to human, while sars-cov was not found to circulate in the intermediate reservoirs, explaining sars being a one-off outbreak and mers a continuing zoonotic disease [179] . aerosolgenerating procedures such as intubation were associated with increased viral transmission of both covs resulting in nosocomial outbreaks [179] . super-spreaders are responsible for large and prolonged outbreaks [181] . the diagnosis for sars and mers include both serological tests and pcr assays that can quantify viral loads [183] . functional genetic polymorphisms leading to low serum mannose binding lectin (mbl) are associated with susceptibility to but not severity of sars in both southern and northern chinese [184] [185] [186] . mbl was shown to bind to sars-cov and inhibit the infectivity [184] , suggesting its role as first-line defense against sars-cov. although no patients with primary immunodeficiency infected with sars-cov or mers-cov were identified, likely due to the limited number of such infections, patients with t cell defect and type 1 interferon pathway defects could suffer a more severe disease course [187, 188] . virus-based and host-based treatment strategies are largely experimental with uncertain benefits. ribavirin, type 1 interferons, small molecules, and monoclonal antibodies that block covs entry have been explored [183] . passive immunotherapy and multiple candidate vaccines have been tested in various animal models. convalescent plasma immunotherapy has been considered, but clinical trials are lacking in mers [189] , while for sars a systematic review concluded convalescent serum may reduce mortality and appear safe [190] . the filoviridae family contains three known genera, the ebolaviruses, marburgviruses, and cuevavirus. ebolavirus and marburgvirus cause hemorrhagic fever syndromes in primates and humans, with high fatality rates. cuevavirus infects only bats. the ebolavirus genus contains five species, with two of the species (zaire ebolavirus and sudan ebolavirus) being responsible for the majority of cases of human disease, while marburgviruses contain two species (marburg virus and ravn virus). filoviruses are capable of replicating in a number of cell types (with the exception of neurons and lymphocytes). upon entry into the body of the host (via breaks in the skin, parenterally, or through mucosal surfaces), filoviruses employ a variety of mechanisms to evade the activity of the immune system [191] . the incubation period (interval from infection to onset of symptoms) varies from 2 to 21 days. symptoms begin abruptly, with high fever, severe headache, malaise, myalgia, diarrhea, nausea, and vomiting. a rash can occur between 2 and 7 days after onset of symptoms. hemorrhagic manifestations occur between 5 and 7 days, and fatal cases usually have some form of active bleeding. in an outbreak setting, the symptoms are unmistakable but confusion with malaria can occur early in the disease or in sporadic cases. since their original descriptions in 1967 and 1976, respectively, for marburg and ebola, there have been a number of sporadic cases and several major outbreaks. the largest marburg virus outbreak occurred in angola in 2005 (with a fatality rate of >80%), while the largest ebola epidemic happened between 2014 and 2016 in west africa (sierra leone, guinea, and liberia) and claiming over 11,000 lives (fatality rate > 40%). although not definitively proven in the case of ebola, bats are believed to be the natural animal reservoir for these viruses [192, 193] . these viruses are transmitted via contact with blood or body fluids from an infected host; notably, certain body fluids can harbor virus for weeks to months after resolution of disease. given the recent outbreak in west africa, there has been renewed interest in understanding the pathogenesis of filovirus infections and possible therapies. literature regarding how the pathogenesis of disease may be altered in patients with pidd is lacking. however, the assumption is that in the absence of an intact cellular and/or humoral immune response, the patient with a pidd may be at increased risk of mortality in the setting where mortality is already high. these viruses induce apoptosis of lymphocytes and macrophages, and there is therefore a profound secondary immune compromised [194, 195] . filoviruses can be detected in multiple body fluids via pcr. although practiced for decades, a study in guinea in 2015 failed to show a decrease in mortality among patients receiving convalescent plasma from previously infected donors [196] . a number of additional compounds (e.g., tkm-ebola, bcx4430, and gs-5734) and biologics (zmapp) have been shown to offer protection in animal models of ebola, but to date, no controlled and appropriately powered clinical trials have addressed their efficacy in humans. finally, a number of vaccines for ebola are undergoing clinical studies (including four in phase iii trials). importantly, in late 2016, the rvsv-zebov vaccine was shown to have displayed high efficacy in protecting immunized adults during the 2015 guinea ebola outbreak, and the data also suggested that the vaccine may even offer bherd immunity^to unimmunized persons in proximity to recipients of the vaccine [197, 198] . hepatitis e virus is a single-strand rna virus of the hepeviridae family. it is an important zoonotic disease in asia and africa, and fecal-oral spread is the usual route of transmission [199] . handling of pig or boar meat is a risk factor, and 2-10% of pig livers sold in grocery stores in japan and the usa are infected [200, 201] . swine represent the major reservoir, although antibodies to the virus have been found in many species [199] . the incubation period is 2 weeks to 2 months, and viremia disappears with the onset of symptoms. the mortality rate is 1-4% and can reach 20% in pregnancy [202] . acute hepatitis usually resolves but can lead to liver failure in severe cases. patients with hepatitis e posttransplant have had severe courses in some cases [203] . in immune compromised patients, the course can become chronic [204] [205] [206] . in these cases, cirrhosis develops. the diagnosis is by serology or pcr, and the treatment is supportive. prevention modalities for infections transmitted by humans are conceptually different than infection prevention for vector-borne infections. hand hygiene is extremely important, and avoidance of clearly infected people can be helpful. recognition of infections with fecal-oral transmission and the importance of water purity are critical for patients with pidd. in contrast, infections transmitted by aerosols require prevention strategies related to droplet precautions. in outbreak scenarios, if the risk to the patient is high, specific chemoprophylaxis may be considered. influenza viruses type a and b cause annual epidemic influenza, while type c causes sporadic mild influenza-like illness. patients present with sudden onset of fever, chills, and myalgia, followed by sore throat and cough. other less common features include diarrhea, acute myositis, and encephalopathy [207, 208] . co-infection with bacteria such as pneumococci results in more severe disease [209, 210] . influenza pandemics occur yearly around the world. influenza viruses infect 5 to 15% of the global population, resulting in~500,000 deaths annually [211] . the viruses circulate in asia continuously and seed the temperate zones, beginning with oceania, north america, and europe, then later seeding into south america [212] . diagnosis of influenza includes direct/ indirect immunofluorescent antibody staining for antigens in nasopharyngeal aspirates and pcr. a patient with compound heterozygous null mutations of the gene encoding irf7, a transcription factor for amplifying ifn-α/β, was reported to have life-threatening influenza during primary infection [213] . fatal influenza-associated encephalopathy in both chinese and japanese children has been reported to be associated with genetic variants of thermolabile carnitine palmitoyltransferase ii [214] . patients with scid will have prolonged viral shedding [215] . severe pandemic influenza a virus (h1n1) infection has been associated with igg2 and igg3 subclass deficiency [216, 217] . in addition, influenza infection can be more severe in pidd patients with underlying lung disease, such as bronchiectasis, and antibiotic coverage of chronic colonizing bacteria (such as pseudomonas) in this setting may be helpful. inactivated seasonal influenza vaccine should be given to pidd patients even those with humoral deficiencies as their t cell response to influenza could be normal and offer protective immunity against severe influenza [218, 219] . antiviral drugs include neuraminidase inhibitors (oseltamivir and zanamir) and adamantanes (amantadine and rimantadine), but resistance to adamantanes is widespread. measles is a single-stranded, negative-sense, enveloped (nonsegmented) rna virus of the genus morbillivirus. measles is highly communicable, transmitted by droplets, and less commonly by airborne spread. patients present with fever, cough, coryza conjunctivitis rash, and koplik spots. complications include pneumonia, acute encephalitis, and subacute sclerosing panencephalitis (sspe) [220] . diagnosis of measles includes serological tests, virus isolation, and pcr. in an outbreak, the clinical features may be sufficient for diagnosis. measles vaccine has caused severe measles in children with stat2 and ifn-α/β receptor deficiency [187, 188] , demonstrating the importance of type 1 interferon pathway in controlling measles. immune compromised of nearly any type is associated with severe disease and higher mortality [221] . t cell deficiency states are the most strongly associated with the development of giant cell pneumonia and inclusion body encephalitis, the most feared complications of measles. sspe is a slow encephalitis due to persistence of replication defective measles virus in the cns. it is most frequently seen when young infants are infected with measles and 6-10 years later, sspe becomes evident. there are case reports supporting the immune compromised as increasing the risk of sspe [222] . treatment of sspe with ribavirin has shown some improvement, but the prognosis in general with sspe is very grave. patients with cgd have defective memory b cell compartment, resulting in lower measle-specific antibody levels and antibody-secreting cell numbers, but severe disease has not been reported [223] . pidd patients may harbor the virus latently for longer than usual, leading to complications at the time of transplant [224] . specific antiviral therapy is lacking, but ribavirin has been given to severely ill and immunocompromised children. for measles post-exposure prophylaxis, intravenous immunoglobulin (ivig) is recommended for severely immunocompromised patients without evidence of measles immunity [225] . this would likely include patients with scid and hypogammaglobulinemia who are not yet on regular ivig. measles vaccine, given in a two-dose regimen, has brought down incidence enormously worldwide and the who is planning for eradication globally. enteroviruses (evs) are among the most common viruses infecting humans worldwide. evs are small non-enveloped, single-stranded rna viruses of the picornaviridae family. human evs are categorized into seven species that include hundreds of serotypes, such as polioviruses (pv), coxsackie viruses a, and b (cv-a and b), echoviruses, and human rhinoviruses (hrvs). of these species, many important serotypes are known to infect human such as pv1-3, cv-a16, cv-b3, ev-a71, ev-d68, and hrv (table 7) . non-polio enteroviruses (npevs) have a worldwide distribution. infants and young children have higher incidence of infection and a more severe course of illness than adults. the mode of transmission is mainly through fecal-oral and respiratory routes. infection occurs all around the year in tropical and subtropical regions, while in temperate climates the peak incidence of infection is during summer and fall months [226] . npevs are associated with diverse clinical manifestations ranging from mild febrile illness to severe, potentially fatal conditions. most cases are asymptomatic or have mild symptoms including fever with or without rash; symptoms of hand, foot, and mouth disease; herpangina; acute hemorrhagic conjunctivitis; upper respiratory infection; and gastroenteritis. more severe symptoms occur in infants and young children [227, 228] . acute flaccid paralysis [229] , neonatal enteroviral sepsis [230] , myocarditis/pericarditis [231, 232] , hepatitis, pancreatitis, pneumonia, and atypical hemolytic uremic syndrome [233] are severe manifestations seen in immunocompetent people. chronic infections have been seen in immunocompromised patients [234] . each virus may produce one or more of the aforementioned manifestations; however, some serotypes are often associated with particular features ( table 7) . the definitive diagnosis of npev infection relies on pcr or virus isolation from the cerebrospinal fluid, blood, stools, urine, or throat swab [229, 235] . treatment of npevs is mainly supportive since most infections are self-limited. high doses of intravenous immunoglobulin (ivig) are recommended in patients with severe symptoms. the efficacy of some new antiviral drugs (pleconaril, vapendavir, and pocapavir) is still under investigation [236] . no vaccine has been licensed yet for npevs. however, phase 3 clinical trials of inactivated monovalent ev-a71 vaccines manufactured in china showed high efficacy against ev-a71 in infants and young children [237] . patients with a variety of pidds are unusually susceptible to ev [238] . the most susceptible groups are patients with primary antibody deficiency such as xla, cvid, and hyper-igm syndrome (higms) as well as those having scid and major histocompatibility class ii deficiency [239, 240] . the most severe form of infection has been described in patients with xla due to the profound deficiency of immunoglobulins essential for viral neutralization during infection. affected patients usually present with indolent but relentlessly progressive non-necrotizing meningoencephalitis. regression of cognitive skills, flaccid quadriplegia, and deafness has been described. the reported non-neurologic presentations in xla include septicemia, dermatomyositis-like disease, hepatitis, and/or arthritis [238, 241] . the incidence of npev meningoencephalitis in large registries of xla cases is 1-4% [242] . unpublished data from the kuwait national pidd registry, which includes 271 pidd patients, showed that nine patients had documented npev infections and two died from these infections. the two deaths were seen in scid patients (personal communication with prof. waleed al-herz, md). in addition, npev meningoencephalitis and/or septicemia were reported in few cases with either primary b cell deficiency such as b cell linker (blink) protein deficiency [243] or acquired b cell deficiency following the administration of anti-cd20 (rituximab) [244, 245] . in all reports, better outcome was attributed to the early administration of high doses of ivig during npev viremia [246] . npev infection in pidd diseases remains a major threat to patients. also, the possible prolonged viral excretion and the emergence of resistant strains runs the risk of spreading infection to the surrounding community. oral polio vaccine (opv) consists of a mixture of three live attenuated poliovirus serotypes. opv induces production of neutralizing antibodies against all three serotypes, in addition to a local intestinal immune response. opv can result in vaccine-associated paralysis (vap) secondary to reversion of the vaccine strain to the neurovirulent wild-type strain. an example for such an event was demonstrated by the 2000-2001 outbreak in the dominican republic and haiti [247] , believed to be driven at least in part by undervaccination of the population, which allowed the spread of the reverted vaccine strain [248] . although rare, patients with pidd have a higher risk to develop vap. reports have shown that pidd patients with antibody deficiency can have prolonged viral replication which can persist for years and therefore theoretically increase the risk for a spontaneous reversion within the immunodeficient host [249] [250] [251] [252] . cases of vap were shown in patients with antibody deficiency and combined immunodeficiency syndromes [248, 253, 254] . therefore, opv is contraindicated in patients with pidd, and this contraindication extends to their household contacts [22] . beyond the obvious risk for the pidd patient, prolonged virus shedding also increase the risk for spreading vaccine-derived paralytic strain in the general population. bacterial infections have molded human behavior and altered societies over human history. today, largely ignored due to antibiotic susceptibility, they continue to cause misery and disease around the world. three infections are highlighted, and additional commonly encountered infections are listed in table 8 . pertussis is a respiratory infection caused by bordetella pertussis that begins after a 7-to 10-day incubation period as a minor upper respiratory infection that progresses with cough. initially intermittent, it evolves into paroxysmal coughing spells usually followed by vomiting in infants and young children. it lasts 6 to 10 weeks and can have many complications such as syncope, weight loss, rib fracture, and pneumonia. infants under 6 months are more severely affected, developing pneumonia, pulmonary hypertension, hypoxia, subdural bleeding, and seizures. death can occur, especially in young infants [255, 256] . adults typically have a prolonged cough with fewer complications [257] . it is transmitted via aerosolized droplets during close contact. people are most contagious during the catarrhal stage and the first half of the paroxysmal phase, totaling 5 to 6 weeks [258] . the introduction of whole-cell pertussis vaccine (dpt) in the 1940s in the usa reduced the incidence of the disease from 250,000 cases to around 1000 cases per year in the 1970s. a resurgence in 2012 was associated with the substitution of the whole-cell vaccine by the acellular pertussis vaccine (dtap) [258] . new strategies such as boosters with acellular pertussis for adolescents and adults with tdap and use of tdap during pregnancy seem to be effective in partially reducing the incidence of the disease [259] ; however, pertussis cases in the usa remain higher than the 1970s. the lack of persistence of antibody in the adult population means adults not only represent a reservoir for the disease but also do not provide sufficient titers to immunoglobulin products prepared from adult plasma pools. a relatively recent requirement in some countries is vaccination of adults every 10 years to maintain immunity. this should, over time, improve titers in immunoglobulin products. culture of specimens obtained by nasopharyngeal swabs is the gold standard of laboratory diagnosis due to the 100% specificity, but polymerase chain reaction (pcr) is gaining prominence due to its higher sensitivity and speed of results; serodiagnosis can be used in the late stages of the disease [259] . filamentous hemagglutinin (fha) and pertussis toxin (pt) antibodies were detected at peak measurements in pidd patients on regular ivig, although some of them had pt antibodies below the protective level as trough measurements [260] . severe pertussis cases have not been reported in pidd patients, but severe disease has been seen in malignancies [261] . antimicrobials such as azithromycin, erythromycin, and clarithromycin, if given during the catarrhal stage, may ameliorate the disease and shorten the contagious period. to avoid cases of pertussis, it is also worth emphasizing the importance of good vaccine coverage rate among the whole population, but especially among healthcare workers and family members of patients with pidd. neisseria meningitidis the onset of neisserial meningitis is associated with sore throat, headache, drowsiness, fever, irritability, and neck stiffness [262, 263] . purpuric lesions are very characteristic. this pathogen can also present with sepsis which has a 20% mortality rate as opposed to 11% mortality with a meningitic presentation. this bacterium can also cause a chronic condition referred to as chronic meningococcemia. this condition is characterized by intermittent fevers lasting 1 week to 3-4 months [264] . a non-purpuric rash is common which may evolve into purpura. arthritis, similar to that seen with gonococcus, is common. meningococcal disease primarily affects children under 5 years of age. n. meningitidis is a global pathogen [265] . there are 12 serogroups, but the majority of invasive meningococcal infections are caused by organisms from the a, b, c, x, y, or w serogroups. the annual number of invasive disease cases worldwide is estimated to be at least 1.2 million, with 135,000 deaths related to invasive meningococcal disease. serogroups b and c are responsible for most infections in europe. serogroup a has historically been the major organism in africa; mass vaccination has led to some improvement, but the emergence of group x disease is worrisome. the hajj in the middle east has seen epidemics of w-135, and vaccination is now required for hajj travelers. b and c serogroups are the most common through the americas. n. meningitidis cases occur at a rate of about 1 case per 100,000 people throughout the world [266] , but across the sahel of africa and in china, epidemics can lead to case rates of 500 per 100,000 [267] . the bacterium is a natural human commensal, with carriage rates of about 10%. diagnosis can be by clinical examination in epidemics or by gram stain and culture. complement-deficient individuals have an increased risk of neisserial disease, but not necessarily increased mortality. hiv is associated with increased disease, suggesting that t cells are also important for defense. thirdgeneration cephalosporins are typically used for treatment. penicillin, ampicillin, aztreonam, and chloramphenicol are alternatives. there is great inter-individual variability in the development of tb disease. roughly, 5% of infected individuals develop clinical disease within 2 years of infection (mostly during childhood). about 90% became latently infected without clinical disease, and the remaining 5 to 10% develop pulmonary tb later in life, either from reactivation of latent infection or reinfection. molecular epidemiology studies from high burden areas suggest more disease results from recent transmission than from reactivation of latent tb, particularly in people living with hiv [268] . acquired or inherited host factors may at least partially account for the variable disease course, resulting in increased susceptibility to mycobacteria infections [269] . pidd associated with tb and ntm infections include t cell deficiencies, gata2 deficiency, cgd, anhidrotic ectodermal dysplasia with immunodeficiency, x-linked (xl) recessive cd40 ligand deficiency, autosomal recessive (ar) stat1 deficiency, ar irf8 deficiency, and ar tyk2 deficiency. in addition, a group of disorders with a strong susceptibility to ntm, named mendelian susceptibility to mycobacterial diseases (msmd), have been recognized since the 1990s. these are rare inborn errors of ifn-γ signaling pathway that present with isolated predisposition to infections caused by weakly virulent mycobacteria such as bcg vaccine and environmental ntm, in otherwise healthy patients. the genetic defects involve impairment in the production of interferons (ar il12rβ1, ar il12p40, autosomal dominant (ad) irf8, ar isg15, xl recessive nemo) or response to interferons (ifn-γr, ad stat1, ad irf8, cgd) [270] . an acquired form exits: adults with ntm infection in thailand and taiwan were found to have high-titer anti-interferongamma antibody [271] . these individuals from southeast asia were found to have hla-drb1*1502/16:02 and dqb1*05:01/05:02. patients suspected of having pulmonary tb should have acid-fast bacilli (afb) smear microscopy and culture performed in three sputum samples. pcr for mtb can be performed [272] . the use of rapid tests facilitates early diagnosis, and the who has recently recommended their use. the only recommended rapid test for detection of tb with and without rifampicin resistance is the xpert mtb/rif assay (cepheid, sunnyvale, ca). the who recommends the xpert test for those suspected of having drug-resistant tb or in hiv; however, culture is still the mainstay and is not replaced by the xpert test. tb skin testing (mantoux testing) uses a purified protein derivative injected under the skin. its advantages are that it can be used for large-scale screening and it is cost effective. skin testing does have several disadvantages when used as a diagnostic test. reading the induration requires training and immunodeficiencies can alter the magnitude of the induration. immunosuppressed patients (hiv, organ transplant) are considered positive if the induration is ≥5 mm. some immunodeficiencies may completely ablate the response. other causes of false-negative tests are malnutrition, concurrent infection, recent live viral vaccine administration, renal failure, malignancy, medical stress, very elderly, young infants, or with a very recent infection with mtb. conversely, the results may be falsely positive if bcg has been administered. interferon gamma release assays can be used in any setting where skin testing would be done but are considered superior in settings where the patient has had bcg vaccination and, in some cases, where skin testing has been sown to have high false-negative rates. in general, tb treatment for patients with impaired immune response, including pidd, hiv infection, and immunosuppressive therapy, is based on the standard 6-month regimen consisting of a 2-month intensive phase of isoniazid, rifampin, pyrazinamide, and ethambutol, followed by 4 months of isoniazid and rifampin. decisions regarding treatment duration can be individualized, taking into account disease severity, organs involved, and response to treatment. significant pharmacological interaction can occur between rifampin-based mtb regimens and immunosuppressive drugs, such as calcineurin inhibitors or rapamycin, requiring strict monitoring of drug plasma concentrations [273] . therapy for ntm is complex with highly variable drug resistance patterns and a need for biological augmentation to effectively clear the organism. aspergillus fumigatus (see above), cryptococcus gattii, histoplasma capsulatum, coccidioides immitis (or c. posadasii), blastomyces dermatitidis, paracoccidioides brasiliensis, emmonsia pasteuriana, and penicillium (talaromyces) marneffei are environmental fungi that are endemic in certain parts of the world (table 9 ). with the exception of penicillium marneffei and emmonsia pasteuriana that only cause disease in profoundly immune compromised individuals, these fungi can cause infection in healthy individuals, ranging from mild, self-limited pulmonary disease to infection that requires antifungal therapy for eradication. on the other hand, patients with acquired defects in cell-mediated immunity such as those infected with hiv, and patients with specific monogenic disorders, particularly those involving the il-12/ ifn-γ/stat signaling pathways, scid, and gata2 depends on underlying state of immunosuppression and magnitude of environmental exposure icl idiopathic cd4 lymphocytopenia, aids acquired immune deficiency syndrome, stat1 signal transducer and activator of transcription 1, gata2 gata binding protein 2, scid severe combined immunodeficiency, cvid common variable immune deficiency, pidd primary immunodeficiency, il12rb1, interleukin-12 receptor subunit beta 1, ifngr1 gamma interferon receptor 1 deficiency, are at high risk of developing life-threatening disseminated infections by these endemic fungi following environmental exposure [42, [274] [275] [276] [277] [278] . diagnosis relies on culture of the corresponding fungus, histopathological demonstration of the fungus-specific characteristic morphologies, and/or surrogate serological and fungal antigen tests. treatment of clinical disease (as opposed to colonization) typically involves an initial induction phase with amphotericin b, followed by long-term azole maintenance therapy and secondary prophylaxis, and prognosis varies significantly depending on the fungal pathogen and underlying pidd. melioidosis is caused by b. pseudomallei, a gram-negative bacteria found in soil and water, in tropical climates of southeast asia and northern australia [279, 280] . melioidosis is an emerging, potentially fatal disease (20% mortality). b. pseudomallei can be transmitted by inhalation, ingestion, or direct contact (through open skin) with contaminated soil or water. animals (sheep, goats, swine, horses, cats, dogs, and cattle) are also susceptible to infection and cases of zoonotic transmission through direct contact of skin lesions with infected animal meat or milk have been described [280] . b. pseudomallei infections are endemic in northern australia and southeast asia. approximately 75% of reported infections occur during the rainy seasons. cases have also been reported in south pacific, africa, india, and the middle east. in temperate areas, infection is extremely rare and is predominantly imported by travellers or immigrants [281] . the incubation period of melioidosis is variable from 1 day to years, although common symptoms develop between 2 and 4 weeks after exposure. melioidosis presents most frequently in adults 40-60 years of age, but can occur in all ages, with one study reporting 5% of cases occurred in children [282] . in australia, the average annual incidence in 2001-2002 was reported as 5.8 cases per 100,000 people [279] . the incidence in indigenous australians was higher at 25.5 cases per 100,000. a case-cluster in an australian community was associated with post-cyclonic flooding. a recent review suggests that b. pseudomallei is increasingly prevalent in the americas, with a mortality rate of 39% [283] . infection in healthy individuals is uncommon, and more than 70% of cases occur in the setting of underlying conditions such as chronic renal disease, diabetes, chronic lung disease, and alcoholism. a recent review of melioidosis in travelers found that 46% of cases were acquired in thailand. symptoms usually started at 23 days (range 1-360 days) after leaving the endemic area. traveller infections were less often associated with predisposing risk factors (37.5%), diabetes mellitus being the most common (21%). melioidosis in travelers had lower mortality (17%) than infection in autochthonous cases in southeast asia [284] . pneumonia (~50-55%) is the most common presentation in adults. there is usually high fever, headache, anorexia, and myalgia. b. pseudomallei infection may also present as localized skin infection, septicemia, or disseminated infection. localized infection results in an ulcer, nodule, or skin abscess. this usually occurs from the bacterium breaching through a break in the skin. patients with renal disease and diabetes are more susceptible to sepsis. in disseminated infection, abscesses may develop in the liver, spleen, lung, and prostate. in children, primary cutaneous melioidosis is the commonest presentation (60%). bacteremia is less common in children than in adults, but brainstem encephalitis has been reported [282] . difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes [285] . diagnosis of melioidosis is primarily by isolation of the organism. identification of b. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. although various serological tests have been developed, they are generally unstandardized bin house^assays with low sensitivities and specificities. pcr assays have been applied to clinical and environmental specimens but are not widely available and sensitivity remains to be evaluated. cases of melioidosis have been reported in patients with acquired or inherited immune deficiency. melioidosis was the presenting complaint in several patients with cgd. bacteremic melioidosis was recently reported in two patients with prolonged neutropenia, who succumbed despite appropriate antibiotics [286] . it is likely that there is increased susceptibility in situations where innate or adaptive immunity is compromised. treatment is with intravenous antimicrobial therapy for 10-14 days, followed by 3-6 months of oral antimicrobial therapy. intravenous therapy with ceftazidime or meropenem is usually effective. oral therapy may continue with trimethoprim-sulfamethoxazole or doxycycline. free-living amoebas (fla) are protozoa found worldwide that do not require hosts to survive. fla do not employ vectors for transmission and are not well adapted to parasitism in humans. however, there are four genera/species that can cause human disease: naegleria (n. fowleri), acanthamoeba (multiple species), balamuthia (b. mandrillaris), and sappinia (s. pedata). all of these amoebae are capable of inducing cns disease in humans, but acanthamoeba species also cause various extra-cns infections, especially in immunocompromised hosts. the fla that are pathogenic in humans are reviewed below. naegleria are a diverse group of fla flagellate protozoans with a large number of distinct species. only one species, n. fowleri, has been shown to cause infection in humans. n. fowleri has a multi-stage life cycle with amoeboid and trophozoite-infective forms as well as a cyst form [287] . n. fowleri is found commonly in warm freshwater around the world including lakes, rivers, and hot springs. humans become can become infected when swimming or diving in contaminated water. in rare circumstances, infections have also been attributed to exposure from contaminated tap water sources when utilized for religious cleansing of the nose or irrigation of the sinuses. thus, tap water should not be used for nasal and sinus irrigation. it is not possible to become infected from drinking contaminated water or from contact with an infected person, and the amoeba is not found in salt water. after entry to the nasal cavity, the amoeba travels through the cribiform plate to the olfactory bulbs and migrates to the cerebellum, resulting in primary amoebic meningoencephalitis (pam), a rapidly fatal brain infection characterized by the destruction of brain tissue. in its initial presentation, pam can mimic bacterial meningitis, further delaying accurate diagnosis and initiation of therapies that may save the patient. overall, n. fowleri infections are rare. worldwide, most cases are reported in the usa, australia, and europe; however, in developing countries, it is suspected that a large number of cases go unreported. between 2006 and 2015, there were only 37 infections reported in the usa with 33 of the cases attributed to contaminated recreational water, 3 infections following nasal irrigation with contaminated tap water, and 1 case where a person was infected following use of a backyard slipn-slide utilizing contaminated tap water [288] . the fatality rate associated with n. fowleri infection is over 95%, and between 1962 and 2015, only 3 of the 138 infected persons in the usa have survived infection. initial symptoms of pam start 1 to 9 days after infection and can include headache, fever, nausea, or vomiting [288] . progressive symptoms can include stiff neck, confusion, lack of attention, loss of balance, seizures, and hallucinations. cardiac arrhythmias have also been observed. the infection progresses rapidly after initial onset and causes death within 1 to 12 days after exposure (mean of 9.9 days). since infection often progresses rapidly to death, there is often insufficient time to mount a robust immune response. however, both the innate (neutrophils, macrophages, and complement system) and the adaptive (both t and b cells) arms of the immune system have been shown to participate in the immune response to n. fowleri [289] . patients with pam have csf with elevated pressure that is often cloudy or hemorrhagic, with neutrophil-predominant pleiocytosis, elevated protein levels, and very low glucose. wet mounts from centrifuged csf will show motile mono-nucleated trophozoites measuring~10-25 μm in size. additionally, trophozoites can be identified with giemsa and wright stains of csf smears combined with an enflagellation test [289] . confirmation can be achieved via a variety of timeconsuming methods including an immunofluorescence assay [290] , culture of csf [291] , or pcr-based methods [292] . the optimal therapy for n. fowleri pam is still debated. the use of intravenous amphotericin b and fluconazole followed by oral administration of rifampin resulted in survival of a 10year-old child with pam [293] . another child was shown to survive following intravenous and intrathecal amphotericin b and miconazole as well as oral rifampin [294] . most recently, an adolescent girl was successfully treated with the combination of azithromycin, rifampin, fluconazole, and miltefosine [295] . prevention is critical for this highly fatal infection and warning pidd patients not to use tap water for nasal irrigation is important. since its original description in 1986, over 200 cases of b. mandrillaris infections have been described worldwidewith most cases occurring in south america and the usa. balamuthia are found in soil, and acquisition of disease has been associated with agricultural activities, dirt-biking, gardening, and swimming in contaminated water sources. b. mandrillaris is thought to enter the body of the host through breaks in the skin and or via inhalation. the organism is believed to access the cns through hematogenous spread, resulting in a chronic, insidious, but often fatal granulomatous amoebic encephalitis (gae), which has been documented in both immunocompetent and immunocompromised hosts [291, 296] . the incubation period from exposure to development of clinical symptoms is not well established and experts believe that this may occur between 2 months and 2 years. finally, an alternative mode of transmission via solid organ transplantation has also gained attention [297] [298] [299] . in many cases, gae is diagnosed post-mortem, due to delayed diagnosis or unawareness of the clinical entity. following infection by b. mandrillaris, two clinical patterns of presentation have been described. in the first pattern, patients initially develop a skin lesion that may resemble a painless plaque that may evolve into subcutaneous nodules and rarely ulcerations [300] . these patients may develop neurologic manifestations weeks to months later. histopathologic examination of these lesions typically reveals granulomatous reactions in the reticular dermis, associated with lymphocytic and plasma cell infiltrates as well as multinucleated giant cells, without distinct epidermal changes. skin lesions will harbor trophozoites, but these are scarce and often easily overlooked as they resemble histiocytes. it is believed that early diagnosis of b. mandrillaris infections in those presenting with skin lesions may prevent subsequent development of cns disease, but there have also been cases in which patients presenting with skin lesions have progressed to developing gae despite treatment. in the second pattern, patients present with cns involvement without a previously recognized skin lesion. patients presenting with gae may initially display fever, malaise, headache, nausea and vomiting, and frank lethargy. later, these symptoms evolve into visual abnormalities, cranial nerve palsies, seizures, focal paresis; as intracranial pressure builds, coma, and eventually death with tonsilar or uncal herniation ensue within 2-3 weeks [301] . upon infection with b. mandrillaris, brain endothelial cells produce the proinflammatory cytokine il-6, thereby initiating an inflammatory response [302] . moreover, the amoebic trophozoites infiltrate blood vessel walls. degradative enzymes, vessel wall infiltration, and the host inflammatory responses result in tissue necrosis and infarctions in the cerebral hemispheres, cerebellum, and the brainstem. in a mouse model of b. mandrillaris infection, cd4+ t cells were shown to be protective [303] , suggesting that patients with lowered number or dysfunction in cd4+ t cells may be more susceptible to disease by this amoeba. however, b. mandrillaris infections have been described in a variety of human hosts [304] , ranging from the young, healthy, and presumably immunocompetent to the elderly, and those with hiv, chronic corticosteroid exposure, on post-transplant immunosuppression and even patients with cvid. as such, further research is necessary to fully delineate the susceptibility of pidd patients. in patients who develop the characteristic skin lesions, recognition, testing, and treatment for b. mandrillaris may prevent subsequent gae. as such, obtaining tissue and looking for granulomas and trophozoites is quite helpful. skin biopsies can be stained via immunofluorescence or immunoperoxidase techniques to identify b. mandrillaris [305] . additionally, a pcr technique that identifies mitochondrial 16s ribosomal rna from b. mandrillaris is also available through the cdc [306] . in patients in whom the diagnosis is confirmed via skin biopsy, wide resection and medical treatment appears to prevent development of cns disease in at least a proportion of patients. in patients presenting with cns involvement, lumbar punctures reveal csf with lymphocytic pleiocytosis, low-to-normal glucose, and mildly to significantly elevated protein levels. trophozoites are not typically found in the csf, but pcr analysis may be performed. ct or mr imaging may show multiple nodules with ring enhancement; some of these nodules may also contain focal areas of hemorrhage. biopsies of brain tissues typically reveal granulomas and foamy macrophages and multinucleated giant cells surrounded by lymphocytic infiltrates. additionally, there will be areas of necrosis filled with neutrophils, multinucleated giant cells, and lymphocytes, with balamuthia trophozoites and cysts interspersed with macrophages [16] . as with the skin biopsies, immunofluorescent and immunoperoxidase stains may aid diagnosis and should be performed. unfortunately, the optimal medical management of cns disease is unknown. in the usa, a few patients have been successfully treated with a combination of fluconazole, flucytosine, pentamidine, a macrolide antibiotic (either clarithromycin or azithromycin), and one of the following agents: liposomal amphotericin b, miltefosine, sulfadiazine, or thoridazine [307] [308] [309] ; others in peru have been treated successfully with fluconazole (or itraconazole), albendazole, and miltefosine [307] . based on these case reports, most experts recommend treatment with a combination of medications (along with partial or complete resection of nodules) for a prolonged period of time to prevent further deterioration and death [307] [308] [309] [310] . acanthamoeba spp. the genus acanthamoeba contains at least 24 morphologically distinct species that live in a diverse array of habitats, including soil, salt, brackish, and fresh water. acanthamoeba spp. have also been found in humidifiers, heating and cooling unit components, jacuzzis, hot water tanks, bathrooms and drains, eye wash stations and dentistry irrigation systems, and more. acanthamoeba spp. have been isolated from reptiles, birds, and other non-human mammals, suggesting a broad distribution in the environment. acanthamoeba trophozoites feed on bacteria, but have also recently been shown to harbor a number of bacteria (including legionella and burkholderia spp., e. coli, listeria monocytogenes, vibrio cholerae, mycobacteria spp., chlamydophila, and others) and at least one virus (mimivirus) as endosymbionts. acanthamoeba infections in humans can present in a variety of ways. of primary importance are cns infections. like b. mandrillaris, acanthamoeba spp. can induce gae (described above). there is a high predilection for gae in those with hiv/aids, patients on chemotherapy, and those receiving broad spectrum antibiotics [301] . acanthamoeba are rarely found in csf, but some case reports indicate isolation of amoebae by culturing csf on bacterized agar plates. similar to gae seen with b. mandrillaris, cns histopathology may reveal edema, multiple areas of necrosis and hemorrhage, and occasional findings of angitis and blood vessel cuffing by inflammatory cells, as well as occasional trophozoites or cysts. cns disease treatment is not standardized, but several patients have been successfully treated with pentamidine, fluconazole, flucytosine, sulfadiazine, as well as miltefosine. acanthamoeba can rarely cause cutaneous infections; these lesions, like gae, are also predominantly seen in immunocompromised hosts. these lesions can start as nodules or papules on the lower extremities and develop into necrotic ulcers. histopathologic examination may reveal granulomatous dermal lesions in immunocompetent hosts, with histiocytes, as well as neutrophils and plasmacytes; trophozoites are typically visible [311, 312] . the optimal management of cutaneous disease is not known, but typically involves combinational therapy with topical (e.g., chlorhexidine, gluconate, or ketoconazole) and systemic (miltefosine, sulfadiazine, flucytosine, liposomal amphotericin b, azole antifungals, etc.) drugs. additionally, nasopharyngeal and sinus infections have been seen in people with severe compromise in immunity [313, 314] . patients typically present with purulent nasal discharge, and examination may reveal erosion of the nasal septum. nasopharyngeal disease can present concomitantly with cutaneous disease. treatment of nasopharyngeal or sinus disease is difficult and involves surgical debridement and combinations of systemic drugs. disseminated disease is also seen in immunocompromised hosts and typically involves concomitant pulmonary and cutaneous disease in the presence or absence of cns infection. keratitis readily occurs in immunocompetent hosts-with the major risk factor being contact lens wearing without proper adherence to recommended cleansing protocols. this infection less commonly presents as a result of direct inoculation with trauma. one of the most common reasons for contact lens wearers to acquire disease is due to the use of non-sterile tap water in preparing contact lens saline solutions [315] , although contaminated solutions from manufacturers have also been identified. patients will have pain and photophobia. physical exam reveals conjunctival injection and epithelial abnormalities (including pseudodendritic lesions) and stromal infiltrates [316] . the proper diagnosis can be made by staining corneal scrapings with calcofluor or wright-giemsa stains and examined by confocal microscopy, culture, or pcr analysis. prompt therapy with a combination of polyhexamethylene biguanide (or biguanide-chlorhexidine) and propamidine or hexamidine [317, 318] is indicated, but misdiagnosis and delayed therapy are common. more severe cases may also require debridement. the use of topical steroids before administration of combinational therapy may result in worse outcomes and should be avoided; however, if scleritis ensues, it may be necessary to use immunosuppressants to reduce the need for enucleation. severe and/or refractory cases may result in the need for cornea transplantation. phenotypes seen in pidd like hsv-1 and candida [337] . patients with ifnar2 deficiency seem highly susceptible to cns disease caused by mmr vaccine, an otherwise extremely rare phenomenon [187] . recently, a case of noroviral cns disease was described associated with a novel, yet unpublished pidd, suggesting that some pidds may lead to susceptibility of the cns to viruses that normally do not exhibit neurotropism (casanova jl, personal communication) . this again favors metagenomic approaches in the study of cns sequelae in pidd patients. in pidds, the cns is also more vulnerable to virally induced immunodysregulation [325] . conditions like primary hemophagocytic lymphohistiocytosis (hlh) may present as isolated cns disease or relapse only in the cns [338] [339] [340] . almost 20% all human malignancies are associated with chronic infections by hbv, hcv, hpv, ebv, hhv8/kshv, htlv-i, hiv-1, hiv-2, jcv, merkel cell polyomavirus (mcpv), helicobacter pylori, schistosomes, or liver flukes [341] . accordingly, pidd patients' malignancies are often associated with chronic infections. mcpv-associated merkel cell carcinoma has now been described in gata2 and tmc8 (ever2) deficiencies as well as other forms of pidd [330, [342] [343] [344] [345] [346] . large follow-up cohorts are needed to refute or confirm associations between novel pidds and malignancies, such as hyperactivating pik3cd and ovarian dysgerminoma or gata2 deficiency and leiomyosarcoma [329, 347] . recently, hymenolepis nana was found to have driven malignant transformation in an hiv patient. likely, other novel pidd-and pathogen-associated malignancies will be found in the future by those with an open and inquisitive mind [348] . understanding the specific infection susceptibility for each pidd allows not only a better understanding of host defense, but also allows the clinician to collaborate with the microbiology laboratory to make definitive diagnoses and provide the best therapy. reviewing all of the infections for each pidd is not within the scope of this article, but there are several infections that are unique for specific pidds and require special attention from the microbiology laboratory. three examples are provided below. g. bethesdensis is a gram-negative bacterium that was identified to cause disease in patients with cgd in 2006 [349] . g. bethesdensis is a member of the methylotroph group of bacteria, which are able to use single-carbon organic compounds as their only source of energy. they are widespread in the environment, but are rare human pathogens, and infections with g. bethesdensis have been limited thus far to patients with cgd. the organism was first detected in an adult patient with indolent and recurrent necrotizing lymphadenitis [349] . subsequently, g. bethesdensis was isolated from nine patients with cgd, primarily causing lymphadenitis, but there have been two fatalities [350] . treatment has been most effective with intravenous ceftriaxone. the microbiology laboratory should be alerted when there is concern for g. bethesdensis infection to allow for proper culture media. charcoal yeast extract (cye) agar and lowenstein jensen (lj) media are appropriate culture media. mycoplasma and ureaplasma spp. as molecular techniques are becoming more widely used to detect pathogens, the spectrum of infections that were previously only detected through serologic assays and research laboratories will increase. this is important especially for patients with pidd who have unique susceptibility to infection and may not have the ability to mount a serologic response. examples of infections in this group are those caused by mycoplasma and ureaplasma [351, 352] . these pathogens have been known to cause osteoarticular infections for those with antibody deficiency, such as xla and cvid. recently, mycoplasma orale, typically an oral commensal, has been isolated from two patients with defects in the activated pi3k delta syndrome, as chronic lymphadenitis in one and chronic splenic abscess in the other (sm holland personal communication). defects in pi3kcd and pi3kr are frequently associated with hypogammaglobulinemia and therefore would fit in the pattern of mycoplasma infections in those with humoral immunodeficiency. mycoplasma orale has also previously been reported as causing bone disease in a patient with cvid [353] . in patients with xla, helicobacter, camplyobacter, and the related flexispira bacteria that are typically isolated to the gi tract can disseminate and often lead to chronic bacteremia, ulcers, and bone infections [354] [355] [356] . xla patients have higher susceptibility than other humoral pidd and are thought to be due to the role that igm is playing in controlling the dissemination of these pathogens and potentially iga in providing mucosal immunity. these bacteria can be fastidious to grow, and therefore, when there is suspicion, identification needs collaboration with the microbiology laboratory. for instance, the blood culture media may allow growth (although with a longer incubation period), but then the organisms may need molecular techniques for identification, such as 16 s sequencing, as they will not grow on the agar plates. treatment is often difficult, requiring combination antimicrobials for prolonged periods (such as 1 year), and relapse is common. this review provides an important perspective for practicing immunologists, namely that we are a part of a global community as are our patients. this overview of emerging infections and infectious concerns for travelers serves as a foundation for practical considerations for clinicians and patients. using prevalence data, an estimation of the number of infected patients with pidd (table 10) can be developed [25, 130, [357] [358] [359] [360] . thus, the concerns addressed in this review are not theoretical but impact a considerable number of patients already. the landscape of emerging infections is by its nature highly dynamic. during the preparation of this manuscript, a mumps outbreak in the usa occurred, a new bunyavirus outbreak causing an hlh picture was reported (severe fever with thrombocytopenia syndrome), a new outbreak of the hantavirus seoul virus occurred, and an enlarging demographic of the h7n9 influenza virus was reported. this review lists several resources, many of which are updated in real time to support efforts to provide information to patients. doctor's guide alerts can be sent weekly to provide updates on current outbreaks around the world. approach to fever in the returning traveler potential changes in disease patterns and pharmaceutical use in response to climate change climate change 2014: synthesis report. contribution of working groups i, ii and iii to the fifth assessment report of the intergovernmental panel on climate change impact of climate change in the epidemiology of vector-borne diseases in domestic carnivores impact of climate change on human infectious diseases: empirical evidence and human adaptation climate change and health: global to local influences on disease risk climate change and mosquito-borne disease climate of origin affects tick (ixodes ricinus) host-seeking behavior in response to temperature: implications for resilience to climate change? drivers, dynamics, and control of emerging vector-borne zoonotic diseases social inequalities and emerging infectious diseases chemical, clinical, and immunological studies on the products of human plasma fractionation. xii. the use of concentrated normal human serum gamma globulin (human immune serum globulin) in the prevention and attenuation of measles the natural history of infectious hepatitis functional antibacterial activity of a human intravenous immunoglobulin preparation: in vitro and in vivo studies giant cell pneumonia due to respiratory syncytial virus outcome of intravenous immunoglobulin-transmitted hepatitis c virus infection in primary immunodeficiency epidemiology of carbapenem resistant klebsiella pneumoniae infections in mediterranean countries west nile virus neutralization by us plasma-derived immunoglobulin products prevention of infection in children and adolescents with primary immunodeficiency disorders recommendations for live viral and bacterial vaccines in immunodeficient patients and their close contacts vaccination of special populations: protecting the vulnerable vaccination in primary immunodeficiency disorders vaccination guidelines after hematopoietic stem cell transplantation: practitioners' knowledge, attitudes, and gap between guidelines and clinical practice vaccine-derived poliovirus from long term excretors and the end game of polio eradication bcg vaccination in patients with severe combined immunodeficiency: complications, risks, and vaccination policies live rubella virus vaccine long-term persistence as an antigenic trigger of cutaneous granulomas in patients with primary immunodeficiency cutaneous and visceral chronic granulomatous disease triggered by a rubella virus vaccine strain in children with primary immunodeficiencies rubella persistence in epidermal keratinocytes and granuloma m2 macrophages in patients with primary immunodeficiencies vaccine use in primary immunodeficiency disorders epidemiology of carbapenemase-producing enterobacteriaceae and acinetobacter baumannii in mediterranean countries worldwide dissemination of the ndm-type carbapenemases in gram-negative bacteria independent emergence of colistin-resistant enterobacteriaceae clinical isolates without colistin treatment bayesian analysis of two diagnostic methods for paediatric ringworm infections in a teaching hospital global fluoroquinolone resistance epidemiology and implications for clinical use worldwide epidemiology and antibiotic resistance of staphylococcus aureus the changing epidemiology of vancomycin-resistant enterococcus (vre) bacteremia in allogeneic hematopoietic stem cell transplant (hsct) recipients centers for disease c, prevention. sexually transmitted diseases treatment guidelines european guideline on the diagnosis and treatment of gonorrhoea in adults the emerging threat of untreatable gonococcal infection update. who guidelines approved by the guidelines review committee mendelian genetics of human susceptibility to fungal infection primary immunodeficiencies underlying fungal infections extrapulmonary aspergillus infection in patients with card9 deficiency voriconazole versus amphotericin b for primary therapy of invasive aspergillosis rapid induction of multiple resistance mechanisms in aspergillus fumigatus during azole therapy: a case study and review of the literature azole resistance in aspergillus fumigatus: can we retain the clinical use of mold-active antifungal azoles? in-host adaptation and acquired triazole resistance in aspergillus fumigatus: a dilemma for clinical management farm fungicides linked to resistance in a human pathogen high prevalence of azole resistance in aspergillus fumigatus isolates from high-risk patients international expert opinion on the management of infection caused by azole-resistant aspergillus fumigatus candida and host determinants of susceptibility to invasive candidiasis immune defence against candida fungal infections card9-dependent neutrophil recruitment protects against fungal invasion of the central nervous system invasive fungal infection and impaired neutrophil killing in human card9 deficiency inherited card9 deficiency in otherwise healthy children and adults with candida species-induced meningoencephalitis, colitis, or both decreased susceptibility of candida albicans to azole antifungals: a complication of long-term treatment in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) patients antifungal drug resistance of pathogenic fungi candida auris sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a japanese hospital investigation of the first seven reported cases of candida auris, a globally emerging invasive, multidrug-resistant fungus-united states first report of candida auris in america: clinical and microbiological aspects of 18 episodes of candidemia candida auris candidemia in kuwait simultaneous emergence of multidrugresistant candida auris on 3 continents confirmed by wholegenome sequencing and epidemiological analyses epa-registered repellents for mosquitoes transmitting emerging viral disease west nile virus in europe: emergence, epidemiology, diagnosis, treatment, and prevention risk factors for west nile virus infection and disease in populations and individuals systems immunology reveals markers of susceptibility to west nile virus infection idiopathic cd4 lymphopenia associated with neuroinvasive west nile disease: case report and review of the literature west nile virus meningitis in patient with common variable immunodeficiency west nile virus central nervous system infection in patients treated with rituximab: implications for diagnosis and prognosis, with a review of literature co-distribution and co-infection of chikungunya and dengue viruses dengue and dengue hemorrhagic fever urbanization and globalization: the unholy trinity of the 21(st) century severe dengue in the early postoperative period after kidney transplantation: two case reports from hospital geral de fortaleza dengue haemorrhagic fever after living donor renal transplantation dengue shock syndrome in a liver transplant recipient clinical presentation and outcome of dengue viral infection in liverelated renal transplant recipients in karachi dengue vaccine: who position paper yellow fever: epidemiology and prevention yellow fever in africa: estimating the burden of disease and impact of mass vaccination from outbreak and serological data zika virus: new clinical syndromes and its emergence in the western hemisphere zika virus: a new threat to the safety of the blood supply with worldwide impact and implications. transfusion (paris) detection of zika virus in saliva potential sexual transmission of zika virus fatal zika virus infection with secondary nonsexual transmission prolonged shedding of zika virus associated with congenital infection zika virus in the americas: a review for clinicians zika virus associated with microcephaly chikungunya disease dengue chikungunya virus infection in man in thailand, 1962-1964. 3. clinical, epidemiologic, and virologic observations on disease in non-indigenous white persons cutaneous manifestations of chikungunya fever: observations made during a recent outbreak in south india persistent arthralgia associated with chikungunya virus: a study of 88 adult patients on reunion island chikungunya fever: an epidemiological review of a re-emerging infectious disease atypical chikungunya virus infections in immunocompromised patients clinical presentations and interactions of the chikungunya viral infection in hiv patients during the chikungunya epidemic in southern thailand persistent and relapsing babesiosis in immunocompromised patients babesiosis and hiv babesiosis in splenectomized adults. review of 22 reported cases the limits and intensity of plasmodium falciparum transmission: implications for malaria control and elimination worldwide the burden of co-infection with human immunodeficiency virus type 1 and malaria in pregnant women in sub-saharan africa is there an interaction between human immunodeficiency virus and plasmodium falciparum? central role of the spleen in malaria parasite clearance parasitologic and clinical human response to immunoglobulin administration in falciparum malaria naturally acquired antibodies to plasmodium vivax blood-stage vaccine candidates (pvmsp-1 19 and pvmsp-3α 359−798 ) and their relationship with hematological features in malaria patients from the brazilian amazon organ-specific immune responses associated with infectious disease taxonomy of the genus leishmania: a review of nomenclature and classification leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans risk factors for visceral leishmaniasis in a new epidemic site in amhara region emerging leishmania/hiv co-infection in africa the relationship between leishmaniasis and aids: the second 10 years clinical aspects of visceral leishmaniasis in hiv infection epidemiologic, clinical, diagnostic and therapeutic aspects of visceral leishmaniasis in renal transplant recipients: experience from thirty cases the lipophosphoglycan of leishmania and macrophage protein kinase c leishmania major infection activates nf-kappab and interferon regulatory factors 1 and 8 in human dendritic cells transcriptional inhibition of interleukin-12 promoter activity in leishmania spp.-infected macrophages a case of interleukin-12 receptor beta-1 deficiency with recurrent leishmaniasis visceral leishmaniasis and other severe infections in an adult patient with p47-phox-deficient chronic granulomatous disease the syndrome of hemophagocytic lymphohistiocytosis in primary immunodeficiencies: implications for differential diagnosis and pathogenesis rickettsiae and rickettsial infections: the current state of knowledge rickettsioses and the international traveler tick-borne rickettsioses in international travellers threats to international travellers posed by tick-borne diseases epidemiology of rickettsial diseases update on tick-borne rickettsioses around the world: a geographic approach tick-borne rickettsioses in europe tick-borne encephalitis in sweden and climate change ehrlichia chaffeensis: a prototypical emerging pathogen national surveillance of spotted fever group rickettsioses in the united states severe ehrlichia infection in pediatric oncology and stem cell transplant patients. pediatr blood cancer infections with ehrlichia chaffeensis and ehrlichia ewingii in persons coinfected with human immunodeficiency virus diagnosis and management of tickborne rickettsial diseases: rocky mountain spotted fever, ehrlichioses, and anaplasmosis-united states: a practical guide for physicians and other health-care and public health professionals clarithromycin versus azithromycin in the treatment of mediterranean spotted fever in children: a randomized controlled trial clinical diagnosis and treatment of human granulocytotropic anaplasmosis human ehrlichiosis and anaplasmosis investigation of tick-borne bacteria (rickettsia spp., anaplasma spp., ehrlichia spp. and borrelia spp.) in ticks collected from andean tapirs, cattle and vegetation from a protected area in ecuador the biological basis of severe outcomes in anaplasma phagocytophilum infection ehrlichia-induced hemophagocytic lymphohistiocytosis: a case series and review of literature diagnosis and management of q fever-united states, 2013: recommendations from cdc and the q fever working group practice guidelines for the diagnosis and management of patients with q fever by the armed forces infectious diseases society q fever and hiv infection coxiella burnetii infection among subjects infected with hiv type 1 in the central african republic rapidly fatal q-fever pneumonia in a patient with chronic granulomatous disease coxiella burnetii infection with severe hyperferritinemia in an asplenic patient bartonella quintana and urban trench fever bartonella sp. bacteremia in patients with neurological and neurocognitive dysfunction bartonellosis (carrion's disease) in the modern era bartonella infection in immunocompromised hosts: immunology of vascular infection and vasoproliferation prevalence of bartonella infection among human immunodeficiency virus-infected patients with fever bartonella henselae-mediated disease in solid organ transplant recipients: two pediatric cases and a literature review bartonella henselae infections in solid organ transplant recipients: report of 5 cases and review of the literature cutaneous bacillary angiomatosis in renal transplant recipients: report of three new cases and literature review refractory bartonella quintana bacillary angiomatosis following chemotherapy for chronic lymphocytic leukaemia molecular epidemiology of bartonella infections in patients with bacillary angiomatosis-peliosis the x-linked hyper-igm syndrome: clinical and immunologic features of 79 patients. medicine (baltimore) granulomatous hepatitis and necrotizing splenitis due to bartonella henselae in a patient with cancer: case report and review of hepatosplenic manifestations of bartonella infection granulomatous hepatitis due to bartonella henselae infection in an immunocompetent patient disseminated bartonella infection following liver transplantation louse-borne relapsing fever (borrelia recurrentis) in an eritrean refugee arriving in switzerland louseborne relapsing fever in young migrants tickborne relapsing fever a case of meningoencephalitis by the relapsing fever spirochaete borrelia miyamotoi in europe meningoencephalitis from borrelia miyamotoi in an immunocompromised patient lyme disease disease-specific diagnosis of coinfecting tickborne zoonoses: babesiosis, human granulocytic ehrlichiosis, and lyme disease lyme disease and human granulocytic anaplasmosis coinfection: impact of case definition on coinfection rates and illness severity burden and viability of borrelia burgdorferi in skin and joints of patients with erythema migrans or lyme arthritis the clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the infectious diseases society of america outbreak of human buffalopox infection laboratory-acquired buffalopox virus infection orf infection in a patient with stat1 gain-of-function a case of human orf in an immunocompromised patient treated successfully with cidofovir cream maternal onset de novo sh2d1a mutation and lymphocytic choriomeningitis virus infection in a patient with xlinked lymphoproliferative disease type 1: a case report pneumonic tularemia in a patient with chronic granulomatous disease nipah virus encephalitis outbreak in malaysia nipah virus encephalitis reemergence sars and mers: recent insights into emerging coronaviruses epidemiology, genetic recombination, and pathogenesis of coronaviruses the role of super-spreaders in infectious disease mers-covoutbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study coronavirusesdrug discovery and therapeutic options mannose-binding lectin in severe acute respiratory syndrome coronavirus infection functional polymorphisms of the ccl2 and mbl genes cumulatively increase susceptibility to severe acute respiratory syndrome coronavirus infection association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection human ifnar2 deficiency: lessons for antiviral immunity stat2 deficiency and susceptibility to viral illness in humans feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis ebola and marburg hemorrhagic fever assessing the evidence supporting fruit bats as the primary reservoirs for ebola viruses investigating the zoonotic origin of the west african ebola epidemic ebola virus infection of human pbmcs causes massive death of macrophages, cd4 and cd8 t cell sub-populations in vitro human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis evaluation of convalescent plasma for ebola virus disease in guinea efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, clusterrandomised trial (ebola ca suffit!) containing ebola at the source with ring vaccination zoonotic hepatitis e: animal reservoirs and emerging risks sporadic acute or fulminant hepatitis e in hokkaido, japan, may be food-borne, as suggested by the presence of hepatitis e virus in pig liver as food inactivation of infectious hepatitis e virus present in commercial pig livers sold in local grocery stores in the united states fulminant liver failure from acute autochthonous hepatitis e in france: description of seven patients with acute hepatitis e and encephalopathy hepatitis e virus: an underestimated opportunistic pathogen in recipients of allogeneic hematopoietic stem cell transplantation hepatitis e virus-related cirrhosis in kidneyand kidney-pancreas-transplant recipients hepatitis e virus and chronic hepatitis in organ-transplant recipients reactivation of hepatitis e infection in a patient with acute lymphoblastic leukaemia after allogeneic stem cell transplantation severe morbidity and mortality associated with influenza in children and young adults-michigan symptomatic predictors of influenza virus positivity in children during the influenza season a further investigation into influenza pneumococcal and influenzal-streptococcal septicaemia: epidemic influenzal pneumonia of highly fatal types and it relation to purulent bronchitis interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness influenza-who cares the global circulation of seasonal influenza a (h3n2) viruses infectious disease. life-threatening influenza and impaired interferon amplification in human irf7 deficiency fatal viral infection-associated encephalopathy in two chinese boys: a genetically determined risk factor of thermolabile carnitine palmitoyltransferase ii variants pigs with severe combined immunodeficiency are impaired in controlling influenza a virus infection association between severe pandemic 2009 influenza a (h1n1) virus infection and immunoglobulin g(2) subclass deficiency igg3 deficiency and severity of 2009 pandemic h1n1 influenza cellular and humoral influenza-specific immune response upon vaccination in patients with common variable immunodeficiency and unclassified antibody deficiency dendritic and t cell response to influenza is normal in the patients with x-linked agammaglobulinemia the clinical significance of measles: a review severe measles in immunocompromised patients subacute measles encephalitis in the young immunocompromised host: report of two cases diagnosed by polymerase chain reaction and treated with ribavirin and review of the literature defective b-cell proliferation and maintenance of long-term memory in patients with chronic granulomatous disease a new complication of stem cell transplantation: measles inclusion body encephalitis centers for disease c, prevention. prevention of measles, rubella, congenital rubella syndrome, and mumps the impact of increased molecular diagnostics persistent coxsackie b encephalitis: report of a case and review of the literature enterovirus infections in neonates enteroviruses in the early 21st century: new manifestations and challenges neonatal enterovirus infections reported to the national enterovirus surveillance system in the united states clinical features of acute and fulminant myocarditis in children-2nd nationwide survey by japanese society of pediatric cardiology and cardiac surgery severe enterovirus infections in hospitalized children in the south of england: clinical phenotypes and causative genotypes role of non-polio enterovirus infection in pediatric hemolytic uremic syndrome enterovirus infections: diagnosis and treatment five years' experience of reverse-transcriptase polymerase chain reaction in daily diagnosis of enterovirus and rhinovirus infections a randomized, double-blind, placebocontrolled trial of pleconaril for the treatment of neonates with enterovirus sepsis efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in china: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial enteroviral infections in primary immunodeficiency (pid): a survey of morbidity and mortality high susceptibility for enterovirus infection and virus excretion features in tunisian patients with primary immunodeficiencies sequential asymptomatic enterovirus infections in a patient with major histocompatibility complex class ii primary immunodeficiency treatment of chronic enterovirus encephalitis with fluoxetine in a patient with xlinked agammaglobulinemia enteroviruses in x-linked agammaglobulinemia: update on epidemiology and therapy enteroviral infection in a patient with blnk adaptor protein deficiency enteroviral meningoencephalitis after anti-cd20 (rituximab) treatment fatal coxsackie meningoencephalitis in a patient with bcell lymphopenia and hypogammaglobulinemia following rituximab therapy enterovirus antibody titers after ivig replacement in agammaglobulinemic children outbreak of poliomyelitis in hispaniola associated with circulating type 1 vaccine-derived poliovirus update on vaccine-derived polioviruses-worldwide prolonged replication of a type 1 vaccine-derived poliovirus in an immunodeficient patient chronic enteroviral meningoencephalitis in agammaglobulinemic patients the primary immunodeficiencies persistence of vaccine-derived polioviruses among immunodeficient persons with vaccine-associated paralytic poliomyelitis vaccine-associated paralytic poliomyelitis in immunodeficient children paralytic poliomyelitis caused by a vaccine-derived polio virus in an antibody-deficient argentinean child pertussis of adults and infants pertussis in infants: an underestimated disease pertussis in adults: epidemiology, signs, symptoms, and implications for vaccination bordetella pertussis transmission rediscovering pertussis. front pediatr antibody levels to bordetella pertussis and neisseria meningitidis in immunodeficient patients receiving immunoglobulin replacement therapy infectious disease report: bordetella pertussis infection in patients with cancer meningococcal disease: history, epidemiology, pathogenesis, clinical manifestations, diagnosis, antimicrobial susceptibility and prevention diagnosis and management of bacterial meningitis in the paediatric population: a review chronic meningococcemia cutaneous lesions involve meningococcal perivascular invasion through the remodeling of endothelial barriers global epidemiology of invasive meningococcal disease epidemic spread and antigenic variability of neisseria meningitidis fit genotypes and escape variants of subgroup iii neisseria meningitidis during three pandemics of epidemic meningitis the transmission of mycobacterium tuberculosis in high burden settings inherited and acquired immunodeficiencies underlying tuberculosis in childhood mendelian susceptibility to mycobacterial disease: genetic, immunological, and clinical features of inborn errors of ifngamma immunity adult-onset immunodeficiency in thailand and taiwan official american thoracic society/infectious diseases society of america/centers for disease control and prevention clinical practice guidelines: diagnosis of tuberculosis in adults and children infectious diseases society of america clinical practice guidelines: treatment of drug-susceptible tuberculosis defects in the interferon-gamma and interleukin-12 pathways gata2 deficiency signal transducer and activator of transcription 1 (stat1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis endemic infections in southeast asia provide new insights to the phenotypic spectrum of primary immunodeficiency disorders endemic mycoses in patients with stat3-mutated hyper-ige (job) syndrome melioidosis: evolving concepts in epidemiology, pathogenesis, and treatment melioidosis: a clinical overview melioidosis: epidemiology, pathophysiology, and management clinical presentation and medical management of melioidosis in children: a 24-year prospective study in the northern territory of australia and review of the literature a review of melioidosis cases in the americas melioidosis in travelers: review of the literature laboratory diagnosis of melioidosis: past, present and future fatal bacteremic melioidosis in patients with prolonged neutropenia pathogenic free-living amoebae: epidemiology and clinical review primary amoebic meningoencephalitis caused by naegleria fowleri: an old enemy presenting new challenges the immune response to naegleria fowleri amebae and pathogenesis of infection production of monoclonal antibodies to naegleria fowleri, agent of primary amebic meningoencephalitis pathogenic and opportunistic free-living amoebae: acanthamoeba spp., balamuthia mandrillaris, naegleria fowleri, and sappinia diploidea multiplex real-time pcr assay for simultaneous detection of acanthamoeba spp., balamuthia mandrillaris, and naegleria fowleri successful treatment of naegleria fowleri meningoencephalitis by using intravenous amphotericin b, fluconazole and rifampicin successful treatment of primary amebic meningoencephalitis successful treatment of an adolescent with naegleria fowleri primary amebic meningoencephalitis centers for disease c, prevention. notes from the field: transplant-transmitted balamuthia mandrillaris-arizona balamuthia mandrillaris transmitted through organ transplantation-mississippi transmission of balamuthia mandrillaris by organ transplantation balamuthia mandrillaris infection of the skin and central nervous system: an emerging disease of concern to many specialties in medicine infections with free-living amebae balamuthia mandrillaris exhibits metalloprotease activities resistance to intranasal infection with balamuthia mandrillaris amebae is tcell dependent balamuthia mandrillaris amoebic encephalitis: an emerging parasitic infection histopathologic spectrum and immunohistochemical diagnosis of amebic meningoencephalitis demonstration of balamuthia and acanthamoeba mitochondrial dna in sectioned archival brain and other tissues by the polymerase chain reaction successful treatment of balamuthia amoebic encephalitis: presentation of 2 cases balamuthia mandrillaris meningoencephalitis: survival of a pediatric patient balamuthia mandrillaris brain abscess successfully treated with complete surgical excision and prolonged combination antimicrobial therapy neurosurgical intervention in the diagnosis and treatment of balamuthia mandrillaris encephalitis cutaneous acanthamebiasis infection in immunocompetent and immunocompromised patients tender ulceronecrotic nodules in a patient with leukemia. cutaneous acanthamebiasis parasitic sinusitis and otitis in patients infected with human immunodeficiency virus: report of five cases and review acanthamoeba: a rare primary cause of rhinosinusitis the epidemiology of acanthamoeba keratitis in the united states acanthamoeba sclerokeratitis. determining diagnostic criteria acanthamoeba keratitis update-incidence, molecular epidemiology and new drugs for treatment comparison of polyhexamethylene biguanide and chlorhexidine as monotherapy agents in the treatment of acanthamoeba keratitis primary immunodeficiency diseases: an update on the classification from the international union of immunological societies expert committee for primary immunodeficiency heterozygous stat1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype clinical and immunologic phenotype associated with activated phosphoinositide 3-kinase delta syndrome 2: a cohort study clinical spectrum and features of activated phosphoinositide 3-kinase delta syndrome: a large patient cohort study the extended clinical phenotype of 64 patients with dedicator of cytokinesis 8 deficiency new clinical phenotypes of fungal infections in special hosts progressive multifocal leukoencephalopathy in primary immune deficiencies: stat1 gain of function and review of the literature kaposi sarcoma of childhood: inborn or acquired immunodeficiency to oncogenic hhv-8. pediatr blood cancer immunologic defects in severe mucocutaneous hsv-2 infections: response to ifn-gamma therapy paracoccidioidomycosis and other unusual infections in brazilian patients hyper igm syndrome: a report from the usidnet registry genetic variation in schlafen genes in a patient with a recapitulation of the murine elektra phenotype warts and all: human papillomavirus in primary immunodeficiencies severe infectious diseases of childhood as monogenic inborn errors of immunity enrichment of rare variants in population isolates: single aicda mutation responsible for hyper-igm syndrome type 2 in finland a homozygous card9 mutation in a family with susceptibility to fungal infections genetic, immunological, and clinical features of patients with bacterial and fungal infections due to inherited il-17ra deficiency primary polyomavirus infection, not reactivation, as the cause of trichodysplasia spinulosa in immunocompromised patients tlr3 deficiency in herpes simplex encephalitis: high allelic heterogeneity and recurrence risk hemophagocytic lymphohistiocytosis with isolated central nervous system reactivation and optic nerve involvement isolated central nervous system hemophagocytic lymphohistiocytosis: case report predominant neurological manifestations seen in a patient with a biallelic perforin1 mutation (prf1; p.r225w) the prevention of infection-associated cancers merkel cell carcinoma in a patient with gata2 deficiency: a novel association with primary immunodeficiency spontaneous regression of merkel cell carcinoma developed in a patient with epidermodysplasia verruciformis merkel cell polyomavirus detection in a patient with familial epidermodysplasia verruciformis merkel cell polyomavirus-positive merkel cell carcinoma in a patient with epidermodysplasia verruciformis merkel cell polyomavirus in merkel cell carcinoma from a brazilian epidermodysplasia verruciformis patient association of gata2 deficiency with severe primary epstein-barr virus (ebv) infection and ebv-associated cancers malignant transformation of hymenolepis nana in a human host granulibacter bethesdensis gen. nov., sp. nov., a distinctive pathogenic acetic acid bacterium in the family acetobacteraceae methylotroph infections and chronic granulomatous disease osteoarticular infectious complications in patients with primary immunodeficiencies increased susceptibility to mycoplasma infection in patients with hypogammaglobulinemia disseminated mycoplasma orale infection in a patient with common variable immunodeficiency syndrome relapsing campylobacter jejuni systemic infections in a child with x-linked agammaglobulinemia bacteremia and skin/bone infections in two patients with x-linked agammaglobulinemia caused by an unusual organism related to flexispira/helicobacter species successful approach to treatment of helicobacter bilis infection in x-linked agammaglobulinemia prevalence and morbidity of primary immunodeficiency diseases, united states epidemiological characteristics of spotted fever in israel over 26 years complications of bacille calmette-guerin (bcg) vaccination and immunotherapy and their management acknowledgements the authors would like to thank thomas krell for information regarding ivig safety and david peden for recognizing global warming as central to medical knowledge. conflict of interest the authors declared that they have no conflict of interest. pidds display wide genetic and phenotypic heterogeneity [319] . similar disease phenotypes may be caused by multiple genes, while patients' phenotypes caused by the same gene and even by the same mutations vary between individuals. importantly, after a novel pidd has been described, subsequent reports often reveal a wider variation in associated infections and cellular findings, often without clear genotype-phenotype correlations [320] [321] [322] [323] [324] . variation may be caused by mechanisms such as other contributing genes or geographical variation in infectious exposures. geographic differences seem most pronounced in intracellular and often chronic infections. while the numbers of described pidd patients increase, at first seemingly rarely pidd-associated infections turn out to be found in a significant subset of pidd patients [325] [326] [327] . for example, patients with cd40l deficiency living in endemic areas display susceptibility to bartonellosis and paracoccidioidomycosis, infections not described in european and us cohorts [157, 328, 329] .often, an infectious phenotype previously only described in secondary immunodeficiencies may reveal the possibility of an underlying primary immunodeficiency [327, 330, 331] . increasing numbers of genetic defects causing early-onset, severe, and recurrent susceptibility to commonly circulating pathogens like pneumococci, tuberculosis, herpes simplex, and influenza viruses as well as endemic protozoans like trypanosomes and fungi are being recognized, and thus, infections with unusual pathogens require a high index of suspicion for pidd [332] . in contrast, pidds may also manifest as suspected infection but sterile inflammation. for example, in inflammatory lesions like granulomas and necrotizing fasciitis where no clear pathogens are found, one needs to rule out aberrant host responses due to pidd [333] .chronic viral and fungal infections may also display novel phenotypes never or rarely seen in secondary immunodefic i e n c i e s . i n f e c t i o n s l i k e d e r m a t o p h y t o s i s a n d phaeohyphomycosis deeply infiltrating the skin and lymph nodes, occasionally extending to bones and central nervous system (cns) as well as predisposition to primary cns candidiasis and extrapulmonary aspergillus slowly revealed the full phenotypic spectrum of card9 deficiency [44, 324, 334] . chronic skin ulcers caused by hsv-1 and severe molluscum contagiosum suggest dock8 deficiency or gain-of-function mutations of stat1 [320, 323] . chronic mucocutaneous candidiasis has revealed a large group of monogenic diseases (il17ra, il17rc, il17f, stat1 (gof), rorx, act1), which may also be associated with recurrent bacterial infections or syndromic features [335] . while novel diseases by newly described viruses are being discovered, one needs awareness to suspect these in pidd patients [336] .interestingly, most novel forms of infectious disease in pidd patients have been described either in easily accessible sites like the skin or in immunologically privileged, normally sterile sites like the cns. this suggests that with the increasing use of invasive sampling and sensitive metagenomic approaches, we might find more novel infectious phenotypes. pathogens highly suggestive of certain pidds, like chronic enteroviral cns infections in xla patients are reviewed above. in hypomorphic mutations, cns seems to be especially vulnerable to chronically active and/or recurrent novel infectious bsmoldering^focal encephalitis lesions by pathogens key: cord-019347-tj3ye1mx authors: nan title: abstract book date: 2010-02-19 journal: ann allergy asthma immunol doi: 10.1016/s1081-1206(10)61294-x sha: doc_id: 19347 cord_uid: tj3ye1mx nan introduction: the effect of anti ige has been described as a function of complexing free ige to reduce mast cell implantation and consequent reduction of mast cell degranulation upon exposure to antigen. theoretically, as total free ige drops below 10 ng/ml, improvement occurs as free and cell bound ige equilibrate and allergic reactions subside. initial observations ( togias a et al. j allergy clin immunol. 1998 ;101:s171)suggested that prick skin tests consistently reached their low point at 98 days with significant reduction in size. laynadier (leynadier f, doudou o, gaouar h, le gros v, bourdeix i, guyomarch-cocco l, trunet p : effect of omalizumab in health care workers with occupational latex allergy.j allergy clin immunol 2004;113:360-1) noted some reduction in skin testing in sixty percent of patients treated over a six month period with monoclonal antiige. but clinicians since approval of the drug in 2002 have noted that even in the presence of good clinical response to asthma, skin test reactivity is still obvious, and in many cases unchanged. methods: following the suggestions for standard clinical follow up by lanier and marshall ( annals may 2003) , routine skin prick skin testing was completed after informed consent on 24 patients at onset of anti-ige therapy, and repeated at 6-7 months. in all instances were skin tests done and photographed in a standard manner. in a few cases, skin tests were repeated on patients receiving anti ige for as long as five years. results: photographic evidence will be presented. in 22 of 24 patients on current therapy, prick skin testing remained at an almost identical photographic level to the baseline analysis, but there was a correlation between the patients with the lowest serum ige and the likelihood of apparent reduction. there was no correlation to reduction of skin testing and clinical response to anti ige since all 24 patients had experience significant quality of life improvements. conclusion: anti ige has variable effects on reducing prick skin testing, with a greater likely hood associated with low or extremely low initial total ige levels. skin testing is more sensitive for detection of allergen-specific ige than is immunoassay for allergen-specific ige. for some assay methods, the qualitative cutoff of 0.35 ku/l is inappropriately high. this study was conducted in order to determine whether a particular assay system can be used to measure lower levels of allergen-specific ige. the pharmacia unicap system was studied. all reagents were purchased from the manufacturer, and the study was approved by a local human assurance committee. to determine background of the calibration curve, the fluorescent unit (fu) response of 6 replicates of assay diluent measured with an anti-ige solid phase was determined. the background responses of 7 arbitrarily selected allergen solid phases (oak, timothy and short ragweed pollen; cat; peanut; yellow jacket venom; penicillin g) were also measured. lower detection limits (lld) were calculated using the mean background measurement + 3 standard deviations (sd). to examine linearity of a low-range calibration curve, 1:2 dilutions to extinction were made, starting with the 3.5 ku/l calibrator. the mean background of the anti-ige solid phase was 15.4 fu, sd 2.7 and coefficient of variation (cv) of 18%; the lld was 24. this lld corresponded to a level of approximately 0.01 ku/l of ige. the lld of the allergen solid phases ranged from 17.2 fu (yellow jacket venom) to 71.6 fu (short ragweed); this corresponded to about 0.08 ku/l ige. the low level dilution curve exhibited parallelism with a curve constructed using a zero (diluent) calibrator as a 7th point in the assay's reference curve. a low level method is capable of measuring specific ige levels lower than the manufacturer's 0.35 ku/l cutoff. this would seem to be particularly important in research applications, and in the analysis of certain highrisk allergens. the clinical significance of very low levels of specific ige in the serum warrants study. allergists/immunologists are often consulted on patients with rashes or recurrent infections. the differential diagnosis can comprise a variety of disorders, some of which are rare syndromes that require early diagnosis and specific management. a 3 mo old wm developed persistent rash and worsening eczema. later, he had recurrent severe skin infections, skin abscesses, recurrent sinusitis, and chronic otitis media that required multiple placement of tympanostomy tubes, yet had a persistent tympanic membrane perforation. recurrent wheezing was noted from 3-7 yr of age. he had several episodes of pneumonia since 3 yr of age affecting different lobes. he failed to shed the primary teeth and had history of periodontitis and oral thrush. he developed scoliosis and multiple fractures of the upper extremities and ribs. at 12 yr he had staphylococcal bacteremia and osteomyelitis of the acetabulum. in spite of antibiotics prophylaxis, he continued to have recurrent skin infections and lymphadenitis. laboratory evaluation revealed eosinophilia (up to 3138/mm3), normal levels of igg, iga, igm & igg subclasses, except for decreased igg2 (51 mg/dl). he had protective levels of anti s. pneumoniae and h. influenzae, but low anti-tetanus and anti-diphtheria titers. at 4 yr, total ige level was 42, 500 iu/ml which supported the diagnosis of hyper ige (hie) syndrome; it decreased to 197 iu/ml by 12 yr. rast was positive to hd mite, cat, dog, egg, milk, and pollens. flow cytomerty, nbt and phagocytic index were normal; ch50 was low (66 mg/dl). dexa scan showed osteopenia. chest x-ray showed bilateral infiltrate, atelectasis and scoliosis of the thoracic spine. during his hospitalization at 13 yr, his skin was fair, lichenified with widespread maculopapular erythematous rash. the palms and fingers showed open cracks and pustules, but no weeping lesions. in addition to coarse facial features, his face was eczematous. conclusion: hie syndrome is an autosomal dominant disorder that mimics atopic dermatitis but has severe course, multiple infections, and several complications. although a markedly elevated total ige level introduction: mannose-binding lectin (mbl) is a serum protein in the lectin complement pathway. it is important in innate immunity, and is thought to be particularly relevant in young children during the development of adaptive immunity. deficiency in mbl has been reported in population-based studies as a risk factor in children for infection and hospitalization. additionally, age-dependent variability of mbl has been previously noted. this study evaluates the prevalence of mbl deficiency in children with established recurrent infection who were referred for evaluation of immunodeficiency. method: we prospectively evaluated mbl status of children referred for recurrent infection. serum was collected from october 2002 to september 2003. children with known primary or secondary immunodeficiencies were excluded. mbl analysis was performed by standardized elisa using mbl oligomer assays. we performed chart and laboratory review for comorbid diagnoses, quantitative immunoglobulin levels and subclasses, and complement function with ch50 and ah50. results: two-hundred thirty five children were evaluated. mean age was 4.5 years (range 0.08-17 years). mean mbl was 1780 ng/ml (range 17-4806 ng/ml). thirty-one of 235 children (13.1%) were mbl deficient, levels <200, and among this group the mean mbl level was 61.3 ng/ml (range 17-163 ng/ml). mean age in this group was 3.6 years (range 0.3-16 years). of the children with mbl deficiency, 13 (5.1%) children had levels <50. the m:f ratio of children with abnormal mbl levels was 1.08. linear regression analysis showed no correlation of mbl level with age. comorbid diagnoses were variable, including asthma, allergy, and atopic dermatitis. none of the children had symptoms compatible with connective tissue disease. no child with an abnormal mbl level was found to have significant deficiency of igg, iga, or igm. several children had low igg4, which were within normal physiologic range. no other concomitant complement pathway disorders were detected. conclusions: our study did not reveal any correlation between mbl level and age. in children with recurrent infections, mbl deficiency was approximately twice the estimated rate of the general population. contrary to previous studies, no other significant immunologic disorders were identified. therefore, mbl deficiency alone is a risk factor for infection in children. t.b. fausnight * , hershey, pa. introduction: the incidence of perioperative anaphylaxis is estimated to be between 1 in 10, 000 and 1 in 20, 000 procedures. approximately 60% of cases of perioperative anaphylaxis are attributed to neuromuscular agents. very little information is reported in the literature regarding pediatric perioperative anaphylaxis. i describe a pediatric patient with suspected perioperative anaphylaxis to rocuronium. methods: a 10 year-old girl with a history of sacral agenesis and neurogenic bladder was scheduled to have bladder augmentation surgery. the patient was taken to a latex-free operating room. during induction of general anesthesia, she was found to be difficult to ventilate. she also became hypotensive. examination of the patient revealed urticaria on her right arm. she was given epinephrine, diphenhydramine, and dexamethasone. the procedure was aborted. the reaction was believed to be from either rocuronium or propofol. results: because of the high incidence of anaphylaxis to neuromuscular agents, allergy skin testing was performed for rocuronium, vecuronium, and succinylcholine. the patient had negative percutaneous skin tests (1:10) for rocuronium, vecuronium, and succinylcholine. she had a negative intradermal skin test to rocuronium at the 1:1000 dilution. she had a positive intradermal skin test to rocuronium at the 1:100 dilution. she had negative intradermal skin tests to both vecuronium and succinylcholine at the 1:1000, 1:100, and 1:10 dilutions. she underwent surgery 2 weeks after skin testing. she received a test dose of vecuronium and had no reaction. she received 3 doses of vecuronium and multiple doses of morphine without any adverse reactions. propofol was avoided. the surgery was completed without difficulty conclusions: this case illustrates the usefulness of skin testing for neuromuscular agents in a pediatric patient. introduction: extrapulmonary pneumocystis jiroveci infection is rare in non-hiv infected individuals and, to our knowledge, it has not been reported before in a patient with good's syndrome. we report a case of extrapulmonary pneumocystis in a patient with good's syndrome. case report: a 44-year-old african american male patient with a history of good's syndrome presented with left flank pain of 3 months duration. the pain was constant and associated with night sweats, fever, and chills. furthermore, the patient also reported a 20-pound weight loss. on exam, patient was found to have multiple hypopigmented areas over his abdomen and left lower extremity, bitemporal wasting, sunken eyeballs, and oral thrush. the patient was also found to have left costovertebral angle tenderness with a palpable solid fixed mass along the left mid axillary line overlying the splenic area. computedtomography (ct) scan of the chest and abdomen showed a 2.2 x 2.9 cm soft tissue mass at the left lateral aspect of the t11 vertebral body and 7.0 x 5.2 cm soft tissue parasplenic mass. the parasplenic mass involved the inferior anterior aspect of the left 11th and 12th ribs (see figure) . the giemsastained biopsy of the parasplenic mass revealed pneumocystis jiroveci and was confirmed using immunohistochemical stain with monoclonal antipnumocystis antibodies. the patient's symptoms of night sweats and loss of appetite improved within 48 hours following initiation of trimethoprim-sulfamethoxazole therapy. a ct of the chest and abdomen, repeated six months post treatment, confirmed the resolution of both the paravertebral and the parasplenic masses. conclusion: to our knowledge, this is the first case of extrapulmonary pneumocystis presenting in a patient with good's syndrome. although it is rare, extrapulmonary pneumocystis should be considered in the differential diagnosis of patients with good's syndrome and chest or abdominal mass. introduction: t cells are regulated by cellular interactions with the environment during development. they play a critical role in the regulation and development of autoimmune diseases. we report inflammatory myositis in a 10-year-old caucasian female with primary t cell-deficiency since infancy. methods: at nine months of age, our patient developed lymphoid interstitial pneumonitis. immunologic evaluation revealed isolated t-cell deficiency. during the first 3 years of life, she had failure to thrive, recurrent sinusitis, oral candidiasis, fungal skin infections, and pseudomonas pneumonia. at 5 years, autoimmune conditions, characterized by a purpuric rash over the nose; face and exposure area of elbows; knees and fingers; raynaud's phenomenon; and nasal septum perforation, developed. inflammatory markers were persistently elevated and autoantibodies detected. by age 9, she developed proximal muscle weakness with distal joint contractures. dry gangrene of the fingers from a thrombotic incident occurred. results: serial immunologic evaluation revealed low cd4 9-36%, # 153-335 (normal 36-61%, # 900-2860 cell/mm3), low cd8 5-23%, #142-237 (normal 16-34%, # 630-1910 cell/mm3) , decreased lymphocyte transformation to antigens but appropriate to mitogens. igg 1320-2560 (normal 667-1179 mg/dl); iga 470-780 (normal 79-169 mg/dl), and igm 215-340, (normal 40-90 mg/dl) with appropriate antibody responses to vaccine antigens. bone marrow and thymus biopsies were normal. further evaluation was not consistent with known primary or secondary immunodeficiencies. several skin biopsies revealed nonspecific dermatitis without vasculitis. mri of lower extremities showed abnormal signals involving bilateral muscles of thighs and calves. muscle biopsy revealed inflammatory myositis with plasma cell predominance, inconsistent with dermatomyositis or polymyositis. aldolase 8-10 iu/l (< 5), crp 8-16 mg/dl (< 0.5), esr 50-70 (< 20), rf 1:1280 (< 1:40) and type ii collagen ab 26-35 ei/ml (< 20) . ana, anca and ace were within normal range. antibodies for myositis and myositis related-antibodies were negative. diagnosis of nonspecific plasma-cell inflammatory myositis was made. a trial of monthly ivig 2 gm/kg resulted in clinical improvement. conclusion: this novel plasma cell myositis occurring in a child with primary t cell deficiency underscores the role t cells play in the development of autoimmune diseases. introduction the incidence of hypersensitivity reactions (hr) is increased in patients treated with multiple courses of carboplatin. the purposes of this investigation were to evaluate the effectiveness of a 6-hour, 12-step desensitization protocol and to characterize the immune mechanism of carboplatin hr. methods we analyzed ten consecutive patients over a two year period with documented hr to carboplatin who required continued treatment with a platinum agent. the patients were treated with carboplatin using a 6-hour, 12-step desensitization protocol. skin tests were performed on five patients. results ten patients successfully completed 35 planned courses of desensitizations to carboplatin, 31 of which were without reactions. four patients had symptoms during their first (n=3) and third (n=1) desensitizations but tolerated the re-administration of infusions without further reactions. for subsequent courses, the protocol was modified for two patients who had extracutaneous symptoms during desensitization and was unchanged for the patient who had mild urticaria. these three patients tolerated subsequent courses of desensitizations without reactions. the fourth patient with symptoms during desensitization no longer required carboplatin. of the five patients who were skin tested to carboplatin, four had positive wheal and flare reactions. in one patient, the skin test response to carboplatin became negative after desensitization. conclusions the 6-hour, 12-step desensitization protocol is safe and effective for treating patients with carboplatin hr. positive skin tests to carboplatin suggest a mast cell/ige-mediated mechanism. conversion of the positive skin test to a negative response after desensitization supports antigen-specific mast cell desensitization. hypothesis: there is an association between food allergies and acid reflux in atopic adults study design: a retrospective chart review of 60 patients was conducted. 30 people who tested positive and 30 people who tested negative for food allergy were included. the prevalence of gastroesophageal reflux disease (gerd) in the total group and each of the study arms was compared to population prevalence. methods: results of 70 allergen specific ige tests (pharmacia immunocap, nj) run for food allergies were reviewed to help locate charts of 60 atopic subjects, 30 with positive, and 30 with negative food allergy results. we reviewed charts for history of heartburn and acid regurgitation or the diagnosis of gerd, laryngopharyngeal reflux (lpr) or peptic ulcer disease (pud). population prevalence (19.8%; ci 17.7-21.9) of acid reflux was estimated from a historical comparison that studied adults in a similar geographical location. results: in the food allergy positive group, 26.7% of the subjects (95% ci 11.8-41.6) had either a history of heartburn and/or a diagnosis of gerd, lpr or pud. in the food allergy negative group, 40% (95% ci 25.1-54) had acid related disorders. in the total study group of atopic subjects, the prevalence of heartburn, gerd, lpr or pud was 33.34% ). on chi square analysis, the prevalence of acid reflux disorders was significantly higher in the total study group when compared with population prevalence (p=0.013). the prevalence of acid reflux disorders between the food allergy study group and in the population shows no significant difference (p=0.41). the prevalence of acid reflux disorders was significantly higher in the food allergy negative group when compared with the prevalence in the population (p=0.003). there was no significant difference in the prevalence of acid reflux disorders between the two study groups with and without food allergy (p = 0.27). conclusions: the prevalence of acid reflux disorders was higher in people with food allergy when compared to the population, but missed statistical significance. patients without food allergy had a significantly higher prevalence of acid reflux disorders compared to the population. atopic individuals have a statistically significant higher prevalence of acid reflux disorders, but there is no statistically significant difference between atopics with and without food allergy. abstract background: analysis of dispensing patterns of injectable epinephrine offers a method to study the characteristics of school children with allergic or anaphylactic reactions. objective: to analyze the demographics of children prescribed injectable epinephrine in massachusetts school districts with diverse racial and ethnic enrollment patterns. methods: school nurses in 44 schools (grades pk-12) enrolling 21, 870 students recorded the characteristics of students prescribed injectable epinephrine including the number and racial mix of students in each school, student age, grade level, race, sex, and allergic disorder requiring epinephrine. surveyed school districts were two predominately white (98%) suburban districts enrolling 5855 students and one urban area with a minority population of 60% enrolling 16, 020 students. the use of epinephrine for peanut allergy was analyzed in detail. results: a total of 181 of 21, 875 (0.83%) students in three school systems were dispensed injectable epinephrine. males outnumbered females (110 to 71). whites outnumbered non-whites 153 to 28. two thirds (n=116) of children dispensed epinephrine had peanut allergy. the second most common allergy was stinging insect allergy (n=34). the rate of dispensed epinephrine for peanut allergy was lowest in the urban school district (0.30%) versus the two suburban districts, 1.1% and 1.26% respectively. whites with peanut allergy outnumbered nonwhites 99 to 19. males outnumbered females 69 to 49. the lowest rate of prescribed injectable epinephrine for peanut allergy in all three school districts was found in 9612 non-white school children-0.17%. eighty-nine of 118 (75%) school children with peanut allergy were enrolled in grades pk through 5. there were twice as many white versus non-white (32 to 16) school children in the urban system with prescribed injectable epinephrine for peanut allergy. only eight (.11%) of 7209 hispanic and asian students in the were dispensed injectable epinephrine for peanut allergy. conclusions: this is the first study to suggest that there may be a racial disparity in the prevalence of childhood peanut allergy. one possible explanation for this disparity is that varied feeding practices in minority infants and children may induce a state of tolerance and lead to a lower incidence of peanut allergy. f.i. hsu * , h.j. burstein, m.c. castells, boston, ma. introduction: trastuzumab is a humanized igg1 kappa monoclonal antibody specific for human epidermal growth factor receptor 2 protein, her2, used in the treatment of her2/neu positive breast cancer. we present the first report of desensitization to trastuzumab in a patient with an ige-mediated reaction to trastuzumab and documented igg-anti-igg1 human antibodies. meth-ods: a 37 year old woman with metastatic invasive ductal breast cancer (er/pr positive, her-2 strongly positive), unresponsive to conventional therapy, presented with an anaphylactic reaction to trastuzumab and was evaluated for rapid desensitization by skin testing and serum specific igg antibodies. the patient had previously received trastuzumab. results: the anaphylactic reaction to trastuzumab consisted of diffuse erythema, urticaria, respiratory distress and laryngeal edema. premedication with steroids, h1 and h2 blockade, and slow infusion did not prevent a subsequent reaction. skin prick testing with trastuzumab (21mg/ml) was positive with 2 negative controls, confirming an ige-mediated mechanism. igg anti-human igg1 antibodies (haha) to trastuzumab were confirmed by elisa. the patient was desensitized to trastuzumab 2 mg/kg with an intravenous protocol that started at 0.5 mcg/h, and increased in rate every 15 minutes until a final rate of 30 mg/h, for a total infusion time of 6 to 8 hours. premedication included diphenhydramine, prednisone, famotidine, and montelukast. desensitization was confirmed by negative skin prick and intradermal tests (2 mg/ml and 21 mg/ml), with positive histamine control. the patient tolerated a total of 10 weekly doses of trastuzumab 2 mg/kg. intradermal skin testing prior to her 5th and 10th courses showed reactivity, indicating resensitization. serum levels of tratuzumab were undetectable at 1 week after desensitization. conclusion: allergic reactions to trastuzumab are rare but can include anaphylaxis. in patients with documented haha and/or ige-mediated reactions to trastuzumab and clinical symptoms of type i/mast cell mediator-related symptoms, desensitization can allow continued administration of this treatment by providing tolerance. the clinical effectiveness of desensitizations in patients with ige and haha antibodies remains to be defined. in contrast to reports in the medical literature, details of the medical aspects were limited. there were 2 attacks on infants bringing the total to 3 reported infant attacks to date. two of the 3 infants suffered long term morbidity and 1 died. like those reported in the medical literature, the majority of adults were in long term care facilities, although 2 were in hospitals. overall, 6 of the 20 individuals stung died within one week of stings. no significant medical consequences of stings were reported in 6 of 10 of the newspaper reports as opposed to 2 of 10 reports in the medical literature. conclusion: our data suggest that increasing numbers of fire ant attacks are occurring in medical facilities, where chronically ill, frequently immobile patients come in contact with foraging ants. unattended infants in private homes in fire ant endemic areas also appear at risk. the factors that determine why individuals are stung and the severity of injury after attacks remain uncertain. the presence of fire ants inside health care facilities and homes is a harbinger for fire ant attacks of disabled or infant occupants. we hypothesize that morbidity is determined by the number of stings, the condition of the patient and the type of treatment administered. purpose: to determine if gender confers a risk for positive penicillin (pcn) skin test. method: rates of positive pcn skin tests, according to gender, were determined in patients with a history of pcn allergy undergoing an allergy pre-operative evaluation from june 2002 to june 2004. during this period, 19, 429 patients were seen in the pre-operative evaluation clinic in which 1921 had a history of pcn allergy and comprise our study population. a univariate logistic regression analysis was employed to calculate the odds ratio (or) and the 95% confidence interval (ci) for gender differences in the rates of positive pcn skin test and a multivariate logistic regression analysis was used to adjust for age and history of multiple drug allergies. p-value of 0.05 or less was considered statistically significant. results: of the 1921 patients, 1759 underwent skin testing for pcn, 157 patients did not, and 5 charts were not available for review. the mean age of the study group was 60 years. sixtyfour (3.6%) patients had a positive skin test to pcn. of these, 53 (83%) were females and 11 (17%) were males (or 3.6, 95% ci 1.9-7.2, p = 0.0001). of those with a negative/equivocal pcn skin test, 960 (57%) were females versus 720 (43%) males. in a multivariate logistic regression analysis adjusted for age and history of multiple drug allergies, female gender again was more likely to have a positive pcn skin test (or 4.7, 95% ci 2.3-10.5 p < 0.0001). 157 patients did not undergo pcn skin testing, 62 (40%) were male and 95 (60%) were female. conclusion: this is the first report showing that a greater risk for a positive skin test to pcn exists in association with female gender. age and a history of multiple drug allergies are unlikely to be confounding factors to the observed gender risk. further studies are needed to identify other possible confounding factors and/or mechanisms that can explain this risk. a. fiocchi 1* , p.a. restani 1 , s. cucchiara 2 , g. lombardi 3 , g. magazzu' 4 , g.l. marseglia 5 , k. pittschieler 6 , s. tripodi 2 , r. troncone 7 , a. vierucci 8 , 1. milan, italy; 2. roma, italy; 3. pescara, italy; 4. messina, italy; 5. pavia, italy; 6. bolzano, italy; 7. napoli, italy; 8. firenze, italy. background: cow milk substitutes for children allergic to cow milk proteins (cmp) include soy-based formula and cow s milk hydrolysates. neither can rule out a sensitisation risk. objective: prospective assessment of clinical tolerance to a rice-based hydrolysate formula by children allergic to cow milk proteins (cmp) who consume rice openly. patients and methods: ninety-seven children aged 4 to 136 months with immediate reactions to cow milk confirmed during dbpcfc were assessed for clinical tolerance to cow milk by spt with whole milk, -lactalbumin (ala), -lactoglobulin (blg), -and -casein ( -cas, -cas) (sigma chemical, st. louis, mo). whole milk, ala, blg and cas specific ige determinations were performed using cap test (pharmacia, uppsala, sweden) . similarly, sensitisation to rice and hrf were investigated by spt and cap test. patients sera were investigated by immunoblotting for cmp, rice and hrf (heinz-plada, milan, italy). dbpcfc was carried our with 24 g rhf powder masked in neocate tm (equivalent to 180 ml reconstituted formula). results: spt: all patients were positive to cow s milk and/or cmp fractions (>3 mm wheal diameter). ige determinations gave positive results with cow milk and/or cmp fractions (72/84 patients tested), rice (16/81) and with hrf (2/85). immunoblots (n=87) were positive for -cas (n=46), -cas (n=34), ala (n=39), blg (n=34) and bovine serum albumin (n=52). similarly, although patients sera largely recognized rice (78/87), only one weakly reacted with rhf. challenge with rhf was negative in all cases. conclusions: we conclude that, despite their frequent sensitisation to rice, children with cma tolerate both rice and rhf clinically. hydrolyzed rice formulas may thus represent an alternative protein source for children with cma. r.c. cartwright * , w.k. dolen, augusta, ga. introduction: conventional treatment of human seminal fluid allergy includes abstinence, barrier protection, or subcutaneous immunotherapy with fractionated seminal fluid. treatment using local intravaginal desensitization with unfractionated seminal fluid has recently been described, but experience with this desensitization method is still limited and little has been reported as to its long-term results. methods: a 30-year-old woman with a history of allergic rhinitis and asthma experienced anaphylaxis following her first unprotected intercourse since the birth of her first child. ige-mediated sensitization was demonstrated through the use of specific ige testing (pharmacia cap system) and skin prick testing using undiluted seminal fluid obtained from the patient's husband. after approval from the human assurance committee, the patient underwent rush intravaginal desensitization with steadily increasing concentrations of seminal fluid, beginning with a 1:10, 000 v/v concen-tration. results: her specific ige level to human seminal fluid was 0.26 ku/l. skin prick testing was positive with a wheal of 10 mm diameter with pseudopods and a flare of 25 mm diameter. desensitization was successful and the patient tolerated local application of whole semen without significant reaction. following desensitization, the patient reported that she and her husband engaged in unprotected intercourse without local or systemic symptoms. over the last year since the desensitization, she has not experienced further anaphylaxis, but she has developed local symptoms if her exposure to seminal fluid was delayed past 5 days. the longest time period between exposures has been 2 weeks. she became pregnant 5 months ago and has not had any pregnancy complications. conclusions: local intravaginal desensitization was a safe and effective treatment for human seminal fluid allergy in this patient. a delay in seminal fluid exposure greater than 5 days was associated with the return of symptoms emphasizing the need for frequent seminal fluid exposure to maintain desensitization. l. terracciano, t. sarratud, a. fiocchi * , p. restani, s. guerci, milan, italy. background kiwifruit allergy in children has been seldom reported and the reactions observed are usually mild. anaphylaxis has not been described. we document the case of an infant who developed anaphylacic symptoms within minutes of his mother eating two kiwifruits and initiating breastfeeding. case history in his fourth month, the boy was admitted into an emergency department with dysphonia, breathing difficulties, generalised urticaria and angioedema of the lips and face. this episode was treated as an anaphylactic reaction with epinephrine, chlorpheniramine and hydrocortisone sodium succinate administered via a percutaneous catheter and symptomatic control was achieved within 30 minutes. after 15 days a second anaphylactic episode of similar severity occurred under the same circumstances. neither episode required critical care. the boy presented two months later for clinical evaluation in our paediatric allergy unit. skin prick tests (spt) were positive only with egg white and yolk while negative to cow s milk (and protein fractions), beef, chicken, pork, codfish, rice, wheat, soybean, maize, potato, carrot, tomato, bean, pea, celery, peanut, dermatophagoides pteronyssinus and d. farinae, grass and banana. spt with kiwifruit was specifically ordered because exquisite contact was suspected, and induced a positive wheal (>3 mm diameter). positive specific ige determinations (cap-feia from pharmacia, sweden) with kiwifruit (0.71 ku/l), cat dander (1.97 ku/l) and egg white (1.97 ku/l) were returned but determinations with other inhalant and food allergens were all below the cut-off point of 0.35 ku/l and total ige levels were 155 ku/l. strict avoidance of kiwifruit by the breastfeeding mother proved effective and nothing untoward happened in the intervening year. currently aged 2.5 years, the boy is free from food-related symptoms. prick-byprick tests with native allergens and spt carried out with commercial extracts of allergens associated in the literature with kiwifruit allergy were all negative. comment there are no reports of immediate reactions to kiwifruit under two years. in this case, severe reactions via breastmilk in an infant monosensitised to kiwifruit indicate that nursing may represent a hidden source of exposure. in older children monosensitisation without prior sensitisation to pollen has been described as the major risk associated with severity of symptoms. m. sikora * , j.w. sleasman, n. tangsinmankong, st. petersburg, fl. introduction: treacher-collins syndrome (tcs) is an autosomal dominant disorder with an abnormality of craniofacial development during early embryogenesis. there is a known relationship between new bone formation and development of lymphocytes and cytokines. the association of tcs and humoral immunodeficiency has not yet been reported. we present a novel case of a 12 year-old caucasian female patient with tcs and common variable immunodeficiency. methods: our patient presented with midface hypoplasia, micrognathia, microtia, conductive hearing loss and cleft palate with a history of recurrent upper and lower respiratory infections. patient had a 10-year history of chronic sinusitis, recurrent otitis media and pneumoniae which resulted in bronchiectasis. haemophilus influenza and staphylococcus aureus were persistently isolated from bronchial fluids. laboratory work-up for immunodeficiency was initiated. results: immunoglobulin analysis revealed low igg 450 (528-2190 mg/dl); low iga 42 (44-441mg/dl); igm 65 (48-226mg/dl); igg 1 354 (165-1440 mg/dl); low igg 2 59 (71-460 mg/dl); igg 3 85 (28-125 mg/dl); and low igg 4 <8.1 (2-143 mg/dl). patient had no detectable response to protein-derived vaccines (diphtheria and tetanus) and to polysaccharide-derived vaccines (pneumococcal) at baseline and at 4-6 weeks after immunization. t and b lymphocyte enumeration, ch50, ah50, and mannose-binding lectin were all within normal limits. laboratory findings were consistent with the diagnosis of common variable immunodeficiency. patient began receiving monthly doses of 550 mg/kg/dose of intravenous immunoglobulin resulting in clinical improvement. our patient no longer requires antibiotic therapy for her respiratory infections; pulmonary function has improved and bronchiectasis resolved 6 months after initiation of ivig. conclusion: our finding shows that tcs can be associated with common variable immunodeficiency which suggests a link between skeletal dysplasia and immunodeficiency. a.s. hartel * , j.w. sleasman, n. tangsinmankong, st. petersburg, fl. introduction: streptococcus pneumoniae is the most common cause of invasive bacterial infection in humans. however, incidence of s. pneumoniae infections in healthy adolescents is low (5/100, 000 per year). mannose-binding lectin (mbl) is a c-type lectin which plays a central role in the innate immune response by activating the classical complement pathway and acting as an opsonin by binding c1q receptors. several studies have demonstrated an association between invasive bacterial infections, including s. pneumoniae and a homozygous mutation of the mbl gene. however, this association has not previously been described in patients with the heterozygous mutation. methods: a 13-year-old caucasian female presented with a second episode of meningitis over a 4-year span. streptococcus pneumoniae was isolated from peripheral blood cultures on both occasions. lumbar puncture revealed wbc 22, 600; 6% bands; 83% pmns; glucose 3 mg/dl, protein 329 mg/dl; these results consistent with bacterial meningitis. extensive evaluation for predisposing causes revealed benign arnold-chiari type1 malformation. further immunological evaluation was initiated. results: evaluation revealed igg 867 mg/dl (normal 528-2190); iga 67 (44-441); igm 181 (48-226), and normal igg subclasses. antibody responses to diphtheria and tetanus vaccines were adequate. antibodies response to pneumococcal vaccine given 4 years prior were protective to all 10 common serotype tests (>1.4 mcg/ml). lymphocyte subset analysis was within normal range. howell-jolly bodies were not detected from peripheral blood smear. evaluation for secondary immunodeficiency was negative, including hiv antibody by elisa. complement analysis showed ch50 88 u/ml (65-95); ah50 71% (66-129%), and markedly low mbl of 74 ng/ml (>1000). genetic analysis of her mbl revealed heterozygous mutation in codon 54 and mutations in two promoter regions (homozygous mutation on h/l variants and heterozygous mutation on p/q variants). conclusion: heterozygous mutation of mbl codon when associated with mutation of promoter regions can result in severe impairment of mbl protein production, and may lead to increased susceptibility to invasive bacterial infections and meningitis. rationale: patients with similar symptoms may have allergic, non-allergic, or mixed rhinitis triggers and can differentially respond to distinct medications. we assessed st in our clinic patient population to characterize factors that influence appropriate diagnosis and treatment of rhinitis type. methods: we used a validated commercial questionnaire and chart review of 230 patients seen in an academic allergy clinic, to assess the number of allergic vs. irritant st and demographic and historical factors (including pharmacotherapy) associated with differences in symptoms. results: the population (n=189: 138 female, 51 male) consisted of individuals with a mixed rhinitis history. women had higher st scores than men, including: total scores [10.44+/-0.51 vs. 7.20+/-0.65, p=0.0001 (unpaired t-test)]: allergen scores (4.56+/-0.30 vs. 3.51+/-0.37, p=0.0055); and irritant scores (5.58+/-0.29 vs. 3.69+/-0.41, p=0.0003). further, patients treated with both azelastine (az) and intranasal steroids (ins) had lower st scores than patients treated with either alone (total st: az 10.06+/-0.94, ins 10.3+/-0.97, both 8.0+/-0.88, p=0.0348; allergen st: az 4.69+/-0.54, ins 5.05+/-0.37, both 3.11+/-0.50, p=0.0416; irritant st: no significant difference). conclusions: female patients have significantly higher symptom scores; total, allergic and irritant. rhinitis monotherapy (ins or az) is minimally effective in reducing st but is more effective when used in combination. these data demonstrate rhinitis population heterogeneity and address differential responsiveness to similar medications. increased response to combined drug therapy support the heterogeneous pathophysiology of mixed rhinitis features and may relate to a combination of multiple aeroallergen and air pollution exposure in the houston area. introduction: interaction between eye & nose warrants attention with regard to the propagation and treatment of allergic reactions. the role of topical therapy for rhinitis and conjunctivitis is becoming more widely considered and questions of therapeutic route have been raised. purpose: to elucidate the anatomic and pharmacokinetic interactions of conjunctival & nasal mucosa leading to more rational selection of routes of medication administration. methods:our studies have investigated the effect of ocularly instilled allergen inducing signs and symptoms of rhinoconjunctivitis. 1 study compared effects of allergen administered via conjunctival allergen challenge (cac) or nasal allergen challenge (nac) and analyzed tear & nasal secretions for ecp & tryptase. studies have also used the ability of the cac model to induce rhinitis signs & symptoms to evaluate the relative effects of medication routes: 1) ocular v. nasal spray v. systemic; 2) ocular + nasal spray v. systemic + nasal spray; 3) ocular v. placebo. results: the nac/cac study revealed that, following cac (n=34), significant ocular and nasal signs & symptoms were noted; following nac (n=39), only nasal symptoms were clinically significant. nasal symptom scores between cac & nac differed significantly at 1 timepoint, representing lag in allergen & mediator movement from eye to nose. measurable levels of ecp & tryptase were found in tears and nasal secretions for cac, but only in nasal secretions (with exception of 1 subject) for nac. in cac studies: 1) ocular therapy exhibited greater efficacy in ocular itching relief (n=73;p<0.05) and was not significantly different from nasal or systemic therapy in nasal symptom relief. 2) eyedrop+nasal spray combination exhibited significantly greater prevention of overall rhinoconjunctivitis signs and symptoms than nasal+systemic therapy (n=80). 3) ocular therapy offered greater protection from nasal signs & symptoms compared to placebo (n=32;p<0.05). conclusion: nac and cac results support the unidirectional flow from eye to nose, the ability of cac to yield nasal symptoms, and the efficacy of topical therapy. tearing, due to inflammation of the inferior turbinate would be the only ocular symptom resulting from nasal challenge. these results offer insight to the nature of the connection between ocular and nasal mucosa and the efficacy of varied medication administration routes for management of rhinoconjunctivitis symptoms. introduction: asthma disease management programs frequently target high utilizers because they are responsible for a large amount of asthma costs. once identified, such "frequent fliers" usually are offered interventions including case management. programs using this approach usually demonstrate reductions in utilization. though the interventions may cause this decline, it could also be due to regression to the mean (rtm) which is a tendency for outliers to become more like the mean over time. in this study we measured rtm in a medicaid hmo asthma population to determine whether targeted case management is effective independent of this phenomenon. we hypothesized that directed case management provides additional utilization reduction beyond rtm. methods: a weighted asthma utilization score was determined quarterly for members of an hmo with asthma from 2000 to 2004. rtm was measured by determining how many patients were persistent high utilizers quarterly for 1 year after baseline. to determine the effect of utilization-directed case management, the number of frequent fliers at baseline for each quarter also was determined. results: a total of 296 asthma frequent fliers were identified on january 1, 2001. by september 1, 2001 only 15 of these individuals continued to be frequent fliers. similar decreases in utilization were seen for frequent fliers identified at the beginning of each quarter of 2001. the mean decrease in the number of frequent fliers is shown in the table. this represents a substantial regression to the mean. the total number of high utilizers at baseline decreased by 27% after implementation of utilization-directed case management in 2002 independent of regression to the mean. this also represented a decrease from 1.6% of health plan members with asthma to 0.6% by the start of 2004 suggesting that the decline is not a result of diminishing health plan membership. the benefit appears to be the result of early intervention with members before they become frequent fliers. conclusions: utilization-directed case management can reduce overall asthma utilization by preventing members from becoming high utilizers at an early stage. programs that claim to intervene with plan members who already are high utilizers are likely to be taking advantage of regression to the mean. studies indicate a high incidence of asthma(as) among school-aged children. with the prevalence increasing, significant numbers remain unidentified. they experience morbidity, including school absenteeism, which may be preventable, in part, by adherence to national asthma guidelines. we implemented a pilot study to detect as, as control and initiation of appropriate health care among 7th grade students in a suburban population. the study was coordinated with the middle school staff and the local county health department. the screen parameters included: student questionnaires, peak flows, exercise with peak flow assessment (frast)2, and spirometry as indicated. environmental tobacco smoke (ets) was recorded. results: 196 students screened. 131 no as history and negative screen (d). 31 as history and negative screen (e). 5 as history and positive screen (a). 23 no as history with suggestive written screen and negative screen on exercise (b). 6 no as history and positive screen (c). ets in groups a, b, c: 40%, 32%, 60%. ets in groups d, e: 23%, 16%. individual student results were mailed home with medical follow-up recommended for a positive screen. parents of the 34 children who failed the questionnaire or exercise screen (groups a, b, c) were phoned at a 6-12 wk interval. no student in-group a or b had medical follow-up. 2(33%) students in group c had medical follow-up. conclusion: this as screen of 7th grade students identified 6(3%) as having significant, undetected as. 5(14%) with known as had uncontrolled as at the time of screen. 23(11.7%) students with strong suspicion of as based on questionnaire had an acceptable exercise screen. only 2 of the 34 children with a screen suggestive of as or uncontrolled as had medical follow-up. ets was increased in the homes of students with a positive screen and remains a serious health issue. school screening for as has merit, but mail and phone follow-up was insufficient to intervene or initiate acceptable as treatment. references 1. redline s. et. al. development and validation of school-based asthma and allergy screening instruments for parents and students. ann allergy asthma immunol. 2003; 90: 516-528 2. tsanakas.j.n., et al. free running asthma screening test. arch diseases in childhood.1988; 63:261-265 3. american college of allergy, asthma, and clinical immunology screening test ages (8-14) acaai.org. introduction. some laboratory evidence suggests that desloratadine is effective in inhibiting of inflammatory mediators, which play an important role not only in allergic but also in virally induced inflammation. the purpose of this study was to assess its efficacy in acute bronchiolitis developed in young children suffering from concomitant atopic dermatitis (ad). meth-ods. participants were 34 young boys and gilrs aged 18-36 who suffered from acute bronchiolitis as established by wheezing, rhinitis and fever. all patients also had concomitant ad as established by hanifin and rafka criteria. patients were allocated to receive syrup formulation of desloratadine (erius, schering plough) 1, 25 mg/day for 5 days (active group n=17) or no desloratadine treatment (control group n=17) using quota allocation system. we calculated physical global symptom score (combination of cough, wheezing, chest retractions, nasal flaring, blocked nose, runny nose, sore throat -0-5 scale) and respiratory distress assessment instrument (rdai) to examine the patient at baseline and on day 5th after therapy was initiated, day of normalization of body temperature, respiratory rate, heart beat rate, and use of albuterol. results. both group of patients were comparable on age, gender, family history of asthma, time of onset of the acute respiratory illness, extent of medication taken prior to entering the study, global symptom score and rdai index. all children were evenly treated with oxygen, oral theophylline (10 mg/kg/day) and adequately rehydrated. on day 5th global symptom score of patients receiving desloratadine was 2, 51â±0, 51 vs 3, 42â±0, 62 in control group (p=0.035). on day 5th it was also significant difference between active and control groups on rdai index (4, 44â±1, 24 vs 7, 23â±2, 30; p=0.046) especially for significant drop in wheezing score in active group. day when respiratory/heart rate normalized, temperature returned to normal, use of albuterol did not differ significantly in these groups. conclusions. our preliminary study is the first to show that desloratadine is an effective agent in viral lower respiratory tract infections of young children suffering from ad. it encourages further gcp trials to explore action of desloratadine in infantile bronchiolitis and concomitant ad. h.j. su * , w.t. lin, p.j. tsai, c.y. huang, p.c. wu, tainan, taiwan. increasing prevalence of childhood asthma has been observed across the world, and found to be associated with, partly, indoor pollution, including bioaerosol exposure. meanwhile, adequate ventilation is shown to be effective in diluting most indoor air pollutants, while only limited data are available addressing directly how the ventilation rate is implicated with childhood respiratory symptoms. this study aimed to examine the concentration distribution of selected indoor air pollutants, including bioaerosols, in domestic environment, and further to assess the effects of ventilation rate, characterized by a co2 trace gas concentration decay method, on concentration variations of the above-mentioned air pollutants. study subjects were chosen from a prior city-wide questionnaire survey based on positive response to inquiry for physician-diagnosed asthmatic status and wheezing symptoms in the past 12 months. environmental assessments, including ventilation and air quality measurements, were conducted twice, 1 in early winter and the other on early summer. respiratory health diary and pefr (peak expiratory flow rate) were recorded for 1 week concurrent with the sampling activity. increasing ventilation rate is statistically associated with decreasing indoor concentrations of co2 and tvocs. after adjustment for sex, age, and selected housing characteristics, the or between tvocs concentrations and reporting cough of study children is 9.47, and 12.84 between co2 and nasal congestion. the or between indoor bacterial concentration and the morning pefr less 80% is 10.98 in the similar multivariate logistic regression for data collected from winter study. in summer, the only significant relationship is between ventilation rate and the morning pefr less 80% of study children, or= 0.17, in a multivariate logistic regression. this study has identified less reporting of childhood respiratory symptoms, especially for coughing and the morning pefr less 80% are associated with increasing ventilation rate, and higher levels of indoor air pollution appear to be present with greater frequency of the above symptoms. this study suggest quantitatively that proper management of ventilation efficiency may be beneficial for the control of childhood respiratory illnesses. *adjusted for: sex, age, environmental tobacco exposure, use of incense and air conditioner **:p<0.05 ns:no statistical significance with whole model test background: moderate-to-severe allergic asthma can have a substantial impact on a patient's asthma-related quality of life (arql). in addition to asthma symptoms, allergic rhinitis, rhinosinusitis and other related comorbidities are often apparent in patients with more severe disease making it difficult to treat. despite guideline-consistent care, many patients still experience variability in asthma control signaling an unmet need within this population. omalizumab (xolairâ®) has recently demonstrated clinical efficacy and safety in treating asthma. objective: the aim of this paper is to summarize the arql outcomes associated with omalizumab therapy in moderate-to-severe allergic asthma. methods: we performed a systematic review of arql data from the clinical study reports and published clinical trials on omalizumab. arql was measured by the juniper-asthma quality of life questionnaire (aqlq). results: statistically significant results for arql endpoints consistently favored omalizumab over placebo. the magnitude of the changes in arql were consistently aligned with clinical endpoints. moderate to large effect sizes in the omalizumab groups were maintained throughout the 28-week clinical trial program and during the 24-week double-blind extension phase. however, the placebo groups also experienced within-group improvements and moderate effect sizes. a meta-analysis indicated a 1.6 to 2.0 fold increase in large (>1.0 point) improvements in overall aqlq scores in the omalizumab-treated group compared with placebo during the stabilization and steroid-reduction phases of the clinical trials. conclusions: the consistently positive impact of omalizumab on arql outcomes during the clinical trial program provides evidence of its value as an adjunct therapy in patients with moderate-to-severe allergic asthma. significant differences and large effect sizes were observed despite the fact that the control group received active, guideline-consistent treatment producing a substantial placebo effect. improvements were observed in overall, symptom, activity, emotional and environmental dimensions of the aqlq indicating that omalizumab produced benefits in arql in patients with moderate-to-severe allergic asthma. background: omalizumab, a monoclonal anti-ige antibody, significantly improves asthma-related quality of life (arql) for patients with moderatesevere allergic asthma who express symptoms despite moderate-high inhaled corticosteroids (ics) doses. mean scores can mask underlying variability in arql outcomes. this investigation examined variability in outcomes to elucidate the specific impact of omalizumab treatment. methods: aqlq data (n=948) from two randomized, double-blind, placebo-controlled clinical trials were pooled to assess underlying variability in the mean scores and to identify key drivers of arql treatment-effect differences (juniper-asthma quality of life questionnaire (aqlq)) between omalizumab and placebo (active control) patients. results: correlations between aqlq and other clinical outcomes were low to moderate at best (r=0.14 to r=0.60). aqlq assessment captures patient benefit that supplements clinical outcome measures. across all component items of the aqlq patients receiving omalizumab improved more than patients receiving placebo (active control) (p<0.05). omalizumab patients reported the greatest improvement for reducing waking with symptoms in the morning (symptoms domain: 2.06 vs. 1.50; p<0.001), limitations in all activities done (activities domain: 1.18 vs. 0.68; p<0.001), the fear of not having medication available (emotions domain: 0.99 vs. 0.60; p<0.01), and symptoms from being exposed to dust (environment domain: 1.31 vs. 0.88; p<0.001), compared to placebo (active control) patients. conclusion: arql assessment provides complementary and non-overlapping information on clinical benefit that is distinct from other clinical outcome measures. examination of underlying variability in aqlq mean scores and item-level response extend previously published results on omalizumab treatment effect by showing that aggregate arql improvements for omalizumab patients are strongly influenced by symptom and activity improvement. a. brimer 1 , k. malhi, c. adams, c. dinakar, kansas city, mo. introduction: the desire to belong to a group is a very powerful motivator and is exceptionally strong in the adolescent population. asthma is a disease that makes people feel different. this perception may impinge on patient adherence with medication regimens. we hypothesize that belonging to a club where every member has asthma may encourage identification of the adolescent asthmatic with their peers, and mitigate the perception of being different. objectives: (1) to investigate the factors that make the asthmatic adolescent feel different (2) to explore the hypothesis that group activities, such as an asthma club, would help adolescent asthmatics feel less different. methods: as part of an ongoing survey, children with asthma between the ages of 8-18 years were offered an anonymous questionnaire in the primary care and adolescent clinics at our hospital. the questionnaire included both multiple-choice and open-ended questions designed to explore the feelings of the respondents. the responses were numerically tallied and reported as percentages. results: at the present time, 31 surveys out of a proposed 100 have been completed. one third of the youth with asthma answering the survey had negative feelings regarding their asthma. nearly forty percent of the respondents reported that their diagnosis made them feel different from their healthy peers. forty-five percent responded that they have felt restricted or excluded from school activities, athletics, and clubs due to their asthma. over one-third of them feel uncomfortable taking their inhaler in front of their friends. most respondents (93.6%) indicated that they enjoy group activities. the majority of respondents (89.7%) rated playing sports as their favorite group activity. conclusion: almost forty percent of asthmatic youth surveyed indicated that having asthma made them feel different and resulted in restriction/exclusion from school activities, athletics, and clubs. an overwhelming majority expressed a preference for participation in group activities, particularly recreational sports. this social preference towards group activities may be incorporated into an intervention, such as an asthma club, to help asthmatic youth adjust to the disease and its treatment regimen. introduction: airway hyperresponsiveness (ahr) is an exaggerated narrowing of the airways in response to stimuli, such as allergens, histamine, and cold air. ahr is a characteristic feature of asthma linked to chronic airway inflammation. phosphodiesterase 4 (pde4) is an enzyme found in key inflammatory cells involved in the pathophysiology of asthma. inhibitors of pde4 prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, once-daily pde4 inhibitor, which has shown anti-inflammatory activity in vitro and in vivo. this study examined the effect of a single dose of roflumilast on changes in ahr following allergen-induced asthmatic reactions in patients with mild asthma. methods: this double-blind, randomized, crossover study consisted of 2 treatment periods separated by a 2-to 5-week washout period. a total of 13 patients (forced expiratory volume in one second [fev 1 ] 70% predicted) who were hyperresponsive to histamine (provocative concentration causing a 20% drop in fev 1 [pc 20 fev 1 ] 16 mg/ml) were randomized to receive a single dose of oral roflumilast 1000î¼g or placebo on day 1 of each treatment period followed by an allergen challenge 60 min after medication. histamine provocation was performed before and 24 h after intake of study drug. results: there was no change in fev 1 60 min after roflumilast administration, suggesting the absence of direct or acute bronchodilation. allergen challenge elicited early and late asthmatic reactions that were attenuated by a single dose of roflumilast 1000î¼g. the histamine pc 20 fev 1 in patients treated with roflumilast decreased to a lesser extent than in those patients treated with placebo. the change in pc 20 fev 1 from baseline after challenge was 2.51 â± 2.95 mg/ml (mean â± sd) with placebo and 1.23 â± 2.75 mg/ml with roflumilast. the magnitude of the change in pc 20 fev 1 was statistically significantly different between the placebo and roflumilast treatment groups (p=0.002). thus, roflumilast decreased the development of ahr by approximately 1.1 doubling-dilutions. conclusions: a single dose of oral roflumilast 1000î¼g attenuated allergen-induced ahr. these data provide further evidence that roflumilast may provide anti-inflammatory activity in vivo and may be an effective treatment for patients with asthma. introduction for the past 7 years we have been tracking pediatric asthma admissions and evaluations at the huntington memorial hospital. during the fall and winter months (oct-march) from 1996 to 2002, we noted a 1.6 x increase in the number of asthma admissions and evaluations compared with the spring and summer (april -september; 4, 993 patient encoun-annals of allergy, asthma & immunology ters in 7 years). we have analyzed potential causes for this increase. diesel particulate was estimated in 1998-9, and a 2.5-fold increase in the fall versus spring was found in burbank, a city adjacent to pasadena. pollutant particles from the combustion of fossil fuels may act as immuno-adjuvants to allergen exposure as has been demonstrated experimentally in mice and in human nasal exposure studies. methods computerized analysis of asthma admissions and evaluations were calculated according to established codes for acute and chronic reactive airways disease. infectious diseases in patients were analyzed by standard asthma questionnaire. also, pollen and mold counts, as well as diesel particulates and meteorological conditions were studied. results similar numbers of viral infections occurred in the fall of 2001 and spring of 2002. among the most prevalent pollens and molds are chinese elm pollen (sept -oct) weed pollens (july -dec), and a newly recognized aureobasidium mold (nov -march). pollen grains can fragment and release respirablesized debris that are loaded with allergens (taylor et al 2002 (taylor et al , 2004 . the conidia of aureobasidium are also of respirable size and were identified by sequence analysis. fine particulate air pollution (pm2.5), containing diesel particles, is increased in the fall and winter in southern california. con-clusions the incidence of asthma outbreaks may vary with air pollution levels in the immediate environment. pollen fragments and particles from fossil fuel combustion can deposit in similar regions of the lower airways. rainfall increases in the fall and winter and this coincides with increased molds and aureobasidium. aureobasidium was recently discovered in high concentrations in pasadena, is known to be allergenic and could be a factor in increased asthma. a mixture of pollens and molds and fine combustion particles may be relevant to this increased incidence of asthma in the fall and winter months in pasadena. background we have previously noted that tree and ragweed pollen grains can be airborne for most if not all daily periods during their respective seasons (aaaai, 2003) , (acaai, 2003) . because grass levels are lower than those of trees & weeds (aaaai / nab), we chose a longer daily interval to examine (six hours) that we had for trees (one hour) and ragweed (three hours). methods twenty-four hour microscope slide samples were collected using a burkard 7 day air sampler from 05/01/04 to 06/30/04. the slides were examined @ 100 x along a single longitudinal traverse. the presence or absence of grass pollen was noted per six-hour segment (six successive fields). results the presence of airborne grass pollen during the 4 daily segments was sparse the first two weeks of may. it gradually increased during the next 10 days, and then was detected during most of the daily segments through to the end of june. # ammophila arenaria (aa) is a highly resilient grass with habitat in europe, north america, australia, new zealand, south africa and the mediterranean, used predominantly for dune stabilization. patients with atopic diseases including allergic reactions to pollen are transferred to coastal regions in order to minimize pollen exposure. in spite of the presence of aa pollens, patients experience allergy relief at the coast. our study examines the contradiction between the presence of strong grass allergens and the absence of allergic symptoms in atopic patients, and whether allergic cross-reactions among different types of grass pollen include aa pollen. 9 adult patients presenting symptoms of grass pollen allergy underwent prick testing and rast testing to grass pollen mix and extracted aa pollen. 4 subjects showed positive results in prick and rast testing to aa extracts and were subsequently tested via rhinomanometry for reactions to aa using prick test solutions. 2 subjects showed positive reactions to prick tests for aa extract and grass pollen mix, but did not show specific antibodies. all other sera showed elevated specific ige levels, with specific reactions on 8 protein bands of aa. in sera incubated with grass pollen extract, almost all specific bands previously detected with aa were now inhibited. 3 patients with high levels of total and specific ige to aa showed weak bands after inhibition, demonstrating the allergenicity of aa. comparing data from coastal and mainland weather stations in june and july from 1961 to 1990, coastal wind speeds average 6.6 to 6.9 m/s while inland wind speeds average only 3.6 to 3.7 m/s. high wind speeds likely dilute pollen concentrations. based on findings of an inverse relationship between wind speed and pollen concentration in the dispersion of ragweed pollen, we hypothesize that concentrations of aa pollens may be similarly influenced. no sensitized patient reported allergic symptoms in coastal areas inhabited by aa plants, indicating that under natural conditions, either the plant modifies its pollen allergenicity or that environmental conditions including salt aerosols, wind patterns, topography or light exposure cause the modification. as such patterns are likely to become more dramatic as the effects of global climate change transform the natural environment, these findings may also become relevant for other grass allergy types. asthma is the most common chronic inflammatory disorders of the airways with increased prevalence in the past decade. it is characterized by airway obstruction, airway inflammation and airway hyperresponsiveness (ahr) to non-specific stimuli. in this study, we examined the effect of a novel class of molecule, ocid1001, in inhibiting antigen-induced early and late allergic response (ear and lar), ahr, and airway eosinophilia in mouse and guinea pig models of asthma. pulmonary functions were measured in conscious unrestrained animals by whole-body plethysmography. animals were sensitized with ovalbumin (ova; 20mg, intraperitoneally) with 2mg alum followed by treatment with ocid1001 (120mg/kg, i.p. twice daily for 10 days). after the last dose, animals were challenged with ovalbumin. pulmonary functions were recorded for ear and lar and 24 hrs later for ahr. this was followed by bronchoalveolar lavage, blood and lung tissue collection. treatment with ocid1001 (120mg/kg) significantly attenuated lar in ova-sensitized and challenged mice or guinea pigs with no effect on ear. ahr to methacholine in mice and to histamine in guinea pigs were prevented by treatment with ocid1001. ocid1001 attenuated the rise in total number of inflammatory cells in the lung and almost ablated the rise in bal eosinophilia in the lung due to antigen sensitization and challenge. effect of ocid1001 on pulmonary functions and airway inflammation in ova-sensitized and challenged mice were comparable to that of dexamethasone in both models of asthma. these data suggest that ocid1001prevents the underlying pathophysiological changes in allergic airway inflammation and therefore could prove beneficial in the treatment of bronchial asthma. ma; 2. madison, wi; 3. portland, or; 4. milwaukee, wi; 5. normal, il; 6. denver, co; 7. los angeles, ca; 8. fremont, ca. introduction. daclizumab (zenapaxâ®), a humanized monoclonal antibody directed against the il-2 receptor chain (cd25), is approved for prevention of renal allograft rejection and is under evaluation for treatment of asthma. daclizumab inhibits activation of human t lymphocytes by blocking il2induced proliferation, and by reducing production of th2-and th1-associated cytokines. we recently reported that daclizumab improved pulmonary function in a phase ii trial of patients with moderate to severe chronic persistent asthma (jaci, 2004, 113 (2):s286). we now report additional data from this trial on the possible role of daclizumab as an anti-inflammatory agent that affects asthma outcomes. methods. 116 non-smoking asthmatics age 18-70 with baseline fev1 50-80% predicted despite use of 1200 mcg daily inhaled triamcinolone (taa) or equivalent were enrolled in a randomized, multi-center, double-blind, placebo-controlled trial. patients were randomized (3:1) to i.v. daclizumab (2 mg/kg followed by 1 mg/kg every 2 weeks) or placebo added to stable dose taa. starting at week 12, patients underwent 25% reduction of taa every 2 weeks while continuing study drug to week 20. results. patients on daclizumab demonstrated a prolonged time to exacerbation requiring systemic steroid rescue compared to placebo patients (p=0.024). exacerbation rates were reduced in the daclizumab group compared to the placebo group for the 20-week steroid-stable and steroid-taper phases (11.6% vs. 28.6%, p=0.09). patients on daclizumab demonstrated decreased peripheral eosinophil counts from baseline to week 20 (-30â±20 /mm 3 vs. placebo +60â±30 /mm 3 , p=0.004). daclizumab-treated patients with elevated baseline serum eosinophil cationic protein (secp) had a significant reduction in secp from baseline to day 56 compared to placebo patients (p<0.01). peripheral eosinophils decreased significantly in daclizumab patients who had no asthma exacerbations compared to an increase in daclizumab-treated patients with at least one exacerbation (p=0.032). conclusions. daclizumab reduces time to and frequency of exacerbation in treated asthmatics. the mechanisms of this effect remain to be fully elucidated, but initial findings suggest that associated reductions in circulating eosinophil and eosinophil products may play a role in these therapeutic effects. hae is a genetic deficiency causing a decrease in c1 esterase inhibitor levels. it is a rare condition (approximate prevalence 1:10000 to 1:50000 individuals). it is manifest by acute attacks of swelling which can involve the larynx (a potentially life threatening condition), the gi tract (causing a syndrome similar to an acute abdominal catastrophe) or the skin and soft tissue of the patient including the limbs, face and external genitalia. there is no approved therapy for acute attacks in the usa. pathogenesis of this potentially fatal condition is thought due to excessive activity of plasma kallikrein. dx-88 is a specific and highly active (ki 40 pm) recombinant inhibitor of human plasma kallikrein undergoing evaluation in hae and cardiopulmonary bypass. it is an animal free product. a double blind, randomised (5:1 drug to placebo) placebo controlled dose ascending study of dx-88 in acute attacks of hae was run in the usa and the european union. four dose groups of 12 patients were to be included in the study. the doses were 5, 10, 20 and 40 mg/m2. the primary outcome variables were proportion of patients achieving a significant clinical response within 4 hours of administration of therapy, and safety. secondary endpoints included site response, dose response, and median time to significant response by site and dose group. dx-88 met its primary endpoint; 72% of dx-88 patients had a significant improvement within 4 hours, (placebo response rate 25%, difference 47% p= 0.0169). patients receiving dx-88 responded in a median time of 70 minutes. there was no difference in proportions of cases that responded within 4 hours between the three anatomical sites. time to significant response did not significantly differ between dose groups and anatomical sites. the safety profile was comparable for patients treated with dx-88 and placebo, in terms of saes and aes. in conclusion, dx-88 in this double blind study was shown to be statistically superior by 47% to placebo and to be at least as safe as placebo. the drug is effective at treating any hae site, including the larynx. dx-88 represents an important advance in the management of hae. ar is among the most common chronic childhood disorders, and is most effectively treated with intranasally inhaled glucocorticosteroids (ics). recent evidence suggests that intranasal ics therapy may suppress hpa axis function and decrease growth velocity. this study reports 1-year follow-up data on 24 children (12 female, 9 african american), aged 6 to 14 years (mean 10.4 years) at entry, enrolled in a long-term growth trial of intranasal taa for treatment of ar. primary measures included height velocity, bone mineral density (bmd), serum osteocalcin and salivary cortisol levels. all subjects followed their expected age-appropriate growth velocities. average growth velocities were 5.6 and 5.8 cm/year for girls and boys aged < 12 years, respectively, and 5.5 and 7.7 cm/year for girls and boys aged > 12 years, respectively. mean (+ std) bmd was 1.27 + 0.12 and 1.22 + 0.14 gm/cm2, mean serum osteocalcin levels were 89.1 + 25.5 and 114.5 + 32.7 ng/ml, and mean salivary cortisol levels were 16.7 + 8.0 and 11.5 + 6.5 nm/l at entry and 1-year follow-up, respectively. these results demonstrate no significant effect of one year of intranasal treatment with taa on growth velocity or hpa function in children with ar. 13 patients 6 males, 7 females ranging in age from 66 years to 12 years, received (o) in doses ranging from 2 to 17 injections over 4 to 34 weeks. patients were evaluated with symptoms scores, pulmonary function (pft), blood eosinophil count (bec), medication use, and allergy skin test. st were initially performed by prick and if negative, by intradermal (id) (1/1000 w/v) and if further negative by id (1/500 w/v). skin tests were performed immediately before and 20 minutes after the administration of o. there was no change in pft or bec. however there were significant reductions in symptom scores, including nocturnal asthma (<.0001), exercise tolerance (<.0001), sense of well being(<.0001). in addition, parenthetically there was a reduction in nasal symptoms (<.0001). there were also significant declines in medication use especially for albuterol (<.0001). st declined in all patients evaluated and dramatically in many. for several individual allergens st went from prick positive to id (1/500 w/v) negative. moreover st declined in all instances further, 20 minutes after the administration of o except for 2 patients. in addition to asthma one of the patients had allergic bronchopulmonary aspergillosis and another had allergic fungal sinusitis. three of the patients tested positive on prick test to mouse antigen and in each instance this became negative after administration of o with all 3 patients tolerating o without problem. we conclude: (1.) administration of o is associated with an improvement in symptoms and decreased use of medications (2.) in addition there is a prominent decline in st which is more marked 20 minutes after administration of o compared to immediately prior to administration the explanation of this finding is not apparent. (3.) o appears to be safe to administer to patients even if they demonstrate specific ige to mouse antigen by st a.p. baptist * , t.s. tang, j.l. baldwin, ann arbor, mi. introduction: academic medical institutions are under pressure to shorten or eliminate resident elective rotations due to federal work-hour restrictions. it is unknown if this will affect knowledge and referral patterns for resident and faculty physicians. the objective of this study was to examine the factors associated with resident and faculty perceived knowledge and referral patterns towards allergy/immunology (a/i). methods: a questionnaire was sent to 375 primary care physicians at one academic center. it addressed past history of a/i referrals, referral intentions for conditions that may be seen by allergists, and perceived a/i knowledge. independent variables included history of an a/i rotation, gender, department, years in training/practice, and history of referral to an allergist. results: 228 (61%) completed surveys were returned. using logistic regression with forward selection, we found in the resident physician cohort those with advanced years in training or history of an a/i rotation were more likely to have increased past history of referral to an allergist (or = 4.09, or = 3.81), increased referral intention for chronic sinusitis to an allergist (or = 3.51, or = 5.18), and increased perceived a/i knowledge (or = 2. . for the faculty physician cohort, those with a history of an a/i rotation were more likely to have an increased perceived knowledge about a/i (or = 5.64-6.18). in addition, pediatric faculty physicians were more likely to refer asthma (or = 5.62) and chronic eczema (or = 17.09) to an allergist than faculty from other departments. finally, faculty physicians with a past history of referral to an allergist were more likely to have refer asthma (or = 10.64) and allergic rhinitis (or = 9.78) to an allergist. conclusion: the factors associated with resident and faculty physician perceived knowledge, referral history, and referral intentions towards a/i differ. for residents, senior training level and a history of an a/i rotation appear most important. for faculty, history of an a/i rotation, pediatric department, and past history of referral to an allergist appear most important. given that a history of an a/i rotation may increase perceived knowledge and referral patterns towards a/i, eliminating a/i rotations in medical institutions may have important consequences for patients and the field of a/i. because recent anecdotal reports suggest that hbv may be an effective treatment for patients with multiple sclerosis (ms), there has been a movement of patients to zealous lay practitioners to receive multiple and repeated bee stings. since this practice has real risk of possible fatal allergic reactions as well as emotional and economic costs, properly conducted studies of safety and efficacy are needed. the purpose of the present phase i pilot study was to evaluate the safety of hbv extract in patients with pfms. a total of 9 evaluable bee venom non-allergic patients with pfms (21-55 years) were enrolled and were randomly divided into 4 groups, each receiving an increasing allergen dose immunization schedule for one year. hyperreactivity to hbv was evaluated by questionnaire, px and hematologic, metabolic and immunologic tests including skin tests. any possible beneficial responses to therapy were evaluated by questionnaire, functional neurologic tests (fnt) and changes in measurement of somatosensory-evoked potentials (seps) prior to and at the completion of the study. no serious adverse allergic reactions were observed in any of the 9 subjects even at the highest doses of bee venom therapy. although 4 of 9 subjects had worsening of neurologic symptoms during the course of bee venom therapy, requiring termination, this response could not be ascribed to side effects of the hbv or to a spontaneous worsening of the neurologic disease independent of treatment since there was no correlation of these events with the bee venom injections. of the remaining 5, 3 felt that the therapy was beneficial.there were no changes either in fnt or seps measured during the study. the present report represents the first controlled study evaluating the safety of hbv as a possible adjunctive treatment for multiple sclerosis. while this preliminary safety study suggests that administration of repeated injections of hbv in a step-up dosage regimen in appropriately selected non-hbv allergic patients with ms had no serious adverse allergic reactions, because of the small number of subjects studied, it does not permit conclusions regarding efficacy and therefore provides little evidence to support the use of hbv in the treatment of ms. much larger carefully conducted multi-center studies would be required to establish efficacy. i. finegold * , new york, ny. rush immunotherapy (rit) has benefits for inducing immunologic desensitization in shorter periods of time than conventional immunotherapy. however, there is an increased risk of systemic reactions utilizing accelerated schedules. in the past radiocontrast media reactions have been significantly reduced using a standardized protocol. a modification of this protocol was used prior to rit. the oral premedication consisted of 50 mgm prednisone 12 hours, 7 hours and 1 hour prior to rit, fexofenadine 180 mgm at 7 hours and again 1 hour prior to the procedure, and ranitidine 300 mgm, 7 hours prior to rit. patients tolerated the premedications well. a signed consent was obtained prior to rit. patients were desensitized in an office setting with appropriate therapy for anaphylaxis available without an indwelling intravenous line. injections generally began with 1/100 dilution of the anticipated maintenance solu-tions and were doubled every 20-30 minutes for about 2-3 hours and then the patients were observed for 1 hour or more prior to discharge. in this manner patients were desensitized in 2-3 half day sessions. the patient was injected again in 1 week, 2 weeks and then 4 weeks. doses were increased if they were not at a full maintenance dose. if reactions occurred this process was appropriately decreased. utilizing this technique 46 patients were treated. the average age was 35.5 years old (14-56 years of age) 72 % were males.72% of patients had allergic rhinitis, 25% asthma, 3% insect allergy. patients with complicated medical illnesses were excluded. nine patients had systemic reactions requiring an injection of epinephrine. among the symptoms experienced were wheezing, nasal congestion and flushing. no patient required a second injection of epinephrine, intravenous fluids, or hospitalization. the patient reaction rate was 21 % of patients treated or 9 in 1073 injections or 1% of all injections given during the rush protocol. these results are typical of the increased rate of reactions to rit. however, these systemics were mostly very mild and responded to therapy and in only one case was there a delayed 4 hour reaction. thus premedication while not preventing systemic reactions seemed to modify them to make rit in an office setting a practical and effective therapy. we have previously shown that oligonucleotides consisting a novel 3'-3'linked structure and synthetic immunostimulatory motif cpr (r is a synthetic purine moiety), referred to as second-generation immunomodulatory oligonucleotides (imos), induce potent th1 immune responses and prevent ovainduced allergic asthma in mouse models. in the present study, we examined local and systemic immune responses of imos following intranasal (i.n.) administration to naive mice. these studies showed that imos produce higher levels of serum and local il-12 compared with il-6. we next examined the ability of s.c. and i.n. administrated imos to reverse ova-induced th2 immune responses in a murine model of asthma. treatment of ova-sensitized and challenged mice with imos by either route of administration suppressed il-5 and il-13 levels with an increase in ifn-gamma secretion in spleen cell cultures and lung homogenates. imos decreased levels of serum ige and igg1 and induced higher levels of total and ova-specific igg2a titers in serum and balf. while both routes of imo delivery showed similar efficacy on serum and balf immunoglobulin levels, s.c. delivery resulted in stronger systemic effects on the spleen cell cytokine production and the i.n. route of administration produced stronger local effects on the lung cytokines. imos also decreased eosinophils in balf and suppressed inflammatory cell infiltration and goblet cell hyperplasia in lungs. these effects in lung were superior with i.n. administration of imo than did with s.c. administration. further studies to understand the effect of low multiple doses against a single higher dose of i.n. administered imos in naive mice suggest that low multiple doses induce strong th1 responses locally while the single higher dose produces higher systemic responses. these findings suggest that second-generation imos containing cpr dinucleotides potently reverse ova-induced th2 immune responses with strong th1 type cytokine induction in ova-sensitized and challenged mice and i.n. delivery is superior to s.c. delivery in reversing ovainduced airway inflammation and mucosal secretion. showed ait was medically inappropriate, lacked documentation of necessity for initiation or continuation and/or had strong contraindications in 12/18 (67%) of the reviewed records. method: we audited a convenience sample of our own records for documentation of necessity for (continuing) ait, using a checklist based on the oig report which included the following 6 criteria: an appropriate diagnosis; specific justification; exclusion of strong contraindications; signed, informed consent; a written physician order, and at least annual re-evaluation for continuing ait. results: the first 50 charts in our alphabetized file of 80 total ait charts were reviewed. the patients (21 m, 29 f) ranged in age from 9-63 yr (mean, 40 yr). the mean duration of ait was 2.3 yr (range, 0.25-12 yr). indications for ait included allergic rhinitis alone (68%) or with asthma (26%) or chronic sinusitis (2%), asthma alone (2%) and venom anaphylaxis (2%). all had initial documentation of necessity. seven charts (14%) had no written order to initiate ait. nine (18%) lacked a signed consent form. no strong contraindication was found. (peak flow documentation showed the asthma cases were well-controlled. three episodes of ait-induced anaphylaxis requiring epinephrine had occurred, but ait was subsequently resumed and well-tolerated.) seventeen (34%) charts had no documentation of re-evaluation during the preceding 6-12 months, including 3 with no documented re-evaluation during the previous 24 months or more, of ait. discontinuation of ait was "considered" for 3 patients after 1.5, 7, and 10 yr of ait, respectively, but all were still receiving maintenance doses. the estimated time for completing this review was 15-20 min per chart. inter-pretation: an internal chart review for adherence to ait practice parameters using a checklist such as ours can quickly and easily assess documentation for medicare reimbursement and patient safety. objective(s): immunotherapy is an accepted mode of treatment for children suffering from allergic rhinitis and allergic asthma. this study demonstrates that rapid allergen vaccination (rav) can be as safe as conventional allergen vaccination (cav) in children. rav may also improve patient adherence. study design: 148 pediatric patients, 1-18 years of age, diagnosed with allergic rhinitis and/or mild to severe asthma underwent rav over a 2 1/2 hour period in an office-based setting. all patients were premedicated with prednisone and h1 antagonists for 3 days prior to the procedure. patients were monitored for reactions during the procedure and continued on a cav schedule. adherence was reviewed subsequently. results: a total of 148 pediatric patients underwent rav utilizing a 2 1/2 hour protocol. of the 148 patients, 88 of them were male (59.5%) and 60 of them were female (40.5%). 143 (96.6%) of the patients had allergic rhinitis, 118 (79.7%) had asthma, and 23 (15.5%) had chronic sinusitis. during the procedure, 8 patients (5.4%) experienced systemic reaction, and none experienced true anaphylaxis. seven of the eight patients who suffered a systemic reaction were patients with asthma on inhaled corticosteroids, three of the eight patients were male (37.5%) and five of the eight were female (62.5%). every systemic reaction occurred within 15 minutes of the injection. treatment usually included one or a combination of the following: nebulized breathing treatment with albuterol, two sprays in each nostril of azelastine, and diphenydramine taken orally or intramuscularly. every patient that was treated was discharged within two hours, and no one required treatment with subcutaneous epinephrine, treatment for recurrent symptoms or hospitalization. all of the patients continued with a conventional allergen immunotherapy regimen following the rapid protocol to reach their maintenance dose. typically, 6 months of build-up period was saved. patients reached efficacious dosages almost immediately. adherence rates were 95.3%, 90.5%, and 79.7% at 3, 6, and 12 months respectively. conclusions: effective doses of allergen vaccine can be safely reached using a 2 1/2 hour protocol annals of allergy, asthma & immunology for children. advantages of rav over cav include improved adherence with almost immediate clinical efficacy, and decreased costs. introduction: studies involving allergen immunotherapy (ait) have furthered our understanding of cat allergen, proteases, and dust mite allergen. the impact of these data has not been assessed. objective: to evaluate ait prescribing trends over 12 years (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) focusing on new prescriptions before and after published data regarding specific prescribing recommendations. methods: a retrospective review of 38, 400 ait prescriptions from a centralized allergy and extract laboratory database was performed with respect to published literature on findings and recommendations regarding the ubiquitous nature of cat allergen, effective dosing of cat antigen, combining protease containing extracts with those susceptible to degradation, dust mite antigen dosing, and the use of house dust versus dust mite antigen. results: 1) in 1993, cat allergen was included in 10% of new allergy immunotherapy prescriptions reviewed; in 2003, it was included in 45% of new prescriptions, a statistically significant increase (p<0.00001). 2) over the past 11 years, the mean quantity of cat antigen included in new prescriptions has been unchanged at 1ml/prescription. only 5% of new prescriptions were written at the manufacturer recommended maintenance dose of 4ml, significantly less than those written below this level (p <0.00001). 3) there was no significant change in the percentage of extracts combining antigens with proteases with antigens susceptible to degradation. in 1992, 64% of prescriptions containing alternaria and/or cockroach antigen were mixed with one or more antigens susceptible to degradation; in 2003, 56% of these extracts were still being combined. 4) the mean volume of dust mite mix contained in these prescriptions was between 2-2.99 ml/10ml. 5) prescriptions containing dust mite rose from 20% in 1992 to 65% in 2003. the use of house dust was unchanged over the same period at 1.5% of prescriptions. conclusions: although the percentage of cat containing extracts has increased significantly, dosing of cat allergen has remained unchanged despite published literature recommending higher volumes. protease-containing allergen prescribing patterns have not significantly changed from [1996] [1997] [1998] [1999] [2000] [2001] [2002] [2003] young stage of life is a crucial period for aeroallergen sensitization, considering the immaturity of the neonatal immune system which may lead to t-cell anergy or a deviation towards th2-type response in mice. the aim of this work was to evaluate the influence of oligodeoxynucleotides containing cpg motif (cpg-odn) on ovalbumin (ova) and blomia tropicalis (bt) immunization in early life compared to adult stage. three days old a/sn mice were immunized with ova in al(oh)3 and boosted on 10th and 30th day after immunization (dai) and bled on 37th dai. groups of mice received 4mg of cpg-odn or control-odn associated with ova on immunization and boost. some animals from the three groups (ova, ova+cpg, ova+co) were killed at 20 days old and the spleen was collected and prepared to culture. eight to 10 weeks old female a/sn mice were immunized with ova in al(oh)3, boosted on 14th dai and bled on 21th dai. groups of mice received 50mg of cpg-odn or control-odn associated with ova on immunization. animals from the three groups were challenged 3 months after immunization and bled 7 days later. similar protocols were performed using blomia tropicalis (bt) extract in the place of ova. neonate and adult co-administration of cpg-odn with ova or bt were able to significantly decrease specific ige antibody levels and increase igg2a production. moreover, cpg-odn decreased specific igg1 levels in the bt immunization. the co-administration of control-odn in neonates decreased anti-ova igg1 production. after ova-challenge 3 months later of adult immunization, a similar response was detected in the group that received cpg-odn associated with ova, which showed a decreased anti-ova ige and igg1 and enhanced igg2a levels. analysis of cell division of neonate lymphocytes by flow cytometry showed a decreased proliferation in ova+cpg mice group under ova stimulation. this effect was seen either in b cells and t cells. the results showed that immunization with both allergens, ova and bt, associated with cpg-odn decreased the type i hypersensitivity response. the pattern of antibody production suggests th1-type response induced by cpg-odn, and the establishment of memory th1 response. this findings may imply that the use of cpg-odn in early life seems to be beneficial as an strategy to modulate or to prevent the development of allergic diseases. n. horne * , a. capetandes, m. frieri, east meadow, ny. introduction: it has been shown that dust mite allergens can affect the functioning of airway epithelial cells (winton et. al. br j pharm 124:1048 , 1998 . previous experiments showed ca549 aggregate in the presence of dermatophagoides pteronyssinus (dp) (capetandes et. al. am j clin path, 121:25, 2004) . it is hypothesized that epithelial damage is associated with airway remodeling characterized by fibroblast dysregulation. to evaluate the response of fibroblasts to damaged airway epithelial cells, the bioactivity of serum-free conditioned media from dp-treated ca549 cells (dpcm) was assayed with nhlf. methods: all experiments used insulin-transferrin-selenium supplemented dmem (its). dpcm (generated with ca549 treated with 300 au/ml dp; alk-abello) was added to 50% confluent nhlf for 24 hours at 37oc and 5% co2. its from cultured ca549 without dp (its cm) was added to 50% confluent nhlf (control). cell morphology and density were evaluated by microscopy and mtt assay, respectively. data were analyzed by anova followed by student-neuman-keuls (snk) at p<0.05 with power analysis. results: 50% confluent nhlf treated with dpcm showed decreased cell density (figure 1) , and increased aggregation relative to its cm or its alone. nhlf in its alone or with direct addition of 300 au/ml dp to its (dp its) showed no or weak aggregation. aggregated nhlf showed 100% viability and grew to confluence when subcultured to serum-supplemented media. conclusion: nhlf aggregation was greater, and cell density was lower with dpcm than with its, its cm, and dp its. this suggests that dp-treated ca549 cells release an unidentified factor or factors that contribute to nhlf aggregation and possible apoptosis. identification of these factors would increase the understanding of the fibroblast response to epithelial cell exposure to dp which may be involved in airway remodeling, a feature of chronic severe asthma. j.t. zimmermann * , y.c. huang, m. frieri, east meadow, ny. introduction: budesonide has been demonstrated to inhibit il-8, tgf and gm-csf in ragweed and dust-mite (dm) stimulated human alveolar epithelial cells (j allergy clin immunol 209:3a, 2002; ann allergy asthma immunology 90: p79, 2003) . rantes production and expression in dm and il-1 -stimulated a549 cells has recently been shown to be inhibited by budesonide (allergy asthma proc, in press 2004). histamine is known to influence immune response by regulating cytokine synthesis (allergy 47:024-1992) . in this study we examined the effect of budesonide in the presence of histamine on il-8 production and expression by a549 cells. methods: a549 pulmonary epithelial cells were cultured in dmem for 24 hours in 5% co2 in the presence of 1:20 of 10, 000 au/ml dm, 10 -6 m histamine and 10 -7 m budesonide. il-8 production was measured by a sensitive elisa and il-8 mrna was measured by qualitative rt-pcr using standardized primers for il-8 with a gapdh control. results: a549 cells were stimulated by dm (65-101 pg/ml) (p<0.01). budesonide alone decreased il-8 levels to 38 pg/ml (p<0.05), and in combination with histamine further decreased il-8 levels to 23 pg/ml (p<0.001). relative intensity of il-8 mrna expression was reduced 12-fold in dm-stimulated cells and 2-fold in control cells by budesonide. conclusion: budesonide in combination with histamine showed greater inhibition of il-8 production by a549 cells than budesonide alone possibly by increasing cell membrane permeability. c.r. oliveira * , a.e. fusaro, j.r. victor, e.a. futata, c.a. brito, a.j. duarte, m.n. sato, sã£o paulo, brazil. antigen-driven bystander suppression induced by oral tolerance could be an interesting approach in the allergy field, considering the high incidence of new allergens sensitization in atopic individuals. to address the influence of non-related allergen exposure on the type i hypersensitivity response to the mite blomia tropicalis (bt) or ovalbumin (ova) in mice and to verify oral tolerance effect in the bt/ova co-immunization model. groups of mice were immunized with bt extract and two weeks later submitted to ova-immunization, or first immunized with ova or co-injected with bt and ova. ova feeding was performed five days prior co-immunization. ige abs were estimated by means of passive cutaneous anaphylaxis reaction and specific ab and cytokines secretion by elisa. mice sensitized with bt and then exposed to ova developed an enhanced ige response to itself and to ova, but such response has not been observed when ova-immunization was prior to btimmunization. co-injection of bt and ova led to a dominant ige response toward ova over bt and vice-versa for the igg1 response. ova feeding prior co-immunization decreased ige, igg1 and igg2a ab levels against ova in parallel to a bystander suppression, which avoided the outcome of bt-sensitization. these mice showed increased ifn-?secretion levels induced by antigen-specific stimulus. furthermore, effectiveness of oral tolerance was also related with ova amounts employed in the co-immunization since ova feeding prior co-immunization with low amounts of ova led to inhibition of ige ab response to both allergens, whereas inhibition of specific and non-related antigen igg ab response was broken. the results evidenced that, depending on allergenic potential, new allergen exposure may exert an adjuvant effect on the primary sensitized allergen. the bystander effect to non-related allergen by oral tolerance should be an interesting mechanism to control new allerrationale. cd4 + cd25 + regulatory t cells from patients with atopic asthma undergo apoptosis when stimulated by specific allergen. antihistamines are used to control allergic inflammatory diseases, but their influence on treg cells is currently unknown. the present study investigates the influence of desloratadine (d) on apoptosis of cd4 + cd25 + regulatory t cells in atopic patients having dust mite induced allergic disease, including allergic asthma. methods. patients (n=6) with positive skin prick tests to house dust mite allergens and a clinical history of allergic upper and/or lower respiratory symptoms were treated with d 5mg (schering-plough, usa) once daily for 10 days. no patients used systemic corticosteroids, while theophylline and other medications were stopped at least 72 hours before blood collection. blood was sampled before and after treatment with d. the control group was comprised of 6 individuals without allergic respiratory symptoms and with negative skin prick-tests. peripheral blood mononuclear cells (pbmc) were isolated over a ficoll density gradient and stimulated by specific allergen (dermatophagoides farinae) and in a control series by phorbol myristate acetate (pma). pbmc were labeled with anti-cd4, cd25, cd95, and bcl-2 monoclonal antibodies. the annex-inv-propidium iodide (anv-pi) test was done. lymphocyte subpopulations and apoptosis were analyzed by flow cytometry. results. after treatment with d, atopic patients had no significant differences in the number of cd4 + cd25 + cells. during co-culturing of pbmc from patients treated with d with specific allergen a significant increase in cd4 + cd25 + cells was observed. simultaneously, treatment with d led to a significant increase in expression of the antiapoptotic protein bcl-2 in cd4 + cells. this data paralleled the decrease of cd95 expression on cd4 + cells. there was also a decrease in treg cells in the late stages of apoptosis (anv + pi + cells) in d treated patients. conclusions. the antihistamine preparation desloratadine prevented allergen-specific apoptosis of cd4 + cd25 + t regulatory cells in patients with atopic asthma. the type 1 histamine receptor may be involved in the regulation of apoptosis and/or survival of cd4 + cd25 + t cells. a.g. palma-carlos * , m.l. palma-carlos, lisboa, portugal. introduction: solar urticaria porphyrin corresponds to sun sensitivity to a 4000 a wavelengths and is due to disturbances of posphorin metabolism. photosensitivity can cause urticaria, erythema, polymorphic solar eruption and in more severe cases vesicles and bullae. photosensitivity was confirmed by light test. methods: protoporhyrin in rbc, uro and coproporphyrins in urines, copro and protoporhyrins in faeces and porphyrin precursors have been studied un all the cases of solar urticaria seen in the last few years. results: in 10 patients, 8 female, 2 males, a diagnosis of porphyria has been confirmed by laboratory methods. clinically 4 patients presented solar urticaria and 6 solar erythema or polymorphic solar eruption. 7 patients, 8 female, 2 males (21-54) presented also neuro visceral symptoms: abdominal pains (7) vomiting (4) asthenia (7) constipation (3) muscle pains and paresia (3) depression (2) anesthesic reactions (2). in 2 cases the symptoms were associated with anticonceptional drugs. the conjunction of clinical history with laboratory data has allowed to confirm the diagnosis of 2 cases of protoporphyria erytheropoietica, one triggered by anticonceptionals, 6 cases of coporporhyria hereditaria and 2 cases of porphyria variegata. this group comprises one case of erythropoietic protoporphyria induced by estrogens not previously reported. conclusions: research of porphyrins must be done in all the patients with solar urticaria and erythema. the incidence of sun sensitivity in mixed hepatic porphyriaalso presenting neuro-visceral symptoms which can be drug dependent suggests that porphyria study must be mandatory in suspected cases. a.r. narayan 1* , j. kaplan 1 , v. sirvan 1 , a. chandrasekaran 2 , c. goodwin 2 , l. goodwin 2 , l. guida 3 , m. frieri 1 , 1. east meadow, ny; 2. manhasset, ny; 3. bayshore, ny. rationale: our division has reported that nitric oxide (no) from tracheal epithelium can mediate induction of il-8 (american journal of respiratory critical care medicine 151:642a, 1995) . increased levels of il-8, ltb-4, and no in mononuclear cells (mnc) from cystic fibrosis (cf) patients were noted.(pediatric asthma allergy immunology 10: 101-7 1996) . no in mnc from cf was increased by stimulation with aspergillus fumigatus (asp) and rhdnase (j. clin allergy immunol 105:320p 2000). tnf-is produced from bronchial epithelial cells (bec) in the presence of mnc of normal controls (nc). rantes, a chemokine that facilitates leukocyte migration, is associated with airway inflammation in cf, and asp sensitization in cf patients could amplify pro-inflammatory cytokines such as il-8, tnf-, and rantes. methods: we studied the mrna and protein expression of tnf-, il-8 and rantes in a 65 year-old cf patient and a nc. 1x10 6 mncs from the patient and nc were stimulated with 21î¼g/ml of asp with bec at 10x10 5 and 10î¼g/ml rhdnase. mrna expression was studied by real time pcr (taqman chemistry abi prism 7700) and protein levels by elisa. results: tnf-production in supernatants of mnc with bec from nc increased from 8 pg/ml to 64 pg/ml with rhdnase. whereas in cf patients the tnf-mrna expression was greatly enhanced over nc but declined in the presence of rhdnase (fold difference: 8.37-7.70). there was no difference in expression levels of nc (fold difference: 4.0-3.9) with rhdnase treatment. il-8 expression in mnc with bec of patients increased 2.4 fold compared to nc (1.5 fold). rhd-nase decreased asp stimulated levels to 1.72 fold in patients compared to nc (2.9 fold). mnc and bec rantes production in patients increased from 49 to 1025 pg/ml and decreased to 570 pg/ml with rhdnase, but increased from 1134 pg/ml to 1775 pg/ml in nc. in contrast, rantes mrna expression in all groups was higher in nc but decreased with rhdnase in both cf and nc (37-26 fold vs. 14-10 fold). conclusion: rhdnase can increase no and decrease pro-inflammatory tnf-and rantes in cf patients stimulated with asp. rhdnase could lead to augmented bactericidal activity and epithelial defense by enhancing no production and decreasing the expression and production of tnf-, il-8 and rantes. this is a case report of two steroid dependent atopic dermatitis patients who both responded to treatment with omalizumab. patient a is a 39 year-old white male who presented with a history of severe atopic dermatitis for 10 years along with concomitant mild persistent asthma and allergic rhinitis. he had previously received monthly triamcinolone injections, methotrexate and doxycycline with limited response. on presentation he had mild improvement on a regimen of alternate day prednisone 30mg, fexofenadine 180mg bid, and zafirlukast 20mg bid. however, attempts at weaning prednisone were unsuccessful. omalizumab was initiated at a dosage of 150mg every four weeks. the patient remains prednisone dependent yet free from atopic dermatitis. patient a's diagnostic work up determined a total ige of 33.1 ku/l. the patient's specific ige tests were positive for grass mix, weed mix, maple, white pine, peanut, strawberry, gluten, soybean, wheat, oat, and dog dander. specific ige tests were negative for cat dander, egg white, milk, goose feathers, chicken feathers, mold mix, dust mix, and fish mix. patient b is a 23 year-old white male who presented with a history of severe full body atopic dermatitis along with mild persistent asthma and allergic rhinitis. he also was receiving triamcinolone injections with limited success. he responded to alternate day prednisone 30mg, desloratidine 5mg bid, and zileuton 600mg bid. attempts at weaning prednisone failed until initiation of omalizumab 375mg every two weeks. his atopic dermatitis is now under control with once daily desloratidine and omalizumab every two weeks. he was successfully weaned off of corticosteroid medication and has begun an immunotherapy regimen. patient b's diagnostic work up determined a total ige level of 1159 ku/l. patient b's specific ige tests were positive for weed mix, tree mix, maple, peanut, strawberry, dust mix, cat dander, dog dander, egg white, milk, oat, wheat, goose feathers, and chicken feathers. specific ige tests were negative for grass mix, white pine, mold mix, gluten, soybean, and fish mix. these two case reports reveal significant response to omalizumab in severe steroid dependent atopic dermatitis. further research is strongly indicated considering the quality of life issues with severe atopic dermatitis and potential side effects to long-term corticosteroid treatment. eosinophilic esophagitis (ee) has been described in children and is characterized by high levels of eosinophils (>20-24 eosinophils/high powered field [hpf]) in the esophageal mucosa. a recent report indicates that the combined prevalence of the eosinophilic gastrointestinal disorders may be higher than that of inflammatory bowel diseases (ibd). the presenting symptoms of ee mimick those of gastroesophageal reflux disease (gerd), and patients of all ages may experience a delay in time from onset of symptoms to diagnosis of ee. there is a paucity of data in the literature regarding the delay in time from symptom onset to diagnosis of ee. we report the data on four children three years of age and under with ee who experienced a significant diagnostic delay. four young children were referred to the pediatric allergy clinic with a diagnosis of ee (table) . three of the patients were male and one was female. the patients were 17-36 months of age at the time of diagnosis, and the diagnostic delay was 15.5-33 months (average delay 23.6 months). the most common presenting symptom was vomiting, and three of the four patients had a history of respiratory obstructive symptoms. all of the patients had received conventional treatment for reflux disease, and one had undergone a nissen fundoplication prior to diagnosis. in all four patients the esophageal biopsy revealed >20 eosinophils/hpf. three out of four patients had a family history of atopy, and two patients had a known history of a food allergy. skin tests to foods were positive in two patients and rast (radioallergoabsorbent) testing to foods was positive in one patient. recent data suggests that the diagnostic delay in ibd is decreasing as much as 50%. it is hypothesized that this may be due in part to an increased index of suspicion by health care providers. there is little data available regarding the diagnostic lag in children with ee. it is currently believed that chronic ee can lead to progressive esophageal scarring and dysfunction. stricture formation has also been described in children less than two years of age. given the reported increased incidence of this allergic gastrointestinal disease over the past decade, more data regarding the diagnostic delay of these conditions may be instrumental improving health care providers' awareness of this disorder. a. kohli-pamnani * , e. cooney, p. huynh, f. lobo, new haven, ct. introduction: cutaneous hypersensitivity reactions to amprenavir, an hiv-1 protease inhibitor, are reported in up to 28% of treated patients, of whom 4% have severe or life-threatening rashes, with treatment discontinuation required in 3% of cases. we report a case of successful desensitization to amprenavir, for recurrent maculopapular exanthem, in an hiv-infected patient with late stage disease and limited antiretroviral (arv) agent options. methods: the patient is a 40 year-old caucasian female with late stage hiv disease (absolute cd4 count 30 cells/mm 3 , hiv rna 200, 000 copies/ml) and multiple arv intolerances, who developed a severe generalized maculopapular eruption sparing mucous membranes six days following initiation of a regimen comprised of amprenavir, lopinavir/ritonavir, zidovudine, and lamivudine. a similar reaction occurred following re-challenge with amprenavir alone (600 mg oral formulation bid via percutaneous endoscopic gastrostomy (peg) tube), despite concurrent administration of oral prednisone 20 mg/day and loratidine 10 mg/day. results: skin prick testing with amprenavir 0.01 mcg/ml was negative, whereas intradermal testing with 0.1 mcg/ml was positive with a 7 mm wheal and 20 mm flare. percutaneous and intradermal testing with normal saline was nonreactive. the positive histamine control (skin prick only) yielded a 5 mm wheal and 20 mm flare. subsequently, incremental doses of 0.025 mg, 0.1 mg, 0.25 mg, 1 mg, 2.5 mg, 7.5 mg, 25 mg, 50 mg, 100 mg, 300 mg, 600 mg, and 1200 mg of amprenavir oral solution were administered via peg tube at 20 to 30 minute intervals (table) . the patient successfully tol-erated amprenavir desensitization and has remained on therapy without recurrence of rash out to 16 months of follow-up. conclusions: desensitization may permit continued use of amprenavir in patients with a history of amprenavirinduced maculopapular eruptions who have limited alternate treatment options. efforts aimed at characterizing the mechanism of amprenavir cutaneous hypersensitivity reactions seem warranted, given the frequency of reactions and the limited number of arv agents available for patients with late stage hiv disease. doses of oral solution were administered by peg tube at twenty to thirty minute intervals. introduction: beta-lactam (bl) allergy is the most common drug allergy. in most cases, ige antibodies are specific to the bl nucleus. however, sidechain-specific ige (scsige) to bl has been described. despite this, skin testing (st) to bl other than penicillin (pcn) is not commonly performed. we report a case of a 31 year-old female with a selective allergy to pip, and describe the utility of st to this agent to confirm scsige. methods: st to prepen (pp) and pcn g was carried out with percutaneous (pc) testing followed by intradermal (id) testing. st was also carried out with ampicillin (amp; 1 mg/ml), pip (60 mg/ml at pc level; 1 mg/ml at id level-a non-irritating concentration), and pip/tazobactam (tbm; 60 mg/ml at pc level; 1 mg/ml at id level) to exclude the possibility of selective allergy to tbm. case report: a 31 year-old white female with crohn's disease was hospitalized for treatment of intra-abdominal abscesses. she had immediate flushing and urticaria during an infusion of pip/tbm approximately 2 years prior. the allergy/immunology service was consulted for pcn st because her primary service preferred to empirically treat with pip/tbm. she had received imipenem approximately 8 months prior without untoward reaction. st to pp and pcn g was performed with negative responses and adequate controls. she subsequently received pip/tbm, but developed flushing and urticaria during her initial infusion, consistent with an ige-mediated reaction. two months later, st to pp and pcn g was repeated. in addition, st to amp, pip, and pip/tbm was performed. she had positive responses to pip and pip/tbm with negative responses to pp, pcn g, and amp, implying selective ige-mediated potential to pip. she tolerated an oral challenge with pcn vk 250 mg immediately following st without untoward reaction. conclusion: we have described a case in which st to pip was useful for diagnosing scsige in our patient, who had 2 prior reactions consistent with ige-mediated pathogenesis. this information will be helpful in identifying antibiotics she can safely receive in the future. this case supports the utility of st to bl in addition to pcn, in evaluation and management of patients with a history of adverse reaction to bl that may reflect presence of scsige. a.r. vaishnav * , b.s. bochner, baltimore, md. we report the case of a 39-year-old caucasian male with a 15-year history of asthma, two episodes of eosinophilic pneumonia and chronic peripheral eosinophilia with baseline eosinophil counts around 1000/î¼l who developed amnesia and elevated cardiac enzymes in february 2004. another physician had added montelukast in january 2004 and because of a good response advair was decreased from 500/50 to 250/50 bid. in february his eosinophil count increased to 3430/î¼l and soon he developed left arm numbness, diplopia as well as left arm and leg weakness. at his local hospital, he was found to have elevated troponins and cpks. cardiac catheterization showed normal coronary arteries and good left ventricular function. soon after discharge, he developed confusion and global amnesia. brain mri showed diffuse uptake consistent with global inflammation and/or vasculitis. upon hearing this story, we told him to take 60 mg of prednisone and urgently come to our hospital for admission. within hours, his mental status and visual symptoms improved. physical examination was unremarkable except for subungual splinter hemorrhages. montelukast was stopped and 1 gm/day solu-medrol iv was started. endomyocardial biopsy 2 days later showed mild hypertrophy and fibrosis without eosinophils. repeat brain mri and mra were normal. chest ct showed multiple patchy infiltrates with a new central cavitary lesion in the right lobe. other labs included a negative anca, ana of 1:40, normal complements and csf. our differential diagnosis included churg-strauss syndrome (css) versus hypereosinophilic syndrome (ihes) with myocardial, neurological and pulmonary involvement. serum tryptase was normal as was fluorescent in situ hybridization for the fip1l1-pdgfr fusion gene. based on this, along with the new pulmonary cavitary lesion on chest ct, the diagnosis of css was made. after 3 days of iv steroids, he was switched to 60 mg/day of prednisone. upon discharge his eosinophil count was 350/î¼l on 60 mg of prednisone. after all procedures were completed, he was also started on aspirin 325 mg daily to prevent thrombotic and thromboembolic complications. he was seen in follow-up in clinic and cytoxan 100 mg daily was started. his prednisone is being tapered. he is tolerating the treatment well and so far has had an excellent clinical and laboratory response. introduction-c1-esterase inhibitor (c1-inh) deficiency is a rare disorder classified into acquired and hereditary forms. both entities are distinguished by recurrent angioedema without pruritus or urticaria. the upper airway, head, neck, extremities and gi tract are typically involved. the inherited form usually presents in the first or second decade accompanied by a family history. the acquired form more commonly presents after the fifth decade. in acquired c1-inh deficiency, serological evaluation reveals low levels of c4, c1-inh, and c1q and a normal c3 level. levels of c1q are normal in the hereditary form. the acquired form may be associated with lymphoproliferative disorders and autoimmune disease. acquired c1-inh deficiency is typically recognized before the underlying malignant condition is diagnosed. clinical regression has been reported in patients whose underlying disorder responds to treatment. methods-a case report of a 57-year old female who developed repeated episodes of facial angioedema requiring hospitalization preceded by one year of vague abdominal pain and cramping. data-laboratory investigation of this patient revealed c4<10mg/dl (16-47 mg/dl), c1q<3.5mg/dl(5.0-8.6 mg/dl), ch50<20u/ml(31-66 u/ml), and c1-inh at 3 mg/dl(6-25 mg/dl) with a 51% activity (>68% normal). c3 was 138 mg/dl(90-180 mg/dl). other laboratory evaluation was notable for a normal cbc with diff, comprehensive metabolic panel, amylase, lipase and spep pattern. ige was elevated at 173 ku/l. hematology was consulted. ct of chest, abdomen, and pelvis were nondiagnostic. peripheral blood flow cytometry revealed a small distinct pop-ulation consistent with a clonal b-cell lymphoproliferative disorder. a repeat test showed skewing towards lamba light chain expression. bone marrow biopsy and aspirate were essentially normal except for erythroid hyperplasia on danazol treatment. the physical examination was without any masses or lymphadenopathy. the patient remained asymptomatic after beginning danazol although elevations in rbc, hgb, hct and absolute lymphocyte count have developed. conclusion-acquired c1-inh deficiency is rare cause of recurrent angioedema that has been reported in small cohorts and case presentations. we report another case report that reinforces the need for physicians to evaluate for concomitant lymphoproliferative disorders. chronic urticaria is a distressing condition usually associated with poor quality of life and poor response to symptomatic therapy. a wide variety of causes and mechanisms have been described and in some patients the cause remains unknown. rare cases have been associated with thyroid antibodies and some responded to thyroxin, even in the absence of overt thyroid disease. case report: a 54-yr-old obese white female presented with a history of persistent urticaria/angioedema for 4 mo. urticaria involved various parts of the body and individual lesions usually lasted < 24 hr. it was often accompanied by facial edema. oral diphenhydramine 25mg qid caused only little improvement. the patient could not suspect any offending factors. review of systems and past medical history were unremarkable, except for hyperthyroidism at age 12 yr that was treated with "medication" for 3 yr. on physical examination there were several urticarial lesions though she has been taking diphenhydramine. the thyroid appeared normal regarding size, shape and texture. various second generation antihistamines and doxepin were of little help. laboratory evaluation revealed total serum ige of 7 iu/ml (normal <200 iu/ml) and ch50 of 73 u/ml (normal 22-60 u/ml), but high tsh of 15.2 iu/ml (normal 0.4-5.0 iu/ml) and low free t4 of 0.58 ng/dl (normal 1.45-3.48 ng/dl). her antithyroglobulin titer was 32 iu/ml (normal <100 iu/ml) but the antimicrosomal titer was highly elevated at 382 iu/ml(normal <20 iu/ml) consistent with hashimoto's thyroiditis. thyroxin therapy was initiated at a dose of 100 mcg/d which resulted in marked improvement within one week and the patient was able to discontinue doxepin and other antihistamines. because of marked drop in tsh to 0.08 iu/ml, the thyroxin dose was reduced to 50 mcg/d. her thyroid function tests became normal within two months. she did not experience any recurrence of urticaria or angioedema during over 6 mo of follow-up so far. conclusion: patients with chronic urticaria/angioedema, especially women, should be screened for thyroid autoantibodies and if positive, thyroxin therapy might bring impressive remission in urticaria. hypersensitivity pneumonitis (hp) results from an abnormal immunologically mediated response to an environmental antigenic trigger. typically patients with hp experience transient fever, hypoxemia, muscle and joint pain, difficulty breathing, fatigue, weight loss, and cough. there are two clinical presentations of hp differentiated by onset and resolution of symptoms. patients experiencing acute hp experience the onset of symptoms 2 to 9 hours following exposure to a particular antigen and resolve in 1 to 3 days without specific treatment. however, patients with chronic hp experience symptoms that persist for months to years when exposed to a recognized cause of hp. a 55 year-old caucasian male was referred to our practice with acute bronchitis. his referral was secondary to a history of allergic rhinitis, chronic asthma, gastro-esophageal reflux disorder, and sinus surgery. this non-smoking male was a machine operator at a local factory. he was doing well until he began working in the presence of a heat induction machine and metal lubricating spray called multan ea20 (hinkle surface technologies), i.d. no.: 238073. multan ea20 is a product containing naphthenic petroleum distillates, amine salts, amine soap, triethanolamine hexyl, hexylene glycol, ethanol, sodium petroleum sulfonate, and triazine. this lubricant is known to irritate the eyes, skin, and respiratory tract. recent studies indicate that thermal decomposition of triazine, which readily occurs during metalworking, results in the production of formaldehyde. formaldehyde is a known carcinogen, as well as respiratory, skin, eye and digestive tract irritant. the patient developed serious respiratory health problems resulting in work absence and eventually hospitalization. while hospitalized he received a high resolution ct scan which showed diffuse ground glass appearance, and multiple attenuation areas of the lung which are consistent with hypersensitivity pneumonitis. the patient was treated with corticosteroids, and antibiotics with eventual normalization of lung parenchyma by chest ct. this represents the first reported case of hypersensitivity pneumonitis resulting from exposure to the metalworking fluid and respiratory irritant multan ea20. a.j. ham pong 1* , f. chan 1 , s.l. bahna 2 , 1. ottawa, canada; 2. shreveport, la. sexual intercourse has been known as the route for semen hypersensitivity reactions to the seminal fluid protein or to a contaminating drug or food. we report a case of anaphylaxis to cephalexin-containing semen by ingestion. history of present illness: a 37-year-old woman developed a systemic reaction after swallowing semen. since the couple frequently practiced fellatio without any reactions, her husband suspected cephalexin that he was taking over 2 days (500 mg qid). her reaction began in less than 10 min and peaked over 50 min; first as itchy oral mucosa followed by wheezing, flushing of the face and upper chest, and nausea. diphenhydramine 50 mg was administered po at 30 min and the reaction subsided over 1 hr. past medical history: she had generalized urticaria to oral penicillin more than 15 yr earlier and had positive penicillin skin test. she also had allergic rhinoconjunctivitis, mild intermittent asthma, and positive skin test to house dust mite, cat and dog epidermals, and pollens of grasses, trees, and ragweed. she also had oral allergy syndrome to certain fresh fruits and tree nuts; hazelnuts caused also diarrhea and colic. neither the patient nor her husband ate any nut-containing food on the days preceding the reaction. family his-tory: allergic rhinoconjunctivitis in the mother, sister and brother. eval-uation: the patient sought allergy evaluation about 2 yr after the reaction. she and her husband gave a consent. her serum ige was 108 iu/ml and skin test positive to penicilloyl polylysine 6x10-5 mol id, but negative to cefazoline 10 mg/ml for prick and 3 mg/ml for id. she had negative prick test to her husband's seminal plasma both before and after his intake of cephalexin 500mg qid for 2 days. using a cephalexin bioassay sensitive down to 6.25 mcg/ml, the medication level in the husband's urine at 120 min post last dose was 2400 mcg/ml and in his serum at 135 min was 12 mcg/ml, but was undetectable in the semen collected at 90 min. conclusion: this is probably the first case report of systemic reaction to ingested semen. the reaction occurred in an atopic, penicillin-sensitive woman and seems to be caused by cephalexin excreted in the semen or through urine contamination (1 drop = 120 mcg). in addition to her avoiding penicillin and cephalosporins, she was advised to avoid her husband's semen while him taking such drugs and for at least 3 days afterwards. background:asthma causes serious morbidity & mortality all over yet most asthma educational evaluations and programs have an urban rather than rural focus.there is lack of data on rural north dakota(nd). this nd survey aimed to study knowledge & awareness of management strategies for asthma like use of metered dose inhalers, spacer devices and peak flow meters. methods:the qualitative survey addressed attitudes and beliefs on asthma & its triggers;knowledge of medication delivery routes & use of hospitals and provider visits.a simple questionnaire was self-administered at 6 rural clinics in small towns in southeastern north dakota.10 questions explored asthma knowledge, extent of family history of asthma & factors aggravating asthma.it was voluntary & the population surveyed included patients, family, teachers, employees at nursing homes, the local hospital & clinics. results:of 297 surveys completed 61% had no asthma in the family.of those with a family history(39%), 59% had 1 person with asthma, 28.6% had 2 & 12.3% had 3 or more.respondents were aged 18-90 years & 81.1% were female.see table. discussion:our survey was rural & comparison with urban areas may help in planning for asthma education & resource allocation.61% denied asthma in their family. similarly, nonurban alaskan natives have a lower incidence of asthma compared to nonnatives.our results show a lack of awareness in our area.grain dust exposure was perceived as an asthma trigger but without evidence, local research is needed.in our study smoking was considered a trigger by 91.5% & this may be real.there was lack of awareness of peak flow meters & their role in asthma care.a study showed that compared to other areas even rural nurses used peak flow meters less often to assess and monitor asthma.this suggests a need for comprehensive asthma educational programs in rural areas that are based on national guidelines.we had only a moderate followup rate at 60%.in comparison, one study had 21% missed scheduled follow-ups. exercise induced asthma was known to only 50% of our group. conclusion:the need for better patient-provider communication has been highlighted by the lack of awareness of proven strategies to combat asthma.the use of a simple survey has revealed many unknown facts about asthma awareness in rural nd.further study to determine cost effective solutions to achieve national targets for asthma management are essential. aim: in most studies of asthma, the emphasis was on changes in large and middle airways. the aims of this study:(1)to observe the morphologic changes in small airways and lung tissue(salt) in guinea pig asthma models(gpam); (2)investigate the role of vcam-1, eotaxin, nf-b and ap-1 in the inflammation of asthma; (3)explore the functional variation of alveolar type 2 cells in asthma; (4)evaluate the effects of inhaled glucocorticoids on the above annals of allergy, asthma & immunology parameters in salt of asthma models. methods: (1)the gpam were established by ovalbumin challenge. five groups were divided: control, asthma day 4 and day 14, intraperitoneal dexamethasone, and budesonide inhalation group. (2)the expression of vcam-1, eotaxin, nf-b and ap-1 was determined by immunohistochemical technology and rt-pcr; the dna binding activity of nf-b and ap-1 by electropharetic mobility shift assay. (3)the balf phospholipide concentration was measured by phosphorus detection. results: (1)significant inflammatiom with infiltration of eosinophils and lymphocytes in salt was observed in asthma groups. (2)the protein levels of vcam-1, eotaxin, nf-b and ap-1 expressed in the salt were significantly elevated in asthma groups than those in control. (3)the mrna expression of vcam-1 and eotaxin of lung tissue homogenate was significantly increased in asthma group. (4)the dna binding activity of nf-b and ap-1 of lung homogenate was significantly increased in asthma group. (5)the balf surfactant represented by phospholipides was significantly decreased in asthma groups. (6)glucocorticoids in different ways of intake provided significant effects on the inflammatiom of salt. conclusion: (1)widespread and significant inflammation with eosinophilic infiltration existed in the salt in gpam, indicating asthma is a disease involving the whole airway and lung system. not only there were structural changes, but also impaired function of alveolar cells. the role of small airway inflammatiom may be of great importance. (2)eotaxin and vcam-1 actively mediated the process of inflammatiom, nf-b and ap-1 played important roles in the regulation of vcam-1 and eotaxin. the up-regulation of the above mediators in gpam was expressed in salt similar to that in central airways. we have recently reported that immunomodulatory oligonucleotides (imos) consisting of a novel structure and synthetic cpr or r'pg (r and r' are synthetic purine moieties) stimulatory motif effectively prevent ovainduced asthma in mouse models. in the present study we examined whether these novel imos (hyb2055 and hyb2087) can reverse established allergic airway inflammation in mice. balb/c mice sensitized and challenged with ovalbumin (ova) were evaluated for airway hyperresponsiveness (ahr) to methacholine. following ova-sensitization, mice were randomized and treated with placebo or 30 mg or 60 mg/dose of hyb2055 or 2087 s.c. during the following 10 days. two days after the final treatment, mice were rechallenged with ova and pulmonary functions were recorded. mice treated with either imo were significantly protected from both early (ear) and late (lar) allergic response, airway hypersensitivity and hyperreactivity to methacholine, bal and peribronchial eosinophilia, bal and serum il-5, and total serum ige as compared to vehicle-treated ova-sensitized and challenged animals. there was a significant increase in immature lung dendritic cells (cd11c+cd45r+) together with increase in serum il-10 levels and significant decrease in lung dc2 type cells (cd11c+ cd11b+cd8a-) as compared to vehicle-treated ovalbumin-sensitized animals in the lungs following treatment with either imo. these data suggest that both imos are effective and potent in attenuating key features of established allergic airway inflammation in bronchial asthma, and this effect could be mediated via decreasing lung dc2 cells and increasing immature dendritic cells. additionally, imos contain a novel 3'-3'-attached structure that provides higher metabolic stability and may permit lower and/or less frequent dosing. hyb2055 was evaluated for its safety and immunopharmacology in a phase 1 clinical trial in healthy human volunteers. introduction -hemoglobinopathies are common in southern europe and mediterranean area the more frequent being thalassemia and sickle cell disease. aside of the major hematological diseases of homozygotic patients minor forms are frequent, thalassemia minor and sickle cell trait. in these cases mycrocytosis with decrease of red cell volume and mean corpuscular volume or abnormal erythrocytes are found and can lead to hemorheologic disturbances in bronchial circulation and bronchial hypereactivity. the rationale of this study is to evaluate the incidence of asthma in hemoglobinopatic patients allergic to house dust mites.methods-from 4 000 patients seen in the last 4 years in an out-patient allergy clinic 44 cases of hemoglobinopathies have been confirmed by red cell count, hemoglobin electrophoresis, assays of hemoglobin a2, f, and s, and sickle cell test.all these patients had allergic disease characterized by clinical history, skin prick test to aeroallergens total and specific ige (rast-cap-feia) and respiratory function evaluation. results -hemoglobinopathies: betathalassemia 39 cases, betadelta thalassemia 2, sickle cell trait 2, hemoglobin c.1. aside of 4 cases of urticaria, all the patients presented respiratory allergy, rhinitis in 12 cases of thalassemia and 1 hemoglobin c, asthma with or without rhinitis in 25 cases of thalassemia and 2 cases of sickle cell trait. therefore asthma was present in 67, 5%. in a group 491 of respiratory allergic patients without hemoglobinopathies, 57% had asthma and 43% only rhinitis. conclusions -the prevalence of asthma is higher in hemoglobinopathies.(p<005 square chi test). hemorheological changes probably greater rigidity of red blood cells and capillary bed can contribute to bronchial hypereactivity. detection of hemoglobinopathies must be done in asthmatic patients with slight anemia or mycrocytosis. we investigated the role of aspirin-exacerbated respiratory disease (aerd) as a risk factor for the development of airway remodeling. patients with aspirin intolerance develop hyperplastic sinusitis with fibrosis and nasal polyposis. we speculated that similar mechanisms could be acting in the lower airway and that these individuals would demonstrate more severe asthma and evidence for airway remodeling. the epidemiology and natural history of asthma: outcomes and treatment regimens (tenor) study is a multicenter observational study of subjects with severe or difficult-to-treat asthma. baseline data were compared between subjects 18 years who reported asthma exacerbation following aspirin ingestion and those who did not. the primary measure of asthma remodeling was the maximally achieved post-bronchodilator spirometry. adult subjects with aerd (n=469) were compared with aspirin tolerant subjects (n=3009). subjects with aspirin intolerance demonstrated evidence for airway remodeling as shown by lower post-bronchodilator predicted fev 1 . in addition, they were more likely to have physician-assessed severe asthma, to have been intubated, and to have required high-dose inhaled corticosteroids or oral corticosteroids in the previous 3 months. we conclude that aspirin intolerance is associated with remodeling of both the upper and lower airways. asthma is a complex and variable disease with two main components, airway inflammation and smooth muscle dysfunction. according to national and international asthma guidelines, subjects with persistent asthma can be classif ied into one of three categories (mild, moderate, or severe) based upon lung function, symptoms, nighttime awakenings, medications and exacerbations. though it is widely believed that there is a high degree of variability in both pediatric and adult subjects with asthma, few studies have compared variability between them. therefore, an analysis of previously conducted asthma studies was undertaken to evaluate pediatric subjects aged 4-11 years (n=276) and adult subjects (n=85) previously receiving short-acting beta 2 -agoinsts alone in seven double-blind, randomized, 12-week trials. the analysis is limited to subjects who were randomized to placebo in these trials. during the study, subjects exhibited marked fluctuations in asthma severity. despite the fact that all subjects met criteria for moderate or severe asthma at baseline, 27%, 18%, 48% and 8% of weeks for pediatric subjects and 9%, 14%, 71%, and 6% of weeks for adults were spent in the intermittent, mild, moderate and severe categories, respectively. a summary of severity classification based upon symptoms and albuterol use is presented below. in addition, based upon pef % predicted, 62% and 52% of weeks were spent in the intermittent/mild category for pediatric and adult subjects, respectively. however, fluctuations in pef occurred frequently, with 46% of pediatric subjects and 30% of adult subjects experiencing 10 changes in severity based on pef over 12 weeks, indicating more variability in pediatric subjects. this analysis clearly demonstrates that asthma is a variable condition and that both pediatric and adult subjects frequently move between severity categories. furthermore, there are marked differences in severity classifications between pediatric and adult subjects. asthma severity, and consequent optimal therapy, cannot adequately be assessed by discrete, point-intime assessments of lung function, frequency of albuterol use, or asthma symptoms, especially in the pediatric age group. studies suggest that there may be an association between single nucleotide polymorphisms (snps) in the beta 2 -adrenergic receptor gene (adrb2) and the response to beta 2 -adrenergic bronchodilators. polymorphisms at codon 16 have been at the center of this debate. therefore, a retrospective analysis of six large, randomized trials was conducted to evaluate clinical responses to salmeterol administered with fluticasone propionate (fsc) 100/50mcg bid for 12 weeks in patients ( 12 yrs) with moderate or severe asthma and differing adrb2 polymorphisms at codon 16. baseline demographics were similar for all genotype subgroups. all measures of asthma improved over baseline and were similar across arg16/gly subgroups at 12 weeks. pairwise comparisons were conducted between genotypes and there were no differences other than fev 1 as noted in the table. findings from this retrospective analysis show that regardless of arg16/gly genotype, clinical response to salmeterol with an ics was similar during chronic dosing. although prospective studies are needed to fully understand the effect of adrb2 polymorphisms on response to therapy with a long-acting beta 2 -agonist, this analysis suggests that therapy with salmeterol and an ics together is appropriate for caucasian patients with differing arg16/gly genotypes. coronary artery disease(cad) and asthma commonly coexist in adults. iv dipyridamole thallium scintigraphy is considered a safe non-invasive technique in the evaluation of cad when patients can't exercise. dipyrdamole is a purine that blocks reuptake of extracellular adenosine increasing serum adenosine levels after iv administration.this results in a transient coronary vasodilation increasing the sensitivity of the thallium study. methylxanthines reverse the effects of endogenous adenosine by competitively antagonizing adenosine at local purinoreceptors. we report a case of a 69 yr.old female with a 30 year history of stable mild persistant asthma treated with daily salmeterol and low-dose fluticasone. she was referred to cardiology for atypical chest pain. within minutes of a standard iv dose of dipyridamole the patient reported chest tightness, cough, and wheezing. all these symptoms, typical of her past asthma flares, were abolished within 5 minutes of an aminophylline infusion and before albuterol was administered.the stress test was completed and was normal. inhaled adenosine induces bronchconstriction and is used as a probe for bronchial hyper-responsiveness. commercial iv adenosine preparations used in treating supraventricular tachycardia are reported to induce bronchospasm in known asthmatics. since serum levels of adenosine are transiently increased following iv dipyridamole, bronchospastic symptoms during dipyridamole stress tests should not be unexpected. an earlier study found an increased incidence of wheezing in 39% of patients with known copd/asthma undergoing dipyridamole stress tests despite pretreatment with beta agonists. the marked decline of theophylline use for chronic asthma in recent years may actually be increasing the incidence of this risk. cardiologists performing thallium stress tests are familiar with the risk of asthma flares after dipyridamole. they use iv aminophylline frequently to reverse several types of clinical dipyridamole reactions, including bronchospasm. on the other hand, we are impressed that allergists are relatively unfamiliar with this association. allergists and asthma specialists as well as asthmatic patients should be aware of risk of acute dipyridamole induced bronchospasm during elective cardiac stress testing. it has been shown that adhesive molecules are involved in inflammatory diseases of the lungs such as bronchial asthma. the purpose of the study was to measure and establish possible difference in serum levels of soluble icam-1 in 42 atopic patients (patients with allergic rhinitis and patients with bronchial asthma) in comparison with 28 patients without atopy (patients with asthma without rhinitis); whether there is a difference in sicam-1 levels between groups of 26 patients with allergic rhinitis and asthma in comparison with group of 16 patients with allergic rhinitis only and also in comparison with 10 healthy controls. results of the study have substantiated statistically significant difference in sicam-1 levels between all groups of patients in comparison to healthy control, but no statistically significant difference in sicam-1 levels between patients with and without atopy (z=-1.738) or between patients with allergic rhinitis and bronchial asthma in comparison with group of patients with allergic rhinitis only (z=0.00). conclusion: icam-1 is an important marker of inflammation in patients with allergic rhinitis as well as in those with bronchial asthma. atopic status does not influence differences in sicam-1 levels. although mean sicam-1 levels were higher in patients with allergic rhinitis and bronchial asthma (312.71 ng/ml) in comparison with mean sicam-1 levels in patients with allergic rhinitis only (279.69 ng/ml), no statistically significant difference was noted in sicam-1 levels between these groups of subjects, i.e. asthma itself did not contribute to statistically significant increase of sicam-1 levels. k. nadarajah * , g.r. green, m. naglak, abington, pa. objective: to study the various clinical outcomes of penicillin skin testing (pst) in a community-based hospital and to determine the percentage of patients who have an antibiotic modification and the choice of antibiotic used following the result of pst. method: this study is a retrospective chart review of all in-patients who were penicillin skin tested during a period of 6.6 years(jan 1997 to july 2003). information was collected on 101 patients using a detailed data collection form. data was summarized using descriptive statistics including frequencies and percentages. results: of the 101 patients penicillin skin tested, 92 had a negative test, five had a positive test and in four patients the test was indeterminate(histamine controls were negative). eighty six percent of the patients had a history of penicillin allergy, 7.9% had a history of cephalosporin allergy and 5.9% had a history of both penicillin and cephalosporin allergies. the duration of antibiotic prior to pst ranged from zero to 18 days. there was a 72.7% (67/92) reduction in the use of vancomycin, a 24.9% (23/92) reduction in the use of floroquinolones and a 7.6% (7/92) reduction in the use of aminoglycosides following pst. aztreonam was used in 19.6% (18/92) of patients before pst and in zero patients after pst. use of penicillin-based drugs was 49% (45/92) after pst and cephalosporin use increased from 4.4% (4/92 (all third generation cephalosporins)) to 47.8% (44/92( second and third generation cephalosporins)). i.e., 97% of patients with a negative pst received a penicillin or a cephalosporin. vancomycin usage was higher among pst positive patients. endocarditis was the diagnosis in 22% of all patients skin tested and staphylococcus aureus (23.8%) and enterococcus (16%) were the most common organisms on culture. there were no serious adverse reactions to the use of penicillins or cephalosporins when used following a negative pst, and there were no adverse reactions to penicillin skin testing. conclusion: in the population studied, pst lowers the usage of vancomycin, floroquinolones and aminoglycosides and increases the use of penicillins. third generation cephalosporin usage was increased by pst in our study. overall, pst results in antibiotic modification that could lower the emergence of multi-drug resistant organisms and vancomycin resistant enterococcus. the aim of the study was to find a new marker which could be easily done to predict about possible allergy development in infants. preventive procedure is always economically better than treatment itself.food allergens are able to stimulate lymphocytes even in prenatal period. immunological maturity of newborns is still in the center of our interest because its dependence on many different factors. the question was if cd30 activity in cord blood and in 1-year-old children correlates with development or not of allergy? the examined group consists of 120 newborns(58, 2% of boys). breastfed were only 87 children until 3-months-old and 62 until they were 6-monthsold. total ige from cord blood and mothers' blood taken at delivery were measured using unicap. immunological detection was done using fluorocytometry(becton nad dako). additionally parents were questionaired( family atopy). our reaults show that positive family atopy history(group a) or elevated level of ige( group b) or symptoms of allergy in first 3 months of life(group c) does not correlate with cd30 antigenicity. in all subgroups cd30 frequency was 1%. our findings show unfortunately that cd30 cannot be a predictive marker for allergy development. rationale: the etiology of eosinophilic esophagitis (ee) is unknown and the relationship to a type i allergic response is unclear. comparison of patients with positive and negative type i allergy testing may further clarify ee and determine what are the most common food allergens. methods: this is a retrospective chart review of medical records from january 2000 to january 2001 of children 1 year of age with biopsy confirmed ee (n = 42). ee was defined as having 15 eosinophils per high power field on esophageal mucosal biopsy. patients were grouped according to positive (n = 26) and negative (n = 16) allergic responses on skin or radioallergosorbent testing (cap rast, pharmacia). skin testing with commercial extracts (hollister-stier laboratories and greer laboratories) was performed in an allergist's office. a wheal of 3mm greater than the negative control on skin testing or ige > 0.35 ku/l on rast was considered positive. we performed fisher exact analysis to determine if associations existed between a type i allergic response and factors such as age, symptoms, peripheral eosinophilia, and personal and family history of atopy (asthma, allergic rhinits, and atopic dermatitis). results: ee patients with a positive type i allergic response were significantly younger than those with a negative response (mean 4.6 yo, median 4.5 yo, range 1 to 10.3 yo versus mean 8.5 yo, median 7.8 yo, range 1 to 21 yo; p = 0.0065). vomiting as a presenting symptom was significantly increased in the allergic population (85%, 44%; p = 0.014) and abdominal pain as a chief complaint was significantly increased in the non-allergic population (31%, 69%; p = 0.026). personal and family history of atopy and peripheral eosinophilia were similar between groups. the most common allergens were cow s milk (54%), peanut (46%), egg (42%), soybean (38%), and wheat (27%). conclusion: patients diagnosed with ee who present at a young age or who present with symptoms of vomiting may have a type i allergic response contributing to the esophageal eosinophilic inflammation. consistent with food allergies in children, milk, peanut, egg, soybean, and wheat were the most common foods found with allergy testing. because the positive predictive accuracy of food annals of allergy, asthma & immunology testing is only about 50%, the challenge remains to develop a specific plan to confirm causative foods such as through oral challenges or elimination trials. rationale: the relationship between specific ige and igg levels in atopic individuals is unknown. increasing antigen exposure is expected to increase igg production, however, the influence on ige levels is unclear. to further clarify this relationship the following studies were conducted. methods: three hundred and sixty tandem determinations of specific ige and igg levels in 65 individuals were collected using the immunocap 100 instrument (pharmacia diagnostics) and commercially available reagents. only subjects who had ige levels > 0.35 ku/l and igg determinations > 2 mg/l to the same specific allergenic species were included in the study. specific ige and igg determinations were to alternaria alternata, aspergillus fumigatus, penicillium notatum, cladosporium herbarium, felis domesticus, canis familaris, dermatophagoides farina, and periplaneta americana. correlation coefficients were calculated using excel (microsoft). results: the review of this data set yielded 55 tandem determinations of specific ige and igg levels in 25 atopic individuals. the most common ige sensitization was to alternaria. sixtyfour percent of individuals had specific ige directed against alternaria. the lowest ige response rate was for cat and roach. only 20% of individuals had an ige response to those specific species. correlation coefficients (cc) were calculated between specific ige and igg levels and were positive for all eight species tested. the highest cc was for dog (n = 6, cc = 0.92) followed by aspergillus (n = 13, cc = 0.71), penicillium (n = 6, cc = 0.69), alternaria (n = 13, cc = 0.62), cladosporium (n = 8, cc = 0.35), cat (n = 5, cc = 0.31), and roach (n = 5, cc = 0.16). the lowest correlation was for dermatophagoides farina (n = 7, cc = 0.03). conclusion: when individuals with measurable specific ige and igg levels are considered, there appears to be a positive correlation between specific ige and igg levels for many allergenic species. factors that influence the correlation are likely to be both genetic and environmental. identifying this link and a clinical application are our next challenge. recently, protein microarray tests are competing with traditional allergenspecific in vitro assays. while the advantages of microarrays in allergy testing are attractive, microarray analysis alone has not been very effective in reducing analysis time and automated microarray equipment typically is much larger than its benchtop counterparts. we have incorporated microarray technology into an automated microfluidic cartridge that provides rapid allergen testing using a compact, low-cost, desktop instrument. the injection molded microfluidic cartridge (fig. a) contains arrays of miniature pumps and valves that direct reagents independently to a solid phase reaction area. standard ige (nibsc) as well as allergens were immobilized within the cartridge to form a protein microarray. a small desktop analyzer actuated the pumps in the cartridge to automatically carry out a chemiluminescence-based elisa reaction. total ige quantitation was performed in a 10 minute reaction. fig.b shows the resultant image of a dilution series of immobilized ige. concentrations ranged from 0.625 to 1000 iu/ml corresponding to 1.9 fg to 3 ng ige per spot. the average cv value for ige quantitation was 12% showing good linearity (r2 = 0.99) and 6 fg sensitivity. this quantitative ige curve is used to eliminate the effects of cartridge variability. specific ige detection was demonstrated using 3 extracts, d. pteronyssinu(dp), a.fumigatus and b. verrucosa(bv). a 2-step, 20 minute reaction elisa technique was employed. fig. c and d show the resultant images for bv and dp positive serum, respectively. the sensitivity of the dp specific ige test was further investigated using a positive class 1 serum sample confirmed by mast. dilution of the class 1 serum with negative control serum could then be detected on the cartridge down to an 8 fold dilution, resulting in a limit of detection in the range of 0.08-0.31 iu/ml for dp specific ige. we have demonstrated total ige and allergen-specific ige detection with total analysis times less than 30 minutes in a convenient, low cost system using a microfluidic-based microarray cartridge. such rapid diagnosis allows physicians to provide treatment while the patient is still in the office. background: accurate allergen skin tests (ast) are the basis of optimal care of the allergic patient. completing these tests on the first visit can expedite the diagnosis and treatment and offer patients(pts.) efficient use of their time and increase satisfaction. appropriate pre-visit preparation is necessary to reduce the variables that affect ast outcomes. both positive and negative skin test controls are necessary to assure the ast are reliable. methods: we conducted a retrospective chart review of 1, 248 sequential pts. skin tested at an allergy practice, in order to examine the success of our pre-visit instructions and to identify other medications that may affect ast. pts. were given verbal and written instructions to discontinue antihistamines and other histamine-1 receptor antagonist medications (h-1a) 5 days before their visit. they then underwent ast only if adequate positive (histamine) and negative (saline/vehicle) controls were obtained. results: only 53(4.2%) of the 1, 248 pts. had inadequate histamine response (ihr). of these 53 pts., 23(1.8%) had used h-1a within the prior 5 days. 18(1.4%) pts. had taken h-1a in the recent past, but stopped them at least 5 days prior to testing. 12(1.0%) pts. had no exposure to h-1a. the use of psychiatric medications in the pts. with an ihr was also examined. of the 18 pts. who discontinued h-1a for more than 5 days, 13(76%) were also taking various medications drugs used in the treatment of psychiatric disorders (ssris, benzodiazepines, atypical antidepressants & antipsychotics) which are not usually associated with h-1a. the group of 12 of the pts. with no prior use of h-1a included 10 (83%) who were also on various psychiatric medications. the high prevalence of psychiatric medication use in these two groups is in contrast to the to 23 pts. with recent use of h-1a in whom 43% were on psychiatric medications. conclusion: pre-visit patient education about discontinuation of h-1a can lead to successful first visit ast in the overwhelming majority of patients (95.7%).failure to stop h-1a at the appropriate time occurred in only 1.8% of the pts. 1.4% of pts. discontinued h-1 a for the recommended 5 day interval, but still had ihr. another 1% of pts. had an ihr yet no obvious use of h-1a. a few pts. taking psychiatric medications not normally associated with h-1a had ihr. further investigation into this issue is warranted. a. blaziene * , a. chomiciene, l. jurgauskiene, n. ciaponiene, vilnius, lithuania. background: sublingual specific immunotherapy (sit) is accepted as an alternative treatment to subcutaneous sit of seasonal allergic rhinitis. the aim of this study was to evaluate changes in allergen-specific ige and basophil degranulation test (bdt) before and after 1 year of sit with a grass pollen mix. methods: 6 patients (3 male and 3 female, aged 25-44 years) having sensitization to grass pollen with seasonal allergic rhinitis were treated with standardized grass pollen extracts. the blood samples were collected before and after 1 year of sit assessing allergen-specific ige using an elisa method and bdt using a direct immunofluorescent method employing monoclonal antibodies. results: the sit was well tolerated by all patients. an increase in total ige after sit was observed in all patients. specific ige concentration was increased in 2 (33.3%) patients, while the bdt result was greater in 3 (50%) patients after 1 year of sit. conclusions: allergen-specific ige and bdt may serve as markers to indicate the clinical efficacy of sit for seasonal allergic rhinitis patients. a.g. palma-carlos * , s.l. silva, m.l. palma-carlos, lisboa, portugal. introduction -the incidence of primary immunodeficiencies in patients attending an out-patient center for clinical allergy and immunology depends on the recruitment and on patients refered. methods -in the last 3 years 3 000 patients have been observed in lisbon clinical allergy center reporting allergic diseases or repeated infections or refered by specialists (ent, gynaecology, internal medicine, pediatrics, or gp.). screening for primary immunodeficiencies has been done by electrophoresis, assay of immunoglobulins and complement, igg subclasses and flux-cytometry for t, b and nk cells. results -102 cases (3, 4%) of primary immunodeficiencies have been diagnosed. humoral: 13 deficits of iga (12, 7% of immunodeficiencies), 7 of igg (6, 8%) 16 of igg subclasses (15, 7%) 11 of common variable immunodeficiency cvi-(10, 8%), 2 of igm (2, 0%). complement: 4 cases of c1 esterase inhibitor deficiency (3, 9%) cellular: 45 cases of mucocutaneous candidiasis (44, 1%) in fertile females, 2 cases of cd4 deficiency (1, 9%). combined: 2 cases of deficiency of the couple cd40/c40 ligand with igg deficiency (1, 9%).conclusions -in the present series chronic mucocutaneous candidiasis is the prevalent primary immunodeficiency, due to the number of patients refered by gynaecologists. aside of this group where a decrease of nk cells was the more common immunologic pattern, humoral immunodeficiencies are frequent, mainly cvi, iga, igg and igg subclasses deficiencies. the search for immunodeficiencies must be done in all patients with repeated infections attending immuno-allergology departments. a case of atypical c2 complement deficiency. c.c. randolph * , waterbury, ct. introduction:c2 complemennt deficiency occurs in 1 in 10, 000 individuals .it results in decreased opsonization and chemotaxis with increased prevalence ofsle and other rheumatic disordersas well as enhanced vulnerability to pyogenic infection.there are two types of c2complement deficiency :type 1(90%associated with sle)with no protein translation and type ii with absence of protein secretion.we present a 15 year old white female otherwise healthy with recurrent urticaria and angioedema with c2 complement deficiency. method:case report:a 15y/o w/f athlete presented with a two month history of recurrent hives and angioedema which she associated with ingestion of halloween candy .one week before evaluation she had hives with coconut as well.her history was othewise unremarkable except for recurrent uti's, annual sinusitis, pneumonia in 1998 as well as migraines.she denied sexual activity.her physical exam was normal.results:an evaluation for autoimmune disease revealed normal esr, ana, dsdna, mono and hepatitis serology as well as lyme titers however her ch50 was low17u/ml(normal 26-58u/ml)and evaluation of complement revealed c4 14mg/dl(normal 16-47mg//dl)and c2 <1.3mg/dl(normal 1.6-3.5mg/dl)with normal c3, c5-c9.her father had nor-malc4 but c2 was 1.4mg/dl (normal 1.6-3.5mg/dl)her sister had c2 of 1.5mg/dl and normal c4 and her mother had normal c2 and c4.her workup included positive prick skin test to ragweed, ash and grass and she was started on rhinocort and clarinex seasonally.she has been followed for one year with resolution of hives and is asymptomatic.her diagnosis had been confirmed by a pediatric rheumatologist.conclusion;we present an atypical case of c2 complement deficiency in an currently asymptomatic individual. background: we diagnosed a 10 month old male with cgd and aspergillus brain abscesses and pulmonary infiltrates. review of the literature suggests he is one of the youngest cases of cgd complicated by cerebral aspergillosis. case report: a ten month old male previously seen for unexplained persistent pulmonary infiltrates since 4 month of age presented to our hospital with lethargy, fevers, and increased irritability. ct scan of the head revealed severe hydrocephalus. multiple brain abscesses were found on surgical exam and cultured for aspergillus fumigatus. he had a history of failure to thrive and hypotonia since birth. family history was significant for parents that were second cousins. there was no family history of recurrent infections or childhood deaths. physical examination was significant for a child with weight and height in the 25% and 75%, respectively with persistent fevers >38.5 c, hypotonia, hepatosplenomegaly, and left lower lung field rales. initial immune workup was normal except for elevated total ige of 215 ku/l. cd4+ (526/m3) and cd8+ (332/m3) t cells were initially low, but later increased to normal levels without intervention. neutrophil oxidase activity assessed by dihydrorotamine 123 (dhr) oxidation was 0% of control. superoxide production assessed by cytochrome c reduction was zero. the patient was treated with interferon ? for presumed cgd. his aspergillus brain abscesses improved with intravenous voriconazole and caspofungun. the responsible organism for his recurrent pulmonary infections was not identified, but improved with antifungal therapy and broad spectrum antibiotics. the dhr test of both parents and siblings were normal. antibody testing showed a complete deficiency of the p67 protein with normal levels of gp91, p22, and p47 proteins. evaluation of the exons of the ncf-2 gene that encodes the p67 protein were normal. conclusion: mutations due to p67 protein deficiency are responsible for 3% of cases of cgd. there has been only one previously reported case of p67 deficiency without ncf-2 exon mutations in which a single point mutation in an intron of the ncf-ii gene was identified. fungal infections may complicate cgd, however cerebral aspergillosis is seldom encountered. this case annals of allergy, asthma & immunology reports a 10 month old with cgd due to a p67 deficiency without exon mutations complicated by cerebral aspergillosis. introduction: lymphoid interstitial pneumonia is an uncommon condition which is considered both as a disease per se or as an inflammatory pulmonary reaction to various external stimuli or systemic diseases; however, at the present time most of the cases remain idiopathic. we present a case of an infant who developed a lymphoid interstitial pneumonia associated to a deficit of interferon-production. methods: we describe a clinical case and review the medical literature. results: we present the case of a five months old male with a history of cough, respiratory distress and four previous hospital admissions regarded as a 'pneumonic' events from two months of age and four siblings dead (two of them with 'abdominal disease' and one of them with candidiasis) during their first year of life. the parents referred no consanguinity. the physical examination showed a malnourished child with signs of bilateral lung consolidation. oxygen supplementation and intravenous antibiotics were started. our patient didn't have neonatal history related to the present compliant. the chest film revealed a diffuse bilateral interstitial pattern which was confirmed by pulmonary ct scan. sweat chloride test, hiv and epstein barr virus serology test were all negative. an initial immunologic evaluation reported slightly increased leukocytic count, hypergammaglobulinemia, lymphocityc flow citometry and nitro blue tetrazolium reduction test in normal parameters. the open lung biopsy showed a thicked pulmonar interstitium with moderate number of lymphocytes with not atypical pattern. our patient was discharged with clinical improvement, ambulatory oxygen supplementation and inhaled fluticasone-salmeterol. in an ambulatory follow-up we evaluated lymphocytic production of cytokines and we found no levels of interferon-. we started weekly administration of oral transfer factor. the patient showed an excellent clinical improvement evidenced by no more oxygen dependence, weight gaining and absence of further hospital admissions. con-clusions: our patient represent a new linkage between lymphoid interstitial pneumonia and primary immunodeficiency characterized by interferonproduction deficit, maybe with a non previously described molecular defect and a probably autosomic recessive inheritance pattern; also we confirmed the transfer factor usefulness to induce gamma interferon endogeous production. objective: marijuana is a schedule class 1 psychoactive control substance more frequently used by the oriental societies( in the event of jubilation) causing altered state of conscience, euphoria, its prolonged use is followed by addiction and dependence.it contains more than 426 chemical entities with over 60 are of the cannabinoid i.e, cannabidiol (cbd), cannabinol (cbn), and delta-9, tetrahydrocannabinol (thc) the later on use has been associated with craving for further use of narcotics just to enhance the euphorient effects .the active ingredients i.e, delta-9-tetrahydrocannabinolthc) and other cannabinoids(thc) are liquid soluble at high concentrations which alters membrane function, resulting in alterations in immune cell response. cannabinoid has immunosuppressant properties causing impaired cell-mediated and humoral immune system activities, cytokine production, leukocyte migration and natural killer-cell(nk) activity resulting in reduction in the host resistance to bacterial and viral infection, (pms)with hiv infection are at higher risk of developing aids, infection by opportunistic bacteria, fungi, or viruses (pms), when compared to non marijuana smokers, have more respiratory ill-ness.cannabinoids also characteristically as being immunomodulators i.e, generally suppressing but occasionally enhance some immunological responses, some have suggested that the immunosuppressive effects of cannabinoids might be useful clinically; for example, in treating multiple sclerosis.as per clinical response cannabinoids had been found exacerbating existing allergies from antigenic complex (eliciting formation of specific antibodies/metabolites as a hapten, combining with a body proteins). methods .in the follow up studies including 18individuals(all males age25-60 years) with(pms).of more than 6 months having serum level of tch >10um, there have been 20 times reduction in the proliferation of t lymphocytes with a proportionate increase in the proliferation of b lymphocytes, reduction in the cytotoxic activity of t lymphocytes, reduction in macrophage activities i.e, phygocytosis rationale: patients with xla are subject to arthritis and cellulitis. these three relatives have had similar courses, suggesting infectious etiology. methods: two brothers and a cousin, from an african-american family known to carry xla, have been diagnosed with xla and chronically treated with ivig. all three have developed arthritis and cellulitis of a lower extremity. the eldest, now 23 years old, had a 5-month waxing and waning arthritis and cellulitis that was refractory to the oral antibiotics attempted. his younger brother had not yet accomplished pubertal changes at the age of 16, and was below 3rd percentiles for weight and height. he also had several months waxing and waning arthritis and cellulitis refractory to oral antibiotics. he and his cousin have had esophogastroduodenoscopies that show gastritis and duodenitis with heavy growth of an organism visualized by specific staining for h. pylori. the cousin has also experienced more recent weight loss and arthritis/cellulitis. results: the younger brother had a blood culture that grew a curved gram-negative rod. a subsequent culture at the state lab grew a similar organism that was urease-negative. the two brothers are at or near completion of a six-month course of ertapenem and gentamicin; they have had resolution of their arthritis and cellulitis. the younger has gained 10.3 kg and experienced his pubertal changes at the age of 17 years. con-clusions: there are prior reports of several different related organisms that can cause arthritis and cellulitis in patients with xla. these include helicobacter, campylobacter, and flexispira species. the urease-negative organism would not be flexispira, but may be a campylobactor species or helicobacter canis. the results from this family suggest that long-term combination iv antibiotics may be indicated for patients with this syndrome. slow virus infections have been reported to affect the central nervous system. parvovirus b19, herpes and cmv are usually not included in the diagnosis of cns diseases nor has been associated with cns manifestation in an immuno-compromised patient. in this abstract, we report on a neurological manifestation that can be linked to ebstein-barr/herpes virus and parvovirus-b19 in an immune deficient patient. a 50 year old female presented to our clinic with a history of chronic fatigue symptoms, daily arthralgias, frequent sinus infections and idiopathic tremors for the past 6 years. the patients neurological evaluation, including mris was found to be within normal limit, despite worsening tremors of her head and arms. a full clinical and labora-tory evaluation was performed which revealed igg subclass, low t-cell numbers and functioning, low response to specific antibodies and positive igm for ebv, parvo and cmv. the patients common immune deficiency coupled with chronic viral infection and worsening tremors led us to try high dose ivig (2g/kg divided over 3 days on cycles of every 3 weeks). within three months of starting ivig, the patient had a large reduction in her tremors and negative igms for parvovirus, herpes and cmv. given our findings, we suggest that ivig may play a role as a neuro-immune modulator, as has been shown in other neuro-immune diseases. we further suggest that the interaction between parvovirus b-19, herpes and cmv as part of the slow viruses, combined with underlying immune disorder may lead to neurological presentation and high dose of ivig can reverse the syndrome. purpose: to date, only two patients older than fifty years of age have been diagnosed with velocardiofacial syndrome (vcfs); this case represents the third such patient. methods: physical and laboratory findings of the patient are presented as a case report. results: we describe a sixty-six-year-old male, recently diagnosed with vcfs, confirmed by fluorescent in-situ hybridization (fish), was referred by his psychiatrist for comprehensive care. he had a history of cleft palate and learning disabilities as a child, with the onset of psychiatric illness in his late teens. although there was no history of cardiac disease, the patient had agenesis of the left renal artery as well as other vascular anomalies, including hypoplasia of the left a1 segement of the anterior cerebral artery. other findings included hypothyroidism, hypoparathyroidism, sensorineural hearing loss, and typical facies. he also suffered from chronic candidiasis. immunologic laboratory studies revealed over a 2:1 ratio of cd8:cd4 t cells with a decreased cd4 percentage, markedly diminished b cell percentage, decreased t cell mitogen responses, and markedly decreased b cell mitogen responses. nevertheless, total serum igg and specific antibody responses remained normal, with a low serum igm. conclusions: this patient meets the criteria for the diagnosis of vcfs; only the third reported case diagnosed over the age of fifty. though his immunologic findings may be consistent with his diagnosis, they may be due in some part to immunologic senescence given his age. background: the classic presentation for patients with xla and mutations in the btk gene includes marked hypogammaglobulinemia, absent b cells, and significant sinopulmonary infections beginning at 4-5 months of age. typically, mds presents with anemia, leukopenia, monocytosis, and thrombocytopenia with recurrent infections, skin rash, and hepatosplenomegaly. many of these subjects demonstrate chromosomal abnormalities including monosomy 7. we describe a case of mds presenting with some features of both conditions. clinical history: we present a 2 year old male with a suspected diagnosis of xla, chronic sinopulmonary disease beginning at 12 mo, diffuse bronchiectasis, and agammaglobulinemia with absent b cells. he had wbc =3750 cells/ul, with 60% monocytes and platelet count of 122, 000 /ul. he had no anemia, hepatosplenomegaly, or skin rash. bone marrow biopsy showed mds with absent plasma cells and monosomy 7 in 95 % of his cells. btk expression and sequence analysis were normal. conclusion: while xla is the most likely primary immune deficiency that would present in a male with the clinical picture described above, this case illustrates the importance of maintaining an expanded differential diagnosis in patients with presumed primary immunodeficiency. j. wang * , l. mayer, c. cunningham-rundles, new york, ny. background: chronic granulomatous disease (cgd) usually results in acute or chronic infections with a defined spectrum of bacteria or fungi. other characteristics include inflammatory disorders, including genitourinary or mucosal inflammation resembling inflammatory bowel disease. gm-csf has been used in the treatment of mucosal inflammation in glycogen storage disease ib and crohn's disease with some success. objective: to explore the use of a novel therapy in the treatment of mucosal inflammation associated with cgd. methods: the patient was treated with daily gm-csf (0.6 mcg/kg/dose subcutaneous injection) along with parenteral antibiotics and total parenteral nutrition. after 4 days on gm-csf, hydrocortisone enemas were added due to continued pain and swelling. results: rectal and abdominal pain significantly improved, blood streaked stools decreased; he tolerated a regular diet and was discharged home 10 days after starting gm-csf. he now has no rectal or abdominal pain and no blood in the stool. conclusions: gm-csf may be a useful therapy for the management of mucosal inflammation in cgd patients. it appears safe and well tolerated and permits avoidance of immune suppressive alternatives. introduction: management of autoimmune cytopenias in patients with cellular immunodeficiency may be very challenging. treatment of these cytopenias with corticosteroids may potentiate the immune dysfunction leading to further infections. methods: we describe a 15 month-old female with a cellular immunodeficiency and autoimmune thrombocytopenia / hemolytic anemia being treated with steroids and ivig without improvement. rituximab, a monoclonal antibody that binds to b-lymphocyte cd20 surface antigens, was initiated. results: this patient presented at 11 months of age with failure to thrive, eczema and recurrent bacterial pneumonias. she was diagnosed with a t lymphocyte immunodeficiency with decreased absolute numbers of cd3 (493), cd4 (365), and cd8 (80) lymphocytes. t cell function was markedly decreased as measured by pha, cona, and pwm. she developed chronic thrombocytopenia with platelets of less than 20, 000 and coombs positive anemia with hemoglobin of 6 g/dl. she was placed on 4mg/kg/day of prednisone with minimal improvement and continued to develop severe pneumonias. rituximab 375 mg/m 2 /week was initiated for 5 doses. her cytopenias improved with an increase in both platelet count (229, 000) and hemoglobin (15.1g/dl). her absolute cd19 count fell from 1319 to 0. she was weaned off steroids permanently. she has also continued on ivig, prophylactic antibiotics, and as needed rituximab pending bone marrow transplant. conclusions: rituximab, a monoclonal anti-cd 20 antibody, may be helpful in treating autoimmune cytopenias in patients with cellular immunodeficiency in which steroids need to be avoided. ivig antibody replacement substitutes for the subsequent humoral antibody depletion due to induced secondary b cell lymphopenia. introduction: the patient is a 3 month old male born at full term. during his prenatal course he was noted to have a pulmonary abnormality in his left lower lobe at approximately 12 weeks of age. serial ultrasounds and fetal mri were consistent with the diagnosis of congenital cystic adenomatoid malformation (ccam). histology from elective resection of the lesion performed at 2 months of age revealed no evidence of ccam. silver stain revealed florid pneumocystis jiroveci (carinii) infection. the patient was completely asymptomatic. the differential diagnosis for this infection in the newborn period includes hiv infection, severe combined immunodeficiency, and x-linked hyper-igm syndrome. the infection has also occurred in newborns with apparently normal immune systems. methods: immunology evaluation was performed to evaluate the patient for the listed diagnoses. a complete blood count was performed. lymphocyte subsets were performed by flow cytometry. quantitative immunoglobulins g,a, m and e were measured by nephelometry. specific antibodies to tetanus and diptheria were determined by elisa. hiv status was ascertained by western blot and dna pcr. dichlorofluorescein assay was performed. fluorescenceactivated cell sorter technique was used to evaluate cd40 ligand. lymphocyte stimulation to mitogens and tetanus were performed. results: the patient had negative hiv1/2 antibodies and a negative hiv dna pcr. the result of the complete blood count was normal with an absolute lymphocyte count of 6, 750. the patient had normal percentages and numbers of cd3, cd4, cd8, cd19, and natural killer cells. lymphocyte stimulation to mitogens and tetanus was normal. dcf assay was normal. facs revealed normal levels of cd40 ligand. at 5 months of age he had protective titers to diptheria and tetanus titers of 0.52 iu/ml (protective >0.6 iu/ml). at 5 months igg, a, m and e were 235 (normal 218-636), <6, 36, and <5 respectively. conclusion: immunologic testing revealed no evidence of severe combined immunodeficiency, hiv infection or x-linked hyper-igm syndrome. the patient remains well clinically with normal growth and development with no subsequent infections. pneumocystis jiroveci pneumonia can be seen in infants with apparently normal immune systems. commercial preparations of igg produced for iv use (ivig), must fulfill regulatory requirements. the method of preparation, however, may produce alterations in the content or function of igg subclasses such as disturbed composition which can be reflected as a lowered clinical effectiveness of the product. we report 2 patients with specific igg3 immunodeficiency, that, when changed to a new ivig preparation, had lessening of clinical effectiveness of the new product but who also experienced recovery after they were changed to a different ivig product. case 1. a 53 y/o male with hx of cervical lymphadenopathy associated with recurrent uri, fatigue, cognitive alterations and hypercholesterolemia. he was dx in 1993 with a specific igg3 immunodeficiency for which he was started on ivig infusions 400mgxkg q/4 w which brought his igg3 levels to normal values 21 mg/dl, (18-95 mg/dl) with good clinical improvement. when he was changed to a different ivig product with lower iga content his initial symptoms returned with decrease of his igg3 to 17 mg/dl (41-129 mg/dl) he was changed to a different ivig product with higher iga content with disappearance of all his previous symptoms. case 2. a 47 y/o female with history of chronic yeast infection, fatigue, joint pain, asthma, ge reflux, depression, hypothyroidism and shunt for hydrocephaly. she was diagnosed with igg1 and igg3 immunodeficiency and started on ivig 400 mg x kg x q/4 w. when changed to another ivig with lower iga content she experienced worsening of her symptoms during the 2 months that she was receiving it. she was changed to a different ivig product with a higher iga content with complete improvement of her symptoms. although the content of iga in product 1 (50-150 ug/ml) was significantly lower than in prod-uct 2 (720ug/ml), the total igg and igg subclass distribution (including igg3) were comparable therefore, the lack of clinical effectiveness of product 1 would appear to be related to qualitative alterations in the product related to methodological preparation possibly in removal of the iga or other physicochemical alterations affecting biologic activity which contributed to a lessened clinical efficacy of the product. we present these cases to alert the allergist-immunologist to these observations when treating patients with igg or igg subclass deficiencies. background: development of atopy has been known to occur in nonatopic recipients of bone marrow transplants ( bmt) from atopic donors. the reverse condition, with atopic recipients losing evidence of specific ige after bmt from non-atopic donors is quite unique. case report: ph is a 37 year old male referred for recurrent nasal congestion. as a teenager, the patient was treated for chronic allergic rhinitis and asthma, atopic dermatitis diagnosed as severe, and multiple food allergy. skin testing done as a teenager showed 3+ reactions to multiple pollen, dog, cat, most seafood, cashew, walnut and pecan. at 16 years of age, he underwent an allogeneic bmt from his non-atopic brother for acute myelogenous leukemia. asthma and atopic dermatitis were observed to clear almost completely and allergic rhinitis improve after the bmt. his blood type changed from 0+ to 0-. skin testing done on his current visit showed unremarkable responses to all pollen, cat, dog, dust mites and foods with good histamine control. discussion: the negative skin test results suggests that his current nasal symptoms are vasomotor in nature. considering the past severity of his symptoms, it appears unlikely that he lost his specific-ige sensitivity spontaneously. transfer of atopy is known to occur in non-atopic recipients of bmt from atopic donors. in a study of 12 patients undergoing allogeneic bmt for hematologic malignancies, nonatopic recipients developed positive skin tests with profiles similar to atopic donors. 1 a possible mechanism is passive transfer of memory b-cells which initiate specific-ige production with a donor pattern. a reverse of this mechanism could occur with the recipient gaining lymphoid precursors with no atopic tendency leading to clearing of atopy. the literature yielded one report of asthma resolving after high-dose chemotherapy and autologous stem cell transplantation the authors suggested that chemotherapy could have resulted in immune system reconstitution, normalization of the t-cell repertoire and resolution of asthma. introduction: primary central nervous system (cns) lymphomas constitute only a small percentage of central nervous system tumors in adults and are seen more frequently in aids patients and immunodeficiency states. they are extremely rare in children, even those with primary immunodeficiency. we present a 4-year-old female with combined immunodeficiency who developed an eber+ (ebv-associated) large b-cell lymphoma that was confined to the cns. methods: the patient is a 4-year-old female that was being evaluated for combined immunodeficiency who presented to our institution with acute fever and seizure activity. results: the patient had a history of recurrent pneumonia, otitis media, sinusitis, and upper respiratory infections. she also exhibited failure to thrive, chronic diarrhea, and sen-sorineural deafness. she was found to have a combined immunodeficiency with severe neutropenia and lymphopenia, low iga, low igm, and poor specific antibody response to protein and polysaccharide antigens. she had no response to a candida delayed hypersensitivity skin test. hiv tests were negative. she was thought to have acute meningoencephalitis causing the fever and seizure activity, however bacterial, viral, and fungal studies were negative. she received multiple anitbacterial, antifungal, and antiviral agents. she died secondary to brainstem compression due to severe cerebral edema. autopsy revealed an eber+ large b-cell lymphoma that involved about 80% of her brain and spinal cord. no tumor cells were found elsewhere. con-clusions: this case represents a rare manifestation of combined immunodeficiency: the development of a primary cns lymphoma. the tumor cells were found to be eber+ (ebv-associated). ebv is known to be associated with lymphoma, especially in aids and immunocompromised patients. only 6.5% of primary tumor sites in severe combined immune deficiency patients involved the cns in the immunodeficiency cancer registry that was instituted in the 1970s. v. litvinova * , g. muzlaev, krasnodar, russian federation. introduction: glyoma is one of the most prevalent diseases among all brain tumors and more than 35% of them are malignant. in the previous investigations it has been shown the immune disorders in patients with glyoma tumor. at the same time it was suggested the possible anti-tumor activity of some proinflammatory cytokines (tnf, il1, il8), which can penetrate via haematoencephalic barrier and induce the lysis of tumor cells. the aim of this investigation was to study the serum and spinal fluid concentrations of one of the key immunoregulatory cytokines -gamma ifn and il18 in patients with glyoma tumor. methods: the concentrations of the gamma ifn and il18 have been studied in serum and spinal fluid of 16 patients with glyoma by eliza method. in control, the same parameters have been studied in 6 patients with brain trauma. results: it has been shown that concentrations of gamma ifn and il18 in serum and spinal fluid was 1.5-2 times higher than in trauma patients (p<0.001). the serum levels of gamma ifn and il18 in glyoma patients were 82.8 and 28.5pg/ml, accordingly. in trauma patients-44.3 and 16.3pg/ml. the concentration in spinal fluid of the studied cytokines was 1.8 time higher than in control patients. conclusion: proinflammatory cytokines gamma ifn and il18 may be involved in pathogenesis of glyoma and can participate as possible anti-tumor factors of immunity in glyoma patients. a. arrey-mensah * , r.u. sorensen, new orleans, la. rationale: immunodeficiencies need to be ruled out in infants that present with failure to thrive. patients with t-cell lymphopenia, hypogammaglobulinemia and pancytopenia may have a primary immunodeficiency such as scid, or a secondary immunodeficiency that may need to be managed differently. methods: evaluation of cellular and antibody-mediated immunity of a 15 month old aaf admitted with failure to thrive and chronic diarrhea. ohistory of duodenal atresia corrected by creating a blind duodenal loop and anastomosis of the stomach to the jejunum at birth. o mass in hypogastrium. results: immunologic evaluation revealed: " lymphopenia ranging 210 to 350cells/ul " anemia: hemoglobin 7.6g/dl, retic 6.8 " ancs ranging 700 to 2100 " thrombocytopenia 54000l to 26500l " cd4+: 80 " cd8+: 50 " cd4/cd8r: 1.5 " cd19: 290 " cd56: 190 " mitogen responses were normal: " pha 46, 397cpm " con-a, 7, 690cpm " pwm, 36, 168cpm immunoglobulins: igg 102mg/dl iga 80mg/dl igm 55mg/dl ige 20 iu/ml total protein was 3.4g/dl, albumin was 1.2g/dl the immunoglobulin-g half-life study was 7 days in our patient: metabolic imbalance: hypokalemia (1.8mmol/l) hyponatremia (127mmol/l) hypocalcemia (4.6mg/dl) blood, stool, urine cultures normal. stool ova, parasite, virus were negative. hiv-pcr (-) radiological evaluation: superior mesenteric vein thrombosis, chronic malrotation and volvulus, dilated duodenal blind loop with possible lymphatic obstruction. the patient improved clinically and all immunological markers normalized after surgical removal of adhesions and obstruction. conclusion: the patient had a secondary immunodeficiency due to lymphangiectasia. lymphangiectasia should be considered in the presence of a lymphopenia with normal lymphocyte function and hypogammaglobulinemia accompanied by hypoproteinemia and hypoalbuminemia. hypogammaglobulinemia and hypoproteinemia were secondary to gastrointestinal losses. the immunological component of 22q11.2 deletion syndrome (also known as digeorge syndrome;dgs), hypoplasia of the thymus, is quite variable among patients and provides the opportunity to determine the relationship between thymic function and t cell receptor (tcr) repertoire diversity. using a novel measure of repertoire diversity based on cdr3 length polymorphism called hamming distance, tcrbv repertoire diversity in dgs was found to differ from control subjects by having an overall skewing of receptor expression. as expected from clinical outcomes, there was also great intra-individual variability in dgs tcr repertoires. the degree of repertoire diversity was directly correlated with thymic output as measured by t cell receptor excision circles (trecs), i.e. greater thymic output resulted in more diverse repertoires (see figure below). this result demonstrates a quantitative relationship between thymic function and repertoire diversity in humans and may reflect the balance between thymic output and peripheral expansion to maintain an adequate tcr repertoire. in addition, it may suggest a basis wherein limited repertoire diversity mediates both immune deficiency and autoimmunity. tcr repertoire diversity in dgs, as measured by hamming distance, correlates with thymic output, as measured by trecs (plotted on a logarithmic scale) a. sabra 1* , s. sabra 1 , h.j. castro 2 , j. malka-rais 2 , j.i. mendez-inocencio 2 , g. santos 1 , j.a. bellanti 2 , 1. rio de janeiro, brazil; 2. washington, dc. a major investigative effort of our laboratory has been directed to the study of non-ige mechanisms and their role in the immunopathogenesis of food allergy (fa). we have previously reported th1 and th2 cytokine alterations associated with several clinical entities that had overlapping disease manifestations affecting the mucosal-associated lymphoid tissues (malt), of the gi tract (galt), skin (salt), nasal (nalt) and bronchial tissues (balt). the objective of the present study was to evaluate specific immunologic alterations in a group of patients with non-ige fa. peripheral blood cd4 and cd8 lymphocyte subset analyses were performed in 10 patients with fa documented by double-blind placebo-control food-challenge (dbpcfc). all patients were treated with an amino-acid based formula (aabf) and then received an open challenge using a panel of commonly offending allergenic food allergens in a normal diet.the clinical picture of all 10 patients with fa were linked to malt-related manifestations; 8 with galt symptoms of diarrhea, vomiting, abdominal pain and ftt, 4 with balt symptoms of asthma, 4 with salt symptoms of eczema, 3 with nalt symptoms of rhinitis and 2 with edema, ascites and anaphylaxis. all subjects had normal serum ige and eosinophil levels, negative ige food rast tests, normal cd4 (mean 1562 + 368) and very low cd8 (174 + 42) levels in peripheral blood. abnormal cd4/cd8 ratios >3.5 were observed in all patients. age-and gender-matched controls revealed cd4/cd8 ratios ranging from 1.5 to 2.5. both patients with anaphylaxis had cd4/cd8 ratios >6. all patients responded well to aabf. open challenge to common offending foods from a regular diet (milk, soy, wheat, egg, nuts, beef and chicken), revealed multiple food allergies. after two years of follow-up on an aabf regimen, the 10 patients remain well but allergic to multiple foods. very low cd8 levels remain as the unique immunological alteration in all 10 patients.these studies suggest that abnormalities of very low cd8 distribution may play a pathogenetic role in non-ige mediated fa. since cd8 lymphocytes play a role in immunologic tolerance (it), it is tempting to speculate that the very low cd8 levels may signal the failure of development of it in our patients predisposing them to the development of allergies to multiple food components. background: mixed connective tissue disease (mctd) is a disease taht have caused controversy, while some authorities consider it a distinct rheumatic disease, others believe that it's an early stage of a fully defined autoimmune disease. the distinctive feature is the presence of the u1 small nuclear rnp autoantibodies. clinical features involve raynaud's is phenomenon, swollen hands, sclerodactyly, esophageal hypomotility, polyarthritis, and myositis, allof them can be present in others well deined rheumatic diseases. only aew cases have been described in the pediatric population. objetive: to determine the frecuency of mixed connective tissue disease in mexican.children in a tertiary level institution according to kasakawa's criteria, alarcon-segovia's criteria and sharp's criteria and establish if a diagnosis of a well defined autoimmune entity was made during the follow up. methods:medical charts were assessed with the diagnosis of mctd between 1988 and 2003 in our hospital. results: between 1988 and 2004, 9 , 919 cases of autoinmune diseases were treated; 310 with systemic lupus erythematosus(sle), 6544 with rheumatoid arthritis(ra), 1, 128 with scleroderma and 1, 937 with dermatomyositis. we found only four cases, 3 of them did not complete the kasakawa's criteria but the diagnosis was probable according to alarcon-segovia and sharp's criteria. one of them developed sle after one year of follow up. the case that fulfilled the diagnosis criteria of mctd; raynaud's phenomenon, rnp antibody positive, arthritis, lymphadenopathy, restrictive pulmonary disease, sclerodactily and muscle weakness developed a full blown sle at 10 years follow up. discussion: the disease is extremely rare in children. we conclude that at least in pediatric age the disease may not exist by it's own, but it's an early stage of a defined autoimmune disease, and the terminology "un differentiated connective tissue disease" is more appropriate to define this cases. a. baysan * , h.y. song, s. gupta, l. yel, irvine, ca. hereditary angioedema (hae) is an autosomal dominant disease characterized by episodic angioedema of the skin or mucosa of the respiratory and the gastrointestinal systems. hae is caused by a quantitative (type i) and/or functional (type ii) deficiency of plasma protein c1 inhibitor, an early component of the classical complement pathway. attacks of hae may be lifethreatening and even cause death when the airway is involved. here, we report a 67 year-old female patient with hae type ii, who has seven affected family members, two of whom died of laryngeal angioedema. the patient had a history of recurrent swelling of the eyelids, lips, tongue and extremities, and respiratory distress since five years of age. she was hospitalized on several occasions one of which required intubation and assisted ventilation. laboratory studies, between the attacks, revealed normal serum ch50 and complement c3 levels. complement c4 level was decreased to 5 mg/dl (n:16-47 mg/dl). c1 esterase function was impaired, 46% (n: greater than 68%) in contrast to normal quantitative c1 esterase. the patient was on long-term danazol treatment for ten years and experienced side effects, most notably hirsutism. currently, there are limited efficacious and safe treatment options for hae. the treatment of choice for acute attacks and prophylaxis appears to be the c1 inhibitor concentrate, which is not yet licensed in the us. the present patient emphasizes the need to establish the correct diagnosis and an appropriate management plan in recurrent angioedema. j. hajsam * , l. ponomarjev, krasnodar, russian federation. background: the previous investigations indicate on the positive effects of ncir in different chronic inflammatory and infectious diseases. nevertheless, the mechanisms of the clinical efficacy of ncir remain still unknown. the aim of this investigation is the study of the immune disorders in patients with chronic tonsillitis and the immunomodulatory effects of the complex treatment in combination with ncir. methods: 55 children with chronic tonsillitis in the age from 7 to 14 years old were under the observation. the t cell receptors (cd3+, cd4+, cd8+, cd16+, cd25+, hla-dr+) have been studied using flow cytometry method. the cytokines (il6, gamma ifn and il18) have been investigated by eliza method. the treatment include traditional methods in combination with ncir. control group (without tonsillitis) consists of 56 children of the same age. results: it has been shown that in chronic tonsillitis was dcrease of the number of cd3+, cd25+ and hla-dr+ cells. the decrease of cd3+ cells was mainly due to decrease of cd4+ cells. at the same time it has been shown the increase in number of cd8+ cells. in chronic tonsillitis have been determined the increase in serum proinflammatory cytokine il6 level in 3.6 times (p<0.001). at the same time the levels of gamma ifn and il18 were lower than in control up to 3-4 times. the studied treatment method including ncir resulted in increase in the number of cd4+ and cd25+ cells. the level of il6 decreased from 95 pg/ml to 28.4 pg/ml. at the same time the serum concentration of gamma ifn and il18 increased up to 2.4 and 1.9 times, accordingly. conclusion: in chronic tonsillitis it has been shown the immune disorders in t cell's subpopulations and cytokine production. the efficacy of ncir mainly depends on the normalisation in t cellular subpopulations and increase in the concentration of gamma ifn and il18. introduction: viral hepatitis is characterized by suppression of cd4+th1cell function related to disease severity. the aim of our research was the determination of total ige concentration and anti-c1q-autoantibodies levels in patients with viral hepatitis c (vhc) and mixed viral hepatitis (vhb+c) and the investigation of their correlation with t cell parameters. methods: 31 patients with vhc and 11 patients with vhb+c confirmed by pcr were examined. evaluation of serum total ige level was performed by elisa kits produced by "vector-best", russia. immunophenotyping of peripheral blood mononuclear cells (pbmc) was done by cytometry with monoclonal antibodies to cd3, cd4, cd8, cd11b, cd16, cd20, cd25, cd95 and hla-dr-antigens. results: the level of serum total ige in patients with vhc and vhb+c was significantly increased in all groups studied, especially in acute hepatitis c (526.8â±129.4 mu/ml, p<0.05) compared with the control group. analysis of pbmc subpopulations in patients with vhc and vhb+c was done related to the total ige level: group i -0-200 mu/ml; and group ii ->200 mu/ml. in patients with viral hepatitis and high total ige level, the content of cd3+t-lymphocytes, cd4+t-cells and cd4/cd8 ratio were significantly decreased compared with the control group (p<0.05). more notable changes were found in patients with acute vhc and vhb+c. the increase in cd20+-cell content was seen in groups with both low and high levels of total ige. the content of cd16+-cells in patients with high levels of total ige was also increased (p<0.05). conclusion: patients with vhc and vhb+c had significantly greater levels of total ige compared with the control group (p<0.05). the increase in total ige levels in patients with viral hepatitis is associated with imbalances of t-lymphocyte subpopulations, including a decrease in cd4+t-cells, and an increase in b-and nk-cells. title: occupational airway allergy among health care workers (hcw) with latex allergy. introduction: for the last years the frequency of latex allergy has increased. health care workers (hcw) is the risk group of this diseases. latex allergy symptoms occure in different organs -including skin, conjunctive, nose and bronchi. study aim:the aim of this study was to estimate the incidence of airway allergy in group of hcw with latex allergy. materials and methods: the investigations were carried out in a group of 252 hcw, aged 21-67 (average age 36, 3). there were two hundred and eight women and forty four men in the group. investigation consisted of questionaire examinations, skin prick tests (latex), patch tests (rubber additives), sige (latex) and spirometry. results: seventy eight (31%) hcw reported undesirable effects after the contact with latex products. fourty (15, 9%) hcw reported symptoms of airway (nose and bronchi). latex allergy (spt and/or sige and anamnesis positive) was diagnosed in 49 (19, 4%) cases. the symptoms concerned with: skin, nose and conjunctive (12 cases, 25%); skin and conjunctive (2 cases, 4%); nose and conjunctive (2 cases, 4%); nose and skin (5 cases, 10%); bronchi, skin, nose, and conjunctive (2 cases, 4%) and only skin in 26 cases (53%). conclusions: the incidence of occupational airway allergy in group of health care workers with latex allergy is high (43% of all cases). latex allergy symptoms usually affect skin, but sometimes it includes also other organs -conjunctive, nose and seldom with bronchi. the incidence of anaphylaxis is increasing. the food allergy and anaphylaxis network wishes to expand the use of injected epinephrine by first responders to allergic emergencies. in indiana, there are 7, 500 first responders, 14, 900 basic emts, 1, 350 advanced emts, and 2, 500 paramedics. the use of injected epinephrine was only permitted by paramedics and advanced emts (17% of all responders). in 2002 a bill was introduced to expand the use of injected epinephrine by ems personnel in the event of an allergic emergency. it was sponsored by the indiana allergy asthma and immunology society and the emergency medical services commission of indiana. on july 1st 2003 senate bill no. 213 took effect permitting all first responders to administer, without restrictions, injected epinephrine. once the legislation passed it became the responsibility of the medical director (md) of each ambulance service in the state to implement the change. in an attempt to assess the impact of this legislation we surveyed each of the mds in the state 9-12 months after the passage of the bill. mds were notified of the legislation by memo and public meetings. in addition each md received a copy of the legislation. of the 266 mds, 56 (25%) responded to the survey. results: 1. unaware of the legislation: 14 (25%) 2. aware of legislation but no implementation: 9 (16%) 3. aware of legislation with implementation restricted to basic emts: 29 (52%) 4. aware of legislation and implementation at all levels: 4 (7%) conclusions: twenty-five percent of mds were unaware of legislation. among those that implemented changes the majority (52%) permitted use by basic emts only. allergists, lay organizations, and ems commissioners need to assure implementation of legislation with all mds within the state. a. khadavi 1* , b. silverman 2 , a. schneider 2 , 1. great neck, ny; 2. brooklyn, ny. rabbit anaphylaxis is extremely rare, with one documented case upon inhalation only. we describe a patient with severe anaphylaxis upon consumption of a rabbit. a 6-year-old female with a 3-year history of asthma and allergic rhinitis was presented for evaluation. the patient complained of worsening asthma and rhinitis symptoms in the home and upon exposure to outdoor pollen. further history revealed the family had a rabbit as a pet. laboratory testing showed her total ige was 865 ku/l, rast results were normal for tree, ragweed and molds (<0.35 kiu/l). positive results were found for ragweed, dust, cockroach, cat, dog, mouse, peanut and rabbit epithelium (8.43 kiu/l, class iv). among other environmental control measures, the family was advised to eliminate the rabbit from the home environment, as it could be a potential trigger for their daughters allergy symptoms. two days later, the patient presented to the emergency room with wheezing, coughing and angioedema of the hands and face. she was treated with albuterol, diphenhydramine and oral steroids. the parents said they followed all of our instructions and did not know why anaphylaxis occurred. further questioning revealed that the family consumed the pet rabbit on the same night preceding the anaphylactic reaction. this was the first time the daughter ate rabbit. the parents assumed that only exposure to the live rabbit would worsen her allergy and asthma symptoms, never expecting an allergic reaction via ingestion. they were advised not to feed their daughter rabbit again and were given a prescription for self-injectable epinephrine. this demonstrates either complete identity, partial similarity or cross reactivity between inhaled and food allergens, as has been noted previously with certain other foods such as garlic and crustacean proteins. physicians need to advise food allergic patients that an allergic reaction can develop upon inhalation of the food, as seen with peanuts. but also respiratory sensitization to an allergen can lead to allergic symptoms upon its ingestion. t. nsouli 1* , j. scheiner 2 , j. malka-rais 2 , j.a. bellanti 2 , 1. burke, va; 2. washington, dc. the safety of selective cyclooxygenase-2 (cox-ii) inhibitors in patients with known nsaid-induced allergic reactions has not been definitely established and is still open to debate. in the present report, we describe two patients with hypersensitivity reactions to naproxen, the first characterized by a systemic anaphylactic reaction and the second by localized urticaria following ingestion of the drug. the first case was a 56 y/o wf with a history of osteoarthritis who, 15 minutes after the ingestion of 375 mg of naproxen, developed generalized pruritus, urticaria, laryngeal edema and hypotension. she was treated in the er with epinephrine, diphenhydramine and iv corticosteroids. epicutaneous and intradermal skin testing to celecoxib revealed negative results similar to a control. an oral challenge with celecoxib was well tolerated by the patient without any adverse responses. the second case was a 57 y/o wf with a known history of chronic urticaria and osteoarthritis requiring regular use of naproxen. a complete allergic and immunologic workup was negative. upon discontinuation of the drug there was resolution of the urticaria. an oral challenge with celecoxib was well tolerated by the patient without adverse sequelae. these two case reports exemplify two extremes in the spectrum of adverse allergic reactions to naproxen, a non-selective cox-i and cox-ii inhibitor, one systemic, the second localized and suggest that the pathogenesis of these symptoms was most likely pseudo-allergic in nature and not immunologically-mediated. the dissimilar chemical structures of naproxen and celecoxib together with their differing mechanisms of action suggest that the absence of allergic reactions to challenge with celecoxib, a cox-ii inhibitor, may be related to a lack of cross-reactivity between the two drugs. although these findings suggest that oral celecoxib could be a possible safe alternative in patients with naproxen-induced drug reactions, it would be prudent to first conduct careful challenges with the drug in a well-equipped medical setting where clinical acceptability and tolerability could be safely assessed. introduction: most fixed and removable dentures are made from casting alloys. many orthodontic appliances are also fabricated from metallic biomaterials. it has been documented in vitro and in vivo, that metallic restorations release metal ions mainly due to corrosion. those metallic ions may be distributed systemically and locally and could pay a significant role in the induction of oral or/and systemic immunoinflammatory conditions. this study determines the frequency of sensitization to metal salts and clinical characteristics in the group of patients with complaints related to adverse effects of dental alloys. methods: 31 patients (29 women and 2 men) aged 35-71 years with symptoms assumed to adverse effects of dental alloys were studied. all patients were studied with base metal salts, including cu 2+ , co 2+ , cr 6+ , mg 2+ , mn 2+ , ni 2+ , ti 3+ , zn 2+ using patch tests. all patch tests substances were 3% salts in petrolatum. the patch tests were conducted in accordance with recommendations of the icdrg. occlusion time was 2 days, and patch tests were read when removing the patches (finn chambers on scanpor, epitest ltd oy, tulusa, finland) and 2 to 4 days later. the total number, percentage of irritant and allergic patch test reactions were calculated. results: in 25 of 31 patients (80%) sensitivity to base metals used in dental restorations was noted. symptoms included burning mouth (45%), metal taste (19%), electrical sensations (11%), dry mouth (9%), and taste irritation (12%), but 16/31 patients (54%) had no symptoms. local gingivitis (19.3%), anomalies of the tongue (12%), stomatitis (12%) and lichenoid reactions (6%) were often seen. the most frequent patch test reactions were caused by ni (45%), cr (41%) and co (19%) . conclusion: this study demonstrated higher frequency of hypersensitivity reactions to ni, cr and co in this group of patients often having symptoms such as burning mouth, metallic taste, electrical sensation, local gingivitis, and anomalies of the tongue. w.y. mak * , s. kearney, b. silverman, a. schneider, brooklyn, ny. the process of intravenous drug desensitization often involves simple but tedious calculations. miscalculations may arise due to human error. for patients who are truly allergic to the drug in question, such errors can be life threatening, if not fatal. we propose the use of a spreadsheet to minimize such calculation errors. spreadsheets, such as microsoft excel, ibm lotus 1-2-3, and corel quattro pro, are used extensively in finance for tedious and repetitive calculations. this property of a spreadsheet makes it an ideal tool to assist with any drug desensitization protocol. we chose microsoft excel for its availability at our institution. a general template was initially created with equations in specific cells which were based on the protocols listed in patterson's allergic diseases, 6th edition and middleton's allergy: principles and practice, 5th edition. by inputting specific numbers, such as the amount of drug and volume of diluent into key cells of the spreadsheet, the program will automatically calculate the protocol for the given intravenous drug. an example of our pipericillin protocol is listed below. we find that the use of a spreadsheet not only minimizes calculation errors and hence reduces the likelihood of avoidable reactions; but also decreases our preparation time for the desen-sitization protocol. we recommend its use for all allergists performing drug desensitizations. states 25 years ago, the infestation of homes across the northeast, southeast and midwestern states with these insects during the fall and winter months has become increasingly common. in the last 5 years, several investigators have published case reports of patients with allergic rhinitis, conjunctivitis and asthma symptoms associated with suspected inhalational exposure to high levels of proteins from these beetles. we report a series of 5 patients who presented with a spectrum of various allergy complaints ranging from symptoms of allergic rhinitis, asthma, urticaria, angioedema and acute anaphylaxis (with documented elevated serum tryptase) on exposure to high numbers of multi-colored asian ladybeetles(malb.) methods: we performed western blotting with the patients' serum and a whole-body ladybeetle extract made from confirmed h. axyridis obtained from one of the infested homes in that region. results: blots with the patients' serum revealed ige binding to 3 different proteins with molecular weights approximately 8, 28 and 78 kd. the serum from two patients bound a 28 kd protein possibly similar to the 30 kd protein previously identified by yarbrough et al. jaci 1999; 104; 704-5 . ige from four patients bound to an 8 kd protein not previously reported. lastly, serum from two patients revealed ige binding to a 78 kd protein possibly similar to the heavier proteins mentioned in an abstract by magnan et al. jaci 2002; 109; 580 . conclusion: we present five patients with specific ige to malb and allergic symptoms on exposure to high levels of malb. we conclude that malb are likely a significant source of several different allergenic proteins and that malb are increasingly becoming a significant cause of a wide variety of hypersensitivity reactions across the united states. as multiple exposed subjects and several entomologists report that these beetles bite humans, we speculate that a wide range of ige-mediated symptoms including urticaria, angioedema and anaphylaxis can occur by exposure to proteins by inhalation, direct contact and possibly by inoculation of these proteins into the skin by the bite or scratch of this beetle. a case report and literature review. c.s. taylor 1* , s. ramesh 2 , 1. getzville, ny; 2. buffalo, ny. introduction: lentils belong to the legume family and are the staple ingredient of the ethnic indian diet. lentils have been shown to cause allergic reactions and even anaphylaxis. however, little research has been done to distinguish the types of lentils used commonly in the indian diet and their various hypersensitivities. some of these lentils include the black gram (phaseolus mungo), mung bean and split chick peas (bengal gram). case report: we report a 19-month-old indian boy who presented with severe atopic dermatitis since the age of four months. his dermatitis was exacerbated by the ingestion of peas and beans and resolved with the avoidance of such. at nine months of age, the child was introduced to boiled mung beans and subsequently developed lip swelling within a few minutes. a similar reaction occured with split chick peas. physical exam at consultation revealed severe eczema. skin tests using commercial extract revealed 5+ response to peanut (he had never eaten peanuts), soybean, pea and egg. subsequent skin tests at the follow up visit to prepared reagents revealed much greater than 5+ response to mung bean, chick pea, split chick peas, and black gram. the patient developed a systemic response during testing which required epinephrine. discussion: legume allergy (other than peanut, soy and peas) is rare in the unites states but has been reported in asia and the mediterranean area. it has been reported in one case series that patients with lentil allergy react to more than one lentil. there is a five percent cross reactivity between peanut and other legumes such as soy and peas. however, the extent of cross reactivity between peanut and the other legume sub-groups (ie. lentils) is not well documented. there also appears to confusion in the labeling of the various lentils used in ethnic diets. conclusion: this case has been presented to make allergists aware that there are many differnt types of lentils that can cause hypersenstivity reactions and the importance of considering ethnicity and dietary habits in evaulating for food allergy. background: epinephrine is the drug of choice for the treatment of anaphylaxis however it is frequently underutilized. one possible reason is the fear of adverse cardiac effects. the most common side effects of epinephrine are palpitations, tachycardia, sweating, nausea, vomiting, respiratory difficulty, pallor, dizziness, weakness, tremor, headache, nervousness, anxiety and arrhythmias. a previous study showed that administration of epinephrine, in a small number of healthy adults did not cause any significant cardiac adverse events and vitals sign changes were not clinically significant. the purpose of this retrospective analysis is to determine the safety profile of epinephrine in a large number of patients that were administered epinephrine for treatment of anaphylaxis that occurred in a physician's office. methods: all patients in an allergy office, that were administered epinephrine for acute anaphylaxis between 1999-2004 were selected for this retrospective analysis. a chart review for adverse events and vital was conducted. vital signs and side effects had been monitored every 5 to 10 minutes for minimum of 30 to 90 minutes after the administration of epinephrine. results: 105 patients between the ages of 8yrs to 55yrs received 128 injections of epinephrine. after an injections of epinephrine 60% of the patients had a pulse between 50-90, 27% between 72-100, and 13% between 102-110. systolic blood pressure determined 81% of patients to be between 80-140, with 10%, between 142-160, and 7% between 162-170, and 2% between 172-180. concurrently, diastolic blood pressure was found to have 94% between 50-90, 3% between 92-100 and 3% betweem 102-110. the most common reported side effects were tremor, headache and pallor. any rise in blood pressure and heart rate after epinephrine was transient and returned to normal within 90 minutes of observation. there were no serious side effects. all patients responded to the epinephrine and were able to go home. conclusions: this retrospective analysis revealed that the administra-tion of epinephrine is safe and effective for the treatment of acute anaphylaxis in an outpatient setting. the use of epinephrine for treatment of anaphylaxis is life-saving and its use to treat non-cardiac or elderly patients who have anaphylactic reactions should be encouraged. purpose: a clinical pathway was established to decrease the use of vancomycin in surgical patients with self-reported penicillin (pcn) allergy. methods: in 2001, our institution developed a preoperative evaluation (poe) clinic for elective surgical patients. in june 2002, on-site, same-day allergy consultation and penicillin skin testing was made available for preoperative patients with self-reported pcn allergy. we reviewed the antibiotic recommendations, the actual administration of vancomycin, and compliance by the surgeons with our recommendations from july 1, 2002-september 16, 2003. results: during this time, 11, 819 patients were seen for their preoperative medical exam at the poe clinic, and 1204 were evaluated for pcn allergy. of these, 1120 patients met irb standards to participate in the study. the mean age of the patients was 60 years. the top three surgical specialties represented in the poe clinic were orthopedic (28.4%), urology (22%), and neurosurgery (15%). of the 1120 patients, 1031 underwent skin testing for pcn allergy. eighty-five percent (85%) or 951 of these patients with a history of pcn allergy were recommended to use -lactams such as cefazolin, and 164 (15%) were recommended to avoid -lactams. forty-three (43) or 4% had one or more positive skin tests to pcn. of the 1120 patients, 957 patients actually received pre-operative antibiotics. of these 957 patients, 718 (75%) of those received cefazolin, 60 (6%) received clindamycin, 33 (3%) received ciprofloxacin, 25 (2%) received levofloxacin. some patients received more than one antibiotic for prophylaxis. only149 (16%) patients received vancomycin. conclusions: establishment of a clinical pathway in a preoperative clinic that includes allergy testing and consultation reduced vancomycin use to only 16% in surgical patients with a history of penicillin allergy. h. yarmohammadi * , a. nowak-wegrzyn, new york, ny. introduction: anticonvulsant hypersensitivity syndrome is a potentially fatal drug reaction with cutaneous and systemic reactions to the arene oxideproducing anticonvulsants. in most cases, the hallmark features of fever, rash, and lymphadenopathy are accompanied by multi organ-system abnormalities. fatal outcomes are most often associated with liver failure. recognition of the syndrome, which may have variable presentations, is the key to prompt discontinuation of the drug, close monitoring, and management. case 1: a 16y/o male received dilantin for seizure prophylaxis following craniopharyngioma resection. fourteen days later he was readmitted for fever and csf leak from surgical site. he was started on ceftriaxone and vancomycin empirically; an lp and pan culture was done. he continued to have low grade fever and, on the 6th day of admission, developed a maculopapular rash on trunk. on day 8th lfts increased and cbc showed eosinophilia. antibiotics were stopped but lfts continued to rise. on day 10, dilantin was stopped and lfts normalized in 2 days, fever stopped and rash disappeared. case 2: a 17 y/o girl was admitted to the hospital with generalized rash, facial edema and fever (39 degrees c). she developed a pruritic maculopapular erythematous rash over her trunk and face 2 weeks prior to admission. she then developed high fever. three months prior to this visit she had been started on phenytoin (300mg/kg) for control of grand mal seizure. physical exam revealed cervical and axillary lymphadenopathy. she had elevated wbc (13, 400), eosinophilia (20 %), and elevated lfts. phenytoin was stopped upon admission but 24 hours later she continued to have fever, elevated lfts and devel-oped erythema in her mouth. treatment with ivig and prednisone was initiated and resolution of symptoms was seen within 2-3 days after therapy. conclusions: the timely recognition of anticonvulsant hypersensitivity syndrome is important, because accurate diagnosis prevents potentially fatal re-exposure and influences subsequent anticonvulsant treatment options. ivig and prednisone should be considered in situations where prompt improvement of the lfts or clinical symptoms is not observed. the recently published results of a telephone survey about the prevalence of seafood allergy in the us indicate that seafood allergy is a significant health concern. aim of the present study was to retrospectively review our data on the topic. we have interviewed 7391 subiects attending our allergy outpatients about any suspected food allergy. over 95% were 14 years or older. any reaction to food was reported by 33% of the patients, more frequently by women. of these 2419 patients, 125 reported reactions to seafood (5%) with an overall prevalence of seafood reactions of 1.7%: 50 to fish (0.7%), 58 to shellfish (including mollusks) (0.8%) and 17 to both (0.2%). in the case of the reactions to fish it is quite possible that shellfish is also included, as the patients were often unable to tell the role of finfish from shellfish apart. this is most probably due to the fact that seafood dishes or a complete seafood meal in italy generally include both shell and finfish. in only less than half cases there was a convincing relationship between the ingestion of seafood and the reaction. the reactions reported were: gastrointestinal (17), non life-threatening angioedema (15), mild oral allergy syndrome (13), acute urticaria (9), asthma (3), rhinoconjunctivitis (2). the gastrointestinal reactions might not all be true (ige-mediated) allergic reactions, but also toxic. in conclusion, even in a selected population like that attending an allergy clinic and using a broad definition of adverse reaction, seafood allergy appears to be a rare phenomenon and of limited clinical impact, the prevalence of moderate to severe reactions (angioedema and asthma) in the whole selected population being 0.2%. clopidogrel (plavix) is an antithrombotic agent currently used to prevent thrombotic cardiovascular events by inhibiting adenosine diphosphate (adp)dependent platelet activation. clinically, it has been used in patients with aspirin intolerance or who developed neutropenia from ticlopidine. there have been reported cases of clopidogrel causing rash and urticaria. often these patients are told they are allergic to clopidogrel and should not take it again, despite the fact that no workup was ever performed to elucidate whether the reaction was indeed immunologic in nature. a review of the literature revealed no reported attempts at desensitizing a patient to clopidogrel after a presumed immunologic drug reaction. we present the case of a 49 year-old female who initially took clopidogrel (75 mg. p.o. daily) for transient ischemic attacks. after tolerating the medicine for five days, it was replaced with an aspirin/warfarin combination, which she took for the next five days. reintroduction of clopidogrel a week later was uneventful, until she developed a pruritic, maculopapular rash on the 5th, through 7th days of restarting therapy. at that time, she was not taking any other medications, and had no cardiopulmonary or gastrointestinal complaints. she had discontinued the clopidogrel and was asymptomatic during our consultation. skin testing was done using clopidogrel prepared at concentrations of 1 mg/ml and 1/10 mg/ml. at the same time, control skin testing on three non-atopic subjects was performed and produced no reaction to either epicutaneous or intradermal testing. by contrast, the patient had a positive skin reaction at both concentrations on intradermal testing. the patient was hospitalized for an oral desensitization where clopidogrel dilutions were prepared by the hospital pharmacy. the patient tolerated an initial dose of 0.02 mg. serial three-fold increases in concentration were given every 15 minutes, until a total cumulative dose of 77 mg. was reached. the patient tolerated the entire procedure and suffered no adverse reactions upon continuation of the treatment. this demonstrates the utility of oral desensitization to clopidogrel in patients with a positive skin test who require this medication. lamotrigine is a non-aromatic anticonvulsant with desirable pharmacological profile. the most common idiosyncratic reaction in children is rash (10-20%). severe cutaneous adverse reactions and systemic hypersensitivity reactions are uncommonly reported. method: case presentation an 11-yearold caucasian female was prescribed lamotrigine in escalating dose for her first generalized seizure. a week later she developed a pink rash on her cheeks gradually progressing to her trunk, hands and feet. three days later she was seen in the er and treated for possible streptococcal-pharyngitis with fever and rash. oral penicillin-v treatment was initiated. the rash continued to spread all over her body with mild pruritis and sores on lips and mouth. few blisters were noted in left ear, hands and feet. later lamotrigine was discontinued. oral steroids were prescribed. she continued to develope new blisters, rash and hemorrhagic plaques and dysuria. she was later transferred to our institute. the physical examination was significant for afebrile girl without pallor and icterus. bilateral conjunctivitis without keratitis evident. tender cervical lymphadenopathy was palpable. multiple ulcers on lips and gingiva were seen. several targetoid lesions on trunk and extremity were noted along with few hemorrhagic plaques on extremities. superficial sloughing of distal fingers and toes was noted. nikolskys sign was not demonstrable. the systemic examination was unremarkable. laboratory tests: hemoglobin 14.2g/dl, wbc-normal range without eosinophilia. normal pt/aptt and inr. liver function tests showed elevated sgpt-1380iu/ml and sgot-225iu/ml with normal bilirubin. urine analysis was normal. serology for cmv, ebv, hsv and hepatitis a and b was negative. skin biopsy showed full thickness epidermal necrosis and sub epidermal clefts. aggressive supportive therapy was initiated. corticosteroids were continued for five more days. prior to being discharged from hospital the rash had dried and most lesions faded. the blisters and oral ulcers had healed. the liver enzymes had decreased. conclusions: we report an occurrence of stevens-johnson syndrome with lamotrigine in a young child. in the literature severe reactions are associated with higher doses or rapid escalation of the dose, and concomitant use of valproate. early recognition and withdrawal of medication is necessary to improve the outcome. introduction: in patients with a history of penicillin (pcn) allergy and negative pcn skin tests to major and minor determinants, 97-99% of patients will tolerate pcn administration without risk of an immediate reaction. in fact, middletons allergy principle & practice reported that no life-threatening false-negative reactions have been reported when pcn was administered after a negative pcn skin test. we describe a case in which a patient with a history of pcn allergy and negative pcn skin tests to the major and minor determinants experienced life-threatening anaphylactic shock when administered piperacillin/tazobactam. case history: a 38-year-old woman with crohns disease was admitted for treatment of an enterocutaneous fistula. three months before admission, the patient reported a severe reaction to either piperacillin/tazobactam or intravenous (iv) lorazepam needing respiratory support in the intensive care unit. in order to delineate this problem, an allergy consultation was obtained. serum ige antibodies to pcn, measured by a commercial cap system radioallergosorbent test (rast) fluoroenzymeimmunoassay (feia; pharmacia and upjohn, uppsala, sweden), and pcn skin tests to major and minor determinants were negative. a skin test (prick and intradermal) to piperacillin/tazobactam at 4.5mg/ml was also negative. five minutes into receiving 3.375 grams of piperacillin/tazobactam iv, the patient reported feeling lightheaded, flushed, nauseated, and diaphoretic. the blood pressure decreased to 80/50 from a baseline of 110/60, heart rate 150/minute and appeared toxic. no wheezing or rash was noted on physical examination. patient was treated with iv epinephrine, diphenhydramine, and dexamethasone and recovered without sequela. serum tryptase, drawn 1 hour after the beginning of the reaction, was elevated at 56.3 ng/ml but decreased to 17.6 ng/ml 12 hours later. complement levels were normal. conclusion: despite a very high negative predictive value of a negative pcn skin test to the major and minor determinants and reports that no life-threatening false-negative reactions have been reported when pcn is administered after a negative pcn skin test, physicians need to be very cautious in administering piperacillin/tazobactam and other b-lactams in patients with a history of severe reactions such as anaphylaxis to past pcn administrations. a. majmundar * , d.a. khan, dallas, tx. introduction: a variety of adverse drug reactions have been reported after therapy with allopurinol. successful protocols for desensitization to allopurinol have been developed. we report a case of a patient who was successfully desensitized to allopurinol only to develop dress after four months of allopurinol therapy. methods: a 57 year old man was referred to our department with a remote history of rash and fever resulting in hospitalization after initiation of allopurinol. due to severe gouty arthritis unresponsive to colchicine and requirement of chronic steroids, it was recommended to attempt allopurinol desensitization. based on the history of his prior reaction, a slow desensitization protocol was performed beginning with allopurinol 10î¼g and increasing the dose weekly to 25î¼g, 50î¼g, 100î¼g, 200î¼g, 500î¼g, 1mg, 5mg, 10mg, 25mg, 50mg, and 100mg. the patient tolerated the entire desensitization protocol without adverse reaction and was initiated on daily allopurinol at 200 mg per day. results: four months after desensitization and daily allopurinol use, the patient developed fevers and an acute maculopapular eruption on his back and abdomen without mucosal involvement. laboratories demonstrated eosinophilia of 936 cells/mm 3 , elevated ast of 101, alt of 102, and ggt of 163 u/l. he was treated with prednisone 80mg a day which was tapered weekly over 5 weeks with successful resolution of his symptoms, eosinophilia and transaminitis. conclusion: while desensitization to allopurinol can be safely accomplished, allergists need to be cognizant of the potential for delayed serious drug reactions such as dress. wiskott aldrich syndrome is an immunodeficiency characterized by eczema, pyogen infections, and mixed immunodeficiency.we describe a male seven-month old, perinatals antecedents without importance who had an ulcer in site of application of bcg. non-blood relatives, healthy parents. at twomonths-old, he initiated with continuous fever, not quantified, treated with multiples antibiotics without resolution, he was hospitalized, plaquetopenia and anemia were diagnosed, he received a red globules and plateles transfusion, one week after he presented a disseminated dermatosis characterized by erythema and desquamation, at four-month-age presented right axillary adenomegaly, hepatoesplenomegaly and recurrent bilateral media otitis for what was hospitalized in our institute. he was malnutrition, bad general condition, febrile, with disseminated dermatosis affecting the head, trunk and extremities characterized by erythema and desquamation, in addition he had left ankle cellulitis, right axillary adenomegaly, hepatoespenomegaly. during his hospitalization he presented left hand cellulitis, hairy skin abscess, oral candidiasis, required surgical treatment by econdary compartimental syndrome because cellulitis of left ankle. persistent hemogram reported thrombocytopenia with normal platelet volume, blood cultives were positive for grampositive bacterias, isoaglutinines, were normal. immunoglobulines were elevated for the age range, he received antimicrobial and antifungic treatment. but he died. in the autopsy timic alymphoplasia was reported, cortical lymphoid depopulation in lymphatic ganglia and spleen, disease by disseminated cytomegalic inclusion, multifocal pulmonary pneumocystosis, bcgitis, disease graft versus guest. . with the previous features we concluded in a compatible mixed immunodeficiency with wiskott-aldrich syndrome with particular characteristics that make this case interesting. the patient course with cellular immune deficiencie with thrombocytopenia and eczema, even when he didn't have platelet sizing diminished, we consider that the patient had a severe of wiskott s aldrich syndrome and at the moment we are awaiting result of genetic study uasp gene. background : hypersensitivity to mosquito bites (hmb) is a disorder characterized by necrotic skin reaction and systemic generalized symptoms subsequent to mosquito bites. it has been suggested that hmb is associated with chronic epstein-barr virus (ebv) infection and natural killer cell leukemia/lymphoma. we describe here a korean child who had hmb associated with chronic ebv infection and natural killer cell lymphocytosis. case : a 5-year-old male was admitted with well-demarcated necrotic skin lesions and severe swelling on right ear lobe developed after mosquito bites. he had suffered several similar symptoms since last summer, which complicated as deep scars on skin. hepatosplenomegaly or peripheral lymphadenopathy was not detected. laboratory tests showed wbc 7, 280/mm3 (neutrophil 39%, lymphocyte 56%), total eosinophil count 156/mm3, ige by prist above 1, 000 iu/m. immunoglobulin levels were normal. specific ige for aedes communis by cap was negative. lymphocyte subset analysis demonstrated increased nk cells (cd16+cd56, 43%) and decreased cd3 and cd4 cells. igm for anti-nuclear antigen (ebna), igm for viral capsid antigen (vca) and igm for anti-early antigen (ea) dr to ebv were negative. but the levels of anti-vca igg (> 200 u/ml), anti-ea dr igg (> 150 u/ml) and anti-ebna igg (62 u/ml) were increased. type a eb virus was demonstrated in blood mononuclear cells by dna pcr method, and eber in situ hybridization was negative in necrotic tissues. immunostaining with nk-cell marker (cd56) revealed many immunoreactive cells with the perivascular inflammatory infiltrates in tissue. skin patch tests for mosquito allergen (aedes togoi and culex pipiens) showed positive response to c. pipiens. introduction: scimitar syndrome is a congenital anomaly resulting in anomalous pulmonary venous return from the right lung to the inferior vena cava. recurrent respiratory infections have been associated with scimitar syndrome. methods: we report on a 16 year-old adolescent female who presented to immunology clinic with recurrent pneumonias. results: the patient presented with numerous recurrent pneumonias, multiple er visits, and hospitalizations. she complained of intermittent chest pain, cough, fatigue and exercise intolerance. the patient had a large secundum atrial septal defect surgically corrected at 5 years of age. a cardiac echo obtained at 7 years of age revealed rvh, but was normal at 9 years of age. she was a known atopic asthmatic. previous immunologic workup was normal with iga 183 (33-202), igg 1290 (633-1280) and igm 97 . at presentation to our clinic pulmonary functions were fvc of 79% and fev1 of 78%. review of previous chest radiographs showed chronic changes with an opacity partially obscuring the right hemidiaphragm. a high resolution chest ct-scan showed a large irregular venous structure extending through the right chest joining the inferior vena cava above the liver consistent with scimitar syndrome. the patient was referred to pediatric cardiology who recommended surgical correction. conclusion: scimitar syndrome may present as recurrent pneumonias and chronic lung disease. a high resolution chest ct scan may be useful in delineating this disorder. background: laryngomalacia is the most common cause of stridor in infants but only rare reports exist of clinically relevant laryngomalacia in adults. objective: to present a case of laryngomalacia in an adult with significant respiratory symptoms initially attributed to asthma. methods: an 18 year-old female with a history of allergic rhinitis and gastroesophageal reflux disease presented to the allergy clinic for further recommendations regarding a prior diagnosis of asthma poorly controlled on inhaled fluticasone, montelukast and albuterol. the patient was clinically diagnosed with asthma at age 13 due exercise related symptoms. the symptoms progressed and intermittent trials of various inhaled steroids provided minimal relief. on evaluation, the patient described constant wheezing which occurred only on inhalation and originated from the throat. symptoms did not respond to albuterol use three to four times a day, rhinitis control and long-term, high-dose, antireflux therapy. baseline spirometry was normal. histamine bronchoprovacation and fiberoptic laryngoscopy were performed for further evaluation. results: histamine challenge was positive with a 33% decrease in fev1 with 1 mg/ml histamine. however, laryngoscopy revealed redundant airway tissue most notable over the right arytenoid cartilage, consistent with laryngomalacia, which prolapsed into the laryngeal vestibule significantly obstructing the airway on inspiration only. the patient was referred to otolaryngology and surgical excision using a carbon dioxide laser was performed with subsequent improvement in symptoms and decreased asthma medication use. conclusions: we report an unusual case of laryngomalacia in an adult presenting as asthma, which was successfully treated with laser surgical excision. laryngoscopy of the patient revealing redundant airway tissue most notable over the right arytenoid. c. so 1* , s. kuhl 2 , 1. davis, ca; 2. mather, ca. c. so, s. kuhl u.c. davis medical center, sacramento, ca & sacramento va hospital, mather, ca background: wheezing is an uncommon manifestation of phrenic nerve dysfunction. objective: we offer a description of wheezing attributable to phrenic nerve dysfunction to remind clinicians that dyspnea and wheezing can be caused by cor pulmonale which can be caused by phrenic nerve dysfunction. methods: a 71-year old male non-smoker presented with chronic dyspnea and occasional wheezing for the last decade. the patient had a remote history of pericardial stripping for presumed tuberculous pericarditis. he also had a non-productive cough and orthopnea. dyspnea was exacerbated by putting his hands over his head and bending over. he had been treated with inhaled corticosteroids and bronchodilators without relief. results: repeated chest x-rays showed a chronically elevated right hemidiaphragm. pulmonary function tests showed a restrictive pattern with: fev 1 1.2 (40%), fvc 1.59 (36%), tlc 3.23 (49%), dlco/va 4.54 (125%) and no bronchodilator response. left heart catheterization was normal while right heart catheterization showed elevated pulmonary artery pressures (56/20) and he was subsequently diagnosed with cor pulmonale. cardiopulmonary exercise testing showed an increase in minute ventilation which was achieved predominantly by an increase in respiratory rate rather than to an increase in tidal volume suggesting restrictive or interstitial lung disease. chest ct showed left ventricular enlargement and no evidence of interstitial lung disease. a fluoroscopic sniff test was performed and showed paradoxical movement of both diaphragms. he was diagnosed as having diaphragmatic dysfunction as a result of phrenic nerve injury from prior pericardial stripping. conclusions: wheezing and chronic dyspnea can be related to phrenic nerve dysfunction. a review and discussion of various causes of phrenic nerve dysfunction, including autoimmune causes, is offered. patients with a history of prior cardiothoracic surgery who present with recalcitrant dyspnea and wheezing may benefit from evaluation for phrenic nerve dysfunction. background: sudden sensorineural hearing loss (ssnhl) is defined by a loss of at least 30 db in 3 contiguous frequencies over a time course of 72 hours or fewer. etiologies of ssnhl include viral infections, ototoxic drugs, autoimmune diseases, trauma, neoplasms, and vascular occlusion, but viral labyrinthitis is the most common cause. in cases of sudden hearing loss, herpes infections can be reported in approximately 70% of cases caused by viral infections. typically, sensorineural hearing loss is not recurrent. we report a case of recurrent ssnhl is which oral herpes lesion preceded the onset of symptoms on three consecutive episodes. case report: a 55-year-old male with a history of hypertension, hypothyroidism and chronic tinnitus of the left ear (since a gunshot wound 18 years prior) presented to clinic with a complaint of recurrent episodes of sensorineural hearing loss. the first episode of bilateral hearing loss occurred 14 months prior. an otolarnolgologist treated with a steroid taper and valacyclovir and the hearing loss resolved after one day of therapy. since the initial presentation, the patient reports three subsequent episodes of ssnhl, with two of the episodes responding to prednisone and valacyclovir and one episode responding to steroids alone. oral herpetic lesions preceded at least three of the episodes one day prior to the hearing loss. laboratory data was significant for an elevation of varicella-zoster igg, and of hsv igg. hsv igm was not performed. rpr was non-reactive and ana was negative. a mri of the head was normal. audiogram demonstrated 30db increase and speech discrimination improved from 80% to 100%. the rest of the laboratory data was unremarkable. the patient has been maintained on daily valacyclovir therapy and has had no further episodes of hearing loss. conclusion: our patient experienced hearing loss with concomitant evidence of hsv-1 stomatitis. this suggests a cause and effect relationship. we found that the antiviral therapy was effective for treatment of the ssnhl in this patient as demonstrated by the absence of further symptoms while on antiviral prophylaxis and conclude the most likely etiology of the recurrent hearing loss was secondary to the recurrent herpes simplex infections. abstract the illness was described for the first time in 1937 in the chinese literature by kimm, and szeto, the definitive histological description was published by kimura in 1948, this illness is endemic in asia, but rare, about 200 cases had been reported. kimura disease is extremely sporadic in the rest of the world. the etiology of this disease is ignored but it is believed that there is an aberrant immune reaction to an unknown antigenic stimulus, however epstein barr's virus, human herpes virus 8 and candida albicans had been involved in certain cases. the mast cells had been implicated in its pathogenesis and a th2 cytokine pattern with the production of interleukin 4, 5 and rantes which regulate the synthesis of ige and orchestrate the eosinophilic infiltration. on the other hand it is suggested that the eosinophils had undergone an accelerated apoptosis in this illness. case report. it is a 7 year-old boy with a 6 months evolution with the presence of bilateral subcutaneous nodules of 4x5 cm in parotid and submaxillary glands, presenting hypereosinophilia (8172 total eosinophils ) and high ige ( 9187 total ige), with normal renal function and negative mycotic and parasitic tests.the histopathologic findings revealed the presence of eosinophilic infiltrates with capillary proliferation and fibrosis. discussion. for the clinical characteristics of the nodules together with the hypereosinophilia, extremely high ige and the characteristic histological lesions the diagnosis is kimura disease. the usual clinical presentation consists on several indolent subcutaneous nodules that grow very slowly in volume, located in the neck and head, accompanied by satel-annals of allergy, asthma & immunology lites adenophaties, and with increment in the salivary glands, there is renal affectation in half of the patients, and the laboratory detects hypereosinophilia and elevation of the total ige. the histological lesions, shows hyperplastic lymphoid tissue with proliferative germinal centers, with infiltration of eosinophils in their interfollicular and perivascular zones sometimes forming an eosinophil abscess and proliferation of poscapillary venules. at the moment he have been treated with prednisone (1mgkdia), with great improvement in the clinical evolution. this is the first case reported in the literature in mexico. introduction: the incidence of cow's milk protein allergy (cmpa) is approximately 2-3% and presents primarily during the first year of life. manifestations of cmpa in the neonatal period include gastroenteritis, colic, lethargy, metabolic acidosis, and hematochezia or melena and appear to be non-ige mediated. ige mediated reactions in the neonatal period, such as urticaria or angioedema, are unusual. case: a 13-day-old african-american male presented with a 3 day history of rash, swelling, and erythema overlying multiple joints. there was no history of fever, diarrhea, or eczema. he had been on no medications prior to admission. besides mother with a history of childhood asthma, there was no family history of atopy or food allergy. he had been fed cow's milk-base formula (similac â® ) since birth exclusively. physical examination revealed a diffuse erythematous, raised, macular-papular rash, with areas of duskiness and exfoliation. there was angioedema overlying the joints and periorbital areas (figure 1). laboratory evaluation included cbc with diff, hgb electrophoresis, lumbar puncture, urinalysis, c 1 q (qualitative and quantitative), c 2 , c 3 , c 4 , and cultures of the blood, csf, and urine which were within normal. percutaneous allergy skin testing for cow's milk allergy was performed revealing a 3 + reaction to cow's milk extract (greer, lenoir, nc) with positive histamine and negative saline controls. rast testing showed 8.23 ku/l for -lactoglobulin and 5.10 ku/l for cow's milk. he was placed on an elemental formula. skin lesions and swelling resolved completely within 36 hours. at 2 week follow-up the patient was thriving without complaint. conclusion: early sensitization to cow's milk protein in the neonatal period may occur, resulting in ige mediated urticaria and angioedema. the rashes may be misdiagnosed as erythema multiforme or anaphylactoid purpura, since hemorrhagic lesions and cockade pattern are common. physicians should be aware that these reactions may occur so that early recognition and management may be initiated. neonate with periorbital angioedema and exfoliating, urticarial rash. rationale: two patients, presenting with invasive fungal cns infections, were found to have nk cell dysfunction and hypogammaglobulinemia. methods: case reports results: patient #1: a 47 year old caucasian male presented with headache, double vision, periorbital swelling, and bilateral sinus disease on ct scan. biopsies showed invasive rhinocerebral mucormycosis. he continued to deteriorate in spite of iv and intrathecal amphotericin b, hyperbaric oxygen, many debridement procedures, and a left orbital exenteration. immune evaluation revealed low igg, low igm and a barely detectable nk cell killing activity. neutrophil respiratory burst was normal. he continued to worsen despite ivig replacement. he had intolerable side effects to ifn-a. gm-csf (500 mcg qod) was initiated, in order to boost nk cell activity. this resulted in stabilization of his infection. he was discharged on oral anti-fungal therapy (posaconazole), ivig, and gm-csf. sixteen months later, he continues to remain stable clinically and radiographically on ivig and gm-csf. patient #2: a 62-year-old caucasian male presented with headache, mental status changes, and ataxia. a head ct scan showed a left mass effect and edema. he underwent surgical debridement for ventriculitis and zygomycetes (the same family as mucormycosis) was found. he was started on iv amphotericin b and oral posaconazole. his immune evaluation revealed low igg and igm and low nk cell activity. neutrophil respiratory burst was normal. he was started on ivig and gm-csf after which his nk cell function improved significantly. he remains clinically stable on ivig and gm-csf with persistent radiographic evidence of enlarged ventricles. conclusion: hypogammaglobulinemia is usually not associated with invasive cns fungal infections, suggesting that nk cell dysfunction was likely responsible for the clinical courses of these patients. nk cell deficiency has been reported to be associated with recurrent mucosal candidiasis, suggesting an important role for these cells in the control of some fungi. the spectrum of the clinical presentations of nk cell deficiency is not known since it is not normally included in immune system evaluations. these patients illustrate the importance of including functional nk cell assessment in any evaluation of immune function. introduction: to report a case of successful systemic hydrocortisone desensitization, since allergic reactions and systemic desensitization to corticosteroids have rarely been documented. method: we present a patient with multiple medical problems who has a history of both radiocontrast induced anaphylactoid reaction and corticosteroid allergy. this patient had to undergo cardiac catheterization and corticosteroid desensitization was performed prior to the procedure. results: skin testing to radiocontrast is not considered helpful, therefore patients with suspected reactions to radiocontrast are generally premedicated with corticosteroids and antihistamines to decrease the risk and severity of a reaction. 1, 2 since this patient experienced an allergic reaction to a corticosteroid previously, she was skin tested to two different corticosteroids. the least reactive skin test revealed a 3+ positive immediate reaction to hydrocortisone. cardiac catheterization with contrast was considered absolutely necessary in this case and corticosteroid desensitization was performed. the half-life of hydrocortisone is the shortest among the tested corticosteroids (80-118 minutes), so a protocol was developed with short intervals of escalating doses. each dose was diluted yielding a total volume of 75ml to avoid fluid overload because patient has renal failure. during desensitization, patient developed pruritus and erythema between the 3 rd and 4 th dose that resolved immediately with 50mg diphenhydramine. after desensitization, the patient continued to be on hydrocortisone 200mg intravenously every four hours. one hour prior to the procedure, the patient was premedicated with hydrocortisone and diphenhydramine and was administered radiocontrast without any adverse reactions. conclusion: we successfully desensitized our patient to a corticosteroid and premedicated her with hydrocortisone and diphenhydramine before administering radiocontrast. this case illustrates that intravenous desensitization may be a suitable approach to therapy in corticosteroid allergic patients who require systemic corticosteroids administration. autoimmune neutropenia is defined as a decrease in the absolute number of peripheral neutrophils caused by an immunologically-mediated mechanism. autoantibodies directed to neutrophils can lead to the peripheral destruction of neutrophils and/or inhibit myelopoesis in the bone marrow. although recurrent aphthous stomatitis is often the heralding sign of neutropenia, the diagnosis of ain requires the demonstration of specific antineutrophil antibody which acts by promoting the immune destruction and clearance of neutrophils by mononuclear phagocytes. the present case report describes the rare clinical association of ras in an adult. a 66 yr-old black male presented with a 3 year history of recurrent and repeated painful multiple oral ulcers. initially the oral ulcers occurred on a monthly basis then over time the ulcerations on the oral mucosa and tongue began to appear weekly and later continuously. past medical history revealed no drug allergies but a positive history of hypertension. physical exam revealed an otherwise healthy male with no lymphoadenopathy or splenomegaly. laboratory workup revealed : hiv negative, viral culture for h. simplex negative, mild increase in cd8, cyroglobulins negative, anti dsdna negative anti-smith and rnp negative, wbc count 3300/ml, neutrophils 29% (anc 957), lymphocytes 54%, monocytes 11%, and platelets: 243, 000. bone marrow biopsy revealed a normal production of neutrophils. a direct neutrophil antibody assay: revealed an elevated value of 6, 576 (n= <4, 000). although following initiation of prednisone (60 mg) an immediate increase in anc was seen, the levels fell as the dosage was tapered. the figure below shows the time course and dose-response relationship of anc and prednisone dosage. this case report illustrates the importance of recognition of the relationship of ras and neutropenia, an association that can masquerade as other clinical entities. background: dyspnea, wheezing, and decreased fev1 are suggestive of asthma. it is essential for the clinician to consider a broad differential diag-nosis as the outcome could be catastrophic if the correct diagnosis is missed. case presentation: we present a case of a 48 y.o. filipino female who was referred to our clinic for the evaluation of cough, shortness of breath, and wheezy respiration associated with changes in voice quality, nasal and palatal pruritus, and postnasal drainage. her initial evaluation revealed mold spore hypersensitivity by prick puncture testing and spirometry with an obstructive pattern with fvc-3.05 l (99%) and fev1-1.25 l (50%) predicted and a 15% reversibility post nebulized albuterol. an initial diagnosis of allergic rhinitis with adult onset asthma was made and she was started on salmeterol, budesonide, montelukast, and pirbuterol. her symptoms persisted and rabeprazole was added to treat possible laryngopharyngeal reflux. repeat spirometry revealed fev1-0.84 l prompting treatment with systemic corticosteroids again with no improvement. fiberoptic laryngoscopy was within normal limits. a high resolution computed tomography was obtained and showed a mass in the left side of the trachea which was obstructing 60% of the airway. bronchoscopy revealed a tumor 4-5 cm below the vocal cords with the appearance of adenoid cystic carcinoma which was confirmed by pathology. the tumor was resected by removal of 7 cm trachea with re-anastomosis, followed by a 6 week course of radiation therapy. all medications were discontinued. her symptoms of wheezing, dyspnea, and cough completely resolved. repeat spirometry was within normal limits and she remained asymptomatic. surveillance bronchoscopies have been negative for any recurrence. discussion: adenoid cystic carcinoma (acc) is an uncommon form of malignant neoplasm that occurs within the salivary glands. tracheobronchial acc typically presents with symptoms of cough, dyspnea, and hoarseness. due to its slow growth, acc has a relatively indolent course. in a recent study of a cohort of 160 acc patients, survival was 89% at 5 years but only 40% at 15 years. standard therapy is surgical resection often followed by radiotherapy. conclusion: in patients who fail conventional therapies for asthma it is important to entertain other diagnosis and have a systematic approach to establish the correct diagnosis. ipex is an extremely rare, hereditary condition characterized by immune dysfunction, polyendocrinopathy, enteropathy and x-linked recessive inheritance that leads to death without prompt diagnosis. patients usually present by 4 months with severe diarrhea, failure to thrive, and early onset iddm. most children die by one year without a bone marrow transplant. immunologic evaluation is typically normal except for elevated ige, eosinophilia, and autoantibodies. we report a case of ipex in an infant who presented at birth and died at 79 days of multi-organ system failure. this male infant was born at 30 weeks due to chorioamnionitis and prom. at birth, copious green fluid appeared from his rectum and ng tube which evolved into a secretory diarrhea. workup for fistula was negative. at 2 weeks, the patient developed ascites and explorative laparotomy revealed an inflamed appendix. after the laparotomy, the patient had no further stool output and never tolerated enteric feeds. he remained intubated and had problems with apnea and coagulapathy. pathology of the appendix showed an excess of lymphocytes. immune system investigation showed elevated ige (5320) and igg (979). serum anti-enterocyte igg antibody was positive in the patient and negative in his mother. based on this data, ipex was suspected which autopsy seemed to confirm. autopsies are scarce in patients with ipex. the findings revealed many organs affected by fibrosis but lymphocyte infiltration of only the pancreas and gi tract. the pancreas revealed almost complete loss of the exocrine structure, with invasion of fibrosis and chronic inflammatory cells. the mucosa of the gi tract from the stomach to rectum showed columnar epithelium taken over by fibrosis, capillaries and chronic inflammation. these inflammatory cells were cd3+ lymphocytes and plasma cells on immunochemistry. the lymphoreticular system was consistent with lymphoid paucity in the thymus, lymph nodes and spleen. preliminary data suggests this patient has a splice mutation of the foxp3 gene. ipex is an extremely rare disease, often difficult to diagnose while a patient is alive. infants will present in early infancy with diarrhea and endocrinopathies. without prompt diagnosis, death is inevitable. as a result, one must be aware of atypical presentations and considered in patients with total villous atrophy and one other clinical feature such as iddm or autoimmunity. introduction: digeorge syndrome (dgs) is characterized by thymic hypoplasia, parathyroid hypoplasia, and conotruncal cardiac defects, but has a wide variety of clinical manifestations. there is also an increased risk for autoimmune phenomena in later life due to thymic hypoplasia. case report: a 13 year-old aa girl with tetralogy of fallot (repair at age 2), developmental delay, asthma, recurrent sinopulmonary infections/skin abscesses, gerd, arthralgias and seizure disorder (due to hypoxic encephalopathy during cardiac surgery) presented to allergy/immunology clinic for immune workup. there was no history of documented hypocalcemia. at age 10, she developed persistent annular patches on her left leg. a skin biopsy appeared consistent with sarcoid dermatitis. there was no other evidence of sarcoidosis except for slightly elevated ace level. given lack of systemic involvement, the skin lesions were not treated, but resolved spontaneously. at age 12, she developed swelling of the right mandible, and a bone biopsy revealed garre's osteomyelitis (sterile hyperproliferative osteomyelitis), likely triggered by dental caries, with elevated esr (40) and polyclonal hypergammaglobulinemia (igg 2640), but normal crp. immune workup revealed a decrease in cd4 and cd8 cells (cd4/cd8 ratio 2.44), slightly elevated b cells, high-normal range nk cells, normal range t cell cytokine production in response to mitogens and il-12, but excessive production of proinflammatory cytokines in responses to lps. in conjunction with facial dysmorphism, dgs was suspected, and fish analysis revealed 22q11.2 microdeletion. a repeat workup did not reveal evidence of systemic sarcoidosis, and autoantibody screening was negative including lupus anticoagulant. she was treated with a cox-2 inhibitor, secondary to gerd and mild thrombocytopenia, and her joint symptoms resolved with a concurrent decline in esr (to 30) and igg level (to 2390) after 2 months. conclusion: recurrent infections with fragmented care and resultant chronic inflammation (hence overstimulation of the immune system) may have led this dgs patient to develop atypical autoimmune phenomena and other unusual clinical manifestations. this case illustrates the importance of recognizing the phenotypic features of dgs early in life, and of providing close monitoring and coordinated care. rationale: we report a case of a patient with celiac disease who continues to have symptoms of fevers, nausea, vomiting, night sweats and fatigue despite being on a gluten free diet whose symptoms have been responsive to antihistamines. methods: case report. results: in 1998, this 41 year-old white male suffered from mononucleosis and episodes of prostatitis. he began suffering from fatigue and fevers and was followed at a chronic fatigue syndrome center in 1999. in november 2000, patient was placed on famvir alleviating his sore throat. he was later placed on interferon gamma which was discontinued due to increased fevers, nausea and vomiting. after stopping medication, symptoms abated for sometime. in 2002, patient's father was diagnosed with celiac sprue. in april of 2003, the patient developed a pruritic rash on his right arm. biopsy revealed subepidermal vesicles with pmn's at the tips of dermal papillae. in may of 2003, he developed oral ulcers as well as joint pains. patient underwent endoscopy with biopsies revealing intraepithelial lymphocytosis in the duodenum. patient has been on a strict gluten free diet since october 2003. he reports that some symptoms have improved: skin lesions, itchy eyes, and oral ulcers. however, he has begun to suffer from constipation and continues to intermittently have night sweats, fevers, nausea and vomiting. in february of 2004, he had pruritus that was not alleviated by hydroxyzine. a regimen of benadryl and pepcid was started to aid with the pruritus. the patient reported in the improvement of his symptoms of pruritis, sweats, and emesis. conclusions: diagnosis: celiac disease(cd) patient with persistent nausea, vomiting, sporadic fevers, and fatigue despite maintaining a strict gluten free diet has shown improvement of symptoms with antihistamines. patient has history of chronic infections: prostatitis and chronic ebv. patient also reported alleviation of symptoms after interferon therapy suggesting some autoimmune component to his current illness. cd prevalence in the united states is much higher than once thought 0.5-1% of the u.s. celiac disease has been associated with increased risk of lymphoma and malabsorption leading to neurological diseases. this is a rare case of cd associated pruritus responding to antihistamines. rationale: eosinophilic cystitis is a relatively rare condition in children and adults with a varied course. it usually responds to short-term nsaid and steroid therapy. we report a man with a severe case who responded to prolonged oral steroids. case report: a 54 year old caucasian man with a prior esophageal cancer s/p esophagectomy, diabetes, hypertension, allergic rhinitis and chronic obstructive pulmonary disease presented with suprapubic pain, urinary frequency, dysuria, and hematuria. urinalysis showed numerous red blood cells and bacteria but no malignant cells or eosinophils. he was treated with antibiotics with resolution of symptoms. several weeks later he experienced severe suprapubic pain and hematuria resulting in a symptomatic drop in his hemoglobin. he underwent a cystoscopy and biopsy that revealed a chronic cystitis with an inflammatory infiltrate containing numerous eosinophils. he was unresponsive to treatment with nsaid and intravesicular dmso and was then referred to our service. we started prednisone 20 mg/day but the dysuria and hematuria persisted. prednisone was doubled plus a third generation quinolone was added. three weeks later the hematuria and dysuria resolved and he was asymptomatic. the antibiotic was stopped and prednisone was gradually tapered over several weeks. whenever his steroid dose dropped below 20 mg/day he experienced an exacerbation of his symptoms. over the last three years this dose of prednisone has kept his symptoms abated and his renal function stable. conclusion: eosinophilic cystitis is an uncommon diagnosis of unknown etiology. we describe a patient with debilitating suprapubic pain and symptomatic anemia from the hematuria associated with eosinophilic cystitis. for over three years since we first saw him, continued therapy with 20 mg of prednisone/day has prevented exacerbations. this case is unique in the severity of the hematuria experienced and the prolonged relatively low steroid dose needed to suppress exacerbations. we propose that in eosinophilic cystitis patients with severe hematuria who may otherwise be candidates for a cystectomy, a trial of prolonged oral prednisone may be beneficial. rationale: allergic reactions to insulin occurred more frequently in the past, with porcine and bovine preparations. in contrast, allergic reactions to human insulin preparations are now reported in <1% of patients treated with insulin. insulin allergy may be manifested as an immediate-type 1 ige-mediated reaction, delayed type hypersensitivity or as serum sickness, varying in severity from mild discomfort to life-threatening events. we present a case to illustrate that an insulin allergy may complicate hospitalization and often be misdiagnosed. methods: a 66 y.o. diabetic female with a history of "insulin allergy" was evaluated. the patient is a long standing diabetic controlled on oral hypoglycemics but required insulin during acute illnesses and hospitalizations. the patient was unable to recall previous types of insulin she had received. during previous hospitalizations, the patient complained of vague symptoms of fatigue, parasthesia, sweating, anxiety, and palpitations. on one occasion the patient had a syncopal episode with hypotension and on another occasion the patient developed a rash and dyspnea after receiving sq insulin. the physcian intrepreted the findings may be due to hypoglycemic. an allergic reaction was not suspected and the patient never underwent any testing. during a recent hospitalization, the patient's history was reviewed and a formal allergy consult was obtained. she underwent epicutaneous and intradermal testing to human insulin preparations. insulin antibody levels were also obtained. results: the patient had negative epicutaneous testing to all human insulin preparations. however, on intradermal testing, the patient had positive reactions to lispro, nph, and lente and negative intradermal tests to insulin glargine and regular insulin. igg and ige insulin antibody test results were < 1. conclusion: hospitalized patients receiving multiple medications commonly experience pharmacologic, adverse and/or allergic reactions. it is necessary to obtain a thorough history and document all reactions that patients experience to determine what type of event occurred. with a suspicion of drug allergy, skin testing and/or rast assay may provide insight into possible ige mediated reactions. in this case we advised the patient that she may use regular insulin during hospitalizations and that insulin glargine can be used to help achieve optimal glycemic long term control. background: hereditary angioedema type iii (hae iii) is a recently described form of angioedema occurring exclusively in females and characterized by normal c4, c1 inhibitor (c1 inh) protein and function. hae iii is thought to have an x-linked or autosomal dominant mode of inheritance. we evaluated a 13 year old female with recurrent facial swelling, abdominal pain and laryngeal edema with a family history of similar symptoms in several female relatives. case report: a 13 year old african american female presented with a two year history of recurrent lip, tongue and facial swelling. she also had abdominal pain, diarrhea and shortness of breath. episodes were not associated with hives. her symptoms predated menarche and did not correlate with her menstrual cycle. at the time of presentation she described an increase in frequency of her symptoms that did not respond to antihistamines (diphenhydramine, cetirizine) or prednisone. the patient had no other medical problems. family history was significant for recurrent episodes of facial, lip and tongue swelling in a maternal aunt, grandmother and great grandmother. the patient's great grandmother required tracheal intubation with ventilator support for upper airway compromise. the patient's physical exam was unremarkable. laboratory values drawn during an acute episode of swelling revealed: c1 inh function = >68% (normal >68%), c1 inh protein = 323mg/ml (normal 158-413 mg/ml), c4= 21.70 mg/dl (normal 20-59 mg/dl). dna sequencing at exon 8 to investigate the possibility of an unusual c1 inh mutation with normal c1s binding but abnormal kallikrein inhibition was negative. other lab tests included a normal mast cell tryptase of 2.9 mcg/l, a negative rf and ana, a normal angiotensin converting enzyme of 62 u/l (normal 6-64 u/l) and positive skin prick tests to a variety of foods which the patient tolerates. based on two case reports of treatment of hae iii with androgens, the patient was started on danazol 200mg daily with symptomatic improvement. conclusion: hae iii is a rare disease that affects females exclusively. clinically it is indistinguishable from c1 inh deficiency. the mechanism of inheritance remains to be elucidated. it is unclear why danazol appears to ameliorate symptoms even though there is no evidence for c1 annals of allergy, asthma & immunology inhibitor dysfunction in this disorder. we believe this to be the first kindred of hae iii reported in the united states. r. dworski * , m. peters, nashville, tn. a four-month-old female identical twin was evaluated for noisy breathing and recurrent cyanosis. she was delivered at 32 weeks of an estimated gestational age after an uncomplicated pregnancy. at birth she was intubated for 3 hours for respiratory distress but the remainder of her neonatal period was uneventful. at age 3 weeks she developed a noisy breathing often associated with cyanosis, particularly in a supine position or while crying. treatments with inhaled albuterol and prednisolone were ineffective. she had no respiratory infections or symptoms of gastroesophageal reflux disease. her growth was normal. her twin sibling was well. the family history was negative for allergies or respiratory conditions. initially she was diagnosed with tracheomalacia. however, the history of cyanotic episodes prompted a search for a definitive diagnosis. she underwent bronchoscopy which showed tracheomalacia when breathing spontaneously and circumferential narrowing of lower trachea likely due to compression. echocardiogram revealed a double aortic arch. the finding was confirmed by computed tomography angiography which demonstrated the presence of a vascular ring composed of double patent aortic arches, each giving rise to the ipsilateral carotid and subclavian arteries. the airway was normal at the aortic inlet, but narrowed markedly at the level of the two arches. a surgical division of the ductus ligamentous and distal anterior vascular arch was performed. aortic arch abnormalities should be suspected in all infants with hoarse coughing or noisy breathing, especially during inspiration but sometimes also during expiration. the diagnosis should also be considered in older children with recurrent bronchitis or pneumonia. respiratory symptoms are more frequent than gastrointestinal manifestations. diagnosis usually occurs in the first year of life. a surgery is often the treatment of choice. postoperative complications are relatively rare. outcome of surgery should be judged after 12 months, including at least one winter season. malacia can delay extubation and recovery following surgery. surgical cure occurs in approximately 70% of patients. surgery is probably less successful in children with double arches and malacia. a. thatayatikom * , a.j. white, st. louis, mo. background: common variable immunodeficiency (cvid) is the commonest symptomatic primary antibody deficiency syndrome in which b lymphocytes produce low levels of immunoglobulin, leading to recurrent bacterial infection. although hypogammaglobulinemia and susceptibility to the recurrent infection are seen in all patients, other associated conditions such as a non-infectious granulomatous disease have been well described in cvid. corticosteroid therapy has been used with improvement in a subset of cvid with granulomatous disease; however, its treatment remains problematic and a new therapeutic agent is needed. tumor necrosis factor (tnf ) has been demonstrated as a primary mediator in granuloma formation and maintenance. therefore, anti-tnf medications are potentially therapeutic agents of the granulomatous disease. case report: a 22-year-old caucasian male with cvid and severe granulomatous disease was treated successfully with infliximab, a chimeric anti-tnf monoclonal antibody. the patient initially presented with high fever, chills and abdominal pain; subsequently, he developed acute respiratory failure and adult respiratory distress syndrome. the patient was hospitalized and he required intensive care with ventilatory support. his diagnostic tests revealed elevated sedimentation rate and positive epstein-barr virus (ebv) capsid igm antibody. imaging studies demonstrated bilateral diffuse pulmonary infiltrates and hepatosplenomegaly. open lung and liver biop-sies revealed non-caseating granulomatous lesions without evidence of ebv or other infections. high dose corticosteroid therapy was given with partial improvement, then high dose infliximab (10mg/kg) was given weekly with remarkable improvement. the patient was able to wean off ventilator successfully within 3 weeks and his prednisone dose was dramatically decreased. infliximab infusion (5mg/kg) every 8 weeks and low dose prednisone were continued. a follow-up liver biopsy after 6 months of the infliximab showed no granulomatous lesions. infliximab was discontinued after 12 months of the treatment. there was no serious infection or complication during the period of treatment. conclusion: anti-tnf therapy may be a safe and effective treatment and may allow corticosteroid dose reduction. future clinical studies of anti-tnf therapy in cvid with granulomatous disease are warranted. background: chrug-strauss syndrome is a disorder characterized by hypereosinophilia and systemic vasculitis occurring in individuals with asthma. objetive: to present a pediatric case suffering from a systemic vasculitis. this case fulfilled the churg-strauss syndrome clinical criteria and the histophatology findings were compatible with the diagnosis. case: a 14 year female came to our institution with the diagnosis of severe asthma, chronic sinusisits and polyps requiring high doses of steroids. there was no history of administration of antileukotriene receptor antagonists. 2 months before her admission she presented weight loss, fatigue, cephalea and cough. on physical examination, pallor, respiratory difficulty and signs of bronchospasm were evident. tachycardia and hepatomegaly were also documented. the laboratory test showed anemia, eosinophilia 1300/dl, anca+, sgot 246, stgop 253, ige elevated 1345 ui/ml. the chest-x ray showed patchy opacities in both lungs and cardiomegaly. pulmonary scintigraphy reported low perfusion in both lungs, predominantly in the left lung.echocardiography demonstrated signs of myocarditis and eyection fraction of 32%. an open lung biopsy was executed and vasculitis with fibrinoid changes affecting small and medium vessels was reported. treatment was started with oral prednisone 2 mg/kg/d and cyclophosphamide pulses with a satisfactory evolution. discussion: to our knowledge this is the first pediatric case of css reported in mexico. she fulilled the following criteria. asthma, eosinophilia and systemic vasculitis involving the heart, liver and lungs. the disease is extremely rare, specially in mexico. aggressive treatment is necessary as in this case, with a favorable outcome. introduction "all that wheezes is not asthma" is a well-known aphorism among physicians. this same principle exists for patients that present with lip swelling in the allergist's office. we describe a patient who presented with fluctuating lip swelling who was ultimately found to have cheilitis granulomatosa. case history a 26-year-old male with a history of allergic rhinitis and asthma presented to the allergy clinic with lower lip swelling and occasional upper lip swelling. he was receiving allergen immunotherapy for dust mites and trees. the swelling had been waxing and waning for 13 years, but became more persistent for the previous six months. he denied tongue/throat swelling, dysphagia, respiratory distress, or any triggers for the swelling. chapstickâ® and vaselineâ® were used topically on his lips. failed treatments included loratadine, ranitidine, cetirizine, fexofenadine, and montelukast. he was placed on a one-week course of prednisone, which decreased his lip swelling, but it recurred after completion of the treatment. physical exam was significant for diffuse, firm lower lip edema to 3-4 times the normal size. there were no oral lesions or tongue swelling. patch testing to a standard panel, preservatives, oral flavors, and dental acrylate was negative. punch biopsy revealed a noncaseating epithelioid granulomatous inflammation consistent with granulomatous cheilitis. he was placed on minocycline 100 mg by mouth twice daily with little benefit. an 8-week course of oral prednisone resulted in improvement of the lip swelling. conclusion melkersson-rosenthal syndrome (mrs) is a rare syndrome that is characterized by a triad of recurrent facial paralysis, chronic edema of the face and lips, and hypertrophy and fissuring of the tongue. cheilitis granulomatosa is considered a monosymptomatic form of mrs and manifests as a chronic swelling of the lips caused by granulomatous inflammation. the swelling is typically not tender and may be either soft or firm. allergists are often consulted for lip swelling thought due to angioedema. however, as this case illustrates, not all lip swelling is angioedema and one must consider other diagnoses such as melkersson-rosenthal syndrome and cheilitis granulomatosa. progressive multifocal leukoencephalopathy (pml) is a disorder of the nervous system that affects individuals with immune suppression. it has been associated with hiv infection and is present in nearly 10% of patients with acquired immune deficiency syndrome. the jc virus, a common human polyomavirus, causes this demyelinating disorder. progressive symptoms reflect the multifocal distribution of brain lesions, and include mental deterioration, vision loss, speech disturbances, ataxia, paralysis, and, ultimately, coma. in rare cases, seizures may occur. there is no known treatment for pml. we report a 21 year-old (y/o) male with 6 months weight loss and a sudden onset of confusion, lethargy, and progressive loss of cognition requiring hospitalization. upon questioning he was found to have had recurrent upper respiratory tract infections since infancy successfully treated with antibiotics. as a child he had atopic dermatitis, exercise induced asthma, and myringotomy tubes placed twice. at 12 y/o, he underwent a nasal polypectomy. in 2002, at 19 y/o, a squamous cell carcinoma was removed, and he developed benign cervical lymphadenopathy and common warts. a year later he developed hsv esophagitis. the remainder of his history was unremarkable with normal development and growth and no history of drug abuse, multiple sexual partners, or homosexual contacts. on physical examination, he was thin, ill appearing, with oral ulcers, generalized scanty lymphoadenopathy, multiple common warts on both feet, and occasional ronchi. a brain mri showed a demyelinating process consistent with pml. pcr for jc virus was positive, while hiv pcr was negative; total immunoglobulins and cd4 counts were low ( we are reporting a 47 year old female with a 10 year history of moderate persistent asthma, who was started on xolair 150mg sq q 4 wks., and who then presented with a presumed allergic reaction. three hours after her second xolair injection, patient reported developing dizziness, shortness of breath, and felt like she was having an allergic reaction. she was evaluated and observed for two hours in an emergency department. the treating physician reported no wheezing and felt there was no need for treatment. to rule out psychogenic factors, she was given a placebo injection at the next scheduled visit. ten minutes later she reported developing throat tightness and shortness of breath. she had no changes in her peak flows and her lungs were clear. her "symptoms" resolved completely within 5 minutes of receiving placebo epinephrine and nebulized normal saline. patient was informed she had reacted to a placebo injection, as well as placebo epinephrine and albuterol, and counseled. the patient returned to the office every week for the next three weeks to receive blinded injections. she subsequently did not react to either doses of placebo or xolair. she has since tolerated her monthly xolair injections without incident. this case illustrates the importance of ruling out psychologic causes of presumed allergic reactions. introduction :latex allergy, type i ige mediated hipersensitivity, occurs specially in high risk populations, like in patients that have undergone various surgeries. case: a 10 year old boy with asthma and allergic rhinitis since 1996, penicillin allergy and retrospectively, his mother refers lip edema with balloon inflating.at 4 years of age (1998): left orchiorrhaphy because of cryptorchis. between 1999-2004: 4 surgeries because of sacral giant melanocitic naevus.during the fourth surgical intervention (0ct 8 -2004) to collocate a tissue expander, the patient presents perioral and fingertip cyanosis, generalized cutaneous rash and severe bronchospasm.ap:40/22 mmhg, hr 140 x/min. he was treated with iv fluids, steroids, antihistamines and inhaled racemic epinephrine.at the icu his final outcome is satisfactory. laboratory: total ige :27.4iu/l, skin prick test with glove extract, raw and natural latex extract and purified latex proteins(pseudoeveine, molecular hevein, hev b 6.02 and modified hevein)all positive +++. western blot with protein extract of latex positive and elisa with purified latex proteins ( same as above) positive. the last surgery to withdraw the tissue expander was performed with latex free surgical equipment without any problems. discussion: the patient's risk factors for latex allergy are atopy and repetitive exposure to latex articles because of surgery. he presents mild manifestations of latex allergy, till he finally develops full-blown anaphylaxis. diagnosis was made based on clinical history, skin prick test, western blot with protein extract and elisa with purified latex protein. he had a favorable outcome withdrawing latex during the last surgery. introduction: churg-strauss syndrome (css) is a form of primary vasculitis that is a rare diagnosis in an elderly patient. case report: a 75 year old woman presented with a 3 week history of fatigue, vomiting, diarrhea, abdom-inal pain, and right lower leg paresthesia. prior to admission she was being treated for left neck erythema and adenopathy presumed to be cellulitis. she was in good health with no history of atopy until the age of 72, when she developed both chronic sinusitis, requiring bilateral sinus surgery and polypectomy, * and new onset asthma, * that required systemic steroid control. prednisone was tapered 1 month prior to admission. objective findings included coalescent non-blanching petechiae on her abdomen, peripheral eosinophilia of 4800(50%), * normochromic normocytic anemia, rf=124, esr=90, ige=232, igg=1645. ana, p and c-anca were negative. ct showed ascites and pleural effusions. egd revealed duodenitis with ulceration and eosinophilic infiltration on biopsy. echo showed pericardial effusion and septal motion abnormality. troponin of 31 without cad was consistent with subepicardial myocarditis. emg confirmed right peroneal neuropathy.* skin biopsy revealed a dense superficial and mid-dermal perivascular and interstitial eosinophilic infiltrate. additional evaluation excluded malignancy, infection, and abpa. treatment with prednisone, 1mg/kg/day was initiated. a rapid clinical improvement ensued and has persisted. {*acr criteria for css}. discussion: churg-strauss syndrome is a rare form of vasculitis with mean age of onset within the third and fifth decades. it is an uncommon cause (<5%) of systemic vasculitis in patients older than 65. the formes frustes of css is a variant in which early manifestations of the syndrome are hidden by oral and systemic steroids employed in the treatment of worsening asthma often associated with css. this variant makes expedient identification of css more challenging. delayed recognition contributes to a relentless progression of this entity resulting in a systemic vasculitis with multi-organ involvement. early diagnosis, especially in the prodromal and eosinophilic phases, is essential. untreated, css has a high rate of morbidity and mortality. therefore, css and the formes frustes variant must be an integral component in the differential diagnosis of patients presenting with adult onset asthma and/or recurrent sinusitis. common variable immunodeficiency (cvid) is the most prevalent of the primary immunodeficiency diseases. cvid is a heterogeneous group of immunologic disorders of unknown etiology, characterized by impaired antibody responses, hypogammaglobulinemia with normal b cells. the common immunologic defect in patients with cvid is defective antibody formation, and many different immune system defects have been reported in this group of patients. most patients, really, have no identified molecular diagnosis. cvid consists of several different genetics defect. the immunologic defect in cvid is a failure of b-lymphocyte differentiation into plasmacells. b lymphocytes from these patients failed to differentiate into ig-producing cells when stimulated with pokeweed mitogen in vitro, even when cocultured with normal t cells. an overwhelming body of literature suggests that most patients with cvid have intact b lymphocytes of immature phenotype. however the functional classification of cvid patients on the basis of in vitro ig production is time consuming. recently has been proposed a new classification based on the quantitative repartition of memory b cell according to the dual expression of igd and cd27. we present a case of a 14 yr old boy. he presented soon in his life frequent infections, of particular severity: pneumonia, meningoencefalitis, sepsis, bronchitis, otitis, linfoadenitis. he also had a -thalassemia intermedia. this clinical manifestations suggested an immunodeficiency. for this reason at 2 years of age serum immunoglobulin and antibody detection showed a reduction in igg2 subclass and in cd19+ cells, with normal total igg, iga, igm, normal cd4/cd8 ratio, normal cd3, isohemagglutinins and in vitro t cell function. there was also a defective antibody production after tetanus, diphtheria, pertussis immunization. these laboratory findings did not allow, however, a sure diagnosis for cvid. a new immunological evaluation at the age of 11 years old, after the onset of splenomegaly, and enlarged lymph nodes, demonstrated a b memory defect, with a severe deficit of t lymphocytes function in vitro. it was also possible to find a severe deficiency of cd 27 + cells, meaning a defect in memory b cells: we can therfore label this condition as a cvid. in the past two decades there have been conflicting views regarding the clinical importance of igg subclass deficiencies in children. igg2 subclass plays a vital role in the immune response to polysaccharide antigen. isolated igg2 subclass deficiency may be widespread and often asymptomatic in children. however, in association with other subclasses and/or other immunoglobulin classes, there may be a significant, symptomatic outcome. the reported patient was diagnosed with familial dysautonomia (fd) at the age of five weeks, presenting with severe hypotonia and tachypnea. hindered by poor pulmonary function, he was hospitalized over fifteen times for recurrent pneumonias, including four lengthy intensive care admissions. daily inhaled-corticosteroids and brochodilator therapies were initiated, along with chest therapy via a high frequency chest wall oscillator. immunoglobulin levels were measured recently and point to low levels of igg2, igg4 and total iga antibodies: igg1 443 mg/dl (n 330-1065), igg2 57 mg/dl (n 57-345), igg3 50.5 mg/dl (n 7.5-125.6), igg4 <2.0 mg/dl (n 1.8-115.5), and total iga 28 mg/dl (n 33-235). since receiving monthly ivig therapy he had no further recurrence of pulmonary infections and was slowly weaned off daily brochodilator therapy. the currently accepted theory is that low igg2 subclass levels may be associated with increased risk of bacterial infections only in selective groups. fd patients may be in a distinctively susceptible population in which igg2 levels are critical. the older brother, who was also diagnosed with fd, demonstrated igg2, igg4 and iga levels that were slightly higher but nevertheless on the lower end of the normal range. he suffers from less invasive and less frequent bacterial infections. this may support a genetic association between the fd and hypogammaglobulinemia. alternatively, it may signal that fd patients may have a prolonged variant of transient hypogammaglobulinemia of infancy. follow-up immune profile studies, post-ivig trough levels and broader investigations of the fd population are necessary to determine the severity and prevalence of these findings. pulmonary failure is the dominant cause of death in patients with fd. prompt diagnosis and effective treatment of the associated immune deficiency may be proven essential in the effort to enhance and prolong their lives. s. hassan * , j.a. grant, galveston, tx. rationale: common variable immunodeficiency (cvid), a rare primary immunodeficiency presenting in young adults with repeated sinopulmonary infections as a result of profound hypogammaglobulinemia, was first described by suri et al. (ann acad. med. singapore, 1988) . we describe a young man with diagnosed but untreated cvid and its eventual course. case description: a 27-yr-old caucasian male hospitalized secondary to chronic pneumonia and respiratory failure was noted to have non-existent levels of immunoglobulins. history revealed ivig treatment at age 12, stopped after a year due to non-compliance. although untreated, he denied recurrent sinusitis, otitis media, or bronchitis for almost 14 years but notes a recent inability in keeping up with baseball practice. he is the last of ten healthy siblings. patient started monthly ivig infusions but was noted to have hypertension (150/90), tachycardia (120-130), tachypnea (25-30) on the 4th month with o2 saturation of 87-93% and po2 of 49% on room air. with a 2-day history of acute dyspnea, calf muscle and right abdominal pain, he was admitted to the hospital and pulmonary thromboembolism was ruled out. laboratory data: humoral functions (pneumococcal, tetanus toxoid, and hepvac) -undetectable t cell function (mumps, candida, ppd) -normal immunoglobulin (igg, iga, igm) -undetectable flow cytometry -b and nk cell markers normal, mild decrease in the cd4/cd8 ratio high resolution ct thorax -bronchiectasis, bronchial wall thickening, and obstructive changes with airtrapping. 6 minute walk -desaturation to 78% on 4l o2 by nc bnp -462 echocardiogramestimated ef 40-45%; severe pulmonary hypertension. cardiac catheterization -normal coronary arteries, severe pulmonary hypertension (pa pressure 70/40). conclusion: cvid patients have a reasonably good prognosis on treatment. untreated cvid is associated with chronic lung infections, bronchiectasis, pulmonary hypertension and right heart failure. although, lung transplant became available during the early eighties (nejm 1982), this extremely invasive but life saving procedure was undertaken in a patient with cvid and end-stage pulmonary hypertension in 1998 (thorax 1998). lung transplant may be the only way of ensuring survival for this patient. introduction chronic eosinophilic pneumonia (cep) is a rare disorder of unknown etiology characterized as a chronic and relapsing interstitial lung disease with blood or tissue eosinophilia. cep occurs more often in women with preexisting atopic disease. patients with cep respond rapidly to systemic corticosteroids, but often relapse with short, low-dose courses of therapy. while uncommon, extrapulmonary involvement, such as arthralgia, cutaneous purpura, pericarditis, and hepatitis, have been reported. we present a case of a patient who has cep with pericardial effusion. case report the patient is a non-smoking 60-year-old woman with a past medical history significant for allergic rhinitis and asthma who initially presented with a four-month history of worsening shortness of breath, dyspnea on exertion, dry, non-productive cough, and weight loss (approximately five pounds). her symptoms were refractory to increased dosages of inhaled fluticasone. she then developed intermittent fever up to 102â°c. chest x-ray revealed bilateral apical infiltrates. treatment with levofloxacin for seven days resulted in no improvement of symptoms or roentographic findings. thereafter, she presented with chest and abdominal pain, hypotension, and hypoxia. blood work revealed a white cell count of 6800 c/mm 3 with a differential significant for 34% eosinophilia (absolute eosinophil count of 2300 c/mm 3 ). chest ct showed dense consolidation predominantly along the peripheral aspect of the upper and superior segment of the lower lobes, and pericardial effusion. moderate pericardial effusion without evidence of tamponade was confirmed on echocardiogram. left upper lobe wedge biopsy findings included significant tissue eosinophilia, scattered foci of active organizing exudates, and no evidence of granulomatous or necrotizing vasculitis. the patient was treated for cep with a six-month course of prednisone, starting at 60 mg daily, resulting in rapid improvement of her pulmonary and systemic symptoms. background: immunologists are consulted for evaluation of immunodeficiency in patients with recurrent skin infections. disorders of the phagocyte system may be associated with cutaneous infections. methods: case report case: a 15-month-old african american girl (twin a) presents with recurrent skin abscesses. at 13 months of age, she had her first buttocks abscess, which required incision and drainage with oral antibiotics. a month later, she developed another abscess and was found to be neutropenic, with an absolute neutrophil count (anc) of 264/î¼l. cyclic neutropenia was considered and her pediatrician monitored cbcs, which all showed persistent neutropenia (ancs between 68 and 264). at 15 months of age, she was hospitalized for fever with neutropenia. immunology was consulted for evaluation of neutropenia. the rest of the past medical history was unremarkable. she was healthy appearing and growth parameters were appropriate for age. her physical examination was unremarkable. immunoglobulins, b-and t-cell markers, nitroblue tetrazolium, complement assay, hemoglobin, platelets and the peripheral smear were normal. antibodies for hiv, cmv, ebv and parvovirus were undetectable. anti-neutrophil antibodies were positive, establishing the diagnosis of primary autoimmune neutropenia (ain). the abscess healed with oral antibiotics. severe neutropenia (anc 0-280) persisted for three subsequent months without further infections. twin (b) was also found to have persistent severe neutropenia without morbidities, suggestive of primary ain. primary ain is less known among physicians and is typically diagnosed after extensive investigations that exclude other causes of neutropenia. the exact incidence of ain is unknown. it is usually seen in children between 5 and 38 months of age, often with severe neutropenia and self-limiting bacterial infections. the clinical course and presence of antibodies to neutrophil antigens (na1, na2 or cd11b/18) is diagnostic. familial occurrence of primary ain is not reported in the literature. conclusion: primary ain may remain under diagnosed due to lack of characteristic clinical features. although a benign clinical course is likely, severe infections, including pneumonia, sepsis and meningitis, have been reported. genetics may play a role in this disease, as we present primary ain in twins. background: atopic dermatitis (ad) is a chronic inflamatory disease of skin that affects 5% of the wordl population.the natural history of ad in some patients, evolve to the coexistence with other allergic diseases: allergic rhinitis, allergic conjunctivitis and asthma. the sublingual immunotherapy has demonstrated utility in some patients; however, semi-rush immunotherapy to pollens has only showed utility in one animal case published few years ago. case report: a 2-year-old infant was referred to us. he began one year before with skin lesions compatible with ad, six months later began perennial rinhorrea and nasal itching. multiple treatments with topic corticosteroids, moisturizing, antihistamines and topic/systemic antibiotics did not demonstrate utility. our evaluation revealed ad lessions that affected 60% of the total body surface and clinical features of allergic rhinitis (ar).the lab tests revealed eosinophylia (600cell/mm3) in blood cell count, normal levels of total ige but specific ige to dermatophagoides pteronissinus (dpt) was high. the skin prick test reveales the same results. we added environmental control, oral costicosteroids and transfer factor. despite our treatment no improvement was observed. we considered dpt as the principal factor in the maintenance of ad lessions and ar episodes. in the absence of sublingual immunotherapy in our hospital, we decided for semi-rush immunotherapy schedule. we began from 0.1 ml of 1:10, 000 w/v concentrations of dpt until 0, 5 ml of 1:100 w/v concentrations in two months receiving three doses per week. no local or systemic adverse events was reported and the ad lesions and ar symptomatology disappeared in the first month of treatment. at this time, no exacerbations have been documented. conclusion: the semi-rush immunotherapy can be useful and safe for treatmente of ad in some patients in whom specific ige to aeroallergens has been demonstrated. introduction: eosinophilic gastrointestinal disorders are a rare group of disorders that can involve the entire gastrointestinal tract. presentations are varied but may include vomiting, dysphagia, abdominal pain, diarrhea and failure to thrive. the diagnosis is made by endoscopic biopsies which reveals eosinophil rich inflammation in the absence of known causes for eosinophilia. peripheral eosinophils and ige may be elevated but can be normal. we report a patient with eosinophilic gastroenteritis associated with an ampullary tubulovillous adnenoma. methods:a 71-year-old white male with allergic rhinitis and a family history of atopy was admitted for profuse watery diarrhea. he denied any new medications, eating raw foods or recent travels. eosinophils were elevated to 2.4(29% of wbc) and ige was elevated to 3259. multiple stool specimens were negative for ova & parasites and enteric pathogens. serology was negative for strongyloides, trichinella, e. histolytica and toxocara. ast, alt & bilirubin were elevated and a ct scan showed dilated billiary ducts with a possible ampullary mass. biopsy of the ampullary mass revealed a tubulovillous adenoma. biopsies of the duodenum, terminal ileum, colon and rectum were remarkable for focal eosinophilic cryptitis & chronic inflammation in the lamina propria consisting of eosinophils, scattered lymphocytes and histiocytes. all specimens were negative for parasitic infections including duodenal aspirates. he was empirically started on metronidazole and singulair with gradual improvement of symptoms, eosinophils and liver tests. two months after discharge, the patient remained diarrhea free with a normal eosinophil count. conclusion: we report a patient with eosinophilic gastroenteritis and an ampullary tubulovillous adenoma with obstruction of the biliary system. this unique presentation illustrates the diverse nature of gastrointestinal manifestations seen in eosinophilic gastroenteritis. background: zonisamide is an anti-seizure medication chemically classified as a sulfonamide and unrelated to other ant seizure agents. we report a case of hypersensitivity to this agent in a child. method: case report results: this patient is a 22-month-old girl who developed "peeling of her lips" three weeks after starting zonisamide. one week later she developed a rash that began on her face and generalized over several days to her neck, trunk and extremities. there was no respiratory distress, joint complaints and no angioedema associated with the episode, but fever to 103 prompted referral to our hospital on day 6 of the rash. the rash was macular-papular without discrete urticarial lesions. the rash coalesced with underlying erythema on face, chest and neck. the patient has a known history of seizure disorder, asthma, mild eczema, gastro esophageal reflux, development delay, lactose intolerance and failure to thrive. her other medications were, lansoprazole, topiramate, and albuterol. labs revealed a normal white cell count, an elevated sed rate ( 51) and elevated lft's, i.e. sgot 80 and sgpt 180. the only new medication was zonisamide that was discontinued. she was treated with iv steroids and hydroxyzine. the rash started fading by day 2 and the patient's fever resolved by the third hospital day. liver enzymes returned to normal by day 7. conclusion: this relatively new anti-seizure agent can be associated with hypersensitivity reactions in children. introduction: kawasaki disease (kd) is an acute chilhood vasculitis. in addition to the diagnostic criteria a broad range of nonspecific clinical features may be observed including aseptic meningitis, vomiting, diarrhea, abdominal pain, sterile pyuria, arthralgia and arthrtis, pulmonary infiltration, pleural effusion and nonspecific paralytic ileum as manifestation of gastrointestinal vasculitis. we describe a child who developed all features of kawasaki disease included the most rarely reported. patient report: a 2 year-old female presented 15 days of high fever, nonsuppurative cervical lymphadenopathy, petequial rash in legs, swelling of feet, distended abdomen, vomiting, obnubilated and hypoactive, incongruent speech, fisured lips and distended abdomen. lab tests showed: anemia (9.8g/dl), high wbc count (24, 200cells/mm3), thrombocytopenia (47, 000/mm3), hypoalbuminemia (1.1g/dl), lactate dehydrogenase (ldh) 209mcg/l, glutamin transferase (ggt) 190. csf total proteins 129, glucose 60, cells 23, pm 5%, mn 95%, seric complement 88, cultures were negatives. she developed myocarditis, aneurysms in the right and left coronary arteries. on the 25th day presented cardiac failure, pleural effusion, paralytic ileum, hydrops vesicular, mechanic ventilatory assistance was required. the first dose of intravenous immunoglobulin (ivig) (2g/k) was infused, heparin and hydrocortisone. on 45 day a second doses of ivig was infused, because of fever, and abdominal vasculitis. steroids ware discontinued, heparin was suspended and aspirin was added as antiaggregant. discussion: our patient presented an unusual and severe presentation of kd with pleural effusion, nonspecific ileum, cardiac failure secondary to myocarditis, aseptic meningitis and thrombocytopenia; all those manifestations had rarely been reported at the same time. she presented with a devastating evolution. complications were resolved. the lastest studies have shown that treatment with ivig plus corticosteroids significantly reduce serum concentrations of proinflamatory cytokines. in this case antiinflamatory doses of aspirin were contraindicated and steroids were used with satisfactory outcome. limited data is available for nonresponding patients to guide therapy. although multiple doses of ivig are sufficient in some patients, some of them remain refractory to therapy and they need corticosteroids to control the vasculitis process eosinophilia is defined by an absolute eosinophil count above 0.7 x 10 9 and can be seen in association with a broad spectrum of disorders ranging from allergic to malignant. idiopathic hypereosinophilic syndrome (hes) should be considered in any patient with an eosinophil count above 1.5 x 10 9 for more than 6 months without commonly recognized causes of eosinophilia and with evidence for organ damage not otherwise explained in the clinical setting. it is potentially an aggressive disease with mean survival of less than a year without treatment. we present a 39 year old male horse breeder, with eosinophilia lasting more than 10 years. prior to coming to our institution, he underwent extensive medical evaluation including bone marrow and gi biopsies, multiple imaging studies with mri and ct scans of the chest and the head as well as detailed evaluation of his cardiac status. all these tests were normal and the patient was empirically started on treatment for possible hes including trials of prednisone, hydroxyurea, interferon alfa, and imatinib, all without lasting resolution of his eosinophilia and causing significant side effects with profound depression of his immune status. finally, in light of the patient's profession, a strongyloides enzyme immunoassay was done and found to be remarkably positive. duodenal drainage confirmed the infestation with this nematode. within a week of starting treatment with ivermectin, his eosinophil count came down by 50% and eventually normalized. we learn that one should be persistent in excluding all common causes of eosinophilia before considering hes. in case of parasite infestation, premature treatment with immunosuppressors can result in worsening of the infection with potential for poor and even fatal outcome. stool evaluation might not be sensitive enough for detection of parasites and it is therefore necessary to complete a diagnostic work up with appropriate serology. doxil is the pegylated liposomal form of doxorubicin and has been used successfully as a cancer chemotherapy agent in many types of tumors. a hypersensitivity reaction can occur, usually during the first infusion, and appears to be rate related. the symptoms include dyspnea, tachypnea, facial swelling, chills, hives, chest pain, and back pain. we report a case of a hypersensitivity reaction to doxil in a 26 year-old woman with hodgkin's lymphoma. during her first outpatient doxil infusion at 1 mg/min, she developed urticaria, chest tightness, dyspnea, and back pain. these reactions persisted despite being medicated with antihistamines and steroids and immediately resolved during pauses in the infusion. after 23 mg of doxil, the infusion was discontinued. six days later, she was admitted to complete the other half of the dose. she had a negative intradermal skin test to a 1:100 dilution of doxil. she was then premedicated with diphenydramine, dexamethasone, acetaminophen, and famotidine. the doxil infusion was started at 0.4 mg/min and was soon stopped due to flushing of the hands and face. the infusion was decreased to 0.2 mg/min. she was able to tolerate this slow infusion with only mild and tolerable symptoms. when her symptoms worsened, the infusion was stopped for 30 to 60 minutes. she completed the 23 mg infusion of doxil after 13 hours. pre-infusion and post-infusion complement levels were drawn during this second administration of doxil. her pre-infusion c2, c3, c3a, c4, c5, c5a, and bb levels were all normal. her pre-infusion c4a and sc5b-9 levels were high, indicating that she might have had some residual or persistent complement activation caused by her first doxil infusion. her post-infusion c2, c3, c3a, c4, c5, c5a, and bb levels were all normal. however, her post-infusion sc5b-9 levels significantly increased, suggesting complement was activated during the second doxil infusion. given her reaction during her first doxil infusion and a negative skin test to doxil, it is highly unlikely that her doxil hypersensitivity was an ige-mediated process. therefore, in patients with a similar doxil hypersensitivity, we suggest a slow rate of infusion of 0.1-0.2 mg/min, toleration of mild symptoms, 30 to 60 minute pauses during more severe symptoms, and continuation of premedication during the entire lengthy infusion. introduction eosiniphilic esophagitis (ee) is an isolated, severe esophageal eosinophilia. patients are usually young males presenting with vomiting, epigastric or chest pain, dysphagia and obstructive respiratory problems. they are often initially misdiagnosed with and treated for gastroesophageal reflux disease (gerd). distinction between the two diseases can be made with biopsies of the esophageal mucosa. while any eosinophils in the esophageal mucosa indicate pathology, gerd typically presents with up to 7 eosinophils per high powered field (hpf, 400x) while ee most often presents with greater than 20-24 eosinophils per hpf. case report the patient is a 3 and one half year old boy who has experienced severe symptoms of gerd from the age of 6 months. he vomits after meals at least 1-2 times per day. he coughs when he lies down at night and regurgitates phlegm in the early morning. he has complained of discomfort in his lower chest after eating. his weight has remained at 35 pounds for the last six months. the patient was diagnosed with asthma at age 2 and a half; however, there is no family history of asthma or allergies. the patient initially experienced improvement on lansoprazole, but his symptoms recurred when the medication was discontinued and subsequent courses were ineffective. his physical exam was normal barring slightly edematous nasal turbinates. an endoscopic biopsy showed "numerous eosinphils" (later clarified to 38 eosinophils per hpf) in his distal esophagus. the patient showed allergy to milk and wheat on radioallergosorbent (rast) testing. the patient was started on swallowed fluticasone 2 puffs twice daily and advised to see a nutritionist regarding a wheat and milk elimination diet. conclusion ee is an important differential of gerd-like symptoms in childhood. to avoid misdiagnosis it is critical to evaluate the number of eosinophils present in a biopsy specimen to help differentiate between gerd and ee. children with ee are at increased risk of developing esophageal dysmotility and esophageal strictures. patients often have positive skin prick or rast tests to foods and aeroallergens. alternative treatments such as food elimination diets and glucocorticoids (systemic or topical) are effective in treating symptoms which may not respond to reflux medications. introduction in 1992 asherton introduced the term catastrophic antiphospholipid syndrome (caps) to describe patients sharing clinical evidence of multiple (three or more) organ involvement and/or histopathological evidence of multiple vessel occlusions. case report we present a 13 year old female, with lumbar pain, arthralgias, weight loss, fever, malaise, raynaud phenomenom, alopecia, oral ulcers and hepatomegaly. on arrival, she was polypneic with tachycardia, basal hypoventilation, and hepatomegaly was evident. hb: 8.8, leucocytes: 39.300, total lymphocyctes: 8, 300, total neutrophyles: 28, 700, platelets 393, 000. coombs positive. urin exam: proteinuria, leucocyturia and erythrocyturia. creatinine 1.3. diagnosed as sle with pericarditis, cardiac failure and acute pulmonary edema, urinary tract infection and pneumonia, requiring mechanical ventilation and inotropic support, intravenous gammaglobulin and hydrocortisone. acute renal failure and hemodialysis was begun with improvement. suddenly she presented seizures crisis, stuporous, livido reticularis skin and acrocyanosis, external opthamalplexia, bilateral facial diplexia, ocular fundus with arteriolar vasospasm. right facio-corporal hemiparesia, cortical and progressive medular annals of allergy, asthma & immunology segment changes. ct showed multiple left fronto-occipital parietal hypodensities suggestive of lacunar infarcts. diagnosed as neurolupus and caps. initially anti b2 glp1 and anti clp were negative with later positivization. urinary tract infection contraindicated high doses of steroids and, intravenous gamaglobulin and anticoagulation were started. with significant improvement. currently the patient is evolving in a satisfactory way. discussion the literature establishes that catastrophic aps is characterized by elevated mortality. in this patient damage was evident to the cns, pns, kidneys and the skin. with the presence of positive antiphospholipid antibodies with an energic antiinflamatory, immunoregulatory and immunosupressive therapy, the function of each affected organ completely recovered. this case exemplifies that the therapy of caps should be undertaken in a sufficient and early manner given the elevated mortality of this syndrome. patient, a 70-year-old white, male non-smoker physician on high-dose regimen of advair and singulair presents with an 8-day history of progressive chest tightness. pulmonary function tests showed normal (fev 1 of 96%) lung function. in the past, whenever inhaled steroid dosages were lowered, patient experienced a reoccurrence of asthma symptoms, despite consistently normal lung function results. to rule out the possibility of a psychologically induced asthma exacerbation, the patient's fractional concentration of exhaled nitric oxide (feno) levels were measured. the patient's feno level was elevated at 48.9 ppb. values greater than 30 ppb have been described as consistent with airway inflammation. with an increase in the patient's inhaled steroids, the patient had a remission of symptoms within a week. this case is illustrative of an increasingly common clinical picture where a symptomatic asthmatic may have normal spirometry but elevated feno. as devices for measuring feno become more available in the outpatient clinic setting, elevated feno may be an excellent diagnostic marker in assessing whether airway inflammation is being adequately treated in situations where spirometry values are within normal ranges. introduction the autoimmune thrombocytopenic purpura (atp) is characterized by thrombocytopenia and megakaryocytic hyperplasia. the first choice of treatment consists of intravenous gamma globulin (ivig), corticosteroids and anti-d antibodies and the second line measures are immunosuppressant drugs, splenectomy and danazol. case report a 4 years old male presented in the first year of life ecchymosis in several parts of the body intermittently. in april 2003 he presented ephistaxis and lower gastrointestinal bleeding. complete blood count showed platelet count of 45, 000/ul. prednisone was started (2 mg/kg) with no improvement. at that time, danazol, anti-d antibodies, and ivig (2 g/kg) were added. subsequently, the platelet average count diminished to 5, 000. the patient was transferred to our hospital. at his admission he presented cushingoid habitus (figure 1) and acanthosis nigricans. the immunological tests (aan, ch50, c3, c4, immunoglobulins, anticardiolipins and b2 glycoprotein) showed no alterations. bone marrow aspirate demonstrated megakaryocytic hypercellurarity. prednisone dose was tampered when the patient presented secondary glaucoma. patient continued his treatment with danazol and ivig (2 dosages of 2g/kg each), hydroxychloroquine and cyclophosphamide with no improvement. thus, 3 months after the immunosuppressant treatment, splenectomy was performed, obtaining partial improvement. at that time ranitidine and hydroxychloroquine were suspended and cyclophosphamide was changed to azathioprine. last platelet count was 106, 000/ul. conclusion chronic atp in childhood as the present case account for approximately 4-5% of the total atp patients. chronic atp that does not respond to conventional treatment is a therapeutic challenge. in this patient we used first choice and second choice drugs with no improvement, consequently a splenectomy was indicated, not obtaining the desired response at first. the use of immunosuppressant drugs is not common for this disease, but it could be a good alternative for atp resistant to conventional treatment. this are the most important laboratory test of our patient. background: budesonide is the only corticosteroid available for inhalation by jet nebulizer and is indicated for the treatment of asthma in children. objective: to evaluate the distribution and clinical efficacy of inhaled budesonide administered by nebulization with a modified commercial device. methods: a 23 year old male with severe persistent asthma underwent a lefort procedure for multiple craniofacial abnormalities. the external device maintained his mouth open impeding the proper use of controller medications. as a result he developed an increase in his asthma symptoms. he was subsequently treated with nebulized budesonide delivered with a modified jet nebulizer through the end of a plastic tube in a flow-by manner. we then performed dynamic ventilation imaging after administering 33.40 mci of nebulized technetium 99m-dtpa diluted in two ml of normal saline. images were obtained for five minutes at three seconds per frame. spirometry monitoring was not possible given the obstructive nature of the facial device. results: the patient demonstrated improvement of his asthma symptoms. the ventilation scan showed that the patient breathed the technetium dtpa through the specialized device with delivery to his full lung volumes within 12 seconds. conclusions: we report the successful treatment of a patient unable to use the commercially available methods for administration of inhaled steroids. rationale: hp is a non-ige mediated inflammatory response in the lungs due to a variety of organic antigens, including metal working fluids. the wideranging clinical features include acute, subacute, and chronic forms. elevated antineutrophilic cytoplasmic antibodies and platypnea, defined as dypsnea induced by the upright position and relieved with recumbency, have not been previously reported in patients with hp. case: 35-year-old white male tool and dye machinist presented with progressive cough, weakness, dyspnea on exertion, and platypnea. symptoms began 15 weeks earlier with coryza and diffuse myalgias. he had lost 12% of his body weight since symptom onset. examination revealed a pulse of 140, respiratory rate 18, and right basilar crackles. resting pulse ox on room air was 95%, but dropped to 91% upon ambulation. pft's revealed severe obstruction (fev1 42% predicted) with significant reversibility (fev1 +19%), and a dlco of 82% (adjusted for va and hemoglobin). hemoglobin was 17.5 with a normal leukocyte count and differential. c-anca (anti-pr3) was 19.3 (<5), and p-anca (anti-mpo) was 15.9 (<15), both performed by elisa. echocardiogram showed an ef of 50-55% with mild pulmonary hypertension; no shunt was present. chest roentogram was normal, but a chest ct revealed a diffuse ground-glass pattern with scattered centrilobular opacities. biopsy was consistent with hp. he was removed from his work exposure to metal working fluids with full recovery of lung function and resoulution of symptoms. conclusions: hp presents as a constellation of symptoms without a single, unique identifying pattern. platypnea and elevated anca's have been observed in a wide range of disorders, all of which were excluded in this patient. platypnea has been associated with hereditary hemorrhagic telangiectasia, pulmonary avm, hepatopulmonary syndrome, recurrent pulmonary emboli, and patent foramen ovale. elevated anca's has been associated with wegener's granulomatosis, goodpasture's syndrome, churg-strauss vasculitis, drug-induced vasculitis, inflammatory bowel disease, and others. elisa is a more specific modality for anca's than indirect immunofluorescence, but it is less sensitive. the possibility of hp should be considered in patients with either of these two abnormalities. rationale: heart disease is the leading cause of death in america and 70% of persons between 70 and 80 years of age have coronary athersclerosis at autopsy. the presentation in the elderly is commonly atypical. 45% of myocardial infarctions were silent or unrecognized in the framingham cohort. in patients 65 and older, it is estimated that 8-25% will present with dyspnea without any associated chest pain. we present a case of unstable angina presenting as exercise-induced asthma. case: an 81 year-old white male with moderate persistent asthma, hypertension, dyslipidemia, and a long history of allergic rhinitis presented having had an abrupt increase in his usual exercise-induced asthma symptoms four weeks prior. asthma had been diagnosed at age 69 with spiromety showing moderate obstruction and significant but incomplete reversibility. he had a remote history of pipe and cigar smoking, but had quit at age 50. he had been well-controlled since that time with his most recent regimen consisting of fluticasone 100 mcg/salmeterol 50 mcg diskus, one inhalation twice daily. his typical exercise-induced asthma symptoms included dyspnea on exertion and chest tightness without radiation, and were relieved with rest and albuterol. the amount of exertion needed to trigger his symptoms, however, was much less than he had previously experienced, and this remained constant during the four weeks prior to his presentation. one week prior he had a normal ecg evaluation by his primary care provider. six months prior he had a normal nuclear cardiac stress test. physical examination was unremarkale except for moderately decreased aeration and 1+ pitting edema of his lower extremities. cardiology performed a nuclear stress test the next day which was abnormal. catheterization revealed 99% steonsis of his proximal lad. he succefully underwent cabg and is doing well on follow-up. conclusion: the elderly present many challenges to the diagnostician; these include multiple co-morbidities as well as atypical and often late disease presentations. the key to raising suspicion for cardiac involvement in this case was the recognition of the patient's cardiac risk factors in the setting of an abrupt onset of exercise symptoms while lacking other asthma symptoms such as nocturnal cough. cold urticaria is an uncommon form of physical urticaria. this case of a 9-year-old with cold urticaria and angioedema provides additional information regarding an unusual disorder in the pediatric population. an otherwise healthy 9-year-old female presented with a complaint of urticaria precipitated by cold exposure over the preceding 5 weeks. she had no recent illnesses and a past medical history significant only for cat allergy. on multiple occasions the patient noted erythema and pruritus of her arms and face after walking through the freezer aisle of a grocery store. urticaria would then develop on regions where she scratched, spontaneously resolving in 2-3 hours. on one occasion, urticaria appeared diffusely while taking a shower after the patient had been swimming. the urticaria resolved within a few hours after the patient was given diphenhydramine by her mother. three days prior to presentation the patient experienced upper lip angioedema with erythema, globus sensation and difficulty swallowing after drinking a strawberry slushy. the patient denied any respiratory complaints at that time and her symptoms again annals of allergy, asthma & immunology resolved spontaneously. family history was significant for a maternal history of seasonal allergies. upon physical exam the patient was well appearing. she had 2-3 discrete urticaria on each calf. the patient's mother noted that recently these would appear on "cold and rainy" days, attributing them to the fact that the patient's pants left her lower legs exposed. the remainder of her exam was normal and dermatographism was absent. laboratory evaluation consisted of cryoglobulins and strawberry rast, both of which were negative. application of an ice cube to the patient's forearm for 5 minutes resulted in a 9x6 centimeter wheal noted 3 minutes after ice removal. a diagnosis of cold urticaria with associated angioedema was made. the patient's mother opted to use diphenhydramine as needed and an epinephrine autoinjector was dispensed. by 3 months after symptom onset, the patient's only complaint was pruritus of her hands if they became too cold. no urticaria were noted. at 6 month follow-up the patient denied any symptoms for the preceding 2 months and had a negative ice cube test. cold urticaria in the pediatric population is a rare entity and not well understood. this case of a 9-year-old with cold urticaria and angioedema offers additional insight into this unusual disorder. a. khuntia * , m. mcmorris, ann arbor, mi. introduction: chronic granulomatous disease(cgd) is a heterogeneous group of disorders characterized by genetic defects in the ability of phagocytes to generate microbicidal reactive superoxide anions and its metabolites. it manifests early in life, primarily as recurrent infections, caused by catalase-producing bacteria such as staphylococcus aureus, burkholderia cepacia, and serratia marcescens and fungus such as aspergillus fumigatus. the disease may be inherited in an x-linked or autosomal recessive manner, with x-linked disease accounting for 65-70% of cases. the us incidence of cgd is 1/250, 000 live births with an average age at presentation of 3 years for xlinked and 7.8 years for autosomal recessive disease. case report: 18 year old male with history of three separate episodes of pneumonia beginning at age 16. each episode resulted in a hospital admission and intravenous antibiotic therapy after failed oral antibiotic therapy. an extensive pulmonary evaluation was initiated after the third episode of pneumonia, including a chest ct, viral, bacterial and immunodeficiency studies. cbc, complement, immunoglobulins, flow cytometry, viral and bacterial studies were all normal. the ct scan revealed dense nodular opacities in the right upper lobe with surrounding ground-glass opacification and mild bronchiectasis. a subsequent bronchoscopy demonstrated necrotizing granulomatous inflammation. open lung biopsy grew burkholderia cepacia on tissue culture. the clinical history, tissue histopathology and atypical organism found on culture were all suggestive of an underlying immunodeficiency. a neutrophil oxidative burst assay was performed which demonstrated minimal neutrophil activity upon stimulation, suggestive of the diagnosis of cgd. a chemilluscence assay verified the diagnosis of cgd, with minimal fluorescence noted on flow cytometry after neutrophil stimulation. dna analysis demonstrated a p47-phox deficiency, resulting in one of the autosomal recessive and less clinically severe forms of the disease. conclusions: this case of cgd is unusual because of the delayed presentation. it demonstrates the importance of a complete immunological evaluation including an evaluation for neutrophil disorders such as cgd in patients of all ages who present with recurrent and recalcitrant episodes of pneumonia, especially when atypical organisms such as burkholderia cepacia are found on culture. autoimmunity may play a role in the development of premature ovarian failure (pof), but the exact mechanism is not well understood. pof is mainly diagnosed after 19 years of age and most women complain of secondary amenorrhea. pof has been reported in combination with presence of ana and autoimmune diseases, but rarely with jra. here we report two cases of pof and positive ana in pediatric patients, one with clinical features of jra. the first patient, an african american 17-year-old girl, height in 50th percentile, weight in 10th percentile, with tenosynovitis of wrists and arthritis of knees and elbows for the past two years, was referred for further evaluation and management. she did not have her menarche yet and laboratory investigation revealed positive ana. the second patient, an african american 17-year-old girl, 50th percentile for height and weight, was referred to the immunology clinic with chief complaint of primary amenorrhea and delayed development of secondary sexual characteristics and was found to have positive ana without clinical findings of jra. both patients were tanner stage 2 for breasts and tanner stage 2-3 for pubic and axillary hair. both had elevated fsh and lh, karyotype 46xx and small uterus and small ovaries on pelvic ultrasound. antiovarian antibody was not detected in any of the two patients. the bone age was significantly delayed. pof in karyotypically normal women is frequently seen in combination with elevated serum ana. the clinical spectrum of rheumatoid disease associated with pof ranges from asymptomatic ana positivity to typical presentation of jra. women with pof should be monitored for the emergence of autoimmune disorders including jra, and women with jra should be followed for menstrual irregularities and signs of pof. introduction: many u. s. military personnel deployed to the middle east continue to develop infection with leishmaniasis, a parasite transmitted by the sand fly. pentavalent antimonials have been used as an effective treatment for leishmaniasis for many years. in the united states, sodium stibogluconate (pentostam) is the pentavalent antimonial of choice, and is currently being administered under an ind protocol. side effects of therapy include myalgias, arthralgias, rash, malaise, abdominal pain, pancreatitis, and hypersensitivity reactions. the true incidence of hypersensitivity reactions is not currently known. case reports: two u. s. soldiers receiving pentostam for the treatment of cutaneous leishmaniasis were evaluated in our clinic at walter reed army medical center after experiencing urticaria, angioedema, wheezing and dyspnea after medication infusion during the 20-day course of daily therapy. due to the concern of a potential ige-mediated reaction, skin testing was performed. after informed consent, skin testing included a prick test at full strength, followed by intradermal (id)testing. soldier #1 was a 23-year old male reporting lip swelling, throat tingling, dyspnea and chest pain 7-8 hours after treatment #10/20. skin testing to both lots used during the treatment course showed positive values of 8x20mm and 9x14mm respectively at id 1:1000. soldier #2 was a 28-year old male reporting diffuse pruritus, hives, dyspnea, and chest pain 15 minutes after infusion #16/20. skin testing showed positive values of 7x14mm and 8x12mm respectively at id 1:1000. due to clinical symptoms and positive skin testing, therapy was discontinued in each case. one control individual showed negative testing to prick at full strength, id 1:1000, and id 1:100. conclusion: skin testing with pentostam may provide an objective tool for accurate classification of adverse reactions as igemediated. reliance on skin prick testing alone may not be sufficient to detect pentostam skin test reactivity, as both of these patients reacted to id testing only. a prospective study including pre-and post-treatment skin testing should provide more information on the value of skin testing in providing objective evidence for an ige-mediated process and determining the incidence of hypersensitivity to pentostam. introduction: a 3-year-old girl with a history of multiple infections was hospitalized with respiratory distress and hypoxia. her past medical history was significant for hypothyroidism, psoriasis, asthma and multiple pneumonias. a cbc revealed an absence of lymphocytes. laboratory: t and b cell subsets showed no b-cells and very low t cells (<100 cells). inadequate lymphocytes were present for mitogen and antigen studies. serum immunoglobulins were normal and she had antibodies to streptococcus pneumoniae and tetanus. antibodies to rsv, influenza and mycoplasma were absent. chest x-ray revealed interstitial infiltrates bilaterally. a high resolution ct scan revealed septal thickening and confirmed interstitial infiltration. open lung biopsy was consistent with non-specific inflammation with extensive lymphocytic infiltration. bal fluids and biopsy were negative for bacteria, fungi and opportunistic pathogens. clinical course: the patient did not improve with antibiotics and steroids were started. the patient improved rapidly and was discharged to home. biochemical analysis demonstrated a deficiency of adenosine deaminase (ada), a form of severe combined immune deficiency (scid). the patient was started on replacement ada, adagen. a repeat high resolution ct scan showed some improvement. however, the patient continues to be steroid dependant to control her pulmonary symptoms. lymphocyte numbers remain low despite effective ada replacement and the absence of serum datp. she remains clinically stable and receives adagen injection twice weekly. discussion: ada deficiency is a condition, which leads to accumulation of the metabolite datp, which is toxic to lymphocytes. this disease most commonly presents in the first year of life as scid and is fatal unless treated. our case is unusual due to the late onset of severe disease, normal serum immunoglobulin and the presence of some protective antibodies. despite adequate replacement of ada the patient continues to be profoundly lymphopenic, most likely due to steroids. although we do not yet completely understand the underlying lung disease we suspect that the patient has an autoimmune process causing interstitial inflammation. background: atopic dermatitis has a broad range of differential diagnoses. human sarcoptic infestation is characterized by severely pruritic lesions of variable appearance and distribution and may masquerade as eczema. infestation may be difficult to confirm and eradicate. animal transmission has been reported as a source of human infection. objective: to report a case of persistent sarcoptic infestation masquerading as eczema and associated with delusional parasitosis. methods: a 58-year-old female was referred to allergy clinic for evaluation with a two year history of recurrent, pruritic rash thought to be refractory atopic dermatitis. previous ineffective treatments included topical steroid creams, lindane, topical anti-fungals and multiple otc antiitch preparations. at initial evaluation, she had widespread excoriated papules in various stages of healing over 70% of her body. she reputed her dog was diagnosed with recalcitrant mange, which necessitated giving him medicated baths twice daily. results: a clinical diagnosis of subacute sarcoptic infection was made and the patient was prescribed two courses of elimite followed by oral ivermectin. her rash quickly resolved except for post-inflammatory hyperpigmentation. three weeks after resolution of primary lesions, she again complained of pruritic eruptions occurring on easily accessible areas and began bringing in medicine bottles of skin debris and scabs for examination. scrapings, koh preparation and skin biopsy examined microscopically showed no evidence of sarcoptic re-infestation. a diagnosis of delusional parasitosis was made. conclusions: the animal to human transmission of sarcoptic infection seen in this patient is rare and responded quickly to appropriate treatment. despite eradication of the infection, she developed delusional parasitosis, a rare psychiatric disorder characterized by fixed, false belief of an infestation by insects or other creatures. she displayed the classic matchbox sign in which samples of skin, scabs and other detritus are brought in for examination. she is currently receiving psychiatric care. background: thimerosal is a mercury derivative that has been used since the 1930s. it is a commonly used preservative in ophthalmic solutions, otic drops, and vaccines due to its bactericidal property. objective: to report the first case of a generalized reaction to thimerosal found in an influenza vaccine. methods: we present a patient who developed a generalized maculopapular eruption after receiving a thimerosal containing influenza vaccine. patch testing was performed to determine if there was an allergy to thimerosal. results: patch testing confirmed a type iv (t cell mediated) sensitivity to thimerosal, further supported by the prior history of a reaction to a thimerosal containing contact lens solution. the patient was asked to avoid thimerosalcontaining products, including vaccinations, unless the benefit clearly outweighed the potential risk of a reaction. conclusion: physicians need to be aware that thimerosal is found in many products including vaccinations. clinicians should also be aware that allergic reactions do occur with exposure to thimerosal even in vaccines. this is the first case report in the literature of a generalized reactions to thimerosal from an influenza vaccine angioedema is a rare condition that has been described in the literature and exists in both inherited and acquired forms. a defect of the innate immune system, more particularly the complement system, is the inciting culprit. the acquired form of c1 esterase inhibitor deficiency has been divided into two classes and is generally not commonly seen until after the forth decade of life. type 1 acquired angioedema has been described in association with lymphoproliferative disorders, while type 2 is related to excessive complement activation and consumption due to autoantibodies. our case is a 67-year old man referred from an outside physician for further management of idiopathic edema. he first experienced facial edema in april 2002 attributed to sweet myrrh root ingestion. subsequently, in november 2002 he developed diffuse swelling of his upper extremities and tongue without airway compromise. a minimal work-up at that time was inconclusive. he fortunately remained asymptomatic until january 2004 at which time he experienced two episodes of tongue swelling without etiology. he was seen in his local emergency department and treated with diphenhydramine, corticosteroids, and epinephrine on both occasions with gradual improvement of his symptoms. the patient was not on medications commonly associated with angioedema and did not report insect envenomation. subsequently, he was seen by his primary care physician who ordered a number of laboratory tests including a ch50, which was significantly suppressed. in light of this finding, he was referred to our clinic for further evaluation. at the time of presentation to our office, the patient was symptom free. his physical exam was within normal limits. further laboratory evaluation was essentially unremarkable with the exception of comple-ment studies, which are listed in the table. our case demonstrates an elderly patient with evidence of idiopathic swelling. we arrived at our diagnosis of acquired angioedema based on clinical presentation and confirmatory serum complement levels. a low ch50 at time of presentation allowed us to further delineate the etiology of complement deficiency. the fact that our patient did not present with swelling until after age 60 and the paucity of a family history of idiopathic angioedema makes the diagnosis of acquired angioedema probable. measure of serum c1q level confirmed the diagnosis. introduction: certain diseases, widely believed to be of allergic etiology, including atopic dermatitis and episodes of wheezing might be the result of interplay of genetic and environmental factors, at least partially. we describe a child with a rare chromosomal disorder presenting with typical features of atopic dermatitis and recurrent mild wheeze. materials and methods: a new born african-american male was noted to have unusual facial features immediately after birth. the baby was born naturally, without antenatal and neonatal problems. on physical examination, the infant had unusual facial characteristics with tight and taut facial skin, relatively diminished facial pad of fat, pointed chin, and markedly hypertonic extremities. additionally, cardiac exam revealed mumur consistent with uncomplicated asd. frequent reassessments of the infant in the outpatient clinic were done. the child had repeated bouts of wheezing attacks, and facial rashes compatible with the diagnosis of atopic dermatitis. the wheezes were treated in the clinic with nebulaized bronchodilators, and the atopic dermatitis responded reasonably to topical steroid applications and moisturizers as needed. chromosomal analysis of the peripheral blood of the chid confirmed the diagnosis of deletion of long arm (q) of chromosome 1 [46, xy, del (1)(q42q43)]. karyotypic analyses of the mother and father were normal. the child exhibited features of developmental delay, and seizure activities. anti-epileptic drugs (aed) were instituted. initial eeg was normal and the subsequent eeg showed findings of static encephalopathy. ct scan of the brain revealed absent corpus callosum. neurologic evaluations and physiotherapies were requested for improvement of fine motor skills. he did not seem to suffer from any serious infectious or immunodeficient illnesses. his cbc was normal. he continued to have fair gains in his weight, height, and head circumference. his atopic dermatitis and wheezing episodes remained under control. there were a few hospitalizations for break-through seizures and dehydration from gastroenteritis. a subsequent echocardiography confirmed closure of asd. con-clusion: a child with chromosomal anamoly, atopic dermatitis, and mild intermittent wheeze is reported. despite multiple congenital problems, the child continued to have a stable clinical course. etoposide is a chemotherapeutic agent used to treat many solid tumor malignancies. hypersensitivity reactions have been well described and there are a few reported deaths from anaphylaxis. some suggest that the hypersensitivity is an anaphylactoid type reaction as it may occur during the first dose. case reports of cutaneous complications include stevens-johnson syndrome, radiation recall and diffuse erythema. there are four cases in which diffuse erythematous papules developed after etoposide therapy. all rashes spontaneously resolved within three weeks and biopsies demonstrated keratinocytes in a starburst pattern. we report the first case of an immediate etoposide induced skin reaction that evolved into long lasting hyperpigmented plaques. pretreatement was able to prevent this immediate and late reaction on subsequent exposure to etoposide. a 27 year old female with ovarian cancer was treated with bleomycin, etoposide and vinblastine. three hours after initiation of the third dose of etoposide, patient developed pruritic, erythematous macules on her chest, abdomen, face and extremities. the infusion was stopped and decadron administered. over forty-eight hours, the pruritic macules became hyperpigmented plaques on her chest, abdomen, extremities and face. pruritus resolved after a slow taper of prednisone. the darken plaques were treated with multiple topical preparations but persisted for about three months. biopsies demonstrated superficial perivascular infiltration of lymphocytes and a few eosinophils suggestive of a drug eruption. etoposide was considered essential for this patient so allergy was consulted. the literature supports cautious readministration of etoposide with pretreatment and slower infusion rate to prevent immediate hypersensitivity. we could not guarantee prevention of the late hyperpigmented reaction. the patient was pretreated with prednisone and cetirizine. etoposide was administered in an icu with a slower rate of infusion. etoposide was tolerated without immediate pruritus or erythematous reaction. the patient did not develop the delayed darkened plaques. cautious administration of etoposide after premedication and a slow rate of infusion prevented both the immediate and late reaction previously experienced by this patient. eosinophils normally comprise 1-3% of peripheral white blood lymphocytes. peripheral blood eosinophilia is defined as an absolute eosinophil count >500 cells/mm3 and is most often caused by atopy, helminth infections, or collagen vascular diseases. less common causes include adrenal insufficiency and neoplastic processes. eosinophilia can be characterized as mild (<1500 cells/mm3), moderate (1500-5000 cells/mm3) or severe (> 5000 cells/mm3). although severe eosinophilia has been reported in association with adult hiv infection, studies of hiv-infected children have not shown peripheral blood eosinophilia to be a feature of pediatric hiv infection. we report an unusual case of severe peripheral blood eosinophilia in an adolescent male who was subsequently found to be hiv-infected. a previously healthy 15-year-old male presented with 2 week history of fever, diarrhea, cough, vomiting, abdominal pain, anorexia and a 5lb weight loss over the previous 2 months. the patient had recently emigrated from guyana and denied sexual activity, intravenous drug abuse, or other hiv risk factors. there was no known maternal hiv infection. repeated stool specimens were negative for ova and parasites. serologies for e. histolytica, t. canis, and s. stercoralis were negative. an abdominal ultrasound, chest x-ray, serum electrolytes, and liver function tests were all within normal limits. total white blood cell was 16, 500 with 59% eosinophils (absolute eosinophil count of 9, 735 cells/mm3). elisa and western blot were positive for human immunodeficiency virus (hiv-1). cd4+ tlymphocyte count was 1277 cells/mm3 with an hiv-1 rna level of 11, 000 copies/ml. the patients symptoms resolved over the next 10 days without treatment. he was started on combination antiretroviral therapy (zidovudine, lamivudine, abacavir, and efavirenz). absolute eosinophil count continues to slowly decrease with the last count of 3, 605 cells/mm3 three months following the introduction of antiretroviral therapy. severe peripheral blood eosinophilia may be a presenting feature of hiv infection in adolescents. hiv testing should be considered in cases where more common causes of eosinophilia such as atopy and parasitic infections are excluded. triad asthma is well described in adults but not in the pediatric literature. this case highlights the successful treatment of a severe pediatric asthmatic with nasal polyps and aspirin sensitivity. a 13 year-old female presented with severe persistent asthma, eib, allergic rhinitis, chronic sinusitis, nasal polyps, and a history of pneumonia. her asthma symptoms were minimal from age 2 until age 9. she then started flovent and required increasing amounts of inhaled and oral steroids. she required 6 courses of oral steroids per year by age 13. in addition, she had snoring, fatigue, chronic nasal congestion, rhinorhea, and sneezing despite treatment withallegra and rhinocort. she had received immunotherapy from age 8 to 10 with no clinical improvement. since age 8 she had recurrent sinusitis and had required 2 polypectomies. she noted aspirin caused nasal stuffiness and mild wheezing and therefore avoided it. on exam she had allergic shiners, dennie lines, boggy turbinates, nasal polyps, and diffuse wheezing. on evaluation she had severe airway obstruction. her fev1 increased from 36% to 51% with albuterol. her chest ct had a mosaic pattern due to severe air trapping, and her no level was 177. a sleep study showed severe hypopneas, and she had pan-sinusitis on ct. she had multiple positive spts, an eosinophil count of 1100 and an ige of 700. she had a normal sweat chloride and ph probe.the differential diagnosis included churg-strauss, abpa, cystic fibrosis, bronchiolitis obliterans, and triad asthma. after a thorough evaluation, she was diagnosed with triad asthma. with 6 weeks of treatment with oral prednisone, her fev1 improved from 36% to 83%. her inhaled controller therapy was increased and she was placed on xolair. she had a polypectomy and then underwent aspirin desensitization. one year after starting treatment her fev1 remains 83%. she requires albuterol 1x/month and has not required prednisone. her eib, allergic rhinitis and congestion are markedly improved. she has had no further episodes of sinusitis, and a repeat sleep study was normal. in conclusion, we treated a severe pediatric asthmatic with nasal polyps and aspirin sensitivity. this is not frequently reported in the pediatric population, and raises questions about the incidence, optimal long term treatment of triad asthma, and the differences from the adult onset of this disease. a.a. white * , r.a. simon, la jolla, ca. background: human disease from mold has traditionally been isolated to infection, allergic disease, or hypersensitivity pneumonitis. specific diseases or pathologic findings other than those listed above have not been well described. we report a case of acute eosinophilic pneumonia related to mold exposure. case presentation: a 50 year old woman developed dry cough and shortness of breath after stachybotrys mold was discovered in her home. a chest radiograph showed bilateral upper lobe infiltrates which worsened two weeks later. treatment with macrolide and flouroquinolone antibiotics was ineffective. a white blood cell count was 12, 000/cumm with 35% eosinophils. an erythrocyte sedimentation rate was 99 mm/hr. aspergillus ige was negative. treatment with prednisone led to complete resolution of the chest radiographic abnormalities and improvement in pulmonary function testing. this patient was given a diagnosis of acute eosinophilic pneumonia with mold as a likely causal factor. discussion: there is dispute amongst health care professionals regarding the significance of environmental mold contamination in the etiology of human disease. this case represents well characterized disease occurring in the setting of mold exposure. while a causal relationship cannot be established, the temporal relationship of mold contamination, symptom onset, and disease progression is compelling. interestingly, a recent report of mold contamination leading to nonspecific interstitial pneumonia/fibrosis has been described. we have observed a patient in our clinic with similar pathology on biopsy and temporal relationship to mold exposure. to our knowledge, acute eosinophilic pneumonia has not been reported in conjunction with mold exposure. conclusion: this case highlights a new condition in which mold may have a causal role. the mechanism is unknown, but likely is through a non-ige mediated immunologic pathway. public awareness of mold as a health concern is increasing. perhaps similar cases will emerge as a history of mold exposure is offered by patients at the time they are evaluated for lung disease. a stronger correlation may then emerge. scuba diving is a commonly practiced activity which normally carries only minimal risks. any severe allergic reaction such as anaphylaxis, however, occurring during this activity could be a particularly dangerous not only because the swimmer is submerged but also because of the lack of proximity to medical care. we report a case of anaphylaxis occurring during scuba diving resulting from an unsuspected hypersensitivity to a latex component of the scuba diving suit. a 21 yr-old white male developed a severe generalized urticarial rash with angioedema of his lips and eyelids, and difficulty breathing within 5 minutes of his applying the suit and entering the water. after being rescued, he was transported to a nearby emergency department where he received epinephrine, antihistamines, corticosteroids and iv fluids with gradual improvement over a 4 hour period. the patient denied being stung by a marine aquatic organism and there was no prior history of allergy or medications usage prior to his reaction. since subsequent investigation revealed that the scuba diving suit contained latex (brazilian rubber), a specific ige rast was found to be strongly positive (class v) to latex. therefore, the patient was advised to use a suit made of neoprene synthetic rubber (polychloroprene) which is a nonlatex containing product. this case report illustrates the importance of a diligent search for hidden sources of latex products which can produce life-threatening allergic reactions in sensitized patients. there has been considerable debate concerning the safety of immunizing egg-allergic children with the combined mmr vaccine. this concern derives from the possibility of an anaphylactic reaction since the mmr vaccine is prepared from attenuated viruses grown on chick embryo fibroblast cell cultures. we have previously reported the safe administration of the mmr vaccine to 6 severe egg-allergic children without development of an adverse reaction (nsouli, tm, et al. ann allergy asthma immunol. 2004; 90:163) , a finding consistent with the current report of the committee on infectious diseases, american academy of pediatrics, 2003. the present case report describes an anaphylactic reaction in an egg allergic 4 yr-old-white male consisting of generalized urticaria, angioedema, wheezing immediately following the administration of a second mmr vaccine. the history of hives following ingestion of eggs was confirmed by positive specific ige rast testing. the patient's anaphylactic reaction necessitated emergency treatment including epinephrine, diphenhydramine, and corticosteroids in addition to iv fluids. although the administration of mmr vaccine in egg-allergic children is not considered as an absolute contraindication, the present case report suggests that caution should be observed when administering the mmr vaccine in such patients and that careful medical observation be included together with the ready availability of emergency medical equipment. p. buddiga * , r. turbin, a. baisre, l. bielory, newark, nj. introduction: sarcoidosis is a chronic granulomatous disease of unknown etiology that may have a multi-organ system manifestation and is characterized by the histopathological evidence of nonnecrotizing granulomas. case report: a 53 year old african american woman with a 10 year history of type ii diabetes mellitus, hypertension and sinusitis recalcitrant to multiple courses of antibiotics over 3 months was admitted to the hospital with complaints of right eye proptosis, diplopia, headache and right facial numbness.her exam was consistent with an ipsilateral mild optic neuropathy, complete sixth (vi)nerve palsy, trigeminal, ophthalmic and maxillary division numbness.ct and mri of the face, orbit and brain revealed an extensive process infiltrating ethmoid, maxillary and frontal sinuses;orbits and deep facial structures.chest ct revealed bilateral interstitial nodules and symmetric hilar adenopathy. differential diagnoses included sarcoidosis, lymphoma/tumor, wegener's granulomatosis or fungal infection.labs-purified protein derivative test=negative(neg), antineutrophil cytoplasmic autoantibodies=neg. angiotensin converting enzyme=83[8-52 u/l], fungal & anaerobic and acidfast bacilli culture of sinus secretions=neg.ethmoid, adenoid and maxillary sinus biopsies=multiple nonnecrotizing granulomas. on the basis of compatible clinical and radiographic findings, histopathological evidence and exclusion of other diseases with similar findings, the diagnosis of sarcoidosis was established. she was started on parenteral methylprednisolone and subsequently tapered to oral prednisone after 5 days.concomitantly she was started on methotrexate as a steroid sparing agent.most recent chest x-ray after 4 months of treatment reveals near complete resolution of the hilar lymphadenopathy.at 5 months her optic neuropathy and facial dysesthesia had resolved, and she was left with mild persistent vi nerve dysfunction. conclusion:sarcoidosis that manifests itself as sinusitis is an uncommon presentation and the mechanism involved is thought to be a consequence of the destruction of cilia and mucus producing glands by the granulomatous process.review of the literature indicates that this is the eighth case reported and illustrates that a high index of suspicion of other etiologies must be maintained when there is a refractory response to multiple courses of antibiotics in the treatment of sinusitis. j.b. hein * , p. patel, l. bielory, newark, nj. introduction: cid may result in opportunistic infections such as cryptococcal meningitis. we present an unusual case of cryptococcal meningitis in an hiv-negative patient with severe cd4 lymphocytopenia. case: a 40 yearold male with a three year history of sarcoidosis presented with acute onset of right-sided body numbness. the patient had been on prednisone 40 mg/day for 5 months prior to presentation. ct and mri scanning showed no vascular defects, meningeal enhancement, hydrocephalus or mass lesions. gallium scanning revealed normal uptake in the liver and spleen, mild uptake in the lungs and nasopharyngeal region, and asymmetric uptake in bilateral kidneys. the patient's initial wbc was 8, 000 cells/mm 3 , composed of 6970 neutrophils/mm 3 , 50 eosinophils/mm 3 , 50 basophils/mm 3 , 530 lymphocytes/mm 3 , and 400 basophils/mm 3 . repeat studies revealed the total lymphocyte count decreased at 240 cells/mm 3 with cd19 (pan b) cells decreased at 29 cells/mm 3 and cd3 (pan t) cells decreased at 189 cells/mm 3 . cd4 count was 144 cells/mm 3 and cd8 count 50 cells/mm 3 with a cd4/cd8 ratio of 2.9. igg levels were normal at 716 mg/dl. elisa was negative for hiv, and hiv-1 rna by pcr was not detected. serum ace level was 19 u/l and csf ace level was 23 u/l. csf obtained by lumbar puncture stained positive with india ink and culture revealed cryptococcus neoformans. the patient responded to amphotericin b lipid complex 5 mg/kg/day, and his neurological status eventually returned to baseline. conclusions: the severe lymphopenia in this patient caused predisposition to infection with cryptococcus neoformans. the etiology of the profound lymphopenia likely was multifactorial, including mild sarcoidosis activity (lung uptake on gallium scan) and chronic corticosteroid therapy. however, once cryptococcus became entrenched in the patient's csf, the infection itself likely depressed peripheral lymphocyte numbers even further. this case demonstrates the importance of exploring a broad differential diagnosis when faced with lymphocytopenia. background: isosulfan blue 1% is a common dye used in sentinel lymph node dissection. the usage of the procedure and dye has increased in numbers, and although rare, several cases of anaphylactic reaction have been reported. objective: we are reporting a patient who had an anaphylactic reaction to isosulfan blue while undergoing breast cancer excision with sentinel lymph node biopsy. methods: the patient is a 44 year-old woman with breast cancer. she underwent breast mass excision with sentinel lymph node biopsy using the lymphazurin 1% blue dye (isosulfan blue). she has a history of penicillin induced hives but has no other drug allergy. as soon as the dye was injected, she became flushed, hypotensive, and tachycardic. hypotension was refractive to fluid challenge. she was then treated with epinephrine as well as intravenous steroids and cimetidine with relief of symptoms. subsequently, she was evaluated in allergy and skin tests with isosulfan blue 1% at 1:100, 1:10 dilution, and undiluted were performed using histamine as positive control and saline as negative control. this procedure was also performed on two control subjects. results: the patient had a positive skin test (wheal and flare) to isosulfan blue 1% (undiluted) with the control being appropriately positive for histamine and negative for saline. control subjects had negative response to dye and saline and positive response to histamine. conclusions: isosulfan blue 1-% dye may cause anaphylactic reactions in patients undergoing sentinel lymph node dissection. . the positive skin test result to the dye plus the negative skin test responses in the controls suggests that the reaction may be immunoglobulin e (ig e) mediated. eosinophilic duodenitis (ed) and gluten-sensitive enteropathy (gse) or celiac disease (cd) are distinct clinical entities with well-defined clinical and laboratory parameters. ed is a rare condition of unknown etiology, which is manifest by eosinophilic infiltration of the gastrointestinal tract and peripheral eosinophilia. gse or cd is a form of non-ige food allergy caused by immune hypersensitivity to ingested gluten. the simultaneous occurrence of the two entities, however, is a rare event. the following presentation is a case report in which both entities were found in the same patient. an 11 y/o white hispanic male presented with severe, chronic, colicky abdominal pain and headache of 5 months duration. hematologic and immunologic studies were within normal limits. serum ige levels were 356 iu/ml (n= < 200 iu/ml), anti-endosomial ab (+), igg antigliadin: 16 u/ml (n=: 0-12 u/ml), skin tests for food and inhalant allergens were weakly positive (1+). biopsy of the inferior third of esophagus revealed chronic moderate esophagitis; biopsy of gastric antrum revealed lymphatic hyperplasia; duodenal biopsy showed shortening of the villi with the presence of eosinophils. following treatment with esomeprazole 40 mg bid, famotidine 20 mg qd, montelukast 20 md qd, lactobacillus, a diet free of gluten, rofecoxib 25 mg qd and betamethasone for 15 days, betamethasone the patient improved with partial resolution of symptoms. the presence of duodenal eosinophils persisted despite a gluten free diet, and continued to require repeated bursts of prednisone. since to our knowledge this is rare finding in which ed occurred in association with gse, a high index of suspicion for simultaneous occurrence of ed should be raised in any case of gse that fails to respond to a conventional gluten free regimen. we report the evaluation of a 6 month old male presenting with a pustular rash and choking episodes from birth. by age 6 weeks, he had experienced two pneumonias, one with fleeting infiltrates requiring intubation. persistent eosinophilia (5000-9000/ml) and eosinophils on pustule biopsy were noted. persistence of these and subsequent rsv pneumonia and recurrent draining otitis media led to referral. physical examination showed a thriving male infant with multiple erythematous papules and pustules along the scalp, face, axilla, and trunk. eosinophilia was confirmed. quantitative immunoglobulins were abnormal for igg 179 and ige 255 iu/ml. peanut cap rast was 23 kua/l. hib post-vaccination titer, t cell enumeration/stimulation, and nbt were normal. hiv testing, stool eosinophils, o&p, and hemoccult were negative. cxr, hrct, ekg, and echocardiogram were normal. pustule cultures for bacteria, virus, and fungus were negative. skin biopsy revealed numerous eosinophils in the pustule, dermis, epidermis, and perifollicular region. cd1a+ and s-100 staining were negative for histiocytic infiltration. nemo defect/incontinentia pigmenti were considered but testing was negative and karyotype 46, xy. bone marrow biopsy revealed numerous eosinophils at different stages of maturation, and no myeloproliferative or neoplastic changes. bronchoscopy was remarkable for 15% eosinophils on balf. 24-hour ph probe and impedence evaluation was negative. egd with biopsy was normal except for mild eosinophilic infiltration of the proximal and distal esophagus. with the clinical picture of recurrent pruritic crops of sterile pustules and characteristic skin biopsy demonstrating eosinophil infiltration, the diagnosis of eosinophilic pustulosis (ep) of childhood was made. despite no longterm sequelae or other end-organ involvement in ep, the degree and duration of eosinophilia, and presence of eosinophils in the airways and esophagus, raises the concern for other eosinophilic syndromes. following cardiac, cns, pulmonary, and gi systems is warranted. the infants pneumonias were felt to be due to aspiration, and ear infections the result of humoral immunity nadir or draining pustules in the external auditory canals. immunoglobulin levels will be monitored. this case illustrates the heterogeneity of eosinophilic diseases and raises the question of what drives the mechanisms behind malignant and benign disease. introduction: behcet's disease is a chronic, relapsing vasculitic disease characterized by recurrent oral, genital, and gastrointestinal ulcerations, a wide variety of skin lesions, uveitis, and arthritis. pediatric cases of behcet's disease are uncommon with an estimated prevalence of 1 in 600, 000 children under the age of 15. although the disease in children shows similar characteristics as adults, the diagnosis of behcet's disease in the pediatric population remains a challenge. this report describes an 18 year old girl referred to our pediatric immunology clinic for evaluation of recurrent painful oral ulcerations since the age of 7 with no genital ulcerations. the oral ulcers would last for two weeks and heal spontaneously but would reappear 2 to 3 weeks later. the patient had a history of raynaud's phenomenon for the last 6 to 7 years and the recent onset of joint pain. physical examination revealed 4-5 white elliptoid lesions 5-7 mm in diameter on an erythematous base on the tongue and soft palate. skin examination was significant for hyperpigmented patches over the neck, abdomen, and back. there were erythematous, serpigenous lesions on the palms and punctuated necrotic lesions on the finger-tips. pathergy test was positive. laboratory investigation was unremarkable and cultures from the oral ulcers remained negative. the patient was diagnosed with juvenile behcet's disease. she was started on immunosuppressive therapy with low dose prednisone and responded. conclusion: behcet's disease is a difficult diagnosis to make in the pediatric population. this case demonstrates that in children, recurrent oral ulcerations may be the only initial manifestation of behcet, and an important clinical marker for the disease. rationale: relapsing polychondritis is an uncommon severe inflammatory condition with unknown etiology that can present in a variety of manners. we report a 14 year old patient who initially presented with signs and symptoms of anaphylaxis to shellfish and was later diagnosed with relapsing polychondritis. case report: a 14 year old african-american boy with a 6 year history of asthma and fish and shrimp allergy presented to the pediatric intensive care unit on 4/23/03 after an episode of severe shortness of breath. his symptoms started shortly after accidental exposure to shrimp. in the intensive care unit, he was noted to be wheezing and stridorous. an initial chest xray as well as subsequent fiber optic laryngoscopy showed sub-glottic stenosis. he was intubated and received mechanical ventilation for greater than a week. after extubation, he stated that he felt fine, but continued to demonstrate audible stridor. pulmonary function tests demonstrated extra-thoracic obstruction, laryngoscopy continued to show sub-glottic stenosis, and esophagogastroduodenoscopy showed erosive esophagitis consistent with reflux. initial lab tests demonstrated a normal eosinophil count, elevated ige (969 iu/ml), and positive immunocaps testing to multiple foods. the patient was discharged home with minimal stridor and no wheezing. over the next nine months, the severity of his stridor waxed and waned, but in general it worsened in spite of a strict elimination diet and anti-reflux therapy. on 9/25/03 he received an emergency tracheostomy. on 1/26/04, the patient was again admitted to the hospital. at this time he complained of bilateral knee pain as well as significant weight loss. physical exam demonstrated arthritis with effusions of both knees as well as unilateral auricular chondritis and bilateral episcleritis. antibodies against type ii collagen were positive (42.1 eu/ml). the diagnosis of relapsing polychondritis was suggested. conclusion: relapsing polychondritis is a rare inflammatory disorder of the cartilage and connective tissue with an unknown etiology. the wide array of presenting complaints as well as the relapsing nature of this disorder often causes significant delay in diagnosis and treatment. in our case there was a period of nine months between the initial presentation and the time when the diagnosis of polychondritis could be made. d.k. geller * , m. ballow, buffalo, ny. introduction: an 11 yo male previously diagnosed with pandas presented to our immunology clinic for ivig. the patient was healthy until age 8 when he developed facial motor tics associated with group a beta hemolytic strep pharyngitis. he was treated with antibiotics and the tics resolved completely. he has had multiple recurrences associated with strep and other viral infections. the tics improve or resolve completely when he is infection free. he has no history of vocal tics, attention deficit/hyperactivity disorder, or obsessional thinking or compulsive behaviors. laboratory: multiple throat cultures positive for group a beta hemolytic strep, positive antistreptolysin and antideoxyribonuclease b titers. discussion: pandas identifies a subgroup of children with an obsessive compulsive disorder and/or tic disorder whose symptoms seem to be triggered by streptococcal infections. the proposed pathophysiology is an immune-mediated mechanism in which antistreptococcal antibodies, antistreptolysin and antideoxyribonuclease b, cross react with the basal ganglia of genetically susceptible hosts. a few studies have looked at immunomodulatory therapies including ivig for neuropsychiatric symptoms including tic disorders secondary to post-streptococcal autoimmunity. we plan a trial of ivig for this patient given the recurrent nature of his tics and the relation to streptococcal and other viral infections. introduction: the chronic granulomatous disease (cgd) is an inheritable disorder of phagocyte cell respiratory burst that result in life-threatening infections. the pulmonary infection is the primary cause of death in greater that 50% of the cases and the role of surgery in management of this entity remains undefined. case report: a lobectomy was performed in a 2 year-old male who presented a persistent opacity of the medial right lobe with fever, cough, malaise an rapid onset of respiratory failure; skin abcesses were concomitant. his familiar history was unremarkable. a chest tube was placed in a first instance in order to drain empyema, but no progress was observed. a ct scan showed necrotizing pneumonia of the right lung, mediastinal and retroperitoneal limphadenopathy and bronchopleural fistula. a second surgical intervention was undertaken to correct the fistula and pleural debridement was done. at this time, serratia marcenses was isolated from blood culture and lung; a primary immunodefiency was suspected. our evaluation showed nbt reduction on 0% and dyhidrorhodamine assay (dhra) didn't demonstrate and effective oxidative burst. the subsecuent management was based on specific antibiotic to serratia and prophylaxis with tmp-smz and itraconazole. transfer factor to improve the ifn levels was initiated. the patient's mother showed on dhra two granulocyte populations, with and without oxidative burst. a x linked cgd was consistent. medical progress was observed in the next days. conclusion: despite the poor utility showed by the surgical procedures in the complicated pneumonia in cgd patients in many series, attending to the unusual nature of the pulmonary infections and the high mortality and morbidity associated with thoracic surgery; the management of this case showed that an aggressive approach in the diagnosis combined with some procedures used in immunocompetent patients pneumonia may improve the clinical condition in cgd patients. background: takayasu arteritis is a large vessel vasculitis primarily affecting the aorta and its branches. it is most prevalent in women in the second and third decades of life. initial symptoms are systemic and often self-limited, but the disease may progress undetected over years until signs of vascular insufficiency develop. arteritis is commonly seen in the aortic arch and its branches, although the disease is a panarteritis and vascular compromise can occur in many organs. pathologically, involved vessels show intimal proliferation and fibrosis, and scarring of the elastic lamina. case report: we report the case of a 31 year old southeast asian woman who presented with diplopia, ptosis, dizziness, and progressive dyspnea. past medical history was significant for rheumatic fever complicated by aortic valve insufficiency and hypertension. physical findings on admission included hypertension, asymmetric upper extremity blood pressure (left arm 95/50mm hg, right arm 200/50 mm annals of allergy, asthma & immunology hg), wide pulse pressure and a harsh diastolic murmur. admission labs showed anemia (hemoglobin: 11g/dl, hematocrit 33%) and an elevated erythrocyte sedimentation rate of 31mm/hr. transesophageal echocardiography revealed severely dilated aortic root aneurysm with secondary severe chronic aortic valve insufficiency and calcified aortic leaflets not typical of rheumatic heart disease. resection of the aortic valve and aneurysm was performed. pathologic examination of the aortic aneurysm revealed intimal and adventitial fibrosis with focal chronic inflammation and partial loss and fibrosis of media, findings compatible with healed takayasu arteritis. the patient was started on prednisone 100mg by mouth daily for takayasu arteritis and discharged in stable condition. discussion: this is a case of takayasu arteritis masquerading as rheumatic heart disease. the episode of rheumatic fever was likely the initial presentation of takayasu arteritis, as the systemic symptoms of these diseases can overlap. takayasu arteritis is an insidious disease that often leads to a delayed diagnosis with considerable morbidity for the patient. since takayasu arteritis may go undiagnosed until ischemic symptoms develop, physicians should be alert to the possibility of this disease in young women to avert end organ damage and achieve early remission with drug therapy. we present a fifteen year old boy with disseminated papillomatosis and idiopathic cd4+ lymphocytopenia (icl). the warts have been present since he was one year old and have progressively worsened. there are more than fifty warts on each of his hands. his legs have multiple warts and about ten warts recently appeared on his lips and around his mouth. his past medical history is significant for no hospitalizations and no blood transfusions. he has never had problems with other infections, and he had a benign course of chicken pox that spontaneously resolved. he has been on cimetidine for six months without improvement. several topical therapies, including imiquimod, have failed. he has a total lymphocyte count of 1484/ul (normal range, 1288-4512/ul) and a cd4+ count of 301/ul (normal range, 361-1715/ul). the cd8+, natural killer cells, and cd19+ cells were normal. his hiv test is negative. the serum immunoglobulins were normal as well. lymphoctye proliferation studies reveal an absent response to antigens (candida and tetanus) with a normal response to mitogens. further studies showed no other cause of cd4+ lymphocytopenia. this case shows that the diagnosis of idiopathic cd4+ lymphocytopenia should be considered in any patient with widespread viral infection whose hiv test is negative. appropriate evaluation of the cd4+ count should be pursued. j. ko * , a. nowak-wegrzyn, new york, ny. introduction: approximately 40% of children with moderate to severe atopic dermatitis (ad) have food allergies. complementary and alternative medicine (cam) is utilized by an estimated 16-27% of patients with allergies. we present a case of a 4 year-old boy with ad referred after receiving a diagnosis of food allergy by a cam practitioner. methods/case: a 4 year-old boy was referred for evaluation of mild ad and possible food hypersensitivity. his ad started at 1 year of age and was limited to the face. he was initially breast-fed and was switched to milk-based formula at 4 months of age. solids were introduced at 5 months of age without difficulty. he had no immediate reactions or noted worsening of ad due to foods. the patient presented to a naturopath for allergy evaluation. based on electrodermal skin testing results, he was reported to be allergic to milk, egg, peanut, rice, and beef (all of which he had tolerated previously). he was restricted from milk and egg for 4 weeks without noticeable change in his ad, which was limited to the cheeks and controlled on topical pimecrolimus. results: his weight and height were both 75th percentile. physical exam was normal except for 2-3 cm dry, erythematous patches on both cheeks and mildly dry skin on flexor/extensor surfaces of both arms. skin prick testing revealed negative tests to milk, egg, peanut, and dust mite. serum ige testing was also negative. since he had no evidence of ige sensitization to commonly allergenic foods, he was recommended to continue an unrestricted diet and discontinue use of a "sippy" cup. his ad subsequently improved on a skin care regimen of postbathing moisturization and topical pimecrolimus prn. asthma and allergies are the #2 reason for cam use in the us. electrodermal skin testing is a method utilized by naturopathic doctors to detect allergen "sensitivity". however, in two double-blinded trials examining atopic and non-atopic patients, electrodermal testing did not correlate with skin prick testing results, regardless of inter-operator variability. conclusions: allergy patients are seeking cam treatment, and it is important for physicians to be aware of the safety and efficacy of alternative medical practices. the use of electrodermal skin testing has not been proven to detect allergen sensitivity and use of this modality should be discouraged. e.e. mcgintee * , k.e. sullivan, philadelphia, pa. introduction: hematopoietic stem-cell transplantation causes profound tcell immunodeficiency due to the rigorous conditioning involved. following transplant, the t-cell compartment is initially reconstituted through expansion of mature donor-derived t-cells. however, thymopoiesis is necessary to generate naive t-cells with a diverse repertoire. factors affecting thymic function impact the ability of the body to reconstitute the immune system. we report a case of severe t-cell immunodeficiency after two stem-cell transplants for neuroblastoma in a patient with a history of thymic hemorrhage secondary to mediastinal surgery. case: a 5-year-old female with a history of high-risk neuroblastoma was referred for immunologic evaluation due to a significant infectious history since completing her neuroblastoma therapy. she initially presented at 21 months of age with a right atrial mass that was presumed to be an atrial myxoma. she underwent mediastinal surgery to resect the tumor, which was found to be neuroblastoma extending from her right adrenal gland along the vena cava. a ct scan following her surgery revealed an area of hemorrhage in her right thymus. she subsequently received high-dose chemotherapy and two autologous stem-cell transplants. her infection history was significant for multiple episodes of upper respiratory illnesses, sinusitis, and otitis media requiring myringotomy tube placement on two occasions. immunologic evaluation revealed a dramatically low absolute lymphocyte count with deficits primarily in the t-cell compartment, and reduced proliferation in response to mitogens. immunoglobulin levels were normal; she formed protective titers to diphtheria and tetanus but not to any of 14 pneumococcal serotypes. conclusion: stem-cell transplantation often results in severe t-cell immunodeficiency. the thymus plays an important role in reconstituting the t-cell compartment with naive t-cells generated from hematopoietic stem-cells, which restores tcr diversity. as this case illustrates, damage to the thymus can significantly impair the ability to reconstitute the t-cell compartment. a.k. knight * , c. cunningham-rundles, new york, ny. rationale: this is the first case report of oxcarbazepine induced immunoglobulin deficiency. methods: a 49 y/o white female was referred for further investigation of low serum immunoglobulin found as part of an evaluation for chronic bacterial vaginitis. she was taking oxcarbazepine for chronic pain. this was discontinued and immunoglobulin levels and b cell numbers were tested at intervals. specific antibody responses to pneumococcal vaccine were tested. results: at presentation, on oxcarbazepine, immunoglobulin levels were low [igg 576, iga <11, and igm <4 mg/dl] and she had a b cell deficiency [1%, 18 b cells (normal 5-15%, 75-375 cells/cu mm)]. antibody response one month after pneumococcal vaccine was poor (protective antibody to 2/12 serotypes). (7%)] with protective antibody responses to 5/12 pneumococcal serotypes. she continued to have iga deficiency. conclusions: the patient's initial evaluation suggested the diagnosis of common variable immune deficiency with hypogammaglobulinemia and specific antibody deficiency. however, these defects reversed when oxcarbazepine was discontinued. it is unclear if persistent iga deficiency was a pre-existing condition, possibly predisposing her to this adverse reaction to oxacarbazepine, or induced by the oxacarbazepine. immunoglobulin deficiency is a known, though rare, reaction to carbazepine, the parent drug of oxacarbazepine; this adverse reaction can occur with its derivative oxcarbazepine as well. secondary hypogammaglobulinemia should be considered before diagnosing primary immunodefiencies such as cvid and committing the patients to lifelong immunoglobulin therapy. introduction: interferon and glatiramer acetate (copaxone) are indicated for the treatment of relapsing-remitting multiple sclerosis (ms). anaphylactic reaction has been reported as a rare complication of interferon and copaxone use. we report a case of interferon -1a (rebif) and copaxone hypersensitivity associated with positive skin tests and desensitization to interferon -1b (betaseron). case report: the patient is a 28 year-old woman with asthma and allergic rhinitis who was diagnosed with ms in oct '04 after an uncomplicated pregnancy and was subsequently placed on rebif. one month after starting therapy, patient developed wheezing and generalized urticaria after receiving a dose of rebif. the symptoms recurred the next day. her neurologist stopped the rebif and started her on copaxone. two months later she developed episodes of generalized urticaria. the patient was then evaluated in our center and underwent skin testing with interferon -1a intramuscular (avonex), interferon -1a subcutaneous (rebif), betaseron, and copaxone. she had a positive skin prick reaction to copaxone and positive intradermal skin tests to avonex (1:10 strength), rebif (1:1), and betaseron (1:1) after 24 hrs. the intradermal reactions to all 3 interferon formulations continued to progress upto 48 hrs. her neurologist felt that she would benefit from betaseron therapy. the positive intradermal skin test to betaseron was sufficient to warrant desensitization to prevent immediate hypersensitivity reactions. she underwent subcutaneous desensitization as shown in the table. the desensitization was halted at 240 minutes after the patient developed itching of the hands.* she returned in 24 hrs and we restarted by administering 0.1 ml of 1:1 strength betaseron. increasing doses were given until the patient received a full therapeutic dose (0.3 mg in 1 ml). she has since done well with daily doses of betaseron (4.5 mil units). conclusion: we report a ms patient who developed urticaria and asthma exacerbation in response to rebif and urticaria to copaxone. skin tests confirmed an ige-mediated allergic reaction. she underwent successful desensitization to betaseron. to our knowledge, this is the 1st report of desensitization to betaseron as well as extension to previous reported cases of systemic reaction to interferon and copaxone. pentoxifylline (ptx) is a phosphodiesterase inhibitor that has been used for ischemic vascular disease because of its effects on red blood cells and platelets. it has been used for systemic inflammatory diseases such as sarcoidosis because of its ability to inhibit production of cytokines such as tumor necrosis factor (tnf-a). we decided to offer a trial to a patient with chronic urticaria since agents that increase intracellular camp have been shown to inhibit mast cell degranulation. we report a case of a 50 year-old female with a history of allergic rhinitis, hypothyroidism, and diabetes mellitus with recurrent episodes of chronic urticaria since adolescence. she had known allergies to various antibiotics but otherwise had an unremarkable history, family history and social history. she was being treated with fexofenadine 180mg qd, azelastine nasal spray, zafirlukast 20mg bid, hydroxychloroquine 200mg bid, levothyroxine 25mcg qd, doxepin 25mg qhs, metformin 500mg bid, and spironolactone 25mg qd at the time of her initial evaluation. her physical exam was notable only for scattered 2-3 cm urticarial skin lesions as well as areas of hypo and hyperpigmentation from previous excoriations. her nasal turbinates were mildly congested with dull membranes. a previous skin biopsy showed only urticaria with increased numbers of mast cells. her workup included normal cbc, urinalysis, spep and complete chemistry profile, as well as negative ana and anti-thyroid antibodies. pentoxifylline 400mg tid was added to the patient`s regimen. her urticaria resolved within 2-3 weeks and was no longer visible at 2 months follow-up. pentoxifylline is a safe medication with few side effects that may benefit patients with chronic urticaria via several mechanisms, including the inhibition of mast cell degranulation and secretion of various cytokines and chemokines. the beneficial effects of pentoxifylline in the treatment of this patient`s urticaria have been seen in other patients. further study is indicated to determine which patients with urticaria are most likely to benefit from pentoxifylline, as well as elucidate the mechanisms of action by which pentoxifylline ameliorates symptoms of chronic urticaria. c.m. mjaanes * , m. boguniewicz, denver, co. we report the case of an 11-year-old male who since the age of 6 years has suffered from recurrent episodes of left tongue swelling. onset of the swelling is usually abrupt, and generally occurs during the night, awakening him from sleep. he describes a sensation of pain in his tongue but denies any pruritus, throat, lip or eyelid swelling. he has no cough, dyspnea, wheezing, rash, or itchy/watery eyes. the episodes occur infrequently, approximately once every spring . they tend to resolve spontaneously within 4 to 6 hours, however, on 2 occasions, the swelling has lasted for 7-21 days. the patient's current episode has been present for 3 weeks and is progressing. today he reports more pain and increased swelling. the child reports no benefit from oral antihistamines. he was treated with dexamethasone during his initial episode and experienced gradual resolution of the swelling. he has never been treated with topical or systemic epinephrine, or additional courses of systemic steroids. there is no family history of angioedema. the child was referred for evaluation of an immunologic/allergic etiology of his unilateral tongue swelling. laboratory studies obtained early in the course of his current episode revealed the following: c3 level 84 mg/dl; c4 level 14.4 mg/dl; c2 level 22.5 mug/ml; c4d level 0.94 mug/ml; c1 esterase inhibitor function 115% of normal. c1 esterase inhibitor level 17.4 mg/dl; ch50 223 units/ml; ana 1:40, negative; beta-tryptase < 1.0 ng/ml; total tryptase 7.0 ng/ml (all normal). initial evaluation revealed marked swelling along with erythematous and violaceous discoloration of the left lateral and anterior portions of the child's tongue. the remainder of his examination was normal. the patient was referred to the pediatric otolaryngology clinic with a presumptive diagnosis of a glossal hemangioma versus lymphangioma. he underwent a magnetic resonance imaging study which revealed a poorly defined, mass-like enlargement of the anterior and left aspect of the tongue. he was treated with a five day course of prednisolone. after initial regression of the lesion a planned surgical excision was carried out without any complications. in conclusion, this rare case illustrates the unique presentation of glossal hemangioma presenting as recurrent angioedema. highlighted are the differential diagnoses, laboratory studies and clinical features of oropharyngeal swelling. severe tongue swelling. rare skin conditions such as leprosy need to be considered in the differential diagnosis of apparent urticaria and angioedema that is atypical in appearance or response to therapy. a 28 year old male originally from laos presented with chronic recurrent nonpruritic indurated plaques on his face and trunk, and was referred to allergy by his primary physician with a diagnosis of urticaria and angioedema to rule out an allergic reaction to foods. he was otherwise healthy and on no medication, and had a negative personal and family history of atopy. he had moved to the united states from southeast asia in 1990, and visited for several weeks again in 2003. he was treated with prednisone and hydroxyzine without benefit; skin testing to foods was negative, and cbc, differential, sedimentation rate, antinuclear antibody, and liver function testing was normal or negative. a skin punch biopsy from an indurated area on the back showed granulomas with clusters of acid-fast organisms on afb stain consistent with tuberculoid leprosy. as the patient was g6pd deficient, he could not be treated with dapsone and was treated alternatively with minocycline 100mg, rifampin 600mg, and clofazimine 50 mg daily. he was subsequently started on prednisone as he was determined to be having a reversal reaction; his lesions continue to improve. this patient had a rare condition, leprosy, which was confused with urticaria and angioedema. in patients presenting with atypical apparent urticaria or angioedema, especially if response to standard pharmacologic treatment is poor, a skin biopsy is indicated, and may be diagnostic as in this case. introduction: cutaneous flushing about the face of a toddler within minutes of eating specific foods prompts parents and clinicians alike to pursue food allergy testing. auriculotemporal syndrome, also known as frey syndrome, is an isolated and transient facial erythema about the distribution of the auriculotemporal nerve following mastication. we report a child who was referred for food allergy testing and was diagnosed with auriculotemporal syndrome. case history: a 2 yo wf, prior full-term forceps-assisted delivery, has a history of facial flushing within minutes following ingestion of specific foods. the flushing can be induced by a variety of foods, specifically spaghetti, crackers, potato chips and hard candies, and typically resolves within minutes after eating. the child has no respiratory or gastrointestinal distress during the flushing episode. there is no angioedema, urticaria, or other rash elsewhere on her body. within ten minutes of eating a lollipop in clinic, an erythematous, warm macular eruption appeared in a band-like region anterior to her ear extending from her temporal bone to her mandible. this response occurred bilaterally, with a mildly greater intensity on the right side of her face. no sweating or other symptoms developed, and the flushing began to diminish after thirty minutes of observation. discussion: auriculotemporal syndrome is commonly seen in patients who have undergone facial surgery, specifically about the parotid gland. it is believed to be secondary to a disruption in the fibers of the auriculotemporal nerve. it is theorized that the nerve regeneration following an injury may join the parasympathetic fibers of the salivary gland with sympathetic fibers of eccrine sweat glands. mastication then could result in flushing and sweating over the cutaneous distribution of the auriculotemporal nerve that extends from the temporal region to the mandible, anterior to the ear. although rare in children, auriculotemporal syndrome has been associated with forceps delivery. therapy is seldom of benefit or needed, and the syndrome often resolves spontaneously over many years. conclusion: auriculotemporal syndrome leads to a cutaneous eruption temporally related to the ingestion of foods. allergists should maintain an awareness of this rare syndrome to minimize food allergy testing or eliminate unnecessary food provocation challenges or restrictive diets. m. al-ahmad * , s.s. mace, toronto, canada. introduction: ranitidine is a well-known h2-receptor antagonist. anaphylactic reactions are seldom reported despite widespread use of the drug. we report a patient with anaphylactic reaction to ranitidine methods: we report a case of anaphylactic reaction with ranitidine. results: a 45-year-old female with a history of urticaria over one year period, presented with 4 episodes of anaphylactic reactions of increasing severity over 1 year, after ranitidine ingestion. the first two episodes, occurred one hour after ranitidine ingestion, began with a sensation of burning, and itching in the head followed by dizziness, hypotension and near loss of consciousness. the patient had no significant pulmonary or laryngeal symptoms. the fourth episode, occurred 12 minutes after taking ranitidine tablet, was characterized by severe hypotension. two episodes were managed at the emergency department. a skin prick test with a crushed ranitidine tablet demonstrated a positive 20 mm wheal response, which was negative in a control. investigations for carcinoid syndrome and systemic mastocytosis were negative. conclusion: an ige mediated allergy to ranitidine is possible. anaphylactoid reaction to ranitidine is a well recognized entity. however, to our knowledge, few cases of anaphylactic reaction to ranitidine have been described. hypersensitivity reaction to ranitidine should be considered in patients suspected of having drug-related allergy. introduction: severe combined immune deficiency(scid) and cystic fibrosis(cf) may both present in infancy with a history of failure to thrive, diarrhea, and recurrent respiratory infections. although cf is the most common genetic disease occurring in caucasians with an incidence of 1 in 2500 births, scid is a rare condition estimated to occur between 1 in 50, 000 to 1 in 500, 000 births. we report a case of x-linked scid where the diagnosis of cf was originally entertained due to similar presentation and misleading laboratory results. case: 5-month-old caucasian male with 3 month history of recurrent respiratory illnesses, fever, loose stools, thick nasal discharge and failure to thrive. this was his second admission for a respiratory infection. relevant lab results consisted of cultures which grew parainfluenza a virus on np wash and pseudomonas aeruginosa on deep throat culture, chest xray showed patchy infiltrates and a borderline sweat test of 51 mmol/l. despite being treated with broad spectrum antibiotic coverage, albuterol and pulmozyme, his respiratory symptoms were not improving. physical exam revealed a thin infant with palpable though small lymph nodes cervical and groin, diffuse scattered rhonchi and liver edge palpable 3 cm below the costal margin. quantative immunoglobulins were obtained and resulted as an igg of 75, iga of <10, and igm of 13. review of his chest x-ray was significant for absent thymic shadow. lymphocyte count since admission ranged from 400-1000. flow cytometry revealed a cd3/cd4 count of 0%, cd3/cd8 count of 0%, cd56 of 1% and cd19 of 99% of all lymphocytes. t-cell stimulation tests were markedly diminished to both antigen and mitogen. a presumed diagnosis of x-linked scid was made and patient undrewent haploidentical bone marrow transplantation. subsequently, cf gene analysis resulted as negative. discussion: as outlined in the case it is essential to recognize the similarities between the presenting symptoms of cf, a relatively common genetic disease and scid, an uncommon one. although pseudomonas is seen in increased frequency in patients with cf, it is essential to recognize that it is also a leading pathogen affecting patients with antibody deficiency. review of the literature did not reveal any previous cases of individuals with scid reported as having an increased sweat chloride, nor any explanations of why this would occur. while immunodeficiency has previously been associated with some forms of dwarfism, we report the first described case of common variable immune deficiency in a patient with seckel syndrome (bird headed dwarfism). the diagnosis of seckel dwarfism, an autosomal recessive disorder, was made at 6 months of age in this boy based on intrauterine growth retardation, proportional dwarfism, microcephaly, micrognathia, and narrow face with "bird-like" large eyes and beaking of the nose. hematologic abnormalities are reported in an estimated 15% of such patients, and this child developed thrombocytopenia at the age of 3 years. by age 7, he had pancytopenia with hypocellular bone marrow. a low cd4 count (117/ul) was noted and both bactrim and neupogen were begun. his only significant infections, rotavirus and salmonella gastroenteritis, occurred at that time along with frequent otitis leading to pe tube placement at ages 7 and 8 but which subsequently resolved. he has never had opportunistic infections or thrush. by the age of 10 years, he required monthly red cell transfusions for severe anemia. after several months of transfusions, the blood bank reported that he no longer had detectable blood group antibodies during crossmatch; this led to measurement of immunoglobulins. igm was undetectable with low iga (35 mg/dl) and low igg (545 mg/dl). he demonstrated no protective titers to any pneumococcal serotypes after either conjugated or unconjugated pneumococcal vaccines. response to diphtheria, tetanus, rubeola, and influenza vaccines was adequate. total hemolytic complement was normal. he remains lymphocytopenic (alc = 552/ul) with low cd4 (211/ul). after five doses of ivig at 400mg/kg monthly, platelets have remained between 15-20 k/ul and there has not been any improvement in the anemia. although the mechanism for the bone marrow failure in these patients is presumed to be chromosomal breakage, this has not been demonstrated in this child. his karyotype is 46xy with no chromosomal fragility or deletions detected to date. hypogammaglobulinemia should be investigated in any patient undergoing frequent transfusions who loses blood group antibodies, and suspicion of immune deficiency should remain particularly high in patients with severe growth retardation or dwarfism. n.l. rider * , t. craig, hershey, pa. the objective of this study was to assess the effectiveness of physicians at a university primary care clinic in diagnosing and managing adult patients with asthma. a retrospective chart review of 63 adult patients from this clinic was performed. each chart was evaluated using a survey instrument developed from the national asthma education and prevention program (naepp), which consisted of 22 queries. of the charts evaluated 50 met our entry criteria. overall compliance with the naepp recommendations was 34%. adherences to the asthma guidelines in the following areas were: diagnosis (42%), treatment/disease control (60%), monitoring (15%), and education (18%). these data suggest that there is an opportunity to improve the care provided to patients with asthma, especially in the areas of asthma education and monitoring, even in a university outpatient facility. these data also suggest that naepp guidelines have not been effectively incorporated into primary care practices. b.m. wahlers * , l. sherwood, t. craig, hershey, pa. objective: our aim was to evaluate adherence to the national heart lung and blood institute (nhlbi), national asthma education and prevention program (naepp) diagnosis and management guidelines, regarding asthma admissions at a teaching hospital. methods: a retrospective chart review of 197 patients over the age of 18 years admitted to the hershey medical center between 1997 and 2002 with a primary diagnosis of asthma or asthma exacerbation was conducted using a 21-item questionnaire focused on key aspects of asthma care as outlined in the naepp guidelines. results: overall compliance to the guidelines was 61.5%. maximal areas of compliance were noted in regards to pharmacologic treatments utilized (99%, 95%). much lower levels of compliance were noted in areas of patient education (58%), instruction in written action plan prior to discharge (11%), provision of peak flow meter for home use (47%), assessment of asthma symptoms and severity (58%), and inhaled steroid prescription on discharge (69%). conclusion: adequate care in terms of pharmacologic treatments is being given but sub-optimal cares in other areas of the guidelines exist. there continues to be room for improvement in regards to asthma management, most notably in the area of education, documentation of triggers, and preparation for discharge with adequate tools to monitor and control symptoms. background: asthma is one of the most common problems of childhood, responsible for a significant proportion of absence from school because of chronic illness. objective: this study was carried out among the school-aged children (7-18 years) in tehran schools during 2002-2003, in order to determine the frequency of asthma. methods: according to the recommendation of who, a questionnaire was designed, containing 8 standard questions, and the students were given necessary information to complete the questionnaires. the guidance and high school students completed the questionnaires but the parents of primary school students completed them by themselves. results: seven hundred and eight children out of 2000 children had asthma (35.4%); this prevalence was higher in the boys (37.1%), when compared to the girls (33.5%). the prevalence of this disease has been estimated about 39.5% in guidance schools, 35.4% in high schools and 31.6% in primary students. based on this survey, the most common clinical manifestations in asthma were included: prolonged cough lasting more than 10 days (22.4%), and exerciseinduced wheezing or dyspnea (16.9%), followed by repeated dyspnea or wheezing (6.4%). based on the drug responses after receiving solbutamol, the prevalence of asthma was evaluated in the range of 32.2% in primary schools, 16.7% in guidance schools and 22.9% in high schools. conclusions: the prevalence of asthma is high among the students of tehran schools and it needs more careful screening programmes and either more information given to the patients and parents about the disease. in the us, asthma is the most common chronic disease of childhood and affects approximately 5 million children. reasons for inadequate asthma control include: inappropriate therapy, incorrect inhaler technique, poor compliance with treatment, and exposure to environmental triggers. rates for asthma prevalence, hospitalization, and death are highest among children residing in inner cities, and important risk factors for asthma-related mortality include being poor and black. the communities of braddock, north braddock and rankin are among the poorest in allegheny county and are designated areas of greatest health risk. recent statistics have demonstrated an increase in the number of asthma hospitalizations in these communities: 13.6 children per 1000, which is double the average for allegheny county and greater than seven-times the overall us rate of asthma hospitalizations. our goals were to assess asthmatic severity and appropriateness of treatment, provide asthma education, and to ensure access to healthcare for asthmatic children living in these medically underserved and predominantly african-american communities. twenty-seven asthmatic children were identified. of the children assessed 7% were under the age of 2 years, 19% were aged 2-5 years, and 74% were 6-14 years of age. based on nhlbi guidelines the severity of each child's asthma was determined: 41% had mild-intermittent asthma, 15% had mildpersistent asthma, 26% had moderate-persistent asthma, and 18% had severepersistent asthma. the appropriateness of treatment based on severity was also determined. we found that 27% of the children with mild-intermittent asthma, 50% of children with mild-persistent asthma, 60% of children with moderate-persistent asthma and 100% of children with severe-persistent asthma were receiving inappropriate treatment. furthermore, of the inappropriately treated children 92% were undertreated. following the assessment, patients and parents were educated with regard to asthma triggers, pathophysiology, use of peak flow meters, inhaler technique and pharmacologic treatment. patients were also scheduled for outpatient follow-up care. these results demonstrate that inappropriate treatment, especially undertreatment, may be a significant cause of the increased asthma morbidity in this population and underscore the need for aggressive education and innovative methods of ensuring health care. w. li-ling * , t. keng-leong, f.c. stephanie, e. philip, singapore. background: asthma admission is an important indicator of asthma morbidity. little is known about the risk factors associated with asthma admission among high-risk asthmatics. purpose: 1) to characterise the profile of high-risk asthmatics who had previous asthma hospitalization. 2) to identify predictors and risk factors that are associated with asthma hospitalization. methods: data for consecutive high-risk asthmatics who were enrolled into our asthma program from october 2001 to may 2004 were retrieved from our prospective database. high-risk asthmatics were defined as patients who had a clinical diagnosis of asthma and who had at least one hospital admission for exacerbation of asthma in the preceding 12 months or at least one severe exacerbation of asthma in the preceding 6 months requiring unscheduled visit for beta2-agonist nebulisation. a comparison was made among high-risk asthmatics who had at least one or more hospitalization for asthma in the past 12 months prior to the asthma program enrolment with those who were not hospitalized. statistical analyses were performed using spss version 10.1 for windows. results: among 521 high-risk asthmatics, 252 (48.4%) had at least one or more asthma hospitalization in the past 12 months prior to the enrolment into the asthma program. hospitalization was significantly associated with female gender (63.1% with asthma admission v 43.1% without asthma admnission). a significantly higher proportion of asthmatics with no formal education (19.4% v 17.7%), primary education (23.8% v 16.6%) and secondary education (32.1% v 29.3%) were hospitalized for asthma as compared to the those who had tertiary education (24.6% v 36.5%). those admitted were predominantly in lower income group, had poor inhaler technique and have no ownership of an asthma action plan (33.3% v 76.6%). those hospitalized were also younger, median age 37years (19, 62) v 35years (15, 84) and had a lower first peak flow reading, 260l/min (130, 500) v 300l/min (150, 480) . conclusion: among high-risk asthmatics, certain population subgroups are at greater risk for hospitalization for asthma. intervention aimed to reduce asthma morbidity should take the above findings into consideration. introduction an association between asthma symptoms and air quality is well established, but controversy exists concerning the role of various air quality indicators. the association of san antonio air quality with emergency department (ed) visits for asthma at our military medical center has not been defined. the objective of this study is to determine the correlation of selected air quality indicators in san antonio with ed visits for asthma for both the general population and for the pediatric population. methods selected air quality indicators were the 8-hr air quality index (aqi) for ozone, 24-hr aqi for particulate matter < 2.5 î¼m, pollen counts, mold spore counts, and daily high temperature. the number of daily ed visits coded for asthma was obtained retrospectively. pediatric visits (patients < 18 years-old) were evaluated as a subgroup. pearson correlation coefficients (r) were calculated for all data pairs. results during the 331 days of the study period, there were 491 visits for asthma. of these visits, 151 were pediatric. the number of daily visits ranged from 0 to 6 for all visits and from 0 to 3 for the pediatric visits. r values for all visits were -0.067, -0.069, 0.110, -0.011, and -0.218; for pediatric visits, -0.055, -0.036, 0.033, -0.010, and -0.074 for the 8-hr aqi for ozone, 24-hr aqi for particulate matter < 2.5 î¼m, total pollen grains/m 3 , mold spores/m 3 , and daily high temperature, respectively. in addition to calculating r values for same-day air quality indicators and ed visits, r values were also calculated for a 3-day average of air quality indicators with the ed visits. for the 3-day averages, r values for all visits were -0.113, -0.117, 0.127, -0.071, and -0.267; for pediatric visits, -0.081, -0.011, 0.025, 0.017, and -0.063 for the 3-day averages of the 8-hr aqi for ozone, 24-hr aqi for particulate matter < 2.5 î¼m, total pollen grains/m 3 , mold spores/m 3 , and daily high temperature, respectively. conclusion there was no significant correlation between the selected air quality indicators and ed visits for asthma, either for the general population or for pediatric visits. this study provides no evidence that air quality is linked to ed visits for asthma in san antonio but the degree to which air quality in san antonio affects asthma symptoms remains unclear. introduction: the aim of this study was to evaluate the level of no in the exhaled air of patients suffering from chronic cough. methods: the study was performed on 213 young (mean age 28.5 +/-8.2 years), nonsmoking patients with chronic cough referred to the allergy clinic for evaluation. bronchial provocation challenge with histamine was performed according to the method of ryan. exhaled no was measured on-line using a chemiluminescence analyzer (sievers, usa). all patients had resting spirometry within normal values. results: significant bronchoconstrictive response to histamine at a concentration equal to or lower than 8 mg/ml (pc20 8mg/ml) was found in 65 patients (30.5%). these patients had a significantly greater mean concentration of no in the exhaled air (80.6+/-59.4 ppb) than those with a pc20>8mg/ml (28.4 +/-29.2 ppb; p<0.0001). receiver operating characteristic (roc) curve analysis revealed that it is possible to identify from among patients suffering chronic cough those who have significant bronchial hyperreactivity (pc20 8mg/ml). using 40 ppb as a cut-off value for the exhaled no concentration, the specificity for bronchial hyperreactivity was 86.5% and sensitivity 72.3%. conclusion: assessment of no concentration is helpful in the assessment of bronchial hyperreactivity in patients having chronic cough. l. abetz 1* , j. bousquet 2 , e. juniper 3 , e. bateman 4 , h. boushey 5 , w. busse 6 , t. clark 7 , r. pauwels 8 , s. pedersen 9 , 1. manchester, united kingdom; 2. montpelier, france; 3. ancaster, canada; 4. cape town, south africa; 5. san francisco, ca; 6. madison, wi; 7. london, united kingdom; 8. ghent, belgium; 9. kolding, denmark. introduction: the acq has been developed as a measure of asthma control for use in clinical practice or in clinical trials. the reliability, validity and responsiveness have previously been demonstrated for the original 7 item acq, the 6 item (minus fev1 question) and 5 item (minus fev1 and short acting bronchodilator use questions) versions. data from the 1-year gaining optimal asthma control (goal) study was used to assess the predictive value of the acq s 7, 6, and 5 item versions in determining asthma control status. method: receiver operating characteristic curves (roc curves), plotting true positive rates versus false positive rates for different acq cut-points, were used to determine optimum cut points for totally controlled and well-controlled asthma. scores for the 7, 6, and 5 item versions of the acq were examined at 0.25 intervals. results: when predicting totally controlled asthma, the areas under the roc curves (arocc) were 0.88/0.88/0.86 for the 7, 6 and 5 item versions, respectively; and when predicting well-controlled asthma the arocc were 0.86/0.86/0.84 for the 7, 6 and 5 item versions. in both predictive models, the 7 and 6 item versions provided the best arocc. for the 7 and 5 item versions, the best cut-off point to predict totally controlled asthma was <=0.75, with arocc of 0.81/0.79, and corresponding sensitivity of 0.74/0.71 and specificity of 0.89/0.88, respectively. for the 6 item version the best cut-off point was <=0.5, with arocc of 0.82, sensitivity of 0.76 and specificity of 0.87. the best cut-off points for predicting well-controlled asthma were <=1.25 for the 7 item version (arocc=0.77; sensitivity=0.70; specificity=0.83), and <=1 point for the 6 and 5 item versions (arocc=0.77/0.75; sensitivity=0.70/0.69; specificity=0.83/0.81, respectively). conclusion: results indicate the sensitivity and specificity of the acq in predicting asthma control status. a 29 yo black male with asthma and allergic rhinitis developed gradually worsening cough productive of clear to yellow sputum despite treatment with high dose fluticasone, salmeterol, monteleukast, and prn albuterol. asthma history was significant for an intubation at age 10 and pneumonia at age 28. physical exam revealed pale turbinates and expiratory wheezes. pulmonary function test showed a severe obstructive lung defect with fev1 1.52 (38%), improving by 4% after bronchodilator and fev1/fvc ratio 54%. a chest ct revealed significant bronchiectasis in the right middle lobe, lingula and both lower lobes. evaluation for bronchiectasis showed normal quantitative immunoglobulins, subclasses, and humoral responses, rendering innate immunodeficiency less likely. the diagnosis of allergic bronchopulmonary aspergillosis was entertained, but ige was 500ku/l and aspergillus titers were negative. a sinus ct failed to show active disease, yet the patient reported chronic yellow nasal discharge despite courses of antibiotics. although he was sexu-ally active with several male partners, hiv testing was negative 2 times, 6 months apart. evaluation for cystic fibrosis (cf) showed no symptoms of malabsorption and a negative sweat test. other tests revealed normal -1 antitrypsin level, p-anca, esr, ace level, and negative sputum for afb and mycobacteria, helping to exclude other conditions such as 1-antitrypsin deficiency, vasculitis, sarcoidosis, and chronic infection. with no definitive answer, the diagnosis of cf was pursued, but genetic screening as well as mapping were negative. the diagnosis of primary ciliary dyskinesia was then entertained; however, a mucosal biopsy of the nose did not reveal any structural abnormalities. finally, a sperm analysis revealing azoospermia confirmed the diagnosis of young's syndrome, a rare disease with features similar to cf including bronchiectasis, chronic sinusitis and azoospermia. in young's syndrome however, there is an obstructive process rather than a lack of vas deferens resulting in azoospermia and normal sweat chloride, genetic, and pancreatic testing. this case demonstrates the importance of investigating a persistently productive cough in an asthmatic patient and the differential diagnosis as well as the appropriate work up for bronchiectasis. a final diagnosis is important since appropriate management in each situation may affect long term outcome. a.g. palma-carlos * , m.l. palma-carlos, lisboa, portugal. introduction: bronchial challenges are currently done in allergic asthma with allergen, histamine or metacholine.bronchial reactivity to allergens and histamine can probably be variable. purpose to evaluate if the dose of allergen and histamine triggering a significant bronchial obstruction are always correlated in asthmatic patients. methods: a vitalograph model compact has been used throughout the study. allergen challenge has been done with an acqueous solution of house dust mite. bronchial challenges with placebo (sodium chloride) histamine or allergens have been done in all patients. for the provocations with histamine or allergens the threshold dose, inducing a decrease in fev1 greater than 15% has been determined in all the cases, as well for histamine as for allergens. a second challenge has been done in all patients with a supra-threshold dose of histamine or allergen corresponding to the double of the threshold. results: 30 provocations have been done in 10 patients either with histamine or allergen. 6 were allergic and 4 non allergic. the threshold dose for histamine were between 5 and 25 ug. in allergic patients and 250 and 500 ug. in non allergic patients.in allergic patients after histamine threshold challenge mean decrease was for fev1 19, 2% and for fev1/vc 17, 3%. for supra-threshold dose fev1 decreases 37, 8% and fev1/vc 32, 8%. after allergen challenge fev1 decreases 20, 3% and fev1/vc 15, 8%. with supra threshold doses fev1 decreases 28, 6% and fev1/vc 29, 3%. in 4 non allergic patients there was no changes after allergen challenge. a good correlation in constriction induced by histamine or allergen either for threshold or supra threshold doses (p<0.001) was observed but not between the threshold doses of histamine and allergen. conclusions: these results confirm that bronchial obstruction can be triggered by allergens and mediators that the sensitivity of fev1 and fev1/vc for evaluation are roughly comparable after challenge and points to a better sensitivity of a supra threshold dose of allergen or mediators. allergen and histamine challenges don't give the same information. introduction: peak expiratory flow rate (pfr) and forced expiratory volume in the first second (fev1) are currently used in functional evaluation of asthmatic patients but his degree of correlation in obstructive, restrictive and mixed spirometric ventilatory patterns are not well known. the purpose of this study was to evaluate if the correlation between pfr and fev1 2 markers of global airways obstruction is comparable in asthmatic patients with obstruction restriction or both. methods: 152 asthmatic patients, 69 males, 83 females have been studied by spirometry with a vitalograph compact in absence of bronchodilatory or anti-inflammatory therapy. patients have been classified in 4 functional patterns according to vc, and fev1/cv: normal : vc greater than 90% of expected value and fev1/vc greater than 70%, obstructive:vc greater than 90% and fev1/vc greater than 70% restrictive vc, less than 90% and fev1/vc greater than 70% and mixed vc less than 90% and fev1/vc less than 55% presenting a so called functional emphysema. results: according to these criteria 15 patients were normal, 44 restrictive, 15 obstructive and 78 mixed. in all the group the correlation between pfr and fev1 was statiscally significant (p<001) but the correlation coefficient variable. in functionally normal patients r =+0, 74 in restrictive patients + 0, 67 in obstructive + 0, 66 in mixed + 0, 84 and in the sub-group with functional emphysema + 0, 96. for this sub-group the correlation coefficient r allows to define fev1 = 91 + 5, 6 pfr. conclusions: the report between the two most usual assays of global bronchial obstruction depends on the balance between restriction, (either by interstial fibrosis or hyperinflation) and obstruction and is more close when a more marked obstruction coexist with a restrictive pattern (functional emphysema). in practice pfr has the same value that fev1 only in more severe cases of ventilatory failure. the dispersion of values and the standard error or regression do not allow to correlate both data in most patients. introduction: the tenor study longitudinally observes the natural history of subjects with severe or difficult-to-treat asthma. this analysis assessed the association between previous and future asthma exacerbations in tenor. methods: subjects eligible for this analysis were 12 years of age at baseline and had at least one follow-up assessment in year 1 and year 2. recent asthma exacerbations in the past 3 months were defined as: asthma-related emergency room (er) visits, nights of hospitalization, unscheduled physician office contacts; or as a composite measure of at least one of the previous exacerbation events. relative risks (rrs) and 95% confidence limits (95%ci) were generated comparing exacerbations in year 2 relative to year 1. exacerbation rates were defined as the number of events divided by total patient follow-up time. no adjustment was performed for baseline characteristics. results: 2826 subjects were eligible for this analysis. rrs in year 2 vs. year 1 were: 3.9 (95%ci: 3.3-4.6) for unscheduled office contacts, 4.1 (95%ci: 3.48-4.82) for the composite exacerbation measure, 10.2 (95%ci: 7.8-13.4) for er visits and 15.1 (95%ci: 9.8-23.3) for nights of hospitalization. over 37% of subjects with 5 or more exacerbations (composite measure) in year 1 had a similarly high rate in year 2 compared to 3% of patients without an event in year 1 who went on to have 5 or more exacerbations in year 2 (see table) . conclusion: tenor subjects who experienced an asthma exacerbation in year 1 were at statistically significantly increased risk of subsequent exacerbations the following year compared to subjects not experiencing an exacerbation in year 1. this increased risk was consistent for all asthma exacerbation outcomes and most elevated for hospitalizations due to asthma. assessment of recent asthmarelated healthcare use is a critical component of the clinical evaluation of subjects with severe or difficult-to-treat asthma. funded by genentech inc. and novartis pharmaceuticals corp. background: preliminary studies investigating yoga and breathwork for asthma have been promising. several randomized controlled trials have shown a benefit from yoga postures and/or breathing versus control, but the control in these cases involved no intervention other than usual care. this study advances the field by providing an active control. methods: a randomized, controlled, double-blind clinical trial was conducted from october 2001 to march 2003 to determine the effectiveness and feasibility of a yoga and breathwork intervention for improving clinical indices and quality of life for adult patients with mild to moderate asthma. random assignment was made to either a 4-week yoga intervention that included both postures and breathwork, or a stretching control condition. outcome measures were assessed at 4, 8, 12 and 16-weeks and included the mini asthma quality of life questionnaire (mini-aqlq), rescue inhaler use, spirometry, symptom diaries, and health care utilization. results: sixty-two participants were randomized into the intervention and control groups, and 45 completed the final follow-up measures. intention-to-treat analysis was performed. significant within-group differences in post-bronchodilator fev1 and morning symptom score were apparent in both intervention and control groups at 4 and 16 weeks; however, no significant differences between groups were observed on any outcome measures.conclusions: iyengar yoga conferred no appreciable benefit in mild to moderate asthma. circumstances under which yoga is of benefit in asthma management, if any, remain to be determined. in order to investigate asthma mortality trends for the u. s. hispanic population, i have collected and graphed data from the national center for health statistics. mortality data for the hispanic population have been available for the entire united states only since 1997 and readily available for asthma only since 1999. these data indicate decreases in deaths from asthma (j45-j46) from 320 in 1999 to 274 in 2001 with decreases in the non-hispanic population from 4324 in 1999 to 3976 in 2001. rates of death from asthma decreased for the hispanic population from 1.0 per 100, 000 in 1999 to 0.7 in 2001, while that for the non-hispanic population decreased from 1.84 to 1.6. rates for non-hispanic whites decreased from 1.5 to 1.4 in 2001. rates of death from asthma have been twice as high among hispanic women as men. rates of death from asthma for non-hispanic blacks have been more than twice as high as those for non-hispanic whites. (national center for health statistics data dictate racial terminology). data for people of hispanic origin include people of any race. these data are affected by both underreporting of hispanic origin on death certificates and undercoverage in the census and resultant population estimates for a net correction of 1.6%. thus, rates of death from asthma have been lower and have been decreasing faster among hispanics than the rest of the population in the united states. allergic rhinitis and asthma are both highly prevalent diseases and often coexist in patients. rhinitis symptoms have been shown to impact the lower airway. as such, the impact of rhinitis on asthma control may be the greatest in patients with the most severe rhinitis symptoms. therefore, this analysis was performed to determine the impact of baseline rhinitis severity on asthma control in patients with both asthma and allergic rhinitis who received either fluticasone propionate aqueous nasal spray (fpans) or montelukast (mon) added to fluticasone propionate/salmeterol 100/50 mcg (fsc). a total of 863 patients (>15 years) with persistent asthma and seasonal allergic rhinitis received fpans 200mcg qd, mon 10mg qd or placebo (pbo) in addition to fsc 100/50mcg bid for 4 weeks. at baseline, total nasal symptom scores (tnss) ranged from 44 to 400. the analysis was restricted to patients whose pre-enrollment asthma therapy did not include fsc. regardless of baseline rhinitis severity, in patients with both asthma and allergic rhinitis, mon added to fsc resulted in no additional improvements in overall asthma control compared with fsc alone. these data suggest that optimal treatment of the individual conditions should be the goal of treatment for patients with coexistent allergic rhinitis and asthma. (sam40066) introduction recent studies have demonstrated that parainfluenza and coronaviruses are as important triggers of asthma. parainfluenza viruses are one of the main triggers of virus-induced asthma at summer. parainfluenza viruses often cause severe low respiratory tract infections in immuncompromised patients and in patients with bone marrow transplantation. a retrospective study found that 41% of pediatric bmt patients with hpiv infection (47% of viral respiratory infections) developed pneumonia and 6% died. both viruses contributed to fatal asthma. materials and methods for amplification of these viruses, primers which anneal to specific regions of viral genomes: hn-gene for piv1, piv2 and piv3 and spike glycoprotein gene for coronavirus oc43 and nucleocapsid glycoprotein gene for coronavirus 229e were used. pcrdiagnostics was performed on nasal and throat swabs taken from children with atopic asthma exacerbation. results from 02/04 to 05/04 a total of 56 samples (including 28 negative controls) were tested by pcr. 11 positive for respiratory viruses samples noted in children having asthma exacerbations including 23.9% of all tested samples. piv3 was detected in 54.5% of all positive samples, piv2 in 18.2%, and coronavirus oc43 in 27.3%. piv2 and coronavirus 229e were not detected during this study. for negative controls samples were taken from children with atopic asthma in remission. all 10 controls were found to be negative by viral pcr. conclusions respiratory viruses are an important factor in asthma exacerbations in children. piv3 was the most frequently detected among the positive samples. although hpiv-2 was less often positive than hpiv-1 and hpiv-3 infections in this study, the frequency of the detected coronaviruses may have been influenced by the seasonal activity of these viruses. introduction. in budapest all children come under the regular supervision of a specially trained paediatrician. each paediatrician is responsible for an average of 950 children, often seen up to the age of 18 years. methods. a questionnaire was sent to the district paediatricians of budapest in 1995 budapest in , 1999 budapest in and 2003 . the total number of children in their practice and the number of the asthmatics was assessed. the diagnosis of asthma in every case was established in a paediatric hospital, or a special allergy or pulmonology outpatient clinic. results. in 1995, replies were received from 118 paeditricians, who were responsible for the supervision of 104, 087 children, of which 1.88 â± 0.87% had been diagnosed as having asthma. in 1999 replies were sent by 153 physicians, who had a total of 142, 684 children under their care, including 3, 228 asthmatics, a prevalence of 2.26 â± 0.95%. at the end of 2003, 151 paediatricians having 141, 276 children answered, noting 3, 831 asthmatics, a 2.71 â± 1.2% prevalence of asthma. conclusion. the current survey of prevalence of asthma in childhood is by far the largest that has been made. an increasing prevalence of the diagnosis of asthma based on clinical investigations was noted, the differences between the investigated years being highly significant. panel report guidelines for the diagnosis and management of asthma have been available since 1991, to standardize and improve the quality of asthma care. however, asthma remains the leading cause of hospitalization for new york city children. implementation of the guidelines has not been fully embraced in the primary care setting. we attempted to increase primary care practitioners' compliance with the guidelines through extensive training. a hospital based pediatric clinic and 4 school based health clinics in the brooklyn inner city, participated from april 2002 to march 2003 in the program as part of new york city's education and quality improvement project (equip). initially an asthma physician specialist and asthma educator trained the primary care physicians and nurse practitioners in the guidelines. the training was reinforced during monthly luncheon sessions. clinical exam rooms were also supplied with asthma education materials, peak flow meters, and asthma action plans. goals were to identify asthmatics, document asthma severity, give persistent asthmatics written management plans, and utilize appropriate controller medications. compliance was measured by tracking asthma visits with an asthma intake form. prior to this project no consistent attempt had been made in the clinics to regularly classify asthmatic severity, provide written asthma action plans or to place patients on controller medications. after onset of the project a total of 216 asthma visits were evaluated; 104 were from school based health centers and 113 from hospital based pediatric clinic. severity was classified in 83%(179/216) of asthma visits. patients classified as persistent received updated written management plan during 90%(100/110) of asthma visits and 92%(102/110) were placed on controller medications. this asthma management program demonstrated success in improving primary care practitioners' compliance with the naepp asthma guidelines. further studies are needed to determine if compliance with the guidelines persisted after cessation of the extensive training and if such compliance improves asthma outcomes. numerous studies have associated asthma and obesity. although obesity has been identified as a risk factor for asthma, there is limited information on the impact of obesity on childhood asthma. this study examines the effect of body composition on asthma severity and pulmonary function in children. we retrospectively reviewed charts from asthmatic children ages 5 to 18 years, referred to a community-based pediatric pulmonology practice between 1999 and 2003. data were included if asthma was the the primary diagnosis and a baseline spirometry was available. asthma severity was classified and body mass index(bmi) was classified as normal, overweight and obese. spirometry results were reviewed. eighty-five patients were reviewed. thirtyone(36.5%) patients were normal weight; 21(24.7%) were overweight and 33(38.8%) were obese. baseline characteristics such as age, gender and race/ethnicity were similar. asthma severity classification did not differ between normal, overweight and obese children. overall, 34% were classified as intermittent, 53% as persistent mild asthma and 13% as persistent moderate-severe asthma. 21% of children with intermittent asthma were receiving controller therapy compared to 62.5% with persistent mild and 81.8% with moderate-severe asthma(p=0.0001). although healthcare utilization for asthma(emergency room visits/admissions) were significantly different between children with intermittent, persistent mild and moderate-severe asthma(p=0.0004), there was no difference when normal, overweight and obese children were compared. spirometry results showed comparable peak expiratory flow rates(pfr) in all groups; 75% of predicted in normal weight and 79% of predicted in both overweight and obese. mean forced vital capacity(fvc) was 93.9%, 88.5% and 95% and the mean forced expiratory volume in 1 second(fev1) was 85.9%, 81.2% and 86.6% respectively. there was no difference in fev1/fvc; 82.3%, 83.1% and 81.8% and forced expiratory flow in mid-lung volumes (fef25-75); 74.4%, 73% and 76.9% of predicted. in this study we retrospectively reviewed asthmatic children and compared bmi, asthma severity and spirometry. we found no difference in asthma severity classification between normal and overweight/obese patients. regardless of the bmi, all indices of spirometry were similar. further studies are needed to examine the impact of obesity on different asthma related disease outcomes. our objective is to further investigate the effect of budesonide, formoterol, and levalbuterol, alone, and in combination, on tgf-1 production and expression in ragweed (ra) stimulated a549 cells. meth-ods: a549 cells were cultured in duplicate for 24 hours at 37 degrees celcius with 5% co 2 in serum free dmem with or without the following: 10î¼g/ml of ra, 1.0 x 10 -7 m of budesonide, 1.3 x 10 -7 m formoterol, and 1.0 x 10 -5 m levalbuterol. tgf-1 production and expression was measured by a sensitive elisa and mrna assay in cell supernatants and pellets. results: tgf-1 production and expression from a549 cells rose markedly in the presence of ra (952.35 -1268 pg/ml) (59 -71 amol/ml) with significant inhibition following the addition of budesonide, and the combinations of budesonide and formoterol, budesonide and levalbuterol, and budesonide, formoterol, and levalbuterol (p<0.05). combined treatment with budesonide and levalbuterol versus budesonide alone reached significance for production and expression (p<0.05). budesonide and formoterol compared with budesonide alone reached significance for tgf-1 production (p<0.05). conclusion: combination therapy with budesonide and a long or short acting beta-agonist showed greater effect of tgf-1 inhibition compared with budesonide alone. this synergistic effect on the distal airways may prevent a subset of asthmatics from developing airway remodeling. g. tamura 1* , y. sano 2 , k. hirata 3 , s. ishioka 4 , m. nakashima 5 , t. miyamoto 2 , 1. sendai, japan; 2. tokyo, japan; 3. osaka, japan; 4. hiroshima, japan; 5. hamamatsu, japan. tulobuterol tape is the world's first long-acting transdermal preparation of a beta2-agonist designed to release tulobuterol in an optimal fashion. when it is applied once daily at bedtime, the blood concentration of tulobuterol peaks early in the morning and is kept at effective levels for 24 hours. tulobuterol tape has milder and less frequent adverse events than conventional oral tulobuterol preparations, and when used at bedtime can prevent marked drop in peak expiratory flow (pef) early in the morning. consequently, tulobuterol tape has been commonly used in japan as a long-acting beta2-agonist. in the present study, to evaluate its efficacy as a long-acting beta2-agonist and to compare its effects at two different doses, we administered tulobuterol tape at doses of 1 or 2 mg/day to patients with persistent asthma already using inhaled corticosteroids. this study was a randomized, double-blind, double-dummy, parallel-group, multicenter trial of 4-week duration. mean pef in the 1 and 2 mg/day groups were significantly increased from the baseline value by 15.1 and 28.2 at week 1, 19.5 and 32.6 at week 2, 22.6 and 35.3 at week 3 and 23.8 and 35.9 l/min at week 4, respectively. the increase in mean morning pef in the 2 mg/day group was significantly higher than that in the 1 mg/day group at every point of determination. the mean evening pef was significantly increased in both treatments groups compared with baseline values at every point of determination. although the increase in the 2 mg/day group was greater than that in the 1 mg/day group at each point of determination, the difference between groups was statistically significant only at week 1. the safety profiles of the two treatments were similar. in patients with persistent asthma who require inhaled short-acting beta2-agonists while receiving inhaled corticosteroids, tulobuterol tape 2 mg/day significantly improved pef compared with tulobuterol tape 1 mg/day. introduction: on an average, 4% of the 1500 children admitted annually for asthma to children s hospital require admission to the pediatric intensive care unit (picu). these admissions are highest in the months of august, september and october. aeroallergens such as alternaria and ragweed predominate during this season, and allergies to these allergens may account for the increased number of picu admissions for asthma. objective: we hypothesized that the seasonal increase in the admission rate could be related to allergen sensitivity, particularly to alternaria and/or to ragweed that predominate during late summer and fall. method: data was collected from a retrospective chart review of all patients admitted to the picu for asthma exacerbation between 1997 and 2003. skin prick testing (spt) within one year of picu admission was the criteria for inclusion in this study. results: there was a higher prevalence of admissions to the picu for asthma exacerbation during august, september and october (168 out of 416 admissions = 40% of all admissions, p<0.001). sixty-one subjects met the one-year spt criteria for analysis. subjects admitted during august, september and october were categorized as group a, and all others were categorized as group b. sensitivity to a total of 11 aeroallergens (tress, grasses, weeds, ragweed, outdoor molds, indoor molds, alternaria, cat, dog, roach and dust mites) was compared between the two groups. there was no statistical significance in aeroallergen sensitivity to alternaria or ragweed between subjects admitted to the picu in august, september and october (group a) and subjects admitted during the other months of the year (group b). conclusion: asthma exacerbation requiring picu admissions is more prevalent during the months of august, september and october. the increased rated of picu admissions did not correlate with the aeroallergen sensitivity to the prevalent allergens during august, september and october. therefore we suspect other factors such as, viral upper respiratory infections, in addition to aeroallergen sensitivity, which may contribute to severe asthma exacerbation during the late summer and fall months. k. kuzume * , shigenobu, ehime, japan. infantile wheezing may be the result of different phenotypes. the aim of this study was to evaluate the risk factors for allergies and the results of allergyrelated blood tests among infants with different types of allergy symptoms. 2130 infants were examined physically at birth and at 1, 3, 6, 9, and 12 months of age, and questionnaires about number of siblings, the family history of allergy, and feeding methods were given. allergic diseases were diagnosed at 12 months of age, and the subjects were divided into 4 groups: infants with atopic dermatitis (ad) only (a group, n=447), infants with both ad and wheezing (a/w group, n=175), infants with wheezing only (w group, n=155), and infants without allergic symptoms (c group, n=1353). risk factors were then evaluated. 332 patients (198 from the a group, 109 from the a/w group, and 25 from the w group) were given allergy-related blood tests, total serum ige levels, and rast with egg white, milk, and house dust mites at 6 and 12 months of age. the results of the blood tests in 36 control subjects at 6 months of age were compared to the other groups. as shown in table1, there were more male infants in the a/w and a groups than in the c group (p<0.001, a/w vs. c; p<0.01, a vs. c). numbers of siblings were greater in the w and a/w group than in the a or c group (p<0.0001, w vs. a and w vs. c; p<0.01, a/w vs. a; p<0.05, a/w vs. c). the rate of positive family history of allergic diseases was high in the a and a/w groups, compared to the w or c group (p<0.0001, a vs. c and a/w vs. c; p<0.05, a/w vs. w). the rate of breastfeeding at 3 months of age was high in the a group compared to the others (p<0.0001, a vs. c). patients in the w group had lower levels of total serum ige at 12 months of age, compared to patients in a or a/w (p<0.01, w vs. a/w and w vs. a). also patients in the w group had lower levels of rast with egg white at 12 months of age, compared to patients a or a/w (p<0.0001, w vs. a/w and w vs. a). the median of total serum ige levels at 6 months of age was higher in a and a/w but not in w, compared to c (p<0.001, a/w vs. c and a vs. c). in conclusion, there were two different phenotypes of wheezing in infants before 12 months of age. wheezing with ad might be "allergy-related", while wheezing without ad may be related to other factors. as compared to c group: a), p<0.05; b), p<0.01; c), p<0.001; d), p<0.0001 as compared to a group: 1), p<0.05; 2), p<0.01; 3), p<0.001; 4), p<0.0001 as compared to a/w group: f), p<0.05; g), p<0.01; h), p<0.0001 a group, infants with atopic dermatitis only; a/w group, infants with atopic dermatitis and wheezing; w group, infants with wheezing only; c group, infants without allergic symptoms. rast, radioallergosorbent test, n/a, data not available r. amelio 1* , c. capristo 1 , a. capasso 1 , m. miraglia del giudice 2 , n. maiello 1 , f. decimo 1 , 1. naples, italy; 2. napoli, italy. forced oscillation technique (fot) is considered a sensible and specific method to estimate breathing functionality in asthmatic children aged 3 to 6 years, because it doesn't need special collaboration. the aim of our study was to value baseline respiratory resistances with the fot in preschool-aged children with asthma under high dose of budesonide and flunisolide treatment. at this purpose, 40 asthmatic children aged 3 to 6 years were selected: at a first examination (t0) all the patients showed basal value fot at 6hz frequency (rr6) changes major or equal than 35% under 200 mcg of salbutamol treatment. 30 days before the study, children selected didn't take systemic and inhaled steroids, cromons, leukotrienes antagonists, teophylline or anti-histamines and they didn't present upper and lower airways infections; then, two weeks before run-in, all selected patients took beta-2-agonists at least 3 times per week. all the patients were divided in two groups: group i (20 children) under inhalation of flunisolide 40mcg/kg/dose + salbutamol 1500 mcg b.i.d. for 7 days and, for the following two weeks, under inhalation of flunisolide 20 mcg/kg/dose + salbutamol 200 mcg by pmdi+spacer prn.; group ii (20 children) under inhalation of budesonide 0, 5 mg b.i.d. + salbutamol 1500 mcg b.i.d. for 7 days, and for the following two weeks, under inhalation of budesonide 0, 25 mg b.i.d. + salbutamol 200 mcg by pmdi+spacer prn. moreover, we gave diary-card with a simple symptoms score (at t0 and t7 ), to get the real perception of the symptoms in the course of study. however, during the treatment, 4 children have ritired from the study. the results obtained were statistically analysed (t student's test). significant rr6 pre beta-2-agonists and ? value reduction was obtained at t7 and t21 vs. t0. group i had (at t7) such a quick action than group ii reducing airways resistances (p<0, 01). children treated with flunisolide had lower frequency of breathless and nocturnal cough at t7 and lower salbutamol use from t7 to t21 than budesonide group (p=n.s.). in conclusion, fot is actually a safety method to value efficacy of inhaled steroids in preschool-aged children with asthma. introduction: approximately 7.5% of u.s. adults and 6.0% of louisiana (la) adults have current asthma, and over 25% currently smoke cigarettes, according to the 2002 behavioral risk factor surveillance system (brfss). the purpose of this study is to compare asthmatic smokers vs. nonsmokers in la and the u.s. here we address whether asthmatic smokers utilize more medical care services in the er and outpatient clinics, and whether they were able to fully participate in activities of daily living compared to nonsmoker asthmatics. methods: brfss is a state-based, telephone survey of u.s. adults. the survey collects self-reported information about modifiable risk factors for chronic diseases. this study analyzed data from the 2002 brfss survey using sas software. data: out of the current asthmatics, 25% are cigarette smokers in the u.s. in louisiana, 31% of asthmatics are currently smoking, which is significantly higher than the national average (p<0.0001). within the past year, the average number of emergency room visits due to asthma was similar for both locations (la 0.53, us 0.48, p=0.46) . nationally, smokers visited the er significantly more frequently than nonsmokers (s 0.61, ns 0.43, p<0.01). in la, smokers visited the er twice as often as nonsmokers (s 0.80, ns 0.40, p<0 .01) the number of annual routine outpatient checkups for asthma was similar based on location (la 1.39, us 1.66, p=0.06). smokers and nonsmokers had a similar number of checkups nationally (s 1.74, ns 1.64, p=0.32) and in louisiana (s 1.79, ns 1.21, p=0.06). the number of days where asthmatics were unable to perform their usual activities was significantly fewer in la than nationally (la 3.6, us 11.6, p<0.001). smokers had a significantly higher number of missed days nationally (s 17.8, ns 9.6, p<0.0001). in la, there was not a significant difference in the number of missed days based on smoking status (s 4.9, ns 2.9, p=0.47). conclusion: even though louisiana has a lower prevalence of asthmatics compared to the national average, significantly more of our asthmatics are smokers. asthmatic smokers in la and the u.s. utilized the er more than their nonsmoking counterparts. smokers and nonsmokers had similar numbers of routine outpatient visits. asthmatic smokers missed more days, but this was only significantly different at the national level. other research has shown that young age is highly associated with improper use in children using a mdi and holding chamber (arch pediatr adolesc med 2002; 156:378) . the objective of this study was to compare health outcomes achieved by children with asthma using inhaled corticosteroids (icss) delivered by a nebulizer compared with outcomes of children using icss delivered by non-nebulized device. methods: using a managed care organization database (pharmetrics integrated outcomes database), we identified children aged 8 years with an asthma diagnosis and asthmarelated hospitalization/emergency department (ed) visit (july 2000 -june 2002 and with a prescription claim for an ics within 30 days of discharge. we compared relative risk of hospitalization/ed recurrence from day 31-180 (cox proportional hazards regression, covariates=sex, age, current and prior asthma medications, prior oral corticosteroid and short-acting 2 -adrenergic agonist use, initial type of index event) for patients receiving nebulized ics vs other ics by age groups (0-4 years and 5-8 years). results: of 1552 patients with claims for ics, 729 received nebulized ics, of which 480 were aged 4 years; 823 received non-nebulized ics, of which 292 were aged 4 years. postindex hospitalization/ed rates were 12.4%. refill rate was higher for patients using nebulized ics. after model risk adjustment, patients using nebulized ics had a 53% risk reduction for hospitalization/ed recurrence vs those not using nebulized ics (hr: 0.47, 95% ci: 0.28, 0.78). in the 0-to 4-year age group, patients on nebulized ics had a 62% risk reduction compared with patients not using nebulized ics (hr: 0.38, 95% ci: 0.21, 0.69). in the 5-to 8-year age group, patients on nebulized ics had a 52% risk reduction (hr: 0.48, 95% ci: 0.16, 1.46). conclusion: in actual practice, treatment with nebulized ics after an asthma exacerbation is associated with a significant reduction of recurrent hospitalization/ed visits in young children, possibly due to improved technique and compliance. introduction: tenor is a 3-year, multi-center, cohort study of patients with severe or difficult-to-treat asthma. the objective of the tenor study is to better understand the natural history of patients with severe or difficult-totreat asthma. this analysis assessed the frequency of skin testing in this population and characterized the differences between the positive (st+) and negative (st ) subjects. methods: subjects were asked whether they had ever been skin tested and, if so, the test results. those who were st+ were compared to both st and those never tested (stnd) using clinical and other asthma-related characteristics. subjects 12 years of age were included in this analysis. results: 2985 subjects were eligible for this analysis. 85.8% were skin tested in the past, with stnd frequency of 5.1% from allergist sites and 33% from pulmonologist/other sites. of those tested, 93.5% were positive (allergist 95.7%, pulmonologist/other 87.3%). baseline ige for st+ subjects was 104.6 iu/ml vs. 32.4 iu/ml for st ; p<0.0001 (table) . as shown, the age at asthma onset, duration of asthma, rate of atopic disorders, and the rate at which asthma was triggered by aeroallergens differentiated the st+ from the st group. disease severity as evaluated by fev 1 , healthcare utilization, and medication use, however, was similar between the two groups. in general, stnd were more likely to have values closer to the st+ group, suggesting that the majority of those not tested would have been st+, if administered a test. conclusions: the prevalence of st+ subjects from both allergy and pulmonary practices was high, demonstrating that the majority of these severe or difficult-to-treat patients have allergic asthma. st+ patients showed differences in clinical characteristics compared to st , including a greater likelihood of their asthma symptoms being triggered by aeroallergens. these data also show that the clinical profile of stnd patients may be similar to that of st+ patients, suggesting the utility of a more universal allergic evaluation in severe asthmatics. funded by genentech and novartis pharmaceuticals corp. background current national guidelines for the diagnosis and management of asthma such as the naepp epr 2002, classify asthma severity based on frequency of asthma symptoms, medication use, and measurements of lung function by spirometry or peak expiratory flow variability. recently, the utility of spirometry measurements for assigning asthma severity has come into question. here we evaluate the correlation of spirometry measurements with asthma severity in school-aged children who are mostly naive to anti-inflammatory therapy. methods children were participants in a school-based lowincome asthma mobile van program, the breathmobile. recruitment was through referrals by school nurses and community public health clinics, parental response to flyers, and asthma screening questionnaires. spirometry was performed on all children greater than 4 years of age as part of a comprehensive asthma evaluation. asthma severity was assigned based on symptoms only, according to the naepp epr 2002 . results from april 2002 to april 2004 of the 620 children evaluated on the breathmobile, 540 patients were diagnosed with asthma. classification of severity by symptom frequency according to naepp epr guidelines resulted in 28% as mild intermittent (mi), 28% as mild persistent (mip), 32% as moderate persistent (mop), and 12% as severe persistent (sp). the percentage of patients and their mean lung functions at baseline evaluation without bronchodilator challenge are shown in the table. there were statistically significant differences (p<.05) between severity groups which was most pronounced for the fef25-75% when comparing the severe persistent group and the intermittent group. however, mean values appear to be "normal" for all degrees of asthma given current accepted cut-off values, fvc 80%, fev1 80%, fev1/fvc 80%, and fef25-75 60%. conclusion in our study, airflow impairment did correlate with asthma severity, although it was "normal" (fev1 80%) even in children with severe persistent asthma. therefore, a "normal" lung function measurement may be misleading as sole criteria for asthma severity classification. perhaps a higher cutoff point (fev1<90%) might be a better indicator of asthma severity in children. asthma is the most frequently chronic disease in childhood.our main was to know the prevalence of bronchial asthma in children (6-7 and 13-14 years) in the north zone of mexico city and to compare the prevalence obtained by the written questionnaire versus the video questionnaire in the group of 13 to 14 years, according to methodology proposed by isaac. material and methods: by means of a validated and standardized questionnaire (isaac) that was applied to 3500 children from 6 to 7 years and 3899 from 13 to 14. the questionnaires of 6 and 7 years were filled out by their parents. the group from 13 to 14 years answered a questionnaire and a video questionnaire. according to isaac specification and based on a studied population, it was calculated by statcal program the size of the sample. it was choose a haz-ardous sample of 3000 children between 6-7 years and 3000 of the 13-14 group years, both sexes in elementary and high school. measures of central tendency and chi2 were used. results: the final sample size for both groups was 7110 children (3211 of 6-7 and 3899 adolescents), 51.5% men and 48.5% women. the prevalence of the asthma diagnosis for teenagers was of 8 % (p<0.05 ic 7.1-8.7) and it was 4.5% in children (p<0.05 ic 3.8-5.2), wheezing in the last 12 months was 6.8% (p<0.05 ic 5.9-7.6) in children and of 9.9% (p<0.05 ic 9-10.8) for 13-14 years. wheezing induced exercise appeared in 13.1% (p<0.05 ic 12-14.1) in teenager and 3.4 % (p<0.05 ic 2.7-4) in the other group. the severity of asthma showed by nocturnal awakness was 4.3% and 2.6% (p<0.05), number of crisis in the last year 6.3% and 9.3% for children and teenagers respectively (p<0.05). in the video questionnaire of teenagers 7.3% (ic 6.5-8.1) answered affirmative on have displayed a shaken breathing on the last year resting and only 6.8% (ic 6-7.6) in the last month. about exercise question 16.5% confirmed have displayed symptoms, 14.6%(ic 13-15.8) in the last year and 11.1% (ic 10.1-12.1) in the last month. wide-awake at night 7.5% (ic 6.7-8.3) in the last year and 5.2% ) in the last month. conclusions: the prevalence of asthma by diagnosis was higher in teenagers group than in children group. the prevalence of asthma was higher also in this group. rationale: we investigated the relationship of pet exposure and healthcare utilization (hcu) in a cohort of pediatric patients with severe or difficult-totreat asthma in the epidemiology and natural history of asthma: outcomes and treatment regimens (tenor) observational study. we hypothesized that pet exposure would increase hcu due to increased airway inflammation. methods: tenor is a 3-year multicenter cohort study of patients with severe or difficult-to-treat asthma. children ages 6-12 were interviewed at baseline regarding asthma-related hcu in the previous 3 months. patients with pet exposure (ppe, n=370) were compared with patients with no pet exposure (pnpe, n=382). responses were analyzed using fisher's exact test. results: ppe were less likely to have severe asthma by physician assessment (32% vs. 40%; p=0.01). in addition, ppe were less likely to have an emergency room (er) visit (18% vs. 26%; p=0.006) or to have been hospitalized (7% vs. 12%; p=0.02) in the previous 3 months. more pnpe had an ige level >100 iu/ml (74% vs. 63%; p=0.001). ppe and pnpe were similar with regard to presence of allergic rhinitis and skin test positivity. compared with pnpe, fewer ppe with two or more pets had er visits and hospitalizations (table) . among ppe, there were no differences in ige levels or in asthma severity related to the number of pets. conclusions: pet exposure was associated with reduced hcu in the tenor pediatric cohort, with the most pronounced effect seen in reduced er visits and hospitalizations needed by those with multiple pets. ppe had significantly lower ige levels compared to pnpe, regardless of the number of pets. these data support the hypothesis that exposure to pets may paradoxically protect individuals from the development of severe asthma. alternatively, these data may reflect a self-selection in patients with severe or allergic asthma who are likely to avoid having a pet. however, there may be other confounding factors in families with pets that confer a protective effect on asthma severity. introduction: in asthma and other chronic obstructive airway diseases, phosphodiesterase 4 (pde4) is involved in the pathophysiology of the disease and is expressed abundantly in key inflammatory cells. inhibitors of pde4 are investigational, anti-inflammatory agents that prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, once-daily pde4 inhibitor with demonstrated in vitro and in vivo anti-inflammatory activity, which may translate into clinical efficacy in asthma therapy. this study examined the ability of roflumilast to exert direct or acute bronchodilatory activity in patients with mild to moderate asthma. methods: this was a double-blind, randomized, placebo-controlled, crossover study consisting of three treatment periods of one day each, separated by a 7-to 14-day washout period. during each treatment period, 15 patients with a forced expiratory volume in one second (fev 1 ) of 50% to 90% of predicted received either oral roflumilast 1000 î¼g, roflumilast 500 î¼g, or placebo. the fev 1 was measured twice prior to administration and periodically up to 6 h following treatment. after 6 h, patients inhaled 200 î¼g salbutamol from a metered-dose inhaler; fev 1 was measured 15 min later. adverse events, vital signs, and electrocardiogram results were monitored throughout the study. results: median baseline fev 1 was similar for the three treatment periods. both doses of roflumilast did not lead to statistically significant differences versus placebo in the time-averaged fev 1 over the first or up to 6 h after drug intake (p > 0.05). thus, there was no evidence of a direct or acute bronchodilatory effect with either single doses of roflumilast 1000 î¼g or 500 î¼g. in contrast, inhalation of the short-acting bronchodilator salbutamol led to a distinct improvement in fev 1 versus baseline in all treatments groups with median fev 1 increases of 19%, 21%, and 14% in the roflumilast 1000 î¼g, roflumilast 500 î¼g, and placebo groups, respectively. roflumilast was well tolerated. conclusions: oral, once-daily roflumilast exhibits no direct or acute bronchodilatory activity in patients with mild to moderate asthma as could be achieved with short-acting 2 -agonists. these data support the proposed mechanism of action of roflumilast as an antiinflammatory agent. south africa; 2. guadalajara, spain; 3. munich, germany; 4. weinheim, germany; 5. budapest, hungary; 6. konstanz, germany. introduction: phosphodiesterase 4 (pde4) is found in key inflammatory cells involved in the pathophysiology of chronic obstructive pulmonary disease and asthma. inhibitors of the pde4 enzyme are anti-inflammatory agents that prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, oncedaily pde4 inhibitor with demonstrated in vitro and in vivo anti-inflammatory activity. this study examined the dose-related efficacy of roflumilast in patients with asthma. methods: patients with stable asthma (forced expiratory volume in 1 second [fev 1 ] 50% to 85% of predicted) were enrolled in this randomized, double-blind, dose-range finding study. after a single-blind placebo run-in period of up to three weeks, patients received oral roflumilast 100 î¼g, 250 î¼g, or 500 î¼g once daily (n = 229, 228, and 233, respectively) for 12 weeks. efficacy was assessed by change from baseline in spirometric lung function fev 1 and forced vital capacity (fvc), as well as morning and evening peak expiratory flow (pef) recorded in patient diaries. safety and tolerability parameters were monitored throughout the study. results: treatment with roflumilast led to dose-dependent and statistically significant increases in fev 1 , fvc (both p < 0.0001), and in morning pef (p < 0.01). at last visit, fev 1 improved from baseline by 260 ml (11%), 320 ml (13%), and 400 ml (16%) in patients treated with roflumilast 100 î¼g, 250 î¼g, and 500 î¼g once daily, respectively. similarly, dose-dependent improvements of 10 l/min, 12 l/min, and 20 l/min in morning pef from baseline were reached. roflumilast was well tolerated at all dose levels tested. the most frequent drug-related adverse events were headache followed by gastrointestinal disorders such as diarrhea and nausea. there were no clinically relevant changes in vital signs, electrocardiogram, or laboratory parameters. conclusions: oral, once-daily roflumilast was associated with dose-dependent, clinically relevant improvements of lung function in patients with stable asthma. roflumilast was well tolerated. j.l. izquierdo 1* , e.d. bateman 2 , p. magyar 3 , u. harnest 4 , p. hofbauer 5 , a. varga 6 , c. schmid-wirlitsch 7 , d. bredenbroeker 7 , t.d. bethke 7 , 1. guadalajara, spain; 2. cape town, south africa; 3. budapest, hungary; 4. munich, germany; 5. weinheim, germany; 6. tatabanya, hungary; 7. konstanz, germany. introduction: phosphodiesterase 4 (pde4) inhibitors are a new class of anti-inflammatory agents for therapeutic use in inflammatory airway diseases. in several studies, the investigational, oral, once-daily pde4 inhibitor roflumilast has provided clinically meaningful improvements in patients with chronic obstructive pulmonary disease and asthma. this study examined the long-term safety and tolerability of roflumilast over 40 weeks in patients with asthma. methods: patients with persistent, stable asthma (fev 1 50% to 85% of predicted at randomization) participated in a double-blind, randomized dose-range finding study and were treated with oral roflumilast 100 î¼g, 250 î¼g, or 500 î¼g once daily for 12 weeks. patients completing the 12-week study per-protocol, then continued treatment in this open-label 40-week extension study. all patients received oral roflumilast 500 î¼g once daily during the extension period. adverse events (aes), clinical laboratory parameters, vital annals of allergy, asthma & immunology signs, and ecg were assessed throughout the extension period. results: a total of 456 patients were enrolled in the 40-week extension study. overall, the most common aes were related to the respiratory system. only a small number of patients (6%) experienced aes that were assessed as at least likely related to study medication. the most frequent drug-related adverse events were headache, diarrhea, and nausea as reported by 3%, 1%, and 1% of patients, respectively. most aes were mild to moderate in intensity and transient in duration; 6% of patients discontinued the study due to aes. most (60%) of these aes leading to discontinuation were assessed as not related or unlikely related to study drug. out of 456 patients, 16 reported serious aes, which were all assessed as not related or unlikely related to roflumilast. no clinically relevant changes in clinical laboratory parameters, vital signs, ecg, or physical examination occurred. conclusions: oral, once-daily roflumilast 500 î¼g administered for 40 weeks was well tolerated in patients with persistent, stable asthma. this study provides evidence that roflumilast is associated with a low incidence of aes, which are mostly mild to moderate in intensity and transient in nature, thus, supporting the potential therapeutic use of roflumilast in asthma. paris, france; 3. madrid, spain; 4. guadalajara, spain; 5. harrow, united kingdom; 6. weinheim, germany; 7. augsburg, germany; 8. munich, germany; 9. kaufbeuren, germany; 10. konstanz, germany. introduction: in asthma, inhaled corticosteroids (ics) have been the mainstay of maintenance treatment. new therapeutic agents are needed that target key inflammatory processes in asthma as effectively as ics but overcome known limitations of ics. phosphodiesterase 4 (pde4) inhibitors are anti-inflammatory agents that prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, once-daily pde4 inhibitor for potential use in anti-inflammatory asthma therapy. this study compared the standard corticosteroid treatment of twice-daily, inhaled beclomethasone dipropionate (bdp) with oral, once-daily roflumilast in patients with asthma. methods: in a randomized, double-blind, double-dummy study, patients (forced expiratory volume in one second [fev 1 ], 50% to 85% of predicted) received either oral roflumilast 500î¼g once daily (n=207) or inhaled bdp 200î¼g twice daily (400î¼g/day; n=214) for 12 weeks. mean change (lsmean and sem) in lung function parameters fev 1 , forced vital capacity (fvc), and morning and evening peak expiratory flow (pef) from baseline was determined after 12 weeks of treatment. further, symptom score and rescue medication use were assessed. results: both roflumilast 500î¼g and bdp 400î¼g improved fev 1 from baseline to clinically meaningful levels (300 â± 40ml and 370 â± 30ml, respectively; both p<0.0001). similarly, roflumilast and bdp improved fvc (300 â± 40ml and 360 â± 40ml, respectively; both p<0.0001) as well as morning and evening pef (p 0.0003). roflumilast 500î¼g was statistically non-inferior to bdp 400î¼g. roflumilast and bdp led to statistically significant and comparable improvements in asthma symptoms and use of rescue medication. adverse events were generally mild to moderate; the most common drugrelated adverse events reported in the roflumilast group were nausea, headache, and diarrhea. there were no clinically relevant changes in vital signs, ecg, or laboratory parameters. conclusions: roflumilast 500î¼g provided clinically meaningful improvement of lung function parameters, reduced asthma symptoms, and decreased the need for rescue medication. oral, once-daily roflumilast was as effective as inhaled, twice-daily bdp in the treatment of asthma. oral roflumilast 500î¼g was well tolerated. rationale: evaluation of medical claims from commercial health plans permits assessment of therapeutic interventions on resource utilization. this study investigated the impact of controller therapy in children starting asthma maintenance medications. the risk of obtaining prescriptions for oral corticosteroids (ocs), short-acting beta-agonists (saba) or adding another asthma therapy was evaluated. methods: this was a retrospective observational study utilizing medical and pharmacy claims from a large representative managed care plan from 1/1/2000-5/31/2003. children aged 4-17 years old with an asthma diagnosis (icd-9, 493.xx) and an initial pharmacy claim for one of the following regimens: fluticasone/salmeterol in a single inhaler, n=1, 168 (fsc), fluticasone propionate alone, n=2, 473 (fp), montelukast, n=4, 246 (mon), any inhaled corticosteroid plus montelukast, n=1, 009 (ics+mon), and an ics plus salmeterol from separate inhalers, n=296 (ics+sal) were included in the analysis. subjects were excluded if they had received any asthma controller medication in the 6 months prior to the initiation of therapy. regression was used to estimate the incidence rate ratio (irr) for ocs and saba use (negative binomial) and the odds ratio (or) of adding a controller (add) and an ed or hospitalization (ip) event (logistic). all models controlled for demographics, pre-controller asthma-related medications and events, and baseline total health care costs. results: the ratios in the table with a confidence interval excluding unity indicate significantly increased use or likelihood of an event compared to the fsc cohort. controller naã¯ve children started on fsc were less likely to add another controller than mon, fp or ics+sal, and had less use of ocs or saba than the 4 cohorts when studied for 12 months after the initiation of controller therapy. in addition, children treated with ics + mon had a significantly greater or of an ed/ip visit compared to fsc. conclusion: use of fp + sal (fsc) in a single inhaler assures that children are getting more optimal inhaled corticosteroid therapy as well as the benefits from the long acting bronchodilator while avoiding the potential for selective discontinuation of ics in the 2 multiple controller cohorts. a.s. nayak 1* , r. nathan 2 , j. williams 3 , s. kundu 3 , m. lloyd 3 , d. banerji 3 , 1. normal, il; 2. colorado springs, co; 3. bridgewater, nj. introduction: inhaled corticosteroids (ics) are recommended firstline therapy for severe, persistent asthma. systemic exposure to ics may suppress hypothalamic-pituitary-adrenal (hpa)-axis function, particularly at doses required to control severe asthma. ciclesonide (cic) hydrofluoroalkane (hfa) metered dose inhaler (mdi) is a novel and effective ics that is converted in the lungs to its active metabolite, desisobutyryl ciclesonide (des-cic). previously, in short-term studies, cic has been shown to have no effect on serum or urinary cortisol levels, which may be attributed to the low oral bioavailability, high serum protein binding and high clearance rate of cic and its active metabolite. this hpa axis analysis was part of a long-term study evaluating the safety of cic and beclomethasone dipropionate (bdp) hfa-mdi in patients with severe persistent asthma. methods: this was a multicenter, double-blind, parallel-group, 12-month extension of a 12-week double-blind trial of patients 12 years with severe persistent asthma. patients were randomized in a 2:1 ratio (cic:bdp) to receive cic (320 g bid) exactuator or bdp (320 g bid) ex-actuator. after 2 weeks, the doses of both medications could be titrated to 160 g bid as needed for asthma control. hpa-axis function was assessed at selected centers in a subset of patients at baseline and end of study (at month 12 or early termination) by measuring both peak serum cortisol induced by low dose (1-î¼g) cosyntropin and 24-hr urinary free cortisol corrected for creatinine. results: data were available for a small number of patients at 13 centers evaluating hpa-axis (table) . mean baseline levels and change from baseline to end of study values in low-dose cosyntropin peak serum cortisol levels for cic and bdp were comparable. likewise, baseline levels and mean change from baseline to end of study values in 24-hr urinary free cortisol corrected for creatinine were comparable among the two treatment groups. conclusion: these findings demonstrate that cic 320 to 640 î¼g daily for 12 months is safe and has no significant effect on hpa-axis function. a. long 1* , a. rahman 2 , 1. boston, ma; 2. wilmington, de. introduction: little information is available regarding the relationships between asthma severity, disease control, and compliance with medications. methods: physicians' perceptions regarding these variables were determined by internet-based general asthma medication usage survey and patient-specific asthma medication usage surveys between december 18 and 29, 2003, for 406 physicians (100 pcps, 106 pediatricians, 100 allergists and immunologists, and 100 pulmonologists). the patient-specific surveys were based on chart information of 3 randomly chosen patients per physician on controller medication. results: of 1218 patients, 12%, 31%, 43%, and 14% were categorized by their physicians as having mild intermittent or mild, moderate, or severe persistent asthma, respectively. overall, 45% of patients were categorized as having "very controlled" asthma and 58% as being "very compliant" with their current regimen. worse asthma control, decreased compliance, and increased physician visits were all associated with increased asthma severity classification. physicians reported that only 5% of patients with severe persistent disease (n=169) had "very controlled" asthma in contrast to 77% of patients with mild intermittent asthma (n=143). patients with mild intermittent asthma, and mild, moderate, and severe persistent asthma, had a mean of 2.9, 3.4, 4.5, and 5.5 physician visits for asthma, respectively, within the past year. physician perception of patient compliance varied less across the groups, with 55% of patients with severe persistent asthma and 66% of patients with mild intermittent asthma being rated as "very compliant." the incidence of allergic rhinitis was similar among all severity levels (range: 50%-56%), but patients with severe persistent asthma were more likely to have comorbid conditions, including hypertension, chronic obstructive pulmonary disorder, chronic bronchitis, and osteoporosis, compared with patients in other asthma severity levels. conclusions: decreased asthma control and rates of com-pliance, as well as increased physician visits and comorbid medical conditions, may be associated with increased asthma severity classification. rationale: children with asthma frequently have co-morbid allergic conditions requiring medications. the purpose of this study was to assess whether the controller asthma medication selected would reduce the utilization of intranasal steroids (ins) and prescription nonsedating antihistamines (nsa) for children treated for co-morbid allergy. methods: this was an observational retrospective study that utilized the pharmetrics integrated outcomes database that contains administrative medical and pharmacy claims data from over 30 managed care plans across the united states. children age 4-17 years old with a new diagnosis of asthma (icd-9, 493.xx) were identified and observed for 12 months after their initial diagnosis and treatment of asthma (post-index). four cohorts were established based on their controller medication dispensed: montelukast, n=1906 (mon), fluticasone propionate, n=2455 (fp), fp+mon n=287, fp/salmeterol in a single inhaler, n=599 (fsc). results: baseline comorbid diagnosis of allergic rhinitis was observed in 14-20% of the children. the patterns of use of ins and nsa were similar in the 12 months before and the 12 months after the initial asthma diagnosis and treatment. children with a diagnosis of allergic rhinitis were nearly twice as likely to receive a nsa or ins. all 4 treatment cohorts used a similar amount of each allergy medication. the presence or absence of a diagnosis of allergic rhinitis did not alter the findings. the adjusted odds ratio (95%ci) of using a nsa or ins relative to the use of fsc in the post-index period was mon: 0.989 (0.809, 1.208); fp: 0.828 (0.682, 1.007); fp+mon: 1.095 (0.804, 1, 489 ). the table below shows the percent of children dispensed a nsa and/or an ins. the number of units of each allergy medication was also similar across all 4 cohorts. conclusion: the use of mon, fp, fp+mon or fsc for asthma did not alter the rate or quantity of either intranasal corticosteroid or nonsedating antihistamines dispensed to children over the 12-month period. although allergic rhinitis is a common comorbidity of pediatric asthma, the selection of a regimen containing montelukast did not reduce the use of either nsa or ins compared to asthma treatments with fp or fsc. rationale: medication compliance is recognized as a significant challenge in pediatrics.the purpose of this study was to compare inhaled corticosteroid (ics) persistence in pediatric patients using a single inhaler, containing both an inhaled corticosteroid (fluticasone propionate, fp) and an inhaled long-acting beta-agonist (salmeterol, sal) (fsc), to patients receiving fp alone, fp + sal from separate inhalers, or ics + montelukast (mon). methods: retrospective 24-month pre-post database study utilizing medical and pharmacy claims. a total of 4946 subjects 4-17 years of age with a diagnosis of asthma (icd9 493) were grouped into 4 cohorts: fsc [n=1168]; fp only [n=2473] ics+sal [n=296]; and ics+mon [n=1009]. patients were controller naã¯ve for 6 months prior to the index event (the first prescription of the medication of interest). subjects were required to have continuous enrollment of 6 months pre-and 12 months postindex. subjects with cystic fibrosis, copd, bronchopulmonary dysplasia or respiratory distress syndrome were excluded. ics refill rates, as a measure of persistence, were compared between fsc and the ics components of the other cohorts over a 12-month follow-up period. results: mean refill rates over the 12-month follow-up period was significantly greater (p=< 0.05) for fsc (4.48) compared with the mean ics refill rate in the fp alone (3.39) cohort, the ics + sal (3.61) and the ics + mon (3.92) cohort. patients on fsc filled 32% more ics prescriptions than patients on fp alone, 24% more than ics+sal and 14% more than ics + mon. mean refills were generally higher than the median fills as a considerable number of children had only one fill: fsc (35.6%), fp (49.1%), ics (48.3%) + sal (50.0%), ics (36.0%) + mon (18.6%). conclusion: patients using fsc, are likely to fill their inhaled corticosteroids more often over 12-months compared with patients using ics + sal in 2 separate inhalers, ics+mon, and fp as monotherapy. improved persistence with ics has been shown in other studies to decrease morbidity and mortality of asthma. use of fsc, a single inhaler, assures that children are getting more optimal inhaled corticosteroid therapy as well as the benefits from the long acting bronchodilator while avoiding selective discontinuation of ics that may occur when two medications are dispensed. the national health interview survey (nhis) estimates 14.5 million united states residents with asthma. noncompliance with treatment has been estimated as between 20 and 80%. the consequences of noncompliance include absences from work or school, increased emergency room visits, more severe attacks, increased drug side effects, greater cost of care, and death. we questioned compliance in a 47yo man with severe, prednisone dependent eosinophilic asthma because he had persistent symptoms despite treatment with fluticasone propionate/salmeterol xinafoate 500/50 bid, prednisone 20mg to 40mg qd, and budesonide (200 mcg/puff) 2 puffs bid. after discontinuation of budesonide and prednisone and initiation of methylprednisolone 16mg bid for 24 hours and then 16mg qd and beclomethasone dipropionate 2 puffs (80mcg) hfa bid with a spacer, clinical improvement was unexpectedly abrupt. fev1 increased from 58% to 90% of predicted. sputum eosinophil counts decreased from 78% to 2%. because of the sudden improvement with the change to methylprednisolone, compliance to prednisone and fluticasone was questioned. to evaluate compliance, the patient's blood, urine, and sputum were tested for synthetic corticosteroids, without his knowledge, using mass spectrometry. the blood level of methylprednisolone was 2.6mcg/dl and prednisolone was 0.35mcg/dl, documenting use of methylprednisolone and the recently discontinued prednisone. the urine levels were 13mcg/dl of methylprednisolone, 1.3mcg/dl of prednisolone and 0.74mcg/dl of prednisone that further confirmed recent use of prednisone. the sputum testing revealed 0.8mcg/dl beclomethasone, 33mcg/dl fluticasone and 1.8mcg/dl methylprednisolone confirming compliance to inhaled fluticasone and beclomethasone at the time of the test. thus, contrary to our clinical suspicion, the patient was indeed compliant with his inhaled glucocorticoids and prednisone and subsequently with the methylprednisolone. to our knowledge, this is the first case report to document compliance to inhaled and oral corticosteroids by analysis of synthetic corticosteroid concentrations in blood, urine, as well as sputum. if compliance could be documented with certainty, it could potentially minimize the adverse effects of needless escalating steroid doses, reduce the cost of treatment, and minimize morbidity and mortality as well. rationale:the burden of pediatric asthma extends beyond those children with an asthma diagnosis. children with wheezing frequently have a delay in the diagnosis of asthma that may impede instituting appropriate therapy and reducing disease morbidity. this observational study was designed to assess the treatment patterns of children 4-17 years old receiving asthma medication(s) without a diagnosis of asthma compared with children diagnosed with asthma and children without claims for asthma. methods:this was a retrospective cross-sectional study that utilized the pharmetrics integrated outcomes database containing administrative medical and pharmacy claims data from over 30 managed care plans across the united states. three cohorts were selected: children with an asthma diagnosis (icd-9, 493.xx) (dx cohort), children with prescription claims for asthma controllers or rescue medications without an asthma diagnosis (rx cohort) and children with neither an asthma diagnosis nor prescription claims for asthma medications (control cohort). utilization of asthma medications, asthma specific costs and total costs of care were assessed. results: a total of 295, 000 children were identified: 6.7% in the dx cohort, 4.4% in the rx cohort and 88.9% in the control cohort. the potential asthma cohort was 11.1%. total annual non-asthma related costs for the dx cohort was $1, 642 for the rx cohort was $1, 306 and for the control was $802. only 19% and 5% of the total healthcare charges in the dx and rx cohorts retrospectively were asthma related. non-asthma charges were significantly higher in both the dx and rx cohorts compared with the control cohort. in the rx cohort, the ratio of saba prescription claims to asthma controller claims were 1.85-fold higher than in the dx cohort. conclusion: children treated with asthma medications without an asthma diagnosis consume greater health care resources resembling the pattern of health care utilization of children with an asthma diagnosis more closely than controls. further, children with treatment in the absence of an asthma diagnosis appear to be under-treated with controller therapy as recommended by national and international guidelines. background: a recent retrospective study assessed asthma variability in inner-city patients 6 months before and 6 months after enrollment into an naepp guidelines-directed clinical management and educational program. while guidelines-directed care improved asthma symptoms and outcomes, significant variability in disease indices were observed over the 6-month period. the present analysis evaluates resource utilization associated with this variability. method: economic end points, including hospital/emergency department (ed) visits, total unscheduled office visits, sick visits, and days lost from work or school, were collected from 125 inner-city patients aged 18 years (74% female, 69% minority, 80% treated by primary care physicians) enrolled in a guidelines-directed asthma clinical management and education program aimed at minimizing barriers to adherence. patients were stratified into 2 groups: those with high variability in asthma (n=62) and those with low variability in asthma (n=63), with variability defined as number of fluctuations in naepp symptom class in the 6-month postintervention period (high variability = patients with fluctuations higher than the mean; low variability = patients with fluctuations lower than the mean). results: guidelines-directed therapy was associated with improvements in asthma during the 6-month treatment period for both groups. patients in the high-variability group had more ed visits, sick visits, total unscheduled visits, and office visits compared with patients in the low-variability group (see table) . conclusions: despite guidelines-directed therapy and overall improvement in asthma control, patients experienced variability in asthma, indicating that even when their disease is stable, their symptoms continue to fluctuate. as a result, asthma variability may contribute to increased resource utilization. introduction: inhaled corticosteroids (ics) are considered first-line therapy for patients with persistent asthma. ics can lead to oropharyngeal adverse events, which may affect treatment outcomes and adherence. the occurrence of these local adverse events is dependent upon several factors, including dosage and duration of ics treatment. ciclesonide (cic) hydrofluoroalkane (hfa)-metered dose inhaler (mdi) is a novel and effective ics, with relatively low oropharyngeal deposition. cic is converted in the lungs to its active metabolite, desisobutyryl-ciclesonide (des-cic). furthermore, cic undergoes limited conversion in the oropharynx, which may account for its improved safety profile. this oropharyngeal safety profile analysis was part of a long-term study evaluating the safety of cic hfa-mdi vs beclomethasone dipropionate (bdp) hfa-mdi in patients with severe persistent asthma. methods: this was a multicenter, double-blind, parallelgroup, 12-month extension of a 12-week double-blind study of patients 12 years with severe persistent asthma. patients were randomized in a 2:1 ratio (cic:bdp) to receive cic (320 g bid) or bdp (320 g bid) (both ex-actuator). after 2 weeks, the doses of both medications could be titrated to 160 g bid as needed for asthma control.) oropharyngeal adverse events were monitored, and suspected oral fungal infections were verified by positive culture. results: the incidence of oral candidiasis was lower in subjects receiving cic (4.1%), compared with those receiving bdp (10.4%) ( table) . the incidence of pharyngitis and hoarseness was 3.6% and 2.5%, respectively, for the cic group, and 5.2% and 1.0%, respectively, for the bdp group. all local adverse events resolved without sequelae, and there were no withdrawals due to oropharyngeal treatment-emergent adverse events. conclusion: after 12-months of treatment with cic 160 î¼g or 320 î¼g twice-daily, the incidence of oropharyngeal adverse events was low. cic treatment resulted in a much lower incidence of oral candidiasis, compared with similar doses of bdp, reflecting a better local tolerance. objective: to determine the prevalence of asthma and asthma-related morbidity, treatment and asthma-risk factors in a selected population. method: a routine health screening, including tests for asthma, was conducted at the new orleans center for creative arts high school. participants (204) identified their medications and asthma tools in addition to completing the isaac questionnaire. prevalence data included lifetime wheezing, 12 month wheezing, and previous asthma diagnosis. asthma-related morbidity included sleep disturbance, wheezing with exercise, asthma attack rate and night awakening. results: " participants were aged 13-9 years (50.5% african american, 45.1% white) with 31.4% male, 68.6% female. " cumulative asthma prevalence was 18.8%, lifetime wheezing (24%), 12-month wheezing 12.7% with girls reporting 3.36 times more often (95% ci: 1.54, 7.36), and wheezing more than 4 times in 12 months 4.7%. " there was a significant association between race and wheezing in the last 12 months (p < 0.024) with whites reporting 3.78 times more often (95% ci: 2.01, 7.10). asthma morbidity was reported as follows: a. night cough -10.5% b. wheezing with exercise -10.9% " parents with a less education (high school or less) were 8.33 times more likely (95% ci: 7.46, 9.30) to have children who developed wheezing in the past 12 months. " higher body mass index (bmi = 25) was associated with wheezing after exercise (p < 0.008) with a 3.27-fold increase (95% ci: 2.46, 4.31). " among those with current asthma (15/204), 4 were not on any medication, 10 were using bronchodilators, and 5 were on anti-inflammatory medications. three reported using a spacer and 6 reported using a peak flow meter. conclusion: asthma prevalence is higher in this school population than the national average. less parental education, female gender, and bmi = 25 were associated with greater asthma morbidity. the majority of the participants with current asthma reported receiving episodic and inadequate treatment with inhaled corticosteroids. objectives: to determine the prevalence of asthma and asthma related symptoms; to assess its severity among new orleans school children. methods: seven elementary, middle, and high schools in new orleans participated in the screening of 1511 students, 54% female, 45% male, ages 5-18 in the fall, 2000. the 8-item questionnaire was designed by the international study of asthma and allergies in childhood (isaac) to determine asthma prevalence and severity in children. the isaac questionnaire is a validated protocol used on over 500, 000 children in 56 countries. asthma is defined as cur-rent wheezing for the purposes of this study. data was entered using spss 10 and sas 8 software for analysis. results: table: presence of asthma symptoms by ethnicity conclusion: asthma prevalence is significantly higher in inner-city school children in new orleans when compared to the national prevalence (24.6% to 26.4% vs. 7.5%). asthma prevalence and severity do not differ significantly between african-american and caucasian children in new orleans even after removing the effect of gender and age. video is an effective method of teaching pollen identification. projected digital images offer three-dimensional views of unique pollen detail similar to focusing a microscope. the national allergy bureauâ�¢(nab) currently provides pollen counts to the media. these reported levels are obtained by standardized counting methods rather than forecasts based on historical pollination predictions and weather patterns. the american college of allergy, asthma and immunology and the american academy of allergy, asthma and immunology have an interest in ensuring consistent counting and accurate reporting practices. both organizations offer pollen identification training in conjunction with their annual meetings and have used the video for initial training sessions and for experienced counters in advanced courses. there are 112 certified pollen counting stations in the united states and canada with approximately 80 currently active locations. the nab requires the demonstration of ability to count and accurately identify pollen on test slides. an updated recertification process using an interactive web site is currently being developed. ongoing pollen identification training is essential for the continued success of this aeroallergen network. most observers found that the video provided a quality emulation of microscopic viewing and enhanced the depth and detail of individual pollen characteristics. the video received the highest possible ratings by course attendees. in conclusion the video plays a positive role in pollen identification training and the continuing education of experienced counters. the diagnosis and treatment of mold allergies are complicated by the genetic diversity of individual fungal species and the influence of endogenous fungal proteases on extract compositions and potencies. studies examining the biochemical comparability of mold extracts from different sources and their compatibility with other allergens in immunotherapy mixtures are essential to the clinical effectiveness of these products. compositional comparisons of extracts prepared from alternaria, aspergillus and penicillium source materials by 7 u.s. allergen manufacturers revealed variable sds-page protein banding patterns and noticeable differences in total protein and carbohydrate concentrations. alt a 1 levels in alternaria extracts did not correlate closely with total protein levels or with ige-binding potencies measured by elisa inhibition. immunoblot profiles of fungal extracts were more closely related to one another compared to sds-page patterns, suggesting that similar allergenic or antigenic epitopes may be retained in molecules of varying size. parallel elisa inhibition dose-response curves provided statistical evidence that a repertoire of ige epitopes is conserved in many of these products. variations in the potencies of fungal extracts from different manufacturers may result from differences in source materials and/or conditions of extraction and storage. the stability and compatibility of allergen mixtures containing mold extracts were assessed after storage for up to 6 months at 2-8â°c using specific immunoblot and elisa procedures. grass and mite allergens compromised by mixing with mold extracts were stabilized by glycerin. alternaria extracts from different sources produced similar effects on grass allergens when added at comparable strengths. degradative effects caused by aspergillus or penicillium products were more closely related to total fungal protein concentrations than to extraction ratios. structural epitopes on cat, ragweed and fungal allergens were stable after mixing with protease-containing mold extracts, indicating the presence of natural protease inhibitors or allergenic protein sequences distinct from those recognized by these enzymes. allergenic extracts labeled as weight/volume or by pnu have no regulatory requirement for determination of activity. alum-adsorbed allergenic extracts are labeled in pnu and are felt to be depot formulations. the purpose of this study was to develop methods to measure important allergen proteins and specific ige binding capability of these alum-adsorbed allergenic extracts and to begin to assess the potency of these non-standardized extracts. allergen proteins are adsorbed to alum particles making their measurement difficult. in this study allergens were released from center-al â® alum precipitated allergenic extracts using 0.3 m citrate buffer, ph 5. the major allergen contents of several lots of each product were measured using monoclonal antibody elisas for group 5 grasses, cyn d 1 bermuda, bet v 1 birch, ole e 1 olive, and pla l 1 english plantain. these assays were developed and validated by alk-abello and optimized for the citrate releasing buffer. direct binding ige was performed by immobilizing serial dilutions of extract to microtiter plates and then adding specific atopic sera and enzyme labeled anti-ige. the methods used were able to measure in vitro allergen activity in the alum-adsorbed extracts. major allergen content was successfully measured in the extracts and the amount was related to the pnu content. as expected, the variability of the major allergens within a particular extract species was higher than in standardized extracts that are adjusted for potency. the extracts demonstrated specific ige binding ability in the binding assay. the ige binding capability at various dilutions was related to the major allergen content. in conclusion, methods were developed to release adsorbed allergen proteins from alum extracts. the extracts were then analyzed for major allergen pro-teins and the ability to bind ige. major allergen content is currently used to monitor the activity of aqueous and glycerinated extracts and this study demonstrates that implementing these testing procedures will improve the consistency and quality of alum-adsorbed extracts. introduction there are over 5000 species of smut and rust fungi. ustilago maydis, or corn smut, is a basidiomycete fungus that infects ears of corn. it is responsible for a percentage of grain loss in the united states but eaten as a delicacy in mexico. it grows into large tumor-like structures, called galls, dispersing soot-like spores responsible for the common name, "smut." it is readily airborne and believed to be a causal agent for allergic rhinoconjunctivitis. "corn smuts" was added to the standard screening panel for patients presenting for evaluation of rhinitis in the region of middle tennessee. methods using greer laboratoriesâ�¢ corn smut extract 1:20 w/v, patients were tested by prick/puncture method with the hollister-stier quintipâ�¢ device. if negative, intradermal testing was performed. positive and negative controls were assessed. based on current national recommendations, wheals 3 mm greater than negative control were considered positive for prick/puncture testing and wheals 8 mm greater than negative control were considered positive for intradermal testing. results 105 patients were tested between november, 2003 and march, 2004 . 14/105 (13.3%) met criteria for prick/puncture positivity. 52/105 (49.5%) met criteria for intradermal positivity. patients prick positive to corn smut had the following prick positive tests: dust mites 12/14 (85.7%); cat 11/14 (78.6%); local grass mix 10/14 (71.4%); local tree mix 10/14 (71.4%); local weed mix 10/14 (71.4%), mold mix 8/14 (57.1%); cockroach 7/14 (50%); and dog 4/14 (28.6%). discussion allergy testing for aeroallergens is available to a limited number of antigens, of which, only a few are standardized. as time passes, relevant antigens are discovered and their importance further elucidated. this assessment reveals a significant presence of corn smut ige largely accounted for by intradermal positivity which may or may not reflect clinical sensitivity. these numbers are provided so that other clinicians may compare to prevalence in their geographical area. further studies are needed to determine the association of corn smut ige with clinical symptoms. h 1 antihistamines are the mainstay of therapy for allergic disorders, including skin diseases. the most common side-effect of marketed antihistamines is sedation, especially when the clinical symptoms require a higher dose than recommended, a condition commonly encountered in dermatological disorders. the present study describes the pharmacology of a new non-sedating h 1 antihistamine, r129160 (hivenylâ�¢). r129160 binds to the cloned human h 1 receptor with a similar affinity (ki: 19 nm) as the reference antihistamines cetirizine (ki: 50 nm) and loratadine (ki: 37 nm). in vivo, r129160 protects rats and guinea pigs from lethal shock, induced by compound 48/80 and histamine, respectively (ed 50 : 0.06 and 0.055 mg/kg respectively). the compound is at least as effective as cetirizine and loratadine. in rats, r129160 inhibits the histamine-and allergen-induced cutaneous reactions (ed 50 : 1.4 and 2.3 mg/kg) with a similar potency as cetirizine (ed 50 : 0.7 and 2.7 mg/kg) and loratadine (ed 50 : 1.8 and 1.6 mg/kg). even so, in guinea pigs the compound inhibits the histamine-and allergen-induced skin reactions (ed 50 : 0.14 and 1.2 mg/kg) to a similar extent as loratadine and cetirizine. however, in dogs r129160 is more potent in inhibiting the ascaris allergen-induced wheal reaction (ed 50 : 0.024 mg/kg) than cetirizine (ed 50 : 0.14 mg/kg) or loratadine (ed 50 : 0.20 mg/kg). r129160 fails to occupy central h 1 receptors in the guinea pig cerebellum up to 40 mg/kg, in contrast to loratadine (ed 50 : 2.2 mg/kg). in vitro and in vivo cardiovascular safety experiments indicate that r129160 lacks the intrinsic capacity to prolong the qt-interval, even at high doses. as such, r129160 (hivenylâ�¢) is characterized as a potent, non-sedating and cardiosafe h 1 antihistamine. it has been selected for further clinical development, mainly in the field of dermatology. the compound will be a suitable tool to explore the activity of a selective h 1 antihistamine in various indications, without the contamination of the sedative activity often observed with other marketed antihistamines when increasing the dose. ciu represents one of the most clinically perplexing disorders which the allergist-immunologist is faced with. we have previously reported the clinical efficacy of fxt (prozac), an ssri widely used for the treatment of depression, in the management of ciu (nsouli tm, et al. ann allergy asthma immunol 2002; 88:60) . in this presentation we report 2 additional cases of ciu which responded dramatically following the use of fxt. a 41 yr-old female and a 21 yr-old male presented with ciu of 18 and 24 months duration, respectively, requiring oral corticosteroid (cs) therapy for control of their ciu after failure of high dose anti-h1 (hydroxyzine) and anti-h2 (ranitidine) agents (table) . testing for food and latex allergy, viral hepatitis, autoimmune thyroid disease and parasitic infection were all negative. a skin biopsy performed in subject #1 was consistent with an urticarial vasculitis. following one week of oral fxt (40 mg qd [subject #1] 20 mg qd [subject #2]) a striking and complete resolution of urticaria in each patient was observed within 7 to 10 days during which time it was possible to successfully taper the cs. both patients have been maintained on fxt therapy with complete control of their ciu. the very favorable response to fxt therapy in these 2 patients in addition to our first case report suggests that this drug may have a new therapeutic application in the management of recalcitrant ciu. background: ipratropium bromide nasal spray (ib) is indicated for treatment of rhinorrhea caused by common cold (cc), seasonal and perennial allergic rhinitis (ar) in adults and children age 5 and up. symptoms of rhinorrhea from cc or ar in children are similar to those in adults, yet there is little data on the use of ib in children under 5 years of age. objective: evaluate the safety and efficacy of ib nasal spray 0.06% in 2-5 year-old children with symptoms of rhinorrhea from cc or ar. methods: a total of 230 children (43 cc and 187 ar) were treated in an open-label, multi-center study. the cc patients received ib nasal spray (84 mcg per nostril) tid for 4 days, the ar patients received ib nasal spray (42 mcg per nostril) tid for 14 days. effectiveness was measured using a global assessment questionnaire and daily symptom scores reported by the parent. results: from the global assessment questionnaire, 91% and 90% of the parents found ib either "very useful" or "somewhat useful" in the cc and ar groups respectively. regarding effectiveness, 98% (cc) and 87% (ar) of the parents reported that ib had either a "good effect" or "excellent effect" in treating rhinorrhea. moreover, 67% (cc) and 91% (ar) of parents found administration of a nasal spray either "extremely easy" or "very easy." eighty-one percent (cc) and 89% (ar) of the parents reported they would use ib again for their child's allergy symptoms. the daily symptom score (0 = none to 5 = unbearable) for rhinorrhea decreased from 2.9 to 1.3 (-55%, p<0.0001) for cc and from 2.1 to 1.1 (-48%, p<0.0001) from baseline compared to the average on-treatment score, with decreases also seen for stuffy nose and sneezing. the nasal spray was well tolerated with adverse events (ae) reported in 28% of cc and 35% of ar patients. the aes were mostly mild to moderate and no potentially systemic anticholinergic or serious aes were reported. conclusions: ib nasal spray 0.06% administered at a dose of either 42 or 84 mcg per nostril tid is an easy to use, safe, and effective therapy for control of rhinorrhea in children 2 to 5 years of age with common cold or allergic rhinitis. chronic idiopathic urticaria (ciu) represents one of the most clinically perplexing disorders which the allergist-immunologist is faced with. the pathogenesis of the condition derives from the release of potent vasoactive substances including histamine, and products of arachidonic acid metabolism, e.g., prostaglandins and thromboxanes formed by the action of the enzymes cyclooxygenase (cox) of which cox-2 is responsible for pseudoallergic and other inflammatory responses. although a number of therapeutic options exist owing to the plethora of mediators produced, treatment has focused largely on the use of h1 antihistamines and, unsurprisingly, at times without complete resolution of symptoms. in this presentation we report the successful use of a cox-2 inhibitor for the treatment of chronic urticaria. a 10 y/o white hispanic female presented with a 4 month history of chronic urticaria predominantly on the soles of the feet, with unknown precipitating factors, and fail-ure to respond to prior treatment with: cetirizine, steroids, benadryl, ebastine and epinastine. the patient received a blood transfusion 3 years ago and there was no prior history of allergies. skin testing revealed 3+ reactions to dust mite, pollen, cockroach, shellfish, 2+ reactions to corn, milk and ige = 262 iu (nv < 90 iu), aso = 400 ui (nv < 200 ui). after 2 weeks of treatment with rofecoxib (vioxx) 25 mg, and benadryl, the rash resolved. vioxx was continued for 2 more weeks until complete resolution of symptomatology. this case illustrates that the addition of a selective cox-2 inhibitor (i.e., rofecoxib) with an h1 antihistamine may be a more effective regimen for patients with ciu who fail to respond adequately to conventional therapy. introduction: elderly asthmatic patients whose symptoms are controlled by inhaled corticosteroid (ics) therapy may still have breathlessness on exertion. we randomized elderly asthmatic patients stabilized by medium-dose ics therapy into two groups, treated one group with medium-dose ics therapy plus montelukast, a leukotriene receptor antagonist, and the other with increased-dose ics therapy, and compared the effects of the treatment regimens on exercise tolerance. methods:the subjects were 24 patients with bronchial asthma (16 males and 8 females, 70.6â±3.9 years) stabilized by ics therapy (with fluticasone proprionate, fp; 400 mg/day) for three months or more with low peak expiratory flow (pef) rates (<80%predicted, variability<15%). they were randomly divided into two groups (12 patients each) to be treated with ics therapy plus montelukast (400 mg/day fp + 10 mg/day montelukast; m group) or with increased-dose ics therapy (600 mg/day fp; f group). pulmonary function tests, a six-minute walking test, respiratory gas analysis during incremental (15w/min) cycle ergometer exercise were conducted, and exhaled nitric oxide (no) levels were measured before and after two weeks of the study treatment. results: pulmonary function tests showed significant increases in maximal mid-expiratory flow (mmf) and forced expiratory flow at 50% vital capacity (v50) in the m group (p<0.01) but no significant changes in the f group. exhaled no levels decreased significantly in both groups (40.7â±13.9 to 27.5â±9.1 ppb in the m group and 53.1â±17.6 to 38.0â±10.4 ppb in the f group; p<0.01). the six-minute walking distance extended from 531â±64 to 575â±58 m in the m group and from 553â±60 to 570â±58 m in the f group.the peak oxygen uptake (peakvo2) increased significantly in the m group (%peakvo2/w from 76.2â±12.7 to 84.4â±14.6%; p<0.01) but not in the f group (%peak vo2/w from 76.2â±10.7 to 76.4â±10.3%). the peak exercise load also increased significantly in the m group (76.6â±22.0 to 96.1â±23.1 w; p<0.01) but not in the f group (81.2â± 16.2 to 82.2â±14.3w). conclusions: the results indicate that concomitant administration of montelukast is more effective than dose escalation of ics on exercise tolerance in elderly asthmatic patients under medium-dose ics therapy. e. meltzer 1* , y. luo 2 , l. shen 2 , z. guo 2 , c. schemm 2 , y. huang 2 , k. chen 2 , p. king 2 , r. nave 3 , d. banerji 2 , s. rohatagi 2 , 1. san diego, ca; 2. bridgewater, nj; 3. konstanz, germany. introduction: inhaled corticosteroids (ics) are first-line treatment for persistent asthma. ciclesonide (cic) is a novel and effective ics under development. freely circulating, unbound ics is available to cause systemic adverse effects, such as hypothalamic-pituitary-adrenal (hpa) axis suppression. hence, it is important to determine the free fraction of ics in plasma. in separate studies, the protein binding of the active metabolite of cic, desisobutyryl-ciclesonide (des-cic), was evaluated, and the effects of inhaled cic on hpa axis function were determined. methods: human plasma protein bind-ing of des-cic (0.5-500 ng/ml) was determined using equilibrium dialysis. dialyzed samples were analyzed by liquid chromatography with tandem mass spectroscopy to determine free and bound des-cic. in 3 separate clinical studies, the effects of cic (hfa; 320-1280 î¼g daily) and placebo (pbo), via metered-dose inhaler (ex-actuator doses), over 9 days, 4 or 12 weeks, on basal hpa axis function (24-hour area-under-the-curve [auc0-24h] serum or urinary cortisol corrected for creatinine) or stimulated (low-dose [1 î¼g] cosyntropin) serum cortisol were investigated in patients ( 18 years) with persistent asthma. results: the mean % of human plasma protein binding for des-cic was 99%. in 2 studies measuring serum cortisol auc0-24h, there was no difference between pbo and cic (320-1280 î¼g). similar results were observed for 24-hr urinary cortisol corrected for creatinine. in the 3rd study measuring low-dose (1 î¼g) cosyntropin-stimulated peak serum cortisol, after 12 weeks of treatment, there was no significant difference in the mean change from baseline versus placebo for either cic320 (p=0.383) or cic640 (p=0.911). conclusions: the favorable pharmacokinetic profile of cic, in particular the high protein binding of des-cic, may explain the lack of hpa-axis suppression. this appears to result in greater systemic safety. purpose: unscheduled office and ed visits for urgent asthma care are an ongoing point of concern. determining preventive variables regarding these unscheduled visits could have a significant impact on asthma costs and quality of life. objective(s): this research addresses the following question: when stratifying by race, gender, age, metropolitan/rural place of residence and comorbidity, do adults with asthma have fewer ed or unscheduled office visits for urgent asthma care if they: a) have an identified primary care provider, or b) have health insurance? method(s): univariate and stratified bivariate contingency table analyses were performed on weighted 2001 behavioral risk factor surveillance survey (brfss) data. result(s): adults with asthma who had an identified primary care provider were more likely to have no unscheduled office visits (or=1.87) or ed visits (or=2.07) for urgent asthma care. this was also true for adults with asthma who had health insurance (or=1.38 for no unscheduled office visits and or=1.71 for no ed visits). these relationships held when stratifying by race, gender and age. the relationships also held for metropolitan residents. the analysis was inconclusive for rural residency and the existence of co-morbidities. conclusion(s): despite race, gender, age and metropolitan residency, having a primary care provider or having health insurance impact whether or not adults with asthma are more likely to have unscheduled office or ed visits for urgent asthma care. further investigations are needed to examine how these factors impact adults with asthma who are rural residents or who have co-morbidities. d. bukstein 1* , g.a. cherayil 2 , 1. madison, wi; 2. brookfield, wi. introduction: costs for plans to process prior authorizations for non-formulary medications, has been estimated to be $20 to $25 per request. costs for physicians to process these requests has not been extensively studied. methods: dr.bukstein, board certified allergist, developed a data collection tool. the form was utilized by physicians and nurses to document time spent on processing prior authorizations. data collected included, class of medicines requiring the pa, nursing time spent on calling the patient, pharmacist, health plan, nursing time spent completing forms, nursing time clarifying the information for the pa, as well as physician time spent completing pa activities. results: data was collected over 8 weeks in 2003 and 117 requests were processed.the class of medicines most often processed was antihistamines, comprising 66% of requests. nursing calls were tracked and calls to and from patients were the most common call documented. they averaged 5.6 +/-6.5 calls per day per nurse. the nurses spent an average of 17 minutes per patient call. calls to health plans averaged 1.8 +/-1.5 calls per nurse per day and time spent on these call was 8.4 +/-9.5 minutes per call. physician calls documented included 154 calls averaging 1.9 +/-1.2 calls per physician per day. these calls averaged 5.8 +/-5.0 minutes per call.often the results of the prior authorizations were not known on the day of the request. originally 30.8% of requests were granted the same day. retrospective review revealed 98.7% were approved the first time they were processed. salary and benefits were calculated for nurses and physicians. the hourly rate was defined as $21.50 per hour for nurses and $150 per hour for physicians. the costs for the time spent on the 117 prior authorizations were calculated. during the 8 week study period, over 40 hours was spent by nurses on 231 calls and over 8 hours was spent by physicians on 154 calls during the same time period. the total nursing and physician cost in this specialty practice was $17.77 per prior authorization. conclusion: there are substantial costs with processing of prior authorization requests for non-formulary drugs on the physician office side of managed care as well as on the insurance side of the process. specialty physicians should have a different process for obtaining non-formulary medications since almost 100% of their requests are granted. introduction: diabetes mellitus can adversely impact the course and outcome of myocardial infarction (mi). one of the mechanisms underlying this phenomenon is alteration of the course of inflammation and the reparative process following myocardial necrosis. abnormal wound healing, tissue reparation and immune responses in diabetic patients have been intensively studied, but the cellular and the molecular mechanisms are still unclear. transforming growth factor-beta (tgf-beta) is a multifunctional cytokine which plays a critical role in coordination of the course of inflammation and reparation, acting as a potent depressor of inflammation and a stimulator of regeneration. this study investigates the dynamics of serum concentrations of the active form of tgf-beta1 during the period up to the 20th day after the onset of a mi in diabetic and non-diabetic patients. results: in non-diabetics a significant increase was observed in tgf-beta1 on day 2 (5-fold greater than in controls; 1370.0â±306.2 and 269.3â±214.5 pg/ml respectively) with further increases reaching a peak on day 7 (1723.1â±127.6 pg/ml). on day 20 tgf-beta1 decreased to levels less than on day 2, but was still greater than in healthy controls (1265.3â±369.8 pg/ml). in diabetics, concentrations of tgf-beta1 on days 2 (939.7â±155.2 pg/ml) and 7 (965.8â±150.4 pg/ml) after mi were similar to diabetics without mi (724.6â±233.5 pg/ml). only on day 20 was tgf-beta1 increased to levels (1502.8â±285.6 pg/ml) which were 2 fold greater than in diabetics without an mi. thus, in diabetic patients serum concentrations of the active form of tgf-beta1 are much greater than in non-diabetics. mi in patients with diabetes mellitus is associated with a reduced and significantly delayed increase in tgf-beta1. conclusion: tgf-beta deficiency may be a factor associated with low activity of tissue reparation after mi in diabetic patients. introduction helicobacter pylori (hp) is the most common gastrointestinal infection worldwide, but only 10-15% of those infected develop chronic gastritis or peptic ulcer disease. the pathogenesis of ulceration, mechanisms of hp lifelong persistence in gastric mucosa and local immune disturbances induced by this infection are well known, but the mechanisms of resistance and elimination of this infection have not been extensively studied. most study is based only on phenomenological findings, such as absence or low hp colonization in subjects spontaneously producing high levels of il-2. the aim of this investigation was the analysis of the efficacy of the recombinant interleukin (ril-2) roncoleukin (biotech, russia) in treatment of hp-associated gastric ulcer disease. methods 108 patients were randomly divided into two groups. the first group of patients was treated with standard therapy including of two antibiotics (claritromycin and amoxicillin), proton pomp inhibitors and h2 receptor antagonists. patients of the second group were treated with the same therapy, but instead of antibiotics they received 0.1 mg roncoleukin into four to six areas submucously using a gastroscopic method and 0.4 mg roncoleukin dissolved in 400 ml of 0.9% nacl with 4 ml 10% human albumin infused intravenously. this procedure was performed three times at an interval of 72 hours. results immunological findings demonstrated that roncoleukin results in an increase of cd25+, hla-dr+ and cd16+cd56+ cell levels. there was an increase in the serum concentrations of il1 (3 fold), il6 (4 fold) and ifn (more than 20 fold) while the level of tnf and il8 profoundly decreased. one month after the end of treatment, the group treated with ril-2 had hp eradication achieved in 95.4% in comparison to 81.5% of the control patients. in the ril-2 treated group, the ulcer epithelization period was 10.79â±0.46 days while in the normal treatment control group it was 35.23â±1.58 days (p<0.001). conclusion immunotherapy with ril-2 is a more effective method of treatment of helicobacter pylori-associated gastric ulcer disease when compared with traditional methods of treatment employing only antibiotics. introduction the role of different cytokines and the growth factors is now appreciated in progression of essential hypertension. the participation of et-1 and tgf 1 in pathogenesis of essential hypertension especially in the process of fibrosis, hypertrophia of vascular smooth muscular fibers, and cardiomyocytes, and the activation of renin-angiotensin system is now recognized, in addition to effects on myocyte cultures. the aim of the investigation was the study of serum et-1 and tgf 1 levels in patients with essential hypertension. materials and methods 60 patients with essential hypertension (40 males and 20 females) with average age 54.0 â± 5.3 years were studied. all patients suffered from left ventricle hypertrophy documented by echocardiography. the control group consisted of 20 healthy volunteers similar to the investigated patients in sex and age. in all patients the serum concentrations of et-1 and tgf 1 were determined using elisa (biomedica, biosource). results an increase in the serum concentrations both growth factors were noted in the studied group when compared with the control group. the average concentration of et-1 in patients with essential hypertension was 0.64 (0.32-0.91) pmol/l. the level of et-1 in the control group was 0.1 (0.05-0.17) pmol/l (p=0.04). the concentration tgf 1 in essential hypertension patients was 224.2 (125.4-314. 3) pg/ml and in control group was 68.2 (42.5-81.8) pg/ml (p=0.02). there was a positive correlation between the concentrations (spearmen's rank coefficient of correlation was 0.42; p=0.01). con-clusion the increase of the serum concentration of growth factors et-1 and tgf 1 and their co-influence in patients with essential hypertension, suggests a role of these growth factors in the pathogenesis of arterial hypertension. u. kaza 1* , c. lauter 2 , 1. bloomfield hills, mi; 2. royal oak, mi. introduction: few studies have examined the referral patterns for allergy and immunology inpatient consultations in a community hospital. consequently, an invaluable part of physician and housestaff education is missing. our objective was to examine the number of inpatient allergy and immunology consultations, the reasons for consultations and the outcome of the patients in order to improve physician education. methods:we performed a retrospective chart review of all inpatient allergy and immunology consultations in the years 1996-1997 and in 2002-2003 to determine the reasons for consultation, the recommendations made and if they were followed and the outcomes of the patient. results:we reviewed a total of 408 inpatient allergy and immunology consultations. in the 1996-1997 time period 26% of inpatient consults were for asthma, 22% for drug allergy, 12% for rash. in the 2002-2003 time period 25% of inpatient consults were for rash, 21% for drug allergy, 16% for asthma. the top three reasons for consultations remained the same although the order changed. consultations for immune deficiency, angioedema and rashes increased, whereas consultations for asthma and allergies decreased. there were a total of 202 consultations in 1996-1997 and 206 in 2002-2003 . the number of consultations remained the same despite an increase in overall number of hospital admissions from 44, 677 in 1996 to 58, 348 in 2003 . in greater than 95% of consultations, allergists' recommendations were followed. in both of the time periods studied, greater than 70% of patients improved, with less than 9% having no improvement, in the remainder of cases improvement was not applicable. conclusion:in conclusion, we believe that identifying the reasons for inpatient allergy and immunology consultations and examining the most common recommendations, as well as the outcomes of patients will be a valuable guide in the education of our physicians. by incorporating this information into grand rounds and resident conferences, physicians will benefit from learning about when an allergist can be helpful and how to manage some of the more common allergic and immunologic problems in patients that are hospitalized. the high percentage of providers who follow the advice of allergists indicate that allergists have a great deal of educational value to offer other physicians. while complementary and alternative medicine (cam) has generally experienced increased popularity, its utilization by allergy/asthma patients remains uncertain. our private allergy practice surveyed the use of cam in allergy/asthma patients in 1998(1). using a similar questionnaire, we assessed the current interest in cam with our allergy/asthma patients and compared the data to our 1998 survey. we analyzed 103 completed questionnaires from 113 sequential surveys administered. the results were compared to 113 questionnaires reported in 1998. they indicated that in both study periods (1998 & 2004) , the majority of patients wanted to discuss cam (65 and 69% respectively). an equal number (18%) in each study period discussed cam with their primary care provider. sixteen percent (16%) of our respondents sought a cam practitioner for general medical needs in 1998 vs. 19% in 2004. however, there was an increase from 4% to 10% of our patients seeing a cam practitioner for their allergies and/or asthma from 1998 to 2004. sixty-two percent (62%) would like to consider pursuing cam through our allergy spe-cialty practice or other provider. when asked regarding preferred treatment, 62% stated combination traditional with cam, 8% preferred traditional only, 3% cam only, and 27% did not know/doctor s choice. more patients are now seeking chiropractic care (36% to 52%) compared to our 1998 results. acupuncture was the first choice cam modality at 48% in 2004 surpassing vitamin/mineral therapy in 1998. currently, the second and third choices were vitamin/mineral therapy and deep tissue massage. while these numbers were small, we were impressed that currently 10% (two and a half times more since 1998) of patients in our practice were seeking cam allergy/asthma care from outside of our practice. these results demonstrated that more than half of our patients were interested in pursuing traditional with cam options within our office. given this trend, we have begun discussing the concept of integrative allergy, which to us means integrating evidenced-based cam modalities within our traditional allergy/asthma practice. (1) introduction: clinical immunization knowledge is complex and demands ongoing training. nationally, basic immunization and vaccine safety education is limited within traditional medical, nursing, and provider education. project immune readiness (pir), a peer-reviewed, web-based, interactive course, was developed in response to this deficit and the need for standardized resources to provide initial and sustainment training for safe and effective immunization services. it is designed for medical personnel with diverse educational preparation. currently, pir offers 21 course modules addressing 42 hours of instruction on specific vaccines, their respective diseases, and general information on immunization healthcare and vaccination procedures. methods: users completed a pre-test (establishes baseline knowledge), an interactive module, followed by a post-test (to observe change from baseline) for each course in sequence. anonymous user and score data were collected as part of a quality assurance and course validation process. learning gain indices (lgi) were calculated based on average mean pre-test and post-test scores for each module lgi of all modules demonstrated substantial increases in user vaccination knowledge. comparing pre and post-testing is an effective method to assess learning gains. the findings support pir as a successful and valid distance-learning tool that establishes and documents core knowledge of medical personnel administering vaccinations. further research is needed to assess the effectiveness of knowledge acquisition and retention, in addition to variance in vaccine delivery after training. this approach to learning may have value as a resource that supports smallpox and influenza pandemic emergency preparedness plans for just in time training. introduction: variances from practice guidelines for the prescription of auto-injectable epinephrine are well documented among practicing physicians, families, and patients. effective patient education requires provider competency. the current study was designed to survey resident physician perceptions regarding auto-injectable epinephrine education, use, and patient education requirements. methods: residents from primary care disciplines at a tertiary care medical center were invited to complete a voluntary, anonymous questionnaire. a total of 58 surveys were distributed and returned: 22 emergency medicine, 11 family practice, 9 internal medicine, and 16 pediatric residents completed the questionnaire. due to the small sample size, responses from physicians in various disciplines were reviewed as a whole. results: 40 respondents (69%) reported that they had previously prescribed auto-injectable epinephrine for allergic emergencies. the majority (63%) of these prescribers reported that their training was inadequate. 14 respondents (35%) reported no training, while 11 respondents (28%) reported that their training was less than that needed to ensure proficiency. 15 respondents (37%) reported that their training was adequate to ensure proficiency. none reported expertise. only 7 of 40 prescribers (17.5%) reported that either they or their staff always demonstrated proper medication use to the patient. interestingly, 2 of these 7 providers reported they had not been trained on proper use. the table below summarizes resident training and patient education practices. additional physician knowledge deficits included the proper site of medication administration, the proper interval for replacing medication, and the proper place for medication storage. conclusions: of the residents surveyed, who have previously prescribed auto-injectable epinephrine, 63% identified training deficiencies. only 17.5% of prescribers reported that the standard of care requirement to demonstrate proper medication use was always met. there is a clear need to improve auto-injectable epinephrine education in all residency training programs. r. bloebaum 1* , r.k. calabrese 2 , m. 1. houston, tx; 2. new york, ny. introduction: pneumocystis carinii pneumonia (pcp) is a major cause of morbidity and mortality in patients with aids. adverse reactions occur frequently to the most effective medication for both the prevention and treatment of pcp, trimethoprim-sulfamethoxazole (tmp-smx). we looked at the immediate safety and efficacy of three protocols for desensitization in aids patients. methods: by retrospective chart review, we identified 49 patients with aids who had experienced previous mild to moderate hypersensitivity reactions to tmp-smx and required desensitization. patients received one of three desensitization protocols based on illness severity or ward attending preference: a 6-hour intravenous (iv) desensitization, an 8-day oral desensitization, or a 10-day oral desensitization. results: of the 49 patients that received desensitization, 38 (77.5%) completed successfully. of these, 33 subjects had no reaction during the desensitization process; however, seven of these subjects were on steroids for treatment of other diseases. the remaining five successful patients had mild reactions which were treated symptomatically with acetaminophen, antihistamines or both. eleven patients failed to complete the desensitization: six stopped by the attending physician and four dropped out voluntarily. one patient expired during desensitization from extensive disease complications related to the admitting diagnosis. all protocols were equally successful when comparing the immediate success rates, 6/7 (86%) of the 6hour protocol, 4/5 (80%) of the 8-day protocol and 28/37 (75.7%) of the 10day protocol. the 6-hour iv desensitization protocol was most frequently used in the intensive care unit on critically ill patients. two of these patients, counted as successfully desensitized, died 5 and 28 days post protocol completion secondary to extensive comorbid conditions unrelated to the desensitization. conclusion: given the insignificant differences between the success of the 6hour iv desensitization and the oral desensitization protocols, we believe that either may be used effectively. further, in patients with mild to moderate hypersensitivity reactions to tmp-smx, an oral desensitization protocol may be used safely in the outpatient setting if given appropriate lab follow up. a. morales * , e. gonzalez, a. contreras, d. lopez, g. lopez, mexico city, mexico. introduction in 1966 davis describes job syndrome in two women, in 1971 dr buckley reports two children, being known as hyper-ige syndrome (job's syndrome or buckley's syndrome). defined as a primary immunodeficiency, dominant autosomic, characterized by multi-systematic alterations (immunological, skeletal, dermal and dental). it's diagnostic criteria are levels of ige 2000 ui/ml, chronic dermatitis, recurring respiratory infections, cold abscesses, pneumatoceles, infections caused by candida, and finally craniofacial alterations. on another front, extraordinary high levels of ige have been reported in patients with allergic illnesses that increase the risk of anaphylaxis, but do not have the job's syndrome criteria. objetive to determine if allergic patients with ige levels higher than 2000 ui/ml have diagnostic of job's syndrome. material and methods a retrospective revision was realized from may 2001 to april 2004 in files of 27 patients treated in allergy services at the instituto nacional de pediatria with a total ige greater than 2000 ui/ml, by means of a page with recollected dates. results nine women and 18 men were included with an age range of 3 to 18 years, and an average age to 9 years; 22 (81.5%) were diagnosed with allergies; of which 55.6% rhinitis allergy, 51.9% asthma, and 40.7% topical dermatitis of which co-existed in patients. 51.9% presented hereditary antecedents of atopic. the cutaneous tests were positive in 11 (40.7%) with a greater reactivity of dermatophagoids. one syndrome of hyper ige was detected. others diagnosed were found as not allergic were hunter's syndrome and toxocariasis. conclusion there are patients with allergies that have total levels of ige as elevated as 49, 560 ui/ml without correspond to job's syndrome. by which a multi-allergic clinical entity is proposed with levels greater than 2000 ui/ml. introduction home monitoring of lung function in asthmatic patients is used extensively in both clinical and research settings, however, little attention is given to device quality control. objective the purpose of this study was to determine the accuracy, precision and usefulness of the airwatch system (enact health management systems, palo alto, ca, usa). methods the subjects included in this study were submitted to spirometry following american thoracic society guidelines (ats 1995), using collins gs 4g pft system. afterwards, peak expiratory flow rate (pef) and forced expiratory volume in one second (fev1) were determined using airwatch. fev1 and pef measures from both devices were compared, by using two sample t-test and pearson correlation coefficient. results a total of 115 patients (75 females) were enrolled, and their mean age was 43.7 years (9 to 76 years). fev1 measures ranged from 0.54 to 5.81 l (mean 2.43 l), and from 0.62 to 7.44 l (mean 2.51 l), in collins and airwatch, respectively. pef ranged from 107 to 871 l/min (mean 384 l/min) in collins and from 61 to 794 l/min in airwatch (mean 353 l/min). significant difference was noted for pef measures (p< 0.05 and pearson correlation r= 0.61) between the two devices; however, we did not observe this difference when fev1 measures were concerned. regarless the degree of obstruction (high or low flow rates), these results did not change. conclusion airwatch showed great utility for fev1 measures when compared to collins spirometer. although airwatch is able to assess lung function at home, on a daily basis, it is not reliable for pef measures. introduction: epidemiologic data show that poorly controlled asthma is a serious public health problem. the degree of implementation of the naepp guidelines in primary care practice remains to be defined. the objective of this survey was to determine if introduction of an assessment tool into primary care practices along with a specially designed program to implement the guidelines would improve diagnosis and therapy. methods: the asthma care network (acn), a program designed to assist healthcare providers in the assessment and management of their patients with asthma, employs a team of specially trained respiratory care associates (rcas), 100 rns and rts, who visit primary care offices to inform staff about various components of the naepp guidelines and assist in their implementation. .a total of 4901 primary care providers in 2876 sites were recruited as part of the acn program. data from more than 60, 000 patient visits were collected and analyzed between march 2002 and january 2004. the program assessment tool surveyed asthma control and medication prescribing patterns. outcome measures included degree of symptom control, limitation of activity, sleep disruption, use of rescue medication and utilization of urgent care services. these data were collected on an office visit assessment form (ova) completed by both patient and physician. the rcas provided information, education, device training in the use of inhalers and spacers, and a ce course for the staff discussing pathophysiology, assessment and management of asthma. results: a total of 60, 248 ova forms were completed. among all patient including adults (older than 18 years of age) and children (<4-17 years of age), 74% (range 69% to 81%) reported symptoms consistent with lack of asthma control. approximately 70% of the survey group had more than 2 markers of uncontrolled asthma. as a result of this assessment, controller medication use increased by over 30%, 52% of which was an ics-containing medication. conclusion: the information provided to the primary care health care providers resulted in a considerable increase in prescription of controller therapy, and in particular, increased use of ics controller medication consistent with naepp guidelines. background: patients with allergic rhinitis (ar) demonstrate symptoms of allergy to fruits, vegetables and nuts in 48-72% of cases. oral allergy syn-drome (oas), typical for hypersensitivity to plant food, is based on cross-reaction of pollen-allergen specific immunoglobulin e (ige) antibodies with homologous food proteins. the production of th1 and th2 cytokines in allergic rhinitis patients with or without sensitization to food (fruits and vegetables) allergens was assessed. methods: fifty five patients aged 18-38 years with allergic rhinitis were observed. group i -20 patients with ar; group ii -35 patients with ar and oas. sensitivity to pollen allergens was tested by skin prick tests. the allergic reaction to food in patients with oas was proved by a positive history of oral symptoms caused by eating fruits and vegetables and a positive skin prick test with respective food allergens. blood eosinophil counts and total ige levels were determined during the peak of allergic rhinitis symptoms. il-5, il-10 and -interferon levels were measured by elisa. results: 24.5% of patients were sensitized only to grass pollen, 23.3% only to tree pollen and 52.2% reacted to pollens of grasses, trees and weeds. in patients with oas, skin tests were more often positive to birch (57.8%), alder (46.3%), and mugwort (26.7%). the most common food products implicated in oas were hazelnut (69.3%), apple (29.2%), carrot (16.4%), and peanut (12.7%). the allergy to fruits and vegetables was confirmed by positive prick test in 78.4%. during the season blood eosinophil count and total ige levels were elevated in all patients. there was an increase in the production of il-5 to 187.3â±5.4 pcg/ml in group i and to 221.5â±6.2 pcg/ml in group ii (nor-mal=74.3â±3.3 pcg/ml). the levels of il-10 increased to 186â±12 pcg/ml in group i and to 197â±10 pcg/ml in group ii (normal=5.8â±0.25 pcg/ml); the level of -ifn decreased to 287.6â±7.4 pcg/ml in group i and to 272.6â±5.2 in group ii (normal=331â±35 pcg/ml). after sit with pollen allergens the clinical manifestations of allergic rhinitis and oas decreased in 84.6% of patients. conclusion: allergic rhinitis patients' sensitivity to food allergens may cause oas in these patients associated with increased functional activity of th2 responses. patient knowledge and improvement with aller-gen immunotherapy. c.c. randolph * , waterbury, ct. introduction: there is no consensus on objective parameters for improvement in immunotherapy. similarly little is known regarding patient knowledge of immunotherapy. utilizing two published questionnaires (1-2), we assessed clinical improvement based on symptom and medication scoring and knowledge of immunotherapy. methods: the charts of 219 patients of an estimated 530(41%), 109(50%) with allergic rhinitis only and 116 (53%) with concomitant asthma, were retrospectively or prospectively reviewed who had been on immunotherapy to inhalants for 6 months to 6 years, mean 2.7 years, age range 7y-64y, mean 25y, 108 male(49%), 111 female(51%) with 213 caucasian (97%), 2 oriental (1%) and 4(2%) afroamericans .they completed symptom and medication survey (1) every 6 months with range of improvement using a decline in likert scale (+)20 to (-)104, mean 24. 186 (85%) indicated improvement in their symptoms and/or medication .111(50%) completed (2) a 7 question survey of knowledge of immunotherapy. there were 7 questions regarding the outcome of immunotherapy, the years to onset of immunity, the duration until onset of immunity, the danger of immunotherapy and the extract in the vial. the correct responses were recorded to 2/7 (2/111=1.8%), 11(9.9%) 3/7, 19(17%)4/7, 10(9%)5/7, 18(16%)6/7.39(35%) had a perfect response to all questions. 12(11%) had no correct responses. conclusion: the majority of immunotherapy patients improved (85%) by symptom and medication scoring over the mean of 2.7 years but only 35% had complete knowledge of the rationale for immunotherapy. further education and repeated surveying of patients on immunotherapy to assure comprehensive knowledge of immunotherapy and achieve better outcomes is recommended by this investigation. as is unique in this study symptom and medication scoring using the rhinitis /sinusitis questionnaire approved by the acaai and a comprehensive questionnaire assessing knowledge should be conducted periodically ie every 6 months to provide objective parameters for improvement. references: 1.santilli j, nathan r, glassheim j .patient receiving immunotherapy report it is effective as assesses by rhinitis outcomes questionnaire(raq) in private (42)) is a protein involved in the parasite invasion of host erythrocytes and is a leading vaccine candidate for the erythrocyte stage of malaria infection. an increasing number of vaccine clinical trials are being undertaken using various formulations of msp-1(42). comparison of humoral responses among these trials has been limited by the lack of a universal reference standard for specific antibody. the purpose of this study was to develop a human reference standard for msp-1(42) antibody measured in absolute quantity units that could facilitate comparison of interstudy vaccine response. method: we formulated the reference standard by pooling human plasma samples known to contain high titers of msp-1(42) antibody. the specific antibody within the pooled plasma was captured by msp-1(42) adsorbed to nickel resin in a process of immobilized metal affinity chromatography (imac). the intact msp-1(42) antibody-antigen complexes were separated from the nickel resin and total igg in the complexes measured by enzyme-linked immunosorbent assay (elisa). results: our antibody quantitation method yielded a concentration of 48.3 mcg/ml of msp-1(42) antibody in the reference standard. conclusion: the reference standard characterized in this study may be useful as a quantitative working standard for msp-1(42) antibody response in future vaccine clinical trials involving msp-1(42). this standardization may facilitate the clinical development of msp-1(42) as a candidate vaccine for malaria infection. background. specific immunotherapy (sit) is currently one of the most effective and widely used treatment methods of allergic diseases including asthma. efficacy of sit depends on the correct choice of patients, severity of asthma and patient's condition when the sit is begun. according to who recommendations, sit is approved for use in patients with mild to moderate asthma. methods. sit with a saline extract of house dust allergen was administered to 30 patients with mild persistent allergic asthma. injections were performed subcutaneously 2-3 per day. the course consisted of 6766 pnu of allergen and lasted 12-14 days. patients were repeatedly surveyed at 3, 6 and 12 months after sit was completed. dyspnea attacks occurring during daytime and at night were assessed, as was the influence of physical exertion on dyspnea and lung function, and also the number of utilizations of short-acting beta-agonists per day. pulmonary function measurements were performed as was assessment of concomitant allergic rhinitis. 50% of patients received treatment including cromones, and 30% utilized inhaled corticosteroids among whom 13.3% had a dose of 400 mcg per day and 16.7% 200 mcg per day. sit efficacy was assessed by clinical symptoms, pulmonary function measurements and amount of concomitant therapy received. results. the number of daytime dyspnea attacks reduced from 10.70 â± 3.40 per month to 1.20 â± 0.09 during the 3 months following sit, and 76.7% of patients reduced concomitant asthma therapy. at 6 months, dyspnea attacks increased to 2.90 â± 0.75 per month, still significantly less then prior to sit. nocturnal symptoms followed the same pattern, occurring 7.20 â± 1.62 per month prior to sit, 1.01 â± 0.01 per month 3 months after sit and 1.99 â± 0.25 per month at the end of the sixth month after sit. 26.7% of patients had no symptoms of bronchial obstruction during the 6 months after sit and their pulmonary function approached normal values, although 36.7% of patients returned to asthma therapy. nasal congestion decreased from 3.28 â± 1.20 points to 1.98 â± 0.08 (p<0.05), clinical improvement still present for 3 months after sit, but recurring at 6 months after sit. conclusion. sit may be an effective treatment method that improves both asthma and allergic rhinitis for more than 3 months following a brief course, allowing a decrease in the amount of symptomatic treatment. a safe therapeutic vaccine that can alter the allergic response to peanuts would serve a serious unmet medical need. peanut allergy responses are largely associated with downstream events related to antigen specific ige crosslinking of ige receptors and subsequent degranulation of mast cells and basophils. our clinical studies with ragweed have shown that linking immunostimulatory dna (iss) to allergens can decrease ige recognition of the allergen and generate an immunogen that generates protective th1 responses and reduces harmful th2 responses. to test this approach to peanut immunotherapy, we focused on the clinically relevant allergen, ara h 2, as a proof of concept. iss oligonucleotides were linked to ara h 2 at two different ratios: pic (2 iss per protein) and hpic (4 iss per protein). immunogenicity of pic and hpic was evaluated in c3h/hej female mice immunized twice with 5 ug of pic, hpic, or ara h 2. sera were analyzed for anti-ara h 2 igg1 and igg2a responses. spleens were harvested and ara h 2-specific ifng and il-5 responses were measured in vitro. mice were immunized with ara h 2 elicited predominantly igg1 and il-5 responses, indicative of a th2-type response. animals immunized with pic showed significantly enhanced igg2a responses and strong ifng responses, indicative of th1-type responses. hpic immunized mice elicited little antibody response, presumably due to iss blocking b cell epitopes, but did induce ifng responses. to assess allergenicity of pic and hpic, histamine release was measured in blood from peanut allergic donors treated in vitro with pic, hpic, and ara h 2. histamine release was detected at very low concentrations of ara h 2 (0. 01 ng/ml), 10-fold higher concentrations for pic (0.1 ng/ml) and was undetectable at concentrations up to 1000fold higher for hpic (10 ng/ml). an ige binding competition assay also confirmed reduction in allergenicity following the same trend for the iss-linked allergens. linking iss to ara h 2 increases th1 responses to the allergen, blocking ige recognition of epitopes on ara h 2. thus, iss-linked ara h 2 appears to be a promising candidate for a safe immunotherapy product for treating peanut allergic subjects. introduction: immunotherapy has been studied for its adverse effects in some sensitive patients as with worsening of therapeutic response in some amounting to withdrawal of the later. methods: in the allergy center therapeutic trials of immunotherapy(house dust, mixmite) had been undertaken since 1998, in therapeutic dose of 0.02pnu/ml, 0.2pnu/ml, 2pnu/ml, 20pnu/ml, 200pnu/ml2000pnu/ml, 4000pnu/ml(housedust), 0.01au/ml, 0.1au/ml, 1au/ml10au/ml 100au/ml 1000au/ml 2000au/ml(mixmite).patients age 2-65 years, both sex after diagnostic scratch/prick tests with appropriate antigenic preparations reported for local/systemic adverse effects the details of which were as under, results: please see the attached table for details, in some cases worsening of the existing allergic status was noted, which on analysis revealed initially a slow rise in the allergen -specific ige to be later on fol-lowed by rise of igg1 & igg4 & gradual fall of ige.only 1/100 patient could not complete immunotherapy for fear of adverse effects. conclusions: with utmost care/follow-up, no incidence of mortality outcome was reported from 1998to late 2003 the incidence of adverse effects were significantly lowered on pre-medication with anti-histamines significantly more with elderly than younger age. even with all these documented hazards the beneficial effects far exceeded the harmful effects. background: the prevalence of allergic disease in most human population is steadily increasing. seafood allergy is a serious food allergy, although hypersensitive reactions caused by seafood has long been know, biochemical and immunological studies on seafood allergies had only begun lately. shrimp and abalone are the most frequently reported causes early asthmatic response. objective: to investigate cross-reactivity of shrimp, abalone and derp1. methods: shrimp and abalone extracts were prepare from raw seafood. sera from 17 patients from hongkong were studied who had asthma after consumption of seafood. ige elisa analyses comfirmed the combined sensitization to shrimp, abalone and derp1. specific-ige elisa assays were accomplished for shrimp and abalone extracts inhibited by derp1 and derp1 elisa and immunoblot assays inhibited by shrimp and abalone extracts. results: elisa inhibition showed that most ige antibodies against shrimp and abalone were cross-reactive with derp1 and the same time, derp1 elisa was inhibition by shrimp and abalone extract. the elisa inhibition percent (%)of shrimp extract (gm:53.28%) and abalone extract(gm:36.75%) by derp1 were significantly higher than derp1 by shrimp extract(gm:26.64%) and abalone extract (gm:17.59%). (p<0.01). furthermore, and there was a significant correlation of elisa inhibition percent between shrimp extract, abalone extract and derp1 inhibited by each other; sds-page and immunoblot of shrimp and abalone is the 38 and 49kd allergen respectively. conclusion: this indicates that derp1 was the sensitive agent. shrimp, abalone and derp1 demonstrate significant cross-reactivity. these findings confirm that the primary crossreactive allergen of shrimp and abalone is the 38 and 49kd allergen respectively. b. sun 1* , a. wu 2 , n. zhong 1 , 1. guangzhou, china; 2. hong kong. background: the house dust mites (dermatophagoides farinae (derf) are a major source of aeroallergens implicated in the expression of atopic disorders, including asthma, allergic rhinitis, and atopic dermatitis . in particular, strong circumstantial evidence suggests that house dust mites antigens are important precipitating factors of asthma. many house duse mite allergens are proteases that can elicit airway inflammation by stimulating the release of cytokines in bronchial epithelial cells. objectives: we have investigated whether der f allergen proteases induced cytokine production from the epithelial cell line beas-2b. methods: cells were exposed to four different concentrations with serial additions of der f (0.02, 0.2, 2, 20ug/ml) were incubated for 24h to 96h. and compare with those without incubation of allergen. cytokine in the supernatants were assayed by elisa, reverse transcription?pcr was also performed. results: cells treated with der f allergen showed serial changes in the cohesiveness of the monolayer. there was a significant increase in the level of cytokine production compared with the untreaed sample. statistically significantly increased with addition of der f caused the release of il-6 and il-8 in time and concentration-dependent manner (p<0.05, respectively). levels of il-6 and il-8 were elevated 24 h and 48 h after allergen exposure, increasing with time, continued increased levels to be present of il-6 and il-8 in the supernatants at 72 h and 96 h. at the same time show the concentration dependence of induction of il-6 and il-8 expression as well as an increase in the expression of il-6 and il-8mrna. conclusion: hdm-induced airway inflammation may include der f-mediated release of inflammatory mediators, and the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium. suggesting that il-6 and il-8 production by bronchial epithelial cells may play a role in the pathogenesis of allergic asthma. the purpose of this study is to delineate the immune injury mechanisms that involved in the autoimmune inner ear disease by introducing plasmid dna encoding of th1 cytokines (inf-g) into the inner ear. b-tubulin is a microtubular protein which we found as an important autoantigenic in meniere's disease as well as other autoimmune hearing loss. hearing loss was induced in mice and guinea pigs when they are immunized with the tubulin molecules. autoimmune hearing loss could be the results of th1 cytokine responses from autoimmune injury. to test the hypothesis, guinea pigs were immunized with 200mg of tubulin in cfa and boosted once more. two weeks later, we introduced 100ug (5ul) of naked dna encoding inf-g was injected into the left side inner ear through round window. same volume of 0.1m pbs was injected into right side as control. abr was recorded before and after the injection. 15 weeks after the injection, the animals were sacrificed and temporal bones were examined with h-e and inf-g immunocytochemical staining. the ears injected with the plasmid dna-inf-g had an enhanced hearing loss (30 db), and degeneration of the spiral ganglion was found in these ears. however, the injection of the naked dna encoding inf-g did not change the expression of the inf-g in the inner ears. these results suggest that autoimmune hearing loss could be the result of th1 responses to inner ear autoantigens. -tubulin is an important molecule in the hair cells supporting cells within the sensory epithelium of organ of corti and found to be an auto autoantigen in autoimmune hearing loss including mã¨niã©re's disease. the object of the study is to induce hearing loss in mice with varying doses of antigen and evaluate the pathogenesis of autoimmune hearing loss induced by -tubulin in mice. mice were immunized with 100, 200 or 300 î¼g of -tubulin and hearing was evaluated by auditory brainstem responses (abr) and distortion product of otoaccoustic emission (dpoae). all mice had hearing loss by abr and dpoae tests and morphological study of temporal bone showed spiral ganglion degeneration and tunel staining positive cells were noted in these immunized mice. thus this study showed that -tubulin is an autoantigen for hearing loss in animal model as in human autoimmune hearing loss patients including mã¨niã©re's disease. supported by nih r0dc005010-01 introduction: oral polio vaccination (opv) in the united states is currently being replaced with inactivated polio vaccination (ipv) given parentrally. while past studies have looked into the relationship of vaccination and asthma prevalence, none have investigated this relationship with regards to vaccination route namely orally versus parentrally. this study investigates the relationship between vaccination rates for the live attenuated orally administered polio vaccine and parentrally administered vaccines(dtp, mmr)and two potentially dependent factors; asthma prevalence rate and asthma-caused death rate. methods: we looked at data from the national center for health statistics yearly publications of health, united states (1983 -1999 and the morbidity and mortality weekly report surveillance summaries . two databases were compiled to cover the 0-4 age population in the united states since this is the primary period of childhood vaccination. one database contained information for asthma related deaths and vaccination rate (dtp, mmr, opv) covering the years 1970-1999 and the other database compiled information for self reported asthma prevalence and vaccination rates(dtp, measles, rubella, opv) covering the period 1983-1999. a t-test was used for statistical analysis. results: statistically significant correlation was found between vaccination rates and asthma prevalence rates. data for dtp (p = 0.012), measles (p = 0.024), and rubella (p = 0.023) indicated a statistically significant positive correlation with asthma prevalence. the oral polio vaccine was the only one of the vaccines that failed to display a significant relationship with asthma prevalence rates (p = 0.133). statistical analysis proved that a correlation between vaccination rates of united states children ages 0-4 and asthma-caused deaths was insignificant (p > 0.05). conclusions: the opv, which is administered orally rather than parentrally, displayed no relationship with asthma prevalence. this could be due to the fact that live attenuated orally administrated polio vaccine may induce mucosal immunity, simulating a normal pathogen route of entry into the body. childhood vaccination had no relationship with asthma-caused death rates. a.s. alfrayh * , riyadh, saudi arabia. introduction: heredity plays a major role in asthma and other allergic diseases, mechanisms underlying the inheritance of these disorders are poorly understood. this study therefore analyzed the risk conferred by family history of asthma and atopy for having childhood asthma. methods : a total of 1601 children between 1-16 yrs selected randomly in three cities in saudi arabia ( hail, taif and gizan )in 1995-1996 . the questionnaire which is similar to the one used in the international study of allergy and asthma in childhood isaac. were self administerd under medical supervision.apart from the demogrphic details, the questionnaire included questions on symptoms and physician diagnosis of asthma, rhinitis, eczema and family history of these conditions. the family members were grouped as immediate family and relatives. asthma and atopy were defined as ever having had physician diagnosis of such conditions information was also available about exposure to cigarette somke at least one member was a smoker in the household and having pets. relative risk for developing asthma was estimated in terms of odds ratio by bivariate analyses using chi square test and p value was considered significant when less than 0.05. results : history of asthma in the immediate family and relatives conferred a 4 fold and 3 fold risk for development of childhood asthma odd ratio ( or )=4.2, 95% confidence interval(ci)=3.3 to 5.5, p=0.00001 and or=3.3, 95%ci=2.5 to 4.3, p=0.00001 respectively. rhinitis in immediate family and the relatives was associated with 3 folds increased risk for childhood asthma or = 3.0, 95% ci = 2.2 to 4.0, p=0.0001 respectively whereas history of eczema conferred over 3 folds risk or=3.4, 95% ci = 2.4 to 4.7, p=0.00001 for childhood asthma when present only in the immediate family. history of eczema in relatives was not associated with any risk. of the environmental factors, exposure to cigarette smoke conferred 2 folds risk of developing childhood asthma or = 2.6, 95% ci = 2.0 to 3.3, p= 0.0001, whereas exposure to pets was not a significant risk foctor. conclusion : presence of asthma and atopy either in the biological parents or relatives constitute a significant risk for childhood asthma.paricularly in the presence of evironmental risk factors. the murine local lymph node assay (llna) has been developed as an alternative to guinea pig models for the assessment of the contact sensitization potential. however, there is a need to develop a non-radioisotopic endpoint for the llna, because of the radioisotopic method's requiring the use of special facilities. in this study, we investigated to evaluate the lymphocyte subpopulations in the lymph node cells following allergen and irritant treatment. female balb/c mice were treated by the topical application on the dorsum of both ears with sensitizers, 2, 4-dinitrochlorobenzene (dncb), toluene diisocyanate (tdi), and a-hexylcinnamaldehyde (hca), and an irritant, sodium lauryl sulfate (sls), once daily for three consecutive days. the lymph node (ln) cells were harvested 72h after the final treatment. phenotypic analysis of lymphocytes subsets was performed with a flow cytometry. the allergens dncb, tdi, and hca and an irritant, sls increased cell number compared to the vehicle. there was an increase in the percentage of b220+ cells in mice treated with dncb and tdi compared to the vehicle control, but not in those treated with sls. mice were treated with dncb, hca and tdi showed a preferential increase in the percentage of b220+cd40+ cells compared with vehicle and irritant-treated mice. there was an increase in b220+cd86+ cells of mice treated with dncb, tdi and hca, but no significant increases were observed in mice treated with sls. mice were treated with dncb, and tdi showed an increase in the percentage of b220+cd23+ cells compared with vehicle and irritant-treated mice. these results suggest that analysis of b cell activation marker, cd40 on b cells may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice. m. frieri * , y.c. huang, east meadow, ny. introduction: nitric oxide (no) is an important biomarker for inflammation in airway epithelial cells and in exhaled breath of asthmatic subjects. we have previously demonstrated no production in antigen-stimulated human bronchial epithelial cells and an effect of omalizumab (monoclonal anti-ige antibody) on no production in those cells [leyko bt et al. j allergy clin immunol 2004; 113(suppl 193) :a668]. in this study, we investigated the potential role of ige and its receptors on a549 cells by characterizing the effect of omalizumab on antigen, egf, and il-1 stimulation of a549 cells in a medium containing atopic serum. methods: a549 human alveolar epithelial cells were stimulated with 100 iu/ml of il-1 , 10 î¼g/ml of ragweed (ra), 1000 au of dust mite (dm), and 40 ng/ml of egf, and exposed to either 10 -7 m budesonide or 0.1 î¼g/ml omalizumab for 6 or 24 hours. no production was measured in duplicate by a highly sensitive elisa. results: omalizumab but not budesonide inhibited no production at 6 hours in a549 cells stimulated with il-1 (17-13î¼m, p<.05)and il-1 +dm (18-14î¼m, p=.08), but not with ra alone. however, at 24 hours omalizumab and budesonide each significantly inhibited no production stimulated by il-1 (29-9î¼m; 29-17î¼m), il-1 +dm (22-12î¼m; 20-14î¼m), and egf (17-9î¼m; 17-13î¼m) (p<.05). conclusion: no production is a marker for inflammation. omalizumab demonstrated a significant anti-inflammatory effect in distal alveolar cells by inhibiting no production stimulated by antigen, il-1 , and egf in a medium containing atopic serum. as persistent inflammation in asthma may play a role in airway remodeling, treatment with omalizumab may have a beneficial effect on chronic airway inflammation in patients with asthma. oral tolerance trigger regulatory mechanisms able to down-modulate antigen-specific t and b cell response. to address the lasting effect of several regimens of oral tolerance to ovalbumin, in naive or antigen primed mice, b-cell function has been focused. furthermore, we analyze specific antibody response up to eight months of post-immunization, proliferative response, b7 (cd80/cd86) expression on b cells and t cell ctla-4 involvement in oral tolerance. a/sn mice were immunized by intraperitoneal route with 50î¼g of ova/0, 1 mg-alum and boost 10 days after priming (dap). ova feeding was done with 25mg at different days before or after antigen priming. in others protocols, mice were fed twice, before and after priming with a total of 50mg of ova. all groups were boosted at 60 or 120 or 180 or 240 dap. the results showed that only mice fed at naive status and those fed twice before and after immunization demonstrated a long lasting of ige ab response inhibition up to 8 months of immunization. these mice showed a marked inhibition of antigen-specific proliferative response that was restored with anti-cd28 mab in vitro stimulation. evaluation by flow cytometry of spleen cells cultured for 48 hours upon ova stimulation, showed an important decrease of b 7.2 expres-sion on b cells of naive fed mice, which remained inhibited until 8 months of immunization. after addition of anti-ctla-4 mab an enhancement of b7.2 expression was detected on b cells of naive fed mice. ctla-4 molecules expression on cd4+ t cells of naive fed mice remained unchanged following ova stimulation while a peak of expression was detected at 96h of ova stimulation in control group. this finding reinforce the t cell anergic status of naive fed mice, due to the less cell division and consequently a low rate of cd4+/cd44high activated cells. the results showed that antigen feeding before immunization induce a long lasting anergy mediated by an impaired t-b cell cooperation and ab production due to the decrease of costimulatory molecules expression on b cells and negative signaling effects by ctla-4 expression. introduction: vascular endothelial growth factor (vegf), basic fibroblast growth factor (bfgf) and fibronectin (fn) can promote angiogenesis, a putative component of airway remodeling. (s)-albuterol can exacerbate airway hyperresponsiveness, bronchospasm and release pro-inflammatory cytokines from small airway and smooth muscle cells. our study evaluated the effects of (s)-albuterol ((s))-and (r)-albuterol ((r)) on secretion of these factors on normal human lung fibroblasts (nhlf) and myofibroblasts (myonhlf) in the presence or absence of tgf 2, il-1 , or allergens. methods: nhlf were stimulated to differentiate to myonhlf with 1000 pg/ml tgf . dose-dependent effects of (s) and (r) [10 -8 to 10 -4 m] were evaluated for secretion of vegf, bfgf and fn by nhlf and myonhlf with and without 1000 au/ml d. pteronyssinus (dp), 1000 pnu/ml ragweed (ra), 100 u/ml il-1 , or 1000 pg/ml tgf 2 into serum-free media (its) at 37 o c with 5% co 2 collected at 24 hr and assayed by elisa. results: in nhlf the following was observed: vegf secretion was 2-fold higher with 10 -7 m (r) relative to (s), p<0.05; bfgf secretion was increased 50%-100% by 10 -5 m (s) relative to (r), p<0.05. a lower concentration of (s) (10 -6 m) in the presence of either dp or il-1 caused a 2-fold increase in secretion of bfgf relative to (r), p=0.02. in the presence of myonhlf the following was observed: 10 -7 and 10 -5 m (s) caused a 50%-100% increase in secretion of fn relative to (r). at 10 -7 m (s), this effect was further increased with the addition of il-1 , p=0.022). conclusion: in a dose-dependent manner, (s)-albuterol stimulated the release of bfgf and fn by nhlf and myonhlf, respectively. this was enhanced by dust mite and/or il-1 , potentially contributing to the matrix remodeling observed in chronic asthma. vegf over-expression can have a protective effect against chronic hypoxia and can recruit immune cells to the alveoli. increased vegf by (r)-albuterol could contribute to such an effect in vivo in asthmatics. bfgf, in bal and sputum of asthmatics, and fn which contributes to subepithelial fibrosis, can promote angiogenesis. increased bfgf and fn by (s)-albuterol could be detrimental over time by enhancing matrix deposition and remodeling in a subset of asthmatics. o. ozdemir 1* , c. moore 2 , y. ravindranath 1 , s. savasan 1 , 1. detroit, mi; 2. new orleans, la. background: mast cells (mc) have been shown to demonstrate natural cytotoxicity against mouse fibrosarcoma cell line in culture when incubated for 24-48h. this effect has been postulated to be mediated through soluble and/or membranous tnf-. more recently; fasl, mc chymase and serine protease granzyme h with its chymase activity were proposed as mediators of mast cell-mediated cytotoxicity. thus, both 'granule-exocytosis' through chymase, granzyme h and soluble tnf-and 'death receptors' through membrane-bound tnf-and fasl pathways appear to be operative in this process. aims: following our earlier observations on long-term liquid culture-grown human bone marrow mast cell cytotoxicity against human leukemia cells in 24-48h co-incubation experiments, we investigated mast cell-mediated cytotoxicity against natural killer/lymphokine-activated killer cell-sensitive cells in short term (12h) cultures without any stimulation for the first time. methods: human bone marrow mononuclear cells were cultured in methyl cellulose supported with il-3, il-6 and scf. mast cell colonies that developed in six weeks were transferred to liquid medium and maintained for 18 weeks before experiments. cytotoxicity was investigated against k562, raji and daudi cells at 12, 24 and 48 hours of co-incubation by our established flow cytometric cell-mediated cytotoxicity assay. results: after 12h co-incubation, 61% (11% early apoptotic and 50% late apoptotic or necrotic death) and 29% (21% early apoptotic and 8 % late apoptotic or necrotic death) target cell kill was demonstrated in daudi and raji cells, respectively. daudi cell killing has stayed stable at 24h (67%; 27% early apoptotic and 40% late apoptotic). despite a small numbers of experiments, daudi cell kill was statistically significant at 12h (p:0.011) and 24h (p:0.011) compared to control. however, k562 cell elimination (18%) has not occurred until 48h. mast cell-daudi cell conjugates were seen on the wright/giemsa slides (figure) . conclusion: our results demonstrate that human mc can cause cell-mediated cytotoxicity against certain cells in relatively short-term. this further suggests possible contribution of 'granule-exocytosis' pathway to mc natural cytotoxicity, indicating a faster mc response in immune surveillance. o. ozdemir 1* , m. buyukavci 2 , y. ravindranath 1 , s. savasan 1 , 1. detroit, mi; 2. erzurum, turkey. background: the effect of melatonin (mlt) on cellular immunity has been controversial. recently, mlt has been demonstrated to activate t and nk cells through its membrane or nuclear high affinity receptors. it was also shown that pharmacological concentrations of mlt (>nm) could be cytotoxic against different human cancers. aims: our aim was to investigate the effect of mlt alone or in combination with il-2 on peripheral blood lymphocytes (pbl), lymphokine activated killer (lak) cell generation and its cytotoxicity. methods: pbl were cultured for 7 days in the presence of mlt at different concentrations (10 -3 , 10 -5 , 10 -7 m) with or without il-2 (100 u/ml). cell viability was determined by trypan blue exclusion test. cell-mediated cytotoxicity of lymphocytes/lak cells against k-562 and daudi cells was studied using our established flow cytometric cell-mediated cytotoxicity assay. results: although 10 -3 m concentration of mlt did not affect cell proliferation much on day 3, it significantly inhibited proliferation by day 7 (p<0.05) consistent with known anti-proliferative effect of mlt. 10 -5 and 10 -7 m concentration of mlt also mildly inhibited proliferation on day 3; however there was a minimal rebound with 10 -7 m concentration by day 7. consistent with the reported mlt-treated pbl's reduced response to mitogens, il-2 and mlt (10 -3 m) combination suppressed proliferation on days 3 and 7; however, with 10 -5 m mlt concentration pbl counts increased gradually from day 3 to 7. although mlt treatment alone did not enhance cell-mediated cytotoxicity, il-2 and mlt combinational treatment at both concentrations (10 -3 and 10 -5 m) increased it significantly compared to baseline activity (figure) . conclusion: il-2 and mlt combination at 10 -5 m concentration resulted in superior lymphocyte proliferation and lak cell-mediated leukemia cell kill. mlt-induced increase in il-2r-expression of pbl shown earlier might be the mechanism for our observations. mlt can be considered in immunotherapy as an adjunct to il-2 treatment. a.e. fusaro * , j.r. victor, c.r. oliveira, c.a. brito, e.a. futata, m. maciel, a.j. duarte, m.n. sato, sã£o paulo, brazil. the maternal exposure to allergens during pregnancy or even in postnatal period may influence the allergy onset to newborns, through antigen or antibody transmission. we sought to verify the effect of maternal antigen exposure before conception, during gestation or in the breastfeeding period on the offspring type i hypersensitivity response. female balb/c mice were immunized or not with ova extract/alum, boosted twice at 10th and 20 th and mated with normal balb/c male on the 21th day after sensitization (das). others groups of immunized mothers also received oral administration with ova along pregnancy, or non-immunized mothers received ova only during breastfeeding. offspring from immunized or normal mice were immunized intraperitoneally with ova at 25 do and boosted on the 10th das. the results showed a important decreased of tgf-levels in the amniotic fluid and milk from immunized mothers before mating in comparison to obtained from normal mothers. ova exposure during pregnancy of immunized mothers decrease significantly the transference of tgf-by breastfeeding, while both tgf-isoforms were founded at high levels in the amniotic fluid. similar levels of tgf-transference by placenta to the newborn was detected in both immunized mother groups. pups from mothers exposed with ag during pregnancy showed an increased spleen cell number, whereas did not produced il-2, il-4, il-10 e ifn-secretion induced by antigen neither altered responsiveness to anti-cd3 or mitogen. maternal ova-immunization induced a marked inhibition of spe-cific ige antibody response in the immunized pups, contrasting to the enhancement of ige responsiveness detected in the immunized pups from mothers which were exposed to ova only at postnatal period. these results showed that preconceptional immunization exert a protective effect on the offspring ige development and an exacerbation of ige responsiveness due to mother antigen exposure during breastfeeding. the findings suggest that rather than in utero antigen priming occurrence, postnatal period may contribute to offspring early life sensitization. financial support: fapesp and lim56-fmusp introduction: the reasons for the increased incidence of allergic diseases in westernized countries are still unknown. mercury is an important pollution factor to which humans are increasingly exposed. prior studies on the effect of mercury on mitogen stimulated human lymphocytes indicated a th2weighted immune response, but the results were inconsistent and difficult to reproduce. phorbol myristate acetate (pma) is a direct activator of protein kinase c, which has been shown to play a role in mercury induced il-4 production in animals. therefore we investigated the effect of mercury on pmaactivated human peripheral blood mononuclear cells (pbmc). methods: pbmc from 8 individuals were cultured for 4 days in culture medium containing pma and ionomycin in the presence or absence of mercuric chloride (hgcl2). il-4 and gamma-ifn concentrations were measured by elisa of culture supernatants. cell death and apoptosis were determined by 7-aad and annexin staining and fluorescence activated cell sorting (facs). cell-proliferation was assessed by 3h-thymidin-incorporation. results: after 4 days of culture, pma/ionomycin-stimulated a small amount of il-4 compared to untreated pbmc (1.7 +/-0.92 pg/ml versus 0.36 +/-1.0 pg/ml). however, mercury induced a more than 30 fold increase in il-4 production when added to pma-activated cells (57.4 +/-45.2 pg/ml, p<0.01). gamma ifn production was strongly increased in pbmc that were treated with pma/ionomycin (>3500 pg/ml versus 417 +/-1045 pg/ml in unstimulated cells) but dropped markedly in cells treated with mercury plus pma/ionomycin. in addition, mercury induced increased cell death, apoptosis and reduced cell proliferation. conclusions: hgcl2 strongly stimulates il-4 production in pma/ionomycin treated pbmc while cell viability and gamma-ifn production drop significantly. these preliminary results suggest that human exposure to mercury may be playing a role in the observed increased incidence of allergic disease in the industrialized world background: mast cells (mc) have been shown to induce natural cellmediated cytotoxicity in long-term (24-48 hrs.) in vitro assay systems. the cytotoxicity is mediated by at least two pathways: secretory via exocytosis of mc granules containing serine proteases such as granzymes, chymase and soluble tnf-and nonsecretory (cell-to-cell contact) via membranous tnfand fasl. chymase induces apoptosis in neonatal rat cardiomyocytes and human vascular smooth muscle cells. the objective of this study was to investigate mc mediated cytotoxicity against nk/lak-sensitive cells in short term (12 hrs.) unstimulated cultures. methods: human bone marrow mononuclear cells were cultured in methylcellulose supported with il-3, il-6, and stem cell factor. mast cell colonies developed at 6 weeks and were transferred to liquid imdm and maintained for 18 weeks. a flow cytometric cytotoxicity assay was used to determine cytotoxicity against daudi, raji, and k562 cell lines at 12 hrs., 24 hrs., and 48 hours. the controls consisted of cell lines without mast cells at 12 hrs., 24 hrs., and 48 hours. results: rationale: to establish the antibody response rate in children with recurrent infections and fully immunized with the pneumococcal 7-valent conjugate vaccine. methods: we have analyzed 39 patients referred to our clinic with recurrent infections despite complete immunization with the pneumococcal 7-valent conjugate vaccine for age, according to acip guidelines. we assessed the patients by checking their immunization status and the antibody titers to all 7 streptococcus pneumoniae serotypes included in the vaccine (4, 6b, 9v, 14, 18c, 19f, 23f) assessed by standardized elisa. the patients were assembled into 3 groups, a non-immunized group with laboratory data prior to the vaccine, and an immunized group consisting of responders and nonresponders according to their antibody titer (<1.3 or >1.3 iu/ml respectively). the data were analyzed using epi info and spss. results: the mean age was 3.3 years for non-responders and 3.8 years for responders. there was no significant statistical difference between the groups regarding age, race and sex. ten patients were identified who failed to respond to all 7 serotypes included in the pneumococcal 7-valent conjugate vaccine. there was no significant statistical difference between the non-immunized and the immunized nonresponders to all 7 serotypes. conclusions: we have identified a special immunological phenotype of specific antibody deficiency (sad) patients with normal total immunoglobulins and normal responses to protein antigens, but who failed to respond to conjugate pneumococcal polysaccharides. a. yates 1* , r. deshazo 1 , j. butler 1 , g. howell 1 , j. farley 1 , h. liu 1 , n. nanayakkura 2 , g.b.yi 1 , r. rockhold 1 , 1. jackson, ms; 2. oxford, ms. introduction: venom from s. invicta consists of 95% disubstituted piperidine alkaloids and is toxic to insects, birds and farm animals. recent reports of morbidity and mortality in elderly patients after massive fire ant stings suggest the potential for systemic mammalian toxicity. we evaluated the toxic responses to systemic administration of two structurally verified, synthetic s. invicta venom alkaloids, solenopsin a (trans-2-methyl-6-n-undecylpiperidine) and its cis-isomer, isosolenopsin a, in rats. methods: sprague dawley rats were anesthetized with isoflurane, paralyzed with gallamine, artificially ventilated and instrumented to record arterial blood pressure (bp; mm hg), heart rate (hr; bpm) and % change in left ventricular contractility (lvc; p/ t). in addition, a group of rats was chronically instrumented to record bp and hr while the animals were conscious and freely-moving. results: solenopsin a at 3 to 30 mg/kg iv dose-dependently lowered bp, hr and lvc. at 30 mg/kg iv, hypotension (-40â±12 mmhg), bradycardia (-27â±8 bpm) and decreased lvc (-41â±17 p/ t) were marked. isosolenopsin a, 15 mg/kg iv, produced responses similar to solenopsin a 15 mg/kg iv. solenopsin a 30 mg/kg iv elicited tonic-clonic convulsions and respiratory arrest in conscious, freely-moving rats. hematuria was seen with solenopsin a, but not isosolenopsin a. superfusion of a working, isolated, perfused rat heart with 10 um of solenopsin a elicited a marked, reversible decrease in lvc, and cardiac arrest occurred with 100 um of solenopsin a. conclusion: the results demonstrate that these alkaloids possess significant depressant activity on the cardiac and respiratory systems of rats. the neurologic and cardiorespiratory effects can account for lethality to small mammals in the wild, and may contribute to adverse cardiovascular effects noted in humans after massive fire ant stings. introduction: during asthma attacks, the ph of exhaled breath condensate (ebc) decreases two log orders (ph=5.0), returning to normal levels after corticosteroid therapy (ph=7.0). ion channels, once thought to participate only in the transport of ions, are now suspected of mediating airway inflammation in asthma as well. to determine whether allergen directly alters airway mucosal ph and ion function, we measured nasal ph and nasal potential difference (pd) before and after nasal allergen challenge (nac). methods: ten allergic rhinitic subjects (meanâ±sem age 28.3 yearsâ±2.9, 7 females) underwent a crossover, single-blinded, placebo-controlled study, where they were challenged with allergen (dust mite, grass, cat) and control diluent in two different occasions in random order via nasal spray. nasal ph was measured on the surface of nasal mucosa with a ph probe. nasal mucosa pd was measured between nasal mucosa and forearm skin. subjects also filled out a nasal symptom scale. measurements were taken at baseline, 1 hour, 4 hours, and 24 hours after nac. results: the nasal symptom score increased significantly immediately after allergen compared to control challenge (13.0â±2.4 vs. 3.7â±1.1 respectively, p=0.003). there were no statistically significant differences in the nasal ph at 1h and 4h, but a significant decrease at 24h compared with baseline (-0.2â±0.1 vs. +0.14â±0.1, p=0.02). the change in ph from 0h to 24h correlated significantly and inversely with change in symptoms (r=-0.59, p=0.007) and number of sneezes (r=-0.65, p=0.002) after challenges. nasal pd did not change significantly after challenges. conclusion: allergen decreases nasal mucosal ph in the very late phase after challenge in allergic rhinitic subjects. decrease in airway mucosal ph during asthma exacerbations may be caused by aggravation of allergic inflammation. nasal potential difference does not change after allergen challenge. funding: niaid, acaai foundation introduction: uv induces differentiation of t-and b-lymphocytes, suppresses natural killer cells, renders a tolerogenic effect and induces apoptosis resulting in local and system immunosuppression. methods: lymphocytes were studied using indirect immunofluorescence methods employing monoclonal antibodies to cd-markers cd4, cd8, cd16, cd25, cd95, hla-i, and hla-ii, from the blood of 14 volunteers (aged 18-24 years) before uv exposure (control), after an exposure of blood in vitro to a dose 1.12 kj, and after 24 hours of exposure of the medial surface of the elbow joint in vivo (s=200 cm2), to a dose of 1.12 kj. results: the direct exposure of blood results in a significant reduction in the number of lymphocytes, probably due to direct phototoxic effects. significant modifications both of the aggregate number of lymphocytes and amount of t-helpers after uv exposure of the skin were not seen. after uv the number of cells which express the marker cd8 is sig-nificantly increased. exposure to uv in vitro induces reduced number of cells bearing cd16. however on in vivo exposure, a significant increase in number of cells which express cd16 was observed. uv exposure of blood reduced significantly the hla-i (9.67â±0.72 reduced to 0.32â±0.54) and hla-ii (dr) (21.56â±1.19 reduced to 18.45â±1.15) expression. uv exposure of skin increased significantly hla-i to 11.56â±0.81, while hla-ii (dr) was insignificantly decreased to 20.65â±2.01. cd95 expression increased from 22.43â±1.12 to 25.95â±1.08 due to blood uv exposure, while there were insignificant reductions in cd4 (39.33â±2.14 reduced to 36.62â±2.34) and cd8 (36.50â±2.03 reduced to 25.75â±2.74) expression. conclusion: uv starts a cascade-like response, including apoptosis, leading to changes in nk cell, helper and suppression lymphocyte numbers consistent with an immunomodulating effect of uv radiation. background: previous investigations have shown the involvement of histamine and histamine receptors (h 1 , h 2 , h 3 , h 4 ) in ige synthesis in atopic and lymphoproliferative diseases. ige responses may depend on the concentrations of histamine and histamine receptor antagonists and agonists. the goal of this investigation was to evaluate the role of the concentration of histamine and h 3 /h 4 antagonists along with the importance of pre-existing levels of ige in determining ige responses. methods: ige synthesis was studied in mnc cultures of patients (7-14 years old) having mild atopic asthma and rhinitis. all patients had high levels of total ige and specific ige to ragweed pollen, house dust mite, epidermal or mold allergens. patients were divided into two groups according to the serum ige and levels of spontaneous ige synthesis: group a -with low levels (4.8â±1.2 iu/ml) and group b -with high levels (9.7â±1.4 iu/ml). the serum level of total ige in group a was 426.1â±24.3 iu/ml and in group b was 570.2â±26.5 iu/ml. fub 181 hydrogenmaleate was used as an h 3 /h 4 specific antagonist. results: histamine in high concentrations (10 -5 m) suppressed and in low concentrations (10 -8 m) stimulated spontaneous ige synthesis. the h 3 /h 4 antagonist fub 181 activity depends on the pre-existing levels of ige. in high concentrations (10 -5 m), the antagonist increased ige synthesis only in group a, but not in group b having high spontaneous levels of ige synthesis. the synthesis in group a increased 1.36 fold (p<0.001), but h 3 /h 4 blockade cancelled the ige suppressive effect of high concentrations of histamine (10 -5 m). the addition of histamine into the mnc culture stimulated ige synthesis 1.56 fold. fub 181 had no effect on ige-stimulatory effects of low concentrations of histamine (10 -8 m). in allergen (ragweed)-stimulated mnc culture, fub 181 had a co-stimulatory effect on ige synthesis induced by histamine. conclusions: the ige stimulating response depends on the concentrations of histamine and the h 3 /h 4 antagonist as well as pre-existing levels of serum ige and ige spontaneous synthesis. introduction: preliminary data has shown that oral contraceptives can precipitate or worsen attacks of hereditary angioedema (hae). it is thought that estrogens affect the synthesis and degradation of bradykinin with resulting edema. objective: our objective is to determine if there is a difference among the various oral hormonal preparations, namely, combined monophasic, combined multiphasic, progesterone only, and hormone replacement therapy, in regards to their effect on frequency of hae exacerbations. methods: patients in this study consisted of women over the age of 18 diagnosed with hae who receive their care at our institution and women meeting the same criteria who are active in the hae foundation and have access to this association's web page. all patients answered a sixteen item questionnaire about past or present birth control pill or hormone replacement therapy usage and its impact on the frequency of exacerbations. results: of the patients who completed the questionnaire, 94% had taken or were currently taking oral contraceptive pills or hormone replacement therapy. of the women whom had taken monophasic birth control pills, 100% worsened with increased number and severity of exacerbations. however, only 75% of the women that had used combined multiphasic birth control pills worsened. all of the women who had tried progesterone only birth control pills worsened. of those on isolated estrogen for hormone replacement, 67% had an increased number of exacerbations. when androgens were used concurrently with oral contraceptive pills or hormone replacement therapy, exacerbations decreased to 60%. conclusion: we are not able to demonstrate a statistical benefit of one oral contraceptive pill preparation over another because of our sample size. however, our preliminary data supports that the oral contraceptive pill which produces less exacerbations and less symptoms is a combined multiphasic pill with concurrent androgen treatment. introduction atopic dermatitis (ad) is associated with multiple immunological abnormalities including imbalances in the subsets of blood circulating lymphocytes forming cellular infiltrates in inflamed skin. many studies of the functional and phenotypic properties of lymphocytes in ad have been limited to either peripheral blood or skin-infiltrating lymphocytes. the purpose of our study was to evaluate and compare the content and phenotypic properties of cd4+ lymphocytes from blood and inflamed skin of ad patients. materials and methods 15 adult (age 18-43 years) patients with chronic ad were selected by the criteria of hanifin. all patients gave written informed consent. the cd4+ lymphocytes of peripheral blood were phenotyped by flow cytometry. skin biopsies were obtained from eczematous areas, then cryosected and double immuno-histochemistry was performed. for phenotyping of lymphocytes in blood and skin a panel of monoclonal antibodies was used including cd3, cd4, cd8, cd25, hla-dr and cla. results immunophenotype analyses of the peripheral blood lymphocytes showed a predominance of cd3+cd4+ cells (86%â±21.3). most cells were cd4+hla-dr+ (14%â±12.6) and cd4+cd25+ phenotype (26%â±13.5). double immunohistochemistry of the skin biopsies revealed in the epidermis rare but constant presence of cd4+cd25+cells (1.0â±6.4 cells/mm 2 ) and cd4+hla-dr+ cells (1.2â±1.3 cells/mm 2 ). in the dermal inflammatory infiltrates the predominant cells were cd4+cd25+ (21.0â±9.7 cells/mm 2 ) and cd4+hla-dr+ (45.0â±6.7 cells/mm 2 ). the dominant inflammatory infiltrate consisted of cd4+cla+ phenotype (62.9â±23.5 cells/mm 2 ) cells. conclusion in ad skin inflammation is associated with the appearance in the circulation of cd4+ lymphocytes in activated form (cd4+hla-dr+) and lymphocytes with regulatory properties (cd4+cd25+). these lymphocytes are recruited from the circulation, having the skin homing properties (cd4+cla+), and form the main constituent of the dermal inflammatory infiltrate. contact dermatitis (cd) comprises a spectrum of inflammatory skin reactions usually caused by exposure to non-immunogenic low molecular weight substances (haptens). failure to diagnose the condition may result in a chronic and disabling condition with impaired quality of life. a 28 yr-old white male presented with a severe, symmetrically distributed facial maculopapular rash with secondary excoriated lesions. prior to the appearance of the rash, the patient had been applying lubriderm as a skin moisturizer for dry skin. a standardized thin layer rapid use epicutaneous (true) test containing 23 chemical agents suspended in a vehicle and attached to an adhesive backing was applied to the patient's back. at 48 hrs, a positive reaction was observed at the skin site of the paraben mix application. the lubriderm preparation used by the patient contained paraben, in contrast to a newer preparation, advanced therapy lubriderm, which is paraben-free. complete resolution of the facial lesions occurred following discontinuation of the lubriderm and the use of oral and topical corticosteroid therapy. this case report illustrates how a commonly used moisturizer can contain a sensitizing agent that could be detected by standardized patch testing which should be a part of the diagnostic armamentarium of every allergist-immunologist. we report how a commonly used and effective moisturizer (lubriderm) can cause severe allergic cd and that the use of a standardized thin layer rapid use epicutaneous (true) patch test panel can be an effective diagnostic tool for the detection of the offending agent. rationale: this study aimed to establish the possible differences of cutaneous sensitization to common aeroallergens in children under 1 years old. methods:the study was conducted between 2003-2004 and included 120 infants from silesia/southern part of poland/.these infants were refered to our allergy unit due to respiratory symptoms like rhinitis, otitis, pharyngitis, cough, bronchitis recurring;eye's symptoms/ conjunctivitis/ and oral symptoms. the skin prick test/ spt/ to major aerollergens in our environment(hdii, birch, alternaria, cladosporium and trees)were done to 6-months-old and 1-yearold children. a spt of 3 mm or larger was considered as positive. patients were classified into three groups: i:positive family atopy ii:smoking ciggarettemother/ or father iii:cat or dog at home iv: coexisting food allergy results:58, 8 % of examined children were boys. the prevalence of sensitisation found were as follows:latex 38, 1%, birch 32, 8%, dpii-28, 6%, grass pollen 28, 1%, alternaria-8, 4%. positive spts to latex were first seen in the 6-months-old group and this prevelance doubled until they finished 1 year. conclusion: in our study, cutaneuos sensitisation start to appear in 6-months-old chlidren. background: propylene glycol (pg) may induce allergic contact dermatitis (acd) and skin irritant reactions. topical formulations frequently contain pg. it is present in low concentration (5%) in pimecrolimus cream 1%, a non-steroid inflammatory cytokine inhibitor. objectives: this study was designed to assess the incidence of cutaneous responses to pimecrolimus cream in patients allergic to pg and to determine their nature (acd or irritation) and severity. methods: in this double-blind, randomized, vehicle-controlled, withinpatient study, 20 subjects allergic to pg underwent 48-hour patch-testing (pg 30 and 100%, pimecrolimus cream and vehicle) followed by a 7-day repeated open application test (roat). application sites were assessed by the investigator using a scale ranging from 0 (no reaction) to 7 (spreading bullous reaction) at 0, 48, and 120 hours after patch removal and at the completion of the roat. results: pg allergy was confirmed by patch-testing in 16 patients. two patients showed a positive patch-test reaction with pimecrolimus cream and vehicle, indicating an acd. in contrast, no patient demonstrated signs of acd when pimecrolimus cream was applied under normal conditions, i.e. without occlusion (roat). the statistical analysis showed that there were significantly less reactions at pimecrolimus patch-test sites than at the pg patch-test sites (p<0.01 for both pg concentrations, one-sided exact binomial test) and that the median severity was lower with pimecrolimus cream vs. pg (p=0.02 and p<0.01 for pg 30% and 100% respectively, wilcoxon signed-rank test). conclusions: this pilot study suggests that pimecrolimus cream 1%, when applied under normal conditions, can be used safely in patients with pg allergy. introduction: chronic idiopathic urticaria is not always responsive to antihistamine therapy. multiple alternative agents have been tried. we report two cases where tacrolimus has been successful in treating this condition. methods: this is a case report of two patients who have chronic idiopathic urticaria and have been treated with tacrolimus. patient #1 is a 27 y/o wm who pre-sented with urticaria that had been going on several months. the duration of his urticarial lesions was <24 hours his urticaria was treated with several medications including: hydroxyzine, cyproheptadine, ceterizine, montelukast, colchicine, and dapsone. he was intolerant to hydroxychloroquine. montelukast seemed to provide some modest benefit but he continued to have daily urticaria with >50 lesions/day after two months of therapy. tacrolimus was added to montelukast at 1mg bid. patient #2 is a 64 y/o wf with a history of biopsyproven urticaria. she had suffered from episodic urticaria since the age of six that was responsive to systemic steroids. after thirty years of having no urticaria, she developed recurrent urticaria at age 62. at this time, she was treated with hydroxyzine, doxepin, montelukast, loratadine, ceterizine, fexofenadine, colchicine, dapsone, and even prednisone with very little improvement in symptoms. she was then started on tacrolimus 1mg bid as monotherapy while she was having daily urticaria. results: after one month of treatment with tacrolimus 1mg bid, patient #1 had a decrease in the number of urticaria. his dose of tacrolimus was increased to 3mg/day, and after four weeks, his urticaria resolved. he remained on tacrolimus for a total of six months which was tapered and discontinued. he continues to be in remission from urticaria. patient #2 had complete resolution of her hives after just two doses of tacrolimus. after two months, she remains free of urticaria on tacrolimus 1mg bid. conclusion: for patients with severe chronic urticaria unresponsive to multiple therapies, tacrolimus, like cyclosporine, may be efficacious. tacrolimus may also be truly immunomodulatory and capable of inducing remission of urticaria. s.v. gerasimov * , lviv, ukraine. introduction. recent studies suggest that probiotics can be useful in prevention and treatment of atopic dermatitis (ad) in children. as clinical effect of probiotics varies greatly depending on the specific strain, we are currently conduct a search for the most promising probiotic to be used as complementary therapy in infants and young children with ad. methods. we studied 23 infants aged 25-51 weeks (36â±8) with ad established using hanifin and rafka criteria. patients were evenly allocated among probiotic (n=11) and nonprobiotic (n=12) treatment groups using quota allocation system. severity of ad was defined using scorad index and infant`s quality of life was assessed using idqol index. patients were examined before and 4 weeks after the treatment with/without probiotic powder formulation (b. infantis, l. acidophilus dds-1, 10 billion cfu/day, dds-junior, uas laboratories). we compared pre/posttreatment values of indices above and amount of mometasone furoate (0, 1%) used employing a paired or unpaired t-test. results. at baseline, infants in either groups were comparable on age, gender, duration of the disease, scorad and idqol indices, and medication taken for the previous 4 weeks. before entering the study all patients were maintained on allergen elimination diet due to milk and egg white allergy. after the treatment with/without probiotics the mean scorad index decreased from 42, 2 to 32, 7 (p=0, 14) and from 51, 5 to 31, 3 (p=0, 06), respectively. however, the mean amount of mometasone furoate used in non-probiotic treatment group was significantly greater (7, 8 g vs 2, 1 g, p=0, 03), despite the same recommendation on the use of the drug was given. additionally, parents of children taking probiotics reported an improvement in infant`s quality of life as seen on the decrease of idqol index from 21, 1 to 12, 0 (p=0, 04). in non-probiotic group decrease in idqol was not significant (24, 2 to 19, 5; p=0, 14) . there were no adverse events in any of the treatment groups. conclusions. our preliminary results suggest that some probiotics may have a corticosteroid-sparing effect and improve quality of life of infants with ad. there is a clear trend towards reduction of scorad index, which did not change significantly, probably due to a limited number of patients involved. introduction : the aim of this study was to analyze the risk factors of severe atopic dermatitis(ad) in the first 6 months of life. methods : the children aged less than 6 months with ad were divided into two groups according to six area six sign in atopic dermatitis(sassad) score. children with score less than 14 was classified as mild ad(n=90) and those with score above 15 as severe ad(n=44). these patients were fed with breast milk or cow's milk formula, and no allergenic food was given except rice and some vegetables. we analyzed the gender, feeding patterns, family history of allergy, number of siblings, total ige and specific ige to common food allergens (egg white, cow's milk, wheat, soy) by cap-feia assay. total eosinophil count was 1863â±2032 /ul in severe ad and 686â±539 /ul in mild ad(p<0.001). results : 1) total ige was 180.6â±245.1 u/ml in severe ad and 32.2â±50.7 u/ml in mild ad(p=0.002). specific ige to egg white, wheat and soy (19.03â±29.72 u/ml, 4.68â±10.62 u/ml, 7.07â±22.01 u/ml in severe ad; 1.78â±3.54 u/ml, 0.15â±0.99 u/ml, 0.09â±0.30 u/ml in mild ad; p<0.05) were associated with severe ad, but cow milk(4.34â±16.6 u/ml in severe ad, 0.80â±4.17 u/ml in mild ad) showed no difference. 2) gender, feeding patterns, family history of allergy and the number of siblings were not significantly associated with severe ad. conclusion : severe ad is associated with sensitization to food allergens in the first 6 months of life, although they are not fed with those foods. background: atopic dermatitis (ad), an inflammatory skin disease diagnosed primarily in children, has been shown to have a negative impact on quality of life (qol). pimecrolimus cream 1% is a non-steroid, topical calcineurin inhibitor with demonstrated efficacy in the acute treatment and longterm management of pediatric and adult ad. in a 24-week, double-blind, vehicle-controlled study, a secondary aim was to evaluate the impact on parent's quality of life of a pimecrolimus-based or corticosteroid (cs)-based treatment regimen of children with ad. methods: 275 children aged 3 months to 11 years (mean 5 years) with mild to severe ad were randomized 2:1 to receive treatment with pimecrolimus or vehicle cream. emollients for dry skin and pimecrolimus or vehicle bid were applied at the first signs of ad. for severe flares, a mid-potency topical cs (fluticasone or mometasone) indicated for once-daily use in ad replaced the evening study drug application for a maximum of 3 weeks or until all ad resolved. parents completed the parent's index of quality of life-atopic dermatitis (piqol-ad), a 28-item validated questionnaire, which measures parent's needs-based qol, at baseline, week 12, and study completion. change in piqol-ad scores at baseline and week 24 were compared between treatments. a negative change indicated improvement. results: parents of patients in both groups reported improvement in piqol-ad scores at week 24, with greater improvement for the pimecrolimus group. the mean change in piqol-ad score from baseline to week 24 was -3.3 in the pimecrolimus group vs. -2.6 in the cs-based treatment group (37.6% vs. 26.8% improvement, respectively). ancova analysis, using treatment and center as main effects and baseline score as a covariate, compared the change in piqol-ad score in two treatment groups. pimecrolimus treatment demonstrated a more favorable change (-1.2) which approached statistical significance [p=0.056; 95%ci= (-2.5, 0.0)]. conclusion: a treatment regimen utilizing pimecrolimus cream 1% had a beneficial effect on parent's quality of life compared to a corticosteroid-based regimen. this benefit was consistent with other measures of efficacy studied. introduction: atopic dermatitis is a chronic, inflammatory and recurrent skin disorder . its prevalence has increased in recent decades but little is known about it in mexico city. the aim of this study was to evaluate the prevalence and severity of atopic eczema in a pediatric population. methods: a cross-sectional questionnaire survey (isaac phase i questionnaire) was conducted on random samples of schoolchildren aged 6 to 7 years and 13 to 14 years from educational centers of four northern counties from mexico city. those children with a positive response to being questioned about the presence of an itchy relapsing skin rash in the last 12 months were considered to have atopic dermatitis. children whose symptoms resulted in sleep disturbance for 1 or more nights per week were considered to have severe atopic eczema. statistical analyses were done with spss 6.0 for windows, chi square . the size of the sample was determined folowing the isaac specifications and calculated with the statcal program. results: complete data was available for 3211 children aged 6 to 7 years in 45 schools and 3899 children aged 13 to 14 years in 47 high schools. 51.5% males and 48.5% females. response rates were high ( 91.4 % for those aged 6 to 7 years and 100% for those aged 13 to 14 years). the prevalence of symptoms of atopic dermatitis in the last 12 months was 9.6 % at age 6 to 7 and 9 % at age 13 to 14 years. medical diagnose of atopic eczema was 4 % and 2.4 % for children aged 6 to 7 years and 13 to 14 years, respectively. reported eczema accounted for 12.4 % and 11.3 % at age 6 to 7 and 13 to 14 years, respectively. children with symptoms of severe atopic dermatitis accounted for 1 % of all those with symptoms of atopic dermatitis. we also found that the majority of the children begun with skin manifestations of atopic dermatitis before the age of 4 years old. conclusions: the prevalence of atopic eczema was very similar for both groups of ages. our results agree with those found in other two studies acomplished in other mexican cities (cuernavaca and chihuahua) and with those from other countries of latin america like brazil, chile and costa rica. although, we found lowest values than those from sweden, japan and australia. studies that include objective skin examination are required to confirm these findings. a. kaplan 1* , s. meeves 2 , y. liao 2 , s.t. varghese 2 , g. georges 2 , 1. charleston, sc; 2. bridgewater, nj. introduction: the symptoms of chronic idiopathic urticaria (ciu) can have a profound impact on patient health and quality of life. the safety and efficacy of fexofenadine (fex) bid for the treatment of ciu has been previously established in two multicenter, double-blind, randomized, placebo-controlled trials. this study evaluated the efficacy and safety of a qd dose of fex hcl 180 mg, as this dosing schedule could offer advantages in terms of patient compliance and convenience. methods: this multicenter, randomized, double-blind, parallel-group, placebo-controlled study consisted of a single-blind placebo run-in period of 2-5 days, followed by a 28 (â±4)-day treatment period. males and females aged 12 years with a diagnosis of ciu and with active disease were enrolled. patients were randomized 2:1 to receive either fex hcl 180 mg qd or placebo qd. the primary endpoints were change from baseline in mean daily number of wheals (mnw score) and mean daily severity of pruritus (measured on a 5-point scale) over the 28-day treatment period, as assessed reflectively by the patient. secondary efficacy measures included a modified total symptom score (motss), comprising sum of number, frequency, size and duration of lesions, and severity of pruritus. mnw and pruritus severity were also assessed instantaneously at trough drug levels (immediately prior to dosing). results: over the 28-day treatment period, patients treated with fex (n=167) experienced significantly greater improvements in mnw and pruritus scores compared with the placebo group (n=92) (p<0.0001 for both). similarly, over the treatment period, and at individual weekly timepoints, the mean reductions in am reflective, pm reflective and mean daily motss were significantly greater for patients in the fex group compared with those in the placebo group (p 0.0052 for all comparisons). the mean reductions in instantaneous mnw and pruritus scores were greater with those who received fex than those who received placebo (mnw: p=0.0154; pruritus score: p=0.00088). there were no significant differences in the frequency of treatment emergent adverse events between the two treatment groups, and no clinically relevant changes were observed with respect to clinical laboratory data, vital signs or ecgs. conclusion: this study demonstrated that a qd dose of fex hcl 180 mg offers effective and well-tolerated relief from the symptoms of ciu. introduction: the wheals and pruritus associated with chronic idiopathic urticaria (ciu) are often so debilitating that they have a profound impact on patient quality of life. specifically, ciu has been shown to negatively affect patient mobility, sleep, energy levels, social interaction and emotional wellbeing. the purpose of this study was to examine the impact of treatment with fexofenadine hcl (fex) 180 mg on health-related quality of life (hrql) among patients with ciu. methods: as part of a multicenter, randomized, double-blind, parallel-group, placebo-controlled study, designed to evaluate the efficacy and safety of a once-daily dose of fex 180 mg in ciu, the impact of treatment on hrql was also examined. patients were asked to complete the dermatology life quality index (dlqi) and the work productivity and activity impairment questionnaire (wpai) at baseline and at weeks 2 and 4 (final visit or early termination). the primary endpoint was mean change from baseline in dlqi total score, using the mean data of evaluations performed at weeks 2 and 4 as the post-baseline measure. secondary endpoints included change from baseline in individual dlqi domains and wpai scores. additional analyses were conducted to examine the reliability, validity and responsiveness of the hrql measures. results: a total of 254 patients were included in the hrql population: n=163 fex, n=91 placebo. patients in the fex group experienced significantly greater improvements in the mean dlqi total score than those in the placebo group (p=0.0029). this pattern was repeated with respect to the individual domains of symptoms and feelings (p=0.0071), daily activities (p=0.013), leisure (p=0.0425) and personal relationships (p=0.0023). both treatment groups reported improvements in productivity, as measured by change from baseline using the wpai. patients randomized to fex experienced significantly less impairment while working (p=0.0455) and performing activities (p=0.0088) than those who received placebo. the dlqi was demonstrated to be reliable (cronbach's alpha=0.87); both the dlqi and wpai were found to be valid and responsive instruments in this ciu population. conclusion: this study demonstrated that a once-daily dose of fex 180 mg improves the hrql of patients with ciu, as assessed by change in dlqi total score. t. algozzine 1* , a. lee 2 , s. wong 3 , l. anzisi 4 , 1. manchester, nh; 2. centerport, ny; 3. syosset, ny; 4. mamaroneck, ny. symptoms of allergic and non-allergic rhinitis may significantly impact a patient's quality of life, by causing fatigue, headache, cognitive impairment and other systemic symptoms. appropriate management of allergic rhinitis is important in the effective management of coexisting or complicating respira-tory conditions (e.g. asthma, sinusitis, or otitis media). in addition, many commonly used over-the-counter (otc) antihistamines can cause performance impairment that may impact daily activities. we designed a brief ten-question allergy survey in an attempt to determine how patients were being treated for their allergies, evaluate a patient's self-assessed impact of their allergies on their daily activities, and identify any difference in treatment outcomes between primary care and allergist practices. patients were invited to complete the surveys anonymously. completed surveys were collected and entered into a microsoft access database for evaluation. minitab was utilized for statistical calculations. four hundred and thirty seven patients completed the survey. fifty-eight percent of survey respondents were women and 89% were adults (age >18). of those patients surveyed, 53% (n=233) were under the care of an allergist. compared to primary care, patients treated by allergists reported more allergies (3.4 vs. 1.8, p <0.001) and received more medications on average (2.3 vs. 1.4, p<0.001). pollen was the most common allergy type reported among all patients. while the majority of all patients (69%) received prescription medications, more primary care patients received no therapy or only otc treatment when compared to allergist patients (46% vs. 17%, p<0.001). seventy-four percent of primary care patients reported their allergy symptoms were controlled compared to 63% of allergist patients (p<0.05). patients receiving treatment from an allergist reported less impact of their allergies on daily activities (a lower score signified less impact) compared to patients seen in primary care (2.27 vs. 3.84, p<0.001). the results of our survey suggest that allergists treat more complex patients. these patients had multiple allergies, more uncontrolled symptoms, and utilized more prescription medications. despite these findings, patients treated by allergists reported less impact of allergies on their daily activities. introduction the patogenic mechanism of nasal polyps are unknow.they frecuently are associated with aspirin intolerance, intrinsic asthma, chronic sinusitis, young sindrome, cystic fibrosis, kartagener syndrome and churg-strauss syndrome.chemical mediators found in nasal polyps are as follows: histamine, serotonin, leukotrienes, norepinefrine and possibly pgd2.recently, leukotrienes have been implicated in mediation of bronchoconstriction and inflammatory leukotriene levels have also been shown to be elevated in some patients with sinonasal polyposis (figure 1 ) hypothesis antileukotrienes might play a significant role in controlling polyposis and symptoms secundary to sinonasal disease, and they might viable alternative to longterm, oral steroid therapy and repeat surgical debridement purpose this study was undertaken to evaluate the potential role of leukotriene receptor antagonist on recurrent polyposis associated with asthma and improvement of some factor implicated with them design of study:clinical assay, prospective, triple blind. materialsand methods:the study involved 30 patients 12 (40%) males and 18 (60%) women, mean age 20.7 years, with a range 16-45 years selection criteria:inclusion criteria: nasal polyps associated with astha, allergic or non allergic.exclusion criteria: patients with any concurrent illness, use of sistemic or local corticosteroidsor any kind of antileukotriene within 1 month prior to the beggining of the study.we made an alleatory selection 10 patients recived montelukast (10 mgs/day), and nasal steroids, beclometasone (600 mgs/day) for 6 months.10 patients recived loratadine plus pseu-doefedrin5mgs/120 mgs/day and nasal steroids, beclometasone, 10 patints were operated of fees and transnasal endoscipic polypectomy plus montelukast and nasal steroids, beclometasone ige serum levels 2.skin prick test 3.sensitivity in vitro 4.ct scan 5. celullarity on nasal lavage (eosinophils and neutrophils)6.proinflamatory cytokines (il4-il5-il8) on nasal lavege by elisa test. results ther was a tendency for more improvement of the group 3 ( surgery-montelukast-local steroid) of symptoms and objective measurments, in second place of improvement the group 1 montelukast-local steroid) and the worse on improvement subjective and objetive was group 2 local steroid and loratadine-pseudoefedrine use as placebo introduction: sensory perceptions of intranasal corticosteroids (ins) vary among products and can be unpleasant and affect adherence to therapy. methods: we conducted a cross-sectional study of 120 patients across 4 allergy and immunology clinics in the united states. respondents were asked to choose between pairs of hypothetical ins that differed by sensory attribute composition. based on prior research, we measured 6 salient sensory attributes: smell, taste, aftertaste, throat rundown, nose runout, and feel of spray in nose/throat. each attribute was described in 3 intensity levels, such as "no taste" (low), "weak taste" (moderate), and "strong taste" (high) ( table) . other outcomes included an importance score for each sensory attribute and patients' willingness to adhere to an ins having the lowest levels of each sensory attribute compared to one with moderate levels. results: preferences decreased with increasing intensity level of each sensory attribute. the most important attribute was aftertaste in 28% of patients, taste in 19%, throat rundown in 18%, nose runout in 12%, smell in 11%, and feel of spray in 7%. only 5% had more than one attribute tied for most important. if instructed to take ins daily for 3 months, 77% of patients stated that they would definitely be able to follow their doctor's advice (willing to adhere) if given an ins containing the lowest level of each sensory attribute compared with 4% for one having moderate levels (p<0.01). conclusions: patients' preferences decrease with increasing intensity levels of each sensory attribute and affect patients' willingness to adhere. tailoring ins to patient preferences may lead to improved treatment satisfaction and adherence. introduction:a high prevalence of rhinitis and asthma comorbidity has been persistently noticed in epidemiological studies in last years. our objective was to evaluate the prevalence of rhinitis and asthma comorbidity in a portuguese population including both atopic and non-atopic patients. methods: a retrospective study was performed. clinical records from patients attending an immunoallergology outpatients'clinic during 39 months (from january 1998 until june 2001) were reviewed. data were collected concerning clinical history of rhinitis and/or asthma, aeroallergens'skin prick tests and respiratory function evaluation. results: among the 3582 patients attending an appointment during the study period, 2448 (68.3%) had rhinitis and/or asthma (56.2% f; 43.8% m;mean age 26 +/-19 years). patients with respiratory disease included 1983 atopic (81.0%) and 465 non-atopic 19%). diagnosis of asthma, rhinitis or of both diseases was made in 170 (8.6%), 581 (29.3%) and 1232 (62.1%), respectively, in atopic patients and in 56 (12.0%, 265 (57.0%) and 144 (31.0%), respectively, in non-atopic patients. rhinitis was diagnosed in 87.9% of atopic asthmatic and in 72-0% of non-atopic patients with asthma. in patients with rhinitis, asthma was also diagnosed in 67.9% of atopic patients and 35.2% on non-atopic patients. conclusions: in this study population rhinitis was frequently diagnosed in patients with asthma and vice-versa, thereby leading to a high prevalence of this comorbodity. this association was more frequent in atopic patients. our data are comparable to those we found in recent literature. these results suggest that clinical investigation of asthma should be mandatory in management of patients with rhinitis as well as asthmatic patients should be routinely submitted to clinical evaluation of rhinitis. introduction: cetirizine hcl (c) has been shown to be more effective than fexofenadine hcl (f) in controlling seasonal allergic rhinitis (sar) symptoms at 21-24 hr post-dose, however, the efficacy of c and f was comparable between 0-5 hr post-dose. response to treatment in the middle of the dosing interval needed to be addressed. methods: this randomized, double blind, placebo (p)-controlled study was designed to compare the efficacy and tolerability of a single dose of c 10 mg, f 180 mg, and p between 5-12 hr postdose in ragweed-sensitive sar subjects. subjects meeting entry criteria were exposed to pre-determined controlled levels of ragweed pollen in the eeu during priming and double blind treatment. subjects assessed rhinitis symptoms at half hr intervals during pollen exposure; at priming, at the qualifying period, at baseline, and from 5-12 hr post-dose. the primary efficacy endpoint was the change from baseline in total symptom severity complex (tssc) score at 12 hr post-dose. tssc score was the sum of severity ratings (0=absent to 3=severe) for 4 symptoms: runny nose, sneezing, itchy nose/palate/throat and itchy/watery eyes. results: a total of 599 subjects (mean age 32.8 y; 58.9% females) were randomized: c, 249; f, 250; p, 100. baseline characteristics were comparable among groups; tssc: c=9.2, f=9.2, p=8.9. c produced a 26% greater reduction in tssc score at 12 hr (-4.3, p=0 .001) and a 14% greater reduction overall (i.e., average over the 5-12 hr post-dose period) (-5.0, p=0.006) compared with f (-3.4, -4.4, respectively). both c and f reduced tssc score more than p (12 hr, -1.9; overall, -2.3, p<0.001) including all individual symptoms (p<0.05). c however, was more effective than f for runny nose and sneezing at 12 hr and overall, itchy/watery eyes at 12 hr, and itchy nose/throat/palate overall (p<0.05). rates of discontinuation due to adverse events (aes) were low: c, 0.4%; f, 0%; p, 1.0%. the incidence of treatmentemergent aes was similar: c, 25.3%; f, 29.6%; p, 35.0%. somnolence occurred in 0.8% of subjects on c, and 0% on f or p. conclusion: c produced a greater improvement in rhinitis symptoms compared with f and p, at 12 hr post dose and over the 5-12 hr post-dose period. all treatments were safe and well tolerated. background: medication utilization patterns of patients suffering from seasonal allergic rhinitis (sar) are not well documented, and although many anti-allergic medications are prescribed for daily use, actual usage is unknown, but recognized to be quite variable. methods: 1821 subjects with positive ragweed skin tests were mailed a survey during the third week of ragweed season, soliciting the nature and severity of sar symptoms, usage patterns and reasons for choice of anti-allergic medication. results: 550 subjects completed the survey (30.2%). the prevalence of symptoms were, in decreasing order: sneezing (91.5%), runny nose (82.4%), itchy/gritty eyes (80.4%), stuffiness (78.5%), itchy nose (71.3%), watery eyes (64.7%), itchy palate/throat (56.9%), post-nasal drip (55.6%), red/burning eyes (49.1%), headache (36.5%), itchy ears (34.0%), cough (30.0%), shortness of breath (15.7%), and wheeze (15.5%). sar patients used antihistamines most frequently (94.7%), followed by decongestants (63.1%), combination (antihistamine/decongestant) products (52.0%), and intranasal corticosteroids (42.5%). medications were mostly taken intermittently rather than daily (antihistamines 68.0%; nasal corticosteroids 71.8%), conclusion: a constellation of nasal symptoms were the most common seasonal allergic manifestations, followed by ocular, palatal and ear irritation. antihistamines were the most frequently used medication to treat symptoms, succeeded by decongestants (alone or in combination). a significant proportion of subjects took their allergy medication, including nasal corticosteroids, intermittently rather than regularly, underscoring the relevance of single-dose evaluations of drug efficacy. a.k. ellis * , e. rafeiro, j.d. ratz, j.h. day, kingston, canada. background: traditional assessment of seasonal allergic rhinitis (sar) medication efficacy utilizes randomized controlled trials over 2-4 weeks in season. an additional study method employs single-dose responses using controlled allergen challenge such as the environmental exposure unit (eeu). a comparison of allergic symptoms generated by controlled allergen challenge to those occurring in ragweed season symptoms has not been done. methods: 1821 subjects with known sar to ragweed were mailed a survey during the third week of ragweed season, soliciting the nature and severity of sar symptoms. subjects participating in a subsequent controlled allergen challenge study using the eeu, were again asked to complete a similar survey that documented symptoms generated in this model. those who completed both surveys comprised the primary analysis group. results: 550 subjects completed the ragweed season survey, 516 subjects completed the eeu survey, and 270 completed both. symptoms generated by eeu exposure were similar to those elicited during ragweed season, with the exception of cough (68% vs. 27%, respectively, p <0.01). subjects reported that symptoms were more severe in the eeu than those experienced on a typical ragweed season day, but less severe than those during peak ragweed season days. conclusion: allergic upper respiratory tract symptoms produced during controlled ragweed pollen exposure in the eeu were similar in nature and degree to those expe-rienced during ragweed season, supporting evidence that the eeu is a valid model for studying sar. r. nave 1 , m.a. wingertzahn 2* , s. brookman 3 , s. kaida 4 , t. shah 2 , 1. konstanz, germany; 2. florham park, nj; 3. princeton, nj; 4. tokyo, japan. rationale: ciclesonide (cic) is a new corticosteroid under development for treatment of allergic rhinitis (ar). cic is a pro-drug that is hydrolyzed to the active metabolite desisobutyryl-ciclesonide (des-cic) in the target tissue. cic has low oral bioavailability and is highly bound to plasma proteins; therefore cic administered as a nasal spray is expected to have minimal local and systemic effects. objective: the primary objective was to evaluate the safety and tolerability of repeated escalating doses of cic (50-800 mcg/day) given as a nasal spray for 14 days to healthy and asymptomatic subjects with sar. secondary objectives were to determine pk of cic and des-cic and to evaluate the effect of cic on endogenous cortisol. methods: this was a single-center, randomized, placebo-controlled, double blind, modified sequential dose study. six cohorts were randomized. cohorts i-v consisted of healthy subjects given doses of cic up to 400 mcg bid. cohort vi (asymptomatic sar subjects) received 400 mcg bid. each cohort was comprised of 6 subjects who received cic and 2 subjects who received placebo. safety assessments were conducted by recording adverse events (aes), clinical laboratory, eye, and nasal examination findings. serum and urine samples were taken for evaluation of cortisol levels. cic and des-cic serum concentrations were determined by lc-ms/ms. results: no trend was observed for aes when comparing cic and placebo treatment. additionally, no subject experienced a serious ae or withdrew from the study due to an ae during the trial. cortisol levels showed no differences between the dose groups or between activetreated subjects and placebo-treated subjects. additionally, serum concentrations of cic and des-cic in the majority of serum samples were shown to be below the lower limit of quantification (lloq; 25 pg/ml for cic and 10 pg/ml for des-cic), therefore no descriptive statistics could be calculated. con-clusions: ciclesonide nasal spray, at the doses evaluated, was safe and well tolerated in healthy and asymptomatic sar subjects with no detectable effects of cic on serum or urinary free cortisol concentrations. additionally, no pk parameters could be calculated for cic nasal spray, as it was virtually undetectable in serum despite the use of a sensitive assay. the preferred treatment for allergies is avoidance. air filtration is logical but room air purifiers have been of limited efficacy. zephyrâ�¢ is a new device which creates an envelope of air 99% free of allergenic particles around the head of the sleeping person. allergic rhinitis frequently causes daytime somnolence. this is a pilot study of the effectiveness of zephyr on symptoms of seasonal allergic rhinitis (ragweed hay fever) and on daytime sleepiness. methods: subjects age 12 to 45 with ragweed hay fever were studied in the ragweed season using each subject as his/her own control. usual allergy medicines were not allowed, except loratadine for rescue. outcome measures were a symptom score, juniper rhinitis quality of life questionnaire, a tolerability rating, and epworth sleepiness scale. during the first week subjects qualified by symptom scores, then entered the one-week treatment period with zephyr. the third week was a post-treatment observation period. results: of 13 participants, 10 (77%) showed symptom improvement. the whole group averaged 26% reduction in morning symptoms and 24% reduction in evening symptoms. sleepiness scores improved 29%. rhinitis qol improved 33%. the zephyr system was well tolerated as evidenced by the response to several statements regarding zephyr use and tolerability: (scoring: 1 = strongly disagree, 5 = strongly agree) "the system did not bother me while i was sleeping." (mean score 4.8) "the noise level did not affect my ability to sleep." (mean score 4.7) "the temperature was just fine for me." (mean score 4.8) "the system did not get in my way during sleep." (mean score 5.0) conclusions: zephyr significantly reduced seasonal hay fever symptoms and daytime sleepiness, improved quality of life, and was well-tolerated by subjects. zephyr may provide maximal environmental control of bedroom allergen exposure irrespective of ambient airborne allergen levels in the room. nasal congestion is an important symptom that is associated with significant morbidity in the rhinitis sufferer. disrupted sleep leading to daytime fatigue, loss of concentration, and decreased productivity are potential results of this symptom. a study employing online interviews with 1200 rhinitis sufferers from harris interactive's online database was conducted in order to understand the symptoms they identify with most and to determine the impact nasal congestion had on their daily activities. respondents were at least 18 years of age and experienced nasal congestion from seasonal allergic, perennial allergic, and/or perennial non-allergic rhinitis. the results showed that 60% of the respondents agreed nasal congestion is the most bothersome symptom of rhinitis; 37% experienced nasal congestion daily, while 35% experienced it several times per week. not surprisingly, sleep was the most important factor affected by nasal congestion. two-thirds (66%) of the respondents felt their sleep had been moderately or significantly impacted by nasal congestion, and more than half (56%) felt it was difficult to get a good night's rest because of congestion. sleep was interrupted or disturbed by nasal congestion approximately 3 nights per week, on average. furthermore, sleep disruption from nasal congestion contributed to daytime fatigue; 62% of the respondents indicated that they were typically tired or fatigued during the day when they experience nasal congestion. in addition, 42% felt that activities requiring concentration, such as reading, were moderately or significantly impacted by nasal congestion. this study confirms that nasal congestion causes the rhinitis sufferer significant problems beyond that of just a stuffy nose. consequently, healthcare providers need to ensure that their rhinitis patients who have nasal congestion as their primary complaint are managed effectively. desloratadine (dl, clarinex â® ) is a non-sedating oral antihistamine that is metabolized to 3-oh dl. however, a phenotypic polymorphism has been observed in some patients that results in reduced formation of 3-oh dl. the prevalence and safety profiles of such poor metabolizers of dl were examined in pharmacokinetic and clinical trials. a poor metabolizer was defined as a subject having a 3-oh dl to dl auc ratio of <0.10, or a dl half-life of 50 hours. in pediatric studies, where a sparse sampling approach was uti-lized to screen for poor metabolizers, a plasma concentration ratio of 3-oh dl to dl of <0.10 at 12 hours classified a subject as a poor metabolizer. a total of 3, 748 adult and pediatric subjects (2-70 years old) were phenotyped with a single dose of dl or loratadine. the overall prevalence of the poor metabolizer phenotype was 6% (228/3748). this prevalence was comparable for adult (70/1194, 6%) and pediatric subjects (158/2554, 6%), and greater in both populations among blacks (16% pediatric, 18% adult) than caucasians (3% pediatric, 2% adult). pharmacokinetic analysis found that exposure to dl was approximately 6-times higher in poor metabolizers than in normal metabolizers. there was no apparent difference in dl exposure among poor metabolizers in different age groups (6-months to <2-years, 2-to <6-years, 6-to <12-years, and 12-years) when treated with age-appropriate doses. the multiple-dose (7-35 days) safety profile of dl was examined in pediatric poor metabolizers (2-11 years) in 5 placebo-controlled trials, and in adult poor metabolizers (20-70 years) in pharmacokinetic trials. pooled ae rates were low and comparable between the poor metabolizer and placebo subjects, as shown in the table below with all aes that appeared in >2% of subjects in any treatment group (or >3% in adult poor metabolizers). there was no difference in cardiovascular safety profile or ecg results (including qtc interval) among these groups. in conclusion, (1) expression of the dl poor metabolizer phenotype is independent of age, but higher in blacks than in caucasians, (2) exposure to dl in poor metabolizers is independent of age when administered at age-appropriate doses, (3) the safety profile of dl poor metabolizers is not different from that of placebo at all ages down to at least 2-years old. these results are consistent with the high therapeutic index of dl. desloratadine (dl, clarinexâ®) is extensively metabolized to 3-oh dl and subsequently glucuronidated. the enzyme responsible for the formation of this active metabolite is unknown. poor metabolizers of dl represent a subset of the population that has a reduced ability to form 3-oh dl. a poor metabolizer was defined as a subject having a 3-oh dl to dl exposure ratio of <10%, or dl half-life of 50 hr. pk parameters from adult and pediatric poor metabolizers following repetitive administration of dl were characterized in clinical pharmacology trials and are summarized in the table below. exposure to dl [auc(0-24hr)] in poor metabolizers was approximately 6-fold greater than the corresponding values in normal metabolizers; cmax was 3to 4-fold greater. the magnitude of the reduction in the formation of 3-oh dl and the concurrent increase in exposure to dl associated with the poor metabolizer phenotype was similar in pediatric and adult subjects at age-appropriate doses. despite the increased exposure to dl in poor metabolizers, there was no increase in adverse event frequency or changes in electrocardiographic parameters. a: dose normalized to 5 mg. b: least squares mean ratio: anova of log-transformed data extracting sources of variation due to age group and metabolizer status. the ratio is a contrast of metabolizer status. c: lower and upper 90% confidence interval based on log-transformed data. introduction: many patients with seasonal allergic conjunctivitis (sac) complain of symptoms of dry eyes. we have previously reported that patients with sac experience discomfort from dry eyes anywhere between 2 to 6 days per week and that the overall severity of the dry eye symptoms tend to range from mild to severe. this preliminary cross over study evaluated the benefits of nedocromil sodium 2% ophthalmic solution used twice daily, compared with nedocromil sodium 2% ophthalmic solution used with refresh (ocular lubricant), for the treatment of dry eye symptoms in association with sac during 8 weeks. methods: patients who had a minimum of two-year history of sac and dry eyes, a positive skin prick test towards grass pollen and between the ages of 18 to 65 were enrolled. patients were evaluated on four visits and completed a daily diary, which included scales for grading allergic conjunctivitis and dry eye symptoms. at each clinic visit they completed the osdi and the rqlq (rhinitis quality of life questionnaire). the osdi (ocular surface disease index) was developed to assess dry eye symptoms and the impact on vision related functioning. the rqlq evaluates quality of life in patients with allergic conjunctivitis. at the last visit, both the patient and the physician assessed the treatment and the effectiveness, if any, of the addition of refresh to nedocromil sodium 2% ophthalmic solution. daily grass pollen counts were preformed using a burkhard sampler. results: 19 patients (7 male and 12 female) were enrolled and 3 dropped out. patients experienced minimal symptoms at the start of the season due to low concentration of grass pollen. preliminary analysis carried out using a paired t-test was preformed on the ocular average scores for the two treatment periods. there was no significant difference between the 2 treatment groups for redness, light sensitivity and tearing of the eyes. a reduction in dry eye symptoms was reported in all patients using refresh eye drops and the number of drops required varied between patients. during the treatment periods there was improvement in patients rqlq. conclusion: patients preferred the addition of refresh eyedrops in alleviating sac and dry eye symptoms. further studies need to be carried using refresh eye drops in larger number of patients with sac and dry eye symptoms. c. laforce * , raleigh, nc. introduction: the objective of this study was to determine the ability of azelastine nasal spray to improve rhinitis symptoms and quality of life parameters in seasonal allergic rhinitis patients remaining symptomatic after treatment with fexofenadine. methods: this placebo-controlled, double-blind study began with a 1-week, open-label lead-in period, during which patients received fexofenadine 60 mg bid. after 7 days, patients who improved less than 25% to 33% on fexofenadine were randomized to treatment for 2 weeks with: (1) azelastine nasal spray, (2) azelastine nasal spray plus fexofenadine, or (3) placebo. the primary efficacy variable was the change from baseline to day 14 in thetotal nasal symptom score (tnss), which consisted of runny nose, sneezing, itchy nose, and nasal congestion scores recorded twice daily in patient diary cards. in addition, quality of life was assessed using the rhinitis quality of life questionnaire (rqlq). results: after 2 weeks of treatment, azelastine nasal spray (p<.01) and azelastine nasal spray plus fexofenadine (p<.01) significantly improved thetnss compared to placebo. based on 90 patients with complete tnss and rqlq data, the overall rqlq score also was significantly (p<.01) improved compared to placebo. conclusions: azelastine nasal spray was an effective treatment for patients with seasonal allergic rhinitis who did not respond well to fexofenadine and significantly improved quality of life parameters compared to placebo. the results of this study indicate that azelastine nasal spray is an important alternative to oral antihistamines and should be considered in the initial management of seasonal allergic rhinitis. w. berger * , mission viejo, ca. objective: to evaluate improvement over time with azelastine nasal spray in the treatment of patients with moderate-to-severe seasonal allergic rhinitis (sar) who remained symptomatic after treatment with loratadine or fexofenadine. methods: the studies were 2-week, multicenter, double-blind, placebo-controlled trials that began with a 1-week, open-label lead-in period in which patients received either loratadine 10 mg qd (study no. 1) or fexofenadine 60 mg bid (study no.2). patients who improved <25%-33% with loratadine were randomized to treatment with: (1) azelastine nasal spray 2 sprays/nostril bid, (2) azelastine nasal spray 2 sprays/nostril bid plus loratadine 10 mg qd, (3) desloratadine 5 mg qd, or (4) placebo. patients who improved <25%-33% with fexofenadine were randomized to treatment with: (1) azelastine nasal spray 2 sprays/nostril bid, (2) azelastine nasal spray 2 sprays/nostril bid plus fexofenadine 60 mg bid, or (3) placebo. the primary efficacy variable was the change from baseline to day 14 in the total nasal symptom score (tnss), consisting of runny nose, sneezing, itchy nose, and nasal congestion. symptom severity was recordedam and pm in diary cards on a 4-point scale (0=none; 1=mild; 2=moderate; 3=severe). results: in both studies, patients treated with azelastine nasal spray experienced increasing improvement intnss over 14 days of treatment.the improvements were approximately 2-fold greater than placebo at each day of the study, and the differences from placebo were statistically significant (p<.05) at days 2, 7, 14, and overall. in study no. 1, 33% of patients treated with azelastine had >30% improvement in tnss compared to 17% in the placebo group. in study no. 2, 29% of patients treated with azelastine had >30% improvement in tnss compared to 12% in the placebo group. conclusions: azelastine nasal spray was effective in treating patients with moderate-to-severe sar who remained symptomatic after treatment with either loratadine or fexofenadine. azelastine demonstrated first-day effectiveness, and patients treated with azelastine experienced increasing improvements in rhinitis symptoms over the 14-day study periods.azelastine nasal spray is an effective treatment alternative to oral loratadine or fexofenadine and an appropriate first-line therapy in the management of sar. introduction: a large, open-label, azelastine (astelin) nasal spray patient experience trial was conducted in patients with sar, vmr, or mixed rhinitis (allergic rhinitis with nonallergic triggers). this analysis evaluated the effect of azelastine nasal spray in treating rhinitis symptoms in a subset of patients with a history of asthma or sinusitis. methods: patients were entered into an open-label protocol and treated for 2 weeks with azelastine nasal spray at a dosage of 2 sprays per nostril bid. after 2 weeks, the patients completed a questionnaire that assessed onset of action, symptom improvement, satisfaction with therapy, and quality of life. results: from a total of 4364 rhinitis patients who received azelastine monotherapy during the 2-week study period, data were analyzed for patients with asthma (n=260) or sinusitis (n=346). a greater percentage of these patients had severe rhinitis compared to the overall population. nasal congestion and postnasal drip were reported as the most bothersome rhinitis symptoms. after 2 weeks of treatment with azelastine nasal spray, >80% of patients with asthma and >85% of patients with sinusitis reported some or complete control of congestion and postnasal drip. in addition, >62% of patients with asthma reported chest tightness, shortness of breath, and wheezing were somewhat or completely controlled, and >77% of patients with sinusitis reported that headache and facial pain were somewhat or completely controlled during treatment with azelastine nasal spray. conclusion: azelastine nasal spray provided effective control of rhinitis symptoms, including nasal congestion and postnasal drip, in patients with a history of asthma or sinusitis. introduction: epinastine is an antihistamine with mast cell stabilization and anti-inflammatory properties. epinastine 0.05% ophthalmic solution was evaluated for treatment of allergic signs and symptoms elicited by feline dander in a cat exposure room. methods: participants (n=30) were aged 18 years, with a history of ocular allergy to cats, a positive skin prick reaction to cat dander, and an ocular itching score of 2 (on a 0-4 scale) within 30 minutes of entering the cat room. subjects wore a tb mask while in the cat room to reduce the effects of inhaled allergen. after 30 minutes of exposure, 1 drop of epinastine hcl 0.05% was instilled in one eye, and olopatadine 0.1% was instilled in the fellow eye. environmental exposure to cat dander continued for another 60 minutes. prior to instillation, and at 5, 15, 30, 45 and 60 minutes after instillation, conjunctival hyperemia and chemosis (scales of 0-3), and ocular itching, tearing, ocular burning, nasal itching and rhinorrhea (scales 0-4) were assessed. results: instillation of a single drop of ophthalmic epinastine significantly reduced ocular itching from a mean pre-instillation score of 2.27 to a mean score of 0.37 at 60 minutes after instillation (p<.001), despite continuous exposure to cat dander during the entire period. similarly, ocular burning, tearing, and hyperemia were significantly reduced by epinastine treatment (p<.002). chemosis was only weakly induced by cat room exposure, with a mean pre-instillation score of 0.167; however, epinastine treat-ment decreased that to 0 (p=.023). instillation of epinastine also significantly reduced nasal itching and rhinorrhea scores (p<.043). results for olopatadine were not statistically different; however, the change from baseline in itching scores in the epinastine-treated eyes was greater than that seen in the olopatadine-treated eyes at the majority of timepoints. conclusion: ophthalmic epinastine is indicated for the prevention of itch associated with allergic conjunctivitis.this study shows that treatment of cat-sensitive subjects with ophthalmic epinastine following environmental exposure to cat dander significantly reduced ocular itching and other signs and symptoms of allergic conjunctivitis. objective: the objective of this study was to evaluate azelastine (astelinâ®) nasal spray, cetirizine (zyrtecâ®), fluticasone (flonaseâ®), and placebo in the treatment of patients with symptomatic seasonal allergic rhinitis. methods: this was a double-blind placebo-controlled pilot trial in 60 patients with seasonal allergic rhinitis. the study began with a 1-week, placebo lead-in period, followed by a 1-week blinded treatment period (day 1 to day 7). efficacy variables were: (1) change from baseline to day 7 in the total nasal symptom score (tnss; consisting of rhinorrhea, sneezing, itchy nose, and nasal congestion); (2) onset of action based on tnss over the 4 hours following initial administration of study drugs; and (3) change from baseline to day 2 (24-hour change) in tnss. tnss was scored twice daily (am and pm) on a 4-point rating scale (0=none, 1=mild, 2=moderate, 3=severe). patients recorded a minimum 12-hour tnss of 8 on at least 3 days during the lead-in period, and a congestion score of 3 on at least 3 days to qualify for entry. qualified patients were randomized to treatment with: (1) azelastine nasal spray 2 sprays per nostril bid plus placebo capsules qd; (2) cetirizine 10-mg tablets qd plus placebo nasal spray; (3) fluticasone 2 sprays per nostril qd plus placebo capsules qd; or (4) placebo nasal spray plus placebo capsules. results: azelastine significantly (p<.05) improved the tnss compared to cetirizine, fluticasone, and placebo beginning 30 minutes after initial administration; cetirizine significantly improved tnss versus placebo at 150 minutes; fluticasone showed no significant differences from placebo over the 4-hour evaluation period. azelastine significantly (p<.05) improved the tnss at day 2 (24-hour change from baseline) compared to cetirizine, fluticasone, and placebo. conclusions: azelastine nasal spray improved the tnss in patients with moderate-to-severe seasonal allergic rhinitis. in addition, azelastine nasal spray demonstrated a 30-minute onset of action and significantly improved tnss versus cetirizine, fluticasone, and placebo 24 hours after initial administration. introduction: epinastine, an antihistamine with mast cell stabilization and anti-inflammatory properties, has been developed for the treatment of allergic conjunctivitis. the efficacy and tolerability of epinastine was assessed and compared with levocabastine. methods: eligible patients for this randomized, double-masked, parallel-group, active-controlled environmental clinical trial were 18-65 years old with a recent diagnosis of seasonal allergic conjunctivitis. patients instilled 1 drop epinastine hcl 0.05% or levocabastine 0.05% ophthalmic solution in each eye bid for 6 weeks. patients and investigators assessed efficacy and tolerability at study visits on days 0 (baseline), 7, 14, 28, and 42, and assessed overall efficacy and tolerability at study exit. adverse events were monitored. results: epinastine provided superior itch relief compared with levocabastine (p=.048; see figure) ; mean ocular itch scores over 4 treatment visits were 0.79 for epinastine and 0.97 for levocabastine (0-3 scale; 3=worst; baseline scores were 2.08 for epinastine and 2.06 for levocabastine). the mean summed score (ocular itching, tearing, and foreign body sensation) over 4 treatment visits was significantly better for epinastine than levocabastine (p=.010).at study exit, 53% of epinastine-treated patients rated overall efficacy "very good" (the highest possible rating), versus 34% of levocabastine-treated patients. at 5 minutes postinstillation, 77% of epinastine-treated and 75% of levocabastine-treated patients rated tolerability "very good". at study exit, 80% of epinastine-treated and 75% of levocabastine-treated patients rated overall tolerability "very good", with investigator ratings being similar. treatment-related adverse events occurred in 7.1% of epinastine-treated patients (most frequent: eye pain and skin itching, 1.2% each) and 10.3% of levocabastine-treated patients (most frequent: double vision, 3.4%; influenza-like symptoms, 2.3%); most aes were mild or moderate. conclusion: our results confirm the therapeutic potential for epinastine as an efficacious treatment suitable for long-term use throughout the allergy season. ophthalmic epinastine was superior to levocabastine for relief of ocular symptoms in patients with allergic conjunctivitis and was well-tolerated. since chronic inflammation is the histopathologic landmark of otitis media with effusion, clinical observations have led us to believe that the combination of a cysteinyl leukotriene receptor antagonist montelukast with an oral antibiotic may be more efficacious than monotherapy with an oral antibiotic in the treatment of serous otitis media. we studied twenty pediatric patients (age 3 years to 6 years) in a randomized open labeled 2-week trial to compare the efficacy of the combination montelukast (4 mg or 5 mg chewables tablets qd dosed according to patient's age) with an oral antibiotic amoxicillin/clavulanate potassium (90 mg/kg/day in 2 divided doses every 12 hours) to a monotherapy with an oral antibiotic amoxicillin/clavulanate potassium for the treatment of otitis media with effusion. the efficacy of treatment options was assessed using pneumatic otoscopy, impedance tympanometry, and audiometry to monitor the clinical course of the middle ear effusion in both treatment groups. in the combination group montelukast and antibiotic a resolution of otitis media with effusion occured at the 7th day. in contrast in the group treated with monotherapy with the oral antibiotic the resolution of otitis media with effusion occured on the 14th day. in conclusion, the combination of montelukast plus an oral antibiotic is more effective than monotherapy with an oral antibiotic.the combination of montelukast plus an antibiotic may be a safer and shorter therapy given the safety issues with long term use of systemic antibiotics. introduction: a randomized, double-blind, placebo-controlled study was conducted in an environmental exposure chamber (eec) to compare the efficacy of three doses of olopatadine nasal spray, a topical anti-allergy treatment for seasonal allergic rhinitis (sar), versus placebo spray. methods: patients aged 17-65 years old with a history of sar were screened, consented, evaluated for a positive skin test to ragweed allergen, and enrolled in this irbapproved study. a total of 320 "primed" patients were exposed to ragweed allergen in the eec and randomized to olopatadine 0.2% (n=80), olopatadine 0.4% (n=80), olopatadine 0.6% (n=80), or placebo (vehicle) (n=80) spray, 2 sprays/nostril once in the morning. symptoms were self-assessed using a 4point scale (total nasal symptom score, tnss, comprised of sneezing, runny, itchy and stuffy nose) via diaries at periodic intervals during the 12-hour study period. safety was also assessed. results: obvious trends indicated a dosedependent response to olopatadine 0.2%, 0.4% and 0.6%, though concentrations were not statistically different from each other. all three concentrations of olopatadine were clearly more efficacious than placebo spray at the first time point, 30 minutes, continuing to the end of the 12-hour session. olopatadine exhibited a safety profile comparable to placebo. conclusions: olopatadine nasal spray 0.2%, 0.4% and 0.6% exhibited dose-dependent responses. onset of action for all three concentrations of olopatadine was apparent at the first post-dose timepoint, 30 minutes, and efficacy was maintained throughout the next 12 hours. olopatadine nasal spray was safe, well-tolerated, and effective for the treatment of sar in the eec chamber. c. slonim * , tampa, fl. objective: to assess patient subjective responses to the treatment of allergic conjunctivitis using azelastine hydrochloride ophthalmic solution. methods: participating physicians selected 20 patients from their practice to receive azelastine hydrochloride 0.05% ophthalmic solution 1 drop per affected eye twice daily for 5 days. patients on prior ocular allergy medications were allowed to participate. after 5 days of treatment with azelastine hydrochloride, patients (n=2, 887) rated their experiences with azelastine hydrochloride via a self-administered survey. not all questions were answered by each patient. results: at baseline, 88% of patients reported that their ocular itching affected their work, school, and/or leisure activities at least "somewhat". after using azelastine hydrochloride, 70% of patients achieved either "moderate" or "complete" relief from ocular itching, including 30% who reported "complete" relief from itching. seventy-one percent (71%) of patients had been treated previously with a topical prescription anti-allergy medication for their ocular itching. seventy percent (70%) of patients (n=836) who had not been previously treated with a topical prescription anti-allergy medication treatment and 71% of previous medication users (n=2, 192) experienced at least "moderate" relief of itching when treated with azelastine hydrochloride. sixty-five percent (65%) of the previously-treated patients rated azelastine hydrochloride as "somewhat better" or "much better" than their previous medication. conclusions: results suggest that azelastine hydrochloride ophthalmic solution is an effective treatment for the ocular itching associated with allergic conjunctivitis as rated by the patient, regardless of whether they had been treated previously with a topical prescription anti-allergy medication. objective: to determine an association between food allergy and acid reflux in adults. method: we conducted a retrospective chart review of 60 atopic adult patients in an academic otolaryngic allergy practice. 30 adults who tested positive and 30 adults who tested negative for food allergy were included in the study. charts were reviewed for a diagnosis of gastroesophageal reflux disorders (gerd). this included a history of laryngopharyngeal reflux (lpr) and peptic ulcer disease (pud). population prevalence (19.8%; ci 17.7-21.9) of acid reflux was estimated from a historical study (locke gr et al. prevalence and clinical spectrum of gastroesophageal reflux: a population-based study in olmstead county, minnesota. gastroenterology 1997; 112:1448-56) that had a similar subject population. the prevalence of gerd in each study arm, as well as in the total study group of atopic adults, was compared to the historical control. results: subjects testing positive for food allergy had a diagnosis of gerd in 26.7% (95% ci 11.8-41.6) of cases. those subjects who did not demonstrate a food allergy were positive for gerd in 40% (95% ci 25.1-54) of cases. in the total study group, the prevalence of gerd was 33.34% (95% ci 22.8-43.8). on chi square analysis, the prevalence of gerd was significantly higher in the total study group when compared with the historical control (p=0.013). those subjects who tested negative for food allergy had a statistically significant higher prevalence of gerd than the control population (p=0.003). the prevalence of gerd between the group testing positive and the historical control did not show a significant difference (p=0.41). when comparing the study arms to each other, there was no significant difference in the prevalence of gerd (p=0.27). conclusions: the prevalence of acid reflux disorders was not higher in subjects with food allergy when compared to a historical control. although a statistical significance was noted between adults who tested negative for food allergy and the control population with regards to the prevalence of gerd, this may reflect the fact that individuals examined in an otolaryngologist's office are more likely to have acid reflux disorders than the general population. overall, the atopic subjects did have a higher incidence of acid reflux disorders, but there was no statistical significance between subjects with and without food allergy. a. suryadevara * , d.l. hamilos, boston, ma. introduction: recent studies suggest that crs without nasal polyposis (crssnp) and crs with nasal polyposis (crscnp) represent distinct pathologic entities. we wished to determine whether these conditions differed in their clinical presentation. methods: over a two-year period, new patients coming to a university based specialty clinic meeting criteria for crs were enrolled in an outcomes study. patients indicated which of four major (facial pain/pressure/headache, nasal obstruction, nasal purulence/discharge, and hyposmia/anosmia) and four minor (fever, halitosis, dental pain, cough) criteria for crs they were experiencing. rhinoscopy was performed to look for nasal polyps or polypoid tissue in any sinus area. the prevalence of each symptom was compared in the groups by chi square analysis. results: the population (n=126) had a mean age of 46 +/-13.6 and was 59% female, 86% caucasian, 8.7% african-american, 1.6% asian and 1.6% hispanic. most patients (87%) were non-smokers. all had at least 2 major criteria or 1 major + 1 minor criteria for crs at enrollment. forty-one patients (32.5%) had crscnp. the mean number of major criteria was greater in the crscnp than crssnp (3.27 vs 2.91, p=0.017). nasal obstruction and hyposmia/anosmia were more prevalent in crscnp (p= 0.05, 0.025 respectively). facial pain/pressure/headache was more prevalent in crssnp (p=).025). the most prevalent symptoms in crscnp were: nasal purulence/discharge (97.6%)>hyposmia/anosmia (85.4%)>facial pain/pressure/headache (82.9%)>nasal obstruction (61%). in contrast, the most prevalent symptoms in crssnp were: facial pain/pressure/headache (95.3%)>nasal purulence/discharge (88.2%)>hyposmia/anosmia (64.7%)>nasal obstruction (42.4%). none of the symptoms were absolutely distinguishing of these conditions. no differences were found between the two groups for fever, halitosis, dental pain or cough. conclusion: we conclude that patients with crscnp have a greater burden of symptoms of crs and a much higher prevalence of hyposmia/anomsia. these findings are consistent with other studies showing that, in comparison to crssnp, crscnp tends to be more difficult to treat and have a higher rate of relapse after intensive medical therapy (subramanian et al, am j rhinology 2002;16:303). l.e. mansfield 1* , e.e. philpot 2 , c. posey 1 , 1. el paso, tx; 2. research triangle park, nc. daytime sleepiness is a common complaint in sar. patients often complain of mental slowness, difficulty in concentrating, and thinking. the present study evaluated whether effective therapy of sar would decrease dss and improve an objective measure of cp. dss was measured using the epworth sleep scale (ess). objective cp was measured using the test of variables of attention (tova), a validated test. thirty two adults (15 males, 17 females with a 2 year history of sar, a compatible physical exam, and corresponding positive allergy testing) volunteered for this 3 week randomized double blind placebo controlled study. after a one week inf placebo (pl) baseline, the subjects received either active inf or continued pl. they maintained daily nasal symptom dairies and ess. the subjects took the tova test at the end of week1 and week3. weekly nasal symptom scores significantly improved with the inf, but not the pl. w1 vs. w3 nasal congestion inf 29, 24 p=.03, pl 29, 26 p=ns; runny nose inf 22.5, 19.9 p=ns; pl 21.75, 21.38 p=ns; sneezing inf 26.5, 19 p=.01; pl 24.9, 21.1 p=ns. total weekly ess was abnormal and decreased significantly in the inf group, but not in pl group. w1 vs. w3 inf 60.5, 46.5 p=.001, pl 52.6, 46.8 p=ns. response time of the tova testing, initially somewhat slow, significantly decreased in inf but not pl treatment. w1 vs. w3 inf 435 msec, 385 msec p=.02; pl 360 msec, 359 msec p=ns. these results demonstrate that sar is associated with dss and cp problems. the mechanism is likely to be sleeping disordered breathing associated with nasal congestion and obstruction. effective treatment of nasal congestion with inf led to decreased dss and improved cognitive performance. l.e. mansfield 1* , c. graham 2 , 1. el paso, tx; 2. new york, ny. there is increasing recognition that sleep disturbance and daytime tiredness occur during active ar. the mechanism appears to be nasal congestion and obstruction leading to sleep disordered breathing and resultant poor quality of sleep. in our practice, as part of the initial history, questions regarding fatigue, snoring, sleep problems, and tiredness are addressed. sleep problems and tiredness are graded according to the following scale: effect on daily activity ; 0=not troubled;1= a little trouble; 2=somewhat troubled ;3= trou-bled a lot ; 4= total disruption. we reviewed 272 consecutive charts of patients with allergic rhinitis documented by history, physical examination and allergy testing. there were 169 females and 103 males; age range 5y to 84y. 127 (47%) of patients or parents recognized they commonly snored. 40 (15%) stated they were chronically fatigued. the graded answers concerning sleep problems and tiredness were even more revealing of ar patient's perception of their problems see table in general, the higher sleep problem responses were associated with higher tiredness scores. national surveys of unselected populations suggest that about 10 percent of adults consider themselves to have sleep problems. the high frequency of sleep related problems and tiredness in our sample has prompted us to add more detailed questions regarding sleep related events to our intake history. it is our opinion that questions specifically related to sleep and daytime tiredness should be included in all evaluations for allergic rhinitis. we conclude that sleep related problems and tiredness may be more common in patients with allergic rhinitis than previous reported. r.w. weber 1* , j. garcia 2 , r. faruqi 2 , d. banerji 2 , g. georges 2 , the study 405 investigator group 3 , 1. denver, co; 2. bridgewater, nj; nj. introduction: the intranasal glucocorticosteroid triamcinolone acetonide (taa) is a safe and effective treatment for persistent allergic rhinitis (par). a new hydrofluroalkane-134a (hfa) propellant delivery system (taa-hfa) has recently been developed. this study primarily assessed the long-term safety of taa delivered via this new device, as well as its long-term efficacy. methods: 396 patients aged 12-69 yrs (mean=32) with par enrolled in this 1-yr, open-label study at 10 centers in the us. patients received taa-hfa 220 î¼g once daily for a 2-week run-in period before adjusting the dose to 440 î¼g or 110 î¼g once daily based on symptom severity. doses were standardized to 440 î¼g once daily across all patients at ~4 months to ensure sufficient long-term safety data at the maximum dose. physical exams, including measurement of vital signs and laboratory measurements were taken at baseline, 6 months and study end. independent patient and physician global symptom evaluations were performed at baseline, week 2 and months 1-12 thereafter. patients recorded any adverse events (aes) on daily diary cards. results: of the 396 patients included in the study, 349 (88.1%) reported aes. the incidence of aes was similar to that of other comparable allergic rhinitis long-term studies. the most frequently reported aes were pharyngitis, rhinitis, local reactions, headache, epistaxis and sinusitis (table 1) . most aes were mild-to-moderate in intensity; 34 patients withdrew from the study due to aes. there were no clinically relevant changes in physical exams, vital signs or laboratory measurements. a total of 4 serious aes (saes) were reported; breast carcinoma (n=1), depression (n=1), post-operative spinal fluid leak (n=1) and staphylococcal infection (n=1). saes were thought to be not related to the study drug. at final visit, 83% of patients had either moderate or marked/complete relief of symptoms using global symptom scores. similarly, 86% of physicians rated their patients as having either moderate or marked/complete relief. conclusions: long-term administration of taa-hfa 440 î¼g exhibited a good safety and tolerability profile, while providing moderate-to-complete symptom relief in more than 80% of patients treated for par. introduction: second-generation, 'non-sedating' antihistamines (ahs) have lower tendency to cross the blood-brain barrier and cause central nervous system side effects than first-generation agents. however, studies have suggested differences between second-generation ahs regarding cognitive function impairment. methods: a medline literature search was performed using the search terms 'antihistamine and impairment', 'antihistamine and psychomotor' and 'antihistamine and central effects', as well as with individual ah names (acrivastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, mequitazine and mizolastine). the findings for second-generation ahs were reviewed and well-designed, placebo-and positive-controlled studies in humans using objective measures of cognitive impairment were included. results: 43 publications were identified as the inclusion criteria. all included objective assessments such as driving performance, critical flicker fusion and divided attention tasks. there was a large variation in the numbers of available well-designed studies; the most rigorously assessed agents were fexofenadine and cetirizine. a number of the ahs (ebastine, acrivastine, loratadine, mequitazine and mizolastine) were not impairing at recommended doses, but were at higher doses. in a small number of available studies, the newer ahs desloratadine and levocetirizine produced no impairment at recommended doses (5 mg); however, higher doses were not investigated. cetirizine studies were variable; impairment at the recommended 10 mg dose or higher was seen in a number of studies. in contrast, fexofenadine hcl, up to a dose of 360 mg, was non-impairing in a large number of studies. only one study indicated impairment in one task (critical tracking) and this was only observed with the first doses of fexofenadine hcl (120 and 180 mg) and not subsequent doses; however, the authors concluded that doses up to 240 mg/day should be safe for patients who drive. conclusion: while second-generation ahs are less impairing than the first-generation, some newer ahs produce impairment at or above the recommended dose. these differences become important to the patient when agents cause sedation or if patients over use beyond the recommended dose. in conclusion, fexofenadine was the only ah found to be non-impairing in all studies, even at double the recommended us dose. s. shaver 1 , r.b. berkowitz 1* , c. lutz 1 , p. jones 1 , c. qiu 2 , s. meeves 2 , s.t. varghese 2 , g. georges 2 , 1. woodstock, ga; 2. bridgewater, nj. introduction: fexofenadine (fex) is a h1-receptor antagonist, with proven efficacy in the treatment of allergic rhinitis (ar). to date, no live cat-room challenge studies have assessed the efficacy of fex in cat-allergen induced ar. this study assessed the efficacy of a single dose of fex hci 180 mg in preventing and controlling cat-allergen induced ar using the cat-room challenge model. methods: this single-center, prospective, randomized, doubleblind, placebo-controlled, two-way crossover study consisted of a screening visit, one or two priming visits and two treatment periods, separated by a 14 (â±3)-day wash-out. qualifying subjects were randomized to treatment sequence 1 (placebo followed by fex) or 2 (fex followed by placebo). baseline endpoints were obtained prior to study drug administration, and 5 minutes before entering the cat challenge room for allergen challenge. allergen challenges were initiated 1.5 hours post-dose, for both treatment periods. the primary endpoint was the change from baseline in total symptom score (tss; sum of rhinorrhea, itchy nose/palate/throat, sneezing and itchy/watery/red eyes) after 30 minutes of allergen exposure at 2 hours post-dose, compared with placebo. other endpoints included changes in individual symptom scores, including nasal congestion. levels of airborne felis domesticus allergen 1 (fel d 1) were determined. results: of 211 subjects screened, 66 were randomized and 63 completed the study; 32 and 31 in sequence 1 and 2, respectively. mean change in tss from baseline was significantly less with fex compared with placebo at 30 minutes after initiation of the cat allergen challenge (p=0.0327). significantly greater percentage reductions in the individual symptom scores for sneezing (p=0.0037) and nasal congestion (p=0.0455) were observed with fex compared with placebo, 30 minutes after challenge. although levels of fel d 1 varied widely, they were balanced between treatment groups. the overall incidence of treatment-emergent adverse events (aes) was low and comparable between groups; no serious aes occurred. conclusion: this study demonstrated that a single dose of fex hci 180 mg is effective and well tolerated as a prophylactic agent for alleviating the ar symptoms induced by exposure to cat allergen. further large-scale clinical trials are warranted to confirm these findings. rationale: allergic rhinitis in children is thought to be associated with several co-morbid disorders. we conducted the following study to assess whether children with diagnosed hypertrophy of tonsils and/or adenoids evaluated by an allergist were more likely to demonstrate objective evidence of allergen sensitization than children similarly evaluated without evidence of these upper airway obstructions. methods: records from the past ten years were identified in the hospital database by presence of an allergy clinic visit and an icd-9 code for hypertrophy of adenoids and/or tonsils. we reviewed these records to confirm that the diagnosis was accurate. we included subjects if reliable skin prick or in vitro testing for specific ige to aeroallergens was performed. for comparison, we randomly obtained an age and testing-type similar sample. first group subjects were excluded from the second group. skin testing was performed with commercial allergen extracts applied with a dermapikâ©. in vitro testing was by unicapâ© feia. positive skin testing had at least a wheal 3 mm or flare 10 mm greater than the negative control. positive in vitro values were 0.7 ku/l or greater or a positive pollen mix. additionally, we performed a search in an insurance database to determine how many local children had a code for allergic rhinitis. results: of 202 pediatric allergy patients with adenoid/tonsillar hypertrophy tested, 41.6% (95% ci +/-6.8%) had at least one positive test. in 802 without adenoid/tonsillar hypertrophy, 50.7% (95% ci +/-3.5%) had at least one positive test. the observed difference was 9.1 % (95% ci +/-7.6%). the standard error of the difference in percentage was 3.94; the z score was 2.3. the corresponding p value is 0.02. the percentage of 70, 356 children with an allergic rhinitis icd-9 was 15.6% (95% ci +/-0.3%). conclusion: the number of allergist-referred children with adenoid and/or tonsillar hypertrophy testing positive for an aeroallergen is slightly less than the number of similar children without these diagnoses testing positive. it is not clear that adenoid/tonsillar hypertrophy is associated with a higher risk of allergic rhinitis. a prospective study with allergy testing of all children with adenoid and/or tonsillar hypertrophy might provide information that could alter referral patterns. w.e. berger 1* , w. storms 2 , s. kimura 3 , m. beck 4 , s. galant 5 , t. westbrook 3 , 1. mission viejo, ca; 2. colorado springs, co; 3. pensacola, fl; 4. miami, fl; 5. orange, ca. background: patients having allergic rhinoconjunctivitis are often treated with nasal spray or systemic allergy therapy, forgoing therapy specifically targeting ocular symptoms. the rhinitis quality of life questionnaire (rqlq) and allergic conjunctivitis quality of life questionnaire (acqlq) instruments can be used to quantify the relative benefit of varying medication regimens. objective: to determine the extent of benefit gained in quality of life when an eye drop treatment for ocular allergy (olopatadine) was added to rhinitis patients' preexisting regimen of nasal or systemic allergic treatment. methods: this was a four week prospective, multi-center, open-label crossover, quality of life study occurring during allergy season. at visit 1, patients completed the rqlq and acqlq questionnaires to assess baseline quality of life. patients were randomized to receive ocular allergy therapy (olopatadine, patanol bid) concomitant with their systemic or nasal rhinitis treatment(s) for 2 weeks between either visit 1 and visit 2 (group a) or between visit 2 and visit 3 (group b). at visit 2 and visit 3, patients completed the rqlq and acqlq. results: a total of 200 patients completed this study: 97 in group a, 103 in group b. of these, 181 (90.5%) experienced eye allergy symptoms at least 1 day during the previous week as reported in the baseline acqlq. baseline scores of the rqlq and acqlq for both groups were comparable. clinically significant improvement from baseline in global rqlq and acqlq was seen following addition of ocular therapy for both groups (rqlq: -1.07, -0.94; acqlq: -1.2, -1.2). similar improvement was seen across all domains of both questionnaires. the acqlq correlated with the rqlq in the applicable domains. conclusion: many allergic rhinitis patients using nasal or systemic medication also suffer from ocular allergic symptoms. for these patients, quality of life improvement is not maximized; the addition of a topical antiallergy eye drop can result in beneficial effects on quality of life. in this study, the addition of olopatadine eye drops to these patients' medication regimens resulted in significant improvement in not only eye symptom related quality of life domains but in overall quality of life. h. milgrom 1* , r. lanier 2 , f.c. hampel 3 , b. kittner 4 , 1. denver, co; 2. fort worth, tx; 3. new braunfels, tx; 4. bridgewater, nj. introduction: fexofenadine, a non-sedating, selective h1-receptor antagonist, is currently indicated for use in children aged 6-11 years with seasonal allergic rhinitis in a number of countries, including the us, and has an excellent safety profile in this age group. this study was designed to assess the safety and tolerability of fexofenadine in children aged 2-5 years with allergic rhinitis (ar). methods: the study had a multicenter, double-blind, randomized, placebo-controlled, parallel-group design. children aged 2-5 years (n=453) with ar were randomized 1:1 to either placebo twice daily (bid; n=231) or fexofenadine hcl 30 mg bid (n=222), for 2 weeks. both treatments were given orally as granulated powders (capsule content) mixed with two teaspoons of apple sauce. treatment-emergent adverse events (teaes) were recorded for all subjects by parents/caregivers and assessed by investigators. clinical laboratory variables, physical examinations, vital signs and ecg evaluations were also assessed in a subgroup of children (placebo, n=79; fexofenadine, n=71). results: baseline demographics were similar in both treatment groups. while approximately 50% of children in both groups experienced at least one teae, no unusual or unexpected teaes were observed in the fexofenadine group. when the total group was analyzed for teaes by body system, or assessed separately by age groups of 2-, 3-, 4-and 5-year-olds, no clinically meaningful differences were observed between the two treatment groups. in both treatment groups, the majority of subjects overall and in each age group experienced teaes rated as mild or moderate in intensity. few subjects experienced teaes considered possibly related to study medication (placebo: 8.2% [19/231]; fexofenadine: 9.5% [21/222]). the percentage of discontinuations due to teaes was also comparable between treatment groups (placebo: 6.9% [16/231]; fexofenadine: 5.0% [11/222] ). in the subgroup assessment, no clinically relevant changes were seen from baseline for laboratory variables, vital signs, ecgs or physical examinations in either treatment group. introduction: the efficacy and safety of fexofenadine hcl 30 mg bid has been proven in two large phase iii studies in pediatric subjects aged 6 to 11 years with seasonal allergic rhinitis. in addition, the safety of fexofenadine has been demonstrated in pediatric subjects 2 to 5 years of age with allergic rhinitis (ar). subsequently, two studies (t/3001; t/3002) have assessed the pharmacokinetics, safety and tolerability of fexofenadine 15 and 30 mg bid in pediatric subjects 6 months to 2 years of age. methods: both studies were of multicenter, randomized, double-blind, placebo-controlled, parallelgroup design and enrolled pediatric subjects aged 6 months to <1 year weighing 10.5 kg and aged â±1 year to <2 years weighing >10.5 kg. all subjects had a diagnosis of ar as assessed by previous medical history, pattern, or suggestive physical findings. subjects were randomized to receive fexofenadine hcl 15 mg (t/3001), or 30 mg (t/3002) granulation powder twice-daily, or placebo for a minimum of 7 days. safety was evaluated based on adverse events (aes), vital signs, 12-lead electrocardiograms (ecgs), and physical examinations. results: a total of 174 and 218 subjects were randomized in studies t/3001 (fexofenadine hcl 15 mg bid: n=85, placebo: n=89) and t/3002 (fexofenadine hcl 30 mg bid: n=108, placebo: n=110), respectively. in t/3001, 40.0% (34/85) of children receiving fexofenadine and 42.7% (38/89) receiving placebo experienced at least one treatment-emergent ae (teae). in t/3002, the incidences of teaes were 35.2% (38/108) and 52.7% (58/110), respectively. in both studies, most of the teaes experienced were mild or moderate in intensity. the incidence of possibly-related teaes was also similar for both treatments in each study. no clinically relevant changes from baseline to study end were observed for vital signs, ecgs and physical examinations. conclusions: the findings of this study show that fexofenadine 15 mg and 30 mg bid are well tolerated and have a safety profile comparable to placebo in pediatric subjects aged 6 months to 2 years. for seasonal allergic rhinitis (sar) patients that remain symptomatic on an h1-receptor antagonist, cetirizine, and a nasal glucocorticosteroid, mometasone furoate, the addition of omalizumab, a recombinant, humanized, chimeric, anti-ige monoclonal antibody, may provide additional efficacy in sub-optimally controlled seasonal allergic rhinitis patients. in this open labeled 12week trial, 20 patients with symptomatic sar currently using cetirizine, 10 mg qd, + mometasone furoate, 100 mcg/nostril qd, were randomized to continue the existing therapy cetirizine + mometasone or to add-on omalizumab subcutaneously (every 2 weeks to 4 weeks) dosed according to patient's weight, and baseline ige levels to the existing therapy cetirizine + mometasone furoate. the endpoints of the trial include: rhinomanometry, nasal symptom score (composite score of nasal congestion, rhinorrhea, sneezing, post nasal drip and itching) and flexible rhinopharyngolaryngoscopy examination. mean efficacy measurements at the end of the 12-week trial revealed a significant improvements in all parameters examined in the treatment group receiving omalizumab (as add-on to the existing therapy), compared to the other group. in conclusion, the addition of omalizumab to the combination of cetirizine plus mometasone furoate, is more effective than the combination of cetirizine plus mometasone furoate, for the treament of seasonal allergic rhinitis patients. it appears that when omalizumab is added to the combination h1receptor antagonist, cetirizine, and nasal corticosteroid, mometasone furoate, the primary end points (rhinomanometry and symptom scores) are significantly improved. background: based on the official statistic data, the prevalence of allergic diseases in russia has increased more than 3 times during the past 10-15 years, but still the lowest of all european countries. in some studies done in some regions of russia based on isaac program, it has been shown that the prevalence allergies is significantly higher than indicated in official statistics of health departments. the goal of this investigation was to study the prevalence of allergic diseases, in children of south russian region. meth-ods: this study was done in two steps according to isaac program guidelines. in the first step a questionnaire was given to a total of 6558 school children, ages 7 to 8 (group a) and 13 to14 years old (group b), from the two cities of krasnodar and novorossiysk in southern russia. in the second step a physical exam, skin prick tests with different allergens (pollens, molds, cat, dust mites, foods etc.), and pulmonary function tests were performed. results: wheezing was reported in 12.9% of group a and 15.3% of group b children within the past 12 months. the severity of asthma reported was mild in 72% (group a)-84% (group b); moderate in 16.7% (group a)-10.7% (group b) and severe in 3.4% (group a)-5.8% (group b) in studied children). majority of the cases of asthma was noted to be allergic asthma. in both groups of children there was a high incidence of allergies to perennial allergens such as dust mites, cats, molds and other indoor allergens, 18.7% (group a) and 77.3% (group b). the percentage of allergies to pollens was 19.1% and 33.0%, accordingly. allergic rhinitis symptoms were noted in 26.6% (group a) and in 38, 6% (group b). atopic dermatitis symptoms with skin itching observed in 8.3% (7-8 years old)and 5.3% (13-14 years old). family history of atopy was noticed in 34.6% of group a and 41.6% of group b children. conclusions: the prevalence of allergic diseases in south russia is similar to the ones in most of the european countries. the prevalence of asthma has been associated with increased incidence of allergies, due to indoor and pollen allergens. the prevalence in rhinitis and atopic dermatitis in south russia is parallel to asthma. acknowledgement: we would like to thank hollister-stier lab., greer and antigen lab. for allergen samples. objective: to examine the cost and effectiveness of telithromcyin vs. azithromycin for treatment of mild acute sinusitis under current levels of antimicrobial resistance. methods: we considered an adult with mild acute sinusitis, and symptom duration of 7 days. a decision analytic model was created to compare 2 strategies of 1st-, 2nd-and 3rd-line therapies: azithromycin/amoxicillin-clavulanate/levofloxacin (azi/amc/lev), and telithromycin/amoxicillin-clavulanate/levofloxacin (tel/amc/lev). we considered 3 outcomes: response to initial therapy, cost, and time to completion of all antibiotic therapy. clinical resolution was due to response to antibiotic, or to spontaneous resolution. we assumed bacteria that were resistant in vitro would only be resistant in vivo 50% of the time. resistance levels were based on the 2000-2001 us protekt surveillance study. model parameters, such as prevalence of bacterial infection, frequency of causative pathogens, and rates of spontaneous resolution, were based on the published literature. those failing to improve after 5 days were switched to next line therapy. we analyzed claims data from 8 managed care organizations to estimate non-drug charges for initial and follow-up care, and applied a 60% cost-to-charge ratio. drug costs were estimated using the wholesale acquisition cost plus $8 for overhead and dispensing. all costs were adjusted to year 2004 $us. results: the model predicted the tel/amc/lev strategy would result in 71.8% improving by 5 days compared to 65.4% for azi/amc/lev. the telithromycin strategy had lower mean cost, $172 vs. $176. although telithromycin cost more than azithromycin, the tel/amc/lev strategy had slightly lower total drug costs ($95 vs $96) due to less need for additional antibiotic courses. mean time to completion of therapy was 8.4 days for tel/amc/lev and 9.1 days for azi/amc/lev. sensitivity analyses showed that tel/amc/lev had superior health outcomes even when only 5% of in vitro resistant organisms were also resistant in vivo. tel/amc/lev also had lower cost if at least 30% of cases were bacterial, or if in vitro resistant organisms were at least 30% resistant in vivo. conclusion: based on results obtained using this decision analytic model, initial treatment of mild acute rhinosinusitis with telithromycin may result in improved outcomes and lower cost than initial treatment with azithromycin. rationale: "delta crud" is the term used to describe rhinosinusitis syndromes in the mississippi delta region. local perception is that it is associated with chemical crop dusting, regional produce, especially cotton, or high rates of mold allergy. the climate is extreme with mild winters, hot humid summers, and large volume rain. thirty-five percent of inhabitants live below the poverty level, 62% are african american. patients from the agriculturally intensive delta suffer from asthma at almost twice the rate of those in the neighboring hills. rates of pesticide use are among the highest in the nation. there are no prevalence estimates for rhinosinusitis in this region. "delta crud" is chronic in many patients with exacerbations occurring in the summer and fall, especially during chemical spraying and defoliation. we conducted this cross-sectional questionnaire based pilot study to begin characterization of rhinosinusitis in this population. method: the sinusitis treatment outcome questionnaire, with two modifications, was given to consecutive patients presenting to the north sunflower regional hospital er and the sunflower outpatient clinic one month prior to the beginning of summer crop dusting. the question, "do you have delta crud?" was added. the survey was anonymous, allowing irb exemption. results: 91 consecutive patients returned completed questionnaires. 46(50%) admitted to having "delta crud". patients who claimed to have delta crud had significantly more sinus headache, nasal pruritus, conjunctivitis, and chest symptoms (see table) . there was no significant difference in antibiotic prescriptions, er visits, and missed work. six patients with delta crud had been hospitalized for "allergy reasons" compared with three patients without delta crud. despite severe symptoms just 4 patients received sinus ct scans. only 10/46 patients were prescribed intranasal corticosteroids. conclusion: in our pilot study, the prevalence of chronic rhinosinusitis symptoms (delta crud) is 50%, with severe sinus, pulmonary and ocular symptoms. evaluation and treatment may be suboptimal in this economically disadvantaged rural region. further characterization of "delta crud", pollen counts, ige mediated disease, environmental contributions and comparative studies of related regions are needed. introduction: patients with rhinitis commonly experience sinus pain and pressure (sp+p). many patients with recurrent acute bacterial sinusitis (rabs) base the presence of a recurrence on the severity of sp+p and other nasal symptoms. the ability of patients to differentiate between rhinitis and rabs was evaluated by reviewing the subject screening logs of 4 previously reported clinical trials of flonase (fluticasone propionate) nasal spray, 50 mcg (fp). methods: the efficacy of fp was studied in two trials in subjects with sp+p due to allergic rhinitis and in two trials of subjects receiving cefuroxime axetil to treat an acute episode of rabs. sp+p screening logs were examined to determine how many subjects who thought they had sp+p were excluded from the study for an upper respiratory or sinus infection. rabs screening logs were examined to determine how many subjects with symptoms of an acute episode of rabs were excluded from the study for a negative sinus x-ray or ct scan. rabs symptoms experienced at the screening visit by subjects who qualified for the study were also evaluated. results: of 505 subjects screened in the sp+p studies, only 4 (0.8%) were excluded for evidence of sinus or upper respiratory tract infections, as clinically diagnosed by the investigator. out of 1502 subjects with screening log data in the rabs studies, 649 (43.2%) were excluded for a negative sinus x-ray or ct scan. of the 704 subjects who qualified for the rabs study, the most prevalent symptoms included nasal congestion (100%), mucopurulent discharge (97%), facial pain or tenderness in the sinus area (93%), sinus headache (90%) and malaise (90%). only 15% of subjects had a fever. only one symptom, nasal congestion, was rated by clinicians as severe for greater than 40% of subjects in either study (46% and 43%). conclusions: while most subjects can determine when sp+p is due to allergic rhinitis, many cannot determine when symptoms progress to a sinus infection. symptoms experienced by subjects with rabs were consistent with inflammation, but did not include fever as may be expected with an untreated bacterial sinus infection. under appreciation by patients for the role of inflammation in sinus disease may result in over self-diagnosis of sinus infection and the demand for antibiotics. liposomes are small particles consisting of lipid bilayer membranes, which are used to deliver drugs including amphotericin b, more efficiently and with less toxicity. very few cases of allergy to liposomal amphotericin b (lab) have been reported to date. although there is a report on conventional amphotericin b (cab) desensitization, to our knowledge no case of lab desensitization has been described yet. a 75 y/o patient with lymphoma was treated with lab for pulmonary aspergillosis. within 30 minutes of infusion he developed urticaria, hypotension and tachycardia for which he was treated with the appropriate drugs for anaphylaxis. a second attempt to re administer lab resulted in a similar anaphylactic reaction. desensitization to lab was then successfully completed using a modified protocol based on desensitization to cab (jaci 1995; 96:425-7) : 0.0005 mg infusion over 10 minutes 0.005 mg infusion over 10 minutes 0.05 mg infusion over 10 minutes 0.5 mg infusion over 30 minutes 5 mg infusion over 30 minutes 50 mg infusion over 30 minutes 100 mg infusion over 30 minutes 200 mg infusion over 120 minutes conclusion: desensitization to lab, a lifesaving drug for invasive fungal diseases, can be performed successfully. adverse events (aes) were recorded throughout the study. therapeutic comparability was defined a priori as a clinical response within 30% for hfa vs cfc for the primary efficacy measures. results: baseline demographics were similar between treatments for the intent-to-treat population. mean 24-h baseline symptom scores for nasal stuffiness, nasal discharge and sneezing were 2.4, 2.3 and 2.1, respectively, out of a possible score of 3. all taa-cfc and taa-hfa doses significantly (p<0.05) reduced 24-h, and am and pm 12-h reflective scores for nasal stuffiness, nasal discharge, sneezing and ni vs placebo (except cfc 14 î¼g for sneezing). taa-hfa and taa-cfc were statistically comparable (within 0.70 and 1.3 for the 2 one-sided 90% confidence interval) at doses of 110 î¼g and 440 î¼g for the primary variables of nasal discharge, sneezing and ni for the 24-h, as well as the am and pm 12-h reflective assessment, over the 2-wk period 36 * , 66 * agrawal p27, p35, 41, p58, p105, p106, p110, p180 41 * , p21, p27, p58 * p105, p106, p110, p180 p7 * finegold, i. 42 * fink p21 * 26 * sienra monge 45 * , 55, 62 * so, c. p52 * soane 70 * , p143 * staveren, a.m rates of cardiovascular mortality are higher during peak pollen times. (brunkreef, lancet 2000) . for this reason, mast cell stabilizing agents and leukotriene antagonists are under development for the treatment of myocardial infarction. conversely, ige mediated disease may be protective against sudden cardiac death.(szczeklik, coron art dis 1993) ar has not been studied in this context. we conducted a retrospective case control pilot study of va patients who were diagnosed with allergic rhinitis and had an acute myocardial infarction. methods:the electronic medical record of a large va hospital was queried for patients diagnosed with an acute myocardial infarction (ami) in the past six years. patients were divided into groups with and without ar. patients with chronic urticaria, asthma, and eczema were excluded from this study. primary outcome was all-cause mortality. secondary outcomes included lv systolic function and peak troponin levels. results: 424 patients were diagnosed with ami, 63 (15%) of which were also diagnosed with ar. patients with and without ar did not differ in terms of age, ethnicity, tobacco status, hypertension, diabetes, or lipid levels. 14/63 (22%) patients with ar died, compared to 152/361 (42%) control patients, (p-value: 0.0028). ar patients had lower mean peak troponin values (7.7 vs. 11.8), but these were not significant. both groups had similar lv function. conclusion: this pilot study suggests an association between allergic rhinitis and a decrease in ami allcause mortality independent of other factors. atopic patients have been shown to have prolonged bleeding times, reduced platelet aggregation, and delayed thrombin generation which can result in delayed clot production. (szczeklik, thromb haem 1986) possibly, patients in our study diagnosed with allergic rhinitis were more likely to monitor their health symptoms or had physicians who carefully addressed multiple issues. rigorous prospective studies are needed to determine the role of ar in myocardial infarction. purpose and methods. a formulation of olopatadine hydrochloride ophthalmic solution (olopatadine 0.2%) was evaluated in a randomized, placebocontrolled, double-masked, hybrid environmental study intended to determine efficacy and safety in 240 subjects with histories of seasonal allergic conjunctivitis or rhinoconjunctivitis. in this 12-week trial, subjects regularly assessed their ocular signs and symptoms. additionally, subjects evaluated both the frequency and severity of their nasal symptoms. daily throughout the study, ragweed pollen counts were obtained from each investigative center. repeated measures analysis of variance was used to compare treatment differences in the slopes for nasal symptoms as a function of pollen counts. results. the nasal results are presented herein. specifically, relative to placebo, olopatadine 0.2% significantly reduced the frequency of pollen effects on sneezing (p=0.0355) and itchy nose (p=0.0032), and reduced the severity of pollen effects on sneezing (p=0.0451), itchy nose (p=0.0178), and runny nose (p=0.0327). in this study, the most frequent ocular adverse event that was related to therapy with olopatadine 0.2% was ocular dryness, occurring at an incidence of 1.7%. no treatment-related, clinically relevant changes were observed for visual acuity, intraocular pressure, ocular signs, or fundus parameters. conclusion. olopatadine ophthalmic solution, 0.2% is safe, well-tolerated, and effective in reducing some effects of pollen on the nasal symptoms associated with allergic rhinoconjunctivitis. author index key: cord-268977-hcg2rrhl authors: feikin, daniel r.; njenga, m. kariuki; bigogo, godfrey; aura, barrack; aol, george; audi, allan; jagero, geoffrey; muluare, peter ochieng; gikunju, stella; nderitu, leonard; balish, amanda; winchell, jonas; schneider, eileen; erdman, dean; oberste, m. steven; katz, mark a.; breiman, robert f. title: etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya, 2007–2010 date: 2012-08-24 journal: plos one doi: 10.1371/journal.pone.0043656 sha: doc_id: 268977 cord_uid: hcg2rrhl background: few comprehensive data exist on disease incidence for specific etiologies of acute respiratory illness (ari) in older children and adults in africa. methodology/principal findings: from march 1, 2007, to february 28, 2010, among a surveillance population of 21,420 persons >5 years old in rural western kenya, we collected blood for culture and malaria smears, nasopharyngeal and oropharyngeal swabs for quantitative real-time pcr for ten viruses and three atypical bacteria, and urine for pneumococcal antigen testing on outpatients and inpatients meeting a ari case definition (cough or difficulty breathing or chest pain and temperature >38.0°c or oxygen saturation <90% or hospitalization). we also collected swabs from asymptomatic controls, from which we calculated pathogen-attributable fractions, adjusting for age, season, and hiv-status, in logistic regression. we calculated incidence by pathogen, adjusting for health-seeking for ari and pathogen-attributable fractions. among 3,406 ari patients >5 years old (adjusted annual incidence 12.0 per 100 person-years), influenza a virus was the most common virus (22% overall; 11% inpatients, 27% outpatients) and streptococcus pneumoniae was the most common bacteria (16% overall; 23% inpatients, 14% outpatients), yielding annual incidences of 2.6 and 1.7 episodes per 100 person-years, respectively. influenza a virus, influenza b virus, respiratory syncytial virus (rsv) and human metapneumovirus were more prevalent in swabs among cases (22%, 6%, 8% and 5%, respectively) than controls. adenovirus, parainfluenza viruses, rhinovirus/enterovirus, parechovirus, and mycoplasma pneumoniae were not more prevalent among cases than controls. pneumococcus and non-typhi salmonella were more prevalent among hiv-infected adults, but prevalence of viruses was similar among hiv-infected and hiv-negative individuals. ari incidence was highest during peak malaria season. conclusions/signficance: vaccination against influenza and pneumococcus (by potential herd immunity from childhood vaccination or of hiv-infected adults) might prevent much of the substantial ari incidence among persons >5 years old in similar rural african settings. compared with other regions, the mortality rate among older children and adults remains several-fold higher in sub-saharan africa, where acute respiratory infections (ari) are a leading cause of this high mortality, as well as associated morbidity [1] . however, data on etiologies and rates of ari among persons $5 years old in africa have been principally focused on few geographic areas and pathogens (e.g., pneumococcus, tuberculosis). few studies have comprehensively examined the etiologies of ari among africans $5 years of age. more importantly, there is even less available data on disease incidence for specific etiologies, which can inform public health policies regarding treatment and prevention. from population-based surveillance in rural western kenya undertaken from 2007-2010, we report bacterial and viral etiologies of ari by age group, hospitalization status, hivinfection status and season. we also provide incidence by etiology, adjusted for health-care seeking and presence of pathogens in asymptomatic controls. written informed consent was obtained for data and specimen collection at the clinics and households. for children ,13 years of age, written informed consent was obtained from parents or guardians for specimen collection. for minors aged 13-17 years of age, written informed consent was obtained from parents or guardians and written assent from the minor him/herself for specimen collection. the protocol and consent forms were reviewed and approved by the institutional review boards of kemri (#932) and cdc (#4566). cdc's international emerging infections program and the kenya medical research institute (kemri) have conducted population-based, infectious disease surveillance since late 2005 in asembo, nyanza province, in rural western kenya [2, 3] . all households in 33 villages within 5 kilometers of the referral facility, st. elizabeth lwak mission hospital (lwak hospital), were offered enrollment -78% were enrolled. the surveillance population on july 1, 2007, included 21,420 persons $5 years old, who had resided permanently in the area for at least 4 calendar months [4, 5] . enrollment was continuous since the project's beginning. malaria transmission was holoendemic [5, 6] . hiv prevalence was high (17% in adults $18 years in 2008) [7] . from march 1, 2007 , to february 28, 2010 , all enrolled participants received free medical care by kemri/cdc-trained nurses and clinical officers at lwak hospital for acute illnesses [2, 3] . lwak hospital has 40 inpatient beds and manages most hospitalizations, only referring complicated patients. no chest radiographs were taken during the study period. ari was defined, using a variation on the definition of severe acute respiratory illness suggested for influenza surveillance, as cough or difficulty breathing or chest pain and documented axillary temperature $38.0 o c or oxygen saturation ,90% or hospitalization [2, 8] . for ari patients, blood, nasopharyngeal and oropharyngeal specimens using polyester-tipped swabs, and urine were collected. giemsa-stained malaria blood smears were performed on patients with history of fever or documented temperature $38.0uc. community interviewers visited enrolled households every two weeks and questioned participants using a standardized questionnaire, in the local language, about all illness episodes in the past two weeks, including symptoms and health-seeking [3] . for this analysis, we defined ari from the household visits as cough, difficulty breathing or chest pain and reported fever. the ari cases from the household visit were only used to assess healthseeking patterns in the area and not directly included in the incidence calculations. no specimens were collected at household visits. from january 1, 2009, asymptomatic controls were enrolled from lwak hospital. eligible controls were those who presented with non-severe illness (i.e., not requiring hospitalization), for immunizations, or for medicine refills. eligible controls could not have had fever, any respiratory symptoms or diarrhea in the past two weeks. each month we attempted to enroll a targeted number of controls, frequency-matched to cases by age and hiv status. the monthly target was 23 controls aged $5 years old (six hivinfected), yielding power to detect a significant difference in detection of a pathogen between 12% of cases and 5% of controls. nasopharyngeal and oropharyngeal swabs were collected on controls. clinic nurses attempted to collect five to ten ml of blood for culture, which was inoculated into commercially-produced blood culture bottles and bacterial growth identified using standard methodology, which we have described previously (bactec tm aerobic plus tm , becton dickinson, belgium) [9, 10] . naso-and oropharyngeal swabs were placed together in 1 ml viral transport media without antibiotics and transported the same day at 2uc-8uc to kemri/cdc laboratories near kisumu, approximately 60 km from lwak hospital, where each specimen was divided into four aliquots and stored at 270uc. in monthly batches, a frozen aliquot was transported on dry ice to the kemri/cdc laboratory in nairobi, where lab technicians, blinded to case-control status, tested it after one freeze-thaw cycle, using quantitative real-time reverse transcription polymerase chain reaction (qrt-pcr). nucleic acid was extracted from 100 ml aliquots of each sample using qiagen's qiaamp viral rna minikit (qiagen inc, california), according to manufacturer's instructions. prior to august 2008, taqmanh universal pcr master mix (applied biosystems, california) was used for qrt-pcr; later qrt-pcr was carried out using agpath-id tm onestep rt-pcr reagents (applied biosystems). samples were tested for the presence of adenovirus, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), influenza a and b viruses, and parainfluenza virus (piv) types 1-3 using qrt-pcr assays using previously published assays [11, 12] . each clinical specimen was also tested for the human ribonuclease p gene to measure nucleic acid integrity and to confirm sample adequacy. a qrt-pcr test result was considered positive if an exponential fluorescence curve was produced that crossed the assigned threshold at c t ,40.0 [12] . between january 1, 2009, and february 28, 2010, enhanced testing was done, which included testing respiratory swabs for three additional viruses (rhinovirus, enterovirus, and parechovirus) and atypical bacteria, and testing of asymptomatic controls. for testing of swabs for these additional pathogens, total nucleic acid extracts were prepared from 100ml of specimen per aliquot using magmax viral rna isolation kit (applied biosystems) in nairobi and transported to the viral respiratory diseases laboratory at cdc atlanta on dry ice. singleplex qrt-pcr for rhinovirus, enterovirus and parechovirus were performed using previously published methodologies [13, 14, 15] . the rhino-and enterovirus assays targeted the 59 noncoding region of an area of high sequence similarity between human rhinovirus and enterovirus and exhibit some cross-reactivity (s. oberste, personal communication). therefore, positive rhinovirus and/or enterovirus qrt-pcr were reported together as rhino/enterovirus positive. for atypical bacteria, multiplex qrt-pcr was performed at kemri/cdc laboratories in nairobi for mycoplasma pneumoniae, chlamydia pneumoniae and pan-legionella species using published assays [12, 16] . urine specimens were tested for pneumococcal urine antigen using the binaxnowh kit (binax inc., maine) starting in may 2007; kits were not consistently available throughout the entire study period. sputum specimens were not collected by protocol, but only based on the clinician's suspicion of pulmonary tuberculosis, and microscopy was done at lwak hospital using ziehl-neelsen staining. hiv testing was performed as part of a home-based testing initiative during 2008 when all persons $13 years in the surveillance area were offered hiv testing (two parallel rapid hiv tests), as described previously [7] . seventy-eight percent of eligible adults agreed to be tested. we assumed that a person's hiv status during home-based testing was the same throughout the study period. hiv-testing was not performed routinely in the clinic on most patients during this period. structured questionnaires on scannable paper forms detailing the current illness were completed for all sick visits at lwak hospital. (teleformh software, cardiff tm , vista, ca). at the household visits, data were collected using personal digital assistants (pdas) [3] . analysis was performed using sas (version 9.2, cary, nc). proportions were compared using chi-square test or fisher's exact test, and medians by wilcoxon rank sum test. rate ratios and 95% confidence intervals were calculated using fisher's method (computer programs for epidemiologists, pepi, version 4.0x) for crude rates and the delta method for adjusted rates [17] . correlation between monthly rates of ari and percent positive for each pathogen were tested by pearson's or spearman's correlation coefficient, depending on normality of the data. we compared detection of each virus by qrt-pcr between cases and asymptomatic controls. odds ratios (or) and 95% confidence intervals were calculated using unconditional logistic regression, adjusting for age group, season when the swab was taken (december-february, hot and dry; march-may, long rains; june-august, cooler and dry; september-november, short rains) and hiv status (positive, negative, unknown). we used the or to calculate pathogen-attributable fractions, which estimated the proportion of cases positive for each virus in which the virus was the likely cause [18, 19] . the pathogen-attributable fractions were calculated as (or-1)/or; pathogen-attributable fractions were only calculated for viruses with ors that were statistically significant (p,0.05). for purposes of this analysis, we assumed the pathogen-attributable fraction was 1.0 for s. pneumoniae based on the negligible probability of detection of s. pneumoniae in blood or urine of asymptomatic controls [20, 21] . ari incidence was calculated as the number of ari clinic visits per 100 person-years of observation. revisits for the same illness were not counted as separate episodes. permanent residence status in the surveillance area was used to determine person-time contribution. adjusted rates of clinic visitation were calculated accounting for the percentage of all clinic visits made for ari that went to lwak hospital as opposed to other area clinics, as determined from the household visits [3] . etiology-specific incidence was calculated by applying the proportions of each etiology to the adjusted incidence of ari ( figure 1 ). inpatients were over-represented among ari patients tested at lwak hospital (35% inpatients), compared to ari patients who visited a health facility in the community (5% inpatients), as determined by the household surveillance. therefore, the inpatient and outpatient etiologic results from lwak hospital for each pathogen were weighted using a 19:1 outpatient:inpatient ratio. after adjusting for the outpatient/inpatient distribution, the pathogenattributable fraction for each pathogen was applied to adjust the etiology-specific proportions. for those pathogens found to have an or that included 1.0, no incidences were calculated because the role of the pathogen as a cause of ari was not supported by our data. during the study period, 3,406 ari patients $5 years old were seen at lwak hospital, of whom 2,212 (65%) were outpatients and 1,194 (35%) were hospitalized. among ari patients, 2,978 (87%) had cough, 980 (29%) had difficulty breathing and 1,209 (35%) had chest pain; 2,602 (76%) had documented temperature $38.0uc, 188 (6%) had oxygen saturation ,90% without fever (among a total of 242 with oxygen saturation ,90%), and 616 (18%) were hospitalized without documented fever or low oxygen saturation. the majority (59%) was aged 5-17 years old and only 10% were $50 years old. fifty-eight percent were female. overall, 73 (2%) ari patients died within 30 days of their clinic visit (0.2% among outpatients, 6% among inpatients). among ari patients $18 years old, 893 (65%) had hiv test results available, of whom 439 (49%) were hiv-infected, compared to 17% hiv-positivity among the entire surveillance population $18 years old [7] . among 3,406 ari patients, 8.6% reported receiving one or more antibiotics before presentation (4.7% cotrimoxazole, 2.3% penicillin or amoxicillin, 2.0% other). epidemiologic characteristics were similar between patients who did (n = 1,677, 49%) and did not (n = 1,729, 51%) have blood cultures taken ( table 1 ). the median blood volume for culture was 4.0 ml. four percent of blood cultures grew contaminants ( table 2 ). among uncontaminated cultures, the most common bacteria isolated were streptococcus pneumoniae (3%) and non-typhi salmonella (3%). the percentage of patients with s. pneumoniae identified increased to 16% when adding urine antigen results to blood cultures (23% inpatients versus 14% outpatients, p = 0.003). among the 131 patients who had s. pneumoniae detected, 10 (8%) were positive by blood culture only, 114 (87%) by urine antigen only, and 7 (5%) by both tests. the case-fatality ratio was higher among those with positive blood cultures for s. pneumoniae (7%) compared with those with only urine antigen detected (0.8%, p = 0.049). s. pneumoniae were detected more frequently among hiv-positive patients (28%) than hiv-negative patients (13%, p = 0.002). mycoplasma pneumonia was detected in 0.7% of patients; no legionella or chlamydia pneumoniae were detected (table 2) . among 47 sputum samples tested by smear for mycobacterium tuberculosis, 7 (15%) were read as positive. epidemiologic characteristics were similar between patients who did (n = 1,216, 36%) and did not (n = 2,190, 64%) have naso/ oropharyngeal swabs taken (table 1) . overall during the time period of full viral testing, 68% of swabs were positive for at least 1 virus, detection being higher among outpatients (71%) than inpatients (58%, p = 0.008, table 2 ). the detection of at least one virus decreased with increasing age (p,0.001). the most commonly detected virus was rhino/enterovirus (33%) followed by influenza a virus (22%). detection of other viruses ranged from ,1% to 9%. influenza a virus was more often detected in outpatients (27%) than inpatients (11%, p,0.001) and among those ,35 years old (23%) than those $35 years old (11%, p,0.001). influenza b virus was also more common among outpatients (8%) than inpatients (2%, p,0.001). all other viruses were similarly detected among inpatients and outpatients. there were no significant differences in viral detection between hivpositive and hiv-negative patients. of 569 patients with full pathogen testing, 29% had .1 pathogen identified (table 3 ). detecting .1 pathogen was more common among hiv-positive patients (40%) than hiv-negative patients (25%, p = 0.04), but in similar proportions among inpatients (24%) and outpatients (31%, p = 0.21). the coinfection prevalence among those who had influenza (41%) was lower than that among those with other pathogens (66%, p,0.001). when limited to only those pathogens significantly more common in cases than controls, 18% of ari patients had .1 pathogen identified; still no differences between inpatients (15%) and outpatients (19%) existed for coinfection (p = 0.51). cases (n = 766) had a younger age distribution than controls (n = 273), with 68% and 42% aged 5-17 years old, respectively (p,0.01, table 4 ). more cases (44%) than controls (33%) were enrolled in the hot, dry season (p,0.01). among those with hivtesting results available, 33% of cases and 39% of controls were hiv-positive (p = 0.28). after adjustment for age, season, and hiv status, detection of influenza a virus, influenza b virus, rsv and hmpv in the naso/ oropharynx was associated with being a case (table 4 ). influenza a virus and hmpv were more strongly associated with outpatients than inpatients, although the confidence intervals overlapped. in contrast, rsv was associated with being an inpatient, but not an outpatient. piv2 and piv3 were also more common among cases, but did not reach statistical significance. rhino/enteroviruses and adenovirus were equally common among cases (33% and 11%, respectively) and controls (24% and 12%, respectively). the number of ari cases tended to peak in june and july each year, with a smaller peak in october-december ( figure 2 ). there was a trend towards increasing ari cases during the three year period (p = 0.002, linear regression). the number of ari cases was highest in january-february 2010 when pandemic h1n1 influenza a virus circulated widely in asembo [22] . of the pathogens associated with case status in the case-control study, the monthly rate of ari was correlated with the percentage positive for influenza a virus (p = 0.018), influenza b virus (p = 0.026), and hmpv (p = 0.012), but not for s. pneumoniae and rsv. of note, when we limited the analysis to the 32 months prior to november 2009, when pandemic h1n1 virus first appeared, the monthly correlation between ari case rates and viral respiratory pathogens was no longer significant. the percentage of malaria blood smears positive among ari patients ranged from (13) 1041 (10) 341 (8) 3406 (11) -1194 (62) 2212 (8) 489 (14) 802 (7) cfr a for ari cases (5) 15 (4) 73 (2) -69 (6) 4 (0.2) 20 (4) 6 (1) blood cultures 965 (48) 556 (53) 156 (46) 1677 (49) -613 (51) 1082 (49) 257 (53) 365 (46) without contaminant 924 (96) 544 (98) 149 (96) 1617 (96) -582 (95) 1035 (96) 249 (41) 353 (59) s. pneumoniae (4) 10 (7) 41 (3) 3 (7) 27 (5) 14 (1) 15 (6) 8 (2) positive urine pneumococcal antigen c 68 (13) 42 (17) 12 (20) 122 (15) 1 (0.8) 37 (19) 85 (13) 26 (23) 21 (12) s. pneumoniae by blood culture or urine antigen c 68 (14) 47 (19) 16 (27) 131 (16) 2 (2) 45 (23) 86 (14) 30 (28) 22 (13) h. influenzae (3) 25 (2) 14 (6) 6 (2) other pathogenic bacteria d 5(0.5) 8 (1) 2 (1) 15 (1) 1 (7) 9 (2) 6 (1) 2 (1) viruses and atypical bacteria naso/orophangeal specimens 716 (35) 386 (37) 114 (33) 1216 (36) -396 (33) 820 (37) 179 (37) 255 (32) influenza a virus 169 (24) 69 (18) 11 (10) 249 (20) 0 29 (7) 22 (27) 27 (15) 53 (21) influenza b virus 45 (6) 21 (5) 4 (4) 70 (6) 0 8 (2) 62 (8) 9 (5) 20 (8) rsv 57 (8) 28 (7) 7 (6) 92 (8) 3 (3) 33 (8) 59 (7) 17 (9) 13 (5) adenovirus 78 (11) 28 (7) 7 (6) 113 (9) 0 29 (7) 84 (10) 19 (11) 16 (6) parainfluenza 1 (1) 34 (3) 1 (3) 6 (2) 28 (3) 4 (2) 5 (2) parainfluenza virus 3 46 (6) 17 (4) 6 (5) 69 (6) 1 (1) 14 (4) 55 (7) 9 (5) 13 (5) human metapneumo 42 (6) 13 (3) 5 (4) 60 (5) 0 10 (3) 50 (6) 10 (6) 11 (4) rhino/enterovirus e 109 (38) 29 (24) 9 (34) 147 (33) 3 (2) 39 (35) 108 (33) 13 (24) 8 to 69% by month, and tended to be higher in the rainy months ( figure 2 ). the monthly ari rate was correlated with the malaria prevalence throughout the 36 months (p = 0.015), as well as in the 32 months prior to pandemic h1n1 (p = 0.007). the adjusted overall incidence of ari resulting in a clinic visit was 12.0 per 100 person-years for persons $5 years of age and 8.4 per 100 person-years for persons $18 years of age (table 5) . among hiv-positive persons $18 years old, the rate of ari was 21.4 per 100 person-years, compared to 5.6 for hiv-negative persons (rr 3.8, 95% ci 3.5-4.2). the highest pathogen-specific incidence among persons $5 years old was influenza a virus at 2.6 per 100 person-years, followed by pneumococcus at 1.7 per 100 person-years ( table 6 ). rates of all pathogens were higher among hiv-positive than hiv-negative persons. our study was unique in several respects. first, we compared etiologies in inpatient and outpatient settings, whereas most previous adult etiology studies in africa have focused on severely ill, hospitalized patients [23, 24, 25, 26] . we demonstrated different etiologic predominance for the same broadly defined ari syndrome based on hospitalization status, with pneumococcus more common among inpatients and influenza viruses and hmpv among outpatients. second, we enrolled a group of asymptomatic controls. as has been noted elsewhere, a positive result from pcr is not necessarily conclusive as to etiology because of its high analytic sensitivity; pcr may detect small amounts of nucleic acid due to past or asymptomatic infections [27, 28, 29, 30] . by enrolling asymptomatic controls, we were able to calculate a pathogenattributable risk, which suggested that several viruses commonly found among ari cases (i.e., rhino/enterovirus and adenovirus), were not associated with illness. third, because we embedded our etiologic study in ongoing population-based surveillance, we were able to calculate incidence of etiology-specific ari. the accuracy of our rate calculation was likely improved by adjusting for several factors that are often neglected, namely health-seeking patterns in the population and the attributable fraction of illness for each pathogen. using the calculated incidence, we estimated that approximately 3% and 2% of adults in nyanza province (approximately 4.5 million persons $5 years, 2009), where lwak is located, will have an episode of ari from influenza viruses and pneumococcus each year, respectively, which translates to approximately 135,000 and 90,000 illnesses, respectively, in the province [31] . as expected, pneumococcus was the most common bacterial cause of serious ari in this population, and was particularly prevalent among hiv-infected persons [2, 25, 32, 33, 34, 35, 36] . there was more than a 5-fold increase in the frequency of positive results for pneumococcus when incorporating the urine antigen test, supporting previous evidence that most pneumococcal pneumonia is non-bacteremic [25, 37] . non-typhi salmonella (nts) was the second most common bacterium identified in ari patients. nts bacteremia commonly presents with respiratory symptoms in children, although its role in causing pneumonia based on lung aspiration studies is doubtful [25, 38] . alternatively, patients could have a mixed infection where nts bacteremia is accompanied by another pathogen causing ari, particularly among hiv-infected persons who are at increased risk for both [23, 25, 38, 39] . the role of atypical bacteria as a cause of ari in african adults is unclear. in south africa, c. pneumoniae and legionella pneumophila table 3 . coinfection among ari cases aged $5 years of age, limited to those that had full testing -nasopharyngeal/oropharyngeal specimens, blood culture and urine antigen testing for pneumococcus. were detected by seroconversion in 21% and 9% of adults with pneumonia [40] . in botswana, 17% of mostly hiv-infected adults had evidence for m. pneumoniae infection [24] . apart from these studies, other studies of adults in less developed african countries showed low prevalence of atypical bacteria, a finding supported by our study [25, 34, 41, 42] . influenza a virus was the most commonly detected virus. a prominent role of influenza viruses as a cause of ari in africa is becoming clear as more surveillance data becomes available [22, 43] . we found influenza viruses more often in outpatients than inpatients. we have previously speculated that even when detected among inpatients with ari, influenza viruses might not be the primary reason for the admission [44] . several other viruses besides influenza virus were associated with ari. we found rsv associated with ari among inpatients, but not outpatients. rsv has been shown to be the leading cause of hospitalized ari in african children [45] . rsv has a less clear etiologic role among african adults. one study among south african adults did not find an excess in hospitalizations or mortality during rsv seasons [46] . in the u.s., several studies have postulated that rsv can cause pneumonia in adults, especially those with underlying disease or the institutionalized elderly, although none of these studies evaluated asymptomatic controls [47, 48] . in contrast to rsv, we found hmpv associated with ari among outpatients, but not inpatients. the only study of hmpv among adults on the african continent was in egypt and found hmpv in 14% of hospitalized adults with lower respiratory tract infections, but no asymptomatic controls were included [49] . coinfection was common, with almost one-third of ari patients having .1 pathogen identified. coinfection could represent a true biologic phenomenon where infectious cofactors are necessary for pathogenesis. alternatively, the frequency of coinfection could reflect the fact that certain pathogens, such as rhino/enteroviruses and adenoviruses, are commonly detected in the upper airways, with unclear clinical significance. of note, ari patients with influenza viruses detected had less coinfection, which might be related to the high pathogen attributable risk of influenza viruses and the lack of need for a coinfection to cause illness. our study cannot be considered a comprehensive assessment of the etiologies of ari. additional specimens likely would have increased the yield of pathogens identified. lung aspirates have been shown to have high diagnostic yield in african children, and one study in african adults, as they sample material directly from the affected sections of the lung [25, 50, 51] . however, lung aspirates are rarely performed in african adults with pneumonia due to the potential risk of pneumothorax in populations with high risk of pneumocystis jiroveci pneumonia (pcp). bronchoscopy with bronchoalveolar lavage has been used in a few hospitals in africa, and has improved diagnostic yield, although its use is usually reserved for severely ill, hypoxic patients in tertiary hospital settings [23, 25, 26, 34, 36] . while good-quality sputum specimens might have a role in diagnosis of some etiologies, if strict criteria for assigning a predominant organism are used, sputum is generally considered too prone to contamination with upper respiratory tract secretions to be useful diagnostically [30, 37, 52, 53] . lack of these additional specimens limited our detection of pneumocystis jiroveci and mycobacteria tuberculosis, two important causes of severe respiratory infection in high hiv prevalence populations in africa [23, 25, 26, 34, 36, 54, 55, 56] . moreover, we did not test for some viruses, such as coronaviruses and bocavirus, which might play a role in pneumonia, although their pathogenicity, particularly in adults, is still debated [11, 34, 57, 58] . while we did not observe discrete seasonality of ari in western kenya as previously shown in temperate climates, there tended to be yearly peaks coincident with the peak malaria season, which usually occur 1-2 months after the rainy season begins. there could be several explanations for this. malaria infection could augment the risk for ari. an association between malaria and bacteremia in african children has been shown, although no direct associations between malaria and pneumonia in children or adults has been proven [59] . second, similar climactic conditions could favor both malaria and pneumonia. we have shown previously that influenza incidence tends to be highest in kenya during a broad wave that mostly corresponds with the southern hemisphere winter, from june to october, a time period that also encompasses peak malaria season [5, 22] . lastly, malaria might cause symptoms that meet the nonspecific ari case definition we used. the overlap in symptoms between malaria and pneumonia in children is well-documented [60] . although similar evidence does not exist for older persons, the non-specificity of the ari definition we used suggests that symptomatic malaria could have resulted in an illness that met the ari case definition. our study had several other limitations. first, not all ari patients were sampled. reasons for not collecting swabs included high patient volume, after hours clinic attendance, and rare refusals (,10%). while we showed similar demographic characteristics and case-fatality ratios between sampled and non-sampled patients, undetected sampling bias might still have occurred that could influence the pathogens detected. second, we used a broad ari definition that likely captured a range of illnesses from influenza-like illness to pneumonia. without further diagnostic procedures, such as chest radiographs, we could not confirm the anatomic location of the respiratory infection. third, although the controls were by definition asymptomatic in the two weeks prior to enrollment, they might have been in the incubation period of a viral infection and subsequently developed symptoms. if this were true, a small number of controls might have been inaccurately classified. fourth, unlike in developed countries, we found lower rates of ari among the elderly, which we speculate is due to lower healthcare utilization by the elderly in rural kenya [3, 61, 62] . fifth, we assumed that a person's hiv status during home-based testing in 2008 was unchanged throughout the study period, which might have led to some misclassification of hiv status. however, we feel that the number of misclassified individuals is small because the annual incidence of hiv among adults in the area has been estimated at 1% (kemri/cdc unpublished data), which at the most would have resulted in 39 individuals among the 1,291 ari patients with hiv results having misclassified hiv status for some period of the three year surveillance period. lastly, despite the lack of a statistical association with ari at the population level for some viruses, an etiologic role of a virus detected in any given individual case cannot be ruled out. a study in thailand showed that rhinovirus was associated with hospitalized respiratory illness among adults, particularly among the elderly [63] . outbreaks of rhinovirus-associated pneumonia have been suspected among the elderly in developed countries [64] . adenovirus has on occasion been implicated as a cause of pneumonia in african adults, but without a control group for comparison [65] . our study demonstrated several opportunities to make an impact on the incidence of ari among older children and adults in africa. the high incidence among hiv-infected persons suggests that expanded testing and access to cotrimoxazole prophylaxis and anti-retroviral drugs will likely decrease the number of ari cases, as has been shown before for pneumococcal infections [66, 67] . vaccines could also decrease the incidence. pneumococcal conjugate vaccine was introduced for infants in kenya in early 2010 and if herd immunity occurs, as it has in developed countries, then decreases in adult pneumococcal disease incidence are expected [68] . moreover, targeted vaccination among high-risk groups, such as hiv-infected adults, might be applicable for both influenza and pneumococcus [22, 32] . participation with kemri/cdc. we thank brett whitaker for assistance in virologic testing at cdc, atlanta. we thank sonja olsen for her critical review of the manuscript. this paper is published with the approval of the director of kemri. the findings and conclusions are those of the authors and do not necessarily represent the views of the centers for disease control and prevention. the pathogen-attributable fraction (paf) is calculated as (or-1)/or using viral prevalence data from cases and controls. for pneumococcus, afe was assumed to be 1. b the percentage of ari was adjusted for the paf and then adjusted for the prevalence among inpatients and outpatients for each pathogen at lwak hospital and the percentage of ari cases in the community who were seen in clinics as inpatients and outpatients (see methods). c only persons with complete testing for s. pneumoniae including blood culture and urine antigen testing were used to calculate the percent of ari due to s. pneumoniae. doi:10.1371/journal.pone.0043656.t006 high rate of pneumococcal bacteremia in a prospective cohort of older children and adults in an area of high hiv prevalence in rural western kenya the burden of common infectious disease syndromes at the clinic and household level from population-based surveillance in rural and urban kenya health and demographic surveillance in rural western kenya: a platform for evaluating interventions to reduce morbidity and mortality from infectious diseases a reversal in reductions of child mortality in western kenya impact of permethrin-treated bed nets on the incidence of sick child visits to peripheral health facilities homebased hiv counseling and testing in rural and urban kenyan communities strategy to enhance influenza surveillance worldwide performance standards for antimicrobial susceptibility testing; nineteenth informational supplement. clsi document m100-s19 manual for the laboratory identification of bacterial pathogens of public health importance in the developing world incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in thailand application of taqman low-density arrays for simultaneous detection of multiple respiratory pathogens real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses clusters of acute respiratory illness associated with human enterovirus 68-asia, europe, and united states detection of all known parechoviruses by real-time pcr detection of mycoplasma pneumoniae, chlamydia pneumoniae, and legionella spp. in clinical specimens using a single-tube multiplex real-time pcr assay mathematical statistics: basic ideas and selected topics epidemiologic research viral respiratory infections in hospitalized and community control children in alaska diagnosis of streptococcus pneumoniae infections in adults with bacteremia and community-acquired pneumonia: clinical comparison of pneumococcal pcr and urinary antigen detection evaluation of a rapid immunochromatographic test for detection of streptococcus pneumoniae antigen in urine samples from adults with community-acquired pneumonia the epidemiology, seasonality and burden of influenza and influenza-like illness in urban and rural kenya spectrum of disease among hiv-infected adults hospitalised in a respiratory medicine unit in abidjan etiology of pulmonary infections in predominantly hiv-infected adults with suspected tuberculosis aetiology, outcome, and risk factors for mortality among adults with acute pneumonia in kenya causes of lower respiratory infection in hiv-infected ugandan adults who are sputum afb smear-negative multiplex real-time pcr for detection of respiratory tract infections frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values breathing new life into pneumonia diagnostics the 2009 kenya population and housing census: volume 1c population distribution by age, sex and administrative units a trial of a 7-valent pneumococcal conjugate vaccine in hiv-infected adults 23-valent pneumococcal polysaccharide vaccine in hiv-1-infected ugandan adults: double-blind, randomised and placebo controlled trial etiology of suspected pneumonia in adults admitted to a highdependency unit in estimating the proportion of pneumonia attributable to pneumococcus in kenyan adults: latent class analysis clinical features and etiology of pneumonia in acid-fast bacillus sputum smear-negative hiv-infected patients hospitalized in asia and africa classical and latent class analysis evaluation of sputum polymerase chain reaction and urine antigen testing for diagnosis of pneumococcal pneumonia in adults nontyphoidal salmonella infections of children in tropical africa nontyphoidal salmonella bacteraemia among hiv-infected malawian adults: high mortality and frequent recrudescence atypical' bacteria are a common cause of community-acquired pneumonia in hospitalised adults mycoplasma pneumoniae and the aetiology of lobar pneumonia in northern nigeria epidemiology of respiratory infections caused by atypical bacteria in two kenyan refugee camps elevated influenza-related excess mortality in south african elderly individuals risk factors for hospitalized seasonal influenza in rural western kenya viral etiology of severe pneumonia among kenyan infants and children influenza-and respiratory syncytial virus-associated adult mortality in soweto respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults respiratory syncytial virus and influenza a infections in the hospitalized elderly study of human metapneumovirus-associated lower respiratory tract infections in egyptian adults reappraisal of lung tap: review of an old method for better etiologic diagnosis of childhood pneumonia transthoracic lung aspiration for the aetiological diagnosis of pneumonia: 25 years of experience from the gambia a preliminary study of pneumonia etiology among hospitalized children in kenya bacteriologic diagnosis of acute pneumonia. comparison of sputum, transtracheal aspirates, and lung aspirates pulmonary complications of hiv-1 infection among adults living in sub-saharan africa evidence for high prevalence of pneumocystis jirovecii exposure among cameroonians high prevalence of pulmonary tuberculosis and inadequate case finding in rural western kenya human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus infections in hospitalized children and adults relation between falciparum malaria and bacteraemia in kenyan children: a populationbased, case-control study and a longitudinal study distinguishing malaria from severe pneumonia among hospitalized children who fulfilled integrated management of childhood illness criteria for both diseases: a hospital-based study in mozambique the epidemiology of hospitalized pneumonia in rural kenya: the potential of surveillance data in setting public health priorities healthcareseeking behaviour for common infectious disease-related illnesses in rural kenya: a community-based house-to-house survey human rhinovirus infections in rural thailand: epidemiological evidence for rhinovirus as both pathogen and bystander two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus respiratory viruses in hospital patients on the witwatersrand. a 7-year study the impact of antiretroviral treatment on the burden of invasive pneumococcal disease in south african children: a time series analysis changes in invasive pneumococcal disease among hiv-infected adults living in the era of childhood pneumococcal immunization sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine we thank oliver morgan for assistance with control enrollment, joe oundo for establishing blood cultures, and joy chebet for graphical assistance. we thank the management of st. elizabeth lwak mission hospital and the people of asembo for their continued support and august 2012 | volume 7 | issue 8 | e43656 key: cord-023346-8sqbqjm1 authors: nan title: monday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00652.x sha: doc_id: 23346 cord_uid: 8sqbqjm1 nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023017-k6edtg58 authors: nan title: aasld abstracts (pp. 282a–382a) date: 2006-02-10 journal: hepatology doi: 10.1002/hep.1840380505 sha: doc_id: 23017 cord_uid: k6edtg58 nan of well-characterized human hepatocyte cell lines could facilitate cell therapies such as htx and bioartificial livers. toward this goal, we have developed a tightly regulated human hepatocyte line. methods human hepatocytes were immortalized with a retroviral vector encoding human telomerase reverse transcriptase (htert) and green fluorescent protein (gfp) cdnas flanked by a pair of loxps. after retroviral transduction, a single cell clone was obtained using flow cytometric cell sorting followed by limiting dilution method. the resultant gfp-positive clones were supertransduced with tamoxifen-inducible cre recombinase expression cassette, mercremer. recovery of the reverted form of htert-immortalized cells was conducted by tamoxifen treatment and subsequent gfp-negative cell sorting with moflo. the expression of hepatocyte markers and albumin secretion was compared before and after tamoxifen treatment. transplantation effect of the reverted cells (lx lo9) into the liver were evaluated in a pig model of liver failure induce by an intravenous administration of d-galactosamine (d-gal). resu1ts:a non-tumorigenic human hepatocyte cell line ttnt-16-t3 was established. albumin secretion and gene expression of liver markers were increased in the reverted ttnt-16-t3 cells. transplantation of the reverted cells via the portal vein of pigs treated with d-gal was effective to prolong the survival by providing liver support until spontaneous hepatic regeneration occurred. conclusion: we here demonstrate reversible immortalization of human hepatocytes using tamoxifen-induced crellox self-excision. the resulting cell line would be highly desirable for research and therapeutic applications. acknowledgmenk the work was supported in part by life science project of 2lst century, japan. background and aim: stem cells play a key-role in tissue homeostasis. haematopoietic stem cells (hscs) could migrate into the liver and transdifferentiate, becoming a "liver stem cell reserve''. we aimed to analyse: human hsc (h-hsc) ability to engraft into the mouse liver and to contribute to its regeneration after a toxic damage. materials and methods: nodiscid mice were so divided: a) chimeric not-damaged mice: mice were submitted to irradiation followed by h-hsc intraperitoneally (i.p.) injection; finally they were killed 22, 27 and 31 days after; 8) chimeric damaged mice: mice were irradiated, injected with h-hscs and, 21 days after, damaged using ally1 alcohol (aa), i.p.; killing was performed 1 , 5 and 10 days after the damage; c) damaged not-chimeric mice: mice were damaged (with aa injection) and killed 1, 5 and 10 days after; d) not-chimeric not-damaged mice: mice were killed without any treatment. spleens (s), bone marrows (bm) and livers (l) were tested by flow-cytometry to detect h-hscs and immunohistochemistry (l only) to localize h-hscs and their hepatic derivatives. results: flow-cytometry: group a) presence of low percentage of human cells; groups c) and d) human cells undetectable; group b) presence of low percentage of human cells in bm and s while in the l they represented up to 20% of whole cellular population (the difference respect to the group a has high statistical significance, being alpha<0.001%). immunohistochemistry showed the human cell differentiation into parenchymal cells. conclusions: our results suggest that hscs could be an option to improve thie liver regeneration after a toxic injury, supporting the feasibility of bm-derived liver stem cell hypothesis. we have previously demonstrated that cd8-mediated immune damage of allogeneic liver parenchymal cells is difficult to regulate due to the activity of "immunoresistant" cd8+ t cells. in prior studies we demonstrated that short-term interference with cd401 cd40l (but not cd281b7) costimulation transiently suppressed cdb-dependent hepatocyte rejection. recently, we have determined that targetintg lfa-1 alone preferentially suppresses cd8dependent versus cd4-dependent hepatocyte rejection. in addition, short-term immunotherapy targeting both lfa-1 and cd40lcd40l costimulation produced synergistic effects and resulted in longterm suppression of cd8-dependent rejection such that hepatocyte survival up to 90 days was achieved in the majority of recipient mice. the purpose of this study was to determine whether targeting both cd40lcd40l and lfa-1 mediated signals results in an immunoregulatory state which controls naive cd8+ t cells. methods: 2 x lo6 fvbln (halat, hepatocytes, were transplanted into cd4 ko or c57bl16.scid (all h-2b) mice, and recipients were treated with anti-lfa-1 mab (0.3mg d0-6) and anti-cd4ol mab (lmg, do, 2,4,7) . hepatocyte survival was monitored by detection of serum halat reporter product by elisa. hepatocyte recipients with longterm survival (>60 days) were challenged by adoptive transfer of 2 million naive cdb' t cells (isolated from cd4 ko mice) which are sufficient to initiate rejection of functioning hepatocellular allografts in scid recipients. results: combined treatment with anti-lfa-1 mab and anti-cd40l mab significantly prolonged hepatocyte allograft survival in cd4 ko mice such that 95% achieved hepatocyte survival >60 days posttransplant (mst>105 days, n= 17) in comparison to either agent alone (anti-cd40l mab mst=35 days, n=4, p<o.ool; anti-lfa-1 mab mst=77 days, n=9, p<o.ool). recipient mice induced to achieve hepatocyte survival >60 days (by short-term treatment with anti-lfa-1 and anti-cd40l mab) received adoptive transfer of 2 million nayve purified cd8+ t cells. continued survival of longterm hepatocellular allografts despite adoptive transfer of cd8+ t cells was observed in 60% (3 of 5) recipients. in 2 recipients, adoptive transfer of cd8+ t cells was associated with loss of hepatocellular allograft function by 31 and 56 days following cell transfer. in contrast, control scid mice with functioning hepatocellular allografts which were reconstituted with 2 million naive cd8+ t cells rapidly rejected hepatocytes with mst of 17 days (n=5). conclusion: targeting of both cd40l cd4ol and lfa-1 not only prevents immunoresistant cdb-dependent immune damage of liver parenchymal cells, but also x lo3 vs. 1.321.3 x lo3 and 3.920.4 x lo3 vs. 0.321.0 x 103, respectively) while at 72 hrs it was 2.4t0.8 x lo3 vs. 2.122.0 x 103 (ns) . in the spleen, significantly higher heparanase treated cells were located within the tissue, showing proliferative activity at 48 and 72 hrs post transplantation. already by 24 hrs after transplantation the proliferating index of sec increased from 150.5 in controls to 2256 in heparanase treated rats (i'<o.o2). conclusions: heparanase increases the long-term presence of transplanted cells within portal radicles, thereby facilitating their engraftment. it has also a significant effect on ec proliferation. disc 1 o s u r e s : yaacov baruch -no relationships to disclose orit goldshmit -no relationships to disclose neta ilan -no relationships to disclose gideon shoshany -no relationships to disclose vladislav tsiperson -no relationships to disclose ella veitzman -no relationships to disclose israel vlodavsky -no relationships to disclose 264 cell activation. arunzazu sanchez, peter n u n , insa s schroeder, valentina m factor, snorri s thorgeirsson, national cancer institute, bethesda, m d substantial evidence suggests that proliferation of hepatic stem cell progenies (referred to as oval cells) is responsible for liver regeneration when the replicative capacity of hepatocytes is impaired. it is well established that activity of the tnf-a , nf-kb, il-6, and stat3 pathways is necessary for the regenerative response of hepatocytes following partial hepatectomy. however, much less is known about the signals mediating oval cell proliferation and differentiation. the aim of the present study was to evaluate the involvement of nf-kbistats signaling pathways in regulation of oval cell activation. high activity of nf-kb was found in regenerating liver after aaflph protocol coinciding with the proliferation of oval cells. urification of the oval cell population by magnetic beads sorting using ov1 antibody confirmed that the activation of both nf-kb and stat3 is specific for this cellular compartment. three distinct subpopulations of oval cells were identified based on intensity of ov1 staining by facs and named as ov1 dim, medium and bright. real time pcr analysis showed that they represent oval cells at different stages of differentiation along the hepatocytic lineage. ov1 dim subpopulation was the closest to the hepatic stem cell, as judged by high expression of c-kit. on the contrary, ov1 bright cells displayed the highest expression of hepatic differentiation markers, ttr, albumin, connexin 32 and hnf-4. nf-kb was uniformly activated in the entire oval cell compartment, whereas stat3 was phosphorylated only in the most differentiated ov1 bright subpopulation. furthermore, activation of stat3 tightly correlated with a high proliferative activity in these cells. together, these data provide the first evidence that the transcriptional activity supported by nf-kb and stat3 is required for oval cell activation. the differential induction of these two transcription factors suggests that they may control different stages of oval cell activation. disclosures: introduction: haematopoietic stem cells (hscs) have been shown to have greater plasticity than previously thought, with studies demonstrating that both human and rodent hscs can differentiate into hepatocytes. our group has also confirmed that human stem cell derived hepatocytes are the result of true differentiation and not the result of cellular fusion (newsome et al, gastroenterology 2003) . the extent to which endogenous hscs are naturally mobilized and contribute to liver repair is however uncertain. aims 1) to establish whether liver injury mobilizes endogenous hscs into the circulation & into the liver. 2) to establish if circulating stem cells in liver injury have altered stem cell potential. patients and methods 4omls peripheral blood was analysed from the following groups (n=8): normal adult controls, patients with alcoholic hepatitis, abstinent chronic alcoholic liver disease and paracetamol-induced liver injury. using a flow cytometry sorting machine, circulating cd34 positive hscs were quantified as a percentage of the number of mononuclear cells according to the ishage flow cytometry protocol. equal numbers of cd34 positive cells from each gmup were collected and cultured, to assess their in vitro stem cell properties, as judged by number of colony forming units (cf'us) produced. for patients with alcoholic hepatitis, age, sex, biochemical parameters, maddrey score and outcome (mortality) was recorded. liver biopsies from paracetamol induced liver injury and alcoholic hepatitis patients were stained for cd34 and c-kit markers to quantitate stem cell numbers (number/ portal triad) and compared with normal adult controls. results 1.levels of circulating cd34 positive hscs were significantly elevated in patients with alcoholic hepatitis (0.19% 20.06; p<0 .05) when compared with all other groups. the corresponding cd34 positive values for the other groups are as follows: healthy controls (0.05% 20.01); paracetamol liver injury (0.08% 20.04) and abstinent alcoholic cirrhotics (0.04% 20.002).there was no significant difference in mean mononuclear cell count between these groups. 2.patients surviving their alcoholic hepatitis had significantly higher levels of circulating cd34 positive stem cells (0.26% 20.09; p<o.o5) when compared with those who had died (0.057% 20.03). the surviving alcoholic hepatitis patients had significantly lower maddrey scores (22.1 92.1; p=o.oos) than those who had died (91.67 228) whilst no significant difference in sex or age distribution was demonstrated between these two groups. the total mononuclear cell counts were lower in the deceased alcoholic hepatih group (0.85xlox9l1 2 0.5) when compared with those that had survived (2.7xlox911 2 0.26). if cd34 numbers are adjusted for the total mononuclear cell counts then the increase in circulating stem cells between these two groups became even more apparent (0.44 ? 0.34 and 4.98xlox6 cellsll 24.8 ; p<0 .05). 3.hscs from alcoholic hepatitis patients produced greater numbers of cfus compared with adult control hscs (245 vs.65). 4.liver biopsies from paracetamol and alcoholic hepatitis patients contained greater numbers of stem cells compared with healthy controls (p<0.05). control (0.65 cellsltriad ?o.l) , alcoholic hepatitis (1 cellltriad 20.17) and paracetamol liver injury (1.9 cells/ triad 2 0.35). stem cells were only identifiedwithin the portal triads and not within the hepatic parenchyma in all of these groups. conclusions 1. alcoholic hepatitis is associated with increased levels of circulating hscs both in the blood and the liver. 2. these mobilized cells display in vifro stem cell potential at a level equal to or higher than control hscs. 3. increased levels of stem cell mobilization are associated with improved clinical outcome. disclosures: background klf6 is a ubiquitously expressed kruppel like zinc finger transcription factor identified as a tumor suppressor gene in prostate cancer (narla et al, science 2001) . in addition, klf6 is frequently lost and/or mutated in hepatocellular carcinomas (tal-kremer, aasld 2002-#924) . klf6 is also an immediate early gene that is rapidly upregulated in hepatocytes following partial hepatectomy and in hepatic stellate cells after injury. its expression is regulated in a tissuerestricted manner during development (laub et al, mech dev 2001) . these broad roles yet defined sites of expression suggested a potentially important role of klfg in development. the aim of this project was to examine the role of klf6 in hepatic differentiation using an embryonic stem (es) cell differentiation system in which undifferentiated cells can be driven along tissue specific lineages under defined stimuli. materials: two targeting constructs were generated in which the second exons of klf6 were replaced by a neomycinllac z cassette or hygromycin cassette. klf6 (+/-) es cells were generated by homologous recombination through selection with a klf6 targeting vector containing the neomy&/lac z cassette. to obtain klf6 (-/-) es cells, klf6 (+/-) clones were subjected to the second round of homologous recombination. a l l es cell dones were confirmed to have a normal karyotype after selection. methods and resulk in the undifferentiated state, klfg (-/-)null es cells grew more slowly than wild type es cells, with doubling times up to 50% longer. following differentiation into embryoid bodies (eb) over 6 days in culture, the number of ebs was reduced by > 80% compared with wild type ebs. the disparity in growth rate between wt and (-/-) es cells was greater in later stages than in the initial 2 days of differentiation. this was accompanied by prolongation of the epiblast-like stage, associated with markedly delayed expression of endodermal mesodermal and ectodermal markers. next, we examined the capacity of klfg (-/-) ebs to differentiate into hepatocyte like cells by withdrawing leukemia inhibitory factor after two days, then culturing ebs under serum free conditions for 8 days, followed by growth on gelatinized dishes. basic fibroblast growth factor and dexamethasone were added into the medium starting day 6 and day 10. whereas expression in klfg (+/+) ebs of alpha-fetoprotein and albumin mrna occurred after 10 days of differentiation, their expression was almost absent and significantly delayed in klf6 (-/-) ebs. an impaired ability of klf6 (-/-) cells to differentiate along hematopoietic and neuronal lineages was also observed. in conclusion, these data indicate a broad and proximal role of klfg in tissue specific development with specific role in hepatocellular development. cell transplantation has been recognized as a possible future treatment for liver cirrhosis. one of the major questions is which cell will be of greatest usespleen might be a major source of stem cell, if exist, since splenomegaly is a common in cirrhotic patients and even splenectomy is performed for treatment. recent advance in the isolation technique of stem cells based on the efflux of fluorescent dyes hoechst 33342 has turned out to be an efficient method to purify stem cells called side population (sp) cells. we utilized this method to isolate stem cells from spleen. we also transfused these sp cells into rat liver treated with carbon tetrachloride (cc14) to see whether these sp cells proliferate and differentiate into both hepatocytes or cholangiocytes. methods. c57/6j male mice and female nodlscid mice were obtained from nihon crea. egfp transgenic (tg) rat were obtained from nihon slc. splenocytes were prepared by digesting in 0.5% collagenase solution and forcing tissue through sterile mesh. splenocytes were then resuspended at lo6 cellslml in hanks balanced salt solution (hbss) containing 2% fbs, 1mm hepes (hbss+), and ug/ml hoechst 33342 w or w/o verapamil and were incubated at 37 degrees centigrade for 90min. splenocytes were then stained for 30min. on ice with fitc or pe conjugated various antibodies against sca-1, c-kit, thyl.1, cd45 and cd34. splenocytes were washed in hbss+ alone 3times and then in hbss+ with 2ug/ml propidium iodide. splenocytes were analyzed and sorted on a dual-laser facstar plus flow cytometer. ( excitation:36nm, emission:424/bp44 & 660/bp20 filter & 640-nm short-pass dichroic mirror). we also prepared spcells from donor egfp tg rats' spleen as described above. these spleen egfp positive sp cells were directly injected into liver of anesthetized recipient nodlscid mice. the recipient nodlscid mice were injected with 20 ug. cc14 intrapentoneally every once a week before and after transplantation. immunohistochemical staining were performed to identify engrafted egfp positive cells, liver specific proteins such as cytokeratinl8j9, afp and albumin. results. initial hst333422 staining of low density splenocytes showed the sp fraction to be readily detectable ( 0.3 -1%). these cells were verapamil sensitive and could be stained with hst33342 after verapamil treatment. the frequency of each surface markers positive cells were as follows (table 1 and 2). two weeks after cell transplantation, donor derived egfp positive cells were engrafted. the some engrafted cells distributed in a cluster indicating donor cell proliferation. hepatic differentiation of engrafted cells were shown by double staining of egfp and rat albumin or cytokeratinld. we observed some of afp positive cells in egfp positive cells, but very few of cytokeratinl9 positive cells. conclusions. the adult rodent spleen had consistently detectable fraction of sp cells. the surface marker profiles were different from those of bone marrow sp cells. the splenic sp cells could be engrafted in liver and differentiate into hepatocyte like cells. our results suggest that splenic sp cells could be exploited for the repair of damaged liver. backgroundlaim : it has been reported that bone marrow cells (bmcs) can replace liver in a murine model of tyrosinaemia and correct this metabolic disease through the fusion of donor bmcs to recipient hepatocytes. the aim of this study was to test whether or not this replacement is a general phenomenon in other liver injury models. the following three models were tested; (1) hepatitis b transgenic mouse (tgn(alblhbv)), (2) albumin-urokinase transgenic mouse (tgn(alblp1au)) and (3) carbon tetrachloride (cc14) treatment model. as the selective liver injury models, irradiated tgn(alb1hbv) and tgn(alblp1au) were transplanted with lx106-lx107 bmcs from green fluorescent protein (gfp) transgenic mice (tgn(actbegfp)) or from @-galactosidase transgenic mice (tgn(mtnlacz)). as a non-selective liver injury model, irradiated c57bl/6 mice were transplanted with 1x106 bmcs from tgn(actbegfp) or tgn(mtnlacz) followed by the administration of cc14 (0.02mllkg animal weight, twice a week for 4 weeks). a cci4 protocol without preparative irradiation was also tested, in which c57bl16 mice were first administered ccl, 8 times, then followed by transplantation of tgn(actbegfp) or tgn(mtnlacz) bmcs. after the analysis of the donor cell engraftment in the peripheral blood, these recipient mice were sacrificed and checked for donor-derived hepatocytes at 2-47 weeks post-transplantation. sixty liver sections for each animal (containing approximately 1 . 5~1 0~ hepatocytes), were analyzed for gfp positive cells and the whole livers were inspected for @-galactosidase expression with x-gal cytohistochemistry. however, there were no gfp positive hepatocytes and no gross blue staining of the livers with x-gal in any of the 18 recipient mice. gfp positive cells were only located in sinusoids or associated with larger vessels. they were readily distinguishable from hepatocytes through their morphology and were negative for albumin by immunostaining. in addition, the livers from 4 female animals with gender mismatched bm transplantation were also tested with y chromosome fish analysis, which might be a more sensitive method to detect donor derived cells than transgenic epitope tagging. total of 5 isolated hepatocytes were positive for y chromosome in 4 . 1~1 0~ hepatocytes analyzed, although it still remains to be elucidated whether these cells arise from spontaneous fusion events or from transdifferentiation. conclusions: these results demonstrate that there is little or no contribution of bmcs to the replacement of injured livers in these models. thus, we do not believe that bm derived cells can generally lead to a cure of liver damage. background &aims: mouse embryonic stem (es) cells are clonal cell lines derived from the inner cell mass of developing blastocysts and have multi-lineage differentiation ability. we previously reported that es cells can be made to differentiate into hepatocytes possessing high metabolic activities by transfection of hepatocyte nuclear factor-3beta (hnf-3b). in the present study, we investigated the expression of hepatobiliary organic anion transporters and bilirubin uridine diphosphate glucuronosyltransferase (ugtlal) in undifferentiated and differentiating hnf-3btransfected es (hnf-3 b-es) cells. materials & methods: hnf3b-es cells were established from a mouse es cell line. undifferentiated hnf-3b-es cells were main-tained in gelatin-coated dishes without feeder cells in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), 0.1 mm of z-mercaptoethanol(2me), 0.1 mm of non-essential amino acids (neaa), 1 mm of sodium pyruvate and 1000 ulml of leukemia inhibitory factor (lif). undifferentiated hnf-3b-es cells were allowed to differentiate in dmem supplemented with 10% fbs, 0.1 mm 2-me, 0.1 mm neaa, 1 mm sodium pyruvate, and 20 nglml of fibroblast growth factor 2 (fgfz) in the absence of leukemia inhibitory factor (lif). differentiating hnf-3 b-es cells were collected for rt-pcr analysis on day 14, and for western blotting on days 14 and 28. results: the expression of organic anion transporting polypeptide 1 (oatpl), multidrug resistance-associated protein 1 (mrpl), mrp2, mrp3, and ugtlal was not seen in the undifferentiated hnf-3 b-es cells by rt-pcr, whereas all were expressed in differentiating hnf-3 b-es cells. protein expression for oatpl, mrpl, mrp2, mrp3, and ugtlal was also observed in the differentiating hnf-3 b-es cells by western blotting. an immunofluorescence examination revealed that oatpl was co-located with desmoplakin, a marker for the basolateral (sinusoidal) membrane, and mrp2 was co-localized with cd26, a marker for the apical (canalicular) membrane, though they were both expressed throughout most of the cell membranes. we have used cdna microarray analysis of clonal murine hepatoblasts to identify genes that are significantly and preferentially expressed in undifferentiated hepatoblasts compared to adult mouse liver. unique stem cell genes were identified by overexpression in undifferentiated hepatoblasts compared to adult liver. 61 genes were identified by these criteria and were designated candidate stem cell genes (cscg). 54 cscg were distributed on all chromosomes of the mouse except chromosomes 12,14 and the y chromosome. however, chromosomes 2, 3 and 19 showed a preferential distribution of cscg compared to the total number of genes mapped on these chromosomes. approximately half of our cscg are commonly expressed in other murine stem cell populations including embryonic stem cells, neural stem cells, mesenchymal stem cells and hematopoetic stem cells, suggesting that hepatoblasts share a molecular signature with other stem cell populations. some of the commonly identified stem cell genes include: sc14a2, c-myc, nek2, phosphodiesterase 7, nicastrin, smarca 3 and smarca 5 and pias3. these genes function in a variety of pathways including ion transport, anion exchange, cell cycle control, chromatin organization, dna binding activity and signal transduction. approximately 25 % of cscg are expressed in early endoderm, fetal liver and/or the pancreatic bud. a search for homology to known proteins indicated that some of these genes contain homology to membrane transporters, zinc finger proteins and spindle apparatus components. this class of genes is of particular interest because they may represent new hepatic lineage markers. the results of this analysis leads us to conclude that a network of pathways some of which are in common with other stem cell populations function together to maintain the liver stem cell phenotype. valentina m factor, aranzazu sanchez, ju-seog lee, tanya n hoang, snom' s thorgeirsson, national cancer institute, bethesda, m d rat liver epithelial cells (rle) were isolated from normal adult rat livers and established as a cell line . rle cells were found to express undifferentiated characteristics resembling adult liver stem cells. we have addressed cellular and molecular mechanisms controlling lineage commitment of hepatic stem cells using rle-13. to induce rle differentiation along the hepatocytic lineage, differentiation protocols were developed consisting of a sequential treatment with the demethylating agent 5-aza-cytidine (5ac), fibroblast growth factors 1 and 2 (fgf), oncostatin m (osm) and hepatocyte growth factor (hgf) in the continuous presence of the synthetic glucocorticoid dexamethasone (dex). these treatments resulted in a remarkable enlargement in cell size and organelle complexity as shown by facs and confocal microscopy. morphological maturation of rle was paralleled by a decrease in cell proliferation. more significantly, rt and real time pcr analysis revealed an induction of hepatocyte specific markers such as tat, ttr, glucose-6-phosphatase, and connexin 32. in addition mrnas encoding liver enriched transcription factors hnf lalp, hnf 3ru, hnf 4, and clebp p were notably induced along with hepatocytic differentiation. the pcr results were supported by a cdna microarray analysis. comparison of the gene expression profiles following the treatment protocols reflected an activation of the hepatocytic differentiation program as judged by increased expression of a differentiation-associated gene set (phosphofructokinase, glutathione-s-transferase) and decreased expression of a cell cycle regulated gene set. currently, transplantation studies are performed to identify the potential of the differentiated rle to repopulate and restore the diseased livers. the results indicate that cell lines derived from adult liver stem cells may provide a renewable resource for transplantation. adult liver stem cells (adulis) can be isolated from rodent bone marrow. when cultured under specific conditions in co-culture with isogeneic hepatocytes, adulis fiom 14 days bile duct ligated (bdl) rats are transdifferentiating into a hepatocyte-like lineage and are able to produce urea from ammonia. the aim of the study presented is to describe the hepatocyte specific metabolic capacity of cultured adulis from normal or bdl rats in single or co-culture with isogeneic hepatocytes, with or without interleukin-3 (il-3). methods: adulis were isolated by a two-step immunoisolation procedure (i.e. beta-2-microglobulin negativity and thy-1 positivity) from rat femoral bone marrow (male wistar rats, 250g) and cultured on a matrigel layer in 24-well polystyrene dishes at a cell density of 50'000 cells/cm* in small hepatocyte media. isogeneic hepatocytes were isolated by a two-step collagenase perfusion technique and seeded (100,000 cells/cm2) on an inlay with a collagen-coated ptfe membrane (poresize: 0.4pm) for co-culture experiments. of note, the culture media was supplemented with 5% isogeneic serum and dexamethason m) was administered after culture day 3. il-3 was added in the corresponding experimental groups (10nglml). after removal of the hepatocyte-inlay (in the co-culture groups) adulis cultures were exposed to 1.5mmol nh4ci for 5 hours in dmem and urea formation determined thereafter with a colorimetric assay. cells were harvested in trizol, total rna extracted and after reverse transcription cdna used for real-time pcr determination of 18s(rm~) content to standardize the metabolic signal for cell number. relative ureagenesis values were compared statistically using jandel scientific. the significance level was set at pc0.05. results: the amount of adulis isolated from normal rat femoral bone marrow (n=lo) was 1.4620.75 x lo6 and 1.8321.97 x lo6 in animals (n=6) after seven days of bdl (p=n.s.). relative urea synthesis in cultures from normal animals was 1.0320.42 in single and 1.3820.41 in co-culture at culture days 3, 6, 9, and 12. with addition of il-3 to the culture media urea genesis was determined to be 1.6820.6 in single and 2.6521.0'2 in co-culture. in cell cultures from seven days bdl rats relative urea formation was 1.5821.43 in single and 2.6351.32 in co-culture. with the addition of il-3 to the culture media values were 2.3920.51 in single and 3.1620.81 in co-culture. addition of il-3 to the culture media increased the metabolic signal in all cell cultures from normal animals (anova on ranks, p<0.05) but not in cultures with cells isolated from bdl animals (p=n.s.). co-culture induced stronger ureagenesis under all culture conditions examined (paired t-test: p<0.05). conclusions: to exclude immunological interference from adult immunocompetent bone marrow cells cultures were strictly isogeneic (i.e. adulis, hepatocytes, and serum from the same animal). co-culturing adulis with isogeneic hepatocytes increased ureagenesis in all paired culture experiments. as there is no direct cell contact between hepatocytes and adulis paracrine soluble, so far undetermined, factors must be involved. interestingly by the addition of il-3 transdifferentiation was inducible in cultures of adulis from normal animals but no additional effect was detectable in the cell cultures from bdl animals. in further studies it will be necessairy to determine if cholestatic serum also induces accelerated and more pronounced hepatocyte specific metabolic capacity in adulis from normal animals. factors responsible for this transdifferentiation should then be isolated from cholestatic serum and might be used to support the failing liver in vivo potentially by activation of the adulis pool in the bone marrow and the liver. disclosures: saji, shinji tamura, yuichi yoshida, shinichi kiso, ayuko iizuka, hitoshi matsumoto, takako kawasaki, yoshihiro kamada, yuji matsuzawa, graduate school of medicine, osaka university, suita, osaka, japan background and aims: recently, the evidence that bone marrow cells (bm cells) have trans-differentiating potential into hepatic lineage cells has been described in animal transplantation experiments and human pathology of bone marrow transplantation recipients. however, the molecular mechanism underlying this phenomenon has remained unclear because of lack of effective culture system. to address this issue, we developed a novel in vitro culture system in which murine bm cells are cultured with growth factors and investigated factors required for the transdifferentiation of em cells into hepatic linage cells. methods and results: (1) bm cells were isolated from the femora of 6-to 10-week-old male c57bl/6] mice. they were cultured in three-dimensional culture system using type i collagen gel and stimulated without or with the growth factors (egf, hgf, hb-egf, osm, bmp-4, bfgf, fgf-4; 2ongiml) for 12 day. to investigate the effect of these growth factors, the gene expression of albumin was examined by quantitative rt-pcr. when bm cells were cultured with hgf, osm, bfgf and fgf-4, albumin was induced effectively. among these growth factors, bfgf was found to induce albumin the most effectively in the cultured bm cells. (2) bm cells were cultured in collagen gel with bfgf (2onglml) for 6, 12, 18 days. the gene expression of hepatocyte-specific markers (albumin, cytokeratin 18, alphal-antitrypsin, glucose 6 phosphates and tyrosine aminotransferase), cholangiocyte-specific marker (cytokeratin 19) and liver-enriched transcription factors (hnflalpha, hnf3alpha, hnf3beta, hnf4alpha, gata4, gata6, clebpalpha, and ciebpbeta) were examined by rt-pcr. upon the stimulation of bfgf, bm cells were found to express cytokeratin 18, alphal-anitripsin and glucose 6 phosphates, cytokeratin 19 and liver-enriched transcription factors including hnflalpha, hnfjalpha, hnf3beta, gata4 and gata6. (3) bm cells were cultured in collagen gel with bfgf (20ngiml) for 6 days. we assessed expressions of albumin and ck18 proteins of cultured bm cells by immunohistochemistry. about 5% cells of them were positively stained for both albumin and cytokeratin 18. conclusions: we established a novel in vitro culture system of bm cells and demonstrated that basic fibroblast growth factor could induce the trans-differentiation of bm cells into hepatic lineage cells the most effectively. furthermore, this conversion was associated with induction of liver-enriched transcription factors including hepatocyte nuclear factors and gata family proteins. these results indicate these liver-enriched transcription factors are the major components, which induce this trans-differentiation. disclosures: ayuko iizuka -no relationships to disclose yoshihiro kamada -no relationships to disclose takako kawasaki -no relationships to disclose shinichi kiso -no relationships to disclose hitoshi matsumoto -no relationships to disclose yuji matsuzawa -no relationships to disclose yukiko saji -no relationships to disclose shinji tamura -no relationships to disclose yuichi yoshida -no relationships to disclose crtteil, france; nadine martin, hbpital henri mondor, criteil, france; dominique couchie, anne m preaux, yannick laperche, elie 5; zafrani, inserm u581, cre'teiz, france liver can regenerate from oval precursor cells when the replication of hepatocytes is inhibited. these cells proliferate in the periportal region, migrate in the lobule and differentiate into hepatocytes. stromal-derived factor-1 (sdf-1) is a chemokine which plays a major role during embryogenesis. since sdf-1 and its sole receptor cxcr4 are involved in the differentiation of progenitor cells (e.g. b lymphopoiesis, digestive epithelial cell renewal), the aim of our work was to study the expression of sdf-1 and cxcr4 in a model of hepatic regeneration from precursor cells. hepatic regeneration from oval cells was induced by treating rats with acetylaminofluorene followed by partial hepatectomy. rats were sacrificed the day of surgery (day 0) and at days 1,5,9,14 and 21 after partial hepatectomy. rare oval cells were observed at day 1. their number was moderate at day 5, marked at day 9 and 14 and declined thereafter. oval cells predominated in the periportal region and usually formed canalar structures resembling cholangioles. individual or cholangiole-forming oval cells strongly expressed sdf-i, as demonstrated at the mrna level by in situ hybridization (ish) and at the protein level by irnmunohistochemistry. interlobular bile duct epithelial cells were also positive for sdf-1 protein but negative for its mrna. cxcr4 mrna hepatic levels, as assessed by quantitative rt-pcr, paralleled the degree of oval cell proliferation. ish showed marked cxcr4 mrna expression in individual as well as in cholangiole-forming oval cells. in order to determine which role the sdf-1icxcr4 couple could play in hepatic regeneration from oval cells, rats were treated or not with fucoidan (25 mglkg ip, twice daily), a sulfated polysaccharide known to bind to sdf-1 and to block its biological effects. as compared to untreated animals, oval cell proliferation was markedly decreased in five of the seven fucoidan-treated rats. in conclusion, oval cells express sdf-1 and its receptor cxcr4 during hepatic regeneration from precursor cells. our results strongly suggest that the sdf-1lcxcr4 couple acts in an autocrinelparacrine way to stimulate the proliferation of these precursor cells. disclosures: dominique couchie -no relationships to disclose background: bone marrow cells and embryonic stem cells are being used for cell transplantation. although these cells are known to be multipotent and capable to become hepatocyte, several problems have been pointed out, such as cell fusion and teratoma generation. thus we paid attention to side population (sp) cells, those with a verapamil-sensitive ability to efflux hoechst 33342. sp cells have been identified in several tissues. the bone marrow sp phenotype appears to be a common feature of stem cells, but hepatic and splenic sp cells have been less well characterized. aim: we tried to separate and culture sp cells from non-parenchymal liver cells (npcs) and splenocytes, and examined whether they would obtain hepatic phenotype. methods: 8-12-week-old female transgenic rats that ubiquitously express egfp were used to isolate non-parencymal liver cells by the standard collagenase two-step perfusion method, and splenocytes were isolated by forcing the tissue through sterile mesh, then mononuclear cells were separated by ficoll density gradient centrifugation. they were resuspended at lo6 cellslml in hanks balanced salt solution (hbss) containing 2% fetal calf serum, 1mm hepes (hbss+), and 5uglml hoechst 33342 (hst) w or wlo verapamil and were incubated at 37 degrees centigrade for 90min. cells were washed twice in hbss+ and then resuspended in hbss+ with 2uglml propidium iodide. cells were analyzed and sorted on a dual-laser facstar plus flow cytometer. rt-pcr was performed with rna extracted from freshly isolated sp cells. isolated sp cells were cultured with male rat primary cultured hepatocytes in collagen gel sandwich. cell morphology was monitored under a phase-contrast microscope and a fluorescence microscope. the expression of albumin, cytokeratin 18, and cytokeratin 19 was analyzed by immunohistochemistry. fluorescence in situ hybridization (fish) for the sry gene was performed to exclude ceil fusion. results: the rate of npc-sp was 0.05% of isolated npcs. 89% of npc-sp expressed cd45, while 55% of npc main population (mp) did. freshly isolated sp cells were smaller than mature hepatocytes. they looked like monocytes or lymphocytes. these cells did not express liver specific markers, such as albumin, alpha-fetoprotein, cytokeratin 18, or cytokeratin 19. during one-week culture with hepatocytes using collagen gel sandwich method, we could find no change in gfp-positive sp cells, but after further culture, we found several colonies consisting of gfp-positive sp cells, whose shape became polygonal. after 2-week-culture, most of gfp-positive cells were stained positive for albumin. npc-sp cells died within one week without hepatocyte co-culture. npc-mp cells formed colonies after 2 weeks culture with hepatocytes, but unlike sp cells, they looked like fibroblasts and were negative for albumin. freshly isolated sp cells from splenocytes were also small and monocyte-like, and expressed no liver specific markers. spleen sp cells could culture for more than 2 weeks in the same way as npc-sp cells, and looked like hepatocytes, but did not proliferate. after having been cultured, most of gfp-positive cells were stained positive for albumin and cytekeartin 18, and negative for cytokeartin 19; on the other hand splenic main population cells after 2 weeks' culture expressed those very little. conclusion: we succeeded in culturing liver and spleen sp cells. sp cells which did not express mrna of liver specific markers, such as albumin or cytokeratin 18, became to express those after the culture with hepatocytes. these results suggest that these sp cells have plasticity and obtain hepatic function. thus, tissue stem cells, especially sp cells, are useful for cell transplantation. spleen sp cell might be a candidate for source of cell transplantation for treatment of liver cirrhosis. miura, takashi goto, ken-ichiro mikami, kunio nakane, kazuo yoneyama, hirokazu nagai, kunihiko terada, toshihiro sugiyama, katsuyuki imai, haruki senoo, sumio watanabe, akita university, akita, japan background epimorphin, a mesenchymal cell surface-associated molecule identified as an epithelial morphogen, is detected on stellate cells in the liver. we have reported that a contact with hepatic stellate cells (hscs) promotes differentiation of hepatic stem-like cells (hslcs). here, we show the involvement of epimorphin in that process.materials and methods: hslcs and hscs were isolated from healthy adult rats using two-step collagenase perfusion and percoll gradient centrifugation, and maintained standard medium, dulbecco's modified eagle's medium with 10% fetal bovine serum. hslcs were cultured in stellate cell-conditioned medium, co-cultured with hscs to maintain cellcell contact or in the presence of epimorphin. phenotypical and morphologic changes were investigated by rt-pcr and with an electron microscopy, respectively. results: hslcs are polygonal in shape and assume a cobblestone appearance when cultured in standard medium. transmission electron microscopy showed that hslcs , 20 to 25 micro-m in diameter, have a round to ovalshaped nucleus, a few vacuoles, scant organelles and a high nucleuslcytoplasm ratio compared with normal hepatocytes. the phenotypic properties of hslcs did not change as well as their size and shape during this experiment. hslcs characterized by rt-pcr are follows: positive, c-kit, musashi-1 (a neural stem cell marker), alpha-fetoprotein, cytokeratinl9, connexin43; negative, albumin, transferrin, tyrosine aminotransferase, gamma-glutamyl transpeptidase. hslcs cultured in stellate cell-conditioned medium had no phenotypical and morphological changes. hslcs co-cultured with hscs expressed albumin, transferrin, and tyrosine aminotransferase, which were inhibited by an anti-epimorphin antibody. furthermore, epimorphin induced the markers not only for hepatocytes including albumin, transferrin and tyrosine aminotransferase, but also for cholangiocytes including gammaglutamyl transpeptidase in addition to increased expression of connexin43 and cytokeratinl9, with decreased expression of c-kit and musashi-1. in addition, clebp beta was enhanced, which has been reported to mediate through morphogenesis by epimorphin. hslcs co-cultured with hscs piled up and subsequent development of bile-canalicui-like structures, which was dramatically inhibited by an anti-epimoirphin antibody. hslcs, close to epimorphin, stated piling up, changed their shape from flat to cuboidal, became rich in mitochondria and rough endoplasmic reticulum and formed bile-canalicui-like structures. conclusions: hslcs have a self-renewing capacity and multilineage differentiation potential. epimorphin is involved in differentiation of hslcs though a contact with hscs. disclosures: takashi goto -no relationships to disclose n order to identify new and differentially expressed genes in fetal rat liver that are specific for epithelial stemlprogenitor cells and genes involved in liver progenitor cell differentiation, we used murine cdna microarrays containing 8,976 cdnas, available at the functional genomic facility, aecom. the expression pattern of fetal liver stemlprogenitor cells was studied from embryonic day 13 through birth, 7 days after birth and in adult liver. the driver rnas were isolated from cells adhered to the dish after plating the cell suspension in order to remove the blood cells. reference rna was isolated from the livers of newborn rats. we found that 511 genes present on the cdna microarrays were developmentally regulated. these genes fall in two major hierarchical clusters, according to their pattern of expression. the 281 genes that are down-regulated during fetal liver development were distributed in functional groups and further analyzed. in this study, special attention was paid to genes that were induced in fetal liver but were not expressed (or expressed at a very low level) in adult liver. these genes are of special interest because they can serve as specific markers for identification and for isolation of liver stemlprogenitor cells. in addition, these genes represent links to understanding the fetal liver specific molecular pathways that govern cell proliferation, survival, apoptosis and differentiation. to determine which of the 281 over-expressed genes in 13-14 day fetal liver that are down-regulated in adult liver are progenitor cells specific, we searched in the available databases whether the expression of these genes in adult liver was previously reported. seventy genes were further analyzed: the clones of interest were hybridized to radioactive labeled 32p cdna synthesized from fetal and adult liver rnas. for 48 of the clones, we found that there was little or no expression in adult liver. the expression level of 25 selected clones was analyzed further by quantitative pcr, and they were confirmed as highly induced in fetal hepatoblasts compared to adult liver. half of the 48 clones are ests. the known genes fall in different categories, the major four being: genes related to transcription; signal transduction; morphogenesis, histogenesis and organogenesis; cell adhesion, de-adhesion and migration. some of the known genes over-expressed in fetal liver that are not expressed or expressed at very low level in adult liver are: grblo (aa260248), fh12 (aa023645), tnc (aa270625), peg3 (aa003064), hey1 (aa049474), enah ((aa217593), pkcd (aa276844), lox (w96914), shcbpl (aa265225), magoh (aa254528), manba (aa200473), klf5 (aa432818), gpc3 (aa274932), pcolce2 (aa153907), ppap2c (aa220316), nfkb2 (aa060802), adam19 (aa051790), akapl2 (aa387076), tagln (bc003795. it should be noted, that most of the 48 clones and all those listed here that we have identified as liver progenitor cells specific, are expressed in stem cells of embryonic, hematopoietic, or mesenchymal origin. two of the presented genes encode cell surface proteins: a disintegrin and metalloproteinase domain 19 (adaml9) (meltrin beta), glypican 3. using in situ hybridization, we are currently verifying whether our putative liver progenitor cell specific genes are expressed in hepatoblasts and in rare progenitor cells that remain in the adult liver. identifying and cloning new genes that are expressed uniquely in liver stemlprogenitor cells will allow us to design a method for isolation of these cells and to study their role in liver development, growth control and regeneration. hepatocyte transplantation has been shown to be an effective treatment for liver diseases including fulminant hepatic failure and metabolic defects in liver function. however, the availability of sufficient numbers of hepatocytes with which to conduct clinical studies limits the wide-spread availability of this therapy. previously, we have shown that human placenta derived stem cells (pdsc) differentiate along neural or hepatic lineages depending on the culture conditions and suggested that these stem cells could be a source of cells for clinical transplantation. here we report on a protocol to further optimize hepatic differentiation of pdsc. a three stage culture system was applied to induce hepatic differentiation. ae cells were propagated in dmem based standard media which contains egf 10 nglml for a week (stage i) and subsequently with growth factors such as fgf-1, 2, 4, 7, 8, oncostatin m, hgf, andlor dexamethasone (dex) and insulin-transferrin-selenium (its) for 2 weeks (stage 11). the cells were then changed to media supplemented with different nuclear hormone receptor agonists for 1 week to stimulate hepatic maturation (stage 111). total rna samples were examined for hepatocyte specific gene expressions with real-time quantitative pcr (rtq-pcr). additional studies examined hepatic differentiation on different culture substrates including collagen, gelatin, fibronectin, arg-gly-asp-ser, and matrigel 1%, 20%, 100% were also examined. in the three stage differentiation system, 20% (vlv) matrigel coated plates and dexlits containing media, and phenobarbital (pb) were identified as the best conditions to upregulate liver specific gene expression. the principal human drug-metabolizing enzymes (cytochrome p-450, cyp) were also examined by rtq-pcr. cypla1, 2c8,2d6, and 3a4 were induced with either rifampicin or pb. in parallel, expression of the relevant liver-enriched transcription factors, hnf-4, clebp-a and ciebp-b mrnas were increased under conditions which induced hepatic differentiation. under slightly different conditions, differentiation of pdsc into cells which expressed pax6, pdxl and nkx2.2, insulin and glucagon was observed. flow cytometric analysis showed isolated nayve ae cells contains a population of cells which express the embryonic stem (es) cell markers, ssea-3, 4, tra 1-60, and tra 1-81. furthermore, the placental stem cells formed embryonic body (eb)-like structure when cells were cultured on matrigel. the eb-like structures retained the expression of ssea-3, 4, tra 1-60, and tra 1-81. these data indicate that cells can be isolated from term placenta which express markers of es cells. under appropriate conditions pdsc expand, and differentiate into cells with characteristics of hepatocytes, neurons, or pancreatic cells. we propose that pdsc could provide a new source of cells for clinical transplantation and regenerative medicine. unlike with es cells, there should be no social, ethical or religious objections to the isolation and use of placental-derived stem cells. using the ssh technology, we have identified previously 643 induced clones in 13 day fetal compared to adult liver; 202 of these clones were independent transcripts, 64 of them represented ests and4 appeared to be new sequences (petkov et al., genomics 6 8 197-209,2000) . applying the rapid amplification of 5' and 3' cdna ends to subtracted genes (generace kit, invitrogen), we have obtained the full-length cdnas for two of the subtracted clones. one of them (clone 2g8) encodes a novel putative serine proteasel subtilase highly expressed in fetal liver and comparatively low in adult. the expression of 2c8 mrna was analyzed by northern blots with rna isolated from different rat tissues: fetal and adult liver, isolated 14 day fetal hepatoblasts, brain, bone marrow, kidney, spleen, intestine, colon, stomach and muscle. on northern blot with total rna, a strong signal was detected with fetal liver/ hepatoblasts rna and a weak signal with adult liver rna, which showed that the expression of this mrna is developmentally regulated. to confirm the liver specific expression of 2g8, quantitative rt-pcr was camed out. the results of this analysis confirmed our previous results and revealed also a very low expression of 2g8 mrna in the colon. the 2g8 cdna is 3345 nucleotides in length and codes for a novel protein of 691 amino acids. we were able to identify in the data bases the sequences of a rat contig which contains the complete gene for 2g8. the gene coding for 2g8 comprises 21,920 bp of the rat genome and includes 12 exons and a long 3'-utr segment of 1,000 nucleotides. the initiation of the translation begins with an aug codon, 188 nucleotides downstream from the 5'-end of mrna. using specific primers designed from the contig sequences, we obtained clones corresponding to the upstream promoter region and downstream of the gene in the pcr4 top0 vector (invitrogen). these two upstream and downstream clones were ligated to the 2c8 cdna upstream and downstream sequences, respectively, so that the whole gene with the 5' and 3' regulatory sequences was obtained in the pcr4-top0 vector. analysis of the structure of 2g8 showed that this protein resembles 8 others mammalian subtilases, named convertases described during the last decade: furin, pc1/3, pc2, pc4, pace4, pc516 and pc7ilpcipc8 and ski-1isip. these proteases are synthesized as proproteins and secreted from the cell. 2g8 contains a putative signal sequence for release from the er located between amino acid 30 and 31 (alanine 4 glutamine). however, it does not share the consensus cleavage sequences characteristic for the other convertases: (k,r)-(x)n-(k,r) for processing, release and secretion of the active enzyme form. at present, we do not know the processing site of the proprotein. in situ hybridization experiments showed that this serine protease is expressed in fetal liver hepatoblasts. convertases are secretory proproteins implicated in tissue specific processing of hormones, growth factors, metalloproteinase, extracellular matrix proteins, viral proteins etc. they show tissue specificity and some are implicated in development and differentiation. 2g8 is a novel convertase, it is developmentally regulated and most likely functions in processing and activation of growth factorslcytokines or extracellular matrix proteins implicated in morphogenesis. further studies of its promoter region will reveal the control sequences and transcriptional activators and repressors regulating its expression during development. college, london, uk introductioniaims: bone marrow cells (bmcs) can contribute to regeneration of the chronically damaged liver but in human studies and animal models the magnitude of this axis is highly variable. in a murine model of hepatitis b we examined whether this pathway of regeneration is enhanced by inhibiting endogenous hepatocyte regeneration. methods: 2 month old female mice transgenic for hepatitis b surface antigen (hbs-tg) received lethal irradiation and were then transplanted with c57b1/6j male bmcs by tail vein injection. 6 weeks later half the mice were treated with retrorsine, a pyrrolizidine alkaloid, to irreversibly block proliferation of endogenous hepatocytes. mice were sacrificed at 3 and 6 months following retrorsine injections. y chromosome containing hepatocytes were identified using in situ hybridisation and phenotype markers (positivity for cytokeratins 8/18, cytochrome p450 and glycogen, negative for cd45). results: in the control mice with chronic liver damage there was an increase in y chromosome positive hepatocytes over time, but the proportion remained <5% of the total number of hepatocytes. however, 19.3% (corrected count) of y chromosome containing hepatocytes could be found repopulating the livers of the mice that had received retrorsine. conclusions: bmcs contribute to the regeneration of the chronically damaged liver, but under conditions where regeneration of endogenous hepatocytes is possible this is minor. however, when chronic liver damage occurs and regeneration of the endogenous hepatocytes is inhibited, the contribution to liver regeneration from the bone marrow is significantly enhanced. beaujon, paris, france; thierry poynard, groupe hospitalier pitie-salpetriere, paris, france background portal hypertension depends in part on the development of hepatic fibrosis. thus, since fibrotest (ft) is a potential biochemical marker of fibrosis, this test was prospectively used to evaluate the presence and severity of portal hypertension in patients with different liver diseases. methods: 89 consecutive patients (56 males; 32% chronic viral hepatitis c, 9% hepatitis 8, 33% alcoholic liver disease, 7% transplanted, 19% miscellaneous) with transjugular liver biopsy had same day measurements done for hemodynamic parameters (gradient, free hepatic, wedged pressures), histological parameters (fibrosis scoring system in 4 stages, necroinflammatory activity grades and modification of architecture in 3 classes), and biochemical parameters via blood sample (ft for fibrosis and actitest for activity). the measurements of histological and biochemical parameters were done blind to any other characteristics. sensitivity (se), specificity (sp), predictive values (npv and ppv), spearman correlation (sc), accuracy (ac), kappa (k) and the area under the roc curves (auroc) were assessed. the main endpoint was the prediction of elevated portal pressure (epp), defined by a gradient of 2 5 mm hg. the secondary endpoint was the prediction of highly elevated portal pressure (hepp) of 212 mm hg (hepp). results: the mean ft value was 0.47 (se 0.05) for 15 patients without epp, 0.73 (0.04) in 23 patients with moderate epp and 0.87 (0.03) in 49 patients with hepp (p<o.ol between all groups, dunnett's multicomparison test) (figure 1 ). high aurocs of ft were obtained for the prediction of epp, 0.86 (0.04) and hepp 0.78 (0.05); a cutoff at 0.70 had a 95% ppv for the presence of epp (se=76%, sp=80%) and a 0.45 cutoff had a 100% npv for the exclusion of hepp (se=100%, sp=28%). there was a significant correlation between ft and gradient (sc= 0.50, p<o.ool). the auroc of ft for the histological architectural disturbance (fibrosis or cirrhosis) was very high 0.94, (0.03). there were also excellent ft diagnostic values for stages f2f3f4 ac=87%; k=0.54 (0.11); sc =0.57, p<o.ool; auroc=0.87 (0.04). therefore histological staging did not give higher diagnostic values than ft for epp, auroc=0.91, (0.03) introduction: the prevalence of gastric varices ranges from 20-25% in cirrhotic patients and is associated with high morbidity and mortality rates. conventional endoscopic hemostatic treatment is not effective for gastric variceal bleeding. tips can achieve initial control of bleeding but it is associated with high cost, shunt dysfunction, and increased encephalopathy. cyanoacrylate injection has been shown to be very effective with 90% hemostasis and low rebleeding rates of 0-28%. in our current series of gastic varix patients at the university of virginia, injection with n-butyl-2-cyanoacrylate (histoacryl) has yielded similar results (scaldwell, fda-ide). however, distal embolization after n-butyl-2-cyanoaaylate injection has been reported and probably results from "beading" upon polymerization. whether other cyanoacrylates with different physical-chemical properties have less embolic risk with equal or greater efficacy remains to be determined. &to investigate and compare the efficacy and safety of two different cyanoacrylates in the management of gastric varices. meth-ods:polymerization properties of two cyanoacrylates, n-butyl-2cyanoacrylate (histoacryl) and octyl-cyanoacrylate (dermabond), were compared in vitro by adding a drop of blood to different concentrations of n-butyl-2-cyanoacry1atel:ethiodol and octyl-cyanoacry-1ate:ethiodol (ethiodol, iodinated poppy seed oil, is radio opaque and delays polymerization). the two cyanoacrylates were then compared for "beading" by injecting each cyanoacrylate solution into a flowing circular system of fresh frozen plasma and then collecting the resultant polymer. lastly, cyanoacrylate injection was tested in an animal model using the femoral vein of a pig to simulate a gastric varix. each femoral vein was injected with n-butyl-2-cyanoacrylate and octyl-cyanoacrylate, respectively, and then both veins were ligated and examined for histologic change. results n-butyl-2-cyanoacry1ate:ethiodol (1:l) and octyl-cyanoacrylate alone had similar polymerization properties as far as initiation of polymerization to formation of a "hard substance"these concentrations were used for subsequent testing. preliminary results from the circular flowing system show that n-butyl-2-cyanoacrylate forms a "beaded" and "crystallized" polymer whereas the octyl-cyanoacrylate polymer is more "stringy" and "rubbery". in the animal model, n-butyl-2-cyanoacrylate had formed a large, extended thrombus in the vein with an irregular surface that appeared granular. in contrast, octyl-cyanoacrylate appeared uniform throughout and occluded the vein wall with only a s m d thrombus in the center. conclusion octyl-cyanoacrylate appears to be a viable alternative with possible lower embolic risk. vessel occlusion appears to involve a combination of polymer and thrombus formation, although interaction with the dotting cascade has not been adequately investigated. further studies in an animal model of gastric varices are in development to assess the relative safety and efficacy of these and other cyanoacrylates. disclosures: stephen evgeny farber, doron fisher, rami eliakim, nira beck-razi, ahuva angel, ella veikman, irit chermesh, camal yassin, diana gaitini, michael libas, rambam medical center, ha+, israel; soboh soboh, porria hospital, tiberia, israel; yaacov baruch, rambam medical center, haqa, israel background: current guidelines recommend that patients with liver cirrhosis, undergo egd screening every 1-2 years for the presence of ev. patients with large ev should be treated with beta-blockers and those with small ev should be rescreened. platelet count and us correlate with high risk patients but are not accurate enough to avoid endoscopy in low risk patients. barium swallow is preferred by most patients but data concerning its sensitivity is not sufficient. to evaluate the accuracy of be to diagnose esophageal varices in patients with compensated cirrhosis and portal hypertension at an early stage. patients and methods: 45 patients with liver cirrhosis where evaluated by egd (japanese general criteria), as a gold standard and be for the presence and size (small, large) of varices. x-rays were taken within 3 weeks of endoscopy, with 98% barium sulfate (e-z-hd), as suspension adjusted for double contrast studies and barium paste (e-z, 60%, esophageal cream) for mucous coating. results: 45 patients diagnosed by liver biopsy; 60% postnecrotic, 20% cryptogenic, 10% nash, 10% alcohol. 90% were child-pugh's score a and 10% were child-pugh's r. the correlation between egd and be was very high; r=0.84, p<o.oool. there were only 4 false negative results with be. overall sensitivity of re was 89%. all large varices (f2-f3) were diagnosed by be. sensitivity declined with f1 varices to 73%. the positive predictive value was 86%. the cost of x-rays was one fourth that of egd. conclusions: re can be used as a non invasive procedure for the screening of patients with varices and compensated liver disease at a minimal cost. using be will allow to avoid universal prophylaxis with beta-blockers. studies dealing with the prevention of rebleeding have shown that hemodynamic response to pharmacological treatment of portal hypertension is adequate when the hepatic venous pressure gradient (hvpg) decreases to ?12mmhg or by ?20% from baseline. with beta-blockers these targets are achieved in only around 30% of cases. however, even with such a high rate of poor hemodynamic response, variceal bleeding occurs in few patients when beta-blockers are used for primary prophylaxis (around 15%). the aim of this study was to assess whether the cut-off value to define response in primary prophylaxis may be defined more accurately and to investigate whether the acute response to beta-blockers may predict evolution (avoiding a second study). methods: a total of 101 cirrhotic patients with large varices and without pre-vious bleeding were investigated. a baseline hemodynamic study was performed in all patients. after initial measurements, propranolol was administered in 55 patients (0.15mg/kg i.v.) and measurements were repeated 20 minutes later. subsequently, nadolol was chronically administered to prevent variceal bleeding. a second hemodynamic study was performed 1 to 3 months later in 72 patients. results: during a mean follow-up of 37 months, 17% of patients had a bleeding episode and 19% died. there was a significant correlation between acute and chronic hemodynamic response to beta-blockers (r=0.6, p=o.ool). using roc curve a decrease of hvpg ?lo% from baseline was the better cut-off point to predict bleeding, both for acute and chronic studies. hemodynamic responders (defining response as a decrease of hvpg to ?12mmhg or ?lo%), had a significantly lower probability of bleeding (p?o.ol) and of requiring hospital admission because decompensations of cirrhosis, while survival probability was higher, both after acute and chronic beta-blocker administration. the hvpg decreased ?20% in 38% of cases and decreased ?lo% in 74% (p?o.ool) after acute beta-blocker. these figures were 31% vs 68% (p?o.ool) after a chronic administration. conclusions: our results suggest that: 1) a decrease of hvfg ?lo% from baseline may be the target to identify hemodynamic responders in primary prophylaxis of variceal bleeding; 2) ,4cute hemodynamic response to beta-blockers may provide an accurate prognostic information on long-term risk of variceal bleeding. ut; william m grosse, corneliu sanda, milton taylor, lndiana university, bloomington, in treatment of chronic hepatitis c patients with type 1 interferon (ifn-alpha) and ribavirin leads to a sustained virologic response in -50% of patients. the elimination of serum hcv rna in an individual patient displays biphasic kinetics with the first order attributed to the direct antiviral effects of ifn-alpha and the second order attributed to an immune mediated clearance of infected cells by induction of th1 cytokines, primarily ifngamma (type 2 ifn). we have postulated that the direct antiviral effects as well as the th1 response of current therapies may be enhanced by the addition of type 2 ifn (ifn-gamma). to study this hypothesis, we examined the activity of a type 1 ifn, infergen; (ifn-alfaconl) in combination with a type 2 ifn, actimmune; (ifn-gamma l b ) in preclinical models of hcv. these included an infectious flavivirus system (west nile virus) and the hcv replicon. in addition, global transcriptional profiling was examined utilizing the hg-u133a genechip; (affymeirix, santa clara, california) system. effective concentration (ec) studies of the l effects of ifn-alfaconl, ifn-gamma l b and the combination against the west nile virus and the hcv replicon are shown below: analysis of synergy for the combination of ifn-alfaconl and ifngamma l b in the hcv replicon using the chau-talalay methodology demonstrated very strong synergistic effects for a range of doses (c1<0.1 for all observations). initial statistical analysis (pv<o.ool; fold change 2 1.2) of global transcriptional profiling revealed that treating hcv replicon containing cells with ifnalfaconl up-regulated 156 transcripts, while 61 transcripts were significantly down-regulated. ifn-gamma l b treated cells had 109 transcripts up-regulated and 32 down-regulated transcripts. most significant was the finding that several transcripts were up-regu-lated by the combination of ifn-alfaconl and ifn-gamma l b that were not up-regulated by either ifn alone. these transcripts were involved in cell-cycle regulation, antigen presentation, apoptosis and immuno-activation. in addition, several known interferon stimulated genes were highly elevated by the combination when compared to the monotherapy treatments. these results demonstrate synergistic effects of the combination of type 1 and type 2 ifns for the treatment of chronic hepatitis c both on a cellular and molecular level. further study in hcv infected patients is warranted. hcv is leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. clearance of hcv is associated with strong t cell immunity to hcv immunogens including the non-structural ns3 protein (ns3). dendritic cells (dcs) play a pivotal role in priming rare antigen specific t cells to initiate protective hcv immunity. therefore, dcs are an important target to elicit broader and more robust immune responses against hcv. to target immunogens to dcs, 12-mer peptides were screened from a phage display peptide library for specific binding to human dcs. a candidate 12-mer peptide (dc-peptide# 3) was identified that bound human monocyte-derived dcs. dc-peptides bind to ligands that are strongly involved in receptor mediated endocytosis. dc-peptides could be linked to hcv ns3 biochemically or fused genetically with preservation of dc targeting specificity and immunogenicity. dcs pulsed with ns3 that has been genetically fused to dc peptide augmented t ceil proliferation >3 fold better thanns3 alone, and skewed t cells towards thl polarization resulting in elevated levels of ifn-.)i (higher than with ns3 alone) but not il-4 or il-10. t cells also acquired an activated phenotype (cd69+). to test in viva efficacy of ns3-dc peptide fusion protein, nod-scid mice were xenotransplanted with hcv-nake t cells, and vaccinated with three separate weekly injections of autolo-gous dcs (106) pulsed with nothing, ns3 alone, or the ns3-dc pep-tide#3 fusion construct. vaccination using dcs pulsed with ns3-dc peptide# fusion protein significantly increased ns3-specific t cell proliferationlactivation and enhanced the expression of ifn-y, and tnf-a compared to ns3 alone but no detectable levels of il-10 or il-4 were observed. these data indicate that targeting hcv subunits to dcs using specific dc-peptides represents a novel vaccine approach that may facilitate targeting of immunogenic antigens to dcs to elicit specific t cell immune responses against ns3 of hcv. this immunotherapeutic strategy can be implemented against the broad range of immunogenic antigens of various pathogens. it has recently been reported that the administration of eta in the setting of chronic hcv infection enhances ifn-induced viral clearance (1). the mechanism of these synergistic effects remains to be understood. our hypothesis was that eta enhances antiviral effects of ifn by increasing t cell reactivity to antigens. methods: peripheral blood mononuclear cells (pbmcs) were isolated in cpt tubes from blood of two healthy volunteers. cells were washed and then cultured in complete media 24-well plates in the absence of any antigen (negative control) and in the presence of immobilized anti-cd3 antibody (i-cd3, 150 nglml, positive control) as a nonspecific t cell stimulant. i-cd3 stimulated cells were treated immediately with peg ifn alpha-2a at two different concentrations consistent with physiologic doses in humans (10 nglml or 100 nglml) with or without the addition of eta at two different concentrations that were also consistent with physiologic doses in humans (1.65 pglml or 3.30 pglml). supernatants from each of the previous conditions were collected and assessed by elisa for ifn-7 secretion as a marker of t cell activation. results: our findings are displayed in the figure below. there was no spontaneous ifn-7 detected in cells that were not stimulated. ifn-y was detected at a modest level after exposure to i-cd3 alone, a response that became greater after exposure of cells to peg ifn alone or eta alone. production of ifn-7 was substantially enhanced in cells that were exposed to a combination of peg ifn and eta compared to those exposed to peg ifn alone or eta alone. conclusion: our findings suggest that eta has a synergistic effect to that of peg ifn in promoting in vitro activation of pbmcs and ifn-7 production in healthy individuals. increased t cell activation with eta may also suggest that tnfa suppress t cell function and may contribute to refractoriness to inf therapy in hcv patients. the influence of absolute dose and dose in mg per kg body weight of ribavirin on sustained virologic response (svr) in interferonbased combination treatment of chronic hepatitis c as well as the influence of dose reduction is still controversial. here, we address this problem by reanalyzing data of 343 previously untreated patients with chronic hepatitis c from a multicenter trial who were treated with interferon alfa-2a plus ribavirin and amantadine or interferon alfa-2a plus ribavirin (berg et al, hepatology 2003 ,37,1359 -1367 and completed therapy. thereby, we used a multivariate approach to account for correlations of the dose of ribavirin with body weight and body mass index (bmi) and known predictor variables from this data set which are low baseline hcv rna, high platelet counts, high pretreatment alt, and low y glutamyl transpeptidase (ggt) as well as hcv genotype non-1. because per protocol dosage of ribavirin was weight-adjusted (1000 mg for body weight below 75 kg and 1200 mg for body weight 75 kg or more) and body weight was positively correlated with ggt and negatively correlated with platelet counts (spearman rank correlation, p<o.ol% and p=0.2'x), respectively, we used a stratification with respect to genotype (gt 1 vs. non-1), ggt, and platelet counts in the analysis of ribavirin dose. when comparing body weight, bmi, the absolute intention to treat (itt) ribavirin dose as well as the itt dose of ribavirin measured in mg per kg body weight and the respective ribavirin doses at the end of treatment (eot), a significant association with svr was found for the eot ribavirin dosage (mg per kg body weight) and bmi (p=1.8% and p=3.0%, respectively). for predicting svr, a weight-based ribavirin dosage threshold of 13.75 mg/kg was calculated using receiver operating characteristics (roc) curves. the svr rate was 66% (1121169) in patients with a ribarivin dose of more than 13.75 mglkg as compared with 46% (79l172) in patients receiving equal or less than 13.75 mglkg ribavirin at eot (odds ratio 2.3; 95% ci 1.5-3.6; p=o.l%). in conclusion, the analysis of ribavirin dosage motivates new considerations of weight-adjustments of the ribavirin dosage to further increase svr in hcv-infected patients treated with combination therapy. effective and approved for treatment of patients with chronic hepatitis c virus (hcv) infection, the medications have many side effects and frequently require dose reduction or discontinuation. improved adherence to the medical regimen increases sustained response rates. data currently available regarding dose reduction, discontinuation and svr are largely derived from registration trials with experienced hepatologists at academic centers. anecdotal information suggests that dose reduction and discontinuation rates are higher and svr lower in community practice, where the majority of hcv patients are treated. actual dose reduction and discontinuation rates and svr in community practice are unknown. aim: we sought to determine dose reduction and discontinuation rates and svr in community practice in comparison with academic centers within the context of a prospective, randomized, controlled, multi-center trial. methods: patients with chronic hcv (+hcv rna) were evaluated at academic centers and community sites within the context of a randomized trial comparing peg ifn a (1.0 pglkglwk) + ribavirin (800-1400 mgld) vs. peg ifn a (1.5 pglkglwk) + ribavirin (800-1400 mg/d). demographic, serological (alt), virological (hcv rna level and genotype) and histological data were obtained. eligible patients were randomized to one treatment group. medications were administered for 24 weeks (hcv gt non-1) or 48 weeks (hcv gt 1). tolerability (dose reduction rates, adverse events and discontinuation rates) through week 24 of therapy was determined and comparisons were made between academic (ac) and community-based centers (cc). results: 294 patients have been enrolled, 74 in ac and 220 in cc. 6 patients in ac and 80 in cc remain on therapy within the initial 24 weeks and are not included in this report. genotype, histology, gender and age profiles were similar in the 2 groups. * designates a significant difference p<0.05. by week 24,7168 (10 %) in ac discontinued medications in comparison to 29/140 (21%) in cc". 3/68 (4%) discontinuations were due to aefsae in ac in comparison to 101140 (7%) in cc. 2155 (4%) patients in ac who did not discontinue by wk 24 received peg ifn dose reduction in comparison to 23/108 (21%) in cc". 14/55 (25%) patients in ac who did not discontinue by week 24 received ribavirin dose reduction in comparison to 31/108 ( the clinical outcome in response to combination therapy for treatment of chronic hepatitis c virus (hcv) infection appears to be different for caucasian versus african american patients. combination therapy of ribavirin and interferon alpha-2b has generally been found to be more effective for eliminating hcv in caucasians. the present study has been undertaken to further define this difference in response to therapy between caucasians and african americans and to determine whether the difference may be related to a difference in t cell-dependent immune responses to hcv in the two patient populations. to this end peripheral blood mononuclear cells (pbmc) were collected from patients prior to the initiation of combination therapy with ribavirin and pegylated interferon alpha-2b, week 0, and at 28 weeks and 44 weeks after initiation of therapy. the pbmc were stimulated in vitro with recombinant hcv-superoxide dismutase (sod) fusion proteins for ns3 (sod-c33c), core (sod-c22-3), ns4 (sod-c100-3), ns5 (sod-nss), and the relevant recombinant sod controls from yeast or bacteria (all generous gifts from dr. michael houghton, chiron). after four days, 3h-tymidine was added to the cultures and incubation continued for 16 hours to measure proliferation. the cultures were harvested on day five. supernatants from identical cultures were collected for assay of th1, il-2 and interferon gamma (infy), and t h~, il-10 and il-4, cytokine production. t cell responses to tetanus toxoid and the mitogen phytohemagglutinin (pha) were used as positive controls for t cell response potential. to date 68 patients have entered the study. week 0 data has been collected from 64 patients and 9 normal controls. data have been collected from only 25 patients that have completed the study through week 44. the results indicate that generally all patients' t cells were stimulated by pha and to lesser extent tetanus toxoid. the major cytokine stimulated by both pha and tetanus toxoid was infy. few of the patients had significant t cell responses to hcv proteins at time 0 measured either as proliferation or cytokine production. over the period of combination therapy, most patients in which serum virus titers were reduced to non detectable levels had significant increases in t cell responses to hcv proteins. although t cell proliferation was varied among the patients, there was generally a marked increase in infy production but little production of other cytokines. responses to hcv proteins were less consistent in those patients that either did not clear the virus or in which there was resurgence of virus after initial clearance. hepatitis c virus (hcv) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer (hcc). the high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for hcv. the hcv ns3 serine protease is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. intracellular biological processes can be specifically manipulated by anti-viral antibodies. we have recently presented the modulation of hcv ns3 serine protease transforming activity by non-neutralizing recombinant intracellular antibodies (intrabodies). we herein report the development of a novel bacterial genetic screen for inhibitors of ns3 serine protease catalysis and its application for the isolation of single-chain antibody-inhibitors. our screen is based on the concerted co-expression of a reporter gene, of recombinant ns3 and of stabilized single-chain antibodies (scfvs) in e. coli. the reporter system had been constructed by inserting a short peptide corresponding to the ns5aib cleavage site of ns3 into a permissive site of the enzyme p-galactosidase. the resulting cleavable lac2 gene is carried on a low copy plasmid that also carries the ns3 coding sequence. the resultant p-galactosidase enzyme when active, conferring a lac' phenotype (blue colonies on indicative x-gal plates) while co-expression of active ns3 results in loss of / 3 -galactosidase activity (transparent colonies on x-gal plates). the identification of inhibitors, as shown here by isolating ns3 inhibiting single-chain antibodies is based on the appearance of blue colonies (ns3 inhibited) on the background of colorless colonies (ns3 active). our source of inhibitory scfvs was a scfv library we prepared from spleens of ns3-immunized mice. once isolated, the inhibitors were validated as genuine and specific ns3 binders by an elisa and as bone fide ns3 serine protease inhibitors by an in vitro catalysis assay. these antibodies are also analyzed for intracellular immunization against hcv ns3 serine protease, as inhibiting intracellular antibodies. single chain ns3 neutralizing antibodies may emerge as useful clinical reagents for the treatment of infectious diseases and cancer. our genetic screen, although applied here for the isolation of antibody-based inhibitors, should be applicable for the identification of candidate inhibitors from other sources. 1ntroduction:genetic heterogeneity is an important feature of hepatitis c virus (hcv) genomes with six known genotypes and more than 50 subtypes. genotypes 1,2 and 3 are broadly distributed in patients around the world, whereas the other types are more geographically restricted. the peptidomimetic inhibitor biln 2061, optimized to have a high affinity for genotype 1 ns3 protease, has been reported to reduce viral load in genotype 1 hcv infected individuals. in this study, the ability of biln 2061 to inhibit the ns3 proteases from the other widespread genotypes 2 and 3 was assessed. methods: the ns3 protease domains and the ns3-ns4a proteins of genotypes la, lb, 2ac, 2b and 3a were cloned, expressed in e. coli and purified. protease activity was evaluated using fluorogenic depsipeptide substrates derived from the amino acid sequence at the ns4a-ns4b and ns5a-ns5b junctions. results: the activity of the ns3 protease domains revealed no major differences among the various genotypes. for the ns3-ns4a proteins, the catalytic efficiencies of the non-genotype 1 enzymes, although higher than the ones observed for the corresponding protease domains, were similar to that observed for genotype 1 (within 3-fold). differences in activity observed among genotypes were mainly related to changes in k,,, values. binding constant (k,) values for biln 2061 were similar among non-genotype 1 proteases with k,'s ranging from 80-90 nm for the ns3-ns4a proteins, up to a 60-fold reduction in affinity when compared to genotype 1. hepatitis-c-infection (hcv) is associated with an increased incidence of the chronic fatigue syndrome and depressive mood changes. beside immunological changes the modulation of the serotonergic system might be related to these psychiatric syndromes. serotonin concentration, 5-ht uptake and the activity of the enzyme mao-b in platelets are useful peripheral parameters to monitor serotonergic activity and function in patients with a chronic hepatitis c (chc). method in a prospective controlled study biochemical measures included assays of 5-ht concentration and 5-ht uptake activity at a low physiological substrate concentration in platelets as well as mao-b activity of 64 patients with a chronic hepatitis c were compared to 21 age and gender-matched healthy subjects. furthermore the effect of interferon-alpha treatment on serotonergic parameters was evaluated in 30 patients before and during treatment. results: the mean serotonin platelet concentration did not differ between controls and hcv-infected patients. however, 5-htuptake and the mao-b activity were significant lower in patients with hcv-infection (p<o.ool respectively, t-test, two-tailed). interferon-treatment led to an increase in 53it reuptake (p=o.ol), to an increase of mao-b activity (p<o.ool) and to a decrease of serotonin concentration (p<o.ol). thus, during hcv-infection serotonin concentration was stabilized by a decrease in re-uptake and reduced metabolism by lowering mao-b activity. treatment with interferon-alpha led to a decompensation of those mechanisms by increasing the mao-b activity and 5-ht-reuptake, followed by a decreased serotonin concentration. conclusion: our data support the hypothesis of a tryptophan depletion and serotonergic deficit in patients with chronic hcvinfection and interferon-alpha treatment. furthermore the results give evidence for a useful role of serotonin reuptake inhibitors for the treatment of the serotonergic deficiency during chronic hcvinfection and interferon-alpha treatment. chronic background: recent large prospective trials demonstrated that the combination therapy of interferon (1fn)-alphalribavirin significantly increased the ratio of a sustained virological response in patients with chronic hepatitis c in comparison with ifn monotherapy, especially in patients with high hcv-rna titer and genotype lb. however, the mechanism of beneficial effect of ribavirin is still unknown. on the other hand, interleukin 18 (il-m), originally termed ifn-y inducing factor, is one of proinflammatory cytokines, which acts on th-1 cells and strongly induces intrahepatic nk or nkt cells as well as activated t cells to produce ifn-y. to clarify the effect of ribavirin in combination with ifn, special emphasis on il-18, we examined the correlation between serum il-18 level and anti-viral effect, and compared it with ifn monotherapy in patients with chronic hepatitis c. patients and methods: all patients in this study are biopsy proven chronic hepatitis c with high hcv-rna titer (hcv-rna>loo kcopieslml before therapy) and serotype 1 (genotype l a or lb). forty-two patients were treated with ifn alpha-2b (6 mu) in combination with ribavirin (600-800 mg) daily for 2 weeks, following 22 weeks with ifn alpha-2b (6 mu) 3 times a week (the combination group). another 40 patients who were treated with ifn alpha-2b alone between 1998 and 1999 (before introduction of ribavirin in japan) with the same schedule are used as control (the monotherapy group). serum samples were taken before, 2 weeks after administration and 12 weeks after cessation of therapy. serum il-18 are examined by enzyme linked immuno-sorbent assay (elisa), using human il-18 elisa kit (mbl, nagoya, japan). the il-18 ratio is defined as serum il-18 level before administration divided by serum il-18 level 2 weeks after administration. hcv-rnas are quantitatively examined before and 2 weeks after administration. sustained viral responses are confirmed 12 weeks after cessation of administration by rt-pcr. results: there are no differences in the background of patients between the combination group and the monotherapy group. in the combination group, the decline of hcv-rna level highly correlates with the il-18 ratio in patients with higher viral titer (hcv-rna>500 kcopieslml before therapy) despite no correlation is observed in patients with lower viral titer (hcv-rna<soo before therapy). similarly, the hcv-rna level 2 weeks after administration controversially correlates with the il-18 ratio in the higher titer group despite no correlation in the lower titer group. interestingly, serum il-18 level itself does not correlate with the decline of hcv-rna level in both the higher and lower hcv-rna titer group. on the contrary, in the monotherapy group, the level of up-regulation of serum il-18 is lower than that of the combination group. furthermore, although the decline of hcv-rna also correlates with the il-18 ratio in the higher titer group, the level of a correlation coefficient is lower than that of the combination group. however, the sustained viral clearance 12 weeks after cessation of treatment is not correlated with the il-18 ratio. it suggests that ribavirin enhances the up-regulation of serum il-18 level in combination with ifn therapy and the effect of ribavirin is critical for the early anti-viral response during therapy, not for sustained viral response. objective: treatment with pegylated interferon alfa and ribavirin has an efficacy in chronic hepatitis c patients with sustained response rates of about 50%. however, response in genotype 1 patients with high viral load is considerably lower and is not significantly improved by pegylation of interferon. methods: in the present study, the efficacy of cifn daily dosing and induction therapy followed by combination with ribavirin in 200 naive patients with chronic hepatitis c and genotype 1 was evaluated. au patients had histologically proven hepatitis, elevated alt values and were viremic, the average weight was 80 kg. patients were treated with cifn dosages of 27 or 18 ug qd for 4 weeks, followed by 18 or 9 ug qd for 8 weeks. treatment was continued with cifn at 9 ug qd with ribavirin (7.5 or 15 mglkgld) for another 36 weeks. results: at 48 weeks therapy an undetectable hcv-rna was observed in 79 % and 72 % in the cifn 27/18 and 18/9 ug groups, respectively. data regarding sustained response rates showed 64 '10 and 58 % for cifn 27/18 and 18/9 ug groups, respectively. due to side effects cifn had to be dose reduced in 11% and discontinued in 6% of patients. addition of ribavirin potentiated the effect of consensus interferon in a dose-dependent manner as has been shown for other interferons previously. when viral response rates were related to the initial viral load, in the genotype 1 i low viral load group, sr rates of 71 and 74% were obtained for the cifn 27/18 and 18/9 ug groups (diff. n.s.). in contrast, genotype 1 / high viral load patients showed sr rates for the cifn 27/18 and cifn 18/9 ug groups of 41 and 49% (diff. sig.), respectively. the viral response rates obtained with daily dosing of cifn were significantly higher than an independently run control arm using cifn at 9 ug tiw with ribavirin irrespective of the initial viral load. four patients (2%) experienced grade i11 thrombocytopenias, while no grade iv neutropenias or thrombocytopenias were observed. the overall tolerability in the cifn 18/9 ug qd arm was comparable to standard therapy with pegylated interferon a2b and ribavirin, while the cifn 2711819 ug arm was less tolerable during the high dosing period. however, the withdrawal rates were not sigruficantly affected by the higher dose of cifn. conclusions: cifn daily dosing l induction therapy in combination with ribavirin thus shows significant response rates with the highest response rates in genotype 1 patients seen to-date. the sustained response rates for genotype 1 i high viral load patients are higher than the ones demonstrated in the peg interferon and ribavirin registration trials. these data suggest that for difficultto-treat genotype 1 / high viral load patients cifn with daily and higher initial dosing may be a worthwhile alternative to standard combination therapy with pegylated interferons. introduction: hcv-induced cirrhosis or hepatocellular carcinoma account for at least a quarter of all patients undergoing liver transplantation. almost 100% of hcv related liver transplant patients are re-infected with hcv after liver transplantation, often within days. in most of these cases, the recurrent infection leads to chronic hepatitis. these patients are in need of preventive therapy against re-infection of the transplanted liver. antibodies may be used as a stand-alone prophylactic therapy to prevent re-infection following liver transplantation or in cases of accidental exposure (e.g. needle stick injury). in addition, it may be feasible to use monoclonal antibodies in combination with other antivirals to treat chronic hcv patients. purpose: to develop an effective therapy to prevent hcv infection in liver transplant patients. methods: several human monoclonal antibodies were generated from peripheral blood b cells isolated from individuals infected with hcv genotype lb. these antibodies are directed against the hcv envelope protein (e2) of the virus and have high affinity and broad reactivity against different genotypes. the antibodies were shown to neutralize hcv in vitro in a cell-based infection assay and in vivo using the hcv-trimera mouse model. hepex-c, a single human monoclonal antibody, was further developed and studied clinically for safety, tolerability, and potential antiviral activity in phase l a and l b clinical studies in chronic hcv patients. results: in a phase l a study, single doses of 0.25, 1.0, 2.5, 10, and 40 mg of hepex-c were administered to a total of 15 chronic hcv patients. these patients had varying levels of hcv-rna (2.6 x lo3 to 1 x lo6 iulml) and endogenous anti-e2 antibodies (5 to 550 pglml). doses of hepex-c were well tolerated and no moderate or serious adverse events (sae) were reported. in 8 out of 15 patients, hcv-rna levels were reduced at least by half immediately following infusion and then gradually returned to initial levels. a multidose phase l b study of 10,20,40,80, and 120 mg of hepex-c in 25 hcv chronic patients was conducted. doses were administered weekly for 3 weeks and then 3 times a week during the fourth week. the patients were followed for an additional 3 months. multiple doses were well tolerated; no drug related saes were reported and no specific pattern of ae noted. 9 out of 20 patients from the 10 through 80 mg cohorts had at least 1 log infusion related reduction in hcv-rna from pre-treatment levels following administration of hepex-c. data from the 120 mg cohort are pending. conclusions: the good safety and efficacy data from this trial provided a basis for a phase 2a pilot study of the safety and efficacy of hepex-c for the prevention of recurrence in hcvinfected patients who have received a hepatic allograft for endstage liver disease. a multicenter, blinded, placebo controlled study is currently underway in 19 liver transplant patients receiving 20, 40, 80, and 120 mg doses of hepex-c. logic response rates to interferon (ifn) therapy in aa patients w i t h chronic hcv infection have been reported to be lower than that for caucasians (ca), but the effect of therapy on hepatic histology in aa patients remains to be assessed. objective: to evaluate histologic progression of disease in conjunction with anti-viral efficacy and safety in non-hispanic aa hcv genotype 1 patients and non-hispanic ca, hcv genotype 1 patients treated with peginterferon alfa-2a (40kd) in combination with ribavirin (rbv). methods an open-label, multicenter study was conducted to evaluate the efficacy and safety of the combination of peginterferon alfa-2a (4okd) and rbv in aa patients and in ca patients. all patients were required to have previously untreated chronic hcv genotype 1 infection, elevated alt levels and. a liver biopsy obtained within the 24 months prior to enrollment demonstrating chronic hepatitis but without cirrhosis. patients received peginterferon alfa-2a (4okd) 180 pg sc once weekly plus rbv 1ooo or 1200 mg orally based on body weight (<75 kg or =75 kg) for 48 weeks, with 24 weeks of treatment-free follow-up. histologic responses based on the knodell hai score were determined from liver biopsies obtained prior to treatment and within 4 weeks of completion of the 24-week untreated follow-up period. hai activity scores range from 0-18 and fibrosis scores range from 0-4. the histology outcome was evaluated for patients with paired biopsies only. improvement in necrointlanunatory activity was defined as 2 2 point decrease, and improvement in fibrosis as 2 1 point decrease. the primary efficacy endpoint was sustained virologic response (svr) with undetectable hcv rna (<50 iulml) at 72 weeks. results: the intent-to-treat population included 106 patients. paired biopsies were available for 53 of the 78 aa patients and 16 of the 28 ca patients. these patients were representative of all patients with respect to knodell hai scores. mean baseline hai activity scores were 7.2 0.32 for aa and 6.9 & 0.69 for ca patients; mean baseline fibrosis scores were 1.8 + 0.14 and 1.9 + 0.3, respectively. the svr rate for all aa patients was 20l78 (26%) and for all ca patients, 11/28 (39%). for those patients with paired biopsies, svr rates were 17/53 (32%) in aa patients and loll6 (63%) in ca patients. the majority of patients in both groups exhibited improvement in activity scores: 64% for the aa group and 69% for the ca group. more importantly, the table beiow shows that the proportion of patients with worsening fibrosis was similar in both groups, while 13/53 (25%) of aa patients and only 1/ 16 (6%) of ca patients showed fibrosis improvement. mean changes in fibrosis scores were -0.4 +-0.14 for aa and -0.1 t 0.14 for ca patients. among aa patients who had paired biopsies, 5/17 (29%) patients who achieved svr had fibrosis improvement; and 8/36 (22.2%) viral nonresponders exhibited fibrosis improvement. the only patient who had fibrosis improvement among ca was a viral non-responder. conclusions: therapy with peginterferon alfa-2a (4okd) plus rbv produced improvement in necroinflanunation and fibrosis in a substantial group of aa patients in this study. although the svr of 26% in aa with genotype 1 hcv is the highest response to combination therapy yet reported in this population, even patients without an svr achieved a benefit with regard to hepatic histology. higher sustained response rates (svr). however, modification of interferon-alfa by pegylation also reduces biologic potency. delivery of a bio-optimized alpha interferon in an unmodified form (consensus interferon, infergen(r)) by continuous infusion can be expected to provide sustained and constant levels of a fuuy potent protein. we hypothesize that such an approach may potentidy result in greater antiviral activity and improved tolerability by avoiding wide swings in interferon levels. methods:we conducted a phase 2 pilot study to assess the safety, tolerability and viral kinetics of mergen (12 fg/day) by continuous infusion using a subcutaneous infusion device (medtronic minimed(r) pump) in combination with ribavirin (loo0 mg or 1200 mg daily). the minimed model 508 micro infusion pump is cleared for use in the treatment of diabetes. hcv rna viral levels were measured at days 1, 3,7,10,14,21,28 and then at weeks 6 and 12. results: among 10 nonresponders treated to date with infergen by continuous infusion with available early viral kinetics data, 7 showed a substantial reduction in hcv viral levels (at least 1 loglo reduction in 4, and 2 loglo reduction in 3). all 7 of these patients were genotype 1 and 6 had less than 1.0 loglo decrease with prior treatment while 1 had a 1.5 loglo decrease at similar time-points with other combination therapy [either pegylated interferonlrbv (4 pts) or interferon-alfa/rbv (3 pts)]. no serious adverse events were reported and 3 patients discontinued (2 due to moderate interferon-related side effects and 1 due to inability to acclimate to the pump). other patients continuing on study experienced mild adverse events that have been tolerable. conclusions: consensus interferon administered by continuous delivery via the medtronic minimed pump shows early substantial reduction of hcv rna levels among nonresponder patients and shows a good safety and tolerability profile. updated data on a total of 22 patients (including 10 na'ive genotype 1 patients) will be presented. background side effects of interferon-ribavirin combination therapy limit the sustained viral response achievable in a given hcv patient population. furthermore, it has been suggested that higher ribavirin doses and longer treatment duration are required to achieve a sustained response rate in patients with genotype 1. coupling of ribavirin to hemoglobin offers the potential of a therapeutic with improved safety and efficacy by targeting the delivery of ribavirin to key tissues infected by viral infections such as hcv. these tissues include liver parenchymal cells and macrophages, both of which possess receptors for binding and internalization of hemoglobin-haptoglobin complexes as part of the natural clearance pathway for cell-free hemoglobin. hemoglobin thus serves as a natural drug carrier for targeted therapy of such receptor-bearing tissues. aim: to evaluate the effect of a ribavirinhemoelobin coniueate (rbv-hb. hrc 203) in a mouse model of viral hepatitis induced by the coronavirus, mhv-3. methods: ribavirin was covalently coupled to highly purified hemoglobin hbao at a mean molar drug ratio of 8 1 (ribavirin:hemoglobin). the rbv-hb was evaluated for retention of haptoglobin binding and cellular uptake in vitro, as well as the ability to recover bioactive ribavirin following enzymatic cleavage from rbv-hb. the rbv-hb was complexed to haptoglobin prior to administration to mhv-3 infected balblc mice, and carried one-third of the drug dose that was used for the ribavirin group. the rbv-hbtreated group was compared to control infected untreated and ribavirin-treated groups for survival, clinical behavior, viral titer, biochemistry and liver histopathology. results: rbv-hb was specifically internalized by cultured human and mouse hepatic cells but not by control non-hepatic cells. ribavirin enzymatically cleaved from rbv-hb retained in vitro bioactivity similar to unmodified ribavirin. untreated and mhv-3 infected mice all developed clinical and biochemical signs of acute viral hepatitis and died by day 4 post infection (altmax 7000 iuil). livers recovered from untreated and infected mice showed greater than 90% necrosis. in contrast, survival was enhanced in both ribavirin and rbv-hb treated and mhv-3 infected mice with a marked reduction in biochemical (alt 1000 iuil) and histologic evidence of hepatic necrosis ( 4 0 % ) . clinically, rbv-hb treated mice behaved normally at all time points, in contrast to ribavirin treated mice which developed lethargy and abnormal fur texture compatible with drug toxicity or ongoing viral infection. conclusions: in this study, a beneficial effect of rbv-hb was shown on the course of mhv-3 infection as demonstrated by prolonged sunival, improved clinical behavior, and a reduction in biochemical and histologic disease. the study provides a rationale for additional studies to examine the mechanism for the beneficial effect. ( introduction: the teravic-4 study targets patients with chronic hepatitis c who do not show a very early viral response at week 4 of therapy, as defined by detectable serum hcv rna (>50 kjlml, pcr methodology). after 48 weeks of therapy, these patients are expected to achieve a lower rate of sustained viral response (svr) than patients with undetectable hcv rna at week 4 (vr4). models based on viral dynamics and data from pilot studies with conventional interferon (ifn) suggest that a treatment period longer than 48 weeks may lead to a higher svr rate in these difficult-to-treat patients. this trial compares 72 versus 48 weeks of treatment with pegasys(') plus copegus('i in treatmentna'ive patients without vr4. methods: this phase 111, randomized, parallel group, multicenter study is being conducted in spain in accordance with the principles of good clinical practice.(l) after signing the informed consent form, all patients received the study treatment (peginterferon alfa-2a [40kd] [pegasys(']] 180 pg once weekly and ribavirin [copegus(')] 800 mg daily 1400 mg bid]) for 4 weeks. at this time, viral response (vr) was assessed using the cobas amplicor hcv(') test v2.0 (detection limit hcv rna 50 iulml). patients achieving vr4 continued therapy for an additional 20-or 44-week period, according to their hcv genotype and viral load. patients without vr4 were randomized 1:l to continue the study treatment for either an additional 44 weeks (total active treatment duration 48 weeks or an additional 68 weeks (total active treatment duration 72 weeks ). a 24-week follow-up period is ongoing to determine the svr rate. results: the mean age of the patients was 42 years, the mean weight was 73.5 kg, and 37% were female. the prevalence of hcv genotype 1 or 4 was 78%. vr4 occurred in 36% out of 511 patients, (23% of those with hcv genotype 1 or 4,83% of those with other hcv genotypes). hcv genotype distribution, viral load, age, and body weight were similar in the randomized study groups (group-48 and group-72). among patients without vr4, hcv rna was undetectable in 62"h in group-48 and in 69% in group-72 at week 24. the biochemical response rate (br) at week 24, defined as normal alt levels, was similar as well: 81% in group-48 and 78% in group-72. the dropout rate after 24 weeks was 9% in group-48 and 6% in group-72. at the end of therapy (eot), hcv rna was undetectable in 93% of patients with vr4. the eot vr rate in group-48 was 74% versus 81y0 in group-72. the rate of br in patients with vr at eot was also higher in group-72 (85%) than in group-48 (77%). dropout rates and reasons for dropping out by the eot visit were different between groups: 17% in group-48 and 36% in group-72. therapy was well-tolerated and no unexpected adverse events were observed. the prolongation of therapy from 48 to 72 weeks did not cause an increase in the frequency of neutropenia or thrombocytopenia. conclusions: pegasys(') 3 80 p g weekly plus copegus(') 400 mg bid administered for 72 weeks offers higher rates of vr and br at eot than 48 weeks of treatment in patients with detectable hcv rna four weeks after treatment initiation. the 800 mglday dose of ribavirin used in the trial has been proven to be optimal in patients infected with hcv genotype 2 or 3, but not in those with genotype 1 who require 100011200 mglday. this evidence was not established when the trial was initiated. "the teravic-4 study group also included v. ripollks background isis 14803 is an antisense phosphorothioate oligodeoxynucleotide inhibitor of hepatitis c virus (hcv). in a previous phase i study, plasma hcv rna reductions 2 1.0 log were observed in 3 chronic hcv patients treated with isis 14803 for 4 weeks. aims: the goals for this phase i1 study were to evaluate the safety, antiviral efficacy, and pharmacokinetics of isis 14803 given for 12 weeks. methods: the study was conducted in noncirrhotic chronic hcv patients. following 2 weeks of thrice weekly intravenous dosing at 2.5 mglkg ideal body weight (ibw), patients were treated for an additional 10 weeks at higher doses given either once (group a) or twice weekly (group b). the two doses studied were 4 (initial 3 patientslgroup) and 6 mglkg ibw. results: of 43 patients enrolled into the study, 41 had genotype 1 hcv, 39 had plasma hcv rna levels 2 2 x lo6 copieslml, and all but two had previously failed interferon-based therapies. two of the 16 group a and 5 of the 18 group b patients treated at 6 mglkg ibw had plasma hcv rna reductions 2 1.0 log. three in group b had reductions of 3.2-3.8 log. hcv levels remained 2.5 log reduced at 110 days after the last isis 14803 dose in one of these patients. seventeen patients had transient elevations in alt including all those with hcv rna reductions. peak alt levels ranged 5-30 times the upper limit of the normal range (x uln). for those with hcv rna reductions, the alt flares were temporally associated with the reductions. the alt flares were not associated with any clinical signs, symptoms, or sequelae. there were no changes in prothrombin time or albumin level. minor alkaline phosphatase (5 2.2 x uln) and bilirubin (5 1.5 x uln) elevations occurred in 2 patients. dosing with isis 14803 was continued during 7 alt flares, all of which resolved to baseline levels despite continued dosing. three flares, ranging from 9-29 x uln, occurred 3-8 weeks after the last dose of isis 14803, when presumably little or no drug remained in the liver. similar alt elevations have not been observed in the clinical studies of other phosphorothioate oligodeoxynucleotides. aside from the alt flares, common adverse events in this study were mild to moderate headache, fever, chills, fatigue, nausea, myalgia and other flu-like symptoms. the fever and chills usually occurred several hours after the end of the 6 mglkg infusions and resolved within 24 hours. two serious adverse events that were considered possibly related to isis 14803 occurred in the study: one patient had cryoglobulinemic glomerulonephritis and one was hospitalized for precautionary monitoring of an altlastlbilirubin flare. the former event was possibly due to an exacerbation of a pre-existing condition. the isis 14803 plasma pharmacokinetics observed in this study were consistent with those seen in the previous phase i study. conclusions: isis 14803 appears to have significant antiviral activity in some chronic hcv patients. the plasma hcv rna reductions (up to 3.8 log) achieved in this study were in difficult-to-treat patients (i.e., genotype 1, high virus level, andlor nonresponders to interferon-based therapy). although, alt flares were observed in some patients, the results suggest the flares may not be due to a direct hepatotoxic effect of the drug. other than the alt flares, isis 14803 treatment was generally well tolerated. this study established the dose of 6 mglkg ibw twice weekly for further clinical evaluation. a study of isis 14803 in combination with peginterferon and ribavirin for patients not achieving an early virologic response during standard peginterferon and ribavirin therapy is in progress. with the long serum half-life of albumin. the objectives of this phase 1/2, open-label, dose escalation study were to evaluate the pharmacokinetics, safety, tolerability, immunogenicity, and pharmacodynamics of albuferon in subjects with hepatitis c virus (hcv) who had previously failed ifna containing regimens. methods: subjects were initially enrolled in 4 sequential dose groups (10-12 subjects per each group at 7 pg, 20 pg, 40 pg, and 80 pg). within each dose group a minimum of 5 and a maximum of 6 subjects per cohort were enrolled sequentially to receive 1 or 2 subcutaneous (sc) doses of albuferon administered 14 days apart. dose escalation beyond 80 pg included single injections of 120 pg, 180 pg, 240 p g 320 pg and 400 pg. plasma albuferon concentrations and antibody to albuferon were measured by elisa. hcv rna was measured using the amplicor hcv monitor kit (roche). 2' 5' oligoadenylate synthetase (oas1) mrna levels in whole blood was measured by a research-based taqman pcr assay. results: of the 63 subjects currently enrolled, 96% were infected with hcv genotype 1 with a mean baseline viral burden of 2 million copies/ml. 78% of subjects enrolled in these cohorts had previously failed pegylated ifna containing regimens. albuferon pharmacokinetics showed linear increases in auco.8 and c, , , with mean terminal half-lives of 139-158 hours with doses of 80 pg and higher. t, , , occurred between days 4 and 5. there is an approximately 40% increase in auc after the second injection in the double injection cohort at 80 pg. albuferon was well tolerated and there were no discontinuations. no subjects developed detectable anti-albuferon antibodies. adverse events were transient and most were mild to moderate. the most common adverse events were injection site erythema (39%), headache (30%), fatigue (26%), mylagia (21%) and arthralgia (19%). the mean reductions in the nadir neutrophil counts in the higher single dose cohorts ranged from 40-60%. there was induction of oasl mrna expression in all cohorts, with approximately 9-fold increase in median values at day 7. in the single dose cohorts (=80 pg), 52% of subjects showed a maximal 0.55 log or greater reduction in hcv viral load during the first two weeks. also, 20% or greater reductions in alt levels were observed in 36% of subjects in these single dose cohorts. conclusions: in the ongoing phase 112 study, albuferon demonstrated a favorable safety and immunogenicity profile. the pharmacokinetic profile supports dosing every 2-4 wks given its reduced clearance and extended half-life of up to 158 hours. all cohorts showed evidence of biological activity, as demonstrated by oasl induction, with anti-viral activity evident in the higher single dose cohorts. disclosures: v balan -human genome sciences, inc.: investigator t bambury -human genome sciences, inc.: investigator g everson -human genome sciences, inc.: investigator w freimuth -no relationships to disclose h mesghali -no relationships to disclose j murray -no relationships to disclose d nelson -human genome sciences, inc.: investigator l novello -no relationships to disclose b osborn -no relationships to disclose j recta -no relationships to disclose g subramanian -no relationships to disclose m sulkowski -human genome sciences, inc.: investigator j zhong -no relationships to disclose introduction pirfenidone (pfd) is an orally bioavailable pyridone derivative that affects a variety of cytokines, including inhibition of tgfbeta, tnf-alpha, pdgf, and egf. pfd has been shown in clinical studies to improve physiologic parameters in patients with pulmonary fibrosis. we have previously shown that pfd decreased hepatic fibrosis in two different rat models of cirrhosis of hepatology 37: [797] [798] [799] [800] [801] [802] [803] [804] [805] 2002) . our objective in this pilot clinical study was to evaluate the safety and preliminary activity of pfd in patients with cirrhosis of varying etiologies. methods all patients had histologic andlor clinical evidence of cirrhosis. pfd was given orally at a dose of 400 mg tid for 12 months. physical examination and labs including alt, ast, bilirubin, albumin and prothrombin time, platelet count were assessed at baseline and on monthly basis. hcv rna levels were measured in patients with chronic hepatitis c (cobas amplicor hcv monitor v2.0). liver biopsies were obtained at baseline and after 12 months of treatment and were read independently by two hepatopathologists who were blinded to the biopsy sequence. modified histological activity (hai) index of knodell and ishak fibrosis stage were used to assess changes in necroinflammatory scores and fibrosis stage, respectively. change in steatosis was also assessed. results a total of 26 patients with cirrhosis due to hepatitis c (15), ethanol (s), amyloidosis (l), autoimmune disease (1) and budd-chiari syndrome (1) were included. the mean age was 57 years (range, 29-75) with 13 males. liver biopsies at end of therapy showed a 2-point or greater reduction in the ha1 necroinflammatory score in 41% of the patients. steatosis decreased in 33% of the patients, was unchanged in 42% and worsened in 25%. while there was no significant reduction in the ishak fibrosis stage, improvement in interstitial fibrosis was noted. evidence of cell regeneration was seen in some patients. hcv rna levels were measured in 15 patients with chronic hepatitis c. at 6 months, 9 patients had a decrease in viral load, 2 patients remained unchanged and 4 patients displayed an increase in viral load compared to baseline. no patient had a sustained virologic response. 4 out of 15 (27%) hcv patients had normalization of alt, 7 out of 15 (47%) had decreased alt values, 1 did not change (7%) and 3 patients showed a modest increase in alt (20%). pfd was well tolerated with the predominant drugrelated adverse events being nausea, photosensitivity rash, and itching occurring in 15% of the patients and which improved after 2 to 3 months of therapy. conclusions: in this pilot study, treatment of cirrhotic patients with pirfenidone for one year was well tolerated. a significant reduction in necroinflammation ( 2 2-point reduction in ha1 grade) and steatosis was observed in a substantial proportion of patients. a subset of patients with chronic hcv infection showed on-treatment reduction in hcv rna levels. longer treatment duration or a less cirrhotic patient population may be needed to demonstrate effects on fibrosis. these data support conducting clinical studies to evaluate a potential role of i'fd in the treatment of steatosis, or in combination therapy for chronic hepatitis c. this work was supported by grants of marnac, inc and intermune, inc. disclosures: arnulfo alvarez -no relationships to disclose gil arechiga -no relationships to disclose juan armendariz-borunda -no relationships to disclose background: viral kinetic modeling of hcv response to interferon-based therapy provides important insights into factors associated with treatment outcomes. hcv/hiv coinfected patients appear to have lower overall response rates than that observed in monoinfected subjects, but the reason for this is not clear. we speculated that kinetic responses in key parameters would be decreased in coinfected subjects. methods: hcvlhiv coinfected patients and hcv monoinfected patients prospectively matched for treatment, genotype, age (k 5 yrs), gender, race and histology were evaluated. coinfected patients were randomized within the context of a large us. multicenter trial (actg 5071) to receive pegylated interferon alfa-2a + ribavirin vs. interferon alfa-2a + ribavirin. quantitative hcv rna (roche cobas amplicor) kinetic testing was performed at 0, 6, 12, 24, 48, and 72 hours and at days 7, 14. non-linear regression and linear models were evaluated in an effort to best predict response and identify prognostic factors. results: twenty-seven subjects underwent viral kinetic sampling and evaluation. these included twelve hcvlhiv case subjects (10 men, 2 women) and 15 matched hcv controls (some patients double-matched). the mean age of coinfected subjects was 46.1 years (range, 38-60). among hiv+ subjects the mean cd4+ count was 325 cells/mm3 (range, 175-855). 11/12 had baseline hiv rna 1400 copieslml. mean hcv viral load was 6.6 log iulml among coinfected vs. 6.2 log iulml in controls. 75% of coinfected subjects had hcv genotype 1. the remainder were genotype 2.25% of coinfected patients had no detectable virus 24 weeks after completion of 48 weeks of therapy (svr). overall svr in control subjects was 46.6%. efficiency ( e ) of phase 1 response (a) slope at 72 hours, lambda2 (slope of phase 2 decline) were calculated. efficiency of clearance ( e ) at 72 hours was highly associated with actual viral clearance across all groups (p=o.ool) but a2 was not. regression analysis failed to demonstrate a relationship between e and baseline cd4+ count, hcv viral load or genotype. in contrast, #955;2 was significantly associated with genotype. viral kinetic parameters were not predictive of svr. conclusion: viral kinetic modeling demonstrated that the efficiency of clearance at 72 hours was significantly associated with viral clearance during treatment. coinfection status did not affect key kinetic parameters. peg-ifn was superior to standard interferon in terms of viral clearance efficiency, particularly in the coinfected group. diminished svr in coinfected patients may be related to immune factors that are operative after reduction of viral loads to undetectable levels. the prevalence of chronic hepatitis c (hcv) infection is greater in african-americans (aa) than in caucasian-americans (ca). however, small studies and post-hoc analyses of larger clinical trials have not found racial differences in disease severity at study entry, though indicate that interferon and ribavirin combination therapy is not as efficacious in aa as in ca. it is important to identify disease and patient characteristics that differ between aa and ca in a large study designed to ascertain whether the apparent racial difference in response to therapy can be explained. aim: the study of viral resistance to antiviral therapy in chronic hepatitis c (virahep-c) is investigating reasons for the lack of sustained viral response (svr) to pegylated interferon and ribavirin therapy in previously untreated patients chronically infected with hcv genotype 1. a key objective is to determine whether previously reported response rate differences between aa and ca exist in a large multi-center cohort. methods: virahep-c will recruit 200 aa and 200 ca from 8 clinical centers in the united states. hypothesizing that aa and ca patients differ by factors that influence svr which could explain racial differences in svr, we compare demographic, clinical, histological, virological and health-related quality of life variables (hrqol) before treatment begins. liver biopsies performed within 18 months of study entry were assessed without knowledge of patient race by a central pathologist. results: based on the first 155 participants recruited, age and sex distributions are similar in the two racial groups with patients averaging 48 years of age and 2/3 being male more than 80% of the cohort has at least a high school education, but aa are more likely to have less than a high school education. body mass indices tend to be higher among aa than among ca (p=.06), though waistto-hip ratios are similar (mean 0.9 in each group). there are no racial differences in mode of hcv transmission, estimated duration of hcv infection, or baseline serum hcv rna levels. aa are more likely to have sub-genotype l b (p=.004).the ishak fibrosis score does not differ by race, but severe lobular inflammation is greater in aa patients (p=.03). diabetes and hypertension are more common in aa. the sf-36 physical component score in ca is similar to the general population and higher than among aa (p=.002). the sf-36 mental component score is similar in the two racial groups and comparable to the general population. summa-wlconclusion: with few exceptions, pretreatment virological, clinical and liver biopsy findings were generally similar in the two racial groups. differences in response are unlikely to be explained by these factors. lshak flbrosls score 58 (%) severa lobular influnmation (%) hypertension (%) diabetes ( bar-llan university, ramat-gan, israel background: combination therapy with peginterferon alfa and ribavirin for 48 weeks achieves sustained virologic response rates (svr) of 54-61%0 in patients with chronic hepatitis c. however, the svr rates are highly variable according to baseline (hcv genotype) and on-treatment parameters (initial viral decline). thus, it must be anticipated that current standard therapy recommendations lead to under-treatment in some and over-treatment in other individual patients. objectives: comparison between a dynamically individualized treatment schedule according to the early virologic response versus the standard of care combination therapy with peginterferon alfa-2a (40kd) (pegasys @) (peg-ifn) (180 pg qw) plus ribavirin (copegus @) (rbv) (1000-1200 mg qd) for 48 weeks. the primary aim of the study was to increase svr, while optimizing the available drugs, treatment duration, quality of life and the socio-economical burden of therapy. the secondary aim of the study was to enable a comprehensive analysis of virallhostlimmune correlates of response to treatment (ongoing). methods: all patients (n=273) were initially treated with peg-ifnlrbv for 6 weeks and initial viral kinetics were defined according to centralized serum hcv rna quantifications (cobas amplicor@ hcv monitor v2, roche molecular systems) on baseline and days 0,1,4,7,8,15,22 and 29 . after classification into viral response categories at 6 weeks, patients were randomized (n=270) to individualized therapy (arms al, a2, b1, b2, c, d) or continuation of standard therapy (std arm). treatment tailoring included: in rapid viral responders (rvr)discontinuation of rbv (al) or shortening of treatment duration to 24 weeks (a2); in slow partial responders (spr)addition of histamine (bl) or prolongation of treatment to 72 weeks (b2); in flat partial responders (fpr)addition of histamine (c); in null responders (nur)retreatment with high-dose of peg-ifn (360 pg qw) plus rbv (d). svr was defined as undetectable serum hcv rna (< 50 iulml) at the end of 24 weeks of untreated follow-up. results: demographic and baseline virologic parameters were similar in the standard and the individualized treatment groups. according to the initial viral decline patients were categorized as rvr (51% for genotype 1 and 94% for genotype 2-3), spr (35% and 5% accordingly), fpr (5% and 0% accordingly), nur (10% and 1% accordingly) or unclassified (1%). the overall svr rates for genotype 1 patients were 49% for the individualized and 56% for the standard treatment arm (ns), and for genotype 2-3 patients 90% and 87% respectively (ns). svr rates (by itt analysis) according to hcv genotype and within each initial viral response category and arm are given in the table. conclusion: the overall sustained virologic response rates of 53% in genotype 1 and 88% in genotype 2-3 patients with chronic hepatitis c treated with peginterferon alfa-2a (40kd) in combination with ribavirin support previously presented data. individualized treatment according to initial viral kinetics appears to be clinically feasible, but did not improve the sustained virologic response rate with the drugs and dosages usable at the time of this study. nevertheless, the possibility that in rapid viral responders discontinuation of ribavirin does not decrease the sustained virologic response rate warrants future prospective trials. supported by the european community (qlk2-2000-00836), hoffmann la-roche and maxim pharmaceuticals. 10% 10% (hepatology 2000; 32(suppl) :844a). the rectally administered ifn is transferred not into blood but into regional lymphatic system and reaches to the thoracic duct via intraperitoneal lymphatics (pharmaceut. res. 1986; 3,116) . therefore, the ifn suppository appears to have different antiviral mechanism from the conventional ifn injection. in this study, we performed a combination of the ifn suppository and ifn injection. the antiviral effect and immune-related markers were examined comparing with the controls (ifn injection therapy). (patients and methods) fourteen patients with chronic hepatitis c were given an ifn suppository and ifn alpha (6-10m units) injection daily for 4 weeks. after that, only ifn injection was continued three times a week for 20 weeks. the control group contains 9 patients with chronic hepatitis c, who received ifn alpha (6-10m units) injection daily for 4 weeks and were followed by the ifn injection three times a week for 20 weeks. the viral loads were 1.52-2400kiulml (median 280kiulml) in the ifn suppository group and 18.7-616kiulml (median 97kiulml) in the ifn injection group. the genotype population (lb/2a,zb) was 5/9 and 3l6, respectively. there is no significant difference in the clinical background in the two groups. serum hcv rna was tested every 4 weeks. peripheral blood nk cell activity and cd4lcd8 ratio were investigated before and 4 weeks after the beginning of the therapy. the ifn suppository contains low dose ifn alpha (ball-1). (results) serum hcv rna turned negative in 73.3% of the ifn suppository group and in 66.7% of the ifn injection group (n.s.) after 4 weeks (end point of ifn suppository administration). the hcv rna seronegative rates of the two groups were 80.0% and 77.6% (n.s.) after 24 weeks, respectively. however, while the nk cell activity was decreased in 78% of the ifn injection-treated patients after 4 weeks, only 36% of the ifn suppository patients had decreased nk cell activity (p=0.049, chi-square test). furthermore, whereas serum alt levels were significantly decreased after 4 weeks in the ifn injection group (83.5+47.9 iull vs. 53.3+30.1 iull, p=0.026, paired t test), no change was seen in the ifn suppository group (80.1iu/1+63.9 vs. 80.5+53.7, n.s.). (con-clusion) the conventional ifn therapy decreased nk cell activity and serum alt levels. however, the ifn suppository combination therapy maintained augmented nk cell activity and elevated alt levels. these findings suggest that the ifn suppository continuously activates the hosts' immunity during ifn treatment. we used the ifn suppository only for 4 weeks; however, longer administration may lead to more frequent elimination of hcv the aim of the study was to evaluate the efficacy and tolerability of induction dose pegylated-interferon and ribavirin vs pegylatedinterferon and ribavirin as treatment strategies in relapsers to standard interferon + ribavirin in chronic hepatitis c patients. methods: 110 patients, virological relapsers after a first treatment with standard interferon and ribavirin for at least 6 months, were randomized to receive either pegylayed-interferon alpha-2b 2 fglkg qw plus ribavirin 800-1000mg qd during 8 weeks then peg-interferon alpha-2b 1 pglkg qw plus ribavirin 800-1000mg qd during 40 weeks (n= 52 pts) or peg-interferon alpha-2b 1.5 pglkg qw plus ribavirin 800 -1000mg qd during 48 weeks (n= 58 pts). efficacy assessments consisted of serum hcv rna level by pcr and serum alt at the end of treatment and after 24 weeks of follow up (week 72) and liver fibrosis with metavir score before treatment and at week 72. safety evaluations included adverse events and laboratory tests. results: patients were not different for baseline characteristics in induction and non induction groups including sex (male 58 % and 71% ), mean age ( . liver fibrosis metavir score decreased in 41% of patients in non-induced group and in 19% of patients in induced group (p=0,16) whatever the response (34% in sustained responders and 25% in non responders or relapsers). conclusion 1) combination therapy with pegylated-interferon is efficient in half of relapsers to standard combination therapy. 2) the response is similar to the response observed in naive patients. 3) 8 weeks induction dose does not increase the virological response. 4) we observed a regression of liver fibrosis in 30% of patients whatever treatment regimen and virological response. backgroundanemia has been shown to be an important factor in impairing hrql in cancer patients receiving chemotherapy, with changes in hb directly correlated with changes in hrql (gabrilove et al., j clin oncol2001). since 29-36% of patients with hcv develop anemia as a side effect of combination ifnlrbv therapy (rebetron@ package insert), it is important to assess the relationship between hb and hrql in this population. the objectives of this analysis were to (1) descriptively correlate changes in hb to changes in hrql in an anemic hcv-infected population; and (2) analyze the independent relationship of hb to hrql. methods: hrql scores and hb values were obtained from clinical trial data of 185 anemic hcv-infected patients (hb 10.8 t-0.9 gldl) who had been receiving ifnlrbv therapy for 12-14 weeks (afdhal et al., ddw 2003) . during the 8-week double-blind phase, patients were randomized to receive either epoetin alfa 40,000 u once weekly or placebo. hrql was measured using the short form-36 health survey (sf-36), an accepted and validated tool that measures 8 domains of hrql (table l) , and the linear analog scale assessment (lasa), which measures constructs of overall quality of life, energy, and activity. patients were categorized into the following groups based on their change in hb levels between randomization and week 9: (1) decrease in hb; (2) hb increase from 0 to <2 gldl; and (3) hb increase 2 2 gldl. changes in hrql (by domain of each instrument) corresponding to the aforementioned hb categories were summarized (table 1) . regression analyses were conducted to evaluate the independent impact of hb change on hrql improvement. the factors included in the regression model (in addition to hb change) were: age, gender, baseline hb, baseline hrql domain, fibrosis status, rbv dose change, duration of hcv therapy, and hcv rna. results: changes in hb values were directly related to changes in hrql for all 8 domains of the sf-36 and the lasa (table 1) . hrql changes were seen in an hb-dependent manner; patients whose hb increased 2 2 g/dl had higher improvements than patients whose hb increased between 0 to <2 gldl. similarly, patients whose hb decreased from randomization to week 9 had mean decreases in hrql for most of the domains (table 1) and minimal increases for others. regression analysis substantiated that the hb change was a significant independent predictor (p=.006 to r.001) of hrql change in all subscales of the lasa and the sf-36, with the exception of bodily pain and general health. conclusions: similar to cancer patients receiving chemotherapy, improvement in hb is a strong independent predictor of improvement in the hrql of hcv-infected patients. patients on combination ifnlrbv therapy should be monitored and considered for anemia treatment to improve hrql and potentially enhance their adherence to hcv therapy. virological response. the current trial was designed to see if better results could be achieved by retreating with higher doses of peginterferon alfa-2b (peg2b) + weight-based ribavirin. aim to compare the efficacy, safety and tolerability of three different doses of peg2b + weight-based ribavirin among interferonlribavirin non-responders. methods: patients were randomized to 1 yr of treatment with peg2b 0.5, 1.5 or 3.0 mcglkglwk, plus ribavirin 12-15 mglkglday. treatment assignment was stratified for sex, race, hcv genotype and histologic fibrosis. treatment was stopped at 24 wk if pcr(+). doses were reduced by 33% for toxicity; growth factors were not allowed. results: patients: enrollment took place between february 2001 and november 2002, with 963 patients recruited from 100 centers; data forms have been received on 794 thus far. enrollment was stopped in the low-dose group after fda approval of higher doses of peg2b. the study population is 31% female, 16% african-american, 93% genotype l, and 63% f2/3/4. efficacy: on-treatment virological response rates were dose-related at 24 wk but less so at 48 wk (see table) . this was partly due to a higher rate of discontinuation after a satisfactory response at 24 wk on the higher dose. on-treatment response rates were lower among african-americans and patients with more advanced fibrosis. sustained response data will be available for most patients by october 2003. 'tolerability: the rate of dose reduction was 33% on peg2b 1.5 mcglkglwk, vs. 45% on 3.0. rates of discontinuation were the same for peg2b 1.5 and 3.0 mcglkglwk: 25% vs. 24% overall, and 13% vs. 14% for adverse events. the frequencies of subjective adverse events were similar between the two groups. some degree of iieutropenia was observed in 27% on 1.5 mcglkglwk, vs. 32% on 3.0; however, only 14 patients in the study discontinued treatment for neutropenia overall. conclusions: 1) thirty to 40 percent of interferon/ of high-dose peginterferon alfa-pb + ribavirin m s l d abstracts 313a ribavirin non-responders achieved initial clearance of viremia on peginterferon alfa-2b + ribavirin. 2) doubling the peg2b dose to 3.0 mcglkglwk resulted in a higher viral clearance rate at 24 wk but a similar rate at 48 wk. 3) the higher dose of peg2b 3.0 mcglkglwk was well tolerated, with a higher rate of dose reduction but an identical rate of discontinuation. beliefs about therapy and psychosocial factors may influence the course of treatment and therefore could be important for developing comprehensive medical care plans that improve treatment adherence and optimize response to therapy. objectives: (1) to describe baseline indices of social support, depression, and self-efficacy (i.e., perceived confidence in the ability to perform health behaviors necessary for successful treatment) of patients with hcv genotype 1 participating in a treatment trial of combination peginterferon and ribavirin therapy. (2) to assess the degree to which baseline psychosocial measures are interrelated. methods: the sample consisted of the first 155 patients enrolled in the virahep-c study, a multicenter, collaborative clinical trial, designed to examine reasons for non-response to hcv therapy in african american and caucasian patients. using a touch-screen computer, patients completed the 21-item medical outcomes study social support survey (mos-sss); the 20-item centers for epidemiological studies depression scale (cesd); 24 questions about self-efficacy, and the sf-36 quality of life instrument. questionnaires were completed during an 8-week period prior to initiation of therapy. mean scores were calculated for each of the 4 subscales of the mos-sss (l=supported none of the time, 5=all the time): a) tangible support (material aid, behavioral assistance); b) affectionate (expressions of lovelaffection); c) emotional-informational (expressions of understandinglencouragement); and d) social interaction (others to have an enjoyable time with). similarly, mean scores were calculated for 5 subscales of the selfefficacy instrument (o=no confidence, 10= high): a) obtaining help; b) communicating with physicians; and managing c) symptoms, d) depression, e) medications. correlation analysis was used to compare each subscale with the cesd and sf-36. results: of the participants in the analysis, 43% were african american and 33% were female. on average, perceived social support and self-efficacy beliefs were high at the initiation of treatment. mean scores for the tangible, affectionate, emotional, and social interaction subscales were 4.2 (sd=0.9), 4.4 (sd=0.8), 4.4 (sd=0.7) and 4.2 (sd=0.8), respectively. for self-efficacy, the mean scores were 8.0 (sd=2.0) for obtaining help; 9.2 (sd=1.5) for communicating with physicians; 7.3 (sd=2.2) for managing symptoms; 8.3 (sd=1.6) for managing depression; and 9.4 (sd=1.3) for managing medications. the distribution of social support and self-efficacy scores were similar for both racial groups and genders. depressive symptoms were common in the sample at baseline with 55% having a score of at least 16 on the cesd and 7% having a score of at least 28. in general, the social support and self-efficacy subscales were unrelated to the cesd and to the subscales of the sf-36. conclusion: african american and caucasian study participants rate their social support and self-efficacy beliefs high in the weeks prior to initiating combination antiviral therapy for hcv, although depressive symptoms are common. social support and self-efficacy instruments may measure different psychosocial constructs as compared with other instruments used in hcv research like the sf-36 and cesd. as a result, social support and selfefficacy may represent new and important predictors of adherence and sustained virologic response (svr) in hcv treatment. baseline data from virahep-c will be used to evaluate factors associated with adherence and svr to determine if racial differences exist among these measures. if so, this information can be used to develop new initiatives for educating patients and families prior to initiating hcv therapy. background and aims: interferon(1fn) a-2blribavirin therapy is an effective anti-viral therapy for the patients with hepatitis c virus (hcv) genotype 1. however, the response to this therapy is not satisfactory because only about 40-50% of the patients become sustained responder(sr)s. further, the patients receiving ifn a-2blribavirin therapy have been frequently withdrawn because of severe adverse effects. the efficacy of ifn therapy is mediated by both genomic type and viral load and to immunological response of the hosts. reportedly, some amino acids were identified to modulate host's immunological response. in order to achieve more higher sr ratio after ifn a -2blribavirin therapy and to decrease the number of withdrawn patients, it seems important to improve the nutritional condition and to upregurate potential immunological response by modifying the balance of amino acid. in the present study, we investigated the dynamics of a panel of 41 amino acids during ifna-2blribavirin therapy, then analyzed the effects of nutritional condition on the clearance of hcv. materials and methods: seventy patients with chronic hcv infection were included after informed consent. six-million unit of ifna-2b in combination with ribavirin (600-800mglday) were given during 6 months. during the first 2 weeks of the therapy, ifn a -2b was given daily. blood samples were obtained after overnight fasting at zweeks, 8weeks, and 24weeks of the therapy and served for the analysis of amino acids. at the same time, peripheral blood mononuclear cells (pbmc) were collected and the number of ifny-and il4-producing cells were measured by facs analysis. thllth2 ratio was calculated as following; thll results: hcv rna was undetectable in 22%, 52%, and 78% of the patients at 2, 8, and 24 week of the therapy, respectively. thllth2 ratio decreased gradually during the therapy. thllth2 ratio at the end of therapy (24 week of the therapy) was significantly smaller than that at the beginning of the therapy (17.8210.9 vs 13.558.0, p=o.o11). however, there was no significant correlation of thll th2 level with the rate of hcv eradication. total amino acids (taas) and branched chain amino acids (bcaas) decreased gradually during the therapy. even at 8week of the therapy, there was a significant decrease in taa and bcaa (p=0.017 and p<o.oool, respectively). fischer ratio (fr) also showed drastic change. it changed from 2.83 (at the beginning of the therapy) to 2.19 (at 8 week of the therapy), indicating that the balance of amino acid rapidly altered to the similar condition to the patients with liver cirrhosis. of interest, the levels of taa and bcaa at the beginning of the therapy were significantly higher in hcv rna-negative group than in hcv rna-positive group at the end of the therapy (taa: 3243+.275 vs 28772252 nmol/ml; p=0.004, bcaa: 470292 vs 377265 nmollml; p=0.024). although we could not find a significant difference, fr at the beginning of the therapy was larger in hcv rna-negative group than in hcv rna-positive group at the end of the therapy (2.8120.51 vs 2.4750.68, p=0.15). analysis of each amino acid showed that there were significant differences in the concentration of citrulline and ornithine, both amino acid are involved in urea cycle, between hcv rna-negative and hcv rna-positive group at the end of the therapy. conclusions: interferon a-zblribavirin therapy induces malnutrition at early stage of the therapy. successful elimination of hcv rna seemed to depend much on nutrition rather than the ratio of thl/th2 during the therapy, suggesting that better response in interferon a-zblribavirin therapy might be possible by improving the condition of nutrition at the beginning of the therapy. in addition, adequate nutrition support would contribute to the improvement of the prevalence of hcv infection in egypt varies between 9% and 24% and approximately 90% are infected with hcv genotype 4.to date limited data exist on the effect of antiviral therapy on genotype 4-infected subjects. the purpose of this study was to assess the effect of interferon (standard or pegylated) in combination with ribavirin in na'ive-to-treatment subjects with chronic hepatitis c who reside in egypt. methods: in a prospective, open-label, randomized clinical trial, 200 egyptian subjects with chronic hepatitis c documented by liver biopsy and detectable hcv rna in serum received interferon alfa 2b (ifn-a2b) 3 mu tiw or pegylated ifn-a 2b (100 mcglweek) in combination with weight-based oral ribavirin (800 or 1000 mg for both groups). the study design was that if hcv rna was detectable at week 24, the treatment was stopped but if hcv rna was undetectable at week 24, treatment was extended for a total of 48 weeks. subjects were observed for an additional 24 weeks and hcv rna status at week 72 determined the response to antiviral therapy. safety laboratory tests were done at regular intervals. the study received irb approval. the hcv rna was done by a pcr technique with a detectability cutoff of 50-copieslmb results: both randomized groups were similar at baseline without statistical significant difference, 190 (90%) were infected with genotype 4, mean age was 39.5 years ? 8.7 years, 158 were men (79%), mean bmi was 27.8 2 4 kglm2, mean inflammatory ha1 score was 7/18 2 2 and fibrosis stage 2.716 t 1.3. of the 190 genotype-4 infected individuals, 156 have finished therapy (week 48) and 78 had completed the 24week follow-up (week 72). the study will be completed by october 2003. the table shows the intention to treat rates of undetectable hcv rna in genotype-4 infected subjects. serious adverse events that led to discontinuation of medications were observed in 6 subjects in the standard ifni'rbv group and in llsubjects in the peg ifnirbv, one death was observed in the latter group. expected adverse events were observed in similar rates in both groups. laboratory adverse events were observed between 2"h and 10% of subjects receiving either treatment regimen depending on the week of therapy. no growth factors were used in this population. in summary: subjects with chronic hepatitis c and genotype-4 infection have a similar antiviral response to standard or pegylated interferon in combination with ribavirin. the safety and tolerability of the medications is similar to what has been observed for other hcv genotypes. patients infected with hcv genotype 4 respond effectively to therapy and should be encouraged to undergo antiviral treatment. daily for 4 weeks, followed by 3 times a week for 20 weeks (week 24) and followed up for 24 weeks after cessation of therapy (week 48). the expression of socs-1 protein in the liver specimen obtained before the treatment was investigated by immunohistochemistry for socs-1. results: patients with the expression of socs-1 protein in the liver were defined as "socs-1 positive group". all other patients without staining were classified as "socs-1 negative group". sixteen patients were socs-1 negative and 61 patients were socs-1 positive. we compared baseline characteristics among two groups. socs-1 positive group had a significant lower rate of svr to ifn therapy than socs-1 negative group (p=0.0014). socs-1 positive group had significantly higher hcv-rna titer than socs-1 negative group (p=0.015). we analyzed factors with ifn svr in univariate logistic regression analysis. not only hcv genotype (p=o.oool) and hcv-rna titer (p<0.0001), but also the expression of socs-1 were found to be significant predictors of ifn svr (p =0.004 background: interferon(1fn) and ribavirin combination therapy is now a standard regimen for chronic hepatitis c infection and achieves a higher sustained virological response than interferon monotherapy worldwide. but the response rate to patient with genotype l b and high viral load is still not satisfactory. some virological factors have been shown to influence the effect of interferon monotherapy in previous studies. however little is known about those influencing the outcome of combination therapy. to examine the genetic basis of the genotype l b virus resistant to combination therapy, we analyzed full-length nucleotide and deduced amino acid sequences of hcv rna. method: among patients infected with genotype l b and high viral load, 6 patients treated with monotherapy : (group a-3 nullresponders, 3 relapsers) and 10 patients treated with combination therapy: (group b-3 nullresponders, 7 relapsers) were studied. the group a consists of 6 naive patients and group b consists of 5 naive patients and 5 patients who failed to previous ifn monotherapy. the group a patients were treated with 6-18miu of ifn, there times a week for 6 months. the group b patients were treated with 6miu-1omiu of ifn three times a week and ribavirin 600mg-800mg per day for 6 months.sera were obtained from the patients at three points: 6 months before the treatment, just before the treatment, and after or during the treatment. complementary dna was reverse-transcribed from total rna extracted from these sera, amplified by polymerase chain reaction and directly sequenced. the mutation rates during natural course of infection and during the treatment were calculated by comparing the nucleotide sequences obtained from the samples just before the treatment and 6 months before the treatment, and just before the treatment and after or during the treatment. the deduced amino acid sequences in different region were also analyzed in each case. result: the mutation rates of natural evolution analyzed in 14 patients were 1.89~10-~nt isitelyear. the mutation rates of hcv rna from nullresponders during the treatment were almost identical with those before the treatment in both groups a and bcu (3.3052.06 vs 3.0421.22, p=ns and 0.681-0.69 vs 0.6420.78, p=ns, respectively).in contrast, those from relapsers during the treatment were higher than those before the treatment in both groups ( table. no major differences were observed in the frequency or intensity of saes and aes. conclusion: the combination of peginterferon alfa-2a (40kd) (pe-gasysb) and ribavirin (copegusb) was safe and effective in patients with hcv genotype 1. despite the use of a dose of ribavirin (800 mglday) that is lower than the recommended dose for patients infected with g1 (100011200 mglday), approximately half of the patients in the study achieved an svr. because the study could not be blinded, a bias might have influenced the outcome of the study as suggested by the unequal discontinuation rate. these preliminary data do not support the hypothesis that higher svr rates can be achieved in patients with hcv g l by extending treatment duration. final data will be presented. *itt population, defined as all patients who took at least one dose of study medication and had at least one hcv rna evaluation postbaseline. **safety population, defined as all patients who took at least one dose of the study medication elusive goal, particularly in previously treated patients who failed or relapsed following prior therapy. we hypothesize that response rates in these patients who are re-treated with pegylated interferon and ribavirin will be improved compared to standard interferon preparations with or without ribavirin. the present prospective study is designed to determine the efficacy and safety of treatment with peg-intron (pegylated interferon alfa-2b) and ribavirin in a group of patients who failed prior therapy with interferon with or without ribavirin. methods: 462 aatients are uresentlv enrolled in this multi-center study. the first250 enrolled patie& were treated with 1.5 mg/kg o~p e g -intron sc q week, and ribavirin 800 mg po qd, which was standard at the time. the intended duration of treatment is 48 weeks. 225 patients have been trcated for at least 48 weeks and week 72 data is available in 141 patients. adverse events: dose reduction was required in 22% of pts most commonly due to leukopenia and anemia. permanent discontinuation of therapy was required in 8% most often due to: depression or anxiety serious adverse events: (1.8% of pts) included one patient each with neutropenialmrsa sepsis, optic neuritis with monocular blindness, perirectal abscess, cellulitis and multi-organ failure, cutaneous and pulmonary sarcoidosis, pneumonia, homicidal ideation, and suicide. conclusions: 1) overall etr was 50% and svr was 41%. 2) svr was significant in genotype non-1 patients and patients who failed or ;elapsed after previous monotherapy 3) 18% a multi-centre, randomised controlled study comparing the combination of interferon and ribavirin with no treatment was undertaken. patients were eligible if they had biopsy proven mild hcv (defined as necro inflammatory scores less than 4 and fibrosis scores less than 3 using the ishak scoring system. interferon was administered at a dose of 3 mu thrice weekly for 48 weeks regardless of viral genotype. ribavirin was administered according to the patient's body weight ( 5 75kg 1000mg/day, > 75kg 1200mgl day in two divided doses). randomisation was stratified according to viral genotype (1 and non-1). the primary end point was the virological outcome of treatment with sustained response defined as an undetectable hcv rna pcr 6 months post cessation of therapy. in addition there were a number of secondary aims: 1. viral kinetics. serum was collected at baseline, days 3,7,10 14 and week 12 for quantitative rt pcr to determine hcv viral load. the ability of early phase kinetics and the presence or absence of a 2 log drop at week 12 to predict eventual outcome is assessed in the context of mild disease. 2. health economics. the collection of resource use data in hcv infected patients who have mild, moderate and cirrhotic disease was performed as an integral part of this study. this data has been incorporated into a markov chain based model to assess likely future costs of the disease and their possible avoidance by treating mild cases. response rates from this trial will form the basis of the model. 3. quality of life assessments. sf36 and euroquol data were collected at baseline, weeks 1 2 2 4 and 48 during treatment and at post treatment weeks 12 and 24 in order to compare quality of life between treated patients and controls before during and after therapy. results. 98 patients received treatment and were matched with 98 controls (no treatment). sustained viral response rates will be available at the end of july when the follow up period of the trial is complete and will be included in the presented results. end of treatment results are shown in table 1. 30% of patients infected with genotype 1 and 61% of patients infected with genotype non-1 achieved end of treatment responses. sustained viral response rates to date are shown in table 2 .20% of patients infected with genotype 1 and 53% of patients infected with genotype non-1 have achieved sustained viral responses so far. 25% of patients in the treatment group were unable to complete at least 6 months of therapy due to side effects of medications. there were 4 hospitalisations in the treatment group, 3 of which were related to adverse events. patients with mild hcv have lower response rates to those with more severe histological change on liver biopsy. side effects are common and frequently result in treatment discontinuation, lowering response rates. the decision to treat patients with mild histological change must be weighed against the side effects. deferring treatment until later in the disease process does not prejudice response to therapy and may increase likelihood of success. the hcv ns5b polymerase is essential for hcv viral replication and infectivity as demonstrated in a chimpanzee model. the enzyme adopts a unique molecular structure that resembles a "thumb-palm-finger" which has some features different from other known dna and rna polymerases, highlighting the attractiveness for this enzyme as a target of antiviral therapy. from an in-house high throughput screening campaign and the subsequent lead optimisation, we have identified a novel chemical series of substituted thiophene-2-carboxylic acids that have good potency against hcv polymerase in vitro. x-ray crystallography studies revealed that these compounds bind to an allosteric site that is -35 angstrom away from the active site. further studies on selected members of the series confirmed the ability of these compounds to inhibit the sub-genomic replication of hcv in huh-7 cells. moreover, a representative compound was also found to have good in vitro safety index and favourable in vitro and in vivo metabolic and pharmacokinetic properties. in an open and prospective trial we investigated, if a pre-treatment with citalopram as an ssri can reduce the frequency of ifn-associated depression in hepatitis-c-infected psychiatric risk patients during methadone substitution. 36 patients with a chronic hepatitis c were treated with pegylated interferonalpha 2b and ribavirin according to body weight. 11 patients without any psychiatric history (group a) were compared to 25 patients during methadone substitution. the methadone substituted patients were separated into two groups: the first group (group b, n = l l ) were only treated with ifn-alpha and ribavirin and the second group (group c, n=14) received a two week pre-treatment with citalopram (20mglday) before combination treatment with ifn-alpa and ribavirin was started. antidepressant treatment was continued over the study period of four months. the hamilton depression rating scale (hamd, 17-item) was used and major depression defined as >20 points. patients were followed over the first 4 treatment months. group differences were calculated with anova for parametric and chi2-test for non-parametric scales. results: hamd scores at baseline were significantly higher in the psychiatric groups as compared to the controls (p=o.olo dallas, t x background: peg plus rbv is the mainstay of chronic hepatitis c treatment. however, an upper limit in terms of safety and efficacy for dosing with peg has not been evaluated. methods: we studied a group of nake patients using either conventional weight-based dosing of peg; (1.5 pglkg) or double the dose (3.0 pglkg) plus rbv 13+2mg/kg/day for 48 weeks. genotype 1 patients were randomized 1:l and to receive medication accordingly in unblinded fashion, with intent to enroll a total of 1,100 nayve patients n, an additional 300 previous nr or r to any type of interferon i rbv in a non randomized arm, using only the 3.0 pglkg peg dose. values for hcv rna at week 12 were compared to those obtained at baseline. results: to date, 573 patients 306 n, 193 nr, 74 r have enrolled in the study. 175 patients 106 n, 48 nr, 21 r have reached week 12. hcv rna results at week 12 are shown in the table below. treatment was well tolerated in most patients. dose reductions were required in 20% of n 1.5pglkg peg and 29% of n 3.0pglkg peg patients. side effects were equivalent between the two groups as shown in the table below. serious adverse events (sae's) were less than 5% and equally observed in both arms of the n patients. none were directly attributed to study drug. there were 25 patients in the wk 12 analysis that discontinued therapy. the reasons these patients were discontinued after reaching wk 12 were side effects (36%), +hcv rna (32%). it is interesting to note that 40% of the patients that were discontinued had 2 2 log drop or negative hcv-rna at week 12 and a positive hcv rna was not a reason for their discontinuation. conclusion: 3.op.glkg peg dosing does not appear to improve 12 wk viral response, but we await 24 wk results and sustained viral response rates. increased side effects as a result of doubling the interferon dose were not observed. more data is needed to verify the long-term efficacy and safety of this dosing regimen for patients with chronic hepatitis c . this study was supported by a grant from integrated therapeutics group a subsidiary of schering plough. background and aim: one of the major adverse effects of the combination therapy for chronic hepatitis c is ribavirin-induced hemolyhc anemia. however, little is known about the mechanism of this anemia. oxidative stress has been suggested as potentially important pathological mechanism in hepatitis c. this burden may cause peroxidation of erythrocyte membrane phospholipid in conspiracy with the accumulation of ribavirin triphosphate in the erythrocyte, which potentially attenuates the mobility of erythrocyte membrane. the aim of this study was to examine the change of fatty acid composition in erythrocyte membrane and the effects of vitamin e and vitamin c on fatty acid composition in combination therapy. methods: thirty-nine patients with chronic hepatitis c were enrolled in this prospective study. they were randomized to receive daily oral vitamin (500 mg of vitamin e plus 750 mg of vitamin c) (vitamin group) or none (control group), in addition to injections of 6 million units of interferon-a-2b daily for two weeks, followed by thrice-weekly for 24 additional weeks, plus daily oral ribavirin (600 or 800 mg) for 26 weeks. blood samples were obtained at 0, 2, 4, 8, and 26 weeks after initiation of therapy. phospholipid was separated by one-dimensional thin-layer chromatography after extraction of total lipid from the erythrocyte ghosts. following transmethylation, fatty acid methyl esters were quantified by gas chromatograph. a-tocophenol concentrations in erythrocyte were determined by high performance liquid chromatography. plasma thiobarbituric acid reactive substances (tbars) were measured by spectrofluorometer. results seven patients were obliged to suspend or cease receiving therapy because of adverse effects including anemia by 8 weeks after initiation of therapy. twenty-one patients have completed the assigned therapy up to now. among the 26 kinds of fatty acid analyzed, the mean content of 205n-3 polyunsaturated fatty acid (pufa) significantly decreased at 8 (0.96 5 0.12 mol% vs. in a cross-sectional study we investigated mxa expression in patients with chronic hcv infection(n = 60 ) and in patients with chronic hcv infection receiving ifn-alpha therapy (n = 23) as well as in healthy controls (n = 22). in a prospective study with 16 chronically infected patients (hcv genotype 1) known to be ifn non-responders, we followed mxa gene expression during combination therapy with pegylated ifn-alpha2b and ribavirin. results: patients with chronic hcv infection showed higher mxa gene expression levels than healthy controls, indicating that hepatitis c virus induces ifn production. however there was no correlation between mxa gene expression and clinical parameters (viral load, alt, liver histology). patients with chronic hcv infection receiving ifn-alpha had significantly higher mxa levels than patients without treatment or healthy controls. further we addressed the question whether mxa expression in pbmcs during therapy in previous non-responders could be a predictive marker of therapy outcome. mxa expression was clearly induced in all but one patient compared to pretreatment values (median: 10.2 fold). importantly, the level and kinetics of mxa expression did not correlate with the response to antiviral therapy. surprisingly, one patient showed an unusually low pre-treatment level of mxa expression and a total lack of mxa induction during ifn therapy, which was associated with complete non-response to therapy. in a neutralization assay, we could detect neutralizing antibodies to ifn-alpha2b in the serum of this patient whereas no neutralizing antibodies were found in all others patients. hepatitis c virus (hcv) infection is a common cause of transfusion acquired hepatitis and is today a major health problem throughout the world. the present study reports the prevalence of hepatitis c virus (hcv) infection in general population and in various high-risk groups from the city of hyderabad in south india. a total of 308 out of 3589 (8.6%) people (both general and risk groups) tested positive for hcv rna by rt-pcr, while anti-hcv antibody positivity, as determined by third generation eia, was found to be 5.8% (209/3589). this suggests that a number of cases go unreported, as screening of blood and blood products is done primarily by elisa. among 124 chronic renal failure (crf) patients with history of either renal transplant or hemodialysis, 46% were infected with hcv alone, 6.5% hbv alone while 37.1% were found to be co-infected with bothe hcv and hbv. our findings implicate these viruses as the major cause of post transplant hepatitis in indian patients with crf and indicate the necessity for immediate implementation of stringent screening procedures for these viral infections. additionally we report here that tattooing and slashing (a cultural practice among one sect of muslims) increase the mode of transmission of hcv infection. in addition, we also report a high incidence of hcv infection ( background in japan, long-term treatment with glycyrrhizin, given as snmc, is used to reduce alt and the risk of hepatocel-glycyrrhizin given as stronger neo-lular carcinoma in patients with chronic hepatitis c. phase 1/11 studies confirmed the alt lowering effect of glycyrrhizin therapy in western patients with chronic hepatitis c. the treatment duration in these studies was limited to 4 weeks. alt response was lost during follow-up. aim 1) to evaluate which dose frequency is required beyond week 4 to maintain the initial alt response. 2) to evaluate the effect of snmc treatment on liver histology. methods: hcv-rna positive patients with alt >2x uln, fibrosis stage 2 3 or necro-inflammation score 2 6 (ishak's score) who were not eligible for interferon therapy (prior non response, absolute contraindications) could enter this multi-center, randomized, open phase i1 clinical trial. all patients were treated 4 weeks with six infusions weekly of 100 ml snmc (minophagen pharmaceutical co. ltd., japan) containing 200 mg glycyrrhizin. patients with an alt response at week 4 (defined by a decrease of more than 50 percent of the baseline value or alt 51.5 uln) were randomized to continue treatment in three dose frequency groups for a total of 26 weeks. snmc was given 6 times weekly (group l), objectives: it is important to maintain reduced serum alanine aminotransferase (alt) levels in cases with chronic hepatitis c (ch-c) that do not respond to interferon (ifn) and in those with no indication of ifn therapy. we reported previously that dietary restriction of iron intake reduces serum alt levels in such patients. we evaluated ch-c patients treated with iron-restricted diet for two or more consecutive years, mainly focusing on the balance of energy intake, physical examination, and changes in hematological indices of nutrition. methods: twenty-two patients with ch-c (males, 18; females, 4; mean age, 56 year-old) that consulted our outpatient department were enrolled in this study. the inclusion criteria were as follows: 1) elevation of alt levels above the upper normal limit for 3 months or more; 2) positive tests for hcv-antibody and hcv-rna; 3) absence of other causes of ch (alcoholic liver disease, drug-induced liver injury, hemochromatosis) and negativity for hepatitis b surface antigen and for serum anti-nuclear and anti-mitochondria1 autoantibodies. twenty cases had received ifn therapy for more than 12 months before the beginning of the study; none of them responded to ifn therapy. dietary prescriptions included iron intake 7 mglday or less, energy intake 30 kcallkglday, protein intake 1.1-1.2 glkglday, and a fat energy fraction of 20%. nutritional balance was evaluated based on meal records, and instructions was given when necessary. results: the average energy intake before dietary prescription was 2184 kcal(36.7 kcallkg)lday, and it was significantly reduced to 1655 kcal(28.5 kca1lkg)lday (p < om), and then maintained stable at 30 kcallkglday. the average protein intake before dietary prescription was 85.7 g (1.45 glkg)lday and it was reduced to 1.1-1.2 glkglday after the prescription. the average fat intake of 66.5 g (1.1 glkg)lday and the average fat energy fraction of 27% before the dietary prescription were significantly decreased to 30.8 g (0.52 glkg)lday; p < 0.01 and 16% (p < 0.001), respectively, after dietary instructions. the fat energy fraction was maintained at a level of 20% or less. carbohydrate intake did not change remarkably during the observation period, although the carbohydrate energy fraction significantly (p < 0.001) increased. the average iron intake decreased significantly (p < 0.001) from 9.6 (before) to 6.1,5.2, 5.1,5.2, and 5.1 mglday 6, 12, 18, and 24 months after die* prescription, respectively. body mass index (bh4i) before diet prescription was 23.9 on average; bmi had no significant change throughout the course. the body fat percentage was 24.6% on average before the diet instructions, and it significantly decreased after the diet. the average values of aspartate aminotransferase and alt before diet prescription were 65 iull and 66 lull, respectively, and they were significantly reduced to 48 iull and 49 iull, respectively, after 24 months (p < 0.01). serum iron levels significantly decreased after 18 (p < 0.01) and 24 (p < 0.05) months, while unsaturated iron binding capacity tended to increase. the average serum ferritin levels were 376, 210, 189, 189, 141 nglml before and 6,12, 38, and 24 months after diet, respectively; there was a significant reduction (p < 0.01) in the values measured before and after the diet instructions. the average levels of hemoglobin, albumin and cholinesterase did not change significantly during the follow-up period. conclusions: restriction of iron intake is safe and well tolerated for a long period. the results of our present study suggest that decreased dietary intake of iron may constitute an important adjuvant therapy in patients with ch-c. chru, nice, france; c henquell, chru, clermont ferrand, france; c dizrcha, s ughetto, p dechelotte, hotel-dieu, clemont ferrand, france; h lafeuille, chru, clermont fewand, france; g bommelaer, hotel-dieu, clemont fewand, france peginterferon (pegifn) background: hcv co-infection is common among patients with hiv disease. it has been documented that hiv co-infection accelerates the course of liver disease in patients with hcv, and that liver failure is higher in co-infected patients. at this moment, there is no effective treatment for hcv non-responders to interferon therapy. mono-therapy and interferon-ribavirin combination is only effective in 2-3% and 5-10% interferon resistant patients respectively. treatment strategies to slow the progression to cirrhosis and to prevent liver failure in this population are needed. objective: this is a report of a study that examines the efficacy of peg ifn alfa 2-a (pegasys) vs pegasyslrbv 800mg in co-infected hcvlhiv patients that have not responded to a previous 6-12 months course ifn-alfa. the study also examines the histological benefit of treatment in this population. patients and methods: 76 patients were randomized 1:l to receive either pegasys 180mcg weekly x 24 weeks (group 1) or pegasys 180mcg weekly plus 800mg rbv for 24 weeks (group 2). patients that have hcv non-detectable or 210g decrease from baseline at week 24 (groupl), were added rbv 800mg. all similar responders (group 2) continue treatment for a total of 48 weeks. baseline demographics were similar between both groups. more than 70% of patients were nonresponders to ifn monotherapy.most patients in both groups (80%) were genotype l. the mean hiv baseline log was 2.92 (sd.64) group 1, vs 2.85 (sd.57) group 2 and most patients had baseline cd4 levels>400 cells, and were in stable antiretroviral therapy. the majority of patients (81%) are non cirrhotic, with mean grading group 1 6.63 (sd 3.24), group 2 7 (sd 3.64), and mean staging, group 1 3.51 (sd 2.2), and group 2 3.48 (sd 1.70 conclusions: this study is completed and pending some results, that will be available for presentation. sustained virological response(svr) (intention to treat), will be of the order of 5-20%. (11 group 2 results are pending). in this study, 10 patients were discontinued because adverse events and 10 were lost to follow up, before week 24th.svr in patients that completed at least 24 weeks of therapy will be of the order of 7-26%.a significant number of end of treatment responders are relapsing at week 72th. histology analysis show improvement in both groups of the mean grading and staging after treatment. in responders and relapsers the fpr becomes static or regressive.these results show that pegasys lrbv therapy is effective in this population.the study also suggests that longer duration of therapy ,and higher doses of rbv should be studied in coinfected nonresponders. * -pending results, will be available for presentation. disclosures: josi. rodriguez-orengo -no relationships to disclose maribel rodriguez-torres -roche laboratories: investigator; other: grant to perform study. adverse events by week 12 of therapy were grade 2 to 3 in severity and were therapy related. see table 2 . sixteen patients who continued therapy to week 24, three dropped out due to adverse events (1 anemia, 1 hyperbilirubinemia and 1 related death due to self-overdose). this patient had a history of hypothyroidism and mild untreated depression. at 48 weeks of therapy, 1 patient discontinued treatment due to suicidal thoughts. there was no difference between the ethnic groups regarding the drop out rate due to adverse events. hivfhcv coinfected population had a poor tolerance to pegylated interferon and ribaiirin. this was markedly increased in the first 12 weeks of therapy. the use of high dose pegylated interferon and ribavirin led to a significant drop out rate in comparison to the low dose pegylated interferon. background during liver injury, poorly characterized factors activate quiescent hepatic stellate cells (hsc) to become proliferative, matrix-synthesizing myofibroblasts. activated hsc are the major source of liver collagen and thus, play a key role in the fibrotic response to liver injury.the sns appears to promote fibrosis in injured livers because hepatic fibrosis is increased in the spontaneously hypertensive rat, which has an overactive sns. conversely, prazosin, an adrenoceptor antagonist, inhibits fibrosis in toxin-damaged rat livers. hsc express several neuronal proteins, including glial fibrillary acidic protein (gfap). they also contain synaptic vesicles and receive autonomic fibers. therefore, our hypothesis is that hsc are hepatic neuroglial cells that produce and respond to neurotransmitters in order to become activated during liver injury. methods: hsc were isolated from normal mice and dbh-lmice that cannot produce norepinephrine (ne) due to targeted disruption of the dopamine p-hydroxylase (dbh) gene. lysates of culture-activated hsc were analysed by rt-pcr, immunoblot and hplc to determine if they express adrenoceptors, catecholamine biosynthetic enzymes andlor produce ne. the effect of adrenoceptor antagonists and ne on hsc growth in vitro was also assessed. then in vivo activation of hsc by hepatotoxic diets was evaluated in control and dbh-lmice by comparing numbers of a-smooth muscle actin (asma)+ cells with immunohistochemistry and the induction of tgfp-1 and collagen a-1 gene expression by ribonuclease protection analysis of whole liver rna. results: hsc express a-and p-adrenoceptors, tyrosine hydroxylase and dbh. hsc from control, but not dbh-i-, mice release ne. endogenous ne is an autocrine growth factor for hsc because a-and p-adrenoceptor antagonists inhibit proliferation of hsc cultured from control mice. moreover, hsc from dbh-lmice, which cannot make ne, grow poorly in culture and are rescued by addition of ne. exogenous ne also promotes hsc proliferation. inhibitor studies demonstrate that the latter effect is mediated via g-protein coupled adrenoceptors that activate mitogen activated kinases and phosphatidylinositol 3 kinase pathways. hsc activation in response to diet-induced liver injury is inhibited in dbh-imice, as evidenced by reduced hepatic accumulation of asma (+) hsc and inhibited hepatic induction of background & aims: hepatic cirrhosis is six times more prevalent in obese individuals than in the general population, and obesity is one of the risk factors for liver fibrosis in which plasma adiponectin levels are decreased. adiponectin is an adipocytokine, which we previously identified by screening adipose-specific genes in the human cdna project. hepatic stellate cells (hscs) play central roles in liver fibrosis. when they are activated, they undergo transformation to myofibroblast-like cells, then proliferate, migrate, produce transforming growth factor-pl (tgf-pl), and various extracellular matrix proteins, and express a-smooth muscle actin (a-sma). we previously reported that adiponectin suppresses not only the proliferation and migration of hscs, but also the tgf-pl-induced fibrogenic gene expression in hscs. adiponectin could have biological significances in liver fibrosis. in this study, in order to clarify the effect of adiponectin on liver fibrosis in vivo, we tested the role of adiponectin on liver fibrosis using adiponectin-knockout (ko) mice and adenovirus mediated adiponectin expression system. methods: (1) to investigate the anti-fibrogenic effects of physiological concentrations of adiponectin, male wild type (wt) mice and ko mice were used. mice were each injected with a dose of carbon-tetrachloride (ccl4) (300 pllkglbw) intraperitoneally twice a week for 12 weeks to induce liver fibrosis. (2) to investigate the anti-fibrogenic effects of excessive concentrations of adiponectin, male wt mice were used. mice were injected cc14 (1000 pl/kg/bw) intraperitoneally twice a week for 12 weeks. mice were divided into 6 groups. control, received an injection of corn oil only; gr.1, mice treated with cc14 for 12 weeks; gr.2, mice treated with cc4 for 12 weeks after infusion of adenovirus producing adiponectin (adadn); gr.3, mice treated with cc4 for 12 weeks after infusion of adenovirus producing p-galactosidase (adlacz); gr.4, mice treated with cc14 for 12 weeks with adadn infusion at 6 week; gr.5, mice treated with cclb for 12 weeks with adlacz infusion at 6 week. results: (1) ko mice showed extensive liver fibrosis with an enhanced expression of tgf-p1 and connective tissue growth factor (ctgf) compared to wild type (wt) mice (p<0.05). the hydroxyproline content and the numbers of a-sma positive cells in mice liver significantly increased in ko mice. (2) the fibrosis areas were significantly decreased in gr.2 and gr.4 compared to those in gr.3 and gr.5, respectively. the hydroxyproline content in mice liver significantly decreased in gr.2 and gr.4 compared to that in gr.3 and gr.5, respectively. moreover, in gr.4, the hydroxyproline content was significantly decreased compared to that in 6-weeks of c c 4 treatment even though ccl, was given for an additional 6-weeks (total 12 weeks). conclusions: the findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention. disclosures: (ttg) is observed in mature scars and might promote wound repair by protecting the neomatrix from degradation by matrix metalloproteinases (mmps).however, such ecm crosslinking has the potential to hinder the matrix remodelling required for resolution of liver fibrosis. we have explored this hypothesis by examining expression of ttg and its crosslink product epsilon-(gamma-glutamyl) lysine in livers of: a) rats administered cc14 for 6 weeks (6wk c c 4 cohort)after which liver fibrosis resolves spontaneously after 28 days; b) rats administered cc14 for 12 weeks (12wk cc14 cohort) which develop micronodular cirrhosis; c) 12 wk ccl4 treated rats allowed to recover for 365 days after last cc14 dose (12 wk+365d cohort) which show partial resolution of fibrosis but with persistence of a macronodular cirrhosis. although ttg was identified by immunohistochemistry within and around fibrotic bands in 6 wk ccld cohort, no crosslinks were detectable. however, both ttg and crosslinks were detected in and around mature fibrotic bands in the 12wk cc14 cohort and in persisting fibrotic bands in the 12wk+365d cohort. western blotting of liver homogenates from these two cohorts using antibody against the crosslink revealed a major product of 160 kd which was degradable to lower molecular weight products following incubation of homogenates with bacterial collagenase but was refractory to mmp-2. to evaluate if ecm crosslinking in livers correlated with its resistance to mmps, 10 micron cryostat sections of livers from the three cohorts were incubated with purified active forms of mmp-1 or mmp-2 for 8 hr at 37 c and any residual ecm was stained with sirius red. ecm of 6 wk cc14 cohort was effectively degraded by these mmps but that of 12wk c c 4 and 12 wk+365d cohorts was only minimally degraded. hsc freshly isolated from normal rat or human liver showed no ttg expression whilst hsc activated for 7-15 days on plastic substrate expressed ttg protein (by western blotting) and mrna (by rt-pcr). there was ttg in cell lysates and in conditioned media of activated hsc. pure rat tail type i collagen incubated with conditioned media of activated hsc incorporated crosslink antigen, confirming that hsc secreted functional enzyme. however, no crosslink was found in type i collagen incubated with media from hsc which had ttg gene silenced by cell transfection with small inhibitory rna. cultured hsc which had ttg gene silenced had enhanced apoptosis (by acridine orange staining) following serum deprivation in culture. this suggests ttg or ttg activity promotes survival of these cells. we conclude that in persistent, incompletely resolving liver fibrosis there is evidence of ttg mediated crosslinking of the liver ecm and resistance to exogenous mmps. both features are lacking in ecm of fully resolving fibrosis. ecm crosslinking by ttg might limit resolution of cirrhosis. activated hsc are a potential source of ttg following liver injury and, as this protein supports their survival, it might contribute to their persistence in cirrhotic liver. we recently defined multifunctional biochemical properties of the long and frizzled variants and hypothesized that they may be inherent in the 192-aa-long-specific and the 235-aa-fz-specific modules, respectively. we thus transfected pcdnas.l-v5-his vectors containing variant-specific sequencer3 in mhat3fls315 hepatoma cells. this cell line stably expresses a truncated hnf3 protein turning off endogenous expression of liver-specific genes, such as albumin or c18. consistently, mhat3fls315 hepatoma cells transfected with an empty vector showed no endogenous c18 expression. by immunohistochemistry, using variant-specific and tag-specific antibodies and after analysis by confocal microscopy, long was localized in large supranuclear vacuolae suggesting protein storage/maturation in golgi structures or in beaded perinuclear vacuolae suggesting rer cysternae. in contrast, fz was strongly detected in close proximity to the plasma membrane of single cells. clusters of fz-transfected cells showed a strong and dense signal at sites of cell-cell contact, outlining single cells. these findings were confirmed by transfection of prep-7 vectors containing full-length long or fz forms of human c18 and confocal microscopy analysis after immunohistochemistry using an antibody directed against an epitope common to all c18 forms. immunoblot analysis of conditioned media showed that the fulllength long form was secreted into the medium but not fz. the latter was detected as a heavily glycosylated protein (> 300 kd) in the cell layers, predominantly in the triton-x-100 insoluble pellet after sonication and 10% sds solubilization, suggesting binding to extracellular matrix proteins. finally, glycosydase analysis of fz and long n-terminal modules showed an important increase in mobility after pngase f plus sialidase a digestion. these data show that, in addition to the previously described plasma (long) and tissue (short) forms of cl8, the fz form constitutes the pericellular matrix form, suggesting that the specific modules of c18 regulate tissue targetting through protein-protein interactions. backgr0und:during liver fibrosis, hepatic stellate cells (hsc) acquire an activated phenotype, proliferate and produce an excess of collagens. mycophenolic acid (mpa) is a known immunosuppressive drug. which inhibits the proliferation of b-and t-lymphocytes by inosine mono phosphate dehydrogenase (impdh) inhibition, causing an intracellular guanosine depletion. in addition, mpa has shown to inhibit growth of mesangium cells in the kidney and of skin-and tenon fibroblasts. therefore, we hypothesize that the proliferation of hsc may also be influenced by mpa. in this study we explored whether mpa is able to inhibit the proliferation of primary isolated rat hsc in vitro. furthermore, we studied the in vitro effects, mechanism of cellular uptake, and in vivo pharmacokinetics of mpa coupled to mannose-6-phosphate modified human serum albumin (m6p-hsa). m6p-hsa is a newly developed, hsc-specific drug carrier, homing towards the upregulated m6pligf-i1 receptor on activated hsc. in this way we hope to achieve cell-specific delivery of mpa to the hsc avoiding undesired effects of mpa on the immune system. meth-odslresults: in primary cultures of hsc a dose dependent reduction in the number of brdu-positive nuclei was observed after 24h incubation with 750,3000 and 6000 nm mpa, as assessed immunohistochemically. coupling of mpa to m6p-hsa via a biodegradable ester linker resulted in a conjugate with a maximum drug: carrier ratio of 1.2:l. analysis of the synthesized constructs was performed by hplc, fplc and ms. this conjugate was able to inhibit 3t3-fibroblast growth, as detemined by brdu-incorporation (elisa) in a dose dependent manner, reducing proliferation to 38.0 2 14.5%, 30.0 t 18.0% and 18.0 2 16.8% of control at 120, 240 and 480 uglml of conjugate, respectively. when cells were co-incubated with an excess of m6p-hsa, a competitor for receptor mediated uptake of the conjugate, the anti-proliferative effect was reduced by 69 yo. the organ distribution of the conjugate, 1251 labeled, was evaluated by iv injection of a tracer dose in rats 3 weeks after bile duct ligation (bdl-3). 47.3 t-4.1 % of the injected dose accumulated in the liver after 10 min of injection. in contrast, spleen and thymic gland accumulated 1.47 f 2.65% of the injected dose. intra-hepatic distribution was assessed in bdl-3 rats by immuno-histochemical double staining for hsa and specific markers for kupffer cells (ed-2), hsc (desminelgfap) or endothelial cells (his52). m6p-hsa-mpa showed a non-parenchymal distribution and co-localization with hsc and kupffer cells. conclusions: mpa is able to inhibit the proliferation of primary isolated rat hsc. this suggests that stellate cells are dependent on intracellular impdh activity to proliferate. coupling of mpa to mcip-hsa, a stellate cell specific drug carrier, resulted in a pharmacologically active construct, able to inhibit fibroblast proliferation in vitro after specific uptake via the m6p/igf-ii receptor. the major part of the injected conjugate accumulates in the hsc and kupffer cells of the liver, and avoiding uptake in the major resident organs for band t-lymphocytes. future studies will assess the advantage of this first hsc-selective compound in animals with liver fibrosis. background: platelet-derived growth factor (pdgf) is the most potent stimulator of migration and proliferation of mesenchymal cells. the expression of pdgf-p-receptor is increased during liver fibrosis. our aim was to investigate the effect of p3-integrin subunit blockade using the specific non-peptidic inhibitor emd409915 on migration and proliferation of hepatic stellate cells (hsc) and human foreskin fibroblast (hff). materials and methods: cell migration of human hsc and hff was assessed using a scratch assay. scratches of 600-700 pm width were made in confluent cell monolayers of cells following 24 h starvation in serum-free medium. after wounding. cells were stimulated with pdgf-bb (10 nglml) with or without emd409915 (merck, darmstadt, germany) at increasing concentrations (10-10m -10-6m). cell proliferation was measured as dna synthesis by brdu-incorporation. mitogen-activated protein kinase (erk112, p38, sapkljnk and akt) phosphorylation was evaluated by phospho-mapk-specific western blotting of cell lysates after 10 min of stimulation. results: stimulation of human hsc cells with pdgf-bb at 10 nglml in the absence of other growth factors resulted in pronounced stimulation of cell migration. pdgf-stimulated migration of human hsc was inhibited dose-dependently between 10-9 and 10-6 m by emd409915, with complete abrogation of migration at 10-6m. surprisingly, human skin fibroblast migration was not affected by b3-blockage. there was no effect of p3 integrin inhibition on cell proliferation as measured by brdu-incorporation, neither in human hsc-nor in hff cells. pre-incubation of hsc cells with the p3 integrin inhibitor at 10-6m did not affect the activation of p38 mapk, p44l42 mapk or sapkljnk. conclusions: 1. pdgf-bbinduced migration is strongly b3integrin dependent in human hsc, but not in human skin fibroblasts. 2. in contrast to cell migration, hsc and hff proliferation is p3-integrin-independent. 3. p38, p44l42, sapkljnk and akt mitogen-activated protein kinase signalling pathways are not modified by p3-integrin inhibition. 4. p3-integrin antagonist may be an adjuvant approach to treat hepatic fibrosis. epithelial to mesenchymal transition (emt) is defined as a process, in which epithelial cells loose their epithelial characteristics and acquire typical characteristics of fibroblasts. epithelial cells are tightly attached to their neighboring cells via cell adhesion molecules and they adhere with their basal side to their underlying basement membrane matrices, whereas the apical side faces a lumen. in contrast to immobile epithelium, mesenchymal fibroblasts are specifically designed to invade extracellular matrix (ecm). this is reflected by their prominent mesenchymal cytoskeleton and by their lack of a typical polarity. in the adult organism, emt occurs in epithelia in response to injury, potentially as a means to replenish fibroblasts, which are required for repair of injury. in organ fibrosis however, enhanced conversion of epithelium into fibroblasts is considered to contribute to progression of disease, as parenchymal epithelial cells acquire phenotypic and functional properties of activated fibroblasts. recent studies provided increasing evidence that parenchymal epithelium can potentially contribute to activated fibroblasts by emt, as in mouse models of kidney fibrosis 36% of activated fibroblasts were found to be of tubular epithelium origin. in liver fibrosis, stellate cells are considered the principal source of activated fibroblasts, whereas the role of hepatocytes is considered minor, even though they constitute for more than 80% of the liver mass. here we provide evidence that the pro-fibrotic growth factors tgf-beta1 and egf induce expression of fibroblast-markers fibroblast specific protein-1 (fspl), alpha-smooth muscle actin (alpha-sma) and type i collagen in primary mouse hepatocytes in vitro. furthermore, tgf-beta1 and egf induce acquisition of fibroblast characteristics, migratory capacity and release of mmp-2, suggesting emt of hepatocytes in vitro. additionally, progression of liver fibrosis in a mouse model of tetracarbon chloride-induced liver disease is associated with appearance of fspl positive hepatocytes, indicating emt, which suggests a role of emt in liver fibrosis. in conclusion, our results for the first time provide evidence for an active role of hepatocytes in liver fibrosis by undergoing emt. human studies -serum mcp-1 levels were significantly elevated in both cfld (929t67 pglml; p<o.oool) and ba (1,2902220 pglml; p<o.oool) vs controls (602240 pglml). in celd subjects serum mcp-1 was negatively correlated with the stage of fibrosis (r= conclusion: these results provide strong evidence for a role for mcp-1 in early fibrogenic events in cholestatic liver injury, ie., ductular proliferation and procollagen q(i) mrna expression, and suggest that serum mcp-1 may be a very good marker of the onset of cholestatic liver disease. this is the first study to clearly demonstrate elevated excretion of mcp-1 into bile in obstructive cholestasis, implying a potential role for mcp-1 in hsc and inflammatory cell recruitment to the biliary tree in cholestatic liver injury. [background and aim] angiotensin i1 (ang 11) is considered to play an important role in hepatic fibrogenesis. however, the molecular mechanism of renin-angiotensin system in hepatic fibrogenesis has not been clarified. we previously reported that ang i1 up-regulated mrna expression of pro a (i) collagen and tgf-p in isolated rat hepatic stellate cells and an angiotensin converting enzyme inhibitor, lisinopril, reduced carbontetrachroride-induced rat hepatic fibrosis. ang i1 type i receptor blocker (arb) have shown a good clinical response in patients with hypertension, and a promising preventive effect has been described in rat hepatic fibrosis. recently, the rholrho-kinase has been demonstrated to be involved in the signal transduction pathway of the ang i1 type i receptor signaling downstream. thus,the aim of this study was to evaluate the role of rholrho-kinase pathway in hepatic fibrogenesis in rat. to that effect, a rat hepatic fibrosis model was produced by chronic administration with cholinedeficient, l-amino acid-defined (cdaa) diet and the effect of an arb and a rho-kinase inhibitor (y27632) in this model was investigated. methods: wistar rats (male, 6 weeks-old) were continuously fed the cdaa diet or the control choline supplemented, l-amino-defined (csaa) diet. the rats were treated with 1 mglkg candesartan cilexetil, an arb (a kind gift by takeda chemical induustries, ltd., osaka, japan), or 1 mglkg y27632, a rho-kinase inhibitor (a generous gift by mitsubishi welpharma co., tokyo, japan), once daily through stomach tube. twelve weeks after the start of administration, the liver was excised and subjected to histological examination for fatty change and fibrosis by hematoxylin and eosin staining. serum alanine aminotransferase (alt) values and hyaluronic acid (ha) levels were also measured. since cdaa diet causes hepatic steatosis and fibrosis via production of free radicals, we also studied the oxidative membrane damage by immunostaining of 4-hydroxynoneal (hne) adduct. results: histologic examinations showed severe steatosis and fibrosis in the liver of cdaa-fed rats and no significant change in csaa-fed rats. the liver weight of cdaa-fed rats was significantly increased than that of csaa-fed rats. both candesartan cilexetil and y27632 remarkably reduced not only fibrosis, but also severe steatosis and the liver weight of the treated rats was significantly reduced in both treated groups. serum ha levels but not alt values significantly elevated in cdaa-fed rats compared to csaa diet fed group. the increased levels of serum ha were significantly reduced by the treatment with candesartan cilexetil or y27632. the number of hne-positive hepatocytes increased by cdaa diet and this increase was attenuated in the candesartan cilexetil-or y27632-treated groups. conclusion: these results suggests that an arb, candesartan cilexetil, and a rho-kinase inhibitor, y27632, show the same inhibitory effect on hepatic steatosis and fibrosis induced by chronic cdaa diet, and this free radicalinduced hepatic injury is a good therapeutic target of arbs or rho-kinase inhibitors. the rholrho-kinase may be a key enzyme system in the ang i1 signaling involved in the hepatic fibrosis. in liver cirrhosis, malnutrion and portal congestive enteropathy facilitate bacterial translocation from the intestine to mesenteric lymph nodes and the development of endotoxemia and spontaneous bacterial peritonitis in ascitic patients. igf-i is an anabolic hormone synthesized in the liver, whose levels are reduced in hepatic cirrhosis. igf-i possesses hepatoprotective properties and displays trophic effects on the intestine. here we investigated whether igf-i therapy could in-fluence portal pressure and bacterial translocation in experimental cirrhosis. methods: liver cirrhosis was induced in sprague-dawley rats (n=30) by oral administration of carbon tetrachloride during 8 weeks. after this time, rats were randomized in two groups to receive subcutaneous injection of recombinant human igf-i (2 microgllo0 g body weight in two separated doses per day) (ci-igf, n=15) or vehicle (ci, n=15), for 21 days. eight healthy rats, receiving vehicle were studied in parallel. at the end of treatment period, animals underwent laparotomy to measure portal pressure and to collect blood and tissue samples. endotoxin quantification in blood samples was carried out with a colorimetric limulus amebocyte lysate test. liver and ileum histopathological changes were assessed in tissue sections stained with masson's trichrome, using a numerical scoring system. bacterial translocation was evaluated by culturing blood samples and mesenteric lymph nodes in appropriated culture media. results: treatment with igf-i significantly improved biochemical markers of liver damage, being the alt values significantly lower in the ci-igf group than in ci rats (p<0.05). in addition we found that igf-i administration to cirrhotic rats reduced both the hepatic histopathological score ( the rate of progression to cirrhosis varies among individuals infected with hepatitis c virus (hcv) cholesteryl ester transfer protein (cetp) is a plasma glycoprotein involved in lipid metabolism which transfers cholesterol esters from hdl to vldl and ldl. obesity and steatosis are emerging as key factors in the pathogenesis of liver fibrosis in hcv. variability in function or levels of cetp may affect lipid transport and therefore steatosis. the uncommon vallval genotype results in decreased cetp plasma activity and may protect against myocardial infarction. we tested for a relationship between possession of cetp ile405val polymorphisms and rate of fibrosis in 327 white european patients chronically infected with hcv. rate of fibrosis was calculated by dividing the fibrosis score (ishak modification of knodell) by infection duration. genotyping was by reverse line blot hybridisation. a significant association was seen between rate of fibrosis and the ile405val polymorphism in cetp (p =0.002 (anova)) (figure 1) . disease association showed patients possessing the val allele (which is associated with lower cetp activity) were more likely to be slow fibrosers (no cirrhosis for >30 years) genotype frequency p=0.0017, allele frequency p=0.0007, odds ratio=1.8. (table 1) . possession of the val allele is associated with slow rate of disease progression in hcv. the functionally less active forms of cetp may result in decreased liver steatosis as a result of altered composition or increased levels of hdl, and therefore less fibrosis when a second insult such as infection with hcv is sustained. backgroudslaims: hepatitis c virus (hcv) is a major cause of chronic liver disease worldwide. however, little is known about exact mechanism of fibrogenesis by specific hcv gene or protein because the study of hcv-induced liver fibrosis has been mainly studied in human or animal model. moreover, the dynamically molecular study of hcv role in relation to liver fibrogenesis or immune response has been hampered due to lack of efficient in vitro hcv culture system. in the present study, we investigated whether hcv core protein directly influence on the liver fibrogenesis through stimulation of hepatic stellate cell(hsc) in in vitro or not. methods human and rat hscs were isolated using collagenase perfusion system and density gradient method, and other human hsc line (l190) was purchased from atcc. we established co-culture system of primary hsc and hepg2-core stable cell line which hcv-core gene was transfected into hepg2 cell. co-culture system was divided into two way, mixed co-culture and separated co-culture system. immunocytochemical staining was performed to identify the cytokines such as transfoming growth factor pl (tgf-p1) and a-smooth muscle actin(a-sma) overexpressed from hsc in the liver fibrogenesis. a-sma, tgfpl, transforming growth factorp receptor i1 (tgforii), collagen type i were also quantitated by western blot analysis. the expression of mmp-2 and collagen type i in the culture media was measured and analyzed by each zymogram and elisa. results the expression of tgf-p1 and a-sma was significantly higher in mixed co-culture of hepg2-core plus hsc than in hepg2 plus hsc as negative control. also the markers related fibrosis such as a-sma, tgf-pl, collagen type i and tgfrii, mmp-2 and collagen type i were highly expressed in separated co-culture of hepg2core and hsc conclusions in conclusion, hcv core protein may play a direct role in the fibrogenesis via upregulation of a-sma, tgf-pl, collagen type i and tgfrii, mmp-2 and collagen type i. further study is needed to clarify exact signal transduction of fibrogenesis by hcv core protein in the co-culture system. background: to current knowledge, transforming growth factor beta (tgf-beta) signaling is mandatory to produce liver fibrosis. various molecular interventions designed to affect the tgf-beta system were successfully used to inhibit fibrogenesis. activated hepatic stellate cells have been considered as a major producer of extracellular matrix proteins in liver injury and fibrosis. in the present study, we wondered whether follistatin, activin inhibitory protein, reduce apoptosis and prevent liver fibrosis and whether activin plays a key role in liver fibrogenesis. methods: wistar male rats weighing around 380g were injected intraperitoneally with dimethylnitrosamine (dmn) (lopg/g body weight) three times a week for three weeks. either follistatin or saline were also injected intravenously three times a week. on the 22nd day, blood was collected and biochemical parameters were measured using the standard methods. the liver was either fixed with 4% bufferedparaformaldehyde for histological examination, or frozen immediately in liquid nitrogen for the rna analysis. tissue sections were either stained with hematoxyline-eosin, masson-trichrome, or subjected to immunohistostaining using antibodies against collagen type iv, alpha-smooth muscle actin (sma), fibronectin, tgf-beta. the mrna expression of activin, tgf-beta, timp were measured by rt-pcr. apoptosis was analyzed by tunel methods. wistar male rats were injected with dmn (10kglg body weight) once and liver tissues were obtained and fixed with 4% buffered-paraformaldehyde 0, 12, 24, 36, 48, 60, 72 hours and 7 days after injection for immunohistochemical staining. hepatocytes and hepatic stellate cells were isolated by collagenase perfusion method and by collagenase-pronase perfusion method and analyzed activin and tgf-beta mrna expression by rt-pcr. results: 50% of control rats died, whereas none of follistatintreated rats died within 22 days. the serum level of hyaluronic acid in follistatin-treated rats were significantly reduced. ast and alp levels were also decreased significantly. apoptosis was reduced by follistatin dose-dependently. the expression of tgfbeta, timp, collagen lv and alpha-sma expression were also decreased in follistatin treated rats. we then examined activin expression in liver after dmn treatment. activin expression was observed at the maximum level in hepatocytes 12 hours after dmn treatment. tgf-beta expression was not detectable 12 hours after dmn administration, and it was sfxikingly increased in stellate cells 48 hours after dmn administration but it was not detectable in hepatocytes. activin appeared in hepatic stellate cells as well as in hepatocytes 72 hours after dh4n administration. inducible nitric oxide synthase (inos) has been reported to play pivotal roles in the development of various types of liver injury and inos expression may be important in the process of fibrogenesis of nonalcoholic steatohepatitis in obesity. no-induced active substance was shown to activate matrix metalloproteinase. the inos mrna and protein activity have been found to be induce in rats on a high-fat diet. objectives we investigated whether 1) induction of nos occurs in liver of mice fed high-fat diet, and 2) nos increases matrix metalloproteinase activity and reduces collagen content, thus attenuates liver fibrosis. we compared fatty and fibrotic changes of hepatic tissues in inos-knockout (inos-'-) and wild-type (inos+/+) mice which were fed with high-fat diet for 12 weeks starting from 8 weeks old. marked induction of inos mrna occurred in inos+/' mice, but not in inos-/-mice. immunohistochemically, nitrotyrosine staining which is a footprint of no-induced active substance, showed positive in inos+'+ mice, and negative in inos-'-mice. in histopathologically showed fatty metamorphosis, but did not have any distinction between groups inos+'+ and inos-/-. the extracellular collagen content with azan staining in inos+/+ mice was markedly decreased compared with that in inos-'-mice. in gelatin zymography of matrix metalloproteinases we found that prommp-2 and prommp-9 were increased both in inos+'+ and inos-'-mice, but their active form was found only in inos+'+ mice. similar results were obtained from in situ zymography of hepatic tissue. the stronger gelatinolytic activity was found diffusely in hepatic tissues of inos+/+ mice than inos-/-mice. (129xc57bl16) were subjected to either sham operation or bile-duct ligation. animals were sacrified two weeks after sur-gery and blood and liver samples obtained. bile duct ligationinduced elevation of serum liver enzymes was similar between wt and atla (-/-) mice. however, the liverlbody weight ratio was greater in wt than in atla (-/-) mice. bile duct ligated wt mice showed severe septa1 fibrosis, as assessed by trichromic masson and sirius red staining. in contrast, atla (-/-) mice showed minor fibrotic lesions, which were mainly located in peribiliary areas. collagen accumulation, as assessed by morphometric analysis of sirius red staining and hepatic hydroxyproline content, was markedly lower in atla (-/-) mice compared to wt mice. positive sirius red stained area in bile duct ligated wt and atla (-/-) mice were 8.150.9 and 4.651.0%, respectively (p<0.05). the increase in hepatic concentration of bioactive tgfb and proinflammatory cytokines (tnfa and illb) was attenuated in atla (-/-) mice compared to wt mice, as assessed by elisa. moreover, immunohistochemistry analysis revealed decreased lipid peroxidation products as well as decreased phosphorylation of c-jun and p42-44 mapk in atla (-/-) mice compared to at1 (+/+) mice. chronic liver failure stimulates the onset of interstitial liver fibrosis, which eventually results in "capillarization" of the sinusoid, impeding clearance of toxic substances by hepatocyte cells. the goal of this work was to search for the therapeutical effect of adjuvant gene therapy using adenoviral vectors containing cd-nas for human urokinase plasminogen activator and human matrix metalloproteinase-8 (ad-hupa plus ad mmp-8) on cirrhosis and its relationship with manganese brain accumulation, striatum dopamine and its metabolites in the rat. mn+2 is a wellknown neurotoxic metal which has been found accumulated in brain and blood of cirrhotic patients with hepatic encephalopathy. mnt2 elimination takes place via the hepatobiliary route. methods 200 gr wistar rats underwent bile-duct ligation (bdl) for 4 weeks and concomitantly treated with 1 mglml of mncl2 in drinking water (bdl/md2). after this point, five animals were sacrificed and serum, liver and brain tissue (striatum) were obtained. of the remaining bdl/mn+2-cirrhotic animals (n=10), 5 were injected with ad-hupa plus ad-mmp-8 (3x10" + 1.5~10~' vp/kg respectively) and 5 rats injected with (4.5~10" vp/kg of ad-p-gal). this treatment lasted 10 days. then, biological samples were recollected as before. an additional experimental model represented by cirrhotic rats injured chronically for 7 weeks with cclb were also monitored for their response to this therapy. results seven wk-cc4-cirrhotic animals treated with ad-hupa plus ad-mmp-8 (total 4.5x101'vp/kg) were monitored at 2,4,6,8, 10 and 12 days after combined gene therapy treatment (n=18). these animals showed improvement in liver fibrosis of up to 55% as compared with their ad-p-gal (n=18) treated cirrhotic counterparts. these results correlated with hydroxyproline detemiinations. furthermore, survival was determinated in 28 additional cirrhotic animals. 14 cirrhotic rats treated with ad-hupa plus ad-mmp-8 had a significantly higher probability of survival at 60 days after beginning of treatment as compared with 14 ad-@-gal cirrhotic rats. bdl/mn+2 injured rats displayed tremors, rigidity, and gait abnormalities. ten days after treatment with combined therapeutic gene therapy, these symptoms decreased. also, liver fibrosis was evidently less (25%) as compared with ad-p-gal treatment rats. brain tissue (striatum) was recollected 10 days after bdl/mnf2 animals were injected with either ad-hupa plus ad-mmp-8 or ad-p-gal alone. dopamine (3.04 mglgr) was decreased in 30% in ad-p-gal treated animals, as compared with 4.79 mglgr of dopamine found in ad-hupa plus ad-mmp-%treated animals. dyhydroxyphenylacetic acid (dopac a main dopamine metabolite) was as high as in ad-hupa plus ad-mmp-%treated animals (13.56 mglgr striatum) indicating a higher dopamine turnover in nontherapeutically treated cirrhotic animals. moreover, animals treated with ad-hupa+ad-mmp-8 showed a 50% decrease in ascitis and gastric varices as compared with ad-p-gal-treated animals. (gastroenterology 1221924,2002) . while ppar-a was reported to be involved in induction of antioxidant enzymes expression and activities (life sci 63:135,1998). furthermore, in respect of hepatic fibrosis, oxidative stress induces hepatic fibrosis and many reports have demonstrated that several antioxidants can inhibit hepatic fibrosis. therefore, in the present study, we examined whether ppar-a ligands, i.e. wy-14,643 and fenofibrates, can suppress hepatic fibrosis by attenuating oxidative stress in experimental rat model and possess a possibility of therapeutic candidate for hepatic fibrosis. methods: fibrosis was induced in male wistar rats by intraperitoneal administration of thioacetamide (taa) (200 mglkg twice weekly for 6 weeks). in the treated groups, ppar-a ligands, wy-14,643 (wy) and fenofibrates, were fed a diet containing 0.1% (wlw), and ppar-y ligands, pioglitazone (pio), were fed containing 0.01% (wlw) all through the experiment. control cirrhotic rats received saline injections for 6 weeks. after killing all rats, histological examinations (he, azan staining, immunohistochemistry of a-sma), serum value of alt and hyaluronic acid, mrna expression of ppars, acyl-coa oxidase (aco), a1 (i) procollagen, and antioxidant enzymes, such as superoxide dismutase (sod) and catalase. we also determined lipid peroxidation (lpo), glutathione levels, and activities of sod and catalase in the perfused liver. results: semi-quantitative analyses of fibrotic area revealed that wy co-administration with taa reduced to only 19% of the area of taa-treated rats. there was no significant difference in azan staining of the liver between taatreated rats and taa-treated rats. wy administration could not lower serum alt value in chronic and acute taa injury. these observations suggest that wy fails to modulate the hepatotoxicity of taa. an increased expression of ppar-cy in taatreated rats were observed, but the expression of ppar-a was abolished in taa-and taa-treated rats. wy intensively enhanced mrna levels of aco which was regulated by ppar-a, increase nearly 50-fold than controls, but the expression of aco diminished in taa-and taa-ppar-a activators, wy-14,643 and fenofibrates, treated rats as well as ppar-a. the mrna levels of a1 (i) procollagen and tgf-/3l were strongly increased in taa-and taa-administrated rats than in controls, while they were dramatically suppressed in taa-treated rats. the catalase mrna was reduced to 15% of the controls in taa-and taa-treated rats, however, taatreatment prevented this decrease to 70% of the control levels. catalase activity increased 2-fold in taatreated rats than in controls, while it decreased 60% of the controls in taa-and taa-treated rats. in taa-and taa-treated rats an increase of lpo was observed, but not in ta-atreated rats. we c o n h e d that fenofibrates treatment could reduce hepatic fibrosis to approximately 16% of taa treatment for 6 weeks by semi-quantitative analysis of fibrotic area. conclusion: our data indicate that war-a activators, not ppar-y, can markedly inhibit hepatic fibrosis in the experimental taa-induced rat cirrhotic model. we suggest that ppar-a and catalase are important in the development of hepatic fibrosis. therefore, we conclude that suppression of hepatic fibrosis by ppar-a activators is probably due to their antioxidant effects via increased activitiy of catalase which reduces hydrogen peroxide, and that fibrates, such as fenofibrates and bezafibrates, might be new candidates for the treatment of hepatic fibrosis. fibrosis is a common end-point in clinical trials of chronic hepatitis c. liver biopsy remains the gold standard for fibrosis evaluation. however, variability in fibrosis distribution within the liver (sampling variability) is a potential limit to liver biopsy. in order to assess the influence of sampling variability on the accuracy of liver fibrosis assessment with biopsy, we measured liver fibrosis on virtual biopsies of increasing length reconstituted from digitalized images of large liver sections using two different methods of fibrosis assessment. method large sections (3cmz) were performed from 17 surgical liver samples with chronic hepatitis c and various degree of fibrosis. measurement of fibrosis on the whole section was considered as the reference value. from the digitalized image of the whole section, virtual biopsies of increasing length (2.5 to 200mm) were reconstituted. fibrosis was assessed independently on each individual virtual biopsy using both image analysis and meta-vir score. results were compared to the reference value. results: a total of 10.659 virtual biopsies were studied. using image analysis, a strong dispersion of area of fibrosis was observed for virtual biopsies smaller than 4omm. only 22% of 1,5 mm length virtual biopsies had a measured of area of fibrosis equal to reference value 2 10%. this percentage increased progressively with increasing size (30%, 50%, 69% for biopsies of 25mm, 4omm, loomm length, respectively). using metavir scoring system, 65% of biopsies of 1,5 cm length were correctly categorized according to the score assessed on the whole large section. it increases to 75% for a 2,5 cm size without any substantial benefit for longer biopsies. a same trend was observed whatever the stage of fibrosis. conclusion: sampling variability of fibrosis is a significant limit for fibrosis assessment with liver biopsy. this study suggests that a length of at least 25mm is necessary to valuably evaluate fibrosis with semiquantitative score. sampling variability become a major limitation for more accurate method such as automated image analysis. (msi-1) , a rna-binding protein, is highly observed in developing central nervous system and thought to be a mammalian neural stemlprogenitor cell marker. we have reported that cells positive for musashi-1 (msi-1) protein appeared during spontaneous resolution of rat liver cirrhosis and that some msi-1-postive cells were also positive for matrix metalloproteinase (mmp)-13, possibly implicating active participation of stem cells in the resolution of collagen. though msi-1 antigen is expected to be expressed during very early stage of differentiation in the cellular lineage of liver, the type of cells appeared in the damaged liver and expressed msi-1 remain to be elucidated. the present study is designed to characterize the msi-1-positive cells in cirrhotic liver from the aspect of differentiation, significance and expected function of stemlprogenitor cells in the liver, especially fibrolytic function. methods: rat liver fibrosislcirrhosis was established by intraperioneal injection of cc14 twice a week. liver samples were obtained 2, 5 and 7 days after the last injection of 12-week cc14 intoxication. liver tissue samples were immunohistochemically stained for msi-1, c-kit, nestin, a-smooth muscle actin (a-sma), ck-19 and mmp-13. gene expression of msi-1 was also observed by reverse-transcription polymerase chain reaction (rt-pcr). results: neural stemlprogenitor cells, as defined by their expression of msi-1 andlor nestin, were not observed either in normal rat liver or after 4 weeks of c c 4 administration. rt-pcr analysis revealed very slight signals of msi-1 mrna at 2 days after discontinuance of 8-week ccld treatment. on the contrary, remarkable increase of msi-1 mrna was observed at 2 days after the 12 weeks ccl4 treatment, then the signals decreased gradually. immunohistochemical analysis showed that a considerable number of cells positive for msi-1 appeared in the early recovery stage from advanced liver fibrosis, especially at day 5 after the last injection of 12 weeks ccl4 treatment. expression of msi-1 in liver tissue was proceeded by and partly overlapped with that of c-kit, a marker of hematopoietic stem cells. some msi-1-positive cells observed around perivenular regions were as very small as oval cells, while the size of msi-1-positive cells present along the resolving fibrous bands varied and some of them expressed mmp-13. some ductal cells were positive for msi-1 but not for ck-19 examined in serial sections. msi-1 positive cells did not express hepatocytes markers such as albumin or afp. cells positive for a-sma and msi-1 were observed in some portions, but most of msi-1 expressing cells did not show positive staining for a-sma or desmin. at day 7, a few msi-1-positive cells were observed around the remaining fibrous bands. s-adenosyl-l-methionine represses the fibrosis, constitute a good model for studying the mechanisms responsible for antifibrotic therapy. to test whether administration of s-adenosyl-l-methionine, a precursor of glutathione and an agent shown to prevent liver toxicity in a variety of settings, could repress the activity of the mouse pro-alpha 2(i) collagen gene in hepatic stellate cells, homozygous transgenic mice harboring the -17 kb to +54 bp of the proximal promoter of the mouse alpha 2(i) collagen gene cloned upstream of the escherichia coli beta-galactosidase reporter gene (lacz) were used. chronic liver injury was induced by injecting intraperitoneally 5 mllkg of body weight of cc4 (25% vlv in mineral oil) three times per week for 4 weeks. s-adenosyl-l-methionine was administered intraperitoneally at a dose of 10 mglkg of body weight every day for 4 weeks. control groups were given either mineral oil or s-adenosyl-lmethionine alone. hepatocellular damage and protection by sadenosyl-l-methionine was confirmed by measuring serum levels of transaminases; s-adenosyl-l-methionine lowered alt levels in the ccl4-treated mice from 143 to 28 ull and ast from 171 to 66 ull (control values in the absence of cc14 were 26 and 58 ull for alt and ast, respectively). hematoxylin and eosin staining in the cc14-treated mice revealed the presence of mallory bodies, lymphocyte infiltration, centrilobular steatosis, and perivenular and pericellular fibrosis, and s-adenosyl-l-methionine minimized the pathology score. masson's trichrome staining showed less collagen deposition in mice treated with cc14 plus s-adenosyl-lmethionine than in the cc4-treated mice. histochemical analysis using x-gal staining allowed for the precise identification of the cell type in which the beta-galactosidase gene was active as driven by the pro alpha 2(i) collagen promoter. results indicated activation of the pro alpha 2(i) collagen promoter in mice treated with ccl, and repression of such activation in mice co-treated with s-adenosyl-l-methionine. immunofluorescence analysis of mice injected with cc14 revealed colocaliiation of alpha-smooth muscle and beta-galactosidase positive cells, suggesting that the activation of the promoter occurred only in hepatic stellate cells. these results suggest that one mechanism by which administration of s-adenosyl-l-methionine, a precursor of glutathione synthesis, could ameliorate liver fibrosis is by decreasing the responsiveness of the promoter of the alpha 2(i) collagen gene to a profibrogenic stimuli such as ccb. these transgenic mice should prove to be useful in further studies on how s-adenosyl-l-methionine exerts this repression of the alpha 2(i) collagen gene and perhaps to studies with other liver toxins such as alcohol. background endothelin-1 (et-i), the most powerful constrictor of the liver vasculature and stimulator of the synthesis, by kupffer cells, of a potent systemic vasodilator, platelet-activating factor, is also implicated in the fibrosis of lung, kidney and liver. thus increased hepatic concentrations of et-1 and its receptors in human and experimental cirrhosis suggest its major role in the pathology of chronic liver disease and its complications (fibrosis, portal hypertension and systemic hypotension). we investigated whether et-1 receptor antagonism, after the development of fibrosis and cirrhosis, arrestslreverses the progression of chronic liver disease. methods: chronic liver injury was induced in rats by cc14 treatment ( of the development of cirrhosis in group 1 (histopathology score of 3.7 ? 0.41 vs 4.8 2 0.16, p< 0.01) and reversal of cirrhosis in group 2 (histopathology score of 3.6 2 0.41 vs 5 2 0, p<o.ol) rats. this was accompanied by reduced a-smooth muscle actin-positive cells (activated stellate cellslmyofibroblasts) in the tak-044treated versus saline-treated rats receiving cc4. tak-044 treatment caused significant amelioration of portal hypertension (p<0.05 in both groups), systemic hypotension (p<0.05 in both groups) and liver injury (reduced activities of serum ast, alt and ldh), and improved hepatic synthetic capacity (increased serum albumin concentration) in both groups of rats relative to the vehicle-treated rats. tak-044 treatment reduced collagen synthesis as evidenced by decreased hepatic hydroxyproline, collagen-a type i mrna expression, and mrna and protein expression of a potent fibrogenic cytokine tgf-p1. conclusions: the results emphasize the role of et-1 in the development of cirrhosis and strongly suggest that blockade of its actions can be a rational therapy for chronic liver disease and its complications. subunits which are reported to be stimulatory. however p50 and p65 can form homodimers which have been described as inhibitors and stimulators of gene expression respectively. the aim of the study was to determine the role of the p50 subunit of nfkb in the progression of fibrosis. methods -chronic liver fibrosis was induced in p50 knockout and wild type control mice by administering twice weekly ccl4 injections for 12 weeks. livers and blood samples were harvested at day 1, 3, 5,7 and 14 after the last ccl4 injection. results -p50 knockout mice developed more severe liver disease as measured by serum alt's and peak fibrosis scores. the increased liver injury in p50 knockout mice was associated with a massive infiltrate of inflammatory cells into the liver compared to the control wild type mice. the majority of the inflammation was neutrophilic as measured by immunohistochemistry, however we also observed an increase in cd3 positive t cells. taqman quantitative rt-pcr analysis was used to measure collagen i, alpha-smooth muscle actin (asma) and timpl gene expression in the p50 knockout and wild type mice. all three genes were elevated in the mice laking p50 subunit of nfkb. immunostaining confirmed an increase in asma positive cells in the livers of p50 knockout mice compared to wild type controls. conclusion: the p50 subunit of nfkb may play an important role in protecting against the development of liver fibrosis and reducing liver inflammation in chronic liver injury. gnainsky, volcani center, bet dagan, israel; orna halevy, hebrew university of jerusalem, rehovot, israel; gadi spira, technion -israel institute of technology, haifa, israel; rafael bruck, wolfson medical center, holon, israel; arnon nagler, sheba medical center, tel hashomer, israel; mark pines, volcani center, bet dagan, lsrael introduction halofuginone is an inhibitor of hepatic fibrosis as demonstrated by prevention of dimethylnitrosamine (dmn) and thioacetamide (taa)-induced fibrosis. halofuginone is also responsible for the resolution of advanced fibrosis and increase in cirrhotic liver regeneration. methodslaims in this study atlas microarray technique was applied in vivo in an effort to identify genes involved in the halofuginone-dependent prevention of liver fibrosis. to that end we compared cdna array hybridization of taa-treated rat liver biopsies to biopsies of livers from rats treated with both taa and halofuginone. results from the 588 genes of the array, 13 were differentially expressed. one of the genes up-regulated by halofuginone was identified as protein tyrosine phosphatase of regeneration liver -1 (prl-l), an immediate early gene known to be induced during liver regeneration. the atlas microarray results were confirmed by northern blot analysis and by in situ hybridization. hepg2 cells in culture expressed high levels of prl-1 gene that was down-regulated upon serum starvation. halofuginone increased the prl-1 gene expression in the serum-starved cells in a dose and time-dependent manner. on the protein level, halofuginone caused translocation of prl-1 from hepg2 and huh-7 nucleus outer membranes to the cytosol as demonstrated by immunofluorescence using prl-1 antibodies. prl-1 translocation is known to be associated with alterations in cell cycle. halofuginone caused accumulation of hepg2 cells in the gym phase as analyzed by flow cytometry suggesting a putative g2im arrest. conclusions these results demonstrate the involvement of halofuginone in the hepatocytes cell cycle through prl-1 gene expression and its cellular translocation. nathalie senant, universitk victor segalen bordeaux 2, bordeaux, france; genevieve freyburger, hdpital pellegrin, bordeaux, france; toulouse, france; alexis desmouliire, universitk victor segalen bordeaux 2, bordeaux, france several lines of evidence incriminate the hemostasis system in liver fibrogenesis. first, experimental and human pathological data suggest a role for micro-thrombosis in fibrosis progression. secondly, several hemostasis proteins, such as thrombin, can regulate the expression of fibrogenic mediators through signaling by cell-surface receptors. the aim of this study was thus to evaluate the effect of direct thrombin inhibition on experimental liver fibrosis. fibrosis was induced in rats by administration of cc14 in olive oil orally 3 times a week for either 3 or 7 weeks. the thrombin antagonist ssr 182289 (sanofi-synthelabo research) was given daily orally at 30 mglkg (a dose that has been shown to significantly inhibit thrombin in vivo) starting 1 week after the beginning of cc14 intoxication. control groups received either the respective solvents or ssr 182289 alone. nine animals were used in each group. fibrosis was quantified with histomorphometrical analysis of sirius red-stained liver sections and expressed as a percentage of the surface of the section. we also measured the level of fibrogenic cell activation with quantitation of the area occupied by alpha-smooth muscle actin (asma)-positive cells. the levels of tissue inhibitor of matrix metalloproteinase-1 (timp-i) transcripts were analyzed by northern blot on total liver rna and expressed as timp-l/gapdh ratios following quantitative measurement with an instant imager. after 3 weeks of cc14, the area of fibrosis increased from 0.285 % 0.08 in controls to 2.62 i 0.68 % and was slightly but non significantly decreased by ssr 182289 (2.26 lr 0.62 %, ns). the area of asma-positive cells was however significantly decreased by ssr 182289 from 3.83 ? 1.04 to 2.86 i 0.79 % (p =0.03). in animals receiving cc14 for 7 weeks, treatment with ssr 182289 resulted in a decrease in fibrosis area from 7.01 t 0.24 to 4.62 t 0.77 % (p =0.05). similarly, the asma-positive area decreased from 11.57 5 5.57 to 7.71 i 3.89 % (p =0.05). the timp-i/gapdh ratio was decreased by ssr 182289 at 3 weeks (from 0.118 2 0.018 to 0.087 2 0.014, p =om). ssr 182289 alone had no effect on the measured parameters at both time points. additionally, it did not alleviate the acute toxicity of cc14 as shown by the levels of serum transaminases and the area of necrosis that were not significantly modified. altogether, these data provide evidence that thrombin antagonism can reduce the extent of liver fibrosis in this model. ongoing experiments should clarify whether this results from an anticoagulant effect or an inhibition of thrombin signaling effects. center, tel aviv, israel background and aims: proliferation and "activation" of hepatic stellate cells (hscs) and production of collagen are involved in the pathogenesis of liver inflammation and fibrogenesis. ligands of nuclear hormone receptors, such as triiodothyronine (t3) and peroxisome proliferator activator receptor y (ppary) ligands have been shown to affect fibrogenesis. levels of ppary are reduced in activated stellate cells and addition of the iigand increases ppary receptor expression and ameliorates fibrosis. in contrast, t3 has the opposite effect on liver fibrosis. low circulating t3 levels reduces liver fibrosis and cirrhosis. in this study, we investigated the in vitro effects of t3, ppary ligand and their combination on proliferation and activation of a rat hsc cell line. methods: transforming growth factor (tgf-pi) was used to induce myofibroblast-like phenotype in rat hscs (hsc-t6). activation of thyroid hormone and ppary receptors was induced with t3 and 15-deoxy-prostaglandinj(2)(pj2), respectively. signal transduction markers and activation marker a-smooth muscle actin were determined using western blotting. collagen i expression was measured by northern blotting. results: both t3 and pj (2) inhibit hsc proliferation induced by platelet-derived growth factor and by tgf-p. t3 inhibits cell proliferation by inducing apoptosis, while pj(2) reduces expression of proliferation marker cyclin d1. both "i3 and pj(2) decrease expression of activation marker a-smooth muscle actin. in addition, t3 strongly increases both basal and tgfeinduced collagen i expression. by contrast, pj(2) decreases collagen expression and inhibits t3-induced collagen production in tgfp-activated hscs. conclusion: both t3 and pj(2) reduce mitogen-induced proliferation of rat hscs, but have different effects on collagen i expression. these results suggest that prolierationlactivation and collagen i expression are two differently regulated processes in kato, sadakazu aiso, hiromasa lshii, keio university, tokyo, japan connective tissue growth factor (ctgf) is a profibrogenic molecule involved in several human fibrotic disorders including liver fibrosis. in human as well as experimental liver fibrosis, ctgf overexpression, through upregulation of ctgf mrna in hepatic stellate cells (hscs), is associated with the degree of fibrosis. it has been known ctgf is produced in many cell types in the live and exogenous ctgf modulates even the activation of hscs. although alcohol is the major cause of liver fibrosis, the role of ctgf in alcoholic liver fibrogenesis, has been poorly understood. cytochrome p-4502e1 (cyp2el)-mediated oxidative stress, as a result of chronic and excessive ethanol consumption, has been implicated as a crucial factor in alcoholic liver fibrogenesis. the goal of a series of our studies was to clarify the effect of cyp2e1mediated ethanol oxidation on ctgf expression. we previously reported the results of the assay using the well-established hepg2 cell line constitutively expressing cypzei, which was kindly provided by dr. a. cederbaum ( mt. sinai school of medicine, ny). ctgf mrna quantitation assay by a real-time reverse polymerase chain reaction (rt-pcr) showed a significant increase in ctgf mrna levels in ethanol-treated e9 cells in a time-dependent manner up to 36 hours after initial ethanol treatment. there was nearly a 2-fold increase in ctgf mrna levels when ethanoltreated e9 cells were coincubated with phorbol myristate acetate (pma), a reagent which enhances expression of cyp2e1. ctgf mrna levels were significantly reduced when ethanol-treated e9 cells were co-incubated with 4-methylpyrazole(4mp), a cyp2el inhibitor, or antioxidants, n-acetylcysteine and trolox. in the present study, we visualized the state of oxidative stress in ethanol-treated e9 cells by using mab5f6, a newly-established marker for oxidative stress. the mab5f6, which was generously provided by dr. k uchida (nagoya university, japan), was raised against the acrolein-modified keyhole limpet hemocyanin to verify the protein-bound acrolein in vivo. the staining, procedure was detailed elsewhere. immunohistochemical detection of proteinbound acrolein revealed e9 cells were stained when treated with ethanol. without ethanol treatment, no staining was observed in ethanol upregulates pro-fibrotic connective cells via cytochrome p-4502e1-mediated e9 cells even at a 24 -hour incubation period. moderate or no staining was observed when ethanol-treated e9 cells were coincubated with antioxidants. staining intensity by mab5f6 is associated with ctgf mrna levels in this cell line. collectively, these data suggest ctgf mrna upregulation in e9 cells seems to be mediated by the state of oxidative stress. our present study first linked cyp2el-mediated oxidative stress with ctgf gene regulation, thus suggesting a possible involvement of ctgf in alcoholic liver fiberogenesis. disclosures: sadakazu aiso -no relationships to disclose hiromasa ishii -no relationships to disclose mikio kajihara -no relationships to disclose (2) human livers with biliary fibrosis. methods: changes in ntp-dase2 expression in rat liver were determined in normal and 2-wk bile duct ligated (bdl) or 6-wk c c 4 rat livers and pflpm using confocal immunofluorescence, immunoblot, and northern blot. changes in ntpdase2 expression in human liver were determined in de-paraffinized liver biopsy specimens using confocal immunofluorescence. results: as was previously reported, ntp-dase2 was expressed in pf surrounding intrahepatic bile ducts in normal and sham bdl controls. in bdl rat livers, however, nt-pdase2 fluorescence was completely lost. no decrease in ntp-dase2 fluorescence was noted in cc14 rat livers within portal areas, although an increase in ntpdase2 fluorescence was seen in central areas. pf from sham bdl control animals expressed nt-pdase2 and the intermediate filament vimentin. "pf" from bdl animals did not express vimentin but were positive for a-smooth muscle actin (sma), suggesting that they were in fact pmf. most bdl pmf did not express ntpdase2; however, occasional cells were seen with sma and ntpdase2 in a pen-nuclear compartment. immunoblot analysis of pf revealed a marked decrease in ntpdase2 detection in bdl cells relative to sham bdl. no change was noted in ccll or c c 4 control cells. northern blot analysis revealed no differences in ntpdase2 mrna levels in sham bdl, bdl, ccl, control, or cc14 pf. confocal micrographs of normal human biopsy specimens revealed ntpdase2 co-localization with vimentin in pf surrounding intrahepatic bile ducts. however, ntpdase2 fluorescence was either markedly decreased or lost in specimens from patients with primary biliary cirrhosis. no loss of ntpdase2 fluorescence was seen in biopsy specimens from patients with hepatitis c. conclusions: ntpdase2 expression is specifically decreased in biliary fibrosis, largely through a posttranslational mechanism. "portal fibroblasts" from bdl rats are in fact portal myofibroblasts, which lose expression of ntpdase2. consistent with findings in rats, ntpdase2 expression is also specifically lost in human biliary fibrosis. work is now underway to determine whether loss of ntpdase2 in the peri-biliary compartment alters nucleotide signaling in bile duct epithelia. myofibroblasts of bone marrow origin have recently been found in a number of parenchymal organs such as the gut and kidney. we hypothesised that marrow derived myofibroblasts contribute to liver cirrhosis. methods. we sought to analyse the origin of myofibroblasts within cirrhotic liver in two scenarios: three male recipients of female liver transplants who have subsequently developed post transplant hepatitis c cirrhosis and a female recipient of a male bone marrow transplant who later developed hepatitis c induced cirrhosis. through the use of in situ hybridisation for the y chromosome we have been able to track cells of male origin. results. we have detected significant numbers of y chromosome positive cells in fibrotic areas. by combining the in situ hybridisation with immunocytochemistry these cells were found to be positive for vimentin, alpha-smooth muscle actin and negative for cd45. these cells were therefore determined to be of a myofibroblast phenotype. in the liver transplant recipients, 15.4% (corrected count 33.6%) of the myofibroblasts were y chromosome positive. in the female recipient of the male bone marrow transplant, 12.5% (corrected count 27.3%) of the myofibroblasts were y chromosome positive. conclusions. within all the livers analysed we found frequent myofibroblasts within and adjacent to the fibrotic bands that are of bone marrow origin indicating an extrahepatic cell contribution to the pathogenesis of human liver cirrhosis. (hsc) is the major fibrogenic cell of the liver and a key target of potential antifibrotic therapies. hepatocyte growth factor (hgf) is antifibrotic in several models of experimental liver fibrosis, but its mechanism of action is uncertain. potential use of hgf as a therapeutic peptide is limited by its size and need for parenteral administration. to overcome these potential limitations, refanlin has been developed as a synthetic small molecule sflhgf mimetic, that has demonstrated antifibrotic properties in renal fibrosis. aim: to study the in vitro and in vivo antifibrotic efficacy of refanlin. methods: serum starved lx2 cells, an immortalized human hsc line, were treated with 12 mglml of refanlin for 24 hours. proliferation studies using tritiated thymidine incorporation were performed. rna was isolated, and a panel of fibrosis markers (al-smooth muscle actin (asma), collagen i, bpdgf-receptor, 9, 14 (mmp2, 9, 14) , tissue inhibitor of metalloproteinase-1,2 (timp1,2), were assessed by real time rt-pcr analysis. cirrhosis was induced in sprague-dawley rats by if thioacetamide (taa) (200 mg/kg tiw) for six weeks. in the refanlin treated group, 1 mglkg of refanlin was administered five days per week for six weeks, concurrently with the taa. control rats received ip pbs five days per week for six weeks, along with taa. liver tissue was formalin-fixed and paraffin-embedded, then stained with 1% picrosirius red and computer morphometric analysis was performed for collagen quantitation using the bio-quanttm software. results: in cultured human stellate cells, there was a 60% decrease in asma, a 70% decrease in the expression of tgfb-receptor and mmp2, and a 50% decrease in timp2. there was no effect on proliferation. in vivo, fibrosis decreased by one metavir stage in rats treated with c6 and thioacetamide for 6 weeks. furthermore, c6 led to a significant decrease in portal pressure compared with non-treated rats with fibrosis. conclusions: refanlin may have antifibrotic efficacy through its inhibition or downregulation of tgfb receptor as well as mmp2. in vivo antifibrotic activity of refanlin is associated with reduced portal pressure. these experiments better define the potential targets of hgf's antifibrotic activity. ongoing studies in mechanistically distinct models of fibrosis will broaden our understanding of this agent's effects and point towards therapeutic trials in humans. kanto, aki sato, michiyo inoue, hideki miyatake, mitsuru sakakibara, takayuki yakushijin, chika oki, tetsuo takehara, norio hayashi, osaka university graduate school of medicine, suita, osaka, japan background and aim hepatitis c virus (hcv) infection induces a wide range of chronic liver injuries. impaired thl response against hcv may be involved in hcv persistence, however, its precise mechanism is largely unknown. dendritic cells (dc) are the most potent antigen-presenting cells that play essential roles in initiating as well as regulating anti-virus immune responses. blood dc are grouped as myeloid (mdc) and plasmacytoid dc (pdc). with respect to their functions, mdc produces il-12 and tnf-a upon stimulation and drives thl response, while pdc releases considerable amount of type-i ifn upon virus infection and mainly skews th2. we previously reported that monocytederived dc is functionally impaired (kanto t. et al. j lmrnunol 1998) , however, the roles of blood dc subsets in the direction of thllth2 in hcv infection are still elusive. to address these issues, we compared the numbers and functions of mdc and pdc between chronically hcv-infected patients and uninfected donors. subjects and methods sixty-six hcv-positive patients (43 chronic hepatitis and 23 asymptomatic carriers) and 19 agematched uninfected donors were enrolled. mdc or pdc was determined by the patterns of lineage markers, hla-dr, cdllc and cd123. each type of dc was magnetically separated or sorted under facs vantage and was subjected to the functional analyses. the ability of dc to polarize naive cd4 t-cells towards thl or th2 was estimated by the pattern of ifn-ylil.-4/il-10 production from mdc-or pdc-primed cd4. results absolute numbers of mdc, pdc and their cd34+ progenitors are lower in hcv-infected patients than in control subjects, most significantly in those with chronic active hepatitis (mdc, pdc, cd34, median, patients vs. controls; 4.2,1.0,0.511*.1 vs. 6.9,3.0,1.9/pl, p<0.05). both types of dc from patients are impaired in the stimulation of allogeneic cd4 t-cells and the production of il-12 p70 or ifn-a compared with healthy donors. on encounter with nayve cd4 t-cells, mdc from the patients are less able to drive thl response, while pdc from them prime more il-10 producing t-cells than those from donors. exogenous il-12 significantly improved the patient mdcdriven thl response, however, it simultaneously increased il-10 producers both in mdc and pdc. in contrast, ifn-y or il-15 stimulated the patient mdc to drive thl without increasing mdc-or pdc-primed il-10 producing cells. conclusion both types of blood dc in chronic hcv infection are reduced and play decisive roles in the development of an impaired thl and an il-10-rich environment, thus favoring hcv persistence. ifn-y or il-15 is feasible to restore the altered t-cell polarizing ability of blood dc in chronic hepatitis c. hepatitis c virus (hcv) replicates its genome in a membraneassociated complex composed of viral nonstructural proteins and rna as well as altered cytoplasmic membranes and other as yet unidentified host cell components. a specific membrane alteration, designated membranous web, was recently identified as the site of hcv rna replication in huh-7 cells harboring subgenomic replicons (gosert et al. j virol 2003; 77:5487-5492) . here, we describe hcv replicons that allow the direct visualization of hcv replication complexes in living cells. methods: a transposon-mediated random insertion screen followed by selection of viable replicons was performed to identify sites that tolerate insertions in nonstructural protein 5a (ns5a). the green fluorescent protein (gfp) coding sequence was subsequently inserted into selected sites. rna was in vitro transcribed from subgenomic replicon constructs harboring these insertions, followed by electroporation into huh-7 cells and selection in the presence of g418. results: two sites in the c-terminal domain of ns5a were found to tolerate the gfp insertion and allowed efficient initiation and maintenance of hcv rna replication. expression of a ns5a-gfp fusion protein of the expected molecular mass could be demonstrated by immunoblot, indicating that the gfp was stably retained and that processing of the viral protein occurred normally. more important, expression levels were robust enough to allow direct visualization of this fusion protein by fluorescence microscopy. gfp fluorescence was found in a cytoplasmic reticular pattern and in brightly fluorescing dots, the typical distribution of hcv nonstructural proteins in replicon cells. by confocal laser scanning microscopy, gfp fluorescence was found to colocalize with other hcv nonstructural proteins in the brightly fluorescing dots, suggesting that these represent hcv replication complexes, previously identified as membranous webs. metabolic labeling of the nascent viral rna with brutp and (immuno)electron microscopy studies are underway to further validate these findings. moreover, live cell imaging studies using these replicons have successfully been initiated. conclusion: we have identified sites in ns5a that allow insertion of gfp in the context of functional hcv replicons and direct visualization of hcv replication complexes in living cells. these findings have implications for the structure and function of the hcv ns5a protein as well as for the architecture of the hcv replication complex. this system will allow to gain a dynamic view of the formation and turnover of hcv replication complexes and may be useful to select for cell populations permissive for hcv rna replication. infection. masahisa jinushi, tetsuo takehara, tomohide tatsumi, takuya miyagi, takahiro suzuki, tatsuya kanto, norio hayashi, osaka university graduate school of medicine, suita, osaka, japan background and aim it has been generally accepted that the pertubation of cellular immunity plays an important role in establishment and persistence of chronicity in hcv infection as well as resistance to ifn therapy. accumulating lines of evidence have unveiled that dendritic cells (dcs), most potent antigen presenting cells, are impaired in patients with hcv infection, which may be involved in disturbance of anti-hcv immune responses. we recently reported that mhc class i-related chain a and b(mical b),ligands of nk activating receptor nkgzd, are induced on dcs upon interferonaand gain their ability to antivate nk cells; their expression is severely impaired in dcs derived from hcv-infected individuals (hcv-dc) (3. immunol. 170 1249 immunol. 170 -1256 immunol. 170 ,2003 . in the present study, we evaluated whether various types of cytokines have an effect for inducing micalb expression on hcv-dcs, thus resulting in triggering nk cell activation by dcs. methods dcs from healthy volunteers (n-dcs; n=15) or hcv-dcs (n=20) were generated from monocyte treated with gm-csf (1000 ulml) and il-4 (500 ulml) for 6 days. after stimulated with various cytokines (ifnalp, ifny, tnfa, il-12, il-15 or il-18) for 24 h, dcs were subjected to flow cytometric analysis of micalb expression. to investigate the involvement of each cytokine in mica/b expression on ifnalp-stimulated dcs, we employed elisa and rt-pcr to examine the cytokine expression (tnfa, il-lp, il-6, il-12~70, il-15 and il-18) in ifnalp-stimulated dcs, and also assessed micalb expression on dcs in the presence of neutralizing antibodies for these cytokines. to further address the stimulatory capacity of dc, allogeneic nk cells were cocultured with n-dcs or hcv-dcs at a ratio of 5:l for 24 h, and applied for flow cytometry of intracellular ifny expression, as well as 51cr release assay for the cytolysis of k562 target cells. in some experiments, masking antibody for nkg2d or micalb was added during nkldc cocultures, and then subjected to nk cell function assays described above. results micalb were induced on n-dcs, but not on hcv-dcs, upon iifnalp(% mic positive: n-dc 42.1% vs hcv-dc 3.4%; p<o.ol). in sharp contrast, micalb expression was increased on hcv-dcs at levels similar to n-dcs in the presence of of 5onglml il-15 (but not other cytokines) (%mic: n-dcs 49.4% vs hcv-dcs 47.6%). moreover, n-dcs, but not hcv-dcs, was capable of producing il-15 in response to ifnalp. little differences were detected in n-dcs and hcv-dcs for the production of tnfa, il-lp, 1l-6, il-12~70, or il-18. in addition, ifnalp-mediated micalb induction in n-dcs were completely inhibited in the presence of neutralizing antibody of anti-il15l il-15 receptorcu. even much lower doses (1nglml) of il-15 restored the ability of hcv-dcs to induce micalb and to activate nk cells in response to ifnalp. the ability of hcv-dcs for nk cell activation was largely diminished by adding the masking antibody of nkg2d or micalb. conclusion the current study demonstrated that il-15 acts as an indispensable mediator to induce micalb expression on ifnalp-stimulated dcs, and that hcv-dcs lack the ability to produce il-15 and to induce micalb expression in response to ifnalp. since defect in il-15 production is responsible for impaired nk cell activities by hcv-dcs, these findings shed light on the possibility that il-15 could improve the ifn-mediated anti-viral immune responses in chronic hcv-infected patients. the cellular immune response contributes to viral clearance as well as liver injury in acute and chronic hepatitis c virus (hcv) infection. understanding the relative immunodominance of hcvencoded peptides is important for understanding pathogenesis as well as potential vaccine candidates. we have developed a comprehensive and sensitive method to screen immune responses to all potential hcv epitopes in hcv-exposed individuals. methods: subjects with evidence of spontaneously resolved infection (eia+/hcv rna-neg, n = 15) and patients with evidence of chronic hcv infection (mean hcv rna level = 23.6 million copieslml; n = 15, all genotype la) had either leukophoresis or one-unit blood draw. we synthesized 750 peptides (15 mer-peptides overlapping by 11 amino acids) that spanned the entire hcv genotype l a polyprotein; responses to 33 pools of peptides were screened directly ex vivo by an ifn-gamma elispot assay. briefly, autologous dendritic cells (dcs) were generated from peripheral blood mononuclear cells selected by plastic adherence then cultured with gmcsf, il-4 and 10% hs for 5 d. dcs were pulsed with peptide pools to stimulate bead-purified cd8+ t cells. the remaining cd4+ t cells (5.0 x lo5 cells/well) were stimulated the same day with the peptide pools; 10 wells with no peptide (or siv peptides) were used as controls. only responses greater mean + 3 sds above controls were considered positive. the frequency and vigor of cd4+ t cell responses were statistically greater in resolved vs. chronic infection (mean 9.4 pools out of 33; median 8 in resolved vs. mean 3.86 pools; median 3.0 in chronics; p =.005). there was an inverse trend between # peptide pools with cd4+ t cell responses and hcv rna level. as shown in the figure, the hcv peptide pools differentially elicited responses; specifically, resolved infection was associated with greater cd4+ responses to pools core b, ela, ns3 protease-2, ns3 helicase-3, ns3 helicase-4, ns3 helicase-5, ns3 helicase-6, ns4b-2, ns5a-2, ns5a-3, ns5b-2. on the average, cd8+ t cells responded to 4.8 pools in resolved infection vs. 1.3 pools in chronics (p =.as). cd8+ t cell responses specifically directed against core 8, ns3 helicase-5, ns3-helicase 6, ns4b-2, ns5a-2 were more frequent in resolved as compared to chronic infection. conclusions: comprehensive mapping of hcv-specific immune responses by overlapping pools reveals significant differences in immunogenicity in resolved vs. chronic infection. in particular, regions core b (amino acids 96-203), ns3 helicase-5 (aa 1369-1479) and helicase-6 (1469-1579), ns4b-2 (1801-1899), ns5a-2 (2076-2191) induce both cd4+ and cd8+ t cell immunity and thus represent potential vaccine candidates. supported by nih r01 dk60590. sud, geoffiey c farrell, priyanka bandara, james g kench, storr liver unit, westmead hospital, westmead, nsw, australia; geoffiey w mccaughan, aw marrow gastroenterology (and liver centre, camperdown ns w, australia; jacob george, storr liver unit, westmead hospital, westmead, ns w, australia introduction: chronic hepatitis c virus (hcv) infection is associated with an increased prevalence of type 2 diabetes mellitus, which is known to accelerate fibrosis in nash. we hypothesized that viral-induced insulin resistance (ir) may be a mechanism for fibrogenesis in chronic hcv infection. methods and results: 1. to assess the influence of hcv infection on ir independent of any effect of hepatic fibrosis, 121 hcv subjects with minimal (stage 1) or no (stage 0) fibrosis were compared with 137 healthy volunteers matched by gender, body mass index (bmi) and waistlhip ratio. although the hcv subjects were younger than the healthy volunteers (p=0.004), they had significantly higher levels of markers of ir including fasting glucose (p=o.ol), insulin (p=o.o02), cpeptide (p<o.ool) and homa-ir (p=o.o02). 2. in 260 hcv patients (fibrosis stage 0 to 4), e examined the viral and diseaserelated factors (portallperiportal & lobular inflammation, viral genotype) which may be involved in the pathogenesis of ir in a multivariate model, controlling for other demographic and biochemical variables. by multiple linear regression analysis, independent predictors of homa-ir included bmi (p<o.ool), failed previous antiviral treatment (p<o.ool), portal inflammatory grade (p<o.ool) and genotype 3 status (p=o.ol). genotype 3 cases had significantly lower homa-ir than other genotypes at each stage of hepatic fibrosis. the adjusted difference in homa-ir between genotype 3 and non-genotype 3 was -0.58 (95% ci: -0.13 to -1.02; p=o.ol). 3. we then assessed if ir was associated with increased fibrotic severity. by multiple ordinal regression analysis, independent predictors for the stage of fibrosis in the 260 hcv subjects were homa-ir (or 1.3, p<o.ool), portal inflammatory grade (or: 5.3, p<o.ool), past alcohol intake (or 1.7, p<o.ool), age (or 1.1, p <0.001), alt (or 1.0, p=0.04), platelet count (or 0.93, p<o.ool), and serum cholesterol level (or 0.65, p=o.ool). as cirrhosis is known to cause ir, a separate multivariate analysis was performed for the 236 non-cirrhotic subjects. the independent predictors for fibrosis were unaltered and homa-ir remained in the model. in a subgroup of 117 patients with estimated duration of infection, multiple linear regression analysis showed that homa-ir was independently associated with an increased rate of fibrosis progression (p=0.03). conclusion: hcv infection induces insulin resistance irrespective of the severity of liver disease, and this effect appears to be genotype-specific. further, increased insulin resistance contributes to fibrotic progression. disclosures: priyanka bandara -no relationships to disclose geoffrey c farrell -no relationships to disclose jacob george -no relationships to disclose jason m hui -no relationships to disclose james g kench -no relationships to disclose geoffrey w mccaughan -no relationships to disclose background: approximately one half of hepatitis c virus (hcv) infected patients will fail to respond to current treatment regimens, pegylated interferon alfa and ribavirin. modeling of hcv rna decay in response to antiviral therapy can provide insight into viral and host determinants of viral response. aims: pharmacokinetic, pharmacodynamic, and immunologic outcomes were evaluated on 24 co-infected patients: 1) to evaluate the relationship between hcv rna decay and peg-ifn alfa2b serum concentrations in hivlhcv co-infected patients and 2) to evaluate association between hcv-specific immune responses and virologic outcome. methods: 24 co-infected, therapy-nake patients were treated with peg-ifn alfa2b 1.5pglkglwk and rbv 13 2 2 mglkglday. serum was obtained for hcv rna, hiv-1 rna and serum peg-ifn alfa2b levels at baseline, after the first dose (6,12, 24 hours) and on days 2, 3, 5, 6; after the second dose (6, 12, 24 hours) and on days 8,9; and after the third dose on days 14,15, and 16. peripheral blood mononuclear cells were obtained at baseline, weekly for the first month, and monthly thereafter. hcv and hiv-1 rna levels were determined by reverse-transcriptase polymerase chain reaction and peg-ifn alfa2b levels were measured by elisa. cd4+ cell proliferative responses to hcv antigens (core, ns3, ns4, ns5) and hiv-1 gag were performed at baseline, day 7 and 14, and weeks 4, 8, 12, and 24. among 24 co-infected patients, 21 are males, the mean age was 46 2 5.8 years, 8 are caucasian, 12 are african-american and 4 are hispanic. 20l24 patients were infected with genotype 1, 3/24 patients with genotype 2, and 1/24 with genotype 3a. results: pharmacokinetics: to date, 21 patients have been evaluated. 10/21 patients were end of treatment responders, but 2 of these were "late responders" (i.e. hcv rna declines after week 8 and is undetectable by week 24). data fitting during the first seven days revealed a free virion clearance rate c of 13.3 (day-l) and an infected cell clearance rate 6 of 0.21 (day-'). the half-lives were 2.7 hours and 4.0 days, respectively. pharmacodvnamics: viron production was blocked after the first dose of peg-ifn alfa with an average maximum effectiveness (emax ) of 89%. we estimated that the fifty percent effective concentration (ec50) of peg-ifn alfa2b, the theoretical in v i m drug concentration at which the drug efficacy is 5070, was five-fold lower in responders compared with non-responders while the maximum (cmax) and minimum (c,,,in) concentrations did not differ significantly ( table i ). the calculated minimum ( € , , , i n ) and maximum (emax) efficacy were also significantly increased in responders. immunolonic: the mean number of cd4+, cd8+ and cd56+ cells did not differ significantly at baseline or during treatment between responders and nonresponders. peripheral blood cd4+ hcv-specific proliferative responses did not differ significantly between responders and nonresponders except that the response to core antigen was increased in non-responders compared with responders at weeks 4 and 12. hiv gag cd4+ proliferative responses were stronger than hcv-specific responses at all time points evaluated. conclusions: the serum peg-ifn alfa2b concentration necessary for an end of treatment response is fivefold lower in responders compared with non-responders while there are no significant differences in the maximum and minimum peg-ifn alfa2b concentrations in responders and nonresponders. these data suggest that host factors responsible for a viral response may be different in responders compared with nonresponders, independent of serum peg-ifn alfa2b concentrations. is a significant predictor of liver disease severity in patients who are coinfected with hepatitis c virus (hcv). this accelerated liver disease is presumed to be due, in part, to hn-related immune suppression. hcv quasispecies variability is likely a marker of immune pressure on the virus. hyuotheses: 1. hcv quasispecies variability is decreased in those with hiv coinfection, and 2. treatment with highly active antiretroviral therapy (haart) is associated with increased variability, methods: hcv-infected patients with or without hiv were enrolled in a cross-sectional study. patients with genotype 1 infection and a parenteral risk factor for hcv were included. those with injection drug use (idu) were estimated to have acquired hcv 2 years after the onset of idu. alcohol use was determined using standard questionnaires. haart data were collected from the hiv clinic's database. only those with complete records were included. hcvquasispecies variability was determined using clonal frequency analysis of the second envelope gene hypervariable region. quasispecies heterogeneity was characterized according to complexity (the normalized number of unique quasispecies variants within a sample) and diversity (the average genetic distance between individual variants within the same sample). groups were compared using the student t-test for unequal variances, anova and the wilcoxon rank-sum test where appropriate. linear regression was used to assess the independent effects of hiv infection and haart on complexity and diversity. results: to date quasispecies data are available on 43 patients, 22 of whom are hivpositive. as expected, hiv+ subjects had significantly lower cd4 counts (mean 508 vs 956 cellslul, p<.oool). mean age and hcv duration were significantly lower in the hiv+ group (40.3 vs 45.5, p=.0017, and 15.7 vs 22.2 years, p=.oo18, respectively). the groups did not differ with respect to alcohol use over their lifetime or in the 6 months prior to enrollment. quasispecies complexity and diversity are detailed in the table below. while there was no significant difference among groups by anova (p=.16), the hiv+ subjects not on haart had less complexity and diversity than the other two groups, and differences between groups 1 and 2 and groups 2 and 3 were significant (table) . both hiv infection and haart were significantly and independently associated with complexity in a linear regression model; hiv infection predicted decreased complexity (p= .001), while haart predicted increased complexity (p=.007). in addition, higher alcohol use in the prior 6 months was independently associated with decreased complexity (p=.007). hiv infection and haart also were independently associated with decreased and increased diversity, respectively (p=.ool and.034). the cd4 count did not predict complexity or diversity, and the effect of haart appeared to be independent of the cd4 count. conclusion: hcv coinfection with hiv is associated with decreased hcv quasispecies complexity and diversity. while this effect is not completely reversed by haart, treatment of hiv is associated with significantly increased hcv complexity and diversity in our sample. furthermore, the effect of haart on hcv quasispecies variability appears to be independent of its effect on the cd4 count. these findings suggest that the immune pressure on hcv is enhanced in hcvlhn coinfected individuals who are on haart when compared to those who are not on haart. diversity" group (mean .t sd) (mean f sd) in the majority of cases, hepatitis c virus (hcv) becomes chronic and is associated with impaired viral-specific t cell immune responses; the mechanisms underlying viral persistence and lack of protective immunity are poorly understood. considering dendritic cells (dcs) play critical roles in initiating and modulating immune responses, we explored the effect of hcv proteins on dc genetic expression, phenotype, and function. methods: human dendritic cells (dcs) were generated following plastic adherence of monocytes from normal subjects and culture with gm-csf and il-4. autologous non-adherent cells were infected with vaccinia constructs expressing various hcv proteins (core, nssa, ns5b) or an irrelevant protein /3-galactosidase @-gal) as the control. following induction of apoptosis with uv irradiation, these vaccinia-infected cells were co-cultured with dcs. the phenotype, gene expression profiles and functions of the dcs were analyzed. dcs were evaluated after 8, 30, and 48 hrs of co-culture as well as after maturation with a cytokine cocktail (tnf-a, il-lp, pge2, il-6) for an additional 48 hrs. results: by facs analysis, we found no alteration in expression of co-stimulatory markers (cdso, cd86, cd40,) or mhc class i or i1 molecules. by elispot assay, we found that the recall response of cd4t t cells for cmv following pulsing of hcv-dc or control (p-gal)-dc was also not altered. however, between 2-10% of the genes probed in the bd clontech nylon array were differentially regulated. real time pcr has confirmed increased expression of scyc 1 (lymphotactin) in the presence of ns5a in 2 normals as well as 2 separate biological replicates. both clontech and affymetrix gene arrays revealed ns5a-induced increases in expression of il-8, a chemokine with pro-inflammatory properties. functional assays reveal that dcs which had engulfed apoptotic cells containing hcv core, ns5a, or ns5b demonstrated comparable ability to macropinocytose as measured by uptake of dextran-fitc or albumin-fitc when compared to p-gal-dcs. however, following uptake, the hcv-dcs died at a significantly greater rate than p-gal-dcs (-30%). conclusions: taken together, these results demonstrate that following engulfment of apoptotic cells containing hcv proteins, differential gene expression occurs in human dendritic cells, although the expression of mhc and costimulatory molecules and ability to stimulate memory (cmv) responses remain intact. the mechanisms underlying the surprising induction of death of dc-hcv in the macropinocytosis assay is currently under further investigation. institutes of health, bethesdu, md steatosis is a common histological feature of chronic hepatitis c virus (hcv) infection. the cause of hepatic steatosis in hepatitis c remains to be elucidated, but its association with obesity, diabetes and hyperlipidemia suggests an underlying metabolic dysfunction similar to that found in patients with nonalcoholic steatohepatitis (nash). additionally, recent data suggest an association between body mass index (bmi) and the degree of hepatic steatosis and fibrosis in hcv infection. insulin resistance (ir) is a key factor in the pathogenesis of nash and may play a similar role in the steatosis seen in chronic hepatitis c &: to compare the prevalence and severity of hepatic steatosis, visceral adiposity, j r and their relation to disease severity in african americans (aa) vs caucasians (ca) with genotype 1 hcv infection being considered for anti-viral therapy. methods: virahep-c is a prospective study of response rates to peginterferon and ribavirin in treatment-na'ive aa vs ca patients with genotype 1 hcv infection. all patients underwent liver biopsy within 18 months of enrollment. biopsies were read blindly by a central pathologist and scored for inflammation and fibrosis using the histologic activity index [i-iaii and for steatosis (0-4+) and features of steatohepatitis (zone 3 ballooning, zone 3 perisinusoidal fibrosis and mallory bodies). waist circumference and waist-hip ratio were measured as determinants of visceral adiposity. ir was determined using fasting glucose and insulin levels based upon the homa method. results: to date, 177 patients have been enrolled and 166 have complete liver biopsy information (75 aa and 91 ca). steatosis was present in 66 patients (40%) with similar rates among aa (39%) and ca (41% ca). grades of steatosis were also similar between aa and ca 1+ (38% vs 41%), 2+ (45% vs 27%), 3+ (14% vs 27%) and 4+ (3% vs 5%) (p = ns). patients with steatosis had significantly higher values for homa index (4.0 vs 2.4, p<o.ol), total hai score (10.3 vs 8.7, p<o.oool), hai inflammation score (8.5 vs 7.1, p<o.oool), waist circumference (40.2 in vs 36.5 inches, p<o.oool) and bmi (30.6 vs 26.7, p<o.oool). there were no significant differences in amount of alcohol consumption between patients with and without steatosis. spearman's non-parametric correlations were performed to assess associations between the ordinal variables (scores for fat, steatohepatitis ballooning degeneration + perisinusoidal fibrosis + mallory bodies], total hai, inflammation and fibrosis) and one continuous variable (homa index). waist circumference, waist-hip ratio, bmi, homa index, total hai, hai inflammation and hai fibrosis all correlated with both the fat score and steatohepatitis score (p<o.ol). median homa index levels were significantly greater among aa than ca (3.1 vs 2.7, p< 0.05), but step-wise logistic regression showed that race was not an independent predictor of steatosis after controlling for alt, ast, waist circumference, waist-hip ratio, bmi and homa index. conclusions: in patients with chronic hepatitis c and genotype 1, steatosis and steatohepatitis correlate strongly with the presence of insulin resistance and visceral adiposity. race is not an independent predictor of steatosis after controlling for other factors. bulk populations of antigen-presenting dendritic cells (dcs) contain myeloid dcs and plasmacytoid dcs. myeloid dcs polarize t lymphocytes to thl phenotype that produce interferon (1fn)-7. plasmacytoid dcs produce abundant amounts of interferon (1fn)-alpha. both ifn-?-producing t cells and adequate amounts of ifn-alpha are essential for viral clearance. in patients with chronic hepatitis c virus (hcv) infection, dcs are infected with hcv, which is presumed to have a role in viral persistence. however the functional implication of this has not been addressed. the aim of this study is to evaluate the functional capabilities of both myeloid and plasmacytoid dcs in patients with chronic hcv infection. myeloid dcs were defined as linage-, cdllc+, hla dr+ and cd123-cells, and plasmacytoid dcs as lineage-, hla dr+, cdllcand cd123+ cells among the peripheral blood mononuclear cells (pbmcs). the frequencies of myeloid and plasmacytoid dcs were measured in 64 patients with chronic hcv infection and 34 agematched control subjects by four-color flow cytometry. to assess the production of ifn-alpha by plasmacytoid dcs, these were cultured with graded doses of herpes simplex virus-1 (10-100 plaque-forming unitsldc). the frequencies of plasmacytoid dcs expressing intracellular ifn-alpha among total plasmacytoid dcs were counted by two-color flow cytometry. to evaluate the capacity of myeloid dcs to induce th polarization, dcs were cultured with autologous naive t cells for 7 days. polarization of t cells to thl phenotype was assumed when na'ive t cells expressed intracellular ifn-y. steatosis has been reported to occur in up to 50% of liver biopsies in patients with chronic hepatitis c virus (hcv) infection.risk factors for steatosis include alcohol abuse and those found in nonalcoholic steatohepatitis: obesity, increasing age, insulin resistance and hypertriglyceridemia. studies have shown an association between genotype 3 and hepatic steatosis in chronic hcv infection, and others have found an association between steatosis and fibrosis stage. previously published studies have been based primarily on cohorts of clinic referral patients and often have not analyzed for confounding variables. we herein report a population-based study of hepatic steatosis in hepatitis c. methods: in a study of 924 alaska nativestamerican indians with chronic hcv infection, 185 patients underwent at least one percutaneous liver biopsy as part of evaluation for treatment. all patients had a positive hcv rna by pcr and were negative for hbsag and hiv. all biopsy slides were reviewed by a pathologist blinded to patient identity, demographic, clinical and biological data. histologic activity was scored using the knodell system and fibrosis using the ishak system. steatosis was graded as 0, 1 (4 so%), 2 (30-65%), and 3 (> 65%). patients were analyzed for a) gender, age, estimated length of infection and bmi at the time of biopsy, b) genotype, current ethanol use, hcv rna within 1 year of biopsy, c) alt within 30 days of biopsy, and d) histologic activity and fibrosis found on biopsy, using univariable and multivariable analysis. results: steatosis was found in 79/185(43%) of liver biopsies; 62 (33%) had grade 1; 13 (7%) grade 2, and 4 (2%) grade 3. genotype distribution was as follows: 114 genotype 1 (62%), 44 genotype 2 (24%), and 27 genotype 3 (14%). steatosis was found in 39% (441114) of genotype 1,48% (21/44) of genotype 2, and 52% (14/27) of genotype 3 patients (p = 0.34). grade 2 or 3 was found in 6% (7/114) of genotype 1, 11% (5/44) of genotype 2, and 19% (5/27) of genotype 3 patients (p = 0.09). there was no significant association found between steatosis and gender (p = 0.93); age at biopsy date (p = 0.09); alt (p = 0.58); histologic activity (p = 0.27), viral load (p = 0.27) and estimated length of infection (p = 0.22). there was a significant association of steatosis with bmi (< 0.01), fibrosis score (p < 0.01) and current ethanol use (p = 0.03). bmi 2 30 occurred in 52% (42/81) of genotype 1, 39% (11/28) of genotype 2, and 27% (6/22) of genotype 3 patients (p = 0.10). after controlling for bmi, there was no significant relationship found between steatosis and genotype. for bmi < 30, 23% (9/39) of genotype 1, 47% (8/17) of genotype 2, and 50%(8/16) of genotype 3 patients had steatosis (p = 0.08). for bmi 2 30,60% (25142) of genotype 1,55% (6111) of genotype 2, and 67% (4/6) of genotype 3 patients had steatosis (p = 1.00). multivariable analysis was performed on 5 variables (p 0.25 in univariable analysis) and genotype. these included age (categories < 30,30-40,40-50, so+), bmi (< 30,30+), current ethanol use (yes, no), ishak fibrosis score (0-1, 2 21, and estimated length of infection (5 10, 11-15, 16-25, 25+ years) . a significant association with steatosis was found in 3 variables: ishak fibrosis score 2 2 (or 4.2, 95% ci 1.5-11.6, p = 0.005), bmi -> 30 (or 3.7, 95% ci 1.6-8.8, p = 0.003), and current ethanol use (or 2.5, 95% ci 1.0-5.8, p = 0.04) after adjustment for hcv infection length, which itself was not statistically significant (p = 0.82). conclusion: contrary to a number of previously reported studies, this population-based study did not find an association between steatosis and genotype, including genotype 3 in chronic hcv infection after multivariable analysis. we did find that ishak fi-brosis score, bmi and current ethanol use were associated with steatosis after adjustment for hcv infection length, but age and histologic activity were not associated. infection results in necroinflammatory liver disease characterized by the insidious progression of hepatic fibrosis and loss of functioning hepatocyte mass. a cell-mediated immune response with prominent lymphocytic infiltration of liver is thought to play a major role in pathogenesis, although little is known about the molecular mechanism underlying the liver injury associated with this viral infection. on the other hand, non-immune mechanisms have also been proposed as another mechanistic candidate for such sophisticated processes. the recent intensive interest is that the expression of hcv proteins seemingly alters lipid metabolism and transport in the liver in association with the reactive oxygen species (ros) mediated carcinogenesis, although the molecular basis of underlying mechanisms as well as responsible elements of hcv gene for this is yet to be established. thus, the aim of this study was to clarify whether hcv core protein expression directly alters lipid metabolism-relating enzymes to (cause steatosis, especially focusing on nuclear receptors and atp binding cassette transporter proteins expression. methods: 1. the complementary dna clone of full length hcv core (aa 1-191) was derived from serum of hcv i b patient by reverse transcription and nested polymerase chain reaction, and hcv-core expression plasmid (pcag-hcvcore) was prepared by a standard procedure. control plasmid was also prepared with p-galactosidase (pcag-lacz). 2. hepg2 cells were transfected with pcag-hcvcore or pcag-lacz, thereafter harvested for enzymatic triglycerides (tg) assay, hcv core antigen rneasurement by elisa at 24 h or 48 h. also, lipid metabolism-related enzymes mrna expression was estimated by semi-quantified rt-pcr; mitochondrial and peroxisomal fatty acids ,&oxidation, o-oxidation, abc transporters (mdr3, mrp2, bsep), microsomal tg transfer protein (mtp), ldl receptor, and nuclear receptors. resultss: 1. hcv core expression was evidenced at 24 and 48 h at the similar level. 2. hepatic tg was increased in hcv core expressing cells, along with down-regulation of carnitine palmitoyl transferase 1 a(cptla), a precious protein in liver mitochondrial fatty acid@-oxidation (50% at 24 h, 85% at 48) and up-regulation of acyl-coa oxidase 1 (acol), a precious protein in peroxisomal fatty acidso-oxidation (130% at 48 h). cyp4a11, a precious protein in microsomal o-oxidation, and mtp, a precious protein in vldl assembly, were not detected or unchanged. superfamily 2 (sf2) helicases and helicase-like proteins share 6 conserved motifs. alignments reveal that several additional conserved motifs are present in the sf2 helicase encoded by the hepatitis c virus (hcv). the roles of two such motifs are examined here using structure-based site-directed mutagenesis. the first motif (yrgxdv) forms a loop that connects sf2 helicase motifs 4 and 5, at the tip of which is arg393. when arg393 is changed to ala, the resulting protein retains a nucleic acid stimulated atpase but cannot unwind rna, unwinds dna poorly, and does not translocate on ssdna. dna and rna stimulate atp hydrolysis catalyzed by r393a like the wildtype but the mutant protein binds dna more weakly than wildtype both in the presence and absence of a non-hydrolysable nucleotide analogue. thus, this "arg-clamp" motif anchors the protein on nucleic acid enabling processive unwinding. the second motif (dfsldptf) forms a beta-loop between sf2 motifs 5 and 6 that connects domains 2 and 3. when f444 in this "beta-arm" is changed to ala, the resulting protein is devoid of all activities. when f438 is changed to ala, the protein retains nucleic acid stimulated at-pase, but unwinds dna and rna poorly. in this case, uncoupling of atp hydrolysis and unwinding is due to the fact that the f438a mutant does not release dna upon atp binding like the wildtype. the f438a mutant also has a lower melting temperature than the wildtype indicating the hydrophobic pocket formed by the beta-arm and residues in domain 3 stabilizes the protein. data support an inchworm model for helicase action and identify two new potential sites for rational hcv drug design. this work was supported by the aasld liver scholar award from the american liver foundation. tat is an early gene product of hiv-1 that is essential for replication and viral gene expression. this transactivating protein is secreted by hiv-infected cells and taken up by neighboring cells. tat modulates expression of specific cellular genes and may be a key player in the interactions between hiv and other infections. one of the most common coinfections observed among hiv patients is hepatitis c virus (hcv). if hiv tat has an effect on hcv replication, this could help explain the rapid development of liver disease and cancer among hiv patients. aim this study was designed to test the hypothesis that tat alters hcv replication. methods: soluble tat was added to the media of huh-7 cells containing a hcv replicon. replicon replication was quantitated using a ribonuclease protection assay. using an ltr-luciferase plasmid to transfect hela cells, we developed a series of controls outlining the relative oxidative state of the tat protein. results exposure to tat protein in the reduced state substantially increased hcv replicon replication. oxidized tat was found to have no significant effect on the replication of the replicon. the increase was dependant on length of exposure to tat and the concentration of tat. present work: further assays seek to define the nature of the effect by tat on hcv replication. conclusion: tat may upregulate hcv replication directly or indirectly, and thereby play a role in modulation of hcv infection and pathogenesis. elucidating the complex interactions between hiv and hcv will be critical in evaluating and developing workable treatments for the increasing number of coinfected hiv/hcv persons with liver disease. acknowledgements: this research was supported by nih grants ai34764, ai54626, ai54238, ca54576 and ca89121. we have found previously that expression of hepatitis c virus proteins in cell lines or in the liver of transgenic mice inhibits interferon alpha induced signaling through the jak-stat pathway (heim et al., j virol, 1999, 73:8469 and blindenbacher et al., gastroenterology, 2003, 1241465) . the aim of the present study was to investigate if the same inhibition takes place in livers cells of patients with chronic hepatitis c. from february 2001 to april 2002, all patients with chronic hepatitis c referred to the outpatient liver clinic of the university hospital basel were asked for their permission to use part of the liver biopsy for this study. for non-hcv controls, patients that underwent ultrasoundguided liver biopsies of focal lesions (mostly metastasis of carcinomas) were asked for their permission to obtain a biopsy from the normal liver tissue outside the focal lesion. the protocol was approved by the ethical commission of basel. written informed consent was obtained from all patients that agreed to participate in the study. after removal of a 20 to 25mm long biopsy specimen for routine histopathological workup for grading and staging of the liver disease, the remaining 5 to 20 mm long biopsy cylinders were immediately incubated in pbs or pbs with human interferon alpha (lo00 iulml) for 10 to 60 minutes at 37°c. the samples were then used for the preparation of cytoplasmic and nuclear extracts and in some cases for immunofluorescence studies. in a first part, the ex vivo stimulation of biopsy tissue was validated. biopsy samples of patient with different degrees of fibrosis were incubated for 0, 10, 20,30 and 60 minutes, and the nuclear translocation (a surrogate marker for activation) of statl was visualized by immunofluorescence. we found a transient activation of stat1, with a peak after 20 minutes of stimulation with interferon alpha. the signal was shut down in all biopsies after 60 minutes. interferon alpha diffused readily in the biopsy cylinders regardless of the degree of fibrosis. in the second part, 44 consecutive biopsies of patients with chronic hepatitis c and 12 consecutive control biopsies of patients who underwent ultrasound-guided biopsy of focal lesions in otherwise healthy livers (mainly liver metastasis of carcinomas) were used for semiquantitative assessment of interferon alpha induced statl activation using gel shift assays. we found a signhcant inhibition of statl dna binding in patients with chronic hepatitis c compared to controls. as in our previous work with hcv protein expression systems, statl phosphorylation was not impaired in human liver biopsies from patients with hepatitis c. we conclude that interferon induced intracellular signaling can be analyzed semi-quantitatively in human liver biopsies ex vivo by gel shift assays. using this newly validated method, we found that signaling is impaired in liver biopsies from patients with chronic hepatitis c. the block in stat signaling is at the level of dna binding in the nucleus, whereas stat activation at the interferon receptor functions normally. methods we studied the intrahepatic and peripheral hcv-specific cds+ t cell response in a cohort of 15 hla-a2 positive chronically hcv infected patients by tetramer staining, intracel-m a r ifngamma staining and cfse labeling. in addition, viral amino acid sequences corresponding to the four ctl epitopes used in the study were deduced by nucleotide sequence analysis to assess the potential role of viral escape variants. results (1) substantial higher numbers of hcv specific cd8+ t cell responses were detectable in the liver compared to the peripheral blood, suggesting compartmentalization at the site of infection. in addition, intrahepatic cds+ t cells were multispecific whereas peripheral hcv specific cds+ 'i' cell responses were primarily monospecific. these results suggest, e.g., the persistence of hcv-specific t cells at the site of disease or the direct priming of t cells in the liver. (2) epitope-specific cd8+ t cells that were detectable in peripheral blood as well as liver were characterized by a good peripheral proliferative capacity after peptide specific stimulation suggesting that proliferation is a prerequisite of peripheral virus-specific cd8+ t cells to accumulate in the liver. this is further supported by the observation in one patient that the lack of proliferation of virus-specific cd8+ t cells in the peripheral blood was associated with the absence of the same cds+ t cell response in the liver. (3) despite the strong accumulation of hcv specific cd8+ t cells in the liver, the majority of those cells was unable to secrete cytokines after peptide specific stimulation indicating that dysfunction of intrahepatic hcv specific cd8+ ' i cells might contribute to viral persistence. importantly, ifn gamma producing tetramer positive cd8+ t cells were only detectable in patients with low viral titers or in patients with high viral titers but with sequence variations in the corresponding viral epitope, suggesting viral escape. the relative contribution of t cell dysfunction versus viral escape to viral persistence is not known. it is important to note, however, that both mechanisms can operate within the same patient. insulin resistance (ir) is a frequent feature in chronic hepatitis c while risk factors of steatosis are body mass index in patients infected with genotype 1 and viral load in those infected with genotype 3. in patients with chronic hepatitis c, we adressed the following issues: 1) is ir the cause or consequence of steatosis and fibrosis ? 2) what are the risk factors of ir ; 3) does ir play a role (and to what extent) in the occurrence of steatosis ? 4) is ir involved in the progression of fibrosis ? therefore, this study was designed to assess relationships between ir, steatosis and fibrosis according to hcv genotypes in non diabetic patients. methods: 152 non-diabetic patients with biopsy proven non-cirrhotic chronic hepatitis c had fasting serum glycemia and insulinemia measurements. ir was evaluated by using homa model assessement. 4 groups of patients were defined according to hcv genotypes (1 or 3) and degree of steatosis (absent or mild vs moderate to severe). results: the 4 groups were similar in terms of age, sex ratio, bmi and disease duration. prevalence of ir (homa higher than1.64) was significantly higher in genotype 1 patients with steatosis than that of other patients (77% vs 27,23 and 21% respectively, p=o.oool). ir was significantly associated with extensive fibrosis in genotype 1 patients (p=0.008) but not in genotype 3 patients. among genotype 1 patients, independent parameters associated with ir were age (p=0.002) and steatosis (p=o.o3) but not fibrosis. independent risk factors for steatosis in genotype 1 and genotype 3 patients were dr (p=o.o4) and viral load (p=0.02) respectively. steatosis was associated with significantly higher fibrosis score whatever the genotype (p=o.o1). among genotype 1 patients, the median progression rate of fibrosis was significantly higher in those having steatosis and ir together than in other patients (0.1 vs 0.05, p =0.02). conclusions : in non-diabetic patients with non-cirrhotic chronic hepatitis c 1) steatosis and fibrosis are associated with ir in genotype 1 patients but not in genotype 3 patients, 2) among genotype 1 patients, ir mainly depends on age but not fibrosis 3) ir is a major risk factor of steatosis in genotype 1 patients, 4) the combination of steatosis and insulin resistance is a risk factor of fibrosis progression. these results suggest that ir is not the consequence but rather the cause of steatosis and fibrosis progression. . however, in the first month of life, in 4 of these 13 newborns, hcv rna was only detected in pbmcs and not plasma. in 2 neonates, the sscp band patterns of pbmc-derived viral sequences were different from maternal sequences in serum or pbmc; however, they were identical to hcv rna negative strand amplified from mothers' pbmc. the latter strongly suggests that the infection was transmitted through maternal infected pbmcs. another infant harbored different hcv strains in serum and pbmc; both strains were present in the mother's serum. only 7 of 13 hcv-rna positive children developed hcv antibody at one year. hiv infection was found in 2 out of 13 hcv rna-positive and in 6 out the 35 hcv rna-negative infants (ns). conclusions: we have found that 1) hcv strains infecting neonates may be derived from strains residing in maternal pb-mcs. 2) hiv+ pregnant women who were hcv rna positive in pbmcs were more likely to transmit hcv to their infants. these data suggest that one mechanism for perinatal transmission of hcv is maternal-fetal transfusion of hcv-infected pbmcs late in pregnancy or during labor and delivery. additionally, hcv infected neonates may have a limited ability to develop hcv antibodies in the first year of life and thus there may be an underestimation of the prevalence of hcv infection by anti-hcv determination among children born to hcv rna positive mothers. (hcc) in this clinical setting is very high. however, the molecular mechanisms of how hcv related proteins may contribute to hcc is not well defined. hcv core protein, a viral structural protein, has been implicated in tumor development. the goal of this study was to characterize the transcriptional regulation induced by core protein expression with respect to generations of a malignant phenotype. meth0ds:a cdna fragment encoding hcv core protein of l b genotype was subcloned into the ptre2hyg vector and co-transfected with ptet-on vector (clontech) into a human huh 7 hepatoma derived cell line using polyamine (mirus) as a carrier. stably transformed clones were selected by growth in culture medium containing g418 and hygromycin. clones positive for hcv core protein expression were screened by immunoblotting from cells grown in the presence and absence of tetracycline. one clone with core expression tightly regulated by tetracycline was chosen for further analysis. cell proliferation was evaluated by a modified mtt assay, at 6,12,24, 36 and 48 hours after induction of core protein expression. twenty-four hrs after hcv core expression, the cellular transcriptional changes were analyzed by microarray analysis using human genome u133 array sets (affymetrix). the microarray data were validated by real-time pcr on selected genes. results hcv core protein expression was tightly regulated by the presence of tetracycline and the protein levels were dependent on the amount of the tetracycline present in the culture medium. core protein expressing cells showed significant increase in growth compared to non-expressing cells at 6,12, 24,36 and 48 hours after addition of tetracycline indicating a proliferation stimulus provided by core protein. microarray analysis using human gene chip u133 revealed that 1072 of 39,000 genes were significantly changed (> 3 fold), with 626 up-and 446 down-regulated. this screen was quite informative since 52 genes involved in regulation of cell proliferation (38 genes) and apoptosis (14 genes) were changed respectively. conclusions: enhanced growth of liver-derived cells by hcv core protein is due to cellular transcriptional changes induced upon core expression; two major molecular pathway(s) are involved 1) those involved in celi growth control and 2) those involved in cell survival. such gene regulation by hcv core protein is important to the molecular pathogenesis of hcc daudi-cd4 cells were infected with pnl4-3, a t-tropic hiv molecular clone. 24 hours later, uninfected and hiv-infected cells were incubated with either 10% high titer hcv-positive patient serum, 10% hcv-negative patient serum, or 10% fbs (no human serum). infections were continued for 3,4,5, and 6 days; cells were harvested, stained with annexin v and propidium iodide, fixed, and apoptosis was measured by flow cytometry. annexin v is a marker of early apoptosis, and propidium iodide indicates cell death either by apoptosis or necrosis. results: six high titer hcv-positive sera and six hcv-negative sera were tested in five separate experiments, and in each case, the percentage of viable cells was higher in the hivihcv-coinfected cells compared to those infected with hiv only. to further elucidate dynamics of the course of coinfection, we did a time course consisting of harvests every eight hours beginning at day 3 (the earliest point at which apoptosis had been observed in prior experiments) and continuing until day 6 plus 16 hours. the results of the five harvests from day 5 plus 8 hours to day 6 plus 16 hours are shown in the figure below. the percentages of viable cells and those undergoing apoptosis are shown as measured by flow cytometry. during this 32-hour interval the percentage of viable (negative for apoptosis) cells fluctuated very little in untreated cells, or in cells incubated with hcv-positive serum, or even in hn-infected cells incubated with hcv-positive serum. in striking contrast, hiv-infected cells that were incubated with hcv-negative patient serum dropped to only 16% viability by the end of the 32 hours, compared to 64% viability in the hiv-infected cells incubated with hcv-positive patient serum. this effect was not observed when apoptosis was induced by either of two chemical inducers of apoptosis in daudi cells: peroxisome proliferator-activated receptor gamma ligand and calpain inhibitor 11. next, we sought to determine if the effect of apoptosis inhibition was dependent on hcv particles. when h ninfected cells were kept separate in culture from hcv-infected cells by a membrane that prevented virus passage, apoptosis in the hn-infected cells was still inhibited. conclusions: collectively, these results suggest that in our experimental conditions: 1) hcv-positive sera, but not hcv-negative sera provide a protective effect to hiv-induced apoptosis in daudi-cd4 cells, and 2) a cellular factor induced by the presence of hcv, rather than hcv particles themselves, is responsible for the inhibition. methods: serum and pbmc samples from an individual with acute hcv-infection (genotype l a ) were collected every two to three months over a time period of 20 months beginning 2 months after infection. pcr amplification of vrna using a set of overlapping primers for the entire hcv genome was performed and pcr-products population sequenced using an abi automated sequencer. cd8+ t cell responses were defined using an ifngamma elispot assay using an overlapping synthetic peptide set spanning the whole hcv-genome (genotype la). based on the hla-type the complete viral genome was also screened for putative cds+ epitopes using a web based epitope prediction program (syfpeithi). results: elispot screening with the whole hcv-genome peptide set detected only two ex-vivo ifn-gamma responses (ns3 1073-1081 and ns5b 2594-2602), although neither epitope was associated with the development of mutations. however, over the 20 month period of observation we detected a total of 25 mutations throughout the genome (2 in el, 4 in e2,3 in ns2,5 in ns3, 2 in 353a ns4 and 9 in ns5), in addition to multiple changes in the hypervariable regions. interestingly, only five of these mutations resided within known hla-restricted optimal epitopes. however, 17/25 mutations (68%) were located within predicted epitopes based on known hla-binding motifs of the subject. in order to begin to define the rate at which these mutations develop an intermediate time point of 12 months was also sequenced. interestingly, 19/25 (76%) of the mutations had already occurred by this time. it is likely that at least some of these mutations are the direct result of immune pressure exerted through cd8+ t lymphocytes. we are currently generating peptide-specific cds+ t cellines against these regions exhibiting viral evolution, providing an opportunity to determine which of these regions are associated with previously undescribed cd8+ responses. conclusions: hypothesizing that evolving mutations in hcv might be the result of immune pressure by ctl, longitudinal full length sequencing of hcv may represent a powerful tool to define additional hcv-specific cd8+ t cell responses, especially those exerting substantial selective pressure. using this approach we were able to identify a number of candidate regions where cd8+ t cell responses may be present and driving viral evolution. determining the extent to which viral escape from cd8+ responses occurs during hcv infection will elucidate its overall impact in preventing proper control of hcv. indicate that the main open reading frame of hcv contains rna structures in the corelarfp (alternate reading frame protein) and nssb (polymerase) genes. we hypothesized that one or more of these structures are cis-acting replication elements (cre)s. methods. we used custom software, thermodynamic rna folding programs, and classical comparative phylogenetic analysis to build secondary structural models. we then used directed mutagenesis in the subgenomic replicon system to seek stem-loop elements in ns5b that are required for viability. the mutations we introduced maintained the sequence of the polymerase protein, i.e., they were silent, synonymous codon substitutions. structural probing was carried out on replicon rna. rnase t1 and lead (ii), and nuclease v1, were used to identify loops and helices, respectively. results. mutations in ns5bsl3.2 blocked replication, indicating that this novel structure is an essential cis-acting replication element (cre the aims of this study were: 1) to correlate mean alt level with hcv rna by pcr positivity or negativity, 2) to compare clinical, demographic and risk factor data as well as viral load and genotype between persons with pnalt, persistently elevated alanine transaminase (pealt) and fluxuating alanine transaminase (fluxalt) who had at least 3 years of data available. methods: for each person in the cohort from whom 2 2 pcr results were available (n=278), we calculated the mean level of all alts measured over a 3-year period after enrollment. for a subset of the cohort, we selected persons who met the following criteria: 2 positive pcr tests 2 1 year apart with the first positive pcr occurring prior to date of the first alt and a minimum of 6 alt levels measured over the subsequent 3 years with a minimum interval of 1 month between alt measurements (n=129). we defined a person as having pnalt or pealt when 2 5 of 6 alt levels were normal or elevated, respectively, during the 3-year follow-up period. persons who did not fit into either of the above 2 categories were defined as fluxalt. we reviewed clinical data via chart review, demographic and risk factor data via interviews, as well as data on viral load (performed by branched dna assay version 2.0) and genotype (restriction polymorphism analysis background: hcv infection rarely presents acutely, but when it does it offers a potential early "window" for effective therapy. it has been suggested that such therapy is efficacious due to enhanced activity of t cell responses, which are most active during acute disease. methods: 8 subjects with acute hcv infection were comprehensively mapped for cd8+ t cell responses with an interferongamma elispot assay using 301 overlapping peptides, spanning the entire hcv polyprotein. responses were confirmed by establishing peptide-specific t-cell lines and intracellular cytokine staining. the screening for hcv-specific cd8+ t cell responses was repeated during and after therapy. responses detected in the screening assay were longitudinally studied before, during and after therapy using single peptide elispot ,as well as tetramer assays. we also studied cd4 proliferative responses over time in some subjects using recombinant hcv proteins. results: cd8+ t cell responses were detected in 6/8 subjects before therapy was initiated and were typically multispecific, with up to 5 epitopes targeted. after the start of therapy, frequencies of hcv-specific cd8+ t-cells declined following suppression of hcv viremia. therapy also did not increase the breadth of the hcv-specific response as no additional specificities were detected at later timepoints, when elispot screening was repeated. in 5/8 subjects viral relapse occurred after therapy was stopped, with 2 subjects being retreated successfully. relapse was not predicted by the presence or absence of hcv-specific cd8+ t-cell responses. during viral relapse, a vigorous expansion of pre-existing hcv-specific cd8+ t-cells was observed for most specificities, with single responses reaching almost 3% of cd8+ t cells as measured by tetramer staining. these responses were unsuccessful in containing hcv. in the two subjects who were retreated after viral relapse, we observed the identical pattern of declining cd8+ t cell responses as in the first treatment course. in contrast to cd8+ t cell responses, cd4 proliferative responses usually became more vigorous after virus was suppressed on therapy. however, such responses also did not protect from viral relapse. conclusions: these data suggest that although multispecific functional cd8+ t cell responses may be present during acute disease, this does not predict a successful outcome of antiviral therapy. rather than boosting cd8+ t cell responses, therapy and viral suppression appear to lead to their attenuation, even though cd4+ t cell responses may be restored. figure) in the fibrous septum of portal tract in cirrhotic liver. significantly less fltl immunopositivity occurred in nd liver. hepatocytes were negative for fltl. conclusion: the greater de of genes involved in immune activation, fibrosis, cellular proliferation and cell signalling indicated that these processes are more active in the cirrhotic liver that has developed hcc than the cirrhotic liver without hcc development. plgflfltl signalling has an important role in neo-vascular development within organs. the decreased expression of plgf and its receptor flt 1 in cirrhosis with hcc implies that vascular proliferative signals although normally active in cirrhosis itself may be decreased in cirrhotic livers with hcc. prospective analysis of cirrhosis prior to hcc development is needed to indicate whether these findings represent a premalignant phenotype. background: hepatitis c virus (hcv) infection often leads to chronic liver diseases including liver cirrhosis and hepatocellular carcinoma (hcc). at least 10 hcv proteins have been identified, which serve as viral structural and non-structural proteins required for viral replication and virion formation. several viral proteins have been implicated in hcv persistence and the development of hcc. we have previously reported that the ns2 protein inhibits gene expression f+om various cellular and viral promoters in liver and non-liver derived cells. thus, expression of endogenous cellular proteins was significantly reduced in ns2 expressing cells. in this regard, the ns2 protein was recently found to be an inhibitor of the pro-apoptotic cide-b protein and therefore may contribute to hepatic oncogenesis. to understand the molecular basis of repressed gene expression, we constructed successive deletion mutants of the ns2 protein and tested their effect on luciferase expression driven by cmv promoter. meth-ods: the cdnas encoding the full-length (1-217), n-terminal part (1-91) and internal parts of ns2 were generated by pcr and cloned into the pcr3.1 vector for expression under control of the cmv promoter. a liver-derived huh-7 cell line was co-transfected with a plasmid encoding the luciferase reporter gene and plasmid encoding various deletion mutants of the ns2 gene. inhibition of gene expression was measured by luciferase activity assay at day two after transfection. cell viability was also evaluated as well. results: deletion constructs 61-121, 61-111, 71-121 and 71-111 exhibited a significant inhibitory effect on luciferase activity comparable to the fulllength ns2 protein, whereas the deletion constructs 81-111 and 81-121 lost the inhibitory effect. therefore, the minimal amino acid residues required for the inhibition of gene expression by this viral non-structural protein was mapped to residues 71-111. interestingly, this region partially overlaps with the binding site of the ns2 to a newly identified cide-b pro-apoptotic protein. il12 plays an essential role in the antagonism of t helper 2 differentiation and in the induction of antiviral host defence. we postulated that genetically determined attenuation of il12 production could provide a plausible immunological mechanism able to determine outcome of hcv infection and have investigated a recently described polymorphism in the il12b gene. methods: we have extracted genomic dna from whole blood taken from 196 hcv antibody positive patients. 124 patients had chronic hcv with detectable hcv rna and 72 had spontaneously resolved infection, testing hcv rna negative on several occasions. genotyping for a single nucleotide polymorphism (snp) at position (a16974c) on the ill2b gene was performed using polymerase chain reaction and restriction digest. maximal in-vitro il-12 production by cultured mononuclear cells in response to sac (staph aureus cowan) stimulation was determined in 24 cases by elisa. results: of the hcv rna positive cases 81 (65%) were homozygous for the 'a' allele, 39 (31%) heterozygous 'ac'; and 4 (4%) were homozygous 'cc' whereas of the hcv rna negative cases 36 (50%) were 'aa', 33 (46%) were 'ac'; and 3 (4%) were 'cc'. hcv rna negative cases were significantly more likely to be heterozygous for lac' than hcv rna positive cases (p=0.04). of the 24 patients studied for il-12 production, 13 were genotype 'aa' and 11 'ac'. 13 were hcv rna positive (8 genotype lac' and 3 'aa') and 11 hcv rna negative (3 genotype 'aa' and 3 'ac'). maximal il-12 production with sac stimulation was lower in the 11 genotype 'aa' cases (mean 104 units) than in the 13 genotype 'ac' cases (mean 228 units) (p= 0.08). hcv rna positive cases produced significantly less il-12 than did hcv rna negative cases (mean 84 units compared to 266 units, p= 0.016). comparison of hcv rna positive or negative cases only, revealed a trend for higher maximal il-12 production with genotype 'ac' regardless of hcv outcome. conclusion: cases heterozygous for the variant 'c' allele at position 16974 of the il-12 p40 gene are significantly more likely to be hcv rna negative than those homozygous for the 'a' allele. recent data found carriage of the variant 'c' allele to be associated with greater il-12 production capacity and our data from a small number of cases supports this. genetically influenced enhancement of il-12 production appears to be a factor influencing the outcome of hcv infection. we have previously demonstrated that hepatitis c virus (hcv) core protein expression in huh-7 hepatoma cells increased reactive oxygen species (ros) derived from mitochondria without inducing apoptosis. the aim of this study was to investigate whether hcv core protein inhibits the rosassociated apoptosis induced by deoxycholic acid (dca). methods:: we measured ros level and 8-hydroxy-2'-deoxyguanosine (8-ohdg) content, and evaluated apoptosis in the presence or absence of dca (500 pm), using a human hepatoma-derived cell line with tightly regulated hcv core protein expression under the control of a tet-offrm promoter. cells were incubated with 5pm of chloromethyl2',7'-dichlorodihydrofluorescein diacetate for 30 min for measurement of ros. cellular 8-ohdg content was quantified with enzyme-linked immunosorbent assay. fragmented nuclei were assessed with 4',6-diamidino-2-phenylindiole staining and dna fragmentation was evaluated by genomic dna laddering. the degree of apoptosis was quantified with enzyme-linked immunosorbent assay. we also examined whether the general caspase inhibitor (zvad-fmk) inhibited the ros-associated apoptosis induced by dca. in some experiments, cells were incubated with 500wm of ursodeoxycholic acid (udca) in addition to dca. the experiments were repeated 3 or 4 times. results:: strong core expression was detected 72 hours after withdrawal of tetracycline from the culture medium. the expression of core protein increased the basal ros level (1.6 2 0.17-fold, p<0.05) and 8-ohdg content (1.13 i 0.005-f0ld, p<0.05) of huh-71191-20 cells without inducing apoptosis. dca stimulation produced a 4.0-fold increase in ros content in the presence of core expression and a 2.5-fold increase in the absence of core expression. similarly, the 8-ohdg content was significantly increased by dca regardless of hcv core expression (1.13-fold, p=0.02 for core expression, 1.15-fold, p=o.o1 for core non-expression). nevertheless, hcv core protein significantly suppressed the ros-associated apoptosis induced by dca (w0.05). also apoptosis induced by dca was almost completely inhibited by zvad-fmk. udca significantly decreased the ros (p<0.05) and the 8-ohdg content (p=o.oos) in the core-expressing cells and attenuated dca-induced apoptosis. on-treatment virological response (otr) was defined as complete loss or greater than 100-fold drop in hcv viremia within 3-6 months of therapy by quantitative or qualitative rt-pcr (roche cobas amplicor v2.0), based on recommended early virological testing for continued therapy. forty-six patients with at least 3 months of continued antiviral therapy were examined thus far, including 30 with genotype 1 (cl) and 16 with either genotypes 2 or 3 infection (c2). on-treatment response (otr) was achieved in 29/46 (63%) patients overall, including 47% among c1 and 94% in c2 groups. positive and negative control groups included 19 healthy hcv seropositive but nonviremic subjects without history of antiviral therapy ("recovered") and 23 healthy hcv seronegative volunteers, respectively. hcv-specific cd4 t cell response was examined using recombinant hcv core, ns3l4, ns5 and control proteins in standard proliferation and ifn-gamma (ifng) elispot assay at baseline (pre-treatment) and at 1, 3 andlor 6 months during antiviral therapy. results: hcv-specific cd4 t cell response was significantly greater in the recovered compared to the chronic patients, consistent with its expected role in natural hcv clearance. among the chronics, c2 patients (genotype non-1) displayed a baseline t cell response to hcv ns3/4 that was modestly but significantly greater than c1 patients ( the progression of fibrosis in chronic hepatitis c virus (hcv) infection differs among individuals and determines the ultimate prognosis and thus the need for therapy. the molecular mechanisms associated with the progression of fibrosis are poorly understood. gene expression profiling technologies allow the analysis of gene networks whose expression is associated with specific pathological conditions. we used real-time quantitative rt-pcr assays to compare the mrna expression of 148 selected genes in liver biopsies of controls (n=10 normal liver samples) and of 57 untreated patients with chronic hcv infection and different stages of fibrosis according to metavir, i.e. stage flal (n=11), f1a2 (n=9), f2a1 (n=10), f2a2 (n=lo), f3a2 ( n = l l ) and f4a2 (n=6). in order to limit the number of pcr experiments, we first studied total mrna pools which were prepared by mixing amounts of individual liver biopsies mrna of each group. this pooled sample analysis allowed the selection of genes displaying significant different expression (> 2-fold variation) in comparison to "normal livers". the selected genes were then studied at the individual level and their diagnostic performance was assessed using roc curves. the most informative genes were used to construct specific gene expression signatures. we identified several genes specifically involved in various stages of fibrosis. the dysregulated genes mainly encoded extracellular matrix proteases, growth factors, cytokineslchemokines, and ifncu-induced genes. we also observed a statistically significant association between hcv rna amount (as determined with the same real-time quantitative rt-pcr technology) and expression of several genes, mainly ifn-a-induced genes including stat1, ifi27, mix1, oas2 and gip2. understanding the correlates of protective immunity in this setting is an important first step in the development of potential vaccine candidates. methods: two recipients of frozen patellar allograft tissue procured from the same donor developed evidence of acute hcv within 6 months of surgery. in retrospect, the donor was confirmed to be hcv antibody negative but hcv rna positive (genotype la). both patients were enrolled in a prospective study of t cell immunity requiring whole unit blood draw at baseline, 2, 4, 6 and 12 months. comprehensive hcv-genomewide analyses were determined by ifn-y elispot responses to 33 overlapping peptide pools that spanned the entire hcv polypeptide (genotype la, 3010 aa, total 750 peptides). peptide responses were defined as greater than mean plus 3 sd compared to 10 control wells. results: patient 1 (51 yo wf) cleared the virus spontaneously within 6 months; when first evaluated, the pt. demonstrated cd4+ t cell responses to 8 of 33 (24%) peptide pools, with effector frequencies as high as 1 in 5,000 (to ns3helicase-7, amino acids 1569-1667). ifn-y cd8+ t cells were detected following stimulation with 6 of 33 (18%) pools (highest frequency to pool ns5b-5, aa 2829-2927). remarkably, 6 months later, the repertoire of the hcv-specific cd4 t t cell response had expanded further, and patient 1 demonstrated responses to 13 of 33 (39%) pools. in contrast, patient 2 (49 yo wm) demonstrated persistent viremia (4.4 million copieslml, bayer assay) and lacked cd4+ t cell responses to any peptide pools when first evaluated; only one peptide pool (ns3-helicase-5, aa 1369 -1478) elicited responses in cd8+ t cells. despite virologic clearance with antiviral treatment (pegylated interferon and ribavirin), elispot screening failed to reveal emergence of new cd4+ or cd8+ t cell responses (6 months later). conclusions: comprehensive analyses of hcv-genome-wide cd4+ and cd8+ t cell responses reveal significant differences in patients exposed to the same hcv innoculum and correlate with spontaneous clearance versus chronicity. in hcv infection that resolves spontaneously, the repertoire and strength of the hcv-specific immune response may continue to expand in the first year after infection (and may target more than one-third of the hcv polypeptide). in contrast, the profile of t cell responses remains narrow, weak, and constant in the acute infection that becomes chronic, even after successful antiviral therapy. alcohol consumption exacerbates liver injury in chronic hepatitis c and enhanced oxidative stress is one possible pathophysiological mechanism. we have previously developed a hepatoma cell line with conditional, stable expression of hcv core protein and constitutive expression of cyp2e1. these cells demonstrate dosedependent ethanol toxicity when core protein is expressed. the aims of this study were to determine whether reactive oxygen species (ros) production and mitochondrial dysfunction are responsible for ethanol-induced cytoxicity. methods: huh-7 cells with conditional expression of core protein and constitutive expression of cyp2e1 (l14 subclone) were exposed to 0.1 micromolar tbooh and/or ethanol (25 mm) for up to 24 hrs. cytotoxicity was measured by mtt assay or trypan blue exclusion. ros production was assayed with the oxidation sensitive fluorescent dye dcfda. mitochondria1 membrane potential was analyzed by flow cytometry using the dye jc-1 as a probe. apoptosis was detected by cellular dna content analysis with flow cytometry. results: in the presence of 0.1 pm tbooh, there was a progressive increase in cell death in huh-7 cells expressing no heterologous proteins (2 2 2%), cypzel only (12 2 l%), core only(l5 2 5%), or core plus cypzel (3927 x). addition of 25 mm ethanol to core/ cyp2e1 expressing cells further increased cell death to 5326%. dna content analysis and nuclear morphology showed that this type of cell death represented necrosis and not apoptosis. effects on mitochondrial membrane potential were also examined. under control conditions, only 250.5% of cells had depolarized mitochondria. expression of corelcyp2el and exposure to tbooh depolarized mitochondria in 47211% of cells. similar to cell death, ethanol (25 mm) addition increased depolarization to 7027% of cells. compared to control cells, core protein increased ros by a factor of 1.220.3, and the combination corelcyp2el by 2.150.3. furthermore, cells expressing corelcyp2el ampiified the effect of exogenous tbooh. incubation with tbooh (0.1 pm) had no effect on ros content of control cells. it approximately doubled the ros content of cells expressing either core or cyp2e1 and it increased ros content of cells expressing both corelcyp2el by 4.120.5 fold. in all cases the increase in ros was completely blocked by the antioxidant n-acetyl cysteine (20 mm). the antioxidant also completely blocked both mitochondrial depolarization and cytotoxicity. conclusions: hcv core protein and cyp2el synergistically enhance ros production in hepatoma cells, amplify the oxidative stress produced by extracellular ros, and sensitize cells to mitochondrial depolarization and necrotic cell death caused by alcohol. since all these effects are blocked by antioxidants, the formation of ros is likely to be the primary event which subsequently produces mitochondrial dysfunction and cell death. sponse associated with different outcomes of hcv infection may provide important insights into our understanding of hcv pathogenesis. for this purpose, we compared the functional features of hcv-specific cd8 cells in patients with chronic hepatitis c (23 patients, ch), chronic asymptomatic carriers of hcv with persistently normal alt and positive serum hcv-rna (12 patients, as) and in subjects with resolved hcv infection, either spontaneously (8 subjects, sp) or following anti-viral treatment (12 subjects, rt). functional analysis was carried out with 5 highly immunogenic peptides corresponding to ns31073-1081 , ns31406-1415 , ns41812-1821 , ns41992-2000 , ns52627-2635 , containing previously identified hla-a2 restricted epitopes. since the different hcv genotypes were represented in the different groups of patients in different proportions, different sets of peptides designed on genotype 1, genotype 2 and genotype 3 sequences were synthesized and used to reproduce more closely the sequence of the viruses infecting each individual patient and responsible for in vivo priming of the cds response. the immunological parameters tested were: a) frequency of hcv specific, cd8+ t cells by tetramer staining, both ex-vivo and after in vitro stimulation with synthetic peptides; b) ifn-7 production by intracellular cytokine staining; c) cytolytic activity by a standard %hromium release assay. the results of our study indicate that subjects recovered from hcv infection following treatment show lower ex-vivo frequencies of circulating hcv-specific cd8 cells compared to ch patients but their cells are able to expand and to produce ifn-7 more efficiently after in-vitro stimulation. indeed, hcv-specific t cell lines were induced in 83% of rt subjects and in 74% of ch patients; moreover, ifn-7 production was induced in 100% of rt subjects compared to 67% of ch patients (p<0.05). in the group of subjects spontaneously recovered from infection, ex vivo cd8 frequencies were below the threshold of tetramer detection; however, peptide stimulation in vitro was able to expand tetramer+ cd8 cells and to induce ifn-7 production in 37% and 63% of the subjects, respectively. these lower levels of reactivity of sr subjects compared to tr patients are likely due to the different time elapsed from recovery, which was approximately 3 years in tr subjects but much longer in sp subjects, who had recovered even decades before the time of immunological analysis. finally, the group of as patients showed the lowest levels of response to hcv, in terms of both frequencies of hcv-specific circulating cd8 cells and cytokine secretion. in conclusion, the hcv-specific, cd8-mediated response has different features in patients with different outcomes of infection, showing a hierarchy of efficiency declining from subjects recovered after treatment who displayed the best responses, to chronic hepatitis patients, subjects recovered spontaneously and asymptomatic hcv-carriers, who appear to be the least responsive as a likely result of a deeper condition of tolerance to hcv. hepatitis c virus (hcv) infection is the most frequent cause of chronic liver disease in western countries and it has been involved in the development of cirrhosis with the risk of hepatocellular carcinoma. cyclooxygenases (cox) are crucial enzymes in the biosynthesis of prostaglandins and cox-2, the inducible isoform, has been implicated in inflammation, fibrogenesis and carcinogenesis. to address whether cox-2 may play a role in hcvrelated hepatic inflammation and fibrosis, we determined the cox-2 expression pattern in the liver tissue from 27 patients with hcv-induced chronic hepatitis (low histological activity=22, high histological activity=5) and 5 with end-stage cirrhosis, searching for correlations between the expression level of this enzyme and the histological activity of liver disease as well as with the intrahepatic expression and activity of metalloproteinases (mmps). we also investigated whether structural and non-structural hcv proteins may promote cox-2 expression in the human hepatocytederived cell line ccl13. western-blot and rt-pcr analysis for cox-2 demonstrated that cox-2 expression levels were higher in mild chronic hepatitis (mch, 2.6 fold), severe chronic hepatitis (sch, 3 fold) and cirrhosis (2.4 fold) than in normal liver. moreover, pge2 levels were also higher in liver samples from patients with sch (1.9 fold) and in those with cirrhosis (3.3 fold) than in normal liver. besides other cell types, hepatocellular cox-2 immunoreactivity was markedly observed in hcv cirrhosis, and although some cox-2 positive hepatocytes were observed at the edge of hepatic lobules, the majority of them were mainly restricted to the regenerative nodules. in contrast, none or few cox-2 expressing hepatocytes located in periportal areas were observed in patients with mch and sch, respectively. interestingly, we found a significant correlation between the intrahepatic expression and activity of cox-2 and mmp-2 and -9 in all the histological groups of patients analyzed. cox-2 mrna, protein and activity was de novo induced in resting ccl13 cells stably transfected with hcv core and ns5a, and this effect was higher (1 fold and 2.5 fold) when activated with cytokines and phorbol esters, respectively. in conclusion, our results provide evidence that a virus-induced hepatocellular cox-2 upregulation could mediate important pathogenic events in the course of chronic hcv infection, suggesting that specific cox-2 inhibitors might be useful for chemoprevention and therapy of hcv-related liver disease. were nahe to hbv. thl responses to hcv proteins ns3, ns4 and core were sought by elispot. as a control thl responses were also sought to hbsag, where results were analysed according to recovery from hbv infection or nayve to hbv. thl responses to each of the 5 proteins was performed before and after depletion of t-regs using anti-cd25 (>80% depletion). results: following treg depletion thl responses were revealed de novo in 3/12 and enhanced in 8112 patients with ns3,2 and 9 with ns4 respectively and 4 and 7 with hcv core respectively. the overall increase in the thl responses with t-reg depletion was significant for both hcv core (median increase 13-fold p = 0.004) and ns3 (5backaround: in both chronic viral hepatitis b and c viral persistence is thought to be due to an inadequate antiviral t cell response, which in turn may be caused by inappropriate priming of t cells by dendritic cells. an important subset of peripheral blood dendritic cells, the plasmacytoid dendritic cell, has been identified to be the major interferon-alpha producing cell in humans. since both hepatitis b and hepatitis c infection can be successfully treated by exogenous interferon-alpha, an impairment of endogenous interferon-alpha producing cells is an attractive hypothesis for the pathogenesis of chronic viral persistence. methods: patients with acute symptomatic ( n = l l ) and chronic hepatitis c (n=25), sustained responders to interferon-alpha therapy (n=22) and patients with acute hepatitis b (n=lo) as well as healthy controls (n=20) were included in this study. plasmacytoid dendritic cells were stained for facs-analysis in peripheral blood mononuclear cells with antibodies against bdca-2 and cd4 and by exclusion of lineage marker positive cells (cd3, cd14, cd16, cd19). in addition pdc were stained with various activation and maturation markers (e.g. cdso, cd83, cd86). production of interferon-alpha was measured by elisa after stimulation with cpg. results: in acute hepatitis c, ifn-alpha production was about 45-fold reduced as compared to healthy controls (15 pg/50000 pbmc vs. 701 pg150000 pbmc). this reduction was caused by both a significant reduction in absolute numbers of pdc (3.8/~1 vs. 8.4/pl, p=0.0032) as well as by a reduction of ifn-alpha production per cell (0.19 pg/pdc vs. 3.45 pg/pdc; p=0.0004). in chronic hepatitis c, ifn-alpha production was still reduced by over 50%, although absolute numbers of pdc were similar to healthy controls. following spontaneous or treatment induced viral clearance both number and ifn-alpha secretion of pdc were not different from healthy controls. importantly, in acute hepatitis b, which runs a self-limited course in the majority of cases, also the ifn-alpha production was reduced (21 pg150000pbmc). this reduction was also caused by both the reduction of absolute pdc count (5.291~1, p=o.l) as well as by the ifn-alpha secretion per pdc (0.32 pg1pdc; p=0.0007). using a panel of dc activation and maturation markers we did not find any evidence of a more immature or more activated phenotype of pdc in acute hepatitis c. exogenous ifn-alpha administration during acute hepatitis c led to a further reduction of ifn-production by pdc. conclusions: acute viral hepatitis has a dramatic impact on frequency and function of plasmacytoid dendritic cells in the peripheral blood, indicating that pdc play an important role during this phase of viral hepatitis. however, no differences were found between patients with self-limited vs. chronically evolving acute hepatitis c or patients with acute self-limited hepatitis b. future studies have to clarify whether the reduced ifn-alpha secretion by pdc contributes to viral persistence or whether this is rather part of a physiological response to acute viral disease. it has been suggested that hepatocellular steatosis, a frequent histological feature of chronic hepatitis c, is more frequent in hcv genotype 3 infection, and disappearance of steatosis in patients who clear hcv genotype 3 infection after antiviral therapy suggests a possible direct role of this hcv genotype. in order to assess the direct causal role of hcv in steatosis, we studied the relationship between hcv rna load and steatosis according to the hcv genotype. methods: we studied 176 patients with chronic hepatitis c (genotype 1, n = 83 ; genotype 3, n = 93). the serum hcv rna level was measured at the time of liver biopsy by means of a third-generation "branched dna"-based assay (versant hcv rna 3.0 quantitative assay, bayer diagnostics). steatosis was graded as absent, mild (< 10% of hepatocytes), moderate (10 to 30% of hepatocytes) or marked (> 30% of hepatocytes). daily alcohol intake during the 6 months prior to liver biopsy, and the body mass index (bmi), were recorded. results: steatosis was more frequent in patients with genotype 3 infection than in those with genotype 1 infection (84.9% vs 74.7%, ns). steatosis was significantly more severe in hcy genotype 3 than in genotype 1 infection (moderate or marked in 54.8% vs 25.3%, p=o.oool). in patients infected by genotype 3, the severity of steatosis was significantly related to hcv rna load but not to alcohol intake or bmi. in contrast, in patients infected by hcv genotype 1, the severity of steatosis was not related to the hcv rna level but was significantly influenced by alcohol intake and bmi. bivariate analysis demonstrated the effect of the hcv genotype on the association between the severity of steatosis and viral load in genotype 3-infected patients, and between the severity of steatosis and exogenous metabolic factors in genotype 1-infected patients. multivariate analysis showed that : (i, in patients infected by hcv genotype 3, only hcv rna load was independently related to the severity of steatosis (odds ratio (or) = 2.5, 95% confidence interval (ci): 1.4-4.4; p = 0.001), (ii) in patients infected by hcv genotype 1, bmi higher than 28.0 kglm2 (or = 5.8, 95% ci: 1.3-26.9; p = 0.016), alcohol intake exceeding 30 glday (or = 29.6, 95% ci: 3.2-272.0; p = 0.009), and histological grade (or = 20.0, 95% ci: 2.6-152.0; p = 0.001) were independently related to the severity of steatosis. conclusion: our results suggest : (i) steatosis is a cytopathic lesion induced by hcv genotype 3 ; (ii) hcv genotype 1 is not steatogenic per se or at the usual in vivo expression levels, and liver steatosis is a feature of associated steatohepatitis in these patients. the effect of hcv genotype 3 sequences and the role of their expression levc4s must be tested in vitro, in order to better reflect the in vivo situation. in animal models, steatosis is associated with oxidative stress and lipid peroxidation which is known to promote fibrogenesis. this study was aimed to assess in patients with chronic hepatitis c whether steatosis contributes to fibrosis through oxidative stress. methods: markers of lipid peroxidation and antioxidant status were measured in blood from 192 chronic hepatitis c patients and age and sex matched healthy controls. intrahepatic levels of these markers were also assessed in a subgroup of 23 patients and controls. results: the lipid peroxidation marker malondialdehyde was significantly higher in the blood and in the liver of patients compared to controls (p=0.05 and p=o.o01 respectively) while the antioxidant glutathione peroxidase was significantly lower (p=0.05 and p =0.0001 respectively). the antioxidant superoxide dismutase was significantly higher in the blood and lower in the liver compared to controls (p=o.o01 and p=0.002 respectively). autoantibodies to soluble liver antigen (sla) are specific for autoimmune hepatitis. the molecular characterization of the sla antigen has allowed the establishment of highly sensitive and specific radioligand assays. to determine serum anti-sla a specific radioligand assay was used. total rna was isolated and reverse transcribed from hepg2 cells. the cdna encoding sla was amplified by pcr and used as a template to express sla protein eukaryotically in a tnt coupled reticulocyte lysate system (promega corporation, southampton, uk). anti-sla was measured retrospectively in 16 consecutive patients (10 girls, median age at transplant 52 months; range 8-160) with dn-aih, in whom sera were available before transplant, 1,2,6,12 and 24 months post-transplant and at the time of the diagnosis of dn-aih. eight patients had biliary atresia, 2 alagille syndrome, 2 cryptogenic cirrhosis, 2 alpha-1-antitrypsin deficiency, 1 druginduced acute liver failure and 1 bsep deficiency. the patient with acute liver failure did not have pre-transplant serum specimen stored. twelve patients had developed smooth muscle andlor antinuclear antibodies, two anti-liver kidney microsomal (one atypical) and two anti-mitochondria1 antibody. before transplantation, anti-sla was negative in 13/15 cases, the two positive patients having cryptogenic cirrhosis and biliary atresia. post transplant, anti-sla remained positive in these two and became positive in further 9 patients. of these, in 7 patients anti-sla became positive at a median of 17 months (range: 1-115) before the clinical diagnosis of dn-aih. in 5 children anti-sla became detectable as early as 2 months post-surgery. in 6 patients anti-sla levels were highest at the time of the clinical diagnosis of dn-aih. the presence of anti-sla in dn-aih supports the autoimmune nature of this condition; the frequent appearance of anti-sla before the clinical manifestation of the disease makes it a potential predictive marker for development of dn-aih. all 38 patients showed positive response to the therapy, with respect to remarkable release of severe meteorism, active diet, and significant improvement of liver and kidney functions. however, no difference was presented in the markers of electrolytes, blood routine and blood gas analysis before and after the mars, while the effects on serum levels of alanine aminotransferase, aspartate aminotransferase and y-gt were remained uncertain since the alt decreased from 941 ull to 690 ullduring a in conjunction with observations from other centres, our data show that mars treatments resulted in a ramarkable removal of bilirubin, uric acid, bun, cr and ammonia, which could therefore decrease the toxic effects that higher concentrations of these compounds exert on liver and kidney function and could thus contribute to improvements in multiple organ dysfunctions. we found additionally that the higher concentration of serum toxin before detoxication therapy, the more efticacy removal could be achieved which presents mars could be {of therapeutic results even in the very serious cases. it remains questionable whether some of useful substances characted in low molecular weight such as trhlthyrotrophin-releasing factor), gnrh), adh(anti-diuretic hormone and calcitonin will be also removed by albumin dialysis, whether the added synthesis function is necessary for an artificial liver support, and whether this encouraging survival rate applies to long term outcome remains open for further investigation. scoring system has recently been introduced to determine priority for organ allocation in liver transplantation (lt). there is limited available data on resource utilization in the post-meld era.aims: 1. evaluate the effect of the meld system on lt-associated resource utilization and post-lt survival. 2. compare the costs of lt in the periods before and after the implementation of meld. methods : patients undergoing lt at our center in the 6 months following meld implementation(cases) were compared to those undergoing lt in the immediately preceding 9-month period (controzs). the outcome parameters studied were: 1) resource utilization: defined as ( there were no significant differences between the two groups in terms of age, sex distribution or presence of hepatocellular carcinoma (hcc). the average meld among cases was 25.9, compared to 24.1 in controls (p =0.19, ns). overall los was similar in the two groups. however, pre-lt los, especially in the intensive care unit (icu), was significantly higher in the pre-meld or control group. the pre-lt cost was also higher in this group(p=0.05), translating into significantly higher total costs of lt in the pre-meld period (p=o.ol). the need for post-lt hd, pmv and pressors was no different between the two groups. this data is summarized in table 1. the meld score was found to be significantly correlated with bile d u d damage is a maior feature in liver graft rejection. ductopenia (dp), defined as loss of more than 50%-of bile ducts, is a major hallmark of chronic liver graft rejection (cr), which usually leads to graft failure within the first post transplant year. in a previous study we have shown that in patients who developed dp and cr a deficient proliferative response of canals of hering (coh) was present in the preceding biopsies showing acute rejection (ar) when compared with a control group who experienced ar but did not progress to cr. these findings support the postulation that coh represent a regenerative compartment of the biliary unit in the liver. aim of the present studv: to analyze whether preservation of coh might contribute to the reversibility of dp. patients and methods we studied 2 groups of patients. the index group (ig, n=5) developed loss of bile ducts without progression to graft failure due to cr during a follow up of 5 years after transplantation. the second group (crg, n=12) were all cr patients, retransplanted at a median time of 7 months (range 2-19 months). reperfusion (rb), 1 week (1-w), 1 month (1-m) biopsies were studied in both groups. in the crg the last biopsies (lb) before retransplantation taken at a median time of 160 days (range 42-329 days) were also studied. additionally, in the ig the 1& (1-y), 2nd (2-y) and 5* (5-y) annual biopsies were also included. bile ducts and coh were identified by immunohistology using cytokeratin 7. ki67 (mib) was applied to investigate the proliferative activity. the number of bile ducts and coh were counted per portal tract and the number of k67+ cells was counted as ratio of the total number of cells in these structures. clinical follow-up of the ig group was studied based on liver function tests at similar time points as the biopsies. the ig group showed damaged bile ducts and decreasing numbers of bile ducts in the course of time, leading to dp at a median of 3 years. contrastingly, the crg developed dp at a median time of 160 days, observed in the last biopsies taken before retransplantation. when compared with the crg, the ig showed significantly less proliferative activity in bile ducts at 1-w (p=o.o43) but not in rb, 1-m and 1-y, the latter was compared with the lb of the crg. coh showed an initial increase at 1-w in the ig, but a progressive decrease in the subsequent biopsies, reaching significant loss at 5-y (p=0.043). in the crg, progressive loss of coh started at 1-w, leading to a significant loss within 1 year. numbers of coh were consistently lower in the crg at all time points except rb, although not statistically significant. there were also no significant differences in proliferative activity both within the ig in the course of time and when compared with crg. all ig patients continuously showed abnormal liver function tests apart from the bilirubin levels, which were elevated during the first month after transplantation but were normal after 1-y. at 5-y, median serum level of alkaline phosphatase was 228 u/l, gamma glutamyl transpherase was 312 ull, bilirubin was 26 micromoll1 and asatlalat were 96193 ull. conclusion: the ig showed a much slower course of loss of bile ducts when compared with crg, leading to graft survival of more than 5 years. a higher proliferative activity in bile ducts at 1 week in the crg did not prevent the progressive development of dplcr in crg. the initial increase of coh at 1 week in the ig might be responsible for the prolonged preservation of coh in the ig. as coh are believed to represent a regenerative compartment of the biliary unit, our findings indicated that prolonged preservation of coh might contribute to the delayed occurrence of dp but apparently not to reversibility of dp. absence of reversibility of dp is supported by the abnormal liver function tests. (ltx) . recurrent hcv poses a significant and possibly increasing threat to graft and patient survival. diagnosis of acute rejection and alterations in immunosuppression (is) may significantly impact the tempo of post-tx hcv. these associations motivated us to re-examine risk factors for early acute rejection (ear) in a large and contemporary cohort of ltx recipients. methods: the study cohort comprised of 282 consecutive adults undergoing primary ltx between 1/1/99 and 10/31/2002 for chronic liver disease with a minimum of 7 day graft and patient survival. recipients received triple is, typically with steroids, tacrolimus, and mycophenolate mofetil. ear was defined as biopsyproven rejection or treatment for presumed rejection within 6 months of ltx. risk factors for ear were determined by cox proportional hazard methods. spearman rank and phi correlations were used to determine associations between factors. results: 177 men (63%) and 105 women (37%) underwent deceased (n = 236; 84%) or living donor (n = 46; 16%) ltx. the etiologies of chronic liver disease were hcv (n=138; 49%), aih i pbc / psc (n=40; 14%), hbv (n= 34; 12%), cryptogenic (n=27; lo%), alcohol (n=24; 9%), and miscellaneous (n=19; 7%). our overall incidence of ear was 45% (126 i282 recipients); 118 recipients (94%) had biopsy-proven rejection while 8 recipients (6%) were treated for rejection without histologic confirmation. cox univariate models showed that hcv, female gender, meld 2 28, year 2000 compared to year 1999, and lower day 5-7 tacrolimus (tac d5-7) level were positively associated with ear while asian compared to caucasian ethnicity was negatively associated with ear (table la) . factors such as recipient and donor age, recipient african american or hispanic ethnicities, donor type (deceased or living), other years (2001 and 2002) , child's score and class, pre-ltx icu location, and cold and warm ischemia times were not significant risk factors. cox multivariate models showed that hcv diagnosis and female gender remained independent and significant risk factors for ear (table lb) . hbv etiology and asian ethnicity were significantly correlated (phi coefficient 0.53; p = <0.0001) and thus, did not remain independent risk factors in multivariate analysis. while lower tacrolimus level tended to predispose to ear, meld 2 28 and year 2000 were no longer associated with ear. lower tacrolimus level was significantly correlated with both higher meld score (spearman rank correlation -0.34; p = 0.0000) and later transplant year (spearman rank correlation -0.17; p = 0.019). conclusions: hcv etiology of liver disease is strongly associated with ear after ltx. risk of ear is higher for hcv than etoh, cryptogenic, and hbv etiologies and comparable to aih / psc i pbc etiologies; this effect is independent of gender, ethnicity, year, meld, and post-ltx is. recipients with high meld scores are also at increased risk for ear, at least partly because of lower post-ltx is. it is unclear whether the strong association of hcv etiology with ear is because hcv infection results in an immunologic environment predisposing to ear or whether we are unable to accurately diagnose rejection in the setting of hcv recurrence. aim: although the donor pool has expanded in response to increasing waiting lists the quality of donor livers has suffered as a result. it remains unclear whether such marginal organs can be safely used in high-risk recipients or whether they should be implanted solely into good recipients. we aimed to define recipient selection criteria for implantation of these grafts. methods: a prospectively collected database containing 397 patients who underwent orthotopic liver transplantation between 1994 and 2002 was analysed. donors were scored using a formula derived by logistic regression that identified 3 covariates (graft steatosis, donor age and cold ischaemia time) that independently correlated with primary graft dysfunction. this enabled them to be stratified into either marginal or non-marginal groups. using logistic regression analysis, recipient factors independently correlated with 1-year post transplant survival in the marginal group (recipient age, plasma albumin and urea) were built into a mathematical model and each patient was given a score accordingly. patients with scores higher than the defined optimum cut-off value were considered as high-risk and the others with lower scores as low-risk recipients. outcomes were then evaluated in these subpopulations results: marginal and non-marginal donor groups consisted of 65 and 332 patients respectively. the recipient score derived from the multivariate analysis was as follows : score = 1.989 x plasma albumin+ 1.22 x age group + 2.076 x urea, with the recipient albumin level coded as 1 for < 3.1 gldl and 0 for 2 3.1 gldl, the recipient age group coded as 2 for > 55 years, as 1 for 43-55 years, and as 0 for 22.4 mgldl, and as 0 for 5 22.4 mgldl. the roc curve analysis showed that the score had a good discriminating power (area under the curve 0.78, p =.001) and the ideal cut-off value demarcating high-risk from low-risk recipients was 3.25. therefore the recipients were classed as high-risk if they had a plasma albumin level below 3.1 gldl with either an age of over 55 or a urea level of above 22.4 mgldl or when they had a urea level over 22.4 mgldl with an age of over 42. of the 65 recipients in marginal group, 28 (43%) were classified as high-risk and 37 (57%) were classified as low-risk. there was a huge 1-year survival difference between high-risk and low-risk recipients (60.7% and 91.9%, respectively, p <.0001 background single-center experience with pretransplant use of rabbit anti-human-thymocyte globulin suggests that despite early depletion of lymphocytes, a third of all pediatric liver recipients develop early rejection, while the remainder demonstrate clinical graft adaptation. purposelmethods: to identify potential mechanisms, 31 pediatric liver recipients, median age 7.8 years, median followup 8 months, received pre-and post-transplant measurements of 1. whole blood concentrations of tacrolimus (tac), 2. interdose changes in mitogen-stimulated t-and b-cell responses (slr), 3. dendritic cell (dc),and peripheral blood mononuclear cell subsets, and 4. cd8+28-suppressor effect on donor antigenpresenting cell types (cd34fmonocytes and cd19+ b-cells). all patients received ratg preconditioning with a total dose of 5 mglkg in two divided doses. the first dose was given before liver transplantation (ltx). maintenance agent was tac without steroids. in 9 patients this was replaced with sirolimus (srl). mitogen-stimulated t and b-cell responses were compared with those seen in a historical population, who had not received ratg pretreatment. results: all measurements were performed at a median interval of 47 days (22-57 days) after ltx. 1. in slr, the frequency of t-cells expressing the cytokines ifn-g, tnf-a, and il-2 decreased with increasing total exposure (auc) to tac in historical controls (n=6). this relationship was markedly dampened in ratg patients (n=15), as suggested by slopes of 0.0023, 0.032, and 0.0006 relating ifn-g, tnf-a and il-2 on the y-axis to tac auc. 2. significant decrease in cd4 absolute counts at 1 week and partial reconstitution at 2 months after ratg pretreatment (mean pretx-3120 vs 1 week-1920 vs month 2-2117 celllmm3). during this period, monocytes and b-cells remained stable. 3. dc2 remained unchanged, while dc1 frequency increased significantly, from 0.15 to 0.23, p=0.05. 3. in coculture experiments, purified cd8+28-subpopulations from two recipients receiving minimal tac doses induced decreased cd86 expression in donor apc, but increased its expression in hla-mismatched apc. eleven of 31 subjects experienced rejection, while 18 subjects are being maintained on daily (n=13) or every other day (n=5) doses of tac or srl. conclusions: despite lymphocyte depletion, rejection in the setting of t-cell anergy can be explained by relative sparing of antigen-presenting cells, which can recruit alternative effector mediators. the appearance of donor-specific cd8 + suppressor cells in recipients on low immunosuppression suggests that these conditions may also foster a favorable immunomodulatory response toward the liver allografts. (dropout) . the objective of this study was to evaluate the impact of the hcc-adjusted model for end-stage liver disease (meld) organ allocation scheme on the intention-to-treat outcome. under the meld scheme, patients with hcc meeting the united network for organ sharing (unos) t1 (single lesion under 2 cm) and t2 (single lesion between 2 to 5 cm, or 2 to 3 lesions none exceeding 3 cm) criteria were eligible for an initial meld priority score of 24 and 29, respectively. they were also entitled to an increase in meld score, corresponding to a 10% increase in mortality, for every 3 months on the waiting list. methods: excluding patients undergoing living-donor liver transplantation, we prospectively evaluated 102 consecutive patients with hcc listed for olt since january 1998. the kaplan-meier probabilities of olt and dropout among 44 patients with hcc listed under the meld system between february 2002 and january 2003 were compared with 58 patients listed between january 1998 and january 2002 under the previous system of organ allocation. follow-up in the pre-meld group was censored on february 27,2002, when the meld system for organ allocation was implemented by unos. for patients under meld, follow-up was censored on february 27,2003, when further refinements of the meld policy for hcc were made. results: the baseline characteristics were not significantly different between the two groups. eighteen of 44 patients (41%) in the meld group and 26 of 58 patients (45%) in the pre-meld group received chemoembolization or various ablation treatments before olt (p=0.89). all patients in the meld group met t1 (5 patients) or t2 criteria (39 patients). in the pre-meld group, 6 patients had hcc stage exceeding t2 but meeting our proposed expanded criteria (single lesion not exceeding 6.5 cm or no more than 3 lesions none greater than 4.5 cm with total tumor diameter not exceeding 8 cm) by the time of olt. the kaplan-meier cumulative probabilities for olt at 3, 6, and 8.5 months of longest followup were 22.5%, 64.0% and 88.0%, respectively, in the meld group, versus 17.2%, 24.7%, 35.8%, and 47.2% at 3, 6, 9, and 12 months, respectively, in the pre-meld group (p=0.0006). under meld, none of the 5 patients with t1 lesion had received olt. the cumulative probabilities for olt for the 39 patients with t2 hcc in the meld group were 25.5%, 69.2%, and 89.2%, respectively at 3, 6, and 8.5 months (p=o.oool versus the pre-meld group). the cumulative probability of dropout was 5.6% at 8.5 months of longest followup without olt under meld, whereas the cumulative probability of dropout increased from 7.2% at 6 months to 37.8% at 12 months in the pre-meld era. the difference did not reach statistical significance (p=0.74) largely due to low dropout rates in the first 6 months for both groups. among the patients who received olt, 3 of 22 patients in the meld group versus 9 of 25 patients in the pre-meld group had pathologic hcc stage in the explant exceeding t2 criteria (p=0.78). unfavorable histologic tumor features in the explant, including either poorly differentiated grade or microvascular invasion or both, were observed in 4 of 22 patients in the meld group versus 5 of 25 patients in the pre-meld group ( i " 1.0). the short duration of follow-up under meld precluded comparison of intention-totreat survival or hcc recurrence between the two groups. con-clusion: the hcc-adjusted meld system significantly improved the probability of timely olt, and was not associated with selection of a greater proportion of hcc with unfavorable explant tumor histology. given the low probabilities for dropout in the first 6 months following listing for olt even in the pre-meld era, patients with hcc might have indeed received too high a priority score in the first year under meld. disclosures: nancy l ascher -no relationships to disclose nathan m bass -no relationships to disclose randomized at d7 to receive maintenance is regimen with ciclosporine microemulsion + prednisone (group 1) or without steroids (ciclosporine microemulsion + placebo, group 2) after a 7 days blinded oral steroid tapering period. results : 193 patients were recruited and a total of 174 were randomized at d7 (group 1 = 90, group = m). there was no difference between the 2 groups for baseline characteristics, proportion of patients with hepatitis c (18.9% and 20.2%), or cyclosporine blood levels. the incidence of treated biopsy confirmed acute rejection at 6 months was 24.4% in group 1 and 38.1% in group 2 (p= 0.03), with a trend for higher incidence of grade 213 rejection ( 18.9% vs 28.6% ; p=0.12). this difference was maintained in hcv pos and hcv neg patients. no difference was observed between the 2 groups in terms of adverse events, infections, incidence of hypertension and renal dysfunction. changes from baseline were similar with regards to metabolic parameters. a trend towards a better glucose tolerability was observed, less patients receiving an antidiabetic treatment in the placebo group ( 2 vs 10). conclusion : peritransplant immunosuppression with basiliximab, cyclosporine microemulsion, and steroids resulted in excellent low rejection rates in lt patients. early steroid withdrawl strategy at day 14 is not supported by the results of this study, which showed an higher incidence of acute rejection and a trend to more severe acute rejection, only balanced by a trend to a lower need of antidiabetic treatment. to determine if an association exists between meld score and post transplant graft loss within 3 months. methods from 2/28/02 through 10/31/02,2,745 patients underwent liver transplantation, 92% of whom were followed for a minimum of three months. cox regression analysis was utilized to assess the relationship between meld score and post transplant outcome. transplantation. seventy one percent of patients were positive for autoantibodies: 52.9 % were positive for anti-nuclear antibody, 23.5% for anti-smooth muscle antibody and 0% for anti-liver kidney microsomal antibody. the average alt elevation was 224 (range 61-486) and ast elevation 166 (range 52-346). an elevated ggtp was seen in 70.6% of patients. all patients were hepatitis c pcr negative. at the time of diagnosis, 12 patients were being treated with cyclosporine, 5 with f'k506, and 7 with steroids in addition to calcineurin inhibitors. after diagnosis, all patients received standard therapy with l-zmg/kg of steroids. in addition, 89% (15117) began hthioprine and 24% had their calcineurin inhibitor dose decreased. after a mean of 2.6 years of follow-up (range 0.53-5.93), 59% remain steroid dependent and 12% are off steroids. one patient was re-transplanted for biliary complications and 1 patient required conversion to sirohus. conclusion: diagnosis of de novo am requires a high index of suspicion as auto-antibodies are often negative. steroid therapy, often with the addition of azathioprine, is effective in controlling disease. many patients, however, become steroid dependent in order to remain in remission. background. liver allografts have an improved outcome compared with other solid organ grafts, and rodent studies have suggested that activation-associated lymphocyte death may play a key role. studies from our laboratory have shown increased levels of apoptotic lymphocytes in human liver grafts compared with renal grafts and native liver. although the levels of apoptosis did not differ significantly between those who developed acute rejection (rej) and those who did not (nr), the earliest timepoint studied was 3 days post-olt. we hypothesized that the very early postoperative period (within 48 hours) would reveal a relationship between leukocyte apoptosis and rejection. aims. the aim of this study was to determine the amount of early leukocyte apoptosis and its relationship with the degree of lymphocyte activation, subsequent rejection status and degree of donor cell chimerism. methods. peripheral blood mononuclear cells were isolated from 76 patients undergoing olt and were collected on the day prior, 5 hrs after reperfusion (day 0) and 24 hrs post-olt (day 1). dna and rna were prepared and real-time pcr used to quantify apoptosis (ligation-mediated pcr), lymphocyte activation (il-2, ifn-gamma, il-10, cd40 ligand, using gapdh as an internal standard) and donor cell chimerism (y chromosome dyz3 or donor-specific drb1). results. the mean level of circulating apoptotic cells in day 1 recipient pbmc was higher than healthy controls (0.9% t-0.2 vs 0.2% ? 0.1, ~~0 . 0 1 3 ) . apoptosis was greater in nr (1.1% 2 0.3) compared with rej (0.3% t-0.1, p=o.o21). on day 1 the pbmc from nr had increased expression of ifn-gamma (p=0.006), il-10 (p=0.016), cd40 ligand (p=0.02) and il-2 (trend) compared with rej, despite no difference in lymphocyte counts. donor cell chimerism on day 1 did not differ between the groups indicating that this was unlikely to account for increased leukocyte apoptosis in the nr group. interestingly, the level of chimerism 5 hrs postreperfusion (day 0) was significantly higher in nr (3.8% f 0.6) compared with rej (1.2% t-0.4, p=0.004) and there was a close correlation between chimerism on day 0 and pbmc cytokine expression on day 1 (r=0.58, p=0.006). conclusions. patients who did not experience rejection had a paradoxical increase in markers of lymphocyte activation at day 1, in association with higher levels of leukocyte apoptosis. this suggests that recipient cell death may have a role in graft acceptance and reduced acute rejection in human olt. the higher donor cell chimerism seen in non-rejectors implicates the passenger leukocytes in the process of heightened cell death. disclosures: andrew clouston -no relationships to disclose wenyi gu -no relationships to disclose julie r jonsson -no relationships to disclose elizabeth e powell -no relationships to disclose daina m vanags -no relationships to disclose amadeo marcos, bridget flynn, paulo fontes, thomas cacciarelli, wallis marsh, michael devera, obaid shakil, noriko murase, anthony demetris, john fun% thomas e stanl, university of pittsburgh, pittsburgh, pa the seminal mechanism of organ engraftment is thought to be immune activation-dependent clonal exhaustion-deletion. the conventional use of heavy immunosuppression may depress the initial step of donor-specific activation to the extent that the treatment is anti-tolerogenic. to avoid this pitfall, we have applied 2 therapeutic principles in management of 78 adults cadaveric liver recipients transplanted between 9/2002-5/2003. the treatment principles were, first, host conditioning prior to transplantation, and second, minimum post-transplant immunosuppression. the host conditioning was done with one gram of methylprednisolon and a single infusion of 30 mg of alemtuzumab, completed before liver revascularization. there were 49 males and 29 females in the group with mean age 52.3110.3 years (32-73). main causes of liver disease were hepatitis c (40%), cholestatic liver disease (18%), alcoholic liver disease (22%). daily post-transplant monotherapy with tacrolimus (trough target 10 ng/ml) was started 12-24 hours postoperatively (starting tacrolimus dose: 10.5+5 mg, median 10). because of suspected neurotoxicity, 5 patients were switched to cyclosporin. in addition sirolimus monotherapy was substituted for tacrolimus in one patient for management of nephrotoxicity. immune activation diagnosed by liver function tests or by mild or equivocal rejection in liver biopsies frequently was not treated, and tended to resolve spontaneously. clinically and pathologically significant rejection was treated with a bolus of methylprednisolone and/or a 30 mg dose of alemtuzumab. nine patients (11.5%) died 5 sepsis; 2 pnf; one coagulation disorder (hypercoagulable state), and one congestive heart failure. of the 69 surviving recipients four experienced rejection during the first two months which was readily reversed with a bolus of methylprednisolon. after demonstrating the absence of immune activation in liver biopsies at 120 days post-transplantation, weaning from monotherapy was initiated in 49 patients (with the aim of completely stopping treatment in selected cases). seven patients experienced one episode of rejection that was reversed with single dose of methylprednisolone and/or infusion of 30 mg of alemtuzumab. presently, 14 patients are on once a day monotherapy, 14 patients on every-other-day, 10 patients on three timedweek, 10 patients on twicelweek and one patient on once-a-week immunosuppressive regimen (latest tacrolimus dose 5.7t3.5 mg, median 5.5). the patients on cyclosporin and sirolimus are also following the same stepwise weaning pattern. no cases of new onset diabetes, renal failure or hypertension was found in these patients. mean serum creatinine level in the study group at the time of transplantation was 1.020.4 mgldl and at the latest follow up 1.220.3 mgldl. cmv infection was seen in 22% of the patient population but was easily treatable with antivirals. no ptld was seen during the follow-up period. hepatitis c recurrence was seen in over 70% of the patients. response to anti-hcv therapy in this group, after reduction of immunosuppression and especially during the weaning process was promissing. conclusion: by applying the principles of immunosuppression outlined above, it has been possible to drastically reduce the amount of total immunosuppression relative to any (of our) pre-vious experience with liver transplantation. to see less side effects of chronic and high dose immunosuppressive therapy, and probably to have a chance to get a better response to treatment in our hepatitis c population. the results suggest the possibility of systematically achieving drug-free tolerance after liver transplantation. background:end stage liver disease as a consequence of hepatic sarcoidosis is an uncommon indication for liver transplantation (lt). consequently, there is a paucity of information on the pre-lt findings and postoperative course of individuals transplanted for hepatic sarcoidosis. the purpose of this study was to evaluate our experience with lt for sarcoidosis. methods: cases were identified by review of the mount sinai hospital lt database. patient records were reviewed; including the pathologic analysis of the hepatic explant, to confirm that sarcoidosis was responsible for liver failure. for each case, two control patients with other causes of liver failure matched for age, gender and date of transplant were selected. data collected encompassed a mean follow up period of 5 years (range 1-8 years) post-lt. two-tailed student's t-test was used for comparison of continuous data, two-tailed fisher's exact test for comparison of categorical data and log rank test for comparison of survival d,ita. results: hepatic sarcoidosis was the indication for lt in 7 of 2016 adult-lt (0.3%) performed from september 1988 -june 200.3. the mean age at transplant was 56 years and 4/7 (57%) were males. the diagnosis of sarcoidosis was established by findings of extensive, non-caseating granulomas in pre-lt biopsy specimens or in the native liver explant. in 2/7 cases, sarcoid had been previously diagnosed by biopsies of lung parenchyma or mediastinal lymph node. 6 of 7 patients were ama negative. the sole exception was a patient with an ama titer of 1:320 in whom sarcoid was still considered the most likely diagnosis based on liver biopsy findings of granulomas present in both portal and lobular areas and granulomas in a lung biopsy performed 10-years prior to lt. extrahepatic disease was limited to pulmonary involvement in 4 patients with radiographic findings of either interstitial infiltrates (2) or interstitial infiltrates and hilar adenopathy (2). the mean age at transplant and gender distribution were identical in cases and controls. the indications for lt in the control group were: hepatitis c (7), pbc (2) and one patient each with hereditary hemochromatosis, cryptogenic cirrhosis, hepatitis b and laennec cirrhosis. no statistically significant differences between the groups were present with respect to pre-lt levels of ast, alt, alkaline phosphatase, total bilirubin, serum creatinine or prothrombin time. cases and controls had a similar prevalence of ascites and anti-hepatitis b core antibodies. in contrast, sarcoid cases were much more likely to have a diagnosis of diabetes mellitus (100% vs. 21%, p=.002) and less likely to have antibodies to hepatitis c (0% vs. 50%, p=.05). standard orthotopic lt was performed (two control patients received right trisegment grafts). rates of acute cellular rejection were 71% in cases and 43% in controls (p=o.44) with cases experiencing a greater number of acute rejection episodes per patient (1.9 vs. 0.6 episodeslpatient). de novo autoimmune hepatitis developed in one case and one control patient. both de novo autoimmune hepatitis and chronic rejection developed in one case patient. recurrence of hepatic sarcoidosis was diagnosed in two patients at 1.5 and 5.6 years of follow-up. among cases, the one-year graft and patient survival rates were 100% and five-year graft and patient survival rates were 86%. there was no statistically significant difference in five-year graft or patient survival between cases and controls. conclusions: end-stage liver disease as a consequence of sarcoidosis is a rare indication for lt. these patients share many features in common with those transplanted for other indications. despite the small number of patients, a relatively high rate of acute rejection and de novo autoimmune hepatitis was observed in patients with sarcoidosis. recurrence of hepatic sarcoidosis was observed. five-year graft and patient survival rates were comparable to those of patients transplanted for other indications. disclosures: sander florman -no relationships to disclose leona kim schluger -no relationships to disclose kevin m korenblat -no relationships to disclose evan j lipson -no relationships to disclose 440 transplantation. james d eason, ari j cohen, safheesh nair, george loss, ochsner clinic foundation, new orleans, la sirolimus has been used successfully in improving renal function in olt recipients previously treated with tacrolimus and steroids. we report our experience with early sirolimus conversion in steroid-free olt recipients to determine safety and efficacy in patients never treated with steroids. methods: we performed 220 olt over a threeyear period. steroid-free immunosuppression with rabbit atg induction and tacrolimus and mmf was used in 140 of these patients. twenty-five of these steroid-free recipients were converted from tacrolimus to sirolimus within one week to ll months of transplant. results of these patients receiving sirolimus conversion were reviewed. results: renal insufficiency was the reason for conversion in18 patients, while seven patients were converted because of neurotoxicity. six patients were dialysis-dependent at the time of conversion. one-year patient survival in this high-risk group was 80% (20/25) compared to 88% in patients who remained on tacrolimus. the four deaths were in patients converted because of renal failure, three of whom were on dialysis. the incidence of rejection was 31% in sirolimus patients compared to 24% in patients who remained on tacrolimus. rejection was treated by the addition of mmf or reintroduction of low-dose tacrolimus. only one patient required steroids to reverse rejection. conclusion: early sirolimus conversion is safe and effective lawal, sandy florman, isabel fiel, ronald gordon, myron schwartz, charles miller, thomas d schiano, the mount sinai medical center, new york, ny introduction: primary non-function (pnf) fter liver transplantation occurs in approximately 5% of cases and is fatal without timely retransplantation. pnf has been associated with many risk factors, however the etiology remains unknown. some evidence suggests that ultrastructural changes in the liver may be causative. aim: we sought to examine the hepatic ultrastructure of donor allografts in patients with and without pnf using electron microscopy. medical center, over 2000 adult orthotopic liver transplants were performed. patients with the classic clinical presentation of pnf requiring retransplant were identified. archived pre-and postreperfusion donor liver biopsies were examined by electron microscopy in 9 patients with pnf and in 9 matched controls. each pnf case was matched by donor age t5years, gender, cold ischemic time klhour and the donor's cause of death. all biopsies were blindly reviewed by the same pathologist. particular attention was paid to abnormalities of the mitochondria, endoplasmic reticulum and sinusoidal endothelial cells. in addition, the glycogen content of the cells was also assessed. the recipient age and creatinine, as well as the donor serum peak transaminases and bilirubin were compared.non parametric tests with p values <0.05 were regarded as significant using the spss 11.5 program. results: overall, patients with pnf were older (55.7 t 10 vs. 48.99 years, p=0.03) and had higher peak alt levels (141 ? 185 vs. 40 2 32 u/l, p=0.04). there was no significant difference in recipient peak serum creatinine, donor peak serum ast, sodium or donor peak serum bilirubin. in all cases, the endoplasmic reticulum and sinusoidal endothelial cells were ultrastructurally normal. the hepatocytes had variable degrees of glycogen pooling and moderate to severe fatty infiltration. in 7/9 (78%) pnf cases vs. 2ly (22%) control had intramitochondrial crystalline inclusions on pre-perfusion biopsy. conclusion: liver allografts from patients with primary non-function have significant mitochondria1 ultrastructural changes on pre-perfusion biopsies, and may thus have some intrinsic mitochondria abnormalities. introduction: the ideal liver allocation system would allocate donated livers to patients who are most likeiy to die without a transplant, but who have the lowest predicted mortality once they have been transplanted. this mode of allocation assesses the condition of both the recipient and the donor at the time of transplantation. in this study we have constructed a self-organising map (som: this is a neural network or non-linear mode of decision analysis suitable for modelling complex multidimensional relationships) and then validated it by using the som to classify survival in an unrelated population. patients and methods: we have previously described a som consisting of 72 (50 recipient and 22 donor) factors (inputs) constructed from 827 (449 male; median age 52 years; median meld score 16) consecutive primary liver transplants undertaken between 01/01/1993 and 31/07/2002 in the queen elizabeth hospital, birmingham, uk. using a 3 neuron version of this som with three output functions (patient survival at 3 months; 1 year and 5 years) we used the som to classify the post-transplant survival of all primary graft recipients (n=200 patients) over a 5 year period from a north american centre (125 male; median age 48 years; median meld score 16). the birmingham (uk) patients were first classified by the som and then the probabilities of survival (p,) at 3 months, 1 year and 5 years calculated by dividing the number of surviving patients by the total number of patients in neuron i. using this som, each of the wisconsin (us) patients was classified to each of the neurons and the distribution of survival probes for each neuron compared between the two populations using the chi squared test. results: there was no significant difference in the survival probabilities of patients in each neuron when the wisconsin population was compared with the birmingham population. thus, the birmingham som was able to classify successfully the survival of the wisconsin patient population at 3 months, 1 year and 5 years following transplantation by using the same donor and recipient factors ( table 1) . analysis of the inter-neuronal survival probabilities demonstrated that for 3 month and 1 year survival, neuron 3 was associated with a significantly lower survival probability than neurons 1 and 2 (p=0.006 for 3 months; p= 0.006 for 12 months). further, if recipients from neuron 1 received livers from patients classified to neuron 3 by "computer simulated transplantation", a proportion of patients then had a lower predicted survival probability (by som re-distribution to neuron 3). this analysis indicates that patient survival post-transplantation is significantly infuenced by both the condition of the recipient and donor factors at the time of transplantation. conclusions: (1)som analysis provides an efficient and automated method for assessing the probability of survival of individual patients in an unrelated population at 3 months, 1 year and 5 years following liver transplantation. (2)the model not only assesses the pre-transplant condition of the patient, but also considers a wide range of donor factors when predicting the probability of survival post-transplant. (3)this unique resource can match a single liver to a population of recipients most likely to die without a transplant whilst ensuring the best possible post-transplant survival for the most suitable recipient (4)som analysis can also assess the probability of survival of individual recipents matched to a range of possible donated livers when they are being considered for transplant programmes. purpose of study: to assess the efficacy of targeted prophylaxis in patients identified to be at highest risk of if1 after orthotopic liver transplantation. introduction: historically, invasive fungal infection (in) has complicated up to 20% of cases of orthotopic liver transplantation (olt). retrospective analysis has identified a variety of risk factors associated with a high risk for the subsequent development of ifi. these include patients undergoing retransplantation, transplantation for fulminant hepatic failure (fhf), requiring haemodialysis and prolonged intensive care unit (icu) stay. prophylactic anti-fungal therapy is safe and can reduce the incidence of ifl in olt recipientri. in recent years our unit has moved towards targeting prophylaxis to higher risk transplants. we assessed the efficacy of our policy in reducing the incidence of invasive fungal infection. methods: a retrospective audit was conducted comparing two groups of adult olt recipients over two 5 year time periods, 1990-1994 when targeted prophylaxis was not used (group 1) and 1997-2001 when targeted prophylaxis was used (group 2). data were collected with respect to risk factors for ifi, anti-fungal prophylaxis use and development of ifi. results: there was no difference in the overall number of risk factors for if1 associated with olt between the two groups. however, a higher proportion of patients in group 2 had high risk factors for if1 compared to group 1 (p=0.07). there was a significant difference in the use of targeted anti-fungal prophylaxis given to high risk patients in group 2 compared to group 1 (52.3% cf 23.8%, p=0.03), and there was a significant reduction in the number of ifis in group2 compared with group 1 (4.5% cf 28.6%, conclusion: a policy of targeting anti-fungal prophylaxis to highest risk liver transplant recipients leads to a significant reduction in if1 this group. there remains a background incidence of infection in low risk or long term recipients in whom prophylaxis is not given. background: only few studies have described risk factors associated with cellular rejection. the diagnosis of cellular rejection requires histology. there are no data evaluating the absence of rejection in a protocol biopsy population. aim: to assess predictive factors associated for the absence of cellular rejection in a protocol liver biopsy population and to evaluate the usefulness of protocol liver biopsies. method 449 consecutive patients transplanted on a data-base at our centre. protocol liver biopsies were performed between 5 and 10 days post transplantation and then when clinically indicated, during the first 3 months. rejection was scored prospectively. the following variables were examined with respect to absence of rejection over 3 months and in the first protocol liver biopsy: a) donor factors: age, gender, race, donorlrecipient gender match, abo match. b) pre-transplantation recipient factors: age, sex, aetiology, ascites, oesophageal varices, tips, encephalopathy grade, renal support, ventilation, total bilirubin, albumin, inr, ast, alt, creatinine, urea, c) graft and surgical factors: surgeon's visual assessment of graft, cold ischaemic time, use of venovenous by-pass, intraoperative blood transfusion and initial maintenance immunosuppression of either cyclosporin or tacrolimus in triple-dual or monotherapy. standard treatment of rejection was 1 g iv daily of methylprednisolone x 3 days. results: absence of rejection over 3 months was in 69 (15%), mild in 103 (21%) and moderatelsevere in 277 (62%). in the first biopsy: absence of rejection in 92 (21%), mild in 178 (39%), moderate in 159 (35%) and severe in 20 (4.5%). univariate analysis showed for both first protocol biopsies and 3 months, that 4 variables were significantly correlated with non rejection: pre-transplantation use of renal support (haemodialysis or hemofiltration), (p=0.042), cold ischaemic time >15 hours (p=0.030), suboptimal graft visually (p=0.042) and blood transfusion <= 7 units cp=0.008). no correlation was found between aetiology of liver diseases, initial maintenance immunosuppression. conclusion: a suboptimal graft with prolonged cold ischaemic time in recipients with previous need of renal support seems to be associated with absence of rejection. these data are in contrast with previous reports. these factors may be helpful in identifying patients who do not need to be submitted to protocol liver biopsy. tables 1 and 2 1 child developed abnormal transaminases and was found to have chronic hepatitis on liver biopsy and was treated with steroids. 2 required adjusting cyclosporine dosage to maintain trough levels in the identified range (range 65-90 microgramll as per protocol). summary: there was good correlation between the trough levels taken on 2 occasions in the stable paediatric post liver transplant group of patients. the range of c2 peak levels were also similar suggesting good bioavailablity conclusion: this preliminary study on long term post transplant recipients suggests that a peak c2 level is within the range of 280-460 microgramll. background hepatocytes and cholangiocytes release substantial amounts of adenosine triphosphate into bile, where it is rapidly degraded by membrane-bound ecto-atpase and 5'-nucleotidase. this degradation process leads to the generation of adenosine and inorganic phosphate (i?#. whereas adenosine is reabsorbed in a sodium-dependent manner back into hepatocytes, little is known about the fate of biliary pi. in rat, biliary pi concentration is 0.01 mm, which is about 100 fold lower than in hepatocytes (1 mm) and 200 fold lower than in plasma (2.3 mm)9 indicating active reabsorption of pi from bile canaliculi andlor from the biliary tree. aim: the purpose of the present study was to functionally characterize canalicular p, reabsorption in rat liver and to identify the involved p, transport system(s). methods: p, transport was determined in isolated canalicular liver plasma membrane (clpm) vesicles using a rapid filtration technique. identification of putative p, transporters was performed with reverse transcriptase-polymerase chain reaction (rt-pcr) from rat liver total mrna using sodiumlphosphate cotransporter specific primers for napi-iib (gggattgggaaattcatccti ttccaacacaaggttggtca), napi-111 subtype pit-1 (catctcggtgggatgtgcitgttgctctctcctccttca) and napi-i11 subtype pit-2 (gctctaccattggcttctcglaca-gaggaagtgcctggaga)3. on the protein level, phosphate transporter expression was confirmed by western blot analysis in isolated basolateral (blpm) and canalicular (clpm) rat liver plasma membrane vesicles. specific polyclonal antibodies were raised in rabbits against antigenic peptides from mouse napi-iib and human napi-iiilpit-2 showing >88% identity with rat homologues. results: transport studies in isolated clpm vesicles demonstrated sodium-dependent p, uptake (najut > nag 3877 pmollmin; k&t > kg 107 pmollmin). initial na+-dependent p, uptake was linear for at least 6 sec and exhibited a clear overshoot indicating transient intravesicular concentration of p, (secondary active p, transport). initial p, uptake rates (4 sec) were saturable with increasing p, concentrations and exhibited an apparent k,,, value of -11 km. furthermore, sodium-dependent p, transport was higher at an acidic (phout = 6.5; ph,, = 7.4) as compared to an alkaline extravesicular ph (8.0). in addition, an intravesicular negative membrane potential stimulated sodium-dependent p, transport indicating a na:p, stoichiometry of > 1. these data are comparable with the transport characteristics of sodiumlphosphate cotransporters napi-iib, napi-iiilpit-1 and napi-iiilpit-23. mrnas of all these three napi's were found to be expressed in rat liver by rt-pcr. however, on the protein level only napi-iib was found to be expressed selectively in clpm, whereas napi-iiilpit-2 was detected in blpm. no clearcut localization of napi-iiilpit-1 protein could be obtained. conclusions: the canalicular membrane of rat hepatocytes localizes the sodiumlphosphate cotransporter napi-iib, which can reabsorb p, from primary hepatic bile back into hepatocytes. in contrast, napi-iiilpit-2 was found to be expressed at the basolat-era1 hepatocyte plasma membrane. the results indicate that napi-iib regulates p, concentration in bile and may play an important role in the overall p, homeostasis in rat liver backgroundlaims. organic anion transporting polypeptides (oatps) are a family of transport proteins of the basolateral hepatocyte membrane. human oatp8 (slc21a8) is predominantly expressed in hepatocytes and is an uptake system for xenobiotics such as digoxin. the oatp8 gene promoter possesses an inverted repeat (ir1) element at nt -82l-70 relative to the transcription start site, that binds and is activated by the farnesoid x receptorlretinoid x receptor (fxrlrxr). ligands of fxr include bile salts such as chenodeoxycholic acid (cdca), previously shown to activate the oatp8 gene. however, because oatp8 is only a poor bile salt but an efficient xenobiotic transporter, we investigated whether xenobiotics such as rifampicin regulate the oatp8 gene. rifampicin is a prototypic ligand of the xenobiotic receptor pxr (pregnane x receptor). methods. endogenous oatp8 mrna levels in caco2 cells were quantified by real-time pcr. an oatp8 gene promoter construct containing nucleotides -120l +38 relative to the transcription start site (luc-120) was assayed for reporter activity in transiently transfected cells. the fxr binding site in the oatp8 gene (ir1 element) was characterized using the luciferase construct ir1-tk-luc that contained a thymidine kinase promoter under the control of the irl element. results. endogenous oatp8 mrna levels in caco2 cells were induced 7fold by cdca (100 pmolll). interestingly, incubation of cells with rifampicin (10 pmolll) resulted in a 4.5fold increase in oatp8 mrna levels. to study the mechanism of rifampicinmediated induction of oatp8, the promoter sequence was searched for potential pxr binding sites. because the ir1 element, previously shown to bind the fxrlrxr heterodimer, was the only nuclear receptor binding site found, we hypothesized that the induction by rifampicin could be mediated through the fxr element. we, therefore, studied the effect of cdca and rifampicin on the ir1 element. in cells cotransfected with the ir1-tk-luc construct and expression plasmids coding for fxrlrxr, pxrlrxr or car (constitutive androstane receptor)lrxr, fxrlrxr induced the irl element llfold in the presence of cdca and 2.5fold in the presence of rifampicin, indicating that rifampicin is capable of activating fxr. in contrast, pxrlrxr or carlrxr did not activate the ir1 element in the presence of cdca or rifampicin. to confirm that an oatp8 promoter construct is also activated by rifampicin, huh7 cells cotransfected with the luc-120 construct and fxrlrxr expression plasmids were incubated with cdca or rifampicin. cdca induced oatp8 promoter activity -zfold, rifampicin -15fold, confirming that rifampicin transactivates the oatp8 promoter. to investigate whether other xenobiotics also activate the fxr element, huh7 cells cotransfected with the ir1-tk-luc construct and fxrlrxr expression plasmids were incubated with rifampicin, rifamycin, ru486, clotrimazol, phenobarbital or pcn. a 1.4fold (rifamycin) to 2.5fold (clotrima-201) induction of the ir1 element was seen in the presence of these xenobiotics. conclusions. the human oatp8 gene is induced transcriptionally by xenobiotics that represent prototypic ligands of the xenobiotic receptor pxr. however, activation does not occur through pxr but through activation of fxr bound to the ir1 element in the oatp8 gene promoter. the data thus indicate that pxr ligands such as rifampicin and clotrimazol can also activate fxr and thereby induce the fxr-regulated transporter gene oatp8. disclosures: may-britt becker -no relationships to disclose michael fried -no relationships to disclose diana jung -no relationships to disclose gerd a kullak-ublick -no relationships to disclose peter j meier -no relationships to disclose epidemiologie de population epi 106 seema sonnad -no relationships to disclose 428 preservation of canals of hering mitigates bile duct loss in liver graft rejection. ivlarius c van den michael angelis -no relationships to disclose jeffery cooper -no relationships to disclose erick edwards -no relationships to disclose richard b freeman jr -no relationships to disclose ann harper -no relationships to disclose abigail mithoefer -no relationships to disclose no relationships to disclose the data was not available. the ca 19-9, cea, and afp levels were obtained using standard commercially available assays results: out of a total of 108 patients with esld fifty-eight patients fulfilled the inclusion and exclusion criteria. of these, thirty-three patients had evidence of ascites and twenty-five did not. the etiology of liver disease in the two groups was similar. the mean levels of ca 19-9, cea and afp levels in patients with ascites were not significantly different from those without ascites (ca 19-9 48.92 % 65 furthermore, 15 out of these 58 patients with esld had levels -> 37 ulml, the upper limit of normal in this assay unitslml] vs 6.53 5 6.13 conclusions: ascites does not seem to make an impact on the serum levels of ca 19-9 chhaya hasyagar -no relationships to disclose savant mehta -no relationships to disclose 445 severe mitochondrial toxicity after liver transplantation in hiv-hcv coinfected patients liver transplantation (lt) may be the only potentially curative treatment available to these patients at this stage. however its feasibility and benefit has still to be established. severe recurrence of hepatitis c on the liver graft may complicate the post operative course as recently suggested by us and others. mitochondrial toxicity of highly active antiretroviral therapy (haart) may also play an active role on the liver graft of htv-hcv co-infected patients. we aimed to study this complication on the liver graft of hiv-hcv coinfected patients. patients and methods: between he had an history of episode of pancreatitis and haart therapy was azt-3tc-nelfinavir. at m10 post lt, microvesicular steatosis was noted. at m18 a low content of liver mtdna was found (mtdnalnuclear dna = 0.28). 3 other patients had microvesicular steatosis respectively at m2, m4 and m6 post lt. one of these 3 patients had low content of liver mtdna (mtdnalnuclear dna = 0.20). severe defect of complex iv of the respiratory chain was noted in 1 of these 3 patients. no deletion of mtdna was observed by southern blot or long range pcr conclusion: mitochondrial toxicity on the liver graft may become a major problem during post lt c o m e and could worsen graft lesions related to hcv recurrence on the liver graft in hiv patients daniel azoulay -no relationships to disclose henri bismuth -no relationships to disclose denis castaing -no relationships to disclose duclos-vallee -no relationships to disclose cyrille feray -no relationships to disclose michelle gigou -no relationships to disclose catherine guettier -no relationships to disclose philippe ichai -no relationships to disclose claude jardel -no relationships to disclose anne lombes -no relationships to disclose bruno roche -no relationships to disclose faouzi saliba -no relationships to disclose didier samuel -no relationships to disclose elina teicher -no relationships to disclose daniel vittecoq -no relationships to disclose 381a 457 identification of sodiumlphosphate cotransporter type iib marek nowicki, usc, los angeks, ca; jorge rakela, tomasz laskus, there is growing evidence that patients with chronic hepatitis c are more likely to have significant changes in their physical and mental well being, commonly manifested as fatigue and depression, than patients with liver disease of other etiology. recently published studies demonstrated also that hcv infection is associated with cognitive dysfunction. hepatitis c virus (hcv) was reported to replicate in monocyteslmacrophages and lymphoid cells. we have recently demonstrated that leukocytes carry hcv across the blood-brain barrier and we found hcv rna in the central nervous system (cns) in some infected patients (j virol 2002, 76, 10064-10068; 76, 600-608) . however, biological basis for neurocognitive abnormalities observed in hcv-infected patients remains unclear. aim: to determine the pattern of gene expression in cns in hcv-positive patients as compared to hcv-negative controls. material and methods we analyzed samples of brain tissue obtained at autopsy from 3 hcv-positive patients and 3 hcv-negative control patients. all were men of similar age, none was infected with hiv. all 3 hcv+ patients and 2 out of 3 controls had liver cirrhosis. only 2 deaths (one in each group) were liverrelated. the analysis of gene expression was conducted using three different techniques: differential display (genhunter inc), reverse northern, and microarray analysis (bd atlas plastic miroarray). reverse northern analysis was used for confirmation of differential display findings. analysis of microarray data was done using atlas image 2.01 (bd) and cluster 2.20 and treeview 1.60 (m.eisen; ucb). only those genes that were up or downregulated 1.8 times in reverse northern and/or microarray analysis were considered differentially expressed. results: the most striking finding was downregulation of mitochondria] oxidative phosphorylation genes in all hcv-infected patients as compared to controls; impairment of brain oxidative/ energy metabolism has been previously suggested to be the proximate cause of many disorders that impair mentation. another consistent finding in differential display and microarray analysis was downregulation of multiple ribosomal proteins genes and several genes involved in transcription regulation. these could indicate reduced metabolic activities perhaps secondary to deficiencies in oxidative phosphorylation. we also observed upregulation of several genes involved in immune response. there was upregulation of mhc class i and class i1 and several lymphokine receptors like interferon (alpha, beta), il-6, il-9, il-17 as well as lfa-1 ligand intracellular adhesion molecule icam-2 and ceacam1. furthermore, upregulation of cd8o and allograft inflammatory factor-1 gene (aif-1) suggested the presence of activated microglia cells and/or activated macrophages. conclusions: we found downregulation of oxidative phosphorylation genes and upregulation of genes involved in immune response in brains from hcv-positive patients when compared to hcv negative patients. our findings provide the likely substrate for neuropsychiatric symptoms and cognitive impairment associated with hcv infection. medicine, ehime, japan; emmett v schmidt, raymond t chung, massachusetts general hospital, boston , m a background/aims: we previously reported cell-based hcv replication using a novel binary expression system in which cells were transfected with a t7 polymerase-driven full length hcv cdna plasmid (pt7-flhcv-rz) and infected with vaccinia-t7 (pnas 989847). to circumvent vaccinia-induced cytotoxicity, we sought to determine whether replication-defective adenoviral vectors expressing t7 or cell lines stably transfected with t7 could support hcv replication. because vaccinia interferes with pkr function, we further sought to define the antiviral activity of interferon-ar (ifn) in these models. methods: 24 hours after transient transfection of cv-1 and huh7 cells with pt7-flhcv-rz, cells were treated with recombinant replication-defective adenovirus vectors expressing t7 polymerase (ad-t7pol). medium with or without 1000 iulml of ifn was changed at day 1 post-infection and every 2 days thereafter. cells were harvested on day 1, 2, 3, 5, 7 and 9 post-infection. for t7-stably transfected cell lines (huh-t7), we transiently transfected with pt7-flhcv-rz, then added ifn to selected cells in analogous manner. hcv positive and negative strand were measured by strand specific real-time rt-pcr. hcv core protein expression was assessed by western blotting. results: in the hcv replication system with recombinant adenovirus vectors and with t7 stable cell lines, no cytotoxicity was observed at 9 days. with pt7-flhcv-rz and ad-t'lpol, hcv positive and negative strand rna expression was strongest in the first 3 days post-infection and diminished thereafter, but were expressed throughout the 9 days. in the 2 days after adding ifn, hcv positive strand was significantly decreased (0.48520.213 vs. 0.162+0.017 in cv1, 0.123t0.051 vs. 0.035?0.012 in huh7, hcv/ gapdh copy ratio, pc0.05) than the samples without ifn. sustained expression of hcv rna and ifn inhibitory effect was also observed in t7-stable huh-t7 cell lines. pkr expression was strongly induced by ifn, suggesting that pkr is appropriately induced by ifn. conclusions: we have successfully substituted recombinant adenovirus vectors or t7 stable cell lines in our cell-based binary hcv replication system. in these systems, ifn inhibits hcv replication, and upregulates the pkr pathway. these improved binary systems are a durable and more authentic model for identification of host cellular processes critical to hcv replication. disclosures:jason blackard -no relationships to disclose raymond t chung -no relationships to disclose yoichi hiasa -no relationships to disclose norio horiike -no relationships to disclose yoshitaka kamegaya -no relationships to disclose morikazu onji -no relationships to disclose emmett v schmidt -no relationships to disclose the meld scoring system for the allocation of cadaveric donor liver for transplantation gives significant priority to cirrhotic patients with a small, t1 or t2, hepatocellular carcinoma. patients on the wait list were given adjusted meld scores equivalent to 3-month mortality rates of 15% and 40% for t1 and t2 lesions, respectively based on theoretical tumor doubling times. frequently these patients have compensated cirrhosis and hence their intrinsic meld score is lower, yet they are not candidates for surgical resection secondary to the underlying cirrhosis. to assess whether the additional meld points for hcc is valid, we sought to examine the impact of the meld scoring system on a single center's waiting list one year after implementation.methods: records of all patients undergoing liver transplantation (oltx) at uthsc-san antonio from feb 27,2002 through feb 27,2003 were retrospectively reviewed. pathology and radiology reports as well as data from unet were collated for analysis and review. results over a 1-year time period following implementation of the meld system, 126 patients underwent oltx at our center. of these 24 were excluded from analysis because of the following reasons: status 1 (lo), living-related recipient (6), pediatric non-tumor (8). the table below summarizes the pertinent findings. of the 102 remaining patients, 36 (35%) underwent oltx with suspected hcc (4 tl, 32 t2).none of the hcc patients had their meld score downgraded because of tumor progression (t2-t3). at transplant, 2 patients were found to have extra hepatic disease(one with known hcc and the other unknown) and the cases were aborted. sensitivity of radiological evaluation for the presence of hcc was 92.5% and specificity 94%. the calculated meld score at the time of listing for oltx and at the time of oltx was significantly lower for those patients thought to have an hcc. exception points for hcc gave those patients a significantly higher average meld score at the time of oltx than patients without hcc. patients wlo hcc had a aupper; true meld of +3.1 at the time of oltx compared to listing as opposed to hcc patients whose a-upper; true meld was -0.3. during the study period 34 patients (none with suspected hcc) died on the waiting list or were too sick to transplant while 215 new patients were listed compared to 24 dying the prior year with 163 new registrants (15.8% vs 14.7%). mean meld score of those dying during the study period was 22.6 8 (median 22.5) and mean time on the list was 284 421 days (median 99 days). of those patients dying. 16 had a meld score of 1 2 4 . conclusion: meld has effectively prioritized cadaveric donor livers to the sicker patients. however, patients with hcc are given too much priority as evidenced by all of the wait list deaths occurring in patients without hcc and lack of tumor progression (to t3) sufficient to loose meld exception points in those with hcc. likewise, the mean meld score of those dying was less than that given to patients with t1 lesions(22.6 vs. 24). the recent downward adjustments of meld exceptions to 20 and 24 for ti and t2 lesions, respectively, are reasonable. ongoing analysis will be needed to accurately place hcc patients in the current allocation system. there was no significant difference (p>0.05) in weight gain between the sexes, those with a bm1>30 pre-transplant and those with a bmi<30 pre-transplant or those on prolonged courses (>3 months) of steroids. weight gain was significantly greater (p<0.05) in patients aged over 50 compared to those under 50 and those transplanted for chronic liver disease compared with fulminant liver failure. a pre-transplant bmi>30 was a strong indicator that the patient would still have a bmi>30 at 3 years. there was no effect of immunosuppression on weight gain: corticosteroids were discontinued in 59.3% by 3 months and long term use of steroids was not associated with weight gain. weight gain was similar in those on cyclosporine and on tacrolimus.conclusions: a bm1>3o is common in the liver transplant population, but seems to unrelated to specific immunosuppression and may be more closely linked to lifestyle. most weight gain occurs after the first 6 months, and intervention with dietary and lifestyle advice at this point could be implemented to minimise the long-term morbidity and mortality risks associated with obesity. serum tumor marker estimations are often performed as a part of evaluation for liver transplantation. there is a paucity of information on the effect of esld on the levels of these tumor markers making interpretation difficult one tumor marker, ca-125, has been consistently reported to be elevated in patients with ascites from liver disease. purpose:-to compare the levels of serum ca 19-9, cea and afp in patients with esld with or without ascites.-to compare the serum ca 19-9 levels in patients with esld vs normal controls methods: all patients referred for liver transplantation between jan 2000 and dec 2002 at our center were included in the study. men with end stage liver disease (esld) develop a plethora of debilitating symptoms, including severe muscle wasting, that render many of these patients non-suitable for liver transplantation. esld-associated hypogonadism, i.e. testosterone deficiency, may, in part, be responsible for the development and progression of many of these incapacitating symptoms. in general, hypogonadism can be effectively treated with topical testosterone replacement (ttr), which avoids the first pass effect via the hepatic system in contrast to oral anabolic steroids. ttr has been shown to effectively improve muscle mass and overall well being in patients with hiv. however, little is known about the effects of ttr in men with esld. the aim of this study was to determine the potential benefits and safety of ttr in patients with esld and muscle wasting. method:the medical records of liver transplant patients treated for at least 3 months with testosterone gel 1% (5 grams per day) therapy for muscle wasting (mw) from january 2002 to march 2003 were reviewed. information collected included; demographics, albumin, pre-albumin, transferring, testosterone, estrogen, lh and fsh. drug safety results were also collected and included, acute cellular rejection, cholestasis, malignancy, and patient or graft loss. tumor markers for afp, ca19-9, cea, psa, prolactin levels and radiographic imaging was reviewed to rule out and potential malignancy. patients not treated with testosterone but with a similar clinical setting were compared (group 2). results: thirteen patients were identified with esld and mw, group 1 (n=8), group 2 (n=5). demographics were as follows: (group 1) 6 males, 2 females, and mean age 56 years; (group 2) 4 males, 1 female, mean age 53 years. disease etiology for group 1: (hcv (n=5), hcv/hiv (n=l), aih (n=l), cryptogenic (n=l)) group 2: hcv (n=4), hcvilaennec's (n=l). most patients in group 1 stated a subjective improvement in muscle strength, and overall wellbeing. in group 1, serum albumin levels increased from day 1, 30, 90 (2.18/2.7875/3.45 g/dl, respectively) while those in group 2 did not (2.30/2.16/2.0 g/dl) (p= 0.0002). similar results were seen for prealbumin (days 1, 30, 90) group 1 (10/18/22 mgldl) versus group 2 (10/8/6 mg/dl). in group 1, 7/8 patients received a liver transplant, while 1 remains listed for olt. in contrast, 3/5 patients in group 2 were transplanted while 2/5 died. two patients in each group were not listed at the time of evaluation due to extreme deconditioning. of those in group 1, one patient was transplanted and another patient remains listed for olt with an albumin greater than 3.5 gldl. this patient presently has no radiographical evidence of ascites. the two patients of group 2, who were not listed for lt due to extreme deconditioning, died on day 28 and 35, respectively. there was no rise in the tumor marker elevations in any of the patients. after transplantation, no patients in either group suffered from rejection or graft failure. ascites accumulation, as assessed by abdominal ultrasound imaging, appeared to be less in patients receiving ttr (group 1) compared to all 3 living patients in group 2, who had persistent ascites. one patient of the treatment group developed marked cholestasis 10 days after starting ttr and received a liver transplant 14 days later. prior to starting ttr, this patient underwent weekly large volume paracentesis and suffered from marked deconditioning and recurrent bouts of hepatic encephalopathy while maintaining a meld score of 8-9 points over a 6-month period. conclusion: our preliminary data suggest that ttr increases muscle strength (subjective measures), stimulates albumin synthesis and improves the outcome of olt. thus, ttr (testosterone gel 1%) appears to be of great benefit in patients with esld and muscle wasting. further studies are needed to determine the efficacy and safety of ttr in patients with esld. percutaneous ethanol injection (pei), radiofrequency tumor ablation (rf), and laser thermal ablation (lta) are percutaneous techniques used in the treatment of unresectable hcc. percutaneous seeding of neoplastic cells along the needle track is a rare complication of these tecniques, and it has been recently suggested to consider with caution these techniques as a "bridge" treatment in cirrhotic patients with hcc candidates to lt. the aim of this study was to verify whether percutaneous ablation treatments represent a risk factor for hcc recurrence in these patients. during the period 1990 -2002,32 patients (mean age 53 yrs) with hcc complicating liver cirrhosis previously treated with percutaneous ablation treatments underwent lt in 3 italian centers. among the 32 patients, 13 had monofocal hcc, 19 multifocal hcc. in the latter group, 5 had monolobar disease, 14 had bilobar disease. considering the 13 patients with monofocal hcc, 7 were treated with rf (associated with transarterial chemoembolization [tace] in one case), 4 with pei (associated with tace in one case), 2 with rf and pei (associated with tace in one case). among the 19 patients with multifocal hcc, 9 were treated with rf (associated with tace in four cases), 9 with pei (associated with tace in four cases), 1 with lta associated with tace. on the whole, 38 nodules were treated in the 32 patients. furthermore, 31 additional untreated hcc nodules were found at pathological examination of the explanted liver. among the 19 rf-treated nodules, 6 showed total necrosis, 12 partial necrosis, 1 absence of necrosis. among the 15 pei-treated nodules 4 showed total necrosis, 5 partial necrosis, 6 absence of necrosis. the 2 nodules treated with rf and pei showed total necrosis in one case and partial necrosis in the other one while the 2 nodules treated with lta, showed partial necrosis. the mean follow-up period was 35 months (range 3-130 months). five patients died during the follow up and the actuarial survival rate was 94% at one year, 85% at 3 and 5 years. three patients (9.4%) had post-transplant recurrence of hcc which was the cause of death in all cases; the diagnosis of tumor recurrence was made at 2 months from lt (peritoneal carcinomatosis), at 8 months from lt (bone), and at 9 months from lt (abdominal liymphnodes and bone), respectively. the 3 patients had all been treated with pei before lt and no one of them showed recurrence of hcc at the abdominal wall level. according to our results, percutaneous ablation techniques performed before lt do not seem significantly affect both survival and tumor recurrence rate in patients transplanted because of hcc complicating liver cirrhosis. in particular, no cases of recurrence at the abdominal wall level were observed in the overall series and no one case of hcc recurrence was observed in patients treated with rf alone or in combination. background: transjugular intrahepatic portosystemic shunt (tips) is valuable in the management of portal hypertension (phtn) in patients awaiting liver transplantation (olt). recurrent phtn after olt can be refractory to medical management and portosystemic shunting may be considered in rare situations, either as a bridge to retransplantation or as definitive therapy. in this report we review our experience and outcomes with tips after olt. methods: records of 309 primary adult olt recipients, between january 1995 and december 2003, were retrospectively reviewed. evidence of refratory post-olt phtn was noted. those who required tips were the subject of this review. demoraphics, indications for olt and tips, evidence of allograft dysfunction and outcomes after tips were described. results: during the study period 6 tips were placed in 6 patients at a mean of 13.5 months after olt (range 2-36 months). there were 3 males and 3 females, age 53.0l7.8 years. hepatitis c was the primary indication for olt in 5 and primary biliary cirrhosis in 1. indications for tips included refractory ascites (6), variceal bleeding (2) and various degree of associated hepatic vein outflow stenosis (3). five patients had resolution of phtn and 1 patient with refractory ascites had severe hepatic vein outflow stenosis and associated hepatitis c in the allograft. two patients required re-olt for recurrent hepatitis c. there were 3 deaths: liver failure 1 month after tips done in the setting of allograft dysfunction, 3 months after tips with subsequent re-olt and organ failure, and lung cancer 5 months after tips. bridging fibrosis was present in 3 patients, 2 needed re-olt and 1 died from liver failure while waiting for olt. currently, 3 patients are alive without evidence of phtn 3, 9 and 58 months after tips. conclusions: 1-tips effectively and safely controls phtn after olt. 2-tips in setting of a moderate to severe allograft dysfunction does not abrogate the need for re-olt and can be associated with a high mortality. 3-timely re-olt should be considered in the presence of fibrosis from recurrent hcv and phtn. 4-tips has been successful in the setting of mild-moderate hepatic vein outflow stenosis and phtn. introduction: biliary tract complications after liver transplantation (lt) are reported to occur in 13% to 35% of patients. the frequency of biliary infections is not well documented. therefore, we prospectivly obtained bile samples during diagnostic and or therapeutic endoscopic retrograde cholangiography (ercp) in all liver transplant patients from 0112001 to 1112002. methods: in 67 patients (mean age 53.6 years, range 24-73 years) a total of 172 ercp's were performed (2.57 ercflpatient). all patients had a lumen adapted, end-to-end biliary anastomosis. lt was performed between 3.2 months and 2.8 years before the intervention. cholangitis was defined as the presence of cholestasis with clinical and biochemical signs of infection not explained otherwise. only in 19 cases of 172 interventions, positive culture results were combined with clinical signs of cholangitis. the follwoing risk factors were identified stenosis of any type, plastic endoprothesis, choledocholithiasis and previous papillotomy. patients with these risk faktors had significantly higher incidence of positive bile cultures (77.6% vs 40%, p<o,ool; chi-square). in addition, they were more often infected with multiresistant bacteria (p<0.05). 49.3% were resistant to gentamycin, 44.0% to ceftriaxone. vancomycin (1.7% resistance) was the most effective antibiotic. imipenem (18.2%), ciprofloxacin (27.5%) and amoxicillin (28.8%) had intermediate incidence of resistant bacteria. conclusion: in patients with biliary tract complications after lt, biliary infection is common. clinical cholangitis, however, occurs predominantly in those with risk factors. gram-positive bacteria were more commonly demonstrated than gram-negative. cocolonisation with anaerobes andlor candida can occur. ciprofloxacin and amoxicillin seem to be appropriate antibiotics for prophylaxis. selection of an empiric antibiotic therapy for patient after liver transplantation with sings of cholangitis should be directed toward agents with a low incidence of resistance or a combination therapy. disclosures: thomas buratti -no relationships to disclose adolescent health related quality of life after liver transplantation. shikha s sundaram, katie neighbors, lisa amaruso, james sinacore, estella m alonso, northwestern university, chicago, il background: adolescence is an important developmental stage, during which children with chronic diseases begin to assume some of the responsibility for their own medical care. many children who received liver transplants as infants are now reaching adolescence and their unique health perceptions should be considered in providing appropriate healthcare. the aims of this study were to 1) quantify the health related quality of life (hrqol) in adolescent liver transplant recipients and compare this to a published healthy population, 2) compare parental ratings of adolescent hrqol to adolescent self-reports of hrqol, and 3) assess adolescent hrqol and it's relationship to disease specific disability. methods: a cross sectional mailing survey of liver transplant recipients between ages 11-18 years, surviving transplant by at least 1 year and receiving care at our center was conducted. primary caregivers of these patients were also recruited. adolescents completed the child health questionnaire, child form 87 (chq-cf87) and primary caregivers the child health questionnaire, parent form 50 (chq-pf50). demographic and medical data was collected using an institutional transplant database and standard demographic survey. a disease specific disability score was calculated using the liver transplant disability scale (ltds). mean scores for 12 chq-cf87 subscales, including general and mental health, physical and social functioning and self-esteem were calculated. these were compared to published normative values from a healthy population and mean chq-pf50 subscale scores. results: thirty-seven of 53 surveys mailed were received for a response rate of 69.8%. adolescent respondents had a mean age of 14.122.13 years, were 65% female and 81% caucasian. cadaveric donors accounted for 67.6% of transplants and 38% of patients were transplanted for biliary atresia. a majority (86.5%) of respondents had private insurance and 86.5% indicated mothers were their primary caregivers. after transplantation, adolescents had a mean global health score of 59.28518.47 as compared to mean normative score of 66.38214.62, p=0.031. they had higher scores than the normative population for role behavioral, 94.89211.29 vs. 86.542 21.45, p=o.ool and mental health, 77.53z10.81 vs. 72.65215.95, p=0.021. all other scores were similar between the 2 groups. significant positive correlations between adolescent and parental scores were observed in relation to physical functioning, (r =0.64, p<0.0005), self-esteem (r = 0.72, p < 0.0005) and mental health (r = 0.59, p<0.0005). the remaining sub-scales lacked statistically significant correlation. an inverse relationship between inpatient hospitalization during the previous 6 months and mean global health score was observed 0 inpatient days had a mean of 76214.77,l-4 inpatient nights had a mean of 65235.5 and greater than 4 inpatient nights 38.3243.1. there were no other significant correlations between subscale scores and ltds scores. conclusion: adolescent liver transplant recipients report their hrqol as equal or better than the normative population. parents may be adequate proxies for some, but not all domains of adolescent hrqol in this population. were quantitated from wild type and fxrnull mice. results:transfection of the moatp4-pgl3 into hepg2 cells led to a 5.5fold ( 2 1.0 ) increase in reporter gene activity over vector controls. co-transfection with the bile acid receptor fxr and addition of its ligand, chenodeoxyacholic acid (cdca), resulted in a further 6.25 ( t 0.35 ) -fold increase suggesting fxr-mediated transactivation.fxrlrxr cotransfection and addition of ligands, cdca and 9-cis retinoic acid decreased transactivation suggesting rxr-mediated antagonism ( jbc, 278: 10028,'03 ). hnf-1 a also transactivated moatp4 promoter as shown previously. mutation of ir-1 sequence in moatp4 promoter abolished fxr-transactivation. emsa analysis using rat liver nuclear extracts showed specific binding of nuclear proteins to moatp4 fxre which was supershifted by fxr and rxr antibodies. mrna levels for moatp4 in fxrnull mice were reduced by 48.3 % compared to wild-type mice ( 0.612 in wild-type vs 0.296 infxr-null mice). conclusion: based on the above data, we suggest that moatp4 is regulated by bile acids by binding to fxr via ir-1 in its promoter. , and g1249a encoded for amino acid changes i1173f, a1450t, and v4171, in the third membrane spanning domain, the second nucleotide binding domain, and the second membrane spanning domain, respectively. the full-length coding regions of the reference mrp2 and three mutants sequences were inserted into the baculovirus genome. sf9 insect cells were infected with the baculovirus stocks, and membranes were isolated from the infected cells expressing mrp2 or the mutants. atpase activity was measured by determining release of inorganic phosphate by a colorimetric assay. atp-dependent transport of 3h-e23g (estradiol-p-(3p-d-glucuronide)) or 3h-e217g (estradiol-p-(17p-d-glucuronide)) was determined by uptake into inside out membrane vesicles collected on filters and counted by liquid scintillation. results: the total expression levels of mrp2 and the three mutants in the infected sf9 cells were comparable by densitometry of western blots of three levels of each protein, indicating total expression in sf9 cells was not influenced by the mutations. mrp2 atpase activity was significantly stimulated by the uricosuric probenecid lmm, the cholestatic 250pm, and the mild choleretic e23g 1mm. although the control and stimulated at-pase activities of the v417i mutant were not different from mrpz, the i1173f mutant had significantly less baseline and less stimulated atpase activities, indicating impaired function. a1450t had less probenecid-stimulated atpase activity, but activity stimulated by e217g and e23g was not different from mrp2. ursodiol saturably stimulated atpase activity of mrpz by 520% with km =72138pm. although the three mutants transported 3h-e23g with affinities not significantly different from mrp2 (km=122223 pm), the vmax of i1173f was only 5.1% (15414 pmollmglmin) and the vmax of v417i was 157% (4701251 pmollmglmin) compared to the vmax of mrpz (29912186 pmollmglmin); the vmax of a1450t was not different (2967287 pmollmglmin). ursodiol activated transport of 3h-e23g (60nm) with a peak effect of 950% at 100pm; higher ursodiol concentrations (up to 1mm) returned transport to near control values. mrpz-mediated atp-dependent transport of 3h-e217g (44nm) in the presence of ursodiol (1oopm) for the mutants differed from mrp2 (lo%, 67%, and 140% for i1173f, a1450t, and v4171, respectively). conclusions: ursodiol markedly activated mrp2 transport of e23g. although ursodiol has several anticholestatic effects, these data show its direct effect on transport of mrpz substrates, and may suggest activation of mrp2 as a target in the treatment of cholestatic liver disease. the mrp2 mutants showed several important differences in activity, including changes in transport vmax, and transport in the presence of ursodiol, and activation of atpase activity by ursodiol, probenecid, and the mrp2 substrates e217g and e23g. further characterization of these mrp2 mutants and others will lead to a better understanding of mrp2 structure and function and may yield new therapeutic approaches. also, although e217g is an established mrp2 substrate, these data show that its noncholestatic isomer e23g is also an mrpz substrate, implicating mrp2 in the disposition of other estrogen metabolites, which can be interconverted to cholestatic compounds like e217g. argentina; nicholas f larusso, mayo medical school, clinic and foundation, rochester, m n previous work from our laboratory is consistent with an important role for aquaporins (aqps), a family of water channel proteins, in canalicular bile secretion principally via agonist-induced exocytic insertion of aqps into the hepatocyte canalicular membrane. to further define the pathways and molecular mechanisms for water movement across hepatocytes, our aim here was to directly assess osmotic water permeability (pf) and activation energy (ea) in basolateral (blmv) and canalicular membrane (cmv) vesicles using stopped-flow spectrophotometry. scattered light intensity was used to follow changes in vesicle volume in response to gradients induced by sucrose solutions of different osmolarities. results: the time course of scattered light for blmv and cmv fit a single-exponential function. in blmv, pf was 10824 pm.sec-' (25 oc) with an ea of 7.7 kcallmol; in cmv, pf was 8615 pm.sec-' (25 oc) with an ea of 8.0 kcallmol. the aqp blocker, dimethylsulfoxide, significantly inhibited the pf of both basolateral (8124 pm.sec-l; -25%) and canalicular (59?4 pmsec-'; -30 %) vesicles consistent with the presence of aqps in both vesicle populations. experimental data for cmv from dibutyryl camp-treated hepatocytes fit best to a double-exponential curve implying the presence of two functionally distinct cmv populations; one population had pf and ea values similar to those of cmv from untreated hepatocytes and the other population had a very high pf (6552135 pm.sec-l, 25 oc) and very low ea (2.8 kcallmol). dimethylsulfoxide completely inhibited the high pf value in this second vesicle population. in contrast, the pf of blmv was unaltered by camp treatment of hepatocytes. conclusions: our data are consistent with the notion that the canalicular but not the basolateral membrane becomes more permeable to water after a secretory stimulus due to the insertion of aqp molecules. the data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation. disclosures: ariel j caride -no relationships to disclose sergio a gradilone -no relationships to disclose bing q huang -no relationships to disclose key: cord-017675-in9r33ww authors: nan title: the way forward: prevention, treatment and human rights date: 2008 journal: global lessons from the aids pandemic doi: 10.1007/978-3-540-78392-3_9 sha: doc_id: 17675 cord_uid: in9r33ww there now is a considerable body of evidence to support the view that an effective hiv/aids strategy integrates prevention, treatment and human rights. in this chapter, we emphasize the importance of each of these aspects and draw upon the conclusions reached in previous chapters to map out the future of hiv/aids. while medicine and science have a crucial role to play in addressing pandemics, whether slow-moving (like hiv/aids) or fast-moving (like influenza), the social, legal, political, financial and economic ramifications of pandemics can not be ignored. well-considered social, legal, political and financial strategies are essential in order to address any pandemic effectively. united kingdom 1998 kingdom -2004 source: global hiv prevention working group (2007) in chap. 2, we discussed how integrated prevention-treatment-human rights strategies aimed at high-risk groups have proved effective in countries like brazil. in chap. 7, we explained that limited resources need to focus on high-risk groups and locations to achieve the best possible results. however, as we showed in the sex workers, who are the source of almost 80% of hiv infections, a negligible amount of funding for hiv/aids is targeted at this group. the mismatch between the most affected group and the allocation of funding in ghana highlights the importance of matching funding to prevailing prevalence and transmission patterns in a given country or region. as we saw in chap. 2, an hiv prevalence rate above 1% is a key threshold for an hiv epidemic to run out of control unless funding for prevention efforts is targeted at high-risk groups, such as commercial sex workers, men who have sex with men, injection drug users and prisoners. however, in chap. 8 we saw that pepfar -the largest bilateral donor of funding for hiv/aids programs in developing countries -prohibits the use of funding for programs for commercial sex workers and needle exchange programs. in chap. 3, we saw that african-americans make up 54% of hiv/aids patients, even though african-americans account for less than 15% of the us population. moreover, black men who have sex with men (msm) have the highest rates of unrecognized hiv infection, hiv prevalence and incidence rates and aids mortality rates among msm in the united states. in five us cities, 46% of african-american msm are infected with hiv. hiv and aids prevalence rates have affected black msm disproportionately since the beginning of the epidemic. black msm are the only group in the united states with hiv prevalence and incidence rates that are comparable to those in the most affected developing countries. however, the vast majority of hiv prevention intervention for african-americans does not target homosexual men and for homosexual men does not target black msm (millett and peterson 2007) . thus, the need to focus prevention efforts on the most vulnerable groups remains an issue not just in developing countries. while prevention strategies need to be tailored to the sources of hiv infections in specific contexts, there are several proven prevention strategies that need to be scaled up. the resources for prevention need to be focused according to the specific nature of the epidemic in different settings, as we showed in chap. 2. figure 9 .2 shows the source of new hiv infections by region. table 9 .2 summarizes the coverage levels of several essential prevention strategies and fig. 9 .3 shows their deployment by region. it is important to emphasize that prevention and treatment are mutually supportive and need to be addressed simultaneously. access to treatment supports prevention by reducing risky behaviors, increasing disclosure of hiv status, reducing stigma and reducing infectiousness (global hiv prevention working group 2007) . prevention supports access to treatment by reducing the number of people that require treatment, thus making universal access to treatgroup (2007) order to enhance the effectiveness of both. ment more affordable. hiv treatment and prevention should be integrated, in hiv prevention strategies fall into four general categories: (1) prevention of sexual transmission; (2) prevention of blood-borne transmission: (3) prevention of mother-to-child transmission; and (4) social strategies. the strategies for preventing sexual transmission are: (1) behavioral change programs (to increase condom use, to delay the initiation of sexual behavior in young people and to reduce the number of sexual partners); (2) condom promotion; (3) hiv testing (knowledge of hiv status decreases risky behavior); (4) diagnosis and treatment of sexually transmitted infections (which significantly increase the risk of hiv acquisition and transmission, particularly in the case of genital herpes); and (5) adult male circumcision (which reduces the risk of female-to-male transmission by about 60%) (global hiv prevention working group 2007) . the effectiveness of these strategies varies. the promotion of condoms has been largely successful with respect to commercial sex and casual sex, but condom use remains low within marriage. as we noted in chap. 4, increasing life expectancy, in areas where it is low due to diseases like malaria, is a cost effective strategy for enhancing behavioral change to lower the risk of hiv infection. a survey by the who, on behalf of the global fund, reviewed anti-malaria operations in ethiopia, ghana, rwanda and zambia. in ethiopia, childhood malaria declined by 60% and the death rate was cut in half within 2 years of the beginning of the mass distribution of mosquito nets. within a single year, both cases and deaths dropped by twodeaths by a third. in many cases, the distribution of free nets was accompanied by free drugs based on artemisinin, a substance to which the malarial parasite has yet to develop widespread resistance, and spraying ddt inside people's houses. free nets and malaria drugs would bring malaria under control in most of africa at a cost of usd 10 billion (economist 2008) . these promising results also bode some studies suggest that treating sexually transmitted infections may not rediseases. moreover, as we noted in chap. 4, oster (2005) argues that the explanation for the substantial difference in the transmission rates between the united tions, which leave open sores from chlamydia, syphilis and gonorrhea that facili-there is significant evidence that male circumcision significantly reduces hiv a similar picture is seen in south and south-east asia, where overall hiv prevalence is much lower, but the countries with highest hiv prevalence have little thirds, in rwanda, and one-third in zambia. in ghana cases fell by an eighth and based on these results, the who believes that a 5-year campaign that distributes well for hiv prevention. association between the risk of infection with hiv and other sexually transmitted prevent as many as 24% of new infections over a decade duce hiv transmission significantly (halperin 2007) . however, there is a strong transmission. box 9.1 discusses the relationship between circumcision and hiv/ 305 tate hiv transmission. thus, treating bacterial sexually transmitted infections could states and sub-saharan africa is due to other untreated sexually transmitted infec-male circumcision (papua new guinea, cambodia and thailand) . conversely, hiv prevalence is extremely low in those countries where most men are circumcised (pakistan, bangladesh, indonesia and philippines). there is ecological evidence that prevalence of circumcision is negatively correlated with prevalence of hiv/aids. specifically, there is a strong inverse correlation between the prevalence of circumcision in countries and the prevalence of hiv in those countries. all the highest hiv prevalence countries are those where circumcision is little practiced. in fact, no country with nearly universal circumcision coverage has ever had an adult hiv prevalence higher than 8%, including higher risk than that in countries with prevalence of around 25%. this fact is illusfig. 9 .4 ecological relationship between circumcision and hiv prevalence. source: bailey (2007) a large, randomized controlled trial in 3,274 men between the ages of 18 and 24 years showed that circumcision resulted in a significant 60% reduction in hiv vulnerability to hiv varies considerably from one epidemic to the next, as do the issues facing vulnerable groups. for example, in a concentrated epidemic, such as in asia and latin america, hiv transmission occurs primarily among vulnerable groups and prevention programs targeted at vulnerable groups would reduce infection (auvert et al., 2005) . these results were confirmed by two other trials. 9 the way forward prevention, treatment and human rights trated in fig. 9 .4. countries such as cameroon, where a 1997 survey found sexual behavior to be overall infection. however, in a generalized epidemic, such as in several countries groups, halperin (2007) argues that transmission would continue unabated despite prevention programs targeted at vulnerable groups. however, as we noted in chap. 4, research regarding the relationship between trade routes, truckers, sex workers and hiv propagation contradicts this idea. in a generalized epidemic, where hiv is spread along trade routes, prevention programs targeted at truckers and sex workers would be effective in bringing down the growth rate of the spread of the disease. having multiple sex partners increases the risk of hiv infection in both concentrated and generalized epidemics, but the impact of this factor on hiv prevalence rates can vary considerably. for example, even though the united states and uganda have similar rates of multiple sex partners, and the number of sexual partners that men and women had over a 10-year period were much higher in the united states than in uganda, uganda's hiv/aids prevalence rate was about 18 times higher than that of the united states (halperin 2007). however, as we noted in chap. 8, a recent study indicates that abstinence-only programs are as effective as providing no information at all when it comes to preventing pregnancies, unprotected sex and sexually transmitted diseases. abstinence-plus interventions, which promote sexual abstinence as the best means of preventing hiv, but also encourage condom use and other safer-sex practices, are more effective than the proven strategies for preventing blood-borne transmission are: (1) to supply injection drug users with clean injection equipment; (2) methadone or other substitution therapy to reduce drug dependence; (3) blood safety programs, including screening of donated blood; and (4) infection control in health care settings, including injection safety and antiretroviral treatment following exposure to hiv. as we noted in chap. 8, the risk of aids infection through the use of blood products was recognized as early as 1982, but countries were slow to adopt measures to ensure the safety of the blood supply and the world health organization (who) passed a resolution on blood products that made no mention of aids as late as january 1987. in the 1990s, chinese health authorities promoted bloodselling by poor farmers to commercial blood collection centers, despite warnings from the who, spreading hiv/aids through the blood fractionation and reinjection process. in 2007, new hiv infections through hospital blood transfusions continued to be reported in china, and illegal underground blood collection centers have continued to operate. box 9.2 recounts the story of the libyan scandal over blood-borne transmission to children. in southern africa, where hiv transmission occurs primarily outside vulnerable 307 abstinence-only programs (underhill et al., 2007) . on december 14, 2007, sixth sense productions, inc., an independent holly-snezhana dimitrova, valentina siropulo) and a palestinian medical intern (ashraf ahmad djum'a al-hadjudj) who were jailed in libya and faced the death penalty for allegedly infecting children with hiv. this news item is a postscript to a long international drama that began to unfold in 1999 when the medics were arrested on charges of injecting 418 libyan children with hiv-tainted blood while at a benghazi hospital. of them, over 50 had died by the end of 2007. one important report was submitted by luc montagnier and vittorio colizzitwo leading experts on hiv/aids. their report concluded that the infection at the infections began before the arrival of the nurses and doctor in 1998. through hospital records, and the dna sequences of the virus, they traced it to patient n.356 who was admitted 28 times between 1994 and 1997 in ward b, iso and ward a. the first cross-contamination occurred during that patient's 1997 admission. montagnier and colizzi both testified in person at the trial of record for the defense. on that the strain of virus was already present before the arrival of the six accused. the accused were tried and retried. the libyans had signed confessions from them -which the accused said were extracted under torture. the final verdict in 2006 sentenced them to death by firing squad. the libyan president likened the scotland for the bombing of pan am flight 103 over lockerbie, scotland, on 21 and political favors in exchange for the release of the six. in the end, bulgaria, qatar and a group of european countries funneled usd 460 million into the international fund benghazi to finance the treatment of the hiv-infected children and the improvement of the libyan health care system. france played a pivotal role in the final release of the accused. in exchange for the release, france agreed to sell antitank missiles and nuclear technology to libya. it was a win-win deal for france: they did multi-million dollar business with libya and got publicity for helping the release of the accused. when the nurses returned to bulgaria, the government endorsed a 10,000 leva reimbursement for each of the nurses. a bulgarian mobile telephony provider donated an apartment for each nurse. 9 the way forward prevention, treatment and human rights 14 december 2006, nature (446, 836-837) published a report that also concluded wood producer, announced plans to make a usd 40 million movie about five event to the case of abdel basset ali al-megrahi, who is serving a life sentence in hospital resulted from poor hygiene and reuse of syringes. they concluded that the bulgarian nurses (kristiyana vulcheva, nasya nenova, valya chervenyashka, december 1988. thus, it became clear that libya was trying to extract economic the proven strategies for preventing mother-to-child transmission are: (1) general hiv prevention for women of child-bearing age; (2) a brief course of antiretroviral treatment in advance of delivery (which can reduce transmission by 50%, but is only received by an estimated 11% of women in need); (3) prevention of undesired pregnancy in hiv-positive women; (4) breast-feeding alternatives; and (5) cesarean delivery where the mother has a high viral load (global hiv prevention working group 2007) . in developing countries, a small but growing number of children are dying of hiv/aids. as fig. 9 .5 shows, some 4% of children died of hiv/aids in 2005. hiv infected mothers carry additional risks for the baby. in table 9 .3, we indicate some of the major risks. some risks like stillbirth or high infant mortality have been found only in developing countries but not in developed countries. for these additional risks, it has been suggested that one way of eliminating vents mother-to-child transmission by 100%. thus, family planning could also help to reduce mother-to-child transmission: o t h e r ( 2 9 % ) mother-to-child transmission is not to have the baby in the first place. this pre-another often neglected aspect of hiv prevention -one prohibited from funding by the bush administration's international aids programinvolves expanding family planning services, including for hiv-positive women who do not want to conceive. reducing unintended pregnancies could greatly decrease the number of infected infants as well as the number of children who eventually become orphans (halperin 2007). if an hiv-positive woman gives birth to a child, there is a risk of transmission of hiv itself, in addition to the other risks listed in table 9 .3. however, the transmission risk of hiv from mother to child is not 100%. it can be minimized through drug treatment of the mother and careful birthing. figure 9 .6 clearly demonstrates this fact, using the data from the united states. the introduction of zidovudine (for the mothers before childbirth) has dramatically reduced the risk of hiv infection of the baby. since 1998, most countries have applied a regimen of zidovudine from 28 weeks, with nvp administered during labor and to the baby, and the addition of a 7 day zidovudine/lamivudine postpartum regime. the result has been a dramatic reduction of infected newborns (see fig. 9 .7). note that the reduction has been evident in europe and the united states since 1994, when this regime was introduced. in thailand, the regime was introduced in 1996 and in most parts of africa 2 years later. once a child is born, the question is whether the infected mother should breastfeed the child. on the one hand, unaids estimated that globally there are 300,000 babies infected through breastfeeding. on the other hand, the unicef estimates that 1,500,000 children die every year from lack of breastfeeding by the breastfeed the baby. key factors that increase vulnerability to hiv include: (1) gender inequality (which reduces women's access to information and services, reduces power to negotiate safe sex with partners, increases the risk of sexual violence and may create the need to depend on sex for economic survival); (2) institutionalized discrimination against vulnerable groups (such as criminalizing drug use and needle possession, commercial sex work and sex between men); (3) poverty (which reduces access to information and services and access to prevention tools, such as condoms); (4) hiv stigma (which discourages individuals from seeking testing, disclosing their status, seeking hiv-related services or using alternatives to breast-feeding); and formation and social support by displacing populations and increase the risk of sexual violence) (global hiv prevention working group 2007). as we noted in chap. 4, there is no clear evidence that reducing poverty and income inequality will necessarily reduce hiv/aids prevalence. moreover, povcondoms and circumcision. significant percentage of men who have sex with men are hiv-infected in many africa; 27% in guyana; 33% in st. petersburg, russia; and 73% in urban ethiobetween vulnerable groups and the general population in bangladesh. the linkages between vulnerable groups, and between vulnerable groups and the general social strategies that address the factors that increase vulnerability to hiv innities and hiv-positive individuals in hiv/aids programs; (4) visible political leadership; (5) engaging a broad range of sectors in hiv awareness and prevention 9 the way forward prevention, treatment and human rights hiv/aids prevention strategy. thus, if poverty reduces access to information and parts of the world (28% in bangkok; 15% in phnom penh; 21.5% in urban sene-vulnerable groups are not compartmentalized. people infected through injection drug use can infect their sexual partners. a significant percentage of men who their clients, who in turn may infect their spouses or other sexual partners. in would be to find innovative ways to improve access to information, provide funding measures; (3) gender equity initiatives to empower women; (3) involving commution working group 2007). human rights are the core of most social strategies to gal; and 46% of african-american men in five us cities). sex workers can infect many areas, sex workers have very high rates of hiv infection (50% in south have sex with men also have sex with women (for example, 20% in asia) and a (5) conflict and humanitarian emergencies (which reduce access to services, inerty reduction is too broad a goal to constitute what might be considered a concrete 312 programs; and (6) legal reforms to support hiv prevention strategies, such as laws clude: (1) hiv awareness campaigns, including in the mass media; (2) anti-stigma services and access to prevention tools, such as condoms, a concrete policy response to enhance access to services and provide free access to prevention tools, such as pia) (global hiv prevention working group 2007). figure 9 .8 shows the linkages decriminalizing needle possession and anti-discrimination laws (global hiv preven-population, make effective prevention strategies for vulnerable groups essential. awareness of hiv status has a significant impact on rates of hiv transmission. when unaware of hiv seropositivity, the transmission rate is estimated at 8.8-10.8 the transmission rate to an estimated 1.7-2.4% (holtgrave 2005). however, inorder to balance the need for more testing with the need to respect human rights, it has been recommended that health care providers offer and recommend hiv testing, in conjunction with counseling (the opt-in approach), rather than rely on the client to initiate this process. however, mandatory hiv tests and routine hiv testand confidentiality (jã¼rgens 2007 ). the major barriers to increasing hiv prevention are: (1) failure to target limited funding where it will have the greatest impact, due to ing unless the client opts out risk violating individuals' rights to informed consent lack of information on the nature of the epidemic or ideological, non-scientific creasing access to hiv testing and counseling also raises human rights issues. in increases in funding; (4) failure to integrate hiv prevention in schools, workplaces and other health care programs, such as tb and reproductive health; and (5) stigma and discrimination against hiv-positive people and vulnerable groups, which deter people from seeking testing and prevention services and discourage political leadership (global hiv prevention working group 2007) . we analyze the problems and solutions regarding stigma and discrimination against hivpositive people and vulnerable groups later in this chapter. we analyzed the issues of inadequate financing, targeted financing and donor coordination in chap. 7. as we noted in chap. 7, a lack of donor coordination is an obstacle to expanding treatment and prevention programs, due to the administrative burden that it imposes on recipients. as we noted in chap. 8, the us government's foreign aids program, pepfar, devotes only 20% of funding to prevention and requires that two-thirds of that amount be spent on abstinence-only programs that do not promote condom use, despite evidence that this approach to prevention is not effective and undermines best practices. pepfar guidelines also undermine hiv/aids prevention by further stigmatizing sex workers and prohibiting funding of needle exchange programs, despite evidence that such harm reduction programs are effective. the pepfar approach assumes that vulnerable groups do not interact with the rest of society. pepfar is perhaps the best example of ideological, non-scientific restrictions on the use of donor funding, although it also serves as an example of two of the other significant barriers to hiv prevention, due to its promotion of stigma and discrimination against vulnerable groups and the percentage of funding that it allocates to prevention. however, it is important to emphasize that pepfar has done more than any other bilateral funding program to address the need for adequate financing. the key point is that the money that has been made available through pepfar could be better spent. on 26 december 2007, us president bush signed legislation that lifted a 1999 ban that had made washington, dc the only us city barred by federal law from using municipal money for needle exchange programs. officials of the district of columbia health department planned to allocate usd 1 million for such programs in 2008 (urbina 2007) . extending this change in policy to pepfar would enhance the effectiveness of prevention programs in the countries that receive pepfar funding. in chap. 3, we provided an overview of the history of drug developments to treat hiv/aids and saw the dramatic impact on survival of triple combination therapy. without this treatment, the chance of surviving 10 years was about 50%. with this treatment, patients have a 50% chance of living another 35 years. in the early 1980s in the united states, the leading causes of death among 25-44 old year men come the leading cause of death in this group. following the introduction of universal access to triple combination therapy, deaths from aids fell to fourth place, behind accidents, cancer and homicide. as a result, whereas 68% of americans considered hiv/aids to be the most urgent health problem facing the united ents became so important, as we saw in chaps. 5 and 6. this is also why access to there are five classes of anti-hiv drugs, which are known as antiretroviral verse transcriptase inhibitors (nnrtis), which began to be approved for use in 1997, stop hiv from replicating within cells by inhibiting the reverse transcriptase protein. (4) fusion or entry inhibitors prevent hiv from entering human immune sert its genetic material into human cells (http://www.avert.org/introtrt.htm). hiv-positive people are prescribed antiretroviral therapy once the number of cd4 cells falls below a certain threshold or when they develop clinical aids 2006, an international panel of experts continued to recommend these guidelines in low-and middle-income countries, which represents 28% of those in need (who/unaids progress report on universal access to treatment). the "3 by lion people on arv therapy by the end of 2005. during this period, the number of people in low-and middle-income countries receiving arv treatment increased from 400,000 to 1.3 million (who 2006) . and human resources, management capacity and the ability to identify new patients through testing and counseling (chai 2007) . in chap. 7, we examined multilateral funding programs that address these capacity constraints in developing ing treatment are just that -estimates. as we have noted, unaids hiv/aids estients that have been identified as requiring treatment. the reluctance of the united states to allow pepfar funding to be spent on who-approved drugs has also been criticized as an obstacle to expanding treatturer, cipla, created a triple-combination drug in a single pill (triomune) that could be taken twice daily, which it offered to sell for about usd 300 per patient per year in 2001. cipla's triomune offer made the 3 by 5 initiative a realizable goal and cipla has the production capacity to produce four million doses of triomune per day. the who approved triomune in december 2003 as a first-line treatment for hiv/aids (hamied 2005) . the pepfar restriction on the use of who-approved drugs had the effect of preventing the use of pepfar funding to buy triomune. moreover, the majority of pepfar funds have been used to purchase patented versions of hiv/aids drugs, rather than generic versions (see chap. 8). figure 9 .9 shows how generic competition has lowered the cost of triple combination antiretroviral therapy. between 2001 and 2007, the price of the generic drugs has brought down the price of the originator substantially -from over usd 10,000 to under usd 350. at the same time, the generic prices have stayed in the 25-30% range of the originator price. pepfar funds can be used to purchase other low-cost generic equivalents of several patented hiv/aids drugs, including some produced by cipla. pepfar requires that generic drugs be approved by the us fda, canada, japan or western europe to be eligible for funding (see chap. 8). if us fda approval is sought for fixed dose combinations of previously approved antiretrovirals for the treatment of hiv, if one or more of the approved drug components are covered by a patent, the fda cannot approve an application until the patent expires. however, the application can receive tentative approval (which recognizes that at the time the tentative approval action is taken, the application meets the technical and scientific requirements for approval, but final approval is blocked by patent or exclusivity). products that receive tentative approval are eligible for procurement under the table 9 .4. lists the generic versions of hiv/aids drugs that have been approved by the fda for purchase with pepfar funds, along with the generic companies that own the patents for the specific generic formulations and the country of manufacture. the fact that the patents for hiv/aids drugs are owned by different companies has delayed combining different hiv/aids drugs in a single pill in markets protected by patents. one such pill, atripla, was created through a joint venture between merck and bristol-myers squibb with gilead sciences and combines efavirenz (bristol-myers squibb, merck) with emtricitabine and tenofovir (gilead sciences) (ib times 2007). atripla was approved for sale in the united states in 2006, several years after the indian generic manufacturer, cipla, had started manufacturing a triple-combination pill and 3 years after cipla's pill was approved by the who. approval to market atripla in the european union was sought in december 2007. gilead sciences and merck have formed a joint venture to market atripla in developing countries (ib times 2007). table 9 .5 shows the us patents, patent owners and patent expiry dates for selected hiv/aids drugs. zidovudine was the first drug to be approved for treatment of hiv infection. as table 9 .5 shows, the patent for zidovudine expired in 2005 and the patent for lamivudine expires in 2009. however, glaxo extended the life of these patents to 2016 by combining the two drugs into one pill (called combivir). while the new combination reduces the number of pills that a patient needs to take, it did not involve the invention of any new chemical entities. the patent history of zidovudine has been cited as a classic case of "evergreening" -the use of the patent system to extend drug monopolies far beyond the term of the original patent (hamied 2005). box 9.3 discussed evergreening. zidovudine was originally synthesized in 1964, as a potential cancer treatment. research in 1984 showed that it was effective against hiv/aids, which formed the basis for glaxo's 1984 patent application. following clinical trials, the us fda approved zidovudine in march 1987 for advanced hiv disease in adults and the patent for zidovudine as a treatment for hiv/aids was granted in february 1988 (cochrane 2000) . while zidovudine alone only extended life by a matter of months, once it was combined with two other classes of hiv/aids drugs, it extended life for years. the fda expanded zidovudine approval in 1990 to include evergreening is a mechanism by which pharmaceutical and other companies can keep extending patents on drugs after the initial patents expire. over a fixed period of time. the intention of providing a monopoly is to provide an incentive to innovate. granting a patent requires three elements: (1) novelty of the product. product; (2) non-obviousness of the new product; and (3) demonstrated utility of the the role of patents is to give exclusive rights to manufacture the patented product 319 less-advanced stages of hiv disease (coffey and peiperl, 2006) . to understand evergreening in the united states, we need to examine the drug price competition and patent term restoration act, informally known as the "hatch-waxman act" . it is a 1984 united states federal law which established the modern system for generic drug approval. hatch-waxman generic. section 505( j)(5)(b)(iv), the so-called paragraph iv, allows 180-day exvolume). for pharmaceutical companies, the hatch-waxman act has created a perverse drugs by making marginal changes than to try the risky strategy of inventing completely new chemicals. thus, the two decades following its passage, the hatch-waxman act has resulted in more me-too drugs than drugs with new chemical case of prilosec -the so-called "purple pill" of astrazeneca (usd 6 billion/year global blockbuster drug), the patent for which expired in 2001 only to be reincarnated as a new patented drug nexium. however, on 30 april 2007 the supreme court of the united states issued a ruling in ksr international co v. teleflex et al., which raises the bar for patent holders to prove that their invention is not obvious, and therefore patentable. this ruling will make many existing patents more vulnerable, make it harder to gain approval for new patents and make evergreening more difficult in the future. if the patent claim extends to what is obvious, it is invalid. for example, a patent's subject matter can be proved obvious if there existed at the time of invention a known problem for which there was an obvious solution encompassed by the patent's claims. the supreme court noted that, "granting patent protection to advances that would occur in the ordinary course without real innovation retards progress and may, in the case of patents combining previously known elements, deprive prior inventions of their value or utility." it is worth quoting in full the court's description of the reason that patents are only granted for non-obvious innovations: "we build and create by bringing to the tangible and palpable reality around us new works based on instinct, simple logic, ordinary inferences, extraordinary ideas, and sometimes even genius. these advances, once part of our shared knowledge, define a new threshold from which innovation starts once more. and as progress beginning from higher levels of achievement is expected in the normal course, the results of ordinary innovation are not the subject of exclusive rights under the patent laws. were it otherwise patents might stifle, rather than promote, the progress of useful arts." 9 the way forward prevention, treatment and human rights ents for branded counterparts. the hatch-waxman act encouraged the growth of generic industry, whose market share rose from 19% in 1984 to 49% in 2002 (by amended the federal food, drug, and cosmetic act. section 505( j) sets forth clusivity to companies that are the "first-to-file" an anda against holders of pat-the process by which would-be marketers of generic drugs can file abbreviated incentive. it has given them more incentive to try to extend the life of existing abbreviated new drug applications (andas) to seek fda approval of the compounds. the most famous documented case of evergreening occurred in the who guidelines for arv treatment regimens provide a basis for a range of treatment protocols in individual countries. in individual countries, factors such as prices, drug efficacy and side effects are also taken into account. stavudine (d4t) recommended that d4t no longer be used, due to toxicity. instead, countries should switch to tenofovir (tdf) or zidovudine (azt). of these two, tdf is preferable, because of its efficacy and safety and because it can be taken only once a day. emtricitabine (ftc), in one, triple-combination pill that can be taken once a day. patients are more likely to adhere to this once-a-day regimen, thereby reducing azt and tdf, compared to d4t, has delayed the shift to the new regimen in many below), the clinton foundation hiv/aids initiative (chai) has negotiated price reductions for several hiv/aids drugs for use in 27 low-and middle-income countries (see table 9 .6). the clinton foundation is discussed in chap. 7. as of may 2007, 750,000 people were benefiting from medicines purchased under chai agreements in 50 countries (chai 2007). table 9 .6. first, the prices are generally higher for middle-income countries than for low-income countries. thus, the pharmaceutical companies are pursuing a price discrimination strategy across different markets, selling drugs at a price that the markets can bear. therefore, there is clear room for generic products in these markets, especially for the low-income countries. compulsory licensing is a distinct possibility (see, however, our discussion in chap. 6 about the difficulties many developing countries faced importing drugs under compulsory license from canada). some companies have used the world bank's country income index or the human development index as their criteria for setting prices. in chap. 6, we developed a much more comprehensive index that takes into account not just the level of development of the country but also level of prevalence of hiv/aids explicitly. second, the price of hiv/aids drugs in many cases in many developing countries is not necessarily lower than in developed countries. for example, in guatemala, between 2000 and 2003, prices of most hiv/aids drugs were consistently higher than in the united states (hellerstein 2003). according to chai, in 2006, 80,000 (4%) of those receiving arv treatment in low-and middle-income countries were taking second-line treatment. the reason that relatively few are on second-line treatment is that most only began treatment within the last 4 years. as a result, relatively few have experienced treatment failure, which is defined as (1) virologic failure (a viral load of more than 400 copies per milliliter), (2) immunologic failure (a declining cd4 cell count in spite of treatment) or (3) clinical failure (progression to aids evidenced by weight loss or the appearance of opportunistic infections). another reason is that poor diagnostic and laboratory capacity in many countries has made treatment failure difficult to diagnose. by 2010, chai estimates that close to 500,000 people will require second-line treatment in low-and middle-income countries (chai 2007) . the higher cost of second-line treatment means that access requires further funding. however, patents are not expected to be an obstacle to acquiring affordable second-line treatments in the most affected low-income countries, due to the delay of trips patent rules on pharmaceuticals to 2016, although patent rules may affect affordability in middle-income countries (chai 2007) . in chap. 6 we analyzed trips rules on patents for pharmaceuticals in developing countries. however, as we noted in chap. 5, the problem of regulatory capture in free trade agreements can undermine trips rules so that patents create obstacles to affordable treatment in some lowincome countries and political pressure on low-and middle-income countries can discourage the use of trips flexibilities to increase access to treatment. unitaid is a global health initiative for hiv/aids, tuberculosis and malaria that is funded by several national governments. with respect to hiv/aids, unitaid funding is focused on pediatric and second-line treatment and the prevention of mother-to-child transmission. unitaid will finance a free supply of second-line hiv/aids treatment in 27 countries for 18 months, after which the reduced prices achieved by chai will enable other funding sources, such as the global fund (discussed in chap. 7) and pepfar (discussed in chap. 8), to fund the purchase of second-line treatments at lower prices (chai 2007) . while high-income countries and middle-income countries with low prevalence rates are in a position to pay for hiv/aids treatment, middle-income countries with high prevalence rates and most low-income countries are not. low-income countries with high prevalence rates in particular will have to depend on external funding sources, such as pepfar and the global fund, to expand access to treatment and then to maintain treatment. medical care for people with hiv/aids in developing countries costs about usd 1,000 a year, in drugs and support facilities. the economist estimated that it would cost usd 6-7 billion a year to provide treatment for the 6-7 million people with hiv/aids in low-income countries that were in need of treatment in 2006. however, expanding treatment means that fewer people will die. moreover, millions more will become infected, and even more so if prevention efforts are not improved. thus, universal treatment in lowincome countries could cost usd 40 billion by the end of the next decade. this highlights the need to ensure that external funding is both increased and sustained and the importance of prevention in making universal access to treatment affordable (economist 2006). scientists have been trying to develop an hiv vaccine for more than 20 years, although some have suggested that an effective aids vaccine may be a biological impossibility (epstein 2007) . in 2007, about 50 experimental hiv vaccines were being tested in clinical trials. most viral vaccines work by generating antibodies that neutralize or inactivate the invading virus. however, unlike other viruses, hiv-1 evades the antibody response, which, together with the large genetic variety found in hiv-1 strains, has made the development of an hiv-1 vaccine difficult. to date, antibody-based hiv-1 vaccines have only succeeded in neutralizing a minority of the copies of the virus that are found in a given patient. hiv-1 antibodies target the mechanism that hiv-1 uses to bind itself to the host immune cells in order to prevent hiv-1 from entering the cell. however, hiv-1 uses shielding mechanisms to prevent the antibodies from recognizing the virus, including a dense coating. current hiv-1 vaccine research therefore seeks to find vulnerabilities in these shielding mechanisms, but this requires research for multiple genetic subtypes of hiv-1 (montefiori et al., 2007) . for example, one recent study identified a place on the outside of the human immunodeficiency virus that could be vulnerable to antibodies that could block it from infecting human cells, which might be targeted with a vaccine aimed at preventing initial infection (dunham 2007) . a new class of hiv vaccines was designed to trigger cell-mediated immunity to create an extended immune defense. however, in 2007, merck reported that its hiv vaccine, v520, had failed. v520 was being tested by merck and the us national institutes of health in a clinical trial involving 3,000 people in highrisk groups in australia, brazil, canada, the dominican republic, haiti, jamaica, peru, puerto rico and the united states (associated press 2007). v520 used the common cold virus (the adenovirus) to transport three synthetic hiv genes into the body's cells (park 2007) . merck halted the trials after 24 of 741 volunteers who got the v520 vaccine later became infected with hiv, while only 21 of 762 participants that received a placebo also became infected (associated press 2007). the v520 vaccine was one of only two aids vaccine candidates in advanced human trials, the other being tested by sanofi-aventis sa (dunham 2007) . other approaches are also being explored. david ho (the inventor of triple combination therapy) and his team at the aaron diamond aids research center are researching the use of different vectors, or not using vectors at all, to produce stronger immune responses. scientists at the international aids vaccine initiative are studying the use of crippled, live strains of hiv and ways to stimulate a special class of antibodies that appear to be able to defuse hiv. the global hiv vaccine enterprise, which is funded by the gates foundation (discussed in chap. 7), wellcome trust, the us national institutes of health and the european union, is seeking to accelerate research on hiv vaccines by linking together independent organizations so that researchers can learn from each other, rather than work in isolation (park 2007). as we noted in chap. 5, there are many subtypes of hiv-1 (the most commonly occurring hiv infection in humans). the major hiv-1 subtypes accounting for most infections in africa are subtype c in southern africa, subtypes a and d in eastern africa, and circulating recombinant form 02_ag (crf02_ag) in westcentral africa (peeters and sharp 2000) . the most commonly occurring form of hiv-1 in north america and in europe is subtype b. the first hiv/aids vaccine ever to reach phase iii trial was for subtype b. the gp120 vaccine was not effective. however, what vaccine trials have indicated thus far is that, in the case of hiv/aids, there is pattern of development of potential vaccines not in the subtypes where the needs are the greatest but in the area where the biggest monetary rewards are expected. the economics of hiv/aids vaccines suggest that funding for vaccines for the worst-effected countries are unlikely to come from the private sector (see box 9.4). hiv/aids affects hundreds of millions and kills several million people every year. the disease was identified several decades ago. two nobel prizes have been awarded in the past two decades for identifying the cause and the transmission mechanism of hiv/aids. yet we still do not have a vaccine for hiv/aids. kremer and snyder (2004) have developed an argument as to why the private sector is very unlikely to develop a vaccine for aids. here, we illustrate the argument with one example. imagine there are 100 people in the world. there are 90 people (type l) who have a small chance of 10% of contracting hiv/aids. there are another ten people (type h) who would develop hiv/aids with a 100% chance. let us suppose that the harm from hiv/aids is usd 100 for each person. let us also assume that for each usd 1 decrease in harm, a consumer is willing to pay usd 1 (technically, each consumer is risk neutral). suppose the drug is perfectly effective, has no side effects and is costless to produce. how much revenue will a pharmaceutical company generate in each of the following scenarios? (1) it develops a drug d that cures hiv/aids (forever). (2) it develops a vaccine v that prevents hiv/aids from developing. we show that under the assumption that the pharmaceutical company cannot distinguish between type h and type l, it is more profitable for the drug companies to produce the drug rather than the vaccine. if the pharmaceutical company develops the drug d, it will be able to sell it to all the people who get hiv/aids. by assumption, all the type h people will develop hiv/aids. thus, there will be ten people from type h who will get hiv/aids. in addition, nine people of type l will also develop hiv/aids. in total, there will be 19 people with hiv/aids, including both types. by assumption, each person contracting hiv/aids will be willing to pay usd 100 to reduce the effects of hiv/aids by 100%. therefore, the pharmaceutical company will be able to earn usd 1,900 in revenue from the entire population. given our assumption of zero cost of production, usd 1,900 will also be the profits of the pharmaceutical company. the vaccine has to be sold before hiv/aids strikes. for type l, there is a 10% chance of hiv/aids. thus, they will be willing to pay the average loss of 100 (1/10) = usd 10 for the vaccine. if the pharmaceutical company cannot distinguish between type l and type h, it can only charge usd 10 to all. in that case, it will generate usd 10 100 = usd 1,000 profits by selling the vaccine to all 100 people. the other possibility is the following. the company sets a price of usd 100 for the vaccine. in that case, no person of type l will buy the vaccine ex-ante (as their expected benefit before hiv/aids strikes is usd 10 but the cost is usd 100). the only people who will buy the vaccine will be of type h. since there are ten of type h, the profits will be usd 100 10 = usd 1,000. thus, in either price strategy, the profits of the company will be usd 1,000. therefore, the profits of the company are bigger in the case of the development of drug d instead of the vaccine v. this argument is extremely general as long as the probability of the type l does not get close to the probability of type h getting the disease and the company cannot distinguish between the types. at the beginning of this book, we highlighted the need to integrate three interrelated issues into any comprehensive aids strategy -prevention, treatment and human rights protection. as we showed in chap. 2, each of these issues must be considered in the context of specific countries or regions, in order to take into account variations in cultural values, affected groups, infection rates, legal systems, economic resources and human resources. in this chapter, we have analyzed prevention and treatment issues in greater detail. the preceding discussion shows that great progress has been made on these two fronts and that greater progress is possible. our analysis of prevention issues in particular has shown the need to integrate prevention, treatment and human rights strategies. the primary reason that human rights need to be addressed is because discrimination keeps people away from both prevention and treatment programs (gruskin et al., 2007) . changing social attitudes in order to overcome stigma and discrimination is not an easy task, particularly given deep-seated fears and prejudices surrounding sex, blood, disease and death and the wide-spread perception that hiv/aids is closely supportive and enabling environment for women, children and other vulnerable to change attitudes of discrimination and stigmatization associated with hiv/aids variations in cultural values and legal systems make hiv/aids-related human rights particularly difficult to tackle on a global basis. however, hiv/aidsrelated human rights are the area where the least progress has been made and need to become a central focus in the global fight against hiv/aids (jã¼rgens and cohen 2007) . in this section, we focus on three categories of laws: (1) laws that discriminate against vulnerable groups; (2) laws that discriminate against hiv-positive people, such as those that criminalize hiv transmission; and (3) laws that prohibit discrimination against vulnerable groups, including hiv-positive people. we review the united nations international guidelines on hiv/aids and human rights and provide examples in each category. 9 the way forward prevention, treatment and human rights to understanding and acceptance (united nations 2007). groups. the guidelines also recommend that states promote the wide and ongoing distribution of creative education, training and media programs explicitly designed united nations international guidelines on hiv/aids and human rights redialogue, specially designed social and health services and support to community commend that states, in collaboration with and through the community, promote a groups by addressing underlying prejudices and inequalities through community 326 tied to deviant or immoral behavior (jã¼rgens and cohen, 2007) . in this regard, the the law plays different roles with respect to infectious diseases. some health risks, such as poor access to sterile injection equipment, can be directly attributed to law, and laws have been used to change unhealthy behaviors, such as smoking and drunk driving. both international and national laws are used in disease control. in addition to the law's role as a source of disease control authority for government, the law has a countervailing role as a source of protection against excessive and unnecessary regulations (burris 1999) . the united nations international guidelines on hiv/aids and human rights acknowledge the inherent limitations in using law reform to enhance human rights. the effectiveness human rights laws depend on the strength of the legal system in a given society and on the access of its citizens to the system, both of which vary considerably from one country to the next. moreover, the law cannot serve as the only means of educating, changing attitudes, achieving behavioral change or protecting people's rights. nevertheless, since laws regulate conduct between the state and the individual and between individuals, they can either support or undermine the observance of human rights, including hiv-related human rights (united nations 2007) . for these reasons, we first consider laws that support human rights. while social attitudes may take time to change, an important first step is to reform laws, policies and practices that institutionalize discrimination against the groups of people who are most vulnerable to hiv/aids: women and girls; men who have sex with men; commercial sex workers; and injection drug users. the united nations international guidelines on hiv/aids and human rights recommend consistent with international human rights obligations and are not targeted against vulnerable groups (united nations 2007). laws in this category include those that prohibit sexual acts between consenting adults in private, laws prohibiting sex work that involves no victimization and laws prohibiting measures such as needle exchange that can reduce the harm associated with illicit drug use (elliot 2002 ). the united nations international guidelines on hiv/aids and human rights recommend the enactment of anti-discrimination and protective laws to reduce human rights violations against women and children in the context of hiv, to reduce the vulnerability of women and children to hiv infection and to the impact of hiv/aids. with respect to women, the guidelines recommend law reforms to ensure the equality of women regarding property and marital relations and access 327 that states reform criminal laws and correctional systems to ensure that they are have a negative impact on hiv-related human rights and then consider laws that to employment and economic opportunity, such as equal rights to own and inherit property, to enter into contracts and marriage, to obtain credit and finance, to initiate separation or divorce, to equitably share assets upon divorce or separation and to retain custody of children. in addition, laws should ensure women's reproductive and sexual rights, including the right of independent access to reproductive and sexual health information and services and contraception, the right to demand safer sex practices and the right to legal protection from sexual violence. with children against sexual abuse and provide for their rehabilitation if abused and ensexual abuse by their husbands. when the husband is hiv-positive or engages in unsafe sex or drug use, this increases the risk of infection for women. child cusdren make it difficult for women to leave abusive relationships. while statutes allow property ownership regardless of sex, in practice women only have user rights under customary laws, not ownership. under inheritance laws, property remains in the man's family after he dies. thus, if a woman wants to leave an abusive husband or her husband dies, she cannot take any property with her, leaving women economically dependant upon their husbands or, as widows, their families. new laws have created inheritance rights for dependants, but are ignored by the man's family and not enforced. as a result, women and children widowed and women must either rely on their in-laws for support or become commercial sex workers (kelly 2004) . laws and cultural traditions thus increase women's vulnerability to hiv/aids, either within marriage or by forcing them to support themselves and their children as sex workers. recommend the enactment of anti-discrimination and protective laws to reduce discriminatory property, divorce and inheritance laws for same-sex relationships. 9 the way forward prevention, treatment and human rights tody laws, customary practice and traditions that favor paternal custody of chil-sure that they are not subject to penalties themselves. protection under disability human rights violations against men having sex with men, including in the context 328 orphaned by aids are left without adequate resources for medical treatment, and the united nations international guidelines on hiv/aids and human rights of hiv, including penalties for vilification of people who engage in same-sex respect to children, laws should provide for children's access to hiv-related inlaws, inheritance laws, and child custody laws. in many african countries marital rape does not exist as a legal concept, leaving women with no recourse against formation, education and means of prevention, govern children's access to volcontext of orphans, including inheritance and/or support. laws should also protect untary testing with consent, should protect children against mandatory testing, particularly if orphaned by aids, and provide for other forms of protection in the in sub-saharan africa, laws of particular concern include marital rape, property laws should also be ensured for children (united nations 2007). one key purpose of such anti-discrimination laws is to reduce the vulnerability of men who have sex with men to infection by hiv and to the impact of hiv/aids. the guidelines also recommend that the age of consent to sex and marriage be consistent for heterosexual and homosexual relationships and that laws and police practices relating to assaults against men who have sex with men ensure adequate legal protection (united nations 2007). in a 2006 internet-based survey of 759 sexually active msm in new york city, 11% reported being hiv-positive and 74% reported being hiv-negative. the majority were white, college-educated and in their 30s. the race of the respondents was white (77%), latino (13%), black (4%) and other (6%). in the previous 12 months, 45% had more than ten male sex partners, 53% had engaged in unprotected anal sex and 37% had used non-injection drugs. fifty percent of the hiv-positive men had unprotected anal sex in the previous 12 months and 71% of the hiv-negative men had unprotected anal sex in the previous 12 months (nyc health 2007). in a 2006 survey of 614 black msm in new york city, 67% were hivhigh school education, 67% were unemployed and 57% had an annual income of less than usd 10,000. fifty-six percent identified themselves as homosexual, 32% as bisexual, 6% as heterosexual and 6% as other. sixty-five percent had previously been diagnosed with a sexually transmitted infection and 30% had been raped (80% before they were 18 years old). eighty-four percent knew that they were hiv-positive. of the 16% that were unaware that they were hiv-positive, 53% reported having been tested for hiv previously. of those who had never been tested for hiv, the reasons they gave were: (1) being afraid to learn that they had the perception of not being at risk because they practiced safe sex (16%); and (4) being afraid that results will be reported to the government (16%). fifty percent reported unprotected anal sex with a man in the previous 3 months and 31% had exchanged sex for drugs, money or a place to stay in the same period. among those who had unprotected anal sex with a man in their last sexual encounter, 84% of the hiv-positive men had an hiv-positive sex partner and 89% of the hivnegative men had an hiv-negative sex partner (nyc health 2007). according to the unaids guidance note on hiv and sex work, despite high hiv prevalence among sex workers, only one in three receive adequate hiv prevention services and even fewer receive adequate treatment and health care (unaids 2007) . the unaids guidance note focuses on the reduction of hiv vulnerability among sex workers, who are defined as adults over the age of 18 years in order to take into account that sexual exploitation of children under 18 years of age is prohibited under international law. the key factors that lead people into sex work include poverty, gender inequality, indebtedness, migration, criminal hiv (48%); (2) being worried that others might treat them differently (26%); (3) positive. the median age of the respondents was 42 years, 22% had less than a coercion, humanitarian emergencies, drug use and dysfunctional families. laws, policies and practices that drive sex work underground make hiv/aids prevention and treatment for sex workers and their clients more difficult. discrimination against sex workers among the police, health care services and other social services impede access to prevention and treatment. the unaids guidance note organizes its recommendations into three categories: (1) reducing vulnerabilities and addressing structural issues; (2) reducing risk of hiv infection; and (3) building supportive environments and expanding choices. the strategies in the first category are to: (1) address poverty and gender inequality by providing alternatives to sex work through micro-finance programs and reforms to property rights; (2) address the demand for paid sex by seeking to changes men's behavior; (3) expand access to education for girls and women; (4) provide alternative job opportunities through employment growth and vocational training; and (5) provide employment and education opportunities and access to social services for refugees, internally displaced persons and economic migrants. the strategies in the second category are to: (1) involve sex workers in hiv prevention and treatment programs; (2) make male and female condoms available for free or at low cost; (3) increase access to antiretroviral treatment; (4) address the specific needs of sex workers in sexual and reproductive health programs, taking into account the different needs of female, male and transgender sex workers; (5) make hiv prevention information and condoms readily available to clients; (6) seek to eliminate violence against sex workers by clients, managers, police and other government officials; (7) seek to change attitudes towards sex workers to reduce stigma and discrimination; (8) promote initiatives to enable sex workers to negotiate safe sex practices; and (9) promote access to drug addiction treatment programs and harm reduction programs, such as needle exchange. the strategies in the third category are to: (1) address sex work stigma and discrimination to reduce economic, cultural and social marginalization in families and communities; (2) improve access to health care, education and training, microfinance and credit, social services, housing support and legal services; and (3) promote community organizations that work with sex workers. the unaids guidance note on hiv and sex work has been criticized for emphasizing alternative livelihoods without offering concrete examples, rather than emphasizing the right to engage in sex work and workplace safety and national laws that undermine sex workers' rights, particularly criminal prohibition of sex work and related activities. the guidance note's strategy of reducing demand for sex work has been criticized as implicitly supporting the criminalization or repression of sex work, which can increase the risk of hiv infection by driving sex work underground, limit sex workers' choices regarding working conditions and clients and increase stigmatization. the guidance note was further criticized for not advocating enhanced human rights protection for those engaged in sex work -as women, men, transgender persons and workers. the process used for preparing the document was criticized for not meaningfully engaging sex workers. unaids' response to criticism of this document -to withdraw it as a public document and restrict it to internal use -was also criticized (canadian hiv/aids legal network 2007b) the united nations international guidelines on hiv/aids and human rights recommend that criminal law prohibiting sexual acts (including adultery, sodomy, fornication and commercial sexual encounters) between consenting adults in private should not be allowed to impede provision of hiv prevention and care services and should be repealed. with regard to adult sex work that involves no victimization, the international guidelines on hiv/aids and human rights recommend de-criminalizing and legally regulating occupational health and safety conditions to protect sex workers and their clients, including support for safe sex during sex work. more generally, criminal law should not impede provision of hiv prevention and care services to sex workers and their clients and should ensure that children and adult sex workers who have been coerced into sex work are not prosecuted for such participation but rather are removed from sex work and provided with medical and psycho-social support services, including those related to hiv (united nations 2007). in eastern europe and central asia, unaids (2006) estimates that the use of contaminated injection equipment accounts for more than 80% of hiv/aid cases and accounts for about 30% of new infections outside sub-saharan africa. the united nations international guidelines on hiv/aids and human rights recommend that criminal law not be an impediment to measures taken by states to reduce the risk of hiv transmission among injecting drug users and to provide them with hiv-related care and treatment. they further recommend that criminal law be reviewed to consider: (1) the authorization or legalization and promotion of needle and syringe exchange programs; and (2) the repeal of laws criminalizing the possession, distribution and dispensing of needles and syringes (united nations 2007) . in saint petersburg, russia, a 2002 study found that 48% of injection drug users had shared needles in the 30 days prior to their first use of a needle exchange program. in early 2004, there were four syringe exchange facilities in saint petersburg -one mobile service (a bus) and three fixed facilities. however, the most important source of sterile syringes for injection drug users was drug stores. human rights watch found that state-supported impediments to access to both needle exchange points and drug stores were important barriers to hiv prevention, including: (1) police patrols of drug stores, which deterred injection drug users from purchasing syringes; (2) police patrols of needle exchange bus stops; and (3) arrests, fines or bribes for possession of syringes, even though carrying syringes is not illegal in the russian federation. however, while police interference with the syringe exchange bus was a problem in the late 1990s, it lessened in the early 2000s. humanitarian action, an ngo that delivers syringe exchange services in saint petersburg, visited with police chiefs to talk about the importance of syringe exchange for hiv prevention and organized a training session in 2003 for police officers that included the participation of former drug users and people living with hiv/aids. however, due to past incidents, the fear of apprehension by the police kept some drug users from using fixed as well as mobile syringe exchange facilities (human rights watch 2004) . table 9 .7 shows the dramatic increase in hiv prevalence among injection drug users in saint petersburg from 1998 to 2001. a 2005 survey of 500 injection drug users (idus) in new york city found that 65% had obtained a syringe from an exchange program in the previous year, 49% at a pharmacy, 10% from a medical provider, 53% from a friend or sexual partner and 25% from a drug dealer. the self-reported hiv prevalence rate in the group was 21%. idus who obtained syringes from sterile sources (exchange, pharmacy or provider) were less likely to share syringes than those who obtained them from non-sterile sources (friends, relatives or the street). those who obtained syringes from exchange programs were significantly less likely to share syringes. nevertheless, 19% of idus had shared a syringe at least once in the previous 12 months and 53% had engaged in unprotected sex. idus that had shared a syringe were 2.5 times more likely to engage in unprotected sex (nyc health 2007). another category of laws discriminates directly against people with hiv/aids, such as laws that criminalize hiv transmission and travel restrictions based on hiv status. there is a concern that the criminalization of hiv transmission will discourage people from seeking testing (tarantola and gruskin 2007) . there is evidence that knowledge of hiv status results in behavioral changes that reduce transmission. in addition, where knowledge of hiv status leads to antiretroviral treatment, treatment also reduces transmission by reducing the amount of virus in the body. thus, the criminalization of hiv transmission may have the effect of increasing, rather than reducing, hiv transmission. one possible response is mandatory hiv testing in health care settings (that is, testing without the informed consent of the patient). however, this policy, too, may be self-defeating if it discourages people from nations international guidelines on hiv/aids and human rights recommendation that public health legislation ensure that hiv testing of individuals should several studies have concluded that the criminalization of hiv transmission is unlikely to serve the goals of public health policy or the goals of criminal law, and ommended that governments and the judiciary take into account the following principles in determining policy regarding the use of criminal sanctions under modes and risk of hiv transmission to rationally determine when and if conduct should attract criminal liability; (2) the primary objective should be to prevent public health and conform to international human rights norms, particularly nondiscrimination and due process; and (4) policy makers should assess the impact of law or policy on human rights and prefer the least-intrusive measures possible to achieve a demonstrably justified objective of preventing disease transmission. with respect to the four functions of criminal law (harm prevention through response to the epidemic: (1) imprisoning an hiv-positive individual does not prevent transmission through conjugal visits or high-risk behavior with other prisoners; (2) criminal penalties are unlikely to change sexual activity and drug use, due to the complexity of these human behaviors; (3) punishment/retribution do not achieve the goal of hiv prevention and risk reinforcing prejudice and discrimination against already stigmatized hiv-positive people; and (4) criminal sanctions are unlikely to act as a deterrent, given that drug use and sexual activity persist even with the risk of criminal prosecution and are more likely to be driven underground when prosecuted, hindering hiv prevention. moreover, overly broad use of criminal laws risks spreading misinformation regarding how hiv is transmitted. in an empirical study conducted in the united states, burris et al. (2007) found that laws prohibiting unsafe sex or requiring disclosure of infection do not influence people's normative beliefs about risky sex and did not significantly influence sexual behavior. the study concluded that criminal law is not a clearly useful in-moreover, given concerns about possible negative effects of criminal law, such as stigmatization or reluctance to cooperate with health authorities, criminal law should be used with caution as a behavioral change mechanism for hiv-positive people. seeking health care. moreover, mandatory hiv testing runs counter to the united thus may do more harm than good. in a unaids policy paper, elliot (2002) bution; and deterrence), elliot (2002) concluded that criminal law is an ineffective imprisonment; prevention of future harm through rehabilitation; punishment/retri-hiv transmission in common law countries. in some cases, courts have applied existing criminal laws to cases involving hiv, where the laws themselves do not refer specifically to hiv. in this context, law reforms could come from the legislature, through amendments that clarify the application of relevant criminal laws to cases involving hiv, or through the evolution of precedents in the courts. the united nations international guidelines on hiv/aids and human rights recommend the reform of criminal laws and correctional systems to ensure that they are consistent with international human rights obligations and are not misused in tization of the judiciary, in ways consistent with judicial independence, on the legal, ethical and human rights issues relative to hiv, including through judicial education and the development of judicial materials (united nations 2007). criminal laws should not include specific offences against the intentional transmission of hiv but rather should apply general criminal offences to these exceptional cases. such application should ensure that the elements of foreseeability, intent, causality and consent are clearly and legally established to support a guilty verdict and/or harsher penalties (united nations 2007) . in the united states, a series of cases involving spitting have gone in different directions. in ohio v. bird (1998) , an hiv-positive man was convicted of felonious assault, which requires the knowing attempt to harm by use of a weapon capable of inflicting death, after spitting in a police officer's face, even though all medical and scientific evidence demonstrated that saliva does not transmit hiv. in state v. jones (2000) , another case of an hiv-positive individual accused of spitting on an officer, the new mexico court of appeals ruled that criminal liability for battery could not be based upon the victims' subjective and unsubstantiated fears that they could develop a disease, and reversed the lower court on this issue. in weeks v. state (1992) , the texas court of appeal sustained the attempted murder conviction of an hiv-positive inmate who spat in a guard's face. the spitting cases show how the application of criminal laws to hiv-positive individualswhen based on hiv status, stigma and discrimination rather than on medical or scientific evidence -can undermine genuine efforts to reduce hiv transmission by spreading misinformation and increasing stigma and discrimination. in cases involving behavior that does carry a risk of hiv transmission, such as unprotected sexual intercourse or sharing drug injection equipment, the central issue is consent. in r v. cuerrier (1998) the supreme court of canada established that there is a duty to disclose one's hiv status before engaging in any activity that poses a "significant risk" of hiv transmission. failure to do so legally invalidates a sexual partner's consent to sexual intercourse. the lack of consent to have intercourse with a partner that is hiv-positive converts the sexual intercourse into a criminal assault. in that case, the complainants did not become infected with hiv as a result of the unprotected sex. however, if the complainants believe that their partner is hiv-free and the accused puts the complainants at significant risk to their health, failure to disclose hiv status vitiates consent to sexual intercourse. 9 the way forward prevention, treatment and human rights there have been numerous cases in which criminal laws have been applied to the context of hiv/aids (united nations 2007). they also recommend the sensi-this decision suggests that there might not be a duty to disclose hiv status prior to engaging in activities that do not pose a significant risk of transmission, such as kissing and oral sex, or where an hiv-positive individual uses a condom. in r v. edwards, a lower court judge ruled that there is no duty to disclose hiv status prior to engaging in unprotected oral sex because it is a low risk activity (canadian aids society 2004) . on 15 november 1991, the defendant learned that he was hiv-positive, but did not reveal his status to the complainant and continued to have unprotected sex with her. the supreme court of canada ruled that the defendant was not guilty of the act itself, but rather the consequences of the act. because it was likely that the defendant had infected the complainant before he learned of his hiv status, it could not be proved beyond a reasonable doubt that he had endangered the life of the complainant. however, the defendant was guilty of attempted aggravated assault for continuing to have unprotected sex with the complainant after having learned of his hiv status. the court ruled that there is sufficient criminal intent for a conviction on a sexual assault charge if a person acts "recklessly". in canadian law, a person acts "recklessly" if they know that their conduct risks committing a crime but they commit the act nevertheless. in this case, the supreme court ruled that criminal recklessness is established once an individual becomes aware of a risk that he or she has contracted hiv, but continues to have unprotected sex without disclosure of hiv status, thereby creating a risk of further hiv transmission. in this case there was no evidence before the court regarding the defendant's awareness of the risk that he might be hiv-positive, prior to 15 november 1991, other than the fact that he had been asked to take an hiv test. this aspect of the ruling raised the issue of whether there is a duty to disclose the mere awareness of a risk that one might be hiv-positive before having unprotected sex. the court also suggested that an hiv-positive person might be held criminally liable for failure to disclose hiv status before having unprotected sex with another hivpositive individual, where this results in the transmission of a different strain of hiv or a drug-resistant strain of hiv. the supreme court of canada cases have been criticized, on the one hand, for discouraging people from seeking testing in order to avoid the possibility of a criminal conviction based on knowledge of hiv status and, on the other hand, for risking undesirable invasions of privacy if courts are required to determine whether an individual was aware that their past activities put them at risk of hiv infection (canadian hiv/aids legal network 2003) . however, in r v. williams, the fact that the defendant had been asked to take an hiv test, because he was on a list of former partners provided by an individual who had tested hiv-positive, was not sufficient to establish that he was aware that his past activities had put him at risk in r v. williams (2003) , the defendant began a sexual relationship with the comthe complainant". what distinguishes aggravated assault from mere assault is not 335 plainant in june 1991, in which they had unprotected sex on numerous occasions. requires that the assault "wounds, maims, disfigures or endangers the life of aggravated assault under section 268(1) of the canadian criminal code, which of hiv infection. nevertheless, the decision has been criticized for extending the without defining the nature of the awareness that might be required. more generally, the use of criminal law to prevent hiv transmission has been criticized for stigmatizing all hiv-positive people because of the conduct of a few individuals, for discouraging those most at risk from seeking testing and for being unlikely to stop people from having risky sex or sharing needles and syringes. moreover, all of the hiv-related criminal prosecutions in canada have occurred in the context of heterosexual intercourse, rather than homosexual intercourse or injection drug use, creating a perception of discriminatory application (or non-application) of the laws (betteridge 2007). sion of hiv/aids between 2003 and 2007, in which eight accused pleaded guilty, two were convicted and one was acquitted (klein 2007) . a new zealand court has ruled that people living with hiv/aids are not required to disclose their hiv status if they use condoms during vaginal sex (klein 2007) . in particular, the use of criminal laws to prevent hiv transmission also has been criticized for not taking into account that hiv-positive individuals living in abusive relationships may fear the consequences of disclosing their status to partners and may not be able to use a condom or insist that their partner use a condom (canadian aids society 2004) . in a literature review of hiv/aids and genderbased violence, the harvard school of public health program on international health and human rights (2006) found that gender-based violence (which is not limited to violence against women) can interfere with safe sex practices and access to treatment. not only is gender-based violence a risk factor for acquiring hiv/ in summary, the use of criminal laws to prevent hiv transmission may undermine overall public health initiatives by: (1) reinforcing hiv/aids-related stigma; (2) spreading misinformation about hiv/aids; (3) creating a disincentive for hiv testing; (4) hindering access to counseling and support services; (5) creating a false expectation that criminal laws eliminate the danger of unprotected sex for people who believe that they are hiv-negative; (6) creating the risk of selective prosecution of marginalized groups; (7) criminalizing behavior that results from gender inequality, in the case of hiv-positive people living in abusive or economically dependent circumstances; and (8) invading privacy through the disclosure of medical records and hiv status in public court proceedings (elliot 2002) . however, the use of criminal laws may be warranted in some circumstances, where hiv status is an aggravating or otherwise relevant factor in cases involving physical assault that would constitute criminal behavior even in the absence of hiv, such as rape or the use of needles as weapons (elliot 2002) . finally, a distinction should be made between criminal laws and public health laws that are quasi-criminal in nature, particularly those regarding quarantine. while quarantine laws, such as isolation, detention or quarantine, may be suitable 9 the way forward prevention, treatment and human rights criminal law beyond cases where individuals know that they are hiv-positive, in the united kingdom, there were eleven prosecutions for reckless transmis-aids, but hiv/aids is also a risk factor for gender-based violence. for casually communicable and curable diseases, such laws run the same risk of misuse as do criminal laws (elliot 2002) . in this regard, the united nations international guidelines on hiv/aids and human rights recommend that public health law provisions applicable to casually transmitted diseases not be applied inappropriately to hiv/aids and that they be consistent with international human rights obligations (united nations 2007). some countries have restricted the entry of people living with hiv/aids, for shortterm or long-term stays, through mandatory testing or a requirement to declare one's hiv status. as we saw in chap. 8, the who international health regulations also contain provisions regarding health measures applied to travelers. these provisions encourage states to base their determinations upon scientific principles, available scientific evidence of a risk to human health and any available specific guidance their dignity, human rights and fundamental freedoms and minimize any discomfort or distress associated with such measures. governments cite two main reasons for imposing travel restrictions on people living with hiv/aids -public health protection and reducing demand on health care and social services (unaids/iom 2004) . in the united kingdom, another source of demands for hiv screening of migrants has been a concern over "health tourism" -hiv infected migrants from developing countries that go to europe to receive health care. however, research shows that access to treatment is rarely the after having arrived in the host country, and there is no uniform policy in european union countries regarding screening of migrants for hiv (carballo 2007) . hiv/aids is not considered to be a condition that poses a threat to public health in relation to travel because hiv/aids is already present in virtually every country in the world and hiv is not transmitted through casual contact. unlike highly contagious diseases with short incubation periods, such as sars, cholera and plague, hiv transmission can be prevented through safe sex and safe drug injection, which can be used by both the infected and the non-infected to prevent transmission. there is no evidence to support the assumption that both the infected and the non-infected will engage in unsafe practices. as a result, the presence of hiv-positive individuals, by itself, does not pose a risk to public health. in addition, travel restrictions are not effective in preventing the entry of hiv-positive individuals, since hiv tests do not detect the virus in newly infected people and nationals that are returning from travel abroad (who may have been infected while outside the country) are not subject to hiv/aids-related travel restrictions and are not prevented from entering their own country. moreover, travel restrictions can undermine hiv/aids-related public health initiatives by increasing stigma and discrimination and mislead the public into thinking that hiv/aids can be or advice from the who. they also require states to treat travelers with respect for reason for migration to europe, since most migrants only learn of their hiv status prevented through border measures, rather than through proven prevention strategies (unaids/iom 2004) . unaids and the international organization for migration (iom) recommend that exclusion on the basis of possible costs to health care and social services only occur on an individual basis, where the following considerations are shown: (1) the person requires the health care and social services and is likely to use them in the near future; (2) the person has no other means of meeting those costs (for example, through private or employment-based insurance or personal resources); and (3) these costs will not be exceeded by the benefits of the person's skills, talents, contribution to the labor force, payment of taxes, contribution to cultural diversity and capacity for revenue or job creation (unaids/iom 2004) . they also recommend that countries treat similar conditions alike, rather than singling out hiv/ aids. one study showed that the 10-year economic impact of admitting immigrants with asymptomatic hiv infection would be similar to admitting immigrants with asymptomatic coronary heart disease (zowall et al., 1994) . the canadian immigration and refugee protection act provides that foreign nationals can be deemed "medically inadmissible" based on a medical condition, danger to public health or public safety; or (2) they might reasonably be expected to cause excessive demand on health or social services. since 1991, canadian danger to public health or public safety by virtue of their hiv status. the issue of excessive demand on health or social services is mainly a consideration in cases of immigration or stays that exceed 6 months, is determined on a case-by-case basis permanent residents (spouses and children). demand on health or social services of health or social services for the average canadian resident; or (2) the demand the united states has had a travel and immigration restriction in place for people living with hiv/aids since 1987 (human rights watch 2006) . under the united states are inadmissible if they have "a communicable disease of public high level meeting on aids a "designated event" for which an hiv waiver would be available. visitors entering the united states on the visa waiver program (which waives the requirement to apply for a visa prior to traveling to the united 9 the way forward prevention, treatment and human rights government policy has been that people living with hiv/aids do not represent a and therefore denied a visa or entry at the border, if: (1) they are likely to be a 338 would add to existing waiting lists for those services and would increase the rate us immigration and nationality act, applicants for a visa or for admission to the health significance", which includes hiv infection, although waivers are available ces by canadian citizens or permanent residents. the social or economic contribu-and does not apply to refugees or close family members of canadian citizens or tions the individual is expected to make to canada are not taken into account. or mortality and morbidity in canada by denying or delaying access to those servihiv status or to be tested for hiv (canadian hiv/aids legal network 2007a) . on a case-by-case basis. for example, the us attorney general named the 2006 people entering canada for less than 6 months are not required to disclose their is considered excessive if: (1) the anticipated costs would likely exceed the costs states, for certain countries) must fill out an i-94w form, which asks, "have you ever been afflicted with a communicable disease of public health significance." if the visitor answers yes to the question or the us border authorities suspect a visitor to be hiv-positive the person may be: (1) placed into secondary inspection; (2) questioned by an official of the us department of homeland security; (3) placed into deferred inspection; (4) asked to withdraw the application for admission into the united states; (5) placed into the expedited removal process; or (6) placed into an us department of homeland security detention center and detained until the case is heard by an immigration judge (gmhc 2006) . hiv-positive non-immigrants seeking to enter the us on a temporary basis for business, pleasure, or education are eligible for a waiver under which they can be allowed to enter the united states. in practice, a waiver is granted in most cases if: (1) they are not symptomatic; (2) it is a short visit; (3) they have insurance or other assets sufficient to pay medical expenses; and (4) they don't appear to be a public health risk. permanent residency and immigration applicants can also apply for a waiver, but they are usually rejected. to receive a waiver as an immigrant, the person must be the spouse, unmarried son or adopted child of a united states citident as their son or daughter. in addition, an hiv-positive immigration applicant must prove that: (1) he will not be a danger to public health; (2) the possibility of spreading the disease is minimal; and (3) there will be no cost incurred by any level of government without its prior consent (tarwater 2001) . june 1987 the us public health service added aids to the list of excludable conditions, noting that the exclusion was not based on any new scientific knowledge and that aids is not spread by casual contact, which is the usual public concept of contagious. in july 1987, republican senator jesse helms also added hiv infection to the exclusion list, through the us congress, together with a prohibition on funding from the us centers for disease control for aids programs that "promote, encourage or condone homosexual activities" (koch 1987; aids treatment news 1991) . senator helms accompanied the introduction of his amendments with the following statement: "we have got to call a spade a spade, and a perverted human being a perverted human being" (koch 1987) . in july 1995, senator jesse helms advocated spending less money on hiv/aids, because it resulted from "deliberate, disgusting, revolting conduct" and was "a disease transmitted by people deliberately engaging in unnatural acts" (associated press 1995). ten years later, he had this to say: "it had been my feeling that aids was a disease largely spread by reckless and voluntary sexual and drug-abusing behavior, and that it would probably be confined to those in high-risk populations. i was wrong" . in 1990, the us centers for disease control (cdc) recommended that all diseases except active tuberculosis be removed from the list of excludable conditions. hiv was left on the list because it had been put on the list by congress. in november the political history of the us hiv travel restrictions is an interesting story. in zen or permanent resident or have a united states citizen or lawful permanent resi-1990, the immigration reform act of 1990 directed the cdc to establish a new list of excludable conditions, based solely on current epidemiological principles and medical standards. in january 1991, the cdc again proposed that only active tuberculosis remain on the list of excludable conditions. religious leaders campaigned to maintain the ban and the us house of representatives opposed removing the hiv ban (aids treatment news 1991) . in august 2007, democratic representatives barbara lee and hilda solis introduced the "hiv nondiscrimination in travel and immigration act". the proposed legislation would restore the authority of the secretary of health and human services to determine whether hiv status is a communicable disease of public health significance. the decision to maintain or remove the ban would then be based on public health analysis instead of a formal ban made by congress (latino commission on aids 2007). in november 2007, the us department of homeland security proposed a new rule that would allow short-term visas to be granted to hiv-positive people by us consulates in their home countries. however, applicants would have to agree to conditions, including ceding the right to apply for longer stays or permanent residency in the united states. democratic members of the us house of representatives objected that the changes would only shift decision-making authority to local consular officers, who may lack the appropriate medical expertise. moreover, there would be no appeal process (werner 2007) . the united states and canada are similar societies, both culturally and economically, but have adopted very different approaches to hiv/aids travel restrictions. the hiv prevalence rate in the united states is higher than in canada. this suggests that the us travel restriction has not been effective in preventing hiv transmission in the united states, and that the lack of such a restriction in canada has not had the effect of increasing hiv prevalence. health care costs, measured as a percentage of gdp, are also higher in the united states than in canada. while this difference is attributable to many factors, making it difficult to determine the impact of the different travel restriction policies on health care costs without further study, it is an indication that the canadian approach has not led to a significant increase in health care costs compared to the american approach. in 2003, americans spent usd 5,711 per capita on health care, compared with usd 2,998 in canada. americans spent 15.2% of gdp on health care compared with 9.9% of gdp in canada. interestingly, this gap was not always there. in 1970, both countries spent exactly 7.0% of their respective gdp on health care (oecd 2006) . another factor that suggests that us travel restrictions are unlikely to prove successful is illegal immigration. there are several million illegal entries into the united states each year. they are obviously not screened. thus, from a practical point of view, travel and immigration restrictions for hiv-positive individuals are unlikely to be effective in preventing the entry of many hiv-positive individuals and may provide additional incentives for some individuals to migrate illegally. the united nations international guidelines on hiv/aids and human rights recommend that states enact or strengthen anti-discrimination laws that protect vulnerable groups, people living with hiv/aids and people with disabilities from discrimination in both the public and private sectors, and provide for speedy and effective administrative and civil remedies (united nations 2007). human rights laws in many jurisdictions prohibit discrimination against vulnerable groups or against people with hiv/aids, as well as providing other rights that are relevant to hiv/aids, such as the right to life and the right to health. human rights laws fall into two categories. the first category applies to governments, prohibiting governments from passing discriminatory laws or requiring governments to uphold certain human rights. the second category of human rights law prohibits discrimination on the part of private actors, for example with respect to employment practices or rental of housing. while it is not possible to eliminate individual or societal prejudices with legislation, human rights laws provide victims of discrimination with legal recourse against acts of discrimination and create economic disincentives through fines or other legal remedies, thereby contributing to social change. canada provides one example of the sources and functioning of human rights laws. section 15 of the canadian charter of rights and freedoms, which is part of the constitution of canada, guarantees equality rights in the following terms: the equal protection and equal benefit of the law without discrimination and, in particular, without discrimination based on race, national or ethnic origin, colour, religion, sex, age or mental or physical disability. canadian courts have interpreted the term "disability" to include hiv/aids, which means that people living with hiv/aids have constitutional protection listed, but also covers analogous grounds, such as sexual orientation. any law that is inconsistent with constitutional provisions may be struck down or interpreted by courts to make it consistent with the constitution. the charter applies to all levels and branches of government, all government acts, government corporations and ment government policies or programs. however, the charter does not otherwise apply to acts by private citizens. instead, discrimination by an employer, a landlord or a private business is addressed under other federal and provincial human rights laws, such as the canadian human rights act, which apply to both the public and private sectors. by virtue of a 1996 policy of the canadian human rights commission and decisions of canadian courts and tribunals, the prohibition against disability-based discrimination in the every individual is equal before and under the law and has the right to against discrimination by the state. section 15 is not limited to the grounds that are private persons or bodies that exercise authority granted by a statute or that imple-canadian human rights act and its provincial counterparts cover discrimination based on hiv/aids status (elliott and gold 2005) . the remainder of this section provides an overview of court cases in a variety of countries that have applied constitutional law, international law and other legislation to uphold the rights of people living with hiv/aids with respect to employment and access to hiv-related medical care and treatment. in march 2007, mexico's national supreme court of justice ruled that a provision in article 226 of the social security institute law for the armed forces (issfam) that required hiv-positive individuals to be discharged from the military was unconstitutional, because it was not based on an individual assessment of the pering people living with hiv/aids. the court ordered that three soldiers be reinduty, which would include an obligation to reinstate their social security benefits with respect to hiv/aids, laws in south africa and latin america that provide a action campaign used this provision to challenge the government's program that limited the use of nevirapine to prevent mother-to-child hiv transmission to 18 test sites. the court ruled that the government's restriction on the use of nevirapine was unreasonable and that the policy should be reformed to meet the government's constitutional obligation (singh et al., 2007; elliot et al., 2006) . in argentina, five court cases between 1996 and 2003 repeatedly ordered the argentine ministry of health to supply antiretroviral treatment to people living with hiv/aids, in accordance with the right to health set out in international treaties, which had been incorporated into domestic law. the failure of the ministry of health to act in a timely fashion, which led to interruptions in the supply of antiretroviral drugs, ultimately led to a court order that would fine the ministry of health usd 1,000 per day (funds which would then be used to implement the national aids plan) until it complied with the courts' previous orders, and the threat right to health care have been used to induce governments to provide access to and equality and was inconsistent with mexico's international obligations regardvides a right to health care that is binding on the government. the treatment mexico's national supreme court of justice ruled for a fifth time that this provision was unconstitutional, thereby creating jurisprudence that is binding on all federal son's ability to work, violated constitutional protections of non-discrimination judges in mexico (avilã©s allende 2007). table 9 .8 summarizes several other antiretroviral treatment (gruskin et al., 2007) . the south african constitution pro-cases involving hiv-related discrimination in employment from various juris(pearshouse 2007; medina and reyes 2007; scjn 2007a, b) . in september 2007, dictions around the world. stated until medical certificates were issued to determine whether they were fit for (elliot et al., 2006) . an argentine court also relied on the right to health set out in international treaties to order the government to produce and administer a vaccine within a set period of time, in order to protect people living in a region affected by argentine haemorrhagic fever (singh et al., 2007) . the constitutional court of ecuador relied on the right to health set out in international treaties to rule that the ministry of health had failed to meet its obligations when it suspended its hiv treatment program (singh et al., 2007; elliot et al., 2006) . in costa rica, the supreme court ruled in 1997 that the costa rican social security fund could not argue that financial constraints justified failure to comply with its very reason for its existence, which is to provide coverage for necessary medical care. shortly after this ruling, the supreme court ordered the social security fund to develop a plan to provide coverage to all persons living with hiv/aids that were in need of antiretroviral treatment. a few weeks later, costa rica became the first central american country to include cov2006) . in india, the courts have interpreted the right to life in the indian constitution to sources to uphold the right to health in a variety of cases (singh et al., 2007) . table 9 .9 summarizes several other cases from various jurisdictions around the world where litigation has increased access to hiv-related medical treatment. these cases suggest that human rights laws can be instrumental in promoting health care reforms through litigation, provided that judicial authorities are independent and competent and governments respect the rule of law (singh et al., 2007) . in addition to laws that institutionalize or prohibit discrimination, institutional policies and practices can represent an important force with respect to stigma, discrimination and access to health care. the united nations international guidelines on hiv/aids and human rights recommend that states ensure that government and the private sector develop codes of conduct regarding hiv/aids issues that translate human rights principles into codes of professional responsibility and practice, with accompanying mechanisms to implement and enforce those codes. in many jurisdictions, the courts have the power to order changes in policies and practices of both governmental and non-governmental institutions. however, litigation is an expensive and time-consuming process that creates additional stress for the people living with hiv/aids who choose to litigate. thus, it is important to promote the voluntary adoption of appropriate policies and practices. one example in this category is the policies and practices of health care institutions. for example, in the mid 1980s, in british columbia, canada, all hospitals refused to treat aids patients, with the exception of st. paul's hospital, which adopted appropriate policies based on the commitment of the founding sisters of 345 include a right to health, and have obliged the indian government to dedicate re-erage for antiretroviral drugs in its national health insurance plan (elliot et al., another example in this category is the policies and practices of employers. as we showed in chap. 3, hiv/aids affects the productivity of workers substantially, making it cost effective for companies to have prevention programs and to provide treatment for employees, from a purely financial point of view. business leaders have an economic incentive to invest resources in fighting the epidemic. moreover, as we saw in chap. 7, firms can have a tremendous impact in promotment. however, it is important to have an overarching framework that ensures the adoption of best practices by individual firms and to minimize overlap between the private sector and the other players that are involved in addressing the pandemic. in this regard, the global business coalition on hiv/aids has provided leadership, particularly in its efforts to identify ways to improve the global business community's response to hiv/aids, including through leadership to dispel myths and stigma, break down workplace barriers and influence community change. given the economic and legal incentives, an effective hiv/aids response 9 the way forward prevention, treatment and human rights providence to care for all who were in need, regardless of financial or social standing (gratham 2007) . in 2006, the city of philadelphia agreed to resolve a complaint regarding the refusal of emergency medical services personnel to touch or lift a patient because of his hiv status, by paying monetary compensation and agreeing to implement a mandatory paramedic/emt training program on hiv and infectious diseases (john gill smith and united states v. city of philadelphia 2006) . ironically, "philadelphia" was the name and setting of the first high-profile hollywood film to take aids seriously, in 1993. we can think of hiv/aids as a disaster from the point of view of a country as a whole. unlike other disasters (such as an outbreak of an influenza pandemic), this kind of risk, we need to measure the severity and the frequency of occurrence of that risk. once we measure the risk, we need to find ways of managing the risk in a dynamic way. that means putting a risk management plan in place, monitoring the plan and modifying the plan as events unfold. most often, at the national level, hiv/aids is seen as a public health problem and is managed as such. thus, various measures are taken to reduce the incidence of hiv/aids by taking steps against the main channels through which the disease strikes: (1) actions to reduce the contamination of the blood supply; (2) special steps to promote health care for key groups, such as sex workers; (3) needle exchange programs; (4) promoting safe sex through the use of condoms; and (5) minimizing hiv transmission from infected mothers to newborns. 346 disaster unfolds over many years. however, the standard operating procedure for ing prevention among employees and their families and providing access to treatdisaster management also applies to managing hiv/aids risk. for managing any must be a core component of an overall business strategy. another approach to risk management is risk avoidance. at the country level, risk avoidance could imply two extreme actions: quarantining people who are already infected and preventing infected people from coming into the country. neither of these policies is feasible for most countries, as they directly go against human rights. thus, extreme forms of risk management and the respect for human rights pose a tradeoff for a country. cuba provides a striking example of how containment of hiv/aids can be conducted at a national level. cuba started promoting public health messages against hiv/aids in 1983, 3 years before the first hiv case was reported in the country. between 1986 and 1989, cuba undertook a massive testing exercise, which tested more than 80% of the adult population. those who were seropositive were quarantined indefinitely in sanitariums. over the years, cuba has relaxed the rule. today, anybody found seropositive is required to attend an 8 week course. after that, they are free to leave. nearly half the people choose to stay in the sanitariums, where they get free food and a place to stay, along with retraining if they choose to help with the logistics of the sanitariums. such a curtailment of freedom of movement without committing a crime is unprecedented anywhere in the world. it has been criticized by many. it did produce a result that is also unprecedented. cuba has an hiv incidence rate of 0.05%. in the neighboring island of haiti, the rate is 120 times as high, at 6.1%. it should be noted that quarantine of individuals who have committed no crime is not unheard of. there was the case of mary mallon in the united states in 1908better known as the "typhoid mary" -who carried typhoid without every showing any symptoms. she was quarantined against her will for a number of years. similarly, during the outbreak of influenza in the united states in 1918, many families were quarantined on public health grounds. individuals with sars were also quarantined in toronto. the future of hiv/aids presents a mixed picture. while hiv/aids incidence has begun to level off in some high-prevalence countries, new infections have increased in many developed countries. while several science-based prevention strategies need to be scaled up significantly, the increase in mother-to-child prevention has dramatically reduced infections among newborns and male circumcision is a promising new prevention strategy. while millions still lack access to treatment, there has been a large increase in funding, drug prices have dropped dramatically, several key drug patents will expire in the near future and efforts to develop new treatments continue. while stigma and discrimination remain obstacles to effective prevention and treatment, human rights laws have proved to be an effective vehicle for addressing discrimination and increasing access to treatment around the world. thus, while hiv/aids continues to pose a significant threat to public health, there are many signs that progress in fighting this pandemic can and will continue, as knowledge gradually replaces ignorance. travel/immigration ban: background senator jesse helms: cut aids funding experimental aids vaccine falls short randomized, controlled intervention trial of male circumcision for reduction of hiv infection risk: the anrs 1265 trial amplã­a corte protecciã³n a militares con vih male circumcision: the road from evidence to practice. division of epidemiology, school of public health, university of illinois at chicago betteridge g (2007) criminal law and hiv transmission or exposure: new cases and developments law as a structural factor in the spread of communicable disease the way forward prevention, treatment and human rights do criminal laws influence hiv risk behavior? an empirical trial hiv disclosure & the criminal law in canada supreme court of canada decision in r v canada's immigration policy as it affects people living with hiv/aids a human rights-based commentary on unaids guidance note: hiv and sex work communicable diseases: challenges for health in the age of migration 2c1fe1c/10624/chpt4eu.pdf. accessed a%20human%20rights-based%20commentary%20on % 20unaids %20guidance % 20 zidovudine's patent history look to the future: the war against aids. the economist net benefits: giving bed nets and drugs away free may be the way to deal with malaria criminal law, public health and hiv transmission: a policy options paper protection against discrimination based on hiv/aids status in canada: the legal framework the effect of pregnancy on survival in women infected with hiv: a systematic review of the literature and meta-analysis global hiv prevention working group (2007) bringing hiv prevention to scale: an urgent global priority history, principles and practice of health and halperin d (2007) evidence-based behavior change hiv prevention approaches for sub-saharan africa harvard school of public health program on international health and human rights hamied yk (2005) trading in death lessons not learned: human rights abuses and hiv/aids in the russian federation family, unvalued: discrimination, denial, and the fate of binational same-sex couples under memoir, jesse helms says he was no racist a salute to the sisters. promise fall/winter literature review increasing access to hiv testing and counseling while respecting human rights reasons why human rights should occupy the center of the global aids struggle. open society institute the way forward prevention, treatment and human rights conspiring to kill: gender-biased legislation, culture, and aids in sub-saharan africa new hiv cases drop but rise in young gay men. www.nytimes senator helms's callousness toward aids victims the latino commission on aids supports the hiv nondiscrimination in travel and immigration act discriminaciã³n por vivir con vih: fuerzas armadas, a punto del revã©s, letra s 128 the known hidden hiv/aids epidemic among black men who have sex with men in the united states plos medicine 4:12 nyc health (2007) hiv epidemiology and field services research unit report accessed 5 november human rights watch (2006) family, unvalued: discrimination, denial, and the fate park a (2007) assessing a failed aids vaccine mexico: supreme court rules discharge of hiv-positive troops unconstitutional genetic diversity of hiv-1: the moving target seeds of change in rwanda sesiã³n pãºblica nãºm the tuberculosis & hiv debate in immigration law: critical flaws cuerrier (1998) 2 scr 371, supreme court of canada new mexico court of appeals, 129 nm 165 ohio state court appellate amicus brief us food and drug administration (2006) guidance for industry fixed dose combina antiretrovirals for the treatment of hiv /nr/rdonlyres/98ef667e-cf50-4abb-ab9f-107 adf012927/0/265demarzode do human rights matter to health? lancet marde2007 state (1992) texas court of appeal united states academic anti-exclusion arguments. georgetown immigration law journal hivlawandpolicy.org /resources /partial %20case %20&resource %20 list-lambda packaged drug products, and single-entity versions of previously approved tarantola d, gruskin s (2007) new guidance on recommended hiv testing and hiv_data/2006globalreport/default.asp. accessed the way forward prevention, treatment and human rights consolidated version new law allows needle exchanges in washington modeling health care costs attributable to hiv infection and hiv prevention programs in high-income countries systematic review of abstinence-plus aids epidemic -planning, policy and predictions limited setting: treatment guidelines for a public health approach scaling up antiretroviral therapy in resouurce lawmakers, gay-rights groups protesting new hiv/aids travel unaids/iom (2004) statement on hiv/aids-related travel restrictions international guidelines on hiv/aids and human rights key: cord-000638-ss1435el authors: beq, stephanie; rozlan, sandra; pelletier, sandy; willems, bernard; bruneau, julie; lelievre, jean-daniel; levy, yves; shoukry, naglaa h.; cheynier, rémi title: altered thymic function during interferon therapy in hcv-infected patients date: 2012-04-16 journal: plos one doi: 10.1371/journal.pone.0034326 sha: doc_id: 638 cord_uid: ss1435el interferon alpha (ifnα) therapy, despite good efficacy in curing hcv infection, leads to major side effects, in particular inducement of a strong peripheral t-cell lymphocytopenia. we here analyze the early consequences of ifnα therapy on both thymic function and peripheral t-cell homeostasis in patients in the acute or chronic phase of hcv-infection as well as in hiv/hcv co-infected patients. the evolution of t-cell subsets and t-cell homeostasis were estimated by flow cytometry while thymic function was measured through quantification of t-cell receptor excision circles (trec) and estimation of intrathymic precursor t-cell proliferation during the first four months following the initiation of ifnα therapy. beginning with the first month of therapy, a profound lymphocytopenia was observed for all t-cell subsets, including naïve t-cells and recent thymic emigrants (rte), associated with inhibition of intrathymic precursor t-cell proliferation. interleukin (il)-7 plasma concentration rapidly dropped while lymphocytopenia progressed. this was neither a consequence of higher consumption of the cytokine nor due to its neutralization by soluble cd127. decrease in il-7 plasma concentration under ifnα therapy correlated with the decline in hcv viral load, thymic activity and rte concentration in blood. these data demonstrate that ifnα-based therapy rapidly impacts on thymopoiesis and, consequently, perturbs t-cell homeostasis. such a side effect might be detrimental for the continuation of ifnα therapy and may lead to an increased level of infectious risk, in particular in hiv/hcv co-infected patients. altogether, this study suggests the therapeutic potential of il-7 in the maintenance of peripheral t-cell homeostasis in ifnα-treated patients. the hepatitis c virus (hcv) causes persistent infection in approximately two thirds of cases leading to chronic liver disease, liver failure, and, eventually, hepatocellular carcinoma in a substantial proportion of infected individuals. the most common therapy for chronic hepatitis c consists of pegylated interferon-a (ifna) and ribavirin administration which results in viral clearance in 43-46% (genotype 1) to 80%, (genotype 3) of treated patients [1] . interferon will continue to be a major component of new direct acting antivirals for treatment of hcv [2] . ifna is produced in large amounts during the acute phase of many viral infections [3, 4, 5, 6] . direct activation of interferonstimulated genes enhances naïve t-cell survival through increased bcl-2 and reduced bax activation [7] and contributes to clonal expansion of antigen-specific t-cells [8] . recent data suggest that early therapeutic intervention with pegylated ifna rescues polyfunctional memory t-cells expressing high levels of the il-7 receptor alpha chain (cd127) and bcl-2, allowing a higher rate of sustained viral response [9] . however, despite good efficacy, ifna-based therapies lead to sustained anemia, thrombocytopenia, neutropenia and lymphocytopenia [10, 11, 12, 13, 14] . moreover, pegylated ifna therapy enhances the risk of infection in older hcv-infected patients and hiv-infected individuals, independently from neutropenia [15, 16, 17] . the mechanisms of action of ifna include inhibition of different hematopoietic growth factors [18, 19] , possibly affecting lymphoid differentiation at an early stage [20] , and modifications in cell homing [12, 21, 22] . the mechanisms involved in ifna therapy-associated leukocyte depletion remain poorly understood. others and we have documented a strong reduction in the ability of hiv-infected patients to sustain thymic production as a direct consequence of a drop in intrathymic precursor t-cell proliferation [23, 24, 25] . similar thymic impact was also seen during early siv-infection in the rhesus macaque model [26] . the capacity of the thymus to produce recent thymic emigrants (rtes) is, in large part, dependent on thymocyte proliferation [27] . indeed, extensive thymocyte proliferation occurs between t-cell receptor beta (tcrb) and alpha (tcra) chain rearrangements. the extent of this proliferation directly correlates with thymic export [28] . the extent of cell proliferation in the thymus can be measured in patients through estimating, in peripheral blood cells, the ratio (sj/btrec ratio) between the frequency of signal joint t-cell receptor excision circles (sjtrec), produced during the excision of the tcrd locus prior to tcra chain rearrangement, and that of dbjbtrec t-cell receptor excision circles (trecs) produced during tcrbd to tcrbj rearrangement [29] . these by-products of tcr rearrangement processes are generated by the circularization of the chromosomal dna excised during tcr rearrangements respectively occurring at the dn3 (dbjbtrec) and dp (sjtrec) stages of differentiation. due to the fact that trecs do not replicate during mitosis, increased proliferation between dn3 and dp leads to the reduction of dbjbtrec frequency in rtes as compared to sjtrec frequency and consequently to an increase of the sj/btrec ratio [23] . the correlation between initial plasma ifna levels and the speed of thymic dysfunction observed during hiv primary infection suggested that ifna, produced as part of the innate immune response to infection, participates in the impairment of thymopoiesis. however, no direct evidence of the relationship between ifna production and thymic dysfunction was provided by these studies. in contrast, arizcorreta and colleagues showed that ifna and ribavirin therapy induces a substantial reduction of circulating sjtrecs, in hiv/hcv co-infected patients, accompanied by sustained naïve cd4 + t-cell defect, suggesting thymic dysfunction [10] . similarly, in the siv-infected rhesus macaque model, we showed that ifna therapy induced a strong decrease of circulating rte numbers as defined either by sjtrec frequency and numbers or by cd31 hi expression on naïve t-cells [30] . interestingly, in these animals, recombinant interleukin (il)-7 therapy more than abrogated the deleterious effects of ifna therapy [30] . il-7 is a key cytokine implicated at various levels of thymocytes differentiation. it allows cell survival during the rearrangement processes, and is implicated in the extensive thymocyte proliferation, in particular in intermediate single positive (isp) and early dp cells [31, 32, 33, 34] . this proliferation participates in the development of naïve t-cell diversity [35] . while up regulated by ifna [36, 37] , the cyclin-dependent kinase inhibitor p27/kip1 is negatively regulated by il-7 [38] , allowing isp and early dp thymocytes to proliferate. moreover, ifna also inhibit il-7 dependent proliferation through down modulation of the common c chain, that participates, together with cd127 to the il-7 receptor [39] . we here investigated the early impact of ifna therapy on thymic function and naïve t-cell homeostasis in both hcv-infected and hiv/hcv co-infected patients who started ifna therapy. we first evaluated the evolution of naïve t-cell subsets in three groups of hcv infected individuals: 1) acute hcv infection (n = 8), defined as ,6 months post estimated date of infection; 2) chronic hcv infection (n = 8), defined as .6 months post estimated date of infection; and 3) hiv/hcv co-infected individuals (n = 10). in all groups, patients were enrolled at the beginning of ifna therapy and were followed for a total of 4 months. while, for any group of patient's, naïve cd4+ and cd8+ t-cell counts were not significantly different from healthy individuals (figure 1a), as early as one month following treatment initiation, naïve cd4+ t-cell counts were significantly reduced in chronically hcv-infected patients (39%, 58%, 46% and 35% decrease at m1, m2, m3 and m4 respectively; p#0.025; figure 1b , top central panel). a similar trend was also observed in the cd8 compartment (40%, 39%, 33% and 33% decrease; figure 1b , bottom central panel). a comparable effect was also observed in most co-infected patients (mean cell count declines were 19%, 32%, 52% and 43% at m1, m2, m3 and m4 in the cd4+ t-cell compartment and 9%, 21%, 41% and 42% in cd8+ t-cell subset; p#0.05 by m2-m3; figure 1b , right panels). in contrast, naïve t-cell counts were only barely affected in acutely-hcv infected patients under ifna therapy ( figure 1b , left panels). similarly, central memory cd4+ t-cells (cd45 ra-ccr7+; tcm) demonstrated 38% and 28% decrease in hcv and hiv/hcv patients respectively (59% and 60% in cd8+ tcm) while effector memory (cd45ra2 ccr7-; tem) cd4+ t-cell counts declined by 45% and 10% in the same groups (61% and 65% in cd8+ tem) ( figure s1 ). within cd4+ naïve t-cells, rtes can be identified by their higher expression of the platelet endothelial cell adhesion molecule-1 (pcam-1 or cd31) [40] . while the number of rtes was similar in hcv-infected patients at study entry and healthy individuals ( these data demonstrate that, as early as one month following treatment initiation, ifna induces stronger alterations of naïve tcell subsets, and more specifically in the rte compartment than in any other t-cell subset, suggesting a specific effect on thymopoiesis. we thus analyzed the evolution of intrathymic precursor t-cell proliferation, peripheral t-cell cycling, il-7 plasma concentration and il-7 receptor alpha chain (cd127) expression, different factors affecting naive t-cell homeostasis. despite differences between the 3 groups at study entry, rte cycling rate, as estimated through measurement of ki-67 expression, did not change significantly during the follow-up period ( figure 3a ). these data demonstrate that the observed changes in sjtrec frequencies were not a consequence of variations of rte proliferation during ifna therapy but more probably due to reduced thymic production. we thus estimated thymic output through quantification of the sj/btrec ratio in all groups of patients ( figure 3b ). the sj/ btrec ratio estimates the extent of thymocyte proliferation between tcrb rearrangement and the excision of the t-cell receptor delta (tcrd) locus [23] . this parameter directly reflects the extent of thymic production and, contrarily to sjtrec values, is independent from peripheral rte proliferation or survival capacity [28] . the sj/btrec ratio was already low in hivinfected patients (p,0.005 as compared to healthy control donors; figure 3b bottom left panel) and did not evolve further under ifna therapy in co-infected patients ( figure 3b , bottom right panel). in contrast, acutely hcv-infected patients demonstrated higher than normal sj/btrec ratio at baseline (p,0.05 as compared to aged matched healthy controls), showed a significant reduction in sj/btrec ratio at m1 (p = 0.014) and m2 (p = 0.001; figure 3b , top panel). finally, a similar decline in the sj/btrec ratio was observed during ifna therapy in chronically hcv-infected patients (p,0.02 at m1, m2 and m3; figure 3b , central panel). precursor t-cell proliferation in the thymus is, at least in part, dependent upon il-7. we thus quantified plasma il-7 concentration in all groups of patients. at study entry, hcv-and hiv/ hcv-infected patients presented with elevated plasma il-7 (median = 10.3 pg/ml, range (6.7-12.9) in acutely hcv-infected patients; 8.3 pg/ml (6.3-10.5) in chronic hcv-infected patients and 7.15 pg/ml (4.3-13.5) in co-infected subjects), as compared to that observed in healthy control individuals (p,0.001 for any patients' group; figure 4a) . surprisingly, while lymphocytopenia established, il-7 plasma concentrations significantly decreased in both groups of hcv-infected patients (30, 54, 18 and 29% decrease at m1 to m4 in acute infection, p,0.05; 25, 46, 26 and 16% decrease at m1 to m4 in chronic infection, p,0.05; figure 4b left and central panels). in contrast, il-7 plasma levels did not significantly evolve in co-infected individuals during the first month of ifna therapy ( figure 4b right panel) . only patients with the highest il-7 plasma levels showed a reduction in the concentration of this cytokine. decreased plasma il-7 concentrations could be a consequence of reduced il-7 production, increased consumption by t-cells or sequestration by soluble il-7 receptor (scd127). in both hcvinfected and hiv/hcv co-infected patients, neither scd127 considering the variations in all the parameters we used to evaluate thymic function, we then sought to evaluate the impact of changes in il-7 plasma levels on de novo production from the thymus and on the number of both sjtrec and circulating cd4+ rtes. in a majority of patients, il-7 plasma level, sj/btrec ratio, sjtrec/ml and blood rte concentration fluctuated in parallel ( figure s2 ). variation of il-7 plasma concentration (dil-7) during the first month of therapy correlated with variations in naïve t-cell counts (cd4+ + cd8+; dnaïve t-cell counts) and rte cd4+ t-cell counts (drte t-cell counts) in both hcv (r = 0.521, p = 0.039 and r = 0.595, p = 0.025; figure 5a and 5b, left panels) and, to a lesser extent, hiv/hcv co-infected patients (r = 0.636, p = 0.048 and r = 0.539, p = 0.108; figure 5a and 5b, right panels). moreover, in hcv-infected patients, dil-7 also correlated with variations in intrathymic precursor t-cell proliferation (dsj/btrec ratio; r = 0.601, p = 0.020; figure 5c ). variations in plasma il-7 levels also correlated with changes in the proportions (d%ki-67+ in cd4+rtes; r = 0.806, p = 0.0002; figure 5d , left panel) and numbers (dki-67+rtes; r = 0.706, p = 0.002; figure 5e , left panel) of cycling rtes in acute and chronic hcv infected patients and with d%ki-67+rte counts in co-infected patients (r = 0.709, p = 0.022; figure 5e , right panel). overall, il-7 concentration was associated with reduced thymopoiesis and rte proliferation, lower consequently leading to limited circulating rte and naïve t-cell counts. these data strongly suggest that changes in il-7 plasma levels during ifna therapy directly impact the homeostasis of rtes. we herein demonstrated that ifna-based therapy leads to major lymphocytopenia in naïve t-cell compartments, in particular in the rte subset. several mechanisms could be implicated in the establishment of such a lymphocytopenia [41] . among these, enhanced apoptosis [42, 43] , cell sequestration in lymphoid or non-lymphoid organs [12, 21, 22] and regulation of peripheral t-cell homeostasis [20] . in our study, no major change in cell survival (bcl-2 expression) or t-cell activation (cd25 and cd69 expression) was observed during the follow-up period (data not shown). moreover, we did not observe any significant modification in ki-67 expression in any t-cell subset during the first month of therapy (data not shown and figure 3 ). finally, ifna-induced t-cell homing, although rapid and massive, is only a transient process [22] suggesting that this mechanism marginally contributes to the observed long lasting lymphocytopenia. interestingly, both sjtrec quantification (sjtrec/ml) and intrathymic precursor t-cell proliferation (sj/btrec ratio) were affected very early on after initiation of therapy ( figures 2b and 3b ). while sjtrec frequency and concentration in peripheral blood can be affected by modifications of parameters that impact on peripheral t-cell homeostasis (cycling, survival/apoptosis, homing), the sj/btrec ratio is a marker of the intrathymic proliferation history of rtes. indeed, this parameter is generated by cell proliferation that occurs between tcrb chain rearrangement and the excision of tcrd locus. further cell cycling after tcra chain rearrangement does not modify the sj/btrec ratio as both type of trecs are similarly diluted upon cell proliferation. accordingly, while exported to the periphery, the sj/btrec ratio of mature t-cells cannot be modified. therefore, while the observed decrease in sjtrec concentration (figure 2) can be a consequence of modifications of circulating t-cell homeostasis, the decline of the sj/btrec ratio observed during the first months of ifna therapy (figure 3) defines changes in thymocyte proliferation, thus in thymic output [28] . acutely infected patients demonstrated a higher sj/btrec ratio at baseline than patients in the chronic phase. however, this group was younger (median = 31.5 (26-47)) versus median = 53.5 (37-61)) than the chronic group (p,0.01; data not shown) and demonstrated normal sj/btrec ratio for their age. similar evolution of thymic function and circulating t-cell subsets were observed in both groups of hcv-infected patients, irrespective of the development stage of hcv pathology. the lack of effect of ifna therapy in hiv/hcv co-infected patients might be due to the fact that, as expected for chronically hiv-infected individuals, these patients already had a low thymic function at study entry. the impairment of thymopoiesis in hcv-infected patients under ifna therapy is reminiscent of that observed during the acute phase of hiv-1 infection [23] which suggested that long term production of ifna, as part of the anti-hiv innate immune response, may play a role in the observed thymic defect. the correlation between decline in il-7 plasma levels under ifna therapy and both thymic dysfunction and reduced t-cell counts, in particular in the naïve and rte compartments ( figures 5a and 5b) , confirms this hypothesis. finally, in a recent study, we showed that ifna treatment leads to decreased sjtrec frequency as well as reduced naïve t-cell and rte counts in siv-infected rhesus macaques [30] . such an effect was accompanied by a 30-40% decrease in il-7 plasma levels in these animals and could be counteracted by injection of recombinant simian il-7 [30] . one could expect that such an effect of type i ifns is not restricted to hiv-infection as many viral infections induce ifna responses and cause transient lymphocytopenia in the infected hosts [3, 4, 5, 6] . moreover, the ifna-induced reduction of thymic function and its probable consequences on naïve t-cell diversity may contribute to the higher infectious risk associated with ifna therapy, in particular observed in older patients [15, 16, 44] . there are multiple sources for circulating il-7 during viral infections including lymphoid organs, epithelial cells and recently the liver was identified as a major source of il-7. moreover, increased plasma il-7 levels can also be observed during viral infection in non-lymphopenic individuals ( [33] and unpublished data), suggesting a role in the development of immune responses. indeed, this cytokine participates to t-cell homing in various lymphoid and non-lymphoid tissues through stimulation of local chemokine productions [45] . increased il-7 plasma levels in lymphopenic individuals is likely due to reduced consumption [46] yet augmented production to counteract lymphopenia cannot be excluded [33] . the recent identification of the liver as an il-7 producing tissue upon tlr stimulation [47] makes it tempting to speculate that hcv-infection can also, through tlr activation, stimulate il-7 production by the liver. indeed, non-lymphopenic hcv-infected patients demonstrate similar il-7 plasma levels than lymphopenic hiv-infected individuals [33, 48] suggesting that most of the il-7 production in untreated hcv-infected patients was not linked to circulating t-cell counts. the reduction of il-7 plasma levels while lymphocytopenia establishes under ifna therapy, the absence of a correlation between il-7 plasma levels and cd127 expression and the concomitance of decreases in il-7 plasma levels and hcv viral load under therapy suggest that viremia might be driving il-7 production before initiation of therapy. our data suggest that, before initiation of ifna therapy, actively replicating hcv leads to the overproduction of il-7. subsequent reduction of il-7 production upon initiation of therapy probably reflects the elimination of il-7 producing hcv-infected hepatocytes. this sudden reduction of il-7 plasma levels may lead to diminished thymopoiesis. the fact that il-7 plasma levels did not reach normal levels when hcv became undetectable may suggest that, after the initial decline that follows the drop in viremia, il-7 plasma levels were regulated, as in hivinfected patients [33] and in ifna-treated siv-infected rhesus macaques [30] , as a consequence of lymphocytopenia through either reduced consumption or increased production in lymphoid organs [49] . future studies with a longer follow-up period, in particular after the end of ifna therapy and recovery from lymphocytopenia are required to further elucidate this point. we herein demonstrated that a substantial reduction in thymic export was observed in hcv-infected patients, during the first months of ifna therapy. this effect directly paralleled ifnainduced lymphocytopenia and decreased il-7 plasma levels, initially high in hcv-infected patients. these data suggest that il-7 production by the liver, a consequence of active hcv replication, was reduced while patients controlled hcv viremia. restricted il-7 plasma levels might, in association with the antiproliferative effect of ifna, limit t-cell production in the thymus. our study highlights the therapeutic potential of il-7 as a complement to the standard ifna based treatment to help hcvinfected patients to sustain normal circulating t-cell counts, and restore the diversity of the peripheral t-cell repertoire through its central thymopoietic effect. restoring the breadth and intensity of t-cell control over the hcv virus might be immediately beneficial for the hiv/hcv co-infected population and offer new promising avenues for chronic hcv in the context of massive drop of hcv viral load after short term treatment with new antiviral compounds that will continue to be administered in combination with ifna [50] . sixteen hcv-infected patients (c-1 to c-16) and ten hiv/ hcv co-infected patients (i-1 to i-10) naïve to ifna therapy were enrolled in this study. a summary of the virological and immunological status of patients at baseline is shown in table 1. all the hiv/hcv co-infected patients but one were under haart with undetectable viremia (,40 hiv copies/ml). chronically infected patients (c-9 to c-16 and i-1 to i-10) initiated pegylated ifna/ribavirin treatment (ifna-2a: pegasys, 180 mg weekly, ribavirin: copegus, 800 mg to 1000 mg daily) and were followed over a 4 months period. patients included in the acute phase of hcv infection (c-1 to c-8) were treated with pegylated ifna (ifna-2a: pegasys, roche, 180 mg weekly) [51, 52] . blood samples were taken monthly on edta. two milliliters of total blood were 2-fold diluted in fcs/20%dmso frozen at 280uc and conserved in liquid nitrogen. these total blood samples were subsequently used for flow cytometry analyses. plasma was separated from the remaining eight milliliters and mononuclear cells were purified on ficoll hypaque (eurobio, courtaboeuf, france) and frozen for further analyses. patients from the hcv mono-infection group were followed at the centre de recherche du chum, hôpital saint luc, montreal, qc, canada and its collaborators as previously described [9, 53] . patients from the hiv-hcv groups were followed at the hôpital henri mondor, creteil, france. clinical protocols conformed to figure 5 . variations in il-7 plasma levels correlate with evolution of rte production. correlations. between variations in il-7 plasma levels (dil-7) and either variations in (a) total (cd4 + + cd8 + ) naïve t-cell counts (dnaïve t-cell counts), (b) rte defined as cd31 hi naïve cd4 + t-cells (drte cd4 counts), (c) the sj/btrec ratio (dsj/btrec ratio), (d) the frequency of ki-67 + cells in the rte cd4 + t-cell subset (d%ki-67 + in cd4 + rtes) or (e) the number of circulating ki-67 + cd4 + rtes (dki-67 + rte counts) between study entry and month 1 of therapy were calculated for acutely (black symbols) and chronically (white symbols) hcv-infected patients (left panels) and hiv/hcv co-infected patients (right panels). correlation coefficients (spearman's r) and the associated probabilities (p) are shown. doi:10.1371/journal.pone.0034326.g005 ethical guidelines of the authors' institutions and the us department of health and human services' human experimentation guidelines. this study was approved by both the ethical committee of centre hospitalier de l'université de montreal (chum) and the ethical committee of hôpital henri mondor, créteil, france. samples were obtained with the written subjects' informed consent. immunophenotyping and flow cytometry analysis facs analyses were performed on cryopreserved samples. after thawing blood cells were incubated for 15 minutes at 4uc with conjugated monoclonal antibodies (mabs). for intracellular labeling, cells were permeabilized with the cytofix/cytoperm kit (becton dickinson) before incubation with specific mabs according to the manufacturer's instructions. samples were then washed, fixed in 2% paraformaldehyde phosphate-buffered saline (pbs/pfa 2%) and acquired using a cyan cytofluorometer (dako) and analyzed with flowjo 8.7 software. the monoclonal antibodies used in this study were: cd3-pacific blue (pb) (clone ucht-1; dako, trappes, france), cd4-peridin chlorophyll protein-cyanine 5.5 (percp-cy5.5) (clone l200; bd, le-pont-de-claix, france), cd45ra-phycoerythrin (pe) (clone hi100; bd), ccr7-allophycocyanin (apc) (clone 150503; r&d systems europe, lille, france); cd8-phycoerythrin-cyanine 7 (pe-cy7) (rpa-t8; bd), cd31-biotin (clone wm59; abdserotec, düsseldorf, germany); ki-67-fluorescein isothiocyanate (fitc) (clone mib-1; dako), bcl-2-fitc (clone 124; dako) and strepatavidin-pe-texas-red (bd). il-7 was quantified in the plasma using the il-7 quantikine hs kit according to the manufacturer's instructions (r&d systems europe). plasma soluble-cd127 quantification soluble plasma il-7 receptor (scd127) quantification was performed as previously described [54] . parallel quantification of the sjtrec and the 13 djbtrecs, together with cd3c gene (used as a housekeeping gene) was performed for each sample using lightcyclertm technology (roche diagnostics) with a technique adapted from [29] . intrathymic precursor t-cell proliferation was evaluated through calculation of the sj/btrec ratio as described [23] . hcv rna quantification was performed using an in-house quantitative real-time reverse transcription-pcr assay as previously described [9] , cobas amplicor hcv monitor test tm , version 2.0 (sensitivity 600 iu/ml)), qualitative cobas ampli-prep/cobas amplicor hcv test tm , version 2.0 (sensitivity 50 iu/ml) or abbott realtime hcv assay tm (sensitivity 12 iu/ ml). statistical analyses (spearmans rank correlations and wilcoxon matched -paired signed-rank tests) were performed using the stata/ic 10.0 (stata corporation, college station, tx u.s.a.). due to the exploratory nature of the study there was no correction for multiple comparisons, and calculated p values are reported herein. mechanism of action of interferon and ribavirin in treatment of hepatitis c a new standard of care for the treatment of chronic hcv infection selective lymphocyte depletion during the early stage of the immune response to foot-and-mouth disease virus infection in swine quantitation of t lymphocyte subsets helps to distinguish dengue hemorrhagic fever from classic dengue fever during the acute febrile stage effects of severe acute respiratory syndrome (sars) coronavirus infection on peripheral blood lymphocytes and their subsets extensive lymphopenia due to apoptosis of uninfected lymphocytes in acute measles patients a dual role of ifnalpha in the balance between proliferation and death of human cd4+ t lymphocytes during primary response type i interferons act directly on cd8 t cells to allow clonal expansion and memory formation in response to viral infection early interferon therapy for hepatitis c virus infection rescues polyfunctional, long-lived cd8+ memory t cells t cell receptor excision circles (trecs), cd4+, cd8+, and their cd45ro+, and cd45ra+, subpopulations in hepatitis c virus (hcv)-hivco-infected patients during treatment with interferon alpha plus ribavirin: analysis in a population on effective antiretroviral therapy efficacy and safety of combination therapy with interferon-alpha2b and ribavirin for chronic hepatitis c in hiv-infected patients efficacy and safety of alpha-interferon treatment for chronic hepatitis c in hivinfected patients. hiv-hepatitis spanish study group future trends in managing hepatitis c treatment of hepatitis c and anemia in human immunodeficiency virus-infected patients use of pegylated interferons is associated with an increased incidence of infections during combination treatment of chronic hepatitis c: a side effect of pegylation? incidence of neutropenia and infections during combination treatment of chronic hepatitis c with pegylated interferon alfa-2a or alfa-2b plus ribavirin opportunistic infections and cd4 lymphocytopenia with interferon treatment in hiv-1 infected patients regulation of cytokine expression by interferon-alpha in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of interleukin-1 receptor antagonist effects of recombinant alpha and gamma interferons on the in vitro growth of circulating hematopoietic progenitor cells (cfu-gemm, cfu-mk, bfu-e, and cfu-gm) from patients with myelofibrosis with myeloid metaplasia impairment of t and b cell development by treatment with a type i interferon cd4+ t-lymphocytopenia in hiv-infected patients receiving interferon therapy for chronic hepatitis c. hiv-hepatitis spanish study group type i interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia hiv infection rapidly induces and maintains a substantial suppression of thymocyte proliferation changes in thymic function with age and during the treatment of hiv infection slow disease progression and robust therapy-mediated cd4+ t-cell recovery are associated with efficient thymopoiesis during hiv-1 infection il-7 induces immunological improvement in siv-infected rhesus macaques under antiviral therapy t cell homeostasis: thymus regeneration and peripheral t cell restoration in mice with a reduced fraction of competent precursors the magnitude of thymic output is genetically determined through controlled intrathymic precursor t cell proliferation estimating thymic function through quantification of t-cell receptor excision circles interleukin-7 treatment counteracts ifn-{alpha} therapy-induced lymphopenia and stimulates siv-specific cytotoxic t lymphocyte responses in siv-infected rhesus macaques interleukin-7 promotes survival and cell cycle progression of t-cell acute lymphoblastic leukemia cells by down-regulating the cyclin-dependent kinase inhibitor p27(kip1) interleukin-7 mediates the homeostasis of naive and memory cd8 t cells in vivo increased production of il-7 accompanies hiv-1-mediated t-cell depletion: implications for t-cell homeostasis the role of interleukin-7 in early t-cell development a direct estimate of the human alphabeta t cell receptor diversity how cells respond to interferons augmentation of antitumor activity of 5-fluorouracil by interferon alpha is associated with up-regulation of p27kip1 in human hepatocellular carcinoma cells down-regulation of p27kip1 expression is required for development and function of t cells endogenous interferon-alpha production by differentiating human monocytes regulates expression and function of the il-2/il-4 receptor gamma chain two subsets of naive t helper cells with distinct t cell receptor excision circle content in human adult peripheral blood tlymphocyte populations in hepatitis c and hiv co-infected patients treated with interferon-alfa-2a and ribavirin interferon alpha augments activation-induced t cell death by upregulation of fas (cd95/apo-1) and fas ligand expression ifn-alpha suppresses activation of nuclear transcription factors nf-kappa b and activator protein 1 and potentiates tnf-induced apoptosis rapid decline of cd4+ cells after ifn alpha treatment in hiv-1 infection injection of glycosylated recombinant simian il-7 provokes rapid and massive t-cell homing in rhesus macaques interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of cd4+ t cells hepatic interleukin-7 expression regulates t cell responses loss of il-7 receptor alpha-chain (cd127) expression in acute hcv infection associated with viral persistence interleukin-7 receptor expression: intelligent design hepatitis c virus (hcv) genotype 1 subtype identification in new hcv drug development and future clinical practice determinants of antiviral treatment initiation in a hepatitis c-infected population benefiting from universal health care coverage response rates to pegylated interferon and ribavirin in hcv/hiv coinfection: a research synthesis rare birds in north america: acute hepatitis c cohorts a validated assay to measure soluble il-7 receptor shows minimal impact of il-7 treatment the authors acknowledge the subjects who participated in this study. we also thank c. chesnel for clinical and logistical support. key: cord-018785-tcr5xlf8 authors: nambiar, puja; silibovsky, randi; belden, katherine a. title: infection in kidney transplantation date: 2018-06-27 journal: contemporary kidney transplantation doi: 10.1007/978-3-319-19617-6_22 sha: doc_id: 18785 cord_uid: tcr5xlf8 infection is an important cause of morbidity and mortality after kidney transplantation. it has been estimated that 70% of kidney transplant recipients will experience an infection episode within the first 3 years after transplantation (dharnidharka et al. 2007). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. 2009). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. pretransplant screening, immunizations, and optimal antibacterial and antiviral prophylaxis can help to reduce the impact of infection. awareness of the approach to infection in the transplant recipient including diagnostic and management strategies is essential to optimizing outcomes. a total of 17,600 kidney transplants were performed in the united states in 2013. as the incidence of acute rejection has declined, the probability of graft and patient survival continues to improve (usrds 2015) . infection, however, remains an important cause of morbidity and mortality after kidney transplantation. it has been estimated that 70% of kidney transplant recipients will experience an infection episode within the first 3 years after transplantation (dharnidharka et al. 2007 ). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. 2009 ). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. the kidney transplant recipient's net state of immune suppression and epidemiologic exposures determine the risk for infection at a given time. a traditional timeline has been used to predict patterns of infection after organ transplantation. this timeline has been altered in recent years with changes in immunosuppressive therapy and the routine use of antibacterial and antiviral prophylaxis. treatment for acute rejection and coinfection with viruses such as cytomegalovirus (cmv) and epstein-barr virus (ebv) may also alter predictable patterns of infection (fishman 2007) . the basic concepts of the traditional timeline, however, are still used to establish a differential diagnosis for infection at varied intervals posttransplantation (fig. 1) . within the first month, infections are noted to include those related to surgical complications, nosocomial exposures, and donor-derived pathogens. multidrug-resistant organisms including methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococcus (vre), and carbapenem-resistant enterobacteriaceae (cre) are important considerations, as is clostridium difficile. urinary tract infections are common within the first 6 months. opportunistic infections are more likely to occur 1-6 months after transplantation, reflecting the greater impact of immune suppression during this time. reactivation of latent pathogens such as polyoma virus bk, hepatitis c virus (hcv), and mycobacterium tuberculosis may also occur. prophylaxis for pneumocystis jiroveci, herpes viruses including cmv, and hepatitis b virus (hbv) makes these infections less common during this time period. beyond 6 months, the degree of immune suppression for most patients decreases. risk remains, however, for community-acquired infection, environmental exposures, recurrent infection, and the late presentation of viral infection, in particular cmv, once prophylaxis has been discontinued (fishman 2007; karuthu and blumberg 2012) . interventions can be undertaken to reduce the impact of infection after kidney transplantation. pretransplant screening of donors and recipients for infection that can be transmitted with organ donation or reactivated in an immune suppressed recipient is essential for optimizing transplant outcomes. guidelines for pretransplant screening are available from the american society for transplantation (fischer et al. 2013) , kidney disease: improving global outcomes (kdigo 2009) and the us public health service (seem et al. 2013) . recommended screening tests for donors and recipients are listed in table 1 . screening of living donors is performed prior to transplantation with varied timing. if there is a hiv(þ) consider if hiv is well controlled hcv: anti-hcv and hcv nat hcv(þ) hcv(à) reject, may be a consideration in the future hcv(à) hcv(þ) consider, hcv(þ) candidates should have a liver biopsy, improved outcomes if hcv is treated pretransplant hcv(þ) hcv(þ) consider (as for dà/rþ) hbv: hbsag, hbsab and hbcab (igm/igg); hbv nat (center dependent) sag(à), cab(à) sag(à), cab(þ), sab(þ/à) accept, vaccinate sab(à) candidates consider, with prophylaxis posttransplant sag(à), cab(þ) sag(à), cab (þ/à), sab (þ/à) accept if donor is cigm(à) and vaccinate sab(à) candidates, offer prophylaxis posttransplant if sab(à) or lost; reject if donor is cigm(þ) sag(þ), cab(þ) sag(à), cab (þ/à), sab (þ/à) reject cmv igg cmv(þ) or (à) cmv(þ) accept; will need posttransplant prophylaxis or preemptive therapy cmv(þ) cmv(à) accept; high risk for cmv infection, will need posttransplant prophylaxis ebv igg ebv(þ) or (à) ebv(þ) accept ebv(þ) ebv(à) accept; at risk for primary ebv and ptld, monitor posttransplant hsv 1/2 igg hsv(þ) hsv(þ) or (à) (fischer et al. 2013) . deceased donor screening, in contrast, is under time constraints and is usually performed within hours of transplantation in coordination with organ procurement organizations. infection with hiv, hbv, and hcv may not be detected in the early stages of infection. many transplant centers now perform more sensitive rapid molecular testing on potential organ donors including nucleic acid amplification (nat) testing for hiv, hbv, and hcv. a comprehensive medical and social history on potential organ donors is required in order to identify risk factors for blood borne pathogens. in efforts to expand the pool of available organs, recipients may consent to receipt of a kidney from a nat negative donor who is deemed "high risk" for blood borne infection based on identified risk factors. recipients of such organs are monitored posttransplantation with testing for hiv, hbv, and hcv between 1 and 3 months and for hbvagain at 12 months (fischer et al. 2013; seem et al. 2013; kovacs et al. 2014; len et al. 2014 ). use of hcv-and hbv-positive organs can be considered in respective positive recipients. furthermore, in 2013 the hiv organ policy equity act lifted a long-standing ban on allowing hivpositive organs to be donated to hiv-positive recipients (mgbako et al. 2013; muller et al. 2015) . donors who have active bacterial infection at the time of kidney procurement may transmit infection to the recipient. screening for bacterial infection in kidney donors includes assessing for urinary tract infection and bacteremia. urine and blood culture data are reviewed. if a kidney donor is known to have a urinary tract or systemic infection with a virulent organism such as staphylococcus aureus, pseudomonas aeruginosa, or candida species, the organ recipient is usually treated with a 10-14 day course of targeted antimicrobial therapy since these bacteria can compromise vascular and urinary anastomoses leading to mycotic aneurysms, anastomotic, and organ failure (fischer et al. 2013 ). allograft contamination can occur during organ procurement or processing. interpretation of organ preservation fluid cultures is challenging. the risk of transmission of infection to the organ recipient from contaminated preservation fluid, however, is low (fischer et al. 2013; len et al. 2014 ). candidates for kidney transplantation should have their vaccine status reviewed and updated in accordance with recommendations issued by the advisory committee on immunization practices with the centers for disease control and prevention (cdc 2012). while vaccinations in end stage renal disease patients may be less effective and durable than in healthy patients, a better response can be anticipated prior to transplantation than after (janus et al. 2008; kausz and pahari 2004) . special consideration should be given to vaccination for pneumococcus, influenza, and hbv. two pneumococcal vaccines are currently licensed for use in the united states: the 13-valent pneumococcal conjugate vaccine (pcv 13, prevnar 13) and the 23-valent-pneumococcal-polysaccharide vaccine (ppsv 23, pneumovax 23). current guidelines recommend that unvaccinated patients with chronic renal failure receive pcv 13 followed at least 8 weeks later by ppsv 23 (kobayashi et al. 2015) . a second dose of ppsv 23 is recommended 5 years after the first dose (cdc 2012). influenza vaccination should be administered annually. there are a number of influenza vaccine formulations available. live attenuated influenza vaccination (flumist) is not recommended in chronic kidney disease patients. an inactivated vaccine option should be used (cdc 2012) . a high dose inactivated influenza vaccine is now available and was shown to induce a higher antibody response than traditional vaccines in adults over the age of 65 (diaz-granados et al. 2014) . the use of this vaccine in transplant candidates and recipients is currently under investigation. transplant candidates not immune to hbv should receive high dose hbv vaccination (40 micrograms antigen per dose) due to decreased response rates with standard dosing (huprikar et al. 2015) . human cytomegalovirus-human herpes 5 (cmv), a member of the family herpesviridae, is an opportunistic pathogen occurring in 20-60% of solid organ transplant recipients (brennan 2001) . cmv is a cause of significant morbidity and mortality in this population (mwintshi and brennan 2007) . the incidence of cmv in the renal transplant population is estimated to be between 8% and 32% (patel and paya 1997) . renal transplant patients are at lower risk for primary cmv compared with other organ transplant recipients owing to a lower burden of latent virus in renal allograft tissue. the risk factors for development of cmv disease include donor seropositivity/recipient seronegativity(dþ/rà), use of induction immunosuppression (antilymphocyte antibodies), donor age >60 years, simultaneous kidney-pancreas transplantation, treatment for acute rejection, impaired transplant function, and concurrent infection from other viruses (like ebv and hhv-6 and 7) (de keyzer et al. 2011) . cmv-seronegative recipients of cmv-seropositive donors (d + /r à ) are at the highest risk, whereas d + /r + or d à /r + transplantations are considered to be moderate risk with d à /r à being lowest risk, with an incidence of cmv disease <5% (de keyzer et al. 2011) . immunosuppressive drugs also influence the incidence and severity of cmv disease. for instance, cyclosporine increases the risk of cmv disease, whereas use of sirolimus seems to have a protective effect (san juan et al. 2008) the use of antilymphocyte antibody (antithymocyte globulin or muromonab-cd3) is associated with a two to fivefold increase in the rate of cmv, but basiliximab and daclizumab do not seem to increase its incidence (de keyzer et al. 2011) . cmv infection may occur in solid organ transplantation recipients as primary infection when a cmv seronegative individual receives cells latently infected with cmv from a seropositive donor, donor-derived reinfection, or reactivation of latent recipient infection (patel and paya 1997) . the following definitions are commonly used in the transplant literature to differentiate cmv infection from cmv disease. cmv infection is evidence of cmv replication regardless of symptoms, and cmv disease is evidence of cmv infection with symptoms, such as viral syndrome, leukopenia, thrombocytopenia, or invasive tissue disease (e.g., pneumonitis, hepatitis, retinitis, gastrointestinal disease) (humar and snydman 2009) . cmv disease and even asymptomatic cmv infection have been shown to be independent risk factors for reduced graft survival and overall mortality beyond 100 days posttransplantation . infection with cmv has been implicated in acute allograft dysfunction and chronic allograft nephropathy (mclaughlin et al. 2002; tong et al. 2002) . cmv disease is also associated with posttransplant lymphoproliferative disorder (ptld), posttransplant diabetes mellitus, and transplant artery stenosis (pouria et al. 1998; hjelmesaeth et al. 2004; manez et al. 1997) . cmv infection can occur as acute infection between the first and 6 months following transplant, when immunosuppression is at its maximum or as delayed infection from reactivation of latent virus after antiviral prophylaxis has completed, later in the first year. given the significant effect of cmv on patient outcomes, prevention plays an important role. serologic screening for cmv should be performed on both donor and recipient prior to transplant to categorize high risk patients. several cmv vaccine candidates are under investigation although none are currently available. universal prophylaxis involves giving antivirals to those recipients at risk posttransplant before the onset of infection, whereas in preemptive therapy patients are monitored at regular intervals and started on antivirals when there is early evidence of replication prior to onset of clinical disease. chemoprophylaxis in high risk patients (dþ/rà) has shown to reduce the incidence of cmv disease by 60% and has decreased cmv associated mortality and opportunistic infection (hodson et al. 2005) . preemptive therapy in high risk patients based on cmv viral load monitoring has not shown reduction in acute rejection or all-cause mortality (strippoli et al. 2006) . a randomized controlled trial by kliem et al. in 2008 comparing oral ganciclovir chemoprophylaxis with viral load monitoring revealed improved graft survival in those who received ganciclovir chemoprophylaxis (kliem et al. 2008) . a recent cochrane review from 2013 concluded that the efficacy of preemptive therapy compared with prophylaxis to prevent cmv disease remains unclear due to significant heterogeneity between studies and that additional head-tohead studies are required to determine the relative benefits and harms of preemptive therapy and prophylaxis to prevent cmv disease in solid organ transplant recipients (owers et al. 2013) . standard prophylactic guidelines recommend therapy in dþ/rà, dþ/rþ, and dà/rþ using oral ganciclovir or valganciclovir for a minimum of 3 months posttransplant and 1-3 months after treatment of rejection with antilymphocyte therapy (humar and snydman 2009; kotton et al. 2013 ). valganciclovir has replaced ganciclovir because of better bioavailability, lower pill burden, and reduced availability of oral ganciclovir (paya et al. 2004 ). the optimal length of prophylaxis is unknown, but recent trials have shown that 6 months of prophylaxis is more effective in decreasing the incidence of cmv disease in dþ/ rà kidney transplant recipients (humar et al. 2010; doyle et al. 2006) . current guidelines recommend dosing valganciclovir at 900 mg daily (adjusted for renal dysfunction) if tolerated in dþ/ rà recipients. some centers have successfully treated patients with half of this dose (450 mg daily) with less drug toxicity. however, dþ/rà recipients may be at higher risk of breakthrough infection and the development of resistance with this lower dosing strategy (kotton et al. 2013) . the diagnosis of cmv disease can be made by several techniques including cmv antigenemia assay, nucleic acid testing (nat), serology, antibody testing, viral culture, and histopathology. nat is generally more sensitive than antibody testing or culture. higher values by nat are suggestive of cmv disease and weekly viremia testing can be used to monitor response to therapy. the interlaboratory variability of nat is expected to be reduced with the recent establishment of international standards, intended to be used in the standardization of nucleic acid amplification technique (nat)-based assays for hcmv (karuthu and blumberg 2012) . patients with gastrointestinal and neurologic cmv disease often fail to exhibit cmv viremia and histopathology is necessary to establish diagnosis in these instances. treatment of active cmv disease requires a combination of immunomodulation, antiviral therapy with or without adjuvant therapy and if possible, reduction of immunosuppression (kotton et al. 2013; green et al. 2004 ). the mainstay of therapy is intravenous ganciclovir. the victor trial (valcyte in cmv disease treatment of solid organ recipients) demonstrated oral valganciclovir was not inferior to intravenous ganciclovir in mild to moderate cmv disease in solid organ transplant recipients (asberg et al. 2009 ). the current guidelines recommend renally adjusted intravenous ganciclovir 5 mg/kg twice daily or oral valganciclovir, 900 mg twice daily for mild cmv disease (kotton et al. 2013) . in severe cmv disease, intravenous ganciclovir is preferred with reduction of immunosupression despite the increased risk of rejection (de keyzer et al. 2011) . the use of adjuvant therapy with cmv-specific hyperimmune globulin or standard intravenous immunoglobulin may be considered in individuals with hypogammaglobulinemia, severe systemic infection, or in failure to respond to standard therapy (humar et al. 2010) . cmv resistance to ganciclovir has been noted in renal transplant recipients due to mutations in ul 97, the gene responsible for the first phosphorylation step in ganciclovir activation and ul 54, the gene responsible for dna polymerase (limaye et al. 2000) . cmv resistance should be considered when patients have worsening disease or persistent, unchanged viremia at 2 weeks of therapy and in such cases, genotype testing for mutations of the genes encoding ul 97 and ul 54 should be performed (weikert and blumberg 2008). treatment options for drug resistant cmv include the use of high dose ganciclovir, foscarnet, and cidofovir; however, no clinical trial data are available regarding optimal therapy options for resistant cmv. the use of novel agents including leflunomide and artesunate has been attempted as salvage therapy with varying success. several new antiviral treatment options are currently under investigation including maribavir and brincidofovir (an oral prodrug of cidofovir with less nephrotoxicity) for use in the treatment of drug resistant cmv (limaye et al. 2000) . epstein barr virus -human herpesvirus 4 (ebv) is a ubiquitous gamma herpes virus that remains latent in lymphocytes following primary infection. it is responsible for posttransplant lymphoproliferative disorder (ptld) which increases morbidity and mortality in the transplant population. approximately 62-79% of ptld cases have been associated with ebv (karuthu and blumberg 2012) . ptld most commonly occurs in the first year posttransplant (cockfield et al. 1993) . the risk factors for ptld include ebv naïve recipients who receive ebv seropositive organs, active primary ebv infection, younger recipient, coinfection by cmv and other viruses, prior splenectomy, second transplant, acute or chronic graft versus host disease, immunosuppressive drug regimen (okt3 or polyclonal antilymphocyte antibody), and the type of organ transplanted. kidney transplant recipients are at lower risk compared with other types of transplants and have an incidence of approximately 1-3% (gulley and tang 2010; allen et al. 2009; taylor et al. 2005) . the majority of symptomatic ebv infections in renal transplant recipients are primary infection likely related to transmission of donor virus. ebv disease can be asymptomatic or presents with a nonspecific febrile syndrome, lymphadenopathy, hepatosplenomegaly, atypicalþ lymphocytosis, hematologic disorders including anemia, leukopenia, thrombocytopenia, and organ-specific diseases like gastroenteritis, hepatitis, or pneumonitis (allen et al. 2009 ). ptld typically follows primary infection and frequently presents as a rapidly enlarging mass in the grafted organ, lymph nodes, bone marrow, or extranodal sites (manez et al. 1997) . ptld is divided into four major histopathologic subtypes as per the world health organization (who): early lesions, polymorphic ptld, monomorphic ptld, and classical hodgkin lymphoma type ptld. definitive diagnosis of ptld requires histopathologic confirmation by tissue excision biopsy with immunologic cell typing, cytogenetics, immunoglobulin gene rearrangements, and ebvspecific staining (allen et al. 2009 ). staging is performed by histologic types (monoclonal versus polyclonal, t cell versus b cell) and location (allograft, other organs, metastasis) (weikert and blumberg 2008) . clinical management of ptld typically involves reduction of immunosuppression which can lead to remission in 23-86% of the patients (weikert and blumberg 2008). antiviral therapy with acyclovir or ganciclovir is controversial and no evidence supports its efficacy (taylor et al. 2005) . rituximab (monoclonal antibody to cd20) is commonly used for treatment of ptld in recipients who failed reduction of immunosuppression alone (allen et al. 2009 ). in isolated graft ptld, surgical resection is an option (weikert and blumberg 2008) . in patients that fail the above strategies, ifn and ivig have been used with varying success and cytotoxic chemotherapy with radiation remains salvage therapy (green et al. 2004 ). there is no standardized therapy to prevent ptld. kdigo guidelines recommend monitoring ebv viral load in high risk renal transplant patients within the first week after transplant, then at least monthly for 3-6 months and then every 3 months for the rest of the first posttransplant year. additional viral load monitoring is recommended after treatment for acute rejection in high risk groups (children, ebv dþ/rà). outcomes with ptld in renal transplant patients vary according to the site involved. patients with isolated graft involvement have a 5-year survival of 68% compared with those patients with ptld extending beyond the allograft whose survival varied between 36% and 38% (weikert and blumberg 2008). human herpesvirus 1herpes simplex virus types 1 and 2 (hsv)and human herpesvirus 3varicella zoster virus (vzv)are alpha herpes viruses. hsv 1 has a seroprevalence of 60% in the adult population, while hsv 2 has a seroprevalence of 15% and vzv rates can be as high as 90% (green et al. 2004 ). the incidence of hsv disease in renal transplant recipients is approximately 53% and vzv 4-12% (patel and paya 1997) . hsv may cause primary infection following which the virus remains latent in the sensory nerve ganglia or more commonly causes reactivation infection. hsv may be seen as early as in the first posttransplant month in the absence of prophylaxis. hsv infection usually presents with oral or genital mucocutaneous lesions, occasionally pneumonitis, tracheobronchitis, esophagitis, hepatitis, encephalitis, or disseminated infection (green et al. 2004) . vzv causes localized dermatomal or multidermatomal or disseminated zoster with or without visceral involvement (pneumonitis, hepatitis, pancreatitis, encephalitis). pretransplant screening for prior vzv infection should be performed, and naïve patients should be vaccinated with live attenuated varicella vaccine before transplant whenever possible in order to avoid primary vzv infection posttransplantation (fehr et al. 2002) . since vzv is a live vaccine, it should not be given if transplant is expected within 4-6 weeks in order to avoid active shedding of virus at the time of transplant. posttransplant prophylaxis is recommended with acyclovir, valacyclovir, or ganciclovir (in those who need cmv prophylaxis) for approximately 1-3 months posttransplant in order to avoid hsv and vzv reactivation (green et al. 2004) . diagnosis of hsv and vzv infection can be made with pcr or direct fluorescence antibody for hsv from vesicular lesions, csf, or visceral tissue samples. serologies are rarely helpful in active infection owing to high seroprevalence. kdigo guidelines recommend that renal transplant recipients who develop less severe hsv or vzv infections can be treated with an appropriate oral antiviral agent (e.g., acyclovir, valacyclovir, or famciclovir), and those with systemic infection should be treated with intravenous acyclovir and a reduction in immunosuppressive medication and subsequently switched to an appropriate oral antiviral agent (green et al. 2004 ). the use of foscarnet, cidofovir, or topical trifluridine may be considered in patients with acyclovir resistant virus with careful monitoring of renal functions (kotton and fishman 2005; tan and goh 2006) . human herpesvirus 6 and human herpesvirus 7 (hhv 6 and hhv 7) are ubiquitous with high seroprevalence in adults. these viruses are common causes of fever in children and remain latent in lymphocytes following primary infection. hhv 6 uses the cd46 molecule as its receptor but may also infect other cell types, such as monocytes, and epithelial and endothelial cells. hhv 7 uses the cd4 molecule as its receptor and is more strictly lymphotropic. infection occurs as a result of reactivation in the first 4 weeks following transplant often in recipients not on cmv prophylaxis (singh and carrigan 1996) . clinical manifestations include fever, rash, hepatitis, interstitial pneumonitis, encephalitis, leukopenia, and myelosuppression. owing to its immunomodulatory effects, it is hypothesized that hhv 7 may act as a cofactor for hhv 6 and cmv reactivation, while both hhv 6 and hhv 7 may act as cofactors in the pathogenesis of cmv disease and acute rejection (kidd et al. 2000; chapenko et al. 2000; dockell and paya 2001) . the diagnosis of hhv 6 and hhv 7 is made by tissue immunohistochemistry or nat testing of peripheral blood lymphocytes. treatment includes reduction in immunosuppression and ganciclovir, but cidofovir and foscarnet have also been utilized (green et al. 2004; kotton and fishman 2005; dockell and paya 2001) . hhv 8 is associated with primary effusion lymphoma, kaposi's sarcoma, and multicentric castleman's disease. infection can be acquired as primary through the allograft or through reactivation of latent virus (diociaiuti et al. 2000; regamy et al. 1998 ). hhv 8 causes kaposi's sarcoma, the most common presentation in renal transplant recipients, through upregulation of vascular endothelial growth factor (vegf) receptor f1 k1/kdr in endothelial cells (stallone et al. 2005) . treatment includes reduction in immunosuppression and cytotoxic chemotherapy. sirolimus, an immunosuppressive drug used in renal transplant patients is thought to inhibit not only the production of vegf but also dampens its effect on endothelial cells (stallone et al. 2005 ). bk polyomavirus (bkv) and jc polyomavirus (jcv) belong to the family polyomaviridae. bkv is responsible for causing polyomavirus associated nephropathy (pvan) in 95% of cases and jcv in less than 5% of the cases. pvan occurs in 1-10% of patients with renal transplantation and causes renal allograft loss in 10-80% of cases dadhania et al. 2008) . the risk factors for bkv associated pvan include the use of potent immunosuppressive regimens, caucasian race, older age, diabetes mellitus, cadaveric renal transplant, and combined kidney and pancreas transplant trofe et al. 2003) . bkv is known to cause interstitial nephritis, ureteral stenosis, and ureteral stricture of the allograft kidney most commonly occurring within the first 3-4 months after renal transplant patients when immunosuppression is at its highest (randhawa and brennan 2006) . jcv less commonly causes pvan and is more frequently associated with progressive multifocal leukoencephalopathy (pml), a demyelinating disorder of the white matter presenting as neurologic impairment and dementia (phillips et al. 2004) . diagnosis of bkv includes the use of viral load assays (blood, urine), detection of viral cytopathic effect (decoy cells), nat, bkv-specific antibody, or histopathology (hariharan 2006) . kdigo guidelines recommend screening all renal transplant recipients for bkv with quantitative plasma nat at least monthly for the first 3-6 months after transplantation, then every 3 months until the end of the first posttransplant year, whenever there is an unexplained rise in serum creatinine, and after treatment for acute rejection. the guidelines suggest reducing immunosuppressive medications when bkv plasma nat is persistently greater than 10,000 copies/ ml (107 copies/l) (kdigo 2009). sustained high bk viremia in spite of reduction in immunosuppression may need additional antiviral therapy, although data regarding optimal treatment options are unknown. there are limited data regarding the effectiveness of leflunomide and/or cidofovir or the use of fluoroquinolones or ivig for treatment of bkv infection (randhawa and brennan 2006; josephson et al. 2006) . to date there is no effective treatment for pml. patients with allograft loss due to pvan have undergone successful retransplantation (hariharan 2006) . patients with chronic renal failure, in particular those receiving hemodialysis, are at increased risk for contracting hepatitis b virus (hbv). the prevalence of hepatitis b surface antigen (hbsag)positive patients has declined because of hbv vaccination, strict segregation of hbsag-positive patients in dialysis units, improved screening of blood products, and the use of erythropoiesis stimulating agents (karuthu and blumberg 2012) . approximately 2-10% of patients with a history of hbv prior to transplant will reactivate posttransplant (weikert and blumberg 2008) . serial monitoring of hbv dna every 3-6 months is required after transplantation as liver enzyme levels do not reflect infection status and elevated viral loads suggest resistance to therapy (levitsky et al. 2013) . in a meta-analysis conducted by fabrizi and his colleagues, hbsag seropositivity was an independent risk factor for allograft loss and posttransplant death (fabrizi et al. 2005) . the treatment options currently approved for chronic hbv include: ifn alpha, pegylated ifn, lamivudine, entecavir, telbivudine, tenofovir, and adefovir (fabrizi et al. 2005; chan et al. 2002; chang et al. 2010) . kdigo recommends that interferon treatment generally be avoided because of the high associated incidence of rejection. tenofovir or entecavir are preferable to lamivudine, to minimize the development of drug resistance, unless medication cost requires that lamivudine be used. during therapy with antivirals, hbv dna and alt levels should be measured every 3 months to monitor efficacy and to detect drug resistance. all hbsagpositive renal transplant recipients should receive prophylaxis with tenofovir, entecavir, or lamivudine. hbsag-positive patients with cirrhosis should be screened for hepatocellular carcinoma every 12 months with liver ultrasound and alpha feto-protein. patients who are negative for hbsag and have hbsab titer <10 miu/ml should receive booster vaccination to raise the titer to >100 miu/ ml (kdigo 2009). hepatitis c virus (hcv) infection has been increasingly recognized in end stage renal disease patients (esrd). donor-derived hcv may uncommonly occur after transplantation. screening of patients with esrd and testing renal transplant patients for newly acquired hcv should include nat (levitsky et al. 2013) . hcv-positive donors can be considered for hcv-positive recipients and possibly will be considered for hcv-negative recipients in the future given improved treatment options for cure of hcv that could be administered post transplant. hcv-infected renal transplant recipients have decreased survival and increased complication rates. posttransplant complications include glomerulonephritis (gn), posttransplant diabetes mellitus, and accelerated progression to cirrhosis with fibrosing cholestatic hepatitis (morales et al. 2010) . liver biopsy within 6-12 months of transplantation and subsequent biopsies are required for evaluation of liver disease posttransplant as 20-51% of patients may have normal liver enzyme levels with abnormal histologic features (ashry ahmed gheith 2011). hcv-infected recipients should be tested for proteinuria every 3-6 months, and patients with new onset proteinuria should undergo allograft biopsy (kdigo 2009) . the effect of immunosuppression on the progression of hcv-related liver injury and the management of immunosuppression in the hcvinfected renal transplant recipient remain uncertain. thus, it is preferable to treat hcv in transplant candidates prior to transplantation given the potential for improved outcomes with successful hcv treatment and the complications associated with treatment posttransplant. patients with a sustained virologic response to pretransplant treatment have a reduced risk for hcv recurrence and decreased posttransplant gn (domınguez-gil and morales 2009). options for treatment include interferon/ peginterferon alone or in combination with ribavirin. the risk of toxicity with the addition of ribavirin has limited the use of combination therapy in chronic kidney disease (ckd) patients. the availability of direct acting hcv protease and polymerase inhibitors has sparked new enthusiasm for treating hcv-infected ckd patients and studies are ongoing evaluating the use of these agents in ckd. if treatment cannot be given prior to transplant, kdigo recommends monotherapy with standard interferon for hcv-infected renal transplant recipients in whom the benefits of antiviral treatment clearly outweigh the risks (kdigo 2009). the use of direct acting hcv antivirals posttransplantation can also be considered and will likely be preferred in the future given improved tolerance and efficacy with these agents with an understanding that drug interactions with calcineurin inhibitors may occur.a study looking at 20 hcv-positive kidney transplant recipients (60% treated pre-transplant with interferon unsuccessfully) treated with direct acting antivirals posttransplant found that 100% cleared the virus and had a sustained virologic response at 12 weeks. the most common agents used were sofosbuvir and simeprevir (sawinski et al. 2016 ). human immunodeficiency virus (hiv) belongs to the family of retroviridae. with the introduction of antiretroviral therapy (art) in the mid-1990s, the incidence of hiv related deaths has been reduced. renal diseases related to hiv infection include hiv associated nephropathy (hivan), immune complex diseases, and thrombotic microangiopathy (frassetto et al. 2009 ). a total of 10% of patients with hiv develop hivan and it remains an important complication of hiv infection, progressing rapidly to end stage renal disease (esrd) (shahinian et al. 2000) . a large prospective clinical trial examining outcomes among 150 hiv + kidney transplant recipients reported 3-year patient and graft survival rates of 88.2% and 73.7%, respectively, which were similar to survival rates among a cohort of unmatched elderly (>65 years) hivnegative (hiv à ) kidney recipients (stock et al. 2010 ). the candidates for transplantation include those with well-controlled hiv infection with undetectable viral loads, cd4 >200 cells per microliter, and absence of untreatable infections or malignancies (blumberg et al. 2009 ). the most significant complications in this patient population posttransplant include increased rejection rates (up to 25%), managing drug interactions between art and immunosuppressive therapy and complications related to cardiovascular risk factors and hepatitis coinfection (blumberg et al. 2009 ). the choice of art should be based on susceptibility results and if possible, the use of protease inhibitors should be avoided owing to significant drug interactions with this class of art. with regards to immunosuppressive therapy, the use of thymoglobulin may result in prolonged depression of cd4 counts, whereas monocloncal anti-il2 receptor antibodies, such as basiliximab/daclizumab, have been shown to increase cd4 cell counts (ciuffreda et al. 2007; carter et al. 2006) . the risks of antilymphocyte therapy should be balanced with the risks of rejection in hiv-infected recipients. of note, hiv-positive donors can now be considered in hivpositive recipients. the various respiratory viruses that cause infection affecting the renal transplant patient population include adenovirus, respiratory syncytial virus (rsv), influenza, parainfluenza, human metapneumovirus, rhinovirus, and coronavirus (green et al. 2004) . clinical manifestations include upper respiratory tract infection, bronchitis, and pneumonia. in addition to respiratory illness, adenovirus is known to cause gastroenteritis, hemorrhagic cystitis, pancreatitis, meningoencephalitis, necrotizing hepatitis, and nephritis/renal dysfunction in renal transplant recipients (pham et al. 2003; alsaad et al. 2007) . infection with these viruses may also be associated with rejection (weikert and blumberg 2008) . prevention involves hand hygiene and the use of droplet precautions for those suspected of having infection. influenza vaccination is recommended prior to transplant and yearly following transplant. treatment of respiratory viral infection involves supportive care and antiviral medications. influenza can be treated with oseltamivir or zanamavir. ribavirin is approved for the treatment of rsv. adenovirus infection is treated with reduction of immunosuppression with consideration of cidofovir (ison 2006) . emerging viral pathogens include newly recognized viruses or previously known viruses that are either increasing or threatening to increase in incidence. some of the emerging viruses causing infections in renal transplant population include human t-cell leukemia virus type 1 (htlv-1), hepatitis e virus (hev), measles virus, rabies virus, lymphocytic choriomeningitis virus (lcmv), dengue virus (denv), west nile virus, and zika virus. case reports of adult t-cell leukemia (atl) following renal transplantation in htlv-1-positive patients have been documented, though in a case series of renal transplant recipients with long-term follow-up, no cases of atl or htlv-1-associated myelopathy (ham) developed (nakamura et al. 2005; tanabe et al. 1998) . hev may induce kidney injury with significant reduction in glomerular filtration rate. glomerular injuries such as membranoproliferative glomerulonephritis have been described in kidney transplant patients with acute and chronic hev infections (kamar et al. 2012) . the incidence of measles in transplant recipients is unclear. cases of subacute measles encephalitis (sme) have developed in renal transplant recipients. the clinical course is one of deteriorating mental status and treatment refractory seizures (waggoner and deresinski 2013) . worldwide, vector-borne viral disease is increasing in incidence and can be transmitted with blood products and organ transplantation. fatal cases of dengue have been reported within the first month following renal transplant (waggoner and deresinski 2013) . west nile virus has also been reported in transplant recipients with a high incidence of neuroinvasive disease and poor outcomes. zika virus is also now a concern. cases of donor-derived rabies in the sot population have been reported. patients typically developed encephalitis between 1 and 2 months posttransplant, and all symptomatic reported patients died (srinivasan et al. 2005) . cases of lcmv causing severe disease in organ transplant patients have been documented. the 4 clusters of lcmv infection occurred in the united states and involved kidney, liver, and lung transplants; symptoms included fever, abdominal pain, nausea, diarrhea, and altered mental status (srinivasan et al. 2005; barry et al. 2008; fischer et al. 2006) . two renal transplant recipients survived lcmv infection. ribavirin has been employed in some cases, though the benefits remain unclear (waggoner and deresinski 2013) . data regarding the incidence, screening and treatment options of the above-mentioned emerging viruses are limited. given the risk of donor-derived viral transmission, organs should not be accepted from donors with unexplained febrile or neurologic illness. in unclear cases, the risk of donor-derived infection should be balanced with the benefit of the transplant. bacterial infections after renal transplantation can be due to surgical complications at the time of transplantation, nosocomial infection, immunosuppression, or community-acquired infection. donorderived bacterial infections from the transplanted kidney or blood stream can occur as well. about 47% of kidney transplant recipients develop bacterial infections (patel and paya 1997) . occurring any time posttransplantation, urinary tract infections account for the overwhelming majority of these infections and are the most common bacterial infections prolonging or leading to re-hospitalization (wyner 1994) . enterococci, staphylococci, enteric gram-negative organisms, and p. aeruginosa are the most common bacteria isolated (wyner 1994) . bacterial pneumonia, postoperative wound infections, and bacteremia or sepsis, although less common, also prolong or lead to rehospitalizations after transplantation (karuthu and blumberg 2012) . common bacterial pathogens for these infections are gram-negative organisms, including multidrug resistant bacteria; gram positive organisms, including methicillin-resistant staphylococcus aureus (mrsa), and vancomycin-resistant entercococci (vre), as well as organisms more typically seen in immunocompromised patients such as listeria. months after the operation, bacterial pathogens include streptococcus species, mycoplasma, legionella, listeria, salmonella, and nocardia. trimethoprim-sulfamethoxazole (tmp-smx) prophylaxis has been shown to reduce the incidence of some of these infections. increased antimicrobial resistance, urgency of treatment, drug interactions, and toxicities, as well as the risk for clostridium difficile colitis all contribute to the complex decision making required for antimicrobial management. risk factors for urinary tract infection after transplantation are a prolonged period of hemodialysis before transplant, prolonged bladder catheterization, female sex, deceased donor transplant, kidney-pancreas transplant with bladder drainage, uretero-vesical stents, and an increased immunosuppressed state (karuthu and blumberg 2012; lapchik et al. 1992) . prophylaxis to lower the risk of infection after transplant with trimethoprim-sulfamethoxazole is routine (karuthu and blumberg 2012) . controversy regarding the exact dosing and duration of prophylaxis exists. typically it is given at a dose of 160 mg trimethoprim and 800 mg of sulfamethoxazole daily for 6-12 months (kdigo 2009 ). trimethoprim-sulfamethoxazole reduces the risk of uti and bacteremia (karuthu and blumberg 2012; patel and paya 1997) . symptoms of uti include frequency, urgency, and dysuria as well as nausea and vague abdominal complaints. some patients are asymptomatic. escherichia coli is the most common pathogen and an increasing number of pathogens are multidrug resistant. sensitivity testing is required. treatment of asymptomatic bacteriuria in the renal transplant recipient is controversial and is not routinely recommended (coussement and abramowicz 2013) . although not well studied, since utis in renal transplant patients are complicated, 7-14 days of antibiotics is a typical duration. removal of stents and catheters as well as drainage of abscesses are frequently required to prevent relapse and for cure. surgical wound infections, occurring at a rate of 3-4%, usually present within the first 4 weeks after transplant (ramos et al. 2008) . obesity, urine leaks, re-operation through the original incision, diabetes, high creatinine levels in plasma, and prolonged bladder catheterization are risk factors for wound infections (humar et al. 2001; khoury and brennan 2005) . improved organ procurement, preservation, and surgical techniques along with preoperative antibiotics all reduce the risk of subsequent postoperative wound infection. bacterial organisms causing these types of infections may be nosocomial and multidrug-resistant making antibiotic treatment difficult due to limited options or toxicities. source control with good wound care is critical in the management of these types of infections. although pneumonia is the most common bacterial infection in all solid organ transplant recipients, its incidence is lowest in those who have received a kidney (khoury and brennan 2005) . occurring early in the posttransplant period, cmv infection and rejection treatment with antilymphocyte preparations increase the pneumonia risk. hospital-acquired pneumonia due to resistant pathogens, such as mrsa, and extended spectrum beta lactamase (esbl) or carbapenemresistant (cre) gram-negative organisms are increasing in incidence and sometimes require nephrotoxic agents for treatment. communityacquired pneumonia can occur any time after transplantation and the incidence of communityacquired pneumonia specifically due to streptococcus pneumoniae can be lowered with vaccination. bacteremia and sepsis are most commonly due to a urinary source, followed by lung, wound, and abdomen (khoury and brennan 2005) . intravenous catheters also play a role. diabetes mellitus and posttransplant dialysis increase the incidence of sepsis which decreases the survival rate in these patients (abbott et al. 2001) . prompt treatment with broad spectrum antibiotics followed by rapid de-escalation to pathogen-specific therapy based on sensitivities is required. removal of foreign bodies such as intravenous catheters and stents is also necessary for cure. nocardia is a rare infection seen in the renal transplant recipient occurring in less than 4% of patients (wilson et al. 1989 ). trimethoprim-sulfamethoxazole prophylaxis used after transplant to prevent pneumocystis jiroveci pneumonia (pcp) likely prevents nocardia infection as well. nocardia asteroides is the most common species and causes pulmonary infections, including cavitary lesions and pleural effusions (patel and paya 1997) . other common sites of infection, due to dissemination, are central nervous system (cns) and cutaneous. all patients with nocardia should be evaluated for cns disease. allograft rejection, high-dose prednisone, azathioprine, instead of cyclosporine based immunosuppression, and neutropenia are risk factors for this infection (patel and paya 1997) . diagnosis is made by the identification of branching and beading rods on gram and modified acid fast staining and cultures of infected sites. antimicrobial susceptibility testing should be performed on all isolates. high dose trimethoprim-sulfamethoxazole sometimes in combination with amikacin is the treatment of choice, but allergic reactions and other side effects sometimes limit their use. alternatives include imipenem, minocycline, and ceftriaxone, but choices should be based on susceptibilities and site of infection (spelman 2016) . nocardia infections can relapse and prolonged therapy up to a year is recommended followed by chronic suppressive therapy (spelman 2016; arduino et al. 1993 ). listeria monocytogenes is a bacterial organism that is transmitted most commonly during summer and early fall to humans via the gastrointestinal tract from contaminated dairy products, raw vegetables, and meat. although more common during the first 2 months after transplantation, infection may occur at any point, and risk is increased with rejection therapy (patel and paya 1997) . infections involving the central nervous system, such as meningitis and meningoencephalitis, are most common and present with headaches, fever, meningismus, altered mental status, and possibly focal neurologic deficits including cranial nerve palsies and seizures (patel and paya 1997) . cerebrospinal fluid examination typically reveals a pleocytosis, mostly polymorphonuclear leukocytes, decreased glucose, and elevated protein, but as the name implies, a mononuclear predominance may occur instead. gram staining has a low sensitivity and may be negative or reveal gram positive bacilli which may be confused with diphtheroids. other sites of infection include bacteremia, pneumonia, endophthalmitis, and septic arthritis. while trimethoprim-sulfamethoxazole, used for p. carinii prophylaxis, may also prevent infection with listeria, the treatment of choice is intravenous ampicillin and gentamicin for up to 8 weeks in those with cns infections to prevent relapses. gentamicin is usually continued for a shorter duration, about 2 weeks if kidney function is stable. (gelfand 2016) . trimethoprim-sulfamethoxazole is an alternative treatment for those who are allergic to penicillin. decreasing immunosuppressive agents is sometimes, but not always necessary. legionella infections in renal transplant recipients most commonly occur early in the posttransplantation period, but can be seen any time, especially during episodes of rejection. legionella pneumophila is the most common species to infect humans, and although more commonly community-acquired, nosocomial transmission occurs (patel and paya 1997) . most infections are pulmonary including pneumonia, and abscess with cavitation. symptoms are typical of lung infections but also may include headache and diarrhea. a legionella urinary antigen test and culture of lower respiratory secretions on selective media are used for diagnosis. empiric treatment for legionella is appropriate while waiting for results. quinolone antibiotics, such as levofloxacin, are preferred over macrolides in renal transplant patients because of drug interactions between macrolides and immunosuppressive medications. initially given intravenously, quinolone antibiotics can be quickly deescalated to oral treatment when the patient has defervesced. renal transplant patients, especially those who are severely ill at presentation, should receive 21 days of treatment (yu 2016) . along with pcp and listeria, as noted above, prophylaxis with trimethoprim-sulfamethoxazole may also prevent legionella infection. immunosuppression increases the risk of developing mycobacterium tuberculosis (tb) disease. although the majority of tuberculosis infections in renal transplant recipients occur in the first 18 months, tb can occur any time after transplantation (khoury and brennan 2005) . its overall incidence is lower in the united states when compared to the rest of the world, and foreign-born recipients are at greatest risk. having a high index of suspicion is important in renal transplant patients because presentation can be atypical and pretransplant screening with tuberculin skin tests or ifn-gamma release assays are unreliable in chronic kidney disease patients due to anergy. extra-pulmonary sites of infection and disseminated disease occur in about a third of cases (karuthu and blumberg 2012). laryngeal, meningeal, skeletal, cutaneous, intestinal, and renal infections are examples of extra-pulmonary disease. fevers are common, but sweats and weight loss may be absent (patel and paya 1997) . screening prior to transplant should include a history regarding prior exposures, and treatment for tb, as well as a chest x-ray and urine afb culture. prophylaxis with isoniazid or rifampin should be offered to patients prior to transplantation with a history of inadequately treated tb, an abnormal chest x-ray suggestive of prior tb exposure, a positive ppd or ifn gamma assay, contact with someone with active tb, or a kidney from a ppd-positive donor in order to minimize reactivation disease after transplantation (khoury and brennan 2005) . patients receiving treatment for latent tb may undergo renal transplantation and complete their defined course afterwards with special attention to potential drug interactions and toxicities (karuthu and blumberg 2012) . diagnosis of tb after renal transplantation often requires a biopsy of the infected site with stains for acid fast bacilli and cultures for sensitivity testing. treatment of active disease after transplantation requires multiple drugs and should follow the american thoracic society, center for disease control, and infectious disease society of america guidelines (mmwr 2003) . special attention to drug toxicities and interactions with immunosuppressive agents is required. rifampin, in particular, decreases cyclosporine levels and increases the risk for rejection. fungal infections in kidney transplant recipients occur less frequently than in other solid organ transplant recipients. most present within the first 6 months after transplantation (hagerty et al. 2003 ) and can represent primary, reactivated, or donor-derived infection. those associated with geographic and environmental exposures include histoplasmosis, coccidioidomycosis, blastomycosis, and paracoccidioidomycosis. others are considered opportunistic and include infections such as candida, aspergillus, and cryptococcus (karuthu and blumberg 2012) . broad spectrum antibiotics, corticosteroids, diabetes mellitus, rejection therapy, cmv infection, and duration of pretransplant dialysis are risk factors (khoury and brennan 2005) . esophageal candidiasis, urogenital candidiasis, and pneumonia are the three most common sites of fungal infections in these patients (abbott et al. 2001) . clinical presentation may be nonspecific and diagnosis difficult due to testing limitations. positive cultures may represent colonization rather than infection with pathogens such as candida and aspergillus. cultures, antigen assays, serum galactomannan assays, and radiography may be helpful, but are not always diagnostic. subsequently, biopsy with pathology and cultures is considered the gold standard for diagnosing fungal infections (karuthu and blumberg 2012) . drug interactions and toxicities as well as immune reconstitution, due to lowering of immunosuppressive medications, further complicate the management of fungal disease in these patients and require expert advice (karuthu and blumberg 2012) . pneumocystis jiroveci (formerly pneumocystis carinii (pcp), protozoa) is a pathogen currently considered a fungus based on nucleic acid and biochemical analysis. presenting as pneumonia with interstitial infiltrates on chest x-ray within the first year after transplantation in those not receiving prophylaxis, mortality may be high. nonproductive cough and shortness of breath with rapid progression to hypoxia is a classic presentation. diagnosis is based on silver staining of deep respiratory specimens from induced sputum, bronchoalveolar lavage, or transbronchial biopsy (martin and fishman 2013) . the treatment of choice is high dose trimethoprim-sulfamethoxazole for 21 days with corticosteroids in hypoxic patients (partial pressure of oxygen of <70 mmhg on room air) tapered over 14 days. atovaquone or clindamycin plus pyrimethamine are alternative agents (martin and fishman 2013) . trimethoprim-sulfamethoxazole prophylaxis for 6-12 months after transplantation is highly effective in preventing this infection and should be administered to all renal transplant patients if tolerated. frequently used alternatives for prophylaxis in allergic patients include dapsone (if glucose-6 phosphate dehydrogenase levels are normal) and atovaquone. infection remains an important concern in patients undergoing kidney transplantation. attention to pretransplant screening of the potential organ donor and recipient is essential to optimizing transplant outcomes. advances in the management of transplant-related infections include the increasing use of rapid molecular diagnostic testing as well as improvements in the approach to prophylaxis and treatment. ongoing challenges include the need for prolonged immunosuppression to prevent organ rejection, drug-drug interactions, and the management of resistant and emerging pathogens. continued awareness of the risks, timing, and presentation of infection posttransplant and strategies to reduce its impact will contribute further to progress in the field of kidney transplantation. hospitalizations for bacterial septicemia after renal transplantation in the united states epstein-barr virus and posttransplant lymphoproliferative disorder in solid organ transplant recipients late-onset acute haemorrhagic necrotizing granulomatous adenovirus tubulointerstitial nephritis in a renal allograft nocardiosis in renal transplant recipients undergoing immunosuppression with cyclosporine long-term outcomes of cmv disease treatment with valganciclovir versus iv ganciclovir in solid organ transplant recipients dilemma of hcv infection in renal transplant recipients lymphocytic choriomeningitis virus transmitted through solid organ transplantation -massachusetts solid organ transplantation in the hiv-infected patient cytomegalovirus in renal transplantation thymoglobulin-associated cd4þ t-cell depletion and infection risk in hiv-infected renal transplant recipients hiv transmitted from a living organ donor recommended adult immunization schedule-united states preemptive lamivudine therapy based on hbv dna level in hbsag-positive kidney allograft recipients entecavir treatment for up to 5 years in patients with hepatitis b e antigen-positive chronic hepatitis b co-infection of two β-herpesviruses (cmvand hhv-7) as an increased risk factor for "cmv disease" in patients undergoing renal transplantation effects of immunosuppressive drugs on hiv infection: implications for solid-organ transplantation post-transplant lymphoproliferative disorder in renal allograft recipients: clinical experience and risk factor analysis in a single center should we treat asymptomatic bacteriuria after renal transplantation? epidemiology of bk virus in renal allograft recipients: independent risk factors for bk virus replication human cytomegalovirus and kidney transplantation: a clinician's update risk factors for hospitalization for bacterial or viral infection in renal transplant recipients-an analysis of usrds data efficacy of high-dose versus standard-dose influenza vaccine in older adults hhv 8 in renal transplant recipients human herpesvirus-6 and-7 in transplantation transplantation in the patient with hepatitis c 24-week oral ganciclovir prophylaxis in kidney recipients is associated with reduced symptomatic cytomegalo-virus disease compared to a 12-week course polyomavirus disease in renal transplantation: review of pathological findings and diagnostic methods hbsag seropositive status and survival after renal transplantation: meta-analysis of observational studies disseminated varicella infection in adult renal allograft recipients: four cases and a review of the literature transmission of lymphocytic choriomeningitis virus by organ transplantation ast infectious diseases community of practice (2013) screening of donor and recipient in solid organ transplantation infection in solid organ transplant recipients renal transplantation in patients with hiv clinical manifestations and diagnosis of listeria monocytogenes infection guidelines for the prevention and management of infectious complications of solid organ transplantation using epstein-barr viral load assays to diagnose, monitor, and prevent posttransplant lymphoproliferative disorder fungal infections in solid organ transplant patients bk virus nephritis after renal transplantation polyoma-virus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations asymptomatic cytomegalovirus infection is associated with increased risk of new-onset diabetes mellitus and impaired insulin release after renal transplantation antiviral medications for preventing cytomegalovirus disease in solid organ transplant recipients ast infectious diseases community of practice: cytomegalovirus in solid organ transplant recipients are wound complications after a kidney transplant more common with modern immunosuppression the efficacy and safety of 200 days valganciclovir cytomegalovirus prophylaxis in high-risk kidney transplant recipients solid organ transplantation from hepatitis b virus-positive donors: consensus guidelines for recipient management adenovirus infections in transplant recipients vaccination and chronic kidney disease polyomavirus-associated nephropathy: update on antiviral strategies hepatitis e virus and the kidney in solid-organ-transplant patients common infections in kidney transplant recipients the value of vaccination in chronic kidney disease kdigo clinical practice guideline for the care of kidney transplant recipients infectious complications in kidney transplant recipients: review of the literature prospective study of human betaherpesviruses after renal transplantation. association of human herpesvirus 7 and cytomegalovirus co-infection with cytomegalovirus disease and increased rejection improvement in long-term renal graft survival due to cmv prophylaxis with oral ganciclovir: results of a randomized clinical trial intervals between pcv13 and ppsv23 vaccines: recommendations of the advisory committee on immunization practices (acip) viral infection in the renal transplant recipient transplantation society international cmv consensus group: international consensus guidelines on the management of cytomegalovirus in solid organ transplantation selecting suitable solid organ transplant donors: reducing the risk of donor-transmitted infections risk factors for nosocomial urinary tract and postoperative wound infections in renal transplant patients: a matched-pair case-control study recommendations for screening of donor and recipient prior to solid organ transplantation and to minimize transmission of donor-derived infections infectious diseases community of practice (2013) viral hepatitis in solid organ transplant recipients emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants posttransplant lymphoproliferative disease in primary epstein-barr virus infection after liver transplantation: the role of cytomegalovirus disease american society of transplantation and american society of transplant surgeons, pneumocystis pneumonia in solid organ transplantation cytomegalovirus seromismatching increases the risk of acute renal allograft rejection allowing hiv-positive organ donation: ethical, legal and operational considerations renal transplantation in patients with hepatitis c virus antibody. a long national experience hiv-positive-to-hiv-positive kidney transplantation-results at 3-5 years prevention and management of cytomegalovirus infection in solid-organ transplantation prognosis of htlv-i-positive renal transplant recipients pre-emptive treatment for cytomegalovirus viraemia to prevent cytomegalovirus disease in solid organ transplant recipients infections in solid-organ transplant patients valganciclovir solid organ transplant study group: efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cyto-megalovirus disease in solid organ transplant recipients fatal disseminated adenovirus infections in immunocompromised patients polyoma nephropathy and progressive multifocal leukoencephalopathy in a renal transplant recipient cmv infection is associated with transplant renal artery stenosis incisional surgical site infection in kidney transplantation bk virus in transplant recipients: an overview and update transmission of human herpesvirus 8 infection from renal-transplant donors to recipients impact of early cytomegalovirus infection and disease on longterm recipient and kidney graft survival resitra network of the spanish study group of infection in transplantation (2008) impact of current transplantation management on the development of cytomegalovirus disease after renal transplantation successful treatment of hepatitis c in renal transplant recipients with direct-acting antiviral agents phs guideline for reducing human immunodeficiency virus, hepatitis b virus, and hepatitis c virus transmission through organ transplantation prevalence of hiv-associated nephropathy in autopsies of hiv-infected patients human herpesvirus-6 in transplantation: an emerging pathogen rates of first infection following kidney transplant in the united states up to date transmission of rabies virus from an organ donor to four transplant recipients sirolimus for kaposi's sarcoma in renal transplant recipients outcomes of kidney transplantation in hiv-infected recipients preemptive treatment for cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ transplant recipients viral infections affecting the skin in organ transplant recipients: epidemiology and current management strategies long-term results in human t-cell leukemia virus type 1-positive renal transplant recipients post-transplant lymphoproliferative disorders (ptld) after solid organ transplantation the association of viral infection and chronic allograft nephropathy with graft dysfunction after renal transplantation polyomavirus in kidney and kidney-pancreas transplant recipients epidemiology of kidney disease in the unites states. national institutes of health, national institute of diabetes and digestive and kidney diseases in: safdar a (ed) principles and practice of transplant infectious diseases nocardial infections in renal transplant recipients the evaluation and management of urinary tract infections in recipients of solid organ transplants treatment and prevention of legionella infection key: cord-017012-yl0vanuh authors: herberg, jethro; pahari, amitava; walters, sam; levin, michael title: infectious diseases and the kidney date: 2009 journal: pediatric nephrology doi: 10.1007/978-3-540-76341-3_52 sha: doc_id: 17012 cord_uid: yl0vanuh the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. in this chapter, a microbiologic classification of the organisms affecting the kidney is adopted. although they are important causes of renal dysfunction in infectious diseases, urinary tract infections and hemolytic uremic syndrome (hus) are not discussed in detail because they are considered separately in chapters 54 and 48 respectively. elucidation of the cause of renal involvement in a child with evidence of infection must be based on a careful consideration of the geographic distribution of infectious diseases in different countries. a history of foreign travel; exposure to animals, insects, or unusual foods or drinks; outdoor activities such as swimming or hiking; and contact with infectious diseases must be sought in every case. the clinical examination should include a careful assessment of skin and mucous membranes and a search for insect bites, lymphadenopathy, and involvement of other organs. a close collaboration with a pediatric infectious disease specialist and hospital microbiologist will aid the diagnosis and management of the underlying infection. a tantalizing clue to the pathogenesis of glomerular disease is the marked difference in the incidence of nephrosis and nephritis in developed and underdeveloped areas of the world. in several tropical countries, glomerulonephritis (gn) accounts for up to 4% of pediatric hospital admissions; the incidence in temperate climates is 10-to 100-fold less. this difference might be explained by a complex interaction of several different factors, including nutrition, racial and genetically determined differences in immune responses, and exposure to infectious diseases. a growing body of evidence, however, suggests that longterm exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. renal involvement in infectious diseases may occur by a variety of mechanisms: direct microbial invasion of the renal tissues or collecting system may take place in conditions such as staphylococcal abscess of the kidney as a result of septicemic spread of the organism or as a consequence of ascending infection; damage to the kidney may be caused by the systemic release of endotoxin or other toxins and activation of the inflammatory cascade during septicemia or by a focus of infection distant from the kidney; ischemic damage may result from inadequate perfusion induced by septic shock; the kidney may be damaged by activation of the immunologic pathways or by immune complexes resulting from the infectious process. in many conditions, a combination of these mechanisms may be operative. in the assessment of renal complications occurring in infectious diseases, the possibility of druginduced nephrotoxicity caused by antimicrobial therapy should always be considered. the nephrotoxic effects of antibiotics and other antimicrobial agents are not addressed in this chapter but are covered in chapter 53. bacterial infections associated with renal disease and the likely mechanisms causing renal dysfunction are shown in > impaired renal function is a common occurrence in systemic sepsis (1) . depending on the severity of the infection and the organism responsible, the renal involvement may vary from insignificant proteinuria to acute renal failure requiring dialysis. the organisms causing acute renal failure as part of systemic sepsis vary with age and geographic location and also differ in normal and immunocompromised children. in the neonatal period, group b streptococci, coliforms, staphylococcus aureus, and listeria monocytogenes are the organisms usually . responsible. in older children, neisseria meningitidis, streptococcus pneumoniae, and s. aureus account for most of the infections. in people who are immunocompromised, a wide range of bacteria are seen, and, similarly, in tropical countries other pathogens, including haemophilus influenzae, salmonella species, and pseudomonas pseudomallei, must be considered. where h. influenzae type b vaccine has been introduced, however, the incidence of severe systemic infections due to this organism has shown a sharp fall. systemic sepsis usually presents with nonspecific features: fever, tachypnea, tachycardia, and evidence of skin and organ underperfusion. the pathophysiology of renal involvement in systemic sepsis is multifactorial (1, 2) . hypovolemia with diminished renal perfusion is the earliest event and is a consequence of the increased vascular permeability and loss of plasma from the intravascular space. hypovolemia commonly coexists with depressed myocardial function because of the myocardial depressant effects of endotoxin or other toxins. the renal vasoconstrictor response to diminished circulating volume and reduced cardiac output further reduces glomerular filtration, and oliguria is thus a consistent and early event in severe sepsis (1, 3) . a number of vasodilator pathways are activated in sepsis, including nitric oxide and the kinin pathways. this may lead to inappropriate dilatation of vascular beds. vasodilation of capillary beds leading to warm shock is common in adults with sepsis due to gram-negative organisms but is less commonly seen in children, in whom intense vasoconstriction is the usual response to sepsis. if renal underperfusion and vasoconstriction are persistent and severe, the reversible prerenal failure is followed by established renal failure with the characteristic features of vasomotor nephropathy or acute tubular necrosis. other mechanisms of renal damage in systemic sepsis include direct effects of endotoxin and other toxins on the kidney, and release of inflammatory mediators such as tumor necrosis factor (tnf) and other cytokines, arachidonic acid metabolites, and proteolytic enzymes (3) . nitric oxide (no) is postulated to play a key role in the pathophysiology of renal failure in sepsis. whether the renal effects of increased no are beneficial or harmful remains unclear. trials of selective no synthetase inhibition did not offer any advantages over saline resuscitation (4) . no in endotoxemia is possibly beneficial because it maintains renal blood flow and glomerular filtration. activation of coagulation is an important component of the pathophysiology of septic shock and may contribute to renal impairment. activation of multiple prothrombotic and antifibrinolytic pathways occurs, together with downregulation of antithrombotic mechanisms such as the protein c pathway. treatment with activated protein c has been shown to improve outcome of adult septic shock, but has not been confirmed to have benefit in pediatric sepsis, and may carry a risk of bleeding particularly in infants (5) . the renal findings early in septic shock are oliguria, with high urine/plasma urea and creatinine ratios, low urine sodium concentration, and a high urine/plasma osmolarity ratio. once established, renal failure supervenes, and the urine is of poor quality with low urine/ plasma urea and creatinine ratios, elevated urine sodium concentration, and low urine osmolarity. proteinuria is usually present, and the urine sediment may contain red cells and small numbers of white cells (1) . the management of acute renal failure in systemic sepsis depends on early diagnosis and administration of appropriate antibiotics to cover the expected pathogens. . in addition, management is directed at improving renal perfusion and oxygenation. volume replacement with crystalloid or colloid should be undertaken to optimize preload. central venous pressure or pulmonary wedge pressure monitoring is essential to guide volume replacement in children in severe shock (1, 2) . the use of low-dose (2-5 pg/kg/min) dopamine to reduce renal vasoconstriction together with administration of inotropic agents such as dobutamine or epinephrine to improve cardiac output may reverse prerenal failure. early elective ventilation should be undertaken in patients with severe shock. if oliguria persists despite volume replacement and inotropic therapy, dialysis should be instituted early, because septic and catabolic patients may rapidly develop hyperkalemia and severe electrolyte imbalance. in most children who develop acute renal failure as part of systemic sepsis or septic shock, the renal failure is of short duration, and recovery can be expected within a few days of achieving cardiovascular stability and eradication of the underlying infection. occasionally, renal cortical necrosis or infarction of the kidney may result in prolonged or permanent loss of renal function. meningococcus n. meningitidis continues to be a major cause of systemic sepsis and meningitis in both developed and underdeveloped parts of the world (6) . in developed countries, most cases are caused by group b and y strains, particularly after introduction of meningococcal c vaccination, whereas epidemics of meningococcus groups a, c and w135 continue to occur in many underdeveloped regions of the world (6, 7) . infants and young adults are most commonly affected, but cases in adolescents and young children are also common. there are two major presentations of meningococcal disease (6) : meningococcal meningitis presents with features indistinguishable from those of other forms of meningitis, including headache, stiff neck, and photophobia. lumbar puncture is required to identify the causative agent and distinguish this from other forms of meningitis. despite the acute nature of the illness, the prognosis is good, and most patients with the purely meningitic form of the illness recover without sequelae. meningococcemia with purpuric rash and shock is the second and more devastating form of the illness. affected patients present with nonspecific symptoms of fever, vomiting, abdominal pain, and muscle ache. the diagnosis is only obvious once the characteristic petechial or purpuric rash appears. patients with a rapidly progressive purpuric rash, hypotension, and evidence of skin and organ underperfusion have a poor prognosis, with a mortality of 10-30%. adverse prognostic features include hypotension, a low white cell count, absence of meningeal inflammation, thrombocytopenia, and disturbed coagulation indices (8) . renal failure was seldom reported in early series of patients with meningococcemia, perhaps because most patients died rapidly of uncontrolled septic shock. with advances in intensive care, however, more children are surviving the initial period of profound hemodynamic derangement, and renal failure is more often seen as a major management problem. approximately 10% of children with fulminant meningococcemia develop renal failure, which usually occurs 24-48 h after the onset of illness (9) . the pathophysiology of meningococcal septicemia involves the activation of cytokines and inflammatory cells by endotoxin (6, 7) . mortality is directly related to both the plasma endotoxin concentration and the intensity of the inflammatory response, as indicated by levels of tnf and other inflammatory markers (10) . patients with meningococcemia have a profound capillary leak leading to severe hypovolemia. loss of plasma proteins from the intravascular space is probably the major cause of shock (11) , but myocardial suppression secondary to il-6 production is also important (12) . intense vasoconstriction further impairs tissue and organ perfusion, and vasculitis with intravascular thrombosis and consumption of platelets and coagulation factors is also present (6) . oliguria is invariably present in children with meningococcemia during the initial phase of the disorder. this is prerenal in origin and may respond to volume replacement and inotropic support. if cardiac output cannot be improved and renal underperfusion persists, established renal failure supervenes. occasionally, cortical necrosis or infarction of the kidneys occurs. children with meningococcemia should be aggressively managed in a pediatric intensive care unit, with early administration of antibiotics (penicillin or a third-generation cephalosporin), volume replacement, hemodynamic monitoring, and the use of inotropic agents and vasodilators. if oliguria persists despite measures to improve cardiac output, elective ventilation and dialysis should be instituted early (6, 7) . because activation of coagulation pathways occurs, severe acquired protein-c deficiency may result and is usually associated with substantial mortality (13) . protein c is a natural anticoagulant which also has important antiinflammatory activity. despite evidence for impaired function of the activated protein c pathway in meningococcal diseases (14) , and adult trials suggesting benefit of activated protein c administration in septic shock (prowess trial) (15) , pediatric trials of activated protein c showed no clear benefit, and were associated with increased risk of intracranial bleeding in very young infants (5) . the role of apc therapy in pediatric sepsis remains unclear. most patients who survive the initial 24-48 h of the illness and regain hemodynamic stability will ultimately recover renal function even if dialysis is required for several weeks. the least common presentation of meningococcal sepsis is chronic meningococcemia. patients with this form of the illness present insidiously with a vasculitic rash, arthritis, and evidence of multiorgan involvement. the features may overlap those of henoch-schonlein purpura or subacute bacterial endocarditis (sbe), and the diagnosis must be considered in patients presenting with fever, arthritis, and vasculitic rash, often accompanied by proteinuria or hematuria. response to antibiotic treatment is good, but some patients may have persistent symptoms for many days resulting from an immunecomplex vasculitis. staphylococcal infections may affect the kidneys by direct focal invasion during staphylococcal septicemia, forming a renal abscess; by causing staphylococcal bacteremia; or by toxin-mediated mechanisms, as in the staphylococcal toxic shock syndrome. staphylococcal abscess. staphylococcal renal abscess presents with fever, loin pain and tenderness, and abnormal urine sediment, as do abscesses caused by other organisms (16) . the illness often follows either septicemia or pyelonephritis. the diagnosis is usually considered only when a patient with clinical pyelonephritis shows an inadequate response to antibiotic treatment. the diagnosis is confirmed by ultrasonography or computed tomographic scan, which shows swelling of the kidney and intrarenal collections of fluid. antibiotic therapy alone may result in cure, but if the patient remains unwell with evidence of persistent inflammation despite use of appropriate antibiotics, surgical intervention may be required. percutaneous drainage under ultrasonographic or computed tomographic scan guidance is often effective and may avoid the need for a more direct surgical approach (16, 17) . staphylococcal toxic shock syndrome. the staphylococcal toxic shock syndrome is a systemic illness characterized by fever, shock, erythematous rash, diarrhea, confusion, and renal failure. the disorder was first described by todd et al. in 1978 in a series of seven children (18) . during the 1980s, thousands of cases were reported in the united states. most cases were in menstruating women, in associated with tampon use. although most cases worldwide are seen in women and are associated with menstruation, children of both sexes and of all ages are affected (19) . the illness usually begins suddenly with high fever, diarrhea, and hypotension, together with a diffuse erythroderma (20) . mucous membrane involvement with hyperemia and ulceration of the lips and oral mucosa or vaginal mucosa, strawberry tongue, and conjunctival injection are usually seen. desquamation of the rash occurs in the convalescent phase of the illness. confusion is often present in the early stages of the illness and may progress to coma in severe cases. multiple organ failure with evidence of impaired renal function, elevated levels of hepatic transaminases, thrombocytopenia, elevated cpk and disseminated intravascular coagulation (dic) is often seen. according to cdc criteria, the diagnosis is made on the basis of the clinical features of fever, rash, hypotension, and subsequent desquamation along with deranged function of three or more of the following organ systems: gastrointestinal (gi), mucous membranes, renal, hepatic, hematologic, central nervous system, and muscle. other disorders causing a similar picture, such as rocky mountain spotted fever, leptospirosis, measles, and streptococcal infection, must be excluded. the staphylococcal toxic shock syndrome is now known to be due to infection or colonization with strains of s. aureus that produce one or more protein exotoxins (21) . most cases in adults are associated with toxic shock toxin i; in children, many of the isolates associated with the syndrome produce other enterotoxins (a to f). the staphylococcal enterotoxins appear to induce disease by acting as superantigens (22) , which activate t cells bearing specific v beta regions of the t-cell receptor; this causes proliferation and cytokine release (23) . the systemic illness and toxicity are believed to result largely from an intense inflammatory response induced by the toxin. the site of toxin production is often a trivial focus of infection or simple colonization, and bacteremia is rarely observed. renal failure in toxic shock syndrome is usually caused by shock and renal hypoperfusion. in the early stages of the illness, oliguria and renal impairment are usually prerenal and respond to treatment of shock and measures to improve perfusion. in severe cases and in patients in whom treatment is delayed, acute renal failure develops as a consequence of prolonged renal underperfusion, and dialysis may be required. in addition to underperfusion, direct effects of the toxin or inflammatory infectious diseases and the kidney mediators may also contribute to the renal damage. recovery of renal function usually occurs, but in severe cases with cortical necrosis or intense renal vasculitis, prolonged dialysis may be required. the management of staphylococcal toxic shock syndrome depends on early diagnosis and aggressive cardiovascular support with volume replacement, inotropic support, and, in severe cases, elective ventilation. if oliguria persists despite optimization of intravascular volume and administration of inotropic agents, dialysis should be commenced early (20) . anti-staphylococcal antibiotics should be started as soon as the diagnosis is suspected and the site of infection identified. initial empiric antimicrobial therapy should include an anti-staphylococcal antibiotic effective against betalactamase-resistant organisms and a protein synthesisinhibiting antibiotic such as clindamycin to stop further toxin production (24) . if there is a focus of infection such as a vaginal tampon, surgical wound, or infected sinuses, the site should be drained early to prevent continued toxin release into the circulation. the intravenous administration of immune globulins may be considered when infection is refractory to several hours of aggressive therapy, an undrainable focus is present, or persistent oliguria with pulmonary edema occurs (24) . with aggressive intensive care, most affected patients survive, and renal recovery is usual, even in patients who have had severe shock and multiorgan failure. relapses and recurrences of staphylococcal toxic shock syndrome occur in a proportion of affected patients because immune responses to the toxin are ineffective in some individuals. panton valentine leucocidin (pvl) producing staphylococcal infection: in recent years there have been increasing reports of severe staphylococcal disease, associated with shock and multiorgan failure, caused by strains of staphylococci producing the pvl toxin. panton-valentine leukocidin (pvl) is a phage-encoded toxin, which profoundly impairs the host response due to its toxic effect on leucocytes (see review (25) ). pvl producing strains are associated with tissue necrosis and increased propensity to cause abscesses in lung, bone, joint, and soft tissue infections. perinephric abscesses have been reported (26) . there are increasing numbers of children and adults admitted with fulminant sepsis, and shock due to pvl producing strains, and renal failure is a significant component of the multiorgan failure. in addition to intensive care support, antibiotic treatment of pvl strains should include antibiotics which reduce toxin production, such as clindamycin, linezolid or rifampicin, as well as vancomycin if the strain is resistant to methicillin. beta-lactam antibiotics should be avoided, as there is some data to suggest that pvl toxin production can increased by these antibiotics under some conditions (27) . immunoglobulin infusion may also be of benefit. aggressive surgical drainage of all collections requires close consultation with orthopedic and surgical teams. the group a streptococci (gas) are a major worldwide cause of renal disease, usually as poststreptococcal nephritis. however, in addition to this post-infection immunologically mediated disorder, in recent years there have been increasing reports of gas causing acute renal failure as part of an invasive infection with many features of the staphylococcal toxic shock syndrome (28) . acute poststreptococcal glomerulonephritis. acute poststreptococcal gn (apsgn) is a delayed complication of pharyngeal infection or impetigo with certain nephritogenic strains of gas. different strains can be serotyped according to the antigenic properties of the m protein found in the outer portion of the bacterial wall. apsgn after pharyngeal infection is most commonly associated with serotype m12. in contrast, in apsgn after impetigo, serotype m49 is most commonly identified (29) . on occasions, other serotypes and non-typeable strains have been described as causing gn. the pathology and pathogenesis of the disorder is discussed in detail in chapter 30. apsgn has a worldwide distribution. epidemiologic differences are observed between pharyngitis-associated and impetigo-associated streptococcal infections. pharyngitis-associated apsgn is most common during school age and has an unexplained male/female ratio of 2:1. it occurs more often in the cooler months, and familial occurrences are commonly described. the latent period is 1-2 weeks, in notable contrast to impetigo-associated cases, which have a latent period of 2-6 weeks. in many developing countries, children have chronic skin infections, and it may be difficult to establish the latent period with accuracy. impetigoassociated cases are more common in the warmer months, sex distribution is equal, and children tend to be younger. introduction of a nephritogenic strain into a family often results in the occurrence of several cases within that family, and in some cases, attack rates of up to 20% have been described (30) . the incidence is linked to poor socioeconomic conditions. renal involvement in apsgn can be mild, and in many patients, the disease may not be manifested clinically. studies of epidemics with nephritogenic strains of streptococci have shown that up to 50% of those infected had subclinical evidence of renal disease (30, 31) . in a typical case a sudden onset of facial or generalized edema occurs. hypertension is usually modest but is severe in 5% of cases, and occasionally may lead to encephalopathy or left ventricular failure. the urine is smoky or tea colored in 30-50% of cases. pallor, headache, backache, lethargy, malaise, anorexia, and weakness are all common nonspecific features. the urine volume is decreased. proteinuria is present (up to 100 mg/dl), and microscopy shows white cells, red cells, and granular and hyaline casts. urea, electrolyte, and creatinine levels are normal in subclinical cases but show features of acute renal failure in severe cases. it may be possible to culture gas from the skin or the throat in some patients. other evidence of infection with a gas can be obtained through the antistreptolysin-o titer (asot), which is increased in 60-80% of cases. early antibiotic treatment can reduce the proportion of cases with elevated asot to 30%. anti-deoxyribonuclease b and anti-hyaluronidase testing has been shown to be of more value than asot in confirming group a streptococcal infection in impetigo-associated cases. measurement of anti-m protein antibodies is of more value for epidemiologic purposes than for the diagnosis of individual cases (31) . decreased c3 and total hemolytic complement levels are found in 90% of cases during the first 2 weeks of illness and return to normal after 4-6 weeks. penicillin should be given to eradicate the gas organisms. erythromycin, clindamycin, or a first-generation cephalosporin can be given to patients allergic to penicillin. antibiotic treatment probably has no influence on the course of renal disease but will prevent the spread of a nephritogenic strain (32) . close contacts and family members who are culture-positive for gas should also be given penicillin, although antibiotic treatment is not always effective in eliminating secondary cases. recurrent episodes are rare, and immunity to the particular nephritogenic strain that caused the disease is probably lifelong. antibiotic prophylaxis is therefore unnecessary. most studies suggest that the prognosis for children with apsgn is good, with more than 90% making a complete recovery. however, 10% of cases may have a prolonged and more serious course with long-term chronic renal failure (33) . other streptococci. apsgn has also been described after outbreaks of group c streptococcus infection (34) . this has occurred after consumption of unpasteurized milk from cattle with mastitis. patients developed pharyngitis followed by apsgn. endostreptosin was found in the cytoplasm of these group c strains, and during the course of the illness, patients developed anti-endostreptosin antibodies. this antigen has been postulated to be the nephritogenic component of gas. in addition, strains of group g streptococci have been implicated in occasional cases of apsgn (35) . isolates possessed the type m12 protein antigen identical to the nephritogenic type m12 antigen of some group a streptococcal strains. streptococcal toxic shock syndrome and invasive group a streptococcal infection. since 1988, there have been several reports of an illness with many similarities to the staphylococcal toxic shock syndrome, occurring in both children (36) and adults, associated with invasive group a streptococcal disease (32, 37, 38) . patients with this syndrome present acutely with high fever, erythematous rash, mucous membrane involvement, hypotension, and multiorgan failure. unlike staphylococcal toxic shock syndrome, in which the focus of infection is usually trivial and bacteremia is seldom seen, the streptococcal toxic shock syndrome is usually associated with bacteremia or a serious focus of infection such as septic arthritis, myositis, or osteomyelitis (36, 38) . laboratory findings of anemia, neutrophil leukocytosis, thrombocytopenia, and dic are often present, together with impaired renal function, hepatic derangement, and acidosis. acute renal failure requiring dialysis occurs in a significant proportion of cases. it is not clear why there are increasing numbers of cases with invasive disease caused by gas, nor why there has been an emergence of streptococcal toxic shock syndrome, and indeed a similar syndrome caused by some pseudomonas and klebsiella strains. the most common antecedent of invasive gas disease is varicella infection, with the streptococcal infection developing after the initial vesicular phase of the disease is subsiding. strains causing toxic shock syndrome and invasive disease appear to differ from common isolates of gas in producing large amounts of pyrogenic toxins that may have superantigenlike activity. another important mechanism is the production by invasive gas of an il8 protease. il8 serves as a molecular bridge between receptors on neutrophils and the vascular endothelium. cleavage of this protein prevents neutrophil attachment to the endothelium, and results in uncontrolled spread of the bacteria through the tissues (39) . in severe cases necrotizing fasciitis occurs with extensive destruction of the subcutaneous tissues, and is often associated with multiorgan failure. the pathophysiology of streptococcal toxic shock syndrome and that of invasive disease is similar in that superantigen toxins that induce release of cytokines and other inflammatory mediators play a role in both conditions. however gas toxic shock is usually more severe, carries a higher mortality, and is more often associated with focal collections or necrotizing fasciitis. treatment of streptococcal toxic shock syndrome depends on the administration of appropriate antibiotics, aggressive circulatory support, and treatment of any multiorgan failure. surgical intervention to drain the infective focus in muscle, bone, joint, or body cavities is often required. antibiotic therapy with beta-lactams should be supplemented by treatment with a protein synthesisinhibiting antibiotic, such as clindamycin, and it is suggested that this limits new toxin production (40, 41) . a number of new therapies are in development. firstly, pooled intravenous immunoglobulins are now in widespread use in the treatment of toxic shock, particularly when caused by streptococcus (42, 43) . the role of steroids remains unclear, with their hemodynamic benefit set against the detrimental effects of hyperglycemia secondary to gluconeogenesis. (44) . the benefit of insulin therapy to control hyperglycemia is unclear. a recent study in adults found that intensive insulin therapy increased the risk of serious adverse events (45) . in contrast to adult patients, in children with severe sepsis, the use of activated protein c (drotrecogin) cannot be recommended, as in a multicenter trial, fatality was increased in the treatment group (5) . recovery of renal function occurs in patients who respond to treatment of shock and the eradication of the infection. leptospirosis is an acute generalized infectious disease caused by spirochetes of the genus leptospira (46) . it is primarily a disease of wild and domestic animals, and humans are infected only occasionally through contact with animals. most human cases occur in summer or autumn and are associated with exposure to leptospiracontaminated water or soil during recreational activities such as swimming or camping. in adolescents and adults, occupational exposure through farming or other contact with animals is the route of infection. the spirochete penetrates intact mucous membranes or abraded skin and disseminates to all parts of the body, including the cerebrospinal fluid (csf). although leptospires do not contain classic endotoxins, the pathophysiology of the disorder has many similarities to that of endotoxemia. in severe cases, jaundice occurs because of hepatocellular dysfunction and cholestasis. renal functional abnormalities may be profound and out of proportion to the histologic changes in the kidney (47) . renal involvement is predominantly a result of tubular damage, and spirochetes are commonly seen in the tubular lesions. the inflammatory changes in the kidney may result from either a direct toxic effect of the organism or immunecomplex nephritis. however, hypovolemia, hypotension, and reduced cardiac output caused by myocarditis may contribute to the development of renal failure. in severe cases, a hemorrhagic disorder caused by widespread vasculitis and capillary injury also occurs (47, 48) . the clinical manifestations of leptospirosis are variable. of affected patients, 90% have the milder anicteric form of the disorder, and only 5-10% have severe leptospirosis with jaundice. the illness may follow a biphasic course. after an incubation period of 7-12 days, a nonspecific flu-like illness lasting 4-7 days occurs, associated with septicemic spread of the spirochete. the fever then subsides, only to recur for the second, ''immune,'' phase of the illness. during this phase, the fever is low grade and there may be headache and delirium caused by meningeal involvement, as well as intense muscular aching. nausea and vomiting are common. examination usually reveals conjunctival suffusion, erythematous rash, lymphadenopathy, and meningism. the severe form of the disease (weil's disease) presents with fever, impaired renal and hepatic function, hemorrhage, vascular collapse, and altered consciousness. in one series the most common organs involved were the liver (71%) and kidney (63%). cardiovascular (31%), pulmonary (26%), neurologic (5%), and hematologic (21%) involvements were less common (49) . vasculitis, thrombocytopenia, and uremia are considered important factors in the pathogenesis of hemorrhagic disturbances and the main cause of death in severe leptospirosis (50) . urinalysis results are abnormal during the leptospiremic phase with proteinuria, hematuria, and casts. uremia usually appears in the second week, and acute renal failure may develop once cardiovascular collapse and dic are present (48) . the clinical features of leptospirosis overlap with those of several other acute infectious diseases, including rocky mountain spotted fever, toxic shock syndrome, and streptococcal sepsis. the diagnosis of leptospirosis should be considered in febrile patients with evidence of renal, hepatic, and mucous membrane changes and rash, particularly if a history of exposure to fresh water is found. diagnosis can be confirmed by isolation of the spirochetes from blood or csf in the first 10 days of the illness or from urine in the second week (48) . the organism may be seen in biopsy specimens of the kidney or skin or in the csf by dark-field microscopy or silver staining. serologic tests to detect leptospirosis are now sensitive and considerably aid the diagnosis. immunoglobulin m (igm) antibody may be detected as early as 6-10 days into the illness, and antibody titers rise progressively over the next 2-4 weeks. some patients remain seronegative, and negative serologic test results do not completely exclude the diagnosis. in one series levels of igm and igg anticardiolipin concentrations were significantly increased in leptospirosis patients with acute renal failure (50) . leptospirosis is treated with intravenous penicillin or other beta-lactam antibiotics. the severity of leptospirosis is reduced by antibiotic treatment, even if started late in the course of the illness (51) . supportive treatment with volume replacement to correct hypovolemia, administration of inotropes, and correction of coagulopathy is essential in severe cases. dialysis may be required in severe cases and may be needed for prolonged periods until recovery occurs. infection with s. pneumoniae is one of the most common infections in humans and causes a wide spectrum of disease, including pneumonia, otitis media, sinusitis, septicemia, and meningitis. despite the prevalence of the organism, significant renal involvement is relatively rare but is seen in two situations: pneumococcal septicemia in asplenic individuals or in those with other immune deficiencies presents with fulminant septic shock in which renal failure may occur as part of a multisystem derangement. the mortality from pneumococcal sepsis in asplenic patients is high, even with early antibiotic treatment and intensive support. the second nephrologi syndrome associated with s. pneumoniae is a rare form of hus. in 1955, gasser and colleagues described hus as a clinical entity in children, and they included two infants with pneumonia among the five patients they described (52) . hus associated with pneumococcal infection is induced by the enzyme neuraminidase released from s. pneumoniae (53, 54) . thomsen-friedenrich antigen (t antigen) is present on the surface of red blood cells, platelets, and glomerular capillary endothelia against which antibodies are present in normal serum. neuraminidase causes desialation of red blood cells, and possibly other blood cells and endothelium, by the removal of terminal neuraminic acid, which leads to unmasking of the t antigen. the resultant widespread agglutination of blood cells causes intravascular obstruction, hemolysis, thrombocytopenia, and renal failure. results of the direct coombs test are frequently positive, either from bound anti-t igm or from anti-t antibodies. the diagnosis of thomsen-friedenrich antibody-induced hus should be suspected in patients with acute renal failure, thrombocytopenia and hemolysis after an episode of pneumonia or bacteremia caused by s. pneumoniae. fragmented red blood cells will usually be present on blood film. association with s. pneumoniae is defined by culture of pneumococci from a normally sterile site within a week before or after onset of signs of hus. clues to a pneumococcal cause, in addition to culture results, include severe clinical disease, especially pneumonia, empyema, pleural effusion, or meningitis; hemolytic anemia without a reticulocyte response; positive results on a direct coombs test; and difficulties in abo crossmatching or a positive minor crossmatch incompatibility (55) . however, when renal disease is seen in the context of severe pneumococcal infection, it is important to maintain a broad diagnostic perspective, because the occurrence of acute tubular necrosis due to septic shock and dic is well described (56, 57) . therapy for this syndrome should be with supportive treatment and antibiotics (usually a third generation cephalosporin); dialysis may be required if renal failure occurs. because normal serum contains antibodies against the thomsen-friedenrich antigen, blood transfusion should be undertaken with washed red blood cells resuspended in albumin rather than plasma (53, 54) . exchange transfusion and plasmapheresis have been used in some patients, with the rationale that these procedures may improve outcome by eliminating circulating neuraminidase (53, 57, 58) . intravenous igg has been used in a patient and was shown to neutralize neuraminidase present in the patient's serum (59) . in comparison to patients with the more common diarrhea-associated hus, s. pneumoniae-induced hus patients have a more severe renal disease. they are more likely to require dialysis. their long-term outcome maybe affected by the severity of the invasive streptococcal disease itself, and a significant proportion of surviving patients (30-70%) develop end-stage renal failure (60, 61) . a recent review of uk cases found an eightfold increase in early mortality as compared to diarrhoea-induced hus (62) . gastrointestinal infections (escherichia coli, salmonella, campylobacter, yersinia, shigella, vibrio cholerae) the diarrheal diseases caused by escherichia coli, salmonella, shigella, campylobacter, vibrios, and yersinia remain important and common bacterial infections of humans. although improvements in hygiene and living conditions have reduced the incidence of bacterial gastroenteritis in developed countries, these infections remain common in underdeveloped areas of the world, and outbreaks and epidemics continue to occur in both developed and underdeveloped countries. renal involvement in the enteric infections may result from any of four possible mechanisms. regardless of the causative organism, diarrhea results in hypovolemia, abnormalities of plasma electrolyte composition, and renal underperfusion. if severe dehydration occurs and is persistent, oliguria from prerenal failure is followed by vasomotor nephropathy and established renal failure. e. coli, shigella, and salmonella (particularly salmonella typhi) may invade the bloodstream and induce septicemia or septic shock. acute renal failure is commonly seen in infants with e. coli sepsis but is also reported with klebsiella, salmonella, and shigella infections. its pathophysiology and treatment were discussed previously. enteric infections with e. coli, yersinia, campylobacter, and salmonella have been associated with several different forms of gn, including membranoproliferative gn (mpgn), interstitial nephritis, diffuse proliferative gn, and iga nephropathy (63) (64) (65) . in typhoid fever, gn ranging from mild asymptomatic proteinuria and hematuria to acute renal failure may occur (64, (66) (67) (68) . renal biopsy findings show focal proliferation of mesangial cells, hypertrophy of endothelial cells, and congested capillary lumina. immunofluorescent studies show igm, igg, and c3 deposition in the glomeruli, with salmonella antigens detected within the granular deposits in the mesangial areas. in the iga nephropathy after typhoid fever, salmonella vi antigens have been demonstrated within the glomeruli. yersinia infection has been reported as a precipitant of gn in several studies (65, 69) . transient proteinuria and hematuria are found in 24% of patients with acute yersinia infection, and elevated creatinine levels in 10%. renal biopsy reveals mild mesangial gn or iga nephropathy. yersinia antigens, immunoglobulin, and complement have been detected in the glomeruli. yersinia pseudotuberculosis is well recognized as one of the causes of acute tubulointerstitial nephritis causing acute renal failure, especially in children; patients have histories of drinking untreated water in endemic areas (70) (71) (72) . the illness begins with the sudden onset of high fever, skin rash, and gi symptoms. later in the course, periungual desquamation develops, mimicking kawasaki disease. elevated erythrocyte sedimentation rate, c-reactive protein level, and thrombocytosis are noticeable, and mild degrees of proteinuria, glycosuria, and sterile pyuria are common. acute renal failure, which typically develops 1-3 weeks after the onset of fever, follows a benign course with complete recovery. renal biopsy mainly reveals findings of acute tubulointerstitial nephritis. antibiotic therapy, although recommended, does not alter the clinical course, but reduces the fecal excretion of the organism (73, 74) . hus is characterized by three distinct clinical signs: acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. it was first described in 1955 and was associated with infection by shiga toxin-producing shigella dysenteriae. a major breakthrough in the search for the cause of hus occurred in the 1980s when karmali et al. reported that 11 of 15 children with diarrheaassociated hus had evidence of infection with a strain of e. coli that produced a toxin active on vero cells (75) . in diarrhea-associated hus in the united states and most of europe, e. coli 0157:h7 is the most important of these strains. e. coli 0157:h7 occurs naturally in the gi tract of cattle and other animals, and humans become infected through contaminated food products. most outbreaks have been associated with consumption of undercooked meat, but unpasteurized milk and cider, drinking water, and poorly chlorinated water for recreational use have also been implicated as vehicles for bacterial spread. hus is discussed in detail in chapter 67. the global epidemic of mycobacterium tuberculosis is growing. several factors have contributed to this increase, including the emergence of the human immunodeficiency virus (hiv) infection epidemic, large influxes of immigrants from countries in which tuberculosis (tb) is common, the emergence of multiple-drug-resistant m. tuberculosis, and breakdown of the health services for effective control of tb in various countries. it is generally estimated that, overall, one-third of the world's population is currently infected with the tb bacillus. there are more than 8 million cases of tb, which result in the death of approximately 2 million people each year. furthermore, 5-10% of people who are infected with the tb bacillus develop tb disease or become infectious at some time during their lives (76, 77) . after respiratory illness in children, mycobacteria are widely distributed to many organs of the body during the lymphohematogenous phase of childhood tb (78) . tubercle bacilli can be recovered from the urine in many cases of miliary tb. hematogenously-spread tuberculomata develop in the glomeruli, which results in caseating, sloughing lesions that discharge bacilli into the tubules. in most cases, the renal lesions are asymptomatic and manifest as mycobacteria in the urine or as sterile pyuria. tuberculomata in the cortex may calcify and cavitate or may rupture into the pelvis, discharging infective organisms into the tubules, urethra, and bladder. dysuria, loin pain, hematuria, and pyuria are the presenting features of this complication, but in many cases, the renal involvement is asymptomatic, even when radiologic and pathologic abnormalities are very extensive. continuing tuberculous bacilluria may cause cystitis with urinary frequency and, in late cases, a contracted bladder (79) . the intravenous urogram is abnormal in most cases. early findings are pyelonephritis with calyceal blunting and calyceal-interstitial reflux. later, papillary cavities may be seen, indicating papillary necrosis. ureteric strictures, focal calcification, hydronephrosis, and cavitation may also be seen. renal function is usually well preserved, and hypertension is uncommon. in some cases, either the infection itself or reactions to the chemotherapeutic agents may result in renal failure with evidence of an interstitial nephritis (79) (80) (81) . classic symptomatic renal tb is a late and uncommon complication in children, rarely occurring less than 4 or 5 years after the primary infection, and therefore is most commonly diagnosed after adolescence (78, 80) . adult studies have shown that 26-75% of renal tb coexists with active pulmonary tb and 6-10% of screened sputumpositive pulmonary tb patients have renal involvement. the diagnosis is established by isolation of mycobacteria from the urine or by the presence of the characteristic clinical and radiographic features in a child with current or previous tb. renal tb is treated with drug regimens similar to those used for other forms of tb, with isoniazid, rifampicin, pyrazinamide and ethambutol administered initially for 2 months, and isoniazid and rifampicin then continued for a further 7-10 months. late scarring and urinary obstruction may occur in cases with extensive renal involvement, and such patients should be followed by ultrasonography or intravenous urogram. mycobacteria, both m. tuberculosis and atypical mycobacteria, have also emerged as important causes of opportunistic infection in immunocompromised patients undergoing dialysis and in patients undergoing renal transplantation. the possibility of mycobacterial disease must be considered in patients with fever of unknown origin or unexplained disease in the lungs or other organs. results of the mantoux test are often negative, and diagnosis depends on maintaining a high index of suspicion and isolating the organism from the infected site. renal involvement has been well documented in both congenital and acquired syphilis, with an estimated occurrence of 0.3% in patients with secondary syphilis and up to 5% in those with congenital syphilis (82, 83) . the most common manifestation of renal disease in congenital syphilis is the nephrotic syndrome, with proteinuria, hypoalbuminemia, and edema. in some patients, hematuria, uremia, and hypertension may be seen. the renal disease is usually associated with other manifestations of congenital syphilis, including hepatosplenomegaly, rash, and mucous membrane findings. nephritis in congenital syphilis is usually associated with evidence of complement activation, with depressed levels of clq, c4, c3, and c5. histologic findings are a diffuse proliferative gn or a membranous nephropathy. the interstitium shows a cellular infiltrate of polymorphonuclear and mononuclear cells (84) . immunofluorescent microscopy reveals diffuse granular deposits of igg and c3 along the glomerular basement membrane (gbm). mesangial deposits may also contain igm. on electron microscopy, scattered subepithelial electron-dense deposits are seen, with fusion of epithelial cell foot processes (84) . good evidence exists that renal disease is due to an immunologically mediated reaction to treponemal antigens. antibodies reactive against treponemal antigens can be eluted from the glomerular deposits, and treponemal antigens are present in the immune deposits. treatment of both congenital and acquired syphilis with antibiotics results in rapid improvement in the renal manifestations (82, 84) . renal involvement is surprisingly rare in mycoplasma pneumoniae infection considering the prevalence of this organism and its propensity to trigger immunologically mediated diseases such as erythema multiforme, arthritis, and hemolysis. acute nephritis associated with mycoplasma infection may occur 10-40 days after the respiratory tract infection (85, 86) . renal histopathologic findings include type 1 mpgn, proliferative endocapillary gn, and minimal change disease (87) . antibiotic treatment of the infection does not appear to affect the renal disease, which is self-limited in most cases (85, 86) . since its recognition in 1976, legionnaires' disease, caused by legionella pneumophila, has emerged as an important infectious diseases and the kidney cause of pneumonia. the disease most commonly affects the elderly but has been reported in both normal and immunocompromised children (97, 98) . renal dysfunction occurs in a minority of patients (98) . patients who develop renal impairment present with oliguria and rising urea and creatinine levels. they are usually severely ill, with bilateral pulmonary infiltrates, fever, and leukocytosis. shock may be present, and the renal impairment has been associated with acute rhabdomyolysis with high levels of creatine phosphokinase and myoglobinuria. renal histologic examination usually shows a tubulointerstitial nephritis or acute tubular necrosis (98, 99) . the pathogenesis of the renal impairment is uncertain, but the organism has been detected within the kidney on electron microscopy and immunofluorescent studies, which suggests a direct toxic effect. myoglobinuria and decreased perfusion may also be contributing factors, however. mortality has been high in reported cases of legionnaires' disease complicated by renal failure. treatment is based on dialysis, intensive care, and antimicrobial therapy with erythromycin (98) . steroid therapy may be effective for tubulointerstitial nephritis (99) . the rickettsial diseases are caused by a family of microorganisms that have characteristics common to both bacteria and viruses and that cause acute febrile illnesses associated with widespread vasculitis. with the exception of q fever, all are associated with erythematous rashes. there are four groups of rickettsial diseases: 1. the typhus group includes louse-borne and murine typhus, spread by lice and fleas, respectively. 2. the spotted fever group includes rocky mountain spotted fever, tick typhus and related mediterranean spotted fever and rickettsial pox, which are spread by ticks and mites, with rodents as the natural reservoir. 3. scrub typhus, which is spread by mites. 4. q fever, which is spread by inhalation of infected particles from infected animals. rickettsial diseases have a worldwide distribution and vary widely in severity, from self-limited infections to fulminant and often fatal illnesses (88) . in view of the widespread vasculitis associated with these infections, subclinical renal involvement probably occurs in many of the rickettsial diseases. however, in rocky mountain spotted fever, tick typhus, and q fever, the renal involvement may be an important component of the illness. rocky mountain spotted fever is the most severe of the rickettsial diseases (89, 90) . the onset occurs 2-8 days after the bite of an infected tick. high fever develops initially, followed by the pathognomonic rash, which occurs between the second and sixth days of the illness. the rash initially consists of small erythematous macules, but later these become maculopapular and petechial, and in untreated patients, confluent hemorrhagic areas may be seen. the rash first appears at the periphery and spreads up the trunk. involvement of the palms and soles is a characteristic feature (88) . headache, restlessness, meningism, and confusion may occur together with other neurologic signs. cardiac involvement with congestive heart failure and arrhythmia are common. pulmonary involvement occurs in 10-40% of cases. infection is associated with an initial leucopenia, followed by neutrophil leukocytosis. thrombocytopenia occurs in most cases. histopathologically, the predominant lesions are in the vascular system (91) . rickettsiae multiply in the endothelial cells, which results in focal areas of endothelial cell proliferation, perivascular mononuclear cell infiltration, thrombosis, and leakage of red cells into the tissues. the renal lesions involve both blood vessels and interstitium, and acute tubular necrosis may occur. acute gn with immune-complex deposition has been reported (92) , but in most cases the pathology appears to be a direct consequence of the invading organism on the renal vasculature (90, 93) . renal dysfunction is an important complication of rocky mountain spotted fever. elevation of urea and creatinine levels occurs in a significant proportion of cases, and acidosis is common. prerenal renal failure caused by hypovolemia and impaired cardiac function may respond to volume replacement and inotropic support, but acute renal failure may subsequently occur, necessitating dialysis. rocky mountain spotted fever is diagnosed by the characteristic clinical picture, the exclusion of disorders with similar manifestations (e.g., measles, meningococcal disease, and leptospirosis), and detection of specific antibodies in convalescence. culture of rickettsia rickettsii, immunofluorescent staining, and polymerase chain reaction (pcr) testing of blood and skin biopsy specimens are available only in reference laboratories. antibiotics should be administered in suspected cases without awaiting confirmation of the diagnosis (93) . doxycycline is the drug of choice for children of any age. chloramphenicol is also effective (94) . intensive support of shock and multiorgan failure may be required in severe cases, and peritoneal dialysis or hemodialysis may be required until renal function returns. before the advent of specific therapy, mortality was 25%. today the overall mortality in the united states is still 5-7%. death predominantly occurs in cases in which the diagnosis is delayed. q fever is caused by coxiella burnetii and has a worldwide distribution, with the animal reservoir being cattle, sheep, and goats. human infection follows inhalation of infected particles from the environment. the clinical manifestations range from an acute self-limited febrile illness with atypical pneumonia to involvement of specific organs that causes endocarditis, hepatitis, osteomyelitis, and central nervous system disease (95) . proliferative gn may be associated with either q fever endocarditis, rhabdomyolysis or a chronic infection elsewhere in the body (96) . renal manifestations range from asymptomatic proteinuria and hematuria to acute renal failure, hypertension, and nephrotic syndrome. renal histologic findings are those of a diffuse proliferative gn, focal segmental gn, or mesangial gn. immunofluorescent studies reveal diffuse glomerular deposits of igm in the mesangium, together with c3 and fibrin. c. burnetii antigen has not been identified within the renal lesions. treatment of the underlying infection may result in remission of the renal disease, but prolonged treatment may be required for endocarditis. tetracycline has been used in conjunction with rifampicin, co-trimoxazole, or a fluoroquinolone. nephritis has been reported in association with the presence of a wide range of microorganisms that cause chronic or persistent infection (> table 52 -2) (63, 100). it is likely that any infectious agent that releases foreign antigens into the circulation, including those of very low virulence, can cause renal injury either by deposition of foreign antigens in the kidney or by the formation of immune complexes in the circulation, which are then deposited within the kidney. nephritis is most commonly seen in association with intravascular infections such as sbe or infected ventriculoatrial shunts, but it is also seen after focal extravascular infections; ear, nose, and throat infections; and abscesses. renal involvement is one of the diagnostic features of bacterial endocarditis. virtually all organisms that cause endocarditis also produce renal involvement ( > table 52 -2). although endocarditis caused by bacteria is the most common and is readily diagnosed by blood culture (100), unusual but important causes of culture-negative endocarditis include q fever (101) and legionella infection (102) . in the immunocompromised individual, opportunistic pathogens such as fungi and mycobacteria are important causes. the usual renal manifestations of sbe are asymptomatic proteinuria, hematuria, and pyuria. loin pain, hypertension, nephrotic syndrome, and renal failure may occur in more severe cases. the renal lesions occurring in endocarditis are variable, and focal embolic and immune-complex-mediated features may coexist (100, 103, 104) . embolic foci may be evident as areas of infarction, intracapillary thrombosis, or hemorrhage. more commonly, there is a focal necrotizing or diffuse proliferative gn. immunofluorescent studies show glomerular deposits of igg, igm, iga, and c3 along the gbm and within the mesangium. electron microscopy reveals typical electron-dense deposits along the gbm and within the mesangium (100, 103, 104) . early reports suggested that the renal lesions were caused by microemboli from infected vegetations depositing in the kidney, a hypothesis supported by the occasional presence of bacteria within the renal lesions. most subsequent evidence, however, indicates that immunologic mechanisms rather than emboli are involved in the pathogenesis in most cases: bacteria are rarely found within the kidney, and renal involvement occurs with lesions of the right side of the heart, which would not be likely to embolize to the kidney. immune complexes containing bacterial antigens are present in the circulation, and both bacterial antigens and bacteria-specific antibodies can be demonstrated within the immune deposits in the kidney. serum c3 level is usually low, and complement can be found within both the circulating and the deposited immune complexes. these features all support an immune-complex-mediated pathogenesis of the renal injury (100, 103, 104) . treatment of the endocarditis with antibiotics usually results in resolution of the gn and is associated with the disappearance of immune complexes from the circulation and return of c3 levels to normal. the prognosis of the renal lesions in sbe generally depends on the response of the underlying endocarditis to antibiotics or, in cases of antibiotic failure, to surgical removal of the infective vegetations (105) . in patients previously treated by shunting for hydrocephalus, there is a well-documented association of gn with infected ventriculoatrial shunts. this condition is another example of an immune-complex nephritis similar to that seen in endocarditis (106) . coagulase-negative staphylococci are the causative organisms in 75% of cases. the clinical and pathologic findings are similar to those in sbe. presenting features are proteinuria, hematuria, and pyuria, and they may progress to renal failure. immune complexes containing the bacterial antigens and complement are present in the serum, and c3 is depressed. histologic findings are those of a diffuse mesangiocapillary gn. immunofluorescent microscopy demonstrates deposits of immunoglobulin and c3 along the gbm, and bacterial antigen can be demonstrated in the renal lesions (107) . the prognosis for the renal lesion is good if the infection is treated early. this usually involves removal of the infected shunt and administration of appropriate antibiotics (106, 108) . the possible progression to end-stage renal disease requires frequent nephrologic monitoring of patients with ventriculoatrial shunts (106) . there are a few reports in the literature of a similar renal complication occurring in chronic infection of ventriculoperitoneal shunts. gn has been reported after chronic abscesses (63), osteomyelitis, otitis media, pneumonia, and other focal infections ( > table 52 -2). acute renal failure has been the presenting feature of focal infections in various sites, including the lung, pleura, abdominal cavity, sinuses, and pelvis. many different organisms have been responsible, including s. aureus, pseudomonas, e. coli, and proteus species. this is probably another example of immunecomplex gn. c3 level is decreased in approximately onethird of reported cases, and immunofluorescent studies reveal diffuse granular deposits of c3 in the glomeruli of all reported instances, with a variable presence of immunoglobulin. the renal lesion is that of mpgn and crescentic nephritis. the renal outcome is reported to be good with successful early treatment of the underlying infection. the role of viral infections in the causation of renal disease has been less well defined than that of bacterial infections. clearly defined associations of renal disease have been made with hepatitis b virus (hbv), hepatitis c virus (hcv), hiv, and hantaviruses, but the role of most other viruses in the pathogenesis of renal disease is not clearly defined. most viruses causing systemic infection may trigger immunologically mediated renal injury. with increasing application of molecular techniques, it may be that a significant proportion of gns currently considered to be idiopathic will ultimately be shown to be virus induced. in children with immunodeficiency states and those undergoing renal transplantation, viruses such as cytomegalovirus (cmv) and polyoma virus have been recognized to be associated with nephropathy. since the discovery of hepatitis b surface antigen (hbsag) in 1964, hepatitis virus has been shown to infect more than 5% of the world's population and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma worldwide. some 350 million people have hbsag in the circulation (who figures). the infection is most common in africa and the orient, where it is acquired in childhood by vertical transmission from infected mothers or by horizontal transmission from other children or adults. in developed countries, transmission in adults occurs more often by blood product exposure, sexual contact, or intravenous drug use. the epidemiology of hbv infection in children is changing following the widespread use of effective vaccination at birth, in both developed and developing countries. hbv is a complex dna virus with an outer surface envelope (hbsag) and an inner nucleocapsid core containing the hepatitis b core antigen (hbcag), dna polymerase, protein kinase activity, and viral dna. incomplete spherical and filamentous viral particles consisting solely of hbsag are the major viral products in the circulation and may be present in concentrations of up to 1014 particles per ml of serum. hepatitis b e antigen (hbeag) can be released from hbcag by proteolytic treatment and may be found in the circulation either free or complexed to albumin or igg antibodies. the presence of hbeag correlates with the presence of complete viral particles and the infectivity of the individual (109) . infection with hbv may result in either a self-limited infectious hepatitis followed by clearance of the virus and complete recovery, or a chronic or persistent infection in which the immune response is ineffective in eliminating the virus. chronic hbv infection with continued presence of viral antigens in the circulation caused by an ineffective host immune response provides the best-documented example of immunologically mediated renal injury caused by persistent infection (110) . development of chronic hbv infection is positively associated with infection at a younger age, particularly in infancy, where the rate of chronic infection is up to 90%. in contrast, the likelihood of an acute, symptomatic illness increases with age, to a level where approximately 40% of infections are symptomatic in children above 15 years. in the early prodromal phase of hbv hepatitis, before the onset of jaundice, some patients develop fever, maculopapular or urticarial rash, and transient arthralgias or arthritis. occasionally, proteinuria, hematuria, or sterile pyuria are observed. the syndrome usually lasts 3-10 days and often resolves before the onset of jaundice (110, 111) . there have been no histologic studies of the renal changes during this prodromal period. since 1970, numerous reports have linked hbv infection with polyarteritis nodosa (pan). most of these cases have been in adults, but the disorder has also been reported in children (112, 113) , where it is estimated that approximately one third of pan cases are caused by hbv (114) . hbv pan appears to be uncommon in africa and the orient, where infection is usually acquired in childhood, and has declined in incidence following the introduction of hbv vaccination (115) . hbv pan presents weeks to months after a clinically mild hepatitis but may occasionally predate the hepatitis. after a prodromal illness, frank vasculitis affecting virtually any organ appears. abdominal pain, fever, mononeuritis multiplex, and pulmonary and renal involvement may occur. the renal involvement may appear as hypertension, hematuria, proteinuria, or renal failure (see chapter 61) . laboratory investigations reveal a florid acute-phase response, leukocytosis, and anemia. transaminase levels are usually elevated, and hbsag is present in the circulation. the pathology consists of focal inflammation of small and medium-sized arteries, with fibrinoid necrosis, leukocyte infiltration, and fibrin deposition. renal pathology may be limited to the medium-sized arteries or may coexist with gn (110, 116) . circulating immune complexes containing hbsag and anti-hbs antibodies are usually present in the circulation (110, 116) . c3, c4, and total hemolytic complement levels are depressed. hbsag, igg, and igm antibodies to hbv and c3 have been identified by immunofluorescence in the blood vessels (116) . a positive anca excludes hbv-pan (115) . although most evidence suggests that the pathogenesis involves an immunecomplex-mediated vasculitis, autoantibody or cell-mediated vascular injury may coexist. if the condition is untreated, the mortality is high (112) . most studies suggest that steroids or immunosuppressants help to suppress the vasculitis but potentially predispose to chronic infection . (110, 112) . successful treatment of hepatitis b-associated pan with nucleoside analogues such as lamivudine or newer anti-viral drugs, either alone or in combination with interferon-alpha and conventional immunosuppressive therapy, has been reported (117) (118) (119) . hbv is now the major cause of membranous gn (mgn) in children worldwide. the proportion of patients with mgn caused by hbv is directly related to the incidence of hbsag in the population, with 80-100% of all cases of mgn in some african and oriental countries being associated with hbv (110, 120) (see chapter 26) . hbv mgn usually presents in children aged 2-12. there is a striking male predominance; in the united states, 80% of patients are males (121) . the virus is usually acquired by vertical transmission from infected mothers or horizontally from infected family members. unlike adults with hbv mgn, children do not usually have a history of hepatitis or of active liver disease, but liver function test results are generally mildly abnormal. liver biopsy specimens may show minimal abnormalities, chronic persistent hepatitis, or (occasionally) more severe changes (121) . the renal manifestations are usually of proteinuria, nephrotic syndrome, microscopic hematuria, or (rarely) macroscopic hematuria. hypertension occurs in less than 25% of cases, and renal insufficiency is rare. hbsag and hbcag are usually present in the circulation, and hbe antigenemia is seen in a high proportion of cases. occasionally, hbsag may be found in the glomeruli but is absent from the circulation. c3 and c4 levels are often low, and circulating immune complexes are found in most cases. immunohistologic study reveals deposits of igg and c3 and (less commonly) igm and iga in subepithelial, subendothelial, or mesangial tissue. hbv particles may be seen on electron microscopy, and all the major hepatitis b antigens, including hbsag, hbcag, and hbeag, have been localized in the glomerular capillary wall on immunofluorescence. immunologic deposition of hbv and antibody in the glomerular capillary wall is clearly involved in the glomerular injury, but the underlying immunologic events are incompletely understood (110, 122) . passive trapping of circulating immune complexes may be involved, but the circulating immune complexes containing hbsag are usually larger than would be expected to penetrate the basement membrane. hbsag and hbcag are anionic and are therefore unlikely to penetrate the glomerular capillary wall. in contrast, hbeag forms smaller complexes with anti-hbe antibodies and may readily penetrate the gbm. this may explain the observation that hbeag in the circulation frequently correlates with the severity of the disease (110) . an alternative mechanism for immunemediated glomerular injury is the trapping of hbv antigens by antibody previously deposited in the kidney. anti-hbe antibodies are cationic and may readily localize in the glomerulus and subsequently bind circulating antigen and complement. the third possibility is that the depositions of hbv and antibodies are consequences of glomerular injury by cellular mechanisms or autoantibodies. little evidence supports this view at present (110) . a transgenic mouse model of hbv-associated nephropathy has been developed, in which hbsag and hbcag is expressed in liver and kidney, particularly tubular epithelial cells, without viral replication. in these mice, gene expression analysis revealed upregulation of acute-phase proteins, particularly c3, although measurable serum c3 levels were reduced. this supports the notion that local persistent expression of hbv viral proteins contributes to hbv-associated nephropathy (123) . hbv infection has been associated with a variety of other forms of gn in both adults and children. in one small series in children, mpgn was found to be equal in incidence to mgn in the spectrum of hbv-associated gns (124) . both mpgn and mesangial proliferative gn may be triggered by hbv. in several countries where hbv is common, the proportion of patients with these forms of nephritides who test positive for hbv greatly exceeds the incidence of positivity in the general population (122) . as with mgn and hbv-associated pan, circulating immune complexes and localization of hbv antigens in the glomeruli have been reported in both mpgn and mesangial proliferative gn, and it is likely that similar mechanisms are occurring (110, 125) . several other forms of gn have been associated with hbv, including iga nephropathy, focal glomerulosclerosis, crescentic nephritis, and systemic lupus erythematosus, but the evidence for these associations is less consistent than for the entities discussed earlier (125) . hbv is normally cleared as a result of cell-mediated responses in which cytotoxic t cells and natural killer cells eliminate infected hepatocytes. it is not surprising, therefore, that the administration of steroids and immunosuppressive agents either may have no effect on hbv disease or may increase the risk of progressive disease (126) . children with hbv mgn have a good prognosis, and two-thirds undergo spontaneous remission within 3 years of diagnosis. steroid therapy does not appear to provide any additional benefit (110, 120, 110) . antiviral therapy with pegylated interferon-alpha and lamivudine shows promise in facilitating clearance of hbv, and in some cases, elimination of the infection with antiviral therapy in both children and adults is associated with improvement or resolution of the coexisting renal disease. there is considerable effort being put into the development of newer anti-viral agents which avoid the common problems of resistance associated with lamivudine (127-129). hcv is an enveloped, single-stranded rna virus of approximately 9.4 kb in the flaviviridae family. there are six major hcv genotypes. hepatitis c is a common disease affecting approximately 400 million people worldwide. in the united states, 4.1 million persons are estimated to be anti-hcv positive, and 3.2 million may be chronically infected (130) . an estimated 240,000 children in the united states have antibody to hcv and 68,000-100,000 are chronically infected (131) . children become infected through receipt of contaminated blood products or through vertical transmission. the risk of vertical transmission increases with higher maternal viremia and maternal co-infection with hiv. acute hcv infection is rarely recognized in children outside of special circumstances such as a known exposure from an hcv-infected mother or after blood transfusion. most chronically infected children are asymptomatic and have normal or only mildly abnormal alanine aminotransferase levels. although the natural history of hcv infection during childhood seems benign in the majority of instances, the infection can take an aggressive course in a proportion of children, leading to cirrhosis and end-stage liver disease during childhood. the factors responsible for this more aggressive course are unidentified (131) . even in adults, the natural history of hcv infection has a variable course, but a significant proportion of patients will develop some degree of liver dysfunction, and 20-30% will eventually have end-stage liver disease as a result of cirrhosis. the risk of hepatocellular carcinoma is significant for those who have established cirrhosis. hepatitis c is currently the most common condition leading to liver transplantation in adults in the ''western world.'' gn has been described as an important complication of chronic infection with hcv in adults. the clinical presentation is usually of nephrotic syndrome or proteinuria, hypertension, or hematuria, with or without azotemia (132) . mpgn, with or without cryoglobulinemia, and mgn are most commonly described. isolated case reports of other, more unusual patterns of glomerular injury, including iga nephropathy, focal segmental glomerulosclerosis, crescentic gn, fibrillary gn, and thrombotic microangiopathy, have also been associated with hcv infection (132, 133) . glomerular deposition of hepatitis antigens and antibodies has been described and is believed to play a role in pathogenesis. cryoglobulinemia is a common accompaniment of gn that is associated with the depression of serum complement levels (132) . renal failure may develop in 40-100% of patients who have mpgn (132, 134) . the presence of virus-like particles as well as viral rna within the kidney sections of patients with hcv-associated glomerulopathies has been reported (135) . the diagnosis should be suspected if glomerular disease is associated with chronic hepatitis, particularly with the presence of cryoglobulins, but renal biopsy is necessary to establish a definitive diagnosis. hcv infection is relatively common in children with end-stage renal disease and is an important cause of liver disease in this population. acquisition of hcv infection continues to occur in dialysis patients because of nosocomial spread (136) . elevation of transaminase level is not a sensitive marker of infection in children and hcv enzymelinked immunosorbent assay or pcr testing should be used to increase sensitivity (137) . hcv-infected renal transplant recipients had higher mortality and hospitalization rates than other transplant recipients (138) , and hcv infection has been reported to be associated with de novo immune-mediated gn, especially type 1 mpgn, in renal allografts, resulting in accelerated loss of graft function (139, 140) . no large randomized, controlled trials of treatment of children with chronic hepatitis c have been performed, although one study (peds-c) is currently recruiting patients into a trial of pegylated interferon +/ã� ribavirin (141) . small heterogeneous studies of interferon monotherapy have reported sustained virologic response rates of 35-40% (131) . in adults, improvement of proteinuria and renal function often follows interferon-alpha treatment (132, 134) , but relapses are common after cessation of treatment. combination of interferon with ribavirin in infectious diseases and the kidney patients with chronic liver disease has been shown to increase the rate of sustained response in these patients (142) . as yet, however, there are few data regarding the use of combination therapy with interferon and ribavirin in children. moreover, interferon-alpha therapy is associated with acute or subacute renal failure in more than one-third of the patients with renal transplants (143) . hepatitis c may be complicated by systemic mixed cryoglobulinemic (mc) vasculitis, and in some cases by a polyarteritis nodosa (pan)-type non-cryoglobulinemic vasculitis (144) . treatment with interferon-a (ifn-a) and ribavirin mostly is associated with an improvement of vasculitic symptoms. in some cases, exacerbation and rarely new onset of vasculitis of the peripheral nervous system have been described after this treatment. in fulminant cases immunosuppressive therapy with steroids, and cyclophosphamide, or rituximab may be needed to control life threatening vasculitis prior to antiviral treatment (144) . cytomegalovirus cmv is one of the eight human herpes viruses. transmission of the virus requires exposure to infected body fluids such as breast milk, saliva, urine, or blood. individuals initially infected with cmv may be asymptomatic or display nonspecific flu-like symptoms. after the initial infection cmv, like all herpes viruses, establishes latency for life but will be periodically excreted by an asymptomatic host. cmv replicates within renal cells, and on biopsy samples from immunocompromised hosts, viral inclusions can be visualized by light microscopy in cells of the convoluted tubules and collecting ducts (145) . glomerular cells and shed renal tubular cells may have characteristic inclusions, but clinically evident renal disease is rare and is seen virtually only in immunocompromised or congenitally infected children (145, 146) . the clinical manifestations of cmv-induced renal disease in congenitally infected infants are variable and range from asymptomatic proteinuria to nephrotic syndrome and renal impairment. in congenital cmv infection, histologic changes of viral inclusions commonly occur in the tubules. in addition, proliferative gn has been reported, with evidence on electron microscopy of viral immune deposits in glomerular cells (146, 147) . in cmv-infected immunocompromised patients, immunecomplex gn has been documented with mesangial deposits of igg, iga, c3, and cmv antigens within glomeruli. eluted glomerular immunoglobulins have been shown to contain cmv antigens (148) . cmv is the most common viral infection after kidney transplantation. experience with pediatric kidney transplant recipients suggests a 67% incidence of cmv infection (149) . the direct and indirect effects of cmv infection result in significant morbidity and mortality among kidney transplant recipients. cmv-negative patients who receive a cmv-positive allograft are at risk for primary infection and graft dysfunction. patients who are cmv seropositive at the time of transplantation are also at risk of reactivation and superinfection. tubulointerstitial nephritis is a well-characterized pathologic feature of renal allograft cmv disease, which can be difficult to distinguish from injury caused by rejection. histologic evidence of endothelial cell injury and mononuclear cell infiltration in the glomeruli has been reported (148) . cmv glomerular vasculopathy in the absence of tubulointerstitial disease, causing renal allograft dysfunction, has also been reported (150) . beyond the acute allograft nephropathy associated with cmv viremia, cmv is known to cause chronic vascular injury. this may adversely affect the long-term outcome of the allograft and may be the explanation for the observed association with chronic allograft nephropathy (151) . newer techniques for rapidly diagnosing cmv infection are becoming widely available and include shell vial culture, pp65 antigenemia assay, pcr, and the hybridcapture rna-dna hybridization assay for qualitative detection of cmv pcr. quantitative plasma pcr testing (pcr viral load) is increasingly used for diagnosis and monitoring of cmv viremia in renal transplant recipients. antiviral agents that have been shown to be effective against cmv include ganciclovir, valganciclovir, foscarnet, and cidofovir. ganciclovir remains the drug of choice for treating established disease. intravenous ganciclovir therapy is preferred in children because of the erratic absorption of oral ganciclovir. major limitations of ganciclovir therapy are the induction of renal tubular dysfunction and bone marrow toxicity, principally neutropenia and thrombocytopenia. dosage adjustments are necessary for recipients with renal dysfunction. oral valganciclovir is now used for cmv prophylaxis post-transplant (152) . use of other antiviral agents such as foscarnet and cidofovir is limited because of nephrotoxicity and difficulty of administration. a number of reports have demonstrated the effectiveness of high-titer cmv immune globulin therapy in reducing severe cmv-associated disease when used in combination with ganciclovir (149, 153) . the association of varicella with nephritis has been known for more than 100 years since henoch reported on four children with nephritis that occurred after the appearance of varicella vesicles. varicella, however, is rarely associated with renal complications (154) . in fatal cases with disseminated varicella and in the immunocompromised individual, renal involvement is more common. cases in which varicella infection caused gn in renal transplant recipients have been reported (155) . histologic findings in fatal cases include congested hemorrhagic glomeruli, endothelial cell hyperplasia, and tubular necrosis. in mild and nonfatal cases and in non-immunocompromised individuals, varicella is occasionally associated with a variety of renal manifestations, ranging from mild nephritis to nephrotic syndrome and acute renal failure (156) . histologic findings include endocapillary cell proliferation, epithelial and endothelial cell hyperplasia, and inflammatory cell infiltration (154) . rapidly progressive nephritis has also been reported. immunohistochemical studies reveal glomerular deposition of igg, igm, iga, and c3. on electron microscopy, granular electron-dense deposits have been found in the paramesangial region, and varicella antigens may be deposited in the glomeruli. the features suggest an immune-complex nephritis. elevated circulating levels of igg and iga immune complexes and depressed c3 and c4 levels support this possibility (154) . fulminant disseminated varicella and varicella in immunocompromised patients should be treated with intravenous acyclovir. renal involvement is common during acute infectious mononucleosis, usually manifesting as an abnormal urine sediment, with hematuria in up to 60% of cases. hematuria, either microscopic or macroscopic, usually appears within the first week of the illness and lasts for a few weeks to a few months. proteinuria is usually absent or low grade. more severe renal involvement with proteinuria, nephrotic syndrome, or acute nephritis with renal failure is much less common. acute renal failure may be seen during the course of fulminant infectious mononucleosis with associated hepatic failure, thrombocytopenia, and encephalitis. it is usually caused by interstitial nephritis that is likely the result of immunopathologic injury precipitated by epstein-barr virus (ebv) infection. however, the identification of ebv dna in the kidney raises the possibility that direct infection might play a role (157) . the renal involvement must be distinguished from myoglobinuria caused by rhabdomyolysis, which may occur in infectious mononucleosis, and from bleeding into the renal tract as a result of thrombocytopenia. renal histologic findings in ebv nephritis are an interstitial nephritis with mononuclear cell infiltration and foci of tubular necrosis. glomeruli may show varying degrees of mesangial proliferation. on immunohistochemical study, ebv antigens are seen in glomerular and tubular deposits. the prognosis for complete recovery of renal function is good. treatment with corticosteroids may have a role in the management of ebv-induced acute renal failure and may shorten the duration of renal failure (158) . ebv-associated post-transplantation lymphoproliferative disease is a recognized complication in renal transplant recipients. latent infection of ebv in renal proximal tubular epithelial cells has recently been described as causing idiopathic chronic tubulointerstitial nephritis (159) . the herpes simplex virus (hsv) causes persistent infection characterized by asymptomatic latent periods interspersed with acute relapses. as with other chronic and persistent infections, immunologically mediated disorders triggered by hsv are well recognized, and it is perhaps surprising that hsv has rarely been linked to nephritis. acute nephritis and nephrotic syndrome have been associated with herpes simplex encephalitis. renal histology shows focal segmental gn with mesangial and segmental deposits of igm, c3, and hsv antigens. as with other herpes viruses, hsv has been suggested as a trigger for iga nephritis, mpgn, and membranous nephropathy. elevated levels of hsv antibodies have been reported in patients with a variety of forms of gn, but no conclusive evidence exists of an etiologic role for hsv (160) . adenovirus and enterovirus, are unrelated ubiquitous pathogens that infect large proportions of the population annually and yet are rarely associated with renal disease. the literature contains scattered reports of acute nephritis after infection with each of these viruses. adenovirus is a major cause of hemorrhagic cystitis and was implicated as the cause of hemorrhagic cystitis in 23-51% of children with this disorder (161) . boys are affected more often than girls, and hematuria persists for 3-5 days. microscopic hematuria, dysuria, and frequency may occur for longer periods. adenovirus types 11 and 21 are the usual strains isolated. picornaviruses, including enteroviruses, echovirus and coxsackieviruses, have been linked with acute nephritis and acute renal failure associated with rhabdomyolysis. coxsackie b virus can be isolated in urine. direct infection of kidney cells is supported by in vitro work demonstrating lytic infection of human podocyte and proximal tubular epithelial cell cultures, although different strains exhibit variable degrees of nephrotropism. renal damage in vivo may have both a direct lytic mechanism and an immune-complex basis (162) . in the newborn, enteroviruses cause fulminant disease with dic, shock, and liver failure, and acute renal failure may occur. renal involvement from measles virus is uncommon, although measles virus can be cultured from the kidney in fatal cases. an acute gn has been reported to follow measles with evidence of immune deposits containing measles virus antigen within the glomeruli. the nephritis is generally self-limiting (163) . mild renal involvement is common during the acute phase of mumps infection. one-third of children with mumps have abnormal urinalysis results, with microscopic hematuria or proteinuria. mumps virus may be isolated from urine during the first 5 days of the illness, at a time when urinalysis findings are abnormal. plasma creatinine concentrations usually remain normal, despite the abnormal urine sediment, but more severe cases in adults have been associated with evidence of acute nephritis with impaired renal function. renal biopsy specimens demonstrate an mpgn with deposition of iga, igm, c3, and mumps virus antigen in the glomeruli, which suggests an immune-complex-mediated process (164) . despite the increasing availability of interventions to limit vertical hiv transmission, an estimated 1,500 children renal involvement in hiv infection was first described in 1984 in adults (165) (166) (167) and in children (168) , and renal involvement occurs in 2-15% of hiv-infected children in the united states (169) (170) (171) . since the development of highly active antiretroviral therapy (haart), however, the incidence of end-stage renal disease in hiv infection in both adults and children in industrialized countries has declined, but it is predicted that the dramatic decline in aids-related deaths will lead to an ageing population of hiv-infected individuals who will be at risk of non-hiv related renal problems, such that the numbers of hiv-positive esrd patients will increase in the united states (172) . hiv infection is associated with a number of renal pathologies. hiv-associated nephropathy (hivan) is a syndrome of glomerular and tubular dysfunction, which can progress to end-stage renal failure. it is discussed more fully below. glomerular syndromes other than hivan include mgn that resembles lupus nephritis and immune-complex gn, with iga nephropathy and hcv-associated mpgn being the most common forms. there have also been several case reports of amyloid kidney (171, 173, 174) . the kidneys may be affected by various other mechanisms. opportunistic infections with organisms such as bk virus (bkv) that give rise to nephropathy and hemorrhagic cystitis have been reported in association with hiv infection (175) . systemic infections accompanied by hypotension can cause prerenal failure leading to acute tubular necrosis. acute tubular necrosis has also been reported in hiv patients after the use of nephrotoxic drugs such as pentamidine, foscarnet, cidofovir, amphotericin b, and aminoglycosides. intratubular obstruction with crystal precipitation can occur with the use of sulfonamides and intravenous acyclovir. indinavir is well recognized to cause nephropathy and renal calculi (176) . mpgn associated with mixed cryoglobulinemia and thrombotic microangiopathy/atypical-cal hus in association with hiv infections have been reported (177, 178) . hivan is characterized by both glomerular and tubular dysfunction, the pathogenesis of which is not entirely known. hivan is a clinico-pathologic entity that includes proteinuria, azotemia, focal segmental glomerulosclerosis or mesangial hyperplasia, and tubulointerstitial disease (171) . in adults in the united states, there is a markedly increased risk of nephropathy among african american persons with hiv infection. this appears to be true in children as well, but the data are sparse. the spectrum of hivan seems to be coincident with the degree of aids symptomatology. it is thought that hivan can present at any point in hiv infection, but most patients with hivan have cd4 counts of less than 200 ã� 10 6 /200 cells/ml, which suggests that it may be primarily a manifestation of late-stage disease (179) . although a spectrum of clinicopathologic entities including mesangial hyperplasia, focal segmental glomerulosclerosis, minimal change disease, and systemic lupus erythematosus nephritis has been described, the classic pathologic feature of hivan is the collapsing form of focal and segmental glomerulosclerosis (180) . in the affected glomeruli, visceral epithelial cells are hypertrophied and hyperplastic, and contain large cytoplasmic vacuoles and numerous protein resorption droplets. there is microcystic distortion of tubule segments, which contributes to increasing kidney size. podocyte hyperplasia can become so marked that it causes obliteration of much of the urinary space, forming ''pseudocrescents'' (173) . capillary walls are wrinkled and collapsed with obliteration of the capillary lumina. the interstitium is edematous with a variable degree of t-cell infiltration (181) . the bowman capsule can also be dilated and filled with a precipitate of plasma protein that represents the glomerular ultrafiltrate. one of the most distinctive features of hivan, however, is the presence of numerous tubuloreticular inclusions within the cytoplasm of glomerular and peritubular capillary endothelial cells (173) . immunofluorescence testing is positive for igm and c3 in capillary walls in a coarsely granular to amorphous pattern in a segmental distribution (180, 181) . the presence of the hiv genome in glomerular and tubular epithelium has been demonstrated using complementary dna probes and in situ hybridization. proviral dna has been detected by pcr in the glomeruli, tubules, and interstitium of micro dissected kidneys from patients who had pathologic evidence of hivan, but it has also been detected in the kidneys of hiv-positive patients with other glomerulopathies (182) . a combination of both proliferation and apoptosis of renal cells may cause the loss of nephron architecture. apoptosis has been demonstrated in cells in the glomerulus, tubules, and interstitium of biopsy specimens from hiv-positive patients with focal segmental glomerulosclerosis. in addition, the role of various cytokines and growth factors, specifically transforming growth factor beta (tgf-beta), in the development of sclerosis has been studied (183, 184) . transgenic murine models provide some of the strongest evidence for a direct role of hiv-1 in the induction of hivan. these mice do not produce infectious virus but express the hiv envelope and regulatory genes at levels sufficient to re-create the hivan that is seen in humans (183) . serial deletion experiments have concluded that the nef and vpr genes are necessary though not sufficient for hivan pathogenesis. additional factors such as genetic predisposition are thought to explain the fact that african americans have a far greater likelihood of developing hivan than other racial groups, and that hivan is more likely in patients with a family history of esrd. hivan can manifest as mild proteinuria, nephrotic syndrome, renal tubular acidosis, hematuria, and/or acute renal failure (168) (169) (170) (171) . nephrotic syndrome and chronic renal insufficiency are late manifestations of hivan. children with hivan are likely to develop transient electrolytic disorders, heavy proteinuria, and acute renal failure due to systemic infectious episodes or nephrotoxic drugs. early stages of hivan can be identified by the presence of proteinuria and ''urine microcysts'' along with renal sonograms showing enlarged echogenic kidneys. urinary renal tubular epithelial cells are frequently grouped together to form these microcysts, which were found in the urine of children with hivan who had renal tubular injury (171) . advanced stages of hivan typically present with nephrotic syndrome with edema, heavy proteinuria, hypoalbuminemia, and few red or white blood cells in urinary sediments. hypertension may be present, but usually blood pressure is within or below the normal range. hivan in adults follows a rapidly progressive course, with end-stage renal disease developing within 1-4 months, but in children this rapid progression does not necessarily occur. definitive diagnosis of hivan should be based on biopsy results, and biopsy should be performed if significant proteinuria is present, because in approximately 50% of hiv-infected patients with azotemia and/or proteinuria (>1 g/24 h) who undergo renal biopsy, the specimen will have histologic features consistent with other renal diseases (179) . when available, haart should be given to children with symptomatic hiv disease. specific treatment of hivan remains controversial. several studies have looked at the role of haart, angiotensin i-converting enzyme (ace) inhibitors, steroids, and even cyclosporin with somewhat encouraging results. however, as yet no randomized case-controlled trials have been undertaken. most of the studies have been small and retrospective, and many have included patients both with and without renal biopsy-proven hivan. cyclosporin has been used to treat hivan in children with remission of nephrotic infectious diseases and the kidney syndrome (169) . similar responses have been reported to treatment with corticosteroids in various studies (185) (186) (187) (188) . ace inhibitors have been used with encouraging results (189) . the general regimen used to treat patients with hiv, including haart, should be applied to children with hivan. the dosages of some medications must be adjusted to the patients glomerular filtration. there are reports of spontaneous regression of hivan with supportive management and treatment with haart, particularly with regimes containing protease inhibitors (190) (191) (192) (193) . it should be emphasized that the improvement reported with other modalities of treatment such as corticosteroids, cyclosporin, and ace inhibitors always occurs when these agents are given in conjunction with antiretroviral therapy. the kidneys of transgenic mice have been found to have elevated levels of tgf-beta messenger rna and protein (184) . furthermore, gene expression analysis on tubular epithelial cells from a patient with hivan found upregulation of several inflammatory mediator genes downstream of interleukin 6 and of the transcription factor nfkb (194) . several other therapeutic options have been suggested, aimed specifically at the presumed role of tgfbeta in the pathogenesis of hivan. treatment directed at its synthesis using gene therapy to block tgf-beta gene expression is being explored. therapy directed at decreasing the activity of tgf-beta using anti-tgf-beta antibodies or other inhibitory substances is also an area of investigation. in addition, blocking renal receptors for chemokines such as rantes (regulated upon derivation, normal t cell expressed and secreted), interleukin-8, and monocyte-chemoattractant protein-1 has been proposed as another possible treatment alternative (195) . in the haart era, the outlook for hiv patients with esrd has improved, but these patients fare worse than esrd patients without hiv (196) . most reports of hivinfected patients on hemodialysis have shown poor prognosis, with mean patient survival times ranging from 14-47 months. mortality is therefore still close to 50% within the first year of dialysis. in general, improved survival is associated with younger age at initiation of hemodialysis and with higher cd4 counts. access complications such as infection and thrombosis tend to occur at a higher rate in hiv-infected hemodialysis patients. cross infection with hiv in dialysis patients is very rare. no patient-topatient hiv transmission has yet been reported in a hemodialysis unit in the united states, although several such cases have occurred in south america (195, 197) . peritoneal dialysis is an alternative for hiv-infected patients. the incidence of peritonitis varies across studies, but some studies did report a higher incidence of pseudomonas and fungal peritonitis in the hiv-positive population (195) . infections with unusual organisms such as pasteurella multocida, trichosporon beigelii, and mycobacterium avium intracellulare complex have also been reported. several studies, however, have suggested that there is no significant difference between the hiv-infected and non-hiv-infected populations. of note is that virus capable of replication in vitro has been recovered from the peritoneal dialysis effluent, and it can be recoverable for up to 7 days in dialysis bags at room temperature and for up to 48 h in dry exchange tubing (195) . previously, long-term dialysis had been thought to be preferable to renal transplantation, primarily because of the concern that the immunosuppressive therapy required after transplantation could promote progression of hiv/ aids. a multicenter prospective study has been addressing these questions (198) . data so far indicate that the outcome for liver and kidney transplantation is not considerably different from patients without hiv, with good graft persistence, and a low rate of development of opportunistic infections in those with well-controlled hiv and relatively high cd4 counts (199) . the human polyoma viruses are members of the papovavirus family and have received increasing attention as pathogens in immunocompromised patients. they are nonenveloped viruses ranging in size from 45-55 nm, with a circular, double-stranded dna genome that replicates in the host nucleus. the best-known species in this genus are the bkv, the jc virus (jcv), and the simian virus sv40. bkv was first isolated from the urine of a 39-year-old man who developed ureteral stenosis 4 months after renal transplantation (200) . the name of the virus refers to the first patients initials, which is also true of jcv. bkv establishes infection in the kidney and the urinary tract, and its activation causes a number of disorders, including nephropathy and hemorrhagic cystitis. bkv-associated nephropathy has become an increasingly recognized cause of renal dysfunction in renal transplantation patients (201) (202) (203) (204) (205) . jcv establishes latency mainly in the kidney, and its reactivation can result in the development of progressive multifocal leukoencephalopathy. there are a few reports of nephropathy in association with jcv infection (see references in (206) ), but bkv poses a much bigger problem in this regard. recent studies have reported sv40 in the allografts of children who received renal transplants and in the urine, blood, and kidneys of adults with focal segmental glomerulosclerosis, which is a cause of end-stage renal disease and an indication for kidney transplantation (207) . seroprevalence rates as high as 60-80% have been reported among adults in the united states and europe. the peak incidence of primary infection (as measured by acquisition of antibody) occurs in children 2-5 years of age. bkv antibody may be detected in as many as 50% of children by 3 years of age, and in 60-100% of children by 9 or 10 years of age; antibodies wane thereafter. bkv infection may be particularly important in the pediatric transplantation population, in whom primary infection has a high probability of occurring while the children are immunosuppressed (208) . primary infection with bkv in healthy children is rarely associated with clinical manifestations. mild pyrexia, malaise, vomiting, respiratory illness, pericarditis, and transient hepatic dysfunction have been reported with primary infection. investigators hypothesize that after an initial round of viral replication at the site of entry, viremia follows with dissemination of the virus to distant sites at which latent infection is established. the most frequently recognized secondary sites of latent infection are renal and uroepithelial cells. secondary infection has been reported to cause tubulointerstitial nephritis and ureteral stenosis in renal transplantation patients. it may be that renal impairment in immunocompromised patients and in non renal solid organ transplant recipients is found to be frequently associated with bkv infection. the reported prevalence of bkv nephropathy in renal allografts is between 1 and 8% (201, 202, 205, 209, 210) . asymptomatic infection is characterized by viral shedding without any apparent clinical features. viruria, resulting from either primary or secondary infection, can persist from several weeks to years. tubulointerstitial nephritis associated with bkv in renal transplant recipients is accompanied by histopathologic changes, with or without functional impairment. ''infection'' and ''disease'' must be differentiated carefully. bkv infection (either primary or reactivated) can progress to bkv disease, but will not always do so (208) . furthermore, not all cases of bkv disease lead to renal impairment. however, infection can progress to transplant dysfunction and graft loss, although the diagnosis may be complicated by the coexistence of active allograft rejection. bkv nephritis is reported to have a bimodal distribution, with 50% of bkv-related interstitial nephritis cases occurring 4-8 weeks after transplantation and the remainder of patients developing disease months to years after transplantation (211) . allograft failure is due mainly to extensive viral replication in tubular epithelial cells leading to frank tubular necrosis (203) . although damage is potentially fully reversible early in the disease, persisting viral damage leads to irreversible interstitial fibrosis. tubular atrophy and allograft loss has been observed in 45% of affected patients (203, 212) . in most cases, bkv nephropathy in adult renal transplant recipients represents a secondary infection associated with rejection and its treatment. in children, however, primary bkv infection giving rise to allograft dysfunction may occur (208) . the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathologic features include severe tubular injury with cellular enlargement, marked nuclear atypia, epithelial necrosis, denudation of tubular basement membranes, focal intratubular neutrophilic infiltration, and mononuclear interstitial infiltration, with or without concurrent tubulins. this constellation of histologic features, particularly severe tubulitis, is often misinterpreted as rejection, even by the experienced pathologist. the presence of well-demarcated basophilic or amphophilic intranuclear viral inclusions, primarily within the tubular and parietal epithelium of the bowman capsule, can help distinguish bkv disease from rejection (202, 203, 205) . additional tests such as immunohistochemistry, pcr analysis, or electron microscopy of biopsied tissue aimed at the identification of bkv may be required. a practical diagnostic approach for identifying bkv in renal transplant patients is summarized in > table 52-3. bkv infection may cause ureteral obstruction due to ureteral ulceration and stenosis at the ureteric anastomosis. bkv-associated ureteral stenosis has been reported in 3% of renal transplant patients and usually occurs between 50 and 300 days after transplantation. ulceration due to inflammation, proliferation of the transitional epithelial cells, and smooth muscle proliferation may lead to partial or total obstruction. high-level bkv replication is implicated in acute, late-onset, long-duration hemorrhagic cystitis after bone marrow transplantation (213) . there are two case reports in children of renal carcinomas arising in the transplanted kidney in association with bk virus nephropathy. it remains unclear whether infectious diseases and the kidney bk virus itself has oncogenic potential in the transplant setting, but this is possible given that the big t antigen (t-ag) expressed by polyomavirus family viruses has been shown to have the ability to disrupt chromosomal integrity (214, 215) . whether patients with asymptomatic viremia or viruria need specific therapeutic intervention is not certain. review of the literature suggests that careful reduction of immune suppression, combined with active surveillance for rejection, will result in clinical improvement. reduction in immunosuppression may precipitate episodes of acute cellular rejection, which need to be judiciously treated with corticosteroids. the outcome of bkv nephropathy is unpredictable, and stabilization of renal function may occur regardless of whether maintenance immunotherapy is altered or not (216) . some reports favor the use of cidofovir. cidofovir has important nephrotoxic side effects in the usual therapeutic dosage recommended for the treatment of cmv infection, and for bkv nephropathy a reduced dosage regime is generally used. the efficacy of cidofovir in reducing viremia has been demonstrated (see review in (210)). however, spontaneous clearance of viral infection after reduction of immunosuppression (without cidofovir) has also been reported. there are also case studies of the use of leflunamide. presence of bkv by pcr or decoy cells in urine signifies bkv replication. decoy cells are caused by infection of the urinary epithelial cells with human polyoma viruses. the nuclei are enlarged and nuclear chromatin is completely homogenized by viral cytopathic effect. positive pcr results for bkv viruria and presence of decoy cells have poor predictive value. specificity is increased if >10 cells/ cytospin along with presence of inflammatory cells. presence of antibody is usually indicative of previous infection; however, positive results for bkv dna pcr on serum signifies bk viremia. bkv pcr testing of plasma has proven to be a sensitive (100%) and specific (88%) means to identify bkv-associated nephropathy in adults. viral load has also been used to monitor infection and clearance. however, because primary infection occurs in childhood, it might not be applicable to the pediatric population. the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathology might mimic rejection or drug toxicity. however, characteristic findings have been described. electron microscopy and immune staining are helpful in confirming the diagnosis. pcr assays of viral load in tubular cells have been reported to be a sensitive marker for diagnosis and monitoring. viral hemorrhagic fever involves at least 12 distinct rna viruses that share the propensity to cause severe disease with prominent hemorrhagic manifestations ( > table 52 -4) . the viral hemorrhagic fevers, widely distributed throughout both temperate and tropical regions of the world, are important causes of mortality and morbidity in many countries. most viral hemorrhagic fevers are zoonoses (with the possible exception of dengue virus), in which the virus is endemic in animals and human infection is acquired through the bite of an insect vector. aerosol and nosocomial transmissions from infected patients are important for lassa, junin, machupo, and congo-crimean hemorrhagic fevers, and marburg and ebola viruses (217) . viral hemorrhagic fevers have many clinical similarities but also important differences in their severity, major organs affected, prognosis, and response to treatment. in all viral hemorrhagic fevers, severe cases occur in only a minority of those affected; subclinical infection or nonspecific febrile illness occurs in the majority. fever, myalgia, headache, conjunctival suffusion, and erythematous rash occur in all the viral hemorrhagic fevers (218) . hemorrhagic manifestations range from petechiae and bleeding from venepuncture sites to severe hemorrhage into the gi tract, kidney, and other organs. a capillary leak syndrome, with evidence of hemoconcentration, pulmonary edema, oliguria, and ultimately shock, occurs in the most severely affected patients (218) . renal involvement occurs in all the viral hemorrhagic fevers, proteinuria is common, and prerenal failure is seen in all severe cases complicated by shock. however, in congo-crimean hemorrhagic fever and hemorrhagic fever with renal syndrome (hfrs), an interstitial nephritis, which may be hemorrhagic, is characteristic, and renal impairment is a major component of the illness. dengue is caused by a flavivirus that is endemic and epidemic in tropical america, africa, and asia, where the mosquito vector aedes aegypti is present (219). classic dengue is a self-limited nonfatal disease; dengue hemorrhagic fever and dengue shock syndrome, which occur in a minority of patients, have a high mortality if not aggressively treated with fluids. after an incubation period of 5-8 days, the illness begins with fever, headache, arthralgia, weakness, vomiting, and hyperesthesia. in uncomplicated dengue the fever usually lasts 5-7 days. shortly after onset a maculopapular rash appears, sparing the palms and the soles, and is occasionally followed by desquamation. fever may reappear at the onset of the rash. in dengue hemorrhagic fever and dengue shock syndrome, the typical febrile illness is complicated by hemorrhagic manifestations, ranging from a positive tourniquet test result or petechiae to purpura, epistaxis, and gi bleeding with thrombocytopenia and evidence of a consumptive coagulopathy. increased capillary permeability is suggested by hemoconcentration, edema, and pleural effusions (219) . in severe cases, hypotension and shock supervene, largely as a result of hypovolemia. renal manifestations include oliguria, proteinuria, hematuria, and rising urea and creatinine. acute renal failure occurs in patients with severe shock, primarily as a result of renal underperfusion. however, glomerular inflammatory changes may also occur. children with dengue hemorrhagic fever show hypertrophy of endothelial and mesangial cells, mononuclear cell infiltrate, thinning of basement membranes, and deposition of igg, igm, and c3. electron microscopy shows viral particles within glomerular mononuclear cells (220) . the diagnosis of dengue is made by isolation of the virus from blood or by serologic testing. there is no specific antiviral treatment, and management of patients with dengue shock syndrome or dengue hemorrhagic fever depends on aggressive circulatory support and volume replacement with colloid and crystalloid (221, 222) . with correction of hypovolemia, renal impairment is usually reversible, but dialysis may be required in patients with established acute renal failure. yellow fever is caused by a flavivirus, and is transmitted by mosquito bites, typically aedes species. it remains an important public health problem in africa and south america. renal manifestations are common and include albuminuria and oliguria. over the next few days after first manifestation of infection, shock, delirium, coma, and renal failure develop, and death occurs 7-10 days after onset of symptoms. laboratory findings include thrombocytopenia and evidence of hemoconcentration, rising urea and creatinine levels, hyponatremia, and deranged liver function test results. pathologic findings include necrosis of liver lobules, cloudy swelling and fatty degeneration of the proximal renal tubules, and, often, petechiae in other organs. the oliguria appears to be prerenal and is due to hypovolemia; later, acute tubular necrosis supervenes. at present, there is no effective antiviral agent for yellow fever. . congo-crimean hemorrhagic fever, first recognized in the soviet union, is now an important human disease in eastern europe, asia, and africa (223) . severely affected patients become stuporous or comatose 5-7 days into the illness, with evidence of hepatic and renal failure and shock. proteinuria and hematuria are often present. the disease is fatal in 15-50% of cases. the virus is sensitive to ribavirin, but in one small trial of i.v. ribavirin versus supportive treatment only, there was no significant improvement in outcome in the treatment group (224) . rift valley fever is found in many areas of sub-saharan africa. in humans, most infections follow mosquito bites or animal exposure. the infection may present as an uncomplicated febrile illness, with muscle aches and . (226) . clinical entities include korean hemorrhagic fever, nephropathia epidemica in scandinavia, and epidemic hemorrhagic fever in japan and china. in general, hfrs due to hantaan, porogia, and belgrade viruses is more severe and has higher mortality than that due to puumala virus (nephropathia epidemica) or seoul virus. hantaan is predominant in the far east, porogia and belgrade in the balkans, and puumala in western europe; seoul has a worldwide distribution (225) . the clinical features of the disease vary. the incubation period is 4-42 days. although hfrs occurs with the same clinical picture in children as in adults, both incidence rates and antibody prevalence rates are very low in children under 10 years of age. men of working age make up the bulk of clinical cases (226) . mild cases are indistinguishable from other febrile illnesses. in more severe cases, fever, headache, myalgia, abdominal pain, and dizziness are associated with the development of periorbital edema, proteinuria, and hematuria. there is often conjunctival injection, pharyngeal injection, petechiae, and epistaxis or gi bleeding. the most severely affected patients develop shock and renal failure. the disease usually passes through five phases: febrile, hypotensive, oliguric, diuretic, and convalescent. laboratory findings include anemia, lymphocytosis, thrombocytopenia, prolonged prothrombin and bleeding times, and elevated levels of fibrin degradation products. liver enzyme levels are elevated, and urea and creatinine levels are elevated during the oliguric phase. proteinuria and hematuria are consistent findings. the renal histopathologic findings are those of an interstitial nephritis with prominent hemorrhages in the renal medullary interstitium and renal cortex. acute tubular necrosis may also be seen. immunohistochemical analysis reveals deposition of igg and c3, and the gbm, mesangial, and subendothelial deposits may be seen on electron microscopy (227) . recovery from hantavirus-associated disease is generally complete, although chronic renal insufficiency is a rare sequela of hfrs. in mildly affected patients, the disease is self-limiting and spontaneous recovery occurs. however, in severe cases, with shock, bleeding, and renal failure, dialysis and intensive circulatory support may be required (228) . mortality rates vary depending on the strain of virus; rates are 5-15% for hemorrhagic fever and renal syndrome in china and significantly lower for the milder finnish form associated with the puumala virus strain. ribavirin is active against hantaan viruses in vitro, and clinical trials indicate that both mortality and morbidity can be reduced by treatment with this antiviral agent if it is administered early in the course of illness. dosages of 33 mg/kg followed by 16 mg/kg every 6 h for 4 days and then 8 mg/kg every 8 h for 3 days have been used (229) . lassa fever is a common infection in west africa, caused by an arenavirus, and usually manifests as a nonspecific febrile illness. in 10% of cases, a fulminant hemorrhagic disease occurs. in severe cases, proteinuria and hematuria are usually present, and renal failure may occur. ribavirin is effective in decreasing mortality. as in other hemorrhagic fevers, intensive hemodynamic support and correction of the hemostatic derangements are important components of therapy (230) . junin and machupo viruses, the agents of argentine and bolivian hemorrhagic fever, respectively, cause hemorrhagic fevers with prominent neurologic features and systemic and hemorrhagic features similar to those of lassa fever. oliguria, shock, and renal failure occur in the most severe cases. marburg and ebola viruses have been associated with outbreaks of nosocomially transmitted hemorrhagic fever. both viruses cause fulminant hemorrhagic fever. onset is with high fever, headache, sore throat, myalgia, and profound prostration. an erythematous rash on the trunk is followed by hemorrhagic conjunctivitis, bleeding, impaired renal function, shock, and respiratory failure. the mortality rate is high. renal histopathologic findings in fatal cases are of tubular necrosis, with fibrin deposition in the glomeruli. there is no specific treatment for these disorders. the important role played by a number of other recently characterized viruses is only now being recognized, as improved molecular diagnostic techniques allow identification of hitherto unrecognized viruses. two examples of recently described viruses are metapneumovirus (237) and bocavirus (238) . while both have significant prevalence, and may make an important contribution to the burden of childhood viral infection, as yet there are no reports indicative of significant renal pathology in association with these infections. influenza virus has been linked with nephritis and acute renal failure. an emerging infectious disease is avian flu, caused by highly pathogenic h5n1 strains which have hitherto been confined to an avian reservoir, and there have been several outbreaks of infection in humans, particularly in the first part of this decade. commonly, these patients develop a flu-like illness with prominent respiratory and gastrointestinal symptoms. renal failure may develop alongside multi-organ failure in the context of acute respiratory distress syndrome (231) . as yet, there is no clear correlation of degree of initial renal insufficiency, and outcome (232) . there is little data available on treatment, but based on the known resistance patterns of h5n1 strains, oseltamivir and zanamivir are the preferred agents to be used for treatment of infection with h5n1. severe acute respiratory syndrome (sars) is a newlyemerged infectious disease which was first seen in south china in 2002. it is caused by a sars coronavirus (sars cov). predominantly, it causes a viral pneumonia, with diffuse alveolar damage; it has considerable mortality (233) . renal effects are not generally significant in the pathophysiology of sars. however, sars cov has been found in kidney tissue at post-mortem (234) (235) . sars cov enters cells via angiotensin converting enzyme 2 (ace2) (236) , and it is thought that the invasion of kidney tissue reflects the virus' tropism for ace2, which is expressed on kidney cells. chronic exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. malaria is the best-documented parasitic infection associated with glomerular disease, but other parasitic infections including schistosomiasis, filariasis, leishmaniasis, and possibly helminth infections may also induce nephritis or nephrosis. malaria is estimated to cause up to 500 million clinical cases of illness and more than 1 million deaths each year (239) . the association of quartan malaria and nephritis has been well known in both temperate and tropical zones since the end of the nineteenth century. epidemiologic studies provide the most conclusive evidence for a role of plasmodium malariae in glomerular disease (240, 241) . chronic renal disease was a major cause of morbidity and mortality in british guiana in the 1920s. the frequent occurrence of p. malariae in the blood of these patients led to detailed epidemiologic studies that implicated malaria as a cause of the nephrosis. after the eradication of malaria from british guiana, chronic renal disease ceased to be a major cause of death in that country (240) . the link between malaria and nephrotic syndrome was strengthened by studies in west africa in the 1950s and 1960s that demonstrated a high prevalence of nephrotic syndrome in the nigerian population (242) . the pattern of nephrotic syndrome differed from that in temperate climates, with an older peak age, extremely poor prognosis, and unusual histologic features. the incidence of p. malariae parasitemia in patients with the nephrotic syndrome in nigeria was vastly in excess of that occurring in the general population, whereas the incidence of plasmodium falciparum parasitemia was similar to that in the general population. the age distribution of nephrotic syndrome also closely paralleled that of p. malariae infection (242) . in some affected patients, circulating immune complexes and immunoglobulin, complement, and antigens were present in the glomeruli that were recognized by p. malariae-species antisera. there is now a view that the patterns of childhood renal disease described in the last century may no longer be representative of the current situation. the variable patterns of renal disease throughout africa may no longer reflect a dominant role for ''malarial glomerulopathy,'' and the relative causative role of tropical infections in nephropathy remains an unanswered question (243) . most patients have poorly selective proteinuria and are unresponsive to treatment with steroids or immunosuppressive agents. the characteristic lesions of p. malariae nephropathy are capillary wall thickening and segmental glomerular sclerosis, which lead to progressive glomerular changes and secondary tubular atrophy (242) . cellular proliferation is conspicuously absent. electron microscopy shows foot-process fusion, thickening of the basement membrane, and increase in subendothelial basement membrane-like material. immunofluorescent studies show granular deposits of immunoglobulin, complement, and p. malariae antigen in approximately one-third of patients. in addition to the histologic pattern, termed quartan malaria nephropathy, p. malariae infection is associated with a variety of other forms of histologic appearance, including proliferative gn and mgn (244) . although quartan malaria nephropathy has been clearly linked to p. malariae infection in nigeria, a number of studies from other regions in africa have not revealed the typical histopathologic findings described in the nigerian studies (245) . furthermore, quartan malaria nephropathy may be seen in children with no evidence of p. malariae infection or deposition of malaria antigens in the kidney. this, together with the fact that antimalarial treatment does not affect the progression of the disorder, raises the possibility that factors other than malaria might be involved in the initiation and perpetuation of the disorder. although there is undoubtedly a strong association between p. malariae infection and nephrotic syndrome on epidemiologic grounds, the direct causal link is not proven. most likely, a number of different infectious processes, including malaria, hepatitis b, schistosomiasis, and perhaps other parasitic infections that cause chronic or persistent infections and often occur concurrently in malaria areas, may all result in glomerular injury and a range of overlapping histopathologic features. the prognosis for the nephrotic syndrome in most african studies has been poor, regardless of whether the histologic findings were typical of quartan malaria nephropathy or whether p. malariae parasitemia was implicated. treatment with steroids and azathioprine is generally ineffective, and a significant proportion of patients progress to renal failure. p. falciparum appears to be much less likely to cause significant glomerular pathology. epidemiologic studies have failed to show a clear association between p. falciparum parasitemia and the nephrotic syndrome. whereas renal failure appears to be a common complication of severe malaria in adults, it seldom occurs in children. renal biopsy specimens from adult patients with acute p. falciparum infections who have proteinuria or hematuria show evidence of glomerular changes, including hypercellularity, thickening of basement membranes, and hyperplasia and hypertrophy of endothelial cells (246) . electron microscopy reveals electron-dense deposits in the subendothelial and paramesangial areas. deposits of igm, with or without igg, are localized mainly in the mesangial areas. p. falciparum antigens can be demonstrated in the mesangial areas and along the capillary wall, which suggests an immune-complex gn. the changes, generally mild and transient, are probably unrelated to the acute renal failure that may complicate severe p. falciparum infection (246) . heavily parasitized erythrocytes play a central role in the various pathologic factors (247) . renal failure occurring in severe p. falciparum malaria is usually associated with acidosis, volume depletion, acute intravascular hemolysis or heavy parasitic infection that leads to acute tubular necrosis. recent studies have confirmed an important role for volume depletion in children with severe falciparum malaria, who characteristically have evidence of tachycardia, tachypnoea, poor perfusion and in severe cases hypotension (248) . volume expansion with either colloid or crystalloid results in improvement in hemodynamic indices and reduction in acidosis (249) . there is growing evidence that volume expansion with albumin is associated with a better outcome than saline or synthetic colloids (250, 251) . treatment with antimalarials, correction of hypoglycemia and infectious diseases and the kidney electrolyte imbalance, and volume expansion reduces mortality to less than 5%. although renal failure is usually associated with infection by p. falciparum, acute renal failure has been described with plasmodium vivax infection and mixed infections (252) . the term blackwater fever refers to the combination of severe hemolysis, hemoglobinuria, and renal failure. it was more common at the start of the twentieth century in nonimmune individuals receiving intermittent quinine therapy for p. falciparum malaria. blackwater fever has become rare since 1950, when quinine was replaced by chloroquine. however, the disease reappeared in the 1990s, after the reuse of quinine because of the development of chloroquine-resistant organisms. since then, several cases have been described after therapy with halofantrine and mefloquine, two new molecules similar to quinine (amino-alcohol family) (253) . renal failure generally occurred in the context of severe hemolytic anemia, hemoglobinuria, and jaundice. the pathophysiology of the disorder is unclear; however, it appears that a double sensitization of the red blood cells to the p. falciparum and to the amino-alcohols is necessary to provoke the hemolysis. histopathologic findings include swelling and vacuolization of proximal tubules, necrosis and degeneration of more distal tubules, and hemoglobin deposition in the renal tubules. recent studies indicates a better outcome with earlier initiation of intensive care and dialysis combined with necessary changes in antimalarial medications. schistosomiasis affects 200 million people living in endemic areas of asia, africa, and south america (254) . the infection is usually acquired in childhood, but repeated infections occur throughout life. schistosoma japonicum is found only in the orient, whereas schistosoma haematobium occurs throughout africa, the middle east, and areas of southwest asia. schistosoma mansoni is widespread in africa, south america, and southwest asia. human infection begins when the cercarial forms invade through the skin, develop into schistosomula, and move to the lungs via the lymphatics or blood. they then migrate to the liver and mature in the intrahepatic portal venules, where male:female pairing takes place. the adult worm pairs then migrate to their final resting site -the venules of the mesenteric venous system of the large intestine (s. mansoni) or in the venules of the urinary tract (s. haematobium). the females release large numbers of eggs, which may remain embedded in the tissues, embolize to the liver or lungs, or pass into the feces or urine. clinical manifestations may occur at any stage of the infection. cercarial invasion may cause an intense itchy papular rash. katayama fever is an acute serum sicknesslike illness that occurs several weeks after infection, as eggs are being deposited in the tissues. deposition of the eggs in tissues results in inflammation of the intestines, fibrosis of the liver, and portal hypertension. with s. haematobium, chronic inflammation and fibrosis of the ureters and bladder may lead to obstructive uropathy (255) . renal manifestations of schistosomiasis occur most commonly in s. mansoni infection. schistosomal nephropathy usually presents with symptoms including granulomatous inflammation in the ureters and bladder, but glomerular disease (probably on an immune-complex basis) may also occur. renal disease usually occurs in older children or young adults with long-term infection, but serious disease may also occur in young children (255) . the early renal tract manifestations of schistosomiasis are suprapubic discomfort, frequency, dysuria, and terminal hematuria. in more severe cases, evidence of urinary obstruction appears. poor urinary stream, straining on micturition, a feeling of incomplete bladder emptying, and a constant urge to urinate may be severely disabling symptoms. the fibrosis and inflammation of ureters, urethra, and bladder may be followed by calcification and may result in hydroureter, hydronephrosis, and bladder neck obstruction. renal failure may ultimately develop, and there is a suspicion that squamous cell carcinoma of the bladder may be linked to the chronic infective and inflammatory process. secondary bacterial infection is common within the obstructed and inflamed urinary tract (254) . the hepatosplenic form of s. mansoni infection may be accompanied by a glomerulopathy in 12-15% of cases, manifested in the majority as nephrotic syndrome (256) . histopathologic findings include mesangioproliferative gn, focal segmental glomerulosclerosis, mesangiocapillary gn, mgn, and focal segmental hyalinosis (257) . immune complexes may be detected in the circulation of these patients, and glomerular granular deposition of igm, c3, and schistosomal antigens are seen on immunofluorescence. usually schistosoma-specific nephropathy is a progressive disease and is not influenced by antiparasitic or immunosuppressive therapy (258) , but isolated case reports of remission after treatment with praziquantel have been reported (259) . the diagnosis is confirmed by the detection of schistosoma eggs in feces, urine, or biopsy specimens. eggs are shed into the urine with a diurnal rhythm, and urine collected between 11 am and 1 pm is the most useful. urinary sediment obtained by centrifugation or filtration through a nuclepore membrane should be examined. in cases in which studies of urine and feces yield negative results in patients in whom the diagnosis is suspected, rectal biopsy specimens taken approximately 9 cm from the anus have a high diagnostic yield for both s. mansoni and s. haematobium infection. biopsy of liver or bladder may be required to establish the diagnosis. antibodies indicating previous infection can be detected using enzyme-linked immunosorbent assay or radioimmunoassay. the tests are sensitive but lack specificity and may not differentiate between past exposure and current infection. praziquantel is the drug of choice for treatment of schistosomiasis. a single oral dose of 40 mg/kg is effective in s.haematobium and s. mansoni infection and is usually well tolerated. the alternative drug for s. mansoni infection is oxamniquine. complete remission of urinary symptoms may occur in renal disease of short duration, but in late disease with extensive fibrosis, scarring, and calcification, obstructive uropathy and renal failure may persist after the infection has been eradicated. there are reports of a drastic decrease in the number of severe hepatosplenic forms of s. mansoni infection after mass treatment of the population in endemic areas with oxamniquine. this also reduced schistosomal nephropathy (256) . visceral leishmaniasis is a chronic protozoon infection characterized by fever, hepatosplenomegaly, anemia, leukopenia, and hyperglobulinemia. proteinuria and/or microscopic hematuria or pyuria have been reported in 50% of patients with visceral leishmaniasis (260) . acute renal failure in association with interstitial nephritis has also been reported (261) . renal histologic analysis in patients with visceral leishmaniasis reveals glomerular changes, with features of a mesangial proliferative gn or a focal proliferative gn, or a generalized interstitial nephritis with interstitial edema, mononuclear cell infiltration, and focal tubular degeneration. immunofluorescence reveals deposition of igg, igm, and c3 within the glomeruli, as well as electron-dense deposits in the basement membrane and mesangium on electron microscopy (260) . circulating immune complexes together with immunoglobulin and complement deposition in the glomeruli suggests an immune-complex cause. renal disease in leishmaniasis is usually mild and may resolve after treatment of the infection. renal dysfunction may be associated with treatment for visceral leishmaniasis with antimony compounds. proteinuria is more common in filarial hyperendemic regions of west africa than in nonfilarial areas. renal histologic analysis has shown a variety of different histopathologic appearances; the most common is diffuse mesangial proliferative gn with c3 deposition in the glomeruli (262) . renal biopsy specimens also demonstrate large numbers of eosinophils in the glomeruli, and microfilariae may be seen in the lumen of glomerular capillaries. filarial antigens have been detected within immune deposits within the glomeruli. echinococcus granulosus causes chronic cysts within a variety of organs. in addition, nephrotic syndrome in association with hydatid disease has been reported. membranous nephropathy, minimal change lesions, and mesangiocapillary gn have been described in association with hydatid disease (263, 264) . immunofluorescence reveals deposits of immunoglobulin, complement, and hydatid antigens within the glomeruli. remission of nephrotic syndrome has been reported with treatment by antiparasitic agents such as albendazole (263, 264) . few reports have been published of renal disease occurring in patients with trypanosomiasis. the trypanosomal antigens can induce gn in a variety of experimental animals (265) . nephrotic syndrome has occasionally been reported as a manifestation of congenital toxoplasmosis. dissemination of previously latent toxoplasma infection in patients undergoing treatment with immunosuppressive drugs has been increasingly recognized in recent years. reactivation of toxoplasmosis or progression of recently acquired primary infection should be considered in patients undergoing renal transplantation or immunotherapy for renal disease who develop unexplained inflammation of any organ. fungal infections of the kidneys and urinary tract occur most commonly as part of systemic fungal infections in patients with underlying immunodeficiency, as focal urinary tract infections in patients with obstructive lesions, or as a result of indwelling catheters. although candida infection is the most common fungal infection in both immunocompromised and non immunocompromised hosts, virtually all other fungal pathogens may invade the renal tract during severe immunocompromise. urinary infection with candida albicans is most commonly a component of systemic candidiasis in patients who are severely immunocompromised. systemic candidiasis is also seen in premature and term infants with perinatally acquired invasive candidiasis. presentation is usually with systemic sepsis, fever or hypothermia, hepatosplenomegaly, erythematous rash, and thrombocytopenia. systemic candidiasis may be seen on ophthalmologic investigation as microemboli in the retina. the first clue to the underlying diagnosis may be the presence of yeasts in the urine (266) . candida involvement of the urinary tract may affect all structures including the glomeruli, tubules, collecting system, ureters, and bladder. microabscesses may form within the renal parenchyma, and large balls of fungi may completely obstruct the urinary tract at any level. acute renal failure caused by systemic candidiasis or obstruction of the renal tracts with fungal hyphae is a wellrecognized complication of systemic candidal infection (266, 267) . indwelling catheters (which form a nidus for persistent infection) should be removed. successful treatment of non-obstructing bilateral renal fungal balls by fluconazole either alone or in combination with liposomal amphotericin b has been reported (268, 269) . in the presence of obstruction, however, percutaneous nephrostomy to relieve the obstruction with antegrade amphotericin b irrigation, coupled with systemic antifungal therapy, is the mainstay of treatment (267) . amphotericin b is the most effective antifungal agent, but it is not excreted in the urine. local irrigation via nephrostomy provides good results, however. for treatment of urinary tract candidiasis, it is usually combined with fluconazole or 5-flucytosine, both of which are excreted in high concentrations in the urine. treatment is required for weeks to months to ensure complete elimination of the fungus, and the ultimate outcome is largely dependent on whether there is a permanent defect in immunity. in 1983, levin et al. first described hemorrhagic shock and encephalopathy, which appeared to be distinct from previously recognized pediatric disorders (270) . other cases have subsequently been reported from several centers in the united kingdom, europe, israel, the united states, and australia, and the syndrome is now recognized as a new and relatively common severe childhood disorder (271) . hemorrhagic shock and encephalopathy usually affects infants in the first year of life, with a peak onset at 3-4 months of age. a prodromal illness with fever, irritability, diarrhea, or upper respiratory infection occurs 2-5 days before the onset in two-thirds of cases. affected infants develop profound shock, coma, convulsions, bleeding and evidence of dic, diarrhea, and oliguria. laboratory findings include acidosis, falling hemoglobin and platelet levels, elevated urea and creatinine levels, and elevated levels of hepatic transaminases. despite vigorous intensive care, the prognosis is poor, and most affected infants die or are left severely neurologically damaged (271, 272) . a small number of patients have been reported to survive without residual sequelae. the renal impairment appears to be largely prerenal in origin, and when aggressive volume replacement and treatment of the shock results in improved renal perfusion, rapid improvement in renal function is usually observed. in patients with profound shock unresponsive to initial resuscitation, vasomotor nephropathy supervenes and dialysis may be required. myoglobinuria in association with hemorrhagic shock and encephalopathy has been reported. following the description of the mucocutaneous lymph node syndrome by kawasaki in 1968, kawasaki disease has been recognized as a common and serious childhood illness with a worldwide distribution. although the etiology remains unknown, epidemiologic features clearly suggest an infective cause. the disease occurs in epidemics, and wavelike spread has been demonstrated during outbreaks in japan. deposition of amyloid within the kidney is an important complication of chronic and persistent infection. amyloidosis is most common in patients with chronic osteomyelitis and chronic pulmonary infections such as bronchiectasis and is seen occasionally in those with persistent infections such as leprosy or malaria (273) (274) (275) . paediatric emergencies, 2nd edn. black ja shock in the paediatric patient vasoactive hormones in the renal response to systemic sepsis renal effects of nitric oxide in endotoxemia drotrecogin alfa (activated) in children with severe sepsis: a multicentre phase iii randomised controlled trial treatment of meningococcal disease in childhood pathophysiology and management of meningococcal septicaemia severity of meningococcal disease: assessment by factors and scores and implications for patient management case records of the massachusetts general hospital. case 4 plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease hemodynamic patterns of meningococcal shock in children role of interleukin 6 in myocardial dysfunction of meningococcal septic shock dysfunction of endothelial protein c activation in severe meningococcal sepsis dysfunction of endothelial protein c activation in severe meningococcal sepsis efficacy and safety of recombinant human activated protein c for severe sepsis renal abscess in childhood: diagnostic and therapeutic progress percutaneous drainage of renal and perirenal abscesses: results in 30 patients toxic shock syndrome associated with phage group 1 staphylococci toxic shock syndrome not associated with menstruation: a review of 54 cases toxic shock syndrome phenotypic distinctiveness of staphylococcus aureus strains associated with toxic shock syndrome the staphylococcal enterotoxins and their relatives toxic shock syndrome: associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumour necrosis factor toxic shock syndrome bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes perinephric abscess caused by communityacquired methicillin resistant staphylococcus aureus impact of antibiotics on expression of virulence? associated exotoxin genes in methicillin, sensitive and methicillin, resistant staphylococcus aureus clinical and bacteriologic observations of a toxic shock-like syndrome due to streptococcus pyogenes acute poststreptococcal glomerulonephritis. a review of recent developments occurrence of acute glomerulonephritis in sibling contacts of children with sporadic acute glomerulonephritis epidemic glomerulonephritis in israel does antimicrobial therapy of streptococcal pharyngitis or pyoderma alter the risk of glomerulonephritis? medium and long-term prognosis of patients with acute post-streptococcal glomerulonephritis infectious diseases and the kidney nephritis caused by streptococcus zooepidemicus (lancefield group c) acute glomerulonephritis following group g streptococcus infection streptococcus associated toxic shock changing group a streptococcus: the reappearance of streptococcal ''toxic shock severe group a streptococcus infections associated with a toxic shock-like syndrome and scarlet fever toxin a specific c-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of streptococcus pyogenes penicillin and clindamycin differentially inhibit the production of pyrogenic exotoxins a and b by group a streptococci improved outcome of clindamycin compared with beta-lactam antibiotic treatment for invasive streptococcus pyogenes infection intravenous immunoglobulin g therapy in streptococcal toxic shock syndrome: a european randomized, double-blind, placebo-controlled trial intravenous immunoglobulin therapy for streptococcal toxic shock syndrome -a comparative observational study. the canadian streptococcal study group a history of adjunctive glucocorticoid treatment for pediatric sepsis: moving beyond steroid pulp fiction toward evidence-based medicine intensive insulin therapy and pentastarch resuscitation in severe sepsis philadelphia renal lesions in leptospirosis leptospirosis and acute renal failure: clinical experiences and a review of the literature clinical profile of leptospirosis in south gujarat evaluation of hemostasis disorders and anticardiolipin antibody in patients with severe leptospirosis placebo controlled trial of intravenous penicillin for severe and late leptospirosis hã¤molytich-urã¤miche syndrome: bilaterale nierenrindennekrosen bei akuten erworbenen hã¤molytischen anã¤mien hemolytic-uremic syndrome associated with neuraminidase-producing micro-organism: treatment by exchange transfusion hemolytic uremic syndrome associated with pneumococcal sepsis recent advances in understanding the pathogenesis of the hemolytic uremic syndromes glomerular and arteriolar thrombosis in pneumococcal septicemia hemolytic uremic syndrome associated with invasive streptococcus pneumoniae infection hemolytic-uremic syndrome associated with neuraminidase-producing microorganisms: treatment by exchange transfusion hã¤molytisch-urã¤misches syndrome bei pneumokok-ken-menigitis und -sepsis. bedeutung der t-transformation prognosis of streptococcus pneumoniaeinduced hemolytic uremic syndrome invasive pneumococcal disease and hemolytic uremic syndrome hemolytic uremic syndrome associated with invasive pneumococcal disease: the united kingdom experience acute renal failure of glomerular origin during visceral abscess glomerulitis in typhoid fever yersinia and chronic glomerulopathy in the savannah region of nigeria typhoid glomerulonephritis acute glomerulonephritis in multi-drug resistant salmonella typhi infection acute glomerulonephritis in salmonella typhi infection urinary tract and renal findings in acute yersinia infection acute tubulointerstitial nephritis in association with yersinia pseudotuberculosis infection acute renal failure associated with yersinia pseudotuberculosis infection in children acute renal failure associated with yersinia pseudotuberculosis infection ampicillin vs. placebo for yersinia pseudotuberculosis infection in children yersinia enterocolitica and yersinia pseudotuberculosis infections sporadic cases of haemolytic uraemic syndrome associated with faecal cytotoxin and cytotoxin producing escherichia coli in stools fact sheet no. 104. revised. geneva: world health organization global burden of tuberculosis: estimated incidence, prevalence, and mortality by country mycobacterium tuberculosis. in principles and practice of infectious diseases genitourinary tuberculosis revisited urogenital tuberculosis in children genitourinary tuberculosis: review of 102 cases the glomerulopathy of congenital syphilis: an immune deposit disease nephropathy of secondary syphilis: a clinical and padiological spectrum immunopathogenesis of syphilitic glomerulonephritis membranoproliferative glomerulonephritis and mycoplasma pneumoniae infection mycoplasma pneumoniae associated with acute glomerulonephritis mycoplasma pneumoniaeassociated nephritis in children textbook of pediatric infectious diseases rocky mountain spotted fever: clinical, laboratory and epidemiological features in 262 cases acute renal failure in rocky mountain spotted fever kidney lesion in rocky mountain spotted fever acute glomerulonephritis in a patient with rocky mountain spotted fever rocky mountain spotted fever rocky mountain spotted fever q fever: current concepts glomerulonephritis associated with coxiella burnetii endocarditis disseminated legionella pneumophila infection in an infant with severe combined immunodeficiency legionnaire's disease and acute renal failure: base report and review tubulointerstitial nephritis associated with legionnaires' disease the immune complex glomerulonephritis of bacterial endocarditis glomerular nephropathy associated with chronic q fever legionella prostheticvalve endocarditis glomerulonephritis in bacterial endocarditis the immune nature of subacute bacterial endocarditis (sbe) nephritis infective endocarditis in children the clinical spectrum of shunt nephritis the role of complement, immunoglobulin and bacterial antigen in coagulase-negative staphylococcal shunt nephritis membranoproliferative glomerulonephritis associated with infected ventriculoatrial shunt: report of two cases recovered after removal of the shunt the hepatitis b virus hepatitis b infection and renal disease: clinical, immunopathogenetic and therapeutic considerations extrahepatic manifestations of viral hepatitis christian cl vasculitis with hepatitis b antigenemia. long term observation in nine patients hepatitis b-associated vasculitis in an infant polyarteritis nodosa related to hepatitis b virus. a prospective study with long-term observation of 41 patients hepatitis b virus-associated polyarteritis nodosa: clinical characteristics, outcome, and impact of treatment in 115 patients immune complexes of heparins b surface antigen in the pathogenesis of periarteritis nodosa. a study of seven necropsy cases successful treatment of polyarteritis nodosa related to hepatitis b virus with interferon alpha as first-line therapy lamivudine in the treatment of polyarteritis nodosa associated with acute hepatitis b successful treatment of hepatitis b virus associated polyarteritis nodosa with a combination of prednisolone, afpha-interferon and iamivudine infectious diseases and the kidney membranous glomerulonephropathy in childhood hepatitis b surface antigenemia in north american children with membranous glomerlonephropathy the clinico-pathologic features of hepatitis b virus-associated glomerulonephritis gene expression profile of transgenic mouse kidney reveals pathogenesis of hepatitis b virus associated nephropathy hepatitis-b virus associated nephropathies: a clinicopathological study in 14 children strong association between iga nephropathy and hepatitis b surface antigenemia in endemic areas approaches to the treatment of hepatitis b virus and delta-related liver disease hbv associated nephrotic syndrome: resolution with oral lamivudine management of chronic hepatitis b virus infection: a new era of disease control lamivudine and hbv-associated nephropathy the prevalence of hepatitis c virus infection in the united states children with hepatitis c hepatitis c virusassociated glomerulonephritis focal segmental glomerular sclerosis among patients infected with hepatitis c virus hepatitis c virus-associated glomerulonephritis. effect of alpha-interferon therapy a comprehensive study of the association between hepatitis c virus and glomerulopathy hepatitis c infection and the patient with end-stage renal disease hepatitis c infection in children and adolescents with end-stage renal disease hepatitis c virus seropositivity at the time of renal transplantation in the united states: associated factors and patient survival de novo membranoproliferative glomerulonephritis in hepatitis c virus-infected renal allograft recipients hepatitis c virus infection and de novo glomerular lesions in renal allografts design of the peds-c trial: pegylated interferon +/ã�ribavirin for children with chronic hepatitis c viral infection effect of combination therapy (ribavirin and interferon) in hcv-related glomerulopathy acute renal failure in kidney transplant patients treated with interferon alpha 2b for chronic hepatitis c vasculitic complications of interferon-alpha treatment for chronic hepatitis c virus infection: case report and review of the literature widespread presence of histologically occult cytomegalovirus glomerulonephritis in congenital cytomegalic inclusion disease immune complexes in congenital and natal cytomegalovirus infection of man glomerulopathy associated with cytomegalovirus virenmia in renal allografts epstein-barr virus, cytomegalovirus and other viral infections in children after transplant cytomegalovirus-induced glomerular vasculopathy in renal allografts: a report of two cases the association of viral infection and chronic allograft: nephropathy with graft dysfunction after renal transplantation antiviral medications for preventing cytomegalovirus disease in solid organ transplant recipients infection in organ transplant recipients acute glomerulonephritis associated with varicella infection varicella infection in a renal transplant recipient associated with abdominal pain, hepatitis, and glomerulonephritis nephrotic syndrome associated with varicella infection epstein-barr virus-associated acute interstitial nephritis: infection or immunologic phenomenon? acute interstitial nephritis secondary to infectious mononucleosis an atypical pattern of epstein-barr virus infection in a case with idiopathic tubulointerstitial nephritis an in situ hybridization study of herpes simplex and epstein barr viruses in iga nephropathy and non-immune glomerulonephritis a review of adenoviruses in the aetiology of haemorrhagic cystitis coxsackie b viruses and the kidney -a neglected topic measles and acute glomerulonephritis mumps associated with nephritis associated focal and segmental glomerulosclerosis in the acquired immunodeficiency syndrome renal disease in patients with aids: a clinicopathologic study glomerular lesions in the acquired immunodeficiency syndrome urinary and renal histological changes in children with the acquired immunodeficiency syndrome (aids) nephrotic syndrome associated with acquired immunodeficiency syndrome in children human immunodeficiency virus nephropathy human immunodeficiency virus (hiv)-associated nephropathy in children from the hiv-1 and hiv-associated nephropathy 25 years later renal pathology of human immunodeficiency virus infection protease inhibitor therapy for hiv infection: the effect on hiv-associated nephrotic syndrome bk virus renal infection in a patient with the acquired immunodeficiency syndrome indinavir-associated interstitial nephritis and urothelial inflammation: clinical and cytologic findings mixed cryoglobulinemia in hiv-1 infection: the role of hiv-1 a typical hemolytic uremic syndrome in human immunodeficiency virus-1-infected children hiv-associated nephropathy is a late, not early, manifestation of hiv-1 infection hiv-associated nephropathy: current concepts hiv-associated nephropathy glomerulosclerosis and viral gene expression in hiv-transgenic mice: role of nef pathogenesis of human immunodeficiency virus (hiv)-associated nephropathy increased levels of transforming growth factor-[beta] in hiv-associated nephropathy a steroid responsive nephrotic syndrome in a patient with human immunodeficiency virus infection clinicopathologic correlates of prednisone treatment of human immunodeficiency virus-associated nephropathy cohort study of the treatment of severe hfv-associated nephropathy with corticosteroids prednisone improves renal function and proteinuria in human immunodeficiency virus-associated nephropathy effect of angiotensin-converting enzyme inhibition in hiv-associated nephropathy spontaneous improvement of the renal function in a patient with hiv-associated focal glomerulosclerosis hiv-1-associared nephropathy and response to highly-active antiretroviral therapy protease inhibitor therapy for hiv infection: the effect on hiv-associated nephrotic syndrome zidovudine is beneficial in human immunodeficiency virus associated nephropathy hiv-1 infection initiates an inflammatory cascade in human renal tubular epithelial cells treatment of human immunodeficiency virus (hiv)-associated nephropathy hivan and medication use in chronic dialysis patients in the united states: analysis of the usrds dmms wave 2 study maintenance hemodialysis in patients with hiv-associated nephropathy organ transplant multi-site study. clinical, immunologic and pharmacologic consequences of kidney transplantation in people with hiv infection hivinfected liver and kidney transplant recipients: 1-and 3-year outcomes new human papovavirus (bk) isolated from urine after renal transplantation polyomavirus disease under new immunosuppressive drugs: a cause of renal graft dysfunction and graft loss diagnosis and management of bk polyomavirus interstitial nephritis in renal transplant recipients polyomavirus infection of renal allograft recipients: from latent infection to manifest disease infectious diseases and the kidney testing for polyomavirus type bk dna in plasma to identify renal-allograft recipients with viral nephropathy nephropathy due to polyomavirus type bk polyomavirus-associated nephropathy: update in diagnosis pathogenesis and management of polyomavirus infection in transplant recipients bk virus infection in renal transplant recipients prospective study of polyomavirus type bk replication and nephropathy in renal-transplant recipients bk virus infection, replication, and diseases in pediatric kidney transplantation occurrence and significance of papovaviruses bk and jc in the urine polyoma-virus induced interstitial nephritis in two renal transplant recipients: case reports and review of the literature late-onset hemorrhagic cystitis associated with urinary excretion of polyoma-viruses after bone marrow transplantation collecting duct carcinoma arising in association with bk nephropathy posttransplantation in a pediatric patient. a case report with immunohistochemical and in situ hybridization study association of renal adenocarcinoma and bk virus nephropathy post-transplantation clinical course of polyoma virus nephropathy in 67 renal transplant patients epidemiology of haemorrhagic fever viruses virus and hemostasis dengue and dengue haemorrhagic fever glomerular changes in dengue haemorrhagic fever intravenous fluids -getting the balance right comparison of three fluid solutions for resuscitation in dengue shock syndrome epidemiology and clinical features of crimean-congo haemorrhagic fever in southern africa viral hemorrhagic fever-induced acute kidney injury hantavirus: an increasing problem? haemorrhagic fever with renal syndrome, virological and epidemiological aspects different pathohistological presentations of acute renal involvement in hantaan virus infection: report of two cases clinical characteristics of haemorrhagic fever with renal syndrome in children prospects for treatment of viral haemorrhagic fever with ribavirin, a broad spectrum antiviral drug lassa fever: effective therapy with ribavirin human infection with highly pathogenic h5n1 influenza virus renal insufficiency on presentation of bird flu infection: is it correlated to outcome? pathogenesis of severe acute respiratory syndrome fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a newly discovered human pneumovirus isolated from young children with respiratory tract disease cloning of a human parvovirus by molecular screening of respiratory tract samples malaria in 2002 malaria and renal disease with special reference to british guiana. ii. the effect of malaria eradication on the incidence of renal disease in british guiana immunopathology of malaria the nephrotic syndrome and other diseases in children in western nigeria malaria-induced renal damage: facts and myths clinicopathological features of childhood nephrotic syndrome in northern nigeria quartan malarial nephrotic syndrome renal disease in acute plasmodium falciparum infection in man current knowledge in falciparum malaria-induced acute renal failure severe p. falciparum malaria in kenyan children: evidence for hypovolaemia response to volume resuscitation in children with severe malaria volume expansion with albumin compared to gelofusine in children with severe malaria: results of a controlled trial randomized trial of volume expansion with albumin or saline in children with severe malaria: preliminary evidence of albumin benefit severe acute renal failure in malaria recurrence of blackwater fever: triggering of relapses by different antimalarials philadelphia schistosomal specific nephropathy leading to end-stage renal failure is glomerulopathy due to schistosomiasis mansoni disappearing? characterisation of kidney lesions in early schistosomal-specific nephropathy schistosoma mansoni-induced mesangiocapillary glomerulonephritis: influence of therapy minimal change glomerulonephritis associated with schistosoma haematobium infectionresolution with praziquantel treatment renal involvement in visceral leishmaniasis acute renal failure in visceral leishmaniasis the nephrotic syndrome associated with filariasis minimal change glomerulonephritis associated with hydatid disease reversible nephrotic syndrome due to mesangiocapillary glomerulonephritis secondary to hepatic hydatid disease immune complexmediated glomerulopathy in experimental chagas' disease anuria in a newborn secondary to bilateral ureteropelvic fungus balls the role of percutaneous nephrostomy in the management of obstructing candidiasis of the urinary tract in infants successful treatment of bilateral renal fungal balls with liposomal amphotericin b and fluconazole in an extremely low birth weight infant renal fungus ball in a premature infant successfully treated with fluconazole haemorrhagic shock and encephalopathy: a new syndrome with high mortality in young children haemorrhagic shock and encephalopathy: reflections about a new devastating disorder that affects normal children haemorrhagic shock and encephalopathy: clinical, pathologic, and biochemical fea-mks use of protein-c concentrare, heparin, and haemodiafiltration in meningococcus-induced purpura fulminans quantitative viral load monitoring and cidofovir therapy for the management of bk virus associated nephropathy in children and adults antiviral therapy and prophylaxis for influenza in children infectious diseases and the kidney key: cord-004501-guiy89x8 authors: cojocaru, florina-daniela; botezat, doru; gardikiotis, ioannis; uritu, cristina-mariana; dodi, gianina; trandafir, laura; rezus, ciprian; rezus, elena; tamba, bogdan-ionel; mihai, cosmin-teodor title: nanomaterials designed for antiviral drug delivery transport across biological barriers date: 2020-02-18 journal: pharmaceutics doi: 10.3390/pharmaceutics12020171 sha: doc_id: 4501 cord_uid: guiy89x8 viral infections are a major global health problem, representing a significant cause of mortality with an unfavorable continuously amplified socio-economic impact. the increased drug resistance and constant viral replication have been the trigger for important studies regarding the use of nanotechnology in antiviral therapies. nanomaterials offer unique physico-chemical properties that have linked benefits for drug delivery as ideal tools for viral treatment. currently, different types of nanomaterials namely nanoparticles, liposomes, nanospheres, nanogels, nanosuspensions and nanoemulsions were studied either in vitro or in vivo for drug delivery of antiviral agents with prospects to be translated in clinical practice. this review highlights the drug delivery nanosystems incorporating the major antiviral classes and their transport across specific barriers at cellular and intracellular level. important reflections on nanomedicines currently approved or undergoing investigations for the treatment of viral infections are also discussed. finally, the authors present an overview on the requirements for the design of antiviral nanotherapeutics. today, we are living through a so-called fourth great transitional period, after the other three waves of epidemiological transitions, namely early agrarian-based settlements, early eurasian civilizations and european expansionism (more details can be found in mcmichael's description [1] ). the current configuration and variety of infectious diseases closely followed the combined evolutions in demography, environment, technology, social change and behaviours. medicine itself has created new opportunities for microbes either through blood transfusions, organ transplants, the use of hypodermic syringes or the excessive use of antibiotics thus contributing to the induction of iatrogenic effects in some treatments for infections such as hepatitis c, hiv and others [1] . in recent decades, old concerns have been reactivated at both the official and the general public levels regarding infectious diseases as a threat to public health. mcmichael [1] analyzed the reflection of this issue in social media and noticed the "emergence and resurgence" of infectious diseases (determined by environmental, sociological and economic changes) and a so-called "public anxiety" set on this topic. despite their widespread and increasing transmission, there is still a poor understanding of global economic impact of viral diseases, which makes difficult to evaluate the societal costs and the cost-effectiveness of preventive efforts. the issue of estimating a general impact of viral infections involves several aspects that hinder this approach, namely: the variety of viral infections, the incidence of associated co-morbidities, social and psychological issues having economic repercussions (hidden costs), the variety of treatments (only direct-acting antiviral (daa) and vaccinations can be used in the evaluation) and the presence of negative externalities [2] , meaning that the disease consequences are not limited to their patients infected or potentially but also to the related families who may experience social distress as well. also, human mobility and long-distance trade have increased; ever-larger cities, often girded with slums, have become highways for microbial traffic; poverty perpetuates vulnerability to infectious disease; and sexual practices, drug injecting, intensified food production and much modern medical technology all create new "audience" for microbial opportunism, and new management issues for public health decision makers [1] . for all these reasons, a global assessment of socio-economic impact seems practically impossible or, if contrary, it could not meet all the criteria to be relevant. rather, the impact can be split by relevant categories of viral infections, were the literature is more precise but even so, there remain some inconsistencies regarding the unification of the methodologies from the various studies that will ensure the comparability of the data. figure 1 presents statistical facts related to the most "burden-generator" viral infection diseases [3] [4] [5] [6] [7] . pharmaceutics 2020, 12, x for peer review 2 of 34 in recent decades, old concerns have been reactivated at both the official and the general public levels regarding infectious diseases as a threat to public health. mcmichael [1] analyzed the reflection of this issue in social media and noticed the "emergence and resurgence" of infectious diseases (determined by environmental, sociological and economic changes) and a so-called "public anxiety" set on this topic. despite their widespread and increasing transmission, there is still a poor understanding of global economic impact of viral diseases, which makes difficult to evaluate the societal costs and the cost-effectiveness of preventive efforts. the issue of estimating a general impact of viral infections involves several aspects that hinder this approach, namely: the variety of viral infections, the incidence of associated co-morbidities, social and psychological issues having economic repercussions (hidden costs), the variety of treatments (only direct-acting antiviral (daa) and vaccinations can be used in the evaluation) and the presence of negative externalities [2] , meaning that the disease consequences are not limited to their patients infected or potentially but also to the related families who may experience social distress as well. also, human mobility and long-distance trade have increased; ever-larger cities, often girded with slums, have become highways for microbial traffic; poverty perpetuates vulnerability to infectious disease; and sexual practices, drug injecting, intensified food production and much modern medical technology all create new "audience" for microbial opportunism, and new management issues for public health decision makers [1] . for all these reasons, a global assessment of socio-economic impact seems practically impossible or, if contrary, it could not meet all the criteria to be relevant. rather, the impact can be split by relevant categories of viral infections, were the literature is more precise but even so, there remain some inconsistencies regarding the unification of the methodologies from the various studies that will ensure the comparability of the data. figure 1 presents statistical facts related to the most "burden-generator" viral infection diseases [3] [4] [5] [6] [7] . hiv/aids, considered as one of the major burdens of disease globally, became a chronic disease after the introduction of multiple antiretroviral therapy (art), and therefore it needs to provide longhiv/aids, considered as one of the major burdens of disease globally, became a chronic disease after the introduction of multiple antiretroviral therapy (art), and therefore it needs to provide long-term care and support for the ill person, demanding a higher level of treatment costs for the hiv-affected households. consequently, hiv/aids causes depletion of savings and productive assets, and increases the indebtedness of the hiv-affected households [8, 9] . moreover, the higher health care expenditure of the households reduces investment for nutritional food for the family members, investment for farming or business, and the children education. death during the working age of the patient is a major factor in the economic impact of hiv/aids. the household level impact of hiv/aids includes direct costs, including medical and non-medical costs, and productivity costs such as loss of labour time, as a result of the morbidity of hiv positive household members, as well as time spent by others caring for them [10] . this evidence suggests that hiv/aids places significant economic pressure on households trying to pay for health care costs, and trying to make up for lost income. the difficulty in accurately quantifying and explaining the morbidity and mortality related to viral hepatitis stems from the fact that hepatitis deaths are caused by five distinct viruses (hepatitis a-e) with different routes of transmission, or from the fact that death occurs decades after infection, and that when people die with hepatitis-related liver cancer and cirrhosis, these deaths are not always linked to the underlying infection. although antiviral therapy appears to be expensive (for example, average cost for a treated hcv case ranges from $26,500 to $94,500 [11] ), is considered to be also cost effective when compared with other well-accepted medical interventions [12] due to sustained viral response to therapy, the cost savings and quality-of-life improvement and prolongation of life expectancy from the prevention of hcv complications. in the era of new daas, the statement "provide treatment to hcv-patients" generates savings compared to not provide it. low and middle-income countries may consider hcv-treatment as a cost-saving intervention for the health system, not only in a long-term horizon, but in 5-10 years [5] . economic impact of no treatment is higher than treatment costs itself. but still, the enthusiasm for daa therapies, however, has been tempered by two major concerns: the price, still very high, of these medications, and, the challenges patients and clinicians face with respect to drug access in many countries. hsv and herpes zoster (varicella-zosterian virus-vzv reactivation) are some of the most common infections in humans, with no effective treatment available at this time. the impact on the health degree varies from a very small impairment to severe, disabling forms, in most cases limiting methods are applied to local infection and reducing side effects (pain, manifestations dermis etc.). also, specific psychological issues are related with this disease, such as negative feelings correlated to the condition following diagnosis, in particular if they have acquired the genital form of the disease. feelings can include depression, fear of rejection, feelings of isolation, fear of being found out, and self-destructive feelings [6] . as mentioned above, all the available reports present crude estimates rather than precise measures of the economic costs of the illness, because most of the costs are calculated directly on interventions or on medical budgetary chapters, without taking into account the societal losses. moreover, different approaches are discrepant (can be explained, at least in part, with the influence of compliance to treatment and possible under sampling of subpopulations in the data set) [7] . also, limitations must be placed on the ability to generalize the results beyond the sample. moreover, not only the cost matters. the costs of an intervention have to be compared with the results of interventions because the effectiveness of treatments and the efficiency can produce societal gains that must be offset by losses [13] . the usual most accurate methodology to estimate a burden (associated with a disease) is the so-called "cost-of-illness" [10] , and measures all the costs of a particular disease, including the direct, indirect, and intangible dimensions. it is widely accepted that estimating the total social cost of a disease is useful in establishing policy decisions [14] . but there are also other methods as the cross-sectional surveys of samples of primary and secondary care physicians, analyzing health care resource utilization or approaches based on the analysis of a large administrative data sets, such as values of spending or drugs consuming lists [15] . other approaches are used to estimate the number of patients seeking medical treatment, the average medical expenditures (as health inputs employed per unit multiplied by number of units) and estimated national costs. these comprehensive studies can often be advantageous in allocating total national expenditures among the major diagnostic categories [16] . however, regardless of the method, such analyzes are not possible otherwise than inside countries (where the impact determined by cultural and social aspects can vary substantially). but even in the absence of global data of this nature, we still can extract from the information presented the relevant issue for the topic of this paper: the current arrangements in the management of viral infections (treatments, prevention and limitations of spread) are costly and less effective, unaffordable in some cases and burdensome for medical systems. for example, according to the current analysis of globe newswire reports and data [17], the global antiviral drugs market was valued at $49.87 billion in 2018 and is expected to reach $71.48 billion by year 2026. sales of antivirals increased by approximately 20% each two years. moreover, thanks to better diagnostics, innovative drugs and new therapeutics, the market is likely to witness even further future growth. however, the list of viral diseases for which antiviral therapies are available is still relatively short [18] . there are several factors that hinder the development of antiviral drugs: • dependence of viruses replication on host cell biosynthetic machinery [19] , that leads to a limited number of virus-specific metabolic functions can be targeted by antiviral drugs without any damage to the host; • the viruses' functions are specific to each virus, preventing the development of a broad-spectrum antivirals fighting against different viruses that cause similar symptoms. antivirals developed for some viruses (as hsv and hiv) can treat the acute illness, but do not cure the latent infection. this leads to recurrent or chronic diseases that require treatment for longer periods of time [18] . all these limitations prompted the need for a paradigm shift. the great challenge of antiviral therapies is to move on to developing new drug formulas. this involves changing the physico-chemical and bio-pharmaceutical properties of antiviral molecules using new scientific strategies during the preparation or in dosage configuration. viruses are sub-microscopic intracellular parasitic particles of genetic material contained in a protein coat, totally dependent by host for cell replication, showing both living and non-living characteristics [20] . living characteristics of the viruses are represented by the high rate of multiplication (only in living host cells) and by the ability to mutate. the non-living characteristics for viruses consist in acellularity (lack of cytoplasm and organelles), the replication only by using host cell's metabolic machinery and the composition with dna or rna [20] . in humans, viral infections are responsible for different diseases as briefly presented in table 1 . according to international committee on taxonomy of viruses (ictv) there are 1 phylum, 2 subphylia, six classes, 10 orders, seven suborders, 89 families, 36 subfamilies, 387 genera, 59 subgenera and 2202 species [43] . currently, viruses are classified based on their type of nucleic acid (dna, rna, single-stranded, double-stranded) and their way of replication, known as baltimore classification [44] , divided as seven baltimore classes: • i-dsdna viruses (e.g., adenoviruses, herpesviruses, poxviruses): enter to the host nucleus and are dependent by host cell polymerases to replicate viral genome. the virus may induce the cell to forcefully undergo cell division, which may lead to transformation of the cell and, ultimately, to cancer. • ii-ssdna viruses (+ strand or "sense") dna (e.g., parvoviruses), consists of viruses that have a single-stranded dna genome of the same polarity as the mrna. excepting parvoviruses, most of them have circular genomes and are replicating within nucleus. • iii-dsrna viruses (e.g., reoviruses): not dependent by host replication polymerases and their replication (monocistronic) is realized into capsid (in cytoplasm). • iv-(+)ssrna viruses (+ strand or sense) rna (e.g., picornaviruses, togaviruses): the rna can be directly accessed by ribosomes of the host to form proteins, and use a simple reproduction pathway (viruses with polycistronic mrna) or a more complex transcription pathway (for which subgenomic mrnas, ribosomal frameshifting, and proteolytic processing of polyproteins may be used). [34] [35] [36] [37] [38] gastroenteritis adenoviruses, rotaviruses, noroviruses, astroviruses, coronaviruses [39] [40] [41] sexually transmitted diseases herpes simplex type 2, human papillomavirus hiv [42] there are distinct stages of viral replication (cell entry, uncoating, transcription of viral genome, translation of viral proteins, post-translational modifications and assembly of virion components) and the classes of antiviral agents that can act at each stage, the correspondence between stage of replication and classes of selective inhibitors being described in detail in reference [45] and their pharmacological properties as it follows in the next section. according to our knowledge (based on found studies) only the antivirals summarized in figure 2 were applied in nanomedicine. the therapy of the large hsv family of double-stranded dna viruses, widely distributed among humans, includes highly selective and effective antivirals, from which only acyclovir (acv) and ganciclovir (gcv) were incorporated into nanomaterials; a complete classification of hsv antivirals pharmaceutics 2020, 12, 171 6 of 34 can be found in described in detail in references [46] [47] [48] [49] [50] . briefly, acv pharmacology can be explained as follows: • the acv selectivity is dependent by interaction with viral hsv thymidine kinase and dna polymerase, therefore the cellular uptake and first phosphorilation are facilitated by hsv thymidine kinase that presents a high affinity for acv; • then, intracellular enzymes convert monophospate to triphosphate acv and this form of acv inhibits viral dna polymerase and, to a much lesser extent, cellular dna polymerase; acv triphosphate is also integrated into viral dna and acts as a chain terminator, as it binds to viral dna polymerase and determines its irreversible inactivation by a mechanism called suicide inactivation [51] [52] [53] ; • occurrence of resistance to acv could be acquired by three mechanisms: impaired production of viral thymidine kinase (the most common), altered thymidine kinase substrate specificity (e.g., phosphorylation of thymidine but not acyclovir), or altered viral dna polymerase (rare). alterations in viral enzymes are caused by point mutations and base insertions or deletions in the corresponding genes [54] . according to our knowledge (based on found studies) only the antivirals summarized in figure 2 were applied in nanomedicine. the therapy of the large hsv family of double-stranded dna viruses, widely distributed among humans, includes highly selective and effective antivirals, from which only acyclovir (acv) and ganciclovir (gcv) were incorporated into nanomaterials; a complete classification of hsv antivirals can be found in described in detail in references [46] [47] [48] [49] [50] . briefly, acv pharmacology can be explained as follows:  the acv selectivity is dependent by interaction with viral hsv thymidine kinase and dna polymerase, therefore the cellular uptake and first phosphorilation are facilitated by hsv thymidine kinase that presents a high affinity for acv;  then, intracellular enzymes convert monophospate to triphosphate acv and this form of acv inhibits viral dna polymerase and, to a much lesser extent, cellular dna polymerase; acv triphosphate is also integrated into viral dna and acts as a chain terminator, as it binds to viral dna polymerase and determines its irreversible inactivation by a mechanism called suicide inactivation [51] [52] [53] ;  occurrence of resistance to acv could be acquired by three mechanisms: impaired production of viral thymidine kinase (the most common), altered thymidine kinase substrate specificity (e.g., phosphorylation of thymidine but not acyclovir), or altered viral dna polymerase (rare). alterations in viral enzymes are caused by point mutations and base insertions or deletions in the corresponding genes [54] . the mechanism of gcv inhibits viral dna synthesis [55] as briefly explained below:  it is monophosphorylated intracellular by viral thymidine kinase during hsv infection and by a viral phosphotransferase encoded by the ul97 gene during cmv infection, while diphosphate and gcv triphosphate forms are produced by cellular enzymes; the mechanism of gcv inhibits viral dna synthesis [55] as briefly explained below: • it is monophosphorylated intracellular by viral thymidine kinase during hsv infection and by a viral phosphotransferase encoded by the ul97 gene during cmv infection, while diphosphate and gcv triphosphate forms are produced by cellular enzymes; • cmv can become resistant to gcv by mutations in the viral phosphotransferase encoded by the ul97 gene and by mutations in viral dna polymerase [56] . the conventional treatment (prophylaxis or therapy) of an influenza virus infection, as a major public health concern worldwide, is designed to target viral proteins and could be used, either alone or in combination [57] . these include also amantadine and neuraminidase inhibitors (zanamivir and oseltamivir), that have been encapsulated into nanoparticles as specified in table 2 . potent vacuolar atpase (v-atpase) inhibitors, namely diphyllin and bafilomycin, previously shown to have broad-spectrum antiviral activity represent another possibility against influenza virus infection [58] [59] [60] . briefly, the antiviral mechanism of amantadine is based on nterference with the viral protein, m2 (an ion channel), the protein needed for the viral particle to become uncoated once it is taken inside the cell by endocytosis [61] . also, oseltamivir carboxylate mechanism implies a selective inhibition of influenza virus neuraminidase enzymes, which are glycoproteins found on the virion surface, very important for viral entry into uninfected cells, for the release of recently formed virus particles from infected cells, and for the further spread of the infectious virus in the body [62, 63] . hepatitis viruses have been the subject of intense study in the last years, with a special attention on therapy. as mentioned in the first section, hepatitis treatment depends upon the type of hepatitis, therefore different antivirals are considered and summarized in detail in references [64] [65] [66] [67] [68] [69] . currently, interferons (ifns) α, β, and γ have antiviral activity, the first two being produced by nearly all cells as response to viral infections, while the third is restricted to t-lymphocytes and nk cells. ifn-induced proteins include 2 -5 -oligoadenylate [2-5(a)] synthetase and a protein kinase, either of which can inhibit protein synthesis in the presence of double-stranded rna. the 2-5(a) synthetase produces adenylate oligomers that activate a latent cellular endoribonuclease (rnase l) to cleave both cellular and viral single-stranded rnas. antiretroviral therapy-art-refers to the treatment with hiv medicines. according to the last updated list approved by food and drug administration (fda) these drugs can be classified as can be seen in figure 2 [70, 71] . in the following paragraph the authors describe the pharmacological mechanism of the medicines for hiv treatment that were included/encapsulated/incorporated into nanomaterials. the representative nucleoside reverse transcriptase inhibitor (nrti) zidovudine (azt) is phosphorylated intracellular by kinases specific to azt 5 -triphosphate, a metabolite responsible for termination in elongation of proviral dna because it is incorporated by reverse transcriptase into nascent dna but lacks a 3 -hydroxyl group. resistance to azt is associated with mutations at reverse transcriptase codons 41, 44, 67, 70, 210, 215, and 219 [72] . also, lamivudine (lam), another nrti agent, enters cells by passive diffusion, and then is converted to the monophosphate by deoxycytidine kinase, and undergoes further phosphorylation by deoxycytidine monophosphate kinase and nucleoside diphosphate kinase to yield lamivudine 5 -triphosphate, which is the active anabolite [73] . tenofovir disoproxil is a derivative of adenosine 5 -monophosphate lacking a complete ribose ring, and it is the only nucleotide analogue currently marketed for the treatment of hiv infection, being active against hiv-1, hiv-2 and hbv. after a rapid hydrolysis, tenofovir is formed, being then phosphorylated by cellular kinases to its active metabolite, tenofovir diphosphate which is a competitive inhibitor of viral reverse transcriptases and is incorporated into hiv dna, causing chain termination [74, 75] . non-nucleoside reverse transcriptase inhibitors (nnrtis) include a variety of chemical substrates that bind to a hydrophobic pocket in the p66 subunit of the hiv-1 reverse transcriptase and induce a conformational change in the 3d structure of the enzyme that greatly reduces its activity, and thus they act as non-competitive inhibitors. unlike nucleoside and nucleotide reverse transcriptase inhibitors, these compounds do not require intracellular phosphorylation to attain activity [76] . also, the binding site for nnrtis is virus-strain-specific and the approved agents are active against hiv-1 but not hiv-2 or other retroviruses and should not be used to treat hiv-2 infection. hiv protease inhibitors (pis) are peptide-like chemicals that competitively inhibit the action of the virus aspartyl protease (a homodimer consisting of two 99-amino acid monomers). the preferred cleavage site for this enzyme is the n-terminal side of proline residues, especially between phenylalanine and proline. these drugs prevent proteolytic cleavage of hiv gag and pol precursor polypeptides that include essential structural (p17, p24, p9, and p7) and enzymatic (reverse transcriptase, protease, and integrase) components of the virus, preventing the metamorphosis of hiv virus particles into their mature infectious form [77, 78] . only sqv, indinavir, atv, rtv, nfv and lopinavir are currently employed in nanotechnology research. there are two drugs available in the entry inhibitors class: enfuvirtide-enf and maraviroc-mcv, with different mechanisms of action, both incorporated into nanoparticles as specified in table 2 . enf inhibits fusion of the viral and cell membranes mediated by gp41 and cd4 interactions, while mcv is a chemokine receptor antagonist and binds to the host cell ccr5 receptor to block binding of viral gp120. as such, mcv is the only approved antiretroviral drug that targets a host protein [79] [80] [81] . raltegravir (ral), the first approved hiv integrase inhibitor, has potent activity against both hiv-1 and hiv-2, and also retains activity against viruses that have become resistant to antiretroviral agents of other classes because of its unique mechanism of action [81] [82] [83] [84] . ral was encapsulated into a polymeric plga nano-formulation and gold nanoparticles (see table 2 ). the first line of defence [85] that any substance encounters is the biological barriers penetration into the organism. the "security" system includes physiological barriers, such as blood-brain barrier (bbb), epithelium, stratum corneum, air-blood lung barrier [86] , reproductive system barrier, etc. all of which control the extracellular and intracellular access and trafficking of foreign substances such as bacteria, viruses, fungi, and chemicals [87] but also provide selective access to "suitable candidates" such as nutrition and/or therapy molecules. it is well established that more than one mechanism may be involved in intracellular drug delivery. the mechanisms involved in nano-based intracellular drug delivery include passive diffusion of free drug, non-specific phagocytosis of the nanocarrier, nanocarrier uptake by pinocytosis, and receptor-mediated endocytosis [88] . in this section, we will discuss in particular how to overcome biological barriers such as mucus, skin, cell membrane and bbb in antiviral therapy. the gastrointestinal tract, respiratory system, the urogenital cavities, eyes and mouth, are all covered with mucosal membranes. the highly adhesive mucus acts as protective layer as well as for lubrication purposes. although large molecules cannot pass, small ones along with viruses can easily penetrate. these are also the reasons that drug delivery is so challenging. mucus contains 95% of water along with mucin fibres, lipids, salts, cholesterol and proteins. it is continuously produced but the thickness, ph and amount differ by its position. two strategies have to be followed for passing through mucus, mainly depending how fast the turnover is: fast mucosal penetration or highly adhesive particles (slow turnover) to increase the drug's residence on the targeted mucosa. mucoadhesion can be the resultant of interactions like hydrophobic, hydrogen bonding, ionic bonding or van der waals ones. other possible attractive interactions can be covalent bond formation between catechols, maleimides, thiols, and acrylates with domains of mucin glycoproteins rich in cysteine. chitosan, alginate, pectin or cellulose polymers are mostly used for achieving adhesion on mucosa. furthermore, it is known that thiolation of the mentioned polymers develops high mucoadhesive properties. mucopenetration is the second strategy found to permeate the mucus layers by two potential mechanisms: active strategy characterized by the interaction with the mucus and chemically shifting the features of the mucus or their own structures and passive strategy that uses hydrophilicity enhancers to penetrate the mucus [89] . the classical hsv therapy includes daily dosing of orally administered acv [90] that is effective in most cases, and of course problematic in other cases, for example, in long-term use of acv patients report resistance against the drug followed by renal injury [91, 92] . another issue produced by standard hsv treatment in this case for the topical form of trifluridine (tft) and ganciclovir (acv analog) gels, is denoted by low retention time on vaginal and corneal mucus followed by multiple doses up to 10 times [93] . the nanotechnology field offers a great deal of drug delivery modalities in order to overpass the biological barriers, deliver efficiently the incorporated active principle in a controlled and targeted pharmaceutics 2020, 12, 171 9 of 34 manner, reduce circulating drug levels and attenuate the renal damage. a recent review points out the nanogels based on the above-mentioned materials capabilities as an adequate example to pass through these types of biological barriers [94] . for example, the synthesized nano-drug delivery micelles based on chitosan-g-oligo (nipaam) copolymers stabilised by ionotropic crosslinking by raskin et al. [95] , gave good results for delivering antiretroviral drugs (efv) through mucosa. more studies on how acv penetrates different barriers can be found in table 2 and detailed in section 5. it is important to mention a "special barrier" namely the ocular mucus that changes completely in 5 to 8 min, making the drug absorption unfavourable. in addition, the eye is protected by blood-anterior chamber and blood-retinal barriers. in case of drug administration, a combination of penetration and adhesion of the substances is necessary. polymers like phosphotyrosine could be the solution. it was demonstrated that if intestinal alkaline phosphatase is present, polymers can manifest a zeta potential change, thus causing their immobilization after penetration. another kind of construct for combining adhesion and penetration is through thiolated systems with mucolytic enzymes, ph dependent. therefore, at acid ph, the absence of disulphide bonds formation with cysteine-rich domains in mucins does not manifest mucosal adhesion unless they are near the epithelium [94] . skin, as the largest organ of our body, protects us from microorganisms and chemicals, regulates our body temperature and maintains hydroelectrolytic balance. the two layers of the skin are the epidermis and the dermis. the first one is avascular and is composed of stratified, keratinized squamous epithelium, in four layers from bottom up: basale, spinosum, granulosum and corneum. thick skin (palms and soles) has a fifth layer (under the most superficial corneum) called lucidum. the dermis consists of two layers (reticular and the more superficial papillary) of connective tissue of elastin and collagenous fibres and has in its component blood and lymphatic vessels network, nerves, touch receptors (meissner corpuscles), adipocytes, phagocytes, hair follicles and sweat glands [96] . topical, through skin drug delivery, has a local effect, requiring less drug for the targeted outcome. transdermal therapy results in fewer side effects with no need of regular treatment but systemic distribution of the drug. both methods of treatment have a common blockage, stratum corneum. to pass through this barrier, different approaches were developed based mostly on disrupting this structure chemically with substances like surfactants, alcohols, esters, amines, terpenes, alkanes phospholipids, or mechanically by using ultrasounds, micro needling, magnetophoresis, iontophoresis, electroporation or lasers. excessive use, though, can damage the skin. analysing the literature data, we can suggest that there are different processes and mechanisms that govern the penetration of small/large molecules through skin barrier. according to schneider et al. [97] review there are two general pathways for skin absorption: through skin appendages or through the stratum corneum and the underlying layers. the lipophilic statum corneum medium determines the first mechanism of skin penetration, namely absorption of lipophilic compounds [98] . the three transport routes of substances across stratum corneum can be classified in transcellular, intercellular and trans-appendageal pathways as defined by liang et al. [98] . more examples are available but the conclusion is the same: "the full understanding of the penetration or absorption processes is still under evaluation" due to the challenges associated with delivering complex burdens through the skin barrie [99] . as specified in section 2, the current antiviral therapy for hsv infection includes topical formulations of acv that is unable permeate stratum corneum and target the virus site at the basal epidermis due to its polarity and solubility, leading to poor clinical efficacy due to delayed antiviral activity and sub-inhibitory concentrations [100] . nanotechnology strategies [97] seem to facilitate the "admission fees" due to the rationally design and innovative functionalities of the synthesized nano-platforms as presented in figure 3 . one more problem in dermal drug passing is related to inflammation skin pathology. the barrier is changed, and the drug resides much less on the targeted site because of fast penetration. the thermoresponsive drug delivery nanogels used in this purpose have encouraging results for overcoming the abovementioned problems. ph sensitive nanogels can also be utilised for controlled medicinal release. epidermis due to its polarity and solubility, leading to poor clinical efficacy due to delayed antiviral activity and sub-inhibitory concentrations [100] . nanotechnology strategies [97] seem to facilitate the "admission fees" due to the rationally design and innovative functionalities of the synthesized nanoplatforms as presented in figure 3 . one more problem in dermal drug passing is related to inflammation skin pathology. the barrier is changed, and the drug resides much less on the targeted site because of fast penetration. the thermoresponsive drug delivery nanogels used in this purpose have encouraging results for overcoming the above-mentioned problems. ph sensitive nanogels can also be utilised for controlled medicinal release. thermoresponsive nanogels can be controlled through irradiation with infrared lamp. another way of skin penetration is the hair follicle. the stratum corneum is less intact in the lower infundibulum, so the nanoparticle's (nps) passage is dependent on size and not on composition [94] . the cell membrane separates the content of a cell from the exterior surroundings. besides the standard protection around the cell, the cell membrane controls what substances go in and out. the composition is based on a bilayer of phospholipids, internally hydrophobic (tails) and externally hydrophilic (heads) with different proteins and cholesterol between them. the membrane is permeable selectively, permitting only some materials to pass through its lipid layer by active (through protein pumps or vesicles) or passive (diffusion) processes of transportation. water passes the membrane through a process called osmosis, which occurs when an imbalance of solutes appears outside and inside the cell [101] . in most of the cases, hydrophobic and small molecules can pass through diffusion. nanoscale drug delivery systems depend upon an active mechanism (endocytosis). over this process, the cell unit takes in ions, solid particles and molecules. there are studies that prove that positive charged nanogels can bind the membrane of the cell (negative charge) through electrostatic intercommunication. more than that, receptor-mediated endocytosis can provide a mechanism through which selectively attracted cell groups are targeted. in addition, hydrophobic nanoplatforms can grow the adhesion to the membrane and the amount of drug entered in the cell [94] . thermoresponsive nanogels can be controlled through irradiation with infrared lamp. another way of skin penetration is the hair follicle. the stratum corneum is less intact in the lower infundibulum, so the nanoparticle's (nps) passage is dependent on size and not on composition [94] . the cell membrane separates the content of a cell from the exterior surroundings. besides the standard protection around the cell, the cell membrane controls what substances go in and out. the composition is based on a bilayer of phospholipids, internally hydrophobic (tails) and externally hydrophilic (heads) with different proteins and cholesterol between them. the membrane is permeable selectively, permitting only some materials to pass through its lipid layer by active (through protein pumps or vesicles) or passive (diffusion) processes of transportation. water passes the membrane through a process called osmosis, which occurs when an imbalance of solutes appears outside and inside the cell [101] . in most of the cases, hydrophobic and small molecules can pass through diffusion. nanoscale drug delivery systems depend upon an active mechanism (endocytosis). over this process, the cell unit takes in ions, solid particles and molecules. there are studies that prove that positive charged nanogels can bind the membrane of the cell (negative charge) through electrostatic intercommunication. more than that, receptor-mediated endocytosis can provide a mechanism through which selectively attracted cell groups are targeted. in addition, hydrophobic nano-platforms can grow the adhesion to the membrane and the amount of drug entered in the cell [94] . the bbb is a highly selective semipermeable structure composed of five parts: the basement membrane, the astrocytes, the immune cells, the pericytes and an endothelial cell layer of capillaries. the area between basement membrane and the neurons is called virchow-robin space. in this region there is interstitial fluid in which reside microglia. all the above components are called neurovascular unit. the kinetics of this unit is crucial to the role of bbb and its states of illness. the capillaries of bbb compose a layer of squamous epithelial cells that fold to form a circular vessel. these cells are linked with strong connections for blocking entrance or exit of materials through central nervous system. protein transportation facilitates the selective flow of molecules through vessel lumens, essential biomolecules being in higher concentrations, like glucose and at the same time eliminating toxins. the bbb is a physical and metabolic "obstacle", physiologically important and active, which survey blood-brain traffic and control it, restricting the paracellular diffusion between the endothelial cells (microvessels) and the efflux pumps activity that quickly expel back into the capillary lumen a wide variety of xenobiotics. bbb integrity and function is critically influenced by what is now referred to as "the extended neurovascular unit" that incorporates not only microvascular endothelial cells and adjacent pericytes, astrocytes and neurons, but also neighbouring smooth muscle cells and microglia in the brain, and blood cells in the capillary lumen such as polymorphonuclear cells, lymphocytes and monocytes [102] . transport at the bbb level is assured by numerous transport mechanisms that provides to the brain the necessary nutrients and also protects from the toxic xenobiotics. the main transport mechanisms are represented by free diffusion of small lipophilic substances or by active transport (carrier mediated, receptor mediated and active efflux transport). active efflux transport is assured by two major types of transporters that extrude metabolic waste, xenobiotics and a large number of drugs from the brain back into the blood. the first superfamily of bbb efflux transporters is the solute carrier proteins (slc) superfamily, being represented at the level of bbb by slc22 and slco (slc21) efflux transporters. the second is the atp-binding cassette (abc) efflux transporter family represented by permeability glycoprotein (p-gp), breast cancer resistance protein (bcrp) and the multidrug resistance associated proteins (mrps) [103] [104] [105] . based on their localisation, the abc efflux transporters prevent lipophilic and amphiphilic environmental toxic compounds or drugs, including anti-inflammatory, immunosuppressive, anti-infectious, antineoplastic drugs, some antiepileptic, antidepressant and psychotropic agents, and drug conjugates by an energy-dependent, unidirectional direct transport mechanism, from entering specific substrates [106] . the bbb's efflux machinery does an excellent job of recognizing xenobiotics, but a poor job on distinguishing between toxicants and therapeutic drugs, creating an important obstacle to treatment of brain cancer, epilepsy and neuro aids [107] . the penetration of the bbb for drug delivery, although challenging, captivates the interest of numerous researchers in antiviral therapy since the mechanisms by which for example hsv-1 penetrates the cns remain unclear. the most likely routes include retrograde transport via the olfactory or trigeminal nerve fibres, occasionally leading to herpes simplex encephalitis (hse) caused by hsv-1 [108] . another studied virus that is involved in encephalitis and bbb disruption is hiv, known to cause severe neurological disorders and leading to hiv-related encephalitis [109] since bbb is impermeable to 98% of antiretroviral drugs [110] . the possible mechanism responsible for bbb disruption in hiv-1 encephalitis is considered a "trojan horse" mechanism, where hiv infects specific t-lymphocytes and circulating monocytes, then entering to cns through bbb gaps and followed by inflammatory reactions [111] , but, in the last years nanotechnology has been intensely explored and several experimental attempts have been carried out in order to enhance the bbb permeability toward antiretroviral drugs, briefly described below based on the nano-based formulation composition, since it is well known that the size and surface functionalization influence transport properties within tissues: • polymeric polybutylcyanoacrylate (pbca) nanoparticles with two incorporeated antiretroviral drugs (azt and lamivudine) showed a 8-20 and 10-18 fold increase in bbb permeation, by three possible mechanisms as presented by the authors: prolonged interaction interval between drug-loaded nanoparticles and brain-microvascular endothelial cells elevated the concentration gradient between blood and the brain, polysorbate 80 covering on the periphery of nanoparticles was able to be absorbed and degraded nanoparticles improved drug absorption [112] ; • spherical transferrin coated-pegylated albumin nanoparticles encapsulating azt prepared by ultra-emulsification method using chemical cross-linking by glutaraldehyde gained an access across the bbb through the transferrin receptor mediated endocytosis on the membrane [113] ; • transferrin-conjugated quantum rod nanoparticles conjugated with saquinavir crossed an in vitro bbb model by exploiting a receptor-mediated transport [114] ; • magnetic liposomal nanoformulations of azidothymidine 5 -triphosphate (the active form of azidothymidine) migrate across bbb in vitro, either directly or by a monocyte-mediated transport, under the influence of an external magnetic field [115] ; • novel nanodrug consisting of an iron oxide nanoparticle coated with pma amphiphilic polymer and functionalized with the antiretroviral peptide enfuvirtide crossed the bbb by a passive diffusion, probably mediated by the absorption of the amphiphilic coating on the cell membrane [116] . as briefly presented the preliminary results are more than encouraging. certainly, future investigations on the mechanisms about bbb disruption are needed along with novel, innovative, safe and efficacious therapeutic approaches. up to now we have concluded that current antiviral therapy has not yet achieved the ideal shape and efficiency and also that the complex biological barriers are major obstacles, but can we critically say that nanotechnology could be the identified solution? search engine queries on "nanotechnology" generate more than 114,000 items on specialized platforms (science direct, for example) that represents potential and challenges in different fields from biosensors and industry-related applications up to nanomedicine and biomaterials. when the search keywords are "nanotechnology as antiviral therapy" the same engine only returns 1404 results starting from 1997. it is a given fact that nanotechnology is defined as the application of materials on the nanometer scale. according to the literature data results, namomaterials designed with different shapes and morphologies display numerous advantages for use in antiviral therapy, namely: nanometric size that permits drug delivery through impermeable barriers [88] , large surface area to volume ratios for large drug payloads incorporation [117] and improved efficacy, surface modification and/or backbone functionalization versatility that facilitates cellular membranes passage [118] or enhancing stability and bioavailability [119] , virucidal activity against a series of viruses (hiv, hsv, hbv, etc.) due to biomimetic properties [120] , increased specificity, improved antiviral delivery and controlled drug release to the target [121] through engineered moieties, decrease the emergence of drug resistance, personalized therapy possibility, protection of the drugs and low adverse drug side effects mainly due to the composition. the mechanisms of nanomaterial-mediated drug delivery are determined by the chemistry, the architecture and the specific properties of each nanosystem (as presented in the schematic representation in figure 4 ). the design of new drug delivery systems for the antiviral therapy is focused on manipulating these features that are relevant in viral diseases where high drug doses are compulsory, implies high costs and the patient is depended on the administration protocol. lembo and cavalli [18] present the current status up to 2010 in the nanoparticulate delivery systems in antiviral therapy area, highlighting their perspective on the challenges that must be tackled before the nanotechnology can be translated into clinical use as safe and effective antiviral formulations. therefore, the nanoparticulate antiviral systems synthesised up to 2010 consisted mainly of micelles, polymeric nps, solid lipid nps (slns), nanostructured lipid carriers (nlcs), liposomes, nanocapsules, vesicles, dendrimers, nanogels, cyclodextrin-based systems and emulsions. tackled before the nanotechnology can be translated into clinical use as safe and effective antiviral formulations. therefore, the nanoparticulate antiviral systems synthesised up to 2010 consisted mainly of micelles, polymeric nps, solid lipid nps (slns), nanostructured lipid carriers (nlcs), liposomes, nanocapsules, vesicles, dendrimers, nanogels, cyclodextrin-based systems and emulsions. in 2016, liu and chen [122] summarized in a review paper an interesting perspective of nanotechnology use in hiv/aids vaccine development. their overview underline the potential of various nanomaterials and nano-architectures to be used as hiv vaccine carriers or adjuvants due to proven capabilities to improve delivery, permeability, stability, solubility and pharmacokinetics of traditional hiv vaccine approaches. the authors exhibit also the desired features of nano-carriers and adjuvants with high benefits-cost ratio. in 2017, milovanovic et al. [123] outlined, beside, the virus replication cycle and mechanism of actions of antiviral agents, an overview of particulate carriers for drug delivery. the review summarized several classes of the mostly considered carriers namely liposomes, micelles, microspheres, dendrimers and nps synthesized as alternative supports for antiviral therapy. table 2 highlights part of their summary and described based on virus classification in section 5. in 2019, cao and woodrow [124] reviewed the nanotechnology solutions used to eradicate hiv reservoirs and also the gene delivery and immunotherapy nanocarriers used in cancer with potential in hiv treatment. in section 4. nanocarriers for eradicating hiv reservoirs" the authors focused mainly on nanocarriers incorporating combination therapeutics developed in order to boost drug effectiveness and minimize toxicity. several examples are presented in table 2 and described in section 5. in 2016, liu and chen [122] summarized in a review paper an interesting perspective of nanotechnology use in hiv/aids vaccine development. their overview underline the potential of various nanomaterials and nano-architectures to be used as hiv vaccine carriers or adjuvants due to proven capabilities to improve delivery, permeability, stability, solubility and pharmacokinetics of traditional hiv vaccine approaches. the authors exhibit also the desired features of nano-carriers and adjuvants with high benefits-cost ratio. in 2017, milovanovic et al. [123] outlined, beside, the virus replication cycle and mechanism of actions of antiviral agents, an overview of particulate carriers for drug delivery. the review summarized several classes of the mostly considered carriers namely liposomes, micelles, microspheres, dendrimers and nps synthesized as alternative supports for antiviral therapy. table 2 highlights part of their summary and described based on virus classification in section 5. in 2019, cao and woodrow [124] reviewed the nanotechnology solutions used to eradicate hiv reservoirs and also the gene delivery and immunotherapy nanocarriers used in cancer with potential in hiv treatment. in section 4. nanocarriers for eradicating hiv reservoirs" the authors focused mainly on nanocarriers incorporating combination therapeutics developed in order to boost drug effectiveness and minimize toxicity. several examples are presented in table 2 and described in section 5. recently, arca-lafuente et al. [163] overviewed nanotechnology-based systems as reliable alternative diagnostic tools for hcv infectious disease. even if our review does not cover screening, it is important to mention that new diagnostic methods are required in order to overcome current drawbacks of hcv under-diagnosed infection as highlighted in the above-mentioned review. the nanotechnology-based tools described in the review seem to fulfil the necessary features for hcv elimination. with the aim of developing new personalized diagnostic tools, farzin et al. [164] summarized current strategies and under-development tools for early diagnosis of hiv. their review combines the use of nanomaterials such as carbon nanostructures, nanoclusters, quantum dots, metallic and metal oxide nps as advanced structures for hiv detection with possible biosensing strategies targeting to offer innovative outlooks for developing intelligent, sensitive and specific nano-objects for in situ and real-time detection of hiv. in this section the authors point out the suitability of nanomaterials (recent data) for antiviral therapy, highlighting the enhanced features pursued to overcome the identified issues as related above. donalisio et al. [155] have reported the preparation by a modified nano-emulsion method of chitosan nanospheres (ns) loaded with 8.5% acv as a topical formulation against both hsv-1 and hsv-2 herpes virus strains. the main component, chitosan, a natural polycationic polysaccharide, was selected as a material for acv release, due to its distinctive properties: hydrophilic character, in situ gelling, mucoadhesion, permeation enhancing, in addition to a low cytotoxicity, biocompatibility and bioresorbability features [165] . the obtained gel formulation based on acv-loaded ns proved an enhanced ability to penetrate porcine skin to about 55% (at 24 h) greater than the commercial cream product (10%). ic 50 values against hsv-1 and hsv-2 were also determined on vero cell cultures infected with above-mentioned strains, displaying significant reduced values of 0.012 µm and 0.100 µm, respectively, when using the ns formulation as compare to 0.156 µm and 1.608 µm for free acyclovir. this nano-technological approach attests the higher efficacy of the described formulation and with promising expectations for further preclinical and clinical experiments. yadavalli et al. [133] have explored the potential of highly porous activated carbon (hpac) particles as a model for restricting hsv-1 and hsv-2 from entering target cells. they have considered this material due to the charcoal surface-active that could provide antiviral effects through virion sequestration approach. furthermore, acv molecules adsorbed or encapsulated inside the hpac pores revealed sustained drug release acting in a synergistic manner to obtain an enhanced therapeutic effect. the hpac compound prove d a 40 to 60% reduction in hsv-1 and hsv-2 entry for concentrations as low as 1 mg/ml. the ic 50 value of hpac corresponding to hsv-1 and hsv-2 infection in prophylactic administration was found of 0.8 and 1 mg/ml, respectively, significantly lower than clinically accepted tc50 value (half maximal toxicity concentration) of hpac. following the promising outcomes from in vitro tests, further determinations of antiviral efficacy on in vivo studies using a murine model of ocular (hsv-1) and genital (hsv-2) infection were performed. as a result, the acv loaded hpac acts by capturing the virus and releasing the encapsulated drug, hindering inflammation and immune cell infiltration in targeted tissue. the strong antiviral activity of this product was assigned to the charged surface of its pores which may interact with the cell's surface, stimulating an active exchange of ions (na + , k + , ca + , cl − , and oh − ), when sustained or slow release of acv has been acquired. moreover, these particles exhibited both prophylactic and therapeutic effects against hsv-1/hsv-2 cells, unlike the free drug that did not demonstrate a prophylactically antiviral response. gold and silver nps using seaweed sargassum wightii with anti-herpetic activity biogenic gold (au) and silver (ag) nps were prepared using the seaweed sargassum wightii (sw) and investigated for their antiviral activity against hsv-1 and hsv-2 strains [166] . the nps synthesis resided in an eco-friendly method, previously described in the literature [167] , replacing the use of different reducing agents. the obtained nps, sw-au and sw-ag, were evaluated concerning both cytotoxic and antiviral effect, using mtt and cpe (cytopathic effect) assays on vero cells. the results showed that cell viability ranged from 93% to 85% when the concentration ranged between 2.5 and 25 µl per sample in sw-au, and from 97% to 84.58% for concentrations of 2.5 and 1 µl per sample in sw-ag. the antiviral assay have shown a 70% decrease of cpe on both hsv-1 and hsv-2 when vero cells were treated with 10 and 25 µl sw-au, whereas sw-ag exhibit similar reduction of cpe at a concentration of only 2.5 µl per sample. higher concentrations of sw-ag are not accepted due to an increased cytotoxic effect. the authors claimed that the obtained results are in agreement with other published research and they inferred that functionalized metallic nps act as antiviral agents by blocking the virus attachments and cell access, depending on particle size. cagno et al. [168] conducted a research study concerning broad-spectrum antiviral products, which usually mimic heparan sulfate proteoglycans (hspg), as well-preserved target of "viral attachment ligands" (vals). the antiviral effect relies on the binding mechanism of the nanoparticles to the virus surface, thus preventing virus-cell attachment. in most cases, the reversibility of these bonds is reported [169] , so that by increasing the dilution viral inhibition is lost, causing those compounds not to be considered antiviral drugs. the aforementioned authors have designed antiviral nanoparticles of virucidal effect based on long and flexible linkers simulating hspg, leading to irreversible viral deformation. of the synthesized compounds, the most notable virucidal effect was found in the aunps coated with a 2:1 mixture of decanesulfonic acid (mus) and 1-octanethiol (ot). mus allows a multivalent binding [170] as a consequence of its structure comprising a long hydrophobic chain, sulfonic acid terminated. the enhanced activity of mus:ot-nps was assigned to the new construct using mus linker that caused local distortions and then a global virus deformation, leading to irreversible loss of infectivity. the mus:ot-nps exhibited efficient virucidal effect against hsv-1 and hsv-2, human papilloma virus (hpv-16), respiratory syncytial virus (rsv), dengue and lenti virus. the in vivo testing on balb/c mice infected with rsv reveals the efficacy of mus:ot-nps treatment that prevented the pulmonary dissemination of the infection. these results are in agreement with previous published data [171] , which assessed the relationship between the surface structure of nano-objects and their ability to cross cell membranes. both in vivo and in vitro tests on cell cultures have proven the lack of toxicity of mus:ot-nps. coumestrol is an isoflavonoid-like compound having the ability to inhibit the replication of hsv-1 (both acyclovir sensitive and resistant strains) and also some strains of hsv-2 [172] . argenta et al. [173] have designed a formulation in an effort to obtain a topical product for coumestrol delivery at the level of mucosa. in this approach, the bioactive compound was entrapped by fluid or rigid phospholipid nanoemulsions (dioleylphosphocholine, dopc and distearoylphosphocholine, dspc, respectively) dispersed in a hydroxyethylcellulose gel. the effectiveness of the proposed antiviral agents was tested regarding permeation and retention ability on intact and damaged porcine esophageal mucosae and for antiherpes activity on cell culture assays using vero and gmk ah1 cell lines. the greatest performance of both coumestrol-loaded nanoemulsions ne-cou/dopc and the same product thickened with hydroxyethylcellulose, hne-cou/dopc, as compared to those based on dspc, relies largely on the physico-chemical properties of the nanoemulsion. the positively charged nanoemulsion showing highest values of ζ-potential may interact with negatively charged surface of mucosa membrane, with beneficial consequences relating to transmucosal delivery of coumestrol [174] . the length of phospholipids alkyl chain, the number of unsaturations, the lipophilic/hydrophilic balance of the active principle also contributed to the global effect, so that the fluid-state of hydrocarbon chain induced by dopc explained the interaction between the oil-water interface and mucosa, increasing coumestrol permeation and retention. the low ic 50 values proved an enhanced antiviral activity against hsv-1 and hsv-2 after coumestrol formulation using nanoemulsions based on dopc, which could be considered for advanced studies in order to be introduced in therapy. the huge socio-economic impact of hiv, as mentioned in section 1, determined a continuously increased trend of studies related with finding an almost perfect treatment. since the seven classes of antiretroviral drugs defined by fda contain a large number of active principles, plenty of studies regarding their incorporation in different types of nanosystems can be found in the literature. several examples are presented below. rtis classes, nrtis and nnrtis, include some of the drugs often used in hiv treatment plans. recently, grande et al. [175] published a complex review on rtis nanosystems for controlled drug delivery and our review complements part of the data presented in this article, emphasizing the penetration of biological barriers in vitro or in vivo by nanosystems containing rtis. azt, a high bioavailable drug, has serious side effects, the most common being bone marrow suppression, toxicity for some organs, neutropenia and anaemia. specific target drug delivery using different nanosystems is a promising solution [140] . atz has been incorporated in hybrid nps based on alginate and stearic acid-poly ethylene glycol. c6 glioma, neuro brain and hela cells have been used to study the cellular uptake and the cytotoxicity of the nps in vitro. the results proved that these nanosystems are nontoxic and have significant brain cellular uptake, suggesting that they can be used for more complex internalization in brain cells studies [176] . in another study, sol-oil chemistry has been used to prepare small nps lactoferrin loaded with azt (50-60 nm in size), stable in biological simulated fluids (gastric and intestinal). antiviral activity of nps has been analysed using supt1 cells infected with hiv-193in101 virus and the results suggested that the encapsulation of azt in lactoferrin does not influence the drug activity. the nps loaded with azt and the drug alone have been orally administrated to wistar rats of both genders, the performed assays (bone marrow micronucleus, histopathological and biochemical analysis) showing that azt loaded in nps is more efficient and less toxic, compared with the soluble form [140] . lamivudine-lam, a water-soluble drug with two main drawbacks: its half-life is only 2 h and has a deficient bioavailability, especially in paediatric patients (68%) [177] . lam has been included in nps based on poly(ε-caprolactone)-pcl [178, 179] , poly lactic-co-glycolic acid-plga [141] , chitosan and sodium alginate [180] , eudragit e100 [177] . the physico-chemical characterization of the obtained nps has shown an adequate size of the nps and a good stability. in vitro drug release tests indicate that nps can support the drug delivery for 24 h, indicating a less frequent administration [179, 180] . sneba et al. [177] have reported a more complex study, where lam-polymeric non-cytotoxic nps have been included in films for drug delivery through the buccal mucosa barrier. four mucoadhesive polymers: polyvinyl alcohol-pva, polyvinyl pyrrolidone-pvp, sodium carboxymethylcellulose-scm, hydroxypropyl methylcellulose-hpmc have been used to prepare the films. moreover, ozturk et al. [141] , obtained plga nps loaded with lam and proved that are physicochemical stable and slowly released the drug, a great property attributed to ester end-group of plga. because these nps were intended for oral administration, the authors evaluated the gastrointestinal stability of the nps in vitro, using different fluids of biological interest with ph in the range 1.2-7.4 phosphate buffer solution, intestinal fluid phosphate buffer solution, physiological serum and distilled water; the tests have been developed at 37 • c for 24 h. the results indicated that plga nps are promising intestinal targeted drug delivery system for lma, being stable in tested media. in clinical practice, lam is frequently administrated together with azt, therefore this combination of drugs is being studied for target delivery using different types of nps. sankar et al. [142] used plga, methylmethacrylate-sulfopropylmethacrylate-mma-spm, poly lactic acid-pla, and poly methyl methacrylate-pmma to prepare different types of nps by emulsion polymerization, as drug delivery nanosystems for atz (52%) and lma (58%). in vivo acute toxicity has been studied in mice; the results proving the fact that the drug doses loaded in the nps are not toxic. atz-lam plga nps seemed to be the most promising nanosystems. efavirenz-efv, one of the most used nnrtis in clinical practice, is a poorly water-soluble drug, and the incorporation in different drug delivery nanosystems being a solution for this drawback. patel et al. [156] obtained nanosuspensions-ns based on povidone polymer-polyvinylpyrrolidone (pvp) k30, poloxamers steric stabilizer (188 and 407) and an anionic electrostatic stabilizer (sodium lauryl sulphate, a steric stabilizer-sls). compared with the drug alone, an important improvement of saturation solubility has been noticed for the ns with efv. the incorneporation of efv in ns increased the absorption of the drug in rat intestine in situ, and very important the oral bioavailability in the studies on albino rabbits. lactoferin used to prepare nps loaded with atz [140] , as described above, has been used also to encapsulate efv [146] , based on the same preparation technique: sol-oil chemistry. compared with free efv, the encapsulation of the drug in nps showed a reduced toxicity to peripheral blood mononuclear cells, jurkat t cells and b16-f10 cells, an increased anti-hiv-1 activity and improved oral bioavailability and pharmacokinetic profile in studies on rats. atv. low brain permeability and antiretroviral drug resistance are two of the most important disadvantages of atv. this pis drug has been encapsulated in slns and studied as nanosystems for brain delivery using hcmec/d3 as a blood-brain barrier in vitro model, hcmec/d3 being human brain microvessel endothelial cell line. average atv had an important increase regarding the cellular uptake once delivered through average nanosystems (around 167 nm) [159] . sqv, another anti-hiv pis, has been incorporated in poly(ethylene oxide)-modified poly (ε-caprolactone) (peo-pcl) nps [143] by a solvent displacement process. human monocyte/ macrophage (mo/mac) cell line-thp-1 has been used for in vitro cellular uptake assay. the drug has been successively released intracellular and a meaningful uptake of the sqv-peo-pcl has been noticed. nfv, used in hiv-1 and hiv-2 treatment as pi, is a promising drug that can be used also for other grave medical disorders like cancer [181] . some studies have showed the ability of different types of nps loaded with nfv to activate latent hiv and to restrict viral spread in vitro. kovochich et al. [160] showed that lipid nps-lnps incorporated with bryostatin-2, a protein kinase c activator (lnp-bry), can be loaded with nfv (lnp-bry-nfv), and proved the above mentioned abilities on j-lat full length cells (10.6). tang et al. [144] have prepared nps based on poly(lactic-co-glycolic acid)-polyethylene glycol diblock copolymers and anti-cd45ro antibody conjugated with suberoylanilide hydroxamic acid (saha) and nfv and tested theirs in vitro properties on ach-2 cells. more complex studies have been performed by venkatesh et al. [145] plga nps loaded with nfv have significantly enhanced the oral bioavailability of the drug studied in vivo in new zealand rabbits, a reduced frequency of dosing being needed in this case. the literature data reports also several studies where combinations of pis drugs have been incorporated in nanosystems and pre-clinically evaluated. duan et al. [161] have included separately atv and drv in lnps, but only atv-lnps proved to form stable drug-lipid concentrations. based on these results, the authors have developed lnps containing atv and rtv and also lnps containing atv + rtv + tenofovir (tfv-an hiv nrtis), the last ones being prepared in a large volume for a preliminary primate pharmacokinetic study. after lnps subcutaneously administration, the three drugs have been detected in plasma for seven days. enf is a fusion inhibitor that is incapable to cross the cerebrospinal fluid. fiandra et al. [116] proved that by including it into a nanosystem composed from magnetic nanoparticles (mnp) synthesized by solvothermal decomposition in organic solvent followed by fluorescent labelled pma coating could solve enf drawback. in vitro model (co-colture of rbmvecs and astrocytes) and in vivo model (balb/c mice) studies proved that nanoconjugated enf could penetrate bbb. mcv, an entry inhibitor acting as a ccr5 co-receptor antagonist, has been also included in some nanosystems in order to increase its oral bioavailability. solid drug nanoparticles-sdns, containing 70 wt % mvc and 30% some polymer/surfactant excipients have been prepared using the emulsion-template freeze drying technique. monolayers of caco-2 have been used as a human gut in vitro model in order to study the absorption behaviour of mvc sdns and in vivo oral pharmacokinetics of the mvc solid drug nanoparticles (sdns) has been analysed on a rat model. both studies indicated an advanced permeability of the mcv nps (based on pva and sodium 1,4-bis(2ethylhexoxy)-1,4-dioxobutane-2-sulfonate (aot) excipients) correlated with the normal drug [148] . bowman et al. [182] proved that small organic monovalent molecules conjugated to aunps acts as fusion inhibitors in vitro, while vijayakumar et al. [183] proved that aunps alone acts as entry inhibitors. moreover, integrase inhibitor, ral, has been functionalized with a thiol group in order to link au-nps. in vitro cellular uptake has been tested on macrophages, human brain microendothelial cells and primary peripheral blood mononuclear cells, the results suggesting that ral-pmba-au-nps penetrate the cells and also can exhibit antiviral activity. in vivo studies performed by injection of ral-pmba-au-nps in female adult balb mice tail proved that the studied nps could cross the bbb [184] . nanomedicine represents a fast-revolutionizing field that faces rapidly and constantly progress assessed by the numerous nanodrugs that have entered clinical practice and also by even more being investigated in clinical trials. table 3 presents the approved antiviral nanomedicines, from which half are vaccines. [193] according to singh et al. [185] review from 2017 and also to available information on the respective websites there are still several nanomedicines under evaluation, namely: • fluquit (stp 702) from sirnaomics inc. currently under preclinical evaluation, a polymer-based nanotherapeutic that incorporates sirna and targeting the h5n1 (avian flu), h1n1 (swine flu) influenza, and newly emerging h7n9; and cervisil (stp909), a nanobased drug candidate, which incorporates sirna for the treatment of hpv16 and hpv18; • dermavir from genetic immunity, a synthetic pathogen-like nanomedicine that incorporates single plasmid dna expressing 15 hiv antigens that assemble to hiv-like particles; dermavir vaccine completed phase i/ii randomized, placebo-controlled, dose-finding, double-blinded, multicenter study to assess the safety, tolerability and immune response in hiv-1-infected adults who are currently receiving anti-hiv treatment (number nct00270205) [194] ; • doravirine (mk-1439), from merck, a novel, next generation nnrti described as solid drug nanoparticle formulation tested for hiv; currently doravirine completed the pharmacokinetic trial of the bioavailability of four mk-1439 nano formulations in healthy adults (number nct02549040) [195] ; • lipid nanoparticles of arb-001467 tkm-hbv containing three rnai therapeutics for hbv genome targeting from arbutus biopharma; in 2018 the company completed the phase 2a, single blind, randomized, placebo controlled, study evaluating the safety, anti-viral activity, and pharmacokinetics (pk) following multiple doses of intravenous arb-001467 (number nct02631096) [196] . as discussed above, nanotechnology started to be a critical player in the antiviral therapy. as mentioned by ross et al. [99] , nanotechnology frees the current therapy payloads in terms of delivery across biological complex barriers, and could resolve the low bioavailability drawback as already stated in section 3. nanomaterials impart many physical, chemical and biological advantages [18, 197] such as: (1) small particle size in order to facilitate drug delivery through biological barriers, (2) large surface area to volume ratios to ensure large drug payloads, (3) tunable surface charge to facilitate cellular entry across the negatively charged cellular membrane, (4) biomimetic properties which result in intrinsic antiviral assets, (5) ability to anchor targeting moieties to increase specificity to desired cell types, tissue or other compartments, (6) improved solubility and pharmacokinetic and/or pharmacodynamics properties translated in longer time to allow greater accumulation, controlled and sustained release, (7) enhanced efficiency gained either by drug molecules entrapment to protect them from physiologically hostile media, or by using surface conjugation to target drugs to specific tissues, (8) reduced toxicity and (9) multifunctionality by combining several beneficial features in a stable construct, designed to simultaneously stimulate the replication of latent virus and deliver an antiviral to the activated cell [198] . several limitations were also acknowledged such as: (1) degradation, for example nanoparticles are degraded in the gut following oral administration, or fail to penetrate the mucus barrier and are thus minimally absorbed [199] , (2) undesired interactions with biological molecules that leads to opsonization, uptake by macrophages and reduced plasma half-life [200] , (3) non-specifically absorption that may induce apoptosis and disrupt cell membrane and adverse immunological responses [201] , (4) large dimension for renal clearance therefore cannot be degraded within the body, and are accumulated, leading to toxicity [202] , and (5) scaling up issues and high costs. in our perspective, the "ideal" nanocarrier for efficient antiviral delivery must take into considerations several key factors namely: clinical outcome, since patients need safe, effective, targeted, available and affordable therapy, as they are our inspiration; • from the clinical perspective, the future antiviral candidates should improve the efficacy of the fused/encapsulated drug, reduce the intake frequency and time, restrict adverse side effects and reduce therapy costs; • design consideration for the nanoplatforms that will allow targeted delivery of the drugs in sustained released manner and improves efficacy, safety and patient convenience; therefore, from a chemist point of view, hybrid nanosystems can gather all the necessary features in terms of composition, shape and size by overcoming limitations of individual systems and offers greater advantages. starting with the composition, the chosen materials should be biodegradable, biocompatible, and non-toxic, for example polymers are very attractive since they offer the possibility for chemical modifications over the surface or backbone. in addition to these advantages, the second component from the hybrid architecture (in the shape of potential liposomes) should offer besides advanced barrier penetration, higher encapsulation efficiency for the intended drug, which in combination with the polymeric piece will be able to modulate the release kinetics, the stability and prolong drug release. when thinking about the shape, we have in mind targeting capabilities as impact. as we already know, the shape is linked with size and surface charge and density, therefore a complex puzzle that must be solved. the surface charge and density should be carefully chosen during the nanoplatforms design through the surface modification possibility. the ideal candidate here from our perspective is peg due to its versatility to exhibit various charges, shapes and sizes but also to enhance tolerability, reduce clearance, and lengthen circulation time. the size influences the biodistribution and the uptake rate therefore the "nominee" has to be in the submicron size range, recommended to be under 200 nm. taking into consideration the performance indicators of nanomedicine, we claim that the development a personalized nanomedicine is possible via a synergistically approach. since the development of "best" viral carriers involves a multidisciplinary team, virologists should be directly implicated in the development, offering specialized support on the following matters: identification of differentially expressed moieties virus cells for targeted delivery, elucidation of the type of desired targeted and the response from the host cells to nanodelivery platforms. therefore, multidisciplinary research-oriented efforts have to be related also to system biology by exploring machine learning for process optimization and pharmacology in order to introduce best appropriate combination of therapeutic agents. in 2018, an interdisciplinary team of virologists and biochemists, which developed low-cost and "cell-friendly" nanogels that can efficiently prevent viral infections, addressed these challenges [203] . here, the flexible, nontoxic and broad-spectrum nanogels based on dendritic polyglycerol sulfate mimic cellular surface receptors where several viral families bind. the designed nanogels can multivalently interact with viral glycoproteins, shield virus surfaces, and efficiently block infection since they act as robust inhibitors for these viruses. when thinking about the translation into the clinical practice, the nano-based future antiviral therapy must follow a specific flow-chart, starting with the optimization and scale-up practices according to the good manufacturing practice, the elaboration of suitable regulatory guidelines and finishing with the development of cost-effective and high quality formulations available worldwide. taken into consideration all these enhanced features, the road to clinical practice still has many addressed issues in order to provide effective and safe antiviral nano-formulations to patients. treating or improving treatment success rate for viral diseases are fundamental responsibilities. the established potential and boosted progress of nanotechnology in antiviral therapy development generates great expectations for new therapeutic innovative strategies for attacking or eradicating viral disorders. at present, studies explored numerous and diverse nano-platforms including nanoparticles, liposomes, micelles, with different compositions, size, with single or combined entrapped drugs that may serve as potential antiviral drug delivery transporters. these nano-based systems have exhibited versatile features to improve the identified current therapy drawback. however, the clinical use of a nano-based antiviral formulation to date based on our knowledge has turned out just a few approved or under clinical trials nanoformulations, mainly vaccines, despite more than 22 years of constant efforts. it is expected in the upcoming years that part of this "success preclinical story" to be scaled-up, translated and applied for better outcome, convenience and access for patients. environmental and social influences on emerging infectious diseases: past, present and future triage nurse application of the ottawa knee rule the joint united nations programme on hiv/aids. available online who. herpes simplex virus key facts the estimated economic burden of genital herpes in the united states. an analysis using two costing approaches socio-economic impact of antiretroviral treatment in hiv patients. an economic review of cost savings after introduction of haart the evolution of three decades of antiretroviral therapy: challenges, triumphs and the promise of the future: three decades of antiretroviral therapy socio-economic impact of antiviral intervention cost and access to direct-acting antiviral agents costs, and affordability of new medicines for hepatitis c in 30 countries: an economic analysis evidence from cost-effectiveness research economic note: cost of illness studies methods for the economic evaluation of health care programmes cost-of-illness studies: concepts, scopes, and methods nanoparticulate delivery systems for antiviral drugs how viral and intracellular bacterial pathogens reprogram the metabolism of host cells to allow their intracellular replication the common cold herpetic eye disease study: lessons learned adenoviral keratoconjunctivitis viral pneumonia la revue de médecine interne viral myocarditis viral infections in type 1 diabetes mellitus-why the β cells? hepatitis a and b infections hepatitis d virus: introduction and epidemiology. cold spring harb hepatitis e: discovery, global impact, control and cure clinical features of varicella-zoster virus infection molecular mechanisms of varicella zoster virus pathogenesis viral skin diseases viral gastroenteritis in the adult population viral gastroenteritis in adults epidemiology of gastroenteritis viruses in japan: prevalence, seasonality, and outbreak: epidemiology of gastroenteritis viruses in japan sexually transmitted and anorectal infectious diseases expression of animal virus genomes the pharmacological basis of therapeutics a new agent with potent anti-herpesvirus activity long-term foscarnet therapy remodels thymidine analogue mutations and alters resistance to zidovudine and lamivudine in hiv-1 docosanol: a topical antiviral for herpes labialis phosphorylation of acyclovir diphosphate by cellular enzymes history, pharmacokinetics, and pharmacology of acyclovir causes of severe pneumonia requiring hospital admission in children without hiv infection from africa and asia: the perch multi-country case-control study antiviral drug resistance: mechanisms and clinical implications an update of its use in the prevention of cytomegalovirus infection and disease in transplant recipients antiviral treatment of cytomegalovirus infection and resistant strains antiviral agents active against influenza a viruses inhibitory and combinatorial effect of diphyllin, a v-atpase blocker, on influenza viruses inhibitors of v-atpases: old and new players suppression of influenza a virus replication in human lung epithelial cells by noncytotoxic concentrations bafilomycin a1 antiviral treatments. clin neuraminidase inhibitors for preventing and treating influenza in adults and children oseltamivir resistance during treatment of influenza a (h5n1) infection mechanism of action of ribavirin in the treatment of chronic hepatitis c amino acid ester prodrugs of nucleoside and nucleotide antivirals clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of dna virus and retrovirus infections an evaluation of entecavir for the treatment of chronic hepatitis b infection in adults telbivudine for the management of chronic hepatitis b virus infection telbivudine: a new treatment for chronic hepatitis b antiretroviral drugs used in the treatment of hiv infection nucleoside and nucleotide analogue reverse transcriptase inhibitors: a clinical review of antiretroviral resistance lamivudine: a review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in the management of hiv infection a review of the toxicity of hiv medications efficacy of tenofovir 1% vaginal gel in reducing the risk of hiv-1 and hsv-2 infection conformational changes in hiv-1 reverse transcriptase induced by nonnucleoside reverse transcriptase inhibitor binding hiv protease inhibitors: a review of molecular selectivity and toxicity pharmacotherapy of hiv-1 infection: focus on ccr5 antagonist maraviroc hiv entry inhibitors and their potential in hiv therapy hiv entry inhibitors: progress in development and application progress in hiv-1 integrase inhibitors: a review of their chemical structure diversity raltegravir with optimized background therapy for resistant hiv-1 infection the first hiv integrase inhibitor the first hiv type 1 integrase inhibitor canton, i. inspired by nature: fundamentals in nanotechnology design to overcome biological barriers air-blood barrier translocation of tracheally instilled gold nanoparticles inversely depends on particle size walking the line: the fate of nanomaterials at biological barriers anti-hiv-1 nanotherapeutics: promises and challenges for the future approaches in polymeric nanoparticles for vaginal drug delivery: a review of the state of the herpetic eye disease study group. oral acyclovir for herpes simplex virus eye disease: effect on prevention of epithelial keratitis and stromal keratitis drug delivery approaches of an antiviral drug: a comprehensive review acyclovir: a decade later comparison of famciclovir, valaciclovir, and brivudine treatments in adult immunocompetent patients with herpes zoster crossing biological barriers with nanogels to improve drug delivery performance mucoadhesive nanogels by ionotropic crosslinking of chitosan-goligo(nipaam) polymeric micelles as novel drug nanocarriers layers of the skin nanoparticles and their interactions with the dermal barrier penetration of nanoparticles into human skin nano-enabled delivery of diverse payloads across complex biological barriers nanocarriers for effective topical delivery of anti-infectives the cell membrane physiology and pathophysiology of the blood-brain barrier: p-glycoprotein and occludin trafficking as therapeutic targets to optimize central nervous system drug delivery efflux mechanisms at the developing brain barriers: abc-transporters in the fetal and postnatal rat multidrug resistance in cancer: role of atp-dependent transporters abc transporters in drug-resistant epilepsy: mechanisms of upregulation and therapeutic approaches abc transporters at the blood-brain barrier regulation of abc transporters at the blood-brain barrier mechanisms of blood-brain barrier disruption in herpes simplex encephalitis disruption of the blood brain barrier is vital property of neurotropic viral infection of the central nervous system towards nanomedicines for neuroaids: nanomedicines for neuroaids analysis of human immunodeficiency virus type 1 gp160 sequences from a patient with hiv dementia: evidence for monocyte trafficking into brain effect of nanoparticulate polybutylcyanoacrylate and methylmethacrylatesulfopropylmethacrylate on the permeability of zidovudine and lamivudine across the in vitro blood-brain barrier targeted brain delivery of azt via transferrin anchored pegylated albumin nanoparticles blood-brain barrier transport of therapeutics via receptor-mediation magnetic nanoformulation of azidothymidine 5 -triphosphate for targeted delivery across the blood & ndash nanoformulation of antiretroviral drugs enhances their penetration across the blood brain barrier in mice unique benefits of nanotechnology to drug delivery and diagnostics. in characterization of nanoparticles intended for drug delivery strategies in the design of nanoparticles for therapeutic applications nanostructured materials for applications in drug delivery and tissue engineering biomimetic and bioinspired nanoparticles for targeted drug delivery targeted nanomedicines: effective treatment modalities for cancer, aids and brain disorders role of nanotechnology in hiv/aids vaccine development nanotechnology approaches to eradicating hiv reservoirs preparation and ocular pharmacokinetics of ganciclovir liposomes recombinant high-density lipoprotein complex as a targeting system of nosiheptide to liver cells liver targeting and anti-hbv activity of reconstituted hdl-acyclovir palmitate complex targeted delivery of sirna against hepatitis c virus by apolipoprotein a-i-bound cationic liposomes rnai-mediated ccr5 silencing by lfa-1-targeted nanoparticles prevents hiv infection in blt mice butyldeoxynojirimycin is a broadly effective anti-hiv therapy significantly enhanced by targeted liposome delivery targeted delivery of indinavir to hiv-1 primary reservoirs with immunoliposomes sustained and specific in vitro inhibition of hiv-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes drug-encapsulated carbon (decon): a novel platform for enhanced drug delivery nanoparticle-based topical ophthalmic formulation for sustained release of stereoisomeric dipeptide prodrugs of ganciclovir inhibition of h1n1 influenza virus-induced apoptosis by functionalized selenium nanoparticles with amantadine through ros-mediated akt signaling pathways inhibitory activity of selenium nanoparticles functionalized with oseltamivir on h1n1 influenza virus reversal of h1n1 influenza virus-induced apoptosis by silver nanoparticles functionalized with amantadine antiviral efficacy of nanoparticulate vacuolar atpase inhibitors against influenza virus infection bioerodible polymeric nanoparticles for targeted delivery of proteic drugs improved safety, bioavailability and pharmacokinetics of zidovudine through lactoferrin nanoparticles during oral administration in rats preparation and in vitro characterization of lamivudine loaded nanoparticles prepared by acid and/or ester terminated plga for effective oral anti-retroviral therapy formulation and in-vitro evaluation of zidovudine-lamivudine nanoparticles intracellular delivery of saquinavir in biodegradable polymeric nanoparticles for hiv/aids plga-peg nanoparticles coated with anti-cd45ro and loaded with hdac plus protease inhibitors activate latent hiv and inhibit viral spread fabrication and in vivo evaluation of nelfinavir loaded plga nanoparticles for enhancing oral bioavailability and therapeutic effect an oral formulation of efavirenz-loaded lactoferrin nanoparticles with improved biodistribution and pharmacokinetic profile the mixed lineage kinase-3 inhibitor urmc-099 improves therapeutic outcomes for long-acting antiretroviral therapy improving maraviroc oral bioavailability by formation of solid drug nanoparticles polymeric nanoparticles containing combination antiretroviral drugs for hiv type 1 treatment pharmacokinetic and tissue distribution profile of long acting tenofovir alafenamide and elvitegravir loaded nanoparticles in humanized mice model triple drug combination of zidovudine, efavirenz and lamivudine loaded lactoferrin nanoparticles: an effective nano first-line regimen for hiv therapy multivalent peptide-nanoparticle conjugates for influenza-virus inhibition amantadine surface-modified silver nanorods improves immunotherapy of hiv vaccine against hiv-infected cells enhancing the delivery of anti retroviral drug "saquinavir"; across the blood brain barrier using nanoparticles acyclovir-loaded chitosan nanospheres from nano-emulsion templating for the topical treatment of herpesviruses infections nanosuspension of efavirenz for improved oral bioavailability: formulation optimization, in vitro, in situ and in vivo evaluation assessment of ocular pharmacokinetics and safety of ganciclovir loaded nanoformulations tissue distribution of borneol-modified ganciclovir-loaded solid lipid nanoparticles in mice after intravenous administration solid lipid nanoparticles enhance the delivery of the hiv protease inhibitor, atazanavir, by a human brain endothelial cell line activation of latent hiv using drug-loaded nanoparticles evaluation of atazanavir and darunavir interactions with lipids for developing ph-responsive anti-hiv drug combination nanoparticles anti-hiv drug-combination nanoparticles enhance plasma drug exposure duration as well as triple-drug combination levels in cells within lymph nodes and blood in primates nanotechnology: a reality for diagnosis of hcv infectious disease hiv biosensors for early diagnosis of infection: the intertwine of nanotechnology with sensing strategies chitosan as a bioactive polymer: processing, properties and applications anti-herpes simplex virus (hsv-1 and hsv-2) activity of biogenic gold and silver nanoparticles using seaweed sargassum wightii a novel extracellular synthesis of monodisperse gold nanoparticles using marine alga, sargassum wightii greville broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism inhibition of human metapneumovirus binding to heparan sulfate blocks infection in human lung cells and airway tissues pathogen inhibition by multivalent ligand architectures surface-structureregulated cell-membrane penetration by monolayer-protected nanoparticles antiherpes evaluation of soybean isoflavonoids topical delivery of coumestrol from lipid nanoemulsions thickened with hydroxyethylcellulose for antiherpes treatment lecithin based nanoemulsions: a comparative study of the influence of non-ionic surfactants and the cationic phytosphingosine on physicochemical behaviour and skin permeation reverse transcriptase inhibitors nanosystems designed for drug stability and controlled delivery novel dendritic structure of alginate hybrid nanoparticles for effective anti-viral drug delivery design of antiretroviral drug-polymeric nanoparticles laden buccal films for chronic hiv therapy in paediatrics influence of solvent evaporation technique parameters on diameter of submicron lamivudine-poly-ε-caprolactone conjugate particles nanoencapsulation of water-soluble drug, lamivudine, using a double emulsion spray-drying technique for improving hiv treatment formulation and characterisation of chitosan based lamivudine nanoparticles the antiretroviral agent nelfinavir mesylate: a potential therapy for systemic sclerosis inhibition of hiv fusion with multivalent gold nanoparticles gold nanoparticles as an hiv entry inhibitor gold nanoparticles to improve hiv drug delivery the role of nanotechnology in the treatment of viral infections epaxal ® : a virosomal vaccine to prevent hepatitis a infection pharmacokinetics and pharmacodynamics modeling and simulation systems to support the development and regulation of liposomal drugs eleven years of inflexal ® v-a virosomal adjuvanted influenza vaccine package insert peg-introntm (peginterferon alfa-2b) powder for injection, schering corporation progress in nanomedicine: approved and investigational nanodrugs tolerability and immune response to lc002, an experimental therapeutic vaccine bioavailability of mk-1439 experimental nano formulations in healthy adults (mk-1439-046)-clinicaltrials.gov identifier: nct02549040. available online subjects with chronic hbv infection receiving nucleos(t)ide analogue therapy-clinicaltrials.gov identifier: nct02631096. available online therapeutic nanomedicine surmounts the limitations of pharmacotherapy nanotechnology and the treatment of hiv infection physiologically based pharmacokinetic modeling of nanoparticles multifunctional nanoparticles-properties and prospects for their use in human medicine state of the art of nanocrystals-special features, production, nanotoxicology aspects and intracellular delivery nanotechnology and drug delivery part 2: nanostructures for drug delivery multivalent flexible nanogels exhibit broad-spectrum antiviral activity by blocking virus entry this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-260191-0u0pu0br authors: haas, w.; krause, g.; marcus, u.; stark, k.; ammon, a.; burger, r. title: „emerging infectious diseases“: dengue-fieber, west-nil-fieber, sars, vogelgrippe, hiv date: 2004-05-29 journal: internist (berl) doi: 10.1007/s00108-004-1199-2 sha: doc_id: 260191 cord_uid: 0u0pu0br some emerging infectious diseases have recently become endemic in germany. others remain confined to specific regions in the world. physicians notice them only when travelers after infection in endemic areas present themselves with symptoms. several of these emerging infections will be explained. hiv is an example for an imported pathogen which has become endemic in germany. sars and avian influenza are zoonoses with the potential to spread from person to person. avian influenza in humans provides a possibility for the reassortment of a potential new pandemic strain. outbreaks of dengue fever in endemic areas are reflected in increased infections in travelers returning from these areas. currently, west-nile-virus infections are only imported into germany. the timely implementation of diagnostic, therapeutic and infection control measures requires physicians to include these diseases in their differential diagnosis. to achieve this goal, good cooperation between physicians, laboratories and the public health service is essential. ▃ die erfahrungen mit sars zeigen, wie rasch sich ein neuer erreger buchstäblich in wochen weltweit ausbreiten kann. epidemiologie aids-erkrankungen wurden 1981 erstmals in den usa beschrieben.retrospektive untersuchungen belegen jedoch,dass die der aids-erkrankung zugrunde liegende hiv-infektion sich bereits seit anfang der 1960er jahre zunächst unbemerkt weltweit ausgebreitet hat,wobei der internationale reiseverkehr wohl eine wesentliche rolle gespielt hat.⊡ abbildung 1 zeigt die aktuelle weltweite verteilung von hiv-infektionen (prävalenz),⊡ abbildung 2 die verteilung der neuinfektionen mit hiv im jahre 2003 (inzidenz). bei den von deutschen im ausland erworbenen hiv-infektionen rangiert als infektionsregion südostasien vor subsahara-afrika,gefolgt von der karibik/lateinamerika, anderen ländern in westeuropa und nordamerika sowie osteuropa. im vergleich zu deutschland, wo ca. 80% der hiv-infizierten männer sind und der altersgipfel in den altersgruppen zwischen 25 und 40 jahren erreicht wird, sind in entwicklungsländern mit vorwiegend heterosexueller übertragung von hiv mehr als die hälfte der infizierten frauen, der altersdurchschnitt liegt unter 25 jahren. der bedeutsamste übertragungsweg für hiv sind ungeschützte sexualkontakte, je nach region kann intravenöser drogenkonsum mit spritzentausch eine erhebliche rolle spielen. in vielen ärmeren ländern muss auch mit der möglichkeit von hiv-übertragungen durch verwendung unzureichend sterilisierter instrumente im medizinischen und paramedizinischen bereich (z.b.beim tätowieren) oder durch nicht auf hiv untersuchte blutspenden gerechnet werden.übertragungen von hiv durch kontaminiertes wasser, lebensmittel oder vektoren wie stechmücken sind nicht beschrieben. insbesondere im bereich der prostitution müssen in den meisten ländern über dem durchschnitt liegende hiv-infektionsraten erwartet werden,zumal andere, zusätzlich vorliegende sexuell übertragbare infektionen das übertragungsrisiko für hiv weiter steigern. von besonderer bedeutung als hiv-infektionsrisiko sind "urlaubspartnerschaften", da in diesen eher auf den schutz durch kondome verzichtet wird. tage bis wochen nach einem infektionsereignis treten bei ca. 50-70% der infizierten symptome einer akuten virusinfektion auf.am häufigsten werden fieber,muskel-und gliederschmerzen, lymphknotenschwellungen, ein flüchtiges, meist stammbetontes virusexanthem, kopfschmerzen und durchfälle berichtet.auf grund der unspezifischen symptomatik ist bei entsprechenden beschwerden eine expositionsanamnese zu empfehlen, bei der vor allem nach sexuellen risiken (ungeschützter vaginal-oder analverkehr) gefragt werden sollte. tions in travelers returning from these areas. currently,west-nile-virus infections are only imported into germany.the timely implementation of diagnostic,therapeutic and infection control measures requires physicians to include these diseases in their differential diagnosis.to achieve this goal,good cooperation between physicians, laboratories and the public health service is essential. abstract some emerging infectious diseases have recently become endemic in germany.others remain confined to specific regions in the world.physicians notice them only when travelers after infection in endemic areas present themselves with symptoms.several of these emerging infections will be explained.hiv is an example for an imported pathogen which has become endemic in germany.sars and avian influenza are zoonoses with the potential to spread from person to person.avian influenza in humans provides a possibility for the reassortment of a potential new pandemic strain.outbreaks of dengue fever in endemic areas are reflected in increased infec-gehäuften erkrankungen bei rückkehrern wieder.west-nil-virus-erkrankungen kommen derzeit nur als importierte erkrankungen in deutschland vor.wichtig ist,diese erkrankungen frühzeitig in die differenzialdiagnostischen überlegungen des klinikers einzubeziehen,um die erforderlichen maßnahmen zur diagnostik,therapie und zum infektionsschutz rechtzeitig einleiten zu können.dies erfordert ein gutes zusammenspiel mit dem labor und dem öffentlichen gesundheitsdienst. [23] . in deutschland wurden 9 wahrscheinliche fälle bekannt, die sich alle in betroffenen ländern infiziert hatten. sars wird im wesentlichen durch tröpfcheninfektion und/oder durch schmierinfektion übertragen.die inkubationszeit beträgt 2-10 tage.das erregerreservoir ist noch nicht eindeutig geklärt,ein tierisches reservoir wird jedoch angenommen [5] . der zentrale ausgangspunkt für die weltweite verbreitung von sars war das hotel metropol in hongkong. dort hatte ein arzt übernachtet,der sich zuvor in sei ein antikörpernachweis gelingt frühestens ca.3 wochen nach dem infektionsereignis. fällt bei vorliegen der geschilderten klinischen symptomatik und klarem anamnestischen infektionsrisiko der antikörpernachweis negativ aus, ist entweder eine wiederholung der untersuchung angezeigt oder es kann ein hiv-nukleinsäurenachweis (pcr-untersuchung) versucht werden, der im rahmen einer frischen infektion ca. 1-2 wochen vor dem antikörpernachweis positiv wird. bei einem negativen antikörpernachweis 3 monate nach dem letzten möglichen expositionsereignis kann eine hiv-infektion in der regel ausgeschlossen werden. die wichtigste präventionsmaßnahme für reisende ist der verzicht auf ungeschützte sexuelle kontakte bzw. die konsequente verwendung von kondomen beim geschlechtsverkehr. aus medizinischer sicht stellt die hiv-infektion keinen hinderungsgrund für reisen dar. je nach immunstatus, reiseland und reisemodalitäten kann jedoch ein erhöhtes infektions-bzw.erkrankungsrisiko für andere erreger bestehen.während impfungen mit totimpfstoffen unbedenklich sind (der impferfolg kann bei bereits bestehendem immundefekt beeinträch[18] . insbesondere innerhalb von krankenhäusern betroffener regionen hat sich sars sehr rasch sowohl auf patienten als auch auf personal übertragen.in deutschland ist es dagegen weder innerhalb noch außerhalb der krankenhäuser zu übertragungen von sars gekommen [20] . nach gegenwärtiger erkenntnis scheint dagegen das risiko einer übertragung von sars zwischen flugpassagieren während des fluges gering zu sein [13, 21] .seit juli 2003 wurden 6 weitere sars-fälle bekannt: 2 infektionen bei laborwissenschaftlern (singapur, taiwan) und 4 infektionen in südchina, bei denen die infektionsquelle unklar ist. bei sars handelt es sich um eine schwere, infektiöse atemwegserkrankung, die sich klinisch und radiologisch entweder als atypische pneumonie oder als akutes atemnotsyndrom (engl. adult respiratory distress syndrome,ards) manifestiert. die klinische symptomatik beginnt mit influenzaähnlichen symptomen mit meist hohem fieber, später kommen husten und rasch zunehmende atemnot sowie durchfälle hinzu [15] . als erreger wurde im märz 2003 das sars-coronavirus (sars-cov) identifiziert [2] . für die frühe diagnostik steht die polymerasenkettenreaktion (pcr) zum nachweis von sars-cov in respiratorischen sekreten,im stuhl und anderen sekreten zur verfügung. dabei muss allerdings beachtet werden, dass ein negativer befund auch in der akuten erkrankungsphase eine infektion mit sars-cov nicht ausschließt. ein indirekter immunfluoreszenztest zum serologischen nachweis von igm-und igg-antikörpern gegen sars-cov und ein elisa bieten sich insbesondere für epidemiologische fragestellungen an, da entsprechende nachweise erst mehrere wochen nach infektion zu erwarten sind. die bekämpfung und eindämmung einer derartigen epidemie ist aufgabe des öffentlichen gesundheitsdienstes. hier ist zum einen eine lückenlose surveillance und eine frühzeitige verfügbarkeit umfassender informationen und empfehlungen erforderlich [10] .für die gesundheitsämter stellt eine derartige epidemie eine herausforderung dar,da sie sehr umfangreiche schutzmaßnahmen umsetzen müssen [19] .diese bestehen zum einen in der frühzeitigen identifizierung von kontaktpersonen und gegebenenfalls in der absonderung von personen nach einem festgelegten schema.dies erfordert auch eine enge zusammenarbeit mit krankenhäusern,niedergelassenen ärzten und krankentransportdiensten [11] . eine impfung wird mittelfristig nicht zur verfügung stehen. die aviäre influenza wurde zum ersten mal im jahr 1878 in italien beschrieben [16] . aviäre influenza wird durch die subtypen h5 und h7 des influenza-a-virus verursacht.das reservoir dieser erreger stellen wildvögel (insbesondere wasservögel) dar, deren infektion in der regel asymptomatisch verläuft. mutationen im hämagglutinin sind jedoch die ursache dafür, dass immer wieder h5-bzw. h7-viren auftreten, die für vögel hochpathogen sind. zwischen 1959 und 2003 wurden insgesamt 21 ausbrüche bei geflügel registriert [24] . schwere respiratorische erkrankungen durch ein rein aviäres virus nach übertragung direkt von erkranktem geflügel auf den menschen wurden erstmals bei dem ausbruch 1997 in hongkong beobachtet.bis zu diesem zeitpunkt war eine direkte übertragung von aviären influenza-a-viren auf den menschen nicht belegt worden. seit 1997 wurde in 6 situationen eine übertragung von hochpathogenen aviären influenzaviren (hpai) auf den menschen nachgewiesen (⊡ tabelle 1). davor war man davon ausgegangen, dass für eine übertragung auf den menschen eine genetische neukombination ("genetic shift") von aviären und humanen influenza-a-viren erforderlich ist. als hauptvehikel für die genetische neukombination, das sog. reassortment, des segmentierten genoms der influenza-a-viren gilt das hausschwein, da es auf seinen zellen sowohl rezeptoren für aviäre als auch humane influenza-a-viren besitzt.ein reassortment konnte als entstehungsweg der pandemischen influenzaviren der "asiatischen grippe" 1957/58 (h2) und der "hongkong-grippe" 1968-70 (h3) nachgewiesen werden. im frühjahr 2003 trat ein großer ausbruch mit h7-viren in den niederlanden auf und breitete sich nach belgien und auf einen betrieb in deutschland aus. mehr als 1000 betriebe waren betroffen, mehr als 30 mio.vögel wurden daraufhin getötet [9] . bei diesem ausbruch wurden bis ende april 266 fälle von konjunktivitis diagnostiziert. fast alle erkrankte hatten engen kontakt mit erkranktem geflügel gehabt. bei 40 (12%) der erkrankten traten zusätzlich grippeähnliche symptome ("influenza-like illness",ili) auf,bei weiteren 23 personen wurde nur eine ili beschrieben. ein tierarzt, der einen betrieb mit infizierten tieren besucht hatte, verstarb kurz darauf an der erkrankung.der nachweis von h7n7 in der bronchoalveolären insgesamt wurden bei diesem ausbruch 453 menschen mit gesundheitsproblemen identifiziert [9] . bei 89 dieser personen war der influenza-a/h7-nachweis positiv. drei kontaktpersonen von an konjunktivitis erkrankten,die an der tötung von influenza-a (h7n7) infiziertem geflügel beteiligt waren, die selbst aber nicht mit infiziertem geflügel in berührung gekommen waren,wiesen ebenfalls symptome auf.in ihren bindehautabstrichen wurden influenza-a/h7n7-viren nachgewiesen. dies wurde als ein starker hinweis für eine mensch-zu-mensch-übertragung gewertet [3, 9] . [7, 25] .in keinem der fälle kam es bisher durch die h5n1-erkrankungen zu einem ausbruch oder infektionsketten mit einer effizienten mensch-zu-mensch-übertragung. nur ein teil der infizierten reisenden entwickelt symptome, nur ein teil der erkrankten sucht einen arzt auf und auch in diesen fällen wird nicht immer eine untersuchung auf dengue-fieber durchgeführt. serologische untersuchungen haben gezeigt,dass bei etwa 7% der tropenreisenden mit fieber eine dengue-infektion vorlag [12] . wie die ersten erfahrungen zeigen,ist die surveillance auf der basis des ifsg jedoch im sinne eines frühwarnsystems gut geeignet,risikogebiete für reisende identifizieren und entsprechende reisemedizinische informationen veröffentlichen zu können. dengue-viren werden durch aedes-moskitos übertragen und sind in über 100 tropischen und subtropischen ländern verbreitet. für die großen epidemien beim menschen ist ausschließlich die virusübertragung zwischen vektor (vor allem aedes aegypti,in südostasien auch aedes albopictus) und mensch von bedeutung. die aedes-mücken sind zeitlebens, d.h. 2 bis 4 monate lang infektiös. das spektrum der klinischen manifestation reicht von inapparenten oder milden formen bis zu schweren, unter umständen letalen verläufen.die inkubationszeit beträgt im durchschnitt 4-7 tage ein wirksamer dengue-impfstoff ist bisher nicht verfügbar. deshalb kommt der prävention die entscheidende bedeutung zu. reisenden sollten gebiete mit massiven dengue-ausbrüchen meiden. eine expositionsprophylaxe gegenüber moskitostichen ist wichtig und muss aufgrund der überwiegend tagaktiven dengue-vektoren konsequent durchgeführt werden. [1] . in deutschland werden derzeit blutund plasmaspender von der spende zurückgestellt, wenn sie sich zwischen dem 1. juni und dem 30. november eines jahres auf dem nordamerikanischen kontinent aufgehalten haben und der tag der rückkehr weniger als 4 wochen zurückliegt. beim menschen nimmt die überwiegende zahl von wnv-infektionen einen inapparenten oder milden verlauf.nach einer inkubationszeit von 3-14 tagen entwickeln etwa 20% der infizierten eine fieberhafte erkrankung, die einige tage andauert. die symptome umfassen kopfund muskelschmerzen, übelkeit, exanthem und generalisierte lymphknotenschwellungen. eher selten kommt es zu einem komplizierten verlauf mit zeichen der meningoenzephalitis. dann ist ein breites spektrum an neurologischen symptomen zu beobachten.über die hälfte der patienten mit enzephalitis leidet an spätfolgen. bei den ausbrüchen in den letzten jahren in usa, rumänien, israel und russland lag die letalität bei den hospitalisierten patienten zwischen 4% und 14%. risikofaktoren für einen tödlichen verlauf sind höheres alter,diabetes mellitus und immunsuppression. bei neurologischen erkrankungen von personen, die sich in den letzten 14 tagen in endemiegebieten aufhielten, sollte unter berücksichtigung der jahreszeit und der aktuellen epidemiologischen situation differenzialdiagnostisch auch an eine wnv-infektion gedacht werden. die diagnose einer wnv-infektion kann durch den direkten virusnachweis (z. b. pcr) oder auch serologisch erfolgen (nachweis wnv-spezifischer igm-antikörper, mindestens 4facher titeranstieg zwischen akutphase-serum und rekonvaleszenten-serum; [14] ). mögliche kreuzreaktionen mit anderen flaviviren müssen berücksichtigt werden (dengue, gelbfieber, fsme, japanische enzephalitis, st.-louis-enzephalitis). dabei spielt auch die gezielte reiseanamnese eine wichtige rolle. zurzeit werden in deutschland epidemiologische untersuchungen (surveys bei vögeln, pferden, ausgewählten humanen populationen) zum vorkommen von wnv bzw.zur prävalenz von wnv-antikörpern durchgeführt, um das gefährdungspotenzial für die bevölkerung besser einschätzen zu können. estimated risk of west nile transmission through blood transfusion during an epidemic in queens identification of a novel coronavirus in patients with severe acute respiratory syndrome ausbruch von aviäre influenza durch influenzavirus a/h7n7 in den niederlanden increase in imported dengue isolation and characterization of viruses related to the sars coronavirus from animals in southern china dengue and dengue hemorrhagic fever avian influenza a (h5n1) in 10 patients in vietnam west nile fever -a reemerging mosquito-borne viral disease in transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands sars-surveillance -wurde sie den anforderungen an die surveillance neu auftretender infektionskrankheiten gerecht? prevalence of infection with dengue virus among international travellers transmission of the severe acute respiratory syndrome on aircraft west-nil-virus: prävalenz und bedeutung als zoonose-erreger the severe acute respiratory syndrome e (1878) epizoozia tifoide nei gallinacei west nile virus sars: retrospective cohort study among german guests of the hotel ‚m' , hongkong arbeitsaufwand des ögd im zusammenhang mit sars -ergebnisse einer befragung zur seroprävalenz von krankenhaus und praxispersonal nach der betreuung von sars-patienten zur übertragung von sars in verkehrsflugzeugen -erfahrungen aus deutschland are we ready for pandemic influenza consensus document on the epidemiology of severe acute respiratory syndrome (sars) avian influenza a (h5n1) da die übertragung überwiegend durch nachtaktive mückenarten erfolgt, ist bei aufenthalten in gebieten mit bekannter wnv-aktivität eine expositionsprophylaxe erforderlich. key: cord-264986-glm2qcuz authors: tam, cheuk chi; sun, shufang; yang, xueying; li, xiaoming; zhou, yuejiao; shen, zhiyong title: psychological distress among hiv healthcare providers during the covid-19 pandemic in china: mediating roles of institutional support and resilience date: 2020-10-21 journal: aids behav doi: 10.1007/s10461-020-03068-w sha: doc_id: 264986 cord_uid: glm2qcuz psychological distress among healthcare providers is concerning during covid-19 pandemic due to extreme stress at healthcare facilities, including hiv clinics in china. the socioecological model suggests that psychological distress could be influenced by multi-level factors. however, limited covid-19 research examined the mechanisms of psychological distress among hiv healthcare providers. this study examined organizational and intrapersonal factors contributing to psychological health during covid-19 pandemic. data were collected via online anonymous surveys from 1029 hiv healthcare providers in guangxi, china during april–may 2020. path analysis was utilized to test a mediation model among covid-19 stressors, institutional support, resilience, and psychological distress (phq-4). thirty-eight percent of the providers experienced psychological distress (phq-4 score > 3). institutional support and resilience mediated the relationship between covid-19 stressors and psychological distress. psychological distress was common among chinese hiv healthcare providers during covid-19 pandemic. psychological health intervention should attend to institutional support and resilience. since the first case emerged in december 2019 in china, the novel coronavirus disease 2019 (covid-19) has spread rapidly across the world and become a worldwide public health emergency [1] . the covid-19 pandemic has led to numerous detrimental consequences, including fatalities and significant socio-economic impacts (e.g., significant medical costs, increased unemployment and financial stress) [2, 3] . in addition to medical and economic consequences, growing literature suggests prevalent psychological distress (e.g., depression and anxiety) during the covid-19 pandemic [4] [5] [6] . healthcare providers may be particularly vulnerable to psychological distress due to a variety of factors. relative to non-medical workers, healthcare providers encounter greater covid-19 threats (e.g., the high risk of infectious exposure, shortage of personal protection equipment, and increasing workloads) detrimental to their psychological health [7, 8] . a recent meta-analysis on 10 covid-19 studies has identified a high prevalence of psychological distress among healthcare providers (23.8% depression, 22.8% anxiety) [9] . psychological distress in healthcare providers during covid-19 have been associated with physical problems, including insomnia and somatic symptoms [10, 11] . however, existing covid-19 studies regarding psychological distress among healthcare providers mostly focused on nurses or surgical staffs [12, 13] , and limited research have focused on other providers. psychological distress among hiv healthcare providers in china could be particularly concerning due to several covid-19 related stressors at hiv clinics. first, as the first country to experience this novel infectious disease, the chinese medical system lacked initial preparedness for the immediate transitions necessary at hiv healthcare services (e.g., in-person to internet-based medical care) [14] . second, medical management system in china is relatively centralized, and hiv patients usually receive hiv care at designated clinics. thus, city lockdowns and quarantine enforcement during the pandemic could lead to massive hiv care disruptions [15, 16] , which may adversely affect providers' psychological health. third, due to hiv providers' background in infectious disease and a shortage of providers on the frontline, many hiv healthcare providers were assigned to provide covid-19 care and control [15] , leading to increased workload. however, despite multiple stressful burdens at hiv clinics during the covid-19 pandemic, no studies have yet attended to the influences of these stressors on psychological distress in chinese hiv healthcare providers. from an ecological perspective, the psychological impact of covid-19 stress among hiv healthcare providers may be shaped by factors from different levels. the ecological systems theory posits that individuals' health outcomes are determined by factors of multi-level systems, such as microsystem (e.g., intrapersonal), exosystem (e.g., organizational), and macrosystem (e.g., contextual) [17] . in addition to highlighting systematic structure of factors, this theory illustrates the underlying mechanism of multi-level factors on individual's psychological outcomes, suggesting that factors from distal levels (e.g., contextual factors) can indirectly impact psychological outcomes through affecting factors from proximal levels (e.g., organizational and intrapersonal) [18] . in other words, distal factors would first affect proximal factors, and, in turn result in psychological consequences for the individual. thus, covid-19 stressors as a contextual factor may indirectly affect psychological distress through organizational and intrapersonal factors among chinese hiv healthcare providers. to better understand the stress-psychological-distress mechanism among chinese hiv healthcare providers, it is worth identifying organizational and intrapersonal factors contributing to psychological health in the covid-19 context. institutional support has been consistently identified as an organizational factor that influences psychological distress in healthcare providers. institutional support can be understood as supportive responses offered by institutes for addressing physical, emotional, and psychosocial needs of the workforce [19, 20] . systematic reviews of literature among healthcare providers documented that institutional support (e.g., organizational climates and infrastructure of health facilitates) was associated with psychological health [21, 22] . in addition to the direct effect, as suggested in the ecological systems theory, previous studies identified an indirect effect of institutional support, such that individuals with a lower occupational stress perceived more institutional support, and, in turn, individuals reported a lower psychological distress [23, 24] . it is particularly worth noting that institutes play a critical role in providing support for healthcare providers during other similar public health crisis (e.g., influenza pandemic) [25] . in the context of covid-19, a focus group among 69 frontline healthcare providers during the outbreak highlighted the lack of institutional supportive responses to covid-19 as a direct source of distress (e.g., 'uncertainty if their organization would take care of their personal and family needs if they become infected by covid-19'), suggesting an importance of institutional support in this unprecedented public health emergency [19] . taken all together, institutional support (i.e., institutional supportive responses to covid-19) could be an important organizational-level factor to directly and indirectly affect psychological distress among hiv healthcare providers in face of covid-19 stressors. as an intrapersonal factor, psychological resilience (or resilience) refers to personal abilities and resources (e.g., optimism, tenacity, tolerance to stress, and self-efficacy) and is a protective factor for coping with or overcoming stressful circumstances [26, 27] . for instance, a meta-analysis on 60 studies found resilience to be a protective factor for psychological distress across diverse samples [28] . similar to institutional support, resilience could have an indirect effect in the association between pandemic stressors and psychological distress [29] [30] [31] . specific to the pandemic context, a recent study found a negative association of resilience with psychological distress among chinese healthcare providers during the covid-19 outbreak [32] . further, as a higher-level factor (exosystem), institutional support could affect resilience (at microsystem), and, in turn, influence psychological outcomes. indeed, a previous study have shown an indirect effect of resilience in the relationship between institutional support and depression among individuals with high occupational stress (e.g., frontline correctional officers) [33] . in line with the institutional support influences on resilience, covid-19 stressors may exert influences on psychological distress by the intermediating effects of institutional support and resilience. that is, the stress-psychological distress mechanism among hiv healthcare providers would occur through a serial association, including a link between covid-19 stressors and institutional support and a link between institutional support and resilience, followed by a link between resilience and psychological distress. understanding this mechanism among hiv healthcare providers in china is important, as it can potentially guide timely and needed interventions and contribute to scientific knowledge on the utility of an ecological perspective in psychological health during extreme stress. the aim of the present study was to investigate the underlying mechanisms among covid-19 stressors, institutional support, resilience, and psychological distress among hiv healthcare providers in china. figure 1 depicts our research hypotheses in the form of a serial indirect model, guided by the ecological systems theory [17] , specifically, we hypothesized that (1) covid-19 stressors would be positively associated with psychological distress; (2) institutional support would mediate the association between covid-19 stressors and psychological distress; (3) resilience would mediate the association between covid-19 stressors and psychological distress; (4) the association between covid-19 stressors and psychological distress would be serially mediated by institutional support and resilience. the present cross-sectional study used data collected by a online survey from a convenience sample of healthcare providers who engaged in hiv care services in guangxi, one of the chinese regions with the fastest growth of the hiv epidemic. since 2014, guangxi reported the third highest number of hiv cases among china's 31 provinces [34, 35] . data were collected from april 2020 through may 2020, a time when the confirmed covid-19 cases exceed 82,000 in china according to chinese center of disease control and prevention (cdc) [36] . the so jump system technology, a popular chinese online survey platform [37] , was used for data collection. eligibility criteria for participants included: (1) currently providing hiv-related care and services in guangxi; and (2) being 18 years of age or older. personnel in guangxi cdc contacted hiv healthcare providers across the region by an email invitation to the 15-min online survey. a total of 1280 hiv healthcare providers from 14 counties (see table 1 ) in guangxi responded the invitations and participated in the survey. participants were provided with an electronic consent prior to the survey started. the consent showed information regarding study purpose, voluntary nature, and confidentiality. after obtaining consent, the so jump system navigated participants to the online survey. data were saved at the so jump system with the password protection. participants were encouraged to share the survey invitation with their colleagues. the research protocol was approved by the institutional review boards at both university of south carolina in the united states and guangxi cdc in china. participants provided their demographic (i.e., age, gender, residence, educational level, and marital status) and occupational information including professional position (i.e., nurse, lab personnel, cdc staff, physician, and other), professional ranking (i.e., no ranking, entry level, middle level, senior level, and advanced level), administrative ranking (i.e., no ranking, department leader, hospital/cdc director, and other), and level of affiliated institution (i.e., province, city, country, and rural). psychological distress was measured through the patient health questionnaire-4 (phq-4; chinese version) [38, 39] . the phq-4 had four items asking mood disorder symptoms (two items for depression and other two items for anxiety) in the past two weeks. all items were rated on a four-point scale ranging from 0 (not at all) to 3 (nearly every day). given that the depressive and anxiety symptoms were highly correlated (r = 0.71), a sum score of all four items was used to represent psychological distress, with higher scores indicating higher levels of psychological distress. in addition, we divided responses into four psychological distress levels (i.e., normal [0-2], mild [3] [4] [5] , moderate [6] [7] [8] , and serve [9] [10] [11] [12] ) according to the diagnosis guideline suggested by kroneke et al. [38] . the cronbach's alpha of the phq-4 was 0.86 in the current study. a self-developed and 20-item checklist was used to evaluate the covid-19 stressors. participants were asked to answer (0 = 'no'; 1 = 'yes') whether 20 potential stressful events occurred at the hospital or hiv clinics during the covid-19 outbreak, such as care of covid-19 patients (e.g., "treated/ provided care for patients infected by covid-19"), occupational overload (e.g., "hospital was overloaded due to the covid-19 outbreak"), difficulties in the delivery of healthcare (e.g., "inadequate personal equipment for covid-19 prevention"), and daily life disturbance (e.g., "guilt because i was unable to take care of my family during the outbreak"). a sum score was calculated, with a higher score representing the exposure to more covid-19 stressors. cronbach's alpha was 0.78 in the current sample. institutional support was measured through a self-developed scale with five items describing supportive responses (i.e., financial, emotional, medical, and technical) from participants' institutes to cope with difficulties at clinics due to covid-19 (e.g., "my institute offers trainings about the provision of hiv care during the covid-19 outbreak" and "my institutes provide sufficient personal preventive equipment"). participants rated items on a four-option scale (1 = strongly disagree to 4 = strongly agree). a sum score was generated, with higher scores presenting higher levels of institutional support. cronbach's alpha of this scale was 0.82. psychological resilience among hiv healthcare providers was assessed using a measure adapted from the connor-davidson resilience scale (cd-risd) [40] . a total of nine items assessed personal capacities in response to stress such as tenacity, tolerance of negative affect, positive acceptance of change, self-efficacy to deal with stress, and positive view of adversities (e.g., "able to adapt to change" and "can deal with whatever comes"). participants were asked to rate items from 1 (not at all) to 5 (nearly all the time). a higher sum score indicated a greater level of resilience. internal reliability for the current study sample was excellent (α = 0.93). first, data were screened for proper coding and random or careless responses. mahalanobis distance (d 2 ) [41] was utilized to identify multivariate outliers. considering that path analysis model can be strongly jeopardized by multivariate outliers [42] , we followed the suggested best practice and removed 251 surveys with multivariate outliers and random responses, resulting in an analytical sample of 1029 participants. kurtosis and skewness tests were used to detect normality of data. descriptive statistics were performed on demographic variables and psychological distress. bivariate analyses, pearson's correlation coefficients, were used to assess correlations among study variables (covid-19 stressors, institute support, resilience, and psychological distress). pearson's (for continuous variables) and point-biserial correlation tests (for categorical variables) were used to examine the bivariate relationship of demographic variables and psychological distress. descriptive statistics and bivariate analyses were employed using spss software version 26. path analysis was employed to examine the hypothesized model (fig. 1) . this model adjusted for significant demographic variables in bivariate analyses. all study variables were entered as manifest factors and the standardized regression weights for all paths between study variables were calculated. given that endogenous variables (psychological distress, institutional social support, and resilience) were continuous and normally distributed (kurtosis and skewness estimates closed to 1) [43] , the path analysis model was tested using maximum likelihood (ml) estimation as suggested by meyers et al. [41] . model's goodness of fit was determined by several indices, including the root mean square of approximation (rmsea), the comparative fit of index (cfi), the tucker-lewis index (tli), and the standardized root mean squared residual (smsr) [41] . according to hu an bentler [44] , the suggested cutoff values were 0.95 for cfi, 0.95 for tli, 0.05 for rmsea, and 0.06 for smsr, with higher cfi and tli and lower rmsea and smsr indicating a greater fit. the indirect effects of institute support and resilience in the relationship between covid-19 stressors and psychological distress were examined using the delta z score [45] . the indirect effect analysis examined whether institutional support and resilience partially or completely mediated the relationship between covid-19 stressors and psychological distress. as suggested by baron and kenny [46] , complete mediation is identified when a mediation factor reduces the regression coefficients between independent variable (covid-19 stressors) and criterion variable (psychological distress) to zero (non-significant). partial mediation occurs when a mediation factor reduces the regression coefficient between independent and criterion variables, but the regression coefficient remains significant. path analysis was performed in mplus version 8 [45] . as shown in table 1 as presented in table 2 , pearson's correlation tests suggested that covid-19 stressors was positively correlated table 2 correlations among covid-19 stressors, resilience, institute support, and psychological distress (n = 1029) *p < .05; **p < .01; ***p < .001 with psychological distress (r = 0.33, p < 0.001) and negatively correlated with resilience (r = − 0.13, p < 0.001) and institutional support (r = − 0.11, p < 0.001). psychological distress was negatively correlated with resilience (r = − 0.43, p < 0.001) and institutional support (r = − 0.21, p < 0.001). resilience was positively correlated with institutional support (r = 0.28, p < 0.001). pearson's and point-biserial correlation tests suggested that psychological distress was significantly correlated with age (r = − 0.11, p = 0.001), gender (r = 0.09, p = 0.006), marital status (r = − 0.16, p < 0.001), and affiliated institute level (r = 0.12, p < 0.001). professional position was not significantly correlated with psychological distress (r = 0.02, p = 0.465). the final path analysis model with standardized regression coefficients is shown in fig. 2 . according to bivariate results, this model controlled for age, gender, marital status, and affiliated institute level. the chi-square test of the model (chi-square = 11.06, df = 7, p = 0.136) and the model-fit indices (cfi = 0.99; tli = 0.98, rmsea = 0.02, and srmr = 0.02) suggested an excellent fit to data. covid-19 stressors were positively associated with psychological distress (β = 0.27, p < 0.001). the paths from covid-19 stressors to institutional support and resilience were negative (β = − 0.10, p = 0.001; β = − 0.10, p = 0.001; respectively). institutional support and resilience were negatively associated with psychological distress (β = − 0.07, p = 0.01; β = − 0.36, p < 0.001; respectively). institutional support was positively associated with resilience (β = 0.27, p < 0.001). the model explained 28.5% of variance in psychological distress, 12.4% of variance in resilience, and 2.4% of variance in institutional support. the indirect effects in the hypothesized model were presented in table 3 . as hypothesized, covid-19 stressors had a direct effect on psychological distress (β = 0.27, delta z = 9.24, p < 0.001, 95% confidence interval [ci] 0.22, 0.31). results suggested significant indirect effects (β = 0.05, p < 0.001, 95% ci 0.03, 0.07) in the association between covid-19 stressors and psychological distress by resilience and institutional support were significant (β = 0.03, delta z = 3.08, p < 0.001, 95% ci 0.02, 0.05; β = 0.01, delta z = 1.97, p = 0.042, 95% ci 0.002, 0.01; respectively). in addition, the total indirect effect of institutional support together with resilience was statistically significant (β = 0.01, delta z = 3.12, p = 0.003, 95% ci 0.01, 0.02). these results suggested partial mediations of institutional support and resilience (solely or jointly) in the association between covid-19 stressors and psychological distress. in the relationship between institutional support and psychological distress, results suggested a direct effect of institutional support (β = − 0.07, delta z = − 2.54, p = 0.011, 95% ci − 0.12, − 0.03) and an indirect effect of resilience (β = − 0.10, delta z = − 7.43, p < 0.001, 95% ci − 0.12, − 0.08), indicating that resilience partially mediated the association between institutional support and psychological distress. guided by an ecological perspective, the current study explored psychological distress among chinese hiv healthcare providers and examined the roles of covid-19 stressors, institutional support, and resilience in psychological distress. to the best of our knowledge, this is the first attempt to document psychological distress and apply an ecological framework for examining the effects of protective factors on our study found 38.7% (33.4% mild, 5.4% moderate, 0.3% severe) chinese hiv healthcare providers exhibited psychological distress during the covid-19 pandemic, which was similar to yet higher than the findings in a previous meta-analysis study (pooled prevalence of 23.2% for anxiety and 22.8% for depression) in chinese healthcare providers [9] . our findings indicated that during the covid-19 pandemic, hiv healthcare providers, similar to nurses and surgical staffs, were highly vulnerable to psychological distress. their psychological health should not be neglected and merits more attention. our serial indirect model plotted a stress influences on psychological distress among chinese hiv healthcare providers in response to the emergent pandemic. results suggested that such stress influences occurred through two intermediating factors, namely institutional support (at organizational level) and resilience (at intrapersonal level). this finding provided a novel insight regarding covid-19 stress management at hiv clinics, particularly on the importance of institutional support. previous studies showed that healthcare providers in supportive working environments were inclined to exhibit greater resilience-related abilities, including emotional regulation [47] , self-efficacy [48] , and adaptive coping strategies [49] . in facing a public health emergency, sufficient institutional support might enable hiv healthcare providers to use their internal coping recourses (i.e., resilience) and facilitate providers' adaptive coping instead of being overwhelmed to deal with covid-19 stressors, resulting in reduced levels of distress. this implied that psychological health among hiv healthcare providers could benefit from effective institute responses to covid-19, such as covid-19 training and guidelines, sufficient supplies of personal protection equipment, and cares of workers' personal and family needs [19, 50] . in addition to institutional support, the protective and mediating role of resilience also highlighted the effects of personal coping abilities on psychological health in chinese hiv healthcare providers during the covid-19 pandemic. in face of the extreme pandemic stress, the availability of cognitive resources (e.g., self-efficacy) might allow hiv healthcare providers to reappraise stressors as controllable or manageable instead of threatening, resulting in reduced levels of perceived stress and a lower likelihood of emotional exhaustion [51] . the role of resilience supports the value of providing resilience-enhancing interventions for hiv healthcare providers during the covid-19 pandemic. for example, group-based resilience-enhancing intervention programs have shown psychological benefits to healthcare providers [52] . after resilience-based cognitive trainings (e.g., selfawareness and meditation practice) at workplaces, healthcare providers demonstrated significant improvements in resilience, perceived stress, and interpersonal abilities (e.g., empathy and understanding of peers) comparing to those in control group [53, 54] . it may be worthwhile to adapt the group-based resilience-enhancing intervention and tailor it to the covid-19 context for hiv healthcare providers in china. there are several limitations in the current study. first, the participants were recruited online using a convenience sampling approach. although the current study has covered 14 counties in guangxi, our findings may not be generalized to other regions in china and may be limited by only reaching participants who are familiar with web-based devices. future research would benefit from using a random sampling approach and recruiting participants from multiple chinese regions. second, given cross-sectional data, the indirect model from covid-19 stressors to psychological distress cannot draw conclusion about causality. future longitudinal research is needed to ascertain the directionality of relationships identified in this study. third, self-report data were subject to bias (e.g., social desirability). fourth, some measures were self-developed (covid-19 stressors and institutional support) and future research is needed to validate these measures. fifth, data were collected in a certain period of the covid-19 outbreak. our results may not be replicated as the pandemic evolves and hiv healthcare providers' experience changes. future research would benefit from collecting data across different phases of the pandemic. despite these limitations, as the first attempt to examine psychological distress and its related factors among chinese hiv healthcare providers during the covid-19 pandemic, our study offered some important insights. first, the current study expanded the knowledge of healthcare providers' psychological health during the covid-19 pandemic by attending to hiv healthcare providers. psychological distress was prevalent (38.7%) among hiv healthcare providers during the covid-19 pandemic in china. second, by using an ecological perspective, we identified that institutional support (as interpersonal factor) and resilience (as intrapersonal factor) mitigated the influences of covid-19 stress on psychological distress among hiv healthcare providers in china. our findings implied that multi-level intervention approaches, such as the establishment of supportive environment at hiv clinics and the resilience-enhancing intervention, should be utilized for promoting psychological health among hiv healthcare providers in china. world health organization. novel coronavirus (2019-ncov) situation report b the potential health care costs and resource use associated with covid-19 in the united states: a simulation estimate of the direct medical costs and health care resource use associated with covid-19 infections in the united states labor markets during the covid-19 crisis: a preliminary view covid-19, unemployment, and suicide suicide risk and prevention during the covid-19 pandemic mental health and the covid-19 pandemic burnout among healthcare providers during covid-19 pandemic: challenges and evidence-based interventions grief during the covid-19 pandemic: considerations for palliative care providers prevalence of depression, anxiety, and insomnia among healthcare workers during the covid-19 pandemic: a systematic review and meta-analysis a multinational, multicentre study on the psychological outcomes and associated physical symptoms amongst healthcare workers during covid-19 outbreak generalized anxiety disorder, depressive symptoms and sleep quality during covid-19 outbreak in china: a web-based cross-sectional survey psychological status of surgical staff during the covid-19 outbreak vicarious traumatization in the general public, members, and non-members of medical teams aiding in covid-19 control covid-19, telemedicine, and patient empowerment in hiv care and research challenges to hiv care and psychological health during the covid-19 pandemic among people living with hiv in china provider burnout and fatigue during the covid-19 pandemic: lessons learned from a high-volume intensive care unit ecological models of human development still misused after all these years? a reevaluation of the uses of bronfenbrenner's bioecological theory of human development understanding and addressing sources of anxiety among health care professionals during the covid-19 pandemic developing and testing a measure of covid-19 organizational support of healthcare workers-results from peru, ecuador, and bolivia prevalence and factors associated with burnout among frontline primary health care providers in low-and middle-income countries: a systematic review organizational climate and employee mental health outcomes: a systematic review of studies in health care organizations the relationship between work-stress, psychological stress and staff health and work outcomes in office workers work-family conflict, perceived organizational support and professional commitment: a mediation mechanism for chinese project professionals local public health workers' perceptions toward responding to an influenza pandemic development of a new resilience scale: the connor-davidson resilience scale (cd-risc). depress anxiety vulnerability and competence: a review of research on resilience in childhood a meta-analysis of the trait resilience and mental health. pers individ diff risk and resilience in family wellbeing during the covid-19 pandemic association between perceived hiv stigma, social support, resilience, self-esteem, and depressive symptoms among hiv-positive men who have sex with men (msm) in nanjing social capital and ptsd among plwha in china: the mediating role of resilience and internalized stigma a cross-sectional study on mental health among health care workers during the outbreak of corona virus disease positive resources for combating depressive symptoms among chinese male correctional officers: perceived organizational support and psychological capital declining trend in hiv new infections in guangxi, china: insights from linking reported hiv/ aids cases with cd4-at-diagnosis data public mental health crisis during covid-19 pandemic up-to-day estimates of the covid-19 outbreak in china investigation on the status of influencing factors for depression symptom of children and adolescents with home quarantine during the prevalence of novel coronavirus pneumonia an ultra-brief screening scale for anxiety and depression: the phq-4. psychosomatics reliability and validity of the chinese version of the patient health questionnaire (phq-9) in the general population psychometric analysis and refinement of the connor-davidson resilience scale (cd-risc): validation of a 10-item measure of resilience applied multivariate research: design and interpretation. thousand oaks: sage publications principles and practice of structural equation modeling statistical notes for clinical researchers: assessing normal distribution (2) using skewness and kurtosis. restor dent endod cutoff criteria for fit indexes in covariance structure analysis: conventional criteria versus new alternatives mplus user's guide. 8th ed. los angeles: muthén & muthén the moderator-mediator variable distinction in social psychological research: conceptual, strategic, and statistical considerations sexual minority status and psychological risk for suicide attempt: a serial multiple mediation model of social support and emotion regulation. front psychiatry cultivating secondary traumatic growth among healthcare workers: the role of social support and self-efficacy aggression and violence against health care workers in germany-a cross sectional retrospective survey factors contributing to healthcare professional burnout during the covid-19 pandemic: a rapid turnaround global survey handbook health, health care, health professional the impact of mindfulness-based interventions on doctors' well-being and performance: a systematic review mindfulness-based stress reduction for residents: a randomized controlled trial stress management and resilience training among department of medicine faculty: a pilot randomized clinical trial acknowledgements we appreciated ms. shuaifeng liu for her assistance in irb application and project coordination. we also greatly thank all the hiv care providers who finished the online survey. research reported in this publication was supported by the national institute of allergy and infectious diseases of the national institutes of health under award number r01ai127203-4s1 and the university of south carolina office of vice president for research covid-19 grant (uscip 80003673). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. work by shufang sun was also supported by national institute of health (t32mh078788 and k23at011173). conflict of interest the authors have no conflicts of interest to report. key: cord-255697-trig04hd authors: cheng, vincent chi-chung; chan, jasper fuk-woo; hung, ivan fan-ngai; yuen, kwok-yung title: viral infections, an overview with a focus on prevention of transmission date: 2016-10-24 journal: international encyclopedia of public health doi: 10.1016/b978-0-12-803678-5.00514-2 sha: doc_id: 255697 cord_uid: trig04hd viruses are obligatory intracellular pathogens with a simple structure consisting of assembled proteins enclosing the nucleic acid genome with or without a lipid envelope. despite increasing availability of rapid nucleic acid amplification assays for laboratory diagnosis, effective antivirals, and safe vaccines, the control of most viral infections depends on time-honored surveillance and infection control measures. moreover most viruses can be readily destroyed by common disinfectants. this article is focused on the epidemiology, diagnosis, and control of common and emerging viral diseases. traditionally, the epidemiological control of most viral infections depends on the isolation of cases, quarantine of contacts, personal protection by infection control measures and mass vaccination, because specific antiviral treatment is generally not available for most viral infections ( table 1) . this scenario is rapidly changing with the increasing availability of rapid diagnostic tests which use nucleic acid amplification, and the development of an increasing number of effective antiviral agents. common acute viral diseases such as respiratory, diarrheal, exanthematous, or neurological infections can overlap with each other and appear as seasonal epidemics, which peak in incidence every few years and coincide with the accumulation of sufficient number of nonimmune hosts in the young population. arboviral disease activity often coincides with arthropod vector activity such as mosquito breeding in hot rainy seasons which are associated with increased incidence of hemorrhagic fever or neurological diseases such as dengue hemorrhagic fever, west nile virus, or japanese encephalitis in southeast asia. many chronic blood-borne viral illnesses such as human immunodeficiency virus (hiv), hepatitis b virus (hbv), and hepatitis c virus (hcv) are still taking a major toll in certain geographical regions due to specific human behaviors or vertical transmission. some of these chronic viral infections such as hbv, hcv, hiv, polyomaviruses, and papillomaviruses are also linked to the genesis of cancers. over 70% of emerging viral infections such as the ebola virus, severe acute respiratory syndrome (sars) coronavirus, and middle east respiratory syndrome coronavirus (mers-cov) are associated with acute explosive outbreaks after the virus jumped the species barrier from bats or other animals into humans ( table 2) . this article will focus on the prevention and control of viral infections, while other articles in this encyclopedia will cover information on specific viruses. unlike bacteria, fungi, and parasites, viruses are too small to be visible under light microscopy. moreover, viruses are obligate intracellular pathogens and do not grow in artificial culture medium. collecting the correct clinical specimens during the peak of viral shedding in appropriate viral transport medium is crucial for accurate diagnosis. electron microscopy is not sensitive and has a limited role in the examination of feces from viral gastroenteritis and vesicular fluid from skin lesions caused by herpesviruses and poxviruses. virus isolation in cell lines or chick embryo is the gold standard for virological diagnosis but seldom alters clinical management due to its long turnaround time. viral antigen detection by immunofluorescence, enzyme immunoassay, and point-of-care rapid lateral flow immunochromatographic assays has significant impact on therapeutic and infection control strategies. the most important rapid virological tests are nucleic acid amplification tests such as real-time or multiplex reverse transcription-polymerase chain reaction (rt-pcr) assays that are useful for accurate diagnosis and subsequent viral load monitoring during antiviral treatment. genotyping by nucleic acid amplification and sequencing for detection of mutations associated with antiviral resistance directly from clinical specimens are now available for many antiviral agents and are routine for antiretroviral drugs used to treat hiv infection. though nucleic acid amplification tests still have practical limitations in the field settings of developing areas, such tests are now routine in most hospitals in developed countries. antibody testing by enzyme immunoassay for igm in acute infection, igg for immune status of exposed individuals, and retrospective diagnosis by the presence of rising antibody titers in paired acute and convalescent sera of symptomatic patients is useful for making clinical and epidemiological decisions. antibody screening is especially important in antenatal visits of expectant mothers, blood donors, and organ donors and recipients before transplantation. next-generation sequencing performed directly on clinical specimens may revolutionize virological diagnosis in the coming decade. the timely and accurate diagnosis of viral infections has important implications for effective epidemiological control in the community and infection control for hospital outbreaks. transmission of blood-borne viruses can result from sexual intercourse and maternal-fetal transmission in the community setting, needle stick injury, and other exposure-prone procedures in the health-care setting. in a study from the usa, the annual death rate of health-care workers from occupational events was estimated to be 17-57 per 1 million workers, and most of these deaths resulted from infection-related complications of blood-borne viruses (sepkowitz and eisenberg, 2005) . the overall risk of transmission of blood-borne viruses by hollow needle stick injury is 33%, 3%, and 0.3% if the source is a hepatitis carrier with positive hbe antigen or high viral load, hepatitis c carrier with viremia, and hiv, respectively. compliance with standard precautions including wearing gloves when handling blood during patient care practice, disposing sharp needles into puncture-resistant box, and avoidance of recapping needles remain the most important ways to prevent nosocomial acquisition of blood-borne viruses (garner, 1996) . active immunization for hbv can protect health-care workers from hbv infection with an efficacy of 80-95% (dienstag et al., 1984; sabido et al., 2007; shim et al., 2011) . postimmunization anti-hbs antibody level should be measured at 4-8 weeks after completion of the 3-dose immunization regimen given at baseline, 1 month, and 6 months. a good responder is defined as a person whose anti-hbs antibody level is greater than 100 iu/l. if the hepatitis b antibody level is between 10 and 100 iu/l, a booster dose of vaccine should be given. for nonresponders whose anti-hbs antibody is less than 10 iu/l, another course of hbv vaccine should be given. the response rate is about 61% in repeated hbv vaccination by the same route as the initial vaccination (struve et al., 1994) . alternatively, immunization with high-dose intradermal recombinant hbv vaccine, given in up to four doses, can achieve immunity in 88% of health-care workers who failed to respond to intramuscular vaccination and boosters (levitz et al., 1995) . the anti-hbs titer was persistently higher than the protective level for at least 10 years after primary hbv vaccination (floreani et al., 2004) . when a health-care worker sustains a needle stick injury, he/ she should be advised to rinse the wound with tap water and allow natural bleeding. the source patient's blood is collected to check for the presence of hiv, hbv, and hcv. if the status of blood-borne viruses of the source patient is positive or unknown, postexposure prophylaxis (pep) should be offered according to current guidelines (kuhar et al., 2013 ). the exposed health-care worker will be closely followed up for counseling, baseline and follow-up hiv testing, and monitoring for drug toxicity. if a newer fourth-generation combination hiv p24 antigen-hiv antibody test is utilized for follow-up hiv testing, it may be concluded 4 months after exposure. otherwise, follow-up hiv testing is performed 6 months after the exposure (kuhar et al., 2013) . for hbv, pep with hepatitis b immune globulin (hbig) and/or hbv vaccination should be considered for occupational exposures after evaluation of the hbsag status of the source, and the vaccination and vaccine-response status of the exposed person (2001). for hcv, pep is not currently recommended. however, an open-label pilot trial was conducted to determine the safety, tolerability, and acceptance of peginterferon alfa-2b as pep. among 213 health-care workers exposed to an hcv antibody-positive source, 51 hcws enrolled in the study and 44 (86%) elected to undergo peginterferon alfa-2b as the study group. seven subjects who elected not to undergo pep were treated as the control group. in this pilot study, peginterferon alfa-2b was proven to be safe without serious adverse rural residents with contact with bats (organ transplantation) dietzschold and koprowski (2004) and kusne and smilack (2005) effects. however, the lack of hcv transmission in both the study and control groups did not support the routine use of pep in health-care workers after hcv exposure (corey et al., 2009) . it is likely that the new polymerase and protease inhibitors used in the treatment of hcv infection will result in new strategies for pep of hcv exposures. blood-borne viruses can also be transmitted from healthcare workers to patients, especially during exposure-prone procedures in dental and cardiothoracic operations. the most well-known example involved an hiv-positive dentist working in florida, usa, who infected five of his patients after performing invasive dental procedures on them . sequencing of the hiv proviral envelope gene showed that the viruses infecting the dentist and the five patients were closely related . however, the overall risk for transmission of hiv from a health-care worker to a patient is very small. in a study conducted by the centers for disease control and prevention (cdc) of 22 171 patients being cared by 51 hiv-positive health-care workers, 113 (0.5%) patients became hiv positive. epidemiologic investigation did not implicate health-care workers as the source of infection in any of these patients (robert et al., 1995) . in contrast, transmission of hbv and hcv from health-care workers to patients was more frequently documented. between august 1991 and july 1992, 19 of 144 (13%) patients who were operated on by a thoracic surgeon with acute hbv infection became hbvinfected. sequencing of 160 bases in the core region of hbv showed an indistinguishable pattern among the strains of the surgeon and nine infected patients (harpaz et al., 1996) . subsequently, numerous health-care worker-to-patient transmissions of hbv and hcv were reported. among all these reported cases, the viral loads of the index health-care workers were more than 10 6 genome copies per ml (buster et al., 2003; gunson et al., 2003) . in this connection, the society for healthcare epidemiology of america (shea) issued a guideline for the management of health-care workers who are infected with hiv, hbv, and hcv to impose restriction on different categories of exposure-prone procedures according to the viral load (henderson et al., 2010) . epidemiologically important respiratory viruses such as influenza a virus are predominantly transmitted by the droplet route. by definition, the virus can spread within 1 m from the index case. however, individuals infected with influenza a virus may produce as many as 40 000 droplets of 0.5-12 mm in size and expel them at a velocity of 100 m s à1 upon sneezing (tang et al., 2006) . droplet nuclei of less than 3 mm may suspend in air and do not settle onto the ground (knight, 1980) . therefore, an explosive outbreak with high clinical attack rate as a result of aerosol transmission may occur under special conditions. in a jet airliner with nonfunctioning ventilation system, 72% of 54 passengers developed influenza-like illness within 72 h after being kept on ground for 3 h due to delay in flight time (moser et al., 1979) . as the virus may survive on inanimate surfaces for 12-48 h, and on the surface of hands for 10-15 min (kampf and kramer, 2004; kramer et al., 2006) , influenza virus can be transmitted indirectly by contact with hands from the contaminated environment to the pharyngeal mucosa. symptomatic influenza may develop after intranasal inoculation of 1 tcid 50 of influenza a virus (tellier, 2006) . hand hygiene is always the core component of infection control measures in both community and hospitals to prevent the transmission of influenza a virus. wearing face masks by either the index case as source control or the health-care workers as contacts has shown to be equally effective in the control of nosocomial transmission of pandemic influenza a h1n1 (cheng et al., 2010) . hand hygiene and face masks have been shown to prevent household transmission of influenza virus when implemented within 36 h of the index patient's symptom onset . oseltamivir pep halted an outbreak of pandemic influenza a h1n1 in a secondary school (asiedu-bekoe et al., 2012), but not in nursing homes (van der sande et al., 2014) . prevention of nosocomial transmission of influenza a virus requires multiple actions. early identification of symptomatic cases by direct antigen detection from nasopharyngeal specimens and initiation of droplet precautions by wearing surgical masks, along with staff education, could achieve reductions in nosocomial pandemic influenza to near zero (cheng et al., 2010) , while a similar protocol was also effective in minimizing the risk of nosocomial transmission of avian influenza a/h7n9 virus (cheng et al., 2015) . to ensure hand hygiene compliance, directly observed hand hygiene was adopted to control the spread of respiratory viruses in hospitals (cheng et al., 2010 (cheng et al., , 2007b . alcohol-based hand rub is delivered to every health-care worker and conscious patient once every 2-3 h in the clinical areas, which may further reduce the spread of respiratory viruses. varicella zoster virus (vzv) and measles are predominantly transmitted by aerosols and deposited in distal airways (roy and milton, 2004) . the exact mechanism of airborne transmission remains to be elucidated. however, an early study demonstrated that nosocomial outbreak of vzv occurred even when the index case was strictly isolated in a single room (gustafson et al., 1982) . there was a lack of nosocomial spread of vzv when all index cases were placed in negative pressure airborne infection isolation rooms (anderson et al., 1985) . measles virus can survive in the air for at least 1 h, as shown in an outbreak where three susceptible children who contracted measles were never in the same room with the source patient and one of the three arrived at the office 1 h after the source patient had left (bloch et al., 1985) . a massive community outbreak of measles occurred in a modern suburban elementary school in new york in spring, 1974, when the index case produced 28 secondary cases in 14 different classrooms. the epidemic subsided after two subsequent generations when 60 children had been infected. from estimates of major physical and biologic factors, it was possible to calculate that the index case produced approximately 93 units of airborne infection (quanta) per minute, which was higher than that of patients with laryngeal tuberculosis (riley et al., 1978 (riley et al., , 1962 . early recognition and placement in airborne infection isolation room of index case of vzv and measles may reduce the risk of nosocomial outbreaks. standard and transmission-based precautions are important to prevent the spread of respiratory and gastrointestinal viral infection ( table 3) . some of the respiratory viruses such as respiratory syncytial virus (rsv), parainfluenza virus, and the gastrointestinal viruses, norovirus, and rotavirus are predominantly spread by direct contact. as an illustrative example, rsv is the most frequent cause of nosocomial infection in pediatric wards and causes lower respiratory tract disease in 40% of young children. prolonged shedding of rsv for 3-11 days has been observed in immunocompetent children (hall, 2000) , and the virus can survive on inanimate surfaces for 6 h (kramer et al., 2006) . all these factors contribute to fomite-mediated transmission of rsv in the hospital. the risk of nosocomial rsv transmission was not related to age or underlying disease, but to length of hospitalization (hall et al., 1975) . contact precautions with cohort nursing and wearing gloves and gowns during patient care resulted in a significant reduction in nosocomial transmission of rsv in three consecutive winters (madge et al., 1992) . in another study, the incidence of nosocomial acquisition of rsv was significantly decreased after implementation of wearing gloves and gowns and isolation of cases even though the duration of rsv shedding remained unchanged before and after the intervention (leclair et al., 1987) . for the gastrointestinal viruses, norovirus is the most famous agent to cause outbreaks in the community and hospital. transmission is predominantly by the fecal-oral route. numerous community outbreaks of norovirus have been reported in restaurants, resorts, cruise ships, schools, and nursing homes (arvelo et al., 2012; britton et al., 2014; kuo et al., 2009; lai et al., 2013; wikswo et al., 2011) . the emergence of a new variant of norovirus, genogroup ii, type 4 (gii.4), in australia, europe, and north america associated with increased acute gastroenteritis activity has been reported since 2006 (bruggink and marshall, 2010; hasing et al., 2013; kanerva et al., 2009; yen et al., 2011) . norovirus is a nonenveloped rna virus which is relevantly resistant to common disinfectants. as norovirus is unculturable, feline calicivirus has been used as a surrogate for in vitro and in vivo testing for different preparations of disinfectants (gehrke et al., 2004; lages et al., 2008) . in the who formulation ahr, formula i preparation contains ethanol (80% v/v) which, based on the above studies, may possess reasonable virucidal activity for norovirus when the contact time is prolonged for up to 30 s. successful control of nosocomial outbreaks of norovirus by directly observed hand hygiene has been reported, especially during high-risk nursing care practices such as changing napkins and feeding . a proactive infection control approach with the provision of 'added test' was implemented to prevent the occurrence of nosocomial outbreak when the new variant of norovirus, table 3 infection control measures for transmission-based precautions in resource-poor areas (cheng et al., 2011) . rt-pcr for norovirus was performed as an 'added test' by the microbiology laboratory for all fecal specimens that were requested for bacterial culture, clostridium difficile culture or cytotoxin, and rotavirus antigen detection without a request for norovirus detection. during the study period, almost 50% of newly diagnosed norovirus infections were detected by the added test. timely implementation of infection control measures by single room isolation of index case with strict contact precautions significantly reduced the incidence of hospital-acquired norovirus infection from 131 (baseline) to 16 cases per 1000 potentially infectious patient-days (p < 0.001) (cheng et al., 2011) . disease outbreak such as sars, pandemic influenza, and ebola the outbreak of sars in 2003 was the first emergence of an important human pathogen in the twenty-first century. sars emerged as an outbreak of atypical acute community-acquired pneumonia in late 2002. the epidemic may have started when a bat sars coronavirus jumped into caged palm civets in a wildlife market and became adapted and amplified to jump from civet to human. infected chefs and animal handlers transmitted the adapted virus to health-care workers and then the epidemic became amplified into the community. the epidemic was rapidly and globally disseminated when a 'super-spreader' of sars, who was a medical professor from a teaching hospital in guangzhou, went to hong kong on 21 february 2003. during his stay in hotel m, he transmitted sars-cov to other residents, and the secondary cases spread the disease to hospitalized patients in hong kong, and to other countries including vietnam, singapore, and canada. eventually, a total of 8096 patients were infected in over 30 countries among five continents and 774 (9.5%) of them died (cheng et al., 2007a) . nosocomial outbreaks were reported in many parts of the world including toronto, hong kong, guangzhou, kaohsiung, singapore, and vietnam during the sars epidemic. there were a total of 716 secondary and tertiary cases of sars as a result of the admission of infected index patients. health-care workers constituted 410 (52.3%) of the secondary and tertiary cases (cheng et al., 2013) . as there were no known effective antiviral agents or vaccine for the treatment and prevention of sars, infection control measures and extensive tracing to quarantine the contact person became the most important interventions for sars control. the longitudinal follow-up of sars patients revealed that the viral load gradually increased on day 5 after symptom onset and peaked at day 10. early isolation of source patients can prevent ongoing transmission of sars in the community. in hospitals, temporary suspension of clinical services in both inpatient and outpatient settings was adopted (gopalakrishna et al., 2004; liu et al., 2006; nishiura et al., 2005; reynolds et al., 2006) , while home quarantine of health-care workers who had contact with sars patients was also mandated in some centers (dwosh et al., 2003) . provision of personal protective equipment (ppe) such as n95 respirators, gloves, gowns, and goggles, and placement of suspected or confirmed cases of sars in airborne infection isolation rooms were enforced when resources were available. the appropriate use of ppe was also important for staff protection. many health-care workers apparently lacked a clear understanding of how best to remove ppe without contaminating themselves. little information about the appropriate sequence of removing ppe was available at that time (puro and nicastri, 2004) . infection control measures are particularly important for emerging viral infections without effective antiviral therapy and vaccine. recently, the largest outbreak of ebola virus disease (evd) in west africa (guinea, sierra leone, liberia, nigeria, and équateur province of democratic republic of the congo) resulted in a total of 21 724 cases and 8641 deaths as of 21 january 2015. ebola virus is transmitted via contact with contaminated body fluid or the contaminated environment, and therefore the practice of contact precautions with appropriate ppe is of utmost importance when handling suspected or confirmed evd cases. health-care workers should preferably work in pairs so as to mutually observed against lapses in infection control measures. they are required to put on the ppe in the following sequence: n95 respirator, water-repellent cap or hood, fulllength shoe cover or boot, water-resistant gown, face shield, and finally long nitrile gloves. if the patient has hemorrhagic symptoms, double nitrile gloves should be worn. in view of the high virulence and mortality, patients suspected to have evd should be isolated in airborne isolation rooms, although the who allows cohorted nursing in designated areas with dedicated instruments, where access should be restricted in developing countries with limited isolation facilities. degowning remains the most critical procedure for healthcare workers. the most contaminated ppe should be removed first, starting with the long nitrite gloves, water-resistant gown, full-length shoe cover or boot, face shield, water-repellent cap or hood, and finally n95 respirator. hand hygiene with alcohol-based hand rub should be performed in each step of degowning. when the hand is visibly soiled, it should be washed with soap and water. health-care workers must be well trained and audited for the proper procedure of gowning and degowning. when the suspected or confirmed case of evd dies, the health-care and mortuary workers are required to wear ppe as described above. the dead body is placed in double bags with leak-proof characteristic of no less than 150 mm thick. absorbent material should be put under the body and placed in the first bag. the surface of each body bag is wiped with 10 000 ppm sodium hypochlorite solution. the bags are sealed and labeled with the indication of highly infectious material (category 3) and moved to the mortuary immediately. viewing in funeral parlor, embalming and hygienic preparation are not allowed. the dead body should not be removed from the body bag and should be sent to cremation as soon as possible. in august 2014, who declared the evd outbreak in west africa a public health emergency of international concern. preparedness and response plans were made available by health authorities in nearly all countries worldwide. the aim was to detect the first imported case for early isolation in order to prevent local transmission in the community and healthcare settings. therefore, risk assessment at ports, emergency rooms, and outpatient clinics for any patient fulfilling both clinical and epidemiological criteria for evd is important. for the clinical definition, patient suffering from elevated body temperature or subjective fever or symptoms including severe headache, fatigue, muscle pain, vomiting, diarrhea, abdominal pain, or unexplained hemorrhage should be alerted, while the epidemiological definition includes close contact with a confirmed or probable case of evd or residence in or history of travel to an affected area or countries (guinea, liberia, sierra leone) within 21 days before symptom onset. for health-care workers working in volunteer medical services or nongovernment organizations, who have had direct contact with patients in the affected areas or countries, should also perform medical surveillance or be placed in quarantine for at least 21 days after leaving the affected areas or countries. medical evaluation should be sought promptly if there are any symptoms of fever, diarrhea, vomiting, or bleeding during quarantine or medical surveillance. with reference to the experience in the community spread of pandemic influenza a virus infection, nonpharmacological interventions with social distancing, such as school closures, have been evaluated in previous modeling and epidemiological studies (bell et al., 2009; bootsma and ferguson, 2007; ferguson et al., 2006; markel et al., 2007) . during the influenza pandemic in 2009, school closures were practiced in the usa and australia (borse et al., 2011; effler et al., 2010) , because school closures were associated with a 65% reduction in the mean total number of contacts for each student as reported in a retrospective questionnaire survey in the united kingdom (jackson et al., 2011) . in hong kong, kindergartens and primary schools were closed when local transmission of influenza a virus was identified in 2009, followed shortly afterward by secondary school closures for summer vacations. influenza a virus transmission was estimated to be reduced by 25% (wu et al., 2010) . home quarantine was also shown to reduce the incidence of pandemic influenza a in the workplace (miyaki et al., 2011) . in fact, home quarantine has been used to control the community spread of sars in beijing, taiwan, singapore, and toronto (centers for disease control and prevention (cdc), 2003; cava et al., 2005; hsu et al., 2006) . home quarantine can be considered for the control of the spread of ebola virus in affected countries although in resource-limited settings effectively implementing these strategies can be challenging. the local government and health authorities have already implemented home quarantine for 3 days as an urgent infection control measure. however, if it is technically and politically feasible, home quarantine may be extended for up to 21 days (one incubation period) for ebola virus disease. however, public health staff is expected to face unprecedented challenges in implementing an extensive quarantine policy, as they have a dual role of monitoring compliance and providing support to people in quarantine. countries in close proximity to the affected areas require implementing border control measures to screen for any suspected case of ebola virus or even considering closing the border for 21 days. although these measures may adversely affect international travel and local economies, it may be worthwhile to implement such strict measures to control this reemerging infectious disease with high mortality and psychological fear in a timely manner. currently available antivirals against influenza a include the adamantanes (amantadine and rimantadine), neuraminidase inhibitors (oseltamivir, zanamivir, and peramivir) and a pyrazinecarboxamide derivative (favipiravir). only the neuraminidase inhibitors and pyrazinecarboxamide derivatives are active against currently circulating influenza a viruses. oseltamivir and favipiravir are available orally. zanamivir is available either as a dry powder that is delivered by oral inhalation or recently, intravenous formulation is available. peramivir is only available in the intravenous formulation. randomized controlled trials in patients with seasonal influenza suggested that the use of neuraminidase inhibitor can shorten the duration of illness by approximately 1 day. a recent meta-analysis had demonstrated that early neuraminidase inhibitor treatment (within 2 days of symptom onset) was associated with a reduction in mortality (muthuri et al., 2014) . two prospective clinical trials have demonstrated that treatment with convalescent plasma or hyperimmune intravenous immunoglobulin for patients with severe influenza infection was associated with lower viral load, cytokine level, and reduced mortality . clinical trials on various antiviral treatments against evd are underway. these agents include bcx4430 (a novel nucleoside analog) (julander et al., 2014) , brincidofovir, favipiravir, tkm-ebola, and zmapp (a chimeric monoclonal antibody) in guinea, sierra leone, and liberia (bishop, 2015) . when there is no highly effective antiviral for the treatment of a severe viral illness, especially in patients at the extremes of age or with medical comorbidities, and infection control measures are difficult to implement or comply with, vaccination is the final option to prevent massive outbreaks. influenza vaccine is the most widely used annual vaccine in the community and health-care setting to protect at risk or any person to develop influenza-related complications and prevent institutional outbreaks. seasonal influenza-related excess hospitalization and death were estimated to be 10 000 and 1100 per year, respectively, in hong kong, a subtropical city (chiu et al., 2002; wong et al., 2004 wong et al., , 2006 . in a meta-analysis assessing influenza vaccine efficacy and effectiveness in elderly patients, the inactivated influenza vaccine could reduce the risk of hospitalization as a result of pneumonia by 21-38%, and cardiovascular disease by 18-30%, and all cause of mortality by 39-56% (nichol, 2008) . control of virus disease outbreak by vaccination is particularly valuable for exposed individuals, when the viral diseases have a long enough incubation period so that the exposed individuals have sufficient time to develop protective immune responses before symptomatic disease set in. measles (incubation period of 7-18 days), mumps (incubation period of 12-25 days), rubella (incubation period of 14-23 days), and varicella (incubation period of 10-21 days) are relevant examples. reactive vaccination for measles outbreak has been shown to be an effective measure to reduce the scale of outbreaks. in the democratic republic of congo, weekly reported cases reduced respectively by 89.3% and 68.9% in the 3 weeks following mass vaccination campaigns (alberti et al., 2010) . similarly, nationwide mass vaccination interrupted the transmission of paralytic poliomyelitis in albania. in 1996, a total of 138 paralytic cases occurred with an attack rate of 10 per 100 000 population among adults aged 19-25 years. the epidemic was controlled by two rounds of mass vaccination with trivalent oral poliovirus vaccine targeted to persons aged 0-50 years (prevots et al., 1998) . while routine laboratory diagnostic tests and specific antimicrobial agents are generally available for the treatment of bacterial, fungal, and parasitic infections, we are just entering the stage when rapid nucleic acid tests and a greater array of antiviral agents are available for tackling viral infections. the broad array of viruses worldwide causes substantial morbidity and mortality, ranging from respiratory viruses, arthropod-related viruses, to the most deadly blood-borne viruses. novel emerging or reemerging viruses are causing major epidemics from time to time especially in densely populated areas where human populations have close contact with wild animals (wildlife markets) and food animals (wet markets and abattoirs). such epidemics such as the ebola virus disease can be explosive in countries with failed governance and poor health infrastructures. currently, there is a lack of antiviral treatment for most of these infections. therefore, prevention by implementing effective infection control and vaccination is of utmost importance to contain these viruses. see also: arboviruses; hiv safety guidelines; hepatitis, viral; influenza; measles; mumps; rabies; respiratory syncytial virus; rubella. reactive vaccination as an effective tool for measles outbreak control in measles mortality reduction settings lack of nosocomial spread of varicella in a pediatric hospital with negative pressure ventilated patient rooms norovirus outbreak of probable waterborne transmission with high attack rate in a guatemalan resort mass oseltamivir prophylaxis halts pandemic influenza a h1n1 2009 outbreak in a secondary school in ashanti region pandemic influenza as 21st century urban public health crisis potential and emerging treatment options for ebola virus disease measles outbreak in a pediatric practice: airborne transmission in an office setting the effect of public health measures on the 1918 influenza pandemic in u.s. cities closing schools in response to the 2009 pandemic influenza a h1n1 virus in new york city: economic impact on households norovirus outbreak at a wildland fire base camp ignites investigation of restaurant inspection policies doctor to patient transmission of hepatitis b virus: implications of hbv dna levels and potential new solutions the experience of quarantine for individuals affected by sars in toronto clinical management and infection control of sars: lessons learned severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection outbreak of human metapneumovirus infection in psychiatric inpatients: implications for directly observed use of alcohol hand rub in prevention of nosocomial outbreaks successful control of norovirus outbreak in an infirmary with the use of alcohol-based hand rub prevention of nosocomial transmission of swine-origin pandemic influenza virus a/h1n1 by infection control bundle infection control preparedness for human infection of influenza a h7n9 in hong kong prevention of nosocomial transmission of norovirus by strategic infection control measures influenza-related hospitalizations among children in hong kong transmission of human immunodeficiency virus in a dental practice pilot study of postexposure prophylaxis for hepatitis c virus in healthcare workers facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial efficiency of quarantine during an epidemic of severe acute respiratory syndrome-beijing, china hepatitis b vaccine in health care personnel: safety, immunogenicity, and indicators of efficacy rabies transmission from organ transplants in the usa identification and containment of an outbreak of sars in a community hospital household responses to pandemic (h1n1) 2009-related school closures ebola virus: from discovery to vaccine strategies for mitigating an influenza pandemic long-term persistence of anti-hbs after vaccination against hbv: an 18 year experience in health care workers guideline for isolation precautions in hospitals. part i. evolution of isolation practices, hospital infection control practices advisory committee inactivation of feline calicivirus, a surrogate of norovirus (formerly norwalk-like viruses), by different types of alcohol in vitro and in vivo clinical features of nipah virus encephalitis among pig farmers in malaysia isolation and characterization of viruses related to the sars coronavirus from animals in southern china hepatitis b virus (hbv) and hepatitis c virus (hcv) infections in health care workers (hcws): guidelines for prevention of transmission of hbv and hcv from hcw to patients an outbreak of airborne nosocomial varicella nosocomial respiratory syncytial virus infections: the "cold war" has not ended nosocomial respiratory syncytial virus infections isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus transmission of hepatitis b virus to multiple patients from a surgeon without evidence of inadequate infection control emergence of a new norovirus gii.4 variant and changes in the historical biennial pattern of norovirus outbreak activity in alberta shea guideline for management of healthcare workers who are infected with hepatitis b virus, hepatitis c virus, and/or human immunodeficiency virus identification and molecular characterization of hendra virus in a horse in queensland confidence in controlling a sars outbreak: experiences of public health nurses in managing home quarantine measures in taiwan convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe school closures and student contact patterns bcx4430, a novel nucleoside analog, effectively treats yellow fever in a hamster model epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs prolonged norovirus outbreak in a finnish tertiary care hospital caused by gii.4-2006b subvariants viruses as agents of airborne contagion how long do nosocomial pathogens persist on inanimate surfaces? a systematic review updated us public health service guidelines for the management of occupational exposures to human immunodeficiency virus and recommendations for postexposure prophylaxis a non-foodborne norovirus outbreak among school children during a skiing holiday transmission of rabies virus from an organ donor to four transplant recipients in-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus a norovirus outbreak in a nursing home: norovirus shedding time associated with age severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats prevention of nosocomial respiratory syncytial virus infections through compliance with glove and gown isolation precautions immunization with high-dose intradermal recombinant hepatitis b vaccine in healthcare workers who failed to respond to intramuscular vaccination epidemiologic study and containment of a nosocomial outbreak of severe acute respiratory prospective controlled study of four infection-control procedures to prevent nosocomial infection with respiratory syncytial virus nonpharmaceutical interventions implemented by us cities during the 1918-1919 influenza pandemic an effective quarantine measure reduced the total incidence of influenza a h1n1 in the workplace: another way to control the h1n1 flu pandemic an outbreak of influenza aboard a commercial airliner efficacy and effectiveness of influenza vaccination rapid awareness and transmission of severe acute respiratory syndrome in hanoi french hospital, vietnam. am molecular epidemiology of hiv transmission in a dental practice outbreak of paralytic poliomyelitis in albania, 1996: high attack rate among adults and apparent interruption of transmission following nationwide mass vaccination sars and the removal of personal protective equipment factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi airborne spread of measles in a suburban elementary school infectiousness of air from a tuberculosis ward. ultraviolet irradiation of infected air: comparative infectiousness of different patients investigations of patients of health care workers infected with hiv. the centers for disease control and prevention database airborne transmission of communicable infection-the elusive pathway timing of hepatitis b vaccination: its effect on vaccine response in health care workers occupational deaths among healthcare workers anti-hepatitis b core antibody is not required for prevaccination screening in healthcare workers seroconversion after additional vaccine doses to non-responders to three doses of intradermally or intramuscularly administered recombinant hepatitis b vaccine. scand effectiveness of postexposition prophylaxis with oseltamivir in nursing homes: a randomised controlled trial over four seasons factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises review of aerosol transmission of influenza a virus updated u.s. public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis disease transmission and passenger behaviors during a high morbidity norovirus outbreak on a cruise ship influenza-associated mortality in hong kong influenza-associated hospitalization in a subtropical city school closure and mitigation of pandemic (h1n1) impact of an emergent norovirus variant in 2009 on norovirus outbreak activity in the united states key: cord-003764-141u6ax7 authors: shrestha, ashish c.; wijesundara, danushka k.; masavuli, makutiro g.; mekonnen, zelalem a.; gowans, eric j.; grubor-bauk, branka title: cytolytic perforin as an adjuvant to enhance the immunogenicity of dna vaccines date: 2019-04-30 journal: vaccines (basel) doi: 10.3390/vaccines7020038 sha: doc_id: 3764 cord_uid: 141u6ax7 dna vaccines present one of the most cost-effective platforms to develop global vaccines, which have been tested for nearly three decades in preclinical and clinical settings with some success in the clinic. however, one of the major challenges for the development of dna vaccines is their poor immunogenicity in humans, which has led to refinements in dna delivery, dosage in prime/boost regimens and the inclusion of adjuvants to enhance their immunogenicity. in this review, we focus on adjuvants that can enhance the immunogenicity of dna encoded antigens and highlight the development of a novel cytolytic dna platform encoding a truncated mouse perforin. the application of this innovative dna technology has considerable potential in the development of effective vaccines. vaccines represent an effective strategy in the fight against infectious diseases and recent estimates suggest that vaccination prevents 2-3 million deaths every year [1] . the need for rapid and large scale vaccine production during epidemics against emerging pathogens is a major challenge in vaccine development [2] , including effective vaccines for antigenically diverse and versatile pathogens that successfully subvert host immunity such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), and malaria [3] [4] [5] . dna vaccines can overcome some of these challenges, as it is relatively easy to produce large number of doses within a short period of time, and they are stable at ambient temperature and do not require cold chain transportation. they are also consistent between lots and have an excellent safety profile allowing for safety evaluations by regulatory authorities and distribution in a large scale [6, 7] . importantly, dna vaccines can induce both humoral and cell-mediated responses in the vaccinated host [8] [9] [10] . although they are safe and well tolerated, they are often poorly immunogenic and inefficacious in humans [11] . therefore, recent studies on the advancements of dna vaccines are focused on effective delivery and increasing the immunogenicity of the encoded antigen/s of interest [12, 13] . effective immunization with dna vaccines requires efficient transfection of host cells which is highly dependent on the delivery route and use of devices. conventional delivery routes to introduce the dna vaccine include intramuscular, intradermal, subcutaneous and oral routes [12] . the preferred delivery route depends on the requirement to activate specific immune cells. the skin is rich in immune cells including local dendritic cells (dcs) and natural killer (nk) cells, and is therefore likely to be a more favorable site for vaccine delivery [14, 15] . attempts to improve dna delivery have been made through other physical methods with the use of 'gene guns' or electroporation, which transiently permeabilizes the cell membrane to efficiently transfer the dna resulting in increased vaccine uptake by skin and muscle cells [16] . although these methods have shown to increase dna uptake [17, 18] , they require optimization to achieve increased efficiency and acceptance for clinical use. an alternative approach to improve transfection efficiency includes formulation of dna with liposomes or nanoparticles [19] . liposomal delivery can be affected by pre-systemic (epithelial) and systemic barriers (enzymatic degradation, binding, and opsonization) [20] . encapsulation of dna with nanoparticles has been reported to increase dna uptake or transfection efficiency [21, 22] . some of the challenges in the use of nanoparticles with dna include encapsulation inefficiency, endocytosis by target cells and toxicity [23] . the use of genetic adjuvants is one approach to enhance the immunogenicity of the antigen and can be used to complement other strategies (e.g., dna delivery) also designed to improve the immunogenicity of dna vaccines. upon immunization with a dna vaccine, the target cells uptake dna by endocytosis [24] and the transfected cells express the dna-encoded protein antigen(s). when antigen-presenting cells (apcs) are directly transfected, the intracellular proteins are processed and immunogenic epitopes are then presented by mhc class i molecules, which can directly stimulate naïve cd8 + t cells [12, 25] . the protein immunogen released from transfected cells can be endocytosed and/or phagocytosed by other apcs and are presented by mhc class ii molecules to activate naive cd4 + t cells [25, 26] . if the proteins are expressed by stromal cells like keratinocytes, apcs can also indirectly capture secreted antigens and cross-present by mhc class i molecules to further stimulate cd8 + t cells [27] . after dna vaccination, cd8 + t cells specific to the vaccine antigen undergo expansion, acquire effector functions and differentiates into memory cd8 + t cells [28, 29] . the memory cells differentiate into effector memory t cells upon re-exposure to the antigen [29, 30] . the ability of a dna vaccine to elicit t cell immunity is thus dependent on activating apcs to present antigen: mhc complexes to t cells [31] and adjuvants can serve as an important costimulatory factor to enhance this process. in this brief review, based on our experience, we discuss the progress in the development of dna vaccines, approaches to improve delivery and genetic adjuvants used to enhance immunogenicity. we focus on an innovative cytolytic dna technology developed and patented in our laboratory. the immunogenicity of dna vaccines is enhanced by cpg motifs present in the plasmid backbone, which can activate apc via toll like receptors (tlr9) [32] [33] [34] . unmethylated cpg motifs have been reported to induce b cell proliferation and secretion of immunoglobulin in vitro and in vivo [33] . activation of macrophages and dcs results in upregulation of antigen presentation and costimulatory molecules, and secretion of cytokines (il-12 and il-18) involved in helper t cells (th1) response [34] . thus, cpg motifs in dna plasmids serve as a 'natural adjuvant' for dna vaccines. plasmid dna can be designed to encode additional adjuvants with the antigen(s) of interest. molecular adjuvants such as fusion proteins including heat shock protein 70 (hsp70), and vesicular stomatitis virus (vsvg) have been developed and used to enhance vaccine immunogenicity [35] [36] [37] . the gene encoding such proteins as adjuvants is either fused with the gene encoding the vaccine antigen to produce a fusion protein driven by a same promoter or as separate proteins driven by different promoters in the same or different plasmid. co-encoding of genes creates a suitable cellular micro environment such as sustained antigen release and/or upregulation of cytokines, enhancing the immunogenicity of dna vaccines [38] . a majority of the studies on experimental dna vaccines with genetic adjuvants have been studied in animal models such as mice (table 1) , and very few of these have been clinically tested ( table 2) . limited published data on clinical trials pose difficulty to compare efficacy between adjuvants in animals and humans. different cytokines, such as interleukins (il-2, il-6, il-12), chemokines, granulocyte/macrophage colony-stimulating factor (gm-csf), costimulatory molecules (cd40, cd80, and cd86), and signaling molecules (interferon regulatory factor -3) have been used as genetic adjuvants with dna vaccines [39, 40, [42] [43] [44] 48, 68] . genes expressing ifn-γ il-2, il-12, il-15, and il-18 have been used to stimulate th1 responses [44, 45, 70] , and il-4, il-6, il-10, il-13, for th2 stimulation [42, 43, [71] [72] [73] . the inclusion of genes encoding cytokines, like il-2 or il-12, as adjuvants for hiv-1 dna vaccines is known to increase cell mediated immunity (cmi) [74, 75] . however, a bicistronic hiv dna encoding gp120 and il-2 elicited weaker specific immune response than monocistronic hiv-1 gp120 dna [76] . combinations of genetic adjuvants like il-2 and il-15 with hiv-1 dna vaccine have also been used but no synergistic effect on the level of total antibody to hiv-1 antigen was reported [77] . a phase i/iia trial showed that coadministration of dna vaccine encoding prostatic acid phosphatase (pap) with gm-csf elicited pap-specific cd4 + and/or cd8 + t cell responses [68] . however, gm-csf was administered as a recombinant protein. hsp70, a class of molecular chaperone, is known to induce maturation of dcs and activation of the th1 pathway [78] [79] [80] . a fusion vaccine for multiple myeloma termed hdkk1-hhsp70 was shown to be effective in inhibiting the targeted tumor and increased survival of vaccinated mice by eliciting tumor-specific humoral and cellular immune responses [80] . however, a dna vaccine encoding hpv16e7 fused with hsp70, targeting hpv16 and cervical intraepithelial neoplasia 2/3 failed to enhance significant t cell responses in a phase i clinical trial [69] . a bicistronic dna encoding hsp70 as a membrane bound or secreted protein has been used to improve the immunogenicity of a hiv gag [60] . in this case, hsp70 expression was driven by a weaker sv40 promoter and hiv gag by a stronger cmv promoter. such a vaccine design enhanced gag-specific t cell responses, providing greater protection in mice challenged with ecohiv [60] . ecohiv is a chimeric virus containing the envelope protein gp 80 of mouse leukemia virus rather than hiv gp 120 that can replicate in mouse leukocytes in vivo, thus representing a viable mouse challenge model for early assessment of hiv vaccines [81] . the proposed mechanism of hsp70 as an adjuvant is that tlr 2/4 on dcs interacts with secreted or bound hsp70, further attracting dcs to the site of antigen expression. this is followed by dc maturation, presentation of antigens by mhc molecules and secretion of cytokines and costimulatory molecules [82] , thus enhancing t cell immune responses against the vaccine antigen. a chimeric version of the oligomerization domain from the chicken complement inhibitor (c4bp) was used to produce an oligomeric form of vaccine antigens [35, 83] . this protein, termed imx313, forms a heptameric structure of the vaccine protein. this has been used to develop dna vaccines for tuberculosis, malaria and hiv to enhance humoral and/or cellular responses [35, 36, 84] . vaccination with a dna vaccine encoding secreted hiv tat (tpa-tat imx313) induced higher humoral and cellular responses and improved protection against ecohiv challenge in mice [36] . a phase i clinical trial of tuberculosis vaccine mva85a-imx313 evaluated the vaccine to be safe and immunogenic, but cellular (ag85a-specific ifn[81] . the proposed mechanism of hsp70 as an adjuvant is that tlr 2/4 on dcs interacts eted or bound hsp70, further attracting dcs to the site of antigen expression. this is by dc maturation, presentation of antigens by mhc molecules and secretion of cytokines mulatory molecules [82] , thus enhancing t cell immune responses against the vaccine en complement inhibitor imeric version of the oligomerization domain from the chicken complement inhibitor as used to produce an oligomeric form of vaccine antigens [35, 83] . this protein, termed forms a heptameric structure of the vaccine protein. this has been used to develop dna for tuberculosis, malaria and hiv to enhance humoral and/or cellular responses [35, 36, 84] . on with a dna vaccine encoding secreted hiv tat (tpa-tat imx313) induced higher and cellular responses and improved protection against ecohiv challenge in mice [36] . a inical trial of tuberculosis vaccine mva85a-imx313 evaluated the vaccine to be safe and enic, but cellular (ag85a-specific ifn-ƴ ) and humoral (mva-specific igg) responses were ficant [85] . thus, the ability of such an adjuvant to enhance immunogenicity of dna n humans is still debated. g is a type iii viral fusion envelope protein, which mediates fusion of the virus envelope ost cell membrane [86] . the use of fusogenic membrane glycoprotein (fmg) gene from vsv vaccine encoding the h7 protein of human papillomavirus type 16 was shown to enhance ell responses and effectively control growth of tumors [87] . the vsv g protein was also induce a t-response at low doses or a t-independent response at higher doses [88] . fmg s into large multinucleated syncytia, which are then killed by a nonapoptotic mechanism ytiosomes are released from the membrane and present antigens efficiently to dcs [90] . thus act as an adjuvant for any antigen expressed by a dna vaccine. vaccination of mice hepatitis c virus (hcv) nonstructural protein 3 (ns3) together with vsvg resulted in frequency of ifn-γ, but not tnf-α-or il-2-producing cd3+ cd44+ cd8+ effector memory lls [57] . this dna vaccine was constructed in a bicistronic vector with vsvg co-encoded e plasmid with hcv ns3. vsvg expression was driven by a weaker sv40 promoter and stronger cmv promoter [57] . however, others have also reported an increase in the specific n the immunogen and vsvg were expressed from different plasmids [37, 87] . tic protein rin (prf) is a pore forming protein released by immune cells including nk cells and t lymphocytes (ctl) [91] . the 67-kilodalton prf protein oligomerizes to form pores that channel to release granzyme into the cytosol of target cells [92] . suicide genes inducing of target cells have been used for cancer therapies, and the role of apoptotic cell death in n, whether immune-stimulatory or immune-suppressive, is debated [93] . recently, a novel hnology has been developed--termed cytolytic dna technology-in which a truncated f is incorporated in a bicistronic dna vector to act as a vaccine adjuvant [55] [56] [57] 94] . lytic dna vaccines are based on a bicistronic dna plasmid constructed on a pvax (invitrogen) with a cytomegalovirus (cmv) promoter and a simian virus (sv40) promoter gene encoding the protein of interest as an immunogen is inserted downstream of the cmv and the gene encoding a truncated version of mouse prf downstream of the sv40 promoter . the sv40 is a weaker promoter compared to cmv and has been shown to result in 10-fold tein expression in transfected hek293t cells in vitro [55, 95] . ) and humoral (mva-specific igg) responses were not significant [85] . thus, the ability of such an adjuvant to enhance immunogenicity of dna vaccines in humans is still debated. vsv g is a type iii viral fusion envelope protein, which mediates fusion of the virus envelope and the host cell membrane [86] . the use of fusogenic membrane glycoprotein (fmg) gene from vsv in a dna vaccine encoding the h7 protein of human papillomavirus type 16 was shown to enhance cd8 + t cell responses and effectively control growth of tumors [87] . the vsv g protein was also shown to induce a t-response at low doses or a t-independent response at higher doses [88] . fmg fuses cells into large multinucleated syncytia, which are then killed by a nonapoptotic mechanism [89] . syncytiosomes are released from the membrane and present antigens efficiently to dcs [90] . fmgs can thus act as an adjuvant for any antigen expressed by a dna vaccine. vaccination of mice with the hepatitis c virus (hcv) nonstructural protein 3 (ns3) together with vsvg resulted in increased frequency of ifn-γ, but not tnf-α-or il-2-producing cd3+ cd44+ cd8+ effector memory t (t em ) cells [57] . this dna vaccine was constructed in a bicistronic vector with vsvg co-encoded in the same plasmid with hcv ns3. vsvg expression was driven by a weaker sv40 promoter and ns3 by a stronger cmv promoter [57] . however, others have also reported an increase in the specific cmi when the immunogen and vsvg were expressed from different plasmids [37, 87] . perforin (prf) is a pore forming protein released by immune cells including nk cells and cytotoxic t lymphocytes (ctl) [91] . the 67-kilodalton prf protein oligomerizes to form pores that serve as a channel to release granzyme into the cytosol of target cells [92] . suicide genes inducing apoptosis of target cells have been used for cancer therapies, and the role of apoptotic cell death in vaccination, whether immune-stimulatory or immune-suppressive, is debated [93] . recently, a novel dna technology has been developed--termed cytolytic dna technology-in which a truncated mouse prf is incorporated in a bicistronic dna vector to act as a vaccine adjuvant [55] [56] [57] 94] . cytolytic dna vaccines are based on a bicistronic dna plasmid constructed on a pvax backbone (invitrogen) with a cytomegalovirus (cmv) promoter and a simian virus (sv40) promoter [55] . the gene encoding the protein of interest as an immunogen is inserted downstream of the cmv promoter and the gene encoding a truncated version of mouse prf downstream of the sv40 promoter ( figure 1) . the sv40 is a weaker promoter compared to cmv and has been shown to result in 10-fold lower protein expression in transfected hek293t cells in vitro [55, 95] . the prf gene was modified to express a truncated version of prf (~60kda) lacking the final 12 amino acid residues of the c terminus (unstructured region of prf) [56, 57, 94] in order for prf to become cytolytic [96] . the final 12 c-terminal amino acids are required to export prf protein from the endoplasmic reticulum (er) to the golgi, from where glycosylated prf is then transported to the prf gene was modified to express a truncated version of prf (~60kda) lacking the final 12 amino acid residues of the c terminus (unstructured region of prf) [56, 57, 94] in order for prf to become cytolytic [96] . the final 12 c-terminal amino acids are required to export prf protein from the endoplasmic reticulum (er) to the golgi, from where glycosylated prf is then transported to secretory granules [96] . removal of the c-terminal abrogates the export of prf from the er and its subsequent accumulation in the er is cytotoxic to the host cell [96, 97] . different recombinant cytolytic dna-prf vaccines (rdna-prf) have been shown to elicit immune responses higher than those elicited by canonical dna vaccines (without prf) and the mechanism underlying this has been established [94] . a previous study showed that coexpression of hcv ns3 and prf elicited nonapoptotic cell death in transfected cells, whilst immunization with ns3-prf dna vaccine increased ns3-specific t cell mediated responses as evidenced by increased ns3-specific ifn-γ responses in an elispot assay and increased numbers of polyfunctional cd8 + t em cells that simultaneously secreted ifn-γ, il-2, and tnf-α [56] . cytolytic dna platform where the expression of immunogen is driven by a stronger promoter allows for sufficient antigen expression and accumulation within the target cells followed by nonapoptotic cell death due to lesser expression of prf driven by a weaker sv40 promoter; thus, balancing the level of antigen expression with the timing of cell death [94] . necrosis is considered as the mechanism of cell death by prf as evidenced by release of lactate dehydrogenase (ldh) and low caspase activity [55, 56, 94] . ldh release occurs after the rupture of cell membrane during secondary necrosis [98, 99] . in contrast, ldh was not released by cells treated with doxorubicin (a proapoptotic drug) or cells transfected with ns3 wild type prf or ns3 12del483a prf (mutant and nontoxic prf) [94] . expression of prf from a cytolytic dna, e.g., ns3 prf vaccine, thus results in necrotic cell death mediated by receptor-interacting protein-1 kinase activity, as evidenced by detection of uncleaved cytokeratin 18 in huh-7 cells [56] . necrosis releases damage associated molecular patterns (damps) which in turn activate dcs to migrate to the site of vaccination [100, 101] . when purified dcs including the cd8α + subset from naïve c57bl/6 mice were exposed to hek293t cells (transfected with ovalbumin-prf), it resulted in upregulation of costimulatory molecules (cd80/cd86), indicating maturation of the immune cells with the cytolytic dna [94] . a significant increase in cd11c + dcs and cross-presenting cd8a + dcs, and upregulation of cd80 has been reported in mice vaccinated with a cytolytic dna hiv 1 gag prf compared to a canonical dna vaccine [55] . local and migrated dcs at the site of inflammation can take up antigens by endocytosis and are also exposed to damps. activated and matured dcs can then prime naïve cd8 + t cells (figure 2) . antigen cross-presentation by dcs to cd8 + t cells has been shown to increase the number of proliferating cd8 + t cells by~2-fold with cytolytic dna compared to the noncytolytic prf dna [94] . thus, a cytolytic dna vaccine has an inbuilt adjuvant to enhance the immunogenicity of the vaccine immunogen. whereas, the immunogenicity of canonical dna vaccines mostly depends on direct transfection of dcs and to a lesser extent cross-presentation of antigens shed from transfected cells and/or derived from transfected cells that have undergone spontaneous cell death [94] . several studies have established that dcs exposed to necrotic or lytic cells expressing antigens mature and cross-present more efficiently than dcs exposed to antigens derived from a cellular milieu that comprise of apoptotic cells [102] [103] [104] [105] [106] . comparative studies evaluating the ability of proapoptotic (e.g., rotavirus nonstructural protein 4 (nsp4) and diphtheria toxin subunit a (dta)) and necrotic proteins (e.g., truncated prf) to enhance the immunogenicity of dna when encoded in plasmid dna vaccines showed that truncated prf is the most effective for this purpose [55] [56] [57] . however, a caveat is that vaccine-encoded antigens need to accumulate significantly inside the cell before necrosis occurs following expression of truncated prf in order to activate dcs to cross-present vaccine-encoded antigens [55, 94] . several studies have established that dcs exposed to necrotic or lytic cells expressing antigens mature and cross-present more efficiently than dcs exposed to antigens derived from a cellular milieu that comprise of apoptotic cells [102] [103] [104] [105] [106] . comparative studies evaluating the ability of proapoptotic (e.g., rotavirus nonstructural protein 4 (nsp4) and diphtheria toxin subunit a (dta)) and necrotic proteins (e.g., truncated prf) to enhance the immunogenicity of dna when encoded in plasmid dna vaccines showed that truncated prf is the most effective for this purpose [55] [56] [57] . however, a caveat is that vaccine-encoded antigens need to accumulate significantly inside the cell before necrosis occurs following expression of truncated prf in order to activate dcs to cross-present vaccine-encoded antigens [55, 94] . rdna-prf technology has been used in the development of hiv and hcv dna vaccines [55, 57, 107] . direct comparison of the effects of the cytolytic prf and the apoptotic protein dta on the immunogenicity of the hiv-1 gag protein showed that prf activated dcs more efficiently, as evidenced by the increase in frequency of cross-presenting dcs and upregulation of activation marker (cd80) [55] . in both dna vaccines, prf and dta were driven by sv40 promoter. immunization of mice with a dna vaccine encoding proapoptotic dta as an adjuvant in a hiv gag dta vaccine resulted in decreased dc activation, suggesting that dta-induced apoptosis attenuated immune response [55] . furthermore, improved protection in the mouse ecohiv challenge model was achieved with rdna-prf encoding hiv gag compared to protection levels in mice vaccinated with a canonical gag dna vaccine [57] . a rdna-prf vaccine encoding the hcv ns3 protein coexpressed with prf was shown to increase ns3-specific cmi in mice and pigs, compared to ns3 coexpression with a proapoptotic protein, the rotavirus nsp4 protein [56] . nsp4 is an enterotoxin that elicits a proapoptotic effect by disrupting the mitochondrial membrane and activating caspase-3, -8, and -9 [108, 109] . this study showed that prf coexpression induced cell death by necrosis, and thus enhanced ns3-specific immune responses, whereas, proapoptotic nsp4 reduced ns3-specific response [56] . importantly, hcv ns3 prf was more immunogenic than the canonical ns3 vaccine in pigs, demonstrating the translational potential of the cytolytic dna vaccines in human clinical trials [56] . likewise, we have shown that a multi-antigenic hcv dna vaccine encoding genotype 3a proteins ns3, ns4a, ns4b, and ns5b coexpressed with prf induced robust cmi against the range of hcv ns proteins, compared to coexpression with vsvg [57] . we also showed that multi-antigenic and multigenotypic (hcv genotype 1 and 3a) dna cocktail vaccines encoding prf can significantly increase the magnitude and breadth of cmi responses to ns3 and ns5b against both genotypes compared to those elicited by a single-genotype vaccine [107] . rdna-prf technology has been used in the development of hiv and hcv dna vaccines [55, 57, 107] . direct comparison of the effects of the cytolytic prf and the apoptotic protein dta on the immunogenicity of the hiv-1 gag protein showed that prf activated dcs more efficiently, as evidenced by the increase in frequency of cross-presenting dcs and upregulation of activation marker (cd80) [55] . in both dna vaccines, prf and dta were driven by sv40 promoter. immunization of mice with a dna vaccine encoding proapoptotic dta as an adjuvant in a hiv gag dta vaccine resulted in decreased dc activation, suggesting that dta-induced apoptosis attenuated immune response [55] . furthermore, improved protection in the mouse ecohiv challenge model was achieved with rdna-prf encoding hiv gag compared to protection levels in mice vaccinated with a canonical gag dna vaccine [57] . a rdna-prf vaccine encoding the hcv ns3 protein coexpressed with prf was shown to increase ns3-specific cmi in mice and pigs, compared to ns3 coexpression with a proapoptotic protein, the rotavirus nsp4 protein [56] . nsp4 is an enterotoxin that elicits a proapoptotic effect by disrupting the mitochondrial membrane and activating caspase-3, -8, and -9 [108, 109] . this study showed that prf coexpression induced cell death by necrosis, and thus enhanced ns3-specific immune responses, whereas, proapoptotic nsp4 reduced ns3-specific response [56] . importantly, hcv ns3 prf was more immunogenic than the canonical ns3 vaccine in pigs, demonstrating the translational potential of the cytolytic dna vaccines in human clinical trials [56] . likewise, we have shown that a multi-antigenic hcv dna vaccine encoding genotype 3a proteins ns3, ns4a, ns4b, and ns5b coexpressed with prf induced robust cmi against the range of hcv ns proteins, compared to coexpression with vsvg [57] . we also showed that multi-antigenic and multigenotypic (hcv genotype 1 and 3a) dna cocktail vaccines encoding prf can significantly increase the magnitude and breadth of cmi responses to ns3 and ns5b against both genotypes compared to those elicited by a single-genotype vaccine [107] . dna vaccines are still a promising option in the development of novel vaccination strategies. although they have many advantages, the ability to induce effective immune responses in humans required for protection has been challenging. these challenges include ineffective delivery and poor uptake of dna. consequently, a recent focus has been in the development of delivery methods and/or inclusion of genetic adjuvants. such genetic adjuvants are generally coexpressed with the antigen of interest or delivered through different plasmids. in the quest to develop and identify effective genetic adjuvants, a range of adjuvants was tested (hsp70, vsvg, imx313, dta, and prf) and a novel and promising cytolytic dna vaccine strategy has been developed. this cytolytic dna vaccine is unique as it is based on a bicistronic plasmid with the ability to coexpress antigen and prf in a balanced mechanism causing necrosis of vaccine-transduced cells, followed by increased activation of immune cells and cross presentation of vaccine immunogen. cytolytic dna vaccines encoding nonstructural proteins of hcv have been tested to enhance immunogenicity of vaccine antigen in mice [57, 107] and in a large preclinical animal model, the pig [56] . likewise, increased immunogenicity and improved protection against ecohiv challenge in mice with hiv gag prf [60] demonstrate the effectiveness of cytolytic dna vaccines. adjuvants that provide effective costimulation for immune responses with specific immunogens may not have a similar effect with other immunogens, and therefore these need to be tested for their efficacy. use of a genetic adjuvant such as prf produces a suitable microenvironment for multiple/different immunogens and thus improves the delivery, immunogenicity and effectiveness of dna vaccines. this strategy has considerable potential in the development of dna-based vaccines against a range of infectious agents. world health organisation. 10 facts on immunization the complexity and cost of vaccine manufacturing--an overview editorial: why vaccines to hiv, hcv, and malaria have so far failed-challenges to developing vaccines against immunoregulating pathogens a 2020 vision for vaccines against hiv, tuberculosis and malaria dna vaccines for emerging infectious diseases: what if? emerg advancements in dna vaccine vectors, non-mechanical delivery methods, and molecular adjuvants to increase immunogenicity effective humoral immune response from a h1n1 dna vaccine delivered to the skin by microneedles coated with plga-based cationic nanoparticles differential humoral and cellular immunity induced by vaccination using plasmid dna and protein recombinant expressing the ns3 protein of dengue virus type 3 vaccines for emerging infectious diseases: lessons from mers coronavirus and zika virus clinical applications of dna vaccines: current progress dna vaccines-how far from clinical use? dna vaccines: recent developments and the future. vaccines dose sparing with intradermal injection of influenza vaccine skin-resident antigen-presenting cells: instruction manual for vaccine development what you always needed to know about electroporation based dna vaccines gene transfer into muscle by electroporation in vivo electroporation-mediated gene delivery liposomes as vaccine delivery systems: a review of the recent advances barriers to liposomal gene delivery: from application site to the target plga nanoparticles for dna vaccination-waiving complexity and increasing efficiency solid lipid nanoparticles mediate non-viral delivery of plasmid dna to dendritic cells production and clinical development of nanoparticles for gene delivery specific endocytosis and degradation of naked dna in the endocardial cells of cod (gadus morhua l.) directly transfected langerin + dermal dendritic cells potentiate cd8 + ; t cell responses following intradermal plasmid dna immunization chemical adjuvants for plasmid dna vaccines programming, demarcating, and manipulating cd8+ t-cell memory duration of antigen expression in vivo following dna immunization modifies the magnitude, contraction, and secondary responses of cd8 + t lymphocytes secondary memory cd8 + t cells are more protective but slower to acquire a central-memory phenotype immunogenicity of infectious pathogens and vaccine antigens bacterial cpg-dna and lipopolysaccharides activate toll-like receptors at distinct cellular compartments cpg motifs in bacterial dna trigger direct b-cell activation cpg-oligonucleotides in vaccination: signaling and mechanisms of action enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing pfs25 to imx313 multimerization technology a hiv-tat/c4-binding protein chimera encoded by a dna vaccine is highly immunogenic and contains acute ecohiv infection in mice enhanced presentation of major histocompatibility complex class i-restricted human immunodeficiency virus type 1 (hiv-1) gag-specific epitopes after dna immunization with vectors coding for vesicular stomatitis virus glycoprotein-pseudotyped hiv-1 gag particles mechanisms of action of adjuvants engineering dna vaccines via co-delivery of co-stimulatory molecule genes dna vaccine for cancer immunotherapy intracellular adhesion molecule-1 modulates beta-chemokines and directly costimulates t cells in vivo coimmunization with ifn-gamma or il-2, but not il-13 or il-4 cdna can enhance th1-type dna vaccine-induced immune responses in vivo coadministration of dna encoding interleukin-6 and hemagglutinin confers protection from influenza virus challenge in mice development of th1 and th2 populations and the nature of immune responses to hepatitis b virus dna vaccines can be modulated by codelivery of various cytokine genes modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with dna immunogens toll-like receptor adaptor molecules enhance dna-raised adaptive immune responses against influenza and tumors through activation of innate immunity activation of innate immunity, inflammation, and potentiation of dna vaccination through mammalian expression of the tlr5 agonist flagellin regulation of dna-raised immune responses by cotransfected interferon regulatory factors novel strategies to improve dna vaccine immunogenicity co-immunization with an optimized plasmid-encoded immune stimulatory interleukin, high-mobility group box 1 protein, results in enhanced interferon-gamma secretion by antigen-specific cd8 t cells dlm-1/zbp1) as a genetic adjuvant for dna vaccines that promotes effective antitumor ctl immunity mda5 can be exploited as efficacious genetic adjuvant for dna vaccination against lethal h5n1 influenza virus infection in chickens development of a molecular adjuvant to enhance antigen-specific cd8(+) t cell responses dna vaccines with single-chain fv fused to fragment c of tetanus toxin induce protective immunity against lymphoma and myeloma induction of antigen-positive cell death by the expression of perforin, but not dta, from a dna vaccine enhances the immune respons intradermal delivery of dna encoding hcv ns3 and perforin elicits robust cell-mediated immunity in mice and pigs a multiantigenic dna vaccine that induces broad hepatitis c virus-specific t-cell responses in mice calreticulin is an effective immunologic adjuvant to tumor-associated antigens development of a dna vaccine targeting human papillomavirus type 16 oncoprotein e6 dna vaccines encoding membrane-bound or secreted forms of heat shock protein 70 exhibit improved potency safety and immunogenicity of an hiv-1 gag dna vaccine with or without il-12 and/or il-15 plasmid cytokine adjuvant in healthy, hiv-1 uninfected adults vaccination with a plasmid dna encoding her-2/neu together with low doses of gm-csf and il-2 in patients with metastatic breast carcinoma: a pilot clinical trial a phase i safety study of plasmid dna immunization targeting carcinoembryonic antigen in colorectal cancer patients timing of plasmid cytokine (il-2/ig) administration affects hiv-1 vaccine immunogenicity in hiv-seronegative subjects safety and tolerability of hiv-1 multiantigen pdna vaccine given with il-12 plasmid dna via electroporation, boosted with a recombinant vesicular stomatitis virus hiv gag vaccine in healthy volunteers in a randomized, controlled clinical trial dna priming increases frequency of t-cell responses to a vesicular stomatitis virus hiv vaccine with specific enhancement of cd8+ t-cell responses by interleukin-12 plasmid dna safety and comparative immunogenicity of an hiv-1 dna vaccine in combination with plasmid interleukin 12 and impact of intramuscular electroporation for delivery safety and immunological efficacy of a dna vaccine encoding prostatic acid phosphatase in patients with stage d0 prostate cancer a phase i trial of a human papillomavirus dna vaccine for hpv16+ cervical intraepithelial neoplasia 2/3. clin antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of ifn-gamma, il-12, or il-18 gene adjuvants genetic adjuvants for dna vaccines intranasal immunization of a dna vaccine with il-12-and granulocyte-macrophage colony-stimulating factor (gm-csf)-expressing plasmids in liposomes induces strong mucosal and cell-mediated immune responses against hiv-1 antigens cytokine molecular adjuvants modulate immune responses induced by dna vaccine constructs for hiv-1 and siv intranasal administration of human immunodeficiency virus type-1 (hiv-1) dna vaccine with interleukin-2 expression plasmid enhances cell-mediated immunity against hiv-1 enhancement of cell-mediated immunity against hiv-1 induced by coinnoculation of plasmid-encoded hiv-1 antigen with plasmid expressing il-12 augmentation and suppression of immune responses to an hiv-1 dna vaccine by plasmid cytokine/ig administration il-15 expression plasmid enhances cell-mediated immunity induced by an hiv-1 dna vaccine a mage3/heat shock protein70 dna vaccine induces both innate and adaptive immune responses for the antitumor activity heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology human dkk1 and human hsp70 fusion dna vaccine induces an effective anti-tumor efficacy in murine multiple myeloma a mouse model for study of systemic hiv-1 infection, antiviral immune responses, and neuroinvasiveness encoded novel forms of hsp70 or a cytolytic protein increase dna vaccine potency fusion of the mycobacterium tuberculosis antigen 85a to an oligomerization domain enhances its immunogenicity in both mice and non-human primates the oligomerization domain of c4-binding protein (c4bp) acts as an adjuvant, and the fusion protein comprised of the 19-kilodalton merozoite surface protein 1 fused with the murine c4bp domain protects mice against malaria a first-in-human phase 1 trial to evaluate the safety and immunogenicity of the candidate tuberculosis vaccine mva85a-imx313, administered to bcg-vaccinated adults vesicular stomatitis virus g protein transmembrane region is crucial for the hemi-fusion to full fusion transition combined administration with dna encoding vesicular stomatitis virus g protein enhances dna vaccine potency vesicular stomatitis virus indiana glycoprotein as a t-cell-dependent and -independent antigen fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth viral fusogenic membrane glycoproteins kill solid tumor cells by nonapoptotic mechanisms that promote cross presentation of tumor antigens by dendritic cells comparison of primary human cytotoxic t-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production the structural basis for membrane binding and pore formation by lymphocyte perforin dna vaccines and apoptosis: to kill or not to kill? cytolytic dna vaccine encoding lytic perforin augments the maturation of-and antigen presentation by-dendritic cells in a time-dependent manner systematic comparison of constitutive promoters and the doxycycline-inducible promoter protection from endogenous perforin: glycans and the c terminus regulate exocytic trafficking in cytotoxic lymphocytes perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack the roles of caspase-3 and bcl-2 in chemically-induced apoptosis but not necrosis of renal epithelial cells apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells tolerance, danger, and the extended family how dying cells alert the immune system to danger tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression natural adjuvants: endogenous activators of dendritic cells consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the nf-kappa b pathway cross-presentation by dendritic cells induction of genotype cross-reactive, hepatitis c virus-specific, cell-mediated immunity in dna-vaccinated mice rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of apoptosis during infection porcine reproductive and respiratory syndrome virus nonstructural protein 4 induces apoptosis dependent on its 3c-like serine protease activity we thank thrf and the donor community for supporting the development of our novel dna vaccines. ashish c. shrestha and danushka k. wijesundara are recipients of early career fellowships from thrf. the authors declare no conflict of interest. key: cord-022128-r8el8nqm authors: domingo, esteban title: molecular basis of genetic variation of viruses: error-prone replication date: 2019-11-08 journal: virus as populations doi: 10.1016/b978-0-12-816331-3.00002-7 sha: doc_id: 22128 cord_uid: r8el8nqm genetic variation is a necessity of all biological systems. viruses use all known mechanisms of variation; mutation, several forms of recombination, and segment reassortment in the case of viruses with a segmented genome. these processes are intimately connected with the replicative machineries of viruses, as well as with fundamental physical-chemical properties of nucleotides when acting as template or substrate residues. recombination has been viewed as a means to rescue viable genomes from unfit parents or to produce large modifications for the exploration of phenotypic novelty. all types of genetic variation can act conjointly as blind processes to provide the raw materials for adaptation to the changing environments in which viruses must replicate. a distinction is made between mechanistically unavoidable and evolutionarily relevant mutation and recombination. genetic change was a prerequisite for the early life forms to be generated and maintained (chapter 1), and it is also a requirement for the evolution of present-day life. we may willingly or inadvertently modify selective pressures, but genetic change is rooted in all replication machineries. the results of genetic modifications, regarding relative dominances of variant forms, are guided by selective pressures and random events. the replicative machinery itself has probably been influenced by natural selection; as an example, polymerases devoid of a capacity to generate variants should have endured a long-term selective disadvantage. however, once the replicative machinery was established, the mechanisms of variation acted independently of the selective pressures applied or to come. viruses use the same molecular mechanisms of genetic variation than other forms of life: mutation (that encompasses point mutations and insertions-deletions of different lengths), hypermutation, several types of recombination, and genome segment reassortment. mutation is observed in all viruses, with no known exceptions. recombination is also widespread, but its role in the generation of diversity appears to vary among viruses. its occurrence was soon accepted for dna viruses, but it was considered uncertain for the rna viruses. pioneering studies of poliovirus (pv) by p. d. cooper, v. i. agol and colleagues, and of foot-and-mouth disease virus (fmdv) by a.m. king and colleagues provided the first evidence of recombination in rna. the present perception is that recombination is more widespread than thought only a few decades ago and that its frequency and the types of genomic forms it generates are varied among viruses. for example, it appears that positivestrand rna viruses recombine more easily than negative-strand rna viruses to give rise to mosaic genomes of standard length. several negative-strand rna viruses, however, can yield defective genomes through recombination, frequently characterized by deletions in their rna. a connection between the structure of replication complexesdas viewed by x-ray diffraction or high-resolution cryo-electron microscopydand the propensity to produce defective genomes has not been established. defective genomes are increasingly perceived not only as unavoidable side-products of blind replicative imperfections but as classes of genome subpopulations that perform relevant biological roles for the standard, infectious viruses. genome segment reassortment, a type of variation close to chromosomal exchanges in sexual reproduction, is an adaptive asset of segmented viral genomes, as continuously evidenced by the ongoing evolution of the influenza viruses. the three modes of virus genome variation are not incompatible, and reassortantrecombinant-mutant genomes are continuously arising in present-day viruses. the potential for genetic variation of rna and dna viral genomes is remarkable, and it is the ultimate molecular mechanism that lies at the origin of the virus diversity delineated in chapter 1. mutation is a localized alteration of a nucleotide residue in a nucleic acid. it generally refers to an inheritable modification of the genetic material. in the case of viral genomes, mutations can result from different mechanisms: (i) template miscopying (direct incorporation of an incorrect nucleotide); (ii) primer-template misalignments that include miscoding followed by realignment, and misalignment of the template relative to the growing chain (polymerase "slippage" or "stuttering"); (iii) activity of cellular enzymes (i.e., deaminases), or (iv) chemical damage to the viral nucleic acids (deamination, depurination, depyrimidination, reactions with oxygen radicals, direct and indirect effects of ionizing radiation, photochemical reactions, etc.) (naegeli, 1997; bloomfield et al., 2000; friedberg et al., 2006) . the basis of nucleotide misincorporation during template copying (defined as the incorporation of a nucleotide different from that expected from the template residue at that position) lies mainly in the electronic structure of the bases that make up dna (adenine, a; guanine, g; cytosine, c; thymine, t) or rna (with uracil, u instead of t). each base includes potential hydrogen-bonding donor sites (amino or amino protons) and hydrogen-bonding acceptor sites (carbonyl oxygens or aromatic nitrogens) that contribute to standard watson-crick base pairs ( fig. 2.1) , as well as wobble base pairs (nonstandard watson-crick, but fundamental for rna secondary structure and mrna translation) ( fig. 2.2) . the conformation of the purine and pyrimidine bases is highly dynamic. amino and methyl groups rotate about the bonds that link them to the ring structure. in dilute solution, hydrogen bonds are established with water, and they can be displaced by nucleotide or amino acid residues to give rise to nucleotide-nucleotide or nucleotide-amino acid interactions. the strength difference between hydrogen bonds established in a polynucleotide chain with water, and their strength between two bases in separate polynucleotide chains determines whether a double-stranded polynucleotide will be formed. purine and pyrimidine bases can acquire different charge distributions and ionization states. as a consequence, in addition to the standard watson-crick and wobble, other base pairs are found in naturally occurring nucleic acids (notably cellular rrna and trna) and in synthetic oligonucleotides (a-u or a-t hoogsteen, and a-g, c-u, g-g, and u-u pairs, as well as interactions involving ionized bases). one of the types of electronic redistribution leads to tautomeric changes, such as the keto-enol and amino-imino transitions, which modify the hydrogen-bonding properties of the base; tautomeric imino and enol forms of the standard bases can produce non-watson-crick pairs. the proportion of the alternative tautomeric forms can be influenced by modifications in the purine and pyrimidine rings, which, in turn, can favor either the syn or anti conformation of a nucleoside, which is defined by the torsion angle of the bond between the 1 0 carbon of the ribose and either n1 in pyrimidines or n9 in purines ( fig. 2.1) . the anti-conformation is usually the most stable in standard nucleotides and polynucleotides. the transition from the anti to the syn conformation may alter the hydrogen-bonding properties of the base, thereby inducing mutagenesis (bloomfield et al., 2000; suzuki et al., 2006) . the understanding of conformational and g-c, with ribose as pentose). phosphodiester bonds of two potential polynucleotide chains of different polarity (outer arrows) are indicated. effects on the base-pairing tendencies of nucleoside analogs in the context of the active site of a polymerase is very relevant to the design of specific mutagenic analogs for viral polymerases in lethal mutagenesis-centered antiviral approaches (chapter 9). base-base interactions are not only responsible for part of the mutations that occur during genome replication, but also for the formation of double-stranded nucleic acids, either within the same polynucleotide chain or between two different chains. transitions from a coil-like into an organized double-stranded (or other) structure are functionally relevant for both rna and dna. in the case of rna, doublestranded regions in the adequate alternation with single-stranded regions, determine key catalytic or macromolecule-attracting abilities, as for example, ribozyme activities (chapter 1), the internal ribosome entry site (ires) of several viral and cellular mrnas, or multitudes of functional rna-protein interactions (denny and greenleaf, 2018) . adjacent base stacking due to electronic interactions (rather than hydrophobic bonds as once thought), contribute also to the stability of double-helical regions in nucleic acid molecules. structural transitions due to alternative stacking conformations, particularly within polypurine or polypyrimidine tracts, can affect nucleic acidprotein interactions. in turn, replication machineries (typically including viral and host proteins gathered in membrane structures) may also be affected by nucleic acid conformations; such effects are important in virology regarding consequences for mutant generation in a given template sequence context. these considerations on structural transitions are relevant to the nonneutral character of silent (also termed synonymous) mutations (those in open-reading frames that do not result in an amino acid substitution), a point to be addressed in the next section. transitions from a single-stranded into a doublestranded nucleic acid structure and the relative stability of the two forms depend on multiple factors that include the nucleotide sequence of the nucleic acid, its being a ribo-or a deoxyribosepolynucleotide, temperature, ionic environment, and ionic strength. positively charged counterions neutralize negatively charged phosphates, and favor duplex stability [as an overview of figure 2.2 examples of a class of non-watson-crick base pairs termed wobble base pairs. the drawing is similar to that of fig. 2 .1, except that the sugar residues and phosphodiester bonds have been omitted. hydrogen bonds (discontinuous lines in red) are shown between i (inosine) and c, u, and a, and between g and u. wobble base pairs are important for codonanticodon interactions, as described in the text. physical and chemical properties of nucleic acids and their nucleotide components, see (bloomfield et al., 2000) ]. mutations resulting from any of the mechanism just summarized can be divided into transitions, transversions (both referred to as point mutations), and insertions and deletions (referred to as indels) (fig. 2.3) . the latter occurs preferentially at homopolymeric tracts and also at short, repeated, sequences which are prone to misalignment mutagenesis ( fig. 2.4 ). an example is an editing mechanism for some viral mrnas, such as the phosphoprotein mrna of the paramyxovirinae [ (kolakofsky et al., 2005) other examples in vivo are hot spots for variation in reiterated sequences in complex dna genomes (yamaguchi et al., 1998; barrett and mcfadden, 2008; mcgeoch et al., 2008) , or the insertion of two amino acids (often ser-ser, ser-gly, or ser-ala between residues 69 and 70 of the hiv-1 reverse transcriptase), in concert with hiv-1 resistance to nucleoside inhibitors (winters and merigan, 2005 ) (chapter 8). hairpin structures in rna and dna may also induce deletions as a result of slippage mutagenesis (pathak and temin, 1992; viguera et al., 2001) . transition mutations occur more frequently than either transversions or indels during virus replication. nucleotide discrimination at the catalytic site of viral polymerases fits this observation because of the more likely replacement of a purine or pyrimidine nucleotide by its structurally more similar nucleotide. in some cases, however, an abundance of indels and similar numbers of transitions and transversions have been recorded (cheynier et al., 2001; malpica et al., 2002) . the molecular bases of such unexpected behavior regarding mutational spectra are not well understood. the generation of point mutations and indels is subject to thermodynamic and quantummechanical uncertainties inherent to atomic fluctuations, rendering mutagenesis a highly unpredictable event, thus introducing stochasticity (randomness) in a key motor of evolution: the generation of diversity at the molecular level (domingo et al., 1995; eigen, 2013) . indicate point mutations, insertion or deletions (known as indels). a genome is depicted as an elongated rod. symbols on the rod (cross, circle, and line) represent mutations. hypermutation is generally associated with a high frequency of specific mutation types (crosses and lines). a region inserted or deleted from the genome is depicted as an empty rod. the effect of mutations on the structure and function of proteins is extremely relevant to penetrate into the mechanisms that drive virus evolution since selection acts on phenotypes that are often embodied in protein molecules. silent or synonymous mutations are those that do not give rise to an amino acid substitution despite being located in a protein-coding region of a genome. their occurrence is due to the degeneracy of the genetic code: the same amino acid can be coded for by two or more triplets (codons), with the exception of aug for methionine and ugg for tryptophan. synonymous mutations are not necessarily selectively neutral, neutral meaning that they have no discernible consequence for any viral function. the assumption that synonymous mutations are selectively neutral, and the fact that the early comparison of nucleotide sequences of homologous genes showed a dominance of synonymous over nonsynonymous mutations, contributed to the foundations of the neutral theory of molecular evolution. this theory attributes the evolution of organisms at the molecular level mainly to the random drift of genomes carrying neutral or quasi-(or nearly-) neutral mutations (king and jukes, 1969; kimura, 1983 kimura, , 1989 . the terms quasi-neutral or nearly-neutral may seem ambiguous to molecular biologists. however, in the formulation of the neutral theory they had a precise meaning of the selection coefficient (a parameter that measures fitness differences) being lower than the inverse of the effective population size, with minor variations in the equations of some formulations (kimura, 1983) . despite random drift of genomes playing an important role in molecular evolution, evidence gathered over the last decades renders untenable the assumption that synonymous mutations are neutral. evidence to the contrary has been obtained with viruses and cells, including mutations in the human genome that may affect enhancer functions (hirsch and birnbaum, 2015) , mrna folding (faure et al., 2017; mittal et al., 2018) , and microrna targeting (brest et al., 2011) , among other processes (novella, 2004; novella et al., 2004; parmley et al., 2006; hamano et al., 2007; resch et al., 2007; lafforgue et al., 2011; nevot et al., 2011 nevot et al., , 2018 supek, 2016) . there are several mechanisms by which synonymous mutations can affect virus behavior: alteration of cis-acting regulatory elements in viral genomes, decrease of the stability of duplex structures within the rna genome or between viral sequences and mirnas or sirnas, or changes of viral gene expression [splicing precision or translation fidelity through the modification of rna-rna or rna-protein interactions; reviewed in (martínez et al., 2016) ]. synonymous codons use different trnas for protein synthesis, and different trnas do not have the same relative abundance in different host cell types. thus, the rate of protein synthesis, an important phenotypic trait for cells and viruses, can be affected by the frequency of alternative synonymous codons present in mrnas (richmond, 1970; akashi, 2001) . not only codon bias, but also specific codons or codon combinations may affect ribosome speed to regulate the folding of nascent proteins during translation (makhoul and trifonov, 2002; rocha, 2004; aragones et al., 2010; brule and grayhack, 2017) . as a consequence, generation of rare codons by mutation of abundant codons (or vice versa) can modify viral fitness (chapter 5). rare codons may also limit the fidelity of amino acid incorporation when the frequency of the required aminoacyl-trnas is low (ling et al., 2009; zaher and green, 2009; czech et al., 2010) . the frequency of codon pairs in rna genomes is also a fitness determinant relevant to the preparation of attenuated viral vaccines. to complicate matters further, a synonymous mutation may be neutral or quasi-neutral in one environment, but it may contribute to selection in a different environment, because of the phenotypic effects of rna structure and codon usage. neutrality is relative to the environment. regarding the effects of mutations (box 2.1), the following general statements are applicable to viruses: • although difficult to prove due to the limited number of environments used for experimentation, truly neutral mutations (i.e., with no influence on the virus in any environment) are probably very rare. this applies to synonymous, as well as to nonsynonymous mutations. • mutations resulting in chemically conservative amino acid substitutions are more likely to be tolerated than those leading to chemically different amino acids. tolerability (quantified by substitution matrices among amino acids in protein evolution) should be distinguished from neutrality. a tolerated mutation may cause a reduction in fitness, which is nevertheless compatible with virus replication. • a conservative amino acid substitution may have important biological consequences. • the effect of any individual mutation is context-dependent in two ways: it may depend on other mutations in the same genome (epistasis, see also section 2.8 and chapter 5) or on the mutant cloud that surrounds the genome harboring the mutation (effects of complementation, cooperation, or interference, discussed in section 3.8 of chapter 3). • the previous points do not deny the influence of random drift of genomes on intrahost and interhost evolution. the currently most accepted view is that positive and negative selection and random drift occur continuously during virus evolution (chapter 3). the proportion of transition versus transversion mutations may depend initially on the specific replication machinery of a virus that tends to produce some mutation types preferentially over others. for a given virus, short-term evolution is often reflected in the dominance of transitions, a dominance which is less apparent when distantly related sequences of the same virus are compared. the effect of evolutionary distance on the transition to transversion ratio was observed in the fmdv genome sequence comparisons carried out in our laboratory over several decades, that ranged from analyses of mutant spectra relative to their corresponding consensus sequence to independent viral isolates from disease outbreaks separated by several decades [review of the work on fmdv evolution in (domingo et al., 1990 (domingo et al., , 2003 ]. these two levels of sequence comparisons (within mutant spectra vs. independent isolates) can be highly significant, as discussed in chapters 3 and 7. the proportion of synonymous and nonsynonymous mutations that have mediated the diversification of viral genomic sequences that belong to the same phylogenetic lineage is often considered informative of the underlying evolutionary forces. probably because of the rooted (albeit uncertain) notion that biological function is more likely to reside in protein than in dna or rna, the ratio of nonsynonymous substitutions (corrected per nonsynonymous site in the sequence under study) (d n ), to the number of synonymous substitutions per synonymous site (d s ), termed u (u ¼ d n /d s ) is calculated to infer the dominant mode of evolution (nei and gojobori, 1986) . mutations may affect stem-loop or other secondary and higher-order structures involved in regulatory processes through nucleic acidnucleic acid or nucleic acid-protein interactions. the primary sequence in nonstructured, noncoding regions may also be functionally relevant. in coding regions the effect of a mutation may be contextdependent in two manners: it may be affected by other mutations in the same genome (epistasis) or by other genomes of the surrounding mutant spectrum. when u ¼ 1 the evolution is considered neutral, when u < 1 purifying (or negative) selection is dominant, and when u > 1 positive (or directional) selection prevails (yang and bielawski, 2000) . the types of selection undergone by viruses are discussed in section 3.4 of chapter 3. there are several reasons to be cautious about the significance of u: (i) synonymous mutations need not be neutral, for reasons discussed in section 2.3. (ii) in the course of evolution, important but transient events of positive selection (termed episodic positive selection) due to one or a few amino acid substitutions may be accompanied by a larger number of synonymous, tolerated mutations. in this situation, u computes as u < 1, thus indicative of purifying selection despite a critical role of positive selection triggered by one or few nonsynonymous mutations in the evolutionary outcome (crandall et al., 1999) . (iii) in a striking proof of the above arguments, statistically significant mutational biases led to a value of u indicative of positive selection in an in vitro evolution experiment simulating pseudogene evolution in which positive selection was not possible (vartanian et al., 2001) ; this study represents a warning which is rarely mentioned when discussing the limitations of conclusions based on the value of u. (iv) a synonymous change may permit the mutant codon to acquire a relevant nonsynonymous change through a point mutation. the term quasisynonymous has been used to describe codons that encode the same amino acid, but that has a different evolutionary potential regarding the amino acids that they can access through a point mutation. alternative codons for a given amino acid approximate a replicative system to points of sequence space from which a phenotypically relevant change has a different probability (chapters 3 and 4). (v) finally, u was initially proposed to compare distantly related rather than closely related genomes, as is often the case in the short-term evolution of viruses (kryazhimskiy and plotkin, 2008) . for all these reasons, u values as a diagnostic of forces mediating dna and rna virus evolution must be regarded only as indirect and suggestive, not as a definitive parameter. despite these arguments, use of u to propose a model of virus evolution continues being surprisingly unchallenged in the literature of virus evolution. we use u only in a limited way in subsequent chapters because, in addition to the limitations just listed, it does not help in the interpretation of critical evolutionary events regarding viruses. related shortcomings apply to other tests of neutrality developed to interpret the origin of dna polymorphisms in the years following the summit of the neutralist-selectionist controversy (fu, 1997; achaz, 2009 ). mutation rates quantify the number of misincorporations per nucleotide copied, irrespective of the fate (increase or decrease in frequency) of the mutated genome produced. a mutation rate for a genomic site measures a biochemical event dictated by the replication machinery and environmental parameters that affect the catalytic properties of the polymerase. in contrast, a mutant (or mutation) frequency describes the proportion of a mutant (or a set of mutants) in a genome population. the frequency of a mutant will depend on the rate at which it is generated (given by the mutation rate) and on its replication capacity relative to other genomes in the population (drake and holland, 1999) (fig. 2 .5). a specific mutation may be produced at a modest rate, but then be found at high frequency because the mutation is advantageous for genome replication in that environment. the converse situation may also occur. some mutational hot spots (in the sense of genomic sites where mutations tend to occur with high probability) may never be reflected among the repertoire of mutations found in a 2.5 mutation rates and frequencies for dna and rna genomes genome population because of the selective disadvantage they inflict upon the genome harboring them. a very significant example is the elongation of an internal oligoadenylate tract located between the two functional aug initiation codons in the fmdv genome. the homopolymeric tract constitutes a hot spot for variation due to polymerase slippage ( fig. 2 .4b). the elongation of the internal oligoadenylate was dramatic because it sextuplicated the number of adenylate residues present at that site; it was only observed when fmdv was subjected to repeated plaque-to-plaque (bottleneck) transfers, not large population passages. in fact, this drastic genetic modification has not been recorded among natural isolates of the virus. the molecular instruction to elongate the oligoadenylate was very strong because it was observed in many independent biological clones subjected to bottleneck transfers (escarmís et al., 1996) . despite qualifying as a hot spot for variation, the first event in fitness recovery when the clones were subjected to large population passages was the reversion of the elongated tract to its original size (escarmís et al., 1999) . the interpretation of these findings, to be further analyzed in chapter 6, is that during plaque-to-plaque transfers the negative selection to eliminate unfit genomes is less intense than during large, highly competitive population passages. again, a clear molecular instruction to elongate a homopolymeric track may not be reflected in a high frequency of the affected genomes. therefore, although mutation rates and frequencies for viruses bear some relationship, rates cannot be inferred from frequencies and vice versa ( fig. 2 .5). the first calculations of mutation rates for cellular organisms and for some dna bacteriophages were carried out by j.w. drake, who pursued comparative measurements that have generally supported a difference between mutation rates for dna and rna viruses. the rates estimated for bacteriophages l and t4 were about 100 times higher than those of their host e. coli. an approximately constant rate of 0.003 mutations per genome per replication round was calculated for a number of dna-based microbes (drake, 1991) , an observation sometimes referred to as "drake's rule." this rather surprising constancy suggests that different dna organisms have accommodated the template-copying fidelity of their replication machineries to achieve a narrow window in the mutational load measured as mutations fixed per genome, a remarkable fitting of biochemistry with evolutionary needs. the basal mutation rate in mammalian cells has been estimated at about 10 à10 substitutions per nucleotide and cell generation [reviewed in (naegeli, 1997; domingo et al., 2001; friedberg et al., 2006) ] (table 2 .1). the synonymous mutation rate measured with experimental populations of bacteria has been assumed to reflect the neutral mutation rate (despite limitations explained in section 2.3). values for e. coli have ranged from 2 â 10 à11 up figure 2.5 scheme that illustrates the difference between mutation rate and mutant frequency. residue a in a template residue (top) can be misread to incorporate a c, a, or g into the complementary strand (discontinuous lines), at a rate of 10 à4 , 10 à5 , and 10 à5 substitutions per nucleotide, respectively. the replicative capacity of the newly generated templates (with g, u, and c, continuous lines) will determine widely different mutant frequencies with g > c > u. to 5 â 10 à9 substitutions per synonymous site per generation (ochman et al., 1999) with 5 â 10 à10 as the most likely estimate (lenski et al., 2003) . the latter value is in agreement with a rate of 3 â 10 à10 to 4 â 10 à10 substitutions per base pair and generation based on wholegenome deep sequencing of an experimentally evolved lineage of myxococcus xanthus (velicer et al., 2006) . there are biological phyla for which no mutation rates have been calculated. from current knowledge, we can assume that mutation rates in cells and viruses depend on the replicative machinery (generally a multiprotein complex that includes the relevant viral polymerase with additional viral and host proteins and membrane structures) and on multiple environmental parameters (template nucleotide sequence context, ionic environment, temperature, metabolites in interaction with components of the replication apparatus, etc.). whether bacteria are in the exponential or stationary phase of growth can affect intracellular metabolites and proton exchange rates which, in turn, may alter the proportion of tautomeric forms in nucleotides and misincorporation tendencies (friedberg et al., 2006) . the sequence context of the template nucleic acids (presence of repeated sequences that can induce misalignment mutagenesis or g-c vs. a-t rich regions in relation to relative nucleotide substrate abundances, etc.) may impel or attenuate mutability. insertion elements may enhance mutation rates at neighboring sites in a bacterial genome (miller and day, 2004) . despite these influences, vesicular stomatitis virus (vsv) displayed comparable mutation rates in several host cells (combe and sanjuan, 2014) suggesting that there is a limited range of average error rates needed for a virus to maintain fitness (chapters 5 and 9). in addition to the general environmental and sequence context consequences for templatecopying fidelity that may affect any genome type, mutation rates for dna viruses will also be influenced by: (i) whether the dna polymerase that catalyzes viral dna synthesis includes or lacks a functional proofreading-repair activity. high copying fidelity is typical of dna polymerases involved in cellular dna replication (bebenek and ziuzia-graczyk, 2018) , and low copying fidelity is generally a feature of dna polymerases involved in dna repair (friedberg et al., 2006; ganai and johansson, 2016) . thus, repair of lesions that by themselves might not be mutagenic may lead to the introduction of mutations during the error-prone repair process. (ii) expression of proteins active in repair encoded in the viral genome, such as uracil-dna glycosylase, dna repair endonucleases, etc. (iii) the mechanism of viral dna replication, particularly the occurrence of double-stranded versus single-stranded dna in replicative intermediates. (iv) intracellular site of replication and the availability of postreplicative dna repair proteins (regarding both intracellular location and concentration) to the viral replication factories. little is known of the spatial relationships and relative affinities of cellular and viral proteins and structures that may critically affect polymerase fidelity. comparative measurements of mutation rates at specific genome sites of dna viruses are needed, as a first step to define the cellular and biochemical influences on the fidelity of dna virus genome replication. general genetic variability affecting the entire virus genome should be distinguished from rna viruses 10 à5 to 10 à3 retroviruses 10 à6 to 10 à4 dna viruses 10 à8 to 10 à3 cellular dna 10 à9 to 10 à11 values are expressed as substitutions per nucleotide. the range of values is the most likely according to several independent studies. no distinction is made between mutation rates and frequencies. see text for comments and references. 2.5 mutation rates and frequencies for dna and rna genomes localized variability at hot spots in a genome. even the extremely complex human genome shows genetic instability at specific loci, some associated with genetic disease (domingo et al., 2001; alberts et al., 2002; bushman, 2002) . genome size is a parameter pertinent to biological behavior, not only because it imposes a commensurate copying fidelity, but also because it affects the impact of genetic heterogeneity within infected organisms and upon the invasion of new hosts (chapter 3). mutation frequencies measured by subjecting virus to a specific selective agent (e.g., mutants that escape the neutralizing activity of a monoclonal antibody or mutants that escape inhibition by a drug) span a broad range of values (10 à 3 to 10 à 8 ) for dna and rna viruses (smith and inglis, 1987; sarisky et al., 2000; domingo et al., 2001) (table 2 .1). the technical details of any procedure used to calculate a mutation frequency should be carefully evaluated to translate its meaning to the genome level. important variables are the efficacy of the antibody or drug (which will be concentration-dependent) or the possibility of phenotypic hiding-mixing in the escape mutants to be quantified (holland et al., 1989; valcarcel and ortin, 1989) . unexpected low levels of escape mutants (that would imply < 10 à6 substitutions per nucleotide) for an rna virus can mean either a general or sitespecific high polymerase fidelity, a selective disadvantage of the genome that harbors the mutation or, when a phenotypic alteration is measured, the requirement of two or more mutations to produce the alteration. conversely, a high mutation frequency for a dna virus whose replication is catalyzed by a high-fidelity dna polymerase may mean that either repair activities were not functional or that the mutant displayed a selective advantage and overgrew the wild type prior to the measurement of its frequency. mathematical treatments that take into account reversion of a low fitness mutant and its competition with wild-type virus have been used to calculate mutation rates (batschelet et al., 1976; coffin, 1990) . despite difficulties and limitations in the calculations, independent genetic and biochemical methods with different viruses support mutation rates for rna viruses in the range of 10 à3 to 10 à5 substitutions per nucleotide copied [as representative articles and reviews see (batschelet et al., 1976; domingo et al., 1978 domingo et al., , 2001 steinhauer and holland, 1986; eigen and biebricher, 1988; varela-echavarria et al., 1992; ward and flanegan, 1992; mansky and temin, 1995; preston and dougherty, 1996; drake and holland, 1999; sanjuan et al., 2010; bradwell et al., 2013) ] (table 2 .1). a few early studies indicated unusual low mutation rates or frequencies for some rna viruses. as discussed in some of the reviews listed above, there are technical reasons to suggest that such values were probably underestimates of the true average mutations rates or frequencies. obviously, it cannot be excluded that some genomic sites or viruses under a given environment might be unusually refractory to introduce mutations, but most evidence supports the range of values listed in table 2 .1. the near million-fold higher mutation rates for rna viruses than cellular dna, whose biological implications were presciently anticipated by j. holland and colleagues (holland et al., 1982) , have been confirmed. that is, for rna viruses of genome length between 3 kb and 32 kb, an average of 0.1-1 mutation is introduced per template molecule copied in the replicating population. unless most mutations impeded viral replication, a continuous input of mutant genomes is expected, as indeed found experimentally (chapter 3). high mutation rates for rna genomes are also supported by measurements of templatecopying fidelity by rna polymerases, reverse transcriptases, and dna polymerases devoid of 3e5 0 proofreading exonuclease (or under conditions in which, such exonuclease is not functional) [ (steinhauer et al., 1992; varela-echavarria et al., 1992; mansky and temin, 1995; domingo et al., 2001; friedberg et al., 2002 friedberg et al., , 2006 men endez-arias, 2002) , and references therein]. in vitro fidelity tests may be based on genetic or biochemical assays using homopolymeric or heteropolymeric templateprimers. measurements include the kinetics of incorporation of an incorrect versus the correct nucleotide directed by a specific position of a template or the capacity of a polymerase to elongate a mismatched template-primer 3 0 end, [these and other assays have been reviewed (men endezarias, 2002) ] (see also section 2.6). differences between related enzymes (i.e., amv rt is more accurate than hiv-1 rt), and the fact that amino acid substitutions in the polymerases affect nucleotide discrimination, demonstrate that proofreading-repair activities together with the structure of the polymerase and replication complexes are determinants of template-copying fidelity. the term mutation rate if often used in a light manner in the literature of virus evolution, probably driven by nomenclature from classical population genetics. it is used to mean mutation frequency, rate of evolution, and sometimes to mean mutation rate in its real sense (as explained in section 2.5). a particularly risky habit is to use mutation rate when what is measured is a mutation frequency. some studies have claimed that they have a replication system devoid of selection, and therefore, the number of mutations counted corresponds to the true mutation rate of the system. this is incorrect. there is no replicative system devoid of selection because at least selection to maintain replication is in continuous operation. furthermore, as taught by quasispecies dynamics (stadler, 2016) , supported by mutational waves in hepatitis c virus upon prolonged replication in a constant cellular environment (moreno et al., 2017) , the mutant spectrum per se is part of the environment. since the mutant spectrum is constantly changing, so is the environment in which replication takes place. unfortunately, some studies have proposed the existence of mutational cold spots (sites at which mutations as a biochemical event occur at a very low frequency) ignoring that negative selection might have eliminated newly arising mutations. these false conclusions imply the existence of genomic regions particularly suitable as drug or antibody targets because they cannot mutate. these are the types of studies and incorrect conclusions that keep perpetuating the problem of control of viral diseases by encouraging antiviral and vaccine designs doomed to failure (chapters 8 and 9). 2.6 evolutionary origins, evolvability, and consequences of high mutation rates: fidelity mutants the amino acid substitutions in the core polymerase that affect fidelity can be located either close to or away from the active site of the enzyme. the change in fidelity can reach almost one order of magnitude, but virus viability is not compromised. thus, error rates themselves can be subjected to selection, as supported by theoretical studies on evolvability (earl and deem, 2004) . early studies documented heterogeneity in the mutation rates among individual plaque isolates of influenza a virus (suarez et al., 1992) . it is not clear whether mutation rates of viruses have evolved to procure a balance between adaptability and genetic stability, or whether other selective constraints have imposed the observed values. it has been suggested that because of the generally deleterious nature of most mutations, the adaptive value of the high mutation rates for rna viruses is debatable and that there might have been a trade-off between replication rate and copying fidelity (elena and sanjuan, 2005) . mutation rates would be a consequence of rapid rna replication, and an increase in copying fidelity would come at a 2.6 evolutionary origins, evolvability, and consequences of high mutation rates: fidelity mutants cost, resulting in a lower replication rate. a connection between elongation and error rate has been suggested by results with some viral and cellular polymerases [review (kunkel and erie, 2005) ]. in an early study with the poliovirus rdrp in vitro, an increase in the error frequency was observed when the ph and mg 2þ ion conditions were modified, and the decreased fidelity correlated with increased rna elongation rate (ward et al., 1988) . a possible connection between elongation rate and copying fidelity cannot be ruled out, but current evidence points to template-copying fidelity as being the result of multiple factors, not necessarily linked to the rate of genome replication (vignuzzi and andino, 2010; campagnola et al., 2015; domingo and perales, 2019) . there is ample support for an adaptive value of high mutation rates for rna viruses, independently of their biochemical origins. a poliovirus mutant, whose rdrp displayed a three-to fivefold higher fidelity than the wild-type enzyme, replicated at a slightly lower rate than wild-type virus in cell culture but displayed a strong selective disadvantage regarding the invasion of the brain of susceptible mice (pfeiffer and kirkegaard, 2005; vignuzzi et al., 2006) . the impediment to cause neuropathology was due at least partly to the limited complexity of the mutant spectrum since its broadening through mutagenesis restored the capacity to produce neuropathology. these and other studies have provided evidence that mutant spectrum complexity, by virtue of its impact on fitness, can be a virulence determinant. the work by m. vignuzzi, j. pfeiffer, r. andino, c. cameron, k. kirkegaard, and their colleagues on poliovirus fidelity mutants opened a much-needed branch of research in virus evolution and quasispecies implications. as proof of this statement, the field of fidelity mutants is rapidly expanding (bordería et al., 2016) , and references to the information they provide will be made in several chapters. theoretical models and experimental observations suggest that mechanisms for error correction had to evolve to maintain functionality of increasingly complex genomes (swetina and schuster, 1982; eigen and biebricher, 1988; domingo et al., 2001; eigen, 2002 eigen, , 2013 ) (here complexity means genome size, provided no redundant information is encoded). the coronaviruses have the largest genomes among the known rna viruses, with 30e33 kb. this is about 10-fold more genetic information than encoded in the simple rna bacteriophages, such as ms2 or qb. coronaviruses are replicated by complex rna-dependent rna polymerases, which include a domain that corresponds to a 3 0 e5 0 exonuclease, proofreading-repair activity. the protein displays exonuclease activity in vitro, and its inactivation affects viral rna synthesis (minskaia et al., 2006) , and results in increases of about 15-fold in the average mutation frequency (eckerle et al., 2007 (eckerle et al., , 2010 . a coronavirus mutant devoid of this repair function is more susceptible to lethal mutagenesis than the corresponding, nonmutated virus , as expected from a connection between replication accuracy and proximity to an error threshold for the maintenance of genetic information (chapter 9). thus, it is likely that a proofreading activity evolved (or was captured from a cellular counterpart) in rna genomes, whose genomic complexity was in the limit compatible with the fidelity achievable by standard rna replicases. it would be interesting to discover new rna viruses with a single rna molecule longer than 30 kb as a genome to analyze whether they have evolved more accurate core polymerases or exhibit a proofreading-repair function during replication. toward the other end of the rna size scale, viroid rnas display a mutation rate higher than (or close to the highest) recorded for rna viruses, consistent with the correlation between genome size and template-copying accuracy (gago et al., 2009) . studies with bacteria have identified some of the factors that successively increase copying fidelity. it has been estimated that during e. coli dna replication the error rate would be 10 à1 to 10 à2 mutations per nucleotide copied if accuracy relied only upon the strength of interactions provided by base pairing (section 2.2). the error rate would decrease to 10 à5 to 10 à6 with base selection and proofreading-repair, to about 10 à7 with the contribution of additional proteins present in the replication complex, and to about 10 à10 misincorporations per nucleotide with the participation of postreplicative mismatch correction mechanisms (naegeli, 1997; kunkel and erie, 2005; friedberg et al., 2006) . reduction of bacterial genome size results in the increase of mutation frequency (nishimura et al., 2017) . the error rate of the bacteriophage f29 dna polymerase is about 10 à6 without the proofreading exonuclease activity, and it decreases to 10 à8 with the correcting activity [(de vega et al., 2010) and references therein]. postreplicative repair pathways act on double-stranded dna, but not (or very inefficiently) on rna or dna-rna hybrids. therefore, the known postreplicative repair systems that operate in cellular dna do not make a significant contribution to error correction in rna viruses (steinhauer et al., 1992) . the importance of copying fidelity for complex genomes is reflected in the fact that more than 100 proteins are directly or indirectly involved in the repair of the human genome . elevated mutation rates in the range of those operating for rna viruses would be lethal for large dna genomes. localized genetic modification occurs physiologically in processes, such as somatic hypermutation and class-switch recombination in b cells of the germinal centers, as mechanisms of diversification of immunoglobulin genes (upton et al., 2011; methot et al., 2017) . chromosomal instability has long been associated with cancer (gatenby and frieden, 2004; stratton et al., 2009) . surveys have been (and are currently being) used to identify genes associated with chromosomal instability and their role in aging and disease (aguilera and garcia-muse, 2013; vijg et al., 2017 ) (see also chapter 10). while uncontrolled high mutability is deleterious for differentiated cellular organisms, it constitutes a modus vivendi for a great majority of viruses. despite its attractiveness, definitive proof of the hypothesis of a direct relationship between error rate and limited genome complexity will require additional functional and biochemical studies. exceptions to the absence of repair activities in simple genetic elements have been described. a satellite rna of the plant virus turnip crinkle carmovirus evolved a 3 0 -end rna repair mechanism. it implicates the synthesis of short oligoribonucleotides by the viral replicase using the 3 0 -end of the viral genome as a template. the mechanism consists probably of template-independent priming at the 3 0 -end of the damaged rna to generate wild type, negative strand, and satellite rna (nagy and simon, 1997) . a reversible, ntp-dependent excision of the 3 0 residue of the nascent nucleic acid product has been described in some retroviruses and hepatitis c virus (meyer et al., 1998; jin et al., 2013) . this activity is important for drug resistance, and it may also modulate the overall fidelity of some polymerases. it cannot be excluded that some type of point mutation correction may operate in rna genetic elements of less than 30 kb. such putative mechanisms may even diminish mutation rates that would otherwise be prohibitively deleterious, and they do not overshadow high mutation rates as a feature of rna and some dna genomes (table 2 .1). limited copying fidelity in the absence of proofreading-correction mechanisms can be regarded as an unavoidable consequence of the molecular mechanisms involved in template copying by viral polymerases. most nucleic acid polymerases share a structure that resembles a right hand, with fingers, palm, and thumb domains (fig. 2.6a ). three-dimensional structures of viral rdrps and rts indicate that interactions between the incoming nucleotide or residues of the template-primer with amino acids of the polymerase must permit displacement of the growing polymerase chain along 2.6 evolutionary origins, evolvability, and consequences of high mutation rates: fidelity mutants on the left, the klenow fragment is represented with colored fingers, palm and thumb domains, next to an open right hand. the structure on the right is that of pv rdrp, next to a closed right hand. courtesy of n. verdaguer and l. vives-adri an (the hand is that of l. vives-adri an). (b) the structure of the ternary complex between fmdv 3d, an rna molecule, and utp as the substrate (pdb id. 2e9z). the left panel is a front view of the complex, depicting the polymerase chain as a yellow ribbon, the rna in dark blue (template) and cyan (primer). the incoming utp and the pyrophosphate product are shown in atom type, and 2 mg 2 þ ions as magenta balls. the right panel is the same complex in a top-down orientation. (figure courtesy of c. ferrer-orta and n. verdaguer). (c) scheme of the minimum number of steps involved in nucleotide incorporation. the first step consists of the binding of polymerase 0 e to the template-primer r n (elongated up to nucleotide n) to form a complex 0 er n . formation of the activated complex er n is governed by the rate of constant k assembly (k a ). the activated er n complex binds a nucleotide ntp with an apparent binding affinity given by k d,app to form the er n ntp complex. catalysis to covalently incorporate the ntp to the growing primer chain to yield er nþ1 and pyrophosphate (pp i ) is governed by the rate constant k pol . other constants depicted in the scheme are the inactivation rate constant (k inact ) of 0 e, and dissociation of e from rna (k off, er n and k off , er nþ1 ). based on arias, a., arnold, j.j., sierra, m., smidansky, e.d., domingo, e., et al., 2008 . determinants of rna-dependent rna polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin. j. virol 82, 12346e12355, and previous studies with pv polymerase 3d by c. e. cameron and his colleagues. the channel located at the palm domain of the polymerase (steitz, 1999; ferrer-orta et al., 2006; wu and gong, 2018) (fig. 2.6b ). the polymerase of classical swine fever virus and other pestiviruses, such as bovine viral diarrhea virus includes an n-terminal extra-domain of about 100 amino acids; its interaction with the palm domain is important for template copying fidelity . if interactions around the catalytic site to ensure the correct nucleotide incorporation were so strong as to preclude misincorporations, the movement of the growing polynucleotide chain would be hampered. again, this compromise suggests a match between biochemical and evolutionary needs. the orientation of the triphosphate moiety of the incoming nucleotide substrate is important for nucleotide incorporation (men endezarias, 2002; graci and cameron, 2004; ferrer-orta et al., 2009 ). one of the several steps involved in nucleotide incorporation is the formation of a ternary complex (polymerase with templateprimer and the incoming nucleotide) that undergoes a conformational change (reorientation of the divalent ion-complexed triphosphate moiety of the incoming nucleotide). this conformational change activates the complex for phosphoryl transfer, to link the nucleosidemonophosphate to the 3 0 -terminus of the primer (or growing chain). steps involved in the nucleotide incorporation are represented in fig. 2 .6c. both the conformational change and the relative rate of phosphoryl transfer for an incorrect nucleotide versus the correct nucleotide influence the error rate at each site of the growing chain. critical kinetic constants in fig. 2 .6c that are determined experimentally to quantify relative nucleotide incorporations and misincorporations are k d,app (expressed as mm), k pol (expressed as s à1 ), and the ratio k pol /k d,app (mm à1 s à1 ) termed the catalytic efficiency. the ratio of k pol /k d,app for the incorporation of an incorrect nucleotide to k pol /k d,app for a correct nucleotide gives the frequency of that particular misincorporation, and an assessment of polymerase fidelity [ (castro et al., 2005) and references therein]. modifications of polymerase residues by site-directed mutagenesis, combined with comparisons of the relevant structures, have identified critical amino acid residues involved in template-copying fidelity. high-fidelity mutants are frequently obtained by selecting viruses resistant to mutagenic nucleotide analogs such as the antiviral agent ribavirin (beaucourt and vignuzzi, 2014) . limited incorporation of a deleterious nucleotide can be attained either through specific discrimination against the analog (ribavirin or other) or through a general decrease of all types of misincorporations, that is, a high-fidelity phenotype. structural modifications of viral polymerases that lead to high fidelity have inspired the design of mutant viral polymerases displaying either an increase or decrease of copying fidelity achieved through a single amino acid substitution (wainberg et al., 1996; men endez-arias, 2002; mansky et al., 2003; pfeiffer and kirkegaard, 2003; arnold et al., 2005; domingo, 2005; vignuzzi et al., 2006; coffey et al., 2011; gnadig et al., 2012; meng and kwang, 2014; rozen-gagnon et al., 2014; borderia et al., 2016) . the capacity of the virus to evolve at higher or lower rates than their ancestors is achievable through modest numbers of mutations (limited movements in sequence space, chapter 3), again emphasizing the evolvability of mutation rates. the rates of mutation and recombination need not be independent. m. vignuzzi and colleagues have shown that a mutator sindbis virus displays a higher recombination rate and enhanced production of di particles than the wild type virus (poirier et al., 2015) . a connection between mutation and recombination rates strengthens the evolutionary consequences of the modifications of template copying fidelity that can be achieved through a single amino acid substitution. 2.6 evolutionary origins, evolvability, and consequences of high mutation rates: fidelity mutants 2.7 hypermutagenesis and its application to generating a variation: apobec and adar activities some viral genomes either isolated from biological samples or evolved in cell culture show biased mutation types (e.g., monotonous g / a or c / u substitutions in the same genome), generally at frequencies of around 10 à2 substitutions per nucleotide (10-to 1000fold higher than standard mutation rates and frequencies) ( table 2 .1). biased hypermutation was first observed in some defective interfering (di) rnas of vesicular stomatitis virus (vsv) (holland et al., 1982) , and in variant forms of measles virus, associated with postmeasles neurological disease, such as subacute sclerosing panencephalitis (cattaneo and billeter, 1992) . hypermutation is mainly due to the activity of cellular deaminases, such as the apolipoprotein b mrna and the editing complex (apobec), or the adenosine deaminase acting on doublestranded rna (adar) families, that are involved in cellular editing and regulatory functions (sheehy et al., 2002; santiago and greene, 2008; nishikura, 2010; stavrou et al., 2014; pfaller et al., 2018; venkatesan et al., 2018) . in the event of a viral infection, such cellular functions can become part of an innate defense mechanism against the invading virus. viral proteins (i.e., vif in hiv-1) bind some apobec proteins, thus inhibiting mutagenesis and permitting virus survival (sheehy et al., 2002) . in oncoretroviruses, retroviruses, and hepatitis b virus (hbv), the apobec-3 cytidine deaminase acts on singlestranded dna and results mainly in g / a and c / u hypermutation, that may affect 40% e100% of the g residues. the preferred sequence context for g hypermutation in hiv-1 observed in vivo is gpa > gpg > gpt z gpc. the specific dinucleotide context of the hypermutated sites provides a means to distinguish genomes that have undergone hypermutation by cellular activities from those that are heavily mutated by other mechanisms, such as the action of mutagenic agents (chapter 9). apobec 3 proteins play a role in cancer through cytidine deaminase mutagenesis and generation of double-strand breaks in chromosomal dna (wang et al., 2016) . apobec 3 levels in the cell may be regulated by cellular and viral proteins, for example, human papillomavirus (hpv) oncoprotein e that stabilizes apobec 3a in human keratinocytes that may promote cervical cancer (westrich et al., 2018) . the adar-associated hypermutation was identified in negative-strand rna riboviruses and results mainly in a / g and u / c hypermutation. it is originated by a / i (inosine) modifications in double-stranded viral rna catalyzed by adar-1 l, one of more than 100 proteins inducible by type i ifn (maas et al., 2003) . inosine can be recognized as g by the replication machinery (valente and nishikura, 2005) , although it can form wobble base pairs also with a and u (fig. 2.2) . hypermutation can contribute to genetic variation of viruses (hirose et al., 2018) . there are additional mechanisms of hypermutagenesis. higher than average mutation frequencies can occur as a result of replication in the presence of biased concentrations of the standard nucleotide substrates; this has been applied to the in vitro generation of genes mutated at frequencies of 10 à1 to 10 à2 (mutagenic pcr), as a powerful tool to study sequence-function relationships and functional robustness of nucleic acids and proteins (meyerhans and vartanian, 1999) . error-prone pcr has been used in experiments of in vitro evolution of nucleic acid enzymes to generate heterogeneous collections of nucleic acid sequences to select for molecules capable of catalyzing specific reactions (joyce, 2004 ) (chapter 1). high mutation rates have practical implications in laboratory studies on the behavior of 2. molecular basis of genetic variation of viruses: error-prone replication virus mutants obtained by molecular cloning of a biological sample, or constructed by site-directed mutagenesis. a transition mutation that causes a strong fitness decrease but that still allows residual rna genome replication will most likely revert following infection or transfection of cells with the mutant construct and subsequent viral replication. double or triple mutants (preferentially including transversions) should be engineered (when possible according to the genetic code) to study the behavior of a viral mutant with an amino acid replacement of interest that may produce a fitness decrease. as an example, a c / u transition found in an open reading frame of an rna virus may convert a pro into a ser (ccg / ucg). since ser will revert to pro through a u / c transition in the triplet (a common type of misincorporation by most polymerases), ser should be engineered to be encoded by agu; in the course of replication, reversion to pro would require at least two transversions since the codons for pro are ccu, ccc, cca, or ccg. thus, if effects derived from the difference in the primary sequence of the rna or codon bias do not intervene in the behavior of the viral genome, codons with a high genetic barrier to reversion should be engineered for studies involving viral replication. in general, deletions revert at a much lower frequency than point mutations, and when appropriate for the question under study, a deletion should be introduced within the gene of interest to probe gene function in reversegenetics studies. high mutation rates also imply that infection or transfection with debilitated mutant viruses may result in progeny with sequences that differ from the input. v. i. agol and colleagues have coined the term quasi-infectious to refer to mutant viruses that are capable of yielding progeny, but the progeny differs from the initial genome (pseudorevertants) (gmyl et al., 1993; agol and gmyl, 2018) . the difference between the input mutant and the rescued progeny virus will depend on the type of genetic lesion in the input virus and its consequences for virus multiplication. a single point mutation that decreases replication is likely to evolve to yield a true revertant (return to the original sequence) upon replication. if the same reversion depends on two or more mutations, a true revertant will require extended replication for exploration of sequence space (chapter 3), and selection of compensatory mutations elsewhere in the genome (sometimes referred to as second site revertants) becomes an alternative for fitness gain. the term compensatory applies to mutations that compensate for the deleteriousness of other mutations. a typical example is a mutation that decreases the stability of a stem in an rna stem-loop that functions as a cis-acting element. a compensatory mutation restores a stable stem needed for the activity. transfection of cells by an engineered virus with some preselected genetic modification (produced either from cdna copies of a viral genome or by chemical synthesis) may yield progeny genomes, which differ from the parent. if a substantial loss of replicative capacity is produced by a drastic genetic change (an indel, loss of a stem-loop structure, etc.) selection of a true revertant becomes extremely unlikely. the compensatory generation of alternative structures (or constellations of point mutations) that restores replication (partially or completely) becomes an interesting and informative possibility. procedures to copy an entire viral rna genome into a cdna for reverse genetics studies are now available (fan and di bisceglie, 2010) . if for technical reasons an infectious cdna clone is constructed from several molecules, which were copied from different genomes present in the mutant spectrum, the ligation product may be transcribed into an rna, which is not infectious. this is because, while some constellations of mutations may be compatible with infectivity, others may not, or may allow limited, suboptimal replication, thus favoring the selection of additional mutations or reversions. the same applies to a synthetic genome based on one of the multiple genomic sequences from a viral 2.8 error-prone replication and maintenance of genetic information: instability of laboratory viral constructs isolate. individual mutations may be detrimental either per se, or by the combined presence of other mutations. the joint effect of different mutations in the same genome is often referred to as epistasis. mutations that reinforce each other with regard to a viral function are said to produce positive epistasis, and those that interfere with each other produce negative epistasis (also mentioned in section 2.3). epistasis in rna viruses may be blurred by the weight of mutant spectra in determining viral behavior through intergenomic interactions (chapter 3). an interesting contrast that recapitulates concepts given in sections 2.5 and 2.6 is the effect of an active proofreading-repair activity in maintaining the infectivity of a viral genome upon its extended replication in vitro (in a test tube, in the absence of cellular extracts). the 19,285 bp bacteriophage f29 dna can be amplified at least 4000-fold without detectable loss of infectivity due to the fidelity of f29 dna polymerase conferred by a 3 0 e5 0 proofreading-repair exonuclease activity (bernad et al., 1989) . engineered f29 dna polymerases provide a powerful amplification tool in genomics (de vega et al., 2010) . in contrast, the 4220 nucleotides long qb rna rapidly loses its infectivity when replicated by qb replicase in vitro due to the accumulation of mutations and deletions in the viral rna (mills et al., 1967; sabo et al., 1977) . the error-prone qb replicase is not adequate to amplify infectious viral rna, but it was at the origin of the quasispecies concept to be discussed in chapter 3. mutagenic dna polymerases (generally those involved in dna repair) are an alternative to mutagenic pcr (section 2.7) to produce randomly mutated collections of nucleic acid molecules (forloni et al., 2018) . recombination is the formation of a new genome by covalent linkage of genetic material from two or more different parental genomes (fig. 2.7) . recombination can also involve different sites of the same genome to yield insertions or deletions, such as in the formation of defective interfering (di) genomes. it is a widespread mechanism of genetic variation in all biological systems, and in cells, it underlies critical physiological and developmental processes (splicing, generation of diversity in figure 2.7 rna recombination and segment reassortment. (a) scheme of replicative and nonreplicative rna recombination. rna polarity is indicated by þ, à symbols. replicative recombination is displayed as the result of template switching during minus-strand rna synthesis. nonreplicative recombination is depicted as the outcome of breakage and ligation (joining) of fragments of plus-strand rna. (b) an example of genome segment reassortment with the formation of a new segment constellation in which six genomic segments originate from one parent (blue) and two from the other (gold). influenza virus is the best-known example (see text). immunoglobulin genes and t cell receptors, transposition events, phase variation in bacteria, repair pathways that promote postreplicative error correction, etc.). cellular dna recombination relates to replication, repair, and completion of dna replication, operations that involve multiple proteins displaying a variety of activities (smith and jones, 1999; alberts et al., 2002; nimonkar and boehmer, 2003; friedberg et al., 2006) . recombination occurs both with dna and rna viruses, often with the participation of the virus replication machinery. several types of recombination have been distinguished in viruses: homologous versus nonhomologous recombination, according to the extent of nucleotide sequence identity around the recombination (crossover) site, and replicative versus nonreplicative recombination, according to the requirement of viral genome replication for recombination to occur (kirkegaard and baltimore, 1986; king, 1988; lai, 1992; nagy and simon, 1997; plyusnin et al., 2002; boehmer and nimonkar, 2003; gmyl et al., 2003; chetverin et al., 2005; agol, 2010; simmonds, 2010; bujarski, 2013; perez-losada et al., 2015; agol and gmyl, 2018; bentley and evans, 2018) . as in the case of cells, homologous recombination in double-stranded dna viruses is intimately connected with dna replication and repair. it implicates multiple viral gene products (dna polymerase, single-stranded dna-binding proteins, processivity factors, helicase-primase, eukaryotic topoisomerase i, etc.), and a succession of protein-catalyzed steps (czarnecki and traktman, 2017) . in the copy choice (or template switching) mechanism, the nascent dna switches from one template molecule to another, resulting in the synthesis of recombinant, daughter dnas. in its basic form, recombination by breakage and rejoining starts with the introduction of a nick at one of the strands of each parental dna, strand invasion of one parental dna by the other, branch migration, ligation at the nicks (linking dna strands from the two parents), and further isomerization and cleavage reactions. dna recombination is responsible for the endonuclease-mediated isomerization of herpesvirus genomes [four isomers defined by the orientation of the long (l) and short (s) regions of the viral genome]. during the late phase of herpes simplex virus-1 replication, the frequency of recombination has been estimated at 0.6% per kb of the genome . integration or excision of proviral dna or temperate bacteriophage dna, are examples of site-specific recombination that involves specific enzyme activities (i.e., retroviral integrases), and requires a short stretch of nucleotide sequence identity. the copy choice mechanism of homologous rna recombination is also associated with genome replication. an rna polymerase molecule with its nascent rna product jumps into the corresponding position of another template molecule, to complete synthesis of the rna product ( fig. 2.7) . given the large numbers of viral genomes often present in replication complexes (also termed replication factories) in each infected cell, it is not surprising that this mechanism may give rise to frequent recombinant progeny genomes. formation of mosaic genomes has long been recognized as an essential feature of the genetics of some retroviruses and plant rna viruses. for hiv-1 and some plant rna viruses recombination frequencies have been estimated at 2%e10% of progeny per 100 nucleotides; for picornaviruses and coronaviruses the number of recombinants amounts to 10%e20% of the progeny (king, 1988; lai, 1992; nagy and simon, 1997; levy et al., 2004; urbanowicz et al., 2005; sztuba-solinska et al., 2011) . using a phylogenetic approach the average recombination rate of hiv-1 in vivo was estimated in 1.4 â 10 à4 recombination events/site/generation, which is about fivefold greater than the average point mutation rate (shriner et al., 2004) . a ten-fold lower value of 1.4 â 10 à5 recombination events/site/ 2.9 recombination in dna and rna viruses generation was estimated from the changes in the genetic composition of hiv-1 within single patients (neher and leitner, 2010) . recombination is required for hiv-1 replication and genome integrity (rawson et al., 2018) . in negative-strand rna viruses recombination may be inefficient or absent, but some of them can display homologous recombination (plyusnin et al., 2002) , and a high rate of generation of di rnas and other types of defective genomes (roux et al., 1991; rezelj et al., 2018) . recombination frequency may be altered by environmental factors that affect viral replication. a decrease of intracellular nucleotide levels as a result of treatment of cells with hydroxyurea may favor template switching reflected in an increase of intra and intermolecular recombination (pfeiffer et al., 1999; svarovskaia et al., 2000) . homologous rna recombination can also be influenced by amino acid substitutions in the polymerase, the primary sequence in the rna (i.e., high frequency of template switching in au-rich regions), the sequence identity between the nascent strand and acceptor template, and secondary structures at or around the crossover sites, among other influences (nagy and simon, 1997; agol, 2010; agol and gmyl, 2018) . since recombination necessitates coinfections of the same cell by at least two parental genomes, the persistence of a viral genome in a cell increases the likelihood of sequential coinfections, unless some reinfection or superinfection exclusion mechanism operates [(webster et al., 2013) and references therein]. without such restrictions, persistently infected cells may be an environment with a higher probability of recombination than transiently infected cells, assuming comparable genome loads at the sites of replication. nonreplicative recombination does not require replication of the viral genome, and has been described upon cotransfection of cells with viral rna fragments that could not replicate by themselves (gmyl et al., 2003; gallei et al., 2004; agol, 2010; agol and gmyl, 2018; bentley and evans, 2018 ). it appears to be a promiscuous event with a required 3 0 -phosphate in the 5 0 partner rna and a 5 0 -hydroxyl residue in the 3 0 partner rna mediated by cellular components whose mechanisms of activity are not understood. the emerging picture is that the frequency of recombination varies among viruses, and that as new tools for genome analyses have become available, recombination has been detected in an increasing number of viruses. recognition of recombination in a viral system is facilitated when a cell culture system is available. controlled infection of cells with genetically marked parental viruses has been essential to estimate recombination frequencies, and to distinguish true recombination from mutationreversion events that may mimic the formation of recombinants. as with the concept of high genetic variation in rna viruses, recombination has often gone from being considered marginal to prominent and relevant; hcv is a typical example (galli and bukh, 2014) , with presently at least one chimera established as a circulating recombinant form in the field. viral replicative machineries may be endowed with features that influence the occurrence of recombination. one such feature is processivity of the viral polymerase (capacity of continued copying of the same template molecule). genome detachment of the polymerase complex from one genome to bind either to a different genome or to a distant site of the same genome is part of the standard replicative cycle of viruses, such as retroviruses and coronaviruses. reverse transcriptase participates in strand transfer during dna synthesis, and coronavirus polymerase switches from one template site to another during discontinuous rna synthesis. it may be significant that they belong to viral families displaying high recombination frequencies (makino et al., 1986) . thus, here again, we encounter a "molecular instruction" that evolved as an essential feature of viral genome replication, and that can be exploited to generate variation, and permit new genomic forms to undergo the scrutiny of selection (neher and leitner, 2010) recombination is diagnosed by discordant positions of different genes or genomic regions in phylogenetic trees, as a result of the transfer of part of a viral genome from representatives of one lineage to representatives of another lineage (chapter 7). a commonly used procedure measures similarity values between sequences using a sliding-window scanning method. the recombination crossover point (where the two parental sequences meet) is identified by the point (or region) where the similarity plot crosses from one sequence into another (salemi and vandamme, 2004; martin et al., 2005; kosakovsky pond et al., 2006 , perez-losada et al., 2015 . crossover points along a viral genome are not distributed at random, either because polymerase detachment from the template is sequence-dependent or because many of the recombination events do not lead to viable progeny. absence of recombinant viability introduces a parallel with the distinction between mutation rate and mutation frequency (section 2.5); that is, a difference between what does occur at the biochemical level during replication and what is subsequently observed upon analysis of the replication products. newly arising recombinants may be subjected to negative selection, and only a viable subset might be detectable in the progeny virus (king, 1988; lai, 1992 ). an elegant study by d. j. evans and colleagues documented "imprecise" enterovirus recombinant intermediates that were lost upon serial virus passage (lowry et al., 2014) . recombination is viewed as a biphasic process consisting of initial imprecise events followed by a stage of resolution in favor of fit recombinants. the distinction between generation and resolution events that applies both to mutants and recombinants has yet another implication for rna virus genetics. some mutants or recombinants that in isolation do not exhibit sufficient replicative fitness to acquire dominance in a population may nevertheless persist as minority genomes. they may display low-level replication or be maintained by complementation by partner genomes (as in the case of two fmdv genome segments that are described in section 2.11). as minority genomes, they may engage in modulatory activities (chapter 3). in viruses whose genomes are composed of two or more rna or dna segments, genome segment reassortment consists in the formation of new constellations of viral genomic segments from two or more parental genomes (mcdonald et al., 2016) (fig. 2.7) . reassortment can produce new phenotypic traits. it is the main mechanism of antigenic shift of influenza a virusesdoften associated with new influenza pandemics (webster et al., 1992; morse, 1994; gibbs et al., 1995; domingo et al., 2001) das opposed to antigenic drift, which is mediated by amino acid substitutions in the surface proteins hemagglutinin and neuraminidase (barbezange et al., 2018) . reassortments occur among the 9e12 doublestranded rna segments of the widespread reoviridae family (tanaka et al., 2012) . fitness differences among all possible segment combinations (2 n , for two types of coinfecting particles with n genome segments) determine the types of genome segment groupings that dominate subsequent rounds of infection. in the laboratory, analysis of reassortant viruses has been applied to map a viral function into one segment or a combination of segments. genomic segments can be encapsidated either into a single virus particle (as in orthomyxoviruses or arenaviruses) or into separate particles 2.10 genome segment reassortment (as in multipartite plant viruses). a multipartite virus can have either rna or dna as genetic material. the plant nanoviruses have 6-8 molecules of single-stranded circular dna of about 900e1000 nucleotides, and each segment encodes a single protein. in the case of the nanovirus faba bean necrotic stunt virus, its eight segments vary in frequency in a hostdependent manner (sicard et al., 2013) . this observation led the authors to propose a "setpoint genome formula," which may reflect the control of segment (gene) copy number that may provide some still unrecognized benefit to the multipartite phenotype (see next section 2.11). in principle, replication of multipartite viruses requires that each cell be coinfected by at least one of each type of particle harboring a different genome type, which in fact represents a remarkable cost for replicative efficiency. the fact that unsegmented and segmented rna viruses are well represented in our biosphere suggests that neither of the two organizations confers a definitive and general advantage for long-term survival. the origin of viral genome segmentation is a debated issue, although there is general agreement that it may confer adaptive flexibility to viruses. most proposals have been based on theoretical studies. segmentation has been viewed as a form of sex that facilitates genomic exchanges to counteract the effect of deleterious mutations (chao, 1988; szathmary, 1992 ). an alternative, not mutually exclusive model is that segmentation confers an advantage because replication of shorter rna molecules is completed earlier than the unsegmented counterparts (nee, 1987) . yet another possibility is that the lifestyle of a virus (in particular, the particle yield in connection with the number of surrounding susceptible cells), shaped over long evolutionary periods, may favor segmentation over intactness of a genetic message or vice versa. an experimental system of genome segmentation is available with the picornavirus foot-and-mouth disease virus (fmdv). its single-stranded rna genome underwent a modification akin to genome segmentation when the standard virus was subjected to 260 passages in bhk-21 cells at high multiplicity of infection. the experiment was originally intended to investigate the limits of fitness gain following prolonged multiplication in a defined environment, in this case, bhk-21 cells in culture. the starting fmdv had not been well adapted to the bhk-21 cell culture environment since it derived from a diseased swine during a disease outbreak, and it was minimally propagated in bhk-21 cells to obtain a biological clone by plaque isolation. upon extensive replication of the clone in bhk-21 cells, the virus evolved toward a bipartite genome (garcía-arriaza et al., 2004) . each of the two pieces of rna that composed the bipartite (or segmented) genome version contained in-frame deletions affecting trans-acting proteins ( fig. 2.8) . each segment in isolation could not infect cells productively, but, when present together, they were infectious by complementation, and killed cells in the absence of standard fmdv. a low multiplicity of infection rapidly selected the full-length genome as a result of recombination of the two parental, defective segments (garcía-arriaza et al., 2004) . the particles containing the shortened rna were thermally more stable than the standard particles (ojosnegros et al., 2011) , but this difference did not explain the initial trigger of the segmentation event. the solution to this question came with the demonstration that the transition toward genome segmentation was possible because of an extensive exploration of the mutational sequence space by the standard virus. indeed, the mutations that accumulated during serial passages enhanced the fitness of the segmented genome version to a much higher extent than the fitness of the standard genome (moreno et al., 2014) (fig. 2.8) . thus, gradual evolution (drift in sequence space) was a requirement for the major transition toward segmentation, thus adding reassortment to mutation and recombination as potential mechanisms of genetic variation of this laboratory-adapted picornavirus. cooperation and complementation are discussed in section 3.8 of chapter 3. it should be noted that segmented forms of rna viruses have been engineered, but little is known of the incurred fitness cost; it is relevant to have shown that segmentation was possible upon unperturbed replication of a virus with an unsegmented rna genome. the experimental result suggests that in evolution there is no unsurmountable barrier that allows the conversion between intact and split forms of the same genome, reflecting remarkable genome flexibility that will be emphasized in chapter 7 in the context of the relevance of virus variation in the emergence of viral pathogens. mutation, recombination, and segment reassortment contribute to the evolution of most dna and rna viruses. sometimes one form of genetic change appears to be more prominent than another, and sometimes the concerted action of recombination or reassortment with the mutation is apparent [i.e., antigenic drift in influenza virus, following the origin of a new antigenic type through reassortment (ghedin et al., 2005) ]. a mutation is a universal form of genetic change. it underlies numerous adaptive responses and critical biological transitions in viruses, and it is a prerequisite for recombination and reassortment to have a biological impact. if mutations were not present in different template molecules during replication, recombinants with the crossover point at equivalent positions of the parental genomes would be "silent," and display the same behavior as the parental genomes. apparently, "silent" recombination events may take place within replicative units; even if some mutations distinguished individual genomes of the same quasispecies swarm, a recombinant would not be distinguished from a mutant genome. the frequency of recombination in hiv-1 was noticed only when the acquired figure 2.8 evolution toward rna genome segmentation in the laboratory. the monopartite, standard fmdv genome (clone c-s8c1 or pmt28, top) was subjected to 260 passages in bhk-21 cells. the resulting population p260 lacked detectable standard genome that could be rescued by low moi passages. the evolved c-s8p260 accumulated 30 point mutations (depicted as vertical lines on the genome at the bottom) and consisted in two segments that were infections by complementation: d417, that lacked most of the l protease-coding region, and d999 that lacked most of the capsid proteins vp3, vp1-coding region. see text for further details and references. immune deficiency syndrome (aids) pandemic had advanced, and the virus had diversified through the accumulation of mutations. similar arguments apply to segment reassortment. genomes necessitate mutation-driven diversification for reassortment to provide a biological difference; detection of a reassortant will be easier the larger the replicative advantage it confers to the virus (see also chapter 10). the evolutionary significance of recombination has been viewed in two opposite ways: as a means to rescue fit genomes from less fit parents (a conservative force that eliminates deleterious mutations), or as a means to explore new genomic forms for adaptive potential (a vast substrate for the exploration of sequence space; chapter 3) [reviewed in (zimmern, 1988; lai, 1992; worobey and holmes, 1999; simmonds, 2010; perez-losada et al., 2015) ]. recombination has been probably at the origin of new viruses that presently occupy a well-established niche, and it is also at play today to expand diversity during the spread of viruses. as a historical event, the coronavirus mouse hepatitis virus appears to have acquired its hemagglutininesterase gene by recombination with an influenza c virus. the alphavirus western equine encephalitis virus originated probably by recombination between sindbis-like and eastern equine encephalitis-like viruses [reviewed in different chapters of ]. several recent poliomyelitis outbreaks have been associated with recombinants between oral poliovirus vaccine (opv) viruses and other circulating enteroviruses (gavrilin et al., 2000; kew et al., 2002; oberste et al., 2004; muslin et al., 2015) . intersubtype hiv-1 recombinants play a key role in current hiv-1 diversification, with around 100 circulating recombinant forms (and the number is growing) displaying complex mosaic structures (multiple crossover sites) (thomson et al., 2002; gerhardt et al., 2005) . in addition, other hiv-1 recombinants have been characterized that are not established epidemiologically. fewer hcv recombinants have been identified, but the number is likely to increase as the virus diversifies in nature. positive selection of hiv-1 recombinants that unite different drug-resistant mutations in the same genome offers an example of the conservative force of recombination to rescue fit viruses in the face of a strong selective constraint (men endezarias, 2002) . recombination is expected to play an increasing role in the spread of drug resistance among viruses for which new antiviral agents are in use, such as hbv and hcv. some defective dna and rna genomes that include indels, notably di rnas, which originate from recombination events can play an important role in the establishment and maintenance of persistent infections in cell culture, and can modulate viral infections in vivo (holland and villarreal, 1974; roux et al., 1991; rezelj et al., 2018) . detailed genetic and biochemical analyses by a. huang, j.j. holland and their colleagues on the generation of vsv di's and their interplay with the standard, infectious vsv contributed to unveil a continuous dynamics of genetic variation, competition, and selection, observable within short time intervals, a hallmark of rna genetics (palma and huang, 1974; holland et al., 1982) , fully confirmed by application of new sequencing techniques. di particles and defective genomes are present in populations of positive-and negative-strand rna viruses as they multiply in their natural hosts (n€ uesch et al., 1989; drolet et al., 1995; li et al., 2011; saira et al., 2013; ke et al., 2013; rezelj et al., 2018) . their widespread presence in vivo may mean that they are an unavoidable side-product of the replication machineries (i.e., instruction to recombine) or that selection might have favored their generation. both possibilities are compatible. an instruction whose result is a means to modulate replication of the corresponding standard viruses or the antiviral immune response will be selected as a consequence of its biological effects. di rnas can be regarded as the tip of the iceberg of many classes of defective genomes with a range of interfering or potentiating capacities that may coexist with standard animal, plant, insect, and bacterial viruses, and that may facilitate persistence and modulate disease symptoms (holland et al., 1982; vogt and jackson, 1999; l opez-ferber et al., 2003; rosario et al., 2005; sachs and bull, 2005; villarreal, 2005; aaskov et al., 2006; rezelj et al., 2018) . noncytopathic coxsackievirus b3 (cvb3) variants with deletions at the untranslated 5 0genomic region were isolated from hearts of mice inoculated with cvb3. the variants replicated in vivo and were associated with longterm viral persistence (kim et al., 2005) . despite the continuous dynamics of the escape of infectious virus to the interfering activities of dis, some authors consider dis as potential antiviral agents (dimmock and easton, 2014) . if defective genomes are competent in rna (or dna) synthesis or are complemented to replicate, they can act as dominant-negative swarms, provided they reach a sufficient load. in this manner, they may underlie the suppressive effects of mutant spectra of viral quasispecies. intramutant spectrum decrease of replicative capacity due to the presence of defective genomes is one of the mechanisms of virus extinction evoked by enhanced mutagenesis (chapters 3 and 9). recombination events must have been the last step in ancestral processes of horizontal gene transfer that mediated the incorporation of host genes (or gene segments) into viral genomes, and vice versa. host genes related to immune responses were probably captured by complex dna viruses at early stages of their evolution (alcami, 2003; mcfadden, 2005) . mosaicism associated with nonhomologous recombination events is the norm among tailed bacteriophages (canchaya et al., 2003) . nonhomologous recombination can give rise to genomic sequences with a viral and a nonviral moiety. they include di rnas of sindbis virus-containing cellular rna sequences at their 5 0 ends, some cytopathic forms of bovine viral diarrhea virus, rna of potato leafroll virus-containing tobacco chloroplast rna, or an influenza virus with an insertion of ribosomal rna into the hemagglutinin gene, mentioned in chapter 5 regarding transient, high fitness levels [reviewed in (domingo et al., 2001) ]. phylogenetic analyses have suggested that recombination between rna and dna viruses might have occurred to give rise to some present-day single-stranded dna viruses (stedman, 2013) . however, the evidence for this attractive possibility is indirect and, to my knowledge, no experimental evidence of viral rna-viral dna recombination in cell culture or in vivo has been reported. viability of mutant and recombinant viral genomes is severely constrained by the evolutionary history of the virus that has shaped viral genomes as coordinated sets of modules (botstein, 1980 (botstein, , 1981 zimmern, 1988; koonin and dolja, 2014) . experimental studies with engineered recombinant viruses have shown that modularity can restrict recombination (martin et al., 2005) . the three molecular mechanisms of viral genome variation (mutation, recombination, and reassortment) are not incompatible, although it may sometimes be difficult to discern their occurrence (varsani et al., 2018) . it would be truly remarkable if a viral system could be proven to be totally devoid of one of the mechanisms of genetic variation. it would imply that there are powerful molecular reasons to dispense with an effective adaptive mechanism. absence of a mechanism is extremely difficult to demonstrate but, if we could, its basis would open a new chapter of molecular virology. there is an ongoing controversy regarding clonality versus nonclonality in biological evolution, particularly concerning the evolution of cellular parasites (heitman, 2010; tibayrenc 2.12 mutation, recombination, and reassortment as individual and combined evolutionary forces and ayala, 2012, 2014; ramirez and llewellyn, 2014; hauser and cushion, 2018) . clonality means asexual progeny from a single ancestor. in the case of viruses, clonal evolution emphasizes reproduction without the exchange of genetic material among two or more parental genomes. sexual reproduction necessarily involves the exchange of genetic material. the question for viruses is interesting because we would be inclined to propose clonal evolution despite considerable promiscuity of recombination and reassortment. a tentative solution was offered based on one assumption and some experimental observations. the assumption is that recombination is not a requirement for viruses to complete their infection cycles. despite the possibility that recombination might be imprinted in the replication apparatuses (rendering it inherent to replication), its occurrence is not a necessity. the experimental observations are of two sorts. one is that historically recombination and reassortment have been at the origin of the emergence and reemergence of viral pathogens [western equine encephalitis virus, pandemic influenza viruses, emergent circulating poliovirus, hiv-1 and hcv recombinants, etc. (section 2.12)]. the second observation is that recombination-based genome segmentation can occur given the adequate population dynamics and competitive environment, as documented with fmdv (section 2.11). in consequence, the distinction was made between mechanistically unavoidable but biologically irrelevant, and meaningful recombination (perales et al., 2015) . the latter form of recombination requires prior diversification of parental genomes by mutation and a number of cellular and epidemiological conditions. despite its relevance to evolutionary transitions and viral emergence, it is not a requirement for virus survival, propagation, and evolution. this marks a contrast with the genomic exchanges associated with sexual reproduction. the proposal is that viruses evolve clonally at widely different time scales (intrahost or within-host evolution vs. long-term evolution at the epidemiological level). similar arguments apply to mutation. this point will be addressed again in the closing chapter 10, concerning implications of clonality. all forms of genetic variation of viruses must be viewed essentially as blind processes despite preferences of nucleotide sequences or structures for mutation and recombination events: hot spots with higher than average rate values, and cold spots with lower than average rate values. mutation originates largely in fluctuations of electronic structure that modify base-pairing properties, and from features of polymerase-template interactions, not subject to regulation, in the sense that we understand the regulation of gene expression or enzymatic activity. absence of regulation is not incompatible with long-term evolution having shaped the molecular interactions that yield a level of mutagenesis compatible with survival and adaptability. given the biological consequences of mutation rates, many additional studies are needed for the biological phyla, to quantity not only basal mutation rates but also the possible presence of mutator strains. similar arguments can be used for recombination and reassortment. the number of segments that enter a new genomic constellation may be regulated but not which variant forms of the individual segments will make up the new viral particles. it is short-term selection acting at the very center of replication and recombinant complexes that preserves some mutant and recombinant forms in detriment of others. subsequent levels of selection occur when variant forms expand in multiple rounds of infection first within cells, then within an organism and then at the epidemiological level. the very nature of life in our planet has been built upon an inherent tendency to instruct variation in an incessant fashion, as necessary and unavoidable as the physical principles that dictate the behavior of our universe. the net result of all mechanisms of genetic variation available to a virus is the generation of repertoires of variant genomes for random drift and selective forces to act upon. in other terms, genetic variation sets the scene for the actors of evolution to play their roles, and secure a continuous input of new forms despite subtle or catastrophic environmental perturbations. the same forces that drive general evolution have produced the dominant virus forms we see in nature, with all their nuances in the interaction with cell components. the adaptation of viruses to participate in intracellular processes with cells dictates that genetic variation of viruses has its limits to prevent deleteriousness. this is currently exemplified by the effects of amino acid substitutions in viral polymerases that either increase or decrease templatecopying fidelity. viruses have reached a compromise between the stability of core information and flexibility for adaptability. although not yet treated in this chapter, viral population numbers are a key parameter in the evolutionary events. next chapters address some of these questions, not only in general conceptual terms but also in the way evolution affects our daily confrontation with viral disease (see summary box). • mutation, recombination, and genome segment reassortment are the mechanisms of genetic variation used by dna and rna viruses. mutations are due mainly to changes in the electronic distributions of the standard nucleotides, to damage of nucleotides by external influences, and by alignment alterations of the template relative to product polynucleotide chains. the effect of a mutation can range from being well-tolerated to highly detrimental or lethal. • mutation frequencies are only an indirect consequence of mutation rates. their values for viruses whose replication is catalyzed by polymerases devoid of proofreading-repair activity are 10 5 -to 10 6 -fold higher than those displayed by replicative cellular dna polymerases. error-prone replication is a hallmark of rna viruses and some dna viruses. the larger the amount of genetic information encoded in a viral genome, the lower the mutation rate must be to maintain the genetic message. • several mechanisms of genetic recombination have been described for dna and rna viruses. the best characterized is homologous recombination whose frequency of occurrence is dependent on the replicative machinery, in particular, polymerase processivity. genome segment reassortment is operative in segmented genomes, and it gives rise to biologically relevant changes, such as an antigenic shift in the influenza type a viruses. • studies with foot-and-mouth disease virus have shown that extensive evolution of an unsegmented rna genome has the potential to undergo a recombination-mediated transition akin to genome segmentation. therefore, segmented and unsegmented forms of rna viruses need not be considered as completely unrelated classes of genome organization. • recombination and genome segment reassortment have been viewed as conservative forces to rescue viable genomes from a damaged pool, and also as a means to explore new genomic compositions that deviate from their parents. all forms of genetic variation give rise to repertoires of variant genomes on which selection and random drift act to produce the viral forms that we isolate and study. long-term transmission of defective rna viruses in humans and aedes mosquitoes frequency spectrum neutrality tests: one for all and all for one picornaviruses as a model for studying the nature of rna recombination emergency services of viral rnas: repair and remodeling. microbiol causes of genome instability gene expression and molecular evolution a universal bmv-based rna recombination systemdhow to search for general rules in rna recombination fine-tuning translation kinetics selection as the driving force of codon usage bias in the hepatitis a virus capsid determinants of rnadependent rna polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a footand-mouth disease virus mutant with reduced sensitivity to ribavirin remote site control of an active site fidelity checkpoint in a viral rna-dependent rna polymerase seasonal genetic drift of human influenza a virus quasispecies revealed by deep sequencing origin and evolution of poxviruses the proportion of revertant and mutant phage in a growing population, as a function of mutation and growth rate ribavirin: a drug active against many viruses with multiple effects on virus replication and propagation. molecular basis of ribavirin resistance fidelity of dna replication ─ a matter of proofreading mechanisms and consequences of positive-strand rna virus recombination a conserved 3'-5' exonuclease active site in prokaryotic and eukaryotic dna polymerases nucleic acids. structures, properties, and functions. university science books herpes virus replication fidelity variants and rna quasispecies a theory of modular evolution for bacteriophages a modular theory of virus evolution correlation between mutation rate and genome size in riboviruses: mutation rate of bacteriophage qbeta a synonymous variant in irgm alters a binding site for mir-196 and causes deregulation of irgm-dependent xenophagy in crohn's disease synonymous codons: choose wisely for expression genetic recombination in plant-infecting messenger-sense rna viruses: overview and research perspectives lateral dna transfer. mechanisms and consequences structure-function relationships underlying the replication fidelity of viral rnadependent rna polymerases phage as agents of lateral gene transfer incorporation fidelity of the viral rna-dependent rna polymerase: a kinetic, thermodynamic and structural perspective mutations and a/i hypermutations in measles virus persistent infections evolution of sex in rna viruses viral rnadirected rna polymerases use diverse mechanisms to promote recombination between rna molecules insertion/deletion frequencies match those of point mutations in the hypervariable regions of the simian immunodeficiency virus surface envelope gene arbovirus high fidelity variant loses fitness in mosquitoes and mice genetic variation in retroviruses variation in rna virus mutation rates across host cells parallel evolution of drug resistance in hiv: failure of nonsynonymous/synonymous substitution rate ratio to detect selection the vaccinia virus dna polymerase and its processivity factor silent mutations in sight: co-variations in trna abundance as a key to unravel consequences of silent mutations improvement of phi29 dna polymerase amplification performance by fusion of dna binding motifs linking rna sequence, structure, and function on massively parallel highthroughput sequences defective interfering influenza virus rnas: time to reevaluate their clinical potential as broad-spectrum antivirals? virus entry into error catastrophe as a new antiviral strategy viral quasispecies nucleotide sequence heterogeneity of an rna phage population genetic variability and antigenic diversity of foot-and-mouth disease virus quasispecies: the concept and the word quasispecies and rna virus evolution: principles and consequences evolution of footand-mouth disease virus viral quasispecies: dynamics, interactions and pathogenesis a constant rate of spontaneous mutation in dna-based microbes mutation rates among rna viruses detenction of truncated virus particles in a persistent rna virus infection in vivo evolvability is a selectable trait high fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing error catastrophe and antiviral strategy sequence space and quasispecies distribution adaptive value of high mutation rates of rna viruses: separating causes from consequences multiple molecular pathways for fitness recovery of an rna virus debilitated by operation of muller's ratchet rt-pcr amplification and cloning of large viral sequences adaptation of mrna structure to control protein folding a comparison of viral rna-dependent rna polymerases structural insights into replication initiation and elongation processes by the fmdv rna-dependent rna polymerase random mutagenesis using error-prone dna polymerases specialized dna polymerases, cellular survival, and the genesis of mutations dna repair and mutagenesis statistical tests of neutrality of mutations against population growth, hitchhiking and background selection extremely high mutation rate of a hammerhead viroid rna recombination in vivo in the absence of viral replication comparative analysis of the molecular mechanisms of recombination in hepatitis c virus dna replication-a matter of fidelity evolutionary transition toward defective rnas that are infectious by complementation information dynamics in carcinogenesis and tumor growth evolution of circulating wild poliovirus and of vaccine-derived poliovirus in an immunodeficient patient: a unifying model in-depth, longitudinal analysis of viral quasispecies from an individual triply infected with late-stage human immunodeficiency virus type 1, using a multiple pcr primer approach large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution molecular basis of virus evolution functional and genetic plasticities of the poliovirus genome: quasi-infectious rnas modified in the 5'-untranslated region yield a variety of pseudorevertants nonreplicative homologous rna recombination: promiscuous joining of rna pieces? rna 9 coxsackievirus b3 mutator strains are attenuated in vivo a single-nucleotide synonymous mutation in the gag gene controlling human immunodeficiency virus type 1 virion production is sex necessary for the proliferation and transmission of pneumocystis? evolution of eukaryotic microbial pathogens via covert sexual reproduction within-host variations of human papillomaviruses reveal apobec signature mutagenesis in the viral genome persistent noncytocidal vesicular stomatitis virus infections mediated by defective t particles that suppress virion transcriptase rapid evolution of rna genomes virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions ntp-mediated nucleotide excision activity of hepatitis c virus rna-dependent rna polymerase directed evolution of nucleic acid enzymes phylodynamic analysis of the emergence and epidemiological impact of transmissible defective dengue viruses outbreak of poliomyelitis in hispaniola associated with circulating type 1 vaccine-derived poliovirus 5'-terminal deletions occur in coxsackievirus b3 during replication in murine hearts and cardiac myocyte cultures and correlate with encapsidation of negativestrand viral rna the neutral theory of molecular evolution the neutral theory of molecular evolution and the world view of the neutralists non-darwinian evolution the mechanism of rna recombination in poliovirus paramyxovirus mrna editing, the "rule of six" and error catastrophe: a hypothesis virus world as an evolutionary network of viruses and capsidless selfish elements. microbiol the population genetics of dn/ds dna mismatch repair tempo and mode of plant rna virus escape from rna interference-mediated resistance genetic recombination in rna viruses rates of dna sequence evolution in experimental populations of escherichia coli during 20,000 generations dynamics of hiv-1 recombination in its natural target cells defective interfering viral particles in acute dengue infections aminoacyl-trna synthesis and translational quality control dna mismatch repair and its many roles in eukaryotic cells a unique intra-molecular fidelity-modulating mechanism identified in a viral rna-dependent rna polymerase defective or effective? mutualistic interactions between virus genotypes recombination in enteroviruses is a biphasic replicative process involving the generation of greater-than genome length 'imprecise' intermediates a-to-i rna editing: recent news and residual mysteries distribution of rare triplets along mrna and their relation to protein folding highfrequency rna recombination of murine coronaviruses the rate and character of spontaneous mutation in an rna virus lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase influence of reverse transcriptase variants, drugs, and vpr on human immunodeficiency virus type 1 mutant frequencies rdp2: recombination detection and analysis from sequence alignments synonymous viral genome recoding as a tool to impact viral fitness reassorment in segmented rna viruses: mechanisms and outcomes poxvirus tropism molecular evolution of the herpesvirales molecular basis of fidelity of dna synthesis and nucleotide specificity of retroviral reverse transcriptases attenuation of human enterovirus 71 high-replication-fidelity variants in ag129 mice molecular mechanisms of somatic hypermutation and class switch recombination unblocking of chain-terminated primer by hiv-1 reverse transcriptase through a nucleotide-dependent mechanism microbial evolution. gene establishment, survival and exchange an extracellular darwinian experiment with a self-duplicating nucleic acid molecule discovery of an rna virus 3'/5' exoribonuclease that is critically involved in coronavirus rna synthesis codon usage influences fitness through rna toxicity exploration of sequence space as the basis of viral rna genome segmentation internal disequilibria and phenotypic diversification during replication of hepatitis c virus in a noncoevolving celular environment the evolutionary biology of viruses evolution and emergence of enteroviruses through intra-and inter-species recombination: plasticity and phenotypic impact of modular genetic exchanges in the 5'untranslated region. plos pathog. 11, e1005266. naegeli, h., 1997. mechanisms of dna damage recognition in mammalian cells new insights into the mechanisms of rna recombination the evolution of multicompartmental genomes in viruses recombination rate and selection strength in hiv intra-patient evolution simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions rna interference as a tool for exploring hiv-1 robustness hiv-1 protease evolvability is affected by synonymous nucleotide recoding reconstitution of recombination-dependent dna synthesis in herpes simplex virus 1 functions and regulation of rna editing by adar deaminases coordinated changes in mutation and growth rates induced by genome reduction negative effect of genetic bottlenecks on the adaptability of vesicular stomatitis virus detection of defective genomes in hepatitis a virus particles present in clinical specimens evidence for frequent recombination within species human enterovirus b based on complete genomic sequences of all thirty-seven serotypes viral genome segmentation can result from a trade-off between genetic content and particle stability cyclic production of vesicular stomatitis virus caused by defective interfering particles evidence for purifying selection against synonymous mutations in mammalian exonic splicing enhancers 5-azacytidine and rna secondary structure increase the retrovirus mutation rate gonz alez-candelas, f., 2015. recombination in viruses: mechanisms, methods of study, and evolutionary consequences extensive editing of cellular and viral double-stranded rna structures accounts for innate immunity suppression and the proviral activity of adar1 p150 a single mutation in poliovirus rna-dependent rna polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity ribavirin resistance in hepatitis c virus replicon-containing cell lines conferred by changes in the cell line or mutations in the replicon rna altering the intracellular environment increases the frequency of tandem repeat deletion during moloney murine leukemia virus reverse transcription transfection-mediated generation of functionally competent tula hantavirus with recombinant s rna segment low-fidelity polymerases of alphaviruses recombine at higher rates to overproduce defective interfering particles mechanisms of retroviral mutation reproductive clonality in protozoan pathogens ─ truth or artefact? recombination is required for efficient hiv-1 replication and the maintenance of the viral genome integrity the defective component of viral populations non-darwinian evolution: a critique codon usage bias from trna's point of view: redundancy, specialization, and efficient decoding for translation optimization functional characterization of the genomic promoter of borna disease virus (bdv): implications of 3'-terminal sequence heterogeneity for bdv persistence effects of defective interfering viruses on virus replication and pathogenesis in vitro and in vivo alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models a guanosine to adenosine transition in the 3' terminal extracistronic region of bacteriophage qb rna leading to loss of infectivity experimental evolution of conflict mediation between genomes sequence analysis of in vivo defective interferinglike rna of influenza a h1n1 pandemic virus the phylogenetic handbook. a practical approach to dna and protein phylogeny the role of the apobec3 family of cytidine deaminase in innate immunity, g-to-a hypermutation, and evolution of retroviruses difference in incidence of spontaneous mutations between herpes simplex virus types 1 and 2 isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein pervasive genomic recombination of hiv-1 in vivo gene copy number is differentially regulated in a multipartite virus recombination in the evolution of picornaviruses coronaviruses as dna wannabes: a new model for the regulation of rna virus replication fidelity the mutation rate and variability of eukaryotic viruses: an analytical review dna recombination and repair coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics evolution of rna-based networks different modes of retrovirus restriction by human apobec3a and apobec3g in vivo mechanisms for rna capture by ssdna viruses: grand theft rna direct method for quantitation of extreme polymerase error frequencies at selected single base sites in viral rna lack of evidence for proofreading mechanisms associated with an rna virus polymerase dna polymerases: structural diversity and common mechanisms the cancer genome heterogeneity of the mutation rates of influenza a viruses: isolation of mutator mutants the code of silence: widespread associations between synonymous codon biases and gene function template properties of mutagenic cytosine analogues in reverse transcription structural determinants of murine leukemia virus reverse transcriptase that affect the frequency of template switching self-replication with errors. a model for polynucleotide replication rna-rna recombination in plant virus replication and evolution mycoreovirus genome alterations: similarities to and differences from rearrangements reported for other reoviruses molecular epidemiology of hiv-1 genetic forms and its significance for vaccine development and therapy reproductive clonality of pathogens: a perspective on pathogenic viruses, bacteria, fungi, and parasitic protozoa cryptosporidium, giardia, cryptococcus, pnemocystis genetic variability: cryptic biological species or clonal near-clades? aid: a riddle wrapped in a mystery inside an enigma homologous crossovers among molecules of brome mosaic bromovirus rna1 or rna2 segments in vivo phenotypic hiding: the carryover of mutations in rna viruses as shown by detection of mar mutants in influenza virus comparison of moloney murine leukemia virus mutation rate with the fidelity of its reverse transcriptase in vitro notes on recombination and reassortment in multipartite/segmented viruses simulating pseudogene evolution in vitro: determining the true number of mutations in a lineage comprehensive mutation identification in an evolved bacterial cooperator and its cheating ancestor perspective: apobec mutagenesis in drug resistance and immune escape in hiv and cancer evolution biological implications of picornavirus fidelity mutants quasispecies diversity determines pathogenesis through cooperative interactions in a viral population replication slippage involves dna polymerase pausing and dissociation genome instability: a conserved mechanism of ageing? essays biochem satellites and defective viral rnas enhanced fidelity of 3tc-selected mutant hiv-1 reverse transcriptase role of the single deaminase domain apobec3a in virus restriction, retrotranscription, dna damage and cancer determination of the poliovirus rna polymerase error frequency at eight sites in the viral genome direct measurement of the poliovirus rna polymerase error frequency in vitro evolution and ecology of influenza a viruses evasion of superinfection exclusion and elimination of primary viral rna by an adapted strain of hepatitis c virus insertions in the human immunodeficiency virus type 1 protease and reverse transcriptase genes: clinical impact and molecular mechanisms evolutionary aspects of recombination in rna viruses visualizing the nucleotide addition cycle of viral rna-dependent rna polymerase genomic heterogeneity maps to tandem repeat sequences in the herpes simplex virus type 2 ul region statistical methods for detecting molecular adaptation fidelity at the molecular level: lessons from protein synthesis key: cord-004575-b0t6bsya authors: staub, roger title: haben hiv-positive eine besondere verantwortung?: ein diskussionsbeitrag date: 2007-03-27 journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: 10.1007/s00103-007-0190-1 sha: doc_id: 4575 cord_uid: b0t6bsya in the 1980s, most western countries decided to opt for a new public health approach based on learning strategies to fight hiv/aids. within the new public health paradigm, every sexually active person protects him-/herself, whereas it is the infected people that bear the burden of prevention according to old public health. the paper begins with a clear statement for the new public health approach that includes self-protection and the protection of civil rights. it argues that this approach is still valid under the changing circumstances due to combination therapies and the developments related to them, such as the introduction of routine hiv screening in some countries. on this background, the paper examines from an ethical perspective to what extent people with hiv have a responsibility in hiv prevention. the paper argues that a person with hiv has a responsibility to protect his/her partner in the case of a relationship of love. on the other hand, in the case of "purely" sexual encounters, each person is responsible for his/her own security and should not rely on the other. this position also helps to clarify prevention messages. in conclusion, the paper shows how morality evolves into ethical positions and then translates into the law. it finally claims for stronger obligations for sex businesses to support hiv prevention. mitte der 1980er-jahre wurde in den meisten westlichen industrieländern der gesundheitspolitische streit zur medizinpolitisch wie bürgerrechtlich entscheidenden frage nach der richtigen aids-politik in fachgremien und teilweise in der öffentlichkeit ausgetragen. dabei standen sich einander weitgehend ausschließende optionen gegenüber: "auf der einen seite stand die klassische seuchenstrategie nach dem ‚old-public-health'-paradigma, die individuelle suchstrategie, im amerikanischen sprachgebrauch ‚control and containment'". sie steht unter der leitfrage: wie ermitteln wir möglichst schnell möglichst viele infektionsquellen, und wie legen wir diese still? ihr gegenüber stand die auf "new public health", also auf moderne gesundheitswissenschaftliche konzepte gegründete gesellschaftliche lernstrategie, im amerikanischen sprachgebrauch "inclusion and cooperation". sie sucht und realisiert antworten auf die frage: wie organisieren wir möglichst schnell, möglichst zeitstabil gesellschaftliche lernprozesse, mit denen sich individuen, soziale gruppen, institutionen und die gesamte gesellschaft maximal präventiv und ohne diskriminierung der betroffenen auf ein leben mit dem bis auf weiteres unausrottbaren virus einstellen können?" [1] . wie die meisten westlichen länder hat sich auch die schweiz 1986 für die lern-strategie und damit für aufklärung der ganzen bevölkerung entschieden und unter dem logo "stop aids" allen die selbstschutzbotschaft "vor aids schützen -präservative benützen" massenmedial während nunmehr 20 jahren vermittelt. das von der schweizer regierung ende november 2003 beschlossene nationale hiv/aids-programm 2004-2008 kommt zu dem schluss: "das ergebnis kann im rückblick als sieg der gesundheitspolitischen vernunft bezeichnet werden: es hat sowohl bürgerrechtlich als auch epidemiologisch zu guten resultaten geführt. so sind z. b. die zahlen der neu gemeldeten positiven hiv-antikörpertests seit 1990 bis zum jahr 2000 kontinuierlich gesunken. in bezug auf diskriminierung von betroffenen gruppen und hiv-infizierten menschen stellen verschiedene studien fest [2, 3] , dass in der schweiz auf der ebene des rechts keine systematische oder regelmäßige diskriminierung stattfindet" [4] . verantwortung ist eine gesellschaftliche zuschreibung: man ist gegenüber jemandem, für etwas, vor einer instanz, in bezug auf standards und ein normensystem verantwortlich. der moderne verantwortungsbegriff bezieht sich heute auch auf gesellschaftliche probleme, gilt auch prospektiv und für positiv bewertete zustände. er bezieht auch die (absichtliche) unterlassung einer handlung ein und bewertet die folgen. heute ist in der ethik die frage, wer ein guter mensch ist, gegenüber der frage, welches handeln richtig ist, in den hintergrund getreten [6] . in einer binären welt des ja/nein und des entweder/oder ist es schwierig, konzepte des sowohl/als auch zu vertreten. angesichts der aktuellen schwierigkeiten der hiv-prävention behaupten wir nicht einfach, die bisherige strategie sei unverändert weiterzuführen. auf der grundlage der forderung, die prävention weiterhin auf 100 %igem selbstschutz zu begründen, folgt hier ein vorschlag zur frage, welche verantwortung hiv-infizierten menschen zugeschrieben werden soll. dies nicht zuletzt, um das outcome der prävention zu verbessern. von hiv-infizierten menschen sollten wir erwarten, dass sie den geliebten partner, die geliebte partnerin nicht gefährden bei sexuellen begegnungen, die im rahmen einer aktuellen oder erhofften zukünftigen beziehung stattfinden. aber dort, wo es "nur" um sex geht, sollten wir auf selbstschutz setzen. auch von einer hiv-positiven prostituierten oder von einem hiv-positiven homosexuellen bei einem one-night-stand ist nicht zu erwarten, dass sie verantwortung für den schutz des anderen übernehmen. es ist offensichtlich, dass es sich bei diesen beiden positionen um extrempunkte eines kontinuums handelt und dass sich sexuelle begegnungen in der regel dazwischen abspielen. gleichwohl lässt sich mit dieser position effizientere und effektivere prävention organisieren, und zwar unter einbezug des hiv-antikörpertests gemäß den regeln freiwilligkeit, vertraulichkeit und informierte zustimmung. voraussetzung ist, dass der rhythmus von neuen partnerinnen es theoretisch zulässt, mittels test nach geschützten und treuen 3 monaten ("verlobungstest") bei beiden beteiligten zweifelsfrei festzustellen, ob sie hiv-negativ sind. lässt sich eine hiv-positive person auf eine so geartete beziehung ein, kann die liebe nach dem verlobungstest nur weiter gedeihen, wenn der hiv-positive oder ungetestete partner von anfang an seine verantwortung wahrgenommen und für konsequenten schutz gesorgt hat. menschen mit häufigem partnerwechsel, bei denen ein testresultat "hivnegativ" durch in den letzten 3 monaten eingegangene ungeschützte sexuelle begegnungen andauernd und wiederholt veraltet, müssen mitgeteilt erhalten, dass sie sich selbst zu schützen haben. ihnen muss klargemacht werden, dass sie nicht erwarten können, dass allfällig hiv-infizierte partner sie schützen werden. der entscheid, im "nur-sex-fall" keine verantwortung des hiv-infizierten menschen zu erwarten, erlaubt klarere präventionsbotschaften. missverständnisse wie "wenn er positiv ist, wird er mich schon schützen" können korrigiert werden, z. b. durch deutliche botschaften am eingang zu darkroom oder sauna ("hier ficken nur positive ohne gummi"). wenn wir es schaffen, auch von einer hiv-positiven prostituierten nicht zu erwarten, dass sie auf das angebot eines freiers verzichten muss, der für "ohne" gar mehr bezahlen will, dann können wir auch mit freiern deutlich kommunizieren und sie zum "immer mit" motivieren. zusammenfassung · abstract sibility in hiv prevention. the paper argues that a person with hiv has a responsibility to protect his/her partner in the case of a relationship of love. on the other hand, in the case of "purely" sexual encounters, each person is responsible for his/her own security and should not rely on the other. this position also helps to clarify prevention messages. in conclusion, the paper shows how morality evolves into ethical positions and then translates into the law. it finally claims for stronger obligations for sex businesses to support hiv prevention. is there a special responsibility of hiv-positive people? a discussion paper abstract in the 1980s, most western countries decided to opt for a new public health approach based on learning strategies to fight hiv/aids. within the new public health paradigm, every sexually active person protects him-/herself, whereas it is the infected people that bear the burden of prevention according to old public health. the paper begins with a clear statement for the new public health approach that includes self-protection and the protection of civil rights. it argues that this approach is still valid under the changing circumstances due to combination therapies and the developments related to them, such as the introduction of routine hiv screening in some countries. on this background, the paper examines from an ethical perspective to what extent people with hiv have a respon-bzw. bei außenkontakten die safer-sex-regeln anzuwenden. in der konsequenz bedeutet die entscheidung für die new-public-health-strategie (also für die selbstschutzbotschaft) auch, dass man hiv-infizierten menschen unter art mit einer viruslast unter der nachweisgrenze mitteilen könnte, dass sie sich über ein möglicherweise verbleibendes infektionsrisiko bei anonymem sex keine gedanken machen müssten, sie ungeschützten sex haben könnten, sofern ihnen das risiko, sich mit anderen sexuell übertragbaren infektionen (stis) zu infizieren, egal ist. konsequenterweise könnte man hiv-positiven männern ohne therapie für den darkroom dasselbe sagen. von vielen beratenden und auch von betroffenen kommt an dieser stelle aber widerspruch. ein eleganter ausweg bietet sich an im moralischen prinzip "du sollst nicht (absichtlich) schaden zufügen". dagegen ist die frage, ob betroffene unter art ihren (ständigen) partner infizieren können, im gespräch zwischen arzt und beiden partnern zu klären. der hiv-negative ständige partner kann nur für sich entscheiden, ob er das restrisiko auf sich nehmen will oder nicht. und allen beteiligten muss durch eingängige beratung durch den behandelnden arzt klar sein, dass unter dem einfluss banaler infekte oder stis die hiv-replikation auch unter therapie wieder steil ansteigen kann. die ganze verantwortungsdiskussion greift aber zu kurz, wenn wir sie auf hiv-positive und hiv-negative beschränken. es muss selbstverständlich sein, dass für ungetestete gilt, dass es zwar ein recht auf nicht-wissen gibt. damit geht aber die verpflichtung einher, sich so zu verhalten, als ob man hiv-positiv wäre, und damit entsprechende anforderungen an die verantwortlichkeit zu erfüllen. neben individueller verantwortung gibt es auch verantwortung von institutionen: niemand wird bestreiten, dass sich die für die hiv-präventionsstrategie zuständigen institutionen und organisationen (staatliche und private) dafür verantworten müssen, ob die prävention erfolgreich war. sie müssen sich fragen lassen, ob sie es geschafft haben, dafür zu sorgen, dass niemand sagen kann, er/sie habe nicht gewusst, dass selbstschutz erwartet wird. aber neben den für die präventionsstrategie zuständigen institutionen und organisationen bzw. den individuen gibt es eine ebene, die bislang sträflich vernachlässigt wurde: die orte, an denen sexuelle begegnungen angebahnt werden und an denen sie stattfinden. so selbstverständlich, wie wir erwarten, dass eine private oder öffentliche toilette mit papier ausgestattet ist (auch dies eine kulturelle errungenschaft), so selbstverständlich müsste es sein, dass an diesen orten (z. b. lokale mit darkrooms, saunen, swingerclubs oder bordelle) auch jederzeit und in genügender menge präservative etc. gratis angeboten werden bzw. im preis inbegriffen sind, falls für den zugang eintritt verlangt wird. präventionsfachleute haben jahrelang mit viel aufwand und nicht im-mer erfolgreich die hier verantwortlichen zu motivieren versucht, für diese selbstverständlichkeit freiwillig zu sorgen. eigentlich sollten die betreibenden solcher orte heute generell und obligatorisch dafür sorge tragen. wir überlassen den einbau von sicherheitsgurten schließlich auch nicht den motivierten autoherstellern. für die überwachung dieser anordnung wird die konkurrenz unter solchen betrieben schon sorgen. dieser diskussionsbeitrag basiert auf der arbeit des autors zur erlangung des masters in angewandter ethik der universität zürich [6] . der beitrag gibt die persönliche meinung des autors wieder. er dankt dem team der sektion aids des bundesamtes für gesundheit und seinem vorgesetzten für die korrekturen und anregungen. bundesamt für gesundheit, sektion aids postfach 3003 bern, schweiz e-mail: roger.staub@bag.admin.ch rosenbrock r, schaeffer d (hrsg) die normalisierung von aids. politik -prävention -krankenversorgung. edition sigma identification des discriminations institutionnelles à l'encontre des personnes vivant avec le vih en suisse. raisons de santé 18 aids, recht und geld. eine untersuchung der rechtlichen und wirtschaftlichen probleme von menschen mit hiv/aids nationales hiv/ aids-programm erneute ausbreitung der hiv-epidemie in der schweiz unter homosexuellen und anderen männern, die sex mit männern haben (msm) strafgesetzbuch art. 231 verbreiten menschlicher krankheiten wer vorsätzlich eine gefährliche übertragbare menschliche krankheit verbreitet, wird mit gefängnis von einem monat bis zu 5 jahren bestraft. hat der täter aus gemeiner gesinnung gehandelt handelt der täter fahrlässig, so ist die strafe gefängnis oder buße hiv-positiv: fertig mit sex? oder: erwächst hiv-positiven menschen eine andere oder besondere moralische verantwortung? diplomarbeit im rahmen des master-studienganges 2001-2003 master of advanced studies in applied ethics (mae). download schweizerisches strafgesetzbuch vom 21. dezember 1937. (kurzkommentar). (2. auflage) key: cord-270726-w59fu9c9 authors: dikman, andrew e.; schonfeld, emily; srisarajivakul, nalinee c.; poles, michael a. title: human immunodeficiency virus-associated diarrhea: still an issue in the era of antiretroviral therapy date: 2015-03-14 journal: dig dis sci doi: 10.1007/s10620-015-3615-y sha: doc_id: 270726 cord_uid: w59fu9c9 over half of patients with human immunodeficiency virus (hiv) experience diarrhea that contributes negatively to quality of life and adherence to antiretroviral therapy (art). opportunistic infectious agents that cause diarrhea in patients with hiv span the array of protozoa, fungi, viruses, and bacteria. with global use of art, the incidence of diarrhea because of opportunistic infections has decreased; however, the incidence of noninfectious diarrhea has increased. the etiology of noninfectious diarrhea in patients with hiv is multifactorial and includes art-associated diarrhea and gastrointestinal damage related to hiv infection (i.e., hiv enteropathy). a basic algorithm for the diagnosis of diarrhea in patients with hiv includes physical examination, a review of medical history, assessment of hiv viral load and cd4+ t cell count, stool microbiologic assessment, and endoscopic evaluation, if needed. for patients with negative diagnostic results, the diagnosis of noninfectious diarrhea may be considered. pharmacologic options for the treatment of noninfectious diarrhea are primarily supportive; however, the use of many unapproved agents is based on unstudied and anecdotal information. in addition, these agents can be associated with treatment-limiting adverse events (aes), such as drug–drug interactions with art regimens, abuse liability, and additional gastrointestinal aes. currently, crofelemer, an antisecretory agent, is the only therapy approved in the usa for the symptomatic relief of noninfectious diarrhea in patients with hiv on art. advances in the treatment of human immunodeficiency virus (hiv) and acquired immunodeficiency syndrome (aids) have transformed this disease into a chronic illness [1] . as a result, individuals with hiv have a longer life expectancy [2] . when hiv is treated early and aggressively, life expectancy approaches that of uninfected individuals [3, 4] . according to the centers for disease control and prevention (cdc), the annual incidence of hiv has remained stable during the past 10 years [2] . in 2010, the prevalence of hiv in individuals aged over 13 years was estimated to be about 1,148,200 [5] . although this number illustrates a dramatic increase in the prevalence of hiv since 1996, it is largely due to the decreased mortality rate associated with the advent of antiretroviral therapy (art) [2] . because most cases of diarrhea in the pre-art era were due to infectious pathogens, it was widely believed that successful treatment would reduce, or even solve, this problem [6, 7] . although art has improved the survival and quality of life of patients with hiv, and even decreased the incidence of diarrhea in this population, diarrhea remains a common complaint among these patients and continues to negatively impact the quality of life [6] [7] [8] [9] . furthermore, while the burden of diarrhea in the hiv-infected population remains high, the causes of diarrhea have shifted toward noninfectious etiologies [6, 7] . in one survey, *60 % of patients receiving art reported having the symptom of diarrhea in the previous month [10] ; clinical trial data would suggest that up to 19 % of these events may have been due to the effects of art itself [11] . in support of this assertion, us historical data of hiv-infected patients with cd4? t cell counts \200 cells/mm 3 were investigated and demonstrated a decline in infectious diarrhea from 53 to 13 % over 3 years (1995) (1996) (1997) ; this correlated with the introduction of art around 1995 [6] . during that same period, the percentage of patients with noninfectious diarrhea increased from 32 to 71 % [6] . a similar trend in etiologic differences has been observed in other countries for patients taking art versus those not taking art [12] . this review explores some explanations for the continued burden of diarrhea among patients with hiv, discusses the diagnostic workup, and provides guidance on how hivassociated noninfectious diarrhea should be treated. gut-associated lymphoid tissue (galt) is the largest collection of lymphoid tissue in the human body [13] . the gastrointestinal (gi) tract is regularly exposed to a complex and diverse assortment of antigens from both microbial and dietary sources [14] . as a result, naïve b and t cells of the gut are constantly interacting with antigens that induce their maturation into plasma cells and memory t cells, respectively. this persistent stimulation of the immune system leads to a baseline inflammatory state that encourages the production of chemokines and adhesion molecules, which mediate the movement of lymphocytes into the mucosal tissues [13, 15] . the gi tract is targeted during all phases of hiv infection, but the effects of hiv on the mucosal immune system are most apparent in the acute infection period. data from simian models with simian immunodeficiency virus [16] and from patients with hiv [13, 17, 18] show that within weeks of infection, the vast majority of mucosal lamina propria cd4? lymphocytes are depleted. this depletion occurs well before any decrease in cd4? t cells is seen in the periphery and likely reflects the greater expression of the cc chemokine receptor type 5 (ccr5), which functions as the primary coreceptor for the majority of infective hiv strains, on mucosal cd4? t cells [19] . the expression of the chemokine receptor on these cells supports the entry of the virus, but their state of physiologic activation is responsible for enhanced hiv replication in the mucosal compartment and subsequent mucosal cd4? t cell depletion. further, despite the use of potent antiretroviral medications, hiv persists within galt lymphocytes, even after repletion of cd4? t cells in the periphery [20] . thus, in patients with chronic hiv infection, the mucosal environment contains a paucity of cd4? t cells but is overpopulated with cd8? t cells and b cells, giving the wrong impression of a healthy mucosal environment at the microscopic level [21] . the etiology of diarrhea in hiv-infected patients can be divided into two major categories: noninfectious and infectious. as treatment of hiv has improved, the increase in peripheral cd4? t cell counts has resulted in a decline in the risk of infection and associated diarrhea (fig. 1) [22] . indicative of this shift, noninfectious etiologies of diarrhea have now surpassed infectious causes [6] . noninfectious diarrhea is defined as pathogen-negative diarrhea and includes art-associated diarrhea, gi damage related to hiv infection (i.e., hiv enteropathy), and many causes mirroring those seen in gender-and age-matched patients without hiv infection. hiv enteropathy is an idiopathic form of diarrhea observed in patients with hiv in the absence of an infectious source with characteristic histologic features (table 1 ) [8, [23] [24] [25] . while the exact mechanisms by which these changes occur in the gi tract are unclear, hiv has been postulated to alter signaling and cellular structure, which may lead to architectural distortion [23] . several studies have demonstrated crypt epithelial proliferation in response to hiv infection, leading to increased crypt height, subsequent crypt cell encroachment onto villi, and relative decreased villous height resulting in diarrhea and malabsorption [26, 27] . a study by keating and colleagues that investigated monosaccharide absorption in patients with hiv and aids demonstrated that patients with diarrhea had significant malabsorption of all monosaccharides tested [28] . evidence of malabsorption was reported in patients with both pathogen-negative (n = 7) and pathogen-positive (n = 27) diarrhea, suggesting that malabsorption may be involved in hiv enteropathy, as well as in pathogen-induced diarrhea. other hypotheses for the mechanism of hiv enteropathy include decreased transepithelial electrical resistance, decreased sodium-dependent glucose absorption, and increased intercellular permeability in hiv-infected cells [29] . in vitro studies have demonstrated that gp120, an envelope protein of hiv, induces microtubule disruption, decreases epithelial resistance, and promotes calcium signaling within the cell to affect these cytopathic changes [29, 30] . however, there was no effect of gp120 on chloride secretion. hiv enteropathy may occur at all stages of hiv infection, from acute hiv to advanced aids [23] . data suggest that hiv may be capable of infecting mucosal epithelial dig dis sci (2015) 60:2236-2245 2237 cells, resulting in direct effects on epithelial cell function [31] . further, microscopic inflammation may be mechanistically involved in hiv enteropathy [23] . while hiv rapidly and severely depletes mucosal t cells, the total mucosal t cell population remains largely unchanged [21] . this reflects greater mucosal infiltration by activated cd8? t cells. these cells are primed to produce proinflammatory cytokines and chemokines, which may directly [24, 25] defined by the acg as abdominal pain or associated with altered bowel habits over a period of c3 months defined by rome iii criteria as recurrent abdominal pain or discomfort for c3 days/month in the last 3 months with symptom onset at c6 months before diagnosis and associated with c2 of the following: onset associated with change in stool frequency onset associated with change in stool form functional diarrhea [24] defined by rome iii criteria as c75 % of stools that are loose (mushy) and without pain for c3 months with symptom onset c6 months before diagnosis reprinted with permission from [8] acg american college of gastroenterology, gi gastrointestinal, hiv human immunodeficiency virus damage the mucosal barrier, resulting in decreased transepithelial resistance and diarrhea [21, 23] . this has been shown to lead to malabsorption of vitamin b 12 , bile acid, and monosaccharides; thus, hiv enteropathy can also be more specifically defined by its physiologic effect on small bowel function [23, 28] . while diarrhea is an adverse effect of art, protease inhibitors seem to be most strongly associated with diarrhea ( table 2 ) [32] . in mouse models, protease inhibitors and reverse transcriptase inhibitors were found to significantly increase water and electrolyte secretion into the intestinal lumen in vivo [33] . rufo and colleagues demonstrated that protease inhibitors in general and nelfinavir in particular potentiate signaling through muscarinic-and calcium-dependent receptors of intestinal cells, resulting in increased chloride secretion into the lumen [34] . this study also showed that sodium and chloride concentrations in stool samples from patients with hiv taking nelfinavir were elevated, consistent with secretory diarrhea. in an in vitro study, bode and colleagues found that protease inhibitors induced apoptosis of human intestinal epithelium, compromising barrier function in the cells and thereby increasing water secretion in the gut lumen [35] . wu and colleagues also investigated the mechanism by which protease inhibitors cause diarrhea and found that lopinavir and ritonavir (notably not amprenavir) induced endoplasmic reticulum dysfunction in the intestinal epithelial cells [36] . when exposed to protease inhibitors, the cells were found to have decreased alkaline phosphatase activity and thereby an increase in unfolded proteins. the accumulation of defective proteins in the cytosol may persistently activate the cell's unfolded protein response, a specific signaling pathway that aims to return the cell's protein folding function back to normal. if the levels of unfolded proteins do not decrease, the cell may induce apoptosis. functional bowel disorders, such as irritable bowel syndrome (ibs), are additional conditions that may present with diarrhea [8] . in a survey conducted in a veterans affairs outpatient setting, ibs was more common in hivpositive patients compared with hiv-negative patients [37] . another cause of noninfectious diarrhea is small intestinal bacterial overgrowth (sibo) [38] , a condition in which increased bacteria in the small intestine leads to multiple gi symptoms, including abdominal bloating and discomfort, diarrhea, and malabsorption [39] . this condition may be more common in those with anatomic abnormalities and motility disorders, but does not appear to be a common cause of diarrhea in those with hiv [40] . given the multitude of differences in standard of living among developed versus developing countries and differences in availability of care for patients with hiv, the incidence and type of various diarrhea-associated infections in immunocompromised patients may vary substantially by region [41, 42] . in addition, diarrhea experienced by hiv-infected individuals can be caused by pathogens that are unique to the hiv population or by pathogens that cause diarrhea in the immunocompetent host [41, 43] . for example, salmonella, shigella, campylobacter, escherichia coli, and enterotropic viruses cause diarrhea in both the immunocompetent and hiv-infected patients. in addition to these pathogens, patients with hiv who are profoundly immunosuppressed, with a cd4? t cell count \200 cells/mm 3 , are also susceptible to opportunistic infections [44] . these opportunistic infections can be organized into four general categories of organisms: protozoa, fungi, viruses, and bacteria (table 3 ) [41, [45] [46] [47] [48] . cryptosporidiosis is caused by an intracellular pathogen, cryptosporidium [49] . infection with cryptosporidium parvum is a common cause of diarrhea worldwide [45] . there is an estimated rate of *60,000 cases of [51] [52] [53] . it spreads from person to person or via contaminated water (e.g., drinking water or recreational water sites) [54] . although the protozoa cause a mild, self-limited disease in immunocompetent individuals, it can manifest as severe, watery diarrhea in hiv-infected patients and it can also occasionally lead to a chronic infection [49] . in the profoundly immunocompromised, cryptosporidium parvum can lead to a malabsorptive syndrome, severe dehydration, and electrolyte derangements [49, 55] . other protozoal infections that commonly affect patients with hiv include microsporidia, isospora, cyclospora, and, less commonly, toxoplasma and leishmania species, which like cryptosporidium, affect the small intestine and can lead to malabsorptive diarrheal illnesses [41] . similarly, giardia also is a very common protozoal pathogen in both hiv-infected and immunocompetent hosts and infects the small intestinal mucosa. entamoeba histolytica is a protozoal pathogen that can cause colitis in the severely immunocompromised hiv-infected patient and in rare and severe circumstances can lead to ulceration, hematochezia, and even toxic megacolon. cytomegalovirus (cmv) is the most common viral gi pathogen in patients with aids [44] . cmv can affect the entire gi tract but classically leads to diarrhea by causing colitis characterized by rectal bleeding, abdominal pain, and systemic signs such as fevers and weight loss [55] . hiv infection itself can cause a diarrheal illness as described below, and multiple other viruses have been implicated as diarrheal pathogens, including adenovirus, coronavirus, herpes simplex virus, rotavirus, and norovirus [44, 46] . rarely have fungi been reported to cause diarrhea in hivinfected patients [44] . of the infections associated with endemic fungi, histoplasmosis is the most common fungal infection requiring hospitalization and can affect any part of the gi tract, causing diarrhea, weight loss, and fever [56] . mycobacterium avium complex (mac) typically causes diarrhea in patients with aids with profoundly suppressed immune systems (i.e., cd4? t cell count \50 cells/mm 3 ) [44] . symptoms are varied, but they usually reflect the systemic nature of the disseminated infection and include fever and weight loss [45] . mac usually involves the small intestine; however, it can affect the entire gi tract [55, 57] . salmonella, shigella, campylobacter, and e coli are bacterial infections of the gi tract that can present with diarrhea [41] . they are usually spread through food and water contamination. patients with an impaired immune system can become asymptomatic carriers of some pathogens (e.g., shigella, campylobacter). clostridium difficile should be investigated in patients who have recently taken antibiotics [47] . it causes pseudomembranous colitis that can progress to toxic megacolon in severe infections. clostridium difficile is more common in patients with advanced aids (cd4? t cell count \50 cells/mm 3 ). in one study evaluating the incidence of bacterial diarrhea in patients with hiv, clostridium difficile was the most common identified culprit, accounting for 53.6 % of 1115 episodes of diarrhea that had a bacterial pathogen isolated [36] . overall, protozoa, fungi, mac, and cmv gi infections are usually seen only in patients with hiv and other immunocompromised patients [58] . as noted earlier, with improved therapy, patients infected with hiv are less prone to develop diarrhea due to infectious pathogens and are more prone to noninfectious causes [48] . the basic algorithm for the diagnosis and evaluation of diarrhea in patients with hiv is similar to the examination of diarrhea in the immunocompetent host and starts with a thorough medical history [55] . this includes the duration of the diarrhea, constitutional and systemic symptoms, the presence or severity of abdominal pain, and questions on the characteristics of diarrhea (e.g., large versus small volume; infrequent or nocturnal versus frequent) to help localize the source to the large or small intestine and narrow the differential diagnosis (fig. 2) [8, 55] . classically, diarrhea from pathology in the small intestine presents as voluminous and watery, occasionally associated with weight loss as well as signs and symptoms of malabsorption [59] . bloating, nausea, and cramping may also indicate a small intestine source. diarrhea from the large intestine is typically characterized by smaller, more frequent bowel movements [48] . if there is inflammation of the rectum and anus, patients may describe tenesmus and painful defecation, which may be associated with hematochezia. a careful review of medications, travel history, contact with ill individuals, recent ingestions, family history, surgical history, and social history is important in evaluating acute and chronic diarrhea in the patient infected with hiv [8] . physical examination and medical history of a patient with hiv and diarrhea may help to evaluate the chronicity of the diarrhea and the severity of illness and thereby focus the differential diagnosis. nutritional status should be assessed by evaluating for bitemporal wasting, weight changes, skin turgor, and mucosal dehydration. basic vital signs and orthostatic vital signs are essential to gauge the severity of the patient's illness and the degree of the patient's volume depletion. documented fever may be an important clue of an infectious etiology, though the absence of fever in an immunocompromised host does not rule out the possibility of an infection. the gi examination may reveal tenderness to palpation, possibly indicative of pancreatic pathology or colitis. hepatosplenomegaly may represent mycobacterial infection, hepatitis, or fungal or malignant causes of diarrhea. a rectal examination is of integral importance because it may reveal signs of sexually transmitted infections, bleeding, tenderness, and masses, all of which may help to establish a diagnosis. a careful, comprehensive examination of the patient is essential to evaluate the severity of disease, narrow the diagnosis, and guide the management. further diagnostic evaluation may also aid in a diagnosis [8] . laboratory tests should include comprehensive metabolic panel, complete blood count with cell differential, blood cultures, hiv viral load, and cd4? t cell count to gauge the level of immune suppression. stool examinations should include fecal lactoferrin, fecal leukocytes, fecal calprotectin, fecal occult blood, and clostridium difficile stool culture, as well as three separate samples of ova and parasites. infection with cryptosporidium parvum is most commonly diagnosed by microscopic identification or enzyme immunoassay of the organisms in the stool of patients but can also be made by biopsy of the gi tract or aspiration from the biliary tree during endoscopy. diagnosis of giardia can be made by immunoassay of the stool but, again, can be identified on endoscopic biopsy or on luminal aspiration during endoscopy. diagnosis of bacterial infections of the gi tract, such as salmonella, is made by stool culture. if no pathogen is identified, further workup is needed with an endoscopic examination [8] . colonoscopy with biopsy is necessary for diagnosis of cmv and hsv and may reveal hemorrhage, colitis, and ulcers [46] . there is some debate as to whether sigmoidoscopy or full colonoscopy would be beneficial; one colonoscopy study (n = 44) showed that nearly 40 % of 18 patients with cmv-associated colitis had disease localized to the cecum [60] . however, a prospective study comparing sigmoidoscopy and full colonoscopy showed that the sensitivity of a sigmoidoscopy was 77 % for enteric pathogens and full colonoscopy only yielded a small increase in sensitivity to 82 %, with no additional cases of cmv-associated colitis identified [61] . diagnosis of mac infection within the gi tract requires endoscopy, which may identify ulcerations, erythema, or normal mucosa but may also reveal foamy macrophages and positive culture via biopsy [45] . diagnosis of fungal infections may be made histologically from biopsies taken for endoscopy or colonoscopy [58] . mucosal biopsy can also help diagnose inflammatory bowel disease. for patients with negative diagnostic results, the diagnosis of noninfectious diarrhea can be made. the treatment of infectious diarrhea is outside the scope of this article, and this section will focus on the treatment of noninfectious diarrhea. if no other cause of diarrhea is [62] . noninfectious diarrhea can be managed by modifying art and controlling symptoms with medications and lifestyle modification. however, clinical guidelines for patients with hiv recommend careful evaluation of the symptoms and symptomatic management of art-related aes (e.g., diarrhea) before switching art, with supportive care (e.g., antidiarrheals) generally allowing for continuation of prescribed art therapy [63, 64] . however, cessation of art (''drug holiday'') is generally not advised as a management strategy. antidiarrheal medications used in noninfectious diarrhea can be divided into several classes: adsorbents, antimotility agents, and antisecretory agents (table 4) [8, 32, 62, [65] [66] [67] [68] [69] [70] [71] . adsorbent drugs bind bacterial toxins, fluids, and other compounds in the intestines to improve stool consistency [65] . drugs in this category include bismuth subsalicylate, kaolin/pectin, and attapulgite (aluminum magnesium silicate purified from clay). data on the use of adsorbents in patients with hiv are limited to one study in which the efficacy of attapulgite was not better than placebo in the management of diarrhea [65, 72] . antimotility agents include loperamide, diphenoxylate/ atropine, and tincture of opium [65] . these agents act upon opioid receptors in the gut to slow motility by decreasing peristalsis and increasing tone in the large intestine. this allows for greater fecal transit time and therefore longer time for water absorption. the antimotility agent diphenoxylate crosses the bloodbrain barrier and can cause central effects analogous to other opioid agonists [62] . to prevent the abuse of diphenoxylate, it is usually sold as a combination agent with atropine. loperamide does not cross the blood-brain barrier and does not have these adverse effects. one disadvantage to loperamide is that it may interact with art agents, such as ritonavir and saquinavir, which can limit its use for long-term therapy [62, 67] . however, loperamide has been shown in a small trial to decrease the number of bowel movements in patients with protease inhibitor-induced diarrhea [73] . the use of the combination of lowdose diphenoxylate/atropine may be efficacious for treating protease inhibitor-induced diarrhea; however, it appears that this combination had no beneficial effect in patients who were refractory to loperamide therapy [66] . although not studied for the treatment of diarrhea in patients with hiv, tincture of opium is considered more effective than loperamide as an antimotility agent, and it can be titrated easily. tincture of opium may be considered early in the treatment algorithm for patients with severe hiv-associated diarrhea. art antiretroviral therapy, hiv human immunodeficiency virus. reprinted with permission from [8] antisecretory agents, such as octreotide, racecadotril, and crofelemer, directly inhibit secretory processes within enterocytes [62, [68] [69] [70] [71] . the mechanism of action of octreotide, administered subcutaneously, is unclear, but it is believed to alter the binding of vasoactive intestinal peptide, therefore acting as an antimotility and antisecretory agent [8, 62, 74] . published data conflict about the efficacy of octreotide in treating diarrhea in patients with aids [75, 76] . oral racecadotril is an enkephalinase inhibitor, which decreases the breakdown of endogenous gut opioids, thereby decreasing the secretion of water and electrolytes into the gut lumen [69] . racecadotril is not currently available in the usa. orally administered crofelemer is a dual inhibitor of camp-stimulated cystic fibrosis transmembrane conductance regulator (cftr) and calcium-stimulated calciumactivated chloride channels (cacc) in the gut [77] . crofelemer is a minimally absorbed gi drug derived from the latex of the croton lechleri tree, and has reportedly been used by amazonian tribes for diarrheal relief [68] . crofelemer is the only agent approved in the usa for the symptomatic relief of noninfectious diarrhea in patients with hiv on art [78] . compared with placebo, crofelemer has been shown, in a randomized, double-blind, placebo-controlled trial with an open-label extension, to significantly reduce symptoms of noninfectious diarrhea in patients with hiv receiving art (percentage of patients with b2 watery stools per week for at least 2 of 4 weeks during placebo-controlled phase: 17.6 versus 8.0 %, respectively; p = 0.01) [70] . in addition, crofelemer has no known drug interactions with art drugs, is minimally absorbed, and does not have a negative impact on the clinical immune parameters (e.g., hiv viral load and cd4? t cell counts) [68, 70, 71] . diarrhea is a common complication of hiv infection that has substantial clinical ramifications. the differential diagnosis for diarrhea in patients with hiv is broad. while infection has historically been the major cause of diarrhea in patients with hiv, with the widespread use of art therapy, noninfectious diarrhea has become a burden in this population. medications used for noninfectious diarrhea include antimotility and antisecretory agents, including crofelemer, which has been shown to significantly reduce symptoms of noninfectious diarrhea. improved treatment of noninfectious diarrhea will likely improve hiv medication adherence and quality of life. hiv/aids review centers for disease control and prevention. hiv surveillance-united states all-cause mortality in treated hiv-infected adults with cd4 [/=500/mm 3 compared with the general population: evidence from a large european observational cohort collaboration life expectancy of recently diagnosed asymptomatic hiv-infected patients approaches that of uninfected individuals centers for diease control and prevention national center for hiv/ aids viral hepatitis std and tb prevention. hiv in the united states: at a glance the changing etiology of chronic diarrhea in hiv-infected patients with cd4 cell counts less than 200 cells/mm 3 prevalence and impact of diarrhea on health-related quality of life in hiv-infected patients in the era of highly active antiretroviral therapy etiology and pharmacologic management of noninfectious diarrhea in hiv-infected individuals in the highly active antiretroviral therapy era global impact of antiretroviral therapy-associated diarrhea the association of hiv/aids treatment side effects with health status, work productivity, and resource use risk factors for gastrointestinal adverse events in hiv treated and untreated patients enteric parasitic infections in hiv/aids patients before and after the highly active antiretroviral therapy the gastrointestinal tract in hiv-1 infection: questions, answers, and more questions! prn noteb the mucosal immune system and hiv-1 infection expression of the chemokine receptors ccr4, ccr5, and cxcr3 by human tissueinfiltrating lymphocytes gastrointestinal tract as a major site of cd4? t cell depletion and viral replication in siv infection primary hiv-1 infection is associated with preferential depletion of cd4? t lymphocytes from effector sites in the gastrointestinal tract mechanisms of gastrointestinal cd4? t-cell depletion during acute and early human immunodeficiency virus type 1 infection enhanced levels of functional hiv-1 co-receptors on human mucosal t cells demonstrated using intestinal biopsy tissue persistence of hiv in gut-associated lymphoid tissue despite long-term antiretroviral therapy hiv infection and the gastrointestinal tract review article: the aetiology, investigation and management of diarrhoea in the hiv-positive patient the mucosal barrier and immune activation in hiv pathogenesis an evidencebased position statement on the management of irritable bowel syndrome jejunal enteropathy associated with human immunodeficiency virus infection: quantitative histology hiv enteropathy: crypt stem and transit cell hyperproliferation induces villous atrophy in hiv/microsporidia-infected jejunal mucosa intestinal absorptive capacity, intestinal permeability and jejunal histology in hiv and their relation to diarrhoea the virotoxin model of hiv-1 enteropathy: involvement of gpr15/bob and galactosylceramide in the cytopathic effects induced by hiv-1 gp120 in the ht-29-d4 intestinal cell line gp120-induced bob/ gpr15 activation: a possible cause of human immunodeficiency virus enteropathy hiv infection in gastric epithelial cells clinical focus: evolving options for managing noninfectious diarrhea in hiv-infected patients. clinical care options ò hiv evaluation of hiv protease and nucleoside reverse transcriptase inhibitors on proliferation, necrosis, apoptosis in intestinal epithelial cells and electrolyte and water transport and epithelial barrier function in mice diarrhea-associated hiv-1 apis potentiate muscarinic activation of cl-secretion by t84 cells via prolongation of cytosolic ca2? signaling the hiv protease inhibitors saquinavir, ritonavir, and nelfinavir induce apoptosis and decrease barrier function in human intestinal epithelial cells hiv protease inhibitors induce endoplasmic reticulum stress and disrupt barrier integrity in intestinal epithelial cells irritable bowel syndrome and hiv: a cross sectional study of the severity of gastrointestinal symptoms and hiv-infected subjects jejunal bacterial overgrowth and intestinal permeability in children with immunodeficiency syndromes gastrointestinal bacterial overgrowth: pathogenesis and clinical significance no relationship between gastric ph, small bowel bacterial colonisation, and diarrhoea in hiv-1 infected patients etiological agents of diarrhea in patients infected by the human immunodeficiency virus-1: a review diarrhea in the immunocompromised patient bacterial diarrhea in persons with hiv infection idiopathic aids enteropathy and treatment of gastrointestinal opportunistic pathogens aids-related diarrhea-pathogenesis, evaluation and treatment early use of tips in patients with cirrhosis and variceal bleeding clostridium difficile in a hiv-infected cohort: incidence, risk factors, and clinical outcomes investigation of diarrhea in aids cryptosporidium pathogenicity and virulence foodborne illness acquired in the united states-major pathogens cryptosporidiosis and isosporiasis among hiv-positive individuals in south ethiopia: a cross sectional study risk factors for intestinal parasitosis among antiretroviral-treated hiv/aids patients in ethiopia waterborne transmission of protozoan parasites: review of worldwide outbreaks-an update cryptosporidium surveillance and risk factors in the united states diarrheal diseases associated with hiv infection disseminated histoplasmosis: clinical and pathologic correlations endoscopic appearance of gi mycobacteriosis caused by the mycobacterium avium complex in a patient with aids: case report and review gastrointestinal infections in patients infected with hiv-1 diarrhoea due to small bowel diseases cytomegalovirus colitis in aids: presentation in 44 patients and a review of the literature a prospective study of endoscopy in hiv-associated diarrhea diarrhea in patients with aids department of health and human services department of health and human services antimotility agents for chronic diarrhoea in people with hiv/aids management of protease inhibitor-associated diarrhea imodium a-d, and imodium multi-symptom relief (loperamide and loperamide-simethicone) pa: mcneil consumer healthcare crofelemer for the treatment of secretory diarrhea efficacy and safety of crofelemer for noninfectious diarrhea in hiv-seropositive individuals (advent trial): a randomized, double-blind, placebocontrolled, two-stage study population pharmacokinetic (pk) analysis demonstrates no drug-drug interactions (ddi) between crofelemer, a novel treatment for noninfectious diarrhea in hiv? individuals, and antiretroviral therapy (art) therapeutic effect of activated attapulgite mormoiron in related diarrhea syndrome of acquired immunodeficiency (aids) the treatment (tx) of nelfinavir (nfv) induced diarrhea (nfvid) [abstract mechanisms of action of long-acting analogs of somatostatin octreotide therapy of large-volume refractory aids-associated diarrhea: a randomized controlled trial multicenter trial of octreotide in patients with refractory acquired immunodeficiency syndrome-associated diarrhea crofelemer, an antisecretory antidiarrheal proanthocyanidin oligomer extracted from croton lechleri, targets two distinct intestinal chloride channels crofelemer: a review of its use in the management of non-infectious diarrhoea in adult patients with hiv/aids on antiretroviral therapy acknowledgments technical editorial assistance was provided, under the direction of the authors, by pratibha hebbar, phd, synchrony medical communications, llc, west chester, pa. funding for this support was provided by salix pharmaceuticals, inc. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. key: cord-259846-oxbmtend authors: naik, parvaiz ahmad; zu, jian; owolabi, kolade m. title: global dynamics of a fractional order model for the transmission of hiv epidemic with optimal control date: 2020-06-18 journal: chaos solitons fractals doi: 10.1016/j.chaos.2020.109826 sha: doc_id: 259846 cord_uid: oxbmtend in this paper, a nonlinear fractional order epidemic model for hiv transmission is proposed and analyzed by including extra compartment namely exposed class to the basic sir epidemic model. also, the infected class of female sex workers is divided into unaware infectives and the aware infectives. the focus is on the spread of hiv by female sex workers through prostitution, because in the present world sexual transmission is the major cause of the hiv transmission. the exposed class contains those susceptible males in the population who have sexual contact with the female sex workers and are exposed to the infection directly or indirectly. the caputo type fractional derivative is involved and generalized adams-bashforth-moulton method is employed to numerically solve the proposed model. model equilibria are determined and their stability analysis is considered by using fractional routh-hurwitz stability criterion and fractional la-salle invariant principle. analysis of the model demonstrates that the population is free from the disease if [formula: see text] and disease spreads in the population if [formula: see text]. meanwhile, by using lyapunov functional approach, the global dynamics of the endemic equilibrium point is discussed. furthermore, for the fractional optimal control problem associated with the control strategies such as condom use for exposed class, treatment for aware infectives, awareness about disease among unaware infectives and behavioral change for susceptibles, we formulated a fractional optimality condition for the proposed model. the existence of fractional optimal control is analyzed and the euler-lagrange necessary conditions for the optimality of fractional optimal control are obtained. the effectiveness of control strategies is shown through numerical simulations and it can be seen through simulation, that the control measures effectively increase the quality of life and age limit of the hiv patients. it significantly reduces the number of hiv/aids patients during the whole epidemic. epidemiology mainly deals with the infectious diseases and predicts their occurrence, transmission as well as control in a population. it identifies the factors responsible for disease spread, facilitates treatment quality and health services, provides necessary measures for prevention, treatment, planning in order to improve the efficiency and effectiveness of health services [1] . hiv is a retrovirus which is discovered in 1981 in usa among the gay community causes an aids a severe life intimidating ailment. at present, there is no vaccine or cure for aids, that makes it an incurable disease with high mortality rate (there are almost 25 million deaths by aids per year worldwide), also it spread quickly affecting about 14,0 0 0 new case/day. the time duration for hiv to as an attractor. wang et al. [4] studied a delayed fractional order sir model with saturated incidence and treatment functions. they have provided the sufficient conditions that guarantee the existence of equilibria and discussed the global stability results for both disease-free equilibrium as well as endemic equilibrium by constructing a suitable lyapunov functions. almeida [5] in his paper studied a fractional seir epidemic model in presence of treatment. he analysed the model and his main focus was on the fractional differential equations in order to describe the dynamics of certain epidemics. further, he proved the local stability for both equilibria. carvalho et al. [6] provided a hiv/hcv coinfection fractional order model to understand the impact of hiv viral load on the coinfection. their main motive in the model was to provide good fits to real data from patients suffering from several diseases such as hiv, hcv, dengue fever and many more. they have numerically suggested that the hiv viral load impacts impressively the severity of the hcv infection. also, by their results they showed that the treatment efficacy is also influential over the natural progression of hcv on the hiv/hcv coinfection. recently, kheiri and jafari [7] analysed a multi-patch hiv/aids epidemic model with fractional order derivatives and investigated the effect of human movement on the spread of hiv/aids epidemic among patches. they derived the basic reproduction number r 0 of the model and studied the local as well as global stability of the equilibria on the basis of r 0 . they have shown that the system is stable if r 0 < 1 and it becomes unstable if r 0 > 1 . they also obtained the sufficient conditions under which the endemic equilibrium is unique and globally asymptotically stable. besides this, they formulated a fractional optimal control problem in which the state and costate equations are given in term of the left fractional derivatives. they incorporated in the model time dependent controls in order to control the spread of hiv/aids epidemics. they also derived the necessary conditions for the fractional optimal control in their proposed model. the effect of varying the fractional order on the disease spread is also studied in their model. researchers have continuously studied the fractional order models of hiv disease dynamics and provided many well-known mathematical techniques for the solution of these models for the dynamics of hiv epidemics [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . besides this, a number of studies on fractional order modeling of other infectious diseases can be found in the literature [20] [21] [22] [23] . the fractional order derivative not only find its application on modeling infectious diseases but in other fields as well like vibration equation [24] and so on. the optimal control theory is developing fast and its various applications are extensively used in many fields of science and engineering [25] . this theory for linear systems has been highly improved [26] , however, the nonlinear optimal control problem (ocp) has become a strong topic and should be deeper investigated [27] [28] . jajarmi and baleanu [29] proposed a new approach based on the modal series method and eigenvalue decomposition technique to solve a class of nonlinear optimal control problems. they have also investigated the convergence analysis of their suggested technique. jajarmi et al . [30] proposed a new approach for the optimal control of time-varying delay systems with external persistent matched disturbances. in their internal model principle, they converted original time-delay model with disturbance into an augmented system without any disturbance. then, they selected a quadratic performance index for the augmented system to form an undisturbed time-delay optimal control problem. the necessary optimality conditions are then derived in terms of a two-point boundary value problem involving advance and delay arguments. at the end they finally provided a fast iterative algorithm for the latter advance-delay boundary value problem. they also investigated the convergence of the new iterative technique. the purpose of dealing with fractional order systems is the memory and hereditary properties which are the complex behav-ioral patterns of biological systems gives us more realistic way to model hiv/aids systems. in the fractional order models, the memory property allows the integration of more information from the past which predicts and translates the models more accurately. also, the hereditary property describes the genetic profile along with age and status of the immune system. because of such properties fractional order calculus have found wide applications to model dynamics processes in many well-known fields of science, engineering, biology, medicine and many other [31] [32] . saeedian et al. [33] formulated sir epidemic model with the inclusion of memory effect and studied its behavior along the memory effect on the disease spread with the help of fractional derivatives. rihan [34] provided a class of fractional order differential models of biological systems with memory, such as dynamics of tumor-immune system and dynamics of hiv infection of cd4 + t cells. communicable diseases have been a cause of global concern throughout the history of mankind. its outbreak severely affects the morbidity and the mortality rates across the globe. it is therefore important to implement the control measures to prevent and control the disease spread among the populations. kheiri and jafari [35] formulated a fractional optimal control epidemic model of hiv/aids with random testing and contact tracing. in their model, they have incorporated the control measures of condom use and antiretroviral therapy for the control of spread of hiv/aids in the susceptible population. they have presented a forward-backword sweep numerical method based on adams-bashforth-moulton method for the solution of their model. agrawal [36] formulated a fractional optimal control problem by using the reimann-liouville fractional derivatives and presented a numerical method for its solution. bashir et al. [37] presented a fractional optimal control for a kinetic model and provided a numerical scheme for its solution. going by the antecedents, we have seen clearly that modeling of physical and real-life scenarios with the fractional order derivatives is much more accurate when compared with the integer order cases. this assertion has been demonstrated a number of research papers, monographs and books, see for example [38] [39] [40] [41] . in view of these achievements, we are motivated in this research work by modeling the control and analysis of sei 1 i 2 r dynamics of hiv disease transmission using the caputo fractional order operator which is most suited for modeling the biological and physical facts [42] [43] [44] [45] [46] [47] . the choice of using the caputo derivative is due to the fact that, if the given function is a constant, then the caputo derivative of that function gives zero. primarily, the caputo operator computes an ordinary differential equation, followed by a fractional integral to obtain the desired order of fractional derivative. more importantly, the caputo fractional differential equation (fdo) permits the use of local initial conditions to be included in the derivation of the model. in the present paper, we propose and analyze a fractional optimal control problem, in which the state and co-state equations are given in terms of the caputo fractional derivatives. this approach simplifies the use of fractional numerical methods to solve the state and co-state equations. fractional optimal control problems can be regarded as a generalization of classic optimal control problems for which the dynamics of the control system are described by fractional differential equations. we incorporate into the model time dependent controls such as condom use for exposed individuals, treatment for infected female sex workers, awareness about the disease among unaware infectives and behavioral change for susceptibles in order to reduce the risk of the spread of hiv/aids disease. conditions for fractional optimal control of the disease are derived and the state and co-state equations are characterized by caputo fractional derivatives. the numerical solution of the proposed fractional optimal control problem is obtained by using generalized adams-bashforth-moulton method. furthermore, the efficacy of order of fractional derivative, the control strategies and the value of objective functional is investigated. the structure of the paper is designed as: in the next section 2 , some preliminary results required for the formulation of mathematical model is provided. development of the proposed mathematical model and its well-posedness is discussed in section 3 . in section 4 , we discuss the mathematical analysis of the proposed fractional order sei 1 i 2 r epidemic model along with equilibrium points and the stability of equilibrium points. in section 5 , the fractional optimal control problem is formulated and discussed. also, in this section, the necessary conditions for the optimality of proposed fractional optimal control problem is provided. furthermore, in section 6 , application of the generalized adams-bashforth-moulton method is performed on the proposed model and the numerical simulations are done to validate the analytical studies. in section 7 , numerical results are given to illustrate the capability of generalized adams-bashforth-moulton method and the behavior of the obtained solutions is also discussed in this section. finally, section 8 concludes all the major findings of the present research study. researchers have continuously extended the definitions of fractional order derivatives like the riemann-liouville, the caputo, caputo-fabrizio, atangana-baleanu, the grunwald-letnikov, the weyl, the marchaud, the riesz, and the miller and ross [48] [49] [50] [51] [52] . recently, many new definitions of fractional derivative [53] have hugely evolved, going from the derivatives with nonsingular kernel and new riemann-liouville fractional derivative without singular kernel to the two-parameter derivatives with non-singular and non-local kernel [54] [55] [56] . definition 2.1. a real function ψ( t ), t > 0 is said to be in the space c η , η ∈ r , if there exists a real number l > η, such that ψ (t) = t l ψ 1 (t) , where ψ 1 ( t ) ∈ c [0, ∞ ) and it is said to be in the space where κ > 0 and (.) is a well-known gamma function. (3) definition 2.4. the caputo fractional derivative of ψ( t ) order κ > 0 is defined as where the operator c 0 d κ t satisfies the following two basic properties: the definition 2.3 and definition 2.4 are not equivalent to each other, and their difference is expressed by the caputo operator c 0 d κ t , has advantages for differential equations with initial values. in the case of riemann-liouville and caputo derivatives, respectively, the initial values are usually given as [57] rl assume that the function d κ t ψ (t) , satisfies some smoothness conditions in every finite interval (0, t ), t ≤ t . choosing the grid 0 = τ 0 < τ 1 < ... < τ n +1 = t = ( n + 1 ) u, τ n +1 − τ n = u, and using the classical notation of finite differences, the laplace transform of the caputo fractional derivative of ψ( t ) order κ > 0 is defined as definition 2.6. the laplace transform of the function where e κ, κ 1 is the two-parameter mittage-leffler function with κ, κ 1 > 0. further, the mittage-leffler function satisfies the following equation [58] e κ, κ 1 to describe the transmission dynamics of hiv epidemics, we have generalized the basic sir epidemic model by including more compartments, to one in which population is divided into five sub-classes, the susceptible population s ( t ), the exposed population e ( t ), the infective population that don't know they are infected i 1 ( t ), the infective population that know they are infected i 2 ( t ), by means of medical screening or otherwise and recovered population r ( t ). the proposed model is considered as the generalization of the original kermack-mckendrick model [8] , where only three compartments were considered, but here the exposed compartment is included contains those susceptible males in the population who have sexual intercourse with the female sex workers as a result by having sexual contact they are exposed to the infection. furthermore, the infected class is divided into two sub-classes namely infected female sex workers who are unaware about their disease status and the infected female sex workers who knows their disease status. thus, the model takes the following form [ 3 , 15 , 18 ] . for the understanding of hiv disease dynamics, the total population n ( t ) is divided into five sub-population compartments namely susceptible, exposed, infected but unaware, infected but aware and recovered such that the following description is associated to the above classical model: the susceptibles are recruited at a rate , β 1 is the per capita rate for susceptibles individuals with unaware infectives, β 2 is the per capita rate for susceptibles individuals with aware infectives, λ is the natural death rate unrelated to aids, σ is the break through into infected class, θ is the rate of unaware infectives to become aware infectives by screening or testing, ρ is the rate by which types of infectives develop aids and d is the aids related death rate. it may further be noted that we further extend the above ordinary differential model to the following fractional order system of order κ, with σ , ρ > 0 being the rate that exposed individuals become infectious and recovery rate, respectively and λ ≥ 0 being the infection related death rate. the purpose of considering the fractional order case is the significant uniqueness of these varieties of fractional order systems with non-local characteristics (memory) and hereditary properties that have not been seen with the integer-order differential operators which widely exists in biology. also, using fractional order differential equations can help us to reduce the errors arising from the neglected parameters in modelling real life phenomena. in each case, we replace the ordinary derivative by a fractional derivative. thus, our proposed fractional order model for hiv disease transmission has the form subject to the initial conditions and if κ = 1 , then system (9) reduces to an integer order system (8) . it is clear that the variable r ( t ) does not appear in the first four equations, thus it is meaningful to consider the reduced system (9) as: (11) subject to the positive initial conditions here, it is assumed that the functions and their caputo fractional derivatives are continuous at t ≥ 0. the existence, uniqueness, and non-negativity of the solution of system (11) are analyzed. the schematic diagram of the proposed fractional order sei 1 i 2 r epidemic model (9) is shown in fig. 1 . in this section, we first prove the existence and uniqueness of positive solution, then the basic reproduction number and the existence conditions for both equilibria (disease-free equilibrium and endemic equilibrium) are obtained, finally, the conditions for the stability of both the equilibria are obtained. let us denote r 4 for the proof of the main theorem about the non-negativity of the solutions, we recall the following lemma [ 3 , 8 , 15 ] . this completes the proof. t for the initial value problem given by (11) along initial conditions (12) on t ≥ 0 in (0, κ) and the solution will remain in r 4 + . furthermore, the solutions are all bounded. proof. according to lin [58] from the theorem 3.2 [58] and remark 3.2 [58] , we can determine the solution on (0, ∞ ) by solving the model (11) along initial conditions (12) which is not only existent but also unique. subsequently, we have to explain the nonnegative domain r 4 + , is positively invariant region. from model (11) , we find on each hyperplane bounding the non-negative orthant, the vector field points into r 4 + . furthermore, from system (11) thus, by lemma 4.1 , in the case of hiv infection, the total population n ( t ), i.e., the subpopulations s ( t ), e ( t ), i 1 ( t ) and i 2 ( t ) are bounded. by positivity means the population survives and boundedness refers as a natural restriction to growth as a consequence of limited resources. this completes the proof of the theorem 4.1 . therefore, the biologically feasible region for the system (9) is for the equilibrium points, setting the right-hand side of the system (11) equal to zero, we obtain equilibrium points as after simplification, the system (13) gives the disease-free equithus, the proposed nonlinear fractional order sei 1 i 2 r epidemic model has at most two equilibria namely disease-free equilibrium in order to study the local stability of the disease-free equilibrium, we first compute the basic reproduction number by using next generation matrix method [61] [62] [63] [64] (11) can be written as by the next generation matrix method, the matrices ғ and ѵ at the disease-free equilibrium point − d 0 are obtained by where ғ is non-negative and v is a non-singular m-matrix. therefore, the basic reproduction number denoted by r 0 which is considered as the spectral radius of the next generation matrix v −1 at the disease-free equilibrium − d 0 is thus given by it shows that if r 0 < 1 , then the disease does not spread in the population and the infection dies. on the other hand, if r 0 > 1 , then the disease persists in the whole population. now, we will discuss the local stability analysis of equilibrium points. for this, we state the results in the form of theorems and prove them. proof. to prove the above theorem 4.2 , the general jacobian matrix and the matrices corresponding to each equilibrium point will be obtained. therefore, the jacobian matrix is given by therefore, by the routh-hurwitz stability conditions for fractional order systems [65] , the necessary and sufficient condition for various fractional order models. therefore, the disease-free equilibrium of system (11) is asymptotically stable if all of the (14) . hence, a sufficient condition for the local asymptotic stability of the equilibrium points is that the eigenvalues γ this confirms that fractional order differential equations are, at least, as stable as their integer order counterpart. by solving the characteristic equation, the eigenvalues can be obtained as the simplification allows us to get the following algebraic equation this implies, therefore, the roots of the characteristic equation are , satisfy the condition given by (14) . therefore, all the eigenvalues have negative real parts if r 0 < 1 . this completes the proof of the theorem 4.2 . in the next theorem 4.3 , we discuss the local asymptotic stability of the endemic equilibrium of the system given by (11) . proof. the jacobian matrix of the system (11) evaluated at endemic equilibrium − d * is given as the characteristic equation of the linearized system is in the form now, the discriminant of the polynomial − p (γ ) = γ 3 + ϑ 1 γ 2 + ϑ 2 γ + ϑ 3 is described by [ 3 , 12 , 15 ] and using the construction of results by ahmed et al. [ 19 , 66 ] , following fractional routh-hurwitz conditions associated with are observed. we have the following result. i if d (− p ) > 0, then the necessary and sufficient condition for the equilibrium point to be locally asymptotically stable is ϑ 1 > 0, the global existence of the solution of the fractional differential equation always becomes a most important concern, which is carry out in the following section. theorem 4.4. [ 12 , 58 ] , assume that the function : r + × r 4 → r 4 satisfies the following conditions in the global space: 1) the function ( t, ψ( t )) is lebesgue measurable with respect to t on r . 2) the function ( t, ψ( t )) is continuous with respect to ψ( t ) on ( t, ψ ( t ) ) ≤ α 1 + α 2 ψ (t) , for all most every t ∈ r and all ψ (t) ∈ r 4 . here α 1 , α 2 are two positive constants and has a unique solution. proof. from the theorem 4.4 , we obtain the unique solution on (0, ∞ ) by solving the system (11) . firstly, lin [58] discussed the proof of theorem and shows that the solution is not only exist but also unique. in theorem 4.1 , we already proved that the solution of model (11) will remain in r 4 + . ( [ 52 , 67 ] ) let ψ (t) ∈ r + be a continuous and derivable function. then, for any time instant t ≥ 0, and where κ ∈ (0, 1). note that for κ = 1 , the inequalities in (20) and (21) becomes equalities. now, we provide the global stability results of the equilibria in the following theorems by considering the lyapunov direct method. (11) is globally asymptotically stable in , if r 0 ≤ 1 and unstable when r 0 > 1 . to prove this, we define a lyapunov function φ 1 ( t ) given using the disease-free steady state condition of model (11) , s 0 = λ , we have from the equation (22) as in addition, we know that c 0 d κ t φ 1 (t) | ( 11 ) = 0 , if and only if s(t ) = s 0 and i 1 (t) = 0 .substituting i 1 (t) = 0 into (11) , one can directly obtain e(t ) = 0 . using i 1 (t) = e(t ) = 0 again in (11) , then i 2 (t) = 0 . therefore, the maximum invariant set for { ( s, e, i 1 , i 2 ) ∈ 0 : c 0 d κ t φ 1 (t) | ( 11 ) = 0 } is the singleton set -d 0 . according to the lasalle's invariance principle [61] [62] [63] [64] , we know that all solutions in 0 converge to -d 0 .therefore, the disease-free steady state of model (11) is globally asymptotically stable when r 0 ≤ 1 . this completes the proof of the theorem 4.6 . (11) is globally asymptotically stable in , when r 0 > 1 . to prove this, we define a lyapunov function φ 2 ( t ) given using the endemic conditions, therefore, φ 2 ( t ) is bounded and non-increasing. further, the limit of φ 2 ( t ) exits as t → ∞ . in addition, we know that c lasalle's invariance principle [61] [62] [63] [64] , we know that all solutions in * converge to − d * .therefore, the endemic equilibrium of proposed model (11) is globally asymptotically stable when r 0 > 1 . this completes the proof of the theorem 4.7 . in this section, we extend the basic model (8) by including some particular control measures aimed at controlling the spread of the hiv infection and formulate the fractional optimal control problem by proposing the control objectives. the aim of the control measures is to reduce the infection in the population and thus there is the need to formulate the optimal control problem to achieve this goal. the first control function v 1 ( t ) represents the behavioral change for susceptibles which reduced the number of exposed by a factor ( 1 − v 1 (t) ) . the control v 1 ( t ) is proposition of the susceptible individuals who change their sexual habits per unit of time. the second control function v 2 ( t ) is the use of condoms to the exposed individuals who are going to have sexual interaction with the female sex workers. the third control function v 3 ( t ) represent the enhancement of the strength of treatment for the infected individuals. the fourth control function v 4 ( t ) is the awareness source among the unaware infectives about their disease status. under these control measures the proposed model (8) all formulas and models should be left aligned . (24) with the non-negative initial conditions the control is completely effective when v i (t) = 1 and the control is not effective when v i (t) = 0 , for i = 1 , 2 , 3 , 4 i.e., 0 ≤ v i ( t ) < 1. our focus is to minimize the number of exposed individuals under the cost of applying control measures which can be done by consider the following fractional optimal control problem to minimize the objective functional given by subjected to the state system given in (24) along non-negative initial conditions (25) . in eq. (26) , q represent the positive weight constant of the exposed population, while -p 1 , -p 2 , -p 3 , and -p 4 are positive weight constants for behavioral change, personal protection, treatment strategy and awareness source respectively. the subjected to the state system given in (24) , where the control set is defined as the lagrangian ɫ and hamiltonian h for the fractional optimal problem (24) (28) are respectively given by [ 35 , 68-69 ] (29) and this further implies, where λ s , λ e , λ i 1 , λ i 2 and λ r are the adjoint variables. we have to prove the necessary conditions for the optimality of the fractional system (24) . for the optimal control v ( t ), that minimizes the performance index (31) subjected to the dynamical constraints (32) with initial conditions π ( 0 ) = π 0 (33) where π ( t ) and v ( t ) are the state and control variables, respectively, l and ω are differentiable functions, and 0 < κ ≤ 1. we have the following theorem. if ( π , v ) is a minimizer of (31) under the dynamic constraint (32) and the boundary condition (33) , then there exists a function λ such that the triplet ( π , v, λ) satisfies proof. for the proof of theorem 5.1 , readers are suggested to see [ 7 , 35-36 ] , where the authors have given the proof in detail. this completes the proof of the theorem 5.1 . and r * be optimal state solutions with associated optimal control variables v * 1 , v * 2 , v * 3 , v * 4 for the optimal control problems (24) and (26) . then there exist adjoint variables λ s , λ e , λ i 1 , λ i 2 and λ r satisfying with transversality conditions or boundary conditions furthermore, the control functions v * 1 , v * 2 , v * 3 and v * 4 are given by all form ulas and mode ls shou ld be left alig ned . proof. the adjoint system (35) i.e., λ s , λ e , λ i 1 , λ i 2 and λ r are obtained from the hamiltonian h as in this section numerical solution for the proposed fractional order sei 1 i 2 r epidemic model (9) is presented. because no analytical solution to the nonlinear fractional system (9) is available, we use the technique so-called generalized adams-bashforth-moulton method [ 3 , 35 , 37 ] to obtain the numerical solution of the system (9) . in this algorithm, we derive the predictor-corrector scheme for obtaining the numerical solution of the nonlinear fdes. to provide the estimated solution by means of this algorithm, consider the subsequent nonlinear fractional differential equation [ 3 , 35 , 37 ] with the following initial conditions now, with operating by the fractional integral operator on the equation (37) , we can obtain on the solution ψ( t ) by solving the following equation: this equation (39) is equivalent to the volterra integral equation. diethelm et al. [70] [71] [72] used the predictor-corrector scheme based on the adams-bashfort-moulton algorithm to integrate (39) . setting h = t n , t n = nh and n = 0 , 1 , 2 , ..., n ∈ z + , the equation (39) can be discretized as follows: where a q,n +1 = all form ulas and mode ls shou ld be left alig ned . (41) and the predicted value ψ p h ( t n +1 ) is determined by the error estimate is in which p = min (2 , 1 + κ ) . in this subsection, we solve numerically the nonlinear fractional sei 1 i 2 r epidemic model using the proposed method. in view of the generalized adams-bashforth-moulton method, the numerical scheme for the proposed model (9) is given in the following form [73] [74] [75] [76] [77] further, the quantities , r h ( t q )), are computed from the following functions, in addition, the quantities are computed from equations (43) (47) respectively, at the points t n +1 , n = 1 , 2 , 3 , ..., m. for the fractional optimal control problem (24) discussed in section 5 , similar procedure is followed for the numerical results. therefore with π = s, e, i 1 , i 2 , r and the control v . similarly, for the adjoint system we have the coefficients a q,n +1 , b q,n +1 are given by equations (41) and (42) respectively. in order to justify our theoretical findings, we introduced in this section some numerical experiments obtained for different instances of fractional power κ for the hiv epidemic model without control (9) and with control (24) along with adjoint variable systems and the control strategies. we present the numerical results for the model (9) when all control measures are absent and also to examine the role of fractional order κ on the hiv disease spread. then, we simulate the fractional optimal control of the model and investigate the effect of the controls introduced in the model on the spread of epidemics. we use the generalized adams-bashforth-moulton method for the simulation of both the systems and use the values of parameters described in table 1 . in this subsection, we present numerical results for fractional system (9) and allow the values of κ to varies from k = 0 . 75 to k = 1 as seen in the fig. 2 . it is clear from the fig. 2 that fractional order has significant effect on dynamic behavior of all the components. we observe that when the derivative order κ is reduced from 1, the memory effect of the system increases, and therefore the infection grows slowly and the number of hiv-infected population and aids people increases for a long time. also, undiagnosed hiv-infected population in some societies refuse to per-form hiv test for reasons such as stigma and fear of identification due to lack of knowledge about the disease. this results in a delay in identification of hiv-infected individuals, an increase in the undiagnosed hiv-infected population, fast progress of aids and an increase in people diagnosed with aids. on the other hand, the experience or knowledge of individuals about the disease causes susceptible and exposed individuals to take different precautions, such as behavioral change, vaccination, treatment and condom use, against infection transmission. this leads to a slow growth of infection among the population. therefore, from the numerical results in fig. 2 , we conclude that the derivative order κ (0.75 ≤ κ ≤ 1) can play the role of precautionary measures against infection transmission, treatment of infection and delay in accepting hiv test. existence of attractors for some fractional order κ for different population groups are given in fig. 3 . thus, the results from fig. 3 shows that there is tendency of each population class to exist and enter into permanence with time. numerical results for the difference of integer-order and fractional order are given in figs. 4-5 . it is clearly visible from figs. 4-5 that the differential equations with fractional order derivative have rich dynamics and describe biological systems better than traditional integer-order models. from the above discussion and numerical results in figs. 2-5 , we conclude that the derivative order κ can play the role of experience or knowledge of individuals about the past of the disease. therefore, the numerical results confirm that differential equations with fractional order derivative have rich dynamics and describe biological systems better than traditional integer order models. as a result, our numerical results are more logical than the results of other articles on the modeling of the hiv/aids epidemic and other models with integer-order derivative due to the presence of the fractional derivative order κ (0.75 < κ ≤ 1). now, we investigate the effect of the control measures introduced in the model on the spread of the epidemic. we consider the following strategies and examine the corresponding numerical results. in this subsection, we present the numerical results for the model (24) when all control measures are present. the results are obtained in different ways by applying control strategies in the following five ways. in the first control strategy, we set the control measures v 3 = 0 , v 4 = 0 and active the control measures v 1 = 0 . 5 , v 2 = 0 . 5 namely the behavioral change for susceptible individuals and condom use for the exposed individuals which is shown in fig. 6 , for different values of fractional order κ. analysis of control strategy-1 predicts that susceptible and exposed individuals greatly decrease after implementing control measure v 1 , v 2 . in fig. 6 , we observe that this control strategy results in a significant decrease in the number of undiagnosed hiv-infected population and aids people for a long time compared with the case without control. fig. 6 shows that, by applying the strategy-1, the value of r 0 will be less than 1 for more time when κ is reduced from 1. this means that by decreasing κ from 1, we can control the spread of disease over a longer period of time. therefore, the presence of the fractional derivative order κ in the model increases the use of condom control and behavioural control in the population. the control v 1 is proportion of the susceptible individuals who change their sexual habits per unit time. the class r , the removed class, represents the number of people who have greatly changed their sexual habits such that they cannot easily be infected through sexual contact. people in the class r take on safe habits and keep these habits in the rest of their lives. the importance of class r is that it emphasizes the need for prevention for a disease like hiv that has no treatment. therefore, increasing the members of this class plays an important role in controlling the spread of disease. strategy-2. using only condom use control in the second control strategy, we set the control measures v 1 = 0 , v 3 = 0 , v 4 = 0 and active the control measure v 2 = 0 . 5 namely the condom use for the exposed individuals which is shown in fig. 7 , for different values of fractional order κ. analysis of control strategy-2 predicts that exposed individuals greatly decrease after implementing control measure v 2 . the results in fig. 7 , further show that condom use is the main control measure which can be helpful in controlling the disease more properly. this is because the control is applied to exposed class which is the main source from which the virus can transmit and spread due to the fact that this class is easily available for virus during their sexual contact with infected female sex workers. strategy-3. using only treatment control in the third control strategy, we set the control measures v 1 = 0 , v 2 = 0 , v 4 = 0 and active the control measure v 3 = 0 . 7 namely the efficiency of treatment given to the aware infected individuals which is shown in fig. 8 , for different values of fractional order κ. analysis of control strategy-3 predicts that infected individuals greatly decreases after implementing control measure v 3 . this is due to the fact that treatment of diagnosed hiv-infected population results in an increase in the level of cd4 + t-cells of this class. therefore, this strategy prolongs the lifespan of hiv-infected patients and delays the onset of aids. fig. 8 shows that differential equations with fractional order derivative have rich dynamics and describe biological systems better than traditional integerorder models. strategy-4. using treatment control and awareness control in the fourth control strategy, we set the control measures v 1 = 0 , v 2 = 0 and active the control measures v 3 = 0 . 5 , v 4 = 0 . 5 namely the efficiency of treatment given to the aware infected individuals and the awareness source for unaware infected individuals which is shown in fig. 9 , for different values of fractional order κ. analysis of control strategy-4 predicts that infected individuals greatly decreases after implementing control measures v 3 and v 4 . in fig. 9 , we observe that this control strategy results in a signif-icant decrease in the number of aware infected hiv people and unware infected people compared with the case without control. in last control strategy, we activate all the control measures v 1 = 0 . 5 , v 2 = 0 . 5 , v 4 = 0 . 5 and v 4 = 0 . 8 namely the behavioral change for susceptible individuals, condom use for exposed individuals, efficiency of treatment given to the aware infected individuals and the awareness source for unaware infected individuals which is shown in fig. 10 , for different values of fractional order κ. analysis of control strategy-5 predicts that susceptible individuals and exposed individuals decreases with the control measures v 1 and v 2 while infected individuals greatly decrease after implementing control measures v 3 and v 4 . by adding the behavioral change control v 1 or condom use control v 2 to the art treatment control v 3 , we see from figs. 6-9 , that the strategies 1-4 result in a decrease in the hiv infected population and aids people compared with the case without control. with implementing all the control effort s, we observe that the strategy-5 results in a significant decrease in the hiv-infected population and aids people compared with the case without control. with comparison of the strategies, we see that the strategy-5 is better than the other strategies in control and reduction of the spread of hiv/aids epidemic. therefore, by applying the strategy-5, we can increase the life time and the quality of life for those living with hiv and decrease significantly the number of hiv-infected population and aids people. on the other hand, in human societies, the process of evolution and control of the epidemic is associated with memory. when a disease spreads in a society, the experience or knowledge of individuals about the past of the disease helps susceptible individuals to take different precautions, such as behavioural change, treatment, awareness and condom use against infection transmission. also, the experience or knowledge can lead to the screening measures of entry and exit between different groups. it is noticeable from fig. 10 that due to the memory property of fractional derivatives, the derivative order κ affects the values of the controls. we see that the maximum levels of the controls are reduced when κ limits to 1. on the other hand, the memory effect characterized by fractional derivative is reduced when κ limits to 1. therefore, by reducing the memory effect, the maximum levels of the controls are reduced. in the current study, we have introduced a nonlinear sei 1 i 2 r fractional order epidemic model for the transmission dynamics of hiv epidemics. the non-negative solution of the model is provided by using the generalized mean value theorem. we obtained the basic reproductive number r 0 , which perform as a threshold parameter in the disease status. the existence of equilibria and their asymptotical stability results using fractional routh-hurwitz stability criterion is discussed. we established and investigated the stability analysis of the fractional order model with respect to the values of r 0 . the disease-free equilibrium is locally asymptotically stable if r 0 ≤ 1 . for r 0 > 1 , using theorem 4.3 and corollary 4.1 , we investigated the local stability of the positive endemic equilibrium state − d * . meanwhile, global asymptotic stability of the disease-free and endemic equilibrium point is investigated by constructing a suitable lyapunov functions. additionally, we investigated the optimal control problem by the application of the optimal control theory. we used the pontryagin's minimum principle to provide the necessary conditions needed for the existence of the optimal solution to the optimal control problem. furthermore, generalized adams-bashforth-moulton method is applied to obtain a numerical solution of the proposed fractional order sei 1 i 2 r epidemic model (9) and the fractional optimal problem (24) . the re-sults obtained shown that the adams-bashforth-moulton method is an accurate and effective technique for obtaining the numerical solution of the proposed nonlinear fractional order sei 1 i 2 r epidemic model. lastly, the theoretical results are verified by numerical simulations to measure the efficacy and impact of controls on the transmission of the hiv/aids disease. from the numerical simulation, the size of the exposed population is significantly reduced under the controlled conditions. this proposes that if all four control measures v 1 (behavioral change for susceptibles), v 2 (condom use by the exposed individuals), v 3 (strength of treatment for the infected individuals), v 4 (awareness source among the unaware infectives) are employed for the same period of time and continue for a considerable period of time, the spread of hiv disease through prostitution could be restricted. in this manner, the fractional order optimal control method can progress the value of the necessary control measures. this recommends that personal precautional measures, periodic monitoring by medical professionals and researchers should be done to control the transmission of the hiv disease dynamics. the recently emerged virus namely novel coronavirus (covid-19) which has originated from wuhan the capital city of hubei providence of mainland china in december 2019 is a major threat to mankind at present in the whole world. application of fractional order derivatives to model the new outbreak of coronavirus and other trending diseases are left for future research. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. modeling the role of acquired immune response and antiretroviral therapy in the dynamics of hiv infection dynamics of an sir model with nonlinear incidence and treatment rate hiv/aids epidemic fractional-order model dynamic analysis of a delayed fractional-order sir model with saturated incidence and treatment functions analysis of a fractional seir model with treatment hcv coinfection model: a fractional-order perspective for the effect of the hiv viral load stability analysis of a fractional order model for the hiv/aids epidemic in a patchy environment contributions to the mathematical theory of epidemics. iii-further studies of the problem of endemicity a fractional-order differential equation model of hiv infection of cd4 + t-cells solving a fractional order model of hiv infection of cd4 + t cells a fractional-order model of hiv infection with drug therapy effect the modeling dynamics of hiv and cd4 + t-cells during primary infection in fractional order: numerical simulation applications of the extended fractional euler-lagrange equations model to freely oscillating dynamical systems local and global stability of fractional order hiv/aids dynamics model a fractional order seir model with vertical transmission numerical behavior of a fractional order hiv/aids epidemic model a fractional-order model for hiv dynamics in a two-sex population analysis and numerical solution of seir epidemic model of measles with non-integer time fractional derivatives by using laplace adomian decomposition method equilibrium points, stability and numerical solutions off fractional-order predator-prey and rabies models a new fractional model and optimal control of a tumor-immune surveillance with non-singular derivative operator a new fractional modelling and control strategy for the outbreak of dengue fever a new and efficient numerical method for the fractional modelling and optimal control of diabetes and tuberculosis co-existence a new fractional sirs-si malaria disease model with application of vaccines, anti-malarial drugs, and spraying on the analysis of vibration equation involving a fractional derivative with mittag-leffler law optimal control with engineering applications the optimal homotopy analysis method for solving linear optimal control problems online identifier-actor-critic algorithm for optimal control of nonlinear systems inverse optimal controller based on extended kalman filter for discrete-time nonlinear systems optimal control of nonlinear dynamical systems based on a new parallel eigenvalue decomposition approach a new approach for the optimal control of time-varying delay systems with external persistent matched disturbances fractional differential equations fractional-order nonlinear systems: modeling, analysis and simulation memory effects on epidemic evolution: the susceptible-infected-recovered epidemic model numerical modeling of fractional-order biological systems fractional optimal control of an hiv/aids epidemic model with random testing and contact tracing a general formulation and solution scheme for fractional optimal control problems optimal control of a fractional-order enzyme kinetic model fractional calculus: theory and applications, differentiation and integration to arbitrary order fractional integrals, derivatives -theory and applications. yverdon: gordon and breach science publishers mathematical modelling and analysis of love dynamics: a fractional approach numerical methods for fractional differentiation on the formulation of adams-bashforth scheme with atangana-baleanu-caputo fractional derivative to model chaotic problems numerical analysis and pattern formation process for spacefractional super diffusive system computational study of noninteger order system of predation behavioural study of symbiosis dynamics via the caputo and atangana-baleanu fractional derivatives mathematical analysis and computational experiments for an epidemic system with nonlocal and nonsingular derivative numerical simulation of fractional-order reaction-diffusion equations with the riesz and caputo derivatives a new definition of fractional derivative without singular kernel portraying the effect of calcium-binding proteins on cytosolic calcium concentration distribution fractionally in nerve cells properties of a new fractional derivative without singular kernel an introduction to the fractional calculus and fractional differential equations lyapunov functions for fractional order systems a new definition of fractional derivative analytical and numerical schemes for a derivative with filtering property and no singular kernel with applications to diffusion characterizations of two different fractional operators without singular kernel comparing the new fractional derivative operators involving exponential and mittag-leffler kernel a fractional order epidemic model for the simulation of out breaks of influenza a (h1n1) global existence theory and chaos control of fractional differential equations estimating the approximate analytical solution of hiv viral dynamic model by using homotopy analysis method modeling the mechanics of viral kinetics under immune control during primary infection of hiv-1 with treatment in fractional order reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission the stability of dynamical systems global stability of infectious disease models using lyapunov functions the construction of nextgeneration matrices for compartmental epidemic models stability results for fractional differential equations with applications to control processing, computational engineering in systems and application on some routh-hurwitz conditions for fractional order differential equations and their applications in lorenz, rssler, chua and chen systems volterra-type lyapunov functions for fractional-order epidemic systems effect of partial immunity on transmission dynamics of dengue disease with optimal control differential equations: classical to controlled, mathematics in science and engineering an algorithm for the numerical solution of differential equations of fractional order analysis of fractional differential equations a predictor-corrector approach for the numerical solution of fractional differential equations spatiotemporal patterns in the belousov-zhabotinskii reaction systems with atangana-baleanu fractional order derivative new approaches to the fractional dynamics of schistosomiasis disease model optimal solutions for singular linear systems of caputo fractional differential equations on the formulation of adams-bashforth scheme with atangana-baleanu-caputo fractional derivative to model chaotic problems numerical solution of some fractional dynamical systems in medicine involving non-singular kernel with vector order the first author is very grateful to xi'an jiaotong university for the assistant professor position provided to him. also, the authors would like to thank the reviewers and editors of this paper for their careful attention to detail and constructive feedback that improved the presentation of the paper greatly. the study was supported by grants from the china postdoctoral science foundation (grant nos. 2019m663653 and 2014m560755), the national natural science foundation of china (grant nos. 11971375, 11571272, 11201368 and 11631012), the national science and technology major project of china (grant no. 2018zx10721202) and grant from the natural science foundation of shaanxi province (grant no. 2019jm-273). the funding body did not play any roles in the design of the study and in writing the manuscript. key: cord-256324-w3bejmy5 authors: hamada, yoshio; kiso, yoshiaki title: new directions for protease inhibitors directed drug discovery date: 2016-07-22 journal: biopolymers doi: 10.1002/bip.22780 sha: doc_id: 256324 cord_uid: w3bejmy5 proteases play crucial roles in various biological processes, and their activities are essential for all living organisms—from viruses to humans. since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, β‐secretase, virus proteases, and dipeptidyl peptidase‐4. previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human t‐lymphotropic virus type i protease, plasmepsins, and β‐secretase, as drug candidates for hypertension, adult t‐cell leukaemia, human t‐lymphotropic virus type i‐associated myelopathy, malaria, and alzheimer's disease. our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 wiley periodicals, inc. biopolymers (pept sci) 106: 563–579, 2016. e nzymes play important roles in several biological processes including digestion, absorption, metabolism, and propagation. their functions are essential for all living organisms and are closely associated with many pathogenic mechanisms. enzyme inhibitors or activators therefore represent important molecular targets for the development of new disease treatments. among enzymes, proteases are important molecular targets because their substrates are proteins, proteases, and peptides, which play crucial roles in all forms of life-from viruses to humans. proteases are categorized into four major classes: aspartic proteases, serine proteases, cysteine proteases, and metalloproteases, which contain aspartic acid, serine, cysteine, and metallic ions, respectively, in their active sites. renin was the first protease for which inhibitors were designed logically, using a computational approach (structure-based drug design; sbdd). renin is an aspartic protease that is involved in the rate-limiting first step of the renin-angiotensin-aldosterone system (raas) and is a molecular target for antihypertensive agents. [1] [2] [3] renin cleaves angiotensinogen to release the decapeptide angiotensin i as shown in figure 1a . angiotensin i is subsequently cleaved by a nonspecific dipeptidyl carboxypeptidase, angiotensin-converting enzyme (ace), to release the potent octapeptide vasopressor angiotensin ii. the pepsin inhibitor pepstatin a, which features the camino acid statine as the transition state analogue, was reported to inhibit the activity of renin, and since then, many renin inhibitors containing a transition state analogue have been developed. 1 boger et al. designed a renin inhibitor, called statine-containing renin inhibitory peptide (scrip; halfmaximal inhibitory concentration (ic 50 ) 5 16 nm; figure 1b ), that had a statine residue at the p 1 position and contained a part of the angiotensinogen amino acid sequence. 2 we also designed and synthesized the potent, small inhibitor, kri-1314 (ic 50 5 2.4 nm) , that contains a norstatine-type residue as a transition state analogue. 3 the first renin inhibitor drug, aliskiren (ic 50 aspartic proteases form a substrate transition state that has no covalent bonds between the substrate and the protease (as discussed later), and as a result, inhibitors can be easily designed using a computational approach. the development of inhibitors for the metalloprotease ace had to wait until the discovery of the bradykinin-potentiating peptide pyroglutamyl-lys-trp-ala-pro (bpp5a; figure 1c ). bpp5a was found in the venom of the snake bothrops jararaca and inhibits ace activity. 4 the first ace inhibitor drug, captopril, was approved by the fda in 1981 and was designed to emulate the c-terminus ala-pro sequence of bpp5a. 5 the proline residue of captopril docks in the s 2 pocket of ace, and another residue docks in the s 1 pocket. the coordination bond that is formed between the sulfur atom of captopril and the zinc ion in the active site of ace appears to contribute to the potent inhibition. subsequently, human immunodeficiency virus type 1 (hiv-1) protease inhibitors were designed using a computational approach to develop therapeutics for the treatment of acquired immunodeficiency syndrome (aids). hiv-1 protease is also an aspartic protease, and its inhibitors can be designed using an approach similar to that used for the design of renin inhibitors. hiv-1 protease is responsible for processing the gag and gag-pol polyproteins and enabling the proliferation of the retrovirus. the first hiv-1 protease inhibitor approved by the fda, saquinavir (figure 2) , was developed using a computational approach. many hiv-1 protease inhibitors, i.e., saquinavir, indinavir, nelfinavir, atazanavir, and darunavir, possess a hydroxyethylamine moiety that acts as a transition state analogue, whereas ritonavir and lopinavir possess a hydroxyethylene moiety as a transition state analogue. the use of these hiv-1 protease inhibitors in highly active anti-retroviral therapy (haart) has contributed considerably to overcoming aids. 6 all hiv-1 protease inhibitors have a hydroxyl group in the transition state analogue residue and can strongly bind to the active site of the hiv-1 protease. we have also reported a series of potent hiv-1 protease inhibitors such as kni-272, kni-727, and kni-764 that contain a norstatine-type residue, allophenylnorstatine (apns), as a transition state analogue ( figure 2 ). 7 furthermore, we have used the transition state concept to design inhibitors of several other aspartic proteases, such as malarial plasmepsins, bsecretase, and human t-lymphotropic virus type i (htlv-1) protease. [8] [9] [10] [11] [12] here, we describe the latest developments in protease inhibitor design, including our own work. hamada and kiso aspartic proteases have two catalytic aspartic acid residues at the active site. first, a water molecule is deprotonated by the carboxyl anion of one of the aspartic acid side chains; the resulting hydroxide ion then attacks the carbonyl carbon of the substrate at the cleavage site to form a tetrahedral transition state as shown in figure 3 . rearrangement of this transition state causes the amide bond in the substrate to be broken. although serine and cysteine proteases cleave peptide bonds via a transition state consisting of a covalent bond between the catalytic serine/cysteine and carbonyl carbon at the cleavage position of the substrate, aspartic proteases form a transition state based on noncovalent interactions between their carboxylic acid/carboxylate groups and the two hydroxyl groups of the substrate. the fact that the transition state in aspartic proteases involves no covalent bond formation makes the tetrahedral structures in the transition state relatively simple. this makes it possible to design aspartic protease inhibitors logically using a computational approach. malaria is a disease caused by parasitic protozoa of the genus plasmodium that feeds on the haemoglobin of an infected person. the protozoa produce a family of aspartic proteases, plasmepsins, which degrade the host's haemoglobin and eventually cause the symptoms of malaria and death of the host. plasmepsins are currently being investigated as a molecular target for antimalarial drugs. plasmepsins are known to consist of at least ten isoforms-plm i, ii, iii, iv, v, vi, vii, ix, x, and histo-aspartyl protease (hap)-that are encoded in the parasite's genome. although most plasmepsins have two aspartic acid moieties in the catalytic site, one of the catalytic aspartic acid residues is replaced with histidine only in the case of hap. among the plasmepsin isoforms, plm i and plm ii are known to readily cleave haemoglobin between the phe33 and leu34 residues of the a-globin subunit (table i) . francis et al. reported that 1 (sc-50083, figure 4 ) selectively blocks the aspartic hemoglobinase, prevents haemoglobin degradation and kills the parasites. sc-50083 was screened from peptidomimetic aspartic protease inhibitors library. 13 an aldehyde-type compound 2 (ro 40-4388) was reported by moon. ro 40-4388 was identified from the inhibitors library and inhibited a chloroquine-resistant strain of p. falcipaeum. 14 silva et al. reported plasmepsin inhibitor 3, which was screened from the inhibitors library of an aspartic protease, catepsin d. 15 carroll et al. found 4 (ps 429694) from the statinecontaining combinatorial library. 16 haque et al. reported 5 that was screened from hydroxyethylamine-type catepsin d inhibitors library. 17 because the cleavage sites of plm i and plm ii are similar to that of hiv-1 protease (as shown in table i ), several potent hiv-1 protease inhibitors have been tested against plasmepsin ii. 18 some hiv-1 protease inhibitors have been shown to inhibit plasmepsin ii with an inhibitory constant (k i ) of 1 lm or less. kni-727 exhibited the highest selectivity for plasmepsin ii among the tested compounds. additionally, when designing inhibitor 6 (kni-10006, figure 4 ), we incorporated a hydroxyl-bearing indan derivative because the p 2 0 position of the a-globin subunit that is recognized by both plm i and plm ii is a ser residue and, as predicted, the hydroxyl group on the indan derivative appeared to mimic the hydroxyl group of the p 2 0 -ser side chain. inhibitors kni-727 and kni-10006 were assayed for inhibitory activity against the four main plasmepsins: plm i, plm ii, plm iv, and hap. 19 although kni-727 showed potent inhibitory activities against plm i, plm ii, and plm iv, it exhibited low inhibitory activity against hap. a molecule that is able to inhibit the enzymatic activity of all the four main plasmepsins might lead to rapid starvation of the parasite and could be effective against a drug-resistant mutant cleavage sites of malarial plasmepsin i and ii of the parasite. kni-10006 was found to be significantly more potent than kni-727 and could effectively inhibit all four main plasmepsins. amyloid b peptide (ab), the main component of plaques in the brains of ad patients, is formed by proteolysis of the amyloid precursor protein (app). [20] [21] [22] an aspartic protease bsecretase, also known as b-site app-cleaving enzyme 1 (bace1), triggers ab formation by cleaving app at the ab domain n-terminus ( figure 5 ). next, c-secretase cleaves between either the val and ile, or the ala and thr residues in the c-terminus of the ab domain to form either ab 40 or ab 42 , respectively ( figure 5 ). ab 42 shows greater neurotoxicity and aggregability than ab 40 and appears to be a key biomolecular marker of ad pathogenesis. based on the amyloid hypothesis, many inhibitors against b-or c-secretases have been designed and synthesized as drug candidates for alzheimer's disease. [8] [9] [10] [11] [12] the fact that b-secretase-knockout transgenic mice live normally suggests that b-secretase is a promising molecular target for ad drug development. 23 most b-secretase inhibitors possess a transition state analogue at the p 1 position and are designed based on the amino acid sequence of swedish mutant app, which has a double mutation around the b-site (at the k670n and m671l residues). inhibition of the b-cleavage of the swedish mutation of app by b-secretase is important because this increases the levels of ab 42 and ab 40 . many app mutations from ad patients have been reported, and these mutations accelerate abs levels or abs aggregabilities. recently, a mutation on the b-site of app-ala(673)thr-has been reported. this mutation was identified from a set of wholegenome sequence data of 1795 icelandic people as a gene with a low risk of ad and protects against ad and age-related decline. this finding appears to verify the validity of the amyloid hypothesis in the ad pathology. 10 in 1999, sinha et al. at elan pharmaceuticals succeeded in purifying b-secretase from the human brain by using an inhibitor with a statine residue as the substrate transition state analogue and then succeeded in cloning the enzyme. 24 since then, many other research groups have reported b-secretase inhibitors. in 2000 and 2001, ghosh et al. reported the potent inhibitors 7 (om99-2, k i 5 1.6 nm) and 8 (om00-3, k i 5 0.3 nm), which contained a hydroxyethylene unit as a substrate transition state analogue, and the first x-ray crystal structure of a complex between recombinant b-secretase and the inhibitor ( figure 6 ). [25] [26] [27] [28] [29] we reported an octapeptidic b-secretase inhibitor, 9 (kmi-008, ic 50 5 413 nm), which possessed a hydroxymethylcarbonyl (hmc) isostere as a substrate transition state analogue and a leu residue at the p 2 position. 30 moreover, a series of small and potent b-secretase inhibitors, such as 10 (kmi-429, ic 50 5 3.9 nm), 11 (kmi-684, ic 50 5 1.2 nm), and 12 (kmi-574, ic 50 5 5.6 nm), were developed as lead compounds based on kmi-008 ( figure 6 ). [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] in kmi-429 and kmi-684, the carboxylic group of kmi-008 was replaced with a carboxylic acid bioisostere, tetrazole ring, and these compounds were found to be more potent inhibitors. 32-34 kmi-574, which possessed a 5-fluoroorotyl group at the p 4 position, showed improved inhibitory activity in cultured cells (hek293 stably expressing human b-secretase). 35 in 2001, the first nonpeptidic b-secretase inhibitors, such as 13 (tak-070; ic 50 5 2.93 lm; figure 7) , 40, 41 were reported by researchers at takeda chemical industries. tak-070 was found by screening a chemical library in the imr32 human neuroblastoma cell line. an in vitro fluorescence resonance energy transfer (fret) assay using recombinant human b-secretase hamada and kiso and a competition study against a statine-containing, substrate-based inhibitor indicated that tak-070 showed bsecretase inhibition in a dose-dependent and noncompetitive manner. recently, fukumoto and co-workers at takeda chemical industries and the university of tokyo carried out surface plasmon resonance (spr) experiments that showed that tak-070 binds to a specific region in the membrane-spanning portion of b-secretase by using c-terminally truncated b-secretase. 42 this result suggests that tak-070 does not have to target the catalytic site of b-secretase but only its membranespanning domain. however, most inhibitors that have subsequently been reported by other research groups target the active site of b-secretase. elan pharmaceuticals reported a series of nonpeptidic bsecretase inhibitors, such as 14 (ic 50 5 20 nm; figure 7 ) that possess a transition state analogue and were designed using an sbdd approach. compound 14 has an isophthalic scaffold. [43] [44] [45] [46] [47] [48] [49] the p 1 residues are preferentially arranged into planar aromatic rings because the s 1 site of b-secretase, which is formed by the cleft and flap domains, is narrow. many inhibitors with an aromatic ring at the p 1 position were subsequently reported; for example, a research group at merck sharp and dohme (msd) reported the potent inhibitors 50 63, 64 although 18 was designed based on peptidomimetic inhibitors by using the sbdd approach, it is notable because it forms a unique cyclic structure at the p 1 position where the hydroxyl and amino groups on the cyclic sulfone ring of 18 appear to interact with two asp residues at the active site of b-secretase as well as acting as a transition state analogue. using a high throughput screening (hts) approach, many inhibitors have been designed. wyeth (merged with pfizer in 2009) reported a series of b-secretase inhibitors based on some hts hit compounds. [65] [66] [67] [68] [69] [70] [71] the potent inhibitor 19 (ic 50 hamada and kiso iii trial of the b-secretase inhibitor (azd3293) developed by astrazeneca. 74 although ad3839 was terminated at the phase i clinical stage, the clinical trial of azd3293-the latest generation b-secretase inhibitor from astrazeneca-was completed with encouraging results from both alzheimer's patients and healthy participants. ad3293 effectively reduced the level of ab in cerebrospinal fluid. we also designed the nonpeptidic b-secretase inhibitors 21 and 22 that featured a transition state analogue, as shown in figure 7 . 37,75-77 these inhibitors have a pyridinedicarboxylic or chelidonic scaffold at the p 2 position and were based on a virtual inhibitor with an isophthalamide moiety at the p 2 position that was designed using an in silico conformational sbdd approach. 39, 75 the oxazolidine ring fixes the turn structure between the phenyl ring at the p 3 position and the p 3 -aromatic ring, and the p 3 -phenyl ring can closely bind to the s 3 -subpocket of b-secretase. the quantum interaction of an inhibitor with the side chain of b-secretase-arg235 plays an important role in the inhibition mechanism as previously mentioned. 37 we speculated that an electron-rich halogen atom could bind to the electron-poor guanidine p-orbital by a quantum interaction and designed the potent inhibitor 22 with a halogen atom on the p 2 -heterocyclic ring. adult t-cell leukaemia (atl) and htlv-i associated myelopathy (ham) are caused by infection with htlv-i, a retrovirus that processes its precursor polyproteins via the unique aspartic protease htlv-i protease. since htlv-i protease is similar to hiv-1 protease, potent hiv-1 protease inhibitors, such as our compounds kni-727 and 764, and ritonavir were tested as htlv-1 inhibitors. unfortunately, these compounds were ineffective against htlv-i protease. 78 therefore, we designed some htlv-i protease inhibitors using the substrate transition state concept as well as the development of renin and hiv-1 protease inhibitors as shown in figure 8 . octapeptidic inhibitor 23 (ic 50 5 159 nm) with a transition state analogue was designed based on a substrate of htlv-i gag precursor polyprotein that is processed at the ma/ca cleavage site as shown in figure 8a . compound 23 exhibited moderately potent inhibitory activity against htlv-i protease. by inserting the non-natural amino acid dimethylthiazolidine (dmt), which is a proline bioisostere, small hexapeptidic htlv-i protease inhibitors 24 and 25 (ic 50 5 353 and 88 nm, respectively) were obtained. 79 subsequently, hydrophobic and branched amino acids were found to be preferred at the p 2 -p 3 position, and the potent htlv-i protease inhibitor 26 (kni-10562, ic 50 5 7 nm) possessing an l-tert-leucine and l-(1)-a-phenylglycine was designed. 80 recently, we revealed the first x-ray crystal structures of inhibitor-htlv-i protease complexes with kni-10562 as a ligand. 81 this crystal structure is very similar to that of the hiv-1 protease complex. this crystal structure is expected to be useful for designing more potent, nextgeneration htlv-i protease inhibitor drugs. serine proteases generally have catalytic ser, his, and asp residues in the active site-the so-called "catalytic triad." in this section, hepatitis c virus (hcv) ns3 protease, and dipeptidyl peptidase-4 (dpp-4) inhibitors are discussed as representative serine protease inhibitors. hepatitis c involves severe liver inflammation and is caused by an rna virus, hcv. interferon, hcv ns5b rna polymerase inhibitor, cyclophilin inhibitor, and hcv ns3 protease inhibitor are well known as anti-hcv agents. among them, ns3 protease is a serine protease that possesses a serine residue at the active site. the polyprotein that is encoded in the hcv figure 9 hepatitis c virus ns3 protease inhibitors. hamada and kiso rna is proteolytically cleaved into four structural proteins (c, e1, e2, and ns2-ns3-ns4a-ns4b-ns5a-ns5b) by the host's signal peptidase. subsequently, six nonstructural (ns) proteins ns2, ns3, ns4a, ns4b, ns5a, and ns5b are released by selfdigestion. [82] [83] [84] [85] [86] [87] [88] the ns3-ns4a complex is responsible for the release of four nonstructural proteins (ns4a, ns4b, ns5a, and na5b) and is essential for hcv replication. the first hcv ns3 protease inhibitor 27 (telaprevir, figure 9 inhibitors possessing an a-ketocarboxamide group would be designed to bind to the active site of the enzyme via covalent bonding between the carbonyl carbon of the aketocarboxamide group and the oxygen atom of the ser139 side chain that is found at the active site. the x-ray crystal structure of the ns3-telaprevir complex (pdb id: 3sv6) is shown in figure 10a . as expected, it is shown that the inhibitor's carbonyl carbon binds to the ser139 side chain via a covalent bond, and the carbonyl carbon forms a tetrahedral structure. it is most likely that the amide carbonyl group next to the carbonyl carbon stabilizes the tetrahedral structure. subsequently, noncovalent inhibitors 29 (bms-650032), 95 30 (vaniprevir), 96 31 (danoprevir), 97 and 32 (simeprevir) 98 were reported by bristol-myers squibb, msd, array biopharma, and medivir/johnson & johnson, respectively. simeprevir was approved by the fda in 2013 for use in combination with peginterferon-alfa and ribavirin. these inhibitors possess a cyclopropanesulfonylamide group at the n-terminus and appear to bind to the active site of the enzyme via hydrogen bonding. in the x-ray crystal structure of the ns3 protease-bms-650032 complex (pdb id: 4nwl), it is shown that one oxygen atom of the sulfonyl group interacts with the catalytic ser139 side chain and the amino group of gly137. the acidity of the sulfonyl group might allow the tight binding between the inhibitors and the hydrophilic active site that consist of ser139 side chain and gly137 amino group. the biggest problem of these protease inhibitors is that drug-resistant variants can emerge rapidly by a single administration of these drugs. hence, for the treatment of chronic hepatitis c, the ns3 protease inhibitors were commonly used in combination with pegylated interferon (peginterferon) and a nucleoside inhibitor (ribavirin). 99 recently, bristol-myers squibb developed the first all-oral, interferon-and ribavirinfree, hepatitis c treatment-the ns3 protease inhibitor (asunaprevir) and ns5a replication inhibitor (daclatasvir) dual regimen. 98 abbvie also developed an interferon-and ribavirinfree multiple dosing regimen consisting of an ns3 protease inhibitor (paritaprevir) with ritonavir in combination with an ns5a replication inhibitor (ombitasvir) and a non-nucleoside polymerase inhibitor (dasabuvir). 100 figure 12 x-ray crystal structure of didpeptidyl peptidase (dpp)24-alogliptin complex (pdb id: 2onc). the crystal structure of human immunodeficiency virus (hiv)-protease-kni-272 complex as determined by highresolution x-ray and neuron diffraction. (pdb id: 2onc). the water molecules were depicted by ball and stick model. hamada and kiso incretins are gastrointestinal hormones that increase insulin secretion in response to glucose levels. there are two main incretin hormones: glucagon-like peptide-1 (glp-1) and gastric inhibitory peptide (gip, also known as glucose-dependent insulinotropic peptide). [101] [102] [103] [104] [105] because these hormones are rapidly cleaved and inactivated by the serine protease dpp-4, its inhibitor is expected to be a drug candidate for type 2 diabetes treatment. 106,107 dpp-4 recognizes the n-terminal xaa-pro residue of peptides, leading to the release of dipeptide xaa-pro. since dpp-4 also recognizes the xaa-ala sequence, dpp-4 cleaves the n-terminus of glp-1 and gip-the n-terminal dipeptides being his-ala and tyr-ala, respectively. because xaa-ala residues were thought to exist at the n-terminus of many proteins/peptides, dpp-4 inhibitors were predicted to have many side effects. additionally, it is known that the dpp family consists of nine enzymes. among them, dpp-8 and dpp-9 have a similar structure and selectivity to dpp-4. indeed, lankas et al. reported some toxicities by the dpp-8/-9 inhibition of nonselective dpp-4 inhibitors-in rats, alopecia, thrombocytopenia, reticulocytopenia, enlarged spleen, multiorgan histopathological changes, and mortality, and in dogs, gastrointestinal toxicity. 108 a dpp-4 inhibitor for use as an antidiabetes drug therefore needs to be highly specific. most dpp-4 inhibitors were designed based on the phe-pro sequence of the substrate. the first dpp-4 inhibitor 33 (sitagliptin, figure 11 ) was developed by msd and approved as an oral antidiabetes drug by the fda in 2006. sitagliptin has a bamino acid unit and a unique cyclic amine corresponding to the phe and pro residues, respectively. 109 inhibitors 34 (vidagliptin), 35 (anagliptin), and 36 (alogliptin) possess a cyanopyrrolidine group corresponding to the pro residue and were developed by novaltis, sanwa kagaku kenkyusho, and takeda pharmaceutical company, respectively. [110] [111] [112] anagliptin was approved for use in japan. the cyanopyrrolidine inhibitors are apparently designed based on the covalent bond formation between the cyano-carbon of the inhibitor and catalytic ser630 side chain of the enzyme. however, alogliptin shows no covalent bonding with the ser630 side chain in the x-ray crystal structure of the complex with the dpp-4 enzyme (pdb id: 2onc) as shown in figure 11 . the cyano group interacts with the arg125 side chain of the enzyme, and the catalytic ser635 side chain does not interact with alogliptin; instead, the binding relies on the docking of the inhibitor's phenyl ring into the hydrophobic s 1 pocket, the ionic bonding of inhibitor's amino group with glu205 and glu206 side chains, and the p-p stacking interaction between the inhibitor's quinazolinone ring and the tyr547 side chain appear to allow a potent binding mode. linagliptin developed by boehringer ingelheim and lilly was approved by the fda in 2011. linagliptin possesses no cyano group. the amino group, the unsaturated alkyl group, and the uracil ring of linagliptin interact with the glu205 and glu206 side chains, the s 1 pocket, and the tyr547 side chain via ionic bonding, hydrophobic interaction, and p-p stacking, respectively, as well as that of alogliptin. many protease inhibitors have been developed as therapies for diseases such as hypertension, aids, adult t-cell leukaemia, malaria, alzheimer's disease, hepatitis, and diabetes. inhibitors of aspartic proteases such as renin, hiv-1 protease, malarial plasmepsin, b-secretase, and htlv-i protease can be designed using the transition state analogue concept. there is no unified design concept in the case of metallo-, serine, and cysteine proteases. for the efficient development of inhibitors for these proteases, new design concepts or technology are required. despite this difficulty, many drugs have been developed that are based on inhibition of the metalloprotease ace, and the serine proteases hcv ns3 protease and dpp-4. although severe acute respiratory syndrome (sars) coronavirus main protease (m pro ) was not discussed in this review article, m pro is categorized as a cysteine protease. 113 as a potential treatment against future viral epidemics, the development of new design strategies for protease inhibitor design technologies is extremely important. aspartic protease inhibitors can be easily designed using a computational approach such as a docking simulation, in silico screening, or in silico de novo design because aspartic proteases form a tetrahedral substrate transition state that is relatively simple since it contains no covalent bonds between the enzyme and the substrate. on the other hand, cysteine/serine proteases form a covalent bond with the substrate in the transition state. it is therefore difficult to rationally design a transition state analogue inhibitor of cysteine/serine proteases using a docking simulation, because of the van der waals repulsion that is generated between the inhibitor and the catalytic amino acid side chain. in the case of the metalloprotease ace, inhibitor development had to wait until the discovery of a peptide in snake venom, and until then, the design of an inhibitor using a computational approach seemed impossible. in the case of the serine protease hepatitis c virus ns3 protease, aketocarboxamide inhibitors such as 27 and 28 were developed at an early stage. these inhibitors formed covalent bonds with the enzyme as shown in figure 10a . covalent inhibitors with a ketone or aldehyde group are generally unstable against nucleophilic agents or enzymes in vivo. next, noncovalent inhibitors 29-32, which feature a cyclopropanesulfonylamide group, were reported. although the inhibitor's sulfonyl oxygen atom interacts with the catalytic ser side chain by hydrogen bonding (as shown in figure 10b ), the attractive force is weaker than that of a transition state inhibitor. these inhibitors showed potent inhibitory activities by elongation in the direction of s 2 sites. compounds 30-32 were cyclized to fix their conformation when docked in the active site of the enzyme. another group of serine proteases, the dpp-4 protease inhibitors 34-36, were designed in anticipation of the covalent bond formed between the inhibitor's cyano carbon and the catalytic ser side chain. however, the x-ray crystal structure of the ns3 protease complex with 36 shows no covalent bond between the enzyme and the inhibitor as shown in figure 12 . as mentioned previously, it is difficult to rationally design metallo-, serine, and cysteine proteases based purely on the knowledge of the amino acid sequence of their substrate. inhibitors such as ace, hcv ns3 protease, and dpp-4 were developed using unique design concepts and bind to the catalytic side chain of the enzyme in a unique manner. in contrast, aspartic protease inhibitors can be designed logically based on the amino acid sequences of substrates. many aspartic protease inhibitors have a transition state analogue at the p 1 position, and their structures are simple. recently, we determined the whole structure of the complex formed between the hiv-1 protease and kni-272, which contains the location of protons, as shown in figure 12 . 114 although protons cannot be directly observed using conventional x-ray crystallography, the successful determination of all coordinates was achieved, for the first time, using high-resolution x-ray (at 1.4 å ) and neuron (at 1.9 å ) crystallography. as shown in figure 3 , in the cleavage mechanism of aspartic proteases, a carboxylic acid proton and a carboxyl anion in the active site of the enzyme interact with the substrate transition state via two hydrogen bonds. the transition state analogue of kni-272 interacts with a carboxylic acid proton and a carboxyl anion in the same manner and accurately mimics the substrate transition state. transition state analogue inhibitors allow potent inhibition of enzyme activity because the transition state analogue can bind strongly to the active site of the enzyme and is not cleaved enzymatically. • proteases are important molecular targets for curing various diseases, because they play crucial roles in various biological processes in all organisms -from viruses to humans. • since aspartic proteases form a substrate transition state that possesses no covalent bond between the active site of the enzyme and a substrate, inhibitors with a transition state analogue can be easily designed using a computational approach. • the inhibitor development of metalloprotease ace had to wait until the discovery of a peptide found in snake venom. • serine/cysteine proteases form a substrate transition state that contains a covalent bond between the catalytic ser/cys residue side chain and the substrate. as a result, their inhibitors were developed using various design concepts, not using a unified design concept as is the case for aspartic proteases. • for the first time, the entire structure of hiv-1 protease in complex with the inhibitor (kni-272) was determined using high-resolution x-ray and neuron crystallography, and its inhibitory mechanism could be revealed. this box summarizes key points contained in the article. expert opi drug discov aspartic protease inhibitors as drug candidates for treating various difficult-to-treat diseases new directions for protease inhibitors key: cord-017600-4e7mw041 authors: pfister, h. -w.; klein, m.; schmutzhard, e.; meyding-lamadé, u.; sellner, j.; menon, s.; martinez-torres, f.; helbok, r.; pfausler, b.; grabowski, a.; kress, b. title: infektionen date: 2008 journal: neurointensiv doi: 10.1007/978-3-540-68317-9_35 sha: doc_id: 17600 cord_uid: 4e7mw041 trotz weiterentwicklung moderner antibiotika in den letzten jahren sind die letalitätszahlen der bakteriellen (eitrigen) meningitis weiterhin hoch; überlebende haben häufig neurologische residuen. die ungünstigen klinischen verläufe der bakteriellen meningitis sind meist folge intrakranieller komplikationen, wie z. b. eines generalisierten hirnödems, einer zerebrovaskulären arteriellen oder venösen beteiligung oder eines hydrozephalus. als folge dieser komplikationen kommt es häufig zu einem anstieg des intrakraniellen drucks. bei schweren, komplizierten klinischen verläufen der bakteriellen meningitis kommen oft adjuvante therapiemaßnahmen (z. b. intravenöse gabe von hyperosmolaren substanzen, externe ventrikeldrainage) zum einsatz. bei nachweis einer meningitisassoziierten septischen sinus-/venenthrombose erfolgt die dosisadaptierte intravenöse heparintherapie. identifi ziert werden, die im verlauf der meningitis von bedeutung sind, wie z. b. die proinfl ammatorischen zytokine interleukin-1ß, il-6, tumor-nekrose-faktor-α, chemokine, arachidonsäuremetaboliten, plättchenaktivierender faktor, reaktive sauerstoff spezies, stickstoff monoxid und peroxynitrit [8] . verschiedene oxidanzien, wie z. b. peroxynitrit, können auf verschiedene art und weise zur entstehung von zns-komplikationen bei der meningitis führen. so kann es zur induktion einer lipidperoxidation oder zur aktivierung des nukleären reparaturenzyms poly-adp-ribose-polymerase (parp) mit anschließendem energieverbrauch kommen; beide signalwege können zu einer endothelialen dysfunktion und zum endothelzellschaden führen. infolge der endothelialen funktionsstörung kommt es zu einer beeinträchtigung der zerebrovaskulären autoregulation , einer störung der kohlendioxidreaktivität zerebraler gefäße und zu einer störung der blut-hirn-schranke. die entstehung eines vasogenen hirnödems gehört zu den wichtigsten ursachen eines erhöhten intrakraniellen drucks im verlauf der meningitis . ein erhöhter intrakranieller druck kann in zweierlei hinsicht gefährlich werden, zum einen durch entstehung einer zerebralen einklemmungssymptomatik, zum anderen durch eine reduktion des zerebralen perfusionsdrucks mit der gefahr zerebraler ischämien. klinische leitsymptome der bakteriellen (eitrigen) meningitis sind: kopfschmerzen, meningismus, hohes fieber, übelkeit, erbrechen und lichtscheu. ferner können ein verwirrtheitssyndrom, eine vigilanzstörung und epileptische anfälle auft reten. etwa 10% der patienten haben eine hirnnervenbeteiligung , der häufi gkeit nach des iii., vi., vii. und viii. hirnnerven. hörstörungen, die meist folge einer eitrigen labyrinthitis sind, fi nden sich bei etwa 10−20% der patienten mit bakterieller meningitis, bei patienten mit pneumokokkenmeningitis sogar bei bis zu 30%. bei etwa 75% der patienten mit einer meningokokkenmeningitis ist bei krankenhausaufnahme ein exanthem (das spektrum reicht von einzelnen petechien bis zu ausgedehnter purpura mit hautnekrosen) nachweisbar [1] . etwa 50% der invasiven meningokokkenerkrankungen verlaufen als eitrige meningitis, 25% schwerpunktmäßig als sepsis, weitere 25% zeigen mischformen (meningitis und sepsis). bei etwa 10−15% der septischen erkrankungen treten besonders schwere formen des septischen schocks auf, die als waterhouse-friderichsen-syndrom bekannt sind und eine sehr hohe letalität aufweisen. otitis, sinusitis 50 (57, 5) chronische abwehrschwächende krankheiten 27 ( zerebrovaskuläre komplikationen im arteriellen (arteriitis, vasospasmus) und im venösen bereich (septische sinus-oder kortikale venenthrombose) können zu infarkten mit schweren irreversiblen zerebralen schäden und zu einem erhöhten intrakraniellen druck infolge eines zytotoxischen ödems führen. weitere ursachen eines erhöhten intrakraniellen drucks sind 3 eine zunahme des intrakraniellen blutvolumens durch eine gestörte zerebrovaskuläre autoregulation oder eine septische sinus-oder venenthrombose sowie eine liquorzirkulationsstörung mit entstehung eines hydrozephalus. neben den zerebralen komplikationen können sich folgende extrakranielle komplikationen in der akutphase der bakteriellen meningitis entwickeln: septischer schock, verbrauchskoagulopathie, »adult respiratory distress syndrome« (ards), arthritis (septisch und reaktiv), elektrolytstörungen, wie hyponatriämie, syndrom der inadäquaten adh-sekretion (si-adh), zerebrales salzverlustsyndrom oder zentraler diabetes insipidus, rhabdomyolyse, pankreatitis, septische einseitige (selten beidseitige) endophthalmitis oder panophthalmitis, blindheit als folge einer vaskulitis und spinale komplikationen (z. b. myelitis oder spinale vaskulitis; [6, 20] ). entscheidend für die diagnose der bakteriellen meningitis ist die liquoruntersuchung . der eitrig-trübe liquor zeigt typischerweise eine granulozytäre pleozytose über 1000 zellen/ μl, eine schwere blut-liquor-schrankenstörung und eine liquorglukoseerniedrigung (meist <30 mg/dl; liquor-/serum-glukose-quotient <0,3) . bei patienten mit extrem niedrigen li häufi gkeit hirnödem mit der gefahr der einklemmung 10−15% . quorglukosekonzentrationen (<5 mg/dl) fi ndet sich in der regel eine sehr große zahl von bakterien im liquor (bakterienrasen im gram-präparat). an einzelnen zentren wird die bestimmung von liquorlaktat (werte meist >3,5 mmol/l) der glukosebestimmung vorgezogen. liquorzellzahlen <1000 zellen/ μl können bei der bakteriellen meningitis sehr früh im krankheitsverlauf, bei antibiotisch anbehandelten patienten, bei fulminanten krankheitsverläufen und bei abwehrgeschwächten (z. b. leukopenischen) patienten beobachtet werden. der erregernachweis im liquor ist mit verschiedenen methoden möglich: mikroskopisch mittels gram-färbung (oder methylenblau-färbung) und bakteriologisch mittels kultur. . diff uses hirnödem 25 (28, 7) hydrozephalus 14 (16, 1) arterielle zerebrovaskuläre komplikation 19 (21, 8) venöse zerebrovaskuläre komplikation 9 (10, 3) spontane intrakranielle blutung 8 (9, 2) subarachnoidalblutung (bei vaskulitis) 5 2 (2,3) subarachnoidal-und intrazerebrale blutung (bei vaskulitis) intrazerebrale blutung (bei sinusthrombose) 5 1 (0, 9) intrazerebrale blutung (unklarer ursache) nierenversagen (hämofi ltration) 10 (11, 5) adult respiratory distress syndrome (ards) 6 (6, 9) meningitisassoziierte intrakranielle komplikationen entwickelten sich bei 65 (74,7%) und systemische komplikationen bei 33 (37,9%) patienten. a bezogen auf alle patienten (bzw. 25,8% der überlebenden) der nachweis von bakterien im liquor ist mit den genannten methoden bei 70−90% der patienten mit eitriger meningitis möglich. bei etwa 50% der patienten mit bakterieller meningitis sind die blutkulturen positiv; blutkulturen müssen deshalb vor beginn der antibiotikatherapie angelegt werden. bei klinischem verdacht auf eine meningokokkenerkrankung und negativem mikroskopischen sowie kulturellen ergebnis kann eine polymerasekettenreaktion (pcr) zum nachweis der meningokokken-dna im liquor und im blut (vorzugsweise ed-ta-blut) in die wege geleitet werden. als indikationen für den einsatz von verfahren zum antigennachweis klassischer meningitiserreger (z. b. antigennachweis von n. meningitidis, s. pneumoniae, h. infl uenzae und streptococcus agalactiae) gelten [13, 20] : bestätigung unklarer mikroskopischer liquorbefunde, liquor bei deutlicher pleozytose und negativem mikroskopischem befund, liquor eines patienten mit antibiotischer vorbehandlung. im blut fi nden sich eine leukozytose sowie eine erhöhung des c-reaktiven proteins (mögliche ausnahme: immunsupprimierte patienten). die bestimmung des serumprocalcitonins ist für die unterscheidung einer bakteriellen von einer nichtbakteriellen meningitis hilfreich [23] . bei jedem patienten mit bakterieller meningoenzephalitis muss am aufnahmetag eine bildgebende untersuchung durchgeführt werden, in der regel ein schädel-ct mit knochenfenster [20] . mögliche befunde, die im schädel-ct oder -mrt bei einem patienten mit bakterieller meningoenzephalitis nachgewiesen werden können, sind in der folgenden übersicht zusammengefasst. [20] ). bei schwer bewusstseinsgestörten patienten und patienten mit fokalneurologischem defi zit (z. b. hemiparese), bei denen der dringende verdacht auf eine bakterielle meningitis besteht, ist vor der liquoruntersuchung ein schädel-ct mit der frage eines erhöhten intrakraniellen drucks (z. b. hirnabszess, hydrozephalus) erforderlich. um keine zeit durch das warten auf das ct zu verlieren, sollen bei diesen patienten bereits unmittelbar nach der blutentnahme (für das anlegen einer blutkultur) dexamethason und antibiotika gegeben werden. danach wird möglichst schnell ein schädel-ct durchgeführt, anschließend (wenn der ct-befund nicht dagegen spricht) eine liquorpunktion [20] . kontraindikationen für die liquorpunktion sind computertomographische zeichen eines erhöhten intrakraniellen drucks (z. b. generalisiertes hirnödem, hydrozephalus, hirnabszess) und klinische zeichen der einklemmung (z. b. komatöser patient, einseitig erweiterte und nicht lichtreagible pupille). allerdings kann eine signifi kante erhöhung des intrakraniellen drucks mittels ct nicht ausgeschlossen werden [16, 31] . es muss möglichst bald nach aufnahme des patienten eine hno-ärztliche konsiliaruntersuchung erfolgen. wenn klinisch (z. b. otitis) oder im ct ein parameningealer entzündungsherd (z. b. sinusitis) als mögliche ursache für die bakterielle meningitis nachgewiesen wird, soll möglichst rasch (wenn möglich am aufnahmetag) die operative fokussanierung erfolgen. in abhängigkeit von der anamnese und vom klinischen befund soll nach anderen infektiösen foci gesucht werden (z. b. th oraxröntgenaufnahme, abdomensonographie/ct, echokardiographie). ist der erreger nicht bekannt , wird empirisch unter berücksichtigung des alters des patienten, der prädisponierenden faktoren und der damit wahrscheinlichsten bakterien behandelt (. tab. 35.4 und . tab. 35.5; [20] ). eine antibiotikatherapie muss bei patienten mit verdacht auf bakterielle meningitis möglichst schnell begonnen werden 3 [2, 22] . eine verzögerung der antibiotikatherapie um mehr als 3 stunden nach krankenhausaufnahme muss unbedingt vermieden werden [2] ; in einer prospektiven multicenterstudie bei 156 erwachsenen patienten mit pneumokokkenmeningitis konnte nachgewiesen werden, dass eine verzögerung der antibiotikatherapie um mehr als 3 stunden mit einer ungünstigen prognose vergesellschaft et ist. ferner wurde in einer retrospektiven datenanalyse (119 patienten mit einem alter ≥16 jahren und einer bakteriellen meningitis, 56% hatten eine pneumokokkenmeningitis), gezeigt, dass patienten, die später als 6 stunden nach krankenhausaufnahme mit antibiotika behancephalosporin der 3. generation + ampicillin b 5 nosokomial (z. b. nach neurochirurgischer operation oder schädelhirntrauma) 5 vancomycin + meropenem (oder vancomycin + ceftazidim) c 5 abwehrgeschwächte, ältere patienten 5 cephalosporin der 3. generation + ampicillin 5 delt wurden, ein 8,4-mal höheres risiko hatten, an der meningitis zu versterben [22] . liegt das antibiogramm vor, muss die intravenöse antibiotikatherapie entsprechend angepasst werden (. tab. 35.6 und . tab. 35.7; [20] ). die intraventrikuläre vancomycingabe kommt für die th erapie einer katheterassoziierten ventrikulitis in betracht, die durch staphylokokken hervorgerufen wird [17] . die empfohlene behandlungsdauer mit antibiotika liegt bei unkompliziertem verlauf einer h.-infl uenzae-meningitis und meningokokkenmeningitis bei 7−10 tagen, einer pneumokokkenmeningitis bei (10−) 14 tagen. in der behandlung der listerienmeningitis und der durch gramnegative enterobakterien verursachten meningitis wird meist über 3 wochen (oder länger) therapiert. eine routinemäßige liquorkontrollpunktion ist nicht erforderlich. bei unbekanntem erreger und fehlender klinischer besserung kann eine erneute liquorpunktion (wenn keine kontraindikationen bestehen) erwogen werden. wenn es innerhalb von 2 tagen nach beginn der antibiotikatherapie zu keiner klinischen besserung kommt, müssen folgende ursachen bedacht werden: auft reten von intrakraniellen komplikationen, persistierender infektiöser fokus (insbesondere ein nicht sanierter oder unzureichend operierter parameningealer fokus wie z. b. eine mastoiditis, sinusitis oder otitis media), inadäquates antibiotikaregime (z. b. unwirksames antibiotikum oder zu niedrige dosis). es müssen dann entsprechende diagnostische maßnahmen (z. b. bildgebung, hno-konsiliaruntersuchung) in die wege geleitet werden. wenn der erreger der eitrigen meningitis nicht isoliert werden konnte, sollte bei fehlendem ansprechen auf die antibiotikatherapie eine erweiterung bzw. ein umsetzen der antibiotika erwogen werden. finden sich zeichen eines erhöhten intrakraniellen drucks, müssen icp-senkende maßnahmen erfolgen (z. b. oberkörperhochlagerung auf 30°, osmotherapie mit mannit, sorbit oder glyzerin, bei beatmeten patienten normoventilation, bei sonst nicht beherrschbarem intrakraniellen druck möglichst kurzzeitige hyperventilation mit einem zielwert des pco 2 um 32 mm-hg, evtl. gabe von tris-puff er, th iopental-narkose, bei hydrozephalus externe liquordrainage; [10, 20] . es gibt keine systematischen untersuchungen zur wirksamkeit einer moderaten hypothermie (33−36°c) zur icp-senkung bei patienten mit bakterieller meningitis. stuporöse oder komatöse patienten können von einem icp-monitoring profi tieren ([31]; 7 kap. 13). für die arteriellen zerebralen gefäßkomplikationen (arteriitis, vasospasmus) gibt es bislang keine gesicherten th erapieformen. der wissenschaft liche beleg für die wirksamkeit einer antikoagulation septischer sinus-/venenthrombosen bei der bakteriellen meningitis ist nicht gegeben. prospektive kontrollierte studien liegen bisher nicht vor. in einer retrospektiven studie zeigte sich allerdings ein günstiger eff ekt der heparintherapie bei patienten mit septischer sinus-cavernosus-th rombose [26] . bei patienten mit meningitisassoziierter th rombose des sinus transversus wurde eine erhöhte blutungsgefahr berichtet [26] . von den meisten autoren wird derzeit die antikoagulation mit intravenösem heparin (ptt-wirksam) bei kernspintomographisch (oder in der dsa) nachgewiesenen septischen sinus-/ venenthrombosen in folge einer bakteriellen meningitis empfohlen (7 kap. 33.1) . eine antiepileptikatherapie (z. b. schnelle intravenöse aufsättigung mit phenytoin) ist indiziert, wenn epileptische anfälle auft reten oder im eeg epilepsietypische muster nachweisbar sind. die wirksamkeit von dexamethason wurde in einer europäischen, prospektiven, placebokontrollierten, randomisierten, multicenterstudie bei 301 erwachsenen mit bakterieller meningitis untersucht [4] . dexamethason (10 mg) oder placebo wurden in dieser studie 15−20 minuten vor der ersten antibiotikagabe appliziert und dann alle 6 stunden für insgesamt 4 tage. in der studie konnte ein günstiger eff ekt der dexamethasonbehandlung gezeigt werden: dexamethason führte zu einer signifi kanten reduktion der letalität und der häufi gkeit ungünstiger klinischer verläufe. eine subgruppenanalyse zeigte, dass dexamethason nur bei den patienten mit pneumokokkenmeningitis wirksam war, nicht bei meningitiden anderer ätiologie, wie z. b. der meningokokkenmeningitis. der günstige eff ekt von kortikosteroiden konnte in mehreren metaanalysen bestätigt werden [28, 29, 30] . in der zuletzt publizierten metaanalyse wurden die daten von 18 klinischen studien (2750 patienten) ausgewertet [30] . insgesamt konnte mit kortikosteroiden die letalität der bakteriellen meningitis gesenkt werden; auch war die häufi gkeit schwerer hörstörungen und neurologischer residualsymptome mit kortikosteroiden vermindert. subgruppenanalysen zeigten den günstigen eff ekt von kortikosteroiden auf die letalität nur für die pneumokokkenmeningitis. bei der meningokokkenmeningitis konnten letalität und häufi gkeit neurologischer residuen nur tendenziell mit dexamethason reduziert werden. zusammenfassend kann aufgrund der zur verfügung stehenden daten die gabe von dexamethason bei erwachsenen patienten mit verdacht auf eine bakterielle meningitis (d. h. klinischer verdacht plus trüber liquor, nachweis von bakterien im liquor in der gramfärbung oder einer liquorleukozytenzahl von >1000/μl) empfohlen werden; dexamethason (fortecortin) sollte in einer dosis von 10 mg i.v. unmittelbar vor gabe des antibiotikums verabreicht werden [20] . daraufh in wird mit 10 die ständige impfk ommission am robert-koch-institut (stiko) empfi ehlt die impfung gegen meningokokken der serogruppe c mit einem konjugierten meningokokken-c-impfstoff für alle kinder im 2. lebensjahr zum frühestmöglichen zeitpunkt [24] . primäres impfziel ist es, die morbidität invasiver meningokokkenerkrankungen der serogruppe c und deren folgen zu vermindern. ferner empfi ehlt die ständige impfk ommission (stiko) am robert-koch-institut eine meningokokkenimpfung für folgende gefährdete personen bzw. konstellationen [24] : per continuitatem entstehende hirnabszesse sind meist polymikrobiell bedingt und sind die häufi gste ursache eines hirnabszesses (50%). in 20% der hirnabszesse erfolgt die infektion primär, entweder durch einbringen der erreger während einer neurochirurgischen operation, oder bei penetrierendem schädelhirntrauma, diese hirnabszesse sind häufi g monomikrobiell, können aber auch polymikrobiell bedingt sein. sekun-tab. 35 bei bis zu 10% der patienten werden, typischerweise innerhalb von wenigen wochen nach beendigung der antibiotischen chemotherapie, rezidive gesehen . abhängig von der größe und der lokalisation der hirnabszesse werden bei 10−70% der patienten zerebrale anfälle im sinne einer residualepilepsie gesehen. die letalität beträgt 5−10% und ist direkt proportional der störung der bewusstseinslage (. tab. 35.15). ungefähr 65% der überlebenden patienten sind nach durchschnittlich 5 jahren zumindest grob neurologisch weitgehend rehabilitiert. jüngste neuropsychologische untersuchungen weisen jedoch darauf hin, dass ein breites spektrum von neuropsychologischen defi ziten, insbesondere einem subkortikalen muster entsprechend, auch noch nach >10 jahren bei der überwiegenden zahl der patienten mit einem hirnabszess, teilweise unabhängig von größe und lokalisation, bestehen. der kürzlich publizierte »imaging severity index (isi)« unterstützt eine frühzeitige potenzielle prognoseeinschätzung. die überwiegende zahl der abszesse im spinalkanal sind epidural lokalisiert, typischerweise thorakal und/oder lumbal sowie häufi g dorsal dem rückenmark anliegend. am häufi gsten werden sie im höheren lebensalter ( therapie eine akute, progrediente neurologische symptomatik, die am ehesten (bildgebend) dem raumfordernden eff ekt des spinalen abszesses/empyems zuzuschreiben ist, erfordert eine unverzügliche notfallmäßige operative entlastung. parallel dazu muss die bestmögliche erregerorientierte antibiotische therapie eingeleitet werden . da staphylokokken in der überwiegenden mehrzahl der spinalen abszesse die erreger sind, wird sich die empirische antimikrobielle chemotherapie primär an den staphylokokken zu orientieren haben. staphylococcus aureus (aber auch gramnegative erreger) sind typisch bei hämatogener ausbreitung, bei perforierenden verletzungen, nach neurochirurgischen eingriff en (auch lokalen infi ltrationen) sowie bei lokaler ausbreitung von wenn die neurologische ausfallssymptomatik, insbesondere die querschnittssymptomatik bereits 2 tage oder mehr besteht, ist nur mehr bei 50% der patienten eine erholungschance gegeben. eine komplette paraplegie, v. a. wenn sie als ausdruck eines vaskulären geschehens plötzlich aufgetreten ist, zeigt nur noch minimale neurologische erholungschancen. bei allen spinalen abszessen zusammengenommen, ist zu erwarten, dass sich nur 40% komplett erholen, 25% mit einer radikulären oder diskreten querschnittsymptomatik und 20% mit einem weitgehend vollständigen querschnittsyndrom verbleiben. die letalität beträgt 10−15%, insbesondere bei meningitis, sepsissyndrom oder intensivmedizinischen komplikationen. ätiologie und pathogenese mycobacterium tuberculosis ist für den überwiegenden teil der zns-tuberkulosen verantwortlich, bei hiv-patienten können andere mykobakterien (mycobacteria others than tuberculosis -mott) eine zns-infektion verursachen, bei denen im rahmen eines »immune reconstitution syndromes« (iris) mit einer akuten verschlechterung der zentralnervösen symptomatik zu rechnen ist. bei 50% der zns-tuberkulosen besteht eine konkommittierende extrakranielle tuberkulose. nur sehr selten ist mycobacterium bovis ursache einer zns-tuberkulose. die durch tröpfcheninfektion aufgenommenen mykobakterien vermehren sich intrapulmonal und werden bereits frühzeitig hämatogen ausgestreut. sie können bereits zu diesem zeitpunkt den subarachnoidalraum erreichen und mit einer langen latenz ausgangspunkt einer zns-tuberkulose sein. mycobacterium tuberculosis ist ein obligat aerobes, nicht sporenbildendes unbewegliches stäbchen, das sich nicht mit konventioneller gramfärbung, allerdings mit ziehl-neelsen-färbung, fluorchromfärbung oder kinyoun-färbung anfärbt. die generationszeit dieser säurefesten stäbchen ist bis zu 20mal länger als die anderer bakterien und beträgt ca. 20 stunden. mykobakterielle kolonien benötigen bis zu 8 wochen um auf löwenstein-jensen-oder middlebrook-medium sichtbar zu wachsen. eine zns-tuberkulose ist typischerweise eine meningitis mit zusätzlicher aff ektion des hirnparenchyms und der intrakraniellen gefäße. es fi ndet sich eine, vorwiegend basal gelegene granulomatöse entzündung der meningen, häufi g aggraviert durch ein dickes geleeartiges exsudat. eine zns-tuberkulose kann sich als chronische basale meningitis , mit zns-tuberkulomen, sehr selten als pachymeningitis sowie assoziiert mit einer spondylitis präsentieren. die manifestation einer tuberkulösen meningitis nimmt typischerweise einen subakuten bis chronischen verlauf, in seltenen fällen kann sie sich jedoch akut manifestieren. charakteristischerweise bestehen über wochen (bis monate) unspezifi sche prodromalsymptome, krankheitsgefühl, übelkeit, kopfschmerzen sowie subfebrile temperaturen. die klassische trias einer tuberkulösen meningitis mit hirnnervenneuropathie, vaskulitis mit zerebraler ischämie sowie hydrozephalus fi ndet sich nur selten und kann auch oligosymptomatisch bestehen. ein meningismus kann vorhanden sein, ist jedoch nur selten massiv ausgeprägt. insbesondere ein hydrozephalus (mit bewusstseinstrübung, koma), aber auch vaskulär ischämische komplikationen (halbseitensymptome, hirnstammsymptome, etc.) führen potenziell zur intensivpfl ichtigkeit eines patienten mit einer zns-tuberkulose. tuberkulome , granulomatöse entzündungsherde, werden in seltenen fällen durch ihre raumfordernde wirkung (hintere schädelgrube), gelegentlich durch obstruktion der liquorzirkulation (hydrocephalus occlusus), häufi ger jedoch als ursache für einen epileptischen anfall (status epilepticus) zu einem intensivpfl ichtigen krankheitsbild führen. symptome einer zns-tuberkulose, die sich für eine bewusstseinsstörung bis zum koma verantwortlich zeigen, sind: multiple raumfordernde prozesse (tuberkulome), multifokale vaskulär ischämische läsionen, insbesondere im bereich der a. basilaris, hydrocephalus occlusus, evtl. diff uses hirnödem sowie zustand nach tonisch-klonisch generalisiertem anfall bzw. status epilepticus. die untersuchung des liquor cerebrospinalis ist für die diagnose einer chronischen meningitis unverzichtbar , der liquor ist typischerweise klar, bei deutlich erhöhtem eiweiß auch xanthochrom wirkend. es fi ndet sich eine geringe bis mäßige gemischtzellige, gelegentlich überwiegend lymphozytäre pleozytose (bis zu 500 zellen/μl), bei akuten verläufen kann auch initial eine granulozytäre pleozytose bestehen. das liquoreiweiß ist auf bis zu 500 mg/dl erhöht, exzessive eiweißwerte (>1000 mg/dl) werden bei liquorzirkulationsstörungen gesehen. die liquorglukose (bzw. liquor-/serum-glukoseratio) ist bei protrahiertem verlauf weitgehend normal, bei eher subakuten (akuten) verläufen gering bis mäßiggradig erniedrigt, sie korreliert mit der nachweisbarkeit von erregern im liquor cerebrospinalis. mittels ziehl-neelsen-färbung gelingt der nachweis von mycobacterium tuberculosis bei 10−25% der patienten mit chronischer tuberkulöser meningitis, bei 30−50% der patienten ist eine liquorkultur positiv. seriell angelegte liquorkulturen erhöhen die ausbeute auf >50%. wenngleich die ergebnisquote des nachweises von mykobakterieller dna (mittels pcr) nicht höher liegt als die der liquorkultur, ist eine pcr trotzdem indiziert, da die ergebnisse schon nach 24 stunden vorliegen. die nested-pcr, insbesondere mpb-64-pcr erhöht die sensitivität auf 90%, bei vergleichbarer spezifi tät. weitere diagnostische methoden, die bereits erfolgreich zum nachweis von mykobakterien im sputum eingesetzt wurden, müssen noch auf ihre tauglichkeit bei einer zns-tuberkulose überprüft werden, die liquoradenosindeaminase kann als eine solche komplementäre diagnostische methode mit einer spezifi tät von >90% und einer sensitivität von ca. 70% gewertet werden. bei patienten mit bewusstseinsstörung und/oder neurologischer herdsymptomatik muss jeder lumbalpunktion eine bildgebende untersuchung vorgeschaltet werden, dies v. a. in hinblick auf vaskulitisbedingte ischämien, auf das vorhandensein einer basal anspeichernden granulomatösen meningitis sowie in hinblick auf einen hydrocephalus occlusus. bei letzterem ist eine lumbale liquorgewinnung kontraindiziert, eine evtl. notwendige liquordrainage erlaubt die untersuchung des ventrikulären liquors. sowohl die typischen entzündungszeichen als auch der erregernachweis sind jedoch beim ventrikulären liquor häufi g unspezifi sch bzw. nicht erfolgreich. eine transkranielle dopplersonographie erlaubt das frühzeitige erkennen einer arteriitis sowie deren monitoring. ein tuberkulintest ist nicht notwendig, da häufi g falsch positiv oder falsch negativ. in seltenen fällen kann eine meningeale biopsie indiziert sein, v. a. zur abgrenzung eines tuberkuloms oder einer granulomatösen lokalen meningitis von einem malignen tumor (z. b. lymphom). in der bildgebung wurden bei kindern und jugendlichen bestimmte computertomographische kriterien defi niert, die in kombination eine spezifi tät von nahezu 100% und eine sensitivität von ca. 80−90% zeigen, bei älteren und alten patienten mit tuberkulöser meningitis sind dieses radiologischen parameter häufi g deutlich weniger ausgeprägt. eine hyponatriämie, am ehesten im sinne eines zerebralen »salt wasting syndromes (csw)«, bedarf engmaschigsten monitorings der elektrolyte und resultiert nicht selten in intensivpfl ichtigkeit. gerinnungsuntersuchungen zeigen nicht selten einen zustand der hyperkoagulabilität , möglicherweise mit einem erhöhten risiko für zerebrale infarkte assoziiert. hiv-positive patienten mit intrakraniellen tuberkulomen können im rahmen des »immune reconstitution syndromes (iris)« eine durchaus dramatische klinisch neurologische verschlechterung erfahren, mit zunahme der neurologischen herdsymptomatik und/oder verschlechterung von epileptischen anfällen. die chronizität der zns-tuberkulose erfordert eine ausreichend lange th erapie. komplikationen , insbesondere tuberkulome, hydrozephalus, vaskulitis können allerdings ein sich rasch veränderndes, sich plötzlich verschlechterndes klinisch neurologische bild verursachen, das unverzügliche adjuvante therapeutische maßnahmen, inklusive neurochirurgischer interventionen und intensivmedizinische betreuung erforderlich macht. der möglichst frühzeitige beginn einer spezifi schen antimikrobiellen chemotherapie verbessert entscheidend die prognose. die spezifi sche chemotherapie einer zns-tuberkulose besteht mindestens aus einer dreifachkombination mit isoniazid, rifampicin und ethambutol. bei klinisch bereits fortgeschrittenem stadium oder bei bildgebend ausgedehnten befunden, wird eine vierfach-, evtl. fünffachtherapie empfohlen und die dreifachkombination mit pyrazinamid und evtl. cycloserin ergänzt (. tab. 35.17) . die dreifachkombination (vierfach-/fünff ach-kombination) wird für mindestens 3−6 monate gegeben, anschließend eine zweifachkombinationstherapie für weitere 6−9 monate. regelmäßige klinisch neurologische kontrollen, neuroimagingund liquorkontrollen sind essenziell. intrakranielle tuberkulome sind ebenfalls primär konservativ zu therapieren, in einzelfällen nehmen sie unter der spezifi schen chemotherapie an größe zu, in solchen fällen ist eine vier-bis fünff achkombinationstherapie bis zum bildgebenden nachweis einer größenreduktion durchzuführen. ätiologie und pathogenese die übertragung erfolgt durch einen zeckenstich, nicht nur die adulten zecken, sondern auch lymphen und larven sind dazu in der lage. nach einer initial lokalen ausbreitung kommt es frühzeitig zu einer hämatogenen disseminierung und zu einer penetration der bluthirnschranke. der durch die borrelien induzierte entzündungsprozess geht mit einer aktivierung der zytokinkaskade, aber auch mit erregerassoziierten und -getriggerten autoimmunmechanismen einher. die lokale infektion (erythema migrans) sowie die frühe disseminierung bedingen nie eine lebensbedrohliche, intensivpfl ichtige erkrankung. die potenziell intensivpfl ichtige, symptomatik wird evtl. durch eine myokarditis, meningovaskulitis, in sehr seltenen einzelfällen myelitis und polyradikuloneuritis hervorgerufen. der überwiegende prozentsatz der neuroborreliosen verläuft im sinne der klassischen trias (bannwarth-garin-bujadoux-syndrom): meningitis, radikulitis/radikuloneuritis, hirnnervenneuritis. das bannwarth-garin-bujadoux-syndrom bedarf keiner intensivpfl ichtigen diagnostischen oder therapeutischen strategien. von den hirnnerven ist sehr häufi g der n. facialis -gerne auch bilateral -betroff en. eine myositis sowie die chronische borrelien-enzephalomyelitis und die eine acrodermatitis chronica atrophicans begleitende polyneuropathie nehmen ebenfalls nie einen intensivpfl ichtigen verlauf. bei atypischen oder seltenen krankheitsbildern und positiver serologie ist immer an eine koinzidenz einer früher durchgemachten borreliose/neuroborreliose und an aktuell andere entzündliche zns-erkrankung zu denken. die klinische diagnose einer klassischen trias ist, bei entzündlichem liquor, weitestgehend pathognomonisch. der liquor cerebrospinalis zeigt eine lymphoplasmazelluläre milde bis mäßige pleozytose, eine deutliche eiweißerhöhung, (in den meisten fällen igg, igm und iga). der nachweis der intrathekalen spezifi schen antikörperproduktion beweist die diagnose »neuroborreliose«. der direkte erregernachweis gelingt aus dem liquor nur sehr selten, auch die pcr konnte sich für die diagnostik der neuroborreliose noch nicht etablieren. in den einzelfällen einer parallel zu neuroborreliose bestehenden erythema migrans kann eine pcr aus einer hautbiopsie diagnostisch sein. grundsätzlich sind folgende labormethoden für die diagnostik einer akuten neuroborreliose derzeit (noch) nicht geeignet: antigennachweis aus körperfl üssigkeiten, pcr aus serum und urin, lymphozytentransformationstest (ltt) und der sog. »visual contrast sensitivity« test (vcs-test = graustufentest). die kernspintomographie sowie elektrophysiologische techniken sind als adjuvante diagnostische strategien durchaus brauchbar, jedoch wenig spezifi sch. die behandlung einer disseminierten bzw. späten neuroborreliose erfolgt mit ceft riaxon (1. tag 4 g, dann 2 g/24 h über mindestens 2 wochen, i.v.). alternativen sind cefotaxim (3×2 g täglich über 2 wochen) oder doxycyclin (2×100 mg täglich p.o., über 14-21 tage). die akute schmerzsymptomatik der klassischen trias der neuroborreliose bildet sich sehr rasch zurück, bestehende paresen brauchen sehr viel länger zur rückbildung. während sich die entzündungszeichen im liquor cerebrospinalis innerhalb von 2−4 wochen weitestgehend normalisieren, ändert sich der serologische befund häufi g nur sehr langsam bzw. überhaupt nicht; d. h. eine serodiagnostik zur th erapie-und verlaufskontrolle ist nicht geeignet, da die nicht protektiven antikörper persistieren. ein frühzeitiger th erapiebeginn ist für eine günstige prognose essenziell. bereits eingetretene zerebrovaskuläre folgen einer myokarditis mit sekundärer embolisierung bzw. einer meningovaskulitis entsprechen in ihrer prognose anderen zerebrovaskulärer ischämien. eine sehr häufi g gesehene jarisch-herxheimer-reaktion kann in einzelfällen eine akute, potenziell lebensbedrohliche symptomatik verursachen und eine intensivpfl ichtigkeit bedingen. die ersten intravenösen antibiotikaapplikationen sollten immer unter stationären beobachtungsbedingungen durchgeführt werden. symptomatik die im zweiten stadium gelegentlich beobachtete meningovaskulitische symptomatik einer treponema-pallidum-infektion kann ebenso im einzelfall eine intensivmedizinische überwachung oder betreuung erforderlich machen, wie die sehr seltene polyradikuloneuritis des sekundärstadiums. tertiäre verlaufsformen der syphilis (tabes dorsalis und progressive paralyse) werden nur noch sehr selten gesehen und können in einzelfällen auch intensivpfl ichtige symptome bzw. syndrome verursachen. neben der jarisch-herxheimer-reaktion, die in diesem krankheitsstadium nur bei 1−2% zu erwarten ist, kann eine statusartige epileptische anfallsmanifestation oder ein passageres enzephalitisches krankheitsbild bei der progressiven paralyse sowie die sog. oblongata-krise (abdominelle schmerzen, tachykardie, bewusstseinsstörung, atemstörung bis atemstillstand), die in früheren jahren gelegentlich die unmittelbare todesursache eines patienten mit tabes dorsalis war, und deren pathomechanismus unbekannt ist, eine intensivpfl ichtige situation bewirken. zur defi nitiven diagnose bedarf es des positiven ausfalls spezifi scher tests. der liquor cerebrospinalis zeigt in der überwiegenden zahl der fälle eine intrathekale igm-produktion , gelegentlich auch eine intrathekale igg-und iga-produktion. bei progressiver paralyse fi ndet sich fast immer eine liquorpleozytose, während bei tabes dorsalis dies nur in 50−75% der fälle gesehen wird. eine meningovaskulitis zeigt ebenfalls in den meisten fällen eine pleozytose. das gesamtprotein sowie eine intrathekale igg-(häufi g auch igm-und iga-)produktion, ergänzen den liquorbefund. die pleozytose ist häufi g lymphozytär, aber auch ein lymphoplasmazelluläres bild wird gesehen. bildgebende befunde (zerebrale ct-oder mr-untersuchung) zeigen unspezifi sche veränderungen im sinne einer arteriitis, zerebraler ischämie, oder unspezifi sche läsionen in der weißen substanz. jede form einer neurosyphilis wird mit hochdosiertem penicillin g (z. b. 3×10 mio e. täglich) über mindestens 2 wochen behandelt. drittgenerationscephalosporine sowie doxycyclin sind alternativen (z. b. bei β-lactam-allergien). während die meningitische symptomatik unter antibiotischer th erapie abklingt, entspricht die prognose von vaskulitischbedingten hirninfarkten der prognose anderer zerebrovaskulärer ischämien. eine komplette remission ist im tertiärstadium in den meisten fällen nicht mehr zu erreichen. ätiologie und pathogenese zu den häufi gsten erregern akuter meningoenzephalitiden zählen in europa enteroviren gefolgt von arboviren (diverse alpha-, flavi-, und bunyaviren). darüber hinaus kommen auch masern-, mumps-, epstein-barr-viren (ebv), humane immunodefi ziente viren (hiv) und »lymphocytic choriomeningitis« viren (lcmv) in diesem zusammenhang vor [11] . die infektion erfolgt meist im rahmen eines systemischen virusinfekts. beim direkten erregerbefall gelangen die viren am häufi gsten auf hämatogenem weg ins zns. im gegensatz zu früheren annahmen scheinen die viren die bluthirnschranke relativ leicht überwinden zu können. der zns-befall hängt wohl vom ausmaß der virämie und die virämie von der verfassung des immunsystems ab. man vermutet, dass die viren die gefäßendothelzellen direkt befallen oder durch pinozytose/ exozytose durch die zellen hindurchtransportiert werden. einige viren (rabies, hsv) können durch retrograden axonalen transport peripherer nerven in das zns gelangen. sicher müssen mehrere ungünstige faktoren zusammenwirken, damit sich aus einer der häufi gen virusinfektionen eine enzephalitis ent-wickelt. in der regel gehen die infi zierten nervenzellen zugrunde. dadurch werden z. b. entzündliche reaktionen ausgelöst, die weiteren schaden anrichten können. bei immundefi zienten treten gehäuft akute virusinfektionen und hierbei gelegentlich zns-manifestationen auf: cytomegalie-virus-(cmv-)retinitis und -enzephalitis (3%), varizella-zoster-virus-(vzv-)enzephalitis (5%), hsv (4%), progressiv multifokale leukenzephalopathie (pml) im rahmen einer hiv-infektion (2%). für eine virale genese eines akut oder subakut entwickelnden zns-prozesses sollten folgende argumente in betracht gezogen eine akute virale enzephalitis sollte gegenüber einer enzephalopathie , die durch eine vielzahl nichtinfektiöser komponenten eine virale enzephalitis vortäuschen kann, sicher abgegrenzt werden. dabei kann die enzephalopathie metabolische veränderungen wie leberinsuffi zienz, niereninsuffi zienz, diabetisches koma, mitochondriale zytopathien, anoxie/zerebrale ischämie, systemische infektionen, intoxikationen, paraneoplastische störungen, maligne hypertonie, nichtkonvulsiver status bei epilepsie sowie bestimmte nährstoff defi zite beinhalten. die anamnese ist hierbei wie bei allen erkrankungen unerlässlich und sollte sicherlich auch eine reiseanamnese beinhalten. bei klinischer konstellation von kopfschmerzen, fieber und bewusstseinsstörung in kombination mit potenziell fokal-neurologischen ausfällen (z. b. krampfanfälle) muss sofort an eine virale enzephalitis gedacht werden, wobei z. b. ein abrupter beginn mit schneller progredienz durch hsv 1 und erkrankungen mit biphasischen verläufen eher durch enteroviren bedingt sind. bei sicherung einer enzephalitis sollte des weiteren eine infektiöse virale enzephalitis von einer akut disseminierten enzephalomyelitis (adem) unterschieden werden, die anamnestisch häufi g eine kürzlich erfolgte impfung bei kindern ergibt, visuelle störungen eines oder beider augen sowie zeichen der spinalen beteiligung und darüber hinaus multifokale entmarkungsherde in der mrt in beiden hemisphären aufweist [11] . die klinische untersuchung sollte auch das aufsuchen möglicher hautveränderungen (vzv) beinhalten. gibt die klinische konstellation hinweise auf eine enzephalitis, muss sofort eine empirische, antivirale therapie begonnen werden. die kranielle computertomographie (ct) und magnetresonanztomographie (mrt) kann charakteristische befunde zeigen und erlaubt oft die abgrenzung anderer krankheitsbilder [5] , allerdings zeigt sich bei bis zu 10% liquorchemisch nachgewiesener hsv-enzephalitisfälle ein unauff älliger kranieller computertomographie-oder magnetresonanztomographiebefund. die mrt kann das ausmaß des entzündlichen prozesses aufzeigen und bereits auf spezifi sche erreger hinweisen: hsv-enzephalitis bei temporalen marklagerläsionen , japanische enzephalitis bei thalamischen blutungen , enterovirus-71-enzephalitis mit t2-gewichteten hyperintensen läsionen im ncl. dentatus des zerebellum und hirnstamm, multiple marklagerläsionen bei der pml . eine entscheidende rolle spielt die zerebrale bildgebung auch zur verlaufskontrolle bei entstehendem erhöhten icp, wenn eine entscheidung über die anlage einer externen ventrikeldrainage getroff en werden muss. bei viralen infektionen des zns ergeben die blutuntersuchungen entweder einen normalbefund oder geringfügig erhöhte entzündungsparameter. typisch ist eine relative leukozytose bei normalen, leicht erhöhten oder sogar erniedrigen gesamtleukozytenzahlen eine liquoruntersuchung muss bei allen patienten mit verdacht auf eine virale enzephalitis durchgeführt werden, wenn keine kontraindikationen (erhöhter intrakranieller druck) vorliegen [3, 17] . der klassische liquorbefund ist von der einer viralen meningitis nicht zu unterscheiden und ist folgendermaßen charakterisiert: geringe bis mäßige zellzahlerhöhung: 20−1500/μl (selten bis 3000 mit der liquorpolymerasekettenreaktion (pcr) können genombestandteile diverser erreger , wie z. b. hsv-1, ebv, cmv, vzv und enteroviren, nachgewiesen werden und bildet den goldstandard in der diagnostik der virusenzephalitiden. sie kann schnell durchgeführt werden, wobei die ergebnisse bereits nach ca. 24 stunden vorliegen. neuere studien mit hsv-enzephalitis haben gezeigt, dass die sensitivität (ca. 98%) und die spezifi tät (ca. 94%) der liquor-pcr gleich oder sogar besser als die der hirnbiopsie ist [7] . dennoch ist meist die pcr nicht rund um die uhr erhältlich, so dass in der akutsituation keinesfalls auf das pcr-ergebnis gewartet werden sollte, sondern bei verdacht probatorisch antiviral behandelt werden muss. eine negative hsv-liquor-pcr eines patienten mit klinischem und labortechnisch hohem verdacht reduziert zwar die wahrscheinlichkeit, schließt aber damit eine enzephalitis nie aus! durch berechnung des antikörperindex (ai) kann eine intrathekale antikörperproduktion festgestellt werden und erlaubt damit den beweis einer erregerbedingten meningoenzephalitis. diese berechnung ist möglich bei generierung exakter laboreinheiten mittels elisa. ein ai >1,5 zeigt eine intrathekale antikörpersynthese an [13] . die intrathekale synthese von antikörpern entwickelt sich meist erst am ende der 1. erkrankungswoche. eine synthese erregerbedingter antikörper fi ndet aber auch in der spätphase neuroviraler erkrankungen statt (»liquornarbe«) und führt im klinischen alltag gelegentlich zur fehldiagnose einer zns-infektion. eine akut aszendierende parese , die einem guillain-barré-syndrom (gbs) ähnelt, allerdings mit einer pleozytose einhergeht, kann durch eine fsme-, hiv-infektion, rabies oder wnv-infektion bedingt sein [7] . verlauf aufgrund der verschiedenen verläufe wird im speziellen auf die wichtigsten enzephalitiden eingegangen. das hsv ist in westeuropa bei kindern die älter als 6 monaten sind und bei erwachsenen die häufi gste ursache einer sporadischen enzephalitis . bei immunkompetenten erwachsenen wird die hsve in 90% der fälle durch das hsv-1 ausgelöst, während hsv-2 meist nur eine benigne lymphozytäre meningoenzephalitis, hervorruft , welche auch mehrfach remittieren kann (früher: mollaret-meningitis) . bei neugeborenen und immuninkompetenten ruft hsv-2 eine diff use enzephalitis im rahmen einer systemischen, hämatogen fortgeleiteten infektion hervor. die durch hsv-1 erzeugte enzephalitis ist mit einer inzidenz von 5/100.000 die häufi gste sporadische enzephalitis in westeuropa [21] , ohne saisonale bevorzugung. ein 1 / 3 aller hs-ve-fälle tritt als primärinfektion auf. die mehrzahl aller patienten hat jedoch bereits antikörper gegen hsv, wenngleich nur 10% aller hsve-patienten klinische zeichen einer rekurrierenden hsv-infektion aufweisen (z. b. herpes labialis). das virus gelangt vermutlich über die mund-und nasenschleimhaut zum bulbus olfactorius oder ganglion gasseri (n. trigeminus) und über durale nervenäste zur vorderen und mittleren schädelgrube. es verursacht eine fokale enzephalitis , die vorwiegend temporo-und frontobasal gelegen ist und durch hämorrhagische nekrosen und eine erhebliche hirnschwellung charakterisiert ist. in einzelstudien gibt es hinweise auf virusunabhängige chronisch-progrediente gewebsuntergänge im langzeitverlauf der hsve [12] . die initialen symptome können sehr vielfältig sein. nach einem 1-bis 4-tägigen prodromalstadium folgt eine variable phase mit bewusstseinsstörungen, persönlichkeitsveränderungen und fokalen neurologischen symptomen. die hsve kann einen schweren verlauf mit erhöhtem hirndruck und letalem ausgang nehmen. ohne spezifi sche th erapie endet sie in 80% der fälle letal. die spezifi sche frühzeitige th erapie kann die mortalität auf 20% senken, ein groß-3 teil der überlebenden (90%) behält jedoch leichte bis schwere kognitive defi zite zurück [20] . in der kraniellen mrt können morphologische veränderungen bereits deutlich früher und sensitiver als in der cct nachgewiesen werden, wobei durch diff usionsgewichtete und sog. flair-sequenzen ein erheblicher informationsgewinn durch frühzeitige charakterisierung enzephalitischer läsionen herbeigeführt werden kann. die frontomesiotemporalen anteile, die insuläre region, der gyrus cinguli, der th alamus sowie der frontobasale kortex sind mit fokalen ödemen, manchmal sogar mit vereinzelter kontrastmittelaufnahme, häufi g betroff en. es gibt jedoch bildmorphologische hinweise darauf, dass bei säuglingen und kindern im gegensatz zu erwachsenen vermehrt extratemporale läsionen entdeckt werden [19] . eeg-untersuchungen zeigen bei liquor-pcr bestätigten hsve-fällen in bis zu 90% fokal auf den temporallappen bezogene spike-und slow-wave-aktivität, die jedoch häufi g unspezifi sch sind. charakteristische liquorbefunde sind eine lymphozytäre pleozytose von 15−200 (selten bis 700) zellen/μl. oft fi nden sich an tag 3−6 auch plasmazellen und eine mononukleäre pleozytose oder auch eine hämorrhagische komponente (erythrozyten, xanthochromie, siderophagen). das liquoreiweiß ist in über 80% der fälle erhöht. mittels pcr kann frühzeitig (tag 1 oder 2) virusspezifi sche dna im liquor nachgewiesen werden. allerdings korreliert die schwere der erkrankung nicht mit der zahl der viruskopien [23] . falsch-negative liquor-hsv-pcr-befunde sind am häufi gsten innerhalb der ersten 24 die antivirale th erapie reduziert die zahl der viruskopien im liquor. in den meisten fällen führt die aciclovirgabe somit zu einer raschen reduzierung des antigennachweises im liquor, so dass in den meisten fällen innerhalb von 15 tagen nach beginn der th erapie die liquor-pcr negativ ausfällt [19] . bei persistierend positiver liquor-pcr sollte an eine zusätzliche oder alternative antivirale th erapie gedacht werden. die tatsächliche inzidenz der herpes-zoster-enzephalitis ist nicht bekannt. gefährdet durch schwere verläufe sind immunsupprimierte patienten, cmv-seronegative transplantatempfänger und malignompatienten während einer chemotherapie. ein besonders hohes risiko besteht für aids-patienten im stadium iv (chorioretinitis). die vzv-enzephalitis tritt in 1−2 von 10.000 fällen einer vzv-infektion auf, meist 1−2 wochen nach dem typischen exanthem. gelegentlich kann sie dem exanthem auch um bis zu 3 wochen vorausgehen. klinisch kommt es entweder zu einer meningoenzephalitis oder zerebellitis im anschluss an eine windpockeninfektion oder zu einer zosterneuritis (gürtelrose) assoziierten enzephalitis, die häufi ger bei abwehrgeschwächten vorkommt und als polioenzephalitis oder seltener als multifokale leukenzephalopathie verlaufen kann. meist beginnt sie 1−2 wochen nach dem exanthem, doch gelegentlich kann sie den windpocken auch um bis zu 3 wochen vorausgehen. neuropathologisch fi nden sich entzündliche läsionen, hämorrhagische nekrosen, vaskulitiden und infarkte durch gefäßstenosen und -verschlüsse. die kranielle mrt zeigt neben multiplen läsionen in der weißen substanz ischämische und hämorrhagische läsionen mit kontrastmittelenhancement. ein normales eeg im akutstadium spricht gegen die diagnose. die eeg-veränderungen können bis zu einem jahr persistieren. im liquor fi ndet sich eine lymphozytäre pleozytose, anfänglich mit einer granulozytose. die th erapie unterscheidet sich nicht von der der hsve. die mit windpocken assoziierte enzephalitis hat eine letalität von 30%, meist bedingt durch die oft vorbestehende immuninkompetenz. zerebrale beteiligungen bei ebv-infektionen sind meist gutartig . das ebv ist ein herpesvirus, welches verschiedene neurologische manifestationen verursachen kann (meningitis, enzephalitis, aids-assoziiertes zns-lymphom, myeloradikulitis und enzephalomyeloradikulitis). die neurologischen erscheinungen der ebv-infektion treten meistens als komplikationen der infektiösen mononukleose (in zirka 5−7% der fälle) auf. die inzidenz der infektiösen mononukleose selbst liegt bei ca. 8/1000. die klassischen symptome einer infektiösen mononukleose sind fieber (76%), pharyngitis (82%) sowie lymphknotenschwellungen (94%) und splenomegalie (52%). neurologische symptome können sich vor, während und nach den klassischen symptomen manifestieren [4] . die ebv-enzephalitis kann als meningoenzephalitis, zerebellitis (insbesondere bei kleinkindern) und hirnnervenausfälle in erscheinung treten. es wurden auch polio-ähnliche krankheitsbilder beschrieben [24] . schwere krankheitsverläufe kommen insbesondere bei kleinkindern und immunsupprimierten patienten vor. kontrollierte studien zur behandlung der ebv-enzephalitis fehlen. neben aciclovir kann auch ganciclovir gegeben werden. das fsme-virus gehört zu der gruppe der arboviren, wobei das erregerreservoir kleine wildnager und vektorzecken darstellen. die entwicklungszyklen der ixodes-ricinus-zecken führen zu einem saisonalen auft reten der erkrankung von märz bis oktober mit erkrankungsgipfel von april bis juli. durchseuchte zeckenpopulationen fi nden sich vornehmlich in süddeutschland, österreich, tschechien, ungarn und slowakei [9] . eine spezifi sche th erapie existiert nicht. es wird lediglich symptomatisch behandelt. menschen, die ein erhöhtes risiko durch vermehrten kontakt mit rabiesinfi zierten tieren haben, sollten eine präexpositionsprophylaxe erhalten. dabei handelt es sich um einen aktivrabiesimpfstoff , der intradermal oder intramuskulär am tag 0, 7, 21 oder 28 appliziert wird. eine vor kurzem erschienene publikation beschrieb fälle, in denen mrt-läsionen bei sspe aff ektierten kindern auch im hirnstamm detektierbar waren [3] . der liquorbefund ist der einzige auff ällige laborparameter, klinik und infektparameter geben keinen hinweis auf eine infektion. im liquor und serum fi nden sich sehr hohe igg-titer gegen masern, der asi zeigt eine intrathekale synthese an. neueste daten zeigen, dass im liquor von sspe-patienten vermehrte plasmazellklone (cd-138+-zellen) krankheitsrelevante antikörper produzieren, die durch humane igg rekombinante ak (mabs) identifi ziert werden können [2] . das eeg ist stetes pathologisch und es fi nden sich alle 5−8 s gruppen von hohen δ-wellen, die von rhythmischen hyperkinesien begleitet sind (rademecker-komplexe: charakteristisch, aber nicht pathognomonisch). es wurden th erapieversuche mit isoprinosin allein oder in kombination mit intrathekaler oder intraventrikulärer gabe von interferon berichtet, die die überlebensrate verlängert hätten und bei manchen patienten eine gewisse klinische besserung gebracht hätten. allerdings gab es hierzu nie eine kontrollierte klinische studie [1] . die erkrankung endete früher in 80% der fälle letal innerhalb von 3 jahren nach diagnosestellung. inzwischen sind durch gezielte behandlung von myoklonien, spastik, anfällen und weiterer komplikationen verläufe über 10 jahre möglich. diff erenzialdiagnostisch muss an eine progressive rötelnpanenzephalitis oder auch an leukenzephalopathien gedacht werden, die einen ähnlichen im verlauf haben können. die progressive rubellapanenzephalitis ist eine extrem seltene chronisch-progrediente rötelnerkrankung des zns, die überwiegend jungen mit kongenitalem rubella-syndrom (retardierung, hörverlust, verzögertem wachstum, mikrozephalie, katarakt und herzfehlern) betrifft . es werden auch wenige fälle berichtet, bei denen eine prp im anschluss an eine rötelnerkrankung während der kindheit auft rat. weniger als 20 fälle sind seit 1980 bekannt. sie tritt meist zwischen dem 8. und 19. lebensjahr auf. es fi ndet sich eine meningeale, perivaskuläre und parenchymale (mehr weiße als graue substanz) entzündung. die pathogenese ist bislang ungeklärt, vermutlich spielen die im serum und liquor vorliegenden immunkomplexe eine entscheidende rolle. die klinik der prp äußert sich zunächst in form einer langsamen intelligenzminderung. im verlauf tritt neben einer globalen demenz v. a. eine ataxie hinzu . kopfschmerzen, fieber 3 3 oder meningismus treten nicht auf. das spätstadium ist charakterisiert durch schwere demenz, spastische tetraparese und hirnstammsyndrome. zur diagnostik bedient man sich der liquoruntersuchung, die eine mäßige lymphozytäre pleozytose, mäßig erhöhtes protein, deutlich erhöhte werte für γ-globuline und rubella-spezifi sche oligoklonale banden aufweist (. tab. 35.22) . eine gesicherte therapie existiert nicht. inzidenz, ätiologie, pathogenese die pml wurde initial bei malignompatienten und bei iatrogen immunkompromittierten patienten beobachtet. vor dem ausbruch von aids war sie eine extrem seltene erkrankung. es wird geschätzt, dass etwa 1% der aids-patienten eine pml entwickeln werden, wohingegen mehr als 60% der heute diagnostizierten pml-fälle aids-patienten sind . erreger ist das jc-virus, ein dna-virus , welches häufi g in der bevölkerung ist, ohne eine infektion zu verursachen. im jahre 2005 sind im rahmen von 2 großen studien für die zulassung von natalizumab (tysabri) für die th erapie der multiplen sklerose 2 fälle von pml aufgetreten, wobei stets eine kombinationstherapie mit interferonen oder azathioprin in diesen fällen bestand. bei alleiniger gabe von natalizumab wurde kein pml-fall bisher beschrieben. mehrere fälle von pml wurden auch nach hochwirksamer chemotherapie bei malignompatienten beschrieben. in den letzten monaten wurden pml-fälle auch bei patienten mit sle und rheumatoider arthritis unter th erapie mit rituxan beschrieben. die klinischen erscheinungen sind kognitive störungen (demenz, verhaltensauff älligkeiten, persönlichkeitsveränderungen) , aphasie, sehstörungen (homonyme hemianopsien) und psychiatrische symptome. die liquor-pcr kann den erreger in 90% der fälle nachweisen. in der kraniellen mrt sind t2hyperintense marklagerläsionen nachweisbar. die pml ist fast immer innerhalb weniger monate tödlich. in einzelfällen sind längere überlebenszeiten beschrieben. defi nition und epidemiologie das retrovirus hiv wird am häufi gsten durch sexualkontakt und durch verunreinigte injektionsnadeln bei drogenabhängigen verbreitet. in ländern, in denen der hiv-antikörpertest noch nicht zur verfügung steht, können blut und kontaminierte blutprodukte zur hiv-übertragung führen. das virus wird auch vor, während oder nach der geburt über die infizierte mutter vertikal übertragen. das infektionsrisiko bei medizinischem personal durch akzidentelle verletzungen ist eher gering und eine serokonversion kommt in ca. 0,3% dieser fälle vor. der aids-erreger ist das humane immundefi zienzvirus (hiv) . hiv gehört zu den rns-retroviren und enthält das enzym reversetranskriptase. es gibt 2 hiv-typen: der weltweit verbreitetste ist hiv-1. seltener ist der typ hiv-2, der seinen ursprung in westafrika hatte, nun aber auch weltweit verbreitet ist. das hi-virus verursacht eine direkte beeinträchtigung des immun-und nervensystems. das immunsystem des hiv-infi zierten bildet antikörper gegen das virus, aber dadurch wird dessen vermehrung nicht gehemmt. die hiv-inkubation wird serologisch und klinisch defi niert. die inkubation wird serologisch als der zeitabstand zwischen der infektion und dem nachweis von hiv-antikörper im se-3 3 rum defi niert und dauert zwischen 1−3 (selten 6) monate. klinisch wird sie als der zeitabstand zwischen der infektion und dem auft reten von aids defi niert. bei erwachsenen dauert dies meist 10±2 jahre. die inkubationszeit wird bei perinataler infektion und bei menschen mit ernährungsmangel verkürzt. hiv zielt auf die cd4-rezeptor-tragenden zellen des immunsystems: t-helferzellen (cd4 + lymphozyten) und mononukleare zellen wie makrophagen, monozyten, mikroglia und langerhanszellen der epidermis. chemokinrezeptoren sind als kofaktoren für die virale penetration in die zellen zuständig. durch zerstörung der t-helferzellen fällt deren absolute zahl unter die normgrenze von 400/μl. der quotient t-helferzellen/t-suppressorzellen wird deshalb auf werte <1,2 erniedrigt (normalwert: 2). dieser prozess ist für eine gravierende immunschwäche zuständig, die im verlauf der infektion lebensbedrohliche opportunistische infektionen und charakteristische tumore verursacht. im frühverlauf der infektion wird das zns von hiv-infi zierten monozyten erreicht. die hi-virionen vermehren sich in den makrophagen und monozyten des zns. nach einer klassifi kation des cdc (centers for disease control 1993) werden die stadien der hiv-infektion aufgrund klinischer befunde und der absoluten zahl der t-helferzellen eingeteilt (. tab. 35.23). für die stadieneinteilung gilt die am weitesten fortgeschrittene kategorie: eine rückklassifi zierung fi ndet nicht statt. hierbei wird nicht berücksichtigt, dass die antiretrovirale th erapie immunrekonstruktionen möglich macht. zu der kategorie a gehören das akute retrovirale syndrom, auch akute hiv-krankheit genannt, die latenzphase oder asymptomatische infektion und das lymphadenopathiesyndrom (las). das akute retrovirale syndrom tritt nach 3−6 wochen bei ungefähr 30% der hiv-infi zierten auf. das krankheitsbild ähnelt dem einer mononukleose und besteht aus fieber, exanthem, myalgien, lymphknotenschwellung, splenomegalie und angina. die infektion kann auch asymptomatisch verlaufen, obwohl sich die hi-virionen im lymphatischen gewebe vermehren. bei ca. 40% der patienten tritt das lymphadenopathiesyndrom (las) auf. dies wird durch eine persistierende, mehr als 3 monate dauernde, generalisierte lymphadenopathie an mindestens 2 extrainguinalen stellen defi niert. zu der kategorie b gehören pathologische prozesse, die nicht aids-defi nierend sind, aber deren pathogenese v. a. durch immunsuppression hervorgerufen wird. zu dieser kategorie gehören chronische diarrhö, subfebriles syndrom, idiopathische thrombozytopenische purpura, zervikale dysplasie oder carcinoma in situ, candidose (oropharyngeal, vulvovaginal), herpes zoster, orale haarleukoplakie, listeriose und bazilläre angiomatose. für eine progression sprechen ein anstieg der viruslast und eine abnahme der t-helferzellen. zu der kategorie c gehören die aids-defi nierenden krankheiten oder aids-indikatorkrankheiten, die in folgender übersicht aufgeführt sind. der nachweis von hiv-antikörper im serum mittels eli-sa und/oder western-blot sichert die diagnose der hiv-infektion. die infektion mit hiv kann während der sog. »fensterzeit« (zwischen 4 und 12 wochen nach der primärinfektion) mittels polymerasekettenreaktion (pcr) der hiv-genomsequenzen festgestellt werden, bevor die hiv-antikörper nachweisbar sind. entscheidende parameter für die prognose der progression der hiv-infektion, die in korrelation mit den klinischen symptomen mitzurechnen sind, sind die virusbelastung (anzahl der hiv-rna kopien in plasma) und die anzahl der cd4+-t-helferzellen [5] . bei bewusstseinsklaren patienten, muss vor durchführung eines hiv-testes das einverständnis des betroff enen eingeholt werden. die behandlung von hiv-aids besteht aus einer spezifi sch antiretroviralen th erapie, die in einer mehrfachkombinationstherapie besteht, der sog. hochaktiven antiretroviralen therapie oder haart , aus einer symptomatischen behandlung der assoziierten erkrankungen und aus der prophylaxe opportunistischer infektionen. indikationen für den anfang einer spezifi schen antiretroviralen th erapie sind der klinische und/oder laborchemische nachweis des immundefekts (. prognose die hi-viruskonzentration im blut 6 monate nach der infektion (»set point«) ist von prognostischer bedeutung ( [11] ; . tab. 35.25). die wahrscheinlichkeit aids 3 jahre nach der hiv-infektion zu entwickeln, hängt mit der hi-viruslast und der t-helferzellanzahl eng zusammen. die statistische wahrscheinlichkeit einer berufl ichen hiv-infektion nach perkutaner exposition mit blut von hiv-infizierten (z. b. nadelstich-oder schnittverletzungen) liegt bei ca. 0,3%. diese wahrscheinlichkeit steigt bei expositionsarten unter den folgenden bedingungen: tiefe verletzungen, frische und sichtbare blutspuren an dem penetrierenden instrument, verletzung durch eine kanüle, die früher in einer vene oder arterie lag und hohe viruslast des quellen-patienten [3] . eine medikamentöse hiv-postexpositionsprophylaxe (hiv-pep) wird deshalb nach perkutanen verletzungen mit injektionsnadeln oder mit anderen hohlraumnadeln und nach schnittverletzungen unter beteiligung von körperfl üssigkeiten mit potenziell hoher hi-viruskonzentration empfohlen [7] . bei oberfl ächlichen verletzungen und bei kontakt mit schleimhaut oder verletzter haut mit flüssigkeiten mit hoher hi-viruskonzentration kann eine hiv-pep angeboten werden. neurologische komplikationen entwickeln sich vor oder nach dem auft reten von hiv-antikörpern. hiv-assoziierte neurologische komplikationen treten entweder primär von hiv verursacht oder sekundär als opportunistische infektionen und neoplasien auf [15] . zerebrovaskuläre komplikationen unterschiedlicher genese sind überdurchschnittlich häufi g bei hivinfi zierten patienten. die häufi gsten hiv-assoziierten neurologischen erkrankungen und deren pathogenese, klinischer verlauf, diagnostik und th erapie werden in den folgenden abschnitten beschrieben. in der hiv-th erapie erfahrenen arzt erfolgen. zu beachten ist die gefahr der entwicklung einer th erapieresistenz. die zerebrale toxoplasmose ist die häufi gste opportunistische infektion des zns bei hiv-infi zierten patienten in westeuropa. diese erkrankung tritt bei 30% der nicht therapierten patienten auf und ist in 10% der fälle die erstmanifestation von aids. die zerebrale toxoplasmose wird von einer reaktivierung einer infektion mit dem protozoon toxoplasma gondii verursacht , dies wird in erster linie durch katzenkot oder unzureichend gebratenes fleisch übertragen. toxoplasma gondii bleibt nach einer primärinfektion als pseudozyste im gehirngewebe. klinisch kommt es bei 80% der patienten zu fokalneurologischen symptomen. fieber und kopfschmerz treten bei ungefähr 50% der patienten auf. im kraniellen computertomogramm (cct) und kraniellen magnetresonanztomogramm (mrt) werden bei 1 / 3 der patienten eine solitäre läsion, bei ca. 2 / 3 der patienten mehrere läsionen mit perifokalem ödem im marklager mit ringförmiger oder nodulärer kontrastmittelanreicherung gefunden. entscheidend für die diagnose ist das ansprechen der symptomatik und der nachweisbaren läsionen nach th erapie. wenn es nach 2−4 wochen th erapie zu keiner verbesserung kommt, oder es eine verschlechterung der klinischen oder neuroradiologischen zeichen gibt, wird eine hirnbiopsie empfohlen. die th erapie besteht in der regel aus pyrimethamin plus sulfalen oder sulfadiazin in kombination mit folinsäure, um eine myelotoxizität zu verhindern. die akute th erapie dauert in der regel 4−6 wochen, mindestens aber bis die nachweisbaren läsionen kein kontrastmittel anreichern. danach sollte lebenslang eine rezidiv-oder sekundärprophylaxe fortgeführt werden. manche autoren empfehlen die reduktion oder beendigung der sekundärprophylaxe, wenn erstens die hi-viruslast dauerhaft unter 20 kopien/μl bleibt, zweitens die t-helferzellzahl dauerhaft über 200/μl bleibt, drittens die rezidivprophylaxe mehr als 6 monate dauert und viertens die zerebralen läsionen im cct oder mrt nicht mehr nachweisbar sind. eine primärprophylaxe ist bei einer t-helferzellanzahl <200/μl zu empfehlen, v. a. bei patienten mit einer positiven toxoplasmaserologie [2] . die gabe von kortison sollte nur im einzelfall durchgeführt werden (z. b. drohende einklemmung), da die histolo die zytomegalievirusenzephalitis wird durch die reaktivierung einer latenten cmv-infektion verursacht und wird immunhistologisch bei ca. 40% der verstorbenen aids-patienten nachgewiesen. überwiegend kommt sie bei einer t-helferzellanzahl <100/μl vor. klinisch ist sie durch eine rasch progrediente enzephalopathie (mit demenz, psychischen veränderungen, gedächtnis-und konzentrationsstörungen) charakterisiert. der nachweis des cm-virus-genoms im liquor mittels pcr sichert die diagnose [17] , dennoch wird häufi g die diagnose erst post mortem durch den nachweis der typischen riesenzellen mit einschlusskörperchen (eulenaugenzellen) im gewebe gesichert. die akuttherapie besteht in erster linie aus ganciclovir (2×5 mg/kgkg/24 h i.v.) und bei unverträglichkeit foscarnet (2×90 mg/kgkg/24 h i.v.). ganciclovir ist myelotoxisch, deshalb sind regelmäßige blutbildkontrollen erforderlich. foscarnet ist nephrotoxisch, aus diesem grund werden eine ausgeglichene flüssigkeitsbilanz und eine bestimmung der kreatininclearance empfohlen. fakultativ kann eine kombination von beiden substanzen indiziert werden [1] . eine sekundärprophylaxe mit ganciclovir (5−6 mg/kgkg i.v. an 5 tagen der woche) oder foscarnet (1×90 mg/kgkg i.v. an 7 tagen oder 120 mg/kgkg i.v. an 5 tagen der woche) wird in der regel nach ca. 3 wochen akuttherapie lebenslang in reduzierter dosis eingesetzt. die kryptokokkenmeningoenzephalitis ist eine opportunistische infektion mit dem pilz cryptococcus neoformans , deren ausbreitung nach einer asymptomatischen besiedelung nach inhalation von vogelkot hämatogen aus dem respirationstrakt erfolgt. sie tritt bei einer t-helferzellzahl <100/μl auf. charakteristisch ist ein progredienter verlauf über tage oder wochen mit kopfschmerzen, fieber, übelkeit und somnolenz. meningitische zeichen treten bei nur 30% der patienten auf. selten kommt es zu epileptischen anfällen und fokalneurologischen zeichen. der erregernachweis mittels tuschepräparat des liquors gelingt in 75% der fälle, der antigennachweis in serum und liquor gelingt in >99% der fälle. die akuttherapie besteht aus amphotericin b + flucytosin + fluconazol und dauert in der regel zwischen 4−6 wochen. danach wird eine konsolidierungstherapie mit fluconazol oder itraconazol eingesetzt, bis der kryptokokkenantigentiter im liquor um 2 stufen gefallen ist [14] . die progressive multifokale leukenzephalopathie (pml) tritt bei hiv-infi zierten patienten, die keine haart bekommen, mit einer inzidenz von 2% auf. pml wird durch die reaktivierung einer latenten infektion mit dem papovavirus jc verursacht, der eine entmarkung der weißen substanz auslöst. das krankheitsbild besteht aus progredienten fokalen symptomen wie paresen , ataxie, gesichtsfelddefekten und aphasie, die innerhalb von wochen bis wenigen monaten zum tode führen. im ct und mrt ist es typisch, einzelne oder multiple nicht raumfordernde marklagerläsionen ohne kontrastmittelaufnahme nachzuweisen. der nachweis des jc-virus-genoms im liquor mittels pcr ist in 80% der fälle möglich. eine stereotaktische hirnbiopsie ist in einzelfällen für die diagnose notwendig. die th erapie der wahl ist haart [6] , in einzelfallberichten sind längere überlebenszeiten als nur wenige monate möglich. das primäre zns-lymphom ist der häufi gste im zusammenhang mit aids auft retende tumor des zns. primäre zns-lymphome sind in der regel hochmaligne b-zelltyp non-hodgkin-lymphome und fast zu 100% mit dem epstein-barr-virus assoziiert. die wichtigste diff erenzialdiagnose ist die zns-toxoplasmose. eine diagnose mittels neuroradiologischen verfahren ist nicht spezifi sch, denn im gegensatz zu den zns-lymphomen bei immunkompetenten patienten können hiv-assoziierte lymphome ringförmig oder unregelmäßig kontrastmittel anreichern. die präsenz einer positiven ebv-pcr ist hoch spezifi sch und macht die diagnose einer toxoplasmose sehr unwahrscheinlich. ohne behandlung beträgt die mittlere überlebenszeit nur wenige wochen. die th erapie der wahl besteht aus ganzhirnradiatio + dexamethason. bei meningeosis lymphomatosa werden intrathekales methotrexat oder cytarabin bis zur sanierung des liquors verwendet. bei patienten in gutem allgemeinzustand eine detaillierte kenntnis der epidemiologie, der infektionswege, und v. a. von prädisponierenden faktoren ist notwendig, um die diagnose einer parasitären zns-infektion rechtzeitig zu stellen und die entsprechende therapie einleiten zu können. . tab patienten mit zns-mykosen zeigen beim schädelübersichtsröntgen oder beim röntgen der paranasalen sinus gelegentlich eine knochendestruierung, schleimhautverdickung sowie weichteildichte gewebsmassen, die sich von den paranasalen sinus in den intrakraniellen raum ausbreiten können. die zerebrale ct-untersuchung zeigt eine meningeale anspeicherung, insbesondere im bereich der basalen zisternen, hydrozephalus, granulome und abszesse. eine begleitvaskulitis führt zu vaskulär-ischämischen ct-veränderungen inklusive hämorrhagischer transformierung. immunkompetente patienten zeigen häufi g eine ringförmige anspeicherung nach kontrastmittelapplikation, diese fehlt bei massiv immunkompromittierender grunderkrankung. die ct-veränderungen sind typischerweise unspezifi sch und müssen immer im klinischen zusammenhang gesehen und interpretiert werden. die kernspintomographiebefunde ähneln der zerebralen computertomographie, wenngleich das mr die weichteilgewebe der kopf-und nackenregion besser visualisieren kann. sowohl die mr-angiographie als auch die konventionelle zerebrale panangiographie bestätigen die diagnose einer vaskulitis, einer arteriellen okklusion, mykotischer aneurysmen oder einer sinusvenenthrombose. elektrophysiologische techniken zeigen unspezifi sche veränderungen. die transkranielle dopplersonographie kann zum monitoring einer zns-vaskulitis und evtl. bei erhöhtem intrakraniellen druck zum einsatz kommen. die analyse des liquor cerebrospinalis zeigt bei chronischer meningitis eine pleozytose von wenigen bis mehrere tausend zellen/mm 3 . typisch ist ein gemischtzelliges bild, mononukleär betont, gelegentlich auch polymorphzellig, insbesondere bei abszedierenden prozessen in der nachbarschaft der subarachnoidalräume. die ruptur eines mykotischen aneurysma resultiert in einem hämorrhagischen bzw. xanthochromen liquor . nicht selten fi ndet sich bei pilzmeningitis eine mäßiggradige eosinophilie. die liquor-serum-glukoseratio ist üblicherweise geringgradig erniedrigt, eiweißgehalt im liquor cerebrospinalis mäßig bis massiv erhöht (bis >1000 mg/dl), letzteres insbesondere bei arachnoiditis und/oder obstruktivem hydrozephalus. c-reaktives protein, laktat-oder aminosäurenbestimmung im liquor sind zur diff erenzialdiagnose einer zns-mykose nicht geeignet. eine zytologische aufarbeitung hilft eine chronische pilzmeningitis von einer meningeosis carcinomatosa oder leukaemica/lymphomatosa zu diff erenzieren. aspergillus ssp. und zygomyzeten erscheinen im liquor als hyphen, während askomyzeten, basidiomyzeten und deuteromyzeten sich als hefepilze präsentieren. histologisch aufgearbeitete biopsate sind häufi g klinisch relevanter als kulturen, insbesondere da ein positives pilzkulturergebnis oft erst nach wochen zu erwarten ist. gomori-methenamin-silberfärbung oder pas (»periodic acid schiff «)-färbung sind zur direktdarstellung von pilzen am besten geeignete färbemethoden. cryptococcus neoformans kann im tuschepräparat gut visualisiert werden, ist typischerweise von einer polysaccharidkapsel umgeben und zeigt häufi g das phänomen der knospung. bei coccidioides-immitis-infektionen wurden laborinfektionen beschrieben, entsprechende vorsicht bei der diagnostischen aufarbeitung ist geboten. die wichtigsten diff erenzialdiagnosen sind in nach folgender übersicht aufgelistet; sie ist in keiner weise vollständig, da fast jede entzündliche hirnerkrankung sich wie eine zns-mykose präsentieren kann. bakterielle ( therapie patienten mit einer zns-mykose sind häufi g immunkompromittiert und zeigen oft eine disseminierte fungämie oder eine sepsis mit multiorganversagen. aus diesem grunde muss bei solchen patienten eine antimykotische th erapie immer mit den entsprechenden supportiven maßnahmen, inkl. intensivtherapie, durchgeführt werden. ausreichende flüssigkeitszufuhr, amphotericin b (amb) trägt ein hohes risiko einer renalen toxizität , führt zu kaliumverlust und erhöhung der transaminasen. 5-fluorocytosin (5fc) führt zu einer knochenmarkssuppression mit anämie aber auch th rombozytopenie . aus diesem grunde muss ein engstes labormonitoring der nierenfunktionswerte, leberfunktionswerte, elektrolyte und des gesamten blutbilds unter einer antimykotischen th erapie gefordert werden. amb-therapie führt häufi g zu akuten fieberreaktionen , diese werden mit unmittelbar vor der amb-therapie verabreichtem kortison kupiert. stereotaktische oder off ene biopsien einerseits, die implantation eines ventrikuloatrialen oder ventrikuloperitonealen shunts bei hydrozephalus andererseits sowie die implantation eines rickham-oder ommaya-reservoirs zur intraventrikulären medikamentenapplikation sind im einzelfall nötige neurochirurgische th erapieschritte. in vielen fällen ist die prognose eines patienten mit einer zns-mykose trotz antimykotischer th erapie ungünstig, zumindest sehr variabel. die mortalität bei kryptokokkenmeningitis beträgt nur wenige prozent, während eine zns-aspergillose mit einer mortalitätsrate von mehr als 80% einhergeht. patienten mit zns-mykose erfordern häufi g eine besonders langdauerende und auch rezidivierende spezifi sche chemotherapie. in vielen fällen ist die zugrundeliegende immunkompromittierende erkrankung entscheidend für das langzeitergebnis und erfordert aus diesem grunde häufi g ein interdisziplinäres management. die akuten spinalen entzündungen sind ein potenziell risikoreiches und je nach erkrankungsentität diagnostisch wie auch therapeutisch schwieriges und teilweise auch prognostisch ungünstiges krankheitsbild und bedürfen einer dringlichen diff erenzialdiagnostischen klärung. die jährliche inzidenz einer akuten transversalen myelitis (atm) wird mit 1-4 fällen/1 mio. einwohner angegeben und sind damit deutlich seltener als entzündliche erkrankungen des gehirns oder der meningen (geschätzte inzidenz der viralen meningoenzephalitis 10-20 fälle/100.000 einwohner; [16] ). die atm betriff t v. a. jüngere patienten, der altersgipfel liegt zwischen 10-19 sowie 30-39 jahren [19] . die spondylitis und spondylodiszitis ist eine erkrankung älterer erwachsener. die inzidenz einer infektiösen spondylitis wird auf 1-2 fälle/100.000 einwohner geschätzt ( [3, 4] ; 7 kap. 35.2). die einteilung der spinalen entzündungen kann zunächst unter anatomisch-morphologischen gesichtspunkten in medulläre entzündungen und extramedulläre entzündungen erfolgen (. tab. 35.33). letztere sind zum überwiegenden teil erregerbedingt, wohingegen die medullären entzündungen in erregerbedingte und nicht erregerbedingte ursachen unterschieden werden können (. tab. 35.34) . bei der akuten myelitis kann in über 50% der fälle keine ursache gefunden werden. häufi ge ursachen sind v. a. die multiple sklerose [5] und virale entzündungen. die extramedullären entzündungen werden insbesondere durch hämatogene und lokale (per continuitatem) bakterienaussaat bedingt -z. b. nach bandscheiben-oder wirbelsäulehäufi gste erreger sind staphylokokken, mycobacterium tuberculosis, e. coli spp., klebsiella spp., streptokokken und pseudomonaden. risikofaktoren für eine spinale infektion sind neben einer immunsuppression (hiv, immunsuppressive medikamentöse th erapie), patienten mit diabetes mellitus, alkoholund drogenabusus, traumata und chronischen hepatischen und renalen erkrankungen [1, 2, 3] . auch im rahmen einer systemischen infektion (sepsis, endokarditis) kann es, v. a. bei den genannten risikogruppen, zu einer zusätzlichen spinalen manifestation der infektion kommen. die symptomatik akuter myelitiden und extramedullärer entzündlicher prozesse hängt im wesentlichen von der zugrunde liegenden ätiopathogenese ab und macht sich meist durch akut bis subakut entwickelnde neurologische defi zite bemerk-3 bar. neben sensibilitätsstörungen (hypästhesien, parästhesien, dysästhesien und hyperpathien) meist kaudal der rückenmarksschädigung können motorische defi zite und autonome störungen (blasen-und mastdarmstörungen, sexuelle störungen) auft reten. die ausfallerscheinungen können lateralisiert sein, aber auch als akute querschnittsymptomatik imponieren. eine aufsteigende myelitis kann zur beteiligung des hirnstamms mit hirnnervenausfällen und ateminsuffi zienz führen und klinisch dem bild einer »landry-paralyse« entsprechen. rückenschmerzen -häufi g ziehend, stechend oder dumpf -sind v. a. bei extramedullären prozessen im bereich der entzündungen zu fi nden, können aber auch bei einer myelitis auft reten. fieber kann bei einer lokalen entzündung zunächst fehlen und sich erst nach hämatogener streuung entwickeln. die symptome einer spinalen entzündung können anfangs sehr unspezifi sch sein und dadurch die diagnosestellung erheblich erschweren und verzögern. die poliomyelitis verläuft klassischerweise in mehreren stadien und beginnt zunächst mit fieber, gefolgt von einem meningitischen stadium, bis sich dann das paralytische stadium anschließt. die mittlerweile seltene lues spinalis mit der tabes dorsalis (hinterseitenstrangmyelitis) als spätstadium der neurolues geht mit einer progressiven lähmung, sensibilitätsstörungen, lanzierenden schmerzen, refl exverlust und blasenstörungen einher. eine fsme-myelitis ist häufi g mit einer »hohen querschnittssymptomatik« mit beteiligung der arme , der hirnnerven und des zwerchfells verbunden und weist eine schlechte prognose auf [17] . die neuromyelitis optica (devic-syndrom) stellt eine autoimmune erkrankung dar, die ist durch das klinische bild einer akuten (transversen) myelitis und einer optikusneuritis, die vorwiegend jüngere frauen betrifft , charakterisiert. die verdachtsdiagnose einer spinalen entzündung sollte zunächst durch das klinische bild erfolgen. die lokalisation der schädigung ist über die untersuchung der sensiblen dermatome, der myotome und der muskeldehnungsrefl exe möglich. hilfreich in der zuordnung der höhenlokalisation ist die untersuchung des vibrationsempfi ndens einschließlich der dornfortsätze. autonome störungen können beispielsweise über den analen sphinktertonus und blasenentleerungsstörungen mit restharnbildung oder inkontinenz erfasst werden. umschriebene entzündungen der wirbelsäule und angrenzender strukturen gehen häufi g mit einem lokalen klopf-und stauchungsschmerz einher. neben dem klinischen bild ist die neuroradiologische bildgebung von besonderer bedeutung für die diagnosestellung und sollte bei verdacht einer spinalen entzündung zeitnah nach erstem patientenkontakt erfolgen. aufgrund der hohen ortsaufl ösung, guten diff erenzierbarkeit der verschiedenen gewebe und sensitiven darstellung entzündlicher läsionen stellt die kernspintomographie (mrt; . abb. 35.6 und . abb. 35.7) die untersuchungsmethode der wahl dar. entzündliche läsionen werden besonders gut in t2gewichteten und t1-gewichteten aufnahmen nach kontrastmittelgabe dargestellt. um die räumliche ausdehnung zuverlässig beurteilen zu können, müssen bilder in mindestens 2 schnittebenen (bevorzugt axiale und sagittale schnittführung) angefertigt werden (. abb. 35.5). zum ausschluss einer zerebralen beteiligung (v. a. hirnstamm) ist, auch unter diff erenzialdiagnostischen aspekten, bei zervikalen prozessen eine ergänzende zerebrale mrt sinnvoll. falls kontraindikationen für die mrt-untersuchung vorliegen, kann bei extramedullären entzündlichen prozessen alternativ eine computertomographie mit kontrastmittel erfolgen. um die strahlendosis zu minimieren ist eine vorherige höhenlokalisation anhand des klinischen bildes sinnvoll. zur weiteren einordnung des entzündlichen prozesses ist neben der bildgebung die zytologische, chemische, bakteriologische und immunologische analyse des liquor essenziell. auch wichtige diff erenzialdiagnosen zur spinalen entzündung (z. b. spinale ischämie) können dadurch abgegrenzt werden (. tab. 35.35) . bakterielle entzündungen gehen typischerweise mit einer deutlichen erhöhung der zellzahl (>1000 zellen/μl) und dem gesamtprotein einher. bei verdacht auf eine bakterielle infektion muss immer eine erregerisolierung mittels liquorkultur oder pcr-diagnostik angestrebt werden. wenn der entzündliche prozess den subarachnoidalraum noch nicht erreicht hat, ist die liquordiagnostik in der regel nicht richtungweisend. in diesem fall gelingt, v. a. bei systemischen entzündungszeichen, ein keimnachweis mittels blutkultur. bei klar abgrenzbaren entzündlichen prozessen (spinaler abszess, diszitis) kann auch eine ct-gesteuerte punktion zum keimnachweis hilfreich sein und sollte rechtzeitig erfolgen. virale entzündungen weisen neben einer leicht bis moderaten zellzahlerhöhung (meist 500 bis max. 1000 zellen/μl) üblicherweise nur eine leichte eiweißerhöhung auf. der nachweis spezifi scher antikörper (igg und igm) im liquor kann auf eine von rheumafaktoren und bei den »seronegativen« spondylarthropathien die häufi ge assoziation mit hla-b27. neben den chronisch-entzündlichen wirbelsäulenerkrankungen müssen auch extramedulläre tumoren (neurinome, meningeome, angiome, sarkome) und metastasen (z. b. bronchial-, mamma-, prostatakarzinom, plasmozytom) in die diff erenzialdiagnostische aufarbeitung einbezogen werden. selten können spinale epidurale blutungen bei gerinnungsstörungen (antikoagulation!), zustand nach trauma, lumbalpunktion, periduralkatheter und vaskulären malformationen eine querschnittsymptomatik verursachen. auch an degenerative erkrankungen mit wirbelkörperfrakturen, spinalkanalstenosen und bandscheibenvorfällen muss bei extramedullären prozessen gedacht werden. neben der (erreger)spezifi schen therapie sollten allgemeine maßnahmen wie anlage eines blasenkatheters bei blasenentleerungsstörungen, thromboseprophylaxe, lagerung, frühzeitige mobilisierung, physiotherapie und schmerztherapie von anfang an durchgeführt werden. die medikamentöse th erapie hängt wesentlich von der zugrunde liegenden ätiopathogenese bzw. dem erreger ab. oft mals gelingt in der initialen phase keine eindeutige ätiologische zuordnung oder erregerisolation, so dass, je nach dringlichkeit bei akuten erkrankungen die wahl der medikamente empirisch, entsprechend dem klinischen verlauf, den ergebnissen der la a. lsp. lsp. lc. double blind placebo-controlled trial of pleconaril in infants with enterovirus meningitis diagnosis and treatment of viral encephalitis molecular analysis of cerebrospinal fl uid in viral diseases of the central nervous system severe neurological complications in association with epstein-barr virus infection encephalitis, cerebritis, and brain abscess: pathophysiology and imaging fi ndings a 10-year follow-up study of tick-borne encephalitis in the stockholm area and a review of the literature: need for a vaccination strategy harrison's neurology in clinical medicine prospective analysis of 61 cases of enteroviral meningitis: interest of systematic genome detection in cerebrospinal fl uid irrespective of cytologic examination results tick-borne encephalitis (tbe) in germany and clinical course of the disease trends in the management of herpes simplex encephalitis viral encephalitis herpes simplex virus encephalitis: chronic progressive cerebral magnetic resonance imaging abnormalities in patients despite good clinical recovery protein transfer at the blood cerebrospinal fl uid barrier and the quantitation of the humoral immune response within the central nervous system viral meningitis and encephalitis: traditional and emerging viral agents craniectomy: an aggressive treatment approach in severe encephalitis diff usion mri in rasmussen's encephalitis, herpes simplex encephalitis, and bacterial meningoencephalitis acute encephalitis laboratory diagnosis of central nervous system infections update on herpes simplex encephalitis the long-term neuropsychological outcome of herpes simplex encepha induction and maintenance therapy of cytomegalovirus central nervous system infection in hivinfected patients meta-analysis of prophylactic treatments againts pneumocystis carinii and toxoplasma encephalitis a case-control study of hivseroconversion in health care workers after percutaneous exposure. centers for disease control and prevention needlestick surveillance group 1993 revised classifi cation system for hiv infection and expanded surveillance case defi nition for aids among adolescents and adults association of initial cd4 cell count and viral load with response to highly active antiretroviral therapy progressive multifocal leucoencephalopathy: improved survival of human immunodefi ciency virus-infected patients in the protease inhibitor era postexposure chemoprophylaxis for occupational exposures to the human immunodefi ciency virus kamps bs (hsg.). hiv.net 2007. www.hiv. net. steinhäuser verlag aids epidemic update goals and milestones during treatment of hiv-1 infection with antiretroviral therapy: a pathogenesisbased perspective prognosis in hiv-1 infection predicted by the quantity of virus in plasma guidelines for the use of antiretroviral agents in hiv-infected adults and adolescents. department of health and human services guidelines for the diagnosis and management of neurological complications of hiv infection practice guidelines for the management of cryptococcal disease. infectious diseases society of america prospects for therapy of hiv-associated neurologic diseases neue entwicklungen in diagnostik und therapie primärer non-hodgkin-lymphome des zentralen nervensystems diagnosis of cytomegalovirus encephalitis in patients with aids by quantitation of cytomegalovirus genomes in cells of cerebrospinal fl uid the aetiologies of epilepsy in tropical africa exchange transfusion in falciparum malaria plasma and in vitro levels of cytokines during and after a plasmodium falciparum malaria attack in gabon melarsoprol versus eflornithine for treating late-stage gambian trypanosomiasis in the republic of the congo problems for the chemotherapy of human african trypanosomiasis clinical characteristics of human babesiosis die echinokokkuserkrankung des nervensystems lethal african trypanosomiasis in a traveler: mri and neuropathology strongyloides stercoralis hyperinfection syndrome; how often is it missed? role of quinine in lifethreatening babesia divergens infection successfully treated with clindamycin toxocara canis meningomyelitis quinine pharmacokinetics in cerebral malaria: predicted plasma concentrations after rapid intravenous loading using a two compartment mode meta-analysis: cysticidal drugs for neurocysticercosis: albendazole and praziquantel uptake and mode of action of drugs used against sleeping sickness encephalitis due to a free-living amoeba (balamuthia mandrillaris), case report with literature review artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial multifocal central nervous system lesions in three patients with trichinosis cerebral sparganosis trichinose des nervensystems a trial of antiparasitic treatment to reduce the rate of seizures due to cerebral cysticercosis cerebral schistosomiasis presenting as a brain tumor conventional and experimental treatment of cerebral malaria a rare case of toxocara canis cerebral vasculitis intracerebral toxoplasmosis in patients with acquired immunodefi ciency syndrome chemotherapy of echinococcus infection with albendazole first case of human babesiosis in germany -clinical presentation and molecular characterisation of the pathogen human african trypanosomiasis of the cns: current issues and challenges severe falciparum malaria: an important cause of multiple organ failure in indian intensive care unit patients cerebral malaria uk malaria treatment guidelines do patients with cerebral malaria have cerebral edema? a computed tomographic study chemotherapy for chagas' disease: a perspective of current therapy and considerations for future research report of a case with arteritis clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose malawian children effi cacy of combination of dfmo and diminazene aceturate in the treatment of late-stage trypanosoma brucei infection in rats ischemic cerebral changes in the chronic chagasic cardiopathy pathology of human african trypanosomiasis with reference to experimental african trypanosomiasis and infections of the central nervous system spinal toxocara abscess treatment of severe malaria by exchange transfusion entzündliche erkrankungen des nervensystems protozoal infections. in: noseworthy jh (ed) neurological therapeutics -principles and practice secondary cerebral amebiasis due to infection with entamoeba histolytica eosinophilic meningitis and radiculomyelitis in thailand, caused by cns invasion of gnathostoma spingerum and angiostrongylus cantonensis overwhelming strongyloidiasis: an unappreciated opportunistic infection trypanosoma cruzi-triggered meningoencephalitis is a ccr1/ccr5-independent infl ammatory process naegleria and acanthamoeba. free-living amebas pathogenic for man acute psychosis after mefl oquine difl uoromethylornithine, an eff ective new treatment of gambian trypanosomiasis dihydroartemisinin-piperaquine against multidrug-resistant plasmodium falciparum malaria in vietnam: randomised clinical trial specifi c chemotherapy of chagas disease : controversies and advances diagnosis of african and american trypanosomiases the course of seizures after treatment for cerebral cysticercosis dexamethasone proves deleterious in cerebral malaria. a double blind trial in 100 comatose patients praziquantel in treatment of cerebral schistosomiasis single-dose phenobarbitone prevents convulsions in cerebral malaria successful chemotherapy of transfusion babesiosis managment of servere malaria: a practical handbook fatal cerebral mycoses caused by the ascomycete chaetomium strumarium rapid diagnosis of candidiasis and aspergillosis fungal infections of the cns: treatment strategies for the immunocompromised patient coccidioidal meningitis ct scanning in rhinocerebral mucormycosis and aspergillosis magnetic resonance imaging and angiography in cerebral fungal vasculitis combined activity in vitro of caspofungin, amphothericin b, and azole agents against itraconazloe-resistant clinical isolates of aspergillus fumigatus central nervous system infections following bone marrow transplantation: an autopsy report of 27 cases prognostic factors in cryptococcal meningitis. a study of 111 cases activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin b against 448 recent clinical isolates of fi lamentous fungi treatment of cryptococcal meningitis with combination amphotericin b and fl ucytosine for four as compared with six weeks overview of the lipid formulations of amphotericin b cryptococcal infection in patients with acquired immune defi ciency syndrome unsuccessful treatment with voriconazole of a brain abscess due to cladophialophora bantiana meningoencephalitis due to blastomyces dermatitidis: case report and literature review mri fi ndings in an immunocompromised boy with cns fungal infection fungal meningitis new antifungal drugs and new clinical trials: interpreting results may be diffi cult voriconazole versus amphotericin b for primary therapy of invasive aspergillosis report of a fatal case and analysis of its confounding factors survival improvement in coccidioidal meningitis by high-dose intrathecal amphotericin b craniofacial mucormycosis: computed tomographic and angiographic fi ndings in two cases rhinocerebral phycomycosis and internal carotid artery thrombosis intraventricular cryptococcal cysts masquerading as racemose neurocysticercosis magnetic resonance imaging of cerebral aspergillosis systemic antifungal therapy central nervous system aspergillosis in patients with human iummuno-deffi ciency virus infection caspofungin: a new therapeutic option for fungal endocarditis oral versus intravneous therapy in the treatment of systemic mycosis systemic fungal infections in hematologic neoplasms. an autopsy study of 1,053 patients syndrome of the anterior spinal artery as the primary manifestation of aspergillosis coadministration of voriconazole and phenytoin: pharmacokinetic interaction, safety, and toleration the pharmacokinetics and safety of intravenous voriconazole -a novel wide-spectrum antifungal agent primary central nervous system phaeohyphomycocis: a review of 101 cases potential fungicidal eff ect of voriconazole against candida spp cerebral pseudallescheria mycosis after neardrowning mycoses central nervous system complications in renal transplant recipients in a tropical environment entzündliche erkrankungen des nervensystems fungal infections. in: noseworthy jh (ed) neurological therapeutics-principles and practice. informa healthcare treatment of cryptococcal meningitis with combination amphotericine b and fl ucytosine, high dose and long duration is cryptococcal meningoencephalitis in the tropics a distinct entity? a retrospective study from thailand poor effi cacy of amphotericin b-based therapy in cns aspergillosis cerebral mucormycosis: proton mr spectroscopy and mr imaging practice guidelines for the treatment of fungal infections emergence of invasive cerebral aspergillosis in an hiv-positive patient on voriconazole therapy novel eff ect of voriconazole on conidiation of aspergillus species cerebellar and medullar histoplasmosis economic evaluation of voriconazole compared with conventional amphotericin b for the primary treatment of aspergillosis in immunocompromised patients a new era of antifungal therapy systemic antifungal therpay: new options, new challenges neuroimaging and neurologic complications after organ transplantation n. tibialis links / cz'-fz 10 tibialis rechts / cz'-fz 10.0 ms/div, 1.00 uv/div n. tibialis rechts / cz'-fz 10 motorischer cortex / rechtes bein 10.0 ms/div, 1000.0 uv/div, 2/0, 0ma re »vertebral osteomyelitis in northern spain »vertebral osteomyelitis in göteborg, sweden: a retrospective study of patients during 1990-95 »vertebral osteomyelitis at a norwegian university hospital 1987-97: clinical features, laboratory fi ndings and outcome »prospective study of patients presenting with acute partial transverse myelopathy »acute myelopathies. clinical. laboratory and outcome profi les in 79 patients »idiopathic transverse myelitis. corticosteroids, plasma exchange, or cyclophosphamid »paraneoplastic neurological syndromes » 14-3-3 protein in the cerebrospinal fl uid of patients with acute transverse myelitis »prognostic predictors of acute transverse myelitis »neurophysiological studies in acute transverse myelitis »is methyl prednisolone useful in acute transverse myelitis? »transverse myelitis: pathogenesis, diagnosis and treatment the neurology of liver failure »viral meningitis poliomyelitic-like illness in central european encephalitis proposed diagnostic citeria and nosology of acute tranverse myelitis »the spectrum of neuromyelitis optica die jeweilig bestmögliche spezifi sche chemotherapie:. tab. 35 der antigennachweis mittels pcr ist eine schnelle und zuverlässige methode. sie kann insbesondere in der frühphase einer infektion, wenn die humorale antikörperantwort noch unzureichend ist, wichtige informationen liefern. autoimmune entzündungen weisen meist nur eine leichte pleozytose (<100 zellen/μl), aber auch schrankenstörungen und eiweißerhöhungen auf.bei der multiplen sklerose fi nden sich bei über 80% der erkrankten oligoklonale banden im liquor.die neuromyelitis optica ist bei über 70% der patienten spezifi sche antikörper gegen aquaporin 4 (nmo-igg) im serum assoziiert [20] .die routine-labordiagnostik mit kleinem blutbild und creaktivem protein (crp) ist bei isolierten spinalen prozessen teilweise nicht richtungweisend und weist oft mals in der initialphase keine oder nur geringe entzündungszeichen auf. dennoch kann die crp-erhöhung bei bakteriellen spinalen entzündungen ein unspezifi scher hinweis sein, der dann eine detaillierte diagnostik nach sich ziehen sollte.besteht der verdacht einer systemischen entzündung aus dem kreis der kollagenosen, rheumatischen erkrankungen und der vaskulitiden, ist der nachweis spezieller serologischer antikörper häufi g hilfreich.vaskulitiden können häufi g erst durch die histologische aufarbeitung von gefäß-und/oder nerven-bzw. muskelbiopsaten und immunhistochemischer färbung diagnostiziert werden.die diagnostik der funktionellen schädigung des nervensystems kann durch elektrophysiologische untersuchungen (v. a. somatosensibel und motorisch evozierte potenziale) sinnvoll ergänzt werden und besitzt in der abschätzung der prognose einen hohen stellenwert (. abb. 35.8). aus klinischer sicht muss bei akuten sensomotorischen ausfällen eine akute polyradikulitis (guillain-barré-syndrom) in betracht gezogen werden. die abgrenzung zur myelitis gelingt meist durch den typischen liquorbefund einer »zytalbuminären dissoziation« -mit erhöhung des liquorgesamteiweiß bei normaler zellzahl (7 kap. 40.1).auch medulläre tumoren (gliome, ependymome, sarkome, lipome, lymphome, abtropfmetastasen) müssen in die diff erenzialdiagnostischen überlegungen einbezogen werden. aber auch paraneoplastische myelopathien (z. b. beim bronchialkarzinom und m. hodgkin) sind beschrieben [8] .eine strahlenmyelopathie kann bei bestrahlungsdosen ab 20 gy mit einer latenz von mehreren wochen bis monaten und jahren als akute inkomplette bis komplette querschnittssymptomatik auft reten.zu den vaskulären spinalen syndromen zählen v. a. die spinale ischämien (z. b. nach aortenoperationen oder aortendissektion) und spinale arteriovenöse malformationen, angiome, kavernome und durale fisteln. letztere gehen häufi g mit einer venösen stauung und blutungen einher.metabolische myelopathien die akut bis subakut verlaufen können sind insbesondere die funikuläre myelose bei vitamin-b 12 -mangel und die hepatische myelopathie bei leberinsuffi zienz [14] .schwierigkeiten können bei der unterscheidung einer erregerbedingten von einer parainfektiösen myelitis auft reten. bei letztgenannten wird häufi g ein symptomfreies intervall zwischen der vorausgegangenen infektion und der myelitis beschrieben.die extramedullären entzündungen müssen von chronischentzündlichen rheumatischen wirbelsäulenerkrankungen abgegrenzt werden [18] . gedacht werden muss an die rheumatoide arthritis und die seronegative spondylarthropathie . zu den letztgenannten zählt man die ankylosierende spondylitis (m. bechterew), psoriasisarthropathie, enteropathische arthropathie, reaktive spondylarthropathie und als sonderform den m. reiter. diagnostisch hilfreich ist bei den chronisch-entzündlichen rheumatischen wirbelsäulenerkrankungen der nachweis 3 tab. 35 im vordergrund der medikamentösen th erapie steht immer der gezielte einsatz der antibiotika bzw. virustatika. die auswahl der präparate erfolgt entsprechend den ergebnissen der blut-und liquorkulturen bzw. punktatergebnissen (antibiogramm anfordern!) und den serologischen bzw. immunologischen resultaten.bei subakut oder chronisch verlaufenden erkrankungen sollte, wenn es die klinische situation zulässt, zunächst eine gezielte diagnostik möglichst mit erregerisolation und ggf. diff erenzialdiagnostischer aufarbeitung angestrebt werden.bei bakteriellen abszessen muss immer (soweit unter anatomischen und funktionellen gesichtspunkten möglich) zusätzlich zur antibiotischen th erapie eine (neuro)chirurgische herdsanierung diskutiert und individuell entschieden werden. auch wenn es für die th erapie der idiopathischen akuten transversen myelitis (iatm) keine randomisierten, placebokontrollierten untersuchungen gibt, die den einsatz einer kortisontherapie sicher positiv bewerten ( [12, 13] , wird in analogie der behandlung anderer entzündlicher erkrankungen und der klinischen erfahrung häufi g eine 3-bis 5-tägige intravenöse kortisonstoßtherapie mit 500-1000 mg methylprednisolon durchgeführt. klinisch schwer betroff ene patienten können evtl. auch von einer aggressiveren th erapie mit cyclophosphamid und plasmapherese profi tieren [7] . prognostisch ungünstige faktoren sind sowohl ein anfänglich rasch progredienter verlauf und ein andauern der neurologischen ausfälle über 3 monate [19] . auch der nachweis von protein 14-3-3 im liquor , als zeichen der neuronalen schädigung [9] , wie auch pathologische motorisch und sensibel evozierte potenziale, aber auch denervierungszeichen im emg sprechen für eine eher ungünstigen verlauf [10, 11] . 30−50% der patienten mit einer atm haben ein schlechtes outcome, mit bleibender schwerer behinderung, wobei die prognose bei multipler sklerose besser ist als bei patienten mit anderen ursachen eines querschnittsyndroms [6] .die prognose der spondylitis bzw. spondylodiszitis und spinaler abszesse hängt von dem ausmaß und der dauer einer schädigung nervaler strukturen ab. der entscheidende faktor ist daher die frühzeitige diagnose und th erapie. key: cord-260604-lz1qd69t authors: singh, ramendra k.; yadav, dipti; rai, diwakar; kumari, garima; pannecouque, c.; de clercq, erik title: synthesis, structure–activity relationship and antiviral activity of 3′-n,n-dimethylamino-2′,3′-dideoxythymidine and its prodrugs date: 2010-09-30 journal: european journal of medicinal chemistry doi: 10.1016/j.ejmech.2010.05.028 sha: doc_id: 260604 cord_uid: lz1qd69t abstract a probable nrti molecule, viz. 3′-n,n-dimethylamino-2′,3′-dideoxythymidine (4) and its 5′-o-carboxyl ester prodrugs – 5′-(n-α-boc-l-phenylalanyl)-3′-n,n-dimethylamino-2′,3′-dideoxythymidine (5), 5′-l-phenylalanyl-3′-n,n-dimethylamino-2′,3′-dideoxythymidine (6) and 5′-decanoyl-3′-n,n-dimethylamino-2′,3′-dideoxythymidine (7) have been synthesized and screened against hiv, hsv-1 and 2, parainfluenza-3, vesicular stomatitis and several other viruses. the compound 6 showed good antiviral activity with ec50 value 0.03μm (si=8) against vsv in hela and hel cell lines. however, the lead compound 4 and its derivatives 5, 6 and 7 showed no remarkable activity against hiv-1 and other viruses. molecular docking studies with hiv-1 rt using ds 2.5 and pymol softwares have shown marked differences in the interaction patterns between the lead compound 4 and azt. hiv-1 reverse transcriptase (hiv-1 rt), a primary target for developing anti-hiv drugs, is the enzyme responsible for generating linear dsdna from ssrna e the genetic material of hiv-1. this dsdna acts as a provirus and leads to viral expression through integration in the host genome, transcription and translation processes. there are several drugs that inhibit hiv-1 rt and they are classified as nucleos(t)ide rt inhibitors (n(t)rtis) and non-nucleoside rt inhibitors (nnrtis). the nrtis have emerged as important therapeutic agents for the development of anti-hiv drugs [1e4] . the nrtis after intracellular phosphorylation to their active triphosphate form by cellular kinases [5] , act as competitive inhibitors with respect to the dntp substrates. these molecules when incorporated by hiv-1 rt in the growing template/primer, cause chain termination [6, 7] since they lack 3 0 -oh group, which is necessary for chain elongation and thus, they inhibit hiv-1 rt [8, 9] . despite their tremendous utility, nucleoside analogues may cause serious cellular toxicity by interacting with human dna polymerase g [10] , present in mitochondria, which results in mitochondrial dysfunctions. thus nrtis, show many side effects [11e13] . in addition to this, four more significant obstacles currently limiting the clinical application of nucleoside analogues are oral absorption, cellular uptake, short halflife and viral resistance [14] . so, there is a need for new nucleoside mimics and their prodrugs to overcome the limitations. we have already reported several nucleosidic [15, 16] and non-nucleosidic molecules [17] with significant antiviral activities. the nrtis, by virtue of acting as chain terminators of (à) strand dna synthesis during reverse transcription, have been developed as potential drugs against hiv/aids. in order to explore such new inhibitors, we report here the synthesis of 3 0 -n,n-dimethylamino-2 0 ,3 0 -dideoxythymidine (dmat) and its 5 0 -o-ester prodrugs with amino and fatty acids. the lead molecule, dmat, was designed keeping azt, an approved nrti drug against hiv/aids, in mind, where azido group has been replaced by dimethylamino function, fig. 1 . thus, this molecule being a dideoxynucleoside is expected to act as nrti against hiv-1 rt enzyme. similarly, the prodrugs, having biodegradable ester linkages, are supposed to enhance the cellular uptake due to their high lipophilic nature. all these molecules have been evaluated for their activity against various viruses, like hiv-1, vesicular stomatitis, hsv-1 & 2, reovirus, parainfluenza virus and many others. q work presented in nucleic acids symposium (series no. 52) held in japan (ref. [37] ). a new thymidine analogue, viz. 3 0 -n,n-dimethylamino-2 0 ,3 0dideoxythymidine has been synthesized from thymidine as shown in scheme 1. 5 0 -o-(4,4 0 -dimethoxytrityl)-3 0 -mesylthymidine, obtained after 5 0 -oh selective protection with dmtcl followed by mesylation with mscl, when reacted with potassium phthalimide witnessed the introduction of nucleophile (phthalimide ion) into the sugar moiety [18] . during conversion of 1e2, the elimination of c3 0 -mesyloxy group was effected through intramolecular displacement by attack of carbonyl group of thymidine at c2, which resulted in formation of 2,3 0 -anhydronucleoside intermediate. this result was well in accordance with the literature data. it is suggested that under this condition, phthalimide ion is introduced into the 'down' (ribo) configuration [19] . the 3 0 -phthalimide derivative 2 was treated with methanolic methylamine, which through methanolysis and aminolysis resulted in formation of compound 3 [20] . reaction of 3 with formic acid and aqueous formaldehyde (40%) yielded 4. in this reaction, formaldehyde used was the source of methyl groups, while formic acid supplied the hydrogens involved in the reduction. this treatment also removed the dmt group from compound 3. as expected, the uv absorption spectra of all intermediates were similar to that of thymidine, while dissimilar r f values showed that all these changes took place in the sugar moiety and not in the aglycon part [19] . all compounds 4e7 have been screened for their antiviral potential against hiv and several other viruses and the results are shown in table 1 . the lead compound dmat and its prodrugs were found inactive against hiv-1 rod and hiv-1 iiib strains and showed toxicity. compounds 4 and 5 have showed similar values of cc 50 and ec 50 against all viruses studied and thus no activity was observed, while 5 0 -l-phenylalanyl derivative 6, having free amino function was potentially active at an ec 50 of 0.03 mm against vsv in hela and hel cell cultures. the ec 50 value of compound 6 has been found to be eightfold lower than its cc 50 value (si ¼ 8). besides this, it has also showed moderate activity at an ec 50 of around 0.05 mm against hsv-1 & 2, coxsackie b4, and rsv in hela cell culture. at the same ec 50 value, it was found active against vv, tk hsv, in hel cell lines; piv-3, rv-1, sv and ptv in vero cell culture and ecv (fipv) and fhv in crfk cell culture. the 5 0 -decanoyl derivative 7 has shown some activity against fcv (fipv) and fhv in crfk cell culture. all compounds were also tested against influenza a h1n1/h3n2 subtype and influenza b in mdck cell lines. however, none of these compounds was able to inhibit cytopathic effects of influenza a or b virus at subtoxic concentrations or the highest concentration tested. it was observed that compounds 6 and 7 only, showed antiviral properties against vsv (ec 50 0.03) and fcv (ec 50 0.05), respectively. however, the antiviral potency of the prodrug 6 was still significantly lower than that of established antiviral drugs e (s)-dhpa, bvdu and ribavirine against vsv as was evident from its cc 50 and ec 50 values shown in table 1 . since nucleosides exhibit only modest bioavailability [21] , we prepared the prodrug molecules using amino and fatty acids to improve their bioavailability [22, 23] and chemical stability. this was done to ensure enhanced cellular uptake and sustained release of drug molecule without compromising its properties [24e27]. the lead compound 4 and its prodrug 5 did not show any antiviral activity, whereas the prodrug 7 showed activity, albeit, too little against fcv and fhv. however, the prodrug 6 showed good activity against vsv with si value equal to 8. the enhanced reactivity of free amino acid ester prodrug may be attributed to its electronic activation by the positively charged amino terminus at physiological ph, which derives support from the fact that l-amino acid esters enhance nucleoside absorption via human peptide transporters (hpept1) of active transport system [28, 29] . furthermore, since bone marrow progenitor cells lack this active transport system for amino acid, prodrug 6 is expected to show reduced toxicity at bone marrow level. therefore, compound 6 has potential to be developed as an effective antiviral agent against vsv. the lead compound 4, being a 2 0 ,3 0 -dideoxynucleoside, was expected to act as chain terminator and thus, interfere with viral cell metabolism and its prodrugs were designed and synthesized to enhance its cellular uptake. however, this molecule showed almost no anti-hiv activity and rather proved cytotoxic. this might be dmat : r 1 = tyrosynyl, lysinyl, phenylalanyl, valyl etc. r 1 = decanoyl, myristoyl, pamitoyl etc. expected because of introduction of dimethylamino group at 3 0 -c of ribose moiety, which did not show any interaction with hiv rt during molecular docking experiments. the lead molecule 4 was designed because of its similarity with azt, but it completely failed in fulfilling the objectives. it was surprising to see that replacement of azido group by dimethylamino group turned the molecule highly toxic. the fact that the lead molecule dmat (and its prodrugs) being a dideoxynucleoside and having structural features similar to azt, did not show any activity against hiv and rather proved toxic, prompted us to study its interaction with hiv-1 rt. 1 we performed elaborate molecular computational studies using the software discovery studio (ds) 2.5. this molecule showed some striking differences with azt e the approved anti-hiv drug as shown in table 2 . the physiochemical studies suggested that both the molecules e azt and dmat, followed lipinski 0 s rule of five but they have marked differences in their molecular volume, total polar surface area (tpsa) and log p values. similarly, the docking experiment also showed quite different patterns of their interaction with hiv-1 rt (figs. 2e4) . although dmat formed one more h-bond with hiv-1 rt than azt, the crucial interactions as shown by azt were missing in the case of dmat. the azido function on azt formed three h-bonds e one with arg72 and two with asp113 (the amino acid constituting the dntp-binding site), whereas the dimethylamino function on dmat formed no h-bonds at all. similarly, the h-bond formed by phosphate moiety of azt (and ttp as well) with val111 was missing in the case of dmat. however, the aglycan part in both the molecules formed two h-bonds with arg72 through oxo group and "o" atom in the sugar ring. the stability of dmattpert complex was also less than that of azttpert or ttpert complexes as is evident from their minimization energies. a comparative account of interaction of ttp, azttp and dmattp and stability of their respective complexes with hiv-1 rt has been shown in table 3 . thus, the physiochemical and docking studies suggested the possible reasons for inactivity of the lead molecule and its prodrugs. in conclusion, we have synthesized 3 0 -n,n-dimethylamino-2 0 ,3 0 -dideoxythymidine 4 and its 5 0 -o-carboxyl ester prodrug derivatives 5e7 and evaluated their antiviral activity against hiv-1 (iii b and rod strains), hsv-1/2, vesicular stomatitis, parainfluenza-3 virus and many others. it was observed that compounds 6 and 7 only, showed moderate antiviral properties against vsv (ec 50 0.03) and fcv (ec 50 0.05), respectively. further mechanistic studies on molecules 6 and 7 are in progress against vesicular stomatitis, feline corona and feline herpes viruses. the inference drawn from the present studies that for a nucleoside analogue to be an effective nrti, being a dideoxy derivative is not sufficient enough criterion and rather it must possess the characteristic features too for requisite interactions at the dntpbinding site of hiv-1 rt enzyme shall definitely help in designing new nrti molecules. chemicals were obtained from e. merck india ltd, india and sigmaealdrich chemical company, usa. melting points were determined on electrothermal apparatus and are uncorrected. silica gel for tlc and column chromatography (60e120 mesh) was obtained from e. merck india ltd. uv measurements were carried out on hitachi 220s spectrophotometer. hplc analyses were performed using 250 â 4.6 mm 2 c18 column and solvent system meoh:h 2 o (6:4) at a flow rate of 1 ml/min on 6 ad binary gradient shimadzu hplc system. 1 h nmr spectra were recorded on drx 300 mhz instrument using dmso-d 6 as solvent and tms as an internal standard. 13 c nmr spectra were recorded in cdcl 3 on a varian xl-300 spectrometer operating at 75 mhz. mass spectra were obtained using a thermofinnigan trace-dsq electrospray ionization (esi) mass spectrometer. elemental analyses were carried out on a perkineelmer 240-c analyzer. all solvents were dried and distilled prior to use. thymidine (2.415 g, 10 mmol) was suspended in dry pyridine (w100 ml) in a 250 ml round bottomed flask and added dmap (61 mg, 0.05 equivalent), tea (1.9 ml, 1.4 equivalent) and dmtcl (4.1 g, 1.2 equivalent) and stirred the reaction mixture for 2.5 h. tlc at this point showed complete disappearance of thymidine. the contents were cooled to 0 c and methanesulfonyl chloride (1.23 ml, 10.89 mmol) was added dropwise. stirring was continued further for 2 h and the reaction mixture was allowed to warm up to 25 c. after stirring the reaction mixture for an additional 30 min, the contents were poured into crushed ice/water and the viscous mass so obtained was extracted in chloroform (50 ml) and washed with water (2 â 10 ml). the organic phase was dried over anhydrous sodium sulfate, filtered and evaporated on a rotary evaporator to obtain the crude product. silica gel column purification with a linear gradient up to 10% ethyl acetate in hexane, afforded the pure compound, 5 0 -o-(4,4 0 -dimethoxytrityl)-3 0mesyl-2 0 ,3 0 -dideoxythymidine, which was refluxed for 12 h with potassium phthalimide (9.0 g) in dimethylformamide (450 ml). the solvent was removed under reduced pressure and the residue extracted with ethyl acetate. the ethyl acetate layer was finally washed with water (3 â 10 ml), dried over anhydrous na 2 so 4 and concentrated to get the title compound in crude form (4.72 g, 70%). the compound 2 (4.34 g) was stirred with methylamine (16 ml) in methanol at 105 c for 20 h. the reaction mixture was evaporated to an oily residue, washed with water, dissolved in ethanol (100 ml) and treated with hydrochloric acid to neutralize the residual methylamine. the acidic solution was concentrated under reduced pressure, which resulted in crystallization. the crystals were separated and the residual water and acids were removed by co-evaporation with benzene under reduced pressure from the mother liquor, which was further subjected to crystallization. the crystalline residue was triturated with ethanol and ether. the title compound was obtained as colorless crystals (3.04 g, 87%). dicyclohexylcarbodiimide (515 mg, 2.5 mmol) was added to a stirred solution consisting of 3 0 -n,n-dimethylamino-2 0 ,3 0dideoxythymidine (269 mg, 1 mmol), dmap (183.25 mg, 1.5 mmol,) and n-a-t-boc-l-phenylalanine (397.95 mg, 1.5 mmol) in pyridine (10 ml) under anhydrous condition. the progress of reaction was monitored by tlc. after the reaction was complete (70e72 h), the separated dcu was filtered off. the filtrate was evaporated to dryness under reduced pressure and the title compound was purified by column chromatography over silica gel using a mixture of ethyl acetate/hexane (8.5:1.5) as eluent. yield: (269 mg, 62%); mp 97 c; r f : 0.46 (dcm:meoh 9.5:0.5); uv (etoac) l max 265 nm. c18 hplc (265 nm) t r ¼ 2.24 min; 1 h nmr (dmso-d 6 ); d 1.23 (s, 9h, t decanoyl chloride (0.20 ml, 1 mmol) was added dropwise to an ice-cold stirred solution consisting of the lead compound 4 (269 mg, 1 mmol), dmap (0.32 g, 2.6 mmol) in pyridine (25 ml) and the reaction mixture stirred overnight at room temperature under anhydrous condition. reaction mixture was concentrated under reduced pressure and partitioned between ethyl acetate and water. the organic fraction was concentrated to a residue and the product was purified by column chromatography over silica gel using ethyl acetate/hexane (9.5:0.5) as eluent. yield: (200 mg, 59%) mp 160 c; 10.04 (s, 1h, nh, thy); 13 cell cultures were prepared in microtiter trays and inoculated with 100 ccid50 (1 ccid50 corresponding to the virus stock dilution that proved infective for 50% of cell cultures). after 1 h virus adsorption to the cells at 37 c, the residual virus was replaced by cell culture medium (eagle minimum essential medium supplemented with 3% fetal calf serum) and various concentrations of the test compounds. virus cytopathogenicity was recorded as it reached completion in the untreated virus-infected cell cultures, i. e., at 1e2 days for vesicular stomatitis virus; 2 days for coxsackie; 2e3 days for vaccinia, herpes simplex type 1 and 2 and sindbis; 4 days for respiratory syncytial virus and 6e7 days for reo and parainfluenza viruses [33, 34] . the antiviral activity of compounds is expressed as ec 50 (the concentration (mm) required to inhibit virusinduced cytopathogenicity by 50%). cytotoxicity of all compounds was assessed on the basis of two parameters: (i) alteration of normal cell morphology, and (ii) inhibition of macromolecule (dna, rna and protein) synthesis. cytotoxicity (cc 50 ) of compounds was examined by trypan blue exclusion test. to evaluate cytotoxicity, uninfected confluent cell cultures treated with various concentrations of test compounds were incubated in parallel with virus-infected cell cultures prepared in plastic trays containing 24 wells (16 mm diameter; falcon plastics). after 2 days of incubation at 37 c in a co 2 incubator, when the cell cultures were confluent, culture medium was removed from each well and 1 ml of maintenance medium containing serial concentrations of the test compounds was added. for cell control, 1 ml of maintenance medium without compound was added. all cultures were incubated at 37 c, and after 2 and 7 days of incubation, compounds were withdrawn and the viability of the cells was determined by the trypan blue exclusion method. antiviral screening against hiv-1 (iiib and rod strains) was monitored by the efficiency of test compounds to inhibit syncytia formation after hiv infection of mt-4 cells following the mtt method molecular docking of compound 4 and its prodrugs 5, 6 and 7 into the dntp-binding site of hiv-1 rt was carried out using ds 2.5 software and pdb code 3e01 of hiv-1 rt. cdocker was used and flexible docking was done. stimulated annealing was done to get the energy-minimized structures of the molecules. the energy-minimized forms were then docked into hiv-1 rt. visualization was done using pymol software. all molecular modeling studies were performed on an intel pentium 2.99 ghz processor, 1.99 gb ram with windows xp professional version 2002 operating system. and references therein proceedings of the 4th international workshop on hiv drug resistance & treatment strategies drugs, microbes, host e the elements of chemotherapy and references therein financial assistance from the department of biotechnology (dbt) and indian council of medical research (icmr), new delhi, government of india for carrying out this work is sincerely acknowledged. key: cord-254187-dcdc6sqi authors: kimball, am; thant, myo title: “what, me worry?” businesses and aids at davos date: 2005-04-05 journal: lancet doi: 10.1016/s0140-6736(05)74792-9 sha: doc_id: 254187 cord_uid: dcdc6sqi nan at the davos summit in february, 2005, the world economic forum released its current survey on businesses and hiv/aids. 1 in 1996, we outlined the case for business in asia to play a pivotal role in preventing the epidemic. 2 at that time, an estimated 3 million people in asia had been infected with hiv. today that number is estimated at 8·2 million. 3 aids is a preventable disease. the survey finds an absence of alarm in the business sector which, if true, bodes poorly for success in controlling this epidemic. beyond the tragic human costs, the economic and financial tolls of this epidemic are well known. costs to employers include: increased insurance premiums, larger expenditure on welfare benefits, decline in productivity, and increases in hiring and training costs. loss of skill base, institutional knowledge, and potential conflict in the workplace because of stigmatisation are more difficult to measure. at the macro level, the business environment, including markets and disposable income of consumers, savings rates, interest rates, and general education of the labour force, are put at risk. the findings in the survey from 8000 firms worldwide are a subset of global business surveyed annually by the world economic forum. firms appear less concerned this year than last year. this lack of concern is accompanied by conjecture rather than science as a basis for decision-making. informal policies prevail except in high-prevalence areas such as sub-saharan africa. why such complacency? the global epidemic has shown its inexorable nature in community after "what, me worry?" businesses and aids at davos pregnancy to any antiasthmatic drug), there is still much be learned. this need for information is particularly the case for systemic glucocorticosteroids 6 and for recently introduced medications, such as salmeterol, formoterol, and leucotriene-receptor antagonists. one negative point is that, when systemic glucocorticosteroids are administered during the first trimester, the risk of an orofacial cleft is increased by about 3, although the power of the studies is poor and many confounding factors persist. one positive point is that more than 11000 women have been exposed to inhaled corticosteroids (mostly beclomethasone and budesonide) during pregnancy with no increase of outcomes in the infant (including congenital malformations). 2 the higher reported risk of hypertension and pre-eclampsia in steroid-treated pregnant women is not related to the drugs but to uncontrolled asthma. 8 thus, because many clinical trials on inhaled corticosteroids have shown efficacy in pregnant asthmatic women in terms of symptoms, lung function, and reduction of asthma exacerbations, maintaining asthma control during pregnancy is the goal; and, when the indication is clear (eg, severe asthma exacerbation), the benefit of systemic glucocorticosteroids is far greater than the risk. the main message of the update 2 is that it is safer for the asthmatic pregnant woman to be treated with asthma drugs than to suffer from asthma symptoms and exacerbations, and that obstetricians and asthma-care providers should work together. guidelines for asthma during pregnancy should not differ from the guidelines for all asthmatic patients, but fetal toxicity should be monitored and an evidence-based search for drug treatment should be continued. community, country after country; will it now be business after business? in asia, the prospective new epicentre of the epidemic, the efforts of the thailand business coalition on aids and the tata group in india highlight roles business can play: prevention and education for workers; workplace programmes to prevent discrimination; and public-private collaboration and funding for effective programmes. the thailand business coalition on aids offers a range of tools for businesses to assure good-quality programming at all organisational levels. the tata group has focused on a full range of workplace programmes to ensure the sexual health of their employees, including an emphasis on nondiscrimination. leadership at the top of firms, coupled with government commitment, is key. the most populous countries in the global community, china and india, are now in the cross-hairs. projections for india are alarming: 20-25 million people living with hiv by 2010, and a potential of 50 million cases by 2015. 4 the epidemic is driven by commercial sex-work, and clients include businessmen who could benefit from education programmes in the workplace. there are peer-to-peer strategies that are effective in condom promotion. 5 thai experience suggests that also addressing the clients ensures success. workplace programmes to secure the safety of the workforce would be a valuable contribution at this critical time. many economies are rural in asia; the effects of hiv on the household level are profound. 6 the health infrastructure in china is crumbling, 7 especially in rural areas, even as market reforms have created unprecedented economic growth. the business sector in china should lead the reinvigoration of preventive and health services. the march 18 summit of the business coalition on aids, in beijing, was a perfect launch. 8 the expansion of access to antiretroviral treatment in asia will not be a panacea. us experience shows that the reprieve from hiv is not forever, efficacy is not 100%, and people living with hiv face many obstacles in returning to work. 9 from a business perspective, proactive prevention coupled with compassionate policies in the workplace and coverage of treatment through insurance is the wisest choice. there is a gulf between the public and private sectors that must be bridged in the fight against aids and other emerging infections. the urgency of robust public-private partnering is clear in the asia-pacific region, where the threat of new human infections, such as severe acute respiratory syndrome and avian influenza, challenge systems that have not received adequate investment to scale-up to successfully meet these new threats. the economics of intervention by the private sector are not always self-evident. much depends on the ability of the public sector to induce private firms to act for the common good. market incentives, such as tax breaks and subsidies, can lower the cost of training and help businesses provide information and education for workers. the reduction in duties and taxes on imports of equipment and reagents could reduce drug costs. the cornerstone of partnership is for each sector to understand the needs and motivations of the other. the world economic forum's survey is a pioneering work in progress. the kind of information it is positioning itself to capture is very important in the fight against the hiv epidemic. business has brought innovation, energy, and investment in creating value in economies across the globe. the ongoing struggle against hiv and other emergent infections requires no less. in south africa, where less than 3% of people who need antiretroviral treatment actually get it, 1 private-sector companies are stepping in to fill the need. a growing number are supplying such drugs directly to employees who are hiv-positive or are facilitating access to drugs via third parties such as non-governmental organisations. while businesses in the rest of the world are becoming less concerned about aids, the contradictory trend in south africa is being driven, on the face of it, by two imperatives. the first is heightened social awareness in the postapartheid society. the second is more bankable: an often-compelling economic argument. behind these two driving factors, however, lies the darker reality of the south african government's slow and reluctant concession that it needed to intervene with drugs in the country's aids crisis, and its underperformance in rolling-out treatment with antiretrovirals since being pressed into doing so by, among other things, court rulings. this is where, increasingly, private companies are stepping in. "the corporate sector is shouldering more responsibility for the health of its workforce than it ever has in the past", says bernie clark of the health-care consulting team of alexander forbes, a firm that advises businesses on employee benefits and has helped several companies to develop aids policies (clark b, alexander forbes, johannesburg, south africa, personal communication). about aids, specifically, clark adds: "the perception is that the state is doing precious little to assist the formal work sector to stay at work." so the lesson is that necessity is the mother of drugs' provision. the unadorned bottom-line is that it is cheaper for a firm to supply antiretrovirals than to have an untreated aids patients die while on the payroll. the bottom-line is especially true when the prevalence of hiv positivity runs as high as 30% 2 -as it does in many of south africa's mining companies which, not surprisingly, have pioneered inhouse aids treatment. for the same reasons of prevalence and financial reckoning, it may be that south african firms are presently leading where others elsewhere in the world will in time follow. a leading example of aids management in asia is thailand, where strong political backing for awareness and prevention campaigns has held the infection rate to 1·5% 3 -compared with south africa's 22%. 4 in thailand, the thailand business coalition on aids has spawned its own umbrella organisation in the asian business coalition on aids. the coalitions' activities, though, are limited to education and prophylaxis, and do not include inhouse treatment. 5 the most recent survey of the world economic forum's global health initiative 6 shows that awareness by business that aids will affect operations and profits reflects the level of efforts to combat the disease. south african business leaders are well ahead of the rest of the world at 72% awareness; south and south-east asia, where the thai and asian coalitions are active, lags at 37%. then the levels fall off even more dramatically: in east asia, where, in china, one of the most worrying prevalences is developing, awareness is at a low 21%, as it is in latin america. 6 the global health initiative worked with several south african firms to organise case studies, which vividly illustrate the imperatives and benefits for companies offering antiretrovirals to their employees. gold fields, for example, a mining house with 48 000 mainly low-skilled workers in south africa, calculated that-with one in three of its miners hiv positive-failure to intervene would lead to losses of up to 10% of 2002 earnings by the year 2006. we declare that we have no conflict of interest. global business surveybusiness and aids: commitment and action? world economic forum a role for business in hiv/aids in asia fact sheet: asia modelling hiv/aids epidemics in bostwana and india india's hiv epidemic the economic burden of illness for households in developing countries: a review of studies focusing on malaria, tuberculosis, and human immunodeficiency virus/acquired immunodeficiency syndrome china must prioritise health opportunities for all ministry of health and global business coalition on hiv/aids joint summit on business and aids in china perceived barriers to employment among persons living with hiv/aids key: cord-001228-4eh22ek7 authors: ofori, leslie o.; hilimire, thomas a.; bennett, ryan p.; brown, nathaniel w.; smith, harold c.; miller, benjamin l. title: high-affinity recognition of hiv-1 frameshift-stimulating rna alters frameshifting in vitro and interferes with hiv-1 infectivity date: 2014-01-05 journal: j med chem doi: 10.1021/jm401438g sha: doc_id: 1228 cord_uid: 4eh22ek7 [image: see text] the life cycle of the human immunodeficiency virus type 1 (hiv-1) has an absolute requirement for ribosomal frameshifting during protein translation in order to produce the polyprotein precursor of the viral enzymes. while an rna stem-loop structure (the “hiv-1 frameshift stimulating signal”, or hiv-1 fss) controls the frameshift efficiency and has been hypothesized as an attractive therapeutic target, developing compounds that selectively bind this rna and interfere with hiv-1 replication has proven challenging. building on our prior discovery of a “hit” molecule able to bind this stem-loop, we now report the development of compounds displaying high affinity for the hiv-1 fss. these compounds are able to enhance frameshifting more than 50% in a dual-luciferase assay in human embryonic kidney cells, and they strongly inhibit the infectivity of pseudotyped hiv-1 virions. human immunodeficiency virus type 1 (hiv-1), the causative agent of acquired immune deficiency syndrome (aids), remains a significant challenge to global health. 1, 2 since its initial identification in 1983, hiv-1 infection has reached the status of a pandemic. in 2009 alone, there were approximately 2.7 million new infections and about 2.0 million deaths from aids related causes. 3 currently there is no cure for hiv-1 infection. while progression of the disease can be controlled by highly active antiretroviral therapy (haart), a combination of drugs designed to inhibit different stages in the virus' life cycle, 4 the complexity of the haart regimen, and the ability of the virus to evolve resistance suggest that alternative drug targets for hiv-1 treatment and prophylaxis are needed. 5 one potentially attractive target for pharmacological interference in the hiv-1 life cycle is the virus' requirement for a programmed −1 ribosomal frameshift (−1 prf) in order to express its enzymes. 6 ribosomal frameshifting is a recoding mechanism common among viruses with polycistronic (multiple open reading frames, or orfs, in a single gene) genomes. it allows viruses to translate polypeptides in different orfs by avoiding the stop codon(s) present in the single mrna transcript. in hiv-1, the pol gene is in the −1 reading frame with respect to gag. gag, the precusor of the viral structural proteins, is produced via normal translational rules, while pol, the precursor of the viral enzymes, is synthesized as a fused gag-pol polyprotein via −1 prf. this occurs with a frequency of 5−10% of ribosomes translating the full-length viral mrna. a critical molar ratio of gag-pol to gag protein is required for hiv-1 replication and infectivity; alterations to this ratio have been shown to be detrimental. 7 −1 prf in hiv-1 is controlled by two cis acting mrna elements: a heptameric slippery sequence (u uuu uua), with the 0 frame indicated by spaces and where the frameshift actually occurs, and a downstream two-stem helix immediately following the slippery sequence, also known as the frameshift stimulatory signal (hiv-1 fss, figure 1 ). 8 while several mechanisms have been proposed to account for the frameshift, 9 it is currently hypothesized that this event results from an incomplete translocation for a limited number of ribosomes, due to resistance of the fss to unwinding. 10−12 these ribosomes then start translation of pol in the new −1 reading frame. modification of the slippery site or stimulatory sequence (either via natural variation or laboratory mutations) in ways affecting frameshifting efficiency translates to a decrease in viral replication. 13, 14 these and other results have led several groups to propose −1 prf as a potential target for developing antiretroviral agents for hiv-1. 6,15−17 nmr structural analyses indicate that the hiv-1 fss rna consists of a g-c rich upper stem-loop structure, 18 separated from a flexible lower stem by a gga trinucleotide bulge ( figure 1 ). 19, 20 the bulge produces a roughly 60°bend between the upper and lower stems. the upper stem-loop is exceptionally stable. this stability is believed to play a vital role in the stimulation of the frameshift, since the ribosome must unwind the stem during translation. the lower stem is thermodynamically less stable. the highly structured acaa tetraloop is uncommon among tetraloops 21 but is conserved among all hiv-1 group m subtypes except the uncommon h and j subtypes. likewise, the heptameric slippery sequence is conserved across all hiv-1 group m subtypes. shape analysis of the intact hiv genome suggests a more complex structure for the fss rna, although the upper stem-loop is retained. 22, 23 since other viruses also rely on frameshifting, 24 targeting frameshift-regulating structures may have general utility beyond the context of hiv. for example, human t-cell leukemia virus type 2 (htlv-2) uses two −1 prf events similar to hiv-1 in order to synthesize fused gag-pro and gag-pro-pol precursor proteins. 25 the rna responsible for the −1 prf essential for expression of gag-pro in htlv-2 also consists of two cis-acting rna elements, a heptanucleotide (aaaaaac) slippery site and a stem-loop shown in figure 1 . 26 this htlv-2 fss served as a sequence specificity control in the experiments we describe herein. the first attempted use of a synthetic molecule to alter hiv-1 frameshifting and thereby influence viral replication was reported by green and co-workers in 1998. 27 the authors showed that 1,4-bis[n-(3-n,n-dimethylpropyl)amidino]benzene tetrahydrochloride, a bis guanidinium-containing compound termed "rg501", was able to stimulate −1 frameshifting, alter the gag-pol:gag ratio, inhibit hiv-1 replication in cem cells (a lymphocytic cell line), and interfere with the formation of viral particles in chronically infected ch-1 cells (a cos cell line stably transfected with hiv-gpt, an hiv derivative in which the e. coli gpt gene replaces hiv env 28 ). increases in reverse transcriptase (rt) following treatment with 1.5 mm rg501 were also observed, as would be expected for an increase in gag-pol production. recently, the butcher group confirmed that this compound indeed binds the hiv-1 fss rna with weak affinity (k d ∼ 360 μm), and they carried out nmr structure analysis, indicating that rg501 binds in the major groove of the upper stem-loop. 29 unfortunately, as noted by the authors, rg501 is a relatively nonselective binder and interacts with other rnas. it stimulates frameshifting in viruses with different fss, 29 and likely that also interacts with the ribosome. 6 rg501 is also toxic, 27 a likely result of its lack of selectivity. moreover, its interference with hiv replication begins at concentrations below those observed to affect frameshifting. 27, 24 other compounds such as guanidinoneomycin, 30 idarubicin, and doxorubicin 31 have been shown to bind the hiv-1 fss. doxorubicin was found to bind with a k d of 2.8 μm, decreased frameshifting in a rabbit reticulocyte assay, and also significantly reduced overall translation. a screen of an arg-rich peptide library revealed a sequence able to significantly reduce frameshifting, but this displayed no selectivity for the hiv-1 fss relative to other frameshift-stimulating constructs and likely also interacts with the ribosome. 32 thus, as far as we are aware, there are no reported examples of synthetic molecules able to alter hiv-1 frameshifting and interfere with viral infectivity via selective, high-affinity binding to the fss rna. building on our laboratory's longstanding interest in understanding the factors that drive affinity and sequence selectivity in small molecule recognition of rna, 33 we previously reported the use of an 11,325-member resin-bound dynamic combinatorial library 34 (designed based on the structure of dna-binding, bisintercalating peptide antibiotics) to identify a compound (1) able to bind the hiv-1 fss upper stem-loop with moderate affinity (k d = 4.1 ± 2.4 μm immobilized on an surface plasmon resonance (spr) chip via one of its amine groups; k d = 350 ± 110 nm in solution as measured by fluorescence 35 ) and good selectivity. 36 subsequent efforts revealed that replacement of the disulfide bridge with an olefin (to produce compounds 2 and 3) could be accomplished without any reduction in affinity. these studies also indicated that affinity was essentially abolished if the π-surface area of the molecule was reduced, while the peptidic portion of 1 was required for sequence-selective binding. 35 (right) htlv-2 fss stem-loop sequence used as a specificity control in this work. note that when the slippery sequence occupies the decoding site of the ribosome, the lower stem is unwound and it is the upper stem-loop that acts as the effective frameshift stimulatory signal. while the binding ability of 2 and 3 was intriguing, subsequent preliminary experiments indicated the compounds were unable to inhibit virus in a pseudotyped hiv-1 assay (data not shown). given these results, we hypothesized that further increases in affinity were essential. we anticipated that increasing the π surface area of 1 could represent a viable strategy for enhancing the affinity for the hiv-1 fss without significant reductions in selectivity. in particular, incorporation of a benzo[g]quinoline moiety to produce 4 and 5 was viewed as attractive. this hypothesis was supported in part by parallel efforts in our laboratory on the design and synthesis of compounds targeting cug repeat rna in which incorporation of a benzo[g]quinoline was found to enhance affinity and to provide compounds with activity in vivo in a mouse model of type 1 myotonic dystrophy. 37 synthesis of 4 and 5 proceeded via cross-metathesis of half-structure 6, by analogy to our previous work. we also synthesized 7 and 8 ("one-armed" benzo[g]quinoline-bearing structures), as well as compounds 9 and 10, to test the effect of sequential removal of the putative intercalators. compound 11 was synthesized in order to ascertain the effectiveness of the olefin bioisostere in improving cellular availability and bioactivity relative to an easily reduced disulfide. synthesized compounds were first analyzed for binding to the hiv-1 fss by surface plasmon resonance (spr). this technique allows the equilibrium constant (k d ) and kinetic rate constants (k on , k off ) to be determined in a label-free format. 38 a 5′-biotin labeled hiv-1 fss rna upper stem-loop (sequence as in figure 1 ) was immobilized on a streptavidinfunctionalized sensor chip. compound solutions in hbs buffer (0.01 m hepes, 0.150 m nacl, ph = 7.4) were flowed over the rna, and the reference-subtracted sensorgrams were recorded. the associative and dissociative phases of the experimental sensorgrams for at least five different concentrations were globally fit to a 1:1 langmuir equation to obtain the binding constants. 39 under these conditions, benzo[g]quinoline-containing compounds 4 and 5 bound the hiv-1 fss with affinities (k d ) of 89 nm and 102 nm, respectively, in each case representing an approximately 50-fold increased affinity over 2 and 3 (table 1) . binding by benzo[g]quinoline containing monomer 6 was 200fold weaker relative to that of the dimers 4 and 5. removal of one heterocycle "arm" also resulted in an approximately 200fold decrease in affinity for 7 and a 30-fold decrease for 8, while removal of both benzo[g]quinoline groups completely abrogated binding between 10 and the hiv-1 fss rna, consistent with our prior results, indicating the importance of the heterocyclic group for affinity. disulfide linked compound 11 bound the target rna with a k d of 0.741 μm, which is ∼7fold weaker relative to the olefin analogs 4 and 5. no binding was observed between the 2-ethyl benzo[g]quinoline carboxylic acid and the hiv-1fss by spr. confirming their anticipated selectivity for the hiv-1 fss, compounds 4−10 showed no binding to the htlv-2 fss by spr at concentrations up to 3 μm (supporting information p s12). the dissociation phase for spr traces obtained at the highest concentrations for compounds 4 and 5 were not well fit by a single-exponential function. to control for the possibility of compound aggregation interfering with the measurement, 40 we therefore reexamined these compounds using a running buffer containing detergent (20 mm hepes, 150 mm nacl, 5 mm mgcl 2 , and 0.005% tween-20). this had only a marginal impact on the fitted kinetic parameters (table 1) , suggesting aggregation is not a significant complicating factor in the assay. an spr chip with a very low density of rna provided similar dissociation constants (supporting information). to confirm thermodynamic dissociation constants measured by spr with a fully solution-phase method, and to further assess sequence selectivity, fluorescence titrations were conducted. in these experiments, saturable quenching of benzo[g]quinoline fluorescence was observed as a function of added unlabeled rna. dissociation constants obtained in this manner ( table 2 ) for 4 and 5 binding the hiv-1 fss rna are in general agreement with those obtained via spr. by fluorescence we observe a 5-fold selectivity for 4 binding the fss rna vs total yeast trna, while selectivity for fss rna vs fss dna was 4-fold. the selectivity of 5 for fss rna vs trna was somewhat lower (2.14-fold). curiously, unlike 4, 5 had no selectivity for fss rna vs fss dna. thus, as 2 and 3 were previously found to have no measurable affinity for trna, we conclude that the increase in affinity obtained on replacement of the quinoline moieties of 2 and 3 with benzo[g]quinoline does come with a cost of some decrease in selectivity. this result is in contrast to what was observed in a related series of compounds optimized for binding triplet repeat rna sequences relevant to type 1 myotonic dystrophy. 37 it is likely that these differences have a structural foundation, and further experiments will be needed to explore this hypothesis. cell permeation and toxicity. before analyzing the effect of compounds on hiv-1 frameshifting in cells, we verified that they were capable of crossing cell membranes, and not toxic at reasonable concentrations. human embryonic kidney cells (hek 293ft) were treated with 4, 5, and 11 at concentrations of 50, 100, 200, and 500 μm for 12 h. cells were then washed using standard methods 41 to ensure that cell staining was not simply due to surface capture. the inherent fluorescence of the benzo[g]quinoline chromophore in these compounds allowed direct visualization of cell penetration via fluorescence microscopy ( figure 2 shows results for 50 μm treatment; additional images are provided in the supporting information), confirming that cells internalized all three compounds. compounds did not appear to localize in any specific cell viability was analyzed using the wst-1 assay. 42 compounds were incubated with hek 293ft cells cultured in dmem for 24 h, after which a 10:1 media to wst-1 cell proliferation reagent mixture replaced the growth media for 2 h. absorbance was then measured at 450 and 690 nm. disulfide linked compound 11 proved toxic at concentrations above 10 μm, while no significant toxicity for compounds 4 and 5 occurred at concentrations below 60 μm (figure 3 ). the higher toxicity observed for 11 relative to 4 and 5 is not surprising and likely reflects the lability of the disulfide bond in the reducing environment of the cell. compounds were next evaluated for their ability to alter hiv-1 fss-dependent frameshifting, using a dual-luciferase reporter assay. 43 in this system, the fss sequence has been inserted between renilla (rluc) and firefly (fluc) luciferase genes. several constructs were employed. in the pdualhiv(−1) construct, fluc is in the −1 reading frame relative to rluc, while, in the pdualhiv(0) construct, fluc is in the 0 reading frame relative to rluc. in both cases, fluc is expressed only as a fused rluc-fluc protein, but in the case of pdualhiv(−1), a −1 frameshift is required. to ascertain if compound-dependent effects on frameshifting are specific to hiv-1, we also carried out an analogous −1 prf assay using constructs in which the htlv-2 frameshift site 44 was inserted between rluc and fluc genes, such that fluc is only synthesized by a −1prf event [pdualhtlv-2 (−1)]. 45 hek 293ft cells were transiently transfected separately with pdualhiv(−1) and pdualhiv(0) plasmids. the frameshift efficiency (defined as the ratio of firefly to renilla luciferase activities) in the pdualhiv(−1) transfected cells was measured in the presence of varying concentrations of compounds (0−50 μm). a statistically significant (p < 0.005) dose-dependent increase in fluc to rluc ratios, >50% at 50 μm, was observed following treatment with 4 or 5 ( figure 4 ). control compound 10, which showed no measurable binding to the hiv-1 fss by spr, had no significant effect on −1 prf in pdualhiv(−1) transfected cells under similar conditions. in hek 293ft cells transfected with the pdualhiv(0) plasmids, the fused renillafirefly luciferase protein is synthesized by conventional translation rules, i.e. without ribosomal frameshifting. therefore, to confirm that the observed effect in pdualhiv(−1) cells is a direct effect of compound on frameshift-dependent translation, pdualhiv(0) transfected cells were treated analogously with compounds 4 and 5. no change in fluc to rluc ratio relative to untreated pdualhiv(0) transfected hek 293ft cells was observed, suggesting that 4 and 5 specifically stimulate −1 frameshift translation in cells. disulfide linked 11 stimulated hiv-1 frameshifting by approximately 25% (supporting information); this lower effect compared to the cases of 4 and 5 is consistent with its lower affinity to the hiv-1 fss and likely lability in the cell. treatment of hek 293ft transfected with the pdualhtlv-2 (−1) construct with compounds 4, 5, or 11 resulted in no change in fluc/rluc ratio compared to the case of untreated cells. this is consistent with in vitro spr results in which no binding was observed between these compounds and the htlv-2 fss rna. compounds 4 and 5 decrease viral infectivity. in order for the observed −1 frameshift effect of compounds to be significant, it was important that it correlate to an equivalent reduction in viral replication. to assess this, we analyzed the effect of compounds in a single-round infection with pseudotyped hiv in hek 293t producer cells and tzm-bl target cells (a hela cell line). 46 the wild type hiv proviral vector (pdhiv3-gfp) codes for all hiv-1 nl4−3 genes except nef (which is replaced with gfp) and env, thus preserving gag and pol, and the frameshift required for production of the gag-pol polyprotein. preliminary experiments with 2 and 3 showed no activity in this assay. however, we observed a statistically significant (p < 0.001) and concentration-dependent decrease in infectivity of the pseudotyped hiv-1 virions when producer cells were treated with 4 and 5 ( figure 5 ). the decrease in infectivity was >90% at a concentration of 20 μm 4, and >90% at a concentration of 40 μm for 5. the core peptide alone (10) showed a modest decrease in infectivity at a concentration of 40 . compounds 4 and 5 increase frameshifting (>50%) in a dual-luciferase assay incorporating the hiv-1 fss but have no effect on frameshifting in analogous assay incorporating the htlv-2 fss. relative frameshift efficiency in hek 293ft cells treated with 4, 5 or control peptide 10 after transfection with pdualhiv (−1) or pdualhtlv-2 (−1). the frameshift efficiency was calculated as the ratio of fluc to rluc in pdualhiv(−1) or pdualhtlv-2 (−1) transiently transfected cells. this ratio was arbitrarily set to 100% for plasmid-transfected cells, but not exposed to compounds. error is sem on three replicates for each concentration. μm, likely via nonspecific effects, given its lack of affinity for the fss. ec 50 values for 4 and 5 were 3.9 and 25.6 μm, respectively. the protease inhibitor indinavir was tested in parallel to calibrate the assay; its ec 50 was found to be 14.8 nm, in line with previously reported values. 47 while virus titer was determined in these experiments using an elisa assay to normalize viral load into target cells, a compound-dependent decrease in viral production was also readily observable via fluorescence microscopy, as the pseudotyped hiv carries gfp as a marker ( figure 6 ). phasecontrast images of these cells showed no morphological changes, consistent with wst-1 results (supporting information). the concentration range required for a decrease in viral infectivity by 4 and 5 was similar to the range at which a significant increase in −1 frameshift was observed in hek 293 ft cells, supporting the hypothesis that these compounds exert their antiviral activity primarily by altering −1 prf. in order to provide further support for this hypothesis, viral particles were isolated from supernatants from these experiments by spinning through a 20% sucrose cushion, and probed via western blot for the presence of reverse transcriptase (rt). as rt is produced only as part of the gag-pol fusion, increasing amounts of rt relative to capsid protein p24 (a structural protein produced as part of gag) would be required given an increase in frameshifting. indeed, that is what is observed as a function of added 4 or 5 (figure 7) . the amount of rt in unprocessed gag-pol (p160) relative to mature rt (p66 + p51) also increased significantly; this may indicate that treatment with compounds 4 and 5 also inhibits pol processing. however, the pattern of p160 cleavage intermediates observed designing small molecules that target specific rna sequences and elicit a desired rna mediated biological response constitutes one of the signature challenges in chemical biology. 48, 49 in this paper, we have demonstrated that a moderate-affinity "hit" compound for the hiv-1 fss rna, obtained from a resin-bound dynamic combinatorial library screen, can be transformed into high-affinity binders. an intriguing observation from our initial screen 36 was the finding that a symmetrical compound was selected, despite the target rna being nonsymmetrical. this work confirms that both putative intercalators are required for high-affinity binding, as compounds 7 and 8 had only weak affinity for the fss. structural analysis will be essential to fully understand the binding mode of 4 and 5, but the recognition of a nonsymmetrical rna binding site by a symmetrical, dimeric molecule is not unprecedented; for example, arya and colleagues have described the use of neomycin dimers for recognition of hiv tar rna, 50 and the hergenrother group designed deoxystreptamine dimers that bound nonsymmetrical rna loops. 51 likewise, structural information should prove useful for understanding the significantly higher selectivity of 4 for fss rna over fss dna and trna relative to 5. these enhanced compounds are able to alter frameshifting in a dual luciferase assay in hek 293ft cells and strongly inhibit viral infectivity in a pseudotyped hiv assay. relative to previously studied compounds targeting frameshifting in hiv, compounds 4 and 5 produce a roughly equivalent increase in rt at a 37.5-fold lower concentration than rg501. as such, compounds 4 and 5 represent promising leads for further study, as well as interesting tools to further investigate the mechanism of −1 prf. since the pseudotyped hiv used in this assay is only capable of one round of replication, we anticipate that stronger effects will be observed with wild-type hiv in a spreading infection assay. of course, further enhancements in affinity and selectivity would likely improve activity as well. as discussed above, previous high-throughput screening efforts targeting frameshift-modulating compounds in which bicistronic reporters provided the assay readout largely yielded compounds acting on the ribosome. given this observation, it has been suggested by others that assays focused on direct binding to the fss rna could be advantageous, as such compounds could potentially increase frameshifting by inhibiting unwinding of the upper stem-loop. 6 the results presented herein support this hypothesis. in the context of hiv biology, our results with compounds 4 and 5 indicate that an increase in frameshifting and a concomitant increase in the gag-pol/gag ratio decreases viral fitness. this is consistent with the work of mak and colleagues, who increased the gag-pol/gag ratio via cotransfection. 52 ■ experimental section reagents. commercially available reagents were obtained from sigma-aldrich chemical co. (st. louis, mo), tci america (portland, or), fisher scientific, emd chemicals (gibbstown, nj), advanced chemtech (louisville, ky), and alfa aesar, and were used without further purification unless otherwise noted. water used for reactions and aqueous workup was glass-distilled from a deionized water feed. reagent grade solvents were used for all nonaqueous extractions. reaction progress was monitored by analytical thin-layer chromatography (tlc) using em silica gel 60 f-254 precoated glass plates (0.25 mm). compounds were visualized on the tlc plates with a uv lamp (dual wavelength; λ = 254 nm, λ =360 nm). synthesized compounds were purified using flash column chromatography on em silica gel 60 (230−400) mesh or alternatively via preparative reversed phase hplc. cells were cultured in dulbecco's modified eagle's medium (dmem), supplemented with 10% fbs and 1% penicillin−1% streptomycin. premixed wst-1 cell proliferation reagent was purchased from clontech, and luminescence assays were carried out using a promega dual-luciferase assay kit following manufacture's instructions. analysis. 1 h nmr spectra were recorded at 25°c on either a bruker avance 400 (400 mhz) or bruker avance 500 (500 mhz) instrument and processed using mestrenova nmr processing software. chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane and referenced to the residual protium signal in the nmr solvents. data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, and q = quartet), coupling constant (j) in hertz (hz), and integration. 13 c spectra were recorded at 25°c on a bruker avance 500 instrument operating at 126 mhz. chemical shifts (δ) are reported in ppm downfield from tetramethylsilane and referenced (except in d 2 o) to the primary carbon resonance in the nmr solvent. ft-ir spectra were recorded on a shimadzu ft-ir spectrophotometer. high-resolution mass spectra (hrms) were acquired at the university of buffalo chemistry department mass spectrometry facility, buffalo, ny. synthesis of compounds. compounds were synthesized following procedures analogous to those previously reported. 35, 37 all compounds were produced in >95% purity, as determined by analytical hplc. briefly, 4 and 5 were synthesized from olefin precursor monomer 6, which was assembled on wang resin by standard fmoc solid phase peptide synthesis (spps) methods. one half of the resin-bound monomer was cleaved using 50% tfa in dcm with 1% tes, and was used as solution phase partner in an olefin metathesis reaction employing grubbs' second generation catalyst. the olefin products (isomer ratio of z/e = 2:3) were isolated using reverse phase hplc on a c18 column (waters, xbridge prep c18 5 μm obd, 19 mm × 250 mm) using gradient elution from 5 to 100% acetonitrile/0.1% tfa in water/0.1% tfa. olefin geometries were assigned by comparing the olefin proton chemical shifts of the e-olefin downfield to the z-isomer and also by the infrared spectra of the compounds as described previously. 37 figure 7 . compounds affect gag:gag-pol ratio in pseudotyped virions. equal loads of viral particles (measured by p24 elisa) were isolated from media of viral producer cells by spinning through a 20% sucrose cushion at 100,000g. the viral particles were western blotted for rt (abcam) and p24 (nih aids reagent program, cat #3537). densitometry of nonsaturated bands was used to calculate ratios for p160/(p66+p51) (listed below the rt blot) and the ratio of total (all bands) rt/p24 are (listed below the p24 blot). compounds 7 and 8 were synthesized by a slight modification to the procedures described above. the tripeptide (phe-pro-allylgly) was first assembled using fmoc peptide coupling chemistry on wang resin. the resin-bound tripeptide was then separated into two equal parts. 2-ethyl benzo[g]quinoline carboxylic acid was coupled to half the material and cleaved with tfa. this was then employed as the solution phase component in olefin cross-metathesis with the remaining bead-bound portion of fmoc-protected tripeptide. surface plasmon resonance (spr) binding analysis. surface plasmon resonance (spr) experiments were performed on a biacore-x instrument (biacore, inc., uppsala, sweden) on a cm5 sensor chip. approximately 2000 ru of streptavidin (rockland immunochemicals) was immobilized in both flow cells using edc/nhs chemistry. 5′-biotinylated-rna (either 500 nm 5′-biotin-hiv-1 fss or 500 nm 5′biotin-htlv-2 fss rna sequences, obtained commercially from integrated dna technologies inc.) was captured onto the streptavidin surface in one flow cell to a density of approximately 300−1200 ru. the streptavidin surface in the second flow cell was then blocked with biotin solution and served as a reference cell to correct for any nonspecific binding. binding constant measurements were carried out for each compound once on a "low density" (approximately 300 ru) chip and once on a "high-density" (approximately 1200 ru) chip in order to ensure binding was not influenced by rna density. kinetic binding experiments were carried out by flowing various concentrations of compound in (a) hbs buffer (0.01 m hepes, 0.150 m nacl, ph = 7.4) at a 60 μl per min flow rate or (b) 20 mm hepes, 150 mm nacl, 5 mm mgcl 2 , and 0.005% tween-20 at a 50 μl per min flow rate over the captured rna sequence. where necessary, a 20 s, 0.5 or 1 m aqueous nacl injection was sufficient for regeneration (compounds displaying fast off-rates did not require regeneration of the chip between injections). binding constants were obtained by global fit (conditions (a)) of association and dissociation phases of at least five referenced-subtracted and blank-corrected sensorgrams to a 1:1 langmuir binding equation, or via individual fits (conditions (b) using biaevaluation software). injection of each concentration was repeated at least twice for consistency. fluorescence titrations. fluorescence titrations were carried out in 20 mm hepes, 150 mm nacl, using a cary eclipse spectrofluorometer. all titrations started at a volume of 500 μl with the compound at 1 μm. rna was then titrated in from a highconcentration stock (10 μm for hiv-1 fss rna; 40 μm for hiv-1 fss dna and trna) in 1−10 μl increments. after rna was added, the solution was thoroughly mixed in the cuvette via pipetting and allowed to stand for 10 min to reach equilibrium. each measurement was taken three times with a 1 min waiting period between scans to confirm equilibrium was reached. intensities were corrected for dilution. cell permeation. hek 293ft cells grown to 80% confluence at 37°c and 5% co 2 were exposed to compounds at a concentration of 50 μm for 12 h in a 96-well tissue culture plate. after removal of the culture media [dmem containing 10% fbs, 1% penicillinstreptomycin (gibco)], the cells were washed twice with pbs to remove excess and surface-bound compounds. cells were then imaged while in buffer under a fluorescence microscope (olympus ix70) in the 96-well plate using 358-nm excitation and 460-nm emission filters. cell toxicity. hek 293 ft cells were plated in a 96-well tissue culture plate in dmem (10% fetal bovine serum, 1% penicillinstreptomycin) and allowed to grow to 80% confluence at 37°c under co 2 . varying compound concentrations (up to 0.5 mm) and control (identical volumes of sterile h 2 o) in triplicate were incubated with cells for 24 h at 37°c. ten microliters of premix wst-1 cell proliferation reagent (clontech) was added to each well including blanks (dmem), followed by a 2 h incubation at 37°c. absorbances measured at 450 and 690 nm were subtracted and blank corrected. the difference in absorbance was plotted against compound concentration. dual-luciferase reporter frameshift assay in 293ft cells. hek 293ft cells were plated in 96-well plates at densities of 1.5 × 10 4 cells/well 6 h before transfection in dmem (10% fetal bovine serum, 1% penicillin-streptomycin). cells were transiently transfected in separate wells with 0.2 μg of plasmid dna (pdualhiv(0), pdualhiv(−1), pdualhtlv-2(0), or pdualhtlv-2(−1)), using lipofectamine 2000 transfection reagent (invitrogen) and following the manufacturer's protocol. five hours after transfection, various compound concentrations (0−50 μm) were added directly to the cells in triplicate and incubated for 36 h at 37°c. the culture media was gently aspirated, washed with 1× pbs, and lysed with 200 μl 1× passive lysis buffer. rluc and fluc activities were measured with 5 μl of cell lysate and 25 μl of luciferase reagent using the dual-luciferase assay system (promega) in the same wells. fluc and rluc luminescence values were measured on a modulus microplate reader (turner biosystems). relative frameshift efficiencies were calculated by comparing the fluc/rluc luminescence ratio of cells treated with compounds to untreated cells. hiv-1 infectivity assay. the antiviral activity of 4, 5, and 10 was measured by single-round infectivity assay with pseudotyped hiv-1 using hek293t producer cells. the hiv-1 proviral vector (pdhiv3-gfp) codes for all hiv-1 nl4−3 genes except nef (replaced with gfp) and env, thus preserving gag and pol, and the frameshift required for production of the gag-pol polyprotein. a single-round infectivity assay was conducted by transient transfection of the viral vector with vsv-g coat protein vector at a ratio of 1:0.5 using fugene hd (promega). the virus producer cells were dosed with compounds four hours after transfection, and viral particles were harvested from the media 24 h after transfecting by filtering through a 0.45-μm syringe filter. viral load was normalized with a p24 elisa (perkin-elmer). the infections were performed using tzm-bl reporter cells that contain stably integrated firefly luciferase that is driven by the hiv-ltr promoter. therefore, luciferase is expressed upon successful hiv infection. 53 triplicate infections in 96-well plates at 10,000 cells/well with 500 pg p24/well proceeded for 48 h before the addition of steadyglo reagent (promega) to each well for 30 min. luminescence was measured as a quantitative metric for changes in viral infectivity in the presence of compound. the human immunodeficiency virus: infectivity and mechanisms of pathogenesis genome organization and transactivation of the human immunodeficiency virus type 2 non-adherence to highly active antiretroviral therapy predicts progression to aids resistance-associated mutations in hiv-1 among patients failing first-line antiretroviral therapy targeting frameshifting in the human immunodeficiency virus decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1 characterization of the frameshift stimulatory signal controlling a programmed −1 ribosomal frameshift in the human immunodeficiency virus type 1 programmed −1 ribosomal frameshift in the human immunodeficiency virus of type 1. in recoding: expansion of decoding rules enriches gene expression coli ribosomes re-phase on retroviral frameshift signals at rates ranging from 2 to 50% a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting the three transfer rnas occupying the a, p and e sites on the ribosome are involved in viral programmed −1 ribosomal frameshift analysis of natural variants of the human immunodeficiency virus type 1 gag-pol frameshift stemloop structure structure and function of the stimulatory rnas involved in programmed eukaryotic −1 ribosomal frameshifting. cold spring harbor symp a novel strategy to interfere with human immunodeficiency virus type 1 propagation new targets for antivirals: the ribosomal a-site and the factors that interact with it identification of a cellular factor that modulates hiv-1 programmed ribosomal frameshifting solution structure of the hiv-1 frameshift inducing stem-loop rna solution structure and thermodynamic investigation of the hiv-1 frameshift inducing element structure of the rna signal essential for translational frameshifting in hiv-1 rna simulations: probing hairpin unfolding and the dynamics of a gnra tetraloop architecture and secondary structure of an entire hiv-1 rna genome shape-directed rna secondary structure prediction programmed ribosomal frameshifting in hiv-1 and the sars-cov comparative mutational analysis of cis-acting rna signals for translational frameshifting in hiv-1 and htlv-2 two cis-acting signals control ribosomal frameshift between human t-cell leukemia virus type ii gag and pro genes importance of ribosomal frameshifting for human immunodeficiency virus type 1 particle assembly and replication structure of the hiv-1 frameshift site rna bound to a small molecule inhibitor of viral replication guanidinoneomycin b recognition of an hiv-1 rna helix selection and characterization of small molecules that bind the hiv-1 frameshift site rna selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1 rna-selective coordination complexes identified via dynamic combinatorial chemistry resin-bound dynamic combinatorial chemistry strategies for recognition of stem-loop rna structures by synthetic ligands: application to the hiv-1 frameshift stimulatory sequence identification of a selective small-molecule ligand for hiv-1 frameshift-inducing stem-loop rna from an 11,325 member resin bound dynamic combinatorial library from dynamic combinatorial "hit" to lead: in vitro and in vivo activity of compounds targeting the pathogenic rnas that cause myotonic dystrophy surface plasmon resonance biosensor analysis of rna-small molecule interactions survey of the 2009 commercial optical biosensor literature surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors cellular uptake of aminoglycosides, guanidinoglycosides, and poly-arginine use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines a dual-luciferase reporter system for studying recoding signals two cis-acting signals control ribosomal frameshift between human t-cell leukemia virus type ii gag and pro genes comparative mutational analysis of cis-acting rna signals for translational frameshifting in hiv-1 and htlv-2 the dimerization domain of hiv-1 viral infectivity factor vif is required to block virion incorporation of apobec3g interactions of hiv protease inhibitors with a human organic cation transporter in a mammalian expression system recent advances in developing small molecules targeting rna targeting rna with small molecules click dimers to target hiv tar rna conformation size-specific ligands for rna hairpin loops maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type 1 rna dimerization and viral infectivity effects of ccr5 and cd4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1 key: cord-010092-uftc8inx authors: nan title: abstract of 29th regional congress of the isbt date: 2019-06-07 journal: vox sang doi: 10.1111/vox.12792 sha: doc_id: 10092 cord_uid: uftc8inx nan in the fin de siecle was heavily concentrated in vienna. freud, boltzmann, schr€ odinger and mach might be the first names to find, whenever one cites austrian scientists. but more related to transfusion are the noble prize winners max perutz and karl landsteiner. landsteiner 0 s fate illustrates the brain drain beginning in the early 30 s escalating in 1939 with the "anschluss", which lead to the forced emigration of many scientists. a loss which was not regenerated in the post war years and was further aggravated by dubious and often undisclosed relations and scandals in the nazi-era. all together this leads to a severe loss of credibility and productivity of universities across decades. opening university access in the early 80 s and intensive historical work-up of scandals transformed the austrian universities to open and effective scientific institutions driving innovation in the country. austria has achieved a great economic deal in recent decades, which was accelerated by the eu membership in 1995. as a result of strong long-term economic performance, the country's gross domestic product (gdp) per capita is the eighth highest among oecd countries and fourth in the eu28. levels of poverty and income inequality are both below the oecd average. investment in research and development (r&d) increased since the eu accession, when austria's r&d intensity (aggregate r&d expenditure as a percentage of gdp) was well below the oecd average and significantly far lower than switzerland -a country to which austria prefers comparison. the eu target of 3% r&d intensity was first met in 2014 and is 2018 the sixth highest among oecd countries and the second highest in the eu28. austria showed the second highest increase in r&d intensity of all oecd countries, exceeded only by korea. the rapid expansion was matched by a similar increase in human resources and scientific output of universities. austrian science in quantum mechanics, quantum communication and information is world renown. vienna is a major biotech hub, as is linz in mathematics and mechatronics and graz in automotive and production technologies. austria has been a net resource recipient in the horizon 2020 and the preceding 7th framework programme. small and mediumsized enterprises show a high propensity to co-operate with universities and other research organisations and more and more included in scientific grant schemes. vienna is the largest student city in the german-speaking world and consistently ranks among the top cities in the world on quality-of-life indices. as austria possesses globally recognised cultural attractions ranging from famed salzburg festival to the vienna new year concert its inhabitants are not aware of the progress made in r&d and how thriving innovation is going on in their country. they still love to show their cultural heritage and events and impress the world with some kind of eternal sound of music. patients with refractory b-cell malignancies as non-hodgkin lymphomas (nhl) resistant to standard therapies have a dismal prognosis. the outcome is even poorer in patients relapsing after autologous stem cell transplantation. most of these patients do not qualify for an allogeneic hematopoietic cell transplantation (hct) due to refractory disease, lack of a suitable allogeneic donor, higher age or cumulative toxicity of previous chemotherapy. despite patients undergoing allogeneic hct normally profit from a graft-versus -lymphoma effect, overall survival in patients with nhl after hct remains short. a similar situation can be observed for patients with acute lymphoblastic leukemia (all). therefore novel treatment modalities are urgently needed. chimeric antigen receptor (car)-t cells, a new class of cellular immunotherapy involving ex vivo genetic modification of t cells to incorporate an engineered car have been used in clinical trials. in the majority of studies b-cell malignancies treated with cd19 targeting car-t cells have been analyzed. austria had the advantage to participate in two international trials in the past and is currently involved in further car-t studies. recently, results from cd19 directed car-t cell trials with an increased follow-up of patients led to fda (food and drug administration) and ema (european medicines agency) approval of tisagenlecleucel and axicabtagene ciloleucel. common adverse events (aes) include cytokine release syndrome and neurological toxicity, which may require admission to an intensive care unit, b cell aplasia and hemophagocytic lymphohistiocytosis. these aes are manageable when treated by an appropriately trained team following established algorithm. in this presentation, results of four large phase ii cd19car-t cell trials for patients with nhl and all and focus on aes is summarized. preoperative anemia is a known risk factor for increased perioperative morbidity and mortality in patients undergoing major surgery. previous studies have not only shown higher in-hospital mortality, but also an increased hospital length of stay, greater postoperative admission rates to intensive care and prolonged use of mechanical ventilation and intensive care resources in patients with anemia compared to those with normal preoperative hemoglobin concentrations. about 30% of patients scheduled for major surgery suffer from preoperative anemia. this figure is even higher in patients requiring orthotopic liver transplantation, where up to 75% of all patients are diagnosed with anemia prior to surgery. transfusion of packed red blood cells (prbcs) is commonly used to correct anemic hemoglobin values. however, transfusion of prbcs has been associated with increased morbidity and mortality in patients undergoing cardiac, orthopedic, and abdominal surgery. additionally, transfusion of prbcs is associated with a greater incidence of postoperative acute kidney injury in patients undergoing orthotopic liver transplantation. as preoperative anemia might increase the perioperative use of prbcs, negative effects observed after prbc transfusions might even be augmented. data on the influence of preoperative anemia on morbidity and mortality after orthotopic liver transplantation are limited. thus, we retrospectively analyzed the association of preoperative anemia and mortality in adult patients undergoing orthotopic liver transplantation at our institution. in addition, we examined the influence of anemia on perioperative parameters such as transfusion requirements, surgical complications, early allograft dysfunction, acute kidney injury, and the need for renal replacement therapy. based on the results obtained in the retrospective analysis, an ongoing prospective randomized clinical trial was initiated. the two suspensive treatments in sickle cell disease (scd) are hydroxycarbamide, inducing the production of the functional hbf, normally repressed at birth, and red blood cell (rbc) transfusion, a critical component of scd management. however, rbc transfusion is not without risk. repeat exposure to allogeneic rbcs can result in the development of rbc alloantibodies which can make it difficult to find compatible rbcs for future transfusions. however, the main concern of alloimmunization is the development of hemolytic transfusion reaction, with in the most severe cases, hyperhemolysis, leading to multi organ failure and death in 4% of the cases. the prevention of this life threatening condition must be based on risk factors. however, although some risk factors, such as alloimmunization, have been identified, much of the mechanism underlying dhtr remains a mystery, particularly in severe cases presenting hyperhemolysis. here we will describe the current and future development to prevent and treat this severe syndrome in order to decrease exposure to transfusion in scd but also improve red blood cell quality, some new products are developed. oxidative damage is one of the parameter that could be diminished. some work is also ongoing to prevent filter blockage during leucodepletion of precious rbcs units from afro-caribbean donors carrying the sickle cell trait. finally, in countries with higher risks of transmission of infectious disease, treatment of red blood cell units against infectious agents can be discussed. the only current curative treatment of scd is hematopoietic stem cell transplantation (hcs). however, the occurrence, frequency, and effects of immune hematologic complications in hcs remain and will be discussed. finally, gene therapy is a real hope as a definitive curative treatment. clinical trials are ongoing in france and will be discussed as well as the remaining place of transfusion in this therapeutic. in the context of the chronic myeloid leukemia (cml), we have hypothesized that quiescent leukemic hematopoietic stem cells (hsc) compartment, escaping to the current tyrosine kinase inhibitors (tkis) treatment, in part associated in the molecular relapse, may be targeted by cart-cells immunotherapy. gene expression profiling studies have established that a cell surface biomarker il-1rap is expressed by the leukemic but not by the normal cd34 + /cd38-hsc. this talk will focus of the whole process of development of a cart-cells starting from recombinant il-1rap protein mice immunization to produce a specific monoclonal antibody (mab), to the proof of concept demonstration, before moving into the clinic. we produced and selected a specific anti-il-1rap mab (#a3c3 clone, diaclone sa, besanc ßon, france). after molecular characterization of antigen-binding domain, nucleotide sequences were fused with 3rd generation t cell activation coding sequences and cloned as a single chain into a lentiviral backbone comprising a safety switch suicide gene icasp9 (inducible caspase 9) and a monitoring/selection cell surface marker δcd19. we demonstrated in-vitro and in an in-vivo xenograft murine model that il-1rap car t cells can be activated in the presence of il-1rap+ cell lines or primary cml cells, secrete pro-inflammatory cytokines, degranulate and specifically killing them. we also demonstrated that multi-tkis treatment over a 4-year period does not affect transduction efficiency of cml patient t-cells by il-1rap car vector and that autologous cart-cells are able to target il-1rap+ leukemic primary hsc. "off-tumor-on target" toxicity prediction, by studying il-1rap expression on a tissue macroarray comprising 30 normal human tissues (3 donors) , with #a3c3, detected various il-1rap intensity staining in only few tissues. regarding the healthy hematopoietic system, #a3c3 flow cytometry staining did not detect hematopoietic cells, except monocytes that express poorly il-1rap. as expected, monocyte subpopulation is targeted by autologous il-1rap cart cells (ratio e: t = 1:1), but at a lower level that il-1rap cml cell line. in-vivo investigation of specific toxicities of autologous il-1rap cart-cells against hsc and/or immune cells on a human-cd34 + cord blood cell engrafted/nog murine model, but also by an in-vitro cd34 + colony forming unit assay didn't reveal any significant toxicities in immunocompetent cell subpopulations, suggesting that healthy cd34 + hsc are not affected. finally, to overcome potential toxicity, functionality of the icasp9/ rimiducid â safety switch was demonstrated in-vitro but also in-vivo in a nsg tumor xenograft model, showing that, when activate, the system is able to eliminate more than 90% of cart-cells, after exposure to ap1903. in conclusion, based on cml model, we demonstrated that il-1rap is an interesting target for cart-cell immunotherapy, with a limited "on target, off tumor" predictable toxicity. next step will be the up-scaling of the process in order to match with the use in human regarding also the regulatory requirements. this strategy may be applied, in the future, in other hematological malignancies. mortality ranges from 20 to 30% for trauma victims with severe bleeding and is largely dependent on the transfusion therapy from which they can benefit. the nature of this therapy has an impact on prognosis with a halving of mortality when the plasma/prbc ratio is greater than ½ and a decrease of about 20% when the proportion of platelets transfused is close to that of whole blood. the speed with which such therapy is actually administered has a major impact as well with an increase in mortality of 5% for each minute of delay in making the entire therapy available. this can be explained mainly by the fact that the probability of death of these patients is greater within minutes of their admission to hospital with a median time to death of 2 h after admission. to allow plasma, platelets and prbc to be made available in a timely manner, north american trauma centers have mandated that trauma centers have massive transfusion packs at the patient's bedside within 15 min. to further simplify and speed up logistics from distribution to transfusion, several trauma centers now use whole blood stored at 4°c. this return of an "old" product is largely inspired by military experience where whole blood is mainly used "warm" immediately after collection with compelling evidence of its effectiveness. its return to civilian practice requires the ability to deleukocyte it while preserving platelets and to store it while maintaining their hemostatic functions. good quality data shows this is achievable and several clinical studies are planned to begin in the coming months. in france, the french blood establishment and the french army are cooperating to initiate the prospective randomized non-inferiority storhm trial (sang total pour la r eanimation des h emorragies massives) which will be comparing whole blood to separate blood components in an 1/1/1 ratio in severely bleeding trauma patients. the primary endpoint will be a thromboelastographic parameter (maximum amplitude) assessed at the 6th hour after admission. secondary endpoints will include early and overall mortality, lactate clearance (reflection of the effectiveness of resuscitation) and 24 h organ failure. this trial will be recruiting 200 patients in 6 french trauma centers and is planned to be initiated second half of 2019. local/neighbours day: innovation in germany 1c-03-01 mesenchymal stromal cells for regenerative therapy bm-msc / asc obtained from these protocols have been characterized in detail in preclinical evaluations. manufacturing licenses for msc and asc and a platelet-derived growth factor concentrate have been obtained and they have been explored in several clinical trials for treatment of bone defects (ortho-ct1: eudract number: 2011-005441-13; ortho-ct2: eudract number: 2012-002010-39; maxillo1: eudract number: 2012-003139-50) . we will summarize results of completed clinical trials which confirmed feasibility and safety of autologous msc /asc treatment and provided evidence for efficacy (gjerde et al, stem cell res. 2018 introduction: in vitro produced megakaryocytes (mks) may serve as source to produce platelets (plts) ex vivo or in vivo. we have established a strategy to differentiate mks from induced pluripotent stem cells (ipscs) in bioreactors. this study aimed at the large-scale production of mks using microcarriers to increase the mk yield and to characterize their phenotype and functionality after irradiation as a method to decrease possible safety concerns associated to the ipsc origin. methods: ipscs were cultured in an aggregate form in presence or absence of microcarriers using 50 ml stirred flasks. cells were differentiated into mks using tpo, scf and il-3 in apel2 medium for a period of 22 days. non-irradiated or irradiated ipsc-derived mks were analysed for polyploidy, phenotype and proplt production using flow cytometry and fluorescence microscopy. also, plt-production was investigated in vivo. non-irradiated or irradiated mks were transfused to nod/ scid/il-2rcc-/-mice and blood was analyzed for human plts. results: differentiation of mks in presence of microcarriers resulted in an 8-fold increase of mks per ipsc in comparison to only aggregates. this resulted in mean of total mk harvest of 18.7 ae 6.8 9 10 7 in microcarrier-assisted bioreactors in comparison to 4.9 ae 1.3 9 10 7 mks collected from bioreactors containing only aggregates. interestingly, mks produced in microcarrier-assisted bioreactors showed higher proplt formation capacity than mks derived from only aggregates bioreactors. mk phenotype and dna content was comparable between mks derived from both types of bioreactors. irradiation of mks did not affect their phenotype and capability to form proplts or plts after transfusion into nod/scid/il-2rcc -/mice. conclusion: microcarriers showed to significantly increase the yield of ipsc-derived mks in stirred bioreactors to clinically relevant numbers. this may facilitate the use of ipsc-derived mk for ex vivo production of plts, direct transfusion or for innovative mk-based regenerative therapies. although the rosetta stone, found by the troops of napoleon in egypt near the city of rosetta (rashid) contains only a small amount of text in three languages it was key in deciphering hieroglyphs. the rosetta mission tried to achieve something similar: by looking at a tiny body its goal was to decipher the origin of the solar system, planets including earth and life. after more than 12 years the rosetta spacecraft softly crashlanded on comet churyumov-gerasimenko on september 30, 2016 . it has travelled billions of kilometers, just to study a small (4 km diameter), black boulder named 67p/ churyumov-gerasimenko. the results of this mission now seem to fully justify the time and money spent in the last decades on this endeavor. where are we from? where are we going? are we alone in the universe? these are some of the big questions. in this talk i will show which answers we got from rosetta and comet chury. we follow the pathway of the material which makes up our solar system from a dark cloud to the solar nebula and finally to planets and life. i will show that indeed we are the result of stardust and that what happened here may happen elsewhere in the universe. cells, tissues and entire organs can collectively be seen as "living drugs". genetically unaltered cells are routinely used in clinical practice to treat diseases as diverse as anemia, bleeding disorders, leukemia and organ failure. ground-breaking advances in genetic and genome engineering technologies are propelling cell therapies to the frontline of medical research and practice. the hematopoietic system is particularly amenable to genetic engineering because specific cell types can be purified based on the expression of specific surface proteins and the ability to culture and expand cells ex vivo. the recent unprecedented clinical success of killer t cells reprogrammed by chimeric antigen receptors (cars) to attack cd19 expressing tumor cells demonstrates the power of immunotherapy with genetically engineered immune cells. however, given the rapid development of novel genome engineering and synthetic biology tools we are likely only at the beginning of a new era of engineered cellular therapies. i will present recent progress in immune cell reprogramming, gene correction, safety aspects and remaining challenges such as manufacturing. 1d-04-03 cell free nucleic acids (cfna) circulate in the plasma of all individuals and are thought to be released by host and foreign cells into the circulation. after fractionation by centrifugation, cfnas can be extracted from the supernatant of whole blood samples or manufactured blood products. these dna or rna sequences can be of human, bacterial, viral or fungal origin. most of them are human double stranded dnas. research on cfnas is increasing, thanks to technological advancements in molecular biology. some of their results are already implemented in clinical practice in the areas of prenatal diagnosis, oncology and infectious diseases. the latter investigation focuses on the exploration of non-human cfnas, the field of metagenomics. high throughput sequencing associated with bioinformatics, the so-called new generation sequencing (ngs), has sped up the investigations of non-human cfnas. this tool provides the opportunity to classify cfnas into a human or non-human category, and then to identify them. it is thus possible to explore simultaneously the whole landscape of bacterial, viral and fungal populations. presently, ngs of human blood has already proven its feasibility and its value in identifying emerging viruses or investigating clinical cases of fever of unknown etiology. ngs of cfnas is also particularly effective in analyzing the different genotypes of a virus in case of a co-infection (e.g. hepatitis c virus). studying cfnas with the new molecular technologies is therefore of great importance in transfusion medicine, especially regarding security and clinical transfusion reactions. first, transfusion transmitted infections are the most feared adverse complications. second, febrile non-hemolytic transfusion reactions are also the most frequently reported adverse events in hemovigilance systems and their physiological mechanism -if only one-remains not clearly elucidated. investigating cfnas could thus improve our understanding and strategy aiming at reducing those two clinical adverse events. surveying comprehensively the composition of circulating infectious agents in a blood product by ngs technology could be very interesting for investigating a severe febrile transfusion reaction. moreover, when the costs of analysis will be reduced, it might be possible to screen prospectively and regularly the whole metagenomics of asymptomatic blood donors, in addition to the classical epidemiological surveillance. for instance, in a study testing a ngs method on manufactured fresh frozen plasmas, an astrovirus (mlb2) has been identified. finally, it is the responsibility of transfusion physicians implicated in the manufacturing of blood products to ensure that cfnas within a blood product do not have a clinical impact on the innate immunity of the recipients. according to recent research in vitro, cfnas purified from blood products can induce the transcription of inflammatory cytokines by mononuclear cells. as nonhuman cfna have an effect on toll-like receptors (tlr-linked inflammatory pathways), it would be also relevant to insure that donor's cfnas have no significant effect on the immune system of the recipient. in conclusion, cfnas are very diverse molecules contaminating blood products. technological progress makes now their investigation more available. besides being useful markers of infection in asymptomatic donors, their impact on the recipients' immunity should be further investigated. an active life and regular training is part of a healthy life style and for many this includes participation in endurance exercise competition at different levels. thus, it is highly relevant to know how a blood donation affects exercise performance and how close this can done to an endurance competition. endurance exercise performance is determined by many factors, but three of the primary are maximal oxygen uptake, the relative load that an individual can sustain over time and finally the efficiency of movement in the given discipline. over the years, a number of studies have sampled blood volumes ranging from 450-1200 ml and applied different methodological approaches to measure maximal oxygen uptake over a recovery period ranging between 3-28 days. overall, the general finding is a reduction in blood haemoglobin and an attenuated maximal oxygen uptake as well endurance performance after blood donation. in normal to well-trained men maximal oxygen uptake and performance was normalized after two weeks in one study after a normal blood donation (450/470 ml), but remained attenuated after four weeks in another study, despite the change in blood haemoglobin concentration was similar and the design and methodology also similar in the two studies. in addition to maximal oxygen uptake the relative load that can be tolerated during exercise is probably also attenuated, through a decreased arterio-venous oxygen extraction, but the available data is very limited. the first part of this talk will highlight the major findings and discuss some of the methodological issues that complicate interpretation and conclusions. there are sex differences in circulating blood volume, haemoglobin concentration, haematocrit and hormone levels and thus it is entirely possible that there is a sex difference in the effect of blood donation on physical performance and the recovery after blood donation. in addition to the basic physiological sex differences, there is also a higher prevalence of iron deficiency in premenopausal women, physically active women and women donating blood. therefore, we studied the influence of a standard 450 ml blood donation on maximal oxygen uptake and endurance performance and the subsequent recovery in physically active women. we observed that in iron sufficient women blood haemoglobin concentration and maximal oxygen uptake were back to baseline 28 days after blood donation, but endurance performance was normalized already after 14 days. the second part of this talk will discuss the sex differences in the effect of blood donation on maximal oxygen uptake and endurance performance. overall, the available data suggest that, with a careful conservative approach, 4 -5 weeks are needed after a normal blood donation to be fully recovered to participate in endurance exercise competitions. more than one in ten attempts to donate blood result in a temporary deferral, due to concerns about the impact of the donation on the donor or recipient. there is well established evidence that temporary deferrals impact negatively on donors, with a large proportion of those deferred failing to return at the end of the deferral period. this presentation provides an overview of deferrals from the donor perspective, describing the likelihood of receiving a deferral for different donor subgroups. the impact of temporary deferrals on the future donation of donors, considering both short-term and longer-term donation patterns, will also be reviewed, outlining which donors are at highest risk of non-return following a deferral, and what is known about the accumulative impact of multiple deferrals on donors. several hypotheses have been proposed to account for the strong negative impact of deferrals on donor behaviour, and there is preliminary evidence of psychological factors, such as emotional reactions, predicting intention to return. research is also beginning to emerge on the effectiveness of tailored interventions to mitigate the impact of deferrals on donor behaviour. the evidence for these preventative interventions, and for strategies to reactive donors once they have lapsed post-deferral, will be reviewed. recommendations for blood centres will be made, as well as suggestions for future research to address continuing gaps in knowledge. in his influential study "the gift relationship" (1970) , richard titmuss coined the idea of voluntary, non-paid, blood donation being the gift of life for a fellow citizen. this metaphor has been powerful in mobilizing donors (busby 2004) . it conveys a direct relationship between blood donation and patients' vitality, as well as a difference between gains and costs. as the gift of life, blood donation is seen to symbolize pure altruism and promoting solidarity between strangers. but can we apply the metaphor as successfully into donating blood for research? we asked a group of finnish blood donors what they would think if the frc blood service invited them to give a blood sample and personal information for research. the blood donors were usually willing to contribute to research for the public benefit, because they saw great potential in science to create solutions to help patients in the future. however, based on our interview data and previous research, we suggest that the analogy between gift of life and donation for research did not work all the way. the metaphor fails to address donors' questions on new types of relationships, interests and risks related to the use of personal data for research. left unanswered these could discourage donating for research. hence, we argue that the gift of life metaphor is not applicable to donor recruitment at the research context. in this presentation we wish to look for a better metaphor for donation for research that blood services collecting research data could apply. academy day: transfusion challenges in patients with sickle cell disease 2a-02-01 immunohaematological features of patients with sickle cell disease (lfa) should be considered as polymorphic antigens in the african population and these lfa are not present in most commercial panels. the situation is even more complicated when recipients lack high-frequency antigens, the most common ones being hr-, hr b -, sec-, u neg , u+ var , js(b-), (hy-), and jo(a-). finally, there is a high rh diversity among people of african descent. because they harbor variant alleles and/or partial rh antigens, they are at risk of developing alloantibodies. in this setting, screening for partial rh antigens makes sense. the figures illustrating this diversity vary with the approach used. one of them is to take into consideration rhd or rhce*ce variant alleles. in several american studies, their prevalence was estimated to be 29-36% and 53-72%, respectively. other teams take into consideration d, c and e partial antigens. their prevalence was estimated to be 8.4-14%, 12.5-27.7%, 3.3-3.5%, respectively, and the alloimmunization rates were 17.6%, 14.3-30%, 7.1%, respectively. as a result of these phenotype discrepancies, scd patients are more likely to be alloimmunized. an overall immunization rate of 2-6% is commonly admitted in the general population. depending on the unit selection policy and/or the study design, the immunization rate in scd patients varies from 7% to 76%, the highest figures being established when an abo/rh1-only matching policy is implemented. in a meta-analysis of 24 publications, the overall alloimmunization rates were around 20%. alloimmunization is thought to be enhanced by an inflammatory state, which is often present in scd patients. they are more prone to develop a new alloantibody. using a stochastic modeling of alloimmunization, they have a 61% increased risk of producing additional antibodies versus 30% in the general population. autoantibodies have been identified as a risk factor of alloimmunization. as a result, scd patients often have complex mixtures of allo and autoantibodies. rh antibodies and those considered as irregular natural antibodies are present in a significant proportion. another characteristic of the antibodies in scd patients is their evanescence; up to 70% of alloantibodies become undetectable within a few years of their initial development. relatedly, about a third of dhtrs are reported to happen in patients with no previous history of immunization. in addition, a third of patients will not develop an antibody after a dhtr. identifying patients at risk of developing a dhtr is key to managing them properly. alloimmunization is a serious risk of red blood cell transfusion in patients with sickle cell disease (scd) and can result in severe (delayed) haemolytic transfusion reactions, exacerbation of clinical symptoms and life-threatening hyperhaemolysis. once alloimmunized, the presence of alloantibodies in the patients' blood further complicates pretransfusion testing and hampers the selection of compatible blood products. numerous studies have shown that scd patients have a relatively high risk of alloimmunization as compared to the 'general' population. this is not only explained by the large number of transfusions given but also by the increased exposure to foreign antigens as a result of differences in the antigen make-up of the scd recipients and the blood donor population. other factors involved in the immune response such as age at first transfusion, inflammatory state, hla typing are under investigation and are starting to unravel. because blood transfusion is still one of the main treatment modalities for scd and some patients have a life-long transfusion dependency it is important to minimize the alloimmunization rate. theoretically, complete matching for all relevant blood group antigens would prevent alloimmunization. this however, is only possible when all donors are comprehensively. matching strategies should be developed to minimize alloimmunization while balancing patients' need and donor availability and is cost effective. to develop a (preventive) matching strategy some factors need to be established; 1) which antibody specificities are clinically relevant 2) which antigens are most immunogenic 3) what is the availability of specific antigen typings in the donor population 4) how should recipients (and donors) be typed, phenotypically and/or genotypically and to what extent. the latter is especially important in scd patients since they are of african descent and the prevalence of genetic variations in this population is relatively high. rhd and rhce variants are common and can remain undetected when serological typing is used but can be discovered with high resolution molecular typing. patients with partial rh phenotypes are at risk for alloimmunization. apart from special rh phenotypes in individuals from african descent, the fy(a-b-) phenotype related to the gata-box mutation in the fyb allele and the u-or u var phenotype resulting from genetic variations in the mns alleles are also common. several studies have shown that in scd patients antibodies directed against rhd, rhe, rhc and k are most frequently found when unmatched transfusions are given. preventive matching for these antigens has proven successful in reducing alloimmunisation. extended matching for all rh antigens fy(a), jk(a) and jk(b) can further decrease the alloimmunisation rate. currently, different countries have preventive matching strategies in place for this vulnerable patient group. as genotyping is more and more available and within reach, optimal antigen typing approaches for patients and donors, combining serology and genetics are being developed. in this lecture several aspects of antigen typing approaches and preventive matching strategies that will most benefit scd patients of will be discussed. artificial intelligence has become a buzzword that will appear about anywhere in the media. we can forget that ai, or the subfield in this computer science field machine learning, has been around for over 50 years. improvements in computing power, abundance of data, progress in computer science, and the arrival of affordable cloud solutions have now brought it to our daily lives. also in health care news about ai has become omnipresent. and some landmark papers have come out on algorithms outperforming (teams of) physicians in the diagnosis of all kinds of skin disease, eye disease from retina scans, and detect cancer in ct scans. however, little of these solutions have actually shown up in our clinical practice yet. in anaesthesia, we worked with the first algorithm to come to anaesthesia practice; hypotension during surgery is associated strongly with poor outcome like myocardial ischemia, surgical complications, renal failure and even mortality. we worked on a machine-learning trained algorithm that predicts hypotensive events using the arterial blood pressure curve up to 15-30 min before the actual event. to get fda and ce approval, however, mere mathematic validation is required. this can be achieved on retrospective datasets. in reality, we need more before we can use these algorithms to support our decision-making; after internal (retrospective) and external (prospective but passive use) validation steps, clinical (i.e. rct validation is needed. moreover, we will need to assess the economic impact too. ultimately this tool has now reached clinical practice and is starting to help us go from reactive to more proactive hemodynamic management. like this, we have started to work on machine-learning tools to predict the incidence of specific types of patients coming into a&e and predicting infections after surgery. we will discuss our approach, essentials to start with machine learning, practical learnings. we will also discuss a first project design to use machine learning in managing bleeding patients to get the best therapy advice for blood product use like plasma, fibrinogen et cetera. how can we start using this tool in unison with our existing tools to improve science and clinical practice in our respective (bio)medical fields? thrombocytopenia is a very common hematological abnormality found in newborns, especially in preterm neonates. two subgroups can be distinguished: early thrombocytopenia, occurring within the first 72 h of life, and late thrombocytopenia, occurring after the first 72 h of life. early thrombocytopenia is associated with intrauterine growth restriction, whereas late thrombocytopenia is caused mainly by sepsis and necrotizing enterocolitis. platelet transfusions are the hallmark of the treatment of neonatal thrombocytopenia. most of these transfusions are prophylactic, which means they are given in the absence of bleeding. however, the efficacy of these transfusions in preventing bleeding has never been proven. in addition, risks of platelet transfusion seem to be more pronounced in preterm neonates. because of lack of data, platelet transfusion guidelines differ widely between countries. in a recent randomized controlled trial (planet-2/matisse study) among preterm infants with severe thrombocytopenia, we found that those randomly assigned to receive platelet transfusions at a platelet-count threshold of 50 9 10 9 /l had a significantly higher rate of death or major bleeding within 28 days after randomization than those who received platelet transfusions at a platelet-count threshold of 25 9 10 9 /l. this presentation summarizes the current understanding of etiology and management of neonatal thrombocytopenia. transfusion-associated circulatory overload (taco) is a severe transfusion adverse reaction that is associated with increased mortality and morbidity. the incidence of taco in adults varies from 1% to 8%, but is probably underdiagnosed and underreported. the incidence in the pediatric population is undetermined. taco usually occurs in patients who receive a large volume of blood product over a short period of time. it is more common in patients with known risk factors such as cardiovascular disease, renal failure, and older or younger age (> 60 years or < 3 years). hospitalised patients and intensive care patients are also more at risk. the typical presentation of taco is respiratory distress (dyspnea, tachypnea) occurring within 12 h of a blood transfusion. associated signs and symptoms are hypoxia, hypertension, tachycardia, positive fluid balance, high central venous pressure, and acute or worsening pulmonary edema on chest x-ray. echocardiography and measurement of brain natriuretic peptide (bnp) or its n-terminal prohormone (nt-probnp) is helpful for diagnosis. several definition criteria have been proposed for taco, but none are adapted for children, particularly critically-ill children who are more at risk. this is probably the main reason why taco is even more underdiagnosed and underreported in the pediatric population. in a recent study, we compared the incidence of taco in a pediatric intensive care unit using the international society of blood transfusion (isbt) criteria, with two different ways of defining abnormal values: 1) using normal pediatric values published in the nelson textbook of pediatrics; and 2) using patients as their own controls and comparing pre-and post-transfusion values with either a 10% or 20% difference threshold. we monitored for taco up to 24 h post-transfusion. a total of 136 patients were included. taco incidence varied from 1.5% to 76%, depending on the definition used. with such wide variability, we conclude that a more operational definition of taco is needed in pediatrics, particularly for critically-ill children. differential diagnosis from other dyspnea-associated transfusion adverse reactions (e.g. transfusion-associated lung injury, anaphylaxis) is important because treatment differs, as do guidelines to the blood bank. treatment for taco is similar to that of any other cardiogenic pulmonary edema: oxygen, diuresis, ventilatory support. prevention is possible by avoiding unnecessary transfusions, transfusing only the necessary amount of blood product, avoiding rapid transfusions, and using diuretics. background: the risk and importance of transfusion-transmitted hepatitis e virus (tt-hev) infections by contaminated blood is currently a controversial discussed topic in transfusion medicine. in particular, the infectious dose is a not finally determined quantity. the different countries have chosen different approaches to deal with this pathogen. one central question is the need of individual nat screening (id) versus minipool nat screening (mp) approaches to identify all relevant viremias in blood donors. aims: comparison and evaluation of the available screening strategies in relation to the infectious dose to minimize the risk of tt-hev infections. methods: we systematically reviewed the presently known cases of tt-hev infections and available routine nat-screening assays. furthermore, blood donation screening strategies for hev ehev in effect in the european union were compared. we also describe our own experiences of hev screening utilising an id-nat-based donor screening algorithm compared to mp-nat in pools of 96 samples. from november 2017 to january 2018, a total of 10,141 blood donations were screened for the presence of hev rna using a mp-nat (in house, realstar hev rt-pcr kit) and an id-nat (cobas 6800 platform). results: the review of the literature revealed a significant variation regarding the infectious dose causing hepatitis e. in the systematic case review, all components with a viral load (vl) greater than 5.00e+04 iu caused infection (definitive infectious dose (difd) . the lowest infectious dose resulting in tt-hev infection observed in general was 7.05e+03 iu (minimal infectious dose (mifd). the infectious dose of the different blood products is mainly influenced by the remaining plasma content. our data comparing the two different hev screening algorithms revealed eight hev rna positive donations using a mp-nat (incidence 1:1,268) , whereas 17 hev rna positive donations were identified by id-nat (incidence 1:597); all id-nat only positive donations had vl < 25 iu/ml. summary/conclusions: taken into account the current knowledge on the required mifd, the difd, and the analytical sensitivities of the screening methods, we extrapolated the detection probability of hev-rna positive blood donors using different test strategies (nat assay, id vs. minipool with different pool sizes). we also considered the amount of plasma in the different blood products and calculated the infectious doses needed to be detected. only id testing would be sufficient to detect the minimum vl in the donor to avoid tt-hev infections based on the currently known mifd, but a highly sensitive mp-nat should be adequate as a routine screening assay to identify high viremic donors and avoid tt-hev infections based on the difd. we have also determined that the incidence of hev infection was approximately 50 % higher if id-nat was used. however, vl were below 25 iu/ml and will most likely not result in tt-hev infection taken into account the currently known mifd or difd. the clinical relevance of and need of identification of these low level hev positive donors still require further investigation. in the last 25 years several pathogen inactivation (pi) technologies have been developed to be applied to blood components. technologies for inactivating pathogens in plasma and platelets are available in the european union, and some others are currently under development. the first pi technology introduced in the market was for plasma, and was based on the addition of methylene blue and the illumination with light (theraflex mb-plasma, macopharma and gr ıfols). for platelets and plasma two technologies are licensed, one is based on the addition of amotosalen and the illumination with ultraviolet light (uv) (intercept â , cerus) and the other one combines the addition of riboflavin (vitamin b 2 ) and the illumination with uv light (mirasol â , terumo bct). currently another technology for platelet inactivation, based on the illumination with uvc light and strong agitation is under development (theraflex, macopharma). for red blood cells one technology based on the addition of one molecule (amustaline, cerus) is being developed. the mechanism of action, and the spectrum and level of inactivation of pathogens varies among the different technologies. in addition, the number of studies with clinically relevant endpoints and the number of patients included in the studies is not homogeneous. there is published evidence for most of them that show that the treated blood components are safe and efficacious for the patients although, for treated platelet concentrates some decrease in the posttransfusion recovery and survival of the transfused platelets occur, with differences between the different technologies. however, cumulative experience on the use in routine, for some of the technologies for almost 20 years, support the concept of the safety and efficacy of the blood components treated with pathogen inactivation technologies without a significant increase in utilization. the use of pathogen inactivation for blood components is not widespread. differences in epidemiology between countries, infectious risk perception, concerns about potential adverse effects associated with its use and economical considerations might explain the differences observed in its implementation. the history of the p1 and p k antigens is complicated and sometimes confusing because of several changes to the nomenclature. the association between the antigens and their genetic home has raised many questions as well as the longstanding enigma regarding the molecular mechanism underlying the common p 1 and p 2 phenotypes. the system (isbt no. 003) currently includes three different antigens, p1, p k and nor. the p1 antigen was discovered already in 1927 by landsteiner and levine while p k and nor were described in 1951 and 1982, respectively. as for the abo system, naturally-occurring antibodies of igm and/or igg classes can be formed against the missing p1/p k carbohydrate structures. anti-p1 is usually a weak and cold-reactive antibody very rarely implicated in hemolytic transfusion reaction (htr) or hemolytic disease of the fetus and newborn (hdfn). however, some antibodies against p1 have been reported to react at 37°c, bind complement and cause both immediate and delayed htrs. the p k antibodies can cause htr and anti-nor is regarded as a polyagglutinin with unknown clinical significance. a higher frequency of miscarriage is seen in women with the rare phenotypes p and p 1 k /p 2 k . the rbc of the fetus as well as of the newborn express low amounts of p1, p and p k antigens but the placenta shows high expression and is consequently a possible target of the antibodies and the cause of the miscarriages. the p k and p1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. furthermore, altered expression of p k antigen has been described in several cancer forms. a longstanding question has been why individuals with p phenotype not only lack p k and p expression but also p1. recently it was clarified that the same a4galtencoded galactosyltransferase synthesizes both the p1, p k and nor antigens and in addition the p 1 and p 2 phenotypes was confirmed to be caused by transcriptional regulation. transcription factors bind selectively to the p 1 allele in the 5'-regulatory region of a4galt, which enhance transcription of the gene. it has been debated whether the p k and p1 antigens exist on glycoproteins in the human rbc membrane or if glycolipids are the only membrane components carrying these epitopes. a recent publication shows that the p1 antigen can be detected on human rbc glycoproteins and thus glycosphingolipids can no longer be considered as the sole carriers of the antigens. the blood group system which started out with one antigen, p1, has now gained two more members namely p k and nor. step by step the biochemical and genetic basis underlying the antigens expressed in this system has been revealed but still many questions remain to be solved. neither gata1 nor klf1 represent a blood group system but mutations in the genes encoding these transcription factors (tfs) have been shown to result in simultaneous altered expression of blood group antigens in certain rare blood group phenotypes. in particular, mutations in the klf1 gene are responsible for the dominantly inherited in(lu) phenotype, commonly referred to as lu(a-b-) because of the gross reduction in lutheran antigens expression. red cells from in(lu) individuals, though, have also weakened expression of other blood group antigens, like the high-incidence antigen anwj, the antigens of the indian blood group system (cd44) and p1, among others. since the first description of klf1 variants associated with the in(lu) phenotype, many other variants of this gene have been reported with an impact on blood group antigen expression and they are listed on the klf1 table of blood group alleles. other than klf1, a mutated gata1 gene has also been found associated with the x-linked form of the lutheran-mod phenotype and has likewise been registered in the gata1 allele table. besides the effect of tf variants on blood group antigen expression, there are transcription factor-binding site polymorphisms in regulatory regions of blood group genes, which also have an impact on the expression of the encoded antigens in red cells. the first example of such type of polymorphisms was described in 1995, when the disruption of a gata motif in the ackr1 gene promoter was found to abolish erythroid gene expression in fy(a-b-) individuals of african descent. the impact of mutations affecting gata1 binding sites has also been described in some abo subgroups, like the am and bm phenotypes. a regulatory element with gata binding sites in the first intron of the abo gene has been found to be altered in individuals with these phenotypes, either by deletion or by a point mutation disrupting the gata motif. recent findings have also revealed that xga expression on red cells is dependent on gata1 binding to a control element located 3.7 kb upstream of the xg gene. a single nucleotide polymorphism (snp) within this region was shown to correlate very well with the expected distribution of the xga negative phenotype in different populations. further work has demonstrated that this g>c snp disrupts a gata1 binding site and consequently abolishes erythrocyte xg expression. overall, these investigations have allowed to elucidate the underlying genetic basis for xga expression and have made xga genotyping possible. similar to xga, the p1 antigen has been known for a long time to be determined by the a4galt gene but the molecular basis underlying the common p1/p2 phenotypes has remained elusive till recently. several cis-regulatory snps had been identified in non-coding sequences around exon 2a, which showed a very good correlation with p1 antigen expression. interestingly, potential binding sites for several hematopoietic tfs were identified in the same region. finally, recent investigations have demonstrated the role of the runx1 tf in the expression of p1 antigen, by selective binding to a regulatory site present in p1 but not in p2 alleles. to summarize, variation in blood group antigen expression may result from mutations or polymorphisms in the regulatory region of blood group genes. recent reports have unravelled the molecular mechanisms underlying the expression of p1 and xga blood group antigens, which involves tf binding to allele-specific regulatory elements. similar mechanisms may also regulate antigen expression in other blood group systems. 2c-06-03 clinical immunology, copenhagen university hospital, copenhagen, denmark since the discovery of cell-free fetal dna (cffdna) in pregnant women's blood, the development of noninvasive prenatal testing (nipt) has provided new diagnostic applications in prenatal care. in transfusion medicine and clinical immunology, cffdna is extracted from maternal plasma to predict fetal blood groups with the purpose of 1) guiding targeted rh prophylaxis in non-immunized rhd negative women and 2) assessing the risk of hemolytic disease of the fetus and newborn (hdfn) in immunized women. i will give an overview of noninvasive prenatal testing of fetal blood groups. based on the literature, i will summarize the current experience with noninvasive prenatal testing of fetal rhd and other blood groups. for rhd negative pregnant women, routine clinical testing is available in several countries world-wide to assess the risk of hdfn in d immunized women, and routine testing to guide rh prophylaxis is now implemented as nationwide service in 6-7 european countries. noninvasive prenatal testing for fetal rhd is highly accurate with sensitivities of 99.9%, as reported from clinical programs. in general, the sensitivity is challenged be low quantities of cffdna, especially in early pregnancy. the specificity is challenged by the polymorphic rh blood group system, where careful attention is needed to navigate among the many rhd variants. rhd variants may complicate cffdna analysis and interpretation of results, especially in populations with mixed ethnicities. despite these challenges, fetal rhd testing is very feasible when implemented with careful attention to these issues. for blood groups that are determined by snps, such as kel or rhc, the main challenge has been interference from the maternal dna when analyzing the fetal dna which has resulted in low accuracy and lower sensitivity, when using qpcr. with the application of more novel techniques such as next generation sequencing and droplet digital pcr, accurate noninvasive prediction of these fetal blood groups has been demonstrated. the success of predicting fetal rhd and its successful clinical implementation into national programs should encourage wide-spread use of cell-free dna based analysis. future work on noninvasive prenatal testing of fetal blood groups determined by snps may consolidate the application for cell-free dna testing for such targets, including human platelet antigens. at isbt, the newly formed cfdna subgroup of the red cell immunogenetics and blood group terminology working party will work to facilitate clinical applications, implementation and evaluation of cell-free dna testing. blood banks in most of the nordic countries all share a vein-to-vein approach which in short means that the collection of blood, the preparation of blood components, testing/release and storing is served by a single actor. on top of that recipient and donor blood grouping, crossmatching, delivery, registration of transfusion and of any complications is usually handled in a single blood banking information system (bbis). this means that blood banks in the nordics are traditionally operated by a single vendor. the needs for process control in a single vendor bbis, the present solutions, unsolved challenges and untapped possibilities of streamlining processes have been scrutinized with the intention to describe separate processes and to acquire best of breed or best of suite it-systems. the aim for integrations, rather than building an integrated it-system, to support the need for a vein-to-vein process is a precondition in the nordic countries. with multiple it-systems supporting isolated processes, we intend to facilitate development in these and furthermore increase the flexibility in the whole process. we set out to reveal any existing knowledge in the literature on it vendor strategies for blood banks, but we didn't succeed in identifying any relevant literature. however, a systematic literature study on vendor strategies when choosing health it was based on the prisma method, and identified 837 studies, but only 10 was eligible for full text review and 5 met the inclusion criteria. even this broader literature study reveals very little evidence. two studies find single vendor strategies poor and conclude "best of suite" solutions to be optimal. one study was not able to correlate vendor strategies to the investigated productivity, but concludes that best of suite and best of breed strategies requires larger organizational changes than a single vendor strategy. in summary, the existing research is contradictory. this paper adds basic knowledge for breaking down the process control of blood banking in smaller processes. this adds the possibility for identifying best of breed or best of suite vendors, instead of relying on single vendor it solutions. furthermore it is a call for more research in the field of vendor selection strategies which this study didn't succeed in identifying. 2d-07-02 applying drones to supply blood to remote areas: rwanda's experience biomedical services, rwanda-ministry of health-national blood services, kigali, rwanda background: in rwanda, blood transfusion services started in 1976. during the 1994 genocide against the tutsis almost all the socioeconomic fabric of rwanda was destroyed as well as its health infrastructure. the healthcare system was suffering in its aftermath, and there were health inequalities between urban and rural areas, including access to blood for transfusion. from 1995, the government started to rebuild all courses of life including the health system and the blood service in particular. the national center for blood transfusion (ncbt) was then mandated to provide safe, effective and adequate blood and blood components to all patients in need. this was pivotal in achieving health related mdgs 4, 5 and 6. today, rwanda has an ambitious vision to put all 12 million citizens within 30 minutes of any essential medical product. while every second matters in emergency management, the use of drones was the perfect solution to many of the last mile challenges that have been traditionally difficult to overcome. it is impossible to forecast accurately down to the need of a single patient. the government has provided an easy solution by centralizing supply and providing on-demand, emergency medical deliveries by drone. doctors are now empowered to provide the quality care with all the supplies on hand, patients can now be treated close to home, and we eliminate waste from potential overstocking when health workers know that they have a quick and reliable source of supply. description: in 2016, the government of rwanda started to operate the world's first national drone delivery program for blood and other lifesaving medical products. these drones can carry two to six units of blood at a time and deliver in 15 -45 minutes depending on a hospital's location. the average duration was between 2 -4 hours round trip with the vehicle system, before the use of drones. drones currently deliver blood to 25 health facilities throughout the country and are set to reach 90% of transfusing health facilities outside kigali by the end of the year. within the first year, healthcare workers saved an average of 3.1 hours per delivery and a total of 10,115 hours of lost time on road pick up they could instead dedicate to patient care. by march 2019, over 11,000 deliveries have been made, with 30% of those being emergency deliveries. a total of more than 19,500 blood units have been delivered. in february 2018, zipline obtained the highest rating from the health facilities being served in a performance evaluation conducted by the national center for blood transfusion. when a doctor or medical staffer needs blood, they place an order through the hemovigilance order portal. they are then sent a confirmation message saying a drone is on its way. the drone flies to the health facility at up to 100 km/h. when it is within five minutes of the destination, the medical staffer receives a notification. the drone then drops the package, attached to a parachute, into a special drop zone. conclusion: supply is not a developing country problem, it is a global issue. rwanda was just the first one to recognize the potential of this technology and decided to do something about it first and fast, to ensure access to universal access to all blood products. 2d-07-03 scottish national blood transfusion service, edinburgh, united kingdom supporting the provision of viable transfusion services in remote/rural areas is more than just a geographical challenge. limited qualified blood bank resource; small throughput volumes; increased regulation are only three of the additional factors combining to threaten safe and sustainable transfusion service delivery. inventory management and out of hours service provision were identified as essential areas where it was thought that technology, in the form of a remotely controlled blood fridge, could provide a key element of the overall solution. a radio frequency identification (rfid) fridge racking system was installed in a standard blood bank fridge and connected to the laboratory information management system (lims) common to both the remote and central blood bank. the central blood bank was enabled to test patient samples from the remote laboratory, identify components located in the remote fridge suitable for the patient and allow correct component issue, even when qualified staff were unavailable in the remote laboratory. testing has concluded that installation of remote fridge management can play a major role in helping to maintain a remote inventory permitting patient compatible components to be issued. by sustaining transfusion services in remote communities we can avoid transportation of patients who require transfusion support to locations miles from home. antibodies to hna-1b, fcgriiib and hna-2 have been reported, too. neonatal alloimmune neutropenia results from maternal antibodies transferred transplacentally to the fetus and is caused by all known hna-antibody specificities, i.e. hna-1a, -1b, -1c, -1d, hna-2, hna-3a, -3b, hna-4a, -4b and hna-5a specificity. hna and hla antibodies can induce mild febrile transfusion reactions and trali. since the introduction of the male only plasma strategy, in many countries the trali incidence decreased but it is still one of the most common causes of severe transfusion reactions. especially hna-3a antibody containing plasma from female donors is responsible for severe or even fatal reactions. but also hna-1a, -1b, hna-2 and hla class i and class ii antibodies were reported. the latter activate monocytes to secrete soluble factors that act on the primed neutrophils in the narrow lung capillaries. laboratory testing: laboratory work-up requires the knowledge of the patient's clinical condition and the methods that are appropriate to detect the relevant antibodies. the classical granulocyte agglutination test (gat) in combination with the granulocyte indirect immunofluorescence test (gift) can detect nearly all relevant antibodies. hna-1, -2, -4, -5 and hla class i antibodies are clearly detectable in the gift while hna-3 antibodies strongly agglutinate neutrophils in the gat. the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) test detects all hnaantibodies except for hna-3 with high glycoprotein specificity and sensitivity but is time consuming and requires highly skilled personnel. for trali diagnostics laboratory testing is completed by methods like the indirect lymphocyte immunofluorescence test (lift) or elisa for hla class i antibodies and hla class ii specific elisas. since several years fluorescent bead based assays (luminex) enable faster and more automated hna antibody detection but to date not all specificities, especially hna-3, can be reliably detected so that still the classical gift and gat have to complete the methodological spectrum. serological typing today is mostly reduced to the determination of hna-2 in the gift because the molecular reason for the hna-2-null phenotype is not completely understood. establishing only one pcr-asp reaction for the main cd177*787a>t polymorphism would comprise the risk to miss other molecular causes. however, for all other hna allelotyping by pcr methods is the first choice. summary/conclusions: granulocyte serology still today is widely based on a variety of manual methods and will be reserved to specialized laboratories as it requires experienced laboratory staff and profound knowledge of granulocyte immunobiology. 2d-08-02 norwegian national unit of platelet immunology, laboratory medicine, university hospital of north norway, tromso, norway maternal alloantibodies against antigens on human platelets can cause severe thrombocytopenia and bleeding in fetus or newborn, identified as fetal/neonatal alloimmune thrombocytopenia (fnait) . although most cases the thrombocytopenia is selfresolving within the two first weeks of life, some infants present bleeding symptoms and thus require platelet transfusion. a set of laboratory analyses are required to confirm the fnait diagnosis. in addition to guiding compatible platelets to the affected newborn, the correct diagnosis will be valuable to assess the risk of fnait in subsequent pregnancies. in addition, platelet alloantibodies may also complicate platelet transfusions by immune-mediated platelet refractoriness, and require proper identification of the patient's antibody specificities prior to selection of donor platelets. the algorithm for laboratory investigations include both serological and molecular assays, and depend on the objective and timing: whether there is an urgent need for platelet transfusion, follow-up of a pregnancy with known risk, or to do full-scale laboratory testing to confirm diagnosis. molecular genotyping should include all hpa systems relevant for the local population (in caucasians hpa-1, -2, -3, -5 and -15), preferably with optional extended panels for systems for low frequency populations due to immigration/mobility and for less frequently seen alloantibodies (hpa-4, -6 to -11 are most commonly included). serological testing of antibody binding to platelets is often initially tested by flow cytometry analysis (direct test and/or cross-match). however, the detection of platelet-specific antibodies is often complicated by the presence of anti-hla class i antibodies and thus require sensitive platelet glycoprotein-specific assays. serological testing for platelet-specific antibodies includes as a minimum panels of antigens on gpiib/iiia, gpib/ix, gpia/iia and cd109 and preferably additional targets for populations with asian/african origin. several methods are available; i.e. bead-based assays and elisa based methods. however, most reference laboratories perform variants of the monoclonal antibody immobilization of platelet antigens assay (maipa), as reported by the 19th international platelet immunology workshop of isbt (2018) . the investigations also include measurement of the anti-hpa-1a by quantitative maipa if present, as this is reported to potentially predict the severity of fnait. for pregnancies with known risk of fnait, there are methods available to perform non-invasive prenatal typing from maternal plasma. the most feasible and so far appropriate for routine testing is fetal hpa-1 typing with quantitative pcr or by melting curve analysis. other sophisticated, yet resource-demanding techniques have also recently been reported -importantly also for typing of other hpa-systems. 2d-08-03 molecular basis of hna-2 expression justus liebig university, institute for clinical immunology and transfusion medicine, giessen, germany human neutrophil antigen 2 (hna-2) is a neutrophil-specific antigen located on gpi-anchored glycoprotein cd177 (also known as nb1). hna-2 is absent on the neutrophil surface of 3-5% of the healthy individuals that divided the population to hna-2 positive and hna-2 null individuals. exposure of hna-2 null individuals to hna-2 positive neutrophils during pregnancy, after transfusion or transplantation, induces immunization against hna-2 and consequently the production of iso-antibodies. the hna-2 iso-antibodies are involved in the mechanism of neonatal alloimmune neutropenia (nain), transfusion-related acute lung injury (trali) and graft failure following bone marrow transplantation. presence of cd177 on a neutrophil surface of hna-2 positive individuals follows a bimodal expression that categorizes the circulating neutrophils to hna-2 positive and negative subsets. the cd177 gene contains 9 exons encoding a protein of 437 amino acids. the lack of hna-2 (in hna-2 null individuals) is associated with the presence of a missense mutation, cd177*c.787a>t in exon 7 of cd177 gene inducing a premature stop codon in codon 263. this mutation alone or in combination with cd177*c.997delg has been introduced as the main reason for the absence of cd177 in hna-2 null individuals. a pseudogene (cd177p1) highly homologous to exons 4-9 of cd177 gene is located downstream of the cd177 gene. conversion of exon 7 of cd177p1 into cd177 gene is responsible for the generation of cd177*c.787a>t missense mutation. in addition, the heterozygosity or homozygosity of cd177*c.787a>t is accounted for regulation of hna-2 negative and positive neutrophils subpopulations. genotyping has revealed the hna-2 null individuals, heterozygous for cd177*c.787a>t mutation without cd177*c.997delg, indicating the presence of a complementary mechanism regulating cd177 expression. newly in hna-2 null individuals and individuals with atypical cd177 expression a cd177*1291g>a polymorphism in combination with cd177*787a>t is described. altogether these data indicate a complex compound mechanism(s) for regulation of cd177 expression on the neutrophil surface. this presentation will summarize recent findings on cd177 expression and highlights the potential genotyping methods for genetic assessment cd177 expression of donors and patients. blood products ordered, transfusion start and end times, whether patients experienced a reaction to the transfusion as well as vitals measurements taken before and during the transfusion, including temperature, oxygen level, blood pressure and heart rate. for the validation process, 100 transfusion nursing notes were sampled and reviewers assessed the accuracy of the information regarding 1) blood product ordered, 2) whether the patient experienced a reaction, and 3) the start and end times of the transfusion. for each of these fields across all 100 sampled notes, the claritynlp tool reproduced these data points with 100 percent accuracy. in addition, the tool supplied transfusion end times for numerous structured records that were missing this key data point. summary/conclusions: claritynlp can very efficiently digest a large number of transfusion nursing notes simultaneously and also does an excellent job of extracting the main characteristics of a transfusion, which can be used in partnership with structured data to produce a more accurate and more complete picture of patient transfusions. immunohematology and genomics, new york blood center, new york, united states antibody-based typing, with a positive result reflected in agglutination of the red cells (rbcs), has served the profession for nearly a century enabling safe and effective transfusion therapy. the power of rbc typing by serologic methods lies in the availability of standardized antibody reagents which target many of the specificities of significance for transfusion, and the ability to directly detect antigen expression on the rbcs. hemagglutination has historically been relatively inexpensive, particularly for abo and rhd as the most important blood groups in most populations. serologic rbc typing is reliable, requires no sophisticated equipment, is generally straightforward to perform, and is fast requiring less than 1 h to results. hence, antibody-based testing has been considered the "gold standard" for blood group typing. with the age of genomics, dna-based genotyping is increasingly being used as an alternative to antibody-based methods. most antigens are associated with single nucleotide changes (snps) in the respective genes. genotyping has been validated by comparison with antibody-based typing and has been shown to be highly correlated. the power of genotyping of rbcs lies in the ability to test for antigens for which there are no serologic reagents, and to type numerous antigens in a single assay using automated dna-arrays. this increases accuracy and weak antigen expression can be revealed. fresh rbc samples are not required for dna extraction, and there is no interference from transfused rbcs or igg bound to the patient's rbcs. dnabased typing is economical in that it provides much more information, but testing requires special equipment, training, and 24-h turn around. what then is the best approach to use? will serologic typing be replaced by dnabased typing? indeed, genotyping will increasingly be used in the practice of transfusion medicine, especially with the growth of whole genome sequencing (wgs). however, because serologic typing for abo and rhd is fast and accurate and often relied on for sample identification, genotyping will not be the sole means for routine typing. genomic sequencing approaches will certainly reveal unrecognized changes and genetic variability in rbc membrane proteins, but not all variation will be immunogenic. a genetic polymorphism must be associated with antibody production to be considered a blood group antigen. the importance of an antibody, and antibody reactivity, then will continue to be the central defining principal in transfusion. as two sides of a coin, both are key to safe and effective transfusion therapy. since the mid-1980s, research in the molecular basis of structural and functional aspects of proteins carrying and producing the antigens has led to an upgraded and modern understanding of blood group variation. most commonly, single nucleotide polymorphism (snp) based basic molecular typing techniques were utilized to test new findings on a smaller subset of samples, and resulted in concordant sero-and genotyping results in general. however, quite commonly a small number of all samples delivered discrepant results, triggering consecutive rounds of analysis and resolution, finally resulting in a better knowledge with respect to the underlying blood group variation. such rounds of repetitions represented the synergistic incremental process of learning, learning for serologists and molecular biologists. inheritance of public, presence of high frequency, or low frequency, or partial antigens and notice of weakly expressed, or almost undetectable antigens marked the path of incremental learning and may best be exemplified by discoveries within the blood group systems abo, rhd and kell. naming for pheno-and genotypes coevolved alongside the permanent discovery of new antigens. at present, antigens and their antithetic counterparts (if tested and if existent), are more commonly reported independently, as exemplarily shown for the following kell phenotype consisting of three antithetic antigen-couples: kk, kp (a+b+), js(a+b+). alternatively, the same phenotype could be stated as: kel:-1,2,3,4, (5),6,7. genotypes, on the other hand, rather mirror the actual biological background, e.g. display the two parental alleles (or "haplotypes") present in an individuals' sample. genotype of the above mentioned example would read: kel*02.03/ 02.06 (italicized). in an idealized diagnostic environment and for most blood group systems encoded by proteins, every single blood group allele would be defined by its full genomic sequence, derived from one parental chromosome (including "some" 5'and 3'-untranslated regions). thereby, every such "ideal allele" would fully declare presence or absence of all its public, low-and high-frequency antigens and possess its "ideal name". by trend, biallelic snps and their immediate relation to antithetic antigen couples might have distracted from the originally intended meaning of "blood group alleles", more recently. finally, genotypes only dependent on (ideal) allele names, and considering mendelian inheritance patterns (dominant, recessive), would allow for fully comprehensive phenotype predictions. more recently, blood group serology, e.g. the "second side of the coin", seems to gain momentum. since the advent of whole genome sequencing and access to many more than 1000 human genomes, it seems that dozens of new blood group alleles are discovered, almost on a daily basis. beside the challenge of naming this multitude of alleles, respective discoveries are frequently made in samples lacking any phenotypic blood group pre-values. clear procedures will be needed to address naming and analyzing the phenotypes resulting from previously unknown alleles. as a consequence, questions asked 30 years ago have changed: today, molecular biologists looking at hundreds of newly discovered blood group alleles find themselves not being asked by serologists any more: "can you confirm my serology?", but instead, pose their question to the experts for the blood group phenotype: "can you confirm my genotype?" 3a-s02-03 background: the screening of blood donors and returning travelers from active transmission areas have highlighted the importance of diagnosis of acute arboviral infections. in the context of co-infections and similar clinical signs in endemic zones, the differential diagnosis of arboviruses is essential to discriminate the causative agent. the detection of viral nucleic acids in serum or plasma provides a definitive diagnosis, however, in most instances, viremia is transient within less than two weeks after the onset of clinical illness. in addition, the cross reactivity due to the high degree of structural and sequence homology between zikv and other flaviviruses is a significant concern. the combination of molecular (identification of viral genomes) and immunological assays (detection of the immune response) is a key challenge to follow the natural history of these infections and to improve the patient management and the epidemiological surveillance. aims: in this context, we have developed an innovative platform based on agglutination of superparamagnetic nanoparticles (npmag) covalently grafted either with nucleic or proteic probes to face the continuing emergence of arboviruses. methods: dengue (denv) and zika (zikv) viruses are selected as models in this study. a pan-flavivirus rt-pcr is used for the molecular assay to amplify the viral genomes. then, biotinylated viral amplicons are captured specifically on complementary original polythiolated probes coated on npmag. for the immunological assay, npmag are grafted with viral ns1 proteins to capture anti-denv or anti-zikv antibodies potentially present in the plasma samples. both tests are performed in disposable cuvettes in a homogeneous format. a magnetic field generated by an electromagnet is applied to the reaction medium to align the npmag into chains to enhance the capture of the targets between two npmag. aggregates formed are detected when the field is turned off. the optical density is measured in real-time at 650 nm during several cycles of magnetization / relaxation. results: in this study, molecular analytical performances were evaluated on human samples from blood donors with no history of infections as negative controls, on viral standards and on clinical samples. using viral references standards, we have observed sensitivities of 10 -10 2 tcid 50 /ml for zikv and denv (serotypes 1/2/3/4) after a detection phase of around 5 min. the first results obtained on 16 zikv (+) clinical samples previously tested by commercial real-time pcr (ct < 36, altona) showed an 88 % correlation between the two detection methods. no false positive results or cross reactions were observed. concerning immunological assays, commercial human plasma from donors tested positive for denv or zikv antibodies were detected positive with our innovative approach in less than 10 min (sampling + detection) instead of 2 h with classical elisas. further assays on clinical samples are planned to confirm these preliminary results. summary/conclusions: this innovative strategy combining molecular and immunoassays on the same analytical platform offers new opportunities for rapid blood testing to improve the surveillance and the prevention of arboviral infections. background: zika virus (zikv) caused a dramatic epidemic in puerto rico (pr) during 2016, with up to~2% of blood donors reactive for zikv rna in id-nat testing at the peak in june 2016. aims: perform a serosurvey for anti-zikv igg using six panels of 500 donor specimens each collected in march 2015, at the beginning, peak and end of the 2016 epidemic, and from march 2017 and april 2018. methods: we employed a commercially available zikv igg elisa antibody (ab) assay based on the zikv ns1 antigen from bio-techne to characterize zikv seroprevalence in the 6 9 500 cross-sectional sample sets (anonymized with selected demographic information). results: 500 pr donor samples collected in april 2015 were initially evaluated using the manufacturer supplied cut-off to confirm that the zikv ab results were largely negative (3 positive, 3 equivocal) despite the high dengue virus seroprevalence (>90%) in pr that could potentially lead to false positive zikv ab results. we then used this dataset, together with known positives collected 1-3 months postdetection from zikv nat yield donors, to set a population-specific cut-off based on receiver operating characteristic (roc) curve analysis. this cut-off yielded sensitivity and specificity values of > 99%, and an area under the curve (auc) of 0.999, demonstrating a highly accurate assay. we used this new cut-off to calculate final rates of seroreactivity in the additional 5 sample sets (2500 samples) and estimate seasonal incidence. rates of reactivity, together with mean net od for only the reactives (shown in parentheses), were calculated for each sample set: background: sex hormone intake in blood donors occurs in three demographic groups: premenopausal women who take contraceptive drugs (progestins with or without estrogens), postmenopausal women who receive estrogen replacement therapies that may be combined with progestins, and testosterone therapies in men. we hypothesized that sex hormone therapies may modulate the quality of red blood cell (rbc) products via alterations of rbc function and predisposition to hemolysis during cold storage. aims: the objectives of this study were to evaluate the association between sex hormone intake and rbc measurements of hemolysis, and to examine possible mechanisms by which sex hormones interact with rbcs. methods: self-reported sex hormone intake and menstrual status were evaluated in 6,636 female blood donors from the national heart, lung and blood institute's rbc-omics study. the associations between hormone intake and donor scores of spontaneous storage, osmotic, or oxidative hemolysis were determined in all women and by menstrual state. the interactions between sex hormones and rbcs were determined by sex hormone (progesterone, 17b-estradiol, or testosterone) potency to inhibit calcium influx or hemolysis during incubations or cold storage. the calcium fluorophore, fluo-3am, was used to define rbc calcium influx in response to treatments with sex hormones or drugs that modulate transient receptor potential cation (trpc) channel activity including hyp9 (a selective trpc6 activator). results: sex hormone intake by menstrual status was higher in premenopausal women (25.3%) than in postmenopausal women (7.4%). female hormone intake was significantly (all p < 0.0001) associated with reduced storage hemolysis in all females (0.32 ae 0.16% versus 0.35 ae 0.29% in controls), enhanced susceptibility to oxidative hemolysis (37.9 ae 9.3% versus 35.8 ae 9.9% in controls), and reduced osmotic hemolysis in postmenopausal women (23.1 ae 10.2% versus 26.8 ae 12.0% in controls). in vitro, supraphysiological levels of progesterone (10 or 20 lmol/l), but not 17b-estradiol or testosterone, inhibited spontaneous or hyp9-induced calcium influx into rbcs, and were associated with lower spontaneous hemolysis after 30 day cold storage (0.95 ae 0.18% versus 1.85 ae 0.35%, progesterone 10 lmol/l versus solvent control (dimethyl sulfoxide, 0.1%), p < 0.0001). co-incubations (2.5 h, 37°c) of rbcs in the presence of progesterone and a trpc6 activator (hyp9, 25 lmol/l) suggested that progesterone protected against hyp9-induced hemolysis (1.45 ae 0.13% and 1.01 ae 0.09% versus 2.63 ae 0.19%; hyp9 + progesterone at 10 or 20 lmol/l versus hyp9 alone, p < 0.05 by one-way anova). summary/conclusions: this study revealed that sex hormone intake in blood donors is capable of modulating rbc predisposition to hemolysis and led us to propose new mechanistic pathways by which progesterone regulates calcium influx and hemolysis in human rbcs. pre-and postmenopausal women respond differently to hormone intake and its effects on rbc responses to osmotic or oxidative stress. progesterone modulates calcium influx into rbcs via a mechanism that may involve interactions with membrane trpc6 channels, activation of which is associated with pre-hemolytic events such as senescence and eryptosis. 3a-s05-01 international cooperation, swiss red cross, wabern, switzerland background: red cross and red crescent societies were playing an important role in setting up blood transfusion establishments in many low resource countries. by the mid-1970s, the red cross was active in the national blood programs in approximately 95% of countriesmostly in blood donor recruitment and education. today, major organizational developments in blood transfusion services were made in high income settings, where nearly 50% of all worldwide donations take place (home to only 17% of the population). who data shows that the median annual blood donation rate in high-income countries is 3.21% of the population compared to 0.46 % in low-income countries. the factors for this low turnout are multilayered, but it is well-known that most resource poor settings suffer from a low rate of regular donors and challenges to set-up and financially sustain a national blood donor program. the red cross and red crescent (rc) societies assume a key role by reaching and retaining donors from the communities and contribute significantly to better safety and availability of blood. partnerships and international collaboration, such as the swiss red cross (src) program, are aiming to strengthen national structures to improve blood safety and to face today's epidemiological, demographical, and technological challenges. aims: the present work aims to review the role, the mandate and the impact of rc societies in improving blood safety through systematic "voluntary non-remunerated regular blood donor" (vnrbd) programming and international partnerships. methods: data and evidence is drawn from the src international cooperation projects over the last 30 years, more specifically partnering with three rc societies, and the data from the global advisory panel (gap) of the ifrc including their 2018 global mapping. results: the promotion of vnrbd has been a specific objective in all src supported programs. through the engagement of the rc society, the training of volunteers and partnering with the health authorities, the projects significantly increased the blood donor rate by recruiting and retaining donors from the communities. for example, the rc societies increased total donations by 25 % in lebanon; vnrbds by 71% annually in kirgizstan, and from practically zero to 3'588 in south sudan. the importance of rc societies was also underlined in the 2018 published global mapping of gap, which showed that 36 (19%) of them provide level a (full blood service), 75 (39%) are level b (systematic blood donor recruitment) and 47 (25%) are level c (vnrbd blood promotion) blood services. gap has also commenced a new three year vnrbd support program aimed at establishing tools and materials for national societies. summary/conclusions: the red cross / red crescent movement has a unique mandate and position in improving global blood safety at all levels; with its huge network of volunteers, even blood donors in remote communities can be reached and retained. rc societies in low resource settings with a level b or level c role should further capitalize on partnerships with local and international actors to leverage technical assistance and funding for their activities. background: it is essential to motivate and encourage the public to donate blood and be eager to help saving lives, in order to maintain safe and adequate national blood supply. aims: "technical assistance for recruitment of future blood donors (europeaid/ 132420/d/ser/tr)" project aimed to avoid problems in supplying the safest blood to contribute to the improvement of public health by (i) increasing the knowledge of primary and secondary school students regarding blood donation, (ii) creating sensitivity in school principals and teachers regarding voluntary non-remunerated blood donation (vnrd), (iii) motivating family members of the students for blood donation and (iv) creating public awareness through media. methods: an effective coordination is established between ministry of health (moh), ministry of national education (mone) and turkish red crescent (trc). the existing curricula and textbooks of primary and secondary schools were reviewed and revised, and corresponding materials on the importance of blood donation were created. the human resources capacity of moh, mone and trc to support raising awareness on blood donation were developed. to raise public awareness on blood donation nationwide, education and recruitment campaigns on were organized in 500 pilot schools. additionally, media and public relation campaigns on blood donation were organized throughout the country. results: (1) existing curricula and textbooks relevant to promoting blood donation were reviewed, revised and reported to the board of education of mone. (2) corresponding educational materials for students and teachers were developed and distributed. (3) blood donation clubs were established in 500 pilot schools. (4) trainings were conducted for 688 personnel of moh and trc on blood donation regarding their responsibilities. (5) cascade trainings were conducted for 3218 personnel of transfusion centers and 4399 school principals in 81 provinces. (6) information seminars were delivered to 251.475 students and 15.739 teachers and family members of students during school campaigns. (7) four animation films on blood donation were produced and broadcasted on the national tv channel (trt). (8) three different computer games targeting different age groups were developed and uploaded to the web portal of the project and distributed to the pilot schools. (9) media spots were produced and broadcasted 3.851 times in 33 different tv and radio channels. (10) billboard posters and brochures were prepared and distributed to 81 provinces for raising public awareness. (11) advertisements about the project and the importance of vnrd were displayed 386 times on national and local newspapers, 1.117 times on online news, and broadcasted on 6 national tv channels. (12) during the campaigns, 28.310 units of whole blood were collected in pilot schools. (13) visibility kits to recruit future blood donors are prepared and distributed throughout the project activities. (14) awareness and knowledge level of students and their teachers/parents on the importance of blood donation are increased to 32.07 % and 35.99 % respectively, assessed through pretest and posttest. voluntary non-remunerated donation rate of national demand increased from 73.66% to 82.54 in two years. summary/conclusions: training and campaign programmes successfully increased the knowledge on blood donation. to achieve national self-sufficient safe blood supply, efforts for recruitment should be continued. background: despite 70% of pakistan's population being under 29 years, only 10% of blood supplies come from voluntary donors while remaining blood is collected from 'family replacement donors'. in pakistan the system has outsourced the mobilization of blood donors to the patient families. as a result many people reach out to their networks including on facebook to locate blood donors. there are thousands of posts each month in pakistan seeking blood donors on facebook. to facilitate needy families, the global social media giant facebook launched a special blood donation feature for pakistan in collaboration with sbtp, pakistan. the feature makes it easier for people to sign up to become blood donors and helps connect these voluntary donors with people and organizations in need of blood. similar features have been launched by facebook in india, bangladesh and brazil to address the problem of blood shortages in those countries. however, among these four countries pakistan has unique position because of the existence of a national counterpart, sbtp which can facilitate facebook in promoting its feature and provide the feedback on the impact of this innovative effort for continuous improvement of the feature. aims: to promote voluntary blood donations and blood safety in pakistan through facebook. methods: the facebook and sbtp teams launched a pilot to study the impact and effectiveness of the facebook blood donation feature as a tool of community engagement. a six months plan has been chalked out to measure the impact of this tool in five selected blood centres. a checklist called "p0 checklist" was shared with these blood centres to fulfill some basic requirements for an official blood bank page including a display picture, cover photo, contact information, directions, etc. regular skype meetings are held between the teams of sbtp, facebook (san francisco and singapore) and the blood centres to monitor the progress of the pilot and generate feedback. results: the facebook blood donation feature has recorded remarkable success with over one million signups within few months in pakistan. the blood centres participating in the pakistan study have experienced enhancement in the voluntary blood donations trend with 3-10 walk-in donors and an average of more than 20 telephonic queries regarding voluntary blood donation per month in each center. the trend is gradually surging as the feature is being refined on the basis of feedback received. the pilot will end in june 2019. the statistics generated since january 2019 are very encouraging and underscore the importance of social media in reaching out to the untapped potential blood donors. the study will be used to plan an effective nationwide strategy to increase donor mobilization, recruitment and retention. background: in our region, an increasing number of patients of african or asian origin with sickle cell disease (scd) or transfusion dependent thalassemia (tdt) require red blood cell (rbc) transfusions, and many have rbc alloantibodies. selecting optimally matched rbc units for these patients is essential for preventing not only acute hemolysis but also further alloimmunisation. beside antigen-matching for abo, rh d, c, c, e, e and k, patients with scd and tdt should ideally receive rbc units matched also for m, s, s, fya, fyb, jka and jkb (extended phenotype). this is the policy at our center, which currently provides rbc products to 31 patients with hemoglobinopathies. because the vast majority of our blood donors are caucasians, the selection of matched rbc units for patients of different ethnic origin can be difficult. therefore, expanding the number of available african and asian blood donors is becoming increasingly necessary. aims: hereby the recruitment strategy of non-caucasian blood donors introduced at our center is described and the results obtained during six years are reported. methods: since 01.01.2013, whenever a first-time blood donor of non-caucasian origin is registered, an alert is entered in the donor file to trigger the determination of the extended rbc phenotype along with routine testing. rbc antigen determination is performed in our laboratory with serologic methods. in selected cases (i.e. suspected rhd or rhce variant), samples are sent for molecular analysis (ssp pcr). rare rbc phenotypes relative to ethnicity are, among others, fy(a-b-), s-s-, lu(b-) and those with uncommon rh phenotypes. if a rare rbc phenotype is detected, a coded comment is entered in the donor data and the donor is listed in the national rare donor file. results: from 01.01.2013 until 01.03.2019, an extended determination of rbc antigens was performed in 352 subjects presenting for blood donation. twenty-nine rare donors (8%) were identified and included in the rare donor file: 25 fy(a-b-), 1 lu (a-b-), 1 lu(b-), 1 fy(a-b-) and s-, 1 ccddee (r'r'). overall, these 29 donors provided 105 rbc units (range . to date, all donors are still active and 14 are reserved for dedicated donations. the internal price of rbc antigen testing per donor is approximately 100. -chf, resulting in a total financial effort of around 35,200.-chf in the time since the project was started. summary/conclusions: in our experience, a "passive" recruitment of non-caucasian blood donors based on ethnicity has an overall low efficiency from a logistic and financial point of view. moreover, african and asian blood donors may require investigations for hemoglobin variants and serology for malaria in addition to routine testing. nevertheless, a targeted determination of extended rbc antigen phenotype does allow the identification of persons with rare phenotypes. currently, measures for the active recruitment of potentially rare blood donors are being implemented at our center. after a pilot phase, a project for a nationwide recruitment strategy will be elaborated. a further goal is to build a national registry of patients with hemoglobinopathies requiring transfusions. blood transfusion is an essential treatment. transfusion safety consists of several components. although all are important, ion richer countries the order of priority is typically: 1.) avoidance of transfusion transmittable infections; 2.) quality of the blood product with a strong focus on component therapy; 3.) prevention of severe transfusion reactions; 4.) avoidance of clerical errors; 3.) sufficient availability of blood. the keynote of this lecture will be that the order of priorities on transfusion safety should probably be different in resource limited environments. 1.) sufficient availability of blood and proper utilisation; 2.) avoidance of transfusion transmittable infections; 3.) avoidance of clerical errors; 4.) prevention of severe transfusion reactions; 5.) quality of the blood product. most important, in regions with limited resources patients suffer from under-transfusion because not enough blood is available. all efforts should be made to reduce wastage of the available blood either by inappropriate storage, handling, or nonindicated transfusions. in addition, prevalence and number of pathogens transmittable by transfusion is much higher than in the richer parts of the world. this is aggravated by the fact that rejection of blood donors based on their history is problematic when the blood bank is empty. the aim to develop centralized national blood services with few sites manufacturing cost effective (low personnel costs) high-quality blood products, which are distributed to regional hospitals is not matching the reality of infrastructure, governmental support, and functionality. in healthcare systems with limited resources usually available personnel (hands) is not a problem, while reagents, equipment and even electricity are precious resources. the lecture will propose to focus on staff training and education, establishing local hospital-based transfusion services, which provide the blood products for the region, based on donor recruitment campaigns adjusted to the available technology, local culture, including replacement donor programs, with retention of safe donors as highest priority. fractionation of blood into components had been standard in transfusion medicine, but recently whole blood has experienced revival for patients with acute blood loss. given main transfusion indications such as postpartum hemorrhage, or severe trauma, in regions with limited infrastructure, whole blood might be the more appropriate product. most patients requiring transfusion in these regions are younger and volume overload by whole blood is not a major issue. in addition, frequent electricity failures do not allow prolonged storage of plasma at -20°c (this is therefore mostly wasted), although this issue can be overcome by solar powered freezers. ideally whole blood should be pathogen inactivated for which two methods are currently available. to reduce frequencies of acute hemolytic transfusion reactions again education and training to minimize clerical errors in the transfusion process are most important. extended testing for other rhesus antigens and k beside abo and rh-d in transfusion dependent hemoglobinopathy patients may help to reduce delayed hemolytic transfusion reactions. currently a leukodepleted pathogen-inactivated whole blood product might mostly serve the needs for blood transfusion in regions with limited infrastructure . the developed world should invest research efforts to develop such a product available at affordable costs. background: in modern transfusion medicine, serological investigations for blood cell antigens are complemented by genotyping arrays and pcr assays. whilst these platforms are informative in the majority of reference investigations, they are limited in their ability to define nucleotide variants associated with rare antigens and unable to detect novel variants potentially affecting antigenicity. next-generation sequencing is increasingly being employed in reference settings, providing information that cannot be obtained through these methods. whole genome and whole exome sequencing have been successfully employed in many investigations of novel and rare antigens, however concerns remain regarding the collection of genomic data unrelated to reference investigations, and reporting clinically significant incidental findings. these concerns can be addressed through the use of targeted sequencing panels. we report on the design, testing and efficacy of a panel providing a comprehensive genotyping profile for red cell, platelet and neutrophil antigens in a single test. aims: design a customised targeted exome sequencing panel for red cell, platelet and neutrophil antigen genes, and benchmark the efficacy against a commercial medical sequencing panel (illumina trusight one -tso). -test the panel and in-house genotype prediction script on sequence outputs from 51 samples with known red cell, platelet and neutrophil genotypes and phenotypes and determine whether predictions are concordant. methods: the panel was designed with 2654 probes covering exons of 64 genes associated with red cell, platelet and neutrophil antigens. using illumina nextera rapid capture technology, 51 samples were tested over five sequencing runs, on standard and micro chemistries, to determine optimal sample plexity per standard run, and the efficacy of smaller flow cells for lower throughput applications. an in-house python script was used to predict star-allele genotypes based on variants listed in isbt and embl databases. these predictions were compared to results from serology, snp array and previous tso data. results: coverage consistently averaged > 2509, with 94% of target at a quality of q30. optimal sample plexity for a standard run was determined to be 14 samples, allowing for sufficient coverage of all clinically significant variants. for red cell samples with previous typing data (excluding rh structural variants), the script correctly predicted 99.5% of snp based red cell genotypes. script predictions were 100% concordant for platelet genotypes, and four of five neutrophil antigen genotypes. hna2 genotypes defined by cd177 could not be reliably determined. the increased target coverage of the panel allowed for detection of a clinically significant heterozygous variant in scianna system, previously undetected by the tso panel due to extremely low coverage. additionally, a variant defining a potentially novel null allele was detected in the p1pk system. summary/conclusions: the panel demonstrates considerably higher coverage, quality and throughput compared to the tso and allows for detection of variants previously overlooked due to low sequencing coverage. up to 14 samples can be reliably sequenced in a single run. our script correctly predicts over 99% of snp based alleles; however, rh structural variants require further manual analysis. background: to ensure the safety of a transfusion it is critical to identify the blood type of both donor and recipient. serological methods for typing abo, rh and kel use monoclonal antibodies, however, typing reagents for rare blood groups are expensive, unavailable or unreliable. dna-based identification of human blood groups has been used to overcome these limitations and its application has reduced rates of alloimmunisation in chronically transfused patients. however, to date, the cost per sample has prevented the universal application of dna-based donor typing aims: to achieve universal adoption of dna based donor typing, the blood transfusion genomics consortium (bgc) set out to develop an affordable dna based platform, capable of typing all red cell antigens, hla class i and ii and human platelet antigens. methods: the uk biobank axiom array, previously used to type 600,000 uk citizens, was redesigned for donor typing using three approaches: i) mining transfusion medicine knowledge, e.g. isbt allele tables; ii) inclusion of loci associated with donor health; iii) extraction of all coding variants in relevant genes with a frequency > 1:20,000 identified in large-scale sequencing data. samples from nhsbt and sanquin blood donors (n = 7,871) were used for performance assessment. red cell and platelet antigens for each donor were inferred from genotypes using the bloodtyper algorithm and concordance with clinical serological typing results assessed. results: concordance between genotypic and serological typing results was 99.9% for 95,022 comparisons; 29 of the 89 discrepancies were serologically negative and genotypically positive for a given antigen (k/k, fy[a/b], lu[a/b]). in all cases dna variants known to modify or weaken antigen expression were detected, displaying the power of genotyping to detect variant 'weak-antigen' expression. across 48 antigens for which serology was available, genotyping provided a 3.6-fold increase in the number of typing results available per donor (47.9 vs 13.2). furthermore, genotyping provided data on an additional 224 clinically relevant antigens, allowing identification of antigen-negative donors and blood group identification for which antibodies are not commercially available. the power of a genotyped panel of donors to support patient management was demonstrated by a retrospective analysis of clinical cases referred to nhsbt. from 3,146 patient referrals with > 3 alloantibodies between 2014 and 2018, 772 unique alloantibody profiles were identified. we found that there was a 2.6-fold greater likelihood of finding o negative compatible donors for these patients when using genotyping data from the 4,721 nhsbt donors. importantly, the number of alloantibody combinations for which no compatible antigen-negative donor could be identified fell from 293 to 246, representing an additional 164 patients that could be provided with directly compatible blood using the same donors. summary/conclusions: through the bgc efforts an affordable fully automated genotyping platform, including the processes for quality assurance and data analysis, has been developed. furthermore, we have demonstrated the real-world benefits more extensive donor characterisation can provide when selecting blood for patients with multiple antibodies. the results of this international collaboration provide opportunities to introduce fully-automated genotype-based donor typing in a safe and cost-efficient manner in blood supply organisations. 3c-s06-04 biobank performs whole-genome sequencing (wgs) from individuals nation-wide. these data are suitable for allele frequency analysis to demonstrate gene expression and genetic profile in our population, also to estimate the significance of each antigen in transfusion practice. aims: we aim to provide and verify population-based blood group antigen profile using wgs and dna samples from taiwan biobank. methods: a near 1500 wgs and demographic data were analyzed. annotations of blood group antigen were performed according to variants from isbt allele tables, including 2 transcription factors; variants for the lewis system were obtained from previous studies. annotations of blood group variants were verified by 4 dna samples with targeted sequencing on illumina miseq, and specific variants were verified by 40 dna samples with the commercial genotyping kit or sanger sequencing. allele frequencies from wgs analysis were compared with population serology data using two-proportion z test. results: population-wide blood group antigens were analyzed, revealed in-depth antigen expression profiles in all systems (except ch/rg). the antigen frequencies from wgs were similar compared with published serology data, except for the antigens and possible explanations listed as follow, 1) m, n: insufficient sequencing reads, 2) c, c: identical rhce exon 2 with rhd exon 2 for c allele, 3) mur: insufficient read length/depth for gypa/gypb hybridization calling and individuals from high prevalence of mur antigen in aboriginal tribes were not enrolled. blood group antigen predictions and variants from wgs were accord to dna verification. furthermore, 13 systems shown no genetic variations, predicting uniform antigen expression in our population, and we can manage transfusion with minimum concerns for antigen mismatch in these systems. moreover, we found weak and null alleles in our population for blood group systems that we had previously no knowledge of, such as lan, jr, and vel. these variants were helped to identify a patient with anti-jr a carrying homozygous jr a null alleles. summary/conclusions: taiwan biobank wgs is suitable for full blood group antigen profile determination with few adjustments required for specific antigens. the population antigen allele frequency provides valuable insight to antigen significance in transfusion practice, matching strategy for our patients, and estimation of the likelihood to obtain for specific antigen negative blood from mass population. also, the genetic variants revealed in this study can help us to locate rare donors, and to integrate variations into routine donor blood group screening to provide suitable blood at a low cost efficiently. background: providing adequate amounts of safe, appropriately matched blood to meet the demands of an expanding and aging patient population presents a challenging global problem. for these reasons, sustainable in vitro sources of red cells may offer a desirable alternative to reliance on donor blood. the first stable immortalized early adult erythroblast cell line, bel-a2, has been shown to differentiate efficiently into mature, functional reticulocytes (trakarnsanga et al., nat commun. 8:14750, 2017) and consequently could provide a readily available tool for diagnostic use and proof of principle for future therapeutic use. aims: at ibgrl, next generation whole exome sequencing (wes) has been used previously to accurately predict blood group phenotype in a number of blood group systems, including abo, rh and mns (tilley & thornton, transfusion medicine 27 (suppl.2) :42, 2017). here we have used it to analyse and document bel-a2 blood group-related genotypes and predict blood group phenotypes. additional genes involved in cell-growth and enucleation were also analysed in order to further elucidate the characteristics of the bel-a2 cell-line. methods: bel-a2 cells (day 196) were cultured in expansion medium and genomic dna (gdna) was isolated from 1 9 10 6 cells on day 5. for wes, gdna libraries were prepared using nextera â rapid capture exome enrichment and sequenced on illumina â miseq. sequence alignments for 41 genes encoding all 36 known blood group systems and 12 further genes encoding transcription factors and cell enucleation-associated proteins were visualised using integrative genomics viewer, whilst illumina â variant studio was used to identify observed mutations. mutations in coding regions were used to determine bel-a2 genotype and predicted phenotype. results: good coverage of most of the selected genes was achieved. alignment of homologous blood group genes including rhd/rhce, gypa/gypb and c4a/c4b was problematic and additional analysis of coverage of these genes was required for accurate interpretation. despite a number of polymorphisms observed across the tested genes, bel-a2 did not express any novel or rare blood group antigens. genotyping results predicted a common antigenic profile, in agreement with previous serological and genotyping results where available. although a number of missense single nucleotide variations were detected in analysed genes, including cr1, cdan1 and tmx4, these were common polymorphic variants and unlikely to be of any functional significance. summary/conclusions: wes was used to determine bel-a2 genotype in relation to blood group genes and selected genes encoding transcription factors and proteins associated with cell enucleation. wes allowed accurate prediction of blood group phenotypes, showing full concordance with available serological data (trakarnsanga et al, 2017) . a small number of mutations were identified which are of unknown significance and require further work to determine any potential phenotypic effects. this complete record of the bel-a2 blood group-related exome will enable reliable gene editing strategies for future diagnostic and therapeutic purposes. additionally, knowledge of the full cell line exome will allow analysis of any emerging genes of interest and provide better insight into the mechanisms of erythroid differentiation and enucleation. background: emerging evidence, especially in neonates, has shown potential harm associated with liberal platelet transfusion strategies. very little evidence exists regarding optimal platelet transfusion thresholds in critically ill children. randomized controlled trials may be difficult due to lack of equipoise from providers. if regional variation in practice exists, comparative effectiveness studies may be an alternative approach. aims: to describe regional variation in platelet transfusion practices in critically ill children. methods: secondary analysis of a prospective, observational study. subjects were grouped according to region (north america, europe, middle east, asia and oceania) and nation. transfusions were analyzed as prophylactic (given to prevent bleeding) or therapeutic (given to treat bleeding). the primary outcome was the total platelet count (tpc) prior to transfusion. sub-groups analyses were performed in children with an underlying oncologic diagnosis and those supported by extracorporeal life support (ecls). the dosing and processing of the platelet transfusions were analyzed as secondary outcomes. results: five hundred and forty-nine children from 16 countries were enrolled (67% in north america, 17% in europe, 7% in oceania, 5% in asia, and 4% in the middle east). overall, the median (iqr) tpc prior to prophylactic transfusions (n = 360) differed significantly on a regional basis (p = 0.04) and ranged from 12 (8-41) x10 9 cells/l in the middle east to 45 (20-66) x10 9 cells/l in asia. the median tpc prior to prophylactic transfusions did not significantly differ between countries (p = 0.08), nor did the tpc prior to therapeutic transfusions (n = 189) differ on either a regional (p = 0.16) or national (p = 0.57) basis. for children supported by ecls (n = 90), there were no regional (p = 0.06) or national (p = 0.40) differences for prophylactic transfusions. however, significant differences in the tpc prior to therapeutic transfusions were observed on both a regional (p = 0.02) and national (0.04) basis with the middle east, in particular israel, transfusing at the lowest median (iqr) tpc [28 (16-81) x10 9 cells/l]. for children with an underlying oncologic diagnosis (n = 233), no differences were seen in the tpc for prophylactic transfusions (n = 175) on a regional (p = 0.19) or national (p = 0.20) basis. nor were differences seen in the tpc prior to therapeutic transfusions on a regional (0.86) or national (p = 0.33) basis. there was significant variability in the dosing of platelet transfusions on both a regional (p < 0.001) and national basis (p < 0.001). the median (iqr) dose based on volume ranged from 8.4 (5.0-11.2) ml/kg in north america to 12.6 (9.8-16.0) ml/kg in europe. the vast majority of transfusions were leukoreduced and irradiated but significant variation exists in storage duration on both a regional (p < 0.001) and national (p < 0.001) basis. summary/conclusions: regional and national variation exists in platelet transfusion practices among critically ill children, especially in those given therapeutic transfusions while supported by ecls. considering this variation, comparative effectiveness studies may be an appropriate approach to gain evidence to optimize platelet transfusion thresholds. background: the optimal threshold for prophylactic platelet (plts) transfusion in pediatric patients with cancer is still controversial and current clinical practice comes from studies on adults and on inpatient setting. the international guidelines (icmtg, 2015) recommend, for all age patients, a prophylactic platelet transfusion when plts count is ≤ 10 9 10 11 /l and a platelet dose of 1.1 9 10 11 per square meter (sm) of body-surface area (bsa) in inpatient and 2.2 9 10 11 /sm in outpatient setting. aims: in january 2018 we started in our children's hospital a prospective protocol in order to evaluate the impact on bleeding risk of current clinical practice of prophylactic platelet transfusion in inpatients and outpatients onco-haematological patients. methods: bsa was calculated from age-standardized weight. inpatients received a dose per transfusion of 1.1 9 10 11 /sm and outpatients a dose per transfusion of 2.2 9 10 11 /sm. platelets were transfused when the count was ≤ 10 9 10 11 /l or in presence of bleeding signs; pediatric aliquots were obtained from buffy coat derived pooled platelet concentrates or apheresis platelet concentrates, according disponibility. results: from january 2018 to december 2018 a total of 9839 platelet pediatric aliquots were transfused: 5534 (56.2%) were obtained from apheresis platelet concentrates and 4305 (43.8%) from buffy-coat-derived pooled platelet concentrates. the majority of platelets pediatric aliquots (7505-76.3%) were transfused to onco-hematological patients undergoing hematopoietic stem cells transplant (hsct) or conventional chemotherapy. among them, 6365 aliquots were transfused in inpatient setting: 1675 (17%) in the hematology unit, 2772 (28.2%) in the oncology unit and 1918 (19.5%) in hsct unit. a total of 1140 (11.5%) aliquots were transfused in outpatient setting: 840 (8.5%) to patients affected by hematological malignancies and 300 (3%) to patients with solid tumors. five major bleeding events (who grade ≥ 3) were observed during the study period and all of them occurred in hospitalized patients. two patients with solid neoplasm developed a who grade 3 bleeding event. two patients with hematologic malignancies and a patient with neuroblastoma (n = 3, 1.2%) developed intracranial bleeding (who grade 4). the platelet count at the time of the event was 22 9 10 9 /l, 25 9 10 9 /l and 8 9 10 9 /l, respectively. summary/conclusions: our results showed the efficacy, in onco-hematological pediatric patients, of a prophylactic platelet transfusion protocol based on international guidelines: a very low incidence of who grade 4 bleeding has been observed in inpatients setting only (1.2% vs 1% of plado trial, sj slichter, nejm, 2010) , while in outpatients setting the double platelet dose prevents the major bleeding event (who grade ≥ 3) occurrence. background: the problem of blood-borne infections remains relevant in transfusion medicine. pathogen reduction technologies (prt) provide a preventive approach to a wide range of transfusion-transmitted infectious diseases. to date, prt widely used for platelet concentrates and blood plasma, however, the use of these technologies for the treatment of red blood cell-containing blood products undergo research. aims: the aim of our study was to evaluate the safety and efficacy of transfusions of pathogen-reduced (test group) red blood cell suspensions (rbcs) and compare these data with gamma-irradiated rbcs (control group). methods: the technology based on the combined action of riboflavin and ultraviolet (mirasol prt, terumo bct, belgium) was used to reduce pathogens in whole blood. subsequently, the rbcs of the test group were derived from pathogenreduced whole blood. the control rbcs were irradiated at the gammacell 3000 elite (best theratronics, canada) at a dose of 25 gray. all rbcs were used for transfusion for 14 days from the harvest day. 70 pediatric patients with various oncological and hematological diseases were randomized to 2 groups of 35 members in each group. the test group of patients received transfusions of a pathogen-reduced rbcs; the control group received transfusions of a gamma-irradiated rbcs. the next day after transfusion were assessed hemoglobin and hematocrit increment, the level of potassium and haptoglobin in the patients' serum, the frequency and severity of transfusion reactions. 3-5 days after the transfusion, the direct antiglobulin test (dat) was performed and after 14-21 days the indirect antiglobulin test (iat) was performed. the interval to the next need for transfusion was also evaluated. results: the increase in hemoglobin and hematocrit (p = 0.2), as well as the concentration of potassium (p = 0.44) and haptoglobin (p = 0.25) in the patients' serum after the transfusion did not differ between groups. none of the patients in both groups had hyperkalemia after transfusion. in each group, two patients had febrile non-hemolytic transfusion reactions of comparable severity (p = 1). all dat and iat tests were negative in both groups. the interval between transfusions were not significantly different between groups (p = 0.39). only in the test group was found the correlation between the increase in the hemoglobin and hematocrit values with the volume of transfusion, with the dose and the adjusted dose of hemoglobin obtained for the transfusion on body weight. and in this group was found inverse correlation between the hemoglobin and hematocrit increment with the level of hemolysis in the rbcs. summary/conclusions: we found that the clinical efficacy and safety of rbcs of the compared groups did not differ. there was no evidence of immune elimination and allo-sensitization caused by pathogen-reduced rbcs. according to our data, the spectrum of efficiency and safety indicators of pathogen-reduced rbcs is no worse than that of gamma-irradiated rbcs, provided that rbcs is used for 14 days of storage. the founded correlation suggests that the efficiency of pathogen-reduced rbcs transfusions is more dependent on the characteristics of the rbcs. background: patient blood management (pbm) programs are expanding at an international level. a recent nationally representative study from united states observed pediatric age group as the only age group showing lack of objective evidence of pbm initiatives (goel et al, jama 2018) . aims: this study aims to identify trends in peri-operative blood utilization in children undergoing elective and non-elective surgeries over 5 years duration from 2012 to 2016. methods: using 5 years data (2012) (2013) (2014) (2015) (2016) perioperative transfusions decreased steadily per year from 6.4% in 2012 to 5.5% (14% cumulative decline) in 2016 for children of all ages (or 0.966; 95% ci 0.957-0.976; p trend < 0.001). the cumulative change in elective procedures was 9.2% versus 27.2% decrease in urgent/emergent procedures (p trend < 0.001). summary/conclusions: in this large prospective registry study of > 350,000 children undergoing elective/non-elective surgeries, a statistically significant decrease in utilization of peri-operative rbc transfusions was seen across 5 years from 2012 through 2016 with more significant decrease in urgent/emergent procedures than elective procedures while these findings need evaluation for non-surgical indications of transfusion, these results may provide first evidence of peri-operative pediatric patient blood management strategies being implemented to optimize transfusions in pediatric population. adverse events -tti, immune interactions and risk 3c-s08-01 transfusion-transmitted infections (tti) are a long-standing and well recognized concern in medicine, which is tackled on the highest level to guarantee the safety of the transfusion procedure for all stake holders. these include the recipient patients, the donating volunteers, the health care workers involved, and their respective contact persons. accordingly, current national and international guidelines including expert societies and the who provide medical, technical, and legal frameworks, which are the basis for the standard operating procedures. nevertheless, there are important challenges, which render tti a "moving target", and reflect the dynamics in three main areas. first, a change in the type and number of recipient patients with past or ongoing immunomodulatory / immunodeficiency component (examples being hiv/aids, sot, allogenic hct, monoclonal antibody therapies, small molecule inhibitors). second, changing exposure to known agents in donors due to global travel, migration and displacement, as well as environmental/climate change. third, discovery and diagnostics of old and new agents with their known or presumed impact as tti. these aspects will require careful review of data and studies, and judicious discussion of the potential action such as selection versus close monitoring to keep tti rates as low as possible, to deliver maximal safety of patients and stakeholders. background: the implementation of nucleic acid testing (nat) and the development of sensitive and specific serologic assays to detect hbsag and anti-hbc antibodies significantly reduced the risk of hbv transfusion-transmission. the apparent redundant testing for two direct viral markers prompted debates on maintaining hbsag screening, particularly in low endemic countries where blood donations are screened for anti-hbc. however, frequencies of 2-20% of hbsag-confirmed positive/nat negative donations have been reported depending on the sensitivity limit of the molecular assays used. the nature of this discrepancy between hbsag and dna remains largely unknown and it is essential to evaluate any potential negative impact on blood safety before considering removing hbsag testing. aims: the prevalence in blood donors and the molecular mechanisms responsible for a persistent undetectable or barely detectable level of viral replication in the presence of a sustained hbsag production were investigated in a collaborative study including five laboratories/blood centers in europe and south africa. discrepancy between viral dna and hbsag levels suggested the presence of mutations that may negatively affect hbv replication and/or infectious viral particle production. methods: donor samples from france, south africa, poland, and croatia were selected for having hbsag levels ≥ 100 iu/ml and being id-nat (procleix-ultrio plus tm [95% lod: 3 iu/ml]) non-reactive/non-repeatable reactive (nr/nrr) with undetectable viral load (vl) or < 6 iu/ml (n = 44) or nat repeat reactive (rr) with vl < 6 iu/ml (n = 32). french samples initially tested nat nr/nrr with procleix-ultrio (lod 95%: 11 iu/ml) were retested with ultrio plus prior inclusion in the study. hbv dna load was quantified (cobas taqman hbv [loq: 6 iu/ml]). hbv dna was purified from 5 to 12 ml of plasma after ultracentrifugation. the whole hbv genome, pre-s/s, precore/core and bcp regions were amplified and sequenced. results: following viral concentration, hbv dna presence was confirmed in 79% of all samples with undetectable or vl < 6 iu/ml. hbv genotypes were a1 (33.3%), a2 (18.4%), a3 (3.3%), b (3.3%), c (1.7%), d (25%), and e (15%). all samples were anti-hbc positive and 73% of ultrio-negative samples tested positive with ultrio plus. unusual 1-2 nt insertions/deletions identified in bcp regulatory elements (tata boxes, pginr, epsilon domain) suggest altered viral replication. amino acid substitutions (n = 16) or deletions (n = 4) at positions reported involved in nucleocapsid formation, particle envelopment and virion formation were observed in the core protein of 30 samples. the replicative properties of the bcp and core variants are currently evaluated in vitro as a surrogate model for direct infectivity testing. preliminary results indicate that the variants tested so far have replicative capabilities similar to those of control viruses. analysis of pol, s, and hbx proteins is ongoing. summary/conclusions: these data confirmed the presence of extremely low level of circulating dna-containing viral particles in id-nat non-reactive or nonrepeated reactive blood donations with concomitant high hbsag levels and anti-hbc reactivity. despite the presence of mutations in the viral genomes potentially affecting virion production, preliminary data indicate that some of the viruses in plasma retain the ability to replicate in vitro and to constitute a potential infectious risk. 3c-s08-03 background: in switzerland highly sensitive nucleic acid screening in an individual donation format for hepatitis b virus (hbv id-nat) and hepatitis b surface antigen (hbsag) detection is mandatorily performed (guidelines of swiss transfusion src, switzerland). since 1998, hbv (hb) vaccination is recommended in switzerland for children and adolescents until the age of 15 and for adults belonging to known risk groups. aims: to highlight that low anti-hbs titers several years following hbv vaccination still confer protection and enable the host immune system to clear hbv dna without development of serologic markers of disease. methods: a retrospective donor interview was conducted to complete information not covered by the questions included in the standard donor questionnaire. routine hbv serological donor screening was performed on a quadriga system (diasorin, former siemens) with the enzygnost hbsag assay (diasorin, former siemens). further hbv tests were performed on the abbott architect i1000 analyser (hbsag neutralisation, hbeag, anti-hbc igg/igm, anti-hbc igm, anti-hbe and anti-hbs). routine id-nat screening for hiv/hcv/hbv was performed with the roche cobas mpx test on a roche cobas 8800 platform. hbv id-nat positive samples were confirmed with a quantitative hbv nat assay (abbott). background: hepatitis b core-related antigen (hbcrag) is a structural antigen of hbv, consisting in hbcag, hbeag and the p22cr precore protein. quantitative hbcrag measurement is a sensitive marker of viral replication reflecting the cccdna content and persistence of disease. hbcrag positivity was found to be a significant risk factor of hbv reactivation in hbsag-, anti-hbc+, hbv dna-patients (occult hbv infection, obi) undergoing immunosuppressive therapy. aims: no data about hbcrag status in apparently healthy subject with obi are available. the aim of this study was to analyse this marker in our cohort of obi blood donors. methods: hbcrag was measured in 69 blood donors confirmed to be carriers of obi (hbsag-, hbv dna+). of them, 59/69 (85.5%) donors were anti-hbc positive, and 10 (14.5%) negative. 18 donors had both anti-hbc and anti-hbe reactivities. a group of 11 young blood donors vaccinated for hbv infection (hbsag-, hbv dna-, anti-hbc-), and 9 patients with chronic hbv infection (hbsag+, hbv dna+) were used as negative and positive controls group, respectively. serum hbcrag was measured using a chemiluminescent enzyme immunoassay on the lumipulse g600 automated analyzer (fujirebio, tokyo, japan). the lower limit of detection (lod) of the quantitative assay is 2 logu/ml and the lower limit of quantification (loq) is > 3 logu/ml, due to nonlinearity results between 2 and 3 logu/ml. levels of hbcrag were tested in the three groups and analysed in comparison to the presence of anti-hbc and anti-hbe. statistical analysis was performed by the ibm statistics spss 19.0.0. results: all donors in the negative control group had undetectable hbcrag levels, whereas all patients in the positive control group have detectable hbcrag (mean value: 3.0 logu/ml, range 2.0-4.9), confirming that individuals without prior exposure to hbv would not have detectable hbcrag. hbcrag was detectable in 34/69 obi donors (49.3%), with a mean value of 2.21 logu/ml (range 2.0-3.20). hbcrag could be measured only in 2 obi donors (3.0 and 3.2 logu/ml), being below the loq of the test in the majority of obi (32/34). considering the presence of anti-hbc, hbcrag was detected in 29/59 (49.1%) anti-hbc+ and in 5/10 (50%) anti-hbc-obi, with no significant difference in their mean levels (2.21 ae 0.32 vs 2.14 ae 0.26; p = 0.44). interestingly, the presence of anti-hbe (18/62) was independently associated with higher hbcrag levels (2.38 ae 0.42 vs 2.14 ae 0.22; p = 0.03). summary/conclusions: identification of donors with obi is critical to prevent the risk of hbv transfusion-transmission. being hbcrag associated with the cccdna content and replication, our results suggest that the presence of hbcrag, even if not quantifiable, could be useful marker to confirm the occult infection status, even in anti-hbc negative donors. the association between hbcrag, anti-hbc and anti-hbe could also be a useful marker to identify obi donors with a higher risk of hbv reactivation. 3c-s08-05 hc group. human peripheral blood mononuclear cells (pbmcs) from blood donors were stimulated with hbv polypeptides pool in vitro. t cell proliferation assays (cfse) was used to detecting t cell proliferation, enzyme-linked immunospot assay (elispot) was used to detecting the frequency of hbv-specific ifn-c secreted t cells. spss 20.0 statistical analysis software was used for statistical analysis. the measurement data of normal distribution were tested by two independent samples t test; and the comparison between multiple groups was analyzed by one-way anova. mann-whitney u test was used for comparison between non-normal data sets. p < 0.05 was considered statistically significant. results: 1. proliferation characteristics of t cells. the proliferation of cd4 + t lymphocytes was mainly stimulated by specific hbv polypeptide pool, and the proliferation rates of obi group and chb group were significantly higher than those of hc group (3.0%, 3.3% vs. 1.7%), with significant difference (3.0% vs. 1.7%, p = 0.016, 3.3% vs 1.7%, p < 0.001). 2. the frequency of specific ifn-c secreted t cells. the response intensity of the obi group (25 sfc/10 6 pbmcs) and chb group (25 sfc/10 6 pbmcs) was higher than that of the hc group (5 sfc/10 6 pbmcs) under the stimulation of hbv polypeptide pool, and the positive rate of t cell response to the stimulation of hbv polypeptide pool was the highest in the obi group (64.0%). summary/conclusions: both obi and chb had higher rates of hbv-specific t effector cell proliferation and ifn-c secretion than the healthy control group. compared with the chb group, obi group had a higher positive rate of t cell response, which may be one of the causes of host immunity resulting in obi. further studies on other immune factors are required. background: western blood transfusion practices are currently changing due to various drivers such as blood management policies, ongoing technological developments, and new therapeutic options. in the netherlands, as in many high-income countries, these have resulted in a diminishing trend of red blood cells. therefore, it is important for blood bank management to anticipate the future demand of blood products for the sake of medium and long term decision making. to support this decision making, we have employed scenario development, which is used in many other sectors (such as finance and transportation) and can also be applied to blood transfusion. building upon a prior literature review and semi-structured interviews of international experts, we gathered experts together for scenario sessions to assess the opportunities and threats for sanquin's medium-term (15-20 years) strategy using an online platform and face-to-face discussions. aims: to assess for opportunities, threats, and the organizational implications thereof for the medium-term future of sanquin, the dutch national blood bank. methods: twenty-one multidisciplinary experts in blood transfusion agreed to participate and were separated into two groups for half-day interactive sessions. using an iterative process through an online platform, experts brainstormed opportunities and threats for sanquin, which were categorized into themes. these themes were ranked according to importance and certainty, and through consensus, experts chose two themes with high impact and high uncertainty. for these chosen themes, specific actions for the blood bank were listed to mitigate and/or enhance the threat or opportunity. discussions were ample throughout. results: with regards to opportunities and threats for sanquin's medium term strategy, experts brainstormed many ideas and categorized them under 10 themes: political context/ changing legislation, novel products and alternative applications, donors, international markets, commercialization, digitalization, change in perceptions, research, demand, and organizational structure. after ranking for importance and certainty, six themes were chosen: change in perceptions, international markets, political context (opportunities), demand (opportunities), research (vulnerabilities), and donor (vulnerabilities). for each of these themes experts provided specific actions for the organizations to mitigate threats or stimulate opportunities accordingly. these actions included increased transparency and improved communication with the (donor) public, lobbying in political spheres, increased activities in educational institutes and large funding organizations, and creating and collaborating on novel blood products on an international level, to name a few. summary/conclusions: these results show that mapping and assessing a blood bank's future using a multi-disciplinary group of experts is conducive as an effective means of collection a diverse range of opportunities and threats. this provides an opportunity for blood bank management to become proactive towards these potential opportunities and threats and possibly evolve future strategies for the organization. showed that iron-deficient female blood donors were more likely to have depressive symptoms than non-iron deficient female blood donors. among participants with depressive symptoms, females with low plasma ferritin levels had significantly increased odds for reporting a "feeling of lacking energy and strength" (or = 2.11; 95% ci: 1.03-4.31). as it is known that blood donors are at an increased risk of iron deficiency, it is important to determine whether those genetically predisposed to lower plasma ferritin levels have a higher risk of experiencing the tiredness/lack of energy symptom. aims: to investigate whether there is an association between polygenic risk scores (prss) based on plasma ferritin levels and the tiredness/lack of energy symptom in blood donors. methods: the dbds is an ongoing nationwide blood donor cohort, of which genome-wide genotype data are available for 72,000 participants. genotyping was performed using the infinium global screening array (illumina â ) and imputation was achieved based on a scandinavian reference genome. ferritin prss, based on an icelandic ferritin gwas (n = 150,000), were calculated for all dbds participants. 12,903 female donors were available for the analysis. data on depressive symptoms were obtained using the validated major depression inventory scale (mdi), a selfreport mood questionnaire, which assesses the presence of 10 depressive symptoms. a donor was classified as "tired" if they responded "all the time" or "most of the time" to the question "how often do you feel that you lacked energy and strength?". logistic regression analysis was performed, adjusting for age. for generating the quantile plots, the participants were distributed evenly into six quantiles based on their prs, whereby quantile 1 contained the donors with the lowest prss (genetically predisposed to lower ferritin levels) and was set as the reference quantile with or = 1 from the age-adjusted regression analysis (tiredness~quantile). results: prss in females ranged between -0.42 and 0.50 (mean 0.01). a total of 12,523 female donors were classified as "not tired" and 380 (2.9%) were classified as "tired". no significant difference in ferritin prs was found between "tired" and "not tired" female donors (tired mean prs: 0.00; not tired mean prs: 0.01). an age-adjusted logistic regression model found this to be insignificant (or: 0.74, 95% ci: 0.33-1.66), p = 0.47). to visualise the lack of association, a quantile plot was created, separating the female donors into six equal quantiles based on their prs. no clear trend was observed; donors with the highest prss (in quantile 6) had or = 1.02 (p = 0.93) of being tired when compared to those in quantile 1 (or set as 1). summary/conclusions: no significant association was found between the ferritin prss of female blood donors and the tiredness/lack of energy symptom. further studies are needed to understand the effect of blood donation versus genetic constitution on tiredness among female iron-deficient blood donors. background: antiretroviral therapy (art) is critical for the control of clinical progression of human immunodeficiency virus (hiv) infections. however, the outcome of art could be limited by drug resistance-associated mutations (drms), even lead to the transmission of drug-resistant hiv to treatment na€ ıve patients such as blood donors, which is a huge concern to art. drms surveillance in hiv infected groups is strongly recommended by world health organization. characteristics of genetic diversity and drms of hiv among blood donors may provide comprehensive data to monitor viral evolution and optimize art, play important roles in blood safety. aims: limited data concerning the epidemic of hiv-1 subtypes and drms of blood donors is available in china. this study is to investigate genetic characteristics and drms of hiv-1 infected blood donors. methods: from 2016-2018, 177 blood donations collected from 24 blood centers, covering almost the whole of china, were confirmed as hiv-1 positive by national centers for clinical laboratories using abbott realtime hiv-1 assay or cobas taq-man hiv-1 test, version 2.0. then hiv-1 gag (973 bp, hxb2: 1074-2044), pol genes (1454 bp, hxb2: 2068-3521) (encoding the whole protease (pr) and a part of reverse transcriptase (rt)) was sequenced after viral rna extraction and amplification. hiv-1 subtype based on gag and pr-rt regions was determined by comprehensive analyses of los alamos hiv blast tool, rega hiv-1 subtyping tool, phylogenetic trees and online jphmm program. drms analysis was performed in the stanford hiv drug resistance database. results: among 177 donations, gag and pr-rt regions of 160 samples were sequenced successfully. the distribution of hiv-1 genotype was as follows: crf_07bc = 66 (38.8%), crf_01ae = 58 (36.3%), b = 9 (5.6%), crf_08bc = 2 (1.3%), crf55_01b = 4 (2.5%), crf59_01b = 1 (0.6%), crf65_cpx = 1 (0.6%), crf67_01b = 2 (1.3%), crf79_0107 = 1 (0.6 %), crf85_bc = 1 (0.6 %), urf_0107 = 6 (3.8%) and urf = 9 (5.6%). 26 of 160 hiv-1 isolates were identified to have drms. there were 4 (15.4%, 4/26) protease inhibitors (pi) accessory drms, 3 pi major drms and 20 (76.9%, 20/26) non-nucleoside reverse transcriptase inhibitors (nnrti) drms. most of blood donors with drms were crf01_ae and crf07_bc (61.5%, 16/26). 3 of 4 pi accessory drms were q58e. the pi major drms included m46l, m46i and n88s. n88s could result in hlr to atazanavir (atv) and nfv, llr to indinavir (idv) and saquinavir (sqv). v179d/e is main nnrti drm (70.0%, 14/20). a combination of v179d and k103r among two samples acted synergistically to reduce efavirenz (efv) and nevirapine (nvp) susceptibility. furthermore, two blood donors with k103n mutation in reverse transcriptase gene had high level-resistance to efv and nvp. summary/conclusions: overall, the most prevalent subtypes among blood donors in the study were crf07_bc (38.8%), crf01_ae (36.3%). besides, other rare crfs and several urf_0107 and urfs were also found in these hiv-1 isolates, which suggested the epidemic of hiv has been shifted from high risk populations into general populations, including blood donors in china. drms were observed in 16.3% donors in the study, which may result in resistance to pis and nnrtis, especially the hiv-1 variants with n88s mutation in pr gene and k103n mutation in rt gene. in summary, our findings indicate that increasing diversity of hiv-1 in blood donors and remind us the necessity of timely genotypic drug resistance monitoring and molecular epidemiology surveillance of hiv-1 among blood donors. background: labeling of platelets is required to measure the recovery and survival of transfused platelets in vivo. currently a radioactive method is used to label platelets. however, its' application is limited, due to safety issues and the inability to isolate transfused platelets out of the circulation. biotin-labeling of platelets is an attractive non-radioactive option, however, no validated protocol to biotinylate platelets is currently available for clinical purposes. aims: the aim of this study is to develop a simple, standardized, reproducible method to label platelets with biotin as a non-radioactive alternative to trace transfused platelets in vivo. methods: six pooled buffy coats derived platelet concentrates (pcs) stored in 100% plasma were biotinylated at day 1 and day 7 of storage. to distinguish the effect of the processing steps from the effects of biotin incubation, 'sham' samples were processed. for the biotinylation procedure, 50 ml of pcs was washed twice and incubated with 5 mg/l biotin, dissolved in phosphate buffered saline-pas-e (1:9), for 30 min. stability of the biotin labeled platelets after irradiation was tested. annexin v and cd62p expression were assessed as measures of platelet activation. applicability of this method to other platelet products was assessed in three pooled pcs stored in 65% pas-e and three single donor apheresis pcs. results: the method was reproducible performed in a closed system. after biotinylation, 98.4% ae 0.9% of platelets were labeled. platelet counts, ph and 'swirling' were within the range accepted by the dutch blood bank for standard platelet products. the number of annexin v positive cells was not significantly altered by the biotinylation procedure in both fresh and stored platelets. in contrast, cd62p expression was increased in biotinylated platelets 48.4% iqr(41.7-56.2%) compared to the control samples 12.3% iqr(9.5-12.7%) on day 1 of storage. however, biotinylated platelets were not more activated compared to sham samples 50% iqr(41.7-56.2%). thus only the procedural steps led to increased cd62p expression and not the biotin label itself. all samples showed maximal response to thrombin receptor-activating peptide. for platelets labeled at day 7, a similar pattern was observed. irradiation of biotin labeled platelets did not alter the stability of the biotin label nor cell quality. furthermore this method is also applicable to pooled pcs stored in pas-e and apheresis pcs, with similar patterns in annexin v and cd62p expression. summary/conclusions: we developed a standardized and reproducible protocol according to good practice guidelines (gpg) standards, for biotin-labeling of platelets for clinical purposes. the procedural steps, which are similar to the steps used for production of hyperconcentrated platelet products, led to an increased cd62p expression, but did not alter the annexin v expression. this method can be applied as non-radioactive alternative to trace and recover transfused platelets in vivo. blocking activity over the prototypic chs4 insulator in cell lines and substantially reducing genotoxicity in a c-retroviral vector-mediated carcinogenesis mouse model. in contrast to chs4, these insulators are small-sized (119-284 bp vs 1.2 kb) and can be easily accommodated in gt vectors without detrimentally affecting vector titers. aims: we aimed to test whether a1, one of the newly discovered cis, could reduce vector-mediated genotoxicity in the challenging context of sin-lvs, by insulating a therapeutic globin-vector. methods: we tested the genotoxicity effect in the il-3-dependent 32d cells, which upon transduction with oncogenic vectors become il-3-independent, leading to transformation. 32d cells were transduced with sin-lvs: the b-globin-τνs9.3.55-, the insulated b-globin-a1-tns9.3.55and the oncogenic sffv-gfp-vector. transduced cells were expanded in 10% il-3 and transduction efficiency was determined by vector copy number (vcn). transduced 32d cells were seeded in methylcellulose with 10% or 0-1% il-3 to detect the il-3-independent and potentially transformed clones. the il-3-independent clones were further expanded in 10% il-3 and infused in partially myeloablated and il-3-treated c3h/hej mice. wbc analysis, blood smears and bone marrow(bm) cytospins were performed. results: the a1 insulator did not negatively affect vector titers (τνs9.3.55, a1-tns9.3.55, sffv-gfp: 2.8, 1.8, 2.5x10^8 iu/ml, respectively). 32d cells were successfully transduced with all vectors (%vcn positive colonies: 40-100%) and expanded up to 400-fold. the a1-insulator decreased the number of il-3-independent colonies by 78-93% over the uninsulated vectors. the uninsulated vector-transduced, il-3-independent colonies, were greatly expanded in culture with 10% il-3 over the a1-transduced colonies (sffv, τνs9.3.55, a1-tns9.3.55: 48, 40, 10 fold change, respectively). il-3 independence as a transformation event was confirmed in vivo by the development of overt leukemia (hyperleukocytosis, splenomegaly, bm-and extramedullary site-infiltration) in mice transplanted with the il-3-independent and expanded colonies. summary/conclusions: under forced oncogenic conditions, the a1 insulator effectively protected a therapeutic vector from vector-mediated genotoxicity. a1 may serve as a safety feature in the construction of globin-sin-lvs. background: novel rare nucleotide substitutions are frequently identified in rhd, the gene encoding the immunogenic d antigen of the clinically-relevant rh blood group system, resulting in d variant phenotype. so far, it has been commonly accepted that substitutions of amino acids located either in a transmembrane or intracellular domain of the rhd protein induce weak d phenotype, i.e. reduced d antigen density at the surface of red blood cells. recently we showed by functional analysis using a "minigene splicing assay" (msa) that a decrease in d antigen expression may be due also to alteration of cellular splicing. aims: here we pay attention to the general disruption of this mechanism and the related phenotypic consequences in novel and previously reported single-nucleotide variations in rhd. we then sought to characterize functionally by msa novel candidate splicing variants in rhd. then we extended the project by studying prospectively all single-nucleotide variations reported in rhd exons, in order to assess globally the correlation between in silico prediction and functional analysis and to gain insights into the reliability of bioinformatics tools in line with the available phenotypic and/or clinical data. methods: seventeen novel or uncharacterized rhd variations, including missense, synonymous and intronic substitutions, were selected for functional analysis by msa in human cell models. a second set, including 46 missense variants reported in rhd exons 6 and 7, was further analyzed. functional data were compared with an algorithm derived from the quepasa method and tools available in the alamut suite. a published 3d protein model was used to visualize the location of missense amino acid substitutions and to assess potentially their respective phenotypic consequence. results: a novel "universal" minigene was validated and used successfully to characterize eleven novel splicing variants. those variants include six intronic and four missense substitutions close to the consensus dinucleotide splice sites, as well as the c.1065c>t synonymous variation associated with a weak d phenotype, which creates a de novo splice site. very interestingly, c.1154g>t (gly385val; d-negative) disrupts totally normal splicing, while c.1154g>c (gly385ala; weak d) and c.1154g>a (gly385asp; d-negative) only partially alter the mechanism. further visualization of amino acid changes in a 3d model suggests that gly385asp, but not gly385ala, dramatically impair rhd protein structure/folding. subsequently the global analysis of mutations in rhd exons 6 and 7 by msa showed that inclusion of whole exon sequence in the mature transcript is significantly reduced in 15/46 (32.6%) variants, which correlates well with the quepasa-like prediction (sensibility = 0.93, specificity = 0.94). additionally, while normal exon inclusion is affected by c.1012c>g (weak d type 70), the associated leu338val substitution does not seem to be deleterious to the protein. summary/conclusions: on the basis of our functional data, this work shows that splicing disruption in the presence of rhd variants is a common and general mechanism that may act independently or synergistically with alteration of protein structure through amino acid substitutions, resulting in a weak d phenotype. it also illustrates the potency of combining functional tests and in silico tools towards the phenotypic/clinical interpretation of rare variants. background/aims: monetary and non-monetary incentives may support blood services in recruiting blood donors but have also been criticized for violating ethical principles and threatening blood safety by attracting donors with a high risk for infectious diseases. although incentives for blood donors have been discussed extensively over the past decades, empirical research on this topic remains limited. the aim of this study was to describe attitudes towards incentives for blood donors in europe and show donor return rates of compensated and non-compensated blood donors in south-west germany. methods: first, we present results of a secondary analysis of the eurobarometer, a nationally representative survey in all member states of the european union. in 2014, participants were asked to evaluate eight potential incentives for blood donations as acceptable or unacceptable. these incentives were refreshments (e.g. coffee), physical check-ups (e.g. blood pressure), free (testing) laboratory parameters, free medical treatment, complimentary items (e.g. first aid kits), monetary travel reimbursements, additional cash reimbursements, and release from work. second, we conducted a retrospective analysis of donor return patterns of 6.210 compensated and 68.205 non-compensated donors who started donating blood at mobile and fixed donation sites. compensated donors received either 25 eur as a regular reimbursement for their expenses (at a fixed donation site), in accordance with the german transfusion law, or a singular free entrance for an amusement park (at a mobile donation site). these compensated donors were compared with noncompensated donors who started either at a fixed or mobile donation site. chisquare statistics were used to test for differences in regular donor status after 24, 36, 48 and 72 months between compensated and non-compensated first-time donors. results: among german participants of the eurobarometer, physical check-ups (51.9%), refreshments (50.0%) and free (testing) laboratory parameters (38.3%) showed the highest acceptance as an incentive for blood donors. travel reimbursements and free medical treatment were rated as acceptable by 28.6% and 25.3%, respectively. the lowest acceptance was for release from work (17.8%), complementary items (13.9%) and additional cash reimbursement (11.8%). interestingly, the acceptance of potential incentives varies considerably across europe. in south-west germany, donor return of first-time donors differed significantly by type of compensation. among compensated first-time donors, who received 25 eur as a monetary reimbursement, the proportion of regular donors after 48 months (19.9%) was significantly higher than among comparable non-compensated donors (9.2%). however, a non-monetary compensation (free entrance) did not increase donor return rates. conclusion: the eurobarometer survey indicates that in most european countries monetary incentives are only accepted by a small minority. refreshments, checkups, free (testing) laboratory parameters and free medical treatment were most popular as incentives for blood donors. however, results of our four non-randomized donor samples from south-west germany suggest that monetary compensation may increase the likelihood of donors returning to fixed donation sites. regular monetary reward may therefore help to recruit regular donors especially in urban settings. incidentally, non-monetary compensation by a free entrance, however, may not affect donor return. background: previous research showed that whole blood (wb) donors that are temporarily deferred on-site are at higher risk of lapsing, yet very little studies have focused on differentiating the effects that different deferral reasons (e.g., travel, hemoglobin [hb]) may have on donor lapse. in addition, donor experience (i.e., firsttime or repeat donor) has also previously been found to affect donor lapse, yet novice (1-5 prior donations) and reactivated donors (returning after years of not donating) may respond differently. finally, it is currently unclear how and why different deferral reasons and donor experience interact in influencing donor lapse. aims: our aims were to understand 1) how deferral reasons and donor experience jointly affect donor lapse, and 2) why donors may lapse after temporary deferral. methods: a mixed methods approach was used. first, we used sanquin's donor database for a quantitative analysis of return behavior of all dutch wb donors between 2013 and 2015 (n = 343,564). the first wb donation for each donor was identified as the target donation. lapse was defined as non-return within a followup period of two years after the target donation. target donations included 25% new donors, 41% novice donors, 30% experienced donors, and 4% reactivated donors. deferral reasons included travel, hb, medical short-term (<28 days duration), medical long-term (>28 days duration), and miscellaneous. next, we interviewed 31 temporarily deferred donors to understand the deferral process from their perspective. semi-structured interviews were used to understand how these donors cognitively and emotionally experienced on-site temporary deferral. we analyzed the interviews (using the framework approach, cf. hillgrove et al., bmc public health, 2012) to identify key topics and underlying themes. results: of target donations, 11% were deferred, mostly for travel (40%), medical short-term (23%), and hb (19%). survival and time-to-events methods showed that the different deferral reasons and donor experience levels differentially impacted donor return or lapse. importantly, experience and deferral interacted in influencing return (rate). for instance, deferred new donors were more likely to lapse than eligible or experienced donors (ors < 0.75, p's<0.001). even though deferral also affected return of experienced donors, this effect was smaller or even non-existent for certain deferral reasons (e.g., travel-and hb-related deferrals). qualitative results showed that almost all donors experienced temporary deferral as disappointing, particularly when it was unexpected (e.g., first-time deferral). not all donors (fully) understood the aims of deferral or how to prevent on-site deferral. donor beliefs about why deferral would lead to lapse were related to recurring deferrals, (mistakenly) interpreting deferral as permanent, or feeling all the effort did not pay off. summary/conclusions: reasons for temporary deferral differently impact risks of donor lapse at different levels of donor experience. for new donors all reasons for deferral are related to higher risks of lapse, whereas some reasons for deferral seem not to affect lapse among more experienced donors. unexpected or recurring deferrals may explain why donors lapse after temporary deferral. blood banks may tackle disappointment after deferral by explicitly showing that the donor is still valued, for instance by using personalized communication or offering an alternative good deed. background: blood donors experience a temporary reduction in their hemoglobin (hb) value after whole blood donation. in the netherlands, the hb value is measured before each donation, and a too low hb value (cut-off values: 8.4 mmol/l (135 g/l) for men and 7.8 mmol/l (125 g/l) for women) leads to a deferral for donation, in order to prevent iron deficiency and anaemia. the minimum interval between two donations is internationally set at 8 weeks, but over time donors exhibit iron deficiency so that blood donors are temporarily deferred from donation each year. in the us 35% -75% of deferrals are due to low hb, especially in women (editorial, transfusion, 2012) . due to the recovery process after each donation and the unobserved heterogeneity of donors, advanced statistical methods are needed to model the longitudinal data of hb values of blood donors. aims: to estimate the shape and duration of the recovery process of hb until the hb value has returned to its pre-donation level, to assess whether one can distinguish between donors with fast and/or slow recovery of their hb level and to predict future hb values. methods: the study is based on data of the donor insight study, which was a prospective cohort study performed by sanquin in the netherlands from 2007 to 2016. we employed three statistical models for the hb value: (i) a mixed-effects models, (ii) a latent-class mixed effects model, and (iii) a latent-class mixed-effects transition model. in each model, a flexible function was used to model the recovery process after donation. the latent classes identify groups of donors with fast or slow recovery times, and donors whose recovery time increases with the number of donations. the transition effect accounts for possible state dependence in the observed data. all models were estimated in a bayesian way, using data of a sample of 1000 new entrant donors (500 males and 500 females). prior information from the clinical literature (boulton, vox sanguinis 2004) about the recovery process three days after blood donation was incorporated into the analysis since these values were not identified in the observed data. results: the results show that the latent-class mixed-effects transition model fits the data best. we also found that the recovery process shows a concave process (initially fast followed by slower recovery). the estimated recovery time is much longer than the current minimum interval of 58 days between donations. namely, depending on the subgroup that the donor belonged to, males showed a recovery time of 100 to 419 days, while the estimated recovery time for females varies between 54 to 503 days. these results suggest that an increase of this interval may be warranted. summary/conclusions: the analysis shows the usefulness of the sophisticated statistical models that make use of historical information to model complex processes in time, in this case the hb trajectory over time across repeated donations. in addition, our results suggest a (much) longer time lag between subsequent donations to avoid anemia. background: complications of blood donation are known to reduce donors' return for future donation. the episode study (experience success in donation) showed that water drinking shortly before donation had an effect of 23% reduction of selfreported vasovagal reactions (vvr) in younger novice whole blood donors (wiersum-osselton, transfusion, 2019) . aims: in this study we analysed the return for a subsequent donation of the donors participating in the episode study. this was a predefined secondary outcome of the episode study. methods: the episode study was conducted in young (<30 years) whole blood donors making their first, second, third or fourth donation in geographically selected collection centres. the study interventions were: 330 ml water drink, 500 ml water drink or squeezing a ball (placebo intervention) during the wait after the screening interview and before phlebotomy, and a control group without intervention. participating donors were sent an online questionnaire about their experience within a week following their donation attempt. in the netherlands donors are usually invited for blood donation in accordance with hospitals' needs; the aim is to invite eligible donors at least once a year. donors were included in the return analysis if they had received at least one invitation within 400 days after the index donation and we analysed their return for a donation attempt within 421 days. associations with the interventions and donors' donation status, gender and reported symptoms at their index donation were analysed by calculating return percentage of eligible donors and by binomial logistic regression. results: out of the 8199 episode participants who had received an invitation, 6538 (79.7%) returned within the study period. there was no difference in donor return between the two water groups. the likelihood of return was significantly increased in both water and placebo intervention donors compared to the questionnaire group (or 1.2, 95% ci 1.06-1.4 and 1.3, 1.12-1.5 respectively). return was slightly lower in women (or 0.88, ) and lower in first-time donors (or 0.86, 0.77-0.96) than after a 2 nd -4 th donation. a staff-recorded or self-reported vvr at the index donation reduced donor return (or 0.47, 95% ci 0.37-0.60 and or 0.53, 0.46-0.61 respectively). other symptoms following donation were also associated with a lower return percentage. summary/conclusions: in this cohort of younger new and novice blood donors, 79.7% returned for a subsequent donation. a vvr (either staff-recorded or selfreported) reduced donor return. donors who received a study intervention, either water or placebo, were more likely to return, whether or not they had suffered a vvr. it is conceivable that the mere fact of study participation could also have increased donor return, even in de questionnaire group; this will be examined in the total population of target group donors. background: the contribution of older blood donors to the blood supply is substantial. in australia, donors aged > 50 years contributed 41% of all donations made in 2017. however, with ageing, the general health status of older donors changes relatively faster, thus progressively affecting their ability to donate. an indepth understanding of the relationship between older donors' health status, future donation patterns, and risk of iron-deficiency could be of a great value to inform the blood service to predict the number of future donations, and manage the risk of iron-deficiency. aims: to understand the relationship between self-reported health, blood donation patterns, and the management of identified iron-deficiency in older blood donors. methods: we linked the sax institute's 45 and up study baseline data collected between 2006 and 2009 to the blood service donation records, inpatient records, and medicare records*. the data-linkage was conducted by centre for health record linkage. using these linked data, we examined the relationship between health, donation patterns, and iron-deficiency and its management. results: we followed up 22,058 active whole blood donors for 200,403 eligible person-years (average age at recruitment 56.4 years, 56.3% female, average follow up 9.1 years per-person). after adjusting for the effect of age, sex, body-mass index, education, non-english language spoken at home, country of birth, smoking, physical activity, regular use of multivitamins, alcohol consumption at enrolment, and total number of whole blood donations in the 2 years prior to enrolment, participants with better self-reported health at recruitment showed significantly higher rates of donation. excellent, very good, good, and fair/poor health status donors made 1066 (95% ci 1056-1075), 987 (981-993), 900 (891-909), and 710 (690-730) donations per 1000 person-years, respectively. iron-deficiency was identified in 8.9% of donors in the study (n = 1964, 95% ci 8.5-9.3) . sixty percent of those with iron deficiency (n = 1,175, 95% ci 57.6-62.0) visited their general practitioner (gp) within 60 days of the identification of irondeficiency, and 48.4% (95% ci 45.5-51.3) of those visiting gp underwent further iron status examination and monitoring. after adjusting for several potential confounders including the total number of donations made during the follow-up period, excellent self-reported health status was independently associated with lower risk of iron-deficiency (p for trend = 0.004). summary/conclusions: information on self-reported health status can be an effective indicator to estimate the future donation yield of an older blood donor panel, and risk of developing iron-deficiency. donors with better self-reported health had a higher number of future whole blood donations and a lower risk of iron-deficiency. donors referred to gps for management of their iron status utilised the health services as expected, however there is an opportunity to improve their contact with their gps. * medicare records was provided by australian government department of human services. anaemia is a major public health issue, affecting 25% of the population worldwide according to the world health organization. iron deficiency is responsible for approximately half of all cases globally, with other causes including anaemia of chronic disease, other nutritional deficiencies, haemoglobinopathies, renal impairment, malignancy and bone marrow disease. in the elderly, where anaemia is even more common, the cause is frequently multifactorial. anaemia is associated with increased mortality, decreased cognitive and physical function, depressive symptoms and fatigue, particularly in older adults. poor outcomes have also been reported in anaemic patients with underlying comorbidities such as cardiac and renal disease, and cancer. within a hospital setting, anaemia is highly prevalent. preoperative anaemia, affecting up to 30% of patients, is associated with poor clinical outcomes including higher in-hospital mortality, longer length of stay and higher icu admission rates. anaemia management requires a proactive and multi-faceted approach, typically involving a multi-disciplinary team in which the transfusion practitioner plays a vital role. this includes screening of high-risk patients and pre-admission clinics to identify and manage patients at high risk of peri-operative anaemia. implementation of patient blood management (pbm) guideline recommendations has been shown to be effective to prevent and optimally manage anaemia within the community and hospital settings. the transfusion practitioner has key roles in the coordination, monitoring and auditing of pbm programs. active patient involvement and engagement of all members of the multidisciplinary team, including primary care clinicians, are also key to enhance the success of such programs. tp01-02 the role of the transfusion practitioner in anaemia assessment and management: processes, tips and resources for creating background: patient blood management (pbm) is an evidenced based integrated multi-disciplinary approach aimed to improve clinical outcomes by effectively managing and conserving the patient's own blood, thus reducing unnecessary exposure to transfusion. pbm has the patient as the central focus with the aim being to improve their outcomes and include them in the process. pbm includes three pillars: 1) optimising the patient's own blood, 2) minimising blood loss and 3) optimising a patient's physiological tolerance of anaemia. delayed assessment/management of anaemia contributes to increased health costs and unnecessary blood transfusions, and transfusion has been recognised to be associated with increase morbidity and mortality. the term transfusion practitioner (tp) includes those known as transfusion nurses, transfusion safety officers, haemovigilance officers, or patient blood management (pbm) coordinators. a key aspect of the role is driving and influencing clinical blood management activities to help align practice to internationally recognised guidelines and standards, including pbm. aim: to demonstrate the tp role in anaemia assessment & management and discuss strategies, processes, tips and resources for creating organisational and cultural change to implement pbm. context: literature outlines the importance of a multidisciplinary team to implement pbm related changes, and tps play a fundamental role within these teams to support 'buy in'. tps are seen as enablers, pulling resources together, engaging with those involved, providing education and facilitating change. they are often the ones to conduct audits, collating data and evaluating outcomes. approaches to implement pbm should be tailored to suit individual organisations. the authors will outline different approaches, highlighting where the tp can support or lead activities. one approach to anaemia assessment is to undertake an audit, examples of available tools will be shown. with this data, the tp along with the pbm team can explore options for corrective action. these could include interventions such as developing a pathway where all or a specific group of patients are assessed and or treated either at a preoperative clinic, or with their local general practitioner; through to more complicated strategies such as establishing anaemia clinics. the skills of the tp are a valuable asset to analyse clinical specialties/patient mix who should be targeted to achieve best outcomes, they know the organisation and as such are well placed to help develop a process/concept that will suit, and they can provide education and support to promote and embed these practices. conclusion: appropriate assessment and management of anaemia requires a multidisciplinary approach. the tp plays an active and crucial role in this team. examples of processes, tips and resources to support change and embed a pbm culture across the clinical spectrum will be shared. 3d-s11-01 department of hematology and central hematology laboratory, inselspital bern, bern, switzerland immune haemolytic anaemia (iha) is characterized by an increased breakdown of red blood cells (rbcs) due to allo-and/or autoantibodies directed to rbc antigens with or without complement activation. clinical and laboratory signs of haemolysis in concert with the presence of a positive direct antiglobulin test characterize iha. alloantibodies formed during pregnancy and/or after prior transfusions may cause acute or delayed haemolytic transfusion reaction after transfusion of a rbc product incompatible with the specificity of the alloantibody. autoantibodies to rbcs reduce the survival of endogenous and hamper the recovery of donor rbcs after transfusion. lymphoproliferative disease, autoimmune disease, infection or drugs often cause autoantibodies to rbc, but frequently no obvious cause can be identified. besides the antigen specificity, the isotype critically determines the biological activity of rbc antibodies in vivo. the isotype defines the affinity to fc-gamma receptors on cells of the reticuloendothelial system as well as the capacity to activate the classical pathway of complement, igm being the most effective. antibody-mediated complement activation results in the opsonisation of rbc with c3bc/c3d with subsequent complement receptor-mediated removal by phagocytes (extravascular haemolysis). occasionally, complement activation proceeds via the activation of c5 to the formation and insertion of the membrane attack complex resulting in intravascular haemolysis. there is growing evidence that the innate immune system plays an important role in the pathogenesis of iha. the process of complement-mediated haemolysis results in systemic inflammation, which contributes to morbidity and mortality of patients suffering from iha. complement activation results in the release of anaphylatoxins, which are strongly vasoactive and mediate chemotaxis, inflammation and formation of radical oxygen species. release of cell-free haemoglobin and cell-free haeme upon haemolysis induces endothelial cell activation, no-depletion, cytotoxicity, ros formation and neutrophil activation. natural plasma scavengers, such as haptoglobin and hemopexin complex with their target molecules, cell-free haemoglobin and haeme, with subsequent removal of the complexes via cd163 and cd91-mediated phagocytosis. although being positive acute phase proteins due to consumption the plasma scavengers become exhausted during chronic haemolysis thereby failing to prevent the adverse biological effects of cell-free haemoglobin and haeme in the circulation. inducible haeme oxygenase-1 (ho-1) is an efficient cellular scavenger by breaking down haeme into biliverdin with subsequent formation of bilirubin, co and ferrous iron with subsequent oxidation to ferric iron and storage by the ferritin h chain. ho-1 has an established role in the systemic protection from systemic inflammation induced by haemolytic and non-haemolytic diseases. the lecture will emphasise the role of innate immunity with a special focus on different plasma-and cellular systems involved in the pathogenesis of systemic inflammation in patients suffering from iha. 3d-s11-02 understanding erythrocyte clearance c roussel, p amireault, p ndour and p buffet research and teaching, institut national de la transfusion sanguine, paris, france the clearance of erythrocytes is essential in physiology, disease and transfusion. elimination of erythrocytes altered because of senescence or pathological processes is expected to protect the microcirculation from obstruction by adhesive or rigid erythrocytes. it also contributes to the harmful consequences of anemia and hemolysis in hereditary and acquired red blood cells diseases as well as in conditions associated with auto-or allo-immunization. immunobiology has explored in great details antibody-mediated clearance of erythrocytes but conventional approaches may not be fully operational to explain delayed hemolytic transfusion reactions. some important clearance processes are independent from the recognition of molecules or antigens on the erythrocyte surface. increased erythrocyte stiffness triggers their clearance in hereditary spherocytosis, malaria and possibly also in the context of autoimmune anemia. knowns and unknowns on the mechanisms and sites of erythrocyte clearance will be presented based on a critical review of old and recent contributions. 3d-s11-03 cardiovascular and endocrine-metabolic diseases and aging, istituto superiore di sanit a, rome, italy existing literature indicates that red blood cells (rbcs), beyond gas transport, exert a complex role in human physiology, being involved in many functions essential to maintain ion, metabolic and immunological homeostasis. rbcs display an immunomodulatory activity on adaptive immune cells by promoting t cell growth and survival and inhibiting activation-induced cell death. the balance between cell death and survival controls t cell homeostasis and anomalies in this balance account for diseases linked to excessive or faulty t cell growth. rbcs are able to modulate innate immunity by binding endogenous molecules such as chemokines and mitochondria-derived dna, as well as external agents such as pathogens. rbcs can also directly modulate innate immune cell activation or tolerance by controlling the maturation of the circulating pro-inflammatory subset of dendritic cells (dcs). these cells are potent inducers of primary antigen-specific t cell responses, produce tnf-a when stimulated by lps and are the principal il-12p70-producing cells among leukocytes. the pro-inflammatory capacity of circulating dcs is controlled by rbcs that are able to inhibit their maturation and il-12 production. in diseases characterized by local th1 inflammatory response such as psoriasis vulgaris and rheumatoid arthritis, pro-inflammatory dcs play a role in the induction and perpetuation of inflammation. collectively, literature data indicate that rbcs exert important modulatory functions that may result in immune activation or quiescence, depending on the environmental conditions. when rbcs encounter a microenvironment characterized by an intense production of ros, the rbc defenses get overwhelmed or are unable to counteract the new pro-oxidant status and become themselves a source of ros, which cause the generation of senescent signals on rbcs. the major feature of oxidized rbcs is the clustering and/or the breakdown of band 3. other features are the complexation of hb with spectrin, the loss of glycophorin a, the externalization of phosphatidylserine and the reduction of the "marker of self" integrin-associated protein cd47. a similar senescence phenotype has been documented in rbcs during the storage period. oxidized, senescent or stored rbcs, due to surface antigen modification and to the release of pro-inflammatory molecules, fail to control immune cell homeostasis thus contributing to the perpetuation of inflammation and to the pathogenesis of immune-mediated diseases associated to oxidative stress, such as autoimmune diseases and atherosclerosis. our research group demonstrated that rbcs from patients with carotid atherosclerosis presented a senescent phenotype similar to that acquired by rbcs from healthy subjects following to in vitro oxidation. oxidized erythrocytes fail to control t lymphocytes apoptosis and lipopolysaccharide-induced monocyte-derived dc maturation, thus representing dangerous signals for adaptive and innate immunity and contributing to the pathogenesis of atherosclerosis. in conclusion, the crosstalk between rbcs and the immune system represents a mechanism to maintain immunological homeostasis. however, in high oxidative stress conditions, that can take place during a prolonged storage period or in particular diseases, rbcs can acquire a pro-oxidant behaviour and lose their functional and homeostatic features. by interfering in immune system homeostasis, rbcs become a potential tool that can be manipulated to improve or reverse pathological situations characterized by anomalies in the control of adaptive and innate immunity. transfusion therapy remains an important treatment modality for patients with sickle cell disease (scd). transfusions are given to lower the percentage of circulating sickle rbcs, and to decrease blood viscosity and have been shown in clinical trials to reduce the risk of stroke by 90%. however, many indications for transfusion in scd remain controversial partly due to insufficient randomized clinical trials data and in part because of our limited understanding of the complex pathologic networks leading to diverse disease complications in scd despite the common single mutation. similarly, we have incomplete mechanistic understanding of why chronic transfusion protocols must be continued for those indications supported by clinical data. the beneficial effects of transfusion therapy in scd need to also be weighed against potential transfusion risks including alloimmunization associated with lifethreatening delayed transfusion reactions, increased iron stores associated with increased oxidative stress and exposure to infectious agents. we believe that a deeper understanding of the benefits as well as harmful effects of transfusions is crucial to optimize our current transfusion therapy protocols in scd. this knowledge may provide highly needed guidance, which is currently lacking, for expansion or limiting existing indications for chronic transfusions in scd. 3d-s12-02 treatment of thalassaemia department of pediatric hematology, ege university, faculty of medicine, bornova/ izmir, turkey thalassaemia is a devastating blood disease with a significant worldwide burden. annually, 60,000 children are born with a major thalassemia. life-time rbcc transfusions and iron chelation remain standard of care treatment in thalassaemia. transfusion therapy still account for significant iron overload related morbidity and mortality despite chelation therapy which is associated with poor adherence, safety concerns and varied efficacy. higher risk for transfusion transmitted infections (ttis) exists for thalassemia patients whose transfusion exposure sustains lifelong. although, the risk of transmission for traditional viruses is exceedingly rare in the modern era, emerging infectious diseases continue to be recognized as potential threats to transfusion safety. the inadequacy of blood safety points to the necessity for an additional layer of security for the blood supply in the developing world. pathogen reduction technologies for rbcc may imply a proactive, more generalized approach against new and re-emerging pathogens in the developed world and may be an ultimate safeguard for transfusion safety in the developing countries. rbc alloimmunization may become a major challenge in thalassaemia management. prevention is the key reducing the burden of alloimmunization. while the recommendation is to transfuse thalassaemic patients with c/c,e/e,kell compatible blood, it is not universally practiced. extended molecular rbc typing may be an appropriate adjunctive test in addition to serological typing before embarking on transfusion therapy. if a complete rbc antigen profile has not yet been performed in an alloimmunized patient, genotyping is the only option for accurate detection of rbc antigens that may guide the antibody identification. allogeneic stem cell transplantation (a-sct) is the only available curative therapy in children with hla matched sibling which is available to approximately 20% patients. in the absence of msd, mud transplant with high compatibility criteria has still limited experience. mismatch related, cord blood and haploidentical donor scts are considered experimental. a-sct carries a substantial risk of saes and mortality, both increasing with recipient age and disease severity. dfs is 88% in paediatric and 65% in adults. gene therapy for correction of the a-globin chain imbalance overcomes the problems of donor availability and immunologic complications associated with a-sct. multicenter clinical studies on gene addition therapy by using self-inactivating lentiviral vector are currently underway. recently, gene editing by either gene disruption or gene correction emerged as a potential alternative to gene addition therapy in beta-thalassaemia. a new era of novel therapeutics is unfolding in thalassemia management. several targets have been identified that can improve alpha/beta chains imbalance, ineffective erythropoiesis, or iron dysregulation and a number of those now have agents in preclinical and clinical development. hydroxyurea may improve globin chain imbalance and be beneficial for reducing or omitting transfusion requirement in selected group of patients. ruxolitinib has shown the limited effect on pretransfusion haemoglobin and reduction in transfusion needs, but allowed steady decrease in spleen volume that may serve for avoiding splenectomy in beta thalassaemia. luspatercept may restore normal erythroid differentiation and improves anaemia and hepcidin mimetics or tmprss6 inhibitors may modulates ineffective erythropoiesis by iron restriction and improves anaemia and organ iron loading. background: thalassaemia major (particularly b-type) and sickle cell disease (scd) are the commonest clinically important haemoglobinopathies, representing major sources of morbidity. recommended therapy is regular transfusion of safe, good quality blood, and monitoring of related complications. thalassaemia international federation (tif) guidelines, in place since 1987, include strategies for precautionary measures and use of scientific progress in detection, inactivation and elimination of transfusion transmissible pathogens. antigen-matching strategies to avoid alloimmunization against rbc antigens and other measures including haemovigilance are key components for safe blood, alongside voluntary, non-remunerated blood donation and laboratory quality assurance programmes. aims: we present the contribution of tif and the greek experience in ensuring safety and availability of blood for thalassaemia patients applying internationally accepted standards and recommendations. methods: tif -a non-profit, patient-driven organization with 204 national thalassemia associations in 62 countries -promotes national control programmes for prevention and management contributing to the achievement of final cure. the main working methods are provision of education, expert support, networking, communications and projects to support improvements in the quality of health, social and other care. in greece, technical standards for blood donor selection and testing are applied in compliance with directive 2004/33/ec as well as haemovigilance programmes and traceability procedures for recording adverse reactions and events associated with the transfusion of rbcs (directive 2005/61/ec). pre-transfusion and transfusion measures recommended by the council of europe are applied. in particular, measures for transfusion of "the right blood at the right time for the right patient", leucodepletion, rbc washing and accurate cross-matching and antigen and antibody screening for an extended matching policy are practised. fresh (up to 15 days old) rbcs are used. molecular testing for abo and rh d is performed in cases with blood group discrepancies. haemovigilance in greece covers 95% of total blood supply. data on ttis in 3,067 patients with thalassaemia and scd-thalassaemia in 2010-2017 are analysed. results: tti prevalence in thalassaemia syndromes was: hbv 1.8% (occult type 1.3%), hcv 54%, hiv 0.3%, htlv 0.8%, wnv 0.5% and hev 3%. most frequent adverse reactions in 2015-17 were allergic (incidence 1:854), non-haemolytic febrile reactions 1:2,631, "other" 1:5,883, alloimmunisation 1:6,667, taco 1:100,013, tad 1:50,006, tt-hev 1:100,013. hyperhaemolysis was diagnosed in two scd patients, delayed haemolytic transfusion reaction in one thalassaemia intermedia patient. trends in 2010-2017 show reduced incidence of alloimmunisation against rbcs. rates of allergic and pyrexial ars remained stable. no major abo incompatibility case was reported and no fatal transfusion reaction of transfusion has been recorded. summary/conclusions: blood safety in transfusion has significantly improved in high and upper-middle income but unfortunately not in lower and low income countries. blood shortages and lack of stringent protective measures for thalassaemia patients is the reality for many developing countries. tif focuses particular attention on the provision of support and the promotion of initiatives promoting the safety and adequacy of blooda key component of the lifelong management of patients with transfusion-dependent thalassaemia. background: b thalassemia is the most common group of hereditary hemoglobinopathy diseases. affected people with major thalassemia are dependent on regular blood transfusion which leads to iron overload. hepcidin is a peptide and an important regulator of iron homeostasis. expression of this hormone is influenced by polymorphisms within the hepcidin gene, hamp. aims: this study aimed to analyze the association of three polymorphisms in promoter of hamp, rs10421768, rs117345431, and rs142126068 with iron overload in major b thalassemia patients who do not respond to iron chelating therapy. materials and methods: a total of 102 samples from major b thalassemia patients were collected. genomic dna was extracted and sequenced for snps rs10421768, rs117345431, and rs142126068. statistical analysis was performed on ibm*spss* statistic 23 using independent t test and fisher test. results: our analysis revealed statistically significantly difference between the level of cardiac iron concentration and c.-582a>g variant (p = 0.02). for rs117345431 statistical analysis was on the edge of significant relationship between minor allele and serum ferritin (p = 0.058). all samples were homozygous for allele t of rs142126068. summary/conclusions: different factors affect iron overload in thalassemia. our findings and others emphasize the role of hepcidin polymorphism as a key component in iron homeostasis. ten to twenty years ago, countries in south eastern africa faced the peak of the devasting hiv/aids epidemic leading to an up to 20 years drop in general life expectancy. with the burden of hiv/aids falling mainly on the economically active population of young and medium-aged adults, the epidemic endangered social and economic stability in nations most heavily affected. today, despite aids still being a major cause of death in south eastern africa, the epidemic has become an example of public health gains that can be achieved through programmatic, evidencebased approaches that are endorsed by globally aligned policy and funding strategies. based on his work from lesotho, where one out of four among adults is infected by hiv, niklaus labhardt will take the auditors through the history of hiv programs in south eastern africa and show how innovative, pragmatic and evidence based implementation brought the region to a stage where the goal to end the aids epidemic by 2030 might be in reach. background: in france, the deferral for men who have sex with men (msm) was reduced from permanent to 12 months in july 2016. since this change has not impacted the residual risk (rr) of undetected hiv among blood donations, the ministry of health is considering a greater access of blood donation to msm. two scenarios have been studied: s1. deferral of msm during the 4 months preceding the donation; s2. deferral of msm who have had more than one sexual partner in the 4 months preceding the donation, similarly to all other blood donors in france. aims: to assess the impact of these two scenarios on the hiv rr estimated over the period july 2016-december 2017 which is the baseline rr with the current 12month deferral for msm. methods: baseline hiv rr was calculated with the classical incidence-window-period method, where hiv incidence was derived from a detuned assay (eia-ri) detecting recent infections (≤180 days) since all hiv-1 antibodies positive blood donations are tested with this test. the assessment of the impact of both scenarios on the baseline hiv rr was based on (i) data obtained from surveys among msm in the general population and in blood donors (compliance survey), to estimate the number of additional msm who would give blood in each scenario, and on (ii) hiv incidence estimate among these additional donors. this incidence was estimated: for s1, from msm blood donors with the current deferral policy (12 months) and for s2, from monogamous msm of the general population. results: from july 2016 to december 2017, 8/28 (29%) hiv-1 positive blood donors tested with the eia-ri were identified as recently infected, allowing to estimate the baseline hiv rr at 0.16 in 1 million donations [95% ci: 0.04-0.34], or 1 in 6,380,000 donations. for s1, the number of additional msm donors was estimated at 733 and the number of additional hiv positive donations at 0.09, resulting in an hiv rr of 0.16 in 1 million donations [95% ci: 0.04-0.35] or 1 in 6,300,000 donations. for s2, the number of additional msm donors was estimated at 3,103 and the number of additional hiv positive donations at 4.92, resulting in an hiv rr of 0.23 in 1 million donations [95% ci: 0.05-0.56] or 1 in 4,300,000 donations. sensitivity analysis shows that if both the number of msm and the hiv incidence were multiplied by 1.5, the risk would be 1 in 6,225,000 donations for s1, and 1 in 3,000,000 for s2. summary/conclusions: for both scenarios, the hiv rr remains very low. for s1 (4-month deferral), the risk is identical to the baseline rr and is very robust to variations in the model parameters. for s2 (no more than one sexual partner, 4 months), the risk is 1.5 higher than the point estimate of the baseline rr and sensitivity analysis shows that this estimate is less robust than for s1, since the risk could be 2 times higher than the baseline rr. for both scenarios, there was a modest increase in eligible msm donating. 3d-s13-03 background: recruiting safe blood donors amongst the largest hiv-positive population in the world is a major challenge for south african blood transfusion services. south african donor deferral criteria and deferral periods for perceived high risk activities have evolved over time, but current risk factors for infection have not been formally assessed. in addition, most studies have reported risk factors for prevalent hiv infection whereas risk behaviours for incident infection are more informative as donations with these infections could occur during the window periods of available screening assays. aims: to identify the demographic and behavioural risk factors associated with incident hiv infection among blood donors in south africa. methods: we conducted a case-control study with incident hiv-infected blood donors compared to infectious marker negative controls. incident hiv cases and controls seronegative for hiv, hepatitis b and c viruses and syphilis were accrued from a donor pool covering 8 of 9 provinces in south africa. controls were frequency matched at a 3:1 ratio to cases on race, age and geography. incident hivinfections were hiv rna positive by individual donation nucleic acid amplification testing (id-nat; procleix, grifols) but antibody (ab) negative (prism, abbott) as well as those rna+/ab+ donors with recently-acquired hiv based on limiting antigen avidity (lag) assay results with normalized optical density values of < 1.5. eligible cases and controls completed a confidential audio computer assisted structured interview (acasi) on motivations for blood donation and behavioural factors, including behaviours in the 6 months before donation. frequencies and measures of statistical association for risk behaviours comparing cases and controls are reported after adjusting for multiple comparisons. results: from november 2014 to january 2018, we enrolled 323 incident hiv cases and 877 controls; 202 (62.5%) cases and 544 (62%) controls were ≤ 29 years old. there were significantly more female cases 230 (71.2%) than female controls 439 (50.1%) (p < 0.0001). significant hiv risk factors (all p < 0.0001) reported within the 6-months before donation included: having a primary sex partner who is male; reporting increasing numbers of male sexual partners for both females and males; frequency of vaginal sex; frequency of vaginal sex without condoms; use of methods to clean, dry, or tighten one's anus before sex; and having visited a traditional healer for medical care. lack of medical aid (private health insurance) and reports of injury or accident with blood loss were also associated with an incident hiv infection. summary/conclusions: our study has identified a set of novel, putative risk factors for incident hiv infection among south african blood donors while confirming a number of previously known sexual risk behaviours. not having private health insurance and being injured may be markers of socio-economic context that place individuals at higher risk rather than behaviours that directly increase hiv transmission risk. the detection of risk behaviours by acasi in donors who passed predonation questionnaires and interviews suggests that acasi has the potential to improve risk behaviour identification. background: in france from 2000 to 2016, among male blood donors (mbds) found hiv-1 positive at blood donation screening, 31% did not disclose any risk factor for hiv infection during post-donation interviews, while 38% reported having sex with men (msm), and 24% and 7 % reported heterosexual sex (hts) and other risk factors, respectively. aims: in order to gain new insights into the risk factors for hiv-1 infection in mbds, we performed an hiv-1 genetic network analysis, including hiv-1 positive mbds and patients included in the french primary hiv infection anrs co6 primo cohort (pc). methods: 284 mbds, who donated blood between 2000 and 2016, and 1340 pcs, included between 1998 and 2014, were studied. epidemiological data were collected by the french blood service (efs) upon blood donation or post-donation interviews for mbds, and upon inclusion for cps. viral strains were sequenced and genotyped in pol gene, and a recent infection assay was performed to date infection in mbds (recent: <6 months). a partial transmission network was computed based on tamura-nei 93 nucleotidic distance (threshold for hiv-1 s/t b = 1.5%; for non-b s/ t = 0.8%) and assortative mixing was evaluated for mbds epidemiological data, including risk factors for hiv infection (msm, hts, others and unknown). selfreported data were then compared to assortativity-enhanced data. results: hiv-1 strains from 81 mbds and 126 pcs were linked into 54 clusters including at least one mbd. primo-only clusters were excluded from the analysis. compared to mbds who did not cluster, those found linked to the network were younger (30 vs. 36 year-old; p < 0.01) and were more likely to have a recent infection (48% vs. 34%; p = 0.03). assortative mixing indexes showed that paired individuals were more likely to live in the same area (p < 0.001) and to have the same risk factor for hiv infection (p < 0.001) compared to a random distribution. imputing msm risk factor to non-msm individuals paired with msm changed the distribution of risk factors as follows: msm: 38% vs. 49%, hts: 24% vs. 21%, other: 7% vs. 6% and unknown: 31 vs. 24%. summary/conclusions: after validating the assortativity of risk factors between paired individuals, and imputing msm risk factor to individuals self-reported as non-msm (including those with no identified risk factor), up to 18% (31/176) of mbds could be reclassified as msm. this is a worst-case scenario, as the network analysis does not exclude the possibility of one or several persons between two paired individuals (missing link). altogether, these results could help reevaluate the hiv residual risk linked to msm mbds, especially in the frame of the evolution of blood donor deferral criteria. background: although most individuals remain asymptomatic, htlv infection can lead to adult t-cell leukaemia/lymphoma (atll) and htlv-1 associated myelopathy (ham). the serious nature of these diseases, evidence of transmission via non-leucodepleted blood, and concern about a high prevalence among donors originating from endemic areas led to the uk blood services introducing universal blood donation screening in 2002. monitoring through routine surveillance commenced and htlvinfected donors were invited to participate in the htlv national register cohort study to assess disease progression. these data together with evidence from lookback to previously untested donations and cost-effectiveness analysis were reviewed by an expert working group in 2013 and 2015. aims: to describe the epidemiology of htlv among uk blood donors and evidence of disease progression from long term follow up of asymptomatic donors. methods: uk blood donations screened, and infected donors identified are reported to a national surveillance scheme. these donors are contacted, their results explained and information about clinical history and possible sources of infection are collected. where appropriate, htlv-infected donors are consented to the register, with participants completing a baseline questionnaire about their health, flagged in registries for cancer or death, and followed up about every 2-3 years. results: in the uk 2002 -2017, 254 htlv-infected donors were identified. prevalence among new donors was steady around 5/100 000 donations. prevalence among repeat donors peaked in 2002 (2.7/100 000 donations), with most in previously untested. from 2004 to 2016, prevalence of 0.07 per 100,000 donations (average of one positive/year) was recorded. in 2017, prevalence among new donors increased to 9.9/100,000 donations (17 positives), with increased numbers associated with asian ethnicity and coinciding with an increase in collections from bame groups. overall, most were women (182/254, 72%), uk-born (125/254;49%) and htlv-1 infections (228/254;90%). mean age was 43 years. almost all positive donations were from previously untested donors (240/254), with seroconversion within a year of previous donation confirmed for only 5 of the 14 previously tested donors. typically, infections were associated with endemic countries (including caribbean region, west africa, iran, india and japan), acquired through breast feeding or from their heterosexual partner originating from these countries. interestingly, three were thought to have been infected through self-flagellation. a total of 114 htlv-positive asymptomatic blood donors have already been recruited to the htlv national register, and during over 800-person years follow-up, none had developed atll or ham. summary/conclusions: over 16 years of testing, few seroconverters were identified, suggesting very little ongoing transmission among uk blood donors. the lack of disease among the cohort study was also reassuring, although it is likely too early to detect associated symptoms of a slow progressing disease. recruitment to this unique dataset continues, also outside of the blood donation setting. as a result of these surveillance data, evidence from lookback, and cost-effective analysis, in 2017 nhsbt ceased to test donations from previously tested donors unless the donation was being used to manufacture a non-leucodepleted component. finland lies in northern europe between the 60°and 70°n latitude. the length of the country is 1150 km and width 550 km. by surface area it is the fifth largest country in eu. the population of the country is 5.5 million resulting in the lowest population density in eu (15.8 inhabitants/km3). the whole country is inhabited, although most of the population is packed in the south. the climate of finland is influenced mainly by its latitude, but the warm waters of the gulf stream and the north atlantic drift current also play a role. due to finland's northern location, winter is the longest season. the southern portions of the country are snow-covered about three or four months of the year, and the northern regions for about seven months. long distances, low population density and the extreme climate give logistical challenges. it is estimated that these logistical costs can be as much as 10-20% of gdp in finland. the finnish red cross blood service (frc bs) has been the nationwide blood service provider in finland since 1948. frc bs collects annually about 200 000 whole blood units of which 50 % are collected in 10 fixed sites and 50 % in mobile sessions around the country. central activities (donor recruitment, medical support, production, testing, supply chain management, digital services and administration) are located in helsinki. management of transfusion is highly dependent on the logistical arrangements from blood donation sites to the central facilities and from the central inventory to the hospitals. the logistics is outsourced to three major partners all of whom have their roots in nationwide public transportation and logistics services. posti ltd is a state owned company having its roots in the national postal and telecom office. today it is the leading postal and logistics service company having the widest network coverage in finland. blood units collected at different fixed sites and mobile sessions are transported overnight by posti ltd to the frc bs central facilities by 8 am on the day following the blood donation. posti ltd is also used for the regular deliveries of blood products to the hospitals. the other important partner is matkahuolto ltd, which was founded in the 1930s to maintain bus stations and to serve as a common marketing company for the bus and coach services in finland. it maintains a nation-wide package delivery system based on the scheduled bus route network. matkahuolto ltd is used to transport donor testing samples from the donation sites to the central laboratory. by this arrangement it is possible to obtain most of the donor samples to the laboratory around midnight, which significantly speeds up the completion of laboratory results. the third logistics partner is jetpak finland ltd, which operates the air freight for the national flight company finnair. blood transfusion services can be managed centrally in a large sparsely populated country in a manner that is of high quality, safe and cost effective. however, the supply chain has to be planned carefully. background: elearning is a divisive topic. it is often criticised as an inferior form of education while simultaneously being promoted as a means to provide education to large numbers of people in a consistent, cost-effective manner. bloodsafe elearning australia (bea) is a government-funded blood transfusion education program that commenced in 2007 and provides courses in clinical transfusion practice and patient blood management (pbm) including: -clinical transfusion practice (4 courses) -pbm: general (7 courses) -pbm: medical (6 courses) -pbm: acute care and surgical (4 courses) -pbm: obstetrics and maternity (3 courses) -pbm: neonates and paediatrics (7 courses) aims: to determine the engagement, outcomes and impact of learning of bloodsafe elearning australia courses. methods: a retrospective analysis of user registrations, course completion records, course evaluation data and red cell usage in australia to determine learner demographics, and the impact on acquisition of knowledge and application to clinical practice. results: in the period from 1 july 2007 to 31 january 2019: -489,600 people registered as learners -1,072,299 courses were completed -these learners came from 182 countries, with 11,141 (2.3%) of them from outside of australia. analysis by profession shows that: -82.4% are nurses and/or midwives -11.2% are medical -6.4% are laboratory, anaesthetic technicians or other. analysis of user evaluation data (n = 2,885) from 1 april 2015 to 31 january 2018 shows that these courses have a positive impact, with 88.7% of respondents stating they gained additional knowledge, 65.2% able to make changes to clinical practice, and 88.2% reporting that these changes will improve patient safety and outcomes. analysis of international participants shows greater benefits with 93.5% gaining knowledge, 76.4% able to change their clinical practice and 91.9% believing this will improve patient outcomes. analysis of red cell usage in australia shows that since 2012 there has been a 21.1% reduction in red cells issued. this has been achieved through a number of pbm activities including development of guidelines, research and audits, education, waste reduction strategies, and promotional campaigns. bloodsafe elearning australia courses on pbm were released in 2012 and are one part of this pbm activity, and it is notable that these courses have the widest reach as they are undertaken by a large proportion of doctors, nurses and midwives in australia who are not directly involved with the blood sector. stakeholder feedback shows that the program provides credible, consistent education that is cost-effective, reduces duplication, is 'best-practice' elearning, is readily accessible, and allows institutions to focus on the development of practical transfusion skills. summary/conclusions: this analysis shows that elearning is a well-accepted, wellutilised form of education for healthcare workers to learn about clinical transfusion practice and patient blood management, and learners gain knowledge that can change their clinical practice and improve patient outcomes. it is also likely that these courses have contributed to better utilisation of a scarce, freely-donated resource. this approach has global reach and availability, and is a cost-effective model for improving transfusion practice in the developing world by providing education for millions of healthcare workers. 3d-s14-03 prospective platelet auditing: analysis of trainee compliance with guidelines pathology, columbia university, new york, united states background: apheresis platelets are a component product with high cost and limited supply. furthermore, there is a potential for severe transfusion reactions associated with this product such as transfusion related lung injury (trali), and sepsis due to bacterial contamination. therefore, transfusion guideline compliance is closely monitored by many centers. this quality assurance analysis describes our experience with prospective platelet auditing performed by physicians in pathology residency training. aims: this study aims to evaluate the ability of physicians in training to perform prospective auditing and compare policy compliance for different levels of experience. methods: this is a quality assurance analysis of a prospective platelet audit program for a 12-month period (january 2018-december 2018). the blood bank paged the on call physician any time an order was placed for a patient with a platelet count of > 50,000/ll, ≥2 doses of platelets with no interim repeat count, or an unknown platelet count. audit records created by physician trainees in their first post graduate year (pgy1) were compared to subsequent years (pgy > 1). information collected included the total number of doses requiring approval, number of products approved, training year for the approving physician, and transfusion indication. cost analyses assumed $500 for a dose of platelets. descriptive statistics and comparative analysis using a pearson's chi-square were used with a difference of p < 0.05 considered statistically significant. results: there were 1446 platelet doses requiring approval with 670 (46%) routed to the pgy1 group and 776 (54%) to the pgy > 1 group. there were 847 (59%) ordered doses that were in compliance with hospital transfusion policy and 599 (41%) that were not in compliance with hospital policy. of the 847 appropriately ordered doses, the pgy1 group declined release of 7 necessitating the clinical team to insist upon release without approval, and there were zero such instances in the pgy > 1 group. when paged by the blood bank, pgy1 physicians approved product release not in compliance with policy for 191/670 (29%) doses while pgy > 1 physicians approved not indicated products for 79/776 (10%) of doses (p < 0.01). products not indicated by hospital policy were held from release by pgy1 physicians for 113/670 (17%) doses and 216/776 (28%) doses by pgy > 1 physicians (p < 0.01). the ordered doses not in compliance with hospital policy had an estimated cost of $299,500. of this cost, there was a calculated $164,500 savings of products not released due to prospective auditing. there was an additional potential savings of $135,000 for products not indicated but released ($95,500 from the pgy1 and $39,500 from the pgy > 1 group). summary/conclusions: despite a higher number of requests being routed to the more senior pgy > 1 group, there were a disproportionately higher number of out of compliance platelet orders being released by the pgy1 group in addition to withholding needed products on several occasions. potential mitigation strategies for this could include a closer level of oversight for pgy1 physicians, and the potential monetary savings could justify a hiring a dedicated patient blood management team or quality assurance manager to monitor compliance and provide feedback to clinicians. 3d-s14-04 what can we learn from how adverse events are detected? norwegian directorate of health, oslo, norway background: the primary aim of reporting systems, such as haemovigilance systems, should be learning and improvement and to identify risk areas, not simply counting errors. to understand and learn from adverse events the description of how, where and why they occur, and how they are detected, is important. to support our understanding, we use a predetermined classification that is required for reporting to eu, supplemented by classification suggested by ihn, who and ourselves. in 2015 we started asking the blood establishments what steps they would take to prevent recurrence of the event, and we added a simple classification to tell how the adverse event had been detected. aims: this study aims to analyze how different types of adverse events reported to the haemovigilance system were detected, whether the current quality management systems used in norwegian blood establishments had effective barriers and whether new barriers should be considered. methods: adverse events reported to the norwegian haemovigilance system in 2017 and 2018 were analyzed with focus on how the adverse event had been detected. in all cases classification had been performed by the reporter of adverse events and were confirmed, or reclassified if necessary, by the haemovigilance team before analysis. for analysis based on classification we used powerbi (microsoft). results: a total of 188 adverse events were reported from norwegian blood establishments. all had been classified according to how the adverse event had been detected. twenty (10.6 %) adverse events were detected because of alarms or warnings from it-systems or equipment. routine checks by blood establishment staff detected 39 (20.7 %) events and formal internal or external reviews detected one event. seven (3.7 %) events were detected because the donor became ill shortly after donation, but the illness was not caused by the donation. sixty-four percent of events were detected in a way that did not fit our present classification and hence were classified as "other". twelve out of 16 wrong blood in tube were detected by an alarm from the it-system or routine check, as were six of 22 events related to blood ordering, two of seven errors in testing, six of 17 events where incorrect blood had been transfused, and eight of 64 events related to donor selection. in 80 reports human error was listed as the cause of the event and 27 of these were detected by alarms or routine checks. summary/conclusions: detection of adverse events by alarms or routine checks are highly efficient when the blood establishment has historic data to check against, as exemplified with wrong blood in tube or a patient require irradiated blood components. when no historical data exists or when the quality management systems do not require routine checks, events are usually detected by chance. further analysis is needed to see if and where the quality management systems should be improved. the wide variety of adverse events can make it difficult to select which area to prioritize in the improvement work. results: the hb measurements from the finger prick were on average 1.2 g/l (0.9%) higher than from the venous blood samples. the range of the difference was -21 -+22 g/l. these results were used in order to add novel information to determine the measuring uncertainty of hb measurement in frcbs. in 1.4 % (12/845) of the donors in this study the venous hemoglobin measurements were below the cut-off point of donor eligibility. in those measurements the difference of the finger prick and venous hemoglobin measurement was at most + 9 g/l. 92 % of the hemoglobin results from the finger prick were in the range ae10 g/l compared to the venous hemoglobin results. 63 % of the results from the finger prick were between ae5 g/l (the precision of the device) compared to venous hemoglobin results. in 13 cases the difference between finger prick and venous measurements was outside 2 standard deviations from the mean i.e. 2.5 % from the bottom (n = 9) or top (n = 4) of distribution. systematic errors were seen in some nurse's results both towards too low or too high hb result in the finger prick measurement and some nurses had random errors in both directions. the batch of cuvettes, donors' age, gender or the time of sampling were not detected to have an impact on the difference between finger prick and venous hb measurements in this study. summary/conclusions: the results of the poc measurements compared to the cell counter were in agreement with published data and with manufacturers' information on the device. the practical skill test is a workable way to develop competence and operations to measure the hemoglobin from the finger prick. it offers an opportunity to give personal feedback to nurses concerning their personal performance in the use of the current hb measurement technic. it also provided data on the accuracy of the poc method in the everyday donor selection process. background: whole blood donation has frequently been related to iron deficiency. a blood donor loses per donation about 8% (men) to 81% (menstruating women) of iron stores. to replenish the iron lost by blood donation in a donation interval of 56 days, a donor needs to absorb 4.5 mg iron per day. this amount exceeds the reported maximal amount of absorbed iron of 3-4 mg/day, eventually leading to iron deficiency, with consequences such as donor deferral and possibly iron deficiency-related symptoms (decreased physical endurance, fatigue, pica, restless legs syndrome, and cognitive functions). since hb levels do not reflect donors' true iron status, measuring ferritin is a better way to detect low iron stores in whole blood donors. studies from usa and denmark showed that on the introduction of ferritin measurement with either extension of donation intervals or iron supplementation in case of low iron stores, deferral percentages for low hb declined in both male and female donors. aims: to gain more insight in iron status of whole blood donors during their donor career, how this affects donor health and which measures may prevent low iron stores in donors. methods: in the netherlands, sanquin blood bank is currently implementing a policy with ferritin-guided donation intervals. in brief, ferritin levels are measured in all new donors and in repeat donors every 5th donation or in case of an hb below the deferral threshold. donation intervals are extended if ferritin levels are < 15 lg/ l, or ≥ 15 and ≤ 30 lg/l (for 12 and 6 months respectively). we anticipate that routine ferritin measurement will ultimately result in a lower prevalence of iron deficiency, less hb deferrals and improved donor retention. this will be further evaluated in a stepped wedge cluster-randomized trial 'find'em', which may also identify subgroups of donors prone to develop (symptoms of) iron deficiency. in addition, implementing ferritin screening may lead to a decreased donor availability. for this purpose, we modeled the impact of the implementation of our ferritin deferral policy on donor availability over time, which provides insight for both the expected size of the impact of the ferritin deferral policy and the time and rate at which this impact is expected to occur. this allows the blood bank to timely plan actions to counterbalance possible donor shortage and ensure an adequate blood supply. lastly, iron supplementation can be an alternative measure instead of donation deferral. as the used and recommended dosage of iron supplementation varies widely across blood services, sanquin is planning to start a new study in whole blood donors to gain evidence on the dosage and frequency of iron supplementation and its effect on ferritin and hemoglobin levels and donor health. results: the before-mentioned studies are ongoing and results will be expected from 2020 onwards. summary/conclusions: iron deficiency is a frequent side effect of whole blood donation. to prevent iron deficiency and its consequences, like donation deferral and health issues, more evidence-based insight in iron management of whole blood donors is being generated. 3d-s15-02 superdonors -genetic risk profile and risk of low hemoglobin deferral background: no reliable method exists for stratifying new blood donors into those who can maintain sufficient hemoglobin (hb) levels and those who will be deferred because of a low hb (<7.8 mmol/l [<12.57 g/dl] for women and < 8.4 mmol/l [<13.54 g/dl] for men). polygenic risk scores (prss) have shown great promise in predicting complex disease risk. prss could also prove useful for identification of donors genetically predisposed to low hb levels, and, thus, to an increased risk of deferral. aims: the objective of the study was to evaluate the association between prs (modelled to predict hb level as a quantitative trait) and risk of deferral as a binary outcome. methods: the danish blood donor study (dbds) is an ongoing nationwide blood donor cohort since 2010 with more than 110,000 participants. extensive genotyping has been performed on approximately 72,000 dbds participants using the infinium global screening array (illumina â ) and extended by use of imputing based on the pan-scandinavian reference genome. based on hb and genetic data on more than 150,000 icelandic individuals (an independent discovery cohort), we constructed 9 different weighted prss for individuals from dbds. information on the donors' whole blood donations following inclusion into dbds unto end of 2017 was obtained from a nationwide donation database, scandat. the best predictor of hb among the nine prss was chosen and used in all subsequent analyses. we performed multilevel mixed-effects linear regression analysis with hb as outcome, and prs as factorized explanatory variable with cutoffs at 5, 10, 25, 40, 60, 75, 90 , and 95 th percentiles, respectively. moreover, the models had a two-level clustering on donor id and donation site and an id-specific random intercept; and further adjusted for: sex(binary), age(continuous), year of donation(factorized), and time since last donation (continuous). lastly, risk of deferral was evaluated in random effects logit models with similar covariables and clustering structure. results: mean number of donations per donor after dbds inclusion was 6.9 donations. generally, we observed a statistically significant positive association between prs(hb) and current hb levels. compared with donors in the 40-60 prs percentile group, donors below the 5 th percentile had lower (-0.23 hb mmol/l (95% ci: -0.25; -0.21)) and donors above the 95 th percentile higher (+0.19 hb mmol/l (95% ci: 0.18; 0.21) hb levels. in the random effects logit models we observed a marked increase in deferral risk with decreasing prs percentile strata. with the 40-60 prs percentile stratum as reference, donors below the 5 th percentile and donors above the 95 th percentile had odds ratios of deferral of or = 2.58 (95% ci: 2.27; 2.93) and or = 0.46 (95% ci: 0.39; 0.54), respectively. summary/conclusions: we found a statistically significant positive association between prs(hb) and hb levels and a markedly increased risk of deferral with decreasing prs(hb). from a scientific point of view, it is unsurprising that a genetic score for hb from an independent cohort is associated with hb in another cohort. however, from a practical perspective, prss may be the first step in a personalized donation approach to donors and their risk of deferral. background: individually calibrated inter-donation intervals for repeat blood donors have the potential to minimize the risk of iron related adverse outcomes (e.g., hemoglobin deferral or collecting a donation from a donor with low or absent iron stores) without unduly impacting the donated blood supply. machine learning has shown promise for personalized clinical risk assessment. aims: our aim is to use machine learning to develop donor-specific, personalized inter-donation intervals that minimize the risk of adverse outcomes while maintaining or improving the adequacy of the donated blood supply. methods: using a public use dataset from the reds-ii donor iron status evaluation (rise) study (cable, transfusion, 2012) we defined donor profiles with physiological measures including hemoglobin, ferritin and soluble transferrin receptor along with questionnaire responses regarding diet, reproductive health indicators, and demographics. we used these profiles (58 features, 3,162 donations from 1,025 repeat donors) and the time until the next donation attempt to predict iron-related outcomes of the next donation attempt. possible outcomes were no adverse outcome, hemoglobin deferral, low-iron donation (ferritin < 20 ng/ml for women and < 30 ng/ml for men), or absent-iron donation (ferritin < 12 ng/ml for men and women). we trained multiple machine learning models on 2,543 of the donations and selected the model with the best performance (lowest cross-entropy loss in cross validation). we assessed the best model's performance on a hold-out test set of 620 donations, which were not used to train or select the model. we then used our model to generate risk estimates for these 620 test donors as a function of days since their last donation, which varied from 56 days to 250 days. to show individual variation, we generated graphical representations of individual donors' risk over time. results: ferritin, log ferritin, body iron, and time since last donation were most useful for predicting iron-related adverse outcomes at the next donation attempt. the estimated risk of adverse outcomes at the next donation attempt varied considerably across donors. as expected, the risk of adverse outcomes 250 days after the last donation was lower than the risk 56 days after the last donation for most donors (risk of hemoglobin deferral decreased for 84% of donors; risk for low-iron donation decreased for 61%; and risk for absent-iron donation decreased for 94%). summary/conclusions: the risk of iron-related adverse outcomes as a function of time since last donation varies considerably between donors. machine learning models trained on relevant donor profiles can effectively estimate how an individual's risk will change over time. individual risk estimates could allow blood centers to protect highrisk repeat donors while continuing to allow more frequent collections from low-risk donors. further study is needed to ensure this approach works well for donor classes that are not well-represented by the rise dataset, to assess risk prediction outside of the physiological measures collected in the rise study, and to determine the viability of assigning an optimal inter-donation interval to a first-time donor using this approach. background: iron depletion is common among repeat blood donors, who contribute a large proportion of the blood supply in many countries. exogenous iron from multivitamins with iron or iron-only supplements helps prevent donation-induced iron depletion, but whether dietary iron protects against iron depletion in repeat donors has not been rigorously evaluated. available data from the reds-ii rise study in the us (cable, transfusion, 2011) and from the danish blood donor study (rigas, transfusion, 2014) suggest minor impact of dietary iron consumption on blood donor iron status in multivariable regression models. both studies, however, analyzed food items singly, such as beef or fish, rather than in aggregate, so precision was limited. aims: to evaluate whether a composite measure of dietary heme iron consumption, weighted for frequency and iron content, was associated with incident iron depletion among repeat blood donors. methods: a re-analysis of the rise cohort was undertaken to test the hypothesis that reported levels of animal protein consumption was associated with lower risk for incident iron depletion among repeat blood donors. the six blood centers participating in rise enrolled first-time and frequent donors for 15-24 month follow-up of donation frequency and iron status. a brief checklist of 8 food categories was administered at baseline to assess frequency of consumption of several categories of animal protein that are rich in heme iron, the biochemical form of iron most readily absorbed. an iron composite score (ics) weighted for frequency and heme iron content was derived and subjects were grouped into tertiles (thirds) of ics. iron status was assayed at enrollment and study completion and at roughly one-third of donation visits in between. modified poisson regression with generalized estimating equations was used to generate risk ratios controlling for donation frequency and other covariates. results: of 2425 enrolled donors, 1406 were iron replete at baseline and completed the food checklist. the median value of the ics for each tertile (lowest to highest) was 7. 2, 13.1, and 22 .0 mg of heme iron weekly. these values are equivalent to approximately 3, 6, and 9 servings of beef per week, or alternately twice as many servings of chicken or pork. across 2236 follow-up visits with iron outcomes assayed, almost 33% of donor visits were associated with intermediate iron depletion (serum ferritin < 26 ng/ml) and 8.5% with complete depletion of iron stores, representing serum ferritin < 12 ng/ml. after controlling for demographic factors and donation frequency, the lowest tertile of ics was associated with a greater than 2fold higher risk for complete iron depletion during all follow-up visits (rr 2.40, 95% ci 1.51, 3.81, compared to the highest tertile). summary/conclusions: in this longitudinal evaluation of dietary iron and iron status, blood donors with low intake of heme iron had an elevated risk for developing advanced iron depletion. these results suggest that blood centers should continue to recommend iron-rich diets to repeat blood donors. background: blood donors lose approximately 250 mg of iron with every blood donation. as a result, frequent blood donors are at risk of iron deficiency and low hemoglobin (hb) levels, which may affect their health and eligibility to donate. lifestyle behaviors such as dietary iron intake and physical activity, may influence iron stores and thereby hb levels. gaining insight into associations between lifestyle behaviors and hb levels is valuable for blood supply organizations, as lifestyle behaviors can potentially be considered to prevent hb deferrals. examining the mediating role of ferritin, a measure reflecting iron stores, in these associations will help to gain insight into whether iron stores could be the limiting or enabling factor that links lifestyle behaviors to hb recovery after donation. aims: to investigate associations between lifestyle behaviors (dietary heme and non-heme iron intake and physical activity) and hb levels, and whether ferritin mediates these associations. methods: donor insight-iii (dis-iii) is a dutch cohort study of blood and plasma donors and included 2,552 donors. participants who were pregnant, had hemochromatosis, used iron supplements/medication, got a hysterectomy or bilateral oophorectomy were excluded (n = 292). hb levels were measured in edta whole blood samples using a hematology analyzer (xt-2000, sysmex, japan) and ferritin was measured in plasma from lithium heparin tubes (architect ci8200, abbott laboratories, u.s.a.). dietary heme and non-heme iron intake (grams/day) were assessed using a food frequency questionnaire adapted to measure iron intake. moderate-tovigorous physical activity (mvpa, minutes/day) was assessed using the international physical activity questionnaire (ipaq)-short form. results: in total, 2,260 (1,186 female) participants were included. donors with higher intakes of heme iron had significantly higher hb levels (regression coefficient (b) (95% confidence interval (95% ci)) in men and women respectively: 0.147 (0.069 to 0.225) and 0.094 (0.013 to 0.175) mmol/l), independent of age, smoking, menstruation, number of donations in previous two years, donation interval, sedentary behavior, the other lifestyle variable (i.e. (non-)heme iron intake or mvpa), and initial hb level. non-heme iron intake was negatively associated with hb levels (-0.015 (-0.026 to -0.004) and -0.018 (-0.032 to -0.005) mmol/l for men and women respectively). ferritin mediated associations between dietary iron intake and hb levels (indirect effect in men and women respectively: 0.073 (0.046 to 0.107) and 0.065 (0.031 to 0.100) lg/l for heme and -0.003 (-0.008 to 0.000) and -0.007 (-0.013 to -0.002) for non-heme). more mvpa was negatively associated with hb levels in men only (-0.004 (-0.008 to -0.001)), which was not mediated by ferritin. summary/conclusions: in conclusion, higher heme and lower non-heme iron consumption are associated with higher hb levels in donors via higher ferritin levels, indicating that donors with high heme iron consumption may be more capable of maintaining iron stores to recover hb levels after blood donation. more mvpa was associated with lower hb levels, although effect sizes were small, independent of ferritin. taking a donor's lifestyle behaviors into account may be useful in preventing low hb levels in blood donors. immune thrombocytopenia (itp) is still diagnosed by exclusion of other causes for thrombocytopenia. sensitive and specific detection of platelet autoantibodies may support the clinical diagnosis and prevent misdiagnosis of itp. for example, the direct monoclonal antibody immobilization of platelet antigens (maipa) assay, performed with in vivo sensitized patient platelets, offers platelet glycoprotein specific autoantibody detection with high accuracy. a drawback is that low platelet counts demand a large blood sample to have sufficient patient platelets available for analysis. circulating platelet autoantibodies are more difficult to detect by maipa; and may demand more sensitive detection platforms, such as those using surface plasmon resonance. in general, the presence of anti-gpiib/iiia, anti-gpib/ix and anti-gpv platelet autoantibodies is investigated. all these antibody specificities have been found in patients with itp. in itp, platelet autoantibody-mediated destruction via the spleen has been proposed; but also other mechanisms leading to low platelet counts in itp may play a role. inhibition of megakaryocytopoiesis by autoantibodies or by t cells has been suggested. in mice, gpib-directed antibodies induce loss of platelet-sugar epitopes, inducing hepatocyte-medicated platelet destruction. platelet autoantibodies can cause complement activation, which may contribute to platelet autoantibodymediated destruction. interestingly, we recently found that lack of detectable platelet autoantibodies is correlated with non-responsiveness to rituximab (cd20 moab) treatment in itp patients. in children with newly diagnosed and often transient itp, platelet autoantibodies of igg class or not often found, but of igm class are present for short duration. in conclusion, testing for platelet autoantibody characteristics and their pathologic effect may be helpful in establishing the diagnosis of itp and in choosing the best individualized therapy for itp patients. 4a-s16-02 thrombopoietin receptor agonist (tpo-ra) treatment raises platelet counts and induces immunomodulation in immune thrombocytopenia (itp) jw semple 1 , r aslam 2 , e speck 2 , j rebetz 1 and r kapur 1 1 lund university, lund, sweden 2 st. michael's hospital, toronto, canada background: itp is an autoimmune bleeding disorder in which autoantibodies and/ or autoreactive t cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. several therapeutic options e.g. corticosteroids, intravenous immunoglobulins (ivig), rituximab and splenectomy are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. for the last 10 years, tpo-ra e.g. romiplostim and eltrombopag have made a substantial contribution to the treatment of itp patient's refractory to first-line treatments. of interest, approximately 30% of patients that are tapered from tpo-ra therapy show a sustained response (e.g. a stable higher platelet count than before treatment). the mechanism of how tpo-ra induce these sustained responses is unknown. aims: to analyze the efficacy and immunomodulatory properties of a murine tpo-ra (amp4, amgen) in a well-established murine model of itp that demonstrates both antibody-and t cell-mediated thrombocytopenia (chow l et al., blood 2010) . methods: platelet glycoprotein (gp) iiia (cd61) knockout (ko) mice were immunized with cd61 + platelets and itp was initiated by the transfer of their splenocytes into mice with severe combined immunodeficiency (scid). the scid mice were treated with either placebo or tpo-ra weekly and platelet counts and serum anti-platelet antibodies were measured weekly. results: in an initial pilot dose escalation study, control na€ ıve scid mice treated with a single subcutaneous bolus of different concentrations of murine tpo-ra (1, 10 and 20 ug/kg) had significantly higher platelet counts by 72 h post infusion. in addition, compared with untreated mice, bone marrow histology revealed significantly increased numbers of megakaryocytes. maximal platelet count increases were observed with the highest tpo-ra dose and this dose was chosen to treat scid mice suffering from itp. when scid mice were treated with weekly injections of tpo-ra, platelet counts began to increase after 2 weeks and were fully rescued to control levels after 3 weeks post splenocyte transfer. of interest, compared with non-treated itp mice, serum igg anti-platelet antibody production in the tpo-treated mice was significantly reduced starting from two weeks post splenocyte infusion. summary/conclusions: these results suggest that murine tpo-ra is not only an efficacious therapy for murine itp but also induces immunomodulation indicative of immunosuppression. thus, this model may be able to elucidate the mechanism of how tpo-ra's induced immunosuppression in patients with itp. background: desialylation, the loss of sialic acid content on platelets (plts) glycoproteins (gps) was recently identified to contribute in immune thrombocytopenia (itp). however, the potential impact of autoantibodies (aabs) on megakaryocyte sialylation remains unclear. aims: to investigate the effect of itp aabs on plts and megakaryocytes (mks) sialylation and the subsequent impact on plt survival. methods: aabs from well-characterised itp patients induced gp-modifications were tested using a lectin binding assay. after incubation of mks or plts with itp or control sera, glycan changes were analysed by flow cytometry (fc). to investigate the impact of desialylation on plts life-span, the nod/scid mouse model was used. results: 112 itp sera were investigated in this study. 35 (31%) sera induced a significant increase in rca signal on plt surface compared to control sera from healthy donors (rca-mean fold increase (rca-fi): 3.23, range: 1.76-13.61, p = 0.0001). in addition, 23 (21%) sera caused higher ecl binding to test plts (ecl-fi: 2.31, range: 1.54-5.7, p = 0.0001). injection of desialylating aabs resulted in accelerated clearance of human plts from the circulation of the nod/scid mice which was significantly reduced by a specific neuraminidase inhibitor that prevents background: autoimmune hemolytic anemia (aiha) is a rare autoimmune disease characterised by hemolysis associated with the presence of immunoglobulins (igg, igm, or iga) and/or components of complement system on red blood cells (rbcs), which is usually demonstrated by a positive direct antiglobulin test (dat). depending on the presence of an underlying disorder, aiha can be subdivided into primary and secondary and, by the temperature at which autoantibodies bind optimally to rbcs, into warm antibody aiha (waiha), mixed aiha (including both warm igg and cold igm antibodies), cold agglutinin disease (cad), paroxysmal cold hemoglobinuria (pch) and dat negative aiha. a frequent finding in immunohematology is the presence autoantibodies on rbcs without clinical symptoms of hemolysis that may later develop. aims: the aim of this study was to analyse serologic findings and transfusion support in patients with aiha and also to analyse dat positive patients without clinical symptoms. methods: we included data for all adult patients with aiha and dat positive patients without clinical symptoms diagnosed and/or treated at the university hospital centre (uhc) zagreb, croatia in the period between 2014 and 2018. the diagnosis of aiha was defined by anemia with features of hemolysis (elevated bilirubin and/ or elevated lactate dehydrogenase and/or low haptoglobin level) and a positive dat. results: the data from 64 patients (52% women) meeting the inclusion criteria was analysed. the mean age at the time of aiha was 63 years (range 22-89 years). the mean hg level at diagnosis was 68.60 g/l. dat results were positive mostly with igg+c3d (59%) or igg (31%). most patients had warm aiha (70%). other types of aiha diagnosed were mixed aiha (15%), cad (11%), pch (1.5%) and dat negative aiha (1.5%). in 6 cases alloantibodies were detected with autoantibodies in the patient's plasma. patients were treated with corticosteroids as 1st line therapy and some with intravenous immunoglobulins (ivig). in severe or refractory patients rituximab and/or splenectomy was applied. a total of 80% of patients were transfused at a mean hemoglobin level of 67.88 g/l. during this period we detected 136 dat positive patients without clinical symptoms. summary/conclusions: most patients from our study were diagnosed with warm type of aiha, followed by mixed type aiha and cad. on the other hand, pch and dat negative aiha were very rare, which is in concordance with relevant literature. most patients were transfused despite therapy used, which is not desirable in patients with aiha and should be better controlled, especially in moderate cases of anemias, where this is rarely necessary. a significant number of patients that were dat positive without clinical symptoms may later develop aiha and should be closely monitored. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to diagnosis, investigation and management of patients with aiha in english nhs trusts. methods: a survey of diagnostic and management practice was designed, piloted and disseminated to clinical transfusion leads in all english acute nhs trusts from november 2017 to march 2018. completion was by a consultant haematologist treating patients with aiha but a response that represented a departmental consensus was encouraged. results: responses represented 42% (58/137) of english acute trusts. median number of adults with aiha diagnosed annually was 4-6. in the preceding 5 years, 31% (18/58) recalled at least one patient who had died due to aiha. although 7% (4/57) undertook a bone marrow biopsy in all patients, 93% required additional features, mainly: neoplasia, age over 60 or being treatment-refractory. for patients with suspected drug-induced immune haemolysis, 59% (34/58) would not organise confirmatory tests, either because it was considered unnecessary (29/34), or because clinicians were unsure how to access tests (5/34). when determining aiha subtype, 29% (17/58) indicated there were no circumstances in which they would undertake cold antibody testing (antibody titre and/or thermal amplitude), with 12 considering this unnecessary and 5 unsure how to access tests. in 4 clinical scenarios of patients with aiha and dat positive to c3d ae igg ae cold associated symptoms, up to 87% (47/54) of respondents would not test for cold antibodies. for first line treatment of primary warm aiha, mean duration of prednisolone 1 mg/kg given before judging the patient refractory and reducing the dose was 3.5 weeks (sd 1.70, range 1-19 weeks). second line treatment of choice was rituximab for 82% (45/55) of respondents and splenectomy for 5%. intravenous immunoglobulin and splenectomy were the most cited rescue therapies. for primary cold haemagglutinin disease (chad), first line treatment was rituximab-based for 88% (49/56) but single agent steroid for 9%. we also explored the potential for future audit and research. 64% (37/58) of respondents were able to identify patients with aiha who previously required transfusion. 96% (55/57) of respondents would consider supporting a registry of patients with aiha requiring transfusion. the key questions that respondents thought a registry should address were: morbidity and mortality, treatment response, and differences in the diagnosis and treatment of aiha subtypes. there was uncertainty over access to cold and drug-induced antibody tests and clinicians do not always conduct bshrecommended cold antibody tests for aiha with c3d positive dat. initial treatment of primary warm aiha and chad broadly matched bsh guidelines although 44% (25/57) would continue prednisolone at 1 mg/kg beyond the recommended 21 days before starting a taper, with greater toxicity risk. summary/conclusions: the findings support the need for a range of research initiatives, including creation of an aiha registry. preoperative anemia is common and is associated with adverse outcomes in the peri-operative period. preoperative anemia also increases the risk of allogeneic blood transfusions, which may lead to increased perioperative mortality, increased hospital length of stay and infections. diagnosis and treatment of anemia is one of the tenets of patient blood management (pbm), along with reduction in unnecessary transfusions and diagnostic phlebotomy, as well as use of hemostatic agents to reduce bleeding among many others. effective pbm is multi-disciplinary, multi-modal, timely, individualized and patient-centered. early referral to pbm and multi-modal pbm interventions are associated with greater improvement in pre-operative hemoglobin. pbm has been shown to reduce transfusions and cost, while system-wide, multi-modal programs may also be associated with improvement in mortality. using examples from our local research and practice, i will discuss three aspects of pbm. iron and erythropoiesis stimulating agents (esa) are effective, safe and used extensively in management of pre-operative anemia. previous studies have questioned whether esa leads to increased risk of thrombosis, however, recent systematic reviews do not support these concerns. another pbm approach is to reduce bleeding during surgery by using hemostatic agents such as tranexamic acid (txa). txa reduces transfusion requirements in knee and hip arthroplasty, and is safe, widely available and relatively cheap. txa is effective in both anemic and non-anemic patients, making it an attractive universal pbm strategy. finally, recommendations and evidence-based guidelines on pbm exist, including the most recent international guidelines developed by the pbm international consensus conference. however, knowledge translation in pbm has been a problem and a number of barriers to its implementation have been identified. these include perceived or actual lack of expertise, time, and resources, as well as lack of physician and patient engagement. one way to address patient engagement is education through character driven animation and we are currently trying this approach. 4a-s17-02 low vs . high hemoglobin trigger for transfusion in vascular surgery (tv): a randomized clinical feasibility trial (the tv trial) background: current guidelines advocate to limit red-cell transfusion during surgery, but the feasibility and safety of such strategy remains unclear as the majority of evidence is based on postoperative stable patients. aims: we assessed the effects of a protocol aiming to restrict red-cell transfusion during elective vascular surgery. methods: fifty-eight patients scheduled for lower limb-bypass or open surgery of abdominal aortic aneurysm were randomized to a low-trigger (hemoglobin < 8.0 g/ dl, 5 mmol/l) vs. high-trigger (hemoglobin < 9.7 g/dl, 6 mmol/l) for red-cell transfusion throughout hospitalization. intraoperative change in cerebral-and muscle tissue oxygenation was assessed by near-infrared spectroscopy. we used a nationwide registry to collect data on death and major cardiovascular events, which encompassed (1) severe adverse transfusion reaction, (2) acute myocardial infarction, (3) stroke, (4) new-onset renal replacement therapy, (5) vascular reoperation, and (6) amputation of the lower limb. results: the primary outcome, mean hemoglobin within 15 days of surgery, was significantly lower in the low-trigger group: 9.46 g/dl vs. 10.33 g/dl in the hightrigger group (mean difference 0.87 g/dl; p = 0.022, longitudinal analysis) as were units of red-cells transfused ( background: controlled non-hemato-oncological studies have consistently demonstrated a single-unit red blood cell (rbc) transfusion policy as well as a stringent hemoglobin (hb) rbc transfusion threshold to be safe and reduce blood product utilization. yet, it is unclear whether these conclusions also apply to the hemato-oncological patient population. aims: to quantify reduction of rbc blood product utilization by the introduction of a restrictive single-unit hb-triggered rbc transfusion policy among the inpatient hemato-oncological population. methods: under the liberal transfusion protocol, applied up till november 1, 2017, standard double-unit rbc transfusion was indicated with a hb threshold ≤ 8.1 g/dl and/or anemia-related symptoms. following this date, the restrictive transfusion protocol was introduced involving a lowering of threshold to 7.3 g/dl and single-unit transfusion. for patients with an asa-score of ii-iii and iv, a hb threshold of respectively ≤ 8.1 g/dl and ≤ 9.7 g/dl applied. we evaluated rbc blood product utilization over a 6 month period starting december 1, 2016 (liberal protocol) and december 1, 2017 (restrictive protocol) in all hemato-oncological patients admitted for chemotherapeutic treatment including hematopoietic stem cell transplantation (hsct) with an expected duration of neutropenia of ≥ 7 days. analysis of categorical and continuous data was performed using the chi-square and mann-whitney test, respectively. results: during both observational periods, 137 patients were admitted who in total received 164 therapy cycles, including 56 acute myeloid leukemia (aml) induction cycles and 69 autologous hscts. distribution of indications of admittance, median age, duration of hospitalization and duration of neutropenia did not differ between both periods. during the restrictive period, in 303/402 (75.4%) of transfusions the assigned hb trigger was adhered to. the percentage of single-unit transfusion episodes increased from 29/112 (29.0%) to 81/111(73.0%) with the introduction of the restrictive protocol. overall, rbc blood product utilization per admittance did not reduce under the restrictive protocol (cumulative number of transfused rbc units 4.0 (interquartile range (iqr) 2.0-8.0) during the liberal versus 2.5 (iqr 0.0-9.0) during the restrictive period (p = 0.36)). however, rbc blood product utilization per neutropenic day demonstrated a trend towards reduction: 0.25 (iqr 0.11-0.33) versus 0.15 (iqr 0.00-0.32) units per day during the liberal versus restrictive period, respectively (p = 0.06). this reduction was mainly attributed to autologous hscts during which rbc blood product utilization decreased from 2.0 (iqr 0.0-2.0) to 0.0 (iqr 0.0-1.5) units (p = 0.06), corresponding to a reduction from 0.13 (iqr 0.00-0.21) to 0.0 (iqr 0.0-0.13) (p = 0.01) units per neutropenic day. moreover, 14/38 (36.8%) patients during the liberal versus 21/31 (67.7%) during the restrictive period did not require rbc transfusion during admittance. consequently, stringent hb thresholds as compared to single-unit transfusions seem to more strongly impact rbc blood product utilization. summary/conclusions: a hb-triggered single-unit transfusion policy results in a strong reduction of rbc blood product utilization in the setting of autologous hsct. no utilization reduction was observed among other hemato-oncological inpatient populations receiving intensive chemotherapy. further improvement of protocol adherence rates could potentially increase the benefit of this blood saving strategy. 4a-s17-04 assessment of hb content of packed red cells (prbc): is it time to label each unit with hb content? r jain 1 , n marwaha 2 and s sachdev 2 1 transfusion medicine, aiims, new delhi 2 transfusion medicine, pgimer, chandigarh, india background: in the current era of evidence based medicine and individualized care of patients, rbc transfusion continues to be administered on the basis of conventional wisdom and the notion of an average benefit per unit. the existing blood transfusion practice based on the "number of units transfused" ignores the fact that the total hb varies markedly among the individual rbc units. aims: the present study was aimed at estimating the hb content in packed red cell unit prepared by three different protocols from 350 ml and 450 ml whole blood collection in three types of blood donors: replacement blood donor (rd), first time voluntary donor (ftvd) and regular voluntary blood donor (rtvd). methods: a total of 900 prospective blood donors were included in this study. three hundred whole blood collections were performed in each of the three groups of donors (rd, ftvd, and rtvd). within each group 100 collections were done in double 350 ml, triple 450 ml and quadruple 450 ml blood bags respectively. a pre-donation venous sample was drawn from sample collection pouch for analysis in hematology analyzer as reference method for hb concentration of donor. the hb content of packed red cell units were estimated after collection of representative sample from the blood unit. volume of prbc unit was estimated by the formula of weight of blood in prbc divided by specific gravity. the hb content in unit was estimated by the formula: hb content in unit = hb value of the prbc unit (g/dl) 9 volume of prbc unit (dl). results: in this study the hb concentration (g/dl) was comparable among three types of blood donors except that rtvd had lower hb values when compared to rd (p = 0.007). hemoglobin concentration of prbc ranged from 14.2-29.6 g/dl; mean hb was 21.02 ae 2.90 g/dl. net hb content of prbc bag was lower in prbc prepared from rd as compared to ftvd (p = 0.0001) and rtvd (p = 0.008). the hb content of prbc units prepared from 450 ml collection ranged from 35.19-87.36 g and from 350 ml collection ranged from 30.77-65.78 g. we observed a wide range of net hb content in the prbc units and the correlation coefficient showed the strongest association of net hb content of the prbc unit with the overall volume of prbc (r = 0.730, p = 0.0001).higher volume prbcs have more hb content. volume of prbc bags in the study ranged from 155 ml to 370 ml (including both 350 and 450 ml collections). summary/conclusions: the present study shows that labelling hb content of the prbc unit help in better inventory management for patients. the hb content may help in decision making for release of units for paediatric/low weight versus adults/ higher weight patients. adopting a policy of optimizing dosage of rbc transfusion could have the potential to significantly improve rbc utilization and decrease patient exposure to allogenic blood. this would help further in the clinical transfusion practices based on evidence. 4a-s17-05 nv more, p desai, s rajadhyaksha, a navkudkar and n deshpande transfusion medicine, tata memorial centre, homi bhabha national institute, mumbai, india background: red blood cell (rbc) transfusion is an important medical therapy benefiting the patient in a wide spectrum of clinical setting. critically ill intensive care unit patients in particular, as well as medical and hemato-oncology patients, are among the largest group of the user of rbc. periodic review of blood components usage is essential to assess the blood utilization pattern in any hospital or health care set up. our institute is a 639 bedded tertiary care oncology centre with approximately 18,000 to 20,000 rbc transfusions annually. these transfusions are required in various stages of patient treatment like chemotherapy, radiotherapy, surgical and palliative care and there are established guidelines by the institute to be followed by clinicians. aims: to study clinical practices of rbc transfusions based on indications and to evaluate appropriateness of rbc utilization practices at the institute. methods: this was a prospective observational study, started after approval from institutional ethics committee. total of 4413 rbc transfusion events in adult patients over a period of four months were included and analyzed as per institutional guidelines for their appropriateness. details of transfusion events in form of pre transfusion hemoglobin, indication of transfusion, type of request, number of unit requested and issued, time of issue, site of transfusion and adverse reactions, etc were obtained from department of transfusion medicine records. overall statistical analysis was descriptive using spss software. chi-square test in cross tables was applied to see the relationship between different variables and considered significant if p-value was < 0.05. results: total 4413 rbc transfusion events for 2012 patients were analyzed. there were 1877 (43%) events in 628 patients of medical oncology and 2536 (57%) in 1384 patients of surgical oncology. maximum transfusions were received by patients in age group of 41 to 60 years (47%). total 83% of transfusion events were appropriate as per institutional guidelines. all transfusions administered in operation theatre were found to be appropriate with p value < 0.05. inappropriateness was more 53%(396/735) and significant in daycare setup (p < 0.05). anemia was the most common indication of rbc transfusion observed in 90% of events (3971/4413). total 62% rbc transfusions were given as planned and 38% as urgent transfusions. most common adverse transfusion event observed was allergic reaction in 0.3% of total transfusion reactions. summary/conclusions: clinical practice of rbc transfusions in our hospital was largely found to be appropriate and rational with adherence to institutional guidelines. blood utilization audits should be conducted regularly by transfusion services and results should be discussed with clinician for ensuring judicious use of the scarce resource. the concept of transfusion safety officer (tso) can be introduced for better coordination between clinicians and blood transfusion services to improve practices. 4a-s18-01 paul-ehrlich-institut, langen, germany on a global scale, blood services are quite diverse in regard to aspects like organisational structure, regulatory background, donor populations, donation rates or pathogen epidemiology. the world health organization (who) recognizes blood and blood products as essential medicines and provides guidance to member states for various aspects like blood regulation, best practices in blood collection and transfusion, or screening parameters. more recently a who guideline on residual risk of transfusion associated infections has been established which may facilitate decision-making for the most appropriate screening algorithms. it emphasizes the need for regional evaluation of screening assays and regulatory control of blood-associated ivds. background: babesia, a protozoan parasite that infects red blood cells, is a leading infectious cause of mortality in u.s. transfusion recipients. babesia is usually transmitted through the bite of an infected tick but may be transfusion transmitted (tt) or transmitted from mother to child during pregnancy. babesiosis is a world-wide disease; the ticks that carry babesia have a global distribution. babesiosis has been reported throughout europe and in canada, korea, india, and japan. prospective testing of blood donations in endemic areas of the u.s. revealed 0.38% of donors were positive for babesia dna or antibodies (moritz, nejm, 2016) aims: -to report results of ongoing babesia clinical trial -to explain significance of babesia as a tt infection methods: in cobas â babesia for use on the cobas â 6800/8800 systems, is a qualitative polymerase chain reaction nucleic acid amplification test, developed to detect in whole blood (wb) donor samples the 4 babesia species that cause human disease: b. microti, b. duncani, b. divergens, and b. venatorum. testing began in october 2017 under a u.s. fda-approved investigational new drug application. wb was collected into a proprietary medium that lysed red blood cells and stabilized babesia rna and dna. donations were collected in states with high, low, and no babesia endemicity and screened as individual blood donor (idt) samples. reactive index donations were retested in simulated minipools of 6 (mp6), plus 3 idt replicates with cobas â babesia. reactive index donations were also tested with 2 validated alternate babesia nat and for b. microti igm and igg antibodies. donors with reactive results were invited to enroll in a follow-up study to test for additional evidence of infection. results: to date, 256,802 valid donations have been screened with cobas â babesia, and 15 (0.006%) were reactive. 13 of 15 (87%) initially-reactive donations were confirmed to be positive for babesia with a positive alternate nat or serology result. 1 of 13 (8%) confirmed-positive donations was collected in a state with low babesia endemicity (pennsylvania), and 1 (8%) was collected in a state where babesia is not considered endemic (iowa). 11 of 13 (85%) confirmed positive donations were collected in states with high endemicity. 8 of 13 (62%) confirmed babesia-positive donations were detected in late fall or winter. all 13 (100%) confirmed babesia-positive donations were reactive in mp6. serology results are available for 12 of 13 confirmed-positive donations: at index, 6 of 12 confirmed babesia-positive donations were only igg-positive, while none were only igm-positive; 3 were positive for both igg and igm. 3 of the confirmed-babesia positive donations were negative for both igg and igm antibodies. cobas â babesia showed an overall specificity of 99.999% (256,787/256,789; 95% exact ci: 99.997%>100%). summary/conclusions: the cobas â babesia test successfully identified 13 babesiapositive donations, including 3 confirmed-positive donations with no igm or igg reactivity. 2 donations were collected in states considered low-or non-endemic for babesia. 8 confirmed-positive donations were collected outside of the summer babesia season, when most clinical cases occur. screening with cobas â babesia continues in several laboratories. cobas â babesia is not fda licensed or available commercially. background: babesiosis in humans is caused by the erythrocytic protozoan parasite, babesia microti which is transmitted by tick bites, but is also transfusion transmitted. although frequently asymptomatic or presenting with flu-like symptoms in a normal host, if immunocompromised infection can lead to severe complications and death. b. microti is endemic in the north eastern/upper midwest united states where partial testing of donations has been implemented. in canada, a 2013 study of~14,000 donors did not identify any b. microti antibody-positive samples, suggesting low risk at that time, but risk should be monitored. aims: to evaluate the prevalence of b. microti-positive donations in potentially atrisk areas in canada. methods: between july and november 2018, 50,752 blood donor samples were selected from sites near the us border. minipools were tested for b. microti nucleic acid by transcription mediated amplification (tma) using the procleix â babesia assay on the panther â system with individual testing on reactive pools. reactive donations were also tested by b. microti-specific: american red cross (arc) igg immunofluorescence assay [ifa] and imugen ifa/pcr. a subset of 14,758 tma-negative samples, primarily from the province of manitoba and eastwards to nova scotia, were tested for b. microti antibody using the arc ifa and if positive, the imugen ifa/pcr. donor age, sex, donation status, residential location and collection site location were recorded. donors who tested reactive/positive were informed, deferred and asked about risk factors (possible tick exposure and travel within canada, the usa and elsewhere, history of symptoms) and a follow-up sample was requested for supplemental testing (tma, arc ifa). reactive donations were removed from inventory. results: the 50,752 donor samples were proportional to collections in target geographic regions. age group, sex and donation status were also similar to the donor base in the collection areas. one sample from winnipeg, manitoba was tma reactive and antibody positive on supplementary testing. the donor did not remember symptoms or spending time in wooded areas. he visited the city of fargo, north dakota, usa. the subset of 14,758 samples tested for antibody were also proportional to collections in the targeted areas. four antibody-positive samples were identified from mid-september to october 1, all in south western ontario near lake erie. none were tma reactive. three were interviewed and none remembered any symptoms, any likely tick exposure, or relevant travel within canada or the usa. summary/conclusions: this is the largest b. microti prevalence study in canada. the results indicate very low prevalence with only 1 tma-confirmed-positive donation of 50,752 tested. the donor was from the only region in canada where one autochthonous human case has been reported and active tick surveillance identified b. microti positive tick populations. seropositive donations in south western ontario may suggest low prevalence in that region, but interpretation is less certain due to lack of corroborating supplementary results or case history. given the close proximity to the us border, forgotten us travel should not be ruled out. 4a-s18-04 background: the protozoan parasite toxoplasma gondii is prevalent in animals and humans worldwide. wild and domestic felids are the definitive hosts, and homoeothermic animals serve as the intermediate ones. after primary infection, the parasite persists lifelong within latent tissue cysts. transmission is by ingestion of undercooked or raw meat infected with cysts, by ingestion of food or water contaminated with oocysts, or transplacentally. however, it can also be acquired by blood transfusion and organ transplantation. toxoplasmosis can be a severe disease in immunosuppressed people and neonates whose mothers have acquired primary infection during pregnancy. aims: there is no information about the specific epidemiology of t. gondii infection in blood donors in portugal. therefore, we sought to determine the seroprevalence of t. gondii and associated risk factors in the population of blood donors in portugal. methods: between september 2015 and july 2017, 520 blood donors who attended the portuguese blood and transplantation institute blood banks located in oporto, coimbra and lisbon, and also at regional blood collection meetings, were invited to participate in the study. a written informed consent was obtained and a questionnaire about socio-demographic and behavioural variables was answered. sera were assessed for igg antibodies to t. gondii by a modified agglutination test (mat) commercial kit (toxo-screen da â biom erieux, lyon, france). results: of the 520 blood donors (mean age 38.55 ae 11.14; range 18-65 years old), 38.1% were positive for antibodies to t. gondii. when questioned about toxoplasmosis, almost half the blood donors did not have any knowledge about the disease. the centre of portugal had the highest seroprevalence (55.1%) followed by the north (37.2%) and the south (25.3%). blood donors living in rural areas had a significantly higher seroprevalence (p = 0.001) than those living in urban areas. seroprevalence increased with age, with the highest seroprevalence (60.2%) found in the age group of 46-55 years old (multiple logistic regression [mlr]: or = 7.68; ci: 4.08-14.46; p < 0.001), and decreased with educational level (p < 0.001). engaging in soil-related activities (gardening or agriculture) was significantly related to t. gondii seropositivity (p = 0.02). regarding water consumption, untreated sources (even though including mineral and tap water) was confirmed as a risk factor (mlr: or = 2.72; ci: 1.27-5.86; p = 0.001). other behavioural and eating characteristics, including cats in the household, eating raw or undercooked meat, processed pork products, or not washing raw fruit and vegetables before eating, were not associated with t. gondii infection. summary/conclusions: the risk of t. gondii transmission through blood transfusion is low, and serologic testing of antibodies, with exclusion of blood donors, appears not to be feasible. immunosuppressed individuals, organ transplant patients and pregnant women, should receive t. gondii antibody-negative blood components for transfusion. this study explored the epidemiology of t. gondii in portugal thus providing useful information on the seroprevalence and potential risk factors for t. gondii transmission. information regarding toxoplasmosis and its prevention could be promoted by medical and public health authorities among blood donors, and also the general population, when addressing policies, and designing screening programs, for monitoring and controlling infection and disease in portugal. 4a-s18-05 who is syphilis testing excluding? c reynolds 1 , c pearson 2 , k davison 2 and s brailsford 1 1 nhsbt/phe epidemiology unit, nhs blood and transplant 2 nhsbt/phe epidemiology unit, public health england, london, united kingdom background: screening for treponemal antibodies to detect syphilis in blood donors has been in place in england since the 1940s. there have been no reported syphilis transfusion transmissions in england since records began in part due to sensitivity of the organism to cold storage. since we have specific tests in place for other sexually transmitted infections such as hiv and hepatitis b virus (hbv), the utility of syphilis screening is often questioned. however, it may be a useful proxy for higher risk behaviours particularly following shortening of deferrals for higher risk sexual behaviours from 12 to 3 months in november 2017 and against a background of increasing infectious syphilis in the general population. aims: here we describe the epidemiology of recently-acquired syphilis in blood donors in england compared with hiv and acute hbv infection between 2009 and 2018. methods: monthly donation testing results are collected from the nhs blood and transplant (nhsbt) screening centres and reference laboratory. the demographics, possible sources of infection, and compliance to donor selection in confirmed positive donors are collected by proforma at post-test discussion with the nhsbt clinical team. recent syphilis is classified as igm positive and/or recent history including a negative donation within 12 months for regular donors. results: between 2009 and 2018 there were 153 recent syphilis cases, 121 hiv and 32 acute hbv infections identified by donation screening. recent syphilis rates per 100,000 donations increased from 0.66 to 1.64 whereas hiv decreased from 1.04 to 0.19 with less than 5 positive donations in 2018. acute hbv rates rose slightly from 0.28 to 0.38 in 2018. males outweighed females accounting for 71.9%, 63.6% and 59.4% of cases of recent syphilis, hiv and acute hbv respectively. nearly a quarter of cases of recent syphilis and hiv were seen in donors below 25 years old. of the male donors with recent syphilis, 58.2% reported sex between men and women (sbmw), 19.1% sex between men (sbm) and 22.7% did not report a risk. this contrasted with hiv where 45.5% of male donors reported sbm, just 2.6% not reporting a risk. overall 19, 32 and 3 males with recent syphilis, hiv or acute hbv respectively were non-compliant to the sbm deferral in place at the time of donation. in 2018, 26 donors with recent syphilis aged 23-65 years (median 36 years) were excluded from the donor pool, including 3 non-compliant to the sbm deferral. there were fewer than 5 hiv cases identified in 2018, all over 40 years old, all compliant, reporting sbmw. of the 6 hbv acute cases in 2018, 5 were male, all but one in the 45 and over age-group. summary/conclusions: over the 10 year period demographics of recent syphilis cases appeared similar to hiv with highest rates in young males, albeit lower proportions reporting sbm. following the switch to a 3 month deferral, hiv case detection continued at low level, while syphilis screening continued to exclude higher numbers spanning all age-groups, potentially at risk of other sexually transmitted infections, including non-compliant donors. background: globally, an estimated 113 million blood donations are given annually. in the blood service we are obliged to monitor donor health and ensure that blood donation is safe. in recent years, large-scale blood donor cohort studies in several countries have increased our knowledge on health effects of blood donation. health concerns relate both to immediate side effects like fainting and to possible long-term health issues related to repeated blood or plasma donation. the studies have provided us with data that can now help us introduce an evidence-based individualised donor care -a parallel to personalised medicine. individualised donor care in the management of iron depletion: studies have shown that a large percentage of our frequent whole blood donors, especially young women, are iron depleted. iron depletion is a strong predictor of deferral for low haemoglobin but has also been associated with e.g. restless legs syndrome and lower birth weight in children of frequent donors. the risk of iron deficiency can be mitigated by ferritin-guided prolongation of interdonation intervals or by iron supplementation. prolongation of interdonation intervals can challenge our inventories. iron supplementation, on the other hand, may give gastrointestinal side effects and other effects have been proposed as well, e.g. the masking of malignant disease and increased iron availability with subsequent risk of infection. in a large study we found that iron supplementation is not associated with increased risk of infection. what is the optimal balance between iron supplementation and prolongation of interdonation intervals? a growing number of blood services have implemented various flavours of iron management regimens generating more results. moreover, genetic studies in e.g. the uk, us, holland, and denmark can help us to find donors at high risk of iron depletion or low haemoglobin. we can use all these data in a big data approach in the pursuit of an individualised risk assessment model. other risks for blood donors: the presentation will also cover other risks associated with donation. new studies identify predictors of fainting after blood donation and also new interventions to prevent fainting. the global demand for plasma derived medicinal products has increased severalfold the last 10 years. plasma donors are bled up to 104 times per year in the us. very little is known about the health effects of frequent plasma donation. we know that immunoglobulin levels decrease with frequent donation but how does this affect health? summary/conclusions: the precautionary principle mitigates risk through early intervention prior to evidence. we tolerate next to no risk of transfusion-transmitted infectious diseases. the health of the blood donors, however, has not been protected similarly. we owe to our whole blood and plasma donors to investigate health effects of blood donation and ensure their safety. while the first attempts may not be perfect, we now have the tools to construct models for individualised donor care. background: in 2014, the isbt, aabb, ihn and eba jointly issued the standard for surveillance of complications related to blood donation which categorized donor adverse events (dae) into 6 categories (16 subcategories) defined by specific criteria. severity and imputability were briefly described but were optional. subsequent validation of these categories demonstrated consistency in categorizing reactions, but wide variation in assignment of severity. in 2018, with international input, the aabb donor biovigilance committee developed a severity grading tool (sgt) using a recognized medical adverse event grading system in which neutral grades replace subjective terms (mild, moderate, severe). aims: a large us blood collection establishment (bce) applied the draft sgt to assess its use in real cases of dae. methods: we performed retrospective analysis of all allogeneic and apheresis needle-in donations between 1/1/2017 to 9/30/2018. severity grading was assigned based on criteria defined by the sgt. database review of dae was performed, and each event was assigned a grade based on the type of outside medical care (omc), and on specific key search terms. search terms for omc included emergency room, emergency medical response, urgent care, healthcare professional, and hospital admission. additional specific key search terms included fracture, concussion, laceration, dental injury, surgery, and hospitalization. since duration and activities of daily living (adl) limitations were not captured in our dae database, cases in our dae claims' database were reviewed. case files of events classified as grade 2 or higher were individually evaluated by a physician for grading accuracy. results: in 1,511,758 needle-in collections, 31,320 dae were graded for severity. the majority (16,143, 51.5%) were vasovagal reactions (vvr), followed by 8,570 apheresis-related (27.4%), 6,572 needle-related (21.0%) and 35 allergic (0.1%) events. the majority of dae were grade 1 accounting for 98.6% of all dae, followed by grade 2 (1.2%), and grade 3 (0.1%). there were 2 grade 4 and no grade 5 dae. among the vvr, 98.1%, 1.6%, 0.2% and 0.01% were grade 1, 2, 3, and 4 respectively. grade 3 vvrs included 14 concussions, 11 fractures, 1 dental injury, and 2 pre-faint and 8 fainting events requiring hospitalization for work-up. two grade 4 vvrs involved falls resulting in intracranial hemorrhage requiring immediate medical intervention. for allergic and apheresis dae, there were only 6 and 5 grade 2 reactions respectively, and no grade 3 or 4 events. needle-related dae included 98.3% grade 1, 1.6% grade 2, 0.1% grade 3, and no grade 4 events. of the six grade 3 needle-related dae, 4 were nerve irritations lasting > 6 months, and 2 were dvts requiring hospitalization. summary/conclusions: the sgt provided consistent assignment of severity for the majority of dae, based on outside medical care and specific key search terms. assignment of severity based on impact on activities of daily living or on duration of injury/condition requires tracking over time making such assignments more difficult; modification of our dae tracking database and claims database to capture adl and duration should improve severity assignment for such cases. background: the international haemovigilance network (ihn) has collected aggregate data on complications of whole blood and apheresis donations from member national haemovigilance systems (hvs) since 2006. aims: we analysed the data collected in 2006-2016 in order to learn from the data and consider future improvement of data collection. methods: national hvs entered annual data on donor complications in the passwordprotected "istare" (international surveillance of transfusion adverse reactions and events) online database. from 2008 the donor complication spreadsheet allowed entry of separate data for whole blood donation (wbd) and apheresis, but also provided an option for entering data for all donation types. annual numbers of whole blood and apheresis donations were also collected. the harmonised international standard definitions were implemented in 2015. reactions were captured according to severity level (mild, moderate, severe) but without distinction between donor sex or first time vs repeat donation. extracted data were used to calculate national and aggregate donor complication rates (generally per 1000 donations). results: twenty-four hvs provided figures for donations and donor complications for one or more years (median years per country was 7, iqr 2-8). the total number of country years (cy) was 138, covering 155 million donations. the overall complication rate was 6.3/1000 donations and the median country rate was 3.2 complications/1000 donations (iqr 1.1-10.1). rates were generally consistent within a hvs from year to year but showed considerable variation between hvs; this was also the case for reactions classed as severe. not all countries differentiated between mild and moderate reactions and some reported all reactions under a single severity level. vasovagal reactions were the most commonly reported complication: overall 4.6/ 1000 donations, median country rate 3.1/1000 donations (iqr 0.6-7.7). rare and apheresis-related types of complications such as generalized allergic reaction (0.10 per 100,000, 40 cy), and major blood vessel injury (category available since 2015; overall 0.12 per 100,000, 6 cy) were only reported occasionally. eighteen of the hvs provided separate data for complications of whole blood and apheresis donations in one or more years (total 89 cy, 101.6 million wbd and 26.3 million aphereses, total 128 million donations). for these hvs the median rate of vasovagal reactions was 3.4/1000 wbd (iqr 1.0-9.1) and 1.5/1000 apheresis procedures (1.0-4.2) . reported haematoma rates were higher for apheresis than for wbd: the median per hvs was 0.39/1000 wbd (iqr 0.31-1.2) vs 4.2/1000 aphereses (0.69-5.6); rates of arm pain and/or nerve injury (not separated in 2006-2013) also tended to be higher: median 0.09/1000 with wbd, iqr 0.03-0.34, vs 0.39/1000 with apheresis, 0.05-0.57. summary/conclusions: international reporting allows hvs to study rates of blood donation complications, to distinguish between wbd and apheresis complication rates and capture information about very rare events. variability of reporting and of severity assessment between countries impairs the feasibility of comparisons between hvs. work is needed to improve harmonisation of classification of donation complications and severity assessment for data comparison and research. background: to prevent iron related hb loss, screening with ferritin testing was implemented in stockholm county (approx. 52000 registered blood donors) during a two-year roll-out. iron supplementation is offered to blood donors but has not prevented hb deferrals resulting in 8000 control visits per year. ferritin testing is hypothesized to increase iron compliance. aims: implementation of ferritin testing for surveillance of iron levels for the entire blood donor population with specific attention to new donors, women returning after pregnancy, donors with low hb and at return visit after low hb. yearly testing of plasma and platelet donors. methods: ferritin testing, following a staff education program, was implemented for applicant donors, donors with low hb, women after pregnancy, apheresis donors, followed by screening of registered blood donors per donation site. after initial screening, donors will be tested at each 4 th (women) or 8 th (men) donation, and with yearly testing of young adult blood donors below 25 years. six nurses were educated to process ferritin and blood count results. donors with aberrant ferritin were contacted by letter. results: establishment of cut-off levels and algorithms for ferritin testing and iron treatment was evidence based but met practical limitations such as number of analyses and results that could be processed per week, limitations in liss set-up, blood demand contra preferred cut-offs, iron supplementation compliance. for applicant donors, hb testing show that 20% of female and 9% of male applicants cannot be registered because of low hb (125 and 135 mg/l respectively). adding ferritin testing, a preferred cut-off level of 30 lg/ml (male reference level), would result in additionally 24% female and 1.3% male applicant donor loss. as this would threat the blood demand, cut-off was set to 15 lg/ml for women, above the female reference 10 lg/ml, with an acceptable 6% loss of female applicant donors. for registered blood donors, 4000 mg of extra iron tablets were offered at low ferritin (10-29 lg/ml). this was sometimes combined with prolonged intervals and often repeated before ferritin was restored above 30 lg/ml. donors with ferritin below 10 lg/ml (in 0.6% applicant donors, 0.8% registered donors) or above 600 lg/ml (0.5% applicant donors, 0.1% registered donors) were deferred and recommended to see their physician. for hb deferral, the interval was prolonged from 3 to 5 months, irrespective of ferritin levels. this, together with iron supplementation, resulted in an increase from 50% to 70% approved hb at return. the team of nurses processing ferritin and blood count results (1½ nurse fulltime weekdays) reacted to approximately 40 donor results daily, representing 20% of test results. summary/conclusions: many female donor applicants have suboptimal ferritin levels although they meet required hb for donation. iron treatment was added to retain donors with low ferritin as only prolonged intervals may danger the blood supply. for implementation of ferritin testing, it is necessary to have a well-functioning and agile organization to create and apply algorithms for testing, extension of intervals and iron treatment. background: since november 2017 a new donor screening regime is introduced in the netherlands where serum ferritin levels in whole blood donors are measured periodically to further control potential iron deficiency in donors. donor deferral thresholds are set at 30 and 15 ng/ml, and donors are deferred for six and twelve months respectively if ferritin levels are below these values. as limited information is available on ferritin recovery in whole-blood donors, the policy is introduced in parts such that adaptations to the implementation may be considered based on intermediate results and the impact of the measure on donor well-being can be evaluated. aims: to assess the effect of donor deferral on donor ferritin levels. methods: ferritin levels are measured in new donors and at every fifth donation in repeat donors. donors with ferritin levels below the indicated thresholds are deferred and ferritin is re-evaluated at their return for donation after six or twelve months. the policy allows estimating long term trends in ferritin levels post donation in repeat donors. as ferritin levels are measured in all new donors a reference distribution of ferritin levels in healthy individuals is obtained as well. results: among repeat donors 46% (44% of 16,433 male donors, and 48% of 13,525 female donors) have ferritin levels below 30 ng/ml and are deferred for their next donation. furthermore, the distributions of ferritin levels in repeat male and female donors are similar and each has an average ferritin level of 43 ng/ml. in contrast, we found that only 27% of new female donors (n = 13,283) and 1.7% of new male donors (n = 6,334) have a ferritin levels below 30 ng/ml. the average ferritin level in new donors was 148 ng/ml for males and 60 ng/ml for females. comparing the ferritin levels in new and repeat donors, a reduction in average ferritin levels between 1.5 and 2.0 was observed in female donors and between 3.2 and 4.4 in male donors. both ratios increased with donor age. at the end of december 2018 2884 donors with low ferritin levels returned for donation after six or twelve months deferral. repeat ferritin measurements show that on average the ferritin levels in female donors increased by 13 ng/ml per year whereas average ferritin levels in male donors increased by 34 ng/ml per year. summary/conclusions: in line with earlier findings in literature our results show that repeat donations substantially reduce ferritin levels in repeat donors. these range from 1.5 to 2.0 in female and from 3.2 to 4.4 in male donors, who generally have higher ferritin levels. deferral of donors with low ferritin levels seems to be effective in increasing ferritin levels in donors, however, further monitoring of follow-up in repeat donors is warranted to see whether the proposed scheme allows for sufficient donor recovery over time. there are~7000 different rare diseases and the genes for half have been identified. approximately 3.5 million uk citizens experience premature ill-health because of a rare disease. a conclusive diagnosis is generally not reached and on average the diagnostic odyssey lasts 2.2 years. the main aims of the 100,000 genomes project are to reduce the diagnostic delay by embedding whole genome sequencing (wgs) to accredited standards in the care path of patients with undiagnosed rare diseases. the project started in 2013 and dna samples from 100,000 nhs patients and their close relatives have been analysed by wgs. here we review the results from the nihr bioresource pilot study for the 100,000 genomes project comprising phenotype and genotype data from 13,037 individuals recruited at 83 hospitals using approved eligibility criteria for 15 rare disease domains. we determined the population structure including ethnicity and relatedness estimation, high level phenotypes collected using human phenotype ontology (hpo) terms and quality control and summary metrics for samples and variants. the sequence resource contains over 165 million unique variants in the 10,258 genetically independent samples, with 47% of variants previously unobserved in other large scale publicly available genome datasets. we summarise the curation of gene lists and pertinent findings in 2,000 unique diagnostic-grade genes for the 15 domains. over 1,000 reports assigning pathogenic or likely pathogenic causal variants have been issued with diagnostic yields varying between domains from 0.5% to 55%, while the proportion of novel causal variants ranged between 25% and 73%. we show the power of the bayesian association test, bevimed, to recapitulate decades of clinical genetics discoveries and by identifying >30 novel genes and novel diseasecausing variants in the non-coding space of the genome. we show how typing data for all red cell, hpa and hla class antigens can be extracted from wgs data. we mined the data from the 100,000 genomes project and similar sequence resources to re-version the probe content of the uk biobank axiom array. we genotyped 7588 donors from england and the netherlands with this new array and observed a 99.92% concordance when comparing 92,387 blood centredetermined antigen typing results with genotype-determined ones. for the 48 red cell and hpa antigens that were available for 7,473 donors, the array typing provided a 3.6-fold increase in typing results per donor (13.2 vs 47.9) and 38 rare donors were identified. using the genotyping data we identified 2.6 times more compatible units among this cohort of donors when blood demand was modelled using referral data from 3,146 english patients with more than three red cell alloantibodies. in conclusion the 100,000 genomes project has shown the feasibility of using wgs across a universal healthcare system to deliver a diagnosis for patients with rare diseases. based on these results the nhs has commissioned the analysis of another 500,000 dna samples from patients with cancer and rare disease. with analysis of dna by wgs and arrays becoming part of routine clinical care, blood services must develop competencies to extract transfusion and transplant relevant information from clinical-grade genotyping data. next-generation sequencing (ngs) enables the sequencing of thousands of genes, the exomes, and even entire genomes by single experiments at a reasonable price. there have also been advances in cytometry: use of antibodies with different fluorescence tags enables simultaneous monitoring of the expression of dozens of antigens. however, immunological methods cannot detect every variant discovered by ngs. genome sequencing reveals not only the exome but also the regulatory elements of transcription/translation, such as promoters and enhancers. rna sequencing determines which genes and spliced transcripts are expressed. it is amazing to realize how much this novel technology has been contributing to the better understanding of various biological phenomena. since the initial cloning of the human blood group a transferase cdnas in the early 1990s, we have been studying the abo genes, a and b glycosyltransferases, and a and b oligosaccharide antigens. various scientific disciplines including genetics, immunohematology, biochemistry, enzymology, and glycobiology have been applied to their study. we have made several important scientific contributions. we demonstrated the central dogma of abo: the a and b alleles at the abo genetic locus encode a and b transferases, which synthesize a and b antigens, respectively. we elucidated the allelic basis of the abo system. we found 4 amino acid substitutions between a and b transferases and inactivating mutations in o alleles. we became the first who succeeded in the abo genotyping, discriminating the aa and ao genotypes, as well as the bb and bo, which was impossible by the immunological approach. we have taken a simple experimental strategy: preparation of eukaryotic expression constructs of a/b transferases and their derivatives, dna transfection to human hela cells or their sublines, and immunological detection of the a/b antigen and/or biochemical examination of the enzymatic activity. we used this to show that the codons 266 and 268 are crucial in determining the sugar specificities of galnac/galactose of a/b transferases. we also identified mutations in several subgroup alleles causing restricted substrate use and diminished transferase activity. we also showed that cis-ab and b(a) alleles specifying the expression of both a and b antigens by single alleles encode a-b transferase chimeras. since then, other scientists have characterized more than 250 abo alleles. recent human genome sequencings have identified many more single nucleotide polymorphism variations. the genome sequences of many species are also available. taking advantage of those sequences and associated information, we have expanded our research to include evolutionarily related a1,3-gal(nac) transferases and their genes and scaled it up from the genetic to genomic level. in this talk, i would like to present the followings. 1: our elucidation of the molecular genetic basis of the abo blood group system (as requested by the organizer); 2: identification of novel abo alleles by others; 3: more snp data from genome sequences and potential problems for abo genotyping; 4: findings obtained from analysis of abo genes from other species; bacteria, vertebrates, to primates; 5: a1,3-gal(nac) transferases and their genes and the crosstalk between a transferase and forssman glycolipid synthase (fs); and 6: the potential causes of generation of abo polymorphism and of species variations of the gbgt1 gene specifying the fors polymorphism. in recent years, there has been a concerted effort to improve our understanding of the quality and effectiveness of transfused blood components. the expanding use of large datasets built from electronic health records allows the investigation of potential benefits or adverse outcomes associated with transfusion therapy. together with data collected on blood donors and components, these datasets permit an evaluation of the effect of donor and blood component factors on transfusion recipient outcomes. large linked donor-component recipient datasets provide the power to study exposures relevant to transfusion efficacy and safety, many of which may not otherwise be amenable to study for practicality or sample size reasons. analysis of these large blood banking-transfusion medicine datasets allow for characterization of the populations under study and provide an evidence base for future clinical studies. knowledge generated from linked analyses has the potential to change the way donors are selected and how components are processed, stored and allocated. however, unrecognized confounding and biased statistical methods continue to be limitations in the study of transfusion exposures and patient outcomes. results of observational studies of blood donor demographics, storage age, and transfusion practice have been conflicting. this review will summarize statistical and methodological challenges in the analysis of linked blood donor, component, and transfusion recipient outcomes. 4c-s20-01 a large deletion spanning xg xg and gyg2 gyg2 constitutes a genetic basis of the xg null phenotype, underlying anti-xg a production background: the xg blood group system comprises the homologous antigens xg a and cd99. the cd99 gene resides within pseudoautosomal region 1 on the short arms of the sex chromosomes and thus mimics autosomal inheritance. xg, on the other hand, is x-linked and straddles the pseudoautosomal boundary; a truncated pseudogene composed of only the first 3 exons remains on the y chromosome and therefore males carry a sole full-length copy of xg. this phenomenon manifests as asymmetric frequencies of the xg(a+) phenotype between the sexes: roughly 11% of women and 34% of men are xg(aà). also, whilst xg a immunization is rare, the vast majority of all anti-xg a makers reported are men. recently, we reported that the rs311103c variant disrupts a gata motif between xg and cd99. this abolishes erythroid xg a expression and causes the common xg(a-) red cell phenotype. however, rare individuals who produce anti-xg a cannot be accounted for by this finding. we hypothesized that a structural defect in the xg coding region causes the true xg null phenotype underlying anti-xg a production. aims: we undertook to determine a genetic explanation for anti-xg a production. methods: genomic dna (gdna) was extracted from two whole blood samples and cell-free dna (cfdna) from 13 archived plasma samples from donors producing anti-xg a ; one cfdna sample was from a female donor and the rest from males. polymerase chain reaction (pcr) experiments, sanger sequencing, and database searches were performed to identify and confirm the deletion. aliquots of gdna from four males reported to carry a similar deletion in the 1000 genomes project were also tested. results: in one gdna sample, exon-specific pcr identified a deletion involving part of xg and the downstream gene gyg2. database searches indicated that the most likely deletion was the infrequent genomic structural variant esv2662319 reported in the 1000 genomes project. further analyses with a short (714 bp) and a long (3555 bp) pcr amplicon across the suspected breakpoint determined that this deletion was approximately 114 kb and corresponded well with esv2662319. this finding was confirmed in the second gdna sample. given the rarity of anti-xg a producers, we decided to test for the same deletion in cfdna extracted from old archived plasma samples. of the 13 cfdna samples, poor quality in four samples prevented amplification even from control reactions and one was contaminated with bacterial dna. in the remaining nine samples, eight could be amplified for the deletion-specific 714-bp short amplicon while one was negative for the deletion. sanger sequencing of the amplicons revealed a heterogeneous repetitive dna element, ltr6b, hinting at a previously-reported recombination event. this deletion was not detected in the samples from the 1000 genomes project which reiterates the previously identified deficiency in data interpretation and reporting for deletions. summary/conclusions: a large deletion disrupting the xg and gyg2 genes accounts for the xg null phenotype underlying the majority (10 of 11) of anti-xg a makers. one sample remained unexplained, indicating further heterogeneity to be explored. our data help to explain why anti-xg a production is rare and has primarily been reported in men. background: s and s antigens encoded by gypb differ by one nucleotide (nt), c.143c>t, p.thr48met. two different genetic backgrounds are associated with silencing of s antigen and a u+ w phenotype. these include the nt change c.230c>t (p.thr77met) causing partial exon skipping and designated gybp*03n.01 (gypb*ny) and c.270 + 5g>t, an intron change causing complete skipping of exon 5, designated gypb*03n.03 (gypb*p2). aims: samples from three individuals, a previously transfused african american sickle cell patient (p1), a blood donor of unknown ethnicity (p2), and an african american patient (p3) (lapadat r. 2014 aabb abstract) were investigated for discrepant serologic and molecular results when determining s and s phenotype. methods: standard methods were used for rbc typing with licensed s and s reagents and rbcs from donor p2 were also tested with monoclonal and polyclonal anti-s and anti-s. dna was isolated from wbcs and hea precisetype performed on p1 and p2. p1 was also tested by gypb*s/s as-pcr, exon 5 pcr-rflp for c.270 + 5g>t and as-pcr for c.230c>t. p3 was tested for gypb*s/s and c.230 c>t and c.270 + 5g>t changes by a real-time pcr-fluorogenic 5' nuclease taqman chemistry. for all, gypb exons 1-6 were amplified and sanger sequenced and aligned to consensus using clustal x. results: rbcs of all three probands typed s-and strongly s+ while dna testing indicated c.143t/c (gypb*s/s). assay for the two common gypb*s silenced alleles, c.230c>t and c.270 + 5g>t, indicated all three samples had both silencing mutations previously reported to be independently associated with a sàu+ w phenotype. hea precisetype could not interpret this novel allele combination and indicated gypb*s as pv (possible variant). samples were confirmed to be heterozygous for c.143c/t, c.230c/t and c.270 + 5g/t by exon specific sequencing and as-pcr, pcr-rflp and real-time pcr. by long range sequencing of gypb, all three were heterozygous c.59t/g and c.60a/g (p.20leu/trp), c.67a/t (p.23thr/ser), c.71a/g and c.72g/t (p.24glu/gly), c.143c/t (s/s), c.208g/t (p.70val/leu), c.230c/t (p.77thr/met), and c.270 + 5g/t. all samples were also c.251g/g (p.84ser) and heterozygous for several previously recognized silent changes in exon 2, c.87t/c, c.96t/c and c.102a/g. summary/conclusions: we report a novel silenced gypb*s allele that can confound gypb genotyping interpretation. the allele was found in three probands associated with a sàs+ phenotype. in these samples, two changes previously reported to be inherited independently and both associated with silencing of s antigen are carried on the same allele. dna-based testing could not rule out that c.230t or c.270 + 5t are separate and that gypb*s was also silenced. robust s+ rbc typing indicates both changes are on gypb*s. gene sequencing confirms the c.270 + 5t change is on a gypb*03n.02 [gyp*he(ny)] background. c.230c>t (rs79492560) and c.270 + 5g>t (rs139511876) have a frequency of 0.0053 respectively 0.032 in the african population (exac). although we identified 3 samples, the frequency of this novel allele is unknown. background: the lutheran blood group system currently consists of 25 antigens. these antigens are of low immunogenicity and may cause mild-to-moderate transfusion reactions and hemolytic disease of the fetus and newborn. the activation of lu-glycoprotein/bcam on red blood cells (rbcs) and its interaction with laminin-5a is thought to play a role in vaso-occlusion in sickle cell disease and other hematological disorders. the two glycoprotein isoforms lu-glycoprotein and bcam are encoded by the bcam gene which consists of 15 exons located on chromosome 19q13.2. a number of rare lutheran phenotypes have been previously recorded in israel, including lu:-6,9 observed among iranian jews, lu:-20 in one thalassemia patient and one case of lu:-21. in this report, a previously transfused pregnant arab patient with b-thalassemia intermedia was investigated because she presented with an antibody to an unknown high frequency antigen (hfa), potentially related to the lutheran system. aims: to characterize a novel lutheran antigen through serological and molecular investigation of a patient with a lutheran related antibody. methods: initially, the red cell phenotype and the presence of a lutheran related antibody in the serum of the patient were detected by standard serological techniques, utilizing enzyme treated and chemically modified cells and rare cells and sera from the nbgrl collection. further serological investigations were carried out using standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were performed using soluble recombinant lu protein (srlu). eluates were prepared using acid elution method (gamma elu-kit ii). genomic dna was isolated from whole blood and all exons of the bcam gene were amplified by pcr and directly sequenced by sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: the patient's plasma reacted with all cells tested, except for three examples of in(lu) cells and cells treated with 2-aminoethylisothiouronium bromide, trypsin and a-chymotrypsin. inhibition studies with srlu protein showed complete inhibition of the antibody, thereby confirming the antibody to be directed toward an epitope on the lu-glycoprotein. in addition, testing of inhibited plasma revealed the presence of underlying anti-e and anti-fy a . an eluate was prepared to isolate the patient's lu-related antibody and this eluate was found to be incompatible with examples of lu:-5, lu:-6, lu:-8, lu:-13, lu:-21, lu:-22, and lu:-23 cells, whereas in(lu) were compatible. results of serological typing of the patient's cells, for lu system hfas, could not be conclusively determined due to the patient having been recently transfused. however, results suggested (through absence of mixed field reactivity) the patient's cells to be lu: -1,2,8,12,13,-19,21 . bcam sequence analysis confirmed the patient to be lu*02, lu*18 and revealed a novel homozygous mutation c.1351a>c in exon 11, encoding p.lys451gln in the lutheran glycoprotein. summary/conclusions: a novel homozygous mutation c.1351a>c (p.lys451gln) in exon 11 of bcam was identified in a patient with an antibody to a lutheran hfa. serological and genetic evidence presented here indicates discovery of a novel antigen of the lutheran blood group system, which we propose to name lura. background: lutheran glycoprotein and basal cell adhesion molecule antigen b-cam are two isoforms of a type i membrane glycoprotein residing on red cell surfaces. both isoforms are adhesion molecules with the main function of laminin binding, and both carry antigens of the lutheran blood group system (lu). the system currently comprises 25 antigens, all encoded by mutations in the alternatively spliced single gene bcam located on chromosome 19. currently, isbt lists 20 high incidence antigens in the system. aims: we report a case study of an individual with an unidentified alloantibody to high incidence antigen present in her plasma. samples from the patient and her family were investigated. we provide here serological and molecular evidence for a novel high incidence antigen of the lutheran blood group system. methods: serological investigations were performed by standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were completed with soluble recombinant lu (srlu) protein. genomic dna was isolated from whole blood of the patient and her family members; all the exons of the bcam gene were amplified by pcr and analysed by direct sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: presence of a lu-related antibody in the patient's plasma was confirmed, reacting moderate strength by liss iat with untreated and papain treated cells. cells from the patient's mother, father and two siblings were all incompatible with her plasma, though weaker than panel cells, reflecting dosage. only in(lu) cells were compatible with patient's plasma. the antibody was successfully inhibited with srlu protein, thereby confirming the epitope recognised by the antibody resides on the lutheran glycoprotein. the patient's cells were found to be lu: -1, 2, 3, 5, 6, 8, 13, 20, 21 . bcam sequencing revealed a novel homozygous mutation c.121g>a in exon 2, encoding p.val41met in the lu glycoprotein. the c.121g>a change appears to be an extremely rare mutation, listed in gnomad database with a frequency of 3.98 9 10 -6 and with no known homozygous examples. homology model of the novel lutheran glycoprotein was subjected to all-atom molecular dynamics calculations to analyse potential conformational changes. summary/conclusions: we report serological and genetic evidence for a novel antigen of the lutheran system, which we propose to name lunu. the evidence will be submitted to the isbt red cell immunogenetics and blood group terminology working party for consideration for allocation of antigen status. the absence of this high incidence antigen arises from a rare single amino acid change p.val41met in the lutheran glycoprotein and the presence of anti-lunu in the patients' plasma was presumed to have been made in response to previous pregnancy. on native, papain-treated (diagast) and trypsin-treated (sigma) rbcs. genomic dna was extracted from peripheral blood cells by an automated method, amplified by sema7a exon-specific primers and sequenced. results: the proband was a 32-year-old female patient of moroccan origin, group a, d+c+e-c+e+, k-, without transfusion history. she was hospitalized at 27 weeks gestation for a blighted ovum requiring a manual vacuum aspiration, with a significant hemorrhage risk. a rbc antibody screening was performed by a first laboratory. the antibody reacted 1 + by iat on all native reagent rbcs, with negative autocontrols, but was nonreactive on papain-and trypsin-treated cells. an anti-ge2 was initially suspected, due to the pattern of reactivity and ethnic background. new blood samples were referred to our national immunohematology reference laboratory. the antibody showed the same profile. anti-ge2 and anti-ch1 could be ruled out. the serum was nonreactive with two jmh:-1 and positive with two jmh:-3 samples. the patient was found to be jmh1 positive. in addition, a soluble recombinant jmh protein (jmh imusyn/inno-train) fully abolished the reactivity of the panagglutinating antibody. the antibody was an igg4. overall, these results were consistent with a probable jmh variant and prompted us to perform sema7a sequencing. three nucleotide changes were found, in homozygous state: a rare nonsynonymous change in exon 7, c.709g>a (p.asp237asn, rs140707085, maf < 0.01, sift score = 1); a common synonymous change in exon 12, c.1545a>g (p.gln515gln, rs741761, maf = 0.5); a rare non-synonymous change in exon 14, c.1865g>a (p.arg622his, rs140128092, maf < 0.01, sift score = 0.36). the analysis of surface accessibility of asp 237 and arg 622 using the 3d structure of sema7a (rcsb pdb-3nvq https://www.rcsb.org/structure/3nvq) showed that only arg 622 was predicted to be an exposed-epitope. interestingly, all other reported jmh variant phenotypes correspond to an arginine substitution. of note, we retrospectively found another individual of algerian ancestry (pregnant woman) with a pan-agglutinating igg4 antibody showing a similar pattern of reactivity, and with the same three changes in sema7a. we unfortunately could not perform a cross-compatibility testing with the proband (no material left and unsuccessful contact). summary/conclusions: serological and molecular studies allowed us to provide evidence for a novel high-prevalence antigen in the jmh blood group system, very likely encoded by the p.arg622his substitution in sema7a. we propose to provisionally assign the name jmh7 for this antigen. interestingly, our two unrelated jmh:-7 individuals were from north african ancestry. background: the abo system was discovered almost 120 years ago and the underlying structures later elucidated as carbohydrates carried by glycoproteins and glycolipids. the terminal trisaccharides galnaca3(fuca2)gal and gala3(fuca2)gal constitute the clinically important a and b epitopes, respectively. clausen et al. (pnas, 1985) showed that the a antigen could be extended to a repetitive glycolipid a epitope, galnaca3(fuca2)galb3galnaca3(fuca2)galb4glcnac-r. however, extended forms of b antigen have not been described. we encountered two related situations with unexplained serological reactivity. firstly, enzyme-conversion to group o treatment of group b (b-eco) red blood cells (rbcs) with a3-specific gh110 family exogalactosidase (bzyme) abolishes b antigens as detected by hemagglutination and flow cytometry with all monoclonal anti-b tested. despite this, 40% of group o plasmas have been reported to give positive crossmatch results with b-eco rbcs. secondly, plasmas from ab and b individuals of the globoside-deficient p k phenotype contain anti-p and anti-px2 but react stronger with bpp-rbc than with app/opp-rbc. based on these findings, we hypothesized the presence of a bzyme-resistant, b-related glycolipid. aims: to identify the molecular basis of the enigmatic serological observations outlined above. methods: plasma and eluates from an a 1 b individual with the p1 k phenotype were investigated by hemagglutination and flow cytometry, as were eluates from b p 1 k and o plasma. rbc membrane glycolipids were extracted from two batches of pooled, expired group b-rbc units (frozen 500-litre reference preparation and confirmatory preparation from 24 freshly collected units). native or enzyme-treated glycolipid fractions were analysed by liquid chromatography electrospray ionizationmass spectrometry (lc-esi/ms) and immunostaining of thin layer chromatography (tlc) plates. antigen expression in the h+bà human erythroleukemia (hel) cell line was analysed by flow cytometry following overexpression of selected glycosyltransferases. results: anti-p-depleted eluates made from a 1 b p 1 k plasma contained anti-px2 and antibodies of unknown specificity that reacted stronger with native or papaintreated bpp-rbcs compared to app/opp-rbcs. anti-px2 was removed by adsorption onto opp-rbcs but reactivity (here designated anti-extb) remained against b/bpp/b-eco rbcs. lc-esi/ms of glycolipid fractions from group b units revealed an unknown hexnac-hex-(fuc-)hex-4hexnac-hex-4hex heptasaccharide. upon b-nacetylhexosaminidase treatment of this candidate structure, a group b type 2 hexasaccharide was produced, demonstrating that the terminal hexnac of the hexnac-gala3(fuca2)galb4glcnacb3galb4glc heptasaccharide was b-linked. since the discovery of the anti-platelet effects of aspirin platelets have been a major therapeutic target for pharmaceutical companies and also a very profitable target. however, the effectiveness of aspirin has also been a challenge as it is an inexpensive drug and any new agent needs to show clear benefit over aspirin. furthermore the risk of bleeding from anti-platelet agents, especially cerebral bleeds, has also presented challenges. in the 1990's orally active gpiib/iiia antagonists were considered to be the 'super aspirin' but clinical trials showed increased mortality and ultimately this class of drugs was relegated to iv use only in high-risk patients. gpib/ix/v antagonists were also a promising drug target but no agent made it to market. the real breakthrough was the discovery of the p2y12 antagonist clopidogrel which, in conjunction with aspirin, proved to be very effective at preventing thrombotic events and as a result it became the biggest selling drug in the world at the time. with clopidogrel now offpatent the combination of aspirin and clopidogrel is a formidable challenge to any new agent both in efficacy terms and pharmacoeonomic terms. so is there a future for new anti-platelet agents? with the growing awareness of the role of platelets in inflammation and an understanding of how the immune activation of platelets differs from the classical haemostatic activation of platelets it is now possible to develop novel anti-platelet agents that target inflammation without compromising haemostasis. it is here that we should look for the next generation of anti-platelet agent. 4c-s21-02 university hospitals of geneva, geneva, switzerland platelet function defects, either congenital or acquired, are associated with increased bleeding risk, particularly in a perioperative setting. the use of platelet function assays is therefore tempting in order to tailor transfusion and limit platelet transfusion to those bleeding patients with impaired platelet function, as assessed by those assays. however, the current guidelines provide only weak recommendations supporting the routine use of these assays. indeed, there are numerous platelet function assays on the market that differ in their method of evaluation of platelet function and agreement between their results is at best moderate. the threshold values beyond which procedure-associated bleeding risk becomes worrisome is not standardized. moreover, observational studies addressing the predictive value of platelet function testing in perioperative or spontaneous bleeding are not consistent. finally, management trials with randomized patients assessing the benefit of platelet function testing are scarce. more recent data identified selected situations where platelet function testing may be useful though. i will review the different platelet function assays as well as selected clinical studies addressing the impact of platelet function testing to improve bleeding and transfusion-related outcomes. the latest recommendation will be addressed too. background: platelet refractoriness complicates the provision of platelet transfusions in management of thrombocytopenia in oncology patients. platelet refractoriness poses challenge due to alloimmunization to hla and human platelet antigens and is associated with adverse clinical outcomes. aims: a prospective study was undertaken to analyse result of platelet compatibility with post-transfusion platelet count increment and to ascertain presence of platelet antibodies as causative factor in platelet refractory oncology patients. pulmonary complication after blood transfusion is the leading cause of transfusionrelated morbidity and mortality, with an incidence reported between 0.05 -15% of all transfused patients. the most important transfusion related pulmonary complications are transfusion associated circulatory overload (taco), transfusion related acute lung injury (trali) and transfusion associated dyspnea (tad). in this presentation the recent changes in the international definitions will be presented and discussed. furthermore, insights in the different underlying pathophysiologic mechanisms will be highlighted. in the past decades only for trali prevention strategies have successfully been designed and implemented. currently no evidencebased treatment strategy is available for any of these life-threatening syndromes. insight in the pathogenesis of pulmonary complications after transfusion should pave the way for future prevention and treatment studies. the issue of the impact of iron overload / toxicity on the hematopoietic stem transplantation (hct) outcome has been firstly addressed in the field of transfusion dependent thalassemia. today the concept has been extended to other diseases characterized by periods of variable duration of transfusion dependence such as myelodysplastic syndrome (mds) and myeloproliferative diseases. patients requiring regular blood transfusions certainly develop iron overload leading to tissues and organ damage. iron burden before transplant significantly impacts outcome and long-life posttransplant. it is well known that iron overload is deleterious for organs such as liver, heart and endocrine glands and it has been postulated could also increases the risk of infections and severe graft versus host disease early after hct. recent preclinical data has shown how increased production of reactive oxygen species (ros) resulting under iron overload condition, could impair the stem cells clonality capacity, proliferation and maturation. also, microenvironment cells could be affected through this mechanism. for this reason, iron overload is becoming an important issue also in the engraftment period early post-transplant. high baseline ferritin levels before hct have been shown to negatively influence clinical outcome, but nowadays, ferritin is considered a steady and not biologically active form of iron, while free iron forms as non -transferrin bound iron (ntbi) and labile plasma iron (lpi) are considered the main trigger of cell damage more representative of the dynamic tissue damage. the scientific community is moving the iron disease from a "bulky" disease, such as classically in thalassemia (based on quantitative iron parameters as ferritin, red blood cell transfusion number, mri) to a "toxic" disease (based on active and dynamic biological markers as ntbi/lpi). at this time in all the studies published on hct setting, only the correlation between direct or indirect estimates of iron overload (mainly serum ferritin) and outcome parameters has been explored, while the duration of exposure to toxic iron species has not been taken into account. the first study that explored the lpi role in relationship with outcome was published by wermke and colleagues in malignancies. they investigated the predictive value of both stored (mri-derived liver iron content) and non-transferrin-bound-iron, defined as enhanced labile plasma iron (elpi) on post-transplantation outcomes in patients with acute myeloid leukemia or mds. their prospective, observational all-ive study showed that patients who had raised elpi concentration at baseline, also had significantly increased incidence of non-relapse mortality at day 100 (33%) compared with those who had normal elpi at baseline (7%) (p = 0.00034). reinterpreting transplant predictive factors in the light of the current advances in understanding iron homeostasis further supports the concept that the key to successful transplantation is regular and life-long chelation therapy to consistently suppress tissue reactive iron species and prevent tissue damage in the years before hct. in transfusion medicine, the role of donor sex was long considered to be limited to the increased risk of trali observed after transfusions from female donors. this risk has been shown to be limited to female donors with a history of pregnancy and to plasma rich products (i.e. excluding red blood cell products, typically containing < 50 ml plasma). until, in 2011, we found that sex-mismatched red blood cell transfusions were associated with increased recipient mortality. since then, several other studies have confirmed these findings, but some studies also did not find an association. all of these studies relied on the analyses of routinely collected health care data, which was not primarily intended to be used for research. as a result, analyses are complex and often difficult to properly appraise based on published descriptions. therefore, the discussion about possible reasons for these discordant findings has largely focused on the methodological approaches of the different studies. other potential explanations include differences in donor or patient populations, production methods, or storage time of blood products. the different potential explanations are expected to be associated with different underlying biological mechanisms. therefore, further delineating which donor, patient, and product characteristics modify the observed association could provide more insight into the underlying mechanism. in 2017, we observed that only transfusions from female donors with previous pregnancies were associated with increased mortality and only in male recipients under 50 years. this leads us to postulate that pregnancy induced long term changes in the female immune system are transferred during red cell transfusion, with negative consequences for young male recipients. the low amount of plasma present in red cell products further lead us to assume a cellular component, like passenger leukocytes, to be involved. it has been shown that micro-chimerism of passenger leukocytes can persist for decades after transfusion, even of leuko-reduced blood products, suggesting long term immune-modulation could play a role. we hypothesized that passenger leukocytes would die during storage of blood products and the negative effect of ever-pregnant female donors, on the survival of young male red cell recipients, would therefore be attenuated by increased storage time. however, our data seem to indicate the opposite. the risk of death was increased over three-fold for young male recipients of old (>24 days storage) red cells from ever-pregnant donors, compared to for young male recipients of fresh (<10 days storage) red cells from ever-pregnant donors (3-year cumulative incidence of death 15.4% versus 4.8%). the negative control group (i.e. young male recipients of red cells from male donors) showed a much weaker association of mortality with storage time (i.e. 7.2% versus 4.7%). these findings seem to falsify our hypothesis that mortality could be caused by passenger leukocytes, establishing long term immune-modulatory effects. another potential mechanism that has been suggested could be the presence of cellfree dna in transfused blood products. this cell-free dna increases during storage. however, more research is needed both to establish if cell-free dna can also be linked to previously pregnant blood donors and by which mechanism it could negatively affect young male transfusion recipients. clinical trials (cts), the gems in clinical research for generating robust evidence in medicine and public health, are costly and complicated undertakings. in resource limited setting like sub-saharan africa (ssa) where the health systems are sub-optimal and where capacity for research is limited, the conducting of cts can be a daunting challenge. the challenges of undertaking cts in rls may be categorized based on the occurrence of the bottleneck(s) in relation to the ethics and regulatory approval process: pre-approval: protocol development: in order to develop a context-specific protocol which is subsequently subjected to an ethics and regulatory approval process, investigators need to review and ensure that the protocol is pragmatic and feasible with respect to implementation. this results into a time-consuming reiterative process of reality-checking the protocol. site selection: in light of the limited research infrastructure, investigators in rls and their developed world partners spend considerable time reviewing and selecting suitable sites for participation in the anticipated protocol for the cts. suitable sites are usually very few and with competing on-going studies. approval: institutional review board (irb) approval: the irb approval process can be quite lengthy (6-9 months) with considerable unpredictability in the periods between the initial and subsequent irb reviews. national regulatory approval: the requirements by national regulators are unusually innumerable with limited flexibility to accommodate specific cts. post-approval: the key post-approval challenges for cts implementation in rls are attaining appropriate participant enrolment and maintaining high retention rates. specifically, for participant enrollment, the challenge may be unforeseen competing cts targeting the same participant pool or community perspectives that may discourage participants from getting screened for the cts. retention may also be a challenge particularly where participants view enrollment as a chance to access healthcare services may therefore not have any incentive to keep in a study after the initial study visits. in conclusion, cts are complex undertakings wherever they are conducted but are doubly challenging in rls like sub-saharan africa. the bottlenecks at the preapproval, approval and post-approval stages are considerable. nevertheless, it is rewarding to perform ctus in rls given that the data generated therein is highly valued by national regulators and may hasten the registration process for medical products. background: interest in an appropriate and effective whole blood (wb) pathogen reduction technology (prt) is growing, especially in sub-saharan africa where the residual risk of transfusion-transmitted infections (ttis) remains unacceptably high and wb is still frequently used. cerus corporation, manufacturer of the intercept tm blood system, and swiss transfusion src are collaborating on a clinical development program to adapt intercept prt using amustaline (s-303) and glutathione (gsh) for red blood cells (rbcs) into an appropriate prt for wb in resource-limited settings in africa. treatment with amustaline/gsh has been shown to inactivate a broad spectrum of transfusion-transmissible pathogens in rbcs. studies with amustaline/gsh in wb have shown effectiveness against a duck hepatitis b virus (>5.3 log reduction) and plasmodium falciparum (>7.5 log reduction), with future studies planned. a wb prt system with amustaline/gsh also has the potential benefit of minimal electricity requirements. aims: to describe the safety and clinical objectives for a phase 1 clinical trial using the amustaline/gsh prt system for wb in africa, and describe research and development efforts to adapt the intercept prt system for rbcs into a robust and appropriate wb system for settings with high burdens of tti and limited resources. methods: the protocol for a phase 1 clinical trial using pathogen-reduced wb treated with amustaline/gsh in an african country is presented, as are current research and development activities related to the development of a prt system for wb. results: in the planned phase 1 clinical trial in africa, 20 clinically stable patients with anemia who require wb transfusion will be randomized into two study arms at a large medical center in a sub-saharan african country. enrolled patients will receive one unit of non-leucocyte-reduced wb treated with amustaline/gsh, or a unit of untreated control wb or rbcs. the primary safety endpoint will be the incidence of high-imputability transfusion reactions (swissmedic ≥grade 2) within the first 24 hours of transfusion. data will also be collected on all adverse events and transfusion reactions (all grades) and the development of treatment-emergent antibodies to pathogen-reduced wb or auto-antibodies within 59 (ae3) days of the study transfusion. clinical efficacy will be characterized by hemoglobin increment 24 hours after transfusion adjusted to hemoglobin dose and body weight. summary/conclusions: a prt system for wb is being developed based on the intercept prt for rbcs that is in advanced development in europe and the united states. intercept-treated rbcs have met efficacy and safety endpoints in phase 3 clinical trials. the amustaline/gsh prt system used to treat intercept rbcs has demonstrated effective inactivation against a broad spectrum of agents that may result in ttis. a phase 1 clinical trial using an adapted prt system for wb in africa is the first step in a clinical development program that includes additional pathogen inactivation efficacy studies and improvements to the wb prt implementation process. together, these developments and evaluations represent progress toward a realistic and appropriate prt for wb in africa and other resource-limited settings. background: in australia, demand for plasma-derived products has increased dramatically, and there is a need to increase plasma collections. first-time donor retention, including the rate at which first-time donors return, is a pressing issue. a quick return is optimal as this increases the overall plasma yield and is associated with long-term retention. however, we lack evidence of effective interventions to encourage first-time donors, particularly those donating plasma, to return and to establish a higher frequency donation routine. working from schultz's (2014) framework, this intervention study was based upon insights from interviews with first-time plasmapheresis donors. participants identified barriers such as time and lack of knowledge about plasmapheresis. facilitators included being able to help more people and to donate more frequently than allowed with whole blood. participants generally favoured donating at a frequency of every 4 weeks. aims: the aim of this study was to test the effectiveness of three intervention conditions compared with the business-as-usual (bau) procedure on the proportion of donors returning to donate plasma and the number of plasma donations. we report on the data from 2 months post-donation. methods: donors were randomly assigned to one of four study conditions. in conditions 1 and 2, donors received an email one day after their initial donation. in the first condition, donors received the bau 'thankyou' email. donors in the second condition received an alternative email with content derived from the interview study. donors in the remaining conditions received either the bau email (condition 3) or the revised email (condition 4) coupled with a telephone call. the phone call was scripted to provide additional information about plasma, including how often plasma can be donated, a suggestion to donate every 4 weeks, and a prompt to forward-book appointments. results: the final sample (n = 6788) comprised 3859 women (57%) and 2929 men (43%) aged 18-70 (mean = 32). after two months 37.2% of donors returned to donate plasma at least once. after controlling for gender, age, and blood group, donors in each of the intervention conditions were more likely to return to donate plasma than were donors in the bau condition. the greatest effect was found between donors randomized to condition 4 (revised email + phone call), or = 1.305, ci = 1.128-1.510, and bau. donors assigned to the two telephone conditions (condition 3 and 4) donated plasma at a higher frequency than bau. summary/conclusions: this study tested the effectiveness of interventions designed to encourage first-time plasma donors to return to donate plasma and to establish a routine of donation. early indicators suggest that the evidence-based email and phone call elements are more effective than bau in bringing donors back to donate plasma, and the revised email combined with a phone call had the greatest positive effect on short-term plasma yield. background: healthy individuals with hereditary hemochromatosis (hh defined as hyperferritinemia and homozygous p.c282y mutation), but also carriers of other hfe mutations (p.c282y/p.h63d or homozygous h63d) with elevated serum ferritin (sf) are accepted as blood donors, if allowed by local regulations and if eligibility is fulfilled. generally, blood components are released for transfusion at normal sf levels (< 200 ng/ml in females, < 300 ng/ml in males). aims: prospective, two-center, randomized study comparing the efficacy and tolerability of double-erythrocyte apheresis (2rbcaph) and whole blood phlebotomy (wbph) for iron depletion in asymptomatic subjects with hh or hyperferritinemia and other hfe mutations in the setting of routine blood donation. methods: eligibility criteria included age ≥ 18-60 years, total blood volume ≥ 5 l, bmi < 35 kg/m 2 , hb ≥ 140 g/l, elevated sf levels and no end organ damage due to iron overload. 2rbcaph (360 ml rbc) were scheduled every 14 days and wbph (450 ml) every 7 days until sf was < 100 ng/ml. a complete blood count and sf were measured at baseline, at every visit and at follow up 8 weeks after completion of the study. adverse events were systematically recorded. the treatment effect was tested by poisson regression, with gender, hfe mutation, bmi and baseline sf as covariates. results: 30 subjects (5 females; mean age 47 years) were randomized to wbph (n = 16; 1 female) or 2rbcaph (n = 14; 4 females). hfe mutations were p.c282/ p.c282y in 17 subjects, p.c282y/p.h63d in 9, and p.h63d/p.h63d in 4. at baseline, mean hb was 149 g/l (sd 7.8) and median sf was 504 ng/ml (iqr 406-620 ng/ml). 222 procedures (wbph n = 146, 2rbcaph n = 76) were completed; 9 were interrupted (local hematoma, insufficient flow); 35 (16 wbph, 19 2rbcaph) were postponed because of low hb and 15 for non medical reasons. there were 2 drop-outs in the wbph arm due to depression and poor compliance, respectively. anemia (hb < 130 g/l in males, <120 g/l in females) occurred after 15 visits in 8 wbph subjects and after 5 visits in 3 2rbcaph subjects. fatigue was reported after 37 phlebotomies and 31 aphereses. only 5 participants (17%) completed the study per protocol. 136 blood components (94 rbc concentrates and 42 plasma units) for transfusion were obtained. overall, a median of 7.5 wbph (iqr 6.2-9.8) was needed to reach sf < 100 ng/ml, corresponding to 1.8 times of 2rbcaph (median 4.0, iqr 3.0-5.8) (p = 0.0001). analyzing separately p.c282/p.c282y and p.c282y/p.h63d carriers, the relation wbph to 2rbcaph was 1.6 and 1.8, respectively. treatment arm and hfe mutation were the covariates with significant effect on the primary endpoint (p = 0.0001 and 0.007, respectively). summary/conclusions: 2rbcaph is more efficient than wbph for iron depletion in healthy subjects with hh or other hfe mutations and moderate hyperferritinemia. intensive treatment schedules, generally recommended for hh, are difficult to keep because of hb drop and compliance. less intensive treatment in asymptomatic individuals with hh and their inclusion in blood donation would avoid negative effects on quality of life and benefit blood collection centers in the long term. background: serum ferritin (sf) measurements in whole blood (wb) donors demonstrated that female sex and intensity of donation are major risk factors for iron deficiency. approximately 200 ml red blood cells (rbc) and 200-240 mg iron are lost with wb donation. double unit rbc (2rbc) collections of 360 ml (ca. 40 ml less than the rbc amount of two wb donations) lead to a loss of about 400 mg iron. in switzerland, the maximal allowed donation frequency for male donors is once every 6 months for 2rbc and once every 3 months for wb donation. aims: to describe and compare the course of hemoglobin (hb) and sf in male subjects donating wb and 2rbc at our institution. methods: we included 294 wb and 151 2rbc donors (n = 445) who donated with the maximal allowed donation frequency over 48 months between 2008 and 2013, yielding 4,704 wb and 1,208 2rbc donations. we excluded subjects with hyperferritinemia and known hfe mutations. hb limits were 135 g/l for wb and 140 g/l for 2rbc donation. with 2rbc apheresis 360 ml rbc were collected. sf was measured on a predonation serum sample; hb was determined from finger prick samples. the donors received no iron substitution. we used generalized estimating equation models for hb and sf trajectories. results: mean age at the first blood donation was 53 (wb) and 48 years (2rbc), respectively. at the first donation, mean hb was 153 g/l (sd 13) in wb and 159 g/l (sd 8) in 2rbc donors; mean sf was 44 (sd 52) and 73 lg/l (sd 56), respectively. on average, hb and sf were higher in 2rbc donors (5.1 g/l and 26 lg/l, respectively; p < 0.001). there were 137 subjects with sf < 30 lg/l in wb and 19 in 2rbc group, and 85 with sf < 50 lg/l (but > 30 lg/l) and 40, respectively. in 2rbc donors, between the first and the last donation, mean hb declined from 159 g/l to 157 g/l (p < 0.05) and mean sf from 73 lg/l to 66 lg/l (ns). in wb donors, mean hb dropped from 153 g/l to 152 g/l (p < 0.05) and sf from 44 lg/l to 35 lg/l (p < 0.001). similar results were found when adjusting for age and season. hb values dropped from baseline until the 11 th donation for wb donors and until the 4 th donation for 2rbc donors with an upward trend thereafter. in both groups, no hb value below the limits of blood donation and no anemia were observed. sf reached a nadir at the 4 th donation in both wb and 2rbc donors (37 lg/l and 60 lg/l) and increased thereafter in 2rbc donors. in wb donors, sf followed a parabolic trend that peaked at the 10 th donation, and then declined until the last donation. summary/conclusions: the maximal allowed blood donation frequency for wb and 2rbc male donors in switzerland is not only protective for the development of anemia, but also for deferral of blood donors because of low hb. this was observed even in subjects with low sf at baseline. background: granulocyte concentrate transfusion is a potentially lifesaving option for patients without functional neutrophils. however, recent studies have failed to demonstrate the anticipated clinical effectiveness of this procedure. granulocyte concentrates are manufactured using sedimentation agents to separate granulocytes from red blood cells and enhance granulocyte collection efficiency. high-molecularweight hydroxyethyl starch (hes) is most commonly used for this. however, authorities recently restricted the use of hes due to its unfavorable risk-benefit-profile. modified fluid gelatin (mfg) is an already used alternative sedimentation agent. as the granulocyte product contains these substances, any impact of the sedimentation agent on granulocyte function may affect the clinical effectiveness of granulocyte transfusion. aims: we tested the hypothesis that mfg is not inferior to hes in terms of the functionality and viability of granulocytes. methods: granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15% or 30% mfg (gelafundin 4%, b. braun melsungen ag) or hes (hespan 6%/450/0.7, b. braun medical inc.), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ros) production, neutrophil extracellular trap formation (netosis), antigen expression of cd11b, cd62l and cd66b, and viability were subsequently investigated in vitro. testing was performed using live cell imaging of the cells embedded into a collagen i matrix for parallel testing of migration, ros production and netosis. in addition, flow cytometric (facs) analysis was utilized for surface marker expression, viability and respiratory burst measurement. results: granulocyte migration decreased in a dose-dependent manner in response to hes and mfg. relative to the controls, all three concentrations of hes lowered migration distances (p < 0.001 respectively), whereas only the higher concentrations (15% and 30%) of mfg showed lower relative migration distances (p < 0.001 respectively). track straightness was reduced with both sedimentation agents at 15% and 30% to the same extent (p < 0.001 respectively). hes resulted in lower cd11b expression (p = 0.028) and higher cd62l expression (p = 0.007) compared to the controls, whereas the differences for cd66b did not reach statistical significance. mfg did not affect the expression of any investigated surface antigen mediating endothelial adhesion and transmigration in comparison to the controls. no significant differences in the timing of ros production or netosis, or in neutrophil viability or respiratory burst were observed. summary/conclusions: these results indicate that mfg is not inferior to hes in terms of granulocyte phenotype and function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with hes. background: plateletpheresis donation leads to a well-known transient decrease of donor's platelets. the question of long-term effects raised with the development of regular donations by some donors in order to satisfy a growing demand. a seminal work (lazarus, transfusion, 2001) stated that there is a sustained thrombopenia in frequent plateletpheresis donors, correlated with the total number of donations. aims: french regulation authorizes up to 12 plateletpheresis donations per year, with a minimum 4 weeks interval between them. we tried to evaluate the risk of sustained thrombopenia under these conditions. methods: we retrieved all plateletpheresis donations occurring between 01/01/2014 and 08/31/2018 from the french civilian blood donors' base and then selected a cohort of donors with at least 24 donations during that period. in order to minimize measurement errors, platelet counts analysed were means of three consecutive donations, i.e. 8 measures for each donor. results: the cohort includes 2,186 donors (384 women and 1,802 men). mean platelet counts fluctuate between 276.4 and 278.6 platelets/ml. analysis of variance does not show any statistically significant difference (f = 0.462), even taking donor's sex or age in consideration. there is no difference if we consider the total duration of the 24 donations, either. donors with the lowest first counts show a significant rise in subsequent measures and donors with the highest counts show a decrease trend, exhibiting a classical regression toward the mean. summary/conclusions: plateletpheresis french regulation does not seem to be at risk of sustained donor thrombopenia. this conclusion is in agreement with recent literature data. the primary biological role of the human leukocyte antigen (hla) system is the regulation of the immune response to foreign antigens. because of this role, hla genes and molecules have an important role in transplantation, etiology of many autoimmune, non-autoimmune and infection diseases, but also in transfusion medicine. an increasing probability of an hla non-compatible blood products, tissues or organs exists due to the extremely high polymorphism of hla genes, with more than 20,000 described alleles to date, and their different frequency distribution in various worldwide populations. the hla system, originally discovered as a result of a transfusion reaction in the 1950s, can cause detrimental immune reactions in transfusion therapy. hla antibodies present in the patient are responsible for some of these reactions, while in other cases hla antibodies or hla reactive cells present in the transfused product are accountable for the immunoreactivity. hla antibodies form as a result of exposure to foreign hla antigens during pregnancy, transplantations and blood transfusions and can cause platelet immune refractoriness, febrile transfusion reaction, transfusion-related acute lung injury, and transfusion associated graft versus host disease. in order to avoid or reduce the development of these transfusion-related events, hla antibody negative or compatible products should be used. almost all existing methods presently used for molecular typing of hla polymorphisms are based on polymerase chain reaction, but with different resolution levels (low resolution -two digits or high resolution -four digits). in addition to providing a more precise detection of polymorphisms at hla classical loci (e.g. hla-a, -b, -c, -drb1, -dqb1), molecular methods can also determine polymorphisms at hla loci which previously could not be typed by serology (e.g. hla-drb3, -drb4, -drb5, -dqa1, -dpa1). the most commonly used method for the detection of hla antibodies was until recently complement-dependent cytotoxicity (cdc) technique, but it is increasingly being replaced by a more sensitive, solid phase based method (luminex technology). in conclusion, an accurate and precise determination of both hla gene polymorphism and hla antibodies presence is essential for the safe and efficient administration of transfusion products. background: in only a minority of pregnancies complicated with anti-hpa1a antibodies serious fetal/neonatal disease develops. the difficulty in predicting which mothers should be treated with ivig hampers implementation of fnait screening. we found that fc-core fucosylation and galactosylation are highly variable in anti-hpa1a igg, and that these glycan features strongly affect binding to fccriiia receptor. the level of fc-core fucosylation of anti-hpa 1a alloantibodies was found to correlate with platelet count and outcome of the newborn, suggesting that antibodyspecific fucosylation might serve as a biomarker in fnait screening. however, at present the fc-glycosylation pattern can only be determined by complicated methods involving purification of the antigen-specific igg, and analyzing trypticly released -igg-derived-glycopeptides by tandem liquid chromatography-mass-spectrometry (ms) techniques. these methods, although powerful, are not yet suited for high throughput clinical screening. aims: our aim was to provide a simplified method to quantify the biological activity of anti-hpa-1a antibodies, and possibly other alloantibodies against blood cells. methods: here we explored if cellular surface plasmon resonance (spr) imaging can replace ms, resulting in less complicated handling of patient sera and donorantigen-bearing cells. the strength of the binding of platelets to fccr on spr sensor was monitored under flow. the spr sensor was equipped with both wt fccriiia (sensitive to fc-glycosylation status) and mutant fccriiia-n162a (insensitive to fcglycosylation status). in addition, the biosensor was prepared with anti-platelet cd61 (c17) and anti-igg to calibrate the number of injected platelet as well as to quantify igg-opsonization. the quality of the anti-hpa 1a glycosylation was monitored as the ratio of the binding of opsonized platelets to the wt and the mutant n162a-fccriiia. platelets opsonized with recombinant glycoengineered anti-platelet antibodies with different levels of fc-fucosylation were used as standards. for validation, 166 plasma samples with anti-hpa1a antibodies, already analyzed by mass spectrometry and with known clinical outcome were tested (sonneveld, bjh, 2016) . results: we found that the ratio between the binding to the wt fccriiia and to the mutant n162a-fccriiia correlated with the level of fucosylation of the hpa1a antibodies, as measured by mass-spectrometry (r = à0.524; p < 0.0001). overall, a similar predictive value for disease severity was obtained as we previously reported for this retrospective cohort. in addition, quantitative information on antibody concentration can also be extracted using the fccriiia-n162a receptor as sensor on the chip, while anti-igg gave aspecific signals, presumably because it recognized cytophilic platelet-fccriia-bound antibodies as well. summary/conclusions: in conclusion, the combined use of wt and mutant fccriiia in a label free spr assay provides both quantitative and qualitative information of platelet bound anti-hpa 1a antibodies, which circumvents the need for purification of specific antibodies and laborious mass spectrometric analysis. this approach might be generally applicable to determine the biological activity of cell bound antibodies not only for anti-hpa1a in fnait, but also for anti-rhd alloantibodies in hdfn or anti-platelet antibodies in itp. background: immunization against the human platelet hpa-1a alloantigen is the most common cause of severe fetal and neonatal alloimmune thrombocytopenia (fnait) in otherwise healthy term newborns. the screening for hpa-1a antigen in pregnant women is an important tool for identification of pregnant women at risk of having a fetus/neonate with fnait. any targeted intervention depends on efficient screening methods as well as sensitive and specific methods for detection of anti-hpa-1a. within the framework of the polish-norwegian project (prevfnait) we have performed hpa-1a screening program in poland. aims: our aim was to assess the frequency anti-hpa-1a antibody detection and the clinical outcome of newborns identified through the study. women who joined the program due to the fnait in the previous child or in the current newborn are not analyzed in this study. methods: hpa-1a screening of 24 244 pregnant women in 8-40 gestational weeks was performed by facs phenotyping or rq-pcr genotyping at ihtm in warsaw. hpa-1a negative/hpa-1b/1b women were tested for hla drb3*01:01 and for anti-hpa-1a antibodies by maipa (followed up at week 17-20, 28, 32, 38-40 and 6 weeks after delivery). if anti-hpa-1a were detected, quantitative maipa was performed. all hpa-1a negative women were contacted for information concerning the newborn. if the baby had thrombocytopenia and anti-hpa-1a were not detected by maipa, the look back samples were tested retrospectively by paklx test (immucor). results: 554 hpa-1a negative women were identified (2.3%). anti-hpa-1a was antibodies were detected by maipa in 44 women (two delivered tweens). in addition, anti-hpa-1a antibodies were later detected by paklx in further 3 women who delivered baby with severe thrombocytopenia and/or ich. total number of immunized mothers was 47 (8.5%). they delivered 49 babies; 35 were boys. three women were treated by ivig: two by 4 and 8 injections since 33 th and 26 th gw respectively. the anti-hpa-1a concentration in the 1 st one was 0.4; 0.1; 5.88 iu/ml in 17, 28, 32 gw respectively and in the 2 nd <0.05 iu/ml in all examined samples. the decision on treatment was based on the low plt count~50 g/l in the fetus in cordocentesis. their newborns (one delivered tweens) were healthy. the 3 rd treated woman entered the program in 35 gw (anti-hpa-1a concentration was high 22.64 iu/ml). she obtained one injection of ivig. her baby was born with mild thrombocytopenia with no ich. severe fnait occurred in 5/49 newborns: in 3 with anti-hpa-1a detected in paklx only and in 2 with antibody concentration in maipa -1 st : 6.86/12.91/ 23.36 at 28/32/38 th gw respectively; 2 nd : 8.85/17.58 at 32/38 th gw respectively. ich was observed in all of them; plt count was < 50x10 9 in four, 56 9 10 9 / in one. summary/conclusions: 1/ the severe thrombocytopenia due to anti-hpa-1a alloimmunisation in our prospective study occurred in 2/10 000 pregnancies 2/ the paklx could improve anti-hpa detection in the screening program and should be considered as an additional diagnostic test, if maipa result is negative 3/ the hpa-1a alloimmunisation frequency is higher in pregnancies with male than female fetus. background: foeto-maternal platelet alloimmunization (fmpai) is mainly characterized by foetal and / or neonatal thrombocytopenia (fnait), sometimes revealed by intracranial hemorrhage (ich) or even by foetal death in utero (fdiu). the experience of the pnil milwaukee (usa) reported in 2014 that the diagnosis of alloimmunization was carried in only 33% of neonatal thrombocytopenia cases with a clinical symptomatology highly suggestive of an alloimmune etiology. aims: the aim of this two-year study was i) to determine the frequency of platelet incompatibilities in fnait, ich and fdiu and ii) to evaluate the frequency of detectable platelet alloantibodies (alloab) and their specificity in cases of incompatibility. methods: platelet genotyping was performed by hpa beadchip genotyping kit (bioarray solutions, immucor, warren, nj). serology investigation was carried out by 3 different methods: complete maipa kit (apdia bvba, turnhout, belgium), pack lxtm assay (immucor gti diagnostics, waukesha, wi) and « in house » maipa. all 2017 and 2018 data were collected using the laboratory information management system. results: 544 patient files were analyzed. no incompatibility is demonstrated in hpa-1 to -9, -11 and -15 systems in 19.3% (n = 105). hpa-1 and / or 3 and / or 5 incompatibilities were found in 271 cases (49.8%), hpa-2 and / or 15 in 87 cases (16%). platelet alloimmunization was globally confirmed in only 10.6% of the cases. 58 platelet alloabs were identified regardless of clinical manifestations: 24 anti-hpa-1a (41.4%), 20 anti-hpa-5b (34.5%), 8 anti-cd109 (13.8%), 2 anti-hpa-5a and anti-hpa-15b (3.4% respectively) and 1 anti-hpa-1b and anti-cd36 (1.7% respectively). 40 alloabs were found in the context of neonatal thrombocytopenia, 6 in ich and 10 in fdiu, and 1 in a follow-up of pregnancy. even if no anti-hpa-3 alloab could be identified, the incompatibility in this system was highly associated with fnait, ich and fdiu (n = 55, n = 32 and n = 17 on 114 cases). summary/conclusions: this study strongly confirmed the known immunogenicity of some hpa systems and highlighted overall the severity of hpa-3 and hpa-5 incompatibilities. the definite diagnosis of fmpai is difficult to make due to the present technical difficulties in the detection of antibodies against the hpa-3 and hpa-15 systems. however, our results suggest that special attention should be paid to the management of pregnancies with these incompatibilities due to the frequency of severe foetal/neonatal adverse events. background: fetal and neonatal alloimmune thrombocytopenia (fnait) is a potentially life threatening disease caused by maternal alloantibody formation against fetal human platelet antigens (hpas), of which anti-hpa-1a is accountable for the fast majority of the cases. population-based screening for fnait has been topic of debate for over decades. logistically as well as financially, the major challenge of such a screening is the typing of pregnant women to recognize the 2% hpa-1a negative women. at present, hpa-1a typing is mostly done by genotyping. for costeffective implementation of anti-hpa-1a screening there is need for a high-throughput, quick and low-cost phenotyping assay. aims: the aim was to develop a high-throughput, quick and low-cost phenotyping assay in order to identify hpa-1a negative pregnant women. methods: an automated sandwich elisa was developed to perform hpa-1a phenotyping using a murine monoclonal anti-gpiiia as coating antibody and horseradishperoxidase-conjugated recombinant igg1 anti-hpa-1a as detecting antibody. to ensure the applicability for high-throughput testing in a potential screening setting, 20 ll of the uppermost plasma of 3 -6 days-old stored edta anticoagulated blood tubes was used, without first swirling or spinning them. in two phases, samples of pregnant women were tested and compared to an allelic discrimination polymerase chain reaction assay as golden standard. in the first phase, samples from unselected consecutive pregnant women were tested. the second phase was part of a prospective screening study in pregnancy and confirmatory genotyping was restricted to samples with an arbitrary set od < 0.500 in the hpa-1a elisa. the developed elisa was optimized to require no additional handling (swirling or spinning) of stored tubes. during phase i, 506 consecutive samples were tested. in phase ii, the hpa-1a elisa was performed in another 62,171 consecutive samples, with confirmatory q-pcr in 1,825. the two phases combined, samples from in total 1,585 hpa-1a negative and 823 hpa-1a positive pregnant women were genotyped. the assay reached a 100% sensitivity with a cut-off od between 0.075 and 0.200, leading to a specificity of 99.6%. summary/conclusions: a quick, low-cost and reliable assay for hpa-1a phenotyping was developed that can be used in a population-based screening setting to select samples that has to be tested for the presence of anti-hpa1a antibodies. because plasma from non-mixed or spinned tubes of three to six day-old samples can be used, this assay is applicable to settings with suboptimal conditions. background: cytomegalovirus (cmv) sero-prevalence in ireland is lower than that which is reported in many other european countries. a study of 1047 pregnant women in 2002 found that 30.4% of irish women were cmv seropositive in comparison to 56% from western europe and 92% eastern europe and 97% from africa. an internal study carried out by the irish blood transfusion service (ibts) in 2010 indicated the rate of cmv seropositivity in irish blood donors was 21.97%. therefore a significant proportion of the irish donor and recipient population are susceptible to primary cmv. this is of particular concern for patients for certain at-risk groups such as very-low birthweight cmv seronegative neonates, cmv seronegative patients undergoing transplantation and other cmv seronegative immunocompromised patients. this results in a demand for the provision of cmv sero-negative blood components. in 2018 the ibts evaluated the abbott alinity s cmv igg assay as a replacement for the cmv mastazyme eia (total ab eia). aims: to assess the performance of the abbott alinity s cmv igg screening assay in comparison to the cmv mastazyme eia (total ab eia). methods: diagnostic sensitivity was determined by testing 48 confirmed cmv igg positive donors from an external laboratory. sensitivity was assessed using three seroconversion panels (n = 54). analytical sensitivity was calculated using linear regression analysis of the who first international standard for anti-cmv igg. diagnostic specificity was determined by testing 6127 donors. further evaluation of discordant results was carried out using the architect anti-cmv igg and igm assays and vidas anti-cmv igg and igm assays. results: the diagnostic sensitivity of the alinity s anti-cmv igg assay was determined to be 100%. the seroconversion sensitivity reported 42 out of 54 samples reactive. the analytical sensitivity of the alinity s cmv igg assay was determined to be 1.12 iu/ml. the validation reported 65 discordant results from 6127 donor samples tested with both the alinity s cmv igg assay and the current mastazyme total assay. 60 discordant results were observed (alinity s anti-cmv igg positive/mastazyme total negative). further testing of these samples classified 27 discordant results as positive, 12 as negative and 21 as indeterminate. 5 discordant results were observed (alinity s anti-cmv igg negative/mastazyme total positive). further testing classified these samples as negative. overall the diagnostic specificity was determined to be 99.80%. summary/conclusions: both the seroconversion and analytical sensitivities are comparable between the alinity s cmv igg assay, the cmv mastazyme total ab assay, the architect cmv igg assay and the vidas igg assay. the slight variations can be attributed to the individual assay cut-off definitions, which can vary greatly between cmv assays. it must be noted that the determination of the diagnostic specificity (99.80%) does not include indeterminate discordant results. further testing will be carried out to try to characterize all discordant samples in collaboration with abbott. this evaluation did not identify any donors with isolated confirmed cmv igm antibodies in a pool of 6127 donors. based on this evaluation the abbott alinity s cmv igg assay is a suitable replacement to the mastazyme total ab assay for blood donor screening. background: africa has a unique set of challenges regarding safe blood transfusion. two of the largest contributing factors are: 1) the most common disease states in sub-saharan africa (ssa) require large amounts of blood as lifesaving interventions e.g. malaria, 2) the highest burden of infectious diseases transmissible through transfusion (tapko, toure, & sambo, 2014) is found in ssa. this has often led to the binary donor base that exists in ssa, consisting of voluntary non-remunerated blood donors (vnbd) and family or replacement donors (frd) as transfusion centres are unable to supply the demand when relying only on vnbd. voluntary non-remunerated donors are the safest blood donors as they have no incentive (other than altruistic motives) and are not under social pressure to donate, both factors that may induce individuals knowing or suspecting themselves to be infected with a blood-borne agent to donate blood. nucleic acid testing (nat) in conjunction with serological testing is the gold standard for testing, however, the vast distances and high temperatures of africa makes transport of traditional plasma samples a logistical challenge. many publications evaluating the stability, suitability, and ease of use of dried blood spots (dbs) for nat have been published. generally, results have been shown to be comparable to traditional plasma samples. dbs is being used successfully in the early infant diagnosis (eid) programs for hiv by means of pcr testing, especially in africa. aims: 1. to demonstrate that dbs and/or dried plasma spot (dps) testing is suitable for blood donor screening and can make nat testing more widely available in africa 2. to determine the diagnostic sensitivity and specificity of testing dps and dbs samples, in comparison to testing of plasma samples. methods: 900 negative new donor samples and 100 confirmed positive donor samples, as defined by routine blood safety screening done at western cape blood service, were screened using a dried blood spot kit. after routine testing was completed, one dbs sample and one dps sample for each blood donor were prepared and analysed with the ultrio elite assay on the panther analyser. summary/conclusions: dbs/dps can be used as a sample for screening blood donors as the invalid rate was 0.56%, and only found on dbs samples. logistically dbs/dps is well suited for the resource-poor countries as samples are: -easy to obtain (fingerpick samples could be used.) -transport is simplified as samples will not leak or haemolyse due to high temperatures. -samples can be stored at room temperature dbs/dps demonstrated acceptable specificity. the ultrio elite performed well with regards to hiv and hcv sensitivity. sensitivity with regard to hbv was not as high but this could be due to very low and erratic viral loads. background: sanquin blood supply is responsible for the blood transfusion services in the netherlands. at the national screening laboratory sanquin (nss) annually more than 750.000 blood and plasma donations are tested, on average 3.000 samples per day. for more than 10 years, infection serology testing was performed using the prism (abbott diagnostics), but since mid of july 2018, serological testing for the hbsag, hiv ag/ab, anti-hcv and anti-hbc is done with abbott's alinity s system. aims: to compare the numbers of initially and repeatedly reactive results of whole blood and plasma donation samples and the rate of non-specific results leading to deferral of donations and donors for prism and alinity s assays using data from 6 months before and 5 months after implementation of the alinity s systems at nss. methods: initial and repeat reactive rate of the assays run by either prism (hbsag, hiv o plus, hcv) or alinity s (hbsag, hiv ag/ab combo, anti-hcv,) were calculated for january to june 2018 (prism) and august to december 2018 (alinity s). due to the lack of a true confirmatory method for anti-hbc, we only compared the rate of repeatedly reactive results for prism hbc and alinity s anti-hbc. results: the rate of repeat reactive results for prism (p) and alinity s (a) assays were as follows: 1) hbsag p 0.01 % (42/390.736) versus a 0.03 % (87/322.394); 2) hiv p 0.07 % (262/390.735) versus a 0.06 % (201/322.470); 3) anti-hcv p 0.11 % (427/390.737) versus a 0.03 % (109/322.476). the rate of anti-hbc reactive samples was not significantly different between prism (0.39 %) and alinity s (0.42%). over the study period, the rate of initially reactive samples for the three main screening assays (hbsag, hiv, hcv) was also comparable between alinity s (0.39 %) and prism assays (0.34 %), mainly attributable to a rather high number of initially reactive alinity s hiv ag/ab results. this was due to initial issues with blood collection tubes that were resolved. as a result in december, the rate of initially reactive samples decreased to 0.16 %, which was significantly lower than for the three prism assays (0.33%). summary/conclusions: the introduction of the alinity s assays lead to a decrease of the average repeat reactive test results (hbsag, hiv, hcv) by 0.17 % as compared to the prism, mainly due to a lower false reactive rate of the alinity s anti-hcv assay. this will be further investigated for first time and multiple time donors. with the implementation of the alinity s at sanquin we aimed to improve not only the operational efficiency but also to further minimize unjustified disapproval of donors. these first data show that the low initial and repeat reactive rates of the alinity s assays indeed have a positive impact on unnecessary deferrals of donations and donors. background: in blood banks, testing all blood donations for markers of infectious diseases plays an important role in maintaining the safety of blood transfusions. mandatory serological testing in switzerland is performed for anti-hcv, hiv ag/ab, hbsag and syphilis. highly specific and sensitive tests with corresponding automation are essential for this purpose. aims: a comparative study was carried out to evaluate the usability of the newly launched alinity s system (abbott) and the specificity of the infectious disease parameters hbsag, anti-hcv, hiv combo and syphilis (abbott) with the currently used elisa methods on the quadriga befree system (all diasorin, formerly siemens healthcare diagnostics). methods: the study took place at the interregional blood transfusion service in berne, switzerland. the specificity of the parameters was studied on 2,748 blood donor sera from both first time and repeat donors. the samples were tested first on the quadriga be free system with enzygnost hbsag 6.0, enzygnost anti-hcv 4.0, and enzygnost hiv integral 4 assays and on the pk7300 with the newbio-pk tpha assay (newmarket biomedical). all samples were retested on the same day with hbsag, anti-hcv, hiv combo and syphilis on the alinity s. initial reactive samples were repeated in duplicate. discriminatory tests were carried out for repeatedly reactive samples using alternative screening tests and neutralisation (for hbsag) on an abbott architect i1000 system and immunoblots (hiv-, hcv-, syphilis-inno-lia, fujirebio). for all samples, results from our routine individual donation nucleic acid testing (hcv, hiv, hbv, roche cobas 8800 system) were available. results: based on the results from testing 2,748 blood donations, the observed specificities of alinity s assays (a) and enzygnost assays ( summary/conclusions: the alinity s system was easy to use by the operators after a very short introductory training and provides good operational efficiency such as high throughput even when selective testing for samples is needed. the observed specificity of abbott alinity s versus siemens enzygnost assays is comparable in a blood donor screening setting. unfortunately, we were not able to analyse statistically the specificity data due to the insufficient number of donor samples tested in parallel. it is worth mentioning that around 90% of the samples included in the study derived from repeat donors who had been previously tested with the enzygnost assays but were "first time donors" for the alinity s assays. all four assays from both systems exhibit a very good specificity and are highly suitable and practicable for routine blood donor screening. background: effective screening for transfusion-transmissible infections is essential to ensure safe blood transfusions. the world health organization recommends mandatory serological testing of blood donations for human immunodeficiency virus (hiv), hepatitis b (hbv)/c (hcv), and syphilis. due to increasing demands on clinical laboratories, there is a need for reliable and accurate automated blood screening tests. the fully automated cobas e 801 analyser can be used with elecsys â infectious disease parameters to screen donor blood samples. aims: to compare the performance of elecsys â infectious disease parameters on the cobas e 801 analyser (roche diagnostics) with other commercially available assays for routine first-time blood donor screening. methods: we provide results from etablissement franc ßais du sang (montpellier), a blood bank which participated in a large, multicentre study of the cobas e 801 analyser. the following infectious disease marker assays were compared: hiv, elecsys â hiv duo versus prism hiv o plus; hcv, elecsys â anti-hcv ii versus prism hcv; hbv surface antigen (hbsag), elecsys â hbsag ii versus prism hbsag; hbv core antigen antibodies (anti-hbc), elecsys â anti-hbc ii versus prism hbcore; syphilis, elecsys â syphilis versus newbio pk tpha assay. specificity was tested using residual fresh serum samples from unselected first-time blood donors, and calculated according to assay package inserts and site-specific cutoffs. samples were tested using comparator assays, then retested the same day using elecsys â assays. initially reactive samples were repeated in duplicate; confirmatory tests were conducted on repeatedly reactive samples. confirmatory tests: hiv, nucleic acid testing (nat), architect hiv ag/ab and inno-lia â hiv i/ii score assays; hcv, nat, archi-tect hcv and inno-lia â hcv score assays; hbsag, nat, architect hbsag and elecsys â /prism hbsag confirmatory assays; anti-hbc, nat, hbsag, anti-hbs, and architect anti-hbc assays; syphilis, architect syphilis tp and inno-lia â syphilis score assays. sensitivity was tested using 30 preselected, anonymised, positive, citrate-phosphate-dextrose-plasma samples (plasmatec laboratory products) and compared with archived data for comparator assays. sensitivity was calculated according to the final nat result. results: across all infectious disease markers, specificity to detect repeatedly reactive samples using elecsys â versus comparator assays was similar (99.81-100.00% versus 99.79-99.98%; n ≥ 5195). in specificity analyses, there were 14 discrepant results for hiv testing, 27 for hcv, two for hbsag, eight for anti-hbc, and five for syphilis. sensitivity of the elecsys â hiv duo assay (83.33%; 95% ci 65. 28-94.36) was higher than the prism hiv o plus assay (76.67%; 95% ci 57.72-90.07), but the difference was not statistically significant. sensitivities of elecsys â and comparator assays were the same for hcv (85.19%; 95% ci 67.33-95.97), hbsag (70.00%; 95% ci 50.60-85.27), anti-hbc (100.00%; 95% ci 85.75-100.00), and syphilis (100.00%; 95% ci 88.43-100.00); three hcv and six anti-hbc samples were classified negative/ indeterminate and excluded from the analyses. in sensitivity analyses, there were two discrepant results for hiv testing, three for hcv, and five for anti-hbc. summary/conclusions: elecsys â infectious disease parameters on the cobas e 801 analyser demonstrate high specificity/sensitivity for screening first-time blood donor samples, with similar clinical performance to other commercially available assays. background: individual plasma and serum specimens from whole blood or plasmapheresis donors are tested for absence of infectious agents by serological assays prior to use for transfusion or production of blood derived therapeutics. the department of plasma analytics (pa), takeda (austria), and haema ag, grifols (germany), both labs with high throughput and a high level of automation, were seeking for alternatives to replace their current serological test systems (abbott prism next). aims: to allow a direct comparison of the two final candidate analyzers alinity s (abbott) and cobas e801 (roche diagnostics gmbh), a side by side evaluation was carried out by the pa and haema with support from abbott and roche (provision of instruments and reagents). the aim was to compare assay specificities as well as handling and performance of the instruments. the outcome should be used to better understand potential specificity differences and practical handling aspects (throughput, etc.) of a next generation serological analyzer. methods: the two candidate instruments were installed in the pa. from march to june 2018, close to 10,000 aliquots from routine preselected repeat donors, provided by haema, were run on both study instruments in parallel. plasma samples were tested for hbs antigen (ag), hcv antibody (ab), hiv ag/ab, and partially for syphilis ab. serum samples were additionally tested on hbc ab. samples with repeat reactive results ("rr", two reactive results out of three tests) not confirmed by confirmatory tests were counted as false reactive. the necessary sample size was calculated based on a one-sided comparison of proportions with the aim to detect potential specificity differences (a = 10%) in the size of those specified by the manufacturers' instructions. two different lots were tested for the three main assays. results: out of 7,389 plasma and 2,242 serum samples, 41 test results representing 37 individual donations were found rr on one or both instruments. two samples were confirmed positive (1 9 hbsag, 1 9 hcv), two others were indeterminate. the sample containing low level antibodies against hcv was pcr negative and only detected by the roche system. the percentage of false reactive results for the five assays on the two systems were (alinity s/e801): hbs ag: 0.04/0.03 % in a total of 9590/9589 samples tested; hcv ab: 0.01/0.09 % in 9620/9585, p < 10%; hiv ag/ab: 0.03/0.09% in 9362/9329, p < 10%; syphilis ab: 0.1/0.07% in 2971/2987; hbc: 0/0% in 1531/1549. no significant difference was found between the calculated specificities in our study and the manufacturers' data. a potential influence of sample matrix and kit lots was assessed. a trend towards more false reactive results in serum vs plasma was found for nearly all assays. no clear-cut statistical difference was seen between lots. summary/conclusions: the study results are in line with the manufacturers' specificity data, showing that the alinity s hcv ab and hiv ag/ab assay show a slightly higher specificity in a population of plasma and serum samples from repeat donors prescreened by prism. a possible influence on the test specificity by the sample matrix was detected but needs further investigation. the possibility to edit complex genomes in a targeted fashion has not only revolutionized basic research but biotechnological and therapeutic applications as well. with the rapid development of genome editing tools, in particular zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens), and the crispr-cas system, a wide range of therapeutic options have beenand will bedeveloped at an unprecedented speed. therapeutic genome editing in hematopoietic cells enable new interventions in the blood and immune system, including novel approaches to treat immunological disorders, infectious diseases, and cancer. we have developed gmp-compliant protocols to manufacture gene edited cd34 + hematopoietic stem and precursor cells (hspcs) as well as chimeric antigen receptor (car) t cells, with the final goal to provide novel cell therapies for patients suffering from primary immunodeficiencies, chronic infection with human immunodeficiency virus type 1 (hiv-1), and some tumor entities. despite great success in improving their specificity, engineered designer nucleases can induce genotoxic side effects by introducing mutations or chromosomal aberrations. we have established novel genome-wide assays that enable us to detect chromosomal aberrations induced not only by off-target activity but also by on-target activity, such as micro-aberrations and translocations, with unparalleled sensitivity. in toto, our developed protocols allow us to achieve genome editing in hematopoietic cells with high efficiency and to assess the genotoxic risk associated with the expression of crispr-cas nucleases and talens in clinically relevant human cells, so forming the basis for planned phase i/ii clinical studies. adoptive t cell therapy (act) has proven a potent means to treat blood-borne tumors and solid tumors. adoptive cell therapies include t cells that are genetically engineered with tumor specific t cell receptors (tcrs), or with chimeric antigen receptors (cars). in addition, tumor infiltrating cells (tils) can be isolated from tumor lesions, which are then expanded and reprogrammed in vitro prior to transfusion into the patient. the anti-tumoral efficacy of act products depends on several parameters, including the capacity of cd8 + t cells to produce cytokines, chemokines and granzymes, a feature that is critical for effective anti-tumoral responses. here i will discuss our efforts to develop and improve act products for future clinical use. i will present pre-clinical work on developing til therapy for non-small cell lung cancers. in addition, i will show that human cd8 + t cells can be divided into different subsets, and that only one of those subsets is highly cytotoxic. this finding may help improve the quality of genetically engineered t cell products, like tcr and car t cell products. background: the baltic states -estonia, latvia and lithuania have a lot in common. we are located side by side, share the baltic sea as a gate to the west, and more importantly, a common history. we were members of the ussr and suffered 50 years of soviet occupation. we held hands in a 600 km long human . . .chain" across the three states to express our mutual support, and later on, even joined the european union on the very same day -june 1 st , 2004. the three differ a bit in size, population and more in the languages spoken in each one, but that does not explain why the path towards voluntary unpaid donation varies as it does. aim: the aim is to describe the journey towards voluntary non-remunerated blood donation in the baltic states after regaining independence from the soviet union. methods: the information was collected from published and unpublished memories, annual reports and written interviews with latvian and lithuanian colleagues. results: in soviet times, all orders came from moscow and quality control was conducted from the capital city of latvia, riga. donors were mostly paid and given an extra vacation day. big factories were the best places to collect blood and people were queuing to donate. in 1991, the soviet union fell apart and the baltic states suddenly got the freedom and responsibility to decide. in estonia the first edition of "guidelines for the preparation, use and quality assurance of blood components" was taken as guidance in 1992. a lot of advice came from finnish colleagues. in 1997, it was decided to move towards non-paid voluntary donations. the process took 6 years. the first couple of years were economically difficult for the reborn state, as money had less value than food. instead of cash, donors were given rapeseed oil, sugar and pasta, for example. as the situation improved, food items were replaced by small symbolic gifts that carry sentimental value. it has been this way for more than 15 years by now. in lithuania, the process started later, the first program for developing a framework for voluntary non-remunerated donations being carried out in 2006-2015. it resulted in 51% of the donations being unpaid. the second program initiated in 2016 is still ongoing, aiming towards 100% non-remunerated donations by 2020. by the end of 2018, they had reached 99.2%. in the beginning, the main obstacle was a private blood center creating unfair market conditions. in latvia, monetary compensation for blood donations still exists, but the younger generation has been encouraged to donate blood for free and some results can already be seen. summary/conclusions: a common starting point does not guarantee the same results, at least not at the exact same time. examining the circumstances leading to the different outcomes could benefit countries yet to start moving towards non-remunerated donations as well as those considering the opposite. haemoglobin (hb) was as expected significantly different between women and men (meanaesem: 13.8 ae 0.01 vs 15.5 ae 0.01 g/dl; p < 0.000). percentage of females with low hb < 12.5 g/dl were 7.1%, 4.9%, 5.2%, 6.7% and 4.2%, percentage of males with hb < 13.5 g/dl were 0.5%, 0.6%, 0.7%, 1.7% and 1.2% for the age groups 1-5 respectively. ferritin values were higher in males compared to females (median; 25 th -75 th %>tile: 51;31-79 vs 157;106-231 lg/l; p < 0.000) and in older age groups compared to younger age groups (median; range in age groups 1-5 in females: 40;3-702, 52;6-619, 60;6-480, 60;8-725, 98; 10-604 and in males: 99;8-661, 161;18-1080, 206;15-1090, 220;26-1430, 221;33-981 respectively) . percentage of females with ferritin ≤ 15 lg/l were 8.6%, 3.8%, 3.7%, 6.7% and 2.0%, while percentage of males with ferritin ≤ 15 lg/l were 0.2%, 0.0%, 0.1%, 0.0% and 0.0% for the age groups 1-5 respectively. white blood cell counts (wbc) were slightly higher in females compared to males (meanaesem: 7.2 ae 0.02 vs 6.6 ae 0.03; p < 0.000). percentage of females with wbc > 12x10 9 /l were 1.8%, 1.3%, 1.7%, 1.4% and 0.3%, while percentage of males with wbc > 12x10 9 /l were 1.4%, 0.9%, 0.5%, 1.2% and 0.9% for the age groups 1-5 respectively. none had wbc < 2x10 9 /l. platelet counts (plt) were higher in females compared to males (meanaesem: 257 ae 0.6 vs 222 ae 0.6; p < 0.000).percentage of females with plt < 150x10 9 /l were 1.0%, 1.5%, 2.2%, 2.4% and 1.0%, while percentage of males with plt < 150x10 9 /l were 2.6%, 3.6%, 2.7%, 2.4% and 1.6% for the age groups 1-5 respectively. among the low plt counts most were caused by edta-dependent pseudothrombocytopenia. extreme deviations from normality were seldom and referred to gps for further investigations. summary/conclusions: first time donors are young with 75% younger than 30 years of age and the female/male ratio was 58/42. of the 16 583 first time donors with data on ferritin available, 14% had low ferritin (≤ 30 lg/l). the typical male first time donors neither had low hb nor low ferritin, even with a significantly lower ferritin in younger donors. in female first time donors the prevalence of low hb (6%<12.5 g/dl) and low iron stores (23%≤30 lg/l) is high. in all, while all first time donors are highly appreciated, campaigns could target the male population to even out the gender imbalance. blood centers must be aware of the higher prevalence of low iron stores in the youngest donors. background: the aim of assessing suitability of prospective blood donors is protection of their health and the safety of transfused patients. selection process is not always effective in obtaining all relevant information from blood donors in a timely manner. for several reasons, some risks remain undetected or they are disclosed at a future donation(s). therefore, recording and management of post-donation information (pdi) are of great importance for improvement of transfusion safety, donor counselling and education as well as overall improvement of the selection process. aims: the aim of the study was to present results of pdi management at croatian institute of transfusion medicine (citm) and the effect of education activities on their trends. methods: we have analyzed reports on pdi recorded in two-year period (2017-2018), according to the types of information obtained, age and sex of blood donors, total number of their donations preceding pdi, and the time of receiving the information. the effect of an information leaflet on pdi launched in november 2017 was assessed by comparing results in two study years. results: a total of 491 pdi were recorded: 266 in 2017 (1/396 donations) and 225 in 2018 (1/452 donations) with the following distribution: nonsexual risk as tattoo and piercing (17.5%), surgical procedures (17.1%), travel history (16.1%), infections/ contact (15.3%), other medical reasons (13.4%), endoscopy/invasive diagnostic procedures (12.2%), malignancy (4.3%), autoimmune diseases (3.7%) and sexual risks (0.4%). majority (76.4%) were late pdi, revealed on the future donation(s): 58.7% on the first next donation, 28.9% on the second and 12.3% after more than 2 subsequent donations. the mean age of blood donors associated with pdi was 40 ae 12 years (median 39 years), while the mean age of all donors in 2017/2018 was 38 years (median 37 years). of all pdi, 81.1% were related to male donors (84% in total pool of citm donors). using chi-square test there were no significant difference between female and male donors in total pdi frequency and in their distribution to early and late pdi (p > 0.05). the median number of all donations preceding pdi was 6 for female donors and 19 for male donors. implementation of education leaflet for blood donors resulted in 15.4% reduction of pdi in 2018 compared with 2017 (p > 0.05). the effect is more pronounced (p < 0.05) when comparing second and first half of 2018 (-25.6%). reduction is observed in all types of pdi with the exception of infections/contact (because they are mostly early pdi) and malignant diseases. the share of early pdi increased from 21.1% in 2017 to 26.7% in 2018, which may suggest better awareness of blood donors on the importance to inform blood bank on changes in their health status. summary/conclusions: our study points to the importance of systematic recording and management of pdi, including education of blood donors about the need of providing all relevant facts related to their health and the safety of donated blood in a timely manner. we are planning further improvements by providing information on this topic on posters and screens on donation sites. background: currently, the transfer of data between organizations and/or computer systems is very limited, and where present is typically proprietary. in the absence of a standardized reference format individual organizations and vendors attempting to integrate disparate databases must develop unique solutions. aggregation of information from multiple sources is complex and costly, constituting a significant barrier to effective analysis of data to improve practice and inform policy. aims: to standardize the definitions and facilitate integration of key data items used in blood donation and transfusion. we report here on an initial effort to map internationally harmonized critical steps in the blood collection/donation process in order to test the approach. methods: through a collaborative process of serial conference calls and correspondence, an informal multi-national consortium of experts across the transfusion industry are attempting to create a vocabulary with sufficiently precise definitions to be usable by automated systems and that can be the foundation of a blood collection/transfusion medicine common data model (cdm), using the following steps: -define the scope of activity to be addressed and segment into key processes. -identify the set of data elements in each segment that are common to all systems. -review and consider existing standards and definitions for each data element. -develop draft definitions for each data element. -release draft to public domain for critical review and refinement with long-term goal of gaining widespread endorsement. results: a standardized approach to blood donation was mapped through identification of common pathways and core mappable data elements. denominator data associated with donor characteristics and blood collection was selected as the first segment to address. a dictionary (or vocabulary) of common terms has been created and will be presented for international comment. summary/conclusions: developing an international consensus on the core elements and their definitions across the transfusion chain is critical for data integration and automation efforts. the expected benefits of this endeavor include that it allows the establishment of algorithms to automate reporting and thus reduce hands-on staff time; reduces time and resources needed to integrate new databases; allows systems to continue to use existing concepts and definitions internally while also providing data output in a standardized format; supports the ability to consistently analyse, interpret and present information regardless of the data source; establishes data definitions against which new systems can be developed; helps to improve comparability of results by providing a common data model for researchers and policy makers; improves confidence in data integrity and reliability of the derived information as a © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 basis for rational decision making; and reduces data gathering effort and cost thus improving opportunities for more efficient/complex data analysis. standardizing the transfusion medicine dataset is the first step in achieving the automation of data transfer and analysis needed globally to drive patient safety, research innovation, and best business practices. further steps must address the precise methods of data exchange, identification of responsible entities for maintenance and further development, and engagement of computer system developers. red blood cell (rbc) alloantibodies develop in a subset of individuals following exposure to non-self rbcs through transfusion, pregnancy, or other activities; these antibodies can lead to difficulty locating compatible rbcs, acute or delayed hemolytic transfusion reactions, or hemolytic disease of the newborn. alloimmunization is underestimated due in part to antibody evanescence, the random nature of posttransfusion antibody screens, fragmented medical care, and the lack of widespread antibody registries. factors that influence who will develop detectable alloantibodies are not well understood. transfusion burden is one risk factor for alloimmunization, though many highly transfused individuals never form alloantibodies despite exposure to many rbc units (and many non-self abo blood group antigens). individuals with sickle cell disease (scd) and myelodysplastic syndrome (mds) are more likely to form rbc alloantibodies than most other patient populations. individuals with rheumatologic and other forms of autoimmunity, though not chronically transfused, are also at higher than average risk of forming rbc alloantibodies. inflammation, in a broad sense, is one common thread among these diagnoses associated with high prevalence rates of rbc alloimmunization. reductionist murine models support some types of inflammation (including viral-like stimuli) around the time of rbc exposure as being associated with an increased likelihood of alloantibody formation. strategies other than transfusion avoidance or extended antigen matching beyond abo/ rh would be beneficial to prevent new rbc alloantibody formation, especially in patients at highest risk. background: the unique genetic makeup of the omani population makes them rich in the genetic blood disorder. 45 % of omani populations are àa/àa gene carriers, 44% àa/aa, and 11% of the population are aa/aa. around 10 % of omani nationals carry the gene for hbs, and 2 -3 % carry the gene for b-thalassaemia. recent statistics show that there are around 400 patients with thalassaemia major and 3000 with scd in oman. the other rbc abnormality that is common in oman is g6pd deficiency which is found in 28 % of males and 12 % of females. omanis are known to have the highest frequency of a thalassaemia and g6pd reported so far in any race. although blood transfusion is one of the supporting treatments of scd, it can cause some serious complications for the patients. alloimmunization of red blood cells is one of the consequences of blood transfusion. alloimmunisation of the rbcs can cause haemolytic transfusion reactions and may trigger hyperhaemolysis, in which transfused and patient's own rbcs are destroyed. alloantibodies can cause delay in the process of transfusion, it can be costly and time consuming. high number of patients developing alloantibodies may indicate a major difference in the patient and donor population. it may also indicate lack of a controlled, generalised sickle patients management policy. in oman the decision of transfusing scd patient is left to physicians attending the patient. aims: this study is aimed to highlight the increasing number of alloimmunised sickle cell patients. in the royal hospital we get 40 new cases of sickle patient with alloantibodies each year. the acknowledgement of these cases may help in is assessing the current practice of transfusing scd patients, or will help to define the donor and patient population difference. methods: 418 patients were recruited in the royal hospital for this study. edta blood samples were taken for antibody screening test and in the positive cases antibody was identified, all tests done by capture technique using immucor neo machine. results: of the 418 scd patients, 49% of the patients were male and 51% female, mean age was 17 years, in the range of 1-72 years. 38 % of the scd cases were positive for the alloantibodies, 72% were female and 28% were male, the age range was from 6-68 years. 78% of the positive were scd, 19% s trait and 3% were s/ bthal. most of the patients developed one antibody, however cases of multiples antibodies were also detected. 51% of the patients were with single alloantibody, 30 % of them with two antibodies, 10 % with three antibodies, 7 % with four antibodies and 2 % with five antibodies. the majority of the cases were igg against rh antigens anti-e is being the majority 23%, followed by anti-d 13%, anti-k 17%, anti-c 14%, anti-c 8%, anti-jk a 5%, anti-jk b 5%, anti-fy a 5%, anti-e 3%, anti-s 3%, antis 1%, anti-kp a 0.7%, anti-fy b 0.3% and igm being 2%. summary/conclusions: rbc alloimmunisation rate is high in oman majority of the patient affected are female. interestingly sickle trait patients were also transfused and 19% of them developed alloantibodies. the practice of transfusing rh and kell matching blood unit is implemented four years ago and still high alloimmunization percentage is achieved. background: in ghana, routine pre-transfusion investigations for patients with sickle cell disease (scd) involve only abo-d typing and immediate spin crossmatch, without screening for irregular rbc antibodies aims: determine the prevalence and specificities of and risk factors for rbc alloantibodies in multi-transfused patients with scd methods: in 2018, a cross-sectional study in multi-transfused patients with scd, from two tertiary hospitals in ghana was performed. participants' data on demography, transfusion and medical history were recorded. antibody screening and identification tests were done at sanquin, the netherlands, with standard serology using liss as enhancer and with papain treated rbc panel cells ('enzyme only'). characterization of rhd genes was done by multiplex ligase amplification assay. logistic regression was used to determine the association of patient characteristics, i.e. sex, age at enrollment (continuous), age at first transfusion (categorized as ≤ 1, 2-5, 6-9 and ≥10), previous pregnancy, number of transfused units (2, 3-5 and 6-10 and > 10), and years after last transfusion (<1, 1-2, 2-5, >5y) with presence of alloantibodies results: 226 patients (100 males and 126 females, median age 17 years, range 1.7-66) were included. the median number of transfusions was 3 (range 2-40). the median years after last transfusion was 2 (range 2 weeks-55.5 years). in 56 patients, anti-rbc antibodies were detected. in 14 of them the antibodies were weakly reactive with enzyme treated cells only or pan-reactive, possibly some of them representing autoantibodies or antibodies against high frequency antigens. in seven patients enzyme-only anti-le a was demonstrated, likely naturally occurring antibodies. thus, in at least 34 patients (15.0%) alloimmunization was demonstrated or suspected; in 11 patients the alloantibodies were 'enzyme only'. besides, the 36 alloantibodies of known specificity (8 anti-d, 2 anti-d+c, 16 anti-e, 1 anti-c, 3 anti-e, 1 anti-k, 1 anti-s, 1 anti-le a , 1 anti-go a ), three antibodies reactive only with fy(a-b-) cells and two antibodies of yet unidentified specificity were detected. in six d-patients (3 had been pregnant) anti-d (together with anti-c in two patients) was found. in three out of four d+ patients with anti-d, an rhd variant gene was demonstrated (2 dau-alleles and 1 diii type 4 or diva-2). logistic regression revealed that none of the risk factors analysed was associated with the presence of antibodies in the 34 patients. immunobiology -red cell alloimmunity 65 fifty-eight patients, had experienced an adverse reaction during or shortly after transfusion (12 patients had dark urine). adverse reactions were associated with the number of units received (or 1.71 (95% ci, 1.25-2.33; p = 0.001), but not with the presence of antibodies (p > 0.7) summary/conclusions: in at least 15% of multi-transfused patients with scd alloimmunization could be demonstrated, mainly (80%) directed against rh antigens. the enzyme only reactivity, coupled with absence of antibodies in seven of 12 patients with probable haemolytic reaction and known evanescence of especially non rh antibodies suggest possible low titre and disappearance of some clinically relevant antibodies. given the high immunization rate together with the high frequency of adverse transfusion reactions, pre-transfusion screening for rbc antibodies should be considered for patients with scd. background: rh blood group system and mainly antigen d is one of the most immunogenic, diverse and clinically important protein-based blood group. antibody anti-d may induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. anti-d prophylaxes become ineffective if an anti-d immunization has occurred. approximately 1% of the d+ population carries rhd alleles associated with reduced d antigen expression. qualitative variants, in which some epitopes are lacking and can produce anti-d antibody, are usually termed partial d. by contrast, d weak is commonly defined as a quantitative variant that have all d epitopes and should not make anti-d. del is a very weak form of d antigen and cannot be detected by routine serological tests. because some of del individuals have already developed an anti-d antibody whereas others did not this group contains both qualitative and quantitative changes. aims: investigation was prompted by finding discrepant results in typing of d antigen in a pregnant woman /3 rd pregnancy, 1 st delivery, 2 abortions in 1 st trimester/. routine serological techniques detected d negativity and the presence of antibody allo-anti-d in clinically significant titre. the non-invasive testing of d status of the foetus from maternal peripheral blood was indicated, but this was not applicable due to presence of the rhd gene in the woman's dna sample isolated from buccal swab. our aim was to investigate the discrepancy and determine the underlying rhd genotype. methods: blood samples, dna from peripheral blood and buccal swab of the pregnant woman were investigated. routine blood grouping and antibody testing were performed by column agglutination. two anti-d sera (id-diaclon anti-d igg (cell line esd1) by biorad and anti-d duo igm+igg, clone: th28 + ms26 by immucor) were used for adsorption/elution test for identification of del phenotype. initial rhd genotyping was performed by rt-pcr (exons 5,7,10) with the dna from buccal swab; further resolution was performed using pcr-ssp (fluogene; inno-train diagnostik gmbh); sequencing was performed by sanger analysis (inno-train diagnostik gmbh). results: genotype was identified as rhd positive by ce-certified pcr-ssp kits (fluogene). sanger sequencing of rhd from exon 1 to 9 revealed presence of a nucleotide deletion in position c.147dela, which is specific for allele rhd*01el.04. this nucleotide change results in the amino acid change p.val50leufs*5 causing the del phenotype. presence of antigen d was proved by adsorption/elution technique. titre of the anti-d was rising during the pregnancy to the 2000 level two weeks before the delivery. the newborn was delivered by s.c. without a sign of hemolytic disease. blood grouping of the newborn revealed blood group a, d negative, dat negative, testing for del was not performed. summary/conclusions: the case reported here shows that females with rhd*01el.04 allele are able develop strong anti-d immunization, so this type of del phenotype belongs to the "partial del subgroup". presence of variant rhd gene in mother disabling antenatal fetal genotyping from maternal blood by current methods requires a more attentive approach to care for such pregnancies. supported by mh cz-dro uhkt 00023736 and rvo-vfn 64165. 5a-s29-05 ea scharberg 1 , s rothenberger 1 , a st€ urtzel 1 , n gillhuber 1 , s seyboth 1 , e richter 1 , g rink 2 and p bugert 2 1 institute for transfusion medicine and immunohematology, drk-bsd ba-w€ u-he, baden-baden 2 institute for transfusion medicine and immunology, heidelberg university, medical faculty mannheim, mannheim, germany background: rb a (di6) is a low prevalence antigen of the diego blood group system. it has been found in few families only. the clinical significance of anti-rb a is unknown so far. the slc4a1*c.1643c>t (p.pro548leu; isbt allele name: di*02.06) allele is the molecular basis of the rb a antigen. in the gnomad database this gene variant was found in only one of 125,742 sequenced genomes (allele frequency: 0.000004). aims: to prove the frequency of the allele in our population and gain an rb a positive donor we performed a molecular screening for di6 in 1,700 blood donors. after our antibody screening test accidentally contained an rb a positive test cell we found out that anti-rb a is a very common antibody specificity. the frequency of the antibody in patients and blood donors was proved. methods: for the molecular screening of the blood donors we developed a pcr-ssp method. the antibody screening test in 3,652 patients and in 964 blood donors was performed in the gel technique (biorad ahg id-cards) using a 3 cell screening panel (drk-bsd src) including an rb a positive test cell. positive reactions with the rb a positive cell were confirmed by an additional rb a positive test cell of different source. additional antibodies were excluded or identified in the same method using an antibody identification panel (drk-bsd irc). results: the molecular screening for the di*02.06 allele in 1,700 blood donors revealed no single positive individual. within the first 2 weeks of usage of our antibody screening test which accidentally contained the rb a positive test cell 84 patients with anti-rb a were found. it was 2.3% of 3,652 patients tested in 21 laboratories in different parts of germany. some laboratories stopped using the rb a positive lot to avoid expensive and time consuming identification and conformation tests. in 18 of 964 randomly tested blood donors (1.9%) anti-anti-rb a was also present. summary/conclusions: despite the very low frequency of the di*02.06 allele, anti-rb a is a very frequent unexpected antibody in patients and blood donors in germany. it is obviously naturally occurring and is even more frequent than anti-wr a and anti-vw we found in previous studies in around 1% of patients and donors. 5a-s30-01 national blood center, ministry of health and sports, yangon, myanmar hemovigilance which detects every event not only for patient' reactions and donor's complications but also incidents and near misses definitely improve quality of blood transfusion services especially for those situations where implementation of all the standards in one time is not possible. healthcare system in myanmar is still in the stage of requiring priority for clinical professions and has limited resources for supportive roles. supportive services including transfusion service are still not a center of interest from prioritization of health care system. blood transfusion service has been practiced in myanmar since 1935. real essence of transfusion service is hidden behind laboratory practice and transfusion is regarded as part of laboratory investigation. hospital laboratories take care of testing of blood donated by replacement donors. this kind of transfusion services under laboratory umbrella is still being practiced in myanmar except national blood center (nbc) which was established in 2003 in accordance with blood and blood product law. this law was formulated cohesively with who strategies of blood safety. in 2002, who global data-based study sent questionnaires for assessment of safety status of transfusion service. nbc noticed that there was no data which can support corrective actions for safety. from that time onward, active retrospective review of existing data and introduction of records, prospective finding of process errors and any events from hospital blood banks were recorded and taking into actions at local level. cost of every unit of blood is supported by government. in 2017, national blood and blood product committee was established. the steering committee is working hard to get cooperation from every service by aiming to prevent those undesirable events before establishment of national level policy, standards and guidelines for sustainable service quality. in conclusion, by using essence of hemovigilance as a tool, quality of transfusion service can be improved step by step to fulfil the gap in spite of limited resources. the system started in local, extends to regional level by getting agreement of importance from hemovigilance results and is finally approaching to national level endorsement. background: erroneous transfusion of abo-incompatible(aboi) blood almost always reflects a preventable breakdown in transfusion protocols and standard operating procedures and can have disastrous consequences, with significant morbidity and mortality. these incidents need to be investigated in a systematic manner to identify system vulnerabilities to mitigate risks and improve patient safety. since 2016, reporters to shot have been asked to score(0-10) the extent to which the cause of incidents can be attributed to key factors: staff, environmental, organisational and government/regulatory which helps recognise the key factors identified whilst investigating these incidents. aims: to understand why unintentional transfusion of aboi blood components continue to happen despite standard procedures and national guidance available. methods: retrospective analysis of unintentional transfusion of aboi blood components reported to shot between 2010-2017(inclusive) was done to identify common themes and recognise areas of improvement. information provided using the shot human factors investigation tool (hfit) between 2016-2017 was reviewed to understand more about why the errors occurred. results: sixty-seven unintentional aboi transfusions were reported between 2010-2017; majority (56/67, 83.6%) were red cell transfusions but aboi plasma (9/67) and platelet transfusions (2/67) were also seen. most errors occurred in the clinical area (45/67, 67%), and could have been detected at point of administration. in 21(31%) cases, the error could not have be detected at the point of administration with a primary laboratory error in 10/21(48%) incidents. reviewing data from hfit for cases in 2016-2017 (13 aboi cases), the total score for staff culpability was 100, compared to a total score of 99 for all the other three organisational and system factors. this disparity is most obvious for the 4 aboi red cell cases, all of which scored the maximum 10 for staff culpability, i.e. 40/40 compared to 8/120 as the combined total score given to the other factors. in the preceding years (2010 to 2015), there were no hf scores available; however, the emphasis on staff-related culpability is demonstrated by 37 cases that included an outcome of the local case review and 14 (37.8%) mentioned staff-related retraining or disciplinary procedures. the risk of haemolysis and serious harm is more likely with aboi red cells than with other components with 2/56(4%) that resulted in death, 14/ 56(25%) major morbidity and 40/56(71%) no or minor adverse reaction. of these cases, one resulted in conviction for manslaughter and at least two staff dismissals. summary/conclusions: transfusion never events continue to occur, and it is evident that investigations into such incidents focus mainly on staff failings and do not consistently identify system wide changes that need to be incorporated to address prevalent issues. national recommendations and a safety alert to 'use a bedside checklist' immediately prior to administration were issued between 2015-2018 to support prevention of such errors but never events continue to persist. current approach is ineffective because it often leads to apportioning blame, rather than understanding the often-complicated and multidimensional factors contributing to the error. this must be replaced by a holistic approach which addresses local work pressures and embraces advances in automated technology like electronic prescribing and barcode scanning. of the confirmed trars, n = 27 were possibly related to treatment, n = 2 trars were probable, and n = 2 were definitely related to treatment; n = 37 trars were grade 1, n = 4 were grade 2, and none were grade 4. in recipients of conventional wb, there were n = 13 (1.12%) ars, n = 32 (2.75%) fnhtrs, n = 1 (0.09%) taco, n = 0 trali, and n = 16 (1.38%) unclassified transfusion reactions. of the confirmed trars, n = 55 were possibly related to treatment, n = 1 trar was probable, and n = 8 were definitely related to treatment; n = 54 trars were grade 1, n = 1 was grade 2 and n = 1 was grade 4. there were 21 mirasoltreated wb transfusions in pregnant women and 2 trars (9.5%), both grade 1 and probably related. there were 80 transfusions of mirasol-treated wb and 84 transfusions of conventional wb in patients < 18 years old resulting in n = 7 (8.33%) trars in recipients of mirasol-treated wb and n = 10 (11.90%) in recipients of conventional wb. summary/conclusions: timely data reporting of trars and expanding the hv infrastructure has helped to improve the hv system in ghana. of 2181 wb transfusions in routine use in ghana, there were 4.02% trars in recipients of mirasol-treated wb and 5.59% in recipients of conventional wb. additionally, mirasol-treated wb was safely transfused in pregnant women and pediatric patients. haematology, monash health, melbourne, australia background: transfusion-associated graft-versus-host disease (ta-gvhd) is rare and usually fatal. it can be prevented by provision of irradiated blood products to at-risk individuals, such as those receiving nucleoside analogues, alemtuzumab, bendamustine or with hodgkin lymphoma (hl). duration of risk is uncertain, so ensuring these individuals correctly receive lifelong irradiated blood components, as currently recommended by anzsbt and bsh guidelines, is challenging. in australia, platelets are routinely irradiated, but red blood cells (rbc) are not. aims: to determine whether patients receiving fludarabine, cladribine, bendamustine, alemtuzumab, or dacarbazine (for hl), appropriately received irradiated rbcs. secondary outcomes included rates of ta-gvhd after unintended exposure to non-irradiated components, factors influencing correct issue of irradiated rbcs such as transfusion management plans, and provision of adequate clinical information on blood requests. methods: we performed a retrospective audit to identify patients receiving therapies indicating risk for ta-gvhd using pharmacy dispensing records from january 2008 to october 2018 at monash health, a multi-campus university hospital in melbourne, australia. diagnosis, treatment dates, group and hold (g&h) requests, rbc transfusions, and follow-up information were sourced from laboratory and medical records. results: we identified 310 patients who received fludarabine (n = 52, 17%), bendamustine (n = 29, 9%), cladribine (n = 17, 5%), dacarbazine for hl (n = 164, 53%) and alemtuzumab (n = 48, 15%). the median age of patients was 46 years (range 8-92) and 171 (55%) were male. median follow-up was 30 months (range 0-132). post-exposure, 42 patients (14%) received transfusions with 33% correctly receiving irradiated rbcs. the remaining 28, all from haematology/oncology, received a total of 192 unirradiated rbcs. in 8 patients, this was rectified on subsequent transfusions. there were no cases of ta-gvhd at median follow-up of 14.5 months (range 0-75) from first rbc transfusion. after medication administration, 99 patients had g&h requests after a median of 3 months (range 0-129). only 23% of requests had sufficient clinical information to prompt irradiation, such as hl or medication details, and only 20% asked for irradiated components. preventive strategies have now been employed. transfusion management plans for haematology patients were implemented in march 2017. for audited patients, these were written from 38 days prior to 104 days after medication exposure. two were written following inadvertent unirradiated rbc transfusion. patients identified in this audit will have a laboratory flag generated and prospectively, pharmacy dispensing records will be sent to blood bank to identify at-risk patients. our hospital is transitioning to electronic medical records (emr). an alert will be generated in emr when ordering transfusions if there has been exposure to these medications. however, clinical awareness and documentation remain vital. additional measures include patient education, alert cards, and ongoing collaboration with medical staff to encourage transfusion planning. summary/conclusions: recognition of patients at risk for ta-gvhd remains low, even among haematology units. we are making progress on ensuring provision of lifelong irradiated blood components in patients exposed to nucleoside analogues or alemtuzumab, as well as hl patients. implementation of an emr and additional strategies in this domain is important to prevent ta-gvhd. background: blood transfusion is considered an essential element in the management of patients globally. it might be risky and transfusion related adverse reactions may occur with the less adherence of transfusion policies. standard guidelines regarding the screening of blood for infectious disease, genuine need of transfusion and abo compatibility are followed and monitored drastically. however, patient assessment during transfusion especially at patient bedside and post transfusion is also equally important. aims: we are a newly established hospital and are working towards the best possible management of patients. in this regard to minimize the transfusion errors and to highlight if any lacking being practiced during transfusion, we conducted this study to observe the compliance rate of documentation of transfusion form by the healthcare staff and also to observe the compliance of line of action taken in case of occurrence of transfusion reactions. methods: this was a observational study conducted at nibd and bmt, pechs campus from february 2018 to february 2019. ethical approval was obtained prior to the study. transfusion form for each transfusion was filled. the form provided information on documentation of blood product receiver name, employee identity number, date and time of receiving blood product, patient name, medical record number on units, on patient's wrist band and on transfusion form. abo compatibility on the unit and on form, medical record number from wrist band, name and employee identity number of two healthcare staff started transfusion, transfusion start and completion time. time, temperature, blood pressure, pulse and initials of staff at the time of order, onset, after 15 minutes and at the completion of transfusion were also included. transfusion reaction form was also filled by the healthcare staff. data was analyzed by using spss version 23.0. results: a total of 500 transfusions forms were analyzed. over all compliance rate was 18%. out of 500, 115(23%) forms were available in source notes and of 115, 88 (76%) were partially and completely filled. higher compliance was seen in the initial months of hospital establishment than later months (p-value = 0.000). highest non compliance was seen in documentation of initials of duty doctors on transfusion form at the completion of transfusion(3%) and highest compliance was seen in documentation of name by healthcare nursing staff at the start of transfusion(89%). a total of 02(0.4%) adverse events were reported from red blood cells and platelets. mean time of start of symptoms was 2 hours and 30 minutes for red blood cells and for platelets it was 1 hour and 13 minutes. transfusion was instantly stopped as the symptoms appeared with no delay of time and actions were taken to resolve the reactions. time of appearance of symptoms and time of start of medication were documented and error free. all blood bags were returned to the blood bank and discarded after 6 hours as per the policy of hospital. summary/conclusions: the study was conducted to highlight the scarce practices that are being implemented by healthcare staff in context of documentation and reporting of transfusion reactions at our hospital. stringent actions should be taken for the adherence of compliance by healthcare staff to avoid morbidities and mortalities. we believe that it will also be helpful to provide baseline information in the process of preparation of a national guidelines and protocol on blood transfusion procedures. 5a-s31-01 as buser, a holbro and l infanti regional blood transfusion service, swiss red cross, basel, basel, switzerland to make blood supply safer, pathogen inactivation (pi) technologies have been developed. they are based on photochemical (amotosalen/uva or riboflavin/ uv) or uv-c light treatment to reduce potential pathogens in blood components. this gain of safety might however be offset by "off target" effects of these technologies. in virtually all clinical platelet transfusion trials, it has been demonstrated that post transfusion increments with pi platelet (plt) components are lower as compared to conventional components, indicating different biological behaviour such as survival/ clearance of nontreated and treated plt. published studies have also suggested shorter survival of platelets in vivo in animal studies. additionally, data of the rates of alloimmunization and refractoriness after transfusion of pi platelets are show discrepant results. animal studies suggest a reduction of the rates of alloimmunization when transfusing (leukoreduced) pi plt as compare to conventional plt. in the clinical setting, published data, including very recent reports, showed different rates of hla class i and ii alloimmunization with the two currently available photochemical based pi technologies. while pi of plt components surely benefit patients regarding pathogen safety, the impact of potential off target effects possibly impairing efficacy of pi plt transfusions need more investigation. background: brucellosis is an endemic disease and still a major health problem in saudi arabia. ministry of health in saudi arabia listed brucellosis as a notifiable disease due to its endemicity. in the last ten years, the incidence has decreased significantly to approximately 15 cases per 100,000 but is still higher than that in developed countries. human-to human transmission is extremely rare including breast feeding, transplacental, sexually and blood transfusion. five cases of brucellosis through blood transfusion have been reported in the literature. brucella transmission through blood transfusion is likely underreporting due to the long incubation time of 2-4 weeks (range, 5 days to 5 months),vagueness of clinical presentation and lack of hemovigilance systems in endemic areas. (allohsct) and 130 (4.6%) autologous (autohsct) hsct patients, with mean corrected count increments (cci) of 8.7 9 10 3 , 8.0 9 10 3 and 7.2 9 10 3 , respectively. mean cci decreased in a linear fashion between day ≤ 2 and day 7 pcs (9.0 9 10 3 , 9.1 9 10 3 and 8.5 9 10 3 at ≤ 2 days; 6.7 9 10 3 , 5.8 9 10 3 and 4.9 9 10 3 at 7 days, respectively), although the number of pc transfused on day 7 to autohsct patients was small (n = 71). background: nipah virus (niv) is a paramyxovirus (genus henipavirus) that emerged in the late 1990s in malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in bangladesh and india. niv infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of niv contaminated foods. nipah virus (niv) belongs to the list of pathogens identified by the who to have the potential for a global pandemic. aims: this study aimed to investigate the efficacy of the theraflex uv-platelets system to inactivate niv in platelet concentrates (pcs). the theraflex uv-platelets system (macopharma) uses uvc light without the need of any additional photoactive compound. methods: plasma reduced pcs from 4 bcs (35% plasma in additive solution ssp+) were spiked with virus suspension (10% v/v). pcs (n = 2, 375 ml) were then uvcirradiated on the macotronic uv machine (macopharma) and samples were taken after spiking (load and hold sample) and after illumination with different light doses (0.05, 0.1, 0.15 and 0.2 (standard) j/cm 2 )). the titre of the niv (malaysia) was determined as tissue culture infective dose (tcid 50 ) by endpoint titration in microtitre plate assays on vero 76 cells (atcc â crl-1587 tm ). the results of the infectivity assay demonstrated that uvc irradiation dosedependently inactivated niv. after spiking a niv titer of 6.2 (bag no. 1) and 6.5 (bag no. 2) log 10 tcid 50 /ml was received in the pcs. at a uvc dose of 0.10 j/cm 2 and higher niv was inactivated down to the detection limit of the system (1.9 log 10 tcid 50 / ml), resulting in log 10 reduction factors of ≥4.3 (bag no. 1) and ≥4.6 (bag no. 2). summary/conclusions: our results demonstrate that the theraflex uv-platelets procedure is an effective technology to inactivate niv in contaminated pcs. vs. 364 ae 20.6 9 10e11 platelets/unit, p < 0.001), whereas the platelet content of apheresis pc did not change (305 ae 9.9 vs. 300, ae16.1, p = 0.57). summary/conclusions: pathogen reduction resulted in the transfusion of older pc on average, but without altering the number of pc ordered or the use of pc per patient. pathogen reduction has improved pc stock management without an increase in platelet demand, despite lower platelet content of buffy coat pc after pr implementation. donors and donation -donor adherence -are we doing the right thing? the transfusion procedure is the last step in a multi-process supply chain. the task of matching supply with demand requires donor managers to consider average consumption rates on a weekly or monthly basis, but to also have insight into variability in order distribution and possible attribute (blood groups) requirements. since hospitals and blood banks are usually not deeply interwoven and often only ex-post data is available, forecasting methods should be implemented. a thorough analysis of order pattern to set weekly target inventories and safety levels is required to close the information gap. a collection plan needs to identify possible bottlenecks which can be prevented through the planning of inter-shipping, changes in message urgency and building of reserve donor pools. constant analysis of collection and mobilization kpis allows donor managers to implement the rolling-wave planning approach and continually adapt to changing requirements, unexpected events and overall systematic variability. the variability happens on the demand side, as order quantities and their attributes, such as blood group distribution, are subject to change. however, also the supply is subject to significant variation, as donor response rates, attrition, deferrals and overall availability of donors are not constant. the data was collected with the face-to-face interview method right after the donation. 1478 first-time donors has attended to the study in 18 regional blood centres in 29 cities in turkey. the survey included items in accordance with the standard tpb predictors of attitude, self-efficacy, and intention. self-identity, anticipated regret, donation anxiety, paraphernalia anxiety, personal moral norm, descriptive norm, satisfaction, motivation also assessed for the first-time donors. the relation between the predictors and intention confirmed with correlation analyses. the predictors' distribution analysed by multiple linear regression. a number of goodness-of-fit indices were calculated and examined for each tested models (ibm, amos spss). the results of goodnessof-fit tests for proposed model provided a better fit to the data than these models (cmin/df = 14). moreover, this result indicated that the fit between the proposed model and the data could be improved with further modifications with the inclusion of paths between motivation and attitude, self-identity and intention. moreover, inclusion the paths between donation anxiety and intention and between self-efficacy and attitude, on contrary to recent analyses suggesting opposite paths. evaluation of goodness-of-fit tests showed good result for revised model with a value of cmin/df = 3.55, close to perfect fit. the revised model revealed that attitude was the strongest positive direct predictor of intention followed by personal moral norm, self-identity, motivation and anticipated regret (path coefficients: 0.47, 0.28. 0.19, 0.17, and 0.5, respectively). donation anxiety was the negative direct predictor of intention (-0.05). satisfaction was the strongest positive indirect predictor of intention via attitude and followed by self-efficacy (0.90 and 0.08). paraphernalia anxiety was the negative indirect predictor of intention (-0.03). descriptive norm did not show any significance. our model accounted for 75.3% of the variance in intention. summary/conclusions: these findings suggest several potential avenues for enhancing donor retention. the results obtained with this study provide important data from the standpoint of donor retention, which should be, implemented in the future strategies of turkish red crescent. background: transpose-transfusion and transplantation: protection and selection of donors, is a european consortium project, including partners from 16 countries, reviewing donor selection and protection policies for substances of human origin (soho).one of the main issues in the current donor selection system, which transpose aims to tackle, is that for many, if not most criteria, is not evidence based. the transpose consortium therefore tries to re-assess selection criteria, revised them where needed and provide recommendations as evidence-based as possible. transpose additionally adds to the current european directorate for the quality of medicines & healthcare (edqm) guidelines by emphasizing donor safety. aims: the aim is to compare existing donor eligibility criteria throughout europe, and to compile a list of risks to consider, with evidence-or consensus-based deferral criteria to provide more uniform donor screening criteria. methods: there are three horizontal work-packages (wps); wp1 coordination, wp2 dissemination, and wp3 evaluation of the project, and four technical ones with specific deliverables and milestones to be regularly produced: -wp4 inventory of donor selection & protection practices; -wp5 development of risk-based guidelines for donor selection and protection; -wp6 development of a standard donor health questionnaire (dhq); -wp7 training course/workshop on the use of the guiding principles, guidelines and the dhq. the transpose project launched in september 2017 and will complete in spring 2020. wp4 has completed its work in october, wp5 will complete its work in june 2019, and wp6 and wp7 have recently commenced. results: with the use of the deliverables created by wp4, we have created an indepth inventory of current practices in donor selection and protection, including overview of similarities and differences across european countries and across soho types. there is an agreement amongst experts that existing guidelines are often based on the precautionary principle rather than on risk assessment. consequently, in the development of wp5's guidelines for donor selection and protection, we now make an effort to also emphasize donor safety, in a more evidence-based way via the use of risk-based assessments. this will result in a standardized dhq with a common trunk and more in -depth questions per soho. summary/conclusions: the impact of the outcomes of transpose will be threefold. first, outcomes are expected to be of help in revising donor selection and protection related eu directives. second, the set of guiding principles and donor selection & protection guidelines will facilitate eu member states to take a next step in implementing donor selection and protection policies in a consistent and clear-cut way to the benefit of both donors and recipients of soho. third, a standard donor health questionnaire with carefully guided local/regional/national adjustments will become available per soho which can be used widely and will consequently enable comparisons of the prevalence of certain risks and risky behaviours throughout europe. background: transpose-transfusion and transplantation protection and selection of donors is a european consortium project, including partners from 16 countries, that reviews donor selection and protection policies for blood, plasma, tissues, assisted reproductive technology (art) and stem cells (together soho). donor selection criteria (dsc) in europe are based on eu-directives, guidelines and countries' own additional criteria. literature shows that particular criteria are outdated or not risk-based, often leading to unnecessary donor deferral or an underestimation of risks for donors. aims: to 1) provide a comprehensive inventory of current systems for selection and protection of donors and donations, 2) critically review them and 3) recommend an over-arching donor health questionnaire (dhq) including all necessary criteria currently used by different eu-member states (eu-ms). methods: in-depth semi-structured interviews with key stakeholders in blood collection were conducted to identify main topics for improvement in the current dsc. these formed the basis for a survey sent to professionals from collection institutions of all soho to get feedback on current systems from as many eu-ms organisations as possible. questionnaires were sent to a total of 163 experts (40 blood; 40 plasma; 27 tissues; 47 stem cells; 9 art) and 39 (24%) completed questionnaires were received. where information was lacking, additional experts were asked to recommend upon dsc. results: for blood and plasma donation four main areas of concern in dsc were identified: risk-based selection, adaptability, flexibility and consistency. the stakeholders agreed that dsc are often outdated and lack evidence, hence leading to unnecessary deferral of donors and underestimated risks for donors. they suggested to base dsc on group risk-assessment (risk-based selection) and on conducting more research to achieve standardized risk perceptions and evidence-based deferrals, either for safety of recipient or donors. criteria could be made more detailed to fit specific groups to defer less donors (adaptability). furthermore, implementing criteria was considered easy, but abolish criteria when not regarded as a risk anymore seems almost impossible (flexibility). additionally, deferral periods are perceived too long, seen as both negative, i.e. jeopardizing donor return intention and positive, i.e. no risk for safety (consistency). changing legislation into guidance was an often-mentioned suggestion to improve dsc. specific feedback on plasma donations revealed that many whole blood topics are not applicable to plasma-only donors, e.g. parasite infections such as malaria (no deferral needed); travel history (no deferral needed), and recent bacterial and viral infections (deferral periods currently too long). a clear need for more research on plasma collection-related issues was identified. summary/conclusions: dsc are perceived redundant on a substantial number of aspects by most stakeholders. besides achieving the goal of save and sufficient soho for patients, many regulations could be improved to diminish deferrals and decrease donor risks. transpose will add to reviewing, improving and harmonising these regulations and criteria. furthermore, transpose will provide suggestions to improve directives and guidelines and a dhq, focusing on both donor health protection and safety of donations, but also removing deferral criteria that are not relevant (anymore), and offer a future research agenda to make dsc more evidence-based. background: transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of 24 stakeholders from both not-for-profit and private blood collecting organizations as well as researchers and officials. the project aims to create new evidencebased donor selection criteria as well as guiding principles for risk assessment of threats to the safety of all substances of human origin (soho) except solid organs. as part of this, an inventory of current donation-related risks was performed, including an investigation of both type and number of adverse events reported. aims: we here aim to present an overview of reported adverse events in plasma and whole blood donation in europe and to compare this to the anticipated risks rated by transpose stakeholders. methods: national or local data on adverse reactions from the years 2014-2017, both serious and mild, in whole blood and plasma donors was collected from the relevant stakeholders (eighteen and nineteen respectively). stakeholders were also asked to grade the most important anticipated donor risks according to severity, level of evidence and prevalence. we then compared the relevant risk categories as evaluated by the stakeholders with the categories of the provided data, as well as the heterogeneity of category numbers. results: thirteen stakeholders provided data on adverse events during whole blood donation in a given year, including in total thirty-three different categories of adverse events, ranging from only one unspecified reaction to seventeen different categories, with an average of nine categories per stakeholder. the most frequently used categories were hematoma (included by 77%), arterial puncture (77%) and nerve damage (54%). vasovagal reactions were also frequently included (75%); however, this was being done variably as vasovagal reactions unspecified, and acute and/or delayed vasovagal reactions. only one stakeholder reported iron deficiency. for plasma donation, seven stakeholders provided data on adverse events. a total of twenty-seven different categories were reported, ranging from one to seventeen per stakeholder, with an average of nine. the most frequently reported adverse events were hematoma (86%), citrate reactions (86%) and arm pains and nerve damage (both 57%, respectively). anticipated risks in blood donation were rated by nine stakeholders rating iron deficiency, vasovagal reactions and hematomas the greatest risks to donors. for plasmapheresis, six stakeholders rated vasovagal reactions, hematomas and citrate reactions as highest risk. summary/conclusions: as shown, categories used to describe adverse events in blood donation vary tremendously across europe, with some countries only being able to provide total numbers of adverse events without further specification. furthermore, there is a gap between perceived high donor risks and reported adverse reaction categories in donor vigilance for whole blood, as reports on iron deficiency are virtually absent despite being considered the most significant risk. our findings show the need for international collaboration on creating an international standardized donor vigilance system, to gather more insight into donor risks to protect the health of donors. plenary session -a glimpse of the future pl-03-01 modern transplantation medicine has made significant progress within the last decades due to a better immunological understanding of rejection and advances in immunosuppression. however, the severe side effects of long-term, typically lifelong, immunosuppression and the shortage of donor organs remain the major restrictions in transplantation. the idea behind all research to improve transplant outcome has always been the modification of the recipient's immune system to ideally induce a specific tolerance towards the donor's graft. in fact, the immunological blindness of the recipient towards the donor's graft is achieved by a general reduction of the immune system's competence and represents a major burden for transplant patients. the idea of invisible organs is an entirely different approach to solve the problem: instead of inducing an immunological blindness of the recipient's immune system an immunological invisibility of the donor's organ is created. this is achieved by genetically engineering the transplant to eliminate the organ's immunogenicity defined by the gene products of the major histocompatibility complex (mhc) and minor histocompatibility antigens. in addition to manipulating the expression of mhc genes required for immune recognition, immune cloaking strategies are used to evade immune rejection. these approaches take advantage of creating an immunosuppressive environment and expressing immune suppressive molecules by immunomodulatory transgenes. mhc engineering and immune cloaking in an entire organ is achieved during ex vivo perfusion by lentiviral transduction of gene expression modifiers and transgenes to induce a permanent immunological invisibility of the organ. importantly, mhc engineering also prevents the presentation of minor histocompatibility antigens, which usually are not possible to match between donor and recipient, but which trigger potent immune responses and graft rejection. eliminating the targets of cellular and humoral rejection as well as creating an allograft-specific immune environment through immune cloaking camouflages the organ and equips it with a powerful set of defense weaponry. immune-engineering of transplants achieved during the inevitable ex vivo period of the allograft after explantation without the need to accept off-target effects allows keeping the recipient's immune system fully functional and capable to combat infections and cancer. in pre-clinical in vivo studies from rodents to minipigs a clear survival advantage of ex vivo engineered transplants could be demonstrated. this approach has the potential of eliminating the burden of organ rejection and immunosuppression, thereby sustainably increasing transplant survival, organ availability and quality of life. gene editing for sickle cell disease: re-expression of the fetal c-globin genes (hbg1/2) could be a universal strategy to ameliorate the severe b-globin disorders sickle cell disease (scd) and b-thalassemia by induction of fetal hemoglobin (hbf, a 2 c 2 ). we have previously identified bcl11a erythroid enhancer sequences, marked by hbf-associated common genetic variants, that are required for repression of hbf in adult-stage erythroid cells but dispensable in non-erythroid cells. recently we have optimized conditions for selection-free on-target crispr-cas9 editing in human hscs as a nearly complete reaction without detectable genotoxicity or deleterious impact on stem cell function. we demonstrate that cas9:sgrna ribonucleoprotein (rnp) mediated cleavage at core sequences of the + 58 bcl11a erythroid enhancer results in highly penetrant disruption of gata1 binding motif, reduction of bcl11a expression, and induction of fetal c-globin. erythroid progeny of edited engrafting scd hscs express therapeutic levels of hbf and resist sickling, while those from b-thalassemia patients show restored globin chain balance. moreover we find that hscs preferentially undergo nonhomologous as compared to microhomology mediated end-joining repair. nhej-based bcl11a enhancer editing approaching complete allelic disruption in hscs appears to be a feasible therapeutic strategy to produce durable hbf induction. in this presentation, i will compare and contrast bcl11a enhancer editing to other autologous curative gene therapy and gene editing approaches at various stages of clinical and pre-clinical evaluation. oxygen is vital for life. without oxygen death is assured for aerobic organisms. although everybody knows this fact a lot of medical acts forget to take care of it, leading to a lot of potential troubles. indeed, during cell respiration the glucose oxidation by oxygen gives carbon dioxide, water and energy. this energy also called atp is necessary for cellular metabolism and consequently for life. we have identified an extracellular hemoglobin coming from a marine worm, called arenicola marina, which is able to deliver oxygen to this animal living in the intertidal areas on the atlantic coast in france between the north sea and biarritz. this molecule called m101 was developed in the medical device named hemo 2 life â . we have showed that this product was very efficient to protect organs before transplantation. a multi centers clinical trial performed under the supervision of pr. le meur from the chu of brest, on 60 patients waiting kidney grafts showed a delay graft function reduced roughly by three between the two kidneys harvested on the same donor with and without hemo 2 life â and grafted on recipients. in 2018, a world first was realized in france by the pr. lantieri to georges pompidou hospital in paris, france. indeed, it was the first time that a patient received a second graft face. this surgery was realized with hemo 2 life â and showed a very nice result according the pr lantieri, the anastomosis were very easy and no edema was observed. furthermore, we have developed dressing incorporating m101 making a product called hemhealing â . preclinical data on diabetic mice showed an increase of healing process. hemoxycarrier â , a therapeutical oxygen carrier, is also in progress of development in order to address ischemic diseases such as the sickle cells disease, myocardiac infraction and stroke. this universal oxygen carrier without blood typing, which is the ancestor of our red blood cell containing hemoglobin showed that it is able to deliver oxygen at different biological levels, cellular, tissues and organs and could address a multitude of medical applications. background: main goal of transfusion is saving life and/or improve the health status of human by "safe blood" which needs regular, voluntary, unpaid blood donors. donor recruitment is being more sensitive and challenging part of the blood supply system in actual global socio-economic conditions. achievement to enough voluntary non-remunerated blood donation (vnrbd) can be established by an efficient donor recruitment. efficiency of the donor recruitment has still close relation with blood donor recruiter although there are so many new tools. occupational specifications, rights and responsibilities of blood donor recruiter have wide range differences between countries which cannot be explained completely by the specific conditions of each country. also, a concrete document which has an international consensus was not existing on this subject. turkish blood foundation (tbf) has been organizing an international workshop since 2012; anatolian blood days (abd). "who is a blood donor recruiter?" was the topic of abd-vii at 9-11 march 2018. aims: main aim of the workshop was to check and evaluate the existing systems of the participant countries. than create a model for clearly defining occupational specifications, responsibilities and rights of blood donor recruiter. methods: experts from 25 countries participated in the workshop. those countries are albania, algeria, bosnia-herzegovina, estonia, france, germany, hungary, india, kazakhstan, lithuania, macedonia, montenegro, oman, portugal, qatar, romania, russia, saudi arabia, serbia, slovenia, sri lanka, tajikistan, turkey, uganda, uzbekistan. these countries reflect almost all religious, ethnical, social, cultural and economic situations of the world. a questionnaire which was analyzing existing systems at participant countries sent before the workshop. after country presentations 4 different discussion groups were organized. below listed topics were announced at final declaration. results: donor recruiter: 1. should have university degree preferably in marketing and business administration field. 2. should have a certificate and/or professional experience in public relation 3. should have efficient skill in conversation, sociability, independence, self-confidence, reliability, resilience and conscientiousness as well as to work in a team 4. should get a special training which includes not only social topics such as public relation, marketing, etc. and medical topics related bb&tm before practicing alone as a donor recruiter 5. should be a permanent staff 6. should have basic salary and performance bonus might be given 7. is eligible to monitor and modify mobile team working period at blood drive 8. should participate the mobile blood drive which he/she has organized 9. should participate the group who will create promotional materials for national blood service 10. number at each blood establishment should be defined based on annual blood collection such as 5 staffs for 50,000 whole blood collection annually in germany. summary/conclusions: in conclusion; both donor recruitment and retention are not easy tasks to undergo while public are aging, and birth rates are decreasing all around the world. dedicated blood donor recruiter whose occupational specifications, rights and responsibilities are clearly defined will be the corner stone of the success for providing enough safe blood for transfusion. ct smit sibinga 1 and j emmanuel 2 background: africa is a large continent with 55 independent states and a total population of 1,275,710,034 (february 2018) . healthcare policies and strategies are developed through who's advocacy, guidance, and support from hq in geneva and the 2 who regional offices; eastern mediterranean regional office (emro) supporting 8 arabic speaking countries and the african regional office (afro) responsible for 47 sub-saharan countries. population distribution is approximately 40.6% urban. there are a large number of different local dialects and languages spoken. the main languages spoken are english, french, portuguese, spanish and arabic. countries are mainly classified by undp as being of low and medium human development index the africa society for blood transfusion (afsbt) has members in most countries, advocates for the development of sustainable and effective blood services, and has developed a stepwise 3 level accreditation program. in 2016 emro held a consensus meeting developing a "strategic framework for blood safety and availability for 2016-2025" with a set of priority interventions focusing on leadership and governance, cooperation and collaboration, provision of safe blood and blood products, appropriate clinical use of blood, and quality system management. in 2001 all 54 member states of the african union (au) countries, in abuja, nigeria, pledged that national budget for health should be at least 15% of the national fiscal budget. in 2013 ministers of health of who member states endorsed that blood and blood products be included in the essential medicines list; these endorsements and who's universal health coverage (uhc), have yet to be fully implemented. aims: to analyze (gap-analysis) to what extend countries in africa implement the world health assembly resolution wha63.12 on availability, safety and quality of blood products, which urges governments to ensure safe, accessible, affordable and available supplies of blood and components from voluntary non-remunerated blood donors, which meet clinical transfusion requirements and achieve national self-sufficiency, following who guidelines and recommendations. methods: to provide an overview of the current status of the blood supply in africa strengths and weaknesses, data from who's 2016 global status report on blood safety and availability were analyzed and used. the study has been descriptive and explorative. results: the 2016 report identified a number of areas requiring attention; principle amongst these were -inadequate funding; -lack of governance and leadership; -ineffective public education on blood donation; -absence of capacity building for clinicians on rational use of blood; -lack of haemovigilance and implementation of quality management systems; -the need for regulatory or oversight mechanisms. summary/conclusions: national authorities should address areas requiring attention if progress towards ensuring a sustainable safe and sufficient supply of blood products is to be achieved. key is the commitment and support of national governments, which should implement resolutions and recommendations agreed by ministers of health at wha and african union. background: the core function of the blood donation testing (bdt) laboratory is to screen every unit of blood collected from a donor for blood group type and infectious disease markers to ensure safety of the national blood supply prior to transfusion. the lab operates daily on two work shifts, comprising of 4 staff on the morning (am) shift (from 8:00 to 17:30) and 5 staff on the afternoon (pm) shift (from 13:00 to 22:00) on weekdays and 3 staff on the am shift and 5 staff on the pm shift on weekends. bdt lab has a staff strength of 15 to be rostered for the 2 work shifts. each staff is on a five-day work week and has to work 11 pm shift and 9 am shift per month on average. the higher number of pm shift leads to staff feedback that they do not get sufficient time in the evenings for family and social or leisure activities. a lean six sigma project was initiated to review the work rostering to improve the work-life balance of the staff. aims: the project aims to reduce the number of staff working on the pm shift without affecting the downstream processes and continues to meet the timely release of blood supply to the hospitals. methods: lean six sigma tools were used to study the bdt lab workflow process and to identify factors that contributes to the higher number of pm shifts that the staff has to take on. data on the turn-around time and the man-effort required for each screening tests performed was analyzed. a survey was also conducted to understand the preference of the staff on the acceptable number of pm shifts per month. results: the main contributing factor for more staff required to perform the pm shift is due to majority of the daily donation samples being received only in the evening. as this factor is beyond the control of the bdt lab, redeploying work from the pm shift to the am shift was eventually selected as the solution to reduce the number of staff needed for the pm shift. the screening test that was shifted was determined based on the test system that has the shortest turn-around-time and is able to allow continuous release of results. at the same time, most of the staff must be trained for that test system. a trial on the new roster involving 5 staff on am shift and 4 staff on pm shift was conducted. the total number of pm shift per month was reduced from 124 to 112 using the re-defined process. the 10% reduction translates to fewer number of pm shifts that the staff has to undertake and was able to meet the staff's expectation. summary/conclusions: with the adoption of the new process workflow, bdt lab was able to reduce the number of pm shifts that the staff needs to be rostered using evidence based process improvement method. most importantly, the lab has a satisfied team of staff with better work-life balance. background: preparedness of blood transfusion services for emergencies and crisis situations is an important issue concerned with patient and transfusion safety. aims: having an experience of delay in blood component supply in an emergency situation due to partial interruption of hospital information system (his), it is aimed to create a crisis kit and constitute an alternative work flow for emergency in crisis situations. methods: it is stipulated that the failure of his which is normally conducts all process for transfusion would be disabled in a disaster or crisis situation. a brain storm was made on possible challenges associated with disability of his during transfusion emergencies. according to the scenarios a kit was developed for the process management of transfusion emergencies. results: a flow chart was designed in proper with transfusion emergency definitions of who and instructions were written to explain the flow chart. all forms categorized with different colour codes are designed to fill with handwriting. the kit consists of flow chart and instructions, analysis request forms (blue coloured), blood component request forms (pink coloured), proceeding forms (green coloured), pens and blood sample tubes with edta were put into a plastic folder labelled as transfusion emergency disaster & crisis (tedc) kit. additionally, the kit is placed in a sealed clear plastic bag and delivered to all inpatient and intensive care units of pediatrics and pediatric surgery. a training programme concerned with transfusion emergency situations and usage of the tedc kit was developed for health care workers involved in blood transfusion process. pre and post-assessment tests were developed for the evaluation of effectiveness of the training programme. summary/conclusions: it's challenging to improve the response capacity of blood transfusion services during emergencies and crisis situations. abstract withdrawn. background: india is a developing country having 2760 licensed blood banks majority have manual documentation which causes inaccuracies and errors in blood bank activities. monitoring is a herculean task. computerisation is the need of the hour but this goal involves many hurdles and challenges aims: the aim of this study is to discuss the challenges faced during computerisation of blood bank activities and the remedial solutions for it methods: department of transfusion medicine, king george medical university, lucknow is one of the biggest blood bank of the country with annual collection of 70,000 blood units. two years ago, the blood bank worked on totally manual system. computerisation involved challenges associated with hardware and software installation and personnel training. hardware was installed in two phases. initially hp system but later shifted to apple imac due to frequent breakdowns. with hp server. software installation (easy software) involved erratic internet connectivity hence changed to lan. customisation involved radical changes according to our needs. at times we had to change our way of working to suit the software. biometrics linking, medical registration, cash id generation, donation, serological crossmatch, automated blood grouping, labelling, chemiluminescence & nat testing, blood component preparation and camps were all included with challenges at every level. remedial actions were taken from small to big. training of the staff was the most essential part of the implementation of computerisation who initially showed considerable resistance and at time faking ignorance due to apprehension that their mistakes will be highlighted and they will be penalised for it. it was a herculean task in creating their password protected identity and enforcing them to use it. gradually the staff realised that computerisation made their task easier as it cut down on paper work and repetition and also prevented serious mistakes from happening. hard copies at certain essential areas were still maintained to continue work in the event of major breakdown of computer results: computerisation aided us in regulating the movement of the donor which at times was repetitive due to manual entry. transfer of data ensured a safe supply and the mistakes could be retraced very easily. implementation which included installation, training and enforcement took a period of 6 months. after overcoming all the challenges we minimised hard copies to 6 registers and started taking printouts of the other necessary details. the turnover time for the employees due to computerisation decreased by 20%. waiting time for attendant decreased by 10%. traceability of all the units became 100%.supervision of the activities being carried out was 90% accurate. identification of the donors was easy due to biometrics which included thumb impression and iris scanning. the decision making time for donors decreased by 50% thus making the system more efficient. summary/conclusions: manual to computerisation involves involvement from source to supply and it is essential to anticipate the challenges and be prepared for solutions in order to make its implementation successful p-007 ct smit sibinga 1 , y abdella 2 and f konings 2 1 iqm consulting, zuidhorn, netherlands 2 who eastern mediterranean office, cairo, egypt background: who defined essential medicines (ems) as medicinal products that satisfy health-care needs of the majority of a population. they should be available at all times, in adequate amounts, in appropriate dosage forms, with assured quality and affordability. in 2013 blood and blood products (whole blood, red cells, platelets, plasma, and plasma-derived products) were added to the who model list of ems. appropriate and effective regulatory framework (legislations, regulations, etc.) and a functioning regulatory authority (ra) is crucial for management of blood products as ems. however, particularly in the less developed world, these prerequisites have barely been implemented. aims: to analyze and advise on existing legislation and regulations. existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. methods: existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. results: various formal legislative documents of only 9/22 countries are put in force by governments [1960 (egypt) till 2017 (pakistan -sindh)]. most are detailed descriptions of ra, operational establishments, and specific requirements. however, none of these legislations complies with who and eu recommended formats and contents, and will not support effective regulatory oversight to promote and enhance quality, safety and availability of these ems. summary/conclusions: government should provide effective leadership and governance in developing a national blood system (nbs, fully integrated into the national health-care system. essential functions of a nbs include an appropriate regulatory framework with legislations, regulations and other non-legislative instruments administered by a ra. these documents should spell out principles and cadres, standard setting, and organization of the blood system to ensure an adequate supply of blood and blood products and safe clinical transfusion for which a model was designed. the structure of nbs will depend on organization and level of development of the health-care system. however, all critical activities within nbs should be coordinated nationally to promote uniform standards, economies-of-scale, consistency in staff competency, quality and safety of these ems, and best transfusion practices. key is formulation of an appropriate regulatory framework administered by a ra responsible for regulating the vein-to-vein chain in the preparation and use of these ems. background: the capacity of blood transfusion service to provide adequate supplies of blood components is the issue of concern for health providers worldwide; longer term observations of trends in this respect are therefore of crucial value. aims: the study aim was analysis of some basic activities of the polish blood transfusion service in 2008-2017 including organizational changes, numbers of donors, donations and blood components as well as activities directed at increasing their safety. methods: retrospective analysis of data supplied by the regional blood transfusion centers (btcs). results: in the discussed period, blood and blood components were collected in 21 regional btcs and local collection sites as well as during mobile collections. although the number of local collection sites decreased from 170 in 2008 to 133 in 2017 in favor of mobile collections, which increased from 8,672 to 13,189, the former is still the number one location for donating blood. on average 47.36% of all donations were performed in local collection sites. the total number of blood donors both at the beginning and the end of the discussed period was similar (583,908 in 2008 and 588,184 in 2017); over 99% of all donors were non-remunerated. however, the number of first-time donors dropped significantly (from 237,658 in 2008 to 143,038 in 2017). the total number of donations increased from 1,076,655 in 2008 to 1,249,655 in 2017; most frequent were whole blood collections (from 1,016,411 in 2008 to 1,171,302 in 2017) . some blood components (mostly plasma and platelet concentrates) were also collected by apheresis. most frequently prepared blood components were red blood cell concentrates -rbcs (996,678-1,154,239 units per year), fresh frozen plasma -ffp (1,090,369-1,289,021 units) and platelet concentrates -pcs (81,692-129,143 units, with significant increasing tendency). additional processing methods such as leukocyte depletion and irradiation were more frequently applied to pcs (52-57.6% in respective years irradiated, 75.18-92.07% leukocyte-depleted), than to rbcs (4.46-8.46% irradiated, 7.65-20.7% leukocyte-depleted). in 2010, the pathogen reduction technologies in plasma and the pcs were implemented. up to date however the use of these technologies is limited in most btcs. in 2017 approximately 11.5% of pcs and 8% ffp units issued for transfusion were subjected to pathogen reduction technologies. summary/conclusions: our study data may contribute to the assessment of some long-term tendencies observed in polish blood transfusion service and may serve practical-benchmarking. this in turn may prove beneficial to the transfusion community as a whole. background: in poland 80% of hospitals depend on blood for the treatment of patients; over 1.5 mln units of blood components are annually transfused. it is therefore purposeful to expand the knowledge on factors impacting on blood transfusion service (bts). the institute of hematology and transfusion medicine (ihtm) as competent authority is responsible for collection of data related to the activity of all polish blood transfusion centers (bcts). this data is exploited to a much lesser degree than the recently available statistical methods and data processing tools would allow. moreover, survey of research in the field of public health indicates a negligible share of issues related to bts. it seemed therefore necessary to "fill in the gap" with true assessment of performance of the polish bcts for improvement of bts activity. 1 st stage of our investigation refers to collection, merging of data from different sources, their unification and preparation (big data) for further analysis to be performed using multidimensional statistical analysis and data mining methods. aims: assessment of the activity of the polish btcs over the 20 year-period in two stages. goals at 1 st stage: 1. data digitalization; scanning of paper documents. 2. development of a uniform template for collecting digital data from various sources. 3. standardization, unification and quality improvement of available data: filling in missing data, elimination of errors, duplicate records etc, that may distort the outcome of analyzes. 4. selection of data for analysis. methods: digitalization and big data methods for processing various types of data: a) stored in paper form (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) , b) digital stored in two file types (.doc and .xls, for the years 2005-2010 and 2011-2017, respectively). for each data-type, a separate excel file model was created. the models were then merged into one analytical table with data processing methods. results: 1. 1344 pages of paper documents were scanned. 2. models developed for data from 3 different sources: a. paper-data were rewritten and ascribed to its model; outcome -3 tables, 272 columns, 10,400 rows. b. .doc and .xls. filesdata were ascribed to 2 other models; outcome -6 tables, 1656 columns, 788 rows. 3. the 3 models were merged into 1 analytical table to create a 588 mb database (comparable to approx. 784 min of music). 4. the data was subjected to standardization, unification and quality improvement: filling in missing data, elimination of errors and duplicate records that may distort the results of analyzes. 5. selection of data for analysis at 2 nd stage. summary/conclusions: the 1 st stage provided a set of selected data for analysis in the 2 nd stage which will rely on multidimensional statistical analysis and data mining methods. the outcome of such analysis will contribute to optimal realization of objectives: a) gaining in-depth knowledge about the fundamental phenomena that shape polish bts, b) identification of potential changes bcts, c) development of overall guidelines for change management. aimed to touch untouched or less touched topics of bb&tm. so far 8 workshops were organized and each year around 30 countries were participated from almost all religious, ethnical, social, cultural and economic situations of the world. 6. supported realization of major changes in bb&tm in turkey; a convincing medical individuals and agencies mainly moh to give the deserved consideration to bb&tm b encouraging the recognition and establishment of national blood program c issuing a new blood act and numerous necessary bylaws, etc. d creating an appropriate standard donor questionnaire form e changing blood transfusion practice from %96 whole blood (at 1996) to 5%. f changing donated blood screening criteria; while anti-hcv screening became obligatory malaria, screening cancelled g preparing national guidelines h promoting haemovigilance nurse post i promoting patient blood management 7. around 11,000 blood bankers attended national courses, 7,000 attended national congresses, 18,000 attended nationwide symposiums. summary/conclusions: bbtst can be accepted as a sample how a scientific nongovernmental organization may give a very positive impact on developing and progressing bb&tm activities with close collaboration moh and other related organizations abstract withdrawn. background: globally there is growing investment in information technology (it) in business. this similar trend has been observed in blood establishment computerized systems (becs). the it investment can be high hence it decisions need to be properly informed. the africa society for blood transfusion (afsbt) encourages the use of its in african blood services as this optimize quality blood services, thereby improving patient's outcomes. afsbt established in 2016 an it working group (afsbt itwg) with the support of the swiss red cross (src) to spearhead the it standards among member blood services. a number of priority it thematic areas were identified. these includes it governance which focuses on creation (strategic alignment) and preservation (risk management) of business value. there is absence of published literature on how a structured it governance framework can be implemented in a resource constrained setting. a review of the national blood service zimbabwe (nbsz) it governance was done based on published it governance framework. aims: to explore how a structured it governance can be developed, implemented and monitored in a resource constrained setting. methods: a published mit-cisr framework which has six components was used to assess the strength, gaps and opportunities of the it governance. results: nbsz has been implementing an evolving structured it governance system. in terms of service strategy and organization there is a well-established it function which is reflected in the nbsz strategic plans. this ensures it annual budgetary support, which averages 4.3% of the total budget. the it governance arrangements are such that decision rights are assigned to different it staff (executives, it specialist, and users). a range of it solutions have been embedded within the nbsz operations such as becs, financial, donor mobile application, social media, temperature monitoring, and human resources. the business performance goals are defined and are congruent across the various business units. it organization and desirable behaviors are documented in the ict policies and procedures and were needed remedial actions are available through the code of conduct. the it metrics are included within the nbsz monitoring and evaluation system which use a four colored traffic lighting reporting system. it was noted that the it accountabilities are undesirably tilted to the it specialist only hence some ict projects tend to have delayed deliverables. the it governance mechanisms are supported with tools such as service level agreements and established communication approaches. simple excel based solutions are used to track critical performance metrics such as on the interactive blood supply management status, which averages 122.8% (2018) based on a 5-day projected stocking and supply levels. nbsz need to properly document the return on investments on all these ict initiatives, which is estimated (2016/2017) to be at 3.2% of annual savings. summary/conclusions: blood services in resource constrained settings can implement a properly structured it governance and this will ensure maximum return on it investments. the nbsz approach will be shared and further developed in the afsbt itwg to support other blood services in improving their it governance. haematology/blood transfusion, alfred health, melbourne, australia background: in october 2018, an integrated electronic medical record (emr) was implemented at an australian metropolitan multi-campus heath service using cerner millennium tm , aiming to achieve himss (healthcare information and management systems society) level 6. prior to implementation, large numbers of blood specimens were collected from patients unnecessarily and sent to pathology without a test request attached (no blood test requested -ntr). these specimens required additional processing in the laboratory. electronic specimen collection using cerner specimen collect tm allowed streamlining of specimen processing by eliminating paper requests. as part of the new workflow, individual specimen labels are printed with the specified blood test and correct tube type. this helps prevent the practice of collecting additional specimens due to uncertainty of the collection requirements. aims: • to quantify the expected reduction in ntr specimens following introduction of electronic specimen collection, and outline the benefits • to determine the impact on collection errors and wrong blood in tube (wbit) events methods: data was obtained directly from cerner millennium tm using a ccl (cerner command language) query which is run monthly by pathology it staff. this data includes all specimens registered for the month with indication of rejected specimens, wbit & ntr samples. 'rejected specimens' includes incomplete specimen and/or request certification, unlabelled specimens/requests and mismatched specimens. further information about wbit events was collated from riskman reports and staff interviews. results: data from the 12 months prior to emr implementation was compared with 3 months post. ntr numbers reduced from 4220/month to 2019/month (52% reduction), freeing up more storage space in fridges. rejected specimens due to inadequate patient request labelling reduced from a mean of 27/mth to 6/mth. wbit numbers have increased slightly: before having median 1 (range 0-2), after with median 2 (range 0-3). although it was hoped that wbit incidence may reduce with the new emr, 4 of the post implementation 6 wbits involved electronic specimen collection. departure from planned protocols involving a lack of working printers, causing staff to print patient labels away from the patient's bedside, as well as multiple patient labels printing on individual printers appear to be a main cause of the emr wbits. summary/conclusions: emr implementation has led to a reduction in ntr, and rejected specimens due to inadequate request labelling, as well as increased storage space in laboratory refrigerators. associated benefits include: • decreased financial costs of the wasted equipment • decreased staff time collecting and processing unusable specimens • decreased environmental impact of manufacture and disposal of unused specimens • decreased potential of iatrogenic anaemia work in preventing the occurrence of further wbits is ongoing, by ensuring that label printers are in working order, are in plentiful supply and easily accessible to staff; and also ensuring positive patient identification and blood collection by the patient's bedside remains a priority. jm mustaffa 1 , k teo 2 , s tsai 1 , p heng 1 , r sagun 1 and m wong 1 1 laboratory medicine 2 khoo teck puat hospital, singapore, singapore background: khoo teck puat hospital (ktph) is a 761-bed general and acute care hospital, opened in 2010, serving more than 800,000 people living in the northern sector of singapore. the blood bank of ktph department of laboratory medicine provides specimen testing and blood transfusion services for ktph as well as the neighbouring yishun community hospital (ych), one of the largest community hospitals in singapore providing intermediate care for recuperating patients including rehabilitative services. the process of ordering transfusion-related test requests in both hospitals is through printed forms. aims: in line with the hospital directive to move towards electronic patient management, the ktph blood bank intended to implement an electronic type and screen (e-t&s) system. the goal of the project is to ensure zero patient identification errors and maintain full traceability and accountability for the blood collection process in all transfusion-related testing. another aim of the system is to reduce repeated venepunctures when specimens are rejected due to missing essential patient information on the printed forms by implementing mandatory fields in the e-t&s form. methods: the e-t&s was implemented in phases. phase 1: an online version of the printed form was signed electronically by the ordering doctor and a witness within the electronic medical record system, sunrise clinical manager (scm) system with the doctor counter-checking by signing on the specimen label to ensure correct patient identification. phase 2: the ordering doctor is not required to sign on the specimen label since fingerprint biometrics are required for the electronic signin. phase 3: elimination of the witness step for blood collection. specimen collection and rejection data from 2016 to 2018 was analysed. specimen rejection rate was presented as percentage of rejected specimens (mislabelled, unlabelled and clerical errors) over total specimen count for each month. results: between january 2016 and march 2017, before the implementation of the e-t&s phase 1, the average rejection rate for blood bank specimens was 0.16% and 1.14% for identification and clerical errors respectively. during phases 1 and 2 of implementation, rejection rate increased due to unfamiliarity to the new work processes. by february 2018, with the implementation of the final phase of the e-t&s system the specimen rejection rate was 0.38% and 0.12% for identification and clerical errors respectively. rejected specimens were mostly from the few locations that had to use paper requisition due to workflow or infrastructure limitations. summary/conclusions: the e-t&s system was implemented successfully in ktph. full traceability and accountability of the blood collection process was maintained with the fully electronic system. the adoption of electronic documentation has also reduced the number of preventable repeated venepunctures that were due to incomplete order information on the printed forms. future developments in technology and full implementation of e-t&s system in all hospital locations may make zero patient identification error achievable and ensure transfusion safety in all patients in the near future. background: blood component administration represents a critical phase due to the possible occurrence of errors during the different steps from the identification of the patient to the infusion of the product. error occurrence can be reduced by the implementation of validated information systems. we tested the scweb â system at the bedside in a transfusion outpatient clinic. aims: the aim of the study is to validate a system designed to assist and to control blood administration step by step using electronic devices to ensure traceability and documentation of the process methods: the scweb â system is based on it monitored checklists which guide the personnel to follow the procedure, according to best practices; the system must initially be activated by the operator which is recognized by an auto-signing system based on bluetooth low energy which avoids the operator having to identify himself/herself beforehand. appropriate privacy protection is provided. thereafter the system takes up the task to give instructions and to verify the adherence, by asking an active confirmation of the proper fulfillment of the activities; a continuous registration and documentation is made by the system. standards and specifications for each step of the procedure have been configured on scweb â system to track in detail operator and patient identification, presence of informed consent to transfusion, blood pressure, pulse and temperature recording, vein access, verification of the blood unit. an alarm has been set after 15 min, to ensure the control of patient's conditions. for each step, an active confirmation of the action is required and nurse and doctor direct involvement must be actively confirmed on the device by both operators. the system has been tested at the bedside on 30 patients admitted to the outpatient clinic for 45 red cell concentrate transfusion; compliance of the personnel and organizational impact has been recorded. results: the system required a very short training: ease of scweb â system allows its implementation without negative impact on organization of transfusion outpatient clinic and without difficulties by operators (nurses and doctors), who appreciated the help given by the it check system. the registration of the electronic check list offered a reliable tool for the traceability of the transfusion procedure, also granting a paperless and timely available documentation of the entire process through a registration in electronic format of all the operator's action in every single phase of the transfusion process. when prescribed, confirmation of the checklist was only possible in the presence and with the active confirmation of two operators (doctor and nurse). summary/conclusions: the scweb â system is useful as a barrier against the mismatch of transfusion (preventive measure), as a traceability and documentation measure and as a tool for training of personnel in blood transfusion administration; it avoids paper registration during the transfusion process, due to the timely registration of the activities performed by operators recognized by the system thanks to the bluetooth low energy auto-signing device. the scweb â system will be connected to the transfusion data management system, to monitor all the process from the arrival of the unit from the blood bank. background: he blood banks aims at reducing cost and increasing customer satisfaction by providing quality in service. the quality in service can be attained by streamlining the processes and restructuring the supply chain of the organization by implementing it tools. aims: aim is to understand the complex flow of information and processes within the supply chain of the blood bank. the requirement of such a study is a part of the integrated erp modeling for the integrated functioning of a blood bank. methods: he approach used to understand and map the sequence of processes, and the work responsibilities of each process and the operational decisions involved at each step is process mapping and data flow diagrams for front end system modeling and analysis. the processes are mapped and represented in a schematic diagram. dfd (data flow diagram) are constructed for representing the system. a context diagram is also constructed for understanding the entities interacting with the system. the emr systems aim at replacing (or supporting) the paper based medical records. the whole model of the system is divided into two parts-front end and back end. the front end design and analysis is done using epc (event-driven process chains), resource views, data flow diagram for data view. reporting was on donor selection, finance and collection of blood bag, blood collection process, component preparation, blood testing and blood distribution results: process mapping using event driven process chain generated a whole view of the processes involved. the resource view gave an organizational structure and the personnel involved. the data view using context diagram and data flow diagram gives a flow of data and amount of data involved. this framework can be used for business process reengineering for the blood banks by conducting a time study and removing non value added activities. data view helps analyze redundant data in each process. it also helps in staff training and orientation within the department. summary/conclusions: a systematic overview presented in this paper facilitates in removal of non value added processes, duplication of data, bottlenecks, reduction of cycle time and thereby improving service quality in blood banks. background: the transfusion of blood components, one of the most prevalent interventions in clinical practice is a major expenditure item in healthcare services which tend to increase in recent years. aims: it is intended to investigate the impact of transfusion associated costs to hospital costs in pediatric intensive care unit (picu) patients. methods: during a year period (january 2017-december 2017) 76 patients, 40 females and 36 males receiving transfusion with blood components along the stay in picu were included in the study. transfusion associated costs and total costs for healthcare services for children treated in picu was collected by using hospital information system (his). statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). mann-whitney u test and kruskal-wallis test was performed for comparison of independent categoric variables and numeric data; chi-square analysis was performed for comparison of two numeric variables and spearman correlation analysis was performed for associations. results: the median age of patients was 12.0 months (interquantile range-iqr 26). the median length of stay was 16 days (iqr 30). in total 400 blood components were transfused in which of 227 red blood cell concentrates, 114 apheresis platelet concentrates, 6 granulocyte concentrates, 51 fresh frozen plasmas, and 1 cryoprecipitate and 1 whole blood. the ratios of transfusion associated expenditures to hospital costs were categorized in intervals of percentages as < 5%, 5-10%, 11-15% and > 15%. most of the patients (63.2%) were ranked in the lowest interval. the medians for hospital cost and transfusion associated cost were 5478.76 euros (iqr = 11280.02) and 130.57 euros (iqr = 354.86), respectively. a significant strong positive correlation between numbers of transfusions and hospitalization cost of picu was detected (r: 0.674, p < 0.01). while it was found a significant weak positive correlation between transfusion associated cost and hospital cost (r: 0.247, p = 0.032) there was also a significant weak positive correlation between the age and transfusion associated cost (p = 0.048, r: 0.227). a significant difference was found between the patients with and without hematological malignancies (p < 0.01) for transfusion associated cost. the reason why pediatric dosages are mostly prefer is that the hospital provides healthcare for only children and splitting of the blood components was common in the hospital. but unexpectedly a significant increase on the transfusion associated costs which is related to split blood components was detected (p < 0.05). summary/conclusions: studies on the economics of blood transfusion have been conducted mostly in patients who require chronic or multiple transfusions. picus, specialized facilities that provide care for patients with severe life-threatening diseases are major departments often necessitate multiple transfusions. there are many variables to evaluate the impact of transfusion associated cost to hospital cost in picu patients, but the major factors are underlying conditions, admitting diagnoses and transfusion strategies. although there are unexpected data in our study demonstrated the increasing impact on transfusion associated cost originated from blood components split for pediatric usage no significant relationship was determined to explain this situation. further studies on the economics of blood transfusions have to be carried out to clarify the variables of transfusion associated costs. background: approximately 55.7% of the transfused blood component is packed red cell (prc). over ordering of prc unit is a common practice and excessive pretransfusion testing was being wasteful of resources and have adverse consequences on cost. high crossmatch to transfusion (c/t) ratio as quality indicator of blood bank implies that crossmatches were performed unnecessarily. aims: the aims of this study were to evaluate the cost effectiveness of strategies for limiting the number of pretransfusion testing of ordering prc. methods: all prc units who ordered from dr. hasan sadikin hospital from january 2016 to december 2018 were collected in this retrospective study. number of ordering prc unit, completed pretransfusion testing of ordering prc units, and prc units that were transfused were recorded. restrictive pretransfusion testing strategies were done based on the hemoglobin level and diagnosis as transfusion indication criteria. cost effectiveness was measured by multiplying the unit cost of pretransfusion testing and number of prc unit. results: out of total 177,370 ordered prc unit, 166,910 (94.1%) were subjected to pretransfusion testing and 63.1% (105,260) of ordering prc unit which are pretransfusion testing were transfused. this means that 5.9% (10,460) of ordering prc unit were not subjected to pretransfusion test. this showed savings of 1,098,300,000 rupiah. c/t ratio was 1.6 which demonstrate a good ordering pattern. however, 16.4% (27,451) of completed pretransfusion testing of ordering prc unit were not transfused, leading to blood bank loss of 2,882,355,000 rupiah. summary/conclusions: strategies for limiting the number of pretransfusion testing on the good c/t ratio was still associated with saving cost effective background: blood is a precious resource for saving patient lives. the purpose of blood and blood component therapy is to provide suitable and safe blood products to achieve best clinical outcomes. nurses have an important role in ensuring safe blood transfusion. it is crucial for nurses to have sufficient knowledge about blood donation and collection, storage, component preparation, possible adverse effects of blood transfusion and necessary management and care. aims: the aim of this study was to assess the impact of an educational intervention on knowledge and awareness of nurses regarding blood transfusion services and practices. methods: the baseline study to assess the knowledge and awareness regarding blood transfusion services and practices of the nurses posted at various areas of the hospital including wards, operation theatres and critical care areas was carried out at our institute hospital which is a tertiary care teaching centre. the nurses were then sensitized and educated regarding blood transfusion services and practices during their day to day activities by referring them to the blood transfusion guidelines of the institute. subsequently, a self-developed questionnaire which was used for the baseline assessment of knowledge and awareness of the nurses was again used to reassess them. a total of 19 questions were included in the questionnaire pertaining to: general awareness (two questions), blood donation (two questions), testing and blood component preparation related (two questions), storage of blood/blood components (two questions) and pre-transfusion checks and bed side transfusion practices (eleven questions). fifty nurses each were included for both the baseline as well as post-sensitization assessment. for different category of questions, the correct response rates were compared with those obtained in the baseline study using mann-whitney test. the entire study duration was spread over a period of three months (december, 2014 to february, 2015 . results: the overall mean percentage of 'correct' responses for 19 questions in the baseline study was 61.75%, whereas post sensitization it was 77.21%. the mean percentage increase in general awareness related questions was 21.49%, 13.95% for storage of blood/blood components related questions, 17.37% for pre-transfusion checks and bedside transfusion practices related questions, 21.33% for testing and blood component preparation related questions and 27.27% for blood donation related questions. the percentage increase in correct response was found to be statically significant for each of the five categories of questions. the overall mean percentage increase in correct response rate was also statistically significant (p < 0.001). summary/conclusions: this study revealed that after sensitization and educational intervention there was a significant improvement in the knowledge and awareness of nurses regarding the blood transfusion services and practices. abstract withdrawn. background: tact, introduced in the uk in 2014 to support managers, provides resource-saving, continual, 'real-time' monitoring of knowledge-based competency of staff in transfusion laboratories. tact is available online 24/7, complementing existing practical competency schemes and external quality assessment. multiple variations on a standard pre-transfusion testing scenario are generated using constrained randomisation; logic rules for automatic assessment of sample acceptance, abo/d, antibody screen and identification (as/id), and component issue are based on bsh guidance. during 2018, tact was offered internationally to transfusion laboratory managers to trial, and saw uptake in five countries. the core tact programme, based upon uk guidelines, is under review for programming conversion, to be customisable for the international community. aims: to assess the feasibility of tact programming conversion to meet the requirements of country-specific pre-transfusion testing guidelines, and to direct future programming in line with feedback from international users. methods: guidelines from 3/5 international users were obtained and translated where necessary. these were compared against the core assessment elements of current tact programming. international users were approached for their feedback on the current version of tact, as it compared to their local policies and practices. results: the following criteria were cross-referenced: specification of transfusion request forms, sample label acceptance criteria, reagents used for abo/d and as/id, resolution of grouping anomalies, alloantibody confirmation/exclusion, and selection criteria of blood components for transfusion-dependent patients and women of child-bearing potential. apparent differences included:-australia:--selection of red cells for patients with immune anti-d. greece:--inclusion of the name of the patient's father on the transfusion request. italy:--testing of all new patients with an anti-a,b reagent and two different monoclonal anti-d reagents. international users in the same three countries supplied feedback. this included suggestions for:-greater complexity of cases presented, provision of patient history, inclusion of follow-on tests e.g. phenotyping and cells for antibody confirmation/exclusion, broader range of reaction strength grading, and official professional cpd credits. the following differences were noted:-nomenclature used, the format and content of the request form, use of english abbreviations of patient clinical details, and the availability, provision and specification of blood components. summary/conclusions: this analysis has shown very few instances where the current tact iteration differs from the guidelines reviewed, and that it is feasible to expand the use of tact on a more international basis. the current iteration of tact has been developed to represent an abbreviated scope of pre-transfusion testing practices, which can be applied to laboratory practice outside of the uk without difficulty. further work is required to enable international users to configure tact such that the system represents all laboratory practice on an international basis. aims: these courses provide education to clinicians on patient blood management and safe transfusion in neonatal and paediatric settings in order to improve patient outcomes and increase awareness of the national patient blood management guidelines. this analysis aimed to investigate the uptake, practical use, and perceived value of the courses by learners. methods: a retrospective analysis of course completion statistics and course evaluation data. results: there have been 2,376 paediatric and neonatal courses completed from 6 march 2018 to 28 february 2019 with 89.6% of learners being nurses and/or midwives. analysis of course evaluation data (n = 33) showed that these courses: -provide knowledge (96.9%) -improve patient safety and outcomes (84.9%) -result in change to clinical practice (69.9%) -are relevant to clinical practice (70.9%) -are easy to use (67.7%) -are readily accessible (58.1%). examples that learners provided of how they can apply this learning to their clinical practice include: -"[i am now] more aware of special requirements for neonatal blood transfusion" -"[i] feel more confident especially when talking with parents" -"[i will now be] checking the patient's blood results and will speak up for unnecessary blood sampling" -"[it's good that] when there is ambiguity in clinical practice [this is] very well shown by explanation from experts in the field" -"we don't do a lot of transfusions [and this is] a reminder that transfusions are not always the first answer to the baby's clinical picture". summary/conclusions: analysis of course and user evaluation data demonstrates that these courses are being used by nurses and doctors working in the neonatal and paediatric settings and that they provide knowledge of pbm that can be applied to clinical practice, thereby contributing to improved patient care. background: blood transfusion is a high-risk clinical activity that must be mastered both theoretically and practically in order to guarantee the required result without any incident or complications. the mastery of transfusion knowledge among nurses represents a very important link in the transfusion chain. the objective of this work is to compare the theoretical and practical knowledge of transfusion among two groups of nurses divided according to their seniority. aims: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. methods: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. results: the participation rate in the survey was 100%. the 2 nd group participants had an average seniority of 9 years . more than half of them (64%) had seniority of less than 10 years. only 12% had more than 20 years of experience. the rate of correct answers for all items combined was 44.4% for students versus 42.5% for practicing nurses. the theoretical knowledge part was more mastered in the 1 st group than that of practicing nurses (44.8% vs 33.4% of correct answers). on the other hand, the control of the transfusion act was better in 2 nd group (44% vs 50.5%). the overall "dangerous" response rate was 47% for students and 41.7% for practicing nurses. false practical knowledge was more common in group 1 (59.5% vs. 41.5%). summary/conclusions: the theoretical as well as the practical knowledge remains not well mastered by the care staff. our study highlighted the best theoretical mastery for young students and practical for practicing nurses. this could be explained by the freshness of knowledge in the first group and the daily practice in the second group. background: the european commission (ec) directive 2004/33/ec on blood donor selection criteria is 15 years old. in the meantime, knowledge on risks related to blood donor selection has progressed and challenged several obligatory rules. transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of more than 24 stakeholders from both not-for-profit and private organizations providing substances of human origin (soho). the project aims to provide evidence-based donor selection criteria and guiding principles for risk assessment of threats to the safety of soho. as part of this work, an inventory of current blood donor selection criteria in europe and an evaluation of the evidence behind current practice was performed by experts working on this project. aims: to identify the gap between the ec directive 2004/33/ec on whole blood donor selection criteria and a current evaluation of the clinical relevance of the criteria based on scientific literature by a panel of european experts within the transpose project. methods: in 2018, we performed an inventory of blood donor and transfusion recipient risks in participating european countries. project members were asked to provide the existing donor selection criteria related to these risks and to carry out a risk-based evaluation for each of them. the evaluation was based on the available scientific literature and on a risk assessment template based on the abo risk-based decision-making framework, developed by transpose. all risks with divergent assessments within the panel were resolved through discussion; in all cases an expert consensus was established. subsequently we compared the results with the content of the ec directive 2004/33/ec for every risk, thereby identifying discrepancies and missing items in the directive. results: the panel identified 64 risks considered to be significant, distributed between donors and recipients. for 35/64 (55%) of them the expert evaluation deviated from the content of the ec directive, or the ec directive provided no information about the decision making. in particular, a discrepancy was observed for 9/20 criteria concerning general health and medication, 12/22 for transfusion transmissible infections, 10/13 for high-risk behaviour and travel, and 4/9 for other diseases. summary/conclusions: our results highlight a significant gap between the whole blood donor selection criteria stated in the ec directive 2004/33/ec and the scientific evaluation performed by a panel of transpose participating experts. this gap includes both new risks not addressed in the ec directive and addressed risks that are however evaluated differently. this involves both blood donor and transfusion recipient safety, and various medical and epidemiological topics covering several aspects of the blood donation criteria. we strongly recommend a change in the european legislation, allowing a procedure to guarantee that blood donor selection criteria are updated regularly within the framework of the european institutions, to keep aligned with scientific progress, epidemiology and changes in medical practice, in order to enable an updated risk-and evidence-based framework for donor selection criteria. the risk-assessment method elaborated in the transpose project is a valuable instrument for this purpose. background: the brazilian health regulatory agency -anvisa has developed the method for assessment of potential risk in hemotherapy services (marpsh) which is based on the data collected during the inspections of blood services carried out by regulatory authorities. using marpsh any blood service can be classified in one of 5 possible potential risk categories: high, medium-high, medium, medium-low and low risk. each category represents a different potential risk level, according to the proportion of compliance with the established regulatory requirements. marpsh has been used since 2007, showing a trend of risk reduction on blood services evaluated all over the country. aims: this work aims to describe the utilization of marpsh as a tool for an integrated risk management model. also, it shows the main results obtained after 10 years of data monitoring and coordination of regulatory actions and policies by anvisa, targeting quality and safety of blood products. methods: the utilization of marpsh follows a network risk management model since the inspections are carried out by decentralized organs in all 27 states and some municipalities. the inspectors fulfill a standardized inspection guide containing the regulatory requirements, where each item is associated with a level of risk, varying from i to iii as the risk increases. at the end of the inspection, after a statistical calculation, the service is categorized. this classification gives an estimate of its quality profile, guiding the adoption of suitable measures for risk management by local authorities and services. these data are send to the states (if realized by municipalities) and to anvisa that perform consolidation in a national level. either states or anvisa use data to coordinate risk management measures in a broader spectrum. data are continuously monitored by anvisa as part of its strategical panel of indicators. anvisa follows up specially blood services in high and medium-high risk with the aim of helping or complementing local authorities' actions. additionally, anvisa periodically sends this information to the brazilian ministry of health and local governmental organs from brazilian national blood system that also support actions to improve quality in their blood services networks. results: since 2007, when the assessment covered 109 blood services, marpsh reached 1218 blood services in 2017 (57% of the blood services registered) what corresponded to almost 100% of the inspection cover in this year. over this period (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) it is possible to notice a dramatic decreasing in trend for proportion of blood services classified as high and medium-high risk, varying from 26% to 9%. summary/conclusions: marpsh generates data necessary to the categorization of blood services into five levels of potential risk. as a result, the regulatory actions are applied by local organs with the purpose of reducing or eliminating risks involved in the production and use of blood components. data have shown a significant risk reduction over 10 years of marpsh's utilization. additionally, monitoring of this data at a national level has been permitting appropriate planning and prioritization of integrated strategies directed to risk management and strengthening of blood services in brazil. background: sub-saharan africa has the highest need of blood transfusion in the world, mainly for childbearing women and children suffering from malaria. meeting basic quality and operational requirements to provide patients with safe blood remains a challenge in these settings. aims: the aim was to identify and prioritize potential hazards for patients in blood bank practices in the democratic republic of the congo (drc). we focused on two subsets: (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products, using failure mode effect analysis (fmea). methods: two risk analysis workshops were organized at the national blood transfusion centre in kinshasa, the democratic republic of the congo. in both workshops, a multidisciplinary team was invited to represent different hospitals and profiles in transfusion management in drc: quality coordinators (n = 2), training coordinator (n = 1), medical doctor for donor selection (n = 2), hemovigilance officer (n = 1), laboratory technicians performing donor sampling, blood qualification and production (n = 7), biomedical scientists (n = 3), microbiologist (n = 1), clinical biologist (n = 1), nurse (n = 3). the principle of fmea was applied, which implies identification of possible hazards (failures) in a process flow, followed by scoring each hazard according to their impact, probability, detection and feasibility. in the both workshops, participants were guided by an external facilitator who guaranteed common understanding of the methodology. main focus of the risk analysis was potential harm to the transfused patient in the process of (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products. all ideas were written on coloured cards and mapped on a chart according to their impact, probability, detection and feasibility score. hazards were ranked according to their final risk score by multiplying these four scores. results: in the process of sensibilisation and selection of donors, the three hazards with the highest final score for impact, probability, detection and feasibility were: (i) a paid donor is recruited after sensibilisation by family members of the patient, (ii) donor selection staff approves a non-eligible donor for blood donation because of personal/financial motivation, (iii) blood donors are not correctly informed about blood donation during sensibilisation by family members of patients. in the flow of qualification of blood products, highest scores were made for: (i) no double check for validation and sorting of qualified blood products, (ii) stock-out of reagents, (iii) no check for match between registered test result and tested blood tube. regarding production of blood products, the top three consisted of: (i) transport time between blood collection and processing is >8 h, (ii) storage of qualified and quarantined blood products in the same fridge (some sites only), (iii) power cuts. summary/conclusions: the risk analysis resulted in three prioritized hazards in the process of donor selection/sensibilisation and blood product qualification/production. some are very specific to the sub-saharan african setting and have been described before (power cut, family and paid donors, stock rupture,. . .). an action plan needs to be put in place to reduce their final risk score. the risk analysis needs to be continued for the remaining blood transfusion flows. background: 19.3 million of germany's population, so just under a quarter of residents, have a migration background. the majority of these has roots in regions where the population has a distribution pattern of blood group and hla-antigens that differs considerably from the predominant one in the german population. sufficient supply of these individuals with red blood cell (rbc) and platelet concentrates (tc) will continue to be a major challenge in the future, as blood donors with compatible blood group antigens are dramatically underrepresented in the local donor pools. many migrants suffer from severe hematological disorders such as b-thalassemia or sickle cell disease and will not only need compatible blood transfusions, but an allogeneic stem cell transplantation in the foreseeable future. as healthy family donors often are not available, at present suitable stem cell donors with a similar genetic background can only be found in international donor registries. aims: this project was initiated to recruit new donors with a migration background for blood donation and to increase the number of blood stem cell donors among this group. methods: serological extended blood group phenotyping was performed by automated gel card technique (fa. grifols, erytra) and included ab0, rh (ccdee), kk, fy (ab), jk(ab), lu(ab), m, n, s, s. hla typing for hla-a, -b, -c, -dr, -dq, and -dp was performed by next generation sequencing. allele frequencies were analysed using genepop version 4.2; the rare and very rare alleles were defined according to the allele frequency database (www.allelefrequencies.net) rbc genotyping using next generation sequencing is currently being established and will include additional antigens with the most frequent distribution pattern differences between migrant and resident populations according to literature. results: so far, more than 8800 blood donors with a migration background have been recruited for a blood donation in this project. amongst this group, over 1000 blood donors from more than 20 non-european countries enrolled as potential stem cell donors. an initial evaluation of the data revealed a very similar distribution of blood groups compared to the current blood donor population in north rhine-westphalia. of 600 migrant donors, ten fy(a-b-) donors were identified, which corresponds to a percentage of 1.6%. amongst 509 hla-typed potential stem cell donors, we found 28 (5.5%) with rare and very rare alleles. summary/conclusions: blood donors with rare blood group and hla phenotypes (e.g. null types such as fy(a-b-)), are in demand for adequate medical care of people with a migration background. the technological development of blood group determination by next generation sequencing will significantly improve the supply for all blood transfusion recipients in germany. this project is funded by the european development fund 2014-2020 (erdf) and the european union. background: mortality due to uncontrolled haemorrhage following trauma is the most important cause of potentially preventable deaths. trauma care systems in low and middle income countries like india, are still in developing phase. also, the role of blood component therapy in improving patient outcomes has been mostly derived from combat settings. application of these protocols in an urban setup has not been well established and marked variation in practice exists. hence this study aims to identify the key components of transfusion practices to optimize the transfusion protocol in trauma settings. aims: to study the current transfusion practices in severely injured trauma patients, admitted to the red area/resuscitation bay after initial triage in ed methods: this prospective observational study was conducted over a period of 1 year starting from june 2017 to may 2018 at the department of transfusion medicine in collaboration with emergency department at jpnatc, aiims, new delhi. the study included severely injured patients (iss ≥ 16) that were admitted within 24 h to the red area/resuscitation bay after triage. data collected included the demographics, injury, laboratory and transfusion details for these patients results: during the study period 885 patients (83.5% males) were enrolled. mean iss scores was 21.89 . mean time to hospital admission after injury was 9:03 (iqr 3.38-13:48) hours. mean time to first rbc transfusion following admission was 2:09 (iqr 0:27-2:45) hours. approximately 49.3% (436) patients were in shock (sbp < 90 mm hg &/or pulse rate > 110/min). whereas, 160 (18.1%) patients were coagulopathic (pt ≥ 1.5 times of normal). during initial 24 h of admission, these patients were transfused with 2929 (69.7%) rbc, 1986 (51.8%) ffp, 2327 (42.9%) rdp and 384 (81.5%) cryoprecipitate of total blood components utilized for these patients. massive transfusion (defined as transfusion of ≥ 5 units/4 h) was given to 190 (21.4%) patients. summary/conclusions: significant quantity of blood components were required during initial resuscitation in severely injured patients. pre-hospital transfusion can significantly reduce the time to transfusion. further studies are needed to assess utility of pre hospital transfusion in severely injured patients. background: allogenic stem cell transplantation recipients are known to be the main consumers of platelet concentrates (pc). the geneva university hospital is one of the three allogeneic hematopoietic stem cell transplantation (hsct) centers in switzerland. since the blood center is also part of the hospital, data of pc consumption are easily available. as needs rose steadily since several years, with an average increase of 9% per year, pc supply is a serious concern for our center. aims: in this study we tried to evaluate if any pre-transplant indicator could help to forecast the number of pc needed after an allogeneic hematopoietic stem cell transplantation. methods: this observational retrospective study was conducted in geneva hospital on 78 patients suffering from various inherited or acquired disorders of the hematopoietic system who were treated by hsct in 2016. pc consumption was examined from january 2016 to december 2018. the five indicators were: gender, stem cell source (bone marrow (bm) vs peripheral blood stem cell (pbsc)), donor type (hla matched (10-8/10) vs haploidentical), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient. results: data for a total of 78 patients aged from 3 to 74 years were analyzed; 48 (62%) were male and 30 (38%) female; 35 (45%) were cmv-negative and 43 (55%) were cmv-positive. out of a total of 78 transplants, 14 (17.9%) were haploidentical and 64 (82.1%) hla-matched. according to the stem cell source, bm was transplanted in 22 cases (28.2%), and pbsc in 56 cases (71.8%). two patients also received a cd34 + stem cell boost. our analysis showed that, with a mean follow-up of 652 days, the number of pc transfused to our patients treated by hsct ranged from 0 to 383 units, with an average of 37 and a median of 15, illustrating a high variability. the results indicated that gender, stem cell source (bm vs pbsc), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient do not have any statistical impact on platelet consumption. however, we observed a tendency of an increased need for platelet transfusion when patients were cmv positive. our results also showed a statistically significant (p = 0.034) higher number of pc transfused for patients treated with a haploidentical (89) versus hla-matched (26) transplant. summary/conclusions: this study points out the high variability of platelet consumption after hsct, which limits the forecast of platelet production needed to support allogeneic hsct recipients. a larger cohort would be required to confirm a potentially higher platelet consumption in cmv positive patients, and to consolidate our results showing a higher pc consumption for patients treated with haploidentical transplant. abstract withdrawn. background: historically at our institution, a minimum of four red blood cell (rbc) units were crossmatched for all cardiac surgery cases regardless of surgical case-type or patient characteristics. two rbc units were packed in validated blood product coolers and brought to the operating room (or); the balance of crossmatched units remained in the blood bank. a retrospective review revealed that very few rbcs were transfused (2016: 20% (371/1813), 2017: 19% (322/1742)). moreover, approximately 8 products were wasted each month as a direct result of this practice. thus, we recognized an opportunity to improve inventory management in terms of personnel activities and blood component utilization. aims: the goal of this study was to reduce advance preparation of coolers in cardiac surgery cases without compromising patient care and safety. we limited our intervention to those patients who were eligible for electronic crossmatch. we maintained the aforementioned historical practice for those patients with history of and/ or those who currently demonstrated clinically significant red blood cell alloantibodies. methods: a multidisciplinary group consisting of representatives from the blood bank, cardiac surgery, cardiac nursing, cardiac anesthesia and surgery quality department was assembled in october 2017 to determine whether a modification of practice was reasonable and safe. group members evaluated site specific society of thoracic surgery (sts) cardiac surgical data between july 2014 and december 2016 to establish intraoperative red cell transfusion rates classified by type and urgency of surgery. the group's main goal was to discontinue preparation of default coolers for patients eligible for electronic crossmatch who were scheduled for all types of non-emergency cardiac surgery cases in which ≤ 25% of historical cases required at least one red cell transfusion. additionally, team members simulated the multiple protocols by which red blood cells could be prepared and delivered to the or and estimated the time for each scenario. results: review of sts data showed that the following cases met the criteria of ≤ 25%: elective primary coronary artery bypass graft (cabg), urgent primary cabg, elective mitral valve repairs, and elective aortic valve replacements. simulation showed that, in patients eligible for electronic crossmatch, preparation from receipt of order to completion of unit packing for delivery took 2.5 min using the pneumatic tube system (maximum of 2 units per tube) and 4.5 min using delivery of a cooler using a human courier. summary/conclusions: based on the simulation results, and with consensus agreement from the multidisciplinary group, default cooler preparation for elective primary cabg, urgent primary cabg, elective mvr, and elective avr was discontinued in december 2017. one year following implementation of the change in policy 940 rbc units were issued to the or (a 54% reduction); 38% (361) were transfused, compared to 19% in 2017. wastage rates decreased from 8 products a month to 1 per month on average. summary/conclusions: the most obvious drawback of pabd is the higher cost in running the program in comparison with collection of allogeneic blood in the areas of additional patient attention and clerical input in labeling, separate storage and so on. in this audit, 34% of the autologous blood components were not transfused into the intended recipients and wasted; in this context, the pabd program could not be considered as a cost-effective approach in protecting blood safety. background: the national blood service zimbabwe (nbsz)'s blood supply management status (bsms) is an integral process of ensuring the availability of a safe and sufficient blood supply provision. nbsz introduced a new daily blood bank statement with improved metrics from 1 may 2018. the new analytics approach focuses on three interactive components of the blood bank statement; the available stock, quarantine stock (as per the desired 5-days stocks level), and the demand versus supply. it is imperative to have a closely monitored blood supply chain because blood has limited shelf life with uncertainties in both supply and demand. the 'blood-for-free' proclamation by the government of zimbabwe in july 2018 set more pressure on the blood demand. these metric-based analytics seek to assess if the nbsz's improved blood bank statement is a realistic model for the bsms. aims: to assess the use of the interactive metrics in monitoring the blood supply management status. methods: a prospective cross-sectional study was conducted. a total of 704 daily blood bank statements which were submitted between may and december 2018 from each of the five branches were analyzed. the bsms which is calculated as the average of the three interactive measures of quarantine stock, available stock and demand versus supply was determined. sub-analysis of branches was done to determine individual branch performance. analysis by month was done to assess seasonal variations. findings and recommendations were shared among key stakeholders to validate the bsms methodology. results: overall the quarantine stock average was 130.4% (sd +/-101.6), the available stock was 142.3%: (sd +/-118.8) and the demand versus supply was at 95.6% (sd +/-11.3).the overall bsms was 122.8%; (sd +/-77.2) for the study period. gweru and masvingo nearly supplied all the demanded blood with 99.2%, overall bsms of 118.1% and 99.7%, overall bsms of 144.3% respectively. bulawayo supplied 98.3% of the blood demanded with an overall bsms of 99.1%. mutare supplied 97.8% with a bsms of 180.1% and harare 85.6% and a bsms of 78.0%. there were monthly variations but the service could supply above 90% of the blood demand. in the month of may the service met 91.6% of the demand and a bsms of 87.8%. in november and december it supplied 92.6%, bsms of 100.8% and 92.8%, bsms 131.8% respectively. august also had a below average supply of 93%, bsms -98.2%. june, october and september recorded above the average values; 97.3%, bsms of 126.8% and 98.7%, with a bsms of 117.2% respectively. summary/conclusions: the overall bsms performance was satisfactory and it was noted that branches capacitated according to demand. the new interactive analytics approach is appropriate for showing the blood bank status and assessing the performance of the branches. this new approach has optimized the decision-making process in blood supply management. the metrics are tracked using excel based model hence this approach is suitable for resource constrained settings with limited ict infrastructure . st vincent's hospital melbourne (svhm), a tertiary hospital supporting medicine, surgery and non-major trauma emergency and itu services implemented a mtp in 2008. subsequent mtp reassessment has led to implementation of regular multi-disciplinary review of all mts to identify areas for improvement in transfusion and other aspects of support for critically bleeding patients. aims: to implement a systematic service-wide stakeholder review of mt events at svhm aiming to identify deficiencies and implement improvements in mt management. methods: a multi-disciplinary mt review team was established as a subcommittee of the hospital transfusion committee (tc) to update the organisational mtp in 2016 and subsequently continued to meet quarterly as the mt review subcommittee (mtrs) of the tc, systematically reviewing all aspects of mts at svhm. instances where 4 or more red cell units are transfused in <4 h are identified from the laboratory information system and reviewed by the mtrs which includes representatives from accident and emergency, intensive care, operating suite (os) and transfusion laboratory staff; the head of the patient's treating unit is also invited to contribute. reviews include: demographics, clinical details, comorbidities, time from patient arrival to pre-transfusion specimen collection/receipt, time from blood request to release/transfusion, regularity of full blood examination (fbe)/coagulation (coag) testing, timing of blood component transfusion, total component provision/ratios, component waste, patient outcome, and communication between various clinical areas and also the laboratory. a discussion summary with actions/ recommendations is provided to the tc and some cases referred to the hospital mortality/clinical review committee. results: cases reviewed: 65 from 10 treating units including cardiothoracic surgery (18) hepatobiliary/gastrointestinal/colorectal surgery (24), vascular surgery (6), neurosurgery (4), orthopaedic surgery (3), endocrine (2) and "other" (encompassing general surgery, urology, general medicine and oncology -8). areas for monitoring/improvement identified: transfusion documentation, regularity of fbe/coag specimen submission, reducing time between patient arrival and specimen collection, reducing specimen transport time, interfacing point of care bloodgas analysers to the central pathology result management system as well as component management/waste reduction and the introduction of viscoelastometry assessment in the os. 18 of 65 reviewed cases involved the transfusion of emergency uncrossmatched o rhd negative red cell units. the appropriateness of the use of this precious resource is also reviewed by the mtrs. summary/conclusions: the svhm mtrs meets regularly to review mt events and formalise multidisciplinary collaboration in identifying possible improvements to support these often critically ill patients. matters highlighted include communication issues, delays in specimen delivery and blood component waste minimisation. areas for further work include minimising delay between mt events and review, and formalisation of key performance indicators for mts. background: the use of radio frequency identification (rfid) technology to manage the blood supply chain is recognized as a major enhancement to the operations of blood banks and hospital transfusion services. to facilitate optimal blood supply management, it is crucial to guarantee the integrity of rfid tags throughout the transfusion chain. since rfid tags can be affixed to blood products very early in the process, these tags undergo the same process-steps as the blood products themselves (e.g. centrifugation, label printing, shock-freezing and irradiation). aims: the goal of this study was to validate the mechanical and functional resistance of biolog-id rfid tags through different blood related processes: centrifugation, label printing, shock-freezing, intensive reading at à40°c, and irradiation. biolog-id tags are passive hf (13.56 mhz) tags. they are compliant with is0 15693, iso 18000-3 and follow the guidelines for the use of rfid technology in transfusion medicine (vox sanguinis, 2010). methods: biolog-id tags were evaluated using a series of rfid encoding and reading tests. before each of the processing steps, each tag was encoded with donation number, site id, product code, blood group and expiry date. the data was encoded using the isbt 128 format. the different processing steps and conditions tested were: -centrifugation: quintuple whole blood bags, filled with 450 ml water. centrifugation at 4,500 rpm for 10 min. 270 tags processed, 15 tags per kit affixed at different positions. -shock-freezing at à80°c: shock-freezer (angelantoni, sf40), 50 units processed, reading immediately after removal from shock freezer. water, 30 tags irradiated at 30 gy and 30 tags at 50 gy results: all biolog-id tags were encoded and read with a 100% success rate in all series of tests. summary/conclusions: biolog-id rfid tags can be encoded and read through common processes used throughout the blood transfusion chain. their mechanical and functional integrity is not affected by centrifugation, shock-freezing, intensive reading at à40°c, printing, eto sterilization and irradiation. background: the provisioning of compatible red blood cells by international cooperation is presented. the units were meant for an 18-year old female, with homozygous sickle cell disease (scd) and multiple complications. patients' blood group was a positive with anti-c, -e, -wr a and an antibody to a high prevalence antigen in the rh system, anti-hr b possibly combined with anti-hr b (rh34). the antibody was not reactive with rh null , -d-or hr b negative cells. the donor center put out an international request for group a or o, rh null or -d-units lacking wr a and possibly k, fy a , jk a , wr a , do a and s (the latter antigens for prophylactic matching). the patient sample had been genotyped for rhd and rhce using mlpa and sanger sequencing and the patient was found to carry rhd*01/rhd*03n.01 and rhce*cevs.01/ rhce*cevs.03. aims: the request was sent to the american rare donor program (ardp). the ardp working with the american red cross national molecular laboratory, used the rh genotype information to identify donors carrying the same or similar rh variant alleles using the rh allele matching approach described previously (keller et al. transfusion 2013 53(2s):174a). methods: a recent blood sample was used to confirm anti-hr b ; no anti-hr b was detected. the patient rhd and rhce alleles were used to build punnett squares for both genes with donors carrying the same and similar alleles that would be predicted to be compatible. tier 1 donors are those predicted to carry the same combination of rhd and rhce alleles as the patient. tier 2 donors are those predicted to be homozygous for one of the allele combinations carried by the patient. tier 3 donors are those predicted to carry alleles similar (but not identical) to those carried by the patient, with similar predicted phenotype. the database of donors in the ardp carrying rh variant alleles was queried against the alleles in the patient-specific punnett square. results: donors of group a or o and matched for rh alleles were identified as follows: 102 tier 1, 357 tier 2 and 100 tier 3 donors. after the clinical team agreed to drop one or more of the prophylactic antigen matches, one tier 2 unit lacking s and jk a was identified at the american red cross. while the request was being processed, the patient experienced a sickle cell crisis, red cell aplasia and recurrent aiha and her hemoglobin level dropped from 7 to 1.9 g/dl. at that time, she was transfused the only compatible units available -2 of the rare -dphenotype and her hb increased to 3.8 g/dl and eventually to 7 g/dl. the tier 2 rh allele matched unit was shipped to amsterdam where it was frozen, and reserved for the transfusion care of this patient. summary/conclusions: this case illustrates how rh allele matched blood can be found for a highly rh alloimmunized patient, and can avoid use of the exquisitely rare -d-or rh null blood. background: blood transfusion has been a complicated and high-risky clinical procedure. any error could cause serious injuries to patients. to better assure the procedure safety. aims: we enhanced and built a blood transfusion database platform and develop inventory management strategies to better guarantee the patient transfusion safety. methods: we designed six new features of the platform (1) assuring the patient identification with barcode techniques; (2) designing a structured order entry; (3) proactively reminding the physicians with patient's previous blood transfusion reaction with related precautions including the use of leukoreduction filter; (4) automatically reminding physicians the happening of reaction and suggesting relevant test; (5) building a complete traceability log system; and (6) supporting data analysis. the blood transfusion safety team includes medical technologists, nurses, physicians, system analysts, and blood transporter and the whole process is electronic management. the new blood transfusion platform integrated the workflow, reduced the incidence of abnormal blood samples collected (0% after implementation, p < 0.01), reduced the time of call for medical technologists with blood component preparation and improved the achievement rate of emergency 30-min blood crossmatch (98.1% after implementation, p < 0.05). the barcode correctly identified patients and monitored the entire transfusion process to reduce the error rate of blood component supply (0% after implementation, p < 0.01). summary/conclusions: after the transdisciplinary team approach with e-monitoring and a better design of clinical decision support module with barcode technology, blood transfusion database platform improve the blood supply efficiency and assure blood transfusion safety. background: in the modern world, terrorist acts are characterized by a multiplicity of combined injuries to a large number of victims. qualified medical care is urgently required for a large number of patients in one locality at the same time. it leads to increase in emergency demand for blood components, mostly red blood cells. the desire to donate blood to the victims is a natural manifestation of society's solidarity in response to tragic events. however, donor activity and patient needs do not always correlate. aims: to analyze the donor activity during the terrorist attacks. methods: a retrospective analysis of donation activity in periods of terrorist attacks in moscow (2004 moscow ( -2011 . the average daily blood donations' number (dbdn) before ta compared with the number of donations in day after ta and with the dbdn during 7 days after ta. also the number of delivered rbc units (d-rbcu) daily before ta and daily in 7 days after were compared. results: in 2004-2011, 5 terrible ta occurred in moscow: 141 people died and more than 629 were injured. with the explosion in subway in 02/2004 42 people died, 250 were injured. the number of d-rbcus increased by 10% on ta-day, and by 30% during next 7 days. dbdn in the 1st day after ta increased 6,7 times, and in the next 7 days -1,6 times. second explosion in subway in 08/2004 resulted in 10 died, 50 injured. the number of d-rbcus increased by 80% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 1,1 times, and in the next 7 days -2,2 times. in 2006 (explosion on market) resulted in 14 died, 61 injured. d-rbcus delivery increased by 20% on ta-day, and by 10% during 7 days. dbdn in the 1st day increased 2,0 times, but decreased to 0,6 times during the next week. with subway explosion in 2010 40 people died, 88 were injured. the number of d-rbcus increased by 10% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 6,5 times, and in the next 7 days -1,6 times. with the explosion in airport in 2011 35 people died, 180 were injured. rbcus delivery increased by 50% on ta-day, and by 40% during next 7 days. dbdn in the 1st day after ta increased 6,1 times, and in the next 7 days -2,0 times. summary/conclusions: an increase in donor activity is observed already the next day after ta and usually lasts for 7 days, but does not correlate with the number of victims. the rbcs' delivery from blood bank increases in all cases on the day of the ta. therefore, the guarantee for patients is the maintenance of rbcs' stock, including cryopreserved ones. it is also necessary to promptly send excess of red blood cells harvested at the peak of activity to the cryobank. background: rh system is the major blood group system besides abo system. even after proper blood grouping and cross matching there is a possibility of alloimmunisation in recipients against the rh or minor blood group antigens like kell, mnss, duffy etc. in medical colleges which cannot bear the financial burden of complete phenotyping of patient and donor, implementation of rh & kell phenotypes match blood transfusion can play a major role in preventing alloimmunisation and adverse events in multitransfusion patients aims: to evaluate the efficacy of rh & kell phenotyping as a cost effective measure instead of extended phenotyping in multitransfused patients methods: study was carried out in the department of transfusion medicine, one of the biggest blood bank of the country with annual collection of 70,000 blood units. 2000 patients of thalassemia, aplastic anemia and leukemia were taken who required multiple transfusions. complete phenotyping was done initially of all the patients before transfusion. 1000 patients were taken as control and the other 1000 were taken as cases. 2482 blood units of healthy donors were chosen (2442 were males and 40 were females). in all the donor units, identification of rh & kell phenotyping was done by the antigen antibody agglutination test by the erythrocyte magnetize technology on fully automated immunohaematology analyzer qwalys. these blood units were transfused to 1000 patients who had been selected as cases. in the control group, patients were transfused blood units which were not phenotyped for rh & kell but gel crossmatching was done. follow-up was done on these patients for transfusion reactions and at the end of six months they were evaluated for any alloimmunisation. results: at the end of 6 months, no reactions were reported in cases receiving rh & kell phenotype blood and no alloimmunisation was seen on repeat phenotyping. the control group on the other hand reported reactions in 5 cases (0.5%) and phenotype at the end of three months showed alloimmunisation with 'e' antibody. the phenotypic frequencies of rh & kell blood groups in the population were comparable with other published studies. amongst the rh antigens (e) was the most common (99.44%) followed by d (95.45%), c (89.65%), c (53.1%) and e (17.65%). thus 'e' was the most common and e was the least common of all the rh types. background: the prevalence of a particular blood group has an uneven distribution in different geographic areas and is largely determined by the national composition of the population. moscow is one of the largest city of europe with population of 12.5 million. the understanding of prevalence of red blood cells antigens (rbc-ag) among the population has great importance for blood banking planning. aims: to determine frequency and distribution patterns of transfusion-significant rbc-ag among donors in the moscow region. methods: the results of immunohematological studies on ab0, rhesus and kell systems were analyzed retrospectively in 352362 blood donors for 14 years (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) in moscow. data collection and processing was carried out using the regional information system for transfusiology. rbc-ag detection (ab0, rh, kell) systems was performed using microplate method (automatic immunohematological analyzer "galileo neo" (immucor, inc., usa)) and "ih-1000" (bio-rad laboratories, usa) with diagnostic cards. results: the most frequent blood group is a (ii) 35.8%, 0 (i) blood group 34.3%, b (iii) 21.3%, ab (iv) 8.6% (n = 352362). rh(d+) was established as positive in the presence of antigen d and as rh(d-) negative in its absence. donors with weak variants of antigen d (du) were determined as rh (d+) positive. the ratio of rh (d+) and rh (d-) was 82.8% and 17.2%, respectively. donor's phenotype detection was routinely conducted from the 2013 year, therefore the number of donors was 152883. the most common phenotype among donors ccdee (32.61%), the second in frequency ccdee (19.33%), the third in frequency rhesus negative phenotype ccddee (15.42%) in the studied population. the ccdee and ccdee phenotypes were 14.04% and 12.17%, respectively. the most rare are ccdee (2.65%), ccdee (1.86%), ccddee (1.54%). other options: ccddee, ccdee, ccdee, ccddee, ccddee, ccdee, ccddee were detected in single cases and amounted to a total of 0.37% (n = 152883). cw antigen was tested in 104230 donors and was detected in 5.28%. cw is most commonly found in donors with ccddee phenotypes (2.36%), ccdee (2.03%) and ccdee (0.79%), with other variants of the data phenotype, the antigen was detected in 0.1% of the examined (n = 104230). antigen k was detected in 6.8% of donors, in 93.2% of this antigen is absent (n = 352362). summary/conclusions: the study of transfusion-relevant antigens distribution in population is necessary for building of effective and flexible model for blood service managing. a differentiated approach in choosing a strategy to form a long-term bank for storing blood components, taking into account the frequency of various antigen variants, contributes to improving the quality, accessibility and safety of medical care. of dislocated division of our blood establishment in orthopaedic hospital valdoltra (obv) in 2014 the number of outdated units at the hospital side dropped considerably. results: since 2008 when the issued number of red blood cell units (rbc) was 5170 the amount of issued units rose to 5678 in 2011 and then dropped more or less steadily to 4850 in 2018. in this period the hospitals' programmes rose for 10% in all areas. number of donated units declined from 6330 in 2011 to 4820 in 2018. after reorganization in 2009 the number of outdated units fell from 3% of stocked units to 1.5%. after setting a dislocated unit of ctdiz on obv location the number of discarded rbc fell from 397 to 41 in 2018. for transfusion specialist who is constantly in contact with the clinician in the hospital the most important day routine is when the stock availability is displayed. it happens 4 times a day; at 10 a.m. when the previews' day collection is released and another three times a day when the updates occur. the central base is led in ljubljana (the capital) and all centres are able to control and order the stock for the blood banking. blood wastage remained low and the traceability of the blood usage in south-western region remains high (99%). though it is not supported by an informational system the traceability form the blood bank to the patient is done on paper. this issue demands a big effort by the staff in blood bank and in hospitals. summary/conclusions: reorganization enabled better stock utilization and traceability of issued units. sometimes it is impossible to predict the peak demand of rbc especially during the summer season when the population of the area doubles and car accidents as well. transfusion specialist's effort in assuring the optimal blood stock represents the crucial daily routine. blood bank, grande international hospital, kathmandu, nepal background: voluntary non-remunerated blood donor consists of 78% blood donor's population in nepal. therefore demographic about the distribution of blood donors according to the age group is important to achieve 100% voluntary nonremunerated blood donors in nepal. aims: to explore the demographic distribution of the blood donor in different age group in the kathmandu nepal. methods: this is retrospective study conducted at nepal red cross society central blood transfusion service. data from january 2013 to january 2017 were collected from donor management software. the data includes socio demographic data. data has been process with spss version -17 results: during 4 years study period, total of 276,290 blood donation happened from both mobile blood collection and in-house blood collection. out of 276,290 collection, 48351 (17.5%) are from 18-24 age group; 106924 (38.7%) are from 25-31 age group 53324 (19.3%) are from 32-38 age group; 34536 (12.5%) are from group 39-45 age group; 23761 (8.6%) are from age group 46-52 and 9394 (3.4%) from age group above 53 respectively. summary/conclusions: the distribution of abo blood group varies regionally and from one population to another. in kathmandu, nepal 18-38 years age group is the most common age group encountered donating blood. the data generated in the present study and several other studies of different geographical region of india will be useful to health planners and future health challenges in the region. background: the information system on hemovigilance sihevi-ins©, coordinated by the national health institute was available in 2018 to all blood banks in the country. this software allows to centralize and record the identification data of a donor, its infectious and immunohematological tests, as well as the fractionation and final destination of each blood component obtained from a donor. aims: to describe the abo and rhd typing discrepancies in blood donors found in the blood group variables registered by each blood bank to sihevi-ins©. methods: retrospective analysis of the information registered by 80 of the 81 blood banks authorized nationwide between january and december 2018. results: sihevi-ins© received information of 854,462 accepted donors, 33% of them with more than one donation in the same blood bank in a period of 12 months. a total of 72 abo or rhd discrepancies were identified in 69 people, who made donations in 20 blood banks (estimated risk: one discrepancy per 11,876 accepted donors). five of the blood banks implicated in these discrepancies are hospital-based (annual average collection of 6,086 ae 3,785 units, representing 3.6% of the national collect). the remaining blood banks are distributors (average collection: 31,389 ae 25,704 units per year, representing 44% of the national collect). 75% of blood group typing discrepancies (n = 36) were related to the abo group. the most common discrepancy was between a typing group and ab typing group (67%) . in 25% of the cases, the same blood bank initially registered in the same donor, an o blood type donation and later an a blood type (n = 10) or b type (n = 4). rhd typing discrepancies account for 25% (n = 18) of the total. additionally, in three donors, a simultaneous discrepancy between abo and rhd typing was detected in the same blood bank. the results could be due to: a) failure in the warning mechanism before the release of the blood component; b) errors in typing the information of the donor registered in the system or c) failures in the identification of the donors at the time of selection. the above shows risk in the process of control of blood components release, which can impact patient safety unless abo and rhd typing blood groups are systematically verified before transfusion. summary/conclusions: despite blood banks have a verification and validation process through software to release blood components, flaws were detected. although sihevi-ins© is not a software to validate the information before the release of blood components, it was through this program that abo and rhd typing discrepancies were identified in donors who attended the same blood bank multiple times. this finding implies increasing the controls that should be used in each blood bank, to avoid lose traceability of the processes and to put at risk the life of the recipients. blood donor testing department, blood transfusion institute of nis, nis, serbia background: the ability to automate blood grouping and antibody detection procedures is a requirement for blood donor testing laboratories. mistakes in the sample identification and testing procedures could be prevented by testing on automated immuno-hematology systems. irregular antibody screening and abo/rhd grouping of blood donors are tests performed routinely in blood transfusion institute of nis. neo iris (immucor, usa) is a fully automated instrument for the abo and rh d grouping using microplate hemagglutination technique and antibody screening and identification using solid phase red cell adherence (sprca). aims: evaluation of the automated neo iris system for abo and d grouping and irregular antibody screening of blood donors in blood transfusion institute of nis. methods: during the evaluation period a total of 4417 edta-anticoagulated samples for abo and d forward and reverse grouping using microplate anti-s, 1 anti-s, 1 anti-jka and 6 anti-k. in one case ih-1000 failed to identify anti-c antibody in very low titer in sample with anti c+d antibody presence. in two samples (0.045%) false-positive result were observed both on ih-1000 system and neo iris and in two cases (0.09%)only on neo iris due to nonspecific reasons. summary/conclusions: abo/rhd grouping results obtained on neo iris system, using microplate method, have a good correlation with results on ih-1000 system as our routine column agglutination method. for antibody screening and identification neo iris showed high sensitivity for detection of clinically significant antibodies which is important step for increasing blood transfusion safety. background: continuing improvement of laboratory quality to provide accuracy test results for precise diagnosis and treatment is the mission of advanced laboratory. immunogenetic testing for histocompatibility including human leukocyte antigen (hla) typing, hla antibody detection and cytotoxicity test is critical for diagnosis and evaluation of transplantation and prognosis monitoring. in order to improve the quality of experiment competency, an external quality assurance schemes with review and education per year program was established and performed during the period from 2017 to 2018 in taiwan. aims: the proficiency testing (pt) held semiannually from 2017 to 2018 were reviewed to investigate the outcome of competency improvement of laboratories participated in the program. methods: the test items in the exercises were classified into 4 groups, hla genotyping (including pharmacogenetics hla typing), cytotoxicity test, hla and platelet antibody. the methods of hla genotyping include ssp (sequence specific primer), sso (sequence specific oligonucleotide), sbt (sequence based typing) and either ssp+sso or ssp+sbt were used, the methods of hla antibody including elisa, flow cytometry and luminex were used and the methods of platelet antibody including sprca and elisa were used. there are four shipments of exercise materials in two years and each shipment include two positive and one negative samples for antibody detection, two each of whole blood and serum for cytotoxicity of t and b cell and three whole blood for hla genotyping. aims: this study aims to survey transfusion related laboratory tests for the quality improvement of hospital's blood bank management. methods: we analyzed survey results of 11 kinds of routine work categories of 841 blood banks that were registered on korean association of external quality assessment service. blood bank worker voluntarily replied this electronic survey. the 11 categories were as follows: 1. characteristics of institution 2. the equipment of blood bank 3. the kinds of tube in blood bank 4. the present kinds of blood bank tests 5. abo and rh type tests 6. the cross-match tests 7. the irregular antibody tests 8. hemovigilance system 9. other blood bank tests 10. massive transfusion protocol 11. quality control issues we analyzed and compared each category data according to considering characteristics of hospitals. results: there were consensus and some differences of current blood bank tests. we presents the result of a pilot survey. especially the cross-match tests were divided by saline phase method added with irregular antibody tests or completion of 3rd step anti-human globulin phase according to institutional environment. automated typing machines or automated irregular antibody test devices were more increased in large-scale hospitals than small-scale hospitals. different kinds of tubes were used such as edta tube for abo and rh typing, plain tube for cross-match test. the retention segments of rbc were reserved for minimum 7 days. most blood bank were registered and regularly listed up transfusion events to korean hemovigilance system for safety transfusion. also, a lot of institution have none or underdeveloped massive transfusion protocol. more specific survey results will be analyzed in further poster presentation. summary/conclusions: this survey will show the current status of transfusion related blood bank test. this institutional blood bank comparison will be helpful to assess the currency of individual blood bank environments. abstract withdrawn. background: we know that quality management is a continuous process, involving implementation, maintenance and improvement. aims: our purpose is to show our experience in implementing the quality management system in the whole institution and our first steps in achieving the jacie accreditation in the stem cell collection facility in order to provide our patients and donors the best possible care. methods: the institute for transfusion medicine of the republic of macedonia (itm) is the main institution in charge of blood transfusion service (bts) in the whole country, which is the national unified system. the stem cell collection facility is a part of the itm. this facility is operational since 2001 year with 844 collections of stem cells (686 in patients and 159 collections in sibling donors) till now. we are obtaining the implementation and maintenance of qms through the establishing of the iso standardization for the whole institution (itm), as well as of implementing jacie standards in the stem cell collection facility. the two of our colleagues became the jacie inspectors and the standard operating procedures (sops) were developed, followed by regular meetings, trainings and self-evaluation of the personnel. we asked for the orientation visit from the independent jacie inspector in order to come one step closer to the jacie accreditation and to improve our overall qms. results: the institute for transfusion medicine of rm was a part of the ipa project "strengthening the blood supply system". this project aimed to ultimately bring the blood transfusion service to european union standards allowing the exchange of blood components and all other types of collaboration with other european union countries in future. the project put the basis for unification of blood transfusion standards and operating procedures in the whole country as well as set up essential education of blood transfusion personnel. although a lot of strengths were found during the orientation visit from jacie inspector, there are still a lot of areas for improvement. our strengths are motivated team and supportive institutional leadership including macedonian ministry of health. areas for improvement are: labeling of cellular therapy products and lack of laboratory for quality control. there is a national regulatory framework in place and who and world bank initiatives in macedonia which support quality in health care and accreditation. summary/conclusions: our institution has in plan to implement isbt 128 standards for labeling of cellular therapy products and to establish a laboratory for quality control of cellular therapy, as well as to meet all the requirements to become jacie accredited facility. working by standards, following the rules and regular self-evaluations will help us to maintain the strong quality management system. every institution will benefit from a quality management system that brings you into line with international standards. ensuring the quality of our services and products is essential to keep safe and strong blood transfusion service. background: implementation of robust quality assurance program is key to high performing blood establishments. quality control and quality assurance systems together constitute the key quality systems and are parts of quality management. effective and efficient quality control policies not only provide guidance that help to increase the reliability of results but also maintains the laboratory's consistence performance overtime. aims: therefore, we established a set of qc limit using historical data which can timely identify unexpected variation in the testing systems and trigger a review of test processes in blood screening laboratories as part of quality assurance system. methods: last two consecutive years (jan, 2017 to dec, 2018) qc data from archi-tect i2000sr (abbott laboratories, chicago) was extracted using abbottlink for philippines red cross tower national blood center total of 6532 data points (3523 data points in 2017 & 3009 data points in 2018) were obtained for 10 different qc levels for four serological blood screening assays (hiv combo, hbsag, anti-hcv, syphilis). the data was sorted for each assay/lot and qc level combination by year. qc limits were calculated using simple mean, standard deviation (sd) and coefficient of variation (cv%) and were validated and compared with manufacturer's recommendation. results: all the six positive quality control levels cv% (5.70-12.05) were within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2018. five out of six positive quality control levels cv% (5.72-15.80) showed within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2017 except syphilis tp positive control (15.8%). all four negative quality control levels showed the sd values within 0.009-0.41 in 2017 and 0.009-0.41 in 2018 respectively. summary/conclusions: excellent qc performance was observed in philippines red cross tower national blood center blood screening laboratory based on historical data and evidence-based laboratory qc limit for blood screening assays were established using historical data which takes into account total variation expected in a test system and offers a more robust and meaningful mechanism for setting control limits, for the first time. background: quality indicators (qi) in transfusion medicine (tm) are 'critically important aspects of transfusion medicine practice that are measured and utilized to gain insight for continuous quality improvement, into the degree to which the tm is capable of providing quality tm care, products or services for the aspect of practice measured following comparison of the measurement against acceptable local or international reference thresholds, benchmarks, standards, or practice guidelines'. the critical control point (ccp) selected for this study is 'administration techniques and monitoring of key elements'. this has been selected since the clinical fraternity plays a larger role in ensuring quality services in administration of blood components. there was a need to follow up compliance to standard protocol for bedside transfusion practices hence was decided to study the same with four selected quality indicators and introduce corrective measures if necessary. aims: 1. to assess the existing transfusion practices in the institute with specific quality indicators 2. to introduce corrective reforms to improve the existing practice 3. to assess the transfusion practices after interventions using the same quality indicators methods: to assess the existing transfusion practices in our centre, 295 transfusions were prospectively followed up with a structured checklist. the quality indicators used were (i) verification of blood components prior to transfusion (ii)initiation of transfusion within 30 min of release from the blood bank (iii) close observation of transfusions for the first 15 min (iv)completion of transfusion within the right time frame for each component. as a corrective measure, a transfusion monitoring format was designed which was distributed in every ward and the nursing officers were informed to monitor and document transfusions using that. in addition, the blood bank staff was made to call up the wards and ensure that the transfusions of every component had been initiated within 30 min of issue. transfusion practices were once again monitored by following up 306 transfusions using the same quality indicators. results: there was significant difference in all the four variables between the two phases. 57.3% transfusions were verified in phase i while 73.6% were verified in phase ii (p < 0.001). 69.5% transfusions were started within half an hour of issue while in the second phase, it rose to 83.1% (p < 0.001). 47.8% transfusions were observed in the first 15 min in phase i and 88.6% were observed in the second phase (p < 0.001). in phase i, 54.6% transfusions were completed within right time while the same in phase ii was 73.9% (p < 0.001). summary/conclusions: we recommend the following as quality indicators for bedside transfusion practices: background: antibody titration consists in performing antibody detection with selected red cells of different sample dilutions. the titer is reported as the reciprocal of the highest dilution that induces macroscopic agglutination. the usual applications of titration are prenatal studies and complex antibodies identification. some publications have demonstrated that more variation in antibody titer and titration score are noted upon repeat testing of the same sample when testing was performed in tubes as compared to repeat testing in gel. aims: to evaluate the efficacy of automated antibody titration versus manual method by using gel microcolumn technology. methods: edta-anticoagulated whole blood donors' and plasma frozen samples containing a known irregular (rh, kidd, duffy, mns, etc.) and regular (a1 & b) antibodies were selected. the titers of 126 samples were determined in parallel by using grifols analyzers (erytra and erytra eflexis) and compared versus grifols gel manual method by using grifols gel microcolumn technology and grifols red blood cell reagents. sixty of these also processed in parallel in erytra and erytra eflexis analyzers for comparison. for the precision study, 12 of these samples were tested in the 2 automated systems for 5 times (120 datapoints for each analyzer) on different testing days. the hands-on (manual intervention) average time required to complete a titration was measured (3 expert technicians) in 2 different sample workload (1 and 10 samples testing). these results were compared with the same number of independent titrations performed in grifols analyzers. for the walk-away time, 2 different sample workload (1 and 10 samples testing) were assessed in manual method (3 expert technicians) and compared to timings obtained when reproduced in grifols analyzers. results provided by analyzers were reviewed and compared to manual method. results: titer obtained by erytra or erytra eflexis was equivalent to the titer obtained manually (differences ≤ 1 titer: 73% ≤0.5 titer). the results proved that both instruments were equivalent in performing titration (differences ≤ 1 titer; 85% ≤0.5 titer). the precision results showed no difference between titers obtained through the 100% of the runs performed with the grifols analyzers (differences ≤ 1 titer: 75% ≤0.5 titer). the manual hands-on in automated system was reduced in a 15% compared to manual method for 1 sample. when the number of samples was increased (10 samples), the difference in hands-on in was even more reduced (65%). in addition, the walk-away was 46% higher in automated system compared to manual method. furthermore, when the number of samples was increased (10 samples), the walk-away difference was increased even more (78%). finally, automated system software demonstrated to increase the standardization of the test as all samples, results and reagents traceability were automatically managed. summary/conclusions: grifols gel system including erytra and erytra eflexis analyzers provided a scalable and efficient solution to perform standardized titrations in the immunohematology lab. the study proved that using grifols gel system, titrations can be run in an automated reliable way (less than one-fold differences versus manual gel), thus reducing at least 15% the hands-on, increasing at least 46% the walk-away, rising the standardization and automating all testing traceability. , and fourth case of use (results) scenarios, tasks were considered "very easy" by 30%>50% of users and "easy" and by 50-80% of users; 90%>100% of the users considered "sufficient" the design to ease the interaction; and 60%>80% of users never founding any situation of not knowing how to proceed. for the fifth case of use (user roles), 40% of users considered tasks "very easy" or "easy"; 40% of users considered "sufficient" the design to ease the interaction; and 40% of users never found any situation of not knowing how to proceed. for the sixth case of use (maintenance plan), 100% of users considered tasks considered "very easy" or "easy"; 90% of users never found any situation of not knowing how to proceed; and 100% of users considered the maintenance plan similar or better than other instruments. reliability analysis ( background: quality control procedures in blood group serology for reagents, techniques, personnel working and automated equipment are essential for the accuracy of the laboratory results. the observation of high number of uninterpreted results during blood donor grouping was a motive for investigation and possible targeting the problem. aims: to identify blood group interpretation problems by analyzing the testing results obtained with the commercial quality control samples routinely used during blood grouping. methods: a microplate (mp) system for performing abo and rhd, as well as rh phenotype and kell blood group determination with two automated analyzers techno (1 and 2) and correspondent two mp-readers lyra (1 and 2) using maestro software from diamed is currently in use for blood donor typing. three types of mp are being used such as: a, b, ab, dvi-, dvi+, ctl/a1, b profile for first time donors, then a, b, d ctl for repeat donors and finally, the c, c, e, e, k, ctl profile. the accuracy and safety of the blood grouping results is ensured by using the diamed q.c. system which consists of 4 + 2 tubes of whole blood and 2 tubes containing serum with known specific antibodies. we analyzed and compared the interpretation of the q.c. whole blood samples' results from both of the analyzers after a new optic camera was installed on the techno 1/lyra 1 system. ward to ward. methods: a prospective observational pilot study was done for around 140 prbc unit issues which were followed in real time for understanding the tat within blood bank & from ward to ward. as per the definitions, the areas where the times are documented perfectly are understood and considered for calculations. based on the conclusions of pilot study a monitoring form has been designed and utilised to monitor the tat within bb & wtw. the data is analysed monthly and an avg tat for bb & wtw is calculated. the common causes of delay in providing the blood components were analysed and strengthened to both reduce & control the tat. results: in pilot study, total wtw tat averaged to 90 min, with highest time taken 795 min, where there were additional processings like leukodepletion, irradiation, saline washing of red cells and holding the transfusion. lowest wtw tat was found to be 10 min where there was a prior information for crossmatch. after the surveillance form has been started, the average time taken for wtw tat came down to 70 min, maximum being 310 min (jan 2019), the areas where delay happened were identified as internal courier delays, technician delays, billing & other logistics delay. the concerned staff are put on regular training to maintain the tat. summary/conclusions: although ethically all the staff work for providing better care for patients, there will be few areas that delay the life supporting blood transfusion. monitoring using tat surveillance forms help in avoiding the delays and hence provide better & timely transfusion support. blood donation -blood donor recruitment p-059 hematological and physiological characteristics of regular blood donors with beta-thalassemia traits background: according to recent evidence, the physiological variability observed in the hematological characteristics of regular blood donors (linked -in certain caseswith genetic factors or the donor's lifestyle) may affect red blood cell (rbc) storage lesion. beta-thalassemia heterozygous (b-thal-het) blood donors represent a group of particular interest because of a) the high frequency of thalassemia mutations in specific geographical areas b) the physiology of the b-thal-het rbcs, which predisposes towards more effective management of storage-associated stress. aims: the goal of the present study was the comparative examination of the hematological and rbc physiological features of regular blood donors with or without beta-thalassemia traits before blood processing for transfusion purposes. methods: 204 healthy blood donors of greek origin (18-24 years old), who met the blood donation criteria were recruited in this study. plasma/serum (uric acid, electrolytes, extracellular hemoglobin, antioxidant capacity), cellular (rbc indices) and biological parameters (corpuscular fragility, proteasomal activity etc) were measured. the results were statistically analyzed and topologically represented in biological networks for both donor groups (+/-b-thal-het). significance was accepted at p < 0.05. results: b-thal-het represented 9% of the donor cohort. no differences in lifestyle (smoking, alcohol consumption, physical exercise) were observed between the two groups. nevertheless, regardless of sex and sex-dependent parameters (e.g. hct, hb concentration), b-thal-het demonstrated: a) reduced hct, mcv and mch (11% p = 0.039, 26% p = 0.008 and 30% p = 0.006, respectively) and b) increased rbc count (20%, p = 0.042) compared to the average donors. moreover, mpv platelet index was found slightly elevated (p = 0.053) and serum total protein concentration slightly reduced (p = 0.054) in the same group. a trend for higher plasma antioxidant capacity (p = 0.052) was evident in the group of b-thal-het, in addition to statistically significant lower levels of osmotic fragility (by 13%, p = 0.022) and hemolysis (by 41%, p = 0.001) compared to controls. finally, analysis of the three proteasome-associated enzymatic activities (n = 10 per group) in the rbc cytosol and the membrane, revealed similar levels in the two groups (p > 0.05). the b-thalhet and control biological networks showed insignificant variations in respect to the amount of connections and their hub profiles. however, differences were observed regarding the number or type of connections, or even their topology in the network, in the cluster of lipids (triglycerides, ldl etc), nitric oxide, clusterin, carbonylated plasma proteins and rbc osmotic fragility (correlated with the concentration of electrolytes selectively in b-thal-het donors) between the two groups. summary/conclusions: b-thal-het who meet the criteria for blood donation are a non-negligible sub-group of the total donor population in greece. they exhibit several similarities to the general cohort, but differ in fine characteristics of rbc physiology, including resistance to hemolysis and extracellular antioxidant capacity. the differential network profile of hematological and redox parameters may be important in respect to the subsequent blood processing and storage of b-thal-het erythrocytes for transfusion purposes. background: blood service in poland is based on voluntary and non-remunerated donations. regional blood donor centre in poznan as well as other regional centres (total of 23) are the only entities authorized to collect, process, store and distribute blood and its components to hospitals in the region of their activity but they are also responsible to provide sufficient amounts of blood and its components. regional blood donor centre in poznan is one of the largest blood centers in poland with the total number of donations exceeding 100,000 per year. in the recent years we have observed a growing popularity of tattoos among various age groups as well as among people registering to donate blood (first time and repeat donors) hence, it is critical to introduce suitable measures to ensure the safety of blood and its components. aims: the aim was to analyse the correlation between the increasing number of donors deferred from donating blood due to having tattoos made and the number of recorded confirmed hcv infections and the effect it may have on the safety of blood and its components. methods: the analysis was made using the data for the years 2010-2017 obtained from the computer system 'blood bank' which is in operation in regional blood centre in poznan, poland. we have analysed the total number of deferrals of donors due to recently acquired tattoo and the total number of recorded confirmed hepatitis c infections. we must note that the category of temporary deferrals due to tattoos is a broad one: it includes so called regular 'artistic' tattoos, permanent make-up procedures as well as medical tattoos. results: we have recorded a significant increase in number of deferrals due to tattoos from 400 in 2010 to 716 in 2017 (+79%). in the group of male donors this trend remained rather stable with a slight decrease: from 181 in 2011 to 151 in 2017 (à16.6%). in the group of female donors the growth was more prominent: from 219 in 2010 to 565 in 2017 (+158%). in terms of the recorded confirmed hcv infections a downward trend can be observed: from 63 in 2010 to 16 in 2017 (à74.6%). in the group of male donors from 43 in 2019 to 12 in 2017 (à72%), in the group of female donors from 20 in 2010 to 4 in 2017 (à80%). summary/conclusions: as we can conclude from the analysis the applied policy of temporary deferrals of donors with recently acquired tattoos (in the last 6 months) proves to be a reliable method of increasing the safety of blood and its components. nevertheless, the current conduct of the qualification of the donors which requires a 6 month deferral following the new tattoo must be complemented by various and numerous educational activities regarding the means of hcv transmission (and other bloodborne viruses such as hbv, hiv) and ways of protection from possible infections. special emphasis must be put on the group of female donors as the growth of deferrals was more prominent among them. at the same time it is vital to ensure for constant availability for all donors of well designed, concise educational materials (hard copies on the premises, articles, infographics, downloadables etc. on the website). background: a temporary deferral has a negative impact on donor retention, with many donors failing to return at the end of their deferral period. anecdotal evidence collected by the australian red cross blood service suggested that many donors do not know when they are eligible to return to donate, suggesting that a reminder message may be effective at promoting donor return once the deferral has ended. aims: the aim of this study is to evaluate the effectiveness of a reminder message on the return rates of deferred donors at the end of their deferral period. this reminder message notified donors that their deferral period was ending and encouraged them to make an appointment to donate. this study also aimed to determine the most effective time to send the message, message content, and mode of communication (sms vs email) in optimising donor retention post deferral. methods: three separate randomised controlled trials were conducted to answer these questions. data on donors' attempted return behaviour and subsequent deferrals, appointments and donations made one month after the deferral end date were collected and analysed. results: overall, 18.3% of donors who received a reminder message attempted to return compared to 12.8% of donors in the control group (p < 0.05). looking at each time point, donors who received the message 1 week before their deferral ended were 64% more likely to attempt to return compared to the control group (p < 0.05). the 1 week prior reminder message was particularly effective with males, with 30.3% attempting to return to donate, compared with 25.2% of females (p < 0.05). there were no significant differences in the return rates of donors who received the recipient versus non-recipient focused message, or donors who received the message via email or sms. summary/conclusions: a reminder message sent to deferred donors at the end of their deferral period is a simple, cost-effective way to promote donor retention, providing clear information regarding the date on which the donors can return to donate as well as a prompt to make an appointment background: our challenge is to provide 100% voluntary donation for safe blood, thus taking into account the current history of family donation, promotion of blood donation, level of awareness and voluntary donations from various institutions, the opinion of 1000 interviewees will give us a clearer idea of what we want to achieve and what needs to be improved in the future. aims: 1. provide 100% voluntary donation for safe blood. 2. establishing a special department within the national blood transfusion center responsible for marketing and promotion of voluntary blood donation. methods: this study was conducted as a combination of qualitative and quantitative methods. the study was a combination and identification of existing data, direct interviews with persons of different age groups, preparation and dissemination of questionnaires and analytical processing of the collected information. the study questionnaire with 60 questions in total was divided into 6 sections out of which 14 questions on blood practices were answered by all 1000 interviewees. 416 people answered 9 questions on the blood transfusion service. 5 questions on blood knowledge were answered by 270 people. 5 questions on the knowledge of the blood transfusion were answered by 220 people, 17 questions on blood donation were answered by 420 people and 10 questions on the communication channels were answered by 500 people. results: out of 1000 interviewees, 19% have never donated and did not intend to donate, due to the fact that most of them were afraid of needles and infections, while the smallest part didn't donate blood because it was not allowed by the religion, 40% did not donate, but expressed the readiness to donate in the future, 10% have donated voluntarily only once, 24% were family donors, 3% regular volunteer donors, and 4% have donated voluntarily several times and did not want to donate anymore. from those who have donated, 59% have donated for one of their relatives, 28% have donated for thalassemic children, 10% have donated to benefit free check-up and 3% have donated because it was valuable for their health. the question as to whether they would voluntarily donate again, 57% have answered yes, 22% no and 21% were still not sure. this means that donation of those who have donated once did not leave a positive impression, did not increase the desire to repeat the donation once again, rather it has restrained or made it unsafe for them to repeat donation. among the causes mentioned by the interviewees were bad conditions in the donation facilities, staff behavior, inadequate treatment, they did not feel good after donation and had hematoma at the venipuncture. summary/conclusions: based on the results obtained from the study, the national blood transfusion center needs the establishment of a genuine promotion department where there is a need for a transfusion doctor who should be an active part of it. the national blood transfusion center should build up and implement a rigorous retention policy for voluntary blood donors, as the study found out that around 60% of donors who have donated once would like to donate again. their attraction through a donor retention policy will surely lead to self-sufficiency with safe blood. the safe blood is a public good and for this reason it is the duty of all state instances, the media and non-governmental organizations to give their support in the promotion of voluntary blood donation. background: smoking, unhealthy diet, sedentary behavior and inability to maintain adequate exercise have significant consequences for several chronic disorders, including obesity. a balanced and equilibrate nutrition may prevent the negative consequences associated to the status of obesity. in italy, overweight and obesity is increasing with 3 adults of 10 overweight and 1 of 10 obese in 2017 with a higher frequency in the south. blood centers can play a public health role in obesity surveillance and interventions. aims: since the quality of life, self-reported by the patient, related to health and adequate quali-quantitative nutrition, are becoming necessary and relevant in the field of nutrition, we conducted a demographic study to evaluate the health status of the blood donors by monitoring the nutritional habits and lifestyle. methods: a descriptive cross-sectional face-to-face questionnaire was developed. it included a 41 item dietary assessment, reporting semi-quantitative food frequency, dietary behavior and questions on self-rated health status. normal weight was established with bmi < 25 kg/m 2 , overweight with a bmi ≥ 25 and < 30 kg/m 2 , and obesity with bmi ≥ 30 kg/m 2 . obesity prevalence was standardized by sex. donors were repeat blood donors, who had made at least 3 donations in the last 2 years, and were eligible to donate. results: of the 2468 blood donors enrolled between july 2017 and january 2018, 1390 were regular repeat donors, 1102 did not wish or chose not to respond at survey for several reasons (i.e. lack of time or privacy) and 288 accepted, of which 83 were deferred from blood donation and were excluded from the analysis. among the 205 included participants 68.3% (n = 140) were male, age ranged from 19-61 years with a mean age of 39.8 ae 11.1 sd and 31.7% (n = 65) were female age ranged from 20-62 years with a mean age of 37.7 ae 11.0 sd. data showed that donors followed mainly a mediterranean diet and had more awareness to lifestyle, women more than men, in comparison with general population. the prevalence of overweight was found 50.7% in men and 16.9% in women. our survey showed that 84.4% of the participants evaluated their health as "good", without gender difference (men, 86.4% vs women, 80.0%). besides, 14.6% reported their health as "very good". summary/conclusions: overweight and obesity are common among regular blood donors and it is more frequent in men than women. our preliminary data showed that women have a better knowledge of the nutritional properties of food and consequently adopt a more balanced and proper diet. furthermore, it is clear that they are aware about the relationship between lifestyle and health putting into practice their information. unfortunately, the survey structure, of observational nature, does not make it possible to establish whether women are more alert to health to participate more in donation programs or if, on the contrary, the status of regular donor could help the improvement of knowledge and healthy lifestyle. background: donor recruitment pose an ongoing challenge to blood banks worldwide. one approach to improve the effectiveness of donor recruitment is to target influencing factors. a yearly league is conducted at the sultan qaboos university (squ) to encourage university students and faculty to donate blood. during this, the colleges are evaluated based on different measures including the number of donors recruited from each college and the efforts made by the students in increasing the awareness of blood donation in the colleges and in the society via different means including the utilization of the social media. the whole competition is organized and ran by an independent group of students. aims: this study aims at studying the impact of the yearly squ college competition on the perception of blood donation among squ students. methods: a comprehensive anonymous voluntary survey was developed and used to assess perception of students aged 18-25 attending squ and other universities (non-squ) over a two years' period. analysis was performed using ibm spss statistics 22.0. categorized variables were presented in numbers with percentages and associations between the groups were analyzed using chi-square test. a p-value of < 0.05 was considered statistically significant. results: a total of 600 students were surveyed (300 squ, 300 non-squ). there was no statistical difference between squ and non-squ students with regard to past history of blood donation and the number of donations made. when comparing between both cohorts, 73% of the squ and 25% of non-squ students reported the university as the main source for information (p < 0.001), while 56% of squ and 45% of non-squ students reported that the social media was the main source respectively (p = 0.048). there was no statistical difference between male and female donors on their perception of level of self-knowledge on blood donation (p = 0.868). about 84% of the youth agreed that blood donation is one of the duties toward the community. squ students reported higher rates of respond to specific requests for blood donation (44.3% vs 29.7%, p < 0.001). squ students reported greater influence of peers (84% vs 60.7%, p < 0.001), personal knowledge (82% vs 74.7%, p = 0.029) and personal experience (79.3% vs 69%, p = 0.005) when compared to non-squ students. they also reported more feeling of commitment to the society (90.3% vs 78%, p < 0.001). squ students reported lower influence of parents (53% vs 65%, p = 0.005), lower rates of fear from needles (18% vs 32%, p < 0.001) and lower rates of fear from blood (16% vs 29%, p < 0.001). there was no difference between male and female genders in any of the discouraging factors. summary/conclusions: these results highlighted the positive impact and important rule of the youth in the promoting blood donations among themselves through this yearly college competition; in recruiting blood donors and in the dissemination of the knowledge of blood donation. distinct promotion strategies should be adopted to increased first time and repeated blood donation among the youth. we advocate for similar initiatives in encouraging blood donation and disseminate knowledge among individuals in the community. dubai blood donation center, dubai health authority, dubai, united arab emirates background: dubai is multicultural city in united arab emirates. only about 15% of the population consists of uae nationals with the rest comprising expatriates from various countries all over the world. approximately 85% of the expatriate population (and 71% of the emirate's total population) are asian, chiefly indian (51%) and pakistani (16%). dubai blood donation centre is the only centre providing blood donation services in dubai. arabic is the national and official language and english is used as a second language. in order to have good quality screening, it is important that blood donors understand the educational material and questionnaire properly. aims: dubai blood donation centre receives donors (nationals, residents and gcc card holders) from various countries. the aim of this study is to analyze the multinational profile of donors and to find out the need to add any third language to meet the customer needs and expectations. methods: a cross-sectional study of blood donors was conducted in dubai blood donation centre in 2019. the donors were asked about their country of origin, languages which they can read & understand and about the preferred mode of communication. results: a total of 1080 donors were surveyed and asked about the languages which they can read and understand and 1860 responses were obtained. the most common languages which can be read and understood by blood donors in dbdc are english (n = 760; 41%), arabic (n = 272; 14.6%), hindi (n = 268; 14.4%) and malayalam (n = 233; 12.3%). the donors come from different countries, most common 591 (54.7%) donors are indian and 73 (6.8%) are from uae. it was found that 45% donors can read and understand only one language. majority 884 (81.9%) donors can read and understand either of the official languages arabic or english. however, 196 (18.1%) donors can't read and understand these two official languages, the other common languages being hindi and malayalam. the donors were asked about the preferred mode of communication, 1290 responses were obtained. the most common mode of communication were sms and telephone (85% together). summary/conclusions: based on the above findings, it can be concluded that the blood donor profile in our centre is multinational which is a unique and almost similar to the population profile of dubai. as 18.1% donors can't read and understand arabic and english, so it has been decided that the educational material and questionnaire need to be prepared in one more language. hindi has been decided as the third language in the centre and donor questionnaire and educational materials in hindi will also be made available to the donors. further,the donors will be communicated through sms for routine messaging and disease notification while telephonic calls will be done only when the blood is urgently required. background: metabolic disorders (metds), including hypertension, dyslipidemia, hyperglycemia, and central obesity, are tightly associated with cardiovascular diseases and type 2 diabetes mellitus. due to the sedentary lifestyle and increased consumption of high-calorie diet in modern society, metds have become serious health problems worldwide. to have a better understanding and possible improvement on blood donors' health condition, we conducted a survey of the prevalence of metds among blood donors in a blood donation station located in the hsinchu science park in taiwan. participants with metds will be provided with health education materials about metabolic risk reduction, in order to prevent the development of future complications. aims: the aims of this study were to determine the prevalence of metabolic disorders among blood donors, and to calculate how much money would be paid to identify a case of hyperglycemia, hyperlipidemia, or undiagnosed diabetes. methods: this study was approved by the institutional review board of taiwan blood services foundation (tbsf). the body weight, body height, waist circumference (wc) and blood pressure (bp) of participants were measured. blood samples were obtained to determine the values of hemoglobin a1c (hba1c) background: the law on blood donation supports the development of the blood service and guarantees the protection of the donor's rights and the maintenance of health during blood donation in the russian federation. national criteria for donor selection for blood donation are used in the activities of blood service establishments and are aimed at ensuring the blood products safety. the study of the characteristics of blood donors allows to predict the development of blood service and to plan the volume of blood products for transfusion and plasma fractionation. aims: the aim of this work was to study the characteristics of whole blood and apheresis donors in the blood service in the russian federation. methods: indicators of activity in the blood service establishments in the russian federation in sectoral statistical observations over the period 2015-2017 and the calculation of indices characterizing the whole blood and apheresis donors were analyzed. data are presented according to the administrative division of russian federation into federal districts (fd). results: the proportion of whole blood donors was 83.7%, plasmapheresis donors -13.2%, blood cell apheresis, including plateletapheresis, donors -3.1%. for the period 2015-2017, the percentage of repeated and regular whole blood and apheresis donors increased from 66.2% to 72.2%. the percentage of first-time donors ranged from 27.8% to 33.8%. the largest proportion of plasmapheresis donors was observed in the volga fd (22.0%). about 28.9% of the total plasma was collected by apheresis from donors. the percentage of plateletapheresis donors increased from 1.9% to 2.3%. the largest percentage of plateletapheresis donors was observed in the central fd (2.9%), where a significant part of medical centers of cardiac surgery, hematology and bone marrow transplantation are located. the proportion of platelet concentrate collected by apheresis increased to 69.9% in 2017. actions to recruit young donors for blood donation and its components were regularly carried out in all federal districts. summary/conclusions: in the russian federation, the structure of donation is characterized by an increase in the proportion of plateletapheresis donors, stabilization of the percentage of plasmapheresis donors and an increase in the proportion of repeated and regular whole blood and apheresis donors. there are significant regional variations of donor's characteristics in the federal districts. background: shortage of blood supply despite continuous blood donation campaigns especially during local festive seasons has been a major issue in our country. thus, our faculty initiated blood donation drives in collaboration with national blood centre in order meet the demand for the blood requirements. however, the pre-donation deferral rate was relatively high among our young blood donors leading to loss of valuable blood units. understanding the causes of donor deferral provides direction on strategies for young donor recruitment and retention of future blood donation. aims: the aim of this study is to evaluate the young donor deferral pattern and to identify factors which could help in minimizing the preventable deferrals. methods: this is a retrospective study of voluntary young blood donors age between 20 to 23 years old recruited during mobile blood donation in faculty of medicine, universiti teknologi mara, malaysia. the study was conducted between january 2016 to december 2018. the data were retrieved from the official reports of each mobile blood donation. results: a total of 828 young blood donors had attended mobile blood donation during the study period. the overall pre-donation deferral rate is 25.6%. the main causes of deferral are low haemoglobin (hb) level (20.8%) followed by low blood pressure (16.0%), upper respiratory tract infection (11.3%) and sleep less than 5 h (9.4%). summary/conclusions: low haemoglobin and low blood pressure are the two common reasons for blood donation deferral among our young blood donors. in our study a significant proportion of deferrals are due to sleep less than 5 h whereby this could be prevented if the donors are aware of the donor selection criteria. strategies to mitigate preventable deferrals and improve blood donor retention particularly young blood donors as source of motivation for future blood donation are urged to avoid additional stress on the blood supply. background: in the modern world, donating blood has become a humane manner for saving of patients life. but there are barriers to blood donation which are designed to ensure both donor and blood recipients' safety. anemia is one of the most common health problems in the world. based on the who estimation, nearly a quarter of the world's population are suffering from anemia, its prevalence varies among the populations and age groups. the prevalence of anemia among men is 12.7% and in non-pregnant women is 30.2%. aims: the aim of this study was to determine the status of hemoglobin in volunteer blood donors referring to fars province blood transfusion service and to determine the demographic status of them during the last two years. our study included blood donors for all blood donors during the last two years. methods: the study is descriptive cross-sectional and our sampling was non-random and simple sampling method. all parameters related to the donors, including age, sex and type of donation were investigated and analyzed in spss software. results: the total number of referrals for blood donation was 454913. repeated blood donors was 44.8% of total population and had the highest number of referrals, followed by first and lapsed donors with 31.2% and 24% respectively. in terms of gender distribution, 6.2% were female and 93.8% were male. the highest rate of hemoglobin level less than 12.5 g/dl was found in first-time donors with 51.6% and the lowest prevalence was observed in lapsed donors, followed by repeated donors with 24.7%. 44.8% of the repeated blood donors have hemoglobin higher than 16.5. there was a significant difference between blood donation type and hemoglobin level. summary/conclusions: according to our findings, low hemoglobin levels are more common among first-time and female donors, and this requires a special training among these groups. because the high share of first time donors in blood supply and the positive impact of female donors on the blood safety, corrective action for that groups is recommended. finnish blood donor biobank j partanen, t wahlfors, m arvas, j clancy, k l€ ahteenm€ aki, e palokangas and n nikiforow background: the increasing need for large, well-characterized cohorts of healthy individuals for modern biomedical research, such as genomics or phenomics studies typically including tens or even hundreds of thousands of subjects, has posed the possibility of using blood services as an option for collecting samples and related data. the possibility to re-contact blood donors for repeated sampling or asking for additional data has further increased interest in collecting large biobanks from blood donors. there is also a need to study more thoroughly the effects of blood donation on donor health. aims: the first-phase goal is to recruit 50,000 blood donors with broad biobank consent for the finngen (https://www.finngen.fi/) project, a large publicprivate effort aiming to collect genome and health-related registry data of 10% (500,000) of the finnish population. (11.58%), dental examination 10(3.32%) and medication history 6(1.98%). permanent deferral namely, risk factor involving transfusion transmitted infections and chronic disease were 5(1.65%) and 8(2.64%) respectively. the prime cause of permanent deferral was risk factor involving transfusion transmitted infections while the temporary deferral was bed side hypertension. gender wise, the leading cause of donor deferral in male was bed side hypertension and anaemia was the major cause in female. summary/conclusions: the findings of the survey aid to evaluate the significant causes of blood donor deferral. this study suggests that the restrictive criteria can be used for blood donor selection. this will in turn increase the blood supply of tertiary care hospital. background: donor selection is the first step towards safe blood but retaining blood donors is also very important for the blood supply. donor questionnaire and the medical interview should provide optimal doctor deferral. aims: to evaluate deferral rate in blood donors in order to identify the main reasons and to target eventual corrective activities. methods: we analysed the data concerning blood donors who were registered in the period of three years (2016-2018). we used data from the information system e-delphyn. background: iron deficiency (id) in blood donors is an underestimated issue in many countries and may cause symptoms to blood donors even without anemia. id prevention is mainly based on the prevention of anemia in whole blood donors, which is done by deferring donors whose haemoglobin level is under defined threshold (120 g/l in women, 130 g/l in men in france). efs (french blood establishment) studies has observed that the rate of deferral for anemia is significantly higher in women than in men, either in french west indies (15.9% versus 3.4%) or in continental france (2.9% and 0.8%). assessing the prevalence of id is of great interest since strategies to counteract it must deal with donor health and self-supply. however, data on id are missing in france. aims: to estimate the prevalence of id in french whole blood donors and to identify risk factors associated with id. methods: this non-interventional, cross-sectional, multicenter study is performed in blood donors of efs and ctsa (blood center of the french military health service). all whole blood donors who met selection criteria are potentially included. donors coming for bloodletting and donors who refuse to participate to the study are excluded. no additional sample is taken for the study, ferritin is tested after blood qualification on surplus amount. samples are selected at random within all the geographical areas and all mobile blood drives and blood centers between march 11 and march 28, 2019. results: this study ferridon has been approved by ethical research committee. nine thousand (9000) whole blood donors will be included in efs centers in continental france. to have information on donors of afro-caribbean origin and comoros origin, 150 donations should be included in the french west indies and 120 in reunion island. additionally, 1500 whole blood donors will be included in ctsa centers. in this study, id is defined by ferritin lower than 26 ng/ml and iron overload is defined by ferritin higher than 300 ng/ml. all donors with iron deficiency or overload will received a letter advising to consult their general practitioner. weights will be calibrated on age, sex and geographical area so the sample will be representative of the french whole blood donors. estimation of id prevalence will take into account the weights and logistic regression model will be used to analyze risk factors associated with id. data will be analyzed during april and may 2019 to get result at the end of may. summary/conclusions: ferridon will be the first study on id in the french blood donors. considering the french health care system and diet, it will be interesting to compare those results to results from other countries. mostly this study will allow to consider various strategies dealing both with donor safety and self-supply. background: in portugal, with an aging population of around 10 million people, only 3.8% are blood donors. the country has a national center of blood supply and some central hospitals with a blood donation center. despite the growing practice of the excellent concepts of patient blood management, it is imperious to attract new donors. this need has been our inspiration to use new approaches towards people, in a constant work of promotion. aims: reach the majority of our local population using radio and telecommunication as well as social networks in an attempt to raise the number of new blood donors in a central hospital of the north of portugal. methods: active communication with the population of our reference area, via the social networks facebook tm and instagram tm , through educational digital posters and messenger service to answer any kind of questions. establish contact with radio and television stations as well as with the mayor of the city, journalists, schools, town hall deputies and celebrities, through email and telephone calls. design posters, flyers and public advertising to distribute in the city. results: through the social networks it has been possible to reach a population of dozens of thousands in our city, in a daily basis. the national and world donor days were celebrated with success, in our health facility, with city mayor and journalists, and also in three television stations with national broadcast, reaching millions of people. celebrities (sport, television, music, stand-up comedy, journalists and a magician) have accepted our challenge through videos or donating blood, appealing to blood donation and sponsoring our cause. these projects and continuous availability to innovate have given our hospital a self-sufficiency of 95% in 2018, instead of 80% in 2016, which implied receiving less blood unities from the national center of blood supply. our most recent project involves high schools, in an attempt to educate our next generation of donors, with meetings in the town hall with deputies and district school delegates. summary/conclusions: the aging population and the low percentage of blood donors are an important issue concerning public health. nevertheless, the good will and continuous advertising and educative work towards the population, appealing to the ethical and civil responsibility since young ages have shown to improve our capacity of response as a central hospital, increasing the auto-sufficiency of blood unities and the interest of younger donors. it is of the utmost importance to understand that this is a continuous and a hard work of the professional team of our hospital, involving countless calls, emails and hours to obtain some positive response, in an endless job. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . the mean interval between donations is shorter for former regular donors (4.9 months, p < 0.01) whereas donors with an interval of 9 to 12 months are more likely to be regular (aor 16; 95% ci 1.6-164). summary/conclusions: at the provincial blood transfusion centre of bukavu, the percentage of regular donors is low and there is a substantial loss of former regular donors. some factors identified to be linked to fidelity are unique to our study: female gender and a longer interdonation interval. other factors that are similar to those found elsewhere have a particular significance in our donor population which consists mainly of young people and people without income. efforts must be undertaken to ensure a supply from voluntary donations; recruitment strategies and target groups must be refined. future qualitative studies are needed to explain the various associated factors and better understand the motivations of regular and non-regular donors to improve donor retention. results: in kazakhstan, the proportion of donors is higher, especially primary. the number of blood and especially plasma donations is higher, which can be explained by the presence of several albumin and immunoglobulin production sites. increased evidence of blood transfusion rules, the development of a patient's blood management in combination with an increase in the quality of blood components cause a reduction in clinical need for red blood cells and plasma for transfusion. at the same time, the need for platelets is growing. it is difficult to assess the correctness of comparing the amount of banked whole blood. it is equally difficult to compare the number of received and distributed donor red blood cells and plasma: in russia they are measured in liters, and in kazakhstan in doses. with a certain degree of conditionality platelet extraction can be compared. in russia, they are counted in equivalent doses isolated from a dose of whole blood (at least 60 9 10 9 cells per dose), and in kazakhstanin therapeutic doses (at least 200 9 10 9 cells per dose). in 2017, the estimated consumption of platelets in kazakhstan exceeded the russian indicator by 28.0%. 86% of platelets in kazakhstan and 69.9% in russia are harvested by the apheresis method. inactivation of pathogens is performed in 92% of platelets in kazakhstan and in 15.4% in russia. pathogen inactivation with amotosalen allows us to abandon the examination of donors for cytomegalovirus and irradiation of platelet concentrate. the main modern trend in the use of cryoprecipitate is using it as a source of fibrinogen, a blood coagulation factor that is first depleted in coagulopathy associated with injury and massive bleeding. its production is growing in both countries, endowment in kazakhstan in 2017 exceeded the russian indicator by 141.4%. a significant plasma percentage in both countries does not pass quarantine due to repeated non-appearance of the donor and is subject to destruction. inactivation of pathogens is performed in 10% of plasma in kazakhstan and in 3.6% in russia. despite the instruction and selection of donors, laboratory screening of infection markers remains effective: russia more often identifies hiv and viral hepatitis c from potential donors, and viral hepatitis b and syphilis are detected in kazakhstan. 0.7% of donors in kazakhstan are exempted due to the results of multiplex screening of nucleic acids of three hemotransmissive viruses. summary/conclusions: the blood services of russia and kazakhstan perform tasks to provide medical organizations with effective blood components. in conditions of decreasing demand for red blood cells and plasma, it is advisable to focus on the efficiency of resource use and improving the quality of blood components produced. blood collection including apheresis p-091 finnish red cross blood service, helsinki, finland background: skin disinfectant must effectively reduce microbes from the arm of the donor. as a result of poor disinfecting microbes may be transferred from skin via venepuncture to the collected blood and contaminate the blood components. aims: to ensure the efficiency of the skin disinfectant used for donor arm disinfecting by validation. the validation has two criteria which the post-disinfection samples must achieve: 1. no bacteria growth near the puncture spot (result 0 cfu) in ≥ 90% of the samples. 2. total amount of bacteria on average is at most 2 cfu/25 cm 2 . at most 10% of samples are allowed to have 2-10 cfu/25 cm 2 . methods: microbiological samples were taken with contact plates from 30 voluntary persons' elbow folds before and after skin disinfection. the disinfecting was performed according to normal procedure by five nurses altogether with ethanol based disinfectant used in blood donation. on the sample plates an x was marked and this was directed to the puncture spot pointed by nurse. post-disinfection sample was taken at the moment the skin would be punctured with needle. results: the amount of bacteria varied from 20 to above 500 cfu/25 cm 2 in the pre-disinfection samples. disinfection reduced bacteria very well; the critical puncture spot was totally clean (0 cfu/25 cm 2 ) in 93.3% of the samples and 86.7% of the samples had 0 or only 1 cfu/25 cm 2 . the average number of bacteria after disinfection was 0,6 cfu/25 cm 2 and the maximum number was 4 cfu/25 cm 2 . most of the remaining bacteria were single colonies at the edges of the plates. summary/conclusions: both main criteria are fulfilled. the sub criteria of the second main criteria is also full filled if the not so critical colonies at the edges of the plates are not taken into account. the skin disinfectant in question is shown to be effective and can still be used in blood donation paying attention to thorough procedure performance according to the instructions and sufficient drying time of the disinfectant. background: research questions involving blood donation and recipient data often require advanced statistical methodologies. while such methodologies may appear in other medical research areas, specific tailor-made statistical tools and approaches are required for the analysis of blood-related data. these toolkits, which often require collaboration, are not always readily available to blood services, especially so in resource-limited settings. an international network of statisticians, epidemiologists and clinical researchers has been established for this purpose, which started with an invited session at the 2018 meetings of the international biometric society. aims: to exchange ideas, experience and knowledge to further improve the quality of blood sector research. methods: currently our network covers four major blood services and members from five different countries. the network has monthly conference calls about past and current research topics. we wish to extend this network further, and establish a subcommittee on statistical and epidemiological methodology with regular face-toface meetings at an international organization such as isbt. results: the monthly meetings have already demonstrated that the members share common problems and interests. for example, we are discussing techniques to analyze data with repeated measurements, e.g. eligibility haemoglobin tests, ways to assess the healthy donor effect, e.g. determining appropriate controls groups, and predictive models of blood supply and demand, e.g. stochastic processes and queuing models. the network also aims to organize training sessions in methodology either on site and/or by developing web lectures. summary/conclusions: an international network on statistical methodology for the analysis of blood donation and recipient data will improve the quality of research in the field of transfusion medicine research. expanding the network to include countries and blood services in research limited settings needs to be actively pursued. background: blood donor hemoglobin concentration (hb) is commonly measured from a skin-prick sample at the donation site, and low hb is the most common reason for temporary donor deferral. while a proportion of the deferrals do reflect true low hb, the skin-prick sample is prone to preanalytical error and variation resulting in false deferrals. aims: we assessed the applicability of a venous blood sample for second-line hb screening in blood donors failing the initial skin-prick test. methods: initial hb was measured from a skin-prick sample with the hemocue hb201 + (hemocue ab) point-of-care (poc) device. donors with hb < 125 g/l for females or < 135 g/l for males or with a decrease > 20 g/l from latest donation were included in the study. in the study group, a venous blood sample was collected for hb measurement with the poc device at the donation site. donation eligibility was based on this hb result. venous hb was also determined with a hematology analyzer (sysmex xn, sysmex co.). the blood service's current workflow served as the control group: two more skin-prick samples were collected and the donor's final hb and donation eligibility assessed with an algorithm based on all three skin-prick hb results. results: in the study (n = 305) and control (n = 331) groups, the proportion of male donors (27% and 26%) and the mean initial skin-prick hb (122 g/l and 123 g/l) were similar. significantly less donors were deferred from donation in the study group (40%) than in the control group (51%; chi-square test p = 0.004). the mean difference in venous hb with the poc device versus the hematology analyzer was à3 g/l (range à12 to + 6 g/l). two donors were incorrectly accepted based on venous sample poc result; however, in both, hb measured with the hematology analyzer was only 1 g/l below the limit of donation eligibility (124 g/l for a female and 134 g/l for a male). interestingly, a further 33 donors (27% of all deferred in the study group) would have been eligible for donation based on the hematology analyzer result. summary/conclusions: utilizing a venous blood sample for second-line screening of donors failing the initial skin-prick hb test significantly decreased low hb deferrals without compromising donor health. blood donors' and blood service nurses' reactions to the new workflow have been favorable. we conclude that valuable donations can be recovered and donor satisfaction increased by implementing a second-line hb screening model utilizing venous sample analysis at the donation site. background: there is a paucity of literature on haemoglobin (hb) reference values for adults above 60 years of age. this age group has been reported to use up to 50% the blood supply. some studies report a decline of mean hb with age, but others have found no change with age. conflicting findings of hb levels in the healthy elderly population may be associated with challenges in accessing data from healthy older adults, small sample sizes, selection bias and recent health population data. to donate blood, each individual is assessed as 'healthy' and must meet the minimum hb criteria. however, the hb criteria across countries vary and many blood collection services have an upper age limit for donors. as many populations around world are aging, restricting the upper age limit for blood donation may potentially affect the size of the donor pool and consequently the nation's blood supply. aims: to explore the hb levels of healthy older adults, through a multi-centre retrospective observational study of blood donors aged 60 years or older. methods: over a one-year period, hb values were collected from blood donors aged ≥ 60 years from blood centres of four countries. the estimated proportion of blood donors aged ≥ 60 years old for each country was 0.81% in south korea (sk); 2.57% in hong kong (hk), <3.18% indonesia (indo) and 9% in japan (jap). the minimum hb criteria varied between each country and ranged from 11.5-12.5 g/dl for women and 12.5-13.0 g/dl for men. hb levels were determined using point of care testing (hemocue, compolab, hemcontrol) or the xe-2100d sysmex dependant on the country of origin. statistical analysis of the mean, standard deviation and cumulative distribution of hb were determined by gender and age. background: medication usage is assessed to determine donor eligibility from the perspective of both recipient and donor safety. different time frames since last taken apply to different medications. assessment of medication use varies by jurisdiction, but most european centres use multiple questions. these often include a general question about recent medication use whereas the usa does not. at canadian blood services there are 6 medication questions on the donor history questionnaire (dhq), including any medication use in the last 3 days, vaccination and specific medications over different time frames (high teratogenicity medications). the name of each medication taken and reason for use are documented by staff at each donation attempt. assessment of the frequency with which this process occurs is the first step in improving efficiency of this aspect of donor screening. aims: to determine the percentages donors answering yes to medication questions by demographic variables. methods: all whole blood donors who completed the dhq (full length or abbreviated) in 2017 were included in the analysis. donors' answers to each of the 6 medication questions were extracted from the national epidemiology donor database, as well as sex and age. the number and percentage of donation attempts in which a donor answered yes to each medication question were calculated. donors who answered yes to any medication question were sorted by sex and by age group, the totals and percentages calculated. results: there were 975,067 donation attempts with a completed dhq. overall, 34% of donors answered yes to medications in the last 3 days, 8% to vaccination, and less than 0.1% to others (38% any). slightly more were female (42 vs 36%) of those who answered yes to any medication question, as well as by individual question. the percentage of donors answering yes to any medication question increased progressively in each age group from 24% of 17-19 year olds to 57% aged 60 + (p < 0.0001 for trend). summary/conclusions: more than one third of all donation attempts answer yes to a medication question and require further questioning and documentation. this is more common in older donors and follows a similar trend to general population medication use. comparison of ways of assessing medication use in different countries may help identify effective but more efficient approaches. in addition, the contribution to donor and recipient safety of assessing all medications should be assessed. blood center experience with trima accel 7 and tomes software j schreier 1 , a davison 1 , j gambarte 2 , y l opez 2 , c calonge 2 and e herranz 2 1 terumo bct, lakewood, united states 2 centro de transfusi on de la comunidad de madrid, madrid, spain background: in the madrid community, more than 4000 apheresis platelet collections were completed in 2018, of which almost 3000 were completed in the blood transfusion center and the remainder in several hospitals in the region. trima accel 7 was implemented to meet the productivity needs of the blood transfusion center while improving the donation experience. tomes (terumo operational medical equipment software), which enables bidirectional communication with trima accel devices, was used to connect and centrally manage all trima accel 7 devices with automated data capture and reporting. aims: the aim of this study was to evaluate operational improvements using trima accel 7 with tomes compared to trima accel version 6. methods: this was a retrospective study analyzing 1372 apheresis procedures on trima accel version 6 during the control period from 2 january 2018 to 10 september 2018 compared to 541 apheresis procedures on trima accel 7 during the test period from 11 september 2018 to 28 december 2018. this was not a paired study. operator interventions, and completed procedure rate comparisons, were analyzed using a 2-proportions test, whereas donor demographic data were analyzed using a 2-sample t-test. results: trima accel was used to collect single and double platelet products stored in platelet additive solution. operators selected either a single (target platelet yield = 3.0 or 3.5 9 10 11 ) or double (target platelet yield = 6.0 9 10 11 ) platelet donation based on desired procedure time not the maximum number of products that could have been collected per donor. no statistically significant differences were observed for donors in the test arm compared to donors in the control arm for total blood volume (control = 5148 ml, test = 5124 ml, p = 0.545), hematocrit (control = 43.7%, test = 43.6%, p = 0.233), or platelet pre-count (control = 249 9 10 3 / ll, test = 253 9 10 3 /ll, p = 0.091). females represented 21% of donors in the control arm compared to 23% of donors in the test arm. platelet split rate (platelet products per procedure) increased from 1.15 with trima accel version 6 to 1.18 with trima accel 7; procedure time decreased from 55.7 min to 53.3 min for single collections and from 63.0 min to 60.9 min for double collections with trima accel 7 (these differences were not statistically significant). the percentage of procedures that completed with no operator interventions due to access alerts increased from 62.4% to 84.8% (p < 0.001) and the rate of completed apheresis procedures increased from 89.8% to 92.4% (p = 0.083) with trima accel 7. manual transcription of data during the procedure was discontinued with the implementation of trima accel 7 with tomes. tomes captured procedural data and operator steps with barcode scanning and tracking of configured events. this information was transferred to tomes post procedure and printed as a final report. summary/conclusions: trima accel 7 significantly decreased operator interventions, and automated data capture with tomes eliminated manual transcription of data. both outcomes freed operators to complete other tasks and focus on donor well-being. background: the european committee (partial agreement) on blood transfusion (cd-p-ts) of the council of europe (coe) has appointed a working group (wg) to focus on issues with plasma supply management (psm). the task of the wg is, among others, to collect and analyze data in order to fill knowledge gaps concerning donor safety in plasmapheresis. in doing so, the working group will gather evidence base data to support the upcoming revision of the 19 th edition of coe's "guide to the preparation, use and quality assurance of blood components", the blood guide. an international survey was conducted sept-dec 2017, distributed to blood establishments (bes) by the cd-p-ts representatives to coe's member and observer states. the questionnaire included sections covering collection practices (volume and frequency), management of red cell loss, donor panel demographics and data on donor adverse events. aims: the aim of this study was to investigate whether collection practices following the recommendations published in the blood guide for maximal collection volumes and number of donations per year were indeed associated with higher levels of donor safety and improved donor base sustainability. methods: from the total of 36 respondents, 24 bes collected plasma for fractionation (pff) by apheresis and the study had a dataset covering 2,888,390 plasma donations in the latest fiscal year (lfy). the parameter used as marker of donor safety was the rate of immediate vasovagal reactions with loss of consciousness (vvr with loc) per 10,000 plasma collections. the parameter used as marker of donor base sustainability was the retention rate of donors, ie % donors active in the previous year returning to make a donation in the lfy. results: in the blood guide, the collection volume per apheresis is limited to 16% of the estimated total blood volume but maximally 750 ml, including anticoagulant. respondents had differing practices and scale of collection program 15 be were aligned or lower, and 9 be had higher collection volumes. altogether 14 reported the immediate vvr with loc rate, which mainly was lower than 10/10000 collections. there was a small trend towards reduced rate with larger collection volumes than allowed by the current blood guide. saline compensation during or after collection did not affect the rate of vvr with loc. no correlation was observed between the annual donor retention rate and the rate of vvr with loc or saline compensation practices, as reported by 16 respondents. the retention rate banded in the range of 50%>80% (mean = 60%, min = 25%, interquartile range = 14%, max = 90%). the association between maximum allowed yearly plasma collection (25 l) appears to be reasonably constant and showed no clear association with the donor retention rate. summary/conclusions: restricting the maximum collection limit according to the current blood guide was not associated with either lower vvr with loc or with higher donor retention rate. this study supports reassessment of current blood guide 0 s limits for collection volume of maximum of 750 ml per donation and 25 l per year per donor. methods: serum ferritin concentrations were established from sera stored at à30°c from repetitive platelet donors between 2014 and 2016, using architect â ferritin assay chemiluminescent microparticle immunoassay (cmia). the hematimetric parameters were evaluated in a total blood sample using the celldyn 1800 â . sixteen samples were obtained from women (age: 35.3 ae 12.4 years, range: 18-61) and 35 samples from men (age: 36.2 ae 9.4 years, range 20-61), corresponding to 55.2% and 37.2% of the total female and male repetitive donors of platelets by apheresis using trima accel â terumo-bct and amicus tm fresenius-kabi. the difference in the concentration of serum ferritin between the last and first donation was established, as well as the change in the predonation platelet count between the last and first event. results: in the study population, 43.8% of women and 40% of men performed repetitive donations of platelets by apheresis with an interval of less than three months. the change in ferritin concentration was evaluated according to the interval between donations. in women ferritin delta was à38.4 ae 52.2 ng/ml when the donations had an interval less than three months, vs 3.3 ae 13.0 ng/ml when the time between donations was higher (p = 0.046). in men the change in the ferritin levels was à38.5 ae 61.2 ng/ml with donation times less than three months vs © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 à3.3 ae 38.9 ng/ml with prolonged donation times (p = 0.042). in women, the change in platelet count was à2 ae 46.2 9 10 3 /ul, when the donations had an interval less than three months vs à22 ae 45.3 9 10 3 /ul when the time between donations was greater (p = 0.43). in men, the delta of the platelet count was à12 ae 37.0 9 10 3 /ul in donation times less than three months vs à13 ae 26.6 with higher donation times (p = 0.93). no correlation was found between the concentrations of serum ferritin and the platelet count (r = 0.05, q = 0.75 for males, and r = 0.34, q = 0.22 for females). summary/conclusions: the data obtained suggest that repetitive donation of platelets by apheresis with intervals between donations of less than three months, significantly reduce serum ferritin concentrations in women and men, although normal levels were maintained in both groups. there was no correlation with platelet count. therefore, it is proposed to develop prospective studies to establish the minimum time interval safety for platelet apheresis donor procedures. background: the demand for platelets concentrates is increasing continuously and becomes a challenge for the blood establishments. apheresis platelet collections may be a solution for this challenge. improving apheresis collection efficiency while maintaining blood donor safety is an important goal for the service du sang of the belgian red cross. aims: our establishment evaluated the improvements of the trima accel automated blood collection system version 7 (ta7) by comparing its routine performance with that of the previous software version 6.06 (ta6). methods: prospective, multi-site, controlled, non-randomized trial. apheresis collections were performed in three sfs sites: liege, mons and namur using the two trima software versions ta6 and ta7 sequentially. data were collected from december 2017 to april 2018 on ta6 and from june to july 2018 on ta7. simple and double doses of platelets (respectively 5.0 9 10 11 , 5.5 9 10 11 and 8.0 9 10 11 , 9.0 9 10 11 ) were collected in platelet additive solution (ssp+, macopharma) with concurrent plasma from the same cohort of donors in accordance to donor's eligibility and preferences. in order to maintain the same final platelets content in platelets concentrates, the trima accel's tool yield scaling factor (ysf) was subsequently adjusted from 1.06 (ta6) to 1 (ta7). platelet yield, duration of procedure, number of alarms requiring operators' interventions were recorded and evaluated. donor's hypocalcemia was avoided by giving preventively oral or intravenous calcium which was documented by the operators. results: five hundred ninety (590) collections with ta6 and 607 with ta7 were recorded, with 92% and 95% complete procedures respectively. mean duration of procedures was 70 min on ta6 against 72 min on ta7, p < 0.05. the mean alerts number per procedure on ta6 was 4.1 against 0.5 on ta7, p < 0.05 whereas the maximum alerts number per procedure was 57 and 12 respectively. on ta7, 76% procedures did not require operator's intervention against 40% on ta6,. with ta7 the inlet flowrate was automatically adjusted in 97.2% procedures. the inlet flowrate was increased in response to access pressure in 45.6% of procedures, for 3% of the procedures the inlet flowrate was decreased and for 48.6% of the procedures the inlet flowrate was increase and decreased on the same procedure by the ta7 autoflow system. summary/conclusions: ta7 with its autoflow function improves apheresis donors experience while decreasing operator' interventions through a significant reduction of access draw alerts. as expected from the trima accel platform, post-donation safety remains high. a weak increase in procedure duration was observed for the same platelet yield which may be resolved with further adjustments. background: trima accel system is an apheresis platform relying on continuous flow centrifugation to collect from a donor platelets, plasma or rbcs based on donor qualification. the latest software version -trima accel 7 (ta7) introduced the autoflow feature which allows for automated flow rate adjustments. moreover, ta7 leverages the mobilization capacity of the spleen increasing potential platelet productivity while maintaining high post-donation safety standards characteristic of trima accel. aims: the objective of this evaluation was to assess the impact of ta7 software by retrospective comparison of procedure data and potential for increased productivity with those of trima accel version 6 (ta6) in the same cohort of platelet donors. methods: eight hundred twenty one procedures, started on ta6 from 4th january 2016 to 10th october 2017 were compared to 995 procedures, started on ta7 from 18th october 2017 to 31 st december 2018. procedural data from the trima devices were captured using the cadence system (terumo bct, lakewood co). parameters investigated were the number of machine access pressure alerts per procedure, the potential for higher platelet yield collections and the actual collected yields within the same cohort of platelet donors. results: both donor populations (ta6 vs. ta7 respectively) were comparable and were characterized by: tbv -5207 vs. 5344 ml; platelet count pre-procedure -252 9 10³/ll vs. 252 9 10³/ll; hematocrit pre-procedure -44% vs. 44%. gender distribution was 11% female with ta6 vs. 2% with ta7. venous access pressure alerts were significantly improved by ta7 with an average of 0.2 alerts per procedure as compared to 2.0 with ta6, i.e. 92% decrease. this decrease went down to 91% if only male procedures were analyzed. the maximum number of pressure alerts went down by 84% from 90 alerts in one particular run in the ta6 cohort to 14 alerts in one ta7 procedure. procedure time for single platelet products was reduced from 51 to 49 min and for double platelet products from 59 to 54 min (ta6 and ta7 respectively). donor qualification possible was 52% of procedures yielding single products and 48% of procedures yielding double products with ta6. the percentage of procedures qualifying for doubles increased to 91% with ta7. in terms of split rates, i.e. how many platelet doses could be produced per apheresis collection, potential split rates increased from 1.48 to 1.91 from ta6 to ta7, respectively. in fact, the observed split rate rose modestly from 1.01 to 1.05, as shorter procedures were generally selected according to donors' preferences. summary/conclusions: in comparable donor populations, implementation of ta7 decreased the number of access pressure alerts significantly compared to previous trima versions. the average procedure duration was also found to be slightly reduced. implementation of ta7 has the potential to increase productivity significantly. the observed modest actual rise in split rate suggested that factors related to donor and inventory management will determine at which extent the potential of the new software will be used. donor compared experience on trima accel 7 to trima accel version 6 august 11 2017 to october 17 2017 or trima accel 7 during the test period from november 28 2017 to january 9 2018. this was not a paired study. donors completed the survey while recovering from the apheresis procedure in the cantina. results from the paper survey were transcribed into excel for analysis. results: 63 donors completed the survey during the control period whereas 86 donors completed the survey during the test period. the mean number of previous donations for the control period was 3.0 (min 1 max 6) and for the test period was 3.2 (min 0 max 7). there were no first time donors during the control period and 20 first time donors during the test period. 91% of donors rated their overall donation experience as good on trima accel 7 compared to 90% on trima accel version 6. zero (0) donor rated their experience on either trima accel device as poor. 100% of donors who responded to the question said they would donate on trima accel 7 again. summary/conclusions: no significant difference was observed in donor experience between trima accel version 6 and trima accel 7 as both versions receive high marks. background: the trend on growth of query for donor platelet concentrates is observed in russia for past few years. as reported by edqm in 2014, a higher number of the platelets was consumed compare to 2011 by 7.8%. patients' with hematological malignancies treatment requires platelet concentrates transfusions during chemotherapy, immunotherapy and hematopoietic stem cells transplantation. according to the data collected in national research center for hematology (nrch) in 2018, 635 (8.6%) of the 7,371 patients, treated within facility, received platelet transfusions as transfusion therapy. the total number transfused units is 14,292, which is 668 higher (by 4.9%) comparing to 2017. platelet concentrates production can be performed either by apheresis process or by pooling individual units recovered from the whole blood. taking into account that the nrch produces blood units for its own needs, the pooling is not suitable method for production because its implementation doesn't cover require for platelets and overproduces rbcs. that is why platelet concentrates in nrch are obtained by apheresis only. in summary, the growth on requirement for platelet concentrates and their safeness explains the need for a comparative study for effectiveness of platelet production using various apheresis systems. aims: the aim of the study is to compare effectiveness of platelet concentrate production using mcs + 9000 (haemonetics), upp and trima accel (terumo bct) version 6.0 protocols. methods: the data for 4868 protocols of platelet donations performed in 2018 were analyzed: 2773 on trima accel and 2095 on msc + 9000. all donors were voluntary and non-paid donors with previous experience of blood donations. the choice of the platelet collection device was random; analysis of the main characteristics of donors did not reveal any significant differences between the groups. the median age of the donor was 32 years old, height -175 cm, weight -76 kg, platelet count before donation -254 9 10 9 /l, hematocrit -42%. detailed data are presented in table 1 . student's t-test for unrelated sets was used for statistical analysis of the data. a value p of less than 0.05 was considered as significant. results: the data obtained showed significant difference (p < 0.01) between average number of platelets collected on trima accel (6.3 ae 1.06 9 10 11 /l) and on mcs + 9000 (5.09 ae 0.78 9 10 11 /l). while cost of consumables are comparable, trima accel demonstrated 23.7% higher efficiency. procedure duration also was comparable and averaged within 94 min for both devices. detailed data are presented in table 2 . it is crucial to mention that proportion of trima accel's donations was significantly increased in nrch during 2018 and reached 56, 7% in total (2017-19.4%). a flexible usage of trima accel's consumables for different procedures (regular platelet collection and collection in pas) allowed to change the pas/regular platelets collection ratio from 7.64% up to 43.7%. summary/conclusions: obtained results proved the effectiveness of the trima accel's use for platelets concentrates production. it allowed to increase the average count of platelets obtained for one procedure by 23.7% compared to mcs + 9000 while the cost of consumables and procedure duration are comparable. the donor's comfort during procedure did not affect either. in long terms increasing of number of platelets collected is reducing the cost of platelet concentrates production. abstract withdrawn. background: apheresis collected platelet concentrate is preferable in terms of reducing the risks of adverse reactions in platelet transfusion when compared to random donor platelet concentrates. aims: the aim of our study is to present our experience in collection donation of single donor platelets with apheresis. methods: this is a retrospective study performed in the institute for transfusion medicine from 2010 till 2019. all donors were fully informed on the donation procedure and signed an informed consent for donation. the optimal platelet count that we want to achieve was ≥ 3.0 9 10 11 equal to 12 random donor platelet doses. minimum preapheresis platelet count in donors requested to start the apheresis collection was 150.000/ll. platelet collection was performed using flow cell separators haemonetics mcs+ and trima accel. acid citrate dextrose formula a was used for anticoagulation. median precollection platelet count of donors was 273.000/ll, with range from 182.000/ll to 397.000/ll. male were 77% of the donors and females were 23%. the single procedure usually took 45-70 min. the median platelet count collected was 4.0 9 10 11 , range 2-6.5 9 10 11 . the median processed blood volume was 3215 ml and median used acd-a was 352 ml. mean total volume of collected product was 312 ml. the adverse effects included vein perforation and the numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and was very mild. summary/conclusions: the collected platelet count was more than the wanted optimum platelet count. the number of apheresis donors is increasing and we are working on expanding our voluntary platelet donors registry and increasing the number of typed donors in the registry. background: to determine value of hemoglobin in blood donors, there are some tools or methods used, such as: cyanmethemoglobin method that can detect of hemoglobin quantitative and methods cupric sulfate solutions (cuso4) can detect of hemoglobin qualitative. according to who (world health organization) to determine the level of hemoglobin in blood donors enough used cuso4 solutions with specific weight (y) 1.053 can to detect value of hemoglobin above or same with 12.5 gr/dl. but, cuso4 solution specific weight 1.053 can not to detect and elimination value of hemoglobin above 17 gr/dl or polycythemia sick. because it, central blood transfusion unit (utdp) as the central of blood service in indonesia to manufacture cupric sulfate solution (cuso4) with a specific weight (y) 1062 to detect value of hemoglobin below 17gr/ dl and determine value of hemoglobin above 17 gr/dl. because it, do the testing the accuracy of the solution cupric sulfate in detecting and eliminating donor with value of hemoglobin above 17gr/dl with the test of 35 samples. aims: to determine accuracy and effectiveness by blood donors unit in indonesia red cross to use cuso4 solution with specific weight 1.062 in to detect and elimination value of hemoglobin donors above 17 gr/dl. methods: used the method cyanmethemoglobin and cuso4 (y) 1.062 determination value of hemoglobin donor. test results were analyzed with spss software version 23 using nonparametric analysis wilcoxon test. results: this research testing the accuracy and effectiveness of using a cuso4 solution with specific weight (y): 1.062 in detecting and eliminating hemoglobin value donors above 17gr/dl. from data processing using spss with the wilcoxon test p value 0.02. summary/conclusions: it was found that the cuso4 solution (y): 1062 can detect hemoglobins value above 17gr/dl and more effective in checking the hemoglobin in blood donors. it can be seen from the data processing with spss version 23 with the wilcoxon test p value < 0.05. it is important to monitor the precise course by which repeated blood donation affects hb and the probability of low hb deferral. zinc protoporphyrin (zpp) is a functional indicator of body iron levels and is hypothesized to predict hb levels among blood donors. advanced statistical methods are necessary to properly analyze the longitudinal associations between zpp and hb in data with repeated donations per donor. aims: to determine whether predictions of future hb levels using current hb levels can be improved by taking zpp levels into account, and to illustrate the use of statistical models for repeated measurements of blood donors. methods: we used data from the zpp and iron in the netherlands cohort (zinc) study. we identified previous zpp levels (log-transformed) as the main predictor and adjusted for previous hb level, age, day and time of donation, donation history, bmi, blood volume and blood pressure. we used linear mixed models, which take into account missing data in the outcome and associations between repeated measurements, to investigate the longitudinal association between previous zpp and current hb levels. the longitudinal analysis with linear mixed models was contrasted with a simpler analysis based on the area under the receiver-operating-characteristic (roc) curve for the probability of low hb deferral. results: in total, 8194 whole blood donors (20,864 whole-blood donations) were included in the zinc study, 63% being female donors. previous zpp showed a statistically significant association (p < 0.001) with hb levels in females, but the size of the association was quite small (regression coefficient, b = à0.17, 95% confidence interval à0.22 to à0.11). the same was true for males, but the size of the association was even smaller. blood volume and age for women were significant secondary predictor variables; blood volume, age and donation interval for men. by comparison, the roc analyses showed relatively larger, but less statistically significant predictive effects of zpp on hb. summary/conclusions: zpp is a statistically significant predictor of hb levels, but the size of the effect after adjustment for previous hb and other variables is small. the results cast doubt on whether zpp is an effective predictive marker for hb level and low hb deferral, and suggest that zpp should not be included in prediction models for hb levels. by properly adjusting for associations between repeated measurements and by using all available data, longitudinal models provide less biased and more precise estimates than simpler cross-sectional analyses. background: finnish red cross blood service (frcbs) is a national blood service and is responsible for all blood collection and component production in finland. the highest age for blood donors was 66 years until the end of year 2013 and since beginning of 2014 donors between 66 and 70 years have been able to donate blood. blood donation after the age 66 is possible if the donor has donated within the last 24 months. the upper age limit was raised up based on adverse event data from frcbs donors up to 66 years and on published data from other blood establishments. aims: the aim of this study is to find out if the new policy from 2013 with upper age limit of 71 years is safe. therefore donor adverse event data was analyzed in order to evaluate if the blood donors older than 66 years have more adverse events compared to other donors. the most common donor adverse event for donors over 66 years, haematoma, was registered 211 times (0.05%). in the other age groups haematoma was registered 4408 times (0.56%) and the difference between the oldest age group compared to all other donors was statistically not significant (chi2, p = 0.012). vvrs with loc were registered 21 times (0.05%), vvrs without loc 25 times (0.06), and the total number of all daes was 293 (0.65%) in the age group 66 years or older. the respective numbers in the other age group were: 1152, 0.15%; 11055, 1.41%; 17550 (2.23%). the number of vvrs with and without loc, and total number of all daes in the age group 66 years and older was smaller than in the other groups and the difference between these groups was statistical significant (chi2, p < 0.0001). summary/conclusions: donors over the age of 66 years have less donor adverse events than other age groups. decision to raise the age limit from 66 to 71 seems proven to be right as the older age group has even less donor adverse events than other donors. background: deep vein thrombosis (dvt) of the donor's phlebotomy arm is a rare, but serious complication of blood donation that needs to be recognised and managed appropriately in a timely manner. post donation dvt will be classed as a 'serious adverse events of donation' (saeds)these are events that either result in donor death, hospitalisation, intervention or significant symptoms persisting for more than one-year post donation. aims: to review cases of dvt post donation reported in uk in 2016-2018 years and identify any common themes for improving practice methods: all data relating to saeds from the four uk blood services reported to shot in the last 3 years (2016-2018 inclusive) were reviewed to look for reports of dvt post donation results: a total of 135 saeds were reported in uk from approximately 5.8 million donations (whole blood and apheresis) collected during this period. three cases of upper limb dvt were reported during this time accounting for 2% of the saeds reported and rate of dvt of 1 in 1.9 million donations collected. -case 1: a regular male whole blood donor in his early 30s reported worsening arm pain 12 days following blood donation, had a painful venepuncture and a small bruise at site of donation. he was diagnosed to have an upper limb dvt extending to the subclavian and brachiocephalic vein and started on oral anticoagulants. no other contributory factor was obvious -case 2: a female donor in her mid-40s gave her sixth whole blood donation without event. 11 days after donation, she developed worsening arm pain in the donation arm and was diagnosed with an upper limb dvt and commenced oral anticoagulation. there was no other identifiable risk factor for the thrombosis -case 3: a female donor in her 20s developed pain, swelling, redness and itchiness in her donation arm and chest wall two days after donation. she also described prominent veins on the affected side compared to her other arm. she contacted the transfusion service one week after donation; by this time she was also breathless on minimal exertion. she was admitted to hospital and commenced on anticoagulant therapy. a diagnosis of dvt and associated pulmonary embolus was confirmed. the donor's only risk factor for thrombosis was use of the oral contraceptive pill. summary/conclusions: rare complications of blood donation, like dvt, can occur. superficial venous thrombosis may occasionally progress into the deeper veins of the donor's arm but dvt can also occur without signs and symptoms of superficial thrombosis. none of our patients had any overt evidence of superficial thrombosis. one patient in our series reported using oral contraceptive pill. no other risk factors for thrombosis was forthcoming. transfusion services should encourage donors to make early contact with the blood service if they experience arm complications so that they can be investigated and managed in a timely manner. staff dealing with such donors must recognise the possibility of this rare complication, explore other additional contributory factors and initiate prompt and appropriate management. background: voluntary blood donation is widely considered to be safe with very minimum chance of adverse reaction, which may occur during or after the end of phlebotomy procedure. aims: to find the adverse blood donor reaction among voluntary blood donors in tertiary care hospital in kathmandu methods: this is a prospective study done among voluntary blood donors at grande international hospital, kathmandu, nepal from february 2013 to march 2015. the outlines of reported and communicated adverse donor reaction were also collected after the blood donation from voluntary blood donors in different locations including outdoor and in-house blood donation drive results: in the present study 6,955 whole blood donors were included, during the period of 2 years, 105 (1.50%) adverse donor reactions were reported. majority 89 (84.76%) of adverse donor reactions were mild in nature such as, sweating; 27 (25.72%), light headedness; 19(18.09%), nausea and vomiting; 15(14.28), allergy and bruises; 11(10.47%), sore arm; 9(8.58%) and hematoma; 8(7.62%) while 16 (15.24%) were severe adverse reactions similarly, anaphylaxis; 11(10.49%), loss of consciousness; 3(2.85%) and convulsive syncope; 2(1.90%). markers of the adverse donor reaction were age, sex, pulse, weight, blood pressure and donation status. age and first time status were related with significantly higher risk of adverse reaction with 18-23 years old at higher risk compared to 24-55 years old. first time donors were at higher risk compared to repeated volunteer donors. summary/conclusions: the results of the study are helpful to identify and understand the complication of adverse donor reactions though the incidence of reactions in the blood donors is lower than in other studies. donor age and donation status were strong possibilities of complications. background: blood donors with pollen-induced allergy and asthma must often refrain from donation in pollen season despite medication, because of symptom severity or similarity to airways infection. extracts of the medicinal mushroom agaricus blazei murill (abm) given orally have been found to reduce ige anti-ovalbumin levels and ameliorate the skewed th1/th2 cytokine balance in mice sensitized to ovalbumin (takimoto, immunopharm immunotox, 2008; ellertsen & hetland, clin mol allergy, 2009). aims: the objective was now to examine whether supplementation with the abmbased extract that we used in the mouse model for allergy, could alleviate allergy and asthma in blood donors by reducing specific ige levels and basophil sensitization. methods: sixty donors at oslo blood bank with self-reported birch pollen allergy and/or asthma were recruited and randomized in a double-blinded, placebo-controlled study with oral supplementation for 7 weeks before the birch pollen season with the abm-based extract andosan tm (immunopharma, oslo, norway). this is a water extract of the bacidomycetes mushrooms abm (82%), hericeum erinaceus and grifola frondosa. the participants filled in questionnaires for allergic conjunctivitis & rhinitis, asthma and medication. serum ige (immunocap â , immunodiagnostics, sweden) and bet v 1-induced basophil activation in whole blood determined by cd63 expression in a flow cytometer (flow cast â , b€ uhlmann lab ag, switzerland), were analyzed before and after the pollen season. (trial record: nct03198455, clinical.trials.gov). results: there was significant reduction in allover allergy-related ailments and types of allergy medication used in the abm extract compared with placebo group during the pollen season and no side effects. also, abm treated asthmatics had fewer symptoms and used less medication than controls. in the abm group, serum levels of specific ige anti-bet v 1 and anti-t3, were significantly reduced during the pollen season as compared with levels in the placebo group. whereas the maximal allergen concentrations needed for eliciting basophil activation before the season changed significantly to lower concentrations (i.e. enhanced sensitization) after the season in the placebo group, these concentrations remained similar in the group given the mushroom extract. summary/conclusions: oral pre-seasonal supplementation with an abm-based extract for 2 months reduced general allergy ailments, asthma symptoms and medication in blood donors with birch pollen-induced allergy and asthma during the pollen season. this was due to reduced specific ige levels and basophils rendered less sensitive to allergen activation. the study suggests that supplementation with abm mushroom extract can have prophylactic effect on aeroallergen-induced allergy and asthma in blood donors. it may therefore reduce such ailments in affected blood donors and impact blood donations in the pollen season. results: dhv started in 20/9/2014. the data presented in this abstract is till 28/2/ 2019. data is collected from total of 114475 blood donors (86.67% male donors and 13.33% female donors). repeat donors accounted for 59.34% against 40.66% of first time donors. of the total number of donor adverse events recorded, 2.73% (2633) reported for male donors and 6.97% (1063) for female donors when the donor adverse events stratified age-wise, the highest incidence reported in age group 18-24 years (male 5.45% and female 13%). among age group 25-40 years, (male 2.4% and female 4.02%), whereas in age group 41-65 years, (male 1.52% and female 2.11%) data analysis of total 6226 reported and registered donor adverse events, are categorized as hyperventilation (864), sweating (1458), dizziness (pre-syncopal 3160), loss of consciousness (416), vomiting (108), convulsions (220), hematomas with re-bleed (255), nerve irritation (8) and off-site reactions (333). many donors showed multiple forms of reactions. summary/conclusions: evaluation of donor side effects helps to improve donation process and donor compliance. most frequently recorded reaction remains dizziness (pre-syncopal). our donor vigilance data show reactions occurred more frequently in younger age, female and first time donors. repeat donation and age are predictors for low rates of adverse events. participation in dhv implies an effort to improve donor care and safety infrastructure and a desire for national and international comparisons to determine best practices and also to look into effectiveness of risk reduction strategies and follow-up trends. pre-donation hydration was implemented as an interventional tool to test the effects of hydration on pre-syncopal reactions to blood donation, specifically targeting those at highest risk such as female, first-time, high school donors. the results are awaited. background: descriptions of deferral categories and a knowledge in the percentage of deferrals in each category are of value in formulating recruitment and retention strategies. this can also help in planning more efficient recruitment strategies and thereby assist in reducing the shortage in blood supply. aims: the aim of the study is to categorize all donors who were deferred during medical checkup and find out the donor deferral rate in dubai blood donation center from january 1 st 2016 to december 31 st 2018 and also to find out whether there is any yearly or seasonal trend in any of the categories of deferral criteria's which can aid in forecasting and managing donor pool. methods: a retrospective study of donors deferred during last three years from january 1 st 2016 to december 31 st 2018 was done in dubai blood donation centre. the donors deferred during pre-donation medical check-up were categorized into 8 categories including low hemoglobin, high and low bp, intake of antibiotic, fever and flu, taking other medications, travel history etc. the deferrals were analyzed monthly and yearly and then were compiled to find any yearly trend or seasonal trend in the donor deferral rate in any of the categories. the data were analyzed using the spss software and a p value of < 0.05 was considered significant. the assessment of donor suitability is in accordance with aabb standards and is consistently applied in every blood donation setting on each occasion of donation to all blood donors. results: during this study, 193,228 donors were registered from january 1 st 2016 to december 31 st 2018 and 41,037 (21.24%) donors were deferred. the common reasons of deferral were low hb, high bp, travel history, intake of antibiotics and cough/flu symptoms. there was a significant decrease in deferral rate from 24.07% (15302/63563) in 2016 to 20.79% (13180/63394)in 2017 and further to 18.74% (12419/66271) in 2018 (p < 0.05). the specific deferral rate due to low hb also significantly (p decreased during these three years (86/1000 in 2016, 68/1000 in 2017, 58/1000 in 2018), though no change was seen in the deferral due to other reasons. the reduction in the rate of deferral due to low hemoglobin may-be linked to the change in the staff performing the hemoglobin testing in dbdc (nurses instead of phlebotomist were assigned to perform hb estimation of donors). there was a seasonal variation in the deferral rate in all the three years-lowest in june (6/1000) and then increasing with a peak in october (19/1000) and plateauing till january. this pattern of deferral corroborated with the rate of deferral due to flu/fever and cough and antibiotics with an average of 4/1000 in june and increasing to 10/1000 in october (p < 0.05). summary/conclusions: staff competency is pertinent in accurately deferring donors. there is also a significant seasonal pattern in flu/fever and intake of antibiotics deferral rate that is reflected in the total donor deferral pattern. seasonal variation of specific category of donor deferral should be taken into account for donor recruitment and retention efforts. background: west nile virus (wnv) is a mosquito transmissible flavivirus. it has been shown (vogels 2017 thesis) that the common mosquito in the netherlands can transmit wnv in laboratory circumstances but presently does not lead to effective transmission. however, the number of outbreaks of wnv is increasing and moving from the eastern and southern european borders towards the traditionally more colder western and northern parts of europe. in order to prevent wnv transmission to blood transfusion recipients, dutch donors that travelled to regions with wnv risk are deferred for a period of 28 days for whole blood, platelet donations and quarantine plasma in order to exclude potentially infected asymptomatic donors. aims: to assess numbers of dutch donors who are deferred for travelling to wnv risk areas within europe, and the return after onsite and offsite deferrals of donors. methods: data from 2015 to 2018 on donation attempts and deferral were retrieved from eprogesa, the blood bank information system. onsite deferral is defined as a donation that was attempted in the deferral period or in the 7 days prior to deferral, all other deferrals are considered as offsite. a generalized estimating equation model was used to assess the association between onsite versus offsite wnv risk deferrals in 2015-2016 and subsequent return rates within two years (after which a donor is inactive according to domaine). results: in 2015-2018, 16,400 donation attempts led to onsite deferral for wnv risk; 87% at whole blood donation attempts, 13% at new donor examinations. in total 19,624 offsite deferrals could not be traced directly to a donation, but based on the next donation more than 90% were probably whole blood donors. the number of deferrals peaks each year during august, the major holiday period in the netherlands, and increased from 920 in august 2015 to 1711 in august 2018. this increase is probably caused by the expansion of wnv risk regions. the return rate of wnv deferred whole blood donors is slightly lower than for donors who are not deferred (92% versus 95%); for wnv deferred new donors the return rate is 75% (versus 87% for no deferral). thus wnv deferral resulted in approximately 400-500 extra lapsing donors during these 2 years. however wnv deferred donors, that are older (odds ratio (or) 1.03; 95% confidence interval (95% ci) 1.02-1.03), of male sex (or 1.16; 95% ci 1.03-1.31) and whole blood donors as opposed to new donors (or 2.01; 95% ci 1.70-2.38) were more likely to return to donate. there was no difference in return rate by offsite and onsite deferral. summary/conclusions: travel-related wnv deferrals are increasing with expanding risk regions, especially in the holiday season where the availability of donors is already low. although the numbers of donors who are permanently lost after wnv deferral are limited, the increasing numbers of lost donations make it important to consider alternatives to donor deferral such as wnv nat testing. background: low haemoglobin due to iron deficiency is increasingly recognized as a serious problem in many blood centers. donor education, iron supplementation, ferritin monitoring, and lengthening of inter-donation interval are currently the main mitigation measures. however, a number of factors in particular donor knowledge could impact their success. locally, iron supplementation programme was implemented since 2012 with target group of donors who have given blood within the last six months. aims: here we look at an online donor survey to gain insight on their view of the programme and knowledge. methods: donors with successful blood donation in the past six months would be given 14 days of one tablet of iron supplementation (100 mg elemental iron) since 2012. an electronic questionnaire was sent to blood donors in 2017 to assess their view on the programme and knowledge which focused on iron store and absorption, compliance and any side effects occurred. results: 3212 donors (male to female was 1:1.9) replied to the questionnaire. of them, 594 received iron supplementation (male to female was 1:1.6). most of the respondents (94%) had one or more donations in the preceding 12 months. of the 594 donors received iron tablets, only 246 (41%) took all; 69 (12%) took more than 50% but not all; 130 (22%) took some but less than 50% and 149 (25%) did not take any. gastrointestinal upset was reported in 113 (25%) donors and constipation seen in 193 (43%) among those who took at least some of the iron supplementation. most respondents answered correctly to the questions on the knowledge on iron store and absorption. when comparing those with better compliance (took more than 50%) to those who did not (took less than 50%), significantly more donors in the former knew vitamin c could enhance iron absorption (p < 0.05). on the other hand, no difference was seen when they were asked if 1) iron can only be absorbed from meat; 2) tea and coffee consumed during meal can enhance iron absorption; 3) everyone can take iron supplementation on their own; and 4) iron store in male is always more than female. summary/conclusions: the results suggested that there is definitely more room to enhance the blood donors' knowledge on iron store and absorption in order to improve the effectiveness of iron supplementation programme. besides, the side effects reported by the donors could be an important limiting factor that better alternatives should be explored and considered. background: vasovagal reactions (vvr) are a well-established deterrent to donor return. however, the correspondence between vvr experience and donor lapse is not perfect. in australia, for example, vvrs only reduce two-year return rates by 27% for whole blood donors and 22% for plasma donors. the elements of a vvr and the donor's interpretation of this event that protect against or encourage lapse have not yet been identified. aims: in this study we explored the views of donors on donating following a vvr, with a particular interest in their emotional reaction to the vvr, their understanding of what caused the reaction, and their intentions to return. methods: semi-structured telephone interviews were conducted with 36 whole blood and plasma donors who had a recent vvr experience. data were analysed using the framework approach. results: donors are generally motivated to give blood to help others and to positively impact on those in their communities. they anticipate feeling good after their donation but in contrast, for many, a vvr leaves them feeling anxious, embarrassed, and disappointed. for donors, the experience of a vvr negatively influences their perceived ability to donate successfully, and many fear it will happen again. however, this effect appears minimised among donors who at least partially attributed their reaction to their own behaviour, such as poor hydration. for donors already juggling multiple demands, a vvr may tip the balance with donating becoming too much of an effort and perceived risk. however, donors appeared more confident to return if they felt supported by staff or if they could donate with family or friends. summary/conclusions: this study provides valuable insight into the vvr experience, which will aid in the improvement of donor safety and retention. the findings highlight the need to improve communication at the time of and following a vvr, to educate donors on how to reduce their vvr risk, and to intervene to help donors maintain their perceived ability to give blood in order to maximise retention following a vvr. background: frequent blood donation depletes the iron stores of blood donors. iron depletion might have negative effects on the health of the general population, but its effect on the blood donor population is not well known. aims: to investigate the iron status of finnish blood donor population and how it relates to donor health, the finnish red cross blood service set up the findonor 10,000 study in 2015. we investigated whether there were changes in donors' selfrated health and if these possible changes could be associated with differences in iron biomarkers (ferritin and soluble transferrin receptor -stfr) or hemoglobin levels during the first study visit. methods: participants were recruited in three donation sites in the capital region of finland between may 2015 and december 2017. participants filled out an electronic questionnaire about their health and lifestyle at the donation site during their enrollment visit. participants were asked by letter to fill out the same questionnaire electronically during the summer 2018. we included the 1435 participants (596 men and 480 premenopausal and 359 postmenopausal women) who completed both health questionnaires. to evaluate self-rated health we used the well-validated single question: "how would you rate your health in general?". participants were able to evaluate their health status on a five-point scale: excellent, very good, good, moderate, and poor. iron biomarkers and venous hemoglobin were measured from blood samples collected at the first study visit. we first computed the odds-ratios of reporting poorer health depending on demographic group. we then compared iron biomarker and hemoglobin levels between donors who reported improved, similar or poorer health rating. results: donors who rated their health in the first questionnaire as moderate (n = 31), good (n = 483), very good (n = 684) or excellent (n = 237) health tended to report improved (58%), similar (63%), similar (55%) or poorer (55%) health ratings respectively in the second questionnaire. pre-menopausal women reported their health poorer in the second questionnaire compared to the first questionnaire more often than post-menopausal women (pre-menopausal 51%, post-menopausal women 38%), or = 1.37 95% ci 1.02-1.85). there were no differences between other groups. there were no significant differences in iron biomarkers levels (ferritin and stfr) or hemoglobin levels between donors whose health ratings were improved, similar or poorer. summary/conclusions: in this cohort, pre-menopausal women rated their health poorer at the end than at the beginning of the study more often than post-menopausal women. no association was found between changes in self-rated health and iron levels (ferritin, stfr) or hemoglobin levels. further studies about the factors relating to blood donors' self-rated health need to be carried out. background: in recent years, the blood donation business has made great achievements, but it still cannot avoid the occurrence of adverse reactions to blood donation which not only brings certain obstacles to the blood donation work, but also affects the enthusiasm of blood donors. aims: to understood the causes and other relevant factors of adverse reaction among blood donors, the information of blood donors at dai autonomous prefecture of xishuangbanna were analyzed in 2018. methods: the data of volunteers from january to december 2018 were analyzed. the causes of adverse reactions were classified, and the incidence of adverse reactions was compared in terms of gender, frequency, age and blood type of blood donors. results: there were 9081 blood donors in 2018, 69 (0.76%) of whom had adverse reactions and 9 causes were induced, among which mental stress was the most common factor that accounted for 78.26% (54 cases). there was no significant difference in the incidence of adverse reactions between men and female (p > 0.05). from the frequency of blood donation, the incidence in the first donor was significantly higher than that in the second donor (p < 0.01). when it comes to age, the incidence was different and the 18-25 age group was the highest (1.61%). among different blood group donations, there was no significant difference (p > 0.05). summary/conclusions: adverse reactions of blood donation is closely related to the psychological state and age of the blood donors. the staff of the blood center should further optimize the service, strengthen the communication and publicize the knowledge of blood donation. the ultimate goal is to increase the blood donation rate on the basis of reducing adverse reactions. background: blood loss due to repeated blood donation can lead to iron deficiency or anemia, but currently there is no management plan for the prevention of iron deficiency in korean blood donors. female and male donors are required to wait at least 8 weeks between blood donations in korea, which is the shortest period among all northeast asian countries. female and male donors are allowed to donate whole blood up to five times per year and platelets up to 24 times per year (if spaced more than 14 days apart for the latter) due to the chronic blood supply shortage. these facts induce concern about the impact of blood donations on the donors' iron status. aims: this study aimed to evaluate the effect of oral iron supplementation in repeat donors based solely on donation history. methods: the high-risk group included male donors with ≥4 whole blood donations or 16 plasmapheresis or plateletpheresis donations, and female donors with ≥3 whole blood donations or 12 component donations, both within the previous year. the control group consisted of first-time or reactivated (ft-ra) donors who had no history of blood donation in the past 2 years. the hemoglobin (hb) level, ferritin level, total iron binding capacity (tibc), transferrin saturation, and soluble transferrin receptor (stfr) of repeat donors at high risk for iron deficiency were compared to those of ft-ra donors. iron deficient erythropoiesis (ide) is defined as present if the log of the ratio of soluble transferrin receptor to ferritin was ≥ 2.07. the repeat donors took iron supplements for 4 weeks and the same tests were repeated after 2 and 4 weeks to evaluate their effects and the side effect and compliance was assessed. results: a total of 53 male and 57 female repeat donors were recruited, and 30 each male and female ft-ra donors were recruited to the control group. after 4week iron supplementation, among male donors, the prevalence of: low hb level (<13.0 g/dl) decreased from 24.5% to 1.9%; low ferritin level (<15.0 ng/ml) decreased from 58.5% to 3.8%; high tibc level (>380 lg/dl) decreased from 66.0% to 37.5%; low transferrin saturation (<20.0%) decreased from 58.5% to 35.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 28.3% to 2.3%. among female donors, the percentage of: low hb level (<12.0 g/dl) decreased from 43.9% to 9.8%; low ferritin level (<15.0 ng/ml) decreased from 78.9% to 11.8%; high tibc level (>380 lg/dl) decreased from 68.4% to 24.5%; low transferrin saturation level (<20.0%) decreased from 70.2% to 22.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 94.7% to 44.4%. in total, 15 male (28.3%) and 29 female (56.9%) blood donors reported undesirable side effects related to iron supplementation. a total of 38 male (71.7%) and 36 female (70.8%) blood donors were administered iron supplementations for 28 days. 89 participants (85.6%) answered that they were willing to take a complimentary iron supplementation. summary/conclusions: ferritin level, considered a reliable indicator of iron status, increased and ide decreased significantly after iron supplementation in female donor group, but not in male donor group, compared to the ferritin levels and ide of control donors. iron supplementation in repeat donors at a high risk of iron deficiency was shown to reduce their risk of iron deficiency or anemia irrespective of gender; however, 4-week oral iron supplement was not enough to restore iron storage level in the male donor group. background: c-reactive protein (crp) is an acute-phase protein and a non-specific maker of inflammation and tissue damage produced by the liver. several prospective epidemiologic studies have demonstrated that high-sensitivity c-reactive protein (hs-crp) is a predictor of future coronary events among apparently healthy men and women, hs-crp level greater than 3 mg/l has been independently associated with a 60% excess risk in incident of coronary heart disease (chd) as compared with levels less than 1 mg/l. frequent blood donation has been associated with a lower incidence of coronary artery disease (cad); however, there is a dearth of information on serum levels of crp in the nigerian donor population. aims: to investigate whether regular blood donation is associated with lower serum hs-crp level in nigerian blood donors. methods: a descriptive cross-sectional study carried out to measure serum levels of high sensitive c-reactive protein (hs-crp) and ferritin among blood donors attending the donors' clinic in lagos university teaching hospital (luth). subjects who did not meet criteria for blood donation were excluded. additional data on sociodemographic characteristics was collected using interviewer-administered questionnaire. serum ferritin was analysed using chemiluminescent microparticle immunoassay performed on the abbott architect ci4100 (abbott laboratories, abbott park, il, usa). serum concentration of hscrp was estimated by immunoturbidimetry method using analytical kits from erba diagnostics mannheim gmbh in semi-autoanalyzer (xl 180, erba mannheim). data was analysed using stata version 15 (stata corp) statistical software. results: in total of 313 blood donors, 278(90.8%) were males and 28(9.2%) were females, the mean age was 32.3 ae 9.3 years. two hundred and thirty four (74.8%) were first time donors and 79(25.2%) were regular donors, serum levels of hs-crp was slightly higher in regular donors compared to first time donors (0.95 ae 3.7 vs 0.35 ae 1.7 mg/l, p = 0.06) though the difference was not significant. serum levels of ferritin was significantly higher in first time donors compared to regular donors (87.56 ae 23.9 vs 46.44 ae 31.4 ng/ml, p = 0.02). interestingly, levels of serum hs-crp were significantly higher in male than female population (0.52 ae 2.4 vs 0.17 ae 0.2 mg/l, p < 0.03) and smokers than non-smokers (1.4 ae 4.6 mg/l vs 0.4 ae 1.9 mg/l, p = 0.02). correlation analysis showed no correlation between serum hscrp and serum ferritin levels in both categories of donors while there was a weak positive correlation between hs-crp levels and white blood cells among the first time donors. summary/conclusions: this present study did not reveal any decrease in baseline levels of serum hs-crp with regular blood donation; smoking status and gender were however associated with an increase in baseline hscrp. this finding suggests that hs-crp level might not be a useful marker of future coronary events in healthy blood donors in nigeria. background: because the blood donation removes 250 mg of iron from the donor, iron deficiency, frequently occurs in regular blood donors leading at a long term to the anemia. aims: to determine the effect of blood donations on ferritin levels in regular blood donors. methods: all prospective donors have been submitted to a physical examination and a health history assessment intended to ensure that the prospective donor is in a good general health and eligible to donate blood. the acceptance criteria are: • hemoglobin > or = 13.5 g/dl for male and > or = 12.5 g/dl for female • inter donation interval = 56 days • 5 donations/year for male and 3/year for female all eligible donors and deferred donors for all reasons except for low hemoglobin who accepted to enroll in this study and signed a consent. in addition to the medical exam, two samples have been collected one for cbc and another for ferritin. donation history, sex, age and weight have been documented. results: 1056 first time and regular donors accepted to enroll in this study. only 38 female donors (3.6%) participated to this study. 11.3% of the participants were first time donor. 21% of male and 26% of female frequent donors are iron deficient 173 out of 1018 male blood donors were iron deficient (17%) with serum ferritin < 30 ng/ml. 98.8% were repeat donors. 7 out of 38 female donors were iron deficient (18.4%) with serum ferritin < 13 ng/ ml, all were repeat donors. 19.2% of repeat donors were iron deficient 3/180 of the deplete donors were first time donors summary/conclusions: frequent blood donors have higher prevalence of iron deficiency than first time donors. female donors have a slightly higher prevalence of iron deficiency than male donors. prevalence of iron deficiency in abu dhabi donor population is lower than the published data. changes need to be done on: increase inter donation interval or restrict the total number of allowable donations in a 12-month period for whole blood and red cells modifying donor hemoglobin requirements testing for serum ferritin iron supplementation donor education abstract withdrawn. background: haemovigilance procedures aim to guarantee not only the safety of the recipients of blood and its components but the safety of the donors as well. every adverse reaction that occurs during the donation of blood or its components can potentially be a threat to the health of the donor which can subsequently lead to the decision of the donor to resign from donating blood. aims: the aim is to analyse the type and the frequency of occurrence of adverse reactions among the donors donating blood or its components independently of the method of the donation. methods: we have analysed the number of collected donations and the number of adverse reactions in the years 2014-2018 in the group donors of aged 18-24. we have specified following adverse reactions: vasovagal response without fainting, vasovagal response with fainting, vascular reactions (bruises) and other (e.g. allergic reaction to the anticoagulant, loss of blood pressure due to hypovolemia). the analysis was made using data obtained from computer system blood bank which is in operation in blood center in pozna n, poland. results: in years 2014-2018 the total number of 1904 adverse reactions among the donors was recorded which is 0.39% of the total number of collected donations. 50% of the adverse reactions occurred in the group of donors aged 18-24. vasovagal response without fainting was the most common adverse reaction in the total number of reactions and totalled 50.2% of all adverse reactions. in the group of donors ages 18-24 it totalled 27% of all adverse reactions. the second most common type of adverse reactions was vasovagal syncope that totalled 29.8%, in the analysed group of donors 15.96%. vascular reactions (bruises) totalled 10.2% of all adverse reaction, in the analysed group 5.6%. the remaining adverse reactions totalled 9.8%. summary/conclusions: 1. vasovagal reactions (with and without fainting) were proved to be 2 most common adverse reactions in the group of donors aged 18-24 i.e. in the groups of donors just starting to donate blood. it seems reasonable to continue with further research into the reasons for the occurrence of this psychosomatic reactions. 2. it seems beneficial to provide constant educational activities of young donors regarding the preparation for the process of donation of blood and its components (proper nourishment, hydration as well as planning the time for scheduled donation long enough for a safe and pleasant procedure. 3. it seems beneficial to provide constant training for the medical staff involved in the process of donation regarding active observation of donors, proper conduct in the situation when the adverse reactions occur during the blood donation, ways to minimize the fear of donors, effective communication with the donors (explaining the process of blood donation, proper behaviour after the donation e.g. avoiding physical exercise or straining the arm). blood products -blood processing, storage and release background: the accumulation of microvesicles (mvs) in rbc concentrates during storage may be responsible for clinical symptoms such as inflammation, coagulation, and immunization. aims: our aims was to determine whether any of cd molecules responsible for important functions are present on the microvesicles, and if their expression level is dependent on the storage period of rbc units. additionally, by using cytometric analysis and phagocytosis visualization in a confocal microscope, we examined the interactions of donor monocytes with erythrocyte microvesicles, depending on their time of storage. methods: erythrocyte microvesicles were isolated from "fresh" (2 nd day) and "old" (42 nd day) stored rbc units. qualitative and quantitative cytometric analysis of these membrane structures was performed using the annexin v-fitc, anti-cd235a-pe antibody, and calibrated beads. the microvesicles were also visualized under a confocal microscope. the expression of the molecules cd235a, cd44, cd47, cd55, cd59, and of phosphatidylserine was analysed using flow cytometry. measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. results: the analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0.5 lm in the "fresh" and "old" blood samples. we observed a statistically significant increase in the number of microvesicles in the "old" units (201 ae 139 mvs per 1 ll), as compared to the microvesicles in the "fresh" (67 ae 53 mvs per 1 ll). at day 2, the microvesicles had elevated expression levels of cd47 and reduced expression levels of phosphatidylserine. significant changes were also observed in the case of cd55 and cd59 molecules. the expression of these molecules of vesicles isolated from "fresh" rbcs was lower than in the case of 42-day vesicles. the phagocytosis index was significantly higher (28.44%) for the microvesicles isolated from 42-day stored rbcs than for microvesicles from the 2background: platelet concentrates (pcs) are conventionally stored at room temperature with a limited shelf-life of 5-7 days. alternative storage methods, such as cold storage and cryopreservation are attractive options due to the potential for extended storage, reduced bacterial growth and improved hemostatic function. cryopreservation of human pcs has been associated with formation of more microparticles and elevated procoagulant activity compared to liquid-stored (room temperature-and cold-stored) pcs. microparticles are submicron plasma membrane particles that have been postulated as potential mediators of adverse transfusion outcomes. similarities in the size and storage-related changes up to 7 days suggest that sheep may be a suitable model in which to investigate the effects of pc transfusion. previous research has established that room temperature stored sheep pcs contain fewer microparticles than human pcs. however, nothing is known of the effect of other storage conditions. aims: this study aimed to determine whether cold storage and cryopreservation contribute to variation in concentration and size of sheep platelet derived microparticles compared to conventionally stored sheep pcs. methods: sheep buffy coat derived pcs in 30% plasma/70% ssp+ were prepared with minor modifications to standard procedures for preparation of human pcs. sheep pcs were split into 3 units (n = 6 of each) on day 2 and stored either at room temperature (rt; 20-24°c with agitation) for 5 days, cold stored for 21 days (2-6°c no agitation) or cryopreserved (à80°c with the addition of 5-6% dimethyl sulfoxide) for 33-109 days and sampled post-thaw. platelet supernatant, prepared by double centrifugation, was stored at à80°c. the mean size and concentration of microparticles were measured using nanosight ns300 nanoparticle tracking analysis system (malvern instrument). results are mean ae standard deviation. storage associated changes overtime were determined using a one-way analysis of variance with bonferroni's post-test. paired t-tests were applied to determine the effect of cryopreservation. a p-value of < 0.05 was considered significant. results: at day 2, sheep pcs had a microparticle concentration of 3.77 9 10 10 ae0.84 9 10 10 microparticles/ml with a mean size of 156.6 ae16.7 nm. storage duration at rt sheep pcs was not associated with significant changes to microparticle concentration or size. cryopreservation of sheep pcs significantly increased the concentration (4.39 9 10 10 ae0.81 9 10 10 microparticles/ ml; p = 0.0087) and the mean size (170.9ae 20.5 nm; p = 0.0008) of microparticles post-thaw. the mean size and concentration of microparticles in the cold-stored pcs at day 21 was comparable to room temperature pcs stored for 5 days (165.4 ae17.5 nm vs. 163.6 ae20.9 nm; p = 0.4081 and 4.11 9 10 10 ae 0.54 9 10 10 microparticles/ml vs. 3.82 9 10 10 ae0.71 9 10 10 microparticles/ml; p = 0.16 respectively). summary/conclusions: cold storage of sheep pcs did not impact formation of microparticles over the 21 days storage period; however, cryopreservation increased microparticle concentration and the size post-thaw. further investigation is required to determine whether these findings are influence hemostatic function. a pre-clinical sheep model of cold-stored and cryopreserved pc transfusions can facilitate mechanistic studies and complement clinical trials. background: during storage, the properties of rbc in storage solution change ("storage lesion"). for instance, ph, atp and 2,3-dpg concentrations decrease upon prolonged storage. these changes can affect oxygen delivery by the cells. the capacity to deliver oxygen is defined as p50: the oxygen tension (po2) at which 50% of the hemoglobin is saturated with o2. an oxygen dissociation curve (odc) represents the non-linear relationship between saturated hemoglobin and po2. this relationship is dependent on temperature, ph, pco2 and 2,3-dpg. due to changes in these factors, the curve will shift along the x-axis. in whole blood, p50 is at a po2 of about 26 mm hg. not much is known about p50 of rbcs in storage solution, and the changes during storage. aims: to determine the oxygen dissociation of rbcs stored in standard red cell additive solution sagm and in pagggm (an experimental red cell additive solution, transfusion. 2010;50:2386-92). methods: rbcs were prepared in sagm (n = 3) or pagggm (n = 3). pagggm is designed to better maintain both atp and 2,3-dpg during storage. rbcs were stored at 2-6°c and sampled on day 1, 35 and 56 for (internal) ph, atp, 2,3-dpg and p50. p50 was determined by hemox analyzer (tcs scientific corp.). the principle of the hemox is based on the measurement of spectrophotometric properties of hemoglobin at different oxygen pressure. rbc samples were brought from oxygen-rich environment to oxygen-poor environment (2%) using n2 gas. p50 was determined from the obtained odc. results: the whole storage period, ph i of pagggm-rbcs was higher compared to sagm-rbcs. 2,3-dpg content of sagm-rbcs decreased during storage and was below the detection limit after day 14. 2,3-dpg content of the pagggm-rbcs increased the first days of storage and slowly decreased from day 7 on. at day 56, pagggm-rbcs still contained 2.3-dpg (2.2 lmol/g hb). p50 values decreased during storage from 26 mmhg at day 1 to 20 mmhg at day 56 for sagm-rbc and from 31 mmhg to 26 mmhg for pagggm-rbc. p50 values of pagggm-rbcs were higher during the entire storage period. summary/conclusions: during storage, the p50 decreased in all rbcs. the p50 was higher for the pagggm-rbcs during the whole storage period. the higher p50 in pagggm-rbcs seems to correlate with the higher 2,3-dpg content in these cells. background: in belgium 100% of the platelets are pathogen inactivated (pi) and legislation requires a minimum platelet content of 3.0 9 10 11 per platelet concentrates (pc). therefore routine pools are produced with 6 buffy-coats (bc). facing increased demand of pc and stable to slightly declining red blood cells (rbc) demand, production of whole blood (wb) derived platelets must be adapted to switch flexibly from 6 to 5 bc per pool. this dual pooling strategy should allow alignment between wb collection forecast, pc inventory, pc demand and pc production. aims: first develop a pooling procedure with 5 bc and 250 ml platelets additive solution (pas) instead of 280 ml for 6 bc, without changing the settings of our wb separators and platelets separators. maintain a content of ≥ 3.0 9 10 11 platelets with a ratio plasma/pas between 32 to 47% required for pi. after validation, deploy a dual pooling strategy (5 or 6 bc/pool). methods: wb is collected with top and bottom kit (composelect; fresenius kabi) and separated (macopress; macopharma) to produce 44 ml bc with 40% haematocrit (htc) and > 90% platelets recovery with average platelets content of 1 9 10 11 . 5 random bc are pooled with 250 ml or 6 bc are pooled with 280 ml of pas-e, platelets are then extracted on tacsi pl (terumo bct) and pc are treated for pi (intercept blood system; cerus). each pc is sampled and platelet content is determined (abx pentra xl 80; horiba). results: during the study 45594 bc were processed into 8225 pools (3756 (45.7%) with 5 bc and 4469 (54.3%) with 6 bc). before tacsi separation, bc mixture with pas-e had volumes of 421 ae 9 ml (5 bc) and 491 ae 8 ml (6 bc) with respectively htc of 19 ae 1% and 20 ae 2%. the plasma/ pas ratio was 37 ae 1% in both cases. tacsi separation was performed with one same program for both types of pools. after pi, platelets content of the pools was 3.8 9 10 11 ae 0.5 with 5 bc and 4.7 9 10 11 ae 0.5 with 6 bc (average ae standard deviation). pools below the limit of < 3.0 9 10 11 were 139/3756 (3.7%) with 5 bc and 1/4469 (0.02%) with 6 bc. the platelets concentrations (10 3 /ll) were 1395 ae 167 (5 bc) and 1491 ae 155 (6 bc). platelets recovery was 85% ae5 for 5 bc and 86% ae 6 for 6 bc. summary/conclusions: 45594 bc could theoretically produce 7599 pools of 6 bc or 9118 pools of 5 bc. this means a maximum potential gain of + 20% pc. in practice during shortage periods we switched from 6 to 5 bc when dictated by the actual inventory levels and hospital needs. the advantage of this dual pooling strategy was a gain in production capacity to cover these shortage periods (626 pc, +8%). the disadvantage of pooling randomly 5 bc is that 139 pools contained less than 3.0 9 10 11 platelets per pool potentially limiting their usage to low weight or paediatric patients. a preselection of the bc based on platelet count could optimize the 5 bc pooling procedure. background: apheresis-derived platelet concentrates (apcs) is a standard medical therapy indispensable to contrast bleeding or hemorrhage. however, bacterial infection caused by storage at room temperature (rt) still remains the major drawback. recently, we showed that cold-stored apcs are associated with better plt functionality but with accelerated clearance (haematologica 2018, pmid: 30115655). cold-induced apoptosis was identified as a potential mechanism of the shorter plt survival aims: to investigate the protective effect of apoptotic inhibitors during cold storage of apcs methods: apcs were collected and stored at rt and 4°c in the presence or in the absence of caspase-3 inhibitor. the phosphatidylserine exposure and the mitochondrial membrane potential (mmp) (tetramethylrhodamine ethyl ester perchlorate [tmre ] staining) were measured using flow cytometry. the protein expression was quantified by western blot results: a higher expression of the apoptotic marker phosphatidylserine was detected in cold-stored apc compared to rt (% apoptotic events meanaesem: 13 ae 1% vs. 5 ae 1% p = 0.018). to verify if the apoptotic signal, observed with phosphatidylserine, specifically involved the intrinsic pathway, the mmp was analyzed as a marker of alive cells. interestingly, after cold storage a decrease of the mmp was observed compared to rt indicating the activation of the intrinsic pathway (mean fluorescence intensity tmre meanaesem: 6.13 ae 1.89 vs. 18.53 ae 3.64, p = 0.038). accordingly, a decrease of the procaspase-3 level after cold storage was detected by western blot analysis. however, when plts were stored in the presence of caspase-3 inhibitor a significant rescue of the cold-stored cells viability was observed (tmre staining: % alive cells meanaesem: 74 ae 3% vs. 22 ae 3%, caspase 3 inhibitor vs. ionomycin, p = 0.005). this indicates that the activation of the apoptotic pathway, induced during cold storage, can be prevented using caspase inhibitor summary/conclusions: our results show that the reduction of cold-stored plt viability can be prevented by a specific caspase inhibitor. consequently, cold storage, associated with a better plt functionality, may become an efficient strategy for apc storage in combination with apoptotic inhibitors background: gamma-irradiation is used to treat red blood cell (rbc) concentrates (rccs) for patients who are immunosuppressed. this treatment is known to damage rbcs and to increase storage lesions. one of the causes of the storage lesions is the presence of oxygen. several studies have shown, based on different strategies to reduce o 2 , a reduction of storage lesions related to metabolism, protein modifications and cell morphology. aims: the present research work investigated the effect of gamma-irradiation on rccs stored under normal condition and hypoxia/hypocapnia. methods: saturation of o 2 (so 2 )-and abo-matched rccs from whole blood donations, leukoreduced and prepared in paggsm (macopharma, france) were pooled and split in two identical rccs within 24 h post-donation. one bag (treated) was submitted to oxygen and carbon dioxide adsorption (oxygen reduction bag, hemanext, usa) for 3 h on an orbital shaker (50 rpm) at 22°cae2 and then transferred to a storage bag impermeable to gas. the other one (control) was left as it is. the two bags were then stored at 4°c. a g-irradiation treatment (25 gy, gammacell 3000 elan, theratronics) was applied at day 2 or 14 and the rccs (expiry dates at day 16 or day 28, respectively) were stored until day 43. hematological parameters, glycolytic metabolites, extracellular potassium level, antioxidant power, morphology and deformability were measured. results: starting so 2 values were of 63.7%ae18.4 (n = 12) in control and of 20.8%ae9.8 (n = 12) in treated bags, and reached 90.8%ae9.1 and 6.6%ae5.9 at day 43, respectively. as expected, an increase in glycolysis rate was observed during deoxygenation without any influence from the irradiation. potassium levels were identical in treated and control, and reached around 70 mm at expiry with an irradiation-dependent kinetic release. antioxidant power and deformability were identical in both conditions. no difference in hemolysis was observed after irradiation on day 2 and the values stayed equivalent through end of storage (at day 16, hemolysis (control) = 0.37%ae0.24, hemolysis (treated) = 0.34%ae 0.15, p-value > 0.9999). when irradiated at day 14, hemolysis was lower (p-value = 0.033) in treated rccs at the end of storage (day 28, 0.67%ae0.16) compared to control (1.06%ae0.33). seven days post-irradiation, two-third of the control rccs were above the limit of 0.8% whereas all the treated rccs remain below the limit. quantification of microvesicles and morphological analysis confirmed these data. summary/conclusions: the storage under hypoxia has a beneficial effect on rbc storage thanks to a decrease in o 2 content and to an improvement of metabolism. this benefit provided equivalent storage when rccs were irradiated at day 2 and was an advantage when irradiated at day 14. importantly, the results show that combining irradiation with hypoxia/hypocapnia retained the improved hemolysis profile of o 2 depleted rbc. in summary, the reduction of o 2 level in rccs enables a better storage of rcc when a late irradiation is applied. background: in vitro blood circuit machines require a constant monitoring of blood flow rate which have to be maintained at a constant value. also, measuring the hematocrit of flowing blood in such machines is essential for performing real-time diagnostics. recently, acoustophoresis has emerged has a promising blood separation technology capable of replacing centrifugation for the preparation of platelet concentrate. to avoid damaging blood cells, the technique is used without infusing pumps thus increasing the need of flow monitoring. however, acoustophoresis chips performs at low flow rates, outside the range of available commercial flow meters. in addition, hematocrit measurement is of a particular interest for acoustophoresis since it is a direct indicator of the separation efficiency. aims: in this study, we present a straightforward doppler ultrasound system designed for measuring blood flow rate and hematocrit in an acoustophoresis chip [bohec et al, platelets, 2017] . we show that the stability of the in vitro environment can be used to obtain high level of accuracy of the doppler method using a basic and low-cost experimental set-up. this improvement allows a precise measurement of flow rates as low as 0.5 ml/min in sub-millimeter tubing. furthermore we evaluate the capability of the system to measure hematocrit of human blood samples coming from different donors. methods: the experimental set-up was constituted of an ultrasonic continuous wave doppler probe mounted on a 3d printed support. the accuracy of flow rate measurements between 0.5 ml/min and 1.5 ml/min was evaluated as well as the optimal measurement time. for 4 different blood bags, the relationship linking the total energy of doppler signals and hematocrit was derived. hematocrit in a range under 8% was estimated from doppler signals for each blood bag. results: the system is able to acquire exploitable doppler signals for the whole flow rate and hematocrit range. flow rate estimation from the signals shows a high accuracy with a mean measurement error under 3% for a measurement time of 2s. the mean error is still under 5% for a measurement time of 0.5s. hematocrit estimation from doppler signals shows a good linear correlation with reference measurements for bags 2, 3 and 4. hematocrit estimation for bag 1 diverges from reference for values above 6%. summary/conclusions: the proposed doppler ultrasound system is capable of measuring low blood flow rate in narrow medical tubing with a high accuracy. it is particularly suited for an acoustophoresis device but the versatility of the system makes it easily applicable to any in vitro blood circuit. we furthermore demonstrated that the system can be used for measuring hematocrit under 8% without additional developments. this finds interesting applications in blood sorting technologies but also demonstrates that doppler ultrasound is a potential simple and low cost method for measuring hematocrit of flowing blood in vitro. background: hereditary hemochromatosis (hh) is the most common genetic disorder in populations of northern european descent manifesting with high levels of storage iron (ferritin) in blood and tissues. the standard treatment is serial therapeutic phlebotomy to decrease iron overload. the collected blood is frequently discarded but some blood banks allow "healthy" hh patients to donate blood for patient use. red cell concentrates from hh donors have been reported safe for transfusion, but little or no data is available on platelet concentrates from hh donors, including the potential contribution of surplus iron to the "platelet storage lesion". aims: the aim of this study was to compare platelet quality, activation and aggregation over seven-day storage in platelet-rich plasma from patients with newly diagnosed hh and from healthy controls. methods: whole blood (450 ml) was drawn into compoflow blood bags containing cpd and sag-m from 10 healthy controls and 10 newly diagnosed hh patients. platelet-rich plasma (prp) was prepared from whole blood and split into four compo-flex bags each containing 20 ml prp (range 270-320 9 10 9 platelets/l). platelet quality tests were performed on days 0, 1, 3, 5 and 7 of storage. platelet aggregation was tested using a chrono-log aggregometer and four agonists (adp, arachidonic acid, collagen, and epinephrine). platelet expression of cd41, cd42b, and cd62p was measured with flow cytometry while ph and metabolites were measured with a blood gas analyzer. scd40l and scd62p in the supernatant were quantified using enzyme-linked immunosorbent assays. results: both hh and control groups included 7 males and 3 females. the mean age was significantly lower in the control group, 35 years (22-55 years), than in the hh group, 55.7 years (29-77 years) (p = 0.004) while ferritin levels were significantly higher in hh patients (median 847.5, range 498-1889) than in controls (median 45.8 ng/ml, range 9.28-97 ng/ml) (p < 0.001). in the hh group, 5 each had the c282y/c282y and c282y/h63d genotypes. results of prp quality control tests were comparable between the two study groups over seven days of storage (p < 0.05) with the exception of glucose (higher in hh patients on all time points, p < 0.05). platelet aggregation and the expression of activation markers (cd62p and cd42b) on platelets and in the supernatant (scd62p and cd40l) were comparable between hh and control prp units over all seven days of storage. the analysis revealed comparable and expected alterations in metabolic and platelet activation markers over seven-day storage in both groups. ph increased, glucose decreased, and lactate increased over time while cd42b expression decreased and cd62p increased. platelet aggregation responses decreased during storage but to a varying degree depending on the agonist, however, the decrease was comparable in cases and controls. summary/conclusions: these results suggest that high iron stores in hh do not adversely affect the quality of platelet units produced from hh patients. furthermore, the data also suggest that blood from hh patients, including platelets, can be donated for patient use. background: platelets are often shipped over long distances from collection centres to blood processing centres and subsequently to hospitals. platelet agitation facilitates oxygen transfer, thus promoting aerobic metabolism, and maintaining platelet ph. during shipment, platelets cannot be agitated continuously, which may promote anaerobic metabolism. previous studies have examined the effects of prolonged periods without agitation on apheresis platelets collected in plasma, but not platelets in platelet additive solution (pas). it is therefore important to determine whether platelet quality and function are maintained during prolonged transport or hold time in a shipper. aims: the aim of this study was to evaluate the effects of prolonged storage without agitation on the in vitro quality of apheresis platelets in pas. methods: triple dose apheresis platelets (n = 16) were collected using a trima accel platform in 40% plasma/60% pas (ssp+). after resting for 1 h, platelets were split equally into three components, packed into a shipper and transported immediately to the blood centre. upon arrival, one of the platelet components was removed (<6 h; t0), and the others remained within the shipper, without agitation. the second component was removed at 6 h post-collection (t6), and the third was removed at 23.5 h post-collection (rounded up to 24 h; t24). platelets were tested on day 1, 5 and 7 post-collection and in vitro quality and function were monitored. data were analysed using a two-way repeated measures anova, where a p-value of < 0.01 was considered significant. results: platelets held without agitation for 24 h consumed significantly more glucose than those removed at 6 h or immediately upon arrival (p < 0.0001), even on day 1 post-collection. this was accompanied by increased lactate production (p < 0.0001), indicating increased anaerobic glycolysis. consequently, the ph was significantly lower in t24 platelets (p < 0.0001), and on average it was 0.4 ph units lower than in platelets held in the shipper for 6 h or less. however, the ph remained above 6.9 in all components. mean platelet volume was also reduced in t24 platelets (p < 0.0001), suggesting acceleration of the platelet storage lesion. phosphatidylserine exposure, surface expression of cd62p and microparticle generation were significantly higher in the t24 platelets throughout the storage period (all p < 0.0001), suggesting platelet activation. release of scd62p was also increased in t24 platelets (p = 0.0006), whereas extended storage in a shipper did not affect release of rantes (p = 0.4488). adp-induced activation of glycoprotein iib/iiia, measured by pac-1 binding, was decreased in t24 platelets (p < 0.0001), indicating reduced platelet responsiveness to agonist stimulation. additionally aggregation in response to collagen (p = 0.0001) and adp (p = 0.003) were significantly lower in t24 platelets, suggesting a decrement in platelet function after prolonged storage without agitation. summary/conclusions: significant in vitro changes were observed in platelets held without agitation for 24 h. these results suggest that the length of time that platelets are held in a shipper should be minimised where possible. background: the shelf-life for platelet products has been restricted to 5 days. this very limited window of time is intended to sustain the quality of platelet and to reduce the risk of bacterial growth. we have recently demonstrated that in suitable platelet bags, the platelet product quality remains high after 5 days of storage. this was proved by examining in vitro, the quality parameters of platelets such as platelet concentration, glucose, ldh, and ph (alexopoulos k. et al., haema, 9, 2018) . our new target is to extend this research in 2 extra days of storage. we also want to determine if there is any bacterial development in this period. aims: the goal is to investigate the capability of storage period for platelet units, from 5 to 7 days. methods: in this study, platelets were collected from 11 normal blood donors in the blood bank department of general hospital of patras "agios andreas". a total of 450 ae 10 ml of whole blood was drawn into triple cpd/sag-m top-top bags blood container systems, lmb technologie (gmbh). the platelet concentrates were prepared by platelet rich plasma (prp) method and then they were placed in a platelet incubator with agitator (helmer pc 1200). samples were drawn aseptically with a needless access coupler (cair-lgl) on days 1, 5, and 7. platelet count was done by ceeldyn ruby (abbott all data shown are reported as mean ae standard deviation (sd). the swirling effect remained positive (+) during the seven days storage period. the bacterial screening was found negative. summary/conclusions: platelet concentration in the bag remained constant between day 5 and day 7, maintaining platelet yield. the decrease in glucose and increase in lactate, along with the decreased ph, show that the platelets remain metabolically active between days 5 and 7 of storage. the ph remained well within the acceptable range. no bacterial contamination was reported. thus, we conclude that platelet concentrates in these specific bags may be used with an extended shelf life of 7 days. further studies are needed with other platelet bags to confirm our hypothesis. abstract withdrawn. aims: we introduce rt-dc as a fast, robust and unbiased quality control tool for pc, rcc and hpsc. utilizing the interdependency between cell deformation and the molecular state of the cytoskeleton, we demonstrate that rt-dc is capable to assess the quality of blood products. methods: by rt-dc we assessed: i) platelets after storage at 4°c or room temperature (rt) over 10 days for 4 apheresis pcs in addition to standard in vitro platelet function assays; ii). red blood cells before and after gamma irradiation in addition to hemolysis; and iii) hpsc after cryopreservation with 5% or 10% dmso in addition to cell count, and in vitro viability. in addition we compared the regeneration time of patients' platelets and leukocytes after transplantation of hpsc products containing either 5% or 10% dmso. results: for pcs standard quality assurance tests did not show a major difference between 4°c and room temperature storage while rt-dc showed a highly significant difference between both start conditions (day 1-7, p < 0.001 and day 10, p < 0.02). for red cells, we found by rt-dc no impact of gamma irradiation with 30 gy over the entire storage period of 42 days assessing 3 different rcc. for hpsc, rt-dc showed that cryopreservation in liquid nitrogen resulted in a significant increase in deformation (0.052 for 5% dmso versus 0.034 for the control without dmso; p < 0.00004). however, this did not differ to high extent whether 5% or 10% dmso were used for cryopreservation (0.035 and 0.041, respectively; p < 0.02). hpsc viability was lower after cryopreservation using 10% dmso in comparison to using 5% dmso. overall, blood cell regeneration is comparable between 5% and 10% dmso. summary/conclusions: studying platelet and red blood cell concentrates as well as hematopoietic stem cells under different, clinically relevant, storage conditions our results demonstrate that intrinsic material properties reveal insights into cell function and allow to predict cellular state in a robust way and using small sample volumes. in order to offer more flexibility to the production process, the storage of bcs overnight (16 h) has been validated in our blood center. aims: the aim of the study was to assess the platelet quality in platelet concentrates derived from overnight stored buffy coats. methods: whole blood collected at day 0 was separated into plasma, bc and red cell concentrates either at day 0 or at day 1. bcs were then stored until the pooling step at 20°c without agitation and pcs were prepared at day 1 by pooling 5 isogroup bcs. seven "16 h-pcs" were prepared from bcs stored for 16 h (whole blood separation at day 0) and six "90 min-pcs" from bcs stored for 90 min (whole blood separation at day 1). standard quality control measurements were performed during the process and the storage. in addition, the quality of the platelets into the prepared pcs was assessed throughout the period of storage by measuring the hypotonic shock response (hsr) and by measuring by flow cytometry the proportions of platelets in apoptosis (marked with annexin v), of functional platelets (marked with cd42) and of activated platelets (marked with cd62 the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment (amotosalen and uva) with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the ipp pooling and leukodepletion set developed by kansuk. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. methods: 32 dd-bc-pc were prepared with 8 bc and 280 ml of pas (intersol, fresenius kabi (germany) are sterile docked to the octopus harness and combined into a 600 ml pooling bag. the pool is centrifuged and the pc supernatant expressed through a bioflex cs leukodepletion filter into a temporary platelet storage container. the obtained dd-bc-pc were tested within 1 h of preparation and after storage for 8 h in the platelet storage container for volume, platelet content, residual leukocytes (wbc), plasma ratio and biological parameters, ph, po 2 , pco 2 , glucose, lactate, mpv, ldh, p-selectin and swirling. results: the platelet content of dd-bc-pc (n = 32) was on average 6.9 ae 0.3.10 11 in a volume of 393 ae 6 ml. the mean of plasma ratio was 42% [min: 39.3max: 43.9]. all pc contain <1.10 6 wbc [min: <lq; max: 0.38]. po 2 decreased from 166 ae 12 mmhg after separation (1 h) to 44 ae 18 mmhg after 8 h, ph (+22°c) and pco 2 was stable during the same time period. glucose decreased from 9.0 ae 0.3 mmol/l (1 h) to 8.7 ae 0.3 mmol/l (8 h) while lactate increased from 6.07 ae 0.46 mmol/l (1 h) to 6.57 ae 0.54 mmol/l (8 h). the vpm was stable with 7.6 ae 0.2 at 1 h and 8 h. ldh increased from 120.4 ae 8.6 u/l at 1 h and 125.8 ae 10.3 u/l at 8 h as p-selectin 45.7 ae 7.6 ng/ml at 1 h and 57.7 ae 7.0 ng/ ml. all units have maintained swirling. the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the platelet pooling and leukodepletion set developed by fresenius. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. background: the irish blood transfusion service (ibts) is the statutory body with the responsibility for ireland's national blood supply. in 2018 the ibts collected 130,183 whole blood units and performed 9,654 platelet apheresis procedures. blood is collected in three fixed and six mobile sites and is returned overnight in temperature controlled vans to a single processing site. in september 2017, the ibts implemented the tacsi automated system for the preparation of buffy-coat derived platelets as an alternative to the orbisac system. aims: the aim of this study is to compare operational aspects and quality data of buffy-coat derived platelets prepared by the tacsi (t-pcs) and orbisac (o-pcs) automated systems. methods: qc data of t-pcs and o-pcs from years 2016 to 2018 were analyzed. in 2016, the ibts prepared 5,609 orbisac platelet pools of which 5,477 were qc tested. in 2017 5,031 orbisac and 1,625 tacsi platelet pools were prepared and tested. in 2018 6,783 tacsi platelet pools were prepared of which 4671 were qc tested. qc data comprised pc volume, platelet content (10 9 /unit), residual leucocyte content (10 6 /unit) and ph at expiry. feedback from the processing laboratory operators was collected comparing operational aspects of preparation of t-pcs and o-pcs. results: from the 10,508 o-pcs tested in 2016 and 2017, mean pc volume was 292 ae 9 ml. initially the t-pcs mean volume was 273 ae 13 ml but more recently the volumes were increased by addition of a larger volume of additive solution to 303 ae 13 ml. platelet content (10 9 /unit) was significantly improved in t-pcs (384 ae 47) compared to o-pcs (356 ae 50) [p < 0.01] as well as residual leucocyte content (10 6 /unit) which was lower in t-pcs (0.05 ae 0.04) compared to o-pcs (0.1 ae 0.2) [p < 0.01]. ph at expiry was lower in t-pcs (7.32 ae 0.07) compared to 0-pcs (7.38 ae 0.06) but remained within the quality requirements. from an operational perspective with two tacsi devices, five operators produce approximately 48 t-pcs per hour compared to a lower throughput of 20 o-pcs per hour with five orbisac devices. the tacsi system is more compatible with batch processing which aligns with our staffing arrangements in routine production and has resulted in greater productivity since its introduction. with tacsi implementation, we reverted to manual pooling of buffy coats and new staff had to undergo training to achieve optimal platelet recovery. once staff were trained during validation, we observed a 10% increase in platelet recovery. processing laboratory staff indicated that tacsi devices were easier and simpler to maintain in comparison to orbisac, but highlighted that some improvements to tacsi system boxes could be made to make the system more robust. summary/conclusions: pcs prepared with tacsi and orbisac automated systems both met national and european quality requirements, however we observed a significant improvement in quality markers of pcs prepared with tacsi. processing staff reported easier maintenance and processing with tacsi compared to orbisac. throughput is higher with tacsi and there is greater additional capacity to increase production in the future if required. a perez aliaga 1 , f puente 1 , a aranda 1 , j domingo 1 , l call en 1 and a bah 2 1 banco de sangre y tejidos de arag on, aragon, spain 2 terumo bct europe n.v., zaventem, belgium background: the blood bank and tissues of arag on (bsta) is responsible for the collection, processing and distribution of blood in the region of aragon in spain. with five fixed and five mobile sites, the bsta collects and processes 44,000 whole blood units per year. to improve quality of processed units and working conditions of personnel, the bsta decided to implement the reveos automated system. validation studies were carried out in november 2012 and routine use started in july 2013. in parallel, the bsta initiated pathogen inactivation of platelet concentrates (pc) in november 2012 to improve transfusion safety. in november 2015, the mirasol prt system was implemented due to simplicity of this platform. aims: the aim of this abstract is to (i) describe the results of the validation studies carried out during reveos implementation (ii) present routine use data of products processed with reveos and (iii) provide information on mirasol implementation. methods: in 2012, 369 whole blood units were processed using reveos. quality parameters for red cell concentrates (rcc), such as: hemoglobin (hb), hematocrit (hct) and volume; plasma: volume and residual platelet content; and pcs: volume and yield, were analyzed. a control group consisting of units processed with a semiautomated platform was used as comparison. a survey was sent to the collection and processing staff for feedback on operational aspects of the use of reveos. from 2013 to 2018, qc data of rccs (n = 3025), plasma (n = 660) and pcs (n = 660) processed with reveos were collected. number of mirasol treated pcs and hemovigilance data from 2015 to 2018 were collected. results: volume, hb and hct of rccs processed with reveos were significantly higher compared to control rccs (volume: 296 ae 21 ml vs 251 ae 19 ml; hb: 59.9 ae 6.2 g/ unit vs 49.2 ae 7.8 g/unit; hct: 59.5 ae 2.7% vs 56.1 ae 6.3%). control plasma units had higher volume (280 ae 17 ml) and lower residual platelet content (9 ae 7 10 9 /l) compared to reveos plasma units (volume: 249 ae 23 ml; residual platelet content 19 ae 13 10 9 /l). for pcs, volume was higher for units processed with reveos compared to control (350 ae 20 ml vs 310 ae 10), but no statistical difference was observed in platelet content (reveos: 361 ae 60 9 10 9 /unit; control: 386 ae 80.10 9 /unit). we observed either an increase or stabilization in qc parameters of rccs, plasma and pcs during the 5 years routine use of reveos. for example, percentage of rccs units hemolyzed dropped from 4% to 0% between 2013 and 2018 and platelet content in pcs increased from 370 9 10 9 /unit (first six months in routine) to 390 9 10 9 /unit. plasma volume increased from 249 ml during validation to 267 ml in 2018. survey results from staff highlighted the user friendliness of the reveos device. number of mirasol treated pcs increased from 10% in 2015 to 59.7% in 2018 with no pathogen transmission nor serious adverse events reported following transfusion of these products. summary/conclusions: with implementation of two user-friendly systems, the reveos whole blood automation system and mirasol prt system, we observed an increased standardization of our processes enabling us to provide higher quality and safer blood products to hospitals and the patients they serve. background: white blood cells (wbc) may be regarded as contaminants in blood components and potentially reduce transfusion safety. there is currently enough evidence showing that wbc reduction through pre-storage filtration reduces the incidence of three major complications of allogeneic blood transfusions: febrile nonhemolytic transfusion reactions (fnhtr), refractoriness to random-donor platelet transfusions and transmission of cmv. leukodepletion is now mandatory in canada and several european countries. aims: the aim of this study was to evaluate leukoreduction performance and the quality of the resulting red blood cell concentrate (rbc) produced with new quadruple system top & bottom imuflex crc with an integrated red blood cells filter which was used in whole blood fractionation under routine conditions of san paolo asl blood bank methods: twenty-seven whole blood donations (450 ml) were collected with using quadruple blood bags top & bottom system with an integrated rbc filter (imuflex crc, terumo bct). centrifugation and processing (using t-ace ii + -terumo bct) of whole blood units followed the blood bank sop to obtain rbcs, plasma and buffy coat. rbcs were diluted with sagm (100 ml) and then filtered by gravity in average 2-3 h after blood collection to obtain leukoreduced rbc. average temperature during filtration was 26°c. samples were collected before and after leukoreduction for calculation of hemoglobin loss through the filtration process. filtration time was recorded. final hb, hematocrit (hct), residual platelets and white blood cells were determined after filtration using standard laboratory methods (blood cell counter) results: wb donations (n = 27) were performed. rbc characteristics post-filtration and separation were as follows: volume (266.8 ae 17.1 ml), hematocrit (57.8 ae 2.1%) hemoglobin (19.8 ae 1.1 g/dl) and hemoglobin per unit (53.0 ae 5.4 g); residual platelets (11.7 ae 2.2 x10³/ll); residual wbcs (0 à 0.004 9 10³/ll). the mean filtration time was 19 ae 3 min and no filter blocks were observed summary/conclusions: the usability and performance of the new quadruple system top & bottom imuflex â crc with integrated rbc filter was evaluated. all quality parameters complied with italian and european guidelines and requirements. in conclusion, the set was found to be a reliable and efficient collection and separation system that performs consistently background: novel devices for automated whole blood processing (reveos tm terumo bct) have been installed in regional center for blood donation and blood treatment in katowice to modernize and automate production of blood components. this system allows to separation of four whole blood units into four buffy coat depleted red blood cell concentrates (rbc), four units of plasma and four interim platelet concentrate (ipu) units in one automatic cycle. an ipu is the starting blood component for the production of pooled, leucoreduced platelet concentrates (lpc). residual leukocytes (wbc) are obtained as a byproduct and discarded. the use of this system shortens production time because it combines otherwise two separate steps: centrifugation and blood separation in press. aims: to evaluate the quality of the blood components obtained with the newly developed reveos nlr kits, which are a whole blood collection kits containing no leucocyte reduction filter for the rbcs. methods: blood [wb] was collected into nlr reveos set (terumo bct), and processed between 2 and 8 h from collection using the 3c protocol. quality control was performed on rbc, plasma and ipu. rbc were tested for hemoglobin (hb) content, hematocrit (ht), residual white blood cells (wbc), volume and hemolysis level on the last day (42 nd day) of storage. the content of wbc and platelet count (plt), protein, factor viii on the day of production and after one month of storage were determined in plasma. for ipu, platelet and leucocyte counts, volume and average platelet yield index (pyi) were displayed at the device after processing was recorded. furthermore, a comparison between fully automated and semi-automated whole blood processing was done, by comparing the amount of time necessary to fractionate 4 whole blood units using the two different processes. background: direct, single step automatic blood separation devices (reveos tm terumo bct) was installed in regional center in katowice in 2018. the system allows for full automation of whole blood fractionation. one of produced blood components is an interim platelet unit (ipu), containing most of the platelets and limited of white blood cells (wbcs). it is then pooled through a filter to form a therapeutic unit of leucoreduced platelet concentrate (lpc). a rough estimate of the platelet yield in the ipu, the so-called platelet yield index (pyi) is calculated and displayed by the device after the end of the separation phase. it is a useful tool to optimize selection of ipu for pools to produce lpc. aims: to determine the quality of polled lpc obtained on the basis of ipu from whole blood processed up to 8 h after collection and the usefulness of this method in daily practice. methods: ipus used for production of lpc were obtained using fully automated whole blood processing single step system -reveos tm (terumo bct). from the beginning of the application of the method, 1005 therapeutic units of lpcs were produced after overnight hold: 38 pools of 4 ipus and 966 of 5 ipus were made using platelet pooling set (terumo bct), using 250 ml ssp+ solution (macopharma). results: 42 therapeutic units of lpc were analyzed. the mean volume of lpc was 387 ae 13 ml, platelet content was 3.67 ae 0.46 x10 11 , and white blood cells (wbc) count was 0.15 ae 0.13 x10 6 , 100% all lpcs had wbc below 1 9 10 6 . ph measurements were made on the fifth day of storage, the ph was determined in 15 units of lpc and was 7.2 ae 0.04. summary/conclusions: the use of interim platelet units in daily practice increases the possibility of producing high quality blood components for patients and is a great alternative to the laborintensive manual methods of lpc's production. background: the development of cold-stored platelets has been hampered by the rapid removal of blood after transfusion by apoptosis and desialylation due to platelet storage lesions caused by refrigeration. previous studies have shown that antioxidants inhibited the activation and desialylation of cold-stored platelets for 5 days and also inhibited rapid elimination after transfusion in animal models. aims: it was tried whether in long-term (10 days or longer) storage conditions, antioxidants showed similar effects inhibiting the activation and desialylation of cold-stored platelets. methods: platelet-rich plasma (about 250 ml) were obtained from a healthy blood donors of type o blood group and divided into a 30 ml platelet storage bag manufactured by the manufacturer, and the conditions including room temperature and refrigerated condition and various kinds of antioxidants were prepared, (ph, glucose, lactic acid) and activation index (cd62p, gpiba (cd42b), annexin v), degree of reactive oxygen species and sialidase activity on the 7th, 10th and 12th day, and phagocytosis using the hepg2 cell line and platelet recovery after in vivo transfusion using scid mice were compared and compared. results: cold-stored platelets showed autoaggregation on the 2nd day of storage, and apparent aggregation on the fifth day of visual observation but not in the antioxidanttreated group. as expected, the ph, glucose and lactate levels of the cold platelets were maintained and the platelets at room temperature changed significantly. nac (n-acetylcysteine) -treated platelets were less active on day 12 and sialidase activity was maintained lower than that of control group on day 12. the degree of phagocytosis by the hepg2 cell line remained at the same level during the storage period, and the in vivo recovery rate in the animal model was superior to that of the antioxidanttreated cold platelets for 2 h and 24 h after transfusion. summary/conclusions: cold platelets could be developed as clinically effective agents for up to two weeks if appropriate preservatives were added, and antioxidants, especially n-acetylcysteine, were effective. background: the reveos 3c procedure produces the following three blood components for transfusion: plasma, rbcs, and a single interim platelet unit (ipu). whole blood is collected into the reveos blood bag set and held at room temperature a minimum of 2 h before processing on the device. after processing on the reveos device, the plasma product is ready to freeze. the rbc product is ready to combine with sag-m additive solution and leukoreduce using the integrated leukoreduction filter. the ipus are rested and agitated for the recommended time. for example, when whole blood is processed within 8 h of collection, the ipu is agitated overnight before pooling. because the ipu is made from one unit of whole blood, approximately 4 to 6 ipus are pooled together and leukoreduced by filtration prior to storage in a platelet storage bag. this leukoreduced platelet pool (4 units) can be supplied to the demand of hospitals like a platelet apheresis product. aims: to assess the usability of the reveos automated blood processing system to collect units of whole blood and to process the units on the reveos device, producing blood components that meet product quality specifications. methods: this study will evaluate approximately 19 units of whole blood using the reveos system and approximately 4 units of pooled platelet products. a total of 19 units of whole blood (wb) have been collected into the reveos blood bag set and then held at room temperature without the use of an active cooling system (or temperature control system), such as cooling plates, or in this case, ice packs. of the 19 units, 10 units have been processed on the reveos device using the fresh procedure (within 8 h of collection). nine units have been processed using the overnight wbhold procedure. all units have processed successfully without alarms, resulting in three components (rbcs, plasma, platelets). we also evaluated four platelet pools created from eighteen interim platelet units (ipus). one ipu has been discarded due to a 22-min whole blood collection time. the leukocyte count on all component using the automatic sysmex-xn 2000 cell blood counter. results: the red blood cells meet the criteria for hematocrit, hemoglobin and residual wbc counts are less than 1 million per unit. the plasma units meet the criteria for displayed volume accuracy compared to measured volume and also for factor viii levels. the average residual platelets in plasma levels meet the criteria. the platelet pools meet the acceptance criteria for platelet yield and volume. limited data (n = 4) suggests that this criteria will be easily met, provided that wb hold conditions are suitable for platelets. summary/conclusions: final results indicate that products produced from the reveos system meet or exceed product quality expectations. staff report that the reveos system is very easy to use. abstract withdrawn. background: thorough manual mixing of overnight-held whole blood (wb) units prior to centrifugation to separate red cell (rc) from platelet-rich-plasma (prp) is currently practised at the hong kong red cross blood transfusion service (hkrcbts). the mixing step is crucial to maximise platelet yield but laborious, and may cause elbow and wrist injuries in operators. a laboratory shaker, reax 20 (heidolph instruments, schwabach, germany), originally designed for mixing solvents, was modified to allow mixing source wb units by the supplier. to enhance the well-being of the operators in the context of occupation health and safety (oh&s) management, feasibility of the application of such blood mixer to replace the manual mixing steps in blood component preparation was explored. aims: to evaluate the in vitro quality control (qc) parameters of the blood components prepared with the use of the modified mixer. methods: fifty-eight wb collections, including 40 units in 450 ml quadruple bags (450q) with/without integrated leucofilter and 18 units in 350 ml quadruple bags (350q), were processed in accordance with hkrcbts' prevailing component processing protocols except using the motor-driven mixer in lieu of manual mixing. wb collections were divided into 2 groups, mixed at 2 revolutions per minute (rpm) for 2 min (n = 30) and 30 min (n = 28), then processed into rc, prp platelet and plasma components. in vitro qc assessments including haematocrit (hct), haemoglobulin (hb) and haemolysis at expiry for rc; absolute platelet count and ph for platelet concentrates (plt); volume and residual platelet count for plasma units were evaluated and compared. results: all components derived from both 450q and 350q processed with the blood mixer fulfilled the qc requirements stipulated by aabb standards and council of europe (coe) guidelines. there were no significant differences for all the qc parameters in rc and plasma components produced when mixed for 2 or 30 min. summary/conclusions: the overall performance of blood components processed with the motor-driven mixer met the acceptance criteria at the hkrcbts in accordance with the aabb and coe standards. higher platelet yield was observed with prolonged mixing for 30 min. however, prolonged mixing will much delay routine component processing. further evaluation on intermediate mixing time such as 3, 5 or 10 min may help determine an optimal mixing duration to maximise platelet yield without compromising processing workflow. to conclude, using the modified blood mixer to replace laborious manual mixing in components preparation is promising to improve the oh&s for blood component preparation operators. background: leukocyte reduction is widely used to reduce the risk of non-hemolytic febrile transfusion reactions and alloimmunization by multiple blood transfusions. around and after year 2000, universal leukocyte reduction was adopted in many developed countries. there are mainly two types of filter, one to filter whole blood and the other to filter red cell concentrate (rcc) used during pre-storage blood processing. aims: in this study, we conducted the filtration tests with sepacell tm rs2 for rcc in comparison with sepacell tm r-s11 and sepacell tm pure rc widely used in the world. background: the cardarelli hospital blood bank processes over 40,000 whole blood units yearly. in order to constantly cope with the huge demand for platelets from oncohematology and intensive care departments, a progressive review of the processing protocols for platelet concentrates (pc) from buffy-coat (bc) pools has recently been implemented to reduce the number of units assembled, from 5 (routine in italy) to 4. the 4-bc pc units have slightly lower volume and plasma content, factors that further limit the transfusion risk. in addition, the 4-bc assembly allows to facilitate the production of the pc units obtained from the same blood type, in order to minimize both the adverse reactions related to the abo/rh incompatibility and the amount of bcs not used. finally, the described innovation also impacts on the workload, with reduction of assembly, centrifugation and separation time. aims: the aim of this work is to define an optimized protocol for the preparation of pc units from 4-bc pools, so that the platelet yield is always higher than the minimum value required by current regulatory (2.0 9 10 11 cells/unit). background: blood is a basic component for human life and hence blood transfusion is an integral intervention in the treatment of patients. the need of blood transfusion is increasing with decreasing blood supply due to limited blood donations. conversely wastage of blood products is also a topic for discussion. to ensure the availability of blood products when they are needed, wastage of blood products should be controlled and minimized. aims: the study was designed to investigate the quantity and reasons of wastage of blood products at our hospital. methods: this was an observational study based on the statistical data available from blood bank department of nibd and bmt, pechs campus, karachi, pakistan. the study period was from february 2018 to february 2019. study was approved from institutional review board. for all the blood products wastage and reasons of wastage were evaluated. frequencies were calculated by using spss version 23.0 results: a total of 2880 blood products were available at our institute including 960 each of platelets, pack red cells and fresh frozen plasma(ffp). the overall wastage rate was 3.5%. pack red cells and platelets were fully consumed yet shortage of supply was observed. however, highest wastage was observed in ffp in our blood bank i.e. 102(10%). results of investigating the common causes of blood product wastage at the hospital revealed that it was highly associated with 02 common causes, including the expiry of unused products 60(59%) followed by broken bags 30(29%). summary/conclusions: this study showed over all diminutive wastage rate. ffp wastage was highest among all the blood products. wastage of blood products is a genuine issue in a hospital setup, strategies and plan of action should be discussed and implemented including enhanced collaboration with other centres to outsource the blood products or even donate at times, education and training of staff for better clinical use of blood products and ensure the availability when and where they are needed most. background: in the early 2000s, the blood service of the republic of kazakhstan functioned along the lines of the soviet period: donation of blood was approached to the place of its use (carried out at hospitals), screening of donor blood was carried out at one stage (enzyme immunoassay) on analyzers of semi-automatic type, there was no mechanism calculating the cost of blood components, the activity of blood centers was funded at the rate of 1 liter of whole blood. donation was not popular in society, household fears of infection were common when donated. aims: research objective is to study the main stages of reforming of blood services of the republic of kazakhstan (rok). the research methods are based on the system analysis of the condition of the rok's blood services based on the indicators of production activity for the period of 2010-2017. results: in order to effectively reform the blood services, the following steps were taken. the blood supply was centralized and optimized. blood collection at hospitals was terminated, more than 200 blood collection subjects were closed. the result was a more efficient use of technical, human and financial resources, as well as ensuring the high quality of the blood components produced. today, there are 18 blood centers in the republicone for each of the 18 administrative territorial units of the republic. the screening of donor blood for transfusion infection markers has been transferred into a two-step principleimmunological analysis and nat testing. a fully automated analytical equipment of a closed type is used for screening, unified for all blood centers of the republic. unification of the equipment choice allowed to provide centralized service maintenance of complex equipment and its uninterrupted operation. since 2012, the national reference laboratory has been operating in the blood service, which conducts an external assessment of the quality of laboratory research in blood centers, as well as research in controversial and complex cases. introduced pathogen inactivation of platelet concentrates in 100% of cases of platelets. erythrocytes are used only in the weighing solution and only after leukoreduction. the use of resource-saving blood harvesting technologies, as well as methods to further enhance the infectious and immunological safety of components, are expanding annually. the foundations for the development of applied scientific research in the field of transfusion medicine were laid, and an own school of transfusiologists was created. as a result of reforming the blood services, the mechanisms of reimbursement of blood services costs from the budget of the republic have been changed, uniform tariffs have been defined by type of blood components for all blood components suppliers in the country. summary/conclusions: the transformations made it possible to create a blood service in kazakhstan that corresponds to the best international practice and ensures the quality, efficacy and safety of transfusion therapy. further development of the blood service is aimed at deepening reforms, studying and developing cellular technologies, new properties of plasma blood components, and improving the clinical practice of using donor blood components. background: reconstituted platelet concentrate (rpc) is a component that contains platelets (thrombocytes) resuspended in plasma that is blood group compatible with the recipient. it is used when transfusion of platelet concentrate is necessary but the fresh platelet concentrates of the specific blood group are not available. group 0 rh-(negative) platelets resuspended in the ab group plasma are of universal use as such components can be used for all recipients independently of their blood group. in the area of activity of regional blood center in poznan rpc is used most often for patients after stem cell transplant incompatible with their blood group. aims: the aim of the analysis is to evaluate the loss in the number of platelets in the reconstituted platelet concentrates in order to determine their quality and usage. methods: we used for the analysis 30 units of pooled platelet concentrate derived from buffy coats and 30 units of pooled platelet concentrate derived from the apheresis. for the reconstitution group ab plasma was used. we investigated the number of platelets prior to and after the reconstitution. following measures were calculated: number of platelets in pooled platelet concentrates derived from buffy coats, number of platelets in pooled platelet concentrates derived from apheresis; average values and the standard deviation as well as the loss in number of platelets due to the reconstitution. the number of platelets was calculated with impedance method using horiba pentra xl 80 analyser. 1. pooled platelet concentrates derived from buffy coats the number of platelets before the reconstitution: 4.12 9 10 11 /unit ae 0.50 the number of platelets after the reconstitution: 3.40 9 10 11 /unit ae 0.50 the loss in the number of platelets: 17.5% 2. platelet concentrates derived from apheresis the number of platelets before the reconstitution: 3.68 9 10 11 /unit ae 0.55 the number of platelets after the reconstitution: 2.91 9 10 11 /unit ae 0.49 the loss in the number of platelets: 20.7% summary/conclusions: 1. reconstitution results in the loss of 17.5% of platelets in comparison to the initial value of pooled platelet concentrate derived from buffy coats. 2. the average number of platelets after the reconstitution is 3.4 9 10 11 /unit, which fulfils the quality requirements. 3. reconstitution results in the loss of 20.7% of platelets in comparison to the initial value of pooled platelet concentrate derived from apheresis. 4. the average number of platelets after the reconstitution is 2.9 9 10 11 /unit, which slightly deviates from the quality requirements. 5. the results that we obtained show that the process of reconstitution always results in the loss of number of platelets and that the quality of platelet concentrates that have not been reprocessed is always higher. because of that we recommend that the reconstitution should only be performed in special cases such as for patients after stem cell transplant incompatible with their blood group and that hospitals should be supplied with platelet concentrates derived from buffy coats and apheresis. methods: pf4 was extracted from human pcs by using immunoaffinity chromatography and specific anti-pf4 antibody on the column. hereafter, pf4 was purified with concentration tubes having 5 kda molecular weight cut-off and dialyzed via dialysis tubing with 3.5 kda cut-off. pf4 concentration was measured by bradford method. specific enzyme-linked immunosorbent assay (elisa) and dot blot analysis were used to determine the pf4 specificity. molecular weight of obtained pf4 was determined by polyacrylamide gel electrophoresis (page). results: our data confirmed clearly that separated pf4 had 30 kda molecular weight which is corresponding with a tetramer pf4. in addition, elisa showed that the pf4 qualified true antigenicity. dot blot analysis helped us to assure the specificity of pf4. summary/conclusions: we used and optimized a homemade protocol to isolate and purify the pf4. nevertheless, there was no any scientific report among the databases that utilized the similar purification method. recombinant pf4 is more feasible and ready-to-use, but its price may discourage the researchers to study the biology of pf4. unused human pcs and specific anti-pf4 antibody can considered as an alternative to separate the pf4 particularly in labs with the shortage of resources. background: whole blood is increasingly used in civilian hospitals as an alternative to blood components in trauma resuscitation with massive bleeding. however, the hemostatic effect of whole blood depends on the initial platelet content and decreases rapidly within the first 2 weeks of storage at 4°c. consequently the problem arises of how to provide wb to trauma centers without losing valuable low titer group o units if the units are not used within the expiry date of use. aims: to assess the in-vitro quality of red cell concentrates (rcc) produced from 7 days cold stored leucodepleted whole blood units filtered with a platelet-sparing whole blood filtration filter in order to save the rcc once the wb units has reached the expiry date. methods: 34 units of whole blood were leucodepleted within 24 h using a platelet sparing filter (imuflex wb-sp/terumo bct) and stored for 7 days at 4 ae 2°c. on the 8 th day of storage, units are centrifuged and the rcc is supplemented with sag-m additive solution for an additional storage of 35 days at 4 ae 2°c. in vitro parameters such as hemoglobin, hematocrit, residual leucocytes, platelet content, hemolysis, glucose, lactate, atp, 2.3 dpg were tested during storage. results: the imuflex wb-sp provided satisfying leucodepletion of the wb units (0.23 ae 0.2.10e6/u)) while maintaining 80% of the initial platelet content (85 ae 20.10e9/u). mean volume (464 ae 24 ml), hemoglobin content (54 ae 6 g/u) and hematocrit (36 ae 3%) was within conformance range. after 7 days of storage, 50% of the initial platelet content at collection is maintained and mean hemolysis is 0.27 ae 0.15%. rcc processing at day 8 was uneventful, without loss of hemoglobin or noticeable hemolysis. mean volume (266 ae 20 ml), hematocrit (56 ae 3%), hemolysis (0.18 ae 0.19%) were in conformance with mandatory standards at day 8. after 7 days of storage (i.e. day 14 after collection), rcc quality parameters were maintained with however an increase in hemolysis (0.34 ae 0.36%) which was above usual values routinely observed with rcc produced from day 1 processed units. 3 rcc out of 34 were above the maximum limit of 0.8% at day 14 after collection such increased hemolysis was more pronounced at day 28 after collection (0.70 ae 0.45%) with a proportion of rcc with a non-conforming hemolysis ratio above the accepted threshold (20% of the tested units). summary/conclusions: rcc units can be readily obtained from leucodepleted wb units with spared platelet contents after 7 days of cold storage. in vitro quality attributes of such rcc are within conforming ranges for hemoglobin, hematocrit, volume and hemolysis. hemolysis increases more rapidly than in standard rcc, thus limiting the possibility of use within 21 days after collection. additional studies are being initiated to reduce hemolysis during storage. g hetland 1,2 , f mahmood 3 , l nissen-meyer 1 and i nentwich 1 1 immunology and transfusion medicine, oslo university hospital 2 immunology, university of oslo, oslo 3 immunology and transfusion medicine, akershus university hospital, lørenskog, norway background: allergen-specific ige in donors' plasma can be transferred from blood donors to recipients via plasma-containing products and consequently sensitize recipient's effector cells bearing receptors for ige (mast cells, basophils, thrombocytes etc.). these cells can then degranulate after exposure to allergen. this can cause allergy symptoms of various degrees (johansson, allergy, 2005) . food allergen-specific ige antibodies, although causing mild or no symptoms in blood donors, can be of clinical importance to plasma recipients due to varying sensitization and activation grade of recipients' effector cells. aims: the aims of the study were 1) to assess sensitization profiles against an array of 55 allergens in 100 randomly selected blood donors and 2) to identify donors with high concentrations of food-specific ige antibodies that could bear risk of clinically important sensitization of plasma product recipients. methods: we tested serum samples from 100 blood donors randomly selected on one donation day outside the pollen season. we used 300 ll serum per sample in a multiplex ige line-blot enzymoimmunoassay for 55 analytes (euroline atopy screen, euroimmun, germany), which comprised 28 food allergen extracts and single allergens, 24 inhalant allergen extracts, 2 insect venom extracts and one carbohydrate allergen ccd. this high-throughput method enables scanning of 44 serum samples for ige against 55 allergens in less than 3 h. results are expressed as east (enzymoimmunoassay) classes: class 0 (no sensitization), 1-2 (weak sensitization), 3 (moderate sensitization), 4-5 (strong and very strong sensitization). results: east class, allergen, number of samples. food allergens: class 5none detected. class 4, sesame seed: 1. class 3, apple 1; hazelnut 2. class 2, apple 1; almond 2; hazelnut 1; sesame seed 1; soybean 1; cow milk alpha-lactalbumin 3. class 1: 26 donors weakly sensitized to one or more allergens as kiwi, apple, almond, hazelnut, sesame, soybean, wheat, mutton, beef, cow milk beta-lactoglobulin and alpha-lactalbumin. we did not find any donors sensitized to apricot, peanut, rice, rye, yeast, cow milk casein and carbohydrate allergen ccd. insect venoms: class 5, 4 and 3 none, class 2, wasp venom 7; honey bee 2. lower classes (0-1) not reported in this abstract. summary/conclusions: we found considerable sensitization to inhalant allergens but such sensitization probably represents low risk of clinical allergies due to passive transfer of antibodies. sensitization to insect venoms was negligible. we disclosed only one blood donor (1%) with high ige to sesame seed with a potential to transfer clinically relevant ige antibodies via plasma products. the need for a broad screening of donors for ige sensitization remains still questionable. background: the platelet storage lesion (psl) is one of the key factors that limit the platelet shelf-life to 5 days. the mechanisms mediating the psl are not well understood, but it is becoming increasingly evident that platelets from some donors do not store as well as others. similarly, assessment of many in vitro quality parameters has been used to predict transfusion outcomes, with varying degrees of success. donor attributes such as age, and body mass index (bmi) may contribute to these differences. aims: to characterise the variability in the quality and function of apheresis platelet donations and to examine associations between donor attributes and platelet characteristics. methods: apheresis platelets (n = 53) were collected, stored at 22°c with agitation, and tested at day 1, day 5 and 7 post-collection. vwf and fibrinogen binding (indicators of platelet adhesion; ae ristocetin stimulation), phosphatidylserine (ps) exposure (annexin v binding) and microparticles (cd61 + /annexin-v + ) were measured by flow cytometry. platelet aggregation was initiated with collagen, thrombin or arachidonic acid. thromboxane a 2 (txa 2 ) was measured by elisa. the proportion of procoagulant platelets (% coated platelets) was assessed by fibrinogen binding following collagen/thrombin stimulation. platelet-attached glycans were measured using flow cytometry and lectins ricinus communis agglutinin-1 (rca-1; detecting galactose-residues) and wheat germ agglutinin (wga; detecting sialic acid and nacetyl-d-glucosamine, glcnac-residues) ae stimulation with ristocetin. donation time, donor age, blood pressure (bp) and bmi were recorded. linear regression was performed using graphpad prism software. results: for many of the parameters measured, there was high inter-donor variability. platelets from subsets of donors showed up to 4-fold higher vwf and fibrinogen binding, formation of coated platelets and microparticle numbers. binding of lectins was also highly variable between the donors, indicating variability in sugar cleavage. following ristocetin stimulation, binding of all lectins increased, and again there was high variability in the responses, with up to 4-fold higher binding in platelets from a subset of donors. of particular interest, aggregation in response to arachidonic acid was observed in only 27% of platelet donations tested; although platelets from non-responders aggregated in response to the collagen and thrombin. txa 2 was also higher in the arachidonic acid responders (p = 0.027). there were few associations between platelet parameters and donor attributes, and these associations were typically weak. ph at day 5 was weakly associated with donor age (p = 0.009, r 2 =0.2613) but not bmi (p = 0.9143, r 2 <0.001), with a trend towards lower ph in younger donors. summary/conclusions: these data indicate that apheresis platelet products are highly variable. whilst donor characteristics may have some influence on the quality of the donated platelet product, there is a high level of inherent inter-donor biological variability. a much larger sample size is required to confirm these findings, and determine whether they have clinical implications. background: the frequency of beta thalassemia mutations, and thus, of beta thalassemia heterozygotes (b-thal-het) is particularly high in certain geographical regions. it has been reported that a significant proportion of donors (occasionally more than 10%) are b-thal-het that meet the criteria for blood donation (hb > 13.5 g/ dl). red blood cells (rbcs) of b-thal-het donors are characterized, in vivo, by particular geometry and redox status. despite sporadic indications that the rbc storage lesion may be milder in b-thal-het, targeted research on this donor group is still missing. aims: the aim of this study was to investigate whether b-thal-het rbcs storage at blood banks leads to a distinctive hemolytic, physiological and redox profile, thus, making b-thal-het a unique blood donor group. methods: blood samples from 10 healthy non-smoker donors (5 b-thal-het carriers and 5 controls) were analyzed before and after preparation and storage of leukoreduced packed rbc units in cpd/sagm at various time intervals. susceptibility in hemolysis (in the presence/absence of oxidative, mechanic and osmotic stimuli), redox status (lipid peroxidation, reactive oxygen species (ros) accumulation, antioxidant capacity), intracellular ca 2+ and proteasomal activities were determined. for statistical analysis, significance was accepted at p < 0.05. samples from the red cell units were collected aseptically, processed (dual centrifugation at 3,000 g for 15 min) and stored at à80°c. processed samples were thawed, and then analysed using the nanosight ns300 nanoparticle tracking analysis system (malvern instruments). samples from all time-points from each unit were analysed on the same day. data were analysed by one-way anova with bonferroni's multiple comparisons test. results: at d2, red cell units contained an average of 8.1 9 10 9 ae 4.0 9 10 9 mvs/ ml. the mean size of these mvs was 112.8 ae 10.5 nm and the mode size was 79.9 ae 10.3 nm. the concentration of mvs increased gradually throughout storage (p = 0.0276), reaching a maximum at d42 of 32.4 9 10 9 ae 1.8 9 10 9 mvs/ml. both the mean (p < 0.0001) and mode (p < 0.0001) size of the mvs increased during storage; however, this size increase primarily occurred in the first week of storage (d2 vs. d7: p < 0.0001 for both mean and mode). by d42, mean and mode size of mvs was 176.1 ae 4.8 nm and 171.8 ae 5.6 nm summary/conclusions: nanoparticle tracking analysis demonstrated the presence of mvs smaller than 400 nm in red cell units. both the concentration and size of mvs present in red cell units increased during the 42 days of routine storage. the concentration of these mvs was approximately 100-fold higher than we had previously detected using flow cytometry (aung, pathology, 2017) indicating the advantages of more sensitive techniques in characterisation of mvs. background: the lack of availability of sterile saline in a format suitable for use in blood centers for manual washing has led to an urgent need for blood services to consider alternative methods. for operational flexibility it would be desirable to be able to produce a washed rbc unit that had a shelf life longer than 24 h. aims: the aim of this study was to validate the manual method for washing rbcs using sagm solution both as wash and storage solution and to ascertain whether an extended storage period for washed rbcs may be feasible. methods: six 14 day old leukocyte depleted red blood cells (ld-rbc) and six 4 day old ld-rbcs were manually washed and stored in sagm, and half of the units were pre-stored irradiated (25 gy). a volume of 350 ml wash solution (sagm) was added to the ld-rbss by sterile connection. after mixing the units were centrifuged for 6.5 min at 2888 9 g at 4°c (hettich roto silenta 630 rs) before removing the supernatant using compomat g5 extractor. wash procedure was repeated twice using 450 ml sagm solution, and after removal of the last supernatant, 100 ml of sagm solution was added. all units were immediately measured for volume, haematocrit, albumin, iga, potassium, haemolysis, haemoglobin, ph, glucose and lactate and tested again after 24 h, 7 days and 14 days storage at 4 ae 2°c. results: all washed ld-rbcs met european specification for haematocrit (0.50-0.70) and all but one for hb content (≥ 40 g/unit). hemolysis increased during storage. the rate of hemolysis in irradiated ld-rbcs was greater over time than in nonirradiated units. all units, both irradiated and nonirradiated, met european specification for hemolysis (less than 0.8%) 14 days after washing. after wash, potassium levels were low and then increased during storage; increase was greater in irradiated than nonirradiated units. potassium concentration 14 days after washing and irradiation did not exceed those levels found at the end of shelf life (day 35) of standard ld-rbcs. ph decreased during storage due to the metabolic activity of red blood cells converting glucose to lactate. the ph level of the supernatant depends on the age of the unit and not on the irradiation. the glucose concentration of the supernatant after washing is high due to sagm solution. the concentration of glucose decreased and lactate increased due to the metabolic activity of red blood cells. there is currently no specification in europe or finland for iga in washed rbcs. aabb and american red cross rare donor program stipulate that level of iga should be less than 0.05 mg/dl (0.5 mg/l). our iga method 0 s lower limit for detection is 0.05 mg/l and all results were below this level. total albumin were well below finnish specification (<250 mg/unit). background: room temperature has been the standard storage condition for platelets since the 1970s, when it was shown that this improved in vivo survival compared to when stored at 4°c. however, storage at room temperature has several disadvantages, including risk for bacterial contamination and short outdating. recently, the interest in cold-stored platelets increased, especially for patients with a hemostatic need. using extensive analysis techniques, we evaluated the in vitro quality of cold stored platelets in additive solution. aims: investigation of the in vitro quality of platelets stored at 4-9°c in pas-e. methods: three experiments were performed, in which two platelet concentrates, prepared from 5 buffy coats and 300 ml of pas-e (pcs) were pooled and split in equal pcs. pcs were stored for 23 days at 4-9°c, one of each pair with agitation on a flatbed shaker and the other without agitation. various parameters were analyzed to study the in vitro quality during storage and compared to routine room temperature storage. results: during cold storage, the swirling phenomenon disappeared within one day. due to the lower temperature, metabolism of the platelets was lower as compared to room temperature storage. the metabolic conditions were acceptable with ph d1-d16: 7.05-6.77 with platelet count 400 9 10 9 /u and glucose still at 2 mm at least until 16 days of storage. platelet activation maintained acceptable levels with cd62p expression < 30%, while ps exposure increased rapidly; >20% after 6 days of storage. aggregation tests showed functional platelets until 13 days of storage. agitation during storage had no effect on any of the tested parameters. summary/conclusions: during storage of platelets at 4-9°c, the hematological parameters and ph met routine requirements, while swirling phenomenon disappeared already at the first day. the functionality of the platelets did not decrease during cold storage, indicating that the swirling phenomenon is not a good surrogate marker under these conditions. the strong increase of ps exposure might be involved in the observed short survival of cold-stored platelets. platelet concentrates stored at 4-9°c are potentially suitable as a hemostatic agent for patients with a bleeding in need of platelets, but more studies are needed. aims: the goal of the study is the reinforcement of platelet reserves for case of emergency events and increasing their availability for treatment, preferentially in patients with massive bleeding. methods: we performed a comparative study with cpp and fp in vitro. buffy coatderived pooled leukoreduced platelets 0 rhd negative were frozen in 5-6% dmso and stored at à80°c for 6 months. cpp were thawed at 37°c, then reconstituted in platelet additive solution ssp+ and compared to fp. we measured these parameters: platelet content, platelet concentration, platelet loss during preparation process, coagulation properties, volume, ph, dmso concentration, titres of anti-a and anti-b antibodies. results: the average platelet loss after the process of freezing and reconstitution was 24%. the amount of platelets and platelet concentration in unit was lower in cpp compared to fp, but high enough (amount 194 9 10 9 /unit, concentration 0.73 9 10 9 /unit). both types of plts (either pcc or fp) maintained an acceptable ph during storage. swirl was on value 3 in fp and on value 1 in cpp. the average plasma content in fp was 31% compare to 3.22% in cpp after reconstitution. measured titres of igm anti-a and anti-b antibodies were very low (0-1:2). cpp had faster clot initiation (rotem clotting time (ct) in cpp 69.2 s, fp 81.8 s). cpp contributes to a sufficient clot (rotem maximum clot firmness (mcf) in cpp 39.8 mm, fp 53.2 mm). summary/conclusions: our results shows, that cpp have higher procoagulation activity and simultaneously lower clot firmness. thawing and reconstitution of platelets are easy and fast processes if platelet additive solution is used. this method helps to increase the availability of platelets in emergency medicine. low plasma content in cpp enables their use as washed platelet product in specific groups of patients. methods: after donation, the whole blood was stored in room temperature overnight before separating next morning by reveos â system. seven abo compatible ipus, each with a target volume of 24 ml, were selected and then they were connected to the pooling set provided by terumo bct. prior to the pooling of ipus, 250 ml of additive solution (t-pas+ provided by terumo bct) was added and distributed evenly between the ipu bags. the pooling set was then kept 1 h on bench in room temperature followed by 1 h on agitator at 22 ae 2°c. after filtration, the pool might be manually adjusted if its volume exceeded the maximum of 420 ml to meet the requirements by the intercept tm blood system. the final products were two pathogen-reduced platelet units with a shelf life of 7 days. results: during validation of the method, 12 pathogen-reduced platelet units were controlled, in addition to the platelet count, for ph, glucose, po2, pco2 and lactate on day 2, 5 and 7 of storage. the platelet count was 2.27 ae 15 9 10 11 per unit on day 2. the ph value was 6.9 on day 2, 7.1 on day 5, and 6.9 on day 7. the glucose concentration decreased from 5.8 to 3.5 and 1.6 mmol/l on day 2, 5 and 7, respectively. the mean po2 level was 25.3, 25.0 and 28.7 kpa while the mean pco2 was 3.8, 1.7 and 1.8 kpa and the lactate concentration was 5.9, 9.6 and 12.9 mmol/l on day 2, 5 and 7, respectively. since routine implementation of the method in april 2017, regular quality controls showed an average of platelet count of 2.63 ae 34 9 10 11 (n = 161) with a volume of 204 ae 7 ml per unit. summary/conclusions: the validation of the method and the following two years of experience in routine shows that the pooling of 7 ipus processed in reveos â system meet the requirements needed for intercept tm ds processing set for pathogen reduction of platelets. the results from the quality controls of the final platelet units were in accordance with the local and eu guidelines. methods: data was analyzed from 11 published and unpublished clinical studies that performed both primary and secondary testing of platelets using the bta system. the studies included apheresis and whole blood derived buffy coat platelets and tested 4-10 ml sample volume per culture bottle. the 11 studies classified results based on aabb bulletin 04-07 definitions with some modifications. the following assumptions were made including: • data was summarized as total number of positive tests, observed by the total number of tests performed on each day post collection; • it was assumed that one test was performed per platelet unit; • all units eligible for secondary testing were negative by the primary test the data needed to demonstrate a benefit for the use of the bta 3d systems for detecting contamination that was not revealed by previous bacterial testing as well as clinical specificity. results: a total of 217,932 platelet units from the studies where secondary testing of platelets was performed were analyzed. platelets were tested on day 3, 4, or ≥ 6, and represented 8.9%, 44%, and 47% of the units tested, respectively. true positives were detected in 174 platelet units representing 0.08% of the total platelets tested. the majority were reported from platelets tested on day ≥ 6 with a total of 128. data showed the bta 3d system used for secondary testing detects the most prevalent contaminates reported, staphylococcus spp., in ≤27 h with the majority detected in ≤24 h after incubation, allowing for interdiction of the units prior to transfusion. instrument specificity was reported in 5 of the 11 studies for platelets tested at 3 days and ≥ 6 days with a total false positive rate of 0.27% (range of 0-1.1%). instrument sensitivity when used for secondary testing could not be determined since subculture of negative bottles is not performed during routine use. during previous validation testing of lrap and lrwbpc, 1,136 culture bottles were confirmed true negatives by subculture. summary/conclusions: data from the studies that tested platelets at 3 to 8 days post collection provided evidence that the bta 3d with bpa & bpn detects contaminants missed in previously tested platelet units. the data supports that the bact/alert 3d system is an effective safety measure for secondary testing of platelet products to extend platelet dating beyond day 3 and up to day 7 when testing is performed using the test parameters described in the bpa and bpn bottle ifus and according to the fda draft guidance. background: magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (hsa). however, the optimal hsa density coated on particles and the uptake mechanism of single particles in platelets remain unclear. aims: we characterize the interaction between single particles and platelets and determine the optimal hsa amount required to coat particles. methods: ferucarbotran iron oxide nanoparticles were coated with hsa in different amounts (0.5-2 mg/ml) and we confirmed successful hsa coating by addition of a crosslinking hsa antibody (dynamic light scattering). we labeled platelets from pooled platelet concentrates with 2 mm ferucarbotran coated nanoparticles and analyzed labeled platelets for iron content (atomic absorption spectroscopy) and particle localization (transmission electron microscopy). single-molecule force spectroscopy was used to determine binding forces of nanoparticles to platelet compartments. we applied hsa-particles via linkers of different length (i.e. short~2 nm, medium 30 nm and long~100 nm) on the cantilever tip and let them interact with a platelet provided on a collagen surface. after interaction we determined the rupture force required for platelet retrieval. results: the iron content per platelet reached a maximum at 0.5-1.0 mg/ml hsa coated particles with 0.57 ae 0.36 and 0.54 ae 0.39 pg/platelet, respectively. however, the 1.0 mg/ml hsa coating resulted in~100-fold higher binding affinity to platelets than particles coated with 0.5 mg/ml hsa. depending on peg length between tip and particle, particles interacted differently with platelets as shown by one, two or three force distributions of 80, 220, and 360 pn, which correspond up to three different binding pathways, respectively. the results indicate that a particle can interact with three targets including platelet membrane, open canalicular system, and platelet granules. summary/conclusions: our results reveal mechanism of platelet-particle interaction on a single particle level and provide an optimal hsa concentration coated on particles to gain maximal platelet labeling efficiency. labelling of platelets by magnetic nanoparticles may substitute radioactive labeling. results: the activation/lesions on total platelets and small and medium-sized platelets platelet population was detected on storage day 3, by the increased expression of cd36. the percentage of cd36-positive cells among the population of large platelets did not change during storage. on the day 3, increased expression of cd42b and cd62p was detected, but only on large platelets. small and medium-sized platelets had increased cd62p expression only on day 5. the expression of cd42a on total platelets increased significantly on day 3, and stayed unchanged until day 5. the same pattern of cd42a expression was detected for small and medium-sized platelets, whereas on large platelets the expression continued to increase until the end of storage. a decreased percentage of cd41-positive cells was detected among the total platelets and populations of medium-sized and large platelets. the storage induced externalization of phosphatidylserine on total platelets or on any of the platelet populations was not detected. the levels of tgf-b and p-selectin in the pc-bc supernatants were unchanged during the 5-day storage period. increased annexin and pf4 concentrations were detected on day 5. the concentration of b-tg increased on day 3 of storage, and continued to rise until the end of storage. summary/conclusions: the evaluation of expression of activation markers on different platelet populations could be used as an additional analysis in quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation i.e. for testing new additive solutions, cryoconservation protocols, and cryoprotectants. background: the primary goal of autologous blood transfusion is to reduce the risks related to allogeneic blood transfusion, including transfusion-associated graft-versus-host disease (ta-gvhd). although downward trends in rates of autologous blood transfusion have been reported in europe and the americas, it still plays a role in eliminating risks related to allogeneic blood transfusion in japan, especially ta-gvhd. since february 2009, the transfusion service in our hospital has managed 3 autologous blood conservation techniques and helped to prevent mistransfusion by employing a bar code-based electronic identification system. aims: the objective of this study was to determine the use of 3 types of autologous blood components in a single institution over an approximately 9-year period. methods: between february 2009 and december 2017, we retrospectively analyzed autologous blood transfusion, including perioperative autologous cell salvage (pacs), pre-operative autologous blood donation (pad), and acute normovolemic hemodilution (anh). we investigated the use of autologous blood components and the rate of complying with electronic pre-transfusion check at the bedside in the operating rooms. we also determined the adverse reactions to autologous blood transfusion, which were categorized according to the definitions proposed by the international society of blood transfusion (isbt) working party on haemovigilance. results: a total of 9,193 patients (9% of whom received operations) received blood transfusion, of which 5,080 (55%) received autologous blood transfusion alone, 2,101 (23%) both autologous and allogeneic blood transfusions, and 2,012 (22%) allogeneic blood transfusion alone. the rate of autologous blood transfusion among patients who received blood transfusion was 78%. pacs units were transfused to 5,830 patients (60%), pad units to 2,845 patients (30%), and anh units to 982 patients (10%), and multiple blood conservation techniques were used for one patient. the overall compliance rate with electronic pre-transfusion check at the bedside in autologous blood components was 98.6%. adverse reactions were observed only with pad transfusion and not pacs nor anh. the number and rate of adverse reactions to pad transfusion were 12 and 0.1%, respectively, of which the most common was febrile non-hemolytic transfusion reaction at 9 (75%), followed by allergic reactions at 2 (17%), and hypotensive transfusion reaction at 1 (8%). the severity of adverse reactions to pad transfusion was grade 1 (non-severe) in all cases, and no serious adverse reactions were observed. among pad units, the rate of adverse reactions to whole blood pad units was 0.55%, that to frozen pad units was 0.05%, and that to autologous ffp units was 0.10%. summary/conclusions: our observations indicate that the rate of autologous blood transfusion among patients who received blood transfusion was 78%, when all types of autologous blood conservation techniques were included. to accurately determine the rate of autologous blood transfusion in a hospital, it may be better for the transfusion service to manage all types of autologous blood conservation techniques. they are now clinically available as a blood product. the residual plasma level of this product, which is prepared using the automated cell processor acp215 (haemonetics corp.), is approximately 1%. recently, a retrospective multicenter study was reported that this product was effective and safety for prevention of recurrent or severe transfusion reactions. plt products are generally stored with continuous agitation to maintain their quality. the interrupted agitation of plt suspended in additive solution (plasma carryover: 3-40%) for up to 24 h was previously found to have only a slight impact on in vitro plt properties. however, in some small hospitals with no agitator, if the initiation of transfusion is delayed by an emergent change in a patient's condition, the interruption of agitation may be prolonged. aims: the aim of this study was to evaluate the effects the interruption of agitation for 48 h (shelf life of wpc in japan) on the in vitro qualities of plt. methods: leukoreduced apheresis platelet concentrates in 100% plasma were washed on day one after blood collection using the automated closed-system cell processor acp215 (n = 6). wpc, which were rested 1 h after preparing, were divided equally into control and test units using polyolefin containers on day one. control units were agitated from days one to seven. test units were stored without agitation from days one to three, and agitation was subsequently performed until day seven. both units were stored at 20-24°c. in vitro plt quality was examined on days one, three, four and seven. results: the plt concentration of prepared wpc was 110.9 ae 1.9 (910 10 /l) and the volumes of the control and test units after the division were 120 ae 11 and 123 ae 12 (ml). the ph values of the test units on day three were lower than those of the control units; however, the ph of both units were maintained at higher than 6.85 during the seven-day storage period. swirling was well maintained and no clumping was visible in both units during storage period. no significant differences were observed in plts concentrates, mpv, hsr, aggregation response. the pco 2 , po 2 , bicarbonate, glucose and lactate mean values of test units were slightly lower or higher than those of the control units on days three or four. the levels of cd62p expression were significantly higher in the test units than in the control units on days three (52.6% ae 6.0 vs 40.9 ae 5.7, p < 0.01); however, this difference decreased in a time-dependent manner after agitation resumed. the levels of cd42b expression of test units were relatively lower than those of the control units until day seven, but no significant difference between the two units. background: monitoring residual white blood cells (rwbcs) is a requirement for quality monitoring (qm) the production of leucocyte depleted blood components. although flow cytometry is widely used for monitoring rwbc, there are no widely accepted methods to accurately and consistently measure rrbcs in blood components. sysmex have developed a novel algorithm, termed the blood bank (bb) mode for their xn-series of haematology analysers which is specifically designed to quantitate the levels of rwbcs and rrbcs in blood components. aims: we have previously assessed the linearity, accuracy and reproducibility of the bb mode on spiked samples in an r+d lab. we sought to further assess the performance of the bb software in a routine, high throughput blood component manufacturing department. methods: units of plasma, platelets (pcs) and red cell concentrates (rccs) were produced according to standard uk specifications within nhs blood and transplant (nhsbt). qm of residual cells was tested using the bb mode whilst rwbc was additionally analysed by flow cytometry using bd leucocount kit. results: during a 2-month field trial over 10,000 data points were collected representing all types of manufactured component. for some pcs, bb mode results from some sample tubes that did not contain edta gave very high rwbc values, indicating a potential large number of ld failures. the results were significantly different from those obtained from pcs using edta samples (p < 0.0001) which did not show the same high values. for rccs or plasma, the range of results from plain and edta tubes were not significantly different (p ≥ 0.3246). the analyses of ld platelet and plasma concentrates by either bb mode or flow cytometry both show more than > 90% of ld components have less than 1 9 10 6 rwbc/unit. for ld failures (n = 14) there was a good correlation (r 2 =0.9848) between flow cytometry and bb mode measurements. spiking studies suggested that the limits of detection and quantitation of rrbc were around 6 and 8 rrbc/ll respectively. residual red cell counts from manufactured components showed a wide variation in their numbers between units. as expected platelet production methods also showed a significant difference (p < 0.0001) in rrbc contamination, with lower levels in apheresis platelets (median = 15 rbc/ll, n = 1820) compared to those produced from buffy coats (median = 783 rbc/ll, n = 77) in our hands, although the time taken to analyse samples is similar for flow cytometry and bb mode, considerable time can be saved on manual handling and the processing of samples for flow cytometry (approximately 2-3 h for 60-90 samples). summary/conclusions: we have been able to embed the sysmex bb mode into a routine production environment and confirm that its performance in spiked samples is mirrored in routine use. for platelets, sample collection in edta is essential. the bb mode offers an opportunity to reduce operator time compared to flow cytometry whilst gaining additional information on rrbc. abstract withdrawn. results: in our experiment, the typical size of a spectrin matrix section (l) was 60 to 220 nm (without oxidation). the heights (h) of dips were 4 to 10 nm. due to oxidation, the junctional complexes between spectrin and membrane proteins can rupture. only 10% to 15% of the spectrin surface has the same structure as in the control group. the values of l and h vary significantly depending on the intensity and time of exposure. we observed significant changes in the spectrin matrix after exposure to uv radiation in a model experiment. the local topological defects in the membrane arise from the action of oxidizing agents on the red blood cells. the mechanism of their appearance is connected mainly with the distortion of the spectrin matrix. as a result of oxidation processes, the spectrin molecules can be damaged. there is a transformation of tetramers to dimers. additionally, it can be easily seen with the afm, that spectrin network structure was essentially destroyed. most parts of the spectrin matrix have damaged structures with mesh breaks and dips after uv irradiation. also the results of network distortions in response to temperature changes were obtained. there are presented preliminary results of spectrin matrix change during long-term storage of prbc. summary/conclusions: atomic force microscopy in direct biophysics experiment allows to observe and to quantitatively measure the disturbances in the spectrin matrix nanostructure in response to oxidation processes in rbcs. these studies are important for the fundamental research of interactions of rbcs on the molecular level during redox processes and the consequences to their structure and function on the cellular level. this is important for the advancement of transfusion medicine, intensive care medicine, and molecular and radiation biology. methods: blood samples were taken from 8 donors during a prophylactic examination and collected with edta-filled microvettes (sarstedt ag and co., germany). all experiments were carried out in accordance with the institute guidelines and regulations. the polylysine-coated glass was used to perform the sedimentation method for formation of native rbc smears. it is important that any fixatives weren't used. the stiffness of rbc membranes was studied in native rbcs (control) and native cells after the application of modifiers: glutaraldehyde, hemin, zn 2+ (heavy metal ions). local stiffness was studied by atomic force spectroscopy (afs) (ntegra prima, (nt-mdt, russian federation). results: experimental kinetic curves i(z) were measured. nonlinear fitting method was used to determine the young's modulus. the experimental dependences of membrane bending were approximated by the hertz model to a depth up to 600 nm. the young's modulus e = 10 ae 5 kpa for control rbc. it was shown that some natural oxidants (hemin), membrane fixatives (glutaraldehyde) modifiers, heavy metal ions (zn 2+ ) significantly increased the absolute value of the young's modulus of rbc membranes up 2-3 times. the biophysical parameter hertz depth (h hz ) was determined for each curve. under the influence of modifiers the hertz depth h hz was changed from 200 nm to 600 nm. there are presented preliminary results during long-term storage of blood. summary/conclusions: the blood rheology is determined by rbc deformability, associated with membrane stiffness. the young's modulus can be used as a quantitative criterion to estimate the membrane state of a native cell. the results of the work can be used in clinical practice, in assessment of the quality of donor blood during storage before transfusion, in biophysical studies of rbc state. abstract withdrawn. immunohemotherapy, centro hospitalar universit ario são joão, epe, porto, portugal background: transfusion of blood and blood components is an essential resource in modern medicine. a proper use of human blood, being an irreplaceable resource, is necessary in order to achieve minimal wastage. blood wastage may occur for a number of reasons, like expiry date, haemolysis, seroreactivity or low volume. monitoring wastage of blood product during collection, testing and processing of blood is used as a quality indicator. aims: to determine the annual rate of discarded blood components due to expiry date in a portuguese university hospital blood bank (bb) from january 2016 to december 2018, in order to implement appropriate measures to minimize the number of discarded blood to a reasonable rate. methods: we retrospectively analysed the rates of blood components discarded after meeting their expiry date of a portuguese university hospital blood bank from january 1st 2016 to december 31st 2018. results: a total of 54,599 whole blood units and 3,166 apheresis platelets were collected during the study period. of the 66,289 blood components (packed red cells, whole blood pooled platelets and apheresis platelets) prepared during the study period, a total of 2,891 (4.4%) blood components were discarded, of those 75.4% due to expiry date. the rate of discarded packed red cells, according to this component production, decreased considerably over the years, in 2016 was 5.6%, in 2017 was 3.8% and 0.2% in 2018. similar tendency was shown in the pooled platelets for 2 consecutive years with 4.8% (2016) and 3.8% (2017), but with an increase in 2018 (7.7%). the rate of apheresis platelets had a more variable behaviour from 2016 to 2018 with rates of 1.3%, 0.6%, and 0.9% respectively. summary/conclusions: blood transfusion is an essential part of patient care. for this reason, the implementation of a quality system and continuous evaluation of all activities of the bb can help to achieve maximum quantity and quality of safe blood. we identified that date expiry was the main reason of discarded blood components, although there was a significant decrease in the rate of discarded packed red cells over these three years. properly implemented blood transfusion policies, donor screening and training staff as well as implementation of automation also helps to improve the process, reducing the discarding rates of blood and blood component. background: storage performance of platelets (plt) is associated with age of the donor. the risk for plt with poor storage performance, characterized by high lactate production and rapid acidification of a plt concentrate (pc), shows a positive correlation with age. we wished to explore whether high lactate production was associated with donor health issues. aims: to investigate high lactate production by stored platelets in relationship to donor health. methods: single-donor pc were collected by apheresis or prepared from 1 buffy coat and donors were evaluated who could be marked as 'rapid acidifiers'. in total, 31 apheresis pcs and 66 pcs from whole blood were included in four studies. information about donor health was obtained either from the blood bank information system or using questionnaires. in some donors, the lipid profile was measured from plasma, and the diabetes marker hba1c from red cells. triglyceride levels > 2.5 mmol/l and hba1c levels > 42 mmol/mol were defined as high. results: twenty two percent (21/97) of the donors were marked as 'rapid acidifiers' and 71% (15/21) of these donors had health issues. 'rapid acidifiers' were of age 57, 31-69 (median, range) years. three groups of donors can be distinguished: a) donors affected by metabolic syndrome, prediabetes and type 2 diabetes, indicated by high cholesterol and/or triglycerides, high hba1c and/or the use of medication to treat diabetes. b) donors affected by vascular diseases who reported or used medication to treat high blood pressure. c) "other" donors who used other medication to treat various other conditions. the remaining 'rapid acidifiers' (29%) did not have high triglyceride or hba1c levels and did not report health issues. summary/conclusions: pcs with rapid acidification by high lactate production are mainly collected from older donors with health issues. we postulate that high lactate production by stored plts is associated with health issues, and we will combine detailed donor information (health and behavior) with in vitro quality for a significant number of donations. background: the development of applied biotechnologies requires a search and creation of new methods of cells' functional completeness analysis. the instrumental assessment of platelets quality for the selection of the most effective donors, quality assessment of platelet concentrates for short and long-term storage and for the selection of platelets for cryopreservation is in demand in blood service. assessment of platelets morphofunctional status is possible using morphological studies of various platelet granules fractions (makarov, med. alfavit, 2012). among the biologically complete platelets there is a special population of cells, the so-called granule-rich platelets (grp). these cells contain the largest number of cytoplasmic granules (more than 10 visually distinct granules). it is established that grp have increased viability and functional activity. earlier we found a correlation between the grp level in blood plasma and shift of the redox potential value in blood plasma after cryodestruction of platelets (tsivadze et al., doklady physical chemistry, 2016). it was suggested that the shift of the redox potential may be partly due to the release of the low molecular weight antioxidants contained in functionally complete platelets outside the cell. in turn, the concentration of low molecular weight antioxidants can be estimated using the cyclic voltammetry. aims: the aim of the study was to estimate of cyclic voltammetry method possibilities for quality assessment of platelets. methods: the functionality of platelets was examined in platelet concentrate (pc) obtained by apheresis (1246.8 ae 90.1/ll). voltammetric analysis in pc before and after the platelets cryodestruction was carried out on platinum electrode in the potential range from à600 mv to +1100 mv using a potentiostat ipc pro l and saturated ag/agcl electrode as reference. for the morphofunctional analysis platelets were vitally stained with fluorochrome stains trypaflavin and acridine orange. microscopic examination of platelets was carried out using confocal microscope nikon eclipse 80i. the following parameters were evaluated: concentration and percentage of platelets with granules and concentration and percentage of grp. results: voltammetric studies in pc show that there are two oxidation peaks of low molecular weight antioxidants on voltammogram at potentials + 500 mv and + 800 mv. analysis of pc before and after the cells cryodestruction showed that changes in the height of oxidation peaks occur, indicating an increase of antioxidant content in blood plasma. at the same time a correlation between the changes in the height of oxidation peaks and the grp content in the sample was found. in samples with reduced initial grp content (less than 10%) after the cells cryodestruction significant changes in the height of oxidation peaks were not observed, regardless of the total number of cells in the pc. summary/conclusions: in conclusion, voltammetric analysis allows to indirectly estimate the population of functionally active platelets that in combination with other methods of analysis can serve to assess the quality of platelet products. background: determination of hemoglobin derivatives in blood is one of the most important studies in clinical laboratory diagnostics, especially during the storage of donor blood and its transfusion. concentration of hemoglobin derivatives can be changed during redox process. aims: to show the possibility of using non-linear fitting method to calculate concentrations of hemoglobin derivatives during reduction-oxidation processes. methods: for this we performed model biophysical experiment, in vitro. blood samples were collected into edta microvettes from 4 healthy donors (sarstedt ag and co., germany) during prophylactic examinations. all the donors gave their consent to participate in the study. a suspension of erythrocytes was prepared in pbs buffer with ph 7.4. we used ultraviolet (uv) irradiation of blood or nano 2 as oxidizing agent. the drug cytoflavin (stpf "polisan", russian federation) was used as an antioxidant. in our study we used digital spectrophotometer (unico 2800, usa) to measure the absorption and scattering of light (0.5 nm step). the method of nonlinear fitting was used to find the concentrations of hemoglobin derivatives. the empirical spectrum d l (k) exp was approximated by the theoretical curve d l (k l ) theor , which fits the experimental curve in the best way. under approximation the light absorption by different hemoglobin derivatives was considered in model. simultaneously effects of rayleigh light scattering on structures with size d<< k (coefficient s) and light scattering on particles with size d≥ k (coefficient k) were taken into account: d l (k l ) theor = e hbo2,l c hbo2 l+ e hb,l c hb l+ e methb,l c methb l+ e hbno,l c hbno l+ e methbno2 -,l c methbno2 -l+ e methbno,l c methbno l+k+s/k 4 l (1), where e h,l is the molar absorption coefficient for each hb h derivative at given wavelengths k l , c h is the concentration of the derivatives hb h , l is the thickness of the solution layer, d l (k l ) is the optical density of the substance, k and s are the parameters of the model. results: we determined the concentrations of hemoglobin derivatives without any additional chemicals in blood. there were measured experimental spectra for different agents action on blood. it was shown that concentration of methb increased after uv irradiation and nano 2 action (up to 90%). there were calculated c h for each hb h derivative. it was established that theoretical curves coincide with experimental data with good accuracy (r 2 = 0.98). incubation of rbcs with cytoflavin leads to reduction of methb to hbo 2 . summary/conclusions: the determination of hemoglobin derivative concentrations by the method of nonlinear fitting (without adding special chemical agents to blood) can be used for measurement of carboxyhemoglobin in blood during toxic state of organism. also it is important for assessment of rbcs quality before blood transfusion. background: the use of in line leukoreduction filters have been highly expanded in iranian blood transfusion centers within the last decade in order to provide sufficient leukoreduced blood fractions from healthy safe frequent blood donations to be supplied to the leukocyte sensitive patients. leukoflex lcr5, the dominant brand of such filters procured by iranian blood transfusion organization, is the most updated generation of the filters used around the world. aims: in this study, it is tried to recover the trapped leukocytes from this novel filter by different buffering systems and having optimized the elution mode, the cell differential of the viable recovered white blood cells were determined by flow cytometry. methods: having passed the routine virological tests, eight leukoflex lcr5 leukoreduction filters freshly used in tehran blood transfusion center were daily collected and each were back flushed by a self-designed mechanical system (a peristaltic pump, a triple junction with regulator part and an air pump) using various conditions and additives for pbs buffer at different phs in order to find the highest recovery yield for leukocytes. the optimized elute was characterized by flow cytometry for subcellular profile to be determined. results: it was illustrated that a system consisting of pbs (without cacl2 and mgcl2) in ph 7.2 containing 2 mm edta and 4%(w/w) dextran 40 without additive amounts of triton x100 was the most optimized buffering system for lcr5 filter back flushing. total cell content was also determined as 6.28*10 8 granulocytes, 4.27*10 8 lymphocytes and 0.8*10 8 monocytes using auto hemoanalysis and flow cytometric methods. summary/conclusions: in addition to partly compensating of the overhead expenses inflicted by application of leukoreduction filters on healthcare system, the results will assist blood organization system to be more classified in rational profile design, future cell therapy strategies and exceptional blood management. also, the recovered cells could be of significance in stem cell science, cellular interaction studies as well as novel molecular developments in drug discovery. vox sanguinis (2019) results: three lines of strategy are in place to pursue self-sufficiency of the largest number of pdmps. first strategy line: maximizing the yield of driving proteins, represented by immunoglobulins (ig) and albumin; this was assured by csl behring with a yield of 5.2 g ig (70% intravenous -privigenand 30% subcutaneous -hizentra) and 26 g albumin (alburex) per kg plasma fractionated, corresponding to 998.000 g ig and 4.992.000 g albumin; based on present demand, this represents 97% and 100% self-sufficiency, respectively, for naip regions. second strategy line: ensuring other products from plasma fractionation; the fractionator granted 6.000 g fibrinogen (riastap) and 6.000.000 iu vwf/fviii (haemate p) per year, which corresponds to the present demand of naip regions for both products, but it is under the full potential of plasma, thus providing a high margin of safety in case of increased demand (now the case of fibrinogen). third strategy line: exchanging cryoprecipitate, fibrinogen and vwf with italian regions whose plasma is fractionated by other companies to obtain prothrombin complex concentrates (pcc -kedcom) and antithrombin (atked) as to satisfy naip regions demand; this strategy allowed a supply of 5.000.000 iu at and 3.000.000 iu pcc, capable of ensuring self-sufficiency for naip regions until 2020. summary/conclusions: in italy, differentiation of plasma contract manufacturing among companies with different portfolios allowed naip regions to obtain a significant contribution to self-sufficiency from vnrd plasma for a variety of pdmps by different and complementary strategies consisting in maximizing the yield and the portfolio of proteins from the fractionator and exchanging products among regions for other pdmps at high demand but not included in the portfolio of a single fractionator. plasma check system: a valuable tool for plasma freezing validation and monitoring. background: the validation of plasma freezing processes may result problematic in the monitoring/control of critical process parameters (cpp). in 2017 in italy 923,944 litres of plasma were produced and frozen. aims: in order to assist plasma freezing validation and cpp monitoring, 151 of the 176 italian bes performing plasma freezing utilize the plasma check system (pcs), a system able to record, store and certify the temperature (t) detected at the core of "surrogate" bags during the entire freezing session, consistently with gmp requirements. pcs is patented and commercialized by expertmed srl, verona, italy (http://www.expertmed.it). methods: pcs consists of 3 parts: a) "surrogate" bags (check-bags) of 250 and 700 ml corresponding to the average standard volumes of the real products, containing a fluid validated to simulate the thermal behaviour of plasma; b) a mobile probe (cryo-med) positionable at the core of the check-bags; c) a dedicated software (memo-track). plasma freezing session data are tracked via barcode/rfid and can be consulted by the pcs that associates blast freezer code, operator code, cryo-med and check-bag. data on plasma freezing are stored in a shared folder and transferred to the be information system. the pcs can also be used to check and monitor the out-of-storage variations of core t of frozen plasma unit, i.e. during labelling and packaging procedures, thus allowing to establish optimal timeframes and operations and suitably validate these procedures. in the period 2016-2018, at the pievesestina be of emilia romagna region 210,871 plasma units were frozen so as to allow complete freezing within 60 0 to a temperature below à30°c, in 8,349 freezing sessions, using the pcs both for process validation, change control and for the systematic monitoring of core t at each freezing session. furthermore, at the bologna be 108 tests on the out-of-storage conditions of plasma units were carried out to revalidate the procedures of labelling and packaging. results: out of 8,349 freezing sessions carried out at the pievesestina be, 27 (0.3%) were detected to fail to reach à30°c at the core of the check-bags within 60 0 . of the latter, in most cases (70%) a technical error in the activation of the cryo-med was identified. in addition, the pcs was systematically utilized for periodical revalidation of the freezing procedures. the tests performed at the bologna be to validate the out-of-storage procedures of frozen plasma labelling and packaging allowed to modify the operating procedures in place so as to establish optimal timeframes and operations. this prompted corrective actions regarding: i) number of units to be taken out of storage sites at each labelling session (<24 units), ii) labelling time (<8 0 ), iii) optimal storage t (à40°c vs. à30°c), iv) optimal time between two openings of storage sites (>30 0 ). summary/conclusions: the pcs is a valuable system for plasma freezing validation and monitoring, as well as to perform monitoring and control of the whole pathway of frozen plasma in the be. it is a technologically advanced, easy-to-use and costeffective tool that can efficiently replace other traditional methods commonly used for the above-mentioned purposes. assessment of blood group matching quality using six sigma metrics background: six-sigma metrics provides a general methodology to evaluate a process performance on a sigma scale. implementation of six-sigma for quality assurance can benefit the health care sectors. one of the most important health care sectors is blood transfusion service. for that reason, maintaining a high quality in blood transfusion service is required. pathogen in activated plasma is one of the main products that are provided by the blood transfusion service. the process of producing pathogen inactivated plasma involves blood group matching step. the quality of this blood group matching is extremely significant for the delivery of plasma that satisfies the recipient need. aims: the aim of this study is to assess the quality of blood group matching of pooled plasma units using six sigma metrics, and to clarify the potential implementation of six sigma metrics as a quality management tool. methods: this retrospective study was conducted in the component preparation lab of kuwait central blood bank. the twelve months (january 2018 -december 2018) data of pooled fresh frozen plasma units were recruited and examined. the data was separated to data without double check (6 months) and data with double check (6 months). data statistics and analysis were conducted by the use of six sigma metrics. results: in a sample size of 6818 from the first six months a 38 mismatch was found which equals 5573 dpmo and 4.1 sigma metric. and in a sample size of 5854 from the second six months a 25 mismatch was found which equals 4277 dpmo and 4.2 sigma metric. out of the whole 12672 pooled units 63 were found to be mismatched. some of which were found to be discarded as abo discrepancy, broken, or expired. other was still available in the system, while the rest of the mismatched units were issued. summary/conclusions: using the six sigma principle the study presents a successful assessment of blood group matching quality. as a 4.1 sigma metrics obtained from the first 6 months, were shifted to a sigma metrics of 4.2 in the second 6 months, after the addition of a double-checking step to the blood group matching of pooled plasma process. the implementation of these metrics in our laboratory quality management has been shown to be very beneficial. in which six-sigma metrics were able to clarify the reduction in blood group matching errors. although six-sigma benefits in major quality improvements and helps to reach an error free laboratory services, yet it presents a new challenge to laboratory practitioners. currently, the hemophilia a patients treated with factor viii concentrated as the first line of therapy but it is more expensive and the supply is not sufficient so for now they have not used factor viii concentrate as prophylaxis therapy. for some cases, hemophilia patients in indonesia depend on subsidy from the world federation of hemophilia. the first handicapped concentrated case is just for therapy not for prophylaxis. big blood centers in indonesia produce routinely fresh frozen plasma (ffp) and cryoprecipitate-anti hemophilic factor (ahf) as replacement therapy for hemophilia a, but its content and safety of factor viii from 150 ml ffp need to be improved. nowadays, there is an available kit for producing minipool cryoprecipitate (mc) that has better safety and quality but it is available as liquid products, stored in very strict and specific temperature (à20°c). prophylaxis therapy for hemophilia patients needs a stable product, easy to use and convenient treatment for patients. aims: to analyze the content and safety of f viii with minipool cryoprecipitate (mc) and lyophilized mc for home therapy. methods: we produced 7 mc; 1 mc as the control, 3 mc were lyophilized with excipient and 3 mc without excipient. we analyzed the number of factor viii, the safety, and stability. we count the erythrocyte, leukocyte and platelet residual in mc using flow cytometry. we also measure the ph, osmolality, solubility to learn its stability after storage at 30 days at room temperature (30-32°c) and blood bank refrigerator temperature (2-8°c) at central blood transfusion services (cbts). results: we found the content f viii with excipient is higher (9.8 iu/ml) than without excipient (4.5 iu/ml) and the storage at blood bank refrigerator (2-8°c) is better than at room temperature (30-32°c) . in both group, there were no residual cells and bacterial found in mc. no significant difference in the ph, osmolality and solubility in both groups. summary/conclusions: the lyophilized mc with excipient stored at blood bank temperature (2-8°c) is better than room temperature. this experiment will be continued to know its stability in extended storage time. background: peptic ulcer disease (pud) is a multifactorial and complex disease, and it affects a wide range of people in the world. however, a perfect therapy for pud has not yet been available at present. therefore, we provided a novel therapeutic approach for pud patients and observed its effect in this study. aims: we provided a novel therapeutic approach for pud patients and observed its effect in this study. methods: in this randomized controlled trial, pud patients residing in chongqing were enrolled from 2016 to 2017. they were randomly assigned to two groups: (a) a control group used only rabeprazole, and (b) a platelet-rich plasma (prp) group that received a combined therapy of autologous platelet-rich plasma (aprp) and rabeprazole. the aggregation rate of aprp was measured via aggregation remote analyzer module. the therapeutic effect was assessed via the ulcer size and the symptom score. all data were recorded and analyzed statistically using spss. results: a total of 27 patients were included (12 patients as control group) and (15 patients as prp group) in the analysis. we found that the aggregation rate of aprp is not affected in ph 2.5 after treatment with pepsin. our results showed that there were no significant differences between the prp group and control group before the treatment, and there was also no significant difference in healing time between the two groups in different variables. however, regression analysis revealed that the healing time was 6.99 d less in the prp group than in the control group, and the patients with higher symptom scores in the initial examination need more time to heal in treatment. summary/conclusions: this study showed an encouraging preliminary result that aprp has a positive result in the peptic ulcer patients, and it seems to be a better choice for refractory pud patients. despite the further follow-up studies are needed to determine the duration of efficacy of aprp, the approach will be helpful for improving the pud treatment in clinical. background: the croatian institute of transfusion medicine (citm) collects, produces and distributes blood components in an area of 4.2 million habitants. annually, it collects about 100,000 whole blood and 3,500 apheresis donations. platelet concentrates (pcs) are more inclined to bacterial contamination due to storage conditions that favor bacterial replication. the citm decided to evaluate the mirasol pathogen reduction technology (prt) system as it offers the possibility to work with a non-toxic, non-mutagenic compound that upon uv illumination induce nucleic acid damage, reducing the risk of septic transfusion. aims: the study objective was to evaluate quality of pcs treated with the mirasol prt system for platelets and stored in tpas+ for 7 days at 22°c on a platelet shaker. methods: pcs were produced according to the citm's s.o.p., either through pooling of 4 bc with tpas+, "wbd", or through apheresis collection using two devices: the fresenius amicus, "ad" and haemonetics mcs+ system, "mcd". pcs were stored in 33% plasma and 65% pas and mcd were subsequently evaluated also in 38% of plasma and 62% pas. identical pcs were produced with a pool-split protocol to be prt-treated or serve as untreated control. pcs were treated with the mirasol system according to manufacturer's instructions. qc parameters, such as yield, ph and swirl were measured at days 1, 5 and 7. bacteria sterility test was performed at day 7 for a sample of all treated platelets. protein content of pcs produced routinely at the citm was determined to assess accuracy of plasma carry-over calculations for all processed pcs. results: mirasol-treated wbd (n = 10) and ad pcs (n = 8) stored in 33% plasma showed at day 7 an average ph ≥ 6.9; swirl ≥ 2.5 and yield = 3.3 9 10 11 . their untreated counterparts showed average values for ph ≥ 7:0, swirl ≥ 2.9 and yield 3.3-3.4 9 10 11 . mcd stored in 33% plasma (n = 6) that underwent prt showed at day 7 average values for ph = 6.7, swirl = 1.2 and yield = 3.05. control mcd showed average values for ph = 7.0, swirl = 2.8 and yield = 3.1 9 10 11 . mcd stored in 38% plasma (n = 8) that underwent prt showed average values for ph = 6.6., swirl = 1 and yield = 3.1. their untreated counterparts had average ph = 7.1, swirl = 2.9 and yield = 3.2. total protein content in pcs derived from wbd (n = 12), ad (n = 15) and mcd (n = 27) was 22 g/l, 20 g/l and 20 g/l, respectively. while the coefficient of variation of wbd and ad ranged from 3% to 4%, plasma products 129 respectively, the one of mcd reached 14%. all prt-pcs were negative for bacterial growth at day 7. summary/conclusions: mirasol treated wbd and ad produced according to citm current s.o.p. were quite similar to untreated controls at expiry, on day 7 and passed the requirements of the eu guidelines (19 th edition). quality of mcd units met eu criteria at day 5; swirl decreased significantly at day 7 which might be explained by the variability in plasma content of mcs+ -derived platelets, challenging the accurate calculation of illumination index for the mirasol treatment. all mirasol treated pcs showed minimal platelet loss at the end of storage. as the implementation of pr had to be cost-neutral it could only be implemented for~25% of the annual produced buffy coat platelet concentrates (bcp) (~40.000 bcp/year) and required a change in the bcp production method. the primary aim of the implementation was to offer increased blood safety to our most vulnerable patients. the secondary aim was to ensure that we built-up enough routine experience with pr to enable us to quickly ramp-up the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. aims: to verify if we could produce~25% pr-bcp without increasing the overall production cost (opc) for bcp. also evaluate the impact of pr on overall scrap rates of bcp, outdate rates and usage of other safety measures. methods: we compared opc for bcp between the pre-pr period (2017) . this cost was offset by substituting a semi-automated production method for bcp, which was used in 2017 to produce 28.7% of bcp-units. a manual double dose buffy coat production method (dd-bcp) in combination with pr enabled us to reduce the bcp-disposables cost by 89.2%. despite the moves from a semi-automated to a manual production method the overall scrap rates during production decreased in 2018 by 0.19%. the extension of max. storage time from 5 to 7 days for 24% of the bcp-units that were pr resulted in decreasing our overall outdating rates by 29% (versus 2017). this reduction in outdating rates reduced our opc in 2018 by 2.2%. in 2018 we gamma-irradiated 25.9% fewer bcp-units, but this had only a minimal impact on the opc. summary/conclusions: results of this study confirmed that we reached our initial objectives of producing~25% pr-bcp without increasing the overall production cost (opc) of bcp. it enabled us to offer increased blood safety to the most vulnerable patients. we built-up enough routine experience with pr so we could quickly rampup the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. background: irradiation of red cell units is undertaken to prevent transfusion-associated-graft-versus-host-disease (ta-gvhd) in immuno-compromised patients. while irradiators using radioactive c-ray sources are primarily found in blood establishments, they require regular recalibration and supplementary safety measures. xirradiation has been shown to have similar biological effectiveness to c-irradiation and does not require a radioactive source. there is international interest in moving away from gamma sources to reduce vulnerability to terrorism. although damaging, impacts of irradiation on red cells are well recognised. only a limited number of studies have compared red cell component quality following cand x-irradiation for both standard volume red cell concentrates (rcc) and neonatal red cell splits (rcs). aims: to compare the in vitro quality of rcc and rcs when subjected to cor xirradiation on day 14 of storage then stored for a further 14 days. rcs were also irradiated on day 5 of storage as that is most common practice in nhs blood and transplant (nhsbt). methods: four rcc were pooled and split into 4 arms on day 1 of storage, with 10 units in each arm. all units received an irradiation dose of 25.9-48.3 gy. two arms remained as standard volume rcc and were either cor x-irradiated on day 14 of storage. the other two arms were both split into 6 rcs on day 4 of storage before being irradiated on day 5 (early arm) or day 14 (late arm) of storage. for each replicate in these arms, 3 splits were c-irradiated and 3 splits x-irradiated. all arms were tested a day prior to irradiation and 1, 7 and 14 days post-irradiation for red cell quality parameters: haemolysis, intracellular atp and 2,3 dpg, supernatant potassium, glucose and lactate, ph and red cell microvesicle release. the rcc arms were sampled over storage; while for the rcs arms, 1 split was tested on each testing day post-irradiation. a 2-way anova was used to detect statistical differences over storage between cand x-irradiation for the same components. results: all components produced were within nhsbt specification for volume, haemoglobin and haematocrit. there were no significant differences in red cell in vitro quality parameters studied over storage between cand x-irradiated units, for standard volume arms or neonatal arms and whether rcs were irradiated early or late in storage. moreover, all arms were within haemolysis specification for the end of storage (>75% of units with < 0.8% haemolysis) and 100% of units had atp levels above the recommended minimum for acceptable post-transfusion survival (2.3 lmol/ghb). both haemolysis and potassium levels at the end of storage for the standard c-irradiated rcc were comparable to our laboratory's historic data for the same component. summary/conclusions: in summary, the storage quality of rcc and rcs post-xirradiation did not differ from c-irradiation in this study, providing reassurance that either method could be used in routine manufacturing. a pajares herraiz 1 , c coello de portugal 2 , m morales 3 , f solano 4 , c perez parrillas 5 , a rodriguez hidalgo 5 , t diaz rueda 5 and m flores 5 1 direccion, regional transfusion center toledo-guadalajara 2 transfusion service, toledo hospital complex, toledo 3 transfusion service, general university hospital of guadalajara, guadalajara 4 transfusion service, hospital nuestra señora del prado de talavera de la reina, talavera 5 regional transfusion center, regional transfusion center toledo-guadalajara, toledo, spain background: the regional transfusion center of toledo-guadalajara (rtc) manages the collection, processing and distribution of blood components for the hemotherapy area of castilla la mancha (spain) that serves 3 general hospitals (hospital complex of toledo (hct), university general hospital of guadalajara (ughg) and hospital nuestra señora del prado de talavera (nspt)) and the needs of 943,000 inhabitants. by also managing the hct transfusion service, it facilitates the handling of stocks. since 2008, rtc has initiated pathogen inactivation (pi) for a part of its platelet components(pc) with the intercept blood system (cerus) using a photochemical treatment with amotosalen and ultraviolet-a. this system allows the inactivation of a broad panel of pathogens and leukocytes, extending the shelf-life of the cp from 5 to 7 days. this affects the expiry and discards of this blood component, allows a better management of the inventory and has an influence on production costs. aims: the objective was to evaluate the influence of pi in the production of cp at rtc and the expiry in the hemotherapy area during the last 8 years divided into four periods ( results: pc were predominantly obtained from whole blood collections with 85% of bc platelets/15% of apheresis platelets. 77% of the available bc were used in production for period a and 88% for periods b, c and d. after wastes of approximately 1.9%, the distribution of pc was stable for the 4 periods studied. 7075 pc were distributed for period a, 7047 pc for b, 7467 pc for c and 7083 pc for d. the % of pi platelets with 7-day shelf life available in the 3 hospitals was limited to 13% during period a. it was then increased to 14.1%, 20.9% and 26% for periods b, c and d respectively. the percentage of wastes was stable at 0.2-0.3% but the discards due to expiry went down from 24.24% (period a) to stabilize at 10.9% in periods b and c and 10.3% in period d. in the 3 general hospitals the expiry went down from 20% to 5.89%(hct), 29.4% to 14.5% (ughg) and 33.36% to 17.68%(nspt) respectively. summary/conclusions: greater control of pc stocks through historical analysis and consumption projection, together with it tools and the use of pi pc with 7-day shelf life allowed reducing discards for expiry from 24.24% to 10.3% in the last period analyzed at rtc and the 3 major hospitals of the hemotherapy area. this has a great value in cost-reduction and improves inventory management and the efficiency of the processes. background: blood centers are faced with many challenges including availability of concentrate platelets as well as ensuring highest quality of the product. overcoming the shortage of platelet apheresis by using pooled platelet derived from whole blood units separated using automated standardized system, which can assist blood banks to meet the increase demand in platelets. the pathogen inactivation (pi) technology can improve the quality of the product by mitigating the risk of transfusion-transmitted diseases (ttd) and residual white cells, resulting in minimizing non -hemolytic transfusion reactions. however, the pathogen inactivation treatment must not impact the platelet quality and functionality significantly, as well as the patient safety. aims: evaluate the quality of pooled platelets derived from whole blood (five interim platelet units), separated using reveos automated blood processing system (terumo bct), pooled in 100% donor plasma and pathogen inactivated by amotosalen/uva technology. methods: five interim platelet units (ipus) produced with reveos device (terumo bct) from single whole blood donations, were pooled with a platelet pooling set (terumo bct) and leucodepleted with a lrf-xl filter (haemonetics). thirty pools have been included in this study, the units were treated using a large volume cerus intercept processing set for platelets according to the manufacturer's instructions and stored until day 5. the swirling was determined by visual inspection. the volume and yield content were assessed preinactivation and after treatment by pathogen inactivation with a cell counter (dxh-800, beckman coulter), rbc contamination was also measured preinactivation with a cell counter (beckman coulter), bacterial contamination was assessed by automated blood culture with a bact/ alert system (biomerieux). the ph of the platelet units was assessed with a phmeter (jenway), and residual amotosalen levels were assessed by an hplc assay. results: the impact of amotosalen/uva pathogen-inactivated pool platelet products quality were assessed. the pre and post-inactivation of the units showed a swirling score of 2-3. the average volume per unit of the pre-inactivation was 288 ml (278-302 ml) and post inactivation was 269 ml (254-284 ml), with average volume loss during inactivation was 36 ml (24-43 ml), corresponding to 13% (8-15%). the average platelet yield per unit pre-inactivation was 3.7 9 10 11 (3.0-4.4 9 10 11 ) and post inactivation 3.2 9 10 11 (2.8-3.9 9 10 11 ) with an average platelet loss of 13% (3-20%) . the average rbc contamination per unit (0.01-0.02 9 10 9 rbc/ml). the culture tests were negative, the average ph at day 1 was 7.4 (7.1-7.6), average ph at day 4/5 was 7.3 (7. 2-7.4 ). the average residual amotosalen concentration post treatment was 0.30 lm (0.23-0.35 lm). summary/conclusions: the quality of pathogen-inactivated pool platelets tested, met the criteria set by aabb guidelines. the volume and platelet loss were in acceptable range, in alignment with previously published data. a residual amotosalen concentration below 2 lm is considered safe and acceptable by french and german authorities. the evaluated data support the reasonable assurance of quality and effectiveness of the device when used in accordance with indication for use. background: the implementation of a pathogen inactivation process (pi) allows the redesign of processes to obtaining safe blood components by reducing the need for additional testing for pathogens detection, minimizing the residual risks (such as the infectious window period for those pathogens that are detected as usual), eliminates the need for selective tests (eg cytomegalovirus serology test) and complements gamma irradiation given its ability to inactivate white blood cells. in addition, the routine implementation of pi reduces the incidence of bacterial infection in recipients of blood components and allows blood services to proactively protect the blood supply against future emerging infections. aims: to verify the functional integrity and viability of platelet concentrates after being inactivated of any pathogenic agent, to be used as safe and functional components for transfusions. methods: a total of 106 independent platelet concentrates were studied. platelets are donated through a process called plateletpheresis according to the established norms, after the process, platelet concentrates were submitted to pi on the intercept blood system tm platform with uv-a illuminator; an immediate sampling of each donation of platelet concentrates was carried out taking a sample of 2 ml pre-inactivation and another sample post-inactivation (16 h after pi). the platelet viability of each sample was evaluated by demonstrating the cd62p expression marker by flow cytometry. once processed, platelet concentrates were released as safe components for donation. compiled the experimental data of the platelet count with platelet activation marker with respect to the total platelet, a comparative, nonparametric test of wilcoxon was carried out between two measurements (pre vs post) and the platelet viability after pi was determined. results: a total of 106 independent platelet concentrates were studied, where the average percentage of pre-inactivated platelets with expression of the cd62p marker, was 18%, while the percentage of functional platelets post inactivation was 19%, this result only shows that the functionality of the platelets is not being altered after the inactivation process. the wilcoxon test confirms that there is no significant difference between platelet activity pre-and post-inactivation, with a 95% confidence level. summary/conclusions: the process of photochemical treatment with amotosalen hydrochloride and long-wavelength ultraviolet light (uva) applied to platelet concentrates provides functional products without alterations in platelet function to be transfused. background: treatment of platelet concentrates (pcs) with pathogen reduction technologies is widely implemented in blood establishments to reduce the risk of bacterial contamination and to face the presence of new emerging agents in blood components. aims: the reduction of antioxidant power (aop) could be a quality control test to prove the complete viro-inactivation treatment. this evaluation has the goal to study the feasibility of the method from "abonnenc et al., transfusion, 2016" in another blood service, assessing the aop of platelet units treated by intercept technology. methods: the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid. repeatability, intermediate precision and accuracy were determined. linearity was evaluated using the linear regression and the calculation of pearson's coefficient (r²). limit of quantification (loq) was determined by measuring aop using nacl samples to define the background. roc curves were used to determine a threshold to discriminate pcs before and after treatment. a distinction was realized between men and women and between apheresis (a) pc and buffy coats (bc) pcs. a one-year evaluation was assessed on pcs before and after treatment on the routine production. results: the coefficient of variation for the repeatability was less than 10%. for the intermediate precision, the coefficient of variation was less than 15%, but for the pcs after treatment, this result rose up to 21%. the r² value for the linearity was 97.7%. the detection limit corresponded to a result of 6 edel and the loq (equal to 10xsd) is 19 edel. concerning roc curves, the men apcs threshold was 58.5 edel compared to women apcs with 62.5 edel. the threshold for bcpc was 54 edel. all of these results had 100% of specificity. below this threshold, intercept treatment was considered to be executed. about the one-year experience on routine pcs production, 404 apcs (182 women and 222 men) and 263 bcpcs were tested. all of the bcpcs and women apcs were under the threshold after treatment. concerning men apcs, 14.0% of the pcs after treatment were not under the threshold. summary/conclusions: the device validation was satisfied. for the one-year evaluation and concerning men group apcs, the threshold found by abonnenc et al. was 89 edel. our study showed a threshold with 100% specificity and 90% sensitivity at 58.5 edel which is much lower. specificity was favored compared to sensitivity but the analysis should be revised to adapt the threshold to get higher sensitivity. this can lead to reduce the non-conformity and allows measuring the aop only after treatment. for women, our threshold was found at 62.5 edel compared to 66.5 edel for abonnenc et al. concerning sex in apcs, results were statistically lower in women group than men group before and after treatment. and for bcpcs, the two populations (before and after treatment) were very distinguishable and our threshold (54 edel) was lower than abonnenc threshold which was at 59.8 edel. in conclusion, edel threshold enables the segregation and depends on the preparation process adapted in each blood service. aims: this study has the goal of measuring antioxidant power (aop) level in plasma units treated by mb technology. the aim is to use such a test as a quality control assay for documenting the execution of pathogen inactivation treatments during the preparation of plasma units. methods: aop measurements were performed using a potentiostat electrochemical analyzer. a 3-ll volume of sample is deposited over the electrodes on a single-use microship. the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid and reflects the redox status of the plasma units. different protocols were established to understand the role of mb, the illumination and the filtration on the aop variation measure: 1) complete treatment, 2) plasmas with mb without illumination, 3) plasmas without mb with illumination and 4) plasmas without mb without illumination. ten dosages on men donor samples, except for protocol 1 where n = 20, were realized during the viro-inactivation process, t1 corresponds to a dosage of plasmas before treatment, t2 the plasma after the mb dry tablet passage, t3 is the time after illumination and t4 corresponds to the final product (after filtration). results: in each protocol with mb, an increase was observed after addition of mb before illumination. after illumination, the edel values decreased for about less than 50%, which was expected because of the degradation of mb in its photoproducts during the illumination. in the series 1 and 3, the illumination seemed to have an effect by itself, with or without mb because the aop increased. the final filtration has the goal to eliminate the residual mb and its photoproducts. after this step, the aop values fell down. the series 2 was a confirmation of the efficacy of the filter to remove the mb as shown by the decreased aop in t4 (238 ae 16 edel at t3 and 209 ae 17 edel at t4). however, in the absence of mb (series 3 and 4), the results at t1 and t4 were not statistically different. summary/conclusions: the filtration decreases the aop rate, except when there was no mb. the results of non-complete viro-inactivation treatment allow concluding that the measure of aop rate may not indicate that the treatment was completed or not since significant differences before and after treatments were found in the non-complete treatment series. vox sanguinis (2019) background: the intercept blood system (ibs), a photochemical treatment with amotosalen and uva, is used to inactivate pathogens and leukocytes in plasma. the intercept tm plasma processing set (cerus bv, netherlands) was modified to incorporate plastic containers in non-pvc materials sourced from alternate suppliers and connecting parts and accessories in non-dehp pvc formulations, making the system dehp-free. the final storage container was modified with a higher contact surface with plasma to limit the thawing time. proportion of units with a fibrinogen concentration ≥ 2.00 g/l was 94% (>70% required). mean recovery fviii fibrinogen after ibs treatment and frozen storage were 75% and 90%, respectively. residual platelets were < 25 9 10 9 /l, leucocytes < 1 9 10 4 /l and red blood cells < 4 9 10 9 /l. all units had a protein content > 50 g/l. residual amotosalen was below 2 lm in all post-cad samples. the concentration of tat complexes was slightly reduced after treatment and frozen storage. concentrations of c3a and c5a were significantly reduced with the cad treatment. the plasma thawing time in a water bath at 37°c was consistently short (6-7 min). summary/conclusions: pathogen inactivated plasma units (ffp-a-ibs and ffp-wb-ibs) prepared with dehp free intercept processing sets retained in vitro characteristics which meet the quality standards for therapeutic plasma. the process did not activate coagulation or complement. reducing ffp thawing time from routine 8-10 to 6-7 min is an important benefit for emergency use. background: plasma coagulation factor concentrations usually differ for individual donors, therefore pooling of whole-blood derived plasma units moderates high or low coagulation factor concentrations and ensures transfusion of more standardized blood components. moreover, pooling contributes to dilution of reactive antibodies and may reduce the risk of non-hemolytic transfusion reactions and trali. additionally pathogen inactivation reduces the risk of transfusion-transmitted infections, and non-hemolytic transfusion reactions as well as gvhd through inactivation of residual lymphocytes. aims: assessment of the impact of plasma pooling and pathogen inactivation on the standardization of blood components and plasma quality. methods: the study included 5 experiments. for each experiment 5 male-donor, abo-compatible whole-blood derived plasma units (≥ 270 ml) were collected from different donors and pooled using the donopack optipool plasma pooling set (cerus europe b.v.). each of the 5-unit pools were split into 2 equal minipools which were subsequently treated with the in intercept blood system (cerus europe b.v.). then, each minipool was split into 3 (≥200 ml) therapeutic units. samples were collected before and after pooling as well as after inactivation to assess the coagulation factor content (fviii, fix, fibrinogen, vwf antigen using elisa) and coagulation time (aptt, pt). the study-analysis included samples from five pools from 5 single plasma units respectively ( background: biotin (bio) is an alternative to radioactive red blood cell (rbc) tracers which allows one to concurrently track in vivo multiple cell populations labeled at different bio densities. in american clinical trials, multi-labeled biorbc have been transfused in man to assess their survival (mock et al, transfusion, 2018) . in these studies, the different biorbc populations were monitored by ex vivo flow cytometry analysis using streptavidin. so far, the biotinylation reagents biosulfonhs was not complying with good manufacturing practices (gmp). moreover biorbc, with bio ≥ 18 lg/ml, have induced immunization of the recipient, in rare cases (schmidt et al, transfusion, 2017) . this represents an obstacle regarding the regulatory european authorities. aims: the aim of this study is to describe a procedure of biotinylation of rbc intended for clinical trials while refining the levels of bio ≤ 18 lg/ml. methods: sterile status is met throughout the process. rbc are taken from standard rbc concentrates and treated with biosulfonhs of gmp-grade (0 to 70 lg/ml) recently commercialized. washing buffer is of injectable-grade. biotinylation efficacy is controlled by flow cytometry with streptavidin conjugated to 2 different fluorochromes: phycoerythrin (pe) or brilliant violet (bv421). results: labeling with biosulfonhs of gmp-grade or non gmp-grade is comparable and 4 populations of rbc could be easily distinguished between themselves and from unlabeled blood cells. biosulfonhs (lg/ml): 0 (mfi 0.4), 4 (gmp mfi 12; non gmp mfi 9.5), 18 (gmp mfi 44; non gmp mfi 42), 70 (gmp mfi 174; non gmp mfi 180). streptavidin-bv421 brighter than streptavidin-pe is a promising tool because it amplifies by 1.7 the signal of fluorescence and allows a good differentiation of the 4 populations of rbc treated with only 0, 1, 4 and 18 lg/ml biosulfonhs. summary/conclusions: this preliminary study explores the feasibility of multilabeled biorbc production for clinical trials. the benefits of this approach are to overcome the need for non-radioactive tracers, to follow simultaneously various populations of rbc and consequently to limit the number of volunteers, and to reduce the risk of immunization using bio ≤ 18 lg/ml. background: rejuvenation is aiming to revert ageing-related disease development. heterochronic parabiosis studies revealed eotaxin in young and old murine blood as a regulator of brain aging and neurogenesis. umbilical cord blood (ucb)-borne factors including tissue inhibitor of metalloproteinases 2 (timp2) and neonatal immune cells also contributed to rejuvenation in animal models. human platelet lysate (hpl) is commonly used by us and others for highly efficient cell propagation in vitro (burnouf et al., biomaterials, 2016) . published data indicate only limited differences between adult and ucb-derived hpl, partly questioning enigmatic rejuvenation effects. aims: to verify candidate regenerative factors in neonatal blood products we compared protein contents of neonatal and adult plasma and platelets, respectively. methods: heparinized ucb samples (n = 9) were centrifuged within 3 h to collect neonatal platelet rich plasma. aliquots from apheresis platelet concentrates (n = 9) were used as adult counterpart. platelet concentration was adjusted to 7-10 9 10 11 / l. plasma supernatants and platelets were obtained by centrifugation and platelet pellets were re-suspended in saline. after two freeze/thaw cycles at à30°c/37°c for platelet lysis (npl; apl) the platelet fragments were removed by centrifugation. the protein content was analyzed with a proteome profiler tm array. nine samples of each group were pooled to avoid individual donor variations. a threshold of 50,000 au spot density was defined as cut-off. data were analyzed by graphpad prism 8 using two-way anova. results: semi-quantitative evaluation of 105 analytes per array revealed significant differences. in plasma samples and platelets 63 and 48 analytes were detected above cut-off, respectively. in neonatal plasma we found more highly prevalent proteins (>200,000 au spot density) compared to adult plasma (6/63 vs. 3/63). thirteen proteins were significantly elevated in neonatal plasma including growth/differentiation factor 15 (gdf 15), platelet derived growth factor aa (pdgf-aa) and serpin e1 (p < 0.0001). more highly prevalent proteins were detected in npl (10/48) compared to apl (1/48), and 20 proteins were significantly elevated including vascular cell adhesion molecule-1 (vcam-1), platelet factor 4 (pf4/cxcl4), epidermal growth factor and lipocalin-2 (p < 0.0001). in adult samples only 10 proteins were significantly higher in plasma and three proteins in apl compared to the neonatal groups (p < 0.05 to p < 0.0001). summary/conclusions: we detected significant differences in regenerative growth factor and cytokine contents of neonatal and adult plasma and platelet samples, respectively. additional experiments are underway to further characterize their impact in distinct functional readouts. background: the production and storage conditions of platelet (pl) products intended for transfusion are constantly evolving and need sometimes in vivo evaluations in clinical trials to ascertain whether the platelets have retained their ability to survive in the circulation. this requires that the transfused platelets can be distinguished from the recipient's circulating platelets. labeling of platelets with biotin (bio) affords to track in vivo and concurrently, multiple cell populations covered with various biotin densities as already described for red blood cells (mock, transfusion, 2018) . surprisingly, there is only one study describing the transfusion of human biopl (stohlawetz, transfusion, 1999) . so far, the biotinylation reagent bio-sulfonhs was not complying with good manufacturing practices (gmp), which represents an obstacle regarding the regulatory authorities. aims: the aims of this study are 1) to describe a procedure to label injectable human platelets with 2 densities of biotin, 2) to evaluate the impact of biotinylation on platelet functions, 3) to track human biopl in the circulation of the mouse. methods: platelets are taken from standard platelets concentrates and treated with 1.2 and 10 lg/ml biosulfonhs of gmp-grade, recently commercialized. main platelet functions are assessed in vitro. human biopl survival is evaluated in immunodeficient nsg-mice treated with liposome-clodronate to eliminate macrophages and to prevent rejection. circulating human biopl are detected ex vivo by flow cytometry with streptavidin phycoerythrin. results: using trap (60 lm), p-selectin externalization reveals a normal capacity of secretion for all biopl. gpiba and gpiibiiia expression is not affected by the biotinylation process. biopl have the ability to aggregate: using arachidonic acid (1 mm), amplitude of aggregation is 81.7 ae 2.5% (bio 0); 82.6 ae 2.3% (bio 1.2 lg/ ml); 79.2 ae 1.6% (bio 10 lg/ml). using collagen (2.5 lg/ml), amplitude of aggregation is 65.6 ae 4.2% (bio 0); 61.4 ae 4.0% (bio 1.2 lg/ml) 40.8 ae 6.6% (bio 10 lg/ml). the 2 biopl populations could be easily distinguished between themselves and from unlabeled blood cells in the mouse circulation during more than 48 h. after 4 h, the mean fluorescence intensities are 0.13 ae 0.02 for unlabeled circulating mouse platelets, 1.01 ae 0.03 and 8.2 ae 0.15 for circulating human biopl covered respectively with 1.2 and 10 lg/ml biotin. summary/conclusions: this labeling approach should be helpful to evaluate new platelet products in vivo and represents an alternative to radioactive tracers. it allows to follow simultaneously different platelet populations and consequently limits the number of volunteers in clinical trials. background: severe ocular surface diseases, dry eye syndrome, persistent and recurrent corneal epithelial defects and diabetic or neurotrophic keratopathy are mainly successfully cured by standard treatment protocols. however, not rarely does refractory to these usual treatments appear, especially with serious forms of disease. in military medical academy, autologous serum eye drops -auto seds and autologous platelet lysate -apl eye drops have been being applied in the treatment of ophthalmological patients in these categories, who were previously resistant to standard therapy. aims: to show the achieved results of therapeutic use of autologous blood products (auto seds and apl) in the treatment of ophthalmological patients who previously had not responded to conventional therapysingle center experience. methods: auto seds are prepared by taking autologous blood into tubes (bd vacutainer, cat, 10 ml) and apl in tubes with anticoagulants (greiner bio-one, acd-a, 9 ml). control on tti of every patient and sterility of every series has been conducted. before and after the treatment, subjective ocular discomfort (ocular surface disease index -osdi), objective parameters of the tear film (schirmer's test, rose bengal, tear breakup time -tbut) and measuring of epithelialization zone were analyzed. apl, obtained from platelet-rich plasma which had been frozen, unfrozen and diluted with nacl solution, up to 30%. auto seds were administered in the form of 20% eye drops. results: auto seds have been applied to 17 ophthalmological patients (10 men and 7 women), previously resistant to standard therapy. in total 44 treatments were performed (each lasted 20 days). for successful curing, one or two treatments per patient, in average, were applied. apl has been used multiple times to one patient with sj€ ogren syndrome and severe multiple tropical corneal changes. all ophthalmological patients had subjective improvements (the average pre and post treatment osdi scores were 71.3 and 19.6 respectively). also, objective progress was present in 83% of all patients (p < 0.001). summary/conclusions: the use of auto seds and apl in the treatment of ophthalmological patients, previously resistant to standard therapy, is in constant increase, because of its simplicity and low expenses. apl has turned out to be better than auto seds for patients with severe trophic changes, because apl contains larger amounts of the nerve growth factor, tgf-b, vegf and platelet derived growth factor. however, a larger number of clinical cases is needed for future conclusions. background: whole blood (wb) has recently regained favor in treatment of massively bleeding patients in military and civilian settings. 1 platelets (plts) are a vital component in clot formation. as a component of wb, it is critical that they maintain functionality throughout storage. red blood cells (rbcs) stored in hypoxic/ hypocapnic conditions preserve high level of 2,3-dpg while reducing storage lesions stemming from oxidative stress. 2, 3 on the other hand, effects of steady hypoxia (pco2~10-20 mmhg) on plts contained in leukoreduced wb is poorly characterized. aims: examine the effects of hypoxic conditions on plt function and microvesicle (mv) formation in wb stored hypoxically (h) and conventionally (c) for 3-week storage at 1-6°c. methods: 11 units of wb were collected at mayo clinic rochester blood donor center from normal healthy volunteers into 70 ml cp2d. wb was leukoreduced using plt-sparing filter (terumo wb-s) then split into control (c) and hypoxic (h). h-wb was processed by the oxygen-reduction bag (hemanext, lexington ma) and unit was stored in o2-free bag. 5 ml of wb were collected from each unit at day 1, weeks 1, 2, 3. plt counts, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation, nonactivated and agonists activated plt surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding), and microvesicles (mv) were measured by coulter counter and digital flow cytometer. paired student t-tests were used to analyzed differences in degradation rates; significance: p < 0.05. results: h-plt counts declined to~60% by the o2-reduction process, while similar decline was observed after 1 week in c, and thereafter remained steady. plt activation (ps) increased over time (h >> c after processing; c increasing more rapidly during storage). p-selectin increased over time (h < c), while pac-1 showed large increase after 1 week, then remained steady (h << c). plt activation by trap or adp declined modestly over 3 weeks (~15%) while h-plt showed additional~10% reduction for all time points. collagen activation for c-plt increased after 1 week (74%) and gradually increased to 100% after 3 weeks (~20% reduction with h compared to c). plt-derived mv (cd61 and cd61/annexin v) increased~4-fold over storage time; day 0 mv levers were significantly higher for h, but subsequent increase rates were similar or lower. total number of plt-derived mv (cd42a) in wb supernatant increased 17-fold after 3 weeks for c, while h suppressed increase to 7-fold. (majority of the trends described above showed significant differences between h and c.) summary/conclusions: plts were activated over 3-week period when stored at 1-6°c in leukoreduced wb, accompanied by a modest loss of agonist-induced activation. oxygen reduction treatment initially activated h-plts, while subsequent increase in activation rates were suppressed compared to c-plts. wb plts retained activatability, and hypoxic condition showed only modest further reduction on the activatability. hypoxic wb may provide higher quality wb for trauma patients if the levels of initial plt activation can improved during oxygen reduction procedure. methods: after informed consent, eligible patients were randomized to either first receive autologous followed by allogeneic seds or first receive allogeneic followed by autologous seds. each sed treatment phase was one month, separated by one month of patient's standard treatment (wash out period) between sed treatment phases. the patients each donated 500 ml whole blood from which the autologous seds were prepared. allogeneic seds were prepared from blood from never-transfused male donors with blood group ab. all serum was diluted 1:1 by adding saline, and aliquoted in an eye drop dispensing system (meise, schalksm€ uhle, germany). at each visit, the osdi was determined using a validated questionnaire, with higher scores reflecting poorer outcomes. the results were analyzed intention-to-treat, and a random effects linear mixed model for cross-over design was used. results: in total, 19 patients were enrolled, 4 of whom were excluded because they failed the autologous blood donation. background: the following blood components for non-transfusional use (bcntu) are produced in our transfusional center (tc): 1) allogeneic platelet gel (pg), derived from buffy-coats (bc) and human cord blood platelet gel (cbpg); 2) autologous serum eye drops (sed). the creation of both types of platelet gel started in 2014 but only in 2017 we confirmed the process for daily production: these blood components are used to treat pediatric patients with epidermolysis bullosa. the sed, produced from 2018, is dedicated to treat patients with dry eye syndrome. aims: production and storage bcntu. methods: the whole process production of bcntu is traced on the transfusional informatic system (emonet-insielmercato), under the same conditions of another blood transfusional components. the process takes place in closed circuit using the laminar flow hood. 1) pg production starts from the bc resuspended in plasma that are not used for daily platelet concentrates, instead the cbpg is produced using cord blood units that are not used for hematopoietic transplant. both have a platelet concentration between 800-1200 10 3 /ll and negative blood cultures, required by the italian law; the units are frozen at à80°c and last 5-year. pg and cbpg must be activated with calcium gluconate or batroxobin to be used. 2) the ophthalmologist's patients, with dry eye syndrome, donate 120 ml of autologous blood; the serum is separated and after the dilution with a balanced saline solution (30%) are divided in 2 boxes containing 30 single-dose vials each: they are stored at à80°c and they last one year. negative blood culture was evaluated. results : background: candida albicans is the most common pathogen detected in fungal infections. aims: in this study, we aimed to evaluate the in vitro antifungal activity of volunteerderived platelet rich plasma (prp) against c. albicans atcc 10231 strain and the possible effects of certain chemokines, kinocidins that might play a role in this activity. methods: prp from nine volunteers were derived by using magellan prp â kit. 10% calcium gluconate was used to obtain autologous thrombin. c. albicans isolates with a final yeast concentration of 1 9 10 3 cfu/ml and 1 9 10 4 cfu/ml were inoculated on sabouraud dextrose agar at the 1 st , 2 nd , 4 th , 8 th and 16 th hours of incubation to reveal the antifungal activity of autologous thrombin-activated prp. the colonies were counted after 18-24 h of incubation at 30°c. chemokines and kinocidins (platelet factor-4, interleukin-8 and thymosin-b4) were also measured simultaneously by elisa method. results: compared with the pbs-control group, the prp-10 3 group showed that the antifungal activity was still going on at the 8 th hour. the difference in colony production between the two groups at 8 th hour was statistically significant (p < 0.05). it was observed that the antifungal activity continued at the 4 th hour, decreased at the 8 th hour in the group prp-10 4 group. although the same amount of prp was used and the same amount of chemokine and kinocidins were released in both groups, the concentration of c. albicans was considered to be important in the detection of more effective prp-10 3 group. although there was an increase in il-8 levels by hours in the two prp groups by elisa method, no antifungal effect was detected against c. albicans. it was observed that decrease in tmsb4 values results from the antifungal activity on the advancing hours in the prp groups. whereas pf-4 did not act an antifungal activity on prp-10 3 and prp-10 4 . summary/conclusions: even in our study group where the highest platelet counts were obtained at the lowest concentration, c. albicans reproduction could not completely eliminated as mentioned in the literature. repeated doses of prp applications, such as drugs used in patients, may have longer duration of action and even complete repression of reproductive outcomes. background: generally, blood is available in developed countries for transfusion. sometimes, transfused or previously pregnant patients form alloantibodies to red cell antigens and rarely, to antigens of high prevalence. this case focuses on a twoyear-old girl, of pakistani descent, diagnosed with neuroblastoma stage iv with anti-in b and -e. although the publications indicate that 4% of the pakistani, indian or iranian populations are in(b-), it was discovered that this blood type is exceedingly rare. an international search was required to ensure blood product availability for chemotherapy and autologous hematopoietic progenitor cell transplants aims: illustrate the response of the public to a powerful story of a child needing rare blood for treatment and international collaboration for provision of very rare units. methods case report: a two-year-old patient's sample was referred for antibody identification. the patient had received four transfusions (883 ml of red cells) in the preceding 10-day period. hgb level fluctuations were consistent with decreased transfused red cell survival. following the last transfusion of 225 ml, the hemoglobin decreased from 8.6 to 5.8. anti-in b , and a ficin-only reactive anti-e was identified in the serum and anti-in b in the eluate. the monocyte monolayer assay predicted the anti-in b to be clinically significant (68% reactivity). transfusion of antigen neg units once obtained, resulted in a stable transfusion response. although it was expected that in(b-) blood would be more easily sourced, only two donors in the usa were in (b-) e-. as 7-10 units of blood were requested for the post-transplant period, a national and international search was initiated, as was a robust media appeal to donors resulting in many donors for an intense domestic screening effort in the usa. the search of the who international rare donor panel by the international blood group reference laboratory revealed three known in(b-) e-donors; two british and one australian. they were contacted, recruited, collected and shipped to the usa with the work of the american rare donor program (ardp) staff and the isbt working party on rare donors (isbt wprd) members in each of the countries. results: the intense media coverage of oneblood (the florida blood center collaborating on treatment with the hospital) included online news outlets (youtube, facebook) resulted in over 27,000 responses from national and international potential donors to be tested for in b . isbt wprd members were sent the web information of potential donors identified in their countries by the ardp. over 3,500 samples from 75 blood centers and associated laboratories tested with anti-in b by oneblood. two new in(b-) donors were discovered (0.06%); but both typed e+, thus were not a match for the child. summary/conclusions: this intense media coverage and the overwhelming donor response was unprecedented in our experience. the coordination and cooperation among the numerous blood centers reflect the deep dedication of the blood banking community to the well-being of special patients in need. this case illustrates the response potential that a powerful story and a medical appeal for exquisitely rare blood utilizing social media and other online news outlets can generate. background: blood platelet units are generally stored in blood banks for 3-5 days, afterwards they are discarded. prepared infusible platelet membrane (ipm) from fresh or outdated human platelets correct the prolonged bleeding times in thrombocytopenic animals such as rabbits. infusible platelet membrane (ipm) as a platelet substitute may be the most feasible approach to reach the target market. our previous experiments have shown that ipm has a hemostatic efficacy to shorten bleeding time without any adverse effects in rabbits. aims: abnormal toxicity is the european pharmacopoeia standard for assessment of biological products which the test material is administered to the mice. in this study, abnormal toxicity of ipm was evaluated in experimental animal model such as mice to assure the safety of ipm without any evidence of serious toxicity. methods: in this experimental study, infusible platelet membrane (ipm) was prepared from outdated platelet concentrates. platelet concentrates were pooled, disrupted by freeze-thaw procedure, pasteurized for 20 h to inactivate the possible viral or bacterial contaminants with a sodium caprylate stabilizer, formulated by sucrose and human serum albumin and finally lyophilized. at first, the test for sterility is carried out under aseptic conditions for ipm vials and then we injected 0.5 ml of ipm (2 mg/kg) intravenously between 15 to 30 seconds into each 5 health mice, weighing 17-22 grams. these tests were performed according to eu pharmacopeia monographs. results: in the sterility test no evidence of microbial growth in our product is found. the abnormal toxicity test will be passed if none of animals die during 24 h after injection. if more than one animal dies, the preparation fails the test. if one of the animals just dies, the test is repeated. in our experiment all five mice were alive after 24 h of ipm injection. summary/conclusions: in this research the results showed that ipm as a platelet substitute is free of abnormal toxicity with adequate safety and it may be used in human clinical trial studies as a feasible approach to develop a platelet substitute in the future. however, further studies are required to confirm the different aspects of its safety as well. the success of such investigations may affect patients' care in transfusion medicine in the future. a substantial number of infants, especially premature infants, are unable to receive adequate amounts of their mothers' milk for a variety of reasons. the world health organization recommends that infants, especially preterm and ill infants are fed with quality-controlled donor milk if they cannot be fed with their own mother 0 s milk. due to the possible transmission of the human immunodeficiency virus many human milk banks closed in the 1980s, therefore the availability of donor milk has decreased. aims: we analyzed the processing of donor milk and the required laboratory tests to establish a human milk bank within our blood donation service in cooperation with the department of neonatology at the frankfurt university hospital. methods: based on the recommendations for promoting human milk banks in germany, austria, and switzerland (efcni) we evaluated the manufacturing steps and the quality controls require to establish a human milk bank. background: for patients suffering from severe ocular surface disorders treatment with blood derived serum eye drops (sed) is a highly effective therapy. autologous sed, prepared from the patient's own blood, is used preferably. for this approach we have more than 6 years of experience. if auto-sed cannot be manufactured due to medical reasons allogeneic sed present an alternative. since 2 years, the allogeneic approach is well established in our center. aims: retrospectively evaluation of our experience with allo-sed. methods: in germany manufacturing of allo-sed is only possible as an "individual healing attempt". for each patient experienced regular ab0-identical male donors without blood borne disease, who never received blood products and not taking any kind of medication are selected. additionally, donors must pass a questionnaire excluding any form of dry eye syndrome. allo-sed are manufactured directed for each individual patient according to the process for auto-sed in a closed system. patient files of our serum eye drops donors were screened for patients receiving an allogeneic treatment. data concerning indication for allo-sed, contraindication for phlebotomy, problems with donor selection and manufacturing, as well as serological and microbiological testing results were obtained. clinical results were evaluated © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 by ocular surface disease index (osdi) and patient's questionnaire, asking for subjective benefit, symptom reduction, possible side effects, consumption and comparison with artificial eye drops or, if applicable, with auto-sed. furthermore patients are undergoing regular ophthalmologic examination within a special consultation for dry eye syndrome at our hospital. results: 31 patients were identified receiving allogeneic sed, 15 patients had been treated autologous previously. in total, allogeneic sed have been produced 54 times since june 2017. indications were ocular gvhd (n = 15.48%), neurotrophic keratopathy (n = 9.29%), mucous membrane pemphigoid (n = 2.7%), sj€ ogren syndrome (n = 1.3%) and secondary keratoconjunctivitis sicca by virtue of chemotherapy, meige syndrome, rosacea, morbus bruton (n = 4.13%). contraindications for autologous donation were underlying disease (n = 19.61%), poor venous access (n = 12.38%), low haemoglobin (n = 8.25%), low body weight (n = 8.25%), very young age (n = 7.22%), circulatory disturbances (n = 3.10%) and lack of response to auto-sed (n = 2.7%). some patients presented more than one contraindication. manufacturing problems were: lipemic donor plasma (n = 2.4%), high donor haemoglobin (n = 2.4%) and unspecific positive serological findings (anti-hbs n = 2.4%). microbiological testing was sterile every time. as side effects one case of allergic reaction, suspected as serum protein allergy, appeared. clinical outcome can be considered equivalent to ased. subjectively, all patients benefited from the therapy and reported an alleviation of their symptoms. for some indications (highly active gvhd) allo-sed might even be the better option. summary/conclusions: considering our previous experience, allo-sed seem to be a safe and equally effective alternative to auto-sed for patients unable to donate blood. in case of urgent indication, timely supply can sometimes be difficult. to overcome this disadvantage licensing allo-sed as a new blood product with the possibility of production and storage in advance would be a desirable goal. in addition supply would become even safer by preparing allo-sed according to a quarantine principle like ffp. abstract withdrawn. background: vernal keratoconjunctivitis is a chronic, recurrent bilateral inflammation of the outer ocular layer. mostly affected are children and young people and the condition is more common in boys. the disease presents with eye pruritus (itching eye), photophobia (sensitivity to bright light), excessive tearing and foreign eye syndrome. severe cases manifest with diffusion of overgrown papillae usually of the upper eyelid, bursting of the connective tissue barriers and appearance of giant papillae that press on the cornea. corneal ulceration is a severe complication of vernal keratoconjunctivitis that may induce scarring, corneal neovascularization and occasionally perforation. treatment of keratoconjunctivitis mainly relies on steroids, mast cell stabilizers, antihistamines, immunosuppressive drugs (cyclosporine), artificial tears, contact lensdressing, cryotherapy and surgical papillae removal. we present the case of a 8 year-old girl with corneal ulceration who was applied artificial tears after traditional methods of treatment proved unsuccessful. aims: the aim was to share our experience on artificial tears therapy applied in ophthalmic disorders. methods: autologous blood (20 ml) was collected into disposable, sterile transfer bags used for routine blood component preparation (no anticoagulant) and incubated for 1 h at 37°c. the clot was then removed by centrifugation and the serum containing erythrocytes was press extracted. centrifugation was applied again to obtain serum free of cellular components. the serum was then divided into 0.3 ml segments (capsules)and the artificial tears applied to the left eye 8 9 daily. results: ulcer healing was reported after 4 weeks of therapy with artificial tears. the dosage was reduced to 4 9 daily. no recurrence of corneal ulceration was observed after subsequent 8 weeks. summary/conclusions: artificial tears are a safe and effective therapy for ophthalmic disorders in children. background: arv non-disclosure among hiv-positive donors who tested hiv antibody (ab) positive but rna negative (ab+/rna-), so-called false elite controllers, was previously described by our group in south africa, with > 80% of ab+/rnadonations since 2016 testing arv positive. the extent of undisclosed arv use at time of donation represents a significant risk to blood safety in a country with a growing treated hiv population. aims: to establish the prevalence of arv non-disclosure among four subgroups of hiv-positive donors in south africa along with demographic correlates of non-disclosure. methods: south african blood donors are screened by a self-administered questionnaire, which includes questions on current hiv status and arv use, followed by a semi-structured personal interview. specimens for hiv, hepatitis b and c testing are collected at time of donation. based on id-nat (procleix, grifols) and antibody (prism, abbott; western blot) testing, hiv-positive blood donations were classified as acute (ab-/rna+), recent (ab+/rna+, limiting antigen avidity [lag] odn ≤ 1.5), longstanding (ab+/rna+, lag odn > 1.5) and potential elite controller (ab+/rna-) cases. stored plasma from these donations were tested for four arv drugs using qualitative liquid chromatography-tandem mass spectrometry (detection limit 0.02 lg/ml). chi-square tests were used to assess associations of hiv case type, gender, ethnicity, age, donor type, and donor clinic (fixed, mobile) type with arv non-disclosure. results: during 2017, 1671 donors tested hiv-positive of whom 1315 had samples available that were tested for arvs. the overall prevalence of undisclosed arv use was 9.3% (n = 122) with efavirenz most frequently detected (115), followed by lopinavir (5) and nevirapine (2) . potential elite controller cases had the highest proportion of detectable arv (68/80; 85%) (p < 0.0001) followed by longstanding (37/741; 4.9%) and recent (17/366; 4.6%) infections. none of 82 acute hiv cases tested positive for arvs. there were no associations between arv use and gender or ethnicity. however, older (35 to 64 years) hiv-positive donors (49/305; 16.1%) were significantly more likely to test positive for arv than younger (15 to 34 years) donors (73/1010; 7.2%) (p < 0.0001). arv use was more frequent among first time (101/ 716; 14.1%) than in lapsed (13/277; 4.7%) or repeat (8/322; 2.5%) donors (p < 0.0001). donors at mobile clinics had significantly higher arv non-disclosure than donors at fixed sites (10.4% vs 5.0%; p = 0.0051). summary/conclusions: the 9.3% prevalence of undisclosed infection and arv use among hiv-positive south african blood donors is alarming. higher rates of nondisclosure among first-time donors was expected, but non-disclosure among repeat and lapsed donors suggests failure in donor education and assessment. the 4.7% prevalence among concordant ab+/rna+ cases may suggest sub-optimal viral suppression. lack of detection of arvs in acute cases should be qualified because the samples were not tested for tenofovir, the most common drug used in pre-exposure prophylaxis. donor motivation for non-disclosure of known hiv infection and arv use needs further investigation, since early arv initiation or infection while on prep could lead to low ab and rna levels, failure to detect hiv-infected donations and transfusion-transmission of hiv. blood bank, rotary blood bank, new delhi, india background: voluntary blood donation ensures safe blood transfusion. careful blood donor selection is of importance to provide safe blood to patients, although new methodologies have also been adopted by blood centers for blood safety and to minimize risk of transmitting infections through blood transfusion. the quality and the availability of blood components depend on the willingness to donate and reliability of the information given by the donors about their own health, including risk behaviour. blood donor history questionnaire is designed to evaluate donor's history in accordance with the guidelines laid down by the fda. donors, once deferred by the blood bank, will be less motivated to return for donation if he is not counseled effectively. it is important to reduce the number of deferrals by good donor comprehension and the centre should have a mechanism to recall temporarily deferred donors aims: the aim of the study is to analyse donor history and test results of those who donated blood with past history of jaundice. based on their history which suggested the type of viral infection they had, these donors were accepted or deferred. data was collected from voluntary blood donors who were screened for blood donation in the year 2018. methods: in this study, donor history was analysed with reference to history of jaundice. jaundice in donors after the age of 11 yrs, history of surgery, blood transfusion, body tattoos and acupuncture treatment within past one year of donation, history of multiple sex partners and related history and intravenous drug abuse history was taken into consideration. donors who revealed past history of jaundice were asked in detail about their illness and recovery. blood was donated by donors from whom the history of jaundice was elicited and it was understood that the type of virus which caused jaundice was not hepatitis b or c. those who could not give the correct history or were not sure of the cause of hepatitis, those individuals were deferred. aims: to assess the performance of this follow-up program in terms of donor participations, successful confirmed positivity rates, and potential reentry rates. methods: eligible donors were tested for hbsag, hcvab, hivab/ag, and tpab with two eias for each marker. samples reactive with at least one assay were tested further with electro-chemiluminescence assay (eca) and reactive samples were considered repeated reactive (rr). tpab reactive donations were re-tested with particle agglutination assay (tppa). samples eca or tppa non-reactive were considered non-repeatable reactive (nrr background: the blood donation service in suhl processes more than 160.000 samples annually from whole blood and apheresis donations, testing on average around 600 samples per day. for the last 5 years, serology screening was performed on the architect instruments (abbott) (arc), but will be changed to the alinity s system (aly) by middle of 2019. although the design of the aly assays is based on those of the arc assays, we undertook a thorough evaluation of the four mandatory screening assays detecting hbsag, hiv ag/ab, anti-hcv and anti-hbc. aims: to validate the 4 mandatory screening assays on the new aly system in our lab in terms of sensitivity and specificity, also including samples with known falsereactive results. determine the rate of false reactive results for hbsag, anti-hcv and anti-hiv that may lead to deferrals of donations and donors. methods: for sensitivity, we used known positive samples confirmed by immunoblot or nat. known unspecific positive samples for arc not confirmed by immunoblot or nat were testes for aly also. close to 2.000 unselected samples (edta plasma) from routine blood and apheresis donors were tested in parallel on both systems, arc and aly to determine the rate of initial and repeat reactive results. results: all known confirmed positive samples were identical detected by aly. samples with known unspecific reactive results were retested by aly with the following results: 19/33 anti-hcv, 19/21 hiv ag/ab and 04/22 hbsag were found reactive by aly to. one donation from an acute hiv infection in the early seroconversion period was detected by both methods in routine testing. there are no reactive results for aly not already known for arc. the specificity for the screening assays on aly versus arc assays were as follows: 1) hbsag aly 100. 00% (1994 00% ( / 1994 00% ( ) vs arc 99.95% (1993 00% ( /1994 ; 2) hiv aly and arc 99.95% (1992/1993); 3) anti-hcv aly 99. 90% (1994 90% ( /1996 90% ( ) vs arc 99.75% (1991 90% ( /1996 . the number of anti-hbc reactive samples did not differ between aly and arc. summary/conclusions: while the switch to the new system is mainly driven by operational efficiency, obviously, the high specificity of the alinity s assay will reduce unnecessary deferrals of donations and donors. abstract withdrawn. background: blood donor selection is the cornerstone for blood transfusion safety, designed to safeguard the health of both donors and recipients. donor safety is targeted by reducing the risk of complications associated with blood donation and transfusion safety by reducing the risk of transfusion-transmitted infections (tti) and other preventable transfusion reactions. there is always a compromise on blood donor safety as well as blood safety during outdoor mega blood donation drives due to various reasons, mainly due to more number of donations within a stipulated time. aims: to compare the blood donor selection patterns between in house blood donations and donations at mega blood donation drives and its influence on donor safety and blood safety in a tertiary care hospital in india. methods: a retro prospective study was done to audit and compare blood donor safety and blood safety over a period of 2 years from january 2016 to december 2017. blood donor safety was analyzed by two indicators: donor health questionnaire (dhq) monitoring and blood donor reaction rates and blood safety through tti positivity rates. (78) during mega blood donation drive. summary/conclusions: a good donor selection is a lengthy process which involves pre-donation information and advice: this is usually provided in a leaflet, especially about transfusion-transmitted infections (and the associated risk factors) and the potential risks of donation, filling of dhqs by the donor himself, donor interview: conducted by a qualified medical specialist trained in donor selection process and health assessment at the end of the interview to declare if the donor is eligible to give blood or deferred temporarily or permanently. it was observed that seroprevalence rates, number of donor reactions and incompletely filled dhqs were more among blood donations at mega blood donation drives when compared to blood donations during in house collections. this is mainly due huge number of blood donations with in a stipulated time where there is limited time spent on proper donor selection. stringent implementation of who strategy: "safe donor safe blood" is the only way for blood donor and transfusion safety. background: safety of blood transfusion is a great concern especially in crisis countries and during humanitarian emergencies. transfusion transmitted infections (ttis) are one of the major health problem in yemen that are associated with blood transfusion complications. aims: the aim of this study is to determine the prevalence of ttis among blood donors at national blood transfusion and researcher center (nbtrc this contributed to an additional reactivity of 0.09%, thereby total reactivity being 2.39%. 55% (22/40) of these were hcv reactive & 45% (18/40) for hbv. the nat yield was 1 in 1086 and the viral loads of nat reactives ranged from 1-9 x10 4 iu/ml for hcv & all the hbv yields had an extremely viral load of < 06 iu/ml. 12/40 nat reactive showed sero-conversion after 4-8 months with follow-up eclia screening, and 7 of these were hcv reactive and 4 hbv reactives. summary/conclusions: incidence rate indicate that the current risk of transfusion transmitted viral infections attributable to blood donation is relatively high in our country. parallel use of both serology and nat screening of donated blood in countries that have high seroprevalence can improve the blood safety. at our centre, by using best in class serology and nat technologies, we were would add an extra layer of safety to blood supply by interdicting samples from donor with recent infections. abstract withdrawn. abstract withdrawn. (1/190,298) . the both hiv-rna and hcv-rna detected donors by nat were identified in the window period. summary/conclusions: in this study, we found that nat could detect 283 infected cases with hbv-dna, hiv-rna and hcv-rna which were forgotten by serological methods therefore, nat is a sensitive screening method to detect low viral load and shorten the window period of the virus infection to ensure the safety of blood transfusions. service du sang, croix rouge de belgique, namur, belgium background: due to enhancement of kits specificity and machines throughput, roche elecsys â technology is a potential partner for blood donations screening laboratories. aims: the aim of the study was to assess the performance of the elecsys serology assays on a cobas e801 equipment for clinical specificity, analytical sensitivity and reproducibility. background: deceased donors are the primary source of organs and tissues for transplantation but the risk of infectious complications in the recipient is high and is the main cause of morbidity and mortality after transplantation. to minimize the risk of infections by organ or tissue transplantation, donors should be tested for anti-hiv-1/2, hbsag, anti-hbc, anti-hcv, and syphilis. further laboratory tests may be required depending on the history of the donor and on the tissue properties. certain grafts can be donated after circulatory death of the donor; however, the absence of the heartbeat may change dramatically the blood composition by e.g., haemolysis and proteolysis. this may have an impact on test performance and lead to false results. therefore, an assay validation is needed for testing of cadaveric samples. aims: a validation study was performed to demonstrate the suitability of elecsys hbsag ii, anti-hbc ii, anti-hcv ii, hiv combi pt, hiv duo, syphilis, htlv-i/ii, and chagas for the use in cadaveric samples from non-heart beating donors. methods: as the basis for validation, we followed the recommendations of the paul-ehrlich-institut (pei) "proposal for the validation of anti-hiv-1/2 or hiv ag/ ab combination assays, anti-hcv assays, hbsag and anti-hbc assays for use with cadaveric samples". comparison of spiked samples from living donors and cadaveric donors was used to demonstrate accuracy. to determine precision, two cadaveric specimens were tested in several replicates. acceptance criteria were implemented according to the pei recommendations. results: results were found to be within specifications requested by the pei recommendations for all tested assays summary/conclusions: the evaluated results support the extension of the use of these assays with cadaveric specimens. background: in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. aims: in this context, metagenomic analyses of viral content in blood donations collected in geographical zones recognized as "hotspot" for viral emergence represents a suitable approach without any a priori for the identification of a potential emerging viral risk that may compromise blood safety. methods: in the framework of a viral discovery program founded by the french national agency for medicine security (ansm in french), more than 900 plasma samples collected in 7 sub-saharan africa countries (2011) (2012) and the amazon region of brazil (2016) have already been analysed by metagenomics. results: although no viral sequence could be described as novel (i.e. new species or even a new genus), we unexpectedly identified a feline bocavirus in two donors from mauritania. a large diversity of known viruses that are not part of the regularly monitored agents were also observed, among which anelloviruses, hpgv-1 (formerly known as gbv-c), papillomaviruses, herpes viruses, parvovirus b19, chikungunya virus, enterovirus, and various small circular viruses (circo-, cycloand gemycircularviruses). while no significative differences was observed in the higher classification of detected virus (above families/genera) between africa and brazil, we observed variations at the sequence level allowing better resolution of the genetic diversity for several viruses (for example characterization of hpgv-1 genotypes). summary/conclusions: overall, the absence of novel viruses in blood samples collected across countries of two distant continents is reassuring regarding threats emergence. however, continuous monitoring of prospective blood banks should be continued. summary/conclusions: after the high peak observed in 2014 during the first period, this study shows that the decrease in the seroprevalence of viral markers is continuous over the next five years. the second period is marked by an irregular evolution of seroprevalence but with lower levels than the first period. the recruitment of new donors allows a quantitative increase in donations. however, improving the quality of blood products essential condition of transfusion safety is achieved through retention of recruited blood donors. background: in blood screening laboratories, samples may be transferred between automated serological and molecular instruments, and the potential for sample contamination is a serious risk to the integrity of nucleic acid testing (nat) results. the sensitive limit of detection (lod) for hiv and hcv nat assays combined with the high viral titers encountered in specimens from patients with acute infections presents a challenge for maintaining the sample integrity of negative specimens. at additional cost per test, this risk can be reduced with single-use filter pipette tips. aims: we evaluate the efficacy of applying induction heated washes to a non-disposable pipettor on serology instruments-alinity s, alinity i, and architect i2000sr (abbott diagnostics)-to preserve the integrity of samples transferred to a downstream molecular instrument, the m2000 realtime (abbott molecular diagnostics), which amplifies viral nucleic acid targets exponentially. methods: in this application of induction heating, the metallic pipettor warms under its own resistance to coil-induced electrical currents. by sweeping the pipettor through an induction coil, temperatures on the pipettor are elevated throughout its length. single donor high viral titer hiv genotypes a (5.78 log iu/ml), b (5.90 log iu/ml), c (5.62 log iu/ml), crf01 (5.61 log iu/ml), crf02 (6.19 log iu/ml), and urf (6.33 log iu/ml), as well as single donor high viral titer hcv genotypes 1a (6.84 log iu/ml), 1b (6.73 log iu/ml), 3a (6.64 log iu/ml), 2 (6.10 log iu/ml), 2q (6.65 log iu/ ml), and 4t (6.07 log iu/ml) were used as potential sources of contamination; these genotypes account for the majority of hiv and hcv infections worldwide. on serology instruments, one high viral titer hiv or hcv specimen and three consecutive susceptible negative samples (hiv/hcv rna negative human plasma, abbott molecular diagnostics) were tested on an hiv ag/ab combo or anti-hcv immunoassay (abbott diagnostics), and this schema was repeated four times per positive specimen. induction heated washes occurred between all samples processed on the serology instruments. the first susceptible negative from each testing block, with approximately 1 ml of residual sample volume, was then tested using the 0.6 ml abbott realtime hiv assay (lod 40 copies/ml) or 0.5 ml abbott realtime hcv assay (lod 12 iu/ml) and an hcv ag immunoassay (lod 1.24 fmol/l; abbott diagnostics). study acceptance criteria required that any susceptible negative sample had no detectable level of hiv or hcv rna. results: all first susceptible negative samples (n = 24 per platform per virus schema) run on alinity s, alinity i, and architect i2000sr using induction heated washes after a high viral titer hiv specimen or hcv specimen were hiv ag/ab combo nonreactive (< 0.20 s/co) and reported no detectable level of the hiv rna target, or were anti-hcv nonreactive (< 0.20 s/co) and reported no detectable level of the hcv rna or core antigen targets. summary/conclusions: while precautions should continue to be taken for samples run on molecular instruments, the integrity of samples originally tested on the alinity s, alinity i, and architect i2000sr was preserved for downstream molecular testing through the use of induction heated washes. aims: increasing the safety of blood and blood products -motivating the blood donors to be regular donors methods: national reporting system showed the high prevalence of ttis among first blood donors in compares with the regular donors. in 2015 per 2.083.914 donations, 54% % of confirmed positive hiv, 87% of hcv, and 93% of hbv cases has been reported among first blood donors. in the end of 2015 a national program named "pre-donation screening tests "has been developed and has been implemented in high prevalence provinces in whole country. based on this program, all first blood donors who accept in donation sites, if after donor selection process are eligible to donate blood, they refer to give just a blood sample for screening ttis tests. after 3 months, the invitation letters and smss send to the donors who have negative results for all screening ttis tests, and they can be eligible to donate blood after another donor selection process. in 2015, about 0.156% of all donations have been rejected because of at least one of hiv, hcv, or hbv confirmed positive results, while this reject rate in 2017 was 0.082%, which shows a significant decreasing the ttis prevalence among blood donation from 2015 to 2017. the prevalence of hiv, hcv, and hbv among donations has been decreased significantly in 2017 compared with the 2015. prevalence of hiv among donations reduce from 0.0032% in 2015 to 0.0024% in 2017, for hcv and hbv the same results have been experienced, respectively from 0.040% and 0.113% in 2015 reduce to 0.026% and 0.053% in 2017. it seems this applied study could effectively scale up the safety of national blood supplies. in addition this intervention could support iranian blood transfusion service to increase the proportion of regular blood donors from 80.19% in 2015 to 86.97% in 2017. it means that with increasing the regular blood donor population sizes, the safety of iranian blood and blood products will be more and more scaled up. summary/conclusions: evidence based reports show there is a high rate of prevalence of transfusion transmitted infections (ttis) among first blood donors. so an effective intervention which can reduce the risk of unsafe first blood donation can effectively increase the safety of blood and blood products. pre donation screening tests program in iran can support the national program to decrease the rate of ttis among blood donations from 0.0156% in 2015 to .082% in 20117. abstract withdrawn. abstract withdrawn. background: despite the universal application of viral inactivation and elimination technologies during the preparation of plasma-derived products, the exclusion of infectious donations before any other procedure remains the first essential step as well as the major determinant for the safety of untreated labile blood products. current selection and screening techniques have reduced the risk of viral transmission to very low levels, but there is still a very low but quantifiable risk of transmission through donations beyond routine detection, particularly during the " seroconversion window". "of an infection in a blood donor that is to say during the period when the recently infected donor has not yet developed a serological response. the level of residual risk, which must be as low as possible, is mainly conditioned by the rates of the infections concerned (hiv and hepatitis b virus (hbv) and c (hcv)) to blood donors. summary/conclusions: the evolution of serologic markers is generally satisfactory with continued regression, which has improved particularly for hiv. on the other hand, hepatitis b is still a concern because of its still high rate among new donors. it is desirable to initiate a regular donor vaccination program to protect against hepatitis b. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hcvab, havab igm and havab igg essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 61 samples were tested (19 for hcvab, 24 for havab igm and 18 for havab igg) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hcvab ranged from 0 to 4.474%. 5 samples were tested for havab igm in a total of 55 essays and the %cv ranged from 0 to 11.475%. havab igg was tested in 3 samples during 33 essays and the %cv ranged from 0 to 4.861%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hcvab, havab igm and havab igg. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hbsag, hbsab, hbcab, hbeag and hbeab essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 85 samples were tested (10 for hbsag, 20 for hbsab, 18 for hbcab, 14 for hbeag and 23 for hbeab) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 30 essays we found the %cv hbsag ranged from 0-4.375%. 3 samples were tested for hbsab in a total of 33 essays and the % cv ranged from 0-5.196%. hbcab was tested in 3 samples during 32 essays and the %cv ranged from 0-2.811%. using 3 samples and a total of 28 essays we found the %cv hbeag ranged from 4.176-5.147%. 4 samples were tested for hbeab in a total of 29 essays and the %cv ranged from 0-4.444%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hbsag, hbsab, hbcab, hbeag and hbeab. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hivag/ab, syphilis and htlv i/ii essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 38 samples were tested (12 for hivag/ab, 14 for syphilis and 12 for htlv i/ii) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hivag/ab ranged from 0 to 3.05%. 2 samples were tested for syphilis in a total of 22 essays and the %cv ranged from 0 to 1.481%. htlv i/ii was tested in 3 samples during 28 essays and the %cv ranged from 3.342 to 7.5%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hivag/ab, syphilis and htlv i/ii. , human immunodeficiency (hiv) and hepatitis c (hcv) viruses' infection in blood donors were 1.27%, 0.20% and 0.50% respectively. consecutive positive results for hbv were 9.20% (63/685), for hcv were 6.0% (16/266) and nil for hiv. there was no sample carry over in this. out of 79 consecutive reactive donors 69 were donated for same patients and 32 were related with infected patient which were statistically significant (p < 0.0001). summary/conclusions: among all tti reactive donors 7.4% (79/1061) were consecutive reactive. the reason for the same may be process related like sample carry over or reagent carry over or donor related. donor related reasons may be, one of the close relative is reactive for virus and that is transmitted to other family members. in our study 32 reactive donors either had close contacts with persons with history of infective disease or were their first degree family relatives. these findings were found statistically significant (p < 0.0001). this study recommends that in analysis of consecutive positive results in elisa along with looking for procedure/sample error, there is also a need to take retrospective history of donors for close contact with infected patient. background: screening for transfusion-transmitted infections (ttis) is critical in ensuring safety of blood products. transmission of infections through transfusion remains a major source of viral hepatitis especially hbv and hcv. the effectiveness of rapid immunochromatographic test (ict) devices for screening of blood is a concern and needs validation through advanced methods like chemiluminescence immunoassay (clia) and polymerase chain reaction (pcr). aims: the current study was conducted to evaluate the performance and screening effectiveness of commercially available rapid screening kits in comparison with clia and pcr. methods: this single centre, cross sectional study was conducted at the department of blood transfusion services, shaheed zulfiqar ali bhutto medical university, islamabad, from january -june 2018. a total of ten commercially available ict devices and one clia kit (liaisonr xl) were tested for their sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy using 100 positive and 100 negative samples each for hbv and hcv respectively, in comparison with the values determined by pcr. the ict kits included hightop, rightsign, wondfo, accu-chek, fastep, abon, immumed, insta-answer, biocheck and ctk. results: the sensitivities and specificities of ict kits for hbsag were 65% and 70% (hightop), 67% and 85% (rightsign), 62% and 73% (wondfo), 70% and 80% (accu-chek), 68% and 77% (fastep), 73% and 85% (abon), 77% and 83% (immumed), 80% and 90% (insta-answer), 67% and 81% (biocheck) and 72% and 83% (ctk) respectively. similarly the sensitivities and specificities of different ict kits for hcv were 69% and 80% (hightop), 76% and 83% (rightsign), 69% and 81% (wondfo), 78% and 79% (accu check), 68% and 68% (fastep), 63% and 73% (abon), 71% and 70% (immu-med), 79% and 68% (insta answer), 62% and 66% (biochek) and 69% and 78% (ctk) respectively. the sensitivity and specificity of diasorin liaison murex assay for both hbv and hcv were found to be 100%, when compared with pcr. the ppv, npv and accuracy were determined accordingly. summary/conclusions: rapid testing ict devices for both hbv and hcv available in pakistan were found to have a variable degree of sensitivity and specificity, when compared with pcr. comparatively expensive but quality methods are more reliable as compared to rapid devices. the data generated will help policy makers to prepare future plan of action and introduce the concept of quality control in blood centres. the analysis has shown that the population of blood donors also included people infected with syphilis. in reference to the number of the tested samples this number is quite significant. the analysis proved the increase in the number of syphilis infections among the blood donors which is consistent with the general trend in the population. summary/conclusions: we have proved that testing blood donors for the treponema pallidum infection increases the safety of recipients of blood and its components and that obligatory testing of donors is fully justified. , 1940 and 1945 , the use of third or fourth generation serological assays is mandatory for screening of blood donor units for hbv and hcv infection before transfusion. routinely, blood banks in india screen the units by the elisa testing. nat is not very common due to cost constraints. aims: the aim of this study is to determine the frequency and load of hbv dna and hcv rna in hbs and hcv reactive blood donors respectively, and hence it was intended to contribute to determining whether routine hbs and hcv screening of blood donors, using elisa method alone, provides any concrete benefits with regard to hbv and hcv risk reduction or whether the implementation of nat will be of great benefit to low-resource countries like india, which has high prevalence of hbv and hcv. abstract withdrawn. ,906 donors were routinely tested for hbv dna by using cobastaqscreen mpx-1 and mpx-2 (roche) or procleix ultrio and ultrio plus id (grifols) assays. obi was confirmed by repeat dna testing and by performing additional serological and molecular investigations on index and follow-up samples. anti-hbs concentrations were determined and anti-hbc antibodies were tested with three distinct commercial clia assays (anti-hbc elecsys roche, architect anti-hbc ii abbott, and hiscl anti-hbc assay sysmex). hbv pre-s/s, precore/core and bcp regions were pcramplified after viral particle concentration and viral amplicons were sequenced. results: 348 hbsag-/dna+ donors (1:1,462) including 294 confirmed obi were identified (1:1,731 prevalence). among obi donors, 160 (54.5%) tested anti-hbc+/anti-hbs-, 108 (36.7%) were anti-hbc+/anti-hbs+, 25 (8.5%) were anti-hbc-/anti-hbs+, and 1 anti-hbc-/anti-hbs-primary obi (0.3%). anti-hbc-/anti-hbs+ obi donors were significantly younger (mean: 21 years [range: 18-57 years]) than those with anti-hbc+/anti-hbs+ (mean: 44 years [range: 19-60 years]) and anti-hbc+/anti-hbs-(median: 45 years [range: 21-55 years]) profiles (p < 0.0001). hbv vaccination was documented for 18 (72%) of these donors and was reported in one donor but without definitive evidence. extremely low hbv dna loads (range: <10-155 iu/ml) were transiently detected in seven donors during follow up. genotypes identified were genotype b (n = 1/20) and genotype c (n = 19/20). preliminary analysis of core protein (n = 16) and bcp (n = 10) sequences showed no particular genetic feature that could be associated with altered antigenicity or core gene expression. follow-up was available for 15/ 25 anti-hbc-/anti-hbs+ donors (2-6 samples/donor; range: 2-56 months). anti-hbc remained undetectable with all clia assays in these donors except one. low transiently detectable levels of hbv dna were observed overtime with anti-hbs levels fluctuating between 12 and 1,000 iu/l. no significant difference in hla-a, -b (except hla-b*46 more frequently detected in anti-hbc negative obi), and -drb1*. summary/conclusions: overall, the 8.5% prevalence of anti-hbs-only in hbv dna positive obi carriers (1:20,355 of total donors) in dalian blood donors confirmed previous reports from south east asia. this phenomenon was not related to core antigenic variations but was significantly associated with younger age of carriers. a particular route and natural history of the infection may be considered. the hypothesis of acute-phase vaccine breakthrough was ruled out in 15/25 donors by the over 50 months stability of this serological profile. breakthrough in immunized donors may still be suspected. further studies are needed to evaluate the potential infectivity of anti-hbs-only/hbv dna+ obi carriers, and to characterize the potential viral and immunological mechanisms responsible for this unusual hbv infection profile. confirmatory laboratory, hungarian national blood transfusion service (hnbts), budapest, hungary background: vaccination against hepatitis b virus (hbv) is an effective tool to avoid the infection. in hungary, population born after 1986 is considered to be immunized, because inoculation has been mandatory for children as campaign vaccination since 2000. hbv vaccine is strongly recommended for healthcare workers, moreover trips to endemic countries and awareness of individuals could also be reasons of vaccination in immunologically na€ ıve age-groups. since the hbv vaccine contains surface antigen, a recent inoculation can cause reactivity of hbsag screening assays and positivity of confirmatory tests for several days resulting in deferral of donors from blood donation. the former regulation of hnbts, which was valid until december 31, 2018, allowed the re-entry of donors whose immunization records and negative hbv confirmation of the second blood samples proved that the previous vaccination had resulted in the hbsag confirmed positivity. aims: the aim of this study was to strengthen that vaccination against hbv before blood donation had resulted in the reactivity of hbsag screening and confirmatory assays between 2012 and 2018. background: in brazil, the introduction of nucleic acid tests (nat) for hbv-dna detection in the routine screening at public blood banks is relatively recent. at fundac ßão pr o-sangue/hemocentro de são paulo (fps-sp), about 130,000 blood donors are submitted to serological screening tests (hbv, hcv, hiv-1/2, chagas disease, syphilis and htlv-1/2) and nat for hiv, hcv and hbv per year. approximately 2% of the blood donations are discarded due to some reactive result; of these, the hbv discard rate was 0.65% in 2016. aims: our study aims to determine the potential infectious cases among samples that had one or more hbv-reactive screening results (anti-hbc, hbsag and mp-nat-hbv) and verify the different categories of hbv infection (acute, chronic, occult hepatitis b infection (obi) and immunological window). furthermore, to characterize the distribution of hbv genotypes, drug resistance and escape mutations and analyze the risk factors. methods: we carried out a cross-sectional study of roughly 74,000 donations from may 2016 to december 2016. hbv antibodies and antigen screening were performed using cmia kits architect â -abbott/hbsag and architect â -abbott/anti-hbc. nat screening was performed in minipools (mp) of six samples using kit nat hiv/hcv/ hbv -bio-manguinhos (sensitivity 95% lod 300 iu/ml for hbv). reagent samples (n = 407) that presented one or more hbv-reactive screening results (anti-hbc, hbsag and/or mp-nat-hbv), were submitted to individual nucleic acid extraction and "in house" quantitative real-time pcr-hbv (id-pcr-hbv) targeting the hbsag region (sensitivity 10-20 ui/ml). the hbv genotypes and mutation analyses were determined by direct sequencing of the hbv pol-gene/surface-gene and use of the online analysis tool geno2pheno [hbv] 2.0. socio-demographic and epidemiological data were also analyzed. financial support: fapesp 2017/16658-6. results: among the 407 hbv reactive samples, 377 were reactive for anti-hbc only (92.6%), 10 for hbsag only (2.5%) and 20 were reactive for both markers and hbv dna (4.9%). routine testing and id-pcr-hbv identified 20 (0.027%) samples of active infections that had all hbv reactive/positive tests results. no hbv dnayield samples or hbsagyield or obi were observed. viral loads for active infections samples ranged from 1.08e+01 to 2.45e+09 cop/ml (median, 2.12e+08cop/ml). hbv sub-genotypes a1, a2, c1, d3 and f1 were found in 70%, 5%, 5%, 15%, and 5% of the donors, respectively. no reverse transcriptase inhibitor-resistant variants were detected. escape mutations in small hbsag protein shb region were detected in 30% (6/20), with the following main substitutions 100c (3x), 120r, 123n and 144g. the mean age of donors with active hbv infection was 40 years, mostly donors were males (80%), mixed (40%) or white (40%) and had concluded high school (45%). summary/conclusions: discard rate due to isolated anti-hbc is high but no obi was found in the blood donor population studied. in addition, no case of immunological window for hbv or hbsagyield was detected. there was a predominance of subgenotype a1 and mutations associated with escape were found in 30% of hbv-dnapositive samples. continuous research and surveillance about hbv prevalence among blood donors are needed to maintain and evenly increase blood safety in brazil. background: screening for anti-hbcore antibodies in blood donors is considered to contribute significantly to blood safety, since it reveals donors with occult or probable occult hepatitis b, with variable results in molecular screening, due to very low viral load. however, universal anti-hbcore testing in blood donors, might exclude a considerable number of blood donors in countries with high hbv prevalence and even in countries with low to moderate prevalence, like greece. aims: the aim was to investigate the percentage of blood donors with natural hbv infection (confirmed positive anti-hbcore) or hbv immunization due to vaccination (anti-hbs+ only, due to vaccination) and predict the impact of generalized anti-hbcore testing. methods: during the period 15-30 november 2018, all blood donors were asked to give their consensus for additional screening for hepatitis b anti-hbcore and anti-hbs antibodies, besides the obligatory serological and molecular screening, the samples of few donors who disagreed, were not examined. all samples with repeated positive anti-hbcore results, were further examined for anti-hbcore igm and anti-hbe antibodies. furthermore, a new donor sample was requested, to confirm reactivity. the serology results were recorded in an excel spreadsheet. additional data, including age, sex, nationality, number of previous blood donations, abo blood group, family history of hbv infection, hbv vaccination, were also recorded and statistically evaluated. donors were informed of the positive results. results: a total of 620 edta samples were tested using the architect anti-hbcore, anti-hbs, nti-hbcore-m and anti-hbe assays (chemiluminescent microparticle immunoassay (cmia). repeated anti-hbcore(+) occurred in 24 (3.87%) samples, among which 7 (19%) were also anti-hbe(+), while anti-hbs was found > 200 m iu/ml in 15 (62.5%), between 100 and 200 miu/ml in 3 (12.5%), and < 100 miu/ml in 6 (25%). among anti-hbcore positive donors, 4/24 were foreigners (16.6%) and 20 were greeks, while foreigners consisted 6,29% (39/620) of donors examined. so, anti-hbcore was found positive in 10,25% of foreigners (4/39, all from countries with high prevalence for hepatitis b infection) and in 3,44% of greeks (20/581). in total, 285 (45.96%) samples had anti-hbs > 10 miu/ml (considered seroprotective for the donor). summary/conclusions: almost half of our blood donors (45.96%) were immunized, 261 by vaccination and 24 (3.87%) by natural infection. the incidence of natural infection was significantly higher in foreigners (10.25% versus 3,44%). if not all anti-hbcore+ donors, 25% with anti-hbs < 100 iu/ml, might be potentially infectious, especially for immunocompromised patients. if we choose to screen all blood donors for anti-hbcore and reject those with positive results, regardless of the anti-hbs levels, we would probably lose a significant number of donors and jeopardize blood sufficiency. alternatively, we could reject only those with anti-hbs < 100 or < 200, or choose to selectively screen pre-donation blood donors from countries with high prevalence of hbv infection. following this pilot study, the prevalence of immunization against hbv in large numbers of blood donors from various parts of greece, must be investigated, in order to decide whether to introduce such screening. aims: the aim of this study was to perform phylogenetic analysis of the donor samples with hcv found in the neighbouring villages to determine the nature of transmission. methods: altogether, approximately 2 million blood donor samples were screened with anti-hcv immunoassay (architect anti-hcv, abbott gmbh, wiesbaden, germany) and reactive results were confirmed with anti-hcv line-immunoassays (inno-lia hcv score, fujirebio europe, gent, belgium). based on lia positivity, in 20 samples an association of hcv infection was supposed, because the residence places of donors were in three neighbouring villages situated less than 20 km to each other. pcr was positive in 15 samples. from these samples, hcv sequencing and phylogenetic analysis were performed. fourteen hcv infected samples of general population and 11 of ivd users were also included into the study. results: phylogenetic analysis detected genetic relationship among the hcv virus sequences in donor samples. the most abundant was the 1a subtype, and it formed two different groups on the phylogenetic tree. according to their genetic distance, a more distant mutual ancestor could be supposed. two samples with 1b subtype originated from the same village, and their difference was only 2 nucleotides. three hcv from the ivd user control group showed close genetic relationship with the viruses detected in the donor samples. summary/conclusions: based on our phylogenetic analysis, hcv transmission in blood donors could be the consequence of the ivd use and the origin might be related to 2 or 3 primary human sources. during 2011 and 2014, a significant increase in the hcv seroprevalence among the ivd users was observed, which was approximately threefold in the rural areas of hungary. our recent findings highlight the importance of the proper donor selection, which can identify the typical signs of the ivd use. moreover, enhancing awareness of blood donors with education is a further significant issue in order to reduce the risk of transfusion transmitted infections. abstract withdrawn. background: in china, the residual risk of transfusion-transmitted hcv has been declining since screening of blood donors for anti-hcv and/or hcv nat from 1993. however, many high sensitivity reagent, using to test blood donors' samples, lead to false-positive results and donors loss. aims: this study intended to establish a donor reentry procedure for hcv screening reactive donors in china. methods: from march 2014 to december 2017, there were 2083 blood donor samples which were screened reactive or belonged to "grey zone" by elisa and/or reactive by nucleic acid test(nat) at the local blood centers were collected from 15 chinese blood centers. all these samples were sent to institute of blood transfusion (ibt) national reference laboratory where anti-hcv and hcv individual nucleic acid test (id-nat) were conducted. if the results were reactive for anti-hcv, then the samples were tested with a recombinant immunoblot assay (riba). results: based on this study, 1178 of 2083 donors in the study who could be classified into two categories for hcv status: 201(17.1%) true positive and 974 (82.9%) false positive. a total of 905 of 2083 donors lost to follow-up, their hcv status cannot be determined with certainty. based on these data, a reentry procedure for hcv screening reactive donors was proposed. summary/conclusions: based on our proposed donor reentry procedure for hcv screening reactive donors, a majority of screening false-positive donors (82.9%) can re-entry safely. abstract withdrawn. background: providing safe blood for transfusion in sub-saharan africa (ssa) is a particular challenge due to a combination of factors; limited resources and infrastructure, suboptimal diagnostics and a high prevalence of the major transfusion-transmissible infections (ttis). average seroprevalence data estimates from the ugandan and kenyan blood transfusion services (bts) for hepatitis c (hcv) currently stand at 6.6% and 0.9% respectively. between january and december 2016, in mbale (eastern uganda) the hcv prevalence amongst blood donors was an alarming 8.1%. with no provision or funds for confirmatory testing, the bts are unable to confirm or refute a diagnosis of active hcv. this results in large quantities of blood wastage, unnecessary anxiety in potential donors and high donor deferral rates limiting the donor pool. aims: we aim to determine the true prevalence of active hcv infection amongst seropositive donors in bts in uganda and kenya. in addition, we aim to compare the performance of locally used serodiagnostics and best available alternative tests and to examine the feasibility of cost-effective additional or alternative tests to help provide accurate results on the infectious status of blood. methods: 235 hcv seropositive blood samples from 3 bts study sites (kampala, mbale, mombasa) will be re-tested using the local serology screening test (abbott architect anti-hcv), an alternative who pre-qualified rapid antibody test (sd bioline) and a confirmatory test (hcv core antigen test). where there is discrepancy in the results or need for clarification, samples will be tested on the cepheid xpert platform by reverse-transcriptase pcr to obtain a quantitative rna result. s/co (specimen to cut-off) values for false positive samples (by screening serology) will be analysed and presented. pre-analytical factors (centrifugation speed, haemolysis check, time delay between collection and testing) will be controlled for and documented. results: pilot data from re-testing quarantined hcv seropositive donor blood (mbale bts) in uganda demonstrated that 45/50 seropositive blood (90%) was rna pcr negative. in december 2018, 156/249 (63%) of seropositive samples (by screening anti-hcv serology) in kampala bts had s/co values between 1.00-1.99 (1.00 is the cut-off indicating a positive sample). data from the re-testing of 235 seropositive samples as true representation of active hcv will be demonstrated and s/co values for the study period concomitantly with a retrospective analysis of january to december 2018. preanalytical factors, cost analysis comparisons of the diagnostic platforms coupled with costs of the donor deferral process in false positive cases will be presented. summary/conclusions: for the bts in ssa there are significant resource and financial implications, as repeat testing and donor deferral counselling is required. evaluating and introducing new and appropriate diagnostics and algorithms in the screening of hcv is crucial in improving the supply of safe blood transfusion services in east africa. background: in november 2017, the blood services of england, scotland and wales reduced donor deferral to three-months for commercial sex workers and individuals with higher risk sexual partners, including sex between men. the change was recommended after a detailed review by an external expert committee (sabto) which recommended that a shortened deferral of 3 months would allow detection of recently acquired infection and maintain residual risk (rr) at a tolerable level. recommendations were accepted by government but with a government commitment to explore a more individualised approach. aims: to assess the impact of a 3-month deferral on blood safety in terms of epidemiology of infections in blood donors and compliance with donor selection criteria, and to explore evidence required to develop a more individualised approach to donor selection policy methods: routine uk blood donation surveillance data for 2010-2018 (2018: preliminary) were reviewed. annual prevalence and incidence of hbv and hiv infection were estimated, with a poisson regression models to test for trends. incidence was calculated from donors seroconverting within 12-months, and/or microbiological and clinical evidence of recent infection. for donors positive in 2018, compliance with the 3-month deferral was determined. uk hemovigilance data were scrutinised for evidence of transfusion transmitted infections (tti) associated with newly eligible donors. results: from 2010 to 2018 among new donors, annual hiv prevalence decreased significantly by an average of 11.3% each year (p = 0.02) to 1.49/100,000 donations in 2018; no significant trend was observed for hbv. annual hiv incidence among repeat donors also decreased significantly by an average of 17.3% each year (p = 0.03) to 0.23/100,000-person years (pyrs) in 2018 (based on 2 seroconverters). there was no significant trend in hbv incidence over the study period, however between 2017 and 2018 incidence increased from 0.35/100,000 pyrs to 0.84/ 100,000/pyrs (based on 3 and 7 seroconverters respectively). with the information available to date, none of the 2018 seroconverting donors were non-compliant, and there was no reported confirmed ttis associated with the policy change. summary/conclusions: hiv prevalence and incidence has continued to decline. hbv incidence in repeat donors increased in 2018 although initial analysis suggests this is not associated with the policy change. monitoring continues, and residual risks will be re-estimated as data post-change accumulate. these data are reassuring, and therefore it is appropriate to scope the evidence for, and feasibility of, a more individualised approach to selection policy. a multidisciplinary steering group has been convened including representation from patient and stakeholder groups. gaps in knowledge are being defined, and a package of work is in development under the project of fair (for the assessment of individualised risk), using the abo rdf for guidance. background: permanent deferral of men who have sex with men (msm), established in the 1980s, primarily to minimise the risk of hiv transfusion-transmitted infections is increasingly challenged. accordingly, blood services in many countries have changed to time-based deferral. in canada, a 5-year deferral was implemented in 2013, reduced to 12-months in 2016; a 3-month deferral is now being considered. aims: to estimate the risk of undetected hiv among screened blood donations under a 3-month deferral since last sex between men. methods: the applied model combines features of previously published english and canadian models to estimate hiv risk under a 12-month deferral. three scenarios varying hiv incidence, prevalence and non-compliance under a 3-month deferral were modelled. assumed constants were the hiv nucleic acid window period, testing procedure error rate and assay sensitivity. model inputs were incidence under the current 12-month deferral, calculated as hiv positive donors with a previous negative within 12 months divided by number of person years, numbers of hiv positive donations, hiv positive msm, hiv msm incident cases and newly eligible msm donors (from donor surveillance and compliance surveys). the risk with a 3-month deferral was estimated for three scenarios, one determined "most likely", where msm donor non-compliance, hiv incidence and hiv positive donations do not change and msm newly eligible to donate are estimated from compliance surveys. this scenario is based on data from two sequential policy changes in canada. an "optimistic" scenario where non-compliance halves and a "pessimistic" scenario where msm hiv incidence, hiv positive donations, non-compliance and new msm donors double were also used. the median hiv residual risk was used as the final estimate. the uncertainty in this estimate was assessed with the 2.5th and 97.5th percentiles over the simulation (95% ci). results: incidence, per 100,000 donations, was estimated to be 0.15, 0.13 and 0.27 for the "most likely" "optimistic" and "pessimistic" scenarios, respectively. for the 3month deferral "most likely" scenario, hiv residual risk was predicted to be 1 in 32.6 million donations (95% ci: 1 in 217,692 million to 1 in 8.3 million). for the "optimistic" scenario, hiv residual risk was estimated to be 1 in 34.3 million donations (95% ci: 1 in 257,692 million to 1 in 9.1 million). finally, for the "pessimistic" scenario, hiv residual risk was estimated to be 1 in 16.0 million donations (95% ci: 1 in 34,091 million to 1 in 5.7 million). with these residual risk estimates, based on the number of donations in canada, one hiv infectious donation would be in inventory every 30 years for the "most likely" scenario, every 32 years for the "optimistic" scenario and every 15 years for the "pessimistic" scenario. summary/conclusions: the risks of hiv entering the blood supply in canada for a 3-month msm deferral are predicted to be very low for all modelled scenarios, including a "pessimistic" doubling of hiv incidence post change. background: safety of blood and blood products is a major concern in pakistan. the prevalence of transfusion transmitted infections among multi-transfused thalassaemia patients is high (above 25%). the hiv epidemic in pakistan is following the asian epidemic model where after establishment among the high risk groups, its transmission to general public is rapid. fear, stigma and ignorance have contributed heavily to hiv transmission in pakistan. the hiv detection among blood donors is on the rise and reports occur in media repeatedly. aims: to investigate the possible transmission of hiv through blood transfusion in punjab, pakistan and to highlight the steps being taken to reduce further transmission of infections methods: in september 2018, a report of hiv transmission through blood transfusion was reported in the media where a mother and her newborn acquired hiv after blood transfusion from a hiv positive donor (confirmed later). the case was referred to and investigated by the punjab blood transfusion authority (pbta). the pbta team took blood samples of both recipients (mother and her newborn) and the blood donor who was a family relative. the samples were tested by highly sensitive chemiluminescence immunoassay (clia). the clia results confirmed the presence of hiv in both recipients and the blood donor. due to maternal hiv antibodies transfer through the placenta, the infection status of the newborn was not re-confirmed as he died within two weeks. the donor informed that he had donated 22 times in the past few years. the pbta was able to trace only one earlier donation three months ago. the recipient (a female) was found, tested by clia and was found to be hiv positive. all these cases occurred in unlicensed private blood banks that were screening for hiv on rapid manual devices. the blood banks were sealed by the authority and infected cases were registered by the provincial aids control programme and are being treated. summary/conclusions: the main reasons for hiv spread through blood transfusion is the use of sub-standard rapid screening devices which are not evaluated and validated at a national level. in addition, the existing system relies on the family/replacement donors. the national safe blood transfusion programme, is implementing blood safety system reforms as recommended by who. under the reform agenda, the blood transfusion authorities have been made functional and grant licenses to only those blood banks with proper systems to ensure quality and safety of blood products. the programme is developing a national system for the evaluation, selection and validation of all assays used for screening of blood in close coordination with the drug regulatory authority of pakistan. to promote the culture of voluntary blood donations, the programme has taken concrete steps initiating with the formulation of a national blood donor policy, interaction with celebrities, celebration of world blood donor day and more recently the launch of blood donation feature through 'facebook'. the promotion of voluntary blood donation concept along with regulation of blood sector will reduce the risk of hiv transmission through blood transfusions in pakistan. mianyang blood center, mianyang 9 urumqi blood center, urumqi, china 10 rti international, rockville 11 national heart, lung, and blood institute, bethesda 12 stanford university, stanford, united states background: the incidence of hiv infections has increased substantially over the past decade in china, especially among young people, who represent nearly half of the chinese blood donor population. this upward trend in hiv infections underscores the importance of monitoring hiv prevalence and incidence in chinese blood donors. aims: to estimate hiv prevalence and incidence rate (ir) among chinese blood donors using blood donation data from five geographically-disperse blood centers in 2013-2016 participating in the recipient epidemiology and donor evaluation study-iii (reds-iii) china program. methods: western blot confirmatory testing was done on samples of blood donations reactive for hiv-1/2 on one or both rounds of routine elisa tests or positive by nucleic acid amplification testing (nat). multiple imputation was used for samples with missing confirmatory test results. hiv prevalence was calculated among first-time donors. to estimate hiv ir in first-time donors, single-well lag-avidity eia testing was conducted with first-time hiv recent (incident) infections defined as being infected within approximately 129 days based on avidity of hiv antibodies. a novel model was derived to estimate hiv ir among infrequent repeat donors who had provided only one donation in the 2013-2016 estimation interval. to derive an overall hiv ir for repeat donors, this estimate was combined with the classical-model ir estimated for repeat donors who had given at least 2 donations in the estimation interval. multivariable logistic regression model was used to examine factors associated with hiv infection. results: a total of 1,276,544 whole blood and apheresis platelet donations with postdonation screening results were collected at the five blood centers between 2013 and 2016, including 648,607 donations from first-time donors and 627, 937 donations from repeat donors. hiv prevalence among first-time donors was 68.04 per 100,000 donors (95% ci, 61.68-74.40). hiv ir was estimated to be 37.93 per 100,000 person-years (95% ci, 30.62-46.97) among first-time donors and 20.55 per 100,000 person-years (95% ci, 16.95-24.91) among repeat donors. hiv prevalence and ir varied across regions with an increasing trend observed at some blood centers. among first-time donors, being male, older than 25 years, minority ethnicity, less than college education, and certain occupations (commercial services, factory workers, retired, unemployed, or self-employed) were associated with positive hiv confirmatory testing results. summary/conclusions: although hiv prevalence and incidence remain low among chinese blood donors, it is important to monitor hiv epidemiology in blood donors on a continuous basis, especially among populations and regions of higher risk. further donor screening and education strategies need to be developed and evaluated to reduce these risks. the ir methods used in this study for first time donors as well as repeat donors who donate very infrequently is readily applicable to other countries who have similar donation patterns. background: in thailand, the national blood centre is responsible for blood donation service which includes follow-up and blood donor counseling in order to indicate the infection status, especially hiv-positive blood donors. currently, although the epidemic of hiv infection in thailand is in decline, the hiv-positive cases still have been found in blood donors screening. thus, monitoring of hiv infection status in blood donors and post-blood donor counseling are important for providing the hiv-positive infected donors lead to access the hiv treatment immediately. aims: to study the hiv follow-up cases on serological testing over 10 years for assessment of the hiv infection in thai blood donors. the retrospective analysis of hiv follow-up cases on serological testing (cmia, ics and western blot) was conducted during 2009 to 2018 at thai national blood centre. results: a total of 6,408,024 voluntary blood donations over 10 years, the repeated reactive results on hiv serological screening were 5,978 (0.093%) cases and only half of these hiv reactive donors returned to follow-up testing for ascertaining their hiv status. for hiv follow-up process, the hiv reactive screening donors must be followed for 6 months and tested by using the different three principles of hiv serological testing. a total of 5,978 hiv reactive results were separated to three patterns including hiv positive results, inconclusive results and negative results which the number of each group was 1,130 (35.7%) cases, 742 (23.5%) cases and 1,292 (40.8%) cases respectively. out of 1,130 hiv positive results, we found that 1,105 (97.8%) cases were positive with all hiv serological testing for the first-time follow-up and 25 (2.2%) cases were tested and become to positive results after follow-up more than one time. in 742 cases of inconclusive results, 539 (72.6%) cases were reactive only 1 or 2 testing(s) which these donors did not return to confirm again leading to temporarily deferred donors in blood donor system. in addition, 203 (27.4%) cases of inconclusive results could not conclude the hiv result although they were repeated several times. for the last pattern, 1,292 negative results cases showed 762 (59.0%) cases were negative results after follow-up over 6 months while 530 (41.0%) cases were inconclusive results before changing to negative results which almost cases of this group were reentry as blood donor after deferral period is over. summary/conclusions: the number of repeated reactive results on hiv screening was constant over 10 years of which returned to follow-up only half of hiv reactive donors leading to accumulation of temporarily deferred donors in blood donation system. hiv follow-up positive cases were informed and counseled immediately then referring to anonymous clinic for treatment. the problem and challenges of hiv follow-up were inconclusive results that were unclear and some of these did not return to retest lead to loss of re-entry donor who might be changed to negative result afterward. hence, the effective counseling and follow-up system need to be taken urgently to encourage the temporarily deferral donors returned to retest for reducing stigma of deferred donors in hiv follow-up cases. . we only analyzed the information that had non-reactive results for infectious markers reported by blood banks to sihevi-ins©, because they represent a risk for blood recipients. results: when loading the information of sivigila in sihevi-ins©, 241 donors were found (80% men); of these people 256 donations were obtained (97% whole blood). 166 donors (69%) had a reactive result for hiv being subsequently reported in sivigila. in addition, five of them were reactive simultaneously for hbv in blood banks and took on average 174 ae 83 days to be reported in sivigila. 25 donors (10.4%) had an hiv reactive result notified by sivigila and subsequently they were reactive in blood banks. this behavior may suggest an attempt to spread the disease. 20 donors (60% men) despite being initially reported in the sivigila database, presented a non-reactive result in a blood bank for hiv; one of them was reactive for syphilis and hbv and only one for hbv. this pattern may suggest false positive or negative results in one of the two databases analyzed. fourteen donors had negative test in blood banks for hiv and in a range of up to 6 months they were reactive by sivigila (93% of them donate whole blood). this conduct may suggest that accepted donations were in a window period and therefore warrant further investigation. considering that two blood components could be obtained on average from each donor, a potential risk is estimated for 28 recipients. summary/conclusions: the donors reported first in the blood banks through sihevi-ins© and later in sivigila allow to estimate an adequate orientation to the health services. the information from general epidemiological surveillance programs could improve the selection of donors and transfusion safety. background: it is assumed that bacterial contamination of blood products most often takes place during the donation process. the number of bacteria at this time point is estimated to be around 10-100 cfu per bag. little is known about the growth behavior of different bacteria species in whole blood (wb) units during storage and the distribution of bacteria to the different blood products. aims: aim of the current study was to determine the growth of different bacteria species in contaminated wb units and to study the distribution of the bacteria to the different blood components. methods: whole blood (n = 4-12 per species) was inoculated with approximately 10 cfu of different bacteria species (escherichia coli, klebsiella pneumoniae, pseudomonas fluorescens, staphylococcus aureus, staphylococcus epidermidis, streptococcus dysgalactiae, streptococcus pyogenes) and stored for 22 to 24 h at room temperature before centrifugation and separation into red blood cells (rbc), buffy coat (bc) and plasma. bcs from spiked wb were each pooled with 3 random bcs to prepare plasmareduced platelet concentrates (pc). samples were taken from wb after storage and from the blood products (rbc, bc, plasma and pc) right after preparation, and the bacterial titer was determined. sterility of pcs was tested by bact/alert after seven days of storage. results: bacterial growth in wb varied remarkably between donations and bacteria species. the highest titers in wb were detected for the streptococcus species, whereas staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas fluorescens did not multiply. bacteria preferably accumulated in the bcs during separation, reaching titers of up to 3.5 9 10 3 cfu/ml in bcs and up to 0.9 9 10 3 cfu/ml in the corresponding pcs right after preparation. in total, 33 out of 48 pcs tested positive for bacteria at the end of storage. the results were dependent on the species used: e.g., 6/6 pcs tested positive after spiking with streptococcus pyogenes, while only 1/8 pcs tested positive after spiking with escherichia coli. bacterial contamination of rbc and plasma units was much less frequent and associated with higher bacterial titers in the parental wb units. summary/conclusions: the growth and distribution of bacteria during processing of wb into the different blood products is species-dependent and remarkably varies between donations. results: both patients were male (69 yo and 65 yo) with a history of acute myelogenous leukemia status-post haploidentical stem cell transplant. the patients were thrombocytopenic and underwent simultaneous transfusion of irradiated, non-pr, day 4 platelets stored in platelet additive solution, from a single apheresis collection. the blood supplier's primary pre-release bacterial cultures were negative, and the on-site point of release secondary safety measure pan genera detection (pgd) testing was negative for both gram positive (gp) and gram negative (gn) organisms. both apheresis units also passed visual inspection prior to release from the blood bank. during transfusion, both patients displayed signs of septic transfusion reaction including rigors, fever, hypoxemia, tachypnea, tachycardia, and hypotension. transfusion reaction evaluations were initiated, and both patients were admitted to the medical intensive care unit and started on broad-spectrum antibiotics. gram stain of one platelet unit demonstrated gram negative rods (gnr) and gram positive cocci (gpc) in clusters, and the second platelet unit demonstrated gnr only. repeat secondary safety measure pgd testing of both units was negative for both gp and gn organisms. direct bacterial cultures of both platelet units grew both gnr and gpc identified as a. baumanii and s. saprophyticus after 18 h of incubation. colonies on the initial bacterial plates were too numerous to count (tntc), and subsequent re-plating of the platelet units showed: unit 1: a. baumanii tntc and s. saprophyticus with 8.5 9 10 4 cfu/ml unit 2: a. baumanii 6.6 9 10 7 cfu/ml and s. saprophyticus 2.2 9 10 6 cfu/ml blood cultures collected from both patients became positive within 8 h with gnr on gram stain, and both blood cultures ultimately grew both a. baumanii and s. saprophyticus. the primary pre-release cultures at the blood supplier remained negative. after days on antibiotics and pressors, both patients stabilized and were discharged home. the blood donor was interviewed, and he was well. no cultures were collected. summary/conclusions: this case documents failure of both primary pre-release bacterial testing and secondary on-site point of release pgd testing to identify two pathogenic organisms. a. baumanii and s. saprophyticus are unusual causes of septic transfusion reactions, and it is possible that these organisms have different limits of detection in the pgd assay compared to other organisms. rapid attention to clinical signs during transfusion and prompt initiation of antibiotics is critical for the management of septic transfusion reactions. we are currently evaluating ways to further reduce septic transfusion reactions, including increasing the utilization of pathogen reduced platelets. background: transfusion-associated infections due to the transmission of bacteria still represents a major risk in developed countries nowadays. despite the refrigerated storage of red blood cells (rbc), fatal reactions of patients receiving contaminated rbc units are repeatedly reported. in order to further increase the safety of blood transfusions, new strategies and measures have to be developed. in this context, transfusion-relevant bacteria reference strains can serve as a valuable tool for the validation, comparison and interpretation of these new developments. aims: conducting a collaborative study to establish the first repository for red blood cell transfusion-relevant bacteria reference strains. methods: six bacterial strains (serratia liquefaciens, serratia marcescens, pseudomonas fluorescens, listeria monocytogenes, yersinia enterocolitica a-105 and yersinia enterocolitica a-176) were distributed from the paul-ehrlich-institut to 17 laboratories in 10 countries for enumeration, identification and growth measurement in a 7-day interval for a total of 42 days after low-count spiking of rbc bags (10-25 colony-forming units (cfu)/rbc bag). results: except for s. marcescens, all other strains showed good-to-excellent growth in rbc. s. liquefaciens, p. fluorescens, y. enterocolitica a-105 and y. enterocolitica a-176 achieved >10 6 cfu/ml at day 14 and 10 9 cfu/ml at day 21. growth of l. monocytogenes was lower reaching a maximum concentration of >10 6 cfu/ml at day 35. in 9 out of 17 laboratories, s. marcescens showed no growth at all. summary/conclusions: five of the six tested strains showed robust growth in rbc independent of donor variability and are promising candidates to be adopted as official rbc transfusion-relevant reference strains by the world health organization. background: the samplok sampling kit (ssk), itl biomedical, is used by nhs blood and transplant (nhsbt) for sampling of platelet components (pc) for bacterial screening using the bact/alert 3d system. inoculation of bact/alert bottles is performed immediately after sampling. validation of delayed inoculation, with retention of the sample within the ssk devices, would allow a contingency for transport to other screening sites in the event of an incident that prevented testing at the sampling site. ssk maintain a closed system for sampling of pc and are compatible with standard blood collection bags. a graduated chamber (10 or 16 ml) ensures that only the required sample volume is collected and an integrated needle allows inoculation into bact/alert bottles. aims: the national bacteriology laboratory, nhsbt, evaluated the impact of prolonged storage of pc samples in ssk devices with regard to bacterial viability and detection. methods: four reference species were assessed: staphylococcus aureus (atcc 29523), streptococcus agalactiae (atcc 13813), escherichia coli (atcc 25922), clostridium perfringens (atcc 13124). a pool and split method was used with apheresis pc suspended in plasma. units were screened using bact/alert 3d prior to spiking to prove the absence of contamination. pc were spiked with a single species (range 4 9 100-2.8 9 103 cfu/ml) and tested on bact/alert with a 1 ml inoculation into anaerobic and aerobic bottles (positive control). enumeration was performed to confirm the bacterial concentration. each unit was sampled using two 10 ml ssk, which were held for a period of 6 h at 20-25°c. the process was repeated with a 24-h period. once elapsed, 8 ml of each ssk was inoculated into an aerobic and anaerobic bact/alert bottle, one ssk per atmosphere per species and the remaining sample was enumerated. all bottles were incubated on the bact/ alert system for a maximum of 7 days (36ae0.5°c) and subcultured if positive. results: positive controls had detectable growth by bact/alert, excluding aerobic bottles with c. perfringens. this was expected as it is an anaerobic organism. after the storage periods, all bottles had detectable growth by bact/alert. s. aureus showed an increase of 0.6-log10 after 6 h (2.0 9 102 to 8.9 9 102 cfu/ml) and 2.0-log10 after 24 h (3.1 9 101 cfu/ml to 3.4 9 103 cfu/ml). s. agalactiae increased by 0.7-log10 after 6 h (2.1 9 102 cfu/ml to 1.1 9 103 cfu/ml) and 0.09-log10 after 24 h (1.0 9 102 cfu/ml to 1.2 9 102 cfu/ml). c perfringens increased by 0.6-log10 after 6 h (1.3 9 102 cfu/ml to 5.0 9 102 cfu/ml) and 2.7-log10 after 24 h (4.0 9 100 cfu/ml and 2.2 9 103 cfu/ml). for e. coli, the concentration after 6 h was reduced by 0.28-log10 (2.8 9 103 cfu/ml and 1.5 9 103 cfu/ml), however this was possibly a result of inherent counting errors. at 24 h, an increase in growth by 2.7-log10 (1.5 9 102 to 7.9 9 104 cfu/ml) was obtained. summary/conclusions: storage of pc samples in ssk devices for up to 24 h does not have a negative effect on bacterial viability and detection using the bact/alert 3d system. background: the intercept tm blood system for platelets efficiently inactivates pathogens and leukocytes in platelet concentrates (pc). the system utilizes amotosalen and uva light and is available for the treatment of apheresis and whole blood (wb) derived platelets (mostly buffy coat pools) in europe in plasma or platelet additive solution (pas), and the treatment of apheresis platelets in the us (trima tm in 100% plasma or amicus tm for 65% intersol pas/35% plasma). acinetobacter baumanii and staphylococcus saprophyticus strains were isolated from a saline flush taken 7 h after successful and complete transfusion of an apheresis intercept-treated pc in 65% pas/35% plasma, from a patient with a suspected septic reaction that occurred 2 h post transfusion. bacterial isolates, and a sample of a gram stain-negative and culture-negative sister split pc were submitted for evaluation. we report here the in vitro inactivation of the fast growing, gram negative bacterium a. baumanii and slower growing gram positive s. saprophyticus. the sister unit was assessed for amotosalen break down products as an indication of successful inter-cept treatment. aims: the objective of the study was to evaluate bacterial inactivation of a. baumanii and s. saprophyticus in apheresis platelets, assessed immediately after treatment and with re-culture at the end of a 7 day shelf-life. methods: a double apheresis pc collected in 65% pas/35% plasma was split into three equal components, yielding approximately 250 ml of platelets/pc. a. baumanii and s. saprophyticus were grown in lb broth and each pc unit was inoculated with either bacterial strain or the combination of both strains, each at~6 log colony forming units/ml (cfu/ml). after inoculation, the contaminated units were treated in small volume (sv) intercept kits. samples were taken: pre and post-inactivation treatment, and at 3, 5 and 7 days of storage. the samples were analyzed by plating on lb plates (100 ll-10 ml). residual amotosalen levels were assessed by hplc. results: initial bacterial titers were 5.9-6.0 cfu/ml. following the inactivation treatment, no viable bacteria were detected by plate culture. no bacteria were detected after 3, 5 and 7 days of storage, corresponding to > 5.9 log 10 inactivation of both bacterial strains. performance of the intercept treatment process was confirmed in the sister pc unit as evidenced by levels of amotosalen and its byproducts characteristic of intercept treatment, as well as by review of the process documentation. summary/conclusions: amotosalen/uva effectively inactivated a. baumanii and s. saprophyticus individually and together below the limit of detection after 7 days storage. no bacteria in the sister pc by gram stain and culture, and the presence of amotosalen byproducts suggested that the pc collection involved in the septic reaction was sterile at the time of intercept treatment and was successfully illuminated. the possibility of only one-of-two split pc being contaminated due to biofilm formation is minimized in the intercept system which decants platelets into virgin storage bags at the end of treatment. contamination of the source pc container likely occurred after intercept treatment, possibly at the time of spiking for transfusion. background: studies in sub-saharan africa have documented bacterial contamination of blood products for transfusion varying between 0,3%>17,5%, up to 5000 times higher than in the north. published data from central africa are lacking. aims: the aim of this study was to determine the proportion of blood products contaminated with bacteria in three hospitals in the democratic republic of the congo (drc). to assure aseptic sampling, we used a closed system of sampling bags. in addition to the presence of contamination, we assessed semi-quantitative colony counts. methods: from july to december 2018, a total of 2411 blood products were sampled, of which 979 in hôpital provincial g en eral de r ef erence, kinshasa (hpgrk), 662 in hôpital p ediatrique kalembe lembe, kinshasa (hpkll) and 770 in hôpital saint-luc, kisantu (hslk). after compatibilization of blood products, 16 ml of blood was transferred from the primary blood bag to an attached sampling bag. sampling bags were sealed off, stored in the fridge and transported once daily to the bacteriology laboratory. using the adapter connected to the sampling bag, 4 ml of blood was inoculated in a blood culture (bactalertpf, biom erieux) and incubated at 35°c for 7 days. cultures were checked daily for signs of growth. in addition, 2 ml of blood was equally distributed on the cled and macconkey agar-coated sides of a dipslide (meus s.r.l.). dipslides were incubated 48 h for semi-quantitative culture, expressed as colony-formingunits (cfu) per ml. in case of blood culture growth, bacteria were identified and a second blood culture was inoculated to exclude contamination during processing. bacteria grown on semi-quantitative culture were also identified. results: a total of 1.6% (39/2411) of whole blood and red cell concentrates were contaminated with bacteria. in hpgrk, 2.7% (26/979) of blood products were contaminated, in hpkll 1.5% (10/662) and in hslk 0.4% (3/770) . the proportion of contaminated blood products was significantly higher in hpgrk compared to hslk (p = 0.00047). there was no significant difference between the other sites (p = 0.051 and p = 0.12). majority of isolated bacterial species were coagulase-negative staphylococcus spp./micrococcus spp. (46.7%) and bacillus spp. (33.3%). the remaining 20% of bacterial isolates were identified as non-fermentative gram-negative rods, klebsiella pneumoniae, staphylococcus aureus, mould, listeria spp., corynebacterium spp./coryneform bacteria. the concentration of all isolated bacteria was lower than 10³ cfu/ml, except for one coagulase-negative staphylococcus spp. found in hpgrk at 10³ cfu/ml. summary/conclusions: to our knowledge, we were the first to reach a sample size of more than 2000 blood products for bacterial culture and the first to use a closed system of sampling bags in sub-saharan africa, which guarantees aseptic sampling of blood cultures. this might explain the low bacterial contamination rate (1.6%) of blood products in three hospitals in drc compared to previous studies in other sub-saharan african countries. moreover, bacterial concentrations in the contaminated blood products were low (<10³ cfu/ml). the different proportions of contamination between study sites suggest that different environments and practices play a role in the risk for bacterial contamination. background: although the screening of the treponema pallidum (tp) is mandatory in blood donations, its necessity is controversial, because there have been no transmissions by blood products documented in the developed countries in the last few decades. aims: based on laboratory markers, active and early tp infected donors (aeid) were determined. the demographics of aeid and the frequency measures of cases were compared with that of early infected syphilis cases (syc) notified from the 18 to 65-year-old general population registered at the nphc. methods: altogether, 474,017 18 to 65-year-old donors were screened with anti-tp immunoassay (architect syphilis tp, abbott, wiesbaden, germany) between 2015 and 2017. reactive results were confirmed with immunoblots (viramed biotech ag, planegg, germany), which discriminated both specific anti-tp (igg, igm) and non-specific vdrl antibodies in five dilutions. meeting the criteria of anti-tp igg positivity with a vdrl titer of ≥ 1:8 and anti-tp igm positivity, donors were considered as aeid. they were stratified by age, gender and residence regions. the proportion of aeid (paeid) and syc (psyc) were calculated in first time (ft), and repeat tested (rt) donors and in the 18 to 65-year-old general population, respectively, in each year studied. the period prevalence (pp) of aeid and syc was estimated in the populations at risk 1,2 across 2015-2017. statistics: weighted proportions and one-way anova with tukey post-hoc test and z score test were applied. statistical significance was defined as p < 0.05. results: anti-tp reactivity was confirmed in 167 blood donors. aeid was proved in 85 cases with 36 ft and 49 rt donors. in that period, 1696 syc were notified. both in aeid and syc, the age group of 25-34 years with approximately 35% and 36% of individuals was the dominant. the proportion of men was 74% and 77% (p = 0.5) in the aeid and syc, respectively. paeid estimated in ft donors was significantly higher (0.020%; 0.023%; 0.032%, p < 0.0001) than that of rt donors (0.007%; 0.009%; 0.009%) and the proportion of syc (0.008%; 0.009%; 0.010%) in the general population. pp of aeid showed a roughly homogenous distribution in the 7 regions (0.011%-0.025%), however, pp of syc had a significant (0.058%; p < 0.0001) central hungary dominance in relation to the other regions (0.008%-0.016%). comparing the pp of aeid to syc, central hungary indicated a significant difference (0.025% vs. 0.058%, p < 0.0001), however, other regions showed no substantial differences. summary/conclusions: donors with anti-tp confirmed positivity are referred to the healthcare system. based on the laboratory markers tested, aeid could be separated and their demographical characteristics are pretty similar to that of syc notified from the general hungarian population. the proportion of early and active infection in ft donors is significantly higher than that of rt donors and the proportion of syc in the general population. given the considerable number of tp infection in background: quality assurance and safety of hematopoietic stem cells (hsc) with emphasis on prevention of bacterial and fungal contamination are the prerequisites for any transplantation procedure. bacterial contamination is of particular significance as it occurs relatively more frequently and bacteria are gradually gaining more antimicrobial resistance. aims: the aim was to determine the incidence rate of bacterial and fungal contamination during processing of transplantation material at the institute of hematology and transfusion medicine (ihtm) taking into account the hsc sourceperipheral blood (pbsc), bone marrow (bm) or cord blood (cb). methods: analysis involved both autologous and allogenic components collected at ihtm and other hospitals and dedicated for ihtm patients. in all, the analysis comprised 4135 donations, including 112 bm (2.70%), 3787 pbsc (91.60%) and 236 cb (5.70%) donations processed in our laboratory in the years 1996-2016. bm was collected in operating theatre-conditions, pbsc with cell separators -cs-3000 (baxter), cobe spectra (gambro) and trima accel (terumo bct) and cb was acquired from umbilical vein at obstetrics and gynaecology wards. aerobic and anaerobic bacteria contamination was determined at various incubation temperatures (room temperature and 35°c) using a variety of culture media. pbsc and bm were tested using 2 samples with trypcase-soy broth (tsb-t) and 1 with schaedler + vit k3 (biomerieux). cb was tested using bactec peds plus/f and bactec lytic/10/anaerobic/f (becton-dickinson). results: in the 1996-2016 period 42 contaminated products were found: 25 pbsc (0.66% of all tested pbsc units) and 17 cb (7.20% of all tested cb units). no infected bm products were determined. the overall percentage of contaminated products was estimated at 0,1%. in 2010, three (3) products were found contaminated with staphylococcus epidermidis; all came from one patient with central venous catheter and were collected on consecutive days. other products were contaminated mostly with staphylococcus epidermidis (61.36%). detailed results to be presented on the poster. summary/conclusions: according to ihtm policy no contaminated product is admitted to clinical use. the outcome of our study identifies processing experience of the staff as the main indicator of product quality. important is also proper assessment of donor health and condition of the injection site as products are usually collected from central venous catheter. the closed system is an additional safeguard against contamination during processing. the sample collecting procedure should help to avoid false positive results. background: syphilis is considered a global public health problem. the world health organization (who) estimates that there are annually around 12 million new cases of syphilis in the world, more than 90% occurring in developing countries. despite significant decrease in syphilis transfusion transmission. the recent increase in worldwide incidence associated with the risk of transmission through platelet concentrates (cp), which are stored at room temperature, have called attention to the potential residual risk of syphilis transmission by transfusion. between 2015 and 2017 we observed in our institution a significant increase of 24% in positivity of syphilis among blood donors from 0.62% in 2015 to 0.73% in 2016 and 0.77% in 2017 (p < 0.0000001). aims: to determine the prevalence of active syphilis in blood donors and characterize the serological profile of syphilis positive donors. methods: each positive sample in a treponemic chemiluminescence assay (cmia, abbott architect) performed during blood donor screening in 2017 was submitted to a treponemic elisa anti-treponema pallidum igm (euroimmun) and a non-treponemic test (antigen-omega diagnostics). samples with positive results for one or two of these tests (indicating recent syphilis) were submitted to a real-time pcr for syphilis. the inno-lia syphilis-fujirebio immunoblot test was also performed for samples that presented a positive result for elisa-igm alone. financial support: fapesp 2017/23028-9. results: among 123,851 samples screened in 2017, 958 (0.77%) presented a positive result for cmia -syphilis. of these, 626 (65.4%) were included in the study. a total of 106 samples (17%) showed vdrl+/igm+; 96 (15%) vdrl+/igmand 28 (4.5%) vdrl -/elisa igm+. the inno-lia syphilis test was performed as a confirmatory test in 28 (4.5%) samples that presented positive results for elisa igm and vdrl negative with 21 (3.35%) positive results, 1 (0.15%) undetermined and 6 (0.95%) negative. none of the 626 samples showed the presence of treponema dna by real-time pcr. the prevalence was 0.77%, the incidence was 0.1% in the year 2017, and the incidence in relation to the total positivity was 20.4%. both, prevalence and incidence were higher in men, white, not married, aging 18-29 years and high school educational level. we observed a 6% a-hbc seroprevalence in the elisa igm-syphilis positive samples and a prevalence of 1.5% htlv coinfection. summary/conclusions: we observed a significant increase in prevalence of syphilis in 2017 (0.77%) with an incidence of 20.4% of the total of cases initially positive in the cmia test. according to our data, we could identify a risk of syphilis transfusion transmission in blood banks that exclusively use the vdrl for donor screening, once we found 22 (3.5%) cases with negative vdrl and elisa igm and inno-lia positive. continuous monitoring of the profile of donors infected with syphilis at this time of reemergence of the disease is useful and important not just for blood banks, as it reflects the epidemiological situation of disease in community, and can contribute to the definition of health policies. background: transfusion related sepsis is a serious concern limiting platelet storage time to 5 days at room temperature. while most units are screened for bacterial contamination when collected, bacterial monitoring methods can take up to 7 days to detect contamination. thus, cold storage of platelets represents an attractive alternative for improving platelet safety. in this study, we assessed bacterial growth in platelets stored either at room-temperature (rt; 22°c) or refrigerated (cs; 4°c). aims: the aims of this study were to 1) assess the effect of storage temperature on platelet function and bacterial growth in "contaminated" platelet units, and 2) identify factors contributing to bacterial growth during rt storage. methods: apheresis platelets in plasma (plt) were obtained from healthy donors using the terumo trima accel automated blood collection system (terumo bct). fresh plasma (fp) was collected similarly. aliquots of plt or fp were transferred to ph safe minibags (blood cell storage, inc) and "contaminated" with acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis, or pbs (uninfected control). minibags stored at rt were agitated using an orbital shaker set to 60 rpm while cs aliquots were stored under static conditions. bacterial growth was monitored daily through dilution plating. lactate levels were assessed by istat (abbott) cg4 + test cartridges. plasma glucose levels were assessed using blood glucose testing strips (germaine laboratories). platelet activation and aggregation were assessed on days 0, 1, 3, and 5 by flow cytometry and multiplate platelet aggregometry, respectively. results: bacterial growth progressed rapidly over the first 3-4 days post-collection in all plt aliquots stored at rt except those challenged with s. epidermidis. significant growth of s. epidermidis was not detected until day 4. bacterial numbers remained unchanged in refrigerated aliquots through day 5. rt storage resulted in significantly (p < 0.05) decreased platelet aggregation over time which was exacerbated by bacterial challenge. plt function was largely preserved with refrigeration regardless of challenge. bacterial growth was significantly reduced, or at least delayed, in fp. fp challenged with gram-negative pathogens exhibited a significant (p < 0.05) delay in bacterial growth at day 1. while growth of e. coli and p. aeruginosa recovered by day 2, growth of a. baumannii was significantly (p < 0.05) inhibited throughout. fp challenged with gram-positive pathogens exhibited significant (p < 0.05) reduction in bacterial growth relative to plt aliquots. bacterial growth correlated with plt lactate production. lactate levels in plts challenged with e. coli showed diminished significantly after day 3, indicative of lactate utilization. with exception of fp challenged with s. aureus, bacterial growth was restored in fp supplemented with lactic acid in all challenge groups. summary/conclusions: refrigeration preserved platelet function while both inhibiting bacterial growth and lactate production. conversely, the opposite was observed with rt storage. these data demonstrate that bacterial growth can be controlled through refrigeration without loss of function and rt storage may potentiate growth of certain bacterial strains through accelerated plt metabolism. background: bacterial contamination of peripheral blood progenitor cell (pbpc) for transfusion has been the cause of serious sepsis and life-threatening infections. however, a standard procedure or choice of test sample(s) has not been established to screen pbpc products for microbial contamination, because these products are not large enough to facilitate inoculation of the recommended volume for the automated blood culture systems. samples taken from by-product plasma and low volume pbpc product were cultured in routine sterility test. aims: to evaluate the residual risk of microbial contamination in pbpc products for transplantation, we cultured sufficient post-thaw inoculation volumes from pbpc products which were discarded for various reasons in our blood center. methods: in routine sterility test, a 20-ml sample of by-product plasma collected with pbpc product was inoculated into bact/alert bpa and bpn culture bottle (10 ml each) within 48 h after collection. the bottles were then placed in the bact/ alert system and incubated for at least 7 days or when a positive reaction was indicated by the automated liquid-media culture system. moreover, a 2-ml postthaw sample would be cultured before transplantation performed. in the residual risk investigation, discarded pbpc products were thawed, and then a 2-ml and a 20-ml aliquot were taken and cultured with the same method. all positive bottles were subcultured for bacterial isolation and identification. results: in september 2008 and march 2018, after maintaining in liquid nitrogen for 1 to 17 years, 205 pbpc products collected from 45 patients, which was preserved in a volume between 35 and 60 ml, were discarded. all of these products had been cultured negative in routine sterility tests with plasma samples. these 205 final products were thawed and cultured with both the 20-ml and the 2-ml aliquot. one of these pbpc products had the positive culture result with the 20-ml retested samples. nevertheless, the same pbpc product had the negative result with the 2-ml post-thaw pbpc sample and the 20-ml by-product plasma sample. propionibacterium acnes was isolated from the bpn positive bottle. summary/conclusions: the residual risk of microbial contamination in pbpc postthaw products still exist after routine sterility test with the plasma sample and the 2-ml volume of pbpc sample. the bacterium isolated from pbpc product was normal skin flora bacterium. an optimal screening method of pbpc products merits further study to increase the safety of the blood supply. background: hospital hygiene tools that serve as a proxy for assessment of microbial contamination are increasingly used. they include adenosine triphosphate (atp) bioluminescence and air particle counting. however, their use for microbial monitoring of blood banks remains underexplored. they could be of particular interest in a sub-saharan african setting (temperatures, dust) to circumvent bacterial culture and provide direct results usable for monitoring over time. aims: the aim of this study was (i) to quantify environmental bacteria in the air and on surfaces that are regularly in contact with blood products, and (ii) to evaluate atp bioluminescence techniques and particle counts as a predictor for bacterial contamination, in three blood banks in the democratic republic of the congo (drc). methods: samples were taken in three blood banks in the democratic republic of the congo: hôpital p ediatrique de kalembelembe (hpkll) (10 surfaces, 1 air), hôpital provincial g en eral de r ef erence (hpgrk) (24 surfaces, 2 air) and the national blood transfusion centre (cnts) (20 surfaces, 2 air). surfaces that are regularly in contact with blood products were selected (sealer, fridge, donor chair,. . ..). regular surfaces were sampled using rodac contact plates (23.7 cm²) containing cled and macconkey agar, irregular surfaces using swabs (nrsii, medicalwire). atp was measured on the same surface (pd30, kikkoman), expressed as relative light units (rlu) per 100 cm². air was sampled by active sampling (500 liter; spinair, iul) on cled and macconkey medium. in parallel, particles > 0.5 lm and > 5 lm were counted using a particle counter (14,15 liter; metone 227a). culture media were incubated for 48 h at 35°c before counting colony forming units (cfu). results: for regular surfaces, the median (range) viable bacterial count was 27 (6-60) cfu/rodac, 69 (6-103) cfu/rodac, 82 (3-132) cfu/rodac for hpkll, cnts and hpgrk, respectively. at hpkll, highest viable counts were found in the sink (plain growth) and cool boxes (43 and 60 cfu/rodac). in cnts the blood processing bench, the donor chair arm support and washing basin showed the highest counts (plain growth). whereas in hpgrk, most bacteria were found in a fridge (plain growth), blood bag trolley (plain growth) and manual separator (132 cfu/ rodac). gram-negative bacilli were isolated from water basins and sink in cnts and hpkll, but also surfaces close to donor chairs at hpgrk. the median (range) of atp per 100 cm² was 2.906 (778-26.497) rlu at hpkll, ) rlu at cnts and 35.235 (1.754-348 .052) at hpgrk. atp results and total viable count were not correlated (n = 49, p = 0.52). median (range) bacterial count in the air was 258 (210-375) cfu/500l for all sites together. there was no correlation found between total bacterial count and particles > 0.5 lm or > 5 lm (r = 0.7 and r = 0.6 respectively; p < 0.05; n = 5) summary/conclusions: total viable bacterial count of surfaces varies over blood bank sites. according to our results, atp and particle counts did not correlate with bacterial counts on surfaces and in the air, respectively. bacterial isolates from blood bank environments in drc need to be identified and seasonal variations need to be evaluated. background: the risk of transfusion-transmitted hepatitis e virus (tt-hev) infections in line with the question of the necessity of hev-nat screening of blood products is currently subject to an ongoing debate on the importance of timely introduction of hev screening of blood donors and the impact of blood safety. different countries have chosen different regulatory approaches. just recently, the german federal authorities have introduced mandatory testing of all therapeutic blood products beginning from january 1st 2020. however, we already decided to voluntarily test all our blood products since january 2015. aims: in this study, we present our results of a 100% screening of therapeutic blood products for hev rna including four years of active surveillance of hepatitis e infection among blood donors in germany. methods: from january 2015 to december 2018, a total of 386,307 allogenic blood donations from 69,956 individual german blood donors were screened in a minipool format of 96 samples of for the presence of hev rna (realstar hev rt-pcr kit, altona diagnostic technologies (adt), hamburg, germany). nucleic acids were extracted from 4.8 ml plasma using the chemagen msm-i extractor (viral 5k, perkin elmer chemagen gmbh). the 95% lod of the assay was determined to 4.66 iu/ml (447 iu/ml per single donation). the presence of hev-specific igm and igg antibodies was determined using the anti-hev igm/igg elisa (euroimmun, luebeck). hev rna concentrations were quantified using the first who international standard for hepatitis e virus rna for nat-based assays. all hev rna positive donors were deferred from donation for 3 months. follow-up samples were tested for the presence of hev rna and hev-specific antibodies. genotyping was performed by sequencing of the hypervariable region (hvr) and orf1. results: in total, 274 hev rna positive donors were identified. of these, 274 hev rna-positive donors, 216 were nat-only positive donations (78.83%, negative for anti-hev igm and anti-hev igg), three donors had a positive igm titer (1.09%), 30 donors showed reactive igm and igg titers (10.95%), 25 donors already had isolated igg titers (9.12%). median values of viral loads were approximately twice as high in index donations that were antibody negative. merely 62 donors showed elevated alt levels (22.63%), mostly within a double increase within the reference range (16.06%), only 6.57% of donors had even further elevated alt levels. significantly higher alt values were found in donors with a viral load > 1,000 iu/ml compared to the group with viral loads between 100 and 1000 iu/ml. available follow-up samples confirmed igg seroconversion for all donors, however we also observed long-term igm positivity in some donors. genotyping revealed genotype 3 in all cases. the month-dependent incidence ranges from 1:719 to 1:3,781 blood donations with a peak in june and july. summary/conclusions: the high number of identified hev rna positive donors emphasizes the need for hev-nat screening to increase the safety of blood products. this study further confirmed that hev infection is common in german blood donors. background: zika virus (zikv) is a mosquito-borne virus that has caused outbreaks in central and south america in february 2016, and has threatened the safety of blood transfusion globally. there is a high risk of zikv transmission by whole blood and blood components transfusion. it was reported that, zikv rna in infected patients plasma can only be detected within 1 to 2 weeks. however, in whole blood, zikv rna might present positive up to day 101 after the symptoms appear in some patients, even if the clinical symptoms disappeared with zikv rna negative in plasma. this phenomenon suggested that the presence of zika is associated with red blood cells (rbcs). moreover, another report showed that viral load in whole blood of type a west nile virus (wnv) patients was higher than type o, implying that the binding of virus to rbcs may be related to blood group glycoprotein. both of zikv and wnv are member of the flavivirus genus. the study is intended to explore whether zikv have the same adherence mechanism to rbcs as wnv. aims: to investigate the distribution of zikv in blood components and adherence of zikv to different blood types of rbcs in whole blood. methods: five units for each blood type of a, b, o and ab whole blood were randomly selected. each unit of 200 ml whole blood was divided into two half-unit. zikv was added to one half-unit in a certain proportion, and incubated at 37°c for 3 days. each component of whole blood was collected for viral load detection. in the other half-unit,rbcs were suspended in the same type pools of plasma with equal volume after the plasma removed from the whole blood after centrifugation. zikv was added with the same certain proportion, and then incubated at 37°c for 3 days. the whole blood samples and the upper plasma by centrifugation were collected detected for zikv rna. meanwhile, rbcs were washed and resuspended with normal saline followed by viral load detection. results: zika rna of these samples which extracted from whole blood, rbcs, and plasma were determined in a quantitative reverse transcription pcr, and viral rna of each component was all up to 10 9 copies/ml. although, zikv rna loads did not show significant difference in distribution between rbcs and their corresponding plasma components, zikv rna quantification were significantly higher than those in plasma (p < 0.05) in type o rbcs and lower than those in plasma (p < 0.05) in type ab rbcs. summary/conclusions: in our study, we detected high viral rna loads in rbcs. it was demonstrated that zikv adheres to erythrocyte in whole blood, and the blood type may have influence on the adherence. background: hong kong is not endemic for dengue virus (denv) with most of the documented cases being imported. the presence of sufficient number of mosquito vectors, aedes albopictus, in the territory has led to two self-limiting indigenous outbreaks affecting 20 residents from 2000 to 2014. during 14 august to 4 september 2018, another outbreak of 29 confirmed cases of autochthonous dengue fever were reported to the department of health, linked to two epidemiological clusters, one in lion rock park near wong tai sin (wts) district (19 cases) and the other in cheung chau, an outlying island (10 cases). aims: we assessed the risk of dengue transmission from blood donors during the 2018 outbreak using a simplified version of the probabilistic model developed by biggerstaff and petersen (b-p) and the european up-front risk assessment tool (eufrat) model (oei, transfusion, 2013) . methods: patient demographic and general population data were obtained from the centre for health protection and the department of census and statistics of the hong kong government for the number of 15-to 64-year-old patients in the 2018 outbreak and residents of the same age range in hong kong and wts district as at mid-2018 respectively (16-65 years old being the eligible age range for first time donation). to apply the b-p model, we estimated denv incidence among donors in hong kong territory and in wts with confirmed denv infection during 12 august to 8 september 2018 after correction for clinical:subclinical infections ratio, the mean length of asymptomatic viraemia and the probability of collecting blood from asymptomatic donors as described previously (seed, transfusion, 2009 ). to estimate the risk using eufrat model, outbreak and blood donation variables were entered into eufrat's web-based interface (https://eufrattool.ecdc.europa.eu/), which provided automatic calculation of risk-related output parameters. results: while using the b-p model, the estimated risk of collecting a denv viraemic donation was one in 778,000 (266,000-1,822,000) territory-wide for the 28-day study period but increased to one in 147,000 (50,000-345,000) in wts. similarly while applying the eufrat model, the risk of encountering a viraemic donor was 1 in 279,000 (118,000-752,000) territory-wide and 1 in 52,000 (21,000-188,000) in wts during the same period. the eufrat also predicted a territory-wide issue of 0.08 unit of denv-contaminated labile blood component during the outbreak period. summary/conclusions: like many mosquito-borne infections such as denv, the risk is characteristically localised and varies geographically and seasonally during outbreaks. the average predicted risk of collecting a denv-viraemic donation territory-wide is low at 1 in 778,000 during the 2018 outbreak based on the b-p model, which was generally considered as tolerable. however, the risk increased by 4 folds when blood donations were collected from wts residents, who had higher chances of paying visits to lion rock park in close proximity. it was then justifiable to institute risk mitigation policies such as geographically-based deferral and/or fresh component restriction, enhanced post-donation reporting, etc. to protect against blood safety. background: hev is a developing threat to blood safety following the reporting of several cases of transfusion transmission hev (tt-hev). transfusion-related hev infection has been reported in several countries but its true frequency is probably underestimated because it is often asymptomatic and testing of blood donors is infrequent. pakistan is classified as a highly endemic region; with sporadic cases of hev occurring throughout the year, mainly affecting the adult population. to the best of our knowledge, no studies have been reported from pakistan on the epidemiology of hev in blood donors. aims: to assess the epidemiology of the hev specific antibodies and serum alt levels in blood donors of capital twin cities of pakistan. methods: this cross sectional study was conducted from july 2017 to december 2017 at three blood banks in the capital twin cities (rawalpindi and islamabad) of pakistan. the blood donors were equally distributed across the three blood banks. only donors who tested negative for hiv, hbv and hcv were included in the study. serum alt levels were analyzed by using automated clinical chemistry analyzer (selctra pro m) using merck kits. all samples were tested for hev-specific antibodies (igm and igg) by using enzyme linked immunosorbent assay (elisa) kits (adaltis, italy). statistical analyses were performed using spss software version 21.0 (ibm). results: in our study population there were 445 (98.9%) males and 5 (1.1%) females. the mean age of recruited blood donors was 28.38 (sd ae 7.34), with a range of 18-55. younger donors were more common with a frequency of 18-27 year olds of 249 (55.3%). we found an overall hev igg prevalence of 17.5% and an hev igm prevalence of 9.1%. there were 10 (2.2%) blood donors who were positive for both igg and igm antibodies. our results revealed that the hev specific antibodies (igg, igm) prevalence increased with age. the mean value of serum alt was 33.8 (sd ae 41.0) with a range of 4-554 iu/l. the serum alt levels were elevated (>45 iu/l) in 73 (16.3%) blood donors. there was significant correlation (p=<0.001) between serum alt level and hev specific antibodies for igg and igm. summary/conclusions: this study shows that a significant proportion of blood donors at our blood centers have been infected with hev and may be able to cause tt-hev. as we have not yet measured hev rna, we have used igm antibodies as a proxy for donors who have active infection. hev is generally asymptomatic, so it is debatable whether mandatory hev screening in blood donors should be required. results of this pilot study show that there is a need to conduct a larger study at national level with highly sensitive assays before making screening for hev mandatory in pakistan. background: hepatitis e virus (hev) is a zoonotic virus. who estimates that there are 20 million hev infections, 3 million acute hev cases and 56 thousands hevrelated deaths worldwide each year. in recent years, the prevalence of hev in european and american countries has increased significantly. the survey results show that the positive rate of hev igg in blood donors is respectively 4.0% in new zealand, 13.5% in britain, 20.6% in denmark, 16% in the united states and 27.0% in the netherlands. hev has become a global public health concern. in addition to the food route of infection, several cases have been reported that hev can be transmitted via blood products. aims: to investigate the prevalence of hepatitis e virus (hev) infection among voluntary blood donors and potential impact on blood safety in guangzhou china. methods: blood samples from 5552 blood donors were collected from april 2017 to april 2008 at the guangzhou blood center and were tested for anti-hev igg antibody (hev igg), anti-hev igm antibody (hev igm) and hev antigen (hev ag)by enzyme linked immunosorbent assay (elisa). hev rna detection was performed on hev antigen positive samples by rt-pcr. the association of age, gender, ethnicity, occupation and alt with hev igg and igm were analyzed by chi-square test. multivariate logistic regression analysis was applied to identify the independent risk factors of hev infection. results: the positive rates of hev igg, igm and hev ag were 20.05% (1113/5552), 0.76% (42/5552) and 0.04% (2/5552), respectively. no positive hev rna was detected. age and ethnicity were independent risk factors for hev igg and hev igm. the rate of hev antibody increased significantly with age (igg or = 1.089, p < 0.001; igm or = 1.055, p < 0.001). donors who were zhuang minority (32.69%, 7.69%) showed higher anti-hev than those who were han ethnicity (19.89%, 0.70%), and the difference was statistically significant (igg or = 2.052, p = 0.023; igm or = 12.029, p < 0.001). in addition, we found that occupation was an independent risk factor for hev infection, where students showed the lowest anti-hev rate. summary/conclusions: the results indicate that the positive rate of hev antibody among blood donors in guangzhou is high, and the infection status differs in different populations. our study provides basic data for the estimation of risk of transfusion-transmitted hev. background: human cytomegalovirus (hcmv) belongs to the viral family of herpesviridae. it is an enveloped double-stranded dna virus, widely distributed in the human population (60-100% seropositive subjects worldwide) and cause of severe disease in immunocompromised patients and upon infection of the foetus. in normally healthy subjects, hcmv persists lifelong without clinical manifestation undergoing alternating phases of active viral replication and latency. since hcmv can be readily detected in blood, as free virus as well as associated to neutrophils and monocytes, hcmv transmission is a complication of blood transfusion. even though leukoreduction of blood products has been shown to significantly reduce the risk of hcmv transmission, higher inactivation standards may be required for high-risk, immunocompromised groups of patients. aims: in this study, murine macrophages infected with murine cytomegalovirus (mcmv) were used as a model to study the inactivation cell-associated cmv in human plasma using the theraflex mb-plasma system (macopharma). methods: mcmv expressing the green fluorescent protein was used to infect murine macrophages. infected macrophages were harvested 20 h after infection, washed and used for spiking of plasma. plasma units (n = 2, 290 ml) were spiked with infected cell suspension (10% v/v) and treated with the theraflex mb-plasma system according to the manufacturer's protocol using the macotronic-b2 illumination device (macopharma). samples were taken after spiking (load and hold sample), after illumination with different light doses (0 after addition of mb, 30, 60, 90 and 120 [standard] j/cm 2 ) and after blueflex filtration. mcmv titers were determined by endpoint titration and large volume plating on murine fibroblasts. infectious virus, which expressed gfp in infected cells, was detected using a fluorescence microscope. results: the results of infectivity assay showed that the treatment of human plasma by the theraflex mb-plasma system inactivated cell-associated mcmv in a dosedependent manner. after spiking with mcmv infected macrophages a mcmv titer of 4.2 (bag no. 1) and 4.5 (bag no. 2) log 10 tcid 50 /ml was achieved in the plasma units. in hold samples, a mcmv titer of 3.8 (bag no. 1 and bag no. 2) log 10 tcid 50 /ml was determined. the illumination step of the theraflex mb-plasma treatment procedure efficiently inactivated mcmv. already three-fourths of the standard light dose decreased infectivity of cell associated and remaining cell-free mcmv to infectivity levels below the limit of detection (≥ 2.9 log). further investigations would be needed to evaluate the log reduction capacity of the blueflex filtration step for cell-associated mcmv. summary/conclusions: the results with the murine model virus suggest that the theraflex mb-plasma system is an effective technology to inactivate cell-associated cmv in human plasma units. background: the use of pathogen inactivation (pi) technologies for platelet concentrates and plasma is slowly but steadily increasing. methods for treatment of red blood cells (rbcs), the most commonly used blood component, are still under development. aims: a novel approach for pi in rbc units employing uvc light was developed. methods: pi treatment was applied to full-scale rbc units after leukodepletion. the pi capacity of the uvc-based method was evaluated by bacteria and virus infectivity assays. a panel of in vitro assays to measure quality, metabolism, functional, morphologic, and blood group serology variables was applied to a pool-and-split approach in which pathogen-reduced rbcs were investigated in comparison to untreated rbcs. results: uvc treatment caused dose-dependent inactivation of bacteria and enveloped and non-enveloped viruses in rbc units. at a full dose, the mean log 10 reduction factors ranged from 4.2 (bacillus cereus) to 6.1 (serratia liquefaciens) for the tested bacteria, and from 3.1 (emcv) to ≥ 4.9 (vsv) for the tested viruses. uvc treatment did not alter rbc blood group antigen expression. quality of uvc-treated rbcs was maintained during storage, e.g. hemolysis in uvc-treated and untreated rbcs were well below 0.8% until day 35 of storage. summary/conclusions: the data obtained until now show that uvc irradiation is a potential new method for pi in rbcs and justify further development of this process. background: histo-blood abh antigens are the mayor allogeneic antigens in human and they are widely distributed in almost all tissues. the expression of a-1,2-fucosyltransferase (fuct2), encoded by fut2 gene, determines the secretor status of an individual. about 80% of caucasian population have a functional copy of fut2 (secretor gene) expressing abh blood group soluble antigens in organic fluids such as saliva and seminal plasma. this individuals are known as "secretors". soluble abh blood group antigens have been associated with several metabolic and infectious diseases as well as reproductive failures. the incidence of infertility related of both male and female factors continues to rise despite many advances in reproductive technologies. it is well known that abo antigens are expressed on sperm membrane and in seminal fluid of secretors as well as abo antibodies are present in cervical mucus. in previous studies we observed significant loss in progressive motility of spermatozoa of non-secretors compared to secretor ones caused by specific cervical mucus antibodies in abo-incompatible couples. in addition, sperm cells are haploid cells, so that a heterozygous individual has two sperm subpopulations, each expressing the corresponding allele. the specific antibody of cervical mucus will attack only its complementary sperm. aims: to evaluate the prevalence of secretor character in men belonging to fertile and infertile couples in order to investigate a possible association with reproductive success. methods: 126 samples of semen, 68 from infertile men and 58 from fertile controls were studied. comprehensive infertility evaluation was performed in all patients according to who 2010 criteria. secretor phenotype was evaluated in seminal plasma by inhibition of hemagglutination assay using saline erythrocyte suspensions, monoclonal antibodies anti-a, anti-b and lectin from ulex europaeus (anti-h). to distinguish between abo genes, genomic dna was extracted by an enzymatic digestion method. pcr was designed with two sets of oligonucleotides that allow to amplificate two different regions of the transferases without use of restriction enzymes. by comparison of bands of the pcr products, the individual genotype was determine. cervical mucus antibodies of their female partners were titrated with the corresponding red blood cells. results: results were analysed in both groups. in infertile couples with abo incompatibility, the frequency of non-secretor phenotype of male partners (76.9%) were significantly higher than those from fertile couples (21.6%) (p < 0.03) the results obtained by pcr in sperm cells correlated 100% with red cells phenotypes. summary/conclusions: incidence of infertility continues to increase. several factors have a negative impact on men's reproductive health. immunological implications are now being studied and considered as a cause of failure in sperm-egg interaction, even among normal gametes. secretor phenotype in male partners could help reproductive success by blocking cervical abo antibodies. furthermore, if the male is heterozygous, cervical mucus antibodies will only affect the corresponding sperm. we propose to evaluate abh antigen expression on sperm membrane and seminal plasma as well as abo antibodies in cervical mucus to contribute to the diagnosis and treatment of human infertility. background: the h blood group contains one antigen, the h antigen, which is present on virtually all red blood cells (rbc) and is the acceptor substrate of both a and b gene-specified glycosyltransferases. in blood group o the h antigen remains unmodified and therefore its rbcs show the highest and the rbcs of blood type ab the least amounts of h antigen. individuals with the so called bombay phenotype carry homozygous h null alleles (h | h) and do not produce any h antigen. the para-bombay phenotype retains some h antigen on rbcs either induced by a weakly active (h+ w | h+ w ) or completely silenced fut1 gene (h | h), mandatory linked with an active fut2 gene. aims: in this study, we aimed to develop an adapted flow cytometric method to quantify the relative amount of h substance present on rbcs in order to distinguish different abo phenotypes in routine diagnostics as well as to capture rare h-deficient phenotypes. methods: analyses were performed on a flow cytometer (facs canto ii, bd biosciences, ch) and measured with identical instrument settings. list mode data were evaluated and visualised using bd facsdiva software. rbcs were incubated with increasing concentrations of monoclonal anti-h antibodies (bric231-pe and a 1:1 mixture of bric231-pe/bric231, ibgrl, uk). after rinsing the cells with pbs, micro-aggregates were mechanically dissolved. rbcs from 29 blood donors with different abo phenotypes (o (5), a 1 (5), a 2 (5), b (5), a 1 b (5), a 2 b (4)) and 2 patients with genetically confirmed bombay and para-bombay phenotype were assessed. results: saturation of h antigen binding sites on type o rbcs was achieved only upon use of a 1:1 antibody mixture (bric231-pe/bric231) covering approx. half of the h-binding sites by unconjugated bric231. in contrast, non-o type rbcs reached saturation of h-binding sites using pure bric231-pe. rbcs coated with bric231-pe at saturation revealed a distinct pattern of mfi (mean fluorescence intensity) depending on the abo phenotype. in addition, mfis of rbcs upon staining with bric231-pe did discriminate bombay-and para-bombay type rbcs, respectively. summary/conclusions: adapted flow cytometry is able to distinguish variant expressions of rbcs h antigen. thus, our flow cytometric method may complement traditional serological and genetic analyses in routine abo phenotype cases or, more intriguing, when the bombay or para-bombay phenotype is suspected. it will be of interest to further prove this method by investigating additional rare h-deficient phenotype cases. s chen 1,2 , x xu 1,2 , x hong 1,2 , k ma 1,2 , j he 1,2 , j chen 1,2 and f zhu 1,2 1 blood centre of zhejiang province 2 zhejiang provincial key laboratory of blood safety research, hangzhou, china background: weakened a and b antigen expression results in abo typing discrepancies. h gene controls the development of h substance from which a and b antigens develop. depressed a and b antigen expression and strengthened h antigen expression are always simultaneously observed in abo subgroups. there are other possibilities for weak antigen expression of abo system such as leukemic change and pregnancy. it is undiscovered whether abnormal expressions of a, b and h antigen stand for abo subgroups in hemopathic patients. aims: the aim of this study is to explore the role of enhanced reactions with anti-h in direction to abo subgroups of hemopathic patients. methods: 109 samples from blood donors and hemopathic patients with nonconcordant abo typing by serological tests were collected after consent information. the agglutination strength of these rbcs with anti-h reagent was recorded. enhanced reactions were determined by comparison with the results from normal abo groups. the genomic dnas of 109 samples were extracted and genotyped for abo system. this work was sponsored by the medical science research foundation of zhejiang province (2018rc029). results: 69 samples in 80 blood donors showed increased expression of h antigen, of which 47 were identified as abo subgroups. there were 22 enhanced reactions in 29 hemopathic patients. however, 19 were finally confirmed as normal abo genotypes. no statistical significance (86.3% vs 75.9%, p > 0.05) in the frequency of strengthened h antigen expressions was observed between donors and hemopathic patients. the total number of subgroups is 50 and 3 respectively in blood donors and hemopathic patients. extremely significant statistical differences (68.1% vs 13.6%, p < 0.005) existed in the frequency of subgroups with enhanced h antigen, which meant the possibility of subgroups in hemopathic patients samples was less. summary/conclusions: the expression of h antigen is comparably enhanced in subgroups and hemopathies. but most of hemopathic patients with strengthened h antigen expression present normal abo genotypes. as a result, the enhanced reaction with anti-h is necessary but not sufficient for serological identification of abo subgroups in hemopathic patients. background: although the use of automated blood bank analyzer with the advantages of speed and efficiency has recently increased, the abo discrepancies in automated blood bank analyzer have caused the reporting delays of the results and increase of the task. aims: we analyzed the causes of abo discrepancies in automated blood bank analyzer and suggested a solution strategy based on the causes. methods: from november 2018 to january 2019, 55 cases (0.6%) of abo discrepancies among 9,550 abo blood type tests performed using the 15-min reaction mode of ih-500 in chonbuk national university hospital blood bank were identified. we compared the test results of 15-min mode with results of immediate mode using different red cell reagents, and analyzed the causes of discrepancies by performing additional tests such as microscopy, auto-control, antibody screening and identification, anti-a1 and abo genotyping. results: in the immediate reaction using different red cell reagents, 45 cases (81.8%) of discrepancies disappeared and 10 cases (18.2%) remained discrepancies. all abo discrepancies observed in the 15-min reaction were due to serum side causes, and one case (1.8%) was due to both of serum and red cells side cause. nonspecific response (28 cases, 50.9%), cold antibody (20 cases, 36.4%), rouleaux formation (3 cases, 5.5%), cis-ab (3 cases, 5.5%), and abo subtype (1 case, 1.8%) were analyzed as causes of discrepancies. one discrepancy due to cis-ab was disappeared in the immediate reaction using different red cell reagents, abo subtype was changed to totally different blood group, a. on the other hand, in cases of the discrepancy corrected by the immediate reaction using different red cell reagents, the intensity of the positive results still observed in immediate reaction was not different from the 15-min reaction. summary/conclusions: ih-500, an automated blood bank analyzer, was considered useful for automation of abo blood typing, and some observable abo discrepancies are expected to be mostly addressed by reexamining with immediate reaction mode using different red cell reagents. abstract withdrawn. background: abo blood group antigens mainly expressed on red blood cells, but along with that they also present on many organs and tissues like epithelia, platelets, vascular endothelia and neurons etc. the importance of abo antigens extends beyond transfusion medicine by association with various systemic diseases like cardiovascular diseases, gastric diseases, cancers, infectious diseases etc has been proven previously. previous researchers also tried to find out the involvement of abo antigens in neurological diseases like alzheimer's disease, parkinson diseases etc. but association with neurological tumours is less explored. aims: this study aimed to analyse the association of abo blood group antigens with neurological tumours. methods: a retrospective study in a tertiary care institute in india analysed the 2 years data from jan 2017 to dec 2018. the carcinoma patient's admitted in neurosurgical department during study period were included in our study. their diagnosis and abo blood groups were collected from hospital information system. data were analysed into microsoft excel 2016 and spss (version 22). results: during study period a total of 1970 patients with neurological tumours were admitted in our hospital. the blood group frequency of these patients were 20.91%, 37.51%, 32.74%, 8.83% for a, b, o and ab respectively. the common neurological tumours found in our study were glioma (33.55%) followed by pituitary adenoma (20.05%), meningioma (18.58%), schwannoma (8.98%), cavernoma (2.54%), neuroma (2.23%) and space occupying lesions (14.06%). the prevalence of abo antigens was almost similar in all neurological tumours except in neuroma. neuroma was found in 47.73% o group patients as compared to other blood groups which was found statistically significant (p < 0.05). summary/conclusions: in this study we tried to analyse the association of neurological tumours with abo blood groups antigens. we found there is no association of neurological tumours with abo blood groups because the prevalence on abo group in general population is almost similar in patient with neurological tumours except neuroma. neuroma group of tumours like neurofibroma, neuroblastoma, nerve tumours etc. were more common in o group of patients while in our population frequency of b blood group antigen (38.2%) is more common as compared to o blood group(33.4%). background: rhd and rhce represent homologous genes in head-to-head position on chromosome 1 (chr1, p36.11). they encode for the proteins rhd resp. rhce which compose together with rhesus associated glycoprotein (rhag), band 3 and ankyrin the ankyrin complex (ac) linking the red blood cell (rbc) membrane to aspectrin of rbc cytoskeleton (s.e. lux, blood, 2016). cooperatively, the proteins of ac are important for maturation and physiologic properties of rbcs. many proteins of the rbc membrane express blood group antigens on their extracellular surface and are therefore of concern in transfusion medicine. cepellini et al. described weakened hemagglutination reactions of rhd+ rbcs in the presence of an rhc+ antigen (cepellini et al, pnas, 1955) . we attempted to further elucidate the expression of rhd/rhag proteins in various rhce/rhce pheno-/genotypes using a sophisticated flow cytometry approach. aims: in this study, we investigated a flow cytometric method for measurement of the antigen-density of various rhce-phenotypes. methods: analysis was performed on a flow cytometer (facscanto ii, becton dickinson (bd)) using bd facsdiva software and identical instrument settings for all samples. optimized number of rbcs was incubated with saturating concentration of pe-conjugated anti-rhd antibodies brad-3/brad-5/fog-1 (ibgrl, bristol, uk). debris was excluded by rbc gating in fsc/ssc plot. quantibrite-pe beats (bd) were applied according to manufacturer's instruction to quantify the relative expression of rhd epitopes. in addition a representative number of samples from common phenotypes were assessed for expression of rhag using bric-69pe (ibgrl). results: a total of 146 samples from healthy blood donors with serologically defined rhcde phenotypes were included into this study (rr(21), r1r(20), r1r1(23), r2r(18), r0r(15), r1r2(27), r2r2 (22)). variant expression of rhd by different rhce phenotypes using brad-3-pe was shown. rhd was weakly expressed in presence of rhc antigen (cepellini effect). effect of rhd gene dose on rhd protein expression is mitigated by rhc/c genotypes. when only samples with molecularly confirmed phenotypes were assessed, the rhdce genotype predicts consistently the strength of rhd protein expression. outlier samples (3) were retrospectively genotyped and revealed rhdce genotypes as expected from the strength of rhd expression falsifying serological rhcde phenotypes. in contrast, rhe/e polymorphic site does not correlate with rhd expression. in addition, rhag protein is equally present across all rhcde phenotypes. similar results were obtained by using alternative anti-d antibodies such as brad-5-pe and fog-1-pe, although different antibody's avidity precludes quantitative comparison of antigen expression on rbcs. summary/conclusions: sophisticated facs methods reveal different expression of rhd on rbcs according to rhce/rhce phenotype/genotype. rhc/c polymorphic sites (c.48g>c, c.201a>g, c.203a>g of exon 1, exon 2 resp. and intron 2) are in linkage with rhd expression, confirming the observation by cepellini et al. in contrast, rhe/e (c.676c>g, exon 5) is not in linkage with rhd expression. based on epigenomic signature it is conceivable that altered transcription factor binding sites (tbs) of rhd mirrored by homologous rhc/c may cause variant rhd expression. rhe/e snp mirroring the homologous sequence of rhd in exon 5 is not recognised as tbs. in addition, although ac comprises all three rh proteins (rhd, rhce, rhag), their transcriptional regulations seem to be distinct. red cell reference laboratory, australian red cross blood service, perth, australia background: the rh26 antigen was first described when an antisera thought to contain a potent anti-c did not react with all c+ cells. these non-reacting c+ cells were classified as c+, rh:-26, and the antibody specificity anti-rh26. most polyclonal anti-c contain anti-c and anti-rh26. previous studies have shown 2 of 10 monoclonal anti-c reagents are actually anti-rh26. these reagents will not detect the c antigen where the red cells are rh:-26. aims: the australian red cross blood service investigated a phenotype discrepancy in a blood donor. the donor's historic phenotype c+ (r 1 r) was inconsistent with the current donation phenotype c-(r 1 r 1 ). we aimed to investigate the cause of the discrepancy so the donor could be assigned the correct phenotype, identify the root cause of the discrepancy and implement any corrective actions. methods: the donor's red cells were phenotyped with all available anti-c reagents as per the manufacturers product insert across both manual and automated testing platforms. following variable results and weaker reactions with some reagents, dna was extracted from the edta sample and was genotyped using immucor bioarray tm hea precise and rhce beadchip tm . targeted dna sequencing of rhd and rhce was also performed using the trusight tm one sequencing panel. a review of the historical phenotype results, including the testing platform and reagents used at the time was also performed. results: on the current sample the donor's red cells gave a 2 + reaction by tube with bio-rad seraclone â (2) [clone ms35] and immulab epiclone tm [clone anti-c reagents. the sample tested negative on the beckman coulter pk7300 using beckman coulter anti-c [clone 951] blood grouping reagent and tested positive (4) reaction on the immucor neo using immuclone â (1) anti-c [clone . immucor bioarray tm hea precise beadchip tm predicted a c+ phenotype and no variants were detected with the bioarray tm rhce beadchip tm . the trusight tm one sequencing panel genotyped the donor as rhd*01/*01n.01 and rhce*01.15/*02 with a predicted phenotype of c+, c+ w , d+, e-, e+, rh:55 (locr+), rh:-26. a review of the donor's historical records indicated the donor tested as c+ on the pk7200, which at the time was being used with an in-house bromelain preparation (sigma-aldrich) and diagast olymp pheno anti-c reagent [clone ms33]. summary/conclusions: results indicated the phenotype discrepancy was caused by the c+ rh:-26 variant associated with the rhce*01.15 allele. reagents containing clones ms-33 and ms35 correctly phenotyped the donor as c+, with the manual tube reagents showing a weaker reaction which may alert the operator to a possible variant which is important in the patient setting. the beckman coulter pk7300 and associated anti-c [clone 951] failed to detect the c antigen. this reagent appears not to detect the c antigen where it is associated with the rh:-26 phenotype, which is in contrast to the previous report by faas et al, transfusion, 1997 where it was demonstrated that clone 951 reacted with c+ rh:-26 bromelain treated red cells. abstract withdrawn. background: although serological rhd typing has always been challenging due to variation of techniques and variable sensitivity of anti-d reagents, most individuals are unequivocally typed as either rhd positive or rhd negative. however, variants of d (weak d and partial d phenotypes) may present typing difficulties. individuals with partial d (missing epitopes of the d antigen) must be typed as rhd negative as blood receivers, but as rhd positive, as blood donors. aims: the aim of our study was to evaluate the algorithm used since 2017 at ahepa university blood center, to resolve rhd typing problems among first time donors. methods: since automatic analyzers may type variants of rhd as rhd+, our practice is to routinely perform two different typing methods in first time donors: an automated microplate method on the neo analyzer (immucor) and the slide test, using a potent reagent (anti-d blend-immunodiagnostika). in case of negative, weak, slow or mixed-field reaction, further testing with an automated microplate weak d method [immucor-modified indirect antiglobulin (anti-igg) test] follows. the next step of the protocol consists of testing with the commercial id-partial rhd typing kit (bio-rad) comprising a panel of 6 monoclonal anti-d reagents, in an indirect coombs gel test assay. the patterns obtained with this kit can distinguish between d weak and partial d and can also differentiate between categories ii, iv, v, vi, vii dfr, dbt and dhar. the last step of our algorithm consists of molecular testing (immucor bioarray rhce and rhd beadchip assays) at the hellenic national blood transfusion center, in case of remaining uncertainty. results: we applied the above algorithm in 32 samples: a) by using the partial d kit, 23 samples were typed: four samples were characterized as "partial d" (3 dfr, 1diii) and 19 as "weak d". four of the weak d samples (all from women of reproductive age) were confirmed by molecular typing ("weak d type 1" three samples, "weak d type 4.0 or 4.3" one sample). b) the nine (9) remaining samples that showed atypical serological pattern, were sent for molecular testing, which characterized 2 samples as "weak d type 1", one sample as "weak d type 3" and another as "weak d type 11". results are pending for 5 samples. summary/conclusions: in our experience some partial rhds may be mistyped as rhd+ if the technologist does not inspect the pattern of the reactions and only takes into account the assignment by the automatic analyzer as d+ or d-. by use of our algorithm, serological characterization was sufficient to distinguish between weak d and partial d in 68,75% of cases. molecular typing was necessary in the rest. the integration of molecular techniques improves the quality and accuracy of d typing of blood donors. if applied to patients, it also allows administration of d positive blood without compromising safety to those carrying prevalent weak d types that have not been reported to produce anti-d. furthermore, it permits withholding rhig in case of pregnant women carrying such weak d types. background: rhd antigen is one of the most clinically significant blood group antigens. except d positive and d negative phenotypes, there are over 200 rhd variants, which represent as serologic weak d phenotypes (swd). patients with certain swd can make anti-d alloantibodies. by serology testing it is not possible to clearly distinguish among different swd. in croatian institute of transfusion medicine (citm) patients and pregnant females with swd are mostly reported as rhd negative and generally did not refer for confirmation, because molecular testing was not part of the algorithm. that remains the risk of shortages of rhd negative blood and overuse of anti-d immunoprophylaxis for pregnant females. according to uk guidelines patients with swd who are likely to require chronic transfusion support and females ≤ 50 years are treated as d negative and refer for confirmation of d type. people who are rhd genotyped as weak d type 1, 2 or 3 are not susceptible for rhd alloimmunisation. one study showed that in croatian population the most frequent variants are weak d type 1, 2 and 3. aims: the aim of this study is to estimate the prevalence of swd in patients and pregnant females and to find out serologic and molecular characteristics of swd referred for confirmation. methods: from 2013/01/01 to 2018/10/01 we analysed 119.845 samples of patients and pregnant females. rhd typing was performed by anti-d igm monoclonal reagents in direct agglutination micromethod on tango (bs232, bs226) (biorad, dreieich, germany), swing maestro [lmh 59/20 (ldm3) + 175-2 and th-28 + rum-1 + ldm1] and ih-1000 [lmh 59/20 (ldm3) + 175-2] (id-card, biorad, cressier, switzerland). cut-off value for tango was determined as ++ and for gel microtyping as +++. the samples with results below the cut-off were reported and treated as rhd negative, all except those which gave discrepant results at current testing or with historical data. these were sent to rhd genotyping for confirmation. dna extraction was done by qiaamp blood mini kit (qiagen, hilden, germany) and rhd genotyping by pcr-ssp kits ready geneweak d and ready genecde (inno-train, kronberg im taunus, germany). results: from 119.845 samples 300 (0,25%) were swd. 71/300 (24%) were referred to rhd genotyping. 55/71 (77%) samples were weak d type 1, 2 or 3, while 16/71 (23%) were weak d type 14 and partial d variants vii and va. serologic reactions with monoclonal igm anti-d reagents showed different pattern for weak d types 1, 2 and 3. clearly negative serologic reactions were given in 27/29 samples with bs 226 and bs 232, in 30/33 samples with lmh 59/20 (ldm3) + 175-2 and in 18/39 samples with th-28 + rum-1 + ldm1. summary/conclusions: the prevalence of swd in this study is rather low (0,25%). after rhd genotyping 77% of referred samples were finally reported as d positive. serologic determination of d variants is inconsistent and only rhd genotyping can resolve rhd status in swd. to define the permanent rhd status of swd female of childbearing potential and patients who are likely to be chronically transfused we will introduce rhd genotyping in the new algorithm. background: among all blood group systems, the antigens of the abo system are by far the most clinically significant. comes second in importance is the antigens of the rh system, which comprise d, c, e, c, and e antigens. another clinically relevant antigen is the k of the kell blood group system, which is known to be involved in both htr and hdfn. the distribution of the major blood group antigens, such as rh, and kell, is well-studied among populations of developing countries. in contrary, a relatively few studies have addressed their frequencies in saudi arabian population this is also the case in jazan province, where only two published studies have analysed the prevalence of abo and d antigens, while the frequency of other clinically important antigens, such as rh and kell antigens, is yet to be explored. aims: to determine the frequency of the following clinically relevant blood group antigens; rh(d, c, e, c, e) and k among saudi blood donors in king fahd central hospital in jazan province. methods: a retrospective, cross-sectional study was carried out in the blood bank of king fahd central hospital in jazan province. the red cell phenotyping records for blood donation of 3243 randomly selected saudi donors, who donated blood between january and june 2018, were reviewed to identify the prevalence for the following antigens: d, c, e, c, e and k. the hospital blood bank routinely performs rh/k1 phenotyping for all blood donation using either bio-rad or ortho diagnostic column agglutination technology (cat) platforms. phenotype frequencies were expressed as percentages. results: this study included a total of 3243 saudi voluntary as well as family replacement blood donors. the d antigen was found to be positive in 93.7%, while k antigen was positive in 11.1%. among other studied rh antigens, e was the most common (98.1%) followed by c (78.2%), c (70.3%) and e(26.9%). dce/dce (34.2%) and dce/dce (5.3%) were the most common phenotypes amongst d-positive and dnegative donors, respectively. surprisingly, dce/dce phenotype was significantly prevalent (11.9%) with almost 5 times higher frequency compared that reported in caucasians (2.0%). the rare phenotype dce/dce was found in 3 donors (0.09%), while dce/dce and dce/dce phenotypes were found in only one donor each. summary/conclusions: this study is the first to determine the frequency of rh and k antigens in saudi blood donors in jazan province. determination of the frequency of these clinically significant antigens in our geographical area will facilitate the selection of antigen-matched red cell units for transfusion in recipients with multiple alloantibodies. it will also help in the management of blood donation processes and planning the estimated need of blood stock of different blood group phenotypes to meet the patient's needs. abstract withdrawn. background: the gerbich (ge) blood group system includes several high-frequency antigens located on glycophorin c and d. with only few reports published on the clinical significance of antibodies directed against these antigens, it is unclear whether blood transfusions have to be antigen negative in the presence of an anti-ge antibody. the monocyte monolayer assay (mma) is an in-vitro method used to estimate the clinical significance of alloantibodies. aims: to illustrate the role of the monocyte monolayer assay (mma) in the transfusion management of a patient with an anti-ge alloantibody. methods: the clinical and transfusion history was retrospectively retrieved from the patient's medical records. serological investigations were performed by indirect antihuman globulin test. papain and trypsin treated cells were also used. the clinical significance of the antibody was assessed by mma. genomic dna was isolated from whole blood and the samples were further characterized by pcr. results: a 58-year-old male patient with lung cancer without previous transfusions was admitted (06/2009) for surgery. his hemoglobin was 13.2 g/l. an anti-ge antibody was detected and it was decided to transfuse ge-positive packed red blood cells (prbcs). however, no blood transfusion was needed. in july 2017, the patient was admitted for colon cancer surgery with a hemoglobin of 11,0 g/dl. the anti-ge alloantibody was still detectable and a ssp-pcr revealed the genotype ge*01.-03. an mma performed on the pre-transfusion sample revealed a monocyte index (mi) of 0.35% and the antibody was considered not to be clinically relevant. the mi was interpreted as following: 0-3% not significant; 3-5% inconclusive; >5% clinical significant. however, due to the clinical background of the patient it was decided to transfuse ge-negative prbcs, which were obtained from etablissement francais du sang (efs), paris, france. two days after surgery, the patient received 2 units of ge:-2,-3 prbcs without any transfusion reaction. one and a half year later (11/2018), peritoneal carcinomatosis, as a complication of colon cancer, was diagnosed. the patient's hemoglobin was 87 g/l and he had a passage disorder, symptoms of deterioration and an adynamia. based on the mma results from july 2017 indicating no clinical significance of the antibody, it was decided to transfuse ge-positive prbcs. in the following 16 days the patient received a total of 5 units of gepositive prbcs no immediate or delayed transfusion reaction were observed following these transfusions. two further mma's, performed on samples drawn on december 12 th and 16 th (12 days after transfusion of a total of three and two days after two further prbcs respectively), showed a mi of 0.8% und 1% respectively and the anti-ge antibody was considered still not to be clinically significant. summary/conclusions: we report the case of a patient with an anti-ge antibody transfused with ge-positive prbc. as ge-negative prbc are not available in switzerland and not easy to obtain internationally the mma can help in the decision on how to transfuse. in this case, the clinical course confirmed the mma-based prediction. transfusions of ge-positive prbcs were tolerated without signs or symptoms of immediate or delayed transfusion reactions. background: abo grouping discrepancies occur when the results of forward grouping are not corroborative to those of the reverse grouping. these may be due to weak subgroups of a and b, missing or weak abo antibodies or red cell alloantibodies. determination of correct abo blood group of a donor is essential for preventing abo incompatible transfusions and to avoid hemolytic transfusion reactions in the recipient. aims: to determine the frequency of abo discrepancies and their resolution to correctly identify the blood group of the donors. we also determined the frequency of 'weak d' positivity in rhd negative donors. methods: this was a retrospective study on donor samples collected from 1 st april, 2013 to 30 th september, 2015 (two and a half years). for discrepant samples, the abo and rhd grouping was repeated using tube technique using commercial antisera {anti-a, anti-b, anti-ab and anti-d (igm), anti-d blend (igm+igg), anti-h and anti-a1 lectins}. adsorption-elution testing was done for detecting weak subgroups of a and b. antibody screen (3-cell) and identification (11-cell) was done by gel technique (bio-rad, switzerland). 'weak d' testing in rhd negative donors was also performed by gel technique. antibody titration was done using tube technique. the donor details including name, age and the registration/unit number of the donation were also checked for all the discrepancies to avoid repetition while data analysis. results: we detected 104 (0.072%) abo discrepancies out of the total 144279 donor samples tested during the study period. out of these, 135043 (93.6%) were rhd positive. the most common cause of abo discrepancies was weak anti-b antibody (33/104; 31.73%), followed by weak anti-a antibody and weak subgroups of a (24/104 each; 23.07% each) and weak subgroups of b (5/104; 4.8%). the remaining 17.3% (18/104) discrepancies were due to agglutination with o cells in reverse grouping. the overall frequency of weak subgroups of a and b collectively was 0.02% ( background: detection of unexpected red blood cell (rbc) antibodies before transfusion is critical for prevention of hemolytic transfusion reaction. ideally, unexpected rbc antibody detection is carried out within 3 days after receiving a patient's sample. however, in some cases, retests could be performed after more than 3 days for evaluation of any transfusion reaction, quality control or research. therefore, it is necessary to determine the stability of antibodies after refrigeration or freezing for a certain period of time. aims: we carried out antibody identification test with fresh, refrigerated and frozen samples using automated analyzer ih-500 and manual tube methods to evaluate the stability of antibodies after storage and compare the results between the two methods methods: antibody identification tests were performed using ih-500 (bio-rad, 1785 cressier fr, switzerland) and manual tube methods. fifty samples showing positive results in antibody screening test by both methods were divided into three and tested immediately, 1 week after storage at 4°c and 1 month after storage at à20°c. the specificities and reactivities of antibodies at each storage state were recorded and compared between the two methods. results: specificities of antibodies identified were concordant between ih-500 and manual tube methods irrespective of the storage state. the results were as follows: anti-e/e+c, 15; anti-le a , 4; anti-di a , 4; anti-c+e, 3; anti-m, 3; anti-d, 2; anti-c, 1; anti-k, 1; anti-jk a , 1: anti-xg a , 1; unidentified antibody, 13; autoantibody, 2 cases. with regard to the changes in reactivity owing to storage, 26 (52%) samples (anti-e+c, 12; anti-m, 3; anti-di a , 3; anti-d, 2; anti-c+e, 2; anti-le a , 1; anti-c, 1: autoantibody, 1; unidentified antibody, 1) showed identical reactivities after 1 week and 1 month storage by both ih-500 and tube methods. however, 19 (38%) samples, comprising 12 unidentified antibodies, 3 anti-le a , 1 anti-c+e, 1 anti-e, 1 anti-e+c, and 1 autoantibody, showed decreased reactivities after storage in both methods. three samples, comprising anti-di a , anti-e+c and anti-k antibodies, showed increased reactivities after storage. one sample with anti-jk a showed increased reactivity only after 1 month storage, while one sample with anti-xg a showed decreased reactivity only after 1 month storage. higher reactivities were observed in all samples detected using the ih-500 analyzer than manual tube methods (p < 0.005, wilcoxon rank sum test). summary/conclusions: the specificities of unexpected antibodies detected by ih-500 and tube methods were the same in all storage states; however, reactivities were higher in ih-500 than in the tube method. twenty-six (52%) of 50 samples showed identical reactivities after 1 week refrigeration and 1 month freezing. nineteen (38%) samples showed decreased reactivities after storage; however, 12 (12/19, 63%) of them were nonspecific antibodies, unable to identify using commercial id panels. therefore, it is suggested that retests for evaluation of transfusion reaction, quality control or research could be reliably performed after more than 3 days, if stored appropriately in refrigerated or frozen states. abstract withdrawn. t gleich-nagel 1 , d huber-marcantonio 1 , n rufer 1 , g canellini 1 and c niederhauser 2 1 unit of transfusion medicine, interregional blood transfusion src, lausanne 2 laboratory diagnostics, interregional blood transfusion src, bern, switzerland background: a positive direct antiglobulin test (dat) is mainly found in patients with warm/cold autoantibodies or alloantibodies directed against transfused erythrocytes. the identification of antibodies fixed on red cells is important for the clinician, allowing the further evaluation of a patient's clinical situation including their current medication. in immunohematology the elution of a positive dat remains a tedious and expensive procedure. the blood transfusion service src (bts src) has derived a flow chart that indicates in which situation an elution of dat positive samples should be performed. in order to follow the bts src guidelines, it is mandatory to obtain additional data related to the patient's condition, such as haemolytic parameters and recent transfusion history. currently, our laboratory is not always able to apply the recommended flowchart, since information is often unavailable. aims: here, we performed a comparative study between the algorithm provided by bts src and our in-house strategy, which is based on the qualitative changes of a positive dat, without the need for additional patient and biological information. methods: details of dat positivity and the patient's transfusion history was taken from the software eprogesa (mak-system) and analysed in excel. we analysed a total of 3'753 dats and evaluated them for their positivity, whether an elution was performed or whether antibodies were detectable in the eluate. furthermore, we performed an additional analysis on those samples, that were derived from recently transfused patients (<14 days). results: a positive dat was found for igg and c3d in 421 out of 3'753 (11.2%) samples, a level similar to previous reports of positive dats for hospitalized patients. among these positive samples, 244 (57%) were eluted because of a qualitative change in their positivity according to our in-house algorithm. identification of warm autoantibodies or alloantibodies occurred in only 10.7% (26/244) of the cases. from the 161 patients transfused within the last 14 days and having a positive dat, 60 (14%) were eluted according to our in-house algorithm. the same samples would have been analysed if the swiss transfusion guidelines had been applied. however, this comparative study reveals a significant discrepancy in regards to overall sample numbers that should have been eluted according to the two algorithms (244 versus 60 samples). this is mainly due to the fact that the swiss transfusion based algorithm does not recommend an elution of positive dats from patients who did not receive a transfusion within the last 14-days, except if there is a significant clinical suspicion (e.g. haemolysis). summary/conclusions: this comparative study indicates that our elution-based algorithm was performed on all clinically relevant samples as recommended by the bts src guidelines. qualitative changes in the dat positivity represent our main parameter for selecting those samples to eluate. besides ensuring that no clinically relevant samples were missed, this strategy also led to a large number of unnecessary elution analyses. in conclusion, a significant reduction in the laboratory workload and economical savings arises if the relevant clinical information and patients history is known prior to laboratory analysis. background: novel anti-cd38 monoclonal antibodies, such as daratumumab (dara) and isatuximab, used in treatment of multiple myeloma, interfere with routine blood bank serologic tests. as part of the strategies to manage these patients, it is recommended to perform extended phenotyping to provide matched units (aabb association bulletin #16-02). many investigations have focused on the interference with iat for the screening and identification of underlying alloantibodies and how to overcome them, but less has been published on the potential interference with extended phenotyping techniques. aims: the purpose of this study is to compare different technologies to type the most important antigens in myeloma patients before and during the treatment with therapeutic anti-cd38 antibodies. vox sanguinis (2019) 114 (suppl. 1), 5-240 methods: edta-anticoagulated whole blood samples coming from 30 patients in different stages of treatment with daratumumab and 5 with isatuximab have been typed in parallel with dg gel microcolumn (grifols) and mdmulticard technology (grifols). the results are also compared with genotyping results obtained with id core xt (grifols). direct coombs, autocontrol and antibody screening has also been performed as complementary tests. results: the study provides that four patients had positive dat and/or ac before therapeutic cd38 antibodies treatment. in these cases, 6 of 7 negative antigens (fy / jk and/or s) turn to positive in gel technology but mdmulticard showed 100% agreement with genotype id core xt results. focusing in the data obtained during the treatment, 8 negative antigens were type as positive in gel technology (12% of the tests). mdmulticard agreed with genotype in 100% of the analyzed antigens. as complementary data, 13 of 66 patient-treated samples had dat or ac positive and 59 showed panagglutination. summary/conclusions: the results demonstrated that mdmulticard is an effective method to type cd38-directed cytolytic antibodies treated samples in addition to dat and or autocontrol positive samples. background: antibody titration is a semi-quantitative method to estimate the strength and concentration of antibodies present in plasma or serum sample. titration methodology should be validated together with clinical data to evaluate the relevance of the titer value in each application. the titer of an antibody depends on different parameters: the antibody concentration in the sample, the density of the corresponding antigen expressed on the red blood cells used, the affinity constant of the antibody-antigen and other parameters regarding the technique used (e.g. gel cards or tube test). gel cards technology reduces the intra and inter-laboratory variation in titration studies comparing with the tube technique. aims: to evaluate the suitability of dg gel coombs, dg gel anti-igg and dg gel neutral (grifols) for titrations using two sample volumes 25 ll and 50 ll. methods: twenty frozen plasma samples containing unexpected antibodies from different specificities (anti-jk a , -fy a , -k, -d, -e and -c) were titrated in dg gel coombs and dg gel anti-igg cards and 20 donor fresh plasmas with natural occurring antibodies (anti-a and -b) were titrated in dg gel coombs and dg gel neutral (saline technique). the titer of the antibodies was determined by testing two-fold dilutions of the plasma with selected red blood cells depending on the antibody tested. plasma samples were diluted in dg gel sol. selected red blood cells serascan diana, serigrup diana or donor blood were added into the card (50 ll at 0.8%). further, sample dilutions were dispensed into the card (25 ll or 50 ll). subsequently, cards were incubated 15 min, 37°c (coombs technique) and 15 min, 18-25°c (saline technique), centrifuged in dg spin and the results read. agglutination intensity was graded visually according to the instructions for use of dg gel cards. the reciprocal of the highest plasma dilution that gives macroscopic agglutination was interpreted as the titer. results: titers obtained with dg gel coombs and anti-igg (n = 80 titrations, titer ranged 0-256) were compared for each sample with unexpected antibodies. no differences were found between gel cards types (differences were ≤ 0.5 titer in the 98% of the cases). differences between dg gel coombs and neutral (saline technique) (n = 80 titrations, titer ranged 2-512) were observed when anti-a and -b antibodies were titrated using the same sample. the titer was similar or higher in coombs in comparison to the saline technique. coombs titers may be a mix of igm antibodies reacting at 37°c and igg antibodies. differences were > 1 titer in 35% of the comparisons and ≤ 1 titer in the rest of the cases (65%). comparing sample volumes of 25 ll and 50 ll in all cards (n = 160 titrations), higher titers were observed using 50 ll, as expected. differences were 1 titer in the 51% of the comparisons, <1 titer in 44% and > 1 titer in the 5% of the cases. background: autoimmune haemolytic anemias (aiha) are characterized by production of antibodies directed against red blood cells and destruction by the mononuclear phagocytic system or complement system. aiha observed in paediatrics is usually self-limiting and often precipitated by viral infections. in some, the condition is secondary to autoimmune diseases, drugs, infections or underlying primary immune deficiencies. appropriate immuno hematological evaluation to characterise the underlying autoantibody can help identify the type of aiha to aid in diagnosis & treatment of these cases. aims: retrospective analysis of immune-hematological evaluation, treatment and outcome of aiha in paediatrics. methods: patients aged 0-18 years, diagnosed with aiha, between april 2017-december 2018 (21 months) were included in this analysis. aiha was defined as positive direct coombs' test (dct) with anemia associated with corroborative evidence of haemolysis in the form of raised indirect hyperbilirubinemia, raised ldh, raised reticulocyte counts or red cell agglutination on peripheral smear. further monospecific dct and evaluation for the specificity of autoantibody was done for all patients using biorad gel cards and panel cells. steroids were given as first line in all; second line agents included cyclosporine and rituximab. red cell transfusion was given in those with severe anemia with cardiac decompensation. results: 10 patients were diagnosed during the study period with autoimmune haemolytic anemia. haemoglobin at presentation ranged from 2.5 to 9 grams/dl. the initial presentation was severe anemia in 7 children and mild-moderate anemia with thrombocytopenia (evan's syndrome) in 3. the trigger for haemolysis was infection in 4 children. rheumatological evaluation was performed for 5 children out of whom 2 were diagnosed as evolving lupus. primary immune deficiency evaluation was advised for 4 and one child was diagnosed as suffering from combined immunodeficiency. dat was positive in 9 out of 10 aiha patients as one of the infant had dat negative iga mediated aiha secondary to viral infection. two out of 9 dat positive cases had igg & c3d positivity on monoclonal dat testing whereas rest 7 had only igg coating the red cells. dat titration was more than 1:300 in 4 patients; where only 1 of these 4 patients had both igg1 and igg3 coating and rest 3 had only igg1. alloantibody screen was negative in all. specificity of autoantibody was found only in one case, which was against rh blood group antigen (anti e). all patients received prednisolone as the primary treatment. three children attained remission following a 4-6 weeks of steroids. in those who were steroid dependent, cyclosporine was used as the second line agent in 2 and rituximab was used in 3. out of these children 5 children are in sustained remission and off any medication, whereas the rest are on low dose steroids with cyclosporine. summary/conclusions: aiha is not an uncommon problem in children and can vary in its clinical severity. the proper diagnosis and management involves efficient immuno-hematological evaluation, as detailed characterization of the autoantibody coating the red cell is very important in guiding the clinician for management and prognosis. abstract withdrawn. background: drug-induced immune hemolytic anemia (diiha) is rare and has only been described once with dexchlorpheniramine (polaramine tm), an antihistaminic agent widely used in the treatment of a variety of allergic reactions. we report a case of diiha complicated with acute renal failure associated with antibodies to dexchlorpheniramine. a 64-year-old woman with no history of transfusion, was treated semimonthly with a combination of chemotherapy and targeted therapy for metastatic colorectal adenocarcinoma. her chemotherapy regimen consisted of oxaliplatin and 5-fu with leucovorin rescue (folfox). panitumumab (monoclonal antibody anti-egfr) was used as targeted therapy. premedication with dexchlorpheniramine iv was systematically given at the beginning of each cycle of treatment to reduce the risk of perfusion reactions mainly associated with panitumumab. the patient developed chills and febrile agranulocytosis during the first and second infusion respectively. the third infusion was not performed due to pyrexia, chills, general discomfort experienced by the patient at the beginning of chemotherapy. probabilistic antibiotherapy was administered and the patient recovered rapidly. during the next infusion (day 1), following premedication with dexchlorpheniramine, a more "impressive" reaction including all the above mentioned symptoms occured along with back pain and dark colored urine. the infusion was halted and no chemotherapy was delivered. bacterial infection at the implantable port was first thought to be the cause of this adverse event but was not confirmed. additional laboratory findings revealed biological signs of inflammation associated with iha and acute renal failure. the patient was treated with hemodialysis (day 5), two units of rbcs (day 6) and was discharged one week later in stable condition. dexchlorpheniramine was then suspected and samples collected on day 24 were sent for a diiha laboratory workup. aims: the aim of this study was to support a clinical diagnosis of diiha. methods: laboratory workup included direct and indirect antiglobulin tests (dat and iat). drug antibodies investigation was performed by incubating patient's serum and eluate from patient's rbcs in the presence of drug against normal donor rbcs that had not been previously treated with the drug (i.e., by the so-called "immune complex" method). control tests were performed in parallel. drug was diluted in pbs and tested at 1 and 5 mg/ml. results: dat was positive (anti-igg 2 + , anti-c3d 2 + ) and no unexpected rbcs antibodies were detected by iat in patient's serum and eluate without the in vitro addition of the drug. an antibody directed against untreated (titer 2) and enzymetreated (titer 8) normal donor rbcs was demonstrated only in patient's serum in the presence of the drug tested at 5 mg/ml by the gel method. the pool of normal sera did not react in the presence of the drug. summary/conclusions: the multi-drug treated patient described in this study was demonstrated to have dexchlorpheniramine dependent antibody detected by the "immune complex" method. the key to the diagnosis was the observation of positive dat with negative eluate tests which prompted a reexamination of the medications administered in temporal relationship with the hemolytic event. although rare, this case report should alert physicians to the need to investigate the possibility of dexchlorpheniramine induced hemolytic anemia in any patient who develop unexpected anemia after hematologic or oncologic procedures p-331 singapore, singapore, singapore background: daratumumab is a monoclonal antibody against cd38 used in the treatment of multiple myeloma and has been known to bind to cd38 on rbc's and interfere with indirect antiglobulin based serologic tests such as red cell antibody screens and crossmatch compatibility testing. in order to negate the interference of daratumumab, our reference laboratory follows the daratumumab protocol recommended by the aabb which uses dithiothreitol (dtt) treated reagent red cells in red cell antibody screening and identification test in patients known to have received daratumumab. aims: the objective of this study is to determine the impact of daratumumab in the turnaround time (tat) for red cell antibody screening and identification. methods: a retrospective review of the tat for red cell antibody screening and identification samples of patients known to be treated with daratumumab from october 2016 to december 2018 was performed. turnaround time is defined as the time the sample is received up the time the results were reported. the tat for routine red cell antibody screen and identifications were also reviewed during the same period and was compared with the tat of samples from patients treated with daratumumab. results: a total of 55 patients on daratumumab had samples sent to our reference laboratory for red cell antibody screen and identification during the study period. information on daratumumab treatment was not provided to the reference lab prior to the start of testing in 23 of the 55 patients while the use daratumumab was mentioned in the serology request form of the other 32 patients. the median tat for red cell antibody screen and identification is 212 min (range: 47-877) if information on daratumumab was provided prior to start of testing and 301 min (range: 133-1417) if information was not provided prior to testing. the median tat for routine testing is 198 min (range: 15-2245). using wilcoxon rank-sum test, turn-around time for antibody screening and identification for daratumumab treated patients was observed as statistically not significant when compared to routine samples (p value 0.72634). however, tat for serologic tests requests with appropriate medical history compared to the testing requests without relevant information was also observed to be significantly difference (p value 0.00694). summary/conclusions: there is no significant impact in the tat of red cell antibody screen and identification in patients known to receive daratumumab as compared to routine testing. however, there is a significant difference in the tat if information on daratumumab treatment is not provided prior to testing. this highlights the importance of providing the relevant medication information in the request form in order to prevent delays in testing and provision of blood to patients on daratumumab, which can result in improved organizational efficiency and have positive impact on cost and resource savings. background: daratumumab, an anti-cd38 monoclonal antibody, has been shown to be highly efficacious in the treatment of multiple myeloma (mm). cd38 is a glycoprotein highly expressed on plasma cells and, to a less extend, on the surface of red blood cells (rbc). when bound to cd38 on rbc, daratumumab interferes with the pretransfusion tests, with positive antibody screening and crossmatch. anti-cd38 interference is an important challenge as many mm patients will require blood transfusions during their treatment. dithiothreitol is a reducing reagent with multiple applications in blood bank testing. treatment of rbc with dithiothreitol irreversibly removes cell surface cd38 tertiary structure, avoiding the binding and testing interference by the anti-cd38 daratumumab. aims: to demonstrate the efficacy, safety and celerity of the protocol between the blood bank (bb) and haemato-oncology of our institutions, using just the crossmatch. methods: a retrospective research was used for the evaluation of the results obtained from the implemented protocol. this comprehends a previous contact by haemato-oncology that leads to a study of the patient before the beginning of daratumumab treatment, and consists in: abo/rhd grouping; rh and kell phenotyping, and other clinically significant antigens; antibody screening; and direct antiglobulin test. genotyping may be required for some patients who received previous blood transfusions. before the beginning of the therapy, a blood sample of the patient is sent to the bb to perform laboratory tests and frozen after. this frozen sample is used for crossmatching in patients that already started therapy, did not have a blood transfusion in between, and have a positive antibody screening and/or crossmatch. in further transfusions, in case of positive tests, the dithiothreitol-treated donor rbc is applied. the donor rbc antigens are always selected accordingly to patients negative clinically significant antigens, when transfusional support is needed. the laboratory tests are executed in gel column agglutination technique. results: since 2016, 32 patients were studied, from which 16 were transfused with 108 blood units, according to the protocol. there were no immunizations or adverse reactions to transfusion registered within the transfused patients, neither delay on the availability of blood units. patient blood sample collected and frozen prior to the beginning of the treatment, has shown to be a good strategy by reducing significantly the waiting time for the blood unit in the first transfusion. summary/conclusions: this protocol, which defines the communication among the involved professionals, has shown to be a secure and effective way of reducing interferences caused by daratumumab. it ensures the previous study of the patients and their transfusion with rbc respecting the patients negative clinically significant antigens. if not adopted, the mitigation measures described in this protocol, delays in the availability of the rbc requested and alloimmunizations, may and will possibly occur. a good communication between the bb and the haemato-oncology is crucial for a good time management when a transfusion is requested for these patients. three methods were used to resolve this dara interference. reagent rbc's were treated with dtt, which know to denature cd38 and then tested with patient plasma. allo-adsorption study was performed using a certain ratio of red cells to plasma. in addition, a selection of phenotyped cord cells were used as an antibody screening panel. results: dtt treatment of reagent red cells was successful at eliminating dara interference and allowing for the presence of underlying antibodies to be identified. in this case, underlying antibodies were not detected by using reagent dtt treated red cells or phenotyped cord cells. adsorption technique was ineffective at elimination the reactivity. summary/conclusions: dara is the first commercial fda-approved therapeutic monoclonal antibody used in treating multiple myeloma patients. • since cd38 is weakly expressed on normal red blood cells, dara attachment to red blood cells can interfere with pre-transfusion iat testing. • dtt treatment of reagent red blood cells and cord cells can abolish the interference of dara to test for the presence of underlying alloantibodies. • to prevent delays in issuing red blood cell units to patients, hospitals should send patient samples to be tested before receiving dara treatment to ensure that clinically significant alloantibodies are not being masked. background: antibody screening (as)is considered superior to antihuman globulin (ahg) cross match during pretransfusion compatibility testing. in spite of knowing the utility and superiority of as, it has not been adopted uniformly in india. therefore, scarce data is available from this subcontinent in terms of optimisation of red cell antibody detection during pretransfusion testing in form of "type and screen" aims: the main objective was to study the benefits of performing simultaneous antibody screening along with the blood grouping during the first hospital visit to the hospital. other objectives were to study the prevalence of clinically significant antibody among the indian population and to follow up the patients who were transfused antibody screen negative but cross match incompatible blood. we also studied some other relevant quality indicators related to efficiency of blood transfusion services methods: this prospective study was carried out at a tertiary healthcare centre in india between july 2014 and dec 2018 (54 months). the study protocol was submitted to institutional review board and permission was granted. blood grouping and as were done during patients' first hospital visit, which we called "type and screen". when the patient got admitted to the hospital and required blood transfusion, a blood request form was generated by the user and sent to blood bank. depending upon the results of antibody detection, further course of action was chosen. if patient was found to have no antibody, immediate spin test (ist) cross match compatible blood was issued and transfused. in such cases the procedure of ahg crossmatch testing was continued even after issue of blood. cases where ahg cross match test was found negative no further follow-up of the patient was done whereas when ahg cross match was found positive, patients were followed after the transfusion results: a total of 22888 patients were "type and screened". majority were from hemato-oncology, bmt, liver transplant, paediatric cardiac surgery, and medical icu units. clinically significant allo-antibody was detected in 145 patients and autoantibody was detected in 53 patients. alloantibody was detected mainly against rh and kell blood group systems. in diagnosed aiha cases, majority were in the form of warm aiha (58%) and 20% of aiha cases were having hidden single or multiple alloantibody. significantly higher proportion of patients in as positive group required blood transfusion than as negative group (45% vs 34%, p < 0.05). in both the groups, in planned cases, most of the time blood was issued immediately within the defined turnaround time except in 21 where either transfusion was delayed or surgery was postponed. it happened only in trauma or surgical bleed cases. expiry of blood was decreased significantly due to no usage of blood (1.2% vs. 5%, p < 0.05). during the period of study we obtained 10 cases where the ist cross match was compatible but the ahg cross match was incompatible. during follow up none of the cases demonstrated any sign of hemolysis summary/conclusions: in developing countries like us, optimization of as during pretransfusion testing increases operational efficiency and significantly decreases the expiry of blood. results: during the period when absc was performed on pk7300, 250,599 donation samples were tested and 4,895 (1.95%) were found absc positive. antibodies to red cells were identified in 250 donations out of 4,895 (5.11%) absc positive samples and in the rest, no irregular antibodies were detected. the prevalence rate for atypical antibody was 0.10%. the top 5 most frequent antibody specificities were: nonspecific cold antibodies (28.8%), anti-e (26.8%), anti-mi a (19.6%), anti-m (16.8%) and anti-le a (4.0%). a total of 225,124 donations were screened for atypical antibodies by ih-1000 and 2,275 (1.01%) were screened positive. among these, anti-red cell antibodies were identified in 1,239 samples (54.46%), which was significantly higher than those identified in pk7300 screened positive samples (p < 0.00001). the prevalence rate for atypical antibody as screened positive by ih-1000 and with confirmed red cell specificities was 0.55%, which was also significantly higher (p < 0.00001). the top 5 most frequent antibody specificities were: anti-mi a (55.3%), anti-m (21.1%), anti-le a (10.2%), anti-e (6.5%) and non-specific cold antibodies (3.6%). anti-fy b was detected in 7 cases, which would be missed detection by enzyme treated reagent cells on pk7300 system. summary/conclusions: the performance of the ih-1000 system using a 3-cell screening panel including one cell with mi(a+) expression and column agglutination technology with iat phase was superior in comparison with that of pk7300 in the context of higher sensitivity in detecting more true positive results and higher specificity in detecting more true negative and less false positive results. this has translated into the advantages of reduction in workload of reference laboratory in performing less antibody identifications in those false positive samples as well as enhanced transfusion safety by removing more irregular red cell antibody positive plasma-containing components from the issuable inventory, which may potentially lead to haemolytic transfusion reactions. the prevalence of irregular red cell antibodies of 0.55% in healthy blood donors in hong kong reflects more the true statistical figure. background: chronic red blood cell (rbc) transfusion is the upfront therapy for thalassaemia patients, however this therapy is featured by several adverse events including rbc alloimmunization. phenotype matched products transfusion policy can prevent alloantibody formation, but it makes routine transfusion more difficult for both the donor center and the transfusion service. a recent systematic review (franchini et al, blood transfus 2019) reported a rbcalloimmunization prevalence of 11.4%, with a higher incidence against rh and kell systems in thalassemia intermedia patients. aims: the aim of our retrospective study is to evaluate the rbc alloimmunization prevalence in thalassemia patients transfused in a single center over a 18 years period with limited phenotype matched rbc (rh and kell system antigens) units. methods: from 1990 to 2018 thalassaemia patients, with a minimum follow up of 1 year and transfused with more than > 10 rbc units, were included in our study. patients were studied for: blood group and rh / k phenotype determination, direct antiglobulin test (dat), irregular antibodies research (abirr). cross-match and detection of alloantibodies were performed using the indirect antiglobulin test by the column agglutination method. six-monthly dat and antibody screening were performed using the indirect antiglobulin test and enzymatic papain-treated rbc test. results: overall 57 patients (38 females, 19 males) were included in our retrospective analysis: 54 patients were affected by thalassaemia major and 3 by thalassaemia intermedia. rbc alloimmunization prevalence was 12.3% (7 patients): 3 patients were found to be positive for rbc alloantibodies, four with alloantibodies and autoantibodies. eleven alloantibodies were detected (1 anti-h, 1 anti-cw, 4 anti-e, 1 kpa, 1 anti-jka, 1 anti-jkb, 1 anti-m and 1 anti-lua). in 2 out of 3 alloimmunized patients we found an anti-e antibody reactive in enzymatic papain-treated rbc test only, in the third alloimmunized patient anti-kpa and anti-lua antibodies were detected, while in the remaining 4 patients, in which auto and alloantibodies were detected, a severe autoimmune hemolytic anaemia (aea) requiring therapy was diagnosed. in these cases the appearance of alloantibodies is concomitant with the presence of autoantibodies. among the 7 patients positive for alloantibodies, 6 were affected by major thalassemia and one by intermedia thalassaemia summary/conclusions: in our experience a limited phenotype matched rbc transfusion policy showed a rbc alloimmunization prevalence similar to literature data: 12.3% vs 11.4%; we didn't find higher alloimmunization prevalence in thalassemia intermedia patients may be due to the low patients number. we believe that introduction, in our department, of an extended-phenotype matched transfusion, including antigens of the main group systems (fy, jk, mns) and the main rare antigens (cw, kp, lu), could reduce the risk of red blood cell alloimmunization in thalassemia patients. abstract withdrawn. background: undoubtedly, preventing alloimmunization has an advantage over overcoming its consequences. however, the high cost of technical and organizational aspects of preventive measures requires their scientific substantiation confirmed by clinical and laboratory data. selection of donors of the rhesus (d, c) and kell (k) antigens for the red blood cell transfusions to hematological patients has been regulated in the russian federation since 1998. recipients with the phenotype c+c-transfuse red blood cell only with the same antigenic combination. for transfusions red blood cell obtained from k-negative donors are used. the compatibility of the donor and recipient with the antigens c, e, e, c w (rhesus system) and k (kell system) is additionally taken into account from april 2013. that is, transfuse red blood cell that do not contain antigens in the phenotype that are not in the recipient's phenotype. aims: to evaluate the efficiency of red blood cell donor selection using antigens of rhesus (c, c, e, e, c w ) and kell (k, k) systems for the prevention of the recipient alloimmunization. methods: immunohaematological studies using equipment and reagents of biorad (usa) were performed in 3642 patients of the hematology clinic. non-hodgkin lymphoma was diagnosed in 759 patients, acute leukemia in 600, multiple myeloma in 390, chronic lymphatic leukemia in 319, chronic myeloid leukemia in 218, aplastic anemia in 147, hemophilia in 105, myelodysplastic syndrome in 73, and other hematological diseases in 1031. the frequency of detection of antibodies to antigen c (0.25% vs 0.06%) and to antigen e (0.46% vs 0.12%) decreased four times. the frequency of detection of antibodies to the c w antigen has not changed significantly (0.10% vs 0.06%, respectively). selection of antigens c (rhesus) and k (kell) has been carried out in the clinic since 1998, therefore the immunization index for these antigens remained unchanged and amounted to 0.10% vs 0.12% for anti-c antibodies; 0.36% vs 0.36%for anti-k antibodies. alloantibodies to the antigens e (rhesus) and k (kell) were not detected for the entire observation period. summary/conclusions: research verified the effectiveness of alloimmunization prevention of recipients by selecting red blood cell for antigens c, c, e of the rhesus system and k (kell). the study concluded that selection of red blood cells for the antigens c w , e (rhesus) and k (kell) does not affect the level of alloimmunization of patients and is not clinically justified. background: in the russian federation, there is an order according to which patients requiring multiple transfusions, who are at high risk of immunological complications are to typed for red blood cell antigens: abo, d, c, c, e, e, cw, k, k. selection of erythrocyte-containing blood components is carried out taking into account the donorrecipient compatibility according to all the listed antigens. aims: analysis of results of immunological evaluation of patients of hematological clinic. methods: the study included 466 first time patients of hematology clinic in 2017-2018. typing of antigens of abo, rhesus, kell systems, screening and identification of antibodies were carried out using equipment and reagents from bio rad (usa). results: interpretation of results of immunohematological screening was complicated in 84 (18.0%) patients. the total number of complex cases was 96. the double population of red blood cell was most often determined in antigens of the rhesus system (10.9% of the total number of patients) as a result of previous transfusion therapy. of those, chimera for the antigen e was detected in 31 cases (60.8% of patients with the chimera for rhesus and kell antigens), cin 23 (45.1%), sin 15 (29.4%), e -5 (9.8%), cw -8 (15.7%), k -5 (9.8%). in such cases, donor red blood cells were chosen not carrying chimeric antigen for transfusions, in the presence of chimeras in both paired antigensred blood cell transfusion with the cc phenotype and / ee. chimera for abo antigens was detected in 0.6% of the examined individuals. the discrepancy between the direct and reverse blood grouping of the abo system in patients (1.1%) was due to a decrease in the production of antibodies -4 cases and the appearance of extra agglutinins -1 case. autoantibodies were detected in 3.9% of all patients, including 0.6% of patients, when they caused panagglutination phenomenon. upon detection of autoantibodies that complicate the individual selection of donors, transfused red blood cells that are compatible with antigens of abo, rhesus, kell, duffy, kidd, mns systems. alloantibodies were detected in 2.8% of patients, including specific anti-din 4 (0.86%), anti-dcin 2 (0.43%), anti-kin 1 (0.21%); antibodies of unidentified specificityin 2 (0.43%), polyspecificin 4 (0.86%). summary/conclusions: the complexity of interpreting immuno-hematological tests in hematological patients is due to intensive transfusion therapy, changes in red blood cell antigens and appearance of nonspecific antibodies due to underlying disease. red blood cell for transfusion in these patients should be selected taking into account the expanded red blood cell antigen profile. abstract withdrawn. background: blood transfusion is an essential part of therapy for many patients. although life-saving for many patients, blood transfusion is not without risk. the main goal of blood transfusion services is that transfused blood should be compatible with the patient. the clinical and serologic evaluation, which allows for the transfusion of the most compatible (or "least incompatible") blood, requires a joint effort between the clinician and the transfusion medicine physician. aims: root cause analysis of incompatible cross matches in patients. methods: in this prospective study, total of 3,49,497 crossmatches were performed over period of last four & half years, out of which 867 units were found incompatible by column agglutination method-cat in polyspecific (anti-igg+ c3d) gel media. a root cause analysis protocol was formulated to resolve incompatibility to ensure safe transfusion. results: on the evaluation of 3,49,497 crossmatches, only 867 units were found to be incompatible (0.14%). the major cause for incompatibility found in patients was aiha-(32.87%). other causes of incompatibility were infections (27.44%), multiple transfusions (17.41%), trauma (11.23%), evan's syndrome (4.15%), rh negative mother (3.57%), sca (2.99%) & incompatibility due to dat positive packed red cells (0.34%).the most common antibody found were anti-'c', anti-'s' & anti-'m'. summary/conclusions: the rca protocol involves a thorough evaluation of the patient's clinical condition and underlying pathology to identify the cause. a logical stepwise approach will enable provision of safe transfusion to the patient. background: antibodies to high-frequency antigens (hfas) are a transfusion hazard, as compatible blood is often very difficult to obtain. other clinically significant alloantibodies represent an additional transfusion risk. in patients treated with allogeneic bone marrow transplantation (bmt) recipient red cell alloantibodies may cause acute or delayed haemolysis of donor red blood cells (rbc) and contribute to morbidity and mortality. aims: the aim is to present the case of a patient with myelodysplastic syndrome (mds), multiple "common" alloantibodies and an additional alloantibody to a highfrequency antigen, treated with allogeneic bmt. methods: a forty-one-year-old caucasian patient with mds (raeb-1) was admitted to our hospital in january 2017 for unrelated allogeneic bmt. she previously received myeloablative conditioning therapy according to the flu / bu4 / atg protocol (5 days of 250 mg iv. fludarabine, 4 days of busulfan 792 mg iv, 2 days of 300 mg iv. antithymocyte globulin). the indirect antiglobulin test (iat), done in august and december of 2016, was negative. according to anamnestic data, the patient had two pregnancies. she received red cell transfusions during childbirth and platelets in december 2016. results: the patient's blood group was o rhd positive, iat positive. the donor blood group was a rhd positive, iat negative. phenotype of the recipient's rbcs, as well as the donor rbcs, was also determined. anti-e and -c w were found in the patient's plasma, but an additional alloantibody was suspected. the autocontrol was negative. column agglutination technology (cat) and tube technology were used to identify rbc antibodies. plasma was tested with pheno-matched rbcs, papain-and 0.2 m dithiothreitol-treated rbcs, as well as cord and autologous rbcs. adsorption and elution tests were done, excluding other "usual" clinically significant alloantibodies, and the patient received three incompatible (xm in iat, cat) yt(a+), e-, c w -, k-red cell units. the sample was urgently sent for an antibody investigation at the international blood group reference laboratory (bristol, uk). in the reference laboratory, anti-e, -c w and an alloantibody to a high-frequency antigen were confirmed, whose specificity was determined to be anti-yt a . anti-jk b was also suspected and later confirmed. before the patient was discharged from the hospital, she received eight more red cell units (yt(a+), e-, c w -, jk(b-)), during which she was serologically closely monitored. summary/conclusions: the results of the antibody investigation in this case study indicate the presence of multiple alloantibodies in a patient who has previously received immunosuppressive myeloablative conditioning therapy. in addition to the "common" alloantibodies (anti-e, -c w , -jk b ), an alloantibody to a high-frequency antigen (anti-yt a ) was detected in the patient. this patient was transfused with incompatible red cell units (yt(a+)) in an emergency, with no ill effects. although anti-yt a is rarely a clinically significant antibody, according to literature, it can cause immediate haemolytic transfusion reaction. additional risk were "common" clinically significant alloantibodies, especially anti-jk b , which was in this case extremely difficult to detect and had further complicated the selection of blood. background: the identification of an antibody against a high-incidence antigen always introduces a challenge due to the difficulty in finding compatible units of red blood cells (rbcs). in patients needing surgery it is important to minimize their transfusional needs by implementing patient blood management programs (pbm). tests that predict the clinical significance of antibodies, such as monocyte monolayer assay (mma) are also useful in guiding clinical decisions. kell blood group system contains highly immunogenic antigens. antibodies against these antigens are immunoglobulin g, and can cause severe hemolytic transfusion reactions and fetal anemia. results: case report we report the case of a 75-year-old female, with non-hodgkin lymphoma, chronic anemia and scoliosis with severe neurological compromise, proposed for lumbar spinal stabilization surgery. she had a total hip replacement surgery in 2004, with unknown transfusion history. her obstetric history was g6p4a2. the patient had no history of thromboembolic or hemorrhagic events. during pre-transfusional tests, she was typed as a 0 rr and had a positive antibody screening test. the identification studies were suggestive of an antibody against a highincidence antigen, so the surgery was delayed until clarification of these results. she was also referred to a pbm appointment where her hemoglobin was improved from 9.0 g/dl to 12.0 g/dl by administration of ferric carboxymaltose iv and darbepoetin sc. the patient was phenotyped as kp(a+b-) with anti-kpb, an antibody against a highincidence antigen (>99% prevalence worldwide). it is a rare antibody with variable reactivity, causing from none to moderate/delayed transfusion reactions. to access the clinical significance of this antibody, a mma was performed, resulting in a reactivity of 1.2%, suggesting no clinical relevance, however it could be altered after transfusion of kpb+ blood. in order to find compatible rbc's, several family members were phenotyped, however they were all positive to the kpb antigen. in portugal there were no 0 rr kp(b-) blood donors, as it is extremely rare, so we searched in the international rare donor panel (irdp) and two donors were found in spain. two units of compatible rbc's were requested prior to the surgery, which was performed successfully four months later without transfusional support. summary/conclusions: anti-kpb is a rare antibody that in some cases can cause hemolysis of the transfused kp(b+) red blood cells. the combination of kp(b-) and o rr, an extremely rare phenotype, presented a challenge in finding compatible rbcs. this case illustrates not only the complex transfusional and logistic problems that an antibody against a high-incidence antigen can pose, but also the importance of an efficient pbm programme to mitigate the transfusional needs in these patients. background: blood transfusion is an integral part of the supportive care for patients with sickle cell disease (scd). allo-immunization is a recognized complication to red blood cells transfusion (rbc) in those patients. this may result in difficulties in providing compatible blood, and may be associated with the risk of acute of delayed hemolytic transfusion reactions. aims: to describe transfusion management in a patient with scd who has multiple alloantibodies with difficulty in obtaining compatible blood, in order to highlight the importance and clinical consequences of this complication and suggest a possible management approach methods: an 18-years-old female patient with scd presented to our hospital with hemoglobin level of 4 g/dl secondary to acute splenic sequestration. she had a history of multiple previous admissions and many previous rbc transfusions. blood grouping and pre-transfusion compatibility testing were performed in addition to phenotyping of the patient's red cells. screening was done using column agglutination technique by automated machine (ortho; usa) and antibody identification was performed manually using commercial 11 cells identification panel. phenotyping for the patient was done using haemagglutination technique with mono-specific anti sera (bio-rad; switzerland). results: the patient was of group o rhd (positive). antibody screening was positive and antibody identification revealed probable anti-e and anti-fya with possible development of anti-k allo-antibodies, in addition to recent development of autoantibodies; giving pan-positive reactivity with the identification panels. phenotyping of the patient's rbcs was found to be r1r and k-negative. other masked allo-antibodies of undefined specificities were suspected and no compatible blood was found. the clinical condition warranted a blood transfusion, so least incompatible phenotypically matched rbc unit was released. the patient developed acute hemolytic transfusion reaction with drop of the hb level to 2.8 g/dl. despite screening hundreds of rbc units, no compatible units were identified, and no transfusion was given. the patient was managed conservatively using hydration, analgesics, hydroxyurea, erythropoietin, intravenous immune globulin (ivig), steroids, and rituximab. hb level increased to 8 g/dl in 2 weeks, and the patient was discharged from the hospital. the sample of the patient was sent to a reference lab (institut fur klinische chemie und laboratoriumsmedizin-regensburg -germany) for further investigations, clarifications and advice for compatible transfusion in case of need. the report of the reference lab revealed the development of additional anti-m and anti-s with confirmation of the presence of anti-fya, anti-k and warm auto-antibodies. phenotyping of rbcs was confirmed by molecular diagnostic testing done in the reference lab; as r1r, k-neg. summary/conclusions: finding compatible blood may be extremely difficult in patients with scd who develop multiple alloantibodies. it is therefore essential to perform an initial extended red cell phenotyping for the patients at diagnosis and to have on shelf ready phenotyped blood units for issuing to the patients, to minimize allo-immunization. transfusion may occasionally be avoided in allo-immunized patients, utilizing alternative options of treatment and reducing the risk of serious complications such as hemolytic transfusion reactions. background: red blood cell (rbc) antigens that are present on less than 1% of most populations are known as low incidence antigens and those present on more than 90% are known has high incidence antigens. the mns blood group system consists of 49 antigens carried on glycophorin a (gpa), glycophorin b (gpb) or on hybrids of these glycophorins. there are 35 low incidence and 10 high incidence antigens in the mns blood group system. an individual that is homozygous for gp.mur will be negative for the high incidence jenu (mns49) antigen. anti-jenu was first described in a thai patient with thalassemia where only 2 compatible units were found following screening of 3600 units. the jenu negative phenotype is a rare phenotype with an estimated frequency of 0.06%. a male patient with a history of previous transfusion presented with an anti-e and a weak auto antibody with no apparent specificity. a donor unit selected for cross match (group o rhd positive, c+e-c-e+, k-) was incompatible with a reaction grade of 4 + by column agglutination technology. the patient's sample and donor unit were referred to the red cell reference laboratory for investigation for a possible antibody to a low incidence antigen. aims: we aim to characterize the phenotype of the incompatible donor unit. methods: standard serological procedures were used to identify the antibody specificities in the patient's sample. blood group phenotyping of the patient and donor was performed by standard serological procedures. genotyping and zygosity testing was performed using polymerase chain reaction (pcr) high-resolution melting (hrm) assay. gp.mur is a gp(b-a-b) hybrid glycophorin resulting from a gene conversion event between gypa and gypb . this disruption to gpb impacts s expression. the donor was negative with anti-s moab (albaclone), positive with anti-s polyclonal (immulab) and negative with anti-s monoclonal antibody (immulab). this s and s phenotype was consistent with the previously reported examples of gp.mur homozygote jenu negative individuals. molecular testing was consistent with serology supporting gp.mur homozygosity and jenu negative phenotype. summary/conclusions: this donor has been added to our rare donor panel and their red cell donations are cryopreserved for future use in our rare donor frozen inventory. there is limited anti-jenu antiserum available to confirm the jenu negative phenotype. we currently rely on the serological profile of red cells presenting with the gp.mur phenotype, s negative and the discrepant s phenotyping to identify jenu negative donors. this case has highlighted the importance of following up unexplained serological incompatibilities. the development of a monoclonal antibody directed against jenu antigen would provide an opportunity to screen for suitable donors for this rare phenotype. background: molecules expressed on tumor cells are a target of interest for drug development by the use of monoclonal antibodies or blocking proteins. however these drugs have the potential to interfere in pretransfusion testing when the target molecule such as cd47 is also expressed on red blood cells (rbcs). recently, many drugs targeting cd47 have been developed but appropriate mitigation strategies and approach to selecting rbcs for safe transfusion is still an obstacle. aims: we describe a case of delayed hemolytic transfusion reaction (dhtr) by anti-jk a in a patient treated previously with cd47 targeted high affinity sirpa fusion protein alx148. methods: a 35-year-old woman diagnosed with nasal cavity squamous cell carcinoma was enrolled in an alx148 clinical trial. her blood type was group ab, rhd positive, and the antibody screening test was negative for the past 8 months. she had no previous transfusion history during the past two years. after two infusions of alx148, two units of apheresis platelets were requested for transfusion. the blood bank noticed that the antibody screening was positive and further investigation was proceeded. results: antibody screening showed trace positivity in both panel cells (i & ii) at room temperature (rt) and 37°c albumin phase, and 2 + at anti-human globulin (ahg) phase by tube method. the auto control was negative at rt and 37°c albumin phase, but 2 + at ahg phase. antibody screening (2 cells) and identification (11 cells) all showed 3 + at ahg phase using gel cards. direct antiglobulin test was 4 + for anti-igg and 3 + for anti-c3d using gel cards. two units of rbcs were requested for transfusion after hemoglobin decrease to 7.8 g/dl. rbc genotyping was unavailable at the moment. as her previous antibody screening was negative (anti-jk a not detectable), e-, c-, fy b -rbcs were given as a second best option, considering the phenotype distribution of major blood groups in the korean population. the hemoglobin level was well sustained between 11.1-12.0 g/dl but it decreased again to 9.2 g/dl twenty days after rbc transfusion. further laboratory investigation was consistent with a dhtr. the patient was no longer being given alx148, and antibody screening and dat decreased to 0-1+ reactivity. we presumed that antigen typing results would be reliable after chloroquine dissociation and cell washing using antisera that did not require ahg for testing. serologic phenotyping showed that the patient's cells were c+, e+, c+, e+, jk a -, jk b +, fy a +, fy b -, s-, s+, m+, n + . antibody identification using papainized panel cells revealed anti-jk a antibody. we concluded that the dhtr was due to anti-jk a , and jk a -, fy b -, s-rbcs were issued for further transfusion requests. the patient's hemoglobin level recovered to 13.6 g/ dl. the patient's genotype was later identified to be the same as serologic typing. summary/conclusions: communication with the physician and blood bank to perform adequate pretransfusion testing before administration of drugs targeted to cd47 is important to achieve safe transfusion for patients. serologic phenotyping using antisera which do not require ahg for testing can be used as a second option when genotyping is unavailable in a timely manner. background: transfusion is still a key treatment for sickle cell disease (scd) patients. as a result, these patients are much more exposed to transfusions' risks, the most feared one being a delayed hemolytic transfusion reaction (dhtr). we investigated a female scd pediatric patient with no known antibody, who was referred to us for a suspicion of two dhtrs. three transfusion episodes were reported (a total of four units collected from four donors). for the last transfusion, a premedication with rituximab was done. the patient was planned to undergo a bone marrow transplant with her brother as her donor. aims: to describe the molecular and serological workups needed to investigate a dhtr in a scd patient. methods: antibody identification and crossmatches were performed by iat gel testing with red blood cells/panels, which were used raw, papain-treated and trypsintreated. rbcs' phenotypes were determined by conventional techniques. semi-quantitative phenotypes were conducted by serial dilutions with a monoclonal anti-jk a (ms15/seraclone â ). dna was extracted using the magna pure compact instrument (roche). sequencing of jk exons 4-11 was carried out by in-house techniques. results: the antibody identification showed a very weak anti-jk a , which was only reactive on papain-treated rbcs. autologous control was also only positive in this technique. dat and the eluate were negative. as the patient had recently been transfused (less than four months earlier), on this first sample we were neither able to perform autologous adsorptions, nor verify her jk a /jk b phenotypes. in order to rule out the imputability of an anti-lfa in the dhtr outcome, crossmatches with her donors' rbcs were undertaken. three out of the four donors were tested. apart from the anti-jk a reactivity, none of them was reactive. because the patient had previously been phenotyped as jk(a+b+), her jk gene was sequenced. her genotype was determined as jk*01(28a,226a,303a,588g)/jk*02. to confirm this jk a variant allele, a family study was conducted. all her siblings were found to harbor the same genotype. her mother's and father's genotypes were jk*01(28a,226a,303a,588g)/ jk*01 and jk*02/jk*02, respectively. subsequently, autologous adsorptions were performed, which proved the anti-jk a to be an autoantibody. considering the weakness of this antibody, internal controls were used, in order to evaluate a possible dilution effect of this technique. finally, serial dilutions with the anti-jk a reagent showed a weakened jk a expression encoded by the jk*01(28a,226a,303a,588g) variant allele. this finding is consistent with the fact that the crossmatches between the proband's serum and her brother's rbcs were weaker than those performed with (jka+b+) rbcs. summary/conclusions: about a third of dhtrs are reported to happen in patients with no previous history of immunization. performing sensitive serological techniques in order to identify antibodies is necessary to select the most appropriate units. molecular work and extra serological testing can be useful to determine whether an antibody is an allo or autoantibody. even though in this case the anti-jk a was the only antibody identified, because it was proven to be an autoantibody, it is difficult to conclude if it was the cause of the dhtr. nevertheless, jk(a-b+) blood was issued, and no adverse events have been reported. luckily, the patient's bone marrow donor harbors the same variant allele. background: according to the aabb, a pre-transfusion sample must be obtained within 3 days of transfusion if a patient has been transfused or pregnant in the preceding 3 months. despite this safeguard, high risk patients (i.e. those recently transfused with a history of pregnancy or transfusion) may develop antibody during this 3 day window. to avoid issuing incompatible red blood cells (rbcs) to these patients, a new antibody screen (abs) sample should be drawn and tested shortly before anticipated transfusion. aims: we report a case of a 60 y/o man who presented to the ed (hospital day 0, hd0) with a post-fall intracranial hemorrhage and multiple fractures. anti-e and anti-jka were identified after admission on a new specimen prior to current specimen expiration (<3 days). methods: specimen #1 (s1) was sent on hd0 for type & abs (t&s) and crossmatch (xm) of 2 rbcs. abs and immediate spin xm were negative; there was no patient history. by hd9, he had 4 negative t&s specimens (hd0: s1; hd2: s2&3; hd6: s4) and had been transfused 4 rbcs (hd2: 2; hd5: 2) via electronic xm (exm). at 1730 hr on hd9, 2 rbcs were requested and could have been issued via exm since s4 was not expiring until midnight. however, given recent transfusions, bb staff first called the patient's nurse to review history. patient was uncommunicative, but had scars suggesting past trauma or surgery. s5 was requested and received at 1801 hr. results: s5 showed anti-e and anti-jk a in plasma and eluate. his hemoglobin/hematocrit (h/h) decreased from 10.2 (14.0-17.5 g/dl)/30.1 (41.5-50.4%) on hd6 to 6.9/ 20.6 on hd9. during this period, he underwent several surgeries without unexpected bleeding, documented jaundice or dark urine. two e-jk(a-) rbcs were transfused on hd9, which he tolerated well with an increase of hemoglobin from 6.9 g/dl to 8.6 g/ dl. he did well post transfusion with stable h/h between 8.1/24.2.0 to 8.5/25.3. he was discharged on hd19. repeat abs on s4 was negative. of the 4 rbcs transfused before s5, one was e+ and four jk(a+). the family reported that he was injured 20 years prior and had been admitted to 3 hospitals, but was unaware of transfusion. hospital #1 (h1) reported admissions 20 years ago (2 rbcs transfused) and 4 years ago; all abs were negative. h2 admission was 5 years ago with positive abs and inconclusive workup. h3 admission 4 years ago showed negative abs. summary/conclusions: the patient developed a significant antibody response in less than 3 days from the specimen collection, likely a secondary immune response to sensitization from a transfusion 20 years earlier. a new specimen was requested prior to transfusion even though the existing sample (which was abs negative) had not expired. this approach identified new antibodies, preventing transfusion of incompatible rbcs, and a potentially serious hemolytic transfusion reaction. this case suggests that for high-risk patients, abs more frequently than every 3 days may be beneficial. it is important to increase clinicians' and laboratorians' awareness of this issue. background: red cells with partial d antigen have historically been classified as such, based on the fact that the red cells type as d positive, but individuals make anti-d antibody when exposed to conventional d antigen. a definitive confirmation of the variant of d antigen is obtained after the rh d genotyping. aims: to present a case study of the patient's alloimmunisation with the present d partial antigen type dnb, most likely on previously received transfusions. methods: the patient's pretransfusion testing included the determination of the abo blood group and rhd type (id card diaclon abo/d dv+, dv-, reverse grouping, monoclonal antibodies, gel method), antiglobulin crossmatch, additional phenotyping (gel and tube methods), antibody screening, identification of the specificity of irregular anti-erythrocyte antibodies by commercially available red cell panels (id dia-panel bio rad gel method, panocell immucor, tube method) through an indirect antiglobulin test (iat) and enzymes. after routine rhd typing we continued further characterisation of the rhd antigen by serologic assay (bio-rad id-partial rhd typing),and finally by rhd antigen molecular genotyping (fluogene method on fluo vista machine). results: our patient is a 75 year old woman with a diagnosis of tu mammae who was preparing for total mastectomy surgery. she had a history of blood transfusions twenty years ago, and she also had two births. the blood group typing was: o, ccdee, k-, fy (a-b +), jk (a+b +), ss, mn, le (a+b -). the agglutination reactions that we tested with anti d serums were strong (4+). the compatibility test with rhd positive donated blood units was positive. the presence of anti-d and anti-fya antibodies in the serum of the patient was determined. we prepared one compatible blood unit, rhd negative and fya negative, for a surgery. interpretation of the id-partial rh d typing set indicated that this is a diii category of d partial antigen. a sample of blood of our patient was sent to the blood transfusion institute of serbia, where molecular typing of d antigen was performed and the presence of partial form of antigen d, dnb type, was found. summary/conclusions: rhd positive patients or donors with anti-d antibody presents in their serum should be tested for d genotyping. the recommendation for further transfusions of our patient with dnb d partial and her alloimmunisation is to prepare d negative, fya negative erythrocytic blood components, and as a possible blood donor it would be labeled as rh d positive. background: the jr blood group system consists of jr a (jr1), a high frequency antigen expressed by the abcg2 gene. the individuals with jr(a-) phenotype are mainly found in the japanese population and may develop anti-jr a when stimulated by blood transfusion or pregnancy. anti-jr a is a dangerous antibody for pregnancy, but also could cause mild or moderate neonatal jaundice. aims: to conduct the antibody specificity identification of the high frequency antibody in a pregnant woman with history of pregnancy but no transfusion. methods: abo, rhd and some special blood group antigens were identified by tube method in saline. antibody screening and blood group specific antibody identification were performed by indirect antiglobulin test (iat) in gel column. the reagent cells treated with trypsin, chymotrypsin and papain, were used to test the antiserum to obtain the characteristic of antibody reaction. the antibody titer in the patient's serum was detected. dna sample was extracted and 16 exons and adjacent intronic sequence of the abcg2 gene were sequenced. the sample of one family member was collected for testing. results: the blood groups of the patient were b, rhd(+), lu b (+) and kp b (+). the negative reaction of the serum reacted with all reagent cells were tested in saline, but positive (2+) in iat test, while the self-control was negative. the antiserums reacted strongly (4+ in iat test in gel card) with the papain-treated cells, but kept the same reaction strength (2+) with trypsin-and chymotrypsin-treated cells, which indicated the possible existence of anti-jr a . the titer of igg antibody in serum was 2. in cross matching test, the red blood cell of the patient's brother with the same abo and rhd blood group with the patient was successfully matched with the serum of the patient. the sequencing analysis of the abcg2 gene in the patient and her brother revealed one homozygous nonsense mutation in exon 4 (c.376c>t, p.gln126x). after the delivery of the pregnant women, no pathological jaundice was seen in the newborn. summary/conclusions: in the condition of the anti-jr a reagent was unavailable for the identification of jr a antigen in the patient, having an indication with anti-jr a by serological test, the alternative genotyping method was used. the most common silencing jr allele reported in asian population, especially in japanese population, was identified to indicate jr(a-) phenotype. immunohemotherapy, centro hospitalar vila nova de gaia/espinho, vila nova de gaia, portugal background: if the investigation of irregular/unexpected antibodies reveals a pattern in which all or most screen and panel cells are positive, with reactions in the same phase and with the same strength, along with a negative autocontrol, an irregular antibody to a high-prevalence antigen may be suspected. high-prevalence antigens are those that are present in almost all individuals (98% or more). fortunately, because these antigens do occur so frequently, it is not common to find a patient with an antibody to one of them. however, when it happens, it may become a troubling situation. aims: clinical case report of panagglutination in assessment of irregular antibodies. methods: collection of clinical data in scl ınico â and sibas â applications. results: woman, 76 years old, o rhd+, previously transfused with 4 red blood cells concentrates in 2006, was proposed to a correction surgery of a periprosthetic hip fracture. pretransfusion serologic tests were requested and irregular antibodies were detected (2+ in all the 3 screening cells). in order to identify the specificity of the antibody, a panel of 11 cells was tested; the result was considered inconclusive, due to positive reactions (2+) with all test cells in liss/coombs and atypical positivity with dragging in all cells in enzymatic environment. autocontrol and direct antiglobulin test were negative. it was decided to send two blood samples to the reference laboratory for a more complete immunohematological study. compatibilization of red blood cells to this patient was also requested. during the waiting period, haematopoiesis was optimized. although the patient did not present anaemia at admission, the analytical study revealed iron deficiency; therefore, supplementation with intravenous iron was performed. the reference laboratory also obtained a panreactive panel (2+ with all cells) in liss/coombs and weak positivity in papain. after allo-adsorption, the search for irregular antibodies was negative. an anti-yt a , apparently without clinical significance (negative igg1 and igg3) was then identified. transfusion was not needed either during or after the surgery, with a good recovery of the haemoglobin value in the postoperative period. summary/conclusions: yt a , which belongs to cartwright system, is a high-prevalence antigen in all populations. anti-yt a , an igg antibody stimulated by pregnancy or transfusion, is not as uncommon as we may think, which suggests that it is reasonably immunogenic. these antibodies are not generally considered clinically significant, but there are reported cases of acute and delayed haemolytic transfusion reactions in which anti-yt a has been implicated. therefore, although the described pattern of panagglutination in assessment of irregular antibodies may suggest the presence of an alloantibody directed against a highfrequency antigen, it is very important to confirm that hypothesis, recurring to a reference laboratory if necessary, to identify the antibody and to determine its clinical relevance. even if the identified antibody is associated with rare haemolytic transfusion reactions, it is crucial to optimize haematopoiesis when it is not an emergent procedure, in order to minimize transfusion and its associated risks. both for emergent and elective procedures, the creation of a national database of patients with already identified irregular antibodies would facilitate the administration of red cells concentrates without the implicated antigen. aims: to investigate the frequency and explore the genomic characterization of jk (a-b-) phenotype in blood donors in harbin, china. methods: all samples were screened for jk(a-b-) phenotype using a direct urea lysis test. and the results were confirmed with by iat using anti-jka and anti-jkb with a standard tube test. additionally, polymerase chain reaction amplification and sequence analysis of the jk gene were performed. results: from 80865 blood samples, four donors with jk (a-b-) were selected, at a frequency of 0.0049%. among these four samples available for sequencing jk gene, a total of two genotypes were discovered: heterozygote of ivs5-1g>a combining with heterozygote of 359g>a (gly120glu) and heterozygote of 896g>a (gly299glu) combining with heterozygote of 956c>t(thr319met). summary/conclusions: the frequency of jk(a-b-) phenotype in blood donors in harbin area was lower than the reported data from the populations in other areas of china and in finland, but higher than that in japan. ivs5-1g>a, 896g>a and 956c>t were common mutations in the before reports, while 359g>a was reported first time. in addition, it is an effective measure which establish the jk(a-b-) phenotype donors in this region, to solve the blood transfusion problem in patients with anti-jk3. background: blood types, indicating the type of blood group antigen expressed in the red blood cells, is determined by the type of allele at the blood group gene locus. therefore, when allele frequency of each blood group gene is determined, it is possible to predict the frequency of a specific blood type donor with a homozygous allele. it is also possible to estimate the proportion of donors within a particular blood type through combination of specific alleles. and because the ratio of blood group allele differs between ethnicity and race, this can be used as basic data for population genetics and anthropology. therefore, we present a study that examined the allele frequencies of 10 blood group systems in the korean population through blood group genotyping. aims: the purpose of this study is to determine the frequencies of blood group alleles in the korean population, to predict the proportion of homozygous donors, and to obtain the basic data of population genetics. methods: 5,213 blood donors from age 19 to 54 were recruited at korean red cross blood centers located nationwide. acquired samples were examined by blood group genotyping methods using the rbc genotyping system id core xt (progenika biopharma). for each donor, genotypes of 10 blood group systems, excluding abo and rhd, were identified. calculation of the frequencies of blood group alleles in the korean population was done. results: we conducted molecular genotyping of the rhce, kell, kidd, duffy, mnss, diego, dombrock, colton, cartwright, and lutheran blood group systems. the allele frequencies of these blood group systems in the korean population were estimated as follows. -rhce*ce 0.3%, rhce*ce 65.0%, rhce*ce 28.8%, rhce*ce 5.9% -kel*k_kpb_jsb allele 100% -jk*a allele 49.1%, jk*b allele 50.8%, jk*b_null allele 0.1% -fy*a allele 92.8%, fy*b allele 7.2% -gypa*m allele 51.1%, gypa*n allele 48.9% -gypb*s allele 5.1%, gypb*s allele 94.8%, gypb*mur allele 0.1% -di*a allele 4.7%, di*b allele 95.3% -do*a allele 10.1%, do*b allele 89.9% -co*a allele 100% -yt*a allele 100% -lu*b allele 100% summary/conclusions: the significance of this study is accumulation of data on the allele frequencies of blood group genes through highly accurate genotyping method in the east asia region. this enables the prediction of the proportion of donors with a combination of specific blood group alleles in the korean population, which accounts for a decent percentage of the population in this region. background: in donors from arabian countries only little is known about blood groups other than abo and rhesus. during the last years increased migration to central europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. aims: blood group allele frequencies should be determined in individuals from syria, other arabian countries, and iran by molecular typing. methods: as most blood groups are defined by single nucleotide polymorphisms (snps) we introduced a maldi-tof ms assay to detect alleles encoding 16 blood groups including kk, fy (a/b), fy null , c w , jk(a/b), jo(a+/a-), lu(a/b), lu (8/14), ss, do (a/b), co(a/b), in(a/b), js(a/b), kp(a/b), rhce*c.697c>g, and rhce*c.733c>g. additional blood groups and polymorphisms like yt(a/b), s-s-u-, vel null , co null and rhce*c.667g>t were tested by pcr-ssp. a total of 1111 probands including 800 individuals from syria, 147 from iran, 123 from the arabian peninsula and 41 from northern african countries were included. results: 2% of the donors were homozygous for the fy null (fy*-67t>c, fy*02n.01) mutation, 16.2% carried the heterozygous mutation. 0.4% of the syrian probands were heterozygous for the do*350c>t mutation (both, do*jo1 and do*jo2; jo(a+/ a-)) that is virtually unknown in caucasian donors. 0.8% of the syrian donors heterozygously carried the kel*02.06 allele coding for js(a) (phenotype js(a+/ b+)) that is very rare in caucasians. however, no homozygous kel*02.06 carriers were identified. 1.8% of the syrian and 1.5% of all donors were negative for yt*a, which is definitely more frequent than in europeans. one donor from northern africa homozygously carried the gypb*c.251c>g, intron 5 + 5 g mutation, inducing the s-u+ w phenotype. 3.6% of all and 29.3% of northern african donors were heterozygous for the rhce*c.733c>g substitution, 0.3% of the syrian donors carried rhce*c.697c>g (heterozygously) and 0.004% of all donors were heterozygous for rhce*667g>t. heterozygosity for vel deficiency (vel*-01) was detected in 21 individuals (2%; 16 of them from syria) whereas only one syrian donor carried the homozygous mutation. none of the donors carried the aqp1*c.601delg (co*01n.06) mutation that induces the co null phenotype. summary/conclusions: the study provides a first overview on a number of different blood group alleles in blood donors from arabian countries. some blood group alleles that largely are lacking in europeans but had been described in african individuals are present in arabian populations at a somewhat lower frequency. in single cases it could be challenging to provide immunized arabian patients with compatible blood. methods: three unrelated individuals (2 blood donors and one pregnant woman) of polish origin who were typed as ab group with a very weak a antigen and normal b antigen expression were subjected to extended abo typing. in one case family studies were performed (blood samples from donor's mother, father and sister). sequencing analysis of this donor dna was performed three times (from two blood samples and buccal swab). serologic investigations were performed with standard methods: 1/gel cards diaclon id abo/d (anti-a: clone a5, anti-b: clone g1/2, anti-a,b: clone es131, es15+ birma 1+ es4; bio-rad) and diaclon id abd-confirmation for donors (anti-a: clone m297/628 = la-2; bio-rad); 2/tube techniques with: anti-a (birma 1; a-11h5, a1 s.pa1m095, c.9113d10), anti-b (lb-2, b-6f9, c.9621a8). genotyping was determined by rbc fluogene abo basic kit (inno-train, germany) and by sequencing of +7.21-kb site of abo gene to cover sequences ranging from the end of intron 1 to 3 0 utr of the abo gene. additionally sequence of exon 1 of the abo gene was analyzed. results: abo typing showed normal b and a very weak a antigens on rbcs of all three individuals (2 blood donors and one pregnant woman). the a antigen was detected by tube technique only using anti-a clones: birma 1 (1+ to 2+), a-11h5 (1+ to 2+) and c.9113d10 (weak+ to 1+); negative reaction of a antigen typing by gel cards was observed. the sera of all individuals contained anti-a1 antibodies. commercial pcr-ssp kit revealed three heterozygous a/b genotypes (absence of 1061delc typical for abo*a2 alleles). in all these individuals abo sequencing of 7.21-kb fragment confirmed the heterozygous genotype with 7 polymorphisms characteristic for abo*b.01 allele (297a>g; 526c>g; 657c>t; 703g>a; 796c>a; 803g>c; 930g>a) and detected a novel abo*a allele sequence with duplication-based insertion of 21 bp after 564 position (abo*a c.dup543_563; gcaggacgtgtccatgcgccg). as a consequence, the online protein translation predicts an in-frame duplication of seven amino acids after codon 188 (p.dup_182_188qdvsmrr), with synonymous change of the repeated codon 182 (cgc>cgg) and 188 (cgg>cgc) but both coding arginine (r). inheritance of abo*a c.dup543_563 allele was confirmed by family studies of one donor: his father and sister had a/b genotype associated with normal a and b antigens expression; his mother had normal a antigen expression. she carried abo*a1.01 allele and the same abo*a c.dup543_563 allele as a son. summary/conclusions: a novel a weak allele at the abo gene detected in three unrelated polish individuals is an in-frame insertion of seven amino acids to the wild-type glycosyltransferase a. the stability of the encoded protein may be affected causing the weak a phenotype. the inheritance of this mutation was confirmed in the family studies. background: since the cloning in 1990 of cdna corresponding to mrna transcribed at the blood group abo locus, polymorphisms and phenotype-genotype correlations have been reported by many investigators. although many subgroups have been explained at the genetic level, unresolved samples are still encountered in clinical practice. we report here the result of an abo investigation of a sample from a swedish blood donor that showed a very weak agglutination of rbcs with anti-a in routine forward typing. aims: to elucidate the genetic basis of the apparent weak a subgroup. methods: routine abo genotyping by pcr-asp and pcr-rflp including pcrbased analysis of the upstream cbf/nf-y-binding enhancer region was carried out. further genetic analysis was performed by dna sequencing of abo exons 1-7 (including 50 base pairs of the adjacent introns) and the proximal promoter. flow cytometric testing of rbcs was performed with monoclonal anti-a, anti-b and anti-h. results: the weak agglutination of erythrocytes with anti-a was accompanied by the expected lack of anti-a and anti-a1 in plasma. abo genotyping gave the genotype abo*a1.01/o2.01 usually consistent with normal expression of a antigen. enhancer analysis resulted in an amplification product corresponding to the expected single cbf/nf-y binding motif. flow cytometric testing of the sample showed a antigen expression with an almost chimeric pattern where the majority of the cells (approximately 85%) expressed the a antigen at a very low level, marginally distinguishable from the group o control. the remaining approximately 15% of the cells displayed an a antigen level ranging from normal to very weak. genomic abo sequencing showed an abo*a1.01-like allele except for a novel mutation located in intron 5, c.239+4g. the o 2 allele had an additional snp, c.689g>a, consistent with the abo*o2.03 allele variant summary/conclusions: a previously unreported variant, c.239+4a>g, likely effecting the 5 0 -donor splice site of intron 5 was found in an a weak sample. this type of mutations is expected to decrease mrna stability and/or cause skipping of the preceding exon in the mrna. however, small amounts of full-length enzyme might still be made, being able to give rise to the weak a antigen expression seen in this individual. interestingly, this mutation is very similar to the genetic variant underlying the weak a subgroup a finn . in this case, however, the c. 374+4a>g mutation is located in the 5 0 -end of intron 6 and is predicted to cause partial skipping of exon 6. strikingly, the a finn phenotype also results in a pseudochimeric pattern by flow cytometry but with only approximately 2% positively staining erythrocytes. due to the well documented lack of a-allele-derived mrna in peripheral blood, further transcript studies could not be undertaken. further studies are needed to investigate the exact mechanisms underlying the pseudochimeric pattern observed by flow cytometry in these two interesting genotypes/phenotypes abstract withdrawn. background: abo is the clinically most relevant blood group system in transfusion and transplantation medicine. abo genotyping is potentially useful in clarifying serologic blood grouping discrepancies. this scenario includes inherited subgroups alleles, temporary acquired variant abo phenotypes in disease or pregnancy, and chimerism due to exchange of progenitor cells early in fetal life or after blood progenitor cell transplantation. aims: to investigate the molecular basis for abo discrepancies detected in clinical samples, including donors and patients, sent to our reference laboratory during the past 3 years. methods: if routine abo grouping showed weak agglutination or forward vs reverse typing discrepancy, further abo typing studies were performed manually. adsorption-elution tests were also performed in some cases with polyclonal anti-a and anti-b to confirm whether a or b antigens were weakly expressed on the rbcs membrane. a pcr approach using sequence specific primers for a2, b, o1 and o2 alleles was used for initial genotype determination. the full abo coding region was analysed as previously described in selected samples for which abo discrepancy was still unexplained. allele specific fragments spanning exon 6, intron 6 and exon 7 were amplified using a forward primer targeting the 261g nucleotide (to exclude o1 alleles amplification) in combination with either b, a2 or a generic reverse primer. analysis was carried out by sanger sequencing. results: a total of 16 samples with suspected inherited abo subgroup alleles were selected for further molecular studies by sequence analysis. a subgroup alleles: in 8 out of 12 samples with suspected a subgroup alleles, the c.804insg insertion was detected corresponding to the abo*ael.01 allele. the abo*aw31.02-05 variant, a hybrid a1-o1v allele, was found in 2 cases. in 1 case we found the c.722g>c change, previously reported associated with weak a antigen expression. finally, a novel c.961c>g change was detected in an a2 allele. b(a) or cis-ab suspected alleles: the abo*b(a)04 variant carrying the c.640a>g change was found in 1 of 3 samples with bo1 genotype but a weak antigen expression. in the remaining 2 cases, a consensus b allele was detected, thus pointing to a potential chimerism as the cause of the results observed in abo grouping. finally, we have identified an abo*b01.02 allele carrying the nucleotidic change c.926a>g in the context of an abo phenotype vs genotype discrepancy. summary/conclusions: the sanger sequencing approach applied in this study have proved to be informative and helpful to determine the molecular basis of abo grouping discrepancies with suspected inherited subgroups. we found mutations, within exon 7 of the abo gene, in 14 out of 16 samples, including 2 novel alleles. chimerism was suspected in 2 cases of a antigen expression in samples with b1o1 genetic background carrying an apparent normal b allele. we are evaluating at the moment a deep sequencing approach by next generation sequencing to determine the presence of a small amount of a minor allele in the presence of a large surplus of the other two alleles. background: recently, the multiple pregnancy rate has been increasing due to advances in artificial fertilization including in vitro fertilization-embryo transfer. most dizygotic twins have dichorionic placenta, but 8% of them share the placenta. monochorionic dizygotic twins can have blood chimerism, leading to double rbc populations in routine abo serologic typing. recently, more sensitive and objective column agglutination tests with automated systems are being widely used. therefore, blood chimerisms in dizygotic twins can be detected more easily by routine abo blood typing. aims: we report congenital blood chimerism in monochorionic dizygotic twins of triplets, found incidentally during abo serological testing and confirmed by abo genotyping and str marker analysis. methods: a 20-year-old male (one of triplets) was admitted to the hospital for medical checkup. he did not have any history of transfusion or bone marrow transplantation. routine abo blood grouping test was performed using automated blood bank system ih-500; however, it showed abo discrepancy. the red blood cells showed double cell populations in a gel column with anti-a and anti-b. we carried out abo genotyping both from the blood and from a buccal swab. for the further evaluation, we performed abo serologic testing, abo genotyping, and str marker analysis in his family members. results: among the triplets, blood chimerism was demonstrated in the patient and his brother. they both showed a 3 b 3 phenotypes in the serologic test and tri-allelic abo genotypes in the blood, a102/b101/o01. however, in buccal swabs, the patient showed a102/o01 and his brother showed b101/o01. other members of the family (father, mother, and dizygotic sister) had regular abo blood types in the serologic test. we performed str analysis in the triplets and parents. eleven loci (d8s1179, d21s11, d7s820, csf1po, th01, d13s317, d16s539, d19s433, d18s51, d5s818, and fga) revealed more than one additional allele in the blood sample, apart from those in the buccal swabs. str marker analysis showed that his brother too had blood chimerism. summary/conclusions: we found blood chimerism in monochorionic dizygotic twins of triplets during routine abo blood typing, and this was confirmed by str analysis. as the application of assisted reproductive technology increases, the incidence of blood chimerism will also increase. blood chimerism can often create confusion during abo serologic typing and microchimerism can be overlooked in routine methods. therefore, it is helpful to use an automated blood bank system to improve sensitivity and blood chimerism should be considered if abo blood grouping reveals double populations. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial host. results: case #1 is a patient with an unclear abo phenotype: forward type b, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.29-5t>g in heterozygosity on an otherwise common a1 allele, and in trans an abo*b.01 allele. case #2 is a caucasian donor with an abo discrepancy: forward type aweak/o, reverse type a. sequencing also detected variant c.29-5t>g in heterozygosity on an a background, and in trans an abo*o.01.01 allele. given that this variant is located near the intron 1 splice acceptor site, abo*29-5g transcripts are postulated to undergo altered splicing, leading to an aweak phenotype. case #3 is a prenatal sickle-cell disease patient with an abo discrepancy: forward type aweak, reverse type a. dna sequencing detected variants c.467c>t (pro156leu) and c.709t>a (tyr237asn), both in heterozygosity on an otherwise common a1 allele, with an abo*o.01.01 allele in trans. thus, the data establish an association of allele abo*467t,709a with an aweak-like phenotype. case #4 is a donor with an abo typing discrepancy: forward type o, reverse type a. sequencing detected variant c.479c>g (pro160arg) in heterozygosity on an a2 background, and in trans an abo*o.01 allele. an interpretation of the data is that variant c.479c>g weakens the activity of the a2 transferase, with allele abo*a2(479g) encoding the aweak-like phenotype detected by serology. case #5 is a 9 year-old patient with an abo discrepancy: forward type o, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.803g>c (gly268ala) in heterozygosity on an a2 background, and in trans an abo*o.01.02 allele. the serology and molecular results suggest that allele abo*a2(803c) encodes a cisab weak phenotype. case #6 is a caucasian donor with an abo typing discrepancy: forward type o with a weak agglutination with anti-ab, reverse type o. dna sequencing detected variants c.739g>a (glu247lys) and c.871g>a (asp291asn), both in heterozygosity, in trans, and on a1 backgrounds. variant c.871g>a by itself constitutes allele abo*a3.01. the phenotype encoded by abo*739a is uncertain. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. results: case #1 is a 35 year-old pregnant female with an abo typing discrepancy: forward type o, reverse type a. pcr-rflp predicted abo*a1/abo*o1. sequencing detected variant c.107insg (val36gly>fs56ter) in heterozygosity on an otherwise common a1 allele, and in trans an abo*o.01.01 allele. it is unclear how the early truncation of the a1 transferase encoded by allele abo*107insg still allows for some residual enzyme activity, as suggested by the reverse a type. case #2 is a recently-transfused 72 year-old black patient with an unresolved abo type. sequencing detected variant c.297a>g (silent) in homozygosity and variant c.1049c>t (ala350val) in heterozygosity, both on an o1 background, with an abo*b.01 allele in trans. although variants c.297a>g and c.1049c>t are likely of no consequence to the abo phenotype of this patient, they are reported here as components of a new abo*o1(297g,1049t) allele. case #3 is a 40 year-old prenatal female with a rhd typing discrepancy. failure to yield an abo genotype on blood-chip (progenika), a genotyping microarray that interrogates polymorphic positions in rhd and abo, prompted dna sequencing. sequencing of genomic dna and cloned abo exon 7 detected variant c.436c>t (arg146cys) in heterozygosity on an abo*b allele background, and in trans an abo*o.01.01 allele. the phenotype encoded by allele abo*b(436t) is predicted to be b, as evidenced by forward typing on immucor neo and reverse manual typing. case #4 is a prenatal black patient with an abo typing discrepancy: forward type o in gel, a 2+ mixed field (mf) in tube. reverse type on a1 cells 1+ in gel, 0/1+ in tube. sequencing of genomic dna and cloned pcr products covering exons 6-7 detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*o.01.09 allele. case #5 is the newborn baby of case #4, with a forward type a 1+ mf in gel, a 3+ mf in tube. sequencing of the baby's dna detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*b.01 allele. from these results it is inferred that the phenotype encoded by allele abo*784c is a3-like. case #6 is an 18 year-old hispanic donor with an abo typing discrepancy: forward type a, reverse type o. sequencing of genomic dna and abo exons 5-6 and 6-7 detected variant c.979c>t (gln327ter) in heterozygosity, and in trans an abo*o.01.02 allele. the truncation of the a1 transferase at such a relatively late position is consistent with the retention of some enzyme activity, explaining the forward a type encoded by allele abo*979t. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. variants reported to date in the intron 1 enhancer include large deletions, small deletions and single-nucleotide substitutions. here we describe four new alleles with single-nucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. adsorption-elution by the heat elution method and testing for h and a substances in saliva were performed by following the procedures in the aabb technical manual. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. background: inactive alleles of the fut1 could be decreased or aborted the activity of the fucosyltransferase, which results in to form the bombay or para-bombay phenotype with weak or no h antigen expression on erythrocytes. now many para-bombay individuals have been found in the chinese population. according to names for h blood group alleles v5.1 170221 of red cell immunogenetics and blood group terminology working group of the isbt, 55 fut1 alleles were identified for bombay or para-bombay phenotype around the world. aims: the study was explored the distribution of fut1 alleles for the 19 chinese individuals with para-bombay phenotype. methods: the samples were come from the blood donors or the patients. the a, b, h antigens were determined using conventional serological method according to the manufacture's instruction. the sequences of the full coding region for fut1 was amplified, then amplicon was purified with enzymes digestion and used as template for sequencing bidirectionally. all nucleotide sequences obtained were analyzed and compared with standard fut1 sequence. results: nineteen chinese individuals with para-bombay phenotype were identified. ten of them were the donors and nine individuals were come from the hospital. the rbcs had a very weak agglutination reaction with anti-h in the most of the individuals. fut1 homozygous mutations were found in the 12 individuals and fut1 heterozygous changes were existed in 7 individuals after bidirectionally sequencing. .05%, 7.89%, 5.26%, 5.26%, 5.26%, 2.63%, 2.63%, 2.63% respectively in the 19 individuals with para-bombay phenotype. according to our previously reports, the fucosyltransferase activity of fut1*01n.06(c.551_552delag), fut1*01w.09 (c.658c>t) and fut1*01w.01 (c.293c>t) were abolished in vitro assay, while fut1 mrna levels of them had no effect compared with wild type. summary/conclusions: the fut1 mutations in the para-bombay individuals were various. the most common fut1 allele in the chinese individuals with para-bombay phenotype was fut1*01n.06(c.551_552delag). background: the regulatory mechanism of the abo gene is complicated and has been investigated extensively.variation in a antigen expression was recognized very early in the twentieth century and the a blood group was divided into a1 and a2. later the a blood group was subdivided further based on characteristic reactivity with human polyclonal antisera, i.e., strength of reactivity and presence of mixed field agglutination; presence of anti-a1, and whether a or h blood group substance was present in the saliva of secretor subjects. mutations critical for abo blood group phenotypes have predominantly been found in exons 6 and 7 of the abo gene, both of which encode the catalytic domain of abo glycosyltransferase. in our case report we show how mutation ranging from single nucleotide in the intron 1 enhancer element can alter the efficacy of enzyme and alter antigen expression. aims: this study aims to investigate the molecular basis of discrepant results of abo forward/reverse typing in blood donor. methods: the abo typing was performed using tube technique and column agglutination tests (bio-rad, grifols). standard tests were completed with adsorption-elution study using o plasma as a source of anti-a, and with saliva testing for presence of a and h substances. we performed quality control for these methods. abo group genotyping was performed using pcr with sequence-specific primer by commercial kit (abo-variant; bag healthcare, lich, germany). pcr-amplified exons and intron 1 enhancer were subject to bi-directional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results: standard serological forward tests identified blood group o, however, only anti-b iso-agglutinins were present. anti-a in adsorption-elution study was successfully adsorbed and eluted from the investigated cells. a and h substances were detected in saliva. abo genotyping using pcr-ssp indicated genotype o1v/a 1 . dna sequence analysis showed result abo*a01 (28+5859a), abo*o.01.02. the specimen was revealed as an a subgroup, probably a m with an unusual genetic variant in the intron region of the abo gene, the enhancer of the gene expression. summary/conclusions: we report the first case of abo*a01(28+5859a), the mutation located in the enhancer region of gene expression in allele a, that causes discrepant results not only in abo forward/reverse typing but also in molecular blood grouping tests. based on our serological findings, this subgroup is considered as a m . background: a chimera is a single organism composed of cells with distinct phenotypes and/or genotypes. several different types of chimeras are described: artificial, twin and dispermic. the artificial chimerism can be seen following hematopoietic stem cell transplantation, or more transiently following blood transfusion. the second type may also be inherited most commonly through blood exchange in utero between twins. dispermic chimerism is induced by the fertilization of two maternal eggs with two spermatozoa and their fusion into one body. this one is also called tetra-gametic chimerism. in transfusion medicine, chimeras are often detected when mixed field reactivity is observed in abo/d typing or, less commonly, when phenotyping for other blood group antigens. aims: this investigation was prompted by finding a double population of erythrocytes in a surgery patient with no transfusion history. our aim was to investigate the chimera and determine the underlying abo genotype of this patient. methods: routine blood grouping was performed by column agglutination. separation of the double cell populations was performed by differential agglutination with igm anti-d (immuclone, anti-d fast igm, clone: d175-2, immucor). initial abo genotyping was performed by pcr-ssp (fluogene; inno-train diagnostik gmbh); further resolution was performed using in-house pcr-asp and pcr-rflp methods. next generation sequencing (monotype abo; omixon using illumina sequencing platform) and sanger sequencing analysis were also performed. identification of reference alleles was investigated by fragment analysis of short tandem repeats (str) polymorphisms. results: double population was found in column agglutination in tests with anti-a and anti-ab, and subsequently when typing for d and c antigens, with approximately 85% of o d+c+ cells. the patient's genotype was identified as abo*o.01/*a by ce-certified pcr-ssp kit (fluogene). routine pcr-asp and pcr-rflp could not resolve the patient's genotype possible abo*a1/*o1 genotype was detected by pcr-rflp, but the pcr-asp analysis gave an apparent abo*a1 homozygote result. sanger sequencing of abo exons 6 and 7 also gave anomalous reactions: no abo*a allele was detected. homozygosity for c.261delg was observed as well as heterozygosity for c768c/a. this result therefore suggests the patient's genotype is abo*o.01/*o.01.26. next generation sequencing (omixon) revealed the same result. however, when pcr amplification of the cbf/nf-y enhancer vntr 3 0 -region was performed, possible heterozygosity was observed, i.e. a weak band representing a single copy, and one representing 4 copies of the enhancer region were present. presence of a single copy of the 43-bp cbf/nf-y enhancer vntr region is unique background: del is a very weak form of d antigen with low density expression of d antigen on the surface of red blood cell, which is generally typed as d-blood group as couldn't form agglutination in routine rhd blood group testing and could only be detected by the non-routine adsorption-elution test. in the east asian and southeast asian population, 9-30% of the individuals with serologically apparent d-phenotype are not these with truly d-phenotype, but del phenotype, which is very rare in caucasian and black ethic groups. and the rhd*01el.01 (rhd*1227a) is most prevalent (>99%) in del people in these regions, so the del carried this allele was commonly known as "asia type" del. in previous studies, no alloanti-d was observed in a large cohort of chinese "asia type" del pregnant women with d+ fetus to indicate no occurrence of alloanti-d immunization against d+ red cell in "asia type" del individuals. aims: to conduct genotyping analysis in the chinese patients having serologically apparent d-phenotype simultaneous with alloanti-d to confirm the existence of the "asia type" individuals to produce alloanti-d or not. methods: from 2017 to 2018, the blood sample of the patients or pregnant women identified with alloanti-d in our reference lab were collected. d antigen was confirmed again using the blend anti-d reagent (clone th-28/ms-26, igm/igg) by tube method in saline and indirect antiglobulin test (iat) in gel card. the zygosity of rhd gene was detected by hybrid rhesus box pcr with psti digestion. for the samples with d or dd genotypes obtained by rhd zygosity analysis, multiplex ligation-dependent probe amplification (mlpa) genotyping was conducted for rhd genotyping analysis. results: a total of 129 serologically apparent d-chinese patients (female, n = 128; male, n = 1) with alloanti-d were identified. different titers of alloanti-d from 1:2 to 1:4096 (≤1:16, n = 60; >1:16, n = 69) were detected including few cases with mixed antibodies (anti-d mixed with anti-c, n = 11; anti-d mixed anti-e, n = 5). serological rhd typing confirmed the serologically apparent d-phenotype. rhd*01n.01/01n.01 (homozygous rhd gene deletion) genotype was identified in majority of them (123/129, 95.3%) by rhd zygosity analysis, while rhd*01n.03/ 01n.01 genotype (n = 5) and rhd*01n.05/01n.01 genotype (n = 1) carried the rhd non-functional hybrid alleles were detected by mlpa. summary/conclusions: compared with the distribution of average 25% frequency of "asia type" del in serologically apparent dpopulation in guangzhou of china, no one case of "asia type" del was identified in the cohort of serologically apparent d-patients with alloanti-d in this study. this also provides evidence to confirm no occurrence of alloanti-d immunization in "asia type" del individuals. aims: a serologically rhd-negative donor was found to be rhd-positive in the routine rhd screen. to solve the discrepancy between serology and molecular screen, the sample was sequenced on dna and rna level. methods: phenotyping on id/iat-cards (bio-rad) was done using commercial anti-d antibodies. the adsorption-elution analysis was performed using an in-house pool of polyclonal anti-d antibodies. furthermore an antibody d-screen was performed (diagast). for rhd genotyping rh-type and partial d-type assays (bag health care) were carried out. the sample was further characterized by exon sequencing including flanking intronic regions. rna was extracted from whole blood, reverse transcribed and the cdna sequenced. for amplification and sequencing, both published (gassner, transfusion, 2005; legler, trans. med., 2001; richard, transfusion, 2007) and in-house primers were used. results: repeated phenotyping of the sample with commercial as well as, in-house anti-d antibodies confirmed the rhd negativity. in addition, the adsorption-elution analysis showed a negative result. however, genotyping, using commercially available kits, yielded a rhd positive result and no variants were detected. to investigate this discrepancy, all 10 rhd exons were sequenced. the sequencing data revealed the mutation c.148+2delt in the splice donor site of exon 1. to confirm the effect of the splice site mutation on transcription, rna from a fresh whole blood sample was analysed. as a positive control, gypb was amplified and sequenced from the same cdna. wild-type gypb (mns4) was found. with rhd specific primers, no product could be amplified. summary/conclusions: we present a serologically rhd negative case, that was identified as rhd positive by standard commercial genotyping kits. sequencing revealed the new splice site mutation c.148+2delt. rna sequencing yielded no detectable product. the donor was classified as rhd negative. this case of a discrepant result between serology and genetics shows the importance of a profound and highly sophisticated genetic investigation of conflicting laboratory results. j stettler, s lejon crottet, h hustinx, c von arx, f still, j graber, c niederhauser and c henny interregional blood transfusion src berne ltd., berne, switzerland background: one of the most immunogenic and clinically significant blood group antigens in transfusion medicine is the rhd antigen. variant rhd phenotypes with weakened or absent antigen expression pose a challenge for rhd status assignment in blood donors. to ensure patient safety, it is necessary to fully characterize these variants at the molecular level. aims: samples from two donors were investigated in our laboratory due to discrepancy in rhd typing. methods: rh blood group phenotyping was done by standard serological column agglutination testing (id-system, biorad). further rhd characterization was performed by an anti-d antibody panel containing 9 monoclonal antibodies (d-screen, diagast) and an adsorption-elution test using an in-house pool of polyclonal anti-d antibodies. molecular investigation was initially performed by ssp-pcr detecting common rhd variants (rbc-ready gene cde inno-train; rh-type bag health care). rhd sequencing was done on either dna or rna using published and inhouse primers for amplification and sequencing. results: by tube testing, the rbcs of donor 1 were predicted to be rh:-1,-2,-3,4,5. however, all ten exons of the rhd gene could be detected by routine genotyping. sequencing of rhd revealed a homozygous mutation c>g at position 1151 which is the second last nucleotide of exon 8 and thus might have an influence on exon splicing. by cdna analysis a transcript with a correctly spliced exon 8 was identified. the mutation c.1151c>g leads to the amino acid substitution t384r located in the twelfth transmembrane domain of rhd using the model of flegel (transfus apher sci., 2011) as reference. adsorption-elution testing using a pool of polyclonal anti-d showed a weak positive reaction, re-classifying the donor as rhd positive. this novel allele, rhd*1151g, could thus be categorized as a del allele. serological results displayed an almost normal rhd antigen expression for donor 2. further serological determination of the rhd antigen with 9 different antisera, however, showed a reaction pattern typically observed with a weak d variant. with commercial available kits no rhd variant could be detected. rhd sequencing revealed a novel homozygous mutation c.526g>c in exon 4. this mutation causes a p.a176p exchange in the sixth membrane-spanning domain of rhd. based on serological data, the donor is rhd positive and in case of transfusion the patient would be treated as rhd negative. summary/conclusions: here we report two novel rhd missense mutations c.1151c>g and c.526g>c harbouring an amino acid substitution within a transmembrane segment. the c.1151c>g variation displayed an unusual low rhd antigen reactivity and would have been mistyped as rhd negative without extensive genotypic testing. molecular analysis of variant c.1151c>g suggests that the t384r exchange causes a del phenotype rather than a miss splicing event. this was also confirmed by adsorption-elution testing. interestingly, variant c.526g>c could only be detected due to comprehensive serological and genetically investigation. background: the rh blood group system is highly polymorphic and one of the most clinically relevant systems in transfusion. actually d antigen is of critical importance due to its involvement in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. rhd gene variants are common in africans and mostly related to partial d phenotype. aims: rhd gene sequence was investigated in two african brazilian samples. we further attempted to take advantage of combining the molecular data and the available in silico tools for the functional interpretation of the variations, in order to get insights into the clinical phenotype that may be predicted a priori from genotyping. methods: sample #id1 is a d-negative donor self-declared as african descent. sample #id2 is a patient with sickle cell disease (scd) typed as d-positive with anti-d in his serum. serologic d typing was determined by manual gel test and by microplate in an automated instrument. sample #id1 was also submitted to adsorption/elution test. after genomic dna extraction, all ten rhd exons and flanking intronic regions from sample #id1 were pcr-amplified with rhd-specific primers and analyzed by sanger sequencing. sample #id2 was investigated by next-generation sequencing on the miniseq platform (illumina) by using a previously published, custom (selected blood group genes) ampliseq panel. a reported three-dimensional (3d) structural model of the rhd-rhd-rhag heterotrimer was used to visualize the position of variations and predict their putative functional/clinical effect. results: in sample #id1, a single nucleotide missense change, i.e. c.1151c>g in exon 8, was identified. this transversion is thought to replace a threonine by an arginine residue at amino acid position 384 (p.thr384arg) of the rhd protein. analysis in the 3d model clearly suggests a dramatic impact of the p.thr384arg substitution occurring in a functionally-critical, conserved motif in terms of interhelix interaction, which is supposed to be highly deleterious to the stability of the protein, and potentially impairs totally its expression at the red blood cell plasma membrane. this predicted functional effect is definitely in accordance with the d-negative phenotype reported in sample #id1. in sample #id2, the single c.325a>g transition was found in exon 2 leading to a threonine-to-alanine substitution at amino acid position 109 (p.thr109ala). amino acid 109 is located in rhd protein extracellular loop 2, and is thus thought to alter d antigen structure, resulting in a partial d phenotype. this hypothesis is in accordance with anti-d found in the serum of sample #id2. summary/conclusions: for the past years, due to the advent of next-generation sequencing and the subsequent identification of numerous rare variants, bioinformatics prediction and modelling tools have evolved and currently help physicians in diagnostics, clinical management and genetic counselling. we took advantage of some of those in silico methods to predict retrospectively the effect of two novel variant rhd alleles, including one d-negative and one partial d alleles. although phenotype and clinical symptoms remain definitely the standard determinants to assess the effect of genetic variations, use of those approaches may soon become valuable for guiding subsequent investigations in immunohaematology. abstract withdrawn. alleles of the weak d type 4 and diva cluster. in africans, the 16 most frequent were typically associated with alleles of the weak d type 4 (including dol and rhdpsi), diva and dau clusters with f223v occurring in > 10% of alleles; in addition the key mutations of weak d type 1 and dii and two inactivating mutations (c.1056_1057inst and c.1060delg) not reported in rhb were among the first 20 polymorphisms. in east asians, rhd(1227g>a) at 0.8% was most frequent, followed by dfv, weak d type 33, dbo-3, key mutations of diva and weak d type 4 cluster as well as rhce-like substitutions and the mutations of weak d type 25, type 15, type 66, rhd(a85v), dvl-1, weak d 119 and rhd(n135s). weak d type 151 and rhd(t148r) were frequent in south asia but not elsewhere. summary/conclusions: data from tgp and gnomad add relevantly to the knowledge on rhd alleles; tgd discloses linked intron polymorphisms, gnomad frequency data not biased by the likelihood of serologic detection. current typing strategies usually start with serology later complemented by molecular typing. in the future, molecular methods will gain importance and frequent alleles currently not distinguished from "standard rhd" may need a rational transfusion strategy. in this respect, the high frequency of weak d type 45 and type 33 in europeans was surprising, might warrant confirmation by alternative methods and should trigger discussion on rational transfusion strategies for these alleles. consistent with an r 0 haplotype and 1 probable dc-. two siblings that were abo compatible including the dc-sibling were incompatible at iat phase. reactivity could be completely adsorbed from the serum using r 1 r 1 , r 2 r 2 , and rr rbcs indicating the antibody is probably a single specificity. the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. aims: the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. methods: serologic testing was performed by manual tube testing using ahg in the indirect antiglobulin phase. rbc phenotyping was performed by standard tube hemagglutination testing from edta anticoagulated blood. rhd and rhce exons were sequenced using genomic dna and standard sanger dideoxy method with the bigdye terminator v3.1 cycle sequencing kit. sequence data was aligned to rhd_ng_007494.1. rhd zygosity was performed using pcr-rflp with mspi. background: according to recent findings in molecular immuno-hematology, rhd genotyping is strongly indicated in rhc+ and rhe+ donors classified in routine as d-negative. among these, one could find a non-negligible share of entirely new genetic alterations or even del alleles, which are often not identifiable with routine serological methods due to the low number of antigenic sites. aims: the present study reports the genotyping data of rhd on 201 rhc+ and rhe+ caucasian donors classified serologically as d-negative, all enrolled by a single transfusion center in italy methods: rhd serological typing was carried out in microplate direct agglutination tests (iris, immucor) by using 2 different anti-d igm clones (clone 1, dvi+: ldm1+esd1m; clone 2, dvi-: rum-1, th28) and 2 different anti-d igg clones (clone 1: ms26; clone 2: d415 1e4). all donors with d-negative results (n = 201, divided into 153 subjects with rhc+, 45 with rhe+ and 3 with both rhc+ and rhe+) were addressed to genotype analysis with rhd beadchip molecular test (immucor), pcr-ssp (bagene, inno-train) and/or rbc-fluogene (inno-train). the discrepant results between serology (d-neg) and molecular biology (wild-type or full-length rhd gene) were further investigated by bi-directional sequencing of the rhd coding regions. results: one-hundred donors have been analyzed retrospectively, as part of a pilot study. following the data obtained in this first phase, the analysis methods described above have been implemented in routine, allowing to include further 101 donors, studied prospectively. in 92.5% of donors (n = 186), the molecular analyses showed the complete deletion of the rhd locus, while in 15 cases (7.5%) a genetic status was found with "non-deleted" rhd. over all, bi-directional sequencing on these 15 donors revealed the presence of 9 negative and 4 weak-d variants. the list of rhd alleles we have identified at the molecular level is as follows: rhd*01n.82 (2 cases), rhd*01n.83 (1), rhd*01n.80 (1), rhd*01n.61 (1) , rhd*01n.05 (3), rhd*01el.08 (1), rhd*01el.18 (1), rhd*01el.17 (1), rhd*01w.54 (1) . moreover we found a donor with a lack of signal encompassing exons 1-5 of the rhd sequence (bioarray rhd beadchip), while 2 additional cases are currently under investigation. summary/conclusions: our study confirms that a non-negligible number of caucasian subjects, classified serologically as d-negative, present rhd gene alterations that differ from the common total deletion. in line with the literature data, we also found a frequency of about 1 in 50 cases (4 subjects out of 201), in which a donor re-classification as d-positive (weak d type) was necessary. hence, a wider use of molecular typing methods is desirable in order to achieve the correct genetic characterization and the appropriate phenotypic classification of "apparently" d-negative donors. background: without evidence of abnormal serological d antigen expression there will be no quest for weak d, partial d or d variant on the red blood cells. according to our blood donor registry we found that out of 43960 serologically typed donors, 85.5% were d+, 14.0% d-and 0.5% weak d. aims: to compare different weak serological reactions of the d antigen to the rhd genotyping. methods: molecular rhd typing using isolated dna and rbc-ready gene cde and rbc-ready gene d weak kits was performed in 22 blood donors, who were serologically typed as weak d using monoclonal blended igm/igg and dvi-and dvi+ anti-d reagents by slide and microplate (mp) technique respectfully, as well as by the antiglobulin test (iat) in gel and with the set of monoclonal partial d typing reagents (biorad). in addition, rhccee phenotyping and genotyping was also performed. results: all of the donors with serologically weak reactions were confirmed to be weak d variants by genotyping except one donor whose iat was false positive due to rbc autoantibodies. the frequency of d variant genotypes was as follows: 62% weak d type 1, 28% weak d type 3, one donor was typed as weak d type 14 and another one as weak d type 11. these weak d types were associated with different degrees of serologically determined weakness ranging from negative to weak positive reactions concerning slide and mp. all of them gave positive reaction ranging from 2+ to 4+ with iat, except for the weak d type 11 with the score of <1+ which gave negative reaction by slide and mp and inconclusive result with the set of monoclonal anti-d reagents for partial d typing. the percentage of donors, who, at serological typing were only found to be d positive in the iat was 19%. one of the weak d type 1 donors was negative with dvi-and positive with dvi+ reagent in the mp. the additional rh phenotype (genotype) was ccee in all of the donors except in the one who was genotyped as weak d type 14, as well as in the d negative donor, being ccee. summary/conclusions: further rhd genotyping is required to estimate the actual frequency of d variants in our blood donors. in practice, current serological methods are sufficient to detect almost all variant d phenotypes. there is a consensus that routine molecular d antigen screening in d negative donors in order to detect del variant when ddccee phenotyped red blood cell transfusion is practiced in all d negative patients does not seem to be cost-effective. background: rh null or rh mod -the so-called rh-deficiency phenotypes-are characterized by a null or severely reduced rh antigen expression (including d, c/c and e/ e), respectively. molecular genetic studies showed that these phenotypes are transmitted in an autosomal recessive manner. rh null phenotype originates from two different molecular events giving rise to the amorph type and the regulator type. the former is caused by homozygosity for silent genes at rhd and rhce loci, caused by inactivating mutations in rhce and deletion of rhd. on the other hand, the regulator rh null type as well as the rh mod phenotype are attributed to mutations in rhag gene when in homozygous state or when in heterozygosity with another rhag allele containing an inactivating mutation. a functional rhag is essential both for the correct rh complex assembly and rh antigen expression in the erythrocyte membrane. aims: the aim of this study was to investigate the molecular genetic basis of an argentinean proband with no detectable d, c, c, e and e antigens by standard serological techniques. methods: blood samples were collected from the proband, her parents and sister. the proband was a 14 year-old young woman with parameters of hemolytic anemia: low hemoglobin level (10 g/dl), reticulocytosis (14%), hyperbilirubinemia, increased ldh and marked spherocytosis. the d, c, c, e and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. genomic dna was isolated using a modified salting-out method. dna samples were initially screened for the presence of intron 4 and the 3 0 untranslated region of the rhd gene using pcr strategies. rhc/c, and rhe/e alleles were studied by allele-specific pcrs to determine the rhce genotype. rhd zygosity was analyzed by pcr-rflp. rhd exon polymorphisms were studied by rhd exon scanning procedure based on pcr-ssp. rhag gene was investigated by exon-specific pcr amplification and sanger sequencing. results: no d, c, c, e and e antigens were detected in the proband's erythrocytes. the father and sister rh phenotype was: d+, c+, c+, e+, e+ whereas the mother rh phenotype was: d+, c+, c-, e-, e+. rh genotyping confirmed the rh phenotypes for all family members except for the proposita who genotyped rhd+, rhc+ and rhe+. all samples showed an homozygous status for the rhd gene and all rhd exons were detected by exon scanning. sequencing analysis revealed an homozygous c.920c>t mutation in rhag exon 6 in the proband whereas the rest of the family showed an heterozygous state in the same nucleotide position. the c.920c>t mutation is responsible for the p.ser307phe amino acid substitution predicted to be in the 10 th rhag glycoprotein transmembrane segment. summary/conclusions: this study described the molecular background responsible for an rh-deficiency phenotype in an argentinean proband. we identified the novel missense mutation c.920c>t in the rhag gene which results in the ser to phe single amino acid substitution that shows to be critical for rh antigen complex assembly within the erythrocyte membrane. further studies are being performed in order to determine whether the proband is rh null or rh mod . background: rh blood group system is the most immunogenetic blood group system and blood donor typing should account for all expressing antigens in order to prevent anti-d alloimmunization. aims: the objective of this prospective study was to investigate rhd alleles among blood donors who typed d-by serologic methods and positive for c and/or e. for this reason we developed an easy-to-perform dna-based screening method for the detection of rhd gene and positive samples were further characterized by two commercial pcr-ssp kits. methods: of 15692 individual blood donors within a 15 month period, 1688 (10.76%) typed as d-with standardized immunohematologic methods including the indirect antiglobulin test (iat). residual edta-anticoagulated blood samples were used to isolate genomic dna using the qiaamp dna blood kit (qiagen, germany) from 112 out of 151 (8.95%) c/e+ and serologically d-donors. all dna samples were tested individually for the presence of rhd-specific dna sequences in the rhd promoter, intron 4, exon 7 and exon 10 by a multiplex pcr-ssp method. the reaction was conducted in a final volume of 20 ll with primers that were applied as described by f. wagner et al. (bmc genetics, 2001 ) except antisense primer for exon 10 and the two primers amplifying an hgh gene fragment as internal control, designed by our laboratory. pcr products were visualized by electrophoresis on a 4% agarose gel with ethidium bromide staining. in case of a positive reaction the sample was analyzed by pcr-ssp d weak and pcr-ssp cde (inno-train, germany). results: out of 112 d-individuals analyzed, 101 were ddccee, 8 ddccee, 2 ddccee and one had a ddccee phenotype. molecular analysis showed that 104 (92.86%) were negative for all four rhd dna regions. among the other 8 samples, all of ddccee phenotype, three were found to be positive for rhd promoter, intron 4, exon 7 and exon 10, three for rhd promoter and exon 10, and two for exon 10 alone. further genotyping revealed five hybrid rhd-ce-d alleles [3 rhd-ce(2-9)-d and 2 rhd-ce(3-7)-d], one allele represented the del(m295i) genotype, while the remaining two samples did not show an allele that could be determined with the pcr-ssp kits. summary/conclusions: serotyping is the standard method to assign transfusion strategies but it is not always capable to correctly define all samples that show weak reactions in d. a rhd genotyping strategy is needed to confirm d-blood donors and thus to avoid anti-d immunizations. for these reasons we suggest the implementation of an easy and possible cost-effective method. background: more than 100 weak d types have been described to date. transfusion recipients with weak d type 1, 2, or 3 are not at risk for forming allo anti-d when exposed to conventional rh d-positive rbcs. molecular analysis of weak d offers a more reliable basis than serotyping to determine the prevalence of weak d types and optimal d transfusion strategies. background: the d antigen, which consists of a mosaic of epitopes, is determined in all the blood donors and patients. most people are either rhd-positive or rhdnegative, but there is a certain number of people who have a variation of the d antigen, which are called weak d, partial d and del phenotypes. aims: the objective is to use molecular methods to determine whether blood donors in republika srpska (with whom a serological weak d antigen has been detected) really have the weak d antigen. in addition, determine whether blood donors, who have been determined as persons who are rhd-negative, with the phenotypes c and/ or e, who have the rhd gene and d antigen on the erythrocyte membrane, so weak that it could not be determined by serological techniques. methods: blood samples were used from regular blood donors, who have been determined as persons with a weaker d antigen, as rhd-negative or as c and/or e positive (based on the agglutination strength) using serological techniques, the test tube method, the microplate method and the gel method. gp.mur was also modelled and shown to closely resemble the tertiary structure of glycophorin a. the predicted structure is 5 anti-parallel b sheets arranged in a "b barrel" also referred to as an ob-like-fold. the regions in which blood group antigens were identified in the predicted stable dimeric structure. summary/conclusions: ob-like-fold structures typically to bind oligonucleotides or oligosaccharides and are associated with cold shock proteins. further modelling is in progress to predict the structure of gpa/gpb heterodimers as a basis for understanding the presentation of blood group antigens. of interest, this finding is consistent with a previous report showing that this gpa binds to carbohydrates. this model serves as a foundation for future work regarding the properties of gpa, which includes identifying locations of specific interactions between gpa and other rbc surface proteins such as gpb and band 3, as well as identifying structural features of antigenic regions on gpa. . even though no significant differences were found among the groups studied, 4 haplotypes containing the mcc b and sl2 polymorphisms were identified in d samples but were not found in tb and l groups. summary/conclusions: this preliminary data obtained suggests that cr1 polymorphisms and haplotypes, especially those containing mcc b and sl2 snps, could be involved in the disease pathogenesis of tuberculosis and leprosy. the entrance of mycobacteria into macrophages is mediated by complement receptors that facilitate their uptake by host cells so the combined haplotypes could be enhancing parasite phagocytosis and inflammation. further studies are being carried out to establish whether cr1 polymorphisms are risk or protective factors and whether other genetic variations in this receptor are also involved. abstract withdrawn. background: the dombrock blood group system consists of two antithetical antigens, do a and do b , and three high-prevalence antigens, gregory (gy a ), holley (hy), and joseph (jo a ). the rare do null or gy(a-) phenotype lacks all dombrock antigens, and the do null alleles vary with both do*01 and do*02 backgrounds. here we report the molecular basis of a novel do null allele in a gy(a-) brazilian patient with anti-gy a . aims: case presentation: an alloantibody to a high-prevalence antigen was detected in the serum of a 42 year old woman from the northeast brazil with a history of 4 pregnancies but no history of previous transfusion. she required transfusion because of a schedule for total thyroidectomy surgery due to a large compressive nodular goiter. the antibody did not react with the autologous rbcs but reacted by the indirect antiglobulin test in liss with all panel rbcs and other rbc samples tested except with the gy(a-) phenotype. the corresponding antigen was resistant to treatment with papain but sensitive to dtt and trypsin. these results suggested that the antibody recognized an antigen in the dombrock blood group system. the purpose of this study was to identify the antibody specificity and to determine the molecular basis of the phenotype detected. methods: the red cells phenotype and the presence of the dombrock related antibody in the serum were detected by standard hemagglutination techniques. rbcs and antibodies were from our in-house collection of rare samples. genomic dna was prepared from peripheral blood of the patient. dombrock genotyping was performed by id-core xt platform (grifols, spain). the 3 exons of the do gene were amplified by pcr and directly sequenced. experimental immunohematology and diagnostic immunohematology 2 diagnostic immunohematology 3 experimental immunohematology, sanquin, amsterdam, netherlands background: typing of blood group antigens is essential to prevent transfusion reactions or haemolytic disease of the foetus and newborn. to date, the isbt recognises 360 blood group antigens. most antigens (322) belong to one of the 36 blood group systems. since the genetic basis of these systems is known, genotyping of these antigens is possible. the molecular background of 38 antigens is unknown and can only be determined serologically. one of these antigens is sd a (sid), first reported in 1967.~91% of the population carry sd a on erythrocytes, but this frequency might be higher since identification is difficult due to variability in expression. in 96% of individuals sd a is present in urine. cells with a high expression of sd a (cad/sda++) are used for detection of antibodies. recently, a 3-cells antibody detection panel of bio-rad contained a sda++ cell and many individuals with anti-sd a were detected. the b4galnt2 gene has been implicated in the synthesis of sd a . we collected individuals with and without anti-sd a to elucidate the genetic background of the antigen. aims: elucidation of the genetic basis responsible for loss of the sd a antigen on red blood cells. methods: routine diagnostics to identify antibodies in patients was performed using a bio-rad 3-cells panel, containing donor 626521 with high expression of sd a . additionally, 200 pregnant women were screened for anti-sd a . dna of eight samples with anti-sd a and eight samples without anti-sd a was isolated for further analysis. sanger sequencing was performed on b4galtnt2 exon 1-11. results: sequencing of b4galtnt2 revealed two homozygous mutations which are present in all eight individuals with anti-sd a , but not present in 6 controls. the remaining two controls are heterozygous for these mutations. the first mutation within exon 10, c.1396t>c (enst00000300404.2, rs7224888) changes a cysteine to arginine at position 466 of the protein. the second mutation in exon 11 c.1590a>g (rs16946912) does not change an amino acid. both snps have a maf of 0.11 and therefore we expect that 2.1% of the population is homozygous for the minor allele. genotyping of a large population of pregnant women and the serological detection of anti-sd a in women with a homozygous mutation is in progress. summary/conclusions: the high frequency antigen sd a has not been linked to a blood group system because the molecular basis for loss of the antigen has not been elucidated. the b4galtnt2 gene has been associated with sd a synthesis and therefore we analysed this gene for mutations in individuals with antibodies against sd a . a single homozygous mutation within exon 10 causing an amino acid change was found in all individuals with anti-sd a , and no individuals without antibodies were homozygous for this snp. from population studies we expected~4% sd a -negatives, but either this frequency is an overestimation because of difficulties to detect low expressed antigens or mutations in other genes are interfering with sd a synthesis. a larger study of individuals with homozygous mutations in b4galnt2 and linkage to sd a -negativity and presence of antibodies will be performed before sd a can be assigned to a new blood group system. abstract withdrawn. abstract withdrawn. background: erythrocyte duffy blood group antigen can scavenge chemokines in whole blood. duffy blood group gene consists of two major alleles: fy*a and fy*b. however, little is known regarding the association of duffy blood group polymorphisms with the red blood cell (rbc) chemokine scavenging. aims: the aim of this study was to determine the association of duffy blood group polymorphism with the rbc chemokine scavenging. methods: the duffy blood group were genotyped by 5ˊ-nuclease assay in healthy chinese han individuals, while erythrocyte chemokine scavenging function and duffy antigen expression from the same samples were measured using erythrocyte chemokine binding assays and quantitative flow cytometry respectively. results: rbc chemokine scavenging of cxcl8 was significantly lower in the individuals with the fy*a/fy*a genotype compared to those with fy*a/fy*b genotype (p = 0.016). similar result was also observed in rbc chemokine scavenging of ccl2 (p = 0.038). the expression of duffy antigen on rbc surface in the individuals with the fy*a/fy*a genotype was significantly higher compared to those with fy*a/ fy*b genotype (p = 0.021). summary/conclusions: duffy blood group polymorphism is associated with the differential rbc chemokine scavenging. it is probable that a change in duffy antigen structure caused by duffy blood group polymorphism is responsible for the differential rbc chemokine scavenging. background: individuals with p-phenotype can develop a naturally occurring anti-pp1pk and has clinical significance, causing hemolytic transfusion reactions or hemolytic disease of the fetus and newborn. finding and procuring blood units of pphenotype is a challenge because of its rarity throughout the world. therefore, acute normovolemic hemodilution (anh) can be an on hand tool in the perioperative successful management of patient with rare blood group. however, this approach has not been commonly used aims: n/a. methods: n/a. results: a 66-year-old korean woman was referred to samsung medical center for surgical management for gallbladder malignancy. her blood type was group a, d-positive. the patient had no known history of transfusion. however, antibody screening and identification test using the column agglutination method (bio-rad, cressier, switzerland) showed panagglutination with negative reactions to autologous red blood cells, indicating the presence of alloantibodies to high frequency antigens. the specimen obtained from the patient was sent to the central laboratory of the swiss red cross (bern, switzerland) and confirmed as anti-pp1pk. at first, the transfusion team of our hospital recommended the surgical team to postpone the surgery. however, anh was planned because postponing surgery was not preferred and the patient's preoperative hemoglobin was 12.9 g/dl. 800 ml of blood was withdrawn through a radial arterial catheter in two 400 ml blood bags containing citrate-phosphate-dextrose-a solution after anesthetic induction. equal volume of 6% hydroxyethyl starch solution was infused during the procedure. the patient underwent radical cholecystectomy and liver wedge resection with lymph node dissection, and two units of autologous blood were returned to the patient during surgery. she was then discharged 48 h later with a hemoglobin level of 12.3 g/dl. later, the family study was performed with the standard serologic method using the proband's plasma containing anti-pp1pk and sequencing of the a4galt gene, which were conducted according to the protocols by koda et al.(transfusion. 2002) . the proband and her brother were homozygous for c.1029dupc, indicating a rare p phenotype. summary/conclusions: we experienced that autologous blood transfusions via anh is an alternative to allogenic rbc blood transfusion in patients who have no blood available because of high alloimmunization antibodies against rare blood groups. " and the third sample as "gypb*s_gyp*[140a], gypb*s_null(ivs5 + 5t)" with a predicted phenotype: s-s+ mi a + and s+s-mi a +, respectively. the gypa specific primers used for discrepancy resolution detected the nucleotide substitution, gyp.c.140c>a, in gypa-b-a hybrid associated to gp.hut allele, thus confirming the id core xt result. the expression of mi a for one of these samples was confirmed using non-commercial anti-sera. hence, these three samples were not gp.mur but gp.hut phenotype. both alleles codify for the expression of mi a antigen since it is expressed on several hybrids between the usual forms of glycophorin a and b. two of these three gp.hut samples are african-american donors. gp.hut was reported in white people with a frequency about 0.06% and in thais with 0.04%. these three gp.hut cases found by id core xt in this study point to a higher frequency of this glycophorin variant and also to the presence in african american population. summary/conclusions: id core xt was able to detect two glycophorin phenotypes, gp.mur and gp.hut, which codify for the expression of mi a antigen. standard molecular methods should be implemented in pre-transfusion testing and obstetrical care routine to detect the most clinically relevant glycophorin variants in mns system. background: serf(+) is a high prevalence antigen in the cromer blood group system, which is encoded by a crom*12 allele. the lack of the serf antigen, serf(à) on red cells is caused by a single nucleotide polymorphism, c.647c>t in exon 5 of the decay-accelerating factor, daf gene. alloanti-serf has been found in thai pregnant woman with serf(à) and a serf(à) individual was found among thai blood donors. anti-serf is not a marketed product; hence, a molecular technique has to be implemented to genotype for the crom*12 allele among blood donors. aims: this study aimed to identify the crom*12 allele among thai blood donors leading to predicted serf(+) and serf(à) phenotypes. methods: dna samples obtained from 1,512 central thai blood donors were genotyped for serf allele detection using in-house pcr with sequence-specific primer (pcr-ssp) and confirmed by dna sequencing. results: the allele frequencies of crom*12(+) and crom*12(à) among 1,512 central thais were 0.992 (3,001/3,024) and 0.008 (23/3,024), respectively. the homozygous of crom*12(à/à) alleles was not found in this study. additionally, the pcr-ssp technique was validated by dna sequencing using randomly chosen 100 samples together with 23 heterozygous crom*12(+/à) samples and the results were in agreement. summary/conclusions: our results confirm a high frequency of the crom*12(+) allele in the thai population and their frequencies were similar to those formerly reported among thai blood donors. this study would be beneficial to predict the serf antigen from genotyping results due to unavailability of commercial antiserum. background: there is increasing interest in the use of molecular methods for predicting abo grouping. though nextgen and sanger sequencing have both been used to predict abo type, predicting abo type from buccal swab-derived dna and from deceased donors benefits from a quick and reliable method. besides a pcr-rflp that has been used by many labs for more than 25 years, there is a commercially-available research use only (ruo) kit, and both interrogate nucleotides associated with o1, o2, a2 and b with a representing the ancestral allele. aims: the aim of this report is to compare two low-resolution polymerase chain reaction (pcr)-based methods, for investigation of samples submitted to a reference molecular immunohematology laboratory for abo typing discrepancies. fifty-six peripheral blood samples were tested, 31 from patients and 25 from blood donors. methods: genomic dna was isolated from peripheral blood mononuclear cells. background: del is the weakest known d positive phenotype in the rh blood group system and detectable only by adsorption and elution tests. the rhd1227g>a change is an important marker for del phenotype in east asians. a rapid and efficient pcr method for rhd gene 1227 g>a genotyping is useful in east asian countries. aims: the aim of this study was to develop a method for rhd 1227g>a genotyping by using single-tube pcr with melting temperature(t m )-shift primers. methods: two allele-specific primer for rhd 1227g>a and a common primer were designed and synthesized. two gc-rich tails of different lengths were attached to 5 0 ends of the allele-specific pcr primers. single-tube pcr with t m -shift primers was carried out with the three primers. after pcr, melting curve analysis was performed. rhd 1227g>a could be genotyped by differences of the t m s of the pcr products. all of genotyping results were compared with those obtained from conventional pcr-ssp. for the discordant results, rhd exon 9 sequencing was performed to determine rhd 1227g>a genotype. results: a total of 84 samples were genotyped for rhd 1227g>a by pcr with t mshift primers. 32 samples were typed as 1227a+/g-, 22 samples were typed as 1227a-/g+, 6 samples were typed as 1227a+/g+ and 24 samples were typed as 1227a-/g-. two samples typed as 1227a+/g+ by pcr-ssp but 1227a+/g-by pcr with t m -shift primers were confirmed as 1227a+/g-by rhd exon 9 sequencing. summary/conclusions: the single-tube pcr with t m -shift primers for rhd 1227g>a genotyping is simple, rapid, accurate, and it is superior to conventional pcr-ssp. abstract withdrawn. background: the rh blood group system has numerous variant alleles, which may affect rh antigen expression, including rhd-rhce (d-ce) hybrid genes. these variant alleles are frequently found in people of african descent, and typically result in either d-negative (d-) phenotype, or partial d antigen expression, including silencing of high-frequency antigens and/or expression of low-frequency antigens. patients carrying those alleles are particularly at risk of alloimmunization, suggesting that their identification is important in diagnostics. quantitative multiplex polymerase chain reaction (pcr) of short fluorescent fragments (qmpsf) has proven successful for genotyping those dna samples carrying d-ce hybrid genes by assessing both qualitatively and quantitatively rhd and rhce gene exons. aims: the aim of this project was to genotype both rh genes in a cohort of brazilian patients with sickle cell disease (scd), which are known to be of african descent, by using the qmpsf approach and report hybrid gene variability in this population. methods: one-hundred fifteen dna samples were selected for the study and analyzed prospectively by the rhd-qmpsf and rhce-qmpsf approaches to investigate the copy number of all exons in both rh genes. genotypes were further confirmed or investigated by sanger sequencing and conventional pcr-rflp assays. results: in the 115 dna samples, 75 (65.2%) exhibited a "wild-type" profile by qmpsf analysis. hybrid genes involving exon 8, which is functionally not relevant as reported before, was found in 28 samples, including 11 and 17 samples carrying respectively rhd-ce(8)-d and rhce-d(8)-ce (two homozygous each). except two samples that require additional studies (1.7%), rhd zygosity was resolved successfully: 60 (n = 2 rhd gene copies; 52.2%), 49 (1; 42.6%) and 4 (0; 3.5%). clinically relevant, i.e. partial d, genotypes were identified in four hemizygous samples (4/115, 3.5%) carrying rhd*dau5, rhd*dv.2, a rhd*diiia-like allele, and a novel rhd*d-ce(7:g329h-y330s-n331i)-d allele, as confirmed by sequencing. other hybrid alleles, such as rhd*03n.01 and rhd*diiic, were also found in trans with a normal rhd*01 allele. in rhce, c/c genotype could be resolved. the rhce*ce16 (rhce*ce (48c)-d(9)-ce) allele, which is commonly cis-associated with rhd*ψ, was observed in four samples. however the clinically relevant polymorphisms in variant rhce alleles, such as those involved in cemo, cear, ceag, and ceti, were mostly identified by other standard methods. summary/conclusions: although most of the brazilian patients with scd investigated in this study did not carry rhd-rhce hybrid genes, qmpsf analysis has been shown to be an efficient tool in the whole genotyping process to investigate rh gene variation. as previously reported, it has been conclusive for characterization of rhd zygosity and identification of rare, as well as novel, variant alleles. additionally, our results show a large diversity of hybrid genes among the brazilian patients with scd. therefore, we suggest that qmpsf may be used as a complementary screening approach for assessing rh genotype in selected patients and donors. = 19) vs. non-bleeding (n = 15) patients. platelet, pmp and cp phenotype and function were evaluated by flow cytometry: activation and granule release were examined by antibodies against granulphysin (cd63), p-selectin (cd62p), activated gpiib/iiia (pac-1) and phosphatidylserine (ps) (lactadherin) unstimulated and adp, trap or collagen stimulated. coated platelets were identified as a highly granulated independent cell population appearing following collagen stimulation, gated on side scatter and gpiba (cd42b). normal healthy reference levels were available. results: the platelet count in bleeding (72 9 10 9 /l) and non-bleeding (68 9 10 9 /l) patients was comparable (p = 0,66). bleeding patients had a higher bat score compared to non-bleeding patients (10 vs. 2, p < 0,01). the proportion of cps was normal in all patients. however, in non-bleeding patients the proportion of ps+cps and per cell ps expression (mfi) (86,16% and 4,96mfi) were higher, compared to bleeding patients (64,08% and 2,44mfi, both p < 0,05), and the proportion of ps+cps correlated negatively with bat score (r 2 =0,26, p < 0,01). cd63 + cp was higher in non-bleeding (97,25% and 10,54mfi) compared to both bleeding patients (94,88% and 6,89mfi) and significantly higher than the reference level (88,44% and 5,36mfi, both p < 0,05). finally, the proportion of ps+pmps was normal in bleeding patients, but their pmps expressed higher than reference ps per cell, both unstimulated and for all agonist (134,12 mfi unstimulated vs 32,38 mfi reference, p < 0,01). summary/conclusions: patients with it exhibited different bleeding tendency despite comparable thrombocytopenia. in non-bleeding patients the proportion and per cell level of ps+ were higher, indicating that generation of cps with high ps expression is a critical factor determining bleeding phenotype. the finding of high pmp ps per cell level in bleeding patients could represent an inadequate compensation for lack of cp function, indicating that procoagulant pmps may be less important than cps for thrombocytopenic bleeding. quantification and characterization of cps may be a useful tool for future assessment of bleeding risk as well as a therapeutic target in it and other conditions with bleeding diathesis and/or thrombocytopenia. more studies investigating this field are warranted. background: alloantibodies against human platelet antigens (hpas) and human leukocyte antigen (hla) are implicated in several immune-mediated platelet disorders. detection of these antibodies is crucial in the diagnosis and management of these disorders. aims: to establish a method detecting hpa-1, hpa-2, hpa-3, hpa-5 and hla antibodies using luminex bead technology. methods: monoclonal antibodies specific for platelet glycoproteins and hla class i molecules were separately coupled to the luminex microbeads. positive anti-hpa-1a, anti-hpa-2b, anti-hpa-3a, anti-hpa-5a samples were used to validate the specificities of the luminex assay. the anti-hpa-1a, anti-hpa-3a standard samples were used to evaluate the sensitivities of the luminex assay by serial dilutions (from neat to 1/1024). results: 44 samples collected from patients or isbt platelet workshop were tested by the luminex assay. the results showed that luminex assay could detect antibodies against hpa-1a, hpa-2b, hpa-3a, or hpa-5a successfully from the known samples. the sensitivities of the luminex assay detecting anti-hpa-1a, and anti-hpa-3a were 1:512 and 1:64, respectively, using the standard samples. no cross-reactivity was observed in the samples containing multi-platelet antibodies, or mixture antibodies against hpa and hla. the results of 44 samples with platelet disorders were agreement with those of monoclonal antibody immobilization of platelet antigens (maipa) assay. summary/conclusions: luminex beads coupled with monoclonal antibodies could be successfully used to detect hpa and hla antibodies with high sensitivity. background: platelet transfusion is important in clinical treatment. the expression of abo antigen on platelet surface is differential, so it is usually need to ensure the consistency of the abo antigen in clinical transfusion. but in many cases, it is difficult to find the platelets that the abo blood type matched between the recipient and donor, and abo-incompatible platelet infusion is required in these cases. to data, the expression of abo antigens on platelets in normal blood group individuals is rarely reported in chinese population. aims: to understand the differential expression of abo antigen on platelet surface in population of zhejiang province, china. methods: total of 358 individuals with normal abo groups (101 group a, 100 group b and 101 group ab individuals, and 56 group o as negative control of abo antigens on platelets) were analyzed. the expression of abo antigens on platelets was determined by flow cytometry using monoclonal antibodies: fluorescein isothiocyanate (fitc)-conjugated mouse antihuman blood group a and pe-conjugated murine igg1 anti-b antibody (9431pe bgrl1). flow cytometric parameters were statistically analyzed by the mann-whitney test or the kruskal-wallis test to observe the difference in two or more groups using graphpad software v5.01. the correlation and regression analysis between a and b antigen in the platelets and rbcs were also performed by the software. population studies were reported as the mean and standard deviation (sd), and p values less than 0.05 were considered statistically significant. results: according to mfi values of abo antigens expression on platelets, the samples were divided into three groups: low expression (le), high expression(he) and moderate expression (me) according to the background mfi observed in group o samples. it was found that about 66.22% of the individuals had a weak expression of abo antigen on the platelet surface in zhejiang province. there was a significant difference in the intensity of antigen expression between these three different groups of the same blood group. for each blood group, there was a positive correlation between the intensity of abo antigen expressed on the platelet membrane and red blood cells of the individuals. results: 40 cases were found with antibody positive. among them, 6 cases (15%) were only anti-hla-i positive, 4 cases (10%) were only anti-hpa positive, 30 cases (75%) were both anti-hla-i and anti-hpa positive. 8 cases were found without anti-hla-i or anti-hpa. among the 34 cases with anti-hpa positive, the distributions of anti-gpiib/iiia, anti-gpia/iia, anti-gpib/ix, anti-gpiv were 73.5%, 61.2%, 50%, 44.1%, respectively., hla antibody positive rate in the female patients was higher than that in the male and hpa antibody positive rate in the female was lower than that in male, but there was no significance difference between them (p > 0.05). summary/conclusions: in ptr patients, the platelet antibody was mainly hla-i antibody combined with hpa antibody. background: human neutrophil antigens (hna) are polymorphic structures located on surface membrane of human neutrophils. alloantibodies against hna are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions. genotyping for human neutrophil antigen (hna) systems is an important in the diagnosis of disorders involving alloimmunization to hna. aims: the aim of this study was to investigate the hna allele frequencies among blood donors and hematological patients undergoing blood transfusions and to estimate possible hna incompatibilities and risk of hna alloimmunization. methods: a total of 303 blood donors and 302 hematological patients from the north-west region of the russian federation were recruited. dna samples were obtained and typed for hna-1, -3, -4 and -5 systems using polymerase chain reactions with sequence-specific primers (pcr-ssp). specific primers for hna were designed and the polymerase chain reaction amplification conditions were optimized. the v 2 test was used to test for the hardy-weinberg equilibrium for the hna systems. the probabilities of the incompatibility and the potential risk for alloimmunization against different hna systems after random transfusions were estimated based on the hna allele and genotype frequencies. results: in blood donors, the frequencies for the fcgr3b*01 (hna-1a), fcgr3b*02 (hna-1bd), and fcgr3b*03 (hna-1bc) alleles were 0.384, 0.584 and 0.032; for the slc44a2*1 (hna-3a) and slc44a2*2 (hna-3b) alleles, 0.804 and 0.196; for the itgam*1 (hna-4a) and itgam*2(hna-4b) alleles, 0.898 and 0.102; for the itgal*1 (hna-5a) and itgal*2 (hna-5b) alleles, 0.708 and 0.292, respectively. in hematological patients, the gene frequencies for hna-1a/1bd/bc, -3a/3b, -4a/4b, and -5a/5b were 0.376/0.588/0.036, 0.795/0.205, 0.887/0.113, and 0.699/0.301, respectively. no statistic significant difference between genotypes in these groups was observed. since the allele frequencies of hna -1, -3-5 for hematological patients and donors did not have statistically significant differences, possible hna incompatibilities and risk of hna alloimmunization were estimated based on the obtained data on the allele and genotype frequencies of hna in a group that combines donors and hematological patients (n = 605). the predicted risk of hna-1, -3, -4, -5 incompatibilities in this cohort were 34.9%, 26.9%, 19%, and 32.9%, respectively. the possible risk of hna-1a, -1bd, and -1bc alloimmunization were 0.233, 0.143, and 0.064, respectively; of hna-3a and -3b alloimmunization, 0.037 and 0.231; of hna-4a and -4b alloimmunization, 0.01 and 0.163; of hna-5a and -5b alloimmunization, 0.080 and 0.250, respectively. summary/conclusions: the information about hna gene frequencies can be used not only in blood services for detection and identification of hna alloantibodies in donors and assessment of alloimmunization risk but also for anthropological studies. background: non-invasive fetal rhd genotyping is performed using circulating cell-free fetal dna from maternal plasma sample and real-time polymerase chain reaction. this antenatal routine dna test is used to target rh-ig administration to prevent hemolytic disease of the newborn. aims: the aim of this study is to characterize maternal rhd variants responsible for indeterminate results during fetal rhd genotyping due to early amplification of at least one of the exons (5, 7 or 10) of the rhd gene. methods: 2004 samples were tested from 01/12/2017 to 01/12/2018 using free dna fetal kit â rhd. 44 samples (2,2%) yielded a premature signal for one or more exons of the rhd gene. after extraction of maternal cellular dna, the maternal rhd was characterized using rhd beadchip assay (immucor/bioarray). rhdiiia-ce(4-7)-d summary/conclusions: greater diversity is observed in the caucasian population rather than in the afro-caribbean. 65% of the identified variants are rhd negative alleles including alleles leading to partial rh2 antigen expression. unexpected alleles are found such as weak d type 1, 2, 5 or 11. these data underline the benefits of maternal rhd genotyping when abnormal early signals are detected during noninvasive fetal rhd genotyping. background: a considerable number of rhd alleles responsible for weak d phenotypes have been identified. serologic determination of these phenotypes is often doubtful and makes genetic analysis of rhd gene highly desirable in transfusion recipients and pregnant women. dna-based methods are useful for enhancing immunohematology typing in doubtful d phenotypes at pregnant women. aims: determination of the rhd gene in a cohort of pregnant women with doubtful d phenotypes. methods: determination of the rhd phenotyping was performed with microagglutination technique biorad and ortho diagnostic simultaneously. rhd genotyping was performed on 20 cases with d typing serological discrepancies with ready-to-use inno-train rbc-ready gene cde and rbc-ready gene d weak test kits based on polymerase chain reaction with sequence-specific priming (pcr-ssp) to unclear serologic findings. results: molecular analyses showed 16 of 20 (80%) pregnant women were rhd*weak d type 1 and not at risk for anti-d. rhd*weak d type 3 were typed in 3 cases (15%) and 1 case was rhd*weak partial 4.0 and potentially at risk for being alloimmunized producing anti-d allo-antibodies. summary/conclusions: appropriate classification of rhd phenotypes is recommended for correct indication of rhig in pregnant women. however, the serologic differences between rhd-negative and rhd-positive pregnant women is a real problem for unnecessary application of rhig prophylaxis in pregnant women with d variants. conclusion: antenatal rhig prophylaxis is useful in rhd negative pregnant women. with genotyping we found that 95% of serological doubtful rhd negative women was d variants that not produce anti d antibodies. in that cases those rhig prophylaxis was unnecessary and harmful as a product of human origin. on other hand there is a save up of a stock of rhig which is any way in deficit. is it time to think about cost benefit of rhig prophylaxis and genotyping in pregnant women. background: in may 2018, uk neqas (btlp) created an external quality assessment (eqa) sample designed to mimic a feto-maternal haemorrhage (fmh) bleed of 3 ml. all material used passed pre-acceptance serological testing; samples were dispatched to 298 participants in 17 countries. post-dispatch testing by flow cytometry (fc) using an anti-d marker showed a bleed volume of 1.1 ml so an investigation was initiated. aims: to determine the cause of the unexpectedly low bleed volume and what lessons could be learnt. methods: production methodology and results of pre-acceptance testing were reviewed. fc testing was repeated, plots examined, and the fmh scientific advisory group consulted for advice. further fc testing was performed at wbs using alternative markers, and the material used was investigated at ibgrl. participant results were examined to determine if the sample should be withdrawn from scoring. a questionnaire on how results were managed was sent to the 36 participants using fc with an anti-d marker. results: a material production methodology review showed no obvious cause of the erroneous in-house result. review of pre-acceptance testing images showed no issues, further d-typing of the cord showed 2 + reactions vs. two reagents by tube, cf. 4 + with two different reagents by column agglutination technology. repeat fc testing using the anti-d marker gave similar results; however, closer examination of the plots showed a left shift in the positive peak, indicating reduced fluorochrome binding, possibly due to reduced d antigen density on the cord cells. further fc testing at wbs demonstrated a marked reduction in fluorescence intensity with an anti-d marker. further investigation using an anti-hbf marker showed a bleed volume of 3.7 ml, indicating the correct proportion of cord material had been used during sample production. additional serology at ibgrl on the cord material showed reactions which were weaker than the control with 4/17 anti-d reagents. overall, the investigation supported the hypothesis that the cord material was d variant. a review of results submitted by participants mirrored the fc investigation and the sample was withdrawn from scoring, as the fc median result is used to calculate scores and the d variant cord was clearly affecting testing with an anti-d marker. the questionnaire showed that all 16 respondents examine fc plots and the gating used, but not all act on them before reporting results, and not all have a back-up plan for anti-d ig dosing in a similar situation. later sequencing of the d gene revealed the cord donor to be dvii which can have a lower than normal d antigen density. summary/conclusions: the use of a d variant cord in an eqa sample was not planned, but allowed uk neqas to highlight some important learning points: -thorough examination of fc plots is essential to avoid underestimation of fmh; a controlled procedure should be in place if modification of gates is required -access to cord/neonatal blood to allow serological investigation may be useful in a similar clinical situation -it is important to have a back-up plan for issuing anti-d ig in the event of an uninterpretable fmh result background: allo-antibodies against fetal blood group and platelet antigens produced by antigen-negative pregnant women can cause hemolytic disease of fetus and newborn (hdfn) and fetal and neonatal alloimmune thrombocytopenia (fnait). prediction of the fetus antigen status in immunized women is important for making decisions concerning further management of pregnancy. nipt is widely used for determination of fetal blood groups but determination of proper specificity in the real-time amplification of a single nucleotide polymorphism (snp), such as k or hpa-1a, requires modified protocols. droplet digital pcr (ddpcr) permits detection of low-grade fetal chimerism in maternal plasma dna with higher specificity using allelic discrimination pcr protocols. aims: to establish ddpcr protocols for non-invasive prenatal diagnostics (nipd) of clinically important blood group antigens. methods: dna was isolated from 133 plasma samples of pregnant women and donors with known genotypes (easymag, biomerieux). allelic discrimination protocols for determination of k/k (n = 29), s/s (n = 13), hpa-1a (n = 49), hpa-3 (n = 8), hpa-5 (n = 14), hpa-15 (n = 20) genotypes were performed using ddpcr method with droplet digital tm (biorad). the results of allelic discrimination performed using ddpcr were concordant with the already known phenotype/genotype of donors and pregnant women. ddpcr enabled the detection of 100-16,000 reads for total dna from plasma in tested samples. all fetal results were in agreement with antigen positive genotype of the neonates and the fetal chimerism was from 0,01% to 26,4% (one case was for advanced pregnancy -38 week of gestation). in 19/133 tested samples false positive results were detected at the level of 1 or 2 unspecific reads. summary/conclusions: the implementation of allelic discrimination protocols for ddpcr allowed detection of fetal-maternal incompatibility in k/k, s/s and hpa-1a, -3a/b, -5a/b, -15a/b antigens encoded by snp. background: in france, for pregnancies complicated by anti-d (rh1) and anti-c (rh4) allo-immunization, the tests currently used to quantitate maternal antibodies are tube method titration and continuous flow analysis determination of the antibodies concentration. recently, an automated assay was developed using the column agglutination technology on the ih-500 system (bio-rad â). aims: we wanted to evaluate the score, calculated from the agglutination profile of the antibodies on the ih-500 system, as a quantitative data to appreciate the level of maternal antibodies. methods: titers from 29 samples containing anti-d and 20 containing anti-c have been established using the semi-automated tube method performed since decades in our lab and the fully automated gel method on the ih-500 system. scores were calculated manually in both cases. antibodies concentrations were also determined for all samples by continuous flow analysis on our auto-analyzer device (evolution iii ams alliance). we looked for a possible correlation between anti-d and anti-c scores and the corresponding concentrations using the spearman correlation test. results: anti-d tube and gel scores were significantly correlated with the anti-d concentration values (p < 0.0001, r = 0.79 and p < 0.0001, r = 0.82 respectively). anti-c scores were also significantly correlated with anti-c concentration values (p < 0.0001) but gel scores have a better correlation coefficient than tube scores (r = 0.78 versus 0.55). it was easier to extrapolate gel score thresholds than tube score thresholds from the autoanalyzer values, with the aim of triggering fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery only for risk pregnancies. the determined gel score thresholds were 75 and 35, corresponding respectively to 5 ui/ml (250 uchp/ml) of anti-d and 7.5 ui/ml (500 uchp/ml) of anti-c. conclusions: calculating the score from the hemagglutination profile displayed by the ih-500 system provides added values compared to the sole reading of the titer. for anti-c immunization, gel scores are more discriminant than tube ones and better correlated to the concentration values established by continuous flow analysis. the proposed score thresholds to trigger fetal antenatal monitoring need, however, to be confirmed on more samples and to be clinically documented. background: hdnf is due to maternal igg alloantibodies directed against fetal antigens that cross the placenta during pregnancy, causing hemolysis in the fetus, anemia that can lead to edema, ascites, hydrops and, in some cases, death. the diagnosis and management of hdnf is based on maternal screening, and middle cerebral artery (mca) doppler monitoring. in severe hdnf intrauterine blood transfusions (iuts) and or exchange transfusion (et) after birth are necessary to correct anemia, to prevent and treat fetal hydrops. aims: we report eight years of experience in our immunohematology reference laboratory (irl) to highlight the importance of red cell antibody detection as a fundamental parameter to identify pregnancies with high fetal risk and to drive a correct treatment. methods: we report laboratory data from 250 pregnant women with a positive indirect antiglobulin test (iat) referred to our irl from january 2008 to december 2016. we performed antibody screening and identification by indirect antiglobulin test (iat) in microcolumn method with biovue system (ortho-clinical diagnostics, raritan, usa), and the title of antibodies in iat by tube method without additive. follow-up tests were also performed in the presence of significant red cell antibodies in order to check antibody title and begin clinical monitoring. threshold values were ≥ 1:8 for anti kell antibodies and ≥ 1:32 for other specificities. results: out of 250 women, 143 (57.2%) displayed clinically significant antibodies, 92 (36.8%) clinically insignificant antibodies and 15 (6%) natural antibodies of different specificities. among women with clinically significant antibodies the most frequent was anti-d (16.8%) also in combination with other rh antibodies (8.8%), while anti-k accounted for 10%, anti-e for 10% and antibodies against high-incidence antigens for 2.4%. anti-m and anti-le a antibodies were also found (16.8% and 8% respectively) but they were not clinically significant. among 143 women with clinically relevant antibodies, 37 showed a critic antibody title and they underwent gynecological and obstetric monitoring. 21 fetuses resulted affected by hdfn, displaying anti-d in 16 cases and anti-kell in 5. 11 fetuses with severe hdfn (anti-d in 7 and anti-kell in 4) required iuts, 2 were treated with et, 8 received red blood cells units at birth. summary/conclusions: the mother screening program led to important improvements in the outcomes of hdfn. the identification of women with clinically significant antibodies allowed an appropriate monitoring program and therapy. background: the hemolytic disease of the fetus and newborn (hdfn) is a severe disease, resulting from maternal erythrocyte alloantibodies directed against fetal erythrocytes. alloimmunization in pregnant women has been found to range from 0,4% to 2,7% worldwide. there are over 400 erythrocyte surface antigens, of which more than 43 have been reported to be associated with hdfn. although anti-rhesus d was once the major etiology of hdfn, the universal introduction of antenatal and postpartum rh immunoglobulin has resulted in a marked decrease in the prevalence of alloimmunization to the rhd antigen in pregnancy. consequently, alloantibodies other than anti-d emerged as an important cause of severe hdnf, in particular anti-k and anti-c. however, there are other antigens that have also been found to be associated with hdfn. aims: retrospective identification of erythrocyte antibodies in pregnant women in hospital de braga in 2016 and 2017. methods: this study was planned to assess the prevalence of erythrocyte antibodies responsible for alloimmunization, excluding abo-immunizations, in pregnant women attending the antenatal clinics of hospital braga during 2 years, from january 2016 to december 2017. in this study, we retrospectively evaluated the erythrocyte antibody screening results of pregnant women. women with positive erythrocyte antibody screening also underwent identification with gel card system following the manufacturer's instructions (diamed â ). the outcomes of infants, whose mother's indirect antiglobulin tests were found to be positive, were examined. direct antiglobulin tests, jaundice and phototherapy history, transfusion and mortality of the newborns were recorded. results: during the study period, 5982 pregnant women were attended in hospital de braga. the laboratory registered 100 positive erythrocyte antibody screening tests. the prevalence of positive erythrocyte antibody screening was 1,7%. anti-d was the most common antibody found (58,5%). anti-d prophylaxis given during pregnancy was responsible for 51 of 62 cases and maternal antibody titer levels did not exceed 8 among these cases. the prevalence of non-rhd immunization was 33%. anti-e (9,4%) was the most frequent alloantibody other than anti-d followed by anti-m (5,7%) and anti-c (4,7%). multiple maternal antibodies were found in 5 pregnant women. four women had 2 types of alloantibodies: anti-c and anti-e; anti-c and anti-d; anti-k and anti-cw; anti-e and a non-identified antibody. one pregnant had 3 types of alloantibodies: anti-d, anti-c and anti-e. of all cases of newborns whose mothers had a positive antibody screen tests, icterus occurred in 45% of them and phototherapy was given in 14%. summary/conclusions: the prevalence of positive erythrocyte antibody screening in hospital de braga was 1,7%. the erythrocyte antibody screening showed that anti-d was the most common antibody found (58,5%) in most of the cases because of anti-d prophylaxis. the prevalence of non-rhd immunization was 33%. the other most frequent alloantibodies were anti-e (9,4%), anti-m (5,7%) and anti-c (4,7%). an increasing prevalence of non-anti-d alloimmunization was found and there are currently no preventive strategies. in contrast to rhd alloimmunization, the main risk factor for non-anti-d alloimmunization is a previous transfusion therapy. thus, it is important to minimize the exposure of women to incompatible erythrocyte antigens through unnecessary transfusions when possible. background: the mns blood group system is one of the most complex blood group systems. although alloanti-m is a common antibody observed in pregnant women and could also be found in the serum of individuals who have not been exposed to m positive erythrocytes, it is rarely clinically significant and has been regarded as an unimportant antibody to cause hemolytic disease of the fetus and newborn (hdfn), especially in caucasian and black ethnic groups, for a long time. however, an increasing number of cases of severe hdfn resulting in fetal hydrops and recurrent abortion caused by alloanti-m have been reported mainly in the asian population, especially in the japanese and chinese populations. aims: to summarize the characters of serological testing in preterm twins newborns suffered with severe hdfn. methods: the blood sample of two newborns with severe hdfn and the mother, who had the history with three hydrops fetus, were collected. abo, rhd, rhce, and mn blood group typing of the twins newborn and their mother were performed in saline with tube or gel card. direct agglutination test (dat), elution test, antibody specificity identification and antibody titer detection were conducted by iat method in gel card. results: o, rhd(+), and ccee blood groups were identified both in the mother and the twins newborn. background: in france, since may 2018, the legislation does not promote anymore the use of the reference tube method for titration of anti-red blood cells antibodies. this opened the way to the use of newly developed automated anti-red blood cells antibodies quantitation by column agglutination technology. aims: we wanted to assess the performance of titration and scoring by the id-gel test on the ih-500 system (bio-radâ) and to compare it with the performance established for the reference tube method, used in our lab since decades. another objective of the study was to determine titer thresholds for the gel method, to trigger fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery. methods: an home-made internal quality control (iqc) prepared and calibrated using the international anti-d standard (01/572) was used to determine the intraassay and interassay imprecisions, regarding the score and the titer results. patients samples for testing were chosen during the 2-months assay period, regarding the specificity of the antibodies and the tube titer in order to cover a wide range of 2 have lower values. the highest differences (more than 2 to 3 dilutions higher) were seen for antibodies directed against rh system antigens. among the other specificities, anti-k (kel1) and anti-m (mns1) antibodies show the most samples with equal or lower titers compared to the tube method. conclusions: automated anti-red blood cell antibodies titration by column agglutination technology on ih-500 system shows better intra and interassay cvs compared to the tube method. it is explained by the fully automated process that includes the reading step. titer results are almost always higher with the gel technology. thus, it seems possible to safely extrapolate the titer thresholds defined for anti-red blood cells antibodies by the tube method to the gel method. however, based on future clinical studies and fetal/neonatal outcomes, it would probably be necessary to increase these thresholds, at least for anti-rh antibodies, in order to avoid heavy, expensive, stressful and useless monitoring of some pregnancies. results: the first case was a 1-day-old female infant, yellowish skin developed the next day after birth. her capillary bilirubin level was 13 mg/dl, the evidence favored neonatal hyperbilirubinemia and the clinical manifestation revealed hemolysis symptoms. her laboratory findings showed elevated reticulocytes (15.2%), ldh (1162 iu/l) and g6pd (25.3 u/ghb); dat (+/-), iat (-), anemia (hb 8.7 g/dl, hct 28%), and blood smear showed anisocytosis, spherocytes, and polychromatic rbc. her mother blood typed o, d positive, while her blood type was b, d positive and anti-b was found from her elution rbcs (3 + ). due to rarely severe anemia with abo incompatibility, maternal plasma was analysed for abo igg antibodies and showed high antibody a and b titre with 1:1024 and 1:2048. the female infant received one unit washed-prbcs for anemia and intensive phototherapy for hyperbilirubinemia. her clinical condition improved significantly, hb rose to 14.6, bilirubin level was within normal range, she was discharged. another 5-days-old male infant was our second case. on the third day after birth, yellowish skin discoloration developed and bilirubin level was 15 mg/dl. two days later, his transcutaneous bilirubin (tcb) measurement data was high and laboratory findings also showed raised reticulocytes (5.2%), dat (+/à), iat (à), hb 12. background: anti-indian b is a rare alloantibody against the high frequency antigen in b . individuals with the in:1,-2 phenotype (in(a+b-)) are observed with a frequency of < 0.1% in the indian population and have not been described in caucasians. the majority of anti-in b antibodies have been reported in individuals without previous transfusions, indicating the possibility of a naturally occurring antibody. anti-in b is considered clinically significant and haemolytic reactions after in b -incompatible transfusions have been reported. haemolytic disease of the foetus and newborn (hdfn) due to anti-in b has not been described. however, a positive direct antihuman globulin test (dat) may be observed. aims: to describe the challenges of managing a pregnancy and childbirth of a woman with an anti-in b . methods: serological investigations were performed by iat (tube and column agglutination). papain and trypsin treated cells were also utilised. soluble recombinant in blood group proteins (in-rbgp) (inno-train, germany) were used in neutralization tests. the clinical significance of the anti-in b antibody was determined by monocyte monolayer assay (mma). genomic dna was isolated from whole blood and the samples were further characterized by pcr amplification and sanger sequencing of exon 2 of cd44. results: in a 27-year-old pregnant (para 1) woman of indian origin without previous transfusions, an alloantibody of the specificity anti-in b with a titer of 1:2 was detected by iat (negative with papain-treated cells) at gestational week (gw) 32 and 36. the mma, performed in duplicate on samples taken at these dates, showed a mi of 0.5%/4.8% and 7.7%/6.3% respectively. the mi was interpreted as follows: 0-3% not relevant; 3-5% inconclusive; >5% clinical significant. the patient's parents were typed heterozygous, in:1,2 whereas her husband was homozygous, in:-1,2. due to the husbands phenotype, the fetus was predicted to be in b positive. doppler flow measurement of the peak systolic velocity in the middle cerebral artery of the foetus was normal. delivery took place at gw 41 without increased bleeding. the neonate presented no clinical manifestation of hdfn. neither the mother nor the baby required blood transfusions. summary/conclusions: we report the case of a pregnant woman of indian origin with an anti-in b alloantibody. the first mma, performed in gw 32, was inconclusive whereas the second mma, performed in gw 36, indicated that the antibody was clinically significant. if the mi-increase is only due to the pregnancy or has also a clinical significance, cannot be stated. in b negative blood components were not available and the patient's relatives were all in b positive. therefore, measures to avoid transfusions, including optimised peripartal management of haemostasis, was of utmost importance. with only few cases published, the risk of hdfn could not be excluded with certainty. an intrauterine investigation by doppler was performed to exclude relevant anaemia of the fetus. no transfusion was needed at delivery as there were no haemorrhagic complications. the neonate presented no clinical signs of hdfn. background: hemolytic disease of the fetus and newborn (hdfn) is a disease which if untreatedcan cause perinatal mortality and morbidity with a substantial risk for long-term sequela. in albania we lack of studies in this field. aims: the aim of this study is to determine the predictive value and the reliability of the "critical titre" during the evaluation of red cells alloantibodies ability to cause the hemolytic disease of fetus and newborn. methods: we conducted a descriptive, cross-sectional study. the data were collected in the university hospital for obstetrics and gynecology in albania. in the study were included 20 immunized pregnant woman for anti-d antibodies and their newborns which were affected from the hemolytic disease of fetus and newborn. the data belong to the period 2013 and 2018. results: the "critical titre" in our study was 8, meaning that this was the minimal value of the titre antibodies that could cause hemolytic disease of fetus and newborn. our study concluded that only 2 newborns were born without the hemolytic disease of fetus and newborn and the titre values were less than 4. moderate hemolytic disease of fetus and newborn were caused between the titre values 8-32. the summary/conclusions: the titre values of the mothers are a predictive option of the high risk of giving birth to a child with the hemolytic disease of fetus and newborn. it is recommended that in this cases the mother should be followed with doppler ultrasonography to measure the blood flow of the middle cerebral artery. also the doctors should recommend in pregnant women with positive coombs test not only the identification of the anti-d antibodies but also the identification of the other antibodies such as anti-e, anti-c, anti-k. background: rhd-negative pregnant women with allo-anti-d are at risk of having a fetus affected by haemolytic disease of the fetus and newborn (hdfn) where the fetus is rhd-positive. the rhd allele is highly polymorphic and many rhd variants give rise to an array of partial d phenotypes. the clinical significance for many partial d phenotypes is not well-established. rhd genotyping by non-invasive prenatal testing (nipt) to assess the fetal rhd status determines whether the fetus is at risk for hdfn. nipt tests also include strategies for detecting maternal rhd variants to provide for accurate reporting. however, the presence of a paternal rhd variant, while having the potential to confound nipt interpretation, is often not recognised. we report a "trio" family study triggered by a request for nipt for an rhd-negative pregnant mother, 16 weeks gestation, who presented with allo-anti-d and anti-jk a antibody. subsequent paternal and fetal rhd genotyping was conducted and revealed a novel variant rhd allele. aims: we aim to characterise the paternal rhd allele and review clinical case features. methods: rh phenotyping was performed by standard serological procedures. nipt tested for fetal rhd exons 4, 5 and 10. rhd genotyping on whole blood/cord blood dna was performed on the immucor bioarray rhd beadchip kit which predicts a rhd phenotypic variant of best fit. dna sequencing was performed using the illumina trusight one sequencing panel. copy number variation (cnv) analysis was used to assess the rhd exon structure and zygosity. results: the paternal red cells typed as group o rhd+c-c+e-e+, (ror). nipt genotyping detected fetal rhd signals for all 3 exons, predicting rhd-positive. no maternal rhd sequences were detected consistent with homozygosity for the rhd deleted haplotype. for both paternal and cord genomic dna (gdna), beadchip genotyping predicted a rhd variant "diiia/cehar". furthermore, signal drop out was observed at 3 nucleotide positions (c.1154, c.1193, c.1227) located in rhd exon 9 suggesting exon 9 was either deleted or rhce-replaced. paternal and cord gdna sequencing detected 4 out of 6 snps (c.186g>t, c.410c>t, c.455a>c, c.602c>g) associated with diiia phenotype plus 2 additional snps (c.604g>a, c.733g>c) on the rhd gene. both were rhd hemizygote by cnv analysis. no rhce variants were detected. clinical case features: the maternal anti-d quantitation increased from 5.3 iu/ml (16 weeks gestation) to 166 iu/ml (33 weeks gestation). the fetus required 3 intrauterine transfusions during the pregnancy to manage the hdfn. summary/conclusions: both father and fetus carry an rhd allele that does not align with alleles encoding diii phenotypes. this putative novel rhd variant allele comprises snps associated with diiia and with a possible exon 9 deletion/rhcereplaced. a similar allele was reported in literature, although based on sequence analysis only, with no phenotype data. the variant allele here encodes rhd-positive phenotype and we predict that there may be a loss of d-epitopes. notwithstanding, the clinical presentation shows that maternal anti-d against this rhd phenotype (presumed partial) is associated with a severe hdfn and that such rhd blood group phenotypes are of clinical significance for alloimmunised pregnancies. abstract withdrawn. background: cd109 is a glycosylphosphatidylinositol (gpi)-anchored protein with apparent molecular mass of 170 kda. in addition to being expressed on human plts, cd109 is expressed on activated t-cells, endothelial cells, cd34 + hematopoietic stem cells as well as on progenitor cells. in the chinese population, the calculated allele frequencies of hpa-15a and -15b are 0.505 and 0.495, respectively. based on these data, the risk of alloimmunization against hpa-15 alloantibodies due to incompatible plt transfusion or pregnancy is expected to occur in relatively high frequency. however, until today there is no report of hpa-15 alloimmunization in the chinese population. in this study, we analyzed sera from hydrop fetus cases by maipa technique and icfa. aims: to detect the anti-hpa 15b alloantibodies by maipa and icfa. methods: a 24-year-old mother, gravida 2/para 0. the mother in the first pregnancy was diagnosed hydrop fetus at pregnancy 33 weeks by ultrasound. in the second pregnancy, fetal hydrops was observed by ultrasound at pregnancy 24 weeks. the mother's irregular antibody test was negative. the maternal platelet specific antibodies and hla antibodies were negative. blood routine and morphological examination of fetal umbilical cord blood showed that plt count dropped to 4.7 9 10 10 /l, wbc count dropped to 2.96 9 10 10 /l, including neutrophil 37%, lymphocyte 16%, mononuclear 43%, eosinophil 2%, basophil 2%, red blood cells were normal, hb was 111 g/l. screening for hla and plt-specific antibodies was performed using a elisa-based plt antibody kit (pakplus, gti diagnostics) as recommended by the manufacturer. plt antibodies were detected by icfa and maipa.hpa genotyping was detected by cpr-ssp. results: the fetus's genotype was hpa-1a/a, -2a/a, -3a/a, -4a/a. -5a/a, 6a/a, 7a/a, 15a/b, naka (+) and the maternal was hpa-1a/a, -2a/b, -3a/a, -4a/a. -5a/a, 6a/a, 7a/ a, 15a/a, naka (+). the paternal genotype was hpa-1a/a, 2a/b, 3a/a, 4a/a, 5a/a, 6a/a, 7a/a, 15a/b, naka (+), which was the only incompatible antigen compared with the maternal hpa. samples were tested using the fresh plt panels consisting of hpa-15aa and -15bb homozygous donors. the reactivity of the negative control and the mother's sera with the plts from hpa-15a/a (donors 1), hpa-15a/b (donors 2) and hpa-15b/b (donors 3) donors by maipa. the mother's serum showed no reactivity against 15a/a plts, weak positive reactivity against 15a/b plts (od values 0.28), but strong reactivity against 15b/b plts (od values 0.38).this finding could be confirmed by one of the reference plt laboratories (japanese red cross kanto-koshinetsu block blood center, japan) using freshly isolated plts from hpa-15genotyped donors (anti-hpa-15b average value 9.8). summary/conclusions: in this study, we found anti-hpa-15b in a case of fnait (patient hpa-15aa, blood group o) using the maipa technique. we were able to detect the presence of hpa-15b alloantibody in one case of nait. background: fetal and neonatal alloimmune thrombocytopenia (fnait) occurs in 1:10000 live births in caucasians. serological and molecular human platelet antigens (hpa) genotyping tests are performed to investigate and conclude to fnait diagnosis. however, in few cases and particularly in case of suspicion of private platelet antigen, some specialized analyzes must be performed in the laboratory (lab). these analyzes can range from sanger or ngs sequencing to platelet serology with transfected cells. aims: the aim of our study was to explore where the frontier between research and care takes place in the field of platelet immunology through the prism of the fnait investigations carried out by the platelet immunology laboratories. methods: a two-part electronic survey have been sent to foreign platelet immunology experts (pie) from platelet immunobiology working party (piwp) members and espgi board members (n = 40). the first part focused on the lab practices and regulatory environment regarding to accreditation, contact with patient, informed consent and patient results. the second part stressed on the investigations performed to discover new platelet antigen and more precisely on the perceived status of these analyzes ( background: haemolytic disease of the fetus and newborn (hdfn) can occur when maternal red cell antibodies, directed against red cell antigens present on the fetal red blood cells, cross the placenta and enter the fetal circulation. in a "traditionally" conceived pregnancy, when hdfn occurs, it is as a result of maternal antibodies directed against fetal red cell antigens in the heterozygous state, whereby the antigen is inherited from the father only. with the advent of donor oocyte (do) in-vitro fertilisation (ivf), the addition of a third person into the reproductive equation allows for the possibility of a more severe form of hdfn when fetal red cell antigens are present in the homozygous state (one copy from father and one copy from donor) and maternal antibodies are directed against these. antigens expressed in the homozygous state will have more antigens sites per red blood cell and therefore are at an increased risk of red cell destruction from the maternal derived cognate antibodies. aims: to raise awareness of increased severity risk of hdfn in donor oocyte conceived pregnancies. methods: we describe two unusual cases of hdfn in our institution of two women whose pregnancy was induced using a donor oocyte and their offspring requiring transfusion support in the postnatal period to treat hdfn. results: the first is a case previously reported (doyle, quigley, fitzgerald et. al. transfusion medicine, 2014 ) of protracted hdfn due to anti-c, managed with phototherapy initially, then intervention with red cell top-up transfusion at 4 weeks post-delivery. the second is an unusual case of severe abo hdfn requiring exchange transfusion therapy (pre-publication). summary/conclusions: given the increased number of pregnancies conceived using do we recommend that antenatal guidelines are reviewed to create awareness regarding the potential increased risk of hdfn in do pregnancies complicated by allo-immunisation. critically, antenatal testing guidelines should highlight that the predicted outcomes associated with quantitation/titres can only be used when do has not been used to obtain the pregnancy. it is also essential that clinicians inform the blood transfusion laboratory when do has been used. abstract withdrawn. 19%) are deceased due to organ rejection, and 8/36 patients (22%) are deceased due to disease not related to rejection. summary/conclusions: the use of therapeutic plasma exchange for the treatment of antibody mediated rejection in solid organ transplant is safe and effective when used along with other treatment modalities. further studies will help determine whether it can be reproduced in larger cohorts and whether it is more effective in certain organs. background: extracorporeal photopheresis (ecp) is an important cellular therapy for the treatment of several (auto-)immune diseases such as graft-versus-host disease. the international standard for the ex vivo treatment of the leukapheresis product is the application of 8-methoxypsoralen (8-mop) and irradiation with uv-a light. however, the addition of 8-mop to the illumination bag is associated with a potential risk of contamination. aims: the basic principle of the ecp is the induction of apoptosis in the leukocytes. our aim was to find an alternative for the conventional apoptosis induction without the need of external substance application. the objective of the study was the investigation of the apoptosis levels and kinetics in leukocytes after treatment with 8-mop+uv-a compared to uv-c treatment without additional 8-mop. methods: we used an in vitro 72 h cell culture approach with human mononuclear cells from healthy blood donors. untreated control cells were compared with 0,2 lg/ ml 8-mop plus 2 j/cm 2 uv-a treated cells and 2 j/cm 2 (effective dose) uv-c treated cells. apoptosis in several leukocyte sub-populations was detected daily with annexin v and 7-aad flow cytometry standings. results: the apoptosis analysis of cd3 cd4 t-helper cells, cd3 cd8 cytotoxic tcells, cd19 b-cells, cd14 monocytes, cd3 neg cd56 nk-cells and cd3 cd56 nkt cells revealed no statistical differences in almost all of these cell types after treatment with 8-mop/uv-a or uv-c light. the apoptosis kinetic as well as the final apoptosis after 72 h were similar in both treatment groups. summary/conclusions: the addition of 8-mop to the photopheresis irradiation bag is a risk for potential infections. the main effect of the 8-mop/uv-a treatment is most probably the induction of apoptosis in the leukocytes. here, we provide information that this induction of apoptosis can also be achieved with uv-c irradiation without the need of 8-mop addition. the apoptosis patterns in most leukocyte subpopulations are very similar after treatment with uv-c compared with 8-mop/uv-a treatment. future in vivo studies are needed to prove the therapeutic effect of uv-c treated cells in the ecp setting. abstract withdrawn. background: therapeutic plasma exchange (tpe) is performed to remove the implicating substances from the plasma causing the disease. a periodic appraisal of tpe data is important to get insight into the procedural related effects and toxicities and overall outcome in order to have a guided future approach. aims: the purpose of this study is to observe the overall profile and outcome of the patients receiving the tpe in the medicine intensive care unit (micu) of a tertiary care hospital in south india. methods: a record based audit was conducted for the all the patients who were admitted to our tertiary care hospital of south india with 16 bedded micu and received tpe therapy between 1 june, 2016 and 31 december 2018. all the tpe procedures were performed using haemonetics multicomponents system (mcs) + ln9000 apheresis system based on intermittent flow centrifugation. we audited our tpe for: number of treatments, clinical indications, treatments prescribed and administered, any procedural or patient complications, and adherence to current best practice recommendations. results: sixty nine patients had undergone 269 tpe procedures. among them, thirty were female patients (43%). the median age 45 (13-75) years. guillain-barre syndrome (gbs) was the most common indication (72%) followed by cases of thrombotic thrombocytopenic purpura, diffuse alveolar hemorrhage, myasthenia gravis, autoimmune encephalitis and hypertriglyceridemia respectively. the tpe regimens received by patients in this icu were not always prescribed in accordance with current best practice recommendations. there were 48 (18%) episodes of patient related complications during the tpe treatments. in 8 (3%) procedures, technical error in the machine was encountered. summary/conclusions: the findings of this audit have identified differences between the current prescription recommendations for tpe and those applied. the infrequency of the therapy and the different indications may present a challenge for medicine intensive care clinicians to provide best care in all cases. background: microangiopathic hemolytic anaemia (maha) encompasses a spectrum of disorders characterised by widely disseminated thrombosis in small blood vessels resulting in formation of schistocytes and concomitant thrombocytopenia. plasma exchange (pe) needs to be considered as empirical and urgent life saving therapy in these disorders irrespective of waiting for specific testing like adamts 13 levels in thrombotic thrombocytopenic purpura (ttp) or complement levels or factor h antibodies in atypical hemolytic uremic syndrome (ahus). aims: to assess the efficacy and safety of plasma exchange in patients diagnosed as having microangiopathic hemolytic anaemias. methods: a retrospective analysis of all pe procedures performed in patients diagnosed as having maha was done over a period of 9 years (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . procedures were done on apheretic device (cobe spectra, terumo bct, lakewood co. usa). patients' pre and post procedural hematological and renal parameters were analyzed by applying paired t test. adverse event if any was recorded. results: pe was performed in 46 patients with diagnosis of maha (27-ahus, 16 -ttp, 1 each of post stem cell transplantation drug induced thrombotic microangiopathy (tma), post thyroidectomy tma and post-partum tma). the mean age of patient was 19.94 ae 19.58 years with m:f as 1.5:1. number of procedures per patient varied from 1 to 27. post pe recovery was observed within 10-14 days with statistically significant increase in mean platelet count from 40.05 ae 5.9 to 82.11 ae 12.10 9 10 9 /l (p = 0.000) and significant decline in mean lactate dehydrogenase level from 48.91 ae 34.73 to 10.98 ae 6.49 lkat/l (p = 0.000). there was also significant decline in mean percentage of schistocytes in peripheral smear from 5.44 ae 3.96% to 0.56 ae 0.89% (p = 0.000). the mean serum urea changed from 48.88 ae 24.56 to 24.50 ae 17.78 mmol/l and creatinine from 266.11 ae 142.59 to 173.09 ae 127.34 lmol/l (p = 0.000 and 0.001 respectively) with significant increase in urine output from 0.71 ae 0.53 to 1.06 ae 0.33 ml/kg/h (p = 0.000). adverse events were observed in 10 patients (21%), allergic reaction to replacement fluid (n = 6) being the commonest followed by hypotension (n = 2), rigors and chills (n = 2). overall survival rate at 6 months was 89%. summary/conclusions: pe had proven its safety and usefulness as life-saving first line treatment modality in maha. prompt and aggressive treatment helps in achieving early and complete remission in these patients. background: neuromyelitis optica (nmo) also known as devic's disease or devic's syndrome is a rare demyelinating disease of the central nervous system that most often results in selective involvement of the optic nerves (optic neuritis) and spinal cord (myelitis)and has female preponderance. neuromyelitis optica (nmo) attacks are poorly controlled by steroids and evolve in stepwise neurological impairments. assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. aims: to study the effect of tpe in neuromyelitis optica. methods: a 17 year old female in the medicine department, civil hospital, ahmedabad admitted with chief complains of weakness and numbness in the arms and legs, blurred vision, reduced sensation, difficulty in controlling bladder and bowels, uncontrollable vomiting and hiccups since 2-3 days in the medicine department, civil hospital, ahmedabad. attacks were treated with short courses of high doses of intravenous corticosteroid -methylprednisolone intravenous. but there was no clinical improvement. results: clinician advised for the trial of tpe in this patient. the procedure was performed by automated device with continuous flow centrifuge machine fresenius kabi-com.tec using double lumen femoral catheter. after obtaining informed consent from the relative of the patient, 6 cycles of tpe were performed on daily basis. after 6 cycles, both subjective and objective clinical response to tpe was estimated by three different sources (the patient, a transfusion medicine physician, and the treating neurologist). [1] for motor performance, patient was assessed on a disability scale (0 = healthy; 1 = minor symptoms; 2 = able to walk 5 meters without support; 3 = able to walk 5 meters with support; 4 = confined to bed or wheelchair; 5 = requiring assisted ventilation; 6 = dead).patient's motor performance was increased to scale 1(upper limb) and 2(lower limb) from scale 5, deep tendon reflexes were normal. visual function began to improve 1 week after the treatment. visual acuity was 6/6 after 4 weeks. summary/conclusions: assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. this suggests that tpe is beneficial in nmo patients during acute attack if there is no response to corticosteroid treatment. background: babesiosis is a tick borne infectious disease caused by the protozoa babesia. while most infections with babesia are asymptomatic, some patients present with a symptomatic infections and rarely this can be a severe life threatening illness. treatment is primarily with antibiotics but red cell exchange (rce) has been used in the more severe cases which are characterized by high grade parasitemia, evidence of severe hemolysis and or multi-organ failure involving the kidney, lung or liver. a threshold parasite level of 10% has arbitrarily been applied as an indication for rce, however, this threshold is not evidence based. aims: to report on patients with babesiosis and high grade parasitemia who were treated with antibiotics only without rce methods: data were collected from july 2014 to july 2018. a case was defined as a patient diagnosed with babesiosis for whom rce was requested on the basis of a parasitemia of > 10% but on clinical evaluation it was considered that rce could be withheld and the patient monitored awaiting response to antibiotics. results: three cases of severe babesiosis in which the use of rce was requested on the basis of a parasite level of greater than 10%, but was not performed. the rce was deferred on account of the good clinical state of the patient and the absence of renal failure. levels of parasite at diagnosis were 10.6%, 11% and 31%. all patients were followed daily until discharge. two of these patients had been splenectomized and each received a single unit of red blood cells during the hospitalization. the third patient had a long history of refractory lymphoma and was pancytopenic requiring multiple transfusions during the years before the diagnosis of babesiosis. she had transfusion transmitted babesiosis from a red blood cell transfused 46 days prior to the diagnosis. all three patients responded well to antibiotics and were discharged between 9-16 days with undetectable parasites. summary/conclusions: this small case series suggests that requests for rce solely on the basis of an arbitrary level of parasitemia should be questioned and the clinical state and evidence of organ failure considered in the decision to perform rce. abstract withdrawn. chronic transfusion program (ctp) remains the gold standard therapy for stroke prevention and for patients with a severe disease who have inadequate response to hydroxyurea treatment. aims: to evaluate the safety, efficacy and cost between scd patients on ctp that underwent both aet and partial manual exchange transfusion (pmet) procedures. methods: retrospective observational cohort study of patients with scd on ctp that have switched between pmet and aet. this study was carried out from 01/01/2017 to 31/12/2018 in a hospital in portugal. data on patient history, haematological values, duration of the procedure, intervals between them, adverse events as well as the cost of material and working hours were collected and compared between both procedures. results: a total of 6 patients met the inclusion criteria described. however, 1 patient was excluded from our study because of the lack of attendance to the ctp. during the study, we recorded 88 exchange procedures (42 pmet and 46 aet), both on peripheral venous access. from all those procedures the major concern was the poor venous access, which was the reason why 2 patients had returned to pmet. no major complication or alloimmunization was observed. the indications for ctp were cerebral vasculopathy (n = 2), stroke (n = 1) and recurrent vaso-occlusive crisis with multiorgan failure (n = 2). for both procedures, target values were to obtain a pre-exchange hbs level ≤ 30% for stroke and cerebral vasculopathy and ≤ 50-60% for other indications. the median hbs level before pmet was 42,6% (31,3-59,1) and 38,4% (18, 9) before aet. we documented a higher hbs level prior to the next procedure in 11,4% of patients (n = 10). despite that all patients remained stable without any major scd related event. both procedures were well tolerated and iron overload was well controlled (median ferritin level pmet: 1356,1 vs. aet: 1314,7 ng/ml). the duration of the exchange procedure was longer and the intervals between procedures were shorter with pmet (median pmet: 360 vs. aet: 60 min and pmet: 21 vs. aet: 24 weeks, respectively). annual rbc requirements per procedure were superior (median 2 vs. 4 units) and the overall costs related with aet were 2,2 times higher -18.180,93€ and 8.174,79€ aet and pmet, respectively (estimated cost per session aet: 790,48€ and pmet: 389,28€). summary/conclusions: our study shows, that the hbs level before both procedures, performed during the same interval, was similar. we verified that pmet has a comparable efficacy with aet in terms of preventing the development or progression of chronic complications and that the cost per procedure is significantly higher with aet. however, in a clinical situation where it is important to rapidly reduce the hbs level, and/or where the control of the target hbs is stricter so that the patients are clinically controlled without an increase in hospital visits, aet is preferred. we conclude that aet is more effective in the rapid reduction of hbs and ferritin levels, as well as being less time consuming. despite this, for the reasons described above, it is more cost-effective to maintain both aet and pmet procedures. background: erythrocytapheresis/red blood cell (rbc) exchange, involves removing of a large number of rbcs from the patient and returning the patient's plasma and platelets along with compatible allogenic donor rbcs. typical indication for rbc exchange is sickle cell disease and its related complications. however, one of the miscellaneous indications of rbc exchange is for the patients of methemoglobinemia who are refractory to treatment by methylene blue. acquired methemoglobinemia is more common than any genetic causes. acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds. for patients failing to respond to standard treatment with methylene blue or in whom its use is contraindicated; hyperbaric oxygen or rbc exchange is indicated aims: case reports on use of rbc exchange in methemoglobinemia are few and indications are based on anecdotal reports. methods: exchange was performed on the cell separator machine, com tec by fresenius kabi. results: we report a case of acquired methemoglobinemia where patient was admitted with peripheral capillary oxygen saturation (spo2) of 87% on air. the patient did not show improvement in spo2 level with effective emergency treatment of methylene blue. since, the patient was refractory to treatment with methylene blue, the decision was made by clinician to proceed with rbc exchange. the patient improved significantly after two cycles of one rbc volume automated rbc exchange, and was discharged with spo2 of 97% on air. summary/conclusions: automated rbc exchange can be used in patients of acquired methemoglobinemia successfully when methylene blue is ineffective, and may be superior to manual one. background: therapeutic plasma exchange (tpe) is known to disturb the ph and electrolyte status. patients with compromised liver functions may be at a higher risk of electrolyte imbalance due to metabolic abnormalities. aims: the aim of this study was to analyze the variation in ph, ionized calcium, sodium, potassium, and bicarbonate in liver disease patients undergoing tpe. methods: patients with liver disease undergoing tpe during the period from july 2016 to august 2017 were included in the study. data on patient demographics, details of the tpe procedure, blood gas analysis report and adverse effects of tpe (if any) were collected and analyzed. results: one hundred and seven procedures were done during the study period; of these 46 (43%) were done on the mcs plus (haemonetics corporation) and rest 61 (57%) were done on the spectra optia (terumo bct). the percentage change in ionized calcium, sodium, and potassium due to the procedure was found to be statistically significant (p = 0.000). the systolic (p = 0.010) and diastolic (0.001) blood pressure also changed significantly with the procedure. the predictors for the change in ionized calcium were found to be pre-procedure ionized calcium (p < 0.001), the age of the patient (p < 0.001) and the pre-procedure ph (p = 0.002). procedurerelated complications occurred during 30 procedures of which 13 complications (12.15%) were categorized as features of hypocalcemia. no association was found between hypocalcemic manifestations and pre-procedure calcium, change in calcium, age or gender of the patient. summary/conclusions: the tpe procedure in liver disease patients causes remarkable changes in ionized calcium, sodium, potassium and bicarbonate ions. the decrease in ionized calcium during the procedure is predicted by pre-procedure ionized calcium levels, ph and age of the patient. monitoring of these parameters and appropriate corrective measures are imperative to patient safety. background: therapeutic plasma exchange (tpe) in pediatric age group is technically demanding because of low blood volume, difficult venous access and poor cooperation of the patient during the procedure. we here present our experience of tpe in pediatric patients from our centre. aims: to assess the challenges during tpe in pediatric patients and formulate appropriate strategies. methods: we did retrospective analysis of all tpe procedures performed in pediatric patients over a period of 16 years (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . tpe procedures were done on two different apheretic devices (cs 3000 plus, fenwal usa and cobe spectra, terumo bct lakewood, colorado) daily or on alternate days depending on clinical condition of the patient. for all procedures, kit was primed with compatible packed red cells. adverse events during the procedure were noted and analyzed. results: a total of 356 tpe (range 1-22/patient with mean of 6.2 procedures) were performed for 55 pediatric patients with different indications like atypical hus (category i as per american society for apheresis (asfa) in total 44 patients, neuromyelitis optica (category ii) in 4 patients, rapid proliferative glomerulonephritis (category i), c3 glomerulopathy in 3 patients each and one patient of infective hemophagocytosis. the average age of patient population was 7.8 yrs (1.2-13 years) . the male:female ratio was 3:1 with an average weight of 25.5 kgs. adverse events were observed during 20 (5.61%) procedures. most commonly observed adverse events were allergic reaction to replacement fluid (1.4%) followed by hypotension (1.1%), line occlusion (0.8%), vasovagal, endotracheal tube blockage and symptomatic hypocalcemia was observed in one procedure each (0.28%).there was no corelation observed between physical parameters of patient with adverse events. all adverse events were managed as per departmental standard operating procedures (sops) and procedures were completed successfully except in one where the procedure was abandoned. no mortality was observed during the procedures. background: the hemoglobin (hb) content of packed red blood cell (prbc) units is heterogenous. the patient's blood volume is also variable which can be calculated based on the weight, height and body surface area (bsa) of the patient. the efficacy of a transfusion episode can be assessed if the hb content of prbc is known and the patient's post-transfusion hb increment is determined. aims: this prospective study was performed to compare the efficacy of the transfusion of prbcs based on hb content versus the standard transfusion practice in thalassemia major patients. we also determined the correlation between hb increment and the hb content of prbc units transfused. methods: a total of 160 registered thalassemia major patients of our institute were included in the study after excluding the patients who had allo-or auto-antibodies. the study was approved by the institute ethics committee. the enrolled patients were randomly divided into two groups: group i (n = 80)they received abo/rhd identical prbcs suspended in additive solution (saline, adenine, glucose, mannitol: sagm-prbcs) after determining its hb content (units with hb content ≥ 50 g); and group ii (n = 80)they received randomly selected abo/rhd identical sagm-prbcs. the hb estimation of the randomly selected units in group ii was blinded. following tests were done on pre-transfusion sample: hb estimation using the hematology analyzer (orion 60, ocean medical technologies, india), blood grouping using tube technique, anti-human globulin (ahg) crossmatch and direct antiglobulin test (dat) using gel technique (biorad, switzerland), antibody screening (abs) using a fully automated immunohematology analyzer (neo, immucor, usa). on the posttransfusion sample collected 1 h after transfusion, hb estimation and dat were performed. results: there was no significant difference among the patient characteristics of the two groups. the mean hb content of the sagm-prbc units was significantly higher (p = 0.000) in group i (mean ae standard deviation: 67.86 ae 8.07 g; range: 50.80-92.13 g) than group ii (60.92 ae 8.29 g; range: 40.86-86.76 g). the mean hb increment in group i patients (3.26 ae 0.83 g/dl) was significantly higher (p = 0.04) than the group ii patients (3.00 ae 0.76 g/dl). in both the groups i and ii, there was a significant negative correlation between hb increment and weight (p = 0.000 in groups i and ii), age (p = 0.001 for group i; p = 0.032 for group ii), body surface area (bsa) (p = 0.002 for group i; p = 0.000 for group ii) and blood volume (p = 0.006 for group i; p = 0.000 in group ii). in both the groups i and ii, there was a significant positive correlation between hb increment and hb dose adjusted for bsa and the hb dose adjusted for blood volume (p = 0.000 in both groups i and ii for both the parameters). summary/conclusions: the efficacy of transfusion is more when patients are transfused with sagm-prbcs having hb content of 50 g or more as compared to those who are transfused with randomly selected units. for optimal hb increment in thalassemia major patients, the transfusion strategy should be based on the hb content of the sagm-prbcs. background: in male transfusion recipients under 50 years of age, receiving red blood cells (rbcs) from an ever-pregnant blood donor has been associated with increased mortality, compared to receiving a product from a male donor. although it has been suggested that older units of rbcs could be associated with increased mortality, there are significant methodological challenges in these studies. other studies indicated the freshest units of rbcs could be associated with increased mortality among transfusion recipients. we hypothesize both the association between ever-pregnant donors, and fresh units, with mortality could be caused by passenger leukocytes in the transfused rbc units, which decay during storage. aims: to quantify modification of the effect of ever-pregnant donors on mortality in young male rbc transfusion recipients, by storage time. methods: data on transfusion recipients receiving their first-ever rbc transfusion in one of six major dutch hospitals between 20/03/2004 and 01/09/2015 was collected. for the current study, male transfusion recipients under 50 years receiving only transfusions from one donor sex exposure category were selected and followup was censored at three years after transfusion. differences in storage time between groups were estimated by linear regression, adjusted for total number of transfusions, patient age, blood group, transfusion year and month. in a single-unit, single-transfusion cohort, cumulative mortality was estimated separately for patients receiving transfusions from ever-pregnant or male donors and for 'fresh' (<10 days storage) or 'old' (>24 up to 36 days storage) rbcs. results: for recipients of only blood from male donors, the storage time of the freshest unit was 0.47 day shorter when comparing the 221 patients who died, to 1,623 patients who survived (ci: à1.41 to 0.48). for recipients of only blood from ever-pregnant donors, the storage time of the freshest unit was 0.64 day longer when comparing the 27 patients who died, to 101 patients who survived (ci: à2.53 to 3.80). in the single-transfusion cohort, 1,280 patients received a fresh rbc transfusion from a male donor, 52 of whom died; 138 patients received a fresh transfusion from an ever-pregnant female donor, 9 of whom died. 193 patients received an old transfusion from a male donor, 7 of whom died; 16 patients received an old transfusion from an ever-pregnant female, 2 of whom died. the 3-years cumulative incidence of death among young male recipients was 4.7% (confidence interval (ci): 3.6% to 6.2%) after a fresh transfusion from a male donor and 4.8% (ci: 2.2% to 10.4%) after a fresh transfusion from an ever-pregnant female donor. the 3-years cumulative incidence of death was 7.2% (ci: 3.8% to 13.6%) after an old transfusion from a male donor and 15.4% (ci: 4.1% to 48.8%) after an old transfusion from an everpregnant female donor. summary/conclusions: prolonged storage of rbcs from ever-pregnant donors was not associated with decreased mortality at 3 years. contrary to our expectations, our results indicate older units may potentiate the effect of ever-pregnant donors. however, due to limited sample size the observed differences were not statistically significant. background: according to the literature review, there was limited impact of premedication (antipyretics, antihistamines and steroids) before transfusion on the prevention of adverse transfusion reactions (atrs). however, the necessity of premedication remains controversial. the premedication before transfusion is still a common clinical practice in pacific-asian countries, along with the premedication rate ranging from 50 to 80%. in our previous investigation, we found that premedication rate was 92.5% in the outpatients in 2017, which was much higher than the reported rate in asia. aims: to investigate the incidence of atrs and decrease premedication rate without increasing the rate of atrs via education and evidence-based clinical practice. methods: the incidence of atrs from april to december, 2017 was retrospectively surveyed. evidence-based clinical practice was initiated since january, 2018. clinical data of the outpatients receiving transfusion therapy were requested and analyzed from january to september, 2018. the incidences of atrs and premedication rates in 2017 and 2018 were compared using chi-square test. a p value less than 0.05 was statistically significant. besides, feedback of the incidence of atrs and premedication rate was given quarterly to the clinicians during the investigation. results: from april, 2017 to september, 2018, a total of 5,018 blood units were transfused in the outpatients with 2,453 transfusion events. of these, 25 cases of atrs, including febrile nonhemolytic transfusion reactions (fnhtr) and minor allergic reactions were reported. the overall premedication rate in the outpatients was 92.5% in 2017, and was significantly decreased to 77.9% in 2018 (p < 0.001). it was reported that the incidences of atrs in 2017 and 2018 were 0.48% and 0.52% per unit, respectively. there was no remarkable difference between the incidence of atrs in 2017 and 2018 (p = 0.642). summary/conclusions: via education and evidence-based clinical practice, we successfully reduced premedication rate without increasing the rate of atrs in the outpatients. furthermore, introduction of computerized provider order entry (cpoe) and clinical decision support system (cdss) could be considered and be expected to prevent unnecessary premedication before transfusion, increasing the compliance with optimized transfusion strategies in the future. methods: a retrospective analysis was done over a period of one year to evaluate clinical efficacy of 19 granulocyte transfusions in 15 hemato-oncology patients with febrile neutropenia. mobilization of granulocyte donors was done as per standard protocol, which included subcutaneous injection of granulocyte colony stimulating factor (g-csf) 5-10 lg/kg and tablet dexamethasone 8 mg, 10-12 h prior to granulocyte harvest by apheresis. all granulocyte products were gamma irradiated before transfusion. patient parameters like white blood count (wbc), absolute neutrophil count (anc), hemoglobin and platelet count were recorded pre-and post-granulocyte transfusion. infection related mortality (irm) within 30 days of granulocyte transfusion was also recorded. results: minimum adequate granulocyte yield of 1 9 10 10 per unit was fulfilled in 90% of granulocyte harvests. clinical indications for granulocyte transfusions were fever, an absolute neutrophil count (anc) < 500/ll, evidence of bacterial and/or fungal infections (i.e. clinical signs of infection, positive cultures and radiological evidence) and unresponsiveness to appropriate antimicrobial therapy for at least 48 h. effects of clinical, microbiological and granulocyte transfusion related variables on infection-related mortality were investigated. the post transfusion anc (within 24 h) increased significantly (median value: 350/ll) as compared to baseline levels (median value: 40/ll) (p < 0.05). infection related mortality was observed in only 20% (3 out of 15) of patients. patients became afebrile within 2-4 days and culture negative within 3-6 days after granulocyte transfusion. for analysis purpose granulocyte transfusion episodes were grouped according to doses of granulocyte transfusions, based on european guidelines (standard dose: 1.5-3.0 9 10 8 cells/kg and high dose: >3.0 9 10 8 cells/kg background: hsa's blood services group (bsg) is singapore's national blood service. in 2016, we conducted our pilot national pbm audit to promote pbm practices. it was agreed that the audit would be performed annually with incorporation of a new indicator to continue promotion of pbm and sharing of good practices. aims: to provide an update on the second national pbm audit for 2017. results are compared to the pilot audit and summarized below. methods: we collected data on 3 performance indicators from 7 acute public care hospitals for 4 weeks each in march and august 2017 (the pilot audit covered 2 weeks in 2016). the performance indicators were: 1). percentage compliance to documentation of red blood cell transfusion indications 2). percentage of patients screened for pre-operative anaemia, 14 to 45 days before surgery 3). peri-operative transfusion rates (3 days before to 3 days after surgery) for 6 commonly performed surgeries: coronary artery bypass graft surgery (cabg), total knee replacement (tkr), total hip replacement (thr), nephrectomy, colectomy and hysterectomy. the first two indicators assess pbm efforts and were measured in the pilot audit. indicator 3) was added to the second audit to assess impact of pbm practices on transfusion in surgical patients. it was an appropriate time to incorporate this indicator as the hospitals would have been familiar with pbm since its introduction in 2012. results and recommendations were shared with the senior management and hospital transfusion committees of the participating hospitals. results: for indicator 1), 2 hospitals had a compliance of 57-61%, the remaining 5 had a compliance of 90-100%. all 7 hospitals incorporated electronic blood ordering but the usage was not compulsory in some. hospitals which mandated electronic ordering performed better as doctors could only order blood products after entering the transfusion indication. we saw compliance increase from 75% in 2016 to 100% in a hospital that had newly mandated electronic ordering. for indicator 2), results ranged from 30% to 92%. 2 hospitals made notable improvements when compared to 2016, achieving 83% and 88% respectively. they had implemented pre-operative workflows screening all elective surgical cases for anaemia at least 2 weeks before surgery. one hospital also started an outpatient intravenous iron service which reduced pre-operative anaemia rates. for indicator 3), mean number of transfused units for each surgery ranged from 1.5 to 2.8 units per patient, lowest being thr and highest being cabg. this suggests that some transfusions were potentially avoidable with more robust pbm practices. the rate of perioperative transfusions was highest for cabg at 46% and lowest for tkr at 7%. summary/conclusions: the annual national pbm audit increases pbm awareness, allowing hospitals to share and learn good practices and implement measurable improvements. based on this audit, a recommendation to mandate electronic ordering of blood products to improve adherence to red cell transfusion indications and implementing pre-operative workflows with consideration for intravenous iron support was made. this audit was more representative than the pilot, with a longer duration of data collection and incorporation of indicator 3) showing impact of pbm practices. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to the diagnosis, investigation and management of patients with autoimmune haemolytic anaemia (aiha) in english nhs trusts. methods: we designed and distributed a survey to the clinical transfusion leads at all english nhs trusts between november 2017 and march 2018. the survey requested information on detailed, simulated clinical scenarios. the first simulated scenario described a young patient with active aiha 3 months after an allogeneic stem cell transplant, who has received multiple transfusions in the last 2 weeks and is hypotensive, tachycardic, with a falling haemoglobin (hb), currently 48 g/l. the second scenario describes a young man with a new diagnosis of warm aiha who has an initial hb of 104 g/l and returns to clinic at a 2-week interval with symptoms of fatigue. he is actively haemolysing and commenced on 1 mg/kg prednisolone. results: there was a 42% (58/137) response rate by trusts. faced with a 4-6 h delay for allo-adsorption studies, 68% (38/56) of respondents would instead transfuse acutely with abo, rh and k matched red cells negative for any previously detected alloantibodies, 7% (4/56) would transfuse with o rh d negative red cells and 25% (14/56) would wait for completion of allo-adsorption studies before transfusing. in this first scenario, a quarter of respondents appeared to delay a potentially lifesaving blood transfusion. 2017 british society of haematology guidelines recommend that when anaemia is life-threatening in the time required for full compatibility testing, abo, rh and k matched red cells should be transfused. in the 2017 serious hazards of transfusion (shot) report, the most serious and fatal of 95 cases of preventable delayed transfusion was a patient with aiha who died untransfused with an hb of 38 g/l, while awaiting alloadsorption studies. a key shot message was that if clinical harm to patients from withholding blood outweighs safety concerns over a possible delayed haemolytic transfusion reaction, emergency blood is essential and should be offered. the second scenario also identified considerable variation in transfusion practice. it can take several weeks for patients with aiha to respond to prednisolone so a transfusion threshold < 60 g/l after an hb fall of at least 40 g/l in the previous 2 weeks is perhaps overly conservative. summary/conclusions: the overall findings support a need for studies to explore barriers to uptake of guidelines, and to identify areas for further audit and research to guide safe and appropriate transfusion practice in aiha. background: balance between supply and demand of o d negative red cells remains a challenge for almost every blood service. with this re-audit, we wanted to collect objective and comprehensive information regarding usage of o d negative red cells supplied by nhs blood and transplant (nhsbt) to private and nhs hospitals in england. aims: the aim was to understand hospital practices, actual needs and possible avoidable usage of o d negative red cells. where possible, comparisons were made with two previous audits (2008) (2009) (2010) . methods: participating hospitals were asked to determine the fate of all group o d negative red cells they received between 14 th and 27 th may 2018 excluding substitutions and complete an organisational survey regarding activities, policies and stockholding practices with respect to o d negative blood. participating hospitals were asked to provide (if available) the prevalence (as a percentage) of o d negative patients in their population. this information, in conjunctions with hospital activities, will be used to estimate appropriate o d negative stockholding levels. background: o rhd-negative (neg) red blood cells (rbcs) are a precious resource, are often in short supply and transfusion of these units in emergency settings carries the potential risk of transfusion-related adverse outcomes such as haemolytic reaction due to minor blood group incompatibility. as such, their use should be closely monitored within health services. most recent australian guidelines (2008) for their use in emergency settings include pre-menopausal females of unknown blood group (mandatory indication) or while the blood group is being established; use should be limited to 2 or less units where possible before a switch to group-specific rbcs (acceptable indication). aims: audit of use of emergency uncrossmatched o rhd-neg rbcs against national guidelines in our institution (an australian tertiary metropolitan public hospital providing acute medical and surgical, emergency and critical care services). methods: use of emergency uncrossmatched o rhd-neg rbcs units over a six-year period was retrospectively reviewed. we collected information about rbcs transfused and discarded, adverse outcomes, patient characteristics, clinical indications and whether use met national guidelines or could have been avoided. results: 105 episodes of emergency uncrossmatched o rhd-neg rbcs were identified, encompassing transfusion of 241 rbc units to 103 patients and the discard of 2 rbcs (due to incorrect transport). of the 105 episodes, 78 episodes (74%) involved an eventual switch to group-specific rbcs (range of emergency units, 1-13 units). the main requester was the emergency department (53%). the most common clinical indication for transfusion was acute gastrointestinal bleeding (49%). of the 105 episodes, 28 episodes (27%) did not meet the guidelines for emergency use because > 2 units of emergency uncrossmatched o rhd-neg rbcs were issued. 2 episodes (2%) were flagged as potentially inappropriate as the patients were clinically stable according to documentation in the medical records. 30 episodes (29%) were identified as potentially preventable due to delay in pre-transfusion sample collection (defined as > 1 h elapsed between patient arrival and group and screen sample collection) in the setting of acute bleeding (6%), receipt of an unsuitable pretransfusion sample requiring sample recollection (5%), delay in pre-transfusion sample processing (4%), no valid pre-transfusion sample being available at the time of the bleeding episode despite having a planned elective procedure or being an inpatient with recent clinical bleeding (10%). only one patient was investigated for potential transfusion-related adverse outcome (1%) which was thought likely due to concurrent sepsis. summary/conclusions: over six years, 105 episodes utilising emergency uncrossmatched o rhd-neg rbcs were identified with 241 rbcs issued and 2 rbcs discarded. a significant proportion of episodes (29%) were potentially avoidable if there had been a valid pre-transfusion sample available in the transfusion laboratory at the time of the episode. efforts to minimise use of this precious resource are ongoing, and include feedback to clinical units regarding importance of valid pretransfusion samples prior to applicable invasive procedures and in bleeding patients, ongoing education to medical and nursing staff, and continuing audit of use of this blood component in the hospital haemovigilance programme. abstract withdrawn. abstract withdrawn. background: platelet transfusions are often given prophylactically to thrombocytopenic hematology patients. to which extent platelet function improves after transfusion, and how this improvement correlates with an increase in platelet count, is not well studied. flow cytometry has been used to evaluate platelet function after transfusion in a few studies and can be performed even at low platelet counts. rotational thromboelastometry (rotem) represents a more physiological measure of platelet function in whole blood that has not been extensively used in transfusion settings. we used these methods to investigate if platelet transfusion improves platelet function in hematology patients and if improvement correlates with increased platelet counts. aims: the aim was to evaluate the relationship between response to platelet transfusion, measured as corrected count increments (cci), and platelet function in thrombocytopenic patients with hematological disorders. methods: blood samples (sodium-citrate anticoagulated) were collected from unselected hematology patients receiving prophylactic platelet transfusions, after informed consent had been obtained. samples were taken at three time-points: within 1 h before transfusion, 1 h after and 14-24 h after transfusion (via a central venous catheter or a subcutaneous venous port). for each time-point, platelet response to adenosine diphosphate (adp) and thrombin receptor-activating peptide (trap-6) was assessed by flow cytometry by measuring p-selectin and pac-1 expression on single platelets. rotem analysis was also performed on all samples, using intem and extem reagents. results: an interim analysis was performed after inclusion of 22 patients. the mean platelet count before transfusion was 8 9 10 9 /l (range 2-34 9 10 9 /l). 1 h cci was 11 9 10 9 /l and 14-24 h cci was 6 9 10 9 /l, but response was highly variable. pselectin expression after stimulation with adp and trap was significantly higher at 1 h after and 14-24 h after transfusion compared to before transfusion (p < 0.05). pac-1 expression after stimulation with adp was significantly higher at 14-24 h after transfusion (p < 0.001), but not at 1 h after transfusion. in rotem, clot amplitude at 10 and 20 min (a10 and a20) as well as maximum clot firmness (mcf) improved after transfusion (p < 0.05). a significant correlation between absolute platelet count and p-selectin expression after trap and adp stimulation was found (r s =0.53 and 0.45 respectively, p < 0.001). absolute platelet count was also significantly correlated with mcf (r s =0.61, p < 0.001), where 84% of patients with a platelet count of more than 20 9 10 9 /l reached mcf values within the reference interval. summary/conclusions: platelet function generally improves after transfusion and was in our patient population correlated to the absolute platelet count, but was also seen at the single platelet level in flow cytometry. a post transfusion platelet count of more than 20 9 10 9 /l might be sufficient to significantly improve coagulation in heavily thrombocytopenic patients, but larger studies are needed to confirm this conclusion. abstract withdrawn. abstract withdrawn. background: sickle cell disease (scd) is a genetic disorder that is frequently referred to as a hypercoagulable state. hydroxyurea (hu) is known to decrease the frequency of vaso-occlusive complications and need for blood transfusions in severely affected individuals. although cross-sectional studies show that treatment with hu is associated with decreased coagulation activation, there are no prospective studies evaluating the effect of hu on coagulation activation. aims: to assess the effect of hu on markers of fibrinolysis (d-dimer) and endothelial activation (soluble vascular cell adhesion molecule-1 [soluble vcam-1]) in patients with scd in their non-crisis, "steady state." methods: patients, at least 10 years of age, with documented hbss or hbsb-thalassemia, eligible for treatment with hu were studied in this prospective, observational study. laboratory investigations were obtained at baseline, prior to commencement of therapy with hu, with repeat evaluations at three and six months of therapy. non-parametric test was applied to observe the association between hu therapy and the biomarkers of interest. results: twenty-five patients with scd (hbss: 15, hbsb thalassemia: 10) were enrolled (females: 15 [60%]), with a median age of 23 years (iqr: 11). following 6 months of hu, median values for wbc count (9.02 9 10 9 /l vs. 7.22 9 10 9 /l, p = 0.007) and d-dimer (1243.8 ng/ml vs. 830.2 ng/ml, p = 0.028) were significantly lower than baseline values, while the mean corpuscular volume (76.0 fl vs. 88.0 fl, p = 0.001) was significantly higher than the baseline value. no significant differences from baseline were observed in the median values for hemoglobin (9.1 g/ dl vs. 9.6 g/dl, p = 0.72), platelet count (250 10 9 /l vs. 196.5 10 9 /l, p = 0.71), lactate dehydrogenase (744 u/l vs. 567.5 u/l, p = 0.23) or soluble vcam-1 (567.8 ng/ ml vs. 526.4 ng/ml, p = 0.33) following 6 months of hu therapy. summary/conclusions: this exploratory study confirms that treatment with hu is associated with decreased coagulation activation in patients with scd, although no effect on endothelial activation was observed. by decreasing coagulation activation, hu may decrease the risk of thrombotic complications in scd. abstract withdrawn. abstract withdrawn. transfusion medicine, apollo gleneagles hospitals, kolkata, india background: reduction of immune responsiveness through blood transfusion has been documented by previous authors. breast cancer is considered as one of the commonest cancer globally and the second main cause of death in females transfusion of allogeneic blood in breast cancer surgery is variable and differences of transfusion incidence have been observed in the literature. where the maximum surgical blood ordering schedule (msbos) dictates cross matching and reservation of blood before surgery, factors deciding their utilization are varied and numerous. our hospital protocol guides that every patient planned for elective breast cancer surgery should routinely have a blood sample sent for reservation of one unit of compatible packed red blood cell (prbc) in the blood bank. aims: in this prospective study we aimed to audit the blood utilization in patients undergoing elective breast surgery and thereby optimize the blood ordering schedule, economic burden and loss of clinical resources. methods: the study included 478 confirmed breast cancer patients planned for elective breast surgeries from january 2012 to december 2017. patient and disease details like age, stage, tnm status, estrogen receptor (er) and progesterone receptor (pr) status, human epidermal growth factor receptor 2 (her -2) expression, triple negative breast cancer (tnbc) status, reproductive and treatment status were documented. patients were divided into younger group [≤40 years] and older group (>40 years). before surgery blood samples for compatibility testing were sent to blood bank for blood reservation. details of test, blood issue and blood transfusion were documented in the blood bank. approximate loss of time in minutes and wastage of resources in terms of money (inr) in the blood bank were noted. all results were calculated as mean ae sd and a 'p' value of < 0.05 was considered statistically significant. results: of the total 478 patients most underwent wide local excision of the breast and modified radical mastectomy. a total of 16 patients received 71 units of blood and blood components in all categories of surgeries. only 103 were younger women (≤40 years) with mean age of 31 years. non-transfused patients were significantly more than transfused ones (p < 0.05). frequency of blood transfusion was more in young patients (4.9%). seven (22.6%) of the total 31 stage iv patients received blood transfusions. frequency of blood transfusion was more in patients undergoing surgery after chemotherapy (8.8%). a significant loss of time and loss of revenue was observed. summary/conclusions: we conclude that routine compatibility test is not justified for all patients undergoing breast surgery. a more targeted approach is needed to reduce blood demand and associated cost to patient and blood transfusion services. background: blood transfusion guidelines are not only essential for the optimal use of blood products, but also help reduce transfusion-related adverse reactions and improve patient outcomes. the korean national transfusion guidelines were developed in 2009 and fully revised in 2016 by the korean centers for disease control and prevention and the korean society of blood transfusion. in our hospital, which is a 700-bed university hospital, a transfusion-indication data-entry program based on the national transfusion guidelines was developed in 2016. it was applied to the electronic medical record system and all transfusion orders, except emergencies, have been performed through this program since then. aims: we planned to record and analyze the reasons for transfusion in order to monitor blood product usage and provide feedback to clinicians. furthermore, we intended to contribute to patient safety through the appropriate use of blood products. methods: we classified transfusion-indications by the blood product requested and created a pop-up window listing these indications, which would appear at each regular transfusion order. indications for transfusion with each blood product were as follows: red blood cells (rbcs)acute blood loss, chronic disease (sub-classified as hb ≤ 7 g/dl, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, respiratory disease, age ≥ 65 years, age ≤ 6 months, chemotherapy), surgery/ procedure, transplantation and 'other'; platelets (plts)present bleeding, bleeding prevention (sub-classified as hematologic disease, solid tumor, peripheral blood stem cell transplantation, disseminated intravascular coagulopathy, infant), surgery/procedure, massive transfusion and 'other'; fresh frozen plasma (ffp)bleeding in coagulopathy, bleeding prevention in coagulopathy, massive transfusion, plasma exchange and 'other'. transfusion indications entered into the data-entry program from sep 2016 to feb 2018 were analyzed. results: the number of transfusion-indications analyzed was 16138 for rbcs, 11158 for plts and 6024 for ffps. the most common indications for transfusion were chronic disease for rbcs (7977/16138, 49.4%), bleeding prevention for plts (5726/11158, 51.3%) and 'other' for ffp (2180/6024, 36.2%). 'hb ≤ 7 g/dl' was the most frequent sub-indication of chronic disease (3570/7977, 44.8%), and hematologic disease was the most frequent sub-indication of bleeding prevention (3432/ 5726, 59.3%). many clinicians entered transfusion indication as 'other': rbcs (2866/ 16138, 17.5%), plts (856/11158, 7.7%) and ffp (2180/6024, 36.2%). however, the free-text supplied by the clinician when 'other' was selected, often corresponded to an indication already categorized in the transfusion-indication data-entry program; 82.9% of rbcs and 54% of plts. of the indications entered as 'other' in ffp, 80.3% were surgery/procedure-related. summary/conclusions: in our hospital, the release of blood products has been dependent on the data-entry of transfusion indications (except in emergencies) since sep 2016. transfusions of rbcs and plts were most common for chronic disease and bleeding prevention, respectively, but many cases entered as 'other' could have been categorized as existing indications in our data-entry program. therefore, we conclude that additional training is needed for clinicians regarding the determination of transfusion-indications and correct use of the transfusion-indication dataentry program, in order to use blood products more appropriately. methods: this was a prospective cohort designed study. subjects were children aged 1-18 years with indication of platelet transfusions in sardjito hospital yogyakarta indonesia. the patient samples were collected before and 1 h post-transfusion, the expression of cd62p on platelet was determined by flow cytometry method. results: there were 102 subjects who were divided into two groups. fifty-one subjects received non-leukodepleted pcs and the other fifty-one transfused by pre-storage leukodepleted pcs. the mean of pre-transfusion platelet cd62p for nonleukodepleted and leukodepleted groups were 26.2% and 27.7%, and the mean increase of post-transfusion platelet cd62p for non-leukodepleted was 10.1% and the mean decrease of leukodepleted groups was 3.3%. it was shown the increase of post-transfusion platelet cd62p for non-leukodepleted group, and it was significantly (p < 0.05) higher than in the leukodepleted groups. summary/conclusions: there was an increase of post-transfusion platelet cd62p expression in patients received non-leukodepleted, but a decrease in leukodepleted pc transfusions. background: preoperative anaemia is a common finding in patients undergoing surgery and often neglected in our country. aims: the objective of this study was to evaluate hb(values and the identification of cardiac patients who entered operation with anaemia. and also to study the correlation between hb values and the number of rbc (red blood cell) transfused unit methods: this is a retrospective, descriptive and analytical study. the data for this study was collected from the files in the statistic's service at qsut (university hospital center "mother teresa"). the object of our study were the files of 158 patients hospitalized in the period january -may 2018 in the cardiac surgery ward, which were subjected to cardiac surgery. from the files were collected data on age, gender, primary diagnosis, accompanying diseases. we also collected hb, rbc, htc (hematocrit), mcv (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration). from the transfusion service at qsut and from the files were pulled out the transfused patients and the number of transfused units. results: based on the who definition for anemia (females < 12 g/dl and males < 13 g/dl), from the 158 patients included in the study, 61 (39%) were anaemic. from 117 males in the study, 40 (34%) of them were anaemic based on hb lab values, whereas from 41 women in the study anaemic were found to be 21 (51%) of them. from the 61 anaemic patients in the study, 35 (57.4%) of them with mild anaemia, 23 (37.7%) with moderate anaemia and 3 (4.9%) with severe anaemia. in the total of anaemic female 38.1% are under 65, while 61.9% are over/or 65 years old. in the total of anaemic males, 35% are under 65, while 65% are over/or 65 years old. it is noticed that most of them are with normochromic normocytic 67.2%, normocytic hypochromic anaemia 16.4%, hyperchromic microcytic anaemia 8.2%, macrocytic normochromic anaemia and macrocytic hypochromic anaemia respectively 1.6% and microcytic normochromic anaemia 4.9%. the average value of preoperative hb decreased from 12.8 g/dl before surgery to 10.3 g/dl after surgery, so there is a decrease of approximately 2.5 g/dl of hb value. in our 158 patients, 48% (76) were transfused and the remaining 52% (82) were not transfused. from 76 transfused patients 41 (54%) patients were anaemic. the correlation between the values of hb, rbc, htc and the number of transfusions shows that with the decrease of these values the number of transfused units increases. summary/conclusions: the diagnose of anaemia is underestimated before surgical intervention in our country and investigation of hb low values do not take the proper importance to find probable cause and correct it before surgical intervention. the lower the hb values, the greater the chance to be transfused and the number of rbc transfused units. failure to correct hb values before surgery results in unnecessary transfusions for the patients or which could have been avoided, eliminating also the risk of transfusion complications. background: alloimmunization after red blood cell transfusion is affected by various factors. it is known that the incidence of alloimmunization increases in certain diseases. extended red blood cell matching can be used to prevent the development of alloimmunization in diseases which the rate of alloimmunization is increased. in asia, extended red blood cell matching is not actively implemented. aims: we tried to investigate whether there is a difference in the disease categories between unexpected red blood cell antibody positive and negative groups. methods: from january, 2003 to december, 2017, the diseases of the patients who had undergone unexpected red blood cell antibody identification test at dong-a university hospital was examined through medical records. from january 2008 to december 2009, the diagnosis was made on patients who had two or more unexpected antibody screening tests. we analyzed the frequency difference of disease category between two groups. results: a total of 988 patients were performed with unexpected antibody identification tests. of 1896 patients who underwent more than 2 screening tests, 25 (1.3%) were positive. 1871 were consistently unexpected antibody negative. the patients with solid tumors (n = 375, 56.3%) and those with hematologic diseases (n = 112, 16.8%) had a higher incidence in unexpected antibody positive group. the patients with myeloid malignancy had a significantly higher frequency than lymphoid malignancy (p = 0.0002). the frequency of patients with liver cirrhosis was significantly higher in the unexpected antibody positive group (65/988, 6.6%) than in the negative group (77/1871, 4.1%) (p = 0.006). the incidence of non-hodgkin lymphoma was significantly higher in the unexpected antibody negative group (28/1871, 1.5%) than in the positive group (5/988, 0.5%) (p = 0.019). summary/conclusions: there was a difference in the distribution of diseases between unexpected antibody positive group and negative group. the patients with liver cirrhosis were more frequent in unexpected antibody positive group, suggesting that extended red blood cell matching would be considered. background: in hematological patients with multiple platelet transfusions (pc) often develop immune response to human leukocyte associated antigens (hla-i) and human platelet-specific associated antigens (hpa). besides, platelet associated immunoglobulins (paig) and complement components (pac) are found on platelet. this leads to increased platelet destruction and development of refractoriness to transfusions of donors' platelets. transfusion therapy using an individual selection of platelets and plasmapheresis, contribute, in the majority of cases, to the realization of efficient transfusion by pc. but, in difficult cases, there is a need to use intravenous immunoglobulin, which may promote the efficient transfusion of pc. aims: evaluate the algorithms of using the complex therapy of refractoriness to transfusions of donors' platelets with additional application of intravenous immunoglobulin (ivig). methods: in 2018 there were three female patients in the clinics of the centre for observation, age between 29 and 51 years (me = 39) with the ineffectiveness of complex therapy for overcoming refractoriness to transfusions of donors' platelets due to selection and plasmapheresis. the diagnoses were as follows: aplastic anaemia (aa)-2, acute myeloid leukemia (aml)-1. individual selection of platelets was carried out by the adhesion method on the solid phase (immucor "galileo neo"). paig and pac3/4 were evaluated by the method of flow cytometry (bd facscanto ii) by the method of double staining with cd41a. the density of fixed paig, pac was © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 evaluated by the median fluorescence intensity (mfi). the two patients with aa received ivig-igg therapy in the standard dose 0,4 g/kg per day, for 3 days. one patient with aml received ivig-iggam therapy in the standard dose 5,0 ml/kg/day for 3. results: under pressure of the complex therapy with the use of ivig in the standard dose there was are decrease in mfi over time in the case of two patients: aa-1 mfi-paigg reduced from 926 to 65; while the patient with aml: paiga reduced from 5506 to 187, and paigm from 1971 to 302, pac3 from 691 to 31, pac4 from 404 to 103. the patient with aa-2 over time, regardless of the treatment, there was an increase of mfi, but the effect of pc transfusions was achieved under pressure of complex therapy. under pressure of complex therapy all the 3 patients also reduced the frequency of reaction of alloantibodies when resorting to an individual selection and increasing the frequency of compatible couples "donor-recipient". summary/conclusions: delivery of complex therapy and the additional application of ivig enables an adequate transfusion therapy of pc, neutralize hemorrhagic syndrome and continue the treatment of the main disease. detection and monitoring of paig/pac during the development of refractoriness to transfusions of donors' platelets are additional markers for prescription of ivig therapy. anaesthesia, tan tong seng hospital, singapore, singapore background: blood transfusion is quite prevalent in paediatric cardiac surgical procedures. we hypothesized that the routine use of rotational thromboelastography (rotem) to guide transfusion decisions would reduce the overall proportion of patients receiving transfusions in paediatric cardiac surgery aims: the aim of the study was to find if the use of blood and blood products in pediatric cardiac surgical cases in a single centre is affected due to rotem. methods: sixty paediatric cardiac surgical patients undergoing cpb were included in this study. thirty patients (study group) were prospectively included and compared with thirty procedure and age-matched control patients (control group). in the study group, rotem, performed during cpb guided intraoperative transfusions. perioperative transfusions of blood and blood products, postoperative blood loss and hemoglobin levels were compared between the two groups. results: the patients in the control group received fewer transfusions of packed cells (60% vs 78%) and fresh frozen plasma (36% vs 84% p<o.oo1). more patients in the study group received platelets (72% vs 60%) and cryoprecipitate (50% vs 36%). the postoperative blood loss was lower in study group compared to the control group. the postoperative hemoglobin did not differ between the two groups. summary/conclusions: the results suggest that the routine use of rotem in paediatric cardiac surgery to guide transfusions is associated with reduced proportions of patients receiving transfusions and is evidence based practice. abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mycoplasma pneumonia (mp) infection has often been implicated in respiratory tract infections. although this agent has also been associated with other non-respiratory diseases, hemolytic anemia is the most common hematologic complication of mp infection. episodes of sudden onset of severe anemia are rarely seen in beta thalassemia. we report a 33-years-old female with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection aims: to report a case of patient with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection and how it was managed with minimal transfusion. methods: a 33-year-old female case of beta thalassemia major who developed autoimmune hemolytic anemia following mycoplasma infection. the patient is on monthly blood transfusion since the age of 3 years at dubai thalassemia center. she was admitted to the hospital with 5 days history of dizziness, severe low back and joint pain and her travel history indicated a four-week visit to pakistan. initial investigations showed a hemoglobin level of 7.8 g/dl the direct and indirect coombs test were positive. patient was transfused with two units leuko-depleted compatible prbc (o, rh +, negative for e and kell antigens.) and discharged next day. three weeks later, the patient was still with lower limbs pain and the hemoglobin was 8.5 g/dl with a positive dat with both igg+4 and c3+4, patient was not transfused as it was difficult to find matching blood; therefore she was given one week follow up. four days later, patient reported to the center with fever, generalized body pain and fatigue. she had significant drop in hemoglobin 5.6 g/dl, mcv 85.0, platelets count 216000, crp 3.6, retic count 2.18, blood culture no growth, total bilirubin 1.5, malaria parasite smear was negative, urine culture and microscopy were normal. mycoplasma pneumonia antibody showed positive with igg 57.1 and igm 21.3, she was started on azithromycin for 5 days and received 2 units prbc least incompatible leuko-depleted (o, rh +, negative for e and kell antigens.). after which her hemoglobin increased to 9.3 g/dl. she did not show any improvement in her condition over the following 2 weeks and repeat mycoplasma pneumonia antibody showed an increased titer, which denoted resistance to azithromycin; hence, we started her with second line of treatment levofloxacin for 10 days. screening for collagen disease were all negative apart from high esr of 130 mm/hr. patient remained afebrile and transfused with least incompatible available blood, post transfusion hb was 11.8 g/dl. three weeks later, during her routine follow up, she was doing well with esr dropping to 85 mm/hr and hemoglobin 10.2 g/dl, which indicated gradual settling of hemolysis, although the last dat remained positive. subsequent visits showed hb drop of 1.2 g/dl/week which is acceptable for transfusion dependant thalassemia. results: with conservative management patient condition improved. summary/conclusions: hemolytic anemia associated with mycoplasma pneumonia is usually self-limited and resolves spontaneously after mycoplasma infection elimination. packed red cell transfusion in transfusion dependent patient may be troublesome and can aggravate the condition; therefore it should be utilized and limited to significant symptomatic anaemia background: early plasma transfusion in women with severe postpartum hemorrhage is often instituted to prevent and treat coagulopathy, but whether maternal outcomes improve with this treatment is unclear. aims: to quantify the association of plasma transfusion within the first 60 min after diagnosing persistent postpartum hemorrhage and adverse maternal outcome. methods: we performed a retrospective cohort study of women with persistent postpartum hemorrhage, defined as postpartum hemorrhage refractory to first-line measures to control bleeding. women were enrolled from january 1, 2011 to january 1, 2013 in 61 dutch hospitals. in a time-dependent propensity score-matched analysis, women with transfusion of fresh frozen plasma within 60 min following the diagnosis of persistent postpartum hemorrhage were matched to women without plasma transfusion or plasma transfusion at a later time point during hemorrhage. plasma transfusion was not guided by coagulation tests. the main outcome measure was adverse maternal outcome, defined as a composite of mortality, hysterectomy and arterial embolization to control bleeding. we performed sensitivity analyses with initiation of plasma transfusion within 120 and within 180 min following diagnosis persistent postpartum hemorrhage, compared with other plasma transfusion strategies. results results: the cohort consisted of 1216 women with persistent postpartum hemorrhage, with uterine atony as cause of hemorrhage in 780 women (64.1%). in this cohort, seven maternal deaths (0.6%), 62 hysterectomies (5.1%) and 159 arterial embolizations (13.1%) were observed. plasma transfusion within 60 min in 114 women was matched on time-dependent propensity scores with 114 women with no or later plasma transfusion. rate of adverse maternal outcome was similar between women with and without early plasma transfusion, 21.2% versus 19.9%, adjusted odds ratio 1.09 (95% confidence interval 0.57-2.08). results of sensitivity analyses were comparable to the primary results. summary/conclusions: among women with persistent postpartum hemorrhage, initiation of plasma transfusion within 60 min following this diagnosis was not associated with improved maternal outcomes, compared with no or later plasma transfusion. whether other transfusion strategies or identification of women with high risk of coagulopathy will improve maternal outcomes remains to be studied. background: preoperative evaluation of increased risk for perioperative bleeding is essential for cardiac surgery patients. platelet aggregation, as a laboratory test for assessment of platelet function, is very efficient for optimal antiplatelet treatment and also for assessment of the increased risk of postoperative bleeding and perioperative application of allogenic red blood cell transfusions. aims: the aim of this study was to establish whether preoperative platelet function testing for the presence of residual effect of acetylsalicylic acid (asa) on platelet aggregation can be correlated with an increased risk for intraoperative bleeding during cardiac interventions. methods: a retrospective study included 60 patients who underwent surgical procedures (cabg ii and cabg iii) at the department of cardiovascular surgery, clinical center in ni s in period november 2017-july 2018. patients were previously taken asa at a dose of 100 mg per day, but therapy was discontinued 5 days before surgery. platelet aggregation was measured on the day of surgery using impedance aggregometer multiplate (multiplate platelet function analyzer, roche), using trap and aspi tests. patients were divided into two groups: control group (cg), which included patients with preserved function of platelets (aspi 790-1410 au*min), and examined group (eg), which included patients with inhibited platelet aggregation in aspi test (<790 au* min background: hemophilia b is an x-chromosome-linked inherited bleeding disorder resulting from reduced levels or an absence of factor ix clinically represented by recurrent prolonged bleeding. mutations can be inherited or acquired de novo. de novo mutations arise in about one-third of patients with haemophilia. the disease affects primarily males, but approximately 30% of carrier females have factor ix clotting activity lower than 40% and are at risk for bleeding. aims: case report of a hemophilia b carrier. methods: the clinical history of the patient was thoroughly studied by analyzing medical records and analytical results using the software available in the health institution and paper records. results: 45 year old woman, with no history of abnormal haemorrhage, healthy, up to 2004 when, after a dystocic delivery, presents with subcutaneous and ocular haemorrhage and dispersed bruises. a basic coagulation study is performed and a level of 20% fix is found. genetic analysis reveals a mutation in fix gene and she is diagnosed as a carrier of hemophilia b. except for a son, no other direct family members carry the mutation. in 2008, after total hysterectomy with bilateral adnexectomy due to an invasive carcinoma in the uterine cervix, the patient suffers a haemorrhagic shock with multiple transfusion support and around six months later undergoes an ureteronephrectomy also with the need for post-surgery transfusion support. the patient is referred to the immunohemotherapy department where she has regular appointments as an outpatient since 2009. in 2012, she requires surgery for the removal of a thyroglossal duct cyst, which is performed under prophylactic treatment with 2000 iu recombinant fix (rfix) daily, for four days, and up to 48 h after discharge maintaining basal levels of fix of 60% and with no major complications reported. in 2013, the patient is admitted to the er in haemodynamic shock and a history of black stools and syncope. at admittance, she receives a bolus of 4000 iu of rfix and requires severe transfusion support due to active bleeding. she is diagnosed with colonic angiodysplasia and undergoes surgery. daily doses of 3000 iu rfix are administered from day 1 (d1) and up to d7 maintaining fix levels around 60-80%. the patient is operated at d8 under the cover of 2000 iu of rfix, twice per day, and up to d14 after surgery reaching fix levels of around 80-110%. at d15 the dose was diminished to 2000 iu of rfix daily and at d17 she is discharged with no rfix replacement therapy necessary in ambulatory. since then, no complications or new occurrences have been reported. the patient is under prophylactic administration of rfix prior to minor invasive procedures (nodule biopsy, dental extractions and colonoscopies). summary/conclusions: hemophilia b carriers usually do not require any major health care for their daily routine or invasive procedures. however, in the case reported, basal fix levels conditioned any minor intervention to be performed. thus, this carrier must be considered as a moderate hemophilia b patient and mandatory prophylaxis with rfix must be performed prior to any invasive procedure. background: blood transfusion is a life saving procedure but transfusion associated complications are equally important that need to be addressed. with limited resources of blood donations and screening, excessive transfusions must be avoided to prevent risk and economic burden on patients. on the other hand discussion on appropriate use of blood product is a matter of debate in medical literature. it is recommended to restrict the liberal use of transfusion. aims: in this regard we aimed to observe the frequency of genuine and non genuine transfusion at our hospital and also to compare the outcomes of liberal and restrictive transfusions. methods: it was a cross sectional study carried out at nibd and bmt, pechs campus, karachi, pakistan, from february 2018 to january 2019. the study was approved from institutional review board. pack red cells (prc) transfusion in case of < 7 g/dl hemoglobin (hb) with cardiac history and < 7 g/dl hb with symptomatic anemia was considered genuine. platelets transfusion in case of sepsis and count < 50 9 10 9 /l, sepsis with bleeding and < 100 9 10 9 /l, patients on active chemotherapy without bleeding but platelet count < 10 9 10 9 /l and in patients presented with bleeding was considered genuine. patients who were presented with bleeding, deranged coagulation profile and with sepsis + bleeding considered genuine for fresh frozen plasma (ffp) transfusion. spss version 23.0 was used for descriptive and inferential statistics. results: a total of 250 transfusions were evaluated for genuine and non genuine transfusion. male predominance was high in our data 194(78%). out of 250, 134 (54%), 100(40%) and 16(6%) prcs, platelets and ffp were transfused respectively. out of 100 platelets, 10(10%) were platelet apheresis. 80/134 (60%) prcs and 22/90 (24%) platelets were non genuinely transfused as per the criteria described in methods. however, ffp and platelet apheresis were genuinely transfused. in non genuine group of platelet transfusion, all patients received more than 1 unit platelet and the increment of platelet count post transfusion was found statistically non significant (p = 0.121). thirty six (45%) of transfusions were done with more than 1 units of prcs in non genuine group. the mean hb level of patients received 1 unit and more than 1 units prcs was statistically same (p = 0.868) and it was found out that there was same increment in hb in both the groups post transfusion. (p = 0.867). summary/conclusions: we concluded that although blood transfusion is an integral procedure for saving life yet correct use of blood products is necessary in the era where blood donations are scarce and blood transfusion has its potential associated hazards. the approach towards the appropriate use of blood products will not only minimize the risk of transmission of infectious diseases but also will reduce the economic burden. longitudinal studies on large sample size are needed to validate this study. uncertain. an ovine model enables comparisons of; (a) invasive and non-invasive methods to measure oxygen delivery and utilisation at a tissue level, and of (b) the efficacy of various resuscitation fluids and transfusion protocols in treating haemorrhagic shock. aims: to develop an ovine model of haemorrhagic shock. methods: sheep (n = 4) were anaesthetised, ventilated and instrumented. instrumentation included blood flow, perfusion, and microdialysis probes placed in the brain, kidney, liver and skeletal muscle. haemodynamic, tissue oxygenation, tissue flow and respiratory data were recorded continuously by data capture software. non-invasive assessments, including brain and muscle oxygen saturation by nirs, and visualisation of sub-lingual microvascular perfusion, as well as arterial blood gas measurements and plasma/serum/urine collections were made at specific timepoints. after a baseline period (1 h) sheep were bled~40% of their estimated blood volume (estimated at 65 ml/kg) to a mean arterial blood pressure (map) of 30-40 mmhg. sheep were then resuscitated with either plasmalyte â (up to 20 ml/kg/ hr; n = 3) or transfused with sheep red cells (n = 1) to achieve a map > 65 mmhg. sheep were euthanised 4 h after resuscitation. data are presented as mean ae standard deviation. results: sheep were haemorrhaged an average of 931.7 ae 135.3 ml blood which combined with iatrogenic blood loss (~300 ml) corresponded to an average 40.2 ae 2.4% blood loss. two out of the four sheep met clinical criteria for haemorrhagic shock (map = 30-40 mmhg, lactate > 4 mm, svo 2 < 60%). across all four sheep the nadir map averaged 40.5 ae 13.1 mmhg, lactate peaked at 3.9 ae 1 mmol/ l, and nadir svo 2 was 41.3 ae 17.9%. all sheep survived to the end of the experimental protocol. summary/conclusions: these data demonstrate the successful induction of haemorrhagic shock in an ovine model. further experiments are planned to improve the protocol and to achieve 100% incidence of haemorrhagic shock, and then to compare invasive and non-invasive measures of oxygen delivery and utilisation as well as the efficacy of different resuscitation fluids and red cell transfusion. adverse events, including trali p-515 bilirubin were recorded within the 28-day period. the clinical parameters were compared against the reaction strength of the antibody reactions. the automated strength was measured by solid phase. the manual testing consisted of a 15-min incubation using liss and adding monospecific igg. the dat was performed manually by adding poly-specific igg and then testing with monospecific igg and c3d. the rh group and non-rh group had 11 and 10 cases performed manually, and results were 2+ or weaker further indicating the manual strength did not correlate with the clinical hemolysis. likewise, in 31/44 (70%) the dat was negative, and did not show any correlation with clinical hemolysis. however, when ldh and bilirubin were measured, the two parameters increased as the automated strength of the antibodies increased. summary/conclusions: most of the dshtr investigation was not associated with overt accelerated red cell destruction. a strong correlation was observed only between the automated immunohematology testing results and other laboratory markers of hemolysis. in our experience, the direct antiglobulin test and manual strength showed no correlation. background: numerous transfused patients present severe, sometimes critical clinical conditions. the occurrence of adverse transfusion reactions (atr) may induce deterioration in the clinical condition with a worsened clinical course and a lifethreatening or fatal outcome as is the case with nervous system impairment. in france, in 2017, out of 7,276 notified atrs, 113 (1.5%) and 6 (0.1%) were life-threatening and death respectively. aims: our aim was to evaluate the notified atrs with neurological signs that occurred in transfused patients over a period of six years and six months in hospitals in the auvergne rhône alpes area. the study included patients with reported atrs in hospitals in this area from january 1 st 2010 to june 30 th 2018. each atr was registered in the national haemovigilance database system. two signs observed at the time of the atr were analyzed: unconsciousness and convulsions. stroke was excluded. the type of atr, its severity, the blood product involved and its imputability were studied. results: during the period under study, 9,544 atr were reported, of which 29 included unconsciousness and/or convulsions (0.3%). of these 29 patients, 13 were females (44.8%) and 16 males (55.2%). unconsciousness alone was frequently observed (21 reports, 72.4%). convulsions were notified in 8 reports (27.6%) and were associated with unconsciousness in 2 of them. the diagnosis of seizure, with no other clinical signs, was established in 2 cases (6.9%). unconsciousness and/or convulsions were present in 8 allergic reactions (27.6%), 4 cases of transfusion-associated circulating overload (13.8%), 3 cases of suspected transfusion-transmitted bacterial infections and 2 hypertensive reactions. in allergic atrs, unconsciousness was notified in 7 cases and unconsciousness associated with convulsions in one. twelve atrs were severe (41.4%), 10 were life-threatening (34.5%) and in 4 cases, they resulted in the death of the recipient (13.8%). of the 8 allergic atrs, 4 were severe and 4 life-threatening. red blood cell concentrate was involved in 15 atrs (51.7%) and platelet concentrate in 9 (31.1%), including 5 cases with apheresis platelet concentrate and 4 cases with pooled platelet concentrate. fresh frozen plasma was involved in 5 atrs (17.2%). nevertheless, the imputability of the blood product was excluded or unlikely in 11 atrs (37.9%). in the 3 suspected transfusion-transmitted bacterial infections, the imputability of the transfusion was ultimately excluded after a negative result was obtained in the bacterial culture of the blood product. the imputability of the blood product was probable or possible in 7 and 9 atrs respectively, but was certain in only 2 atrs. summary/conclusions: unconsciousness and/or convulsions were rarely observed in atrs notified in transfused patients. nevertheless, the presence of these signs highlights the seriousness of the atr (26 ars, 89.7%). lastly, the imputability of the blood product was often excluded or unlikely. in the multivariate cox model for the effect of lpi on overall survival, adjusted for age and ipss-r category, elevated lpi levels were associated with inferior overall survival (hr 3.0, 95% ci 1.5-5.7, p = 0.001). this effect was most pronounced in the td-rs subgroup (hr 6.0, 95% ci 2.2-16.2, p < 0.001). similarly, elevated lpi levels were associated with inferior pfs (hr 3.4, 95% ci 1.9-6.3, p < 0.001) for the whole study population and the td-rs subgroup (hr 8.2, 95% ci 3.4-21.0, p < 0.001). in total 16 patients received iron chelation during the sample collection period (11 patients deferasirox, 5 patients desferrioxamine). lpi levels were normal in 14 out of the 17 samples collected during deferasirox treatment and in 2 out of 5 samples collected during desferrioxamine treatment. summary/conclusions: transfusion dependency is associated with the presence of toxic iron species and inferior overall and progression-free survival in lower-risk mds patients. in td-rs patients the effects were most pronounced indicating ineffective erythropoiesis leading to additional iron toxicity. background: post-transfusion immunomodulation has been reported to contribute to poor patient outcomes. clinically relevant transfusion models are needed to improve our understanding of underlying mechanisms. sheep transfusion models are of increasing importance in blood transfusion research as they provide several advantages over small animals, including their size, anatomy, physiology and similar blood volume compared to human. a current limitation of sheep transfusion models is the lack of characterisation of the sheep immune system. understanding the sheep immunology is necessary to advance sheep transfusion models, identify mechanisms that contribute to post-transfusion immunomodulation and facilitate the translation of findings into clinical settings. aims: to characterise the sheep leukocyte inflammatory responses to in vitro lipopolysaccharide (lps) challenge in edta and heparinized whole blood. methods: edta and heparinized sheep whole blood (n = 4 of each) was cultured with rpmi media (37°c, 5% co 2 ) alone or with the addition of lps (1-100 lg/ml; derived from escherichia coli 055: b5). the inflammatory response was assessed after 2 h (h), 6 h, 12 h, 24 h, 36 h and 48 h. supernatant was harvested at each time point and stored at à80°c. inflammatory cytokine/chemokine production was determined using sheep specific in-house elisa (il-1b, il-6, il-8 and il-10). twoway analysis of variance with bonferroni's post-test was used to measure the effect of incubation time and concentration compared to no lps matched samples. results: when edta was used as an anticoagulant, addition of lps resulted in production of sheep il-1b and il-10 but not il-6 or il-8. il-1b production was significantly increased following stimulation of 100 lg/ml lps for 6 h (p = 0.043) and declined following 36 h incubation. release of il-10 was significant 12 h post-lps stimulation with 100 lg/ml (p = 0.030) and reached a maximum at 24 h. the use of heparinized blood resulted in a different immune profile as all inflammatory markers tested were detected following stimulation with much lower concentrations of lps (1 lg/ml), although the incubation times differed. il-1b was significantly increased following 2 h incubation (p = 0.002), with increasing levels observed up to 24 h post-lps stimulation. il-8 production was evident from 6 h and reached significance at 24 h post-lps stimulation (p = 0.009). il-10 was significantly increased following stimulation of 5 lg/ml lps for 6 hr (p = 0.043) with lower concentrations of lps resulting in il-10 production at 24 h (p = 0.008). release of il-6 was significant after 6 h of 50 lg/ml lps stimulation (p = 0.046), with lower concentrations of lps resulting in il-6 production at 24 h (p = 0.015). in heparinized whole blood an lps concentration-dependent effect was evident for all cytokines. summary/conclusions: using a time-and concentration-approach our findings indicate that sheep are more tolerant and have a delayed response to lps stimulation compared to previous research using similar human in vitro whole blood culture models. in addition, data suggest that sheep have greater immune responses using heparin as anticoagulant for the collection of blood samples. improving our understanding of sheep immunology and development of relevant sheep transfusion models will provide a bridge between sheep models of transfusion and clinical settings. 1. rhdig inappropriately administered (unnecessary exposure) (n = 17, 40%) administered to: -rhd positive woman (n = 9) -rhd negative mother with rhd negative neonate (n = 5) -woman with immune anti d (n = 1) -administered in error (instead of other ig) (n = 2) 2rhdig delayed/omitted/wrong dose (risk of sensitisation to the d antigen) (n = 17, 40%) -omitted (n = 14) -delayed (n = 1) -inadequate dose (n = 2) 3administration without correct patient identification (n = 6, 14%) 4storage & handling (n = 3, 7%) failure to check the maternal and neonatal blood groups prior to administration was identified as a source of error. misinterpretation of blood results also led to women receiving product inappropriately. e.g. reading a negative antibody screen as the mother being rhd negative. patient identification was raised as an issue. rhdig is often stored in satellite blood fridges for easy access. collection from these areas did not always require confirmation against patient identifiers and there was no register of women who received product or link to the batch number to ensure traceability. two incidents involved the administration of rhdig when the prescription for other immunoglobulin products was not clear, leading to a child and a baby receiving rhdig instead of the intended immunoglobulin. summary/conclusions: these incidents indicate problems with the processes of appropriate identification of women who need rhdig, the use and interpretation of pathology tests and requirements for prescription and administration. these resulted in omitted and inappropriate doses of rhdig. blood matters has made a number of recommendations regarding rhdig administration: -all health professionals involved in rhdig administration should be appropriately trained in the use of rhdig -confirmation of the maternal rhd status is essential prior to prescription or administration -positive patient identification must be used prior to administration of rhdig -health services should consider regular auditing to identify areas for improvement relating to rhdig blood matters continues to work with maternity care providers to improve practice. centro comunitario de sangre y tejidos de asturias, oviedo 2 agencia gallega de sangre, organos y tejidos, galicia 3 banco de sangre y tejidos de cantabria, cantabria 4 banco de sangre de la rioja, la rioja 5 banco de sangre y tejidos de navarra, navarra 6 banco de sangre y tejidos de arag on, aragon 7 fundaci on de hemoterapia y hemodonaci on de castilla y le on, castilla y leon 8 fundaci on banco de sangre y tejidos de las islas baleares, islas baleares, spain 9 terumo bct europe nv, zaventem, belgium background: hemovigilance, a long-term monitoring process made mandatory by national and supranational regulations, begins with a systematic whole blood or blood component collection and ends with an examining period after transfusion of blood components into the patients. in spain, organized in 17 autonomous regions, the hemovigilance system is structured in three levels: (1) the local level comprised of transfusion centers and hospital based transfusion services that monitor and collect all transfusion related adverse events (ae) and level them up to (2) the regional hemovigilance coordinator, who communicates all the region's data to the (3) spanish ministry of health which issues an annual report and corresponds with european institutions. to ensure safer blood supply, pathogen reduction technology (prt) was approved and implementation started in spain in 2008. the mirasol prt system for platelets and plasma was introduced in 2010 and is currently being used in 10 of the 17 spanish regions. aims: to monitor the safety of the system, a passive hemovigilance study on mirasol treated products was initiated in the region of asturias and collaboration was extended to other regions (baleares, galicia, la rioja, cantabria, navarra, castilla y leon and aragon). methods: collected data included allergic and febrile reactions, trali and all other adverse event observed. severity of the event and level on imputability of the transfusion were also assessed using the who grading scale. hemovigilance data of mirasol treated products (platelets or m-pc and plasma or m-p) are included from 2012 to 2017 as blood centers started to apply the technology in routine. results: increase adoption of the mirasol system is observed between 2012, when 4,196 mirasol treated blood products were issued to hospitals and 2017 with 56,229 mirasol products issued. due to low number of transfusions of mirasol-treated blood components in 2012 and 2013, notification rates began to be analyzed in 2014, showing ae rates of 0.2%, similar to reports at the national level. stable transfusion reaction rates were observed with m-pc (around 0.23). rate of ae after transfusion of m-p is fluctuant between 0.06 and 0.17. this fluctuation could be due to the inconsistent numbers of m-p transfused from one year to the other. most of transfusion reactions (around 80%) were of grade i severity and grade ii level of imputability. allergic reactions accounted for most of the adverse events, with g&i > 2 reactions in 2016 and 2017 of respectively 0.18 and 0.07 no bacterial nor viral transfusion transmission was recorded on mirasol products during the study period (2012) (2013) (2014) (2015) (2016) (2017) . at the national level, nine cases of bacterial transfusion transmission (with g&i > 2) were reported. these transmissions were probably due to transfusion of non-pathogen reduced products. summary/conclusions: the observed notification rate of ae is similar to the national rate but allergic reactions with g&i > 2 is inferior with mirasol treated products. also, we found no reports of transfusion transmission infections nor cases of transfusion associated graft-vs-host disease, demonstrating safety of mirasol treated products. were attributed to human error (75%) with the lowest frequency in equipment failure (10%), compared to 21% and 29%, respectively, in the following three years. root cause analysis demonstrated failures in the quality management system including failures in administration, inadequate staffing for blood collection as well as in distribution and processing, and failures arising from institutional constraints and system failures in hospital management. high numbers of "other" aes (25%) in distribution and whole blood collection call for further investigation to indicate measures necessary for prevention and correction. errors related to incorrect blood component transfused (ibct) in 2007-2017 were 80 in 8,385,448 blood units (1/104,818) issued for transfusion. these resulted in 27 serious reactions (34%) (1 fatal, 11 life-threatening) . another 21 (26%) were related with ibct that did not cause a reaction. near misses (component not transfused) were 24 (40%) summary/conclusions: our data demonstrate increasing compliance with reporting requirements. questions about the initial factors for deviations in certain activities specifying failures in equipment and materials due to system as well as human errors, highlight the need for further specifications beyond "other" and "human error". background: the weakest link in the transfusion chain currently is the handling of blood components after their issue and the bedside blood administration practices. aims: to evaluate compliance with standard procedures for bedside blood transfusion practices by analysis of the "transfusion feedback forms" in a tertiary care multi-specialty hospital setting. methods: during the study period of 24 months, the transfusion feedback forms received from various clinical areas of the hospital were studied with special reference to the transfusion times. the data was categorized based on the patient's location as well as the time of transfusion, whether done in routine or emergency hours. results: 42,403 blood components were issued during the study period, while transfusion feedback forms for 37,816 components (89.1%) were received in the transfusion medicine department. delay in starting the transfusion (more than 30 min after issue) was observed in 2965 transfusion events (6.9%). the component transfusion time exceeded the permissible limits for 930 component (2.1%).the overall total permissible time for completion of components exceeded permissible limit in 2217 (5.2%) of transfusion events. the pediatric ward (23.9%), icu and ot complex (25.4%) were found to be the most non-compliant delay in transfusion, transfusion time and total transfusion time. amongst the 2965 delayed transfusions after issue, 2120 (71.5%) were during the routine hours i.e. between 7 am to 7 pm and 845 (28.5%) were in the non routine hours i.e. between 7 pm to 7 am. summary/conclusions: the audit of bedside blood transfusion practices has given us a good insight into various areas of noncompliances as well as the predominant locations in the hospital where the practices need to strengthened further. focused training program on safe blood administration practices for all staff involved in handling and transfusion of blood components is now planned to combat this issue. background: the international surveillance of transfusion adverse reactions (ars) and events (aes) (istare) of the international haemovigilance network (ihn) collects aggregate data from member national haemovigilance systems (hvs) in order to estimate the morbidity and mortality of blood transfusion in a holistic approach. the ultimate goal is to contribute to improving the safety of transfusion by close monitoring throughout the chain "from vein to vein". aims: we analyse recent istare data on suspected transfusion transmitted infections (sttis) for 2013-2016 in comparison to previous years of surveillance, [2006] [2007] [2008] [2009] [2010] [2011] [2012] methods: annual aggregate data from ihn member hvs on transfusion associated bacterial, viral and parasitic infections collected online in istare are analyzed by incidence in blood components (bcs) issued for transfusion, by severity and imputability as well as by blood component. ars with definite, probable or possible imputability were included in the analysis. trend analysis is performed to allow comparisons and to collect information on established and newly emerging infectious threats of blood transfusion. results: for 2013-2016 89 sets of annual aggregated data from 25 countries covering 85,521,393 bcs issued were analyzed. all ars totalled 76,907 and infectious ars amounted to 285 (0.4%). the overall incidence of the infectious ars was 0.33/ 100,000 units of bcs issued. bacterial infections were the most frequent (188, 66%), next viral (84, 29.5%) and then parasitic (13, 4.5%). serious were 46% and there were 11 fatalities (3.9%, incidence 0.013/100,000). nine deaths were attributed to sepsis and the other two were associated with non-malarial parasitic pathogens. one geotrichum clavatum fungal infection associated with apheresis platelets was reported as a free text comment. this very rarely recognized fungal pathogen caused a very severe infection in a patient but the route of transmission is inconclusive. the 84 viral sttis included 17 hbv (20%), 10 hcv (12%) and 2 hiv (2.5%). the 55 recorded as "other" (65.5%) including 24 cases of hev, one case of parvovirus b19, one cmv and one ebv. no case of tt-malaria was reported. other stt-pi were 15 (two fatal). the prevailing bcs were in general rbcs followed by platelets. comparison with corresponding data for 2006-2012 shows a consistent overall incidence in total sttis (0.33 vs 0.4/100,000). however, considerable differences were seen in separate categories, such as bacterial infections (significantly increased rate in 2013-2016, p < 0.001) and an almost doubled rate of parasitic infections (p < 0.001). compared to the earlier period, there were many fewer hbv infections (17 vs 160) and many more hev. a similar reduction in the rate of hcv and hiv was observed in 2013-2016 in comparison with previous years. this may be explained by the fact that nat testing for hcv/hiv/hbv has been implemented in many countries in the last decade. summary/conclusions: the infectious risk of transfusion overall remains very low. the rate of bacterial cases has increased and among other viral sttis the frequency of hev is increasing. the mortality of transfusion due to sttis is lower than in the previous period of surveillance. abstract withdrawn. background: one of the main aspects of haemovigilance system in hospitals is following of adverse events and reactions related to blood transfusions. aims: it was intended to analyse the adverse reactions related to transfusion of blood components in pediatric patients. methods: over a four year period (january 2015-december 2018), the haemovigilance records of all patients receiving blood transfusions procedures were reviewed and transfusion reactions were analysed. statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). majority of blood components were provided by regional blood center organized by national red crescent society. but granulocytes collected by apheresis after donor mobilization and reconstituted whole blood for exchange transfusions were prepared in the transfusion center of the hospital. results: the median age of patients who developed transfusion reactions was 72 months (interquantile range-iqr 108). the median for the numbers of individual transfusions in children in a year was 12 (iqr 17). the median for the numbers of blood components individually transfused to patients was 18 (iqr 24). patients, anaphylactic transfusion reactions in 4 patients and transfusion-related lung injury (trali) in a patient. the overall incidence of transfusion reactions was estimated at a rate of 2.7 per 100000 units. summary/conclusions: it was reported that adverse effects related to blood transfusion, especially allergic reactions and fnhtrs are common in pediatric patients than adults. in a multinational study concerned about the transfusion reactions related with red cell concentrates, allergic transfusion reactions and fnhtrs were reported at a rate of 11 and in 100000 units and 26 in 100000 units, respectively. while the incidence of transfusion reactions in children was found 0.62% in a study from the u.s.a., the overall incidence of transfusion reactions in our study which was estimated at a rate of 2.7 per 100000 units represents a lower rate. hospital gran canaria dr. negr ın, gran canaria 6 hospital general universitario, ciudad real, spain 7 hospital nostra senior de meritxell, andorra, andorra 8 banco sangre y tejidos, santander 9 banc de sang i teixits, barcelona, spain 10 fundaci on hematol ogica colombia, bogot a, colombia 11 centro regional de transfusi on de almer ıa, almeria 12 complejo hospitalario de navarra, pamplona 13 fundaci on banc de sang i teixits illes balears, palma de mallorca 14 hospital de cabueñes, gij on, spain background: root analysis cause is defined as the cause of an error that, if it is treated, eliminates the repetition of the error aims: describe types of human and latent errors detected by a work group in the root analysis cause of transfusion incidents, analyze the concordance between the individual responses of the members and propose recommendations in order to improve transfusional safety. methods: in 2018 fifteen participants (nurses and hematologists dedicated to transfusion and component preparation) studied some incidents of administration of nonirradiated components and tried to approach the root causes by applying the classification of errors in mers-tm transfusion medicine. they transferred the answers to a questionnaire (simple or chain error, initial process affected, human and latent errors and measures derived from the analysis to correct the errors). the communication was made by mail and by the spanish transfusion society web forum, which contained the consultation documents. data and percentages are exposed for each type of error and the answers of the participants are tabulated. results: cases corresponded to 3 patients. two patients of 55 years of age diagnosed of acute myeloblastic leukemia (case 1 and 2) and chronic lymphatic leukemia (case 3). in one case, the hematologist of the transfusion service canceled an irradiation prescription; in another, a patient with fever was transfused in the emergency room without the irradiation requirement and it was later discovered that he had received a transplant of hemopoietic progenitors 1 month earlier; in the last case, neither the requesting doctor nor the laboratory technician nor the following doctor (prescriber) detected the alerts located in their respective computer applications. in all 3 cases, the story was judged as sufficient for analysis. the majority of reviewers (93%) diagnosed a chain of errors. there was agreement of 95% with respect to the initial process affected. the initial error was communication (42%), monitoring (45%) and compliance (62%), in cases 1, 2, and 3. 2-3 human errors were detected per case (average: 2.2, 2.7 and 2.4 errors respectively) and 2-3 latent errors per case (average: 1.8, 2.7 and 2.1, respectively). the latent errors most punctuated were: failures in the quality of the protocols (24%), in the transfer of important knowledge (22%), in the available technology (21%) and in the information to the patient (10%). all the participants contributed feasible measures of improvement according to root causes: 1) improve the quality and drafting of work procedures and their compliance, including procedures of effective communication between professionals, 2) train staff in knowledge important for safety, 3) communicate with computer application providers to improve the effectiveness and visibility of the alerts and 4) involve the patient with essential information to ensure transfusion safety. the measures were processed later as recommendations. summary/conclusions: the root analysis shows agreement between participants and allows the elaboration of useful recommendations to increase patient safety. this strategy can contribute to the comprehensive prevention of errors. background: in transfusion-associated circulatory overload (taco), pulmonary oedema develops primarily due to volume excess. data from the uk haemovigilance scheme, serious hazards of transfusion (shot) suggest that either the incidence of taco, or the recognition and reporting of taco, has increased over time. from 2007 to 2017, reports of taco increased from 6 to 92; deaths from 1 to 7, major morbidity from 3 to 20. known risk factors include pre-existing cardiac and/or renal dysfunction, low body weight, extremes of age (eg, <3 years, >60 years), concomitant fluid administration, positive fluid balance, peripheral oedema and hypoalbuminemia. in a small subset of cases reported to shot, taco developed following transfusion for severe anaemia in the absence of other risk factors. this may be an under-recognised independent risk-factor. aims: to raise awareness of severe anaemia as an under-recognised risk factor for taco and is potentially life-threatening transfusion. methods: cases of taco submitted to shot over the last 3 years were reviewed to identify cases where transfusion for severe anaemia was a key identifiable patient risk factor. results: the following are illustrative cases: -case 1: a patient in their 50s weighing 67 kg was prescribed six units of red cells for iron deficiency anaemia after being admitted with hb 37 g/l. the patient had no risk factors for taco except for profound anaemia. during transfusion of the fifth unit the patient became dyspnoeic, hypoxic and hypertensive. the patient recovered after diuretic therapy and had a post-transfusion hb level of 100 g/l. -case 2: a patient in their 50s presented with a 4-week history of weakness and dizziness and had felt unwell for 6 months. the hb was 34 g/l, ferritin 26 lg/l and ecg showed cardiac ischaemia. two units of red cells were transfused. after the second unit oxygen saturations fell despite supplemental oxygen, post-transfusion hb of 51 g/l. a third unit was transfused over 125 min and the hypoxia worsened with dyspnoea and crackles on chest auscultation. the chest x-ray showed an enlarged cardiac silhouette and pulmonary congestion. the patient improved with diuretics. -case 3: a patient in their 90s with severe megaloblastic anaemia, hb 41 g/l and peripheral oedema developed taco after transfusion of 3 units and recovered with diuretic therapy. summary/conclusions: chronic and acute anaemia are associated with compensatory cardiac changes irrespective of the aetiology of anaemia. this is further compounded by the underlying cause of anaemia particularly haematinic deficiencies (iron/b12 deficiency) that independently affect myocardial function. hyperdynamic circulation related to anaemia increases the load on the heart, causing myocardial ischaemia and hypoxia and if the anaemia is not corrected, eventually leads to heart failure. clinicians need to make an accurate diagnosis and avoid excessive transfusion in patients with severe anaemia with or without other additional risk factors. patients with chronic iron/folate/b12 deficiency without haemodynamic instability should be given the appropriate haematinic replacement. haematinic deficiency responds rapidly to appropriate vitamin/mineral. blood transfusions are to be given only when clinically indicated and even then, only the minimum volume needed for symptomatic relief transfused with consideration for diuretic therapy. methods: legal forms for reporting transfusion reactions were used in the retrospective analysis, which were adjusted by the department of quality assurance and quality control in the electronic form and distributed to clinics using blood components. clinicians were trained to report transfusion reactions through the hospital's transfusion board and through the "service for improvement of the quality and safety of health services" at the clinical center of sarajevo. analysis of the reported reactions in the institute include immunohematological and microbiological examination based on which the guidelines for further treatment with blood components are being made. users of registered services are obliged to report since 2015. results: total of 121,076 different blood components were applied in the period between 2015-2018. department for quality assurance and control has received 4 serious adverse reactions, 1 serious adverse event, 157 reports of transfusion reactions, of which 11 (7%) were inadequately filled, in the same period. from the above, 54 (34.39%) were transfusion reactions to erythrocyte blood components which were applied, 86 (54.77%) to platelet components and 17 (10.82%) were transfusion reactions after the application of fresh frozen plasma. the analysis has shown that the most frequent were febrile non-haemolysis reactions (88 or 56.05%), followed by allergic reactions (50 or 31.84%). two transfusion reactions (1.27%) were characterized as circulation overload. summary/conclusions: the frequency of serious adverse reactions and events was 0.004% (5 of 121,076) and 0.12% were reported transfusion reactions (157 of 121,076). with the establishment of the hospital transfusion board and with the increase of collaboration with the clinical center, significant progress has been made. it is necessary to increase awareness among clinicians in regards to the safe transfusion practice. reporting transfusion reactions should be a mandatory procedure, a path to the proper selection of blood components, monitoring adverse reactions, and for us, transfusiologist, guide to the safest, most efficient blood components. j garc ıa-gala, e martinez-revuelta, a caro-g omez, c castañ on-fern andez and i fern andez-rodriguez hospital universitario central de asturias, oviedo, spain background: elderly patients are the main group of transfusion recipients in our country. given their comorbidities are a risk group for the development complications related to transfusion. aims: analyze the incidence of adverse effects related to transfusion in the elderly population and to assess what factors may influence its appearance methods: transfusions were reviewed in patients > 70 years old. the variables analyzed were: sex, age, diagnosis/reason for transfusion, pre-transfusion hemoglobin (hb), number of transfused units, infusion rate and transfusion side effects, as well as the measures used to prevent or treat the transfusions. effects of circulatory overload results: a total of 93 patients were analyzed (48 women, 45 men), with a median age of 86 years (70-97). in total, 189 ch were transfused. 70 patients received 2 ch, 3 patients 3 ch, 10 patients received 1 ch. 10 patients were transfused at two different times. the median hb prior to transfusion was 7.6 g/dl. in the patients who received 2 ch was 7.5 g/dl, those who received 3 ch: 6 g/dl and those who received 1 ch: 7.75 g/dl. the infusion time could be estimated in 84% of the patients. in those who received 2 ch was 223.62 min; 249.33 min in those who received 3 ch and 109.75 min in those who received 1 ch. 13 patients (14% of the total) suffered some type of adverse effect related to the transfusion. in 9 patients there was an increase in posterior ta, in 2 an increase in hr, in 1 an episode of hypotension and in another one episode of acute respiratory failure. 70% of those who had an adverse effect were older than 85 years. patients with aht after transfusion, 75% received 2 ch and the remainder 1 ch. among their background, 66% had a history of ischemic heart disease. 33% also had a positive balance. the average previous bp was 137/65 mmhg and the subsequent one was 182/72 mmhg. 55% of patients did not receive diuretic treatment. in the case of the patient with acute respiratory failure was in oligoanuria, with positive balances. 2 ch was transfused in total. she was treated with oxygen therapy and with intensification of the diuretic treatment, recovering later. summary/conclusions: -patients > 85 years have a higher risk of suffering some type of adverse effect related to transfusion because they have pre-existing risk factors such as ischemic heart disease or heart failure. -we see that the risk of suffering some type of adverse effect in the elderly population is greater when we transfuse 2ch than 1ch. -we have appreciated that in those patients receiving 3ch, the infusion rate is higher. -the study highlights the lack of methods to prevent the development of circulatory overload. background: iron deficiency anemia is the commonest cause of anemia worldwide. weakness, fatigue, reduced physical activity and difficulty in concentration are the symptoms which are associated with its deficiency. the forefront treatment available is oral iron replacement therapy which is convenient, cost effective and has substantial outcome. another option is intravenous (i/v) iron when oral is not tolerable. despite of potential transfusion associated hazards and limited availability of blood due to shortage of voluntary blood donations, it is insisted by the patients prior to iron therapy. aims: the aim of conducting this study was to observe the impact of administration of oral iron, i/v iron and transfusion on hemoglobin levels in patients presented with iron deficiency anemia. methods: this was an observational study carried out at nibd and bmt, pechs campus, karachi, pakistan from february 2018 to december 2018. the study was approved by the institutional review board. diagnosed ida patients presented at our hospital were recruited for analysis who were given oral iron, i/v iron and transfusion for the correction of anemia. informed consent was taken from the participants. descriptive and inferential statistics was applied by using spss version 23.0. results: a total of 108 ida patients were analyzed in which 74(69%) were females and 34(31%) were males. the most common symptom in females and males was fatigue followed by body aches in females 12(16%) and pallor in males 10(29%). menorrhagia was present in 24(44%) of females of reproductive age. surgical history was present in 12(16%) of females while there was no surgical history in males. mean hemoglobin, mch and mcv of females at baseline was 8.0 ae 2.2, 60.5 ae 9.6, and 20 ae 8.3 while in males it was 7.8 ae 2.3, 63 ae 14.3 and 18.4 ae 6.1 respectively. sixty two (84%) females were advised oral and i/v iron and 12 (16%) received transfusion. however, in males 8(24%) received transfusion and 26 (76%) were advised oral and i/v iron therapy. it was observed that the increment of hemoglobin after oral/iv iron at average of 3 months follow up in males and females was same as that the transfusion (p > 0.05). summary/conclusions: in our society where blood donations are scarce especially voluntary blood donations that are considered to be the safest type of blood donation. we would like to draw attention towards the alternatives to correct anemia such as oral and i/v iron replacement therapy as our results revealed that there was no difference in the increment of hemoglobin between the two groups. we need to educate our society especially the older age adults and young women who are more vulnerable of getting ida to opt oral and i/v iron therapy. it will be cost effective, convenient and also has less risk than transfusion. cellular therapies -stem cell and tissue banking, including cord blood background: the differentiation of megakaryocytes plays an important role in the production of platelets. however, the underlying mechanisms regulating megakaryocytes differentiation have rarely been studied. aims: to identify candidate genes involved in megakaryocytes differentiation and investigate the potential regulatory mechanisms of megakaryocytes differentiation from human cord blood hematopoietic stem cells in vitro. methods: cb-derived cd34 + cells were isolated using density gradient centrifugation and magnetic activated cell sorting (macs). cultures were stimulated with only recombinant human tpo (100 ng/ml). after 12, 14 and 18 days, the mk fraction was selected by immunomagnetic sorting from the non-mk fraction using an anti-cd41a monoclonal antibody. rna-seq-derived gene expression data was performed on uncultured samples (day 0), cultured but unselected samples (day 7), and cultured, selected samples (day 12, 14 and 18) by using the next-generation sequencing (ngs) platform, and rq-pcr technology was used to verify the expression of transcription factors. results: the comparison of the transcriptome profiles among the five stages showed that a massive gene expression change occurred in megakaryocytes differentiation. a total of 65140 genes were detected, of which 16612 showed up-regulation and 48528 down-regulation. among these genes, 8 differentially expressed genes (degs) (fold change ≥ 10; false discovery rate < 0.05) were selected were further validated with rq-pcr, including gabre, cdhr1, wasf3, pkhd1l1, thbs1, pf4v1, lrrc32 and lgals12. the rq-pcr result indicated that the mrna expression level increased with the prolongation of culture time. however, pf4v1 mrna expression level was highest at day 14, lgals12 was highest at day 18. summary/conclusions: conclusion: our study identified a series of genes that may participate in the regulation of megakaryocytes differentiation. these results should serve as an important resource revealing the molecular basis of megakaryocytes differentiation and thrombocytopoiesis. preoperative anemia and blood transfusion requirement during hip and knee surgery rambam health care campus, haifa, israel background: blood transfusion (bt) is independently associated with increased morbidity, mortality and hospitalization length across different patient populations. due to bt-related risks, the concept of "patient blood management" (pbm) has been introduced to clinical practice. the three pbm pillars are: optimizing red cell mass, minimizing blood loss and optimizing physiological reserve. bt indications during orthopedic surgery include excessive bleeding or hemodynamic instability and not the hemoglobin (hb) level. in most clinical scenarios, a restrictive transfusion threshold (hb level: 7-8 g/dl) appears to be non-inferior to the liberal transfusion strategy in terms of blood use, morbidity and mortality. similar results are observed in highrisk patients after hip surgery. we hypothesize that preoperative anemia may lead to blood product overuse and its complications. aims: evaluating potential correlation between preoperative anemia and bt requirement during hip or knee surgery. methods: we reviewed medical files of patients who underwent hip or knee replacement surgery at rambam between 2011-2018. patients with hb level measurement within 90 days pre-surgery were included. receiving > 1 blood unit was considered a surgery complication and such patients were excluded. patient demographic and clinical data, including comorbidities, surgery type, length of hospital stay, were collected. we created a synthetic data cohort using mdclone healthcare data sandbox, an environment enabling fast data extraction and producing synthetic data for analysis that does not require irb approval. results: during the evaluated period, 2591 patients underwent hip or knee surgery; 231 were excluded from the analysis due to receiving > 1 blood unit. hb measurement within 90 days pre-surgery was available for 1202 patients. hip or knee surgery was performed in 588 (49%) and 614 (51%) patients, respectively. women comprised 60% (n = 356) of patients who underwent hip surgery. in the hip-surgery group, 14.5% of patients required bt, with this need being slightly higher among women (31.5% vs. 26.2%; p-value=ns). only 50 (8%) patients were transfused during knee surgery and this cohort was not further analyzed. patients receiving bt had a significantly lower mean hb level than those who didn't require it (11.93 g/dl versus 12.8 g/dl for women and 12.3 g/dl vs. 13.8 g/dl for men; p-value < 0.001). hospitalization was longer in transfused patients compared to non-transfused ones (mean 7.52 vs. 6.91 days, p-value = 0.018) and in patients with a low hb level (female < 12, male < 13.5) than in those with a high hb level, irrespective of receiving bt (p-values < 0.00045). patients with at least one of the following diagnoses: diabetes, renal failure, ischemic heart disease, were significantly more likely to have a lower preoperative hb level (p-value < 0.05). no other factors (e.g., patient's weight, rdw value or blood pressure) were predictive of transfusion need. the probability of a need for 1 blood unit was 0.43 in the hb 11 g/dl group and 0.15 in hb 13 g/ dl group (35%>reduction). summary/conclusions: anemia presence before elective hip surgery is a risk factor for bt requirement and longer hospitalization. diagnosis and management of anemia using timely pre-surgery consultations may minimize intraoperative bt, particularly in women and patients with comorbidities. real-patient data and prospective trials are warranted. abstract withdrawn. abstract withdrawn. background: cd36, known as platelet glycoprotein iv, belongs to type b scavenger receptor and is related to the pathogenesis of many diseases. type i cd36 deficiency was cd36 not expressed on platelets and monocytes. individuals with type i deficiency can produce homologous antibodies and cause related immune diseases. in recent years, it has been reported that cd36 deficient individuals cause fetal immune thrombocytopenia with fetal edema syndrome in asia. cd36 is not expressed in mature rbc, but exists in hematopoietic stem (progenitor) cells. anemia is an important cause of edema. in view of the phenomena of clinical and animal experiments, cd34 + hematopoietic stem (progenitor) cells were cultured in vitro to observe the effect of anti-cd36 monoclonal antibody on cd34 + hematopoietic stem (progenitor) cells. aims: to investigate the effect of anti-cd36 monoclonal antibody on proliferation and differentiation of human cd34 + hematopoietic stem (progenitor) cells in vitro. methods: choose 3 healthy full-term maternal women without various obstetric complications, take cord blood 20 ml. after density gradient centrifugation of ficoll cell separation solution, cd34 + hematopoietic stem (progenitor) cells were sorted by flow cytometry and cultured for 2-3 generations. mtt was used to examine the effect of anti-cd36 monoclonal antibody on the growth of hematopoietic stem (progenitor) cells. flow cytometry analysis was used to detect the apoptosis and cell cycle of cd34 + hematopoietic stem (progenitor) cells. the effect of anti-cd36 monoclonal antibody on the formation of cfu-e/bfu-e in hematopoietic stem (progenitor) cells was analysis by cfu-e/bfu-e account after 10-14 days culture. results: after umbilical cord blood was isolated by ficoll to obtain mononuclear cells, the hematopoietic stem (progenitor) cells of cd34 + were sorted by flow cytometry, and about 0.44% of cd34 + hematopoietic stem (progenitor) cells were isolated. different concentrations of anti-cd36 monoclonal antibody and hematopoietic stem (progenitor) cells were cultured in vitro. the od value of value (0.9 ae 0.15) of anti-cd36 monoclonal antibody group (2 mg/ml) was decreased than normal group (1.05 ae 0.12) (p < 0.05), and the od value (0.81 ae 0.11) was significantly decreased at the cd36 monoclonal antibody concentration of 32 mg/ml (p < 0.01). there was no significant difference between the hematopoietic stem (progenitor) cells culture group and the igg control group (p > 0.05). in the annexin v flow detection, the apoptotic rate of anti-cd36 monoclonal antibody group (2 mg/ml) was statistically increased than the normal group (p < 0.05). anti-cd36 monoclonal antibody significantly induced hematopoietic stem (progenitor) cells to undergo s phase cell reduction, g1 phase cells increased, and g1/s phase cell arrest occurred. the number of cfu-e/bfu-e clones in the normal group was 169 ae 12, the number of cfu-e/bfu-e clones in the control group was 172 ae 12, and the number of cfu-e/bfu-e clones in the anti-cd36 monoclonal antibody culture group was 85 ae 6. the number of colonies formed by hematopoietic stem (progenitor) cells in the anti-cd36 monoclonal antibody culture group was significantly lower than that of the other groups (p < 0.05). summary/conclusions: anti-cd36 monoclonal antibody can reduce the proliferation of human cd34 + hematopoietic stem (progenitor) cells and reduce the ability of erythroid differentiation. background: recently the new modern collection techniques were introduced in the apheresis procedures. cobe spectra system was replaced with spectra optia, and it was necessary to verify the efficiency of spectra optia in pbpc collections. aims: the aim of the study was to evaluate and optimize the new cmnc protocol spectra optia v. 11 (terumo) for pbpc collections in patients with haemato-oncological malignant diseases. methods: the results of 159 autologous pbpc collections were evaluated in: (a) well mobilized patients with precollection cd 34+ cells concentration in blood higher than 20/ll, (b) from only the first collections, which were performed either by the use cmnc spectra optia v. 11 or cobe spectra v. 6, v. 7, terumo (c) collections were performed in the standard and large volume leukapheresis regimen, lvl. engraftment data in 56 transplanted patients were assessed. results: standard collections were performed in 52 patients. the yield of cd 34+ cells was high, and no significant differences were found between the numbers of cd 34+ cells prepared from spectra optia 8,6 (1,3-41) 9 10 6 and cobe spectra 10,9 (1, 6 ) 9 10 6 /kg b. w. (a = 0,05; pval 0,619). the dependence of cd 34+ cell yield on the precollection concentration of cd 34+ cells in blood can be considered as linear with high correlation coefficients in cmnc spectra optia r = 0,95, and cobe spectra r = 0,93. lvl collections were performed in 107 of patients, and there were no significant differences between the numbers of cd 34+ cells prepared by cmnc spectra optia 10,9 (2-61,2)9 10 6 and cobe spectra 9,3 (2,4-86) 9 10 6 /kg b.w. (a = 0,05; pval 0,35). the relations between the precollection cd 34+ cells concentration in blood and the numbers of cd 34+ cells from collections can also be considered as linear with the correlation coefficients in cmnc spectra optia r = 0,93, and cobe spectra r = 0,78, respectively. in lvl, the median platelet loss was significantly lower in cmnc spectra optia (45%) than in cobe spectra (57%). a group of 12 patients was transplanted by means of pbpc prepared in the standard regimen. median time in the neutrophil reconstitution was in cmnc spectra optia as well as cobe spectra 11 days, while in platelets from cmnc 14 days, and from cobe spectra 12 days, respectively. the number of 44 patients obtained pbpc from lvl. the median time in neutrophils and platelets reconstitution was in cmnc spectra optia as well as cobe spectra the same, and corresponded with 11 and 13 days, respectively. summary/conclusions: cmnc protocol spectra optia is a modern, efficient and the safe system, which can be used for both standard and lvl procedures. in well mobilized patients the sufficient dose of cd 34+ cells for transplantation could be prepared from one standard or one lvl procedure. no serious adverse reaction have been observed. background: peripheral hematopoietic stem cells are collected from patients/donors after mobilization with g-scf. the aim of the collection is a fixed number of cd34 + cells/kg. this number depends on the pre-apheresis cd34 + number, the blood processed and the collection efficiency of the procedure. the aim should be to collect all the requested cells in 1 day, whenever possible. this is in order to reduce the dose of g-csf given to donor/patient and the resources used in the collection centre. the only parameter that can be adjusted is the volume of blood processed, if this is increased, the likelihood of collecting the requested amount of cells is increased, but only if the pre-apheresis cd34 number is high enough. therefore, you need to know, when it is feasible to increase the volume and thereby increasing the time of the procedure with the intention to collect all the requested cells in 1 day. it can also show if it is possible to reduce the volume of blood processed, thereby reducing the time of the procedure. aims: to develop an easy tool to calculate the volume of blood processed in order to collect the requested cells in 1 day. methods: the mean collection efficiency (ce) was calculated. ce is calculated as cd34 + cells collected/(pre-apheresis cd34 + number*processed volume)*100%. based on the mean ce, an excel sheet was generated to calculate the volume of blood that should be processed in order to collect all the requested cells. the excel sheet is designed so the user enters the pre-apheresis cd34 + number, patient weight and the requested number of cd34 + cells. the ce is fixed according to the mean ce calculated. the result is the volume of blood processed in order to collect the requested yield. based on that result, the apheresis machine will provide time for the procedure, thereby it is possible to evaluate if the collection can be finished in 1 day or not, e.g. by increasing the volume of blood processed. spectra optia â was used for all collections, cmnc for allogeneic donors and mnc for autologous patients. results: mean ce = 64% (n = 348). a ce of 40% was chosen as the cut-off for the cd34 calculation tool. using the cd34 calculation tool: allogeneic donors (n = 61): mean ce = 61%, mean blood volume processed = 3.3 9 tbv, mean time: 253 min, 37 donors were finished in 1 day collection (61%) autologous patients (n = 205): mean ce = 65%, mean blood volume processed = 2.4 9 tbv, mean time = 218 min, 173 patients were finished in 1 day (83%). the calculation failed in only 1 case (0.4%). in this case the volume of blood processed was reduced according to the calculation, but because of unexpected low ce, the requested number of cells was not achieved. summary/conclusions: the cd34 calculation tool based on an excel sheet has shown to be simple and easy to use in order to personalize the stem cell collection. immunotherapy products: blood product, pharmaceutical, or a new category all together? from 2001 till 2019. all donors were hla typed and matched; they were fully informed on the donation procedure and signed an informed consent for donation. minimum dose required to ensure successful and sustained engraftment was 2 9 10 6 /kg cd34 + cells and 2 9 10 8 /kg mono-nucleated cells (mnc). pbsc harvesting was performed with continuous flow cell separator baxter c53000, cobe spectra and spectra optia using conventional-volume apheresis processing the 2-2.5 total blood volumes per apheresis. a femoral catheter was used for harvesting and acid citrate dextrose formula a is used for anticoagulation. recombinant human granulocyte colony-stimulating factor (g-csf) is used to mobilize pbpc for collection. harvesting of pbsc is usually performed after 4 to 5 days of g-csf subcutaneous administration at a dose of 10 lg/kg body weight. results: all the donors were siblings of the patients treated at the university hematology hospital. there were 159 apheresis procedures performed in 106 healthy sibling donors. there were 75 males and 31 females, aged 20-55. one to two apheresis procedures were required to collect adequate graft. the single procedure usually took 3-4 h and the volume of collected stem cells was 50-220 ml. the needed number of mnc and cd34 + cells was successfully collected by 1.5 apheresis. there were 29 abo incompatible donors. procedures for mobilization and collection of pbpc from healthy donors are generally well tolerated. the only adverse effects of the apheresis procedure were bone pain as reaction of g-csf and numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and were very mild. the collected pbsc were used in allogeneic stem cell transplantation in patients with: acute myeloid leukemia -57 patients (53.7%), acute lymphoblastic leukemia -14 patients (13.2%), chronic myeloid leukemia -9 patients (8.5%), myeloproliferative disorders -8 patients (7.5%), myelofibrosis -5 patients (4.7%), severe aplastic anemia -5 patient (4.7%), non-hodgkin lymphoma -3 patient (2.8%), multiple myeloma -3 patient (2.8%), chronic lymphoblastic leukemia -1 patients (0.9), hodgkin disease -1 patient (0.9%). summary/conclusions: the apheresis collection of pbsc in healthy donors is an effective and safe procedure. we are developing our national stem cell donors registry as a part of bone marrow donors worldwide. in that way we hope we will help widen the world network of stem cell donors and enlarge the possibility for each patient to find the right match. background: leukocyte-removing filters for blood are being used widely as a universal leukocyte reduction policy is being adopted progressively throughout the world. filtration is one of the most effective methods for preventing various adverse transfusion effects caused by leukocytes included in blood components, such as febrile reaction, alloimmunization, and transmission of leukotropic viruses. aims: the goal was to evaluate whether the new domestic blood filter finecell, developed by kolon industries, gumi, korea, is appropriately suited to the international standards. and to reveal its efficacy and safety in the settlement of leukocyte reduction system in korea. methods: thirty-two units of packed red blood cells obtained from 400 ml whole blood collected from 32 healthy donors were used. this was done by analyzing the filtration time, residual leukocyte count, rbc recovery, and hemolysis rate during a storage period of 35 days after leukoreduction. results: the standards commonly used for the evaluation of leukocyte-removing filters are set by the food and drug administration of the usa and the council of europe. the results of our study satisfied these international standards. summary/conclusions: the newly developed domestic leukoreduction filter was, thus, found to be efficient and will contribute to the improvement of the quality of isolated blood components used in korea. faculty of science, humanities and education, technical university of liberec, liberec, czech republic background: as polymeric fibrous scaffold fabrication techniques strive to create structures that more closely replicate tentative extracellular matrix form and function, the need for increased scaffold bioactivity becomes more pronounced. the fibrous structure made from biocompatible and nontoxic polymers ensures mechanical stability, however cell proliferation requires further stimulation. platelet-rich plasma, which has been shown to contain over 300 bioactive molecules, has the potential to deliver a combination of growth factors (gfs) and cytokines capable of stimulating cellular activity. aims: the presented work deals with the preparation of nanofibrous materials with platelet growth factors incorporated into the internal fiber structure. polyvinyl alcohol (pva) was used for the preparation a material providing a progressive release of native gfs without need of subsequent crosslinking. methods: materials were prepared from pva (mw 125,000, 98% of hydrolysis) using electrospinning technology (nanospider tm 1ws500u). platelet lysate (pl) was prepared from thrombocyte rich solution (obtained from regional hospital in liberec, the concentration of 700-900 x10 6 plt/ml, freeze-thaw method with subsequent centrifugation). nanofibers were electrospun from 10% pva solution using water: ethanol (8:2) solvent system. materials with proteins were electrospun from solution containing 10% of pva and 10% of pl. morphological analysis was performed by scanning electron microscopy. protein release was monitored using spectrophotometry (bradford method) and chromatography. results: the prepared fibrous materials consisted of random oriented end-less fiber with smooth surface with minimal defects in structure. the morphology of materials was not altered by the addition of proteins. the average fiber diameter was: 340 ae 120 nm for pristine pva fibers and 350 ae 148 nm for pva with incorporated proteins (pva/pl). pva/pl layers contain 5-10 mg of protein per gram of pva. 60% of the proteins are released during the first day (burst release) followed by a gradual release of up to 2 weeks. summary/conclusions: nanofibrous pva-based nanofiber materials were prepared with native growth factors. the process used for the preparation of solutions and subsequent spinning does not affect the activity of the incorporated proteins, which are being gradually released. therefore, we believe that the developed material has great potential for use in tissue engineering e.g. to promote healing of chronic wounds. acknowledgements: supported by the czech health research council, project no nv18-01-00332. background: human a-defensins are small cationic peptides with antimicrobial and anticancer activity. up till now, six a-defensins have been described in humans. they include the human neutrophil peptides (hnp) 1, 2 and 3 which present in large amounts in neutrophil azurophilic granules and differed from each other only in the first amino acid. a fourth defensin, termed hnp-4, comprises less than 1% of the total defensins in neutrophils and has a distinct sequence from hnp1-3. the other two, human defensin 5 and 6, are synthesized mostly by intestinal paneth cells. neutrophil defensins (hnp 1-3) are 3.4 kda peptides that are characterized by three disulfide bridges. the pattern of disulfide bonds in the mature forms is crucial for the functional properties. due to this structural feature, synthesis of defensins using the chemical and recombinant approach presents quite a challenge. moreover, purification from the natural source can be very difficult because the large number of neutrophils is needed to obtain a sufficient amount of protein. in blood banks, leukocyte reduction filters are used to remove leukocytes from blood components in order to prevent adverse transfusion reactions. leukofilters contain high numbers of normal human cells and discard after use. aims: the aim of this study was to purify a-defensins from neutrophils trapped in leukocyte reduction filters. methods: blood bags from healthy blood donors were collected after written consent. all donors were screened for infectious diseases (hbv, hcv and hiv) and negative samples were included in the study. blood bags were filtered at 22°c by leukoflex lst-1 filters. the cells were extracted from the filters by back-flushing with cold phosphate buffered saline (pbs), ph 7.4, without mgcl2 and cacl2, containing 5 mm edta and 2.5% sucrose. the pbs was homogenized with the filter content and then collected in a sterile tube. neutrophils were separated from mononuclear cells by ficoll. isolated neutrophils resuspended in pbs 1x at a concentration of 1 9 10 7 cells/ml. for degranulation, cells were stimulated with 100 nm of formylmethionyl-leucyl-phenylalanine (fmlp) for 5 min followed by stimulation with 10 lm of cytochalasin b for 5 min. supernatant was collected by centrifugation at 200 9 g for 6 min. supernatant was incubated with mouse monoclonal antibody to hnp 1-3 and purification of hnp 1-3 was performed by lmacs protein g microbeads system. the presence of protein was confirmed by western blot. results: the presence of the 3.4 kda band was confirmed by western blot, which corresponded to the size of the a-defensins. summary/conclusions: the development of defensins as therapeutic products requires access to a steady supply of neutrophils. our results indicated that lst-1 filters are economical source for purifying a-defensins. anatomical sciences, abadan school of medical sciences, abadan, iran background: epigenetic reprogramming of terminally differentiated cell can modify somatic cells to a pluripotential state. there are several approaches that induce pluripotency in somatic cells. exposure the cells with the embryonic stem cell extract is an easy way, and some investigations were done on fibroblast cell line. however, its efficiency was low aims: the purpose of this study was to increase the number of reprogrammed granulosa cell as a full differentiated cell into pluripotential state methods: the human granulosa cells were cultured in the medium containing 5-aza-deoxycytidine and trichostatin a. then, the cells were exposed to mouse escs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor (lif). alkaline phosphatase test and also immunohistochemistry staining for oct4, sox2 and nanog were performed after 24 and 72 h and 1 week results: the results indicated that after 24 h the granulosa cells were revealed a round and expanded morphology. the cells in all groups except in negative control, were showed alkaline phosphatase activity. the cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a and exposed to the extract had the most numbers of alkaline phosphatase positive cells. immunocytochemistry showed the granulosa cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a with extract expressed oct4 with weak intensity after 24 h. no expression of oct4, sox2 and nanog were observed in other groups at the same time. after 72 h, oct4, sox2 and nanog were over expressed in the cells that were treated with 5-aza-deoxycytidine, trichostatin a and extract. furthermore, there was high expression of oct4 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract. after one week, the expression of oct4 and sox2 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract was continued while its expression ceased in the other groups. the expression of nanog were ceased in all groups after one week summary/conclusions: present study revealed that the inhibitors of the methyl transferase (5-aza-deoxycytidine) and histone deacetylase (trichostatin a) could delete the epigenetic markers and improved cells reprogramming by administration of the extract abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mesenchymal stem cells (mscs) are adherent spindle shape cells expressing different surface markers. they show special criteria including, paracrine effects, differentiation to several tissue cells, migration, immunomodulatory and regenerative potentialities. mscs are isolated from different sources and employed as therapeutic tools to treat several diseases and injuries. however, some of mscs properties including secretion of growth factors and migration ability are controversial especially during remission or in presence of tumor. interestingly, msc-derived compartment could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. exosomes are kind of extracellular vesicles (evs) characterized via their size and releasing mechanism. usually they defined as less than 150 nm in diameter vesicles. they secreted from different cells and are also found in urine, blood, breast milk, cerebrospinal fluid and other body fluids. exosomes contain genetic material including dna, mrnas, micrornas (mirnas) and other biomolecules. mirnas are single stranded non-coding rnas transcribed from dna. immature mirnas are subjected to two known cleavages to modify to mature mirna that involve to either mrna degradation and gene expression process or cell-cell interaction and communication via secretion as the part of exosomes. aims: this study was aimed to discuss some aspects of exosomal micrornas derived from mscs in progression, diagnosis and treatment of some diseases. methods: different scientific data bases including pubmed, google scholar and scopus were used to find and review related articles. results: evs play important role either in intercellular communication related to pathological and physiological situation or intracellular communication, angiogenesis, immune system modulation and metastasis progression. mirnas could regulate expression of multiple mrnas then they play important role in different biological processes and contribute cell-cell interaction as well as influence in the progression of different disease. exosomal mirna-derived mscs are involved in cancer procession, tumor growth, angiogenesis and metastasis. they are used as diagnosis and therapeutic tools to treat different diseases such as renal failure, liver fibrosis, myocardial infarction. summary/conclusions: due to controversial aspect of using of intact mscs especially during remission or in presence of tumor, msc-derived exosome could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. aims: the aim of study try to use sybr green i based real-time pcr to identify homozygous, heterozygous, gene deletion or wild type for rhd exon 1, 5, 10 and 1227a. methods: for this study, we used real-time pcr with high resolution melting curve mode, and matrix mix containing sybr-green i were used for sequence specific primers of 1227 g>a and rhd exon 1, 5, 10. samples with rhd gene deletion homozygous/heterozygous, 1227 g>a heterozygous with rhd gene deletion and normal rhd, normal rhd homozygous/heterozygous and rhd1-rhce(2-9)-rhd10 homozygous/heterozygous were enrolled in our study. all samples were screened using rhd exon genotyping, sanger sequencing and rhesus box analysis. concentration and mass of dna samples were 1 in alleles of normal rhd/rhd gene deletion. the tm ratio of rhd exon 1 (87°c) to internal control (77°c) were 2.33 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.09-2.21 in alleles of rhd gene deletion/normal rhd, 2.53-2.66 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 10 (81°c) to internal control (77°c) were 3.35 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 4.29-4.39 in alleles of rhd gene deletion/normal rhd, 4.67-6.10 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 5 (81°c) to internal control (77°c) were 3.64 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.49 in alleles of rhd gene deletion/rhd1-rhce(2-9)-rhd10 3.47-3.59 in alleles of rhd gene deletion/normal rhd, 3.97-4.77 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. summary/conclusions: using the tm ratio of sequence specific primers to internal control is an effective way to detect the rhd gene deletion or rhd weak d types 1, 2 and 3 not detected") were tested with a method based on next generation sequencing (ngs) using the illumina miseq platform to detect a possible rhd variant not interrogated by id rhd xt. results: in total 583 dna samples were tested in 87 pools. fifteen (15) pools (181 samples) gave rhd deletion genotype and seventy two (72) pools (402 samples) resulted to the presence of rhd gene. the 72 positive pools were also analyzed individually. the genotype results obtained were: rhd exon 1 no amplification (1), rhd exon 6 and the genotype results obtained with id rhd xt (in pools and individually) were concordant with the results provided by the centers. hence, 100% accuracy was obtained using id rhd xt with dna pooled samples. the results of rh ngs for the samples with inconclusive results by id rhd xt showed rhd variants previously described: 1 sample rhd*93-94inst (del), 1 sample rhd*ivs3+1g (del), 9 samples rhd*weak d type 11 (partial d), 1 sample rhd*weak d type 5 (weak d), 1 sample rhd*weak d type 61 (weak d) and not described: 1 sample rhd*del 1-5 (unknown) summary/conclusions: id rhd xt is a high accurate tool for genotyping the most common rhd alleles associated to weak d and d negative phenotype in up to 20 pooled dna samples. use of rhd genotyping improve rhd typing in blood donations variant rhd alleles generate qualitative/quantitative alteration in serological expression of d antigen such as weak and partial rhd phenotypes which are clinically important in transfusion setting. population studies have shown varied distribution of the variant d alleles in caucasians, africans, east asians and indians. many countries have developed their own population-specific strategy for identifying d variants. our previous study in indian population showed absence of weak d type 1, 2, and 3 which are commonly found in caucasians d variant individuals. instead, a novel population-specific pattern i.e.~12-kilobase duplication event, including exon 3, was predominantly identified in 58.3% d variant samples. functional analyses showed that this genetic variation results in the expression of several transcripts, including a wild-type product. commercial genotyping assays available, mainly detect common d variants found in caucasians and africans, thus limiting its usefulness in india. hence, based on our findings we have designed a multiplex pcr assay specific for indian population that can be easily implemented at the laboratory level for genotyping variant rhd. aims: to characterize rhd variants using "indian-specific, rhd genotyping assay". methods: seventy samples referred to our laboratory for molecular characterization of rhd variants were included in this study. all rhd variant samples were serologically typed for results: out of the 70 rhd variants included in this study, 49 samples (70%) showed presence of indian specific allele i.e. exon 3 duplication. seventeen rhd variants samples showed presence of both exon 5 and 10. qmpsf analysis of these samples excluded involvement of rhd-rhce-rhd hybrids. sixty of the seventy d variant individual had r 1 r genotype this assay thus can be used routinely in indian laboratories to identify and characterize rhd variants. -128 non-invasive fetal kel1 genotypes from allo-immunized anti-kel1 women were done (34 positive confirmed fetuses, 6 undetermined, 16 positive non-confirmed, 22 negative non-confirmed and 50 negative confirmed). -190 non-invasive fetal rhc genotypes from allo-immunized anti-rh4 women were done non-invasive fetal rhe genotypes from allo-immunized anti-rh3 women were done (66 positive foetuses, 1 undetermined for 21,5% of the allo-immunized women, the pregnancy was compatible and no specific antenatal monitoring was necessary summary/conclusions: non-invasive fetal red blood cell genotype is a powerful tool to diagnose a feto-maternal red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring s purchla-szepioła 1 , m krzemienowska 2 , m pelc-kłopotowska 2 , m jurkowska 3 , m debska 4 , m uhrynowska 2 and e brojer the test developed by ihtm has been offered to clinicians and pregnant women since 2016 but it is not covered by the health care system. rhd nipt is not informative for mothers with rhd variant. in such cases further analysis of the molecular background is offered to exclude from immunoprophylaxis the women with weak rhd type 1, 2 and 3. aims: summary of a 3-year period of routine rhd nipt available at ihtm. methods: cffdna isolated using easymag, biomerieux from plasma of 366 pregnant women determined with standard serology as rhd-negative (in 8-38 week of gestation) was examined for the presence of exons 5 and 7 of rhd and ccr5 by realtime pcr using lc480ii (roche). maternal dna from whole blood was tested for identification of rhd variant using rbc fluogene rbc-dweak/variant (inno-train, germany) or the home-made protocol. results: in 123 cases the rhd gene was not detected in cffdna and the administration of rhig was not recommended. in seven cases ct-values for rhd and ccr5 indicated a maternal d variant (d ct ccr5-rhd >2); the genetic follow-up of six of them identified: rhd*01w.2 in 4 cases, rhd*01w.1 and rhd*15. in 235 cases the rhd nipd results indicated that a fetus is rhd positive and rhig administration was recommended it was recommended in the remaining 67% of mothers. in 1.9% cases with maternal d variant rhd nipt was not possible. however, in 5/6 such cases the test is unnecessary because follow up analysis revealed maternal rhd variant of the weak d type 1 and 2 in switzerland extended antigen-matching for duffy, kidd and mnsbesides rhesus and kell -is recommended for sickle cell disease (scd) patients. the ethnic diversity of red blood cell (rbc) antigen polymorphism engender that these patients are often transfused with rbcs from donors of african origin. this strategy, however, increases the likelihood of being exposed to certain low-prevalence antigens, such as rh23 (d w ), as these are almost exclusive to african populations. rh23 is encoded by several types of rhd*dv as well as by dau-5. anti-rh23 is associated with delayed hemolytic transfusion reactions (htr) aims: here we report a specific low-prevalence antibody newly formed by the same patient, meanwhile gravida 4, para 1, causing positive crossmatches with the rbcs of two of the four selected donors. subsequently, advanced serologic and genetic workup and close international collaboration enabled optimal patient care. methods: standard serological methods were used for antibody specification (biorad, cressier, ch and in-house). crossmatches were carried out by indirect antiglobulin test at 37°c. molecular typing of donors' and parental blood group antigens was performed by further serological analysis (institute national de transfusion sanguine, paris) revealed an anti-rh23 in addition to anti-fy 5 , anti-e and anti-jk a . genotyping of the two donors causing positive crossmatches presented heterozygosity for rhd*10.05 which encodes rh23. the newborn's phenotype was a r0r k-, fy(a-b+) and most likely rh23-and jk (a+b+), considering both maternal and paternal (a r0r, k-k+, fy(a-b+), jk(a+b-), rh23-) predicted phenotypes. the neonatal serum contained maternal anti-a1, anti-rh23 and anti-e. the direct antiglobulin test was positive but elution only showed nonspecific reactions with papain-treated cells. latter might have been caused by anti-fy 5 during her present pregnancy we were able to demonstrate that two positive crossmatches of two former compatible donors were caused by a new alloantibody against a low-prevalence antigen, namely anti-rh23, derived from several rh23 + rbc transfusions during the previous pregnancy. despite this challenging blood supply we were able to support the patient with a total of ten antigen compatible and crossmatch negative rbc units from french and swiss donors until delivery with increasing age, the relative number of women in the study population raise from 22% in the patients younger than 60 years to 58% in the patients older than 80 years. our study showed that cardiovascular diseases were the commonest indications for warfarin use in older patients with 59%. only 48,5% achieved target therapeutic range while the risk of thromboembolism and the subsequent need for proper anticoagulant therapy increases sharply with age, the bleeding risk rises as well. older patients are more sensitive to any given dose of warfarin and need a significantly lower total weekly dose. a well-informed patient provides one of the best defenses against bleeding complications. recent data demonstrate doacs advantages over warfarin, especially for older population: more predictable dosing, fewer drug interactions and reduced risk of intracranial bleeding although vast majority of fh cases are caused by mutations in ldl-r gene 17%-33% patients do not harbor genetic cause in the known loci. patients with homozygous/severe heterozygous fh are unresponsive (ldlc above 200 mg/dl with diet and drug therapy) and require additional extracorporeal therapy to reduce ldlc concentrations to prevent the development/progression of cad. ldl apheresis techniques remove apolipoprotein b-containing lipoproteins from blood and include heparin-induced extracorporeal ldl precipitation(help), immunoadsorption, dextran-sulfate adsorption methods: a 28y iraqi male visited cardiac-opd. ct coronary angiogram showed cad-dvd. he had multiple tendinous xanthomas and xanthelasmas. family history was significant for death of elder brother from coronary event at 30y, a sister with similar profile age 26y and one sister apparently normal. he was taking medical treatment for dyslipoproteinemia (ecosprin 75 mg od, ticagrelor 90 mg bd, rosuvastatin 40 mg od). despite dietary and medical treatment his dyslipoproteinemia was refractory. therefore cascade-filtration was planned with evaflux5a plasma fractionator. one procedure of cascade plasmapheresis was done on com.tec apheresis system (fresenius kabi, germany) separating patient's plasma as the first step and passing it through a pore sized based filter column 5a20 (evaflux, kawasumi, japan) as the second step. a total of 1.5x plasma volume(4470 ml) was processed. the patient was given continuous calcium infusion. the flow rate of 50 ml/min was maintained immunoglobulins) were not assessed. summary/conclusions: the procedure successfully met the requirement of reduction of cholesterol by 60%. the patient became responsive to the medical treatment. follow up of the patient up to a year has been uneventful with no additional procedure requirement actions included development of major haemorrhage protocols with improved communication and required instances of delayed transfusion to be reported to the uk national haemovigilance scheme (serious hazards of transfusion, shot) methods: delayed transfusion data have been collected from 2010. hospitals identify incidents and report them via an online database. mh may also result in avoidable or overtransfusion. reports are analysed and collated data published in the shot annual reports in july each year. results: the total number of reports of delayed transfusion has increased with time: 101, 95, 111 in the last 3 years. delayed transfusion in relation to mh was reported for 54 cases 2016-2018 contributing to death in 6 patients 7%) miscommunication was noted between clinical different teams, between emergency departments, porters and the transfusion laboratory, failure of bleeps, failure to communicate the urgency, failure to confirm the patient location. failures to follow mhp correctly occurred in 31/71 (43.7%): incorrect activation including failed alerts to porters, wrong contact telephone details and wrong components in the mhp packs most transfusions included red blood cells (median, 2 units); 14% of women were transfused with fresh frozen plasma (median, 2 units) and 2% with platelets. mean pre-and post-transfusion hemoglobin levels were 6.7 g/dl and 9.0 g/dl, respectively, representing an increment of 1.0 g/dl per rbc unit transfused (1.09 g/dl in soweto and 0.83 g/dl in durban). indications for transfusion included obstetric hemorrhage (31%), chronic anemia (29%), surgery or anesthesia (13%), other (9%) and not specified (17%). transfusion for chronic anemia (vs. hemorrhage) was associated with gestation ≥ 26 weeks (odds ratio = 7.07, 95% confidence interval 3.52-14.24). surgical blood loss was a common indication in trimester 1 (21%) that declined to 7% then 1% in trimesters 2 and 3. summary/conclusions: hemorrhagic complications accompanying spontaneous abortions and ectopic pregnancies in the first and second trimesters were the most common reasons for antenatal transfusion surveillance and analysis of blood transfusion reactions represents inseparable part of hemovigilance. aims: summarization of data on reported cases of transfusion reactions. methods: analysis of serious undesirable reactions to blood products administration in the czech republic (cr) during period 2016-2018. results: there were evaluated 1,281 688 of blood products administrations in 117,414 patients in the cr during defined three years period. announced 1,456 (0,11%) transfusion reactions including 50 severe transfusion reactions (20 9 adjudged with grade 3). the most frequent types of severe transfusion reactions: anaphylaxis 20, trali 7x, taco 5x, hcv 4x, hbc 2x, bacterial infection 1x, delayed hemolysis 1x. transfusion reactions incidence according to administered bp: red blood cells products: 882,017 administrations, 883 transfusion reactions (fnhtr 386x, allergy 227x, circulatory overload 66x, anaphylaxis 6x, trali 4x, hbv 2x, hcv 1x) platelets: 88,438 thrombocyte administrations, including 178 transfusion reactions (allergy 129x, fnhtr 30x, anaphylaxis 5x, circulatory overload 8x, delayed immune hemolysis 3x, acute circulatory overload 8x. granulocytes: 203 administrations, 2 transfusion reactions plasma: 293,801 administrations, 359 reactions reported (allergy 278, fnhr 25, circulatory overload 14, anaphylaxis 17x, trali 4x, hbv 3x, ards 1x. summary/conclusions: conclusions: comprehensive analysis and data processing help to appropriate prospective setting of blood products (bp) production and hemotherapy. concrete outputs from processed data triggered undermentioned changes in many departments in the cr: 1. plasma for clinical uses from male blood donors, 2. prestorage of leucocyte reduced blood products, 3. production of platelets in additive solutions, 4. implementation of pcr testing method for blood donors screening. background: skae's basic activities include epidemiological surveillance of all adverse events (aes) associated with deviations in the collecting, testing, processing, storage and distribution of blood and blood components that may affect quality and safety near misses" and "uneventful transfusion errors" are collected to identify preventable causes. incorrect blood component transfused (ibct) events are reported following ihn instructions. results: a total of 5 they were mainly associated with deviation in processing (22.4%) and attributed to equipment failure and materials (71%) whole blood collection, materials and distribution, as a result of product defect, equipment failure, human error and other. trend analysis showed a significantly increasing (p < .001) annual rate of total aes by 28% (95% confidence interval 26-30) ) 10% fibrinogen-depleted phpl or (3) 10% fibrinogen-depleted phpl plus heparin. internalization of fluoresceinamine-labeled heparin in stcs was investigated by flow cytometry and immunocytochemistry. all stromal cells were subjected to whole genome expression analysis (affymetrix human gene 2.1 st array) and data were analyzed using r/bioconductor and panther analysis tools. confirmative qrt-pcr was performed and protein levels of selected pathways were analyzed by a bead-based western blot system (digiwest â ). immunophenotyping, in vitro differentiation, longterm proliferation and colony forming units (cfu) assays were done for all cell types. results: in vitro exposure of heparin induced differential internalization and lysosomal accumulation in stromal cells, as well as regulation of distinct gene sets, both in a tissue-source dependent manner. affected signaling cascades were mainly involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis. the influence of heparin on protein expression and phosphorylation of four pathways (wnt, pdgf, notch and tgfbeta) was further analyzed, revealing most alterations in bm-stcs. independent of origin and medium composition, flow cytometry analysis revealed the characteristic fibroblastoid phenotype profile (cd73+/90+/105+ and cd14-/19-/34-/45-/hla-dr-). in addition a comparable osteogenic and adipogenic differentiation capacity was found summary/conclusions: internalization of heparin in lysosomes by stromal cells, differential gene and protein expression and phosphorylation changes were observed in a tissue-source dependent manner. however, stromal cell characteristics as immunophenotype pattern, long-term proliferation, clonogenicity and in vitro differentiation were unaffected, putatively by post-translational protein modifications. in this respect, application of porcine heparin is compatible with efficient manufacturing of stromal cell based medicinal products abo incompatibility may have no effect on the clinical outcome after allogeneic hematopoietic stem cell transplantation. however, it carries additional risks of hemolytic reactions, delayed red blood cell (rbc) engraftment and incidence of graft-versus patients were categorized according to abo compatible and mismatched groups; these were further sub-categorized into major, minor and bidirectional. direct coombs test (dct) was performed when hemolysis was suspected in the post-transplantation period along with serum lactate dehydrogenase (ldh) 5%) were male and 31(36.5%) female. mean age of abo matched and mismatched groups were (6.99 ae 4.6) years. most common indications for transplant included beta thalassemia major 53(60.2%), aplastic anemia in 25(28.4) and pure red cell aplasia 2(2.35%). source of stem cell was bone marrow in 45 and peripheral blood 40 patients abo matched while abo mismatched group comprised of 28(32.9%) patients with further subdivision into major (n = 13), minor (n = 13) and bidirectional in the post transplantation period, packed red blood cell and platelets were transfused in matched group (n = 401) and (n = 1837) comparably(n = 102) and (n = 480) in mismatched group. primary and secondary graft failure in matched group was 8.77% and 12.28% patients while in mismatched group graft failure was observed in 4(14.28%) patients respectively. positive dct in abo matched group in 1(1.75%) patient, whereas 2(7.14%) patients with major and minor abo mismatch group with raised ldh levels and deranged lfts were found. episodes of acute and chronic gvhd in abo compatible and incompatible groups were insignificant. overall survival in abo summary/conclusions: these results indicate that abo incompatibility does not seem to influence outcome of the patients undergoing allogeneic hematopoietic stem cell transplantation. careful monitoring of patients can help detect problems early and treat them efficiently, thus, reducing the number of life threatening events a picascia 1 , c sabia 1 , f cavalca 1 , g nicoletti 2 and c napoli 1 in our routine work with one lambda sab class ii reagents, we observed non-specific reactivity with some beads bearing dr4 and dr16 in patients without sensitizing events. this pattern was not confirmed by testing same sera with screening-and pra-beads suggesting non-specific reactions. aims: here, we sought to determine if fetal bovine serum (fbs) treatment would be effective in reducing/eliminating non-specific reactivity. methods: we tested 5 sera pre-treated with fbs from non-sensitized patients that showed the dr4/dr16 pattern. in particular, 5 ll of fbs was added to 95 ll of patient serum; incubated for 30 min at 37°c; centrifuged and subsequently tested in the sab assay. as controls, we treated sera from 5 patients with documented dsa including dr4/dr16 and 5 patients without hla antibodies. results: dr4/dr16 non-specific reactivity was eliminated or significantly reduced after fbs treatment. we found that patients with dr4 and dr16 dsa had no change in mfi values and additional reactivity was not observed in negative fbs treated sera transfusion medicine, national blood transfusion centre 2 transfusion medicine, national blood transfusion center 3 transfusion medicine, national blood transfusion centre, tirana, albania background: abo blood group, has been associated with many diseases, although the explanation for abo's blood group association and some illnesses is still unclear. aims: to find the distribution of cases by blood groups in patients with malignant pathology compared to donors in order to assess the presence of the abo blood group as an epidemiological indicator to identify populations exposed to different malignant pathologies methods: we conducted a case-control study. abo blood group and diagnosis of all patients have been studied. the control sample was collected from 17,992 healthy donors from which group a (36,8%), group o (41,7%), b (16,2%) and group ab (5,3%) resulted. the study was conducted in 3727 patients who have been transfused and submitted a request to determine the blood group at the blood bank at qsut during the period 2011-2017 results: among the 3727 patients, when all malignant pathologies were taken together, the highest frequency was seen in blood group a (39.7%), followed by 0 (39.5%), b (15.2%) and ab (5.4%). group a frequency was higher and o was lower compared to controls. a high incidence of blood group a is seen in: pancreatic cancer a (42%), in gastric cancer a (43%), colorectal cancer a (39, 6%), breast cancer a (41%), cervical cancer a (43%) and ovarian cancer a (42%) versus a (36.8%) in the control group. a high incidence of blood b is seen in multiple myeloma b (22%) and cervical cancer b (20%) versus b (16.2%) in the control group. blood group ab has a high incidence in malignant lymphoma ab (10%) versus (5.3%) in the control group summary/conclusions: it appears that individuals with blood groups a, b and ab are more at risk of developing malignant pathologies and individuals with blood group o are more protected. background: the high homology and opposite orientation of rh genes promote many rearrangements between them and generate a large number of rhd and rhce variants which can be inherited together. several studies have shown that those rh variants in patients with scd represent an additional risk for alloimmunization and delayed hemolytic transfusion reactions (dhtrs), but little clinical or biological evidence related to alloimmunization and dhtr are presented for all the rh variant alleles. it is well established that transfusion recipients with the most common weak d types 1, 2 and 3, are not at risk for forming alloanti-d when exposed to conventional rhd-positive rbcs. aims: we report here a case of a 12-year-old patient typed as weak d type 3, receiving d+ rbc units who presented anti-d in his plasma detected three weeks after the last transfusion. methods: rhd beadchip (immucor, nj, usa), was performed to identify the rhd variant allele associated with the weak expression of d. rhce genotyping was performed by laboratory developed tests. sequencing of rhd, rhce and rhag were performed to determine if there were other mutations that could explain the production of alloanti-d. serologic testing was by standard hemagglutination methods. the clinical significance of the antibody was evaluated by monocyte monolayer assay (mma). results: serological analysis showed a negative dat and the presence of anti-d in plasma (2+ by gel). anti-lw was ruled out. rhd genotyping revealed the patient was rhd*weak d type 3. rhce genotyping predicted the d+c+c+e-e+ phenotype. sequencing of rhd, rhce and rhag found no additional changes and confirmed the presence of rhd*weak d type 3. mma showed the anti-d was clinically significant (>5%). summary/conclusions: we report the production of alloanti-d in a scd patient with rhd*weak d type 3 allele. weak d type 3 patients are not considered to be at risk for allo anti-d but our results show that there are exceptions and that these anti-d can be associated with clinically significant rbc destruction. background: the mns blood group system is located on glycophorin a (gpa), glycophorin b (gpb) and hybrid glycophorins on the surface of the red blood cell (rbc). these glycoproteins are involved in complex structures interacting with other rbc surface proteins including the band 3/diego protein. the glycophorins are heavily glycosylated and contains multiple clinically significant blood group antigens. it has proved difficult to model the gpa extracellular structure due to its heavy glycosylation, and lack of homology with existing modelled proteins. aims: to develop an in silico model of gpa as a basis for improved predictions of structure function relationships methods: prediction of secondary structure and disorder: 1.1. predictprotein (https://predictprotein.org); 1.2. spider2 (http://sparks-lab.org/server/spider2/); 1.3. dsc (discrimination of protein secondary structure class): using an in-house implementation; 1.4. jpred4 (http://www.compbio.dundee.ac.uk/jpred4/); 1.5. raptorx (http://raptorx.uchicago.edu). prediction of secondary structure: 2.1. robetta (http://robetta.bakerlab.org/submit. jsp); 2.2. falcon (http://protein.ict.ac.cn/treethreader/); 2.3. itasser (https://zha nglab.ccmb.med.umich.edu/i-tasser/) threading methods to evaluate the quality of protein structures: 3.1. verify3d (http://servicesn.mbi.ucla.edu/verify3d/); 3.2. prosa (https://prosa.services.came.sb g.ac.at/prosa.php) protein-protein docking: 4.1. gramm-x protein-protein docking web server (http://vakser.compbio.ku.edu/resources/gramm/grammx/); 4.2. gramm (http://va kser.compbio.ku.edu/main/resources_gramm1.03.php) results: using in silico modelling we derived a stable tertiary glycosylated structure for gpa both as an individual protein and a homodimer. the hybrid glycophorin background: non-invasive prenatal testing of fetal antigen using cell-free fetal (cff) dna from maternal plasma of immunized women is widely implemented into clinical routine but the sensitivity and specificity of the method, especially for genotyping antigens encoded by single nucleotide polymorphisms such as k antigen, is limited by low proportion of cffdna in maternal plasma dna. according to literature reports detection of circulating tumour (ct)dna can be improved by selection of short ctdna fragments using automated electrophoresis methods. aims: the aim was to assess the feasibility for enrichment of cffdna fraction in maternal plasma dna by size selection using the pippin prep gel selection system. methods: plasma dna isolated using easymag (biomerieux) from rhd negative and k-negative pregnant women (n = 10) carrying fetuses with known genotype was loaded into 3% agarose gel casette (3% df marker q3, sage bioscience) and size selection of fraction was performed on a blue pippin tm (sage bioscience) with the elution from 45 min to 2 h 30 min of electrophoresis. results for real-time pcr detection of fetal rhd, kel*1 and ccr5 (as a marker of total plasma dna) in dna fraction after gel selection were compared to results obtained from non-processed plasma dna. results: the total dna level (measured by ccr5) was significantly lower in dna samples tested after gel selection (from 8.4 to 25.9geq/pcr) versus the level obtained from non-processed plasma dna (from 130 to 133geq/pcr). the results for fetal fraction (measured by rhd) from dna samples of rhd-negative pregnant women carrying rhd positive fetus tested after gel selection were from 1,5 to 6.6geq/pcr versus 10.8-11.2geq/pcr for non-processed plasma dna. results for kel*1 detection in plasma dna from k-negative pregnant women carrying k-negative fetus were kel*1-negative in dna samples tested after gel selection comparing to nonprocessed dna samples were false kel*1 positive amplification was observed. however, kel*1 detection in plasma dna from two k-negative pregnant women carrying k-positive fetus gave false kel*1-negative results in dna samples tested after gel selection comparing to non-processed dna samples were kel*1 positive genotype was obtained. the total dna level in samples from k-negative women was from 18.3 to 18.7geq ccr5/pcr after gel selection versus from 52 to 746geq ccr5/ pcr in non-processed dna samples. summary/conclusions: using the pippin prep gel selection system increases the proportion of cffdna fraction in total plasma dna by retaining long maternal dna fragments in the gel cassettes, but the protocol of gel separation dilutes the separated material decreasing the concentration of fetal dna and leading to false negative results of nipt. anti-rh1 quantification assay using ih-500 (bio-rad â ): promising results for monitoring rh:-1 pregnant women j beaud, h delaby, c toly-ndour, a mailloux and s huguet-jacquot centre national de r ef erence en h emobiologie p erinatale (cnrhp), hôpital saint-antoine, paris, francebackground: the generalization of immunoprophylaxis by anti-rh1 immunoglobulins since 1970 complicates the interpretation of the anti-red blood cell antibodies screening during pregnancy. to distinguish an alloantibody from a passive one, many laboratories in france use anti-rh1 microtitration. it is a column agglutination technology using red blood cells rh: 1, -2, -3,4,5 (r0r) . it permits to quantify low levels of anti-rh1 in comparison to a range of an anti-rh1 standard. performed since 1999 at the cnrhp and automated on evo clinical base tecan in 2008 (dilutions and distribution), anti-rh1 microtitration is well adapted to rh prophylaxis. aims: the aim of this study was to evaluate this technique on the ih-500 system from bio-rad â . methods: on ih-500, the reactivity of the bio-rad â reagents was compared with the cnrhp reagents (red blood cells r0r, anti-rh1 standard). the performances of the method were evaluated using three internal quality control (icq) (2 cnrhp home-made at 2 and 12 ng/ml and 1 bio-rad â at 12 ng/ml) on papainized r0r (plc) and native r0r (nlc). a comparison of results from patient sera ranging from 1.5 to 48 ng/ml was done between ih-500 and evo clinical base tecan. results: the results of the 3 qci are similar between the different reagents used. there is no significant difference between the 2 types of red blood cells except for the limit of detection: 1.5 ng/ml in plc -6 ng/ml in nlc. for the 3 qci, the intra and interassay imprecision based on the dilution degree show coefficients of variation between 0 and 15%, similar to those found with the evo clinical base. the correlation with the cnrhp technique performed on 50 samples in plc and 44 samples in nlc was satisfactory (deming plc: r2 = 0.80 y = 0.89x + 0.78 -nlc: r2 = 0.86 y = 1.08x-0.25). summary/conclusions: the anti-rh1 microtitration on the ih-500 offers similar performances to the method conducted at the cnrhp. the ih-500 allows automated reading of gel cards. however, it does not have a calculation or interpretation algorithm and does not directly give the concentration of anti-rh1. this final part remains manual and requires trained staff. background: haemolytic disease of the foetus and newborn (hdfn) due to maternal-foetal incompatibility has been perfectly framed for decades from the etiologic, pathogenetic and therapeutic point of view. the anti-d alloantibody is most frequently responsible for the most serious form of hdfn due to rhd incompatibility (rhdi hdfn). although immunoprophylaxis (ip) has reduced the number of cases of rhdi hdfn, this disease continues to occur and red blood cell alloimmunization still remains the most common cause of foetal anaemia. hdfn due to abo incompatibility (ab0i hdfn) is currently the most common neonatal haemolytic disease in the western world. however, only in about 1.5-2% of cases haemolytic disease demands transfusion support. aims: analysis hdfn from 2011 to 2018. methods: the hdfn's transfusional support is: intrauterine transfusion (iut) in the antenatal period; exchange transfusion (et) for severe hyperbilirubinaemia and neonatal transfusion of small volumes red cells for the newborn's late anaemia in the postnatal period. our policy for iut, for et and for the neonatal transfusion requires the use of a concentrated blood cells (ec) preferably group 0 rh negative (cde/cde) or negative for any red cell antigens if the mother has antibodies, fresh (<5 days), preferably cmv safe. for iut, the ec must be compatible with mother's plasma, must have hematocrit 70 + 5%, and irradiated. the unit for et must be compatible with the newborn's plasma, whit hematocrit 40% -60% and irradiated. the ec used in post-natal transfusions is usually divided into rates of 70 ml, hematocrit 55 ae 5%. results: in last 7 years, we calculated 59 neonates with hdfn (28 males and 31 females): 31 with rhdi hdfn, 19 with ab0i hdfn and 9 with hdfn due to incompatibility for other red blood cell antigens. we have produced 81 iut: 48 for our hospitalized patients and 33 for patients located in other hospitals. 25 of these 48 patients, who received iut, were immunized: 19 showed anti-d antibody and 6 antibodies different from anti-a and anti-b. 7, of the 31 infants with rhdi hdfn, were transfused in utero. 4 neonates on 59 (6.8%) have performed et: 1 with ab0i hdfn and 3 with rhdi hdfn; the latter had also been transfused in utero. 27 neonates on 59 were transfused after birth: 18 with rhdi hdfn, 3 with ab0i hdfn and 6 with hdfn due to incompatibility for other antigens. summary/conclusions: our case studies reflect the literature data. neonates with rhdi hdfn are the most numerous (52.7% of the total) and are those who have requested the highest blood supply both in the antenatal period (39.6%) that postnatal (9.6% performs et, 66.6% performs postnatal transfusions). neonates with aboi hdfn are 32.3%: nobody has received iut, only one has been subjected to et, and 11% has transfused after birth. patients with hdfn due to other antigens are 15%, have undergone iut 12.5% and were transfused after birth 22.2%. background: according to british guidelines on neonatal transfusion, since 2012 we shared with neonatologists a transfusion protocol for preterm babies. patients are anemic premature newborns with a gestational age ≤ 30 weeks and/or a birthweight lower than 1500 g, until 4 months of age. aims: reduce the incidence of iatrogenic anemia. methods: pre-transfusion tests were based on ab0 direct test, rh phenotype, direct and indirect antiglobulin test (dat, iat). a second blood sample was required for ab0/rh confirmation. blood transfusions were performed with 0 negative kell negative (0 cde/cde/kk) cmv negative irradiated erythrocyte concentrates (ec) of less than 5 days. ec were subdivided in 70 ml aliquots with a hematocrit of 55 ae 5%. according to the definition of "small volume transfusions", our protocol established that further four transfusions had to be delivered free of testing. after the fifth ec transfusion the supplementary release of ec was provided of type and screen (t&s) test with 72 h of validity. serological investigation and full compatibility testing were applied in the presence of a iat and/or dat positivity and in the case of mother alloimmunization. results: from october 2012 to the end of 2018, 694 premature newborns received 2328 ec transfusions within their first 4 months of life. in 51% of cases (n = 1186), transfusion requirement was comprised within the 'small volume transfusions'. another 44% of cases (n = 1035), requiring further ec administration, was requested of a blood sample for t&s determination and 5% (n = 107) for a cross-match test. in 79.4% of newborns (n = 551), being transfused within the " small volume transfusions", blood requirement of 1401 ec was fulfilled by the initial blood test (1102 blood samples). 13.3% of newborns (n = 92) received more than 5 transfusions (6-21; median = 8) accounting for 820 ec released and for this group 381 blood samples were required. summary/conclusions: with the exception of babies requiring crossmatch test, 381 blood tests were performed to sustain 643 infants transfused with 2221 units. the alternative option of omitting crossmatch test (otherwise suggested by italian directives), allowed a reduction of 75% of blood drawn without any adverse effect or incident reported. due to the relevance of anemia in premature babies, we suggest the application of this transfusion policy in all newborns in the first 4 months of life. background: glucose-6-phosphate dehydrogenase deficiency (g6pdd) is a sexlinked enzymopathy which is usually asymptomatic unless individuals are exposed to oxidative stress agents. the g6pd 202 genotype is the most common g6pd genotype in sub saharan africa (ssa). some studies have linked blood from g6pdd donors to poor outcome of a transfusion. however, there are no genetic screening programmes for blood donors in the region hence the contribution of g6pdd to the donor pool in the ssa setting had not been described.aims: this study aimed to describe the prevalence of g6pdd 202 genotype among donors in two regions in uganda. it also described the effect of g6pdd and the coinheritance of haemoglobin s and a-thalassaemia on the haematological quality of blood. methods: 3,255 blood samples from donor packs were utilized in a transfusion trial conducted in uganda, were genotyped for g6pd 202, haemoglobin s and a-thalassaemia. haemoglobin and haematocrit measurements for the donor units (packs) at the time of transfusion were used to describe the effect of g6pd 202 and co-inheritance with a-thalassaemia (n = 2,546) and haemoglobin s (n = 2,642) on the haematological quality of blood packs. a subset of 142 donor blood packs was utilized to determine the sensitivity and specificity of the carestarttm rapid diagnostic kit (rdt) for g6pdd. results: based on g6pd 202 genotyping, 10.3% (n = 274) of the blood samples used in the trial were deficient for g6pd enzyme while 5.3% (n = 142) were heterozygous. significant lower hemoglobin values were observed in red cell concentrates (p = 0.010) and whole blood (p = 0.009) donations of heterozygous g6pd 202 genotype. co-inheritance of g6pdd and a-thalassaemia resulted in significantly lower haemoglobin levels. the carestarttm rdt test was 83.3% sensitive and 0.8% specific for detecting donor blood packs with g6pdd. summary/conclusions: the prevalence of g6pdd among ugandan blood donors was similar to that in the general population. the heterozygous genotype resulted in lower haemoglobin concentration of the blood units. the use of carestarttm rdt for screening of stored blood units was not as efficient in this study hence further testing for the determination of g6pdd needs to be done on fresh samples from donors. transfusion medicine, jaypee hospital, noida, india background: during last two decade there has been a continuous remarkable improvement in desensitization therapy in high risk hla sensitized kidney recipients. in india there has been a tremendous increase in the number of kidney transplantations in patients having anti-hla antibodies (hla sensitized) with excellent success rate. aims: in present study, we are describing successful role of desensitization in 39 hla sensitized patients having preformed donor-specific hla antibody (dsa). methods: all patients were preconditioned with combined modality of a standard dose of rituximab, therapeutic plasma exchange (tpe) and low dose ivig. tpe was started using com. tec (fresenius kabi, germany) after 21 days of administration of rituximab. complement dependent cytotoxicity cross match (cdc-xm), luminex cross match with donor lysates (lm-xm, immucor inc., ga, usa) and flow cytometry cross match (fc-xm, bd facs canto ii).) was done in all cases. if any of the three tests was positive, single antigen bead assay (sab) was performed. desensitization therapy was given in all cases where dsa was detected. pretransplant tpe procedures were done until dsa (mfi < 1000) and cdc-xm became negative. cdcxm labeled positive at ≥ 10%. t-cell fcmx was considered positive above 42 mfi and b-cell fcmx was considered positive above 120 mfi. lmxm was considered positive above 500 mfi. sab was performed using lifecodes single antigen (lsa) class i and class ii kits (immucor, usa). if the specificity of anti-hla antibodies was against donor hla antigen(s) it was called as donor specific antibody (dsa). results: present study demonstrated the diagnostic and clinical superiority of adding fc-xm and lm-xm in pretransplant compatibility testing algorithm over cdc-xm. cdc-xm alone was not able to detect anti-hla antibody in 8 patients (20.5%). among the three pretransplant compatibility tests, fcxm demonstrated highest sensitivity. among the 39 cases initially screened 35 showed dsa positivity in sab. desensitization was done in those 35 cases only. in our study, sab was positive for class ii alone in 13(37%) while in remaining 22 (63%) cases it was positive for both class 1 and class ii. the number of pre transplant tpe procedures required was 6.7 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) . the mean number of post-transplant tpe sessions required was 0.7 (range, 1-6). during pretransplant and post transplant tpe procedures, five (14.3%) patients presented with allergic or hypotensive reactions which were managed conservatively. most of the patients were discharged after seven days of hospital stay whereas patients who required post-transplant tpe were discharged after a relatively longer hospital stay (mean-8.5, median-7 days). after three months, protocol biopsy was done in those cases only where post transplant tpe was required. protocol biopsy showed normal findings. in present study, the mean duration of follow up was approx 17 months with the longest duration of follow up of 36 months. summary/conclusions: in a country like india where there is a huge gap in the demand and supply of kidney and a large no. of patients waiting for a suitable organ, transplant across hla barrier could a good doable option. thorough pretransplant compatibility and tpe are essential tools to make these transplants program successful background: most transfusion-dependent chronically anemic patients are managed by simple red cell transfusions. however, some patients are not able to tolerate the additional volume associated with simple transfusions and are at a high risk of developing transfusion associated circulatory overload, if transfused with multiple red cell units. plasma-to red cell exchange (prx) is a modified procedure wherein an apheresis machine is used to remove patient's plasma, while simultaneously replacing with donor rbcs. this procedure allows for a rapid euvolemic transfusion of rbcs to patients that are severely anemic and intolerant to excess fluid volume. others as well as our group have previously described this procedure. we now summarize our institutions nearly seven years of experience performing this procedure on a routine basis. aims: to document patient experience with prx. methods: we performed a retrospective chart review of all patients that underwent prx at our institution in the last seven years. our protocol for prx has evolved during this period. currently, we perform the procedures using spectra optia (terumo bct, lakewood, co) machine using the plasma-exchange program and tubing set. if the patient's pre-procedure hematocrit (hct) is < 22%, we custom prime the tubing set with 5% albumin. the number of red cell units transfused to the patient depends on their pre-procedure hematocrit and is individualized to the patients. results: we have treated four patients with prx procedure. patient #1 is a 56-year-old transfusion-dependent male with beta-thalassemia major. the patient had experienced multiple congestive heart failure exacerbations secondary to simple transfusions and we consequently performed prx procedure, every 4 weeks, starting in 2012. the patient has completed 84 procedures with 3-4 units of washed red cells transfused to achieve a target hct goal of 45 to 57%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #2 was a 25-year-old female who had symptomatic anemia secondary to sickle cell disease (hb ss complicated by end-stage renal disease (esrd). she had progressively become intolerant to simple transfusions, including an episode of severe dyspnea, which required intubation. she underwent 9 prx procedures with 2-3 units of washed red cells. patient tolerated the procedures without any significant complications. however, during a different surgical procedure, she experienced cardiac arrest and subsequently passed away. patient #3 is a 33-year-old transfusion-dependent male with severe anemia secondary to sickle cell anemia (hb ss). he was intolerant to excess fluid because of esrd and congestive heart failure. he has undergone 38 prx procedures with 3-5 red cell units transfused to achieve a hct goal of 30%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #4 is a 48-year-old male with a sickle cell disorder (hb ss) complicated by esrd, heart failure and chronic hypoxemic respiratory failure. the patient has undergone two prx procedures with 4-5 red cell units. other than an episode of non-bloody emesis that was symptomatically treated, he tolerated both procedures. he continues to be managed on this regimen. however, the patient remains noncompliant with treatment. summary/conclusions: prx is a safe and efficient method to transfuse multiple red cell units to volume-intolerant anemic patients. background: transplanted organ failure due to antibody mediated rejection in abo-compatible organs is a serious complication with a bad prognosis. the goal treatment in these cases encompasses the following strategies: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. the 2016 american society for apheresis has assigned a category i to the use of therapeutic plasma exchange for the treatment of abo-compatible antibody mediated rejection in kidney, but a category iii to all other abo compatible organs: liver, lung, and heart. at our institution, a standardized approach for the use of therapeutic plasma exchange as a supportive intervention for abo-compatible immune mediated rejection, regardless of the organ type, has been in place since 2011. aims: a retrospective review was performed to evaluate our patient outcomes using therapeutic plasma exchange for the treatment of antibody mediated allograft rejection in abo-compatible solid organ transplantation. methods: patients used for the retrospective review were selected from an existing therapeutic apheresis list. the therapeutic plasma exchange protocol consists of: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. it is performed as follows: one plasma volume exchange is performed on days 1, 2, 3, 5, 7, 9 along with one or more of the above strategies followed by an ivig infusion. cases with allograft rejection in which plasmapheresis was not used were excluded. and t devos 7 aims: this study aimed to explore the possible causes of the decreased transfusion rate for all adult cardiac surgery patients. methods: data were collected from adult cardiac surgery patients during the mentioned time frame and were extracted from electronic patient files and a database of the department of cardiac surgery. a set of 63 variables was defined as possible confounders by a panel of experts. after discussion, 9 global variables (age, gender, duration of surgery, use of cpb (cardio-pulmonary bypass), american society of anesthesiologists (asa) risk score, type of surgery, urgency, attending cardiac surgeon and attending anesthesiologist) and 7 cpb-related variables (administration of cardioplegia yes/no (cpg), duration of cpb, circulatory arrest, hypothermia, duration of aortic cross-clamp, baseline hemoglobin and cpb-priming volume) were retained. negative binomial models for counts were used for data analysis. all analyses were performed with spss. results: 1018 patients were extracted from databases and further analyzed. the mean age of this group was 65,9 years (sd +/-14,1 years) and 67.3% of them were male. the mean duration of surgery was 256 min (sd +/-88,3 min). the decrease of perioperative rbc transfusion rate over four years was statistically significant (p < 0.001). in 2011, the mean use was 2,34 units per operation (sd +/-2,277), which changed to 1,38 units (sd +/-2,158) in 2014. three variables (urgency, attending cardiac surgeon, attending anesthesiologist) changed significantly over 4 years and were used in a multivariable model as confounders together with rbc transfusions and year. even after adjustment for these factors, the decrease in rbc transfusion rate was still statistically significant (p < 0.001). in the specific group of patients undergoing cardiac surgery with cpb (n = 640), the use of rbc was also significantly reduced (p < 0.001). in 2011, the mean use was 3,06 units per operation (sd +/-0,448) and this changed to 1,94 units (sd +/-0,167) in 2014. after correction for the 3 cpb variables that notably changed over the 4 years (cpg, priming volume and hypothermia) and the three previously defined confounders (urgency, attending cardiac surgeon and attending anesthesiologist) the reduction of rbc transfusions over 4 years still remained statistically significant (p < 0.001). summary/conclusions: our study shows evidence for a decreased rbc transfusion rate in adult patients undergoing cardiac surgery between 2011 and 2014. this tendency was also seen in the subgroup of patients undergoing surgery with cpb. possible explanations of the decrease are implementation of various established parts of patient blood management. however, a unique reason could not be identified in this study. background: growing worldwide demand for immunoglobulin products such as intravenous immunoglobulin (ivig) and subcutaneous immunoglobulin (scig) is driving plasma collection. patients with primary immunodeficiency (pid) or secondary immunodeficiency due to haematological malignancy or its treatment (sid) rely on these products to maintain therapeutic serum igg levels to minimise recurrent infection. efficacy of immunoglobulin replacement therapy (irt) in pid is well established but information on sid is limited. the different aetiologies of hypogammaglobulinaemia between pid and sid raised the question of whether sid patients on irt experience similar clinical and quality of life (qol) benefits as reported in pid patients. aims: to assess whether sid patients experience similar clinical and qol benefits while on irt as pid patients. methods: following ethics approval, data on dosage, serum igg trough levels and infection (bacterial, viral and fungal requiring treatment such as antibiotics) was collected from 14 adult pid and 13 adult sid patients from medical records and pathology reports, for their last 12 months of ivig and their first 12 months of scig. the starting and maintenance dose was 0.4 g/kg/month for ivig, transitioning immediately to 0.1 g/kg/week for scig without a washout period. a study specific questionnaire was developed to gather data on patient perceived side effects, treatment satisfaction and impact of irt on social/family life, work/study and their overall qol. paired t-test was used for parametric data and the wilcoxon signed-rank test for non-parametric data. results: sid patients were significantly older with a mean age of 62.9 years versus 43.4 years in pid patients (p = 0.007). a mean of three training session was required to reach competency in scig administration in both cohorts. there was a trend of reduced side effects on scig for pid and sid patients compared to ivig, with a significant reduction of headaches in the pid cohort (p = 0.041). the majority of patients experienced infusion site reactions, which were predominantly perceived as manageable. 55% of infections were respiratory tract infections. pid patients had slightly higher mean serum igg trough levels with scig (9.3 g/l) compared to ivig (8.4 g/l), and fewer infections on scig than ivig (mean annual infection rate of 1.64 vs 2.14 respectively). sid patients had higher mean serum igg trough levels on scig (8.4 g/l) than ivig (7.1 g/l) (p = 0.009) but experienced more infections while on scig versus ivig (mean annual infection rate of 2.15 vs 1.62 respectively). the number of hospitalisation due to infection decreased in both cohorts with scig. pid patients perceived that switching from ivig to scig improved their health and qol. in contrast sid patients perceived no improvements in health and qol. summary/conclusions: data from this pilot study suggests that the clinical and qol impact of irt in sid patients is different to that of pid patients. to support evidence based irt management and effective use of this limited and expensive blood product in sid, larger studies which account for different stages of malignancy and associated treatment regimes are required. background: there is an increasing platelet transfusion for treatment and prophylaxis of bleeding in patients with hematologic disorders and malignancies. because of limited resources, leukoreduced platelet concentrates is not yet implemented in most indonesian hospitals. in vitro platelet activation may cause morphology, functional, and ultrastructure changes. those changes will reduce the platelet viability, in vivo functions, and clinical efficacy. high platelet cd62p expression is the cause of faster platelet destruction in the reticuloendothelial systems. post-transfusion in vivo hemostatic efficacy can be determined by the measurement of corrected count increment (cci), recovery, and platelet cd62p expression. aims: to analyze the increase of platelet cd62p expression in patients of non-leukodepleted compared to pre-storage leukodepleted pc transfusion.background: haemorrhage is a leading cause of preventable death not only in the military trauma care, but also for civilian population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. resuscitation using blood products and limited infusion of normal saline improves survival for critically bleeding patients. nowadays there are hems programs (helicopter emergency medical system) carrying blood products around the world. the hems in castilla-la mancha, with physician and nurse, is the first out-of-hospital emergency service in spain that provides packed red blood cells (prbc) transfusion where the accidents happen without delaying the transport to the proper hospital for definitive treatment. this program has been developed between the blood center of ciudad real and the hems team ('gigante 2', emergency service castilla-la mancha). the goal of the designed protocol was to preserve the properties of the product to be transfused in out-of-hospital environment by hems teams. aims: to describe the process for out-of-hospital prbc transfusion in hems of ciudad-real. the protocol for out-of-hospital blood transfusion was developed according to criteria of medical indications and security, monitoring, and tracking of the product. methods: data for the observational retrospective study were collected from june 2014 to august 2018. the medical helicopter (ec 145t2) was provided with two prbc o rh(d) negative. the shock index was selected for the indication for transfusion according to the literature revised and as a simple rate to obtain out-of-hospital data. to achieve the feasibility and preservation of the prbc a prospective monitoring of volume was established, haematocrit, haemoglobin, leucocytes, coulter, hemolysis and microbiological culture. blood center established two groups: the case group for the prbc kept in the hems base and helicopter and the control group for the units remaining in the blood center with standardized blood conservation. for both groups, control and comparison of immediately obtained hematologic analyses, and 35 days after collection, were performed. the statistical analysis used spss 23.0 version (significance p < 0,05). results: 138 prbc samples were evaluated, 48,5% (67) from case group and 51,5% (71) from control group. analyses were tested day 1 and day 35 after collection. haemolysis was not observed. all cultures were negative. although significant differences were found between the parameter in the value of before-after in the value of the hematocrit, leukocytes and coulter, there are no differences between the prbc that flew and those conserved in the transfusion-service. all results comply with current legislation and blood transfusion standards. there have been administered 35 prbc transfusion to 28 patients during out-of-hospital advanced medical assistance. no post-transfusion reactions have been registered. prbc units have a 35-day rotation to allow the use of the units in the hospital after achieving their optimal status. summary/conclusions: the out-of-hospital transfusion protocol designed to transport blood (prbc) in the helicopter for hems has demonstrated to keep the standard conditions and properties of the product to be considered useful in the treatment for critically bleeding patients in the out-of-hospital. background: early and adequate treatment of major bleeding is important for survival and a good outcome. blood and plasma are given increasingly early including before hospital admission in trauma based on successes reported from combat experience. in 2010 the national patient safety agency issued a rapid response report requiring national health service hospitals in england to take actions to improve provision of blood in an emergency including provision of major haemorrhage protocols (mhp) and drills. the national reporting and learning scheme had identified reports of 11 deaths and 83 instances of harm due to delay over a 4-year period. aims: the aim of the study was to monitor the acid-base status of the patient by means of abg and to administer the blood component therapy based on teg results. methods: this study was a prospective observational study of 18 adult patients over a period of 7 months. serial monitoring of the abg in the intra-operative period was done. teg guided resuscitation was performed in all 18 cases. results: the abg analysis of all 18 patients showed decrease in the ph, increase in pco 2 , decrease in serum bicarbonate level and elevation in negative base excess. these components of metabolic acidosis can be attributed to massive transfusion. increased lactate, an independent parameter, which reflects poor tissue perfusion or shock and predicts need for massive transfusion was observed in all patients. all the 18 cases showed a decrease in ionized calcium levels which could be a result of citrate related toxicity. increased glucose was observed in all patients which may be due to increase in the catecholamine release as a response to haemorrhagic shock. electrolyte correction was given depending on results of the abg analysis wherever appropriate. two out of 18 cases showed an increase in r time indicating deficient coagulation factors, which was corrected with fresh frozen plasma (ffp). three cases showed elevation in k time indicating deficient fibrinogen levels, which was corrected by ffp. fresh frozen plasma was also given in 4 cases, which showed decrease in the alpha angle, indicating deficient fibrinogen, and cryoprecipitate was given in 2 cases. platelets were transfused in 5 patients showing a decrease in the maximum amplitude (ma), which indicates deficient platelets. summary/conclusions: teg as poc testing is an important tool in appropriate blood component therapy in massive transfusions. serial monitoring of abg helps in monitoring acid-base status of the patient and therefore is a guide in the correcting electrolyte level in patients undergoing massive transfusion. background: massive blood loss is encountered in various situations like trauma, major surgeries, gastrointestinal bleeds and obstetric haemorrhages. haemorrhage is an important cause of mortality and morbidity in massively bleeding patients. early recognition of haemorrhage and intervention is essential for survival. massive transfusion (mt) of blood is required to replenish blood losses and is a lifesaving treatment in these patients. a variety of haemostasis and pathophysiological changes occur during massive haemorrhage and massive transfusion. all of these changes contribute to the vicious cycle of progressive coagulopathy due to the 'lethal triad' of refractory coagulopathy, progressive hypothermia and persistent metabolic acidosis. aims: the aims of the study included understanding management of cases of massive blood transfusion in surgical patients, impact of mt of blood components on patient outcome, evaluating post-operative complications of massive transfusion and the development of institutional massive transfusion protocol (mtp).methods: this prospective observational study commenced after institutional ethics committee (iec) approval. it comprised of 40 adult surgical oncology patients who received massive transfusions and was conducted for a period of 7 months. every case of a massive transfusion was studied under the following headings (1) patient's details (2) patients base-line laboratory tests (3) resuscitation with transfusion (4) intra-operative laboratory tests (5) thromboelastography (teg) (6) post-operative complications (7) duration of stay in the hospital (8) 30 day mortality rate. results: complete blood count, serum electrolytes, arterial blood gases, coagulation profile and teg were used to monitor transfusion therapy in the intraoperative period. intraoperative laboratory parameters of patients showed dilutional coagulopathy, metabolic acidosis, hypocalcaemia, hypomagnesaemia, hyperkalaemia and hypokalaemia, increased lactates and glucose. electrolyte correction was done based on the derangement. the derangements were on a decreasing trend in the postoperative period and returned to baseline level by 2 nd or 3 rd post-operative day with no requirement of correction in the post-operative period. the post-operative outcomes were evaluated in terms of the surgical site infection (ssi) as per the centers for disease control (cdc) criteria, surgical complications as per modified clavien-dindo classification and respiratory complications. a total of 13 (32.5%) patients had ssi, 14 (35%) had surgical complications and 22 (55%) patients had respiratory complications. the length of the stay in the hospital was longer for patients who had postoperative complications. despite complications, owing to excellent peri-operative care, 38 (95%) patients were discharged alive. summary/conclusions: surgeries associated with massive transfusion are at an increased risk of ssi as well as morbidity and mortality. complications associated with rapid transfusions of blood, acute haemorrhage and associated risk of the surgery lead to a prolonged icu stay and increased length of stay in the hospital. a well-developed massive transfusion protocol optimizing the ratio and dose of the blood component therapy results in excellent patient outcome with minimal postoperative morbidity and mortality. background: despite the introduction of new oral anticoagulants (dabigatran, rivaroxaban, apixaban), vitamin k antagonists (vka), such as warfarin and acenocoumarol are still the most widely used oral anticoagulants for the treatment of non-valvular atrial fibrillation (nvaf). the use of vka must be regularly and often laboratory controlled in order to ensure the adequacy of therapy and to avoid subdosing or drug overdose. the most commonly used test for the control of oral anticoagulant therapy is the prothrombin time (pt), expressed in inr system, which provides an internationally standardized monitoring of the treatment. time in therapeutic range (ttr) represents a measure of the quality of the anticoagulant effect of vka and estimates a percentage of time a patient's inr is within the desired therapeutic. aims: the aim of this study was to evaluate of the effectiveness of vka therapy in patients with nvaf and to identify factors affecting the anticoagulation efficacy. methods: a retrospective study was conducted on a population of 725 outpatients with nvaf, treated with vka and followed in blood transfusion institute of ni s from january to december 2017. laboratory control of inr was done from capillary blood of patients on thrombotrack solo (axis shield, norway) and thrombostat (behnk elektronik, germany). targeted 15 ae 17.52%, but 49.72% of patients had a ttr less than 60%. patients were at high risk of thrombosis in 6.15% of time (inr < 1.5) and high risk of bleeding in 2.2% of time (inr > 4.5). the most significant independent factors affecting the quality of vka therapy are gender, arterial hypertension, diabetes mellitus and the use of amiodarone and antiplatelet drugs (aspirin, clopidogrel). summary/conclusions: the ttr is undoubtedly useful indicator of the effectiveness of vka treatment. the most important predictors of poorer efficacy of vka therapy are arterial hypertension, diabetes mellitus, patients' gender and the use of amiodarone and antiplatelet (aspirin, clopidogrel) drugs. to improve the quality of vka therapy, education of patient and better collaboration with them, as well as a successful teamwork with clinicians are also imperative. background: an estimated 1.9 million deaths per year result from haemorrhagic blood loss. at a cellular level, haemorrhagic shock develops when oxygen delivery is insufficient to meet oxygen requirements to maintain aerobic metabolism. successful resuscitation prevents further oxygen debt and repays the prior oxygen debt. this includes the administration of fluids and blood components (e.g. plasma, red cells and platelets). measurement of oxygen delivery and utilisation at a tissue level requires invasive monitoring not possible clinically, meaning that surrogate markers such as lactate and venous oxygen saturation (svo 2 ) are used instead. new technologies such as incident dark field imaging and near-infrared spectroscopy may offer a non-invasive alternative; however their utility in haemorrhagic shock remains background: transfusion-induced red cell alloimmunization is still a major challenge in transfusion practice. besides logistic problems for the transfusion laboratory, it may compromise available blood supply, and when undetected and unanticipated, it may risk haemolytic transfusion reactions. knowledge about risk factors can help to optimize preventive matching strategies. severe renal failure and subsequent renal replacement therapy influence the immune system and could therefore modulate the risk of alloantibody formation against foreign red cell antigens subsequent to transfusion. aims: this study aims to quantify the association between renal failure, according to its degree and its treatment with renal replacement modalities, and transfusioninduced red cell alloantibody formation. methods: we performed a multicenter case-control study within a source population of patients receiving their first and subsequent red cell transfusion between 2005 and 2015 in the netherlands (risk factors for alloimmunization after red cell transfusion, r-fact study). using a conditional multivariate logistic regression, we compared first-time transfusion-induced red cell alloantibody formers (n = 505) with two similarly exposed non-alloimmunized control recipients (n = 1010) during a five-week alloimmunization risk period. degree of renal function was categorized as: 'no renal failure' i.e. glomerular filtration rate (gfr) > 30 ml/min/1.73 m 2 , 'moderate renal failure' i.e. gfr ≥ 10-30 ml/min/1.73 m 2 during a continuous period of minimally seven days, 'severe renal failure' i.e. gfr < 10 ml/min/1.73 m 2 and/or use of any type of renal replacement therapy during at least one day of the alloimmunization risk period. odds ratios were interpreted and presented as relative risks (rr). adjusted rrs were conditioned on the matching variables and identified confounders. results: no renal failure was observed among 441 (87.3%) cases versus 838 (83.0%) controls; moderate renal failure among 24 (4.8%) cases versus 54 (5.3%) controls; and severe renal failure among 40 (7.9%) cases versus 108 (11.7%) controls. among the latter, 30 cases and 97 controls underwent renal replacement therapy. moderate renal failure and severe renal failure without renal replacement therapy were not significantly associated with red cell alloimmunization (adjusted rr 0.82, 95% ci 0.67-1.01 and adjusted rr 0.81, 95% ci 0.58-1.11, respectively). however, patients undergoing renal replacement therapy had a two-fold lower alloimmunization risk (adjusted rr 0.48, 95% ci 0.39-0.59) as compared to transfusion recipients without renal failure, unrelated to type and duration of renal replacement therapy. summary/conclusions: these findings suggest that patients undergoing renal replacement therapy have strongly diminished red cell alloimmunization risks. further research should confirm these results and elucidate the underlying pathophysiological protective mechanism. background: the ability of allogeneic hematopoietic stem cell transplantation(allo-hsct) to prevent relapse depends partly on donor natural killer (nk) cell alloreactivity. nk effector function depends on specific killer-cell immunoglobulin-like receptors (kir) and hla interactions. thus, it is important to identify optimal combinations of kir-hla genotypes in donors and recipients that could improve hematopoietic transplantation outcome. aims: to obtain the optimal combinations of inhibitory kir and its ligand between donor and recipient which is helpful for the guidance of selecting donors and recipients in hsct. methods: the pcr-sbt method was used for kir2dl1, kir2dl2/kir2dl3, kir3dl1, kir3dl2 and hla-a, -b, -c, -drb1, -dqb1 genotyping. 58 pairs of hla 10/10 identical donor/recipients matching samples were retrospectively analyzed. three different models of kir were established. there were kir-kir gene model, kirligand model and haploid model. in kir-ligand model, patients were divided into three groups: c1/c1 homozygote group (48 cases), c1/c2 heterozygote group (8 cases) and c2/c2 homozygote group (2 cases). according to the expression of 3dl1, 53 cases were 3dl1 positive and 5 cases were 3dl1 negative. there were 5 cases of bw4/bw4, 24 cases of bw4/bw6 and 24 cases of bw6/bw6 in the 3dl1 positive samples. according to the expression of a3/a11, they were divided into three groups: a3/a11 negative group (28 cases), a3/a11 heterozygous group (28 cases) and a11/ a11 homozygote (2 cases). according to kir genotyping, kir haploidentical group (38 cases) and kir haploid mismatched group (20 cases) were divided. the clinical data on neutrophil and platelet remodeling time, gvhd and os of 58 cases were statistically analyzed by the mann-whitney test or the kruskal-wallis test using graph-pad software v5.01. results: there was no significant difference in the time of neutrophil and platelet remodeling, the incidence of agvhd and the survival time after transplantation in the kir genotype model. in haplotype model, there was no significant difference in neutrophil and platelet remodeling time and survival time after transplantation. the incidence of agvhd was low when the kir haploid mismatched and kir3dl1 was positive. it was conducive to neutrophil and platelet remodeling when bw4/bw6 and a3/a11 was heterozygosity. summary/conclusions: it is important to establish the three different models of kir genotypes, haplotypes and receptor-ligand mismatches for analyzing the effect on the prognosis of allo-hsct. kir-ligand model plays an important role in hla unre-background: transfusion of platelet concentrates (pcs) has been associated with adverse outcomes including transfusion-related acute lung injury (trali). the underlying mechanism of trali has been related to the accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in pcs. current room temperature storage limits the shelf-life of conventional pcs to 5-7 days. alternative storage conditions, including cryopreservation, offers extended storage and a solution for blood banking logistics. cryopreservation of human pcs has been associated with higher concentrations of immunomodulatory mediators compared to room temperature stored pcs and it has been suggested that cryopreserved pcs may be immunomodulatory. to investigate the effects of cryopreserved pcs, a transfusion sheep model would be a beneficial approach. aims: to characterize immunomodulatory mediators in supernatants of sheep conventional and cryopreserved pc and to investigate whether storage duration and cryopreservation impacts the accumulation of these mediators. methods: buffy coat pooled sheep pcs (n = 3), prepared in 30% plasma/70% ssp+ with minor modifications to standard human procedures, were stored room temperature (rt) for 7 days (d) and sampled on d2, d5 and d7. cryopreserved sheep pcs (n = 3), prepared by the addition of 5-6% dimethyl sulfoxide, were stored at à80°c and sampled pre-freeze and post-thaw. supernatant was prepared at each time point with double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), anti-inflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferongamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of non-polar lipid mediators, such as arachidonic acid (aa), 12-hete and 15-hete were assessed in the stored sheep pc-and cryo-pc supernatant using commercial elisa. results shown as mean ae standard deviation. the effect of storage was determined at p < 0.05 using paired t-test. results: in rt stored sheep pc supernatant il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete were detected at d2, d5 and d7. storage duration significantly increased accumulation of ip-10 at d5 (253.4 ae 266.7 pg/ml compared to 569.7 ae 272.7 pg/ml, p = 0.008) and further increased at d7, and il-8 at d7 (3477 ae 937.4 pg/ml compared to 5092 ae 521.1 pg/ml, p = 0.0460). cryopreserved sheep pc supernatant pre-freeze and post-thaw contained equivalent or higher concentrations of il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete than rt stored d5 pcs. however, cryopreservation did not impact levels of any of the platelet derived mediators. summary/conclusions: several platelet-derived cytokines/chemokines, including high concentration of il-8 with neutrophil priming activity, and non-polar lipids were found in stored sheep pc supernatant. these immunomodulatory mediators may contribute to adverse outcomes associated with pc transfusion. storage at rt, but not cryopreservation was associated with increased accumulation of immunomodulatory mediators in sheep pcs. most importantly, similar to human pcs, sheep cryopreserved pcs contained at least if not higher concentrations of majority of cytokines as pcs stored at rt, therefore making sheep a suitable model in which to investigate immunomodulatory effects of cryopreserved pc transfusion. background: transfusion, despite being a lifesaving therapy, has been associated with adverse transfusion outcomes. transfusion related acute lung injury (trali) remains one of the leading causes of transfusion-related mortality. accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in blood products, including packed red blood cells (prbcs), have been implicated with the development of non-antibody mediated trali. however, how specific mediators contribute to the underlying mechanism is yet to be defined. during routine storage of human prbcs fewer than 10 cytokines/chemokines and several biologically active lipids have been identified. a sheep model of trali has successfully been developed using human prbc supernatant, however transfusing sheep prbc has not been investigated. to support the use of sheep prbc in the trali model and to better understand the precise mechanism, characterization of the potential mediators in sheep prbc is required. aims: to characterize immunomodulatory mediators in sheep prbc supernatants and to investigate whether storage duration impacts the accumulation of these mediators. methods: sheep prbcs (n = 5), prepared according to standard human procedures with minor modifications, were stored (2-6°c, 42 days (d) ) and sampled at d2 and d42. supernatant was prepared by double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), antiinflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferon-gamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of potential non-polar lipid mediators (arachidonic acid (aa), 12-hydroxyeicosatetraenoic acid (hete) and 15-hete) were assessed in the sheep prbc supernatant using commercial elisa. paired t-test was used to compare fresh and stored prbc supernatant (p < 0.05). results are mean ae standard deviation. results: at day 2, aa (75,142 ae 47,205 pg/ml), 12-hete (849.1 ae 235.6 pg/ml), 15-hete (87.6 ae 32.7 pg/ml) and il-1b (144.7 ae 198.9 pg/ml) were detectable in sheep prbcs supernatant. at day 42, storage duration significantly increased concentrations of aa (136,254 ae 60,433 pg/ml, p = 0.0425) and 15-hete (380.9 ae 116.3 pg/ml, p = 0.0022) in sheep prbcs supernatant. summary/conclusions: similar to reported findings of human prbcs, the predominant type of immunomodulatory mediators present in sheep prbcs were non-polar lipids. the concentration of these non-polar lipids increased during storage. these immunomodulatory mediators may contribute greatly to adverse outcomes associated with prbc transfusions. further investigation is required to determine whether stored sheep prbcs supernatant induce immunomodulation in sheep in vitro and in vivo transfusion models. background: dshtr incidence is reported as 1 in 2,500 transfusions, presenting days to months after the transfusion. the published data addressing the correlation between the strength of the antibodies detected after a dshtr has taken place and the corresponding clinical symptoms as measured by laboratory parameters that assess the presence of hemolysis is limited. aims: the aim of this study is to evaluate the correlation between the results of the dat, automated and manual antibody reactivity strength with the corresponding clinical parameters of hemoglobin, lactate dehydrogenase (ldh), bilirubin, and haptoglobin. methods: a dshtr is defined as discovering a new antibody within 28 days of a transfusion. for all positive antibody screens, a work-up is initiated consisting of identification panels, dats, antigen typing of the red cells transfused, and eluates at the discretion of the transfusion medicine physician. additional laboratory testing for hemolysis is requested when indicated. a retrospective review was conducted of patients who were identified as having a dshtr. levels of hemoglobin, ldh, and transfusion safety background: rhd immunoglobulin (rhdig) has been available for 50 years in australia. since its introduction for routine antenatal and postpartum prophylaxis, alloimmunisation has decreased from 16% to 0.2%, reducing the number of australian deaths from haemolytic disease of the newborn over a hundred-fold, to approximately 0.01 deaths per 1000. blood matters serious transfusion incident reporting (stir) system has been collecting transfusion incidents and adverse events across four australian jurisdictions since 2007. since january 2015, rhdig administration errors have been reported. aims: to understand incidents relating to the administration of rhdig and increase safety and awareness of risks. methods: health services registered with stir (n = 93) were notified of the inclusion of reporting rhdig incidents. when an incident is identified, the reporter sends an online notification to stir, prompting the appropriate investigation form to be sent for completion. the completed incident data are reviewed and validated by an expert group. data is de-identified and collated for reporting. results: during the period january 2015-december 2018, 45 reports were received; 43 reports were validated, with 2 reports excluded (reactions rather than administration errors). reports were categorised as below: background: following the nice transfusion guidelines, recommending offering iron before and after surgery to patients with iron-deficiency anaemia (ida), we worked collaboratively with the anaesthetic and pre-operative team to implement a clear and robust anaemia pathway for pre-operative haemoglobin (hb) optimisation. oral iron was started, where appropriate, and our anaemia pro-forma was sent for review in a virtual clinic to assess eligibility for intravenous iron. we performed a retrospective evaluation of the patients who received iv iron during the anaemia pathway. aims: the aim of this retrospective evaluation was to look at the cohort of patients who had received iv iron in 2017 and assess the effect of iv iron on haemoglobin levels for different defined groups. methods: we classified patients, as described in munting and klein, 2019, depending on their iron parameters as having either: -idaserum ferritin < 30 mg/l -chronic inflammation with idaserum ferritin 30-100 mg/l with transferrin % of < 20%/crp > 5 mg/l -anaemia of chronic inflammationserum ferritin > 100 with transferrin % of < 20%/crp > 5 mg/l patients were considered eligible for iv iron if the following criteria were met:1. an inadequate response to oral iron, or were unable to tolerate oral iron or the interval between diagnosis and surgery was short 2. the anaemia pro-forma was completed 3. hb was ≤ 120 g/l 4. they were classified as either having ida or chronic inflammation with ida or anaemia of chronic inflammation hb was measured prior and on average, 20 days following the iv iron infusion. we excluded patients who had their post iv iron follow up blood tests done after surgery. results: this retrospective evaluation included 80 patients. 48 patients were classified as having ida and 32 patients classified as having chronic inflammation with ida. those classified with ida had a mean hb of 96 g/l (55-120), a mean mcv of 77.4 fl and a mean serum ferritin of 16 lg/l. those with chronic inflammation with ida had a mean hb of 106 g/l (77-120), a mean mcv of 87.9 and a mean serum ferritin of 57 lg/l. follow-up hb was measured on average twenty days post iv iron infusion in both groups. the average hb post iv iron infusion in the ida group was 119 g/l (100-149) with an average increment of 23 g/l and in the group with chronic inflammation with ida the average post iv iron hb was 117 g/l (85-129) with an average increment of 11 g/l. summary/conclusions: in conclusion the group with ida had, on average, a lower starting hb that the group with chronic inflammation with ida and the average increment in hb 20 days post iv iron infusion was greater in the group with ida. however, the group with chronic inflammation with ida cases also responded to iv iron and therefore we strongly consider the use of iv iron in both groups. further studies to evaluate the ongoing effect of iv iron would help assess whether the same level of increment seen with ida can also been seen for the group with chronic inflammation with ida over a longer period and how long the increment was sustained. background: the expansion of personalized genomic medicine has led to the development of targeted therapeutic approaches for patients. one example is sipuleucel-t, an autologous cellular immunotherapy product used to treat prostate cancer manufactured from the patient's white blood cells. this study describes our experience with incorporating autologous cellular immunotherapy products into the workflow of the blood bank. the policies supporting the workflow are outlined and compliance with them is assessed. aims: this study aims to evaluate the process and method used to dispense and track the infusion of sipuleucel-t. methods: this is a retrospective analysis of the dispensation and administration of sipuleucel-t from january 2012-august 2018, which was handled exclusively by the blood bank. standard operating procedures and hospital policies were reviewed and compliance with these policies evaluated. included were patients who had the sipuleucel-t product dispensed and administered. information collected included the total number of products dispensed, patient age, adverse reactions/incidents, premedication, and patient outcome. descriptive statistics were used for data analysis. results: there were 154 products dispensed to 53 patients. the recipients were male patients diagnosed with prostate cancer with a mean age of 74 years. there were 3 doses (a complete course) administered to 50/53 (94%) recipients and a partial course (1-2 doses) administered to 3/50 (6%) recipients, for a total of 154 products. the blood bank workflow treated sipuleucel-t as a derivative in the computer system, listing the manufacturer (dendreon corporation) as the supplier. health care providers were instructed to follow the nursing policy for the administration of blood products and derivatives for the infusion of sipuleucel-t. this policy required documentation of the infusion in a transfusion nursing note and reporting adverse events to the blood bank as transfusion reactions. there were no adverse events reported to the blood bank, yet there were 5 adverse events described in provider notes; 2 of them necessitating transfer to the emergency department, and 1 requiring hospital admission. of the 154 infusions, 6 infusions were documented in a chemotherapy note rather than a transfusion note (4%), and 59 (38%) were documented as both a transfusion and a chemotherapy administration. there were 6 additional deviations from the blood product administration policy: 2 cases where the consent check was not performed, 1 case where the product was infused with ringer's lactate rather than normal saline, and 3 cases where the 2-person 3-way check erroneously indicated the product was irradiated. summary/conclusions: this study describes one approach to managing cellular therapy products as an extension to existing blood products dispensed by a blood bank. the findings demonstrate noncompliance with hospital policy with this new product as evidenced by failure to report adverse events and failure to follow hospital practices regarding administration. although sipuleucel-t is a product manufactured from an autologous blood product donation, the administration and perceptions of this product may be more similar to a pharmaceutical. as the field of immunotherapies derived from blood product donations continues to expand, these products may necessitate an entirely new approach to ensure proper management. abstract withdrawn. (ref 10310) . while the mnc procedure is fully automated, cmnc requires frequent interface checks to ensure the collection of the correct cell layer. at the rambam health care campus, a tertiary care center, solely the mnc procedure had been employed till 2017, at which point, the cmnc has been introduced for the use in patients with a white blood cell (wbc) count of ≥ 20,000/ll on the collection day. aims: the current study aimed to compare various parameters of peripheral blood stem cell (pbsc) collection, using the cmnc protocol in allogeneic donors and patients undergoing autologous stem cell (autosc) transplantation. additionally, data on autosc collection using mnc (n = 31) and cmnc (n = 88) procedures were compared. methods: data were retrospectively obtained from pbsc collection reports in 134 consecutive cmnc procedures, including 88 autologous and 46 allogeneic donors. the following comparisons were made: cmnc results of allogeneic versus autologous donors, a sub-analysis of cmnc results for autologous donors with a pb cd34 + count ≥20/ll versus allogeneic donors as well as mnc versus cmnc results in autologous donors. the collection efficiency-2 (ce-2) was defined as the total cd34 + amount in the collection bag divided by the amount of cd34 + cells in the pb processed by the collection apparatus. results: in the cmnc, the following parameters significantly differed between autologous and allogeneic donors: mean collection time (333 ae 59 and 289 ae 73 min, respectively; p = 0.001), the total blood volume processed (3.4 ae 0.8 and 2.4 ae 0.8, respectively; p = 0.001) and the final volume in the collection bag (337 ae 77 and 291 ae 84 ml; p = 0.001). the mean ce-2 in autologous versus allogeneic donors was 49 ae 24 and 66 ae 22, respectively (p = 0.003). using cmnc, the collection was effective in 94% of allogeneic and 63% of autologous donors. in autologous donors, a significantly lower collection bag volume (341 ae 84 and 396 ae 132, respectively; p < 0.01) and increased total wbc in the collection bag (263 ae 119 versus 149 ae 36, respectively; p < 0.000) were obtained using cmnc compared to mnc protocol. thirteen patients were treated with plerixafor due to a low pb cd34 + count following g-csf therapy; 7 of them achieved a cd34 count ≥20 and their collection was considered effective. summary/conclusions: the cmnc protocol is highly effective in terms of the cell yield in both allogeneic and autologous donors with a pb cd34 + count ≥ 20/ll. significantly superior collection results are obtained in allogeneic donors versus autologous ones. cmnc provides a significantly higher wbc and a lower final collection volume than mnc. similar total cd34 + cell counts are obtained with both methods. . tbv processed ranged from 1-4.8 tbv with mean of 2.6, average was 2.65 tbv for females and 2.74 tbv for males mean pre-apheresis cd34 + count was 92.89 cells/ll (range 33.14-152.64). mean postapheresis cd34 + count was 1402.3 cells/ll (559.02-2245.5). mean cd34 + cells x10 6 / kg recipients body weight was 7.5 (range: 2.77-12.33). our target yield was ≥3 9 10 6 cd34 + cells/kg body weight of the recipient and in only 2/34 (6%) cases, the yield was <3. 18/34 (52.9%) procedures were lvl and 16/34 (47.1%) were svl. summary/conclusions: most of our pbsc were done for haematological indications (85.3%) and the target dose was 3 9 10 6 cells/ll in single leukapheresis. in 32 cases (94%), target yield was achieved, only 2 cases had <3 but >2 yield. in our study donors <5 years have shown to mobilize better than the older children. hematocrit (hct) and weight showed correlation with cd34 + cell yield but they cannot be taken absolute predictors. wbc count cannot be taken as a predictor for cd34 yield as high wbc count did not convert into high cd34 yield or vice versa. high prepheresis cd34 + count gave higher postpheresis cd34 + count. large volume leukapheresis (lvl), >3 tbv gave higher yield as compared to standard volume leukapheresis (svl). blood volume processed related to prepheresis cd34 + count and/or the weight difference between the donor and recipient. other parameters like hematocrit, wbc count, age etc did not show correlation to the volume processed. in our study, younger age and prepheresis cd34 + count were found as the most relevant predictors for stem cell yield. background: allogeneic hematopoietic stem cell transplantation is an established therapy for many hematologic disorders. since the discoveries of the potential of peripheral blood stem cells (pbsc) in the hematopoietic reconstitution mid 1980s and early 1990s pbsc gradually replaced bone marrow as the preferred source of stem cells. the introduction of hematopoietic cytokines that can mobilize large number of progenitors into circulation accelerated pbsc usage. aims: the aim of our study is to present our 18 year experience with apheresis collecting of pbsc in donors. methods: this is a retrospective study performed in the institute for transfusion medicine of republic of macedonia and university hematology hospital for period background: obtaining unambiguous results of hla typing plays an important role in the transplantation of hematopoietic stem cells. appropriate selection of alleles in the level of hla between recipients and unrelated bone marrow donors reduces the risk of transplant rejection and graft-versus-host disease. new generation technology ensures the highest possible resolution and obtaining unambiguous genotyping results due to the high complexity of the hla system. currently, this is the selection method for obtaining hla test results at the high resolution level. aims: the aim of this study was to determine 11 hla loci (hla-a, -b, -c, drb1/3/ 4/5, dqb1, dpb1, dpa1, dqa1) in potential bone marrow donors from poland. the research included 18,500 potential bone marrow donors registered between 2017 and 2018. a novelty of this paper was that the amplification of all 11 hla loci was performed by using multiplex pcr primers in a single tube. that solution completely eliminated the need to pool amplicons. methods: the typing of the 11 hla loci (hla-a, -b, -c, drb1/3/4/5, dqb1, dpb1, dpa1, dqa1) of potential bone marrow donors was made by using the alltype tm ngs 11-loci amplification kit (one lambda). genomic dna was isolated from peripheral blood of 18,500 donors. hla genotypes were determined according to the manufacturer's protocol on the miseq illumina platform. the obtained sequencing data was evaluated by using the typestream tm visual ngs analysis software. results: the ngs method allowed to obtaining unambiguous results of genotyping of potential bone marrow donors, and also provided the identification of rare alleles, such as: c*12: 143, c*12: 30, c*05: 37, c*07: 151, drb1*11: 28, c*14: 04, b*51: 22, c*15: 13, dqb1*03: 12, drb1*14: 87, drb1*11:69. summary/conclusions: 1. new generation sequencing technology (ngs), which is based on pcr, ensures the highest possible resolution. 2. the ngs method allows to obtain more accurate sequencing results compared to the conventional methods. 3. the research has confirmed the superiority of the ngs method over conventional methods in obtaining unambiguous hla genotyping results at the high resolution level. background: the accurate results of hla typing are significant for ensuring the success rate of hematopoietic stem cell transplantation. currently, hla typing is mainly based on sanger sequencing, which has a high proportion of ambiguous combination results indicating potential errors for hla typing. it is necessary for finding a more accurate typing method to reduce the risk. next-generation sequencing (ngs) method could provide clonal sequencing of single molecules, which has been used for hla genotyping and improved the scope and precision of hla study. aims: to establish a full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology and be evaluated by classical sangersequencing method, which can effectively improve the accuracy of hla typing for donor and recipient in hematopoietic stem cell transplantation. methods: hla-i (hla-a, -b, -c) gene-specific primers were screened, and the amplification parameters were optimized to obtain full-length sequences of hla-i gene under the same condition. the sample library for the amplicon was prepared with transngs tn5 dna library prep kit and the sequencing step was carried out with illumina miseq platform according to the manufacturer' protocol. all the sequencing data in fastq format were analyzed by typestream visual software version 1.2.0(one lambda inc.)with the default setting. 94 cord blood samples were collected for hla typing with the mentioned above next-generation sequencing method in our study. in parallel, all the sample were also tested with the sanger sequencing method according to the previous study in our laboratory. results: 94 samples were successfully tested with two methods and the coincidence rate between two sequencing methods was 100%. with the next-generation sequencing method, the probability of ambiguous results among 94 samples in our study is 1.06%(1/94) for hla-a, 5.31% (5/94) for hla-b and 0% (0/94)for hla-c. however, the probability of ambiguous results with the sanger sequencing method is 96.8% for hla-a, 95.7% for hla-b, 100% for hla-c. summary/conclusions: the full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology was established, which could greatly reduce the probability of ambiguous results and effectively improve the accuracy of existing hla typing techniques. key: cord-022141-yxttl3gh authors: siegel, frederic r. title: progressive adaptation: the key to sustaining a growing global population date: 2014-08-23 journal: countering 21st century social-environmental threats to growing global populations doi: 10.1007/978-3-319-09686-5_9 sha: doc_id: 22141 cord_uid: yxttl3gh adaptation is an evolving long-term process during which a population of life forms adjusts to changes in its habitat and surrounding environments. adaptation by the global community as a unit is vital to cope with the effects of increasing populations, global warming/climate change, the chemical, biological, and physical impacts on life-sustaining ecosystems, and competition for life sustaining and economically important natural resources. the latter include water, food, energy, metal ores, industrial minerals, and wood. within this framework, it is necessary to adapt as well to changes in local and regional physical conditions brought on by natural and anthropogenic hazards, by health threats of epidemic or pandemic reach, by social conditions such as conflicts driven by religious and ethnic fanaticism, and by tribalism and clan ties. the principal problems with growing populations do not involve space although population density is a problem unto itself for reasons discussed in previous chapters. the main problems are how to nourish people with food and water. the chronic malnutrition that about 1 billion people suffered from in 2013 is likely to grow in number in some regions due to global warming/climate change because humans cannot adapt to less food if they are already at subsistence rations. for example, the 2012 population in sub-saharan africa is 902 million people. the population is projected to increase to about 1.25 billion in 2030, an increase of about 38 %. within the same time frame, the united nations estimates that acreage under maize cultivation in the region will decline by 40 % because of heat and drought brought on by climate change. the loss of arable land for food production can be countered in sub-saharan africa if marker assisted hybridization of maize or maize genetically modified to withstand heat and drought come onto the seed market together with modified seeds for other food staples and if african nations that do not now accept gmo seeds do so in the future. if not, nations favored for food production by climate change will have a moral obligation to provide food staples to people in nations with declining food production at accessible costs based on their economies. it is clear that what happens in sub-saharan africa and other regions with declining cultivation acreage or that will bear other effects of climate change (e.g., drought, shifting rain patterns) will affect the rest of the worldwide community politically, economically, and socially. the earth's problems that associate with global warming/climate change will be further discussed in the last section of this chapter. water is the staff of life. it keeps the body hydrated and is necessary to grow food crops, hydrate food animals, and grow feed grains. chemically or biologically polluted water does not serve these ends. if ingested, contaminated water can result in sickness as discussed in chap. 2. water stokes industry and manufacturing as well, thus keeping economies in many countries contributing to a population's wellbeing by providing employment, goods, and services. ideally, these businesses contribute their fair share to a tax base that supports social needs (e.g., education, healthcare, maintenance of infrastructure). factory owners adapt and plan against water shortages by having a water recycling system in place but may also slow or stop production until operational water conditions return. citizens with a reliable water supply can adapt to periods of water shortage by limiting use according to mandates by government officials but still have water for basic daily needs. however, persons in nations with a chronic per capita water shortage may not have this option to serve their daily needs unless water is imported or new water sources are found (see chap. 2). if imported water is not an option to meet immediate essential needs, an alternative adaptation for people (and animals) is to try to reach a location where water would be available to them. with growing populations, per capita water availability is greatly diminished (table 2. 2), water shortages become endemic, and people are at risk of existing at subsistence levels or dying. most at risk from the lack of a basic water ration are pregnant women, infants, young children, and old people. water wars are a future possibility as nations battle for their peoples' survival unless political differences are set aside and projects are supported to develop and share water sources. in a welcome effort, jordan, israel, and the palestinian authority signed a memorandum of understanding in the world bank, december 2013, with specific aims: (1) produce millions of cubic meters of drinking water for a water-deficient region; (2) pipe 200 million cubic meters of water annually *180 km (110 mi) from the red sea to the dead sea; (3) build a desalination plant at aqaba that would supply water to aqaba and eilat; (4) the israeli water utility would supply 20-30 million cubic meters of drinking water to the palestinian authority for the west bank population at a reduced cost; and (5) there would be an inflow of water to slow and in the future perhaps abate and reverse the shrinking of the dead sea. funding for the estimated $400 million, 5year project will come from the world bank, donor nations, and philanthropic groups. as the global population increases and more people in developing and less developed nations have more disposable income, there will be a growing draw on natural resources other than water and food to service their industrial, agricultural, and manufacturing needs and wants. competition can force economic wars among national and multinational corporations for the resources necessary to provide goods and services and thus drive up prices for resources. industries and manufacturing units that cannot compete economically for natural resources will shut down, thus contributing to unemployment and downturns in economies because of falling domestic demand. to keep order in the increasingly interdependent world economy, accommodation for shared natural resources (or substitutes for them) at affordable prices is the adaptationnecessary. this can be mandated by the world trade organization backed by other practical-minded international groups. another adverse effect of growing populations that is a national resource that can be lost at the expense of some countries to the benefit of others is brain power. this brain power has been cultivated at excellent universities in developing countries, often times at little or no cost to students (e.g., in medicine, science, engineering, economics, the arts) who attend and graduate in increasing numbers. where there are too many well-educated professionals but lack of employment opportunities for them in their fields of expertise, educated people have the option of relocating to another country that can nurture and use the expertise. many adapt to the employment problem by taking up this option. this may mean moving from a developing country to a developed country or from a less developed country to a developing or developed country. ultimately, this loss of citizens with special skills can hurt a country. to counter this brain drain or reverse it, a country can adapt by investing in its future to create programs and conditions that keep talented professionals home, or if they have emigrated, entice them to return. china and india are examples of countries that have successfully taken this tact. when there are increases in a population because of immigration, problems can ensue between immigrants and a general population. adaptation to diversity and the multicultural experiences it brings to a community is often not a comfortable change. the antipathy of some in a host country is based on slowness of the immigrants to learn the language and inability of host country citizens to understand what immigrants are saying among themselves. this makes citizens feel uneasy. some view immigrants as a threat to their own or a family member's employment or advancement. race difference is a factor that some cannot readily adapt to as is ethnicity with its traditions and customs unfamiliar to the general public. religion can be divisive if adherents to its beliefs engage in acts of hatred detrimental to the host country fueled by fundamentalists and zealots who interpret religious writings as giving them license to commit crimes or absolving them of the crimes. sadly, many citizens paint an entire religious community with the taint of the relatively few evildoers. adaptation to diversity is essential for our earth's citizenry with joint efforts by all to resolve worldwide issues (e.g., global warming/climate change) so as to become the keys to providing a sound future for coming generations. there has to be a shared attack on global threats, no matter what the language, race, ethnicity, or religious beliefs are, no matter social or economic status, no matter whether a threat affects less developed, developing, or developed countries. adaptation is a progressive process when dealing with natural hazards because as each type of natural hazard impacts global communities over time, lessons are learned from each one that give direction to the methods of adjustment. adaptation to living where hazards can be expected to strike and where populations continue to increase is dependent on what we learn from the study of past hazards. we can use this evaluation of measured and observed data to minimize the immediate effects and aftermaths of hazards and protect citizens from injury, death, and from damage or destruction of property or infrastructure when hazards strike in the future. in areas prone to earthquakes, we know that earthquakes do not kill and injure people but that collapsing buildings and infrastructure do. earthquakes are not predictable so that there is no adaptation by a timely evacuation to minimize deaths and injury. however, building structures to make them more earthquake resistant can save lives, reduce injuries, and protect property. thus, after a high-magnitude earthquake, forensic engineering teams come to assess the damage and determine where and why damage and destruction took place within the context of the magnitude of an earthquake, the type of motion it originated (shaking, jarring, rolling), its duration, the area it affected, and the geologic properties of rocks underlying structures' foundations. hazard assessment teams also evaluate other factors that contributed to additional damage such as ruptured gas lines that feed fires and ruptured water lines that inhibit fire control. the engineers establish how construction can be improved in the future in terms of construction techniques and materials to prevent the types of collapses and utility failures they investigated. municipalities revise building codes accordingly to direct reconstruction and future building projects. where possible, structures that withstood an earthquake with minor or no visible damage should be retrofitted to improve their resistance to the next "big one." with each event, we gain more data on how to better construct earthquake-resistant structures and alter building codes to more stringent specifications. in theory, this adaptationto an irregularly recurring global event is good, but in practice it is most applicable to nations with the economic resources for reconstruction according to revised building codes and where there is no corruption to allow a bypass of the code. the same can be stated for retrofitting to give more resistance to earthquakes to existing structures. many developed nations and nations rich in commodity exports (e.g., oil) have a moral obligation to donate funds, material, and expertise to help citizens in economically disadvantaged nations recover from a destructive earthquake. some commodity-rich and economically sound nations do not do so directly, whereas others, big and small, rally to help disaster victims. for example, immediately after megatyphoon haiyan devastated many regions in the central philippines in 2013, israel sent 250 medical doctors and nurses and field hospitals to help philippine citizens recover from the impacts of the typhoon. as discussed in an earlier chapter, volcanoes are predictable in terms of becoming active by emitting wisps of smoke, bulging on a slope, warming of the soil or nearby pond or lake waters, emitting increasing concentrations of gases, and showing increased low-frequency seismicity. however, this activity does not always result in an eruption. a marked increase in measurements and observations, especially the low-frequency seismic activity, suggests that an eruption is imminent. adaptation to living and working on or near a volcano means investing in equipment to monitor volcanic activity and listening to alerts from scientists monitoring its activity and being ready to evacuate by gathering important papers and precious mementos and prepared to load into transportation for evacuation to safe locations. governments adapt by charging geologists to map out areas considered as high-, moderate-, and low-hazard zones in the volcano environs. geologists do this by studying rocks deposited from past eruptions and assessments of the topography. municipalities then pass zoning regulations applicable to the hazard level. governments have adapted to repeated periodic flooding in areas by creating flood control systems described in chap. 4. dams hold water during times of heavy and/ or extended rainfall and release any overflow into channels that move water away from urban or rural population centers. levees increase the volume of water that can move through a channel, thereby keeping it from spreading into populated areas and cultivated farmland. for smaller waterways that flow through cities, municipalities may invest in deepening, widening, and straightening channels as well as erecting walls so that more water can flow through the area more rapidly without coming out of a channel. governments define zones on flood plains according to a recurrence interval of damaging floods (e.g., 100 years) as being off limits for residential and factory/plant construction. as much as we plan to adjust to living in an area prone to flooding, there is always the possibility of a megaevent that can overcome in situ control systems. therefore, as described chap. 4, governments adapt to this possibility by installing flood prediction equipment in drainage basins to provide warning to those at risk from rising and sometimes raging waters. the warning gives people time to gather important documents and personal treasures and evacuate to safe areas. the apparent increase in the frequency and magnitude of storms and resulting flooding in recent years is thought by many weather scientists to be related to global warming and the increased amount of moisture in the atmosphere from warmer oceans that gathers in clouds and precipitates during storms. this will be discussed further in this chapter. adaptation to extreme weather events such as an extended period of drought, heat waves, and frigid weather means preparation to wait them out. some municipalities adapt to repeated, sometimes seasonal, times of short-term drought by storing a 3-6 month water supply in surface or underground reservoirs during periods of normal precipitation that can be tapped (conservatively) as needed. others may plan to move water via pipes or water tankers from where it is plentiful to where drought conditions exist. otherwise, to survive, people move as best they can to where they have access to water. in instances of years long drought, crops and livestock and other life forms may be lost. heat waves can kill. adaptation to heat wave conditions means that water has to be available to people to avoid dehydration. where possible, homes should have air-conditioning or fans to keep people comfortable and municipalities should have cooling centers to which people can go. personnel should check on senior citizens and escort them to cooling centers if necessary. clearly, economically advantaged nations have the resources to give support to citizens during natural hazards such as these. these nations, international organizations, and ngos have a moral obligation to help economically disadvantaged nations as is possible when hazard conditions such as these threaten populations. the most extreme of weather conditions that can injure and kill people and destroy housing and infrastructure are tropical storms that evolve into violent hurricanes (typhoons, monsoons) by increasing wind speeds and sucking up moisture (water) as they track across oceans toward land. when these storms make landfall, they drive storm surges that can wreak havoc onshore communities, and as they move inland precipitate heavy rains that cause life-threatening and destructive flooding. these violent storms are destructive to coastal populations and island nations and have regional reach inland as they move along paths until they finally spend their energy or move out to sea. on november 8, 2013, the typhoon named haiyan, the strongest recorded typhoon ever to make landfall smashed into the central philippines killing more than 2,600 people, injuring about 12,500, and displacing almost 600,000 people. there was a 4-m (*13 ft) storm surge driven by winds measured at over 312 km/h (195 mi/h) with gusts reaching 380 km/h (235 mi/h). the typhoon flattened the city of tacloban that was home to 200,000 residents, and there was major flooding inland. the weather alerts led to a government call for evacuation away from the predicted path of the storm, and about 1 million people followed the evacuation warning, surely saving many lives. access to aid typhoon-ravaged areas was difficult, and there were shortages of water, food, and medical care for many evacuees for several days. the philippine central government and local officials were not prepared to deal with a storm of this magnitude but help started arriving from many nations worldwide. there was a post-event concern of attending to sanitation needs of survivors to prevent outbreaks of diseases such as cholera, typhoid fever, hepatitis, and dysentery. if the philippine government had adapted by adopting better policies with respect to response to high-category typhoons in addition to the call for evacuation, the impact of haiyan would have been ameliorated. one would hope that this deficiency would be dealt with to limit the effects of future like disasters. evacuation to prevent injury and death in coastal zones that could be struck by high winds, heavy sustained rains, and storm surges is dependent on weather bureau forecasts and warnings from police, firefighters, or other government-authorized personnel. homeowners adapt to hurricanes by securing roofing with additional nails or special fasteners as a retrofit precaution and by boarding up windows on structures before an incoming storm hits. governments have adapted to the onslaught of violent high-energy storms by constructing seawalls of varying designs and heights to protect population centers by damping the force of storm surges. in china, for example, a seawall 6.72 m (*22 ft) in height and that has been heightened in the past protects shanghai from the full damaging effects of high-category typhoons. as a result of rising sea level, the shanghai seawall and other that protect coastal cities from being flooded by surges from high-energy tropical storms will have to be heightened to afford a greater degree of protection to people and property. wildfires can be a natural hazard when ignited by a lightening strike. however, most wildfires are started by human carelessness such as tossing a lit cigarette on a forest floor or failing to completely extinguish a campfire, or by arsonists. one may adapt to living in an area with a history of wildfires in two ways, neither of which is practical or promises 100 % protection. first would be to clear an area of vegetation in a 30-m (100 ft) swath around a dwelling or site for building. second would be to build with nonflammable materials so that embers propelled during a wildfire could not ignite a structure. adaptation to the advance of a wildfire would be to heed warnings to evacuate carrying a prepared case with important documents and other items of personal value. to delay evacuation by going back to retrieve something from then home can be fatal as it was for two people in a recent (june, 2013) wildfire that destroyed almost 500 homes in colorado springs, colorado, usa. when there is a hazard event coming that calls for evacuation, responsible and often economically advantaged governments have adapted to the threat by designating evacuation routes, by providing transportation for people who need it, by having evacuation centers stocked with water and food, cots and blankets, basic medical supplies and medical personnel, and by having phone service available for people that need it. in the case of a primary or triggered hazard that happens with little or no warning (e.g., an earthquake, a tsunami, a volcanic mud flow), search and rescue teams should be ready to move in soon after dangerous conditions ease and they can move with safety. there should be medical attention to treat injured survivors, and stations set up as soon as possible to provide water, food, and other essentials available to those that survived with little or no physical hurt. these first steps at adaptation are the keys to survival. recovery after a shock phase can be long and drawn out, depending in grand part on a nation's social and economic resources and physical and economic assistance from other nations, international institutions, and ngos. change on our earth's inhabitants global warming is a fact attested to by an overwhelming majority of the scientific community and unwaveringly supported by a february 2014 joint publication of the us national academy of sciences and the royal academy in the uk on the causes and evidence for global warming [1] . as noted in earlier chapters, during the past century, measurements show that the earth has warmed by *0.8â°c (*1.44â°f). global warming is an ongoing process that is attributed in grand part to a slow but continuous and increasing buildup of greenhouse gases in the atmosphere. the greenhouse gas most associated with global warming is carbon dioxide (co 2 ). a plot of the increase of co 2 content in the atmosphere with time against the increase in global temperature shows an excellent correlation of one with the other. additional lesser contributors include methane (ch 4 ), nitrous oxide (no 2 ), and chlorofluorocarbons (cfcs). with the beginning of the industrial revolution and the increased use of coal as the principal energy source, the content of co 2 in the atmosphere was 280 parts per million (0.028 %). the combustion of coal and later oil (petroleum) and natural gas emits co 2 to the atmosphere. initially, and for many years thereafter, the added greenhouse gases were taken up by vegetation for photosynthesis and was also absorbed by the oceans and other water bodies. this kept the atmosphere co 2 close to the 280 ppm pre-industrial level. however, with increased industrialization, the need for electrical power, and the use of internal combustion engines, the amount of co 2 generated was greater than what could be absorbed by nature and the content of co 2 in the atmosphere increased. during june 2013, its concentration reached more than 398 ppm, an increase of over 40 % over the pre-industrial concentration (scripps institute of oceanography mauna loa measurement). the increasing co 2 content, other greenhouse gases, aerosols, and particles acted as a media that admitted sunlight (heat energy) to the earth's surface but did not let all of the heat escape back into the atmosphere. this abets global warming. in the past two to three decades, the rush to industrialization in developing countries (e.g., china, india, and brazil) and their growing power needs and vehicular use has thwarted the implementation of international agreements to reduce emissions from coal-fired power plants, other industrial and manufacturing operations, and the transportation sector. a direct consequence of global warming is sea level rise (slr) caused by the progressive melting of icecaps and ice sheets in greenland, the arctic, and antarctica, and of mountain glaciers in the himalayas, the alps, the rocky mountains, and the andes. the *20-cm (*8 in) sea level rise during the past century may see a rise of another *50 cm (*20 in)-1 m (*39 in) during this twenty-first century. one-third of the rise would be from the expansion of warmer sea water, one-third from icecap and ice sheet melt, and one-third from mountain glacier melt [2] . in 2012, other researchers used computer models on existing data and proposed that 50 % of sea level rise between 1903 and 2007 was from glacial melt [3] . following the same line of investigation, other scientists studied satellite data and ground measurements from alaska, the canadian arctic, greenland, the southern andes, the himalayas, and other high mountains of asia and estimated that glacier contributions to sea level rise from 2003 to 2009 was 29 % and together with ice sheet melt explained 60 % of slr [4] . a publication in 2012 estimated that ocean thermal expansion 0-300 m deep and 300-700 m deep contributed up to 35 % to sea level rise [5] . these latter two estimations are in line with the ipcc prediction for melting ice and ocean thermal expansion contribution to the estimated rise of sea level by the end of the century [2] . with a rise in sea level, marine waters encroach on land. as the rise continues, possibly at an increasing rate, it threatens habitation in lowlying islands, coastal villages and farmland in lowlying zones, and heavily populated cities worldwide settled on inshore terrain close to sea level (e.g., bangkok, ho chi minh city, jakarta, manila, miami, new york, boston, buenos aires, london, rotterdam). rising sea level and warming of ocean waters have other ramifications that affect coastal communities as well as inland areas. as explained in chap. 7, the warmer surface water releases more water vapor with heat energy into the atmosphere. when the water vapor molecules condense in clouds, heat energy is released. this energy gives more force to tropical storms as they form, track to shore, and move inland, or storms that move close to and along a coast. these storms may transition to hurricanes (typhoons, monsoons) with the violent winds that cause destruction, and heavy rainfall that triggers flooding if they move onto land. we recognize that rising sea level means that tropical storms that impact a coast with storm surges have a farther reach inland with their destructive energy that is more pronounced when the surge occurs at high tide. the surges also saturate farmland they reach with salt water that harms crops. they also carry salt water into fresh water marshes and ponds, thus disrupting ecosystems there. the increase in the number of these extreme weather events and the increase in violence and destruction they wreak on land compared with like weather events in the recent past (e.g., during the past 30 years) strongly suggest that they are fueled to a significant degree by global warming. there are two possibilities for adapting to the effects of rising sea level on coastal urban centers, one impractical, the other very costly but doable. the impractical adaptation possibility is to move at-risk population centers inland, out of the reach of the destructive tropical storms. this does not lessen the threat of flooding. the move is possible in some cases where land is available, but such a move is not economically feasible. one practical but costly adaptation to mitigate encroachment from sea level rise and the effects of tropical storm surges is to surround cities at risk within place seawalls 2-3 m higher than recorded high tides or higher depending on historical records and contemporary published data. the walls can have a concave configuration so that surging waves lose energy when their lower parts hit and are curled back on themselves damping some wave energy or there can be a different configuration best for the site(s) to be protected. similarly, gates buried at strategic locations where there is ship access to consider can be built to be hydraulically driven so that they can rise from a near shore seabed site to mitigate the effects of storm surges. both techniques have been used at different global locations. we have read that climate change affects land-based agricultural production, both for crops and animal husbandry. the warming climate at higher mid-hemispheric latitudes and at higher altitudes does not favor the growth and normal yield and/or quality of many crops. depending upon the degree of climate change and the linked change(s) that may follow it, farmers can adapt in several ways to maintain or increase crop yield and nutrition value. for example, when warming starts diminishing the productivity of a traditional crop, farmers can sow crops that are known to grow well in warmer temperature and give a satisfactory economic benefit. however, new groups of weeds, pests, and diseases will migrate to the warmer growth environment and will have to be dealt with in order to protect the new crops. where the effect of global warming reduces water supply for rain-fed agriculture, for crops irrigated with surface waters, and for groundwater-irrigated crops when aquifer recharge does not balances discharge, agriculturalists can adapt in two ways. first is the use of a more efficient irrigation method that delivers water directly to a growing plant (e.g., drip or focused irrigation). this minimizes runoff and loss to evaporation. second and similar to what was mentioned earlier is to sow a crop that needs less water to thrive and that delivers a good yield, good-quality product. another result of global warming for some farmlands is a longer growing season. in this situation, growers can adapt by planting earlier and have the possibility of double cropping. they can also grow a cultivar that is later maturing and that gives a product that brings a good market price. however, switching to new crops in a warmer growth environment means that there will be an invasion of a new set of weeds, pests, and diseases to ward off. in any efficient operation, and as emphasized in earlier chapters, farmers adjust to a changing growth environment for a given cultivar by applying the optimum amounts of fertilizer and other agricultural chemicals as might be needed that nurture and protect it most effectively. this reduces agricultural costs and lessens runoff of these chemicals to ecosystems where they can be harmful. global warming can bring on abnormal weather extremes that affect agricultural productivity. in these cases, farmers have to plan ahead based on recent history of these conditions in their regions. drought, heat waves, and long-term rain or heavy rain in a short time present problems for both cultivars and food animals. periods of less than average precipitation may last months or years. depending on the amount of the deficit precipitation, adaptation can include storing water in reservoirs and cisterns during times of rainfall to be tapped during a drought to sustain food animals and crops during a short-term, not too severe drought. there is also the option of trucking in water to sustain livestock. long-term droughts when precipitation deficits are high take their toll on plants and animals to the detriment of agriculture in a region especially when accompanied by heat waves. they have caused recent disasters for crops and food animals on all continents less antarctica. farmers either wait out the "bad times," change the type of cropping they do, the livestock they tend to, or change careers. the adaptation from crops that have been grown successfully before the effects of global warming reduced yields and quality of a harvest, to those "same" crops that can grow successfully under the advancing warming changes just described generally means that hybridized species have to be developed and used as warming increases at a location and slowly tracks to higher latitudes and higher altitudes. thus, growers turn to plants that are created by hybridization as described in chap. 3: traditional methods and marker-assisted selection methods within the same species, and genetically engineered (-modified, -manipulated) methods using different species. hybridization is a slow process, sped up markedly by genetic engineering, a method that yields foodstuff not accepted by the european union and many nations outside the union, especially in africa. bred species are developed to carry one or more characteristics that favor crop resilience against the effects of climate change. these include resistance to disease, weeds, and pests, and tolerant of drought (water stress), heat, short-term inundation, and short-term saline exposure (see chap. 3). hybrids have also been developed to give higher yields and more nutritious crops. thus far, research has been focused mainly on improving seed for world staples such as rice, maize (corn), wheat, sorghum, and soybean. there have been great successes where hybrid crops were agriculturalists' adaptation so that the possibility exists that we can feed the earth's growing populations and reduce chronic malnutrition. when this is coupled with the opening of additional arable acreage and the use of improved farming methods for seeding, watering, and harvesting, global food security can be strengthened for the existing world population and the future generations on earth. however, this will require economic and technical input by developed nations and international groups. without basic sustenance, people will have less resistance to diseases and there may be local or regional population crashes if diseases evolve into epidemics or pandemics that invade susceptible populations. warming of the open ocean water, enclosed aquaculture operations in ocean waters and on land water bodies has affected marine fisheries and marine and estuarine aquaculture that grow food fish and shellfish, and lakes that sustain fisheries. in marine fisheries worldwide (e.g., in the north atlantic, off the coast of peru, off the coast of the philippines), some food fish or fish captured for other purposes (e.g., to use in pet food, to use to make fertilizer) have migrated to cooler water in ecosystems with conditions conducive to their spawning and growth. in some cases, predators follow fish they prey upon that have migrated to cooler waters, but in other cases they find new prey to sustain them. in other situations, they may become prey for larger fish in an ecosystem. fishing fleets adapt by following the fish they hunt into cooler waters where ideally they capture the hunted species in quantities allotted them by national and international fishery governing body regulations. if the quota system is followed, this will allow recovery of fish populations and sustainable harvesting. aquaculture operations that provide important supplies of food fish worldwide can adapt to warming waters by raising food fish or shellfish that will grow and multiply under the changed range of day/night temperature conditions if the fish they are farming cannot survive in the warmer waters. aquaculturalists also have the option to move their facilities to cooler-temperature waters, but the economic feasibility of doing this has to be evaluated by a benefit to cost analysis. this analysis has to be for the time frame during which the cooler-ecosystem waters are estimated to remain stable within the framework of a time range against global warming/climate change. another adaptation is that food fish currently being raised can be genetically engineered to be resistant to a warmer growth environment without changing their nutrition yield, growth rate, and ability to reproduce. there are diseases that are global threats, others that put regions at risk, and yet others that menace smaller political divisions. humans adapt to the threat of sickness in a population or a sickness itself in several ways. scientists develop methods to eradicate a virus or bacterium health threat, or a chemical/radioactivity threat. failing this, health professionals act to control a disease, to slow or minimize its transmission, and to apply approved therapies and support research to find therapies to treat an illness if one is transmitted. the following discussion draws strongly on the 2013 disease fact sheets put out by the world health organization. vaccines provide a main line of defense against many diseases. smallpox has been eradicated on earth by vaccination. polio has all but been eradicated globally except for a few pockets of the disease in pakistan, afghanistan, and nigeria where, in some cases, religious fundamentalists have beaten and killed health workers tasked with giving the vaccine to children, and in other cases where parents have been warned by the zealots against allowing their children to be vaccinated. recently, 81 polio cases were diagnosed mainly in somalia but also in kenya. this is attributed to the fact that by 2013, 500,000 children in somalia have not received the vaccine and are at risk from this highly contagious disease. it is also attributed to crossborder migration of infected persons into kenya. both governments are stepping up their vaccination programs. there were 59 cases of polio diagnosed in the rest of the world in 2012. measles is a global disease that can be prevented by a vaccine that is safe and cost-effective. measles may soon reach the near-eradication stage. in 1980 and subsequent years, 2.6 million people, mainly children under 5 years of age, died from measles. since 2000, 1 billion children were vaccinated, 225 million in 2011. by 2011, 84 % of the world's children received the measles vaccine, up from 72 % in 2000. from 2000 to 2011, deaths from measles dropped to 71 %, from 548,000 to 158,000. when the vaccination rate reaches 95 %, mainly in low-income countries, the world will have brought another disease close to elimination [6] . seasonal influenza is a global viral illness that afflicts 3-5 million people. the sickness kills 250,000-500,000 people with severe symptoms annually. transmission of the virus takes place when an infected individual coughs or sneezes without covering his/her mouth and releases droplets that can be inhaled by someone up to a meter away. transmission can also be from hands carrying the virus. seasonal influenza affects all age groups, but children less than 2 years old, people over 65, and those with complicating medical problems are most at risk. influenza is a disease to be controlled. the principal control is by safe and effective vaccines that can prevent 70-90 % of influenza cases in healthy adults. secondary controls are obvious for infected persons: cover the mouth when sneezing or coughing, and wash the hands frequently. the influenza vaccine is taken once annually. because strains of the influenza virus change from year to year, adaptation is needed. the adaptation is via a vaccine that is prepared with 3 or 4 strains that scientists determine will be most common during a coming season [7] . other types of influenza and respiratory illnesses have the potential to cause an epidemic or pandemic. they include avian flu and its strains and swine flu if the strains develop the ability for person-to-person transmission after infection, and sars (severe acute respiratory syndrome) and middle east respiratory syndrome (mers) because there is human-to-human transmission of the sicknesses. to the present, the outbreaks of the animal influenza diseases have been contained by quarantining infected people during treatment and by culling flocks and herds, or if available, vaccination of healthy animals. the latter two respiratory illnesses are caused by the coronavirus, and infected people have been in isolation wards. for sars, an illness that broke out in 2003 and spread to 24 countries, isolation of victims and treatment with antiviral drugs and steroids stopped the disease during 2004. mers is a recent (2012/2013) illness that has been confined to jordan, saudi arabia, qatar, and the united arab emirates. the mers virus has been found in camels. infected persons are quarantined in hospitals, but an effective drug treatment is still being sought to complement the normal hospital care-afforded patients. hiv/aids is a global epidemic that killed 25 million people in three decades since 1981. worldwide, in 2011, there were 34 million people with hiv, mainly (33 million or 97 %) in sub-saharan africa and south/southeast asia. the illness is caused by the exchange of body fluids (semen, vaginal excretions, blood, breast milk) from an infected individual with an uninfected person. more than 50 % of the cases of hiv are from heterosexual activity. there is no vaccine against hiv/aids, no cure for it, but there is a cocktail of medicines (antiretroviral treatment) that control viral replication and allow an infected person's immune system to strengthen. this keeps the illness at bay and afflicted people in general good health and productive in their communities. in 2012, only 9.7 million (less than 30 %) of those with hiv in low and middle economies received the antiretroviral treatment. this is changing as more hiv carriers have access to antiretroviral therapy and there are more donations from economically advantaged countries to support hiv stabilization and reduction programs. the number of new cases of hiv is not exploding because more than 50 % of those infected are following protocols that reduce the transmission of the disease. the prevention of transmission methods include access to male and female condoms, blood screening before transfusions, and needle and syringe exchange programs for sterile injections by drug users. hiv testing and education programs and hiv treatment help prevent transmission because individuals in continuous treatment have a very low probability of passing on the disease. male circumcision reduces the infection in men by about 60 %. there is still much progress to be made because there were 2.5 million new cases of hiv in 2011, with 1.8 million of that total in sub-saharan africa. the hiv/aids is a global sickness that is slowly coming under control because of generous donations from governments and foundations in developed countries added to what low-and middle-income countries themselves provide to lower the prevalence and incidence of hiv in their populations [8] . tuberculosis (tb) infected 8.7 million people globally in 2011, killing 1.4 million persons. it is a bacterial disease that spreads among people when infected individuals cough, sneeze, or spit, releasing bacteria into the air where they can be inhaled by others a meter away. although tb occurs worldwide, developing countries carry the largest burden of cases and deaths (95 %). the bulk of new cases are regional in asia (60 %) with sub-saharan africa reporting a large share as well with 2,600 new cases per million inhabitants. there is no vaccination for tb, but the disease can be treated and cured. the treatment is a half-year course of four antimicrobial drugs that must be taken without fail and thus requires continual supervision by healthcare personnel. more than 51 million people have been treated and cured of tb since 1995 and perhaps 20 million lives saved by following the who stop tb strategy protocols including securing adequate, sustained financing, ensuring early reliable detection and diagnosis, and providing approved treatment with a secure effective drug supply. the number of people infected with tb is declining, and from 1990 to 2011, the tb death rate dropped more than 40 %. the success in dealing with tb is muted somewhat because a strain of the bacterium that causes tb has evolved to be multidrug resistant (mdr-tb). in 2011, 310,000 cases of this variant were reported (of the 8.7 million cases worldwide), mainly from india, china, and the russian federation. these are treated with, but do not always respond to, the most effective anti-tb drugs. research into new drugs to deal with this problem is ongoing [9] . there is the question of whether people visiting or immigrating from these countries should be screened before a host country issues them entry visas. regional illnesses threaten the health of 100s of millions of people mainly in tropical and subtropical areas and often affecting children. one of these, the guinea worm disease, is trending toward elimination, if not eradication. this is a parasitic disease caused when people swallow water contaminated with infected water fleas (microscopic copepods) carrying worm larva. the worms release, penetrate the intestines, and move through the body migrating under the skin until they emerge causing swelling and blistering. people infected with guinea worm disease cannot contribute to their communities for months. during the mid-1980s, there were 2.5 million cases mainly in 16 african nations. but attention to where the sources were so that they could be avoided and treated, and assistance in generating clean water, were adaptations that brought the number of cases down to less than 10,000 in 2007. the number of cases continued to decline and was reduced to 542 in 2012 in four african countries: south sudan, chad, ethiopia, and mali. there is no vaccine against guinea worm disease. health officials adapt to counter this sickness in several ways. as noted above, access to clean drinking water is the best way to prevent infection. the prevention or transmission of the worms from infected individuals to healthy persons by proper treatment and hygiene and the use of the larvacide temephos to eliminate the parasite-infected water flea vector and other prevention protocols are important in the control and effort to eliminate/eradicate the disease [10]. the (jimmy) carter institute, atlanta, georgia, usa, has been a principle force since 1986 in the fight to rid the world of guinea worm disease. in tropical and subtropical regions, there are three mosquito-vectored diseases that put millions of people at risk: yellow fever, malaria, and dengue fever. yellow fever is an endemic viral disease in tropical regions of africa and latin america with 200,000 cases reported annually that cause 30,000 deaths. there is no set treatment for afflicted people, but there is an adaptive preventive measure. a vaccine against yellow fever is available that is safe, affordable, and that gives lifelong immunity to the disease with one dose after 7-10 days for 95 % of the people vaccinated. when there is the onset of a yellow fever outbreak where the population lacks vaccination protection, mosquito control is an essential first step in adaptation to prevent or slowdown transmission of the yellow fever virus. spraying insecticides to eliminate breeding sites and kill adult mosquitos is the control used during epidemics to make time for vaccination campaigns in a population and for immunity to take hold. there are limitations to the application of the yellow fever vaccine. first is that babies less than 9 months of age should not be vaccinated or, during an epidemic babies less than 6-9 months of age should not receive the vaccine. second, pregnant women should not be vaccinated except when there is an outbreak of the disease. third, people with a strong allergy to egg protein or those with a marked immunodeficiency or with a thymus problem should not receive the vaccine [11] . malaria is a parasitic disease caused by the bite of an infected mosquito. there is no vaccine against malaria, but one is undergoing a clinical trial in seven african nations with results expected in 2014. a use or no use decision as a control method for malaria will be made in 2015. promising results from an early-stage clinical trial of an unconventional vaccine prepared with live, weakened sporozoites of the malaria parasite were published in 2013. plasmodium falciparum was given to healthy 18-45 year-old volunteers intravenously. the volunteers were grouped to receive 2-6 doses and subsequently exposed to bite by five mosquitoes carrying the parasite. none of the six that received five doses were infected with malaria. three of the 12 that received four doses became infected, whereas 16 of the 17 that received lower doses became infected. of 12 that received no vaccine, 11 became infected. those that became infected were treated with malarial drugs and cured. clearly, higher dosages give protection against infection by malaria [12] . more research and extensive clinical trials are necessary to determine how children respond to the vaccine with adjusted dosages and whether the results from earlystage trial are reproducible in larger volunteer populations. if the results of additional clinical trials go well, the hurdle of producing enough vaccine and adapting it to injection has to be faced. forty percent of the deaths from malaria are of african children in the democratic republic of congo and nigeria. in addition to sub-saharan africa, populations in asia (especially india and the greater mekong region) and latin america suffer from the disease. the effort to deal with the disease that is preventable and curable now centers on control and treatment to reduce the number of cases. in 2010, the who reported that there were 219 million cases and 660,000 deaths (with an uncertainty range of 490,000-836,000). in a 2012 report, researchers suggested that the number of deaths was understated and that their computer model gave a figure for 2010 almost double, 1,238,000 deaths (95 % uncertainty interval of 929,000-1,685,000) [13] . the who stood by its figure stating that much of the data in the cited study were based on verbal testimony of how people had died, not on laboratory diagnosis of samples. either figure represents too many deaths from the disease and have to be reduced. mosquito control is the adaptation that can reduce the transmission of the disease greatly. this includes personal protection by use of proper clothing and/or the application of mosquito repellent, the use of longlasting insecticidal (pyrethroids treated) nets to kill mosquitos and prevent nighttime bites, and indoor residual spraying (remains effective for months). those people infected can be treated with oral artemisinin monotherapy followed by a second drug. failure to complete the treatment as prescribed leaves parasites in a person's blood. no other antimalarial treatment is available so that parasite resistance could become a serious problem. for visitors to a malaria region, antimalarial drugs taken before, during, and after a trip can protect them from the disease. many countries in tropical and subtropical areas have used the above-cited strategies and others to work toward the elimination of malaria. malaria eradication is the goal of the who [14] . dengue fever is a female mosquito-borne virus that infects people with an influenza-like disease in tropical and subtropical regions worldwide. the disease can kill if it evolves to severe dengue. it is endemic in latin america and asia where most cases now occur. since the 1970s, the sickness has spread to more than 100 countries putting about 35 % of the world's 2013 population (2.5 billion people) at risk. dengue fever is especially endemic to urban/semi-urban environments. humans are the main carrier of the virus. after a mosquito bites an infected person, each subsequent bite by the infected mosquito creates another carrier. a mosquito can bite many people each time it feeds. in the americas alone, there were 1.6 billion cases of dengue fever reported in 2010 with 49,000 being severe dengue. there is no vaccination against dengue fever, but research continues to develop one. the main treatment for afflicted persons is to keep them hydrated. adaptation to deal with slowing or stopping the spread of dengue fever involves three main tracks in addition to spraying insecticide to kill mosquitos. the best control method to prevent the transmission of the virus is to deprive mosquitos of sites with shallow, standing water where they can lay eggs and multiply. control can be improved if communities cover and clean water storage containers regularly, and use proven insecticides on them as necessary. finally, individual protection such as the use of mosquito repellants and insecticide-impregnated bed nets can help reduce the incidence of dengue fever as it has with malaria [15] . although controls are known, they are not always applied because of economics and other factors that prevent access to protection methods. the result is that the number of cases of dengue fever reported continues to grow globally. as populations increase in urban locations, the incidence of dengue fever can be expected to increase as well unless strict controls are enforced until a safe and cost-effective vaccine is developed. a positive aspect of the dengue fever problem is that recovery from one serotype of the virus gives immunity for life. however, there are four serotypes of the infectious virus so that recovery from one leaves a person susceptible to the others [15] . chagas is another regional disease. it infects 7-8 million people annually, mostly in 21 latin american countries. it is a parasitic illness that evolves after the bite of a blood-feeding triatomine bug, often on the face, where it defecates close by leaving parasite-bearing feces. parasites access the body when the feces are inadvertently smeared into the bite, the eyes, the mouth, or any skin lesion. the parasites circulate in the blood expressing their presence as a purplish swelling of one eyelid or as a skin lesion. there are several other symptoms as well in this acute stage of the illness, but these may be absent or mild. if diagnosed early during this stage, chagas disease is treatable. the parasite is killed with the medicines benznodazole and nifurtimox taken for 2 months. there are limitations as to who can take these medicines (e.g., not by pregnant women or people with kidney or liver problems). the untreated sickness can cause cardiac alterations and digestive problems that show up 20-40 years after an untreated infection. chagas disease can be spread by blood transfusion and by organ transplant, making blood screening for the parasite essential before a procedure. it can also pass to a fetus from an infected woman. there is no vaccination against the illness so that control of the vector (triatomine sp.) is necessary. the controls adapted by many municipalities include insecticide spraying inside a home, the use of treated bed nets, and hygiene practices that protect food, its preparation, and its storage before eating it [16] . the sickness may recur if control practices become lax. chagas disease is spreading as populations emigrate from latin america to northern countries. blood screening of visitors or immigrants from the countries where chagas is endemic may be necessary, and treatment followed by an infected individual before a host country issues an entrance visa. this would prevent the ingress and possible spread of chagas. outbreaks of diseases in town and cities is most often caused by bacterium-contaminated water or food and poor sanitation. sicknesses such as cholera, typhoid, and various other diarrhea types are examples of such diseases. they are all highly infectious if good hygiene practices are not followed. these diseases are endemic in many countries where populations do not have access to safe water and adequate sanitation. there are vaccinations for some of these sicknesses that may require more than one dose, but they may not be completely effective or long lasting and require revaccination at times specified by medical personnel (e.g., after 2-5 years). otherwise, infected persons can be treated with medicines such as oral rehydration pills or antibiotics. adaptation for prevention is easier called for than realistically available: washing hands with soap and clean water after visiting the toilet, and as noted above, access to safe water and good sanitation. given the millions of people infected by these bacterial diseases and the hundreds of thousand that die from them annually, generally in economically disadvantages countries, there should be an expanding global priority to eliminate the disease-causing conditions, and preparedness to combat an outbreak when it is reported. there are important factors to consider when adopting plans to halt or meliorate the effects of health threats to people in the near and extended future. one is the climate change-driven spread of tropical and subtropical diseases discussed earlier to newly warmer and moister higher-latitude and higher-altitude zones. another is the growth of populations mainly in tropical and subtropical regions in africa, asia, and latin america. together with this latter factor are the increasing populations and population densities in urban centers especially in the regions just cited. an additional factor to consider is whether there is accessibility to populations by healthcare workers or by people to healthcare clinics or hospitals, well-staffed and well-stocked with necessary pharmaceuticals. certainly, future planning has to include funding to support research to develop vaccines for diseases that do not have vaccination as an option against an illness (e. g., malaria, dengue fever) . in addition, improvement of vaccines that are available but that are not completely effective in terms of protection or the length of time they are effective should be a priority in pharmaceutical and biotechnology laboratories. scientists presented a fine review of the status of vaccine research from the design and development of vaccines to discussion of vaccines and infectious diseases (e.g., hiv, malaria, tuberculosis, pneumococcal disease, and influenza) [17] . they also discuss vaccines against enteric infections and viral diseases of livestock as well as vaccines against non-infectious diseases (e.g., cancer) and against chronic noninfectious diseases. continued and repeated education classes on how to prevent the transmission of diseases and free supplies of materials that work to this end (e.g., insecticide-treated bed netting, male and female condoms) are essential to reducing the prevalence and incidence of diseases as are safe water and uncontaminated food. as new medicines or combinations of medicines are developed, tested, and found to be effective in controlling diseases, they become part of the protocol for either curing disease or controlling disease to reduce transmission while allowing persons to carry on with their lives. in these times of easy and rapid migration, one wonders whether screening of visitors or immigrants for diseases known to be endemic or active in the countries or regions from which they come should be required so as to prevent a carrier from infecting others and spreading a disease (e.g., chagas disease, cholera, tuberculosis). this was done at airports during the sars scare for people leaving or entering a country (e.g., china) and likely limited the transmission of the sars virus and spread of the disease. preparedness for a disease outbreak, response to an outbreak, and management of resources during and post-outbreak are the keys to adapting to health threats that could affect future generations. this means developing the capability to extend the reach of health services to regions where climate change brings warmer, moister conditions to higher-latitude and higher-altitude ecosystems that are now reached by disease vectors that have expanded into these formerly cooler and drier environments as a result of global warming. adapting to this reality and planning ahead makes it possible to deal with and stem an incipient outbreak of disease before it is transmitted and spread to the general population. this becomes essential when there is a future disease outbreak in large, dense populations in tropical and subtropical urban centers as well as those in regions warmed and humidified by climate change to subtropical and tropical settings. remember that urban populations worldwide, especially in africa, asia, and latin america, are where much of the global population growth will take place during the next few generations. under these conditions, diseases can spread rapidly in many ways. these include from bites of vectors, by respired droplets after an infected person coughs or sneezes, and by touching surfaces bearing viruses, bacteria, or parasites. diseases are also spread by ingestion of contaminated water and/or tainted food, and by other methods of infection transmission. disease transmission can be checked by rapid response teams with appropriate and sufficient supplies to treat (and perhaps places to quarantine) those in the infected population. lastly, it must be noted that there are many other diseases in addition to those cited previously for which prevention, treatment, and cures are research priorities in laboratories worldwide. in addition, there are addiction diseases that can trigger health problems in important segments of society. these include smoking (e.g., emphysema, lung cancer), alcoholism (e.g., cirrhosis of the liver), drugs (e.g., various psychological and physical ills), and overeating (obesity, diabetes, high blood pressure). adaptation to these health threats involves public education forums through various media outlets, counseling, and sponsored groups with their individual group meeting, and programs are assisting many in breaking from an addiction to the benefit of a healthier life. adaptation to meet the health challenges in the past, and in contemporary times has been a slow, progressive adventure with many successes but with much yet to be done. this is the planned path for the future: meet the challenges of societal health threats, resolve many, and keep researching to resolve others. a special ipcc report in 2012 examines in a general way adaptation to a changing climate as a risk management approach [18] . it uses pre-planning to reduce exposure and vulnerability to extreme hazard events by preparing for them beforehand, responding to their impacts on people, structures, and infrastructure, and having in place recovery systems that can act when a danger condition eases. in this way, there will be an ability of populations to cope with future risks brought on by a changing force with which a hazard impacts a community, changes in the frequency of an occurrence, and extension of the spatial reach of its destructive power. much of this has been discussed in the chapters of the book you are reading. an understanding of what is being done now to adapt to the various problems society faces during the second decade of the twenty-first stimulates proposals of how to adapt to them as global conditions change in the future. to this end, the world bank commissioned a study on the effects global warming as it increased from 0.8â°c that exists on our planet now to what can be expected if the warming reached 2â°c, a change that many scientists believe we can adapt to, and then reached 4â°c as warming continues [19] . the study centered on regions with high population growth and great susceptibility to be negatively impacted by climate changes: (1) sub-saharan africa where food production is at risk: (2) southeast asia where coastal zones and productivity are at risk; and (3) south asia where there could be extremes of water scarcity and excess. the effects of higher temperatures from global warming and climate change included what has been discussed in previous chapters of this book: heat, drought, sea level rise, coastal zones, typhoons, flooding, river runoff, water availability, ecosystem shifts, crop yields, fishing, aquaculture, livestock, health and poverty, and tourism. projections such as those published in the world bank study give impetus to governments, international institutions, multinational companies, private foundations, and ngos to think now, to invest now, and to research now for adaptations that can be realized in good time and that will provide global citizenry with a good quality of life where needed. in this book, we have examined existing human populations and the problems they are experiencing in the second decade of the twenty-first century and have also considered growing populations globally and additional problems future generations will experience. we have discussed strategies on how to cope with manyfaceted threats to citizens. these include how to nourish those who need food and water, how to shelter people safely from natural and anthropogenic hazards, how to provide them with healthcare, education, and employment, and how to prepare them for the evolving global warming and the physical and biological dangers that ensue from climate change. given the present global conditions with about 14 % of our earth's population suffering from malnutrition and more than 21 % not having access to safe water, our capability of nourishing a billion and a half more people by 2035 is in question. also problematical is our capability to provide for an additional billion people 15 years later, or a total of at least 10.3 billion people by the turn of the century, that is, if we reach those population figures or have population crashes such as from pandemics that can kill scores of millions if a disease is not immediately treatable, or an unlikely but possible nuclear conflagration that could do the same. less likely yet is an explosion of a small asteroid or comet in the atmosphere such as happened in a poorly inhabited area of siberia in 1908. here, an exploding mass more than 60 m in size knocked down millions of trees in an area greater than 2,000 km 2 (close to 800 mi 2 ) with energy thought to be 1,000 times greater than the hiroshima atomic bomb. clearly, such an event could kill the population of a megacity if it were to occur. another question is whether national governments are economically strong enough and have the will to set priorities that adopt strategies to protect citizens from natural (e.g., earthquakes) and anthropogenic (e.g., pollution) hazards as well as from extreme weather conditions that are supported by global warming (pollution of the atmosphere) but are naturally occurring. countries can also improve social and economic conditions by investing in health care and education for their citizens in order to form a sound and knowledgeable cadre that would be attractive to investors interested in locating a development project that would provide employment. again, this is in question given limited national economic capabilities and the increasing numbers of people to be accommodated, especially in several developing and less developed countries in africa, asia, latin america, and the middle east. at this point, we must ask, "what is the carrying capacity of the earth?" have we reached it at 7 billion given the billions who are today under served in developing and less developed countries? some scientists will answer yes, whereas others believe that advances in agriculture and technology can allow population expansion although to what point is not defined. can countries that are poisoning their environments do a turn around to save their citizens from grief? can they exert controls on operations that create unhealthy conditions that sicken people, lessen agricultural production, and otherwise disrupt local, regional, and global climate change: evidence and causes (36 p) intergovernmental panel on climate change (2007) climate change 2007 (as four part report). part 1. the physical science basis mitigation of climate change past and future sea level change from the surface mass balance of glaciers a reconciled estimate of glacier contributions to sea level rise ocean thermal expansion and its contribution to sea level rise tuberculosis. fact sheet no. 104 5 p 10. world health organization (2013) dracuncukiasis (guinea-worm disease) protection against malaria by intravenous immunization with a non-replicating sporozoite vaccine global mortality between 1980 and 2010: a systematic analysis world health organization (2013) malaria. fact sheet no. 94 7 p 15. world health organization (2013) dengue and severe dengue chagas disease (american trypanosomiasis). fact sheet no vaccines and global health 2012) ipcc special report summary for policy makers. managing the risks of extreme events and disasters to advance climate change adaptation turn down the heat: climate extremes regional impacts and the case for resilience. a report for the world bank prepared by potsdam institute for climate impact research and climate analytics key: cord-265699-0socw0hp authors: ortega, miguel ángel; guzmán merino, alberto; fraile-martínez, oscar; recio-ruiz, judith; pekarek, leonel; g. guijarro, luis; garcía-honduvilla, natalio; álvarez-mon, melchor; buján, julia; garcía-gallego, sandra title: dendrimers and dendritic materials: from laboratory to medical practice in infectious diseases date: 2020-09-14 journal: pharmaceutics doi: 10.3390/pharmaceutics12090874 sha: doc_id: 265699 cord_uid: 0socw0hp infectious diseases are one of the main global public health risks, predominantly caused by viruses, bacteria, fungi, and parasites. the control of infections is founded on three main pillars: prevention, treatment, and diagnosis. however, the appearance of microbial resistance has challenged traditional strategies and demands new approaches. dendrimers are a type of polymeric nanoparticles whose nanometric size, multivalency, biocompatibility, and structural perfection offer boundless possibilities in multiple biomedical applications. this review provides the reader a general overview about the uses of dendrimers and dendritic materials in the treatment, prevention, and diagnosis of highly prevalent infectious diseases, and their advantages compared to traditional approaches. examples of dendrimers as antimicrobial agents per se, as nanocarriers of antimicrobial drugs, as well as their uses in gene transfection, in vaccines or as contrast agents in imaging assays are presented. despite the need to address some challenges in order to be used in the clinic, dendritic materials appear as an innovative tool with a brilliant future ahead in the clinical management of infectious diseases and many other health issues. infectious diseases are produced by pathogenic microorganisms, mainly bacteria, viruses, parasites, and fungi. since the dawn of civilization, these diseases have persisted as sources of human morbidity and mortality, representing the second cause of death worldwide and the main reason of b groups are dormant/protected and will react in a subsequent step after deprotection/activation. iterative growth and activation steps lead to the desired generation, while the end-groups are available for further postfunctionalization. the divergent growth is the most viable approach, as it employs an excess of inexpensive reagents, but could lead to structural defects at high generations due to incomplete substitutions. the convergent growth approach was initially developed by hawker and fréchet, to improve the weaknesses of the divergent approach. this outside-in strategy relies on the coupling of monomers to generate monodisperse dendrons, which are finally attached to a multifunctional core through their focal points. while the risk of structural defects is minimized, the synthesis of higher generation dendrons and dendrimers are challenging due to steric hindrance, leading to low yields. importantly, new strategies, namely "accelerated growth" approaches, are continuously evolving to simplify the synthetic routes while keeping their perfection. these include the orthogonal chemoselective strategy and the one-pot approaches, among others. the number of steps is thus reduced, as the chemoselective moieties avoid the need for protection/deprotection steps. the reader is referred to excellent reviews on this topic [23, 27] . pharmaceutics 2020, 12, x for peer review 4 of 28 routes are prevalent ( figure 1 ): the divergent strategy and the convergent strategy [23] . in the divergent growth approach, first developed by tomalia et al. [26] , the dendrimer synthesis proceeds inside-out from the core. the core reacts with abn monomeric units through the a functional group, while the b groups are dormant/protected and will react in a subsequent step after deprotection/activation. iterative growth and activation steps lead to the desired generation, while the end-groups are available for further postfunctionalization. the divergent growth is the most viable approach, as it employs an excess of inexpensive reagents, but could lead to structural defects at high generations due to incomplete substitutions. the convergent growth approach was initially developed by hawker and fréchet, to improve the weaknesses of the divergent approach. this outside-in strategy relies on the coupling of monomers to generate monodisperse dendrons, which are finally attached to a multifunctional core through their focal points. while the risk of structural defects is minimized, the synthesis of higher generation dendrons and dendrimers are challenging due to steric hindrance, leading to low yields. importantly, new strategies, namely "accelerated growth" approaches, are continuously evolving to simplify the synthetic routes while keeping their perfection. these include the orthogonal chemoselective strategy and the one-pot approaches, among others. the number of steps is thus reduced, as the chemoselective moieties avoid the need for protection/deprotection steps. the reader is referred to excellent reviews on this topic [23, 27] . a broad variety of dendritic scaffolds have been described in the literature, with a purpose-driven design. for details on their preparation, the reader is referred to excellent books and reviews on the literature [28] [29] [30] . in the biomedical field, the dendritic families which stand out are poly(amino amide) (pamam) [31] , poly(propylene imine) (ppi), poly(l-lysine) (pll) [32] , carbosilane [33] , poly(phosphorhydrazone) (pph) [34] , and polyester dendrimers [35] (figure 2 ). the biocompatibility, flexibility, and commercial availability are behind their prevalence in this field. pharmaceutics 2020, 12, x for peer review 5 of 28 a broad variety of dendritic scaffolds have been described in the literature, with a purpose-driven design. for details on their preparation, the reader is referred to excellent books and reviews on the literature [28] [29] [30] . in the biomedical field, the dendritic families which stand out are poly(amino amide) (pamam) [31] , poly(propylene imine) (ppi), poly(l-lysine) (pll) [32] , carbosilane [33] , poly(phosphorhydrazone) (pph) [34] , and polyester dendrimers [35] (figure 2 ). the biocompatibility, flexibility, and commercial availability are behind their prevalence in this field. pamam dendrimers are probably the most studied dendritic architectures, reaching up to the tenth generation, with different cores and terminal groups (mainly nh2 or oh), figure 2a . pamam dendrimers exhibit appealing properties for biomedical studies [31] , such as a high water solubility, a peptide-mimicking backbone, and readily modifiable amine termini. ppi dendrimers, also known as popam or dab, present multiple tertiary amines on the scaffold and primary amines as terminal groups, figure 2b . they are comparatively smaller than pamam and present a more hydrophobic scaffold, but are also commercially available, prevalent in the biomedical field and similarly cytotoxic due to the peripheral amino groups [36] . pll dendrimers, which comprise the amino acid lysine in their entire structure ( figure 2c ), stand out due to their biocompatibility, biodegradability, and the ability to maintain its activity in environments with high and low salinity [32] . pll differ from other dendrimers such as pamam and ppi in the asymmetry of their branching cell, which inevitably influences the encapsulation properties as they possess no interior void space [37] . however, they share the presence of multiple nh2 peripheral groups, which can cause certain cytotoxicity. carbosilane dendrimers comprise carbon-carbon and carbon-silicon bonds in their scaffolds, conferring flexible, non-polar, inert, and thermally stable properties, very interesting in biomedicine [33] , figure 2d . they are often decorated with polar groups in order to achieve water solubility. they are pamam dendrimers are probably the most studied dendritic architectures, reaching up to the tenth generation, with different cores and terminal groups (mainly nh 2 or oh), figure 2a . pamam dendrimers exhibit appealing properties for biomedical studies [31] , such as a high water solubility, a peptide-mimicking backbone, and readily modifiable amine termini. ppi dendrimers, also known as popam or dab, present multiple tertiary amines on the scaffold and primary amines as terminal groups, figure 2b . they are comparatively smaller than pamam and present a more hydrophobic scaffold, but are also commercially available, prevalent in the biomedical field and similarly cytotoxic due to the peripheral amino groups [36] . pll dendrimers, which comprise the amino acid lysine in their entire structure ( figure 2c ), stand out due to their biocompatibility, biodegradability, and the ability to maintain its activity in environments with high and low salinity [32] . pll differ from other dendrimers such as pamam and ppi in the asymmetry of their branching cell, which inevitably influences the encapsulation properties as they possess no interior void space [37] . however, they share the presence of multiple nh 2 peripheral groups, which can cause certain cytotoxicity. carbosilane dendrimers comprise carbon-carbon and carbon-silicon bonds in their scaffolds, conferring flexible, non-polar, inert, and thermally stable properties, very interesting in biomedicine [33] , pharmaceutics 2020, 12, 874 6 of 27 figure 2d . they are often decorated with polar groups in order to achieve water solubility. they are classified as "inorganic dendrimers" and exhibit relevant differences compared to traditional "organic dendrimers" such as pamam. furthermore, they have a great variability by altering the core and the amount and length of the branches. pph dendrimers, which can be quantitatively prepared up to generation 12 [38] , present phosphorus atoms throughout the entire dendritic scaffold and have been widely studied for biomedical applications [34] , figure 2e . like carbosilane dendrimers, pph are also "inorganic dendrimers" with a huge variability in cores, branches, and peripheral groups, and require the attachment of polar groups in the periphery to become water-soluble. polyester dendrimers attract widespread attention in the biomedical field due to their biocompatibility and biodegradability. in particular, dendrimers based on 2,2-bis(hydroxymethyl) propanoic acid (bis-mpa, figure 2f ) are commercially available. since the first reports in the early 1990s, bis-mpa dendrimers have undergone an extraordinary increase in their structural complexity and control, which capitalized on a constant evolution of the synthetic strategies, from the traditional divergent and convergent routes to accelerated approaches based on chemoselective reactions [35, 39] . since their first reports, dendrimers have been tested in a large number of in vitro and in vivo studies for multiple biomedical applications. the most explored use of dendrimers is their ability to carry drugs to the desired site of action, being an important resource in precision medicine [24] . dendrimers protect the encapsulated or bound drug and allow the delivery to the desired site of action. as they can be customized, dendrimers improve the drug pharmacokinetics and solubility, control the drug release, enable more comfortable administration routes, and reach target sites with difficult accessibility such as the ocular system [40] . another application that has raised great interest is the use of dendrimers in gene therapy. several dendrimers have been explored as non-viral vectors for dna and rna, enabling gene transfection to specific cells. this has been especially useful in in vitro cancer studies, where rna transfected to tumor cells can alter their mechanisms, making them more susceptible to treatment or hindering their uncontrolled division [41] . on the other hand, dendrimers can act as immunomodulators, by either reducing or enhancing the immune response [42] . the first approach can be very useful towards autoimmune diseases and allergies [43] , while the second has been employed, for example, in cancer immunotherapy [44] . the attachment of multiple antigen copies to the dendritic scaffold produces an increase in the immune response related to the multivalency and the decrease in the conformational freedom of the antigen. in infectious diseases, dendrimers can support the development of vaccines by acting as antigens carrier, providing stability, safety, and a sustained release. in addition, they can serve as adjuvants or can promote the uptake of the antigen by the antigen-presenting cells, thus enhancing its recognition and improving the effectiveness of the vaccine [45] . another outstanding application is the use of dendritic materials in diagnosis [46] , such as iron oxide magnetic nanoparticles decorated with dendrimers, which can be monitored through magnetic resonance, or the oxygen sensors, very useful in pathologies such as diabetes [47] . dendrimers also enable a combined therapeutic and diagnostic action in a single platform, the so-called "theranosis" [48] . the diagnostic capacity is provided by a specific molecule (e.g., a radionuclide) which, bound to the surface or encapsulated inside the nanoparticle, serves to detect its position in vivo by means of diagnostic imaging such as single photon emission computed tomography (spect). in order to fully benefit from the use of dendrimers as nanocarriers, it is essential to understand the mechanisms of interaction between the dendrimer and the different cargo ( figure 3 ) [49] [50] [51] : • encapsulation. the drug is physically trapped within the dendritic scaffold due to the spheroidal or ellipsoidal hollow cavities found between the different branches. these cavities are frequently hydrophobic, so they exhibit affinity towards drugs with poor water-solubility, and can also lead to h-bonding due to the presence of oxygen and nitrogen atoms. the main drawback of this approach is the tendency of the drug to rapidly leak in biological fluids, compared to a covalent conjugation approach [52] . • electrostatic interactions. the multivalent structure of the dendrimer enables the formation of multiple bonds in the periphery, which depend on the nature of the end groups. a common example are electrostatic interactions between the drug and a dendrimer bearing cationic (e.g., ammonium groups) or anionic (e.g., carboxylate) moieties. pamam and ppi dendrimers frequently employ this mechanism, due to the multiple ionizable amino groups in the periphery as well as in the interior of their scaffolds. the ph, the ionic strength and the presence of proteins such as albumin have a remarkable impact on dendrimer-cargo electrostatic interactions [52] . this approach is widely employed in gene therapy to generate dendrimer-nucleic acid complexes, or "dendriplexes" [53] . • covalent conjugation. drugs and other molecules can be attached to dendrimers through covalent bonds. sometimes labile or biodegradable bonds are employed, such as amide or ester bonds, to enable the release under chemical or enzymatic scission. other strategy relies on the use of spacers, such as poly(ethylene glycol) (peg), which also generates a hydrophilic surface with a hydrophobic interior, an amphiphilic unimolecular micelle to improve drug encapsulation. furthermore, the attachment of peg reduces the interaction with blood proteins and cells, prolongs the circulation in blood and increases the overall molecular weight, improving the permeability and retention of the drug [54] . other types of ligands have also been covalently bound, such as antibodies or contrast agents. this type of interaction increases the stability of the drug towards degradation, alters the release kinetics, and improves the therapeutic efficiency. pharmaceutics 2020, 12, x for peer review 7 of 28 • electrostatic interactions. the multivalent structure of the dendrimer enables the formation of multiple bonds in the periphery, which depend on the nature of the end groups. a common example are electrostatic interactions between the drug and a dendrimer bearing cationic (e.g., ammonium groups) or anionic (e.g., carboxylate) moieties. pamam and ppi dendrimers frequently employ this mechanism, due to the multiple ionizable amino groups in the periphery as well as in the interior of their scaffolds. the ph, the ionic strength and the presence of proteins such as albumin have a remarkable impact on dendrimer-cargo electrostatic interactions [52] . this approach is widely employed in gene therapy to generate dendrimer-nucleic acid complexes, or "dendriplexes" [53] . • covalent conjugation. drugs and other molecules can be attached to dendrimers through covalent bonds. sometimes labile or biodegradable bonds are employed, such as amide or ester bonds, to enable the release under chemical or enzymatic scission. other strategy relies on the use of spacers, such as poly(ethylene glycol) (peg), which also generates a hydrophilic surface with a hydrophobic interior, an amphiphilic unimolecular micelle to improve drug encapsulation. furthermore, the attachment of peg reduces the interaction with blood proteins and cells, prolongs the circulation in blood and increases the overall molecular weight, improving the permeability and retention of the drug [54] . other types of ligands have also been covalently bound, such as antibodies or contrast agents. this type of interaction increases the stability of the drug towards degradation, alters the release kinetics, and improves the therapeutic efficiency. these three strategies have also been exploited in the treatment of infectious diseases, as previously reported [55] [56] [57] [58] . the present review, however, focuses on a broader overview to cover the prevention, treatment, and diagnosis of these diseases, as detailed in section 3. the interest in the dendrimer field has continuously increased over time. as recently reported by tomalia (2020) [59] , more than 60,000 articles/patents have been published on dendritic materials, with an approximate increase of 1500 publications and 3000 patents per year since 2013. key commercial successes include the stratus cs acute care diagnostic system (siemens healthcare gmbh, erlangen, germany), for emergency diagnosis of cardiovascular infarctions; vivagel ® products (starpharma, melbourne, australia), for the prevention and treatment of sexually transmitted infections (stis); targeted dep ® and priostar ® (starpharma), for the delivery of anticancer drugs and agrochemical products, respectively; or spherical (polymer factory, stockholm, sweden), as mass spectrometry standards [59] . these three strategies have also been exploited in the treatment of infectious diseases, as previously reported [55] [56] [57] [58] . the present review, however, focuses on a broader overview to cover the prevention, treatment, and diagnosis of these diseases, as detailed in section 3. the interest in the dendrimer field has continuously increased over time. as recently reported by tomalia (2020) [59] , more than 60,000 articles/patents have been published on dendritic materials, with an approximate increase of 1500 publications and 3000 patents per year since 2013. key commercial successes include the stratus cs acute care diagnostic system (siemens healthcare gmbh, erlangen, germany), for emergency diagnosis of cardiovascular infarctions; vivagel ® products (starpharma, melbourne, australia), for the prevention and treatment of sexually transmitted infections (stis); targeted dep ® and priostar ® (starpharma), for the delivery of anticancer drugs and agrochemical products, respectively; or spherical (polymer factory, stockholm, sweden), as mass spectrometry standards [59] . from the low rate of issued patents turning into commercial products, it becomes apparent that dendritic materials must face several challenges for the bench-to-bedside translation in the biomedical field. mignani et al. (2017) summarized the requirements to become a clinical candidate [60] . to secure a successful development, the authors highlight the importance of complying with the good laboratory practice (glp) requirements to ensure the quality, reproducibility and reliability of in vitro and in vivo data. furthermore, the good manufacturing practice (gmp) is desirable, but emerges as one of the main challenges in dendrimer translation. indeed, dendrimer defects have been related to the failure of relevant preclinical trials [61] . an accurate dendrimer synthesis and a thorough purification process are deemed necessary to ensure monodispersity and batch-to-batch reproducibility. this is a highly demanding challenge especially for high-generation dendrimers, multipurpose platforms, and large-scale production. many different strategies are currently explored to overcome these challenges in dendrimer translation, including engineering through critical nanoscale design parameters (cndps) [37] ; accelerated synthetic approaches [27] ; the improvement of analytical techniques, such as mass spectrometry; the accurate and simplified design of multipurpose platforms [62] ; or the dendronization of materials to expand their uses [63] . viruses are simple acellular organisms, which have coevolved with living beings to replicate and reproduce inside their cells, after the binding to specific receptors [64] . nearly 5000 species of viruses have been reported; many of them can cause human diseases [65] . some viral infections, such as respiratory ones, represent an important economic burden and a serious public health concern [66] . antiviral drugs are the most common clinical tool to address these pathologies. to date, 86 different drugs have been approved for the treatment of 17 viral infections [67] . however, some drugs have serious side effects, including nausea, insomnia, vomiting, allergic reactions, behavior disorders, cardiovascular complications, and dependency [68] . opening new therapeutic windows, decreasing the side effects while maintaining their efficacy, is a key action. dendrimers contribute to the fight against viral infections [69] , acting as microbicides per se or as drug nanocarriers, with relevant properties such as low systemic absorption, biocompatibility, water solubility, or simple formulation [70] . the main uses of dendrimers in viral infections are herein addressed, the human immunodeficiency virus (hiv) being one of their most important targets. stis are highly prevalent worldwide, despite the efficient preventive tools (e.g., preservatives) [71] . one of the most illustrative examples is the human immunodeficiency virus (hiv). hiv is responsible for the deterioration of immune cells, especially the target cd4+ t cells [72] , thus aiding the entry of opportunistic pathogens that cause the acquired immunodeficiency syndrome (aids) [73] . hiv transmission mainly occurs through body fluids exchange, mostly by sexual contact, but blood, breastfeeding or vertical transmission have also been described. according to the who, 37.6 million people were infected by hiv in 2015 [74] . fortunately, around 23% decrease in infections was registered from 2010 to 2019, but the treatment, diagnosis, and prevention remain as a global challenge, especially for developing countries with lack of resources. to date, antiretroviral therapy has shown an excellent outcome in the clinical management of aids. however, these drugs produce important side effects, including hiv resistance to the treatment [75] . dendrimers represent an interesting alternative to minimize these side-effects and prevent the transmission of hiv and other viral or bacterial stis, figure 4 [76] . a promising approach relies on the use of dendrimers bearing anionic, sugar, or peptide moieties to prevent the entry of the virus in the target cell. these dendrimers block either the host cell or the viral receptors ( figure 4 , top), such as the glycoproteins gp120 and gp41 located at the hiv envelope, two key proteins for the interaction and fusion of hiv with cd4 t cells. the most representative example is the anionic pll dendrimer spl7013 [77] . spl7013 is a component of two approved and marketed products (starpharma): vivagel ® antiviral condom, for the treatment and prevention of hiv and hsv (herpes simplex virus); and vivagel ® bv for bacterial vaginosis (a second product under phase iii clinical trial nct01577537). furthermore, it also shows significant activity against other viruses, such as the coronavirus sars-cov-2 [78] , enabling a fast-track development of tools to fight covid-19. polyanionic carbosilane dendrimers are also promising microbicides against hiv infection, as shown in different animal models [79] . besides their own antiviral activity, muñoz-fernández et al. showed that their combination with tenofovir and maraviroc (two antiviral agents) produce almost complete inhibition of hiv infection and transmission [80] . carbosilane dendrimers are also efficient towards hiv-hsv coinfection [81] and can be employed in the development of fast diagnostic assays based on dendronized magnetic nanoparticles [82] tools. dendrimers can also contribute to the design of efficient vaccines against hiv, such as the peg-citrate g2 dendrimer bearing multiple hiv epitopes which produced a significant cellular immune response in vivo and a higher th1 response compared to th2 [83] . pharmaceutics 2020, 12, x for peer review 9 of 28 fusion of hiv with cd4 t cells. the most representative example is the anionic pll dendrimer spl7013 [77] . spl7013 is a component of two approved and marketed products (starpharma): vivagel ® antiviral condom, for the treatment and prevention of hiv and hsv (herpes simplex virus); and vivagel ® bv for bacterial vaginosis (a second product under phase iii clinical trial nct01577537). furthermore, it also shows significant activity against other viruses, such as the coronavirus sars-cov-2 [78] , enabling a fast-track development of tools to fight covid-19. polyanionic carbosilane dendrimers are also promising microbicides against hiv infection, as shown in different animal models [79] . besides their own antiviral activity, muñoz-fernández et al. showed that their combination with tenofovir and maraviroc (two antiviral agents) produce almost complete inhibition of hiv infection and transmission [80] . carbosilane dendrimers are also efficient towards hiv-hsv coinfection [81] and can be employed in the development of fast diagnostic assays based on dendronized magnetic nanoparticles [82] tools. dendrimers can also contribute to the design of efficient vaccines against hiv, such as the peg-citrate g2 dendrimer bearing multiple hiv epitopes which produced a significant cellular immune response in vivo and a higher th1 response compared to th2 [83] . besides hiv and stis, dendrimers are effective towards other virus such as the enterovirus a71 (ev71). ev71 belongs to the picornaviridae family, associated with the hands-feet-mouth disease in children, a syndrome characterized by the presence of cutaneous vesicles and ulcerations and frequently with severe neurological manifestations [84] . currently, neither vaccines nor therapies have been approved to prevent or treat ev71 infection, representing an important global problem but specially in the asian southeast [85] . a tryptophan-decorated pentaerythritol dendrimer was especially active towards ev71 in some clinical isolates in the low nm-high pm range [86] . as ev71 is mainly transmitted through fecal-oral route, these dendrimers could be used as a prophylactic method after their oral administration, thus avoiding the transfer of ev71 from the gut to the bloodstream. similar dendrimers have also shown a dual activity towards ev71 and hiv [87] . dendrimers and dendronized materials, such as fullerenes and carbon nanotubes, have also promising activity against other viruses like sars-cov-2 [78] , figure 5a ; ebola virus [88] , figure 5b ; zika and dengue viruses [89] , figure 5c ; hsv [90] ; cytomegalovirus [91] ; some flavivirus, such as the responsible of the japanese encephalitis [92] ; and different human or aviary flu viruses [93] . besides hiv and stis, dendrimers are effective towards other virus such as the enterovirus a71 (ev71). ev71 belongs to the picornaviridae family, associated with the hands-feet-mouth disease in children, a syndrome characterized by the presence of cutaneous vesicles and ulcerations and frequently with severe neurological manifestations [84] . currently, neither vaccines nor therapies have been approved to prevent or treat ev71 infection, representing an important global problem but specially in the asian southeast [85] . a tryptophan-decorated pentaerythritol dendrimer was especially active towards ev71 in some clinical isolates in the low nm-high pm range [86] . as ev71 is mainly transmitted through fecal-oral route, these dendrimers could be used as a prophylactic method after their oral administration, thus avoiding the transfer of ev71 from the gut to the bloodstream. similar dendrimers have also shown a dual activity towards ev71 and hiv [87] . dendrimers and dendronized materials, such as fullerenes and carbon nanotubes, have also promising activity against other viruses like sars-cov-2 [78] , figure 5a ; ebola virus [88] , figure 5b ; zika and dengue viruses [89] , figure 5c ; hsv [90] ; cytomegalovirus [91] ; some flavivirus, such as the responsible of the japanese encephalitis [92] ; and different human or aviary flu viruses [93] . bacteria are unicellular prokaryote organisms with great implications in human health, as they compose the core of microbiota [94] , but also in human disease. certain bacterial populations can colonize and infect different tissues, leading to the development of a wide range of pathologies [95] . antibiotics have long been one of the most effective solutions to fight against bacterial infections. however, their improper use drove a global public health problem: bacterial resistance. only in europe, a total of 672,000 multiresistant bacterial infections were estimated, being responsible for up to 33,000 deaths in 2015 [96] . dendrimers emerge as a potential solution, as they employ an unspecific mechanism that prevents the development of resistances (figure 4 bottom) . some representative examples against resistant bacteria are herein collected. biofilms are one of the most important adaptive mechanisms of bacteria, enabling them to survive in an adverse environment. it is activated under different stress conditions, like limited oxygen or iron levels or the presence of some antimicrobial agents in sublethal concentrations [97] . p. aeruginosa represents one of the most important biofilm-forming bacteria, with outstanding impact in some chronic diseases like cancer [98] or cystic fibrosis [99] . this is the main problem associated with p. aeruginosa infection, especially in non-immunocompetent patients, hindering the clinical management. p. aeruginosa represents a clear example of how dendrimers can address resistant bacteria infection, including the inhibition of biofilm formation. it has been described that a bacterial specific lectine (lecb) plays a key role in biofilm formation by promoting the adhesion to cells [100] . lectines are proteins which show a high specificity for sugars and bacteria are unicellular prokaryote organisms with great implications in human health, as they compose the core of microbiota [94] , but also in human disease. certain bacterial populations can colonize and infect different tissues, leading to the development of a wide range of pathologies [95] . antibiotics have long been one of the most effective solutions to fight against bacterial infections. however, their improper use drove a global public health problem: bacterial resistance. only in europe, a total of 672,000 multiresistant bacterial infections were estimated, being responsible for up to 33,000 deaths in 2015 [96] . dendrimers emerge as a potential solution, as they employ an unspecific mechanism that prevents the development of resistances (figure 4 bottom) . some representative examples against resistant bacteria are herein collected. biofilms are one of the most important adaptive mechanisms of bacteria, enabling them to survive in an adverse environment. it is activated under different stress conditions, like limited oxygen or iron levels or the presence of some antimicrobial agents in sublethal concentrations [97] . p. aeruginosa represents one of the most important biofilm-forming bacteria, with outstanding impact in some chronic diseases like cancer [98] or cystic fibrosis [99] . this is the main problem associated with p. aeruginosa infection, especially in non-immunocompetent patients, hindering the clinical management. p. aeruginosa represents a clear example of how dendrimers can address resistant bacteria infection, including the inhibition of biofilm formation. it has been described that a bacterial specific lectine (lecb) plays a key role in biofilm formation by promoting the adhesion to cells [100] . lectines are proteins which show a high specificity for sugars and their derivatives, being able to successfully recognize and agglutinate cells with glycosylated proteins or lipids. lecb binds to fucose, a mucin located in the epithelial mucosa, playing a key role in p. aeruginosa biofilm formation, in conjunction with leca, specific to galactose, although this binding is weaker and less important [100] . in this context, some peptidic dendrimers have been developed to directly inhibit lecb-fucose interactions at low concentrations, such as fd2 (ic 50 = 0.14 µm) depicted in figure 6a . dendrimers decorated with fucose-derived groups prevent the formation of p. aeruginosa biofilms (ic 50~1 0 µm) and even disperse formed structures, by inhibiting the agglutination of the pathogen and acting as antimicrobial nanocarriers, thus increasing the efficacy of the established treatments [100, 101] . pharmaceutics 2020, 12, x for peer review 11 of 28 less important [100] . in this context, some peptidic dendrimers have been developed to directly inhibit lecb-fucose interactions at low concentrations, such as fd2 (ic50 = 0.14 µm) depicted in figure 6a . dendrimers decorated with fucose-derived groups prevent the formation of p. aeruginosa biofilms (ic50~10 µm) and even disperse formed structures, by inhibiting the agglutination of the pathogen and acting as antimicrobial nanocarriers, thus increasing the efficacy of the established treatments [100, 101] . one of the most representative examples of resistant bacteria is gram-negative bacteria. these microorganisms present a peptidoglycan wall located between the inner and the outer membranes, which is responsible for the higher resistance of gram-negative bacteria to immune system, and in case of lysis, promotes the release of proinflammatory substances known as lipopolysaccharides (lps), exacerbating the infection [106] . this structure also confers gram-negative bacteria resistance to some external agents through multiple mechanisms, which are heterogeneous between species [107] . examples of common mechanisms of bacterial resistance are the expelling of toxic residues that eliminate antibacterial agents; a decrease in bacterial permeability, through the alteration of the membrane channels; or the production of antibiotic inactivating enzymes [108] . cationic dendrimers may be a solution to fight against resistant bacteria, as they can bind efficiently to the negatively-charged walls, destabilize it by displacing sodium and calcium ions and increase the membrane permeability [109, 110] , figure 4 bottom. however, despite their promising biocide activity, cationic dendrimers exhibit high toxicity towards mammal cells and require structural modifications to reduce their cytotoxicity, without affecting the efficacy [111] . cationic antimicrobial peptides (amp) arranged as multiple antigen peptide (map) dendritic structures can also exert a potent antibacterial activity, decreasing the minimum inhibitory concentration (mic) and minimum bactericide concentration (mbc) of the peptide alone, while dramatically increasing the peptide stability to proteolysis [112] . for example, a dendritic map structure based on the peptide qkkirvrlsa effectively inhibited diverse gram-negative bacteria (mic = 4-8 g/ml for e. coli atcc 25922, p. aeruginosa atcc 27853, and a clinical isolate of k. pneumoniae) [112] . the higher local concentration of amp allows a multivalent binding and enhances the destabilizing effect of the bacterial membrane. dendrimers may also be used in the rapid diagnosis to discern between gram-negative or gram-positive bacteria, through a ph-dependent bacteria-selective aggregation occurring within 5 min of adding the dendrimer to a bacterial suspension [113] ; and as carriers of different drugs such as vancomycin or agents like led209, both specific to gram-negative microorganisms [114, 115] . chorioamnionitis is an infection in the amniotic liquid, which may cause neurological problems in the fetus due to the production of proinflammatory cytokines, escherichia coli being one of the most important etiological agents [116] . this disease often occurs due to the ascent of microorganisms from vagina to uterus, although other pathways have also been reported like transplacental infection, retrograde seeding from the peritoneal cavity through the fallopian tubes or accidental invasive procedures [117] . the use of antibiotics such as penicillins, cephalosporins, macrolides, and corticosteroids reduce the risk of developing chorioamnionitis [118] . in this sense, dendrimers can increase their efficacy. for example, a study conducted by wang et al. (2010) in a guinean pig model demonstrated that hydroxyl-and amino-functional pamam dendrimers successfully encapsulate ampicillin [119] . both dendrimers significantly decreased the uterus cytokines, compared to the usual therapy, but the amino-dendrimer exhibited a higher toxicity. dendrimers have been studied against other bacterial infections like chlamydia trachomatis, increasing the efficacy of vaccines by conjugating a peptide mimic of a chlamydial glycolipid antigen to a g4-pamam-oh dendrimer [120] ; some opportunistic agents, such as s. aureus, using different generation gn-pamam-nh 2 dendrimers [121] ; or genital infections, through a sustained and localized delivery of amoxicillin in the cervicovaginal region by pamam-peg dendritic hydrogels [122] . overall, these studies show the potential role of dendrimers in bacterial infections, although a long road is still to cover, especially to decrease their cytotoxicity and increase their specificity. fungi are eukaryotic organisms responsible of a wide range of human infections. the prevalence of these diseases has increased in some countries, particularly in hospital areas and immunocompromised patients, candida, cryptococcus, pneumocystis, and aspergillus being the most representative families [123] . data collected by the national nosocomial infections surveillance system (nnis) from january 1990 to april 1996 showed that up to 9% of nosocomial infections in eeuu were due to fungi, mainly candida species [124] . however, current trends indicate the prevalence of aspergillus in different european states [125] . the treatment of fungal infections is performed through antifungal (antimycotic) drugs, which produce some alterations in their cellular structures, thus inhibiting their development, viability and survival in a direct or indirect way. the most representative antifungal drugs include [126] : polyenes (e.g., amphotericin b); azoles (e.g., imidazole, triazole); allylamines; lipopeptides; and miscellaneous agents, as griseofulvin, which inhibits microtubules and mitotic fuse, affecting cell division. unfortunately, the resistance developed by some fungi may hinder the clinical management of these infections [127] . in addition, the great similarity between mammal and fungal cells can lead to cytotoxicity problems, being necessary to find molecules which selectively target fungal cells in a particular tissue [126] . in this context, the use of nanoparticles like dendrimers may be an effective method to carry all these substances, maintaining their benefits and reducing their side effects. candida albicans, which is responsible of more than half of total fungal infections around the world [128] , is often treated with ketoconazole, a dual-action drug capable of inhibiting both ergosterol synthesis and the transformation of spores to micellar infectious forms [129] . however, ketoconazole is poorly water-soluble and can greatly benefit from the use of nanocarriers, which increase its bioavailability in the bloodstream. gn-pamam-nh 2 dendrimers improve the administration of ketoconazole (up to 16-fold increase of antifungal activity using g2 dendrimer, compared to the drug alone), being even more efficient when used as hydrogel formulation [129] ; as well as clotrimazole (up to 32-fold increase with g2 dendrimer) due to its hydrophobic and electrostatic interactions [130] . similarly, these dendrimers have significantly improved the antifungal activity of amphotericin b, overcoming the low water solubility and nephrotoxicity issues [131] . on the other hand, peptide dendrons have shown efficient anti-candida activity per se [102] . the representative example shown in figure 6b , which displays four tryptophan residues in the periphery and a dodecyl chain in the focal point, produced 100% growth inhibition at 16 µg/ml, as well as affected the biofilm viability and the hyphal and cell wall morphology. on the other hand, dendrimer-assisted gene therapy can prevent fungal infections. cationic pamam dendrimers, bearing -nh 2 , -nme 2 , and -nme 3 + peripheral groups, were used with a ribozyme extracted from an intronic region of c. albicans, an rna molecule capable to cut other rna chain or even itself [132] . these dendrimers inhibited the catalytic activity of candida ribozymes, with a generation-dependent activity (g4 > g3 > g2). however, the nature of the peripheral group did not produce a significantly different inhibition. consequently, the construction of the dendrimer depended on the size of the rna to inhibit and the charge ratio between dendrimer and rna [133] . the use of rna:dendrimer complexes may have multiple applications, such as inhibiting protein synthesis, splicing or even rna delivery in an era where non coding rna are beginning to be used, thus showing the potential of dendrimers in targeted therapy [134] . unfortunately, the diagnosis of fungal diseases is currently a challenge. the current golden standard for the detection of these infections relies on poorly sensitive and invasive methods such as cell culture and histopathological study of the infected tissue [135] . new diagnostic tools are demanded, such as polymerase chain reaction (pcr), immunoassays, or tests capable of detecting specific fungal antigens such as beta-glycans [136] . in this context, the use of nanostructures can play a key role in the development of new techniques that are more sensitive and effective in the early diagnosis of fungal infections. at present, very few studies report the use of dendrimers in the diagnosis of fungal diseases. takano et al. (2003) performed cdna microarray analysis using highly sensitive dendrimer-based technology in the detection of the rice pathogen magnaporthe grisea and the stage of infection in this pathogen [137] . another potential approach is the dendrichips ® technology (dendris), relying on a phosphorous dendrimers coating which dramatically increases their sensitivity. this tool is capable of discerning up to 11 respiratory bacterial pathogens from a single sample [138] , and could be refined and targeted to other types of infectious agents such as fungi. parasites are organisms characterized by the need of other living organism or "host" in order to survive. parasites comprise an important variety of species of diverse complexity, from the simplest organisms such as protozoans to the more complex ones, such as plants [139] . helminths and protozoans entail the main threat of human parasitosis; the clinical expression and its severity depend on the condition of the immune system of the host, as part of a tight interrelation [106, 140] . prevention could be the most efficient mechanism of controlling parasitic infections but, despite the considerable efforts, there are no effective vaccines against any of the main parasites. accordingly, antiparasitic drugs are the pillar in protozoan control, when the simple prevention measures fail. however, the drug resistance of protozoans is becoming an alarming public health problem [141] . dendrimers could be an effective tool in the early diagnosis or prevention of parasitosis, as well as a new treatment for some of these infections. protozoans comprise a diverse group of eukaryotic unicellular microorganisms that belong to protista kingdom. most common human infections caused by protozoans are related to plasmodium spp. and toxoplasma gondii, as well as trypanosoma and leishmania spp. protozoan parasites are responsible for a considerable mortality and morbidity all over the world, that affect more than 500 million people [142] . malaria is par excellence the main parasitic infection and it is caused by intracellular plasmodium parasites transmitted by mosquitoes of genus anopheles. approximately, 40% of world population lives in areas where malaria is transmitted, causing 300-500 million infections and 2.7 million deaths per year. in specific regions, such as sub-saharan africa, children below 5 years-old conform 90% of the total deaths from malaria [143] . an important epidemiological study of the prevalence of malaria in salomon islands revealed the high rate of asymptomatic patients, highlighting the need for a diagnostic tool with high sensibility and specificity to detect plasmodium [144] . this study relied on pcr and rapid diagnostic tests (rdt), which are more sensible than a simple inspection with a microscope, but also far more expensive. in order to find a sensible detection method with a lower cost, a dendrimer-based assay was approved in south korea [145] . the coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) could detect two specific antigens of malaria: histidine-rich protein ii (hrp2) and lactate dehydrogenase (ldh). flisa has good spectroscopic properties, such as photostability [146] , and a better performance than traditional elisa, enabling the quantification of the number of parasites in a sample even if they are present in low concentrations. accordingly, flisa method could be useful to detect asymptomatic cases at a modest price and with a high capacity. the process is depicted schematically in figure 7 . the role of dendrimers against helminthic schistosoma parasites has also been tested. the disease, known as schistosomiasis [147] , begins when the larvae form penetrates in the organism through the skin and settle in mesenteric and pelvic veins of the host, turning into the adult form. here, the female parasites lay eggs, which could be eliminated through feces or urine or lead to complications like granulomas or intestinal, hepatosplenic and urogenital damage. this highlights the need for an early diagnosis. wright et al. (2019) confirmed the promising activity of magnetic particles coated with g4-pamam-nh 2 to concentrate the schistosoma circulating anodic antigen (caa), resulting in a 200-fold improvement in caa limits of detection for lateral flow assays [148] . preventive measures like vaccines are key to stop the impact of schistosoma parasites in specific regions where they cause endemic infections. for example, infections by s. haematobium, s. japonicum, or s. mansoni affect over 200 million people worldwide [149] . lysine-decorated pamam dendrimers showed excellent behavior as vaccine vector, enhancing the immunoreactivity and efficacy of dna vaccine against s. japonicum infection [150] . along the same lines, anderson et al. (2016) developed a dendrimer-based vaccine platform which encapsulate antigen-expressing replicon mrnas and generate a protective response towards others parasites such as toxoplasma gondii, and relevant viruses like ebola and h1n1 influenza, with a single dose [151] . the vaccine nanoparticle comprised an ionizable g1-pamam dendrimer, a lipid-anchored peg and rna. these studies show the role of dendrimers in the development of new generation vaccines against different infections. specificity to detect plasmodium [144] . this study relied on pcr and rapid diagnostic tests (rdt), which are more sensible than a simple inspection with a microscope, but also far more expensive. in order to find a sensible detection method with a lower cost, a dendrimer-based assay was approved in south korea [145] . the coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) could detect two specific antigens of malaria: histidine-rich protein ii (hrp2) and lactate dehydrogenase (ldh). flisa has good spectroscopic properties, such as photostability [146] , and a better performance than traditional elisa, enabling the quantification of the number of parasites in a sample even if they are present in low concentrations. accordingly, flisa method could be useful to detect asymptomatic cases at a modest price and with a high capacity. the process is depicted schematically in figure 7 . leishmaniasis is a parasitic disease produced by leishmania protozoans that infects and multiplies in macrophage-rich organs and tissues of the reticuloendothelial system, mainly the liver and spleen. estimations indicate an increase of 1.5 to 2 million cases per year [152] . for decades, leishmaniasis has been treated with pentostam and glucantime, leading to drug resistance and serious side effects like pancreatitis. alternative broad-spectrum drugs with less toxicity, such as amphotericin b, have been used but they present disadvantages such as the high cost, low solubility, the side effects, and its less efficacy as antiparasitic agent. mannose-decorated g5-ppi dendrimers improved the activity of amphotericin b for the treatment of leishmaniasis, reducing the toxicity by increasing the targeting in macrophage-rich organs [153] . other nanocarrier, a dendritic-linear-dendritic hybrid based on peg and citric acid, figure 6c , also improved the solubility of amphotericin b (478 times) and reduced the in vitro/in vivo toxicity. it resulted as potent as free amphotericin and glucantime in reducing the parasite burden and number [103] . toxoplasmosis is a zoonosis caused by the ingestion of the parasite toxoplasma gondii. this disease has a chronic and silent course in the majority of population without immune system disorders, mainly causing symptoms such as low fever and muscular pain. this infection is usually treated with sulfadiazine [154] , which has two disadvantages: it requires a high dose of the drug, which can produce severe side effects like high fever or allergic reactions; and it cannot reach target tissues where the parasite is typically localized. cationic g4 pamam dendrimers and anionic g4.5 dendrimers efficiently solubilize sulfadiazine (up to 30 molecules per dendrimer) and improve the penetration into the parasite, thus greatly reducing the required sulfadiazine dose and localizing the drug in muscle and brain, where t. gondii is usually present [155] . the main dendrimer-drug interactions found were electrostatic, for cationic dendrimers, and hydrogen bonding, for anionic counterparts. furthermore, the anionic dendrimer showed intrinsic antiparasitic effect. other successful examples of antiparasitic dendrimers include pegylated pll dendrimers coated with chondroitin sulfate a, as targeted unimolecular micelles for the delivery of the antimalarial drug chloroquine phosphate [156] ; and pamam dendrimers decorated with ethynil estradiol against trypanosoma cruzi, the parasite responsible for chagas disease, where the g2 dendrimer is 8 times more effective than benznidazole at 24 h and 48 h (ic 50 = 1.25 µm) [157] . amoebae are eukaryotic protozoa extensively distributed in nature and human habitats, often acting as a host and reservoir of other microorganisms like giant viruses and some class of bacteria [158, 159] . amoebae infecting humans are classified as parasitic, such as entamoeba organisms, or opportunistic free-living amoebae, such as acanthamoeba spp., balamuthia mandrillaris, sappinia diploidea, and naegleria fowleri (known as the brain-eating amoebae) [160] . free-living amoebae do not require human infection for their life cycle, which presents two stages: trophozoite (active form) and cyst (inactive form). acanthamoeba and other free-living amoebae are responsible of serious diseases such as keratitis, encephalitis, and infections in immunocompromised patients in the central nervous system, skin, and lungs. fortunately, amoebae infections are relatively rare, although the high mortality associated with meningoencephalitis are of concern, mainly due to the late diagnosis and the lack of effective antimicrobial treatments [161] . low generation cationic carbosilane dendrimers bearing ammonium or biguanide moieties have shown strong amoebicidal activity against trophozoites and cysts of acanthamoeba spp., figure 6d (ic 50 = 2.4 mg/l; minimum cysticidal concentration mcc = 256 mg/l) [104, 162] . furthermore, they exhibit a synergistic effect with traditional drugs such as chlorhexidine, decreasing the required drug concentration 5-10 times using g1 dendrimers [163] . the dendrimers target the amoeba membrane and produce inhibition and cell death. in this context, dendrimers could be a promising therapeutic alternative to properly manage amoebae infections. prions (prp) are infectious pathogens that cause neurodegenerative transmissible diseases such as spongiform encephalopathies, after a long period of incubation from 2 to 10 years [164, 165] . prp sc are glycoproteins with an abnormal folding that are originated from a conformational change of normal prion proteins (prp c ), acquiring pathological properties [166] . prion diseases can occur for two reasons: the infectious prion agent can transmit the pathological folding to the prp c ; or the prnp gen mutates, leading to a genetic variation of the prion disease [167] . infectious prion diseases have gained importance since the epidemic bovine spongiform encephalopathy in united kingdom in 1986 that was transmitted to humans as a variation of the creutzfeldt-jakob prion disease [168, 169] . the spongiform encephalopathies are named after the shape the brain acquires at final stages of the disease, caused by an increase of vacuoles and the holes that appear in the tissue. it is a rare but severe pathology, which leads to neuronal loss and, eventually, dementia. nowadays, there are some pharmacological interventions that delay the progression of prion diseases; however, there is no effective treatment to stop or avoid it in a significant way [170] . in this field, nanomaterials such as dendrimers can exert a relevant activity, preventing the conversion of prp c to prp sc [171] . due to the high affinity of amine groups to prions, phosphorus dendrimers decorated with tertiary amines in their surface are promising agents in the therapy against prion diseases, figure 6e [105]. these dendrimers showed an inhibitory effect in the generation of prions (ic 50 = 10 (g3), 1.5 (g4) and 5 (g5) µg/ml) and they had anti-infectious action in vitro and in vivo for some spongiform encephalopathies, some of them causing creutzfeldt-jakob disease [105] . other dendritic families with even higher anti-prion activity include g4-pamam-nh 2 and g4-ppi-nh 2 (both ic 50 = 80 ng/ml) [172, 173] . for example, ottaviani et al. (2012) showed that ppi glycodendrimers, comprising maltose or maltotriose units, prevented the aggregation of prp and aβ(1-28) proteins due to the interference of dendrimers with the latency phase (nucleation) of the prion protein [174] . dendrimers can also be useful for the diagnosis of prion infections. usually, elisa is used to determine whether the causing agent of the disease is a prion, but this method has important limitations as it cannot distinguish between prion chains [175] . the nature of the prion agent will determine the etiology and course of the disease, changing the pathology parameters like the incubation period or the type of lesion [176] . importantly, different generation pamam and ppi dendrimers were capable of distinguishing between the different types of prions [177] . this study demonstrated that the susceptibility of a prion towards a particular dendrimer could be used to diagnose and predict the course of a disease. on the other hand, korri-youssoufi et al. developed an electrochemical biosensor comprising multiwalled carbon nanotubes modified with dendrimers and aptamers to detect prion proteins with a high sensibility (min. 0.5 pm) [178] . in conclusion, dendrimers constitute a promising research line for fighting against prion diseases as they could slow down their progression and allow a reliable diagnosis of the responsible etiological agent. dendritic nanosystems could have a principal role as excipients in the pharmaceutical industry, providing that they are compatible, safe and effective nanocarriers in several administration routes [179] . for example, ocular administration is still a challenge due to the unique physiology of the eye and the presence of numerous barriers. in this context, recent studies showed that dendrimers improved the time that a drug remains in the cornea after topical administration and they supply directed and sustained neuroprotection for retina [180] . moreover, dendrimers could act as dna vectors, providing safety and reducing cytotoxicity compared to the viral vectors and intraocular injections used at present [53] . dendritic polymers can easily improve the properties of different materials, such as cotton, to generate antimicrobial activity. for example, the addition of amine-functional dendrimers confers antibacterial activity against gram-positive bacteria (e. coli, p. aeruginosa) and gram-negative bacteria (s. aureus) and fungi (c. albicans) [181, 182] , even after several washing cycles. sepsis is a life-threatening response to an infection, which happens when the immune system overreacts and starts to damage the patient's own tissues and organs. peptide-decorated pamam dendrimers inhibited the acetylation of transforming growth factor β-induced protein, thus improving the mortality and organ damage in the septic mouse model [183] . additionally, gadolinium-containing g4 dendrimers can be used as mri diagnostic and prognostic biomarker of sepsis-induced acute renal failure [184] . these studies open up a field in the treatment and diagnosis of sepsis with a not very high cost and that could be protective against infectious pathogens in certain circumstances like during hospital stays, surgeries or risky exposures. the design of metallodendrimers is a promising field to explore, which can open new avenues in the fight against infectious diseases [185] . dendrimers are excellent platforms to control the attachment of a great variety of metal complexes with antimicrobial activity, such as silver(i), copper(ii), and zinc(ii), among others. the resultant metallodendrimers often exhibit a synergistic activity and improve the therapeutic response of the dendrimer or the metal complex alone. for example, demonstrated the impact of a single metal ion in the prevention of hiv infection [186] . a carboxylate-decorated g2 ppi dendrimer, which inhibited hiv-1 infection of hec-1a cells around 30%, reached 90% inhibition by attaching a single cu(ii) ion through its ethylenediamine core. the remarkable control on the dendritic structure enables an optimized antimicrobial activity, difficult to accomplish with other nanomaterials. the reader is referred to a recent review on this topic [185] . dendrimers are an innovative tool in the treatment, prevention and diagnosis of serious infectious pathologies, as summarized in table 1 . they emerge as a unique opportunity to overcome problems of traditional approaches, such as microbial resistance. nevertheless, the transition from the laboratory to the clinical practice still requires facing several challenges, such as the big scale production, the batch-to-batch consistency, the purity for clinical tests, or the regulatory obstacles. it should be kept in mind that most investigations have been conducted in experimental models in vitro or in animal models, so it is not possible to extrapolate these results to humans yet. however, the outstanding results obtained by vivagel ® in the prevention of viral and bacterial infections predict a brilliant future of dendritic materials in the fight against infectious diseases. [171] prevention of prp aggregation [174] diagnosis detect and distinguish between prion diseases [177] author the importance of infection control and prevention in anesthesiology emerging infectious disease prevention: where should we invest our resources and efforts? las enfermedades infecciosas emergentes y reemergentes, un tema de interés para todos climate change and infectious diseases: from evidence to a predictive framework climate change: challenges and opportunities for global health contact structure, mobility, environmental impact and behaviour: the importance of social forces to infectious disease dynamics and disease ecology infectious disease-related laws: prevention and control measures emerging infectious disease laboratory and diagnostic preparedness to accelerate vaccine development reassured diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes a guide to aid the selection of diagnostic tests drivers of irrational use of antibiotics in europe antibiotic resistance antimicrobial resistance: a global emerging threat to public health systems quantum dots in biological and biomedical research: recent progress and present challenges the consolidation of nanomedicine multifunctional nanoparticles for multimodal imaging and theragnosis combatting infections with nanomedicine advances in integrative nanomedicine for improving infectious disease treatment in public health designed synthesis and surface engineering strategies of magnetic iron oxide nanoparticles for biomedical applications nanogel: a versatile nano-delivery system for biomedical applications recent progress in dendrimer-based nanomedicine development discovery of dendrimers and dendritic polymers: a brief historical perspective introduction to dendrimers and other dendritic polymers dendrimers: a novel approach for drug targeting characterization of dendrimers a new class of polymers: starburst-dendritic macromolecules simplifying the synthesis of dendrimers: accelerated approaches dendrimer chemistry: synthetic approaches towards complex architectures boas, u. dendrimers, dendrons, and dendritic polymers: discovery, applications, and the future advances in the chemistry of dendrimers poly(amidoamine) (pamam) dendrimers: synthesis and biological applications poly(lysine) dendrimers and other dendritic molecules from naturally occurring monomers poly(carbosilane) dendrimers and other silicon-containing dendrimers poly(phosphorhydrazone) dendrimers and other phosphorus-containing dendrimers bis-mpa dendrimers and other dendritic polyesters a review on comparative study of ppi and pamam dendrimers the role of branch cell symmetry and other critical nanoscale design parameters in the determination of dendrimer encapsulation properties large dipole moments of phosphorus-containing dendrimers chemistry of multifunctional polymers based on bis-mpa and their cutting-edge applications dendrimers: a novel carrier system for drug delivery pamam dendrimers as efficient drug and gene delivery nanosystems for cancer therapy functionalized branched polymers: promising immunomodulatory tools for the treatment of cancer and immune disorders peptide glycodendrimers as potential vaccines for olive pollen allergy targeted nanosystems: advances in targeted dendrimers for cancer therapy nanovaccines formulation and applications-a review dendrimers for diagnostic applications dendrimers with tetrabenzoporphyrin cores: near infrared phosphors for in vivo oxygen imaging dendrimers for theranostic applications dendrimer-protein interactions versus dendrimer-based nanomedicine dendrimers as drug carriers: applications in different routes of drug administration dendrimers as drug delivery vehicles: non-covalent interactions of bioactive compounds with dendrimers association of chemotherapeutic drugs with dendrimer nanocarriers: an assessment of the merits of covalent conjugation compared to noncovalent encapsulation dendrimers for gene delivery-a potential approach for ocular therapy? pegylated pamam dendrimers as a potential drug delivery carrier: in vitro and in vivo comparative evaluation of covalently conjugated drug and noncovalent drug inclusion complex function oriented molecular design: dendrimers as novel antimicrobials dendrimers and dendritic polymers as anti-infective agents: new antimicrobial strategies for therapeutic drugs dendrimers-revolutionary drugs for infectious diseases application of dendrimers for the treatment of infectious diseases in dendrimer chemistry: synthetic approaches towards complex architectures bench-to-bedside translation of dendrimers: reality or utopia? a concise analysis dendrimer drugs for infection and inflammation synthesis of heterofunctional polyester dendrimers with internal and external functionalities as versatile multipurpose platforms dendronization: a useful synthetic strategy to prepare multifunctional materials virus entry paradigms virus classification-where do you draw the line? update in viral infections 2014 novel activities of safe-in-human broad-spectrum antiviral agents a systems approach to study immuno-and neuro-modulatory properties of antiviral agents dendrimers and antivirals: a review evaluations of unformulated and formulated dendrimer-based microbicide candidates in mouse and guinea pig models of genital herpes diversification of the prevention of sexually transmitted infections réservoirs cellulaires et tissulaires du vih-1: dynamique au cours de l'infection. virologie frequent detection and isolation of cytopathic retroviruses (htlv-iii) from patients with aids and at risk for aids accountability for the global health sector strategies the pharmacology of hiv drug resistance the potential of hiv-1 nanotherapeutics: from in vitro studies to clinical trials vivagel (spl7013 gel): a candidate dendrimer-microbicide for the prevention of hiv and hsv infection astodrimer sodium, dendrimer antiviral, inhibits replication of sars-cov-2 in vitro development of sulphated and naphthylsulphonated carbosilane dendrimers as topical microbicides to prevent hiv-1 sexual transmission triple combination of carbosilane dendrimers, tenofovir and maraviroc as potential microbicide to prevent hiv-1 sexual transmission carbosilane dendrons with fatty acids at the core as a new potential microbicide against hsv-2/hiv-1 co-infection dendronized magnetic nanoparticles for hiv-1 capture and rapid diagnostic conjugated anionic peg-citrate g2 dendrimer with multi-epitopic hiv-1 vaccine candidate enhance the cellular immune responses in mice immunohistology of infectious diseases antiviral drug discovery for the treatment of enterovirus 71 infections optimization of a class of tryptophan dendrimers that inhibit hiv replication leads to a selective, specific, and low-nanomolar inhibitor of clinical isolates of enterovirus a71 structure-activity relationship studies on a trp dendrimer with dual activities against hiv and enterovirus a71. modifications on the amino acid nanocarbon-based glycoconjugates as multivalent inhibitors of ebola virus infection synthesis of highly efficient multivalent disaccharide/[60]fullerene nanoballs for emergent viruses dendrimers functionalized with membrane-interacting peptides for viral inhibition peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in japanese encephalitis antiviral potential of 3 -sialyllactose-and 6 -sialyllactose-conjugated dendritic polymers against human and avian influenza viruses the healthy human microbiome bacterial-host interactions: physiology and pathophysiology of respiratory infection attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the eu and the european economic area in 2015: a population-level modelling analysis the significance of infection related to orthopedic devices and issues of antibiotic resistance review of the incidence and prognosis of pseudomonas aeruginosa infections in cancer patients in the 1990s cystic fibrosis and pseudomonas aeruginosa: the host-microbe interface inhibition and dispersion of pseudomonas aeruginosa biofilms by glycopeptide dendrimers targeting the fucose-specific lectin lecb adaptive and mutational responses to peptide dendrimer antimicrobials in pseudomonas aeruginosa urbańczyk-lipkowska, z. design and studies of multiple mechanism of anti-candida activity of a new potent trp-rich peptide dendrimers reduction toxicity of amphotericin b through loading into a novel nanoformulation of anionic linear globular dendrimer for improve treatment of leishmania major ultrastructural study of acanthamoeba polyphaga trophozoites and cysts treated in vitro with cationic carbosilane dendrimers cationic phosphorus-containing dendrimers reduce prion replication both in cell culture and in mice infected with scrapie diagnostic microbiology of the immunocompromised host resistance to antimicrobial peptides in gram-negative bacteria an overview of the antimicrobial resistance mechanisms of bacteria mesoporous silica nanoparticles decorated with polycationic dendrimers for infection treatment synthesis and in vitro evaluation of monodisperse amino-functional polyester dendrimers with rapid degradability and antibacterial properties the influence of surface modification on the cytotoxicity of pamam dendrimers antimicrobial activity of novel dendrimeric peptides obtained by phage display selection and rational modification rapid and selective discrimination of gram-positive and gram-negative bacteria by boronic acid-modified poly(amidoamine) dendrimer a potent and selective antimicrobial poly(amidoamine) dendrimer conjugate with led209 targeting qsec receptor to inhibit the virulence genes of gram negative bacteria pamam-dendrimer enhanced antibacterial effect of vancomycin hydrochloride against gram-negative bacteria development of a guinea pig model of chorioamnionitis and fetal brain injury micronutrients and intrauterine infection, preterm birth and the fetal inflammatory response syndrome mid-trimester preterm premature rupture of membranes (pprom): etiology, diagnosis, classification, international recommendations of treatment options and outcome inhibition of bacterial growth and intramniotic infection in a guinea pig model of chorioamnionitis using pamam dendrimers dendrimer-conjugated peptide vaccine enhances clearance of chlamydia trachomatis genital infection antimicrobial efficacy and mechanism of action of poly(amidoamine) (pamam) dendrimers against opportunistic pathogens injectable pamam dendrimer-peg hydrogels for the treatment of genital infections: formulation and in vitro and in vivo evaluation general epidemiology of invasive fungal disease international surveillance of bloodstream infections due to candida species: frequency of occurrence and antifungal susceptibilities of isolates collected in 1997 in the united states, canada, and south america for the sentry program. the sentry participant group the changing face of epidemiology of invasive fungal disease in europe tissue penetration of antifungal agents alastruey-izquierdo, a. the global problem of antifungal resistance: prevalence, mechanisms, and management antifungal therapy in european hospitals: data from the esac point-prevalence surveys hydrogel of ketoconazole and pamam dendrimers: formulation and antifungal activity poly (amidoamine) dendrimers increase antifungal activity of clotrimazole prolonged drug delivery system of an antifungal drug by association with polyamidoamine dendrimers polycationic dendrimers interact with rna molecules: polyamine dendrimers inhibit the catalytic activity of candida ribozymes importance of size-to-charge ratio in construction of stable and uniform nanoscale rna/dendrimer complexes the cellular delivery of antisense oligonucleotides and ribozymes diagnosis of invasive aspergillosis: recent developments and ongoing challenges glycobiology of human fungal pathogens: new avenues for drug development large scale parallel analysis of gene expression during infection-related morphogenesis of magnaporthe grisea innovative dendrischips®technology for a syndromic approach of in vitro diagnosis: application to the respiratory infectious diseases one health: parasites and beyond human intestinal microbiota: interaction between parasites and the host immune response biochemical and molecular mechanisms of drug resistance in parasites a review of recent patents on the protozoan parasite hsp90 as a drug target genome sequence of the human malaria parasite plasmodium falciparum a large proportion of asymptomatic plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of temotu province, solomon islands: challenges for malaria diagnostics in an elimination setting performance of coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) to detect malaria antigen development of a novel fluorophore for real-time biomonitoring system poly(amidoamine)-coated magnetic particles for enhanced detection of schistosoma circulating anodic antigen in endemic urine samples schistosomiasis: drugs used and treatment strategies pamam-lys, a novel vaccine delivery vector, enhances the protective effects of the sjc23 dna vaccine against schistosoma japonicum infection dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h1n1 influenza, and toxoplasma gondii challenges with a single dose leishmaniasis impact and treatment access surface-engineered dendrimeric nanoconjugates for macrophage-targeted delivery of amphotericin b: formulation development and in vitro and in vivo evaluation antibiotics for human toxoplasmosis: a systematic review of randomized trials nanomolar cationic dendrimeric sulfadiazine as potential antitoxoplasmic agent pegylated peptide dendrimeric carriers for the delivery of antimalarial drug chloroquine phosphate pérez-campos, e. in vitro activity of steroidal dendrimers on trypanosoma cruzi epimastigote form with pamam dendrons modified by "click"chemistry genetic and physiological interactions in the amoeba-bacteria symbiosis giant virus vs amoeba: fight for supremacy human infections caused by free-living amoebae pathogenic and opportunistic free-living amoebae: acanthamoeba spp., balamuthia mandrillaris, naegleria fowleri, and sappinia diploidea in vitro evaluation of the effectiveness of new water-stable cationic carbosilane dendrimers against acanthamoeba castellanii uah-t17c3 trophozoites in vitro anti-acanthamoeba synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst forms transmissible spongiform encephalopathies and prion protein interconversions the prion concept and synthetic prions prion disease a new variant of creutzfeldt-jakob disease in the uk prion disease genetics toward therapy of human prion diseases nanomedicine for prion disease treatment: new insights into the role of dendrimers elimination of prions by branched polyamines and implications for therapeutics influence of surface functionality of poly(propylene imine) dendrimers on protease resistance and propagation of the scrapie prion protein kinetics of amyloid and prion fibril formation in the absence and presence of dense shell sugar-decorated dendrimers rapid typing of transmissible spongiform encephalopathy strains with differential elisa adaptation of the bovine spongiform encephalopathy agent to primates and comparison with creutzfeldt-jakob disease: implications for human health differentiating prion strains using dendrimers electrochemical aptasensor of human cellular prion based on multiwalled carbon nanotubes modified with dendrimers: a platform for connecting redox markers and aptamers dendrimers as pharmaceutical excipients: synthesis, properties, toxicity and biomedical applications dendrimeric systems and their applications in ocular drug delivery the antimicrobial activity of the cotton fabric grafted with an amino-terminated hyperbranched polymer preparation, characterization, and antimicrobial property of cotton cellulose fabric grafted with poly (propylene imine) dendrimer. cellulose dual peptide-dendrimer conjugate inhibits acetylation of transforming growth factor β-induced protein and improves survival in sepsis dendrimer-enhanced mri as a diagnostic and prognostic biomarker of sepsis-induced acute renal failure in aged mice metallodendrimers as a promising tool in the biomedical field: an overview hiv-1 antiviral behavior of anionic ppi metallo-dendrimers with eda core the authors declare no conflict of interest. key: cord-031907-ilhr3iu5 authors: nan title: isev2020 abstract book date: 2020-07-15 journal: nan doi: 10.1080/20013078.2020.1784511 sha: doc_id: 31907 cord_uid: ilhr3iu5 nan introduction: primary tumours secrete large amounts of extracellular vesicles (evs), which play critical roles in preparing distant sites for a pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism. whether biogenesis, secretion rates and organotropism of evs are linked remains unknown. we have recently shown that ral gtpases control evs secretion in nematodes as well as in mouse mammary tumour cells (hyenne et al. jcb 2015) . since both rala and ralb are overexpressed or over-activated in various human cancers, we aimed to investigate the mechanisms by which these two gtpases control evs secretion and to determine how this affects metastatic progression, with a focus on breast cancer. methods: we used 4t1 mouse mammary carcinoma cells knocked down for either rala or ralb and determined their ability to induce orthotopic tumours and metastasis in a syngeneic mouse model. in vitro, we investigated ev secretion mechanisms using confocal and electron microscopy (em). evs were isolated either by uc or sec and characterized by nta, em, rna sequencing and mass spectrometry. the function of evs was assessed using a transwell assay. finally, we tracked the organotropism of fluorescently labelled evs and their capacity to induce pre-metastatic niches in mice. results: we show that rala and ralb promote lung metastasis of breast cancer cells in mice without affecting their invasive behaviours. we found that rala and ralb control the biogenesis of exosomes, by acting on the formation of multi-vesicular bodies though the phospholipase pld1. as a consequence, knock down of rala or ralb reduces the levels of secreted evs and modifies their rna and protein contents. these differences alter the pro-tumoural function of evs, as demonstrated with an in vitro permeability test. importantly, we show in vivo that evs from rala or ralb depleted cells have a decreased lung organotropism and, as a consequence, are less efficient in priming lung metastasis. finally, we show that high expression of rala or ralb is associated with a bad prognosis in human breast cancer patients. summary/conclusion: altogether, our study identifies ral gtpases as central molecules linking the mechanisms of evs secretion, their dissemination and their capacity to promote metastasis. nuclear proteins are recruited into tumour-derived extracellular vesicles upon expression of tetraspanin tspan8 introduction: tetraspanin tspan8 is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in most healthy tissues. although there is increasing evidence of an effect of tspan8 in invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular mechanisms of tspan8 are yet not fully understood. methods: to study the function of tspan8, we have established a fibrosarcoma model consisting of the parental cell line (ht1080) and its derivatives expressing tspan8 (ht1080-tspan8) either fused with different fluorescent tags or tag-free. life imaging, sted and storm microscopy were used to determine the intracellular localization of tspan8. co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect interacting partners of tspan8. small evs were purified from cell-conditioned media using sec and subjected to mass spectroscopy and ngs for a comprehensive comparative analysis of the proteome and transcriptome of the evs. results: the results of the proteome analysis showed a strong effect in the protein cargo of evs upon tspan8 expression. remarkably, among 20 of the most regulated targets, several histones and ribosomal proteins were enriched in the evs derived from ht1080-tspan8 cells. in line with this finding, life imaging and super-resolution microscopy revealed that, while a majority of the intracellular tspan8 is located on the cell membrane or intracellular membranes, -as it is known for other tetraspanins-, a portion of tspan8 is located on the nuclear envelope. in fact, several histones co-immunoprecipitated with tspan8, indicating their interaction. summary/conclusion: our data show that the expression of tspan8 in the tumour cells greatly impacts ev cargo. moreover, localization of tspan8 on the nuclear envelope, together with the enhanced recruitment of nuclear and ribosomal proteins to the evs, suggests a new mechanism of action of tspan8. introduction: extracellular vesicles (evs) modulate tissue development, regeneration and disease through the transfer of proteins, nucleic acids and lipids between cells. currently, the mechanism of cytosolic delivery of ev cargo is largely unknown. here, we unravel how evs release their cargo in recipient cells. methods: evs were isolated from gfp-cd63 and cd63-rfp expressing hek293 t cells by ultracentrifugation. gfp-cd63 and cd63-rfp evs were added to hek293 t cells stably expressing anti-gfp fluobody and fluorescently tagged galectin-3, respectively. clem and fluorescence microscopy were employed to visualize fluorescent markers in recipient cells. bafilomycin a1 and u18666a were used to inhibit endosomal acidification and cholesterol export from lysosomes, respectively. results: fluorescent galectin-3 which binds to betagalactosides present at the luminal side of endosomes was used to detect endosomal permeabilization. the absence of galectin-3 recruitment to endosomes in presence of cd63-rfp evs showed that endosome permeabilization is not the mechanism behind ev cargo release. gfp-cd63 ev addition to cells expressing anti-gfp fluobodies resulted in the formation of fluobody punctae, reflecting cytosolic exposure of ev cargo. subsequent clem of the fluobody punctae revealed endosomes as the underlying cellular compartments from where cargo release takes place. neutralization of endosomal ph and accumulation of endosomal cholesterol blocked cargo release, showing that ev cargo release is dependent on endosomal ph and cholesterol level. summary/conclusion: we show that genetically encoded cytosolic probes and clem offer an excellent approach to study both the mechanism and efficiency of ev cargo release in cells. we provide experimental evidence that ev cargo release occurs from endosomes. funding: the research was supported by dutch technology foundation ttw and netherlands organization for scientific research nwo, de cock-hadders stichting, and erasmus mundus namaste scholarship. healthy humans. to determine cut-off values for diagnoses, reference intervals of evs in plasma are needed. to establish such reference intervals, (1) a significant number of healthy donors should be included, (2) the presence of non-ev particles, residual platelets, lipoproteins, and haemolysis should be quantified, (3) flow cytometry signals should be in si units. the long-term aim of this study is to determine reference intervals of ev concentrations in human plasma within known dynamic ranges of the detectors. methods: (1) to establish a clinical reference, we collected blood from 224 healthy volunteers and prepared platelet-free plasma. (2) we performed quality control measurements including residual platelet count, serum index, and lipid spectrum. (3) we measured all samples by flow cytometry (apogee a60-micro) and used custom software (matlab r2018b) to automate calibration of all signals and data processing. scatter signals were calibrated in comparable units of scattering crosssection (nm2) and diameter (nm). fluorescence signals were calibrated in units of molecules of equivalent soluble fluorophores (mesf). results: the quality controls showed that most residual platelet concentrations ranged from 10^5 to 10^6 per ml except for one outlier, while the serum index and lipid spectrum were normally distributed. preliminary results of the first 21 donors analysed, show that within the ev size range of 162-1,000 nm, the median concentration of cd61+ evs is 3.9•10^8 per ml (apc>150 mesf), cd62p+ evs is 1.1•10^7 per ml (pe>83 mesf), cd235a+ evs is 5.2•10^7 per ml (pe>123 mesf), and cd45+ evs is 1.8•10^7 per ml (apc>91 mesf). summary/conclusion: we have developed reliable procedures for establishing reference intervals of ev concentrations, within a well-defined size and fluorescence intensity range, in human plasma by flow cytometry. we are currently applying these procedures to 224 samples to obtain, for the first time, ev reference intervals for human plasma. funding: pol, e. van der is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. introduction: the use of extracellular vesicles for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. isolation of a pure population of evs is the first step in studying their physiological functions since contamination of ev preparations with non-ev proteins can lead to incorrect conclusions about their biological activities. we have developed a new method termed tangential flow for analyte capture (tfac) using ultrathin nanomembranes to purify extracellular vesicles from pure, highly complex biological fluids such as blood plasma, resulting in a new method for extracellular vesicle purification. methods: the tff microfluidic devices are assembled through a layer stack process using patterned polydimethylsiloxane (pdms) sheets with the membranes sandwiched between top and bottom channels. undiluted plasma was tested in both normal flow filtration (nff) and tangential flow filtration (tff) modes on ultrathin nanomembranes. we have utilized a pore patterning technique called nanosphere lithography (nsl) that uses close-packing of nanoscale beads to pattern pores in an ultrathin membrane. results: nff of undiluted plasma resulted in a protein cake of~8 μm on the membrane, which prevented further transport across the membrane and evs were buried in the formed cake that were impossible to identify. however, tfac as a modified version of tff, led to capturing cd63 positive evs on the pores of the membrane with little evidence of protein fouling. nsl allows us to fabricate nanopockets (bowls with a single pore at the base) with various diameter, depth and pore diameter. using nsl, we further utilize nanopocket membranes to purify ev samples in tfac devices. this nanomanufacturing technology will allow us to pattern nanopockets with various diameter, depth and pore diameter which increases the efficiency of capturing of evs. furthermore, nanopockets can be modified and coated by specific ev markers to capture different subpopulation of evs based on size and affinity and further allows identifying the phenotypic subsets of evs by combining both size and affinity-based techniques. summary/conclusion: we have developed a method for the capture and release of nanoparticles such as evs called tfac using ultrathin nanomembranes. nsl technology can be applied to fabricate nanopockets with different physical and biochemical properties. utilizing nanopocket membranes in tfac system will allow us to separate different subpopulations of evs based on size and affinity. funding: this project was supported in part by the national science foundation (iip 1660177) to j.l.m and t.r.g., department of defence (ca170373) to j. l.m., and the national institutes of health (r35gm119623) to t.r.g. the addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small rna profile introduction: urinary extracellular vesicles (evs) and their rna cargo are a novel source of biomarkers for various diseases, however non-vesicular rna (e.g. associated with proteins) is also present within urine. this study aimed to identify the optimal method for isolating and enriching evs from human urine prior to small rna analysis. methods: three ev concentration methods, ultracentrifugation (uc); a precipitation-based kit (pk); and ultrafiltration (uf), were compared using 50 ml aliquots of pooled healthy volunteer urine. evs were then separated from protein contaminants by size-exclusion chromatography (sec). presence of evs was confirmed by transmission electron microscopy and western blotting, and evs were quantified using nanoparticle tracking analysis (nta). small rna content of concentrated urine and fractions obtained by sec (evs and proteins) were evaluated with the agilent bioanalyzer small rna chip. results: ev recovery following sec of concentrated samples was 35-78%, however particle: protein ratio (indicating ev purity) was approximately 10x greater after sec, regardless of the concentrating method used. uf+sec yielded the highest number of evs (per ml of urine) compared with pk+sec and uc+sec. small rna analysis from uf-concentrated urine (prior to sec treatment) identified peaks at 20 nucleotides (nt) and 60 nt. following sec, rna analysis indicated that ev fractions contained mostly small rna of~60 nt, whereas the protein factions contained small rna of 20 nt in size (consistent with mirnas). summary/conclusion: uf+sec provided the best balance between ev recovery (per ml urine) and particle: protein ratio. these data indicate that most of the 20 nt sized rnas, presumably mirnas, are not within evs in urine. ev preparations obtained after uc, pk and sec (regardless of concentrating method) contain pre-dominantly~60 nt sized small rna. these data outline the importance of removing non-vesicular proteins and rna from urine ev preparations prior to small rna analysis. funding: this research has been funded by petplan charitable trust. the use of rev for the optimization of ev separation and characterization by af4 introduction: the reproducibility of extracellular vesicle (ev) research has been hampered by the infinite number of separation and measurement techniques and the lack of appropriate reference materials (van deun et al., nat methods, 2017) . recombinant extracellular vesicles (rev) were developed as a biological reference material to overcome these limitations (geeurickx et al. nat comm 2019) . since rev have ev-like physical an biochemical characteristics and as they are trackable and distinguishable from sample ev they can be used as a spike-in material for data normalization and method development, and as a quality control. we used rev to optimize ev separation by asymmetrical flow field-flow fractionation (af4). methods: an af4 long channel column with a frit inlet driven by the eclipse system (wyatt) was coupled to a uv detector (shimadzu), mals dawn helios-ii (wyatt) and fluorescent detector (agilent). a spacer of 350 µm and a regenerated cellulose membrane of 10 kda were used. pbs supplemented with 0.02% nan3 was used as a running buffer. light scatter profiles and uv profiles were analysed as well as the fluorescent emission spectrum as the rev are gfp positive. fractions were collected and analysed by nanoparticle tracking analysis (nta) and western blot. we also estimated the repeatability and reproducibility of the af4 technique by light scatter and fluorescence profiles as well as the recovery efficiency by nta. results: in a first step 4*10^10 rev isolated from conditioned medium by a velocity gradient were injected in the af4 system to optimize the ev characterization protocol. later concentrated conditioned medium was spiked with 1*10^11 rev and injected in the af4 column to optimize ev separation from non-ev contaminants. the most optimal separation protocol was obtained by varying detector and cross-flow settings. this protocol shows elution of monodisperse particles at each time point and size distribution estimations by af4 correspond to size determination by nta and electron microscopy. summary/conclusion: we were able to optimize the af4 protocol for characterization of ev by af4 as well as for separation of ev from crude conditioned medium samples by using rev. we demonstrate that rev are suitable for method development and that af4 has high potential as an ev separation technique. comparative evaluation of ev isolation methods for ev subpopulation analysis in human urine, plasma and cell culture media liang dong, richard zieren, kengo horie, sarah amend and kenneth pienta the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: extracellular vesicles (evs) are membrane-enclosed particles of variable sizes that are released by any cell types to the extracellular space and are identified in all body fluids. a shortcoming in ev research is the lack of standardized isolation protocol for various sample types, resulting in heterogeneous outcomes in downstream analyses. in this study, we compared the ev isolation purity and efficiency among ultracentrifugation (uc), precipitation, sizeexclusion chromatography (sec) and a microfluidic tangential flow filtration device (exodisc) in human plasma, urine and cell culture media (ccm). methods: all evs were isolated by different isolation methods and characterized per misev2018 guidelines. single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence and nanoflow (nfcm) were used for single particle analysis. results: in ccm, total particle yield of exodisc was about 5 times higher than those of the rest three methods. size distribution differed per sample, but the ranges were comparable between the different isolation methods. the total protein amount of sec, precipitation and exodisc were similar which were 6-10 times higher than that of uc. uc had the highest particle-toprotein ratio followed by exodisc. precipitation and sec had low ratios. when loading 9ug of total protein for western blot, cd9, cd81, cd63 and flot1 could only be detected in uc and exodisc samples, but not precipitation or sec. sp-iris and nfcm demonstrated consistent purity findings. in urine, total particle yields of exodisc and sec were about 4 times higher than those of the rest two methods. the total protein amount of precipitation was 3 times higher than exodisc and sec, 10 times higher than uc. sec had the highest particle-to-protein ratio followed by uc and exodisc. precipitation had low ratios. in plasma, total particle yields of exodisc and precipitation were 100 times higher than those of the rest two methods. and so were the total protein amount. sec had the highest particle-to-protein ratio followed by uc. exodisc and precipitation had low ratios. western blot, sp-iris and nfcm demonstrated consistent purity findings in urine and plasma. to evaluate particle capture efficiency, we spiked a known number of density-gradient uc purified evs to each method and the recovery rate of uc, precipitation, exodisc and sec was 8.6%, 31%, 50.1% and 42%, respectively. summary/conclusion: the order of ev isolation purity in ccm is uc, exodisc, sec and precipitation. in urine it's sec, exodisc, uc and precipitation. and in plasma, this order is sec, uc, exodisc and precipitation. exodisc and sec have similar high isolation efficiency followed by precipitation. uc has low efficiency for ev capture. a capillary-channelled polymer (c-cp) fibre spin-down tip approach for the isolation and biomarker characterization of extracellular vesicles of ovarian cancer origin kaylan d. kelsey, rhonda r. powell, terri f. bruce and r. kenneth marcus clemson university, clemson, usa introduction: extracellular vesicle (evs) profiling has shown promise for disease detection through less invasive sampling (liquid biopsies). current diagnostic tools for ovarian cancer are invasive or only semiinformative. thus, use of evs could prove useful in early disease detection. demonstrated is a hydrophobic interaction chromatography (hic)-based capillarychannelled polymer (c-cp) fibre tip spin-down process for the isolation of ovarian cancer evs for use in diagnostics. methods: polyester c-cp fibre micropipette tips are employed in the isolation of evs from biological matrices including cell culture media, urine, and blood plasma in a spin-down solid-phase extraction (spe) approach. evs were isolated from standards of healthy urine origin and from skov3 cells (human ovary adenocarcinoma). the c-cp fibre isolation method (taking less than 5 mins and 10 μl sample volumes) preserves the morphology and functionality of evs as confirmed by sem, tem, and confocal fluorescence microscopy. results: the dynamic binding capacity of ev standards on a 1 cm pet c-cp fibre tip was found to be~7e11 particles (50%). the release of evs was confirmed using dot blot analysis for cd9, cd81, and cd63 tetraspanin proteins. immobilized evs were subjected to immunolabeling to allow the positive identification of a profile of ovarian cancer biomarker proteins (her2, cd24, egfr, epcam, ca125). summary/conclusion: this new ev isolation method introduces a simple capture mode, allowing for direct immuno-characterization and imaging on the fibre surface. this offers a unique and cost-effective opportunity for clinical analyses related to early detection and diagnosis of ovarian cancers (and others). the longterm goal is the creation of a rapid ev isolation and biomarker detection platform. funding: support from the national science foundation, eppley foundation for scientific research, gibson foundation, prisma health system and itor biorepository are gratefully acknowledged. development and optimization of purification method of exosomes by tangential flow filtration and ion-exchange chromatography approach tek lamichhane, ali navaei, sandeep choudhary, yonatan levinson and senthil ramaswamy cell & gene therapy r&d, lonza inc, rockville, usa introduction: extracellular vesicles (evs) such as exosomes have significant therapeutic potential, however, translation of ev-based therapies has been slowed down because of the biomanufacturing challenges. the isolation of evs, especially exosomes, is inherently challenging due to their small size, and heterogeneity in the mixture. the current isolation methods either have low recovery rate, aggregation, damaging the structure, time consuming or co-precipitation of contaminants. specially, it is difficult to process larger sized samples by centrifugation-based or immunoaffinity based methods because of the time and cost associated with these methods. methods: to overcome these roadblocks, we developed and optimized alternative purification techniques to isolate evs with higher purity and yield by using tangential flow filtration (tff) coupled with ion-exchange chromatography. we used bioreactor platform to produce evs from serum-free medium using bm-msc and hek293 s cells. bm-mscs were cultured on stirred tank bioreactors using microcarriers which provide a high surface area to volume ratio for the optimal cell growth and evs production. impellers were used to enhance mixing and maintain homogeneous culture conditions that can be easily monitored and controlled. results: depth filtration was applied for clarification of conditioned medium. we screened different types of filters during depth filtration for the best recovery of evs. tff membranes with different pore sizes were used to optimize the purity and yield of evs. because of the negatively charged nature of evs, anion exchange chromatography was chosen to capture and separate tff purified vesicles by their surface charge characteristics. we compared monolith based and membrane-based anion exchange columns to remove contaminants and purify exosomal fractions. the purity, size and presence of exosomal markers in isolated evs at each step of purification was evaluated by f-nta, nano-fcm and tetraspanins based elisa kits. summary/conclusion: in summary, our optimized methods improved the speed of isolation and purity of evs to the clinical grade. the production and isolation methods of exosomes that we developed here will be easily expandable to support large-scale and cgmp compatible bio-manufacturing in the future. use of an alternating current electrokinetic microelectrode chip to positively identify oncology, neurology, and infectious disease samples through plasma extracellular vesicle analysis juan pablo hinestrosa, jean lewis, david searson, orlando perrera, alfred kinana, heath balcer and rajaram krishnan biological dynamics, inc., san diego, usa introduction: cancer, neurological, and infectious diseases are leading causes of death, with early detection needed to improve outcomes. extracellular vesicles (evs) in the blood contain disease biomarkers, but current methods do not allow rapid analysis, and are often limited to one biomarker type. methods: we developed methods using alternating current electrokinetics (ace) to isolate evs from bloodbased samples and analyse the evs in situ with downstream assays for protein and nucleic acid biomarkers. we investigated if we could identify tuberculosis (tb) donor samples, protein and nucleic acid biomarkers in evs derived from cancer cell lines, and alzheimer's disease (ad) protein biomarker levels. results: ev isolation was confirmed by positive identification of the proteins cd9, cd63, and cd81 and measurement of ev mrnas using a direct rt-ddpcr assay. different disease models were analysed following method development. tb was used as a model for infectious disease, with 20 tb positive and 20 tb negative samples isolated on ace chips and analysed for levels of lipoarabinomannan and ag85. using a cut-off above the negatives, the auc of roc curves were 0.9975 and 1.0, respectively. for oncology, cancer cell lines were cultured and evs isolated from supernatants were spiked into human plasma for analysis. levels of pd-l1 or glypican-1 on evs were able to be measured following ace capture. additionally, dna and rna mutations known to be present in the cell lines were able to be detected using ngs and qrt-pcr, respectively. using ad samples as a neurological disease model, tau and phospho-tau t181 (p-tau t181) in human donor plasma were detected. in 8 ad and 5 healthy donor samples, p-tau t181 signal increased 134% in diseased versus healthy donors. summary/conclusion: ace chips are an innovative ev isolation and analysis platform that allow rapid disease sample detection in a wide range of studies with high sensitivity and specificity. introduction: colorectal cancer (crc) is one of the most frequent causes of cancer-related death. in the majority of crc patients, mutation in the apc gene is among the first genetic events. it leads to uncontrolled activation of the wnt pathway, and thus, to adenoma formation. some of these adenomas may then further progress to crc with the accumulation of other mutations. the 3d organoids maintain the cellular and genetic heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model of human cancers. here we analysed the ev-based communication between cancer cells and fibroblasts by i) identifying factors that substantially increase ev release from intestinal cancer cells and ii) by determining cargo components of evs that enhance tumour cell proliferation. methods: we used commercially available and patientderived fibroblasts and crc organoids. the medical research council of hungary approved all experiments with human samples and informed consent was obtained from patients. evs were studied by using antibody-coated beads, trps, nta, tem and western-blotting. we introduced apc mutation into wild type murine small intestinal organoids by crispr-cas9. results: we found that in crc patient-derived organoid cultures, small evs were preferentially secreted. we observed that apc mutation and the accumulation of the extracellular matrix component collagen critically enhanced ev secretion in intestinal organoids. furthermore, we showed that amphiregulin, present on fibroblast-derived ev, contributed to the maintenance of the intestinal stem cell pool and to cell proliferation in epidermal growth factor-dependent crc organoids. summary/conclusion: by proving the key role of mutations, collagen deposition and ev-bound amphiregulin in the release intensity and functions of the evs, we identified novel mechanisms in the progression if crc. funding: this work was funded by otka-nn 118018, by the national competitiveness and excellence program nvkp_16-0007 (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). prostate cancer-derived evs induce a pro-inflammatory phenotype in the stroma blandine f. victor a , dolores di vizio b , andrew chin c , tatyana vagner b , javier mariscal b , mandana zandian a , catherine grasso a , roberta gottlieb a and helen goodridge a a cedars-sinai medical center, los angeles, usa; b cedars-sinai medical center, west hollywood, usa; c cedars-sinai, los angeles, usa introduction: since 90% of patients with metastatic prostate cancer (pc) develop bone metastasis, identifying the mechanism that drives this process is essential. most ev research has been focused on the role of exosomes in mediating the pre-metastatic niche formation. however, most of these studies do not separate exosomes from large evs. our preliminary studies have demonstrated that a subclass of evs known as large oncosomes (lo) can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis and enhancing the migration and invasion of pc cells in vitro and tumour growth in vivo. the bone marrow is the initial site of entry into the bone microenvironment for disseminating tumour cells (dtcs) and is a rich source of nutrients that houses various cells types including bone marrow derived mesenchymal stem cells (bm-msc) and immune cells such as neutrophils, which have been implicated in metastasis. here we investigate the role of lo in reprogramming bm-mscs and driving bone metastasis in pc. methods: differential centrifugation, density gradient centrifugation, trps, rna sequencing, qpcr, migration assay, invasion assay, chemotaxis assay. results: we report that pc-derived evs induce distinct gene expressions changes in bm-mscs. rna-seq analysis identified inflammatory and immune regulating cytokines as top differentially expressed genes (deg) in bm-msc. moreover, lo induced a more potent response in bm-msc in comparison to exo and to non-treated controls. the genes enriched in lo treated bm-msc were associated with tumour cell motility. in agreement with the gene expression data, lo-treated bm-msc attracted migration and invasion of significantly more pc cells than exo -treated bm-mscs. in addition, the top deg expressed in ev treated bm-msc were identified as potent neutrophil chemoattractant proteins. in line with the rnaseq findings, the lotreated bm-msc demonstrated enhanced chemotaxis of neutrophils towards them in comparison with exo or vehicle-treated bm-msc. finally, we show that the observed differences in bm-msc's response to lo and exo may be mediated by distinct molecular pathways. summary/conclusion: the results from this study provide novel insight into how tumour derived evs alter the bone marrow microenvironment and how they may drive bone metastasis in prostate cancer. the αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis introduction: prostate cancer (prca) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sevs). sevs isolated from prca cell media, express the epithelial-specific αvβ6 integrin, a surface receptor for fibronectin and vitronectin. the αvβ6 integrin is not detectable in healthy prostate tissues but is highly expressed in prca. in this study, we hypothesized that αvβ6 in cancer sevs plays a crucial role in angiogenesis. methods: the sevs isolated from prca cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sev markers. the αvβ6-negative endothelial cells (hmec1) were incubated with αvβ6-positive sevs from prca cells to evaluate the transfer of αvβ6 by immunoblotting (ib) and facs. the effect of αvβ6-positive sevs on motility, tube formation and angiogenic signalling were assessed by boyden chamber, angiogenesis assays and ib in hmec1. results: we demonstrate for the first time that the αvβ6 is de novo expressed on endothelial cell surface by sevmediated protein transfer. prca cell-derived αvβ6-positive sevs, significantly promote the motility and the formation of nodes, junctions and tubules by hmec1. mechanistically, we demonstrate that hmec1 treatment with sevs from pc3 cells that endogenously express αvβ6, decreases pstat1(y701), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. hmec1 treatment with sevs isolated from pc3 cells harbouring crispr/cas9-mediated downregulation of β6, or shrna-mediated downregulation of β6, results in increased levels of pstat1(y701). this sev treatment also results in a decrease of survivin in sevs and hmec1. summary/conclusion: overall, our findings show that αvβ6 in prostate cancer sevs regulates a novel proangiogenic signalling pathway. funding: this study was supported by nci r01-224769 (lrl); p01-140043 (lrl and dca). introduction: advanced prostate cancer (pca) is asso-introduction: extracellular vesicles (evs) are secreted from cells, and carry bioactive proteins and rna cargoes. increasing numbers of studies have identified key roles for exosomes in driving aggressive tumour behaviours, including metastasis. however, the detailed mechanisms and responsible factors in the ev cargo are still unclear. recently, immune system has been considered as an important factor in establishing and maintaining metastasis. our goal is to identify the role of head and neck squamous cell carcinoma (hnscc) derived small evs (sevs) in tumour metastasis from the study analysing the effects of sevs on metastasis and tumour immunity. methods: sevs were collected from the conditioned media of hnsccs and purified through cushioned density gradient ultracentrifugation. an orthotopic mouse model was used for the assessment of tumour angiogenesis and metastasis. moc1 (inflammationinducing rarely metastasizing murine hnscc line) and moc2 (highly metastasizing murine hnscc line) were used for this study. moc1 and moc2 cells were transplanted into mice tongues orthotopically, and moc1/moc2 derived sevs or pbs were injected into the tumour twice in a week. two weeks after tumour transplantation, mice were sacrificed and tumours were sectioned for pathological analysis and facs analysis. in facs analysis, the number and species of tumour-infiltrated immune cells were measured. results: injection of sevs from moc1 into moc2 tumours suppressed frequency of lymph node metastasis. on the other hand, injection of sevs from moc2 into moc1 tumours didn't promote metastasis. cd4 positive t-cell distribution in moc2 tumour was significantly changed by moc1 sev injection. t-cell deprivation treatment using anti-cd3 antibody increased the frequency of metastasis in moc1-sev treated moc2 tumours. from the result of proteomics analysis on moc1 and moc2 sevs, immune-regulated proteins and metastasis-suppressing proteins were observed in moc1 sevs. summary/conclusion: we find that low aggressive hnscc sevs affect metastasis of highly metastasized hnscc, and also find that changing immune cell distribution may be related to the result. this mechanism and finding contributes to understanding the possible role of hnscc sevs on metastasis as well as on the tumour immune microenvironment. funding: this work was supported by the nih under award numbers r01ca163592 and r01ca206458 to aw. desmoglein 2 enhances squamous cell carcinoma tumour development through extracellular vesicles in an il-8/mir-146a-dependent mechanism introduction: the cadherin dsg2 is a stem cell marker that is upregulated in many different cancers, including sccs, and its expression correlates with poor prognosis. dsg2 activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. we recently showed that dsg2 enhances ev release, but the mechanism by which these evs modulate tumorigenesis is not fully understood. methods: we established scc cell lines stably expressing wildtype dsg2 or a palmitoylation deficient mutant, dsg2cacs. evs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qev izon column, and analysed by nta and bca. tumour xenografts were established by subcutaneous injection of 106 cells in scid mice and monitored up to 4 weeks. cytokine profiling was determined by antibody array. mirna expression was analysed by rnaseq and confirmed by qpcr. results: dsg2 enhanced ev release by 40% and promoted a~fivefold increase in tumour size in xenograft models. tumour growth was increased when control cells were treated with a single 20 µg dose of evs. loss of palmitoylation, which altered membrane trafficking of dsg2, reduced ev release (~55%) as well as tumour development. plasma evs from xenograft mice reflected in vitro particle counts from scc cell lines. a cytokine array analysis was performed revealing that dsg2-evs were enriched with pro-inflammatory cytokines including il-8, a potent chemotactic and angiogenic factor. most importantly, il-8 was surface-bound on evs. furthermore, rnaseq revealed mir-146a, a negative regulator of il-8, to be significantly downregulated in response to dsg2. treatment with mir-146a mimic or mir-146a inhibitor decreased or increased, il-8 expression in scc cells, respectively. summary/conclusion: in summary, dsg2 plays a key role in scc tumour development by increasing ev biogenesis and downregulating mir-146a, which in turn upregulates il-8 synthesis and release which can promote invasion, angiogenesis and metastasis. funding: nih r01 introduction: stem-and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (evs). indeed, cardiac progenitor cell (cpc)-derived evs have been shown to protect the myocardium against ischaemia/reperfusion injury in several preclinical models. however, the underlying mechanisms for cpc-ev-mediated cardioprotection remain elusive. here, we utilized the proteomic composition of cpc-evs released during different culture conditions, to unravel protein-mediated effects of cpc-evs on the endothelium. methods: cpcs were stimulated with calcium ionophore (ca ion-evs) or vehicle (control-evs) for 24 hours and evs were isolated from serum-free conditioned medium using size exclusion chromatography. ev concentration and size was assessed using nta. evs were functionally characterized based on endothelial cell activation by western blotting and an endothelial cell scratch assay. the proteomic composition of both ev conditions was profiled using mass spectrometry. cpc-ev knockouts for specific proteins were generated using crispr/cas9 technology. results: we found enhanced phosphorylation of erk1/2 and akt in endothelial cells and increased wound closure after stimulation with control-evs, but not after stimulation with ca ion-evs. proteomic analysis identified a total of 1411 ev-associated proteins, with 79 proteins uniquely expressed in control-evs. another 68 proteins were revealed as candidate proteins, based on their relative enrichment in control-evs compared with ca ion-evs. go analysis demonstrated that differentially expressed proteins were involved in vascular endothelial growth factor signalling, extracellular matrix organization and angiogenesis. to investigate the involvement of the individual candidate proteins on endothelial cell activation, knockout evs of multiple proteins were generated and functionally characterized. summary/conclusion: a specific set of ev proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to cpc-evs. generating knockout evs for each of these proteins will help to investigate their individual roles. this may lead to a better mechanistic understanding of the use of cpc-evs as therapeutics for cardiac repair. funding: erc-2016-cog-725229 evicare grant. hypoxia enhances the therapeutic potential of human cd34+ stem cell exosomes in ischaemic hindlimb repair ischaemic cardiovascular disease. we have previously shown that human cd34+ cell-derived exosomes (cd34exo) improve perfusion and function of the ischaemic tissues. hypoxia is shown to modulate the secretion and content of exosomes in both cardiovascular and cancer research. therefore, we hypothesized that hypoxia can modulate the content and regenerative efficacy of human cd34exo. methods: cd34exo were isolated from primary human cd34+ stem cells cultured under hypoxia (1.5% o2, or normoxia (20% o2, n-cd34exo) using density gradient ultracentrifugation. cd34exo size was measured using trps, nta, and dls and surface protein expression was determined using imaging flow cytometry. function of cd34exo was assessed using cell viability, migration and matrigel tube formation assays in vitro and a mouse hind limb ischaemia model (hli) in vivo. protein content of hypoxic or normoxic cd34exo was evaluated via lc-ms/ms and 2-d -dige followed by lc-ms/ms. results: we did not observe any significant differences in size or in quantity of exosomes secreted from h-or n-cd34 cells. both h-and n-cd34exo expressed cd9, cd81 and cd63 surface markers. interestingly, h-cd34exo significantly improved cell viability, migration and tube formation of huvecs in vitro compared to n-cd34exo. in the same line, h-cd34exo also significantly improved perfusion (ratio: 0.93 ± 0.05 v 0.77 ± 0.02) and prevented ischaemic limb amputation (0% v 37.5%) as compared to n-exo (p < 0.05; n = 7-8) in a murine (balbc nude) model of hind limb ischaemia. flow cytometry and confocal microscopy indicated that h-exo was uptaken by endothelial cells in the ischaemic limb. remarkably, we detected several proteins (including a fragment of hemopexin) and mirnas (mir-210) that could be responsible for the proangiogenic and beneficial function of h-cd34exo. we have also demonstrated that removal of surface proteins diminished the pro-angiogenic function of cd34exo. summary/conclusion: hypoxia enhanced the proangiogenic and regenerative potential of cd34exo, and thus, may represent a more efficient clinical strategy for cd34exo therapy. our research is clinically important to improve therapeutic angiogenesis in diabetic and cardiovascular patients with compromised stem cell populations. hyun-ji park a , jessica r. hoffman b and michael davis b a emory university, decatur, usa; b emory university, atlanta, usa introduction: exosomes, a subset of membrane nanovesicles, transfer cellular information by passing proteins and nucleic acids between cells. exosomes have been implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome synthesis abrogates the effects of cell therapy following cardiac injury. more importantly, increasing evidence indicates that mirnas (mirs) within exosomes serve as important signalling molecules to regulate inflammation, recruit stem cells, and repair diseased tissue. among exosomal mirs, mir-192 and −432 are known to decrease angiogenesis, cell migration, and increase inflammation in various types of cells. here, we investigated the inhibition of these negative mirs as a means to improve the reparative capacity of c-kit+ progenitor cell (cpcs) exosomes. methods: cpcs were isolated from three paediatric patients using magnetic-bead sorting. 2ʹ-o-methylated rna duplexes inhibited mir-192 and −432 expressions in cpcs. exosomes (inhexos) were isolated from mirinhibited cpc conditioned medium. mir expression in exosomes and cpc was quantified by qrt-pcr. migration and proliferation of mesenchymal stem cells (mscs) were assessed two days post-exosome treatment. for inflammation analysis, thp1 cells with/without tnfα exposure were treated with exosomes and the expression of il-6, −8, and −10 was quantified by qrt-pcr. finally, the angiogenic potential of inhexos was tested by tube formation of cardiac endothelial cells. results: inhibitor treatment of cpcs decreased exosomal mir-192 and −432 expression. treatment with inhexos enhanced msc migration and proliferation compared with normal cpc exosome (norexo). moreover, inhexos showed promising results for immune regulation, as tnfα-induced inflammation was decreased in thp1 exposed to inhexos for 4 h. however, tube formation capacity is slightly decreased (~20%) by inhexo compared to norexo. summary/conclusion: exosomes from mir-192 and −432-depleted cpcs may be a promising strategy for the treatment of various cardiac diseases, as they enhanced stem cell recruitment and proliferation, and regulated inflammation and angiogenesis. while other studies focus on boosting the reparative potential of exosomes by increasing positive mir and mrna cargo, the inhibition of negative mir in exosomes could be an overlooked strategy for the treatment of cardiac disease. endo-lysosomes as an alternative intracellular location for ev cargo delivery with disease relevance introduction: extracellular vesicles (ev) are lipidbilayer nanovesicles that carry macromolecules and act as paracrine vectors for cell-to-cell communication. the processes regulating ev biogenesis are largely known, whereas how ev cargo is delivered to recipient cells remains poorly understood. a simple mechanism proposed is direct ev fusion with the cell membrane that liberate cargo into the cytosol. in this study, we observed that cargo release occurs also at an alternative intracellular location and that this acquires a disease relevance. methods: ev were isolated by serial centrifugation and characterized. for uptake studies, ev were traced by labelling donor cells with a lipophilic dye or by overexpressing gfp-cd63. uptake was assessed by cytofluorimetry or by live confocal imaging. co-localization studies were performed with ectopic marker expression or by immune staining. protein-protein interaction was analysed by bi-molecular fluorescence complementation (bifc). prion-like transmission was studied using a pro-fibrillogenic tau fragment in donor cells and full-length tau in recipient cells. for quantification of subcellular localization, an automated algorithm based on machine learning was developed. lysosomal stress was monitored by nuclear translocation of tfe3 and lysotracker staining. antibodies directed against pathogenic epitopes of tau were employed to assess prionlike transmission. results: ev were taken up by recipient cells through an endocytic process and accumulated in endo-lysosomes (el). when cells were exposed to ev carrying a profibrillogenic tau, recipient cells accumulated tau within el by an autophagic process. direct interaction of ev-tau and cellular tau in el favoured the appearance of pathological epitopes. cells displaying this condition showed an increased el stress and cytotoxicity. summary/conclusion: in this study, for the first time we report that el represent a critical subcellular location where transcellular prion-like transmission mediated by ev of a neurodegeneration-associated protein occurs. thus, the degradative pathway most likely involved in the recycle of ev and endogenous proteins is highjacked in disease. these findings represent a novel mechanism for ev acting as vector for transcellular propagation of tau, which opens up new therapeutic interventions trying to halt the disease. funding: supported by gelu foundation. anti-human fab fragment of cd9 antibody prevents the endocytosis of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer of their cargos introduction: interfering with the mechanisms regulating intercellular communication mediated by extracellular membrane vesicles (evs) may find relevance especially in oncology where cancer cell-derived evs have an implication in the malignant transformation of tumour microenvironment. our laboratories recently demonstrated a novel intracellular pathway in which a fraction of endocytosed ev-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of rab7+ late endosomes entering into the nucleoplasmic reticulum. here, we have investigated the effect of a monovalent fab antibody against the tetraspanin cd9 (referred hereafter as cd9 fab), on the internalization of evs and nuclear transfer of their cargo proteins. chair: david r f. carter -oxford brookes university chair: neta regev-rudzi -weizmann institute of science methods: to monitor the intracellular transport of ev-associated proteins, we used bioengineered fluorescent evs containing cd9-gfp fusion protein from femx-i melanoma, sw480 colorectal cancer and bone marrow-derived mesenchymal stromal cells (msc) as donors and the same cell types as recipients. evs were enriched by differential centrifugation from 72 h serum-free conditioned media and characterized by zetaview nanoparticle tracking analysis, zetapotential and immunoblotting. cd9 fab was prepared from 5h9 hybridoma cells using the pierce fab purification kit. results: we previously demonstrated that silencing cd9 both in evs and recipient cells strongly decreased the endocytosis of evs and abolished the nuclear transfer of their cargos. here we show that cd9 fab significantly reduced the cellular uptake of cd9-gfp+ evs and the nuclear transfer of their proteins in melanoma, colorectal cancer and msc used as receptor cells in a dose-dependent manner. the effect on the nuclear transfer is probably a direct consequence of the endocytosis inhibition of evs. in contrast, the divalent, intact cd9 antibody stimulated both events. summary/conclusion: the effect of cd9 fab appears independent of the used ev-donor cell types or receptor cells, probably due to the widespread expression of cd9 both at plasma membrane and ev surface. in conclusion, by impeding intercellular communication in the tumour microenvironment, cd9 fab-mediated inhibition of ev uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic strategies. a bright, versatile reporter for multivesicular body trafficking and exosome secretion and uptake bong hwan sung, ariana von lersner, jorje guerrero, evan krystofiak, david inmann, roxanne pelletier, andries zijlstra, suzanne ponik and alissa weaver vanderbilt university, nashville, usa introduction: live imaging of exosomes is one of the required tools to understand the function of exosomes. our previous live-cell reporter, phluorin-cd63 allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. however, there were some caveats to its use, including dim fluorescence and the inability to make cell lines that stably express the protein. methods: a stabilizing mutation, m153 r is incorporated in the phluorin moiety and now exhibits stable expression in cells and superior monitoring of exosome secretion. a dual-tag reporter was created by incorporating a further ph-insensitive red fluorescent protein, mscarlet to the c-terminus of phluo_m153 r-cd63. cancer cells stably expressing the constructs were imaged using a variety of microscopy techniques in vitro as well as in vivo. purified small evs labelled with phuo_m153 r-cd63 were imaged using immunogold transmission electron microscopy (tem) and quantitated for the half-life in the blood circulation using flow cytometry. results: phluo_m153 r-cd63 and phluo_m153 r-cd63-mscarlet are exclusively detected in exosomeenrich small ev preparations. immunogold tem visualizes the phluo_m153 r tag is located on the surface of small evs. live cell imaging reveals phluo_m153 r-cd63-positive puncta left behind migrating cells suggesting the deposition consists of exosomes. those puncta and trails are not only positive for exosome markers such as cd63, alix, and tsg101 but also correspond to small evs observed by a scanning electron microscopy. the dual-tag reporter allows visualization of the exosome lifecycle, including multivesicular body (mvb) trafficking, mvb fusion, exosome uptake and endosome acidification. summary/conclusion: using phluo_m153 r-cd63 construct, we demonstrate superior visualization of exosome secretion in multiple contexts and a role of exosomes in promoting leader-follower behaviour in collective migration by observing that exosomes are secreted at the front of migrating cells and left behind in exosome trails. the dual-tag reporter allows visualization of the entire exosome lifecycle. we anticipate that these reporters will be broadly useful to investigate regulation and functions of exosome secretion and uptake in diverse physiological conditions. funding: r01gm117916, r01ca206458, 3u19ca1795 14-05s1, r01ca216248. uncovering novel genes regulating ev-mediated functional rna transfer using a crispr/cas9-based reporter system introduction: extracellular vesicles (evs) play a pivotal role in intercellular communication through functional transfer of bioactive cargo, including rna molecules. despite increasing interest in ev-mediated rna transfer, our understanding of the pathways and mechanisms regulating ev-mediated rna delivery and processing is limited due to a lack of suitable readout systems. we recently developed a novel crispr/cas9-based reporter system that allows study of ev-mediated rna transfer at single-cell resolution. here, we further validate this system by studying the role of known targets involved in ev uptake and intracellular membrane trafficking, and subsequently employ this system to uncover various novel genes that play a regulatory role in functional rna transfer. methods: we employed a novel crispr/cas9-based stoplight reporter system, in which egfp expression is activated upon functional delivery of targeting single guide rnas (sgrnas) stably expressed by donor cells. intercellular functional rna transfer was assessed by measuring egfp expression in acceptor cells using fluorescence microscopy and flow cytometry after direct co-culture, transwell co-culture, and upon addition of isolated evs. potential roles of various genes in intercellular rna transfer were assessed by rnaimediated target knockdown in acceptor cells, prior to co-culture experiments. rnai knockdown was confirmed by qpcr analysis. results: a significant activation of egfp expression was observed in acceptor cells after direct co-culture and transwell co-culture with donor cells expressing sgrnas, as well as after addition of evs from cells expressing sgrnas. reporter activation was substantially decreased after knockdown of multiple targets involved in ev uptake through endocytosis and/or intracellular membrane trafficking. based on these results, a potential role of various novel genes in intercellular rna transfer was studied in acceptor cells. these experiments uncovered various novel targets involved in ecm binding, endocytosis, intracellular membrane trafficking, as well as various rho gtpase interactors. summary/conclusion: we previously demonstrated a crispr/cas9-based reporter system that allows the study of functional delivery of small non-coding rnas with single-cell resolution. here, we show that this novel approach allows the study of specific genetic targets and pathways in ev-mediated functional rna delivery, and unravel the regulatory pathways that dictate the underlying processes. quantitative characterization of extracellular vesicle uptake and content delivery within mammalians cells gregory lavieu a , emeline bonsergent b , eleonora grisard c and clotilde théry d introduction: extracellular vesicles (evs), including exosomes, are thought to mediate intercellular communication through the transfer of biomolecules from donor to acceptor cells. occurrence of ev-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. methods: we developed a cell-based assay in which evs containing luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. measurement of luciferase activity associated with acceptor cells revealed ev uptake efficacy. additional cell fractionation procedure that separates membranes from cytosol revealed the occurrence of ev-content release within the cytosol of acceptor cells. results: results from dose-responses, kinetics, and temperature-block experiments suggest that ev-uptake is limited (1% spontaneous rate at 1h), does not depend on bona-fide ev-receptor, at least for the tested acceptor hela cells. yet, further characterization of this limited ev-uptake, through cell fractionation that separates membranes from cytosol, revealed the occurrence of ev-content release within the cytosol of acceptor cells. cytosolic release is inhibited by bafilomycin-a and overexpression of ifitm proteins, which prevent virus content delivery. summary/conclusion: our results show that ev-content release requires endosomal acidification and suggest the involvement of membrane fusion. funding: anr18-ce15-0008-01 and arc pja 20171206453 and pga1 rf20180206962. introduction: glioblastoma is a highly malignant brain tumour with a poor prognosis. its ability to develop therapeutic resistance result in devastating clinical outcomes. to solve the intractable problem, we need highly sensitive diagnostics that can detect the molecular changes during treatments. extracellular vesicles (evs) can be a potential biomarker to monitor treatments and the host cell ev mapping can better reflect molecular changes in the tumour immune microenvironment. we have developed a droplet-based single ev protein sequencing platform that overcomes limitations of current bulk measurement technologies, which make it difficult to discover a rare ev population in the presence of high background. methods: we multiplex protein measurements to profile hundreds of proteins at a time by using an antibody-dna conjugate and sequencing. we barcode each ev in droplets and make amplicons that are comprised of both ev barcodes and antibody barcodes for sequencing. barcoded antibodies are made using tco-tetrazine click reaction and evs are labelled with these barcoded antibodies. the labelled evs are encapsulated into droplets with barcoded beads that serve as a template for ev barcodes. we then perform extension to make amplicons that contain both ev barcodes and antibody barcodes for sequencing. results: we successfully fabricated barcoded beads using a split-pool approach and validated by observing a fluorescence decrease of the sybr green after dna strand denaturation. we used a 3-channel droplet maker to encapsulate barcoded beads, single ev, and master mix into droplets. close packing of barcoded beads allowed >95% encapsulation into droplets. both droplet and tube-based methods achieved a similar high amplification efficiency (ct < 30 for 300 evs). we confirmed the amplicon size by running a gel, which showed the right amplicon size (~150 bp) from the droplet and tube prepared samples and no signal from the negative control. summary/conclusion: the droplet-based single ev profiling platform has the ability to identify rare immune ev subtypes in the peripheral blood, which would otherwise be impossible to detect due to the copresence of abundant normal evs. this cutting-edge technique has the potential to revolutionize treatment monitoring of high-cost immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine capabilities. funding: schmidt science fellows, in partnership with the rhodes trust po1 ca069246, ro1 ca204019, r21 ca236561 quantbio graduate student award at harvard university. introduction: in this study, we compared four orthogonal technologies for sizing, counting, and phenotyping of evs. the platforms were: single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence, nanofcm nanoflow (nf), nanoparticle tracking analysis (nta) with fluorescence, and microfluidic resistive pulse sensing (mrps) . results from these platforms were compared with results from standard ev characterization techniques such as transmission electron microscopy (tem) and western blot (wb). methods: human t lymphocyte h9 (high cd81, low cd63) and promonocytic u937 (low cd81, high cd63) cells were chosen for their distinct tetraspanin profiles without abnormalities that might result from genetic manipulation. evs were isolated from culture conditioned medium (ccm) by differential ultracentrifugation (duc) and size exclusion chromatography (sec) and characterized per misev2018 guidelines. synthetic particles (silica and polystyrene spheres) with known concentrations and mixed size distributions were also tested. results: particle counts from nf and mrps were consistent, while nta detected approximately one order of magnitude lower for ccm derived evs, but not for synthetic particles. sp-iris events could not be used to estimate particle concentrations. for sizing, nf, mrps, and sp-iris returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. nta detected a population of particles with a mode diameter above 100 nm. additionally, sp-iris, nf, and mrps were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. finally, for tetraspanin phenotyping, the sp-iris platform in fluorescence mode and nf were able to detect at least two markers on the same particle. summary/conclusion: based on the results of the study, we can draw conclusions about existing singleparticle analysis capabilities that may be useful for ev biomarker development and mechanistic studies. funding: this project is funded by mh118164 and ug3ca241694. importance to ev organotropism. yet, most techniques rely on bulk characterization, or are severely restricted by the diffraction limit. the exoview r100 (nanoview biosciences) combines interferometry, immunocapture, and immunofluorescence, introduced as an alternative technique to multiplex protein detection on single evs below the limit of diffraction. here, we use this technique to characterize tetraspanin multiplexing on evs and to identify spatial patterning of tetraspanins using steric hindrance of antibodies (abs). methods: evs were isolated from conditioned media from skov-3 cell culture or human serum. evs were incubated overnight on chips to allow immunocapture by anti-cd9, anti-cd63, or anti-cd81. chips were then incubated with three fluorescent abs against the same epitopes and imaged on the exoview r100. following concentration optimization, evs were tested after preincubating with carboxy-fluorescein diacetate succinimidyl ester (cfse) or fluorescent abs against tetraspanins. results: using different concentrations of evs, binding curves could be fit to characterize binding kinetics of abs. maximum concentration of evs could be identified that minimized fluorescent overlap. bright-field interferometry (detection limit~50 nm) distinguished 10x fewer bound evs than fluorescent detection, while pre-labelling evs with cfse produced 10x more detectable evs than immunofluorescence. interestingly, evs captured by one tetraspanin did not necessarily show high fluorescent detection of the same tetraspanin. upon pre-incubating evs with a single ab, vastly different expression profiles were identified, indicating significant steric hindrance between abs. furthermore, pre-incubating evs with anti-cd9 ab significantly decreased detection of cd81 with less impact on cd63. this discrepancy indicated possible spatial patterning of tetraspanins with cd9 and cd81 closely colocalizing on the ev surface. summary/conclusion: this combination of interferometry, immunocapture, and immunofluorescence produces unique information about size distribution of evs and single ev protein profile. this data corroborates that evs have distinct subpopulations of tetraspanins and indicates that tetraspanins may be spatially patterned. regulation of liver homoeostasis, regeneration and diseases by mesenchymal stem cell-derived apoptotic extracellular vesicles university of pennsylvania, philadelphia, usa introduction: billions of cells undergo apoptosis and produce apoptotic extracellular vesicles (apopevs) each day, whereas the roles of apopevs in regulating the organismal health and disease remain poorly understood. mesenchymal stem cells (mscs) emerge as critical contributors to tissue homoeostasis, while mscs suffer from apoptosis in regenerative transplantation. in this study, we investigated the function and mechanisms of msc-derived apopevs in regulating the organismal homoeostasis. methods: fas mutant (fasmut) and caspase 3 knockout (casp3-/-) mice were applied for apoptotic and apopev deficiency. mouse bone marrow mscs were cultured and apoptosis was induced by staurosporine (sts). msc-derived apopevs were collected by serial centrifuges and were infused into mouse circulation via caudal vein. tracing of apopevs were performed by radioisotope or fluorescent labelling. liver homoeostasis was evaluated at the histological and functional aspects. liver regeneration was induced by partial hepatectomy (phx). acetaminophen (apap) was used to establish acute liver drug injury. high-fat diet (hfd) was used to establish type 2 diabetes (t2d) and non-alcoholic fatty liver disease (nafld). results: after systemic injection, msc-derived apopevs migrate to liver and can be uptaken by liver macrophages and hepatocytes. fasmut and casp3-/mice develop hepatomegaly with structural disorders, which particularly reveals hepatocyte polyploidization. furthermore, fasmut and casp3-/-mice demonstrate liver glucose and lipid metabolic disorders. importantly, msc-derived apopev infusion significantly rescues structural and metabolic dysfunction in fasmut and casp3-/-mice. mechanistically, apopevs use the soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor (snare) protein for interactions with recipient organelles thus transferring signalling molecules. moreover, msc-derived apopev infusion promotes liver regeneration after phx, prevents apap-induced liver injury, and ameliorates nafld in t2d. summary/conclusion: msc-derived apopevs serve as crucial regulators of liver homoeostasis, regeneration and diseases. these findings indicate potential significant roles of apopevs in maintaining the organismal health and in developing therapeutics for diseases. (msc-sevs) mediate osteochondral regeneration in rats. however, the therapeutic effects of these msc-sevs/exosomes in restoring the mechanical competence of the repaired cartilage for joint function in a clinically relevant animal model remain to be addressed. to investigate this, we compared the structural and mechanical properties of the repaired cartilage in a rabbit model after intraarticular administration of msc-sevs and hyaluronic acid (ha) with that of ha alone, which is widely used as visco-supplementation. methods: bilateral osteochondral defects were surgically created on 17 rabbits. immediately after surgery and at days 7 and 14 post-surgery, 9 rabbits received 1ml injections of 200 µg msc-sevs and ha in both knees, and 8 rabbits received 1-ml injections of ha in both knees. at 6 and 12 weeks, macroscopic evaluation, histological scoring and compressive testing at different points on the repaired cartilage were performed. results: defects treated with msc-sev/ha showed improvements with time in macroscopic and histological scores and mechanical properties than defects treated with ha alone. in contrast, ha treated defects showed some repair at 6 weeks, but this was not sustained, as evidenced by significant deterioration of histological scores and a plateau in mechanical properties from 6 to 12 weeks. by 12 weeks, the msc-sev/ha repaired tissues demonstrated significantly better macroscopic score (10.33 vs 7.5; p < 0.001) and histological score (21.87 vs 11.11; p < 0.001). mechanical strength as measured by the young's modulus was significantly higher in the msc-sev/ha repaired cartilage than that in ha repaired tissues [defect centre (13.41 vs 3.95mpa; p = 0.001) and overall periphery (12.22 vs 3.37mpa; p = 0.012], and approximated that of the adjacent native cartilage. summary/conclusion: our findings demonstrated that msc-sevs and ha not only improved tissue morphology of the repaired cartilage but also promoted functional mechanical competence. this study establishes a clinically translatable protocol for use of msc-sevs for cartilage repair. introduction: mesenchymal stromal/stem cell (msc)exosome (mex) treatment has shown considerable promise in experimental models of bronchopulmonary dysplasia (bpd) and pulmonary hypertension (ph). mechanisms by which mex afford their beneficial effects remain incompletely understood and here, we embark into investigating them through assessment of mex biodistribution and impact on immune cell heterogeneity. methods: newborn fvb mice were exposed to hyperoxia (hyrx, 75% o2) at birth and returned to room air at postnatal day (pn) 14. mice received a bolus mex dose at pn4. adoptive transfer studies were used to determine the role of mex-educated myeloid cells in vivo. mice were harvested at pn4, 7, 14, or 28 to characterize mex biodistribution and for assessment of pulmonary parameters. results: mex therapy effectively ameliorated core features of hyrx-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. exercise capacity testing and assessment of ph showed functional improvements following mex therapy. biodistribution studies demonstrated that mex localize in the lung, where they interact with lung monocytes/macrophages. whole lung mass cytometry (cytof) revealed that mex treatment promotes a pro-homoeostatic shift in lung immune cell apportion, replenishing the early hyrx-induced depletion in pulmonary cd45+ immune cells, restoring alveolar monocyte and macrophage populations and suppressing cellular inflammation. ex vivo and in vivo analysis showed that mex promotes a "pro-resolving" ccr2-monocyte phenotype. notably, adoptive transfer of mex-educated bone marrow-derived myeloid cells (bmdmy), but not naïve bmdmy, restored alveolar architecture, blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss. summary/conclusion: mex treatment ameliorates core features of experimental bpd, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating ph and improving exercise capacity. the beneficial actions of mex are associated with modulation of immune cell phenotypes, arising from mex-monocyte interaction. furthermore, adoptive transfer of mex-educated bmdmy rescued, at least in part, alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary blood vessel loss. funding: this work was supported in part by an american thoracic society foundation grant (grw); the little giraffe foundation (grw); charles h. hood foundation major grants initiative (sk), nih r01hl146128 (sk) and united therapeutics research grant (sk and sam). immunomodulatory small extracellular vesicles derived from mesenchymal stem cells: a potential cell-free therapy for acute and chronic pulmonary vascular diseases introduction: vascular inflammation plays a critical role in acute respiratory distress syndrome (ards) and pulmonary arterial hypertension (pah). despite decades of research, there is no curative therapy for either condition. mesenchymal stem cells (mscs) have shown preclinical efficacy, mediated by release of extracellular vesicles. hence, msc-derived small extracellular vesicles (sevs) can harness the benefits of mscs with advantages in cost and safety. this study aims to evaluate the immunomodulatory effects of sevs in preclinical ards and pah. methods: msc-sevs were characterized by nanoparticle tracking analysis, electron microscopy and western blot. live fluorescence imaging measured in vitro and in vivo distribution of sevs. using a lipopolysaccharide (lps)-induced mouse model of acute lung injury (ali), a time course study of inflammatory response guided endpoint analyses. cell count and cytokines were measured in bronchoalveolar lavage fluid (balf) and histological lung injury was assessed. in ali mice, saline, mscs, msc conditioned media or sevs were administered 0.5 h post-lps. using a monocrotaline (mct)-induced rat model of pah, animals received saline or sevs at day 3. haemodynamic changes and right ventricular hypertrophy were evaluated at 3 weeks. results: msc-sevs were 72 nm in size with cd63/81 expression. pkh26-labelled sevs were taken up by endothelial cells. in the ali time course study, cell count and il1b in balf peaked at 24h post-lps, whereas il6 peaked at 10h. histology showed significant intra-alveolar cell infiltrate at 72h. msc conditioned media attenuated il1b in balf, whereas a trend towards reductions in il1b and cell count were seen from delivery of mscs and sevs. using fluorescence imaging, lung accumulation of dir-labelled sevs was highest when administered 1 h post-lps as compared to 5 h, 10 h or 24h. for pah rats, sevs reduced right ventricular systolic pressure (51.4 ± 8.3 mmhg) as compared to control (68.1 ± 0.4 mmhg; p = 0.012), whereas no changes were observed for right ventricular remodelling. summary/conclusion: these findings demonstrate the potential of msc-sevs to be used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases. additional live and ex-vivo biodistribution studies will determine optimal timing of sev administration, tissue distribution and clearance in both ali and pah. changes in extracellular vesicle protein cargo after pro-inflammatory priming of umbilical cord mesenchymal stem cells (ucmscs) have been shown to suppress inflammatory responses in studies of autoimmune diseases. these therapeutic effects can be attributed to paracrine signalling, by which extracellular vesicles (evs) are one of the essential components. this study looks at how the culture conditions of ucmscs affects the type of evs they secrete. it also aims to identify an ev population with an anti-inflammatory potential for the treatment of autoimmune diseases. methods: ucmscs were isolated and culture expanded in a quantum® cell expansion system, then grown at 21%o2, 5%o2 and primed with a pro-inflammatory cocktail. evs were isolated from ucmsc conditioned media by differential ultracentrifugation using a sucrose cushion and characterised by transmission electron microscopy and nanoparticle tracking analysis. ev markers were analysed using a europium-based immunoassay, macsplex exosome detection kit and immunoblotting. a proximity-based extension assay was used to identify 92 inflammatory proteins in the evs. results: there was no difference in evs cultured at 21%o2, 5%o2 or with pro-inflammatory conditions when analysed for size and morphology. all evs displayed the tetraspanin markers (cd9/63/81) and internal proteins (alix, hsp70). evs from primed cells showed a > twofold increase of 5 cc chemokines and a > sixfold increase in cxcl5 and csf-1. protein cargo did not differ in evs from 21%o2 and 5%o2. summary/conclusion: this study showed that proinflammatory culture conditions alter ev protein cargo, evidenced by the increased production of chemotactic and angiogenesis associated proteins. upcoming rnaseq analysis will show if ucmsc culture conditions also affect mirna expression in evs. ongoing functional studies will determine how changes in ev cargo correlates with changes in t-cell proliferation and polarisation. funding: this work is fund by the orthopaedic institute ltd, keele university and the rjah orthopaedic hospital charity. alzheimer's disease biomarkers in plasma extracellular vesicles of neuronal origin correlate with brain pathology in mice introduction: multiple studies have shown that neuronal-derived extracellular vesicles (ndes) in blood contain alzheimer's disease (ad) biomarkers, especially tau. however, the convergent validity of tau in blood ndes in relation to brain pathology is yet to be determined. to address this, we measured total and phosphorylated tau levels in matched nde and brain tissue samples from ad mouse models. methods: we collected the cortex, hippocampus and plasma of 4 2xtg-ad, 15 3xtg-ad, and 9 wild type (total of 28 mice; 14 female, 14 male; age: mean = 8.17, sd = 1.61, 5-11 months) . plasma samples were collected retro-orbitally for 5 weeks and at euthanasia via heart puncture. ndes from the pooled serial blood collections (nde1) and the single endpoint (nde2) were immunocaptured by targeting the neuronal marker l1cam. we measured human total tau and pthr181-tau (p-tau) in ndes and cortex and hippocampus homogenates using a luminex multiarray. results: overall, there were strong positive correlations for both total tau and p-tau between ndes and brain tissues across mice types. total tau in ndes showed positive correlations with levels in the cortex and hippocampus (r = 0.6885 and 0.7026, p < 0.0001, cortex vs nde1 and nde2; r = 0.4958, p = 0.0073, hippocampus vs nde1; r = 0.6058, p = 0.0006, hippocampus vs nde2). levels of p-tau in nde1 showed positive correlations with levels in the cortex (r = 0.4640, p = 0.0155) and hippocampus (r = 0.6086, p = 0.0008); however correlations were not observed for nde2 (r = 0.1152, p = 0.6273 vs cortex; r = 0.0531, p = 0.7926 vs hippocampus). summary/conclusion: tau levels in circulating ndes reflect levels in cortex and hippocampus across ad model mice, supporting their convergent validity as "liquid biopsy" biomarkers for ad. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. exosomal ceramide mediates neurotoxicity of amyloid beta (aβ) in alzheimer's disease. ahmed elsherbini a , simone crivelli b , alexander kirov c , michael dinkins d , zhihui zhu a , haiyan qin a , sanjib karki a , priyanka tripathi a and erhard bieberich a a university of kentucky, lexington, usa; b university of kentucky, lexington, usa; c augusta university, augusta, usa; d augusta university, augusta, usa introduction: amyloid beta is a pathologic hallmark of alzheimer's disease (ad), however, the mechanism of aβ neurotoxicity is not fully understood. it has been reported that exosomes associate with aβ, but it is not clear how this association affects aβ neurotoxicity. methods: here we utilized several techniques to isolate exosomes from the sera of wild type (wt) and ad transgenic mouse model. (5xfad) as well as alzheimer's patients and healthy controls. we used exoquick, exoeasy, sequential ultracentrifugation, and size exclusion chromatography. particles' size and number were characterized by nanoparticle tracking analysis (zetaview). results: we report that the sphingolipid ceramide mediates neurotoxicity of aβ. we show that sera from ad transgenic mouse model (5xfad) and ad patients, but not the wt or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. when taken up by introduction: multiple sclerosis is the most common chronic inflammatory demyelinating disease of the central nervous system, affecting more than 2 million people worldwide. ms is a multifactorial, immunemediated disease caused by complex genetic and environmental interactions. in recent years, extracellular vesicles (evs) have been described as powerful mediators of the modulation of biological processes (e.g. inflammatory and immune response) following environmental exposures such as particulate matter (pm), and have been described altered in ms. we characterized evs in 48 patients with ms and 20 healthy subjects matched for age and gender and evaluated the effects of pm exposure on ev release patients with ms compared with controls. methods: evs isolated from blood samples were characterized by nanotracking analysis and by flow cytometry after labelling with the following markers: cd14 + (monocyte), cd61+ (platelet), cd66+ (neutrophil), cd25+ (t-reg), and cd105+ (endothelium). pm10 and pm2.5 concentrations at the residency of each subject were obtained from the regional air quality monitoring network. results: we observed decreased concentrations of cd14+ (p < 0.05), cd61+ (p < 0.003), cd66+ (p < 0.009), cd25+ (p < 0.003), and cd105+ (p < 0.003) in patients compared with controls. in cases, pm was inversely associated with cd14+ evs (pm2.5, β = −0.03; p < 0.05), cd66+ evs (pm2.5 β = −0.03; p < 0.03), and cd25+ evs (pm10 β = −0.02; p < 0.04; pm2.5, β = −0.03; p < 0.02). on the contrary, in controls pm was positively associated with cd14+ evs (pm10 β = 0.02; p < 0.03; pm2.5, β = 0.03; p < 0.02). summary/conclusion: our findings showed a different composition of blood-derived ev subpopulations in patients compared with controls. moreover, we observed that patients and controls react differently to pm exposure in terms of blood-derived ev release, suggesting the involvement of this mechanism in the modulation of both inflammatory and immune responses, and thus in ms pathogenesis. plasma neuronal and astrocyte-derived exosomes serve as biomarkers of neurodegeneration and systemic bioenergetic effects in male cynomolgus monkeys self-administrating oxycodone ashish kumar a , yixin su a , david soto-pantoja a , jingyun lee a , ravi singh a , cristina furdui a , michael nader b and gagan deep a a wake forest baptist medical center, winston-salem, usa; b wake forest baptist medical center, winston-salem, usa introduction: opioid use disorder (oud) is currently a health emergency in the usa affecting millions of people. oud is a complex issue requiring a multipronged strategy. at the biological level, there is an urgent need to understand the dynamic molecular changes and adverse effects associated with opioid addiction. here, we aimed at identifying the biosignature of brain cells-derived exosomes associated with opioid addiction in a non-human primate (nhp) model of oud in which cynomolgus monkeys perform cognitive tasks and self-administer (sa) intravenous oxycodone daily. we also characterized the systemic adverse effects of the brain cells-derived exosomes from drug-naïve and oxycodone sa monkeys. methods: we isolated total exosomes (te) by ultracentrifugation and exoquick methods from the plasma of male monkeys self-administrating oxycodone for 3 years and naive monkeys. subsequently, from the te population, we isolated neuron-derived exosomes (nde) and astrocytes-derived exosomes (ade) using surface biomarkers l1cam (l1 cell adhesion molecule) and glast (glutamate aspartate transporter), respectively. this novel method involved streptavidin coated magnetic beads and photo-cleavable (pc) biotin, providing us biologically intact exosomes useful for co-culture studies. we characterized the exosomes by nanoparticle tracking analyses (nta), western blotting, flow cytometry, immunogold labelling, transmission electron microscopy (tem), elisas and mass spectrometry. respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment was performed by seahorse xf. results: the quality of isolated exosomes (te, nde, and ade) was confirmed by nta (size distribution and concentration), western blotting (e.g. cd9) and tem (size and shape). nta did not show any significant difference in exosomes size and concentration (number per ml) between control and oxycodone sa groups. flow cytometry (e.g. l1cam and glast) and immunogold labelling (cd9, cd63 and l1cam) confirmed the purity of nde and ade isolated from te. proteomics analyses of te, nde and ade identified several unique proteins present in exosomes from the oxycodone sa group. interestingly, we observed significantly higher expression of neurodegeneration markers neurofilament light protein (nfl) and alpha-synuclein in nde and ade of oxycodone sa group compared to controls. furthermore, te treatment of h9c2 cardiac myoblasts and raw264.7 monocytes significantly compromised their mitochondrial metabolism (basal and maximum respiratory capacity). summary/conclusion: these results suggest the utility of plasma exosomes as biomarkers for better understanding of the neurodegenerative and systemic effects of oxycodone addiction. funding: da049267, da017763. vesicles released during mycobacterium tuberculosis infection: immunomodulatory (glyco)lipids and role in host-pathogen interactions emilie layre, pierre boyer and jerome nigou cnrs-université paul sabatier, toulouse, france introduction: the tuberculosis disease remains one of the top 10 causes of death worldwide. mycobacterium tuberculosis (mtb) has evolved strategies to evade immune responses and to persist within the hostile intracellular environment of alveolar macrophages. the current lack of efficient anti-tuberculosis strategies is largely due to our incomplete understanding of the host-pathogen interactions of mtb infection. vesicles released by the bacillus itself (bacterial membrane vesicles, bmv) and by infected cells (host extracellular vesicles, hev) have immunomodulatory properties in vitro and when administered to animals. if vesicles likely play key role in host-pathogen interactions of the tuberculosis infection, their content in bacterial factors, their uptake, trafficking and interaction with host cells receptors remain incompletely deciphered. methods: bmv and hev have been purified by combining differential centrifugation, density gradient and exclusion chromatography. after characterization by microscopy, nanosight and western blot, their content in bacterial (glyco)lipids has been characterized by the use of high sensitivity mass spectrometry-based lipidomic approach. bmv have been tested for their capacity to activate reporter cell lines of pattern recognition receptors. in addition, fluorescent-labelled bmv have been used to study their uptake by host cells thank to super-resolution microscopy. results: we have undertaken to characterize the content, the trafficking and interaction with pattern recognition receptors of bmv and hev released during infection by mycobacteria of variable virulence. we have importantly optimized the purification of bmv showing that lipoproteins aggregates are co-purified with vesicles on density gradient. sfc-ms lipidomic analyses allowed the characterization of the repertoire of immunomodulatory bacterial lipids released by bmv and hev, which excluded a continuum between these two release pathways. preliminary, assays have shown that these vesicles are capable to interact with different pattern recognition receptors including tlr and lectins. finally, we have been able to visualize fluorescent-labelled vesicles uptake by macrophages using superresolution microscopy. summary/conclusion: during m. tuberculosis infection, the bacillus as well as infected cells release vesicles that harbour different content in immunomodulatory bacterial (glyco)lipids, including strain-specific lipids. these vesicles likely play important role in host-pathogen interactions by modulating immune response beyond the infected cells, in part through their interaction with different pattern recognition receptors. funding: fondation pour la recherche medicale, fondation fonroga. introduction: conventional diagnoses of mycobacterium tuberculosis (mtb) rely on quantifying bacteria in sputum samples, which make it incapable of measuring the body's total bacterial load and diagnosing patients that have difficulty producing sputumsuch as children and those that are hiv-positive. nanoscale (30-200 nm) outer membrane vesicles (omvs), which are shed from their bacterial cells of origin and circulate in the bloodstream, have been found to contain rich molecular information from their mother cells. despite the diagnostic potential, their nanoscale size in the presence of high background has complicated the use of these promising biomarkers for clinical diagnosis of tuberculosis. chair: amy buck -the university of edinburgh chair: cherie blenkiron -the university of auckland methods: here we report two complementary approaches to systematically discover and clinically detect mtb-derived omvs using protein and rna biomarkers. first, we employ a digital droplet elisa on whole, unprocessed samples to detect and quantify the presence of these omvs using surface protein markers. second, we have developed a platform to specifically enrich for mtb-derived omvs using our previously developed magnetic nanopore platform, wherein millions of nanofluidic devices are operated in parallel, increasing throughput relative to a single nanofluidic device by a million-fold. using this approach, we identify rnas that are specifically enriched in mtb-derived omvs and can be used to identify tb strain, infectious activity, and total body burden. results: using these platforms, we enriched for mtbderived omvs from plasma and profiled their cargo, both proteins and rna. we first determined a panel of protein biomarkers for multiplexed detection of omvs through a digital droplet sandwich elisa. we then tested our protein markers on spiked plasma samples as models for clinical tb samples. simultaneously, we performed rna sequencing and discovered a panel of rna biomarkers that are preferentially enriched in omvs. we picked ten of the most highly-expressed rna biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore platform. summary/conclusion: these results demonstrate the capability of omv biomarkers in the development of novel liquid biopsy based mtb diagnostics. building on this work, we are working with clinical collaborators to test our assays on clinical samples from philadelphia and west africa. funding: nih ot08.3 introduction: a dearth of knowledge exists regarding the molecular mechanisms by which host exosomes regulate immune response to infections caused by gram-negative pathogens. to address this gap in knowledge, our laboratory has been using two wellestablished model organisms; yersinia pestis (yp), and burkholderia pseudomallei (bp). yp and bp cause the emerging human diseases plague and melioidosis respectively. currently, no licenced vaccines or highly effective therapeutics are available for either disease. methods: ex were purified from naïve u937 monocytes (exu) and infected u937 (exi) by serial centrifugation followed by sucrose density gradient purification, and characterized by tem, zetaview nanoparticle tracking, and exosome markers (cd63, tsg101, . immune responses of naïve u937 cells and response mechanisms were analysed following treatment with equivalent amounts of exi or exu (as control). these included macrophage differentiation assays, multiplex measurements of inflammatory cytokines, bacterial clearance assays, quantitative protein microarray analysis of 173 host signalling proteins, sirna knockdown of exi-induced cytokines in recipient cells, and mass spectrometry (ms) analysis of exi contents. for all assays, at least four biological replicates were performed. results: exi induce monocyte differentiation to macrophages and dramatic release of il-6, il-8 and il-10 cytokines, effects that are also seen when monocytes are infected with the bacteria. the exi also induce a substantial increase in the capacity of the recipient monocytes to clear bacteria in an il-6-dependent manner. specific host signalling molecules are strongly modulated by the exi, including p38, jak2 and alk for exi-yp, influencing the observed phenotypes. ms analysis showed lack of lps in exi-yp and demonstrated the presence of specific bacterial proteins that have antigenic properties. summary/conclusion: we have identified some of the molecular mechanisms by which exi assist the host in clearing infection. exi prime distant naïve monocytes through modulation of distinct pathways such as p38 to mount immune responses similar to when they become infected. these include differentiation to macrophages and migration to infection site for increased il-6-dependent bacterial clearance. introduction: recruitment of monocytes to sites of infection is important in restricting growth and invasion of various microorganisms such as pathogenic fungi c. albicans. beside complement supported phagocytosis and extracellular trap formation, human monocytes secrete extracellular vesicles which are crucial in cellular communication in physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids. the current study attempts to shed light on immune evasion mechanisms by c. albicans via extracellular vesicles. methods: human monocytes were isolated by magnetic beads technique and extracellular vesicles were isolated using polymer precipitation or ultracentrifugation or size exclusion chromatography. vesicles were characterized by elisa, lc/ms-based proteomics, confocal laser scanning microscopy (clsm) as well as electron-and dynamic light scattering microscopy, etc. crispr-cas9 based genome editing was performed to knockout cd11b in human monocytic thp-1 cells. effect of isolated vesicles were determined by using proximity ligation assay (pla), elisa, western blot, next generation rna sequencing, qpcr, immunohistochemistry, etc. results: here we show for the first time that human blood derived monocytes alone and in a whole blood model strongly induced and released extracellular vesicles in response to the pathogenic fungus c. albicans. one induced population carried the anti-inflammatory cytokine tgfβ-1. release of these vesicles is triggered by binding of soluble β-glucan from c. albicans to the cr3 receptor on monocytes as demonstrated by crispr-cas9 based cr3 genome editing in thp-1 cells, and by using cr3 knock out mice. isolated tgf-β1-transporting vesicles reduced the inflammatory response in human m1 macrophages and in a whole blood model. the anti-inflammatory effect by tgf-β1transporting vesicles is investigated in detail and results in inhibition of il-1β gene transcription. summary/conclusion: showing that human apoptotic bodies similarly induced tgf-β1-transporting vesicles from human monocytes we hypothesize that c. albicans hijacks this new cr3-dependent anti-inflammatory vesicle pathway for immune escape. funding: this work was supported by the "deutsche forschungsgemeinschaft" transregio funginet 124 projects c4, c5, c6 and z2. introduction: to date, most research involving extracellular rnas has focused in rnas encapsulated inside extracellular vesicles (evs) or in total unfractionated biofluids. it is known that exrnas also exist outside vesicles or in lipoprotein particles. however, nonvesicular exrnas remain widely uncharacterized despite being a feasible source of contaminants in ev preparations. our interest in nonvesicular exrnas arises from the observation that some small rnas, such as specific trna-derived fragments, have much higher relative representation in this extracellular fraction. at least in part, this enrichment seems to be a consequence of their differential extracellular stability. methods: to get a representative picture of the whole set of rnas released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of rna release and degradation. high-resolution iodixanol gradients were used to separate evs from extracellular rnps or vesicle-free rna. the conversion rate between parental ncrnas and their fragments was studied by high-throughput sequencing and northern blot. results: the inhibition of extracellular rnase activity revealed the presence of full-length trnas and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. extracellular ribosomes co-isolate with evs purified by ultracentrifugation or size-exclusion chromatography, but not with evs purified by density gradients.these ncrnas are substrates of extracellular rnases, demonstrating an extracellular biogenesis route for the formation of ncrna-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. we also highlight the immunoregulatory potential of purified rna-containing extracellular complexes. summary/conclusion: in conclusion, ribonuclease inhibition dramatically shapes extracellular rna profiles and uncovers a population of extracellular ribosomes, trnas and other coding and noncoding rnas which exists outside evs. although these rnas are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. this dynamic view of exrnas impacts our understanding of rna secretion mechanisms and may offer a window to new molecules with biomarker potential. in contrast, evs confer an rnase-protected environment and contain more full-length ncrnas (trnas, yrnas, 7sl rnas, rrnas depending on vesicle size) than their fragments. introduction: cd47 is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin-1 and the counter receptor for signal regulatory protein-α in phagocytes. high expression of cd47 is associated with a poor prognosis for some cancers. conversely, cd47 blocking agents are in clinical trials for enhancing innate and adaptive antitumor immunity in cancer patients. these studies suggest utility of cd47 as a diagnostic and prognostic biomarker and as a therapeutic target. cd47 is also expressed on extracellular vesicles (evs), and we reported that cd47 expression identifies a distinct population of evs from those that express the traditional ev markers cd63 or mhc1. cd47-, cd63-and mhc1-enriched vesicles contain distinct small rna populations (pmid: 29416092), and these differ in rna content from evs that lack any of these markers. the mechanisms by which cd47 directly or indirectly regulates which rnas are packaged into ev remain unknown. methods: to elucidate the mechanism by which cd47 regulates ev rna composition and function, we performed global mirna microarray analysis between evs produced by wild type and cd47-deficient t cells. results were further validated using real-time pcr and rna-immunoprecipitation. interactions between cd47 and exportin-1/ran complex was identified by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy. results: ev released from cd47-deficient human t cells and in cd47-/-mouse plasma were enriched in 5ʹ-7-methylguanosine-capped micrornas and mrnas that depend on the exportin-1/rangtp pathway. knockdown of cd47 in wild type cells or thrombospondin-1 treatment correspondingly enhanced levels of capped-rnas released in ev and re-expressing cd47 in null cells decreased their levels. mass spectrometry and co-immunoprecipitation identified specific interactions of cd47 with components of the exportin-1/ ran nuclear export complex and its known cargos and between the cd47 cytoplasmic adapter ubiquilin-1 and the exportin-1/ran complex. interaction of cd47 with exportin-1 was inhibited by leptomycin b, which inactivates exportin-1 and increased levels of cap-dependent rnas in ev released from wild type but not cd47-deficient cells. summary/conclusion: these findings indicate that cd47-dependent thrombospondin-1 signalling regulates cytoplasmic levels of cap-dependent rnas in t cells at least in part through ubiquilin-1-and gtpdependent physical interactions with the exportin-1/ ran transport complex, which regulate levels of specific pre-mirnas and mrnas available for sorting into evs. funding: this work was supported by the intramural research program of the nih/national cancer institute (zia sc 009172). role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma introduction: desmoglein 2 (dsg2), is a palmitoylated cadherin that is involved in cell-cell adhesion. interestingly, dsg2 promotes mitogenic cell signalling and is upregulated in many cancers, including scc, contributing to poor prognosis and survivability. we recently demonstrated that dsg2 promotes ev release, but the mechanism by which dsg2 enhances ev biogenesis and role of palmitoylation is poorly understood. methods: pharmacological drug inhibitors 2-bromopalmitate, gw4869, and bafilomycin a1 were used. stable scc cell lines were established by retrovirus infection expressing gfp, wild type dsg2/gfp, or palmitoylation deficient dsg2cacs/gfp. evs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by nta. proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using imagestudio. results: here we demonstrate that the effect of dsg2 on ev release was reduced by the palmitoylation inhibitor 2-bromopalmitate. furthermore, mutations that prevented palmitoylation (dsg2cacs) dramatically abrogated ev release by targeting of un-palmitoylated dsg2 to the lysosomes for degradation. dsg2 increased expression and subcellular localization of flot1, a membrane lipid raft protein critical for membrane invagination. dsg2 also altered membrane localization of several early (eps15 and eea1), but not late (rab7, rab11, and hrs), endocytic pathway proteins. loss of palmitoylation in the dsg2cacs mutants abrogated these effects. finally, dsg2-induced ev release was abrogated by the sphingomyelinase inhibitor gw4869 or augmented by the v-atpase inhibitor bafilomycin a1. summary/conclusion: the combined results of the drug treatments and functional mutations of dsg2 suggest that dsg2 plays a critical role in ev biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. introduction: the translation initiation factor eif4e (4e) is an oncogenic protein that is upregulated in 30% of cancers including a subgroup of acute myeloid leukaemia (aml) patients. eif4e regulates post-transcriptional rna processing including the nuclear export and/or translation of mrna transcripts. in particular, it selectively increases the expression of genes that have a prominent role in cancer progression such as myc, cyclin d1, and mcl1. furthermore, our lab pioneered studies demonstrating that a subset of 4ehigh aml patients is clinically responsive to treatment with a 4e inhibitor (ribavirin) indicating the importance of 4e in aml progression and its relevance as a therapeutic target. we investigated an as yet unexplored perspective of 4e-whether the oncogenic role of 4e is in part mediated by its function as a master regulator of vesiculation. methods: to assess mrna export and identify 4ebound mrna targets that correspond to vesiculationrelated genes and associated cargo, we used cellular fractionation and rna immunoprecipitation techniques. to determine whether 4e regulates the number of extracellular vesicles (evs) released as well as their protein and rna cargo we used nanoparticle tracking analysis (nta) as well as mass spectrometry, antibody microarrays, and rna sequencing technologies. results: eif4e upregulates cellular protein levels of the vesiculation marker cd63 by increasing its nuclear export. in addition to increased cellular expression, cd63, cd81, cd9, and flotillin-1 proteins are elevated in evs released from 4e-high cells. this is also associated with an increased release of vesicles that are 50-200 nm in size. currently, we have validated the upregulation of several receptors and cytosolic proteins in evs isolated from 4e-overexpressing cells that function in cell growth, migration, invasion, and stemness. the most abundant rnas in our ev preparations are micrornas (mirs) and we have confirmed downregulation of several of these. summary/conclusion: our work shows that 4e reprograms the vesiculation of cancer cells changing the release and cargo of evs. this may impact cellular communication and tumour biology, which we are currently addressing in functional studies. we hope that these studies will highlight novel therapeutic strategies for aml patients. intranasal administration of neural stem cells-derived extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid plaques in a mouse model of alzheimer's disease introduction: cognitive and memory impairments worsen with time in alzheimer's disease (ad), likely due to a progressive loss of hippocampal neurogenesis, and escalation of neuroinflammation. these changes are also accompanied by increased deposition of amyloid plaques in the brain. methods: in this study, using the 5xfad mouse model, we examined the efficacy of extracellular vesicles (evs) shed from the rat subventricular zone neural stem cells (svz-nscs) for disease modification. we first purified evs from the rat svz-nsc cultures through ion-exchange chromatography and then administered intranasally to 3-months old 5xfad mice (~75 billion/week for two weeks). two months later, the functional effects of ev treatment were quantified through a series of behavioural tests, and animals were euthanized for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation, and amyloid plaque deposition. results: in comparison to ad mice receiving vehicle, ad mice receiving nsc-evs displayed improved cognitive function to discern minor changes in the environment in an object location test, better spatial recognition memory in an object-in-place test, and improved pattern separation ability in a pattern separation test. besides, ev-treated ad mice displayed no anhedonia in a sucrose preference test. analyses of neurogenesis using the birth-dating marker 5ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin revealed maintenance of a higher level of hippocampal neurogenesis in ad mice receiving evs, in comparison to vehicle-treated ad mice. moreover, analyses of brain tissues from ev-treated ad mice revealed decreased concentrations of oxidative stress markers malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase and superoxide dismutase. also, the concentration of proinflammatory cytokines tumour necrosis factor-alpha and interleukin-1 beta and the extent of amyloid plaques were significantly reduced in ev treated ad mice. immunohistochemical analysis showed reduced hypertrophy of astrocytes. summary/conclusion: intranasal administration of nsc-derived evs restrains the deterioration of cognitive and mood dysfunction of ad by maintaining higher levels of neurogenesis and curtailing the progression of neuroinflammation. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) ot10.2 the case of mesenchymal stromal cells, opening new perspectives in the use of ipsc in regenerative medicine. the aim of this study is to evaluate the potential of ipsc-ev in the treatment of kidney disease. methods: the ipsc were generated from skin fibroblast after informed consent of healthy donors (cytotune®-ips 2.0 sendai reprogramming kit -protocol: clementino fraga filho uh 38583914.7.0000.5257/933.018). the ev were isolated from ipsc supernatants (cultured 24 h in mtesr-1 medium) by ultracentrifugation (100,000 g for 2 h at 4°c). characterization of ipsc-ev was performed using zetaview, tem, exoview™ tetraspanin kit and macsplex exosome kit. for in vitro injury, renal epithelial cells were cultured under hypoxia (1% o2). for in vivo injury, male wistar rats were submitted to bilateral renal arterial clamping (45 min) followed by reperfusion without or with injection of ipsc-ev (protocol approval: federal university of rio de janeiro -043/19). kidney damage was assessed by histological and immunohistochemistry analyses (pcna, tunel and ed-1). modulation of rna levels was assessed by rt2 profiler pcr array. results: the results show that ipsc-ev reduce renal cell death, tissue damage, macrophage infiltration, promote mitochondria protection and ameliorate renal function. the ipsc-ev mechanism of action is related to the regulation of key genes known to prevent damage caused by oxidative stress like gstk1, sod1, sod3, txn1 and txnrd2. characterization of ipsc-ev showed that ipsc-ev can carry important molecules that can support renal recovery as epcam and prominin-1. summary/conclusion: ipsc-ev presents renoprotective properties, acting on different aspects of aki. this presents a new relevant application of ipsc as a source of ev for therapeutic purpose in kidney diseases. the hospital for sick children, toronto, canada introduction: incomplete lung development, also known as pulmonary hypoplasia (ph), is a recognized cause of neonatal death. we have previously shown that experimental ph can be rescued by the administration of extracellular vesicles derived from amniotic fluid stem cells (afsc-evs) through an rna-mediated mechanism. this effect was not observed with evs derived from mesenchymal stromal cells (msc-evs) . the aim of this study was to 1) evaluate which rna species were responsible for ph rescue, and 2) to define the mechanism behind this effect. methods: evs were isolated and characterized from conditioned medium of rat afscs and rat mscs (control group) using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd63, hsp70, flo-1, and tsg101 (western). to identify the mediators of afsc-evs, we used deseq (fdr<0.01) to differentially analyse rna from: a) asfc-ev and msc-ev cargo, isolated with seramir and sequenced with nextseq. b) lung epithelial cells from rat ph lungs treated with vehicle or afsc-evs. epithelial cell rna was isolated with mirvana and sequenced with nextseq. we correlated afsc-ev cargo mirna with validated mrna targets that were downregulated after ev conditioning in lung epithelial cells. results: of the rna species contained in asfc-ev and msc-ev cargo, mirnas were the most proportionally different between the two ev populations. afsc-evs were enriched for mirnas that are critical for lung development, such as mir17~92 and their paralogues that control lung branching morphogenesis. afsc-ev administration to ph lung cells significantly downregulated 35 genes, which formed 415 mirna-mrna reported interactions. summary/conclusion: afsc-evs contain many rna species in their cargo, but mirnas are the main effectors of their ability to rescue underdeveloped foetal lungs. we have identified for the first time that afsc-ev biological effect on underdeveloped foetal lungs is in part due to the release of mir17~92 cluster. funding: cihr-sickkids foundation grant. bottom-up assembly of fully-synthetic extracellular vesicles oskar staufer a , franziska dietrich a , jochen hernandez a , martin schröter a , sebastian fabritz b , heike böhm a , ilia platzman a and joachim spatz a a max planck institute for medical research, department for cellular biophysics, jahnstraße 29, 69120 heidelberg, germany, heidelberg, germany; b department for chemical biology, max planck institute for medical research, jahnstraße 29, 69120 heidelberg, germany, germany, germany introduction: extracellular vesicles (evs) are considered as key elements for future therapeutic and diagnostic procedures. however, despite enormous research efforts to understand their physiological relevance and several greatly successful clinical trials, evs are currently not authorized for clinical routines by american or european regulation and approval agencies. this is especially because therapeutic evs are produced or isolated from cell cultures or biofluids, both of which are subjected to batch-to-batch variations and ill-defined contaminations. therefore, complementary technologies that produce evs as reproducible and defined as state of the art nanotherapeutics, would revolutionize the application of evs in clinical settings and provide the scientific community with a holistic understanding of ev-mediated signalling processes. in our study, we achieve de novo bottom-up assembly of fully synthetic evs (fsevs) that comprise identical physiological and therapeutic functionalities to natural evs. methods: we applied droplet-based microfluidic synthesis to sequentially amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that display analogous therapeutic capabilities to natural evs. fsevs were characterized by electron and confocal microscopy, dynamic light scattering and mass spectrometry and tested on organotypic models or in vivo. results: using previously described evs as "naturegiven" blueprints, we assembled several fsevs in their exact molecular composition. in particular, we produced wound-healing promoting evs composed of several exosomal proteins, lipids and micrornas and showed that their therapeutic performance on human skin wounds is equivalent to that of natural evs. besides their high molecular complexity, being composed of dozens of different molecular building-blocks, the presented fsevs are completely defined on a quantitative level. based on this, we achieved a stoichiometric understanding of cell-vesicle interactions. summary/conclusion: by applying bottom-up synthesis of fsevs for quantitative studies on ev signalling, we not only provide innovative and safe compounds for ev-therapeutics but also a vastly new perspective on the application spectrum of extracellular vesicles in fundamental research. introduction: small extracellular vesicles (sevs) contain functional molecules from their cell of origin and can enter recipient cells for intercellular communication. ifnβ has been shown to induce some lncrnas to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (nk) cells. here, we aim to clarify whether ifnβ induced sevs can regulate the cytotoxicity of nk cells by transferring specific lncrnas into nk cells. methods: evs were purified from a549 with/without ifnβ treatment by serial centrifugation followed by sucrose density gradient purification. elisa assay were performed to demonstrate the cytotoxicity of nk cells. qpcr and western blot were used to verify the expression of nkp46. results: surprisingly, ifnβ induced sevs can strengthen the cytotoxicity of nk cells. through human transcriptome array (hta) we found the expression levels of 69 lncrnas were significantly changed within sevs isolated from a549 cells following ifnβ treatment. additionally we found a specific sev cargo, linc-epha6-1, acted as a competing endogenous rna (cerna) for hsa-mir-4485 which subsequently up-regulate the natural cytotoxicity receptor (nkp46) expression. furthermore, we verified over-expression of linc-epha6-1 significantly enhance the cytotoxicity of nk cells against zika virus-infected a549 cells. summary/conclusion: our results demonstrated that ifnβ-induced linc-epha6-1 wrapped in sevs can regulate the cytotoxicity of nk cells. our study provides a novel link between type i ifn and nk cells, which are two major players for the host innate immunity against pathogen infections. introduction: hiv-infected t cells release simultaneously viral particles and small extracellular vesicles (sevs) including mvb-derived exosomes and plasma membrane-derived evs. sevs and hiv share many physical and chemical characteristics, which makes their separation difficult. although several approaches have been used to obtain sevs free of virus they leave a majority of sevs within hiv preparations. for this reason, the function of sevs during hiv infection remains unclear. methods: we have developed a novel un-biased proteomic profiling approach to identify specific markers of the virus or sev subtypes released by a human t lymphoma cell line. our approach was to combine differential centrifugation of medium/small evs contained in the ccm with quantitative mass spectrometry to generate protein abundance profiles across the different sub-fractions. we generated an interactive database to define groups of proteins with similar profiles, suggesting their release in the same evs. results: we thus identified different categories of evs, which bear different surface proteins, e.g. different combinations of t cell surface markers, integrins or tetraspanins. in evs released by infected cells, we identified cellular proteins behaving like hiv proteins, and several that changed behaviour after infection, either moving towards or away from the hiv cluster. we identified two cell-derived proteins that are included in the viral particles and one that is specific of non-viral sevs that are modified by infection, and analysed their respective roles in controlling ev composition or virus infectivity. summary/conclusion: our approach presents a powerful tool for identification of common cargoes of given ev subtypes, and could be now used to identify modifications of ev composition in any given physiological or pathological situation. the encephalomyocarditis virus leader modulates autophagic pathways to promote the release of virions inside extracellular vesicles introduction: recent data indicate that naked viruses belonging to the picornaviridae family can be released from host cells via enclosure in extracellular vesicles (ev). ev cloak virus particles in a host-derived "envelope" and can thereby affect antiviral immune responses and disease severity. a better understanding of the formation and function of ev-enclosed viruses is therefore required. previously, we showed the presence of the autophagosome marker lc3 in ev isolates from encephalomyocarditis virus (emcv) infected cells, suggesting the involvement of a secretory autophagy pathway in ev-mediated virus release. however, little is known about the viral and host factors that regulate this process. here, we have assessed the role of the emcv leader, a viral protein that is dispensable for replication but is required for symptomatic disease. methods: cells were infected with wildtype virus or a mutant carrying an inactive leader. ev produced during the infection were isolated using differential ultracentrifugation and density gradient purification. ev were characterized by high resolution flow cytometry and their infectivity determined using end-point dilution assay. in addition, the fate of autophagosomes in infected cells was monitored using a reporter assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal proteins. results: inactivation of the emcv leader strongly reduced the release of ev-enclosed virus. whereas autophagosomes are typically degraded, we show this is blocked by the leader. instead, autophagosomes fuse with the plasma membrane, as indicated by the secretion of autophagy marker lc3 during infection with wildtype but not the mutant virus. pharmacological reactivation of degradative autophagy in infected cells resulted in a strong reduction in the release of ev and ev-enclosed virus. similarly, the reduction in evenclosed virus release in the absence of the leader could be partially reversed by drugs that promote the secretion of autophagosomes. summary/conclusion: our data supports a role for secretory autophagy in the release of viruses in ev, a pathway that is regulated by the emcv leader. these findings highlight an unconventional route for ev formation that intersects with autophagosomal compartments and contributes to viral pathogenesis. introduction: zika virus (zikv) causes a public health emergency of international concern because of its correlation with microcephaly. during viral infection, the innate immune response quickly to produce some endogenous functional molecules which can prevent viral invasion or replication. extracellular vesicles (evs) contain molecules from their cell of origin under virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv induced evs can regulate viral pathogenicity by transferring specific rna. methods: evs were purified from a549 with/without zikv infection by serial centrifugation followed by sucrose density gradient purification. human transcriptome array (hta) was used to found rna expression within evs. flow cytometry was used to determine cell cycles. zikv replication was assayed by qpcr and western blot. flow cytometry was used to determine cell cycles. results: through hta we found the defensin alpha 1b (defa1b) expression was significantly increased within evs isolated from zikv infected a549 cells. additionally, we found that the extracellular defa1b but not the intracellular defa1b exerts anti-zikv effect mainly before entry step. surprisingly, up-regulate defa1b can retard cell cycles of host cell. we verified defa1b could bind with the origin recognition complex 1 (orc1) which is required to start dna replication during the cell cycle. furthermore, up-regulate defa1b decreased the orc1 level in nuclear. interestingly, evs with defa1b can internalize into recipient cells and inhibit their cell cycles. summary/conclusion: together, our results demonstrated that zikv infection can induce defa1b wrapped in evs, and defa1b not only exerts anti-zikv effect but also regulate cell cycles which may affect neurodevelopment. our study provides a novel viewpoint that defa1b act as first-line anti-viral molecules during zikv infection also correlate with neurodevelopment by retarding cell cycles. extracellular vesicles mediate bacterial-immune cell interactions during respiratory viral-bacterial co-infections sidney w. lane a , matthew hendricks b and jennifer bomberger a a university of pittsburgh, pittsburgh, usa; b university of washington, seattle, usa introduction: respiratory infections are a major cause of morbidity and mortality worldwide and host-derived extracellular vesicles (evs) play important roles in mediating these infections. during respiratory infection, evs are shown to have a modulatory effect: promoting or suppressing infection dependent on the pathogen and cell type. in the age of next-generation sequencing, we now appreciate that many respiratory infections are polymicrobial in nature, with viral-bacterial co-infections correlating with worse disease outcomes. epidemiological studies correlate acute viral infections with the increased likelihood and severity of both acute and chronic secondary bacterial infections; however, the exact mechanisms of these interactions remain poorly understood. evs have been understudied in the context of respiratory viral-bacterial coinfections; thus, their role in mediating these infections is relatively unknown. unpublished data from the lab shows that in airway epithelial cells (aecs), viral infection induces the release of evs that associate with pseudomonas aeruginosa (pa) and promote biofilm growth. here, we aim to expand upon these findings and determine how aec evs mediate pa-immune cell interactions during respiratory viral-bacterial co-infection. methods: to determine how exposure to evs impacts pa-immune cell interactions, evs were isolated from the apical secretions of aecs and co-cultured with pa. ev-treated pa was then co-cultured with macrophages to evaluate ev impact on pa uptake and survival. results: in preliminary experiments using control evs, we observed that evs associate with pa. interestingly, during co-culture with macrophages, ev-treated pa are more susceptible to phagocytosis in comparison to non-treated pa. however, after 4 hours of co-culture with macrophages, ev-treated pa are able to survive and replicate, while nontreated pa are effectively controlled by the macrophages. summary/conclusion: these findings suggest that while pa-ev association promotes pa uptake, it may ultimately enhance pa immune evasion and survival. ongoing experiments in the lab are evaluating the mechanism of pa-ev association and how evs from virus-infected aecs affect the phenotypes observed with control evs. notably, this is one of few reports of a mammalian ev influencing the pathogenesis of a bacterium; thus, results from these experiments will define the function of aec evs in regulating bacterial-immune cell interactions during respiratory co-infections. using machine learning with neuronal ev target proteins and clinical data to predict cognitive impairment in hiv infection lynn pulliam a , michael liston b , bing sun c and jared narvid d a university of california, san francisco, san francisco, usa; b veteran affairs, san francisco, usa; c ncire, san francisco, usa; d ucsf, san francisco, usa introduction: objective biomarkers are needed to assess and predict neuronal function and cognitive impairment. in people ageing with chronic infections, such as hiv, determining the mechanism of impairment will be important when therapies are available. methods: sixty plasma samples from hiv-infected people were obtained from nih-sponsored aids banks. clinical and epidemiological data were collected. all underwent neuropsychological testing and 40 were considered impaired. neuronal extracellular vesicles (nevs) were isolated from plasma and assayed for high-mobility group box 1 (hmgb1), neurofilament (nf-l) and phosphorylated tau-181 (p-tau) proteins. results: using 3 different algorithms, support vector machines (svm) performed the best with an area under the curve (auc) value of 0.82 ± 0.16. using 4 different combinations of clinical data and the 3 nev protein targets, selected clinical data and hmgb1 best predicted cognitive impairment (auc = 0.82). the most important features included cd4 count, hmgb1, nf-l and education. summary/conclusion: specific clinical features plus nev hmgb1, an inflammatory marker, were the best predictors of cognitive impairment. previous published data showed nev p-tau-181 elevated in alzheimer's disease and in this study p-tau had no importance in assessing hiv-associated cognitive impairment. nev target discovery can be improved to better identify neuronal damage, possibly to differentiate other neurodegenerative diseases and hopefully recovery after therapies are identified. in recent years, we have been able to separate and characterize extracellular vesicles (evs) from several different viruses including hiv-1, htlv-1, rift valley fever virus and ebolavirus. however, to date it is not clear whether there is a timing difference between ev and virus release from infected cells. methods: ev isolation by nanoparticle capture and differential centrifugation, ev quantification by nanoparticle tracking analysis, western blot, rt-qpcr, virus rescue assay. results: we have attempted to address the kinetics of ev and virus release from multiple-infected cells using serum starvation experiments from infected (100%) cells. these infected cells were initially put in g0 quiescent stage using serum starvation. both supernatants and cell pellets were collected postinduction release (20% fbs + pma/pha) at 0, 3, 6, 12, and 24 hours and examined for the presence of ev, autophagy and viral proteins as well as viral rna expression. results from supernatants of uninfected cells showed a peak of tetraspanin proteins (cd63, cd81, and cd9) at 6 hours and a gradual decrease of all ev associated proteins by 24 hours. however, the ev from hiv-1 infected cells showed all three tetraspanins present at 3 hours and expression gradually increased up to 24 hours. when compared to htlv-1 infected cells, the three tetraspanin proteins peaked at 6 hours and expression continued to decrease up to 24 hours. htlv-1 infected cells also showed a unique pattern of cd81 expression. autophagy associated proteins (lc3a, lc3b and p62) from uninfected cells and htlv-1 infected cells plateaued at 6 hours, whereas in hiv-1 infected cells their expression continued to increase and peaked at 24 hours. hiv-1 viral proteins (p24, gp120, nef) expression was present at 6 hours and continued to increase and peaked at 24 hours. htlv-1 proteins (p19 and gp46/61) peaked at 6 hours and gradually decreased overtime. hiv-1 and htlv-1 rna gene expression analysis was performed, and data correlated with viral protein expression. additionally, evs release was quantified and showed significant increase of ev concentration overtime in both uninfected and infected samples. finally, experiments of infectivity from 6-and 24hour supernatants were performed on three naive cells. hiv-1 supernatant 6-hour sample was found not to be infectious. however, hiv-1 was successfully rescued from 24-hour sample. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = 31). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and 10 ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine 37 distinct uev surface markers of cd9+/cd63+/cd81+ vesicles in a multiplexed bead-based manner including respective controls. the accuri c6 (bd) was utilized for data acquisition. for further misev2018-compliant characterization, cd9+/cd63+/cd81+ uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd209, all other 36 surface markers could be identified. the most abundant markers were cd9 and cd63, which were detected in 93% of samples, followed by cd133/1 (84%), cd326 (81%), cd81 and cd24 (77%, each). cd3 (42%), cd9, cd45 (39%), cd49e (32%) and cd81 showed similar relative median fluorescent intensities (rmfi), while cd63 yielded significantly higher (p = 0.009) and all other markers significantly lower rmfi (p < 0.011). tem and f-nta revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 e + 10 particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg101, cd9, cd63 and cd81 without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a ,esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = 40) aged 40-70 yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a 3-colour panel, which included annexin v (for the majority of circulating evs), cd41 (for platelet-derived evs) and cd105 (for endothelial-derived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and 10-yr cvd risk detected by qrisk2. results: ev numbers, as determined by nta, were strongly associated with bmi (r = 0.602, p < 0.001), blood pressure (systolic bp: r = 0.359, p = 0.023; diastolic bp: r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = 0.330, p = 0.038). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining 49.4% of the variance for total ev numbers. an additional 9.3% of the variance in total ev numbers was predicted by diastolic bp. ancova of the 10-yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with 10-yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd4 + t cells were isolated from secondary lymphoid organs of foxp3-rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th1, th2, and th17), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, 15-day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for 5 days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th1, th2, th17) showed either no change in proliferation (th1) or decrease in proliferation (th2, th17), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < 0.0001). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th1), il4 and il13 (th2), or il17a and il17 f (th17). in contrast, exposure of tregs to cdc-evs resulted in~1000-fold increase in expression of il10, a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il-10 in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il-10. this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t32hl116273. prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from 42 patients treated with prostanoids (study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with prostanoids (control group; n = 38, 55 ± 19 years, 65% female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; 0.5 mm), adenosine diphosphate (adp; 6.5 µm) and thrombin receptor-activating peptide (trap; 32 µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd61+ and cd62p+; pevs), leukocytes (cd45+, levs) and endothelial cells (cd146+, eevs) were measured in plateletdepleted plasma by flow cytometry (a60-micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = 0.01) and lower concentrations of pevs and levs (p ≤ 0.05). furthermore, thrombus formation was delayed (p ≤ 0.003) and thrombus size was decreased (p = 0.008) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd61+ pevs (p = 0.04). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ 0.05) and the concentrations of pevs (p ≤ 0.08). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ 0.04). introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from 17 cf children (<6 years of age). for nanoflow cytometry, evs were stained with di-8-anepps, and with epcam, cd66b and cd115 (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. results: the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = 0.003, spearman's rho = 0.689). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = 0.001, rho = 0.857). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv15a0), emory pediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: 142 individuals were enrolled into our study, subdivided into a training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer n = 20, pneumonia n = 18, sepsis n = 37). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq2) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative realtime pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed 29 significantly regulated mirnas in pneumonia compared to volunteers, and 25 mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by 12 mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992). mir-193a-5p (log2fc = 1.86, padj = 1.49e-6) and mir-542-3p (log2fc = 1.67, padj = 3.29e-5) differentiated between pneumonia and volunteers and mir-1246 (log2fc = −2.41, padj = 1.78e-04) between pneumonia and sepsis. expression levels of mir-193a-5p and mir-1246 were related to disease severity. mir-542-3p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was 73.33%. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study,3healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd63 and cd81 were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and 9 mirnas (mir-103a-p, mir-191-5p, mir-320a, mir-200b-3p, mir-185-5p, mir-223-3p, mir-21-5p, mir-155-5p, let-7 g-5p) , were evaluated by rt-qpcr. after the optimization of the methodology, 10 healthy adults subjects and 10 patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd63 and cd81. however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna-320a showed a significant increased (p = 0.02) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir-320a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf2-active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds joseph kuhn a , absara hassan b , sonali sharma b , jennifer kwong b , montaha rahman b , salma adam b , jasmine lee b , alvaro villarreal ponce b and piul rabbani b a nyu langone health, new york, usa; b nyu langone health, new york, usa introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid 2-related factor 2 (nrf2) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf2-active exosomes, mscs were incubated with potent nrf2 activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive msc markers (cd44, cd73, cd90, and cd105) and <5% express negative markers (cd45, cd31, cd14, cd19, or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd81, cd9 and tsg101. nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of 168.0 ± 6.5nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf2-active human mscs increase exosome secretion by 54%, compared to nrf2-baseline mscs (p < 0.05). both nrf2-baseline and nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05). this reduction eliminated the delay in closure time compared to wounds of c57/b6 mice. nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated c57/b6 wounds. at day 10, exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd31+ vessels compared to vehicle-treated wounds. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist1 is a key mediator of tumour cell metastasis.. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist1. methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc3-ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc3-ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc3-ml lines stably overexpressing or deficient in twist1. results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc3-ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc3-ml overexpressing twist1 showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist1 expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. funding: american heart association of12 symposium introduction: as researchers continue to explore the therapeutic potentials of extracellular vesicles (evs) for the treatment of many diseases, there is a growing unmet need for real-time in vivo monitoring of these therapeutic evs after they are injected into a subject to understand their safety, targeting, and effectiveness. while current optical imaging solutions like bioluminescence and fluorescence are useful for ev tracking studies in animal models, there is limited utility in clinical applications. here we present a novel ev tracking solution utilizing clinically applicable mri technology. methods: to generate trackable evs, cells were labelled with a clinically applicable novel magnetic agent. evs secreted by the labelled neural stem cells and amniotic fluid stem cells (afscs) were isolated by differential ultracentrifugation. the viability and morphology of labelled-cells were evaluated, and the in vitro mr properties of their derived evs were analysed by magnetometer. a proof of concept in vivo biodistribution study was conducted by injecting labelled evs into wt and alport mice (a model of chronic kidney disease) via retro-orbital and intra-cardiac routes and tracking them via mri at 10 min and 3 hr postinjection. results: the magnetic label did not affect the physiological characteristics of the cells. the mr detectability of labelled-evs was confirmed by in vitro/ex vivo mri phantoms. mri studies showed that homing of afsc evs to the kidney injected intra-cardiacally into alport mice were more efficient versus the retro-orbital route, and prussian blue staining of kidney sections confirmed the mr findings. introduction: a central question in ev biology is the fate of circulating ev. this can be evaluated by developing non-invasive ev bioimaging techniques in mice in order to benefit from transgenic and knock-out models. recent reports described ev biodistribution in vivo using optical (fluorescence) and nuclear imaging. but the physicochemical properties of the probes impact ev integrity, labelling efficiency, background signals and observation timecourse. methods: we developed the radiolabeling of red blood cells (rbc) and ev with [18 f]fluorodeoxyglucose (18 f-fdg). we used rbc-derived ev in their native, intact form, without pre-experimental processing (no centrifugation or filtration). we tracked 18 f-fdg in vivo by pet-scan, within seconds of ev, rbc or free 18 f-fdg injection, and during their dissemination in blood and recruitment by organs over one hour. ev and rbc biodistribution were confronted to the kinetics of free 18 f-fdg. results: we collected images of the biodistribution of rbc, and rbc-derived ev. nuclear imaging was well suited for accurate studies of ev organotropism, with high sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues and an inherent quantitative tomographic nature. ev-specific signals were mostly accumulated within minutes of injection (tail vein), in the spleen and liver, with a small part in the bone marrow (femurs). signals in other compartments were largely transient and linked to tissue perfusion and blood volume. we selected the most drastic control conditions to secure a correct interpretation of the data. this made kidneys, hearts and brains unavailable for analysis. hence the new approach came with limitations, but we describe how "free" 18 f-fdg signals can be used to draw sound conclusions for ev. summary/conclusion: we propose that three types of compartments coexist in control mice at rest: active ev-capturing organs with high capacity and specificity including the spleen, and to a lesser degree the bone marrow; passive ev-retaining organs with high capacity, including the liver; and ev-neutral organs where transient signals only mirror tissue perfusion. we also report how ev biodistribution patterns are altered in ageing animals, as an example. we hope that this novel, non-invasive, quantitative, dynamic wholebody imaging approach will help characterize native cell-derived ev and help set standards for the reproducibility of ev bioimaging in mice. funding: frm grant "biface", inserm copoc, cnrs. introduction: extracellular vesicles (ev) are important mediators of intercellular communication; however, basic principles of ev biogenesis and loading remain largely unknown. a limited repertoire of tools has thus far made these processes challenging to research. the development of an ev-transfer reporter in a genetically tractable organism such as drosophila has allowed us to study mechanisms of cargo loading in vivo and has provided us with a platform to explore fundamental aspects of ev biology. methods: we have developed a bioinformatic pipeline to analyse the properties embedded in the 3ʹutr of mrnas enriched in evs released by drosophila cells. in parallel, we have adapted a cre-loxp system for use in fruit flies that appears to be proficient to reveal the exchange of bioactive molecules between secretory and recipient cells. results: taking advantage of computational methods, we uncovered sequence motifs that preferentially appear in combinations along the 3ʹutr. these sequence motifs occur within characteristic secondary structures, in a way that is more variable and motif dependent than previously reported. identified motifs also show similarities to known binding sites for rna binding proteins; a feature potentially important for ev-loading. in parallel, we developed a drosophila in vivo system to detect cell communication in complex tissues and between different cell types. using this system, we studied the biological significance of specific sequence motifs and identified their ability to modulate mrna ev-transfer in a context dependent and evolutionarily conserved manner. summary/conclusion: in summary, we have developed a novel tool to study cell communication in complex tissues, and shown its effectiveness to study principles of ev biogenesis and loading. beyond improving our understanding of ev biology and providing a novel tool to the scientific community, we hope this knowledge will pave the way to harnessing evs as a means of remotely manipulating cell communication in many biological contexts. introduction: the idea of cross-kingdom, species and inter-individual transfer of bioactive compounds via extracellular vesicles (evs) is a recent avenue. however, the bioactivity and bioavailability of these dietary compounds upon consumption is highly debated. it has been proposed that evs from diet can absorbed by consuming organisms, be bioavailable in various organs and exert phenotypic changes. milk is the most vastly consumed beverage and is an abundant source of evs that may act as signalosomes. whether these milk-derived evs can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. methods: bovine milk-derived evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. the evs were characterised by tem, nta, quantitative proteomics and rna-seq. evs were orally administered to various mice models of colorectal, breast and pancreatic cancer. primary tumour burden was monitored, and the rate of metastases was measured by imaging and qpcr. immune cells were analysed by facs. mechanistic insights were obtained using quantitative proteomics, confocal microscopy and biochemical experiments. results: we demonstrated that upon oral administration, bovine milk-derived evs were able to survive the harsh degrading conditions of the gut and be bioavailable in peripheral tissues. interestingly, oral administration of milk-derived evs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. intriguingly, despite the reduction in primary tumour growth, milk-derived evs accelerated metastasis in breast and pancreatic cancer mice models. timing of ev administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milkderived evs in breast cancer. biochemical and quantitative proteomics analysis highlighted the induction of epithelial-to-mesenchymal transition and senescence upon treatment with milk-derived evs. summary/conclusion: taken together, we were able to demonstrate the capacity of bovine milk-derived evs in mediating cross-species communication and regulating cancer progression in a context-dependent manner. bacterial membrane vesicles (mvs)a bacterial innate defence system against viral infection xiaomei yan, qian niu and ye tian xiamen university, xiamen, china (people's republic) introduction: in order to survive the constant onslaught of phage, bacteria have evolved diverse defence mechanisms that act at every stage of the phage life cycle. it has been suggested that bacterial membrane vesicles (mvs) may play a key role in innate bacterial defence against phage infection by acting as a decoy to prevent phage adsorption. nearly a decade has passed since mvs were first proposed as a decoy, but details of how bacteria utilize mvs to defend against phages remain poorly understood. here we use the laboratory-built nano-flow cytometer (nfcm) to reveal details of the interaction between mvs and phages at the single-particle level, and to provide new insights into innate defence mechanisms of mvs. methods: s. typhimurium was used as the model system. differential ultracentrifugation and density gradient centrifugation were used to isolate and purify mvs and bacteriophage p22. cryo-tem was used to determine the morphologies of mvs and phage p22. the purity of mv isolates was validated by measuring the particle concentration before and after triton x-100 treatment. monodisperse silica nanoparticles were used as the size reference standards to measure the size distribution of mvs via single-particle light scattering detection. the purity of phage p22 was verified by concurrent detection of side scatter and fluorescence signals of single phages upon nucleic acid staining by syto 82. results: by incubating mvs and af532-labelled p22, the number of phages adsorbed on single mvs were accurately quantified. we found that s. typhimurium and mvs it secretes express different affinity for phage p22 attachment. the binding ability of p22 to mvs is greater than that of bacteria. we confirmed that p22 can inject their nucleic acids into mvs, and these nucleic acids can be degraded by non-specific nucleases inside mvs for the first time. besides, by labelling the nucleic acids of mvs with syto 82, we were able to distinguish three different subpopulations of mvs. summary/conclusion: taking advantage of the superior sensitivity of nfcm in single-particle analysis, we developed a novel approach to the characterization of the interaction between mvs and phages. our study revealed that bacteria produce mvs as bait to attract viral adsorption and nucleic acids injection. funding: this research was supported by the national natural science foundation of china (grants 21934004, 21627811 and 21475112). introduction: the development of evs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. in this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo ev tracking method. methods: evs were purified from expi293 f cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following misev guidelines. engineered expi293 f cells were used to generate evs carrying mcherry or nanoluc (nluc) proteins. the membrane of naïve ev was labelled with indium111(in111)-dtpa or xenolight dir post-ev isolation. ct26 tumour-bearing balb/c mice were intravenously dosed with 1 × 1011 evs followed by imaging at 1 h, 4h and 24h using spec/ct and ivis systems. tissue distribution and blood circulation profile of evs were analysed from ex vivo samples up to 24h post-injection. results: xenolight dir and (in111)-dtpa were the most suitable ev labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mcherry evs were found not suitable for in vivo tracking studies due to high background signal fluorescence. ex vivo organ quantification of in111-dtpa and dir showed that naïve evs mainly accumulate in liver, followed by spleen, kidneys, and lungs at 24h post-dose, with less than 5% ev exposure to the tumours. interestingly, nluc-evs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. blood circulation profile of in111-dtpa and nluc evs showed rapid clearance of vesicles from circulation, with 15% of injected dose detected in blood after 30 min and less than 5% after 16 h. summary/conclusion: radionuclide imaging is an excellent technology to detect evs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. the optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. these results contribute to the understanding of the biodistribution and pharmacokinetics of evs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo. symposium introduction: new methods for quantifying extracellular vesicles (evs) in complex biofluids are critically needed. we report the development of a new technology combining size exclusion chromatography (sec), a commonly used ev purification technique, with fluorescence detection of specifically labelled evs (flu-sec). methods: flu-sec was validated using red blood cell derived evs (revs). size and concentration measurements were performed by microfluidic resistive pulse sensing (mrps) using the ncs1 instrument (spectradyne llc, usa). pe-cd235a (anti-glycophorin a) and alexa647-wga (wheat germ agglutinin) were used to label revs. flu-sec experiments were performed on a liquid chromatography system using a tricorn 5/200 glass column filled with sepharose cl-2b gel (ge healthcare). results: a log-normal size distribution was obtained for revs with a mean diameter of 163.5 ± 0.7 nm and standard deviation of 30.6 ± 0.6 nm. the concentration of revs measured by mrps was 3.14*10e11 particles/ ml. the fluorescence chromatograms of the rev samples labelled with pe-cd235a and with alexa647-wga show the typical features of the separation of evs from soluble proteins with sec and enables the determination of the labelling efficiency of the markers. the linear range for quantification of evs in our experiments spans over two orders of magnitude ranging from 10e9 particles/ml to 10e11 particles/ml. the lod depends on the type of the label. in our experiments the lowest lod was 10e9 particles/ml for alexa647-wga. summary/conclusion: the results indicate that flu-sec is a quantitative technique with very good linearity over a wide range of concentrations, though the limit of detection depends largely on the employed label (sci. rep. 9, 19868, 2019) . moreover, the ratio of ev-bound and free-antibody molecules can be also determined by flu-sec, which can be used to calculate the labelling efficiency of the used marker. funding: this work was supported by the national research, development and innovation office (hungary) under grant numbers pd 121326 and nvkp_16-1-2016-0007. zv was supported by the janos bolyai research fellowship. the conan assay: purity grade and concentration of ev microlitre formulations by colloidal nanoplasmonics. (evs). control over such properties is constantly experienced by researchers to be critical for ev proper manipulation, engineering and translation. however, the need for characterization methods that strike the balance between robustness, working volume, cost and accessibility remains unmet. methods: the colorimetric nanoplasmonic (conan) assay we developed consists of a solution of gold nanoparticles (aunps) into which 1-2 μl of the ev formulation is added. the solution turns blue if the formulation is pure, while stays red if soluble exogenous single and aggregated proteins (saps) are present. the colour shift is visible by the naked eye and can be quantified by conventional uv-vis spectroscopy, providing a quantitative index of purity and an estimation the ev molar concentration (particle number). results: the assay specifically targets saps, and not the ev-related proteins, with a detection limit < 50 ng/μl (an order of magnitude higher resolution than the bradford protein assay). for pure solutions, the assay also allows for determining the ev number, as the colour shift is linearly dependent to the aunp/ev molar ratio. instead, it automatically reports if the solution bears sap contaminants, thus avoiding counting artefacts. experiments, conducted on ev separated from milk and ascaris suum culture medium, are repeatable, with an error below 20%. summary/conclusion: conan proves to be robust and reliable, while displaying appealing performances in terms of cost (inexpensive reagents, run by standard microplate reader), working volumes (1-2 μl) and time (the procedure takes less than one hour). the ability to assign a quantitative purity grade is, up to date, a unique peculiarity of this assay. finally, the assay is potentially extendable to all classes of natural and artificial lipid micro-and nanoparticles. funding: evfoundry project, horizon 2020 -future and emerging technologies (h2020-fetopen), id: 801367. marina cretich a , roberto frigerio b , alessandro strada b , greta bergamaschi b , marcella chiari c and alessandro gori c a consiglio nazionale delle ricerche (cnr), istituto di scienze e tecnologie chimiche (scitec), milano, italy; b consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy; c consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy introduction: small extracellular vesicles (sev) present fairly distinctive lipid membrane features in the extracellular environment. these include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sev membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. here we introduce the use of membrane sensing peptides as new, highly efficient ligands to directly integrate sev capturing and analysis on a microarray platform. methods: we designed and synthesized membranesensing peptide ligands as molecular baits for small ev and we demonstrate their use in a microarray platform as valuable alternative/complement to antibodies. evs from blood serum and plasma were isolated by ultracentrifugation, characterized by tem, nta, wb. samples were analysed by label-free, single particle counting and sizing on peptide microarrays coupled to fluorescence co-localization immune staining with fluorescent anti-cd9/anti-cd63/anti-cd81antibodies. results: peptide microarrays were realized using a click-chemistry strategy for optimal peptide surface orientation and used to analyse evs from human blood. membrane sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies. in addition to purified vesicles, peptide ligands were tested with pure serum showing capacity to capture evs even from complex samples. in order to get insights into the ev-peptide binding mechanism and verify whether it is directly mediated by the lipid membrane, trypsin-treated evs were captured on peptide microarrays demonstrating that binding is not directly mediated by surface associated proteins. summary/conclusion: we introduced the use of membrane sensing peptides as a novel class of molecular ligands for integrated sev isolation and analysis, reporting for the first time on peptide microarrays for extracellular vesicles. given their affinity to the membrane of small ev, these molecules can serve as general baits, enabling vesicles capturing unbiased by differential surface protein expression. these new class of molecular probes may be integrated with the use of protein markers towards improved small ev isolation and characterization. compared to proteins and antibodies, peptides are characterized by low cost of preparation, remarkable stability and ease of chemical manipulation, offering virtually unlimited possibilities for experimental design. we anticipate that this new class of ligands, may greatly enrich the molecular toolbox for ev analysis. funding: hydrogex (regione lombardia&fondazione cariplo, grant n. 2018-1720) and index (european union's horizon 2020 research and innovation programme under grant agreement n°766466) projects are acknowledged for partial financial support. high-resolution size-based profiling and morphological analysis of extracellular vesicles by scanning electron microscopy sara cavallaro a , federico pevere b , petra hååg c , kristina viktorsson c , rolf lewensohn c , jan linnros a and apurba dev b a kth royal institute of technology, stockholm, sweden; b uppsala university, uppsala, sweden; c karolinska institute, stockholm, sweden introduction: extracellular vesicles (evs) have been found to mediate intercellular communication in physiological and pathological processes. nevertheless, the understanding of evs bio-functionality remains elusive, mainly because of their high heterogeneity in molecular content, but also in size (30-2000 nm) . therefore, accurate size measurements of evs are highly desired, particularly for exploiting their full diagnostic/therapeutic potential. currently available techniques, such as nanoparticle tracking analysis (nta), cannot accurately measure evs smaller than 70 nm and are not capable to distinguish them from protein aggregates. on the contrary, electron microscopy (em) techniques allow high-resolution size-profiling and morphological analysis of evs over their whole size range. however, their low throughput combined with several long preparatory steps have prevented em from being routinely used for ev size profiling. methods: we shall present a method improvement in throughput and reproducibility of ev size-analysis by scanning em (sem). the technique is based on covalent ev capture onto a silicon wafer, using the protocol reported by cavallaro et al. up to the glutaraldehyde step. after immobilization, critical point drying (cpd) is performed to dehydrate evs before sem, while preserving their shapes. results: sem images, showing the comparison in densities of evs prepared by covalent and non-covalent coupling to substrate, indicated a good capture efficiency of our covalent protocol. the size distribution analysis showed good agreement between nta and sem for evs >80 nm. for smaller evs, sem is more sensitive than nta, thus more suitable to check the purity of ev-isolation techniques. last, atomic-force microscopy (afm) measurements was also used to validate our measurements. introduction: extracellular vesicles (evs) are membrane vesicles secreted into extracellular space, by almost all cellular populations, playing a major role in cell-to-cell communication. it has been already demonstrated that changes in luminal or surface protein cargos of these vesicles, may reflect the status of producing cells. for this reason, evs are considered as potential biomarkers in several types of diseases ranging from cancer diagnosis to heart rejection. periostin (postn) is a matricellular protein associated with evs, and its level is considered a possible biomarker, which indicate malignancy and poor clinical outcome in different types of cancer. here we extensively characterize the presence of postn associated on evs, showing how different isolation methods can drastically affect the amount of postn content in extracellular vesicles fraction. methods: serial ultracentrifugation steps or size exclusion chromatography were used to isolate evs from primary culture of cardiac progenitor cells. evs were characterize, according to misev guidelines, by western blot, nta, facs and cryotem analysis methods. postn amount, associated with evs, was analyses by western blot and elisa. furthermore, functional tests were performed on h9c2 cardiomyoblast cell line, treated with the same amount of evs from different isolation methods; cells response were analysed by western blot. results: evs, from both the isolation methods, showed tsg101, syntenin1, cd63 positivity while grp94 was absent. nta showed no differences, in terms of amount and size of particles. by facs analysis evs resulted enriched with cd63, cd9 and cd81. cryotem showed a similar morphology in the two preparations with presence of protein contaminant in the ultracentrifuge pellet. in vitro, h9c2 treated with evs showed activation of pfak after 10ʹ of treatment, this induction was 1.5 times higher in cells treated with evs isolated with ultracentrifuge compared to evs isolated with sec, confirming a drastic effect of postn protein contamination. furthermore, by phospholipase-c treatment, we found that postn is bound to evs surface through a gpi anchor. summary/conclusion: these results suggest that selection of a proper isolation method is critically relevant in evs studies, in particular when protein analysis is considered. different isolation methods dramatically influence protein amount in extracellular vesicles and consequentially their function. furthermore, in this study we show for the first time, that postn is actually bound to evs surface and not carried in their lumen as previously believed. members of the y-rna family have been detected in ev from various cell types and are among the most abundant non-coding rna types in plasma. we previously showed that shuttling of full-length y-rna into ev is modulated by tlr-activation of ev-producing immune cells. this suggested that y-rnas may have potential as biomarker for immune-related diseases. methods: we separated rna-containing structures in plasma based on differences in size, density, and resistance to protease/rnase treatment. using rt-qpcr, we quantified full-length y-rna subtypes (y1, y3, y4) in ev from various blood-related cell types cultured with or without lps-stimulation. inflammationinduced changes in y-rna were assessed in plasma samples from a human endotoxemia study. results: full-length y-rna in plasma was mainly found in ev (early sec-fractions, density 1.11-1.18 g/ml). in contrast, specific mirnas were either enriched in lpp (e.g. mir-122), in both ev and lpp (e.g. mir-16 and mir-21), or in ev (e.g. mir-150). evenclosed full-length y-rna was resistant to enzymatic degradation, while lpp-bound mirnas were degradation sensitive. we discovered that ev released by different blood cell types varied in y-rna subtype ratios. these ratios remained stable upon lps-stimulation of the ev-producing cells. in endotoxemia plasma samples, the neutrophil-specific y4/y3 ratios and pbmcspecific y3/y1 ratios changed significantly during systemic inflammation. importantly, the plasma y-rna ratios strongly correlated with the number and type of circulating immune cells during the inflammation process. summary/conclusion: cell type specific "y-rna signatures" in plasma ev can be determined without prior ev-enrichment, and may be further explored as biomarkers to diagnose inflammatory responses or other immune-related diseases. mining public ev small rna-seq data with mirev -insights into potential reference transcripts and abundant mirnas recently, extracellular membrane vesicle (mv) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. s. aureus produce membrane-bound, spherical, nano-sized, mvs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. the present study sought to examine the nature of the association between nucleic acids and mvs produced by s. aureus. we also sought to analyse the immunostimulatory potential of mvassociated rna and dna, and to evaluate receptormediated recognition of mv-associated rna and dna molecules by innate immune cells. methods: by following a stringent purification protocol, we characterized the rna and dna content of mvs produced by actively growing s. aureus. nuclease protection assays were performed to determine whether mv-associated nucleic acids are protected from degradation. we assessed the immunomodulatory potential of mv-associated rna and dna by treating cultured mouse macrophages with mvs and measuring the induction of interferon-β mrna using qpcr. introduction: urinary extracellular vesicle (uev) transcriptome could potentially reflect the kidney gene expression profile and serve as virtual/liquid biopsy. in order to explore this possibility, we performed mrna sequencing of uevs from individuals with type 1 diabetes to assess whether it can capture a "kidney enriched genes" expression signature that could lead to novel biomarker discovery for diabetic kidney disease. methods: the study included 75 type 1 diabetic individuals (41 normoalbuminuric, 15 microalbuminuric and 19 macroalbuminuric). urine samples were collected either overnight (n = 38) or during 24-hours (n = 37) and uevs were isolated from 20-30 ml of urine by differential centrifugation. the evs quality was ensured by electron microscopy (em), western blotting and ev-rnasprofiling with the bioanalyzer. isolated rnas were subjected to rna sequencing after cdna library preparation (ultra-low amount protocols) using hiseq 2000 (illumina) pair-end protocol. the association between kidney specific gene expression levels (>4 fold higher compared to other tissues, n = 53) and degree of albuminuria or glomerular filtration rate was explored. results: isolated ev quality appeared good by em and western blotting. rna quantity and quality were sufficient for sequencing of all samples with >5 million pair end reads. we detected on average expression of 12,642 genes. principal component analysis (pc) of the expression of all genes did not reveal any systematic batch differences between the overnight and 24-hour urine collections. comprehensive look-up of 53 kidney-enriched genes revealed expression of >73% (total 39) of these genes in urine evs with high expression of five kidney-specific genes (slc12a3, slc12a1, nphs2, aqp2 and slc22a12). pc analysis combining the impact of 39 kidney-enriched genes revealed that most macroalbuminuric patients clustered together along the pc1 axis, and the axis also correlated with the albumin-to-creatinine ratio (p = 0.0004) explaining 11% of the variance (p = 0.005) in the whole data set. the pc1 axis also showed correlation with hba1c (p = 0.003), but not with diabetes duration, bmi, age and egfr. introduction: due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (aavs) have become the vector of choice for human gene therapy. however, pre-existing neutralizing antibodies (nabs) to many aav serotypes pose a critical challenge for the translation of gene therapies to clinic. here, we describe the use of exosomal aavs (eaav) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid. methods: we developed an ultracentrifugation-based purification strategy to obtain eaav specimens from aav-producing hek-293 t cells, and used electron microscopy-based visualization, confocal microscopybased colocalization studies, qpcr, immunoblotting, dynamic lights scattering, exoview technology and protease assays to characterize eaav morphology, contents and mechanism of action. we then evaluated efficiency of heart targeting for eaav9 or eaav6 and standard aav9 or aav6 encoding for egfp, mcherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, langendorff perfusion system and methods: hlhs patients (n = 3) after glenn procedure and swine (n = 3) after pab were given rv injections of allogeneic/xenogeneic mscs. donor-specific, hla-i+, exosomes were isolated from plasma. in swine, exosomes were collected and rv fractional area change (fac) was measured post-msc-injection. in the elpis patients, exosomes were collected and outcome measurements (fac, stroke volume (sv), rv mass) were recorded 6 and 12-months post-injection. exosomal mrna, microrna (mirna), and proteins were quantified and partial least squares regression (plsr) reduced the dimensionality of the datasets to build a swine model, upon which elpis outcome predictions were made. results: multiomics analysis of swine exosome cargo revealed mirna to be the largest contributor to overall variance. in swine and elpis patients, mirnas were similarly expressed (98%, fold-change<2). plsr reduced the dimensionality of the swine mirna dataset to 50 mirnas with the highest weighted coefficients for changes in fac. pathway analysis of mirna targets revealed links to smooth muscle cell proliferation and cardiac chamber development. importantly, the swine mirna plsr model predicted elpis patient improvements in fac, sv, and rv mass with strong correlation (r > 0.9). summary/conclusion: these findings support the use of: (1) swine pab model for rv failure in hlhs, (2) circulating donor-specific msc-exosomal mirna as a novel, non-invasive biomarker of patient outcomes, and (3) introduction: evs have been shown promising potential as a drug delivery vehicle, especially nucleic acid therapeutics. however, the overall short of specificity to target cancer cells has led to low therapeutic efficacy and potential toxicity. rna nanotechnology is the bottom-up self-assembly of nanometre-scale rna architectures. we previously discovered a stable phi29 prna three-way junction (3wj) motif and used it to construct multivalent rna nanoparticles with high chemical and thermodynamic stability. the resulting arrow-shape rna nanoparticles are homogenous, uniform in size and shape, and can harbour different functionalities while retaining their tertiary folding and independent functionalities both in vitro and in vivo. this flexible platform using rna nanotechnology to achieve tumour-specific targeting has been demonstrated over the last decade. here we introduce a strategy to take advantage of both evs and rna nanotechnology to develop a versatile platform for efficient target-specific delivery of sirnas for cancer treatment. methods: we design membrane-anchoring arrowtail 3wj rna nanoparticles to display tumour targeting ligand (psma rna aptamer or egfr rna aptamer or folate) on birc5 sirnas loaded evs (fig.1 ). nanoparticles were characterized by nanoparticles tracking analysis (nta), transmission electron microscopy (tem), dynamic light scattering (dls) and atomic force microscopy (afm). evs were produced by hollowfiber bioreactor and purify by tangential flow filtration (tff) follow by ultracentrifugation. cell binding were evaluated by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity reverse transcription-pcr (qrt-pcr). formulated evs were introduced to tumour (prostate, triple negative breast cancer, colon pdx) xenograft mice by tail-vein injection and evaluate in vivo tumour inhibition. results: 1) we found the orientation of arrow-shaped rna can be used to control ligand display on evs membranes for specific cell targeting. 2) by placing membrane-anchoring cholesterol at the tail of the arrow results in display of rna aptamer or folate on the outer surface of the evs and enhance cancer cell binding and uptake. 3) taking advantage of the rna ligand for specific targeting and evs for efficient cytosolic delivery, the resulting ligand-displaying evs or plant derived evs-like nanovesicles were capable of specific delivery of sirna to cells, and efficiently blocked tumour growth in three cancer models. summary/conclusion: we developed an rna-evs based nanoparticles platform and shown the flexibility for different cancer type treatment. related publications: pi f et.al. nature nanotechnology. 2018 , 13:82. li z et.al. sci rep. 2018 introduction: extracellular vesicles (evs) contain plasma membrane surface markers that provide insights into their cell source. until now, our understanding of the circulating ev-biome has been limited by the lack of celland size-specific ev quantitation methods. we have developed and validated a multiplex nanoscale flow cytometry approach to image cell-and size-specific ev populations using a novel human "ev-lyoplate" with 3 differently coloured monoclonal antibodies per well in a 96 well plate format (n = 242 separate antibodies with isotype, stained pbs, unstained plasma, and quant-beads controls per plate). we hypothesized that platelet poor plasma samples from patients diagnosed with pancreatic cancer would have significantly different ev-biome profiles than screen negative study subjects. methods: study subjects were enrolled and sampled before clinically scheduled endoscopic ultrasoundguided biopsy (eus-fna) procedures to screen patients with symptoms of pancreatic duct obstruction who later had at least two years of clinical follow up, including surgical resection in cases of pancreatic neoplasia (n = 36) or at least one follow up clinic visit to confirm resolution of symptoms. blood samples were uniformly collected, processed, and banked per isev recommended guidelines. uniform machine (facsymphony) settings to standardize light scatter and fluorescence detection were based on commercially available beads (eg. megamix). samples were coded and randomized for testing and results were reported as the mean cell-and size-specific ev events/ul of plasma. results: clinical outcomes confirmed 10 cases of cancer and 26 screen negative controls. principle component analysis suggested that a number of different celland size-specific evs were significantly more common in the cancer cases (adjusted p-value < 0.05, with aucs >0.80), including epcam+/cd63+ events likely from cancer cells and cd41+/cd62p+/cd9+ microvesicles from platelets, among others. summary/conclusion: in this proof of principle study employing an ev-lyoplate design and nanoscale flow cytometry, we could reliably discriminate the ev-biomes in patients with cancer from negative controls. ongoing studies will determine whether these discriminators will be validated in larger cohorts and provide at least noninferior predictive value compared with the current gold standard clinical testing assay (eus-fna introduction: small cell lung cancer (sclc) is an aggressive tumour type, usually metastatic at diagnostic leading to poor overall survival. interestingly, sclc tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem to drive tumour survival. since the subtype of sclc may have prognostic significance, the aim of this study was to identify surface marker proteins as biomarkers of sclc. methods: a linear discriminant analysis (lda) model, implemented in python via sci-kit learn, was used to choose the best 4 markers for distinguishing subtypes. this analysis was based on rna-seq data from a previous study. in order to identify ev-based biomarkers that would identify sclc evs and not normal evs, we excluded from this analysis proteins without a verified transmembrane domain and proteins associated with evs expected to be present in white and red blood cells, and endothelial cells (according to exocarta and vesiclepedia databases). we also prioritized proteins that could be pan markers for sclc and that might have prognostic significance. to validate our findings, we performed western blotting and flow cytometry in sclc cell lines from different subtypes. results: our rna analysis indicated that the best 4 surface markers to distinguish sclc subtypes were ceacam5, fam174a, lrfn1, epha2. immunoblot analysis validated ceacam5 and epha2 but not fam174a or lrfn1. we also found that ncam1, a commonly used sclc marker, only marks some of the subtypes. for further analysis, we chose proteins with antibodies validated for flow cytometry as our chosen biomarker platform. flow cytometry analysis of cd24 is suitable as a pan-sclc marker. however, the expression of non-ne cell lines was decreased compared to rna-seq data. summary/conclusion: protein analysis of ceacam5 and epha2 corresponded to rna-seq data. ncam was not detected as a pan marker for all sclc subtypes. however, we could see cd24 expression in all sclc subtypes, indicating it may be a useful pan marker for sclc. future studies will be performed to validate the expression of other surface markers in cells, purified evs, and plasma of sclc patients. funding: nih u01 ca224276 and nih u54 ca217450. leukobiopsyexploiting extracellular vesicle-mediated leukocyte sequestration of cancer-specific signatures introduction: in cancer, extracellular vesicles (evs) act as a unique exit mechanism for mutant and oncogenic macromolecules (proteins, rna and dna) en route from malignant cells to blood1. while this process has inspired major liquid biopsy efforts, the biology of circulating evs that carry oncogenic mutations (oncosomes)2 is still poorly characterized. it is also unclear what part (if any) of the tumour-related cell free dna (ctdna)3,4, a major liquid biopsy analyte, is linked to circulating evs and what is their fate, receptacles and biological activity. methods: we employed as series of cancer cell lines carrying mutations in major oncogenes (hras, her2, egfrviii). ev-dna was analysed by digital droplet pcr (ddpcr), along with nuclear anomalies in donor cells (dapi, electron microscopy) and transfer of dna to recipient cells of endothelial (huvec, mmbec), astrocytic (nha) or myeloid (hl60) origin. blood underwent fractionation into red blood cells (rbc), white blood cells (wbc), platelets (plt), evs (100,000g ultracentrifugation) and soluble plasma (sup)5. results: hras-mediated cellular transformation (in ras-3 cells) triggers profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm and released as cargo of evs. oncogenic dna is detectable in blood fractions of tumour bearing mice. while evs, ctdna and plts contain intermediate levels of mutant dna, rbcs contain only traces of this material. the highest hras copy number per ml of blood is found in wbcs (monocytes and neutrophils), which contain more cancer dna/cell than liver, spleen and bone marrow. depletion of neutrophils using anti-ly6g antibody results in an increase in ev-and ctdna-associated mutant dna in blood, suggesting the role of these cells in regulating the circulating levels of cancer cell-derived particles. uptake of dna-containing evs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties. these cells also retain cancer-specific transcripts and other cargo. finally, normal astrocytes treated with oncogenic evs also exhibit phenotypic changes and signs of genomic instability including formation of micronuclei. summary/conclusion: we propose that the process of leukocyte sequestration of circulating particles containing tumour-related nucleic acids renders these cells potentially usable as a novel liquid biopsy platform (leukobiopsy) in cancer. introduction: early diagnosis of colorectal cancer (crc) and precancerous adenoma patients is of vital importance. previously we profiled small extracellular vesicles (sevs) derived mirnas isolated from plasma, proposed a new promising biomarker category of crc patients. here we further gave a full landscape of circulating sevs derived rnas to explore and evaluate sevs based rna biomarkers for early detection of both crc and adenoma patients. methods: plasma sevs were isolated from 60 participants, including 31 early-stage crc patients, 19 adenoma patients, and 10 normal controls (nc), and characterized according to misv2018 guideline. the total sevs derived rna expression profile of all participants was investigated by next-generation sequencing (ngs). weighted gene coexpression network analysis (wgcna) was performed to categorize differentially expressed rnas, and t-distributed stochastic neighbour embedding (tsne) was adopted to distinguish crc, adenoma, from nc samples with the top-ranked genes in wgcna modules. rt-qpcr validation was performed in a cohort of 120 additional participants. results: a total of 1615 rna species (including mirnas, mrnas, and lncrnas) were found differentially expressed between plasma sevs in crc and nc participants. additionally, 888 rna species were differentially expressed between plasma sevs in adenoma and nc participants. 1160 rna species were differentially expressed between plasma sevs in crc and adenoma participants. wgcna categorized all rnas into 6 modules, which exhibited different expression trends during the carcinogenesis of crc. a 60-gene combined tsne model consists of the top 10 genes in each module could perfectly classify crc, adenoma, and nc samples. a 6-gene combined tsne model consists of the top 1 gene in each module could roughly distinguish crc and adenoma from nc, with only 1 sample misclassified. rt-qpcr assays also confirmed the potential classification ability of those genes in another validation cohort of 120 participants. introduction: although the concept of systematic "liquid biopsy" using bodily fluids is simple and elegant, the path of clinical reality has been challenging. recently, numerous tissue-specific biomarkers have been discovered in evs derived from blood, urine, cerebrospinal fluid, cell culture media, and a variety of other fluids. however, tracing the lineage of evs to their tissue of origin remains challenging due to their minute amount of cargo and unavailability of matching biopsied tissue and bodily fluids from the same patient. we recently demonstrated in three separate publications (dogra et. al; smith et. al; murillo et. al) , a new device (nanodld) for ev isolations, it's comparison with current technologies, bioengineered vesicles, and a detailed study of rna types present in small/ large vesicles, lipoproteins, and ago2 protein in different biofluids. in the present study, we aim to investigate the lineage of prostate derived evs in biofluids. methods: using our chip technology, we have isolated exosomes from prostate cancer cell lines and patient tissue, blood and urine samples. after exosome isolation, small rna libraries were prepared, and sequencing is carried out at icahn school of medicine and new york genome center using illumine sequencer hiseq2500. our nanofluidic pillar array is manufactured in an sio2 mask using optical contact lithography and deep ultraviolet lithography. results: our study revealed i) rna markers, which are exclusive to their prostate tissue of origin and are secreted in evs; ii) approximately 1-3% of prostate tissue-specific rna were discovered in evs; iii) over 77% (17 of 22 rna) of literature curated prostatespecific rna signatures were detectable in serum and urine evs from pca patients; iv) evs contained over 50-70% of noncoding rna (40-60% was mirna), while tissue predominantly yielded rrna (>60%); v) finally, gene set analyses generated that over 90% of evs rna were enriched for signalling pathways, yielding mirna-associated, non-canonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively monitor prostate tissue-specific biomarkers, identify tumour-specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. summary/conclusion: in summary, we have investigated patient matched tissue, serum, and urine derived evs in prostate cancer. we present a set of 68 prostatic rna in evs, which are enriched in noncanonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively track prostatic biomarkers, identify tumour specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. a multi-model, liquid biopsy approach for diagnosing and staging pancreatic adenocarcinoma introduction: pancreatic ductal adenocarcinoma (pdac) is the third largest contributor to cancerrelated death in the usa. since there is not yet a feasible technology to diagnose pdac early in the disease, 80% of patients are diagnosed at an advanced stage. moreover, for patients with confirmed pdac, standard imaging method has low sensitivity to detect early metastatic disease, which complicates the selection of therapy. to address these challenges, there has been great interest in developing minimally-invasive, extracellular vesicle (ev) based blood tests for pdac. to this end, we have integrated measurements of tumour derivedev rna cargo with circulating proteins and cell free dna (cfdna), and use machine learning algorithms to distill this multiplexed diagnostic to 1. diagnose pdac patients from healthy and disease controls and 2. distinguish pdac patients with distance sites of metastasis to guide their treatment. we make use of our lab's magnetic nanopore isolation technique to specifically enrich for tumour derived evs directly from patient plasma. methods: we have developed a high throughput nanofluidic sorting platform, which immunomagnetically isolates individual evs from plasma using magnetic nanostructures. however, our architectures is uniquely designed for massive parallelization allowing high throughput, robust processing of ml of plasma in minutes. we performed sequencing on a discovery set of patients and controls (n = 29). subsequently, we trained our panel of biomarkers using a training set of n = 47. finally, we validated the performance of our platform using an independent blinded test set of n = 145. results: the results of a blinded test set achieved an accuracy = 92% and an auc = 0.95 on binary classification of pdac patients versus those that were healthy or disease controls. in addition, we achieved an auc = 0.85 and accuracy = 89% with sensitivity of 90% and specificity of 88% on detecting occult metastasist. summary/conclusion: we developed a highly sensitive pancreatic cancer diagnostics by combining our nanomagnetic isolation platform for tumourderived ev isolation, rna sequencing, and machine learning. we isolated tumour-derived evs and profiled their rna cargo, combined with cfdna and ca19-9 for pancreatic cancer diagnosis. the predictive panels successfully distinguished non-cancer patients from pdac patients, and nodistant metastasis patients(m0) from distant metastasis patients(m1) for appropriate treatment. the resulting auc and accuracy from the independent blinded test set outperformed any individual biomarker, showing both the benefits and the robustness of combining multiple orthogonal biomarkers for pdac diagnosis. introduction: both hypertension and diabetes exhibit significant molecular changes to the vasculature that are associated with increased cardiovascular risk. here we examined the protein composition of large evs (l-evs) isolated from the plasma of hypertensive, diabetic and healthy mice to identify common and diseasespecific molecular changes. methods: we examined circulating l-evs isolated from transgenic mice expressing active human renin in the liver (ttrhren, a model of hypertension), ove26 type 1 diabetic mice, and their wild-type (wt) littermates. at 20 weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. l-evs were isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (ms). results: ttrhren mice exhibited increased blood pressure compared with ove26 mice or their wt littermates. (144.2 ± 7.6 vs 123.5 ± 4.9 [ove26] vs. 114.6 ± 5.7 mmhg [wt], p < 0.05). ms identified 297 independent proteins with at least 2 peptides per protein. of these, 163 proteins were found in all groups studied, 48 were exclusive to wt mice, 23 were exclusive to ove26 mice and 4 were exclusive to ttrhren mice. in addition, 22 proteins were observed with >1.5 fold change (fc) compared to wild-type mice, and 68 proteins were reduced by >50%. amongst the top ten differentially expressed proteins, fibrinogen was upregulated in both ove26 and ttrhren mice compared with wild-type controls. similarly trem-like transcript 1, sarcoplasmic/endoplasmic reticulum calcium atpase 3 and junction plakoglobin were all downregulated in both ove26 mice and ttrhren mice suggesting molecular changes common to both conditions. conversely, arginase was up-regulated in diabetic, but not hypertensive mice while carboxypeptidase was upregulated in hypertensive but not diabetic mice. summary/conclusion: taken together, these results show that the protein composition of circulating l-evs is altered in diabetes and hypertension and that both common and disease-specific changes may be detected. further analysis of these changes may lead to the identification of novel pathways associated with the pathogenesis of vascular injury in hypertension and diabetes. funding: this study was supported by grants (to db) from the canadian institutes of health research, an ontario early researcher award, and the canada foundation for innovation. understanding the role of endothelial cell-derived apoptotic bodies in inflammatory signalling and cell clearance in an atherosclerosis model of inflammation. introduction: apoptotic bodies (apobds) are a class of large (~1-5um) evs formed during apoptotic cell disassembly, that are becoming increasingly recognized as potential mediators of intercellular communication, e.g. via the transfer of proteins and other cargoes to target cells. during the inflammatory vascular disease atherosclerosis, endothelial cell (ec) apoptosis contributes to loss of barrier function and promotes the formation of plaques in regions of ec damage. although, experimentally, ecs generate an abundance of apobds, a specific role for ec-derived apobds (ec-apobds) in the progression of atherosclerosis remains poorly defined. methods: in the present study, a detailed in vitro characterization of ec disassembly was performed via flow cytometry, confocal live cell imaging and cytokine profiling, followed by function analyses of ec-apobds using a murine in vivo model of dead cell clearance. results: characterization of ec disassembly revealed that apobd formation in ecs is regulated by rho-associated, coiled-coil-containing protein kinase 1 (rock1), a process that can be pharmacologically inhibited using a rock-1 inhibitor, thereby providing tools for functional in vivo studies. the specific cargo and role in clearance of ec-apobds were then investigated. profiling of ec-apobds was performed via cytokine antibody array to reveal that ec-apobds generated under inflammatory conditions contain high levels of pro-inflammatory cytokines including mcp-1 and il-8, suggesting a potential role for ec-apobds in the propagation of inflammation during vascular disease. furthermore, the ability of ec-apobds to be cleared from the vasculature via phagocytosis was investigated, revealing that ec-apobds can travel to distal organs to undergo clearance. summary/conclusion: these findings provide important insights into the potential functions of ec-apobds generated under both non-inflammatory and inflammatory conditions and may contribute to future studies involving the therapeutic targeting of ec disassembly for the treatment of atherosclerosis. funding: this work was supported by grants from the national health & medical research council of australia (gnt1141732, gnt1140187) adipose mesenchymal stromal cell derived evs foster cardio-renal protection in the doca-salt hypertensive rat model introduction: cardio-renal syndromes (crs) are disorders of the heart and kidneys whereby "acute or chronic dysfunction in one organ may induce dysfunction of the other". stem cell-derived extracellular vesicles (evs) mediates the protection of the kidney from development of chronic kidney disease (ckd). we here investigated the potential of adipose-mesenchymal stromal cells derived evs (asc-evs) as therapeutic tools for the treatment of crs. methods: adult wistar rats were uninephrectomized and treated with a high-na+ diet and deoxycorticosterone-acetate (doca-salt) for 8-weeks (038/15; a02/16-61-15). evs were isolated by ultracentrifugation method. ev dimension, concentration and surface markers were characterized by nta, cytofluorimetric analysis and transmission electron microscopy. to characterize the role of evs in crs, doca-salt rats were injected weekly with asc-evs. systolic blood pressure was measured by the tail-cuff method. plasma creatinine and urinary protein excretion were determined by colorimetric assays and microalbuminuria by immune turbidimetric assay. qrt-pcr and western blot were conducted to evaluate fibrosis and inflammatory-related genes and proteins in the kidney and heart of doca-salt rats. immunohistochemistry was used to confirm matrix accumulation (a-sma) and immune infiltrate (cd68+ cells). results: multiple administration of asc-evs in doca-salt rats induced a protective effect on the kidney, by reducing tubular and vascular damage. kidney function was also conserved by ev treatment as detected by the normal glomerular filtration rate and the absence of proteinuria with respect to doca-salt untreated rats. ev administration significantly decreases the pro-inflammatory molecules mcp-1 and pai1 and reduce the recruitment of macrophages in the kidney. the mitigation of the inflammatory response by asc-ev infusion consequentially affected the development of fibrosis, as detected by the decrease in collagens (col1a1, col4a1) and fibronectin (fn) expression in respect to doca-salt animals. asc-evs were able to act in multiple organs, preventing fibrosis and inflammation also in the heart, therefore alleviating blood pressure rise during the 8-weeks of treatment in doca-salt rats. summary/conclusion: our results indicate that asc-ev administrations in hypertensive-induced ckd rats promote protection from renal damage, reduction of the inflammatory response and prevention of interstitial fibrosis in the kidney. asc-evs are also able to protect the cardiac tissue and to control blood pressure increase, displaying complex and multiorgan beneficial effects. introduction: alveolar macrophages (ams) tonically secrete extracellular vesicles (evs) containing suppressor of cytokine signalling 3 (socs3) protein. uptake of socs3-containing evs by alveolar epithelial cells is critical for restraint of cytokine-induced janus kinasesignal transducer and activator of transcription 3 (jak-stat3) signalling to promote homoeostasis in the distal lung. at steady state, ams exhibit suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function. whether this glycolytic restraint is critical for am secretion of socs3 is unknown. in fact, to our knowledge, metabolic control over release of any ev cargo has never been explored in any cellular context. methods: immortalized mouse ams (mh-s) were treated with various doses of 2-deoxy-d-glucose (2-dg) and oligomycin, inhibitors of glycolysis and oxidative phosphorylation, respectively. primary rat ams collected by lung lavage were treated with an aqueous extract of cigarette smoke (cse) with or without 2-dg. metabolic activity was measured by seahorse assay, evs were quantified by nanoparticle tracking analysis, and vesicular (>100-kda) socs3 secretion was determined by western blot of conditioned medium. additionally, ams collected from wild-type (wt) and lsl-krasg12d mice bearing lung tumours 16 weeks after intrapulmonary ad-cre were cultured ex vivo in the presence or absence of 2-dg. vesicular (>100-kda) socs3 secretion was measured by elisa. results: in a dose-dependent manner, oligomycin inhibited, whereas 2-dg enhanced, socs3 and ev release by mh-s cells. treatment of rat ams with cse (1%) attenuated secretion of socs3, an effect that coincided with increases in glycolytic activity, and co-treatment of ams with 2-dg abrogated the inhibitory effect of cse on socs3 release. finally, ams collected from lsl-krasg12d mice exhibited a deficiency in socs3 secretion relative to wt ams, an effect that was reversible by overnight culture in the presence of 2-dg. summary/conclusion: in tandem, our data generated using in vitro and in vivo approaches demonstrate that am secretion of vesicular socs3 is down-regulated by glycolysis. we speculate that metabolic control over release of ev cargoes is a phenomenon of broad biologic relevance within and outside of the lung. introduction: bacterial extracellular vesicles (ev) are described to play roles in defence and resistance, pathogenesis and stress responses. cyanobacteria pioneered oxygenic photosynthesis, and are the ancestors of modern chloroplasts. we previously described that by deleting the gene encoding tolc (δtolc) in the model cyanobacterium synechocystis sp pcc6803 (s6803), a key player in protein-mediated secretion systems, a hyper-vesiculating phenotype could be obtained. the goal of this work was to understand why δtolc hyper-vesiculates. methods: isobaric tag for relative and absolute quantitation (itraq) was used for quantitative proteomic analyses of total cell extracts. ev were isolated as follows: cells were separated from the extracellular medium (em) by centrifugation (4400 g, 10 min) and filtration (0.2 µm pore-size filters). cell-free em was concentrated using centrifugal filters (mwco of 100 kda), and later ultracentrifuged for 3 h at 100000 g. the final ev fraction was suspended in growth medium. ev characterization was performed using tem, dls, nanosight, and by the detection and quantification of lps (lipopolysaccharides). detection of specific proteins in ev was carried out by western blot. copper (cu) levels were quantified by atomic absorption spectrometry (aas). results: a large-scale quantitative proteomic analysis was performed, resulting in the identification of several metal-related proteins with differential regulation in s6803 δtolc. both wild-type (wt) and δtolc cells were then challenged with different metals. compared to the wt, δtolc showed impaired growth only when exposed to cu, a co-factor for several proteins with roles in primary metabolism. the intracellular cu levels were quantified and δtolc accumulates threefold more cu than wt cells. we then asked whether the hyper-vesiculating phenotype observed could be linked to the stress induced by cu accumulation. in ev isolated from δtolc we detected the metallochaperone copm, a periplasmic cu-binding protein involved in cu-resistance mechanisms in s6803. in addition, cu could also be detected in isolated δtolc-ev. in addition, more ev were detected when s6803 wt cells were challenged with cu, in a cu-concentration dependent manner. summary/conclusion: these results support the idea that bacterial ev represent an alternative cu-secretion mechanism to deal with cu-induced stress. funding: fct phd grant sfrh/bd/130478/2017; feder-compete2020-poci-fct project: poci-01-0145-feder-029540. juan wang and maureen barr rutgers university, human genetics institute of nj, piscataway, usa introduction: extracellular vesicles (evs) function in intercellular communication. despite their physiological importance and biomedical relevance, knowledge of ev fundamental biology is not well understood, in part due to a lack of tractable animal systems. our analysis of environmentally-released c. elegans ciliary evs provides strong evidence that nematodes package cargo in evs that mediate inter-organismal communication, in analogy to intercellular signalling in mammals. we predict that conserved mechanisms underlie ev cargo sorting, biogenesis and signalling. cilia act as cell towers to both receive extracellular signals and to send information via ciliary evs. ciliary defects result in human ciliopathies including autosomal dominant polycystic kidney disease (adpkd). adpkd is a life-threatening disease that affects 1/800 and is caused by mutations in pkd1 and pkd2, which encode polycystin-1 and −2. in c. elegans and humans, the polycystins are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to cilia, and are shed in evs, suggesting ancient conservation. moreover, ciliary ev biogenesis and shedding is an evolutionary conserved process from algae to worms to humans. by studying how cilia make and receive evs, we aim to uncover fundamental principles of how cells communicate using evs. methods: to study ciliary ev cargo sorting and biogenesis, we use genetically-encoded fluorescent-tagged ev cargo and superresolution zeiss airyscan confocal microscopy in living animals. results: we find that cargoes are sorted into distinct populations. in cilia, kinesin-2 motors and kinesin-3 klp-6/kif28 transport different ev cargoes to the ciliary tip and generate an ev cargo enrichment zone. from here, evs are shed and released into environment in a spatially and temporally regulated manner. ciliary ev biogenesis and release is regulated by mechanical pressure and ph. our work revealsat the single cell levelthat different evs are made in response to environmental stimuli, which may be important for ev signalling properties. summary/conclusion: cells exploit the spatiallyrestricted cilium and its sophisticated transport system to generate distinct populations of ciliary evs. how these ciliary ev communicate cellular messages awaits decoding. introduction: we recently demonstrated that recycling endosomes marked by rab11a generate exosome subtypes distinct in cargos and functions from late endosomes, which we collectively term rab11-exosomes. these exosomes are preferentially released from cancer cells in response to metabolic stress and promote adaptive changes in a xenograft model. here we use comparative ev proteomics in hct116 colorectal and hela cervical cancer cell lines to identify rab11-exosome signature proteins and screen for functional effects. methods: we analysed ev preparations by mass spectrometry using tandem mass tag® labelling to identify changes in ev protein cargo in response to glutamine depletion. candidate genes were subsequently knocked down in drosophila secondary cells, which permit visualisation of rab11-exosome biogenesis using fluorescence microscopy, and in human cancer cell lines. results: we show that accessory escrt-iii proteins, chmp5, chmp1 and ist1, are enriched on glutamine-depletion-induced evs and play a selective and conserved role in generating rab11-exosomes. they are, however, not required to traffic ubiquitinated cargos into late endosomes and lysosomes. escrt-0 components, thought to regulate trafficking of ubiquitinated cargos into intraluminal vesicles, are also required to make rab11-exosomes. in flies the escrt-0, hrs, localises to the limiting membrane of rab11-endosomes. comparative proteomics reveals other proteins enriched in rab11-exosomes, which also appear to be needed to mediate this novel exosome formation mechanism. summary/conclusion: we conclude that rab11-exosome subtypes are formed via a distinct mechanism requiring accessory escrt-iii components, suggesting a route to selectively target these exosomes. introduction: the tumour microenvironment consists of a complex network of host cells embedded within extracellular matrix. communication between these cellular compartments is critical for tumour progression and exosomes have emerged as important regulators of intercellular communication. while a number of studies have implicated exosomes in cancer progression, mechanisms controlling exosome transfer are not well understood. we developed three-dimensional (3d) culture models to evaluate the role of cues provided by the extracellular matrix in exosome release and uptake. methods: exosomes were isolated from cells in two-and three-dimensional culture via ultracentrifugation and characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. exosomes were labelled with fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry. results: cells cultured in 2d display decreased exosome release and increased uptake compared to 3d cultured cells. exosome release in 3d culture was inhibited with the exosome release inhibitors brefeldin a and gw4869, but was not significantly altered by knockout of rab27b. in addition, disruption of polarity signals provided by 3d culture did not impact exosome release or uptake in 3d, but induction of oncogenic hras increased both secretion and uptake of exosomes through activation of pi3 k signalling. summary/conclusion: release and uptake of exosomes is altered in 3d environments. these studies help provide insight into exosome production and uptake in vivo and have potential implications for therapeutically targeting exosome release and the development of exosome based therapeutic delivery vehicles. introduction: previous studies in our lab found that expression of r345 w-fibulin-3 induces rpe to undergo emt. the purpose of current study was to characterize the extracellular vesicles (evs) in rpe cells expressing wt-fibulin-3 versus rpe cells expressing r345 w-fibulin-3 and investigate the effects of these evs on rpe cell differentiation. methods: arpe-19 cells were infected with lentivirus with luciferase-tagged wild-type (wt)-fibulin-3 or luciferase-tagged r345 w-fibulin-3. evs were isolated from the media of arpe-19 cells by conventional ultracentrifugation or density gradient ultracentrifugation. transmission electron microscopy (tem) and cryogenic electron microscopy (cryo-em) were performed to study the morphology of the evs. the amount and size distribution of evs were analysed by nanosight tracking analysis (nta). ev protein concentrations were quantified using the dctm protein assay (bio-rad). ev cargo were analysed by unbiased proteomics using lc-ms/ms with subsequent pathway analysis (advaita). migration ability was evaluated in arpe-19 cells with or without the exposure of evs by conducting scratch assays. results: morphologically, tem imaging showed concave-appearing vesicles and cryo-em imaging showed spherical vesicles with two subpopulations of evs: a small group with diameters around 30 nm and a large group with diameters around 100 nm. moreover, tem and cryo-em showed an increased amount of small evs (~30 nm) in the mutant group compared to the wt group. this result was further confirmed by nta showing that, in the mutant group, the particle size distributions were smaller than the wt evs. no significant differences were shown in ev protein concentrations per particle between wt and mutant groups. our previous data suggest that the expression of r345 w-fibulin-3 causes rpe cells to undergo emt as evidenced by upregulated emt drivers and an increased migration ability. proteomic studies showed that evs derived from arpe-19 cells overexpressing wt-fibulin-3 contain critical members of sonic hedgehog signalling (shh) and ciliary tip components, whereas evs derived from rpe cells overexpressing r345 w-fibulin-3 contain emt mediators, indicating that ev cargo reflects the phenotypic status of their parental cells. ev transplant studies showed that exposing native rpe cells to mutant rpe cell-derived evs containing emt drivers, including tgf-β-induced protein (tgfbi), vim, and smad4, leads to an enhanced migration ability of rpe cells in a dosedependent manner. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms (2d static tissue culture flask vs 3d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in 2d t-flasks and 3d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both 2d and 3d culture systems. introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies methods: pca patients and age-matched healthy controls (hc) plasma (n = 6 in each group) were used to isolate sevs with 3 different isolation methods including commercial exoquick ultra kit, qev 35 and qev 70 size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd9 and cd81 commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev35 demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev70. furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods summary/conclusion: qev 70 method demonstrated better performance in isolating relatively pure sevs from human plasma; qev35 has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev35 but with the highest non-sev protein contaminations. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd9, cd63 and cd81, it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: 1) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and 2) the assessment of subpopulation target enrichment relative to the population average by leveraging tim4 as an unbiased, lipidbased ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasisassociated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr3 (her2+), t47d and mcf-7 (er+pr+), bt549 and mda-mb-231 (triple negative) breast cancer cell lines, as well as five mda-mb-231-derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her2 was broadly detected in her2+ skbr3 evs. interestingly, her2-t47d and mcf-7 evs also expressed her2 where it was highly enriched in its epcam+ subpopulations. itg α6, β3 and β4 were only found in triple negative and organotropic evs with itg β3 and β4 differentially enriched based on the organotropism. the population average of mda-mb-231 and lung-tropic evs had high expression of itg β4, where subpopulations of cd44+ evs showed positive enrichment while cd9+ and cd63+ evs showed negative enrichment. itg α5, β3 and β4 were absent in the bone-tropic cd81+ ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb-231 evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. op2.06 = pf17.06 heparan sulphate proteoglycans are required for ev-mediated delivery of multiple growth factors sara veiga, alex shephard, alex cocks, aled clayton and jason webber cardiff university, cardiff, uk introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du145 prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified 49 targets that bind to hs-gag chains, and also 108 different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of evintroduction: methamphetamine (ma) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. neuroimaging studies have revealed that chronic ma abuse can indeed cause neurodegenerative changes in the brains of human ma abusers including prominent microglial activation throughout the brain. it is still unclear how chronic inflammation caused by ma abuse leads to long-term damage to the brain. with this in mind, we are particularly interested in studying the role of extracellular vesicles (evs) in eliciting chronic inflammation in ma exposed brains. in the present study, we focus on the role of a mirna, mir-29a-3p (mir-29a) in chronic ma exposure. here, we present novel data that shows for the first time how chronic ma impacts not only the biogenesis but also the ev associated mirna cargo thereby affecting the overall health of the neurons and glial cells in the brain. methods: -density gradient centrifugation for isolation of brain-derived vesicles -characterization of bdes by western blotting, nanoparticle tracking analysis and transmission electron microscopy -quantitative rt-pcr -digital droplet pcr -confocal imaging of dendritic spines and synapses results: in the present study, we show from both in vivo and in vitro studies that chronic methamphetamine (ma) treatment alters ev biogenesis and microrna (mirna) cargo. brain-derived evs (bde) isolated from frontal grey tissue of rhesus macaques that were administered ma in a chronic regimen revealed a significant increase in both number and size. further analysis revealed increase in biogenesis genes and increased levels of mirna, mir-29a-3p (mir-29a). in situ hybridization of the frontal brain area revealed that mir-29a was exclusively expressed in microglia and neurons. further, in vitro studies revealed that ev associated mir-29a elicited not only neuronal damage but also was able to activate microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory cycle. finally, we show that an anti-inflammatory drug was able to rescue inflammation, mir-29a levels and synaptodendritic injury. summary/conclusion: in summary, our results present for the first time show that chronic ma exposure in the brain affects ev biogenesis and mirna expression. we further confirm that mir-29a can serve as potential marker to diagnose synaptic deficits for chronic ma addiction in humans. finally, we reveal that anti-inflammatory drug could rescue the ev biogenesis and reduces the secretion of mir-29a, thereby rescues synaptodendritic injury. our data further supports the use of the anti-inflammatory drugs as therapeutic interventions for ma addiction. funding: nida funding # r01da042379 blood-borne and brain-derived ectosomes/microparticles in morphineinduced anti-nociceptive tolerance deepa ruhela, veena bhopale, ming yang, kevin yu, eric weintraub, aaron greenblatt and stephen r. thom university of maryland school of medicine, baltimore, usa introduction: opioid pain treatment is impeded because chronic administration decreases analgesia, a condition called tolerance that prompts dose escalation contributing to morbidity and mortality. inflammatory interleukin (il)-1β is required for tolerance development, so we hypothesized that pro-inflammatory extracellular vesicles (evs) play a role. methods: evs with opioid administration were assayed in mice and humans. annexin v-positive, 0.1-1 µm diameter microparticles (mps) were assessed by flow cytometry in murine and human blood and in murine deep cervical lymph nodes that drain brain glymphatics. blood-borne exosomes (<100 nm) were assayed by tunable-resistance pulse sensing (trps). anti-nociceptive tolerance following morphine administration to mice was assessed by speed of tail removal from warm water. results: repetitive morphine dosing of mice to induce anti-nociceptive tolerance increased blood-borne mps by eightfold, and by tenfold in cervical lymph nodes. mps expressed proteins specific to neutrophils, microglia, astrocytes, neurons and oligodendrocytes. il-1β content of mps increased 68-fold. administration of an il-1β antagonist to mice diminished blood and glymphatic mps elevations and abrogated tolerance induction. intravenous polyethylene glycol telomer b that lyses mps and intraperitoneal methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation factor-2 to inhibit toll-like receptors, inhibited mps elevations and tolerance. neutropenic mice did not develop anti-nociceptive tolerance, elevations of blood-borne mps or cervical node mps expressing microglial proteins. elevations of blood-borne exosomes were not identified based on trps analysis. patients entering treatment for opioid use disorder exhibited similar mps elevations as do tolerant mice. summary/conclusion: neutrophil-derived mps containing il-1β are required for morphine-induced antinociceptive tolerance. funding: this project was supported by grant n00014-16-1-2868 from the office of naval research and an unrestricted grant from the national foundation of emergency medicine. evs are a conveyor of toxic dipeptide repeat proteins in c9orf72 als/ ftd models thomas jefferson university, philadelphia, usa introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterized by loss of motor neurons. in als, motor symptoms initiate focally and then progress gradually, distal from the initial focus. abnormal forms of als-associated proteins are physically exchanged between neuronal cells. pathogenic als proteins like sod1, fus and tdp43 are transmitted between cells by assisted mechanisms, mainly extracellular vesicles (evs), spreading toxicity and misfolding of native proteins within the recipient cells. an intronic g4c2 aberrant nucleotide repeat expansion in c9orf72 gene is the most common genetic cause of als. translation of this expanded region occurs by a process called repeat associated non-aug (ran) translation that produces five dipeptide repeats proteins (dprs), polyga, polygp, polygr, polypa and polyga. polyga, polygr and polypr are associated with toxicity in neurons. in this work we study the recruitment of these aberrant proteins into extracellular vesicles (evs) and the potential role of these evs in spreading toxicity between cells of the central nervous system. methods: to isolate the evs from cell culture media we isolated by ultracentrifugation the larger vesicles at 21,000xg and the smaller evs at 100,000xg. number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack analysis (f-nta) and by cytoflex. the protein content of the vesicles was analysed by western blot (wb). to evaluate the potential toxicity of the evs, a transwell system (tw) was employed. neuron viability was assessed using live imaging techniques. results: nsc34 were transfected with reporter constructs expressing dprs tagged with gfp protein. by f-nta, cytoflex and wb analysis we assessed that all the five dprs were loaded in both the large and the small vesicles isolated from cell culture medium. by tw, nsc34 transfected with the dprs were put in contact with primary cortical neurons (cns) transfected with synapsin driven td-tomato for live imaging purposes. we observed that polygr+ nsc34 were able to cause a significant decrease in cns viability. we also observed that polygr+ evs associated toxicity was directly dependent on polygr length. this effect was reverted reducing the number of polygr+ evs treating nsc34 with gw4869. to understand the downstream effect of polygr+ evs in recipient cells we studied tdp43 mislocalization, ran-translation and activation of the integrated stress response, finding a dysregulation of all these potentially toxic pathways in neurons treated with polygr+ vesicles. summary/conclusion: concluding, dprs are actively secreted in evs and polygr+ vesicles cause the activation of toxic mechanisms in the recipient cells, possibly contributing to the spreading of als introduction: pregnancy is the a condition that profoundly mitigates symptoms of multiple sclerosis (ms) a complex disease characterized by immune dysfunction and neurodegeneration affecting 2.3 million people worldwide. serum exosomes, released by specific cells during pregnancy, modulate the immune and central nervous system function and contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis (eae), an induced preclinical model of ms. extracellular vesicles (evs) are the new means for communication among cells. the aim of our study was to characterize the ability of human amniotic fluid stem cells-derived evs (hasc-evs) to antigen presenting cell function thus correcting immune dysfunction in eae. methods: amniotic fluids were obtained from human 16-17-week pregnant women. hasc-evs were collected by ultra-centrifugation. evs were characterized for their specific proteins, lipids and nucleic acids expression. the ability of evs to modulate immune responses was performed in vitro, testing the ability of evs to induce a tolerogenic phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential to suppress eae, induced by immunization c57/b6 female mice with mog35-55 peptide. results: we found that hasc-evs expressed high levels of galectin-1 and promoted a significant increase of the immunoregulatory enzyme indoleamine 2,3dioxygenase-1 enzyme in dcs. moreover in in vivo experiments administration of hasc-evs significantly reduced disease severity in eae. such effect was associated with reduced neurological deficits and suppression of pathogenic t helper 17 (th17) cells, and increased percentage of regulatory t cells (treg-foxp3+) cells. summary/conclusion: our findings unravel immunoregulatory effects of evs secreted by hascs. evs may represent a novel cell-free immune regulatory and regenerative therapeutic approach that can potentially mitigate immune dysfunction and promote remyelination. association of neuronal-derived extracellular vesicles cargo with cognitive decline in late middle life introduction: alzheimer's disease (ad) is characterized by a long preclinical stage during which phosphorylated tau pathology spreads in the brain leading to clinical symptoms. pathogenic tau spreads, in part, via extracellular vesicles (evs). we and others have demonstrated that tau cargoes of neuronal-derived evs (nevs) from blood can serve as biomarkers for ad. we aimed to examine whether nev tau cargo can predict cognitive decline in late middle age by leveraging samples from participants in the wisconsin registry for alzheimer's prevention (wrap) study. methods: we blindly immunoprecipitated nevs using antibody against neuronal l1 cell adhesion molecule (l1cam) from serum samples of 146 wrap participants who were cognitively unimpaired at baseline (mean age 62.4 ± 6.3 years old; 71.2% females; 42.5% apoe4 carriers), of whom half subsequently developed cognitive decline. we measured phosphorylated (p181 and p231) and total tau in nevs using electrochemiluminescence assays. we used linear regression models to identify differences between cognitive status groups including age, sex apoe status and the cognitive status*age interaction in the model. results: at baseline, we found trends for higher p181-(p = 0.07) and p231-tau (p = 0.05) levels in future decliners compared to stable participants. further, there were significant cognitive status*age interactions for ptau231 (p < 0.01), total tau (p < 0.001) and ptau181 (p < 0.05) with higher levels with increasing age in future decliners summary/conclusion: nev tau cargo differs between late middle-aged individuals at risk for ad with and without future cognitively decline even before decline occurs, presumably due to subclinical spread of tau pathology. further nev biomarker development may allow preclinical ad diagnosis. introduction: in the brain, circulating extracellular vesicles (evs) in the cerebrospinal fluid (csf) contain a variety of signalling factors, including proteins, enzymes, and rna transcripts. while evs have been implicated in many cell-to-cell signalling contexts, the vast majority of these studies are based on findings derived from cell culture conditions. thus, the ability to identify cell typespecific ev release from cellular subpopulations within the brain represents a critical barrier in the field. methods: to address this knowledge gap, we utilized a novel transgenic mouse model to determine the release of cell-type specific evs. here we report the exomap-2 mouse, which is designed to express an exosomal green fluorescent protein in response to expression of cre recombinase. specifically, the exomap-2 transgene was inserted at the mouse h11 locus and consists of (i) a broadly expressed cag promoter/enhancer, (ii) a floxed orf encoding mts-tdtomato, (iii) an orf encoding the exosomal protein acyltya fused to mneongreen (mng), and (iv) a 3ʹ utr containing the wpre element and polyadenylation signal from the bovine growth hormone gene. results: intracranial ventricular injections of the viral vector aav-ttr-cre, which drives cre recombinase expression from the choroid plexus-specific promotor of the transthyretin gene, leads to acyltya-mng expression in the choroid plexus. moreover, we observed that these mice released mneongreen-positive evs into the cerebrospinal fluid and also visualized the vesicles in the blood. furthermore, these mice displayed an accumulation of acyltya-mng fluorescence in the medial habenula. summary/conclusion: the results indicate that choroid plexus-derived evs are trafficked to the csf and the medial habenula, and more generally, that the exomap-2 mouse can be used to follow the trafficking of tissuespecific evs into biofluids and between tissues in vivo. introduction: large-scale colorectal cancer (crc) sequencing studies have shown that 93% of all tumours had at least one mutation in proteins implicated in the wnt signalling pathway. mutations in β-catenin have often been associated with the constitutive activation of wnt signalling pathway and has been established as a major driver of crc. one of the proposed mechanisms of activating wnt signalling involves extracellular vesicles (evs) as cellular couriers to transfer wnt ligands from one cell to another. however, the association of oncogenic mutant β-catenin with evs has not been studied. subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity methods: integrative proteogenomic analysis showed the secretion of mutant β-catenin via evs. evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. silac-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qpcr and xenograft models were employed to unveiling the role of evs carrying mutant βcatenin. results: an integrative proteogenomic analysis identified the presence of mutated β-catenin in evs secreted by colorectal cancer (crc) cells. follow up experiments established that evs released from lim1215 crc cells stimulated wnt signalling pathway in the recipient cells with wild type β-catenin. silac-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (rko crc) cells. in vivo tracking of dir labelled evs in mouse implanted with rko crc cells revealed its bio distribution, confirmed the activation of wnt signalling pathway in tumour cells and increased the tumour burden. introduction: there has been a significant increase in incidence of human papillomavirus 16 (hpv16) driven oropharyngeal cancer (opc) in developed countries. there is evidence that hpv alters the molecular cargo of exosomes released by opc. emerging evidence suggests that hpv integration within the human genome is associated with both genomic and transcriptomic alterations. consistent with previous studies, the genomic viral-cellular junctions were identified using dips-pcr method in 15 (88%) saliva samples collected from hpv16-driven opc. methods: morphology and molecular features of exosomes derived from three different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and salivary oral rinses were examined using transmission electron microscopy (tem), nanoparticle tracking (nta) and western blot analysis. hpv-16 dna detection in salivary exosome was determined by using qpcr method. proteome profile of salivary exosomes derived from both cancer-free controls and hpv16-driven opc patients was characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (lc-ms/ms). results: we demonstrate that unstimulated saliva had greater abundance of exosomes when compared to the other sampling methods. three common exosome markers (cd9, cd63 and cd81) were higher in unstimulated saliva. only salivary exosomes derived from hpv-driven opc patients had a detectable level of hpv-16 dna. the proteomic signature of salivary exosome was significantly (p < 0.01) different between cancer-free controls and hpv-driven opc. we found elevated protein abundance of five main glycolytic enzymes (i.e. phosphoglycerate kinase 1 (pgk1), glyceraldehye-3-phosphate dehydrogenase (gapdh), aldolase (aldoa) and lactate dehydrogenase a (ldha) in salivary exosomes derived from opc patients, suggesting a functional role of salivary exosome in the reciprocal interplay between hpv-driven opc and glucose metabolism. summary/conclusion: our data suggest that the development of a low-cost non-invasive saliva-based test using both salivary exosomal dna and protein may offer an opportunity to detect hpv-driven opc, that may be clinically useful in managing these patients. continuous in vivo release of mast cell derived extracellular vesicles from an implanted device spreads pro-inflammatory response in mice introduction: mast cells are important players of the immune system and they secrete a wide range of mediators during bacterial infections. mast cells are also able to release extracellular vesicles (evs). here, we report that mast cells communicate with each other in vivo by evs. methods: we isolated bone marrow-derived and peritoneal mast cells from gfp-transgenic and wild type mice. evs were separated from the conditioned media of these cells cultured in the presence or absence of lipopolysaccharide (lps). evs were characterised according to the misev2018 guidelines by flow cytometry, electron and fluorescent microscopy, trps, the spv lipid and the bca protein assays. separated ev-s were cultured with naïve mast cells, and tumour necrosis factor (tnf)-α production was tested by elisa and intracellular flow cytometry. gfp+ mast cells were seeded in diffusion chambers which were implanted into the peritoneal cavities of mice enabling us to investigate the continuous in vivo release of evs. uptake of gfp+ evs and tnf-α expression of peritoneal mast cells were tested by flow cytometry and fluorescent microscopy. results: here, we showed that bacterial lps-sensing mast cells release evs that in turn, induce tnf-α expression in resting mcs in vitro. moreover, we confirmed that evs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory response by inducing tnf-α secretion in peritoneal mast cells. summary/conclusion: ev communication between members of the mast cell network, play an important role in spreading and escalating pro-inflammatory responses to immune stimuli. our data may provide an explanation how the relatively rare tissue resident mast cells can play key roles in diseases such as autoimmune arthritis. the ability of small extracellular vesicles (sevs) to reprogramme cancer cells is known. integrins, receptors for extracellular matrix proteins, are major players in mediating sev functions. previously, we have reported that the αvβ3 integrin is detected in sevs of prostate adenocarcinoma (prca) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of prca, such as neuroendocrine prca (neprca). neprca does not express androgen receptor (ar) but does express neuron-specific proteins, such as aurora kinase a, synaptophysin and neuron specific enolase, that activate pro-tumorigenic pathways independently from the ar. methods: we isolated sevs from prca c4-2b cells using iodixanol density gradients and characterized them by immunoblotting and exoview. the experiments were performed in vivo by injecting subcutaneously, in nude mice, du145 cells treated with sevs expressing or lacking the αvβ3 integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sevs. discarded human tissues from prca metastasis were analysed by immunohistochemistry (ihc). results: we demonstrate that a single treatment of prca cells with sevs significantly stimulates tumour growth and anchorage-independent growth. moreover, we show that one treatment with sevs, shed from c4-2b cells that express αvβ3, but not from the control cells that lack αvβ3, induces differentiation of prca cells towards a neuroendocrine phenotype and downregulates ar. finally, our ihc analysis shows coexpression of αvβ3 integrin and synaptophysin in neprca metastatic lesions. summary/conclusion: in conclusion, our current study shows, for the first time, that αvβ3 integrin expression in donor cells generates sevs that reprogramme recipient cells towards an aggressive tumour phenotype. funding: this study was supported by nci-p01-140043, r01-224769 to lrl. introduction: exosomes are small extracellular vesicles (sevs) that carry a variety of cargoes and have been shown to promote tumour cell motility and metastasis. cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. however, how filopodia are induced and controlled by extracellular factors is poorly understood. here, we describe a role for sevs in regulating filopodia formation and tumour cell motility. we utilized b16f1 melanoma cells and ht1080 fibrosarcoma cells for fixed-and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. itraq proteomics was used to identify sev protein cargoes that contribute to filopodia formation. in vivo experiments were performed using a chick embryo model for metastasis. results: inhibition of exosome secretion in cancer cell lines, via rab27a or hrs knockdown, led to decreased filopodia numbers. specificity to sevs was demonstrated by rescue experiments in which purified sevs but not large evs rescued the filopodia phenotypes of exosome-inhibited cells. live imaging of hrs-kd cells revealed that exosome secretion regulates formation and stability of filopodia. proteomics data and molecular validation experiments identified the tgf-beta coreceptor endoglin as a key sev cargo regulating filopodia formation, cancer cell motility, and metastasis. summary/conclusion: in this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. these data are relevant to cancer as endoglin expression is altered in many cancers. in addition, endoglin is the disease gene for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. overall, our data implicate sev-carried endoglin as a key cargo regulating filopodia. astrocyte-derived ev-mediated blood-brain barrier disruption shilpa buch, ke liao, susmita sil, fang niu and guoku hu university of nebraska medical center, omaha, usa introduction: the breach of the blood-brain barrier (bbb), resulting in ensuing neuroinflammation, is a key feature of hiv-associated neurological disorders (hands). while combination antiretroviral therapy (cart) has successfully suppressed peripheral viraemia, cytotoxicity associated with the presence of viral tat protein in tissues such as the brain, remains a significant concern. our previous study has demonstrated that hiv-1 tat can induce disruption of bbb by downregulation of tight junction (tj) proteins in human brain microvascular endothelial cells (hbmecs) and that this is regulated by the autophagic pathways. methods: evs were isolated from hiv tat-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, nanosight & western blot analyses. among the various mirs dysregulated in hiv tat -stimulated astrocyte ev cargo, mir-7 was found to be upregulated by realtime pcr. confocal microscopy identified uptake of astrocytic evs by hbmecs. functional assessment of astrocytic ev uptake by hbmecs involved cell permeability using transepithelial electrical resistance as well as trans-well endothelial cell monolayer permeability assays. results: hiv-1 protein tat-mediated induction of micrornas (mirs) in astrocyte-derived extracellular vesicles (adevs) regulated the permeability of bbb by targeting the expression of tj proteins in the hbmecs. exposure of hbmecs to tat-adevs resulted in down-regulation of the tight junction protein claudin 5, resulting in increased endothelial cell monolayer paracellular permeability. microarray data of tat-adevs demonstrated upregulation of several mirs compared to that of controls, among which upregulated mir-7 was identified to target the tj proteins using ingenuity pathways analysis. increased expression of mir-7 was validated in tat exposed astrocytes and tat-adevs. adevs loaded with mir-7 oligos showed similar effects as that observed with tat-adevs in inducing permeability in hbmecs. increased expression of mir-7 with downregulation of claudin-5 was also recapitulated in microvessels isolated from the brains of doxycycline-inducible hiv-1 tat transgenic mice (itat) mice and in lysates isolated from the frontal cortices of siv+ macaques/hiv+ autopsied brains. summary/conclusion: our findings demonstrated that tat-adevs containing mir-7 as an important mediator underlying tat-mediated disruption of the bbb. introduction: endogenous exosomes and related extracellular vesicles (evs) are potent nanoparticles released by all cells tested to date. the exploitation of their unique scaffolding for engineering next-generation drug delivery systems represents a major area of academic and commercial interest. the lag in exploiting this potential is in part due to our inability to measured extent and efficiency of modification, e.g., composition and drug loading. here we report a robust pipeline of optical tweezing combined with raman spectroscopy to molecularly characterize engineered evs and quantitatively assess extent of drug loading at single particle resolution. methods: evs derived from cell culture and isolated by ultracentrifugation were fused with synthetic liposomes to create engineered evs (eevs). these eevs were formed via well-established vesicle fusion techniques, namely (1) mechanical extrusion, (2) freeze-thawing, or (3) probe-tip sonication. prior to formation, calcein was encapsulated in the liposomes and used as a surrogate for soluble drug loading. laser trapping raman spectroscopy (ltrs) was used to optically trap single evs, before and after synthetic manipulation. raman spectral analysis was used to assess trapped eevs compared to pure standards to quantify ratiometric variation in chemical composition. results: raman laser trapping experiments confirmed that each formation method results in largely varying (1) extent of fusion between evs and synthetic calceinloaded liposomes, (2) efficiency of calcein loading, and (3) particle size. we could also quantify the molar amounts of liposome vs. ev molecules for single particles, revealing a great amount of variation from particle to particle. functional membrane proteins we left intact to varying degree across fusion methods. summary/conclusion: given the rising importance of analytical tools able to characterize extent of molecular loading for engineered evs, we believe this technology will be very useful, thus warrants further investigation for eev characterization across a variety of clinical applications. funding: randy carney, phd was supported by a research scholar grant, rsg-19-116-01-cdd, from the american cancer society. extracellular vesicles containing host restrictive factor ifitm3 inhibited zika virus infection of foetuses in pregnant mice through trans-placenta delivery allen z. wu nanjing university, nanjing, china (people's republic) introduction: zika virus (zikv) infection can lead to neurological complications and foetal defects, and has attracted global public health concerns. effective treatment for zikv infection remains elusive and a preventative vaccine is not available yet. therapeutics for foetus need to overcome blood brain barriers to reach placenta and require higher safety standard. methods: in the present study, we engineered mammalian extracellular vesicles (evs) to deliver a host restrictive factor, interferon-induced transmembrane protein 3 (ifitm3), for the treatment of zikv infection. results: our results demonstrated that the engineered ifitm3-containing evs (ifitm3-exos) were overall safe to the animals and suppressed zikv viraemia by 2 log10 s in the pregnant mice. moreover, the engineered evs effectively delivered ifitm3 protein across placental barrier and suppressed overall zikv viraemia in the foetuses to the basal level with significant reduction of viraemia in key foetal organs as measured by q-pcr. mechanistic study showed that ifitm3 was delivered to the endosomes/lysosomes where it inhibits viral entry to the host cells. summary/conclusion: our study demonstrates that exosomes can act as a cross placenta drug delivery vehicle to foetus and ifitm3, an endogenous restriction factor that is highly expressed in placenta, is a potential treatment for zikv infection during pregnancy. introduction: extracellular vesicles (ev) are natural and abundant nanoparticles capable of transferring complex molecules between neighbouring and distant cell types. translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. important strategies to maximize the therapeutic potential of evs include payload loading, functionalization of the ev surface with pharmacologically active proteins, and delivery to target cells of interest. methods: through comparative proteomic analysis (lc/ms) of purified evs, we identified several highly enriched and ev-specific proteins, including a transmembrane glycoprotein (ptgfrn) belonging to the immunoglobulin superfamily. leveraging ptgfrn as a scaffold for surface display, we generated evs with functional targeting ligands, including single domain antibodies (sdabs), single chain variable fragments (scfvs), single chain fabs (scfabs), and receptor ligands, on the surface to direct ev uptake to cell types of interest. biological activity of these engineered evs was assessed in an array of in vitro and in vivo assays and compared to untargeted controls. results: we engineered evs displaying anti-clec9a scfabs to target conventional type 1 dendritic cells (cdc1s), anti-cd3 scfabs to target t cells, and cd40 ligand to target b cells. in mice, systemic administration of anti-clec9a evs resulted in a 75% increase in the percentage of cdc1 cells that take up evs over controls. anti-cd3 evs resulted in both an increase in the percentage of ev positive t cells (3.75 and 3-fold for cd4+ and cd8+) and the number of evs per cell (15 and 7-fold for cd4+ and cd8+) in the blood. furthermore, in primary mouse dendritic cells, anti-clec9a evs loaded with sting agonist achieved a 15fold greater pathway induction compared to untargeted controls. preliminary in vivo data suggest that anti-clec9a evs reduce the required sting agonist dose 10-fold to achieve efficacy and induce anti-tumour responses, compared to control evs. summary/conclusion: these results demonstrate the potential of our ev engineering platform to generate novel ev therapeutics targeted to cell types of interest for pharmacologic payload delivery. a novel method for the delivery of cell-free therapy to foetuses with congenital anomalies: a proof of principle study lina antounians, louise montalva, gabriele raffler, maria sole gaffi and augusto zani the hospital for sick children, toronto, canada introduction: antenatal cell-based therapies are currently considered invasive for the foetus. a promising cell-free strategy that holds great regenerative potential for several organs is the administration of stem cell derived evs, whose cargo contains bioactive molecules that epigenetically regulate target cells. herein, we aimed to 1) assess the ability of evs to reach foetal organs when administered to the mother intravenously or intra-amniotically; 2) compare these administration routes on normal foetuses and foetuses with a congenital anomaly. methods: evs were isolated from rat amniotic fluid stem cell conditioned medium using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd63, hsp70, flo-1, and tsg101 (western). we injected rat dams with evs stained by exoglow™-vivo or saline (control) via maternal tail vein (iv) or intra-amniotically (ia) at e20.5. ia and iv injections were performed on dams carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic hernia. after 24h, dams and pups were sacrificed. 3d high-sensitivity optical reconstructions of whole foetuses or micro-dissected foetal organs were imaged using the ivis® spectrum imaging system. ev fluorescence signal was compared between normal (n = 27) and nitrofen-exposed (n = 45) foetuses. results: both iv and ia injection routes were successful in delivering evs to foetal organs. no fluorescent signal was detected in saline only control. ia injections yielded higher signal than iv, and evs reached more organs with ia than iv injections. ia injected evs were detected in the lungs, gastrointestinal, and urinary tract of normal and nitrofen-exposed foetuses. nitrofen exposed foetuses had higher signal than normal foetuses. summary/conclusion: this proof of concept study shows that antenatal administration of stem cell evs is feasible with different routes. although maternally administered evs cross the placenta, ia injection is more effective at reaching foetal organs. further studies are underway to reproduce these findings in experimental models of various congenital anomalies. funding: cihr-sickkids foundation grant os22.5 introduction: safe, efficient and specific nano-delivery systems are essential to the current cosmetic, nutraceutical and therapeutic medicine sectors. the ability to optimise the bioavailability, stability, and targeted cellular uptake of bioactive molecules while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. ves4us is a european project, which aims to develop an innovative platform for the efficient production of extracellular vesicles (evs) from microalgae, which constitute a promising renewable bioresource (www.ves4us.eu). here we present characteristics of evs from several microalgal lineages, which offer the opportunity for a potentially developing a new and scalable tailor-made biogenic nanotechnology. methods: we cultivated a number of ev-producing microalgal species and developed protocols for ev isolation both at laboratory (differential ultracentrifugation) and pilot scales (tangential flow filtration). the physico-chemical characterization of microalgal evs was carried out according to the minimal information for studies of extracellular vesicles 2018 (misev-2018 guidelines): biochemical methods to verify the presence of specific ev-biomarkers, tuned for microalgal evs; dynamic light scattering (dls) and nanoparticle tracking analysis (nta) to assess the particles number and size distribution; electronic scanning microscopy (sem), atomic force microscopy (afm), and cryo transmission electron microscopy (cryo-tem) for imaging analyses; bilayer-specific fluorescence staining (f-nta) to test the purity of ev preparation. results: we identified microalgae as a novel natural source of evs that could constitute a cost-effective and sustainable way of mass-producing them. we screened 20 strains of microalgae and generated an "ev identity card" for each, which contained a variety of ev features relating to their biophysical, biochemical and biological characteristics in line with the misev-2018. our approach will next focus on the scalable production, surface functionalization and bio-engineering of selected microalgal evs. at the same time, their bioactivity will be explored using both in vitro and in vivo biological models. summary/conclusion: the ves4us consortium is investigating the potential of microalgae as novel ev bioresources. this research will attempt to bioengineer novel naturally-derived nanocarriers, microalgal evs, suitable for the development of future cosmetics, nutraceutical or therapeutic formulations. funding: this project has received funding from the european union's horizon 2020 research and innovation programme under grant agreement no 801338. sequence-specific rna trafficking to extracellular vesicles is conserved across cell types several sequences have been identified that act as a zipcode for preferential rna targeting into ev (evtropic) or for retention in parental cells (cell-tropic) . in this work, we aimed to compare the ev-tropic capacity of specific rna sequence motifs in promoting loading into ev, across different cell models representing the main cell types found in the body. methods: immune, epithelial and mesenchymal cell lines were transiently transfected with xenogeneic c. elegans micrornas (mirnas) containing ev-tropic or cell-tropic sequences and grown in culture. ev were isolated from the supernatant by differential (ultra)centrifugation. rna was extracted from both cell pellets and isolated ev fraction, and target mirnas were quantified by digital droplet pcr. distribution of cargo mirna across cells and ev was also analysed for chimeras of ev-and cell-tropic sequences. results: the mirnas containing an ev-tropic sequence were highly enriched on the ev fraction, with 1000-10,000 higher levels than in parental cells. contrarily, cell-tropic mirnas were only 10-100 times higher in ev. no significant differences were observed in the ev loading efficiency for the various ev-tropic motifs tested. mutations in the ev-sorting motif resulted in reduced ev loading. ev-tropic sequences consistently promoted mirna loading into ev across all the cell models evaluated, suggesting conserved biological mechanisms. summary/conclusion: we showed that rna loading into ev is dependent on the presence of defined evtropic rna motifs, and that sorting mechanisms are conserved across the major cell types tested. the highest loading efficiencies resulted in 0.001 mirna copies per particle on average, suggesting a limited scope for ev-tropic motifs for therapeutic rna loading into ev. funding: as, os and eli are fellows of the astrazeneca postdoc programme. introduction: coordinated activity between pancreatic islet cells is critical for the regulation of glucose homoeostasis. chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes. extracellular vesicles (evs) derived from cytokine-exposed β-cells modulate physiological and pathological responses to β-cell stress. however, the mechanisms governing this process remain largely unknown. we set out to test the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory evs which promote inflammation and inhibit βcell function. methods: pro-inflammatory cytokine-exposed evs (cytoevs) were generated using conditioned media from mouse min6 β-cell line treated with diabetogenic cytokines (tnfα, il-1β, ifnγ, 48h). evs were also isolated from human type 2 diabetic (t2dm) and lean non-diabetics (lnd) plasma. gw4869 (n-smase inhibitor) was used in the presence of cytokines to determine the effect of reduced ev concentrations on the restoration of β-cell function. proteomic and rna-seq analysis was conducted on min6 β-cell cytoev (vs. control ev) and cytoev treated mouse islets, respectively. results: assessment of ev concentrations from cytoev and human t2dm plasma revealed a~twofold increase (p < .05, vs. control (ctl) and lnd ev). immunofluorescence staining of cd9 and cd63 expression was significantly elevated in human t2dm pancreas (p < .05, vs. lnd). while acute inhibition of ev formation with gw4869 (5 µm) showed significant restoration in β-cell function (glucose stimulated insulin secretion assay, gsis) in cytokine-exposed mouse and human islets (~7 and 2 fold vs. cytokines alone, p < .05). moreover, functional assessment of mouse islets exposed to cytoev (48h) resulted in suppression of gsis (~55%, vs. untreated, p < .05). identification of cytoev content through proteomic analysis revealed a significant upregulation of the chemokine, cxcl10 (~40 fold vs. ctlev) and rna-seq analysis of cytoev treated mouse islets depicted a marked upregulation of transcripts associated with cxcl10-cxcr3 signalling (p < .001) and downstream pathways (e.g. nfκb; p = .016 and jak/stat; p = .021). furthermore, inhibition of cytoev (gw4869) with cytokines markedly decreased cxcl10 (~30%) and cxcr3 receptor (~65%) expression in min6 β-cells. summary/conclusion: these data suggests that cytokines elevate cxcl10 expression in β-cell ev to enhance inflammation-induced diabetes. this is mediated through ev-autocrine release of cxcl10 consequently activating cxcr3 signalling and downstream pathways to impair β-cell function in diabetes. synergy between 15-lipoxygenase and secreted pla2 promotes inflammation by formation of tlr4 agonists from extracellular vesicles introduction: damage associated molecular patterns (damps) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. during oxidative stress, stress-derived evs (stressevs) were found to activate toll-like receptor 4 (tlr4), but the activating ligands were not fully determined. additionally, several enzymes, among them 15-lipoxygenase (15-lo) and secreted phospholipase a2 (spla2) are induced during inflammation and were suggested to promote damp formation. methods: stressevs were produced from hek293 cells exposed to 10um a23187 and isolated with ultracentrifugation. 20:4 lysopi was oxidized for 10 min with 15-lo. additionally, synevs were prepared from phospholipids (pls), oxidized with 15-lo and hydrolysed with spla2. activity was measured by qpcr and elisa on wt and tlr4-ko macrophages. 15-lo oxidized 20:4 lysopi was analysed by mass spectrometry. spla2 activity was measured in synovial fluid from rheumatoid and gout patients using fluorometric assay. k/bxn serum transfer induced arthritis model on wt and tlr4 ko mice (c57bl/6 mice) with spla2-iia injection was used (approval no. u34401-14/2019/8 by mkgp of slovenia). results: stressevs released after oxidative stress were found to activate tlr4 with a gene profile different from bacterial lipopolysaccharide (lps). stressevs, 15-lo oxidized synevs, but only 15-lo oxidized lysopls activated cytokine expression through tlr4/md-2. hydroxy, hydroperoxy and keto products of 20:4 lysopi oxidation were determined by ms and they activated the same gene pattern as stressevs. furthermore, spla2 activity, which we detected in the synovial fluid from patients, promoted formation of tlr4 agonists after 15-lo oxidation. injection of spla2-iia into mice promoted k/bxn serum induced arthritis in tlr4-dependent manner. summary/conclusion: both 15-lo and spla2 are induced during inflammation, therefore these results imply the role of oxidized lysopls in stressevs in promoting sterile inflammation through tlr4 signalling. the formation of tlr4 agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens. funding: h2020-msca-itn project tollerant (grant no. 642157), slovenian research agency (project no. j3-9257 to mmk, research core no. p4-0176 to rj). monocytes traffic extracellular vesicles to damaged muscle and adopt a novel immunophenotype to support muscle regeneration russell g. rogers, akbarshakh akhmerov, weixin liu, lizbeth sanchez and eduardo marbán smidt heart institute, cedars-sinai medical center, los angeles, usa introduction: extracellular vesicles (evs) are secreted membrane vesicles that carry bioactive molecules such as mirnas, mrnas, proteins, and lipids to modify recipient cell behaviour. we recently demonstrated evs secreted by cardiosphere-derived cells (cdc-evs) augment endogenous muscle regeneration in mdx mice, a model of duchenne muscular dystrophy, when delivered intravenously. in parallel, macrophages preferentially accumulate surrounding small regenerating myofibers in cdc-ev treated mdx muscle. however, it is currently unclear how intravenous cdc-evs home to dystrophic muscle and exert their therapeutic bioactivity. methods: fluorescently-labelled and unlabelled cdc-evs were infused into the contralateral femoral vein of wild-type mice with unilateral muscle injury induced by bacl2. injured and uninjured muscles were dissected 24h following infusion and subjected to optical imaging, immunohistochemistry, and confocal microscopy. this experiment was repeated using clodronate liposomes to deplete endogenous monocytes/macrophages. next, rna-seq was preformed on bone marrow-derived m1, m2, and cdc-ev (mcdc-ev) polarized macrophages from mdx mice. conditioned media (cm) from these macrophages were tested in an in vitro model of myogenesis. lastly, small rna-seq was performed on evs secreted by m1, m2, and mcdc-ev macrophages. results: when delivered intravenously, cdc-evs naturally home to injured, but not uninjured, skeletal muscle. cdc-evs were detected in the interstitium adjacent to non-muscle cells, macrophages, and within surviving myofibers. after depletion of monocytes/ macrophages by clodronate liposomes, the presence of cdc-evs in the injured muscle was attenuated. bioinformatic analyses indicate cdc-evs confer a novel immunophenotype to mdx macrophages with features of both m1 and m2. indeed, mcdc-ev cm promotes myoblast proliferation and supports myogenic differentiation. interestingly, mcdc-ev evs have a unique mirna signature and contain several mirnas with known roles in myogenesis. summary/conclusion: these data indicate circulating monocytes traffic cdc-evs to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration. we propose mcdc-ev macrophages mediate their pleiotropic effects via paracrine factors, possibly including evs. introduction: microglia, the immunocompetent cells of the cns, play an important role in maintaining cellular homoeostasis in the cns. these cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. the contribution of microglial-derived extracellular vesicles (m-evs) to the maintenance of cns homoeostasis is unclear. in addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to m-evs is scarce. methods: here, we analysed the effects of m-evs produced in vitro by either tnfα-activated or non-stimulated microglia bv2 cells. we showed that m-evs are internalized by both mouse bv2 and human c20 microglia and that the uptake of m-evs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. consistently, exposure of microglia to m-evs increased the protein expression of the autophagy marker, lc3b-ii, and promoted autophagic flux in live cells. to elucidate the biological activities occurring at the transcriptional level in c20 microglia exposed to m-evs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted rna sequencing. results: inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia exposed to m-evs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. summary/conclusion: we demonstrate that in vitro produced microglial evs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homoeostasis. funding: this work was financed by hasselt university and by efro through the interreg v grensregio vlaanderen nederland project trans tech diagnostics. evaluation of plasma extracellular vesicles as biomarkers for longevity xin zhang a and virginia kraus b a laboratory medicine center, nanfang hospital, southern medical university, guangzhou, guangdong, 510515, p. r. china, guangzhou, china (people's republic); b division of rheumatology, duke molecular physiology institute, duke university school of medicine, durham, usa introduction: extracellular vesicles (evs) have emerged as key indicators and effectors of ageing. although plasma concentrations of evs decline with age, the ev biomarkers associated with ageing and longevity are not fully understood. recently, our group found an age-related decline of plasma evs associated with immune cells during normal human ageing. our study aims to evaluate the association of plasma evs with longevity. methods: plasma samples were selected from the established populations for epidemiologic studies of the elderly study subjects (n = 48): half dying within 2 years (short-lived group) and half surviving ≥10 years (long-lived group) after the blood draw; all matched for age (median age 77.3 ± 1.7 years, range 72-80), gender (50% female), and race (50% white/50% black). the samples were acquired under donor consent and irb approval of duke university. evs were separated from the plasma samples, and profiled based on the surface markers of haematopoietic stem cells (hscs), mesenchymal stem cells, immune cells, skeletal muscles, cardiac muscles and adipocytes (cd81, cd9, cd29, cd63, cd8, cd4, cd68, cd14, cd56, cd15, cd19, cd235a, cd41a, cd34, cd31, hla-abc, hla-g, hla-drdpdq, cd90, cd73, cd105, m cadherin, ryr1, ryr2, fabp4, dlk1). the percentages of evs expressing each tested molecule were determined using a high-resolution multicolour bd lsr fortessa x-20 flow cytometer as we recently reported. graphpad prism 8.0 software was used for statistical analysis. results: we found significantly increased percentages of cd9+, hla-abc+, cd31+ and cd41a+ large evs (1000-6000 nm) in the long-lived compared to the short-lived group. none of the tested surface marker expressing medium (100-1000 nm) or small (<100 nm) evs showed differential percentages between the shortand long-lived groups. summary/conclusion: evs carry surface markers from their parent cells. cd9 is expressed by hscs and immune cells. cd9 regulates homing of human cord blood cd34+ hscs, and delivers a potent cd28independent costimulatory signal to activate t cells. hla-abc, the key human immunogen, is expressed by nucleated cells and platelets. cd31 is expressed by hscs, immune cells and epithelial cells, and cd31 + plasma evs declined with age in healthy people. cd41a is expressed by hscs, megakaryocytes and platelets, and is functionally relevant for hsc maintenance and haematopoietic homoeostasis. our preliminary data suggest that hscs and immune cell associated plasma evs (cd9+, hla-abc+, cd31+, cd41a+ large evs) inform on health status related to longevity. introduction: it is anticipated that stem/progenitor cells-derived extracellular vesicles (spc-evs) will rapidly progress towards clinical studies, and the development of reproducible, efficient, scalable and costeffective process for their production is expected to boost the therapeutic applications of evs-based products. in addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for spc-evs production in terms of reproducibility, stability and quality, while ensuring the approval of regulatory agencies. the main goal of this work is to develop a full-controlled manufacturing platform for the spc-evs production. methods: human mesenchymal stromal cells (msc) were expanded in a xeno-free microcarrier-based bioreactor culture system operating in fed-batch feeding mode and after 10 days the conditioned medium was collected. different methods for spc-ev isolation/purification from the msc-derived conditioned medium, including chromatography were compared and the the quality of the final product obtained was characterized by different methods according to misev, including nanoparticle tracking analysis, lipidomics and western blot. moreover fourier-transform infrared (ftir) spectroscopy was evaluated in terms of its implementation as a standard technique for the identification and characterization of evs. results: after 10 days of msc expansion under dynamic conditions, we collected 1.3 l of conditioned medium with approximately 0.5 million evs/msc. a combination of a pretreatment with a nuclease for the digestion of dna/chromatin with a purification using strong anion exchange chromatography led to the best results so far in terms of evs isolation. of notice, by ftir spectroscopy, it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture conditions tested. summary/conclusion: the platform established herein could be applied to the production of wellcharacterized spc-evs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as evs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer. ultrasensitive protein detection for quantification of extracellular vesicles in human biofluids enables comparison of isolation techniques dmitry ter-ovanesyan, maia norman, wendy trieu, roey lazarovits, george church and david walt wyss institute, boston, usa introduction: extracellular vesicles (evs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. one of the main challenges in studying evs and using them in diagnostics is a lack of suitable methods to quantify evs that are sensitive enough and can differentiate evs from similarly sized lipoproteins and protein aggregates. we propose using ultrasensitive single molecule array (simoa) assays to quantify evs by immunoisolating and detecting ev transmembrane proteins in microwell arrays. we developed single molecule array (simoa) assays using the quanterix hd-x analyser for the quantification of evs using the tetraspanins cd9, cd63, and cd81. simoa allows for the detection of single proteins using arrays of femtoliter wells, turning elisa into a digital immunoassay. we then used these assays, together with an additional assay for albumin, to compare commonly used ev isolation methods from plasma and cerebrospinal fluid (csf): ultracentrifugation, precipitation (exoquick), and size exclusion chromatography (sec) using the izon qev columns. we further used these assays to rapidly optimize and improve sec by comparing different sec resins and column dimensions in both plasma and csf. results: in comparing our simoa assays to traditional elisa with the same antibodies, we found that the simoa assays were more than 100 times more sensitive, detecting the tetraspanins in samples where the proteins were undetectable by elisa. given the high dynamic range and high-throughput capabilities of simoa, we were able to comprehensively compare relative ev yields and ev purity for different isolation methods of evs from plasma and csf. we provide average tetraspanin and albumin levels to directly compare the methods. we also tested different sec resins and provide data for custom sec columns that outperform izon qev and allow for fine tuning of different ratios of evs to albumin. summary/conclusion: our results highlight the utility of quantifying evs using ultrasensitive simoa assays for tetraspanins. we were able to rapidly simoa to rapidly evaluate different ev isolation methods in csf and plasma. in general, the experimental framework we present could be easily applied to evaluate new ev isolation methods, or applied to any other biological fluid. thus, we think simoa is a powerful new tool for relative ev quantitation. introduction: the protein profile of extracellular vesicle (ev) subpopulations has been shown to contain valuable disease information, notably in cancer. currently, techniques aiming to find ev proteins that associate together mainly focus on transmembrane proteins, while methods that also probe cytosolic proteins generally resort to a combination of affinity capture, elution, and lysis, which limits throughput. to allow the high-throughput analysis of both membrane and cytosolic ev proteins, we optimized a total extracellular vesicle antibody microarray (tevam) incorporating fixation and heat-induced epitope retrieval (hier), then leveraged it to perform combinatorial protein profiling of evs from colorectal cancer (crc) cell lines ht29 and sw403. methods: arrays of iggs targeting surface protein markers were incubated overnight with evs purified from cancer cell line supernatants. hier optimization was carried out through variation of buffer contents, presence or absence of prior permeabilization, as well as incubation time and temperature, for a total of 38 conditions. a431 evs, previously profiled with other methods, were used as a model during the optimization. cytosolic protein hsp90 and membrane marker egfr, both with high expression in a431 evs, were probed and the results used to compare hier conditions. following hier treatment, protein targets were detected through incubation with primary antibodies and fluorescent secondary antibodies or streptavidin. the resulting optimized tevam workflow was used to phenotype ht29 and sw403 evs through probing of trios of surface (2) and internal (1) protein targets. results: the selected tevam protocol successfully maximized hsp90 signal while minimally affecting egfr detection, enabling simultaneous analysis of surface and internal proteins. profiles of more than 450 combinations, featuring integrins, claudins, cytokines, and other key actors of cancer-relevant pathways, were obtained for ht29 and sw403 evs, revealing coexpression patterns that highlight the biomolecular heterogeneity both within and between crc cell line evs. summary/conclusion: using tevam, intra-and extravesicular proteins can be detected simultaneously in evs immobilized based on surface protein content, yielding extensive combinatorial protein profiles with significance for health and biomarker research. characterization of evs using orthogonal techniques identifies discrete ev populations from a mouse dendritic cell line bryce killingsworth a , timothy traynor b , joshua a. welsh c , aleksandra dakic a , jason savage a , kevin camphausen d , kenneth aldape a and jennifer jones a a laboratory of pathology, national cancer institute, national institutes of health, bethesda, usa; b laboratory of pathology, national cancer institute, national institutes of health, gaithersburg, usa; c laboratory of pathology, national cancer institute, national institute of health, bethesda, usa; d radiation oncology branch, national cancer institute, national institutes of health, bethesda, usa introduction: extracellular vesicles (evs) have the potential to serve as valuable biomarkers for patient response to cancer therapy. however, development of robust ev-based clinical assays relies on knowledge of ev concentration and diameter distribution. many different methods exist to measure the size and concentration of evs, and each method exhibits strengths and limitations. it is important to use orthogonal methods for determination of these important properties of ev preparations. here, we use dendritic cellderived evs to demonstrate that some ev analysis methods can give a biased interpretation of both diameter and concentration. through comparison, we highlight why orthogonal assays are essential in providing measurement reliability. methods: dc2.4 mouse dendritic cells were cultured in flasks containing a total of 1.2 l of ev-depleted media (10% fbs, centrifuged 18 hr. x 100,000 g.) when cells reached 80% confluency, conditioned media was collected, depleted of debris with two 10 min. x 2,500 g spins, and concentrated down to~5 ml using a pall jumbosep 100 kda mwco filter. the ev concentrate was purified from protein using an izon qev-10 column, with 5 ml fractions collected. the protein content of the ev-containing fractions was analysed by a280, pierce bca, and bioanalyzer. the diameter distribution of the evs was determined by nanoparticle tracking analysis (nta), resistive pulse sensing (rps), flow cytometry (fcm), and electron microscopy (em.) concentration was compared using nta, rps, and fcm. evs were further analysed by protein mass spectrometry and rna sequencing. results: we have identified two distinct populations of evs with our dc2.4 preparation, one highly abundant population with a power-law distribution, whose peak diameter is below 60 nm, and a second, less abundant population with a peak diameter at approximately 140 nm. these two distinct populations and their relative concentration were not detectable with all analysis techniques. based on cross-platform measurements, these populations appear to have distinct compositions that warrant further investigation. summary/conclusion: the use of orthogonal methods allowed the detection of two discrete populations of evs which was not possible on some platforms and would have resulted in a biased perspective of the sample composition. this work has highlighted the need for orthogonal measurements to be conducted by pairing techniques that do not have the same biases. introduction: extracellular vesicles (evs) are nanosized vesicles shed by all cells that serve vital roles in cell-to-cell communication. tumour-associated ev subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules), enabling minimally invasive spectroscopic analysis for a wide variety of cancers. here, we use surface-enhanced raman spectroscopy (sers) in combination with a novel plasmonic substrate for global chemical composition analysis of cancerous and non-cancerous populations of evs to determine distinguishing surface characteristics. methods: evs were isolated from ovarian cancer (ovca) patient serum samples by differential ultracentrifugation. a new hybrid nanoplasmonic scaffold comprised of a microscale biosilicate diatoms embedded with silver nanoparticles (agnps) was used for sers measurements. the substrate was incubated with cysteamine to positively-charge the agnps (responsible for the sers enhancement) so that evs could attach (evs are naturally anionic). in a typical experiment, 40 μl of~108 particles/ml evs per sample were incubated with the porous substrate surface, which was inverted on a glass cover slip for raman interrogation. principle component analysis (pca) was used to compare the spectra and determine distinguishing characteristics between populations from tumour and non-tumour sources. we also trypsinized evs before sers analysis to see the extent of influence the surface molecules play in localizing the evs to the agnp "hot spots." results: a total of 8 clinical samples (7 ovca and 1 non-malignant control) were tested in combination with ovca skov-3 cell line evs. simple pca was able to separate clinical samples according to disease subtype and major peaks were identified to provide chemical content analysis. each sample exhibited inherent heterogeneity but clustered together in a distinguishable way from the others. summary/conclusion: despite innate heterogeneity within single samples (i.e., evs isolated from a single patient sample), evs isolated from clinical samples could be easily distinguished from each other using our hybrid sers substrate, with minimal sample processing, a label-free approach, and only a few microlitres of sample. our study using this novel plasmonic material demonstrates its potential for use as a component in next-generation diagnostic platforms. introduction: single-particle analysis is critical for understanding extracellular vesicle (ev) heterogeneity. yet such techniques remain technically challenging due to low detection sensitivity and presence of variable amounts of "contaminants," including lipoproteins. the high degree of structural similarity between evs and lipoproteins in size, density, and chemical composition, results in their co-isolation using any of the standard ev isolation techniques. here we introduce laser trapping raman spectroscopy (ltrs) as a wellsuited, label-free, and non-destructive tool to distinguish evs from various lipoprotein species at single particle resolution. methods: ev samples were isolated from skov-3 cell culture supernatant by differential ultracentrifugation and their raman spectra measured. as the most abundant lipoproteins in ev isolations from human biofluids are sub-micron low density lipoprotein (ldl), very low density lipoprotein (vldl), and high density lipoprotein (hdl) particles, these were purchased as pure components and also measured by ltrs. ldl and vldl were then spiked-in to isolated evs to mimic "contaminated" post-isolation ev samples. raman spectra were analysed by principal component analysis (pca) using a custom matlab script. results: ldl and vldl have been observed to adhere to ev surfaces in vitro after standard isolation techniques. we could readily distinguish pure vldl, ldl, and hdl standards according to their raman spectra. pca revealed distinction of skov-3 evs from both ldl and vldl. pca also differentiated skov-3 evs incubated with ldl from skov-3 evs incubated with vldl. extent of ldl and vldl adherence to evs could be observed and quantified. summary/conclusion: through raman and pca, classes of lipoprotein and evs can be identified and quantified when co-incubated. ltrs is a quantitative single-ev analysis technique that can be used to differentiate between lipoprotein classes and evs when incubated together. this technique allows for analysis of evs where standard isolation methods fall short. introduction: extracellular vesicles (evs) are endogenous membrane-derived vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the cns and contributing to neurodegenerative conditions. although evs hold great potential as cns theranostic nanocarriers, the specific molecular factors that regulate neuronal ev uptake and release are currently unknown. methods: we used a combination of patch-clamp electrophysiology and ph-sensitive dye imaging to examine stimulus-evoked ev release in individual neurons in real time. results: whereas spontaneous electrical activity and the application of a high-frequency stimulus (hfs) induced a slow and prolonged fusion of multivesicular bodies (mvbs) with the plasma membrane (pm) in a subset of cells, the neurotrophic factor bfgf (basic fibroblast growth factor) greatly increased the rate of stimulusevoked mvb-pm fusion events and, consequently, the abundance of evs in the culture medium. proteomic analysis of neuronal evs demonstrated bfgf to increase the abundance of the v-snare vesicle-associated membrane protein 3 (vamp3, cellubrevin) on evs. conversely, knocking-down vamp3 in cultured neurons attenuated the effect of bfgf on ev release. introduction: heat shock proteins (hsps) function as chaperones under both normal and pathologic conditions. as chaperones they assist in protein folding, in holding protein complexes for current or future activation, and in degradation of senescent proteins for recycling of components and display for immune surveillance. during stressful situations, hsp quantities and/or activities increase as cells and tissues seek protection from insults. these insults can result in the cell surface display of hsps, which can then lead to the surface display of hsps on extracellular vesicles (evs). hsps present on the cell surface or in the extracellular space are regarded as "danger signals" in an ancient biologic paradigm. hsp-accessorized evs may act as "danger boli", carrying not only the hsps, but hundreds of components of the stressed parental cell, capable of prompting differential responses depending on the status of the recipient cell. methods: clarified/filtered plasma from patients suffering from neurologic maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed to bind hsps. the evs congeal under these conditions and are pelleted (microfuge) and washed with increasing-stringency buffers. we lysed the evs and subjected them to metabolomic analyses (focused on lipids) or assayed them on phosphokinase arrays. results: we show that evs from the blood of patients suffering from brain tumours, or from tbi, or from ms, possess distinct metabolomes compared to blood evs from healthy donors. we found hundreds of differentially-expressed lipids amongst the patients vs the healthy donors. the levels of annotation and identification for these compounds ranges from level 4 (low, no matches in databases) to level 2 (high, annotation matches to known database components). in addition, we found differences in phosphorylated kinases as cargo in these evs between patients with matched primary vs recurrent gliomas, and among tbi/stroke patients compared to healthy donors. summary/conclusion: hsp-accessorized evs present different metabolomic and phosphokinase content which may serve as biomarkers in a "liquid biopsy" setting, but may also play roles in the pathobiology of neurologic diseases. introduction: methamphetamine (ma) has deleterious effects to both peripheral organs and the central nervous system. the rewarding properties and addictive potential of ma are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. in rodents, ma alters brain mirna expression and the mirna content of serum extracellular vesicles (ev). here we examined plasma evs isolated from human subjects actively using ma (ma-act) for size, concentration, protein markers, and mirna content. methods: plasma samples from 10 ma-act, and 10 controls (ctl) were obtained from the methamphetamine abuse research center. plasma evs were evaluated by vesicle flow cytometry (vfc) for size, concentration, and surface protein markers. vfc antibodies included markers for a pool of tetraspanins (cd9, cd63, and cd81), platelet evs (cd41), pro-coagulant evs (annv), and red blood cell evs (cd235). next plasma ev isolated by size exclusion chromatography were analysed by qpcr on taqman® array human microrna a + b card set v3.0. fold change was calculated by δδcq between ma-act and ctl for mirna expressed in ≥ 60% of samples in at least 1 group. we identified the top 20% of ranked mirna by fstatistic; of these, the mirna of interest for ma-act were identified by at least a (i) 1.2 fold change in expression, (ii) area under the receiver operating characteristic curve of 0.75, and (iii) glass's δ of 1. for mirna of interest correlations to additional ma variables were conducted, along with ingenuity pathway analysis of predicted gene targets. tobacco use was controlled for. results: vfc data show that the size (~110 nm) and concentration (~7.5 x 1010 particles/ml) of all plasma evs is comparable between ma-act and ctl groups. in addition, the plasma evs primarily consist of tetraspa-nin+, annv+, or cd41+ evs, and to a much lesser extent cd235+ evs. of the 207 mirna expressed in ma-act and/or ctl plasma evs, there were 110 mirna that have at least a 1.2 fold increase or decrease in ma-act. 10 mirna were identified to be of interest in ma-act based on fold change, effect size and diagnostic potential, compared to ctl. further, 5 of the 10 mirna correlate with a ma associated variable, including frequency of use and age of first use. together the 10 mirna best cluster subjects based on ma-act status, not tobacco use. finally, the predicted gene targets of the 10 mirna are associated with canonical pathways linked to ma. summary/conclusion: ev mirna expression in ma-act subjects was unique to ctl participants, suggesting that ma may affect ev communication among cells. the differential mirna expression also implicates a role for evs in behavioural and physiological effects specific to ma, and suggests that there may be changes in expression of mirna that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. bone marrow-derived extracellular vesicles may alter the ageing phenotype of murine haematopoietic stem cells. sicheng wen a , jill kreiling b , mark dooner c , elaine papa c , michael del tatto c , yan cheng c , mandy pereira c , peter quesenberry c and laura r. goldberg d in natural ageing of haematopoietic stem cells (hscs) is unclear. we tested the hypothesis that bone marrowderived evs (bm-evs) can modulate the ageing hsc phenotype. methods: we flushed bone marrow from old (24-26month old) and young (6-8-week old) c57/bl6 mice and collected bm-evs by differential centrifugation (2000 × g for 30 min, supernatant collected and centrifuged 100,000 × g for 1 hour, bm-ev pellet collected and quantified by nanoparticle tracking analysis). we injected old mice with 2 x 10^9 young bm-evs via tail vein, daily x 3 days. control mice were injected with age-matched bm-evs or vehicle alone. we euthanized the mice one month post-injection, harvested whole bone marrow (wbm) and highly purified hscs (lineage negative/c-kit+/sca-1+/cd150+; lsk-slam) and tested stem cell function in competitive bone marrow transplants (4-5 recipients/group). results: at 6 months post-transplant, wbm from old mice exposed to young bm-evs exhibited a statistically significant decrease in engraftment when compared to wbm exposed to age-matched bm-evs (percent average donor chimerism ± sem: 15% ± 5% (young evs) vs. 61% ± 14% (old evs)). for lsk-slam from old mice, we observed a trend towards decreased engraftment when exposed to young bm-evs and a trend towards increased engraftment potential when exposed to old bm-evs (percent average donor chimerism ± sem: 7% ± 4% (young evs), 27% ± 10% (old evs), 15% ± 2% (vehicle)). these findings are consistent with our previous data showing that, in contrast to highly purified hscs which develop impaired stem cell function with ageing, total un-separated old wbm actually has increased engraftment capacity when compared to young wbm. of note, we found that the classic myeloid skewing by old lsk-slam was partially reversed by exposure to both young and old bm-evs. finally, consistent with the known increase in highly purified hscs with age, our preliminary data showed that old mice exposed to young bm-evs had an approximately 7-fold decrease in the number of lsk-slam cells in marrow, indicating that bm-evs may influence agerelated changes in hsc number. summary/conclusion: these preliminary data suggest bm-evs may play a role in modulating hsc ageing phenotypes, potentially altering engraftment capacity, lineage fate, and lsk-slam population size. future studies delineating the molecular mechanisms underlying these ev-mediated effects could provide key insights into normal haematopoietic ageing. funding: this work was supported by the nih grants 5p20gm119943, dk112808-01a1 and by the savit foundation. oral with poster introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr. results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom20. mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the biogenesis of evs is neutral sphingomyelinase 2 (nsmase2), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase2 is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase2 inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles. methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo [1,2-b] pyridazin-8-yl) pyrrolidin-3-yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated 5xfad mice with 10 mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay. results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, 5xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting 20% of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human post-mortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of 20 mdd subjects and 20 hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed. results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd9. tem images showed the typical cup-shaped morphology with sizes mostly between 30 and 200 nm. preliminary sequencing results revealed that mir-33a-5p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir-33a-5p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. op3.04 = ps15.04 combining nanomagnetic isolation and artificial intelligence to guide the treatment of traumatic brain injury zijian yang a , kryshawna beard b , david meaney b , danielle sandsmark b , ramon diaz-arrastia a and david issadore c a university of pennsylvania, philadelphia, usa; b university of pennsylvania, philadelphia, usa; c department of bioengineering, university of pennsylvania, philadelphia, usa introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of 7 protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores (600 nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the 7 protein biomarkers (tau, uchl-1, nfl, gfap, il6, il10, and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl1 were elevated in mtbi patients compared to controls (p < 0.05), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il6 and tnf-with tau from glur2+ evs showed 88% accuracy with 80% sensitivity and 100% specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. l1cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l1cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l1cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l1cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l1cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd9, cd63 and cd81) as well as l1cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l1cam appears. we also immunocapture l1cam from csf and plasma and perform western blots for the internal and external domains of l1cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l1cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l1cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l1cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l1cam in plasma is likely not coming from the brain. this data call into question the utility of l1cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant 3d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed 3d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in 6-month-old wt mice, (n = 6/groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for 15% of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy5y cells with n-myc amplified sk-n-be2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. op3.08 = ps15.08 introduction: quantification and characterization of single extracellular vesicles (sevs) based on surface markers can aid in dissecting the heterogeneous landscape of ev subpopulations. we and others have demonstrated the potential of imaging flow cytometry (ifc) to perform sev characterization. we recently showed release of protoporphyrin (ppix) positive sevs by 5-aminolevulinic acid (5-ala) dosed glioma cells, in vitro and in vivo. rickfels et al. also used ifc to demonstrate the enrichment of cd63+/cd81+ evs in the plasma of glioma patients. herein, we performed in vitro studies to characterize ev subfractions using 5-ala as well as ev and cns specific surface markers. methods: we use ifc to characterize evs released by glioma using 5-ala, fluorescently labelled ev (cfda-se, cd81) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd81) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that 58% are tenascin c positive, 2.7% are egfrviii positive and 1.6% are 5-ala positive. there was only a minor overlap (<16%) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs (2.5-fold), tumour specific evs (2.8-fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: f. graminearum (fgr) and f. oxysporum f. sp. vasinfectum (fov) are severe fungal pathogens of cereals and cotton, respectively. fgr and fov cause economic losses and threaten food and fibre supplies worldwide. understanding host-pathogen interactions is crucial for developing new strategies for disease control. we are determining whether extracellular vesicles (evs) have a role in the interaction between fungal pathogens and their host plant. methods: we isolated evs from fgr and fov by sizeexclusion chromatography and characterized them by nta and tem. evs from fgr and fov are between 100-300 nm and have morphology similar to evs reported for other fungi. we performed label-free quantitative proteomics to describe the protein cargo of evs from fgr and fov, including a comparative study of evs from fov grown on different media: czapek dox (cd) and saboraud's dextrose broth (sdb). results: a total of 658 proteins were detected in fgr evs and, according to prediction software effectorp, 12.5% of these were potential effectors. similarly, 70% of ev proteins do not contain signal peptide indicating that packaging into evs is a novel mechanism of secretion for these proteins. notable fgr ev proteins include lipases, proteases and synthases for toxins and chitin. fov produced evs in similar quantities in both growth media tested, but ev protein cargo differed between them. there was a 39% overlap in proteins identified in the 465 cd and the 658 sbd ev proteins. in general, ev proteins were involved in metabolism, cell wall architecture and oxidoreduction, with 15.4% and 12.9% of potential effectors, respectively. polyketide and toxin synthases, proteases and effectors were present in both types of fov evs. summary/conclusion: this new fungal ev isolation method was rapid, yielded high-quality evs, and did not submit particles to high centrifugal forces. our data revealed that both fgr and fov produce evs enriched with proteins that could alter host immune responses or facilitate fungal infection. furthermore, the protein composition of fov evs was dependant on culture conditions. this supports a potential role for fungal evs in disease progression in plants and provides the foundations to pursue the role of evs in plant-fungal interactions with the potential to identify new targets for disease control. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll 2 receptor (tlr2) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il-12 and il-6, which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells. objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow fieldflow fractionation (af4) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp-1) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk-2 epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with 50 to 50 nm (ev1) and another with 100 to 120 nm (ev2). ev1 induced more tnf-alpha, il-6, ip-10 and ccl20 than ev2. it was also more effective in promoting t. cruzi infection in epithelial cells. summary/conclusion: t. cruzi released two ev populations that affects differently host cells. identification of these evs composition might help to better understand the role of evs in the modulation of t. cruzi infection funding: fapesp, cnpq and capes op3.11 = ps15.11 commensal bacterial extracellular vesicles act as carriers for norovirus sutonuka bhar, melissa jones, annalise galbraith and mariola edelmann university of florida, gainesville, usa introduction: human norovirus (hunov) are one of the most common causes of gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have a massive economic impact resulting in approximately 60 usd billion each year in healthcare costs and missed worker productivity. development of anti-viral therapies for hunov has been hampered by the lack of robust in vitro cultivation systems. several cell types support viral replication but only produce modest amounts of virus due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays. noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr. isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and 0.2um filtration. co-purification of norovirus with the evs was checked. ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid50 assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems. detection of bacterial extracellular vesicles in blood from healthy volunteers kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of 11.5 µg/ml of plasma, as geometric means ≥11.5 µg/ml were nearly equal. hevs were detected at 48 µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f200x electron microscope and measured between 40-90 nm, and hevs were between 60-160 nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: new zealand (nz) has a population of just 4.8 million people with a remote geographical location in the pacific ocean. its unique culture, food-based industries and ethnic population make nz an invaluable place for extracellular vesicle research into all areas. however, as for many places in the world, standardization of methodologies, training and access to appropriate equipment is challenging. methods: the hub for extracellular vesicle investigations (hevi) is a virtual research centre established in 2017 with three-year seed funding from a university of auckland strategic research initiatives fund. two staff members are employed to support training, education and optimization of methods. the hevi is guided by a governance group providing valuable input from australasian experts in evs. results: since 2017 the hevi has organized 2 research symposia, 2 hands-on training days, hosted 2 international students as well as providing one-on-one training for 41 individuals from universities and institutes across nz. training is provided on multiple isolation and characterization methods and tailored to individuals access to essential equipment without bias towards individual manufacturers or techniques. travel funding has supported 38 people to attend conferences and workshops for the purposes of education, networking and research dissemination. the hevi also provides support for project design with 10 grants awarded to hevi members and a number of manuscripts in submission for publication. the embo practical course "extracellular vesicles: from biology to biomedical applications" is organized each year by a group of 4 researchers active in the ev field in collaboration with the embl advanced training center in heidelberg. the course focuses on training phd students and postdoctoral researchers who enter or are already active in the field of ev research. given the large number of methods and protocols that is being used by researchers in the ev field, the organizers aim to provide practical guidance to new researchers and teach them appropriate skills. methods: participants obtain theoretical knowledge and hands-on experience on different ev purification and characterization techniques, such as fluorescent labelling, density gradient centrifugation, size exclusion chromatography, electron microscopy, flow cytometry and nanoparticle tracking analysis and on databases like ev-track and funrich. in addition, the organizers and invited lecturers from several different research areas explain which strategies are used to understand the role of ev in biomedical applications and give an overview of the current state of the field of ev research. results: the course therefore covers a broad range of topics important in the ev field, including heterogeneity in ev subpopulations, mechanisms of ev cargo selection, ev biogenesis, pre-analytical variables, therapeutic and diagnostic use of ev, and in vivo functions of ev. group discussions are facilitated and stimulated via assignments to analyse data obtained during the practicals and to critically evaluate literature. participants also have the opportunity to present their own research during poster presentations and ask for feedback from organizers and invited lecturers. summary/conclusion: among the participants selected for the course, a large geographical distribution is reached, including researchers from the newer eu member states and from outside of europe, to ensure a broad geographical distribution of the knowledge gained during this course. introduction: on 25 october 2019, we organized the 1st lugano exoday, first initiative in the southern switzerland to bring together resident researchers and european experts in the field of extracellular vesicles (evs). the workshop, centred on "technical challenges of extracellular vesicle research" aimed to highlight technical requirements and advances in the evs area, focusing on isolation, characterization and tracking. methods: the workshop started with a lecture by dr. cecilia lässer, from the university of gothenburg. the rest of the workshop was divided in two working groups (wg), each introduced by a keynote lecture followed by presentations by young researchers and a round-table discussion. wg1, introduced by dr. mercedes tkach, from the institute curie in paris, focused on recent advances on evs characterization and isolation. wg2 was centred on evs tracking and introduced by dr. frédérik verweij, from the institute of psychiatry and neuroscience of paris. results: dr. lässer opened the workshop with a comprehensive review and introduced recent developments in the evs field. the first wg discussed different isolation methods, focusing on ultracentrifugation, size exclusion chromatography and immunoprecipitation-based techniques. supported by the keynote speakers, the participants agreed that the best approach to optimize the isolation process consists in the combination of different techniques. wg2 shared insights about new strategies to visualize and tracking evs, focusing on how to improve the routinely approaches used, defining optimal criteria for evs labelling and imaging. all the participants had an in-depth overview on the requirements and the state-of-the-art techniques currently in use for the isolation, characterization and tracking of evs. summary/conclusion: the transferable knowledge acquired during the workshop ensures participants to remain up-to-date with the advances in the field of evs. as our ultimate goal is to create a competence centre in southern switzerland around the field of evs, the workshop was an invaluable opportunity to intensify collaborations between resident laboratories and broaden the scientific exchange with laboratories of the experts hosted during the event. given the success of this first workshop we are already working to prepare the second edition and make the event a recurring appointment. funding: supported by the swiss national science foundation the role of core facilities and emerging technologies in maximizing rigour and reproducibility of ev quantification and characterization and following misev guidelines rachel derita a and andrew hoffman b a thomas jefferson university, philadelphia, usa; b university of pennsylvania school of veterinary medicine, philadelphia, usa introduction: it remains very clear in the field of extracellular vesicle (ev) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical ev interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. as the first core facility in the usa with the sole intention of creating a space where users can both isolate and characterize evs, we provide a central location for the facilitation of ev research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of evs and additional immune or fluorescence-based ev characteri zation techniques. methods: we surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the mid-atlantic region of the us. the survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. results: the current demand is highest for isolation services, ultracentrifugation and nta, with a gradually increasing demand for immunophenotying analyses such as the exoview chip array, fluorescent nta and flow cytometry. we additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous ev characterization in accordance with misev guidelines, and encourage collaboration among ev researchers. summary/conclusion: in order to answer the larger remaining questions in the ev field such as the isolation of specific ev subsets, ev tracking between cells and the use of evs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress. programme to assess the rigour and reproducibility of extracellular vesicle-derived analytes for cancer detection national cancer institute, rockville, usa introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par20-053, is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par16-267/ 277, successfully funded 7 r01 and 4 r21 grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, 2018); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, 2019). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, 2019). summary/conclusion: drs. sudhir srivastava and matthew young are the program directors for the par which began accepting applications on 5 january 2020. this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute introduction: early detection of cancer as well as monitoring cancer treatment are important to improve cancer care. diagnostics for cancer are mainly based on tissue biopsies and re-biopsy during treatment is challenging. moreover, current diagnostics are expensive, time-consuming and have low-throughput. therefore, liquid biopsies are expected to bring the next breakthrough in cancer diagnostics. in liquid biopsies tumour-secreted material is isolated from body fluids and subsequent analyses thereof allow for non-invasive diagnostics. one type of tumour-secreted materials are extracellular vesicles (evs), which are schedded from tumour cells. evs are surrounded by a lipid bilayer, whichs composition resembles the plasma membrane of their parental cell. as many tumours are driven by over-expression or upregulation of transmembrane proteins e.g. growth factor receptors, detection of the later on evs holds promise for early tumour detection and treatment monitoring. methods: for the immuno-pcr evs were first affinitycaptured on magnetic beads, allowing immobilization of purified evs as well as evs secreted into cell culture medium or spiked into plasma. afterwards each sample was divided and affibody-dna-conjugates directed against different targets were added. affibodies are small affinity proteins, which often are developed as high affinity binders for tumour imaging, making them suitable probes in the presented assay. after washing, the bead-ev-affibody-dna-complexes were analysed for the immobilized dna-amount via qpcr. results: via the presented immuno-pcr evs secreted from the non-small cell lung cancer cell line h1975 as well as the ovarian cancer cell line skov3 were analysed. the immuno-pcr method allowed the detection of the tumour-associated membrane receptors epidermal growth factor receptor (egfr), receptor tyrosineprotein kinase erbb2/her2 and insulin-like growth factor 1 receptor (igf1r). different levels of membrane receptors depending on the ev source and concentration were detected. summary/conclusion: the presented immuno-pcr showed to be a comparably fast and robust method for detection of tumour-associated membrane receptors on evs derived from cancer cell lines with medium through-put and is currently further developed into a method for liquid biopsy for non-small cell lung cancer patients. introduction: introduction. evs produced by cells can originate from different cellular compartments and evs in complex biofluids may originate from many different cell types. traditional biochemical analysis, which reports on the total composition of all evs in a sample can't adequately resolve this heterogeneity. single vesicle analysis methods can, if they have the necessary specificity, sensitivity and speed. flow cytometry (fc) is capable of rapid and quantitative analysis of individual particles, but conventional fc-based assays lack the specificity and sensitivity to measure individual evs. assays that combine sensitive instruments with ev-selective sample staining can measure individual evs with accuracy and precision. to better understand the nature and origins of ev diversity, we used single vesicle fc (vfc) to quantitatively measure vesicle number, size, and surface cargo expression on individual evs. methods: methods. evs in culture supernatants (293 t, a431, u87, thp-1, sh-sy5y) were used neat or enriched by standard methods including differential centrifugation or ultrafiltration. evs from platelets (plt) and red blood cells (rbc) were induced by ionophore treatment of washed cells, and measured in diluted supernatant. evs were stained with a membrane-selective dye and fluorescence-labelled antibodies using a commercial vfc assay kit (cellarcus biosciences), measured using a commercial flow cytometer (cytoflexs, beckman coulter), and data analysed using fcs express v6 (de novo software). vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate vesicle and beadbased standards and essential controls performed as recommended by the miflowcyt-ev reporting guidelines. results: results. to assess the compositional heterogeneity of evs, we first characterized the expression of tetraspanins (tss; cd9, cd63, cd81, cd82, cd151, cd53, cd231) on evs released from cultured cell line and primary cell cultures. we find quantitative differences in the expression of ts on evs from different cell types that generally reflected the expression on the cell of origin, with most ev types expressing detectable amounts at least one of the common ts molecules (cd9, cd63 or cd81) but generally not all three. in evs from some cell types, ts expression was uniform across the ev population (cd9 on evs), but evs from other cell types differentially expressed tss, with some evs expressing no detectable ts (rbc evs). intracellular cargo labelled genetically using fluorescent proteins (egfp or mneongreen) or fluorogenic enzyme substrates (cfse) were measured in individual evs and revealed distinctive associations between ev surface and internal cargo. summary/conclusion: conclusions. high resolution measurement of cargo on/in individual evs can help interpret ev heterogeneity in terms of cell of origin, signals carried, and effects on target cells. integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles introduction: fewer than 50% of patients develop both vascular and valvular calcification, implying differential pathogenesis. while circulating extracellular vesicles (evs) act as biomarkers of cardiovascular diseases, tissue-entrapped evs are implicated in early mineralization but their contents and function are unstudied. we developed an innovative method to isolate and study evs from fibrocalcific tissue and investigated entrapped ev cargoes in human cardiovascular diseases. methods: human carotid artery endarterectomies and stenotic aortic valves were obtained from 27 donors under irb-approved informed consent. tissues underwent enzymatic digestion, ultracentrifugation, and a 15-fraction optiprep density gradient. global proteomics was performed on intact tissue, each optiprep fraction, and ev-enriched pooled fractions; the latter also underwent mirna-seq. fractionated samples were also studied by cd63 immunogold electron microscopy (tem) and nanoparticle tracking analysis (nta). high confidence mir targets were predicted by targetscan, pathway analyses utilized the biocarta/kegg/reactome databases, and protein-protein interaction networks were built in string. results: vesicle-associated pathways were increased 5.1x (p < 0.01; 15/30 vesicle-related top go terms) in proteins common to intact arteries and valves (n = 1,411). proteomics found 24 ev markers to be highly enriched in the four least-dense optiprep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by tem/nta. proteomics and mirna-seq of tissue evs quantified 1,104 proteins and 123 mir cargoes linked to 5,182 target genes. pathway networks of proteins and mir targets common to artery and valve tissue evs revealed a shared regulation of rho gtpase and mapk intracellular signalling cascades. 179 proteins and 5 mirs were significantly altered between artery and valve evs (q < 0.05); multi-omics integration found that evs differentially modulated cellular contraction and p53mediated transcriptional regulation in vascular and valvular tissue. summary/conclusion: our findings delineate a novel strategy for studying tissue-entrapped ev protein and mir cargoes and identify critical roles that tissue-resident evs play in mediating cardiovascular disease. funding: this study was supported by a research grant from kowa company (ma) and nih grants r01 hl136431, r01 hl141917 and r01 hl147095 (ea). mir-20a regulates exogenous cd82 expression on proliferation, invasion, migration and angiogenesis of gastric cancer zhengzhou university, zhengzhou, china (people's republic) introduction: to investigate the possible mechanism of mir-20a regulating the expression of exosome cd82 on proliferation, invasion, migration and angiogenesis of gastric cancer, and to study the application value of cd82 in the early diagnosis and prognosis of gastric cancer. methods: the gastric cancer cell line mgc-803 was used as the research object. the exosomes were extracted from the culture supernatant of mgc-803 by exosome extraction kit. the extracted exosomes were identified by transmission electron microscopy and western blotting. the expression of cd82 in exosomes was detected by elisa. the expression of cd82 in exosomes and cd82 in whole blood and serum were detected by western blot. they were randomly divided into blank group (mock) and mir-20a lentivirus experimental group (mir-20a group). the lentivirus control group (mir-20a/con) was transfected into cells. qrt-pcr was used to verify the status of mir-20a after transfection; western-blot was used to detect the expression of cd82 and downstream erk1/2, akt and m tor proteins; mtt assay, cell colony formation assay, transwell migration assay for cell proliferation, invasion, and migration. a nude mouse xenograft model was constructed to observe the growth of transplanted tumours,microvessel density (mvd) was detected by immunofluorescence, and distant metastasis was recorded. results: the expression of cd82 in exosomes was detected by elisa and western blot. the expressions of cd82, akt, erk1/2 and m tor in mir-20a group were significantly lower than those in mir-20a/con and mock groups. cd82 protein is positively correlated with downstream protein levels.the growth rate and cell invasion ability of mir-20a group were significantly lower than those of mir-20a/con group and mock group. the weight of the nude mice in the mock group and the mir-20a/con group decreased, while the weight loss in the mir-20a group was not significant. the tumours in the mir-20a/con group and the mock group showed invasive growth accompanied by abundant microvessels, while the mir-20a group had smaller tumour volume and uniform cell distribution. only a small amount of microangiogenesis was observed, and no obvious necrotic area was observed. summary/conclusion: mir-20a affects the proliferation, invasion, migration and angiogenesis of gastric cancer mediated by akt/erk/m tor signalling pathway by regulating the expression of exosome cd82. streamlined detection and quantification of plasma extracellular vesicles and their protein cargo by high-performance nanoscale flow cytometry and label-free mass spectrometry introduction: nanoscale flow cytometry (fc) and mass spectrometry (ms) are useful for profiling ev surface proteins and performing bulk ev proteomics, respectively. this study sought to develop pre-analytical and analytical pipelines for ev protein profiling that are applicable to clinical studies. methods: to optimize plasma ev detection and quantification by fc, modifications of instrument settings and serial dilutions of platelet-free plasma (pfp) and antibodies were tested for improved separation of signal from noise and reduction of event coincidence and swarming. the high-performance flow cytometry (hpfc) platform was used to assess the effect of time (1, 2, 3, 5, 9, 24, 48 or 120hrs) between blood draw (into acd, nacit, edta or heparin) and blood processing, on ex-vivo release of evs from blood cells. label-free ms was used to examine the intensity and breadth of identified proteins in plasma evs purified using several density and size separation methods, either manually or automated, along with various buffer conditions. results: ev event aborts were minimized at a pfp dilution, prior to staining, of 1:10 and by using a narrow cytometer window extension. target ev signals were distinct from noise and were triton x-100 labile. the most significant changes in plasma evs were associated with platelet-derived fractions, use of heparin and >5-hour delay before blood processing. yet, platelet ev numbers did not significantly change for up to 24 hrs in citrated and edta plasma. higher overall coverage of known ev proteins and a fivefold increase in number of uniquely identified proteins were observed in ms profiling of evs prepared by a combination of ultracentrifugation (uc) and manual size-exclusion chromatography (sec) compared to preparation by fplc on capto core 700/superose 6 resins. uc/sec was better than direct sec at reducing contamination by excipient plasma proteins. column buffers with trehalose increased ev protein recovery while adding protease inhibitors had minimal effect. summary/conclusion: with our optimized hpfc protocol, we established that blood ev numbers remain stable for up to 24 hrs in acd or edta and that uc+sec with trehalose-containing buffer result in high canonical ev protein recovery. we are applying these workflows to investigate cancer-associated changes in plasma ev protein cargo. the value of exosomes as a potential biomarker for devil facial tumour disease. university of tasmania, hobart, australia introduction: the tasmanian devil (sarcophilus harrisii), the largest living carnivorous marsupial is endangered because of two transmissible cancers: devil facial tumour disease (dftd) one and two. current efforts to manage dftd are hindered by the lack of a preclinical diagnostic test for dftd. detecting dftd infection is only possible once tumours are noticed, too late to stop dftd progression. a preclinal test could tell us about unknown components of dftd pathogenesis, such as latent period and host-tumour dynamics. exosomes are extracellular vesicles released by most types of cells under both physiological and pathological conditions. exosomes have utility as diagnosis and prognosis biomarkers in a range of diseases, including cancers. the aim of this study is to investigate exosomes-based approaches towards a preclinical and progression biomarker for dftd 1 and 2 in tasmanian devils. methods: exosomes were isolated from three different dftd-1, dftd-2 and devil fibroblast cell lines by sizeexclusion chromatography. likewise, exosomes were isolated from plasma of healthy and diseased devils. to determine the size and morphology of exosomes, samples were imaged with transmission electron microscopy. exosomes isolated from cell lines and devil plasma were analysed with mass spectrometry to characterise proteins and determine their differential expression between the cell origins, and healthy and diseased animals. results: this study identified the presence of myelin proteins in exosomes from dftd cells relative to fibroblasts, which are diagnostic of dftd. additionally, we found that exosomes derived from dftd-2 abundantly express the inhibitory checkpoint molecule cd200 relative to exosomes from dftd-1 cells and devil fibroblasts, indicating a potential candidate for a differential diagnosis between tumours. moreover, exosomes from dftd cells present a greater amount of proteins related with metastasis in comparison with fibroblast exosomes, such as integrins. finally, we report the protein expression profile of exosomes from healthy and diseased devils, showing clear differences between them and the presence of immunosuppressive and metastasis proteins in animals in late stages of the disease. summary/conclusion: dftd-exosomes may provide a non-invasive diagnosis tool to detect early stages of dftd in tasmanian devils to facilitate the prevention of the disease. furthermore, dftd-exosomes may have utility as a prognosis biomarker, determining late stages of the disease using a simple a blood test, which would facilitate monitoring of wild populations. this project will provide long-term benefits for the future of the devils and encourage exosome-based solutions for other future wildlife disease outbreaks. introduction: despite the increased understanding of evs, from involvement in disease pathophysiology to therapeutic delivery, improved molecular tools to track biodistribution are largely lacking. current approaches used for ev labelling lacks sensitivity and specificity. here, we have explored bioluminescent labelling of evs to achieve a highly sensitive system for absolute in vivo quantification and tracking of exogenous evs at low cost and in a high throughput manner. methods: ev-producing cells were genetically engineered to express various tetraspanin-luciferase fusion proteins. evs purified by uf-sec from these cells were characterized by nta, multiplex bead-based array, tem and wb, followed by luciferase assay to determine the labelling efficiency. for in vitro applications cell lysate from treated cells or the conditioned medium were subjected to luciferase assay. for in vivo applications two different methodologies were applied to determine biodistribution; either by non-invasive real time in vivo imaging using ivis or by luciferase assay on harvested tissues for absolute quantification of injected evs. results: we initially performed a systematic comparison of five different luciferases for endogenous labelling of evs and identified nanoluc and thermoluc as lead candidates. we applied this technology to monitor in vitro cellular uptake and observed cell type differences in cellular uptake of engineered evs. in addition, we also observed an effect of different culturing conditions on exocytosis kinetics. for in vivo application, we applied the nanoluc labelling strategy to determine the pharmacokinetics and effect of different routes of injection on ev distribution. our results indicated a rapid uptake profile of administered evs in different tissues with liver, spleen, and lungs being the primary recipients. we also observed similar results upon tracking in vivo biodistribution in real time immediately after administration. finally, we show how different subpopulations of evs differ in their in vivo biodistribution. summary/conclusion: overall, nanoluc and thermoluc labelling of evs holds great potential for various in vivo and in vitro applications. in addition, it can enable the simultaneous detection of different subpopulations of evs in vivo, which may aid in our understanding of different sub-populations and their behaviour in vivo. apart from monitoring therapeutic evs, with one simple modification this platform offers great potential for tracking tumour derived evs both in vivo and in vitro and thus could aid in the development of anti-tumour therapies. biofunctional peptide-modified extracellular vesicles for cell targeting, macropinocytosis induction, and effective intracellular delivery ikuhiko nakase department of biological science, graduate school of science, osaka prefecture university, sakai-shi, japan introduction: [introduction] in our research group, developing therapeutic techniques based on extracellular vesicles (exosomes, evs) by effective usage of peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been focused. in this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides [1] , artificial coiled-coil peptides with receptor targeting [2] , and cell-penetrating sc18 peptides [3] derived from cationic antimicrobial protein, cap18 for cancer targeting with macropinocytosis induction, on the ev membranes will be introduced. i will also show effects of lyophilization of the peptidemodified evs on their biological activity [4] . methods: [methods] cd63 (ev marker)-gfpfusion protein expressed evs were used for cellular evs uptake assessments. all biofunctional peptides were synthesized by fmoc solid-phase method. results: [results] macropinocytosis with actin reorganization has been shown to be crucial for cellular ev uptake [5] . we developed the methods for modification of arginine-rich cpps or sc18 peptides on ev membranes using chemical linker techniques, and for example, arginine-rich cpps modification can induce proteoglycan-clustering (e.g. syndecan-4) and macropinocytosis signal transduction [1] . the artificial leucine zipper peptide-modified evs recognize the peptide-tagged epidermal growth factor receptor (egfr) on targeted cells, leading to macropinocytotic cellular ev uptake [2] . in addition, lyophilization is a useful technique for long term storage, however, we found that lyophilization negatively affected biological functions of encapsulated proteins in the evs after their cellular uptake [4] . summary/conclusion: [conclusion] these techniques and findings will contribute to development for the ev-based intracellular delivery systems. reference: [1] sci. rep. 6, 34937 (2016), [2] chem. commun. 53, 317 (2017), [3] chrmmedchem. 12, 42 (2017) [4] anticancer res. 39, 6701 (2019), [5] sci. rep. 5, 10300 (2015) os28.3 multi-compartmented microvesicles: novel extracellular secretory organelles that release exosomes and extracellular vesicles introduction: extracellular vesicles (ev) bud from the plasma membrane (pm) as microvesicles (mv) or arise from the fusion of multivesicular bodies (mvb) with the pm to release intralumenal vesicles (ilv) as exosomes. the variety of bioactive molecules carried by ev imparts diverse functionality to ev in intercellular signalling. the biogenesis and extracellular release of these specialized messenger organelles is not well understood. to investigate, we studied endothelial cells that line the inside of blood vessels, known to release ev that support angiogenesis. methods: cultured human umbilical vein endothelial cells (huvec) were examined by thin-section electron microscopy (em), serial sectioning and immunogold labelling to study the structure and composition ev release sites. to obtain optimal views of cellular ultrastructure, cells were preserved by fast-freezing and a freeze-substitution. results: a potential release site was identified in em thin sections as a discrete domain, up to several microns long, on the otherwise smooth huvec pm, where numerous bulbous membrane protrusions with thin necks were clustered. the cytoplasm in these protrusions was enriched with mvb and other vesicles and appeared to be on the verge of pinching off to release multi-compartmented mv (mcmv). consistent with this notion, in the neighbouring extracellular space, a plethora of mcmv of 300-1200 nm with ultrastructural features matching the bulbous protrusions were observed, supporting the concept that mcmv bud from the release site. serial sections confirmed that these extracellular mcmv were independent of cells and not linked by nanotubes or other processes. remarkably, fusion of mvb with the mcmv membrane was directly observed, presumably caught in the act of releasing ilv (exosomes) from the mcmv. immunogold labelling for ev markers is being used to identify proteins enriched at release sites and on released mcmv. summary/conclusion: in summary, 1) mcmv bud from localized sites on the endothelial pm, 2) mcmv contain mvb, and 3) fusion of mvb to mcmv to release exosomes occurs extracellularly. mcmv can now be evaluated as a potential source of exosome and ev release that occurs after budding from the cell of origin, adding new layers of regulation to when, where and how ev are assembled and released. funding: this work was supported by the division of intramural research of the nih. one size does not fit all: overcoming barriers to successful discovery and scaled manufacturing of therapeutic extracellular vesicles jieun lee a , wei guo b , hal sternberg c , mike west d and dana larocca d a stem cell team, seoul, republic of korea; b university of pennsylvania, philadelphia, usa; c agex therapeutics inc, alameda, usa; d agex therapeutics inc., alameda, usa introduction: extracellular vesicles have tremendous intrinsic therapeutic potential. however, the limited availability of production cell lines presents a barrier to scaled ev production and novel ev discovery. indeed, ev sources have been largely confined to a handful of cell types with the vast majority consisting of msc evs. to overcome this limitation, we developed a diverse library of hundreds of clonally pure and scalable progenitor cell lines that provides an alternative resource for ev drug discovery and production. methods: we harnessed the capacity of human pluripotent stem cells (hpsc) to differentiate into virtually any cell type by subjecting hpsc to a wide variety of media and culture conditions to maximize the diversity of partially differentiated cells. the resulting heterogeneous "candidate cultures" were plated at clonal density and further selected for self renewing and scalable clones. transcriptomic analysis indicated >200 distinct progenitor lines. cell fate potentials were mapped by screening for cell type specific marker expression in various differentiation conditions. evs were produced using cgmp methods (tff and sec) and characterized by nta, trps, surface marker analysis, rna and protein content. bioactivity assays included proliferation, migration, vascular tube network formation, senolysis, and oxidative stress. results: the progenitor library contained >200 distinct lines with diverse lineage fates including various types of bone, cartilage, muscle,and fat cells, as well as all blood vessel cell types. the lines displayed much longer replicative lifespans (70-100pd) than primary cell lines like msc. clonal purity minimized phenotypic drift resulting in maintenance of cell identity, genome integrity, differentiation capacity and bioactive ev production over extended culture. evs were highly diverse in their rna and protein cargo and bioactivity displaying various degrees of migratory, proliferative, angiogenic and senolytic activity. library screening identified evs with higher angiogenic potency than primary adult stem cell evs. summary/conclusion: we demonstrated scalable and stable production of bioactive evs from a large progenitor cell library. library screening resulted in discovery of novel angiogenic and senolytic evs having diverse rna and protein cargo. we are currently creating a corresponding library of progenitor cell evs to accelerate discovery of novel evs and their production cell lines. funding: the initial establishment of the cell library was funded in part by grants from the california institute of regenerative medicine and national institutes of health. introduction: besides extreme potential in biomedical applications, extracellular vesicles (evs) are also promising candidates to expand biophysical understanding of membrane active biomolecules. their complex bilayer composition allows the better understanding of adsorbed proteins and protein coronas as well, which sets of macromolecules will likely be key for advanced ev targeted delivery. considering cargo, membrane active peptides are interesting as these can be both drugs to be delivered, but can also facilitate cargo insertion through lipid bilayers. however, at present very little is understood regarding interactions between the peptides and the ev lipid bilayer, and between peptides and membrane associated proteins on evs. methods: we have recently demonstrated, that ev membrane adsorbed proteins and their interactions can be studied by techniques such as polarized light spectroscopy, microfluidic resistive pulse sensing measurements and freeze-fraction transmission electron microscopy [1] . furthermore, initially we studied several peptides with known antimicrobial properties and found that these strongly interact with the ev surface proteins, resulting in efficient removal of some from the lipid bilayer [2] . results: here we present investigation of further evpeptide interactions also focusing on anticancer peptides, which may be promising drug candidates for targeted delivery. these studies allowed to gain insight to novel functions of several peptides, such as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed understanding on how ev protein coronas, or ev bilayers are affected, to such extent that they cannot exert their potential function as delivery systems. summary/conclusion: the above interactions are expected to be interesting both for applicability, i.e. for selecting suitable compounds for ev processing, and also for curiosity-driven understanding of peptide functions, and ev-biomolecule interactions. based on these we promote that peptide -ev interactions will receive increased focus in ev-engineering. introduction: our late-breaking finding is the identification of a non-coding rna (ncrna) in extracellular vesicles (evs) from neuronal cells that is a natural antisense transcript for the dbh gene and associated with epigenetic changes and gene silencing. dna methylation in neurons is involved in memory and neurological disorders (science 2018 361 (6409)). earlier work found that during chronic brain infection with toxoplasma gondii induced a decrease in norepinephrine levels and expression of the host dbh gene; and the decrease is correlated with behaviours linked to noradrenergic signalling (infect immun. 2019 87 (2); infect immun. 2016 84 (2861)). dbh catalyzes the production of norepinephrine from dopamine in noradrenergic neurons. we found that evs from infected cultures suppress transcription of the dbh gene and hypermethylation of the gene in noradrenergic cells in vitro. in this study, we identify a ncrna in the evs from infected neuronal cells. methods: neuronal cells were induced by infection with toxoplasma gondii and evs purified on sucrose gradients. evs were characterised by electron microscopy and used to treat rat and human neuronal cells and expression levels of dbh mrna and nascent dbh gene transcription were measured. induced evs were injected into the locus coeruleus of rats and dbh gene expression was monitored. rna purified from evs was screened for natural antisense transcripts (nats) by strand-specific rt-pcr. results: we found that evs purified from infected neuronal cultures induced transcriptional gene silencing (tgs) and dna methylation of dbh in recipient neuronal cells. the induced evs down-regulated dbh gene expression >200-fold and induced dna hypermethylation of the dbh gene. this could be induced in the brains of recipient rats by intracerebral injection of evs. using a panel of strand-specific primers, antisense transcripts for the dbh gene were identified in infected cells. this permitted us to examine the rna in purified evs and identify a lncrna in evs selective for evs from infected cultures. summary/conclusion: this is the first study to find a specific neurotransmitter antisense lncrna in evs associated with transcriptional gene silencing and epigenetic changes in the gene. this represents a different type of neuron-to-neuron signalling than the classic chemical and electrical neurotransmission. the findings will enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy, drug addiction) and how memory works. human cd4 + t regulatory-derived extracellular vesicles and associated micrornas: role in cell-to-cell communication and involvement in the loss of immune tolerance during multiple sclerosis introduction: an impairment of immune tolerance is a determining factor in multiple sclerosis (ms) and dysregulation of cd4 + t regulatory (treg) cell function is believed to be a major pathogenic factor. micrornas (mirnas) released by treg cells in association with extracellular vesicles (evs) have been shown to participate in the block of pathological immune responses by inhibiting the growth and cytokine production of cd4 + t conventional (tconv) cells, but the molecular mechanism is still poorly characterized. aim of the present work was to evaluate whether treg cell-derived ev-associated mirna signature is dysregulated in ms and whether this defect may play a role in the development of autoimmunity. methods: human treg cells isolated from blood of naïve to treatment relapsing-remitting ms patients and healthy controls were in vitro stimulated and released evs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry. evassociated mirnas were quantified by traditional rt-qpcr and droplet digital pcr for absolute quantification. the actual ev-mediated passage of rna molecules from cell to cell was followed through rnaspecific fluorescent staining and treg-derived ev effect on tconv cell transcriptome was evaluated by rnaseq. results: in healthy conditions, the treatment of tconv cells with treg-derived evs was shown to cause the specific repression of genes involved in the proteasome-dependent proteolytic process, known to be crucial for t cell activation. in ms, treg-derived evs may have lost this capability as a direct consequence of a significantly decreased expression of mir-142-3p, able to target key factors of the proteasome system. summary/conclusion: our results unveil a novel molecular mechanism for treg-mediated maintenance of self-tolerance based on ev-associated mir-142-3p and its potential alteration in human autoimmunity. funding: fondazione italiana sclerosi multipla, fism, # 2016/r/10 and # 2018/r/4 revealing the proteome of brain derived extracellular vesicles isolated from human amyotrophic lateral sclerosis post-mortem tissues. introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterised by the deposition of misfolded proteins in the motor cortex and motor neurons. although a multitude of als-associated proteins have been identified, few have been associated with extracellular vesicle (ev) trafficking, a form of inter-cellular communication. additionally, the role of evs in als is undetermined, specifically in relation to pathogenic stress granule formation, a response to cellular stress involving aggregation of non-coding rnas and their rna binding proteins. therefore, this study aimed to determine the proteome of brain derived small extracellular vesicles (bdevs) isolated from als subjects and identify novel alsassociated deregulated proteins and their potential contributions to pathogenic pathways in als. methods: bdevs were isolated from human post-mortem als (n = 10) and control (n = 5) motor cortex brain tissues through an ultracentrifugation protocol (vella et al., 2017) . following thorough characterisation, bdevs that successfully met the minimum criteria required by the international society for extracellular vesicles were classified as evs. the bdevs subsequently underwent mass spectrometry analysis on the thermo scientific q-exactive hf with ultimate 3000 rslcnano. proteins identified to be statistically significant differentially expressed then underwent validation by western blotting. results: a panel of 16 statistically significant differentially packaged proteins were identified in the als bdevs. this included several up-regulated rna binding proteins and a down-regulated cell adhesion molecule; dhx30, stau1 and vcam1, respectively. pathway analysis revealed that the bdevs were enriched in proteins associated with stress granule dynamics, exosomal and lysosomal pathways. summary/conclusion: the identification of the rna binding proteins in the als bdevs suggests there may be a relationship between als-associated stress granules and als bdev packaging. the packaging of stress granule associated rna binding proteins into als bdevs may be an attempt by the cells to compensate for lysosomal dysfunction caused by stress granule accumulation, a feature of als. thus, these results highlight a potentially novel role for evs in the pathogenesis of als for long-term cultivation . the whole cultivation process of tissue preparation, cultivation, and cryopreservation has been established using strict serum-free conditions under a good manufacturing practice. long-term-cultivated hmnpcs retained stemness and hmnpcs have excellent differentiation efficiency into dopaminergic neurons. hmnpcs reversed impaired motor function in a rodent model of parkinson's disease (pd). based on the promising results in animal experiments, the clinical trial is under way (nct01860794). multiple-system atrophy (msa) is one of fatal neurodegenerative diseases with a combination of progressive autonomic nervous system disorders, parkinson's syndrome, and cerebellar pyramid syndrome. there are three types of msa such as msa-a, msa-c, and msa-p. in case of a msa-p type, it is difficult to diagnose due to the similarity of symptoms with parkinson's disease (pd). methods: in vitro and in vivo animal msa model were established and rotational behavioural was performed. npc cells were isolated and cultured based on moon et al. mirna sequencing (bgi) was performed and several bioinformatics analyses were done. results: based on the finding that hmnpcs exhibited therapeutic effects on pd, we hypothesize that hmnpcs will have a therapeutic effect on msa-p, where sympotoms are largely common with pd. as expected, transplanted hmnpcs survived, integrated, and differentiated in to dopamine neurons in the host brain, consequently leading to the functional recovery in the msa-p model. to further investigate the therapeutic key factors of hmnpcs in msa-p, mirna sequencing of the extracellular vesicles (evs) secreted from hmnpcs was performed. we found that mir-451a highly expressed in the npc-derived evs is one of key regulators of inflammatory response via nfkb pathway. we further experimentally demonstrated that mir-451a had anti-inflammatory effect on cells of msa-p condition such that the level of cx3cl1 expression and its receptor, cx3cr1 were both decreased in the msa-p modelled cells and in severe inflammatory environment in msa brain. summary/conclusion: our study first showed that mir-451a in hmnpcs-evs is one of key therapeutic factors for the recovery of brain damage through immuno-modulation in msa-p. introduction: oxidative insults are known to be involved in the pathophysiology of alzheimer's disease (ad). we have previously demonstrated that some blood-based redox-signature were associated to the cognitive scores in mild cognitive impairment patients and in ad (perrotte et al., 2019) . the aim of this study was (1) to evidence the presence of some oxidative markers in circulating extracellular vesicles (evs), and (2) to compare to their plasma levels. methods: plasma samples from healthy, mci and ad patients were from the memory clinic of sherbrooke (québec, canada). ad patients were stratified in three groups (moderate, mild and severe) according to the mmse and moca scores. total plasma extracellular vesicles (pevs) were isolated from plasma with the total exosome isolation reagent (invitrogen™ by life technologies inc.). pevs were then characterized by electronic microscopy, nta, dls and western blot. antioxidants apolipoprotein j, d (apo j, apod), the glyoxalase-1 and protein carbonyls were determined by western blot. results: in pevs, we found that apo d levels were higher in mci patients but not in ad patients. protein carbonyls levels were higher later, in pevs from moderate and severe ad while apo j levels were not different in pevs from the five groups of patients. in plasma, the pattern of apo j and apo d was different. the levels of apo d was not different in the five groups of patients while apo j levels were elevated in mci and in all ad groups. protein carbonyls were higher earlier from mild ad group, earlier than in pevs. the levels of the detoxifying enzyme glyoxalase-1 were higher in pevs than in plasma and were significantly decreased in early ad as compared to control subjects and mci summary/conclusion: these results demonstrate a differential regulation of redox homoeostasis in plasma and in pevs from ad patients. funding: acknowledgements: this work was supported by the chaire louise & andré on alzheimer's disease, foundation armand-frappier (cr) and cihr grant (tf). carlos j. nogueras-ortiz a , pavan bhargava b , sol kim b , francheska delgado-peraza a , peter calabresi b and dimitrios kapogiannis a a laboratory of clinical investigation, national institutes of ageing, baltimore, usa; b department of neurology, johns hopkins university school of medicine, baltimore, usa introduction: multiple sclerosis (ms) is a neurological disorder characterized by white matter demyelination and extensive synaptic pathology. recent studies have shown synaptic loss in the grey matter of ms brains in the absence of demyelinating lesions which could account for disease progression independent of demyelinating episodes. opsonization of synapses with complement components is a mechanism by which phagocytic cells normally prune synapses, but, when occurring in excess, it may underlie pathologic synapse loss. we sought to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss in ms using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (nevs and aevs). methods: nevs and aevs were immunocaptured in parallel from the plasma of 60 ms patients (45 with relapsing remitting, 15 with progressive ms) and 31 healthy controls, targeting the neuronal-specific marker l1cam and the astrocyte-specific marker glast, respectively. we measured the protein levels of preand post-synaptic proteins synaptopodin and synaptophysin in nevs using elisas and multiple complement cascade components (c1q, c3, c3b/ic3b, c4, c5, c5a, c9, factor b, factor h) in aevs using a luminex array. results: synaptopodin and synaptophysin protein levels in nevs of ms patients compared to controls were markedly reduced (2.5-fold; p < 0.0001 for both), whereas multiple complement components in ms aevs were markedly increased (c1q: 2.5-fold change; c3: 1.25-fold change; c3b/ic3b: twofold change; c5: 1.4-fold change; c5a: 1.4-fold change; factor: 1.5-fold change; p < 0.0001); differences were not observed in total circulating evs or neat plasma. strikingly, we found the nev-associated synaptopodin/synaptophysin and the aev-associated complement levels to be negatively correlated in people with ms (synaptopodin vs: c1q, r = −0.7 and p < 0.0001; c5, r = −0.6 and p < 0.0001; factor h, r = −0.46 and p < 0.0002/synaptophysin vs: c1q, r = −0.75 and p < 0.0001; c5, r = −0.65 and p < 0.0001; factor h, r = −0.52 and p < 0.0002), but not in controls. summary/conclusion: circulating evs provide markers of synaptic loss and complement activation in ms and suggest a link between astrocytic complement production and synaptic decline. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. methylglyoxal and glyoxal affect the protein cargoes in neuronal-derived extracellular vesicles introduction: advanced glycation end-products (ages) and their receptor rages are known to be involved in the pathogenesis of alzheimer's disease (ad). methylglyoxal (mg) or glyoxal (go) are the precursors of ages and particularly n-(1-carboxymethyl)-l-lysine (cml), the most abundant ages. mg induced tau hyperphosphorylation and causes hippocampal damage and memory impairment in mice. the aim of our study was to analyse the effects of mg and go on the neuroprotective, neurotrophic factors, inflammatory and neurodegenerative markers in the human cell line sk-n-sh and their release into the neuronal derived-evs. methods: briefly, sk-n-sh cells were incubated in fbs free media with mg and go (0.5 mm) for 24 hours. neuronal derived-evs (nevs) from culture media were isolated as previously described (haddad et al. 2019 ). nevs were characterized by electronic microscopy, nta and by western blot. cellular and nevs concentrations of bdnf, prgn, nse, app, mmp9, angptl-4, lcn2, ptx2, s100b, rage, dj-1 and alpha synuclein were determined by a luminex assay from r&d systems, inc. aβ1-40, aβ1-42, ptau t181 and total tau levels were measured also with luminex assay from emd millipore corp. results: we found that both ages precursors, at non toxic concentration, reduced the neuronal levels of nse with no effect on bdnf, ptrx-2, lcn-2, dj-1, on neurodegenerative markers and on cml. go decreased the levels of prgn, app, angpl-4 while the expressions of mmp-9 and angpl-4 were, respectively lower and higher in the presence of mg. mg and go greatly reduced the release of lcn-2 by neuronal cells in nevs. bdnf and prgn in nevs were reduced in the presence of go. both mg and go did not modify the release of nse, app, mmp9, agntl-4, ptx-2, dj-1, aβ, ptau and cml in nevs. summary/conclusion: our data demonstrated that mg and go differently affect the content of some protein cargoes in nevs and suggest that targeting mg and go may be a promising therapeutic strategy to prevent neurodegeneration. introduction: peripherally circulating brain-derived extracellular vesicles (evs) and their encapsulated rnas may serve as biomarkers for hiv-associated neurocognitive disorders (hand). however, rates of cigarette smoking are significantly higher in hiv+ individuals than the general population, and smoking can modulate the expression of these markers. to better understand how cigarette smoke might modulate rna expression and ev release, we examined several cnsderived cell lines, representing astrocytes (u87 mg), microglia (sv40), and oligodendrocytes (hog). methods: cigarette smoke extract (cse) was prepared by bubbling through culture medium using a standardized and published method. all cell types were exposed to either 0% or 50% cse for 24 hours. cell viability was assessed by musetm cell analyser, and evs were isolated from culture conditioned media (ccm) by size exclusion chromatography. the void (fractions 1-6), ev (7-10), and protein (11-14) enriched fractions were pooled and concentrated. evs were characterized by transmission electron microscopy (tem), microfluidic resistive pulse sensing, and western blotting. total rna was isolated from cells and circular rna (circrna) expression was assessed with a circrna microarray. results: in response to cse exposure, cell viability was only slightly reduced for all cell types. tem images validate the presence of vesicles in the ev fractions, and their absence in the void and protein fractions. spectradyne particle counts indicated cse exposure substantially increased the ccm particle count in the ev fraction when compared with control. the presence of expected ev markers (cd63, cd81, and tsg101) in the ev fractions, and their absence in the void and protein fractions was observed via western blot. intracellular circrna expression was significantly altered in all three cell lines. summary/conclusion: cns cells display physiologic responses to cse that include vesiculation pathways and significant alterations in circrna expression. we are now studying the effects of cse exposure on circrna expression in released evs. funding: this work is supported by da040385, da047807, and ai144997. a method for exosomal rna extraction from paired human brain and blood specimens emily n. moya a , lillian wilkins a , esther cheng a , lisa linares b , brian kopell b , navneet dogra c , bojan losic a and alexander charney a a icahn school of medicine at mount sinai, new york, usa; b mount sinai hospital, new york, usa; c department of genetics and genomic sciences, department of pathology, icahn school of medicine, mount sinai, new york, usa introduction: diagnosis and treatment of neuropsychiatric disorders has made little progress in the last half-century likely in large part due to the absence of a scalable technique to profile the complex biological activity of the brain in a living person. exosomes are nanovesicles 30-150 nm in size that mediate intercellular communication and contain proteins, lipids, and nucleic acids. it has been shown that brain derived exosomes can be found in peripheral blood, but determining whether peripheral exosomes truly reflect ongoing brain processes has to date not been possible due to the absence of paired living brain and blood specimens. here, we present a novel method for paired sampling of the dorsolateral prefrontal cortex (dlpfc) and peripheral blood from living human subjects for exosomal rna profiling. methods: informed consent, approved by the irb at the icahn school of medicine at mount sinai, was obtained for patients undergoing deep brain stimulation (dbs). paired brain and blood specimens were collected from 8 patients at two deep brain stimulation (dbs) electrode implantation procedures: left hemisphere followed by right hemisphere (total of 30 samples). we developed protocols to profile rna from exosomes of brain tissue extracellular matrix (ecm) and peripheral blood. exosomes were isolated via our in-house protocol using ultracentrifugation. rna was then extracted from the exosomes using the qiagen mirneasy mini kit protocol. quality control (qc) was performed to determine whether rna obtained was sufficient for next-generation sequencing. results: we demonstrate the safety of a novel procedure to sample the brain in living human subjects. bioanalyzer traces and qc data show a mean total rna of 14.83 ng (range 1.72-137.17 ng) and no samples fell below the threshold required for library preparation and sequencing (10 pg) determined by inhouse optimization on the smart-seq v4 ultra low input kit. summary/conclusion: to our knowledge, we have performed the first study to sample pairs of dlpfc and blood from living human subjects for exosomal rna for subsequent next-generation sequencing. ongoing analyses by our group promise to establish peripheral exosomal rna transcripts reflective of brain activity. this non-invasive approach to probing neurobiology in the living human brain may facilitate the development of exosome-based diagnostics for neuropsychiatric disorders. introduction: the relationship between obesity and dementia is complex. while obesity in middle age triples the risk of dementia 30 years later, many patients with alzheimer's disease (ad) are cachectic, and a decline in adiposity portends progression of dementia. this suggests adipose-derived factors are important to nervous system homoeostasis. we previously showed that adipocyte-derived small extracellular vesicles (ad-sevs) induce pathologies critical to developing obesity-related diseases and may provide a mechanistic link between adiposity and dementia. we hypothesized that altered expression of ad-sev micrornas involved in neurodegenerative pathways is associated with more severe cognitive impairment methods: we studied serum and cerebrospinal fluid (csf) from 19 participants with ad and 14 non-ad controls. ad-sevs were isolated from samples by precipitation and immunoselection. ad-sev microrna expression was profiled in both biofluids and compared. results: serum and csf microrna expression correlated strongly (r2 = 0.98). in serum, 189 micrornas were differentially expressed by a fold change ≥|1.1| in the ad and control groups (p ≤ 0.1) and 251 micrornas were differentially expressed in csf. using ingenuity pathways analysis, we identified mrnas expressed in nervous system tissue that are targeted by the differentially expressed micrornas. the mrnas map to 145 diseases and functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental pathways are highly represented. of the 189 differentially expressed micrornas in serum, 6 were moderately correlated with participants' score on the mini-mental state exam, a test of cognitive function (rs = |0.488-0.609|). as validation, rencell cx cortical derived neuronal stem cells had decreased doubling time when exposed to ad-sevs from obese adipose tissue in vitro. summary/conclusion: these findings support our hypothesis that altered expression of circulating ad-sev micrornas are involved in neurodegenerative pathways associated with cognitive impairment. these findings support using serum ad-sevs as a surrogate for csf ad-sevs. functional validation is underway to define the connection between ad-sevs and ad. understanding the link between obesity and ad is crucial as the population ages and the global obesity epidemic grows. funding: supported by uw adrc (nih:p50ag005136) expression of extracellular vesicles after acute traumatic brain injury: an exploratory flow-cytometry study introduction: coagulation derangements related to disseminated intravascular coagulation (dic) are common after tbi and contribute to secondary neural injury. extracellular vesicles (evs) are released from all cell types, including platelets, endothelium, and lymphocytes, which are responsible for dic. we hypothesized that specialized flow cytometry techniques could identify a unique ev signature of dic in acute tbi. methods: using a modified flow cytometry instrument for detection of small particles, fluorescence panels were created to assess for evs from endothelial cells (cd144, cd105), platelets (cd31, cd62p, cd41a, cd42b), and erythrocytes (cd235) as well as brain biomarkers (s100b, uchl-1, gfap, tau and nse) and t-lymphocytes (cd3, cd4, cd8, cd31). samples were prepared in trucount tubes to determine volume and treated with triton to confirm presence of evs. results: 13/17 study patients and 16/20 controls were male. 76% of study patients presented with a glasgow coma scale of 15. in the hypercoagulability panel, of the 10 subsets with statistically significant differential expression, 4 involved s100b+ and were elevated in patients. platelet-derived cd41a evs and uch-l1 evs were significantly elevated in controls in 7 ev subsets identified in the brain-specific panel. finally, cd3+/31+ evs, derived from t-cells and identified in the endothelial/t cell panel, are significantly lower in patients suggesting cns recruitment. summary/conclusion: endothelial and platelet/erythrocyte evs may be elevated early after tbi. s100bcarrying evs are significantly elevated in circulation of tbi patients; if reproducible, this signature profile may be informative for diagnosis and risk stratification. further study is warranted to evaluate whether this expression correlates with secondary microvascular brain injury. funding: intramural award from the university of pennsylvania enrichment of mir-451a in cns extracellular vesicles following impairment of the blood brain barrier nasser nassiri koopaei a , ekram-ahmed chowdhury b , lais da silva a , jinmai jiang a , behnam noorani b , ulrich bickel b and thomas d. schmittgen a a university of florida, gainesville, usa; b texas tech university, amarilo, usa introduction: extracellular rnas (exrnas) are present in essentially all biofluids and include all types of rna including mirna. to enhance their stability outside of the cell, exrnas are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (evs). the blood brain barrier (bbb) is a dynamic interface between the systemic circulation and the cns and is responsible for maintaining a stable extracellular environment for cns cells. the intent of this study was to determine if evs and their contents are transferred from the peripheral circulation to the cns under conditions of an impaired bbb. methods: the bbb of mice was disrupted by hyperosmolar mannitol injections. to validate that the bbb has been disrupted with mannitol, intravenously-dosed [13 c]-sucrose was increased in the forebrain by 14fold with mannitol compared to sham treated mice. evs were isolated from the forebrain, hindbrain and spinal cord following gentle tissue lysis and differential ultracentrifugation. evs were validated by nta, tem and western blotting. mir-451a, a mirna that is highly abundant in erythrocytes, was measured in the evs by qpcr. results: qpcr showed that mir-451a in cns tissue evs increased with mannitol treatment in the forebrain, hindbrain and spinal cord by 15-, 1.6-and twofold respectively. qpcr analysis of mrna from reported mir-451a target genes showed reduced target gene expression with mannitol. summary/conclusion: we demonstrate that evs containing mir-451a, a highly abundant mirna present within erythrocytes and erythrocyte evs, is enhanced in the cns upon bbb disruption. astrocyte-derived extracellular vesicles in morphine tolerance guoku hu, rong ma, naseer kutchy, yuetong zhao, susmita sil and shilpa buch university of nebraska medical center, omaha, usa introduction: opiates, such as morphine are used extensively in the clinical setting owing to their beneficial effects. paradoxically, however, the prolonged use of morphine often results in the development of tolerance, drug addiction, and ultimately leading to various comorbidities associated with drug abuse. although great efforts have been made, at present there is no treatment. the sonic hedgehog (shh) plays a key role in brain development, and brain cells fine-tuning processes such as their proliferation, patterning, and fate specification recent findings have demonstrated that inhibition of the shh signalling prevents morphine tolerance in rodent models. we thus hypothesize that extracellular vesicles (evs) derived from morphine exposed astrocytes and their cargo such as shh are critical for the development of morphine tolerance. methods: mice were received either saline or chronic morphine injection with escalating doses of morphine for 5 days (subcutaneously; 10 mg/kg, day 1, 20 mg/kg days 2-3, and 40 mg/kg days [4] [5] . the development of tolerance was assessed by measuring the tail-flick latency using tail flick analgesia metre (le7106, harvard apparatus). evs were isolated using either differential ultracentrifugation from astrocyte conditioned media or gradient ultracentrifugation from brain tissues. western blotting and qpcr were performed to determine the expression/activation of shh signalling pathway components. results: our data showed that the levels of shh protein were upregulated in morphine exposed astrocytederived extracellular vesicles (morphine-adevs). furthermore, shh containing morphine-adevs activated shh signalling in astrocytes. our in vivo study further demonstrated the upregulation of shh, as well as the activation of shh signalling, in astrocytes of morphine-administered mice. summary/conclusion: these findings thus demonstrated an autocrine mechanism for shh pathway activation in astrocytes associated with morphine tolerance. these findings could pave the way for the development of shh signalling pathway targeted strategies in the prevention and treatment for substance use disorders. biophotonics-based platforms for the evaluation of circulating extracellular vesicles as biomarkers of neurodegeneration in alzheimer's disease silvia picciolini a , cristiano carlomagno a , alice gualerzi a , monia cabinio a , francesca baglio a and marzi bedoni b a irccs fondazione don carlo gnocchi, milan, italy; b irccs fondazione don carlo gnocchi, milano, italy introduction: in the search for novel and non-invasive biomarkers of alzheimer's disease (ad), both circulating brain-derived extracellular vesicles (evs) and whole serum represent a valuable integration of the currently used classification system. to face the technological challenge of evs and serum analysis, we propose the use of biophotonics techniques as reliable, sensitive, fast and label free methods, potentially useful in tailoring pharmacological and rehabilitation treatments. methods: circulating evs, isolated by sec, and serum samples were collected from 10 healthy subjects (hc) and 10 ad patients. all subjects were asked to complete montreal cognitive assessment scale and mri examination. surface plasmon resonance (spr) was performed in order to detect evs coming from neurons, astrocytes, oligodendrocytes and microglia and to characterize each of them for the amount of ganglioside m1 (gm1), aβ and tspo expressed on their surface. serum analysis was performed using a raman microscope through the surface enhanced raman spectroscopy (sers) effect by mixing serum with ag nanoparticles. the pearson's correlation index was used to assess the linear correlation between spri data and clinical, mri data and data obtained from multivariate analysis (mva) of sers spectra. results: the spr analysis of evs showed that the selected bioactive molecules are differently loaded on neural ev populations and that their amount is increased on total evs in ad patients compared to hc. we observed a significant correlation between mva data from sers and the presence of aβ on neuronal and microglial evs and of tspo on neural evs, measured with the spr array. summary/conclusion: thanks to our methodological innovation we have verified the potentiality of evs as ad biomarkers, correlating biophotonics blood-based analysis with clinical data. this platform could provide a powerful tool for the evaluation of ad neurodegeneration. funding: the study was supported by the italian ministry of health (ricerca corrente 2017-2018 to irccs fondazione don carlo gnocchi). raman profiling of extracellular vesicles as new blood-based biomarker for brain disorders: focus on parkinson's disease introduction: extracellular vesicles (evs) play a pivotal role in brain homoeostasis and intercellular communication in both physiological and pathological conditions. in parkinson's disease (pd), evs are key players in the transfer of α-synuclein, with blood evs reported to undergo proteomic modifications. nonetheless, the detection and characterization of the ev cargo is technologically challenging, limiting the use of evs as biomarkers so far. herein, we propose raman spectroscopy for the label-free, bulk characterization of blood evs in pd patients. methods: evs were isolated by sec and ultracentrifugation from the serum of 18 healthy subjects (hc) and 22 pd patients. in all patients, the severity of pd was evaluated with the unified parkinson's disease rating scale (updrs) part iii and with hoehn and yahr scores (hy). after proper ev characterization following misev2018 guidelines, raman analysis was performed. the raman microspectroscope was used with a 532 nm laser in the spectral ranges 600-1800 cm-1 and 2600-3200 cm-1. data from hc and pd patients were compared by multivariate statistical analysis (pca-lda). results: the raman analysis of evs highlighted differences in the biochemical profile of the two groups, with the main variations in the spectral regions related to proteins, lipids and saccharides. a preliminary estimate of the accuracy of raman profiling of blood evs for pd diagnosis was obtained, demonstrating an accuracy of 71%. even more interestingly, we demonstrated the correlation between the raman spectra and the clinical scales (updrs and hy) used to stratify pd patients. summary/conclusion: in conclusion, the biochemical signature of blood evs can be detected by raman spectroscopy in pd patients and the ev spectral modifications can be related to their clinical status. these data suggest the possibility to use the raman profile of circulating evs as a biomarker for brain disorders, complementary to other specific molecular markers. funding: the study was supported by the italian ministry of health (ricerca corrente 2017 to irccs fondazione don carlo gnocchi) impact of circulating extracellular vesicles on brain functions and behaviours introduction: peripheral immune alterations have been described in psychiatric disorders such as schizophrenia, depression, and autistic spectrum disorders. in addition, behavioural changes have been observed in various immunodeficient animal models. however, the mechanisms by which peripheral immune system influences brain development and function are not well understood. in this study, we explored the mechanisms by which circulating extracellular vesicles (evs) mediate immune-brain communication and influence mouse behaviours. methods: mice deficient for rag1 or rag2 gene (rag ko mice) were used as a model to study the effects of loss of adaptive immune cells (t and b cells) on brain cellular phenotypes and behaviours. circulating evs were collected from their sera and analysed by using electron microscopy, nanoparticle tracking assay, and western blotting. brain cellular phenotypes were assessed by immunofluorescent staining and gene expression analysis. behavioural phenotypes of rag ko and wt mice were examined in social interaction test. in vivo transfer of evs was performed to see its effects on behavioural alterations of rag ko mice. results: rag ko mice displayed social behavioural deficits, accompanying by enhance c-fos immunoreactivity and altered microglia morphology in the medial prefrontal cortex (mpfc). circulating evs were also affected in these mice and lacked the expression of markers for t cells. a set of micrornas (mirnas) in circulating evs were diminished in rag ko mice. in vivo transfer of circulating evs rescues the social behavioural deficits of rag ko mice and ameliorate the c-fos immunoreactivities in mpfc of rag ko mice. summary/conclusion: our data showed that circulating ev profiles were altered in mice lacking adaptive immune cells and, accordingly, showing social behavioural deficits. notably, our in vivo experiments suggest that circulating evs may contribute to social behaviours. further study will provide a novel biological insight into the mechanisms underlying peripheral-to-brain immune communication via evs. introduction: the involvement of neuroinflammation on ageing process is widely recognized. extracellular vesicles (evs), such as exosomes, are able to cross the blood-brain barrier and were related to neuroinflammation. in this context, evs have been considered a potential mechanism of spreading molecules, including micrornas (mirnas) that can promote mrna degradation or inhibit translation of their targets. our aim was to investigate the mirna profile of circulating total evs during ageing process and their impact on canonical pathways. methods: the local ethics committee (comissão de ética no uso de animais -ufrgs; n 29818) approved all animal procedures and experimental conditions. plasma was obtained from wistar rats (3 and 21 months-old) and total evs were isolated. ev microrna isolation and microarray expression analysis was performed to determine the predicted regulation of targeted mrnas. results: the analysis of global microrna expression revealed 48 differentially expressed micrornas (p < 0.05; fold change of ≥ |1.1|); 18 mirnas were up-regulated and 30 were down-regulated in circulating total evs from aged animals compared to youngadult ones. a conservative filter was applied on ingenuity pathway analysis (ipa) and only experimentally validated and highly conserved predicted mrna targets were used. ipa showed that neuroinflammation signalling is ranked among the top canonical pathway impacted by differentially expressed micrornas and is upregulated in aged animals (p < 0.0001; z-score: 3.413). the differentially expressed mirnas impacted 32 molecules in the neuroinflammation pathway. interestingly, the ion channel grin2b is predicted to be up regulated and is a target of many evs mirnas; in accordance with our results grin2b was previously related to neurodegenerative diseases. moreover, let-7a-5p is predicted to be downregulated and target all the 32 molecules of the neuroinflammation signalling pathway. previous studies have correlated let-7a-5p and neurodegenerative diseases. summary/conclusion: our data suggest that circulating total evs cargo, specifically mirnas, are altered by ageing and impact neuroinflammation pathway, suggesting the involvement evs mirna on ageinginduced susceptibility of neurodegenerative diseases. introduction: bidirectional cell-cell communication via paracrine mechanisms is critical for wound healing. a new paradigm involving exosome-borne distinctive repertoire of cargo such as mirnas has emerged as a predominant mechanism of cellular communication at the site of injury. unlike other shedding vesicles of similar size, exosomes selectively package mirna by sumoylation of heterogeneous nuclear ribonucleoprotein (hnrnp). methods: keratinocyte-derived exosomes (exoκ) were genetically labelled with fluorescent reporter (gfp) using tissue nanotransfection. purified, gfp-labelled exoκ were isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation followed by affinity selection using magnetic beads. distributions of intact exosome were analysed using a prototype jarrold-geometry charge-detection mass spectrometer to directly measure differences in particle mass and charge distributions. complementary ms and ion mobility spectrometry (ims)-ms experiments have been used to characterize surface glycans and glycopeptides. to selectively inhibit mirna packaging within the exoκ in vivo, ph-responsive targeted sirna functionalized lipid nanocarriers (tlnκ) were designed using materials that have prior history of fda approval for human use. results: an increase in mass/charge ratio with glycan binding sites on the surface of wound-edge exoκ were observed compared to dorsal skin exoκ. wound-edge exoκ were selectively taken up by the macrophages in the granulation tissue (n = 6). keratinocyte targeting sirnahnrnp functionalized lipid nanocarriers (tlnκ) were designed with encapsulation efficiency of 94.3%. application of tlnκ encapsulating sirna of hnrnp (tlnκ/si-hnrnp) to murine dorsal woundedge significantly inhibited the expression of hnrnp by 80% in epidermis compared to control (tlnκ/sicontrol)(n = 10). moreover, mice treated with tlnκ/si-hnrnp showed impaired barrier function, with significant presence of macrophage in granulation tissue at day 10, suggesting impaired conversion of macrophage in the granulation tissue. summary/conclusion: this work provides a novel insight wherein exosomes of keratinocyte lineage are recognized as a major contributor that directs macrophage conversion in granulation tissue for wound healing. multifaced effects of milk-exosome (mi-exo) as modulator of scar-free wound healing gna ahn, hyo-won yoon, yang-hoon kim and ji-young ahn chungbuk national university, cheong-ju, republic of korea introduction: recently, milk exosome (mi-exo) has been focused particularly on the possibility of oral distribution for therapeutic agents. however, studies related to the cosmeceutical effects associated with mi-exo are fairly limited. the purpose of this study is to suggest the anti-oxidant and antiinflammatory effect of mi-exo and possibility that can be induced by scar free healing by micro rna in mi-exo. methods: the characteristics of the extracted mi-exo were verified by size measurement, morphological characteristics through cryo-em and western blot. for antioxidant experiments, an abts assay was performed. next, mrna expression through four major cytokines (tnfα, il-6, cox-2, inos) was used to evaluate anti-inflammatory effects. finally, cell migration assay was performed to confirm the effect of scar-free healing and the detection of mir-30b in mi-exo and vegf mrna expression confirmed. results: mi-exo using 1% acetic acid extraction showed the highest yield. the average size of the exosomes is approximately 110 nm, confirmed the typical double membrane vesicle. as a result of antioxidant experiments, it was confirmed that the treatment of exosomes of 10^10 particles showed about 65% antioxidant activity. when 10^10 particles were treated, rna expression of cytokines showed about 2 times more inhibitory effect than control. elisa test results also confirmed that the concentration-dependent decrease. the activation of the raw cell less proceeded as the treated mi-exo increased. the cell scratch assay cells did not close the cells as the number of milk exosomes increased (wound closing % of 10^10 particle = 4.7%). and mir-30b in milk exosomes was detected at ct value = 22. summary/conclusion: the antioxidant and antiinflammatory effects of mi-exo showed the greatest efficacy when 10^10 particles were treated. in addition, it induced to scar free healing rather than wound healing. mi-exo has great potential as a superior natural material in the future cosmeceutical field. extracellular vesicles in human milk expose tissue factor and promote coagulation introduction: tissue factor (tf), a transmembrane protein, initiates coagulation by binding and activating coagulation factor vii (fvii). tf is associated with extracellular vesicles (evs) in saliva and urine, but it is unknown whether also human milk (hm) contains evs exposing coagulant tf. methods: hm was collected from six healthy nursing women with informed consent. evs were isolated by ultracentrifugation and size exclusion chromatography (sec). the presence of tf antigen exposing evs was studied by western blot, flow cytometry, cryo-electron microscopy (cryo-em), and surface plasmon resonance imaging (spri). the ability of tf exposing evs to trigger coagulation was investigated with a plasma fibrin generation test (fgt), performed in the absence or presence of antibodies against tf or fvii(a). results: addition of hm to plasma shortened the plasma clotting time, even when hm was highly diluted. after ultracentrifugation of hm, both tf antigen and tf activity were detected in the ev-containing pellet. after sec, tf antigen and tf activity were present in the ev-containing fractions 8 and 9. the presence of tf-exposing evs in these sec fractions was confirmed by western blot (cd9, cd63 and tf), flow cytometry, spri, and fgt. in addition, the presence of evs in hm was confirmed by cryo-em. scalable isolation of evs from different probiotic strains with potential as cosmetic ingredients laura soriano-romaní, joaquin espí and begoña ruiz ainia, paterna, spain introduction: extracellular vesicles (evs) are increasing their application in a number of fields. recently, it has been shown that skin health may be affected not only by commensal skin bacteria, but also by the evs that they secrete. however, because most of the efforts have been directed to the characterization and evaluation of evs, the scaling up of the production process remains a bottleneck at the industrial level. in this work, the goal was to evaluate the potential applications of evs produced by different probiotic strains commonly used in the cosmetic field, considering the economic and technical viability of the process. methods: to meet our goal, a standardized workflow was defined to isolate evs from probiotic strains such as lactobacillus and bifidobacterium species, that have demonstrated cutaneous immuno-regulatory effects. the different bacterial strains were produced under standard culture conditions. to isolate the secreted bacterial evs, different chromatographic techniques were performed starting from clarified growth medium. then, evs were evaluated in vitro for a number of biological effects related with skin health. results: the ev yields obtained after downstream processing were calculated for each strain and isolation technique by means of nanoparticle tracking analysis (nta) and total protein content. moreover, evs were visualized by electron microscopy. the in vitro evaluation of isolated evs was based on changes in the expression of five biomarkers related with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell types to identify possible cosmetic claims that could be associated to each probiotic source. summary/conclusion: the potential of evs obtained from probiotic strains as cosmetic active cell-free ingredients was preliminarily assessed with this work, where the process yield and cosmetic function were evaluated. however, additional experiments will be needed in order to increase and optimize the productivity of each step of the ev manufacturing process. acerola derived exosome-like nanovesicles enhances the repair of ultraviolet b-induced dna damage in cultured skin fibroblasts tomohiro umezu, masakatsu takanashi, yoshiki murakami and masahiko kuroda tokyo medical university, shinjyuku, japan introduction: acerola (melpighia emarginata dc.) is a fruit is known to contain not only high amounts of ascorbic acid but also various nutritional components such as carotenoids and polyphenols. previous reports showed the acerola juices are able to confer protection against ultraviolet radiation b (uvb), to improve barrier function of skin. uvb is the main cause of dna damage in epidermal cells, generating several types of pro-mutagenic lesions, like cyclobutene prymidine dimers (cpds) and prymidine (6-4) prymidinone photoproducts (6-4pps): if not repaired, this dna damage leads to skin cancer. in this study, we investigated the biological property of the acerola derived exosome-like nanovesicles (adens), aiming to clarify the involvement of adens in repair of uv-induced dna damage. methods: normal human dermal fibroblasts (nhdfs) were purchased from lonza inc. the exosome-like nanovesicles were isolated from acerola juices using exoeasy maxi kit (qiagen). the morphology and size distribution of adens were checked by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta, nanosight lm10, malvern). nhdfs were exposed to uvb (1 mj/cm2) with pre-or post-adens. effect of uvb was assessed by examining cell viability, cell morphology, and dna damage levels through biochemical assays, microscopy and protein expression studies. results: purified adens were compatible with nta or tem for assessing the nanovesicle size range and concentration (200-400 nm). when nhdfs were added with adens and incubated at 37°c for 48 h, there was no effect of adens on cell proliferation of nhdfs. we found that adens treatment to uvb exposed nhdfs significantly reduced cpds and 6-4pps dna adduct formation. present results showed that aden treatment prevented uvb induced dna damage in nhdfs. summary/conclusion: we confirm that adens have the effect of repairing dna damage caused by uvb. these results provide that adens can be a new source to protect human skin from uv-induced skin cancer. introduction: introduction: despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. however, despite their clearly established potential, evs have limitations that may limit clinical relevancy, such as low potency. hypothesis: increased expression of pro-angiogenic lncrna hotair within msc evs enhances their proangiogenic effects and thus their wound healing properties. methods: methods: hotair was overexpressed in human dermal microvascular endothelial cells (hdmecs) to determine any molecular or functional pro-angiogenic effects. anti-angiogenic mirnas and angiogenic mrna levels were quantified by rt-qpcr. effects of hotair on proliferation of hdmecs was also determined. hotair was then loaded into msc evs by delivering a cmv-based hotair plasmid to mscs for endogenous loading via a concentration gradient. evs were collected by differential centrifugation. hotair content within evs was confirmed by gel electrophoresis and rt-qpcr. effects on migration of hdmecs by hotair-loaded msc evs were determined using a scratch assay. results: results: overexpression of hotair decreased mir-29 c and mir-107, while increasing vegf and hif-1a. hdmec proliferation was also increased in hdmecs overexpressing hotair (p < 0.01). hotair was visually confirmed in hotair-loaded msc evs by gel electrophoresis, but was undetectable in unmodified msc evs. rt-qpcr confirmed a 900-fold increase of hotair compared to control msc evs. hdmecs showed a more statistically significant rate of gap closure when treated with hotair-loaded evs (p < 0.01) than compared to control msc evs (p < 0.05). summary/conclusion: summary: loading lncrna hotair into msc evs is achievable by a concentration gradient-dependent method and offers potential to enhance the angiogenic properties of msc evs. nanomaterial labelling of exosomes for cell biology introduction: exosomes are vesicles secreted by many, if not all, cell types and have been known about for decades. among larger micro vesicles that are produced directly from the cell membrane, the small (30-150 nm), exosomes are similar in size to a virus surrounded by a lipid bilayer. we and others have demonstrated that exosomes contain proteins, lipids, rna, and dna, making them promising materials for diagnosing and treating diseases, including many cancers such as brain cancer. in addition, exosomes from neurons and glial cells represent a novel type of intercellular communication. however, their size makes them hard to track with traditional fluorescence microscopy. to address this, we developed photothermal microscopy (ptm), which uses gold nanomaterial labelling to track exosomes' interaction with and effect on cells/tissue. methods: exosomes secreted by tumour cells and general exosomes found in the blood were isolated using differential ultracentrifugation or a commercially available kit (invitrogen). next, the exosomes were characterized by (tem), (nta), and western blotting to determine shape, size, morphology and the protein profile in the exosomal membrane. after characterization, the exosomes were labelled with gold nanoparticles via sonication. next, the samples were washed, and the exosomes were labelled with fluorescence dye to stain the membrane. after staining and labelling, the exosomes were added to u87 cells in culture and incubated for 3 h. they were then fixed by 4% paraformldehyde and imaged by ptm. results: ptm found that exosome-cell interactions are exosome-type dependent, as u87 cells took up exosomes from other u87 cells but not human serum exosomes. this suggests that exosome uptake is a selective process and depends on the source of the donor cells. summary/conclusion: exosomes can be labelled with gold nanoparticles via sonication then successfully tracked by ptm to study the effect of exosome source on exosome-cell interactions and communication. cells incubated with u87 exosomes took the vesicles up rapidly, while cells incubated with serum exosomes had little uptake. ptm will help us design selective exosome-based strategies to treat different conditions, including brain cancer and cns damage. funding: nsf epscor riii award 1457888. loading of goat´s whey extracellular vesicles with spiked microrna and curcumin as an strategy for developing new nanocarriers for acellular therapies introduction: extracellular vesicles (ev) are involved in cell signalling and are present in a variety of cell secretions such as milk, from which enormous amount of ev can be purified, thus milk is an attractive raw material for scaling up ev production for therapeutic, cosmetic or other uses. here we isolated evs from the whey fraction of goat´s milk and demonstrated that such evs can be loaded with molecules like polyphenols and mirna. methods: to achieve this, milk was collected from lactating goats and fractionated by acidification and centrifugation into whey and caseins. evs were purified from the former fraction by serial centrifugation and precipitation with commercial kit (total isolation/ thermo fisher) and characterized by electron transmission microscopy (tem), western blot to identify surface markers and measurement of size through nanotracking analysis. once isolated, evs were loaded with different concentration of a spiked synthetic mir39 or with the polyphenol curcumin. mirna or curcumin were co-incubated over night with evs at 4oc, precipitated and purified as described above, with an additional washing and precipitation for curcumin. concentration of mirna uploaded by evs was quantified using mir39 specific qpcr. curcumin was measured using a spectrophotometer at 420 nm. results: evs isolated from whey had an average size of 120 nm, were positive for hsp70, cd9 and alix. in tem, evs were identified with their natural conformation and corresponding size to exosomes. qpcr showed a significant difference of expression of mir39 in relation to control (loaded with shame) and the negative control (p < 0.05). curcumin presence was also confirmed after washing and precipitacion. summary/conclusion: in conclusion, milk evs and exosomes can be loaded with mirna and a polyphenol and can be used as alternative nanocarrier for acellular therapies. introduction: extracellular vesicles (evs) are cellderived lipid membrane nanoparticles that serve as messengers of intercellular communication, transferring bioactive molecules to recipient cells. evs have a natural therapeutic potential with high flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic delivery vehicles. considering the importance of evs, their isolation methods are still a bottleneck. to get insights into the tissue-specific cargo in vivo for complete exploitation of evs as therapeutic, biomarker and diagnostic tools, ev purification methods are critical. the aim of the study was brought about to develop an efficient ev purification method both in vitro and in vivo and to further investigate function of evs in cellular senescence. methods: to isolate tissue-specific evs in vivo we developed recombinant evs by genetically fusing snorkel-tag to the cd81. the snorkel-tag enables on-column protease treatment for purifying evs which does not rely on traditional immunoaffinity purification protocols using low ph or high salts solutions. results: we systematically evaluated the purification of evs harbouring snorkel-tag by employing different methodologies. our findings suggest that evs harbouring snorkel-tag indeed can be purified at high purity without altering ev biophysical properties. furthermore, we expressed cd81-snorkel-tag under p16ink4a promoter and were able to purify evs derived from senescent cells. summary/conclusion: finally, we are developing an in vivo model with cd81-snorkel-tag under p16ink4a promoter. this will provide us detail insights into the ev cargo secreted from senescent derived cells, by purifying evs harbouring snorkel-tag under pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools. summarized, we have here developed novel tool for studying content and function of evs in the context of ageing and disease. this tool will now pave the way for studying the molecular mechanisms underlying these ev functions in vivo. funding: this work was funded by the austrian science fund phd program biotopebiomolecular technolgy of proteins (w1224). engineering exosomes with gata-4 jie xu, christian paul, yi-gang wang and meifeng xu university of cincinnati, cincinnati, usa introduction: exosomes, are small vesicles (30-150 nm) secreted from cells that can transport and deliver of their components such as lipids, proteins, dna, mrna, and mirna to target cells. gata-4, a cardiac transcription factor, has been shown to regulate differentiation, proliferation, and survival of a wide range of cell types. delivering gata-4 protein into ischaemic tissues may be one of the most straightforward approaches to improve cardiac function following myocardial infarction. here, exosomes were engineered with gata-4 by infusing gata-4 with exosome targeting peptide. methods: the open reading frame of mouse gata-4 cdna was ligated to xpack lentivirus vector (xpack-gata-4) and plvx-ef1-ires-pouro lentivirus vector (plvx-gata-4), respectively. hek 293 cells were transduced by lentivirus, then exosomes were isolated from conditioned medium of hek293 cells using ultracentrifugation. exosomes were identified using transmission electronic microscope (tem), and the expression of gata-4 was semi-quantified using western blot. the internalization of exosomes was tracked via treating bend3 cells with exosomes pre-labelled with pkh26. introduction: chinese hamster ovary (cho) cells have dominated as the mammalian cell host for the manufacture of humanized biologics, in part owing to their genomic plasticity and robust growth in suspension culture. there is great interest surrounding the use of extracellular vesicles (evs) as novel therapeutics owing to their capacity to deliver bioactive molecules. however, much remains unknown about the mechanisms involved in ev cargo loading, limiting their development as novel biologics. to this end, we have engineered cho cells to stably express constructs enabling loading of gfp into evs. methods: tetraspanins are established markers of ev identity. accordingly, cd81 was selected as a tethering point to generate evs with gfp cargo and constructs were generated via golden gate assembly. cho cells were stably transfected by electroporation and expression was verified with fluorescence microscopy and western blotting. growth in batch culture was monitored to establish maximum viable cell densities for ev harvest and recovered evs were characterized by nanoparticle tracking analysis (nta). finally, uptake of gfp-evs was studied using time-lapse fluorescence imaging in co-culture experiments. results: strong localization of cd81-gfp was observed at the cell membrane and blotting confirmed intact tetraspanin fusion present at the expected molecular weight. additionally, cells were confirmed to retain high gfp expression post-cryopreservation. stable cell pools were able to reach viable densities greater than 7 million cells/ml in batch culture and nta allowed for detection of gfp cargo even prior to ev isolation. evmediated transfer of functional gfp to recipient cells was found to occur over a period of hours. introduction: extracellular vesicles (evs) are considered promising for therapeutic applications. evs resemble the cell membrane, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. despite the promising potential of evs for therapeutic applications, robust manufacturing processes that would increase the scalability and consistency of ev production are still lacking. methods: in this work, evs were produced by mesenchymal stromal cells (msc), isolated from different human tissue sources (bone marrow, umbilical cord matrix and adipose tissue). msc were selected as these cells allow for a scalable production of evs, while displaying low immunogenicity. a vertical-wheel™ bioreactor system was implemented for the production of msc-derived evs and compared with traditional static systems. the obtained ev products were characterized by nanoparticle tracking analysis, atomic force microscopy, zeta potential and western blot. results: the bioreactor system allowed to obtain evs at higher concentration and productivity, as well as more homogeneous size distribution profiles, when compared to traditional static culture systems. functional studies were performed using breast cancer and lung cancer cell lines. proliferation assays allowed to determine the dose-response profiles of these cell lines when exposed to msc-derived evs. a bell-shaped profile was observed for most cases, since raising the ev concentration lead to increased cell proliferation until a certain point (25-50 µg/ml), after which cell proliferation was attenuated with increasing ev concentrations. summary/conclusion: the bioreactor culture system allowed a substantial improvement in the production of msc-derived evs, while the obtained dose-response profiles will be valuable to determine the most appropriate ev concentrations for anticancer drug delivery. overall, we demonstrate that this culture system is able to robustly manufacture human msc-derived evs in a scalable manner towards the development of novel therapeutic products such as anticancer drug delivery systems. biodistribution and cellular location of inhaled exosomes and liposomes in the lung introduction: increasing evidence reveals the potential role of extracellular vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment of lung diseases. encapsulation and delivery of potential rna and microrna targets into liposomes and exosomes are attractive drug delivery methods, but remain difficult to deliver to the pulmonary parenchyma to reach target lung cell types. here, we demonstrate effective delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma via inhalation treatment in a murine model of idiopathic pulmonary fibrosis. methods: human lung stem cells (lscs) were generated and expanded from healthy whole lung donors. lsc-exosomes were purified via ultrafiltration and diilabelled using vybrant☐ labelling solution according to the manufacturer's instructions. dsred-labelled liposomes were generated using lipofectamine™ rnaimax transfection reagent and block-it™ alexa fluor™ red fluorescent control according to the manufacturer's instructions. lsc-exosomes and liposomes were delivered via nebulization to cd1 mice with bleomycin-induced pulmonary fibrosis. exosome and liposome delivery and biodistribution were visualized 4-and 24-hours post-treatment through histological analysis. the study was approved by the institutional animal care and use committee of north carolina state university and complied with all national and state ethical standards. results: exosome and liposome delivery to the pulmonary parenchyma was confirmed by the presence of dii and dsred fluorescence in lung histological sections that penetrated the mucus-lined respiratory epithelium. more exosomes and liposomes surpass mucus-lined surfaces 24-hours post-treatment compared to 4-hours post-treatment. fluorescent colocalization of exosomes and liposomes with alveolar type i cells, alveolar type ii cells, basal lung cells, and cd68 + macrophages was observed through immunohistochemistry analysis. more exosomes and liposomes colocalize with these cell types 24-hours post-treatment compared to 4-hours post-treatment. summary/conclusion: lsc-exosomes and liposomes penetrate the mucus-lined respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment. lsc-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells, and interstitial macrophages with improved biodistribution 24-hours post-treatment. funding: this study was supported by the nc state chancellor's innovation fund. transfection reagent artefact accounts for some reports of extracellular vesicle function codiak biosciences, cambridge, usa introduction: extracellular vesicle (ev) functions are frequently investigated by transiently transfecting cells with plasmid dna to produce evs modified with protein(s) or nucleic acid(s) of interest. however, evs and the dna-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common ev isolation procedure. activities attributed to evs may therefore be due to contaminating dna -transfection reagent complex. methods: ev producing cells were transiently transfected with plasmid dna encoding gene-editing or split enzymes fused to ev-targeting protein sequences. differential and density gradient ultracentrifugation were used to purify evs from cell culture supernatant or dna lipoplexes from cell-free culture media. protein expression and localization to evs was confirmed by western blot. cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. results: reporter cells treated with ultracentrifuge pellet material (ucp) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the ucp with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in evs. ucp isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not evs. serial media changes on ev producing cells post-transfection did not significantly reduce ucp activity on reporter cells. treatment with nucleases did not digest complexed dna, did not significantly reduce dna levels in the ucp as measured by qpcr, and did not decrease activity in reporter cells treated with ucp from either transfected cells or no-cell controls. summary/conclusion: we find that dna-transfection reagent complexes are not separated from evs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. due to the pernicious nature of the dna-complex in these cellular assays, it is likely that some reports of ev function are likely artefacts produced by contaminating dna-complexes. we find that density gradient centrifugation can effectively separate evs and dnacomplexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate ev function. chair: suresh mathivanan -la trobe university cancer stem cell-derived exosomes: potential biomarkers for early diagnosis and prognosis in pancreatic cancer introduction: pancreatic cancer (paca) is the most deadly manlignancy, due to late daignosis and early metastatic spread, which prohibits surgery. it is urgently for relaible, early detection. research shows that tumour-derived exosomes, which had been present in the blood in the early stage of tumour formation and before metastasis, is the vanguard forces of tumour formation and metastasis; cancer stem cell-derived exosomes (csc-exos) has stronger migration ability, so the detection of blood csc-exos for early diagnosis and monitoring of progress for paca has great research potential and the value of application. methods: protein markers were selected according to expression in exosomes of paca cell line culture supernatants, but not healthy donors' serum-exosomes. according to these preselections, serum-exosomes were tested by flow cytometry for the pancreatic cancer stem cell marker cd44v6 and tspan8. results: the majority (95%) of patients with paca and patients with nonpa-malignancies reacted with anti-cd44v6 and anti-tspan8. serum-exosomes of healthy donors' and patients with non-malignant diseases were not reactive. recovery was tumour grading and staging independent including early stages. introduction: chronic traumatic encephalopathy (cte) is a tauopathy that affects individuals with a history of mild repetitive brain injury frequently seen in contact sports. initial neuropathologic change of cte include perivascular deposition of phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation of neurofibrillary tangles in neurons throughout the brain. extracellular vesicles (ev) are known to carry neuropathogenic molecules in neurodegenerative disease and able to cross the blood brain barrier. we therefore examined the protein composition of ev separated from cerebrospinal fluid (csf) and plasma in former national football league (nfl) players with cognitive dysfunction, and an agematched control group with no history of contact sports. methods: evs were separated from csf and plasma from former nfl players (n = 4, 14) and controls (n = 5, 12) by affinity separation method or size exclusion chromatography, respectively. the ev protein profiling was characterized by simoa for tau and ptau and mass spectrometry. the protein data was analysed for ev enrichment, differentially expressed proteins, pathway analysis and correlation with cognitive function, head impact and tau/p-tau levels by biostatistics and bioinformatics. results: the level of total tau and p-tau in csf evs was not significantly changed, but significantly elevated in plasma evs from former nfl players. the 95 proteins were commonly identified between the paired plasma-csf from the same patients, but there was no significant correlation with disease status. collagen alpha-3(vi) chain (col6a3), −1(vi) chain (col6a1) and reelin (reln) were differentially expressed in former nfl players' plasma evs. a combination of these 3 proteins in plasma ev can distinguish former nfl players from controls with 85% accuracy by machine learning. summary/conclusion: the interacting plasma-csf ev proteomes provide an original resource to ev biomarker development for neurodegenerative disease, and col6a3, reln and col6a1 in plasma evs can be potential biomarker for monitoring the cte development. density-based fractionation of urine to unravel the proteome landscape of extracellular vesicles in prostate cancer introduction: current diagnostic tests are unable to discriminate indolent from aggressive prostate cancer (pca), leading to overdiagnosis and overtreatment, and an intense interest in biomarkers to improve clinical decision making. urine is considered an ideal proximal fluid for biomarker identification in pca due to its direct contact with the urogenital system. the discovery and translation of extracellular vesicle (ev) content into pca biomarkers remains challenging due to the difficulty of obtaining urinary ev (uev) with high specificity. methods: we developed a step-by-step protocol to separate uev by orthogonal implementation of ultrafiltration and bottom-up density gradient centrifugation (bu-odg). we implemented complementary particle and protein measurements to identify uev (lower density) and protein rich fractions (higher density) and assess the performance of bu-odg (specificity, efficiency and reproducibility). using mass spectrometry-based proteomics we interrogated uev and protein rich fractions from matched urine and radical prostatectomy tissue samples from pca patients (n = 4), and urine from men with pca prior to (n = 12) and after local treatment (n = 10), benign prostatic hyperplasia (n = 12) and other urological cancers (n = 11). results: bu-odg separated uev from soluble proteins and tamm-horsfall protein (thp) complexes with high specificity and reproducibility, outperforming differential ultracentrifugation, exoquick and size-exclusion chromatography. comparison of the uev proteome from men with benign or malignant prostate disease, allowed us to expand the known human uev proteome and identify a pca specific uev proteome not uncovered by the analysis of the protein rich fraction. proteomic analysis of ev separated from prostate tumour interstitial fluid and matched uev confirmed pca specificity of the uev proteome. analysis of the uev proteome from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uev reflecting their respective cancer tissues of origin. summary/conclusion: we identified hundreds of previously undetected proteins in uev of pca patients and developed a powerful toolbox to map uev and protein rich fractions, ultimately supporting biomarker discovery for urological cancers. immunoglobulin a coating of faeces-derived bacterial vesicles as a marker of inflammatory bowel disease in humans nader kameli a , frank stassen b , heike becker c , john penders c , daisy jonkers d and paul savelkoul b introduction: iga is the most abundant antibody in mucosal secretions and plays a crucial role in maintaining the balance between the host and the gastrointestinal microbiome. recent studies suggested that pronounced iga coating is especially prominent among inflammatory commensals which drive intestinal disease. membrane vesicles (mvs, nano-sized particles released by bacteria) have also been found to interact with the host and modulate development and function of the immune system. however, their interaction with iga has not been studied yet. here we developed a method to isolate and characterize the mvs from faecal samples and checked for possible differences in iga coating patterns of mvs in health and disease. methods: mvs were isolated by using a combination of ultrafiltration and size exclusion chromatography from faecal samples of 6 healthy controls (hc), 6 patients with active crohn disease (cd) and 6 cd patients in a remissive state. quantification and verification have been done with tunable resistive pulse sensing (trpsbased analysis) bead-based flow-cytometer (bbfc) and transmission electron microscope (tem). mvs were selected with specific antibodies for capturing (gram +: lta, gram-: ompa) followed by pe-conjugated anti-human iga antibodies as detection. results: we could successfully isolate 1*109-5*109 particles/ml from 500 mg of faeces. bbfc in combination with trps provide a valuable method for (semi-)quantitative measurements of mixed populations. intriguingly, remarkable differences were found between iga coating mvs derived from healthy controls and active and remissive cd patients as mvs derived from healthy controls were significantly more coated compare to both cd patient groups. in details, for selected g-ve derived mvs: 60% of the total population of mvs derived from hc were coated, 20% from remissive cd patients, and <5% of active cd patients; and for selected g+ ve derived mvs: 55% of the total population of mvs derived from hc were coated, 34% from remissive cd patients, and 25% of active cd patients. (data are represented as the mean). summary/conclusion: here we demonstrate for the first time that mv isolated from the faecal samples are also coated with iga, and surprisingly mvs from healthy volunteers were more densely coated than mvs from diseased patients. the possible consequence of this difference remains to be determined in future studies. monitoring altered tetraspanin and psma expression in prostate cancer derived extracellular vesicles via advanced image flow cytometry (isx) lukas w. prause a , christopher millan b , natalie hensky c , tullio sulser c and daniel eberli c a universityhospital zurich, zurich, switzerland; b university of zurich hospital, schlieren, switzerland; c university of zurich hospital, zurich, switzerland introduction: new diagnostic and therapeutic options for patients with prostate cancer are urgently needed. prostate-specific membrane antigen (psma)-based imaging and therapy are increasingly used for prostate cancer management. unfortunately, as a membrane protein, psma is not found as a soluble protein in the blood and therefore has limited utility as a diagnostic biomarker. however, psma has reportedly been observed as a cargo protein of prostate cancer-derived extracellular vesicles (evs). we demonstrate altered psma expression on evs derived from prostate cancer cell cultures (c4-2, lncap) in response to novel next-generation androgen receptor inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental nonsteroidal antiandrogen (epi-001) that binds covalently to the n-terminal domain of the androgen receptor and dihydrotestosterone (dht). additionally, evs were isolated from the plasma of prostate cancer patients who participated in the prococ biobank campaign at the usz. plasma was taken and stored from patients both pre-and post-prostatectomy. results: transmission electron microscopy, nanoparticle tracking analysis and simple western (wes) analysis show stable size distribution and amount of evs produced by treated and non-treated cells. using advanced image-based flow cytometry, altered tatraspanin and psma expression could be detected in evs isolated from cell culture supernatants of lncap and c4-2 prostate cancer cells following their treatment. summary/conclusion: measuring psma expression on extracellular vesicles might pave the way to use image flow cytometry of evs to develop a blood based diagnostic test for prostate cancer patients with a wide range of possible applications including: 1) monitoring response to therapy and, 2) early indications of potential relapse. funding: vontobel fondation. proteomic profiling of human neural cells derived extracellular vesicles to identify human brain cell-type specific markers introduction: alzheimer's disease (ad) is a common neurodegenerative brain disease which affects appropriately 30 million patients worldwide. one of the major challenges in ad is to develop reliable biomarkers for early diagnosis and disease-modifying therapies, especially before the clinical symptoms. extracellular vesicles (evs) carry cargos of proteins, lipids and nucleic acids. there was no comprehensive characterization of evs isolated from specific brain cell types, which may be useful for cell type-specific biomarkers. the purpose of this study is to isolate evs from human induced pluripotent stem cell (ipsc)derived brain cells for proteomic profiling and characterization of cell type-specific molecules. methods: human ipscs-derived neurons, microglia and primary cultured astrocytes were differentiated in ev-depleted media. the evs were isolated by differential centrifugation combined with size exclusion chromatography, followed by characterization using nanoparticle tracking analysis and mass spectrometry. the proteomic data were subjected to bioinformatics analysis results: we identified 153 proteins from neuronderived ev (nde), 215 proteins from microgliaderived ev (mde) and 380 proteins from astrocytederived ev (ade) by proteomics. gene ontology analysis indicated that most of these proteins are associated with evs. furthermore, 15, 48 and 251 proteins are present individually in ndes, mdes and ades. among them, high levels of atp1a3 and syt1 in ndes, itgam and cd300a in mdes, and eaat1 and gfap in ades were found, all of which are typically and highly expressed in the original cells. summary/conclusion: our results provide us the potential candidates for cell-type specific ev markers, which will be helpful to develop non-invasive tools to enrich ev originating from specific brain cells and may lead to the development of new biomarkers for neurodegenerative disorders. ) are a tremendous resource for extracellular vesicle (ev) research, but they are heavily focussed on mammalian evs, i.e. evs from humans and laboratory animals, where protein cargoes are well characterised, and a wide selection of antibodies are commercially available. protein markers can be used to identify and define the types of mammalian ev and to determine the presence of any contaminants that might confound functional studies. similar resources are not as readily available for bacterial evs as these are not as well characterised, commercially available antibodies are much less abundant and immunological variation between different bacterial species (and there are 1 trillion bacterial species on planet earth!) means that each species, strain, or group of related species may require different antibodies. methods: to identify quality markers for bacterial evs, we have characterised the proteome of cells, crude evs (ultracentrifuge pellet from cell free culture supernatant) and size exclusion chromatography or density gradient centrifugation purified evs from two different (pathogenic vs probiotic) strains of escherichia coli grown under two different environmental conditions, and one strain of mycobacterium marinum grown in one medium. results: our results identify a selection of proteins enriched in purified ev preparations, and proteins that are depleted after purification steps. summary/conclusion: our results allow the identification of potential markers for ev purity and non-ev contaminants, but also highlight the variability in bacterial ev preparations and suggest potential targets that can be used to investigate the heterogeneity of bacterial ev populations. introduction: recent findings indicate an increase in mid-life mortality rates in the usa and persistent, significant race-related health disparities exemplified by differential mortality rates. this suggests that exploring new molecular markers that may be linked to mortality could provide novel insights into factors that are driving mortality rates. accumulating data suggests that extracellular vesicles (evs) circulating in blood may be potential biomarkers of age-related disease. evs are nano-sized membranous vesicles that bear molecular cargo and mediate intercellular communication between different cells and tissues. little is known about whether ev characteristics differ by race or whether evs are associated with clinically relevant mortality risk factors. methods: in this cross-sectional study, plasma evs were isolated from middle-aged african american (aa) and white males and females. results: we report no significant differences in ev size or concentration with race or sex. there were significantly higher ev levels of phospho-p53, total p53, cleaved caspase 3, erk1/2 and phospho-akt in whites compared to aas. higher ev levels of phospho-igf-1r were found in females compared to males. we examined ev characteristics and protein cargo in the context of well-established clinical mortality risk factors. ev concentration was significantly, and positively, associated with several mortality markers including, high-sensitivity c-reactive protein (hscrp), homoeostatic model assessment of insulin resistance (homa-ir), alkaline phosphatase, pulse pressure, body mass index, and waist circumference. the relationship of ev concentration and cargo with mortality markers differs by race. summary/conclusion: our data show that ev-associated proteins can differ by race and sex and are associated with mortality risk factors. this study provides insight into the characterization of evs in middle-aged aas and whites, which may aid in the development of ev-based diagnostics. funding: this study was supported by the national institute on ageing intramural research program of the national institutes of health. repurposing specialised cell-free dna blood collection tubes for extracellular vesicle isolation introduction: liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. however, many plasma processing protocols have been designed with a single biomarker in mind. here we investigate whether specialised dna blood stabiliser tubes could be repurposed for the analysis of extracellular vesicles (evs). methods: peripheral blood (n = 3) was collected into k3-edta, roche or streck cell-free dna (cfdna) blood collection tubes and processed using sequential centrifugation immediately or after storage for 3 days. microev were collected from platelet poor plasma by 10,000 g centrifugation and nanoevs isolated using size exclusion chromatography. particle size and counts were assessed by nanoparticle tracking analysis, protein by bca assay and dot blotting for blood cell surface proteins. results: major variations in micro and nanoevs were seen with delayed time to processing. nanoev counts did not change with processing delay or tube collection type but the associated protein amount increased, indicative of cell lysis or activation. the protein was predominantly derived from from platelets (cd61) and red blood cells (cd235a). the increase in associated protein was seen more in the k3-edta and streck tubes indicating that the roche tubes may offer improved cell stability. conversely, microevs increased in both quantity and protein content with delay to processing indicative of both lysis and cell activation, irrespective of tube type. epithelial cell surface marker epcam abundance remained the same across conditions in both micro and nanoevs demonstrating that epcam+ evs were stable. summary/conclusion: specialised cfdna collection tubes can be repurposed for micro and nanoev analysis, however simple counting or using protein quantity as a surrogate of ev number may be confounded by pre-analytical processing. the evs would be suitable for disease selective ev subtype analysis if the molecular target of interest is not present in blood cells. introduction: nutrigenomics and nutrigenetics have been defined as the effect of nutrients on gene expression and genetic variation on dietary response, respectively. here, we propose the isolation and characterization of exosomes from donors carrying different alleles of hla-dqa1 and hla-dqb1, to investigate their involvement in coeliac disease (cd) management. methods: a chilean population (n = 30) was investigated for snps mutations in hla class ii alleles associated to cd predisposition (as well as other mutations related to other food intolerances), using the genochip food technology. exosomes have been isolated from donors' serum by ultracentrifugation and characterized by sds-page, western blotting (cd63 and cd9), and transmission electron microscopy. exosomes were also studied for their interleukins (il-6 and il-1ra) content. results: among the studied population, 86% present at least one of the alleles leading to cd development and 60% carry alleles encoding for αand β-chains heterodimers associated with very high risk to develop cd. in parallel, isolated exosomes from donors with low to extremely high risk for cd showed high il-1ra content (108.8 ± 15.91 to 148.8 ± 12.37), as the persons were not following any treatment. however, values of il-1ra decrease in exosomes isolated form persons receiving treatment for cd. a relationship between exosomes' content and genetic susceptibility for cd has been observed, which may suggest their possible use as biomarkers for cd as the diagnostic of this disease is still a big issue. summary/conclusion: until this point of this underway project, we demonstrate the existence of a relationship between the exosomes' content in il-1ra and genetic susceptibility for cd. furthermore, the genetic predisposition to cd could also modulate the gut colonization process, another important player in intestinal homoeostasis. in the next step, extracellular vesicles from gut microbiota will be isolated and analysed to determine their role in cd management. nasibeh karimi a , razieh dalir fardouei a , jan lötvall a and cecilia lässer b a krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, göteborg, sweden; b krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, gothenburg, sweden introduction: the ability to isolate extracellular vesicles (evs) from blood is vital in the development of evs as disease biomarkers. both serum and plasma can be used but few studies have compared them in terms of amount and type of evs. we have previously developed a method to isolate evs from plasma with minimal contamination of lipoprotein particles (karimi et al 2018) . the aim of this study was to compare the presence of different subpopulations of evs in plasma and serum. methods: blood was collected from healthy subjects, from which plasma and serum were isolated. evs were isolated using a combination of density cushion and size exclusion chromatography (sec) (protocol 1) or a combination of density cushion and density gradient (protocol 2) or immune-capturing (anti-cd63, anti-cd9 and anti-cd81 beads) (protocol 3). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, electron microscopy (em), exoview, flow cytometry and mass spectrometry (lc-ms/ms). results: as determined by nta and protein measurement more evs could be isolated from plasma with protocol 1 and the majority of the vesicles were cd9/ cd41a positive as determined with exoview and western blot. additionally, flow cytometry and western blot showed that more cd9/cd41a positive evs where also identified with protocol 3. furthermore, western blot showed increased amount of cd41a in plasma samples in protocol 2. when labelled evs were spiked in freshly collected blood, no difference in recovery was seen for plasma and serum. summary/conclusion: this study shows that a larger amount of evs could be isolated from plasma compared to serum when three different isolation methods were used. firstly, this suggests that more evs are present in plasma. secondly, it suggests that these vesicles are probably released by platelets and that evs are not trapped in the clot during serum formation. future studies are needed to answer how this affects the use of blood-derived evs as biomarkers from serum and plasma. tumour-derived extracellular vesicles contain distinct integrin proteins stephanie n. hurwitz a and david g. meckes b a university of pennsylvania, philadelphia, usa; b florida state university, tallahassee, usa introduction: cargo profiling, including proteomic analyses, of tumour cell-derived extracellular vesicles (evs) may provide ripe opportunities for further understanding cancer growth, drug resistance, and metastatic behaviour. accumulating data suggest that cancer-derived evs contain membrane-bound integrin proteins which may aid in cell detachment, migration, and homing to future metastatic niches. we have previously published an extensive proteomic profile of secreted vesicles from the nci-60 panel of human cancer cells. methods: here, we further examine the distinct integrin components in these cancer-derived evs, and additionally profile evs released from benign epithelial cells by liquid chromatography and tandem mass spectrometry for comparison. results: we demonstrate the enrichment of integrin receptors in cancer evs compared to vesicles secreted from benign epithelial cells. total ev integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumour stage across a variety of epithelial cancer cells. in particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumours. other integrins including α4, αl, and β2 are enriched in vesicles derived from leukaemia cells, and may provide a means to distinguish haematopoietic cell-derived evs. summary/conclusion: this study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumour stage. differential expression and selective packaging of integrins into evs may contribute to further understanding the development and progression of tumour growth and metastasis across a variety of cancer types. effect of nicotine and menthol on cytochrome p450 and antioxidant enzymes in rat plasma-derived extracellular vesicles introduction: tobacco products such as e-cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine, flavorants such as menthol, and other particulates. here, we aimed to study the hypothesis that whether nicotine and menthol modulate nicotine-metabolizing cytochrome p450 2a6 (cyp2a6), antioxidant enzymes (aoes), sod1 and catalase in plasma extracellular vesicles (evs). modulation of these enzymes would eventually lead to nicotine-induced toxicity and hiv-1 pathogenesis via evs-based cell-cell interactions. methods: we isolated and characterized evs from rat plasma before and after nicotine self-administration (nic) with audiovisual cue (av) and menthol and characterized using ev markers according to the isev guidelines. protein associated with cyp2a6, sod1, and catalase were quantified by western blot. results: we measured size, total protein, and acetylcholine esterase activity of evs and found no significance difference in these characteristics before and after nic. to investigate the effect av, menthol alone or in combination in the absence and presence of nic, first we evaluated the expression of ev markers cd9 and cd63. the results showed menthol and av together increased the levels of cd9 (p ≤ 0.05), the marker of small vesicles, in the presence of nic. the nic with menthol and av showed a pattern of increased levels of small vesicle but could not reach to significance. next, we demonstrated that the nic with av increased the level of sod1 (p ≤ 0.05), which showed a pattern of increased levels of catalase and cypa6, though statistically non-significant. the expression of nicotine receptor did not change under any conditions used. the results showed an increased level of cyp2a6 (p ≤ 0.01), sod1 (p ≤ 0.05), and catalase (p ≤ 0.05) in plasma evs in the menthol-nic group compared to menthol group only. nic group with a combined av and menthol, showed further increase in the levels of cyp2a6 (p ≤ 0.01), and catalase (p ≤ 0.05). further analysis of plasma evs on inflammatory cytokines/chemokines in these groups, and the effect of plasma evs on nicotine-induced toxicity and hiv pathogenesis are underway. summary/conclusion: nicotine administration increased, though not statistically significant, the levels of circulatory evs. moreover, the study provided evidence that nicotine in the presence of menthol, av, and/or menthol+av increased nicotine-metabolizing cyp2a6 in all the groups and aoes in specific groups. funding: we thank the national institute on drug abuse (grant #da047178, da-047638) for supporting our work. introduction: biomarker discovery in breast cancer (bc) is a clinical need for therapeutics and non-invasive diagnostics. tumour exosomes are involved in premetastatic niche formation and drug resistance and represent a source of non-invasive biomarkers. the identification of tumour exosomal biomarkers provides, not only, the possibility to discriminate patient groups also potential targets to control cancer progression that could be exploited to develop innovate bc therapeutic strategies. methods: we have performed a comparative differenti. al proteomic profile of four bc cell lines and their derived-exosomes, representative of the most relevant bc subtypes in clinic to search non-invasive biomarker candidates. then, we have carried on two bioinformatics approaches: 1) protein association network analysis interaction (string) and 2) pathway inference analysis (hipathia), to characterize the functional profiling for each bc subtype. results: we have found differentially-expressed proteins, in both cells and exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance. exosome proteome profile reflects their different bc cell origin suggesting potential indicators of bc subtype. further, bioinformatics analysis reveals a differential role of exosomes in bc signalling pathways in recipient cells, according to their protein cargo and cell origin. summary/conclusion: our results show a set of cells and exosome proteins that highly discriminate bc subtypes and may significantly contribute to further studies for the design of bc biomarker predictor to stratify bc patients and the development of novel therapeutic strategies. funding: a set of potential biomarkers to discriminate breast cancer subtypes. circulatory evs as potential biomarkers of hiv-drug abuse interactions and neurological dysfunction in hiv-infected subjects and alcohol/ tobacco users sunitha kodidela a , kelli gerth a , namita sinha a , asit kumar b , prashant kumar a and santosh kumar a a uthsc, memphis, usa; b university of tennessee health science center, memphis, usa introduction: abuse of alcohol and tobacco can exacerbate hiv pathogenesis and its associated complications. further, the diagnosis of neurocognitive disorders associated with hiv infection and drug abuse using csf or neuroimaging are invasive or expensive methods, respectively. therefore, extracellular vesicles (evs) can serve as reliable non-invasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. hence, we aimed to study the specific evs proteins, which are altered in both hiv and drug abusers to identify a physiological marker to indicate the immune status and neuronal dysfunction of hiv-positive drug abusers. methods: evs were isolated from plasma of the following subjects: a) healthy b) hiv c) alcohol drinkers d) cigarette smokers e) hiv+alcohol drinkers f) hiv +cigarette smokers. quantitative proteomic profiling of evs was performed by mass spectrometry and potential ev proteins associated with neuronal dysfunction were quantified by westernblot. results: the evs were characterized according to the isev guidelines. a total of 343 proteins were detected in evs of all the study groups. comparison of proteins among all the study groups revealed that hemopexin was significantly altered in hiv+drinkers compared to drinkers and hiv subjects. further, our study is the first to show properdin expression in plasma evs, which was decreased in hiv+smokers and hiv+drinkers compared to hiv patients. though we couldn't identify the few other cns-specific proteins, g-fap and l1-cam, associated with neuronal dysfunction in plasma evs by mass spectrometry, we could detect those by westernblot. the protein expression of gfap (p < 0.01) was significantly enhanced in plasma evs obtained from hiv-positive subjects and drinkers compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. the l1cam expression was found to be significantly elevated in smokers (p < 0.05). both gfap and l1cam levels were not further elevated in hiv+smokers compared to hiv+nonsubstance users. summary/conclusion: the present findings suggest that hemopexin, and properdin show potential as markers for hiv-drug abuse interactions. further, astrocytic and neuronal-specific markers (gfap and l1cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco and thus may represent as potential biomarkers for neurological dysfunction in those subjects. funding: we thank the national institute on drug abuse (da047178) for supporting our work. . electrochemical detection of mirna-21-5p introduction: micrornas (mirnas) are small, single-stranded, non-coding rna species that regulate gene expression post-transcriptionally, and are transported by extracellular vesicles (evs). they play an essential role in biological processes, such as development, cell proliferation, apoptosis, stress response and tumorigenesis. thus, mirnas are considered relevant biomarkers in health. more particularly, mirna-21-5p is expressed in neurons after traumatic brain injury, being expectably transported to peripheral fluids by brain evs that cross the blood-brain barrier. the main goal of this work is to develop an electrochemical biosensor for the detection of mirna-21-5p in serum. methods: overall, the experimental assembly of the biosensor was made in three stages. the first one consisted in the electrodeposition of aunps, the second one in the incubation of anti-mirna21-5p on the carbon screen-s printed electrodes and the final stage in the incubation of mercaptosuccinic acid for blocking unspecific bindings. the probe was hybridized with the target mirna21-5p by a consecutive incubation of several standard solutions. each modification was evaluated with cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis) and square wave voltammetry (swv). the electrochemical behaviour of the biosensor was followed in all steps by monitoring the electron transfer features of a standard redox system. the redox probe selected for this purpose was [fe (cn)6]4-/[fe(cn)6]3-. results: the results indicated that the electrodeposition of gold was more effective for −1.5 v for 600 s and could lead to better signals upon anti-mirna-21-5p hybridization. summary/conclusion: in general, the experiments showed increasing charged transfer resistance upon the incubation of higher concentrations of mirna-21-5p. in these experiments, ev concentration is a critical variable that must be carefully controlled to ensure scientific rigour and reproducibility: without controlling for concentration (dose), experimental outcomes will exhibit excess variability that could mask important biological discoveries. in this study, three orthogonal methods are compared for accuracy in ev quantification: microfluidic resistive pulse sensing (mrps) and nanoparticle tracking analysis (nta) were compared to each other and relative to the gold standard method, transmission electron microscopy (tem). the ability of nta to accurately measure particle concentration is shown to depend on the polydispersity of the sample itself. results validate the accuracy of mrps and emphasize the importance of using orthogonal techniques to quantify evs. methods: reference urinary vesicles were prepared and analysed with the three methods and the relative concentration accuracy of nta and mrps were compared as a function of particle size. the hypothesis that nta concentration accuracy was impeded by sample polydispersity was tested using polystyrene bead mixtures having a range of polydispersity. a theoretical argument based on fundamental physics explains the experimental observations. results: tem and mrps measurements of the evs were in excellent agreement and showed a broad, polydisperse particle size distribution with no peak on the measured size range (50 nm -400 nm diameter). nta differed significantly from tem and mrps by reporting a steep decrease in measured concentration below about 150 nm that resulted in a peak in the reported particle size distribution. bead measurements confirmed the hypothesis to be tested: sample polydispersity significantly affects the ability of the nta method to accurately measure concentration, even for particles as large as 150 nm diameter. summary/conclusion: these experiments validate mrps as an accurate method for quantifying evs and highlight the importance of using orthogonal measurement methods in accordance with misev2018 guidelines. clinically relevant synthetic reference materials to standardize concentration measurements of extracellular vesicles: state-of-the-art and future prospects introduction: there is an unmet need to standardize concentration measurements of extracellular vesicles (evs). flow cytometry is the clinically most applicable method, but the currently available reference materials for calibration are insufficient. for example, the refractive index (ri) between standard particles and evs substantially differs, whereas concentration and fluorescence calibration particles are too bright. the goal of this study is to ascertain the most desired properties of reference materials to standardise ev measurements. methods: an online survey was prepared within the meves ii project to measure the desired size, concentration range, optical properties, choice of fluorochromes, and stability of synthetic ev reference materials for flow cytometry (fcm) measurements. besides the desired properties of ev reference particles, also the available instrumentation was assessed in the survey, which was sent to the members of the stakeholder committee of metves ii project and members of the ev flow cytometry working group. results: the most desired size, concentration, and ri range for ev reference particles is 50 nm to 500 nm, 10e7 to 10e10 1/ml, and 1.35-1.45, respectively. based on mie-theory evaluation of the sensitivity of the available instruments, none of the respondents would be able to detect 50 nm particles with ri = 1.35 with their current instruments. regarding fluorescence intensity, the most desired range according to the responses is from 10 molecules of equivalent soluble fluorochromes (mesf) to 10 000 mesf. considering the sizes of evs and fluorescent labels, the maximal mesf that can be obtained for ev reference particles with 50 nm diameter and high molecular mass fluorescent dyes is in the range of several hundreds. typical antigen densities on evs fall below 100 copies per ev with 50 nm diameter, i.e. mesf values above 100 are probably not physiologically relevant in this size range. summary/conclusion: a part of the desired properties of ev reference materials precludes either their physical feasibility of production or their detection at most currently available fcms, meaning that the intended reference materials will be future-proofed. funding: this work was supported under 18hlt01 metves ii project by the european metrology programme for innovation and research (empir). the empir initiative is co-funded by the european union's horizon 2020 research and innovation programme and the empir participating states. comparison of production and activity of amniotic fluid stem cell extracellular vesicles from 3d hollow fibre bioreactor and 2d culture. culture conditions may affect ev composition and potency. here we compare production, potency, identity and therapeutic potential of evs collected from cells grown in culture dish (2d) versus hfbr (3d). methods: human clonal afsc were derived from patient-consented amniotic fluids. 1x10e6 hafsc were seeded in 2d (145cm2), and 1.6x10e7 hafsc on a small 20kd mwco hfbr (fibercell-c2025d, 450cm2) with fibronectin coating; both cultured in chang medium with 20% of es-fbs, starved for 24 hr and then evs collected. the effect of harvest frequency was tested (8hrs, 24 hr, 72hrs,1 wk). 2d-evs and 3d-evs were compared by nanosight, potency assay (by wb), identity (by exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, alport syndrome). results: 2d production was~5.5x10e9 ev/ml/24 hrs while 3d was~2.8x10e10 ev/ml (first four 24 hrs) and 4.4x10e10 ev/ml (two days of hourly harvests). very little difference in ev concentration and very similar size distribution (~130 nm) were observed during harvest intervals; possibly indicating either significant ev re-uptake or inhibition of ev secretion dependent upon free ev in the supernatant. 3d-evs trapped vegf (an in vitro established potency assay) as efficiently as 2d-evs, and expressed cd9, cd81, cd63, cd80, cd86 and vegfr1 as 2d-evs. summary/conclusion: 3d-evs had comparable properties and bio-activity to 2d-evs, but the hfbr produced 10x more evs. hfbr cell culture conditions for hafsc still need optimization, however an available 1.2 m2 cartridge provides a 50x scale up potential. the hfbr, a cgmp closed system, can produce sufficient numbers of ev to support pre-clinical and clinical applications with at least similar properties to evs produced by conventional 2d methods. funding: -intramural funding -intramural ev core pilot funding demonstration of high gain mode in combination with imaging flow cytometry for improved ev analysis luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. in recent years, the importance of evs has become apparent, as they are key mediators of intercellular communication. however, quantifying and characterizing evs in a reproducible and reliable manner is challenging due to their small sizeexosomes range from 30 to 100 nm in diameter. it is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize evs using flow cytometric methods. however, these systems have not been designed to measure objects smaller than a cell. methods: here, we describe the use of high gain mode on the amnis® imagestream® imaging flow cytometer to address the challenges of measuring small particles. in this new high gain mode, the charge-coupled device (ccd)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the ev. object thresholds and masking have also been adjusted to better identify and detect small particles. results: preliminary results using murine leukaemia virus-sfgfp reference particles have shown up to a fivefold increase in the number of gfp-positive objects collected in high gain mode, when compared to standard gain on the imagestream system. summary/conclusion: in this study, we demonstrate improved small particle detection, including evs, using this new high gain mode on the imagestream imaging flow cytometer. distance-controlled accelerated catalysed hairpin dna circuit for multiple and sensitive detection of exosomes-associated mirnas introduction: sensitive and simultaneous monitoring of multiplexed exosome-associated rnas is of great value for early cancer diagnosis remains a challenge. methods: here, we report a simple, multiple and sensitive exosomes-associated multiplex mirnas detection method that uses distance-controlled accelerated catalysed hairpin dna circuit (chdc) system without any complex operation or enzymatic amplification. the distance-controlled accelerated chdc can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting mirnas without any transfection means. results: we show that distance-controlled accelerated chdc strategy with signal amplification capability could selectively and sensitively identify low level rnas in serum evs, distinguishing patients with early-and late-stage breast cancer from healthy donors and patients with benign breast disease. summary/conclusion: this simple, accurate, sensitive, and cost-effective liquid biopsy by the distance-controlled accelerated chdc method is potent to be developed as a non-invasive breast cancer diagnostic assay for clinical applications. impact of isolation methods on biophysical heterogeneity of single extracellular vesicles university of california los angeles, ca, los angeles, usa introduction: current biophysical analysis of extracellular vesicles (evs) typically encompasses particle density and size distribution determinations using various techniques. however, variabilities in ev isolation methods and the structural complexity of these biological-nanoparticles (sub-100 nm) necessitate more rigorous nanoscale biophysical characterization of single evs to facilitate more reliable and comparable evbased assays. methods: combining atomic force microscopy (afm), super-resolution optical and conventional particle sizing light scatter and microfluidic techniques, we compared the unique sub-nanometre scale biophysical properties of breast cancer cell-derived ev isolates obtained using different isolation methods. results: afm and dstorm particle size distributions showed coherent unimodal and bimodal particle size populations in centrifugation and immune-affinity isolates respectively. more importantly, afm imaging revealed striking differences in nanoscale morphology, surface undulations, and vesicle-to-non-vesicle ratios among ev isolates from different isolation methods. our findings demonstrate the effectiveness of orthogonal high-resolution biophysical characteristics of single evs, not discernable via particle size distributions and counts alone. summary/conclusion: the identified nanoscale biophysical characteristics of ev isolates represent a strategic and complementary framework to resolve differences in the heterogeneity and purity of evs from introduction: extracellular vesicle (ev) concentrations measured by flow cytometry are incomparable. to improve comparability, the metves ii consortium is developing traceable reference materials and procedures, which require validation by test samples. in previous interlaboratory comparison studies, however, a main source of variation was introduced by pre-analytical variables and measurement artefacts introduced by test samples. to minimize variation introduced by test samples, our aim is to develop off-the-shelf biological test samples containing pre-labelled evs. methods: human urine and plasma were collected from healthy donors. evs were labelled with lactadherin-fitc, isolated by size-exclusion chromatography to remove free dye and minimize swarm detection, and mixed with dimethyl sulphoxide (dmso), exocap or trehalose, frozen in liquid nitrogen and stored at −80°c . after thawing, ev concentrations were measured by a calibrated flow cytometer (apogee a60-micro). results: compared to the ev concentrations measured in fresh plasma and urine, the concentrations decreased 27% in plasma (p = 0.04; mean of the 3 cryopreservation agents) and 35% in urine (p = 0.05) after one day of storage. after 5 months of cryopreservation, the concentration of plasma evs decreased 2% (dmso and exocap) and 8.5% (trehalose) compared to one day of storage, whereas the concentration of urine evs decreased 6% (exocap) and 18% (dmso and trehalose). summary/conclusion: we have developed ready-touse, pre-labelled human evs that are stable up to 5 months and dedicated for use in interlaboratory comparison studies. to further increase stability, other cryopreservation agents will be tested. our biological test samples will be key to validate the new reference materials and procedures developed by metves ii in 2021. funding: this project has received funding from the empir program co-financed by the participating states and from the european union's horizon 2020 research and innovation program. understanding intracellular fate of ev-delivered content introduction: despite much work performed on evaluating the potential effects of extracellular vesicles (evs), the functional uptake of their cargo is still controversial. this project aimed to demonstrate that ev content (protein and mrna) is protected and can be subsequently transferred with functional activity into recipient cells, while also developing a tool to assess and quantify functional ev uptake. methods: fusion proteins used were mitochondrial localized coxviii-cfp-nanoluc(cox) and nuclear localized h2b-rfp-nanoluc(h2b). results: hek293 t cell-derived evs protected cox proteins from proteinase k digestion while demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by bioluminescence that was still detectable in recipient cells 96 hrs post-ev-exposure. to confirm functional uptake, recipient cells exposed to evs containing h2b for 72 hrs were imaged and some recipient cells manifested fluorescent red nuclei. to demonstrate the presence of functional mrna within evs, producer cells were transfected for such a duration as not to have detectable levels of protein in the evs while still containing detectable levels of mrna (qpcr) even after rnasea treatment. transfer of these evs to hela cells showed an increase in expression of h2b which was blocked by cyclohexamide, confirming translation of the mrna (2.2kb). to determine if recycling of ev delivered proteins occurs, recipient hela cells were exposed to evs containing cox for 72 hrs. all extracellular evs were removed and cells were trypsinized (0.25% for 30 min) to remove any non-internalized cox protein. 48 hrs later, evs (cd63+ and cd9+) released from cells contained cox suggesting recycling of protein or possibly recycling of entire evs. lastly, an assay was developed to measure functional ev uptake. nanoluc protein was split in two and fused to mturquoise2 (n65) or mscarlet-i(66 c). expression of each fragment alone exhibited non-detectable levels of luminescence while expressing both together had a significantly increased signal. delivery of either fragment within an ev to a cell expressing the corresponding fragment worked as confirmation and quantification of ev uptake (hek293, u87, hela cells). summary/conclusion: this study robustly demonstrates ev delivery of functional mrna and protein to cells, while also establishing a simple assay to quantify and validate functional ev uptake. theoretical model of ev losses due to adsorption on the tube walls. application for immunomagnetic detection of the vesicles introduction: short-term storage of unfrozen samples of vesicles, mainly at 4°c, overnight or during a couple of days is rather common laboratory practice. however, it was found to lead to significant losses of vesicle concentration supposedly due to adsorption on the walls of the tube. the present work develops a theoretical model intended to describe the vesicle adsorption process. the experimental validation of the model was made using method of immunomagnetic precipitation. methods: the theoretical model considers the "diffusion-limited" case of vesicles storage. the maximal adsorption capacity of the surface of contact between the tube and the solution is given as the number of vesicles in hexagonally packed monolayer. for experiment, the vesicles were purified from ht29 cell culture supernatant by differential centrifugation, aliquoted and kept at −80 c. further the aliquots were consequently unfrozen, and placed into the tubes with different surface treatment and kept at +4 c. the kinetics of vesicles loss was measured by anti cd9 immunomagnetic capturing followed by cd81, epcam and cd166 staining and flow cytometry. results: the model allows the estimation of the adsorption-associated losses as dependent on initial vesicles concentration, volume of the solution, tube geometry, the storage temperature and duration case of quiet vesicles storage (without mixing) and also accounts an expected effect of active agitation of the solution (ev-beads complexes formation). theoretical calculations were illustrated by analysis of ev at different storage conditions and during reaction of immunomagnetic precipitation of the vesicles. summary/conclusion: it was demonstrated that application of tubes surface treatment allows increasing sensitivity of immunomagnetic precipitation method to 2x10^5 for cd81+, 5x10^5 for epcam+ and 2x10^8 for cd166+ vesicles. introduction: it is now largely accepted that the intestinal microbiota plays a key role in intestinal bowel diseases (ibd). an imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. there are also indications that extracellular vesicles produced by bacteria and exosomes produced by epithelial cells might be increased in this family of diseases. methods: in order to differentiate healthy and ibd faecal samples on the basis of their vesicle profiles, we want to develop a means to enumerate rapidly particles in faecal samples, based on interferometric microscopy. the videodrop technology, developed by myriade, relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles. it will permit to compare on large scales the viral load of healthy subjects and ibd patients. results: this fast and easy-to-use device was compared to the nta on several types of eukaryotic and prokaryotic vesicles and our preliminary results are encouraging. introduction: small extracellular vesicles (sevs) produced by mesenchymal stromal cells (msc-sevs) may be useful in cell-free therapies for immunomodulation and tissue regeneration. methods: to characterize msc-sevs produced ex vivo, human bone marrow mscs were cultured in mesencult-acf plus (macfp), an ev-free and animal component-free culture medium for 3 days and spent medium collected to isolate sevs by ultracentrifugation (uc). analyses of sevs were performed by nanoparticle-tracking analysis (nta), western blot (wb), and human umbilical vein endothelial cell (huvec) tube formation assay. results: analysis of fresh uncultured macfp by uc, nta and wb for cd63, cd81, and cd9 confirmed the absence of sevs. msc-sevs isolated from spent macfp by uc ranged from 80-150 nm in size and were positive for cd63, cd9, and cd81 proteins. these sevs could be stored at −80°c for >4 months in solution or lyophilized with minimal loss based on nta and wb analysis. the msc-sevs contained the msc-associated micrornas let7a, mir21, and mir26a as per qpcr analysis. the biological function of ex vivo isolated msc-sevs was assessed using a human umbilical vein endothelial cell (huvec) tube formation assay. huvecs treated with msc-sevs generated tubes as early as 6 h after seeding, which were not observed in control huvec cultures until 15 h. moreover, the number of branch points present in such tube structures was >fourfold higher in huvec cultures (n = 5) supplemented with msc-sevs versus control, with the former lasting >60 h and the latter lasting <50 h in culture. direct comparison of the performance of macfp medium to media containing non-depleted or ev-depleted foetal bovine serum demonstrated that only mscs cultured in macfp (n = 3) were able to expand robustly with a doubling time of 1.1, 2.1 and 8.9 days in these media, respectively. lastly, methods for isolating sevs using newly developed easysep-ev™ magnetic separation kits and size exclusion columns will be presented. summary/conclusion: taken together, these data demonstrate that msc-sevs can be produced in high yield in macfp medium and that these possess similar physical, phenotypic and functional characteristics as sevs in vivo. funding: this work was privately funded by stemcell technologies inc. introduction: extracellular vesicles (evs) are heterogeneous group of small vesicular structures released by different types of cells, including stem cells (scs). as recent studies demonstrate that they may enclose bioactive content and transfer it into the target cells, growing interest is placed on the utilization of evs in the field of biomedical research. however, there is still lack of standardized methods of evs characterization. as an example, typical flow cytometry-based protocols, commonly used for cells phenotyping, may be inadequate for the characterization of evs as particles with size close to the detection limit of conventional cytometers. thus, the aim of this study was to optimize and compare the use of different flow cytometry platforms for the multiparameter analysis of evs isolated from different types of scs populations. methods: ev samples were obtained by ultracentrifugation of conditioned media collected from selected scs types, including human induced pluripotent scs (ips) and mesenchymal scs (mscs). next, several high resolution flow cytometry systems: cytoflex, apogee (a50 and a60 micro-plus) and image stream mk ii were employed to compare their sensitivity and resolution, as well as influence of "swarm" effect. furthermore, we examined evs phenotype, including expression of tetraspanins and other surface markers. results: our results have revealed that tested flow cytometry systems may be utilized for the phenotypic characterization of evs secreted by scs populations. however, the conventional staining and gating strategy protocols have to be thoroughly optimized. additionally, depending on a type of tested cytometer, we have demonstrated the difference in a "swarm" effect and its influence on obtained results regarding evs phenotype. finally, imaging flow cytometry platform was also employed to visualize evs on the single particle level. summary/conclusion: in conclusion, we have demonstrated that tested high-resolution flow cytometry platforms are convenient methods for the multiparameter characterization of evs produced by different types of scs populations. however, careful selection of particular measurement parameters should be performed depending on a type of employed system. funding: this study was funded by ncbr grant strategmed iii (strategmed3/303570/7/ ncbr/2017) to ezs. evaluation of atcc's exosomes from cell culture supernatant as reference standards in research and development. introduction: exosomes are subcellular particles 30-150 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. exosomes are stable carriers of cell-free cargo in the form of dna, rna, and proteins, thereby making them an attractive candidate for diagnostic and therapeutic applications. however, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards in extracellular vesicle research (ev). methods: exosomes were isolated from cell culture supernatants of different atcc cell lines including stem cells and cancer cell lines representing the most prevalent cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using tangential flow filtration (tff). these exosomes underwent sterility and mycoplasma tests as a part of their quality control. the morphology and size distribution of these exosomes were evaluated through multiple strategies including nanoparticle tracking analysis (nta), asymmetrical flow field-flow fractionation (af4), cryo-electron microscopy (cryo-em) and spectra dynetm particle analyser. exosome surface markers were also analysed through multiple strategies such as electro chemiluminescent elisa, flow cytometry and western blotting. also, stem cell exosomes and cancer exosomes were further evaluated for functionality through in vitro functional assays including migration assay, angiogenesis and anchorage independent growth assay. results: our optimized tff method resulted in high yields of > 1 × 1010 exosomes/ml and average protein equivalent of more than 1 mg/ml. more than 90% of the exosomes population had an average size distribution of 50-200 nm and median size of 110 nm confirmed through a number of different size distribution instruments. although cell line dependent, we were able to obtain similar expression levels of different cell surface markers including tetraspanins (cd63, cd81, cd9) when evaluated through different methods. our functional data demonstrated stem cell exosomes were functionally active in promoting cell migration and tubule formation. additionally, cancer cell exosomes were found to promote a malignant phenotype in an anchorage independent growth assay. summary/conclusion: collectively, we demonstrated our ability to reproducibly manufacture production-scale batches of exosomes from multiple different cell types. our purified exosomes are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. therefore, they can serve as ideal reference materials that can support different ev-based research applications. exo-cise: extracellular vesicles enriched from plasma post-exercise promotes myogenesis and neurogenesis bianca paris a , yaomeng liu a , vicente pagalday-vergara a , julie davies b , priya samuel a , ayman abu seer a , johnny collett a , laura gathercole a , ken howells a , karl j. morten b , zhidao xia a , daniel anthony b , david r f. carter a , helen dawes a and ryan c. pink a a oxford brookes university, oxford, uk; b university of oxford, oxford, uk introduction: physical activity brings about a widespread physiological response and elicits the beneficial adaptation of several tissues and organs. furthermore, regular participation in physical activity reduces the risk of developing major non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis, and dementia. two important processes known to occur following physical activity are myogenesis and neurogenesis; both of which involve the activation and proliferation of specialised tissue-resident stem cells. the molecular mechanisms regulating these processes following exercise are poorly understood to date. here, we investigated the contribution of extracellular vesicles, which are released into the circulation after exercise, to benefit adult myogenesis and neurogenesis. methods: small extracellular vesicles were enriched from the blood of healthy participants before and following maximum and moderate intensity exercise. differentiation and proliferation using a range of methods was measured following vesicle treatment onto primary myoblasts and neuronal primary exvivo stem cells. activation of key cellular pathways were measured. results: we show significant proliferation and differentiation changes of both stem cell types. this is independent of extraction method, extracellular vesicle depleted fractions and is interestingly conserved across mammalian species. remarkably, we see an age-related effect. summary/conclusion: this advocates that short single bouts of exercise may promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles which opens an interesting field in endogenous ev therapies. show promise as a cell-based therapy for retinal degeneration. while clinical trials are ongoing, the potential of extracellular vesicles (evs) as biomarkers for monitoring eye health and disease is not well studied. this study characterized the ev surface profile and cargo of hipsc-rpe to offer a baseline assessment in normal and disease conditions. moreover, we evaluated the importance of pnpla6, a gene involved in membrane integrity and when mutated causes retinal degeneration, in ev biogenesis and secretion. methods: evs were isolated from serum-free culture medium of hips-rpe and identified with nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of exosomal markers, including alix, tsg101, and cd63. surface marker detection and proteomic profiling were completed using an ev surface marker kit and mass spectrometry, respectively. small interfering rna targeting pnpla6 was used to knockdown the expression in hipsc-rpe and evs were characterized. results: nanoparticle tracking analysis confirmed the presence of both microvesicles (>150 nm) and exosomes (<150 nm) by size distribution and the concentration of evs (1x108 particles/ml) from rpe. tem displayed typical morphological characteristics of evs. the presence of known ev markers, alix, tsg101, and cd63 was confirmed via immunoblot and flow cytometry. surveillance of ev surface markers revealed enrichment of epithelial markers (cd326) and stem cell markers (cd133/1) that depict donor cell origin and functional proteins including integrin-binding (cd29) and tgf-beta receptors (cd105). in addition, proteomic analysis revealed regulators of inflammation and rpe function, including hemopexin, clusterin, complement factor i, and pigment epithelium-derived factor. furthermore, reduction in pnpla6 expression reduced vesicle secretion and vesicle size compared to non-targeting controls. introduction: vascular endothelial growth factor (vegf) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. vegf plays a role in normal physiological functions such as bone formation, haematopoiesis, wound healing, and development. mesenchymal stem cell (msc) was found to secretes potential growth factors such as vegf when cultured in vitro. however there are some beliefs that foetal bovine serum (fbs) which usually used as serum in cell culture content vegf. methods: msc seeded in in 96-well plate in with concentration of 5,000 cell/well. cells were incubated for 24 hours and fasted for another 16 hours using only dmem. cells were treated with complete medium consist of dmem and 10% fbs. culture medium were collected after 5, 12, and 24 hours after treatment. cell were culture in 37ºc dan 5% co2. vegf concentration was detected using elisa technique. results: vegf concentration was not found in fbs which do not contact with msc. an increasing of vegf concentration in time-dependent manner was shown when culture medium was used in msc cell culture in normoxic condition. the result of vegf concentration when culture 5, 12, and 24 hours were 74.022 pg/ml, 76.67 pg/ml, and 93.58 pg/ml, respectively. the mechanism of msc release growth factor is still under investigated. however, the classic growth factors and cytokines serves paracrine control molecules which were important in regenerative medicine. vegf was found to be an important molecules in angiogenesis process and determine the fate of cells. summary/conclusion: msc secreted vegf and concentration increased in time-dependent manner. isolation and characterization of exosomes from canine stem cells introduction: unlike induced disease models using laboratory animals, naturally occurring disease models display pathophysiologic attributes that are more similar to human diseases. unfortunately these models are underutilized in translational regenerative medicine research. this is partly due to the slow development of species-specific experimental therapeutics to investigate comparative efficacy. thus, we set out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem cells (cad-msc) to use as a comparative therapeutic in dogs. to accomplish this, we optimized an isolation and purification strategy and characterized their molecular properties. methods: exosomes were isolated by sequential centrifugation and subsequent ultrafiltration. the proteome was characterized by tandem mass tag (tmt) mass spectrometry and the mirna cargo was identified using a canine specific pcr array with subsequent target and enrichment analysis using targetscan and the panther platform, respectively. also, nanoparticle tracking analysis and transmission electron microscopy were used to determine exosome size and structure. to investigate bioactivity, we measured the ability of exosomes to inhibit collagen production in an in vitro model of fibrosis. results: exosomes were purified by ultrafiltration using a 100 kda cut-off. proteomic analysis by tmt mass spectrometry identified 1262 unique proteins. 90% of the exocarta top 100 were identified from this list. additionally, we identified the mirna cargo within exosomes and found 27 highly expressed mirnas. enrichment analysis identified multiple pathways of probable regulation including angiogenesis (fold enrichment = 3.1; p < 0.0001) and transforming growth factor-beta (tgfb) signalling (fold enrichment = 4.1; p < 0.0001). exosome size was quantified to be 119.7 ± 3.4 nm with a modal average of 98 nm. lastly, in the presence of exosomes, tgfb stimulated fibroblasts deposited 32.2% less collagen than vehicle controls (p = 0.036). summary/conclusion: in summary, cad-mscs exosomes display structural and functional features comparable to stem cell derived exosomes from other species. use of these exosomes in naturally occurring disease canine models may provide superior predictive value for human clinical trials. funding: support provided by the ccah, school of veterinary medicine, uc davis. mesenchymal stem cells-derived exosomes promote in vitro the progression of triple negative breast cancer cells introduction: mesenchymal stem cells (mscs) are multipotent stromal cells and have been described as key regulators of different aspects of tumour physiology. in tumour pathogenesis, mscs can integrate the tumour microenvironment after recruitment and are able to interact with cancer cells to promote tumour modifications by affecting epithelial-tomesenchymal transition (emt). it was revealed that exosomes derived from mscs are critical players in the tumour niche. exosomes are a novel way of cellto-cell communication and play crucial roles in the majority of pathways that contribute and affect response to therapy, cell-adhesion molecules and the progression of tumour cells. because of the known importance of this communication we decided to investigate the implication of mscs with triple negative breast cancer (tnbc) cell lines as well as exosomal profiles between the experimental conditions. methods: the interactions of mscs with triple negative breast cancer cell lines (mda-mb-231 and hs578t) was performed by coculturing mscs (or tnbc cell lines) with exosomes derived from tnbc cell lines (or mscs). physical characterization of isolated exosomes was performed followed by their molecular investigations. cell proliferation was detected by mtt assay and migration was analysed by wound healing assay using 2d cultures. moreover, we also used 3d culture to assess the exosomes uptake and to observe their capability of internalization into a 3d structure. the alterations in expression level of some transcripts (mrnas and mirnas) and protein profile were investigated by qrt-pcr, western blot and immunofluorescence staining. results: we found that mscs-derived exosomes are actively incorporated by triple negative breast cancer cell lines (2d culture). in coculture, in tnbc cells the expression level of mesenchymal markers and emt markers (e-cadherin, vimentin) at mrna and at protein levels, as well as mirna-derived exosomes targeting mesenchymal genes were significantly affected. using bioinformatics tools, we highlighted the important biological processes which were activated by promoting tumour modifications. in addition, using 3d culture we provided a comprehensive understanding regarding exosomes internalization in 3d structures, which closely mimics in vivo conditions, compared to 2d culture. summary/conclusion: in this work, we focus on the investigation of mscs-derived exosomes in order to highlight their implication in several biological processes, including tumour proliferation and progression of triple negative breast cancer cells. all these alterations affect the response to therapy and should be considered for developing efficient therapeutic strategies. natural killer cell-derived extracellular vesicles have a potent anti-leukaemic effect and selectively target the cancer stem cell subpopulation introduction: natural killer (nk) cells of the immune system recognize and kill tumour cells. extracellular vesicles (evs) secreted from nk cells are capable of killing tumour cells independent of the cell to cell contact required for nk cell activation. cancer is a leading cause of death, primarily due to metastasis and recurrence. cancer stem cells (csc) within tumours are resistant to chemotherapy and immune attack, and cause metastasis and relapse. identification of the cancer types killed by nk evs is limited, and the effect of nk evs on cscs has not been described. here we determine whether nk-derived evs kill a myeloid leukaemia cell line and its csc subpopulation. methods: nk evs were isolated from our nk cell line, nk3.3, derived from normal human lymphocytes. nk3.3 evs were characterized by immunoblotting, proteomics, and next generation rna sequencing. human k562 leukaemia cells were treated with nk3.3 evs in vitro and analysed for proliferation and markers of cell death. results: nk3.3 evs contain ev-associated proteins alix, cd63, hsp70, and tsg101, nk effector molecules perforin, granzymes a and b, granulysin and nklam/ rnf19b, an e3 ubiquitin ligase required for maximal nk cytotoxicity, and tumour suppressor mir-186. nk3.3 ev treatment of k562 significantly decreased its expression of proliferation markers cd71 and ki67, and increased the frequency of apoptotic and necrotic cells, paralleled by elevated levels of active caspases −3 and −7. non-tumorigenic cells were unaffected by nk ev treatment. most notably, nk3.3 ev treatment significantly reduced the frequency of k562 cells highly expressing aldh, a csc marker. summary/conclusion: nk3.3-derived evs have a robust anti-tumour effect on k562 myeloid leukaemia cells and selectively target the csc population, suggesting they may circumvent the evasion and resistance mechanisms used by cscs. nk3.3 evs therefore have introduction: due to their potential as a key bioactive agent in regenerative medicine applications, mscderived extracellular vesicles (msc-evs) are increasingly being investigated as a clinical therapy. manufacturing that generates enough evs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. moreover, a complementary approach that increases the ev productivity, i.e. the number of evs produced per cell, could further help to accelerate the development of msc-evs as a therapy. methods: we developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of msc-evs. briefly, human bone marrow-or umbilical cord-derived mscs were rapidly expanded under xeno-free conditions (i.e. >150x expansion within 10 days). cultures were then switched to our proprietary ev collection medium and evs were harvested for up to three additional days. at the end of culture, the evs in the conditioned media were concentrated using a tangential flow filtration (tff) system. to increase the productivity of mscs, two medium supplements were developed that increased ev yield by either increasing the number of evs generated per cell in a shortened culture process or increasing the number of collected evs by lengthening the ev collection culture period. results: this scalable msc-ev manufacturing method was implemented in both 2d flask and 3d bioreactor culture and generated over 2,000 particles per cell in 2d and over 4,000 particles per cell in 3d. with the addition of a medium supplement to increase evs produced per cell, the ev productivity was increased >2x after 24 hrs. alternatively, ev productivity was also increased >2x by addition of the medium supplement that extended ev collection culture period. summary/conclusion: msc-ev success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of evs. these results present one such solution. furthermore, increasing ev productivity, for instance by medium supplements that increase evs per cell or lengthen culture times could further address the limitation of generating the evs required for development and translation of clinical therapies. simplifying scalable msc ev production in a microcarrier-based bioreactor system divya patel, josephine lembong, katrina adlerz, jon rowley and taby ahsan roosterbio inc, frederick, usa introduction: the growpt0ing numbers of msc-ev clinical applications drives the need for a scalable msc-ev production platform. while most msc-evs are generated while cells are attached to tissue culture plastic, such 2d cultures cannot be scaled up to meet the yields necessary for commercialization of ev-based therapeutics. we have shown that 3d bioreactors can be used to generate msc-evs and that paradigm can be scaled directly in terms of yield from the 3 to 15 l scales. the technical expertise of seeding cells onto microcarriers for expansion in bioreactors, however, requires technical expertise not available to all those in the ev field. therefore, our goal here is to simplify and expedite the ev collection process in bioreactors by cryopreserving cells on microcarriers, such that end users can merely thaw and then collect msc-evs. methods: mscs were expanded in 2d and then seeded on three different microcarriers and cultured in a bioreactor for 5 days. when confluent, cells on microcarriers were cryopreserved. to evaluate the microcarriers and the cryopreservation protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth media for 24 hours, then in ev collection media for 3 additional days. cell recovery and ev production upon thaw was evaluated and compared to ev collection from fresh, non-cryopreserved cells. results: total cell counts 24 hrs post thaw were comparable to those before cryopreservation and to fresh samples prior to ev collection. following 3-day ev collection, concentration of particles collected from cryopreserved cells on microcarriers were similar to those collected from the fresh cells (2e9 particles/ml). this process was validated for two different microcarriers using two separate cryopreservation solutions. summary/conclusion: our results show that cryopreserved hmscs on microcarriers can support ev collection in a 3d bioreactor process with a particle yield that is comparable to those collected from fresh cells. this cryopreserved product can simplify ev production, reducing cost and time by removing process steps associated with the hmsc expansion, with in a paradigm suitable for scale-up. the whitening, anti-wrinkle, and wound-healing effects of extracellular vesicles from orbicularis oculi muscle-derived stem cells. introduction: skeletal muscle-derived stem cells possess potent therapeutic activities in the treatment of muscle-related disorders. in our study, we tried to isolate and characterize orbicularis oculi muscle (orm)-derived stem cells (orm-scs) from the discarded human tissues which were obtained from the ocular surgery-subjected patients. we also prepared the natural extracellular vesicles (evs) from the cultured orm-scs and assessed the their therapeutic actitities including the skin whitening, anti-wrinkle, and wound healing effects. methods: we isolated the orm-scs from the patients subjected to ocular surgery and characterized the orm-scs by analysing cell morphology, proliferation, expression levels of the cell surface and stemness-associated markers, and tri-lineage differentiation and colony-forming capacities, confirming the stemness properties of the orm-scs. then, we prepared the natural evs from the orm-scs via the centrifugation and filtration of the media supernatants and their therapeutic activity was investigated. results: the isolated orm-scs showed spindle-like morphology and positive expression of cd105, cd 90, and cd73, but they were negative in expression of cd45 and cd34. the orm-scs showed the capacity of osteogenic, adipogenic, and chondrogenic differentiations. the evs from orm-scs (orm-sc-evs) possessed the apparent inhibitory effect on the melanin synthesis in b16f10 cells by blocking the tyrosinase activity, although orm-sc-evs treatment did not dramatically change the expression level of melanogenesisrelated genes, such as microphthalmia-associated transcription factors (mitf), tyrosinase (tyr), tyrosinaserelated protein1(tyrp-1), and tyrp-2. in addition, we confirmed that orm-sc-evs could stimulate skin cell migration and increase the expression level of antiwrinkle related genes and wound-healing properties. summary/conclusion: this study revealed the stem cell property of orm-scs and the whitening, antiwrinkle, and wound healing effects of orm-sc-evs, suggesting that orm-scs and orm-sc-evs can be successfully used for stem cell-based ev therapy and cosmetics, by regulation the melanogenesis, wrinkle, and wound. funding: this work was supported by grants from the national research foundation (nrf) funded by the korean government (2019m3a9h1030682). use of stem cell extracellular vesicles as a holistic approach towards cns repair introduction: neurological diseases and disorders are leading causes of death and disability worldwide. many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. we have previously shown that extracellular vesicles (evs) from infected cells contain viral by products (noncoding rnas and proteins) and that these evs can exert deleterious effects on recipient cells1-3. therefore, in the context of neurotrophic viruses evs may contribute to or perpetuate processes relating to neuroinflammation and neurodegeneration. due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. in recent years it has been well-established that stem cell evs play a critical role in the functionality associated with stem cells. the diverse biological cargo contained within these vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem cell evs have been demonstrated in a wide range of cell types. while a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the central nervous system (cns). methods: we have isolated and recovered high yields of evs from large scale cultures of both induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) using tangential flow filtration. our ev characterization includes both phenotypic (size, tetraspanin expression) and biochemical assays. ev functionality has also been assessed in vitro utilizing several cellbased assays related to cellular viability, migration, angiogenesis, and immunomodulation in both healthy and damaged recipient cells with relevance to the cns. results: our data suggests that evs from different sources of stem cells display unique phenotypes, exhibit differential association with various cytokines, proteins, and long non-coding rnas, and have the ability to significantly enhance processes that are critical for cellular repair4. lastly, utilizing an ipsc-derived neurosphere model, we have observed a robust uptake of stem cell evs and have found that these evs are able to effectively penetrate these 3d structures. summary/conclusion: collectively, these results highlight the "holistic" properties of stem cell evs by demonstrating their ability to partially reverse or reduce damage in various cell types. funding: this work was supported by national institutes of health (nih) grants ai078859, ai074410, ai127351-01, ai043894, and ns099029 to fk and r33 ca206937 and r01ar068436 to lal. the effect of cell culture media on extracellular vesicle secretion from mesenchymal stem cells and human pluripotent stem cell-derived neurons introduction: cell culture media and its supplements are known to affect the secretion and isolation of extracellular vesicles (evs) from cell cultures. identification of these effects is crucial especially when planning to use evs as therapeutic agents. here, we investigated the effect of cell culture media on ev yield from human mesenchymal stem cells (mscs) and human pluripotent stem cell (hpsc)derived neurons. methods: evs were collected from cell-conditioned media (ccm) and no cell control (ncc) media using size-exclusion chromatography (sec). mscs were cultured in dmem/f12:neurobasal medium or in opti-mem reduced serum medium, both supplemented with exosome-depleted foetal bovine serum (fbs). the ev yield from hpsc-derived neurons was compared at two maturation time points (day 46 and 60), in dmem/f12:neurobasal or in opti-mem, with and without 3-hour kcl stimulation. sec fractions were analysed by nanoparticle tracking analysis (nta), protein concentration assay and blinded transmission electron microscopy (tem). results: ccm samples had a clear peak of evs in sec fractions 7-10, which was not detected with ncc. interestingly, a second population of evs eluted in sec fractions 13-17 in both ccm and ncc, indicating presence of evs in exosome-depleted fbs. moreover, this second population differed largely between used media batches. culture medium had no significant effect on msc ev yield (dmem: 8.30e+08 particles/ ml, opti-mem: 9.61e+08 particles/ml). with neuronal cultures, no significant differences in ev yield were found between culture media or cell maturation time points. in contrast to earlier findings, 3-hour stimulation of neurons by kcl resulted in significantly smaller ev yield compared to non-stimulated controls (stimulated: 5.28e+10 particles/ml, non-stimulated: 1.43e+11 particles/ml, p < 0.02). summary/conclusion: our results indicate that exosome depleted-media are not entirely devoid of vesicles, which can cause bias in downstream analyses. however, sec is a good method to separate cellsecreted evs from the contaminating medium-derived evs. culture medium did not affect the number of evs secreted by mscs or neurons; instead, we observed larger differences between media batches. this data emphasizes the importance of analysing the ncc as negative control in all cell culture experiments. mouse mesoangioblast stem cell extracellular vesicles are able to influence macrophage cell activity maria magdalena barreca a and fabiana geraci b a dept stebicef university of palermo, palermo, italy, palermo, italy; b dept stebicef, university of palermo, italy, palermo, italy introduction: it is largely demonstrated that stem cells release extracellular vesicles (evs) that are able to modify target cell behaviour. interestingly, there is a bidirectional signalling exchange between stem cell evs and damaged cells. moreover, it is well known that macrophages, could also play a role in wound repair and tissue regeneration. it was also demonstrated that stem cell evs are involved in immune cell regulation. for this reason, today takes hold the idea that evs could replace stem cells in regenerative medicine. the aim of our work was to evaluate if evs released by mouse mesoangioblast stem cells (a6) could have a role in immune cell regulation. specifically, we have investigated the possible a6 ev effect on murine macrophages (raw264.7) in terms of cell proliferation, migration and phagocytic ability, and cytokines/chemokine release. methods: a6 evs were collected from conditioned milieu by ultracentrifugation. raw 264.7 cell proliferation with or without a6 evs was evaluated via cfse assay. scratch test was performed to assay their migration ability. to study raw264.7 cell phagocytosis they were treated with 2 μm beads. finally, cytokine array was used to monitor their secretion after ev treatment. results: we have found that a6 evs inhibited macrophage proliferation as proved by a proliferation index significantly reduced after ev treatment. simultaneously, we have noticed that evs increases raw264.7 migration ability. furthermore, a6 evs are able to increase macrophage phagocytic activity. as it is known that hsp70 is involved in for macrophagic activity increase and a6 evs express hsp70 on their surface, we performed phagocytosis assays assay by blocking the protein or its receptor tlr2, tlr4 and cd91. our data demonstrated that a6 evs increase phagocytosis through hsp70 and its receptors. we have also proved that a6 evs modify the expression pattern of cytokines/chemokines released in the extracellular milieu by raw264.7 cells. in particular, we observed an increase in anti inflammatory cytokines, and a decrease in some inflammatory ones, suggesting that evs could polarize macrophages towards an anti inflammatory m2 phenotype. summary/conclusion: in conclusions, our data show that a6 evs influence macrophage activity and additional studies could provide a new insight into understanding the underlying potential of evs in tissue regeneration. 1 and 2) . in a healthy kidney, the polycystins localize to renal cilia. mutations that abrogate ciliary localization of pkd2 (yet preserve its channel function) also cause cysts. besides cilia, pkd2 is also found in other subcellular locations including extracellular vesicles (evs) of human urine. how dysfunction of pkd2 trafficking and localization leads to the kidney pathology remains unknown. pkd2 is evolutionarily conserved across all members of eumetazoa. in c. elegans, pkd-2 is exclusively expressed in ciliated male-specific neurons, where it is trafficked to cilia and evs. gfp-tagged pkd-2-containing evs play a signalling role in inter-organismal communication between animals. conservation of polycystin-2 cellular localization between worm and human suggests that their network of molecular interactions may also be conserved. we propose that pkd-2 plays distinct roles in cilia versus ciliary evs. methods: to understand the role of evs in c. elegans inter-organismal signalling, we aim to identify the pkd-2-associated ev proteome, transcriptome, and metabolome. we established a pipeline for fluorescent labelling and tracking specific ev cargoes in a living animal using super-resolution microscopy. we used fluorescence of the pkd-2 carrying evs to optimize biochemical procedures for their enrichment. results: our initial analysis revealed two populations of pkd-2-carrying evs that differ in their densities: 1.11-1.12 versus 1.14 g/ml. we are currently characterizing these two distinct populations using transmission electron microscopy and refining our enrichment procedure for protein identification by mass spectrometry, sequencing of their rna cargoes and metabolome analysis. summary/conclusion: what function human pkd2 plays within the cilia and within the urinary evs is not well understood. identification of molecular mediators of c. elegans pkd-2 ev signalling will inform on the interactome of human pkd2 and its function in cilia versus evs. introduction: ectosomes play roles in many physiological and pathophysiological processes, and their precise is dependent on molecular cargo and parent cell type. a single cell can release distinct subpopulations of evs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. in the nematode c. elegans, ectosomes bud from sensory neuron cilia and are released into the environment to modulate animal behaviour. methods: c. elegans is genetically tractable and optically transparent, allowing for live imaging of fluorescently tagged ev cargo. we express all tagged cargo at endogenous levels, adding physiological relevancy. results: we discovered that the calcium homoeostasis modulator ion channel clhm-1 localizes to cilia of ev-releasing neurons and observed gfp-tagged clhm-1 in ciliary evs. using super resolution microscopy, we imaged evs released from animals coexpressing tdtomato-tagged clhm-1 and gfp-tagged pkd-2 (another vesicle cargo) in the same neurons. while the two proteins colocalize in the cilia, clhm-1::tdtomato and pkd-2::gfp rarely colocalize in evs. this indicates that separate subpopulations of evs are being released from the same neurons. to determine how the clhm-1 subpopulation is formed, we are investigating candidate genes. anoh-1, a homolog of the ca2+ scramblase tmem16f, localizes to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet. in anoh-1 mutants, the number of clhm-1::gfp evs released is significantly decreased but the number of pkd-2::gfp evs does not significantly change. in addition, i am using facs to isolate clhm-1 and pkd-2 containing evs and analysing the respective proteomes with lc-ms/ms. summary/conclusion: we are elucidating mechanisms that give rise to distinct subpopulations of ciliary evs in c. elegans and defining cargoes being enriched in these ev subpopulations to gain insight into ev cargo sorting and biogenesis mechanisms in ciliated neurons. ceramide accumulation induces exosome secretion through lysosomal protein laptm4b kohei yuyama, hui sun and yasuyuki igarashi hokkaido university, sapporo, japan introduction: exosomes, a type of extracellular vesicles originated from multivesicular bodies (mvb), are important carriers of cellular molecules and have critical roles in intracellular communication in both health and disease. ceramides (cer) are implicated in biogenesis of exosome, however the molecular machinery that mediates exosome secretion remains obscure. lysosome-associated protein transmembrane-4b (laptm4b) is a lysosome/late endosome-resident transmembrane protein, which has been reported to bind cer. we demonstrate here that laptm4b is involved in the exosome secretion, which are induced by exogenous cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells. methods: neuroblastoma sh-sy5y cells were treated with cer (porcine brain-derived cer or synthetic d18:1/c2:0~c24:0 cer) for 24 h. exosomes were isolated from the culture supernatants by sequential centrifugation and their amounts were measured using ps capture exosome elisa kit. to analyse mvb transport, mvb and recycling endosomes are visualized with gfp-cd63 and rab11 immunostaining, respectively. results: we found that exogenous treatment of cer, especially those with c16 and c18 fatty acids, resulted in a marked increase in exosome secretion. in addition, lysosomal cer accumulation induced by acid ceramidase inhibition also accelerated exosome production. knockdown of laptm4b significantly prevented the ceramide-dependent exosome release. in addition, we showed that these cer loading promoted colocalization of cd63-positive mvb with rab11-positive recycling endosomes, further demonstrated that laptm4b knockdown cancelled the cer-dependent increase of the colocalization. summary/conclusion: these data suggest that lysosomal cer binds to laptm4b and promote the transport of mvb to plasma membrane, resulting in an increase of exosome secretion in neuronal cells. chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles jing xu a , kevin yang a , shane colborne a , elham hosseini-beheshti b , gregg morin a , emma guns b and sharon m. gorski a a bc cancer, vancouver, canada; b the vancouver prostate centre, vancouver, canada introduction: small extracellular vesicles (sev) are signalling entities released by many types of eukaryotic cells. sev are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating cancer cell invasion. macroautophagy (hereafter autophagy) is a catabolic process well-known for the recycling of cytosolic cargos through lysosome-mediated degradation. in this study, we profiled the changes in sev content and function under lysosome inhibition and investigated the involvement of autophagy machinery in sev content. methods: chloroquine (cq) was used to inhibit lysosomal degradation and autophagy turnover in triplenegative breast cancer (tnbc) cell lines. sev were collected via precipitation after pre-clearing and concentration of conditioned media. western blotting, nanosight and transmission electron microscopy were used to profile sev. quantitative mass spectrometry was used to characterize cq-induced changes in the sev proteome. antibody-conjugated magnetic beads were used in immunoprecipitation of sev. results: cq treatment did not substantially alter the physical properties of tnbc-derived sev. however, cq treatment altered the sev proteome and growth effects of sev on normal and endothelial recipient cells. cq treatment induced co-localization of mammalian atg8 proteins with endolysosomal markers in the cytoplasm, which coincided with an enrichment of atg8 s and their adaptor proteins in sev. cq-induced enrichment of atg8 s in sev required lipidation, and occured preferentially in one subset of sev. summary/conclusion: our study reveals changes in the content and function of cancer cell-derived sev in response to perturbation of intracellular trafficking pathways, demonstrates the flexibility and heterogeneity of sev composition, and has implications for cq efficacy in therapeutic settings. introduction: introduction: argonaute 2 (ago2) is the essential component of the rna-induced silencing complex (risc) that binds mirnas and promotes mrna degradation. extracellular vesicle (ev)-carried mirnas have been shown to influence gene expression and functional phenotypes in recipient cells. many investigators have found ago2 in evs and it is postulated that ago2 is a major transporter of mirnas into small evs (sevs), such as exosomes. others have reported extracellular ago2 that is non-vesicular. we set out to evaluate the effect of growth factor signalling and serum contamination on the detection of ago2 in sevs. methods: methods: wildtype kras colorectal cancer cells, dks8, were conditioned with 3 different culture media (serum-free dmem, dmem supplemented with ev-depleted fbs, and opti-mem). evs were purified from conditioned media by cushion-density gradient ultracentrifugation. western blot analysis of dks8 total cell lysates, large evs and density gradient fractions was performed, probing for ago2 and ev marker proteins. the size and concentration of the evs were determined by particle metrix analysis. results: results: in all conditions, we found the highest abundance of sevs in fractions 6 and 7, as assessed by western blot analysis. ago2 was detected in the same fractions as sevs in both the serum-free dmem and opti-mem conditions, although the levels of ago2 was higher in the serum-free dmem fractions compared to that of opti-mem. in contrast, ago2 was present in both vesicular and non-vesicular fractions in the dmem supplemented with ev-depleted fbs condition. no significant differences were observed in the size and number of evs collected in the three conditioning methods. summary/conclusion: summary/conclusion: the presence or absence of ago2 in evs has been controversial. multiple factors may affect the ability to detect vesicular ago2, including serum and growth factors in the conditioned media that may provide sources of extravesicular ago2 and also regulate the trafficking of ago2 into vesicles. introduction: cancer-associated glycosphingolipids have been utilized as tumour markers and targets of cancer therapy. we have investigated roles of gangliosides in cancers, and clarified that cancerassociated gangliosides enhance malignant properties of cells by forming complexes with membrane molecules in lipid rafts. in this study, we analysed contents of gangliosides and membrane molecules on extracellular vesicles (ecvs) secreted from melanoma cell lines. methods: melanoma cell lines with various ganglioside patterns were used for isolation of ecvs. gangliosidemodified melanomas with genetic engineering were also used. genetic modification was done by cdnas of ganglioside synthase genes. ecvs were collected by ultra-centrifugation, or by tim4-beads. contents in ecvs were analysed by immunoblotting or flow cytometry. roles of lipid rafts in the generation and secretion of ecvs were analysed by treating cells with 1 mm methyl β-cyclodextrin. results: using 4 melanoma cell lines, ecvs were isolated by ultra-centrifugation, and their sizes were analysed by nanosight. all samples showed uniform sizes between 100 and 200 nm. protein amounts in ecvs were measured, showing heterogeneous levels at 10~80 μg/100 ml. then, gangliosides expressed on ecvs from these cell lines were analysed using tim4beads and flow cytometry. gd3 and gd2 were detected on ecvs almost proportionally with expression levels of those gangliosides on the cell surface. then, immunoblotting was performed to analyse integrin levels in ecvs from transfectant cells expressing high levels of gd3, showing increased levels of integrins in ecvs from gd3+ cells compared with those from gd3-cell lines. integrin levels in cell lysates from these cells (gd3+ and gd3cells) were almost equivalent. treatment of a gd3-expressing melanoma cell line by 1 mm methyl β-cyclodextrin resulted in marked reduction of secreted ecvs and amounts of tsg101 in them. summary/conclusion: ganglioside expression patterns on melanoma cells were well reflected in the expression of gangliosides on ecvs. these results as well as increased levels of integrins in ecvs from gd3 + cells suggest that gangliosides and lipid rafts are involved in the generation and secretion of ecvs. introduction: hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. however, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. we sought to evaluate how hypoxia alters metabolite and metabolism-associated mirna packaging in exosomes. methods: exosomes were isolated from 4t1 breast cancer cells cultured in normoxia (21% o2) and hypoxia (5% o2) via ultracentrifugation, optiprep gradients, and size exclusion chromatography. exosomes were further characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. metabolite and mirna profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. results: secretion of exosomes was increased under hypoxic conditions. metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. profiling of exosomal mirna showed packaging of metabolism-related mirna into exosomes derived from hypoxic cells. summary/conclusion: hypoxia alters the metabolite and mirna profiles of cancer cells, with selective packaging of these molecules into exosomes. we identified metabolites and mirna that are depleted and enriched in exosomes compared to cells. these studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function. lysosomotropic agents prompts the release of extracellular vesicles carrying autophagy-associated markers: evidence of a general mechanism of secretion driven by lysosomal impairment introduction: drug-induced lysosomal storage disorders (lsds) are due to the transient intracellular accumulation, mostly of phospholipids, into multilamellar inclusion bodies within late endosomal/lysosomal compartment. they represent a major side-effect for many drugs of several pharmacological categories. most lsds inducers are cationic amphiphilic drug (cad), but the molecular mechanisms leading to accumulation of undigested substrates are unknown. extracellular vesicles (evs) have been implicated in cell waste disposal, but it is unclear whether they might be involved in extracellular release of undigested substrates. methods: to investigate this aspect, we developed hek cells stably expressing the fluorescent fusion proteins egfp-cd63 and mcherry-cd63, separated evs by differential ultracentrifugation and quantified by evassociated fluorescence and nta particle count. results: evs released by these models upon treatment with drugs inducing the accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed the release of fluorescent medium/large evs (10k fraction) and small evs (100k fraction), whose size and distribution were similar to the same vesicles released by control cells, but enhanced the recovery of medium/large evs and to a lower extent of small evs, analysis of evs associated markers revealed a dosedependent increase of autophagy-associated markers in medium/large and small evs. similar results were obtained when autophagic flux was impaired by drugs raising lysosomal ph by different mechanisms, such as chloroquine and bafilomycin, but not when autophagic flux was stimulated by drugs such as curcumin or overexpression of the endosomal/lysosomal regulator tfeb. summary/conclusion: overall results show that impairment of autophagic flux, either by indigested substrates or higher lysosomal ph, is associated with an increased release evs enriched in autophagy markers, compatible with autophagomes and/or amphisomes, unravelling a connection with secretory autophagy. tomofumi yamamoto a , yusuke yamawaki b , yutaka hattori c and takahiro ochiya a a tokyo medical university, shinjuku-ku, japan; b national cancer center research institute, chuo-ku, japan; c keio university faculty of pharmacy, minato-ku, japan introduction: multiple myeloma (mm) is a haematological tumour. last decade, the prognosis of mm has improved by the development of therapeutic drugs; however, mm cells acquire drug resistance by longterm exposure of these therapeutic drugs. one of the possible explanations of drug resistance is that cells with drug resistance transmit information to other mm cells and their microenvironmental cells. although the elucidation of the mechanism of drug resistance in mm have been desired, it remains poorly understood. methods: in order to understand the mechanism of drug resistance in mm, lenalidomide resistant cell lines were established by long-term exposure of low concentration of lenalidomide. drug resistance was assessed by mts assay and caspase assay. the amount of ev was measured by exoscreen, which is ultra-sensitive detection method of evs by measuring surface protein of evs, such as, cd9 and cd63 (yoshioka et al., nat commun., 2015) . to identify the genes which involved in drug resistance, rna sequence among the drug-resistant cell lines and their parental cell lines was performed. results: firstly, characterization of these cells was confirmed. we found that all of the lenalidomide resistant cell lines secreted more evs than their parental cell lines. in addition to this, the size of ev derived from resistant cells are smaller than those of parental cells. next, we collected evs from resistant cells and parental cells by using ultracentrifugation, and added them to parental cells in the presence of lethal dose of lenalidomide. compared with ev derived from parental cell lines, the evs derived from lenalidomide resistant cell lines increased a number of living parental cells. these results suggested that the evs derived from lenalidomide resistant cells can affect the lenalidomide sensitive cells. as a result of rna sequence, several genes highly expressed in resistant cell line we found, which associated with lysosome pathway. among them, attenuating the sort1 and lamp2 genes could significantly reduce the ev secretion in mm cells, leading to enhance the lenalidomide sensitivity. summary/conclusion: our results showed that ev secretion via sort1 or lamp2 could induce the drug resistance in mm. study on biological stimulate mechanism of stem cell-derived exosome generation by nanoparticles introduction: mesenchymal stem cells (mscs) are pluripotent stromal cells known to release extracellular vesicles (evs) containing various growth factors and antioxidants that can positively affect surrounding cells. nanoscale msc-derived evs, such as exosomes, have been developed as bio-stable nano-type materials, but had low yield and were difficult to quantify. we hypothesized that the mechanism of nanoparticleenhanced exosome production would stimulate intracellular molecules. the aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified positively charged nanoparticles and exosome generation from mscs. methods: mesenchymal stem cells (mscs) were cultured in mem-alpha with 10% fbs and 1× antibiotics. the positively charged nanoparticles were synthesized by poly-lactide-co-glicolide (plga) and polyethylenimine (pei) with cy5.5 for tracking nanoparticles. all of the exosome image were identified using an electron microscope. additionally, it was confirmed the internalization of the nanoparticles by if. the primary antibodies used were anti-eea1, anti-rab7 and anti-gm130. in order to prove the development of exosomes, rt-pcr using autophagy-related mrna was performed. real-time rt-pcr was performed using the applied biosystems sequence detection system 7900. lastly, mirna from msc-derived exosome analysed automatically in the affymetrix data extraction protocol using the provided affymetrix genechip® command console® software (agcc). all statistical testing and visualization of differentially expressed genes was conducted using r statistical language 3.3.3 results: we determined that rab7, located in the mvb and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation. these results suggested that nanoparticles migrated to lysosomes during treatment; however, intracellular exosome-forming factors were stimulated during endosomal maturation simultaneously. summary/conclusion: therefore, msc-derived exosome research using nanoparticles is useful for increasing exosome yield and the discovery of nanoparticleinduced genetic factors. theoretical description of formation of extracellular vesicles by budding of membrane introduction: understanding mechanisms of extracellular vesicles (evs) formation is of utmost importance for their effective use in science, medicine and technology. in particular, the discovery of universal mechanisms explaining the phenomena taking place in vesiculation appears to be crucial and highly warranted. mammalian erythrocytes and giant phospholipid vesicles have been largely used as model systems to study principles of membrane budding and vesiculation. the mechanisms conveniently studied in these simple systems are then generalized to other types of biological membranes. we present a theoretical description of membrane budding and compare the theoretically obtained shapes with the observed ones. methods: in accordance with the fluid crystal mosaic model, membrane is considered as composed of constituents (inclusions) subjected to the local curvature field created by surrounding constituents. constituents can attain different in-plane orientations in the membrane which correspond to different energies. the thermal motion oposes the complete orientational ordering. the single-constituent energy expresses a mismatch of the curvature of the membrane at the position of the constituent and the intrinsic principal curvatures of the constituent and inplane orientation of their principal axes. the free energy of the whole membrane is obtained by summing up (integration) the contributions of the constituents and using methods of statistical physics, and minimized by using numerical methods. results: to outline the principle of (outward and inward) budding, respective sequences of shapes corresponding to a formation of one (outward and inward) spherical bud were calculated by minimization of the free energy. also the corresponding shapes observed in evs (imaged by electron microscopy) and in erythrocytes and giant phospholipid vesicles (imaged by optical microscopy) are shown. it can be seen that theoretically calculated shapes and experimentally observed ones agree well over up to 3 orders of magnitude (the order of the size of giant phospholipid vesicles is between 1 and 100 micro metres, in erythrocytes it is about 5 micro metres and in evs it is about 100 nanometres). summary/conclusion: budding of the membrane is an universal mechanism in formation of external and internal vesicles. introduction: the release of extracellular vesicles (evs) from cells is important for many cellular mechanisms both in normal physiology and in disease. arrdc4 (arrestin domain containing protein 4) is an adaptor protein known to facilitate the ubiquitination of target substrates by nedd4 family ubiquitin ligases. it also traffics cargo to extracellular vesicles. previous studies show the involvement of arrdc4 in the trafficking of the divalent metal ion transporter dmt1 to evs in a ubiquitin-dependent manner, and we aimed to further understand this mechanism. methods: we performed mass spectrometry to identify ubiquitinated lysine residues in arrdc4. we then generated arrdc4 wt and lysine mutant clones and expressed these in cells to determine the effect on ev biogenesis and protein trafficking. results: mass spectrometry data identified 5 potential ubiquitinated lysine residues. out of these, lysine 270 appeared to be the most important for arrdc4 function. arrdc4k270 r mutation caused a decrease in the number of ev released by the cell compared to arrdc4 wt, and a reduction in trafficking of dmt1 to evs. furthermore, we also observed a decrease in dmt1 activity and an increase in its intracellular degradation in the presence of arrdc4k270 r. k270 also appeared to be ubiquitinated with k29 polyubiquitin chains by the ubiquitin ligase smurf1. summary/conclusion: our data suggests that k29 polyubiquitin chains are the signal for arrdc4mediated ev biogenesis and protein trafficking, and loss of this signal causes cargo to be rerouted to intracellular degradation mechanisms. chair: tanina arab -department of molecular and comparative pathobiology, johns hopkins university school of medicine a 3d-printed model to represent the structure and nature of extracellular vesicles, for public engagement and education events. christian burton a , sara veiga a , jason webber a , kate milward a , muireann ni bhaoighill a , lauren evans a , andreia de almeida b , rachel j. errington a and aled clayton a a cardiff university, cardiff, uk; b cardiff university, research associate, uk introduction: explaining the field of extracellular vesicles to the lay public and young audiences can often be challenging. whilst diagrams and images of evs may be helpful, conveying clearly the shape and composition of an ev by these means is not always a success. whilst many members of the audience may be familiar with concepts of cells and related structures, others will find such discussions very abstract and challenging. in order to aid interactions with lay audiences we embarked on the design of a physical hand-held plastic model, representing a typical ev. incorporating flexibility in the design allowing the community to adapt it to showcase their own research. the second goal was to ensure manufacturability using widely available 3dprinting technologies. methods: the basic model design was conceived by dr c. burton, and iteratively developed using solidworks, 2019, then exported for use in any cad environment (stl format). a model showing a halved ev hemisphere, with a visible lipid-bilayer was developed. attachable rings allow trans-membrane-molecules to be represented, current designs include mhc class-i, hspgs, integrins, tetraspanins and supported by handouts accompanying the models. intraluminal cargo is included via removeable "pegs", and examples representing rna or simple globular proteins, and a template has been created. results: the design is free and open source, and available to the community at: https://www.thingiverse. com/thing:3986565. instructions for 3d printing are available from the uk extracellular vesicle society website; https://www.ukev.org.uk/public-engagementmaterials/. models have been produced using entrylevel 3d printers and trialled at engagement events with good early responses. summary/conclusion: the authors hope the community will use and develop this 3d-model design and that the approach provides an additional and helpful tool for educating audiences about the complexities and roles of evs in biology and disease. centrifugal filtration-sec is promising for extracellular vesicle isolation from d492 and d492her2+ breast epithelial cell lines introduction: despite recent developments in breast cancer therapy, there is still need for a more targeted approach. extracellular vesicles (evs), endogenous nanovesicles released from human cells, are an attractive choice as nanodrug carriers due to their size, stability and their unique targeting specificity. the aim of this study was to determine if centrifugal filtration (cf) combined with size exclusion chromatography (cf-sec) would be useful for ev isolation from two epithelial breast cell lines d492 and d492her2+, representing the tissue of interest, and the amount of cell culture needed to get measurable ev concentrations. methods: cell culture media (without serum) from the immortalized breast epithelial cell lines d492 and d492her2+ was concentrated with centrifugal filtration (cf) followed by isolation with size-exclusion chromatography (sec) using hiprep 16/60 sephacryl s-400 column run with äkta start (280 nm), 240 min runs. each fraction (4-5 ml) was collected with fraction collector. dulbecco's particle free pbs was used as mobile phase. the resulting particles were analysed with nanoparticle tracking analysis (nta, nanosight ns300, camera gain 10, static mode, capture time 90 sec), western blotting (wb), microbca and transmission electron microscopy (tem, samples fixed with 2% formaldehyse and stained with 2% uranyl acetate, run at 80kv). results: although sec did not show any prevalent peaks from early eluting regions previously shown to contain extracellular vesicles, these fractions (f1-f3, 40-130 min) were collected from d492her2+ cell culture medium. interestingly, both nta and tem suggest that f2 and f3 contained evs as the isolated particles measured 65 and 58 nm, respectively and tem revealed spherical particles 20-50 nm in diameter. wb was unable to detect the ev associated protein alix (but was present in the whole cell lysate). soluble proteins and protein aggregates eluted late in the sec chromatogram (180 min), with protein analysis (microbca), tem and wb confirming their presence. summary/conclusion: cf-sec is a promising method for ev isolation for pharmaceutical applications, but further work is needed to optimize the isolation process using äkta start for these cell lines. customer stories from the ev core of university of helsinki introduction: the ev core, world's first ev-dedicated technology platform established in 2016, is a joint venture of two extracellular vesicle (ev) research laboratories at university of helsinki. as an academic research/service facility, the ev core provides infrastructure, state-of-the-art and emerging ev-technologies for research groups, hospitals, companies and authorities in the ev-field. the ev core provides ev isolation, purification and characterization services and offers contacts to downstream analyses in other core facilities based on optimized ev protocols. here, we present and discuss the customer experiences and prospects with the aim to further develop ev core services. methods: our most wanted services are nanoparticle tracking analysis, electron microscopy, ev isolation and rna isolation and consultation. currently, the key down-stream analysis methods are (mi)rna sequencing, metabolomics, flow cytometry and functional assays. results: we present the stories from our customers starting with their research questions and need for the ev expertise/consultation and equipment. next, we show how the projects advanced and what types of ev core -derived or other downstream services helped them to achieve their aims. in the end, we will acknowledge the customers experience and current status of their research. summary/conclusion: narratives of customer stories are an effective starting point for fruitful discussions about the current status and next developments in the young ev service field. recent isev workshops: open, reproducible and standardized ev research (ghent, 2019) and evs in immunology (buenos aires, 2020) introduction: since its founding in 2012, isev has sought to further extracellular vesicle research in various ways including scientific meetings. these events encompass annual meetings as well as smaller, topically focused workshops, with the first isev workshop (on rna and evs) organized in new york city in october, 2012. in december, 2019, the workshop "open, reproducible, and standardized ev research" was held in ghent, belgium. in march, 2020, the workshop, "evs in immunology" was held in buenos aires, argentina, with a preceding education day. methods: the international organizing committees of the 2019 and 2020 isev workshops prepared scientific programs around key themes of ev rigour and standardization (ghent, belgium, 2019 workshop) and evs in immunology (buenos aires, argentina, 2020 workshop). abstract and application submissions were invited. applications were reviewed and ranked by panels of ev experts for each event, and participants were invited. results: the 2019 and 2020 workshops assembled a total of more than 100 individuals for talks and discussions around the themes of rigour and standardization and evs in immunology. the buenos aires workshop was preceded by an education day, coordinated by the isev executive committees for education and science and meetings. during these two workshops, poster presentations were permitted for the first time, affording additional presentation and interaction opportunities. the rigour and standardization workshop also featured real-time discussant polling to facilitate discussion. summary/conclusion: isev workshops such as those addressing rigour and standardization (ghent, 2019) and evs in immunology (buenos aires, 2020) continue to provide opportunities for focused discussion of small groups of experts on key topics in the field. often followed by published products, isev workshops help to lead and coordinate progress in ev science. for future isev workshops, educational activities may again expand the reach of each event, while poster sessions and app-driven real-time responses should be considered for enhanced interactions and participant canvassing. ev journal club: exchanging pizza for a worldwide audience during covid-19 kenneth w. witwer johns hopkins university school of medicine, baltimore, usa introduction: a monthly journal club focused on extracellular vesicle science was established at johns hopkins university in 2016, featuring lunch and presentations by academic and industry participants. when covid-19 prevented in-person meetings beginning in march, 2020, the journal club was converted to a virtual, weekly format on the popular online meeting app zoom. the journal club has persisted despite initial problems with online vandalism. most sessions are also made public on a youtube channel, https:// www.youtube.com/c/extracellularvesicleclub. methods: weekly ev club sessions are arranged by the host. most focus on a specific manuscript related to evs, but some weeks feature presentations of published or soon-to-be-published research by the presenting authors. sessions are advertised one week to several days in advance on social media platforms such as linkedin, twitter, and facebook, asking interested parties to sign up to join a mailing list via surveymonkey. the log-in information is then sent to the mailing list. upon clicking the link, participants are placed in a virtual waiting room for vetting by the host and volunteers. after admission, all parties but the host and presenter are muted to avoid distractions. questions and comments may be placed in a chat box. contributions are monitored and compiled by the host and volunteers to build a question-and-answer session at the end of the presentation. recorded sessions-with or without editing as needed-are placed on the youtube channel for additional access. results: despite initial problems with online vandalism known as "zoombombing," the journal club has continued weekly during the covid-19 shutdown in the host country (us). an audience of between 80 and 150 individuals is typical. participants typically ask more questions than can be answered in a one-hour time frame. the online format also allows for debate-style events and polling of the audience. summary/conclusion: this ev journal club is an example of how online tools can be used to facilitate international scientific interactions. further development of such formats could provide alternative approaches for isev activities in the science, education, and communication areas. the study aim is to assess whether the exposure to pm10 and pm2,5, chosen as paradigmatic environmental stressors, could modify the composition of nasal microbiota (nm) and extracellular vesicle (ev9 signalling network, showing a role in allergic ar exacerbation). methods: nm analysis were performed on v3-v4 16 s rrna gene regions amplified from upper-airway tracts of 25 ar cases and 25 healthy individual controls to perform nm analyses. ev size, concentration and cellular origin for each subject were assessed by nanoparticle tracking analysis (nta) and flow-cytometry (fc). information on daily pm10 and pm2,5 concentrations at the municipality of residence in the 7 days preceding nasal sampling (i.e. day −1 to day −7) was assigned to each subject by arcgis software. multivariable and logistic analyses were applied on nm, nta and fc outcomes. results: when taxonomy composition was considered, in controls actinobacteria (50.8%) was the most represented, followed by firmicutes (34.7%) and proteobacteria (12.8%) while in cases proteobacteria were 38.8%, actinobacteria were 37.1% and firmicutes were 23.4%. cases showed a higher concentration of all the investigated ev types, derived from platelets (cd61 +), activated endothelium (cd62e+), monocytes (cd14+), eosinophils (cd294+), neutrophils (cd177+), mastocytes (cd203 c+), epithelial cells (epcam+), gram+ bacteria (lipoteichoic acid+), gram-bacteria (lps+). the effect was greatest in the case of mastocytes evs which were increased 2.5fold in cases versus controls (p < 0.001). evs were modified by pm exposure at several time lags. in particular, a negative association between pm10 and eosinophil evs was observed (beta = −0,016; pvalue = 0,017). as we clustered subjects according to their nm, we observed this variable was a strong effect modifier of the association between pm exposure and ev release. summary/conclusion: our findings start to provide an insight on the effect of air pollution on evs, taking into account the effect of nm, in patients with ar. further research is necessary to disentangle the mechanism exerted by inhaled pollutants in modulating evs and nm, and therefore ar exacerbation. funding: gsk investigator sponsered study aryl hydrocarbon receptor activation induces the expression of specific microrrnas in th17 cells that are release into extracellular vesicules and associated with arthritis introduction: in rheumatoid arthritis (ra), an autoimmune disorder characterized by a chronic sinovial inflammation, smoking is a major risk factor contributing to disease progression, and poor response to therapy. th17 cell is actively involved in worsening smooking-associates inflammation mediated by aryl hydrocarbon receptor (ahr), a cytoplasmic transcription factor involved in xenobiotic metabolism. both, ahr and th17 cells, has important implications during ra development. considering that cigarette smoke is a potent epigenetic modifier, we hypothesized that ahr activation, by cigarette components, would transcribe specific micrornas in th17 cells as a molecular mechanism to exacerbate inflammation in arthritis. methods: microrna expression was evaluated by largescale approach or real-time pcr. c57/bl6 and ahr null mice were submitted to arthritis experimental models and exposed or not to cigarrete smoke (ethical committee approved 048/2012). extracellular vesicles (evs) were isolated by ultracentrifugation, and characterized by western blot and nanosight. rankl-induced osteoclasts (ocs) differentiation in vitro was stained for trap. inhibition of mirnas were performed using anti-mirs transfection. results: we identified a specific group of mirnas induced in th17 cells after ahr activation. during arthritis progression, the micrornas are expressed and increases after exposure to cigarette smoke. in the absence of ahr their levels were drastically reduced. interestingly, we found that these micrornas are released by th17 cells into evs, and are able to promote osteoclastogenesis. ocs differentiation in vitro increases in the presence of th17-derived evs, and this process is reduced in the absence of micrornas. summary/conclusion: microrna-mediated gene regulation plays crucial roles in the immune system functions, and their abnormal expression is highly correlated with the pathogenesis of ra. evs are known to function in cell-to-cell communication and are able to transmit their contents and cause changes in the target cell. our findings demonstrate a new molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis; through the activation of ahr receptor in th17 cells, inducing the transcription of specific micrornas that are released into evs, and act as pro-inflammatory mediators. introduction: chagas disease (cd) is caused by the flagellated protozoan t. cruzi. trypomastigote forms are capable of releasing extracellular vesicles (evs) that contain the major surface molecules of the parasite. the parasite has a complex life cycle that leads to it a rapid adaptation in the environmental changes in the hosts. however, the effects of stress on on evs release are not completely understood. objetive: we evaluated the release of evs by trypomastigotes incubated under different stress conditions and the immunomodulatory role of these evs in pre-activated bone marrow-derived macrophages (bmdm). methods: nanoparticle tracking analysis (nta) and scanning electron microscopy (sem) showed an increase in evs releasing by trypomastigotes at 4°c under acidic conditions, evs released was affected and triggered amastigogenesis process. results: treatment with sodium azide (nan3) also caused changes in the release of evs regarding size and concentration. nitrosative stress caused by sodium nitrite (in culture medium mildly acidic, ph 5.5; in this condition nano2 releases nitric oxide) stimulated an increase in production of evs by t. cruzi. when the parasites were treated with 100 nm s-nitrosoglutathione (snog), we observed a reduction in size and concentration of vesiculate material by trypomastigotes. at a higher snog concentration (100 µm), the concentration of the vesiculate material increased. t. cruzi-derived evs exposed to stress conditions increased the expression of inos, arg 1, il-12 and il-23 genes in ifn-γ and lps pre-activated bmms. summary/conclusion: results suggest that the viability and/or integrity of the parasite are necessary for the evs releasing. in those in vitro conditions they triggered a proinflammatory response in host cells. this may be a strategy developed by the parasite to favour its establishment in the host. funding: fapesp, cnpq, capes and fapemig ppm-x 00102/6. immuno-toxicological evaluation of human mesenchymal stem cellintroduction: mesenchymal stem cells (mscs) have been widely used to the field of autoimmune diseases or tissue regeneration therapy. recently, many research groups have reported that mscs showed their ability via secreted paracrine mediators including extracellular vesicles (evs) rather than cell-to-cell contact. mscs mainly exist on bone marrow, peripheral blood, umbilical cord and adipose and can mostly secrete evs. it has emerged that evs alone are responsible for the therapeutic effect of mscs in plenty of animal diseases models. hence, msc-derived evs may be used as an alternative msc-based therapy in regenerative medicine. methods: as part of safety programme for human therapeutics, we performed immunotoxicological assessment of evs obtained from human mscs (hevs) in mice and human peripheral blood mononuclear cells (hpbmcs). firstly, mice were treated intravenously with a negative control, a positive control (lps; 0.4 mg/kg), or low-dose (1x10e8 paticles/head) and high-dose (1x10e9 paticles/ head) of hevs every other day for 10 days and then analysed lymphocyte subsets from collected spleen by facs. next, we treated the evs on hpbmcs for 3 days with low conc. (1x10e8 particles/ml), high conc. (1x10e9 particles/ml), pma/ionomycin as a cell activator or cpt (10 μm) as an apoptotic inducer. annexin v/pi and csfe were analysed by facs. results: as a result, splenic nk cells and b cells were slightly increased about 2~7% in hevs-treated mice, without biological significance, compared with a positive control (lps) as an immunogenicity inducer. and there were no effects on serum levels of inflammatory cytokines in mice. in addition, hevs had no cytotoxic effect on hpbmcs at both low and high conc. under the culture medium with evs-depleted fbs, the hevs appeared minimal anti-apoptotic effect on hpbmcs. for the cfse assay, the hevs showed slight proliferation on hpbmcs and pbmc activation induced by pma/ionomycin. summary/conclusion: in conclusion, the hevs have little immuno-toxicological effects in mice and hpbmcs. further detailed studies to elucidate immunological response of hevs for development of human therapeutics are needed. funding: this research was supported by a grant (18172mfds173) from ministry of food and drug safety. investigation of immune response to mesenchymal stem cell-derived extracellular vesicles in the cancer setting introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) are thought to be a fingerprint of the secreting cell and therefore may retain the cancer targeting and immune privilege of mscs. thus msc-evs hold immense potential as tumour-targeted therapeutics for breast cancer. the aim of this study was to determine whether msc-ev administration in tumour bearing immunocompetent animals would initiate an immune response. methods: evs were isolated from conditioned media of both human and murine bone marrow-derived mscs through sequential differential centrifugation, microfiltration and ultracentrifugation. evs were characterized by nanoparticle tracking analysis (nta), western blot and transmission electron microscopy (tem). 1 x 10(8) human or murine msc-evs were administered intravenously into 4t1 breast tumour bearing balb/c mice (n = 6) and healthy controls (n = 6). tumour tissue, draining lymph node and spleen were then harvested, dissociated and flow cytometry performed targeting markers associated with a range of immune cells including t-cells, macrophages and natural killer (nk) cells. results: evs were successfully isolated from murine and human mscs with the appropriate size of small evs (sevs: 30-150 nm) and morphology including a lipid bilayer observed by tem. evs expressed tetraspanins cd63, cd81, cd82; cytosolic protein tsg101 and were negative for calnexin. ev concentrations ranged from 4.08 x 10(9) -6.6 x 10(9)/ml. in order to study a range of immune cell populations two antibody panels were created using complimentary fluorescent dyes. the proportion of t-cells (cd4+, cd8+, cd25+), neutrophils (gr-1+, ly-6 c+), dendritic cells (cd11 c+), macrophages (cd11b+, mhci+, mhcii+), nk cells (cd27+) and b cells (cd19+) remained stable in the tumour, draining lymph node and spleen of all tumour-bearing animals that received either human or murine msc-evs, with no significant change observed in any category. summary/conclusion: the data presented supports the hypothesis that msc-evs retain the immune privilege of the secretory cell, with human cell-derived evs illiciting no immune response in mice. this is encouraging and reinforces the potential for use of msc-evs in the therapeutic setting. introduction: mycobacterium avium (m. avium) is a slow growth rate non-tuberculous mycobacterium (ntm). m. avium infection is a severe global health problem. but the mechanisms of pathogenicity of m. avium are poorly understood. outer membrane vesicles (omvs) that traverse the cell wall and contain a varied bioactive components inculding dna, rna, protein and toxins. previous studies have suggested that these omvs are produced in vitro and during animal infection, but the role of omvs secretion during the interaction of m. avium with host cells remains unknown. methods: in this study, m. avium were grown in middlebrook 7h9 medium (m7h9) supplemented with 10% (v/v) oadc enrichment and 0.5% (v/v) glycero. m. avium omvs were isolated by ultracentrifugation method. characterization of omvs by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). the raw 264.7 murine macrophages were incubated with the m. avium omvs to analyse inflammatory response and production of nitric oxide (no) and reactive oxygen species (ros) of macrophage. results: in this study, we demonstrate by fluorescence microscopy that murine macrophages can phagocytosis omvs produced by m. avium. incubation of m. avium omvs with murine macrophages resulted in increased levels of extracellular tumour necrosis factor alpha (tnf-α), interleukin-1β (il-1β), terleukin-6 (il-6) and interleukin-12 (il-12). meanwhile omvs stimulated macrophages produce no and ros. introduction: hospital associated venous thromboembolism (ha-vte) in paediatric patients is the second most common contributor to harm in hospitalized children. platelet-endothelial interactions are integral to the formation of vte, especially in inflammatory conditions such as sepsis. small extracellular vesicles (sevs) have the ability to reprogramme target cell phenotypes via their microrna contents and are known to contribute to vte formation. we hypothesize that sepsis alters platelet-derived sev micrornas capable of net upregulation of vascular endothelial procoagulant and downregulation of anticoagulant pathways. methods: using a precipitation solution and size exclusion chromatography, we isolated sevs from platelet poor plasma of children admitted to the paediatric intensive care unit for sepsis and from healthy controls. we positively selected platelet-derived sevs using immunomagnetic isolation for cd42b platelet antigen and confirmed selection using flow cytometry. microrna was profiled using affymetrix genechip mirna 4.0 array. results: microrna from 9 sepsis patients (median age 7.0 years; iqr:1.2-13 and 77% female) with a median psofa score of 6 (iqr:2.5-10) and from 5 healthy controls (median age 9 years; iqr:6.5-13.5 and 20% female) was isolated and compared. in septic vs. healthy patients 30 mirnas were differentially expressed (false discovery rate (fdr)<0.05; fold change ≥|1.5|) affecting 921 mrna pathways. in septic children, pathways affecting chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores ≥2. summary/conclusion: we developed a method to successfully isolate platelet-derived sevs. sepsis alters the platelet-derived sev microrna profile in paediatric patients with sepsis. these micrornas are predicted to target chemotaxis and cell movement pathways, important contributors in the formation of ha-vte. further analysis into specifically targeted pathways should be conducted as a potential target for the prevention of ha-vte in sepsis. introduction: sjögren´s syndrome (ss) is a systemic autoimmune disease that mainly affects salivary and lacrimal glands. mechanisms of ss pathogenesis are poorly understood. it is thought that inflammation leads to destruction of exocrine glands, however the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology are not characterized. our work identifies t cell-exosomederived mir-142-3p as a pathogenic driver of immunopathology in ss. micrornas (mirnas) are endogenous small noncoding rna molecules that regulate the expression of target genes through translational repression of mrnas. through transcriptomic profiling studies our group had previously documented a significant upregulation of mir-142-3p in patient ss tissues and in serum exosomes. methods: structured search for target genes of mir-142-3p involved in salivary gland (sg) physiology was performed with mirdip 4. serca2b, ryr2 and ac9 were selected for further validation and functional analysis. binding of the mirna was confirmed by luciferase reporter assays in hsg cell lines and human-derived primary epithelial cells. the mrna and protein levels of serca2b, ryr2 and ac9 were determined by qpcr and western blot, respectively. to investigate the cell-specific distribution of mir-142-3p in relation to the expression levels of serca2b, ryr2, and ac9, a double fluorescent in situ hybridization was performed. ca2+ signalling and camp levels were measured using fluorescent sensor. to isolate exosomes, the t cell medium and serum of ss-patients and healthy volunteers (hv) were collected. results: we show that mir-142-3p is over-expression in the sgs of ss-patients. next, we demonstrated that mir-142-3p is contained in exosomes in serum of sspatients significantly more than serum of hv. we also show that activated t cells secrete exosomes containing mir-142-3p which transfer into glandular cells and affecting intracellular ca2+ signalling, camp production and protein production by mir-142-3p targets (serca2b, ryr2 and ac9). summary/conclusion: this study provides evidence for a functional role of the mir-142-3p in ss pathogenesis and promotes the concept that t cell-activation directly may impair epithelial cell function through secretion of mi-rna containing exosomes. treg-derived il35-coated extracellular vesicles promote infectious tolerance (p35) subunits, yet the forms that il35 assumes and its role in peripheral tolerance, remain elusive. methods: we induce cba-specific, il35-producing t regulatory (treg) cells in tregebi3 wt c57bl/6 reporter mice, and identify il35 producers by expression of ebi3tdtom gene reporter, plus ebi3 and p35 proteins. results: curiously, both subunits of il35 were displayed on the surface of tolerogen-specific foxp3 + and foxp3neg (itr35) t cells. furthermore, il35 producers, although rare, secrete ebi3 and p35 on extracellular vesicles (ev) targeting a 25-to 100-fold higher number of t and b lymphocytes, causing them to acquire surface il35. this surface il35 is absent when ev/exosome production was inhibited, or if ebi3 is genetically deleted in treg cells. summary/conclusion: the unique ability of ev to coat bystander lymphocytes with il35, promoting exhaustion in, and secondary suppression by, non-treg cells, identifies a novel mechanism of infectious tolerance. funding: nih grants r01-ai119140-03 (to w.j.b.), r01 ca203689 and p30 ca047904 (to d.a.a.v.) and the university of wisconsin carbone cancer center support grant p30 ca014520. unique formulated dual targeting antigen specific and delivered mirna-150 gene regulating exosomes acting at the immune synapse to induce apc-derived secondary suppressive exosomes introduction: an exosome-apc circuit we uncovered may be applicable beyond skin immunity we study in mice. methods: high antigen dose tolerized cd8 + t cells make suppressive antigen-specific exosomes due to chosen surface antibody light chains that enable targeting antigen presenting cells (apc) antigen-specifically for delivery of also chosen inhibitory mirna-150 to mediate specific functional gene alterations. results: both antigen and gene specificity aspects are lent to naïve but activated exosomes by simple in vitro incubations alone. for mechanism, these primary exosomes bind antigen peptides in mhc on apc that in turn make secondary suppressive exosomes that act peptide/mhc-specifically on the effector t cells at the immune synapse. they transfer another mirna for strong prolonged inhibition of active delayed-type hypersensitivity (dth) for days even, when the primary mirna-150-pos exosomes are administered orally at the height of the in vivo response, in a physiological dose. summary/conclusion: it is shown possible to induce therapeutic exosomes with ag targeting of choice due to placed ab on the surface and that also target specific gene functions of acceptor cells due to carriage of a selected mirna. this dual ag and gene-specific therapy has applications in treatment of cancer, autoimmunity and allergies. introduction: previously, our group characterized distinct populations of extracellular vesicle (ev) released from neutrophilic granulocytes: ev formed spontaneously (sev) and upon activation with opsonized particles (aev). the aev differs in protein cargo and its ability to inhibit bacterial growth. we described that mac-1 integrin (cr3 receptor) plays key role in the aev production and extracellular calcium supply is crucial in this signalization. in the present work, our aim was to investigate whether mac-1 activation or casignalling on their own are sufficient for the initiation of the aev biogeneis. methods: we isolated neutrophil derived evs from peripheral human blood and murine bone marrow by two-step centrifugation and filtration. we tested the effect of ca-ionophore and examined the ev production on c3bi coated surface and in soluble form. we quantified the vesicles by flow cytometry and determined their protein content by bradford assay. we examined their antibacterial effect in parallel with optical density-based measurement and our flow cytometry based method. results: on c3bi coated surface, we observed an increased ev production, and these evs possessed antibacterial capacity. however, in soluble condition, c3bi did not induce further ev production, and these evs did not show any antibacterial property. we found that ca-ionophore initiated ev formation, but these ev did not show antibacterial effect. we observed ev production increase after ca-ionofore treatment both in the presence and in the absence of extracellular ca. the ca-ionophore slightly increased the opsonized particle induced ev production, but did not potentiate their antibacterial capacity. summary/conclusion: mac-1 activation is not just crucial, but sufficient in initiation of the aev biogenesis. clustering of this receptor is required. while the ca-signal is crucial, it is not sufficient in the generation of aevs. extracellular vesicles and their microrna cargo in retinal health and degeneration: mediators of homoeostasis, and immune modulation yvette s. m. wooff, adrian cioanca, riemke aggio-bruce, joshua chu-tan, ulrike schumann and riccardo natoli the australian national university, canberra, australia introduction: photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (amd). however, the molecular mechanisms regulating these biological processes are largely unknown. extracellular vesicles (ev) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. evs, including exosomes, encapsulate and transfer microrna (mirna) to recipient cells and in this way can modulate the environment of recipient cells. dysregulation of evs however is correlated to a loss of cellular homoeostasis and increased inflammation. in this work we investigated the role of isolated retinal small-medium sized ev (s-mev) in the regulation of homoeostasis and immune modulation in both the healthy and degenerating retina. methods: isolated s-mev from healthy and degenerative (photo-oxidative damaged) mouse retinas were characterized using dynamic light scattering, transmission electron microscopy and western blot, and quantified using nanotracking analysis. small rna-seq was used to characterize the mirna cargo of retinal s-mev isolated from healthy and degenerating retinas. finally, the effect of exosome inhibition on s-mev-mediated immune modulation was investigated using systemic daily administration of exosome inhibitor gw4869 and analysed by in situ hybridization of s-mev-abundant mirna. electroretinography and immunohistochemistry were performed to assess functional and morphological changes to the retina as a result of exosome depletion. results: our results demonstrated an inverse correlation between s-mev concentration and photoreceptor survival, with decreased s-mev numbers following retinal degeneration. small rna-seq revealed that s-mevs contained uniquely enriched mirnas, however no differential composition in s-mev mirna cargo following photo-oxidative damage was observed. exosome inhibition using gw4869 exacerbated photoreceptor degeneration, with reduced retinal function and increased levels of inflammation and cell death seen following photo-oxidative damage. further, reduced translocation of the photoreceptor-derived s-mev was demonstrated following exosome-inhibition in photo-oxidative damaged mice. summary/conclusion: taken together, we propose that retinal s-mev and their mirna cargo play an essential role in maintaining retinal homoeostasis through immune-modulation, and have the potential to be targeted using gene therapy for retinal degenerative diseases. impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles introduction: agricultural dust is considered a high-risk occupational hazard by the cdc, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. lung-resident mesenchymal stromal/stem cells (lr-msc) have an important role in maintaining homoeostasis in the lung, and mediating pro-and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. one way in which these lr-msc promote lung homoeostasis is through the release of extracellular vesicles (ev), with a variety of cargo that elicit changes among target cells. we hypothesize that exposure to agricultural dust modifies the quantity and cargo of ev released by lr-msc to promote lung tissue homoeostasis. methods: primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (de) for 8 or 48hrs and the media from these exposures were collected and enriched for ev by opti-prep density gradient ultracentrifugation. the quantity of these ev were assessed by nanoparticle tracking analysis. additionally, cytokine and chemokine release by lr-msc were analysed by enzyme-linked immuno assays. results: as assessed at 48 hr following treatment, deexposed lr-msc released pro-inflammatory cytokines, il-6 and il-8, with il-8 release reaching statistical significance at 0.1%, 0.5%, and 1% de concentrations (p = 0.0367, <0.0001, and <0.0001 respectively) and il-6 trending a similar dose response but only statistically significant at 1% de (p = <0.0001). de exposure of lr-msc also induced changes in the lr-msc-derived ev populations when compared to vehicle control, where lr-msc released significantly more ev in the 5 and 10% iodixanol fractions (p = <0.0001 and 0.0219, respectively) at 8 hr following de treatment. alternatively, there were significantly less ev in the 20 and 40% density fractions in the media of deexposed lr-msc versus vehicle control. summary/conclusion: following exposure to agricultural dusts, lr-msc-derived ev populations more likely consist of exosomes and ectosomes, which play an important role in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation. introduction: during analyses of single extracellular vesicles (evs) by flow cytometry (fcm), particles below the detection limit may exceed the trigger threshold, which is called swarm detection and generates false-positive counts. serial dilutions are recommended to find the minimal dilution for which swarm detection is absent. however, because particle concentrations in plasma vary, the optimal dilution differs >100-fold between donors, but it is unfeasible to do serial dilutions for each clinical sample. therefore, our aims are to (1) develop a faster method to avoid swarm detection, and (2) increase the number of detected evs per second. methods: we measured serial dilutions of cd61stained evs in platelet free plasma (pfp), with and without spiking of fitc beads, by fcm (apogee a60-micro). we triggered either on side scatter or fluorescence. results: for scatter triggering with our fcm, swarm detection consistently occurred for plasma samples exceeding a (total particle) count rate of 4,100-4,200 events/s. the cd61+ evs concentration scaled linearly over 1.5 orders of magnitude of the dilution and most donors required >1000-fold dilution to avoid swarm detection, thereby reducing cd61+ ev counts. for fluorescence triggering, the cd61+ evs concentration scaled linearly over >3 orders of magnitude of the dilution. for all donors, swarm detection was absent after 32-fold dilution (relative to pure plasma). the count rates of cd61+ evs were 30-100-fold higher compared to scatter triggering. the spiked fitc beads confirmed that the median signals remained constant. summary/conclusion: we have developed two clinically applicable ways to avoid swarm detection. for scatter triggering, the count rate provides direct feedback on the presence of swarm detection in plasma samples. for fluorescence triggering, swarm detection was absent for all plasma samples diluted ≥32-fold and compared to scatter triggering, count rates of cd61 + evs were 30-100 fold higher, thereby improving statistical significance. funding: edwin van der pol is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. benchmarking flow cytometric analysis of nanoparticles: a cross-platform study for single extracellular vesicle detection introduction: despite flow cytometry being widely used to analyse cells in suspension, most commercial instruments lack sensitivity when measuring nanoparticles (nps) and extracellular vesicles (evs). furthermore, the use of appropriate reference materials (rms) for calibration and quality control are essential to compare results acquired with different instruments. to work towards successful clinical applications for ev biomarker profiling, benchmarking studies including state-of-the-art flow cytometers are required. we here investigated the ability of three different flow cytometers to detect nps and evs. methods: the instrument sensitivity of light scattering detection was evaluated by using synthetic nps of different sizes and refractive indices. fluorescent calibration was investigated by using molecules of equivalent soluble fluorophores (mesf) beads. biological recombinant evs (revs) were used to validate the detection and quantification of fluorescent evs in a side-by-side cross-platform study using an n30 nanoflow analyser (nanofcm), an optimized bd influx and a cytoflex lx. results: we found that when light scatter based detection was used, the nanofcm detected the smallest non-fluorescent nps, the bd influx was able to provide reliable fsc information from the smallest detected nps and the cytoflex performance was greatly improved by the use of violet-ssc. biological revs showed that the nanofcm could clearly resolve fluorescent evs while the bd influx and cytoflex were unable to fully resolve revs from background, although fluorescence threshold improved detection. in addition, our findings revealed that different concentrations are required to ensure single ev detection in these platforms. summary/conclusion: we identified several strengths and limitations for each platform with respect to single ev analysis. furthermore, our results showed that proper calibration and rms are of utmost importance to ensure reliable interpretation of ev flow cytometric data. caution when using membrane dyes for sequential extracellular vesicle analysis diana pham, michael wong, desmond pink and john lewis nanostics, edmonton, canada introduction: confirmation that particles detected by microflow cytometry are actually extracellular vesicles (evs), or at least membranous in composition, can be achieved through a variety of methods. positively staining particles with a membrane dye strongly suggest that the particle contains a membrane; loss of stain (or detection) after detergent solubilization of the membrane-dyed particles provides even stronger evidence that the particles were evs. it is important to recognize that the labelling protocol provided by the membrane dye manufacturer may not be ideal for all types of evcontaining biological samples, such as blood, urine, semen etc.). removal of excess dye from stained evs is very difficult and can be impractical depending on the nature of the experiment. however, this means that the potential for excess dye to contaminate subsequent sampling is high. therefore, it is important to determine optimal working concentrations and labelling conditions when using membrane dyes for ev detection to understand properties that may impact your analyses. methods: to assess the utility of membrane dyes, titration curves were generated to determine the optimal working concentrations of membrane dyes for ev detection in conditioned media and human serum samples. once the optimal concentration was determined the potential of dye carry-over from sample to sample during microflow cytometry detection was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline (pbs) blanks and matched, unlabelled, sample replicates. results: we found that optimal concentration of any membrane dye is dependent on sample type. even with the inclusion of system washes to prevent sample carryover, there was carryover of low amounts of dye+ particles into sequentially analysed pbs blanks. if unstained samples were analysed following a stained sample, excess dye (or at least dye+ events) appeared in the data. a sample concentration effect was also seen; samples of lower concentrations were more susceptible to dye carryover. summary/conclusion: when using membrane dyes to stain evs in biological samples, especially if an autosampler is employed to run a series of tests, it is critical to determine the optimal concentration of dye for each type of sample, as excess dye can carry over to the next sample in the queue. in addition, determining the necessary steps to clean any excess dye following each sample run will improve the accuracy of ev detection and analyses. funding: nanostics alberta innovates alberta cancer foundation correlation between size and protein expression of single exosomes by combined atomic force and fluorescence microscopy introduction: there are no universal markers of extracellular vesicles, but often they are identified by the presence of tetraspanins in their membrane. based on this, products have been developed to precipitate or quantify evs by acting upon cd9, cd81, and cd63. however, evs also carry proteins from their parent cells, and capturing evs based their presence allows for a more complete understanding of vesicle heterogeneity from a single cell type, and for evs derived from specific tissues to be enriched from other biofluids in support of biomarker assessment. for example, evs derived from the brain could be captured from the general population of serum evs for better assessment of cargo associated with proteinopathy. the goal of this study was to identify specific antibodies to capture and label evs bearing the neural markers cd171, snap25, α-synuclein, tau, and ncam. methods: the targets were overexpressed in hek293 t cells through transient transfection of plasmids (origene). media was conditioned for 24-48 hours, and then centrifuged to remove cell debris. cell lysates and concentrated conditioned media (cm) were analysed by western blot. unpurified cm, or cm after performing size exclusion chromatography (sec, izon), were analysed in the exoview r100 system. diluted cm was incubated on custom antibody microarray chips overnight. then the chips were labelled with a cocktail of 3 labelled antibodies, washed and imaged. vesicles were counted, sized, and phenotyped. next, commercially available pooled human csf was analysed in a similar fashion to determine their abundance in a relevant biofluid. results: multiple antibody clones were tested in different combinations for capture and labelling for the five different neuronal enriched proteins of interest, and optimal combinations were identified. some markers were identified on particles > 50 nm in size that were negative for tetraspanins, while others colocalized with tetraspanins. through comparing permeabilized and intact evs with and without sec to remove non-vesicular proteins, we found that tau could be on the vesicle surface, within the vesicle, and free in solution. summary/conclusion: the exoview platform can be customized to enable the detection of proteins of interest and to determine whether they are on the ev surface, intravesicular, or non-ev associated. methods: forty non-smoking male and female subjects (40-70y) at moderate risk for cvd were recruited for the study. evs from platelet-free plasma (pfp) were isolated using size exclusion chromatography (sec). the concentration and size distribution of evs were measured by nanoparticle tracking analysis (nta) and flow cytometry (fcm). three ev markers, including annexin v for the circulating phosphatidylserinepositive (ps+) evs, cd41 for platelet-derived evs and cd105 for endothelial-derived evs were used for phenotyping. in addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser (hemacore). platelet aggregation to determine platelet function was assessed by a high-throughput platelet function assay with a wide range of concentrations of agonists, including adenine diphosphate (adp), collagen-related peptides (crp-xl), epinephrine, thrombin receptor activating peptide (trap-6) and u46619. the association between thrombogenic risk markers for cvd and ev numbers was tested by pearson's correlation coefficient and linear regression model using the statistical program, spss. results: circulating ev concentration with threshold of 71 nm, measured by nta, were positively associated with coagulation-related risk markers, including rate of clot growth (r = 0.568; p = 0.01) and clot size at 30 min (r = 0.480; p = 0.002). ps+ evs derived from endothelial cells, determined by fcm, were negatively associated with lysis onset time (r = −0.410; p = 0.009), whereas they were found positively correlated with lysis progression (r = 0.417; p = 0.007). both mean and mode size of cevs, detected by nta, were significantly correlated with u46619-induced platelet aggregation (r = −0.338; p = 0.033, r = −0.382; p = 0.015, respectively). summary/conclusion: in subjects at moderate risk for cvd, cev numbers were positively related to rate of clot growth and clot size and size of cevs was negatively related to platelet activity. higher numbers of endothelial cell-derived ps+ cevs were associated with lower rates of fibrinolysis. this suggests that cevs promote clot growth and reduce fibrinolysis, and may therefore be an indicator for greater risk of cvd. beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hipsc) and hipsc-cardiomyocytes as therapeutic approaches for heart failure introduction: heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests extracellular vesicles (evs) play a major role in cardiac repair. within this framework, evs from human induced pluripotent stem cells (hipsc) and hipsc-derived cardiomyocytes (hipsc-cm), hold a tremendous potential to treat cardiovascular disease. we isolated evs from conditioned culture media at key stages of the hipsc-cm differentiation and maturation processes, i.e. from hipsc (hipsc-ev), cardiac progenitors (cpc-ev), immature (cmi-ev) and mature (cmm-ev) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. methods: hipsc were differentiated into cardiomyocytes in a 3d culture approach, using the protocols developed by our group. ev isolation was performed on an iodixanol density gradient, and the evs were characterized in terms of particle size and particle size distribution, presence of ev-specific markers, and imaging through transmission electron microscopy. functional studies were performed using human umbilical vein endothelial cells (huvecs) to evaluate evuptake, cell migration and angiogenesis. results: evs from all hipsc and cardiac derivatives presented a typical cup-shaped morphology and expressed cd63 and cd81. ev yield varied along differentiation, with a minimum for cpc and a maximum for cmi. pkh26-labelled evs were uptake by huvecs, and colocalized with calnexin, a protein from the endoplasmic reticulum. wound healing assays showed an increased cell migration in huvecs treated with cardiomyocyte-derived evs, in comparison with control evs isolated from foetal bovine serum. summary/conclusion: our findings suggest a different ev secretion profile along cm differentiation and maturation, with preliminary assays showing ev functionality. ongoing work aims at elucidating the possible differences in function and cargo amongst these types of evs. endothelial cells differentially load and secrete extracellular vesiclederived micrornas into apical and basolateral compartments this may play a role in microcalcification in calcific aortic valve disease (cavd), but this is poorly understood. annexin a1 is thought to be a marker of membrane-derived evs, but because it can be found on the cytoplasmic or extracellular side of the plasma membrane, its localization within or on the surface of evs is unclear. the goal of this study was to determine whether annexin a1 is found on the surface of evs in two cell lines relevant to cavd, and develop an assay that can be used to determine whether this changes under pathogenic conditions. methods: evs were isolated by differential ultracentrifugation from the conditioned medium (cm) of smooth muscle cells (smc) and valvular interstitial cells (vic). total protein in the cell lysates and ev pellets was analysed by western blot. evs from cells treated with control sirna or anxa1-sirna were enumerated and phenotyped using the exoview r100 platform. evs with surface expression of cd9, cd81, cd63, and annexin a1 were captured using a customized antibody microarray chip. then evs were labelled with fluorescent antibodies to assess ev number, size, and colocalization of ev proteins. the knockdown of annexin a1 allowed us to assess the specificity of the selected annexin a1 antibody. results: the ev fraction was positive for cd9, and lacked markers of other vesicle types. western blot on the ev pellet and supernatant in ± edta indicated that there is annexin a1 both on the surface of and within the evs. using the antibody microarray chips, numerous annexin a1+ evs were captured on the annexin a1 spots from the control cm, and there was a marked decrease in capture and labelling from anxa1-sirna treated cells. under both conditions, vesicles were also captured on tetraspanin probes, with the greatest number captured on cd9, then cd63 and cd81. there was a significant population of annexin a1+ evs that was negative for tetraspanins. summary/conclusion: annexin a1 is found on the surface of evs. the assay developed in collaboration with nanoview biosciences is well suited for assessing the number and phenotype of annexin a1+ evs derived from smc and vic cell lines, which could provide a useful method for understanding ev populations in cavd patient cell lines. funding: this work was supported by hl136432 and hl147095. possibility of exosomal micrornas associated with chronic limb-threatening ischaemia, the end stage of atherosclerosis, as a promising biomarker introduction: chronic limb-threatening ischaemia (clti), the end stage of peripheral artery disease (pad), has poor prognosis and is attributed to lifestyle disease. with increasing of atherosclerotic disease all over the world, establishment of biomarker for should play a pivotal role for early detection and preventing aggravation of the disease. the aim of this study is to explore the possibility of liquid biopsy for atherosclerotic disease by analysis of clti-associated exosomal micrornas. methods: clti due to pad was diagnosed by anklebrachial blood pressure index, skin perfusion pressure (<40 mmhg) and angiography. ten preoperative clti patients and 10 control patients without pad were analysed (all patients with diabetes and 50% of patients had end-stage renal failure [esrd] ). to identify biomarkers associated with clti, exosomes were extracted from patient's serum after ultracentrifugation and total rna including small rna was isolated from the exosomes. the expression profile of exosomal micrornas associated with clti were evaluated using a next generation sequencing. results: forty-three exosomal mirnas associated with clti were identified. intriguingly, these mirnas were clearly categorized with esrd, which was well known as end-stage of life-style disease: these were stratified into 20 micrornas for esrd patients and 23 micrornas for non-esrd patients. since esrd is the most important factor significantly related to patient's prognosis in clti, exosomal micrornas reflected patient's comorbidity onto the expression profile. summary/conclusion: a portion of the expression profile of exosomal micrornas associated with clti was identified. exosomal microrna could be a biomarker to stratify patient's condition along with their comorbidities and is very promising for individualized diagnosis in atherosclerotic diseases with risk diversity. postoperative plasma exosomal mir-21 and mir-133a signature in patients with left ventricular reverse remodelling after surgical mitral valve repair underwent implantation of a prosthetic mitral ring. lv remodelling was assessed by cardiac magnetic resonance imaging and pexos were isolated by optimized ultracentrifugation before surgery (t0) and six months after surgery (t1). isolated pexos were quantified by nanoparticle tracking analysis and mir-1, mir-21, mir-133a, and mir-208a were measured by rt-qpcr. the same analysis was performed on healthy subjects with normal cardiac function (n = 8). local ethical committee approved the study (emigrate study, approval n°1529) and informed consent was obtained from all patients. results: pexos levels at t0 were lower (−32%, p = 0.02) in patients with worst postoperative lv function, while they were higher at t1 (+31%, p = 0.03) in patients with reversed lv remodelling after surgery. at t1, the increase in pexos levels was associated to decreased heart mass index (−13%, p = 0.02) and higher levels of exosomal mir-21 (+78%, p = 0.02) and mir-133a (+69%, p = 0.05) were detected in patients with improved lv function. summary/conclusion: higher postoperative levels of pexos delivering mir-21 and 133a depict lv reverse remodelling after surgical mitral valve repair. monitoring of exosomal micrornas cargo might predict postoperative outcome in patients with mr. expression of lipocalin-2 (lcn2) in circulating extracellular vesicles (evs) and femoral plaque-derived evs of peripheral arterial disease patients. introduction: clinically, the drug resistance situation of acinetobacter baumannii is becoming increasingly serious, and its drug resistance has become a difficult problem for nosocomial infection and clinical treatment. in view of the relatively slow development of antibacterial drugs, exploring the resistance mechanism of acinetobacter baumannii is of great significance to improve bacterial resistance and help clinical treatment. studies have shown that outer membrane vesicles (omvs) can transmit resistance genes to mediate the spread of drug resistance, and recent studies have confirmed that high expression of efflux pumps play an important role in the multidrug resistance of a. baumannii. in this study, we want to explore whether the outer membrane vesicles of acinetobacter baumannii can transfer the efflux pump related substances. methods: first, ultracentrifugation and density gradient centrifugation were used to extract the omvs of acinetobacter baumannii antimicrobial-sensitive strains (atcc19606) and antimicrobial-resistant strains. then, nanoparticle tracking analysis (nta) technology was used to analyse the particle size and distribution range of omvs. transmission electron microscopy (tem) was used to identify their morphology and structure. bradford method was used to determine the protein concentration of omvs. next, the omvs of antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains and then the drug susceptibility test was done to determine whether omvs of antimicrobial-resistant strains could transmit antimicrobial-resistance information to the antimicrobial-sensitive strains. finally, pcr, qpcr and mass spectrometry were used to determine whether the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. results: nanoparticle tracking analysis (nta) detected the concentration and size distribution of omvs of acinetobacter baumannii strains. it showed that the extracted omvs have a relatively uniform particle size and a size between 100-250 nm. tem showed that omvs had a typical vesicle structure. omvs coculture experiments showed that omvs of the antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive strains. and the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. summary/conclusion: omvs of the antimicrobialresistant strains can indeed pass resistance to the antimicrobial-sensitive strains. the cause of acquiring antimicrobial resistance in sensitive strains may be caused by resistant strains passing efflux pump-related genes or proteins to sensitive strains. characterization of melanocytic extracellular vesicles during ageing of the choroid kelly coutant a , léo piquet a , nathan schoonjans b , philippe gros-louis a , julie bérubé c , stéphanie proulx a , alain r. brisson d and solange landreville a a université laval, quebec city, canada; b université de lille, lille, france; c centre de recherche du chu de québec-université laval, quebec city, canada; d université de bordeaux, bordeaux, france introduction: the choroid is located at the backside of the light-sensitive retina and is highly vascularized. it contains pigmented melanocytes, and their melanin protects them against oxidative stress. since ageing reduces the number of melanosomes in melanocytes and generates a stiffer extracellular environment, our hypothesis is that surrounding choroidal cells and the retinal pigment epithelium (rpe) are subject to more oxidative stress-related damages. this study aimed to characterize evs released by human choroidal melanocytes in the context of intercellular cooperation during ocular ageing. methods: melanocytic evs were recovered from the conditioned culture medium of young/old melanocytes grown on hydrogels of varying stiffness (0.5-20 kpa) by differential centrifugation. the concentration and size distribution of melanocytic evs were determined by high-sensitivity flow cytometry. cryo-transmission electron microscopy combined with receptor-specific gold labelling were used to reveal their morphology, size and phenotype. the relative abundance of 8 surface markers was evaluated with the exo-check exosome antibody array. the uptake of fluorescent melanocytic evs by the rpe and choroidal endothelial cells was assessed by confocal microscopy. results: choroidal melanocytes released evs positive for annexin-5 and the tetraspanin cd63. young melanocytes produced more annexin-5 positive evs and evs larger than 500 nm compared to older donors. the stromal stiffness impacted the concentration and size of melanocytic evs. we confirmed the uptake of melanocytic evs by endothelial and rpe cells. summary/conclusion: evs from choroidal melanocytes are internalized by surrounding endothelial cells and rpe. age-related stressors modify the phenotype of melanocytic evs. the identification of melanocytic factors that can protect retina/choroid cells from oxidative stress-induced cell death could lead to more efficient therapy for patients suffering from dry agerelated macular degeneration. introduction: owing to their proposed biocompatibility and ability to cross biological barriers, evs represent an attractive therapeutic delivery platform. however, evs are eminently heterogeneous. a better understanding of ev heterogeneity and its origins will allow for improved design of ev-based therapeutics. ev heterogeneity is mainly studied by focusing on distinct ev subpopulations. other sources of heterogeneity, such as heterogeneity within ev secreting cells themselves, have been investigated in lesser detail. in this study, we assessed the phenotypic drift of cell derived evs to explore the origins of ev heterogeneity and its potential impact. methods: three independent samples of two mda-mb-231 breast cancer cell sub-clones were cultured for six weeks. evs were harvested weekly and analysed using the macsplex exosome flow cytometry kit. at two time points the proteome of evs was analysed by lc-ms/ms mass spectrometry with subsequent gene ontology and reactome pathway analysis. results: the expression of over 600 proteins was deregulated in evs derived from the two different cell clones. many de-regulated proteins were associated with biological processes predicted to affect potential ev toxicity (platelet activation, neutrophil degranulation, blood coagulation) and ev biological activity (antigen presentation, inflammation, tgf-beta/ mtor/wnt signalling). more surprisingly, within only two weeks, over 400 ev proteins, many associated with immune modulation, apoptosis, interleukins, cytokines and cell signalling pathways (including those affecting t-cell/b-cell receptors) were de-regulated between the two ev isolation time points. summary/conclusion: results suggest that temporal changes can be observed in the ev proteome (potentially by clonal drift, epigenetic changes or cellular genomic instability) over short time periods. these changes could cause significant differences in biological effects and delivery capabilities between evs harvested from the same cells at different time points and conditions. in vivo tracking and biodistribution analysis of mesenchymal stem cellderived extracellular vesicles in a radiation injury murine model introduction: recent studies indicated that extracellular vesicles (evs) play key roles in intercellular communication and have great potential for clinical application. understanding the biodistribution of evs is therefore essential. our previous works have shown the ability of mesenchymal stem cell (msc)derived evs to protect haematopoietic cells from radiation damage. in this study, we evaluated the biodistribution of msc-evs in a radiated mouse model. methods: human msc-evs were harvested by ultracentrifugation and labelled with did lipid dye. the reliability of the labelling evs was confirmed by sucrose gradient fractionation analysis. the distribution of evs in radiation-exposed mice after ev intravenous administration were evaluated by fluorescence molecular tomography and further confirmed by flow cytometry and confocal microscopy analysis. results: we observed that did labelled msc-evs appeared highest in liver and spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in heart, kidney and lung. we found the significantly increased msc-ev accumulation in spleen and bone marrow post-radiation appeared with an increase of uptake of msc-ev by cd11b+ and f4/80 + cells, but not b220+ cells, compared to those organs from non-irradiated mice. however, there was a predominant ev accumulation in lung and less accumulation in spleen and liver; in mice infused with human lung fibroblast cell derived evs (lfc-evs) and there was no significant lfc-evs accumulation change in the spleen or liver after radiation. we further found that increasing levels of irradiation caused a selective increase in vesicle homing to marrow and spleen. this accumulation of msc-evs at the site of injured bone marrow could be detected as early as 1 hour after msc-ev injection and was not significantly different between 2 and 24 hrs. post-msc-ev injection. summary/conclusion: this study indicated the specific accumulation of ms-evs at the site of injury of haematopoietic tissue in radiation injury mice. funding: this work was supported by the nih grants 5uh2tr000880, 3uh3tr000880-03 s1, 5p20gm11 9943, and 5t32hl116249. linking fat to colorectal cancer: extracellular vesicle crosstalk sheffield hallam university, sheffield, uk introduction: colorectal cancer is the third most common cancer worldwide, and fourth leading cause of malignancy related mortality. understanding the mechanisms of its growth and metastasis is key to elucidating new therapeutic targets and developing treatments in the clinical setting. epidemiological evidence indicates an increased risk of cancer in obese patients, pointing to bidirectional communication between colon and adipose cells. extracellular vesicles (evs) are small membrane enclosed packages released by cells, capable of transporting bioactive cargo from donor to recipient cells and inducing phenotypic changes. adipocytes are a key component of the tumour microenvironment and interactions between adipose tissue and tumour cells may be important in the growth and metastasis of cancer. in this study, we investigate the effects of colorectal cancer evs on adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic, pro-inflammatory phenotype. methods: evs were isolated from sw480 and ht29 human colorectal cancer cell lines by differential ultracentrifugation and mature adipocytes generated by differentiation of the sgbs human pre-adipocyte cell line. adipocytes were treated with evs and their lipid content measured by oil red o to determine loss of lipids. inflammatory cytokine profile was measured by elisa to assess any increase in pro-inflammatory behaviour, and expression of late adipogenesis markers were determined by western blot. results: ev treatment was shown to reduce lipid accumulation in adipocytes, with up to 80% reduction in lipids observed at the 50 µg/ml dose. treatment was also shown to reduce the expression of late adipogenesis markers, and increase secreted levels of proinflammatory cytokines il-6 and il-8 by over 3 fold and 10 fold respectively. these results provide evidence for colorectal cancer derived ev involvement in the dedifferentiation observed in cancer associated adipocytes in vivo, displaying an altered phenotype, releasing lipid energy stores to fuel tumour growth and increasing pro-inflammatory signalling. summary/conclusion: studies have shown colorectal cancer evs may be involved in signalling which induces functional changes in cells within the tumour microenvironment. our work indicates that ev mediated dedifferentiation of resident adipocytes may potentially contribute to a microenvironment favouring cancer cell growth and metastasis. further work aims to elucidate the specific ev cargo which mediates these effects. introduction: ageing is a major risk factor for many human diseases. it is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. senescence is a cellular phenotype characterized by a stable cell cycle arrest. senescent cells are accumulated in the body during ageing. it contributes to develop age-related diseases and cancer. the alteration in intercellular communication with age has been demonstrated to be due to senescent cells developing a phenomenon denominated senescenceassociated secretory phenotype (sasp). exosomes are small extracellular vesicles (sev) (30-120 nm) of endocytic origin whereas microvesicles are formed by shedding of the plasma membrane. they contain nucleic acids, proteins and lipid that generally reflect the status of the parental cell and can influence the behaviour of neighbouring cells. methods: in this study, we demonstrated that the small extracellular vesicles (sev) contribute for transmitting paracrine senescence to proliferative cells firstly, we evaluated the presence of exosome-like particles in the sev from senescent cells by detection of exosome markers (alix, tsg101 and cd63), transmission electronic microscopy (tem) and nanoparticle tracking analysis (nta). to determine that sev from senescent cells are mediators of the paracrine senescence, we performed functional assays using cre-loxp reporter system and high-throughput results: besides, we confirmed at a single-cell level that the proliferative cells internalizing sev from senescent cells activate senescence process using the cre-reporter system. sev protein analysis from senescent cells by mass spectrometry (ms) and validation of top candidates using a functional sirna screen identify interferon induced transmembrane protein 3 (ifitm3), a component of non-canonical interferon (ifn) pathway, as partially responsible for transmitting senescence to proliferative cells. summary/conclusion: in conclusion, we found that sev are regulators of paracrine senescence and ifitm3 contained in senescent sev has an important role in the intercellular communication mediated through sev during cellular senescence1. bin wu a , lei guan a , ye xu a , likang chin a , ting li a , youhai chen a , gordon mills b , jinqi ren a , ravi radhakrishnan a , rebecca wells a and wei guo a a university of pennsylvania, philadelphia, usa; b oregon health & science university, portland, usa introduction: extracellular matrix (ecm) remodelling and stiffening are associated with solid tumour progression. stiff ecm promotes cell proliferation, epithelial-to-mesenchymal transition (emt), metastasis and chemoresistance. hepatocellular carcinoma (hcc) appears frequently in patients with liver cirrhosis or fibrosis while the mechanism remains unclear. exosomes have been determined to serve as messengers to mediate intercellular communication and influence the extracellular. tumour-derived exosomes have been shown to influence tumour progression, metastasis, drug resistance, angiogenesis and immune regulation. thus, determining whether exosomes provide a mechanism by which stiff matrix modulates tumour microenvironment for tumour progression opens a new way to understand cirrhosis and oncogenesis. here we identified the molecular mechanism of matrix stiffening induced exosome secretion and showed the different effect of exosomes induced by soft or stiff matrix on tumorigenesis. methods: huh7 cells were cultured on acrylamide gels with the stiffness was modulated to 500 pa (soft) or 10 k pa (stiff). the exosomes in conditioned media were collected and analysed by nanoparticle trafficking analysis (nta) and immunoblotting. protein expression level in cells was screened by reverse phase protein array (rppa). inhibitor or shrna were used to inhibit target proteins function. in vitro phosphorylation and gef assay were used to verify rabin8 phosphorylation and activation. exosomes from cells on soft or stiff matrix were injected into mice to study their effect on tumour growth. results: (1) stiff matrix promoted exosomes secretion. (2) akt was activated by stiff matrix and was required for exosome secretion. summary/conclusion: matrix stiffening promotes exosome secretion via akt-rabin8-rab8 pathway, contributing to tumorigenesis. tridimensional fibroblast culture revealed a novel exososome-dependent extracellular matrix secretion mechanism vincent clément a , bastien paré b , cassandra goulet a , thiéry de serres-bérard a , stéphane bolduc a , françois berthod a and françois gros-louis a a université laval, québec, canada; b norgen biotek corp., thorold, canada introduction: the extracellular matrix (ecm) is constituted of a variety of proteins and polysaccharides that are secreted locally and assembled into a thick 3d meshwork to provide biophysical and biochemical support to the surrounding cells, and regulate numerous cellular functions such as adhesion, migration and proliferation. dysregulation of ecm components or aberrant ecm remodelling can lead to various pathologies, as well as to play important roles in wound healing. although ecm secretion pathways are still largely unknown, the current paradigm is that ecmassociated proteins are synthesized in the endoplasmic reticulum and transported via the endosomes to the golgi apparatus en route to the cell surface and released by exocytosis. methods: to study ecm secretion pathway, we used 3dimensional (3d) cultured fibroblasts. this culture method technique has been used widely to generate tissue-engineered self-assembled stromal tissues, free of exogenous materials, and rely on long-term supplementation of sodium ascorbate into the culture medium. non-cancerous fibroblasts, grown in conventional two-dimensional (2d) cellular cultures, are known to be a poor source of secreted exosomes when compared to cancerous fibroblasts. results: here, we provide evidence that non-cancerous dermal fibroblasts can secrete high amounts of exosomes, containing different ecm proteins, when cultivated in a 3d fashion. we also demonstrated that dermal fibroblast-derived exosomes had the capacity to travel from one cell to another, induce cellular migration and promote wound healing. summary/conclusion: altogether, these findings reveal a novel exosome-dependent ecm deposition mechanism and suggest that the use of 3d-fibroblast cellular culture may emerge as an innovative approach in precision medicine to better study the role of patient-derived exosomes and ecm proteins in the establishment of cellular microenvironment in health and disease. anthony yan-tang. wu a , charles lai, yun-chieh sung b , steven t. chou c , vanessa guo c , jasper c. chien c , john j. ko c , alan l. yang c , ju-chen chuang c , hsi-chien huang b , syuan wu c , meng-ru ho d , maria ericsson e , wan-wan lin f , koji ueda g , yunching chen h , chantal hoi yin cheung i and hsueh-fen juan j introduction: bionanoparticles including extracellular vesicles and exomeres (collectively termed evs), have been shown to play significant roles in diseases and therapeutic applications. however, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a limited suitable method. methods: we developed a bioluminescence resonance energy transfer (bret)-based reporter, palmgret, to enable pan-bionanoparticle labelling ranging from exomeres (< 50 nm) to small (< 200 nm) and medium and large (> 200 nm) evs and larger evs (> 50 nm). results: palmgret emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, bret, and fluorescence. using palmgret, we show that evs released by lung metastatic hepatocellular carcinoma (hcc) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. ev proteomics identified hcc-ev lung tropic protein candidates associated with cancer progression, in which slco2a1 and clic1 expression on non-tropic evs conferred lung-tropism, while cd13 gave spleen tropism. our results further demonstrate that redirected lung tropism decreases ev distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs distribution including the liver and kidney. summary/conclusion: we established a multimodal and multi-resolution palmbret method to enable pan-bionanoparticle labelling and imaging and therefore quantification in live cells, whole animals, and preserved tissues. the method can resolve the intricate spatiotemporal dynamics of evs. palmgret revealed that evs derived from lung metastatic hcc are lung tropic, and the tropism can be conferred to non-lungtropic ev-293 t by decorating evs with identified hcc-ev membrane proteins. importantly, the enhanced ev delivery to tropic organs also significantly alters its distribution to other major organs. our findings suggest that the dynamics of ev biodistribution and targeted design should be investigated at the organ systems level in ev biology and therapeutic developments, respectively. tracking mesenchymal stem cell-derived extracellular vesicles (evs) in a in vivo cancer model introduction: small extracellular vesicles (sevs) are nanoparticles (30-120mn) encircled by a phospholipid bilayer, derived from the endocytic pathway and released by all cells. sevs have an inherent role in cell communication and deliver cargo to target cells. mesenchymal stem cells (mscs) and have a natural ability to home to tumours and metastases while avoiding the host immune response. it is hypothesised that msc derived sevs (msc-sevs) also possess tumourhoming and immune-evading capacities therefore could provide a novel targeted delivery vehicle for treatment of cancer. it is imperative to elucidate msc-sevs migratory itinerary in vivo to support translation to the clinical setting. methods: this study aimed to image the interaction of labelled msc-sevs with cancer cells in real time in vivo. sevs were isolated from wildtype mscs and mscs with stably expressing red fluorescent protein (rfp) (via lentivirus) by the combined techniques of differential centrifugation, microfiltration and ultracentrifugation. isolated sevs were extensively characterised by transmission electron microscopy (tem), nanoparticle tracking analysis and western blot. nod scid gamma (nsg) mice with dorsal skinfold window chamber (dsfwc) were injected with either mda-mb-231 luciferase (luc) expressing cells or ht-29-luc cells. bioluminescence imaging was performed to confirm tumour formation. a dose of 1x10^7 msc-rfp-sevs was directly added to the window chamber and rfp expression detected using a microscope with rfp filter attachments. 8x10^7 evs were incubated with the radionuclide, technetium-99m tagged duramycin (99 mtc-dur) for 30 minutes at room temperature. excess radiolabel was removed using exosome spin column (invitrogen™). the 99 mtc-dur-sevs were then added directly to the window chamber and charged particle imaging carried out. results: 18 hours post-administration; the rfp signal was localised at the tumour site. radiolabelled sev signal could be detected 10 minutes and 4 hours after administration. msc-sevs were successfully detected at the tumour site following direct administration using two different tagging and imaging approaches. summary/conclusion: this promising preliminary data supports the potential of this approach for tracking msc-sev migration in vivo. future studies will investigate systemic tracking of msc-sev migration. vaughn garcia 1 ; aejez sayeed 2 ; rachel derita 1 ; shiv ram krishn 3 ; peter a. introduction: tumor-derived small extracellular vesicles (sevs) have emerged recently as mediators of tumorigenesis. however, the role of sevs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. methods: our study involved the tramp mouse model of prostate cancer. we used plasma sevs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. we also used nanoparticle tracking analysis (nta) to analyze sevs. mouse pelvises were irradiated using 10 gy, for 5 consecutive days. results: we first observed that upon pelvic irradiation of tramp mice, the levels of the signaling oncogene c-src are reduced in plasma-derived sevs, while the average size of sevs is increased from 50-100nms to 70-250nms. furthermore, we show that the sevs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. additionally, sevs from irradiated mice increase the amount of dna damage in recipient cancer cells. summary/conclusion: overall, our data show that irradiation of tramp mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sevs when tested on human cells. changes to the composition and behavior of a cancer cell sev population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. introduction: there are emerging physiological and pathological functions of extracellular vesicles (evs) in neurodegenerative diseases including alzheimer's disease (ad). brain derived-evs contain pathogenic proteins, such as tau, amyloid beta (aβ), which have been reported to contribute to cell-to-cell propagation in those diseases. investigation of the brain-derived ev cargo, therefore, is important to further understand the mechanisms of progression in neurodegenerative diseases. we developed the ev separation method from unfixed frozen mouse and human brain tissues and assessed the protein composition. methods: to establish the ev separation method, we separated evs from frozen mouse brain tissue using sucrose density gradient ultracentrifugation (sg-uc) or size exclusion chromatography to compare the results from the particle number, morphology and protein profiling by nta, tem and mass spectrometry. evs were then separated from cortical grey matter of ad (n = 20) and control (n = 18) by sg-uc. tau and aβ in the evs were measured by immunoassay. differentially expressed ev proteins were observed by quantitative proteomics employing machine learning. results: the separated evs were enriched in ev molecules and devoid of contaminant proteins by sg-uc, showing our method was successful. the levels of ps396 tau and aβ1-42 were significantly increased in ad evs. annexin a5 (anxa5), neurosecretory protein vgf, neuronal membrane glycoprotein m6-a (gpm6a), and alpha-centractin (actz) were differentially expressed in ad evs. a combination of these 4 proteins were confirmed to predict ad with the 88% accuracy by machine learning. summary/conclusion: these data suggest our method were suitable for the separation of brain-derived evs and ev anxa5, vgf, gpm6a and actz can be potential biomarkers for monitoring the progression of ad. edta stabilizes the concentrations of extracellular vesicles during blood collection introduction: to establish reliable biorepositories for research on extracellular vesicles (evs) as disease biomarkers, the release of evs during blood collection and handling must be avoided. currently, citrate is recommended as the anticoagulant for blood ev research, but citrate does not inhibit the release of evs from activated platelets. the release of platelet-derived evs excludes pneumatic tube transport and makes assays time dependent, thereby limiting clinical compatibility. therefore, we aim to stabilize the release of platelet ev concentrations. methods: blood samples were collected from healthy individuals and subjected to common circumstances known to induce platelet activation. blood was (i) incubated with or without thrombin receptor-activating peptide 6 (trap; n = 7), a potent platelet activator, (ii) send to the lab by a routine blood transport (pneumatic tube system; n = 3), and (iii) stored at room temperature or at 4°c for 6 hours (n = 3). the concentrations of evs from platelets (cd61+), activated platelets (p-selectin+), erythrocytes (cd235a+), and leukocytes (cd45+) were determined by flow cytometry (apogee a60-micro). results: following activation by trap, concentrations of platelet-derived and activated platelet-derived evs increased 5.8-fold and 10.4-fold in citrate-anticoagulated blood, compared to 1.4-fold and 2.0-fold in edta-anticoagulated blood (edta vs citrate: p = 0.018 and p = 0.043, respectively). preliminary data show that during pneumatic tube transport and routine sample handling, both platelet-and activated platelet-derived evs were more stable in edta compared to citrate. the concentrations of evs from erythrocytes and leukocytes were unaffected under all studied conditions. summary/conclusion: to conclude, edta stabilizes platelet ev concentrations during and after blood collection, which would facilitate pneumatic tube transport, enhance reliability and thereby improves the establishment of reliable biorepositories for ev research. introduction: cancer-cell secreted extracellular vesicles, called exosomes, are an emerging biomarker for cancer liquid biopsy. profiling of cancer-associated exosomes usually required lengthy, and multi-step procedures; therefore simple and easy-setup sensing methods are urgently needed for diagnosing cancer in a timely manner. chirality, the foundational property of all biomolecules, including exosomal proteins, can be utilized for exosome detection and differentiation using recent advances in chiral nanostructures. we found that microfluidic sensors can be successfully implemented for successful detection of cancer-associated exosomes taking advantage of unusually high circular dichroism (cd) of chiral gold nanoparticles (aunps). circular dichroism-based exosome (cdexo) detection utilizes chiroplasmonic enhancement of cd signatures of cancer-associated exosomes. we first synthesized donut-shaped aunps conjugated with l-cysteine and immobilized the aunps on a glass slide using a layer-by-layer assembly. the aunps on slide glass were surface functionalized by the standard biotin-avidin reaction after mua treatment. biotinylated annexin v marker, targeting phosphatidylserine (ps) expression on cancer-associated exosomes, was conjugated to the aunp surface. 5 μl of exosome samples from cancer cells (a549 and h3255) or normal cells (mrc5) were injected into the pdms microfluidic device and incubated for 30 minutes. the cd signal before and after exosome exposure was monitored, compared, and systematically analysed as a rapid technique for the detection of exosomes with high sensitivity. results: we showed that the cdexo signals from cancer exosomes showed 26.6 folds absolute cd peak value change and 3.41 folds shift, respectively, compared to that of healthy exosomes. importantly, the cdexo sensing method takes less than 10 mins in terms of total scanning time and requires minimal sample volumes. from the preclinical studies using 7 blood samples from cancer patients and healthy donors, we found that cancer patients show stronger band shift and signal change comparing to that of healthy donors, implying our platform could be used for cancer diagnosis. summary/conclusion: this new versatile and sensitive method based on chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes in a timely manner for minimal volumes of liquid biopsies. ev classification and fractionation strategy using surface charge labelling takanori ichiki a , hiroaki takehara a , hirofumi shiono b and hiromi kuramochi a a the university of tokyo, bunkyo, japan; b innovation center of nanomedicine, bunkyo, japan introduction: the development of new classification technology is required based on the evaluation of physicochemical properties of exosome surfaces and the diversity of constituent molecules. in this presentation, we present the electric charge activated exosome sorting platform comprising microfluidic device technology and electric charge labelling technique. methods: the single nanoparticle analysis platform, which has been developed by our research group, images rayleigh scattered light (elastically scattered light) obtained by irradiating nanoparticles with convergent laser light and provides information of individual particles by image processing. the method that utilizes electrokinetic phenomena, unlike the method using fluorescent labels, measures the properties of the particle surface without serious difficulty in principle even if the particle size is on the order of tens nanometres, and further enables to perform fractionation. since the number of particles usually handled in exosome research or its envisioned application is enormous, it is not realistic to take an approach such as a cell sorter in which particles are sequentially manipulated one by one following the measurement results of individual particles. results: particles receive attraction or repulsion by an external field according to the charge density on the surface, so there is no need to control the external force, and it is possible to design a device that can autonomously fractionate particles according to the difference in zeta potential. summary/conclusion: in conclusion, we have proposed and demonstrated the new concept of electric charge activated ev sorter. funding: this research was partially supported by the center of innovation program (coi stream) from the japan science and technology agency. high throughput exosome analysis by using reversible microfluidic electrochemical sensor system introduction: exosome is one of the important extracellular vesicles (evs) released from parental cells and it contains various types of molecular cargos from its original cell including proteins, messenger rna (mrna), and micro rna (mirnas) [1] . the exosomes have recently emerged as biomarkers for early stage cancer detection because the number of exosomes originated from cancerous cells are significantly higher than those from normal cells [2] . since many different types of exosomes exist in the whole blood, it is necessary to isolate and detect disease-specific exosomes. for this reason, the isolation and the detection of exosomes is an important research issue and has been studied by many groups. however, limitations such as low throughput and low recovery still make it difficult to use exosomes in diagnostics and therapeutics. methods: in this study, we developed an integrated microfluidic electrochemical biosensor to extract plasma from whole blood and subsequently detect cancer related exosomes in a continuous manner. this consists of two parts. the first part is a channel for extracting plasma containing exosomes from whole blood, and the second part is a channel combined with an electrochemical sensor for multiple detection of various exosomes in the extracted plasma. previously, a multi-orifice flow fractionation (moff) channel that consists of a series of expansion and contraction structures has been developed in our group. in this channel, the blood cells are moved to sides of channels by hydrodynamic forces and then are eliminated to outlets. at this time, the plasma is moved to the electrochemical sensor part, the exosomes in the plasma are captured to the electrodes immobilized with the specific antibodies and are quantified the amount of cancer-related exosomes. results: using this chip, blood cells were eliminated from the whole blood with over 90% of separation efficiency at 225 µl/min flow rate and exosomes were collected continuously with high recovery (~70%). in order to quantify various types of exosomes, a labelfree electrochemical biosensor with electrochemical impedance spectroscopy (eis) was used for the continuous detection of exosomes. the limit of detection was 1x10^6 exosomes/ml. summary/conclusion: the developed device is an integrated device capable of separating exosomes from whole blood with high purity and quantitating exosomes through the electrochemical sensor in a continuous manner. , 1800528, 2019) . the development of highthroughput techniques capable of simultaneously monitoring physical and biochemical properties of evs would significantly simplify and accelerate the characterization process. in this context, microfluidic technology is emerging as an attractive platform. here, we present a microfluidic device based on the combination of diffusion sizing and multi-wavelength fluorescence detection to simultaneously provide information on ev size, concentration and composition. methods: the diffusion of evs in the microfluidic channel provides information on their size distribution, and four different staining protocols with high signalto-noise ratios track different ev native molecules. evs are separated from unbound fluorophores directly during the microfluidic analysis, therefore avoiding the need for sample pretreatments and allowing to operate the device as a single-step immunoassay. results: the microfluidic device coupled with complementary staining techniques allows to individually detect and size particle populations with different ev components such as lipids, primary amines and the ev marker cd63. we demonstrate that this approach can probe the abundance of ev-specific markers and impurities such as lipoproteins with high throughput and low sample consumption. summary/conclusion: we present a microfluidic technique capable of characterizing and quantifying evs at low costs, in a time-scale of minutes and requiring only up to 2 µl of non-pretreated sample. this method is an important complementary tool to the current array of biophysical methods for ev characterization, in particular for high-throughput screening applications. funding: h2020-eu.1.2.1-fet open programme via the grant agreement 801338. immunomagnetic isolation of specific subpopulations of exosomes for liquid biopsy via nano-architected porous materials introduction: exosomes offer the potential to reveal significant biological information in many areas of clinical importance by virtue of their rna contents and protein surface markers. this abstract reports the fabrication of a device for high throughput targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected porous metal lattice materials. methods: we have invented a fabrication technique to precisely make millions of nanoscale exosome sorting devices that can operate on unprocessed plasma. each nanoscale device can precisely sort targeted exosomes from background vesicles but is too slow for practical use individually. however, the operation of millions of these devices in parallel preserves the precision of nanoscale sorting while also enabling high throughput and robust use on raw plasma samples. the metal lattice within which these devices are contained is assembled via metal electroplating onto a selfassembled polystyrene bead lattice with face-centred cubic (fcc) symmetry with 600 nanometre pores. the devices feature a conformally-coated layer of nickel-iron with gold passivation atop a base layer of nickel, resulting in a lattice of millions of nanoscale pores capable of magnetic sorting of exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic nanoparticles. results: compared to our previous work on immunomagnetic exosome capture via commercial track-etched membranes (tempo), this device offers superior capture due to increased surface pore density (>25x) and three-dimensional pore density (>1000x) alongside lower required sample volume due to decreased noncapturing volume in the device. finite-element analysis simulations show that strong magnetophoretic traps emerge at the pore boundaries in this structure between higher-permeability metals such as nickeliron permalloy and the lower-permeability sample fluid in the device. preliminary experimental data shows that this device can isolate iron nanoparticles in solution with >100x enrichment from input and 49x capture efficacy versus tempo. summary/conclusion: current methods of exosome isolation such as ultracentrifugation and column chromatography all suffer from low throughput and limited yield. the application of inverse opal materials towards exosome capture offers the potential for isolation of specific exosome populations from very low clinical sample volumes or sparse biological signals. micropatterned growth surface topography affects extracellular vesicle production colin l. hisey a , james hearn b , yohanes nursalim a , vanessa chang a , cherie blenkiron a and lawrence w. chamley a a the university of auckland, auckland, new zealand; b university of auckland, grafton, new zealand introduction: extracellular vesicles are micro and nanoscale packages released by all cells and play an important role in cell-to-cell communication by shuttling biomolecules to nearby and distant cells. however, producing enough evs for many in vitro studies using conventional tissue culture techniques can be challenging, and despite the success of some bioreactors in increasing ev-production, it is still unknown how many independent culture conditions like growth surface topography can alter the production and content of evs. methods: standard 150 mm petri dishes were patterned with 2 µm tall polystyrene microtracks spaced by 5, 10 and 20 µm across a 100 mm area using standard microfabrication techniques including photolithography, soft lithography and microtransfer printing. the micropatterns were characterized with sem and profilometry, then activated with oxygen plasma and uv sterilized. mdamb231 cells were seeded onto patterned and smooth (control) dishes and grown in serum-free media for the final 48 hours of culture. evs were isolated using sequential ultracentrifugation of conditioned media and characterized using nta, tem and western blot. cell morphology was imaged using immunocytochemistry and single cell migration was characterized using time-lapse microscopy and manual single cell tracking in fiji. results: we demonstrate the simple and repeatable fabrication of microtracks across a large surface area in order to culture cells on topographically patterned growth surfaces. furthermore, we show that the 5 µm spacing produced significantly more evs than other patterns as well as the highest cell aspect ratio and average single cell migration speed (p < .05). summary/conclusion: these findings have implications in both biomanufacturing of evs and potentially in enhancing the biomimicry of evs produced in vitro. however, further experimentation to assess the differences in cargo on patterned growth surface topographies compared to conventional methods is still required. funding: this project was funded by the maurice and phyllis paykel trust. using miscroscale thermophoresis and surface plasmon resonance to measure the interactions of extracellular vesicles mst is a quick method, easy to handle, has a low sample consumption, has no limitation on molecule size, and enables measurements in solution, either in various buffers or complex biological liquids. these properties make mst an interesting tool for research of extracellular vesicles (evs); therefore, our aim is to apply this method to evs. methods: evs were isolated from jurkat cell line by differential centrifugation. microscale thermophoresis (mst) and surface plasmon resonance (spr) were used to analyse the interaction between antibody and evs. results: we have demonstrated that interactions of evs with antibodies could be analysed by mst. however, the tiny glass capillaries for sample mounting represent a challenge due to adhesion of evs to their surface. we have tested commercial capillaries as well as prepared capillaries in house coated by liposomes or bovine serum albumin. the interactions between evs and antibodies were confirmed by surface plasmon resonance (spr), which is an established method for studying the interactions of evs. introduction: the isolation of extracellular vesicles (evs) from cell culture supernatants and complex body fluids, such as blood and urine, is of high importance for ev research as well as for future medical applications in diagnostics and therapy. nevertheless, it is still challenging to reach the desired recovery, purity and specificity due to many manual and time intensive sample preparation steps. conventional centrifugation for ev isolation or sample preparation prior to affinity-based separation methods can damage evs and cells, leading to misinterpretation of results or inactive evs. alternative field flow fractionation methods employing acoustic fields are highly promising, but so far limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly reproducible. here, we present an innovative surface acoustic wave (saw)-based acoustofluidic device for gentle sorting of cells and particles. methods: our device consists of interdigital transducers patterned on a piezoelectric substrate generating saw propagating on the substrate surface. upon interaction of saw with our on-chip structured, fluid-loaden microchannels, an acoustic pressure field is developed across the fluid wherein particles are suspended. this pressure field can be employed to simply manipulate cells and particles based on their intrinsic properties, such as size, density and compressibility in continuous flow. the device is manufactured using precise and low-cost microtechnological methods and is suitable for reproducible mass fabrication. results: we demonstrated the separation of blood components, i.e. the sorting of erythrocytes and thrombocytes. furthermore, we could also show results on thrombocyte activation indicating a gentle separation without damaging these shear-sensitive cells, as well as first results on plasma separation from whole blood samples and nanoparticle sorting. summary/conclusion: our unique acoustofluidic sorting technology for complex suspensions has the potential to overcome the need for time-effective, cheap and gentle separation of evs. funding: this work was supported by efre infrapro project "champ: chip-based acoustofluidic medtech platform". nanophotonic platform for cancer-associated exosomal microrna detection introduction: exosomes have an important role in intercellular communication at physiological and pathological processes. their cargo includes micrornas (mirs), single-stranded non-coding rnas, involved in alterations on recipient cells, such as development of tumourous phenotype and metastasis. more particularly, mir-21 excels due to its association with several cancers. determining exosomal mirs as cancer indicators demands selective and accurate methods, which are not currently available or entail high costs. colorimetric photonic-based assays are a promising label-free alternative, which dismisses complex apparatus for signal reading since biorecognition is detected by colour change. moreover, the clinical and economic systems have also been demanding a decrease on the green footprint of biosensors, requirement fulfilled with naturally derived biomaterials. methods: herein, the biosensor is constructed on a biopolymer matrix to meet the requirements of an eco-friendly disposable device, and it is based on a photonic structure obtained by imprinting a nanopattern on the polymer surface. then, the surface is functionalized with the complementary oligonucleotide sequence of mir-21 as sensing probe. a labelfree detection is thus envisioned and the sensor performance is evaluated by changes in the optical properties when the target is present. results: the combination of biological materials conducted to a biosensor support with great flexibility and low water permeability, allowing easy surface functionalization. the self-reporting ability of the photonicbased sensor enables high intensity colours detected by naked eye. summary/conclusion: the alliance with the high selectivity of oligonucleotide hybridization is expected to offer great exosomal mir-21 recognition ability and an optimistic perspective for utilization in clinical setups. funding: the authors acknowledge the financial support from the european commission/h2020, through mindgap/fet-open/ga829040 project. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = 31). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and 10 ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine 37 distinct uev surface markers of cd9+/cd63+/cd81+ vesicles in a multiplexed bead-based manner including respective controls. the accuri c6 (bd) was utilized for data acquisition. for further misev2018-compliant characterization, cd9+/cd63+/cd81+ uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd209, all other 36 surface markers could be identified. the most abundant markers were cd9 and cd63, which were detected in 93% of samples, followed by cd133/1 (84%), cd326 (81%), cd81 and cd24 (77%, each). cd3 (42%), cd9, cd45 (39%), cd49e (32%) and cd81 showed similar relative median fluorescent intensities (rmfi), while cd63 yielded significantly higher (p = 0.009) and all other markers significantly lower rmfi (p < 0.011). tem and f-nta revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 e + 10 particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg101, cd9, cd63 and cd81 without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a , esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = 40) aged 40-70 yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a 3colour panel, which included annexin v (for the majority of circulating evs), cd41 (for plateletderived evs) and cd105 (for endothelialderived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and 10-yr cvd risk detected by qrisk2. results: ev numbers, as determined by nta, were strongly associated with bmi (r = 0.602, p < 0.001), blood pressure (systolic bp: r = 0.359, p = 0.023; diastolic bp: r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = 0.330, p = 0.038). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining 49.4% of the variance for total ev numbers. an additional 9.3% of the variance in total ev numbers was predicted by diastolic bp. ancova of the 10-yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with 10-yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd4 + t cells were isolated from secondary lymphoid organs of foxp3-rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th1, th2, and th17), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, 15-day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for 5 days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th1, th2, th17) showed either no change in proliferation (th1) or decrease in proliferation (th2, th17), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < 0.0001). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th1), il4 and il13 (th2), or il17a and il17 f (th17). in contrast, exposure of tregs to cdc-evs resulted in~1000-fold increase in expression of il10, a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il-10 in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il-10. this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t32hl116273 prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from 42 patients treated with prostanoids (study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with prostanoids (control group; n = 38, 55 ± 19 years, 65% female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; 0.5 mm), adenosine diphosphate (adp; 6.5 µm) and thrombin receptor-activating peptide (trap; 32 µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd61+ and cd62p+; pevs), leukocytes (cd45+, levs) and endothelial cells (cd146+, eevs) were measured in plateletdepleted plasma by flow cytometry (a60-micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = 0.01) and lower concentrations of pevs and levs (p ≤ 0.05). furthermore, thrombus formation was delayed (p ≤ 0.003) and thrombus size was decreased (p = 0.008) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd61+ pevs (p = 0.04). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ 0.05) and the concentrations of pevs (p ≤ 0.08). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ 0.04). summary/conclusion: patients with pah treated with prostanoids have increased risk of bleeding both due to impaired platelet aggregation, ev release and thrombus formation, compared to patients not treated with prostanoids. antiplatelet effect of prostanoids varies: whereas epoprostenol decreases the release of pevs, treprostinil and iloprost impair platelet aggregation. funding: ag is supported by the national science centre, research project preludium 2018/31/n/ nz7/02260. evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. nanoflow cytometry identifies an imbalance of epithelium-and neutrophil-derived extracellular vesicles in the airway environment of paediatric cystic fibrosis patients brian dobosh, vincent giacalone, milton brown, lucas silva, lokesh guglani and rabindra tirouvanziam emory university, atlanta, usa introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from 17 cf children (<6 years of age). for nanoflow cytometry, evs were stained with di-8-anepps, and with epcam, cd66b and cd115 (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = 0.003, spearman's rho = 0.689). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = 0.001, rho = 0.857). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv15a0), emory paediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: 142 individuals were enrolled into our study, subdivided into a training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer n = 20, pneumonia n = 18, sepsis n = 37). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq2) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative real-time pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed 29 significantly regulated mirnas in pneumonia compared to volunteers, and 25 mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by 12 mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992). mir-193a-5p (log2fc = 1.86, padj = 1.49e-6) and mir-542-3p (log2fc = 1.67, padj = 3.29e-5) differentiated between pneumonia and volunteers and mir-1246 (log2fc = −2.41, padj = 1.78e-04) between pneumonia and sepsis. expression levels of mir-193a-5p and mir-1246 were related to disease severity. mir-542-3p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was 73.33%. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study,3healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd63 and cd81 were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and 9 mirnas (mir-103a-p, mir-191-5p, mir-320a, mir-200b-3p, mir-185-5p, mir-223-3p, mir-21-5p, mir-155-5p, let-7 g-5p), were evaluated by rt-qpcr. after the optimization of the methodology, 10 healthy adults subjects and 10 patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd63 and cd81. however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna-320a showed a significant increased (p = 0.02) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir-320a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf2-active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid 2-related factor 2 (nrf2) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf2-active exosomes, mscs were incubated with potent nrf2 activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive msc markers (cd44, cd73, cd90, and cd105) and <5% express negative markers (cd45, cd31, cd14, cd19, or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd81, cd9 and tsg101. nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of 168.0 ± 6.5 nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf2-active human mscs increase exosome secretion by 54%, compared to nrf2-baseline mscs (p < 0.05). both nrf2-baseline and nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05). this reduction eliminated the delay in closure time compared to wounds of c57/b6 mice. nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated c57/b6 wounds. at day 10, exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd31+ vessels compared to vehicle-treated wounds. summary/conclusion: enhancing nrf2 function in mscs multiplies exosome yield. our results demonstrate exosome-based therapies hold tremendous promise and warrant further investigation for rapid translation. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist1 is a key mediator of tumour cell metastasis. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist1. methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc3-ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc3-ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc3-ml lines stably overexpressing or deficient in twist1. results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc3-ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc3-ml overexpressing twist1 showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist1 expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. introduction: exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. however, a strong heterogeneity in the adaptive response to exercise training exists. differential response to exercise training might be mediated by myokines (proteins, nucleic acids, metabolites) that can be released directly into the systemic circulation, or packaged within extracellular vesicles (evs). the objective of this study was to evaluate if changes in evs after acute aerobic exercise (ae) were associated with the responders phenotype following 6-week resistance exercise (re) training. methods: this is a secondary analysis of plasma samples from the exit trial (clinical trial #02204670). eleven sedentary obese youth (15.7 ± 0.5 years, bmi ≥ 95th percentile underwent an acute bout of ae (60% heart rate reserve, 45 min). blood was collected before [time (at) −15, 0 min], during [at15, 30, 45 min], and after [15 min (at60), 75 min (at120)] exercise. afterwards, youth participated in 6-week re programme, and were categorized into responders or non-responders (nr) based on changes in insulin sensitivity (above or below 50 percentile). primary outcome: evs were isolated using size exclusion chromatography (izon®) at baseline (at0), immediately after ae (at45) and after recovery (at120). ev protein concentration, size, and zeta potential were analysed in a single-blind fashion. results: responders had larger evs (~141.1 nm) as opposed to nrs (~97.3 nm) at at0 (p < 0.05) and this pattern was maintained at at45 and at120, though not significant (p = 0.1). nrs displayed differential ev size distribution (peaks at 100 nm or 300 nm), while ev distribution was highest at 250 nm in responders. no difference in average zeta potential or total ev protein yield was observed between groups. an increase in ev yield with exercise time and recovery was observed in both groups. summary/conclusion: our preliminary data suggest that ev size is significantly increased after an acute bout of ae in obese youth responders. further research to delineate the role of evs as predictors of exercise adaptation is warranted. funding: funded by dream and research manitoba. using dual-fluorescent reporter mice to track tissue-specific extracellular vesicles andrea estrada, gabriella hehn, zackary valenti, christopher allen, nicole kruh-garcia and dan s. lark colorado state university, fort collins, usa introduction: extracellular vesicles (evs) from tissues like skeletal muscle (skm) and adipose tissue (at) have been implicated in human disease but are understudied. skm is likely a major player in ev biology as it accounts for~35% of total body mass. tools to define cellular ev origin are needed because tissues like skm are comprised of a variety of cell types. here, we describe our ongoing efforts using the dual fluorescent mg/mt mouse as a tool to analyse skm-myocyte derived evs. methods: wild-type (wt) and mg/mt mice were used for these studies. mg/mt mouse cells express membrane-tagged red (mt) or green (mg) fluorescent protein in the absence or presence of cre, respectively. we made skm myocyte mg expressing mice using a mouse expressing cre on the human skeletal actin promoter. blood was collected via cardiac puncture and platelet-free plasma was obtained via centrifugation. plasma evs were isolated using exoquick, exoquick-tc or size exclusion chromatography. skm and at were dissected into~5mm chunks, placed in serum-free dmem and incubated for 24 hours. tissuederived evs were isolated using exoquick-tc. ev abundance was determined with a horiba viewsizer. individual evs were analysed with a cytek aurora spectral flow cytometer. settings were optimized using polystyrene beads and spectral unmixing was performed to allow detection of mg and mt. results: in wt mice, skm releases >100 times more evs than adipose tissue per unit of mass (p < 0.01 using paired student's t-test). since skm is also a major component of total body mass, these data further emphasize the importance of skm-derived evs. skmderived evs from wt mice were not fluorescent (<0.1% of events). evs from mg/mt mouse skm overwhelmingly expressed mg (>95% of events) with negligible (< 1%) expression of mt. at-derived evs robustly expressed mt but lacked mg. summary/conclusion: these data provide "proof-ofprinciple" that mg and mt are readily incorporated into evs secreted ex vivo. surprisingly however, plasma evs from mg/mt mice expressed very little mg (~3%) or mt (~4%). this observation was confirmed with three separate isolation techniques. we are currently exploring possibilities to explain this finding, including: 1) modification of evs post-secretion, 2) clearance of fluorescent evs by the liver or 3) that evs secreted from tissues remain predominantly in the interstitial space. funding: this work was supported by an innovative project award from the american heart association (18ipa34110052) to dsl. endothelial cd36 delivery of fa loaded extracellular vesicles is critical for thermogenesis. introduction: membrane cd36 facilitates tissue fatty acid (fa) uptake. we recently found that endothelial cell (ec) cd36 controls muscle and adipose tissue fa uptake, and influences the tissue's metabolic phenotype. the mechanism for cd36-facilitated fa uptake is unknown. here we examined the role of ec cd36 in thermogenesis and in fa delivery to brown fat tissue. methods: adult male mice were housed individually, restricted from food during acute (4 hr) cold exposure (4°c) with core temperature monitored every 30 minutes. after 4 hours, animals were sacrificed and samples collected for analysis. for cellular studies, human microvascular (lonza) or primary murine microvascular ec were used. for primary cells, crude cell pellets from lung homogenates were purified using mouse-cd31 magnetic beads (miltenyi). for microscopy studies, alkyne fa (cayman) was added to cells and to enable visualization of internalized fas, click chemistry (invitrogen) used to label alkyne-fa with alexa 568. for radioactive studies, primary lung ec were serum starved for 5 hrs and incubated overnight with 3h-oleic acid bound to fa-free bsa (2:1 ratio). media was collected, clarified by centrifugation to remove microvesicles and debris. small extracellular vesicles (sevs) were isolated from clarified media using total exosome isolation reagent (invitrogen) and counted for radioactivity. results: basal core body temperatures are similar in mice lacking ec cd36 (eccd36-/-) compared to controls (cd36fl/fl). however, during cold exposure at 4°c , eccd36-/-are unable to maintain body temperature (p < 0.001). plasma free fa are higher in cold exposed eccd36-/-indicating fa clearance by brown fat is impaired. mitochondrial function and expression of thermogenic and mitochondrial genes in brown fat from eccd36-/-and cd36fl/fl mice were similar. these data suggested that endothelial delivery of fas is necessary for thermogenic maintenance of body temperature. to examine fa handling by ecs we used alkyne fas to visualize the process. we found that fas are transferred by microvascular ec through caveolae-mediated transcytosis involving src signalling and cav-1 phosphorylation. the internalized cav-1 and cd36 positive vesicles containing fas are released as sevs. to determine the dependence of cd36 on this process, we treated primary microvascular ec with radiolabeled fa and found that sevs secreted by cd36-/-cells contain less labelled-fa (p = 0.05). summary/conclusion: endothelial delivery of fa is critical for thermogenesis. our working model for the mechanism of fa uptake by brown adipose tissue is the following: endothelial cells transfer the fa through caveolae-mediated transcytosis and secrete small extracellular vesicles (sevs) that help deliver fas to brown adipocytes. funding: this work is supported by nih grants dk111175 and dk056341. introduction: diet-induced obesity modifies intestinal permeability leading to bacteria infiltration and to a decrease in the number of immune cells protecting mucosa. as orange consumption is beneficial for human health and used in preventive medicine, we determined whether orange juice-derived nanovesicles (onv) might be recommended as nutritional strategies for the treatment of intestinal complications associated with obesity. methods: onv isolated from fresh orange juices were characterized by lipidomic, metabolomic, microscopy, nta and for their stability during digestion. intestinal barrier (ib = caco-2 cells+ht-29 cells differentiated with oleic acid) were treated with onv and co-cultured with adipocytes to monitor ib fat absorption and release. obesity was induced in mice fed for 12 weeks with a high-fat high-sucrose diet (hfhs mice vs standart chow diet mice). then half of the hfhs mice were gavaged with 150 micrograms/day for 4 weeks. results: onv did not modify high-fat high-sucrose diet-induced obesity and insulin resistance but reversed diet-induced gut modifications. six hours post-gavage, onv accumulated preferentially in jejunum involved in lipid absorption. in jejunum, and no other intestinal region, onv increased villi size, restored immune response and decreased barrier permeability in hfhsd mice. in addition, onv-treated mice had increased expressions of acat2, angptl4 and dgat1, but a decreased expression of fabp2, fatp4, mtp vs hfhsd animals, which indicated that fat absorption, tg synthesis and chylomicron release were strongly reduced. similarly to other plant-derived nanovesicles, these results were likely associated with onv lipid and metabolite compositions (strong enrichment in bioactive phospholipids: pe, pa, pc, pi and leucine) as onv did not resist to harsh digestive conditions in vitro and were poorly incorporated in enterocytes. as the effects of onv on the decrease in tg content and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate to these effects. summary/conclusion: onv are important bioactive compounds of orange juice and for the first time we demonstrated that they can modulate lipid metabolism in the intestinal barrier associated with morphological changes. interestingly onv treatment targets mtp and angptl4 mrnas, 2 therapeutic intestinal targets to reduce plasma lipids and for attenuating inflammation in gastrointestinal diseases. therefore, onv might be used to reduce the development of dyslipidemia-associated diseases and to restore intestinal functions in obese patients. funding: olga triballat institut; benjamin delessert institut, inrae institut. association, structure, and function of fibronectin in extracellular vesicles from hepatocytes xinlei li, ruju chen, sherri kemper and david brigstock nationwide children's hospital, columbus, usa introduction: we have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (hscs, the principal fibrosis-causing cell in the liver) and hepatocytes. in this study, our goal was to determine the molecular nature of the ev components involved in cell binding. fibronectin (fn1) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. two types of fn1 are present in vertebrates, of which the soluble plasma fn1 is derived principally from hepatocytes, while cell-associated fn1 is produced by numerous cell types. here we describe a novel function of plasma fn1 in facilitating binding of hepatocyte evs to target cells. methods: differential ultracentrifugation was used to collect evs released by parental mouse aml12 hepatocytes, fn1 ko aml12 cells in which fn1 was ablated using crispr-cas9, primary human or mouse hepatocytes, or human hepg2 cells, or from human or mouse serum. evs were characterized by nanosight tracking analysis (nta), western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. the binding efficiency of pkh26-labelled evs from parental (ev-hep) or fn1 ko (ev-hepfn1 ko) aml12 cells was analysed in hepatocytes or hscs. swiss webster mice were injected with ccl4 for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of ev-hep or ev-hepfn1 ko over the last two weeks, followed by determination of hepatic fibrogenic genes by qrt-pcr. results: ev-hep or ev-hepfn1 ko were 50-200 nm in diameter and positive for common ev markers (cd63, cd9, flotillin-1). mass spectrometry showed that fn1 was the most abundant protein in ev-hep and comprised principally the plasma form. the abundant presence of ev fn1 was verified by western blot and co-immunoprecipitation with anti-cd9 or antiflotillin-1. western blot showed that fn1 was also abundant in evs from primary human or mouse hepatocytes, hepg2 cells, and human or mouse serum. fn1 and ev-hep co-sedimented at a density of~1.15 g/ml. ev-hepfn1 ko yield and size-range were similar to those of ev-hep, suggesting that ev biogenesis is fn1-independent. as compared to ev-hep, the binding of ev-hepfn1 ko to target cells was highly reduced whereas ev binding was independent of fn1 expression by the target cells themselves. both ev-hepfn1 ko and ev-hep were anti-fibrogenic in vivo but only ev-hep attenuated collagen 1⍺1 expression in mouse hscs in vitro. summary/conclusion: fn1 is abundantly associated with hepatocyte evs and facilitates ev binding to target hepatocytes or hscs. additional studies are needed to clarify the functional role of fn1 in mediating ev-hep anti-fibrogenic actions in vitro or in vivo. elevated glucose increases soluble and aggregated forms of human islet amyloid polypeptide in islet-derived extracellular vesicles -implications in type 2 diabetes and islet transplantation introduction: type 2 diabetes (t2d) is characterized by reduced beta cell mass and function. islet amyloid, formed by aggregation of human islet amyloid polypeptide (hiapp), contributes to progressive beta cell loss in t2d. amyloid also forms in human islets during pre-transplant culture and following transplantation in patients with type 1 diabetes (t1d) which is associated with graft failure. the cellular mechanisms underlying islet amyloid formation are still unclear. in this study, we examined the potential role of islet-derived extracellular vesicles (ev) in the clearance of soluble and aggregated (pro)iapp species from beta cells and amyloid formation. methods: human islets isolated from cadaveric pancreatic donors (n = 4 donors) and wild-type or hiappexpressing (hiapp+) transgenic mouse islets (n = 4/ group) were cultured in normal (5.5 mm) or elevated (11.1 mm) glucose to form amyloid. ev (exosomes) were isolated from culture medium using classical centrifugation and ultracentrifugation. purified ev were analysed by nanoparticle tracking analysis. western blot analysis and double immunogold transmission electron microscopy were performed to verify the presence of ev markers as well as (pro)hiapp species and oligomers (aggregates). results: human islets formed amyloid during culture with elevated glucose which was associated with progressive beta cell apoptosis. (pro)iapp species were detectable in ev released from human islets cultured in normal and elevated glucose. the latter markedly increased (pro)iapp content in islet-derived ev. interestingly, hiapp aggregates (oligomers) were present in the majority of ev released from human islets cultured in elevated glucose but were not detectable in islets cultured with normal glucose. similarly, ev released from hiapp+ mouse islets which formed amyloid during culture had higher (pro)iapp content compared to wild-type islet-derived ev. moreover, hiapp oligomers were present in ev derived from hiapp+ islets but not wt islets. summary/conclusion: in summary, our data show that (pro)iapp species are present in islet-derived ev and that elevated glucose increases (pro)hiapp and its aggregates in ev released from islets. islet-derived ev may play a key role in the process of amyloid formation in t2d and human islet grafts. funding: university of manitoba research grants program (urgp). on. contraction, but not glycolysis, regulates the size of skeletal muscle evs secreted ex vivo. colorado state university, fort collins, usa introduction: skeletal muscle (skm) is a metabolically active tissue and accounts for~35% of total human body mass. acute exercise increases secretion of extracellular vesicles (evs), but the mechanisms responsible are unknown. muscle contraction increases the demand for atp which requires intercellular communication in order to adapt. we hypothesized that this "metabolic stress" during contraction increases skm ev secretion. methods: we tested our hypothesis using an ex vivo ev secretion assay. all studies were approved by the colorado state university institutional animal care and use committee. vastus medialis muscle (skm) from male c57bl/6 j mice (n = 6) or female mt/mg mice (n = 6) was cut into~5 mg pieces and added to 12 well plates (~50 mg/well) filled with 1 ml of serum-free dmem and placed in a cell culture incubator at 37 c for 24 hours. skm from male mice was treated with 2-deoxyglucose (2-dg) (0.1 nm -100 mm) to induce metabolic stress via inhibition of glycolysis. skm from female mice was treated with 10um of blebbistatin (bleb), a contraction inhibitor. after incubation, skm mass was measured and conditioned media was centrifuged (3,000 x g for 15 min) to remove cell debris. evs were isolated using exoquick-tc. nta was performed on isolated evs using a horiba viewsizer 3000. ev secretion was normalized to tissue mass and culture media volume then reported as ([particle]/ml/mg tissue). statistical comparisons for 2-dg experiments were made using a repeated measures 1-way anova. bleb experiments were analysed using a paired student's t-test. results: there was a trend towards greater ev abundance (p = 0.07) as a function of 2-dg treatment, but no effect on ev diameter (p = 0.37). bleb treatment did not alter ev abundance (p = 0.69), but significantly reduced ev mean diameter (p = 0.007; 11% decrease; dmso: 114.9 ± 5.1 vs. bleb: 103.9 ± 4.2). summary/conclusion: contrary to our hypothesis, inhibition of glycolysis with 2-dg did not stimulate skm ev secretion. however, bleb did appear to promote the release of small evs and/or inhibit secretion of larger evs. ongoing efforts are focused on testing other metabolic stressors and defining how blebbistatin promotes small ev secretion. funding: american heart association grant to dsl (ipa34110052). introduction: extracellular vesicles (evs, exosomes) are nanovesicles (30-200 nm) secreted from various types of cells. because of vesicular encapsulation of mirnas and enzymes, the evs play crucial roles in cell-to-cell communication by delivering these functional molecules to other cells [1] . on the other hand, the evs are highly expected as next generation therapeutic tools due to pharmaceutical advantages such as controlled immunogenicity, effective usage of cell-tocell communication routes, artificial modification and encapsulation of functional molecules. however, cellular targeting and uptake efficacy of the evs are insufficient to be utilized as therapeutic tools [2, 3] . in this study, we newly developed evs decorated with cellpenetrating sc18 or (sc18)2 peptides, which are derived from the c-terminal domain of the cationic antimicrobial protein, cap18, because the peptides can be efficiently internalized by breast cancer cells. [4, 5] . methods: all peptides were prepared by fmoc-solid phase synthesis. secreted evs from cd63-gfp stably expressing hela cells were isolated by ultracentrifugation. cellular uptake of evs was analysed using a flow cytometer and a confocal laser microscope. encapsulation of saponin in the ev was conducted by electroporation. results: sc18 peptide is known as one of cell-penetrating peptides, and branched structure of sc18 peptides, (sc18)2, further enhances the cellular uptake [5] . in this research, we examined the effects of the peptide modification on cellular ev uptake, and modification of the sc18 or (sc18)2 peptides on ev membranes was conducted via stearyl moiety. as our results, increased macropinocytotic cellular uptake by modification of the peptides was successfully attained. especially, the modification of (sc18)2 peptides showed higher cellular uptake and macropinocytosis induction efficacy than that of sc18 peptides. in addition, anticancer protein, saporin toxin-encapsulated evs modified with the (sc18)2 peptides significantly enhanced their biological activity with dependency of glycosaminoglycan expression on targeted cells. summary/conclusion: the cell-penetrating (sc18)2 peptide-modified evs shows high abilities to be effectively internalized by cells and are applicable for intracellular delivery of therapeutic molecules. this study is expected to contribute to development of intracellular delivery techniques based on evs. [ introduction: rna therapeutics possess high potential which is yet to be realised, largely due to difficulties involved in delivery to the cytoplasm of target cells. extracellular vesicles (evs) possess numerous features that may help overcome this hurdle and have emerged as a promising rna delivery vehicle candidate. despite extensive research into the engineering of evs for rna delivery, little is known about how their intrinsic rna delivery efficiency compares to current synthetic rna delivery systems. using a novel crispr/cas9 based rna transfer fluorescent stoplight reporter system, we here compared the delivery efficiency of evs to state-of-the-art dlin-mc3-dma lipid nanoparticles (lnps). methods: evs were isolated from mda-mb-231 cells expressing either a targeting or non-targeting control sgrna and applied to hek293 t stoplight+ reporter cells. lnps containing targeting sgrna were titrated onto hek293 t stoplight+ reporter cells to determine the minimum effective dose. lnp and ev particles were characterized using nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis. sgrna copy number was determined using rt-qpcr. results: evs were 140 ± 7 nm in diameter as measured by dls and possessed a negative surface charge of −25.3 ± 3.3 mv. rt-qpcr and nta analysis indicated that sgrna ev loading was low, with only 1 in 3.8e05 ± 3.5e05 evs containing a single sgrna copy. nevertheless, evs containing targeting sgrna induced significant reporter activation while evs containing non-targeting sgrna did not. lnps were 51 ± 0.6 nm in diameter and possessed a neutral charge. these particles also induced significant reporter activation when loaded with targeting sgrna. when delivered via evs, only between 4 to 54 sgrna copies per cell were required to induce statistically significant reporter activation. in contrast, the minimal effective sgrna dose when delivered by lnps was considerably higher at approximately 9e03 copies per cell. summary/conclusion: mda-mb-231 evs deliver rna in a highly efficient manner and are functional at sgrna concentrations several orders of magnitude lower than those required for lnp mediated delivery. this underlines the potential of evs as rna delivery vehicles and highlights the need to study the mechanisms by which evs achieve their efficiency in order for improved development of rna therapeutics. the role of circulating extracellular vesicles in patients with chronic chagas disease introduction: chagas disease is a neglected tropical disease (ntd) caused by the flagellated protozoan trypanosoma cruzi. it is a major public health problem in latin america, and it is now expanding over the globe through immigration of infected individuals. eukaryotic cells release extracellular vesicles (evs) that circulate in body fluids and have an important roles in intercellular communication, both in physiological and pathological conditions. objectives. our study proposes to characterize and to compare the circulating evs isolated from plasma of the chronic chagas disease (ccd) patients with healthy individuals (controls). methods: peripheral blood was collected from patients and controls in the presence of edta and evs enriched from plasma by differential ultracentrifugation. the obtained evs were characterized and quantified by nanoparticle tracking analysis (nta) and added to human thp-1 cells. after 48 h, the cell supernatants were analysed by elisa for the presence of cytokines. results: lower amounts of evs were obtained from ccd patients in comparison with control individuals. however, the same amount of evs of ccd were more capable of inducing cytokines such as ifn-gamma and il-17 in relation to controls. summary/conclusion: although less evs are present in the blood of ccd, these evs induce high inflammatory reactions on macrophages suggesting a possible role of these evs in the establishment of chronic disease. funding: supported by fapesp, cnpq and capes. extracellular vesiclesa trojan horse for therapeutic agent delivery introduction: extracellular vesicles (evs) may prove to be one of the optimal payload carriers for therapeutic agents. while they travel through the extracellular space, the ev's lipid membrane layer shields their luminal cargo from deleterious external factors. when autologous evs are used to protect this therapeutic cargo, little immunogenic effects are expected compared to viral vectors and artificial structures, such as liposomes. their usage is potentially manifold, and they are ubiquitously present in all body tissues and fluids. the key is to develop a manageable ev loading agent for adoptive transfer therapies. methods: to exploit the unique properties of evs, highly positively charged proteins were used to load them with multiple biomolecules, such as a cas9 protein or dicer substrate dsrna as a functional payload and to improve their apparently inadequate natural ability to deposit cargo into the cytoplasm of recipient cells. results: highly positively charged proteins can associate with and/or diffuse through a phospholipid bilayer (thompson et al. 2012) . when these kinds of charged proteins are mixed with isolated evs in vitro, they are loaded into the evs. the positive charge of the protein has the advantage that it can associate with negatively charged agents, such as rna species, and aids the associated molecule to also incorporate into the ev. moreover, the positive charge of the protein helps with cargo delivery, and thus overcoming the bottleneck of the ev's cargo to escape the endosome post-uptake in a recipient cell. self-quenching fluorescent lipid dyes demonstrated that discharge of the highly positive ev cargo into the cytoplasm is concomitant with lipid mixing between the membrane of evs and the membrane of the recipient cell. when egfp-expressing microglia were exposed to evs loaded with a dicer substrate dsrna able to silence egfp via the positively charged protein, the uptake of dicer substrate dsrna was concomitant with a decrease in egfp expression in the microglia. a similar result was achieved when evs were loaded with cas9 protein conjugated to the highly positively charged protein. post-uptake of these cas9-loaded evs, microglia expressing anti-egfp sgrna (single guide rna) lead to decreased egfp expression. summary/conclusion: our ev delivery technology has the capability of delivering multiple biomolecules, such as protein and rna cargo and demonstrates postuptake of the ev functionality of the ev delivered cargo in the recipient cell. hybrid extracellular vesiclesbiomimetic tool for drug delivery to repair endothelial cell dysfunction introduction: traditional drug delivery systems (dds) are usually based on liposomes, micelles or dendrimers. unfortunately, many dds cause side effects including organ toxicity and/or unexpected immune response. in living organisms, extracellular vesicles (evs) are responsible for delivering biologically active molecules to distant cells. in vitro loading of therapeutic compounds into evs is still not effective and needs developing new strategies. for these reasons we aimed to design hybrid extracellular vesicles (hevs) with high loading capacity for dds. methods: for hev synthesis, we used human endothelial derived evs. using freeze/thawing method we fused them with liposomes composed of cholesterol and one of the three lipids: dopc, sphingomyelin or phosphatidylserine. to confirm membrane fusion, we applied a spectroscopy ruler -fret (förster resonance energy transfer) and cryotem imaging technique. we characterized hevs using nta (for size distribution evaluation), dls (zeta potential) and western blot (for detection of evs markers). we evaluated loading efficiency using calcein as a model drug. additionally, we performed cytotoxicity tests. results: in the cryotem imaging, pure and homogenous hev population with a diameter of 65 ± 15 nm was detected. additionally, we observed changes in zeta potential and in size distribution after fusion. fret measurements showed increased fusion efficiency with the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein ratio in evs. additionally, hev had higher loading efficiency than liposomes and sole evs and that their internalization by endothelial cells did not cause a cytotoxic effect. summary/conclusion: based on cryo-tem and fret, we confirmed that our fusion method of hybrid evs is effective and can be applied as a delivery platform for dds to endothelial cells. response to a range of stressors. the functional activity of these evs in recipient cells may, in part, be driven by changes to their biological cargoes. however, the molecular details of the underlying ev biogenesis and loading processes, and how this may vary in different conditions, is poorly understood. methods: we first studied the effect of oxidative stress on the functional activity of evs in recipient cells using cell viability and mitochondrial membrane potential assays in drosophila s2r+ cells. we then carried out total rna sequencing of ev and cellular rna under three stress conditions and compared results to existing data in mouse cells. further to this we have used a bioinformatic pipeline to identify sequence motifs enriched in evs under stress. results: functional assays indicated changes to cell viability and mitochondrial membrane potential in recipient cells, which were donor cell-stress dependent. subsequent characterisation of rna showed an enrichment of ribosomal rna in evs relative to cells, but no significant changes to other biotypes. comparative analysis has also uncovered a set of genes enriched in evs under oxidative stress, and a further subset whose enrichment may be evolutionarily conserved in mouse. we also identified potential ev-loading motifs which may assist in rna loading specifically under stress. summary/conclusion: we have shown that evs derived from oxidatively stressed cells show dose-dependent differences in rna cargo and identified potential sequence motifs that may have a role in its loading. we are now validating the biological significance of these findings by combining different in vivo approaches in drosophila. this will enable us to gain insights into the basic mechanisms which govern ev loading in different contexts, and ultimately the molecular mechanisms underlying ev-mediated intercellular communication. ishai luz a , bibek bhatta a , kanaga sabapathy b and tomer cooks c a ben-gurion university of the negev, beer-sheva, israel; b national cancer centre, singapore, singapore, singapore; c ben-gurion university, beer sheva, israel introduction: mutations in tp53 are considered one of the most frequent genetic alterations in human cancer. besides the abrogation of the wild-type (wt) p53-mediated tumour suppression, a distinct set of missense mutations was reported to endow mutant p53 proteins with novel activities termed gain-of-function (gof). even though mutations in tp53 are typically thought to arise in the tumour cells rather than in the stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment are poorly understood. in the presented studies, focusing on colon cancer as well as on lung cancer microbiome, we investigated intercellular interactions mediated by exosomes and outer membrane vesicles (omvs) in the context of cancers harbouring mutant p53. methods: p results: in the colon, tumour cells harbouring mutp53 were found to exert a non-cell-autonomous effect over macrophages. when exposed to tumour cells harbouring mutp53, monocytes became polarized towards a distinguished subset of macrophages characterized by tams-related markers. the mutant p53 affected tam were characterized as tnf-αlow/il-10 high, over expressing cd-206 and cd163, with decreased phagocytic ability and increased invasion and matrix degradation potency. investigating the exosomal transfer from mutp53 tumour cells to macrophages, revealed a mutp53-specific mirs signature led by mir-1246 promoting the tam phenotype and creating an invasive front together with tumour cells. mir-1246 was also found to be the top mutp53-associated mir in a cohort of 57 human colorectal resected tumours. separately, in two lung cancer cohorts, we identified a signature of microbiome members associated with p53 mutations. acidovorax temperans, a gram negative bacterium, was found to be abundant in tumours of patients with mutant p53. we found a significant increase in tumour volume in animals inoculated with acidovorax temperans as compared to sham treated animals, and increased lung weight as a percent of total body weight. these preliminary data indicate that acidovorax temperans contributes to lung tumorigenesis in the presence of activated k-ras and mutant p53. omvs shed by acidovorax temperans promoted inflammatory signalling in lung carcinoma cells and elevated cd47 expression on tumour cells and sirpα levels on macrophages. summary/conclusion: altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" gof p53 mutants tightening the interaction between the tumour cell and the immune compartment in colon cancer. in both colon and lung cancer, mutant p53 facilitates cellular interactions within the tumour microenvironmets mediated by vesicles. funding: intramural funding from the national cancer institute, national institutes of health. lori zacharoff and mohamed el-naggar university of southern california, los angeles, usa introduction: the metal respiring bacterium s. oneidensis creates outer membrane extensions and outer membrane vesicles that are sculpted by the novel bar domain protein bdpa. these vesicles and extensions incorporate mutliheme cytochromes involved in extracellular electron transfer to metals and electrodes. however, the physiological relevance of incorporating these cytochromes into the higher order 3d architecture of a vesicle or extension is unknown. given that bar domains serve as a protein sorting mechanism in eukaryotes, we investigated the pathway crosstalk between bdpa and outer membrane multiheme cytochromes as means to understand the physiological significance of membrane architecture. methods: o this end, vesicle morphology and content was measured using dry weights, dynamic light scattering, fluorescence microscopy and comparative proteomics from wild type s. oneidensis and deletion strains. results: cells lacking bdpa make large amorphous vesicles that are dense with protein. in contrast, a strain lacking outer membrane cytochromes recruits less total protein into smaller vesicles. proteomics to show that both bdpa and multiheme cytochromes are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal relationship. summary/conclusion: in the absence of bdpa, protein crowding has to become the main driving force of vesiculation and bdpa is essential for efficient incorporation of cytochromes. however, multiheme cytochromes are not only vesicular cargo, but are also important for shaping and loading vesicles. both of these situations make it clear that vesicles play a role in increasing the respiratory surface area of s. oneidensis cells. moving forward, we hope to be able to control bdpa and cytochrome levels for selective recruitment of technologically relevant payloads. introduction: fascioliasis caused by fasciola hepatica represents a major economic loss and clinical burden in cattle farming worldwide. extracellular vesicles (evs) contain pathogen-derived molecules that represent novel biomarkers of disease. in the present study, we have identified potential new biomarkers of f. hepatica infection in evs present in sera of infected cattle. methods: parasites and sera were obtained from local abattoirs (valencia, spain, and medellin, colombia, respectively). 38 sera from infected and 32 from healthy animals. parasites were cultured, and evs obtained by sizeexclusion chromatography (sec) and characterized by nta, tem and proteomic profiling. recombinant proteins from f. hepatica evs (enolase and fh16.5 tegumentary protein) were produced, and coupled to magnetic beads. measurement of bovine igg antibodies was performed using luminex bead array technology. results: a total of 239 proteins were identified associated with evs as shown by the presence of typical ev-markers (tsg101, alix, cd63). two parasite proteins, enolase and the fh16.5 tegumentary protein were produced as recombinant proteins and used for detection of cattle igg employing luminex bead array technology. interestingly, significant differences were found in the fluorescence values of both recombinant proteins allowing discrimination between sera from infected and non-infected cattle. the use of the fh16.5 protein generated a highly significant difference between the two groups (p value = 0.003614); as did enolase (p value was 0.02294). summary/conclusion: this study demonstrates the usefulness of ev proteins as new biomarkers for early diagnosis of helminth infections using multiplex assays, a technology that may also be applied to other parasite ev molecules. life stage-specific glycosylation of schistosome-derived extracellular vesicles introduction: glycans play an essential role in pathogen-host interactions. larvae and adult worms from schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (es) products. extracellular vesicles (evs) are also among the es products. we recently found that schistosomuladerived evs are glycosylated and bind human dendritic cells via c-type lectin receptor (clr) dc-sign, leading to increased il-10 and il-12 release. here we investigated the glycosylation profile of evs released by s. mansoni adult worms, compared this to schistosomula evs, and addressed how this may affect parasite-host interactions via clrs. methods: evs from cultured s. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. isolated evs were analysed by nta and cryo em. n-glycan and lipid glycan content was determined by mass spectrometry. density gradient fractions with evs were loaded onto sds-page gels followed by western blot (wb) analysis using anti-glycan monoclonal antibodies (mabs). results: cryo em showed that adult worm evs lacked the long thin filaments that are characteristic for schistosomula evs. additionally, in contrast to schistosomula evs, glycolipids could not be detected in the adult worm evs. mass spectrometry analysis showed that the most abundant n-glycans in the adult worm evs contained galnacβ1-4glcnac (lacdinac, ldn) motifs, which correspond to previously published overall glycan profiles of this specific life stage. other differences in ev glycosylation between the two life stages were observed by wb using anti-glycan mabs: adult worm evs showed a paucimannosidic glycan motif whereas in the schistosomula evs galβ1-4(fucα1-3) glcnac (lewis x) was detected in line with previous ms analysis. introduction: phloem plays a central role in plant function, as it is the responsible for the translocation of photoassimilates from source-to sink-organs, and a long-distance route for signals distribution. due to the sap high nutrient content, sieve elements are primary target for plant pathogens and pests. in this work we aimed to isolate and characterize extracellular vesicles (evs) from cucumis melo phloem sap, derived from plant either exposed or not to the melon aphid, aphis gossypii (hemiptera: aphididae). methods: phloem exudates from 5-week-old melon plants, either uninfested or infested with adults of a. gossypii (n = 15, 4 replicates each), were collected by cutting the stem with a sterile razor blade between first and second expanded leaf from the top. evs were isolated by size exclusion chromatography, and analysed by nanoparticle tracking analysis (nta) and transmission electron microscopy. evs proteome was determined by quantitative mass spectrometry. results: evs from phloem sap were successfully isolated in every condition. no significant differences were detected among distinct samples, neither in particle concentration and size by nta, nor in protein concentration. most importantly, a total of 381 different proteins were identified in phloem sap evs, including 152 present in exosome databases (exocarta). on top of that, 44 differentially expressed proteins were identified in evs derived from aphid infested or uninfested plants (p value < 0.05). summary/conclusion: understanding how plants trigger their defences against pests and pathogens is important to develop new control measures. the characterization of several proteins in evs from the phloem sap provide valuable information on long distance signalling in plants. moreover, as plants lack an immune system comparable to animals, the different protein content in phloem sap evs after exposure to aphids could indicate their important role in delivering inducible defences against invading pests and pathogens. extracellular vesicles from nematode species heligmosomoides bakeri and trichuris muris contain distinct small rnas that could enable niche specificity in the host introduction: gastrointestinal nematodes are extremely prevalent parasites that infect most animals and 24% of human population. their success as parasites is attributed to their ability to secrete diverse molecules that modulate the host immune system. extracellular vesicles (evs) are one of the immune modulatory compounds they release that directly modulate host cells. our goal is to understand how the small rna (srna) cargo underpins ev function, using a comparative analysis of ev cargo from diverse nematode species. methods: we first compared how different ev isolation methodologies (ultracentrifuge (uc), size fractionation, sucrose gradient floatation) effect the small rnas detected in h. bakeri evs using different library preparation kits (cleantag, truseq), with or without polyphosphatase treatment. we then compared this to small rna libraries from t. muris evs using comparable methods, uc ev purification, with or without polyphosphatase treatment and using the cleantag library preparation kit. results: evs from both species contained mirnas, however the mirna gene familes in h. bakeri and t. muris evs are distinct. the mirna content detected in ev samples collected by different purification protocols is robust. the largest difference in detected mirnas was found when comparing different library preparation kits. although both h. bakeri evs and t. muris evs were dominated by srnas derived from intergenic or repetitive elements in the parasite genomes, only in h. bakeri evs were these secondary sirnas. summary/conclusion: h. bakeri and t. muris evs contain distinct small rna cargos, which may underpin their ability to colonise different host niches, and/or modulate the host immune system differently. t. muris evs do not contain secondary sirnas, in contrast to h. bakeri, however they are dominated by srnas derived from intergenic or repetitive regions. comparative analysis of helminth evs could help pinpoint the srnas involved in cross-species communication. please provide any keywords if applicable.: nematode, cross-species communication, small rna introduction: extracellular vesicles (evs) are secreted from various cells including cancer cells and known to contain protein and small rnas including mirna isoforms (isomirs). therefore we also focused on isomirs including other small non-coding rnas for biomarker discovery. although liquid biopsies using small rnas are promising biomarkers for early detection of cancer, current approaches to detecting and analysing mirnas in the blood are still inadequate. artificial intelligence (ai) data analysis may provide better algorisms for diagnosing cancer. methods: small rnas were isolated from serum or purified evs using a mirneasy mini kit (qiagen) and quantified by using the ion s5™ next-generation sequencing system. (thermo fisher scientific). evs were purified using total exosome isolation reagent (invitrogen™). ai data analysis was performed using jmp® genomics and datarobot enterprise ai platform. results: three small rnas, isomir of mir-21-5p, mir-23a-3p, and trf-lys (ttt) were significantly upregulated in breast cancer patients compared with the healthy cancer-free individual. the combination algorithm using these three small rnas allows for a more accurate diagnosis of the area under the curve (auc) 0.92. to test the possibilities that these small rnas are derived from cancer cells, we isolated evs from the serum and performed ngs analysis to profiled serum small rnas in evs. interestingly we found that two small rnas, mir-21-5p and mir-23a-3p, also high in breast cancer evs, indicating that these small rnas were expected to be derived from cancer cells. in oesophagus cancer, we also performed ngs analysis and identified twenty-four mir/isomirs candidates for diagnostic biomarkers. a multiple regression model selected mir-30a-5p and two isomirs (mir-574-3p and mir-205-5p) . the auc of the panel index was 0.95. we also performed ai data analysis and discovered the novel algorisms that can diagnose breast and oesophagus cancer more accurately. summary/conclusion: we demonstrated combinations of circulating non-coding rnas containing evs potentially useful for the detection of early-stage breast and oesophagus cancers. in addition, the datarobot enterprise ai platform enables us to the more accurate diagnosis of cancers at the early stage. identification of novel ev-associated mirnas as toxic biomarkers in mouse introduction: recent findings reveal that extracellular vesicles (evs), secreted from cells, are circulating in the blood. evs are classified into exosomes (40-120 nm), microvesicles (50-1,000 nm) and apoptotic bodies (500-2,000 nm). evs contain mrnas, micrornas, and dnas and have the ability to transfer them from cell to cell. recently, especially in humans, the diagnostic accuracy of tumour cell type-specific evs as biomarkers is more than 90%. in addition, micrornas contained in the evs are being identified as specific biomarkers in blood for chemical-induced inflammation and organ damage. therefore, micrornas contained in the evs released into the blood from tissues and organs in response to adverse events such as chemical substances and medicine are expected to be useful as novel biomarkers for toxicity assessment. in this study, we aimed to identify target organs by comprehensive analysis of ev rnas in the blood of mice after chemical exposure to establish a highly sensitive "next generation type" toxicity test for chemical substances and medicine using ev rna in blood as a biomarker. methods: all animal studies were conducted in accordance with the helsinki declaration and the guidelines approved by the animal care committee of the national institute of health sciences. c57bl/6 j male mice (12 weeks) were orally dosed with ccl4 (vehicle, 7, 70 mg/kg). serum were separated from blood after 2, 4, 8 and 24 hours after ccl4 administration. the serum was centrifuged at 10,000 x g to remove cellular debris and subsequently ultracentrifuged 100,000 x g. the pellet is resuspended in pbs and ultracentrifuged 100,000 x g again. the comprehensive small rna-seq of collected evs were performed according to the manufacture's protocols. results: we succeeded in isolating more than 10 novel small rnas, which could be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride (ccl4: 7 mg/kg & 70 mg/ kg). well known hepatotoxicity biomarkers, mirna-122 and mirna-192 were upregulated more than 1000-fold in the administration of 70 mg/kg ccl4, but not responded in the administration of 7 mg/kg ccl4. summary/conclusion: these results suggest that mir-122 and mir-192 are mainly released from liver to blood directly only in the administration of 70 mg/kg ccl4, while novel more sensitive hepatotoxicity biomarkers which responded in the administration of both 7 mg/kg and 70 mg/kg ccl4 should be included in the ev. our novel biomarkers will accelerate a rapid evaluation of chemical substances and medicine in nonclinical safety evaluation. introduction: advancements in sequencing technologies have allowed analysis of the genomic landscape of cancer using circulating cell-free(cf) dna. however, cfdna does not originate only from tumour cells. we recently demonstrated that most of the dna circulating in plasma of cancer patients is associated with large evs (l-evs), and that l-ev-associated dna reflects genomic aberrations of the cells from which l-evs arise. since l-evs are specifically released by tumour cells, we explore their potential to report cancer-specific genomic alterations in patient plasma and compare it to cfdna. methods: differential ultracentrifugation, tunable resistive pulse sensing, qubit dsdna high sensitivity assay, capillary electrophoresis, whole exome sequencing (1500-2000x), targeted sequencing (qiaseqtm), flow cytometry. results: we show here that l-evs in the size range of >1 micrometre are present exclusively in plasma obtained from cancer patients and absent in plasma from healthy donors. in agreement with this finding, double-stranded(ds) dna is detected only in l-ev fractions of patient plasma and not in those obtained from healthy donor plasma using the same protocol. we also demonstrate that the fragments of dsdna associated with circulating l-ev are larger in comparison with cfdna (>10,000 bp versus~170 bp). a large-scale analysis of l-ev dna obtained from plasma of patients with metastatic castration-resistant prostate cancer (mcrpc) as well as with non-small cell lung cancer (nsclc) demonstrates that dna associated with circulating l-evs reports cancer-specific genomic alterations in both types of cancer. we further investigate if l-evassociated dna is intra-or extravesicular and demonstrate that it is present in both forms. we finally compare the purity of the tumour signal in intravesicular l-ev dna, total l-ev dna, and cfdna obtained from patient plasma. summary/conclusion: our results demonstrate that circulating l-evs contain high quality, large molecular weight dna that contains cancer-specific genomic alterations, supporting the use of l-evs as a source of tumour-derived dna in plasma. introduction: epidermal growth factor receptor (egfr) mutation driven lung adenocarcinoma (ac) represents a unique subgroup that lends itself to treatment with oral egfr tyrosine kinase inhibitors. current methods that are used to detect these mutations (e.g. l858 r or the resistance mutation t790 m) involve invasive tumour biopsies or blood circulating tumour dna (ctdna) and cell free dna (cfdna). the sensitivity of blood ctdna and cfdna is limited by the frequency of genomic alterations in the egfr gene; additionally, ctdna does not reflect changes in the egfr protein, against which novel therapies are in development. there remains a need to develop bloodbased biomarkers that can circumvent these disadvantages and replace the more standard, invasive tumour biopsies. we propose the study of exosomes for treatment monitoring as well as to identify egfr resistance related genomic and proteomic changes. methods: we enrolled 10 patients with metastatic lung ac: 8 with egfr mutations and 2 without (control). from the patients with egfr mutant lung ac, we processed 25 blood samples through the patients' treatment course, using ultracentrifugation to isolate exosomes. we then used both droplet digital pcr (ddpcr) to test exosomal rna (exorna) for the mutation of interest and western blots to test protein resulting from exon 19 deletion or l858 r mutations. results: from 6 patients with egfr exon 19 deletion mutations, we detected identical mutations in exorna from 15/19 samples. exorna based mutational load increased and mirrored clinical progression in 2 patients. three patients whose cancer remained stable demonstrated a decrease in their exorna. one patient had blood drawn only at 2 points and was therefore not plotted. exorna from 2 patients with l858 r and t790 m mutations demonstrated the corresponding mutations; however, exorna did not mirror their disease course. we also demonstrated mutant egfr protein presence in exosomes from 3 patients. finally, we tested cfdna for egfr mutations from four matched samples using ddpcr. we detected matched mutations in exosomes in all four, while cfdna mutations were only detected in 2/4 patients. summary/conclusion: in summary, we detected egfr mutations in 19/23 exosome samples isolated from metastatic lung ac. our results set the stage for optimization of exorna methods and inform future experiments relating to exosomal cargo in patients with egfr mutant lung ac. identification of plasma-derived, ev-based biomarkers for glioblastoma introduction: glioblastoma multiforme (gbm) is the most malignant and aggressive primary brain cancer in adults, with an incidence of 3.2 per 100,000 people. currently, diagnosis is only performed via histopathological investigation of a tissue sample from a gbm lesion, complemented with molecular diagnostics for identification of select biomarkers. mri is the standard of care for follow-up and monitoring of treatment response. therefore, development of a "liquid biopsy" to obtain disease-relevant information from patient's body fluids is highly desirable. methods: we present the results from a clinical study in which extracellular vesicle (ev)-derived mrnas and long non-coding rnas were profiled from the plasma of gbm patients and control individuals. we obtained plasma from 8 patients at the time of initial diagnosis, and 8 matched controls by sex and age. ev-associated rna was isolated from 1-2 ml plasma and rna-seq was performed using our proprietary pipeline. sequencing data was analysed for differential gene expression. results: we observed 95 mrnas as differentially abundant between gbm and control samples, with 82 mrnas enriched in gbm samples and 13 mrnas enriched in control samples (p < 0.05). correlation based on differentially abundant mrnas separated gbm and control samples into two unique populations. eight differentially expressed mrnas were previously identified as part of the mesenchymal gbm subtype. these data, while preliminary, provide a potential basis for the further development of a noninvasive gbm gene panel test. summary/conclusion: we have identified a novel rna signature for gbm from plasma derived evs, which differs from previously identified biomarkers isolated from tissue. further work will refine this signature to enable detection, characterization, and patient monitoring for gbm with minimally invasive techniques. introduction: sjogren's syndrome (ss) is a systemic autoimmune disease in which inflammation progressively damages the moisture producing glands of the afflicted. 4 million americans are estimated to be suffering from the disease, 90% of which are women with an average age of 40. overlapping symptoms with other health conditions and co-morbidities make ss particularly difficult to diagnose, with average time to diagnosis of 3 years. saliva exosomal rna profiling has been primarily focused on small rnas and has been limited thus far due to the large contribution of sequencing reads from the oral microbiome. a noninvasive saliva exosomal rna (exorna) based test capable of diagnosis would be highly desirable. methods: we began by first developing a novel long rna-seq workflow to selectively enrich and profile human exosomal mrnas and long non-coding rnas (lncrnas) from saliva. we then profiled salivary exorna obtained from 7 ss patients and 7 healthy matched controls. finally, we performed differential gene expression analysis to obtain an exorna signature for ss. results: rna-seq data analysis demonstrated highly efficient enrichment of human transcriptome, with over 80% of reads mapping to the transcriptome. further rna biotype analysis showed over 75% of transcriptome reads mapped to protein coding genes and lncrnas. we detected over 10,000 mrnas and approx.1000 lncrnas. differential expression analysis (dex) of ss vs. healthy control exorna identified 455 upregulated genes, including 415 mrnas and 37 lncrnas (p < 0.05). 120 genes were found to be downregulated in ss, including 114 mrnas and 6 lncrnas. gene ontology analysis of dex genes revealed enrichment of genes involved in various immune system related pathways. most importantly, principal component analysis (pca) resulted in clear separation of ss patients from healthy controls. summary/conclusion: our optimized rna-seq workflow enables saliva-based liquid biopsy for biomarker discovery. the gene signature identified in this ongoing study could potentially provide a non-invasive molecular means of diagnosing sjogren's syndrome. introduction: increasing embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. usually an endometrial biopsy is done to identify a receptive endometrium, which prevents embryo transfer in the same cycle, as it is detrimental for the implantation. the implantation is a complex process, which requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial cross talk. the presence of extracellular vesicles (evs) as mediators of this communication has been describe in the endometrial fluid. therefore, we hypothesize that the molecular analysis of the content of the evs and companion molecules from endometrial fluid could be a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. methods: the objective is to define a simple, sensitive and reproducible non-invasive ev-based method that allow the quick identification of an implantative endometrium by means of mirna analysis. for the establishment of a robust methodology for analysing evs from endometrial fluid in clinical settings, where the sample is limited and no sophisticated equipment is available, five different methodologies were compared in triplicate. two of them consisted in the direct extraction of rna while in the other three, before the rna extraction an enrichment of evs was done. smallrnaseq was performed to determine the most efficient method. once the best method was selected, it was applied in a set of real samples with different implantation outcome. the content of mirnas (mainly associated with evs) of endometrial fluid samples from women in whom the implantation was successful (n = 15) and unsuccessful (n = 15) were analysed. results: our results show that the protocols with a previous enrichment step of evs obtained a higher mirna expression. the results obtained from the differential analysis of the set of samples with different implantation outcome are being analysed and it is expected that the results will be available by the time this communication is presented. summary/conclusion: this work demonstrates that it is possible to obtain and analyse evs and evs-associated mirnas from a small volume of endometrial fluid samples, which allows the use of ev-mirnas as a low-invasive biomarkers for the detection of an implantative endometrium. funding: jip is supported by a predoctoral grant from the basque government. small rna cargo of evs is affected by hormone treatment in prostate cancer introduction: small rnas are recently reported as a regulator for prostate cancer progression to castration-resistant disease. our previous work has shown that evs protein cargo is affected by male steroid hormone, dihydrotestosterone (dht). in this study, we assess the small rna cargo of evs in response to androgen manipulations. methods: androgen receptor-positive lncaps are grown in css medium to deplete the androgens. media were then replaced with vesicle-depleted css medium ± 10 nm dihydrotestosterone (dht) ± 10 µm enzalutamide (enz) for 48 h. evs were isolated using sequential ultracentrifugation (2000 g for 20 min, 10,000 g for 30 min, 100,000 g for 2 h), washed once in pbs. protein and rna were collected from both parent cells and conditioned medium to allow direct comparison between s-evs cargo and cells. small rna ngs libraries were prepared using the illumina's truseq small rna library prep kit and single-end sequenced at a read length of 50 nucleotides (nt). fastq library files were processed using a custom-designed pipeline. adapters were removed using the cutadapt tool, trimmed reads were mapped with high stringency against ribosomal sequences using bowtie2. snorna and trna fragments were identified using the flaimapper software. remaining reads were mapped against the human genome hg38 using bowtie2. results: we found that the presence or absence of androgens does not significantly change the amount of total rna in small evs (s-evs). however, hormone stimulation altered the small rna content of s-evs, in parallel with our previous published data on ev protein cargo. dht increased the abundance of snorna in cells, while a reduction of snornas was observed in the s-evs fraction. interestingly, dht induced the formation of cell filopodia that are not inhibited by androgen inhibitor enzalutamide. pathway analysis indicates the p53 mediated regulation driven by mirnas found in s-evs upon exposure to dht. the expression profile of snorna and trna fragments in dht treated cells resembles results from clinical prostate cancer specimens. summary/conclusion: our findings show that androgen manipulation alters both s-ev derived protein and rna cargo. changes in the s-ev rna profile due to treatment with androgens are not identical to small rna profiles in parental cells, indicating a specific sorting mechanism of s-ev small rna upon androgen manipulation. further, dht induces the formation of cell filopodia irrespective of enzalutamide, suggesting cargo selection of s-evs. we conclude that small rna ev cargo can be utilised to as prostate cancer biomarkers in androgen targeted treatments. introduction: cancer immunotherapy, such as pd-l1 blockade, is a method to eliminate cancer cells. ectopic expression of pd-l1, on the surface of tumour cells, has been associated with tumour persistence and as an important predictor of therapy response. a test that, specifically and accurately, detects pd-l1 is critically important in order to identify patients that would benefit from these treatments. emerging evidence has shown that extracellular vesicles (ev) can carry immune checkpoint molecules, such as pd-l1, and whose expression have been correlated with tumour immunity response. with a multitude of commercially available antibodies identifying appropriate clones and associated assay is important in order to standardize the diagnostic modality used. methods: pd-l1 expressing cancer cell lines were used to generate evs. pd-l1-myc vector was transfected to generate an overexpression system. exoview® sensors containing different anti-pd-l1 clones were generated. samples (cell derived and plasma) were incubated on chips to allow the antibody to bind the antigen on the ev. after incubation, chips were immunolabeled with fluorescently labelled antibodies against pdl-1 or ev associated markers. exoview r100 reader was used to enumerate the evs captured on the sensor surface and analyse the expression of pdl-1 on single vesicle through fluorescence imaging. immunoprecipitation and mass spectrometry (ip/ms) were employed as an orthogonal method to verify the specificity of the assay. results: to study the detection efficiency of the antibodies, engineered pd-l1-myc evs were used. under these circumstances, all the tested antibodies were able to capture evs. when testing endogenous pd-l1 positive evs from different cancer cell lines, only 28.8 and 73 10 clones consistently bound to evs. in addition, evs derived from plasma demonstrated to be positive for pd-l1, however, only clone 28.8 was able to immobilize these evs. the results suggested that clone 28.8 could be a potential pd-l1 antibody to detect pd-l1 positive evs originating from various sources. to confirm these results, and assure the specificity of the antibody targeted ip/ms was employed. summary/conclusion: in combination with the exoview platform, anti-pd-l1 antibodies can be screened and potentially used to generate a non-invasive ev-specific assay that could detect this protein in patients. differences in extracellular vesicle protein cargo is dependent on head and neck squamous cell carcinoma cell of origin university of michigan, ann arbour, usa introduction: head and neck squamous cell carcinoma (hnscc) is the sixth most common, eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity. in 2017, there were over 49,000 new cases and 9,700 deaths estimated in the usa alone. despite recent advances in treatment, including radiation, chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours develop resistance and progress. patients develop metastases or tumours recur locally or regionally; the 5-year overall survival rate for hnscc is only 40-50%. factors that contribute to poor survival for patients with hnscc include late stage diagnosis, lack of reliable markers for early stage detection, high level of biologic heterogeneity, and local recurrence and distant metastases after treatment. methods: this study used 8 representative hpv-positive and hpv-negative hnscc cell lines, one hpvtransformed cell line. and two non-cancer oral keratinocyte cell lines. evs were isolated using differential ultracentrifugation and peg precipitation/ultracentrifugation. evs were characterized by tem, nta, and wes protein analysis for reported ev markers. ev and whole cell lysates were assessed by lc-ms/ms analysis using the tandem mass tag-10plex kit. cluster analysis was performed on the fold-change peptide spectrum matches (psm) for the evs from the hnscc lines compared to the evs from the normal keratinocyte line (noksi). protein was measured using a capillarybased electrophoresis instrument. results: cd9 and annexinv were detected in all of the ev lysates tested, while calnexin was detected in all of the whole cell lysates and none of the ev samples tested. selected proteins stat3, hla-a, tenascin, e-cadherin, β catenin, cytokeratin 19, epha2, and cd59, and hpv-related markers p16, p53, rb, cyclin d1, and egfr were tested using the wes platform. evs from hpv-positive cell lines showed higher protein levels compared to evs from hpv-negative cell lines in stat3, hla-a, and tenascin. only kert19 demonstrated lower protein levels in evs from hpvnegative cell lines. of the common hpv-associated hnscc markers: egfr, p53, rb, cyclin d1 and p16, only egfr was positive in any the evs tested. the remaining proteins queried, e-cadherin, β catenin, epha2 and cd59 showed varying protein levels in evs from both hpv positive and hpv-negative cell lines. summary/conclusion: our findings suggest that these proteins may be potential hnscc ev markers that may be 1) selectively included in ev cargo for export from the cell as a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment, or 2) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. validation of antibodies on western blot for extracellular vesicles from biological human samples and cancer cell conditioned media the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: one of the major challenges in extracellular vesicles (evs) research is to prove the particles that are isolated are true evs, rather than other co-isolated contaminants, like lipoproteins. isev recommends using multiple assays to characterize evs. this study aims to validate the positive and negative protein markers for extracellular vesicles from plasma, urine and prostate cancer cell conditioned media (ccm). methods: membrane and cytosolic fractions of mcf7 cells served as positive and negative controls for all antibodies validated. evs were isolated from plasma of healthy volunteers, urine of healthy volunteers and ccm of pc-3 cells using differential ultracentrifugation. eight protein markers were assessed: positive markers cd63, cd81, cd9, flotillin1 (flot1), alix and tumour susceptibility gene 101 (tsg101), negative marker calnexin (canx), and contaminant markers apo-a1 for plasma and thp for urine. tetraspanins are small transmembrane proteins expressed in evs. flot1 is membrane protein that forms microdomains in the plasma. alix and tsg101, an accessory protein of the endosomal sorting complex required for transport, are involved in the biogenesis of evs. they are positive markers for evs. canx is in the membrane of the endoplasmic reticulum. apolipoprotein-a1 (apo-a1) is the protein components of lipoproteins, therefore it is marker of contamination for plasma ev. tamm-horsfall protein (thp) is contamination marker for urine ev, because it is most abundant protein in human urine. results: all antibodies were validated in the correct positive and negative control, thus confirmed as usable and reliable antibodies for western blot. in plasma ev, cd63, cd9, cd81 and flot1 were positive and canx and apo-a1 were negative. in urine evs, cd9, cd63, flot-1, alix and tsg101 were positive and canx and thp were negative. in ccm evs, cd9, cd63, flot1, alix and tsg101 were positive and canx was negative. summary/conclusion: we confirmed a high degree of ev purity from 3 sample types: urine, plasma, and ccm. of particular importance, we confirmed that evs isolated from biologic patient samples, plasma and urine, had low contamination. future work will use these methods to confirm purity of ev samples prior to addition analysis, such as examining ev cargo and biologic significance. proteomic study of mesenchymal stem cells derived exosomes modified using mir. introduction: the project we are working on is to modify the immunogenic profile of human cmms from the umbilical cord stroma through its stable transfection with anti-mir-21-5p, and therefore of the exosomes that these cells generate, for use in free-cell therapy to treat inflammatory process. methods: evs released from a primary culture of human umbilical cord mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal stem cells mir21-/-modified through stable lentiviral transfection were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (tem) and measured by nanoparticles tracking analysis (nta). protein extraction from evs was made using ripa buffer and after checking protein integrity the total ev proteins. we performed a shotgun proteomic study using a tmt (10-plex) label of the total mir21-/-exosomes protein comparing it with normal exosomes. after labelling the ltq-orbitrap platform of proteored was needed for fraction injections and data acquisition. proteome discoverer 2.2 (thermo) was used for protein processing and quantification. results: a total of 1.861 proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of mir21-/-exosomes against normal exosomes. we found out several protein modulated by mir21 and related to inflammation. summary/conclusion: we have able to establish the proteomic profile of mir21-/-exosomes against normal exosomes focusing on proteins involving inflammation process. all those results seem indicate that exosomes could be modified, which could be used as an anti-inflammatory free-cell therapy. funding: proteored concept test project grant. a novel extracellular vesicle isolation method used to discover urine liver disease biomarkers introduction: hepatocellular carcinoma (hcc) is the 6th most common cancer worldwide and the 3rd most common cause of cancer death; additionally, its incidence is increasing. while outcomes for early hcc are superior to those for late stage disease, early detection of hcc remains a challenge. current guidelines have suboptimal sensitivity and specificity. in this pilot study, we hypothesize that urine extracellular vesicles (evs) may identify candidate biomarkers towards the development of an inexpensive, widely accessible screening assay for the early detection of hcc. methods: urine samples from 20 healthy subjects, 19 subjects with cirrhosis, and 19 subjects with cirrhosis plus hcc were collected and processed using ymatrix columns to isolate ev-associated protein and mirna. protein was analysed using a tandem mass tag method on a thermo scientific orbitrap fusion mass spectrometer with comet/paws and edger processing. mirna was analysed using a targeted firefly microarray from abcam. differential expression and predictive modelling for the presence of hcc and cirrhosis was performed to identify candidate mirna and protein biomarkers. results: for mirna, 39 samples were eligible for analysis after low expression filtering. we used pair-wise ratios of 34 cancer-associated mirnas by gradient boosting of decision trees to develop a predictive model for hcc. our best model had a sensitivity and specificity of 0.93 and 0.92 respectively using 6 mirnas to distinguish hcc from cirrhosis. all samples were eligible for protein analysis. based on differential expression and biologic relevance, we identified 10 protein candidate biomarkers. interestingly, we found liver-selective proteins and known hcc/cirrhosis plasma/tissue markers, demonstrating proof-of-concept for the method. summary/conclusion: urine extracellular vesicles contain liver-selective proteins and known liver disease serum biomarkers as well as novel mirna and protein biomarkers that are significantly up-regulated in disease samples. the described candidate biomolecules may be easily accessible biomarkers with which to develop a sensitive and specific universal screening diagnostic for the early detection of cirrhosis and hcc. introduction: the peptidergic g-protein coupled receptors (gpcrs) are cell-signalling transmembrane proteins, which in their native form comprise of seven segments embedded in the cell membrane. this structural advancement is believed to be maintained in extracellular vesicles (evs). in autoimmune diseases, the presence of autoantibodies towards gpcrs is not uncommon, and to detect plasma autoantibodies, evs carrying gpcr will be used as template in a novel microarray screening tool. methods: purified evs from hek293 cells were printed on different types of surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic 96 well plates. five different print buffers were tested in a multiplex assays. spots containing evs were stained with biotinylated antibodies (cd9, cd63, cd81, adrβ2, hsp70, epcam and flotilin-1) followed by binding of cy5-labelled streptavidin and visualized microarray scanner. results: the outcome of these experiments was promising, as some of the chosen printing buffers showed increased tendencies to bind evs. the ev presence was verified with a panel of markers known to be present on small evs. in addition, the ev content of the adrenergic beta-2 receptor (adrβ2-receptor), which is a gpcr of interest in autoimmune diseases, was verified in some of the experimental setups. summary/conclusion: the approach of using evs as template in a screening tool possesses the potential to easily screen for autoimmune illness markers in diagnostic purposes. using the microarray technology allows the screening to be multivariate, specific and highly sensitive. circadian variation of extracellular vesicles secreted in urine: analysis of time point collection and normalization strategy. introduction: urinary extracellular vesicles (uevs) are an ideal source of biomarkers for kidney and urogenital diseases. despite the great deal of interest generated by uevs, little is known about its collection time and normalization approach. the majority of the studies on uevs focus on spot urine collection based on the assumption that it accurately reflects the renal function, although time point of collection is not standardized. therefore the practice to collect spot urine does not allow for calculating and standardizing accurately the uev excretion rate which may vary during the day. in addition, no research has been carried out yet to show the quantitative and qualitative difference of uevs between spot urine and 24 h collections.the aim of this study is to compare uevs excreted in all single voids during a 24 hour collection period and compare it with 24 hour collection performed. methods: uevs were enriched by differential centrifugation and electron microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse sensing and imaging flow cytometry were used to quantify uevs and associated markers variation during the 24 hour. creatinine, urine osmolality and particle concentration were used to normalize the assessed analytes. results: electron microscopy showed a heterogeneous population of evs and western blot confirmed the presence of ev markers (tsg101, alix and cd9). rna was extracted by a column-based method (mirna extraction kit qiagen) and cel-39 mirna was spiked in each sample. a multiparametric detection of nephron markers podocalyxin, aquaporin-2 and uevs pan tetraspanins (cd9 + dc63 + cd81) was performed utilizing imaging flow cytometry. whereas the uev composition did not change across the 24 hours analysis, the quantity of uevs and related markers fluctuated during the day depending on the hydration and excretion rate.the results of a 24 hour urine collection reflected the average results of all single voids over a 24 hr period. creatinine and particle count normalization failed to normalize "outliers". summary/conclusion: this study represents the very first report which compares single void urine versus 24 hour uev analysis. we concluded that the 24 hour collection is the preferred choice for a robust and rigorous assessment of uevs and its associated markers. porcine body fluids differ in small extracellular vesicle counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle sources for proteomic analyses helena kupcova skalnikova a , jakub cervenka b , jaromir novak a , karolina turnovcova c , bozena levinska a , jana juhasova a , stefan juhas a and petr vodicka a a institute of animal physiology and genetics, czech academy of sciences, libechov, czech republic; b institute of animal physiology and genetics cas, v. v. i. libechov, libechov, czech republic; c analysis tools were used to identify in silico biological pathways and functions governed by detected mirnas. expression of putative targets of selected mirnas was tested using qpcr after in vitro delivery of uterine evs to ptr cells. results: careful characterisation confirmed that uterine lumen is enriched with a diverse population of evs caring mirnas. interestingly, 36 out of 79 detected mirnas showed difference in abundance between tested days of pregnancy and half of them was exclusively detected on d16. identified mirnas were characterized as potent regulators of cellular development, growth, proliferation, and movement, in addition to their involvement in organismal and embryonic development. the expression of 20 genes identified as a possible mirna targets was tested after evs delivery to ptr cells in vitro. both down-(e.g., ptger4) and up-regulated (e.g., lifr) genes were found (p < 0.05); involved in the same molecular and cellular functions enriched by detected mirnas. methods: evs were harvested from wild type and arrdc4-/-epididymal cells using differential ultracentrifugation, then characterised using nanoparticle tracking analysis and transmission electron microscopy. sperm motility was measured using computer assisted sperm analysis and imagej. fertilisation capacity was measured using the following assays: capacitation-associated tyrosine phosphorylation, calcium ionophore induced acrosome reaction, zona pellucida binding assay and in vitro fertilization with time-lapse imaging of embryo development. immunohistochemistry was also used to visualise two pronuclei formation and blastocyst morphology. arrdc4-/-sperm was supplemented with wild type evs in the above assays to assess whether they could restore function. results: sperm from arrdc4-/-mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and production of two-cell embryos. we observed a significant reduction in ev production by arrdc4-/-epididymal epithelial cells, and addition of wild type evs to arrdc4-/-sperm dampens the acrosome reaction and restores zona pellucida binding. introduction: gestational diabetes (gdm) is among the most common pregnancy complications. despite treatment, up to 25% of pregnancies complicated by gdm result in infants being born large-for-gestational-age (lga). this not only causes problems at birth but predisposes offspring to developing cardio-metabolic disease in adulthood. there are no treatments for lga as the cause is unclear, although it is associated with altered placental vascular development. micrornas (mirnas) regulate placental development; they are produced within cells but can be released into the circulation inside evs, which in turn can be transported into target cells and tissues to influence cellular processes. we aimed to characterise circulating evs in pregnancies complicated by gdm-lga and determine if ev-derived mirnas have the potential to influence placental development. methods: maternal serum and plasma samples were collected from women with pregnancies complicated by gdm at 24-32 weeks gestation; placental tissue was collected at delivery and birth outcomes recorded. serum and plasma evs were isolated and characterised by electron microscopy (shape), nanoparticle tracking analysis (nta; size/concentration), and western blotting (ev-enriched proteins). mirna qpcr arrays were performed on evs. mirnas were quantified in placental tissue via qpcr. results: em and western blotting confirmed isolation of evs and nta revealed no significant difference in size/ concentration in gdm-lga pregnancies (n = 7) compared to gdm-aga (n = 13; p > 0.05). several ev mirnas were altered in maternal circulation in gdm-lga compared to gdm-aga (n = 7/group; >twofoldchange; p < 0.05), including four skeletal muscle-specific "myomirs": mir-1-3p, mir-133a-3p, mir-133b, and mir-499a-3p (all increased). all four myomirs were present in placenta but only mir-1-3p was significantly altered in gdm-lga compared to gdm-aga (n = 12-14/group; p < 0.05). summary/conclusion: ev-bound myomirs could have predictive value for aberrant foetal growth in cases of gdm. mir-1-3p regulates vascular development in other systems, so we propose that mir-1-3p contributes to lga by influencing placental vascular development, however further work is required to establish this. introduction: seminal plasma is particularly rich in extra cellular vesicles. myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. the aim of this study was to look for the presence of myelinosome vesicles in human seminal plasma. methods: because of the viscosity of seminal gel and its water-holding capacity, classical transmission electron microscopy does not seem to be an optimal technique to reveal the presence of myelinosomes in this fluid. cryo-electron microscopy is a technique that allows visualization of nanosized structures without prior fixation or addition of heavy metals for contrast. the sample is therefore visualized as close to its native state as possible. using standard myelinosome preparation from tm4 sertoli cells, we first analysed the appearance of "standard" native myelinosomes by cryo em and then compared it with the vesicles from human seminal plasma samples. results: we have specified by cry-em the morphological aspect of "standard" myelinosomes isolated from the culture media of tm4 sertoli cells. the vesicles with the same morphological appearance were revealed in human seminal plasma specimens. summary/conclusion: myelinosomes are membranous organelles found in the seminiferous epithelium of the testis and secreted by the somatic sertoli cells in the lumen of the seminiferous tubules.the preparations from human seminal plasma contains a population of large ev (average diameter 200 nm) whose morphological appearance resemble those of myelinosomes. defining the specific biomarkers and functionalities of myelinosomes in human seminal plasma are the concerns to be addressed in our further research. introduction: more than one million patients worldwide suffer from tuberous sclerosis complex (tsc) and have mutations in either tsc1 or tsc2 genes. together, the tsc proteins regulate mtorc1 activity. all tsc patient post-mortem samples exhibit renal disease and 40% of patients with tsc experience a premature loss of renal function. mouse and human studies are incongruity with the second somatic hit mechanism of disease, because of the low percentage of cystic cells exhibiting loss of tsc expression. we posited that the loss of a tsc protein expression may alter extracellular vesicle (ev) biology and contribute to disease. methods: we used crispr/cas9 to disrupt the tsc2 gene in mouse inner medullary collecting duct (mimcd) cells, and isolated evs using gel filtration from the isogenic cell lines. we characterized the evs using tunable resistive pulse sensing (trps), dynamic light scattering (dls), transition electron microscopy (tem), and wester blot analysis. we further performed mass spectroscopy on the ev proteins. results: loss of the tsc2 gene in mimcd cells induced a greater than three-fold increase in ev production compared to the same cells having an intact tsc axis. electron microscopy confirmed the purity and spherical shape of evs. both trps and dls demonstrated that the isolated evs possessed a heterogenous size distribution. approximately 90% of the evs were in the 100-250 nm size range. western blot analysis using proteins isolated from the evs revealed the cellular proteins alix and tsg101, the transmembrane proteins cd63, cd81 and cd9, and the primary cilia-related hedgehog signalling-related proteins arl13b. proteomic analysis of evs identified a significant difference between the tsc2-intact and tsc2-deleted cells that correlated well with the increased production. summary/conclusion: evs may be involved in tissue homoeostasis and cause disease by overproduction and altered protein content. the evs released by renal cyst epithelia in tsc complex may serve as a tool to discover the mechanism of tsc cystogenesis and in developing potential therapeutic strategies. introduction: we have shown that evs derived from amniotic fluid stem cells (afsc) of mouse origin present therapeutic effect in an animal model of chronic kidney disease, alport syndrome (as). in light of clinical translation, we isolated afsc-evs of human origin, characterized their cargo and evaluated thier therapeutic effect in vivo. methods: human clonal afsc were derived from amniotic fluid collected after volunteer donors provided consent. evs were obtained from afsc and identity and purity were assessed by rna-seq and proteomics. potency of hafsc-evs was evaluated by performing in vivo studies. ev biodistribution was evaluated by mri and therapeutic effect by measuring renal function and mice life-span. bulk rna-seq was performed on glomeruli obtained from injected and non-injected mice to identify potential ev regulating targets. results: proteomic profiling identified 675 intact proteins and rna-seq data identified 2,535 mirs in hafsc-evs. hafsc-ev "fingerprint" was assessed by performing go analysis on the 100 most highly expressed proteins and mirs. the results identified pathways involved in tissue homoeostasis such as mtor pathway, tgfβ and vegf pathways. when injected in vivo into as mice, biodistribution studies showed that hafsc-evs localized in the kidney, corrected proteinuria. no side effects (including teratoma) were noted in the treated mice. rna-seq of glomeruli obtained from treated as mice showed similar gene expression patterns to wilt type mice, by cluster analysis. our data indicated that hevs highly modulated pathways involved in collagen and matrix deposition remodelling, in addition to downstream targets of vegf, fgf, tnf, angiotensin and preserved glomerular cells structure and function. summary/conclusion: our protocol for hevs derivation is reproducible and allows derivation of ev lots with the same identity (specific cargo of proteins and mirs) and potency (present therapeutic effect in as). hafsc-evs modulated signalling pathways that are central to maintaining glomerular homoeostasis and preserved glomeruli structure with improved kidney function. this suggests the possibility of using hafsc-evs as a new therapeutic option for treating renal failure in humans. introduction: recent studies have shown that stem cell-derived extracellular vesicles (msc-ev) therapy improves renal outcomes in models of acute ad chronic renal disease. however, to better investigate the molecular mechanisms of ev-induced regeneration, and to define new ev sources, devices that mimic 3d organ architecture and flow conditions are needed. the aim of our work is to evaluate the regenerative potential of naïve and engineered ev in a millifluidic in vitro 3d model of glomerular damage in continuous perfusion. methods: methods: we set a millifluidic in vitro 3d model of glomerular filtration, a three-layers structure composed by human podocytes and glomerular endothelial cells, and, in between, of a basement membrane of collagen type iv. the barrier thus formed is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. we reproduced different pathological conditions and tested the localization and effect of evs in a dynamic system. : results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit subject to continuous reperfusion. renal damage was induced by doxorubicin or by hypoxia-reperfusion injury. we evaluated uptake, cargo transfer and effect of naïve and mirna engineered msc-evs or of klotho engineered ineffective evs administered into the dynamic co-culture system. evs were able to pass through the system and to deliver to podocytes proregenerative factors, promoting survival and limiting permeability. introduction: worldwide, renal cell carcinoma (rcc) is 8th most common cancer in men and 10th most common in women. new biomarkers are needed to aid rcc-diagnosis, provide prognostic information, and to predict response to modern targeted therapies. extracellular vesicles (evs) are an emerging source of cancer biomarkers because all cells, including cancer cells, secrete evs into biofluids as blood and urine. however, benign cells contribute to ev populations isolated from blood and urine reducing the diseasespecificity. we have developed a protocol for ev isolation directly from human rcc tissue that can increase tumour-specificity of biomarkers. methods: we obtained technical and biological replicates from normal kidney tissue and clear cell rcc tissue. serum-free media was incubated with the specimens. a combination of differential centrifugation, filtration, and ultracentrifugation was used for ev isolation. evs were quantitated using two methods, allowing for comparison between nanosight ns300 and nanofcm. tem was used to determine presence of intact vesicles in the ev samples. presence of ev introduction: urothelial carcinoma (uc) is a malignant cancer that affects the urothelial cells, representing 90% of all bladder tumours. at diagnosis 75% of bladder cancers are non-muscle invasive tumours. importantly, upon transurethral resection of the bladder tumour, nearly 35-80% of these patients will experience disease relapse and 10-20% will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive follow-up. currently, this is performed through the frequent use of highly invasive cystoscopy and the low sensitivity urine cytology. thus, innovative liquid biopsy-based biomarkers that circumvent these drawbacks are highly desirable for improved uc clinical management. here, we aim to implement a protocol for the isolation and characterization of extracellular vesicles (evs) from uc patients' urine samples. methods: a two-step protocol involving ultracentrifugation (uct) and by size-exclusion chromatography (sec) was optimized for urine samples. the isolated urine-derived evs from 9 uc patients were then characterized according to their size, concentration (nta), morphology (tem), protein amount (lowry method), presence of ev-associated and disease-associated protein markers (western blot). results: isolated urinary evs from uc patients had a size ranging from 50nm to 400 nm with characteristic ev morphology, express ev-associated markers as cd63 and hsp70 and were negative for cell debris markers. the recovery yield and purity of isolated evs following each isolation technique was characterized. upon uct, sec was required to deplete most of the ev-associated thp and albumin protein contaminants. some disease-associated protein markers were highly enriched in isolated urinary evs compared to crude urine. summary/conclusion: taken together, these results indicate that a two-step ev isolation protocol was properly implemented and validated in uc patients' urine samples. notably, several ev-associated disease biomarkers were detected in the urine of uc patients. this ev-based liquid biopsy might provide the means for real-time monitoring of residual disease and relapse in uc patients. introduction: glioblastoma multiforme (gbm) is a very aggressive type of brain tumour. different gbm molecular subtypes (proneural, mesenchymal and classical) often co-coexist within the same tumour, with the mesenchymal subtype driving the tumour progression. recently, our lab demonstrated that the cargo of extracellular vesicles (evs) could mirror the molecular background of the gbm cells from which they were derived. altogether, we believe that gbm cell-derived evs can be directly involved in the expansion of the mesenchymal signature in tumours, thus supporting gbm aggressiveness. methods: non-mesenchymal (t98 & u138) gbm cells were "primed" using evs derived from mesenchymallike (u87 & ln18) gbm cells. ev-primed gbm cells were then co-cultured with their non-primed counterparts to determine whether the mesenchymal signature can "spread" from cell to cell via evs. effect on cell proliferation, migration and invasion (in hyaluronic acid hydrogels) was assessed following ev treatment and co-culture. the expression of mesenchymal gbm markers was measured by western blotting. further mass spectrometry analysis of cell and ev content was undertaken to describe potential underlying mechanisms. results: co-culture with ev-primed gbm cells significantly increased proliferation and hydrogel invasiveness of non-mesenchymal cells. interestingly, the stimulating effect of co-culture was even stronger on the proliferation of ev-primed gbm cells. moreover, further proteomic analysis revealed that expression of mesenchymal gbm markers such as cd44 was increased in non-mesenchymal cells following coculture. summary/conclusion: our data suggest that evs from mesenchymal gbm cells can be uptaken by gbm cells from different subtypes, thus stimulating tumour progression. overall, we think the present study provides with new insights for the understanding of gbm recurrence and the development of potential therapeutic strategies. introduction: triple-negative breast cancer (tnbc) is the most aggressive form of breast cancer. previously we reported that the heterogenous population of evs released from tnbc cells promotes the growth and aggression of recipient cells. here we investigated if, by using compounds proposed to inhibit ev release i.e. calpeptin and y27632 (to block those budding at cell membrane) and gw4869 and manumycin a (to block evs from mvbs), we could reduce the associated transmission of aggressive phenotype. methods: evs were separated from medium conditioned by tnbc cell line hs578ts(i)8, using a discontinuous optiprep density gradient, after the cells were treatment for 48 hrs with the compounds listed above. evs (pooled fractions 3-9 with a density range of 1.03-1.16 g/ml) were characterised by nta, bca, lipid assay, immunoblot, tem and flow cytometry. to investigate the functional effects of the evs released, proliferation and migration assays were performed on hs578t and mda-mb-468 cells using the ev to cell ratios of 1 × 105 evs/3x103 cells, 1 × 106 evs/3x103 cells, 1 × 107 evs/3x103 cells to evaluate doseresponse. ev-track id ev190109 (score of 88%). results: gw4869 significantly (p = 0.035) decreased ev release from hs578 ts(i)8 cells. manumycin a and a combination of calpeptin and y27632 (combo) decreased ev release, but significance was not reached. conversely, calpeptin and y27632 actually increased ev release; but not significantly. of the reduced numbers of evs released following gw4869 treatment, hla-dr+ evs were significantly (p = 0.032) enriched. none of the evs analysed significantly changed hs578 t or mda-mb-468 growth rates. however, evs from cells treated with calpeptin (p = 0.007), gw4869 (p = 0.025), manumycin a (p = 0.01) and combo (p = 0.001) caused significant reduction in mda-mb-468 migration compared to the effects of evs from untreated cells. similarly, ev from cells treated with gw4869 (p = 0.011), and combo (p = 0.018) caused significant reduction in hs578 t migration. summary/conclusion: while gw4869 was the only compound that caused a significant decrease in quantities of ev released, the evs that continued to be released following treatment with gw4869 or calpeptin and y27632 significantly reduced migration of both recipient cell lines. funding: phd funding: 1252 tcd scholarship and carrick therapeutics ltd extracellular vesicles from highly metastatic lung cancer cells induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity in a 16-day mature bronchial epithelium purdue university, west lafayette, usa introduction: epithelial-to-mesenchymal (emt) transition plays an integral role in cancer metastasis, which is responsible for as much as 90% of cancer mortality. cancer exosomes induce emt in bronchial epithelial cells, however, the epithelial cells inhibit emt when allowed to form a mature epithelial barrier with apicalbasal polarity. it is not known if cancer-derived extracellular vesicles (evs) can induce emt and more importantly, barrier disruption in a mature epithelium. here, we show that evs from a highly metastatic lung cancer cell line (calu6) are) are not only sufficient to induce emt in non-tumorigenic bronchail epithelial cells (beas-2b), but are also capable of disrupting a 16-day mature bronchial epithelial barrier by significantly reducing teer, inducing sixfold increase in permeability and complete loss of e-cadherin at cellcell tight junctions. methods: beas-2b and calu6 evs were characterized using electron microscopy, nanosight and western blotting for exosome-specific features. for permeability studies, beas-2b cells were cultured in transwell for 16 days to establish an intact epitheliumconfirmed by measuring teer (trans-epithelial electrical resistance). intact beas-2b monolayers were treated with calu6 evs at 1, 10 and 20 μg/ml for 24 hrs, and barrier intactness and permeability were evaluated by measuring teer, apical-basolateral translocation of dextran beads and confocal imaging of tight junctions (e-cadherin). for emt experiments, beas-2b cells treated with calu6 evs at 1 and 10 μg/ml were evaluated for ecadherin and vimentin levels by qrt-pcr and western blot after 48 hrs. results: beas-2b and calu6 evs were enriched in 50-200 nm size range, and cd9 and cd81 were enriched in the ev fraction in contrast to the cell lysate and vice versa for gp96. calu6 evs significantly impaired 16day mature beas-2b monolayer's barrier properties, which at the highest dose caused 33% reduction in teer from 27.0 ± 2.5 to 18.2 ± 3.2 ω.cm2 (n = 4). this was further confirmed by~sixfold increase in dextran beads' apical-basolateral translocation in 30 min (14.8 ± 7 ng/ml in control vs 87.3 ± 51 ng/ml in treated) (n = 3) and complete loss of e-cadherin expression at cell-cell tight junctions (n = 3). at the transcript level, calu6 evs induced significant downregulation of e-cadherin by 36% and upregulation of vimentin (mesenchymal marker) twofold (n = 3) in beas-2b cells, indicating transition into mesenchymal phenotype. summary/conclusion: we demonstrated the involvement of evs derived from highly metastatic lung cancer cells in inducing emt in bronchial epithelial cells and epithelial barrier disruptionthe initial stage of the intravasation process. grp78 plays a crucial role in the extracellular vesicle-promoted radioresistance of irradiated head and neck cancer cells introduction: small evs released from irradiated head and neck squamous cell carcinoma (hnscc) cells increase resistance of recipient hnscc cells to radiation in vitro. we have identified the glucose-regulated protein 78 (grp78), a chaperone protein of the hsp70 family which is involved in cellular stress responses and associated with worse survival in head and neck cancer patients, as an essential component of the ev-mediated radioresistance. methods: small evs were isolated from conditioned medium from irradiated and non-irradiated bhy hnscc cells by combined microfiltration (0.22 µm) and differential ultracentrifugation. grp78 surface expression was measured by proteomic analysis, immunoblotting and bead-facs. radiation resistance of bhy cells was determined by a clonogenic survival assay. results: increased grp78 was identified on the surface of evs from irradiated cells. the increase in ev grp78 correlated with increased grp78 expression at the donor cell surface. the grp78 content of recipient cells also increased upon transfer of evs from irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival. to check a potential role of elevated grp78 in radiation resistance we overexpressed grp78. here the modest (3x) overexpression of grp78 was sufficient to confer an enhanced radioresistant phenotype to the bhy cells. a correlation between grp78-dependent increase of radioresistance and activation of the akt pathway is yet to be determined. summary/conclusion: our results suggest a pivotal role for ev-transferred grp78 in modulating the radiation response of recipient hnscc cells. radiation directly increases the cellular and vesicular grp78 levels, and subsequent ev-mediated transfer leads to enhanced grp78 levels and radioresistance in recipient cells. this study provides new mechanistic insights into the effects of evs in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy. 3d modelling of ev release in progressing prostate cancer introduction: the modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. however, the extracellular vesicles (evs) isolated from 2d cell models are commonly exploited in research. taking into account the specificity of the prostate cancer (pc) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (crpc) patients, we suggest in-depth profiling of different ev subtypes isolated from 3d culture as a new tool to model the progressing pc. methods: cells from hormone-resistant prostate carcinoma 22-rv1 line were cultured in 2d and 3d conditions, using 3d coseedistm. acd plasma controlled for haemolysis and remaining platelets was taken from patients with pc and crpc. the 40 fractions of 4 ev subtypes from cell culture and plasma were obtained by differential centrifugation (dc) followed by iodixanol density gradient purification. each of the fractions was measured by nanoparticle tracking analysis (nta), tunable resistive pulse sensing (trps) followed by elisa. for that, cd63 and cd9 were used as ev markers, apob and apoa1 for lipoprotein contaminants control, and cd3, cd41 and psma as tissue-specific biomarkers for determination of fractions containing evs of different origin. ev-contained fractions were subjected to next generation sequencing (ngs). results: in 3d conditions, the 22-rv1 cells produce up to 1000-times higher ev number than in 2d. size and density distribution of evs derived from 3d cultures but not of 2d resembled plasma evs. size distribution and biomarker expression among different ev subtypes allowed distinguishing between pc and cprcderived samples, indicating a potential to translate these results into clinics for early cprc detection. summary/conclusion: this work demonstrates a new approach to study the secretome of a progressing pc under 3d conditions. the profiles of ev subtypes produced by cancer cells growing in a 3d spatial architecture resemble the profiles of plasma evs and can serve a useful tool for the establishment of new biomarkers. introduction: renal cell carcinoma (rcc) is the most common primary renal neoplasm, with over 80,000 cases in the us alone each year. early detection of rcc leads to consistently better patient outcomes, and extracellular vesicles (evs) isolated from patient samples may prove to be a valuable clinical tool in the future. evs are abundant in blood and urine and show a large amount of heterogeneity but are difficult to analyse due to their small size and difficulty in isolation. here, we employ a multiparametric analysis of ev surface markers to identify a set of markers that may prove clinically relevant in future studies. methods: rcc cell lines vok111, vok130, and vok151 were cultured in flasks containing 500 ml of ev-depleted media (10% fbs, centrifuged 18 hr x 100,000 g). when cells reached~75% confluency, the conditioned media was collected and spun at 2,500 g for 10 mins two times to deplete any remaining debris, leaving~450 ml of media. this media was concentrated to a final volume of~7 ml using a pall jumbosep 100 kda mwco filter. this concentrate was purified from protein by using an izon qev-10 column, collecting 5 ml fractions. protein content of each fraction was analysed using a280 absorbance while concentration and diameter distribution were determined through nanoparticle tracking analysis (nta). pooled samples made of the three most concentrated fractions were concentrated to a final volume of~190 µl using the pall microsep 100 kda filter and then used for analysis in the miltenyi macsplex exosome kit. flow cytometric data were generated by the cytoflex s and analysed using flowjo and mpapass software. these positive signals were verified through bead-only controls and titrations. results: the mpapass software allowed for heatmap generation, data reduction, clustering and visualization of expression patterns. of the 11 detection antibodies used across 39 capture beads, cd276, cd26, cd82, beta-2 microglobulin, and cd151 were found to be prevalent in these rcc evs. these markers were found to be co-expressed particularly with cd63, cd81, and cd29. summary/conclusion: the use of multiplex analysis allowed for detection of five distinctive surface markers found to be prevalent in evs collected from rcc cell lines. these results demonstrate the utility of multiplex analysis and mpapass software for identifying potential markers of interest and provide proteins that are worth exploring further. the next steps to this work will be developing custom multiplex arrays that tailor capture and detection of evs specifically for rcc pathology. low molecular weight protein tyrosine phosphatase (lmwptp) carried by colorectal cancer cells-derived extracellular vesicles as a player in tumour-educated human fibroblast university of campinas -unicamp, campinas, brazil introduction: extracellular vesicles (evs) are doublemembrane-bound nanovesicles released by cells playing a key role as mediators of intercellular communication. low molecular weight protein tyrosine phosphatase (lmwptp) is upregulated in several cancers type, including colorectal cancer (crc), and it has been correlated with aggressiveness, chemoresistance and poor prognostic. methods: the aim of this study was to determine whether crc cells release lmwptp-enriched-evs and influence tumour microenvironment-associated cells as a representative tumour education. crc cells, hct116 and ht29, were cultured in serum-free medium for 24 hours. conditioned medium was concentrated by ultrafiltration (mwco 10 kda) and evs were isolated by total exosome isolation reagent (invitrogen). evs were characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and western blotting (wb). lmwptp levels were analysed by wb and sandwich-elisa. to evaluate tumour education, hff-1 fibroblasts were used as recipient cells. the uptake of evs (pkh26 fluorescently labelled evs), proliferation (viability) and migration (wound healing assay) were analysed in a co-culture model of crc-derived evs and hff-1. results: nta showed a higher concentration of evs released by ht29. hct116 and ht29 evs displayed a mean diameter around 140 nm and a cup-shaped morphology. isolated evs were positive for evs-markers cd81 and tsg101 and negative for gm130 a non-evs marker. ht29 lineage as well as derived-evs are lmwptp-enriched in comparison to hct116 cells and evs. upon incubation, fluorescently hct116 and ht29 derived evs were internalized into hff-1 cells in a perinuclear region. evs derived from both cells increased the viability and proliferation of hff-1 cells. intriguingly, evs derived from ht29 promoted cell migration. summary/conclusion: in conclusion, for the first time, we showed that lmwptp can be carried by evs derived from crc cells and lmwptp-enriched-evs can modulate biological aspects of hff-1 fibroblast. overall, our findings point lmwptp out as important player in tumour-educated fibroblast. exosomal mir-181a inhibition by vincristine and prednisone in paediatric acute lymphoblastic leukaemia. introduction: vincristine and prednisone are standard agents in treatment of paediatric acute lymphocytic leukaemia (p-all). mechanistically, vincristine induces apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic receptors and inhibits dna synthesis, both of which lead to apoptosis. the effect of these agents on exosomal micro-rna expression and its functional regulation is not yet investigated. elevated levels of mir-181a in circulating exosomes (nanoparticles) has been shown to lead to progression in several cancers, including all. we have previously shown that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating of mir-181a expression and silencing of exosomal mir-181a reverses this exosomeinduced cell proliferating effect. the objective is to investigate the effect of vincristine and prednisone on exosomal mi-r181a expression in all. methods: jm1, sup-b15, and nalm-6 leukaemic cell lines were treated in vitro with vincristine (0.1 to 4.0 µm) and prednisone (0.1 to 12.0 µm) in exo-free medium and apoptosis was measured by mts assay. total rna of exposed cell lines was isolated and cdna was prepared for mir-181a analysis. expression of mir-181a was analysed by q-pcr. exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation method. purity and particle size of exosomes were confirmed by western blot and nanoparticle tracking analysis (nta) assay respectively. total exosomal rna was isolated from exosomes (exo-rna) by trizol method. synthesis of cdna was carried out with the miscript ii rt kit (qiagen). results: vincristine and prednisone promote apoptosis in leukaemia cell lines (jm1 and sup-b15) in a dosedependent manner. both cellular and exosomal mir-181a expression was down-regulated by vincristine and prednisone exposure in all three leukaemia cell lines (jm1, sup-b15, and nalm-6). these observations demonstrate that cellular mir-181a down regulation in the parental cells is stable and can be transferred to exosomes, confirming the concept that exosomes are the fingerprint of parent cells. summary/conclusion: our data suggest that the vincristine and prednisone anti-proliferative effect in p-all maybe induced by another yet unexplored pathway, that suppresses mir-181a at a cellular and exosomal level in p-all, resulting in apoptosis. funding: this project is supported by the dimartino family foundation. secreted extracellular vesicles from renal cell carcinoma cells anatoliy samoylenko, artem zhyvolozhnyi, eslam abdelrady, naveed ahmad, genevieve bart and seppo vainio oulu university, oulu, finland introduction: clear cell renal cell carcinoma (ccrcc) represents the most common form of kidney cancer and is among the most lethal of all genitourinary cancers. despite surgery and medication therapy, most patients with metastatic ccrcc have a poor prognosis. intratumoural hypoxia is a key factor involved in renal cancer progression and it is known to promote secretion of evs by many types of tumour cells. methods: rcc-derived renca cells, embryonic kidney derived ub cells, and primary mouse hepatocytes were used in the study. evs were purified from cell culture media by gradient ultracentrifugation, sequential ultracentrifugation and exo-spin™ columns. before ev isolation cells were kept for 24 h either under normoxia or hypoxia (1% oxygen). evs were analysed by transmission electron microscopy with negative staining and immunolabeling, by nanoparticle tracking analysis (nta) and western blotting. cells proliferation and viability were assayed by live cell imaging using incucyte zoom (essen bioscience), cell metabolic activity by seahorse xf analyser (agilent), rna expression by qpcr and ddpcr. proteins were identified by ultra-performance liquid chromatography-mass spectrometry (uplc-ms). rna libraries were made using nebnext small rna library prep kit, and sequenced on nextseq550 (illumina). results: we showed that hypoxia induced production of evs by rcc cells, and characterized differences in protein and rna content of evs generated by renca cells cultured under normoxic and hypoxic conditions. we also showed that rcc-produced vesicles modify key features of tumorigenesis (gene expression, metabolic activity, motility, and growth) of target cells. these data were obtained by using two target cell types: model mouse kidney cells and primary mouse hepatocytes, which represent typical site of rcc metastasis with an exceptionally poor prognosis. we proposed that a possible mechanism of ev action in rcc is related to changes in caveolin-1 function. we also tracked renca-derived evs in a chick embryo model and in a novel kidney organoid co-culture assay developed by our group (xu et al., 2017) . summary/conclusion: hypoxia may influence tumorigenic properties of rcc by changing rates of production and composition of evs. funding: the study was supported by finnish cancer foundation grants. exosomes synthesizing her2 mirna and engineered to adhere to her2 on tumour cells surface exhibit enhanced anti-tumour activity introduction: exosomes are small extracellular vesicles averaging 100-150 nm in diameter. they serve as a means of intercellular communication. typically they consist of structural proteins as well as selected proteins, mirnas, mrnas, and long noncoding rnas. thus in an earlier report this laboratory designed a mirna targeting a major herpes simplex virus regulatory protein. as predicted by the nucleotide packaging signal the mirnas were packed in exosomes and on exposure to infected cells significantly reduced virus yields. her2 (human epidermal growth factor receptor 2) plays an important role in the neoplasia of some breast cancers. the protein is exhibited on the cell surface and is the target of therapeutic antibodies. methods: firstly, we report on the construction of a mirna targeting the synthesis of her2 both in cells constitutively expressing her2 and in cells transfected with a plasmid encoding her2. secondly, we report that the mirna targeting the synthesis of her2 reduced the viability of her2 positive cancer cells both in cell culture and in implanted tumours. lastly, we enhanced the anti-tumour activity of the exosomes by binding to the exosome surface a ligand with affinity for the her2 on the surface of tumour cells. the 293-mir-her2 exosomes package with mirna designed to block her2 synthesis and deliver to cells. these exosomes kill cancer cells dependent on her2 for survival but have no effect on cells lacking her2 or which were engineered to have her2 but do not depend on it for survival. the 293-mir-xs-her2 exosomes carry in addition a peptide which enables the exosome to adhere her2 on the surface of the cancer cells. in consequence, these exosomes preferentially enter and kill cells exhibiting her2 on their surface. the exosomes with 293-mir-xs-her2 are significantly more effective in shrinking the size of her2-positive tumours implanted in mice than the 293-mir-her2 exosomes. summary/conclusion: our studies indicate that exosomes carrying mirna against her2 have no effect on her2 negative cells it was nevertheless desirable to increase the uptake of exosomes carrying the her2 mirnas by her2-positive tumour cells. to this end we modified the exosomes to exhibit on their surface a peptide that bound the exosomes to the her2 on the surface of cancer cells. in consequence, we significantly enhanced the uptake of exosomes carrying the mirnas directed against her2 by her2 positive cells. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd2015071414385495, shenzhen science and innovation commission project grants jcyj20170411 094933148, jcyj20180306173333907 to shenzhen international institute for biomedical research. systematic characterization of ovarian cancer-derived exosomes unveil mirnas interfering with cd8 + t cell activation introduction: cd8+ tumour-infiltrating lymphocytes (til) have been widely reported to correlate with cancer patient survival, including ovarian cancer. even with the presence of tils, immunotherapy has limited success in ovarian cancer. understanding the interaction between cd8+ til and tumour cells is thus important. our hypothesis is that tumour-derived exosomes are released and taken up by cd8+ til such that specific mirnas contained within modulate physiological processes that inhibit cd8 + t cell activation. we aim to identify mirnas carried in tumour-derived exosomes that inhibit cd8 + t cell activation in ovarian cancer. methods: we purified exosomes from nine ovarian cancer cell lines and stocked in high concentration. interferon-gamma (ifn-gamma expression screening was performed after 3 days of co-incubation of tumour derived exosomes, cd8 + t cells, and activators in conditioned medium. cell counts and viability were tested by trypan blue staining at day 0 and day 3. rna-seq for exosomes were generated to identify mirnas critical in differentiation effects on cd8 + t cell activations. microrna target matching uncovered target mrnas while enriched pathway analysis predicted potential signalling pathways involved. results: our ifn-gamma screening results indicated the exosomes exhibit different behaviours in interfering cd8 + t cell activation owing to different donors. exosomes derived from peo.1 and ovca432 cells have consistent polarized results in ifn-gamma expression. exosomes derived from peo.1 remained a low ifn-gamma expression and from ovca432 stayed at relatively high level. small rnas profiling analysis between the two cell lines identified 56 mirnas (p < 0.05), and 13 mirnas have been reported with validated targeting information, and 10 out of 13 have targets involved in immune signalling. 210 mrna targets were uncovered by target matching. cmap search identified complex connections among mrnas with the top 20 enriched pathways actively involved in cell cycle and immune related behaviours. summary/conclusion: our ifn-gamma screening identified crucial mirnas in ovarian cancer exosomes interfering cd8 + t cell activation. computational modelling on both experimental and public multiomics datasets predicted promising signalling pathways of tumour-immune crosstalk for functional validation. irradiation of breast cancer cells alters the quality of dna cargo in the exosomes that they produce sheila spada, paul zumbo, doron betel, tuo zhang, nils-petter rudqvist and sandra demaria weill cornell medicine, new york, usa introduction: irradiation of breast cancer cells with an immunogenic dose (8gyx3) leads to accumulation of cytosolic dna that is sensed by cgas leading to interferon type i (ifn-i) signalling via cgas/sting pathway [1] [2] [3] . we previously showed that tumour-derived exosomes (tex) secreted by irradiated (8gyx3) (rt-tex) but not untreated (ut-tex) tsa carcinoma cells carry dna that stimulates the production of ifn-i in recipient dendritic cells (dc) via the cgas/ sting pathway [4] . moreover, mice vaccination using rt-tex, but not ut-tex, elicited anti-tumour immune response inhibiting tumour growth [4] . here, we hypothesized that the differential ability of rt-tex and ut-tex to activate ifn-i in recipient dcs is due to qualitative differences in dna cargo of rt-tex compare to ut-tex. methods: the length of dna purified from tex and from the cytosolic fraction of tsa cells was measured by agilent bioanalyzer. the dna cargo of tex was analysed by whole-genome sequencing (wgs) and whole-genome bisulphite sequencing. the percentage of methylation of total dna in tsa cells was quantified by 5-methyl cytosine dna elisa kit. results: dna fragments with size between 60 and 250 bp were enriched in rt-tex compared to ut-tex, as well as in the cytosolic fraction of irradiated compared to mock-treated tsa cells. wgs revealed that the entire genome was represented in tex dna cargo, regardless of rt. more than 99% of tex dna was of nuclear origin, but mitochondrial dna was increased in rt-tex. interestingly, we found that rt decreases the level of methylation in both exosomal and total dna in tsa cells compared to the controls. summary/conclusion: these data support the hypothesis that immunogenic rt alters some characteristics of the exosomal dna cargo, mirroring molecular changes occurring in parent irradiated breast cancer cells. the enrichment in dna fragments of 60-250 bp in rt-tex is intriguing considering that cgas is optimally activated by dna in this length range [5] . we are currently investigating which features of the cargo dna that differ between ut-tex and rt-tex may explain the differential ability to induce ifn-i pathway activation in recipient dcs. the identification of a dna signature associated with the ability of tex to activate the cgas/sting pathway could provide a circulating biomarker of the rt-driven immunogenic tumour response. introduction: triple negative breast cancer (tnbc) is among the most difficult cancer subtypes to treat and continues to cause a high number of cancer-related deaths annually. extracellular vesicles (evs) transfer cell type-specific cargo and have important implications in disease initiation, therapy and outcome. upon treatment of cancer cells with low-dose chemotherapy, released evs are able to transfer phenotypic traits to other cancer cells. new treatment strategies for tnbc, like inhibitors of the er stress pathway (ire1) might impact on ev biogenesis, cargo delivery and response of cells in the cancer microenvironment. our aim is to identify immune modulatory alterations in breast cancer cells and cancer derived evs upon treatment with inhibitors of the er stress pathway. methods: human tnbc cell lines were treated with ire1 inhibitor mkc8866 and cells were analysed for immune modulatory surface markers, like hla-i, b7-h molecules and different integrins. mitochondrial and lysosomal activities were investigated by the use of a mito-and lysotracker and analysed by imagestream (isx) technology. extracellular vesicles were isolated from cell culture supernatants by sequential centrifugation, quantified by nanoparticle tracking (nta) and characterized by exosome bead array. single ev analysis of total cell free supernatants and of isolated evs was performed by isx and marker positive evs were quantified for absolute fluorescence signals and total amount by objectives/ml. ev uptake into t cells was investigated by the use of different ev labelling strategies. results: several immune relevant surface markers (hla-i and cd54) are downmodulated by ire1 inhibition across different cell lines. cell surface expressed cd63 and b7-h3 show cell line specific downmodulation profiles upon ire1 inhibitor treatment. other immunomodulatory marker such as b7-h1 and b7-h4, integrin cd29, cell adhesion-promoting cd146 and stemness/metastasis marker (cd44 and ssea) are unaltered on ire1 treated breast cancer cells. cancer cell derived evs were tetraspanin positive (cd9, cd63, cd81), similar in number and showed differential expression of immune markers upon ire1 treatment. mitochondrial and lysosomal activities were unaltered under ire1 inhibition, whereas cell proliferation was diminished. no breast cancer-derived ev uptake of externally labelled evs into healthy t cells could be detected. summary/conclusion: ongoing analyses focus on the multicolour analysis of multiple markers on single evs by imaging flow cytometry and on the functional impact of cancer derived evs on t cells delivered by ev receptor binding. funding: dagmar quandt is supported by the sfi (cúram research centre, 13/rc/2073), the european regional development fund and the dr. werner jackstädt-stiftung. chair: uta erdbrügger -university of virginia chair: larry harshyne -thomas jefferson university comparison of three isolation protocols to search extracellular vesicles signature in sickle cell disease patients introduction: sickle cell disease (scd) is an inherited disorder characterized by chronic haemolysis and continuous activation of different cell types. extracellular vesicles (evs) were described to be at increased levels in scd patient's plasma compared to healthy subjects and were associated with several clinical manifestations such as leg ulcers and stroke. scd patient's plasma has increased concentrations of haem, free-hb and other proteins and lipoproteins as chronic haemolysis consequence. here, we report the comparison of three mostly used isolation protocols to search ev signature in scd patient's plasma by flow cytometry. methods: blood samples were obtained from scd patients (n = 3) following wisgrill et al., (2016) protocol. three different ev isolation protocols were used: differential centrifugation (dc), ultracentrifugation (uc) and size-exclusion chromatography (sec). lactadherin and calcein-am were used to detect phosphatidylserine (ps)+ vesicles and membrane integrity, respectively. platelet-derived evs (pevs), endothelialderived evs (eevs), leucocyte-derived evs (levs) and monocyte-derived evs (mevs) were quantified. silica beads were used to define evs gate and samples were acquired in the cytoflex cytometer platform. results: the quantification of pevs in uc, dc and sec samples was, respectively, 31x106, 8,5x106 and 9,7x106 events/ml mean, eevs was 6,4x106, 1 × 106 and 4,3x106 events/ml mean, levs was 2x106, 6 × 105 and 1,3x106events/ml mean and mevs 5,7x105, 6,5x105 and 3,7x105 events/ml mean. uc samples demonstrated a higher concentration of evs, which could be more useful to functional studies than dc and sec, however, it took more time to separate than dc. dc was the fastest method to separate evs from plasma, being useful to study large patients cohorts, but showed the smallest overall number of evs. sec also demonstrated high capability to detect evs in plasma and the possibility of obtaining a purer sample, although it is the most expensive and time-consuming method among all tested. all evs populations were detected in the three protocols tested. summary/conclusion: in summary, all protocols tested were efficiently to detect evs in scd patient's plasma and the definition of the best protocol may vary based on the research aim and time and budget available. funding: fapesp 2014/00984-3. gabrielle lapping-carr, joanna gemel, yifan mao and eric beyer university of chicago, chicago, usa introduction: aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (scd), including acute chest syndrome (acs), a deadly and unpredictable complication. we previously demonstrated that the plasma of scd patients contains increased circulating small extracellular vesicles (evs) compared to controls and that those vesicles can disrupt endothelial integrity in vitro by affecting adherens junctions and ve-cadherin. the current study was designed to examine the effects of those evs on other cellular junctions including tight (zonula occludens 1, zo-1) and gap junctions (con-nexin43, cx43) and to test the hypothesis that the junctions would be more severely affected by evs isolated from patients during an episode of acs than by ones isolated from the same patient at baseline. methods: we identified subjects with scd in our biobank who had plasma isolated at baseline and at the beginning of an admission for acs. evs were isolated from platelet free plasma using established methodologies. to determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with evs for 48 h and studied by immunofluorescence, immunoblotting and rt-qpcr. gap junction-mediated intercellular communication was assessed following microinjection of lucifer yellow and neurobiotin. results: the distribution and abundance of zo-1 at the plasma membrane were minimally affected by scd evs. while baseline evs did not affect the distribution of cx43, evs isolated during an episode of acs caused loss of cx43 from the plasma membrane. the integrated intensity of cx43 membrane staining was decreased bỹ 20% following treatment with acs evs. cx43 protein decreased on average by 32%, cx43 mrna levels by 21% and neurobiotin transfer by 67-94% in cells treated with acs evs, compared to baseline evs. summary/conclusion: circulating evs in scd affect multiple components of endothelial junctions. gap junctions composed of cx43 are the most sensitive of the cellcell junctions, since their abundance and function are reduced by acs evs even when the endothelial monolayer appears intact. cx43-mediated intercellular communication may be an early and sensitive event in the endothelial disturbance caused by evs in scd patients. funding: nih ul1 tr000430, comer hospital rbc race funds, ted mullin fund. the effects of platelet concentrate storage time on extracellular vesicle interactions associated with fibrin clot formation in-vitro jamie nash a , christine saunders b , amanda davies a and philip james a a cardiff metropolitan university, cardiff, uk; b welsh blood service, velindre university nhs trust, cardiff, uk introduction: platelet concentrates (pcs) have been utilised for decades to prevent bleeding in thrombocytopenic patients and to stop active bleeding. the storage of pcs however is a logistical challenge due to the limited 7 day shelf life under standard conditions. during storage, platelets undergo a number of mechanical and biochemical changes contributing to the short shelf life of a pc. these changes are collectively known as the platelet storage lesion. platelet extracellular vesicles (pevs) are known to increase throughout pc storage, due to an increase in platelet activation. as pevs have previously been shown to be pro-coagulant and increase in annexin v binding over pc storage. the aim was to investigate the effect of pc storage time on extracellular vesicle interactions on fibrin clot formation. methods: pcs were sampled on alternate days up to 10 days of storage and centrifuged to achieve acellular plasma. the plasma was subjected to ultracentrifugation (100,000xg) to pellet evs. the size and concentration of evs was assessed using nanoparticle tracking analysis software, followed by a western blot to confirm evs were of platelet origin. the pevs were added at a fixed number to a control pooled plasma sample with added thrombin and tissue plasminogen activator. the time to clot and 50% lysis time were recorded by using the turbidometry of the plasma over time. results: evs isolated from the pc were confirmed to be of platelet origin by western blot using cd41 as a marker of platelet origin and cd9 as an ev marker. pevs caused a significant increase effect on the fibrin clot formation (p < 0.001) when compared to the control plasma. pevs also had a significant effect (p < 0.01) on the fibrinolysis time, extending the time taken to lyse the clot. characterization of mirna from serum derived exosomes in a mouse tibia fracture model of introduction: complex regional pain syndrome (crps) is a debilitating chronic disease that occurs after trauma to the periphery and is intimately associated with nerve injury. its presentation is often described as an injury that is disproportional to the inciting event and manifests neuropathic pain, systemic inflammation, and immune dysregulation. owing in part to our poor understanding of disease aetiology, current treatments for crps are insufficient and as a disease of exclusion there is a lack of quantitative diagnostic markers. exosomes are small extracellular vesicles (sevs) 30-100 nm in size which provide a means of cellular communication through their cargo molecules (protein, mirna, mrna, lipids) , and have demonstrated promise in uncovering mechanisms of disease manifestation and identifying potential diagnostic markers. we have shown previously that crps patients have differential expression of several mirnas in serum derived sevs as compared to healthy controls, but little is known on how this compares to the established mouse tibia fracture model of crps. methods: mice undergoing fracture were anesthetized and subjected to a unilateral tibia fracture followed by casting of the injured limb. after confirming the establishment of pain hypersensitivity, serum samples were collected from fracture model and control mice three weeks post-injury. sevs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna-seq analysis is being performed to identify differentially expressed mirnas. results: nanoparticle tracking analysis showed no significant difference in the number or size of sevs present in the serum from the fracture model and control mice. rna-seq is ongoing and differential mirna expression in sevs from fracture model will be compared to control samples. comparative studies identifying mirnas that are common between crps patients and the rodent model will facilitate the development of correlational outcomes between preclinical and human studies. summary/conclusion: identification of similarities and differences between crps patients and animal models will aid in directing future studies at clinically relevant aspects of crps aetiology and identifying potential diagnostic markers for crps patients. extracellular vesicle-based liquid biopsy in acute myeloid leukaemia: a reliable source of residual disease biomarkers? introduction: acute myeloid leukaemia (aml) is an haematopoietic stem cell disorder with a poor 5-year survival rate. monitoring of measurable residual disease (mrd) in aml patients receiving chemotherapeutic treatment is useful to assess therapy response and predict relapse. indeed, many different leukaemia associated immunophenotypic protein markers (laips) are presently useful to detect mrd. nevertheless, their analysis currently requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. therefore, alternative peripheral blood-based methods are highly desirable for an easy, real-time and costeffective monitoring of aml progression. this work aims was to assess the feasibility of a peripheral blood ev-based liquid biopsy method for aml disease monitoring, based on the detection of laips with a known negative impact on the prognosis of aml. methods: the profile of evs isolated from 12 paired samples from aml patients' blood plasma collected at diagnosis, complete remission (and some at relapse) was compared and correlated with clinical data. for that, a size-exclusion chromatography (sec) method was optimized to isolate the circulating evs from the blood plasma. the evs of the 12 paired aml patients' blood samples were then characterized according to their size (dls/nta), morphology (tem), proteinto-lipid ratio (lowry/sulpho phosphovanillin assay), surface charge (zeta-sizer) and protein cargo (western blot). results: sec allowed the isolation of size-resolved plasmaderived evs from the peripheral blood of aml patients. isolated evs had a size ranging from 30 nm to 300 nm with an intact morphology, expressing ev-associated markers such as hsp70, cd63, cd81 and cd9. size-resolved evs also had a differential expression of mitofilin, actinin-4, syntenin-1 and annexin-xi proteins. several laips were detected in the isolated evs and their relative abundance changed throughout the stage of the disease. summary/conclusion: our preliminary data shows that aml patients' circulating evs carry relevant immunophenotypic protein markers, which might predict aml clinical outcome. introduction: cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (emt) and met is critical in the metastatic cascade. extracellular vesicles (evs) may play an important role in this balance by shuttling molecular cargos into recipient cells. this study aims to evaluate the feasibility of profiling mrnas of parental prostate cancer (pca) cells with different phenotypes and their daughter evs using the nanostring low rna input ncounter assay. methods: pc3-epi and pc3-emt cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original pc3 cell line. the cell culture supernatant was first pre-cleared for any dead cells and debris by centrifugation at 1000 × g for 20 min. without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at 10,000 × g for 20 min at 4°c. the remaining supernatant was then centrifuged to isolate the evs at 100,000 × g for 120 min at 4°c. the evs pellet was further washed in 1× pbs followed by a second centrifugation at 100,000 × g for 120 min at 4°c. the final evs pellet was resuspended in 1× pbs for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and western blot) and ncounter assays. the total rna of cells and their daughter evs were assayed by the ncounter pancancer progression panel to determine expression of 770 selected mrnas. the nanostring ncounter low rna input kit with the multiplex 770gene primer pool was used for the pre-amplification of mrna and overnight hybridization with the pancancer progression panel. each sample type was submitted to the assay in biological triplicate. results: when comparing all 12 samples, eisen cluster analysis separated all the cells and all evs into two groups, regardless of their phenotypes. in subgroup analysis, the expression patterns between pc3-epi and pc3-emt cells were significantly different. clec2b, kdr, crip2, il13ra2, cc2d1b were significantly upregulated in pc3-emt cells, while cxcl8, epcam, esrp1, tgfb2, cdh1, s100a14, ovol2 were significantly downregulated in pc3-emt cells. the expression patterns between pc3-epi and pc3-emt evs were also significantly different. tbx1, cav1, col4a1, slc35a3, myc, itgb2, timp4, camk2b, ptgds, p3h2, itgb6, vim, stat3 were all significantly downregulated in pc3-emt cell derived evs. summary/conclusion: the nanostring low rna input ncounter assay can provide reliable mrna expression profiling of evs. the mrna expression patterns are very different between cells and their daughter evs. both cells and evs with different phenotypes have different gene expressions. cancer cell-derived evs containing alphav beta6 integrin regulate cd163, il-6 and il-10 levels in peripheral blood mononuclear cells introduction: extracellular vesicles (evs) mediate communication in the tumour microenvironment and play an important role in cancer progression. previously, we have shown the enrichment of alphav beta6 integrin in small extracellular vesicles (sevs) isolated by differential ultracentrifugation and iodixanol density gradient from pc3 prostate cancer cells. we have also shown in the past that alphav beta6-positive sevs induce peripheral blood mononuclear cell (pbmc) polarization by increasing the expression of pro-tumorigenic m2 markers, such as cd163 and cd204. finally, we have demonstrated that down-regulation of alphav beta6 integrin up-regulates the stat1-interferon stimulated genes (isgs) pathway in cancer cells and in sevs released by them. methods: in order to investigate whether prostate cancer cell-derived vesicular stat1 has a causal effect in pbmc polarization, we down-regulated alphav beta6 and stat1 in prostate cancer cells derived sevs using sirna as well as crispr-cas9 strategies. the sevs isolated from these cells were used to analyse m2 polarization by measuring the levels of cd163 in pbmc. the results show that sevs lacking alphav beta6 inhibit cd163 levels in pbmc in a stat1-independent manner. analysis of cytokines released by pbmc upon incubation with sevs lacking alphav beta6, show that pbmc selectively up-regulate the levels of il-10 and il-6, which are predominantly anti-tumorigenic cytokines. in contrast, sevs lacking alphav beta6 do not upregulate pro-angiogenic cytokines, such as vegf. summary/conclusion: these findings suggest that cancer cell-derived sevs containing alphav beta6 integrin promote a pro-tumorigenic pbmc phenotype in the tumour microenvironment by regulating cd163, il-6 and il-10 levels. introduction: the recognition of donor-mhc molecules by recipient t cells triggers the immune response leading to rejection of allografts. our recent studies have documented the presence of high numbers of recipient apcs displaying donor-mhc molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. in addition, we have reported that acquisition of allogeneic mhc molecules by host apcs (mhc crossdressing) is mediated by donor-derived extracellular vesicles (evs) trafficking through blood and lymphatic vessels (marino et al. science immunology, 2016) . in the present study, we investigated the ability of allogeneic evs and allo-mhc-cross-dressed cells to initiate a t cell alloresponse in vitro and in vivo. methods: evs were isolated (using differential centrifugation) from balb/c bone marrow derived dendritic cells (bmdcs). these evs were used to cross-dress b6 splenocytes in vitro. the transfer of donor mhc class i and ii on b6 cells was analysed by imaging flow cytometry. next, t cells from b6 mice were cultured in vitro with either allogeneic bmdc-derived balb/c evs or b6 spleen cells crossdressed with allogeneic balb/c mhc. alternatively, 2 × 109 balb/c or b6 bm derived evs or 20 × 106 balb/c bm cells were injected iv to b6 mice. in both cases, the t cell response was assessed by activation markers detection, infg production and cell proliferation. results: apcs cross-dressed with allogeneic mhc molecules can trigger a pro-inflammatory direct alloresponse by t cells in vitro and in vivo. on the other hand, allogeneic evs alone were only able to induce early t cell activation but not proliferation in vitro. furthermore, injection of mice with allogeneic evs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete freund's adjuvant. summary/conclusion: blocking donor evs release and subsequent recipient apc cross-dressing may represent a promising target to selectively inhibit anti-donor t cell inflammatory responses thus achieving long-term allograft survival. funding: r01dk115618. antifungal antibiotic activity of outer membrane vesicles from adherent lysobacter enzymogenes c3 against therapeutic and biocontrol targets. rutgers university, new brunswick, usa introduction: lysobacter enzymogenes is a predatory gram negative bacterial species being studied for biocontrol activity against fungi. planktonic l. enzymogenes c3 produces outer membrane vesicles (omv) harbouring small molecule antifungal antibiotics (meers et al. 2018) . we show here that the more biologically relevant surface-associated c3 exerts remote antifungal activity via omv as well. the results have important consequences regarding the natural mechanism of biocontrol of fungal pathogens by c3 as well as isolation and delivery of therapeutically relevant antifungal compounds. methods: omv were isolated from scraped adherent c3 culture on agar by similar methods to meers et al 2018. omv were stained in some cases with fluorogenic syto 9 dna stain for microscopic observation. fungal growth was monitored via turbidity readings in liquid culture or photomicrographs on agar. c3 was also grown on polycarbonate filter membranes with defined pore sizes to monitor growth of fungal cells on the opposite side. vesicles were also labelled with an amine-reactive probe alexa-555 and washed 4x by sedimentation. binding of labelled omv to fungal cells was observed by epifluorescence microscopy. results: syto 9-stained vesicles from surface-adherent c3 were similar to previously observed~130 nm vesicles (meers et al., 2018) . the isolated vesicles inhibited growth of saccharomyces cerevisiae or candida albicans in liquid cultures at similar potency and were active against the filamentous species fusarium subglutinans grown on agar or maize leaves. c3 cultures grown on filters with 400 nm pore size but not 30 nm were able to inhibit the hyphal growth of f. subglutinans on the opposite side. similarly c3 on filters with a 200 nm pore size were able to inhibit growth of c. albicans. observation of fluorescently-labelled c3 omv after interaction with c. albicans showed binding specifically to hyphae or pseudohyphae and for f. subglutinans to the growing hyphal tips. summary/conclusion: the omv of c3 specifically bind and inhibit the growth of fungal hyphae of various species without direct c3 cell contact. these data elucidate mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal antibiotics. meers et al. elucidating the cellular uptake and tissue distribution mechanism of cell derived vesicles, a novel therapeutic carrier hui-chong lau a , jae young kim a , jin-hee park a , jun-sik yoon a , min jung kang a and seung wook oh b a mdimune inc, seoul, republic of korea; b mdimune inc, seattle, usa introduction: cell derived vesicles (cdvs) are emerging as a novel therapeutic carrier. one of the crucial factors in the development and therapeutic applications of cdvs is to understand the precise mechanism by which vesicles find and enter the target cells. in this study, we aim to investigate the uptake mechanism of cdvs produced from natural killer (nk) cells using a manufacturing process established at mdimune inc. both in vitro uptake assay and in vivo distribution analysis were performed to provide precise insights into how cdv exert its effect at the cellular level. methods: nk cells were mainly used to produce cdvs. breast cancer cells, bt549, and human and rodent endothelial cells, with a varying degree of icam-1 expression, were used to determine the effect of lfa-1 expressed on the surface of nk-cdvs in cellular uptake using facs and confocal imaging analysis. next, various inhibitors for uptake pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated endocytosis, were used to understand the underlying mechanism of cellular uptake of cdvs. biodistribution profile of cdvs were characterized using both normal and tumour xenograft models by ivis imaging. results: using a recently established manufacturing process, we demonstrate that nk-cdvs can efficiently enter the target cells. this study also shows that the cellular uptake depends on the molecular interaction between icam-1 and lfa-1. in vivo distribution profile of nk-cdvs are also assessed using various tumour models. furthermore, we present a cellular uptake mechanism involved in the entrance of cdvs into the target cells. summary/conclusion: this study demonstrates that the cdvs produced at the manufacturing scale can be easily taken up by cells via specific cellular pathways. this finding will facilitate the development of more efficient therapeutics for cancer and other debilitating diseases. myofibroblasts-derived microvesicles increase dermal fibroblasts collagen production through plgf-1 syrine arif, sebastien larochelle and véronique j. moulin chu de québec -université laval, loex, québec, canada introduction: a proper wound healing of the skin involves angiogenesis, extracellular matrix (ecm) remodelling and re-epithelialization. these three mechanisms require well-organized interactions between different cell populations. a key role in this context is played by myofibroblasts (wmyo), a cell population mainly differentiated from dermal fibroblasts. these cells contract wound edges and synthesize new ecm. we previously showed that myofibroblasts predominantly produces microvesicles (mvs) and can favour angiogenesis. however, proteomic analysis of mvs from our previous studies indicated some molecules that can potentially be implicated in ecm remodelling. in this study, we evaluated whether myofibroblasts-derived mvs could affect dermal fibroblasts who are highly responsible for ecm regulation. methods: mvs were isolated by differential centrifugation of medium collected from wmyo cells. number and size of mvs were characterized by transmission electron microscopy and nanosizer. multiplex assays of 45 cytokines were evaluated in mvs samples, wmyo and mvs-depleted medium. to examine the interaction of mvs with fibroblasts, we evaluated the uptake of mvs isolated from wmyo transduced with a fluorescent protein. we then treated fibroblasts cultures with mvs or a selected cytokine for 5 days and evaluated collagen production. lastly, we neutralized the selected cytokine in mvs samples before evaluating collagen production. results: plgf-1 was the cytokine detected in mvs samples in large amount (0.88 ± 0.63 pg/µg proteins in mvs). fibroblasts treated with mvs or plgf-1 significantly stimulated pro-collagen i level production with a fold change of 1.80 ± 0.18 and 2.07 ± 0.18. moreover, the neutralization of plgf-1 present in mvs significantly inhibited the production of pro-collagen i by dermal fibroblasts. summary/conclusion: our results indicated that mvs influence fibroblasts pro-collagen production through plgf-1 signalling. funding: this work was supported by natural sciences and engineering research council of canada (nserc) (rgpin2014-04404); les fonds de recherche du québec-santé (frqs) via the research centre funding grant; the quebec cell and tissue and gene therapy network-thécell (a thematic network supported by frqs). structural insights on fusion mechanisms of extracellular vesicles with model plasma membranes introduction: extracellular vesicles (evs) represent a potent intercellular communication system. while their functional biological properties are more and more investigated, the biophysical aspects of their interaction with recipient cells are often overlooked. small size (30 to a few hundred nanometres in diameter) of evs and their heterogeneous origin still pose a great challenge for their isolation, quantification and biophysical/biochemical characterization. in particular the complex network of interactions between differently classified evs and recipient cells remains to be further revealed. here we deeply investigate the fusion mechanism between evs and a model plasma membrane system by an interplay of different structural/morphological techniques to get a molecular description of the interaction helping to clarify the role of different membrane compartments on the evs uptake mechanism. standardized protocols and good manufacturing practice conditions were employed to derive highly stable vesicles of defined size and reproducible molecular profiles from umbilical cord multipotent mesenchymal stem (stromal) cells. after a thorough biophysical and biochemical characterization of evs non-contact liquid imaging atomic force microscopy (afm) and, in parallel, neutron reflectometry (nr), as well as small angle neutron scattering (sans) experiments were performed on evs to determine their interaction with model plasma membranes in the form both of supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. results: we observed that evs tend to fuse with the model membranes with a preferential interaction with the external layer of the fluid membrane. moreover we revealed a stronger interaction with the liquid ordered domains, strengthening the hypothesis of a critical role of lipid rafts in fusion mechanisms. summary/conclusion: our results on the analysis of the interaction of evs with artificial lipid membranes could provide insights on the internalization mechanisms of evs. the approach shown here can be further extended to convey incremental complexity, adding glycolipid and membrane proteins to the model lipid bilayers. this approach combined with data on the specific biological function of each ev subpopulation as retrieved by standard functional assays, will turn useful to select the crucial molecular aspects of evs internalization by cells. introduction: platelet-derived extracellular vesicles (pev) are the most abundant circulating extracellular vesicle (ev) and exhibit platelet-like properties, hence the original term "platelet dust". direct phenotyping of ev surface markers within biofluids is challenging often requiring time-intensive purification steps that can significantly alter resultant ev population characteristics. the exoview™ (nanoview biosciences) specifically captures ev sub-populations and was used to characterise the ev content of platelet free plasma (pfp) and a potential novel haemostatic agent designed for the treatment of severe trauma and haemorrhage, platelet enhanced plasma (pep). methods: freeze-thaw cycling of platelet rich plasma/ expired platelet concentrates was followed by centrifugation to remove platelet remnants and yeilded pep. pfp controls were prepared by double centrifugation (2000 g for 20 minuntes followed by 13,000 g for 2 minutes). rotational thromboelastometry (rotem) and calibrated automated thrombography (cat) were used to assess ev driven haemostasis and thrombin generation. a dilutional and hypothermic model of coagulopathy was designed to assess pep. ev capture arrays comprised of anti-cd41, anti-cd63, anti-cd81 and anti-cd9 were used (exoview™, nanoview biosciences). captured vesicles underewent interferometric imaging and were quantified, sized and further probed with fluorescent tetraspanin markers, annexin-v and intravesicular markers. results: pep is highly procoagulant, exhibits enhanced thrombin generation and can restore haemostasis in a dilutional model of coagulopatic whole blood. pep can be generated from expired platelet concentrates, potentially allowing for upscalable production. the predominant vesicle population were pev with a large cd41/cd9 population that contained a smaller subpopulation of phosphatidyserine positive procoagulant vesicles. pfp as expected has a much lower number of pev and a cd81 positive ev population. summary/conclusion: pep is a unique resuscitation fluid containing high pev levels for the potential treatment of severe trauma and haemorrhage. exoview measurements can be performed in unpurified plasma and may be useful for measuring circulating ev in health and disease. funding: defence and security accelerator, dstl therapeutic effect of exosomes in mice model of autism daniel offen a , reut horev a , nisim perets a , ehud marom b , uri danon b and yona gefen b a tel aviv university, tel aviv, israel; b stem cell medicine ltd., jerusalem, israel introduction: during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. we have previously shown that intranasal administration of msc-exo, cross the bbb and significantly ameliorate autistic-like behavioural phenotype in btbr and shank3 animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (asd). our objective is to study the mechanism of action and the cellular pathways in which the msc-exo activate their target, we performed rna sequencing analysis of primary neurons isolated from shank3 mice treated with msc-exo. methods: primary neuronal cell cultures were prepared from newborn shank3 homozygotes mice model of autism. cultures were treated with msc-exo (10^7 particles/ul), isolated from human adipocytes, followed by rna sequencing. the alterations in gene expression between the treated and intact neurons were analysed for gene ontology and pathways and were also compared to proteomics analysis of the msc-exo in order to find regulatory proteins that may lead to these differences. results: bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with msc-exo. one of them is bdnf which is known as an essential growth factor responsible for neuroprotection and neurogenesis. importantly, no difference in the genetic expression of cancer-related genes was identified following msc-exo treatment indicating for their safety. summary/conclusion: our data suggest that adipocytederived msc-exo carry therapeutic potential in asd via alternation in gene-expression related mainly to immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis. the beneficial effects of the exosomes treatment in mice models is being translated into a novel, easy to administer, a therapeutic strategy to reduce the symptoms of asd. introduction: autologous blood-derived products gain increasing focus in regenerative medicine, especially in orthopaedics and osteoarthritis therapy. this disease is characterised by cartilage degradation and inflammation among other symptoms, which are targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved. citrate-anticoagulated platelet rich plasma (cprp) is often clinically applied to stimulate soft and hard tissue healing. recently, cell-free alternatives to cprp including hyperacute serum (hypact™ serum) have been developed. cprp and hypact™ serum contain specific profiles of growth factors, however, they also contain extracellular vesicles (evs) that harbour signal molecules including mirna. methods: evs were enriched by ultracentrifugation (uc) followed by size exclusion chromatography (sec) to obtain purified evs. particle size and concentration of each fraction was measured by nanoparticle tracking analysis (nta). fractions with the highest amount of particles were pooled and concentrated via uc, before mirna expression was assessed via screening with a panel of 372 mirna-specific primer pairs by rt-qpcr. presence of evs was confirmed by cryoelectron microscopy. results: the ev concentration tended to be lower in hypact™ serum than in cprp as determined via nta. similarly, lower diversity of mirna species was found in hypact™ serum than cprp evs. around 90% of detected mirnas were found in both blood products, whereas only 30% of mirnas were shared between evs from cprp and hypact™ serum. while mirnas such as mir-101 were consistently depleted in evs compared to the corresponding blood product, others like mir-27a were in enriched in hypact™ evs, but not cprp evs, indicating release of specific mirnas via evs in response to clotting. summary/conclusion: although the purification resulted in high loss of evs, we identified specific mirnas enriched in evs from cprp and hypact™ serum. their functional spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation, inhibition of tissue remodelling via matrix degrading enzymes as well as preventing senescence. this renders blood product derived evs as interesting candidates for in vitro and in vivo testing with respect to cartilage regeneration. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst3-f-5030664/003-2017. protective role of shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice baolong liu, xingyi chen and jiujiu yu university of nebraska lincoln, lincoln, usa introduction: fulminant hepatic failure (fhf) is a rare, life-threatening liver disease with poor prognosis. new therapeutic interventions are urgently needed to treat this disease. administration of d-galactosamine (galn) and a low dose of lipopolysaccharide (lps) triggers acute liver damage in mice, which simulates many clinical features of fhf in humans and therefore is widely used to investigate the molecular mechanisms and potential therapeutic interventions of fhf. recently, suppression of the nucleotide binding domain and leucine rich repeat related (nlr) family, pyrin domain containing 3 (nlrp3) inflammasome was shown to alleviate the severity of lps/galn-induced liver injury in animal models. therefore, the goal of this study was to identify food-derived exosome-like nanoparticles (elns) with anti-nlrp3 inflammasome function to potentially control fhf. methods: seven commonly consumed mushrooms were used to extract elns, which were examined for anti-nlrp3 inflammasome activities in primary macrophages. results: it was found that these mushrooms contained elns composed of biomolecules including rnas, proteins, and lipids. among these mushroom-derived elns, only shiitake mushroom-derived elns (s-elns) strongly inhibited nlrp3 inflammasome activation by blocking the inflammasome assembly. this inhibitory effect was specific for the nlrp3 inflammasome because s-elns had no impact on activation of the absent in melanoma 2 (aim2) inflammasome. s-elns also inhibited the secretion of interleukin (il)-6 and both protein and mrna levels of the il1b gene in macrophages. remarkably, pre-treatment of s-elns protected mice from lps/galn-induced acute liver injury. summary/conclusion: therefore, s-elns, identified as potent inhibitors of the nlrp3 inflammasome, represent a new class of agents with the potential to combat fhf. approaches to assess clinically available exosomes' quality and safety introduction: recent adverse events resultant from an exosome product use in a nebraska clinic, highlight the importance of assuring product quality and safety standards. an often-overlooked safety risk is ancillary reagents remaining within a finished product. when processes to obtain exosomes utilize cow proteins such as fbs or bovine sera albumin, failure to adequately remove these can result in significant adverse allergic reactions. we evaluated 3 different exosome products to test the hypothesis that purity of some products may not be consistent with actual product quality and safety profiles claimed. methods: three different exosome products (manufacturer a, b, and c) were prepared per their instructions for use. sample source identity was blinded from assaying scientists. an independent cro service was used to conduct the experiments to ensure unbiased assay execution and data collection. exosome suspensions were sampled undiluted for bovine protein content using commercially available bovine secretome protein arrays from ray biotech. a total of 30 different proteins found in bovine serum were quantified. results: six of 30 proteins were not detected in any sample. 8 of 24 array antibodies were found to cross react with human antigens. of the 16 bovine proteins that were acceptable for analysis, manufacturers a, b, and c exosomes contained 15 of 16 proteins,13 of 16 proteins, and 0 of 16 proteins, respectively. concentrations of individual bovine proteins ranged from 0.2 to 1,220.1 ng/ml. summary/conclusion: these results indicate manufactures a and b are selling potentially dangerous products. the successful implementation of exosome products into the clinic requires equivalent demonstrations of safety and quality. this requires adopting strict quality standards and safety testing during their production. physicians must require safety data prior to clinical use. engineering pro-healing ev cargo using a closed-system bioreactor. introduction: chronic wounds, including diabetic ulcers and pressure ulcers, are difficult and expensive to treat. while tissue engineering approaches have largely failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived extracellular vesicles (evs) may provide several unique advantages. zenbio, inc has developed a methodology to generate commercial-scale stem cell-derived exosomes using a closedsystem hollow fibre bioreactor capable of continuous ev production. additionally, we have shown that by manipulating the cellular environment, we can improve the pro-healing capacity of the evs.this technology leverages the complex healing capabilities of stem cells without the obstacles of replicating cells. methods: we have demonstrated that a mild heat shock resulted in evs enriched for stress-response proteins and increased pro-healing activities in vitro. we extended this innovative approach to include stimulating adipose stem cells with combinations of heat shock and growth factors to generate differential extracellular vesicle packaging that enhances pro-healing activity. to monitor reproducibility across lots and batches, we rigorously characterized tuned evs for particle size and number as well as surface marker and cargo composition. results: our results using tuned evs showed efficacy using cellular models of inflammation, motility, vascularization, collagen production and metalloprotease activity. we utilized an established murine model of pressure ulcers to assess the in vivo efficacy of the tuned evs. these studies showed a single injection into the wound site activated a more rapid wound closure, increased collagen deposition and reduced dermal thickness compared to saline control. summary/conclusion: these data strongly support our hypothesis that evs may be selectively modified to improve their wound healing activity by modulating the culture or tissue microenvironment. future studies will use chronic wound models to determine optimal dosing and routes of administration. introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) can reduce inflammation, promote healing and improve organ function thereby providing a potential "cell-free" therapy. prior to clinical translation, there is a critical need to synthesize existing preclinical evidence supporting their efficacy. this systematic review provides the most comprehensive evidence map of methods, safety and efficacy for msc-ev research to date. methods: medline and embase were systematically searched for in vivo interventional studies using msc-evs. two reviewers extracted data for: 1) methodology, 2) study design, 3) intervention details and 4) efficacy/ adverse events. results: after screening 754 articles, 206 studies met our eligibility criteria. msc-evs were used to treat a variety of diseases including renal (16%), neurological (12%) and cardiac (10%) conditions. benefits were described in 95% of studies across all organ systems and adverse effects were seen in only three studies; two showing tumour growth. however, several key methodological concerns were evident. based on size criteria for ev subtypes (exosomes/small evs~30-150 nm, microvesicles~150-1000 nm) only 60% of studies used appropriate nomenclature. ultracentrifugation (70%) and isolation kits (23%) were the most common isolation methods despite marked differences in yield and purity. evs were inconsistently dosed by protein (68%), particle number (16%) or cell count (4%), hindering inter-study comparisons. two-thirds of studies used xenogeneic evs suggesting immunocompatibility. techniques to determine size, protein markers and morphology was highly heterogeneous, and only 12 and 4 studies met the characterization standards recommended in the misev 2014 and 2018 guidelines, respectively. finally, 50% of studies did not incorporate randomization which represents a high risk for bias and only a quarter performed biodistribution studies. summary/conclusion: this systematic review reveals extensive heterogeneity in methods and intervention details for animal studies of msc-evs. nonetheless, nearly all studies showed significant benefits in a wide range of distinct conditions. the knowledge gaps we identified highlight important opportunities for improving preclinical design and the need for more standardized approaches in this growing field of ev therapeutics. msc-exosomes as next generation therapeutics for atopic dermatitis exocobio inc, seoul, republic of korea introduction: atopic dermatitis (ad) is a systemic inflammatory disease with unknown cause. recent approval of a targeted therapy, dupilumab, opens new era of ad management. however, current therapeutic options for ad are only targeting inflammation, a component of ad vicious cycle including itching and barrier disruption. human mesenchymal stem cells (mscs) have been highlighted as a novel therapy for suppressing allergic progress of ad in clinical studies. unfortunately, phase iii clinical study of human umbilical cord blood mscs for ad was failed with unknown reason. previously, our group reported that exosomes derived from human adipose tissue-derived mscs (asc-exosomes) alleviated the pathological symptoms in a murine ad model with concomitant reduction of inflammation. methods: our group has further investigated the therapeutic effects of human asc-exosomes in an alternative murine ad model with skin barrier defects. large scale isolation of asc-exosomes was performed by tangential flow filtration and isolated asc-exosomes were characterized according to the recommendation by the isev. the protein and lipid cargo were also analysed. results: we found that asc-exosomes induced restoration of skin barrier by inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines. in addition, asc-exosomes suppressed the expression of itching-causing cytokines. transcriptomic analysis of ad skin lesions revealed that asc-exosomes reversed the abnormal expression of genes functioning in skin barrier function, lipid metabolism, and cell cycle. summary/conclusion: taken together, asc-exosomes could be a promising cell-free therapeutic option for the treatment of ad, which affecting inflammation, skin barrier function, and itching. cell derived vesicles: unravelling the science of novel vesicles with therapeutic promises introduction: cell derived vesicles (cdvs) are nanosized vesicles produced by serially extruding cells through small pores. a growing number of studies have implicated their therapeutic potentials, with superior yield compared to other extracellular vesicles (evs). however, two key objectives remain to be accomplished to demonstrate the utility of cdvs in clinical applications. first, a manufacturing process has to be developed to allow a large-scale production of cdvs. next, these novel vesicles need to be thoroughly characterized at multiple levels. methods: manufacturing-scale extruders were developed to allow extrusion of large volume of cell suspension in a single process. cdvs with approximately 150-200 nm in diameter were obtained by a serial extrusion. crude samples were then purified using the tangential flow filtration method to further remove cellular impurities. finally, physical and biochemical characteristics of purified cdvs were analysed using dls, nta, cryo-em, and facs analysis. additionally, cdvs were subject to multi-omics profiling to comprehend our understanding in molecular contents of cdvs. both mesenchymal stem cells (mscs) and natural killer (nk) cells were used for this study. results: in this study, we first demonstrate that the large-scale extruder efficiently produce cdvs with consistent quality at the scale that are compatible for clinical applications. surface marker and membrane composition analyses show that the cdvs are primarily formed using plasma membrane of source cells, with characteristic cellular markers enriched on the surface. comprehensive profiling of molecular components reveals the unique properties of cdvs as well as the underlying mechanism of formation of cdvs. summary/conclusion: recently, we have established a manufacturing process to enable clinical applications of cdvs. this study also highlights key molecular features of cdvs that can be harnessed to offer a powerful tool for regenerative and anticancer medicine. antifibrotic properties of extracellular vesicles derived from human induced pluripotent stem cells introduction: fibrosis is a pathological condition resulting from abnormal healing of various tissues. it is triggered by activation of fibroblasts and their subsequent transition to myofibroblast. in consequence, excessive deposition of extracellular matrix proteins leads to impaired organ function. to revert this process, we employed extracellular vesicles (evs) derived from human induced pluripotent stem cells (hipscs). as a model system, we used human cardiac fibroblasts (hcfs), since heart fibrosis constitutes a serious socioeconomic problem worldwide. methods: we isolated evs from conditioned media from three hipsc lines using ultrafiltration combined with size exclusion chromatography methods. next, we analysed the evs by nanosight, transmission electron microscopy, mass spectrometry and western blot methods. finally, we treated tgf-b-stimulated hcfs with hipsc-evs and evaluated expression of fibrosisrelated genes using real-time qpcr, western blot and fluorescence microscopy. results: we detected anti-fibrotic properties of hipsc-evs exerted on hcfs pre-stimulated with tgf-b. the evs significantly decreased the expression levels of acta2, fn, tnc, snai2, col1a1 and reduced the number of myofibroblasts. the canonical profibrotic tgf-b-dependent smad2/3 pathway was significantly attenuated in response to ev-treatment. summary/conclusion: in this study we demonstrated strong anti-fibrotic function of hipsc-evs. our findings can further be exploited for future medical applications to treat fibrotic diseases, such as heart fibrosis. funding: this work was supported by the project sonata12: umo-2016/23/d/nz3/01310 from the national science centre of poland to sbw. induced pluripotent stem cells-derived extracellular vesicles ameliorates d-galactosamine and lipopolysaccharide induced acute liver failure tianjin third central hospital affiliated to nankai university, tianjin, china (people's republic) introduction: liver failure is among the most causes of death in patients with liver disease. promoting liver regeneration will help patients with liver failure recover on their own. extracellular vesicles (evs) can released by induced pluripotent stem cells (ipscs) through paracrine effects and play a pivotal role in inter-cellular communication in the treatment of disease. in this study, we investigated whether the ipscs-evs have therapeutic effects on acute liver failure. methods: the ipscs-evs were isolated by ultracentrifugation and identified using nanoparticle tracking analysis, transmission electron microscopy and western blotting. the isolated ipscs-evs were administrated d-galactosamine-injured heprg cells in vitro and tail intravenously injected into d-galactosamine and lipopolysaccharide induced acute liver failure model mice in vivo, respectively. the anti-apoptosis role and potential mechanism were evaluated using flow cytometry and immunofluorescence staining. and alanine transaminase (alt) and aspartate transaminase (ast) in serum, h&e staining and tunel staining were explored the effect of ipscs-evs on liver injured and liver function. finally, high throughput sequencing of small rnas was performed to investigate mirna expression profiles in ipscs-evs and ipscs. results: the ipscs-evs that were all 50-200 nm, doublelayered and oval or round cellular vesicles and expressed the marker proteins cd63, tsg101 and hsp70. in vitro, the ipscs-evs treatment inhibited heprg apoptosis induced with d-galactosamine in a time-and dosedependent manner and promote the proliferation of hepatic stem cells. in vivo results showed that ipscs-evs significantly alleviated liver failure, improved liver function and prolonged the survival period. tunel assay showed that ipscs-evs suppress apoptosis of hepatocytes. moreover, mirna expression profiles analysis found that mir17-92a cluster and mir302-367 cluster were enriched in ipscs-evs and ipscs. summary/conclusion: these findings indicated that ipscs-evs could ameliorate d-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte apoptosis, which will be benefit for therapy of liver disease in the future. msc-derived extracellular vesicles promote human cartilage regeneration by control of autophagy introduction: osteoarthritis (oa) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. recently, allogeneic human mesenchymal stromal/stem cells (msc) entered clinical trials as a novel therapy for oa. increasing evidence suggests that therapeutic efficacy of msc depends on paracrine signalling. here we investigated the role of bone marrow msc-derived extracellular vesicles (bmmsc-evs), an important component of msc secretome, in cartilage repair. methods: to test the effect of bmmsc-evs on oa cartilage inflammation the tnf-alpha-stimulated human oa chondrocytes were treated with bmmsc-evs and inflammatory gene expression was measured by qrt-pcr after 48 h. to access the impact of bmmsc-evs on cartilage regeneration the bmmsc-evs were added to the regeneration cultures of oa chondrocytes, which were analysed after 4 weeks for glycosaminoglycan content by dmmb and qrt-pcr. paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-o) and type ii collagen (immunostaining). results: we show that bmmsc-evs promote cartilage regeneration in vitro. treatment of oa chondrocytes with bmmsc-evs induces production of proteoglycans and type ii collagen and promotes proliferation of these cells. msc-evs also inhibit the adverse effects of inflammatory mediators on cartilage homoeostasis. our data show that bmmsc-evs downregulate tnfalpha induced expression of pro-inflammatory cox-2, pro-inflammatory interleukins and collagenase activity in oa chondrocytes. the anti-inflammatory effect of bmmsc-evs involves the inhibition of nfκb signalling, activation of which is an important component of oa pathology. autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is essential to maintaining chondrocytes survival and differentiation. the expression of autophagy regulators is reduced in osteoarthritic joints, which is also accompanied by increased chondrocyte apoptosis. our preliminary data indicate that bmmsc-evs carry mrna of natural autophagy inducers and promote autophagy in oa chondrocytes. therefore, we hypothesize that msc-evs exert their beneficial effects on cartilage regeneration by restoring the expression of autophagy regulators. summary/conclusion: in summary, our findings indicate that bmmsc-evs have ability to promote oa cartilage repair by reducing the inflammatory response and stimulation of oa chondrocytes to produce extracellular matrix, the essential processes for restoring and maintaining cartilage homoeostasis. thus, msc-evs hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis. large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established graft-versus-host-disease model in batch dependent manners introduction: extracellular vesicles (evs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (mscs) can suppress acute inflammatory cues in a variety of different diseases, including graft-versus-host disease (gvhd) and ischaemic stroke. furthermore, they can promote regeneration of affected tissues. following a successful clinical treatment attempt of a steroid refractory gvhd patient, we intend to optimize msc-ev production strategies for further clinical applications. as we observed functional differences of independent msc-ev preparations in vitro, we aimed to adopt an in vivo gvhd model for the more advanced functional testing of different msc-ev preparations. methods: to this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (iir). gvhd was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine t cells. if not treated otherwise, myeloablated mice developed severe gvhd symptoms. results: the gvhd symptoms were effectively suppressed, when msc-ev preparations were applied at 3 consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. msc-ev preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the gvhd mice. thus, our results demonstrate that not all msc-ev preparations harvested from adult bone marrow-derived mscs contain the same therapeutic potential. summary/conclusion: thus, successful transplantation of msc-evs into the clinics requires a platform allowing identification of msc-ev preparations with sufficient therapeutic, most probably immune modulating activities. funding: this research was funded by sevrit leitmarkt lifescience.nrw ls-1-1-051 g. introduction: malnutrition impacts approximately 50 million children worldwide and is linked to 45% of global mortality in children below the age of five. severe acute malnutrition (sam) is associated with intestinal barrier breakdown and epithelial atrophy. extracellular vesicles including exosomes (evs; 30-150 nm) can travel to distant target cells through biofluids including milk. since milk-derived evs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. methods: mice were fed either a control (18%) or a low protein (1%) diet for 14 days to induce malnutrition. from day 10 to 14, they received either bovine milk evs enriched using differential ultracentrifugation and sucrose gradient purification or control gavage and were sacrificed on day 15, 4 hours after a fluorescein isothiocyanate (fitc) dose. tissue and blood were collected for histological and epithelial barrier function analyses. results: mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. despite continued low protein diet feeding, milk ev administration improved intestinal permeability, intestinal architecture and cellular proliferation. summary/conclusion: our results suggest that evs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality. funding: cb was generously awarded a catalyst grant from the centre for global child health at the hospital for sick children to support this work. the impact of spheroids culture on mesenchymal stem cells and ev production introduction: mesenchymal stem/stromal cells (mscs) are now widely believed as bio-factories releasing bioactive products responsible for their therapeutic effect, i.e. cytokines, chemokines, and extracellular vesicles (evs). mscs are highly sensitive to physical stimuli from their surrounding microenvironment and can change their characteristics in response to their environment. the application of 3d spheroids cell culture allows mscs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. we aim to determine how 2d and 3d culture microenvironments can modulate the ev production and investigate their anti-fibrotic activity. methods: for 2d culture, bone marrow-derived mscs were cultured on standard tissue culture plastic. for 3d culture, mscs were aggregated into spheroids using non-adherent 96-well plates and cultured with addition of 0.25% methylcellulose. to collect conditioned media, both 2d and 3d mscs were cultured using serum free medium for 4 days. evs were isolated by serial ultracentrifugation and were characterised on exoview platform which allows simultaneous detection of particle size and expression of cd81/cd63/cd9. cell lysates were collected for mirna isolation and qrt-pcr was performed to analyse expression of candidate mirnas. to model the progress of lung fibrosis, human lung fibroblasts (hlfs) were cultured with tgf-β1 to induce fibroblast activation, subsequently exposed to 2d and 3d evs, and collagen production was measured. further, 2d and 3d msc-evs were added into human lung mscs isolated from healthy and ipf patients and cell proliferation was assessed using mts assay. results: 2d and 3d msc-evs have similar ev characteristics in terms of particle size and ev tetraspanin markers expression. exoview analysis showed expressions of cd81/cd63/cd9 and average particle diameters of <100 nm. on a cellular level, we identified a panel of anti/pro-fibrotic mirnas which are differentially expressed in 2d and 3d mscs. 2d and 3d msc-evs have similar anti-fibrotic activity shown by their ability to reduce collagen deposition in hlf cultures. both 2d and 3d msc-evs could promote cell proliferation on ipf lung mscs but no overall effect on healthy lung mscs. summary/conclusion: this concept of engineering the cellular microenvironment to promote ev production is as yet untouched and we foresee that in 3d cultures, we can culture mscs for longer timeframe and therefore maximising the overall ev production process. the outcome presents future potential for 3d culture of msc to increase the efficiency and feasibility of scalable ev production. outer membrane vesicles from photobacterium damselae subsp. piscicida: characterization and antigenic potential introduction: photobacterium damselae piscicida (phdp) is a gram-negative bacterium that causes a septicaemia in > 20 fish species worldwide. it represents a major drawback for aquaculture, whose importance has been sharply growing as a food supplier. given the phdp massive mortality and widespread antibiotic resistance, an effective vaccine is highly needed. extracellular products (ecps) have an essential role in phdp virulence, containing important antigens. however, the ecps' identity remain undisclosed. in our efforts to dissect their composition, we found that they contain high amounts of outer membrane vesicles (omvs). these particles are potent weapons for bacteria and are being explored in the field of vaccinology, since omvs present antigens in native conformations and are strongly immunogenic, without requiring adjuvants. this potential associated to the urgent need for an anti-phdp vaccine prompted us to isolate and characterize the omvs shed by phdp. methods: in order to harvest high amounts of pure phdp omvs, a reproducible optimized protocol was developed: the bacteria-free supernatant from a phdp overnight culture is concentrated, dialysed and ultracentrifuged to collect the omvs. results: analysis of the obtained omvs preparations by transmission electronic microscopy and dynamic light scattering indicate that the main population of vesicles has sizes around 30-50 nm. proteomic analysis of the vesicles revealed the presence of the apoptogenic ab toxin aip56 that is known to play a major role in phdp virulence, a putative pore-forming toxin, a putative adhesin/invasin and several outer membrane proteins (omps), including a 64kda omp, predicted to be involved in iron acquisition, and 5 other omps (18-34kda), with an ompa-like structure that may act as adhesins. moreover, preliminary in vivo studies suggest that some of those proteins may have important roles for virulence, since injection of knock-out strains in sea bass induced a decreased mortality comparing to the wt strains. summary/conclusion: our findings suggest that omvs are a promising vaccine candidate and we are currently studying their biological activities and determining the antigenic potential of the identified proteins. introduction: whole body exposure to high doses of ionizing radiation (ir) can potentially be lethal if radiation injury is not diagnosed and treated expeditiously. when considering a non-invasive approach for the identification of biomarkers of ir exposure, we and others have studied molecules in plasma, serum, saliva, and urine. however, these matrices can potentially have significant background noise, obscuring potential biomarkers of biological importance. extracellular vesicles (evs) are fast becoming a platform for biomarker discovery in radiation research as well as in other pathologies. however, no groups have investigated the use of metabolomics to analyse evs derived from urine in the context of ir exposure. furthermore, the dominant protocols for ev isolation from urine require a large (up to 30 ml) amount of starting volume, which may not be available for many studies. the aim of this study was to optimize ev isolation from rat urine and assess radiation-induced alterations in urine ev number and metabolic content. methods: as a proof of concept, we compared and optimized several ev isolation methods on small volumes of urine from male wag/rijcmcr rats exposed to 0 gy or 13 gy x-rays to the whole body except the hind leg. starting with either 500 µl or 1000 µl of urine, we isolated evs using ultracentrifugation (uc) with filtration, size exclusion chromatography (sec), and a proprietary bead-based isolation method developed by a 3rd party provider. ev samples were characterized using nanoparticle tracking analysis. metabolomics profiles were measured using lc-qtof-ms. results: we found that sec resulted in the highest yield of evs from as little as 500 µl of urine, while uc was the poorest performing. lc-qtof-ms analysis revealed that sec and uc had the most consistent identification of features, whereas the bead-based method contained artefacts likely as a result of the extraction method. we next used sec to isolate evs from a larger cohort of rats exposed to ir and analysed with ms. ev metabolic content will eb related to differences in survival and organ function between sham and irradiated groups. summary/conclusion: we conclude that sec is the preferred method for isolating evs from small volumes of urine for broad-based mass spectrometric analysis, and that the ev metabolome may be a sensitive and specific early indicator of radiation injury. introduction: there is growing evidence that contents (including rna and proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction of cancers. here we aim to identify potential protein markers for oesophageal cancer. methods: using our newly developed label-free exosome automated preparation system (leaps), exosomes were isolated from 20 ml culture medium of various oesophageal cancer cells with different differentiation profiles and different sources of metastasis. exosomes from 20 µl plasma of cancer patients at different clinical stages or with/without relapse and healthy controls were also prepared by leaps. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) was employed to directly analyse exosomes. protein identities of exosomal fingerprint peaks were tentatively assigned by correlation with top-down and bottom-up proteomics. results: start from 20 ml culture medium or 20 µl plasma, high-quality exosomes rapidly isolated by leaps are sufficient for maldi-tof mass spectrometry. it seemed that poorly differentiated cells showed more exosome release. maldi-tof ms fingerprints of exosomes in cells is cell line specific. ms profiles from poorly differentiated cells showed more peaks than that from highly differentiated cells. fingerprints also allowed classification of cancer cell lines through software mathematical analysis. we identified different numbers of significantly differentially expressed peaks in exosomes of various cancer cells. fingerprints of exosomes derived from the poorly differentiated cells showed more elevated peaks. top four peaks (2,427 m/z, 3,596 m/z, 4,458 m/z, 4 ,537 m/z) were commonly down-regulated in exosomes of most cancer cells. top four protein peaks (2,268 m/z, 5,713 m/z, 6,255 m/z, 6,337 m/z) that might be correlated to the differentiation profile of cancer cells were also identified. maldi-tof ms detection of exosomes in the plasma and clarifying identities of potential biomarker peaks will be done in the future. summary/conclusion: the combination of leaps and maldi-tof mass spectrometry provides a fast and high-throughput tool for exosomal marker discovery. potential biomarker identified in exosomes derived from oesophageal cancer cells or from plasma of cancer patients by this tool might be useful in cancer diagnosis and prognosis. fraction-based proteomic profiling of serum extracellular vesicles derived from cervical cancer patients introduction: current evidence indicates that extracellular vesicles (evs) can release from most of cell types and affect adjacent or distant cells by circulating in all bodily fluids. proteomic analysis of evs from clinical samples is complicated by the low abundance of ev proteins relative to highly abundant circulating proteins. size exclusion chromatography (sec) has been overcome as a method to deplete protein contaminants and enrich evs. methods: we collected serum of healthy women and cervical cancer patients with stage i-iii and then counted concentration and size distribution of the evs using nanoparticle tracking analysis (nta). differential ultracentrifugation combined with sec was used to isolate and purify evs from contaminant proteins. isolated evs were investigated their characteristic based on morphology using transmission electron microscope (tem) and on expression of cd63, cd81, cd9 protein markers using western blot analysis. fraction no.8-10 of isolated evs in among sample groups were profiled by nano-liquid chromatography tandem mass spectrometry (nanolc-ms/ms) analysis. results: nta shows that the concentration of evs is increased in patients compared with healthy women. proteome profiles of evs isolated by sec were compared in each fraction. moreover, we detected molecular evidence for fraction-specific molecular pathways in connection with cancer progression and complied a set of protein signatures that closely reflect the associated clinical pathophysiology. summary/conclusion: these unique features in each fraction among sample groups would be the informative considering in order to select for further analysis as in vitro. introduction: recently, diagnostic biomarkers from exosomes by proteomic analysis have been reported, but it is required to optimize the isolation protocol to screen out more effective biomarkers. for serum-originated exosomes, it has been also reported to isolate them selectively, however, it is observed that a different method resulted in different protein profiles in 2-d gel electrophoresis. methods: we isolated exosomes by two discrete methods, using ultracentrifugation and magnetic separation. before ultracentrifugation and magnetic separation, precipitation using polymer materials was perforemd. the isolation of exosomes by these two methods followed by comparison of their size, total vesicle number, morphology, and protein markers. to identify protein biomarkers, proteomic analyis using 2-d gel electrophoresis was performed. results: both methods induced enrichment of exosome-specific proteins, but protein profiles in each exosome fraction was totally different. the protein profiles showd that the magnetic seperation following a polymer-based precipitation step was more efficient to screen out candidate biomarkers, which showed nearly 200 protein profiles originated from exosomes. summary/conclusion: in our study, magnetic separation of exosomes from serum fraction was optimized for 2-d gel electrophoresis to observe identifiable biomarkers. an extracellular small rna-seq data processing pipeline optimized for high-performance computing chenghao zhu and angela zivkovic department of nutrition, uc davis, davis, usa introduction: a variety of rna species is found in extracellular biofluids such as blood, bile, and urine, carried by extracellular particles including extracellular vesicles (evs) and lipoproteins (e.g high density lipoproteins (hdls)). the extracellular rna (exrna) carried by evs and hdls is of great interest for two reasons: 1) the exrna within different carriers could be diagnostic of the state of the tissues from which the particles originate, and 2) exrna has been shown to affect gene expression in target cells. although the origin and functions of exrnas remain largely unknown, there is growing interest in exrna research for the development of diagnostics and new therapeutic targets. small rna sequencing is widely used to estimate the abundance of exrnas in biofluid samples. here we present a data processing pipeline for extracellular small rna sequencing. sequencing data are pre-processed through quality control, and then aligned to the endogenous genome to obtain the gene counts for various rna biotypes, including microrna, trna, rrna, piwi-interacting rna, long non-coding rna (lncrna) and protein coding rna. it also aligns sequencing reads to exogenous databases, including the ribosomal rna sequence database silva, and all sequenced bacteria genomes available on ensembl, to estimate the abundance of exogenous genes. results: we analysed a publicly available small rna-seq dataset of hdl from three systemic lupus erythematosus (sle) patients and three healthy controls using this pipeline. the mirna hsd-mir-93, lncrna al137186.2 and ac108050.1 were elevated in sle patients compared to controls. exogenous rna reads mapped to bacteroidetes were also elevated in sle patients. summary/conclusion: our pipeline is able to process exrna sequencing data and estimate the abundance of major exrna species, as well as exogenous rna taxonomy. the pipeline is optimized for the job scheduler slurm, and can therefore utilize the full computational power of high-performance computers. the pipeline is publicly available on github (www.github. com/zhuchcn/excernapipeline). introduction: ibd is a chronic hyperinflammatory disorder that severely compromises the intestines. the aetiology of ibd is poorly understood. however, it has been associated with a dysregulation of the immune system and gut microbiota and with genetic and environmental factors. cumulative evidence indicates that evs play an essential role in modulating immune responses. recent research suggests that evs derived from dendritic cells, saliva and intestinal epithelial cells may be involved in the progression of ibd inflammation. however, little is known about the contribution of immune cells-derived evs with this pathology. the goal of this study is to shed light on the contribution of pbmc-derived evs on ibd pathogenesis. here we characterized and compared the composition of evs derived from pbmcs of 3 ibd patients and 1 healthy control. evs were isolated by differential centrifugations from the supernatant of pbmc activated with cd3-cd28 beads for 6 days in serum-free media. size and concentration were analysed using a nano sight 300 instrument, while the presence of known evs markers (cd63, cd81, hsp70) was analysed by immunoblotting. whole evs proteome was performed by ms/ms and functional-enrichment analysis was done using funrich with uniprot database. results: proteomics analyses identified a total of 1299 proteins in the four groups. of those, 673 (51.8%) were present in both the ibd patients and control. this group of protein was composed of several ras-related proteins, eukaryotic initiation factors, granzyme, cd9, tubulin, and serpins among others. patients' evs shared 46 proteins in common such as proteasome subunit beta type-10, t cell receptor beta, and the amine oxidase containing copper 3. interestingly, each patient sample had a unique group of proteins. among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type-3, and signalling lymphocytic activation molecule (slamf1). summary/conclusion: these preliminary studies show that the ev composition from pbmcs of ibd patients is specific and differs from a healthy control. this exclusive composition has the potential to be used as a biomarker for diagnostics and progression of the disease, and it could also provide new insights into our understanding of the cellular pathways involved in the pathogenesis of ibd. the studies were performed with corresponding irb approvals. proteomic analysis of exosomes isolated using precipitation and columnbased approaches introduction: exosomes are a subtype of small extracellular vesicles (evs) involved in various physiological and pathological processes with huge potential as biomarker resources or as therapeutic tools. although several exosome isolation approaches are available, complementary studies focusing on optimizing the methods for human bloodderived exosomes isolation and method-specific comparative exosomal proteomic profiles will be of clinical value. methods: blood-derived evs were isolated through precipitation-and column-based methods and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. serumderived exosomal proteomes were analysed by mass spectrometry (ms). the resulting proteomes were then overlapped with the proteomes obtained from exosome-related databases, to determine the % of similar content. in addition, bioinformatic analysis, including gene ontology (go) was carried out. results: both methodologies tested isolated particles with the expected morphology and size range, although the column-based method isolated a higher number of particles. about 75% of the exosomal proteins identified through ms overlapped with the proteomes extracted from the databases. go terms were similar for the proteomes isolated from the column-and precipitationbased methodologies. the top 3 go terms identified for molecular function were ion binding, peptidase activity and enzyme regulator activity and for biological process were immune system process, transport and response to stress. further, partial least square analysis revealed a clear segregation of proteomes obtained by the distint methodologies and complementary statistical analysis revealed the proteins differently expressed. summary/conclusion: no major differences were found in the top 3 biological processes and molecular function based on go analysis. nonetheless, the two approaches result in different evs yields and significant proteome differences were identified. characterization of distinct methods for blood-derived exosomes isolation can be useful in the context of evs potential in disease diagnostics/therapeutics. introduction: we and others are developing biomarkers for neurodegenerative diseases using neuronalenriched evs immunocaptured from a suspension of total plasma evs. here we assess how the isolation method for total evs affects the yield, purity and enrichment of neuronal evs. methods: for n = 5 subjects, total evs were isolated by ev precipitation solution (exq), ev precipitation solution plus bipartite resin columns (exu) and size exclusion chromatography (qev) from 0.5, 0.5 and 2.7 ml plasma, respectively. then, neuronal-enriched evs were immunoprecipitated using anti-l1cam antibody. in total and l1cam evs, we measured particle concentration by nanoparticle tracking analysis, protein concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins cd81, cd63 and cd9 on intact evs. results: for total evs, yield followed the order of exq > qev > exu, assessed by particle (p < 0.0001) and protein concentrations (p < 0.001). l1cam evs immunocaptured after exq showed 16-fold higher particle (p < 0.001) and fivefold higher protein (p < 0.01) concentrations compared to l1cam evs after exu, and 41-fold higher particle (p < 0.001) and 11-fold higher protein (p < 0.001) concentration compared to l1cam evs after qev. l1cam evs after ev precipitation (exq) showed 48, 35 and 30-fold higher cd81, cd63, and cd9 concentrations (p < 0.001) compared to l1cam evs after exu, and 59, 43 and 36-fold higher cd81, cd63, and cd9 concentrations (p < 0.001) compared to l1cam evs after qev. l1cam evs following 3 different methods had equal purity assessed by ratios of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations (p = ns). summary/conclusion: l1cam ev immunocapture preceded by exq exceeded the yield of immunocapture preceded by exu or qev. recovered l1cam evs showed equal purity by particle/protein and tetraspanin/particle metrics. neuronal enrichment results will be available by the time of isev. immunoprecipitation following exq, often considered impure, purifies final isolates as effectively as more onerous methods typically considered purer. balancing sensitivity, purity and scalability is essential for implementation of blood biomarkers in the clinical setting and may be achieved by combining techniques. funding: this research was supported in part by the intramural research program of the nih, national institute on aging. characterisation of breath exosomes: towards non-invasive diagnosis deanna ayupova a , renee goreham b and paul teesdale-spittle c a school of chemical and physcial sciences, victoria univeristy of wellington, wellington, new zealand; b university of newcastle, newcastle, australia; c school of biological sciences, victoria univeristy of wellington, wellington, new zealand introduction: breath-derived exosomes present new potential for non-invasive diagnosis of lung cancer. however, breath-derived exosomes have not been well characterized and methodology for their purification has not been optimised. in order to exploit their potential for diagnosis, it is first necessary to develop methods that reproducibly provide high quality pure exosomes from breath. in this study, we optimise methods for their isolation and characterise them in comparison to exosomes derived from cell culture models. methods: in order to characterize exosomes from exhaled breath condensate (ebc) it was first necessary to optimize methods for isolation of pure, intact, and high quality exosomes. to this end, isolation methods were optimised on cell-derived exosomes and then applied to ebc, yielding high quality exosomes from size exclusion chromatography (sec). ebc exosomes were compared with those from a549 and wi-cells using dls, tem, and cryo-sem. an immunoblotting-grid technique was used to validate the presence of exosome-specific markers cd63 and cd81. protein content of exosomes were quantified and compared. results: sec-based isolation was more effective at isolation of pure and intact exosomes than ultracentrifugation, with the highest purity exosomes obtained in the middle fractions of the exosome-containing eluate. exosomes from ebc had a size range (45-100 nm), protein content (20-40 ug/ml) and molecular markers typical of cell-derived exosomes. summary/conclusion: breath-derived exosomes isolated through size exclusion chromatography are sufficiently pure for diagnostic purposes and are phenotypically similar to exosomes derived from other sources. we foresee their use in non-invasive diagnostics for lung cancer as an important future application. ligand-based exosome affinity purification (leap) is a rapid and reproducible method for the enrichment of functional evs introduction: platelet-derived extracellular vesicles (pevs) represent the next generation of therapeutic biologics as they enable a more refined and targeted approach when compared to crude blood derivatives currently used for treating diseases such as cancer, thrombocytopenia and chronic wounds. however, development of an ev-based therapeutic is hindered by the lack of a scalable, validated and reproducible purification process. in this study, pevs were isolated from activated platelet concentrates and purified using exopharm's ligand-based exosome affinity purification (leap) technology to produce a functionally active ev therapeutic. methods: platelet concentrates (n = 20) were obtained from the australian red cross blood service and were activated by exopharm's proprietary process. activation was verified by measuring cd62p using flow cytometry. the resulting platelet releasate (500 ml) was subjected to leap purification to isolate pevs. for characterization, protein concentration was determined by a bicinchoninic acid assay, microfluidic resistive pulse sensing (mrps) was used to perform a particle count and transmission electron microscopy (tem) enabled visualization of ev morphology. key ev markers were detected using mass spectrometry (ms) and western blots. to confirm biological activity, human dermal fibroblasts were subjected to serum starvation for 16 hours before treatment with pevs (15 µg/ml). cell growth was recorded by the real-time xcelligence system and differences in proliferation were statistically analysed using a one-way anova. results: mrps and tem both revealed isolated pevs to be 100-300 nm in size. the final product was positive for platelet markers (cd41, cd62p) and key ev markers (tsg101, alix, cd63). treatment with purified pevs significantly increased proliferation in serumstarved fibroblasts over 48 hours. summary/conclusion: exopharm's leap technology is a rapid and reproducible purification process which produces pevs that adhere to misev 2018 guidelines and are functionally active. funding: all funding was through exopharm ltd (asx:ex1) a novel but simple method to obtain purified exosomes by one-step ultracentrifugation introduction: exosomes are extracellular vesicles (evs) that are derived from endosome membrane. they are usually 50-150 nm in diameter, actively secreted in most living cells. originally, exosomes were thought to act as cellular garbage disposals. recent studies showed that exosomes not only can serve as biomarkers for diagnosis, but also can be used as an ideal delivery vehicle for drugs in therapeutics. exosomes are natural carrier for mrna, mirna, sirna, protein, dna and peptide for long distance intercellular communication. isolation of exosomes is challenging due to their small size and heterogeneity. traditional differential ultracentrifugation method is still the gold standard for exosome purification. to further explore the potentials of exosomes being as the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to purify them in high quality at high yield. methods: here, we report a novel method to obtain intact shape, high-quality and high purity exosomes with one-step ultracentrifugation by using "exojuice". results: data of nanoparticle tracking analysis (nta) and western blotting showed "exojuice" can yield exosomes with a simpler method to obtain higher purity exosomes in comparison to previous method of cushion ultracentrifugation using optiprep. summary/conclusion: our method can be used to purify exosomes from cell culture medium, serum, urine, saliva, and other biofluids. a straightforward device to extract apoplastic fluid from succulent fruits for higher purity of extracellular vesicles introduction: edible plants are emerging as a sustainable source for extracellular vesicle (ev)-based drug delivery vehicles. however, current isolation methods (e.g. grinding or squeezing) may cause destruction of plants' biostructures, and in turn leads to unwanted effects in downstream applications and complicates the study of nanovesiclescell. therefore, we designed a simple device that allows the extraction of apoplastic fluid (af) from succulent fruits, facilitating ev isolation as well as effective downstream applications. methods: an inner filter tube was designed to extract af with a determined membrane pore size. af was collected by low-speed centrifugation method and then filtered to eliminate the impurities from the cytoplasm and damaged cells. minced juice (mj) was homogenized by a blender and then centrifuged to remove large fragments. subsequently, the differential centrifugation method was employed to extract evs from af and mj. fourier-transform infrared spectroscopy (ftir), nanoparticle tracking analysis (nta), and transmission electron microscopy (tem) were performed to discriminate af, mj and their evs. results: the "spectroscopic" protein-to-lipid (p/l) ratio of af (1.23 ± 0.03) is significantly lower than that in mj (1.27 ± 0.01), showing the higher lipid contents in af, which may result from the loss of lipids in mj obtained from grinding or juicing methods. similarly, ftir showed the difference in p/l ratio between af and its evs (1.23 ± 0.03 and 1.29 ± 0.02, respectively). nta showed the sharper peak and smaller vesicle size in the following order: mj (173.0 ± 21.3 nm), af (154 ± 11.0 nm), af-derived evs collected at 40,000 × g and 10,000 × g (108.5 ± 2.4 nm and 95.7 ± 3.1 nm, respectively). furthermore, tem study indicated that the collected evs exhibited a typical lipid bilayer of extracellular nanovesicles. summary/conclusion: by using a reusable filter device, we successfully isolated af from succulent fruits, paving the way to collect plant evs without an interference of significant biodestruction or damaged cells, hence improving the purity of evs and facilitating downstream applications. moreover, this method is straightforward, reproductive, and can be potentially used in a large-scale production. method to simultaneously capture multiple classes of intact extracellular rna carriers including extracellular vesicles and lipoprotein particles introduction: extracellular particles including extracellular vesicles (evs), lipoproteins, and free proteins are carriers of extracellular rna (exrna), which has been shown to regulate cellular function. because these particles have different physiological origins, they have different rna signatures, so the first step to understanding the biology of exrna is to isolate individual particle fractions with high purity and efficiency. current methods for isolating evs are optimized for increased yields and purity of ev fractions but typically require multiple millilitres of starting plasma and do not capture the other exrna carrier particle types. methods that can capture evs from low starting plasma volumes and can also capture other exrna carriers simultaneously are needed for analysing samples from previously conducted large cohort studies, biorepositories, and in populations where sample volume is limiting. methods: we have developed a method adapted from lipoprotein isolation that requires only 500 µl of starting plasma, and uses brief ultracentrifugation (uc) followed by fast protein liquid chromatography (fplc) to capture 6 classes of purified exrna carriers including evs, ldl, hdl, lipidated albumin, proteins, and vldl/chylomicrons. we have validated successful capture of evs by microfluidic resistive pulse sensing (mrps, spectradyne), transmission electron microscopy (tem), and single particle interferometric reflectance imaging system (sp-iris; exoview) with optional fluorescence. results: we have observed 1.02 × 1010 particles per ml from a 1 ml fplc fraction of evs measured from 25,000 events by mrps, confirming that evs are being captured by this method. there were also 1.26 × 1010 particles/ml and 2.70 × 1010 particles/ml in the two subsequent 1 ml fractions that are known to contain lipoprotein particles, though these were measured from 1,500 events each. by tem we confirmed these observations that evs are eluting before lipoprotein particles with some evs eluting later in fractions containing lipoproteins. summary/conclusion: these results confirm the efficacy of the method in isolating multiple exrna carrier fractions simultaneously from a single 500ul plasma sample, making it amenable for the analysis of exrna in samples from large cohort studies, biorepositories, and vulnerable populations such as the elderly and young children. funding: nih/nia r01ag062240; nih ug3 ca241694-01 optimizing the isolation of placental mesenchymal stromal cell-derived extracellular vesicles in a 3d bioreactor system leora goldbloom-helzner a and aijun wang baaaaa a uc davis, davis, usa; b uc davis medical center, sacramento, usa introduction: extracellular vesicles (evs) derived from placental mesenchymal stromal cells (pmscs) have the potential to provide neuroprotection at sites of injury. however, a rate limiting step in ev research is the low yield, high technical time, and high cost of current isolation procedures. to address this inefficiency, we cultured pmscs in a unique bioreactor system to increase the absolute yield of evs per ml of media and per cell. future studies will determine if this system can improve pmsc ev yield without altering the demonstrated neuroprotective properties of pmsc-evs. methods: pmscs were cultured in the bioreactor for ten weeks. ev-conditioned media was collected weekly and evs were isolated through differential centrifugation. nanoparticle tracking analysis (nta) measured ev size and concentration. western blots tested for normal ev markers (cd9, cd63, and cd81, calnexin(-)) and enzyme-linked immunosorbent assays (elisa) measured levels of characteristic growth factors in conditioned media including vascular endothelial growth factor (vegf), brain-derived neurotrophic factor (bdnf), and hepatocyte growth factor (hgf). results: evs remained consistent until week eight, after which a decrease in both ev size and concentration was seen. western blots revealed normal positive expressions of cd9, cd63, and cd81 and negative expressions of calnexin. levels of vegf, bndf, and hgf were comparable after 10 weeks. cost analysis revealed an overall increase in ev yield for shorter labour time and lower material cost. summary/conclusion: this initial study uses a bioreactor system for a unique source of cells and has brought us closer to optimizing pmsc ev isolation protocols for increased yield, lower cost and time commitment, and maintained sample purity. preliminary data suggests the ev phenotype and cell secretome are consistent with those present in current culture settings. future experiments will assess the preserved neuroprotective properties of the pmsc evs. a novel method for isolating extracellular vesicles from cell culture media and plasma using polyethylenimine introduction: due to their ability to transport dna, rna, and protein cargoes between cells, extracellular vesicles (evs) are becoming popular for biomarker discovery as well as for therapeutic delivery. here we describe the development of a novel precipitation method for the isolation of evs from cell culture media and plasma that is based on polyethylenimine (pei), an inexpensive, water-soluble, and biocompatible cationic polymer. pei is a group of hydrophilic cationic polymers that are synthesized as either linear or branched forms of varying molecular masses (1,000 to 750,000 da) and are widely used in the biomedical field as a coating and transfection agent. methods: linear and branched pei of varying molecular weights (mw) were tested for their ability to precipitate evs from either conditioned culture media (ccm) or human plasma. isolated evs were characterized by western blotting and nanoparticle tracking analysis (nta). the small rna profile of evs isolated using pei from human plasma was analysed by ngs and ev-specific mirnas were confirmed by digital droplet pcr (ddpcr). mass spectrometry (ms) was used to analyse the proteome of pei-captured evs from plasma. hek293 cells producing gfp+ evs were used to optimize conditions for release of evs from both linear and branched pei by fluorescent spectrophotometry and flow cytometry measure-ments. results: linear and branched pei were both able to precipitate evs as determined by western blotting for ev protein markers; however, branched pei with mw > 10,000 da and linear pei with mw > 25,000 da were more efficient for ev precipitation than lower mw forms. despite its known ability to bind nucleic acids pei was unable to capture cell-free dna from plasma, although rna and in particular ev-associated mirnas such as mir-142-3p were recovered. ms revealed that pei enriches extracellular exosome proteins from plasma. evs captured from ccm by pei could be released from the complex using heparin or high salt conditions. summary/conclusion: pei has an unexpected preference for associating with evs compared to nucleic acids in complex biological samples and has a hitherto unrecognized application for ev precipitation. introduction: there is ongoing debate about which is the most appropriate method for isolation of evs, with most labs using some combination of differential ultracentrifugation (uc), size-exclusion chromatography (sec), and/or density gradient ultracentrifugation (dg). here we applied a surface-enhanced raman spectroscopy (sers) analysis platform to compare chemical composition of the isolate from each method against lipoprotein standards to assess the relative purity of the ev preps. methods: 2-3 ml of plasma was separated from whole blood collected from head and neck cancer patients. each sample was split into 3 batches and evs were isolated by either uc, sec, or dg. following isolation, samples were incubated on commercial sers substrates and raman spectra were collected. lipoprotein standards were purchased and also measured for comparison. using principle component analysis (pca), spectra were analysed for chemical variability. results: sers analysis of sec, uc, and dg isolated evs were chemically distinguishable using simple pca. the chemical changes could in large part be attributed to fitting the differences in spectra to lipoprotein standards. we found that uc isolated populations clustered with the high-density lipoproteins (hdl), sec populations with the low-and very low-density lipoproteins (ldl, vldl), and dg populations were more variable, but mainly clustered together with the highdensity-lipoproteins (hdl). summary/conclusion: this set of experiments matches our expectation that various lipoprotein would contaminate ev preps according to their relative size and density distributions. no single isolation method could separate pure ev samples. this study also illustrates the utility of label-free sers analysis for rapid chemical characterization of evs. bioreactors: lessons to develop an extracellular vesicle factory vanessa chang, priscila dauros-singorenko, lawrence w. chamley, colin l. the university of auckland, auckland, new zealand introduction: high density mammalian cell culture systems (bioreactors) provide valuable advantage for large scale production of secreted products such as extracellular vesicles (ev). however, optimisation of design selection, handling and operational costs can be quite challenging. here we provide our experience with a celline bioreactor system. methods: cultures of 6 adherent cell lines were established in celline ad 1000 bioreactors and propagated for up to 19 weeks. media was changed twice weekly and cells shed into serum-free conditioned medium were counted and assessed for viability. nanoevs were isolated by sequential centrifugation (2000 g -10,000 g -100,000 g) and size exclusion chromatography (sec). nanoevs were characterised in their protein (bca) and particle (nanoparticle tracking analysis) amount, ev markers (western blotting) and morphology (transmission electron microscopy, tem). results: the viability of shed cells varied between cell lines and through time, suggesting a changing dynamic during reactor establishment and continuous growth phases, that was specific to each cell line. hdfa, bt20 and bt474 consistently shed mainly dead cells (60-100%), as opposed to mcf7 and mda-mb-231 which predominantly shed live cells. sec fractionation of nanoevs identified a dominate ev-rich peak and significant quantities of smaller proteins, highlighting the need for further purification. nanoev yields from each 3-4 day culture averaged 2-7 × 1010 particles, representative of yields obtained from cells grown in 8 to 28 conventional t175 tissue culture flasks. ev markers and tem confirmed the protein profiles and morphology of evs obtained from bioreactors. summary/conclusion: high density bioreactor cultures offer a physiologically relevant, cost and space efficient approach to produce significant amounts of evs, providing sufficient material for numerous experimental uses. in our hands, with careful twice weekly management, they can be propagated for up to 19 weeks without significant changes to the evs. introduction: extracellular vesicles (evs) have potential applications for clinical theranostics. ultracentri-fugation is most commonly adopted to the evs isolation, which is recommended as a gold standard method. however, ultracentrifugation is time-consuming and expensive equipment requirement, resulting in the coisolation of contaminants such as protein aggregates. therefore, our aim is to develop a rapid and efficient platform to isolate heterogonous evs based on the insertion of lipid molecules into the evs membrane to avoid co-isolation of non-membranous protein particles. methods: herein, a defected nanoscale functional metal organic framework (mof) was constructed as an efficient platform for evs isolation. typically, one single-stranded dna was designed and modified with a phosphate group at the 5ʹ-end and cholesterol at the 3ʹ-end to form a capture dna named phosphate−dna−cholesterol (pdc). the phosphate group forms a strong covalent bond with the designed defeated site of zr (iv) in mof uio-66-nh2 and the cholesterol inserts into the phospholipid bilayer to capture evs without non-membranous particles contamination. the formed mof−phosphate−dna −cholesterol−evs (mof@pdc@evs) system was further treated with dnase i for dna hydrolysis to give high pure evs. results: a rapid and efficient isolation platform of evs based on a defected mof functionalized with phosphate-dna-cholesterol (mof@pdc) has been constructed successfully. compared with ultracentrifugation, mof@pdc platform promises to isolate size heterogeneous evs i) without non-membranous particles contamination, maintaining evs intact membrane structure, protein components, and biological functions; ii) with the ability to capture evs with 78% isolation efficiency; iii) makes evs isolation process simple and fast, which could be finished in 40 minutes without requirement of the expensive equipment. summary/conclusion: in conclusion, this rapid and efficient platform is suitable for isolation evs from biological fluid for downstream protein analysis. this work opens a new perspective in mof-based separation researches and may shed light on further studies towards evs isolation. introduction: incorporation of pharmacologically active molecules on the surface or the lumen of extracellular vesicles (evs) is an important strategy for maximizing the therapeutic potential of evs. genetic engineering of producer cells by introducing dna through random or site-specific integration are promising strategies for creating engineered evs. longterm stability with consistent transgene expression in the ev producer cells and therapeutic potency of resulting engineered evs are crucial for biomanufacturing. we present a comprehensive study to investigate stability of transgene expression and potency of two potential therapeutic engineered evs derived from stably selected pools transfected by either random integration (ri) or site-specific integration (ssi). methods: producer cells were engineered to make evs displaying interleukin 12 (il12) or interferon gamma (ifng) by ri or nuclease-mediated ssi into aavs1 locus. following puromycin (puro) selection, longterm cellular stability and transgene expression without selective pressure was investigated. evs were generated from stable cell pools at 0, 1, and 2 months post-thaw and purified by density gradient ultracentrifugation. purified evs were biochemically characterized by nta, bca, western blot, and cholesterol quantitation. transgene expression and biological activity of evs displaying il12 and ifng were assessed by alphalisa and in vitro reporter assays. results: transfection by ssi resulted in faster recovery in puro selection compared to ri. all stable cell pools, regardless of integration method, resulted in comparable cell culture performance, ev yield, and lipid and protein content at all time points tested. the engineered evs also demonstrated long-term stability of il12 and ifng transgene expression and in vitro activity from both integration strategies. summary/conclusion: both methods for generating stable cell lines were comparable in terms of cell stability, transgene expression, ev titre and potency, with ssi having the advantage of speed, allowing for more rapid iteration cycle times. thus, both methods are suitable for the precision engineering of therapeutic evs. this work demonstrates feasibility to manufacture therapeutic engineered evs from stable cells from either integration strategy for clinical development. transport of outer membrane vesicles as a model therapeutic delivery system in pathogenic and commensal bacteria introduction: outer membrane vesicles (omvs) in gram-negative bacteria have been shown to be important carriers of biomolecules, including toxins and other virulence factors, peptidoglycan, and nucleic acids. it has been shown that omvs play an important role in the delivery of these biomolecules to host cells and bacterial cells. while many thorough studies have explored omv delivery to host cells, few studies have explored the mechanisms of delivery of omvs to bacterial cells. our goal was to study the delivery of omvs to other bacterial cells. specifically, we were studying the oral pathogen aggregatibacter actinomycetemcomitans (a.a.), a gram-negative organism associated with localized aggressive periodontitis, to study the process by which vesicles from this organism communicate with other bacterial cells. overall, we want to understand the roles specific surface components of omvs play in the transport of these omvs to other bacterial cells. methods: we studied omvs from two strains of a.a.: jp2, a highly pathogenic strain, and 33384, a natural commensal strain. af488-labelled omvs were incubated with fresh bacterial cultures. association of the omvs with the bacterial cells was quantified using flow cytometry. to examine the role of surface-associated dna in this process, dna was digested with dnase, and the amount of surface-bound dna was quantified with the membrane impermeable dna stain, toto-1. results: using flow cytometry, we observed jp2 omvs were delivered to 33,384 cells, and at a lesser amount to jp2 cells. alternatively, 33,384 omvs associated readily with jp2 cells, more than to 33,384 cells. this suggests that the delivery of omvs to bacterial cells may be a targeted delivery mechanism. furthermore, we hypothesized surface-associated dna may play a role in this interaction. we next digested the surface-associated dna on the omvs with dnase, and observed a decrease in association between the omvs and bacterial cells. this supports our hypothesis that dna on the surface of the omvs plays a role in association. current experiments are investigating this interaction in more detail. summary/conclusion: we have demonstrated that omvs are selectively delivered to bacterial cells, and surface-associated dna plays a role in this process. we propose to investigate this process to further understand omvs delivery to bacterial cells. funding: r03de025275 & r21de027769. utilizing a gaucher's disease cell line for the evaluation of a novel exosome-based replacement therapy annie k. brown a , jiayi zhang b , brendan lawler b and biao lu b a santa clara university, san jose, usa; b santa clara university, santa clara, usa introduction: engineered nano-scale exosomes have great potential as new and targeted delivery vehicles for the treatment of gaucher's disease, the most common lysosomal storage disease. recently, we have reported the design, production, and isolation of exosomes loaded with lysosomal β-glucocerebrosidase (gba). people suffering from gaucher's disease do not have functional gba, which results in toxic build-up of undegraded substrates within the cell. methods: to evaluate the efficacy of this exosomebased therapy, a human gaucher's disease model is required. here, we have utilized near-haploid human cells (hap1) modified via crispr-cas9 to model gaucher's disease in vitro. these cells contain a 479bp insertion in the 6th exon of the gba gene, resulting in non-functional gba. pcr, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. results: characterization of gba-knock out cells shows a total loss of gba enzyme activity. further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. summary/conclusion: the utilization of a human gba-knock out cell line will enable the evaluation of the efficacy of our engineered exosomes. disease models will be an important resource for the evaluation of new biologic therapeutics, including exosomes. funding: we would like to acknowledge the santa clara university school of engineering for their support. thrxosomes: a novel exosomes based theranostic for lung cancer introduction: chemotherapy is the first-line of treatment for lung cancer. however, inefficient bio-distribution and reduced accumulation of drugs in the tumour results in treatment failure. therefore, improved drug delivery and diagnostic systems are warranted. herein, we propose a novel theranostic system "thrxosomes" where exosomes are loaded with super paramagnetic iron nanoparticles (spions) conjugated to an anticancer drug via a phresponsive linker for controlled release. we hypothesize that thrxosomes will exert profound anticancer tumour activity that can be concurrently be monitored by magnetic resonance imaging (mri). methods: thrxosomes were produced by combining normal human lung fibroblast (mrc9) cell-derived exosomes with spions conjugated to and anti-cancer drug (chemodrug or mirna) via a ph cleavable linker. the physical and biological properties of thrxosomes were determined using transmission electronic microscopy (tem), nanotracker-analysis (nta), inductively coupled plasma mass spectrometry (icpms), western blotting, cell viability, and mri. results: exosomes used in preparing thrxosomes were 130 nm in size with a typical lipid bilayer structure, and were positive for cd63, cd81, flotillin and negative for annexin a1 confirming presence and purity of exosomes. charge analysis, tem, and icmps data showed successful loading of spion-drug conjugate. biological studies showed selective and enhanced drug release under acidic condition (ph 5.5) compared to drug release at ph 7.2. cell uptake and viability studies demonstrated increased uptake and killing of thrxosome-treated human a549 lung cancer cells compared to mrc-9 cells. in vivo studies demonstrated accumulation and detection of spions by mri in in-situ tumours of a549 tumour-bearing mice. summary/conclusion: our study demonstrates thrxosomes will produce profound anticancer activity in lung cancer that is measurable by mri. exosome-modified nanoparticles as an alternative delivery system for small rnas in cancer therapy petro zhupanyn a , alexander ewe b , thomas büch c and achim aigner a a independent division for clinical pharmacology at rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany; b dr., leipzig, germany; c rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany introduction: gene knockdown by rna interference (rnai) is an alternative, non-invasive method for inhibiting proliferation or promoting apoptosis in tumour cells. this technique allows the specific targeting of key signalling proteins or mutated genes. most of the available transfection compounds suffer from rather profound cytotoxicity in vitro. the aim of our study was to establish a novel targeted small nucleic acid delivery system to the cells, with good cellular biocompatibility and applicability for in vivo studies. for this aim, we used native, cell own vesicles-exosomes. since exosomes are known to transport peptides and different rnas between cells and tissues, these unique, small extracellular vesicles (ev) may also be useful as transport vehicles for therapeutic sirna. methods: as detected by multiple cell surface protein expression analysis, exosomes carry specific surface expression markers, allowing the cellular uptake by the most of tissues. we established an ev purification protocol from tumour cell culture supernatants and a strategy for the efficient ev loading with our test sirnas or antimirs. here we used the combination of polyethylenimine (pei)-complexation of the rnas with ultrasound treatment for their loading into the evs. our ev-modified, ultrasound-treated nanoparticles were tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines or by rt-qpcr gene expression analysis. results: more efficient cellular sirna uptake was observed upon ev-modification of our pei/rna nanoparticles, accompanied by efficient inhibition of gene expression. biological efficacies were retained also after storage for several days at room temperature. the monitoring of the ev-based particles by facs revealed a different time resolution of cellular uptake and nucleic acid release compared to the classically formulated peinanoparticles. in an in vivo therapy study in tumour xenograft-bearing mice, high biocompatibility, significant biological knock-down and tumour inhibition were observed after injection of anti-survivin sirnas formulated in our ecv-modified pei nanoparticles. summary/conclusion: our data demonstrate the usability of ecv-modified nanoparticles as efficient delivery system for small rnas in cancer therapy. microglial extracellular vesicles as therapeutic vector for neuroinflammation giulia marostica a , annamaria finardi b and roberto furlan a a san raffaele scientific institute, milan, italy; b san raffaele scientific institute, milan, italy introduction: microglia is considered an eligible target against the progressive multiple sclerosis (ms), but currently available therapies do not allow its efficient targeting. as many cell types, microglia communicate with the neighbouring cells through a complex system of extracellular vesicles (evs) exchange. recently my group described that microglia derived-evs, engineered to encapsulate il4, are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. in vivo studies suggest that these evs can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target cns in ms. my project focuses on understanding the mechanism of action and the signalling pathway of evs delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. for this purpose, we decided to characterize the evs through trps technology. methods: a murine microglia cell line (bv2) was engineered to stably overproduce endogenous il4. this cell line was cultured in exosome-depleted rpmi and stimulated with pma(20 mg/ml) for 30 min. evs isolation was carried out by collecting supernatant and subjecting it to consequential centrifugation of 300 g,10 min, rt and 2000 g,20 min, 4°c. the resulting supernatant was filtered (5 µm) and ultracentrifuged at 100,000 g for 2h at 4°c. the evs pellet was re-suspended in ice-cold pbs. the evs analysis with trps shows two populations of evs, one with a mean diameter of 60-80 nm and a broad zeta potential ranging from −10 mv to −60 mv, while the second population has a mean diameter of 120-140 nm and a zeta potential of −10/-20 mv. this difference can be consistent with the different pathway formation of exosomes and microvesicles. we demonstrated in vivo the strong phenotypic change induced by our evs to resting microglia in a dose-and time-dependent effect. then, impairing the physiological procedure of the endosome acidification, the effect of our evs on recipient cells is higher. thus, suggesting an endocytic pathway for the internalization of the vesicles. we further demonstrate with gradient ultracentrifugation the capability of our formulation to vehicle endogenous il4 inside the vesicles. even if some protein is co-purified in the procedure, we know that the half-life of this cytokine is too short to elicit a strong in vivo response. consequently, we assume that the anti-inflammatory effect of our evs in vivo is a result of the il4 internalized in our formulation. summary/conclusion: these data help us understand more in detail the process of internalization and phenotype change mediated by these evs. our next goals are to discriminate between different internalization pathways and further validate the efficacy of our therapy on the eae mouse model. targeting il-3rα on tumour-derived endothelial cells blunts metastatic spread of triple negative breast cancer via extracellular vesicle reprogramming introduction: the lack of an approved targeted therapy and the early onset of metastasis highlight the need for new treatments for triple-negative breast cancer (tnbc) patients. interleukin-3 acts as an autocrine factor for tumour-endothelial-cells (tec), and exerts pro-angiogenic paracrine action via extracellular vesicles (nevs). il-3rα blockade on tec changes tec-ev (anti-il-3r-evs) microrna cargo and promotes the regression of established tumour vessels. as tec are the doorway for "drug" entry into tumours, we have aimed to assess whether il-3r blockade on tec impacts tumour progression via their unique ev cargo. methods: 27 human tnbc samples, mda-mb-231, mda-mb-453 and mcf10 cell lines were evaluated for the expression of il-3rα. nevs and anti-il-3r-evs were characterized by electron-microscopy, macsplex-exosome-kit and western blot. proliferation, migration, apoptosis and sphere formation were evaluated. scid mice were used for in vivo experiments. results: we noticed that, besides tec and inflammatory cells, tumour cells from 55.5% of the human tnbc samples expressed il-3rα. mda-mb-231 and mda-mb-453, but not mda10 cells, expressed il-3rα. in vitro, nevs provide survival and migratory signals, while anti-il-3 r-evs promoted apoptosis as well as reduced cell viability and migration of human tnbc cell lines. in vivo anti-il-3 r-ev treatment induced vessel regression in established tumours formed of mda-mb-231 cells and almost abolished the spread of liver and lung metastasis. moreover, decreased β-catenin and twist1 were found in tumours from animals treated with anti-il-3 r-evs. in addition, anti-il-3 r-evs reduced lung metastasis that was generated via the intravenous injection of mda-mb-231 cells. nevs that were depleted of mir-24-3p (antago-mir-24-3p-evs) were effective as anti-il-3 r-evs in down-regulating twist1 and reducing lung-vessel density and metastatic lesions in vivo. summary/conclusion: overall, these data provide the first evidence that il-3 rα is highly expressed in tnbc cells, tec and inflammatory cells, and that il-3 rα blockade on tec impacts tumour progression. introduction: high-grade serous ovarian cancer (hgsoc) is the deadliest gynaecologic cancer. its lethality is explained for late diagnosis at advanced stages and frequent recurrences despite achieving complete response with standard therapy. most of recurrences occurs at abdominal cavity with multiple metastasis. therefore, identifying key determinants of metastatic process remains as priority to find better therapies. current evidence assigns a central role of the exosomes in conditioning the metastatic niche in epithelial cancers. recently, we demonstrated that statins reduce metastasis in hgsoc in preclinical models. here, we decided to study the effects of statins on hgsoc-derived exosomes and its capability to condition the metastatic niche. methods: exosomes were isolated from heya8 ovarian cancer cell line and primary tissue cultures established from advanced-stage hgsocs (with signed informed consent and irb approval) by differential ultracentrifugation and quantified by nanoparticle tracking analysis (nta). enriched-cancer initiating cells (cic) spheroids were established from heya8 cells by using stem-selecting conditions. the paracrine effect of exosomes on cic migration/invasion was studied using either 3d migration or boyden chamber invasion assays. previous to exosome isolation, heya8 cells were treated with simvastatin (5um, 24 h) or solvent and proteins involved in exosome biogenesis/uptake (alix, tsg101), its trafficking (rab7a, rab27a) and in conditioning the metastatic niche (emmprin) were measured by immunoblotting. results: exosomes isolated from heya8 cells or hgsocs enhance the metastatic potential of heya8established spheroids in 3d migration or boyden chamber invasion assays. upon simvastatin treatment, we observed a significant reduction in migration/invasion induced by equivalent number of exosomes in heya8-derived cics. under same treatment, we observed a significant decrease in protein levels of alix and tsg101 and an increase in the inactive forms of rab7a and rab27a in heya8 cells. we also observed a decrease in emmprin levels in heya8-derived exosomes. summary/conclusion: here, we demonstrated a paracrine effect of hgsoc-derived exosomes that favour the metastasis process. in addition, we demonstrated that simvastatin reduces metastasis induced by cancerderived exosomes. such an effect is partially explained by changes in the expression of proteins involved in exosome biogenesis/uptake, its endocytic trafficking and in the content of proteins conditioning the metastatic niche. thus, simvastatin arises as potential therapeutic target to improve outcomes in this disease. funding: this research was supported by fondecyt granted to mauricio a. cuello label-free optical imaging and characterization of cancer-associated extracellular vesicles in tissues introduction: cancer-associated extracellular vesicles (evs) visualized in the tumour microenvironment have been identified as a potential biomarker for cancer-related tissue changes. analyses of evs have traditionally been performed in cells or isolated evs, with no temporal or spatial information that could be critically important for elucidating their roles in carcinogenesis. since the unperturbed distribution and organization of evs in the tumour microenvironment is associated with their cellular function and can potentially serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing evs in freshly isolated tissue specimens. currently, only fluorescent labelling methods enable visualization and tracking of evs. we used a custom label-free multimodal multiphoton optical imaging system to detect and characterize evs and classify them using their optical signatures both in isolated tissues and in situ tumours. methods: label-free multimodal multiphoton imaging was used to provide simultaneous, co-registered structural and functional images of evs in untreated samples. heterogeneous populations of evs could be identified from their unique optical signatures. results: the intrinsic metabolic and structural properties of evs enabled reliable visualization and optical characterization of evs from cell cultures and in situ imaging of tumour-bearing rats. unique optical signatures were then used for identification of cancer-related evs in tissues from human breast cancer patients, and their density was found to be highly correlated with clinical diagnosis. in the current study, evs were isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer patients. analysis of ev content showed higher concentration of nad(p)h in evs isolated from cancer subjects, than from healthy subjects, which reflects the reprogramming of cellular metabolism in carcinogenesis. summary/conclusion: these results suggest a potential label-free optical methodology to detect and characterize evs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. funding: this research was conducted under protocols approved by the iacuc and irb at the university of illinois and carle foundation hospital, and supported by funding from nih. novel potential anticancer therapies based on interference with nuclear entry of cancer cell-derived extracellular vesicle components in recipient cells introduction: the intercellular communication mediated by extracellular vesicles (evs) in the tumour microenvironment plays an important role in tumour progression. experimental evidence indicates that evs derived from highly metastatic cells influence the behaviour of less aggressive cancer cells. we have previously described a novel intracellular pathway where a fraction of endocytosed ev-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum (nr). here, we better characterize this pathway and report that it is required for the induction of an aggressive behaviour induced by evs released from highly metastatic sw620 colon cancer cells in isogenic primary cancer cells. methods: super resolution-structured illumination microscopy and magnetic-based co-immunoisolation studies were employed to identify the protein components of the nuclear pathway and to monitor the entry of ev-containing late endosomes into the nucleoplasmic reticulum. human sw480 carcinoma cells expressing er-gfp and rab7-rfp were exposed to evs from sw620 cells and then live imaged. results: we have previously reported that the tripartite protein complex, containing vap-a, orp3 and rab7 orchestrates the localization of ev-carrying late endosomes into nr. we now report that silencing of orp3 or vap-a, but not its homologue vap-b, reverses the pro-metastatic changes induced by evs isolated from metastatic cells on their non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding and blebbing. moreover, we found that certain nuclear pore complex proteins and importin-beta1 are co-immunoisolated with orp3, vap-a and rab7 suggesting the formation of a large protein complex at the entry of nuclear pores. summary/conclusion: interfering with the mechanisms regulating this novel intracellular pathway may find therapeutic applications particularly in ev field and oncology. educated osteoblasts regulate breast cancer proliferation via small extracellular vesicles thomas jefferson university, philadelphia, usa introduction: breast cancer commonly traffics to bone, where breast cancer cells (bccs) can survive undetected for years before metastatic outgrowth. in bone, bccs interact with surrounding stromal cells, including osteoblasts (obs), to shape the metastatic niche. our lab discovered there are at least two subpopulations of obs in the bone-tumour niche, based on protein marker expression. one group, "educated osteoblasts" (eos) have engaged in crosstalk with bccs whereas another group, naïve obs, have not. we have novel evidence that eos regulate bcc proliferation. the purpose of this study was to determine if extracellular vesicles (evs) produced by eos play a role in regulating bcc proliferation. we hypothesized evs produced by eos would decrease bcc proliferation. methods: eo-derived small evs from culture media were isolated via ultracentrifugation and characterized evs for size, protein marker expression, and density floatation to validate the purity of ev samples. the functionality of eo-derived evs on bcc proliferation was examined using edu and checkpoint proteins p21 and p27. bcc protection from chemotherapy induced cell death was also examined. results: we found that evs produced by eos, but not naïve obs, decreased both triple negative and erpositive bcc proliferation in a concentration dependent manner. furthermore, using an edu assay, we found that exposure to eo-derived evs induces bccs to undergo cell cycle arrest. interestingly, the cell cycle arrest was reversible and bcc proliferation was restored upon removal of eo-derived evs. in addition, exposure to eo-derived evs leads to increases in bcc expression of the g1 checkpoint proteins, p21 and p27. we next wanted to investigate proliferative signalling pathways that may be deregulated in bccs following exposure to eo-derived evs. we found that eoderived evs reduce bcc levels of erk1/2. because our data indicate eo-derived evs induce sustained cell cycle arrest in bccs, we desired to know if eo-derived evs protected bccs from chemotherapy-induced cell death. we found that bccs exposed to eo-derived evs and the chemotherapy drug, doxorubicin, have decreased cell death compared to bccs exposed only to doxorubicin. summary/conclusion: altogether, our data suggest eos play a crucial role in bone-tumour microenvironment by regulating bcc proliferation. funding: supported by nih r00 ca178177 and commonwealth of pennsylvania -department of health sap 4100072566 for kmb. phosphorylation of tyrosine 23 in annexin a2 is essential for its association with exosomes and for imparting invasive and proliferative capacity to other cells priyanka prakash desai a , pankaj chaudhary b , xiangle sun b and jamboor vishwanatha a a unt health science center at fort worth, fort worth, usa; b unt health science center, fort worth, usa introduction: triple negative breast cancer (tnbc) accounts for 15%-20% of all breast cancer cases. the lack of targeted-based therapies highlights the importance of studying tnbc. elevated levels of annexin a2 (anxa2), a ca+2-dependent phospholipid binding protein, has been correlated with worse overall survival in tnbc patients. our previous data implicate that exosomal anxa2 is involved in creating a pre-metastatic niche and facilitating metastasis in tnbc. moreover, n-terminal phosphorylation of tyrosine (tyr) 23 in anxa2 has been implicated in regulating several anxa2 activities in cancer progression. here, we demonstrated that n-terminal phosphorylation of anxa2 at tyr23 is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. hence, dissecting the regulatory pathway will be critical for verifying the value of anxa2 as a therapeutic, diagnostic or prognostic marker in tnbc. methods: pn1-egfp plasmids expressing the constitutive phosphomimetic (anxa2-y23e) and non-phosphomimetic mutant (anxa2-y23 f) gene expressing mutation at tyr23 site were overexpressed in mda-mb-231 tnbc cells. mutant cells were experimentally validated for anxa2 specific functions like migration, invasion and proliferation. exosomes were isolated from the mutant phosphomimetic (exo-anxa2-y23e-gfp) and non-phosphomimetic (exo-anxa2-y23 f-gfp) cells and were analysed for exosomal surface expression of anxa2 by immunoprecipitation and flowcytometry. cal-148 breast adenocarcinoma epithelial cells were treated with exo-anxa2-y23e-gfp and exo-anxa2-y23 f-gfp to analyse the rate of invasion and proliferation by transwell invasion and proliferation assay, respectively. transfer of exosomal anxa2 in cal-148 was studied using immunofluorescence and its implications on signalling pathways were studied by western blot. results: mda-mb-231 phosphomimetic tnbc mutant cells showed increased migratory, invasive and proliferative capacity compared to non-phosphomimetic tnbc mutant cells. exo-anxa2-y23e-gfp had elevated surface anxa2 expression compared to exo-anxa2-y23 f-gfp. cal-148 cells treated with exo-anxa2-y23e-gfp showed high migratory, invasive and proliferative characteristics, with a higher expression of p-anxa2(tyr23), p-src(tyr416) and p-paxillin(tyr31) compared to exo-anxa2-y23 f-gfp treated cells. summary/conclusion: n-terminal phosphorylation of tyr23 in anxa2 in mda-mb-231 tnbc cells (phosphomimetic mutant cells) is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells. funding: the above study is funded by national institute of health ro1ca220273 and nimhd's u54md006882 to dr.j.k.vishwanatha. a novel method for epithelial-derived extracellular vesicle isolation and enrichment in patients with advanced prostate cancer arpit rao, helene barcelo and bharat thyagarajan university of minnesota, minneapolis, usa introduction: evaluation of changes in prostate cancer biology is difficult due to presence of lymph nodal or bony metastatic disease in a majority of patients. a number of liquid biopsy assays have shown clinical utility in prostate cancer, but are limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability to perform transcriptome sequencing (cellfree dna-based assays). epithelial-derived extracellular vesicles (epi-ev)-based assays are uniquely positioned overcome both these limitations as evs are abundantly secreted into the blood and have rnacargo that mirrors the cell of origin. however, a reliable method to enrich for epi-evs is currently lacking. methods: plasma was isolated from the peripheral blood collected from 15 patients with metastatic prostate cancer enrolled in an institutional biobanking study before initiation of systemic antineoplastic therapy. evs were isolated from 500 µl of plasma using a commercially available qev 35 size exclusion column (izon inc.). without subjecting the evs to any physical stressors such as centrifugation, cd61 magnetic beads were used to fractionate the evs into cd61+ (platelet derived) and cd61-(non-platelet derived) fractions. multiparameter flow cytometry was used to evaluate evs that expressed cd9 and epcam and were negative for calnexin. nanotracking analysis (nta) was used to quantify both total ev and cd61+ and cd61fractions in all patient samples. the average ± standard deviation of total evs obtained from the 15 patients was 6.39x10^11 ± 4.60x10^11 evs/ml of plasma (coefficient of variation [cv] : 72%) while the average and standard deviation of cd61-evs was 1.24x10^10 ± 3.37x10^10 (cv: 271%). the cd61-ev fraction represented a variable amount of the total evs in prostate cancer patients ranging from 0.0001% to 32.21%. multiparameter flow cytometry showed that over 80% of total evs were cd9+ and calnexin-, suggesting an endosomal origin for a vast majority of the evs in these plasma samples. however, the proportion of evs expressing epcam (marker of epi-evs) was higher among the cd61-fraction (5% -30%) as compared to the cd61+ fraction (0.04% -1%). summary/conclusion: our novel method was able to isolate and enrich the epi-ev from the plasma of advanced prostate cancer patients. correlation between clinical characteristics and ev quantity is being evaluated to identify the reason(s) for large variations in cd61-ev fraction. future studies are planned to use our method in improving the sensitivity of ev-based assays and increase the rna yield to facilitate transcriptome sequencing. funding: this work was funded by grants from randy shaver community and research fund, minnetonka, mn. exosomes drive medulloblastoma metastasis in a mmp2 and emmprin dependent manner introduction: recurrent/metastatic medulloblastoma (mb) is a devastating disease with an abysmal prognosis of less than 10% 5-year survival. the secretion of extracellular vesicles (evs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. matrix metalloproteinases (mmps) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. we hypothesise that exosomal mmp2 and its inducer emmprin could enhance metastasis of mb. methods: proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. gelatin zymography and western blotting were performed to confirm mmp2 functional activity in cell lines and exosomes. nanoscale flow cytometry was used to measure surface exosomal emmprin levels. exosomal mmp2 and emmprin were modulated at the rna level. results: number of exosomes is directly related to the migratory behaviour of parental mb cell lines (p < 0.01). notably, functional exosomal mmp2 and emmprin levels also correlate with this. furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair bỹ 3.8-fold (p < 0.01). exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. summary/conclusion: together this data suggests that exosomal mmp2 and emmprin may promote medulloblastoma metastasis and supports analysis of exosomal mmp2 and emmprin levels in patient cerebral spinal fluid samples. introduction: exosomes secreted from cancer cells harbour the potential to regulate intracellular signalling and promote metastasis. wherein, metastasis suppressor genes (msgs) play a pivotal role in regulating such signalling cascades. however, the regulation gets hampered due to low expression of msgs under metastatic conditions. nm23-h1, product of first identified metastasis suppressor gene nme1, is significantly downregulated under metastatic conditions. nm23-h1 serves as a regulator of small gtpases. several evidences have highlighted an involvement of small gtpases (such as rab5, rab7 and rab27) in the biogenesis of exosomes. in addition, bacterial homolog of nm23 has been shown to interact with rab5 and rab7. however, experimental evidence supporting a relationship between exosomes and nm23-h1 is lacking. our current focus is to deduce the relationship between exosomes and msgs. methods: breast cancer cell lines were used to assess the effect of exosomes isolated from highly metastatic cells (mda-mb-231 cells) on lower/non metastatic cells (mcf-7 cells). nme1 was overexpressed in mda-mb-231 cells and subsequently used to isolate exosome fractions. equivalent amount of isolated exosome fractions from mda-mb-231 cells and mda-mb-231/nme1 cells were utilized to access their effect on migration and difference in exosome markers. results: we observed an enrichment of nm23-h1 in the exosomes isolated from mda-mb-231 cells upon overexpression of nme1. proteinase k protection assay confirmed the packaging of nm23-h1 inside the exosomes isolated from mda-mb-231/nme1 cells and excluded the possibility of membrane association of nm23-h1. additionally, overexpression of nm23-h1 led to a significant reduction in the ability of mda-mb-231 exosomes to stimulate movement of mcf-7 cells as confirmed by wound healing assays. our data also highlights a clear reduction in the protein levels of exosome markers such as cd63, cd9 and alix in the exosome fraction isolated from mda-mb-231/nme1 cells as compared to mda-mb-231 cells. interestingly, rab7a, a protein involved in the endosome-lysosome fusion was also present in lower amount in the exosomes isolated from nm23-h1 overexpressing cells. summary/conclusion: our data highlights an antimigratory effect of nm23-h1 via exosomes. these findings support a regulatory role of nm23-h1 in the packaging or release of exosomes in highly metastatic breast cancer cells, and further suggest that metastasis suppressor proteins may be involved in the regulation or packaging of exosomes. additional studies will be required to decipher the downstream signalling of nm23-h1 which affects the biogenesis of exosomes as well as to assess the effect of nm23-h1 overexpression on the content of exosomes. these insights could help us delineate the complex exosome biogenesis pathway and provide new potential drug targets for exosome regulation. introduction: exosomes (exs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. our recent study has revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived exosomes (cepc-exs) on protecting endothelial cells against hypoxia injury. in this study, we aimed to investigate whether exercise-regulated cepc-exs contribute to the beneficial effects of exercise on ischaemic stroke (is). methods: c57bl/6 mice performed moderate treadmill exercise (10 m/min, 4-wks) before is induced by middle cerebral artery occlusion surgery. acute injury was evaluated at day 2 by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra and neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor functions at day 28. the correlations of cepc-exs and their carried mir-126 with neurological parameters were analysed. the underlying mechanism of the effects of cepc-exs isolated from exercised mice was explored in a hypoxia neuron model. cellular mir-126 level, apoptosis, axon growth ability and gene expressions (cas-3, bdnf and akt) were measured. results: 1) exercised mice had a smaller infarct volume on day 2, which was associated with decreased cell apoptosis and cleaved cas-3 level, and a higher microvessel density than those in control; 2) the elevated cepc-ex level positively correlated with tepc-exs in ischaemic brain of exercised mice on day 2. the upregulated mir-126 level positively correlated with the numbers of tepc-exs in ischaemic brain; 3) the numbers of cepc-exs and their carried mir-126 level negatively correlated with the infarct volume, cell apoptosis and positively correlated with the microvessel density in the peri-infarct area on day 2; 4) exercised mice had decreased infarct volume, increased microvessel density, promoted angiogenesis/neurogenesis and improved sensorimotor functions on day 28, accompanying with upregulated levels of bdnf, p-trkb/trkb and p-akt/akt; 5) cepc-exs of exercised mice protected neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by mir-126 and pi3 k inhibitors. summary/conclusion: our data suggest that the protective effects of moderate exercise intervention on the brain against mcao-induced ischaemic injury are ascribed to cepc-exs and their carried mir-126. funding: this work was supported by american heart association (18post33990433) and nih (1r01ns 102720). syndecan-1 regulates alveolar type 2 epithelial cell senescence mediating through extracellular vesicles during lung fibroproliferation tanyalak parimon a , changfu yao a , adam aziz a , stephanie bora a , marilia zuttion1 zuttion a , dianhu jiang a , melanie koenigshoff b , cory hogaboam a , paul nobel a , barry stripp a and peter chen a a cedars-sinai medical center, los angeles, usa; b university of colorado, denver, usa introduction: alveolar type 2 epithelial cell (at2) senescence is implicated in the pathogenesis of lung fibrosis, a progressive fatal condition. syndecan-1, a heparan sulphate proteoglycan, is overexpressed by at2 cells of human idiopathic pulmonary fibrosis (ipf) and bleomycin-injured wt mice and the overexpression of syndecan-1 is profibrotic. moreover, syndecan-1 deficient (sdc1-/-) mice had less lung fibrosis after bleomycin injury. we reported that extracellular vesicles (evs) in bronchoalveolar lavage (bal) of bleomycin-injured wt mice augmented lung fibrosis whereas the sdc1-/--bal-evs attenuated the process. moreover, wt-bal-evs expressed lower level of anti-fibrotic mirnas (mir-34b-5p, −142-3p, −144-3p, and −503-5p) compared to the sdc1-/-bal-evs. these mirnas targeted genes in the cellular senescence pathway indicating that syndecan-1 altered microrna profiles in the bal-evs to promote cellular senescence during lung fibrogenesis. we investigate how syndecan-1 regulates at2 senescence through evs. methods: bleomycin was intratracheally given into wt and sdc1-/-mice. at day 21, lungs were processed for single-cell rna sequencing (scrnaseq) and western blot (wb). evs were isolated using ultrafiltration centrifugation method. human (a549) and mouse (mle-15) lung epithelial cell lines were used for in vitro experiments. results: scrnaseq analysis indicated while bleomycin stimulated an overexpression of cellular senescencespecific genes on at2 cells of wt mice, these genes were significantly downregulated on sdc1-/-at2 cells. senescence proteins, p16 and p21, were also less expressed in the lungs of sdc1-/-than of the wt mice by wb. to determine the functional effects of evs in bal, a549 cells were treated with human ipf or control lung wash-evs and evaluated for beta-galactosidase activity. we found that ipf-evs markedly increased beta-galactosidase enzymatic activity. corroborating with these data, bleomycin-injured bal-wt-evs also significantly upregulated senescence marker, p21, by wb on mle15 cells whereas sdc1-/--bal-evs inhibited p21 expression. summary/conclusion: our data indicate that syndecan-1 regulates lung fibrosis through the senescence signalling pathway on at2 cells. furthermore, syndecan-1 controls at2 senescence mediating through extracellular vesicles in the bal. lastly, the most likely cargo molecules mediating this process are micrornas. immortalized cardiosphere-derived cell ev-associated pirna, imev-pi, protects against ischaemic injury in the heart alessandra ciullo, ahmed ibrahim, liang li, chang li, weixin liu and eduardo marbán smidt heart institute, cedars sinai medical center, los angeles, usa introduction: cardiosphere-derived cells (cdcs) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. cdcs function by secreting extracellular vesicles (evs), lipid-bilayer nanoparticles laden with bioactive molecules. recently our group developed a strategy for immortalizing cdcs (imcdc) that retains their therapeutic potential and enhances cdc function indirectly through their secreted evs. imcdc show a different rna content(mirna, mrna, rrna, trna and pirna) compared to primary cdc. in particular, we focus on piwi rnas (pirnas), small rnas bound by piwi proteins, important regulators of both the epigenome and transcriptome. we seek to explore the role of a pirna highly enriched in imcdc-evs (imev-pi). methods: evs are prepared by conditioning cells for 24 hrs in serum-free basal media, in hypoxic culture. after 24 hrs conditioned medium is cleared of cellular debris and evs isolated using ultrafiltration by centrifugation (ufc). fractions were analysed in terms of particle size, number, and concentration and pirna content. in vitro, bone marrow derived-macrophages (bmdm) were exposed to imcdc-ev, imev-pi and control and transcriptomic profile and potentially activated pathways were assessed. in vivo, 8-10 week-old wistar-kyoto female rats received 10^10 imcdc-ev, imev-pi, scramble or vehicle intracoronary 20 minutes after ischaemiareperfusion(i/r). cardiac troponin i levels, scar size and monocytes were assessed at 24 and 48 hrs. results: by small-rna sequencing analysis we found that pirnas are enriched in both cdc-ev and imcdc-ev. imcdc show a different pirna composition compared to primary cdc. imev-pi was identified as one of the most highly-expressed non-coding rnas (the number of reads were 35x higher in imcdc-ev compared to cdc-ev). in vitro, imexo-pi-conditioned bmdm exhibit a different transcriptomic profile compared with control, with upregulation of pathways involved in the inflammatory response, cell death, and cell-to cell signalling. in vivo, imev-pi is cardioprotective, as shown by reduced scar size and lower cardiac troponin levels compared to vehicleand scramble-injected animals at 48 hrs post i/r. imev-pi only minimally alters neutrophil counts profile in blood but it alters monocytes profile with a decreased number at 24 hrs and an increase at 48 hrs. summary/conclusion: we posit that imev-pi is a key determinant of imcdc-ev therapeutic efficacy. our results indicate that target cells may be macrophages/ monocytes, given that imev-pi exposure modifies their composition and mrna profile both in vitro and in vivo. introduction: extracellular vesicles (exosomes, evs) are cell membrane particles (30-200 nm) secreted by virtually cells. during intercellular communication in the body, secreted evs play crucial roles by carrying functional biomolecules (e.g., micrornas and enzymes) into other cells to affect cellular function, including disease progression and tissue regenerations. literature previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of evs. the activation of growth factor receptors, such as epidermal growth factor receptor (egfr), induces macropinocytosis. in this study, we demonstrated the effects of evs on demal papilla and hair follicle regeneration. methods: identification of distinct nanoparticles and subsets of extracellular vesicles from umbilical cord blood stem cell by asymmetric flow field-flow fractionation. results: the effects of evs from umbilical cord blood stem cell on the propagation of demal papilla and hair follicle regeneration were observed. summary/conclusion: the enhancement of extracellular vesicles from umbilicalcord blood stem cell the propagation of demal papilla and hair follicle regeneration were observed and confirmed. mechanisms of host resistance to plasma membrane damage induced by pneumolysin attack introduction: bacterial pore-forming toxins (pfts) are major virulence factors produced by pathogens. pfts target host plasma membrane (pm) and create transmembrane pores, which allow uncontrolled flux of ions and small molecules across the pm disrupting cellular homoeostasis. to survive, cells display poorly understood repair mechanisms to recover the cell homoeostasis. several mechanisms were proposed to participate in cell recovery: exocytosis of cortical lysosomes; endocytosis of pfts pores; pm blebbing and shedding. methods: we used increasing concentrations of purified ply to intoxicate cells. pm permeability was assessed by flow cytometry using propidium iodide dye. cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy. extracellular vesicles released during pm repair were isolated by high-speed centrifugation and characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and mass spectrometry/ liquid chromatography analysis. results: ply triggers a complete reorganization of the actomyosin cytoskeleton inducing the formation of cortical actomyosin bundles at sites of pm remodelling. these structures assemble upon loss of pm integrity and disassemble as pm recovers. we detected the release of microvesicles during the recovery of pm integrity. vesicle population is heterogeneous with sizes ranging from 100 to 500 nm, with the majority of them measuring 100-200 nm. vesicle proteomic analysis revealed that they contain ply, suggesting they participate in pore removal, proteins involved in vesicle trafficking, pm repair and exosome biogenesis. summary/conclusion: our data demonstrate that cells are able to recover from the damage induced by sublytic concentrations of ply. actomyosin cytoskeleton undergo massive changes with the assembly of cortical bundles possibly at sites of pm damage. we showed that cells produced extracellular vesicles during the process of repair. we are now focusing on understanding the biogenesis of those vesicles and its importance during the process of repair. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms (2d static tissue culture flask vs 3d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in 2d t-flasks and 3d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both 2d and 3d culture systems. msc-ev were characterized according to misev 2018 guidelines, using techniques as nta, protein and lipid quantification, western blot, imaging and fourier-transform infrared spectroscopy (ftir). the results indicate that msc-ev derived from different sources/donors have similar size distribution, however, ev yields tend to be higher for the 3d culture system. of notice, several spectral regions were identified by ftir, enabling the detection of differences in the biomolecules present in msc-ev, msc-conditioned media and cells produced under different conditions. summary/conclusion: in summary, this study contributes to the establishment of a scalable process for msc-ev production. evaluation of three different isolation methods for small extracellular vesicles from human plasma in prostate cancer diagnosis introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies. methods: pca patients and age-matched healthy controls (hc) plasma (n = 6 in each group) were used to isolate sevs with 3 different isolation methods including commercial exoquick ultra kit, qev 35 and qev 70 size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd9 and cd81 commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination. results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev35 demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev70. furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods. summary/conclusion: qev 70 method demonstrated better performance in isolating relatively pure sevs from human plasma; qev35 has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev35 but with the highest non-sev protein contaminations. people can choose higher sev content or higher sev purity according to the downstream analysis. moreover, sevs may also be used for treatment monitoring, as recent studies suggested that the expression levels of certain markers may change during therapy, reflecting tumour response. for cancer diagnostics and therapeutic purposes in clinical settings, it is important to have a device which allows multiplexed measurements, in order to scan a large number of markers simultaneously and compare the expression levels of different patients, or same patients at different treatment stages, in a time efficient manner. methods: herein, we propose a multiplexed platform for label-free detection and surface protein profiling of sevs. the technique is based on the electrokinetic phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta* change upon sev binding on functionalized microcapillary surfaces. for the purpose, we used sevs derived from lung cancer cells. in its current form, the platform can measure up to 5 channels simultaneously, however, it can be further expanded. results: having demonstrated that our electrokinetic sensor successfully detects sevs in a specific way, we tested its ability to measure the expression level of membrane proteins. the analysis showed that it could detect differences in the expressions of egfr on sevs, with a sensitivity of 10%. we then extended the platform for multiplexed analysis, by connecting and measuring four capillaries, functionalized with different capture probes, simultaneously. for the purpose, we targeted specific tumour markers, i.e. egfr, and exosomal tetraspanin family proteins, such as cd9 and cd63. the results showed successful multiplexed ev detection. summary/conclusion: being the sensor suitable for multiplexed sev detection, we shall present our investigation on a set of pleural effusion samples collected from a cohort of lung-cancer patients with different genetic makeup. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd9, cd63 and cd81, it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: 1) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and 2) the assessment of subpopulation target enrichment relative to the population average by leveraging tim4 as an unbiased, lipid-based ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasis-associated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr3 (her2+), t47d and mcf-7 (er+pr+), bt549 and mda-mb-231 (triple negative) breast cancer cell lines, as well as five mda-mb-231-derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her2 was broadly detected in her2+ skbr3 evs. interestingly, her2-t47d and mcf-7 evs also expressed her2 where it was highly enriched in its epcam+ subpopulations. itg α6, β3 and β4 were only found in triple negative and organotropic evs with itg β3 and β4 differentially enriched based on the organotropism. the population average of mda-mb-231 and lung-tropic evs had high expression of itg β4, where subpopulations of cd44+ evs showed positive enrichment while cd9+ and cd63 + evs showed negative enrichment. itg α5, β3 and β4 were absent in the bone-tropic cd81+ ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb-231 evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du145 prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified 49 targets that bind to hs-gag chains, and also 108 different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev-associated hspgs will result in attenuation of ev induction of a tumour-supporting fibroblast phenotype. introduction: ovarian cancer (oc) is the fifth leading cause of cancer-related death in women, partly due to difficulty in early diagnosis. extracellular vesicles (evs) show promise for use in early diagnostics of oc. here, evs from cervical mucus (cm) of ovarian cancer patients were used for discovery of oc biomarkers for diagnostics. machine learning was used to mine ev mirna data to develop an oc biomarker panel (validation via the cancer genome atlas). examination of the mirna targets reveal that the panel is a sufficiently accurate predictor of oc. methods: evs from the cm of 48 patients (15 highgrade serous, 24 low-grade, 7 benign) were isolated for small rna-sequencing. the top differentially expressed mirnas were used in a random forest and "voom" (variance modelling at the observational level) model. unsupervised approaches were used and then vetted against patient symptomology data. a tcga ovarian cancer dataset (n = 100) was used for validation. results: an oc biomarker panel of 10 micrornas (voom: 96.55% accuracy; random forest: 88% accuracy) was generated. the panel consists of members from the mir-200 family and the mir-16 family, among others. the mirna targets are associated with molecular functions and pathways specific in oc progression. summary/conclusion: our method has identified ev mirna biomarkers that may be crucial for early, noninvasive detection of oc. data science has been used to develop a feedback system integrating biochemical experiments, smaller datasets, and previously available data to identify and verify a biomarker panel for oc diagnostics. introduction: liver disease has become a significant cause of morbidity and mortality among hiv patients. alcohol exposure can further exacerbate liver damage by activating hepatic stellate cells (hscs), leading to hepatic fibrosis or cirrhosis, often seen at all levels of alcohol exposure among people with hiv. due to the potentiating effects of alcohol on hiv-induced hepatocytes (hep) damage, as well as the effect of ethanol in hsc-mediated extracellular remodelling, it is imperative to understand the interplay of hep and hscs. here, we focus on the exosomes released by hiv-and ethanol exposed hep and how these exosomes modulate the functional behaviour of hscs. methods: human hepatocyte huh7.5cyp2e1 [hepatoma cells stably transfected with cyp2e1 designated as rlw cells] were infected with hiv in the presence or absence of alcohol metabolite, acetaldehyde using the acetaldehyde-generating system (ags). the conditioned medium was collected from 4 groups of cells: untreated, hiv-, ags-and hiv+ags. quantification of exosomes number and size were evaluated with zetaview or nanosight and further characterized for exosome markers following the guideline from minimal information for studies of evs 2018 (misev2018). the human hepatic stellate lx-2 cell line was exposed to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory markers il-1β, il-6, tnfα, and fibrotic markers acta2, and timp1 using quantitative pcr. we also analysed exosome mirna content in primary human hepatocytes (phh), which potentially regulates the function of recipient cells by "programming" their inflammation/fibrosis status. the network analysis for mrna and mirna were carried out using gene ontology consortium, and mirror 2.0 and david bioinformatics resources 6.8. results: ags treatment further enhanced the release of hiv-induced exosome from hepatocytes. size distribution assessed by zeta view or nanosight revealed that approximately 85-90% of particles distributed in the range of 50 to 200 nm, with a peak at~90 nm. enriched expression of hiv protein p24 was observed in fractions f9-f12. western blotting of hepatocytederived exosome demonstrated positivity for exosome-enriched proteins alix, tsg 101 and cd9 specifically in f2-f8 fractions and negative for endoplasmic reticulum protein calnexin. the uptake of hepatocytederived exosomes by hscs was apparent as demonstrated by immunofluorescence. the internalization of hepatocyte-exosome induced activation of hscs as evidenced by increased expression of pro-inflammatory il-1β, il-6, tnfα markers in the latter cells. summary/conclusion: we conclude that ags treatment in hiv-infected hepatocytes potentiates the release of exosomes, which, following uptake by the hscs, leads to their activation. funding: this work is supported by nih-1 r01 aa027189-01a1. antimicrobial peptide ll-37 induces neutrophil-derived extracellular vesicles with antibacterial potential and protects murine sepsis yumi kumagai, taisuke murakami, kyoko kuwahara and isao nagaoka juntendo university, bunkyo-ku, japan introduction: extracellular vesicles (evs) released from immune cells or other host cells upon microbial infection modulate the immune response and thereby regulate the infection. sepsis is a life-threatening multiple organ dysfunction caused by systemic dysregulated inflammatory response to infection. nevertheless, numerous therapeutic trails concerning immune dysfunction have still been disappointing outcomes. we have previously shown that ll-37, a human cathelicidin antimicrobial peptide, improves the survival of caecal ligation and puncture (clp) septic mice. here, we investigated the induction of ev release by ll-37 and functions of ll-37-induced evs in murine sepsis. methods: evs were isolated from peritoneal exudates of clp mice and the supernatant of ll-37-stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography. isolated evs were analysed by flow cytometry, western blotting, and nano particle analysis. neutrophil-derived evs were injected into clp mice to assess the protective function of evs against septic mice. the antibacterial activity of evs was evaluated by incubating with escherichia coli. results: in clp mice, ll-37 augmented the level of evs. evs from ll-37-injected clp mice contained higher amounts of neutrophil-derived antibacterial proteins (lactoferrin and cramp, cathelicidin-related antimicrobial peptide) and exhibited higher antibacterial activity compared to evs from pbs-injected clp mice. furthermore, ll-37 stimulated mouse bone marrow neutrophils to release evs with antibacterial potential, and administration of the ll-37-induced evs reduced the bacterial load and improved the survival of clp mice. summary/conclusion: ll-37 induces the release of antimicrobial evs from neutrophils in clp mice, thereby reducing the bacterial load and protecting mice from lethal septic condition. identification of mirna profiles of serum exosomes in active tuberculosis introduction: tuberculosis (tb) has exceeded hiv as the most lethal infectious disease globally for two consecutive years, mainly due to difficulties in achieving early and definitive diagnosis, and timely treatment. exosomes carrying rna, particularly mirna, have demonstrated their functional and diagnostic potential in diseases including tb. however, few published studies have explored whether exosomal mirnas could be used for diagnosis of tb. thus, more systematic and comprehensive study of exosomal mirnas with regard to their potential as non-invasive tb biomarkers is still urgently needed. methods: we searched the gene expression omnibus database for datasets published before december 2019, and performed meta-analysis on available exosomal mirna profile data for healthy control (hc) and active tb clinical specimens . reprocessing next generation sequencing data under uniform parameters and utilizing state-of-the-art bioinformatics analysis. results: we identified many distinct up-regulated and down-regulated differentially expressed exosomal mirna across multiple studies, and further screened the top 10, which might provide a potential panel for differentiation of hc and tb. we classified all differentially expressed mirnas into six expression patterns and identified two persistently up-regulated mirna (hsa-mir-140-3p, and hsa-mir-423-3p) as potential markers during tb progression. moreover, the differential expressed exosomal genes that we screened from the datasets were consistent with the genes overlapped with predicted mrna targets of differentially expressed mirna. pathway and function analysis further demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism and apoptosis/necrosis. introduction: trypanosoma cruzi is a protozoan parasite that causes chagas disease, a relevant source of morbidity in latin america, which has spread to many countries as result of immigration of the people from endemic areas. many studies have been showed that trypomastigote forms of t. cruzi release extracellular vesicles (ev) that increase parasite infection. objectives. here, we aim to test if previous immunization with evs in adjuvant can generate a protective immune response by decreasing the effects of evs in experimental chagas disease. methods: female balb/c mice were immunized by intra peritoneal (ip) administration with 5 × 105 or 107 evs isolated from trypomastigotes forms, with aluminium hydroxide adjuvant (aloh). injections were administered intravenous in 3 doses during 45 days (15 days interval). after immunization, mice were infected intra-peritoneally with 500 trypomastigotes forms. parasitaemia was quantified by counting motile parasites in fresh blood sample drawn from lateral tail veins. mortality and weight were analysed during the infection. in control group, the mice were immunized with aioh. results: the immunization with evs with aloh decreased the blood parasitaemia and the animals survived, while all animals died in the group aloh alone. the animals immunized with evs had an increase of f4/80+ cd11b+ and cd80/cd86 expression in cells isolated from the peritoneum. summary/conclusion: these results indicate that t. cruzi ev antigens can induce an immune response that controls the development and establishment of the experimental chagas disease. introduction: acinetobacter baumannii (ab) is a nosocomial pathogen, of major concern due to its multidrug resistance (mdr) and the recent appearance of hyper-virulent strains in the clinical setting. the world health organization included ab as a critical priority pathogen for the development of novel antibiotics. ab pathogenesis is associated with a multitude of potential virulence factors (vf) that remain poorly characterized. there is growing evidence that outer membrane vesicles (omv) are used as vehicles to transport bacterial proteins that contribute to set up the conditions for the infections. in the present work we studied the physiopathology of mdr ab. we focused on the contribution of non-characterized outer membrane proteins (omps) associated to omvs, with special focus on lipoproteins (lp). methods: we conducted a bioinformatic prediction using available datasets to construct a list of omv-associated omps putatively acting as vf in ab5075. seven genes were selected and the corresponding mutants were obtained from manoil lab collection. physiological analyses of the mutants were performed, and the involvement of the selected proteins in ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity assays on human lung cells a549. results: biochemical analysis indicated similar growth rates in rich media, as well as similar levels of omv production for all the mutants as compared to wt. also, no differences in susceptibility to chaotropic agents were observed, indicating no alteration of the om function as a general permeability barrier. all mutants similarly reduced a549 cell viability, but to a lesser extent than the wt. moreover, three of them exhibited less adhesion and invasion compared to the wt, and omv isolated from these mutants displayed variable levels of cytotoxicity. summary/conclusion: these results suggest roles for the mutant gene products in ab pathogenesis and contribute to the better understanding of ab virulence mechanisms, revealing novel possible targets for therapeutic development. funding: agencia nacional de promoción científica y tecnológica (anpcyt, pict 2017-3536) medicine, nanfang hospital, southern medical university, guangzhou, 510515, china, guangzhou, china (people's republic); d zhujiang hospital, southern medical university, guangzhou, china, guangzhou, china (people's republic) introduction: talaromyces marneffei (t. marneffei) grows as a mycelial form in the environment but multiplies rapidly as a yeast form in the host and within macrophages. the yeast can cause disseminated and progressive infections or lethal talaromycosis. but the mechanisms of pathogenicity of t. marneffei are poorly understood. fungal extracellular vesicles (evs) have previously been shown to transmit a proinflammatory message to macrophages. however, the characteristics and effects of t. marneffei evs on the progress of infection have not yet been investigated. methods: in this study, evs of t. marneffei yeasts were isolated by ultracentrifugation method. evs were detected and confirmed by electron microscopy and nanoparticle tracking analysis (nta). the raw 264.7 murine macrophages were incubated with the t. marneffei vesicles to observe the changes of macrophage morphology and function, especially in inflammatory response. the proteins, dnas, rnas of t. marneffei vesicles were respectively removed with protease, dnase and rnase. all treated evs were used to incubate with murine macrophages observe the effect on macrophages in inflammatory response. results: we observed that evs secreted by t. marneffei have a typical spherical shape with a diameter of 30 to 200 nm. t. marneffei evs were internalized by raw 264.7 murine macrophages and promoted the production of no and proinflammatory cytokine by macrophages in a dose-dependent manner. t. marneffei evs stimulate macrophages to generate reactive oxygen species (ros). addition of t. marneffei evs to macrophages also promoted transcription of the m1-polarization marker cd86 and diminish that of the m2 markers cd206. incubation of t. marneffei vesicles with murine macrophages resulted in increased levels of extracellular interleukin-1β(il-1β), interleukin-6 (il-6) and interleukin-12 (il-12). the proinflammatory effect of vesicles was weakened when the proteins of the vesicles were destroyed. in contrast, no similar changes were observed in degraded dna and rna. summary/conclusion: our results indicate that the extracellular vesicles of t. marneffei can stimulate macrophage towards to m1 polarization phenotype and promote proinflammatory function. plasma-derived extracellular vesicles as potential biomarkers in chronic chagas disease patients introduction: chagas disease (cd), caused by the parasite trypanosoma cruzi (t. cruzi), is a neglected tropical disease affecting about 8 million people worldwide. currently, one of the main clinical problems is the lack of effective biomarkers for therapeutic response and disease prognosis during chronic infections. in that context, extracellular vesicles (evs) are raising attention as novel, minimally invasive, and inexpensive method for diagnostic and screening of diseases, as well as a new source to identify new biomarkers. the main objective of this study is to use evs derived from biological fluids of chronic cd patients for identifying novel biomarkers, specifically in the context of therapeutic response and disease prognosis. methods: plasma, saliva and urine from a cohort of chronic cd patients are being collected before and at the end of benznidazole treatment. as negative controls, healthy donors have been also included. the purification and characterization of the evs was performed by size exclusion chromatography, followed by nanoparticle tracking analysis, bead-based flow cytometry assay and transmission electron microscopy. a proteomic analysis of the evs was also performed. results: proteins associated with evs secreted by infective t. cruzi have been previously identified in cell culture, but never in human samples. our results, based on the analysis of a single heart-transplanted patient with chronic cd, showed the presence of t. cruzi and human proteins specifically associated with plasma-derived evs. noticeably, several human and parasite proteins identified in evs obtained from plasma samples, were present or upregulated before chemotherapy and were absent or downregulated following treatment. currently, proteomics analyses are being performed with higher numbers of cd plasma samples. summary/conclusion: to the best of our knowledge, this is the first proteomic profiling of plasma-derived evs from a heart-transplanted patient with chronic cd. these results thus open the possibility of using evs from biological fluids as a tool for the identification of new biomarker candidates in chronic cd. these biomarkers are essential for assessing disease introduction: eukaryotic cells communicate with one another through multiple pathways. an established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling "packages", called extracellular vesicles (evs). evs are produced by all domains of life and carry proteins, nucleic acid (rna and dna), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. bacteria can also produce evs that communicate with each other to coordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. here i investigate the poorly explored axis where evs are the vehicle for communication between eukaryotic cells and bacteria. methods: as a first step, i have isolated evs from tissue cultured eukaryotic cells grown in advanced rpmi media with minimal ev-depleted fbs. nanoevs were isolated from spent culture media using sequential centrifugation (2,000 × g, 10,000 × g) and concentration (100 kda filter) before purifying using size exclusion chromatography columns. nanoev-rich fractions were pooled based on particle (nanoparticle tracking analysis) and protein quantity data. nanoevs were characterised by electron microscopy and expression of exosomal markers. eukaryotic nanoevs were then characterised in their effect upon the growth of escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions. results: further experiments with increased dosages are required to determine the effect of human evs on bacteria. summary/conclusion: our work will investigate whether human evs communicate with the resident and pathogenic microbiota, while examining the mechanisms behind this communication. escherichia coli pathogenic bacteria commensal bacteria hydrogen sulphide (h2 s) derived extracellular vesicles: a potential protective role in response to respiratory syncytial virus (rsv) infection methods: evs were isolated from untreated (control evs) and gyy4137 treated (gyy-evs) a549 cells, a human alveolar type ii-like epithelial cell line. evs were purified using a two-step enrichment procedure. evs were characterized using particle sizing (size and concentration) and western blot for the ev markers. electron microscopy and immunofluorescence staining were used to investigate presence of multivesicular bodies (mvbs), evs precursors, in both groups. recipient a549 cells were cultured for 24 hours in the presence or absence of control-or gyy-evs, then infected with rsv for 24 hours. viral titres by plaque assay were measured in recipient infected a549 cells. results: we confirmed the presence and purity of our evs. we found that gyy4137 reduced the particles number of evs, but did not change ev size. a549 cells treated with gyy4137 showed an accumulation of mvbs/lysosomes-like structures, as well as an increase in cd63 expression, a mvbs marker, compared to untreated cells. recipient a549 cells treated with gyy-evs showed lower viral replication than control ev-treated cells in response to rsv infection. we are currently investigating the potential mechanism for this observation and characterizing the rna cargo composition of gyy-evs. summary/conclusion: no vaccine or effective treatment is currently available for rsv. cellular pretreatment with gyy-evs reduced the rsv replication in airway epithelial recipient cells, suggesting that h2 s could exert its antiviral activity in the context of rsv infection potentially through modulation of ev composition. therefore, gyy-evs could represent a future novel pharmacological approach for ameliorating virus-induced lung disease. effects of extracellular vesicle-mediated transmission on reoviridae infection results: taken together, these data suggest that multiple particles of reovirus and rotavirus egress in large, virus-modulated evs, and that transmission in evs increases segment complementation compared to transmission as free particles. summary/conclusion: these discoveries may be broadly applicable to viruses that travel in evs and will contribute to general principles of virus transmission and diversification. continued studies will illuminate the specific cellular pathways reovirus and rotavirus utilize for successful egress. these pathways may prove to be critical targets for the improvement of vaccines and oncolytic therapy. multiparameter flow cytometry analysis of the human spleen and its interaction with plasma-derived evs from plasmodium vivax patients introduction: the spleen is a secondary lymph organ that filters blood and elicits immune responses against blood-borne pathogens, such as malaria parasites. extracellular vesicles (evs) are membrane-bound particles involved in intercellular communication. evs play several roles in malaria ranging from modulation of immune responses to induction of vascular alterations. here, we report the first integrated characterization of human spleen cells using multiparameter flow cytometry (mfc) describing subpopulations of splenic leukocytes and red blood cells (rbcs), and studied their interaction with plasma-derived evs from p. vivax patients (pvevs). methods: human spleens were obtained from organ transplantation donors. myeloid, lymphoid, erythroid and haematopoietic stem cells (hscs) were immunophenotyped by mfc. t cells, dendritic cells (dcs) and rbcs were enriched by density centrifugation and immunomagnetic isolation. pvevs and healthy donors evs (hevs) were purified by size-exclusion chromatography (sec) and characterized by bead-based flow cytometry. enriched evs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes or enriched populations. evs-cells interaction was assessed by flow cytometry. results: human spleen immunophenotyping showed that cd45+ cells included b (30%), cd4 + t (16%), cd8 + t (10%), nk (6%) and nkt (2%) lymphocytes. myeloid cells comprised neutrophils (16%), monocytes (2%) and dcs (0.3%). erythrocytes represented 70% whereas, unexpectedly, reticulocytes were 0.93% of total cells. in addition, we also detected hscs, which accounted for 0.02%. sec separated evs from the bulk of soluble plasma proteins as shown by the enrichment of cd63, cd5 l and cd71 markers. interaction studies showed an increased proportion of t cells (cd4 + 3-fold and cd8 + 4-fold), monocytes (1.5-fold) , b cells (2.3-fold) and erythrocytes (threefold) interacting with pvevs as compared to hevs. summary/conclusion: the integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and evs. a larger proportion of monocytes, t and b lymphocytes as well as erythrocytes was found to interact with pvevs compared to hevs. future functional studies of these interactions can unveil pathophysiological processes involving the spleen in vivax malaria. neuroblastoma-secreted exosomes carrying mir-375 promote osteogenic differentiation of bone marrow mesenchymal stromal cells introduction: bone marrow (bm) is the major target organ for neuroblastoma (nb) metastasis and its involvement is associated with poor outcome. yet, the mechanism by which nb cells invade bm is largely unknown. tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (mscs) have been recognized as a fundamental part of the associated tumour stroma. here, we explore the potential role of nb-derived exosomes in induction of a pro-osteogenic phenotype on bm-mscs. introduction: extracellular vesicles (evs) are nanosized particles delimited by a lipid bilayer which transfer functional molecular cargos from the cells of origin to target cells. this intercellular crosstalk controls both physiological and pathological conditions. given their presence in body fluids and their characteristics, these nanocarriers might be potentially used in diagnostics and/or therapy. breast cancer is the most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality in women worldwide; the triple negative breast cancer, in particular, is the most aggressive subtype with a poor prognosis. since it is recognized that cell stiffness of cancer cells play a crucial role during the metastatic spreading, we set ourselves the goal of clarify the effects and the activity of small-evs (i.e. with a diameter below 200 nm) in metastatic breast cancer, with a special attention on their correlation with the biomechanical properties of cells. methods: functional assays were performed on the non-invasive mcf7 breast cancer cell line, before and after the cellular uptake of small-evs originating from the invasive mda-mb-231 triple negative breast cancer cell line. the mechanical properties (cell stiffness, cytoskeleton organization and focal adhesions) of mcf7 cells were investigated before and after the vesicle uptake. results: the uptake of small-evs derived from mda-mb-231 significantly reduces the young's modulus values of mcf7 cell line making them more invasive. moreover actin and focal adhesion variations were observed in mcf7 cells before and after small-ev's uptake, suggesting a molecular rearrangement inside mcf7 cells upon uptake. summary/conclusion: our results evidence that small-evs play a key role in altering biomechanical properties of target cells and underline their relevance in cell-cell crosstalk. our approach is very promising to identify new molecular mechanisms through which evs perform their oncogenic function. stratification of angiogenic or non-angiogenic lesions in colorectal cancer liver metastases patients using extracellular vesicle mirna introduction: colorectal carcinoma (crc) is the second leading cause of cancer death in the western world. over 50% of the crc patients develop liver metastasis (lm) and 90% will die from metastatic disease. in the current clinical setting, liver resection provides the only possible cure, but only 20% of crclm patients are resectable. the combination of angiogenic inhibitors with chemotherapy is used to downsize crclm with the goal of converting unresectable patients to resectable ones. however, only 10-15% of these patients can be successfully converted to a resectable state. we have no way of identifying those crclm patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. bevacizumab: bev)). proper stratification of patients into angiogenic inhibitor responders and non-responders will permit a proper assessment of the efficacy of angiogenic inhibitors. crclm forms 2 distinct histopathological growth patterns (hgp): angiogenic (desmoplastic) and nonangiogenic (replacement) hgp. we demonstrated that crclm patients with predominant angiogenic lesions receiving bev plus chemotherapy have a more than double 5-year overall survival compared to patients with non-angiogenic lesions. therefore, nonangiogenic lesions do not respond to angiogenic inhibitors. our study focuses on stratifying angiogenic vs non-angiogenic lesions of crclm through extracellular vesicle mirnas. we are using two approaches in the selection of mirnas to target: 1. text mining of published ev mirna from crclm patients; and 2. differentially expressed mirnas present in tumour tissue from both lesion types, we have obtained by sequencing 50-60 patients. these two strategies will generate a list of mirnas that we will target using qpcr on plasma-derived ev mirna from the patients used in approach 2, where we have classified the lesions in the patients. preliminary data on 10 patients will be presented. methods: ev isolation was performed using the gold standard centrifugation method. rnaseq and qpcr are used to generate the expression profile for angiogenic vs non-angiogenic type of crclm. results: the research is under progress. summary/conclusion: the research is under progress. the introduction: it is known that bone metastasis causes a reduction in the quality of life of cancer patients due to fractures and nerve compression. therefore, it is important to elucidate the mechanism of bone metastasis and develop new treatments. metastatic bone tumours occur at particularly high rates in cancers of the prostate, breast, and lung. in this study, we focused on extracellular vesicles (evs) in bone metastasis, and investigated that the role of evs derived from cancer cells in osteolysis. methods: the prostate, breast, and lung cancer cellderived evs were added to osteoclast precursors with rankls. the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (trap) stain and by measuring the expression level of osteoclast markers using by qrt-pcr. a proteome analysis (lc-ms/ms) and sirna approaches were used to identify molecules which are responsible for promotion of osteoclast differentiation in the prostate cancer cellderived evs. to investigate whether the molecules are suitable for the detection of bone metastasis in serum evs, we isolated evs from serum of prostate cancer patients, and analysed the protein level of the molecules by western blot analysis. results: we found that the prostate cancer and lung cancer-derived evs significantly promoted the rankl-stimulated osteoclast differentiation. our analysis revealed that cub domain-containing protein 1 (cdcp1), which is a membrane protein on the prostate cancer cell-derived evs, was responsible for promotion of osteoclast differentiation. moreover, cdcp1 was markedly detected in the evs-derived from serum of prostate cancer patients who had bone metastasis than that of normal subjects. we also found that cdcp1 exits on the breast and lung cancer cell-derived evs. summary/conclusion: we showed that the evsderived from bone metastatic tumours have a role in activation of osteoclastogenesis. moreover, we revealed that cdcp1 in the evs is responsible for promoting of osteoclast differentiation. these evs could be the novel diagnostic and therapeutic target for bone metastasis. increased expression of chemokine receptor cxcr4 in non-invasive colorectal cancer cells after incorporation of platelet-derived extracellular vesicles. introduction: blood platelets and platelet-derived extracellular vesicles (p-evs) play a crucial role in tumour growth and metastasis. p-evs, also referred to as platelet microparticles, are recognized as a carrier for proteins and nucleic acids that control cell-to-cell communication, mediate the formation of metastatic niches and affect tumour invasion and metastasis. among the other factors, p-evs contain the chemokine receptor cxcr4, known as a co-receptor for hiv entry but also regarded as important in cancer development due to the importance of cxcr4/cxcl12 signalling. overexpression of cxcr4 was reported in various, especially in invasive cancers, including colorectal cancer (crc). crc, the third most commonly diagnosed cancer, is usually diagnosed at the late stage and patient's death is mainly related to metastasis. increased levels of cxcr4 has been reported as a poor prognostic factor for survival of crc patients and its blocking has been suggested as therapeutic approach. the aim of this study was to analyse the effect of p-evs on the levels of cxcr4 in crc cells on various epithelial-to-mesenchymal transition stage. methods: we used crc cell lines ht29 and sw620, which represent distant invasive potential and different phenotypes, epithelial and strongly mesenchymal, respectively. p-evs were isolated from outdated concentrates of human blood platelets after activation by thrombin in the presence of calcium ions, by subsequent centrifugation and ultracentrifugation. the p-evs were labelled using pkh67 fluorescent dye to visualize their uptake into cell lines by confocal microscopy. we also quantified the levels of cxcr4 in ht29 and sw620 by western blot analysis. the effect of p-evs uptake on the migration of crc cells was studied by "wound healing" method. results: we found that the levels of cxcr4 in crc lines used in the study were correlated with their emt stage. we show here that p-evs released by activated platelets were incorporated into both ht29 and sw620 cell lines. the expression of cxcr4 in ht29 was increased after the uptake of p-evs. additionally we observed that migration rate of ht29 cells with incorporated p-evs was elevated as compared to control cells. summary/conclusion: we posit that circulating p-evs can be incorporated into yet not invasive crc cells to significantly increase the level of cxcr4 receptors and that may lead to the their more invasive characteristics. introduction: for cancer therapy it is important to identify markers and key processes induced during cancer progression. one of them is epithelialmesenchymal transition (emt) which is associated with cell acquisition of invasiveness, stem cell characteristics and resistance to apoptosis and therapy. also the extracellular vesicles (evs) released from tumour cells, which can be taken up by cells constituting pre-metastatic niches, can alter cancer progression by promoting cells' reprogramming. our group has recently reported that snail transcription factor, a key factor of emt, when overexpressed in crc ht29 cells, drives their early emt and alters the expression of microrna (mirs). in the present study we analysed the mirs profile of evs released from those cells. methods: evs from three ht29 clones stably overexpressing snail and from control ht29-pcdna were isolated by differential centrifugation and ultracentrifugation of conditioned media after 24 h of culturing in serum-free medium. total rna was isolated and nextgeneration sequencing (ngs) analysis of the mirnas was performed followed by gene ontology ( introduction: prostate cancer (pca) is the most common malignant tumour in male urinary system and osteoblastic bone metastasis is the most observed metastasis in prostate cancer patients. it has been demonstrated that circulating micrornas contained in extracellular vesicles are potential early biomarkers and therapy targets for many diseases. however, the potential role of micrornas in prostate cancer bone metastasis, is not yet to be fully explored. methods: after isolation and purification evs using ultracentrifugation from conditioned media of bone metastatic co-opting prostate cancer cells and normal cells, total rna was extracted. subsequent to library preparation and small rna-seq, differential gene expression analysis was performed. data were filtered by mean mirna expression of ≥ 50 reads, two fold up or down regulation between 2.5 − 7.5 and adjusted pvalue ≤ 0.05. the uptake of pca-sevs was performed. three candidate mirnas (has-mir-200 c-3p; has-mir-1275; has-mir-383-5p) were internalized and osteoblast differentiation were detected by qpcr, histochemical staining and protein activity detection. results: total reads of mirnas in bone metastatic co-opting pca-evs exceeded significantly than that in normal evs (p < 0.001), indicating that mirnas delivered by pca cells play critical role in pca bone metastasis. pca-cm enhanced osteoblast differentiation and can be reversed by gw4869. the uptake of pca-evs by mc3t3-e1 was efficient. the high expression of the three candidate mirnas in pca-evs was verified by qpcr. all the three candidate mirnas promoted osteogenesis, verified by mrna expression of osteoblastic markers (alp, ocn, runx2, osx), alp activity, alp staining and aliza red s staining. summary/conclusion: these findings suggest that mirna cargos in pca-evs play a pivotal role in the development of osteoblastic bone metastasis of pca, which can be potential early biomarkers and therapy targets for prostate cancer bone metastasis. funding: this work was supported by grants from the national natural science foundation of china (81872347); xijing hospital science and technology foundation project (xjzt19ptk14). introduction: retinoblastoma (rb) is the most common intraocular cancer of childhood. despite recent advances in conservative treatment have greatly improved the visual outcome, local tumour control remain difficult in presence of massive vitreous seeding. thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. traditional biopsy has long been considered unsafe in rb, due to the risk of extraocular spread. exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumour-associated biomarkers. the aim of this study was to determine the protein signature of exosomes derived from rb tumours (rbt) and vitreous seeding (rbvs) primary cell lines. methods: exosomes from 4 rbt (hsjd-rbt1, hsjd-rbt2, hsjd-rbt5, hsjd-rbt14) and 3 rbvs (hsjd-rbvs1, hsjd-rbvs3, hsjd-rbvs10) cell lines were isolated by high speed ultracentrifugation. vesicles number and size were confirmed by nanosight and scanning electron microscopy. protein content was analysed by bicinchonic-acid assay and high resolution mass spectrometry. results: a total of 5666 proteins were identified. among these, 5223 and 3637 were expressed in exosomes rbt and one rbvs group respectively. gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified identified in rbvs exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ecm) remodelling and interaction, resistance to anoikis and metabolism/catabolism of glucose and aminoacids. summary/conclusion: in conclusion, in this study, we isolated exosomes from rb primary tumour and vitreous seeding cell lines and characterized their content with a proteomic approach. this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in rb. this characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in rb as well as therapeutic targets. activation of hepatic stellate cells by extracellular vesicles released by uveal melanoma cells introduction: uveal melanoma (um) is the main intraocular tumour in adults, and is particularly resistant to treatments when disseminated to the liver. our hypothesis is that extracellular vesicles (evs) released by the primary tumour are priming the liver stroma for metastatic cell colonization by activating hepatic stellate cells (hstecs). this study aimed to characterize evs from um cells, and to determine their interactions with liver cells. methods: evs were isolated from cell lines derived from ocular tumours and liver metastases by differential centrifugation. their concentration/diameter range were determined by high-sensitivity flow cytometry. cryo-tem combined with receptor-specific gold labelling was used to reveal the morphology/size of melanomic evs. the presence of melanoma and ev markers was assessed by western blotting. the internalization of fluorescent melanomic evs in hstecs and their subsequent activation were assessed by confocal imaging using alpha-smooth muscle actin (alpha-sma) and phalloidin stainings. ev impact on invasion was measured with a tumour spheroid model embedded in extracellular matrix. melanomic evs were inoculated into the retro-orbital sinus of immunodeficient mice to study their selective organ distribution. results: melanomic evs were positive for annexin-5, tetraspanins, as well as some melanoma markers. stellate cells with internalized melanomic evs expressed more alpha-sma, reflecting their activation. adding evs on tumour spheroids increased the invasion process. melanomic evs were localized into different murine organs, but mainly into the liver, as observed by in vivo fluorescent imaging. introduction: exosomes are being tested for their use as therapeutic agents in degenerative and chronic diseases. however, the optimal source of exosomes is currently under investigation. amniotic fluid (af) is a naturally-rich source of exosomes that is easily obtained for use in regenerative medicine. organicell flow™ is a minimally-manipulated, acellular product derived from human af and consist of over 300 cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding tissues. we characterized the exosome fraction of our product to elucidate the protein cargo of af exosomes and demonstrate the therapeutic potential as a novel regenerative therapy. methods: the exosome fraction of our product was analysed using nanosight nanoparticle imaging and macsplex exosome surface marker array analysis. exosomes were precipitated using size-exclusion filtration followed by ultracentrifugation from 3 independent products (in triplicate) and subjected to protein lysis and preparation for mass spectrometry analysis using the easy nlc 1000 and q exactive instruments. tune (version 2.9) and xcalibur (version 4.1) was used to collect data while proteome discoverer (version 2.2) was used to analyse data. protein expression lists were created by merging the 3 sample replicates together and commonly expressed proteins were determined using vinny 2.0 vin diagram analysis. webgestalt tool kit classification system was used to identify top protein function and pathway hits. results: organicell flow™ contain a mean concentration of 5.24x10^11 particles/ml (n = 12) with a mean mode size of 125.2nm (n = 12). surface marker analysis confirms the presence of exosome associated proteins cd63, cd81, and cd9 in addition to a high expression of cd133 (n = 3). the completed analysis revealed 1225 commonly detected proteins across 3 products. the top molecular functions of identified proteins included protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription regulators, and transporter proteins representing the most abundant protein groups. pathway enrichment analysis revealed top hits for integrin, pdgf, and p53 pathways. a deeper dive into the enzyme category of the protein cargo further demonstrates the presence of proteins that promote dna repair such as dna polymerase (beta and lambda), telomerase reverse transcriptase, and brca1. summary/conclusion: organicell flow™ characterization demonstrates the therapeutic potential of afderived exosomes. proteomic analysis revealed protein cargo that may regulate various growth factor and cellcycle associated pathways. furthermore, the presence of dna damage response proteins suggests a possible mechanism for induction of cellular repair. generation of car-t and γδt cell-derived exosomes for future cell free immunotherapies γδt cells are a subset of t cells with dual innate and adaptive qualities. this duality provides various advantages over their more studied and used counterpart, αβt cells. in the present study, we sought to compare the immunotherapeutic potential of car-t cell and γδt cell-derived exosomes as novel cell-free based alternatives. methods: cd19-targeting car-t cells were obtained following the isolation, expansion and transduction of αβt cells using a lentiviral vector bearing the car construct. γδt cells were isolated and expanded from peripheral blood mononuclear cells (pbmcs) following innate or adaptive stimulation. exosomes from both cell sources were isolated after a 3-day culture in serum-free media using ultracentrifugation-based methods. exosomes were characterized by nanoparticle tracking analysis (determination of size) and western blot assays (detection of the appropriate surface markers). nalm-6 (b cell precursor leukaemia) cells were used as target cells for assessment of exosome cytotoxic/ killing function. car-t cell and γδt cell-derived exosomes were incubated at 1000 particles/target cell for 24-hours. total viable cell counts were assessed via imaging-based cytometry (nc-3000) utilizing acridine orange and dapi staining. results: exosomes derived from γδt cells activated via innate mechanisms showed significant killing of nalm-6 as compared to exosomes from non-activated or adaptively activated γδt cells. in comparison, car-t cell-derived exosomes showed minor killing capabilities of the target cells. summary/conclusion: here, we report for the first time that exosomes derived from cd19 car-t cells and innately activated-γδt cells show/exert inhibitory action on nalm-6 cells. further studies are currently underway to identify the underlying mechanism(s) responsible. introduction: age-related cognitive dysfunction is associated with increased oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis in the brain. from this perspective, biologics capable of modulating oxidative stress and neuroinflammation, and stimulating neural stem cell activity in the brain might be useful as anti-ageing interventions. methods: we investigated the efficacy of intranasal administration of extracellular vesicles (evs) generated from cultures of rat subventricular zone neural stem cells (svz-nscs) in the middle-aged mice to alleviate cognitive and mood dysfunction, increased oxidative stress, neuroinflammation, and neurogenesis decline in old age. mice were treated intranasally with nsc-evs once weekly for three weeks (50 billion per administration) starting from 18.5 months of age. a month later, the animals were examined for cognitive, memory, and mood function using multiple behavioural tests, and brain tissues were examined for oxidative stress, neuroinflammation, and neurogenesis. results: object-based tests revealed that aged animals receiving vehicle displayed cognitive impairments for discerning minor changes in the environment as well as for distinguishing similar but not identical experiences. these animals also exhibited spatial memory dysfunction and anhedonia. in contrast, aged animals receiving nsc-evs showed improved cognitive and mood function. biochemical analyses of brain tissues revealed that nsc-ev treatment normalized elevated concentrations of oxidative stress markers malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin-1 beta. moreover, nsc-ev treatment stimulated increased production of antiinflammatory protein interleukin-10 and the antioxidant superoxide dismutase. immunohistochemical analysis revealed modulation of neuroinflammation typified by reduced activity of reactive astrocytes and activated microglia and improved hippocampal neurogenesis. summary/conclusion: the results suggest that the intranasal administration of nsc-evs is a promising approach for maintaining better cognitive and mood function in ageing through modulation of oxidative stress, neuroinflammation, and neurogenesis. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) chemically modified myocytes-derived evs for the treatment of cardiac fibrosis. marta prieto-vila a , asao muranaka a and takahiro ochiya b a tokyo medical university, tokyo, japan; b tokyo medical university, shinjuku-ku, japan introduction: myocardial fibrosis is a disorder that may occur after cardiac injure due to a malfunction of the cardiac remodelling. fibroblasts resident in myocardium are erroneously activated causing an excessive accumulation of extracellular matrix, which decreases cardiac function and eventually, leads to death. it is known that cardiomyocytes communicate with the surrounding cells such as fibroblast and endothelial cells by extracellular vesicles (evs). the loss of this communication is thought to play a central role in cardiac fibrosis. therefore, cardiomyocytes-derived evs may be a promising a cell-free system for the treatment of fibrosis inhibition. methods: a novel culture medium was stablished to improve the expansion of primary cardiac myocytes. this was tested using two commercially available primary myocytes cell lines. evs were collected by serial ultracentrifuges, and their effect on fibrosis was tested. for that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast were activated overnight with tgfβ. results: by the use of a defined conjunct of chemicals, mature cardiomyocytes culture was highly improved to ensure a high collection of evs. terminal differentiation markers, as well as senesce apparition was delayed in comparison to predetermined culture medium. interestingly, those primary cells secreted a rather large amount of evs, which expressed common evs membrane marker. tgfβ-treated cardiac fibroblasts were co-cultured with myocytes showing a decrease of fibroblast activation markers both at mrna and protein levels. similar results were found when activated fibroblast were treated with evs. summary/conclusion: our findings indicate that the use of evs derived from chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis. however, further molecular experiments have to be done to identify the molecules within evs responsible for the inactivation of fibroblast. evaluation of osteoinductive and anti-inflammatory properties of spinederived exosomes renaud sicard a , tania del rivero b , jonathan messer c , shabnam namin c and timothy ganey c a vivex, biologics, inc., miami, usa; b vivex, biologics, inc., miami, usa; c vivex, biologics, inc., miami, usa introduction: over the last 2 decades, mesenchymal stem cell-derived exosomes have been shown to play a crucial role in a myriad of cell function such as extracellular matrix synthesis, proliferation, differentiation or cell migration. biological sources of exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct influence on the content of their cargo and their therapeutic application and potential. in this study, we evaluated exosomes excreted from cadaveric spine-derived cells. we hypothesized that exosomes derived from a bone source such as the spine, will drive the osteogenic differentiation of progenitor cells. we also investigated their effects on inflammation in nucleus pulposus cells using an in-vitro assay. methods: after their isolation and characterization, exosomes derived from cadaveric human spines were assayed for osteoinductive properties. a c2c12 myoblast cell line was treated with different concentrations of exosomes and expression of alkaline phosphatase was measured after 5 days incubation. treatment with bmp-4 was used as positive control. anti-inflammatory properties were assessed by incubating tnf-treated nucleus pulposus cells with exosomes for 3 days. qpcr analysis of mrna expression of inflammatory cytokines (il-6, il1-beta, il-8) metalloproteinases (mmp3 and adamts4), and apoptotic genes (bax, bcl2) was used to determine the effects of exosomes on inflammation. results: spine-derived exosomes positively expressed the exosome flow cytometry markers tested (cd81, cd63 and cd9). the mean number of exosomes per microgram of protein was 3.31 ± 2.33 x 108 indicating a relatively high purity. osteoinductive (oi) testing was performed using different concentrations of exosomes. the oi index of treatment of c2c12 cells with bmp-4, 2 x 108, 1 x 109, 2 x 109, 5 × 109 or 1 × 1010 exosomes alone was 28. 5, 1.0, 3.7, 7.4, 11.8 and 27.6 respectively. anti-inflammatory properties of exosome are currently being assessed and will be presented at the time of the poster presentation. summary/conclusion: administering exosomes alone or in combination with an exogenous scaffold has the potential to repair injured tissue and to restore bone function. the clinical significance of this application is aimed to promote the patients' bone healing process and provide a cell-free therapeutic platform that is safe and effective. administration of human mesenchymal stem cell derived extracellular vesicles modulates the abnormal plasticity of newly born neurons and neuroinflammation in a rat model of status epilepticus maheedhar kodali a , daniel gitai b , dong ki kim a , mariam atobiloye a , bing shuai c , sahithi attaluri c , raghavendra upadhya c , leelavathi n madhu a , olagide w. castro a , darwin j. prockop a and ashok k. shetty c decline in the percentage of newly born neurons displaying basal dendrites. besides, ev treated animals displayed higher percentages of resting microglia (ramified microglia), reduced percentages of activated microglia (microglia expressing iba-1 and cd68), in comparison to animals receiving vehicle after se. interestingly, diminished abnormal plasticity of newly born neurons was accompanied by the preservation of interneurons positive for reelin; a protein believed to guide newly born neurons to their correct locations. summary/conclusion: the results suggest that even a low dose in administration of msc-derived evs after se can limit neurons loss, dampen the abnormal plasticity of newly born neurons, and modulate the activation of microglia. introduction: autism spectrum disorders (asd) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. they have been steadily increasing in children over the past several years, with no effective treatment. two percent of all asd patients are suffering from a disorder caused by a mutation in the shank3 gene. shank3 is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. here we test three different autistic mice models. btbr as a multifactorial mice model of autism and two different shank3 mutated mice. the first is a complete deletion of exon 22 (22q13.3) and the second is a specific insertion mutation of guanine to 3680 position in the gene (insg3680) that leads to stop codon. methods: exosomes were isolated using differential centrifugation protocol and characterized using the 2018misev guideline recommendations. each animal received an intranasal administration of 20ul containing 107 exosomes/µl. for intravenous administration, the same number of exosomes, were used, injected in 100µl. results: all three animal models showed significant improvement in their autistic behavioural phenotypes following intranasal administration. the improvement seems to be dose-dependent and was better achieved via intranasal vs intravenous administration. biodistribution of msc-exo showed accumulation in the brain within 72 hours, yet the reduction of the signal was observed in the kidneys, heart and lungs. summary/conclusion: our data suggest that exosomes derived from adipose msc, carry a therapeutic potential in asd, via non-invasive intranasal administration in three different mice models. these data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials. funding: stem cell medicine ltd. israel. equine tendon injury treatment by evs: an in vitro study introduction: current treatment options for tendinopathies (chronic, painful tendon disorders), are not able to restore the functional properties of native tendons. hence, new treatment options are sought. the efficacy of mesenchymal stem cells (mscs) therapies, which combined with a rehabilitation programme including controlled exercise is the current gold standard in equine tendon treatment, has been shown to be largely due to the cells´paracrine activity. the aim of this study was therefore to evaluate the effect of bone marrow msc derived autologous and allogeneic conditioned medium (cm, full secretome) and their extracellular vesicles (evs) on "tendon healing" in vitro. methods: to compare the "therapeutic" effect of msc derived evs and cm, a standardized scratch assay (wound healing assay) was performed. cm from equine tenocytes, ev depleted medium and medium with or without fcs served as controls. tendons and bone marrow aspirates were obtained from three horses (6, 19 and 23 years) which were euthanized for reasons unrelated to this study. mscs were isolated by ficoll density gradient centrifugation and tenocytes were obtained by migration from tendon explants. for cm and ev production, cells were cultured in ev depleted medium. evs were harvested by a stepwise ultracentrifugation approach and characterized by nanoparticle tracking analysis (nta), western blot (cd9, cd63) and transmission-electron microscopy (tem). results: western blot, nta and tem confirmed successful isolation of evs from equine mscs. the strongest positive effect on wound healing (fastest gap closure) was achieved by msc-cm (p < 0.0071). the gap closure achieved with msc-evs was slower than with msc-cm (p < 0.7985) but faster than with cm of tenocytes (p < 0.8289). donor specific differences in wound healing capability were shown for both autologous and allogeneic application. summary/conclusion: treatment with msc-cm resulted in significantly faster wound healing of adult tenocytes in vitro than msc-evs or tenocyte-cm. mscs donor age shows a significant effect on gap closure following autologous but not allogeneic administration. ev-enriched secretome fraction from gmp-compatible, scalable, human ipsc-derived cardiac progenitors improve heart function in chronic heart failure mice introduction: we have shown that research-use-only grade (res) human ipsc-derived cardiac progenitors (cpcres) can produce a secretome whose small-evenriched fraction (svf) can treat chronic heart failure (chf) in mice. gmp-compatible, scalable processes for a cpc-derived svf suitable for human therapeutic use is needed. methods: ipsc-derived cpc were produced and cultured using gmp-compatible, scalable processes (cpctx). media without cells were "cultured" in parallel for "virgin media" controls (mv). cpcres were cultured as previously described. as a proof of concept, svfs were isolated from conditioned media by ultracentrifugation: cpctx-ev, cpcres-ev and mv. particle size distributions/concentrations (nanoparticle tracking analysis), protein levels (bsa), and the presence of cd-63 (elisa) were determined. in vitro activity was assessed by huvec scratch wound healing assay, and by rat and human cardiomyocyte (cm) survival assays. c57bl/6 mice in chf received echoguided myocardial injection of pbs vehicle control (60ul, n = 11), cpctx-ev (60ul, n = 11), or cpcres-ev (45ul, n = 11). change in cardiac function was assessed by echocardiography. results: cpctx-ev particle sizes were polydisperse (mode~72 nm) at a concentration of~1.6e11 particles/ml (~2,600 particles/cell) and~0.975mu cd63/ug protein. cpctx-ev increased wound healing, human cm survival, and rat cm survival in vitro by 4.75x, 1.4x, and 2x, respectively over mv controls. in chf mice, significantly less cpctx-ev mice, and less cpcres-ev mice had severely progressive heart failure (left ventricular end systolic volume, lvesv, increased >14%) than pbs control mice (pbs vs cpctx-ev, p < 0.05; pbs vs cpcres-ev, p < 0.056), and the average ejection fraction of the pbs group deteriorated 2.5x more than the cpctx-ev group (−4% vs −1.6%, respectively; ns). summary/conclusion: we have a process for cpc differentiation and production of conditioned media suitable for use in human clinical trials from which can be made an svf with the potential to treat chf, possibly through re-vascularization or preservation of cm viability. introduction: exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo. mesenchymal stem cell (mscs) modification towards an osteogenic path can occur by uptake of exosomes from other cells. it is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. an electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable and be used in a biologically neutral collagen carrier. comparisons were made of standard laboratory assay of osteoinductivity (oi), and in vivo expression in a mouse calvarial defect model. methods: electrospun type-i collagen was prepared with and without hydroxyapatite (ha) (spinplant gmbh, leipzig) as a foundation base for application of the bone marrow-derived exosomes. individual discs of the collagen enhanced scaffolds (3-mm) were prepared and placed in a mouse calvarial skull defect. animals were followed for 4 and 8 weeks. exosomes were isolated from qualified cadaveric human spines by differential ultracentrifugation. microscopic observation, quantitative assessment of oi with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition to the nano-collagen fibres. a fluorescent protein reporter transgenic mouse model expressing osteocalcin, type-i collagen, phex, and sp7 (osterix) was evaluated at 4 and 8 weeks to determine bone formation across the defect. results: alp activity on the scaffold with ha demonstrated an approximate tenfold increase to that of the collagen scaffold alone. while a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the 50ng bmp-4 control. dose escalation from 1.5, 2, and 4e9 resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. bone formation in the mouse calvaium did not demonstrate gap closure at 4 or at 8 weeks, but did demonstrate enhanced osteoclastivity and robust bone remodelling at the margins of the defect. summary/conclusion: bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. with the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. exosomes loaded with pten-interfering rna enables functional recovery in rats after complete spinal cord transection daniel offen a , nisim perets a , shaowei guo b , oshra betzer c , rachela popovtzer c and shulamit levenberg b a tel aviv university, tel aviv, israel; b technion, haifa, israel, haifa, israel; c bar ilan university, israel, ramt gan, israel introduction: complete spinal cord transection is a debilitating disease that usually leads to permanent functional impairments, with various complications and limited spontaneous recovery. the current investigation of molecular mechanisms controlling axon regeneration, (e.g., signalling networks and environmental cues), led to new strategies to enhance axonal regeneration. we have previously shown that intranasal administration of mesenchymal stem cells derived exosomes (msc-exo), cross the blood-brain barrier and significantly ameliorate motor and behavioural phenotype in several animal models of neurotrauma and neuropsychiatric disorders. methods: msc-exo were isolated from human bone marrow and were loaded with phosphatase and tensin homolog small interfering rna (pten-sirna). the exosomes were given intranasally to rats two hours after complete spinal cord transaction. eight weeks later we followed the motor function and histology and electrophysiology study was performed in order to reveal the connectivity and the biochemical changes in the treated rats. results: we demonstrate that intranasal (in) administrations of msc-derived exosomes could penetrate the blood-brain barrier, home selectively to spinal cord lesion via chemotaxis, and integrated in neurons within the lesion. furthermore, in rats with complete spinal cord transection, msc-exo loaded with pten-sirna silenced pten protein expression in the lesion and promoted robust axonal regeneration and angiogenesis, companied with decreased astrogliosis and microgliosis. moreover, the intranasal treatment partially restored electrophysiological and structural integrity, and most importantly, enabled the remarkable functional recovery and significant improvement in their movements. summary/conclusion: this rapid, non-invasive, approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel strategy for clinical translation to spinal cord injury and beyond. a novel umbilical cord derived wharton's jelly formulation for regenerative medicine applications introduction: musculoskeletal injuries have traditionally been treated with activity-modification, physical therapy, pharmacological agents and surgical procedures. these modalities have limitations, as well as potential side-effects. over the last decade, there has been an increased interest in the use of biologics for regenerative medicine applications (rma), including umbilical cord (uc) derived wharton's jelly (wj). despite this increase, there is insufficient literature assessing the amount of growth factors, cytokines, hyaluronic acid (ha) and extracellular vesicles (ev) including exosomes in these products. the purpose of this study was to develop a novel wj formulation and evaluate the presence of growth factors, cytokines, ha and ev including exosomes. methods: wj was isolated from human-uc obtained from consenting c-section donors and formulated into an injectable form. randomly selected samples from different batches were analysed for sterility testing and quantified for presence of growth factors, cytokines, ha and particles in ev size range. the results showed all samples passed the sterility test. growth factors including igfbp 1, 2, 3, 4 and 6, tgfα, pdgf-aa were detected. expression of several immunomodulatory cytokines, rantes, il-6 r, il-16, were also detected. expression of pro-inflammatory cytokines mcsfr, mip-1a; anti-inflammatory cytokines tnf-ri, tnf-rii, il-1 ra; and homoeostatic cytokines timp-1 and timp-2 were observed. cytokines associated with wound-healing, icam-1, g-csf, gdf-15, and regenerative properties, gh were also expressed. high concentrations of ha were observed. particles in the ev size range (30-150 nm) were detected and were enclosed by the membrane, indicative of true ev. summary/conclusion: our results confirmed the presence of numerous growth factors, cytokines, ha and ev in the wj formulation. more studies are underway to confirm the presence of exosomes in detected ev using exosome-specific markers. we believe the presence of multiple factors within one wj formulation may play a role in reducing inflammation, pain and augment healing of musculoskeletal injuries. this offers a potential expanded use for rma. funding: this study was funded by biointegrate llc, new york, ny, usa. collagen sponge loaded with mesenchymal stem cell-derived small extracellular vesicles promote robust bone regeneration shang jiunn chuah a , chee weng yong a , jacob ren jie chew a , ruenn chai lai b , yi ann cheow a , raymond chung wen wong a , asher ah tong lim a , sai kiang lim c and wei seong toh d introduction: mesenchymal stem cell (msc) therapy has demonstrated effective bone regeneration in clinical studies. however, the therapeutic efficacy of mscs have been attributed to the secretion of extracellular vesicles (evs), particularly 50-200 nm small evs (sevs). here, we investigate the efficacy of msc-sevs loaded in collagen sponge in the regeneration of critical-sized calvarial defects in immunocompetent rats. methods: sevs were isolated from conditioned medium of human mscs and stored at −20 c. calvarial defects of 8-mm diameter were surgically created on thirty-two 10-week-old male sprague-dawley rats. these rats were then randomly assigned to 2 groups (n = 16 rats/group): defects treated with collagen sponge containing 100 μg of sevs in 100 μl saline (cs/sevs) and defects treated with control collagen sponge containing an equivalent volume of saline (cs/control). at 1 and 8-week post-surgery, the calvarial bone samples was harvested for analyses by micro-computed tomography (micro-ct), histology, immunohistochemistry and histomorphometry. results: at 1-week post-surgery, micro-ct analysis showed little bone formation at the defect site in both cs/sevs and cs/control groups. no statistical differences were observed in micro-ct and histology scores in both groups. interestingly, cs/sevs group showed significantly higher osteocalcin (ocn)+ area of 5.7 ± 2.4% than that of cs/control group (2.1 ± 1.3%; p = 0.003). cd31+ microvessels at sizes ≤50 µm and >50 µm in cs/sevs group (34.4 ± 2.9 and 8.6 ± 2.1 microvessels/hpf) were also significantly higher than that of cs/control (18.8 ± 1.1 and 5.2 ± 1.4 microvessels/hpf; p = 0.002 and p = 0.038 respectively). by 8 weeks, cs/sevs group displayed enhanced new bone formation that completely bridged the calvaria defect. in contrast, rats in cs/control showed limited bone formation. consequently, cs/ sevs group displayed a micro-ct score of 3.9 ± 0.2 which was significantly better than that of cs/control group (2.5 ± 0.8; p = 0.001). cs/sevs group also exhibited >twofold increase in bone volume, and improved bone quality with higher trabecular thickness and number, and smaller separation (p < 0.01), compared to cs/control group. consistently, cs/sevs group displayed a significantly better histology score of 5.4 ± 1.0 than that of cs/control (2.6 ± 1.8; p = 0.001). moreover, cs/sevs group showed significantly higher ocn+ area of 48.9 ± 7.9% than that of cs/control group (20.4 ± 4.4%; p = 0.004). summary/conclusion: this study demonstrates that single-stage implantation of collagen sponge loaded with ready-to-use msc sevs can promote robust bone regeneration in a rat calvarial defect model. funding: national university of singapore, r221000114114, national medical research council singapore, r221000123213. immunomodulatory potential of extracellular vesicles derived from mesenchymal stromal cells introduction: extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) are promising new agents in regenerative medicine and immunotherapy. considering that independent msc-ev preparations might differ in their therapeutic function, we have set up a functional assay allowing testing for the potential immunomodulatory properties of independent msc-ev preparations. methods: human peripheral blood-derived mononuclear cells (pbmcs) were pooled from up to 12 different healthy donors warranting high allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. after thawing, mixed pbmcs were cultured for 5 days in the absence or presence of msc-evs. thereafter, cell morphologies were documented, supernatants were harvested for cytokines quantification and cells were phenotypically characterized by flow cytometry. by analysing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by msc-ev preparations considered to be therapeutically active. results: we observed that in the presence of active msc-ev preparations more cd14+ (monocytes) are recovered from the mlr assay than in corresponding control samples. focusing on t cells, we learned that active msc-ev preparations reduced the content of cd4 and cd8 t cells expressing activation markers like cd54 and cd25. summary/conclusion: the mlr assay allows elaborated functional testing of immunomodulatory activities of given msc-ev preparations. currently, we are comparing the immune modulatory capabilities of evs derived from distinct sources and optimize the marker panel to distinguish discrete immune cell subtypes such as different cd4 cell types, i.e. th1, th2, th17 and tregs. extracellular vesicles in platelet-rich plasma: dependency on sample processing zala jan a , saba battelino b , darja božič c , matej hočevar d , ales iglič e , marko jeran c , manca pajnič a , ljubiša pađen a , domen vozel f and veronika kralj-iglič a introduction: platelet-rich plasma (prp) proved effective in regenerative medicine. numerous protocols for its preparation and application are available in the published literature. prp possesses important immune, haemostasis and regenerative factors, however, the mechanisms of their action are yet poorly understood. extracellular vesicles (evs) could be one of the important factors that would contribute to the beneficial effects of preparations. this study was performed as a part of a registered randomised controlled clinical trial (nr: nct04281901). prp was used to treat chronic middle ear inflammations. here we present the results of prp analyses from 29 blood samples of volunteers with no record of disease. methods: plasma obtained from 20 ml of blood was depleted of erythrocytes and enriched with other particles by repetitive centrifugation of samples. flow cytometry (fcm) was employed to monitor particle contents (cells and smaller particles) throughout the sample processing. the platelet gate was divided into two parts: intact platelets and smaller particles. identity and morphology of particles in the preparations were examined by scanning electron microscopy (sem). standard laboratory tests of blood were performed. results: sem images revealed the presence of heterogeneous population of particles in the preparation of prp, most of which were activated and partially fragmented platelets. the population of smaller particles measured with fcm, was identified as evs. the erythrocyte sedimentation rate was statistically significantly correlated to the volume of plasma obtained in the initial centrifugation step (r = 0,70, p < 0,001) and to the concentration of evs (r = 0,82; p < 0,0001). time from sample collection to the preparation of prp was negatively correlated with the concentration of platelets in prp and positively with the concentration of evs (r = 0,75, p < 0,001). platelet concentration in preparation samples was found to depend on the concentration of platelets in the blood and parameters of sample processing connected with larger centrifugal and shear forces on the samples during centrifugation. these include: sample volume, the size and shape of the centrifuge tube and the distance of the sample from the rotor axis. summary/conclusion: evs are gradually forming upon activation and degradation of cells in the sample throughout the sample processing. optimal processing may importantly contribute to the healing properties of preparation. funding: authors acknowledge support from the european union's horizon 2020 research and innovation program under grant agreement no. 801338 (ves4us project) and slovenian research agency (arrs, grants p2-0232, p3-0388, j1-9162). satellite cell-derived extracellular vesicles as a therapeutic for mitochondrial dysfunction in duchenne muscular dystrophy duchenne muscular dystrophy (dmd). sc-derived extracellular vesicles (sc-evs) may unlock the therapeutic potential of scs by overcoming these limitations. to investigate their therapeutic potential, we assessed the ability of sc-evs to reverse mitochondrial dysfunction, a key pathological feature of dmd, in oxidatively-damaged c2c12 and primary dmd myotubes. methods: scs from c57 mice were isolated and cultured. evs were isolated from the supernatant of scs via polyethylene glycol precipitation and characterized using nanoparticle tracking analysis. the ability of sc-evs to deliver protein cargo to c2c12 myotubes, and the localization of the cargo once delivered, were analysed using fluorescence microscopy. to examine sc-ev potential to restore the function of damaged mitochondria, c2c12 myotubes were treated with 50 µm h2o2 for 24 h followed by treatment with 3.12x108 sc-evs for 24 h. separately, cultured dystrophic myotubes were treated with 3.12 × 108 evs every 24 h for 72 h. in both sets of experiments, maximal oxygen consumption rate (max ocr) was measured via seahorse xf cell mito stress test. where appropriate, a t-test was performed to test for statistical significance (p < 0.05). results: based on estimated cell number and ev quantification, each sc released approximately 2.35 × 105 ± 3.10x104 evs/day. evs delivered protein cargo into myotubes within 2 h. fluorescent labelling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. incubation of myotubes with h2o2 resulted in a 42% decline in max ocr relative to untreated myotubes. subsequent treatment with sc-evs resulted in a 76% increase in max ocr. treatment of undamaged myotubes with sc-evs had no effect on max ocr. primary dmd myotubes treated with sc-evs showed a 78% increase in max ocr relative to untreated dmd myotubes. summary/conclusion: sc-evs rapidly deliver proteins into myotubes, much of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged and dystrophic myotubes. introduction: flow cytometry has been used extensively for analysis of ev particles stained with fluorescent antibodies directed to the known cell surface markers. quantitation of the surface markers in terms of the number of molecules or the number of antibodies bound per specific marker has remained one of the largest challenges in the ev research field. changes in instrument setup as well as changes in fluorescent antibodies from different vendors, all impact the relative mfi values for the same ev sample. in this work we report a standardization method of quantitating extra-cellular vesicle surface markers with mesf liposomes. methods: liposomes labelled with fitc fluorescent dye were prepared with a bd proprietary technology. dynamic light scattering analysis was used for size determination of the liposomes. bd facsaria™ fusion system, modified with a small particle side scatter module (sp ssc), was used for analysis of the labelled liposomes by flow cytometry. results: we created a set of 180 nm fitc-modified liposomes of various fluorescent intensities with a known number of fitc molecules incorporated in each liposome intensity. the mfi values of each liposome population (intensity) had a linear relationship to the amount of fitc used for labelling the liposome nanoparticles, suggesting that no self-quenching of fitc fluorescence had occurred. the number for the fitc fluorophores for each liposome intensity was expressed in the units of molecules of equivalents soluble fluorochrome (mesf). a plot of mesf vs. the fluorescent intensity of the liposomes (mfi values) obtained from flow cytometry analysis provided a calibration curve, from which the fluorescent intensity (mfi value) of a stained ev sample can be converted to the number of fluorophores bound (mesf value) to the surface of the ev particles. summary/conclusion: by this approach, the mfi values of stained ev particles are converted to standardized mesf values that are independent of instrument variation, resulting in further improvement of inter-laboratory standardization. furthermore, utilization of liposomes with similar size and refractive index to ev particles simplifies the data evaluation and improves the accuracy of ev surface marker quantitation by flow cytometry. currently, other fluorescent dyes are being explored to expand the utility of mesf liposomes with other fluorescent colours. measuring cholesterol as a high-throughput method for quantifying extracellular vesicles introduction: the extracellular vesicle (ev) field currently lacks a high-throughput method for accurately quantifying evs in solution. ev quantification has traditionally relied on nanoparticle tracking analysis (nta), which is time intensive and indiscriminately counts non-ev particles, such as membrane fragments and protein aggregates. we have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the ev lipid bilayer, and evaluated the utility of these assays to quantify evs in minimally processed samples. methods: the amplex® red cholesterol assay and cedex bio ht were used to quantify cholesterol in ev samples via enzymatic oxidation, with dynamic ranges of 80-8,000 ng/ml and 4-800 µl/ml, respectively. samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified evs separated on an iodixanol gradient. we evaluated several pre-processing methods, to remove non-ev cholesterol content prior to analysis. results: the amplex® and cedex bio ht assays were found to perform comparably for quantifying cholesterol in purified evs (r2 = 0.92). importantly, cholesterol quantification on purified ev samples, ranging from 1e11 to 4e13 particles/ml, correlated well with nta measurements (r2 = 0.96). both 45 µm filtration or an additional 16,000 rcf centrifugation step following clarification removed cholesterol associated with cellular debris or other non-ev sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (ucp) instead of needing to rigorously purify samples with an iodixanol density gradient. summary/conclusion: cholesterol quantitation can be used to accurately estimate ev concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified evs. this highthroughput analytical capability may enable more comprehensive assessment of methods to boost ev yield through mass screening of cell culture conditions. optimization of nanoparticle tracking analysis of extracellular vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small cell lung cancer introduction: recent studies show that tumourderived extracellular vesicles (evs) greatly influence the tumour microenvironment and impact the therapy. in non-small cell lung cancer (nsclc), bronchopulmonary lavage fluid (balf) appears to be a good source of tumour-derived evs, providing more accurate information about the tumour microenvironment than evs from plasma. so far there is a lack of accurate and standardized methods for ev quantification. fluorescence nanoparticle tracking analysis (fl-nta) is an emerging method of ev-analysis, allowing discrimination of evs and exosomes from impurities. here we perform an optimization of the fl-nta method to compare evs from plasma and balf of nsclc patients and healthy controls (nc). methods: evs were isolated using homemade sizeexclusion chromatography (sec) columns (plasma) and ultrafiltration or differential ultracentrifugation (balf). nta was performed using zetaview pmx220 (particle metrix) after ev-staining with membrane dyes or fluorescence-labelled antibodies against typical ev-marker (cd9, cd81, cd63). results: nta scatter measurements showed a higher total particle concentration in plasma than in balf. however, membrane-specific staining showed a much greater purity of ev-preparations from balf, where nearly 100% of the particles detected in scatter mode showed positive membrane-staining. in contrast, only around 40-50% of particles in the plasma ev-preparations were positive for the membrane dyes. fluorescence-staining for ev surface marker requires further optimization to obtain reproducible results. summary/conclusion: classical nta using only the scatter mode fails to discriminate between evs, lipoproteins and protein aggregates. for ev-analysis from complex biofluids like plasma, fla-nta and staining for specific ev marker is necessary to receive reliable data. balf seems to be a better source of tumourderived evs than plasma, since the obtained ev-preparations show a higher purity. improving conditions for fluorescence-staining and nta measurement of evs from plasma and balf of nsclc patients will provide an additional method for quantifying and phenotyping of evs. introduction: the exoviewer platform currently enables the user to capture extracellular vesicles (ev) by means of surface antigen-specific antibodies (e.g. targeting tetraspanins), making possible the enumeration of individual particles using single-particle interferometric reflectance imaging sensor (sp-iris, interferometric) imaging as well as fluorescence. currently, through interferometric imaging particles smaller than 50 nm cannot be detected, while fluorescently stained ev smaller than 50 nm can be well resolved. further, it is conceivable that small ev contain antigen numbers in the single digits, making antigen-specific immunostaining a challenge. to further characterize ev populations of different sizes and surface marker composition, it would be highly advantageous to target the vesicular nature of the detected particles linked to a fluorescence readout. methods: the goal of this project is to detect ev with a probe that is ubiquitously distributed across the surface (or lumen) of the vesicle. small (30-150 nm) ev present fairly distinctive lipid membrane features in the extracellular environment, turning the ev membrane into a "universal" marker, and as such may serve as an alternative marker that is complementary to canonical ev surface markers. results: here we present data on successfully staining ev with the membrane dye di-8-anepps (di-8) and the luminal dye calcein-am. we demonstrate that ev from different sources can be efficiently stained with either dye, allowing the quantitative characterization of ev in an unbiased manner using exoviewer's fluorescence mode. while both dyes certainly have their own unique strengths, they exhibit the wanted linear correlation of ev staining versus concentration. further, both dyes are compatible with subsequent immunostaining applications, allowing the user to target specific surface or luminal markers (di-8). summary/conclusion: while a large-panel screening featuring other powerful dyes is continuously ongoing, the current data support the notion of providing the experimenter with a reference for total particle count and at the same time fully exploring the larger dynamic range of the fluorescence mode. moreover, the universal probe will enable the user to correlate intensity and particle size measurements, thereby significantly improving the exoviewer platform and its applications. membrane labelling is essential for the identification and quantification of extracellular vesicles via facs introduction: extracellular vesicle (ev) research is challenged by the lack of standard protocols to identify and distinguish between exosomes and ectosomes being released via exocytosis or plasma membrane shedding, respectively. analysis of small ev populations requires high-resolution technology and can be further improved using fluorescent labels such as carboxyfluorescein diacetate succinimidyl ester (cfse). at the inner leaflet of the plasma membrane, cfse is cleaved enzymatically resulting in covalent binding of the dye. in this study we optimized the conditions for membrane labelling of evs and their subsequent detection by flow cytometry to obtain a maximum yield of intact evs. methods: using sequential centrifugation, we separated ev subpopulations from supernatants of colo 357 pancreas carcinoma cells based on size and mass. after 10,000x g centrifugation, we reconstituted evs from the pellet. we used cfse for ev detection and analysed the expression of tetraspanins by facs to confirm the lipid bilayer structure. furthermore, we determined size distribution of evs by nanoparticle tracking analysis (nta) and electron microscopy. detecting evs as cfse+ events, we quantified our samples and investigated the impact of threshold adjustment on ev quantification. results: after high speed centrifugation of cell free supernatants, we identified cfse+ events as evs, which appeared as round structures under the microscope, and ranged from 80 to 40 nm in size. interestingly, tetraspanin markers cd9 and cd81 were detectable only on a subpopulation of purified evs, suggesting heterogeneity of our preparations. for sufficient labelling of evs, minimal temperature variations and short incubation times correlated with ev stability. of note, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of labelled evs and hence, is central for data comparability. summary/conclusion: protocol standardization is of major importance for the use of evs as diagnostic markers in liquid biopsies. funding: this project has been supported in part by annelise-asmussen foundation, luebeck (grant 180802), leo pharma germany (grant 180208). surface plasmon field-enhanced fluorescence spectroscopy (spfs) system for quantitative and qualitative extracellular vesicles total evaluation without any sample pretreatment introduction: the function of extracellular vesicle (ev) is interested in the immunology and oncology fields as a key transmitter for cellular communication. however, the conventional ev evaluation methods are required complicated evs preconcentration from the sample, its leads ev analysis uncertainty. in this study, we applied the spfs highly sensitive automated system for quantitative and qualitative ev evaluation without any sample pre-concentration and preparation step. methods: spfs automated system and plastic disposable sensor had been developed by konica minolta corporation in house. anti-membrane protein (cd9, cd63, cd83) antibody was chemically bonded on hydrophilic polymer which was immobilized through the gold thin film on the spfs sensor. the concentration of standard ev materials was evaluated by the qnano system before using. ev detection without preconcentrating was achieved by sandwich immunoassay step in microchannel round-trip flow reaction (tat 120 min) with the spfs system, and elisa was adapted as a conventional standard method. after spfs highly sensitive fluorescent measurements step, extracted and detected ev were effectively recovered by using the recovery buffer reaction. results: the ev sensitivity performance between spfs and elisa clearly showed a significant difference, and the lod of spfs (8.3 particles/μl) method was estimated 3000 times superior to the lod of conventional elisa (26,000 particles/μl). the spfs calibration curve showed a wide dynamic range at least over 5 logs as an additional specificity. spfs method also showed fine results in the dilution linearity test with high reproducibility under the serum/plasma sample condition. the data for recovery test of ev expected us that highly accurate measurement can be guaranteed under the condition of dilution about 10 times or less even in the whole blood sample. after the spfs measurement, extracted ev on the spfs sensor chip could be effectively recovered and could be analysed nucleic acid which contains micro rna. summary/conclusion: spfs system might have great potential for quantitative and qualitative ev evaluation. our strategy with spfs system for ev proteomic and genomic profiling will be possible for applying to ev quality control as well as a novel biomarker development. identification of a novel compound that inhibits small ev secretion and tumour progression by a sensitive elisa screening. yunfei ma a , takeshi yoshida a , duc tuan nguyen a , kazutaka matoba b , katsuhiko kida b , taito nishino b and rikinari hanayama c a kanazawa university, kanazawa, japan; b nissan chemical corporation, tokyo, japan; c wpi nano life science institute, kanazawa university, kanazawa, japan introduction: small evs from tumour cells are known to promote tumour progression, therefore, it is expected to develop drugs that regulate small ev secretion, which can be used in clinical applications. methods: to identify such regulators, we first developed a sensitive elisa system for the quantification of small ev secretion using a high-affinity ev binding protein tim4. by using this elisa system, we screened for small compounds that promote or inhibit small ev secretion using a drug-repositioning compound library (about 1,600 compounds). results: as a result, we identified eight promoters and two inhibitors, including compound a, which significantly reduced small ev secretion from various cell types without affecting cell growth. we further investigated the effects of compound a on a mouse model of osteosarcoma and found that compound a suppressed tumour progression efficiently. summary/conclusion: these data suggest that compound a would be useful not only for the characterization of small ev function but also for the clinical therapy against tumour progression, by inhibiting small ev secretion. introduction: for many years it was believed that several proteins such as cd63, cd9 and flotillin-1 were unique for exosomes, however recent studies have shown that several of these markers also can be present in other subpopulations of evs (kowal et al pnas 2016) . furthermore, few markers have been identified as uniquely present in microvesicles. the aim of this study was to in depth compare the proteome of microvesicles and exosomes. methods: mda-mb-231-luc-d3h1, -d3 h2ln and -bmd2a were cultured in ev-depleted media. microvesicles (16,500 x g, 20 min) and exosomes (118,000 x g 2.5 h) were isolated using a combination of differential ultracentrifugation and a density cushion (~1.1 g/ml). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, and electron microscopy (em). quantitative mass spectrometry (tmt-lc-ms/ms) was used to identify differently enriched proteins in microvesicles and exosomes (n = 3 x 3 cell lines). results: in total 6493 proteins were quantified, with 4851 being quantified in all samples. in total 818 and 1567 proteins were significantly upregulated in exosomes and microvesicles, respectively. go terms associated with the proteins significantly upregulated in exosomes were "extracellular exosome" and "plasma membrane", while the microvesicle proteome was associated with "membrane" and mitochondrion". in exosomes tetraspanins, annexins, escrt and rab proteins were significantly upregulated. in contrast, proteins that were upregulated in microvesicles were involved in protein translocation into the mitochondrial membrane (timm and tomm proteins), in cytokinesis, and in micos complex. however, flotillin-1 was not differently expressed in the ev subtypes. summary/conclusion: this study identifies several proteins to be differently enriched in exosomes and microvesicles. several of the proteins suggest recently by kowal and colleagues, such as adam10 and mitofilin could be validated. additionally several novel proteins could be identified. identifying markers separating microvesicles and exosomes is of high importance for the ev field and future studies will have to validate them also in other cells to determine if they are generic. introduction: the cellular elements composing the lining of brain ventricles have drawn much attention from neuroscientists, especially the role of subependymal cells in neurogenesis, but the role of ependymal cells in brain function and disease is still neglected. our objective is to study the morphological aspects of rat brain ventricles and the ependymal cells as analysed by transmission and field emission scanning microscopy in normal or ischaemic rats. methods: for this purpose, male wistar rats were submitted to 10 minutes of global brain ischaemia and divided into two groups: a) sham-operated animals and b) saline-treated ischaemic animals. all animals were allowed to survive for seven days. all procedures were approved by the ethics committee of the federal university of são paulo (2018/7633081117). transmission and scanning electron microscopic analysis of lateral brain ventricles were done in buffered 2,5% glutaraldehyde/2%formaldehyde perfused brains. cerebrospinal fluid was collected for nta analysis. results: the morphological characterization of brain ventricle revealed a slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared to normal animals. field emission electron microscopy revealed the secretion of vesicles by the ependymal cilia in the lateral ventricle. size and concentration of particles in the cerebrospinal fluid was confirmed by nta and transmission electron microscopy. summary/conclusion: our results are unprecedented and bring innovative potential regarding the role of extracellular vesicles in both the physiology and pathogenesis of the nervous system. these data may also contribute to the development of new technologies for diagnosis and therapy of chronic degenerative diseases. introduction: the function of mitochondria relies on precise and effective quality controls. neurons have high metabolic demands and employ multiple mechanisms to ensure functional mitochondria. we investigated mitochondrial vesiclesa less understood quality control mechanism for mitochondriaand assessed the effect of cellular stress. methods: we surveyed mitochondrial vesicles in rat and planaria brains with electron microscopy. we quantified these vesicles with serial-section electron microscopy (fib-sem). we also conducted confocal microscopy with airyscan analysis of cultured neurons expressing fluorescently tagged mitochondrial markers. results: electron microscopy showed the ultrastructure of various types of mitochondrial vesicles. serial-section electron microscopy revealed the 3d ultrastructure of mitochondrial vesicles and their prevalence in neurons. confocal microscopic analysis showed increased numbers of mitochondrial vesicles in neurons under mild stress. summary/conclusion: our findings provide direct structural evidence for mitochondrial vesicles in neurons and their abundance in response to neuronal stress. their detection in the extracellular compartment (evidence for which is expected to be presented by the time of isev) may allow for development of biomarkers for mitochondrial health, with relevance to numerous pathologic conditions. from endosomes, might be involved in the impairment of rna, specific feature of als disease. combining high-resolution flow cytometry and surface marker analysis using an automated platform to study extracellular vesicle in cerebrospinal fluid unity health toronto, toronto, canada introduction: there is growing enthusiasm that extracellular vesicles (evs) carry the potential for a variety of applications in medicine. as biomarkers, evs may aid clinicians in the evaluation of diagnoses, disease progression, or even response to therapy. however, proper characterization of the amount, size, and phenotype of evs in a given sample remains challenging due to their sub-micrometre size and heterogeneity. over the last years, technologies, including high-sensitivity flow cytometry and automated platforms that simultaneously assess ev amount, size, and phenotype, have matured, providing new opportunities to study evs for future clinical applications. using such technologies to analyse cerebrospinal fluid (csf), which is in direct contact with the brain and spinal cord, may yield valuable insights into neurological disease processes. while there is often uncertainty about the exact source of evs in a biological sample, cd171 has emerged as a surface marker that suggests a neuronal origin. methods: csf samples that had been stored at -80 degrees celsius for advanced biomarker studies were analysed using two distinct approaches. a becton, dickinson and company (bd) aria iii flow cytometer was converted into using violet side scatter (ssc) for improved detection of evs with 405 instead of 488 nm ssc. for the combined analysis of amount, size, and phenotype, samples were analysed with the nanoview bio r100 platform. phenotype analysis included probing for the classic tetraspanins associated with exosomes (cd9, cd63, cd81) and the neural cell adhesion molecule l1 (cd171). results: flow of csf samples showed similar vesicle counts in control vs. disease and an increase of counts in later disease stages when neurodegeneration is thought to be more prominent. all csf samples showed some binding to classic exosomal markers (cd9, cd63, cd81). the sample taken at the latest time point showed relatively high vesicle counts, overall larger vesicle size, and abundant cd171 binding. interestingly, the cd171 positive evs were not positive for any of the classic exosomal markers (cd9, cd63, and cd81). summary/conclusion: this data supports the notion that analysing the amount, size, and surface markers of evs in csf can reveal intriguing dynamics in such basic ev characteristics over time and suggests important differences between ev populations in different disease stages. while previous studies indicated that cd171 could identify an ev to be of neuronal origin, it remains to be determined whether such specific surface markers will emerge as clinically relevant tools to support the evaluation of people affected by neurological diseases. a distinct microrna signature in plasma derived small extracellular vesicles of different neurodegenerative diseases introduction: exploring identifying robust biomarkers is essential for early diagnosis of neurodegenerative diseases. blood stream transports large (levs) and small extracellular vesicles (sevs), which are extracellular vesicles of different sizes and biological functions that are transported in blood. aim of our study was to investigate mrna/mirna signatures in plasma derived levs and sevs of amyotrophic lateral sclerosis (als), alzheimer's disease (ad), parkinson's disease (pdpd), fronto-temporal dementia (ftd) and alzheimer's disease (ad) patients. methods: levs and sevs were isolated from plasma of patients and healthy volunteers (ctr) by ultracentrifugation and rna was extracted. whole transcriptome and mirna libraries were prepared with truseq stranded total rna kit and truseq small rna library kit (illumina). results: our data suggested that the rna cargo in levs and sevs varies among different diseases. mirna analysis in sevs provided the most informative disease specific signatures, while whole transcriptome analysis did not show any specific signature. als was characterized by a small but specific group of circulating mirnas. mirnas profiling revealed that pd and ftd can be subgrouped in two classes while ad appears to be a homogeneous disease population. furthermore, mirnas profiling show the presence of overlaps in the signatures between the analysed diseases. mirna profiling in levs is similar to that observed in sevs, although in levs the overall differences between diseases are less marked. summary/conclusion: in this study we have demonstrated that mirnas are the most interesting subpopulation of transcripts transported by plasma derived sevs since they discriminate a disease from the other and they can provide a signature for each neurodegenerative diseases. may be linked with apoe genotype, we investigated the possible effect of apoe genotype on brain-derived evs (bdevs) and their protein and rna molecular cargo. methods: cortical brain tissues of ad patients with different apoe genotypes [ε2/ε3 (n = 5), ε3/ε3 (5), ε3/ε4 (6), ε4/ε4 (6)] and non-ad controls (n = 7) were obtained. bdevs were separated by size exclusion chromatography plus ultracentrifugation (uc) and characterized per misev2018. proteins were analysed by mass spectrometry. after protein identification, data were normalized using the cyclicloess method and analysed by principal component analysis (pca). nested factorial design highlighted differentially expressed proteins. rna from bdevs was extracted by mirneasy mini kit. small rna libraries were constructed using the ion total rna-seq kit and sequenced on the ion torrent s5™ using ion™ 540 chips. reads were aligned to human reference transcriptomes using bowtie. differential gene expression was quantified by edger and limma. results: among 28 proteins dysregulated in ad bd-sevs, several have reported roles in ad, e.g., microtubule-associated protein tau and peroxiredoxin-6. regarding apoe genotypes, 16 proteins were differentially expressed between ε4 carriers (ε3/ε4 and ε4/ε4) with non ε4 carriers (ε2/ε3 and ε3/ε3). however, ev markers did not differ by apoe genotype. in contrast to protein cargo of bdevs, the overall small rna expression pattern was similar among ad patients with different apoe alleles and non-ad patients. only a few mirnas showed different abundance level between ε2/ε3 and ε4/ε4 groups, or between ad and non-ad groups. summary/conclusion: bdevs carry proteins and mirnas related to ad development and apoe genotypes. further verification of protein and rna expression in brain and plasma derived evs may reveal mechanisms of ev function in neuroinflammation and develop biomarkers for ad disease. funding: this project was funded by mh118164. efficient pathology spread by extracellular vesicles from human brain tissues in mouse brain and tissue cultured neurons: transmission and propagation to gabaergic neurons however, whether human brain-derived evs induce tau pathology has not yet been characterized in the mouse brain. here, we assess the mechanisms of disease spread after intrahippocampal injection of human brainderived evs into the aged mouse model. methods: ev-enriched fractions were isolated from unfixed frozen human brain samples from ad, prodromal ad (pad), control (ctrl) cases, and tau knockout (tko) mouse brains. isolated evs containing 300 pg of human total tau were sterotaxically injected into the right outer molecular layer of the dentate gyrus of 18 months-old c57bl/6 female mice. 4.5 months after the injection, hippocampal slices were prepared for whole-cell patch clamp recordings of ca1 pyramidal neurons were undertakent. hippocampi were analysed with immunohistochemistry using phosphorylated-tau (p-tau) epitopes including at8. evs were examined for protein composition by protein mass-spectroscopy, the neuronal uptake in vitro, and structural analysis by the atomic force microscopy (afm). results: semiquantitative brain-wide immunohistochemistry of p-tau revealed that inoculation of ad or pad-evs induced tau propagation throughout the hippocampus, including the dentate gyrus, ca3 and ca1 subregions. at8 was localized primarily in gad67 + gabaergic neurons in pad and ad evs groups, accompanied with reduced amplitude of inhibitory postsynaptic currents and excitatory-inhibitory ratio in amplitube of postsynaptic currents in ca1 pyramidal neurons in pad evs. afm analysis showed higher density of tau oligomers in both ad and pad evs while only ad evs showed significantly higher neuronal uptake compared to ctrl evs. finally, proteomic analysis showed that ad evs are enriched in disease and glia-related molecules compared to ctrl evs, which may contribute to their enhanced neuronal uptake. summary/conclusion: intracranial injection of ad or pad evs induced p-tau accumulation primarily in gabaergic neurons throughout the hippocampus, resulted in higher uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency as seeding factors. gabaergic neuronal dysfunction in the hippocampal neuronal circuitry reported in early ad brains could be attributed to specific ev mediated tau propagation in this cell type, a phenomenon meriting further investigation and validation. funding: nih rf1ag054199, nih r01ag054672, nih r56ag057469, cure alzheimer's fund, brightfocus foundation, curepsp, coins for alzheimer's research trust introduction: extracellular vesicles (evs) are released by cells of the central nervous system as a result of injury, including mild traumatic brain injury (mtbi). since mtbi may alter circulating levels of evs, this study aimed to investigate differences in circulating ev numbers between contact sport athletes with and without acute mtbi. methods: circulating evs containing cd63 (cd63 + ev), cd81 (cd81+ ev), and neural cell adhesion molecule (l1cam+ev) were analysed in young, male athletes with or without mtbi (18-29 yo, n = 6 per group). sodium citrate-treated blood samples were obtained from athletes with mtbi within 48-hours of injury and from control athletes free of mtbi for one year. athletes were best matched for age and history of prior mtbi. samples were double-centrifuged to obtain platelet-poor plasma and stored at −80°c until analysed. quantification of evs was performed using a spectral flow cytometer. the study was approved by temple university's irb, and all athletes provided written informed consent. results: mann-whitney u tests showed that population percentages of small size (179-304 nm) cd63 + ev, cd81+ ev and l1cam+evs were significantly higher in mtbi athletes (mean rank: 8.0, 9.5, 9. 3) than controls (mean rank: 3.6, 3.5, 3.7) (u = 3.0, p = 0.03; u = 0.0, p > 0.01; u = 1.0, p > 0.01, respectively). population percentages of large size (500-800 nm) cd63+ ev, cd81+ ev and l1cam+evs were also significantly higher in mtbi athletes (mean rank: 8. 2, 9.2, 9.5) than controls (mean rank: 3.4, 3.8, 3.5) (u = 2.0, p = 0.02; u = 2.0, p > 0.01; u = 0.0, p > 0.01, respectively). there were no significant differences between percentages of evs associated with blood brain barrier function (cd144+ ev) or platelets (cd42a+ev) among mtbi athletes or controls. introduction: parkinson's disease (pd) is characterized by clinical heterogeneity, different rates of progression and absence of definitive biomarkers. extracellular vesicles (evs) are easily isolated from plasma and play a central role in intercellular communication which is highly relevant for inflammatory processes implicated in protein misfolding-related neurodegenerative disorders. thus, we characterized distinctive plasmatic ev subpopulations of pd and atypical parkinsonisms (ap) patients, with the aim to identify candidate biomarkers among evs surface membraneproteins. methods: plasmatic evs were collected from 27 pd, 19 matched healthy controls (hc), 9 ap with multiple system atrophy (msa) and 9 ap with tauopathies (ap-tau). evs were quantified by nanoparticle tracking analysis. the expression of 37 ev-surface markers, related to inflammatory and immune cells, were measured by macsplex and correlated to clinical scales. a diagnostic model based on ev markers expression was built via supervised machine learning algorithms and validated in an external cohort (10 pd, 20 hc, 5 msa, 5 ap-tau). the cantonal ethics committee approved the study protocol. all enrolled subjects gave written informed consent. results: pd showed the highest ev concentration compared to others groups. pd and msa displayed a greater pool of overexpressed immune markers compared to ap-tau. ev antigens correlate to cognitive impairment and disease gravity in pd and msa. the roc curve analysis of a compound ev marker showed optimal diagnostic performance for pd (auc 0.908; sensitivity 96.3%, specificity 78.9%) and msa (auc 0.974; sensi-tivity100%,specificity94.7%)andgoodaccuracyforap-tau (auc 0.718; sensitivity 77.8%, specificity 89.5%). a diagnostic model based on ev markers expression, cor-rectlyclassified88.9%ofpatientswithreliablediagnostic performance after validation in an external cohort (77% of accuracy). summary/conclusion: this analysis of multiple immune surface markers of circulating evs in pd and ap well captured the clinical heterogeneity of pd and showed optimal diagnostic performance. furtherly it suggests a different immune dysregulation in pd and msa vs. ap-tau, to be confirmed by functional analysis in experimental models of disease. funding: supported by abreoc. separation and characterization of extracellular vesicles from human cerebrospinal fluid introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off 3,000), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions (3 + 4) after the sec void volume as evaluated by detection of cd63 and cd9 markers (immunoblotting) and annexin a2 (peptide mapping by nanolc-ms/ms). there, nanoparticles around 150 nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between 55 and 165 nm, with average diameter 94 ± 31 nm. cd63 was visualized by immunocytochemistry at the surface of ev around 80 nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin-3 binding protein (lgals3bp or 90 k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those nanoparticles had ring-like appearance. occasionally 90 k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of 90 k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from 90 k nanoparticles to investigate their individual physiological relevance and biomarker potential. introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off 3,000), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions (3 + 4) after the sec void volume as evaluated by detection of cd63 and cd9 markers (immunoblotting) and annexin a2 (peptide mapping by nanolc-ms/ms). there, nanoparticles around 150 nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between 55 and 165 nm, with average diameter 94 ± 31 nm. cd63 was visualized by immunocytochemistry at the surface of ev around 80 nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin-3 binding protein (lgals3bp or 90 k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those nanoparticles had ring-like appearance. occasionally 90 k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of 90 k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from 90 k nanoparticles to investigate their individual physiological relevance and biomarker potential. release of extracellular vesicles from platelets requires platelet-platelet interaction aleksandra gąsecka a , naomi c. buntsma b , sytske talsma c , krzysztof j. filipiak d , rienk nieuwland e and edwin van der pol f introduction: arterial thrombosis is a major and global cause of human death and disability, but a biomarker for early-diagnosis of thrombosis is absent. platelet activation and aggregation are the first steps of plateletrich thrombus formation, but their relative contribution to platelet extracellular vesicles (pevs) release is unknown. methods: to study the relation between pev release and platelet interaction (aggregation), citrate-anticoagulated whole blood (wb) from healthy donors was diluted 2, 4, 8, 16 and 32-fold and activated by 30 μm thrombin-receptor activating peptide (trap). in addition, undiluted wb and 10-fold diluted wb, which totally blocked pev release, were activated with various trap concentrations. concentrations of pevs (cd61 + and cd61+, cd62p + >1000 nm) and activated platelets (cd61+, cd62p+ >1000 nm) were measured by flow cytometry (apogee a60-micro). platelet aggregation was assessed using impedance aggregometry. results: a 10-fold dilution of wb blocked both aggregation and the release of pevs. compared to baseline, activation of undiluted wb with trap increased the concentrations of cd61 + 2.2-fold and cd61+-cd62p + pevs 7.2-fold. the concentration of cd61+ (r2 = 0.71) and cd61+-cd62p+ (r2 = 0.78) pevs as well as platelet aggregation (r2 = 0.8) scaled inversely (reciprocal) with the dilution of wb. further, we found a linear correlation between the % of activated platelets and the concentration of cd61+ (r2 = 0.80) and cd61 +, cd62p+ (r2 = 0.64) pevs in undiluted wb, which was absent in 10-fold diluted blood (r2 < 0.05). summary/conclusion: the absence of aggregation and pev release upon platelet activation in 10-fold diluted blood shows that aggregation directly depends on the distance between platelets, which is confirmed by the reciprocal relationship between pev release and blood dilution. because pevs are only released when platelet activation is followed by aggregation, pevs are a potential early biomarker of thrombosis. funding: ag is supported by the national science centre, research programme preludium 2018/31/ n/nz7/02260. evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. age-dependent alteration in concentration and size distribution of extracellular vesicles in plasma of normotensive and hypertensive rats kosuke otani, muneyoshi okada and hideyuki yamawaki laboratory of veterinary pharmacology, school of veterinary medicine, kitasato university, towada, japan introduction: spontaneously hypertensive rats (shr) are the most widely used animal model of human essential hypertension. we previously reported that plasma small extracellular vesicles (sevs) in shr regulate systolic blood pressure, however, the mechanism has not been clarified. in the present study, we compared the concentration and size distribution of plasma evs (sevs and large evs) from young and aged normotensive wistar kyoto rats (wky) and shr. methods: heparin-anticoagulated plasma was collected from male wky and shr at 5~7-(young) and 15-(aged) week-old. large evs were isolated from the plasma by centrifugation (10000 x g). sevs were isolated by ultracentrifugation (164,071 x g) following precipitation with polyethylene-glycol. the concentration and size distribution of sevs and large evs were measured by a tunable resistive pulse sensing analysis. results: there was no significant difference in the total concentration of plasma sevs between wky and shr or between young and aged rats. the mean diameter of plasma sevs from aged rats was larger than that from young rats in both wky and shr. also, the number of particles with a diameter of smaller than 150 nm in plasma sevs from aged rats was lower than that from young rats. the concentration of plasma large evs from aged rats was higher than that from young rats in both wky and shr. there was no significant difference in the size distribution of plasma large evs between wky and shr or between young and aged rats. summary/conclusion: the present results for the first time demonstrate that the concentration of plasma large-sized evs is increased by ageing, while there is no difference in the concertation and size distribution of evs between wky and shr. further research is required to clarify the cause of age-dependent alternation in plasma ev size distribution and its physiological meaning. microrna profiling of circulating extracellular vesicles is involved with susceptibility to age-related diseases: relevance to cardiovascular signalling in ageing process ionara rodrigues siqueira a , laura cechinel b , rachael batabyal c and robert freishtat c a universidade federal do rio grande do sul (ufrgs), porto alegre, brazil; b universidade federal do rio grande do sul, porto alegre, brazil; c children's national hospital, washington, usa introduction: ageing represents a central risk factor for several diseases, such as cardiovascular diseases. our hypothesis is that extracellular vesicles (evs) can be potential mechanism of spreading molecules, such as micrornas, involved with susceptibility to chronic age-related diseases and geriatric syndromes. in this context, the role of micrornas in age-induced detrimental changes in the cardiovascular system has been suggested. although evs can protect micrornas from endogenous rnases and internalization of these vesicles into cells is involved with cell communication, delivering micrornas even to distant tissues, the relationships between evs micrornas profile and chronic age-related diseases has not been evaluated. our aim was to investigate the microrna profile of circulating evs during ageing process and their downstream signalling pathways. methods: the ethics committee (ceua -comissão de ética no uso de animais -ufrgs; nr. 29,818) approved all animal procedures and experimental conditions. male wistar rats of 3-and 21-month-old were used, and plasma was obtained from the trunk blood. evs were isolated with exoquick following the manufacturer's instructions. microrna was isolated from evs and then amplified. microrna was labelled using the flashtag biotin hsr rna labelling kit and profiled on affymetrix genechip microrna 4.0 arrays. ingenuity pathway analysis (ipa) was used to identify pathways regulated by significantly altered micrornas. results: microarray analysis revealed 728 micrornas. of these micrornas, 48 were differentially expressed between aged and young-adult animals, 18 micrornas were significantly upregulated and 30 were downregulated in aged animals compared to young adult (p < 0.05; fold change of |1.1|). a conservative filter was applied on ipa and only experimentally validated and highly conserved predicted mrna targets for each microrna was used. ipa analysis showed that cardiac hypertrophic signalling is ranked as highly predicted targets for these differentially expressed micrornas (p < 0.0001). moreover, ipa demonstrated that this canonical pathway is upregulated in aged animals when compared to young adult. in addition to cardiac hypertrophic signalling, other relevant cardiovascular canonical pathways, such as endothelin-1 signalling and intrinsic prothrombin activation pathway have predicted targets. summary/conclusion: our results showed for the first time that micrornas profile in circulating evs has a potential role to drive heart senescence and consequent cardiac diseases which represents the leading cause of death. introduction: introduction: the vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. here, we examined the extent to which extracellular vesicles (evs) vesicles participate in endothelial-vascular smooth muscle cell communication. methods: methods: evs were collected from rat aortic endothelial and smooth muscle cell serumfree media by ultracentrifugation. vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. endothelial cell and vascular smooth muscle cell cultures were subjected to various concentrations of evs for various times. functional assays were performed. results: results: western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial-and smooth muscle-derived ev as well as proteins unique to each vascular cell type. functionally, endothelial-derived evs stimulated vascular cell adhesion molecule-1 (vcam-1) expression and enhanced leukocyte adhesion in vascular smooth muscle cells while smooth muscle evs did not elicit similar effects in endothelial cells. evs from endothelial cells also induced protein synthesis and senescenceassociated β galactosidase activity in vascular smooth muscle cells. proteomic analysis of vascular smooth muscle cells following exposure to endothelial cellderived evs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (hmgb) 1 and hmgb2. pharmacological blockade of hmgb1 and hmgb2 and sirna depletion of hmgb1 in smooth muscle cells attenuated nfkb (p65) phosphorylation and nuclear translocation, vcam-1 expression and leukocyte adhesion induced by endothelial cell evs. summary/conclusion: conclusions: these data suggest that endothelial cell-derived evs can enhance signalling pathways that induce a pro inflammatory in vascular smooth muscle cells. introduction: graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (cabg). biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. many studies have generated compelling data on the sensitivity of mvs as biomarkers of cardiovascular disease progression and events. the mv usefulness in cabg has been tested only in a study that highlighted their importance in surgical haemostasis. no information is so far available on the association between the amount or pattern of circulating mvs and cabg outcome. we aimed to evaluate whether mv pre-operative signature could predict mid-term graft failure. methods: this was a nested case-control substudy of the coronary bypass grafting: factors related to late events and graft patency (cage) study that enrolled 330 patients undergoing elective cabg. of these, 179 underwent coronary computed tomography angiography 18 months post-surgery showing 24% graft occlusion. flow cytometry mv analysis was performed in 60 patients (30/group with occluded [cases] and patent [controls] grafts) on plasma samples collected the day before surgery and at follow-up. results: before surgery, cases had two-fold (p = 0.020) and four-fold (p = 0.042) more activated platelet-derived and tf+ mvs, respectively than controls. the mv thrombin generation capacity was also significantly greater (p < 0.05). this mv signature predicted graft occlusion (auc of 0.897 [95%ci: 0,77-0,96], p = 0.02). by using a mv-score (0-6), the or for re-occlusion for a score above 3 was 16.3 (95% ci 4.1-65.3, p < 0.001). summary/conclusion: the pre-operative signature of mvs is an independent predictor of mid-term graft occlusion in cabg patients and a cumulative mvscore stratifies patient's risk. since the mv signature mirrors platelet activation, patients with a high mvscore would benefit from a personalized antiplatelet therapy. exosomes from engineered immortalized human heart cells improve ventricular function and attenuate fibrosis in mice with arrhythmogenic cardiomyopathy yen-nien lin, lizbeth sanchez, rui zhang, thassio ricardo ribeiro mesquita, chang li, ahmed ibrahim, eduardo marbán and eugenio cingolani heart institude, cedars sinai medical center, los angeles, usa introduction: arrhythmogenic cardiomyopathy (ac) is characterized by progressive loss of cardiomyocytes and fibrofatty tissue replacement. currently, there is no effective treatment for this disease. exosomes (imexos) secreted by heart stromal cells, engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and anti-fibrotic effects and improve ventricular function in models of ischaemic injury (ibrahim et al., nature bme 2019). methods: to investigate the effectiveness of imexos in a murine model of ac, four-week old homozygous dsg2 knockout (dsgko) mice and wild type (wt, age-and strain-matched) mice were compared. dsgko mice were randomized to receive weekly imexos or vehicle via intravenous injection for 4 weeks. neonatal rat ventricular myocyte (nrvm) proliferation and apoptotic assays were performed to explore potential effects of exosomes. results: biodistribution studies of dir-labelled imexos revealed some cardiac uptake, along with strong signals in spleen. at 4 weeks, dsgko mice which had received intravenous imexos showed improved cardiac function (echocardiographic ejection fraction 73 ± 2 vs 59 ± 5% in vehicle mice, p = 0.03), with an underlying attenuation in myocardial fibrosis by histology. electrophysiology test showed shorter qrs duration (4.0 ± 2.0 ms imexo vs 6.5 ± 2.9 ms vehicle, p = 0.02) and effective refractory period. programmed ventricular stimulation showed dsgko mice which had received imexos were remarkably less prone to ventricular tachycardia induction (10.0 ± 32% vs 55.0 ± 52% in vehicle, p = 0.031). in vitro study showed nrvm exposed to imexos for 2 days exhibited higher brdu expression relative to vehicle group, and less annexin-v expression after oxidative stress induced by 10-minute illumination with 254 nm uv. summary/conclusion: intravenous administration of imexos improved cardiac function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in ac. our findings motivate clinical testing of imexos in ac, an orphan disease with great unmet medical need. funding: nih r01 hl124074 (to em) cardiac-derived extracellular vesicles contribute to communication between heart and brain in chronic heart failure (chf) and target nrf2/ are signalling changhai tian a , lie gao b and irving zucker b a department of cellular and integrative physiology, university of nebraska medical center, omaha, usa; b department of cellular and integrative physiology, university of nebraska medical center, omaha, usa introduction: mirnas regulate the translation of proteins that are involved in redox homoeostasis in the heart and brain. intra-and/or inter-organ communication takes place by multiple mechanisms including extracellular vesicular (ev) transport. our previous studies suggested that cardiac derived mirna-enriched evs contribute to the dysregulation of nrf2/antioxidant enzyme (are) signalling in the myocardium via intercellular cross-talk, and result in the decreased nrf2/are signalling in the sympatho-regulatory areas of the brain in chf. however, it is unclear if cardiac derived evs circulate to the central nervous system evoking sympatho-excitation by disrupting central redox homoeostasis. methods: cardiac-specific membrane gfp+ mice were generated to track the brain distribution of cardiac evs in rats with chf (coronary ligation). the isolation and characterization of evs were carried out by differential ultracentrifugation, tem, nanosight, western blotting, and qrt-pcr. transfection, labelling, and microinjection of evs into the rostral ventrolateral medulla (rvlm) were performed. results: nrf2 protein was reduced in the rvlm of chf rats consistent with an upregulation of nrf2-targeting mirnas. nrf2-targeting mirnas were enriched in cardiac and circulating evs of chf rats. nrf2-targeting and cardiac-specific mirnas were abundant in brain-derived evs. circulating evs were taken up by neurons in sympatho-regulatory areas of the brain. mirna-enriched evs from chf animals increased sympathetic tone which was prevented by a cocktail of nrf2-targeting mirna inhibitors. summary/conclusion: myocardial infarction-induced mirna-enriched evs mediate the inter-organ crosstalk between heart and brain in the oxidative regulation of sympathetic outflow through targeting the nrf2/ are signalling pathway. these findings suggest that cardiac-derived ev mirnas targeting nrf2/are signalling may act as an endocrine signalling mediator of chf that has potential as a novel therapeutic target. introduction: a fine-tuned communication between cardiac cells is vital to maintain myocardial integrity and contractility. not only an impairment of gap junction (gj)-mediated intercellular communication, but also defects in ev-mediated communication have been associated with ischaemic heart disease, a major causative factor of heart failure. we have previously shown that cx43, the main ventricular gj protein, assembles into channels at the evs surface, mediating the release of vesicle content into target cells.the main objective of this work was to characterize the signals underlying protein sorting into extracellular vesicles (evs) in a human pathophysiological context, using connexin43 (cx43) as a model substrate. methods: animal models of ischaemia/reperfusion (i/ r) injury by ligation of the left anterior descending coronary artery, ex vivo and in vitro ischaemia models and human patients were used to investigate the secretion of ev-cx43. results: release of cx43 was downregulated in circulating vesicles from i/r-injured mice and patients with st-segment elevation myocardial infarction, as well as in intracardiac and cardiomyocyte-derived evs. additionally, we show that ubiquitin signalled the release of cx43 in basal conditions but appeared to be dispensable during ischaemia. depletion of the autophagy adaptor p62 partially restored the secretion of cx43, suggesting an interplay between ischaemiainduced cx43 degradation and secretion. summary/conclusion: overall, we demonstrated that ischaemia impairs the sorting of cx43 into evs, which may ultimately affect long-distance communication. through the identification of the underlying molecular mechanisms and players, these results pave the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders. introduction: remote ischaemic conditioning is a cardioprotective intervention which protects the heart against ischaemia/reperfusion injury. transient activation of toll-like receptor 4 (tlr4) and its downstream regulators (tnfα and il-6) have been implicated in cardioprotective interventions. extracellular vesicles (evs) play a role in cardioprotection through the activation of the tlrs. however, since isolation of evs in high amounts with suitable purity from blood is a challenge, our aim was to develop a cellular model system from which tlr-inducing, cardioprotective evs can be isolated in a reproducible manner. methods: ev release from hek293 cells was induced by calcium-ionophore a23187. evs were characterized, cytoprotection by evs against simulated ischaemia/ reperfusion injury and its mechanism were investigated in h9c2 and ac16 cell lines. results: a23187 induction of hek293 cell induced ev release and the isolates contained mostly large evs. evs decreased cytotoxicity and apoptosis due to 16 h ischaemia followed by 2 h reperfusion in h9c2 and ac16 cells in a dose-dependent manner. evs activated tlr4 and its downstream signalling pathway in h9c2 and ac16 cells as well as the expression of cytoprotective haem oxigenase 1 (ho-1) in h9c2 cells. summary/conclusion: a23187-induced evs exert cytoprotection in h9c2 and ac16 cells by inducing tlr4 signalling and ho1 expression. therefore, evs released via calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective therapy. introduction: biliary strictures may be benign or malignant. the major malignant causes of biliary stricture are a primary cholangiocarcinoma (cca) or pancreatic ductal adenocarcinoma (pdac). there is ongoing debate about adequate diagnostics in biliary strictures of unknown aetiology. micrornas (mirnas) are small non-coding rnas important in tumourigenesis. mirna have been found to be enriched in exosomes, small membrane-bound extracellular vesicles (ev) of endocytic origin, which is a novel pathway for intercellular signalling within the tumour microenvironment and have been implicated in loco-regional pre-metastatic niche formation. this project aims to investigate circulating-free and ev mirnas as biomarkers that can aid diagnosis in patients with a biliary stricture. we will (1) isolate and characterise evs in plasma and bile from patients with benign and malignant biliary strictures (i.e. pancreaticobiliary cancers); and (2) identify differentially expressed circulating-free and ev mirnas in plasma and bile suitable for detecting malignancy. methods: sample size (n = 126) was calculated for a study power of 90% and α error of 5% for the ability of extracellular mirnas to discriminate benign from malignant biliary strictures. prospective matched plasma and bile samples will be collected from patients with benign (n = 63) and malignant (n = 63) biliary strictures undergoing endoscopic retrograde cholangiopancreatography (ercp). evs will be isolated from the biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised (tem, nta and immunoblotting). circulating-free and ev-associated mirnas will be profiled using small rna sequencing. extracellular mirna "signatures" will then be validated by rt-qpcr, and diagnostic accuracy confirmed (sensitivity, specificity, auc). results: evs derived from patient samples have been characterised using nta, western blotting and tem. sec derived evs appear to be more well-defined than uc evs with marker positivity for cd63, cd81 and cd9. ongoing work will be focused on rna profiles of evs from both malignant and benign cohorts. summary/conclusion: there is currently no effective method to differentiate benign from malignant biliary strictures. novel plasma and bile circulating-free and ev-associated mirna biomarkers may improve the speed and accuracy of diagnosis, resulting in considerable patient benefits. furthermore, as little is known about the ev-associated function of these tumours, candidate ev-mirnas could be taken from "bedside to bench" and their function further investigated using in vivo, vitro and silico models. introduction: urine is a source of extracellular rna (exrna) biomarkers that can be obtained non-invasively throughout pregnancy. several studies have profiled extracellular mirnas in biofluids during pregnancy, but few have profiled extracellular mrnas (ex-mrnas) in urine. objective: to optimize methods for ex-mrna isolation and rna-seq library preparation from urine of healthy pregnant and non-pregnant females. methods: rna was isolated from pooled non-pregnant urine using kits based on ev precipitation (mircury exosome kit for csf/urine, seramir), ev affinity purification (exorneasy), and protein precipitation (mirneasy serum/plasma advanced). next, long (>200nt) and short rnas were isolated from ev enriched urine of pregnant (n = 5) and non-pregnant (n = 5) individuals using the mircury kit followed by the mirneasy micro kit. rna-seq libraries were prepared using the smart-seq v4 ultra low input rna (oligo(dt) priming) and the smarter stranded total rna-seq kit v2 -pico input (random priming) methods (takara). preliminary data were obtained using the illumina miseq, and aligned using star v.2.7.3.a. results: overall, rna isolation using mircury followed by the smart-seq v4 library preparation kit yielded the highest % of mapped reads: 42% in pooled non-pregnant, 27% in individual non-pregnant, and 31% in individual pregnant urine. for rna extracted using the mircury kit, the smart-seq v4 libraries had higher % of mapped mrna reads compared to pico libraries (p < 0.05, t-test). in contrast for mirneasy advanced it was reversed (38% vs 21%). summary/conclusion: early results from low-depth sequencing show the highest mrna mapping rates for mircury followed by the smart-seq v4 kit. high-depth sequencing data are now being generated, which will enable us to perform detailed comparisons of different rna species from the rna profiles obtained using different library preparations and rna isolation methods from urine of pregnant and non-pregnant subjects. funding: this study was funded by nih 7 k99 hd096125-02, nih u01 hl126494, and a ucsd igm-illumina mini-grant. il-2 mutein-induced changes of exosomal mirna cargo in a humanized mouse model emily lurier, erik sampson, patrick halvey, mike cianci and katalin kis-toth pandion therapeutics, cambridge, usa introduction: regulatory t cells (tregs) are key contributors to immune homoeostasis. decreased number and/or function of these cells are frequent features of many autoimmune diseases linked to the development of tissue inflammation. while interleukin-2 (il-2) is essential for pan t cell proliferation and performance, low dose il-2 treatment has been shown to preferentially affect tregs and is being evaluated as an intervention in autoimmune diseases. pt101 is a novel il-2 mutein fc fusion molecule (il-2 m) designed to selectively engage with tregs. using a humanized nod-scid il2rn-null (nsg) mouse model we have shown that pt101 expanded tregs without significant effects on other immune cells. we have also shown that tregs from pt101-dosed humanized mice exhibit increased expression of foxp3 and cd25, and demethylation of foxp3 and ctla-4 genes, suggesting enhanced function and stability. in the current study we investigated the mirna content of plasma exosomes isolated from pt101-or vehicle-treated mice in order to identify treg specific mirnas from the il-2 m treated animals. methods: cd34+ haematopoietic stem cell humanized nsg mice were dosed once subcutaneously with pt101 or vehicle. plasma samples from 8 mice were collected at day 7 and exosome isolation was conducted using the exoquick method. small rna was extracted and quantified using the bioanalyzer small rna assay. an illumina nextseq instrument was used for library preparation and sequencing with 75bp single end reads at an approximate depth of 10-15 million reads per sample. raw sequences were mapped to human genome grch37 and analysed via a pipeline provided by the university of california santa cruz. results: rna within the exosomes from vehicle and il-2 m-treated groups was mostly comprised of mirna and trna. plasma was pooled from 8 animals per treatment group and differential expression was determined using a twofold change cut-off. we found that pt101 treatment actively altered the mirna content of plasma exosomes, compared to exosomes from vehicle-treated mice. many of the differentially expressed mirnas are involved in immunoregulation. summary/conclusion: plasma exosomes from pt101treated humanized mice encapsulated treatment-specific mirnas which can potentially be used as systemic biomarkers of treg expansion and function. identification of potential biomarkers in microglial specific exosomes isolated from prion-infected serum introduction: transmissible spongiform encephalopathies (tse) are neurodegenerative disorders caused by the misfolding of the cellular prion protein (prpc) to the beta-sheet rich abnormal prion protein (prpsc). prpsc aggregates in the brain and causes amyloid plaques, neuronal loss, spongiform degeneration and microglial activation. currently, definitive diagnosis of tse diseases is only confirmed post-mortem thus a diagnostic test in accessible body fluid is of interest. exosomes are a good resource for biomarker discovery since they cross the blood-brain barrier easily and contain protein, lipids and nucleic acids from the cells of origin. the goal of this study was to look at biomarkers from brain-originating exosomes (specifically microglia) isolated in the serum of prion-infected animals. methods: westerns and nanoparticle tracking analysis (nta) were used to look at the composition of microglial-specific exosomes. as proof of principle, exosomes were isolated from a microglial cell line (bv2 cells). a cd63 antibody was labelled with a fluorophore and binding to exosomes was visualized via nta. exosomes were isolated from serum of both prioninfected and mock-infected mice throughout disease course. a macrophage specific antibody (f4/80) was bound to beads which were used to isolate exosomes which includes those of microglial origin. microrna was extracted from these exosomes and next-generation sequencing (ngs) was performed using the illumina platform. clc genomics workbench was used for bioinformatics analysis. results: microglial and macrophage proteins (tmem119 and iba1) were identified in exosomes isolated from bv2 cells and prion-infected mouse serum. macrophage exosomes were isolated via a novel antibody-bead based system. results of the ngs analysis of the microrna isolated from these exosomes indicated a series of mirna that could differentiate between control and infected samples as well as age-specific markers. summary/conclusion: to our knowledge, this is the first time microglial-specific exosomes have been isolated from prion-infected serum from early and end stage disease. the results of this analysis could facilitate the diagnosis of prion disease in easily-accessible biofluids pre-mortem. comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease introduction: urinary extracellular vesicles (uevs) are emerging as a source for early biomarkers of kidney damage, holding the potential to replace the conventional invasive techniques including kidney biopsy. several methods are available for uev isolation. our aim was to compare different workflows and isolation by hydrostatic filtration dialysis (hfd), ultracentrifugation (uc) and a kit based isolation method for their subsequent use in mirna-seq and rna-seq for biomarker discovery in diabetic kidney disease. methods: type 1 diabetic patients (t1d) with macroalbuminuria and normoalbuminuric healthy controls were included in the study. sample collection and all experiments were performed in accordance with the declaration of helsinki. evs were isolated from 10-50 ml of 24 h urine collections by uc, hfd, or a commercially available kit (purification based on spin column chromatography, urine exosome purification and rna isolation midi kit, norgen biotech, canada) each with different established urine clarification steps. quality control of the evs was performed with negative staining em, nta and western blotting. isolated rnas were profiled with bioanalyzer pico kit and subjected to mirna and mrna sequencing. for rna-seq, cdna library was prepared using smart-seq v4 ultra low input rna kit for sequencing (takara bio, japan). rna-seq was performed using hiseq 2000 (illumina). mirna-seq library was prepared using qiaseq mirna library kit (qiagen, germany). mirna-seq was performed on the illumina hiseq 4000 platform (illumina). results: our data showed that uev yield, morphology and size distribution were closely similar in hfd and uc preparations, while lower yields were obtained using the kit. by western blot, ev markers were detectable in samples isolated by hfd and uc but not readily in samples isolated with the kit. tamm-horsfall protein was detected in all the samples and albumin levels appeared higher in hfd and kit isolated samples relative to uc samples. the number of paired-end reads for rna-seq in hfd and uc samples (in both > 5 m) were closely similar. instead, rna reads were lower than 2 m for the kit samples. for mirna-seq, the number of reads as well as the molecular biotype distribution were similar for the three methods. by principal component analysis of the rna-seq data, we observed that hfd and uc grouped together showing similarities. however, for mirna-seq data such similarities were not obvious. this suggests that the three different workflows and isolation principles may enrich different mirna-rich uev preparation components. summary/conclusion: our transcriptomics data shows that hfd and uc are suitable methods to isolate uevs for mirna-seq and rna-seq. the kit based method appears better suited for mirna-seq. introduction: exosomes contain a variety of biomolecules including dna. knowledge of cfdna distribution and localization in bioliquid is important for understanding both biological function of cfdna and exosomes. some publications state that a large proportion of plasma cfdna is localized in exosomes. to quantify cfdna content in free vs. exosomal form in human plasma, urine, and saliva, we employed subx technology, which allows affinity capture dna via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. subx is a proprietary compound that can simultaneously bind to both cfdna and exosomes in bioliquids, thus allowing precipitation of the [subx-dna/ subx-exosomes] complexes without ultracentrifugation. methods: detection of subx-dna and exosomes binding was done by measurement of particle sizes using zetasizer nano zs and nanosight ns300. the samples were processed with the subx exo-dna isolation kit following the standard protocols. dna, protein and lipid concentrations were measured by fluorescent assays using qubit fluorometer. results: subx efficiently and selectively captures and co-precipitates cfdna and exosomes directly from bioliquids. exosomes are easily extracted from the pellet in exosome reconstitution buffer (erb), followed by subsequent isolation of tightly bound cfdna from the subx pellet. erb does not extract dna form the [subx -dna] pellet and thus does not contaminate reconstituted exosomes with cfdna. thus, we separate two distinct types of extracellular materialintact exosomes and purified cfdna in a single protocol from the same sample. over 90% of dna in plasma and urine exist as a free circulating pool, while in saliva up to 30% is associated with exosomes. thus, cfdna distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfdna-outer exosomal membrane aggregation. summary/conclusion: subx technology is suitable for simultaneous isolation of both cfdna and exosomes from the same bioliquid sample. subx separates cfdna fragments non-specifically attached to the outer lipid layers of the exosome membrane from the true intra-exosomal cfdna. in contrast, salting-out peg technique is associated with aggregation of macromolecules and vesicles and thus leads to overestimation of exosome-associated polymers content, including cfdna. tracing extrachromosomal dna inheritance patterns in glioblastoma using crispr eunhee yi, amit gujar, hoon kim, albert cheng and roel verhaak jackson laboratory for genomic medicine, farmington, usa introduction: glioblastoma multiforme (gbm) is the most lethal brain tumour; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of 10%. recurrence after therapy for gbm is unavoidable. there are substantial differences among the cells of gbm tumours in the abundance and types of genetic material. this heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. a recent study has suggested that the amount of a particular type of dnaextrachromosomal dna (ecdna)differs substantially among different gbm tumours, and differs within a given gbm tumour over time. despite the speculation that ecdna is a key factor of tumour heterogeneity, how ecdna is propagated and distributed amongand how it behaves withincancer cells is completely unknown. methods: to address this gap in knowledge, this study focused on developing a novel cytogenetic crisprbased tool that enables visualization and tracking ecdna behaviour in live gbm cells. results: we found breakpoint sequences resulting from genome rearrangements during ecdna formation by performing computational analysis from whole genome sequencing data. and each breakpoint was regarded as a unique target sequence for ecdnaspecific labelling. the uniqueness of each breakpoint was validated by breakpoint-pcr (bp-pcr). furthermore, the location and the amount of each breakpoint were observed by breakpoint-fish (bp-fish) analysis in gbm cells. summary/conclusion: this results will be strong evidence to make ecdna-specific crispr system in further research. tracing ecdna dynamics will provide new insight into the impact of ecdna on cancer evolution. introduction: small extracellular vesicles (sevs) are 30-150 nm vesicles that mediate intercellular communication by transferring rna and proteins to the recipient cells. these cargo molecules are selectively sorted into sevs and mirror the physiological state of the donor cells. given that sevs can cross the bloodbrain barrier and their composition can change in neurological disorders, there is an increasing interest in elucidating the molecular signatures of sevs in circulation as disease biomarkers. however, circulating sevs are derived from multiple cellular sources and determining their source is challenging. information on sev composition can be beneficial in predicting whether these sevs are released predominantly from central nervous system cells. we hypothesized that differentially expressed mirnas between neuronal sevs and astrocytic sevs could be used as cell-typespecific signatures. methods: small extracellular vesicles were isolated from cell culture media of postnatal mouse primary neurons and astrocytes using differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna from neurons, astrocytes, and their respective sevs were used for transcriptome and small rna sequencing. results: we observed that only a subset of cellular mirnas was packaged into sevs; differential expression of specific mirnas between sevs and their corresponding cells suggest that cells employ special mechanisms to sort mirnas into sevs. these mechanisms could be celltype specific since neuronal sevs showed a different mirna profile compared to astrocytic sevs. exomotifs, the short sequence motifs that control the loading of rna into sevs, were present in differentially expressed mirnas. we also observed that five rnabinding proteins, which are associated with passive or active rna sorting into sevs, were differentially expressed between neuronal and astrocytic cells. summary/conclusion: mirna signatures of sevs from neurons and astrocytes could be beneficial in determining if these cell types contribute to the alterations of sev composition in circulation in neurological disorders. cell-type-specific selectivity in rna loading might be attributed to the differential expression of rna-binding proteins. introduction: analytes present in the extracellular fraction of bodily fluids (ex. blood, urine) have utility as a tool for uncovering the molecular landscape of tumours and hold great potential for discovery of individualized cancer medicine. urine, being noninvasive as a sample type, has an obvious advantage over blood when used for liquid biopsy purposes. however, potential for microbial proliferation and the labile nature of host cells and extracellular vesicles (evs) at the point of sample collection/transport to the lab drives the need for stabilization of urine samples. development of such sample stabilization opens up capability for the detection of various biomarkers present in the extracellular fraction to be used in liquid biopsy. this is of particular concern as studies around urinary analytes for cancer diagnosis, progression and therapeutic effect are rapidly expanding in cohort sizes. multi-site collections and at-clinic collections are increasingly prohibitive for large scale recruitment and also lead to variability in the time between collection and processing. methods: in this study, we have analysed two commercially available ev extraction kits and compared them with ultracentrifugation technique for size, concentration and specificity of the isolated evs from human urine samples with and without our proprietary preservation solution using nanoparticle tracking analysis and western blot analysis for exosomal membrane markers. ev rna contents in various urine fractions (first morning first void, random first void and midstream) were compared using rt-qpcr assay to provide better understanding of the collection techniques and fractionations that are ideal for ev research work. results: in our current work, we have bench-marked human urine collection and ev extraction in order to provide recommendations in standardization of sample acquisition and processing for urinary ev studies. we have utilized these standardization in order to develop a novel and efficient sample stabilization principle for preservation of evs and ev rna in urine samples during an ambient temperature hold. summary/conclusion: taken together, we have established a framework for evaluating technologies and techniques in the ev sample processing space, which can be utilized by other research groups. vn96-isolated plasma extracellular vesicles improve tumour mutation detection by next-generation sequencing compared to cell-free dna and correlate with tissue biopsy of nsclc patients introduction: liquid biopsy is a minimally-invasive diagnostic method that detects circulating biomarkers and has the potential to improve access to molecular profiling for nsclc patients when tissue biopsy material is unavailable or insufficient. although isolation of cell-free dna (cfdna) from plasma is the standard liquid biopsy method for detecting dna mutations in cancer patients, the sensitivity can be highly variable. vn96 is a 27 amino acid peptide with an affinity for heat shock proteins that are exposed on the surface of extracellular vesicles (evs); peptide-ev aggregates readily sediment using a benchtop centrifuge and therefore the vn96 peptide provides a rapid, clinically-amenable procedure for ev isolation. in this study, we determine whether isolation of evs from nsclc patient plasma improves the sensitivity of single nucleotide variants (snvs) detection compared to cfdna and correlate genetic changes observed by liquid biopsy with tumour ffpe tissue biopsy. methods: blood was collected from stage iii/iv nsclc patients with informed consent in either edta or cell-free dna bct® collection tubes and plasma was harvested within 30 minutes. total nucleic acid (tna) was extracted from either vn96-isolated evs from edta plasma or directly from plasma collected in edta or cell-free dna bct® tubes (cfdna). snvs were detected by next-generation sequencing (ngs) results: vn96 isolation of evs from plasma resulted in higher recovery of dna than cfdna isolation. the snvs detected in both ev-dna and cfdna correlated well with those reported in matched ffpe tumour tissue using ngs, including 100% specificity for egfr mutations. no improvement in snv detection was observed using cell-free dna bct® collection tubes compared to edta tubes. isolation of evs with the vn96 peptide prior to sequencing improved a number of ngs parameters including library yield, total reads, median read coverage and molecular coverage, resulting in improved sensitivity of snv detection. summary/conclusion: in summary, our research demonstrates that vn96-based ev isolation is useful for molecular profiling of nsclc patients for whom tissue biopsy is not an option, thereby improving access to molecular profiling and targeted therapies. funding: atlantic canada opportunities agency novel markers for neuroendocrine prostate cancer divya bhagirath a , michael liston b , theresa akoto a and sharanjot saini a a augusta university, augusta, usa; b veteran affairs, san francisco, usa introduction: prostate cancer (pca) is fuelled by androgens and androgen receptor (ar) signalling. therefore, ablation of ar signalling by androgen deprivation therapy (adt) is the goal of first-line therapy that results in cancer regression initially. however, two to three years post-adt, the disease develops into castration-resistant prostate cancer (crpc). as a second-line of therapy, next generation of ar pathway inhibitors (api) such as enzalutamide (enz) are used that are effective initially followed by emergence of drug resistance. a subset of api-resistant tumours emerges to an ar independent state via undergoing a trans-differentiation to neuroendocrine lineage, a process referred to as neuroendocrine differentiation (ned). due to lack of ar signalling, these pca variants, referred to as neuroendocrine prostate cancer (nepc), are impervious to anti-androgen therapy and constitute an aggressive variant of advanced crpc with poor prognosis. currently, there is a lack of effective molecular biomarkers for predicting api therapy resistance and emergence of therapy-induced ned. methods: exosomes/evs were isolated from sera of a patient cohort with/without ned. the study was conducted in accordance with ethical guidelines of us common rule and was approved by the institutional committee on human research. written informed consent was obtained from all patients. following extensive characterization of evs by electron microscopy, nanosight tracking analyses and western blotting of exosomal markers, small rna sequencing was carried out on illumina hiseq platform to identify differentially expressed transcripts. machine learning algorithms were applied to clinical sequencing data to train a "mirna classifier". further, we probed the proteomic profile of exosomes isolated from nepc cellular model nci-h660 and enzalutamide resistant crpc cell lines by mass spectrometry. results: we identified that transition from crpc-adenocarcinomas to neuroendocrine states is associated with significant ev-mirna dysregulation, with a specific dysregulation in certain mirna families. with the application of machine learning algorithm, we identified an ev-based "molecular classifier" that can robustly stratify crpc-ne tumours from crpc-adenocarcinomas. proteomic analyses identified novel nepc-specific, glycosylated proteins that can be exploited for nepc diagnosis. summary/conclusion: our data suggest that ev mirna and protein profile can predict neuroendocrine differentiation in advanced castration-resistant prostate cancer patients. exosomal mrna in diagnosis strategy for hepatocellular carcinoma aleksandr abramov, alisa petkevich, vadim pospelov and pavel ogurtsov peoples' friendship university of russia (rudn university), moscow, russia introduction: exosomal cargo is informative source illustrating the genetic events happening in cells, what can be especially advantageous in case of cancer development for disease progression or treatment effectiveness monitoring. methods: 10 plasma samples of hepatocellular carcinoma (hcc) patients, 10 plasma samples of patients with liver cirrhosis 3-4 on the hepatitis c virus (hcv) background, 5 healthy donors' plasma samples. exosomes were isolated with ultracentrifugation, western blot (cd63, cd9) was performed. total mrna was isolated with exosomal rna isolation kit, norgen biotec corp. sequencing was carried out on a minion sequencer. housekeeping genes (gapdh, b2 m, actb, tuba1a). detected mutations were confirmed by real-time pcr with specific highly sensitive lna probes. results: significant changes in expression levels were identified for 50 genes in hcc and liver cirrhosis groups (increasing up to x200 compared to control samples and decreasing up to no detected expression). in 6 out of 10 patients with hcc mutant burden was significant increased compared to mutant burden in groups with cirrhotic samples. in 8 out of 10 patients with hcc increased expression for mrna line-1 was identified compared to cirrhotic patients. summary/conclusion: exosomal mrna expression levels may serve as a prognostic and diagnostic marker for patients with liver cirrhosis caused by hcv for hcc risk development. funding: research is supported with federal funds "5-100" circulating extracellular vesicle signatures in small cell lung cancer michela saviana a , giulia romano a , giovanni nigita b , robin toft a , patricia le a , kai wang c , mario acunzo a and patrick nana-sinkam a a virginia commonwealth university, richmond, usa; b the ohio state university, columbus, usa; c institute for systems biology, seattle, usa introduction: lung cancer is the leading cause of cancer deaths worldwide and classified primarily as either non-small cell lung cancer (nsclc) or small cell lung cancer (sclc). compared to nsclc, sclc has a faster growth rate, earlier widespread metastasis, and shorter overall survival. the early diagnosis of sclc and the development of novel therapeutics have proven challenging. thus, progression and recurrence rates remain high. non-invasive methods for cancer detection are increasingly being used to inform clinical decision making. extracellular vesicles (evs) have recently emerged as potential carriers of genetic contents such as micrornas (mirs) to induce reprogramming of components of the microenvironment in cancer initiation and progression. moreover, extracellular mirs expression profiles have been shown to have signatures related to tumour classification, diagnosis, and progression. methods: we selected a cohort of patients divided into 4 groups: high-risk smokers, adenocarcinomas, squamous carcinomas, and sclc. we extracted total circulating ev and plasma rna from plasma (38 patients in total) and rna from plasma in a separate group (24 patients in total). utilizing both next-generation sequencing (ngs) and nanostring platforms, we analysed for global microrna (mirs) expression patterns. candidate mirs were then validated by qrt-pcr. results: we identified several deregulated mirs in both evs and plasma of sclc patients compared to the other groups. for evs, we validated mir-1285-5p as a significant biomarker for the late stage of sclc compared to controls. in the case of plasma, we validated the upregulation of mir-375 in sclc compared to controls. summary/conclusion: our results indicate that a potential combination of plasma (mir-375) and ev-based (mir-1285-5p) mirs be valuable biomarkers for sclc detection and serve as a basis for a non-invasive sclc classifier. funding: virginia commonwealth university, doim -nih/nci introduction: the isolation of evs from milk is technically challenging due to the complexity of milk. currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of edta) of casein micelles. using these protocols the integrity, composition and targeting of bovine milk evs has been evaluated and has led to believe that milk evs might withstand these conditions. however, the effects on functionality of milk evs (i.e. immunomodulatory properties) after processing and isolation have not been studied. therefore, we have set up an in vitro culture system using a human t cell line that allows for the rapid screening of milk ev functionality. methods: fresh bovine milk was defatted and cells were removed after 2x3,000 g centrifugation, followed by differential centrifugation at 5,000 g and 10,000 g. this milk was either subjected to acidification with hcl, or edta was added, or the milk supernatant remained untouched. top down optiprep density gradient separation followed by sec was used to further purify evs. these highly purified milk evs were added to human jurkat t cells, which were simultaneously stimulated using anti-cd3 and anti-cd28 antibodies. after 24 h t cell activation was measured by il-2 cytokine production. results: precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated evs, which aids in the purification of milk evs. in vitro analysis revealed that in the presence of directly isolated, or edta isolated milk evs, jurkat cells were suppressed in their activation as measured by il-2 production. remarkably, evs isolated from hcl-acidified milk were impaired in their suppressive capacity to inhibit il-2 production. summary/conclusion: although casein removal from bovine milk greatly improves purity of isolated milk evs, the detrimental effects on ev functionality should be considered. interestingly, evs exposed to acidic conditions lost their ability to modulate t cell activation, which is in contrast with the general believe that milk evs could withstand the gastro-intestinal tract. funding: this work is funded by the european union's horizon 2020 framework programme under the grant fetopen-801367 evfoundry. optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula introduction: infant milk formula (imf) is intended to impart nutrition to infants, similar to breast milk. however, although industrial imf production involves harsh treatment, potential consequences on extracellular vesicles (ev) in imf are not yet established. this study aimed to optimise methods for separating evs from imf and skim milk (sm) and to characterise the evs in accordance with misev2018. methods: sm and imf were either not treated (nt) or treated with acetic (aa) or hcl acid (isoelectric precipitation, ip), to remove caseins. samples were then subjected to differential ultracentrifugation (duc) or gradient ultracentrifugation using iodixanol solution (guc). for duc, 38 ml samples were centrifuged at 12 k g, 35 k g, 75 k g, 100 k g and 200k g sequentially for 75 min each and pellets re-suspended in 1 ml pbs. preparation of agarose microspheres for high-efficient separation of extracellular vesicles cheng-tai chen, chien-an chen, carolyn yen and nien-tzu chou industrial technology research institute, chutung, taiwan (republic of china) introduction: size exclusion chromatography (sec) is becoming a widely used technique for separating of extracellular vesicles. various commercially available products were launched on the market, however, their separation efficiencies were not fully disclosed. herein, novel porous agarose microspheres with the tunable diameter and pore size were synthesized by emulsion reaction. the performance was evaluated and compared with commercial products. the modified sec column packing materials were shown to exhibit advantages for rapid, high-recovery and high-purity separation of extracellular vesicles from cell culture-conditioned medium and human plasma. methods: the homemade sec column was packed by gravity flow. 100 μl of the sample was loaded and the pbs buffer was used as eluent. 24 factions were collected and analysed by cd9/cd9 sandwich elisa assay and by micro bca assay for determining respectively extracellular vesicles and total protein content. results: agarose microspheres were prepared by emulsification. the particle size can be controlled by the types and concentrations of surfactants. the product was collected by desired screen meshes and used as packing materials of the sec column. our results showed that the extracellular vesicles were clearly separated from proteins. more than 99.8% of proteins were removed while the recovery of extracellular vesicles was close to 70%, which is much higher than 32% of the commercial product. the total separation time was less than 15 min. summary/conclusion: we have established an approach for generating spherical agarose microspheres as packing materials of homemade sec columns, which are capable of separating extracellular vesicles from complex samples with high efficiency. further validations with additional samples are currently ongoing. immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from large and small volumes of biofluid eduardo reategui, jingjing zhang, luong t. h. nguyen, richard hickey, nicole walters and andre f. palmer the ohio state university, columbus, usa introduction: evs derived from tumour cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. however, compromises have to be made when using a particular technology/methodology for the isolation of evs. currently, there is a trade-off between sample volume and specificity in ev isolation technologies that limits quantitative molecular analysis of ev contents, ultimately impacting the utility of evs in cancer diagnostics. here, we present an approach called immunomagnetic sequential ultrafiltration (isuf). our platform combines ultrafiltration and immunoaffinity separation. using isuf, we demonstrate that small or large volumes of biofluid can be processed (~100 µl or > 100 ml) while concomitantly removing 99.9 % contaminating proteins. we also processed serum from breast cancer patients enabling the characterization of different tumour and immune biomarkers on the isolated evs. methods: human samples were collected under an approved irb. size distribution and concentration of evs were measured using a tunable resistive pulse sensing (trps) method. ev proteins and rnas were extracted and quantified using a bca protein assay and uv spectroscopy. isuf and other ev isolation methods were compared for ev concentration, protein, and rna quantity. results: 50 ml of cell culture media (ccm), 0.5 ml serum, and 100 ml urine samples were processed with the isuf platform and recovered in 100 µl. for all cases, evs were enriched with recovery efficiency greater than 95%. the processing time for a 100 ml sample was 120 min with over 99% of purity. we compared ev concentration and purity isolate from 0.5 ml serum using isuf and other commercially available methods, isuf demonstrated superior performance on isolating evs at high concentrations and purities. analysis of total rna amounts in the isolated evs using different methods was corresponding to higher ev recovery efficiency of isuf. we also compared protein and rna levels of evs enriched with isuf present in urine and serum samples from the same donors (n = 10), and we found that for the same number of evs, the ev rna concentration from both biofluids showed no significant difference. finally, we have processed serum samples from 10 metastatic breast cancer patients and demonstrated that their isolated evs have expression levels of her2, cd24 and mir21 biomarkers at significantly higher levels than healthy controls. summary/conclusion: the isuf platform can be scale-down or -up to work with small or large volumes of biofluids for the isolation of evs. using the isuf platform with clinical samples shows the potential of our platform to be used for cancer diagnosis or monitoring treatment response. funding: national institutes of health (nih) grants ug3tr002884 (e.r.); r01hl 126945, r01hl138116, and r01eb021926 (afp). challenges in exosomes isolation from primary biological samples derived from multiple myeloma patients introduction: multiple myeloma (mm) remains incurable despite advances in its treatment and research progress on the crosstalk between mm and surrounding host cells. exosomes are important regulators of the cellular niche. their importance for diagnostic and therapeutic applications has been proven in many cancers. in this context we hypothesized that a better understanding of the molecular role and features of mm-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-mm drugs already in use in clinical settings, given the optimization and validity of their isolation/purification method. methods: exosomes were isolated from human mm cell lines (hmcls) supernatants and peripheral blood plasma (pbpl) isolated from healthy donors, mm and mgus (monoclonal gammopathy of undetermined significance) patients. both fresh and frozen samples were tested. we evaluated 3 commercially-available kits, density-based separation and ultracentrifugation. results: higher purity and recovery, evaluated by western blotting, nanoparticle tracking analysis and electron microscopy, were observed for supernatant density-based purification and for pbpl resin-based isolation. exploring the function of mm-derived exosomes, we observed an increase in proliferation of the immortalized stromal cell (sc) line hs5 treated with exosomes when compared to untreated cells, and a higher increase in proliferation of scs treated with mm-exosomes when compared to exosomes derived from normal and mgus pbpl samples. summary/conclusion: the method of isolation represents a critical step in the study of exosomes as many factors can affect the purity, yield and downstream application. our data demonstrated that density and resin-based isolation methods provided functional mm-derived exosomes with proliferative effects on scs. altogether our findings may serve as a guide to choose exosome isolation methods for mm studies. further optimization steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications. funding: australian government rtp and monash departmental scholarship. a rigorous method for exosome isolation from post-mortem eyes introduction: in order to determine and validate the tissue-specific content of extracellular vesicles (evs) in biofluids, robust ev isolation methods from tissues must be developed. however, to date very few rigorous methods to isolate or enrich for intact evs from tissues have been reported. we present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true ev identity from ex vivo eyes. methods: iodixanol (optiprep) buoyant density gradient ultracentrifugation (dguc), cushioned dguc (c-dguc), and our newly developed c-dguc immunocapture (c-dguc-ip) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between 3 to 5 hours post-mortem. yield was assessed by nanoparticle tracking analysis (nta) and immunoblotting for exosomal markers along with total protein quantitation. enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. results: high enrichment of posterior eyecup small evs (sev) were achieved by dguc and c-dguc, with c-dguc resulting in an eightfold increase in yield by nta and two to fivefold increases of exosomal protein markers such as syntenin-1 and cd81 by immuno-blotting compared to dguc. interestingly, in-depth proteomic analyses revealed that a majority of these sevs with densities of 1.07-1.11 g/ml isolated by dguc and c-dguc were likely of endoplasmic reticulum (er) and golgi origin, suggesting er-to-golgi transport vesicles resulting from post-mortem tissue cell rupture. in order to enrich further for sevs (including exosomes) we subjected sevs isolated by c-dguc to anti-cd81 immunocapture. the resulting sev proteome was enriched 1.5-to 4-fold for bona fide sev and exosome markers compared to c-dguc. summary/conclusion: the c-dguc method provides an enhanced yield and purity of sevs and exosomes from ex vivo eye tissue. however, to avoid significant contamination with er and golgi-derived vesicles from postmortem eyes, a final ev-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. characterization of the extraction of extracellular vesicles using a lab-ona-disc filtration system introduction: personalized treatment for cancer is a promising way to face the multiplicity of the disease, to increase the efficacy of drugs and to decrease their toxicity. as part of this strategy, liquid biopsy explores a new non-invasive approach to diagnose cancer, guide treatment and monitor its efficacy. extracellular vesicles (evs) are nanometric lipid bilayers micelles with high potential as biomarkers. they are involved in the transfer of information (proteins, rna and dna) between cells. evs include a broad spectrum of particle sizes, from the tens to thousands of nanometres. the isolation of evs from complex matrices is the first step of any protocol and is particularly important for the reproducibility and fidelity of the results presented, as it could bring bias in further analysis. in order to explore the heterogeneity of evs, a full characterization (physical and biological) of the extracted evs is needed. we evaluate and compare 3 evs purification methods, including ultracentrifugation, sizeexclusion chromatography (sec) column and an emerging microfluidic technology: labspinner filtration labon-a-disc device isolating evs between two filters of 600 and 20nm. methods: a431 cell supernatant was used as a model matrix. we compared three methods of extraction of evs: ultracentrifugation with two cycles of 1 h10 at 110,000 g at 4 degrees celsius (rotor type 70ti, beckman floor ultracentrifuge optima l90 k), qev size exclusion chromatography columns from izon (qevoriginal/70 nm) and lab-on-a-disc filtration system (labspinner, exodisc c). evs characterization was conducted with nta (nanosightns500), trps (izon), nanodrop (spectrometernd1000), tem (fei tecnai 12 120 kv) and custom micro-immuno-assay. results: in this study, we characterize a filtration system made of two serial filters of 600 nm and 20 nm pores for isolation of evs. compared to ultracentrifugation and chromatography columns, yield of extraction is up to 10 times higher and the size of the extracted particles is smaller. tem imaging was used for assessment of the quality of the extracted evs. however, albumin concentration measurement tends to show that the purity of the solution is decreased. the immuno-labelling analysis shows that the proteomic signature of the extracted evs differs according to the extraction methods. the new filtration technology seems to give us access to a broader range of evs compared to standard methods. summary/conclusion: in this study, we characterized 3 purification methods including lab-on-a-disc filtration, and were able to demonstrate an increase of the concentration of evs by a factor of 10, a decrease of the size of the accessible extracted particles and access to new proteomic signatures. funding: we acknowledge the support of génome québec and action marie skłodowska-curie. effects of sample processing on isolation of extracellular vesicles from blood plasma by centrifugation darja božič a , matej hočevar b , veno kononenko c , marko jeran a , urška štibler d , immacolata fiume e , manca pajnič f , ljubiša pađen f , ksenija kogej g , damjana drobne c , ales iglič h , gabriella pocsfalvi i , veronika kralj-iglič f and darja bozic j introduction: the isolation of extracellular vesicles (ev) from body fluids is still controversial and the poor understanding of vesicle stability and effects of sample processing is probably one of the core issues preventing the breakthrough of this field into applicative practices. methods: we performed an in-depth study of sample changes in blood, blood plasma and samples throughout the increasing speed of centrifugation, considering the number, size, contents and shape of particles in the isolates. flow cytometry, light scattering, mass spectrometry and scanning electron microscopy were employed to reveal the properties of material in the samples. results: the particles of size about 100-500 nm with characteristic topology of membrane vesicles without internal structure were observed by the scanning electron microscope only in ev isolates prepared from fresh blood sample. inspection of the tube surface in which the isolation took place suggests that those particles are likely formed from activated platelets tearing at the tube wall due to the centrifugal pull. the isolates prepared from frozen blood plasma prepared by centrifugation with different forces contained different amounts of particles with similar protein contents, predominated by highly abundant human plasma proteins, including albumins and immunoglobulins. some lipoprotein clearance and fibronectin precipitation were however observed through increased speed and time of centrifugation. summary/conclusion: the results of this study [1] contribute to the understanding of stability and dynamics of membrane particles. the reported evidence provides the support for viewing ev isolates as a product, shaped by uniqueness of the starting samples and the thermal and mechanical stress applied upon processing. we believe this kind of insights strengthen our ability of reading the story of evs. introduction: apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data show that, besides its role in tumour suppression, it can also promote oncogenic proliferation. highly aggressive tumours such as burkitt lymphoma (bl) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. we hypothesize that amongst other elements, extracellular vesicles (ev) are key mediators of apoptotic cell-derived tumour microenvironment signals. here, we report on ev released in vitro by apoptotic bl cells (apo-ev) in relation to their potential use as cancer biomarkers. methods: basic physical properties of apo-ev such as structure, size distribution, surface charge and membrane fluidity are discussed using cryo electron microscopy (em) and tomography, nanoparticle tracking analysis, dynamic light scattering and fluorescence anisotropy respectively. for phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission em, fluorescence microscopy and quantitative pcr. in addition, we study the interaction of apo-ev with blood components such as platelets, leucocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. results: looking at the differences between apo-and non-apo-ev, apo-ev have larger diameter, while structurally are not different. however, we have identified distinct apo-ev markers such as active caspase 3 and histones, or dna and small non-coding rna-y. there is also strong interaction of ev with platelets and leucocytes but not with red cells, indicating potential routes of transfer of ev cargo in the circulation. summary/conclusion: it is concluded that for the characterization of the heterogenous ev populations, combination of multiple techniques is often required, and also, understanding the strengths and limitations of each method is essential for choosing the appropriate set of analytical tools. finally, we consider that monitoring free circulating apo-ev or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response. casting a small netrin: functional roles of a novel surface factor on stroma-derived extracellular vesicles in pancreatic cancer kristopher s. raghavan a , ralph francescone b , janusz franco-barraza b and edna cuckierman b a drexel university; fox chase cancer center, philadelphia, usa; b fox chase cancer center, philadelphia, usa introduction: pancreatic ductal adenocarcinoma (pdac) is a devastating disease driven and supported by changes in its microenvironment, or stroma. here we dissect the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (cafs) and pdac. pdac communicates with its microenvironment, in part, through the exchange of specific types of extracellular vesicles (evs). specifically, we focus on the mechanism by which caf-secreted evs support pdac survival, with an additional goal to identify biomarkers suitable to generate a future "liquid biopsy" test for early pdac detection and prognosis. methods: evs are isolated from patient-derived pdac-associated fibroblasts via differential ultracentrifugation and validated by isev standards. human pdac cell lines used as recipient cells are treated with caf-evs to assess their role in supporting pdac survival. recombinant proteins, neutralizing peptides, and non-functional mutant proteins are used to block ev interaction with target cells. results: we observe sub-types of caf-evs containing unique surface receptors. one ev sub-population of interest contains a novel surface protein (nsp) expressed on the plasma membrane of pancreatic cafs, but not their healthy counterparts. further, pdac cells up-regulate nsp's lone binding partner, suggesting a role for these factors in pdac-selective ev uptake. functional assays designed to test pdac viability suggest these nsp(+)-evs protect pdac cells from programmed cell death as a result of physiological stress. this ev-mediated survival benefit can also be inhibited by blocking the interaction of nsp and its binding partner, suggesting the engagement of these two factors is necessary for cafs to support pdac via evs. pursuing our biomarker goal we confirm stromal nsp expression increases during early panin stages prior to tumour development, and we are currently seeking to validate nsp(+)-evs in blood of pdac patients. summary/conclusion: this research shines light on a novel mechanism of tumour-stroma communication that may be crucial for cancer progression during early disease stages and a potential target for disrupting the supportive role of the tumour microenvironment. additionally, we describe a sub-population of nsp (+)-evs that have the potential to serve as biomarkers for identifying pdac development. exosomes carry distinct mirnas that drive medulloblastoma progression introduction: extracellular vesicles (evs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including rna, from degradation. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the bloodbrain-barrier. here we characterised the rna of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal rna cargo as potential functional biomarkers for medulloblastoma. methods: exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by nanosight, electron microscopy, western blotting and nanoscale flow cytometry. exosomal mirna and mrna from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by rna-sequencing technology. results: based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. mirna profiling revealed several established oncomirs identified in our malignant derived exosomes compared to control samples. using interaction pathway analysis, we identified that our malignant exosomes carry numerous mirnas implicated in migration, proliferation, cellular adhesion and tumour growth. several previously identified oncomirs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-mir-455-3p and hsa-mir-92a-3p. summary/conclusion: this study shows that exosomes from mb cells carry a distinct mirna cargo which could enhance medulloblastoma progression. the use of circulating exosomes as markers of metastatic disease could be an innovative and powerful noninvasive tool. introduction: inflammatory changes in the bone marrow (bm) and suppression of haematopoietic stem and progenitor cell (hspc) function during acute myeloid leukaemia (aml) significantly contribute to patient morbidity and mortality. our laboratory has previously shown that aml-derived extracellular vesicle (ev-aml) trafficking confers a state of enforced quiescence and leads to lasting dna damage in hspcs. here we explore the underlying cause. specifically, we hypothesize that ev-aml incite inflammatory regulators as potential mediators of dna damage. methods: as a validated model of aml, we utilized the murine tib49 cell line as a source of ev-aml. ev-previous work has indicated that mirnas, notably mir-146a and mir-155, play a critical role in scc tumour development. evs are membrane-bound vesicles involved in cell-cell communication carrying actively sorted cargo, protected from degradation. the potential pathways these vesicular mirnas modulate and the implication they have on cancer biology is under active investigation. we have previously shown that the cadherin dsg2, a stem cell marker, modulates ev release. dsg2 is upregulated in a number of cancers, including scc, and correlates with poor prognosis. here we aim to elucidate the impact of ev-associated mirnas in sccs by bioinformatic analysis. methods: scc cells stably expressing dsg2 were generated and evs isolated by sequential ultracentrifugation. total cellular and ev rna was isolated by mirneasy, analysed using rnaseq and identified by grch37 alignment. results were confirmed by qpcr. altered pathways based on targets were identified using mirnet and kegg pathway analysis. potential cancerassociated cytokine targets were confirmed by antibody array. results: rnaseq revealed 87 cellular and 15 ev mirnas that were differentially expressed in response to dsg2 with 7 overlapping. the highest altered mirnas were validated by qpcr. kegg pathway analysis determined that these mirnas have the highest number of shared targets in cancer, cell cycle, and p53 signalling pathways. interestingly, mir-155 was upregulated while mir-146a was dramatically downregulated in evs. targets of mir-146a, icam-1, il-6, and il-8, cytokines critical for cancer progression were upregulated. summary/conclusion: these results suggest that the mirna content of evs is tightly regulated. by altering the mirna profile, dsg2 contributes to the pathogenicity of these evs by increasing levels of cytokines important for cancer stem cell renewal and metastasis. in addition, these mirnas may serve as non-invasive diagnostic markers for sccs. funding: nih r01 cancer cells grown in 3d release distinct extracellular vesicles during tumour growth and invasion jens c. luoto, sara bengs, leila coelho rato, lea sistonen and eva henriksson åbo akademi university, turku, finland introduction: cancer cells secrete extracellular vesicles (evs) that affect tumour progression. the characteristics of evs produced during tumour growth and invasion are however poorly understood. in this study, we identify the composition and characteristics of evs produced by noninvasive and invasive tumours and correlate these characteristics with the invasive status of the tumour. for that purpose, we established a protocol for isolating evs from extracellular matrix (ecm)-based three-dimensional (3d) cancer cell cultures. methods: human prostate cancer pc3 cells were grown in 3d cultures using ecm-based hydrogel, in standard 2d culture conditions and in bioreactor. evs were isolated from these cultures with differential and density gradient centrifugation. the isolated evs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (ms). results: our results demonstrate that 3d ecm-based hydrogel cell cultures secrete evs that can be isolated from both the conditioned media and the hydrogel. the invasive 3d cultivated pc3 organoids were found to secrete large amounts of evs compared to the non-invasive organoids. interestingly, our ms results revealed that non-invasive and invasive organoids secrete evs with partially distinct protein cargo. summary/conclusion: we have established a novel protocol for ev production in a 3d cell culture system utilizing ecm-based hydrogel, in which invasive tumour growth can be mimicked. our method allows the specific isolation and characterization of evs derived from different stages of 3d culture, such as non-invasive and invasive organoids. importantly, we found that tumour-derived evs change in composition during the tumour progression. taken together, our method can be used to define the distinct ev characteristics involved in cancer invasion. we previously showed extracellular vesicles (evs) to be causally involved in transmitting drug resistance. this study aimed to evaluate compounds proposed to reduce/block ev release. specifically, we selected calpeptin and y2763 (proposed to inhibit evs budding from the cell membrane) and manumycin a and gw4869 (proposed to inhibit evs deriving from mvbs). associated effects on -and consequences of-ev release were then investigated. methods: suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (fc). conditioned medium (cm) was collected from docetaxel-resistant pc3 (pc3 rd) cells after 48 h incubation in dfbs-medium with or without the 4 compounds. evs were separated from tangential flow filtration concentrated cm using optiprep density gradient. 1.03-1.16 g/ml fractions were then pooled and washed. evs were characterised using nta, immunoblot, tem and lipid assay and fc. influences on growth and migration, of evs continuing to be released (at 1x105evs/3x103cells, 1x107evs/3x103cells), were evaluated on recipient du145 and 22rv1 cells. evtrack id ev190113, score 88% results: calpeptin and y27632, alone and in combination, did not significantly affect quantities of evs released. however, gw4869 significantly (p < 0.004) increased quantities of released evs, of a larger size; very high protein to lipid ratio; and carrying grp94 compared to control evs (p < 0.006). this effect was reverted when gw4869 was combined with manumycin a (p < 0.004). following all compounds treatments, 1x105evs/ 3x103cells inhibited 22 rv1 proliferation (p < 0.0014), while at 1x107evs/3x103cells only evs from manumycin a (p < 0.05) and y27632 (p < 0.00036) treated cells reduce 22 rv1 proliferation. evs following gw4869 treatment significantly (p < 0.001) inhibited du145 migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of evs (p < 0.001). summary/conclusion: while none of the 4 proposed inhibitors significantly reduced ev release, the resulting evs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. patient-derived organoids represent a novel tool to study the effect of intra-tumoral heterogeneity on ev release in non-small cell lung cancer introduction: lung adenocarcinoma (luad) is the leading cause of cancer-related death with a low 5-year survival. although the importance of intra-tumoral cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging, proper models to study its effects on ev release and cargo in human tissues still lack. the 3d organoid technology maintains the cellular and genetic heterogeneity of in vivo tissues and has proved to be so far the best ex vivo model of human cancers. by using patient-derived and mouse organoids we set out i) to compare the ev release from normal and tumor tissues and ii) to follow changes in ev secretion when the relative ratio of tumor cell subpopulations is shifted. methods: we used mouse and luad patient-derived normal and tumor organoids. the medical research council of hungary approved our experiments with human samples and informed consent was obtained from patients. evs were detected by antibody-coated beads, nta and tem. intra-organoid heterogeneity was proved by immunostaining and rt-qpcr. results: we provide evidence that both mouse and human normal organoids contain all the bronchiolar cell types. interestingly, luad organoids selected for tp53 mutation contained not only ki67+ proliferating cells, but differentiated cell types as well. furthermore, all the lung organoid cultures produced evs and this was shifted to the smaller size range. interestingly however, when modifying the proportion of organoid cell types, we observed an increased ev release when more ki67+ proliferating cells were present both in normal and in luad samples. summary/conclusion: our data show that patientderived lung organoids represent a novel model to study the role of intra-tissue heterogeneity in ev functions in the humans, leading to improved diagnosis. funding: this work was funded by the national competitiveness and excellence program nvkp_16-0007 (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). exosome mediate heart-adipocyte communication after myocardial ischaemia/reperfusion and impairs adipocyte endocrine function yajing wang, lu gan, dina xie, wayne lau, theodore christopher, bernard lopez and xinliang ma thomas jefferson university, philadelphia, usa introduction: by incompletely understood mechanisms, mi patients sustain systemic metabolic disorder. adipocytes are an important cellular type regulating energy homoeostasis. the impact of mi upon adipocyte function remains unknown. exosomes (exo) are critical vehicles mediating organ-organ communication. however, whether and how exo may mediate post-mi cardiomyocyte/ adipocyte communication have not been previously investigated. methods: adult male mice were subjected to mi/r. serum exo were isolated 3 hours after r and incubated with 3t3 l cells for 24 hours. the effects of exo upon adipocyte function were determined. results: compared to control, mi/r exo significantly altered the expression of 17 genes known to be important in adipocyte function. go analysis revealed that genes associated with endoplasmic reticulum (er) function and adipocyte endocrine function are the primary two pathways altered by mi/r exo. venn analysis identified 11 mi-rnas as cardiac-enriched, adipocyte-poor, and er function-related mirnas. rt-qpcr confirmed the mir-23a/27a/24-2 family members are the most markedly increased mi-rnas in mi/r exo. incubation of 3t3 l cells with mi-r27a mimic significantly downregulated edem3, dsba-l, and pparn, and upregulated perk and chop. conversely, mi-r27a inhibitor significantly decreased the impact of mi/r exo upon er function genes. additional studies demonstrated edem3 and pparγ (two critical molecules maintaining er function and adipocyte endocrine function) to be direct targets of mi-r27a. one of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by pparn at the transcriptional level and by dsba-l at the post-translational level. we next determined whether mi/r exo may affect adiponectin expression/ assembly. incubation of 3t3 l cells with mi/r exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by mir27a mimic. finally, in vivo administration of gw4869 (exo biogenesis inhibitor) or mir27a inhibitor attenuated adipocyte er dysfunction and restored plasma adiponectin level in mi/r animals. summary/conclusion: we demonstrate for the first time that mi/r causes significant adipocyte er and endocrine dysfunction by exo mediated cardiomyocyte/adipocyte communication via mir-23a/27a/ 24-2. funding: nih and american diabetes association pancreatic cancer cell extracellular vesicles drastically alter the behaviour of recipient normal pancreas cells charles p. hinzman a , yaoxiang li a , meth jayatilake a , jose trevino b , partha banerjee a and amrita cheema a a georgetown university medical center, washington, usa; b university of florida health science center, gainesville, usa introduction: pancreatic cancer (paca) is predicted to become the 3rd leading cause of cancer-related deaths by 2030. patients diagnosed with pancreatic ductal adenocarcinoma (pdac) have a 5-year survival ratẽ 9%. detection of pre-neoplastic lesions can potentially improve survival. however, there is currently no screening test for early stage detection. importantly, paca tumours are 90% non-tumorigenic cells. a better understanding of early paca oncogenesis is needed. cancer cells shed extracellular vesicles (evs) that are internalized by neighbouring and distant cells to induce a myriad of cancer progression events. we hypothesize that in early paca oncogenesis, evs mediate a behavioural change in surrounding normal cells, leading to the formation of this unique stroma. the purpose of this study was to develop a model to examine the phenotypic changes undergone by normal human pancreas cells when they are exposed to paca cell evs. methods: evs were isolated using differential ultracentrifugation with filtration from established (panc-1, sw-1990, capan-1 and miapaca-2) and patientderived xenograft (ppcl-46 and ppcl-68) paca cell lines. cells were grown using ev-depleted fbs. ev isolations were validated and quantified using transmission electron microscopy, quantitative elisa, immunoblot and nanoparticle tracking analysis. normal pancreas cells (htert-hpne and hpde-h6c7) were co-cultured with cancer cell evs for 24-48 hours. metabolic activity was measured using a mito stress test on a seahorse xfe96 extracellular flux analyser. results: we discovered that normal cells undergo vast behavioural transformations, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability, when co-cultured with paca cell evs. these responses were ev dose dependent. further, paca cell evs metabolically reprogrammed normal cells, causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic and glycolytic profile. summary/conclusion: our results indicate that paca cell evs confer enormous transformational properties to normal human pancreas cells in vitro. we hypothesize that evs impart distinct transformational properties to normal cells in vivo and this influence could unveil novel mechanisms regulating cancer onset and progression. these signals may be detectable before progression of early-stage paca to pdac, leading to the development of assays for earlier diagnosis in patients. further studies are underway to identify the biochemical mediators of these changes. plasma extracellular vesicles-mirnas released by hypoxic cells are associated to pro-tumorigenic and immunosuppressive microenvironment in lung cancer introduction: extracellular vesicles (ev) containing specific subset of functional biomolecules, such as micrornas (mirnas) are released by all cell types. it has been widely demonstrated that ev-mirnas from cancer cells can manipulate the tumour microenvironment modulating the gene expression of recipient cells. we previously identified a three levels risk classifier (msc) based on 24 plasma-mirnas associated with lung cancer development and prognosis. the aim of this study was to investigate the potential role of ev-24 mirnas as mediators of pro-tumorigenic features. methods: evs were isolated from plasma of heavysmoker individuals with high (mscpos) or low (mscneg) risk of lung cancer by differential centrifugation method. purity of evs isolated was confirmed by sizing using nta and tem analysis and expression of ev-enriched proteins. mirna levels were analysed by dpcr. in vitro and in vivo analyses were used to assess the biological effect of plasma evs on different recipient cells. results: levels of mirnas in evs correlated with determination of whole plasma (80% of correlation with msc classifier). mscpos-evs stimulated 2d and 3d proliferation of non-tumorigenic epithelial cells through c-myc transfer into recipient cells. furthermore, mscpos-evs increased the ability of huvec to form tubular structures compared to mscneg-evs. in vivo co-injection of mscpos-evstreated huvec with a549 lung cancer cells resulted in an increase of tumour growth compared to mscneg-evs-treated huvec. mir-126 modulation in evs with mirna mimics or in recipient cells using mirna inhibitors demonstrated that this mirna is implicated in the autocrine proangiogenic modulation of huvec phenotype. mscpos-evs induced m2 polarization of macrophages both in vitro and in vivo. we demonstrated using synthetic oligonucleotides that mir-320 is responsible for the immunosuppressive modulation of these cells. regarding the potential origin of evs in mscpos individuals, we observed that hypoxia stimulated the secretion of evs containing c-myc from fibroblasts, mir-126-evs from endothelial cells and mir-320-evs from granulocytes. summary/conclusion: these data show that plasma evs of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of evs in lung cancer development. exploration of ev-associated metastatic targets on melanoma cells introduction: cancer cells secrete evs that may harbour metastatic proteins. previous studies have demonstrated the decrease of cd63 tetraspanin expression in melanoma cells are correlated with enhancing its metastatic potential. however, other proteins, such as cd44, cd47, met and nrp2 which are overexpressed in melanoma cells, are also associated with the spread of cancer. in this study, we sought to investigate the expression of metastatic proteins in evs derived from murine melanoma b16f10 lineage. methods: b16f10 cells were cultured in dmem supplemented with 10% fbs and antibiotics. the cells supernatant were harvested each 24 hours, filtered through 0.22 µm and ultracentrifuged at 110000 x g for 90 min at 4ºc to pellet evs. next, size and concentration was determined using nanoparticle tracking analyses technique, and the morphology of evs were analysed by negative-staining transmission electron microscopy (tem). the ev's surface protein were characterized by flow cytometry and protein content was profiled by mass spectrometry. results: our flow cytometry results have shown the presence of tetraspanins markers cd63, cd9 and cd81 on vesicle´s surface. in addition, our assay demonstrated a diminished expression of cd63 molecule in comparison to cd9 and cd81. we have profiled melanoma-evs by mass spectrometry, identifying the presence of proteins that may be associated to metastasis, such as cd44, cd47, met and nrp2. summary/conclusion: these preliminary results are consistent with the literature and suggest that melanoma-derived evs harbour proteins, which may contribute to promote tumour metastasis. in our next step, we plan to generate b16f10 lineages harbouring shrna vectors, in order to knockdown gene expression of cd63, cd44, cd47, met and nrp2 to investigate the metastatic potential in vitro, in comparison to parental cells. we also may use syngeneic mice models to explore metastatic potential of genetically modified b16 f10-derived cells. introduction: overexpression of her2 occurs in2 0% of breast cancers and confers aggressive behaviour and poorer prognosis. thankfully, a number of drugs such as neratinib have been developed to target her2, potentially providing substantial benefit for many patients. nevertheless, it is estimated that up to 70% of patients with her2-overexpressing tumours do not gain benefit, as a result of innate or acquired drug-resistance. this study aimed to investigate if nano-sized membrane-surrounded extracellular vesicles (evs) released from drug-sensitive and drug-resistant cells reflect the her2 status of their cells of origin and thus have potential as minimallyinvasive biomarkers. methods: evs were isolated from conditioned media (cm) of her2-positive cell lines (hcc1954 and skbr3) and their neratinib-resistant counterparts (hcc1954-nr and skbr3-nr) that we developed in our laboratory. evs from cm of a triple-negative breast cancer (tnbc) cell line variant, hs578 ts(i)8, were evaluated as negative control for her2. in brief, cm was centrifuged at 300 g, for 10 min x3 to get rid of any cells. the supernatant was then centrifuged at 200,000 g for 6 h at 4ºc to collect evs. the resulting evs were washed in pbs, centrifuged as before, and resuspended in 100 μl pbs. ev quantities were estimated by nanoparticle tracking analysis (nts). ev lysates were characterised by immunoblots, for established positive and negative ev markers. particle concentration as well as size distribution of evs were measured using the zetaview (particle metrix, germany). surface proteins on single evs were analysed by highresolution flow cytometry (fc), using an amnis imagestreamx mark ii. all data was submitted to ev-track (ev-track id: ev190112). results: neratinib-resistant cell line variants were found to have reduced her2 protein expression compared to their respective drug-sensitive counterparts. neratinib-resistant cell line variants released fewer evs, when normalised per number of secreting cells, compared to their-drug sensitive counterparts. furthermore, evs from drug-sensitive cells carried her2, while those from drug-resistant cells lacked her2 (similar to the evs from the tnbc cells); reflecting the her2 status of their cells of origin. summary/conclusion: this study indicates that a reduction in her2 protein expression is a mechanism by which cancer cells manifest resistance to her2-targeted drugs (i.e. by making fewer her2 receptors available on the cells surface to accommodate the drugs activity). furthermore, ev-carried her2 seems to reflect the her2 status of their cells of origin. this suggest that analysis of her2 on evs in the peripheral circulation may help predict response to her2-targeted drugs. thus, analysis of evs in appropriate cohorts of patients' specimens is warranted. introduction: rab27a, a small gtpase involved in exosome biogenesis by regulating mve docking at the plasma membrane, and its expression level highly correlated with ohsv replication ability in vitro. oncolytic viruses is a newly promising therapeutic agent for cancer treatment. however, more than 50% of tumours naturally showed highly resistant to oncolytic viruses for unknown reasons. uncovering the underlying mechanisms of resistance to ohsv can offer potential therapeutic targets to enhance ohsv activity to kill tumour cells. in addition, it will give new insights into the identification of therapeutic biomarkers, which can be used to predict patient response to ohsv in clinical. methods: deep-sequencing data showed that lower expression level of rab27a is present in ohsv resistant tumour cells compared to that in sensitive tumour cells. then an ohsv resistant mc38 tumour cell line was established by repeated injections with ohsv in mc38 tumour-bear mouse model. lastly, it was verified that ohsv resistance is associated with a downregulation of rab27a and overexpression of rab27a can rescue ohsv replication. results: 1) the lower expression level of rab27a is shown in ohsv resistant tumour cells compared to that in sensitive tumour cells shows in deep-sequencing data. 2) furthermore, we established an ohsv resistant mc38 tumour cell line by repeated injections with ohsv in mc38 tumour-bear mouse model. similarly, in ohsv resistant mc38 cell line, rab27a expression was lower than parental mc38 cells. and the release of exosomes and virus was decreased in ohsv resistant mc38 cell line. these results were confirmed by rab27a sirna knockdown. 3) we verified that in ohsv naturally resistant human cancer cell lines, ohsv resistance is associated with a downregulation of rab27a and overexpression of rab27a can rescue ohsv replication. summary/conclusion: downregulation of rab27a expression restricts the efficiency of ohsv replication and the spreading ability of the released progeny virus which also provide rab27a as a potential target and biomarker for ohsv cancer therapy. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd2015071414385495, shenzhen science and innovation commission project grants jcyj201 70411094933148, jcyj20180306173333907 to shenzhen international institute for biomedical research. inactivation of emilin-1 by proteolysis and secretion in extracellular vesicles favours melanoma progression and metastasis introduction: studies have demonstrated that melanoma-derived extracellular vesicles (evs) home in distal organs and sentinel lymph nodes favouring metastasis. although lymph node metastases are themselves rarely life threatening, they could be considered as one of the first step of metastasis in many cancer types. therefore, defining the mechanisms involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes could bring the clue to block the metastatic process from the beginning. methods: we have characterized secreted exosomes from a panel of mouse melanoma models representative of low metastatic potential (b16-f1), high metastatic potential (b16-f10) and lymph node metastasis (b16-f1 r2). we analysed the rna expression in cells and protein content of exosomes derived from mouse melanoma lymph node metastatic models by rna sequencing and mass spectrometry respectively. we validated expression by western-blot, qpcr and immunofluorescence. to define the mechanism of emilin-1 secretion cells were treated with the ev secretion inhibitor (non-competitive inhibitor of sphingomyelinase), gw4869. cell viability and cell cycle assays with an overexpression model (b16-f1-emilin-1) were also performed. in vivo experiments based on subcutaneous and intrafootpad injection for studying the role of this protein during melanoma progression were performed to define the relevance of our findings in vivo. human paraffin samples were analysed for emilin-1 expression by immunohistochemistry. results: we found a signature of over-expressed genes and hyper-secreted proteins in exosomes related to lymph node metastasis in the b16 mouse melanoma model. among them, we found that emilin-1, a protein with an important function in lymph node physiology, was hyper-secreted in exosomes. interestingly, we found that emilin-1 is degraded and secreted in exosomes as a mechanism favouring metastasis. further, we found that emilin-1 has a tumour suppressive-like role regulating negatively cell migration. importantly, our in vivo studies demonstrate that emilin-1 overexpression reduced primary tumour growth and metastasis in mouse melanoma models. analysis in human melanoma samples showed that cells expressing high levels of emilin-1 are reduced in metastatic lesions. summary/conclusion: overall, our analysis suggests that the inactivation of emilin-1 by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive activities in melanoma favouring tumour progression and metastasis. funding: this work was supported by grants from mineco (saf2014 54541 r), asociación española contra el cáncer, fundación de investigación oncológica fero and mineco-severo ochoa predoctoral program. introduction: the amplification of erbb2 gene and the consequent overexpression of the encoded protein her2 have an important role in breast cancer classification at diagnosis and subsequent treatment decision with the anti-her2 monoclonal antibody trastuzumab. fish and ihc have been used so far to detect erbb2 gene amplification and her2 protein overexpression respectively in tissue biopsies. in this context, a major goal for liquid biopsies is to take advantage of the information carried by circulating tumourderived materials (such as extracellular vesicles (evs) and cell free dna (cfdna)) to noninvasively detect erbb2 gene status in the blood. however, the isolation of diverse tumour-derived materials from a single aliquot of patients' plasma and the accurate detection of cancer biomarkers is still challenging. methods: by adopting a recently published nickelbased evs isolation (nbi) protocol1 that allows for recovery of cfdna after evs isolation, we generated a high-sensitivity molecular assay to accurately detect erbb2 amplification and consequent her2 overexpression on a limited volume of plasma (1.5 ml) collected from breast cancer patients (stage i, ii and iii) at diagnosis. results: 1) we detected erbb2 amplifications by droplet digital pcr (ddpcr) on the cfdna isolated from the plasma of erbb2 positive patients; 2) we confirmed her2 overexpression on a subset of patients by setting up an antibody-based affinity reaction designed to detect her2 protein on the surface of the isolated evs; 3) we succeeded in the quantification of her2 transcripts enclosed within evs by performing ddpcr in samples of patients showing a range of circulating tumourderived material. the specificity and sensitivity of these novel methodological assays were tested on a cohort of healthy individuals (n = 20) and on a cohort of her2 positive (n = 20) or her2 negative breast cancer patients (tnbc; n = 20). summary/conclusion: here we report a pilot study on a novel multimodal method for erbb2 detection from a minimal amount of plasma. this approach integrates information from cfdna with evs-derived rna and proteins analysis. this proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. introduction: the biological and medical importance of exosomes recognized over the last decade has given rise to a crucial need for the discrimination between true evs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris. additionally, it is imperative to develop methods to identify and characterize ev sub-populations. considering ev biology and the reliability of labelled biomolecules, we developed both exogenous and endogenous labelling protocols, taking advantage of different dye properties which can target a multitude of compartments. this approach reveals key aspects of ev structure and integrity. methods: nanosized evs/exosomes were purified by size exclusion chromatography (sec) from model cell lines and human plasma. diverse dyes were orthogonally evaluated through different single particle and bulk analysis technologies, such as high-resolution cytometry, nanoparticle tracking analysis and plate fluorimeter. concomitant profiling of specific ev subpopulations was optimized using antibodies (abs) against tetraspanins and cell type specific targets and assessed by single particle analysis and elisa. to assess specificity of labelling protocols we used specific controls such as recombinant rfp expressing vesicles and purified lipoproteins. results: our ev staining protocols allowed for high labelling efficiency and unprecedent ev discrimination, quantification and characterization by combining single particle analysis and bulk measurements in simple matrices (saline buffers) and in complex biofluids (i.e. plasma). different approaches have diverse and complementary advantages (costs, capacity, sensitivity, informative readout) for implementation in research and diagnostic development flows, directly feeding in-house r&d and qc pipeline. summary/conclusion: overall, fluorescent evs are versatile and valuable tracers that can be applied in the optimization of pre-analytical and purification protocols, selection of target biomarkers and diagnostic assay calibration and validation. funding: endevor (por-fse 2014-2020) region tuscany and exosomics r&d program. subpopulations of tissue-derived extracellular vesiclesmethodological evaluation for vesicle size measurement introduction: introduction: subpopulations of extracellular vesicle (evs) are commonly classified by their different size, however, the ev size cut off is still under discussion. the aim of the study was to evaluate size range of six ev subpopulations using three methods: electron microscopy (em), nanoparticle tracking analysis (nta) and exoview™. methods: methods: ev subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. large and small evs (levs and sevs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (ld and hd). size of ev subpopulations was then evaluated by: em introduction: small-extracellular vesicles have an important role in cell metabolism and cell-to-cell communication. moreover, sevs when secreted from cells are capable to act as mediators of various neurological diseases. however, sevs show heterogeneity and this may impact their functions. therefore, to characterize sevs at a single-particle level is important to better detail the associated biological activity. in this scenario, we innovatively propose the structured illumination microscopy (sim) as a technique able to complement the non-optical methods (transmission electron microscopy, tem) to analyse single sevs and their markers. methods: human plasma sevs were separated from healthy cognitive control (ctrl), mild cognitive impairment (mci) and demented subjects. the sevscontaining pellet was resuspended in 4% paraformaldehyde or 2% glutaraldehyde solutions. for sim, sevsenriched preparations were washed with the blocking solution and incubated with the primary antibody (l1cam). the secondary fluolabelled antibody was then added. between the steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge steps were performed. the image acquisition was done on a nikon sim system with a 100x oil immersion objective. sevs were imaged with a 3d-sim acquisition protocol. tem was performed on 300 mesh formvar copper-coated grids. sevs-enriched preparations were incubated first with the blocking solution and then, immunogold-labelled for cd63. samples were counterstained with uranyl acetate and observed under a jeol 1010 ex electron microscope. data were recorded with a morada digital camera system. participation to the present study was approved by relevant local ethics committee of mendrisio and lugano hospital and written informed consents were obtained from subjects. results: for sim methodology, only vesicles in the range from 180 to 190 nm were detected, as the final resolution achieved was 160 nm. instead, for tem, sevs under 100 nm were identified. none of these methods provided relevant information about possible difference in morphology of the ctrl-, mci or demented subjects-derived sevs. summary/conclusion: even if both methods identified cd63 or l1cam-positive vesicles, sim resolution and the complexity of the protocol represent some disadvantages respect to tem, that may be the first choice screening technique for evs analysis to be then completed by sim for particular tasks. fabrication of nanopore structures via conformal metal-film deposition for ev sensing kwanjung kim a , seung-min han b , soo-hyun kim c and sung-wook nam d luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. these evsreleased under normal physiological conditions as well as in the pathogenesis of neurological, vascular, haematological, and autoimmune diseaseshave been shown to transfer biological molecules such as protein and rna between cells, potentially transmitting signals. to understand more about these signalling mechanisms, there is a need for detecting and quantifying evs with cargo protein and rna in a reproducible and reliable manner. however, this has been challenging due to the small size of evs (ranging from 30 to 100 nm in diameter), and the lack of specific staining reagents. methods: here, we utilize the amnis® cellstream® flow cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. we demonstrate that a charge-coupled device (ccd)-based, time-delay-integration image capturing system can be used to detect and quantify evs and their cargo labelled with exoglow™-protein or exoglow™-rna. results: in this study, we show flow cytometry data quantifying ev samples that have been labelled with cargo markers for proteins and rna. the ev cargo contents along with the appropriate control samples will be shown. summary/conclusion: the ccd based detection of the cellstream flow cytometer has the sensitivity to quantify evs and their cargo. single ev imaging reveals novel ev biomarkers and dna cargo siobhan m. king a , ricardo bastos a and andras miklosi b a oni, oxford, uk; b oni (oxford nanoimaging ltd), oxford, uk introduction: extracellular vesicles (evs) are cellderived membrane-bound particles that range in size from 30-1000 nm and carry active molecules such as dna, rna and proteins. upon secretion, evs can execute many biological functions such as initiating intracellular communication or regulating immune responses. depending on their origin evs have different characteristics and cargo, making them attractive candidates for early diagnostic and therapeutic applications. however due to their small size and heterogeneity, direct visualization and characterization of the surface markers expressed remains a challenge since these vesicles are below the resolution limit of standard light microscopy. methods: here, we describe a method that provides size analysis of single-evs, which falls below the diffraction limit of light. this was done with purified evs, immunostained using fluorescently labelled primary antibodies raised against ev surface markers (cd63, cd81, cd9), specific cargo such as dna and probed for tissue specific cargo. characterization of the molecular content and structural properties of surfaceimmobilized evs was performed using single-molecule localisation microscopy (smlm) on the nanoimager platform. results: multicolour smlm was used to detect up to three ev biomarkers showing successful characterization of the molecular signature for different ev subpopulations. the distribution of novel components on urinary evs were visualized for the first time using this approach. in addition, smlm revealed the presence of dna on both the surface and also as a cargo inside evs isolated from tumour cell culture media, which was validated using complementary biochemical characterization. summary/conclusion: smlm is a powerful technique for single-ev analysis and characterization. visualization of single-urinary evs enabled accurate sizing and further insights into novel components expressed on the subpopulation's membrane surface. together, the data demonstrates that the quantitative abilities of smlm can significantly enhance our understanding of evs, as structure, phenotypes, and cargoes can now be successfully resolved. introduction: working skeletal muscle is a common site for injury due to unaccustomed exercise with or without underlying pathology. direct analysis of skm injury requires invasive tissue biopsies. circulating extracellular vesicles (evs) are abundant in blood and have been shown to be enriched in microrna; profiles of which may reflect the state of tissues. evs may therefore serve as a non-invasive indicator of muscle injury and regenerative processes in vivo. methods: two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by 9 healthy male volunteers. serum creatine kinase (ck) and plasma evs were analysed at baseline, 2 and 24 hr post-exercise. perceived muscle pain (pmp) was assessed at 2 and 24 hr post-exercise. large evs were isolated using a 10 000 g centrifugation step, and small evs were isolated using qev columns. ev-enriched isolates were visualized using tem, and size and numbers were quantified using nta. based on nta results the highest particle fractions (7-10) were pooled for rna analysis. qpcr was done on plasma, large evs and small evs. a group of muscle and immune cell-important mirs were analysed by means of normalization to an exogenous control. results: ck and pmp increased post-exercise, providing evidence for muscle damage. tem revealed an abundant and heterogeneous pool of evs. a concomitant abundance of evs was seen with nta (mean = 1.17 x 1010 particles/ml plasma). mean ev diameters were 194 ± 22 nm across all timepoints. no change in ev size nor number was seen over time, however, mir-31 decreased at 24 hr when compared to 2 hr in the small ev isolate only. plasma displayed an immediate increase in myomirs-1 and −206 at 2 hr, which returned to baseline at 24 hr. in contrast, myomirs-208b and 486 remained elevated over the 24 hr period. myomir-133b and 486, as well as immune-mirs, did not change in evs or plasma as a result of the intervention. summary/conclusion: the decrease in mir-31 in small evs at 24 hr is consistent with previous data. no decrease in mir-31 in large evs suggests specific packaging and hence a specific response to the muscle damage in small evs. more changes occurred in plasma myomirs suggesting less specific passive leakage into circulation from damaged cell membranes. funding: south african national research foundation pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration yingnan wu a , dinesh parate a , eng hin lee a , zheng yang a and alfredo franco-obregón b a national university of singpaore tissue engineering program, yll school of medicine., national university of singapore, singapore, singapore; b biolonic currents electromagnetic pulsing systems laboratory, biceps, national 11 university of singapore, singapore, singapore introduction: the mesenchymal stem cell (msc) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. mscs are "environmentally-responsive" to local microenvironmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. we have previously shown that exposures of mscs to pulsed electromagnetic fields (pemfs) enhanced msc chondrogenesis. here, we investigate the influence of pemf exposure over the paracrine activity of mscs and its significance to cartilage regeneration. also, the subsequent extracellular vesicles analysis and isolation are processed for the understanding of how the pemfs affect stem cell evs and consequent differentiation induction. methods: conditioned medium (cm) was generated from mscs subjected to either 3d or 2d culturing platforms, with or without pemf exposure. the paracrine effects of cm over chondrocytes and msc chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and mscs was assessed. the cms which have significant effects during chondrogenesis will be analysed by protein and mirna studies. results: we show that the benefits of magnetic field stimulation over msc-derived chondrogenesis can be partly ascribed to its ability to modulate the msc secretome. mscs cultured on either 2d or 3d platforms displayed distinct magnetic sensitivities, whereby mscs grown in 2d or 3d platforms responded most favourably to pemf exposure at 2 mt and 3 mt amplitudes, respectively. ten minutes of pemf exposure was sufficient to substantially augment the chondrogenic potential of msc-derived cm generated from either platform. furthermore, pemf-induced cm was capable of enhancing the migration of chondrocytes and mscs as well as mitigating cellular inflammation and apoptosis. the cms protein results in the significant promotion chondrogenesis condition showed an increase in proliferation and anti-inflammatory cytokines. summary/conclusion: the findings reported here demonstrate that pemf-stimulation is capable of modulating the paracrine function of mscs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. the pemf-induced modulation of the msc-derived paracrine function for directed biological responses 1 in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical application. effects of extracellular vesicles from blood derivatives on osteoarthritic chondrocytes within an inflammation model introduction: the degenerative disease osteoarthritis (oa) is one of the leading causes of disability especially of elderly people. besides various treatment options depending on the severity of the cartilage degradation, the application of blood derived products such as platelet rich plasma (prp) are getting more and more popular in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. drawbacks of using prp include high donor variability, discrepancies among preparation protocols and the presence of cells (platelets, leukocytes) which can evoke cellular processes, especially inflammation, when injected into the diseased tissue. one possibility is to isolate only extracellular vesicles (evs) from blood derivatives to overcome these problems. in the current study the effects of evs isolated from blood derivatives on oa chondrocytes within an inflammation model was investigated. methods: cd14 positive primary monocytes were isolated from citrate anticoagulated whole blood by magnetic bead sorting. monocytes were differentiated into resting m0 macrophages and activated into m1 macrophages according to published protocols. elisa measurements verified successful differentiation and activation as il1β and tnfα levels increased. as control, thp1 monocytes were used. patient-derived oa chondrocytes were grown in 6 well plates and co-cultivated with activated m1 macrophages which were seeded into thincerts and added to the 6 wells representing the inflammation model. furthermore, cells were treated for 48 hours with media containing fcs, ev depleted fcs or evs isolated from prp or hypact serum. results: successful differentiation and activation of monocytes (thp1 and primary monocytes) into m1 macrophages was demonstrated by elevated levels of the inflammatory cytokines il1β and tnfα. within the inflammation model (co-culture of oa chondrocytes with m1 macrophages), addition of evs isolated from prp or hypact serum resulted in decreased secretion levels of il1β and tnfα compared to media supplemented with either fcs or ev depleted fcs. summary/conclusion: taken together, evs from blood derived products might be chondroprotective and anti-inflammatory mediators which protect cartilage from being degraded during oa. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst3-f-5030664/003-2017. 1α,25(oh)2d3 regulates growth cartilage matrix vesicle micrornas niels asmussen, michael mcclure, zhao lin, zvi schwartz and barbara boyan virginia commonwealth university, richmond, usa introduction: matrix vesicles (mvs) are small (50-150 nm in diameter) lipid bound extracellular organelles isolated from calcifying tissues including the growth zone (gc) of growth plate cartilage. 1α,25 (oh)2 vitamin d3 (1α,25) is a regulator of gc chondrocytes and the mvs they produce. these mvs are key players in the mineralization process and are selectively enriched with enzymes and growth factors. we found that mvs are also selectively enriched with micrornas (mir), including mir-22, mir-122 and mir-451. the aim of this study was to determine the regulatory role of 1α,25 in the packaging of mirna in mvs by gc cells. methods: gc cells were isolated by enzymatic digestion from costochondral gc cartilage harvested from 5 wk-old male sprague dawley rats (iacuc approved). confluent fourth passage gc cell cultures were treated with 10-8 m 1α,25 or vehicle for 24 h. media were removed, cell monolayers digested with trypsin and cells and mvs isolated by differential ultracentrifugation. rna was precipitated from cells and mvs. small rnaseq data were trimmed, aligned and counted before undergoing differential expression analysis. experimental groups had an n = 3 per variable. significant differences (p < 0.05) were determined using r v 3.6.2. results: 1α,25 treatment altered expression of 30 mv mirs compared to control mvs, whereas 5 cell mirnas were differentially expressed. 62.1% of significantly up or down regulated mir found in mvs overlapped between 1α,25 and vehicle groups with the remaining being uniquely differentially expressed. 1α,25 increased mv mir-150 and decreased mir-384-3p two mirs known to regulate osteoblast proliferation (150 increases, 384 decreases). summary/conclusion: 1α,25 regulates gc chondrocyte and mv behaviour and this study demonstrates that it also impacts the mir packaging within mvs. mir discovered in mvs have been demonstrated to impact chondrocyte behaviour and the present study indicates that 1α,25 regulates the growth plate through mir delivered by mvs. introduction: increasing evidence has proposed extracellular vesicles (evs) as mediators of many of the therapeutic features of mesenchymal stromal cells (msc) that have been widely studied in clinical trials over the last years. these evs have been recognized as nanocarriers of important biological information, which play a central role in cell-to-cell communication. in this context, evs can be used as an alternative to a cell-based therapy, with reduced risks. the present work aimed to evaluate the impact of different culture conditions on the msc-derived evs molecular composition through fourier-transform infrared (ftir) spectroscopy. methods: evs derived from msc from different sources, expanded in two different culture media ((xenogeneic -free (xf) vs serum-containing medium (fbs)) were characterized by ftir spectroscopy, a highly sensitive, fast and high throughput technique. moreover, principal component analysis (pca) of preprocessed ftir spectra of purified evs was conducted, enabling the evaluation of the replica variance of the evs chemical fingerprint in a reduced dimensionality space. for that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. results: evs secreted by mscs cultured with serumcontaining medium presented a more homogenous chemical fingerprint than evs obtained with xf medium. the regression vector of the pca enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. ratios between these spectral bands were determined, since these attenuate artefacts due to cell quantity and baseline distortions underneath each band. statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of 5%. it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture medium used for msc expansion and the msc donor. summary/conclusion: this work is a step forward into understanding how different culture conditions affect msc-derived evs characteristics. funding: fundação para a ciência e tecnologia (ptdc/equ-equ/31651/2017, uidb/04565/2020). performance qualification for microflow cytometers: understanding technical limitations to improve your research desmond pink a , michael wong a , diana pham a , renjith pillai a , leanne stifanyk b , sylvia koch b , rebecca hiebert a , oliver kenyon c and john lewis a a nanostics, edmonton, canada; b dynalife, edmonton, canada; c apogeeflow systems, hemel hempstead, uk introduction: as microflow cytometry and other techniques mature as validated modalities for analysing extracellular vesicles (ev), there has been a concerted effort to improve reproducibility . in order for this reproducibility to occur there has to be a critical understanding of advantages and limitations for each technology. for microflow cytometry, several instruments are available to analyse evs. each platform has different limitations as well as advantages over other platforms. to provide the optimal data for your specific research, it is critical to understand the limitations of your platform. to accurately define these limitations, a performance qualification (pq) of your instrument should be undertaken. methods: an apogee a60 platform was used in these experiments. experiments were designed with expected ranges and cut-offs for acceptance criteria.initial tests included autosampling of a 96 well plate with either single or double aspiration, single sample reproducibility and linearity proportional to flow rate. other experiments designed to show machine performance included minimal time to achieve valid data, sample volume required for double aspiration, determination of coincidence; detection sensitivity using a spiked sample; flow rate stability for extended periods (5-30 minutes) . tests should also be performed to determine carryover at a range of sample concentrations. if present, the means to remove contaminating samples should be determined. any performance tests should be applicable to any instrument in the field. results: auto-sampling helped demonstrate consistent data; reproducibility of total events and biomarkers was 2-8% c.v. detected bead concentrations were linear with flow rates between 0.75 and 10.5 ul/min. double well aspirations provided similar data with aspirations between 60-80ul. valid data was achieved for a low abundant target (~200-400 events/ul) after only 30 s, <10%c.v. detection sensitivity was determined to be~1/400,000. carryover ranges were determined in the presence of nominal unstained serum. an optimal number of machine washes was determined. some membrane stains, such as cell mask and cfse require much more rigorous cleaning to remove stain carryover. summary/conclusion: to improve data reproducibility, performance qualification of any instrument is key. operational limitations help define optimal performance parameters of any technology. understanding the types of experiments to perform for your particular type of characterization technology depends on the requirements you set for your research. a good performance test should be applicable to any related instrument in the field. funding: funding provided by nanostics, the alberta cancer foundation, and alberta innovates. introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par20-053, is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par16-267/ 277, successfully funded 7 r01 and 4 r21 grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, 2018); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, 2019). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, 2019). summary/conclusion: drs. sudhir srivastava and matthew young are the programme directors for the par which began accepting applications on 5 january 2020. this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute. introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of 7 protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores (600 nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the 7 protein biomarkers (tau, uchl-1, nfl, gfap, il6, il10, and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl1 were elevated in mtbi patients compared to controls (p < 0.05), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il6 and tnf-with tau from glur2+ evs showed 88% accuracy with 80% sensitivity and 100% specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. ps15.05 = op3.05 l1cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma maia norman, dmitry ter-ovanesyan, wendy trieu and david walt wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l1cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l1cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l1cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l1cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd9, cd63 and cd81) as well as l1cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l1cam appears. we also immunocapture l1cam from csf and plasma and perform western blots for the internal and external domains of l1cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l1cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l1cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l1cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l1cam in plasma is likely not coming from the brain. this data call into question the utility of l1cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant 3d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed 3d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in 6-month-old wt mice, (n = 6/groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for 15% of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and nonamplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy5y cells with n-myc amplified sk-n-be2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. ps15.08 = op3.08 results: murine ctl evs were broadly divided into two populations that were eluted at low salt (l-s: 0.15 m-0.3 m nacl) and high salt (h-s: 0.3 m-0.5 m nacl) concentrations. l-s ctl evs were abundant in late endosome-related proteins, integrins, rabs, and effective mirnas, indicating exosome characteristics, and had biological activity for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations by intratumoral administration (see seo et al., nat. commun. 9: 435, 2018) . contrary, h-s ctl evs were rich in dna, core histones, ribosomal proteins, cytoskeleton proteins, and housekeeping proteins, considering microvesicles and apoptotic bodies, and easily phagocytosed by a kupffer cell line (kup5: kitani et al., results immunol. 4: 68-74. 2014 ). in addition, there were noticeable differences between ls and h-s ctl evs in the negative zeta potential width and membrane glycan structure. summary/conclusion: thus, ion exchange can be an optimal mass fractionation method for discriminating bioactive exosomes from cargos for nucleic acids in evs. funding: cryotem was conducted in nara institute of science and technology (naist), supported by nanotechnology platform program (synthesis of molecules and materials: 2019 #04) of the ministry of education, culture, sports, science and technology (mext). this work was supported by grants from the japan agency for medical research and development (translational research network program (nagoya univ. seeds a64)) and the japan science and technology agency (crest [jpmjcr17h2]). clic4 is essential for breast cancer metastatic competence and predicts disease outcome introduction: metastatic breast cancer is a consequence of complex interactions between cancer cells and the host. clic4, a member of a conserved gene family in the glutathione-s-transferase superfamily, mediates crosstalk between tumour and host in breast cancer. tcga and metabric data indicated that elevated clic4 expression was associated with breast cancers from young women, those with poor prognosis, and those with early stage metastatic disease. methods: since bulk tumour analysis does not distinguish between cancer and host stromal cells, we used genetic modifications of established syngeneic breast cancer mouse models to evaluate the contributions of clic4 in the host or tumour cells to develop metastases. results: experimentally, the essential clic4 host contributions for metastatic competence were related to circulating levels of pro-metastatic soluble factors, neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation, and leukocyte migration. clic4 was detected as cargo in circulating extracellular vesicles (evs) from breast cancer patients. similarly, circulating evs from tumour-bearing mice have abundant clic4 in comparison to those from mice bearing tumours that lack clic4. tumour cells released evs that induced myofibroblast conversion of wildtype but not clic4 ablated lung fibroblasts. summary/conclusion: these results illuminate clic4 expression as a prognostic marker for breast cancer patients, and experimentally, clic4 is a critical host factor for metastatic competence and potential target within host tissues for anti-metastatic therapy. funding: this work was supported by the intramural program of the national cancer institute under project zia bc 005445. the application of flow cytometry in an ev-based liquid biopsy for the detection of cancer multidrug resistance in myeloma gabriele de rubis a , krishna sunkara a , sabna rajeev krishnan and mary bebawy b a laboratory of cancer cell biology and therapeutics, discipline of pharmacy, graduate school of health, the university of technology sydney, australia, sydney, australia; b the university of technology sydney, sydney, australia introduction: multiple myeloma (mm) is an incurable cancer of bone-marrow plasma cells. it is characterized by unpredictable and highly variable therapeutic response and poor survival, attributed to the development of multidrug resistance (mdr) to chemotherapy. presently, no clinical procedures allow for a continuous, minimally invasive monitoring of mdr. we identified unique extracellular vesicle (ev) populations in the blood of myeloma patients, which serve as biomarkers of disease evolution and mdr to combination chemotherapy. we describe approaches used to optimise the use of flow cytometry (fcm) for ev summary/conclusion: although further investigation is required, our results potentially promise an effective and inexpensive priming agent (i.e., ethanol) for the production of anti-inflammatory msc-evs. this, combined with the significant increase in yield via 3d dynamic culture, presents practical solutions to both ev manufacturing scalability and potency issues. donor source affects potency of mesenchymal stem cell-derived extracellular vesicles introduction: mesenchymal stem cell (msc) therapies have been heavily investigated for their utility in applications such as wound healing and regenerative medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. recently, msc-derived extracellular vesicles (evs) have been implicated as primary effectors in msc-based therapies via protein and nucleic acid cargo transfer to patient cells. msc evs represent a superior alternative to msc-based therapies, as they lack the ability to replicate and are much smaller in size, circumventing related safety concerns such as immunogenicity, teratoma formation and blood vessel occlusion. however, a key drawback with msc therapies in general is their variable therapeutic potency, which is dependent on donor source. as a cell derived therapeutic, this crucial limitation is hypothesized to exist in msc evs as well. here, we demonstrate the varying bioactivities of isolated msc evs from differing donors and tissue sources. methods: six separate msc lines were obtained from different donors, with three msc lines derived from donor adipose tissue, and the other three from the bone marrow of separate individuals. evs were isolated from each msc line at passage 3 via differential centrifugation and ultrafiltration. these isolated msc evs were then characterized for size/concentration via nanoparticle tracking analysis, and ev markers (tsg101, alix, cd63) via western blot. pro-vascularization capacities of msc evs were determined by a gap closure assay using human umbilical cord vein endothelial cells (huvecs). results: characterization of msc evs revealed similar sizes and ev marker expression across donor groups, frontiers in chemistry, submitted funding: this work was funded by the momentum programme (lp2016-2), by the national competitiveness and excellence program catalan institution for research and advanced studies (icrea) proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding angel montero carcaboso g , andrea petretto b , franco locatelli a and angela di giannatale a a department of paediatric haematology/oncology and cell and gene therapy, irccs, ospedale pediatrico bambino gesù ps08: separation and concentration a laboratory of clinical biophysics, faculty of health sciences ps10: diverse ev biomarkers chair: pia siljander -faculty of biological and environmental sciences urinary evs were isolated using low vacuum filtration method followed by ultracentrifugation. raman spectra of urinary evs were recorded using a renishaw invia raman spectrometer. data analysis was performed using principal component analysis (pca) and hierarchical cluster analysis (hca). the size distribution and morphology of evs were analysed by transmission electron microscopy and nanoparticle tracking analysis methods. results: average raman spectra obtained for urinary evs from studied groups showed differences in intensities of specific bands in the region of 400-1800 cm-1. we found significant correlations between mean area under curve (auc) calculated for raman bands (phenylalanine, dna, proteins, lipids and amide i) and selected clinical parameters such as: egfr, serum creatinine, glucose, urine creatinine. chemometric methods showed spectral pattern responsible for separation between studied groups. nta measurements visualized evs with size of 204.7 ± 96.5 nm. summary/conclusion: our results showed that characteristic raman spectra of urinary evs are promising candidates for new, non-invasive biomarkers for dkd isolation of circulating extracellular vesicles and cfdna allows for erbb2 detection in a single aliquot of breast cancer patients plasma michela notarangelo a , mattia barbareschi b , antonella ferro c , orazio caffo c , vito d'agostino a and francesca demichelis d a department of cellular results: results: tissue-derived large and small evs showed difference in size (mean 142 nm vs 75 nm) when examined by em, whereas nta and exoview™ were unable to show a clear difference between the populations (nta: mean 125.7 nm vs 122 nm nta can only detected vesicles above 70 nm and exoview™ only measures vesicles between 50-200 nm. of the three different methods, em analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish ev subpopulations by size. funding: funding: swedish research council knut och alice wallenberg foundation imaging of human plasma-derived small-extracellular vesicles using transmission electron microscopy and structured illumination microscopy mitovesicles: a new extracellular vesicle of mitochondrial origin altered in ageing and neurodegeneration alldred b , chris goulbourne b , hediye erdjument-bromage d , monika pawlik b , mitsuo saito e , mariko saito f , stephen d. ginsberg b an in vitro and in vivo perspective on the role of erythrocyte-derived extracellular vesicles in parkinson's disease pathology frédéric calon c , éric boilard f and francesca cicchetti b a centre de recherche du chu de québec and faculté de médecine, département de psychiatrie & neurosciences département de microbiologie-infectiologie et d'immunologie evidences on microalgal extracellular vesicles: a morphological assessment antonella bongiovanni i , ales iglič j and veronika kralj-iglič j a laboratory of clinical biophysics, faculty of health sciences a faculty of dentistry, national university of singapore, singapore, singapore, singapore; b institute of medical biology, agency for science, technology and research, singapore, singapore, singapore; c exosome of cancer-associated fibroblast induce anti-cancer drug-resistance of nsclc so-young kim a and yeon-ju lee b a chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea; b chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea introduction: the understanding of interaction mechanisms between cancer cells and the tumour microenvironment (tme) is crucial for developing therapies that can arrest tumour progression and metastasis. cafs are the major constituent of the tme in many cancers. recent studies indicate that exosomes harbour the potential to regulate proliferation, survival and immune status in recipient cells. most of the current studies are focused on cancer cell secreted exosomes; and little is known about cafderived exosomes and their influence on cancer cells. methods: nsclc cell lines (pc9gr) and mrc5 (normal fibroblast cells) were grown in culture with exosome-free fbs. cutured media was filtrated by tangential flow filtration systems. exosomes in supernatant were isolated with the exoquick-tc™ system. considering the important role of cell extrinsic factors on cell growth and survival, we assessed whether factors contained in the mpa exosome could affect proliferation and survival of recipient cancer cells. cells were then treated with 1 μm osimertinib or pbs for 3 days prior to cell quantification of live cells.to investigate mechanisms of resistance to osimertinib mediated by ma or mpa-exosome in nsclc cell lines, we test cell viability by crystal violet assay in trametinib or osimertinib treated after pretreated ma or mpa-exosome, pc9gr during 6 days. we will investigate how mpa-exsomes activate erk signalling pathway in pc9gr cells to induce antitumor effects by western blot. results: mpa exosome increased proliferation of pc9gr cells by more than 50% compared to control pbs. pc9gr cells grown in mpa-exosome and subsequently treated with osimertinib showed a significant increase in cell survival compared to pc9gr cells grown in ma-exosome. osimertinib is used to treat egfr-mutant non-small cell lung cancer (nsclc) with tyrosine kinase inhibitor resistance mediated by the egfr t790 m mutation.these data show that "mrc5-pc9gr-crosstalk factors" affect proliferation and adaptive drug resistance of cancer cells. mpa-exosome mediates erk signalling activation and attenuated after treatment of 1um osimertinib. summary/conclusion: cafs support cancer growth and invasion. co-cultured nsclc with mrc5 lung fibroblast increased cell viability and exosomal mir-21 through the tgf-ß pathway in treatment osimertinib. exosomal mir-21 up-regulation in cocultured nsclc with mrc-5 induced drug resistance to drug-induced apoptosis. thus, exosomal mir-21 expression in co-cultured nsclc with mrc5 may support drug tolerance persister cells. introduction: neural stem cell (nsc) therapy has shown promise for brain repair after injury or disease mostly through bystander effects. nevertheless, the translation of nscs derived from human induced pluripotent stem cells (hipscs) to the clinic remain constrained due to safety issues, which include immunogenic risks, tumorigenesis potential, and incomplete differentiation. a way to avoid these issues is by using extracellular vesicles (evs) generated from nscs, as nsc-evs likely have similar neuroprotective properties as nscs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. however, this would require reliable purification and characterization processes, and testing of evs for composition and biological properties. methods: we generated evs from hipsc-derived nscs using a combination of ion-exchange chromatography (iex) and size-exclusion chromatography (sec) and investigated their composition through small rna sequencing and proteomics. we also performed in vitro and in vivo experiments to determine their biological and functional properties. results: iex and sec facilitated purification of hipsc-nsc evs nearly to homogeneity, which expressed ev markers such as cd9, cd63, cd81, and alix with a mean size of 145 nm. small rna sequencing revealed enrichment of mirnas related to different neuroprotective signalling pathways and diverse metabolic functions consistent with their role in cell-cell communication. the proteomic analysis identified >1,000 proteins, including ev markers and many other proteins involved in central nervous system function and cellular processes. the evs also displayed antiinflammatory activity in an in vitro mouse macrophage assay. intranasal (in) administration of nsc-evs resulted in their rapid incorporation by neurons, microglia, and astrocytes in virtually all regions of the brain. functionally, in administration of nsc-evs reduced inflammatory activity in the brain in a model of status epilepticus, and increased hippocampal neurogenesis in the adult brain. summary/conclusion: biologically active evs with antiinflammatory and neurogenic properties could be purified and harvested from hipsc-nscs. such evs also contain many mirnas and proteins that are of interest for brain repair after injury or disease. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) introduction: extracellular vesicles (evs) generated from human bone marrow-derived mesenchymal stem cells (hmscs) display anti-inflammatory and neuroprotective properties. our recent study has shown that intranasally (in) administered hmsc-evs incorporate into significant percentages of neurons and microglia in virtually all regions of the intact as well as the injured forebrain within 6 hours (kodali et al., int j mol sci, 2019) . in this study, using a rat model, we investigated the efficacy of a low dose of hmsc-evs administered intranasally for alleviating the abnormal plasticity of newly born neurons and the activation of microglia after se. methods: approximately 10 billion evs were dispensed bilaterally into both nostrils of young f344 rats that experienced two hours of kainate-induced se. animals were euthanized seven days after se, and brain tissue sections were processed for immunohistochemical staining of neun (a neuronal marker), dcx (a marker of newly born neurons), iba-1 (a microglial marker), and parvalbumin (pv) and reelin (markers of subclasses of interneurons). in addition, activated microglia were quantified using iba-1 and cd68 dual immunofluorescence. results: in administration of evs reduced the seinduced loss of pyramidal neurons in the hippocampal ca1 subfield. also, ev administration after se maintained higher levels of pv+ interneurons in the dentate gyrus. furthermore, ev treatment after se modulated abnormal neurogenesis, which was evidenced by a the role of small extracellular vesicles in chronic neuropathic pain zhucheng lin a , renee jean-toussaint b , yuzhen tian b , ahmet sacan a and seena ajit b a drexel university, philadelphia, usa; b drexel university college of medicine, philadelphia, usa introduction: chronic pain is the most prevalent, disabling, and expensive public health condition in the usa. exosomes are 30-150 nm extracellular vesicles that can transport rnas, proteins, and lipid mediators to recipient cells via circulation. exosomes can be beneficial or harmful depending on their source and contents. we hypothesized that the composition of small extracellular vesicles (sevs) can be altered following nerve injury and these alterations can provide insight into how the body responds to neuropathic pain. methods: to characterize changes following nerve injury, small extracellular vesicles (sevs) were purified by ultracentrifugation from mouse serum four weeks after spared nerve injury (sni) or sham surgery. mirna profiling and proteomics analysis using tandem mass spectrometry were performed to determine differential expression of mirnas and protein cargo respectively. for in vivo studies, sevs were administrated intrathecally into the mouse lumbar region. animals were evaluated for mechanical and thermal hypersensitivity over 21 days after injection. results: our mirna profiling showed a distinct mirna signature in sni model compared to sham control. proteomics analysis detected 274 gene products. of these, 24 were unique to sni model. neuropathic pain can induce the activation of the complement cascade and we observed significant upregulation of complement component 5a (c5a) in sevs from sni model. intercellular adhesion molecule 1 (icam-1), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in sevs from sni model compared to sham control. administration of sevs from sni model increased paw withdraw threshold in naïve recipient mice and inflammatory pain model, indicating a protective role for sevs in attenuating chronic pain. summary/conclusion: our preliminary studies suggest a critical role for sevs cargo in regulating pain. additional studies are ongoing to determine the functional significance of alterations in sevs composition using mouse models of pain. introduction: amyotrophic lateral sclerosis (als) is a progressive adult-onset neurodegenerative disease caused by selective motor neurons (mns) death. the rapid disease progression strongly suggests that cell-tocell spreading of noxious factors could take place in als pathogenesis. extracellular vesicles could potentially spread the disease. in this study, we characterized large (levs) and small extracellular vesicles (sevs) isolated from plasma of sporadic als patients and healthy controls and determined their different composition in order to understand their neuroprotective or neurotoxic role in als pathogenesis. methods: levs and sevs were isolated from plasma of 40 als patients and 30 healthy volunteers by differential centrifugation and characterized by nanosight ns300. cd45, cd31, cd61, cd235a and annexin v were used for flow cytometry. sod1, tdp43, fus protein level was investigated by western blot. for raman spectroscopy, evs were dried on top of a caf2 slide and raman spectra were acquired using a 633 nm laser line. mirna libraries were prepared by truseq small rna library kit (illumina). results: the mean size both for levs and for sevs resulted increased in als patients compared to controls. levs derived from als patients were enriched in sod-1, tdp-43 and fus proteins compared to ctrls. sevs showed a distinct spectral pattern from levs. in addition, levs of als patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. we also found a great presence of leukocyte derived levs (lmvs) in als patients compared to ad patients and healthy donors and significant correlation with the progression rate of the disease. on the other hand, mirna and rna whole transcriptome sequencing identified a specific signature of mirnas in plasma derived sevs of als patients compared to a group of healthy controls and three neurological groups of control. summary/conclusion: these data may suggest that levs derived from als patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of als disease, while sevs, deriving ps05.07 introduction: dendritic spines are actin-rich structures at the postsynaptic sites of most excitatory synapses in the central nervous system. they are highly important structures for higher brain functions such as learning and memory. several live imaging studies have shown that long, thin, actinrich protrusions called dendritic filopodia are precursors of dendritic spines in hippocampal and cortical neurons. so far, many intracellular factors that regulate filopodia formation have been identified. however, extracellular mechanisms of filopodia formation are largely unknown. also, detailed molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are not well understood. small extracellular vesicles (sevs)/exosomes have potential to regulate filopodia, spine and synapse formation in autocrine or paracrine manner due to their unique cargo composition. here, we examine role of exosomes in filopodia, spine and synapse formation. methods: primary rat hippocampal and cortical neurons were transiently transfected with the multivesicular body (mvb) docking regulator gfp-rab27b or with shrnas against the exosome secretion and biogenesis regulators rab27b and hrs. transfected neurons were immunostained for synaptic proteins and analysed for filopodia at day in vitro (div) 6 or spines at div12. for rescue experiments, exosomes were isolated using differential ultracentrifugation method from conditioned media of div9 cortical neurons or primary astrocytes and characterized for their size, common protein markers and morphology. results: here, we find that mvb docking factor gfp-rab27b localizes to both the tips and bases of actin-rich filopodia and spines in primary neurons. furthermore, genetic regulation of exosome secretion by overexpression or knockdown of rab27b or hrs leads to respective increases or decreases in the number of filopodia, spines and synapses. the defects of exosome-inhibited neurons in filopodia density are rescued by add-back of neuronal exosomes. additionally, treatment of primary neurons with exosomes isolated from primary astrocyte cultures leads to enhanced spine and synapse formation. summary/conclusion: these results indicate that autocrine and paracrine communication via exosomes are a key part of the process of neuronal filopodia, spine and synapse formation. effects of apolipoprotein e genotype on protein and small rna profiles of brain tissue-derived extracellular vesicles of alzheimer's disease patients introduction: multiple sclerosis (ms) is the most frequent chronic inflammatory disease of the young adult central nervous system. nevertheless, the pathogenesis remains largely unknown. it is therefore relevant to better characterise in cerebrospinal fluid (csf), which irrigates the brain, novel bioactive compounds whose dysregulation could be involved in ms pathology. the concentration of extracellular vesicles (evs) has been already found affected in ms patient fluids but the content in bioactive molecules, particularly the micrornas (mirnas), remains barely investigated. the mirna are short oligonucleotides that are major posttranscriptional regulators and we previously showed the dysregulation of specific mirnas in csf of ms patients. evs can potentiate mirna effects by allowing remote action through the shuttling within biological fluids such as csf while providing a protection from circulating rnase. nevertheless, csf remains a challenging fluid to analyse due to limited access, low volume and presence of lipoproteins (other putative mirna carrier) that can be co-isolated with evs. methods: we performed a comparative analysis of ev isolation from csf by size-exclusion chromatography (sec), density-gradient ultracentrifugation, ultrafiltration or chemical precipitation (chemp) to determine the optimal technique(s) to enrich ev. results: sec applied on csf of control patients showed optimal ev purification with sufficient evs from 0.5 ml of csf for downstream ev characterization. furthermore, we were able to isolate mirnas from csf and determined their enrichment in evs by rnase-sensitivity treatments. finally, we have combined chemp and sec to enable a fast and largescale isolation of evs from > 5 ml of csf, which successfully provided an increase in particles detected by nanoparticle tracking analysis. we are currently characterising the particles to confirm that they are purified evs, cleared from contaminants. summary/conclusion: this work opens perspective to analyse evs from ms patients and to determine whether mirnas participates in ms pathogenesis through their transit in evs. funding: fondation louvain, charcot foundation. differences in circulating number of extracellular vesicles between contact sport athletes with and without acute mtbi: a pilot study meghan rath a , jacqueline sayoc a , soo-young choi a , karlee burns b , aja corchado c , jane mcdevitt b , jingwie wu d , ryan tierney b , michael selzer e , xiaoxuan fan f and joon-young park a for bottom-up guc, increasing iodixanol gradients with 2.3 ml of samples were centrifuged at 186 k g for 18 h. fractions were then pooled based on densities (1.1-1.2 g/ml). bca and sds-page were used to analyse total protein; nanoparticle tracking (nta) and transmission electron microscopy (tem) for ev presence; and immunoblotting and imaging flow cytometry (ifcm) to evaluate ev specific markers. (ev-track id: ev190096). results: immunoblotting showed absence of actinin4 from all samples, while cd63 and tsg101 were detected for all samples; apart from imf_ip. nt_samples were not analysed reliably by nta and ifcm, due to the high concentration of casein micelles present (~10^14/ml in milk) that otherwise would be co-counted with evs. as expected, following ip, which most efficiently removed casein micelles, bca showed that samples had lowest total protein. this was confirmed by sds-page. thus, most effects were then focused on the ip casein-depleted samples. ifcm indicated that, post-guc, sm_ip evs had significantly (p < 0.05) more cd81-positive particles/ml of milk vs all other guc and 200 kduc samples. while there were no significant differences in sizes of ev separated from sm or imf, directly comparing the ip pre-treated samples, sm had significantly (p < 0.01) higher quantities of evs when compared to imf. additionally, tem indicated that evs separated from sm by guc were intact with limited background debris, whereas those separated from sm by duc and all imf evs were not. summary/conclusion: in conclusion, regardless of the method used, imf has fewer intact evs compared to sm. also, to obtain purest sm evs, ip followed by guc separation is optimal. introduction: extracellular vesicles (evs) exist as subpopulations with heterogeneous content. the surface heterogeneity of evs may reflect differences in functionality between ev subpopulations, as interactions with recipient cells may differ between ev subpopulations with different surface profiles. however, it is currently challenging to study functional differences between ev subpopulations due to the lack of suitable techniques to purify intact evs based on their surface signature. here, we showcase a novel capture-and-release platform to enrich intact ev subpopulations by their surface profile and compare their characteristics. methods: mda-mb-231 and skov-3 cell-derived evs were isolated using size exclusion chromatography. ev subpopulations were enriched based on surface markers cd9, cd63, cd81 or phosphatidylserine (ps) using a novel magnetic bead-based capture-and-release platform. obtained evs were characterized by transmission electron microscopy (tem), nanoparticle tracking analysis (nta) and western blotting. evs were fluorescently labelled using pkh67 and celltracker deep red (ctdr) and their uptake by recipient cells was examined using flow cytometry. results: western blot analysis showed that ev subpopulations enriched for the selected tetraspanins and ps were successfully isolated using a novel capture-andrelease platform. interestingly, evs isolated based on ps exposure (ps+) lacked most canonical ev markers. all ev subpopulations showed intact, cup-shaped morphology when analysed by tem, but contained less protein contaminants compared to the initial ev isolate. ps+ evs were slightly larger than other ev subpopulations when analysed by tem and nta. to test the capacity of ev subpopulations to interact with recipient cells, evs were labelled with pkh67 and ctdr prior to subpopulation fractionation. after fractionation, ps+ evs showed a significantly higher ctdr/pkh67 ratio than other ev subpopulations as determined by fluorescence spectroscopy, suggesting higher esterase activity of ps+ evs compared to other tested subpopulations. furthermore, mda-mb-231derived evs isolated based on cd9 and cd81 expression were taken up more efficiently by hmec-1 and mda-mb-231 cells than evs isolated based on presence of cd63 or ps. summary/conclusion: using a novel technology to isolate ev subpopulations based on their surface profile, we here show that composition and cellular uptake efficiency differs between ev subpopulations. theoretically, this technology is applicable to any surface marker of interest, allowing its use to further establish ev surface-functionality relationships and enrich evs with desirable characteristics for therapeutic purposes. funding: this work was supported by a veni grant (no. 17296) of the dutch research council (nwo). aml were harvested from tib49 cells cultured in evfree medium using serial ultracentrifugation. hspc (ksl; lin-sca1+ ckit+) clonogenicity and inflammatory responses were assessed using colony-forming unit (cfu) assay and real-time polymerase-chain reaction, respectively. ifn-alpha receptor 1 (ifnar1) expression and intracellular reactive oxygen species (ros) levels were assessed by flow cytometry. dna damage were assessed by quantifying nuclear γ-h2ax using immunofluorescent microscopy. results: similar to evs derived from aml patients, tib49 ev-aml elicited double-stranded breaks in hspcs, and actively suppressed hspc clonogenicity. transcriptional profiling revealed that exposure to ev-aml induced the upregulation of several inflammatory mediators in hspcs, including isg15, il-6, ifnα, ch25 h. inflammatory signalling triggered by ev-aml did not depend on ifnα signalling as evident from suppression of clonogenicity in ifnar1-null hspcs as well as the lack of evs-induced stat1 phosphorylation or ifnar1 downregulation. instead, we found increased levels of ros following ev-aml exposure. summary/conclusion: our findings support a model whereby ev-aml inflammatory signalling and oxidative stress lead to dna damage in hspcs. introduction: basic leucine zipper atf-like transcription factor 2 (batf2) is implicated in inflammatory response and anti-tumour effects. although the tumour suppressive function of batf2 has been reported, its extracellular role in maintaining a non-supportive cancer microenvironment has not been explored. methods: in this study, we established gbm orthotopic and subcutaneous tumour models in nude and balb/c mice and flow cytometry analysis determined the batf2 inhibitory effects of mdscs recruiting. we used transwell assay to determine batf2-positive evs (evs-batf2) inhibitory of the chemotaxis of myeloid-derived suppressor cells (mdscs) in vitro. in addition, exo-counter detection during the development of the gbm-batf2 model to demonstrate evs-batf2 crosstalk with distant tissues. amd3100 blocking in tumour model confirms that evs-batf2 dominated by the sdf-1a/cxcr4 signalling pathway. in addition, exo-counter detection of evs in 32 pairs of gliomas in different stages proposes plasma-evsbatf2 (plevs-batf2) as a prognostic marker. results: we found that tumour-derived evs-batf2 regulate crosstalk between glioma cells and tumour microenvironment by inhibiting mdscs recruitment. evs-batf2 can be detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence that glioma-derived evs can communicate with distant site by crossing blood-brain barrier. besides, evs-batf2 injection significantly reduced sdf-1α expression in the tumour tissues. after blocking sdf-1α signalling by amd3100, the inhibitory effects of batf2 overexpression on mdscs recruitment were rescued. evs-batf2 inhibit mdscs recruiting and secreting mmp2, mmp9, and vegfa which promote gbm progression. strikingly, exo-counter detection of evs in 32 pairs of gliomas in different stages reveals that the number of plevs-batf2 can distinguish stage iii-iv glioma from stage i-ii glioma and healthy donors. summary/conclusion: our results suggest that evs-batf2 may be an effective circulating biomarker associated with glioma progression. of note, we are the first to determine the regulatory role of evs-batf2 in regulating tumour microenvironment and propose plevs-batf2 as a prognostic marker predicting glioma progression and candidate target for gbm therapy. introduction: electrofluidics is an emerging technology of combining electronics and nanofluidics. one important device in electrofluidics is an ion transistor in which the ionic current through a nanopore is regulated by gate voltage bias. here, we suggest a fabrication method of nanopore by introducing focused ion beam (fib) and atomic layer deposition (ald) to sense extracellular vesicle (ev) via metal electrode structures. methods: we deposited 100 nm-thick silicon-nitrite layers on both sides of silicon wafer by low-pressure chemical vapour deposition (lpcvd). we fabricated rectangular patterns by photolithography followed by reactive ion etching (rie) on the backside of the wafer. anisotropic silicon etching by koh was performed. the front side of the chip was patterned by photolithography followed by ti/au deposition for the fabrication of electrode structures. we drilled 50~100 nm pores in the si3n4 membrane by fib. by the ald process, we deposited highly-conformal metal film, either platinum (pt) or ruthenium (ru) to shrink nanopores by a self limiting process. results: we expect that the ion current through the nanopore is efficiently controlled by the gate-surrounding structures. the nanopore ion transistor can be used to count the number of evs. summary/conclusion: we suggest a fabrication method of nanopore ion transistors by introducing focused ion beam (fib) and atomic layer deposition (ald). this device will be applicable for single ev sensing. introduction: extracellular vesicles (evs) are key players in cell-cell communication and increasing evidence has shown that evs function in cancer by promoting cancer cell motility and metastasis. analysing tumour-derived evs in biofluids is attractive because it would be a novel approach to a non-invasive liquid biopsy. unfortunately, evs are highly heterogeneous. they vary greatly in size, lipid composition, and cargo and are difficult to distinguish from other small particles in complex biofluids. we have developed a novel flow cytometry method to generate a distinct ev fingerprint to profile biological specimens. methods: evs from cell culture media (purified and unpurified) and biological fluids (plasma and urine) were detected by flow cytometry using features on individual evs produced by intrinsic (cd63-phluorin) and extrinsic (lipophilic dye, di-8-anepps, and antibodies) fluorescent labels. ev subpopulations were visualized with dimensional reduction (t-sne and umap) of 20-150 features that defined the vesicle size, shape, and fluorescent emission spectra associated with the fluorescent marker. unsupervised density based clustering (hdbscan) in conjunction with supervised machine learning (xgboost) was subsequently used to define subpopulations. we refer to this method as "ev fingerprinting". results: ev fingerprinting was successfully used to detect evs in complex biological specimens and trace their differential enrichment through conventional purification methods. evs were readily distinguished from protein complexes, lipoproteins and non-lipid particles. calibration with externally validated purified ev, as well as size, lipid, and fluorescence standards enabled ev fingerprinting as a rigorous and reproducible method for resolving heterogenous ev samples. ev fingerprinting applied to conditioned medium from tumour cells and biological fluids from cancer patients reveals unique ev profiles generated by cancer, further supporting the potential of ev fingerprinting as a liquid biopsy. summary/conclusion: our single-ev analysis approach characterizes whole ev populations in complex biological fluids without the need for purification, reducing time intensive purification protocols and subsequent sample loss, permitting efficient analysis of liquid biopsy samples. detection and quantification of extracellular vesicles with cargo protein and rna using the amnis® cellstream® flow cytometer introduction: the particle size distribution (psd) of extracellular vesicles (evs) is commonly measured by tunable resistive pulse sensing (trps) and nanoparticle tracking analysis (nta). both trps and nta have limitations that hamper the accurate measurement of the psd of evs, specifically in the size range from 50 to 100 nm. an alternative technique for measuring the psd of evs is micro-fluidic resistive pulse sensing (mrps). because a standard operating procedure (sop) for characterizing evs by mrps is absent, we aim to establish a reliable sop to ensure reproducible psd measurements of evs by mrps. methods: measurements (n = 3) of red-blood cell, prostate cancer cell line supernatant, and human urine and plasma evs were acquired in 10 × 10 s acquisitions. two microfluidic cartridges were used to study a dynamic range of 50-450 nm. samples were diluted into phosphate buffered saline with different concentrations of tween 20 or bsa. because the excess of particles affects the detection limit, serial dilutions were performed to find the optimal dilution for each sample. data were evaluated using data viewer software. results: the optimal dilution was determined for each sample by maximizing the particle rate and minimizing the measurement time while preserving a robust detection limit of 50 or 65 nm. moreover, we developed a procedure to optimize the peak filter settings of data viewer by fitting data to normal distributions and identifying threshold values for signal-to-noise ratio, symmetry, and transit time within 99% confidence. summary/conclusion: we recommend to use 0.1% w/ v bsa in dpbs as sample diluent, because tween 20 affects evs as confirmed by flow cytometry. by using orthogonal techniques and well-characterized biological test samples, we developed and validated a sop for ev detection by mrps, thereby making mrps a valuable tool for ev researchers. real-time measurements of extracellular vesicles binding kinetics achieved through interferometric imaging in a multiplexed microarray modality introduction: extracellular vesicles are very promising diagnostic biomarkers. as a matter of fact, the properties of these biological nanoparticles depend on the health conditions of each individual. however, experiments that involve evs phenotyping are time consuming, due to 12 h-or overnight incubations. in order to get accurate results, maximizing binding efficiency is a necessity; that normally involves ensuring the saturation of the capture reaction, which can result in an unnecessarily long incubation time. with the ability of labelfree kinetic binding measurements using interferometric reflectance sensing in a microfluidic chamber, we perform an optimization of the incubation time in different flow conditions, while demonstrating a new way of multiplexing for real-time evs specific capture and detection.methods: all the real-time binding measurements were performed with the interferometric reflectance imaging sensor (iris). iris chips were first coated with an organic polymer (mcp-2), which provides an active surface for probe immobilization. then, antibodies against cd9, cd81, cd63 markers were spotted at different densities in a microarray modality. the chips were then encapsulated with a glass window to form a microfluidic chamber that allows for imaging the sensor surface. samples of hek-derived extracellular vesicles were flowed across the sensor surface in the iris system and real-time images were acquired. incubation was performed at different flow rates, and in static and stopflow modalities. results: in this work, we focus on the specific capture of evs under different flow conditions to achieve an optimization of the incubation time. indeed, through the acquisition of real time binding data, we are able to precisely monitor the equilibrium point of the capture reaction. in this configuration of iris, low magnification optics allow for simultaneous detection of binding on hundreds of capture ligand spots. therefore, surface probes (surface density and specificity) as well as assay conditions can be optimized. we report on the optimization of antibodies against cd9, cd81, and cd63 markers. since the sensor chips are identical to the single-particle detection assays developed by nanoview biosciences, the optimization of binding assays will directly impact the phenotyping of individual exosomes. summary/conclusion: our method proved to be very efficient in optimizing the most crucial aspects concerning evs captureflow conditions, incubation time, surface density and sample concentration. introduction: diabetes is a life treating diseases extending its impairing influence on more than 500 billion of people around the world within upcoming 20 years. the most harmful complication generating high treatment and social costs is diabetic nephropathy, which develops in about 10% of patients suffering diabetes. still we do not have an effective and direct prognostic biomarker to diagnose renal complications in the primary stage of renal disease. methods: extracellular vesicles were concentrated from diabetic patients' urine and washed to perform spectral analysis: fourier transform infrared spectroscopy (ftir), based on the molecular absorption of electromagnetic radiation in the infrared region of the spectrum in a range from 400 cm-1 to 4000 cm-1 and raman spectroscopy (rs) as a technique based on inelastic scattering of monochromatic light. both techniques provide information on the chemical structure of compounds by identifying functional groups with high molecular specificity. results: average spectral signature obtained for evs from urine samples of patients in the different stage of kidney damage allowed distinguishing specific bands, representative for amide (i/ii), lipids, cholesterol and nucleic acids. spectral parameters correlated with a clinical stage and a commonly used indicator of renal function (creatinine) in diabetic patients. summary/conclusion: infrared and raman spectroscopy are promising tools to diagnose and monitor renal function in diabetes. introduction: several existing bioanalytical strategies for purifying and characterizing exosomes have allowed for fundamental progress to be made. mixtures of evs can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion chromatography. but, these processes require large amounts of material that are often difficult to obtain and many different types of particles have similar sizes and densities. it is likely that unique subfractions within enriched samples exist, particularly in complex biological matrices such as blood, urine or milk which remain difficult to characterize and isolate with existing analytical technologies. methods: bovine milk exosomes were isolated via differential ultracentrifugation and resolubilized in 100 mm ammonium acetate. these data were recorded using charge detection mass spectrometry (cdms). in cdms, individual particles are reflected back and forth through an electrostatic ion trap where they pass through a sensitive charge detector. each time a trapped particle enters and exits the detector, its charge (z) and mass-to-charge (m/z) ratio is measured. mass distributions are generated by multiplying the m/z values by the charge measured for each ion and binning the resulting masses.results: the masses of particles in a bovine milk extracellular vesicle (ev) preparation enriched for exosomes were directly determined for the first time by cdms. particle masses and charges span a wide range from m~2 to~90 mda and z~50 to~1300 e and are highly dependent upon the conditions used to extract and isolate the evs. in total, 57,350 particles were detected from eight cdms measurements. a simple two-dimensional gaussian model suggests that eight unique subpopulations of particles may be resolvable based on charge and mass. complementary em and proteomics analyses confirm that samples are enriched for exosomes. particles associated with the s1, s2, and s3 families that are centred at~3.5,~5.9, and~8.3 mda, respectively, appear too small to be ascribed to exosomes. the remaining 45,229 (79%) particles detected by cdms are within the mass range expected for exosomes. while cdms measurements are at an early stage of development, this approach appears to provide a new physical basis for separating and characterizing ev particles. summary/conclusion: this work describes a novel biophysical approach for measuring and characterizing the masses and charges of the extremely heterogenous population of exosomes and other extracellular particles enriched in bovine milk. as new sample preparation methods, aimed at purifying specific types of exosomes from different cell lines, tissues, and other body fluids continue to evolve, rapid and sensitive cdms measurements of the physical properties of mass and charge may become an important means of assessing the efficacy of different protocols. funding: nih (r01 gm131100-01). bab is supported by indiana university quantitative chemical biology fellowship (t32gm131994). in situ detection of exosomal microrna-10b by fusion with liposomeencapsulated nanomotor introduction: breast cancer is the most common cause of cancer-associated death in women and has raised global health concerns. early diagnosis and treatment are crucial to improve the prognosis and survival rate of breast cancer patients. liquid biopsy is expected to provide a strategy for early diagnosis of breast cancer. exosomes have been regarded as novel liquid biopsy biomarkers due to their stable cargo of rnas, lipids, and proteins from their origin cells. exosomal micro (mi)rnas have recently been recognized as promising indicators of cancer occurrence and progression. however, most of the reported exosomal mirna detection methods require the lysis or extraction process, which increases the possibility of sample loss. in situ detection strategies avoid interference from body fluid. in this study, we developed a gold nanomotor fluorescence platform based on liposome fusion for breast cancer exosomal mirna in situ detection. the exosomal mirna detection platform was constructed using a gold nanomotor (detector) and liposomes (carrier). the dnazyme amplification sequences which could be especially triggered by mirna-10b were identified by sds-page before modified on gold nano-motor and the capacity of the nanomotor was assessed using synthetic target sequence, breast cancer cell mda-mb-231, mirna-10b-encapsulated anionic liposomes, and mirna-10b-expressing exosomes. three kinds of liposomes were synthesized, characterized, and assessed for loading ability. membrane fusion effect was evaluated by confocal laser scanning microscopy (clsm) and nanoflow cytometry. the performance of this method to discriminate between breast cancer patients and healthy individuals was investigated. results: the chosen dnazyme amplification sequences transformed "locked" status to "cleavable" status on target addition, releasing a fluorescence signal. the modified gold nanomotor showed a ten times higher fluorescence signal in the presence of mirna-10b than the background and no noticeable fluorescence changes from a single-base-mismatch sequence. moreover, among the three different liposomes, cationic liposomes exhibited great stability, high loading efficiency, and excellent membrane fusion effect. furthermore, the fluorescent experiments confirmed that cationic liposomes could load and transfer the nanomotors into exosomes for mirna-10b detection. finally, we were able to distinguish breast cancer patients and healthy individuals by sensing exosomal mirna-10b directly from plasma samples without exosome isolation. summary/conclusion: a separation-free and sensitive assay based on dnazyme amplification technique and membrane fusion effect was established for breast cancer-derived exosomal mirna-10b detection, which could be a promising tool for the liquid biopsy of breast cancer. isolation of exosomes by membrane affinity column increases non-exosomal rna recovery in comparison to differential ultracentrifugation introduction: exosome-based liquid biopsy is a potential aid in the diagnosis and prognosis of cancer patients. however, in order to incorporate exosomes into clinical routine, there is a need to compare different isolation methods. here we analysed the impact, in exosomal rna yield, of two intermediate recovery/ intermediate specificity methods: differential ultracentrifugation (ucd) and a membrane-affinity column (mac) kit. although mac has a faster performance which is more suitable to the clinic, we found that ucd results in a higher recovery of exosomes and less contaminating non-exosomal rna.methods: exosomes were enriched by mac and ucd from identical volumes of human plasma (12,000xg, 34 min/0.22 ) m filtration/110,000xg, 5 h)(n = 7) and lymphoma conditioned medium(300xg, 10 min/200xg, 20 min/1000xg, 30 min/120,000xg 1.5 h/120,000xg, 1 h) (n = 3). all exosomes were characterized by nanoparticle tracking analysis (nta), immunoblotting of cd63/cd9/flotilin/alix and electron microscopy (tem). exosome pellets were pre-treated with proteinase k (1 mg/ml/56°c/10 min) and rnase a (2 mg/ ml/37°c/20 min) before phenol-chloroform/glycogen rna extraction. rna yield was measured by both fluorometer and bioanalyzer.results: isolation of exosomes by ucd, in both plasma and medium, resulted in a higher yield in comparison to mac. this was shown by an augmented intensity of marker bands in the ucd samples (p = 0.005, n = 4) as well as by an increased number of exosomes in tem.in contrast, mac final exosomal fraction (from both plasma and medium), resulted in a 13-fold and 200fold increase in rna, respectively, in comparison to ucd when measured by fluorometer. this was confirmed by bioanalyzer. introduction: there is a need for better techniques for characterizing ev populations. we developed a sensitive multiplexed electrochemiluminescence (ecl)based assay format to characterize evs in cell-conditioned medium (ccm) and human biofluids. here we use the format to analyse ev samples for the presence of 66 ev surface proteins, and to identify changes in ev phenotype associated with different cell lines, purification methods and growth conditions. methods: multiplex plates were prepared on msd's u-plex® platform with antibodies for 66 putative evsurface proteins. each well displayed an array of nine specific capture antibodies and a negative control antibody. evs from samples were captured on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (cd9, cd63 and cd81) conjugated to an ecl label. three distinct cell types were grown at two sites, msd and atcc. resulting ccm were each purified by four common methods: tangential flow filtration, peg-based precipitation, size-exclusion chromatography and centrifugal ultrafiltration. all samples were also assayed without purification.results: fifty-five of the surface markers were detected on intact evs from at least one evaluated cell type. datasets were analysed using correlation matrices, hierarchical clustering, and machine learning. for each cell type, when comparing unpurified ccm grown at different sites or evs prepared by different purification methods, we typically observed correlations above 0.9, indicating that the purification methods did not introduce bias to ev phenotypes, and that the assay format can provide robust phenotypic information without any purification of evs. two unsupervised clustering analyseshierarchical clustering and t-distributed stochastic neighbour embeddingboth generated wellseparated clusters for each of the cell types, regardless of purification method or source. summary/conclusion: we developed multiplex ev surface marker assays and demonstrated their use for multimarker ev phenotyping. this flexible format enables rapid assay development for new ev subpopulations with or without sample purification. these results also demonstrate ev surface marker phenotyping via multiplex ecl assays may be used to distinguish ev populations from various cell types, and characterize bias introduced by purification. detection of misev recommended ev protein-markers using automated western blotting method for isolation of evs and a simple western blotting platform for automated protein separation and immunodetection of misev-recommended proteins.methods: total evs were isolated by affinity-membrane spin columns from pre-filtered 0.5-4 ml plasma or 2-20 ml urine, respectively. intact vesicles were eluted and the ev-depleted biofluid fraction was collected from the flow-through. a small fraction (4 μl) was analysed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of misevrecommended proteins.results: a range of specific antibodies were identified and the ev fractions were shown to be enriched in evproteins, whereas contaminating non-ev proteins were significantly reduced. isolation of evs was necessary to allow detection of the low abundant ev protein markers, whereas non-ev proteins were readily detectable both in the neat biofluids and in the ev-depleted flowthrough. we characterized the effect of washing on the purity of ev isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine ev isolations. summary/conclusion: simple western blotting protocols were established for quality control of isolated evs in accordance with misev-guidelines. evs isolated using affinity-membrane spin columns were shown to be enriched in ev markers and depleted for non-ev proteins. al-pha beads: a library of extracellular vesicle-associated metalloproteinase biosensors (adams) and a disintegrin and metalloproteinase with thrombospondin motifs (adamtss) are highly promising cancer biomarker candidates that have complex roles in cancer pathogenesis and metastasis. importantly, within the context of lung cancer, the detection of adam10 proteolytic activity might be more informative than the level of adam10 protein.therefore, the development of low-cost metalloproteinase biosensors could serve as useful biomarker research tools. methods: to this end, we developed advanced proteolytic detector polyhydroxyalkanoates (al-pha) beadsa library of biodegradable, biopolymer-based protease biosensors. broadly, these biosensors utilise phac-reporter fusion proteins that are bound to microbially manufactured bioplastic beads. these phac-fusions also incorporate specific protease cleavage sites. in the presence of a specific protease, reporter proteins are cleaved off of the al-pha beadsresulting in a loss of bead fluorescence that can be measured using flow cytometry. these biosensors were assayed using either metalloproteinases, conditioned media or evs from in vitro cancer models.results: human metalloproteinase recognition motifs were identified in the literature and a total of 70 different al-pha bead biosensors were designed. a control, tev-specific biosensor detected 0. introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr.results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom20. mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the 374 isev2020 abstract book biogenesis of evs is neutral sphingomyelinase 2 (nsmase2), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase2 is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase2 inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles.methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo [1,2-b] pyridazin-8-yl) pyrrolidin-3-yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated 5xfad mice with 10 mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay.results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, 5xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting 20% of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human postmortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of 20 mdd subjects and 20 hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed.results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd9. tem images showed the typical cup-shaped morphology with sizes mostly between 30 and 200 nm. preliminary sequencing results revealed that mir-33a-5p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir-33a-5p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. methods: we use ifc to characterize evs released by glioma using 5-ala, fluorescently labelled ev (cfda-se, cd81) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd81) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that 58% are tenascin c positive, 2.7% are egfrviii positive and 1.6% are 5-ala positive. there was only a minor overlap (<16%) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs (2.5-fold), tumour specific evs (2.8-fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll 2 receptor (tlr2) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il-12 and il-6, which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells.objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow field-flow fractionation (af4) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp-1) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk-2 epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with 50 to 50 nm (ev1) and another with 100 to 120 nm (ev2). ev1 induced more tnf-alpha, il-6, ip-10 and ccl20 than ev2. it was also more effective in promoting t. cruzi infection in epithelial cells. due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays.noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr.isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and 0.2um filtration. co-purification of norovirus with the evs was checked.ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid50 assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems.ps15.12 = op3.12 kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of 11.5 µg/ml of plasma, as geometric means ≥11.5 µg/ml were nearly equal. hevs were detected at 48 µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f200x electron microscope and measured between 40-90 nm, and hevs were between 60-160 nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: nasopharyngeal carcinoma (npc) is characterized by a large presence of regulatory t cells (tregs) and the production of tumour-derived exosomes with immunosuppressive properties. our team showed that npc-derived exosomes favour the suppressive activity and recruitment of human tregs via ccl20 chemokine, thus contributing to npc immune escape (mrizak et al., jnci, 2015) . more recently, our team has shown that npc-exosomes could induce tregs by altering the maturation of dendritic cells (dcs) and promoting tolerogenic dendritic cells (tdcs) (renaud et al., herpas congress 2017). our main objectives in this study are (i) to define and compare the metabolic status of mature dendritic cells (mdcs), control tdcs and tdcs generated in the presence of npc-exosomes (exocnptdc) and (ii) to evaluate the chemoattractive potential of npc-exosomes on exocnptdcs, and notably to investigate the involvement of ccl20 in this recruitment. methods: dcs are generated from human monocytes in the presence or absence of npc-exosomes. the maturation status of dcs was evaluated at a phenotypic level by studying the expression of maturation markers using flow cytometry and at a functional level by analysing cytokines secretion using elisa. this cytokine analyse has been performed in both conditions, on treated dcs and during co-culture assays of autologous cd3 t lymphocytes with treated dcs. in a second step, a mitochondrial metabolic and glycolytic study was performed using the seahorse technology (ocr and ecar measurement). finally, the chemoattractive potential of npc-derived exosomes on the different induced dcs was analysed (i) using boyden chamber chemoattraction assays or real-time videomicroscopy (chemotaxis µslide ibidi) and (ii) using rt-qpcr analysis of the receptor expression of ccl20 (ccr6).results: npc-exosomes alter dc maturation, which gives rise to tolerogenic dcs that favour the induction of tregs. in addition, the metabolic analysis of dcs seems to put foward a specific metabolic signature of the tdcs induced by npc-exosomes. and finally, chemoattraction assay suggests that npc-exosomes preferentially attract tdcs and exocnptdcs in a ccl20dependant manner. summary/conclusion: taken together our results should allow us to characterize the major role of npc tumour exosomes on the maturation and the recruitment of dc and so identify them as anti-tumoural therapeutic targets. cytotoxic t lymphocyte ev that prevents tumour metastasis by collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle or apoptotic body.naohiro seo a , junko nakamura a , tsuguhiro kaneda a , takanori ichiki b , asako shimoda c , kazunari akiyoshi c and hiroshi shiku a a mie university graduate school of medicine, mie, japan; b the university of tokyo, bunkyo, japan; c kyoto university, kyoto, japanintroduction: recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (evs). in this study, we propose a novel method for large scale preparation of highperformance extracellular vesicles focusing on membrane negative charge. methods: murine cytotoxic t lymphocyte (ctl) evs in supernatant were concentrated more than 20 times at over 97% purity without leaking by 750 kda mwco ultrafiltration, and subjected to ion exchange deae column chromatography after replacing with pbs. after ion exchange, evs were characterized by bca assay, nta assay, cryotem observation, proteome analysis, dna content measurement, mirna microarray analysis, zeta potential measurement, lectin array analysis, and target cell analysis.biomarker detection and analysis and detail strategies for cross-platform analytical validation. methods: we conducted a cross-platform analysis using two commercially available flow cytometers designed for ev detection. scatter resolution, enumeration accuracy and precision were determined across both platforms by analysing submicron silica beads (apogeemix, 180-1300 nm) of known concentration.we detected large evs, as established by reference size beads, electron microscopy, expression of phosphatidylserine and the presence of integral membrane proteins of cell of origin. we analysed evs isolated from plasma by high-speed centrifugation (18,900 g) as well performing analysis by direct plasma labelling followed by validation by detergent lysis of vesicular constituents. a clinical operating range was defined which ensures linearity and avoids swarm detection. we observed comparable scatter resolution, enumeration accuracy (error ≤15%) and precision (cv ≤5%) across both platforms used. we defined two ev size gates: a "latex" gate (300 to 1100 nm polystyrene latex beads), and a "silica" gate (180 to 1300 nm silica beads) for evs at the lower end of our size range of interest. to improve detection sensitivity, we identified common contributors to signal noise and applied workflow strategies to minimize these. finally, we identified linear ranges which avoid swarm detection, and which ensures reproducible ev counts (cv <20%) across both instruments. summary/conclusion: we present an optimised, standardised and cross-platform reproducible working protocol which supports the use of fcm in an ev-based liquid biopsy application. funding: the project is funded by spark oceania and uts innovation commercialisation seed fund scheme to mb. metabolomic profiling of serum and exosomes isolated from head and neck cancer patients after radiotherapy introduction: cancer radiotherapy (rt) induces the response of the whole body that could be detected at the blood level. searching for new molecular signatures which could correlate with treatment response in cancer patients is of particular importance. radiation-induced changes in proteome and transcriptome of serum have been widely described. however, metabolomic changes in serum, exosomes and other classes of small extracellular vesicles (ev) of cancer patients after rt have not been given as much attention. metabolomics of serum and ev of cancer patients could provide a valuable insight into the response of both tumour and whole organism to the treatment. the aim of the study was to compare serum and ev metabolomic profiles in head and neck cancer (hnc) patients before and after rt. methods: serum samples from 10 hnc patients were taken before (a) and after (b) rt. 10 healthy volunteers were used as a control group (c). ev were isolated from 1 ml of serum using size-exclusion chromatography (sec). selected sec fractions were subjected to extraction of metabolites. a mixture of meoh/h2o was used for extraction of metabolites from serum and ev samples. samples were analysed by gas chromatography-mass spectrometry (gc-ms).the study protocol adhered to the tenets of the declaration of helsinki and was approved by the bioethical committee of the maria skłodowska-curie national research institute of oncology, branch gliwice, poland (permit nr. do/dgp/493/4/06/1/2016/g). results: an untargeted gc-ms-based approach allowed the detection of 189 metabolites in serum samples and 50 exosomal small molecules, of which 32 joint. the identified compounds included amino acids, fatty acids, carboxylic acids, sugars, and others. there were 49 metabolites which levels discriminated compared groups (a,b,c) of serum samples and 12 compounds that discriminate the ev isolated from hnc serum before and after rt from hc. summary/conclusion: rt caused significant changes in levels of serum and ev metabolites witch are involved in amino acid metabolism, lipids metabolism, energy metabolism and oxidative stress response. capable of contributing to intercellular communication and metastasis. numerous studies have focused on elucidating their role in cancer progression. we recently showed that sevs isolated from pancreatic cancer cells can function as an initiator in malignant cell transformation. here, using a mass spectrometry (ms)-based proteomics approach, we analysed the differences in the protein cargo of sevs secreted from normal pancreatic and cancer cells to better understand their biological characteristics. methods: sevs were isolated from 3 human pancreatic cancer cell lines (capan-2, mia paca-2, and panc-1) and normal pancreatic epithelial cells (hpde) using a combined ultrafiltration-ultracentrifugation method coupled with a sucrose density gradient purification. proteomic profiling of sevs was carried out using an lc-ms/ms method. protein identification from resulting ms/ms spectra was conducted using proteome database search software followed by gene ontology (go) enrichment and reactome pathway analysis.results: a total of 4,907 unique proteins were identified confidently across the combined samples. the proteins present in all four sev types (1,135 proteins) consist of general housekeeping proteins. 348 proteins were uniquely found in all cancer sevs but not in the normal hpde sevs. this group contains an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion and suggest their important role in driving the increased production of sevs from cancer cells relative to normal cells. moreover, this group includes a set of proteins that have been implicated in malignant cell transformation, consistent with our previous work showing that each of the cancer sevs analysed here could initiate malignant transformation of nih/3 t3 cells. conversely, there were 313 proteins uniquely found in normal hpde sevs. this group includes a number of immune response proteins that are not found in any of the pancreatic cancer cell sevs. summary/conclusion: the differences in the proteomes of cancer and normal sevs may be indicative of their varying roles in cell transformation and helpful in delineating the types of evs that are being produced. in addition, these differences point towards their potential value as cancer biomarkers. proteomic profile of tumour-derived exosomes in plasma of melanoma patients introduction: in the past years, extracellular vesicles (evs) have attracted considerable interest due to their ability to provide valuable diagnostic information from liquid biopsies. the high abundance in all bodily fluids and their cargo stability confers evs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible tissues, therapy response and monitoring, but also to reduce infection risks of conventional highly invasive biopsies. virtually all cells continuously release vesicles into the extracellular environment, diverse in size, content and features depending on the biogenesis, origin and function. this heterogeneity adds a layer of complexity when attempting to isolate and characterize tissue-specific vesicles. methods: hence, we aimed to use a immunomagnetic capture approach for prostate-derived evs from cell culture supernatants, with further investigation into human plasma and urine samples. analysis was performed by nanoparticle tracking analysis, western blotting and electron microscopy. additionally, an in-house spotted antibody microarray is in development. here, we intend to detect different ev sub-populations based on their surface markers. results: isolated immunocaptured ev populations based on the classical ev marker cd9 show an increased signal for the luminal protein tsg101. ev populations targeting the tissue-specific marker prostate specific membrane antigen (psma), were found positive for tsg101 in a lower extent indicating a subpopulation of evs. the microarray uses less than 100 µl of sample (concentrated cell culture supernatant, human plasma, urine) and leads to a faster characterization within 3 h for ev surface marker as compared to western blot. summary/conclusion: immunomagnetic isolation might be a promising approach for liquid biopsy and thereby the microarray could be valuable to identify potential capture targets. the current design for 6 different surface marker from 16 samples simultaneously could be easily extended for sample size and surface profiling allowing for a more economical way to multiplex samples. paving the way for implementing a feasible and reliable technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer in clinical practice introduction: extracellular vesicles (ev) in urine have been proposed as biomarkers for bladder cancer (bc). however, at present there are no standardized methods for ev isolation or urine sampling. our goal was to evaluate the ev isolation performance between different methods, the effect of the sampling time and the importance of urinary creatinine (ucr) normalization. methods: two urine samples of 120 ml were collected from 5 patients with non muscle-invasive bc: one from the first micturition and another from any time of the day. twenty ml were used for ucr measurement and 100 ml were used for ev isolation by either precipitation with polyethylene glycol (peg), concentration by filtration (uf, centricon plus-70, 10 k, millipore), sepharose size exclusion column (sec), or combinations of these methods. additionally, the effect of protease inhibitors (pi) and dtt treatment after collection or during processing was analysed. size and number of particles were evaluated by nanosight and the presence of exosomal markers was evaluated by western blot. results: among the methods evaluated, uf + sec showed the best performance retrieving the highest number of particles in the range of 50-200 nm, and the highest protein expression of exosomal proteins. uf alone showed the highest concentration of ev, but with a tendency to isolate larger particles. particle concentration was positively correlated with ucr, reflecting the importance of ucr normalization before journal of extracellular vesicles 385 comparing between patients. finally, no differences in the performance according to the time of collection, nor in the use of pi or dtt were observed. summary/conclusion: uf + sec gave the highest ev yield and was not affected by the time of urine collection. the use of pi and dtt can be avoided, and normalization to ucr should be considered when implementing this technique for assessing evs as biomarkers for bc in clinical practice. funding: pida 2019. the introduction: human tumours, including pancreatic ductal adenocarcinoma (pdac), often harbour a subpopulation of cancer cells with extra centrosomes. we found, that these cells secrete an increased number of small extracellular vesicles (sevs), within the 20-120 nm size range. sevs play a role in cancer signalling and progression and are widely studied for their diagnostic potential. we aim to understand the role of sevs secreted by cells with extra centrosomes in shaping pdac-associated stroma, particularly fibrosis. methods: to study the sev mediated changes in the pdac microenvironment, we purified sevs through serial ultracentrifugation and size exclusion chromatography, characterised the content through silac-based proteomics, and assessed phenotypic changes in pancreatic stellate cells (pscs) and extracellular matrix (ecm) production through immunofluorescence staining. results: our data indicates, that the sevs secreted by cells with extra centrosomes are exosomes due to their endocytic origin, and we found, that they can activate pscs, key mediators of fibrosis in pdac. indeed, we observed an increased level of collagen i produced by pscs activated by sevs from cells with extra centrosomes as compared to cells without extra centrosomes. interestingly, we found, that psc activation through sevs is not mediated by tgf-β, assessed by the level of nuclear smad2 accumulation downstream of tgfβ activation, suggesting a novel mechanism of pscs activation. summary/conclusion: pdac cells with extra centrosomes contribute to a novel type of psc reprogramming, which could alter their ecm deposition and contribute to the extensive fibrosis observed in pdac. we are currently characterising the signalling pathways associated with sev mediated psc activation and how it impacts padc progression to better understand the role of centrosome amplification in the cancer-stromal crosstalk. exosomal carboxypeptidase e confers and cpe-shrna loaded exosomes inhibit growth and invasion of hepatocellular carcinoma cells. methods: exosomes were isolated from the culture media of high metastic hcc97 h cells and incubated with low metastatic hcc97 l cells. in other experiments, cpe-shna loaded exosomes from hek293cells were incubated with hcc97 h cells. the recipient cells were analysed for proliferation using mtt assay, colony formation, and matrigel invasion. results: analysis of exosomes derived from hcc97 h cells revealed cpe-wt mrna and protein. exosomes released from hcc97 h cells were able to enhance proliferation and invasion of hcc97 l cells. when cpe expression was suppressed in the hcc97 h cells before exosome isolation, the exosomes had no effect on proliferation and invasion. these data demonstrate the ability of exosomes to confer growth and invasion in hcc cells and the role of exosomal cpe in driving the process.previously it was shown that down-regulation of cpe expression by shrna can reverse tumour growth and metastasis in an hcc mouse model. we therefore loaded cpe-shrna into exosomes by infecting hek293 (human embryonic kidney) cells with adenovirus carrying cpe-shrna-gfp. these modified 386 isev2020 abstract book exosomes were used to transfer cpe-shrna to hcc97 h cells, resulting in significant reduction in proliferation and colony-forming ability of these cells. cpe-shrna loaded exosomes were found to down-regulate the expression of cyclin d1 and c-myc, two genes with high relavance to tumour growth and metastasis. summary/conclusion: our results demonstrate the ability of exosomal cpe to enhance proliferation and invasion in low metastatic hcc cells and the potential to use shrna loaded exosomes to target cpe as a therapeutic strategy to treat liver cancer.funding: intramural program of the eunice kennedy shriver national institute of child health and human development, and national cancer institute, national institutes of health, bethesda, md. 20892. stress hormones promote prostate cancer aggressiveness through modulation of mir-628-5p expression and exosome release north carolina central university, durham, usa introduction: despite proactive screening and steady declines in mortality, prostate cancer (pca) remains one of the most prevalent cancers among men. evidence suggests that chronic activation of stress signalling pathways can result in an altered mirnas transcriptome and affect exosomal content and release. here, we study the interaction between leptin and mir-628-5p expression, previously shown to be downregulated in pca patients. in addition, explored the effect of stress hormones cortisol and leptin on exosomal release and content from pca cells.methods: we utilized normal prostate cell line rwpe-1, and pca cells pc3, lncap and mda-pca-2b. proliferation of cells treated with leptin in the presence or absence of mir-628-5p mimic or negative control was assessed by mtt, colony formation, wound healing, and expression of targets affected by mir-628-5p was assessed by western blotting. moreover, exosomes were isolated via differential centrifugation from pca cells treated with leptin or cortisol and exosome number was determined by nanotracking analysis. exosome content was determined by western blotting and proteomic analysis by mass spectrometry.results: we observed that leptin significantly decreased expression of mir-628-5p in rwpe-1 cells.co-treatment with mir-628-5p mimic and leptin abrogated these effects in a cell dependent manner. we also observed that co-treatment with leptin affected mir-628-5p target jag1 and other molecules involved in epithelial to mesenchymal transition. in parallel, we demonstrated that cortisol increases exosome secretion particularly in pc3 cell exosomes with a 2.6-fold increase at 5 nm cortisol compared to untreated. western blotting revealed the presence of gr in exosomes particularly at 5 nm cortisol. summary/conclusion: understanding epigenetic regulation through mirnas and exosomes may be the key to understand stress hormone influence in pca progression. these findings suggest that stress hormones effectively affect mir-628-5p expression and exosomal release and signalling.introduction: extracellular vesicles (evs) are promising drug delivery vehicles for therapeutic microrna (mirna). for the loading of exogenous cargo, researchers broadly seek to either manipulate the evs directly or the cell that produce them. electroporation, sonication, and direct ev transfection are common methods that work by physical disruption or irreversible chemical addition, which may irreparably damage the molecules intended for therapy. on the other hand, transfection into the producer cells is a simple option that does not imperil ev integrity.methods: there are multiple factors that contribute to ev loading efficiency, including transfection reagent used, timing, and dosage. thus, we sought to establish a basic protocol and improve understanding of the underlying dynamics involved in a basic system consisting of hek293 t cells and mir-146a-5p mimic.results: in this work, we examined how different reagents lead to variable ev loading. then we looked at variable dosages, specifically the relationship between rna amount added to reagent, amount present in cell, and amount exported to evs. summary/conclusion: these results will help future studies produce evs with exogenously loaded small rna, and suggest future optimizations. funding: national institutes of health. r01 and t32 (host pathogen interactions at university of maryland). we report a single ev trapping method via aptamermediated assembly between au nanoparticle (aunp) and au superlattice template. we propose a chip-based ev trapping technique based on semiconductor processes. methods: we introduce aptamer coated au nanoparticle (aunp) and au superlattices as a template to capture evs. first, we fabricated poly(methyl methacrylate) (pmma) hole pattern on au-coated si substrates by using electron beam lithography (ebl). we designed 200 nm-diameter hole patterns to capture one ev in each hole. to connect the aunp and the au superlattice template, we used an aptamer molecule as a linker strand. also, to capture individual evs, the aptamer molecule is designed to have a hairpin structure to specifically bind to cd63, a protein marker of ev. we modified 5ʹ-terminal and 3ʹ-terminal of the cd63 aptamer with thiol group for the formation of self-assembly monolayer (sam) on both aunp and au superlattice surface. results: first, we coat the cd63 aptamer on the surface of aunp. afterwards, we load the aptamer-coated aunp into au superlattice template. ev solution is specifically bound to cd63 aptamer. after washing step, each ev is expected to locate within a single hole due to the size confinement of the hole. to separate the evs from the aptamer, we use restriction enzyme, bamhi, to recognize specific dna sequence and cleave them. summary/conclusion: in this report, we propose a aunp -linked au superlattice chip by aptamer molecules for trapping evs. we selected cd63 aptamer for specifically binding with cd63 in evs. in addition, we designed cd63 aptamer as a linker strand to connect introduction: a hallmark of platelet activation is the release of internal granules as extracellular vesicles/ microparticles. thrombolux is a dynamic-light-scattering-based (dls) instrument that was developed for use in clinical setting to check for platelet activation before transfusion. compared to traditional dls, the thrombolux requires no cleaning (single-use capillary) and requires very little sample (70 µl). hence the thrombolux may be a useful instrument beyond platelet pack test in blood transfusion laboratory. we have evaluated its use as an in-process monitoring tool for industrial ev manufacturing, for both quantifying cells (input) and evs (output). methods: the thrombolux was used to test the activation status of expired platelet packs (donated by arcbs for research purpose). the readout was compared with platelet swirling test and flow cytometry data (surface marker). furthermore, the thrombolux was also tested for process development and ev manufacturing monitoring purposes at different stages of the process for its ability to rapidly obtain particle presence and size information on evs. time to result was also compared between different particle analysis methods. results: the thrombolux was a better predictor of platelet packs variability compared to the traditional platelet swirling method. however, we did not observe a strong correlation between the activation status and the flow cytometry-based activation marker data. the thrombolux was able to provide a useful estimation of particle presence and sizing of evs in-process.results are obtained rapidly, within minutes, with minimal sample prep. summary/conclusion: although we did not observe a significant direct correlation between flow cytometry activation data and the % microparticles (within a small sample size), the thrombolux has shown potential to become a useful tool for in-process monitoring for ev manufacturing and other ev research, in particular through its speed and ease of use. funding: all funding was through exopharm ltd (asx:ex1). secreted introduction: a major manufacturing challenge related to exosome bioprocessing is that of robust and scalable purification. as efforts to translate exosomes into clinics grows, the more important the design of quality systems which can reproducibly purify the product becomes. the current gold-standard, ultracentrifugation, was adopted from the viral vaccine industry, but remains imperfect in terms of scale up and manufacturing due to labour and time intensive process requirements. in order to follow the preferential adoption of more standard bioprocesses, as previously achieved by the viral vaccine industry, we show the development of two monolith chromatography steps which can be used to purify exosomes from a clinically relevant, allogeneic stem cell product (ctx0e03). methods: t-flask expansion of ctx0e03 cells was performed to yield batches of 5-15 l of conditioned medium. the medium was subsequently clarified by benchtop centrifugation, and concentrated into a crude concentrate by tangential flow filtration [tff], using a combination of 0.22 µm dead-end filtration prior to concentration in a 300kda hollow-fibre tff system. tff retentate was loaded onto 1 ml hic or aex monoliths, for further purification. potency was assessed by a fibroblast wound healing assay in vitro. results: exosome presence was verified in the tff material by detection of cd 81 and cd 63. exosomes recovered in this manner could achieve full wound closure in vitro over 72 hours, when dosed at 20 µg. further purification by monolith chromatography showed high levels of reduction of albumin, detected by western blot, as well as heightened ratios of particles to both total protein, and total dna. the results indicate that neither aex nor hic steps cause detrimental loss to product function, either alone or in combination with one another. introduction: custom-made platelet pellet lysate (ppl) and heat-treated ppl (hppl) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic luhmes neuronal cell culture. this effect is significantly enhanced using hppl, which was also highly protective of th-expressing neurons in mice parkinson's disease (pd) model. introduction: there is a critical unmet medical need for new therapies to treat age-related diseases including cardiovascular diseases such as stroke. exosome derived from stem cells have shown intrinsic therapeutic potential in a variety of animal models of ischaemic diseases. we have identified scalable exosome production cell lines (purestem) as a source of angiogenic exosomes and are aiming to generate good manufacturing practice (gmp) grade therapeutic exosomes that can effectively mediate angiogenesis and tissue regeneration. we are developing exosome production and purification protocols that combine methods of tangential filtration flow (tff) and size exclusion chromatography (sec). the particle number and size were measured by both tunable resistive pulse sensing (trps) as well as nanoparticle tracking analysis (nta) for comparison. exosomes were characterized by detection of exosome surface markers and absence of cellular markers. purity was assessed by measuring particles per ug of total protein content. the angiogenic activity of purestem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing and tube formation assays. we further investigated the molecular cargo of purestem-exosomes by screening mirnas targets, rna-seq analysis, and mass spectrometry analysis.results: the isolated purestem-exosomes using our developed protocols were highly purified, resulting purity in the range of 1e10-5e10 particles/ug. we selected angiogenic exosome-producing cell lines from our purestem library by screening for functional activity and characterizing their molecular cargo. we found that purestem progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal stem cell (msc)-derived exosomes. furthermore, angiogenic micrornas such as mir-126 were enriched in purestem-exosomes from certain producer cell lines. summary/conclusion: these data demonstrate the potential for using purestem lines as a highly scalable source of therapeutic exosomes. we were able to obtain highly pure exosomes that retain their angiogenic activity. we anticipate that purestem-exosomes will be a valuable resource for developing ev therapies for stroke and other ischaemic diseases. we have developed purification methodologies aimed at achieving a robust and scalable exosome production compatible with gmp for clinical grade purestem-exosomes. these developments have great potential as therapeutic agents for future preclinical in animal model of stroke and clinical trials. neuronal introduction: the hallmark of parkinson's disease (pd) is a-synuclein accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. pd is also associated with insulin resistance, a condition characterized by phosphorylated insulin receptor substrate-1 (irs-1). besides motor symptoms, some pd patients develop mild cognitive impairment (pd-mci) or dementia (pd-d). given the importance for prognosis, there is an urgent need to develop biomarkers for distinguishing pd with normal cognition (pd-n) from pd-mci/d. neuronal-origin extracellular vesicles (nevs) contain cell signalling and pathogenic proteins (including a-synuclein), which may serve as biomarkers for alzheimer's disease, pd and other dementias.methods: from 0.5 ml of plasma from 104 pd-n, 83 pd-mci, and 39 pd-d patients, we immunocaptured nevs using anti-l1cam antibody. then, irs-1pser312 and irs-1ptyr20 and a-synuclein were measured in nevs using electrochemiluminescence immunoassays.results: a-synuclein was lower in pd-mci and pd-d compared to pd-n (p < 0.005) and significantly decreased with increasing motor symptom severity measured by mds-updrs iii score (p = 0.005). irs-1pser312 was lower in pd-d than in pd-n. irs-1ptyr20 significantly decreased with increasing mds-updrs iii score (p < 0.005). no biomarker was associated with disease duration. summary/conclusion: pd patients with cognitive impairment exhibited lower nev levels of a-synuclein than cognitively intact pd patients, whereas a-synuclein and irs-1ptyr20 were inversely associated with pd motor symptom severity. additional biomarkers and measurements will be available by the time of isev. plasma nevs is a valuable tool for discovering biomarkers in pd and investigating aspects of disease progression. introduction: despite decades-long advancement in transplant medicine, there is a necessity for personalized approach regarding early kidney allograft injury recognition and immunosuppression therapy towards improved transplant outcomes. biopsy, a gold standard for assessment of kidney allograft injury, cannot be serially used for the diagnosis of subclinical injury due to it's invasiveness and possible sampling errors. instead, urine is easily obtainable and bearing extracellular vesicles (evs), potential carriers of pathological signals related to kidney injury. our aim was to set up a urinary ev (uev) isolation protocol that would allow consistent and reliable identification of their characteristics and cargo. methods: second morning urine sample (25 ml) was collected from 7 patients and processed within 4 hours. oxalate precipitation, ph and dilution variability, uromodulin polymerization and high protein content were taken into account. isolated evs were defined by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). uev specific proteins and mirnas were analysed by western blot and qpcr, respectively. results: the optimal protocol relied on low speed urine centrifugation (2.000 x g, rt) for cell removal and storage at −80°c prior to further analyses. after urine thawing at rt, added edta averted cryoprecipitate and uromodulin polymer formation, while concentrated pbs neutralized the ph. filtration through 0.22 µm pores was used for large particle removal, while centrifugal 100 kda membrane units (amicon®, milipore) served for sample concentration followed by particle separation on sizeexclusion chromatography (sec; qevoriginal, izon q). protein vacant sec fractions (as rated at a280) were pooled and concentrated to a volume of 70 µl. tem micrographs revealed high sample purity and cup-shaped morphology of uevs. as per nta results, the average mean size of evs was 129,9 nm with concentration range of 1 × 109 particles/ml of starting urine. uevs were positive for the tested marker proteins hsc70, flotillin, tubulin, gadph and cd63. qpcr verified mirna presence in uevs, with ct for mir let-7i at 20. summary/conclusion: we successfully isolated pure uevs. the set up protocol will be used to assess uevs as non-invasive biomarkers of allograft injury in kidney transplant recipients. astrocyte-derived extracellular vesicles regulate dendritic spine formation and neuronal network connectivity introduction: recent advancements in the biology of extracellular vesicles have begun to implicate glial released microvesicles as mediators of glia to neuron communication, suggesting that alterations in the release and/or composition of astrocyte microvesicles could impact neuronal function. methods: astrocytes were allowed to constitutively release extracellular vesicles (adev-cr), or stimulated with atp (adev-atp). adevs were isolated by ultracentrifugation followed by proteomic analysis. we developed a normative whole transcriptome database using primary neurons exposed to adev-cr, and identified changes in neuronal gene expression produced by exposure of neurons to adev-atp. we identified a number of pathways associated with the biological response of synapse, spine and neurite outgrowth that were regulated by adev-atp. the molecular cargo of adev-atp responsible for regulating synaptic functions in neurons were characterized by biochemical, molecular, and functional assays. results: adev-atp enhanced the maturation of dendritic spines and produced functional enhancements in neuronal activity and network connectivity. the mechanism for this effect involved the delivery of integrin-4 1 and epha2 that were enriched in adev-atp. integrin-4 1 facilitated binding of adevs to the neuronal surface, and epha2-receptor signalled through ephrin to the tyrosine kinase erbb2/ 4 that regulated the phosphorylation and activation of trkb without increasing expression of the natural ligands bdnf or ntf3. this direct activation of trkb increased the expression of the synaptic scaffolding proteins disc1, arc, and cplx3 to promote the maturation of dendritic spines. this increase in mature dendritic spines was associated with increased neuronal activity and network connectivity demonstrating a functional strengthening of synapses. summary/conclusion: these data identify a molecular mechanism whereby modifications in adev protein cargo produced by the stimulation of astrocytes with atp regulates synaptic maturation through activation of trkb in a manner independent of growth factors. stephanie kronstadt and steven m. jay university of maryland, college park, college park, usa introduction: mesenchymal stem cell extracellular vesicles (msc-evs) have been shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders. despite this, clinical translation of ev therapies is hindered by potentially low potency in vivo and the lack of a scalable biomanufacturing process. cell culture parameters are critical in modulating both yield and bioactivity of evs. thus, we hypothesized that the combination of chemical priming and 3d dynamic culture would enhance the yield and potency of immunosuppressive msc-evs. methods: bone marrow-derived mscs cultured in flasks were chemically primed using ethanol or curcumin. mscs were also cultured using a 3d-printed scaffold-perfusion bioreactor using a flow rate of 5 ml/min. anti-inflammatory effects were assessed following application of msc-evs to lipopolysaccharide (lps)-stimulated murine macrophages. subsequent inhibition of the production of the pro-inflammatory cytokine il-6, quantified using an elisa, was used to characterize evs as anti-inflammatory. in addition, both chemical priming and the bioreactor will be simultaneously utilized to potentially uncover any synergistic effects on ev immunomodulation abilities. nanoparticle tracking analysis (nta) was used to assess ev size and concentration while protein mass was measured via a bca assay. results: preliminary data suggests that priming mscs with 100 µm ethanol for 24 hours prior to ev collection results in a strong inhibition of il-6 production in stimulated murine macrophages. nta revealed that msc-ev yield increased by about two orders of magnitude in the bioreactor (1.40e12 ± 7.92e10) when compared with flasks (2.28e10 ± 2.81e9). protein measurements also indicated that ev production in the bioreactor (~7600 µg) was much greater compared with production in the flasks (~2400 µg). additionally, average protein content per ev was reduced in the bioreactor when compared with flask evs. regardless of tissue source. furthermore, comparison of adipose tissue-derived (ad) msc evs from three donors indicates varying pro-vascularization bioactivity between those donors evaluated in vitro via gap closure assay. similar results were observed for the bone marrow-derived (bm) msc ev donor groups. summary/conclusion: this work highlights the need for screening of donor derived-mscs before use for therapeutic ev production. additionally, standardized criteria for msc donor selection are needed before isolated msc evs can be used as a large-scale, repeatable therapeutic treatment. analysis of extracellular vesicle populations from malaria-infected erythrocytes by field-flow fractionation reveal distinct sub-sets alicia rojas a , paula abou-karam a , anna rivkin a , yael fridmann-sirkis b , yifat ofir-birin c and neta regev-rudzi c a department of biochemical sciences, weizmann institute of sciences, rehovot, israel, rehovot, israel; b wis, rehovot, israel; c weizmann institute of science, rehovot, israel introduction: malaria is one the most devastating infectious disease in the world and plasmodium falciparum (pf) represents the deadliest species. this parasite invades human red blood cells (rbcs) and releases extracellular vesicles (evs) carrying dna, rna and protein cargo components which are involved in the pathogenesis of the disease. recently, it has been shown in mammalian systems that evs are subdivided into different subpopulations, each with a distinct biological function. however, it is still unknown whether pfinfected rbcs (pf-evs) release different ev subpopulations with distinct cargo. methods: we isolated evs from pf-infected and uninfected rbcs, pf-evs or ui-evs, respectively, using differential centrifugation. the ev pellet was subjected to field flow fractionation (fff). the different subpopulations were collected, concentrated with size-exclusion filters and evaluated by nanoparticle tracking analysis. additionally, the presence of ev markers (sr1 and hsp90) were examined by western blot analysis. results: the fff analysis showed four particle subpopulations derived from the pf-evs and five in the ui-evs. the first three subpopulations were similar in their detection signals in both samples, but the fourth subpopulation was consistently higher in ui-evs than in pf-evs. moreover, hsp90 was detected in subpopulations 3 and 4 of both pf-evs and ui-evs, whereas sr1 only in subpopulation 3. isev2020 abstract book summary/conclusion: pf-ev and ui-ev have similar separation profiles and proteins markers in their subpopulations, consistent with the fact that both samples are derived from host rbcs. additional data regarding the dna and rna cargo, as well as microscopic observations of the pf-ev and ui-ev subpopulations is necessary. this will clarify how malaria parasites sort their components into evs and which fractions are associated to immune evasion and pathogenesis. we have established a small size laboratory production of the microalgae culture in order to harvest the extracellular vesicles (evs) for pharmaceutical and medical uses. in this work we report on globular particles in the isolates from media of microalgae of two types, that we recognize as evs. we observed changes in their production at different temperatures and conditions. methods: samples were fixed by various combinations of aldehyde fixatives and/or osmium tetroxide. they were dehydrated in a graded series of ethanol, hexamethyldisilazane, and air dried. they were au/pd coated for inspection with scanning electron microscopes (sem) crossbeam 550 fib-sem gemini ii (zeiss, germany) and jsm-6500 f field emission scanning electron microscope (jeol ltd., tokyo, japan). results: microalgae were incubated overnight at 22°c and 37°c in growth medium and in growth medium supplemented with detergent. the samples obtained from the microalgae culture contained particles that we recognized as extracellular vesicles, however, these particles do not correspond to characteristic shapes of membrane enclosed entities without internal structure. increased temperature and/or presence of surfactant (triton x-100 and sodium dodecyl sulphate) stimulated formation of evs of different shapes and sizes. the isolates of these samples were rich with evs. in the presence of surfactant, the cell-walls detached from the cell and collapsed upon dehydration. this was documented by sem. summary/conclusion: focused ion beam technique revealed complex internal structure of the algae. it seems from the shapes of the observed structures that the particles deposited on the surface of the microalgae do not derive from budding of the membrane surface, but are instead shed by the cells from the cell interior upon the rupture of the cell wall. key: cord-023168-cd7adns8 authors: thachil, jecko; owusu-ofori, shirley; bates, imelda title: haematological diseases in the tropics date: 2013-10-21 journal: manson's tropical infectious diseases doi: 10.1016/b978-0-7020-5101-2.00066-2 sha: doc_id: 23168 cord_uid: cd7adns8 nan haematological disorders are common in low-income countries. they make a substantial contribution to morbidity and mortality of individuals in these regions and have a negative impact on the growth and development of under-resourced nations. genetic red cells abnormalities are common in lowincome countries because they provide protection against malaria and they often co-exist with other causes of anaemia such as malnutrition and chronic illnesses. there is a close association between haematological abnormalities and infections which are a major cause of illness and death in these populations. morphological abnormalities of blood can often provide clues about the underlying diagnosis and blood film examination is particularly important where diagnostic facilities are limited. abnormal blood counts can manifest as various combinations of alterations of numbers of red cells, white cells or platelets. this section will outline some of the most common causes of abnormal blood counts likely to be encountered in clinical practice in low-income countries. anaemia is one of the most common causes of morbidity in the world and its impact is reflected in several of the health-related millennium development goals. although anaemia by itself is not a diagnosis, it suggests that there is an underlying disease state which needs to be recognized and treated. it is also a useful indicator of the general health of the population. the causes of anaemia may be identified systematically by considering the life cycle of the red cells (figure 65 .1). nutrients necessary for red cell production are absorbed from the gastrointestinal tract and carried through the portal vein to the liver and ultimately reach the bone marrow where erythropoiesis occurs. this process is regulated by erythropoietin, a hormone released from the kidneys mainly in response to hypoxia. mature • africa and asia have more than 85% of the world's anaemic populations and anaemia burden is highest among children and women of reproductive age. • the accurate diagnosis of anaemia has been neglected; clinical assessment of anaemia is unreliable unless the anaemia is severe. • in low-income countries, anaemia in an individual is often due to multiple interdependent factors. removing or treating a single factor may not resolve the anaemia. • early diagnosis of sickle cell disease and rapid access to a specialist centre for emergencies such as severe pain crises, strokes and acute chest syndrome, can help to prevent permanent long-term complications. • beta-thalassaemia major is fatal in the first few years of life unless regular blood transfusions are given; unless they are accompanied by iron chelation, these transfusions will eventually cause death due to irreversible organ damage from iron overload. • malarial anaemia is a particular problem for children and pregnant women and severe anaemia can be caused by p. falciparum and p. vivax. malarial anaemia can be reduced with chemoprophylaxis and intermittent treatment, and by anti-mosquito measures such as insecticidetreated bed nets and vector control. • anaemia occurs in 70% of hiv-infected patients and is an independent risk factor for death. prompt treatment of factors associated with anaemia, such as infections and poor nutrition, and commencement of antiretroviral treatment will reduce deaths. • blood shortages are common in tropical countries. to increase the availability of blood, transfusions should be prescribed in accordance with guidelines and efforts made to encourage blood donors to donate regularly as repeat donors are the safest type of donor. reaction' , characterized by circulating myelocytes and metamyelocytes, can be mistaken for leukaemia but, unlike leukaemia, there is an orderly maturation and proliferation of neutrophils. leukaemoid reactions have also been described in patients with tuberculosis, juvenile rheumatoid arthritis and dermatitis herpetiformis. 2, 3 decreased margination of neutrophils with egress of cells into the circulation can occur with exercise, adrenaline (epinephrine) injection, emotional stress and postoperatively or in response to drugs (e.g. steroids, β-agonists). other drugs, such as lithium and tetracycline, produce neutrophilia through increased production. neutrophilia is also a feature of bone marrow proliferation which occurs in myeloproliferative neoplasms, particularly chronic myeloid leukemia and myelofibrosis. teardrop cells and nucleated red blood cells are features of myelofibrosis on the blood film; basophilia and eosinophilia are common with chronic myeloid leukaemia. molecular testing for the jak-2 mutation or bcr-abl fusion gene can also help to differentiate between myeloproliferative neoplasms. rebound neutrophilia can occur following treatment of megaloblastic anaemia or after recovery from neutropenia induced by drugs. acute haemorrhage can cause neutrophilia, especially if bleeding occurs into the peritoneal cavity, pleural space, joints or adjacent to the dura. this is possibly due to the release of adrenaline and chemokines in response to local inflammation. the presence of neutrophilia can be useful in raising suspicions about the onset of complications in infections that are not primarily associated with neutrophilia. examples include meningitis in tuberculosis, orchitis in mumps, bowel perforation in typhoid fever and superadded bacterial infection in measles. the absence of neutrophilia can be helpful in differentiating typhoid and paratyphoid fever from pyogenic infections. neutropenia is defined as an absolute neutrophil count <1.5 × 10 9 /l. it is usually classified into severe (<0.5 × 10 9 /l), moderate red cells are released into the circulation from the bone marrow and percolate through the tissues and organs. anaemia can result from defects in any of these stages. inadequate production of red cells in the bone marrow can be due to lack of nutrients (e.g. iron, b 12 , folate, vitamin a, copper or zinc), abnormal haemoglobin synthesis (i.e. haemoglobinopathies) or ineffective erythropoeisis from myelodysplasia or infections. red cells can be lost from the body (e.g. gastrointestinal bleeding) or removed prematurely if they are abnormal or the spleen is enlarged (i.e. haemolysis). kidney disease can result in decreased erythropoietin. anaemia of chronic disease (or 'anaemia of inflammation') is due to an inadequate response to erythropoieitin or to increased cytokine-induced hepcidin release in inflammatory states which interferes with iron absorption or iron utilization. diagnostic algorithms to determine the cause of anaemia are usually based on a combination of the mean cell volume of the red cells, the reticulocyte count and blood film appearance (figures 65.2, 65.3 ). this approach is based on the availability of a haematology analyser and an experienced microscopist. several conditions which cause anaemia may co-exist in the same individual (e.g. intestinal parasites, malaria and sickle cell disease) and hence a thorough investigation is crucial to identify all potential causes of anaemia. neutrophils released from the marrow after maturation can either enter the 'circulating pool' or they can remain in the 'marginal pool' where they are loosely attached to the blood vessel wall. cells in the marginal pool are not sampled when blood is taken for a full blood count. 1 neutrophilia can therefore result from increased bone marrow synthesis and also from decreased margination which increases the circulating pool. there are many causes of neutrophilia (box 65.1) but the commonest is bacterial infection in which there is increased bone marrow production of neutrophils and release of neutrophil precursors into the peripheral blood. this 'leukaemoid (0.5-1.0 × 10 9 /l) or mild (1.0-1.5 × 10 9 /l). the propensity to develop infections is related to the degree and duration of neutropenia, with higher risk associated with counts below 0.5 × 10 9 /l. africans, african americans, yemenite jews, palestinians and saudi arabians generally have slightly lower neutrophil counts compared with other races. this is thought to be due to an increase in the bone marrow storage pool as ethnic neutropenia is associated with good neutrophil responses to infections. neutropenia can be due to impaired or ineffective (intramedullary death of neutrophil precursors despite normal bone marrow production) synthesis by the bone marrow (e.g. myelodysplasia, megaloblastic anaemia, treatment with phenytoin or methotrexate); a shift from the circulating pool to marginated pool (pseudoneutropenia) and increased peripheral destruction (e.g. secondary to antibodies against the neutrophils or increased reticulo-endothelial activity in sepsis or haemophagocytic syndrome) (box 65.2). increased consumption of neutrophils can result from increased attachment of cells to endothelium or other leukocytes in inflammatory states. neutropenia is often the result of a combination of several of these mechanisms. infants of hypertensive mothers may have moderate to severe neutropenia, which can last for several days. this is probably related to bone marrow suppression. moderate to severe neutropenia can also occur in newborn infants as a result of the transfer of maternal igg anti-neutrophil antibodies in a manner similar to rhesus haemolytic disease of the newborn. 4 although neutropenia has been described with typhoid fever, minimum neutrophil count seldom falls below 0.6 × 10 9 /l and the box neutropenia may not develop until after the first week of illness. infectious hepatitis and yellow fever can both cause neutropenia. overwhelming infections can lead to a failure of bone marrow production of neutrophils, especially in undernourished individuals and alcoholics. individuals with severe neutropenia can develop lifethreatening septicaemia, often from endogenous flora (e.g. oral cavity), and stringent measures should be taken to avoid situations which may predispose these individuals to infections. they may need prophylactic antimicrobials and should have rapid access to medical care. fungal infections are less common than bacterial infections in neutropenic individuals, and viral or parasitic infections rarely occur with isolated neutropenia. granulocyte colony stimulating factor (gcsf) injections can be helpful in raising the neutrophil count in patients with complicating infections since it stimulates the release of neutrophils from the marrow, but gcsf is only useful if there is some bone marrow reserve. patients with some congenital or immune forms of neutropenia can tolerate persistently low counts without any increase in the incidence of infections. monocytosis occurs in chronic infections and inflammatory conditions. protozoan infections such as typhus, trypanosomiasis and kala-azar may be associated with monocytosis. chronic and juvenile myelomonocytic leukaemias are malignant disorders in which monocytosis may be severe; acute monocytic leukaemias may present with mild to moderate monocytosis. monocytosis, and particularly a monocyte : lymphocyte ratio greater than 0.8-1.0, may indicate active progression of tuberculosis and an unfavourable prognosis. the normal ratio of 0.3 or less is restored when the healing process is complete. a decreased absolute monocyte count occurs in bone marrow failure states such as aplastic anaemia or after chemotherapy. low monocyte counts can occur with overwhelming sepsis and with splenomegaly. monocytopenia is a characteristic feature of hairy cell leukaemia and is considered to be a diagnostic hallmark of this disease. peripheral blood contains only around 2% of the total body lymphocyte population since these represent the cells present in the blood during their transit into secondary lymphoid organs. wide variations exist in lymphocyte counts between individuals especially in childhood. lymphocyte counts exhibit a diurnal pattern; peaking at night with a nadir in the morning. lymphocytosis is characteristic of infectious mononucleosis and many atypical and large lymphocytes can be seen in the peripheral blood film. these atypical cells can also occur in cytomegalovirus infection and infectious hepatitis. absolute lymphocytosis can occur with chronic infections such as brucellosis and in the recovery stages of tuberculosis. lymphocytosis is unusual in bacterial infections except in the case of pertussis. heavy smoking is also an often overlooked cause of lymphocytosis and is probably one of the commonest reasons for a mild to moderate increase in the lymphocyte count. malignant bone marrow disorders, predominantly acute lymphoblastic and chronic lymphocytic leukaemia and non-hodgkin's lymphomas, can cause lymphocytosis. these lymphocytes may have characteristic morphological changes identifiable in the blood film (e.g. smear cells with chronic lymphocytic leukaemia) and the correct diagnosis can be confirmed by immunophenotyping for specific combinations of cell markers. lymphopenia is due to decreased production, redistribution or increased rate of death of lymphocytes. decreased production usually results from cytotoxic drugs and radiotherapy, while increased lymphocyte death can occur in infections such as influenza and hiv. occasionally, an isolated low lymphocyte count in the context of an otherwise normal full blood count can be a clue to the diagnosis of hiv. this reflects the destruction of cd4+ t cells by the virus although an expansion of cd8+ t cells may raise the total lymphocyte count to normal levels. redistribution rather than depletion of total body lymphocyte numbers occurs with steroid treatment or with endogenous secretion of corticosteroids during acute illnesses due to the retention of lymphocytes in secondary lymphoid organs. eosinophilia eosinophils are involved in innate immunity and hypersensitivity. their number in the circulation is relatively small compared to other leukocytes because they predominantly reside in tissues such as the gut, skin and lungs which are entry points for allergens and infections. the commonest causes of eosinophilia are helminthic infections, atopy and allergic diseases, and adverse drug reactions. less common causes are classified under the umbrella term of hypereosinophilic syndromes (table 65 .1). since parasitic infections are likely to be the commonest cause of eosinophilia in the tropics and in returning travellers, an extensive search for such infections should be undertaken in patients with persistent eosinophilia; initial investigations should be determined by the patient's history of geographical exposure (figure 65.4) . [5] [6] [7] the absolute number of eosinophils in the peripheral blood may not correlate with their tissue distribution or with their potential to cause tissue damage from their granule release. this is because the degree of eosinophilia depends on the extent of tissue invasion and is therefore modest with tapeworms and roundworms resident in the bowel but much higher where invasion occurs, for example with, toxocara canis or filaria. schistosomiasis almost always causes eosinophilia. strongyloides stercoralis has the capacity to remain in the host for decades after initial infection and causes varying degrees of eosinophilia, with or without other symptoms. steroid treatment, which may be necessary in cases of eosinophilic tissue damage, can exacerbate clinical problems in patients with strongyloides infection so this parasitic infestation should be excluded before starting steroids for hypereosinophilia. mild to moderate eosinophilia is common in asthma although a very high count should prompt a search for churg-strauss syndrome or allergic bronchopulmonary aspergillosis. most drugs including penicillins can cause eosinophilia but the diagnosis can only be made by noting recovery when the drug is discontinued. eosinophilia can be a feature of hodgkin's lymphoma. it signifies a more favourable prognosis and may precede the original diagnosis of lymphoma or relapses. in immunocompromised patients, such as those with hiv infection, the finding of eosinophilia may be crucial since the success of antiretroviral treatment may depend on concomitant eradication of parasites. thrombocytopenia is often discovered incidentally in patients during full blood count estimation. a platelet count above 20-30 × 10 9 /l is usually not associated with any symptoms such as bleeding. if clinically evident haemorrhage does occur at counts above this level, other conditions such as coagulation defects, vascular problems or rarely platelet dysfunction should be suspected. although the prime role of platelets is in haemostasis, several other important roles have been recognized in recent years including wound repair, tissue healing, antimicrobicidal properties, lymphangiogenesis, tumour metastasization and maintenance of blood vessel integrity. congenital platelet disorders are often part of a syndrome. patients with wiskott-aldrich syndrome have small platelets in association with eczema and recurrent infections. other congenital platelet disorders, such as myh9-related disorders, can present with deafness or cataracts while skeletal deformities and oculocutaneous albinism are common in other syndromic presentations. blood film morphology can provide important clues about the causes of thrombocytopenia (figure 65 .5). fragmented red cells (schistocytes) increase the possibility of microangiopathic haemolytic anaemia, where an altered vessel wall and fibrin formation in the blood vessels shred the erythrocytes and consume platelets. thrombotic thrombocytopenia purpura, haemolytic uremic syndrome and disseminated intravascular coagulation can all present with thrombocytopenia. dysplastic red or white cells should raise the suspicion of myelodysplasia which can be confirmed by bone marrow examination and cytogenetic analysis. it is important to exclude in vitro platelet agglutination as a cause for apparent thrombocytopenia. this can be an anticoagulant (edta)-dependent phenomenon so a repeat sample should be examined using citrate anticoagulant. rarely, platelet satellitism where the platelets clump round the neutrophils, can cause artefactual thrombocytopenia. anaemia affects nearly two billion people globally with a much higher prevalence in developing countries compared with more wealthy nations (43% vs 9%). 8 the continents of africa (highest prevalence) and asia (greatest absolute burden) account for more than 85% of the anaemic population. anaemia burden is highest among children and women of reproductive age. anaemia contributes to more than 115 000 maternal deaths and 591 000 perinatal deaths globally per year. 9 who have defined anaemia according to various haemoglobin concentrations (table 65. 2) 10 but the appropriateness of these thresholds has been questioned because there are wide variations in haemoglobin concentration among people of different races. 11 the prevalence of anaemia can be a useful indicator of public health status of a nation because: • the prevalence of anaemia is objective and quantifiable • anaemia is a major complication of several infections, including malaria, hiv, tuberculosis, and the neglected tropical diseases, which are among the commonest problems in most tropical countries • the incidence of anaemia changes in a predictable fashion with alterations in disease burden • the prevalence of anaemia can be used to assess whether an intervention has reached the poorest communities. haemoglobin concentration of <90 g/l has been recommended for disease surveillance in high-prevalence countries where changes in haemoglobin are used for monitoring the impact of interventions. 11 anaemia in tropical countries (box 65.3) is often due to infections but chronic health problems, such as diabetes and chronic respiratory disease, and cancer and related complications are increasing as causes partly due to lifestyle changes. the body to compensate for the drop in haemoglobin content. for this reason the haemoglobin level can drop to extremely low levels before symptoms develop. anaemia presents with symptoms such as exertional breathlessness, palpitations and in some cases, syncopal attacks. patients with chronic anaemia may also have a multitude of nonspecific symptoms including poor concentration, decreased work performance and easy exhaustion (table 65. 3). a thorough history and clinical examination may provide clues about the cause of anaemia but further investigations are often necessary to confirm the diagnosis and guide treatment. however, in many resource-poor settings, access to routine biochemical and haematological testing is scarce, so much reliance is placed on clinical examination. the international guidelines for the integrated management of childhood illness recommend that a diagnosis of anaemia in sick children is based on the assessment of palmar pallor. for pregnant women, symptoms of fatigue and dyspnoea, combined with signs of conjunctival and palmar pallor, and increased respiratory rate suggest anaemia. however, making a diagnosis of anaemia based on clinical assessment alone is unreliable unless the anaemia is severe. 12 no specific anatomical site is particularly accurate for the prediction of anaemia 11 though sensitivity may be increased by using multiple sites. 12 most central laboratories in low-income countries have automated haematology analysers and several manual methods exist for assessment of haemoglobin concentration, which are suitable for rural areas where there is no mains electricity (e.g. haemoglobin colour scale; hemocue technique). [13] [14] [15] haemoglobin colour scale 16 principle. the colour of a finger-prick blood sample, soaked into special chromatography paper, is compared with the clinical symptoms and signs of anaemia vary and depend on the cause and the speed of onset. a rapid drop in haemoglobin is much more likely to cause symptoms of anaemia than chronic anaemia. slowly developing anaemia allows time for in many cases, there will be more than one of these conditions coexisting in the same individual. an adequate response to the treatment of anaemia requires management of all the contributory factors. intrauterine growth. thus, low birth weight and prematurity are both associated with iron depletion in the postnatal period. several interventions have been suggested to improve infantile iron deficiency, including: [17] [18] [19] • delayed cord clamping at delivery; the short delay of 2-3 minutes allows a small but important amount of blood to continue to flow to the foetus from the placenta • improvement of infant feeding practices • prevention and treatment of infectious diseases • interventions to prevent low birth weight, such as maternal nutritional supplementation, the control of infections and chronic health problems in pregnancy. anaemia in young children can be due to increased nutrition requirements during periods of rapid growth; these requirements may be up to 10 times higher per kilogram of body weight than that of an adult male. in addition, infant and toddler diets often lack bio-available iron. a case-control study of preschool children in malawi with severe anaemia (haemoglobin concentration, <50 g/l) identified bacteraemia, malaria, hookworm, hiv infections and deficiencies of vitamins a and b 12 as the commonest causes of anaemia. lack of folate and iron were uncommon. in low-income countries multiple interdependent causes of anaemia often operate in one individual so rectifying a single factor is unlikely to make a big impact on resolving anaemia. interventions which are useful in preventing anaemia in younger children include micronutrient supplementation (food fortification), de-worming, prevention and treatment of infectious diseases, school nutrition programmes and community-based nutrition promotion. who defines anaemia of pregnancy as a haemoglobin level less than 110 g/l, or haematocrit less than 33%, at any time during pregnancy. about one-fifth of maternal mortality is attributable to anaemia in pregnancy 20 and anaemia affects nearly half of all pregnant women worldwide. maternal anaemia is associated with many factors that might also be causally associated with mortality including poverty, infections and inadequate health-seeking behaviour. globally, the most important cause of anaemia in pregnancy is iron deficiency although hookworm, malaria, hiv infection, and deficiencies in folate and other micronutrients may contribute. pregnancy-associated complications, including septicaemia, pre-eclampsia and other obstetric problems can precipitate anaemia. 21 it is important to note that a diagnosis of iron deficiency in pregnancy which relies on ferritin measurements may be misleading because of high-quality digital examples of known haemoglobin concentration. the colours are represented in 20 g/l increments from 40 g/l to 140 g/l. this method is inexpensive, does not depend on skilled scientists, is durable in dusty, hot, dry and humid conditions and is probably better than clinical diagnosis for detecting mild and moderate degrees of anaemia. the disadvantages are that it requires specific chromatography paper and good natural light and it cannot detect changes in haemoglobin less than 10 g/l. this is a small battery-or mains-operated machine, which uses a drop of blood in a plastic cuvette to produce a direct read-out of haemoglobin in a few seconds. it is simple to use, produces accurate and consistent results to one decimal place and it has an in-built quality-checking mechanism. the hemocue hb-301 has been specifically designed for tropical conditions and operates in temperatures up to 50°c, in dusty and humid conditions. however the recurrent costs associated with disposable plastic cuvettes mean there is little opportunity for cost-saving with high-volume workloads. the iron status of an infant is directly proportional to its body mass and blood volume, both of which are reflections of the major cause of anaemia in most of these cases is iron deficiency. some of the effects have been described in individuals with iron deficiency without obvious features of anaemia. there are three intervention strategies recommended by who to prevent anaemia in pregnancy: 1. weekly iron and folic acid supplementation in women of reproductive age 22 2. daily iron and folic acid supplementation during pregnancy 3. presumptive treatment of hookworm infection during pregnancy in areas where hookworm infection is known to be endemic. 23 several factors may interfere with the efficacy of these interventions. under-participation in antenatal care may be common due to factors such as geographic distance, low motivation and poor interpersonal skills of health staff, poor quality of supplies and facilities, insufficient supply of iron and folic acid pills and womens' poor understanding about the daily use of supplements, especially in the face of common side effects. 23 in sub-saharan africa, the acute shortage and high turnover of health workers, and lack of time have also been shown to contribute to ineffective antenatal measures for reducing anaemia. 24 interestingly, a study from bangladesh showed that the first 20 pills (whether taken on a daily basis or less frequently) yielded most of the benefit for raising haemoglobin levels, which suggests that currently recommended doses may be higher than necessary to achieve optimal outcomes, except when anaemia is very severe. 25 the global burden of iron deficiency has been estimated from anaemia prevalence surveys, which include many different causes of anaemia so data may be unreliable as they are often not based on proven cases of iron deficiency. who estimates that globally 41% of women and 27% of pre-school children are affected by iron-deficiency anaemia, making it number 15 of selected risk factors for preventable death and disability worldwide. 20 iron deficiency begins in childhood, worsens during adolescence in girls and is aggravated during pregnancy. poor iron stores at birth, low iron content of breast milk and low dietary iron intake throughout infancy and childhood result in high prevalence of anaemia in childhood. anaemia is exacerbated by increased requirements during adolescence and iron loss from menstruation and is often compounded by the lack of adequate nutrition. the situation is worsened by pregnancy when iron requirement is approximately two times higher than in a nonpregnant state. iron deficiency should not be considered a diagnosis but a secondary outcome due to an underlying medical condition. although it may be a physiological response to rapid growth or increased requirements during childhood and pregnancy, it still requires treatment due to potential deleterious consequences. many of the chronic effects of iron deficiency may develop before the clinical and laboratory evidence of anaemia becomes apparent. the biochemical evidence for iron deficiency occurs in several steps. 26 initially, iron stores in the bone marrow are depleted as reflected by a decreased serum ferritin. the total iron-binding capacity then starts to rise, while the serum iron saturation begins to fall before microcytosis and a drop in haemoglobin ensue. there have been attempts to identify this early iron deficiency before anaemia develops in order to improve neurological and psychomotor functions in children and work performance in adults through widespread iron supplementation. however, there are concerns that iron excess may promote infections, especially in malarious areas. a range of laboratory investigations are usually necessary if iron deficiency is suspected (table 65 .4) 27-30 because once the diagnosis is confirmed, a search for the precise cause is necessary. a systematic approach to the investigation of iron deficiency (see below) is required based on an understanding of alterations in the iron absorption and transport cycle. • deficient intake (cow's milk has poor iron content and can cause gut blood loss in some infants) • rare defects of haem biosynthesis and iron transport. iron-deficient individuals may have no symptoms. excessive fatigue and other nonspecific signs of anaemia become more pronounced as anaemia develops. consumption of unusual 'foods' such as ice and paint or 'pica' only occurs in a minority of individuals. physical examination may reveal stomatitis, glossitis, koilonychia (spoon-shaped nails) and hair loss. oesophageal webs have been described in the plummer-vinson syndrome but are rare and may respond to iron replacement. since iron is important in neuromuscular development, several features of anaemia described in table 65 .3 may be related to iron deficiency. treatment of iron deficiency is with dietary modifications and oral or parenteral iron. 31 blood transfusions should be reserved for those with severe symptoms especially if the anaemia developed rapidly. haemoglobin levels alone should not be considered as a criterion for transfusion since very low levels (e.g. 10-30 g/l) may be appropriately treated with oral iron if anaemia has developed slowly. intravenous iron should only be considered in cases of poor response or intolerance to oral iron. cereals, poultry and green leafy vegetables, contain non-haem iron, which is often poorly absorbed. if dietary history suggests a deficiency, diet with foods rich in haem iron, such as red meat or liver should be recommended if social and religious customs and financial status allow, ideally with a drink containing vitamin c to facilitate iron absorption. absorption is also facilitated by taking supplements on an empty stomach although side effects of dyspepsia may not always allow this strategy. heavy tea intake can interfere with iron absorption and should be avoided. multivitamin or dietary supplements containing calcium, zinc or copper can also interfere with iron absorption. absorption may be delayed by tetracyclines, milk and soft drinks. since acid is necessary for iron absorption, antacids may account for a poor response to oral iron. iron is usually prescribed as a daily dose of 150-200 mg of elemental iron, commonly ferrous sulphate, 1 tablet three times daily. the dose in children is 3-6 mg/kg per day split into divided doses. assuming good compliance and absorption, this should result in an increase in haemoglobin within 4 weeks. once the haemoglobin is normalized, iron should be continued for 3 months to replenish the iron stores. the major problem with oral iron is upper gastrointestinal side-effects, which can be dose-dependent. a reduction in the dose or change in the formulation to gluconate or fumarate or even liquid forms, may be successful. liquid iron preparations may stain the teeth and should therefore be taken through a straw. oral iron can also cause constipation or diarrhoea which is not dose-dependent. parenteral iron is best given intravenously because intramuscular iron is painful and has been associated with development of soft tissue sarcomas. 32 high-molecular-weight iron dextran carries a low but significant risk of anaphylaxis, but the newer formulations including low-molecular-weight iron dextran, iron sucrose, ferumoxytol and iron gluconate have minimal risks. vitamin b 12 or cobalamin deficiency is a well-recognized cause of macrocytic anaemia (box 65.5). although some microorganisms can synthesize cobalamin, humans need to obtain this essential vitamin from foods, mainly meat, poultry and dairy products. vitamin b 12 is an essential co-factor in dna synthesis, serving as a co-factor in two key biochemical processes involving methylmalonic acid and homocysteine as precursors. consequently vitamin b 12 deficiency can interfere with dna synthesis. clinical manifestations include haematological (megaloblastic anaemia and pancytopenia), and neuropsychiatric disorders (paraesthesia, peripheral neuropathy, psychosis and dementia) and an increased risk of cardiovascular disease because of hyperhomocystinaemia. [32] [33] [34] a systematic approach to the investigation of vitamin b 12 deficiency requires an understanding of the absorption cycle. 35 ingested vitamin b 12 is broken down in the acidic environment of the stomach. it binds to r-binders in gastric secretions and saliva which stabilize the vitamin b 12 . in the alkaline environment of the small intestine, vitamin b 12 is released from r-binders to bind to intrinsic factor, synthesized in the gastric parietal cells. this vitamin b 12 -intrinsic factor complex is absorbed from the terminal ileum. recently, an alternative absorption system independent of intrinsic factor and the terminal ileum has been postulated which provides a rationale for mean cell volume useful as a diagnostic clue but not confirmatory can also be low in thalassaemia, sideroblastic anaemia and rarely lead poisoning can be falsely normal in the presence of iron deficiency in older people or with coexistent megaloblastic anaemia anaemia of chronic disease can occasionally cause microcytosis serum ferritin the most useful laboratory measure of iron status low value is diagnostic in the presence of anaemia very high values (>100 µg/l) usually exclude iron deficiency' being an acute-phase protein, it increases in inflammatory conditions, and certain malignancies, making it unreliable also increased in tissue damage especially of the liver levels are falsely decreased in vitamin c deficiency and hypothyroidism erythrocyte zinc protoporphyrin an intermediate in haem biosynthesis and elevated concentrations indicate interrupted haem synthesis due to iron deficiency when zinc is incorporated in place of iron can be measured on a drop of blood with a portable haematofluorometer small sample size makes it very useful as a screening test in field surveys, particularly in children, and pregnant women where inflammatory states may not co-exist red cells should be washed before measurement (serum bilirubin and fluorescent compounds like some drugs can give falsely high values) although not often done lead poisoning can give falsely high values rarely acute myeloid leukaemia and sideroblastic anaemia give slightly high values useful in that it is not increased in thalassaemias who recommends normal level >70 µmol/mol haem iron studies serum iron concentration represents the iron entering and leaving the circulation. its range varies widely with age, circadian rhythm, infections and iron ingestion total iron binding capacity measures iron bound to transferrin. raised levels are suggestive of iron deficiency transferrin saturation is the ratio of serum iron and the tibc expressed as a percentage -it is probably more useful in detecting iron overload rather than low levels. sensitive indicator that falls within days of onset of iron-deficiency reduced levels shown to be predictor of iron deficiency especially in the setting of renal insufficiency false normal values can occur when mcv is increased or in thalassaemia serum transferrin receptor it is not increased in inflammatory conditions may be upregulated by increased erythropoiesis (haemolytic diseases) giving falsely high values -serum transferrin receptor to ferritin ratio has been suggested in these cases bone marrow examination with special iron staining (perl's) absence of stainable iron in a sample that contains particles can establish the diagnosis without other laboratory tests a simultaneous control specimen containing stainable iron should also be assessed useful in differentiating from anaemia of chronic disorders or α-thalassaemia or milder forms of thalassaemia can help in identifying the sideroblastic anaemias (ring sideroblasts with perls stain), and some forms of congenital dyserythropoietic anaemia which can also cause microcytosis. an improvement in haemoglobin and clinical symptoms with iron replacement is probably the simplest way to diagnose iron deficiency. peripheral smear may help by demonstrating pencil cells, anisopoikilocytosis and high platelet number in cases of blood loss. the treatment of vitamin b 12 deficiency can be by the oral or parenteral route. 37 increasing evidence suggests that oral supplementation may be adequate even in the presence of malabsorption or pernicious anaemia. 38, 39 the recommended initial oral replacement dosage is 1-2 mg but higher doses may be needed for malabsorption or pernicious anaemia. 40 for patients with severe anaemia and/or neurological disease, daily or alternate day intramuscular injections should be initiated for the first 2-4 weeks before reverting to the maintenance threemonthly dose. reticulocytosis is an early marker of response to treatment and is noticeable within 1-2 weeks. folic acid deficiency causes similar haematological manifestations to vitamin b 12 deficiency though neuropsychiatric manifestations are less common. the ability of nerve tissue to concentrate folate to levels five times greater than those in the plasma has been suggested as a reason for the absence of neuropathy in folate deficiency. folic acid deficiency is associated with fetal neural tube defects, and possibly with an increase in atherosclerosis and arteriovenous thrombosis, dementia and colonic cancer. dietary folic acid is present in the form of polyglutamates, which are converted to folate monoglutamates by the enzyme folate conjugase in the intestinal brush border, prior to absorption. the monoglutamates function as a carbon transporter and are essential for dna biosynthesis. folate is found in green vegetables and fruits and deficiency can result from decreased intake, impaired absorption and increased utilization, although the commonest cause is dietary insufficiency. 41 in some wealthy countries, cereals have been fortified with folic acid to successfully prevent vitamin deficiency. however folate deficiency continues to be a problem in less wealthy countries and particularly among children and pregnant women. 42, 43 exclusive feeding of goat's milk to infants can lead to folate deficiency. other causes include alcoholism, excessive cooking of vegetables, and malabsorption (e.g. abnormalities of the small bowel). increased demand for folic acid occurs in pregnancy because the growing foetus has a high avidity for folate. for this reason, folate supplementation has been widely recognized as an essential part of routine antenatal care to reduce the risks of neural tube defects. high folate utilization also occurs in haemolytic anaemias such as sickle cell disease due to high red cell turnover and exfoliative dermatitis. several drugs, including sulfasalazine, trimethoprim, methotrexate, pyrimethamine and phenytoin, can also interfere with folate metabolism. folate-deficient individuals develop a macrocytic anaemia with peripheral blood and bone marrow findings similar to that found in vitamin b 12 deficiency. 32 diagnosis of folate deficiency is confirmed by the presence of low serum folate. red cell folate levels decrease more slowly than serum levels during the 120-day turnover of the red cells. red cell folate levels may be a better indicator of tissue folate levels than serum folate, although red cell folate can be more expensive and falsely low in vitamin b 12 deficiency. 45, 44 treatment of folate deficiency is with oral folate (5 mg daily) which is sufficient even in malabsorptive states. it is crucial that any co-existing vitamin b 12 deficiency is ruled out before initiating folic acid therapy, otherwise the neurological manifestations of b 12 deficiency may deteriorate rapidly. it is also important increasingly popular oral replacement therapies. once absorbed, vitamin b 12 binds to transcobalamin ii to be transported around the body. the diagnosis of vitamin b 12 deficiency is based on the measurement of serum vitamin levels in a patient with clinical evidence of deficiency. a note of caution is that folic acid deficiency can cause falsely low serum vitamin b 12 levels. diagnostic clues for vitamin b 12 deficiency include marked macrocytosis (often >130 fl), neutrophil nuclear hypersegmentation and oval macrocytes in the peripheral blood film. blood tests may demonstrate increased lactate dehydrogenase and low haptoglobin levels due to haemolysis within the bone marrow. the cause of the macrocytosis can be confirmed by bone marrow examination which reveals a megaloblastic picture. although macrocytic anaemia is a typical feature of vitamin b 12 deficiency, it can be absent in older individuals who may only have neuropsychiatric features. measurements of methylmalonic acid and homocysteine levels, two markers which are very sensitive for detecting b 12 deficiency, have shown that vitamin b 12 deficiency can occur with normal haemoglobin levels and without macrocytosis. 36 pernicious anaemia is probably the commonest cause of vitamin b 12 deficiency. the presence of parietal cell or intrinsic factor antibodies supports a diagnosis of pernicious anaemia. [33] [34] [35] [36] schilling tests are rarely performed because of the unavailability of the radio-labelled vitamin b 12 and the difficulty in interpreting the results in the presence of renal insufficiency. • pernicious anaemia (begins after 40), increased risk of gastric carcinoma and carcinoid tumours • rare congenital disorders, e.g. imerslund-grasbeck syndrome. the neglected tropical diseases are a group of infections which are endemic in developing countries. several of these neglected tropical diseases cause anaemia and many can be managed using inexpensive interventions to treat the underlying parasitic infections. 14 the mechanisms of anaemia in these conditions are predominantly blood loss from the gastrointestinal or genitourinary tracts but also poor nutrition, bone marrow suppression, inflammation, hypersplenism and haemolysis. 58 anaemia is a common consequence of infections with soiltransmitted helminths or schistosoma with a strong correlation between haemoglobin level and worm load or faecal egg count. even mild infections can lead to anaemia. 59 polyparasitism (i.e. infection with several parasites simultaneously) can be responsible for unresponsiveness of the anaemia to eradication of one organism. 60 treatment of communities at high risk of soiltransmitted helminths improves growth and iron stores in children and reduces anaemia in pregnant women. 61 the treatment of anaemia due to neglected tropical diseases depends on eradication of the parasite with drugs such as albendazole and praziquantel though anaemia resolution may be less successful if it is due to trichuriasis. 62-64 the addition of iron to anthelmintic treatment has met with variable success rates probably because there is associated anaemia related to inflammation. however it is still generally recommended that iron supplementation should be included with anthelmintic therapy in treatment programmes for neglected tropical diseases. [65] [66] [67] introduction haemoglobin s (hbs) has a prevalence of 25-30% in many parts of africa and also some areas in the middle east ( figure 65 .6). hbs tends to be common among ethnic groups that have traditionally had high exposure to plasmodium falciparum malaria. in sub-saharan africa approximately 230 000 infants are born with sickle cell disease each year, mostly with hbss. sickle cell disease (scd) is an autosomal recessive disorder characterized by production of an abnormal haemoglobin, sickle haemoglobin. sickle haemoglobin (hbs) arises from a mutation in codon 6 of the β-globin gene resulting in replacement of the normal glutamic acid residue by a valine. 68 scd is most commonly caused by the co-inheritance of two sickle cell genes (homozygous hb ss disease) but patients who are heterozygous for hbs and for another haemoglobin mutation such as hbc (haemoglobin sc disease) or β-thalassaemia (sβ 0 and sβ + ) can also present with features of scd. 69 ss disease and sβ 0 disease are more severe than sc disease and sβ + disease (box 65.6). 70 scd can affect multiple organs and its clinical course is punctuated by episodes of acute illness on a background of progressive organ damage, especially of the central nervous system and the lungs. 70 the first description of scd was in 1910 in an anaemic grenadian dental student 71 and over the next 30 years it was that the underlying cause of folate deficiency is identified and treated. vitamin a is important in erythropoiesis, iron metabolism (enhances iron absorption and its release from stores to the bone marrow) and for decreasing the risk of infections. 45 vitamin a deficiency is a major public health problem in lowincome countries, with an estimated 200 million preschool children affected. 47 pregnant women and women of childbearing age also constitute high-risk groups for vitamin a deficiency. 46 vitamin a given to thai school children with conjunctival xerosis led to a significant increase in haemoglobin level 47 and in anaemic school children in tanzania, vitamin a supplementation produced a marked increase in haemoglobin which was enhanced by co-administration of iron. 48 vitamin a can also improve anaemia in pregnant women, depending on the local prevalence of deficiency [49] [50] [51] [52] though the response may be suboptimal in pregnant women infected with hiv. 53 copper is a trace element necessary for normal haematopoiesis and myelopoiesis. anaemia in copper deficiency is due to decreased activity of the copper-dependent enzymes, hephaestin, ceruloplasmin and cytochrome c oxidase. these are important in ferrous-ferric iron conversions and their decrease leads to abnormalities in iron absorption and its incorporation into the haemoglobin molecule. acquired copper deficiency occurs with malnutrition and gastrointestinal malabsorption syndromes. coeliac disease, cystic fibrosis and individuals who have had gastrectomy or surgery resulting in 'short bowel' are also at risk. copper deficiency has also been described in persons ingesting excessive amounts of zinc-containing supplements and those who have swallowed zinc-containing coins. 54, 55 anaemia related to copper deficiency is normocytic or macrocytic and can be associated with neutropenia; thrombocytopenia is rare. bone marrow findings are characteristic with cytoplasmic vacuolization of both erythroid and myeloid precursor cells with ringed sideroblasts and an unusual finding of iron granules in plasma cells. these findings may be misdiagnosed as myelodysplastic neoplasm. measurement of serum copper levels is helpful in confirming the diagnosis although the test is fairly insensitive. since almost complete haematological recovery can occur with copper replacement, this may be a useful diagnostic test. oral copper supplements can be started with 8 mg of elemental copper a day slowly decreasing over the next few weeks to 2 mg until a good response is noted. although low zinc levels do not cause anaemia they have been linked to growth retardation, heightened susceptibility to infection and male hypogonadism in relation to sickle cell disease. 56 zinc deficiency has been described in nearly half of children and 70% of adults with sickle cell disease possibly due to increased loss of zinc in the urine and high cell turnover with decreased dietary intake. 57 in contrast zinc excess can cause anaemia through interference with copper absorption by sequestering it in the gut lumen. for this reason, zinc compounds have been used to treat wilson's disease which is characterized by copper excess. however repeated sickling and unsickling eventually causes irreversible changes, 77 so early management to avoid repeated crises is important to prevent disease progression. polymerization, and therefore the clinical features of scd, are influenced by three main factors 78 ; hypoxia, the intracellular hbs concentration and the co-existence of other genetic haemoglobin abnormalities (e.g. α-thalassaemia or hereditary persistence of fetal haemoglobin-haemoglobin f). 79 sickled red cells lead to vaso-occlusion and haemolysis due to the entrapment of sickled erythrocytes in the microvasculature and upregulation of adhesion receptors. 76, 80, 81 white blood cells contribute to this process by providing an inflammatory discovered hypoxia led to red cell sickling 72 scd arises from the tendency of hbs to polymerize in hypoxic states. this phenomenon occurs where there is deoxygenation and is due to the binding between β1 and β2 chains of two haemoglobin molecules, a property unique to haemoglobin variants that have the glu-6-val substitution. 74 the polymerized haemoglobin fills the erythrocyte and deforms its architecture and flexibility to form a sickle shape. this alteration in the structure promotes cellular dehydration, 70, 75, 76 upon reoxygenation, the polymers dissolve thus reversing the sickling process. 90 exposure to cold, fever, menstruation, alcohol intake and dehydration can precipitate pain crises. unlike acute pain crises, chronic pain in scd usually has an identifiable basis such as femoral head necrosis, osteoarthritis or chronic skin ulcers. sickle erythrocytes have an average life span of 17 days and anaemia can be due to several causes (box 65.8). red cell haemolysis causes anaemia and gall stones and can cause fatigue out of proportion to the anaemia. 91, 92 there are suggestions that patients with low haemoglobin concentrations and high haemolytic rates are more likely to develop vascular problems compared with those with higher haemoglobin concentrations. splenic sequestration with a sudden rapid drop in haemoglobin occurs in those who have not yet developed autosplenectomy so it can occur in young children with hbss and adults with hbsc disease or sickle cell-β + -thalassaemia. treatment may require blood transfusion and in rare cases, sequestration can be fatal. splenectomy may be needed for recurrent severe sequestration. parents can be taught to feel their infant's abdomen for an enlarging spleen and report to hospital if there is a sudden increase in spleen size. red cell aplasia can develop due to secondary parvovirus infection which has a predilection for erythroid progenitors. alloimmunization is common in scd patients who have had frequent transfusions so, if possible, extended red cell phenotyping should be undertaken. hyperhaemolytic crisis is suspected when there is sudden exacerbation of anaemia with increased reticulocytosis and bilirubin level. infectious complications of scd are a major cause of morbidity and mortality, even with adequate vaccination and prophylactic antibiotic regimens. this propensity to infection is related to impaired splenic function although tissue ischaemia, especially in the lungs and renal system, can contribute. hyposplenism is demonstrable in the peripheral blood film by the presence of howell-jolly bodies. most children with scd have undergone autosplenectomy by the age of 5 years and therefore have increased risk of infection from encapsulated microorganisms. 93 typical infectious complications include pneumococcal sepsis, neisseria meningitis, osteomyelitis caused by salmonella species, urinary tract infections and pyelonephritis due to escherichia coli. anatomical abnormalities such as renal papillary necrosis can predispose to urinary complications which may require long-term antibiotics. acute chest syndrome (acs) is defined as a new pulmonary infiltrate on the chest radiograph combined with one or more environment. activation of platelets and the coagulation system also contribute to the vaso-occlusion in scd. [82] [83] [84] [85] [86] infants with scd are protected during the first few months of life by the high levels of haemoglobin f in the red cells. anaemia usually develops by 3 months. at all ages, chronic haemolysis of abnormal red cells means that scd is associated with steady state haemoglobin levels of 60-80 g/l. although any organ can be affected by scd and complications can occur at any age, certain features tend to predominate in different age groups (box 65.7). pain is the hallmark of scd 87 and four different patterns of pain have been described with scd each with different underlying mechanisms: 88 • vaso-occlusive (acute and intermittent) • pain from bone and tissue necrosis (chronic) • neuroplasticity (chronic, neuropathic) -functional brain changes • opioid-induced hyperalgesia (acute or chronic). painful crises often start in young children as dactylitis or handfoot syndrome, in which painful swelling of the hands and feet results from the inflammation of metacarpal and metatarsal periosteum. these crises are the result of vaso-occlusion of the bone marrow causing bone infarction and release of mediators that activate pain receptors. 82 the number, severity and frequency of painful episodes vary widely in individuals. half may never have any episodes whereas about 3% may need hospital admission up to 6 times a year. 89 more than three pain episodes requiring hospitalization per year is associated with increased mortality among patients over 20 years old. in under-resourced settings, hospital visits underestimate the frequency of pain box manifestations such as fever, cough, sputum production, tachypnoea, dyspnoea or new-onset hypoxia. 94 acs is the most common cause of death in scd patients and a frequent cause of hospitalization, second only to painful crisis. mortality in patients with acs in a wealthy country setting is 1% in children and 4.3% in adults. 95 the peak incidence for acs is 2-4 years of age and gradually declines to 8.8 per 100 patient-years in subjects older than 20 years. 96, 97 fever and cough are more common in children with acs and chest pain and dyspnoea are more common in adults. 96 acs is often preceded by febrile pulmonary infection in children and by vaso-occlusive pain crisis and lung infarction in adults. it is important to note that although tachypnoea, wheezing and features of chest infection may be identified, a third of the patients may have a normal physical examination. more than one-third of patients with acs are hypoxaemic (oxygen saturation <90%). 98 chest radiography is essential although infiltrates may lag behind clinical symptoms by up to 3 days. repeat chest x-rays are recommended if there is a strong clinical suspicion of acs. bilateral infiltrates or involvement of multiple lobes may predict a poorer prognosis. risk factors for acs (box 65.9) include fat embolus which can be confirmed by finding stainable fat in pulmonary macrophages. 99 chronic complications such as pulmonary hypertension occur in as many as 60% of patients and do not appear to be associated with prior episodes of acs. high serum phospholipase a2, and the surrogate marker c-reactive protein, have been noted in patients admitted with vaso-occlusive crisis 24-48 hours before the development of acs. 100, 101 stroke neurological complications occur in at least 25% of patients with scd and scd is one of the most common causes of stroke in children. 102, 103 in scd, the risk of having a first stroke is 11% by the age of 20, 15% by age 30 years and 24% by age 45 years. both thrombotic and haemorrhagic strokes occur, although the former is more common in children and those over 30 years of age, whereas the latter is more common between the ages of 20 and 30 years. 108 this age-specific pattern may be related to the higher cerebral flow rates in early childhood. although the prevalence of clinically overt stroke is of the order of 11%, clinically silent infarction, detectable by magnetic resonance scans, affect nearly double this number by the age of 20. silent infarcts are associated with cognitive impairment and the majority of these children require lifelong specialist care. 104 cerebral thrombosis, which accounts for 70-80% of all strokes in scd, results from large-vessel occlusion whereas silent infarcts are the result of microvascular occlusion or thrombosis or hypoxia secondary to large-vessel disease. in a third of scd patients, major-vessel stenosis is accompanied by collateral vessels that appear as 'puffs of smoke' (moyamoya) on angiography. risk factors for ischaemic strokes in scd include increased cerebral blood flow velocity, previous silent infarcts, nocturnal hypoxaemia, severe anaemia, acute chest syndrome and elevated systolic blood pressure. an elevated leukocyte count is a risk factor for haemorrhagic stroke. [105] [106] [107] [108] diagnosis often the family history and clinical findings clearly point towards a diagnosis of scd and during an acute crisis, abundant sickled red cells can be seen on a blood film. white cell counts are higher than normal in scd disease, particularly in patients under age 10 years. the presence of sickle haemoglobin in different sickle syndromes (e.g. hbas, hbss, hbsc) ( table 65 .5) can be confirmed by a simple sickle slide or solubility test. haemoglobin electrophoresis will distinguish between many of these variants but high-performance liquid chromatography and iso-electric focusing are preferred for a definitive diagnosis. haemoglobin mass spectrometry and dna analysis are being increasingly used. antenatal screening is available to women in some countries to help to identify couples who are at risk of having a baby with scd. community acceptance of reproductive genetic services however depends on the effectiveness of education and counselling. the use of prophylactic penicillin and the provision of comprehensive medical care during the first 5 years of life have reduced mortality related to scd from 25% to less than 3%. 109 management (box 65.10) individuals with scd are best managed by a multidisciplinary team as they may require a variety of specialist inputs including haematology, ophthalmology, nephrology, obstetrics, orthopaedics and physiotherapy. the cornerstones of scd therapy are disease modification and prompt and effective management of crises. severe pain crises generally require intravenous fluids and adequate, often opiate, analgesia (box 65.11), while disease modification is based on interventions to increase hbf levels. in steady state it is usual practice to give sickle cell patients folate supplements (1-5 mg/day) because their high rates of haemopoiesis put them at risk of deficiency. scd is associated with functional asplenia so patients should also receive prophylactic oral penicillin (250 mg twice a day) and vaccinations against encapsulated organisms. hydroxycarbamide is the main agent used to increase hbf (box 65.12) and is associated with significant reductions in acute pain crises, hospitalization rate, time to first and second pain crises, episodes of acute chest syndrome, and the need for transfusions and the number of units transfused. 110 other beneficial effects of hydroxycarbamide, which are independent of the increase in hbf, include reduced neutrophil count, increased cellular water content, decreased hbs concentration, changing expression of adhesion molecules and nitric oxide generation. 111 hydroxycarbamide may also be an alternative to frequent blood transfusions for the prevention of recurrent stroke in children as it can lower transcranial doppler velocities. 112, 113 under-use of this cheap, effective drug is related to concerns about leukaemogenicity but this has not been shown to be a problem when used for a non-malignant condition like scd. the two main approaches to transfusion 74 in scd are simple top-up transfusion and exchange transfusion. target haemoglobin level in scd therapy is 100 g/l or a haematocrit of 30%; higher target levels are associated with hyperviscosity and box 65.10 management of complications of sickle cell disease • inability to maximally concentrate urine (hyposthenuria) in response to water deprivation is an early finding • renal tubular acidosis • increased urinary tract infections • glomerular hyperfiltration, increased creatinine secretion, and a very low serum creatinine are characteristic of young patients with sickle cell anaemia, so renal dysfunction can be present even with normal serum creatinine values • microalbuminuria is common in childhood and up to 20% of adults develop nephrotic-range protein loss • gross haematuria can develop due to microthrombin in renal vessels, renal medullary carcinoma, and nocturnal enuresis • treatment is based on the early use of hydroxycarbamide and angiotensin-converting enzyme inhibitors in children with clinically significant albuminuria. • noted in up to 60% of scd cases • no relationship to acute chest syndrome (different pathophysiology) • mortality risk with even mild pulmonary hypertension is high • regular blood transfusions and long-term anticoagulation have been tried • hydroxycarbamide may decrease the risk • prostacycline analogues (epoprostenol, and iloprost), endothelin-1 receptor antagonists (bosentan), phosphodiesterase inhibitors (including sildenafil), and calcium channel blockers are being evaluated. • brief but recurrent (stuttering); may occasionally last for many hours and can lead to impotence • usually ischaemic, or low-flow, priapism • patients should be educated to seek medical attention if more than 2 hours duration • detumescence within 12 hours is necessary to retain potency • intravenous hydration and analgesia initially with consideration for α-adrenergic agonists (etilefrine or phenylephrine) • penile aspiration and irrigation with saline and α-adrenergic agents or shunting may be required in severe cases in combination with an exchange transfusion. • assess pain intensity • choose the analgesic, dosage, and route of administration • paracetamol and hydration should be considered in all patients • oral, sustained-release morphine is as good as intravenous morphine infusion in children and young adults • manage mild pain with rest, hydration, and weak opioids (such as codeine). admit patients in whom pain that does not subside promptly or require opioid treatment; fever, pallor, or signs of respiratory compromise; a low likelihood of receiving appropriate care at home • pain management should be individualized and dosing should take into account prior pain management and use of opioids • the pain pathway should be targeted at different points with different agents, avoiding toxicity with any one class • always look for a cause, e.g. infection, dehydration, etc. • education about avoiding exposure to precipitants • be empathetic, reassuring, and supportive • benzodiazepines may be helpful to reduce anxiety • re-examine the patient often to ensure adequate pain relief, to assess sedation and respiratory rate (to avoid opioid overdose). in assessing patient responses to conventional doses of analgesia, it must be remembered that those with sickle cell disease metabolize narcotics rapidly • re-search for evidence of any complications such as acute chest syndrome or anaemia • always look for a cause, e.g. infection. of multi-organ failure. 116 both simple transfusion and exchange transfusions have been used and neither appears to be superior. a short course of steroids may attenuate acs but it may also increase the risk of re-hospitalization. 117 bronchodilators may help patients with wheezing 118 but inhaled nitric oxide has not shown any clear benefits. 119 since coagulation activation is important in the pathophysiology of acute chest syndrome, treatment with low-molecular-weight heparin may reduce clinical complications. 120 transcranial doppler measurement of cerebral blood flow has been a major step forwards in identifying individuals with an increased risk of ischaemic stroke. a value more than 200 cm/ second imparts a 40% risk of stroke within the next 3 years. 121 regular blood transfusions can reduce the incidence of stroke in children. 105 due to a high recurrence of stroke (60%) on stopping transfusions, continuation of transfusions should be guided by transcranial doppler measurements. 122, 123 once a stroke has developed, the best therapeutic strategy is exchange transfusion which probably needs to be done monthly. 70, 73 neurosurgical re-vascularization should be considered for moyamoya-like syndromes when new strokes occur despite transfusion. haemoglobin sc results from the co-inheritance of hbs and hbc and has its highest prevalence in west africa. clinical features and disease management are similar to those of hbss disease but splenomegaly, splenic infarcts and splenic sequestration may occur into adulthood. proliferative retinopathy necessitates regular ophthalmic review in those aged over 10 years. compared with hbss, anaemia is less marked in hb sc (8-140 g/l) and there are fewer sickle cells and more target cells on the blood film. the diagnosis can be confirmed by haemoglobin electrophoresis, hplc or iso-electric focussing. worsening of complications. in exchange transfusion, the aim is to achieve an hbs% of <30%. complications of transfusion in scd include alloimmunization, 114 delayed haemolytic transfusion reactions and iron overload. the high rates of red cell antibody formation (30%) noted in wealthy countries are due to minor blood group incompatibilities between the recipient and the blood donor who is often of a different ethnicity. leukocyte reduction of transfused blood, routine abo, rh and kell matching for all patients and extended phenotype matching for those with alloantibodies may be useful for reducing transfusion reactions. treatment for acs is predominantly supportive and includes adequate pain relief, antibiotics (e.g. a macrolide with a cephalosporin), continuous pulse oximetry and delivery of supplemental oxygen to patients with hypoxaemia. incentive spirometry can prevent atelectasis and infiltrates 115 and blood transfusion is indicated when a patient develops respiratory distress, a clinically significant fall in the haematocrit or signs the following predict a more severe clinical course and are additional reasons to consider offering hydroxyurea: hb <70 g/l, wbc >15 × 10 9 /l, hbf <6% and renal insufficiency due to scd. • start at 10-15 mg/kg per day (to the nearest 500 mg/day) • if no or poor response, increase dose by increments of 5 mg/ kg per day every 4 weeks (max: 30 mg/kg per day). most good responses require about 1-2 g/day in adults • monitor fbc, hbf%, and reticulocytes every 1 or 2 weeks initially, then every 4 weeks when on a stable dose • monitor biochemistry profile (hydroxyurea has renal excretion and hepatic toxicity). • less pain • persistent increase in hbf (usually measured every 6-8 weeks) or mean cell volume • persistent increase in haematocrit if severely anaemic • decrease in ldh • acceptable toxicity. improvement in symptoms and blood parameters may take 3-4 months of therapy, but can be seen after approximately 6 weeks. if the reticulocyte count is less than expected for the degree of anaemia, erythropoietin deficiency should be considered. • aim in all cases to reduce hbs level to <30% • exchange transfusions may be considered in cases of stroke, acute chest syndrome not responding to top-up transfusion and major surgeries • target haemoglobin concentration of 100 g/l may be considered in cases of organ failure and surgery. individuals with sickle cell trait (hb as) have 10-fold protection against severe malaria compared to individuals with normal haemoglobin (hbaa) probably due to both innate and immune-mediated mechanisms. individuals with sickle cell trait (hbas) are generally asymptomatic and they have a normal haemoglobin and normal life expectancy. uncommonly, complications such as poor perfusion of the renal papillae and increased bacteruria may occur. 124 the blood film is generally normal and the diagnosis can be confirmed by haemoglobin electrophoresis, hplc or iso-electric focusing. the original descriptions of thalassaemia originated from areas round the mediterranean and the term derives from the greek thalassos (sea) and haima (blood). [125] [126] [127] epidemiology thalassaemia is one of the most common single gene disorders and approximately 5-7% of the global population are carriers. α + -thalassaemia occurs throughout the tropics, whereas α 0thalassaemia, which is responsible for haemoglobin bart's hydrops fetalis, is concentrated predominantly in south-east asia and to a lesser extent around the mediterranean. 128, 129 β-thalassaemia is common in the mediterranean countries, parts of africa, throughout the middle east, the indian subcontinent and south-east asia. haemoglobin e prevalence is highest in cambodia, laos and thailand and can reach 50-60% with lower prevalence rates in indonesia, malaysia, singapore and vietnam. β-thalassaemia β-thalassaemia is an inherited quantitative deficiency of β-globin chains which are required to make normal adult haemoglobin. 130 more than 200 mutations have been associated with the development of β-thalassaemia (a complete list is available at the globin gene server website, at: http://globin.cse. psu.edu) and they affect protein synthesis 130, 131 leading to reduced (designated β + ) or absent (designated β 0 ) production of the β-globin chains. the clinical severity of thalassaemia can be lessened by co-existing haemoglobin abnormalities such as the co-inheritance of α-thalassaemia and increased production of haemoglobin f. 132, 133 α-thalassaemia normal α-globin synthesis is regulated by duplicate α-globin genes on chromosome 16. the genotype is usually represented as αα/αα and α-thalassaemia usually results from deletion of one or both α-genes. occasionally point mutations in critical regions of the α-genes may cause non-deletional α-thalassaemia (α t ). 130 mutations can completely abolish expression of the αgenes (i.e. α 0 -thalassaemia) or partially down-regulate expression (α + -thalassaemia). 130 both α 0 and α + thalassaemias can occur in the heterozygous or homozygous state or as a compound α 0 /α + heterozygote form (table 65 .6). underproduction of α-globin chains due to three or four gene deletions gives rise to excess γ (fetal) or β (adult) globin chains which form tetramers, called hb bart's (fetal) or hbh (adult). 134 rare forms of α-thalassaemia occur in association with other conditions such as mental retardation and myelodysplastic/ leukaemia syndrome. 135, 136 pathophysiology β-thalassaemia (figure 65 .8) thalassaemias 131, 137, 138 cause an imbalance of αand β-globin chain synthesis. in homozygous β-thalassaemia, excess α-chains precipitate in the red cell precursors and up to 75% of cells are destroyed in the bone marrow resulting in ineffective erythropoiesis and a shortened red cell survival. the red cells released from the bone marrow contain abnormal α-chains and these inclusions promote destruction of the cells by the spleen leading to clinical symptoms and signs of haemolysis. in heterozygotes, the α-chain excess and the degree of inadequate erythropoiesis is much less than in homozygous β-thalassaemia. hbf production normally tails off within a few months of birth but in β-thalassaemia hbf production can continue into adulthood. the effect of increased hbf production is to prevent precipitation of the excess globin chains and consequent ineffective erythropoiesis. however hbf has a high oxygen affinity, which can lead to increased erythropoietin production and thus, increased bone marrow expansion. the pathophysiology of α-thalassaemia, and hence the clinical manifestations, is quite different from β-thalassaemia. the excess non-α-globin chains form soluble tetramers rather than precipitates so there is only minimal ineffective erythropoiesis. the only clinical abnormality in those with hbh may be splenomegaly secondary to increased work load from destruction of red cells containing inclusions. rarely anaemia may be severe enough to require blood transfusions. classification of α-thalassaemia divide β-thalassaemia into thalassaemia major (transfusiondependent), thalassaemia intermedia (able to maintain adequate haemoglobin without transfusions or requiring less than 8 units/year) and thalassaemia minor (asymptomatic). 139 infants with β-thalassaemia are protected from severe anaemia by the presence of haemoglobin f and are usually asymptomatic. clinical manifestations of thalassaemia major depend on whether adequate blood transfusions are available and the stringency with which iron chelation is undertaken. untreated patients with thalassaemia major will die in late infancy or early childhood from the effects of severe anaemia. those who receive sporadic transfusions may survive longer but suffer from the secondary effects of anaemia, bony deformities and growth retardation. the clinical features of β-thalassaemia major are divided into those resulting from anaemia, bony changes and iron overload. anaemia from defective erythropoeisis, decreased red cell survival and increased haemolysis in thalassaemia major leads to cardiac decompensation, failure to thrive and growth retardation in children. splenomegaly, from the increased work load of culling red cells with inclusion bodies, can cause dilutional anaemia and a further drop in haemoglobin. compensatory extra-medullary haematopoiesis can lead to hepatomegaly and occasionally vertebral compression and neurological defects. haemolysis from increased red cell destruction is associated with gall stones in up to 20% of individuals with β-thalassaemia. 140 another consequence of accelerated haemolysis is the increased incidence of thromboembolism (4% in thalassaemia major and 10% with intermedia) from the exposure of negatively charged phospholipids on the red cell membrane and the generation of red cell and platelet microparticles. 141 splenectomy with postoperative thrombocytosis is a risk factor for thrombosis especially if combined with endothelial oxidative stress from iron overload, or procoagulant co-morbid conditions such as diabetes mellitus, hormone therapy, thrombophilic mutations and atrial fibrillation. 142 folate deficiency, hyperuricaemia and occasionally gout have been observed in thalassaemia major due to the high turnover of red cells. the enhanced erythropoietic drive from anaemia in thalassaemia can lead to increased marrow expansion with in homozygous β-thalassemia, β-globin synthesis is markedly reduced or absent. the excess α-chains cannot form a tetramer but form a precipitate in the red cell precursors leading to intra-medullary destruction of these cells. this destructive process of the red cell membrane occurs from the formation of α-chain hemichromes (shown as red cell inclusions) and degradation products of the excess α-chains. the red cells which may be released from the bone marrow are destroyed by the spleen leading to clinical symptoms and signs of haemolysis. since only the β-chain is affected in these individuals, the synthesis of hbf and hba 2 continues unabated. these haemoglobins have very high oxygen affinity, which can lead to increased erythropoietin production and thus, increased bone marrow expansion splenomegaly, which may be massive, and growth retardation in children. bony changes are unusual. other complications include infections, leg ulcers, gall stones and acute haemolysis in response to drugs and infections. the severity of the clinical features is related to the molecular basis with non-deletional types of hbh disease more severely affected. haemoglobin bart's (−/−) occurs almost exclusively in asians, especially chinese, cambodian and thai populations. an infant with hb bart's hydrops fetalis syndrome has pallor and gross oedema with signs of cardiac failure, marked hepatosplenomegaly and skeletal and cardiovascular deformities. there is often gross hypertrophy of the placenta. many of the clinical manifestations of this condition can be explained by the characteristic bossing of the skull and overgrowth of maxillary region, radiologically noted as 'hair on end' or 'sun-ray' appearance. metatarsal and metacarpal bones are the first to expand so measurement of the metacarpal bones has been considered a good indicator for initiation of transfusion therapy. 143 other skeletal deformities include shortening of long bones due to early epiphyseal fusion and overgrowth of the maxilla causing dental malocclusion. the marrow expansion can also lead to pathological fractures, early bone thinning and osteoporosis 144, 145 while ineffective drainage of the sinuses and middle ear from skull bone overgrowth can cause chronic sinus and ear infections. growth retardation is primarily the result of anaemia with contributions from iron overload, hypersplenism, deficiencies of thyroid and growth hormone, hypogonadism, zinc deficiency, chronic liver disease, malnutrition and psychosocial stress. 146 patients with β-thalassaemia have increased iron absorption mediated by reduced hepcidin and those who receive regular transfusions may also develop transfusion siderosis if they are inadequately chelated. the iron is deposited in the parenchymal tissues with a variety of clinical consequences (box 65.14), [147] [148] [149] [150] [151] [152] [153] [154] a process which may be modulated by variants in the haemochromatosis (hfe) gene. 155 thalassaemia intermedia is characterized by haemoglobin concentrations of 70-100 g/l and children usually present at around 2-4 years of age with symptoms of anaemia, jaundice and hepatosplenomegaly. 156 there may also be skeletal changes such as expansion of the facial bones and obliteration of the maxillary sinuses. 157 several molecular factors including: (a) coinheritance of α-thalassaemia; (b) hereditary persistence of haemoglobin f; (c) δβ-thalassaemia and (d) the specific gγxmn1 polymorphism contribute to the 'conversion' of thalassaemia from major to intermedia type. 158 in contrast to patients with thalassaemia major, iron loading in thalassaemia intermedia occurs mainly as a result of increased intestinal iron absorption rather than transfusion therapy. ineffective erythropoiesis with resultant chronic anaemia and hypoxia can suppress hepcidin, the regulator of iron metabolism, leading to increased iron absorption. 158 the excess iron tends to accumulate in the liver rather than the heart. 159 other clinical complications in thalassaemia intermedia include gallstones, extramedullary haemopoiesis leg ulcers, thromboembolic events and pulmonary hypertension, which is the major cause of heart failure in these individuals. 160 although individuals with thalassaemia intermedia do not usually need regular blood transfusions, there is some evidence that complications, particularly later in life, may be less common in regularly transfused patients. 159 α-thalassaemias 130, 134 carriers of α-thalassaemia (traits, with loss of 1 or 2 α genes) are usually asymptomatic and may only be detected through a routine blood count which shows mild to moderate microcytic, hypochromic anaemia. antenatal counselling may be indicated if the mother has αα/− as there is a possibility that the fetus may be at risk of having haemoglobin bart's. haemoglobin h disease occurs mainly in asians and occasionally in the mediterranean population. it is the result of deletion of three α genes (α−/−) and can produce anaemia varying from 30-130 g/l. there is usually associated • hypogonadism is the most frequent complication in patients with prevalence over 50% in both males and females. it is usually hypogonadotrophic suggesting iron damage to the anterior pituitary or hypothalamus. the features range from total absence of sexual development to delayed puberty. in females with normal menstrual function, fertility is normal with the ovarian function preserved in most although secondary amenorrhoea can develop. damage of the ovaries is rare and is more likely to appear in older women (around 30) because of high vascular activity on the ovaries at this age. secondary hypogonadism is common (50%) in older men. serum ferritin >2000 ng/ml is a risk factor. • hypothyroidism is the second most common endocrine disorder (about 20%) although many of them may have the subclinical variety. most commonly hypothyroidism is of the primary type with secondary, central hypothyroidism increasingly being diagnosed in recent years. • the prevalence of diabetes mellitus is around 5% with the mean age of diagnosis being 18 years. impaired glucose tolerance occurs first with microvascular damage like retinal changes being less common than the conventional form. erythroid precursor destruction. osmotic fragility is reduced, sometimes strikingly so since in some cases the red blood cells do not haemolyse even in distilled water. 161 for this reason, if sophisticated tests are not available, osmotic fragility can be used as a screening test for thalassaemia trait. serum zinc levels may be low and this may be related to abnormal growth. 162 vitamin c levels may also be low due to its increased conversion to oxalic acid in the presence of iron overload. 163 care may be needed if folic acid is commenced on a background of bone marrow failure due to folate deficiency as it may precipitate painful erythropoietic crises. 164 management a comprehensive management plan for patients with thalassaemia may involve transfusion therapy, iron chelation, splenectomy, prevention or early treatment of complications and stem cell transplant. the mainstay of treatment for the severe forms of thalassaemia is blood transfusion with the aim of reducing anaemia and erythropoietic drive. however, in many low-income settings blood supplies are inadequate and many thalassaemic patients are chronically under-transfused (table 65 .7). 165 transfusion frequency should be guided by clinical symptoms and signs such as poor growth and facial or other bone abnormalities, and should take into account any potential disease-modifying comorbidities. 156 although the decision to transfuse should not be based purely on haemoglobin levels, a value of <70 g/l is often used as a trigger for regular transfusions. to prevent alloimmunization, extended red cell antigen typing for c, e and kell in addition to abo and rh(d) typing should be carried out prior to the first transfusion, and before each transfusion, full cross-match and screening for new antibodies should be undertaken. 166 the risk of alloimmunization appears to be greater in patients who begin transfusion therapy after the first few years of life. 128 development of alloantibodies and autoantibodies may result in increased transfusion requirements or haemolysis. use of leukodepletion techniques can result in less alloimmunization and fewer febrile transfusion reactions. since storage of red cells in anticoagulant solutions may decrease their efficacy, the use of blood that has been stored for less than 7-10 days may be beneficial for patients who require frequent transfusions. the use of 1st-degree relatives as blood donors should be discouraged, especially if the patient is a candidate for stem cell transplant. patients with thalassaemia major need lifelong regular blood transfusions, 15 ml/kg per month or 1-2 units of blood every 2-5 weeks, to maintain the pre-transfusion haemoglobin level above 90-105 g/l. the clinical benefits of this regular transfusion programme include normal growth, suppression of erythropoiesis and bone marrow expansion, reduced hepatosplenomegaly and an overall sense of wellbeing, which allows normal age-appropriate activities. a higher target pretransfusion haemoglobin level of 110-120 g/l may be necessary for patients with heart disease or other medical conditions and for those patients who do not achieve adequate suppression of bone marrow activity at the lower haemoglobin level. shorter intervals between transfusions may reduce overall blood requirements but need to be balanced against the patient's work or school schedule and other lifestyle issues. iron chelation therapy 167, 168 has improved survival rates for thalassaemic patients, and prevented hepatic fibrosis and ironinduced cardiac disease; most patients who are compliant with chelation therapy have normal growth and sexual development. iron chelators (box 65.16) are usually initiated in children over 2 years who have received 10 units of blood and/or have a steady-state serum ferritin level above 1000 ng/ml on at least two occasions. this level of iron overload typically occurs after 1-2 years of transfusions. desferrioxamine is started at 25-30 mg/kg per day in these children initially, to avoid toxicity due to over chelation. marked splenomegaly, often treated with splenectomy, was common in thalassaemia patients before the advent of regular transfusion programmes. severe haemolysis in thalassaemia is related to a hyperactive spleen, which aggravates anaemia and can increase transfusion requirements. although early the initiation of regular transfusion therapy for severe thalassaemia usually occurs in the first 2 years of life. some patients with thalassaemia intermedia who only need sporadic transfusions in the first two decades of life may later need regular transfusions because of a falling haemoglobin level or the development of serious complications. haemoglobin should be monitored to assess the rate of fall in the haemoglobin level between transfusions and this can be used to indicate the frequency of transfusions. exchange transfusions have been tried as a way of reducing iron loading and are associated with a reduction in blood requirements by about one-third. (table 65. since each unit of red cells can contain up to 200 mg of iron, cumulative iron burden is an inevitable consequence of a longterm transfusion programme. in addition there is increased iron absorption from the gut (0.3-0.6 mg/kg per day) as a response to severe anaemia and down-regulation of hepcidin. transfusion therapy can avert splenomegaly, hypersplenism still can develop, usually in children between 5 and 10 years of age. in these individuals, splenectomy can limit the complications from extramedullary hematopoiesis. splenectomy should be considered when the annual transfusion requirement reaches 200-250 ml red blood cells/kg per year and usually results in a halving of the transfusion requirements. splenectomy complications include opportunistic infections with encapsulated organisms. patients should therefore receive appropriate vaccinations preoperatively and should be advised to seek medical advice at the first sign of infection. it is advisable to delay splenectomy until patients are at least 5 years old because of the increased risk of overwhelming sepsis below this age. thalassaemia patients can develop thromboembolic complications and pulmonary hypertension after splenectomy so partial splenectomy and splenic embolization have been attempted to minimize these complications but have not been studied in large trials. iron overload can occur in any organ in thalassaemia patients but particularly affects the heart, liver, the endocrine system, the bone and occasionally the pancreas and lungs. iron overload needs to be detected early and treated to prevent long-term damage. annual assessment of the iron loading of the liver and heart can be achieved using non-invasive methods such as magnetic resonance scanning to detect early changes. children should have regular growth and endocrine assessments and appropriate investigations should be carried out if there are any signs of developmental delay or hormonal deficiencies. osteoporosis is increasingly being recognized and should be prevented by ensuring adequate dietary calcium intake and sun exposure. vitamin supplementation with folic acid, zinc, vitamin e and vitamin c may be useful although the combination of vitamin c and desferrioxamine carries a risk of cardiac toxicity. allogeneic stem cell transplant 169, 170 is currently the only means of curing thalassaemia. the outcome in carefully selected patients, measured by overall event-free survival, is around 90% with a transplant-related mortality of 3%. hepatomegaly, liver fibrosis, and inadequate iron chelation therapy predict a poor outcome. the best results from transplant have been obtained with hla-matched siblings. umbilical cord blood is a useful source of stem cells for young children. other potential treatment options for thalassaemia are outlined in box 65.16. [171] [172] [173] [174] prevention of severe thalassaemia births by prenatal diagnosis and termination of pregnancies has been successful in countries with a high prevalence of thalassaemia. 175 early identification of couples at risk and culturally sensitive genetic counselling facilitate decision-making for termination or continuation of pregnancy. the mean corpuscular haemoglobin (mch) is used to screen for the presence of thalassaemia using a cut-off of less than 27 pg. rarely, silent β-thalassaemia mutation may present with an mcv over 27 pg and should be considered in those with a positive family history. at-risk couples should be referred for detailed counselling on the options for prenatal diagnosis. these include chorionic villous sampling or amniocentesis, which are used to obtain fetal dna samples for genetic analysis. polymerase chain reactions and precise hybridization assays to detect single point mutations using very small dna samples have also been developed. a less invasive and less risky option is to isolate fetal dna circulating in the maternal blood for genetic analysis. pre-implantation genetic diagnosis is a newer technique where dna from the blastomere is used for genetic diagnosis. ultrasound can be used from the 2nd trimester for fetuses suspected of having α-thalassaemia to detect signs of hydrops fetalis and enlarged placenta (figure 65.9) . 175 • hydroxyurea -helpful in some patients with β-thalassaemia intermedia, but not as effective in thalassaemia major • histone deacetylase inhibitors -derivatives of butyric acid; intermittent pulses with hydroxycarbamide has been tried • kit ligand • decitabine • knockdown of bcl11a (regulator of γ-globin expression) • erythropoietin. • vitamins c and e • fermented papaya preparations. • successful in β-thalassaemia animal models using a retroviral vector transferring the human β-globin gene sequence and its promoter region into mice stem cells • β-globin gene transfer into progenitor hematopoietic cells of humans is also being studied • other molecular approaches being tried include using different mutations of stop codons and aberrant splicing. partner testing in all cases β-thalassaemia trait protecting red cells. red cells lack any other source of nadph and are solely dependent on the pentose phosphate pathway so g6pd deficiency leaves these cells with no defence against oxidative damage. oxidative damage results in denatured haemoglobin aggregates which form heinz bodies (denatured haemoglobin precipitates). these damaged cells bind to the membrane cytoskeleton resulting in decreased cellular deformability, and are also destroyed in the spleen, resulting in haemolysis. the level of enzyme activity is higher in young erythrocytes than in more mature cells so older cells are more susceptible to haemolysis. the global distribution of g6pd deficiency mirrors that of malaria, and where malaria has historically been prevalent, and it provides a degree of protection against malaria. 181 • class iv -normal (60-150% enzyme activity) • class v -increased activity (>150% enzyme activity). g6pd enzyme variants can be distinguished by their electrophoretic mobility. 184 g6pd b, the wild-type enzyme, and g6pd a + , a common variant in populations of african descent, demonstrate normal enzyme activity and are not associated with haemolysis. g6pd a − is the most common variant associated with mild to moderate haemolysis with approximately 10-25% hb e is caused by a substitution of glutamic acid by lysine at codon 26 of the β-globin gene. 177 this causes reduced synthesis of the β-e chain and leads to a thalassaemia phenotype. hb e β-thalassaemia affects at least a million people worldwide and is an important health problem particularly in the indian subcontinent and south-east asia. in some areas, it has replaced β-thalassaemia as the most common thalassaemia disorder. the frequency of hbe reaches 60% in many regions of thailand, laos and cambodia with estimates of at least 100 000 new cases of hbe β-thalassaemia expected in the next few decades in thailand alone. the natural history of hbe thalassaemia is highly variable; some patients are asymptomatic (e.g. heterozygotes, hbe 20-30% or homozygotes hbe, 80-90%) while others (e.g. hbe with β-thalassaemia) may be transfusiondependent. pathophysiology glucose-6-phosphate dehydrogenase (g6pd) deficiency was originally recognized through its association with haemolysis related to eating fava beans ('favism') and primaquine ingestion. 178 g6pd deficiency is the most common enzyme defect in humans and is present in about 400 million people worldwide (figure 65 .10). 179, 180 it is an x-linked, hereditary defect caused by mutations in the g6pd gene. g6pd is an enzyme that catalyses the first reaction in the pentose phosphate pathway, to produce nadph, which is an important antioxidant used to preserve the reduced form of glutathione. 178, 181 reduced glutathione acts as a scavenger for oxidative metabolites thereby <0.5% 0.5-2.9% 3-6.9% 7-9.9% 10-11.9% 15-25% of africans carrying this variant. g6pd mediterranean, present in all countries surrounding the mediterranean sea, middle east, india and indonesia, has the same electrophoretic mobility as g6pd b but the enzyme synthesis and catalytic activity are reduced. in several populations, g6pd a − and g6pd mediterranean co-exist. the clinical manifestations of g6pd deficiency can be classified into: (i) asymptomatic; (ii) acute haemolytic anaemia; (iii) favism; (iv) neonatal jaundice; and (v) chronic non-spherocytic haemolytic anaemia. acute haemolytic anaemia in g6pd deficiency can be secondary to infection (e.g. pneumonia, hepatitis a and b, and typhoid fever) or oxidant drugs, or may be precipitated by diabetic ketoacidosis, myocardial infarction and strenuous physical exercise. 185, 186 a list of the drugs which may cause haemolysis in g6pd-deficient individuals (table 65 .9) 187 can be obtained from: http://www.g6pd.org/favism/english/index.mv. a drug which is deemed to be safe for some g6pd-deficient individuals may cause haemolysis in others due to the heterogeneity of the underlying genetic variants. haemolysis typically occurs within 1-3 days after commencing the drug and can produce intense haemoglobinuria. fortunately, the disorder is self-limiting and most patients do not develop renal impairment or anaemia requiring transfusion. the spontaneous recovery reflects replacement of the older, enzyme-deficient red cells by younger reticulocytes which can withstand oxidative injury. 186 if the precipitating cause has been removed the haemoglobin begins to recover after 8-10 days. acute renal failure due to acute tubular necrosis and tubular obstruction by haemoglobin casts can develop as a complication of haemolysis in g6pd deficiency. this occurs more often in adults than children and may require haemodialysis. this occurs predominantly in boys aged 1-5 years in mediterranean countries, but it has also been observed in the middle east, asia and north africa. both intravascular and extravascular haemolysis, occasionally severe enough to cause renal impairment, can occur after eating fresh or cooked fava beans, and favism has been reported in breastfed babies of mothers who have eaten fava beans. 179 divicine and isouramil have been implicated as the toxic components of fava beans. 188 neonatal jaundice this occurs in one-third of male babies in areas where g6pd deficiency is common and is likely due to g6pd deficiency. 179 it presents 1-4 days after birth and can lead to kernicterus. 189, 190 maternal exposure to oxidant drugs, and even naphthalenecamphor mothballs, can precipitate haemolysis in affected babies. breast-feeding mothers should therefore be warned to avoid offending drugs, umbilical potions containing fava, triple dye or menthol, and should not apply henna to the skin or use clothes that have been stored in naphthalene. 190 premature infants and babies who have co-inherited the mutation for gilbert's syndrome are at particular risk. phototherapy and exchange transfusion therapy may be required to reduce the level of unconjugated bilirubin. the diagnosis may be easily missed so assessment of g6pd status should be undertaken for any jaundiced infant whose family history or ethnic or geographic origin suggest the likelihood of g6pd deficiency, and in infants who respond poorly to phototherapy. 191 this is an unusual manifestation of g6pd deficiency and usually presents in childhood. 179, 184 there may be a history of severe neonatal jaundice, episodic or worsening anaemia which requires blood transfusions, and complications from gallstones. although these individuals usually have a well-compensated anaemia, and require transfusions only for exacerbations, rarely some may become transfusion-dependent. antioxidants such as vitamin e and selenium may be of benefit in some cases. the haemolysis does not resolve following splenectomy. folic acid supplementation is necessary to support the increased compensatory erythropoiesis. the diagnosis of g6pd deficiency is usually suspected when neonatal jaundice occurs in an area where g6pd deficiency is drugs which may cause haemolysis in g6pd-deficient individuals common or when an episode of non-immune haemolytic anaemia occurs in association with an infection or drug. the appearance of the red cells on the blood film is characteristic because denatured haemoglobin concentrates in one area within the cell creating 'helmet' or 'bite' cells. 192 denatured haemoglobin precipitates in peripheral red blood cells as heinz bodies which can be detected by staining with methyl violet. definitive diagnosis of g6pd deficiency is by quantitative spectrophotometric analysis of the rate of nadph production. point of care tests for g6pd deficiency are being developed but have not yet been validated for routine use. measuring enzyme activity during an episode of acute haemolysis is not helpful since reticulocytosis, which is a feature of acute haemolysis, produces a false-negative result because of the high enzyme levels in younger erythrocytes. 179, 186 management the most effective management strategy for g6pd deficiency is to prevent haemolysis by avoiding triggering agents like infections, drugs and fava beans. for the milder variants (e.g. class iii and iv), drugs known to trigger haemolysis may be given to individuals with g6pd deficiency if the benefits outweigh the risks and the blood count is closely monitored (e.g. use of low-dose primaquine for individuals with g6pd avariant). screening programmes have been established in some mediterranean and other populations where g6pd deficiency is prevalent. 193 haematological complications of malaria (see chapter 43) the pathophysiology of anaemia in malaria is multi-factorial and influenced by the age of the individual and their antimalarial immune status. anaemia mechanisms in malaria involve: • haemolysis with increased red cell destruction of both infected and bystander erythrocytes • dyserythropoiesis • hypersplenism • haemolysis • co-existent conditions which can cause anaemia. haemolysis is more common in non-immune individuals with acute malaria, whereas dyserythropoiesis is the predominant mechanism for anaemia in recurrent falciparum malaria. 207, 194 haemolysis is the result of red cell phagocytosis by the reticuloendothelial system and is triggered by damage to the red cell membranes and exposure of abnormal surface antigens on their surface. [195] [196] [197] [198] ten uninfected red cells are removed from the circulation for each infected red cell destroyed, 199 possibly related to loss of red cell complement regulatory proteins and increased levels of circulating immune complexes. 200 this may partly explain the persistent or worsening anaemia following parasite clearance and the poor correlation between parasitaemia and the severity of anaemia noted in some studies. 207 an increased incidence of anaemia has been noted in malaria vaccine trials possibly due to enhanced clearance of uninfected red blood cells. 201 decreased erythropoeisis with abnormalities in red cell precursors and reticulocytopenia is found consistently on examination of bone marrow from malaria-infected patients. 202 the decreased erythropoiesis is due to many factors including low levels of tnf-α, high levels of interleukin-10, abnormalities of erythropoietin, a decrease in burst colony forming units, cytokine-induced suppression of red cell production and the inhibitory effect of the malarial pigment haemozoin. [203] [204] [205] [206] epidemiology malaria-related anaemia is most commonly seen in children and pregnant women. the prevalence of malarial anaemia in sub-saharan africa in children is 30-90% and in pregnant women it is 60-80%. 207 the highest prevalence is in infants and children less than 3 years of age. infants may acquire malaria through the placenta. 208, 209 individuals living in malarious areas may have multiple reasons for anaemia such as bacteraemia, hookworm infections and vitamin a deficiency 208 making it difficult to assign anaemia solely to malaria. however, animal studies and the fact that anaemia improves with anti-malarial treatment suggest a direct relationship between malaria infection and anaemia. 210, 211 for example, in tanzanian children about 60% of anaemic episodes were thought to be caused by malaria. 212 who defines severe anaemia attributable to malaria as: (i) haemoglobin concentration <50 g/l or haematocrit <15%; (ii) parasitaemia with >10 000 parasites/µl of blood and (iii) normocytic blood film (to exclude other common causes of anaemia). 213 however, aspects of this definition have been criticized because blood films are not examined routinely and parasite density varies with endemicity and age. 207 although traditionally it is p. falciparum that has been associated with the most severe malaria-related anaemia, p. vivax is also a major risk factor for severe anaemia especially in young children or those with chronic and recurrent infections. p. vivax anaemia is associated with recurrent bouts of haemolysis of predominantly uninfected erythrocytes with increased fragility. 214 symptoms of malarial anaemia can vary from negligible to profound depending on the degree of anaemia and the rapidity of onset. splenomegaly is a common feature of malarial anaemia because of the role of the spleen in the removal of both infected and uninfected red cells. 215 blackwater fever, characterized by intense intravascular haemolysis with haemoglobinuria and occasionally renal failure in a patient with malaria, may be related to underlying glucose-6-phosphate deficiency. 216, 217 factors such as poor nutrition, deficiencies of vitamins and micronutrients, bacteraemia, and hookworm or hiv infection may co-exist with malaria and contribute to anaemia 209 so nonmalarial causes of anaemia should be considered in patients whose anaemia does not respond to malaria treatment. the management of severe malarial anaemia involves supportive care and treatment of the malaria and any other underlying conditions. recovery from malaria-associated anaemia can be slow, taking 6 weeks or even longer if there are episodes of re-infection. 212 in children, blood transfusion is usually reserved for those with haemoglobin levels of less than 40 g/l (<50 g/l if there are complications such as respiratory distress 207 ). there of parasitized red cells 227 and the release of von willebrand factor multimers which cause widespread platelet aggregation leading to thrombocytopenia 228, 84, 231 platelet synthesis by the bone marrow is relatively well maintained during infection 231, 229 but antiplatelet antibodies, immune complexes and splenomegaly all contribute to thrombocytopenia. 230 thrombocytopenia occurs in 60-90% of individuals infected with malaria irrespective of the species of plasmodium. 214, 231 thrombocytopenia in febrile patients in an endemic area increases the probability of malaria by a factor of 5 232 and in individuals returning from tropical countries with a fever, thrombocytopenia is highly specific for malaria infection. 233 profound thrombocytopenia is unusual and malaria-associated thrombocytopenia is rarely associated with haemorrhagic manifestations. 234 the clinical consequences of platelet aggregation and endothelial binding are primarily microvascular ischaemia. this may manifest as renal impairment, cerebral ischaemia, and occlusion of retinal vasculature or even in some cases, skin necrosis. bleeding is unlikely, although in severe thrombocytopenia, petechiae or purpura may develop which denotes extravasation of red cells into the subcutaneous tissue. 235 continued platelet activation and consumption can exacerbate bleeding and decreased circulating platelets are associated with increased vascular leakage and the development of oedema. 236 platelet transfusions are rarely required because the platelet count generally rises rapidly on treating the underlying malaria. coagulopathy is a disturbance of the whole coagulation system involving not just coagulation factors but platelets, anticoagulant factors, fibrinolytic system and, in the case of malaria, the parasitized red cells and the vascular endothelium. parasitized red cells induce expression of tissue factor on endothelial cells and monocytes, release of microparticles, cytokine release and platelet clumping, all of which initiate blood coagulation and tilt the balance towards the pro-coagulant state ( figure 65.12) . 234, [237] [238] [239] [240] [241] [242] anticoagulant factors are severely depleted in malaria. protein c and antithrombin levels are inversely correlated with severity of falciparum malaria and return to normal with treatment of the malaria. 245 have been some concerns about a possible increased risk of infection associated with iron supplementation for children in malarious areas 218, 219 but current recommendations advocate that where iron deficiency and malaria are common, iron supplements should not be withheld and appropriate anti-malarial treatment or prevention should also be offered. 220 the best way to prevent malarial anaemia is to prevent malaria infection by avoiding mosquito bites (e.g. through the use of bed nets) or through chemoprophylaxis. malaria chemoprophylaxis during infancy can reduce both malaria and anaemia. 221 children who have been hospitalized with severe malarial anaemia may benefit from intermittent preventive malarial therapy after discharge to prevent recurrence of anaemia. 222 daily co-trimoxazole prophylaxis which is used for hiv-infected individuals has been shown to reduce malaria parasitaemia and anaemia. 223 the normal platelet life span of 7-10 days is reduced to less than 4 days in malaria infection. 224 several factors are responsible for thrombocytopenia in malaria infection, the most common being increased platelet activation and aggregation ( figure 65 .11). 225 platelet activation is by parasitized red cells which express surface tissue factor and initiate coagulation and platelet aggregation. the resultant activated endothelium binds platelets and sequesters them in vascular beds including in the cerebral vasculature. 226, 234 these platelets facilitate the adhesion and to 46% after a year. 253, 247 although transfusions may be required in severe life-threatening cases of anaemia, aggressive transfusion therapy has been associated with fatal pulmonary emboli due to accelerated haemolysis and disseminated intravascular coagulation. 254 in those who do not respond to art, erythropoietin may be considered since reduced responsiveness to this hormone and antierythropoietin antibodies have been noted in hiv patients. erythropoietin is particularly useful in individuals whose erythropoietin levels are less than 500 iu/l 255 because in addition to increasing the haemoglobin it can also improve the quality of life. 256 erythropoietin may take several weeks to achieve full effect and patients should be replete in haematinics. erythropoietin can very rarely be associated with thrombosis or pure red cell aplasia. thrombocytopenia is a common finding in hiv-infected patients and it may be the initial manifestation of hiv infection in as many as 10% of patients. data from wealthy countries demonstrate platelet counts less than 150 × 10 9 /l in 11% of patients, and less than 50 × 10 9 /l in 1.5%. overall the 1-year coagulopathy in malaria infection is unusual, occurring in less than 5% of cases. it appears to be most common in adults with cerebral malaria who may present with gastrointestinal bleeding 243 or with microvascular ischaemia in the brain, kidneys, retina and occasionally, the dermal vasculature. 244 prolongation of prothrombin time and activated partial thromboplastin time only occur in 4-8% of patients with p. falciparum infection and coagulopathy does not appear to be a feature of p. vivax infection. 245 since coagulation factors need to be depleted to less than 20% of normal to prolong the clotting times, these tests can be normal despite active coagulopathy. management of coagulopathy aims to restore the balance between pro-and anticoagulant processes. this is complex and requires input from a coagulation specialist and ideally, access to plasma, heparin and factor concentrates and a well-equipped coagulation laboratory. anaemia anaemia is very common in hiv-infected individuals occurring in up to 20% at initial presentation and about 70% at some stage during their disease. 246 thirty-seven percent of patients with clinical aids have a 1-year incidence of anaemia (haemoglobin <100 g/l) 246 and high rates of anaemia persist despite combination anti retroviral treatment (art). 247 anaemia is directly related to mortality in hiv infection and is independent of other risk factors including cd4 count. 248 there are multiple reasons for anaemia in hiv-infected patients (box 65.17), which often co-exist in individual patients. bone marrow infection by mycobacteria species, histoplasma, cryptococcus and penicillium marneffei can all decrease red cell production 249 and can be detected by bone marrow examination and cultures. parvovirus has a predilection for the erythroid progenitor cells and can cause severe anaemia in hiv-infected patients. serological tests for parvovirus are unhelpful in hivinfected patients and viral polymerase chain reaction is needed to confirm the diagnosis. 250 the likelihood of parvovirusinduced anaemia increases with the severity of anaemia and has been found in 31% of individuals with hiv and haemoglobin less than 70 g/l. 251 haemophagocytosis occurs in hiv infections and may be secondary to co-infection with mycobacteria, cytomegalovirus, epstein-barr or other herpesviruses. poor nutrition due to socioeconomic reasons, hiv-related anorexia, malabsorption from conditions affecting the gastrointestinal tract, and achlorhydria may contribute to anaemia. haemolytic anaemia occurs secondary to drugs or concomitant glucose-6 phosphate dehydrogenase deficiency and because reticulocytopenia is common in those with hiv infection, reticulocyte counts cannot be used to exclude haemolysis. although the direct coombs test may be positive in patients with hiv infection, autoimmune haemolysis is not a common cause of anaemia. a reduction in red cell precursors has also been noted in children in africa with severe anaemia. 252 treatment of hiv-related anaemia should focus on starting art and eliminating any other factors, such as infections or vitamin deficiencies, which may contribute to the anaemia. in wealthy countries art has been shown to reduce anaemia prevalence from 65% to 53% within 6 months of starting treatment, non-hodgkin's lymphoma (nhl) was noted to be associated with hiv infection early in the epidemic and is an aidsdefining illness. 270 the incidence of nhl is up to 200 times greater in hiv-infected adults than in those who are not infected, and it is responsible for nearly one-sixth of the deaths attributable to aids. since the introduction of haart, the incidence of all types of nhl has decreased by approximately 30-50% 271, 272 and the outcome of hiv-infected patients with lymphoma has improved. in the setting of clinical trials, the 60% 1-year survival rate is comparable to those without hiv infection. 273 the incidence of hodgkin's lymphoma has increased in the post-haart era, possibly due to immune reconstitution and increased cd4 cells. 274, 275 evidence of epstein-barr virus (ebv) infection can be found in virtually all cases of hodgkin's disease. 276 hiv-related lymphomas (box 65.18) 277 (see also lymphomas, below), are broadly divided into systemic lymphomas (80%) and primary central nervous system lymphomas. 278 the incidence of highly aggressive lymphomas, either burkitt's lymphoma (approx. 25%) or diffuse large b-cell lymphoma (approx. 75%), is much higher in hiv-infected patients than in those without infection. 279 although t-cell lymphomas are uncommon in hiv disease (1%), there has been an increase in recent years. the incidence of primary central nervous system lymphoma in hiv-affected individuals is 2-6% and it is 1000 times more common than in the general population. 280 the pathogenesis of nhl in hiv infection is related to the inadequate host immune responses to viruses with oncogenic potential, predominantly ebv and human herpesvirus 8 (hhv8)/kaposi's sarcoma-associated herpesvirus. this allows unregulated lymphoid growth and an accumulation of genetic abnormalities in b cells. 272 markers of b-cell activation such as serum immunoglobulins and free light chains, and cd4 cell count have been suggested as predictive markers for the development of nhl in hiv infection. 281, 282 extranodal and leptomeningeal involvement, and b-symptoms occur in the majority of hiv-infected patients with nhl and the bone marrow is commonly involved. the most common extranodal site to be involved is the incidence of moderate thrombocytopenia (<50 × 10 9 /l) is 3.7%, though this is higher in those with clinical aids (8.7%). 257, 258 thrombocytopenia is more common in those who abuse drugs, have opportunistic infections and malignant disorders of the bone marrow (e.g. lymphoma), and it may also be a side-effect of therapeutic drugs. 259 the most common cause of thrombocytopenia in hiv infection is immune thrombocytopenia which may be associated with hepatitis c co-infection, and produces decreased platelet survival, particularly at cd4 counts below 200/µl. the anti-platelet antibodies, immune complexes and cross-reacting antibodies to hiv envelope proteins and platelets, which occur in hiv-associated thrombocytopenia 259, 260 may also contribute to generation of reactive oxygen species. 261 platelet production can also be affected in hiv infection and may explain the high levels of thrombopoietin that have been documented in hivrelated thrombocytopenia. 262 some cases of hiv-related thrombocytopenia may undergo spontaneous remission so treatment of thrombocytopenia is usually only initiated if it is associated with bleeding, which is unusual. 259 the first line of treatment involves antiretroviral therapy with the aim of achieving undetectable plasma hiv viraemia. 263, 264 any drugs that may be associated with causing thrombocytopenia should be withdrawn and opportunistic infections or secondary malignancies treated. the treatment of immune thrombocytopenia is the same as in non-hiv cases and options include a short course of steroids, intravenous immunoglobulin (short-lived response), anti-d, interferon-α or splenectomy. although there are multiple causes of thrombocytopenia in hiv-positive individuals, one of the most devastating is the thrombotic microangiopathy of thrombotic thrombocytopenic purpura (ttp). this is because the combination of haemolytic anaemia and microthrombi has a very poor prognosis. symptoms are nonspecific and may include fever, headache, bleeding and changes in consciousness. if ttp is suspected, an urgent blood film should be requested and the combination of thrombocytopenia with red cell fragmentation is highly suggestive of ttp. ttp associated with hiv infection was more frequent before the introduction of art and is more common if adherence to treatment is poor or resistance to therapy has developed. 265 ttp is thought to be due to endothelial damage, but unlike the situation in non-hiv-infected individuals, low levels of adamts-13 are not a useful predictor of outcome. 266 treatment of ttp involves plasma exchange, and although refractoriness may occur, this can be corrected by art in some cases. 266 if art is administered in these cases it is important to maintain adherence throughout the period of plasma exchange. if apheresis facilities are limited, plasma infusions alone (30 ml/ kg per day) may also produce a response. 267 art should also be administered immediately after plasma exchange to minimize drug removal. patients with a viral load of less than 500 000 copies/ml generally require fewer plasma exchanges for remission than those with a higher load. 265 survival of patients with hiv-associated ttp in the pre-art era was rarely longer than 2 years, even with plasma exchange and steroid treatment, but for patients who are compliant with art the mortality is around 4%. 268 which immediately limits the amount of blood loss. exposure of the subendothelial space releases factors such as von willebrand factor multimers which bind to platelets and initiate platelet adhesion to the endothelium. the adherent platelets release their granules and attract more platelets, which in combination with fibrinogen, form an aggregate. the activated platelets also attract coagulation factors thereby promoting the clotting process. the critical parts of clot formation are the conversion of prothrombin to thrombin and the thrombinfacilitated conversion of fibrinogen to fibrin (figure 65.13) . haemostatic control mechanisms operate throughout the clotting process to prevent excessive clot formation and involve proteins c and s, and anti-thrombin and antifibrinolytic systems. any alteration in these regulatory pathways can lead to either bleeding or thrombotic complications. bleeding can result from: • inadequate vasoconstriction, due to vascular problems which can be acquired (e.g. viral haemorrhagic fevers or immune vasculitis) or congenital (e.g. collagen vascular disorders) • qualitative or quantitative abnormality of von willebrand factor causing von willebrand's disease • decreased number or function of platelets which can be either acquired (e.g. aspirin, nsaids) or congenital (e.g. platelet function defects) • qualitative (e.g. caused by inhibitors to coagulation factors, commonly factor viii) or quantitative (e.g. haemophilia) abnormality of coagulation factors • increased fibrinolysis (e.g. viral haemorrhagic fevers, snake bites). acquired bleeding disorders are commonly caused by vitamin k deficiency, disseminated intravascular coagulation (dic) or platelet disorders (box 65.19) but may sometimes be due to acquired inhibitors of coagulation factors. the initial laboratory tests in a patient with excessive bleeding should therefore include a platelet count, clotting screen (prothrombin time (pt) and activated partial thromboplastin time (aptt)), and gastrointestinal tract, often the stomach or the perianal region. 283 hepatic involvement, seen in a quarter of cases, is associated with a particularly poor prognosis. cns disease may be asymptomatic so diagnostic lumbar puncture may be required. 284 hiv-related lymphomas frequently present with poor prognostic features such as elevated serum lactate dehydrogenase levels. 285, 286 older age, lowest nadir cd4 cell counts prior to nhl diagnosis, developing nhl while on art, and cumulative hiv viraemia are also poor prognostic features. 282 a formal prognostic scoring system has been developed which takes into account the cd4 count (<100 cells/µl). 287 some types of hiv-related lymphoma are associated with characteristic clinical and laboratory features. primary effusion lymphoma is an aggressive lymphoma characterized by effusions in serosal cavities in the absence of any other tumour masses. 288, 289 it is strongly associated with hhv8 infection and the virus can be identified in the nuclei of the malignant cells. plasmablastic lymphoma mainly affects the oral cavity and the mucosa of the jaw and is typically associated with epstein-barr virus. 290 histological examination of biopsied tissue is necessary to confirm the diagnosis and type of lymphoma. diagnostic difficulties may arise because hiv-related hyperplasia in lymph node biopsies may be confused with lymphoma, the histological appearance of hiv-related lymphomas may be different from those of non-infected individuals 291 and many opportunistic pathogens may mimic the appearances of nhl, or co-exist with it, and will need to be identified or excluded before making a diagnosis of lymphoma. prior to the widespread use of art, conventional lymphoma chemotherapy resulted in considerable toxicity, increased opportunistic infections and high mortality. art has facilitated the use of conventional doses of chemotherapy in conjunction with haematopoietic growth factor support. this has markedly improved the outcome of patients with hiv-related lymphomas who now have overall response rates of 60%. 292 the concomitant use of art and chemotherapy is therefore recommended, especially in those with cd4 counts of less than 100/µl. anti-cd20 antibody is now included in treatment regimens for nhl, and studies that include patients with hivrelated lymphomas all report favourable outcomes. 293, 294 some antiretroviral agents such as zidovudine are best avoided in combination with chemotherapy, because it adds to the myelosuppression of chemotherapy. didanosine may worsen the peripheral neuropathy caused by taxanes and vinca alkaloids. hiv-infected patients undergoing chemotherapy should receive adequate anti-infective prophylaxis due to the high risk of opportunistic infections such as pneumocystis, herpes simplex and zoster and candida. consolidation chemotherapy and stem cell transplant have been used successfully in relapsed hivrelated lymphomas. haemostasis is maintained by interactions between vessel walls, platelets and a balance between pro-and anticoagulant factors. although the process of haemostasis is usually considered to occur in a stepwise fashion, in vivo the steps happen virtually simultaneously. activation of the lining of the endothelium by trauma, cancer cells or cytokines triggers vasoconstriction, the tests for each pathway is given with arrows corresponding to each box complications, malignancies and infections and is a serious condition with a high mortality. patients present with spontaneous bruising or excessive bleeding from minor wounds such as venepuncture sites, and they may also have signs of complications such as renal failure, acute respiratory distress syndrome and microangiopathic haemolytic anaemia. dic is associated with a combination of depleted clotting factors (i.e. prolonged pt and aptt), a falling platelet count, red cell fragments on the blood film, raised d-dimers or fibrin degradation products, and reduced fibrinogen levels. management of disseminated intravascular coagulation includes treating or removing the underlying cause, and correcting the haemostatic abnormalities with combinations of platelets, cryoprecipitate and fresh frozen plasma. although bleeding due to thrombocytopenia is unusual unless the platelet counts falls below 10-20 × 10 9 /l, bleeding may occur with a normal platelet count and normal clotting screening tests (i.e. pt and aptt) if platelet functions are impaired (e.g. myelodysplastic syndromes). platelet transfusions are generally not required unless there is active bleeding or prior to surgery. idiopathic thrombocytopenic purpura. idiopathic thrombocytopenic purpura is due to immune destruction of platelets. it is usually primary but can be associated with conditions such as lymphomas and infections including hiv. it may present incidentally or with petechiae, bruising or bleeding from the nose or gums, especially if the platelet count is less than 20 × fibrinogen levels, which may be helpful in cases of excessive fibrinolysis (table 65. 10). a difficult venepuncture can cause in vitro activation of the clotting system resulting in a shortened pt or aptt. similar findings may occur in chronic dic due to in vivo activation. the pt and aptt are not necessarily good predictors of the bleeding risk because some clotting disorders associated with thrombosis (e.g. anti-phospholipid antibodies) can prolong the aptt. a shortened aptt can be associated with marked elevation of factor viii levels (e.g. pregnancy) and may be a predictor of deep vein thrombosis. a prolonged thrombin time is caused by quantitative or qualitative fibrinogen deficiency, heparin and fibrin degradation products. reptilase time is helpful to distinguish between fibrinogen abnormalities (prolonged reptilase time) and heparin therapy (normal reptilase time). deficiency of vitamin k can be due to poor diet, small bowel disease or bile flow obstruction. clotting factors (ii, vii, ix and x) are dependent on vitamin k which is a fat-soluble vitamin. vitamin k deficiency therefore causes prolongation of the pt and aptt. in newborn infants, vitamin-k-dependent clotting factors can drop precipitously within a couple of days of birth. this causes haemorrhagic disease of the newborn which particularly affects infants that are premature, exclusively breast fed or have been exposed to drugs for tuberculosis, convulsions or anticoagulation in utero. these babies develop bleeding into the skin and gut, or bleeding from the umbilical stump or circumcision. vitamin k deficiency will respond to intravenous vitamin k (10 mg/day for 3 days orally or by intravenous injection) and in severe bleeding the clotting abnormality can be treated with fresh frozen plasma. haemorrhagic disease of the newborn can be prevented with 1 mg of intramuscular vitamin k given at delivery. disseminated intravascular coagulation (dic) is characterized by activation of haemostasis with widespread fibrin formation, activation of fibrinolysis and consumption of platelets and clotting factors. it may be precipitated by tissue injury, obstetric desmopressin (ddavp) is a relatively inexpensive drug that increases fviii levels and vwf activity within 30 minutes of administration. it is useful in mild haemophilia and mild von willebrand's disease. 299 the major side effects are headaches and hyponatraemia so fluid intake should be restricted to 1.5 l/ day. tranexamic acid mouthwashes may be helpful for oral mucosal bleeding. danazol can increase both factor viii and ix levels within 5-7 days 300 and has therefore been recommended for patients with recurrent haemarthrosis or with central nervous system bleeding which both carry a high risk of recurrence. most thromboembolic episodes are single events and may be associated with precipitating events or underlying risk factors. thrombophilia is the clinical state of hypercoagulability and should be suspected in patients who have a strong family history of thrombosis, or who have recurrent or unusual thromboses. increasing affluence and consequent lifestyle changes mean that the prevalence of thromboembolism is rising in some low-and middle-income countries. risk factors such as sedentary work, obesity, excessive alcohol intake, smoking and additional cardiovascular risk factors are compounded by other 10 9 /l. spontaneous recovery occurs less commonly in adults than in children. it is important to exclude other causes of thrombocytopenia such as drugs, dic or sepsis. the diagnosis can be suspected from a bone marrow examination which shows increased numbers of platelet precursors. treatment with prednisolone (0.25-0.5 mg/kg) is usually only necessary if there is bleeding or excessive bruising and the dose should be reduced slowly once the platelet count improves. second-line treatments include immunosuppressive agents and danazol. splenectomy may also be beneficial but carries an increased risk of infection. platelet transfusions or intravenous gammaglobulin can temporarily increase the platelet count in an emergency or prior to surgical procedures. inherited bleeding disorders can be classified broadly into coagulation factor deficiencies (e.g. factor viii and factor ix deficiencies), von willebrand's disease and platelet disorders. the frequency of genes for inherited bleeding disorders is the same throughout the world. haemophilia a has a prevalence of about 10/10 000, von willebrand's disease of >10/10 000 and haemophilia b of <0.1/10 000. these conditions occur more frequently among populations where consanguineous marriage is common and where prenatal diagnostic facilities are unavailable. in general, individuals with inherited coagulation factor deficiencies present with soft tissue bleeds such as haemarthroses or intramuscular bleeds. those with platelet disorders or von willebrand's disease tend to present with mucosal bleeds, however severe (type iii) von willebrand's disease can present with severe soft tissue bleeds. many of these conditions are diagnosed following excessive and uncontrolled bleeding after trauma or surgical procedures. menorrhagia and delayed severe postpartum haemorrhage may be presenting features of bleeding disorders, particularly von willebrand's disease or hypothyroidism, which can cause decreased synthesis of von willebrand factor. some inherited platelet function disorders are associated with characteristic syndromes (e.g. oculocutaneous albinism or skeletal defects) which may provide a clue to the diagnosis. early recognition of symptoms by clinicians, teachers and the public is important so that early treatment can be established. patients with inherited bleeding disorders are usually managed with blood products (box 65.20) or chemotherapy designed to reduce bleeding and associated complications. 295, 296, 298, 299 clotting factor concentrates may be imported or produced locally by fractionation of plasma and are included in the who list of essential medicines. 297, 298 one international unit (iu) of fviii clotting factor concentrate per capita is recommended as the minimum requirement for countries wishing to achieve optimal survival for their haemophilia population but only about 25% of the estimated 400 000 people in the world with haemophilia receive adequate treatment. management of patients with bleeding disorders relies on a wellequipped and quality assessed laboratory for accurate diagnosis and monitoring of treatment and access to plasma and components for replacement therapy. appropriate support services such as physiotherapy, orthopaedics and counselling should also be available. in many countries inherited bleeding disorders are associated with stigma, which is particularly directed against the mothers of affected children, 297 acute and chronic leukaemias are usually associated with a high white cell count but acute leukaemias can present with normal or even sub-normal white cell counts. morphology of peripheral blood and bone marrow specimens is crucial to confirm the diagnosis. this is particularly important in the case of acute leukaemia in children which may be mistaken for an acute viral infection. staining methods including sudan black b, myeloperoxidase and nonspecific esterase are important to distinguish between the different subtypes of acute myeloid and lymphoid leukaemias and therefore to guide treatment. acute myeloid leukaemia (aml). prevalence of this increases with age and the success rate with chemotherapy protocols is not high even in the most sophisticated centres. neutropenia and myelosuppression requiring intensive blood component support occur during chemotherapy 314 and bone marrow transplantation offers the best option for cure for patients who relapse. management of aml is therefore complex and expensive. hydroxycarbamide or subcutaneous cytarabine may be used as a palliative treatment. acute promyelocytic leukaemia (aml subtype m3). this must be distinguished from other types of acute myeloid leukaemia because it has a high cure rate with early treatment. it predominantly affects young adults and it has a high incidence in certain ethnic groups especially those of latin american descent. 315 a treatment protocol which includes all-transretinoic acid with combination chemotherapy has been developed which is feasible in low-income countries. 315, 315 another regimen based on intravenous arsenic trioxide has been developed in india, 316, 317 which has an 86% response rate with good diseasefree and overall survival. conditions that are associated with thrombosis such as hiv infection, and chronic infections including tuberculosis 301, 302 and helminth-induced eosinophilic myocarditis. 303 african americans are more likely to be diagnosed with pulmonary embolism rather than deep-vein thrombosis compared to other racial groups 304 and african patients with thrombosis tend to be younger than those reported in literature 305 with higher mortality rates (around 28%) possibly due to late presentation and poor access to health facilities. asian populations 306-308 seem to have a lower prevalence of symptomatic venous thrombosis compared to african americans. 304 very little is known about the prevalence of prothrombotic factors such as mutations of the prothrombin gene or deficiencies of antithrombin, protein c and protein s in tropical countries, although high rates of factor v leiden, a risk factor for venous thrombosis, have been described in tunisia. 309, 310 lupus anticoagulant and anti-phospholipid syndrome, which are associated with increased thrombosis risk, are increased in afro-caribbean populations, especially in the presence of hiv, and have also been described in nigerian women with pre-eclampsia. 311, 312 the management of venous thrombosis is initially with heparin and then with warfarin for 3-6 months. compliance may be difficult in low-resource settings because of the requirement for regular monitoring of warfarin. it is therefore important to try to prevent thromboses by removing any underlying risk factors and by treating individuals at risk of thrombosis with a short course of prophylactic heparin to cover procedures known to be associated with thrombosis risk. this can present as venous or arterial thromboembolism and it may be inherited (e.g. deficiencies of thrombin, protein s or protein c) or acquired (e.g. antiphospholipids). the patient's personal and family history, and the results of clinical and imaging examinations to confirm thrombosis, may suggest the diagnosis. the laboratory tests needed to determine the cause and classify the type of thrombophilia, and their interpretation, are complex, so patients with recurrent or unusual thromboses should be referred to a specialist centre. haematological malignancies are predominantly leukaemias, lymphomas and myelomas. some of the general approaches for managing these conditions in low-income countries are outlined in box 65.21 313 but definitive treatment should be undertaken by a specialist haematology unit. leukaemias can be broadly classified as acute or chronic, and lymphoid or myeloid. the presenting symptoms and signs are related to the disturbed blood cell production from the bone marrow due to the effects of the malignant cell clone (box 65.22) . acute leukaemias are characterized by rapid progression and poor prognosis if left untreated whereas chronic leukaemias generally follow a much slower course. • mobilization of the community (especially parents and families) to raise awareness among local councils and government bodies about the treatability of the cancers and benefits from curing them • find an external partner unit locally, nationally or internationally which is already well-established and willing to help but will not dictate terms • improvement of supportive care facilities, especially protection from those with infectious diseases • development of a safe and reliable blood transfusion service • provision of subsidized travel, and satellite clinics to lessen the burden • development of appropriate protocols for each disease entity which is locally practicable with minimum cost and maximum efficacy • development of medical, nursing and paramedical expertise in the diseases to be treated -initially by offering visiting fellowships and in the long term for the trained individuals to arrange regional and local teaching programmes • formation of a cooperative group bringing together all the professionals involved in the speciality within a country or region to share expertise and develop training programmes. acute lymphoblastic leukaemia (all). this is the most common type of leukaemia in children. it has a good prognosis when treated with modern chemotherapy protocols with cure rates in the best centres exceeding 80%. 318 in low-income countries, cure rates are much lower at around 35% 319 primarily because of failure to complete therapy and deaths caused by treatment. considerable improvements in all outcomes have been achieved by twinning institutions in developing countries with specialist centres elsewhere in the country or internationally. 313 measures that may improve outcomes focus on preventing abandonment of therapy (e.g. providing funding for transport, satellite clinics and support groups) and prompt treatment of infection. 320 treatment in a dedicated paediatric oncology unit using a comprehensive multidisciplinary team approach and protocol-based therapy, is also associated with improved outcomes in resource-poor settings. 321 chronic myeloid leukaemia (cml). management has been revolutionized by tyrosine kinase inhibitors (e.g. imatinib) which can produce complete remission in over 80% of cases. once the diagnosis of cml is established, hydroxycarbamide can be used to reduce the white cell count, followed by treatment with a tyrosine kinase inhibitor. manufacturers will provide the drug free of charge to patients in low-income countries with confirmed cml and generic forms of tyrosine kinase inhibitors are now becoming available. chronic lymphocytic leukaemia (cll). this occurs predominantly in older people and usually presents with lymphadenopathy and recurrent infections. treatment is with chlorambucil and prednisolone although aggressive forms require combination therapy with rituximab, fludarabine and cyclophosphamide. treatment is generally not curative but the disease may be indolent and drugs may only be required if the patient has symptoms or if there is a risk of hyperviscosity from a very high lymphocyte count. approximately 30 000 cases of non-hodgkin lymphoma (nhl) occur in the equatorial belt of africa each year (table 65 .11). 322 there are marked geographical variations in prevalence but up to 50% are thought to be related to hiv infection. 322 burkitt's lymphoma, a b-cell nhl, was originally described in children from africa and has an estimated incidence of 30-70 per million. lymphomas are broadly classified into hodgkin's lymphoma and nhl; nhl are divided into b-cell, t-cell and nk-cell, and immunodeficiency-associated types. the clinical presentation of lymphomas is characterized by enlargement of the lymphoid organs and subsequent compression of the adjacent structures, infiltration of organs by the malignant lymphoid cells and a dysfunctional immunological system which can manifest as immunosuppression or excessive but dysregulated immune activation associated with, for example, autoimmune conditions. the diagnosis and management of the various types of lymphomas are complicated and should be undertaken in a specialist box 65. 22 clinical features of leukaemias • fatigue and cardiac symptoms from anaemia • bleeding from thrombocytopenia • increased risk of infections despite a higher number but dysfunctional white cells • lymphadenopathy and hepatosplenomegaly occur with all although lymphadenopathy may be observed in the monocytic variety of aml • blindness due to hyperviscosity from hyperleukocytosis • tumour lysis syndrome due to spontaneous cell lysis presents as renal failure • pustules or pyogenic infections of the skin from minor wounds • bleeding gums are a characteristic feature of acute monocytic leukaemia • disseminated intravascular coagulation can occur with acute promyelocytic leukaemia • gout can arise from breakdown of the excess white cells and release of uric acid • oral aphthous ulceration is seen with severe neutropenia in both aml and all • granulocytic sarcoma or chloroma represent extramedullary deposits of leukaemic cells in any organ but mainly the skin. this may occur in the absence of peripheral blood involvement and is more common with chromosomal translocation (8; 21) of aml • central nervous system manifestations due to sludging of the cerebral circulation by the malignant cells or increased intracranial pressure due to ventricular blockade can occur. monocytic myeloid leukaemia can also involve the meninges • intracranial haemorrhage can occur in all with very high white cell counts (>400 × 10 9 /l) • bone pain and arthralgia can be a presenting feature of all in children in more than a quarter. these children may present with a limp or unwillingness to walk due to marrow infiltration by leukaemic cells. rarely, they may have normal blood counts delaying the diagnosis of all • anterior mediastinal mass (thymus enlargement) can also occur in children and young adults with all which may present as superior venocaval obstruction • painless enlargement of scrotum is a sign of testicular leukaemia or hydrocele from lymphatic obstruction. priapism can result from hyperleukocytosis rarely. • most often asymptomatic and usually suspected on blood counts • the chronicity of cml or cll tends to cause gradual-onset symptoms since the patients get adjusted to the slowly developing anaemia • abdominal discomfort and early satiety are a feature of cml due to excessive splenomegaly compressing the stomach and reducing the luminal volume • sternal tenderness may be noted in cml • hyperleukocytosis in cml can occur more often than with aml or all due to the gradual increase in white cells. this can cause symptoms like hyperuricaemia and gout, tinnitus, priapism or central nervous system disturbances • left shoulder tip pain can arise from splenic infarction from the massive splenomegaly in cml • cml can rarely present with features of thyrotoxicosis (heat intolerance, weight loss and excessive sweating) due to hyper-metabolism • cll is often associated with lymphadenopathy and rarely with mild to moderate splenomegaly. all, acute lymphoid leukaemia; aml, acute myeloid leukaemia; cll, chronic lymphocytic leukaemia; cml, chronic myeloid leukaemia. centre. diagnosis depends on clinical history and examination, radiological investigations to document the extent of disease, and morphology, immunohistochemistry and molecular studies on tissue samples to confirm the lymphoma subtype. guidance on the diagnosis and treatment of lymphoma in settings where resources are limited includes recommendations about panels of immunostains and chemotherapy regimens that minimize the need for supportive care. 322 tele-pathology, which involves transmitting histological images via the internet to experts overseas, may be helpful in certain circumstances though it is dependent on the quality of the histology preparations and the images of appropriate diagnostic regions in the sample. treatment regimens for lymphomas differ according to the subtype but may involve chemotherapy and radiotherapy. high remission rates can be achieved in burkitt's lymphoma with a combination of cyclophosphamide, vincristine and methotrexate and progressive disease can be managed with ifosfamide, mesna and cytosine arabinoside. 322, 323 adult t-cell leukaemia-lymphoma (atll) adult t-cell leukaemia-lymphoma (atll) is an uncommon lymphoid malignancy which occurs in patients infected with human t-lymphotropic virus type i (htlv-i). 324 htlv-1 is endemic in the caribbean, western africa, peru and southern japan. less than 5% of those infected with htlv-i develop atll and up to 30 years can elapse between the primary infection and the development of atll suggesting additional factors are needed for malignant transformation. 325 atll presents acutely in approximately 60% of cases, although chronic forms have also been described. 326 the clinical presentation is with generalized lymphadenopathy in most cases and hepatosplenomegaly in over half. 324 atll is associated with a high risk of hypercalcaemia which occurs in more than two-thirds of patients during the course of their disease and may be associated with central nervous system disturbances and renal impairment. lytic bone lesions occur as a para-neoplastic types of lymphomas identified from selected countries in sub-saharan africa phenomenon due to production of parathormone-like peptides. as with other t-cell disorders, atll can involve the skin, producing, e.g. erythrodermic plaques. the diagnosis of atll can be suspected from a high peripheral blood white blood cell count in combination with hypercalcaemia and characteristic lymphocytes with convoluted and hyperlobulated nuclei. 324 the diagnosis is confirmed by histological examination of a tissue (lymph node or bone marrow), immunophenotyping for specific cell markers and proof of htlv infection, usually by serological methods. management of atll is primarily with combination chemotherapy with intrathecal prophylaxis. 327,328 a combination of zidovudine and interferon, as agents against htlv, has also been tried with some success. 329 hypercalcaemia and opportunistic infections should be sought and treated early in these patients. the high white cell count is associated with a significant risk of tumour lysis syndrome and should be prevented by adequate hydration and the judicious use of allopurinol and other urate-reducing agents. myeloma is a monoclonal proliferation of plasma cells and it particularly affects older people. myeloma appears to be less common in asian countries than elsewhere, although during the last 25 years, an almost four-fold increase in incidence of myeloma has occurred in taiwan. 330 in the united states, the incidence of multiple myeloma in the black population is twice that of the white population. the abundant plasma cells infiltrate the bone marrow and interfere with normal haematopoiesis. this leads to anaemia, which is a presenting feature in 70% of individuals. bony infiltration by the malignant plasma cells can produce osteoporosis, lytic lesions and pathological fractures in 60% of patients with myeloma. involvement of the bones can lead to hypercalcaemia, which may be a presenting feature, and vertebral fracture leading to spinal cord compression. the malignant plasma cells produce a paraprotein which can cause renal impairment in 20-50% and hyperviscosity may ensue in 10% of patients if the paraprotein production is not controlled. patients with myeloma may need a variety of supportive interventions including management of anaemia, renal failure, hypercalcaemia, hyperviscosity, infections and bone pains. specific anti-myeloma treatment should be managed within a specialist unit and has undergone a radical change in the last decade with the use of thalidomide and its newer formulations, and the more expensive, proteasome inhibitors (e.g. bortzomib). thalidomide is relatively safe and effective although somnolence and constipation can sometimes be troublesome. there is a risk of thrombosis with thalidomide especially at the initiation of therapy, and prophylaxis with heparin, warfarin or antiplatelet agents, depending on an assessment of the risk, may be warranted. melphalan may also be useful, particularly if resources are limited and there is no specialist centre. however it is myelosuppressive, so regular monitoring of the blood count is essential. maintaining an adequate blood supply is a major challenge for low-income countries. only 39% of the global blood supply is donated in the poorest countries where 82% of the world's population lives. 331 blood transfusion is a vital component of every country's health service. it can be a life-saving intervention for illnesses such as severe acute anaemia, but mistakes in the transfusion process can be life-threatening, either immediately or years later through transmission of infectious agents. clinicians need to understand how blood is acquired and its risks and benefits, and to use it appropriately. governments and transfusion services need to put measures in place to ensure that blood is safe for transfusion and that it reaches those who need it in a timely manner. only 16% of member states meet all the world health organization's (who) recommendations for a national quality blood transfusion system. 331 at the national level the transfusion service should have a director, an advisory committee and clear transfusion policies and strategies (table 65 .12). 331 who recommend standardization of blood collection, testing and distribution. although centralization of these services may offer the best guarantee of quality, it is often not practical in countries with poorly developed communications and transport infrastructure. two systems, centralized and hospital-based, exist in lowincome countries for managing blood supply. in the centralized system, voluntary blood donors are recruited, screened and bled by regional centres and the blood collected is distributed to peripheral hospitals. hospital-based systems are the predominant source of blood across sub-saharan africa. hospital-based systems obtain blood predominantly from relatives of patients, and blood is screened and used within the local vicinity. 332 blood from the centralized system costs at least three times as much per unit as that from a hospital-based system. 333 although centralized systems can save costs through batching and bulk purchasing, the quality assurance processes and donor recruitment components are expensive and difficult to maintain without dependence on external funds. in hospital-based transfusion services, testing quality is variable and the families of patients bear the cost of finding blood donors. the vast majority of blood in low-income countries is transfused as whole blood. in high-income countries it is standard practice to optimize the use of each donation of blood by separating it into individual components but whether this approach is cost-effective in low-income countries, where indications for transfusion are different, is not known. these components, which may include plasma, platelets and cryoprecipitate, are prepared by centrifugation using a closed, sterile system and each component has different storage requirements. plasma and cryoprecipitate are kept frozen, red cells are stored at 1-5°c, and platelets at 18-22°c with constant agitation. recent evidence suggests that warm, fresh, whole blood may be better than component therapy for resuscitation of acidotic, hypothermic and coagulopathic trauma patients 334 and for patients needing massive transfusions. 335 many infections can be transmitted through blood transfusions and transfusion of infected blood causes morbidity and mortality in the recipients, and has an economic and emotional impact on their families and communities. those who become infected through blood transfusion are infectious to others and contribute to the spread of disease thereby increasing the burden on health services and reducing productive labour. strategies for recruiting blood donors have to provide blood for all who need it in a timely manner while ensuring that the blood is as safe as possible. the safest type of blood donor is one who donates regularly (i.e. repeat donors). who states that the safest source of blood is altruistic, voluntary, unpaid donors. only 32% of who member states report having at least 90% of their blood supply from voluntary donors, and low-income countries have not been able to increase the recruitment of voluntary donors for several years. 331 recent evidence from sub-saharan africa indicates that the focus on voluntary donors may be misplaced since first-time voluntary donors have a similar prevalence of transfusion-transmitted infections as family replacement donors. 336 in order to limit blood shortage and maintain constant blood supply in poorer countries, both voluntary and replacement donors should be accepted and encouraged to donate regularly. mechanisms to convert family replacement donors into repeating voluntary donors have the potential to significantly increase blood donations in africa. political will and open-mindedness about ways to improve the supply and safety of blood are essential to promote more evidence-based approaches to blood transfusion practice in poorer countries. 337 supporting strategy in wealthy countries, the majority of transfusions are carried out electively. by contrast, in poorer countries, and particularly those where the malaria transmission rate is high, most transfusions are given for life-threatening emergencies. in low-income countries, 50-80% of transfusions are administered to children, predominantly for malaria-related anaemia, and pregnant women. transfusion can significantly reduce the mortality of children with severe anaemia within the first 2 days of hospital admission 347 and successful malaria control can reduce paediatric transfusion requirements. 348 in sub-saharan africa, 26% of in-hospital maternal deaths from severe bleeding were due to lack of blood for transfusion. 349 other specialities which are significant users of blood are surgery, trauma, emergency medicine and general medicine. in low-income countries the most effective way to avoid transfusions is to reduce the prevalence of anaemia. more studies on the efficacy and cost of combinations of interventions including insecticide-treated bed nets, nutritional supplements and anthelmintic drugs to prevent anaemia are needed. when resources are very limited, governments may need to make some difficult decisions in order to achieve an equitable balance between investing in a transfusion service and public health measures to reduce anaemia. whether a patient needs a blood transfusion or not is ultimately a clinical decision. emergency transfusions can be lifesaving for patients in whom anaemia has developed too quickly to allow physiological compensation, as in severe malariarelated anaemia in children, and sudden, severe obstetric bleeding. in contrast, if the anaemia has developed slowly, for example due to hookworm infestation or nutritional deficiency, patients can generally be managed conservatively by treating the cause of the anaemia and prescribing haematinic replacements. iron supplements should be continued for at least 3 months after the haemoglobin has returned to normal, so that body stores can be replenished. clinical guidelines. it is possible to avoid unnecessary transfusions by adhering to clinical transfusion guidelines. most institutions have developed guidelines to help clinicians make rational decisions about the use of blood transfusions (box 65.23) 344, 350 and strict enforcement of transfusion protocols can significantly reduce avoidable transfusions. 351 the principles underlying most transfusion guidelines are similar and combine a clinical assessment of oxygenation, with haemoglobin measurement being used as a surrogate measure for intracellular oxygen concentration. increasingly, transfusion guidelines are making use of evidence which shows that adequate oxygen delivery to the tissues can be achieved at haemoglobin levels that are significantly lower than the normal range. 352 implementation of transfusion guidelines is particularly difficult if clinicians do not have access to reliable haemoglobin high-risk donors, such as commercial sex workers and their contacts, intravenous drug abusers, or those with an itinerant lifestyle such as traders, drivers and military personnel, should be deterred from donating. 338 even in areas where hiv infection rates in the general population are high, donor deferral can be effective in excluding hiv-infected donors. 339 the whole donation process, including tests for hiv and other infections, should be explained to the donor before blood is collected and donors should have the option of knowing the results and receiving counselling. it is imperative that complete confidentiality is maintained throughout all procedures. infections with organisms such as hiv, hepatitis viruses, cytomegalovirus, syphilis, lyme borreliosis, malaria, babesiosis, american trypanosomiasis (chagas disease) and toxoplasmosis can all be acquired through blood transfusions. some 5-10% of hiv infections worldwide are thought to have been transmitted through the transfusion of infected blood and blood products. there have also been reports of transmission of variant creutzfeldt-jakob disease through blood transfusion and there is a theoretical risk of transmission of severe acute respiratory syndrome (sars). 340, 341 who recommends that all donated blood should be screened for hiv, hepatitis b and syphilis and, where feasible and appropriate, for hepatitis c, malaria and chagas disease. malaria can be transmitted by blood transfusion and, depending on the local infection prevalence, 2-55% of blood donors in africa screen positive for malaria. 342 however, there is very little evidence to suggest that these donors transmit malaria to transfusion recipients. although who recommends screening donors in endemic areas for malaria, none of the screening methods that would be practical for transfusion services are sufficiently sensitive. furthermore, in some countries with high malaria transmission, exclusion of parasitaemic donors could result in deferral rates exceeding 50% which would have a major impact on blood supply. 343 there is no evidence to support the widespread practice of routine treatment of transfusion recipients for malaria. fresh blood is potentially infectious for syphilis, but storage at 4°c for more than 5 days can inactivate treponema pallidum. the high demand for blood in low-income countries means that blood is generally not stored for long enough to inactivate t. pallidum and syphilis seroconversion associated with transfusion has been reported from africa. 344 globally, the prevalence of hepatitis c, htlv-1 and -2 and chagas disease is variable and the decision to introduce donor screening for these infections should be based on local assessments of the risks, benefits, feasibility and costs. blood should not be separated into components if the residual risk of infection is high, as this will increase the number of potentially infected recipients. a unit of blood is usually stored until screening tests for infections have been completed. this means that potentially infected blood may be mixed up with units that have already been screened, and costly blood collection bags are wasted. screening potential donors before venesecting a unit of blood may therefore be a more cost-effective way of ensuring safe blood. 345 tests for screening blood donors need to be highly sensitive, and infected blood should be rejected. before informing the donor of the outcome, all positive results should be confirmed using a test with a high degree of specificity. where blood or that the blood may become infected with bacteria during the process. intraoperative blood salvage. this involves collecting blood lost during the operation and reinfusing it into the patient either during or after surgery. although this technique is practical and safe, and reduces the need for donor blood by 27-53%, 355 it requires specialized equipment and training, and may be more expensive than routinely donated blood. 356 other measures. normal saline or intravenous replacement fluids can be used judiciously in acute blood loss, and in certain circumstances may be as effective as whole blood, red cells or plasma. erythropoietin, which stimulates endogenous red cell production is well-established for use in chronic anaemias such as those due to renal failure, cancer and hiv infection but its delayed action makes it unsuitable for use in acute anaemias. synthetic oxygen carriers, such as perfluorocarbons, are not yet routinely available. 357 in low-income countries, the recommended haemoglobin threshold for transfusions is often well below that which would be accepted in more wealthy countries. randomized controlled studies in wealthy countries indicate that for most adults and children undergoing critical care, a haemoglobin threshold of 70 g/l for transfusion is safe 358 whereas paediatric blood transfusion protocols in sub-saharan africa often recommend transfusions for stable children only when the haemoglobin level is less than 40 g/l. 351 complications such as cardiac failure or infection may necessitate transfusion at a higher haemoglobin level. transfusion should be combined with adequate haematinic replacements and underlying conditions should be treated. 359 early evidence suggests that intermittent preventive treatment with anti-malarials may reduce the high hospital readmission rates experienced by children post-transfusion. 360 complications can occur immediately during transfusion, within a few hours of its completion, or be delayed for many years, as in the case of viral infections (box 65.24). measurements. when they doubt the haemoglobin result, clinicians rely entirely on clinical judgement to guide transfusion practice which can lead to significant numbers of inappropriate transfusions. 353 a lack of investment in the quality of a critical test, such as haemoglobin measurement, can waste significant resources downstream in the transfusion process, and unnecessarily expose recipients to the risk of transfusion-related infections. minimizing surgical blood loss. where blood is in short supply, it is particularly important to ensure that the best anaesthetic and surgical techniques are used, to minimize blood loss during surgery. drugs which improve haemostasis or reduce fibrinolysis, such as aprotinin and cyklokapron, and fibrin sealants, can be effective in reducing perioperative blood loss. these drugs can therefore reduce the need for blood transfusion but they may be too expensive for use in low-income countries. a cost-effectiveness study of surgical bleeding in four sub-saharan countries indicates that the antifibrinolytic, tranexamic acid, could save lives in countries with blood shortages, reduce healthcare costs and prevent transmission of infections. 354 preoperative autologous blood deposit. patients undergoing planned surgery who are likely to require a blood transfusion can have units of their own blood removed and stored in case they have significant intraoperative blood loss and need a transfusion. this process, known as preoperative autologous donation, can reduce the need for allogeneic transfusions by 46-74% 355 but it requires careful organization: the surgeon needs to predict how much blood will be required, the patient has to be fit enough to withstand removal of one or more units of blood over the weeks preceding the surgery and the surgery must take place within the shelf-life of the blood. as the blood has to be stored in the blood bank there is still a risk that the patient may receive blood which is not their own box 65.23 prescribing blood: a checklist for clinicians always ask yourself the following questions before prescribing blood or blood products for a patient: 1. what improvement in the patient's clinical condition am i aiming to achieve? 2. can i minimize blood loss to reduce this patient's need for transfusion? 3. are there any other treatments i should give before making the decision to transfuse, such as intravenous replacement fluids or oxygen? 4. what are the specific clinical or laboratory indications for transfusion in this patient? 5. what are the risks of transmitting hiv, hepatitis, syphilis or other infectious agents through the blood products that are available for this patient? bacterial contamination and should be investigated and managed accordingly. allergic reactions are due to infusion of plasma proteins and manifestations include erythema, rash, pruritus, bronchospasm and anaphylaxis. the transfusion should be stopped and the patient treated with antihistamines. if the reaction is mild and the symptoms and signs completely disappear, the transfusion can be restarted. if this type of mild reaction occurs repeatedly with more than one unit of blood, the red cells can be washed before transfusion. this should only be done if absolutely necessary, as it carries the risk of introducing potentially fatal bacterial infection. severe allergic reactions with evidence of systemic toxicity should be managed as acute anaphylaxis. blood should always be transfused slowly to avoid overloading the circulation, unless the patient has active and severe bleeding. fluid overload may be a particular problem when paediatric blood bags are not available, as children may be over-transfused due to miscalculation of the required volume, lack of accurate infusion devices or inadvertent administration of an adult-sized unit of blood. four units of blood contain the equivalent amount of iron stored in bone marrow (approx. 1 g). repeated transfusions for chronic haemolytic anaemia, as in thalassaemia major and sickle cell disease, lead to iron deposition in parenchymal cells. eventually failure of the heart, liver and other organs supersedes. adequate doses of iron chelators, such as injectable desferrioxamine or oral deferiprone, are able to maintain acceptable iron balance in patients with chronic anaemia who need regular transfusions. it is not usually necessary to warm blood unless large quantities are transfused rapidly. this may lower the temperature of the sino-atrial node to below 30°c at which point ventricular fibrillation can occur. if blood needs to be warmed, an electric blood warmer specifically designed for the purpose should be used. this keeps the temperature below 38°c and avoids the haemolysis associated with overheating blood. graft-versus-host disease occurs when donor lymphocytes engraft in an immune-suppressed recipient. the lymphocytes recognize the recipient's bone marrow as foreign and induce aplasia. graft-versus-host disease is almost universally fatal and can be prevented by irradiating the donor blood, which inactivates the donor lymphocytes. transfusion of blood into a recipient who possesses antibodies to the donor's red cells can cause an acute, and occasionally fatal, intravascular haemolysis. this could occur, e.g. if group a cells are transfused into a group o recipient who has naturally occurring antibodies to group a cells. the profound haemolysis induces renal vasoconstriction and acute tubular necrosis. treatment involves stopping the transfusion, cardiorespiratory support and inducing a brisk diuresis. in addition to abnormalities indicating renal failure, laboratory findings include haemoglobinuria and haemoglobinaemia. proof of the diagnosis involves rechecking the whole transfusion process including all documentation stages, regrouping the donor and the recipient, and screening for antibodies on red cells with a direct antiglobulin test. these tests are usually available in any hospital laboratory capable of providing a transfusion service. delayed haemolysis has a similar physiological basis to acute intravascular haemolysis but it tends to be less severe, it occurs 7-10 days after the transfusion and it is less likely to present as a clinical emergency. limited data from sub-saharan africa show rates of bacterial contamination in donated blood of around 9% 361,362 but the clinical consequences for transfusion recipients are unknown. bacteria can enter the blood bag during venesection or if the bag is breached, e.g. when reducing the volume for a paediatric recipient or during component preparation. gram-negative bacteria, including pseudomonas and yersinia, grow optimally at 4°c and infected blood may not necessarily appear abnormal to the naked eye. reactions following infusion of infected blood are often due to endotoxins and may occur several hours after the transfusion has finished. although these reactions are rare, they can be severe and fatal. if bacterial contamination is suspected, the transfusion should be stopped and samples from the patient and the blood bag sent to the laboratory for culture. cardiorespiratory support may be needed and broad-spectrum antibiotics should be started immediately and continued until culture results are available. non-haemolytic febrile reactions are episodes of fever and chills associated with transfusion and for which no other cause can be found. they are due to the recipient's antibodies reacting against antigens present on the donor's white cells or platelets. these reactions are most common in patients who have had transfusions in the past and have therefore been exposed to allo-antigens. mild febrile reactions usually respond to simple antipyretics such as paracetamol. more severe reactions may be the first indication of a haemolytic transfusion reaction or anaemia in low-income and middle-income countries sickle cell disease the inherited diseases of hemoglobin are an emerging global health burden how i treat thalassaemia blood donors and blood collecton: web interface-supported transmission risk assessment and cost-effectiveness analysis of postdonation screening: a global model applied to references 1. dale dc. neutropenia and neutrophilia diagnosis and outcome of 100 consecutive patients with extreme granulocytic leukocytosis the diagnostic value of the neutrophil left shift in predicting inflammatory and infectious disease congenital and acquired neutropenia practical approach to the patient with hypereosinophilia eosinophilia in returning travellers and migrants from the tropics: uk recommendations for investigation and initial management investigation of tropical eosinophilia; assessing a strategy based on geographical area worldwide prevalence of anaemia, who vitamin and mineral nutrition information system comparative quantification of health risks: global and regional burden of disease attributable to selected major risk factors. geneva: world health organization worldwide prevalence of anaemia 1993-2005: who global database on anaemia. geneva: world health organization anaemia in low-income and middle-income countries accuracy of clinical pallor in the diagnosis of anaemia in children: a meta-analysis clinical pallor is useful to detect severe anemia in populations where anemia is prevalent and severe anaemia: a useful indicator of neglected disease burden and control estimating the prevalence of anaemia: a comparison of three methods haemoglobin colour scale for anaemia diagnosis where there is no laboratory: a systematic review late vs early clamping of the umbilical cord in full-term neonates: systematic review and meta-analysis of controlled trials what works? interventions for maternal and child undernutrition and survival iron in fetal and neonatal nutrition global health risks. mortality and burden of disease attributable to selected major risk factors weekly iron-folic acid supplementation (wifs) in women of reproductive age: its role in promoting optimal maternal and child health iron interventions for women and children in low-income countries how much time do health services spend on antenatal care? implications for the introduction of the focused antenatal care model in tanzania efficacy and trial effectiveness of weekly and daily iron supplementation among pregnant women in rural bangladesh: disentangling the issues diagnosis and management of irondeficiency anaemia iron deficiency and overload iron deficiency anemia iron deficiency: a concise review individualized treatment for iron-deficiency anemia in adults clinical update: intravenous iron for anaemia laboratory diagnosis of vitamin b 12 and folate deficiency plasma homocysteine levels and mortality in patients with coronary artery disease how i treat cobalamin (vitamin b 12 ) deficiency vitamin b 12 deficiency sensitivity of serum methylmalonic acid and total homocysteine determinations for diagnosing cobalamin and folate deficiencies oral or parenteral therapy for b 12 deficiency effective treatment of cobalamin deficiency with oral cobalamin high prevalence of cobalamin deficiency in elderly outpatients vitamin b 12 (cobalamin) deficiency in elderly patients causes of vitamin b 12 and folate deficiency importance of folate in human nutrition review of the magnitude of folate and vitamin b 12 deficiencies worldwide red cell or serum folate? results from the national pathology alliance benchmarking review the anemia of vitamin a deficiency: epidemiology and pathogenesis vitamin a or b-carotene supplementation reduce symptoms of illness in pregnant and lactating nepali women vitamin a intervention: short term effects of a single, oral, massive dose on iron metabolism supplemental vitamin a improves anemia and growth in anemic school children in tanzania supplementation with vitamin a and iron for nutritional anaemia in pregnant women in west java effect of vitamin a supplementation on haemoglobin and vitamin a levels during pregnancy randomised trial of vitamin a supplementation in pregnant women in rural malawi found to be anaemic on screening by hemocue double blind randomised trial of low dose supplements with vitamin a or beta carotene on mortality related to pregnancy in nepal. the nnips-study group randomised trial of effects of vitamin supplements on pregnancy outcomes and t cell counts in hiv-1-infected women in tanzania hematologic manifestations of copper deficiency: a retrospective review update on anemia and neutropenia in copper deficiency zinc deficiency in patients with sickle cell disease effect of zinc supplementation on incidence of infections and hospital admissions in sickle cell disease (scd) human schistosomiasis and anemia: the relationship and potential mechanisms hemoquant determination of hookwormrelated blood loss and its role in iron deficiency in african children functional significance of low-intensity polyparasitehelminth infections in anemia impact of mass chemotherapy on the morbidity due to soil-transmitted nematodes haemoglobin concentrations and concomitant infections of hookworm and trichuris trichiura in panamanian primary schoolchildren anthelmintic treatment improves the hemoglobin and serum ferritin concentrations of tanzanian schoolchildren impact of a national helminth control programme on infection and morbidity in ugandan schoolchildren anthelmintic treatment and haemoglobin concentrations during pregnancy low dose daily iron supplementation improves iron status and appetite but not anemia, whereas quarterly anthelmintic treatment improves growth, appetite and anemia in zanzibari preschool children epidemiology of iron deficiency anemia in zanzibari schoolchildren: the importance of hookworms sickle cell disease management of sickle cell disease sickle cell disease peculiar elongated and sickleshaped red blood corpuscles in a case of severe anemia sickle cell anemia and related abnormalities sickle cell anemia, a molecular disease hemoglobin s polymerization: primary determinant of the hemolytic and clinical severity of the sickling syndromes hemoglobin s gelation and sickle cell disease pathogenesis and treatment of sickle cell disease polymer structure and polymerization of deoxyhemoglobin sickle cell anemia and other sickling syndromes levels of fetal hemoglobin necessary for treatment of sickle cell disease sickle cell vaso-occlusion: multistep and multicellular paradigm blockade of adhesion of sickle cells to endothelium by monoclonal antibodies thrombospondin from activated platelets promotes sickle erythrocyte adherence to human microvascular endothelium under physiologic flow: a potential role for platelet activation in sickle cell vaso-occlusion sickle erythrocyte-endothelial interactions in microcirculation: the role of von willebrand factor and implications for vaso-occlusion the clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease nitric oxide decreases cytokine-induced endothelial activation. nitric oxide selectively reduces endothelial expression of adhesion molecules and pro-inflammatory cytokines coagulation activation and inflammation in sickle cell disease-associated pulmonary hypertension the crisis in sickle cell anemia; hematologic studies sickle-cell pain: advances in epidemiology and etiology pain in sickle cell disease. rates and risk factors guidelines for the management of the acute painful crisis in sickle cell disease pulmonary hypertension as a risk factor for death in patients with sickle cell disease deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical sub-phenotypes functional asplenia in sickle-cell anemia acute chest syndrome: sickle cell disease acute chest syndrome in sickle cell disease: clinical presentation and course. cooperative study of sickle cell disease national acute chest syndrome study group. causes and outcomes of the acute chest syndrome in sickle cell disease the acute chest syndrome in sickle cell disease: incidence and risk factors acute chest syndrome in sickle-cell disease bronchoalveolar lavage in adult sickle cell patients with acute chest syndrome: value for diagnostic assessment of fat embolism secretory phospholipase a(2) predicts impending acute chest syndrome in sickle cell disease serum c-reactive protein parallels secretory phospholipase a2 in sickle cell disease patients with vasoocclusive crisis or acute chest syndrome cerebrovascular accidents in sickle cell disease: rates and risk factors pathophysiology and treatment of stroke in sicklecell disease: present and future lesion burden and cognitive morbidity in children with sickle cell disease prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial doppler ultrasonography silent infarction as a risk factor for overt stroke in children with sickle cell anemia: a report from the cooperative study of sickle cell disease nocturnal hypoxaemia and central-nervoussystem events in sickle-cell disease natural history of blood pressure in sickle cell disease: risks for stroke and death associated with relative hypertension in sickle cell anemia prophylaxis with oral penicillin in children with sickle cell anemia. a randomized trial effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. investigators of the multicenter study of hydroxyurea in sickle cell anemia effects of hydroxyurea on hemoglobin f and water content in the red blood cells of dogs and of patients with sickle cell anemia hydroxyurea as an alternative to blood transfusions for the prevention of recurrent stroke in children with sickle cell disease hydroxyurea therapy lowers transcranial doppler flow velocities in children with sickle cell anemia alloimmunization in sickle cell anemia and transfusion of racially unmatched blood incentive spirometry to prevent acute pulmonary complications in sickle cell diseases exchange versus simple transfusion for acute chest syndrome in sickle cell anemia adults corticosteroids for acute chest syndrome in children with sickle cell disease: variation in use and association with length of stay and readmission airway hyperreactivity detected by methacholine challenge in children with sickle cell disease nitric oxide for inhalation in the acute treatment of sickle cell pain crisis: a randomized controlled trial reduction of painful vaso-occlusive crisis of sickle cell anaemia by tinzaparin in a doubleblind randomized trial the use of transcranial ultrasonography to predict stroke in sickle cell disease discontinuing prophylactic transfusions used to prevent stroke in sickle cell disease risk of recurrent stroke in patients with sickle cell disease treated with erythrocyte transfusions complications associated with sickle cell trait: a brief narrative review thalassaemias and related disorders: quantitative disorders of hemoglobin synthesis a series of cases of splenomegaly in children with anemia and peculiar bone changes mediterranean disease -thalassaemia (erythroblastic anemia of cooley): associated pigment abnormalities simulating hemochromatosis global epidemiology of haemoglobin disorders and derived service indicators the inherited diseases of hemoglobin are an emerging global health burden the thalassaemia syndromes pathophysiology of beta thalassaemia -a guide to molecular therapies beta-thalassaemia intermedia: is it possible consistently to predict phenotype from genotype? relationship between genotype and phenotype. thalassaemia intermedia alpha-thalssemia hemoglobin h disease and mental retardation: a new syndrome or a remarkable coincidence? acquired haemoglobin h disease in leukemia: pathophysiology and molecular basis pathophysiology of thalassaemia the beta-thalassaemias thalassaemia minor, the gilbert mutation, and the risk of gallstones thromboembolic events in beta thalassaemia major: an italian multicenter study optimal management of β thalassaemiaintermedia a simple index for initiating transfusion treatment in thalassaemiaintermedia osteoporosis in β-thalassaemia: clinical and genetic aspects osteoporosis in beta-thalassaemia major patients: analysis of the genetic background endocrine investigation and follow up in thalassaemia italian society for the study of thalassaemia and haemoglobinopathies; italian association for the study of the liver. management of chronic viral hepatitis in patients with thalassaemia: recommendations from an international panel effects of iron overload and hepatitis c virus positivity in determining progression of liver fibrosis in thalassaemia following bone marrow transplantation cardiac iron and cardiac disease in males and females with transfusion-dependent thalassaemia major: a t2* mri study guideline recommendations for heart complications in thalassaemia major cardiac t2* magnetic resonance for prediction of cardiac complications in thalassaemia major hypogonadism, diabetes mellitus, hypothyroidism, hypoparathyroidism: incidence and prevalence related to iron overload and chelation therapy in patients with thalassaemia major followed from 1980 to 2007 in the ferrara centre diabetes mellitus and impaired glucose tolerance in thalassaemia major: incidence, prevalence, risk factors and survival in patients followed in the ferrara center complications of thalassaemia major and their treatment h63d mutation in the hfe gene increases iron overload in beta-thalassaemia carriers how i treat thalassaemia guidelines for the clinical management of thalassaemia insight onto the pathophysiology and clinical complications of thalassaemia intermedia overview on practices in thalassaemiaintermedia management aiming for lowering complication rates across a region of endemicity: the optimal care study thalassaemia intermedia: revisited laboratory techniques for the identification of abnormalities of globin chain synthesis. haemoglobinopathy diagnosis changes of trace minerals (serum iron, zinc, copper and magnesium) in thalassaemia effects of iron overload on ascorbic acid metabolism bonemarrow failure due to relative nutritional deficiency in cooley's haemolytic anemia: painful erythropoietic crises in response to folic acid global epidemiology of haemoglobin disorders and derived service indicators guidelines for the clinical management of thalassaemia real-world use of iron chelators optimizing iron chelation strategies in beta thalassaemia major marrow transplantation for thalassaemia allogeneic stem cell transplantation for thalassaemia major fetal globin stimulant therapies in the beta-hemoglobinopathies: principles and current potential hydroxyurea in thalassaemia intermedia: a promising therapy therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human betaglobin future alternative therapies for beta-thalassaemia thalassaemiascreening in pregnancy ultrasound measurement of placental thickness to detect pregnancies affected by homozygous alphathalassaemia-1 enzymatic deficiency in primaquine-sensitive erythrocytes glucose-6-phosphate dehydrogenase deficiency glucose-6-phosphate dehydrogenase deficiency glucose-6-phosphate dehydrogenase deficiency: a historical perspective natural selection of hemi-and heterozygotes for g6pd deficiency in africa by resistance to severe malaria the molecular biology of enzymes of erythrocyte metabolism diabetic ketoacidosis does not precipitate haemolysis in patients with the mediterranean variant of glucose-6-phosphate dehydrogenase deficiency hereditary hemolyticanemias due to red blood cell enzyme disorders glucose-6-phosphate dehydrogenase deficiency active involvement of catalase during hemolytic crises of favism glucose-6-phosphate dehydrogenase deficiency: a hidden risk for kernicterus the need for neonatal glucose-6-phosphate dehydrogenase screening: a global perspective clinical practice guideline: management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation diagnosis from the blood smear g6pd mediterranean accounts for the high prevalence of g6pd deficiency in kurdish jews red blood cell deformability as a predictor of anemia in severe falciparum malaria phosphatidylserine as a determinant of reticuloendothelial recognition of liposome models of the erythrocyte surface adhesion of normal and plasmodium falciparum ring-infected erythrocytes to endothelial cells and the placenta involves the rhoptry-derived ring surface protein-2 destabilization and subsequent lysis of human erythrocytes induced by plasmodium falciparum haem products anaemia of acute malaria infections in nonimmune patients primarily results from destruction of uninfected erythrocytes loss of red blood cell-complement regulatory proteins and increased levels of circulating immune complexes are associated with severe malarial anemia anaemia in a phase 2 study of a blood stage falciparum malaria vaccine the anaemia of p. falciparum malaria hemozoin-and 4-hydroxynonenal-mediated inhibition of erythropoiesis. possible role in malarial dyserythropoiesis and anemia tumor necrosis factor may contribute to the anaemia of malaria by causing dyserythropoiesis and erythrophagocytosis low plasma concentrations of interleukin-10 in severe malarial anaemia compared with cerebral and uncomplicated malaria suppression of erythropoiesis in malarial anemia is associated with hemozoin in vitro and in vivo malariarelated anaemia prevalence of and risk factors for anemia in young children in southern cameroon severe anemia in malawian children malarial anemia: of mice and men intermittent preventive treatment with sulfadoxinepyrimethamine against malaria and anemia in pregnant women randomised placebo-controlled trial of iron supplementation and malaria chemoprophylaxis for prevention of severe anaemia and malaria in tanzanian infants severe and complicated malaria the pathophysiology of vivax malaria dynamic alteration in splenic function during acute falciparum malaria resurgence of blackwater fever in long-term european expatriates in africa: report of 21 cases and review glucose-6-phosphate dehydrogenase (g6pd) mutations and haemoglobinuria syndrome in the vietnamese population effect of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: a communitybased, randomized, placebo-controlled trial effect of routine prophylactic supplementation with iron and folic acid on preschool child mortality in southern nepal: community based, clusterrandomised, placebo-controlled trial oral iron supplementation for preventing or treating anaemia among children in malaria-endemic areas factors contributing to anemia after uncomplicated falciparum malaria intermittent preventive therapy for malaria with monthly artemether-lumefantrine for the postdischarge management of severe anaemia in children aged 4-59 months in southern malawi: a multicentre, randomised, placebo-controlled trial marked reduction in prevalence of malaria parasitemia and anemia in hiv-infected pregnant women taking cotrimoxazole with or without sulfadoxinepyrimethamine intermittent preventive therapy during pregnancy in malawi platelet kinetics and scintigraphic imaging in thrombocytopenic malaria patients the role of platelets in the pathogenesis of cerebral malaria erythrocytes infected by plasmodium falciparum activates human platelets plateletmediated clumping of plasmodium falciparuminfected erythrocytes is a common adhesive phenotype and is associated with severe malaria willebrand factor propeptide in malaria: evidence of acute endothelial cell activation thrombopoietin in plasmodium falciparum malaria immunemediated thrombo-cytopenia of malaria thrombocytopenia in malaria: who cares? thrombocytopenia in malaria can hematological parameters discriminate malaria from nonmalarious acute febrile illness in the tropics? dysregulation of coagulation in cerebral malaria the anatomic basis of purpura platelets, petechiae, and preservation of the vascular wall plasmodium falciparum infected erythrocytes induce tissue factor expression in endothelial cells and support the assembly of multimolecular coagulation complexes monocyte tissue factor expression induced by plasmodium falciparum-infected erythrocytes blood coagulation, inflammation, and malaria thrombocytopenia and release of activated von willebrand factor during early plasmodium falciparum malaria angiopoietin-2 is associated with decreased endothelial nitric oxide and poor clinical outcome in severe falciparum malaria elevated thrombomodulin plasma levels as a result of endothelial involvement in plasmodium falciparum malaria the pathogenic basis of malaria differentiating the pathologies of cerebral malaria by postmortem parasite counts fibrinolysis, inhibitors of blood coagulation, and monocyte derived coagulant activity in acute malaria epidemiology of anemia in human immunodeficiency virus (hiv)-infected persons: results from the multistate adult and adolescent spectrum of hiv disease surveillance project impact of highly active antiretroviral therapy on anemia and relationship between anemia and survival in a large cohort of hiv-infected women: women's interagency hiv study anemia is an independent predictor of mortality and immunologic progression of disease among women with hiv in tanzania human immunodeficiency virus hematology prevalence of parvovirus b19 infection in patients infected with human immunodeficiency virus clinical relevance of parvovirus b19 as a cause of anemia in patients with human immunodeficiency virus infection erythropoiesis in hiv-infected and uninfected malawian children with severe anemia anemia in hiv-infected patients receiving highly active antiretroviral therapy fatal disseminated intravascular coagulation and pulmonary thrombosis following blood transfusion in a patient with severe autoimmune haemolytic anemia and human immunodeficiency virus infection epoetin alfa for treatment of anemia in hiv-infected patients: past, present, and future once-weekly epoetinalfa improves quality of life and increases hemoglobin in anemic hiv+ patients epidemiology of thrombocytopenia in hiv infection surveillance for thrombocytopenia in persons infected with hiv: results from the multistate adult and adolescent spectrum of disease project an overview of the mechanisms of hiv-related thrombocytopenia role of molecular mimicry to hiv-1 peptides in hiv-1 related immunologic thrombocytopenia complement-independent, peroxide induced antibody lysis of platelets in hiv-1 related immune thrombocytopenia ineffective platelet production in thrombocytopenic human immunodeficiency virus-infected patients effect of highly active antiretroviral therapy on thrombocytopenia in patients with hiv infection response of severe hiv-associated thrombocytopenia to highly active antiretroviral therapy including protease inhibitors human immunodeficiency virus associated thrombotic thrombocytopenic purpura -favourable outcome with plasma exchange and prompt initiation of highly active antiretroviral therapy is it hiv ttp or hiv-associated thrombotic microangiopathy? adamts13 activity and the presence of acquired inhibitors in human immunodeficiency virus-related thrombotic thrombocytopenic purpura thrombotic microangiopathy in patients with acquired immunodeficiency syndrome before and during the era of introduction of highly active antiretroviral therapy collaborations in hiv outcomes research/us cohort. hivassociated thrombotic microangiopathy in the era of highly active antiretroviral therapy: an observational study aidsassociated non-hodgkin lymphoma prognosis of hiv-associated non-hodgkin lymphoma in patients starting combination antiretroviral therapy therapeutic options for hiv-associated lymphomas changes in aids-related lymphoma since the era of highly active antiretroviral therapy hodgkin lymphoma and immunodeficiency in persons with hiv/aids why would the incidence of hiv-associated hodgkin lymphoma increase in the setting of improved immunity? dynamics of epstein-barr virus in hiv-1-infected subjects on highly active antiretroviral therapy world health organization classification of tumours, pathology and genetics of tumours of haematopoietic and lymphoid tissues characterization of lymphomas in a high prevalence hiv setting acquired immunodeficiency syndrome-related lymphoma aids primary central nervous system lymphoma improvement of systemic human immunodeficiency virus-related non-hodgkin's lymphoma outcome in the era of highly active antiretroviral therapy incidence and outcomes of malignancy in the haart era in an urban cohort of hiv-infected individuals evolving characteristics of aids-related lymphoma leptomeningeal disease in aids-related non-hodgkin's lymphoma aids-associated malignancies human immunodeficiency virus-related lymphoma: relation between clinical features and histologic subtypes a prognostic index for systemic aids-related non-hodgkin lymphoma treated in the era of highly active antiretroviral therapy clinical features and outcome of primary effusion lymphoma in hiv-infected patients: a singleinstitution study primary effusion lymphoma prognostic factors in chemotherapy-treated patients with hiv-associated plasmablastic lymphoma molecular profile of epstein-barr virus in human immunodeficiency virus type 1-related lymphadenopathies and lymphomas chemotherapy for human immunodeficiency virus-associated non-hodgkin's lymphoma in combination with highly active antiretroviral therapy rituximab plus infusional cyclophosphamide, doxorubicin, and etoposide in hiv-associated non-hodgkin lymphoma: pooled results from 3 phase 2 trials rituximab plus concurrent infusional epoch chemotherapy is highly effective in hiv-associated b-cell non-hodgkin lymphoma factor replacement therapy in haemophilia -are there models for developing countries? hemophilia treatment in developing countries: products and protocols management of haemophilia in the developing world products for clotting factor replacement in developing countries management of hemophilia with minimal factor replacement in developing countries: role of ancillary therapy danazol increases factor viii and ix in classical hemophilia and christmas disease hiv and thrombosis: a review venous thromboembolic disease in the hiv-infected patient systemic thromboembolism in endomyocardial fibrosis (emf): clinical observations, aetio-pathogenesis and treatment effects of race and ethnicity on the incidence of venous thromboembolism pulmonary thromboembolism in an east african tertiary referral hospital epidemiology of postoperative venous thromboembolism in asian countries risk factors for symptomatic venous thromboembolism in thai hospitalised medical patients high incidence of symptomatic venous thromboembolism in thai hospitalized medical patients without thromboprophylaxis prevalence of factor v leiden in south tunisian blood donors prevalence of factor v leiden mutation in patients with thrombosis in tunisia lupus anticoagulant in nigerian patients living with human immunodeficiency virus/ acquired immunodeficiency syndrome lupus anticoagulant in nigerian women with preeclampsia translation of cure for acute lymphoblastic leukaemia to all children an approach to the management of leukemia in the developing world management of apl in developing countries: epidemiology, challenges and opportunities for international collaboration arsenic trioxide in the treatment of newly diagnosed acute promyelocytic leukemia: a single center experience treatment of children with newly diagnosed acute promyelocytic leukemia with arsenic trioxide: a single center experience treatment of childhood leukemias in underprivileged countries descriptive epidemiology of lymphoid and haemopoietic malignancies in bangalore, india outcome of childhood acute lymphoblastic leukaemia in resource-poor countries establishment of a pediatric oncology program and outcomes of childhood acute lymphoblastic leukemia in a resource-poor area lymphomas in sub-saharan africa -what can we learn and how can we help in improving diagnosis, managing patients and fostering translational research? long-term experience with burkitt's lymphoma in uganda adult t-cell leukaemia/lymphoma human t-cell leukemia virus type i and adult t-cell leukemia identification of subtype-specific genomic alterations in aggressive adult t-cell leukemia/ lymphoma treatment of adult t-cell leukemia/lymphoma: past, present, and future current management of adult t-cell leukemia/lymphoma meta-analysis on the use of zidovudine and interferon-alfa in adult t-cell leukemia/ lymphoma showing improved survival in the leukemic subtypes epidemiology of multiple myeloma in taiwan: increasing incidence for the past 25 years and higher prevalence of extramedullary myeloma in patients younger than 55 years reducing replacement donors in sub-saharan afirca: challenges and affordability laboratory costs of a hospital-based blood transfusion service in malawi from whole blood to component therapy: the economic, supply/ demand need for implementation of component therapy in sub-saharan africa warm fresh whole blood transfusion for severe haemorrhage: us military and potential civilian applications moving on from voluntary nonremunerated donors: who is the best blood donor should we neglect or nurture replacement blood donors in sub-saharan africa joint ukbts/nibsc professional advisory committee's (jpac) donor selection guidelines. guidelines for the blood transfusion services in the uk excluding blood donors at high risk of hiv infection in a west african city possible transmission of variant creutzfeldt-jakob disease by blood transfusion world health organization. who recommendations on sars and blood safety transfusiontransmitted malaria in countries where malaria is endemic: a review of the literature from sub-saharan africa the prevalence of malaria parasitaemia in blood donors in a nigerian teaching hospital transfusion-transmitted syphilis in teaching hospital predonation screening of blood donors with rapid tests: implementation and efficacy of a novel approach to blood safety in resource-poor settings blood donors and blood collecton: web interface-supported transmission risk assessment and cost-effectiveness analysis of postdonation screening: a global model applied to ghana effect of blood transfusion on survival among children in a kenyan hospital changing trends in blood transfusion in children and neonates admitted in kilifi district hospital maternal mortality in sub-saharan africa: the contribution of ineffective blood transfusion services press release who/25. 2000. whd/3 information sheet for clinicians development and evaluation of a new paediatric blood transfusion protocol for africa electrocardiographic st-segment changes during acute, severe isovolemic hemodilution in humans use of clinical judgement to guide administration of blood transfusions in malawi giving tranexamic acid to reduce surgical bleeding in sub-saharan africa: an economic evaluation autologous transfusion techniques: a systematic review of their efficacy intraoperative autologous blood management artificial o2 carriers: status in 2005 red blood cell transfusions in acute paediatrics survival and haematological recovery of children with severe malaria transfused in accordance to who guidelines in kilifi intermittent preventive therapy for malaria with monthly artemether-lumefantrine for the post-discharge management of severe anaemia in children aged 4-59 months in southern malawi: a multicentre, randomised, placebocontrolled trial bacterial contamination of pediatric whole blood transfusions in a kenyan hospital bacterial contamination of blood and blood components in three major blood transfusion centres in accra, ghana access the complete references online at www.expertconsult.com key: cord-263157-8jin6oru authors: martínez, miguel angel title: progress in the therapeutic applications of sirnas against hiv-1 date: 2008-10-13 journal: sirna and mirna gene silencing doi: 10.1007/978-1-60327-547-7_17 sha: doc_id: 263157 cord_uid: 8jin6oru therapeutic options against the human immunodeficiency virus type 1 (hiv-1) continue to expand with the development of new drugs and new therapeutic strategies. nevertheless, management of hiv-1 infected individuals has become increasingly complex. the emergence of drug-resistant variants, the growing recognition of the long-term toxicity of antiretroviral therapies and the persistence of viral reservoirs justify the continued efforts to develop new anti-hiv-1 strategies. recent advances regarding the utility of rna-mediated interference (rnai) to specifically inhibit hiv-1 replication have opened new possibilities for the development of gene-based therapies against hiv-1 infection. here, the recent advances in sirna-based therapies are reviewed. although a few hiv-1 infected individuals have remained healthy for nearly 18 years (2, 3) to date not a single spontaneous cure of hiv-1 infection has been confirmed, and infected individuals have either died or remained infected. therefore, hiv-1 poses a greater challenge to the development of classic vaccination or antiviral strategies geared to ultimately lead to the eradication of the virus. a safe and effective hiv-1 vaccine to stop the spread of hiv-1 has not yet been developed and the possibility of developing a successful vaccine in the near future remains to be seen (4) . the introduction of potent antiretroviral therapies has been an important achievement towards the control of hiv-1 infection and aids (5 , see table 17 .1 ). therapies to combat hiv-1 infection have resulted in a dramatic decrease in aids-associated morbidity and mortality in developed countries (6) . despite the development of successful therapeutic strategies employing treatment combinations, or highly active antiretroviral therapy (haart), it has not been possible to eradicate hiv-1 in infected individuals, mainly due to the persistence of viral reservoirs (7, 8, 9, 10) . the hiv-1 reservoir is not eradicated even after extended antiretroviral therapy that can reduce viremia to undetectable levels (<50 viral rna copies per milliliter of plasma). therefore, individuals infected with hiv-1 need to receive antiretroviral therapy for many years, if not for life. current treatments target viral enzymes, such as the reverse transcriptase (rt) and protease, as well as the envelope glycoprotein gp41 (table 17 .1 ). the most recent antiretroviral approved for clinical use blocks the binding of hiv-1 to its co-receptor ccr5 in host cells (11) . despite the success of potent combination regimens, the development of hiv-1 drug resistance constitutes a major hurdle towards longterm efficacy of current antiretroviral therapies (12) . moreover, just several years after the implementation of haart, the increase of hiv-1 drug resistance has required the initiation of a continuous effort in the development of new drugs, therapy strategies and viral targets. so far, 22 antiretroviral drugs have been approved and several others are in clinical or preclinical development (table 17 .1 ). current limitations of haart are not restricted to drug resistance. toxicity and side effects, such as hyperlipidemia, hyperglycemia, hypersensitivity, pancreatitis, lipoatrophy, anemia and neutropenia, rash, diarrhoea, gastrointestinal distress, insulin resistance, immune reconstitution syndrome, renal dysfunction, hepatotoxicity and an increased risk of liver cirrhosis and myocardial infarction have all been described in response to treatment with antiretroviral drugs (13) . moreover, the likelihood of side effects increases due to the improved lifespan as a result of the success of haart. finally, coinfection with hepatitis c, hepatitis b, tuberculosis or malaria complicates the treatment regimens for hiv-1 infected individuals. the limitations and challenges of current antiretroviral therapy justify the continued effort to develop new anti-hiv-1 strategies. fire et al. first discovered that introducing long double-stranded rna (dsrna) into the nematode caenorhabditis elegans led to the targeted degradation of homologous mrna, revealing the existence of a fundamental mechanism now known as rnai through which gene expression can be regulated (14) . later findings by elbashir et al. showed that rnai also occurs in mammalians cells (15) . importantly, this study made the extraordinary demonstration that cell transfection of synthetic 21 base pairs (bp) short interfering rna (sirna) duplexes can mediate rnai in a sequence-specific manner; this finding enabled the specific regulation of gene expression in a variety of biological systems, including diseased cells. in this review, i will focus on the progress in research employing rnai to disrupt the disease process caused by hiv-1. given that an exogenous rna can be targeted without affecting cellular functions, one of the most promising applications of rnai is in the treatment of infectious diseases. nevertheless, there are some obstacles to the development of an anti-hiv-1 rnaibased therapeutic agent. one major problem of all antiretroviral therapies is the emergence of resistant variants, and the enormous genomic heterogeneity of hiv-1 may hinder the efficacy of single defined sirnas. coexpression of multiple sirnas could reduce the emergence of single-sirna-resistant viruses, with an effect comparable to that achieved by haart, which combines three-or four-anti-hiv-1 drugs in treatment. an alternative strategy to counter the possibility of sirna-resistant viruses would be to target transcripts of cellular cofactors essential for hiv-1 infectivity or replication (e.g., specific cell surface receptors). the side effects of downregulating cellular targets over the long term, however, are currently unknown. delivery remains a major hurdle for rnai therapy, as sirnas are unable to cross the mammalian cell membrane without aid. gene-therapy vectors are capable of stably expressing sirna precursors, although well-documented hazards have been observed after the introduction of foreign vector sequences into chromosomal dna. finally, off-target effects, such as the targeting of genes sharing partial homology to the sirna and the stimulation of the innate immune system, need to be considered and avoided. although the innate immune system is efficiently triggered only by dsrnas more than 30 bp, high concentrations of smaller sirnas may be able to activate this pathway. nevertheless, there is no evidence that the activation of the innate immune system influences the degree or specificity of sirnas. the hiv-1 life cycle begins by viral binding via the viral envelope to the cellular receptor cd4 in conjunction with a coreceptor, either cxcr4 in the case of t-cell-tropic virus or ccr5 for macrophage-tropic virus ( fig. 17.1 ). hiv-1 infects cells of the immune system, specifically cd4+ cells, which include t lymphocytes, monocytes and macrophages. as a result, in the absence of effective vaccination or therapy, a slow and continued depletion 2. targeting hiv-1 replication by rnai fig of cd4+ t cells ensues and there is a progression towards aids. fusion of the viral and cellular membranes provides the hiv-1 genome, two rna molecules of positive polarity, access to the interior of the cell. the genomic viral rna is then converted into double-stranded dna by the hiv-1 rt (fig. 17.1 ). the viral dna forms a pre-integration complex along with the viral integrase, which is then transported to the nucleus where the dna is integrated into the host genome as a provirus. hiv-1 replication can be divided into early and late phases. during the early phase, spliced transcripts encoding two essential proteins, tat and rev, are synthesized. tat further activates viral transcription, whereas rev interacts with rev-responsive elements (rre) to facilitate nuclear export of unspliced and singly spliced mrnas. these late mrnas encode the remaining viral proteins, including gag, pol and env, that assemble at the cell surface together with the viral genome to form virions that are released from the cell by budding. the viral protease continues to process the viral polyproteins into their mature form, condensing the viral genomic rna core and yielding infectious particles ( fig. 17.1 ). soon after the demonstration that synthetic sirnas were able to induce the rnai mechanism in mammalian cells (15) , several studies reported that hiv-1 gene expression and replication ex vivo could be inhibited by virus-specific synthetic sir-nas (16 22) or expressed sirnas (16, 18) that were targeted to early or late phases of virus replication. sirnas against several hiv-1 components (gag, env, pol, the hiv-1 long terminal repeat, vif, nef, tat and rev) were confirmed to effectively target these genes. moreover, cd4+ t cells (18, 23) and macrophages (24, 25) , which are the natural targets for hiv-1 infection, were also found to be functional for rnai. however, an important question arose from these pioneer studies: is the virion-associated incoming genomic viral rna targeted by rnai? since hiv-1 is capable of being integrated in the host genome as well as infecting resting cd4+ t cells to create a pool of latently infected cells (4) , the capacity to target incoming viral rna by sirnas has important therapeutic implications ( fig. 17.1 ) (26) . if it is not possible to target incoming viral genomic rna, it will be virtually impossible to prevent the formation of integrated provirus and, as a result, to sterilize cells from infection. targeting the incoming viral rna may have several advantages, as the rnai machinery would only have to deal with two or a few (in the case of superinfection) genomic viral rnas. however, after a provirus is established, several thousands of viral transcripts are generated de novo in the infected cell and the targeted degradation of these may be more difficult for the rnai machinery. data has been conflicting as to whether rnai can target the incoming genomic viral rna of infecting hiv-1 particles. several studies have reported degradation of the incoming rna genome in cells transfected with artificial sirnas or stably expressing sirnas (18 -20, 27) . however, other studies have reported an absence of rnai-mediated degradation of the genomic viral rna in sirnas-transfected or sirna-producing cells (22, 28, 29) . moreover, a study with the non-segmented, negative-strand rna virus respiratory syncytial (rsv) showed that genomic and antigenomic viral rnas encapsidated in the virus nucleocapsid protein were not susceptible to sirna-mediated silencing, but viral mrnas were highly susceptible (30) . the mechanisms underscoring the efficiency of specific sirnas in targeting genes is not completely known. rnai efficiency is known to be influenced by local rna structure of the target sequence (31) . randomly selected sirnas against a target sequence showed a large variation in their efficiency (32) . it has been argued that some regions of genomic viral rna might be less accessible to sirnas when the rna is contained in the virus reverse transcription complex; therefore, some sirnas might be more effective than others in inhibiting this early step in virus replication (26) . recently, a more exhaustive study was performed to address this issue (33) . in this study, the transduction efficiency of a lentiviral vector, as measured by the number of successful integration events, was determined in a cell line stably expressing an sirna targeting the hiv-1 nef sequence. the results indicated a similar transduction efficiency for vectors regardless of the presence or absence of the nef target sequence in their genome. moreover, no reduced transduction efficiencies were observed in the presence of multiple other stably expressed sirnas targeting the vector genome, or when synthetic sirna targeting nef was transiently transfected prior to transduction. these results highlight the difficulties of designing therapeutic rnai strategies aimed at preventing hiv-1 proviral integration. silencing tat, a factor that must be expressed before efficient provirus transcription can occur, may make it easier to target other viral transcripts by virtue of their lower abundance when tat function is compromised (26) . synthetic sirnas mediate strong and specific but transient suppression of gene expression (18 -20, 28) . the transient reduction in gene expression by sirnas severely restricts its applications in a therapeutic setting of a persistent infection, such as hiv-1 infection. however, this limitation was quickly overcome with the use of plasmids or viral vectors that expressed sirnas from polymerase-iii transcription units. the sense and antisense strands of an sirna were expressed from two different promoters and rnai was triggered upon annealing of the two strands (16) . a more potent gene silencing was achieved when the sense and antisense strands were expressed as a single transcript with the ability to form a duplex hairpin structure (34) . indeed, hiv-1 replication could be efficiently inhibited with such short hairpin rnas (shrnas) in stably transduced cell lines (34 -37 ) . an additional benefit in using shrnas is that these can be processed by dicer like the endogenous micrornas (mirnas). tat sirna delivered as a pre-mirna precursor was 80% more effective in reducing hiv-1 p24 antigen production than tat sirna expressed as a conventional shrna. modeling shrnas after endogenous mirnas can increase the antiviral potency of rnai (38) . as discussed in this section, an exogenously induced rnai response in mammalian cells mediated by sirnas or shrnas can inhibit hiv-1 replication. these results raise the question of whether hiv-1 interacts with the cellular rnai machinery, and understanding the potential physiological interaction between hiv-1 and the rnai machinery could significantly contribute to the design of an effective rnai-based therapy. rnai constitutes a key component of the innate immune response to viral infection in both plants and invertebrate animals (39) , and has been postulated to have a similar protective function in mammals (40) . in plants and invertebrate animals, long dsrna serves as the initial trigger for the rnai mechanism; the initial dsrna is then processed by dicer into sirnas. however, in mammalian cells, long dsrna sequences (more than 30 bp in length) are potent inducers of the interferon (ifn) response (41) . it remains unclear whether the introduction of long dsrna into mammalian somatic cells is capable of resulting in the production of sirnas (39) . nevertheless, rnai can be induced in mammalian cells by the nuclear endogenous expression of rnas that are intermediates in the mirna biogenesis pathway (i.e., sirnas and shrnas). the human genome encodes several hundred different mirna molecules that are believed to be key players in the posttranscriptional regulation of many aspects of cellular differentiation, and rnai has been postulated to exist in mammals only as a mechanism of post-transcriptional regulation "programmed" by endogenously encoded mirna, rather than being involved in intrinsic antiviral immunity (39) . all rna viruses, except retroviruses, produce long dsrna molecules in infected cells that represent essential intermediates in genomic rna production. many dna viruses also generate long dsrna, as their small packed genomes have convergent transcription promoters. some studies have attempted to identify sirnas in virus-infected human cells. pfeffer et al. (42) failed to identify any viral sirna in cells infected by dna viruses, such as human cytomegalovirus, kaposi sarcoma-associated herpes virus, epstein-barr virus and mouse herpes virus, as well as hiv-1 and the rna viruses for yellow fever and hepatitis c. this report, however, did identify several virally encoded mirna molecules in the dna virus. in contrast, bennasser et al. (43) reported that hiv-1 gives rise to an sirna that can inhibit hiv-1 replication, and additionally proposed that the hiv-1 tat protein relieves this inhibition by blocking the function of the cellular dicer, which plays a key role in both sirna and mirna biogenesis. moreover, the ebola virus vp35 protein has been shown to be a suppressor of rnai in mammalian cells and its suppressor activity is functionally equivalent to that of the hiv-1 tat protein (44) . vp35 can replace hiv-1 tat and thereby support the replication of an hiv-1 variant deficient for tat. these results support the hypothesis that rnai is indeed part of the innate antiviral response in mammalian cells. however, in a recent report, the stable expression of physiological levels of tat did not globally inhibit mirna production or expression in hiv-1 infected human cells (45) . strong evidence for rnai as part of the antiviral innate immune in plants comes from the demonstration that almost all plant viruses encode one or more suppressor of rna silencing (srs) proteins, which target several key steps in the rnai response (40) . similarly, several viruses that target invertebrates (specifically nematodes and insects) also encode srs proteins. flock house virus, a member of the nodavirus family that infect both insects and vertebrate cells, encodes a viral srs that inhibits dicer function (46) . further evidence of the controversial relationship between hiv-1 and cellular rnai machinery comes from the recent report by triboulet et al. (47) ,which provides evidence for a physiological role of the mirna-silencing machinery in controlling hiv-1 replication. this study showed that hiv-1 infection and replication were more efficient in peripheral blood mononuclear cells from hiv-1-infected donors and latently infected cells knocked-down for dicer or drosha, a mirna processing factor. moreover, hiv-1 actively suppressed the expression of the polycistronic mirna cluster mir-17/92, and this suppression was found to be required for efficient viral replication. as suggested in this study, these findings may help address the current challenge of how to activate latent viral reservoirs in hiv-1 therapy. virally encoded mirnas have been discovered in herpes viruses (39) , and human cytomegalovirus has been shown to evade the host immune system by targeting host genes with virally encoded mirnas (48) . whether rna viruses, including hiv-1, encode mirnas still remains controversial (48) . the therapeutic implications of this molecular feature are intriguing, as targeting these viral mirnas might constitute an antiviral therapy while mimicking their role could provide a means of immunosuppressive therapy (48) . latent infection is one of the most important characteristics required for the in vivo survival of all hiv-1 strains and the major obstacle in eradicating virus infection with haart (7-10) . a recent study showed that cellular mirnas potently inhibit hiv-1 production in resting primary cd4+ t cells (49) ; the 3 ′ ends of hiv-1 mrnas were found to be targeted by a cluster of cellular mirnas including mir-28, mir-125b, mir-150, mir-223 and mir-382, which are enriched in resting cd4+ t cells compared to activated cd4+ t cells (see chap. 20) . because specific inhibitors of these mirnas substantially counteracted their effects on the target mrnas (49) , a combined mirna inhibitor panel could be used to activate latent hiv-1 for therapeutic purposes. in agreement with the former report, the cellular mirnas mir-196, mir-296, mir-351, mir-431 and mir-448 have been shown to be capable of inhibiting hepatitis c virus replication and infection (50) . these mirnas are upregulated by ifn α / β , suggesting that cellular mirnas may be components of the mammalian innate immune response (50) . this review highlights the evidence showing that rnai provides a robust method for specifically inhibiting the expression of targeted hiv-1 genes, and its promise as a novel and broadly applicable approach to antiviral therapy. however, clinical application of rnai faces several challenges, specifically the potential for viral escape. one of the main advantages of the rnai mechanism is that it is highly sequence specific. pioneer studies confirmed the specificity of rnai by evaluating the activity of an sirna with one or more mismatches relative to the target rna sequence (18) ; these studies showed that a mismatch may be sufficient to reduce the silencing effect, suggesting that an unspecific antiviral response induced by sirna was not involved in the silencing effect, but also showing that hiv-1 may easily escape inhibition by sirnas. indeed, hiv-1 has been observed to promptly escape suppression by effective sirnas (36, 51, 52) . unlike eukaryotic dna polymerases, the hiv-1 rt lacks proofreading activity, and its error rate has been estimated at 10 −4 to 10 −5 mutations per nucleotide and cycle of replication (table 17 .2 ) (53) . if one assumes that 10 9 -10 10 viral particles are produced each day in an infected person (54, 55) , these must be the product of at least 10 7 -10 8 replication cycles. given the length of the hiv-1 genome (approximately 10,000 nucleotides) and its high recombination rate (56) , it is likely that every single possible point mutation (and likely many double mutations) will occur at least once each day in an infected individual (57, 58) . although specific combinations of multiple mutations may be rare, it is clear that the degree of potential genetic change drives the diversification of hiv-1 in response to the selective pressure of host immune responses or antiretroviral therapy (table 17 .2 ). hiv-1 strains have diversified extensively through mutation and recombination since their initial transmission to humans many decades ago in central africa. phylogenetic analysis of numerous isolates obtained from diverse geographic origins has allowed the division of hiv into types, groups, subtypes, sub-subtypes, circulating recombinant forms (crfs) and unique recombinant forms (urfs). hiv-1 is divided into three groups, m, o and n (59) . most sequences within hiv-1 group m, which accounts for the majority of infections worldwide, fall into a limited number of discrete clades, allowing the classification into subtypes and sub-subtypes. near 30 circulating genetic forms of the hiv-1 group m are presently recognized, including 11 subtypes and sub-subtypes, and nearly 20 crf. the diverse hiv-1 subtypes, crfs and the inter-subtype mosaic genomes further exacerbate the problem of designing effective sirnas. boden et al. (51) characterized the potency and durability of virus-specific rnai in cell lines that stably expressed shrna targeting the hiv-1 transactivator protein gene tat. they found that the antiviral activity of tat shrna was abolished due to the emergence of viral species harboring a point mutation in the shrna target region. during the first three weeks, hiv-1 replication was reduced by 95% in cells expressing tat shrna compared to control cells. by day 25, however, viral titers increased, indicating loss of tat shrna-mediated antiviral activity, and sequencing of this virus stock revealed that the emerged viral species contained a nonsynonymous mutation at nucleotide position 9 of the targeted sequence. das et al. (36) used retroviral transduction to stably introduce vectors expressing sirnas directed against generally this averages at 1-100 (if not longer in some cases) mutations per genome. error rate has been estimated at between 10 −4 to 10 −5 mutations per nucleotide and cycle of replication. 10 kb variable but 10 9 -10 10 viral particles can be produced each day in an infected individual. every single possible point mutation (and probably many double mutations) will occur at least once each day in an infected individual. many recorded adaptive changes depend on one or a few mutations. based on (57, 58) the hiv-1 nef gene into human t cells; hiv-1 escape variants that were resistant to sirna-nef appeared after several weeks of culture, and these rnai-resistant viruses contained point mutations, double point mutations or partial or complete deletion of the nef gene target sequence. the complete inactivation of the accessory nef gene has a relatively minor impact on the replication capacity of hiv-1 ex vivo. interestingly, westerhout et al. (52) used human t cells that stably express an sirna-nef target sequence to show that hiv-1 can also escape sirna-mediated nef inhibition by a point mutation outside the target sequence. this mutation changed local rna folding, such that the target sequence becomes inaccessible to the rnai machinery. recently, sabariegos et al. (60) showed that optimal hiv-1 gene silencing by sirna requires precise complementarity with most of the target sequence and that only a few substitutions at the 5 ′ and 3 ′ ends are partially tolerated. viral escape was simulated by systematically introducing single-nucleotide substitutions in all 19 hiv-1 residues targeted by an effective sirna directed against the rt coding region. all mutant viruses that were tested replicated better in the presence of the sirna-rt than in the presence of the wild type virus. the antiviral activity of the sirna-rt was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitutions at 3 of the 19 positions rendered nonviable viruses). with the exception of one substitution, substitutions at either the 5 ′ or 3 ′ end were better tolerated by the rna interference machinery and only partially affected sirna-rt inhibition. since the emergence of resistant virus variants poses a serious problem for using rnai in a therapeutic setting, different strategies have been developed to counteract viral escape. one obvious strategy that will successfully both inhibit a diversity of hiv-1 isolates and protect against the emergence of viral escape would be to identify viral conserved sequences for targeting. targeting highly conserved sequences may hamper the development of escape mutants, as these variants may be highly compromised for viral fitness. indeed, lee et al. (37) targeted highly conserved hiv-1 vif sequences and successfully suppressed a variety of primary viral isolates from five different viral clades. this study also showed that tolerance to target sequence mismatches may depend on the sequence of the sirna tested (37) . nevertheless, our knowledge of hiv-1 variability and experience from over one decade of antiretroviral therapy have taught us that is unrealistic to try to target hiv-1 with a unique sirna because the rapid development of resistance will occur. the high error rate of hiv-1 rt and the rapid viral turnover means that for each of the 19 nucleotides targeted by an si-or shrna, several potential escape variants will be already present before the start of therapy (57) . moreover, hiv-1 may escape rnai through silent mutations, which, in most cases, will not have any cost on the viral fitness. to counteract this strategic weakness, co-expression of multiple sirnas or shrnas that target conserved rna sequences could reduce the emergence of single sirna-resistant virus with a comparable effect to that achieved by the multiple anti-hiv drug combination approach employed by haart. delivery of multiple sirnas could induce highly active antiretroviral gene silencing (haags) (61) . the combination of multiple shrnas against conserved hiv-1 genomic regions effectively inhibits hiv-1 replication. expression of three different shrnas from a single lentiviral vector resulted in similar levels of inhibition per shrna compared to single shrna vectors (62) . in this study, the three shrnas targeted gag and pol hiv-1 coding regions; moreover, when cells transduced with a double shrna viral vector were infected, virus escape was delayed (62) . there are several methods currently in use that are capable of expressing multiple effective sirnas. one possibility is by inserting multiple shrna-expression cassettes into a viral vector. however, repeats of the same regulatory sequences (e.g., the u6 or h1 polymerase iii promoter) may cause genetic instability and reduced titer of the vector system (63) . moreover, some studies have shown that it is possible to saturate the rnai pathway by high levels of expression of shrnas (64, 65) resulting in cellular toxicity, particularly with the u6 promoter [64] . polymerase iii promoters other than u6 and h1 has been used (66) . another approach has been the use of long-hairpin rnas (lhrnas), from which multiple sirnas can be produced. lhrnas greater than 50 bp in length can be expressed in cells and create multiple sirnas via dicer-mediated processing without inducing the ifn pathway (67) . several reports described efficient rnai induction by lhrnas against hiv-1 (68, 69) . nishitsuji et al. (69) showed that a 50 bp lhrna against a conserved hiv-1 integrase region suppressed hiv-1 replication in a variant resistant to a shorter shrna for the same target. similarly, lentiviral vectors expressing 50, 53, or 80 bp lhrnas targeting contiguous sequences within the tat and rev genes inhibited viral replication against both non-mutant and mutant variants of hiv-1 (70) . an alternative to target multiple conserved viral genomic regions is to use a second generation of sirnas that recognize the mutated target sites (60, 69, 71) by using sirnas or shrnas that target the most likely escape variants. sabariegos et al. tested a second generation sirna that compensates for a fully resistant mutation at position 9 of the target sequence (60) . nishitsuji et al. isolated single point mutation resistant viruses that emerged in lentiviral vector transduced cells expressing shrnas against the hiv-1 u3 region, integrase and tat genes (69) and restored viral inhibition by using a second generation shrna that matched escape mutants (69) . the use of cell culture infections to determine which target site mutations result in replication competent escape variants will allow the design of sirnas capable of inhibiting these escape variants. to reduce the number of second generation sirnas to be designed, this strategy should also target a conserved region of the hiv-1 coding sequence and the new sirnas should also be capable of inducing rnai. recently, escherichia coli endoribonuclease iii (rnase iii) or mammalian dicer was used to cleave dsrna into endoribonuclease-prepared sirna (esirna) (72) , which generate a variety of sirnas that efficiently and specifically target multiple sites in the cognate rna (73, 74) . in this study, esirnas targeting the hiv-1 rt coding region reduced viral replication by 90% in a dose dependent and sequence specific manner. importantly, esirnas obtained from the prototypic rt sequence of the hxb2 strain and from highly mutated rt sequences showed similar degrees of viral inhibition, suggesting that the heterogeneous population of esirnas could overcome individual mismatches in the rt sequence. these results demonstrated the ability of esirnas to function as potent hiv-1 inhibitors. moreover, sequence targets do not need to be highly conserved to reach a high level of viral replication inhibition. nevertheless, this work was performed in cell culture and its translation to an in vivo setting may be difficult. another recently explored strategy has been to combine an shrna with an anti-hiv-1 ribozyme and an rna decoy. a triple combination lentiviral construct comprised of a u6-driven tar rna decoy with a u16 snorna for nucleolar localization, a u6 promoted shrna targeted to both tat and rev, and a va1 promoted chimeric anti-ccr5 trans-cleaving hammerhead ribozyme efficiently transduced human progenitor cd34+ cells and improved suppression of hiv-1 over 42 days compared to a single anti-tat/rev shrna or double combinations of shrna/ ribozyme or decoy (75) . this triple combination is about to enter human clinical trials for aids/lymphoma patients using autologous hematopoietic progenitor cells as the targets for vector insertion. a second trial in which the same construct will be inserted in autologous t lymphocytes will most likely initiate in late 2007 or early 2008 (66) . in order to prevent provirus establishment and to augment the genetic barrier for viral escape, cellular receptors or co-factors involved in the initial phase of infection may be targeted. the combination of sirnas or shrnas directed against both hiv-1 and cellular transcripts may result in a more potent and robust inhibition of viral replication. nevertheless, targeting a cellular cofactor required by the virus should be performed with caution as this approach has the potential to harm host cells. several cellular factors involved in the hiv-1 life-cycle have been explored as possible rnai targets: the cd4 receptor and coreceptors ccr5 and cxcr4 (17, 76, 77) , integration factors like baf-1, emerin and ledgf/75 (78, 79, 80) , transcriptional factors such as nf-κ b, pak-1 and cyclin (22, 81, 82) , and furin which is involved in env maturation (81) . since hiv-1 actively suppressed the expression of the polycistronic mirna cluster mir-17/92 and this suppression was found to be required for efficient viral replication (47) , nuclear expression of some mirnas from this cluster may be another alternative to inhibit hiv-1 replication (66) . in vivo suppression of genes such as cd4 could be limited due to roles in normal immune function, making hiv-1 coreceptors more attractive alternatives to target host proteins. ccr5 may be a potential coreceptor target, as a homozygous mutation in ccr5 effectively conferred protection from hiv-1 without any serious deleterious effects in human immune function, and heterozygous individuals with 50% decrease in ccr5 surface expression have lower plasma viral load and a substantially prolonged course of disease (83) . a potent and noncytotoxic shrna directed to ccr5 stably down-regulates ccr5 when introduced via cd34+ hematopoietic stem cell transplant in non-human primates (84) and these cells were less susceptible to simian immunodeficiency virus (siv) infection ex vivo. recently, several ccr5 antagonists examined in clinical trials were able to reduce plasma viral loads by more than one order of magnitude during treatment (11) . although, ccr5 antagonists hold great promise, the long-term toxicity associated with the impairment of ccr5 function remains to be addressed. targeting cellular factors will require extensive toxicity studies. delivery remains a major hurdle for rnai therapy, as sirnas are unable to cross the mammalian cell membrane without aid. most of the ex vivo transfection methods used for delivering sirnas can not be used in vivo. there are two strategies for delivering sirnas in vivo. as discussed in the previous section, one is to stably express sirna precursors, such as shrnas, from viral vectors using gene therapy technology; the other is to deliver synthetic sirnas naked or by complexing or covalently linking the sirna with lipids, aptamers, peptides or proteins (85) . intranasal administration of naked sirnas either in saline or with excipients such as 5% dextrose or lung surfactants reduced the viral load of respiratory syncytial virus (rsv) and parainfluenza virus in pediatric or immuno-compromised individuals by more than three orders of magnitude (86 , see chap. 15). similarly, sirna intranasally administered in a non-human primate model of severe acute respiratory syndrome (sars) corona virus infection significant inhibited viral replication in the lung (87) . these studies clearly demonstrated the utility of rnai to treat viral respiratory infections. liposomes, vesicles with an aqueous compartment enclosed in a phospholipid bilayer that can fuse with cell membranes and enhance drug delivery into cells, have been extensively used to deliver sirna in vitro and in vivo. in vitro transfection of sirna using lipid-based delivery agents is a routine laboratory procedure. lipid-based formulations effectively delivered sirna to the liver in animal models of hepatitis b and ebola virus infection (88, 89) . locally injected liposomes have also delivered sirna effectively to target cells in the eye and nervous system, and to tumors (85) . topical delivery of sirnas using liposomes may be especially effective. intravaginal application of sirnas protected mice from lethal herpes simple virus type 2 (hsv-2) sexually transmitted infection (90) . the cervical/vaginal mucosa is the main port of hiv-1 entry in women, thus an effective topical microbicide against hiv-1 may be useful to prevent sexual transmission. since the cellular coreceptor ccr5 is required for infection by the majority of primary hiv-1 isolates, a lipid-formulated sirna against the ccr5 transcripts will be an excellent candidate for anti-hiv-1 microbicide. sirnas can be complexed with cationic peptides and polymers to form stable nanoparticles via ionic interactions with their negatively charged phosphate backbones (91) . similarly, one study showed that a protamine antibody fusion protein delivered non-covalently bound sirna to hiv-1-infected primary cd4+ t cells in vitro with high efficiency (92) . in this approach, the fab fragment of an hiv-1 envelope antibody mediates receptorspecific binding to cells expressing the hiv-1 envelope protein, illustrating the potential for antibodies to also direct sirna selectively into cells in vivo. given the hiv-1 life cycle, in which the persistence of a latent reservoir of resting infected cells makes eradication of the virus from infected individuals extremely difficult, the most promising strategy for in vivo delivery of sirna is the use of gene therapy approaches. because hiv-1 predominately infects t lymphocytes and macrophages, blood cd34+ hematopoietic stem cell transplant could be the best strategy, in which progeny cells would stably express sirnas that down-regulate viral or cellular genes that are targets for hiv-1 infection. as noted before, hiv-1 can infect resting non-dividing cd4+ t cells, thus lentiviral vectors should be employed over murine retroviral vectors, as hiv-1-based lentiviral vectors have been shown to be particularly suited for the transduction of non-dividing cells, such as hematopoietic progenitor cells (93) . these hiv-1-based vectors seem to be preferable candidates for gene therapy development in the treatment of hiv-1-infected individuals (94) . however, shrnas targeting highly conserved hiv-1 sequences may be an obstacle in vector production, as it has been reported that expression of shrnas that target also the delivering vector can result in a reduction of the transduction titer (23) , suggesting that these shrnas could cross-react with critical sequences of the vector. nevertheless, new lentiviral vectors have been developed to overcome this issue, and the vector backbone may be modified by point mutations to resist rnai-mediated degradation during vector production (95) . however, introducing rnai-resistant mutations within the lentiviral vector backbone adds the potential risk of transfer of resistance to the wild type hiv-1 genome. one solution that has been suggested would be to incorporate stop codons within these vector sequences that should not affect the rna/dna function, yet would confer full resistance to rnai (96) . recombination will result in an rnai-resistant yet replication-defective hiv-1 variant due to the stop codons. alternatively, it has also been shown that the incorporation of target sequences for endogenous mirnas within the lentiviral genome can severely reduce potential recombination or mobilization (97) . the hiv-1 genome is highly prone to recombination, and recombinants arise very frequently (56) . lentiviruses, unlike murine retroviruses, are more prone to integrate distally from promoters within intron sequences, potentially limiting their overall oncogenicity (98) . one of the main concerns with current gene therapy approaches is insertional mutagenesis from preferential integration of retroviral vectors into actively transcribed genes, including proto-oncogenes. this has led to hematological malignancies in young individuals with severe immunodeficiency (scid) disease treated with retroviral-based gene therapy (99) . this problem could be addressed by designing vectors that integrate into specific, well-defined regions of the genome. the partially random insertion of transgenes into chromosomal dna of hematopoietic cells may induce clonal competition, which could potentially trigger leukemia or sarcoma (100, 101) . lentiviral vectors derived from hiv-1, hiv-2/siv, or feline immunodeficiency virus (fiv) have been shown to be capable of stably transducing many cell types, including hematopoietic stem cells (102, 103) . interestingly, hiv-1 vectors have been shown to cross-package by fiv and are capable of stably transducing and protecting human primary blood mononuclear cells from hiv-1 infection (104) . fiv packaged hiv-1 vectors reduce the likelihood of immune recognition, or seroconversion, due to exposure to hiv-1 structural proteins. a continuous effort is focused on producing effective, safe and high-titer lentiviral vectors to deliver rnai. several important issues related to in vivo safety, toxicity or side effects warrant close attention before rnai may be considered a valid alternative for treating hiv-1 infection or other diseases. in mammalian cells, dsrna is recognized by dsrna sensors such as toll-like receptors (tlrs), the dsrna-dependent protein kinase r (pkr) and retinoic-acid-inducible gene-i (rig-i), which are components of the innate immune system. this recognition leads to the activation of the ifn-regulatory transcription factors and nf-kb, which in turn results in the expression of the ifns (41) that subsequently activate the transcription of hundreds of ifnstimulated genes (isgs) through the jak-stat pathway. many isgs encode proteins with antiviral activities, including pkr. the ifn response plays a crucial role in antiviral immunity in vertebrates, particularly against rna viruses that generate dsrna molecules during their life cycle. the ifn response could significantly influence the in vivo application of sirna owing to the off-target effects and toxicities associated with immune stimulation. sirna molecules less than 30 bp in length are generally considered incapable of inducing ifn pathways (15) . however, synthetic sirnas formulated in non-viral delivery vehicles can be potent inducers of ifns and inflammatory cytokines both in vivo in mouse and ex vivo in human blood cells (105) . the immunostimulatory activity of formulated sirnas and the associated toxicities may be dependent on the nucleotide sequence (106) . although some of the off-target effects can be reduced using lower concentration of sirnas, the ifn response was observed even at low concentrations (107) . these studies emphasize the relevance of examining the immunostimulatory effects of any potential therapeutic sirna in human immune cells prior to clinical applications. replacement of the 2 ′ -hydroxyl uridines with either 2 ′ -fluoro, or 2 ′ -deoxy or 2 ′ -o methyl uridines can abrogate immune recognition of sirnas by tlrs without compromising sirna silencing potency (108, 109, 110) . in addition, modified nucleotides may protect sirnas from nuclease degradation and ameliorate their pharmacokinetic parameters in vivo (111) . similar to synthetic sirnas, endogenously expressed shrnas also activated the ifn pathway (112) . of particular concern is the development of a multiple shrna or lhrnas approach against hiv-1 because these molecules are longer than 30 bp. endogenously (nuclear) expressed shrnas were recently shown to evade detection by tlrs, rig-1 and pkr when integrated in cd34+ progenitor hematopoietic stem cells (113) . endogenously expressed lhrna encoding an effective nef-specific shrna was capable of inhibiting hiv-1 production without inducing the type i ifn genes (68) . endogenously produced dsrna is suggested to be less active than exogenous dsrna in inducing the ifn response (114) . extended shrnas (e-shrnas) of 40-44 bp that encode two effective sirnas against conserved hiv-1 sequences, pol and nef, did not induce the ifn response in e-shrna transfected 293t cells (63) . a simple strategy for avoiding activation of the ifn response by dsrna has been described (67) , where modified hairpin-rnas (mhrnas) of more than 100 bp with multiple specific point-mutations within the sense strand and transcribed from the u6 or trna(val) promoters can produce rnai without inducing the ifn pathway genes (67, 115) . the expression of lhrnas also appears to be well tolerated and does not induce ifn gene activation in vivo when delivered to mice via by hydrodynamic tail-vein injection (116) . another potential side effect of sirnas and shrnas is lethality associated with overdosing. a previous study in which mice were treated with high doses of a viral vector expressing shrnas that resulted in high copy numbers per cell led to fatality due to oversaturation of the mi/sirna pathways (65) . this potential risk should be assessed prior to an eventual clinical rnai application. cells should be transduced with few or a single vector copy to avoid high expression levels that may induce unwanted side effects. overdosing could also induce off-target effects in which sirnas or shrnas silence partially complementary transcripts through a mirna-like mechanism. cytotoxic effects of shrnas in human t lymphocytes as a result of overexpression have also been reported (64) . the risk of adverse effects of sirnas would increase in situations where shrna expression is to be maintained in a living organism for a long period, such as for intracellular immunization against hiv-1. in addition, transduced t cell lines and differentiated primary human t cells are relatively different from the hematopoietic cd34+ stem cells that will be transduced in an ultimate gene therapy therapeutic setting. stem cells will develop into different lineages, and expression of shrnas could influence their development. as suggested before (96) , saturation of the mirna pathway may disturb hematopoiesis, and indeed, mirnas are involved in the regulation of genes that control hematopoiesis in the mouse (117) . whether in vivo gene silencing by sirna or shrnas can disrupt the endogenous mirna pathway remains to be addressed. recently, target genes were shown to be effectively silenced in the mouse and hamster liver by systemic administration of synthetic sirna without any demonstrable effect on mirna levels or activity (118) . sir-nas targeting two hepatocyte-specific genes (apolipoprotein b and factor vii) were administered to mice and achieved efficient (80%) silencing of mrna transcripts without significant changes in the levels of three hepatocyte-expressed mirnas (mir-122, mir-16 and let-7a) (118) . moreover, multiple administrations of an sirna targeting the hepatocyte-expressed gene scap in hamsters achieved long-term mrna silencing without significant changes in mir-122 levels (118) . this study may advance the use of sirnas as a safe therapeutic alternative. another potential side effect for sirnas and shrnas is the silencing of cellular mrnas that share partial homology to the sirna or shrna sequences through an mirna-like mechanism. this off-target effect requires complementarity between the sirna seed region and the 3 ′ untranslated region of a target mrna (119, 121, 122) . any effective sirna or shrna may in theory have numerous potential off-target mrnas, thus off-target silencing may not be easily eliminated by sirna sequence selection. moreover, the combining of multiple shrnas would increase the number of off-target mrnas potentially affected. off-target transcript silencing may limit the specificity of sirnas for therapeutic applications. one study showed that 2 ′ -uridine modifications of sirnas can reduce sirnas off-target effects (123) , and similarly, chemical modification also reduced off-target phenotypes in growth inhibition studies. key to the modification was a 2 ′ -o -methyl ribosyl substitution at position 2 in the guide strand, which reduced the silencing of most off-target transcripts exhibiting complementarity to the seed region of the sirna guide strand (124) . ex vivo selection of sirnas to verify the absence of unwanted off-targets should help to define strategies to either enhance or avoid the non-specific effects of sirnas in order to develop safe therapeutics. studies with an appropriate animal model should also help preclinical assessment of safety and efficacy. hiv-1 research is largely restricted to in vitro, ex vivo or clinical studies, all limited in their ability to rapidly assess new strategies to treat virus infection. the humanized rag2(−/−) gammac(−/−) scid mouse model sustains long-term multi-lineage hematopoiesis and is capable of mounting immune responses, achieved by the intraperitoneal injection of cd34+ precursor cells into a newborn rag2(−/−) gammac(−/−) mouse (125, 126) . in this model, injecting p53 shrna-transduced cd34+ cells resulted in stable expression and down-modulation of p53 in the mature t-cell offspring. infection of hiv-1 in this mouse model resulted in high viremia and cd4+ t cell depletion (127) . therefore, this in vivo animal model and/or others developed in the near future should be valuable to evaluate rnai-based strategies aiming to prevent or treat hiv-1 infection. the discovery of rnai has provided us with powerful new tools for biological research and drug discovery, and rnai is currently advancing from basic research to clinical trials. it has also expanded the direction of a new field of therapeutic strategies with the potential to treat a wide range of different diseases, including hiv-1. several decades ago, adaptive immunity allowed the possibility of preventing the spread of many pathogenic human viruses. nevertheless, the discovery of an effective preventive or therapeutic vaccine against hiv-1 remains elusive. rnai therapeutics is rapidly arriving into clinical studies for many diseases, including several which are currently untreatable or difficult to treat with conventional drugs. although promising studies and results have emerged over the recent years, the challenge remains as to whether rnai will be suitable for treating or preventing hiv-1 infection. mam is supported by the spanish minsiterio de educación y ciencia (mec) project bfu2006-01066/bmc, fondo de investigación sanitaria (fis) project pi070098, and fundación para la investigación y la prevención del sida en españa (fipse 36549/06). sirna-directed inhibition of hiv-1 infection modulation of hiv-1 replication by rna interference potent and specific inhibition of human immunodeficiency virus type 1 replication by rna interference inhibition of hiv-1 infection by small interfering rna-mediated rna interference prevention of hiv-1 infection in human peripheral blood mononuclear cells by specific rna interference rna interference directed against viral and cellular targets inhibits human immunodeficiency virus type 1 replication inhibition of hiv-1 by lentiviral vector-transduced sirnas in t lymphocytes differentiated in scid-hu mice and cd34+ progenitor cell-derived macrophages sustained small interfering rna-mediated human immunodeficiency virus type 1 inhibition in primary macrophages inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using tat-or ccr5-specific small interfering rnas expressed from a lentivirus vector dissecting hiv-1 through rna interference aptamers directed to hiv-1 reverse transcriptase display greater efficacy over small hairpin rnas targeted to viral rna in blocking hiv-1 replication inhibition of retroviral pathogenesis by rna interference expression of small hairpin rna by lentivirus-based vector confers efficient and stable gene-suppression of hiv-1 on human cells including primary non-dividing cells phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering rna and its application in the reverse genetics of wild type negative-strand rna viruses a systematic analysis of the effect of target rna structure on rna interference positional effects of short interfering rnas targeting the human coagulation trigger tissue factor the virion-associated incoming hiv-1 rna genome is not targeted by rna interference a system for stable expression of short interfering rnas in mammalian cells efficient gene transfer of hiv-1-specific short hairpin rna into human lymphocytic cells using recombinant adeno-associated virus vectors human immunodeficiency virus type 1 escapes from rna interference-mediated inhibition lentiviral delivery of short hairpin rnas protects cd4 t cells from multiple clades and primary isolates of hiv second-generation shrna libraries covering the mouse and human genomes is rna interference involved in intrinsic antiviral immunity in mammals induction and suppression of rna silencing: insights from viral infections type 1 interferons and the virus-host relationship: a lesson in detente identification of micrornas of the herpesvirus family evidence that hiv-1 encodes an sirna and a suppressor of rna silencing the ebola virus vp35 protein is a suppressor of rna silencing analysis of the interaction of primate retroviruses with the human rna interference machinery induction and suppression of rna silencing by an animal virus suppression of microrna-silencing pathway by hiv-1 during virus replication host immune system gene targeting by a viral mirna cellular micrornas contribute to hiv-1 latency in resting primary cd4(+) t lymphocytes interferon modulation of cellular micrornas as an antiviral mechanism human immunodeficiency virus type 1 escape from rna interference hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase rapid turnover of plasma virions and cd4 lymphocytes in hiv-1 infection multiply infected spleen cells in hiv patients hiv population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy viruses as quasispecies: biological implications hiv sequence compendium sequence homology required by human immunodeficiency virus type 1 to escape from short interfering rnas rna interference of hiv replication silencing of hiv-1 with rna interference: a multiple shrna approach design of extended short hairpin rnas for hiv-1 inhibition optimization and functional effects of stable short hairpin rna expression in primary human lymphocytes via lentiviral vectors fatality in mice due to oversaturation of cellular microrna/short hairpin rna pathways optimization and characterization of trna-shrna expression constructs escape from the interferon response associated with rna interference using vectors that encode long modified hairpin-rna inhibition of human immunodeficiency virus type 1 by rna interference using long-hairpin rna effective suppression of human immunodeficiency virus type 1 through a combination of short-or longhairpin rnas targeting essential sequences for retroviral integration expression of long anti-hiv-1 hairpin rnas for the generation of multiple sirnas: advantages and limitations a novel approach for inhibition of hiv-1 by rna interference: counteracting viral escape with a second generation of sirnas endoribonuclease-prepared short interfering rnas induce effective and specific inhibition of human immunodeficiency virus type 1 replication sirnas generated by recombinant human dicer induce specific and significant but target site-independent gene silencing in human cells recombinant dicer efficiently converts large dsrnas into sirnas suitable for gene silencing lentiviral vector delivery of sirna and shrna encoding genes into cultured and primary hematopoietic cells suppression of chemokine receptor expression by rna interference allows for inhibition of hiv-1 replication inhibiting hiv-1 infection in human t cells by lentiviral-mediated delivery of small interfering rna against ccr5 an essential role for ledgf/p75 in hiv integration ledgf/p75 is essential for nuclear and chromosomal targeting of hiv-1 integrase in human cells the inner-nuclear-envelope protein emerin regulates hiv-1 infectivity unpaking" human immunodeficiency virus (hiv) replication: using small interfering rna screening to identify novel cofactors and elucidate the role of group i paks in hiv infection inhibition of human immunodeficiency virus type 1 replication by rna interference directed against human transcription elongation factor p-tefb (cdk9/cyclint1) chemokine receptors as hiv-1 coreceptors: roles in viral entry, tropism, and disease stable reduction of ccr5 by rnai through hematopoietic stem cell transplant in non-human primates interfering with disease: a progress report on sirna-based therapeutics inhibition of respiratory viruses by nasally administered sirna using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque potent and persistent in vivo anti-hbv activity of chemically modified sirnas postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by rna interference an sirna-based microbicide protects mice from lethal herpes simplex virus 2 infection peptide-oligonucleotide conjugates for the delivery of antisense and sirna antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors in vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector lentivirus-mediated rna interference therapy for human immunodeficiency virus type 1 infection lentiviral vectors that carry anti-hiv shrnas: problems and solutions development of an rnai based gene therapy against hiv-1. amsterdam : doctoral thesis endogenous microrna regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer transcription start regions in the human genome are favored targets for mlv integration a serious adverse event after successful gene therapy for x-linked severe combined immunodeficiency insertional mutagenesis in gene therapy and stem cell biology retroviral vector insertion sites associated with dominant hematopoietic clones mark "stemness gene therapy targeting peripheral blood cd34+ hematopoietic stem cells of hiv-infected individuals design of hiv vectors for efficient gene delivery into human hematopoietic cells transduction of cell lines and primary cells by fiv-packaged hiv vectors rna interference and innate immunity sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna cationic liposome-mediated delivery of sirnas in adult mice sirna function in rnai: a chemical modification analysis vivo activity of nuclease-resistant sirnas single-stranded small interfering rna are more immunostimulatory than their double-stranded counterparts: a central role for 2 ′ -hydroxyl uridines in immune responses on the delivery of small interfering rnas into mammalian cells small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3 stable expression of shrnas in human cd34+ progenitor cells can avoid induction of interferon responses to sirnas in vitro trans-inhibition of hiv-1 by a long hairpin rna expressed within the viral genome intracellular-diced dsrna has enhanced efficacy for silencing hcv rna and overcomes variation in the viral genotype effective anti-hepatitis b virus hammerhead ribozymes derived from multimeric precursors micrornas modulate hematopoietic lineage differentiation effective rnai-mediated gene silencing without interruption of the endogenous microrna pathway expression profiling reveals off-target gene regulation by rnai widespread sirna "off-target" transcript silencing mediated by seed region sequence complementarity 3 ′ utr seed matches, but not overall identity, are associated with rnai off-targets sirna-mediated off-target gene silencing triggered by a 7 nt complementation gene expression analysis in blood cells in response to unmodified and 2 ′ -modified sirnas reveals tlr-dependent and independent effects position-specific chemical modification of sirnas reduces "off-target" transcript silencing monitoring the effect of gene silencing by rna interference in human cd34+ cells injected into newborn rag2−/− gammac−/− mice: functional inactivation of p53 in developing t cells experimental models to study development and function of the human immune system in vivo disseminated and sustained hiv infection in cd34+ cord blood cell-transplanted rag2−/−gamma c−/− mice key: cord-023354-f2ciho6o authors: nan title: tuesday plenary session 3 tuesday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00654.x sha: doc_id: 23354 cord_uid: f2ciho6o nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-314528-5yq95giq authors: hirayama, makoto; shibata, hiromi; imamura, koji; sakaguchi, takemasa; hori, kanji title: high-mannose specific lectin and its recombinants from a carrageenophyta kappaphycus alvarezii represent a potent anti-hiv activity through high-affinity binding to the viral envelope glycoprotein gp120 date: 2015-12-12 journal: mar biotechnol (ny) doi: 10.1007/s10126-015-9684-2 sha: doc_id: 314528 cord_uid: 5yq95giq we previously reported that a high-mannose binding lectin kaa-2 from the red alga kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. in this study, the full-length sequences of two kaa isoforms, kaa-1 and kaa-2, were elucidated by a combination of peptide mapping and cdna cloning. they consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium oscillatoria agardhii and a red alga eucheuma serra. using an escherichia coli expression system, an active recombinant form of kaa-1 (his-tagged rkaa-1) was successfully generated in the yield of 115 mg per a litter of culture. in a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-hplc method with 27 pyridylaminated oligosaccharides, his-tagged rkaa-1 and rkaa-1 specifically bound to high-mannose n-glycans with an exposed α1-3 mannose in the d2 arm as the native lectin did. predicted from oligosaccharide-binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of hiv with high association constants (1.48–1.61 × 10(9) m(−1)). native kaas and the recombinants inhibited the hiv-1 entry at ic(50)s of low nanomolar levels (7.3–12.9 nm). thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose n-glycans, and the cultivated alga k. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s). human immunodeficiency virus (hiv) infects cd4+ cells, where a viral envelope glycoprotein gp120 interacts with a receptor cd4 and a co-receptor cxcr4 or ccr5 of the host cells, and subsequent insertion of a viral envelope glycoprotein gp41 into the host cell membrane triggers the fusion followed by entry of the virus (kilby and eron 2003) . gp120 is a highly glycosylated protein, as half of its total mass is accounted by about 24 n-linked glycans including complex and high-mannose types (geyer et al. 1988 ). these densely attached n-glycans shield the viral surface proteins to escape from neutralizing antibody recognition (wei et al. 2003; bonomelli et al. 2011 ). on the other hand, carbohydratebinding agents that target those glycans on the viral envelope exhibit potent anti-viral activities via interfering the interaction between gp120 and cd4 (hansen et al. 1989; balzarini et al. 1991; charan et al. 2000; dey et al. 2000; swanson et al. 2010) . several high-mannose n-glycan-binding lectins are newly found and marked as potential microbicides having anti-hiv activities, such as cyanobacterial lectins of cyanovirin-n (cv-n) from nostoc ellipsosoprum (boyd et al. 1997) , scytovirin (svn) from scytonema varium (bokesch et al. 2003) and mvl from mycrosystis viridis (bewley et al. 2004 ), a bacterial lectin of actinohivin (ah) from actinomycete longispora albida (chiba et al. 2001) , and algal lectins of griffithsin (grft) from a red alga griffithsia sp. (mori et al. 2005) and bca from a green alga boodlea coacta (sato et al. 2011b ). these lectins show strong anti-hiv activities through their binding to mannoside structures on gp120. however, high-mannose recognition mode subtly differed among them, as well as the primary structures were clearly distinct (sato et al. 2011b; koharudin and gronenborn 2014) . high-mannose nglycan structures are also present on the surfaces of the other enveloped viruses such as influenza virus, hepatitis c virus (hcv), human herpes virus (hhv), west nile virus, severe acute respiratory syndrome-related coronavirus (sars-cov), and ebola virus (okuno et al. 1992; vigerust and shepherd 2007) . this suggests that high-mannose-binding lectins could also inactivate the entries of those viruses into host cells (barrientos et al. 2003; helle et al. 2006; o'keefe et al. 2003 o'keefe et al. , 2010 . a lectin esa-2 from a red alga eucheuma serra (kawakubo et al. 1997) was recently found to possess the unique binding specificity with the preferential affinity for high-mannose n-glycans bearing the nonreducing terminal α1-3 man in d2 arm ). since then, several lectins with similar binding specificity were isolated from some lower organisms, including red algae (kawakubo et al. 1999; sato et al. 2011a; hung et al. 2011 hung et al. , 2015 , a cyanobacterium (sato et al. 2000 , and bacteria (cumsky and zusman 1979; sato et al. 2012; koharudin et al. 2012; whitley et al. 2013) . interestingly, these lectins commonly had tandem-repeated structures of a similar domain composed of about 67 amino acids, and grouped into two types of four and two repeats sato et al. 2007; sato and hori 2009 ) as exemplified with esa-2 (four repeats) and a lectin oaa (two repeats) from a freshwater cyanobacterium oscillatoria agardhii. thus, we demonstrated the occurrence of a new lectin family which is widely distributed in lower organisms, indicating that esa-2 and oaa are orthologous to each other. among them, oaa has been characterized in details. the structural analyses of oaa and its complexes with oligomannosides revealed that this lectin recognized a core unit, manα1-6(manα1-3)manα1-6(manα1-3)manβ-of the branched mannoside in high-mannose n-glycan gronenborn 2011, 2014) . this recognition mode of oaa was novel as the other highmannose-binding lectins mostly recognized the nonreducing end mannoses. potent anti-hiv activities of this lectin family, including esa-2, oaa, pseudomonas fluorescens agglutinin (pfa), myxococcus xanthus agglutinin (myxobacterial hemagglutinin, mbha) (koharudin et al. 2012) , and burkholderia oklahomensis agglutinin (boa) (whitley et al. 2013 ) were represented as expected by their specific binding nature to high-mannose sugar moiety. strong anti-influenza virus activities through binding to the envelope hemagglutinin protein were also shown in pfl (identical to pfa) (sato et al. 2012) and kaa-2 isolated from a cultivated red alga kappaphycus alvarezii (sato et al. 2011a) . algae belonging to genus eucheuma and kappaphycus are widely cultivated as food and carageenophyte. k. alvarezii cultivation was originally started in the philippines in latter half of the 1960s using the local varieties selected from the wild (doty 1973; parker 1974; bindu and levine 2011) . after four decades, the kappaphycus became the most widely cultivated commercial eucheumoid (bindu and levine 2011) and has globally been introduced for cultivation and some experimental purposes in tropical and subtropical area, including asia, africa, the pacific island, and american countries (ask et al. 2003; bindu and levine 2011) . we recently reported that kaa-2 isolated from the cultivated sample of k. alvarezii preferentially recognized high-mannose-type oligosaccharides bearing an exposed α1-3 man at the nonreducing end in d2 arm and inhibited infection of various influenza virus strains with ec 50 s of low nanomolar levels through direct binding to the viral envelope hemagglutinin protein, which has several high-mannose n-glycans (sato et al. 2011a) . the results also suggest that kaa-2 may belong to a new lectin family mentioned above. however, the primary structure of kaa is unknown yet as well as its anti-hiv activity. in this study, we elucidated the primary structure of kaa-2 using peptide mapping and complementary dna (cdna) cloning and prepared its active recombinants using an escherichia coli expression system. the native and f1 ori pet28a-rkaa1 t7 terminator lac operator t7 promoter thrombin cleavage site kaa-1 lac i fig. 1 a diagram of recombinant kaa-1 (rkaa-1) expression vector pet28a-rkaa1. rkaa-1 was expressed as a 6-his-and thrombin cleavage site-fused protein recombinant lectins were examined for sugar-binding specificity and anti-hiv activity, including binding nature to gp120. materials k. alvarezii lectins kaa-1 and kaa-2 (previously declared as eca-1 and eca-2) and eucheuma serra lectin esa-2 were prepared as described previously (kawakubo et al. 1997 (kawakubo et al. , 1999 3)manα1-6(manα1-3)man-oh) which were previously prepared . a recombinant glycosylated hiv-1 i i i b g p 1 2 0 ( b a c u l o v i r u s ) w a s p u r c h a s e d f r o m immunodiagnostics (ma, usa). jurkat cells and t lymphocyte-derived cells were propagated in rpmi 1640 medium (life technologies) supplemented with 10 % fetal calf serum (nichirei biosciences, tokyo, japan). kaa-2 and esa-2 were subjected to s-pyridylethylation according to the method described previously ). s-pyridylethylated (pe-) kaa-2 and pe-esa-2 were purified by reverse-phase (rp-) hplc on ymc-pack protein-rp (6.0 × 250 mm, ymc, kyoto, japan). briefly, after injection of sample to the column equilibrated with 10 % (v/v) acetonitrile in 0.05 % (v/v) trifluoroacetic acid (tfa), the column was washed with the same solvent for 10 min and then eluted with a linear gradient (10-70 %) of acetonitrile in 0.05 % tfa for 30 min at a flow rate of 1.0 ml/min. the eluate was monitored by absorbance at 280 nm (a280), and the active fractions were recovered. sds-page for purified proteins was performed using a 15 % (w/v) gel (schägger and von jagow 1987) . one hundred micrograms each of pe-kaa-2 and pe-esa-2 were digested with l-1-tosylamido-2-phenylethyl chloromethyl ketone (tpck)-treated trypsin (roche diagnostics, basel, switzerland) (enzyme/substrate=1:50 (w/w)) in 1 % (w/v) ammonium hydrogen carbonate at 37°c for 24 h. for the isolation of peptide fragments, each digest was separated by rp-hplc on tskgel ods-120t column (4.6 × 250 mm, tosoh, tokyo, japan). briefly, the digest was applied to the column equilibrated with 5 % (v/v) acetonitrile in 0.1 % (v/v) tfa. the column was washed with the same solvent for 5 min and then eluted with a linear gradient (5-65 %) of acetonitrile in 0.1 % tfa for 60 min at a flow rate of 1.0 ml/min. the eluate was monitored by absorbance at 220 nm (a220), and the active fractions were recovered. the molecular masses of purified pe-kaa-2, pe-esa-2, and their trypsin-digested peptides were determined by electron spray ionization (esi)-mass spectrometry (ms) using finnigan lcq (thermo fisher scientific, ma, usa). the masses of purified recombinants were determined by esi-ms using ltq orbitrap xl (thermo fisher scientific). the n-terminal amino acid sequences of pe-kaa-2 and the peptide fragments produced by trypsin digestion of pe-kaa total rna was extracted from 2.5 g of the rnalater-treated alga of k. alvarezii using 25 ml of a plant rna isolation reagent (life technologies). messenger rna (mrna) purification from the total rna was performed using a nucleotrap mrna purification kit (macherey-nagel, düren, germany). full-length cdnas were synthesized from 50 ng of a mrna using generacer kit (life technologies) according to the manufacturer's instruction. the first pcr for rapid amplification of the cdna 5′end (5′race) was performed with a 50 μl reaction mixture containing 5 μl of 10× blend taq buffer (toyobo, osaka, japan), 10 nmol each of dntp, 30 pmol of generacer 5 ′ p r i m e r ( l i f e te c h n o l o g i e s ) ( 5 ′ -c g a c t g gagcacgaggacactga-3′), 250 pmol of a degenerated p r i m e r k a a _ 5 ′ r a c e _ r 1 ( 5 ′ -at i g g i c c y t c i ccyttrtaytgc-3′), which was designed from the partial amino acid sequence of kaa-2 predicted by peptide mapping, 1 μl of 10-fold diluted synthesized cdna and 1.25 u of blend taq dna polymerase (toyobo). thermal cycling conditions consisted of denaturation at 94°c for 3 min, followed by 30 cycles consisting of denaturation at 94°c for 30 s, annealing at 64°c for 30 s, and extension at 72°c for 1 min, and the final extension step at 72°c for 5 min. the nested pcr was performed by the same method, except that 1 μl of a 100-fold diluted solution containing the first pcr product was used as a template, 10 pmol of generacer 5′ nested primer (5′-ggacactgacatggactgaaggagta-3′) and 250 pmol of a degenerated primer kaa_5′race_r2 (5′-ayt grttytciacrttrtaigtrtc-3′) as a primer pair, and that the annealing of the reaction was performed at 58°c. nested pcr products were subcloned into pgem-t easy vector (promega). dna sequencing was performed using bigdye terminator cycle sequencing kit ver. 3.1 with abi 3130xl dna sequencer (life technologies). 3′race was performed by the same method of 5′race as described above, except that a primer pair of generacer 3′ primer (5′-gctgtcaacgatacgctacgtaacg-3′) and a degenerated primer kaa_3′race_f1 (5′-ayaci tayaaygtigaraaycartgggg-3′) for the first pcr and generacer 3′ nested primer (5′-cgctacgtaa cggcatgacagtg-3′) and a degenerated primer kaa_3′ r a c e _ f 2 ( 5 ′ -a rtaya a r g g i g a r g g i c c i a thgg-3′) for the nested pcr were used. at last, the fulllength cdnas encoding two kaa isolectins were amplified using a high-fidelity dna polymerase kod fx neo (toyobo) with primer pairs of kaa1_5′end_f (5′-tatagctgagtcaagttacaccaac-3′) and kaa1_3′ end_r (5′-agagggtgatcacgtttttac-3′) or kaa2_5′end_f (5′-accaccagcaccgtgctact cgct-3′) and kaa2_3′end_r (5′-gaatgtccttacg tggcctttac-3′), which were designed from the 5′ and 3′ terminal sequences of kaa cdnas obtained by 5′ and 3′ races. homologous sequences were searched with the basic local alignment search tool (blast) program. amino acid sequence comparison with homologous proteins from various organisms was performed using clustal omega (sievers et al. 2011 ). the recombinant kaa-1 gene was designed by backtranslation from its amino acid sequence and synthesized by integrated dna technologies (ia, usa). the kaa-1 coding region flanked by the restriction enzyme recognition sites was amplified by means of the pcr using a primer pair of rkaa1_f (5′-gatagctagcggccgctatacagtt caaaacc-3′) and rkaa1_r (5′-gatcctcgagttagc tcgccacgcctttaaag-3′), where underlined nucleotides represent the recognition sites for nhe i and xho i, respectively. the pcr using primestar hs dna polymerase (takara bio) consisted of denaturation at 98°c for 5 min, followed by 30 cycles consisting of denaturation at 98°c for 10 sec, annealing at 60°c for 5 s, and extension at 72°c for 70 s, and the final extension step at 72°c for 5 min. an amplified dna fragment was subcloned into pet-28a(+) (merck) to yield pet28a-rkaa1, which can express recombinant kaa-1 (rkaa-1) with the n-terminal hexa-his tag followed by the thrombin cleavage site (fig. 1) . e. coli shuffle t7 express strain (new england biolabs), which is an enhanced bl21 derivative engineered to express a chromosomal copy of the disulfide bond isomerase dsbc (chen et al. 1999) in the cytoplasm constitutively, was transformed with the rkaa-1 expression vector pet28a-rkaa1 by a fig. 4 the predicted amino acid sequence of native kaa-2. the results of sequence prediction by peptide mapping in kaa-2 are shown using esa-2 sequence as a template. the peptide fragments produced by trypsin digestion of pe-kaa-2 are represented with arrows. the peak numbers examined by edman degradation and their determined sequences are underlined. the sequences whose peptide masses were consistent with those of esa-2 are represented in bold. unidentified amino acids in kaa-2 are represented as lowercase letter of corresponding sequence in esa-2. shaded regions in the sequence were used to design the degenerated primers for cdna cloning conventional method. a transformant was cultured with 1 l of lb media at 37°c until od 600 reached 0.5, and then, iptg was added at a final concentration of 0.1 mm. after continuously cultured at 20°c for 16 h, the bacteria cells were harvested by centrifugation at 10,000g for 20 min. after fracturing the cell by sonication, the his-tagged rkaa-1 (his-rkaa-1) expressed into soluble fraction was purified by his gravitrap (ge healthcare, buckinghamshire, uk) according to the manufacturer's instruction. after purification, his-rkaa-1 was dialyzed thoroughly against ultrapure water to remove imidazole which was contained in the elution buffer. twenty-five milligrams of his-rkaa-1 was then applied to thrombin treatment using a thrombin cleavage capture kit (merck) to disconnect the tag peptide from the recombinant according to the manufacturer's instruction. after removal of thrombin by the above kit, the digested fraction was applied to his gravitrap to exclude the cleaved his-tag peptide and undigested his-rkaa-1. generated rkaa-1 was then dialyzed thoroughly against ultrapure water. the a280 of solution was measured with nanodrop 1000 spectrophotometer (thermo fisher scientific). crude protein concentrations were represented as a280 at 1.0 mg/ml is 1.0. the purified protein concentrations were determined by calculating from a280 and their extinction coefficients which are computed from amino acid sequences (gill and von hippel 1989; pace et al. 1995) . hemagglutination assay was performed using a 2 % (v/v) suspension of trypsin-treated rabbit erythrocytes (trbcs) as described previously (hori et al. 1986 ). hemagglutination activity was expressed as a titer, the reciprocal of the highest 2-fold d i l u t i o n e x h i b i t i n g p o s i t i v e h e m a g g l u t i n a t i o n . hemagglutination-inhibition assay was also performed using a 2 % (v/v) suspension of trbc and following sugars and glycoproteins as described in hori et al. (1986) ; d-glucose, dgalactose, d-mannose, d-xylose, n-acetylglucosamine, nacetylgalactosamine, n-acetylneuraminic acid, l-fucose, lrhamnose, lactose, transferrin (human), asialo-transferrin, fetuin (type iii, fetal calf serum), asialo-fetuin, yeast mannan, porcine thyroglobulin (ptg), asialo-ptg, bovine submaxillary mucin (bsm), and asialo-bsm. the inhibitory activity was expressed as the minimum concentration of sugar (mm) or glycoprotein (μg/ml) that completely inhibited the activity of a lectin solution of hemagglutination titer 8. the oligosaccharide binding activity of rkaas was determined by a centrifugal ultrafiltration-hplc method as described in hori and hirayama (2014) . briefly, 90 μl of 500 nm his-rkaa-1 or rkaa-1 in 50 mm tris-hcl (ph7.0), and 10 μl of 300 nm pa-oligosaccharide were mixed and kept at room temperature for 60 min. subsequently, unbound pa-oligosaccharides were collected by centrifugation (10,000g, 30 s) using nonosep 10 k device (pall, ny, usa). an aliquot of the filtrate was applied to a tskgel ods 80tm column (4.6 × 150 mm, tosoh), and unbound paoligosaccharide was quantified from the peak area. the amount of bound pa-oligosaccharide was obtained by subtracting the amount of unbound oligosaccharide from the amount of added oligosaccharide, which was determined from the filtrate of the reaction solution without a lectin. the binding activity (%) was calculated as the ratio of the amount of bound oligosaccharide to that of added oligosaccharide. interaction of kaas, including his-rkaa-1, rkaa-1, kaa-1, and kaa-2, with the hiv envelope gp120 was analyzed by surface plasmon resonance (spr) analysis using a biacore 2000 system (ge healthcare) as described previously (sato et al. , 2011b . the recombinant glycosylated hiv-1 iiib gp120 (baculovirus) was immobilized to give 300 resonance units on a carboxymethylated dextran-coated sensor chip (cm5, ge healthcare). ninety microliters of each concentration of his-rkaa-1 and rkaa-1 solution (0.98, 3.9, 7.8, and 15.6 nm) was injected into the flow cells at 30 μl/min for 3 min to observe the association, and then, the same amount of the running buffer hbs ep (ge healthcare) was injected into the flow cells at 30 μl/min for 3 min to observe the dessociation. the data of rkaas were fit globally to a simple langmuir 1:1 binding model with local r max (maximum response) using biaevaluation 3.1 software. for kaa-1 and kaa-2, lectin solutions of 0.98 nm were applied to the system and the obtained sensorgrams were compared with those of the recombinants at same concentration to evaluate the functional similarities among the natives and recombinants. the hiv-1 virus stock was prepared from the jurkat cells transfected with an hiv-1 genome-encoding plasmid, pnl4-3, originally constructed from the ny5 and rav proviruses (adachi et al. 1986 ). infectivity of hiv-1 was measured with the median tissue culture infectious dose (tcid 50 ) method using jurkat cells. hiv-1 (100 tcid 50 ) was incubated with 2-fold serially diluted lectins, in which their final concentrations were from 1 μm to 1 nm, at room temperature for 3 min in four or eight wells of a 96-well plate for each dilution. jurkat cells (5 × 10 4 cells/well) were further added and incubated for several days until cytopathic effects developed. fifty percent endpoint of lectin concentration to neutralize virus infectivity was determined according to the behrens-kaerber method, and 50 % inhibitory concentration (ic 50 ) was determined. cell viability in the presence of lectins was examined by using the cell proliferation kit i (mtt assay; roche diagnostics, tokyo, japan). before peptide mapping, kaa-2 and esa-2 were pyridylethylated and then purified as single peaks by rp-hplc on ymc-pack protein-rp column (fig. 2a, b) . the molecular masses of pe-kaa-2 and pe-esa-2 were determined to be 28,124.0 and 28,052.0 by esi-ms. these values were consistent with those estimated by sds-page (fig. 2c ). peptide fragments generated by trypsin digestion of pe-kaa-2 and pe-esa-2 were separated into a total of 26 peaks, including 18 common peaks between both, 5 specific peaks in pe-kaa-2, and 3 specific peaks in pe-esa-2, by rp-hplc on tskgel ods 120t (fig. 3 , table 1 ). the molecular masses of the isolated peptide fragments were determined by esi-ms, except for a peak 22 in pe-kaa-2 and peaks 10 and 21 in pe-esa-2. the determined masses were compared to those of the peptide fragments that are theoretically generated by trypsin digestion of pe-esa-2 (table 1) . all peptides whose masses were successfully determined were found in the pe-esa-2 sequence, except for a peak 19, mass of which was not found in both sequences of pe-esa-2 and trypsin used for digestion. the sequence of 167 amino acids (aa) of pe-kaa-2 was successfully predicted by comparing the sequences of 18 peptides with the same masses, which were commonly generated from both pe-kaa-2 and pe-esa-2. moreover, the n-terminal sequencing of the specific peptide peaks (11, 12, 15, 16, and 23) from pe-kaa-2 showed that there are at least 3 amino acid substitution for pe-esa-2 (fig. 4) . the peptide sequence of a peak 10 (determined mw 2550.8) in pe-kaa-2 could be an incompletely digested product of 197 gklsgannysvenqwgg ssapwnk 220 (calculated mw 2551.7). thus, a total of 230 aa in the whole sequence of pe-kaa-2 was successfully predicted by peptide mapping. fig. 7 preparation of rkaas. a nonreducing sds-page for purified his-rkaa-1 (lane 1) and rkaa-1 (lane 2). five micrograms of each protein was loaded. m molecular weight marker. esi-ms multiply charged spectra of purified his-rkaa-1 (b) and rkaa-1 (c) were shown nucleotide and amino acid sequences of kaas cdna cloning by race methods resulted in isolation of two full-length cdnas encoding kaa isoforms (fig. 5) . compared the deduced amino acid sequences of the two isoforms and the partial sequence of kaa-2 predicted by peptide mapping, it was found that there are substitutions of a few amino acids to one another, further indicating that kaa-2 isolated from the cultivated alga was not encoded by the two cloned cdnas. this may be attributed to the difference of algal samples used for lectin isolation and cdna cloning. here, we defined the isolectin cdna whose deduced amino acid sequence was closer to the predicted kaa-2 by peptide mapping as kaa-2 (ddbj/embl/genbank accession no. lc007081) and the other as kaa-1 (lc007080) (fig. 5) . kaa-1 cdna was composed of 1027 bp containing 77 bp of 5′ untranslated region (utr), 143 bp of 3′utr, and 807 bp of open reading frame (orf) which encoded 268 amino acids including an initiating methionine. kaa-2 cdna was composed of 1161 bp containing 94 bp of 5′utr, 257 bp of 3′ utr, and 810 bp of orf encoding 269 amino acids including an initiating methionine (lc007081). the predicted nterminal amino acid sequence of the mature kaas was completely comparable in the sequence determined by edman degradation for pe-kaa-2 (underlined sequence in fig. 5a) , indicating that kaa cdnas do not encode signal peptide and other propeptide regions. these two kaas showed 89.7 and 98.5 % identities to each other in nucleotide and protein levels, respectively. amino acid sequence identities between esa-2 and kaa-1 or kaa-2 are 98.1 and 98.9 %, respectively. kaas also contain four tandem repeats of around 67 amino acids (fig. 6 ) as well as esa-2 ). these repeated sequences are highly conserved between one another and among related proteins from other organisms including bacteria (burkholderia oklahomensis agglutinin (boa, 4 repeats), myxococcus xanthus agglutinin (mbha, 4 repeats), pseudomonas fluorescens lectin (pfl, 2 repeats)) and cyanobacteria (o. agardhii (oaa, 2 repeats)) (fig. 6) . similar sequence was not found in higher plants or animals by blast search. the sequence identities between kaa-1 and boa, mbha, oaa, or pfl were 58.1, 60.2, 60.6, and 62.9 %, respectively. the amino acids that interact with mannopentaose (manα1-6(manα1-3)manα1-6(manα1-3)man), which is a branched oligomannosyl core structure of m8/9, in boa (koharudin et al. 2012) are absolutely conserved in all repeated domains of the lectins (indicated with asterisks in fig. 6 ). his-rkaa-1 was successfully generated using pet system and a shuffle t7 express strain (fig. 7) . his-rkaa-1 was prepared in the yield of 115 mg per 1 l lb media, and rkaa-1 was generated in the yield of 15 mg from 25 mg of his-rkaa-1. the molecular masses of his-rkaa-1 and rkaa-1 were calculated as 30,202.7 and 28,451.9, respectively, based on their amino acid sequences. although the recombinants ran at about 30-35 kda in sds-page (fig. 7a) , the molecular masses determined by 30, 203.7 da for his-rkaa-1 (fig. 7b) and 28,452.1 da for rkaa-1 (fig. 7c) , were in good agreement with the calculated ones. hemagglutination actvities of his-rkaa-1 and rkaa-1 (each 10 μm) were 2 12 with trbc as well as those of native kaa-1 and kaa-2 (data not shown). hemagglutination inhibition test revealed that the recombinants (his-rkaa-1 and rkaa-1) and native kaa-1 and kaa-2 preferentially bound to glycoproteins containing high-mannose-type n-glycans such as yeast mannan and ptg, and weakly to the other glycoproteins without highmannose n-glycan (table 2) . they had not affinity for any monosaccharide examined, including mannose. a relatively strong binding to bsm and asialo-bsm with o-glycans was also observed for the recombinants and native kaas. thus, the hemagglutination inhibition profiles of the recombinant kaas were consistent with those of native kaas, regardless of the presence or absence of the his-tag peptide in its n-terminus. in oligosaccharide binding experiments by a centrifugal ultrafiltration-hplc method with 27 pa-sugars (figs. 8 and 9 ), his-rkaa-1 and rkaa-1 strongly bound to five oligosaccharides (oligosaccharides 7, 8, 10, 12 and 15) belonging to highmannose-type n-glycans (oligosaccharides 7-17), but hardly to complex type n-glycans (oligosaccharides 1-6), oligomannoses (oligosaccharides 18-22), core structures (oligosaccharides 23 and 24), or oligosaccharides from glycolipids (oligosaccharides 25-27) (fig. 9 ). comparing the binding activities between oligosaccharides 7 (80.8 % in his-rkaa-1) and 16 (17.1 %), 8 (100 %) and 11 (19.3 %), 10 (100 %) and 13 (20.7 %), 12 (95.6 %) and 14 (36.7 %), or 15 (100 %) and 9 (12.7 %) revealed that rkaas recognized the nonreducing terminal α1-3 linked man of the d2 arm of branched oligomannoside, and the nonreducing terminal α1-2 linked man in the d2 arm negatively affects for binding of rkaas. the presence of his-tag hardly influenced to the oligosaccharide binding preferences of the recombinants although his-rkaa-1 and rkaa-1 were approximately 35 % apart in binding activities to oligosaccharides 7 and 17. interaction between rkaas and hiv envelope glycoprotein gp120 was performed by spr analysis using biacore2000 system. his-rkaa-1 and rkaa-1 bound to the gp120 immobilized on the chip dose-dependently (fig. 10a, b) and binding kinetics of the interactions was well fitted to each sensorgram (overlaid red lines in fig. 10a, b) . rkaas (fig. 10a, b) showed high association constants (k a ) (1.48-1.61×10 9 m −1 ) and low dessociation constants (k d ) in the picomolar range (6.22-6.77 × 10 −10 m) to hiv gp120, regardless of presence of his-tag (table 3 ). the sensorgrams of kaas including his-rkaa-1, rkaa-1, kaa-1, and kaa-2 at same concentration of 0.98 nm were very similar as shown in fig. 10c . although the resonance unit of the sensorgram in his-rkaa-1 appeared slightly higher than those of the other kaas, it might be caused by the molecular weight difference, because the molecular weight of his-rkaa-1 having n-terminal his-tag peptide is approximately 2000 da higher than those of the other kaas. in vitro anti-hiv activities of his-rkaa-1, kaa-1, and kaa-2 against hiv-1 strain (derived from pnl4-3) in jurkat cells were determined by a neutralization assay. serially diluted lectins and hiv-1 were reacted for 3 min at room temperature, and then, jurkat cells were added and further incubated until cell fusion fully developed. experiments were repeated at least three times for each lectin. cell viability of jurkat cells without hiv-1 was not affected by the same concentrations of lectins as judged by the mtt assay (data not shown). the results showed that kaas prevented infection of hiv-1 at low nanomolar ic 50 of 12.9, 9.2, and 7.3 nm for his-rkaa-1, kaa-1, and kaa-2, respectively (table 4) . his-rkaa-1 rkaa-1 b a fig. 9 interaction analysis between rkaas and various oligosaccharides by a centrifugal ultrafiltration-hplc method. a binding activities of rkaas to pa-oligosaccharides. binding activity was expressed as a ratio (%) of the amount of a bound oligosaccharide to that of an added oligosaccharide. the assay was performed in duplicate for each pa-oligosaccharide, and the activity is expressed as the average value from duplicate assays. the activities less than 10 % were cut off for their insignificance. b the detailed binding activities of rkaas with high-mannose-type n-glycans. dashed boxes represent the nonreducing terminal α1-2 linked mannose residue in the d2 arm since kawakubo et al. (1999) reported that the n-terminal amino acid sequences of kaas were identical to those of esas, the whole primary structure of kaa-2 was considered to have high similarity with that of esa-2. peptide mapping of kaa-2 using the sequence of esa-2 as a template has elucidated 85 % (230/268 aa) of the full-length sequence. a calculated molecular mass of the predicted sequence by peptide mapping, including the corresponding sequences of esa-2, was 28,022, which gave close agreement with that of pe-kaa-2 (28,124 including an s-pyridylethyl group (105 da) (fig. 2a) ). although cdna cloning by race method elucidated the full length cdnas encoding kaas, both of their deduced amino acid sequences were not identical to that of the native kaa-2 predicted by peptide mapping (fig. 5b) . the calculated molecular mass from their sequences were 27, 881.24 and 28,007.41, respectively, and were also inconsistent with that of native kaa-2. we defined one of the two kaa cdnas as kaa-2, whose amino acid sequence was more similar to native kaa-2 and esa-2, and the other as kaa-1. the slight sequence differences between native kaa-2 and deduced kaa-2 might be individual variability in algal samples used. cdna cloning has also elucidated that the transcripts of kaas do not contain the region encoding a signal peptide sequence (fig. 5) , which target the proteins from the cytosol to the cytoplasmic membrane (prokaryotes) or to the endoplasmic reticulum membrane (eukaryotes) (imai and nakai 2010) , as well as that of oaa from a cyanobacterium does not (sato and hori 2009 ). it has reported that cdnas encoding a lectin from euonymus europaeus (eea) and eul protein from arabidopsis thaliana (aratheuls3) do not encode a signal peptide and these translated product located in the nucleus and cytoplasm (van hove et al. 2011 ) as well as a gna ortholog from rice that lacks the signal peptide and propeptide sequence (fouquaert et al. 2007 ). in addition, there are not any known cellular localization signals in the primary structure of kaas (data not shown). it suggests that kaas are also synthesized on free ribosomes and located in nucleocytoplasm. there is only a report as to the cellular localization of kaa orthologs; nelson et al. (1981) reported that mbha, a kaa ortholog from a myxobacterium, localizes at the specific sites on the surface of developmental cells but not vegetated cells and suggested their function in cellular interactions during aggregation. detailed cellular localization of kaa should be elucidated and will help to predict their functions in the algal bodies. kaas contain four tandem repeat domains in a molecule (fig. 6) . all the lectins of this family from algae are supposed to compose of four tandem repeat domains commonly, contrary to those from cyanobacteria and bacteria; in cyanobacteria, oaa composes of two repeats sato and hori 2009 ) and a hypothetical protein from lyngbya sp. (accession no. zp_016222182) does four repeats (sato and hori 2009 ); in bacteria, pfl composes of two repeats (sato et al. 2012 ) and boa (whitley et al. 2013 ) and mbha (koharudin et al. 2012 ) do four repeats (fig. 6) . interestingly, several amino acid residues (23 aa per a single repeat domain) are absolutely conserved in all the domains among seven lectins shown in fig. 6 . the amino acid residues (7 aa per a single repeat domain) which interact with mannopentaose of a branched oligomannosyl core of m8/9 in boa (whitley et al. 2013 ) are also conserved (fig. 6) . this structural kinship suggested that lectins belonging to this fig. 10 interaction of kaas with an hiv envelope glycoprotein gp120. spr analyses for interaction between gp120 and his-rkaa-1 (a) or rkaa-1 (b) were analyzed with biacore2000. each sensorgram represents the binding activity of rkaas to gp120 on sensor chip. ninety microliters of rkaas solutions of each represented concentration were injected into the flow cells at 30 μl/min for 3 min. the binding response (black line) in resonance units is plotted against time (s). binding kinetics of the interactions between rkaas and gp120 (overlaid red line) were calculated by fitting the data to langmuir model for 1:1 binding. c comparison of binding activity to gp120 among kaas including his-rkaa-1, rkaa-1, kaa-1, and kaa-2. spr analysis was performed using 0.98 nm of each lectin solution by the same method as described above (color figure online) lectin family might have similar carbohydrate-binding specificities. a detailed oligosaccharide binding analysis by the centrifugal ultrafiltation-hplc method using pa-sugars revealed that the recombinants, his-rkaa-1 and rkaa-1, preferentially recognized exposed α1-3man in the d2 arm of highmannose n-glycans (figs. 8 and 9 ) as previously reported for native kaa-2 (sato et al. 2011a ). the nonreducing terminal α1-2 linked man in the d2 arm tended to more negatively affect for binding to rkaas than native kaa-2. the recognition mode of kaas is absolutely different from the other high-mannose specific antiviral lectins including cv-n, grft, svn, mvl, and ah . we also reported the same oligosaccharide binding properties in esa-2 ) and oaa , and recently in ksa from k. striatum (hung et al. 2011) and eda from e. denticulatum (hung et al. 2015) . a lectin pfl from pseudomonas fluorescens pf0-1 belonging to this lectin family also tends to preferentially recognize highmannose glycans with α1-3 man exposed in the d2 arm but not to other sugar types including complex n-glycans and oglycans, as revealed by screening with a glycan array having 611 glycan targets (sato et al. 2012) . three-dimentional structure analyses for glycan-bound oaa , pfa (identical to pfl), mbha (koharudin et al. 2012 ) and boa (whitley et al. 2013 ) supported their strict binding specificities; the amino acid residues which were revealed to interact with branched mannoside in the high-mannose glycans by tertiary-structural analyses were absolutely conserved in all the lectins of this family as mentioned above (fig. 6) . in previous (sato et al. 2011a ) and present studies, native kaa-2 and rkaas bound to high-mannose-type oligosaccharides but not or little to oligomannoses including pa-mannopentaose (manα1-6(manα1-3)manα1-6(manα1-3)man-pa) (oligosaccharide 22 in fig. 8) , suggesting a possibility that the reducing terminal disaccharide glcnac-glcnac is necessary for kaas to bind to high-mannose n-glycans. on the other hand, showed that oaa binds to this type of mannopentaose (not pyridylaminated), by nmr binding studies, and suggested that the reducing terminal disaccharide was not essential for oaa to bind to those glycans. exactly, pa-oligosaccharides used in this study possessed the reducing terminal residues with ring-opening structures by pyridylamination, including free oligomannoses (pa-oligosaccharides 18-22 in fig. 8) . thus, it might be attributable to such ring-opening of the reducing terminal residues that kaas did not bind to the pa-oligomannoses, including mannopentaose (manα1-6(manα1-3)manα1-6(manα1-3)man-pa) (oligosaccharide 22 in figs. 8 and 9 ). although we have not elucidated the structure of a kaamannopentaose complex, it is strongly predicted that kaa, as well as oaa , requires the reducing terminal mannose having the ring structure for binding to mannopentaose, unlike that cv-n and grft do not directly interact with the reducing terminal mannose . spr analyses revealed that rkaas bound to hiv envelope glycoprotein gp120 with high association constants (1.48-1.61 × 10 9 m −1 ) as well as native kaas did (fig. 10) . gp120 is extensively glycosylated with n-linked complex and high-mannose carbohydrates accounting for about half of its molecular weight (geyer et al. 1988) , suggesting that kaas can bind gp120 with a high affinity for the highmannose sugars. supposed from their strong binding to the gp120, kaas represented potent anti-hiv activities at ic 50 of nanomolar levels (7.3-12.9 nm, table 4 ) in virus neutralization assay with jurkat cells. the activities were comparable to those of oaa (12 nm of ic 50 against hiv-1 (r9) in tzm-b1 cells (koharudin et al. 2012) , 45 nm of ec 50 against hiv-1 x4 in mt-4 cells )), mbha (15 nm of ic 50 against hiv-1 (r9) in tzm-b1 cells (koharudin et al. 2012 )), boa (12.2 nm of ic 50 against hiv-1 (r9) in tzm-b1 cells (whitley et al. 2013) ) and esa-2 (165 nm of ec 50 against hiv-1 x4 in mt-4 cells ). there are a lot of hiv-1 strains, being suggested that there may be distinct sugar structures on their envelope gp120 (zhang et al. 2004) . the difference of anti-hivactivities, such as the activities of oaa against hiv-1 (r9) in tzm-b1 and hiv-1 x4 in mt-4 cells, may be caused by the difference of virus strains and cells (huskens and schols 2012) used in the experiments. the anti-hiv activities of cv-n and grft were reported as 0.4 nm (against hiv-1 (r9) in tzm-b1 cells) and 0.04 nm (against hiv-1 x4 in mt-4 cells) of ic 50 (or ec 50 ), respectively (koharudin et al. 2012; mori et al. 2005) . the interaction between grft and hiv-1 gp120 iiib (x4) was also elucidated by xue et al. (2013) via a spr analysis using a biacore t200; the dissociation constant of grft with the gp120 is 6.93 × 10 −11 m, which is 10-fold lower than those his-rkaa-1 12.9 ± 2.2 sd standard deviation table 3 summary of binding kinetics of the interactions between rkaas and gp120 lectin k a (m −1 s −1 ) k d (s −1 ) k a (m −1 ) k d (m) his-rkaa-1 8.71 × 10 5 5.90 × 10 −4 1.48 × 10 9 6.77 × 10 −10 rkaa-1 8.50 × 10 5 5.29 × 10 −4 1.61 × 10 9 6.22 × 10 −10 k a association rate constant, k d dissociation rate constant, k a association constant, k d dissociation constant of kaas (6.22-6.77 × 10 −10 m) (fig. 10) . these relatively strong anti-hiv-1 activities of cv-n and grft and the robust interaction of grft with gp120 come from the multivalency of binding sites on the lectins and that of epitopes on the highmannose oligosaccharides . although the anti-viral activities of kaas are lower than those of cv-n and grft, the oligomannose epitopes recognized by kaas are different from those recognized by cv-n and grft. since high mutation rate in hiv-1 can rapidly produce the resistant viruses against these anti-viral lectins (witvrouw et al. 2005; huang et al. 2011 ), multilateral and synergistic use of the anti-viral lectins whose recognition epitopes in oligosaccharides are different from one another can be effective as férir et al. (2014) represented. we searched higher plant lectins similar to kaas by in silico screening for genome sequences of soybean glycine max, rice oryza sativa, and arabidopsis; however, higher plant genomes encode no lectins similar to this lectin family (data not shown) as jiang et al. (2010) reported a genome wide screening of higher plant lectins. this lectin family is found to date only in lower organisms including eukaryotes of red algae and prokaryotes of cyanobacteria and bacteria. the biofunctional mechanism(s) where this lectin family preferentially binds to high-mannose carbohydrates having an exposed α1-3 man in the d2 arm of branched oligomannoside is still uncovered. as yoshiie et al. (2012) reported that the n-glycans of glycoproteins in red algae are predominantly highmannose types, the internal ligands of kaas could exist in the cells, implied from the predicted nucleocytoplasmic localization of the lectins as described before. however, the lectins peculiar to these lower organisms may have common function(s), such as host defense against infections of invasive viruses, bacteria, or fungi to these organisms as well as several cytoplasmic/nuclear plant lectins, including amaranthins, euonymus lectin-like (eul)-related lectins, jacalin-related lectins, and nictaba-related lectins, have an important role in plant defense against pathogens van damme 2010, 2014; dias rde et al. 2015; de schutter and van damme 2015) . this study showed that the lectins from the cultivated alga k. alvarezii inhibit the hiv virus entry to the host cells through binding to the virus envelope glycoprotein gp120 and confirmed their activities using its recombinants. these results suggested that kaas have potentiality to represent broad anti-viral activities against viruses possessing highmannose glycans on their envelope such as hcv, hhv, sars-cov, and ebola virus, as well as native kaa-2 did against influenza viruses (sato et al. 2011a) . it is important that anti-viral lectins are isolated from a globally cultivated edible algal species in contrast that any other homologous anti-hiv lectins are derived from bacteria (boa, pfl, mbha), a cyanobacterium (oaa), and an uncultivated alga (esa-2); kaas can be purified in a relatively high yield of 270 mg per 100 g of freeze-dried alga (kawakubo et al. 1999 ). these functional lectins will become a strong tool by being supplied in bulk as both native and recombinant forms, which are almost identical in their carbohydrate specificities and anti-viral activities. production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone three decades of kappaphycus alvarezii (rhodophyta) introduction to non-endemic locations alpha-(1-3)-and alpha-(1-6)-d-mannose-specific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro cyanovirin-n binds to the viral surface glycoprotein, gp1,2 and inhibits infectivity of ebola virus new carbohydrate specificity and hiv-1 fusion blocking activity of the cyanobacterial protein mvl: nmr, itc and sedimentation equilibrium studies the commercial red seaweed kappaphycus alvarezii-an overview on farming and environment a potent novel anti-hiv protein from the cultured cyanobacterium scytonema varium the glycan shield of hiv is predominantly oligomannose independently of production system or viral clade discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development isolation and characterization of myrianthus holstii lectin, a potent hiv-1 inhibitory protein from the plant myrianthus holstii chaperon activity of dsbc actinohivin, a novel anti-hiv protein from an actinomycete that inhibits syncytium formation: isolation, characterization, and biological activities myxobacterial hemagglutinin: a development-specific lectin of myxococcus xanthus protein-carbohydrate interactions as part of plant defense and animal immunity multiple antiviral activities of cyanovirin-n: blocking of human immunodeficiency virus type 1 gp120 interaction with cd4 and coreceptor and inhibition of diverse enveloped viruses insights into animal and plant lectins with antimicrobial activities farming the red seaweed, eucheuma, for carrageenans broad anti-hiv activity of the oscillatoria agardhii agglutinin homologue lectin family localization and topogenesis studies of cytoplasmic and vacuolar homologs of the galanthus nivalis agglutinin carbohydrates of human immunodeficiency virus. structures of oligosaccharides linked to the envelope glycoprotein 120 calculation of protein extinction coefficients from amino acid sequence data correlation between carbohydrate structures on the envelope glycoprotein gp120 of hiv-1 and hiv-2 and syncytium inhibition with lectins cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans centrifugal ultrafiltration-hplc method for interaction analysis between lectins and sugars preliminary characterization of agglutinins from seven marine algal species strict specificity for high-mannose type n-glycans and primary structure of a red alga eucheuma serra lectin removal of two high-mannose n-linked glycans on gp120 renders human immunodeficiency virus 1 largely resistant to the carbohydrate-binding agent griffithsin high-mannose n-glycan-specific lectin from the red alga kappaphycus striatum (carrageenophyte) purification, primary structure, and biological activity of the high-mannose n-glycan-specific lectin from cultivated eucheuma denticulatum algal lectins as potential hiv microbicide candidates prediction of subcellular locations of proteins: where to proceed? evolutionary history and stress regulation of the lectin superfamily in higher plants the marine red alga eucheuma serra j. agardh, a high yielding source of two isolectins occurrence of highly yielded lectins homologous within the genus eucheuma novel therapies based on mechanisms of hiv-1 cell entry structural basis of the anti-hiv activity of the cyanobacterial oscillatoria agardhii agglutinin antiviral lectins as potential hiv microbicides novel fold and carbohydrate specificity of the potent anti-hiv cyanobacterial lectin from oscillatoria agardhii structural insights into the anti-hiv activity of the oscillatoria agardhii agglutinin homolog lectin family nucleocytoplasmic plant lectins lectin domains at the frontiers of plant defense isolation and characterization of griffithsin, a novel hivinactivating protein, from the red alga griffithsia sp localization of myxobacterial hemagglutinin in the periplasmic space and on the cell surface of myxococcus xanthus during developmental aggregation potent anti-influenza activity of cyanovirin-n and interactions with viral hemagglutinin broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae analysis of human herpesvirus 6 glycoproteins recognized by monoclonal antibody ohv1 how to measure and predict the molar absorption coefficient of a protein the culture of the red algal genus eucheuma in the philippines cloning, expression, and characterization of a novel anti-hiv lectin from the cultured cyanobacterium, oschillatoria agardhii purification and characterization of a novel lectin from a freshwater cyanobacterium, oscillatoria agardhii primary structure and carbohydrate binding specificity of a potent anti-hiv lectin isolated from the filamentous cyanobacterium oscillatoria agardhii high-mannosespecific lectin (kaa-2) from the red alga kappaphycus alvarezii potently inhibits influenza virus infection in a strain-independent manner high-mannose-binding lectin with preference for the cluster of α1-2-mannose from the green alga boodlea coacta is a potent entry inhibitor of hiv-1 and influenza viuses highmannose-binding antiviral lectin pfl from pseudomonas fluorescens pf0-1 promotes cell death of gastric cancer cell mkn28 via interaction with α2-integrin tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kda fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega a lectin isolated from bananas is a potent inhibitor of hiv replication lectin activity of the nucleocytoplasmic eul protein from arabidopsis thaliana virus glycosylation: role in virulence and immune interactions antibody neutralization and escape by hiv-1 burkholderia oklahomensis agglutinin is a canonical two-domain oaa-family lectin: structures, carbohydrate binding and anti-hiv activity resistance of human immunodeficiency virus type 1 to the highmannose binding agents cyanovirin n and concanavalin a the griffithsin dimer is required for high-potency inhibition of hiv-1: evidence for manipulation of the structure of gp120 as part of the griffithsin dimer mechanism structural features of n-glycans of seaweed glycoproteins: predominant occurrence of high-mannose type n-glycans in marine plants tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin structural studies of algal lectins with anti-hiv activity domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding acknowledgments jurkat cells and pnl4-3 were kindly provided by dr. tsutomu murakami in aids research center, national institute of infectious diseases, japan. we thank dr. tomoko amimoto in the natural science center for basic research and development (n-bard) at hiroshima university for measuring the esi-ms data. this work was partially supported by the program for promotion of basic and applied researches for innovations in bio-oriented industry of japan. conflict of interest no conflicts of interest are declared. key: cord-311679-m6poosn3 authors: santos, glenn-milo; ackerman, benjamin; rao, amrita; wallach, sara; ayala, george; lamontage, erik; garner, alex; holloway, ian w.; arreola, sonya; silenzio, vince; strömdahl, susanne; yu, louis; strong, carol; adamson, tyler; yakusik, anna; doan, tran thu; huang, poyao; cerasuolo, damiano; bishop, amie; noori, teymur; pharris, anastasia; aung, max; dara, masoud; chung, ssu yu; hanley, marguerite; baral, stefan; beyrer, chris; howell, sean title: economic, mental health, hiv prevention and hiv treatment impacts of covid-19 and the covid-19 response on a global sample of cisgender gay men and other men who have sex with men date: 2020-07-11 journal: aids behav doi: 10.1007/s10461-020-02969-0 sha: doc_id: 311679 cord_uid: m6poosn3 there is an urgent need to measure the impacts of covid-19 among gay men and other men who have sex with men (msm). we conducted a cross-sectional survey with a global sample of gay men and other msm (n = 2732) from april 16, 2020 to may 4, 2020, through a social networking app. we characterized the economic, mental health, hiv prevention and hiv treatment impacts of covid-19 and the covid-19 response, and examined whether sub-groups of our study population are disproportionately impacted by covid-19. many gay men and other msm not only reported economic and mental health consequences, but also interruptions to hiv prevention and testing, and hiv care and treatment services. these consequences were significantly greater among people living with hiv, racial/ethnic minorities, immigrants, sex workers, and socio-economically disadvantaged groups. these findings highlight the urgent need to mitigate the negative impacts of covid-19 among gay men and other msm. electronic supplementary material: the online version of this article (10.1007/s10461-020-02969-0) contains supplementary material, which is available to authorized users. the covid-19 pandemic poses a serious health threat worldwide, with over 4.4 million confirmed cases and over 300,000 deaths as of may 15, 2020 [1] . globally efforts to curb the spread of covid-19 have slowed the exponential growth of new cases, but have also led to unprecedented disruptions in society, with vast social, economic and health electronic supplementary material the online version of this article (https ://doi.org/10.1007/s1046 1-020-02969 -0) contains supplementary material, which is available to authorized users. care consequences [2] [3] [4] [5] . unintended impacts of mitigation strategies to curb covid-19 include exacerbating health disparities and deepening social inequities among marginalized groups including gay men and other men who have sex with men (msm), msm living with hiv, racial and ethnic minority msm, and other vulnerable groups [6] [7] [8] [9] . indeed, emerging reports have documented the unique concerns and challenges experienced by gay men and other msm during the covid-19 pandemic, including mental health impacts resulting from anti-gay community backlash, arrests under false pretexts, and loss of privacy during contact tracing and monitoring for covid-19 [10, 11] . furthermore, many gay men and other msm may be in industries that are more prone to covid-19 disruptions, or at increased risk for unemployment, unstable housing, and food insecurity, making them more vulnerable to covid-19′s economic and health impacts compared to the general population [12] [13] [14] [15] [16] [17] . moreover, gay men and other msm also have disproportionately higher unemployment rates relative to the general population [14, 18] . therefore, covid-19 disruptions can further heighten the economic barriers faced by many gay men and other msm. in addition, covid-19 may amplify existing barriers that impede access to hiv prevention, testing, treatment, and care, potentially complicating slow-moving efforts to achieve global hiv targets among gay men and other msm who remain disproportionately impacted by hiv [19, 20] . gay men and other msm are 22 times more likely to acquire hiv than the general population worldwide [21] . reductions in access to effective hiv prevention tools including condoms, hiv testing, pre-exposure prophylaxis (prep), and post-exposure prophylaxis (pep) for gay men and other msm could increase hiv seroconversion, a life-altering event with no cure [21] [22] [23] . for gay men and other msm living with hiv, unstructured treatment interruptions can lead to increased hiv viral load, lower cd4 count, hiv disease progression, and increased risk of developing an opportunistic infection [24, 25] . such interruptions can also cause side effects, difficulties with adherence upon restarting hiv treatment, and even drug resistance [26] [27] [28] [29] . lastly, unsuppressed viral loads caused by treatment disruptions can contribute to increased risk of hiv transmission among partners of gay men and other msm living with hiv [30, 31] . hence, documenting how covid-19 has impacted access to hiv prevention and testing, and hiv treatment and care services is of high public health importance. there is an urgent need to examine the economic, mental health, hiv prevention and testing, and hiv treatment and care impacts of covid-19 among gay men and other msm in order to understand how this marginalized population is uniquely affected by this pandemic and the covid-19 response, and to help inform the targeting of strategies to ameliorate these impacts. furthermore, there is a need to examine whether sub-populations of gay men and other msm are disproportionately impacted by covid-19, including among people living with hiv, racial/ethnic minorities, immigrants, sex workers, and socio-economically disadvantaged groups. we aim to fill the gaps on the economic and health impact of covid-19 by conducting a study among a sample of gay men and other msm living with or at high risk for hiv worldwide. this cross-sectional study is based on a covid-19 disparities survey implemented by the gay social networking app, hornet. hornet is a free, smart-phone based "gay social networking" app with over 25 million users worldwide [32] . therefore, we believe that the cisgender men who use this are gay men and other msm. hornet has been used as a means for conducting research on gay men and other msm worldwide [20, [32] [33] [34] . data collection for the current study occurred from april 16, 2020 to may 4, 2020, during which time hornet users were invited to participate in a brief questionnaire with 58 questions regarding the impact of covid-19 on economic vulnerability, mental health, hiv prevention, testing and treatment and care impacts. hornet users were eligible to participate in the survey if they were age 18 and over and provided informed consent. a total of 4031 respondents participated; however, for this study, we have focused the analysis among participants who reported either being assigned male sex at birth or identify as intersex, and self-identify as male gender, and have available data on our outcomes and characteristics of interest (n = 2732). study procedures were reviewed by the [blinded for review] institutional review board, which determined that the protocol qualified for exempt status under category 4. eligible, consenting participants responded to general demographic questions on age, country of residence, sex assigned at birth, gender identity, and sexual orientation. participants were also asked about their hiv serostatus; membership to a racial or ethnic minority group (e.g., do you consider yourself a member of an ethnic or racial minority?); immigrant status (e.g., "are you, or is one or more of your parents, a migrant to the country in which you currently live?"); and history of engaging in sex work (e.g., "have you ever engaged in sex work (i.e. being paid to have sex)"). additionally, participants were asked a series of questions regarding the effects of covid-19 on the following areas: (1) economic impacts; (2) mental health impacts; (3) hiv prevention and testing impacts; and (4) hiv treatment and care impacts. the survey assessed the impact of the covid-19 pandemic on participants' economic status through questions regarding their employment status; ability to make ends meet (e.g., "thinking of your household's total monthly income, is your household able to make ends meet?"); income level (e.g., "how much are you expecting your income to reduce because of the covid-19 crisis?"); receipt of government financial assistance (e.g., "are you receiving any additional financial benefits from work or government because of the covid-19 crisis?"); loss of health insurance coverage (e.g., "do you expect to lose your health insurance coverage because of the covid-19 crisis?"); and food security (e.g., "since the covid-19 crisis began, have you had to cut the size of your meals or skip meals because there was not enough money for food?"). the survey also asked participants regarding their mental health using validated items from the patient health questionnaire-4 (phq-4), a tool developed and validated by kroenke, spitzer, williams and löwe (2009) used to measure and screen for psychological distress, and particularly, depression and anxiety [35] . participants at risk for hiv were asked whether they experienced changes in access to hiv prevention interventions including condoms, hiv in-person testing, hiv home testing, prep and pep due to covid-19, using likert-type questions (e.g., "do you feel you have access to hiv prevention strategies during the covid-19 crisis?" with the following response options: "definitely yes", "probably yes", "might or might not", "probably not", "definitely not"). these hiv prevention and testing questions were re-coded as binary to compare responses of "definitely yes" to the other response options. for participants living with hiv, questions were also asked regarding their access to hiv providers (e.g., "since the beginning of covid-19 related social isolation in your country, by that or any other name, have you been able to see your hiv provider if you needed to?") and medication (e.g., "do special measures related to covid-19 impact your ability to access or refill your hiv medicine?"), as well as on their perception of being vulnerable to covid-19 due to their hiv status (e.g., "do you feel that you are more vulnerable to covid-19 because you are living with hiv?"). we performed descriptive analyses to characterize the economic, mental health, and hiv-prevention and hiv-care impacts of covid-19 on survey participants. outcomes were also stratified by key sociodemographic and clinical characteristics, including hiv status, being a racial/ethnic minority, immigrant status, health insurance coverage, and engaging in sex work. we examined between-group differences using fisher's exact and chi-squared tests, as appropriate, with a significance level of α = 0.05. for this study, analyses were conducted using a complete case approach, whereby only the fully-completed survey responses were included in the analyses. all analyses were conducted in r (r foundation for statistical computing, vienna, austria). we collected 2732 total responses from cisgender gay men and other msm reported from 103 countries, with the largest numbers of responses from brazil (n = 559), france (n = 381), mexico (n = 181), taiwan (n = 177) and russia (n = 151) (see e-file fig. 1 ). most respondents identified as gay (n = 2294, 84%), and about 1 in 5 respondents identified as a racial or ethnic minority (n = 485). additionally, 274 respondents (11%) reported having ever engaged in sex work, starting either before or after the pandemic. a total of 473 participants (17%) reported that they are currently living with hiv. while the demographics of hiv-positive respondents were fairly similar to those who reported being hiv-negative, there was a greater proportion of individuals above the age of 50 living with hiv compared to those living without hiv (30% vs. 19%, χ 2 = 44.8, p < 0.001). there was also a greater (though not statistically significant) proportion of individuals living with hiv who have reported engaging in sex work (14% vs. 10%, χ2 = 5.7, p = 0.06) (see e-file table 1 for additional demographics information). eleven percent of all participants reported losing employment as a result of changes due to covid-19. regardless of employment loss, a large proportion of participants (38%) also indicated an inability to receive covid-19-related financial benefits when needed, and 19% of participants reported reducing meal sizes or cutting meals completely due to covid-19 (see e-file table 2 ). additionally, 4 out of every 10 respondents anticipated an income reduction of 30% or more due to covid-19, and men living with hiv reported greater anticipated reductions in income compared to those not living with hiv (46% vs. 38%; χ2 = 10.7, p = 0.01). covid-19 has also had a substantial impact on the mental health of cisgender gay men and other msm. overall, 31% of participants reported moderate to severe psychological distress as measured by the phq-4 scale (16% moderate, 15% severe). based on the anxiety and depression subscales, 887 participants (35%) screened positive for depression, and 856 participants (34%) screened positive for anxiety. while these rates did not differ by hiv status, individuals who lost employment due to the pandemic screened positive for anxiety and depression at statistically significantly higher rates compared to those who did not: 50% of those who lost their jobs screened positive for depression compared to 31% of those who did not (χ2 = 37.3, p < 0.001), and 48% of those who lost their jobs screened positive for anxiety compared to 30% of those who did not (χ2 = 35.9, p < 0.001). additionally, 27.6% of participants who lost employment specifically reported feeling depressed nearly every day in the prior two weeks, compared to 11.4% of participants who did not lose employment (χ 2 = 65.5, p < 0.001). similarly, 27.3% of those who became unemployed felt anxious nearly every day over the prior two weeks compared to 13.7% of participants who did not become unemployed (χ 2 = 42.2, p < 0.001) (see e-file fig. 2 ). more difficulties accessing prevention services were reported by study participants as a result of the covid-19 pandemic (see table 1 ). among the 2247 participants not living with hiv, 1459 (65%) felt they definitely still had access to condoms, though substantially fewer participants felt they had similar levels of access to onsite hiv testing (30%), hiv athome testing (19%), prep (21%) or pep (17%). respondents who identified as a racial or ethnic minority reported feeling less definite in their ability to access condoms (62% vs. 68%, χ 2 = 37.1, p < 0.001) and ability to access hiv self-tests (17% vs. 20%, χ 2 = 16.7, p = 0.03) compared to those who did not identify as a racial or ethnic minority. gay men and other msm from immigrant backgrounds reported less definite access to condoms when compared to participants with parents who were born in their current country of residence (61% vs. 67%, χ 2 = 25.7, p = 0.01). less definite access to condoms was also reported by respondents who had ever engaged in sex work when compared to those who hadn't (56% vs. 67%, χ 2 = 15.6, p = 0.048). access to hiv treatment and care for study participants living with hiv has become difficult during the pandemic (see table 1 ). overall, 23% of participants living with hiv indicated that they have lost access to their hiv providers as a result of covid-19 social isolation measures, while 17% reported that they were able to communicate with providers via telemedicine. there were also significant differences in provider access by type of health insurance: uninsured individuals living with hiv reported greater rates of lost access to hiv providers compared to those with government insurance or other insurance types (χ 2 = 10.1, p = 0.04). out of the total number of respondents living with hiv, 455 (96%) indicated that they were currently taking antiretroviral treatment (art) for hiv. of those taking art, 18% (around 1 in 5) indicated either an inability to refill or access their medication, or a difficulty in doing so. additionally, individuals who identified as a racial or ethnic minority reported significantly more difficulties in accessing or refilling art (25%) compared to those who did not identify as a racial or ethnic minority (15%) (fisher's exact p = 0.004). participants living with hiv who also reported having ever engaged in sex work reported significant challenges in accessing hiv treatment. approximately 39% reported losing access to their hiv provider (vs. 23% among those who have not engaged in sex work) (fisher's exact p = 0.01), and 24% reported an inability or difficulty in refilling or accessing medication (vs. 17% among those who have not engaged in sex work) (fisher's exact p = 0.001). covid-19 and the efforts taken to stem its spread have resulted in interruptions to hiv prevention, testing and treatment services, severe economic consequences, and deleterious effects on mental health and quality of life among gay men and other msm globally. these negative effects among gay men and other msm are felt particularly strongly among sex workers, men belonging to racial/ethnic minority and immigrant groups, and men lacking financial means to access healthcare, reinforcing the intersectional vulnerabilities that pre-existed the covid-19 pandemic. our findings highlight the severe economic impact experienced by gay men and other msm worldwide due to covid-19 and the response to this pandemic. many gay men and other msm have reported loss of employment and anticipated reductions in income. gay men and other msm living with hiv were also more likely to anticipate reductions in income compared to those not living with hiv. furthermore, one in five gay men and other msm have reported food insecurity as a result of covid. despite these negative economic consequences, about two in five gay men and other msm indicated an inability to receive covid-19-related financial benefits when needed. these economic impacts and unmet need for financial assistance will likely exacerbate existing disparities in employment and economic opportunity experienced by gay men and other msm [11, 14, 18] . among gay men and other msm at high risk for hiv, a third report less than definite access to condoms, with those who identified as being part of a racial minority, reported being an immigrant, and those who engaged in sex work reporting significantly less access. the majority of gay men and other msm not living with hiv also report less than definite access to hiv testing (seven out of ten for onsite hiv testing, and eight out of ten for hiv self-tests), with racial minorities reporting significantly less access to hiv self-tests. moreover, more than four in five gay men and other msm not living with hiv reported less than definite access to prep and pep during the covid-19 pandemic, with access for these hiv prevention tools being similar across all groups, including among racial minorities, immigrants, and people who engage in sex work. the low access for these hiv prevention tools due to covid-19 observed are very alarming because gay men and other msm continue to be disproportionately impacted by hiv worldwide, with prevalence of hiv for gay men and other msm greatly exceeding the prevalence among the general population in many countries [21] . furthermore, the disparities in access observed is also a great concern because it may exacerbate the heightened vulnerability to hiv among racial minority gay men and other msm, immigrant gay men and other msm and gay men and other msm who engaged in sex work, driven by their exposure to structural risks and intersecting stigmas [36] [37] [38] [39] [40] . additionally, it is incorrect to assume a diminished sexual risk for hiv among gay men and other msm, especially for men in sero-different domestic partnerships or men who are employed as sex workers [31, 40] . new data on from the u.s. has not only reinforced this point but also underscored the ongoing need for uninterrupted access to hiv prevention tools gay men and other msm may need [13] . otherwise, consequences of the reductions in hiv prevention tools can be catastrophic in the hiv prevention response, particularly if they are not reversed. in addition, among gay men and other msm living with hiv, those who lack health insurance were more likely to experience disruptions in access to their hiv care during covid-19. for example, one in five men living with hiv reported being unable to refill or access their hiv treatment medication during covid-19, with those who identified as being part of a racial minority disproportionately affected. lack of access to healthcare for other chronic health conditions (e.g. diabetes, hypertension) also presents a potential double jeopardy for participants with disrupted hiv care and prevention. as mentioned above, men living with hiv reported greater anticipated reductions in income as a result of the slowing of the economy and job loss due to the covid-19 pandemic. the combination of the interruptions to care and reductions in income for gay men and other msm living with hiv may also lead to subsequent barriers to hiv care, and in turn lead to treatment failure and increased hiv transmission [41] [42] [43] [44] . although currently, there is no evidence indicating that people living with hiv who are virologically suppressed are more vulnerable to acquiring covid-19 or having greater severity of this illness than those not living with hiv, experts generally believe that those with high hiv viral load and low cd4 counts may be more susceptible to negative covid-19 outcomes [45] [46] [47] . therefore, to maintain the health of gay men and other msm living with hiv and sustain the benefits from treatment as hiv prevention, efforts to reverse the hiv care and treatment disruptions occurring due to covid-19 need to be implemented with the utmost urgency. the results of this study highlight intersectional vulnerabilities related to hiv and covid-19 among gay men and other msm. structural and social risk factors, like lack of health insurance and racial discrimination, have been shown to increase the risk of hiv acquisition and limit viral suppression [48] [49] [50] [51] [52] . interruptions in care due to covid-19 appear to be more acute in individuals with these same risk factors. arguably, those who were already at greatest risk of poor hiv outcomes stand to be among the worst affected by this pandemic. in turn, poor immune functioning resulting from disruptions to hiv treatment, for example, may further vulnerabilities to covid-19. there is a need to further understand these overlapping vulnerabilities among gay men and other msm. our findings have several important implications. clearly, interventions with the ability to circumvent the need to see patients in-person are needed to ensure delivery of hiv prevention and treatment services, maintain continuity of prep and art treatment access (e.g., multi-month dispensing of treatment), mitigate new transmission events, and secure long-term health outcomes. telemedicine may allow for sustained provider-patient interactions, even in the context of social distancing. additionally, mobile health (mhealth) strategies, which have long been used to keep regular tabs on patients and ensure they are supported in order to maintain their health and well-being, will be even more important now that in-person interactions are limited [53] . in addition, strategies that limit the need for individuals to travel in order to access testing, medications, and other services will be needed, e.g., mobile delivery of medications, drivethrough testing, etc. innovative strategies will be needed to (1) account for disparities in access to technology (both digital devices and internet access), and (2) monitor the efficacy and safety of medication use if regular testing and viral load monitoring are limited [53] [54] [55] . importantly, our findings underscore the need to develop more robust strategies for sub-populations of gay men and other msm-including racial and ethnic minorities, immigrants, the uninsured, those who engage in sex work-to narrow the disparities we observed. specific targeted and tailored interventions designed with the unique needs of these sub-populations may be required to increase their access to economic support, as well as hiv prevention, care, treatment and mental health services. individual-level targeted and tailored interventions are necessary, however, they are likely not sufficient. ultimately, structural and policy changes that prioritize public health and address systemic barriers that maintain racism, xenophobia, and criminalization of sex work are required to alleviate the disparities observed in the long-term, and ensure economic and health equity among all gay men and other msm. notably, there are some limitations of this study. first, individuals have to be users of hornet to participate in the survey and therefore must have internet and smartphone access, which may limit the generalizability of the study findings to the target population of interest. this is a convenience sample and is not necessarily representative of all gay men and other msm globally. based on the sociodemographic characteristics of our sample, those engaged with the app and willing and able to take the time to fill out the questionnaire may likely be gay men and other msm who are less affected by the negative consequences of covid-19. therefore, it is possible that we may be underestimating the true magnitude of the challenges faced by gay men and other msm as a result of covid-19. nevertheless, prior studies have also documented the ability of social networking platforms to efficiently reach hidden and stigmatized populations. therefore, it is also plausible that this sample may have reached a more diverse group of gay men and other msm compared to venue-based sampling or other convenience sampling strategies. additionally, 892 individuals who initially agreed to take the survey left most of the survey incomplete and were therefore excluded. it is possible that certain factors, such as language barriers or stigma, may have led particular subgroups of these participants to not complete the survey, resulting in non-response bias. we are currently translating the survey into multiple languages to mitigate these limitations in future iterations of this study. another limitation stems from the limited information we collected regarding the factors that may be driving the disparities in access to services. further studies, including qualitative interviews, are needed to explore the issues that may be contributing to the unequal levels of access and the cause of these disparities. finally, the results rely on data that is cross-sectional in nature, which precludes our ability to examine temporal changes in the measures we analyzed. despite these limitations, our use of a rapid online survey among existing users of a social networking app provides a snapshot of the effects felt by gay men and other msm in real-time, when it would otherwise be infeasible to collect new data. key strengths of this dataset are, first, that it includes data on 2732 cisgender gay men and other msm across 103 countries and, second, that it captures information on a range of domains that can be harnessed for future areas of research related to the implications of covid-19, including individual financial security, health access, mental health, sex work, issues pertaining to immigration, domestic violence, and a range of others. finally, while different countries are being impacted by covid-19 at different times, these data also represent samples from some of the countries currently most affected by covid-19, including russia, brazil, france and mexico. this study highlights covid-19′s negative impact on gay men and other msm's access to hiv treatment and prevention services, as well as its economic consequences, and impact on mental health status among gay men and other msm globally. the data presented underscore how covid-19 may function to deepen health disparities and social inequities by disproportionately impacting sub-groups of gay men and other msm with intersecting vulnerabilities. as the covid-19 pandemic continues, with vaccine development still in progress, it is imperative to expand efforts to mitigate the reductions in access to hiv prevention, testing, treatment and care services observed in this study, particularly among racial minority gay men and other msm, immigrant gay men and other msm, and gay men and other msm who engage in sex work. it is also imperative to develop novel and creative strategies to support the health, economic security, and well-being of gay men and other msm that can be deployed in the context of current efforts to curb the covid-19 pandemic. efforts are also needed to address the causes of economic and health disparities among sub-populations of gay men and other msm with intersecting vulnerabilities, not just at the individual-level, but also at the structural level. in addition, continued monitoring of covid-19′s impact among gay men and other msm is of great importance to better understand the evolving and longer-term impacts of the pandemic on this vulnerable population, and to ensure that responses are adjusted appropriately to sustain the gains made toward the goal of ending the hiv epidemic. an interactive web-based dashboard to track covid-19 in real time how an epidemic outbreak impacts happiness: factors that worsen (vs. protect) emotional well-being during the coronavirus pandemic mental health consequences during the initial stage of the 2020 coronavirus pandemic (covid-19) in spain strong social distancing measures in the united states reduced the covid-19 growth rate covid-19, unemployment, and suicide understanding covid-19 risks and vulnerabilities among black communities in america: the lethal force of syndemics the covid-19 pandemic: a call to action to identify and address racial and ethnic disparities. j racial ethn health disparities this time must be different: disparities during the covid-19 pandemic covid-19): u.s. department of health & human services unaids and mpact are extremely concerned about reports that lgbti people are being blamed and abused during the covid-19 outbreak vulnerability amplified: the impact of the covid-19 pandemic on lgbtiq people the lives & livelihoods of many in the lgbtq community are at risk amidst covid-19 crisis. human rights campaign foundation characterizing the impact of covid-19 on men who have sex with men across the united states in sexual orientation-related disparities in employment, health insurance, healthcare access and health-related quality of life: a cohort study of us male and female adolescents and young adults a review of the literature on lgbtq adults who experience homelessness homelessness among youth who identify as lgbtq+: a systematic review covid-19 resources for lgbtq employees (and those who have been laid off or furloughed) the williams institute. lgbt demographic data interactive will the global hiv response fail gay and bisexual men and other men who have sex with men? howell s. blue-ribbon boys: factors associated with prep use, art use and undetectable viral load among gay app users across six regions of the world switzerland: unaids successes and challenges of hiv prevention in men who have sex with men a call to action for comprehensive hiv services for men who have sex with men cd4+ count-guided interruption of antiretroviral treatment predictors of unstructured antiretroviral treatment interruption and resumption among hiv-positive individuals in canada global epidemiology of drug resistance after failure of who recommended first-line regimens for adult hiv-1 infection: a multicentre retrospective cohort study emergence of drug-resistant hiv-1 variants in patients undergoing structured treatment interruptions risk factors for antiretroviral therapy (art) discontinuation in a large multinational trial of early art initiators effects of political conflict-induced treatment interruptions on hiv drug resistance antiretroviral therapy for the prevention of hiv-1 transmission risk of hiv transmission through condomless sex in serodifferent gay couples with the hiv-positive partner taking suppressive antiretroviral therapy (partner): final results of a multicentre, prospective, observational study use of, and likelihood of using, hiv pre-exposure prophylaxis among men who have sex with men in europe and central asia: findings from a 2017 large geosocial networking application survey population size estimation of gay and bisexual men and other men who have sex with men using social media-based platforms factors associated with prep awareness according to age and willingness to use hiv prevention technologies: the 2017 online survey among msm in brazil(.). aids care an ultra-brief screening scale for anxiety and depression: the phq-4. psychosomatics syndemic conditions associated with increased hiv risk in a global sample of men who have sex with men using syndemics theory to examine hiv sexual risk among latinx men who have sex with men in philadelphia, pa: findings from the national hiv behavioral surveillance reductions in access to hiv prevention and care services are associated with arrest and convictions in a global survey of men who have sex with men sexual stigma, criminalization, investment, and access to hiv services among men who have sex with men worldwide male sex workers: practices, contexts, and vulnerabilities for hiv acquisition and transmission unemployment as a risk factor for aids and death for hiv-infected patients in the era of highly active antiretroviral therapy hiv and employment among black men who have sex with men in baltimore a scoping review of employment and hiv a critical review of health, social, and prevention outcomes associated with employment for people living with hiv presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the why aren't people living with hiv at higher risk for developing severe coronavirus disease 2019 (covid-19)? aids patient care stds stigma and racial/ethnic hiv disparities: moving toward resilience stress and coping with racism and their role in sexual risk for hiv among african american, asian/pacific islander, and latino men who have sex with men correlates of sexual risk for hiv among us-born and foreign-born latino men who have sex with men (msm): an analysis from the brothers y hermanos study impact of insurance coverage on hiv transmission potential among antiretroviral therapy-treated youth living with hiv population-level effects of uninterrupted health insurance on services use among hiv-positive unstably housed adults use of telemedicine in the management of infectious diseases mhealth for hiv treatment & prevention: a systematic review of the literature using a mobile health intervention to support hiv treatment adherence and retention among patients at risk for disengaging with care we would like to acknowledge the following lankey: cord-264713-38dlh3wg authors: vernet, guy title: molecular diagnostics in virology date: 2004-08-20 journal: j clin virol doi: 10.1016/j.jcv.2004.06.003 sha: doc_id: 264713 cord_uid: 38dlh3wg molecular biology has significantly improved diagnosis in the field of clinical virology. virus discovery and rapid implementation of diagnostic tests for newly discovered viruses has strongly beneficiated from the development of molecular techniques. viral load and antiviral resistance or subtyping assays are now part of the biological monitoring of patients chronically infected by human immunodeficiency virus (hiv), hepatitis b virus (hbv), hepatitis c virus (hcv) and cmv. it will be important to add to this panel assays for other viruses of the herpesviridae family. qualitative assays for the detection of blood-borne viruses have increased safety of blood donation and organ transplantation. screening of other blood-borne viruses (parvovirus b19, hav), multiplexing of detection and test automation to improve practicability and reduce costs will be the next steps. a major evolution in the near future will be the generalization of nat for the diagnosis of viral etiology in patients, mostly with respiratory, cns or gastro-intestinal diseases. major technical improvements have been made to avoid obstacles that still limit this generalization, i.e. genetic variability of viruses, multiplex detection, contamination risk. commercial offers already exist but menus must be extended to limit the validation and documentation work associated with home-brew assays. real-time amplification has allowed the development of new nat platforms but automation and integration of all steps of the reaction are still required to reduce hands-on-time, time-to-result and costs, and to increase throughput. molecular biology has revolutionized all domains of viruses diagnosis including the rapid identification of emerging or re-emerging viruses, viral safety of blood products or organ transplants and viral disease management. one of the major driving forces for the introduction of molecular techniques in virology has been the absence of easy and performing multiplication techniques similar to those developed for bacteriology. the most striking illustration of the power of molecular techniques concerns blood transmitted viruses-human immunodeficiency virus (hiv), hepatitis b virus (hbv) and hepatitis c virus (hcv) for which spectacular progresses in the detection and treatment of viral diseases have been made following the introduction of qualitative and quantitative nucleic acid tests (nat). the recent discovery of a new human coronavirus responsible for severe acute respiratory syndrome (sars) epidemic is another example. it is obvious that nat will encourage the development of antiviral drugs which, compared to antibiotics, has been delayed partly because performing efficiency assessment techniques were lacking. during the last 15 years, many new human pathogens have been discovered among which eight were viruses with various pathogenicity. molecular techniques have played a central role in their discovery (table 1) . the construction of cdna libraries by cloning techniques has been used to identify hcv and hepatitis e virus. representational difference analysis (rda) was successful in identifying human herpes virus 8 (hhv-8), and the hepatic viruses gbv-c and ttv. rda allows the detection of viral sequences by comparing whole nucleic acids sequences in cells from humans or animals before and after infection. reverse-trancription polymerase chain reaction (rt-pcr) with random or degenerate primers has been used to identify human metapneumovirus (hmpv), the virus sen and the coronavirus associated to sars (sars-cov). molecular techniques alone have allowed the characterization of very important human pathogens like hhv-8, responsible of kaposi sarcoma or hcv which induce acute or chronic hepatitis, cirrhosis and hepatocarcinoma. however, other viruses detected in a similar way are still waiting for the demonstration of their clinical importance. this illustrates the need to verify koch's postulate and the importance of keeping laboratory competencies for classical virology-tissue culture, electron microscopy and animal experiments-which plays a major role in virus discovery, together with epidemiological studies. large epidemiological studies are also required to assess the clinical interest of new pathogens. molecular diagnostics have been very rapidly implemented in clinical virology laboratories following the discovery of hmpv (van den hoogen et al., 2001; peiris et al., 2003; boivin et al., 2003) and sars-cov (ksiazek et al., 2003; drosten et al., 2003; anderson, 2003) . a prospective study on the prevalence of hmpv could be initiated as early as during the 2000-2001 winter, although the virus has been discovered in 2000 only. there has been only a few weeks lag between sequencing sars-cov and availability of the first commercial nat. nat are more and more used to exclude blood donations from patients infected by viruses (allain, 2003) . hcv and hiv testing has been implemented as part of routine screening in blood banks in 14 and 7 european countries respectively. in france, two commercial offers -procleix (gen-probe, inc. usa) and nuclisens extractor (biomerieux, france) associated with cobas ampliscreen (roche diagnostics, switzerland)-are used to screen donations for hiv and hcv infections. this allowed the reduction of residual risk from 1 in 400,000 to 1 in 2.5 millions for hiv and 1 in 760,000 to 1 in 5 millions for hcv (assal et al., 2003) . however, there is room from improvement as few contaminated blood units are still missed due to the lack of sensitivity induced by pooling strategies. cost constraints must also be considered if further improvements are to be considered. cost effectiveness of hiv and hcv nat addition to serology testing is already very low: in usa it has been calculated that the cost of each saved life is 4.7-11.2 millions us$ per year (jackson et al., 2003) . hepatitis b virus screening using molecular biology should also be included as even the most recent antigen assays (hbsag) assays miss infected blood units. as an example, single-sample hbv testing would allow the detection of 35-50 additional contaminated units among 10 7 units tested (biswas et al., 2003) . monovalent or trivalent assays (hiv, hbv, hcv) are proposed by roche diagnostics (ampli nat) and gen-probe (procleix ultrio) but blood units should not be pooled to provide sufficient sensitivity. procleix ultrio detects single seroconversions 20 days earlier than abbott prism-hbsag but only 13 days in pools of 8 and 11.5 days in pools of 24 (cambié, 2002) . alternatively, an ultracentrifugation step could be introduced following pooling to obtain a higher sensitivity compared to current antigen assays (roth et al., 2002) . screening for west-nile virus contamination has been implemented in us blood banks. from late june to mid-september 2003, approximately 2.5 million donations were screened. twelve hundred eighty-five (0.05%) were initially reactive for wnv by using nucleic acid-amplification tests and 601 (0.02% of the total donations) are considered presumptive viremic donations (i.e. a donation that is repeatedly reactive by the primary and/or alternate nat assay or a primary nat assay with a very high signal) (cdc, 2003) .other viruses (parvovirus b19, hepatitis a virus) are also transmitted by blood donation and may be part of the screening in a near future. however, nat may not always be the best diagnosis approach and antigen tests or antibody tests may be efficient and less expensive alternatives. automation and integration of nat is necessary to reduce costs and quarantine delays for blood units supply. in this respect, the recent approval by us food and drug administration (fda) of the tigris molecular diagnostic system (gen-probe, san diego, usa) is a major breakthrough as it has been designed to process 500 samples in 8 h. integration and time-to-result are also very important parameters to be considered to insure viral safety of transplant organs, especially lung, heart and liver. it is very important to determine the status of transplants regarding infection by hiv, hbv, hcv and viruses from the herpesviridae family. nat have significantly improved identification of viruses as etiologic agents of human diseases affecting various organs, especially respiratory and gastro-enteric tracts and central nervous system (cns). as a consequence, rapid antiviral treatments can be initiated and considerably reduce morbidity and mortality as, for example, in the case of herpes encephalitis. the increasing number of available antiviral drugs will even accentuate the need for positive viral identification. similarly, unnecessary antibiotic treatments can be avoided or reduced and hospitalisation durations shorten. treatments of chronic viral diseases are very efficiently monitored by viral load assays. however there are still obstacles that prevent a wider dissemination of molecular assays. the extreme genetic variability of some viruses, especially rna viruses (which rna-polymerases have no proofreading activity), often makes their diagnosis difficult. the most striking examples are found in the norovirus family which contains viruses responsible for the vast majority of gastro-intestinal epidemics in adults. hiv diagnosis is also quite difficult to achieve due to its high genetic variability. gardner et al. (2003) have deduced from sequence alignments that a real-time taqman assay should contain not less than nine primer and probe sets to detect with the same sensitivity all hiv strains in a geographically representative panel. to reduce the impact of variability on amplification and detection efficiency, one can use primers and probes with 2 o-methyl bases, degenerate bases or "universal" bases, such as inosine or nebularine. touchdown pcr protocols, in which the annealing temperature slightly decreases during the successive amplification cycles to bracket the melting temperature tm of the reaction, provides sensitivity even when primers have mismatches with target sequences of divergent species in a viral family. finally, degenerate primers or probes, with mixtures of the bases found in sequence databases among various species, may be useful to detect all species of a viral family. it is often desirable to provide the capability for panel detection, i.e. to detect several viruses that can be responsible for a disease. for example, coyle et al. have described at the winter meeting of european society for clinical virology (copenhagen, january 2004) a molecular viral respiratory strip for the detection of 12 common respiratory viruses. whenever possible, consensus primers able to detect all viruses from a family or genus must be used. there are several examples of such consensus primers for enterovirus (kammerer et al., 1994) , flavivirus (scaramozzino et al., 2001) or herpesviridae (tenorio et al., 1993) . however, their ability to amplify all viruses with the same efficiency must be carefully evaluated. if such an approach is not possible, two different possibilities exist for panel detection: multiplex detection in single tubes or parallel detection in individual tubes. mixing primers in a single amplification tube to achieve multiplex detection of viruses usually results in decreased sensitivity of assays compared to single tests. for example, we have observed, using a dna-microarray assay (see below), that the analytical sensitivity of multiplex rt-pcr detection of six viruses, i.e. influenza a, influenza b, rsv a/b, parainfluenza 1, 2 and 3 is reduced by a factor of <1-2 logs compared to single detections, depending on the virus. nevertheless, this multiplex assay was able to identify correctly 21/22 infections in respiratory specimen (one rsv b infection was misidentified as rsv a; unpublished data). the formation of primer dimers is generally considered as the major cause of sensitivity loss but careful optimisation of all parameters of amplification including primer, enzymes, nucleotides and salts concentrations as well as protocol conditions are required to obtain expected performances. realtime assays (see below) that monitor signal apparition during the amplification step are also limited in their capacity to realize multiplex detection by the number of available wavelengths in existing equipments which currently allows the detection of three viruses only. instead of mixing several pairs of primers in a single tube, nucleic acids purified from the original clinical specimen can be distributed into several tubes for independent amplification and detection. major drawbacks of this approach are the reduction of sensitivity because of lower amounts of nucleic acid available for each individual amplification, higher hands-on times required to manipulate all the different tubes, difficulty to automate the distribution of small volumes of purified nucleic acids without introducing cross-contaminations and higher costs due to the need for enzymes in each tube. internal controls (ic) are important components to monitor each step of the assay from extraction of nucleic acids to detection. because inhibitors of the amplification reaction, which are frequent in some specimen types, will also impact its amplification, the presence of an ic is a strong validation in case of negative result. niesters (2002) has described an original approach for internal control of nat: the use of viral universal controls that can be added to each specimen and be amplified with specific primers, preferably in a multiplex format with primers for the virus to detect. seal herpes virus 1 and phocine distemper virus can be used to control nat for dna and rna viruses respectively. of course, animal viruses which can not infect humans are required. other strategies for ic involve synthetic materials, i.e. plasmids or transcripts which contain sequences able to bind the test primers and a specific probe. clinical virology laboratories have high expectations in term of automation and integration of molecular assays. a major bottleneck in the workflow of these laboratories is at the level of sample preparation. table 2 shows automated systems for nucleic acid purification that are currently commercialised. most of them use the nucleic acid binding properties of silica (boom et al., 1990) . as many as 96 samples can be handled in a single run on the biorobot 9604 (qiagen gmbh, germany). time-to-result and, more important, hands-on-time tend to decrease, with the most recent equipments requiring no longer than 20 min of technician time for more than 24 samples. new labelling technologies that do not need solid-phase separation have allowed the development of real-time molecular assays in which the detection of amplicons is done as soon as they appear during amplification. the most simple real-time detection chemistry uses the sybr green dye which specifically binds during double-stranded dna generated during pcr. several probe technologies (fig. 1 ) have also been designed for real-time assays like taqman ( fig. 1a ) or molecular beacons (fig. 1b) in which a quencher molecule is removed from the vicinity of the fluorescent marker upon binding to rna or dna generated during amplification cycles. in the fret technology (fig. 1c) , two probes, one with a fluorescence donor and one with a fluorescence acceptor molecule are designed to bind adjacent sequences of the amplified material to generate signal (mackay et al., 2002) . real-time techniques have been designed for pcr or nasba amplification and have several advantages which facilitate automation and reduce time-toresult (30-120 min) and hands-on-times. they are performed in closed devices which do not need to be opened to transfer amplified material for end-point detection, thus reducing the risk of laboratory or samples cross-contamination. the use of real-time platforms makes the general organisation of molecular biology laboratories easier by reducing the constraints on activities segregation in different rooms to control contaminations. table 3 shows existing real-time automates. the next generation of nat platforms will integrate sample preparation, amplification and detection in a single test device genexpert (cepheid, usa) is the first fully integrated system which allows the detection of bacillus anthracis or group b streptococcus in approximately 45 min directly from clinical specimen. several viral assays based on real-time nasba on this platform will soon be available from biomerieux. several ivd companies offer commercial nat for the diagnosis of viral diseases. besides technical difficulties, another obstacle to the development of molecular assays is the importance of resources needed to optimise, produce and validate home-brew assays and to build up documentation required for qualification of techniques and laboratories. new european community regulations will even increase this need. it is the role of in vitro diagnostic (ivd) companies to provide reagents which are the results of careful optimisation and are produced according to high quality manufacturing procedures. they have clinical and regulatory affairs departments that conduct validation studies and assemble documentation required to get approval of the reagents. however, even for ivd companies, the conception and validation process is time-consuming and timeto-market may be long. this is especially a problem when a diagnostic tool is urgently needed in case of emergence of a new virus. one possibility to reduce time-to-market is to release "research use only" (ruo) assays or assays that have the "ce analytical" approval in europe or the status of "analyte specific reagent" (asr) in the usa. in this case, commercial products that have excellent analytical sensitivity and are manufactured according to quality standards of the ivd industry can be used by clinical virology laboratories, which have the responsibility to validate their use as diagnostic tools and obtain authorization to use them. infections by hiv and hepatitis b and c viruses are usually well diagnosed using serology. only diagnosis of early primo-infections may benefit from nat. platforms like amplicor amplicor from roche diagnostics or easyq from biomerieux that are usually used for viral load measurement during therapy (see below) are also suitable for the early detection of hiv or hcv infections. many other infections and especially acute infections for which igm appear only several days after onset of symptoms cannot be efficiently diagnosed using serology assays. forty to sixty percent of community acquired pneumonia that require hospitalisation 2 have no known aetiology despite intensive investigation and this percentage is even higher when less severe lower respiratory tract infections (lrti) are considered (l. kaiser, personal communication). in a recent study, henrickson et al. (2004) , have shown, using multiplex rt-pcr that 40% of children hospitalised for lrti are infected with the seven most common respiratory viruses. similarly, a recent survey of encephalitis leading to hospitalisation in the usa from 1988 to 1997 has revealed that nearly 60% had no aetiology (khetsuriani et al., 2002) . absence of specific antiviral treatments for most viruses, which limits prescription of biological tests and weak performances of diagnostic tests based on viral culture or serology are major explanations of this situation. nat, which have been implemented by many large european hospitals, significantly improve viral diagnosis. however, there are several obstacles to the generalization of molecular diagnostics in smaller, decentralized laboratories. a major obstacle is the fact that, except for hiv, hbv, hcv and cmv, virology nat are most often home-brew assays which sometimes suffer from bad performances and poor batch to batch consistency. however, quality of nat is the percentage of false-positive results which reflects laboratory or cross-contaminations and used to be high, dropped to 5.5% (wallace et al., 2003) . table 5 shows some products currently commercialised by major companies for viruses other than hiv, hbv and hcv, although the list may not be exhaustive. most of them are asr or ruo kits although some are ec marked. the majority of these reagents have been designed to run on realtime platforms. results shown by liolios et al. in 2001 illustrate the interest of multiplex detection of respiratory viruses by nat. one hundred forty-three clinical specimen were tested using the hexaplex assay from prodesse inc. (usa) which detects six viruses in a single tube using pcr and detection with microplate capture and peroxidase-labelled probes. 9 samples were detected with the prodesse assay only and not with immunofluorescence or viral culture (table 6) . table 6 multiplex nat for the detection of respiratory viruses (hexaplex, prodesse inc.) dna-microarrays or dna-chips are very powerful detection tools that can be combined with amplification techniques to detect viruses or virus variants (reviewed in clewley, 2004) . wang et al. (2002) have described a microarray spotted with 70-mer oligonucleotides which represent the five most conserved sequences (more than 20/70 bases are conserved among all representative sequences in a virus sequence alignment) of each virus of interest. the chip contains 1600 different probes. following pcr amplification of genetic material in the clinical specimen using random primers, hybridisation onto the microarray was able to identify respiratory viruses in the enterovirus, rhinovirus, paramyxovirus, adenovirus and herpesvirus families. however, this technique has not yet been extensively validated for routine diagnosis in a clinical virology laboratory. we have developed assays combining rt-pcr and dna-microarrays for the detection of viruses. the arrays are manufactured using the photolithography in situ synthesis technology (affymetrix, usa) and contain 20-mer oligonucleotides. sequence signatures are identified using extensive sequence databases because they are conserved in all viruses of a genus or family or in all subtypes or isolates of a virus and are not found in other viruses. for each base of the signature, probes perfectly matching the target and probes with a mismatch at the interrogated position are present on the array. if polymorphisms are present in or near the signature, variant probes may also be present. consensus primers have been designed for enterovirus, flavivirus, herpesviridae, parainfluenza and influenza virus, rsv and adenovirus. they can be combined in multiplex detection pcr or rt-pcr assays for the diagnosis of viral respiratory or cns infections. following amplification, dna is labelled using a newly developed diazomethyl chemistry (laayoun et al., 2003) . a complete line of instruments (sample preparation, thermocycler, hybridisation station and laser scanner) is available to perform the assay. as described above, a respiratory assay designed to detect six major viruses (parainfluenza 1, 2 and 3, rsv a/b, influenza a and b) in a single specimen has demonstrated high clinical sensitivity in preliminary evaluation. fig. 2 illustrates the discriminatory capacity of this technology for cns viruses. assays for the identification of human papilloma virus (hpv) are commercialised by several companies. the detection of a highly pathogenic hpv type has a very high predictive value for cervical carcinoma. however, the number of hpv types that are more or less closely associated to cervical cancer is high. dna-microarrays may thus be appropriate for the multiplex detection of all these types. such an assay has been described by and is distributed by biomedlab co. (south-korea). it is based on a consensus amplification and on 30-mer probes that are able to detect and discriminate 22 hpv types and has shown an association of 92.5-97.5% between hpv positivity and lesions of different severity or carcinoma whereas hpv infection was only found in 35.1% of cases when cytology was normal. viral load platforms are available from several ivd companies (versant from bayer diagnostics; cobas ampliprep/ amplicor from roche diagnostics, minimag/nuclisens easyq from biomerieux). significant progress have been made in the ability of most hiv assays to detect all subtypes of hiv-1 but no commercial assay exist for hiv-2. the analytical sensitivity of hbv viral load assays should be increased to reach the same performances as those of hiv assays, especially in the case of infections by variants with low replication competencies. for efficient treatment monitoring of immunosuppressed patients, for example in the case of organ transplantation, viral load assays should also be developed for epstein-barr virus, varicella-zooster virus and hhv6. genotyping tests are now commercially available and are part of the biological follow-up of treated patients although home-brew assays are still used in most laboratories (korn et al., 2003) . hiv and hbv resistance assays as well as hcv subtyping assays are generally based on the sequencing technology (trugene hiv and hbv from bayer healthcare; vi-roseq hiv-1 from celera diagnostics; 5 genotyping hcv kit from bayer healthcare). hybridisation techniques, such as lipa assays (innogenetics, belgium) are also used. however, the number of probes that can be spotted on nitrocellulose strips is limited and only few polymorphisms can be detected which is convenient for hcv subtyping but does not for resistance tests which are based on the detection of a high number of mutations. in addition, hiv and hbv have highly variable genomes and naturally occurring polymorphisms that are present in the vicinity of resistance mutations may affect the binding efficiency of probes. dna-microarrays are an alternative for resistance or typing reagents for viruses or bacteria . we have developed an assay based on rt-pcr and detection with fig. 2 . biomerieux dna-microarray for the detection of neurotropic viruses. following nucleic acid purification, amplification is performed by pcr using a single touchdown protocol in three tubes, one for herpesviridae (one primer pair for hhv 1, 2, 3, 5 and 6), one for enteroviruses (one primer pair for all serotypes) and one for flaviviruses (one primer pair for all viruses). amplification products are combined and labelled using diazomethyl chemistry and hybridised on a dna-microaaray which contains 20-mer oligonucleotides. two or four probes are used for the detection of each base of sequence signatures determined for each virus. a total of 20,000 probes are synthesised on this dna-microarray which has been designed for the simultaneous detection of viruses from the herpesviridae family and from the flavivirus, enterovirus, paramyxovirus, poliomavirus, bunyavirus and orthopoxvirus genus. a: amplicons generated resolved using the bioanalyzer (agilent technologies). b: image of the dna-microarray obtained with a confocal laser reader. c: resolution capability of the array. closely related viruses hybridise with very different efficiency on heterologous probes. high density probe arrays, designed to detect 204 antiretroviral resistance mutations simultaneously in gag cleavage sites, protease, reverse transcriptase, integrase and gp41. this assay has been tested on a panel of 99 hiv-1 patients on a total of 4465 relevant codons in comparison with the classic sequence-based method. key resistance mutations were correctly identified in 95 and 92% of codons in protease and reverse transcriptase, respectively (gonzalez et al., 2004) . we have also developed a similar assay for the detection of polymorphisms in the complete hbv genome: 78 antiviral resistance mutations in pol gene, 146 vaccine, diagnostic or immunotherapy mutation in s gene and 209 mutations in basic core promoter, pre-core, core, x, pre-s1 and pre-s2 regions that may have an impact on disease evolution or treatment efficiency. this assay is currently under evaluation in the frame of hepbvar a european collaborative group for the study of emerging variants of hepatitis b virus. in the coming years, more and more laboratories will offer to clinicians viral diagnosis based on nucleic acid tests. many biological and instrumentation problems that have slowed the generalization of molecular assays have been resolved but several others remain and need to be addressed. major im-provements are expected in the integration and automation of nat diagnostic platforms to reduce hands-on-time, time-toresult and costs and to increase throughput. ivd companies have engaged in development programs to provide clinical virologists with equipments and application menus adapted to their diagnosis needs. technical constraints and recent improvements of molecular assays transfusion risks of yesterday and of today correlation of cervical carcinoma and precancerous lesions with human papillomavirus (hpv) genotypes detected with the hpv dna chip microarray method a novel coronavirus associated with severe acute respiratory syndrome application de la biologie moléculaireà la sécurité virale transfusionnelle: le dépistage génomique viral comparative sensitivity of hbv nats and hbsag assays for detection of acute hbv infection human metapneumovirus infections in hospitalized children rapid and simple method for purification of nucleic acids update: detection of west nile virus in blood donations-united states a role for arrays in clinical virology: fact or fiction identification of a novel coronavirus in patients with severe acute respiratory syndrome limitations of taq-man pcr for detecting divergent viral pathogens illustrated by hepatitis a, b, c, and e viruses and human immunodeficiency virus detection of hiv-1 antiretroviral resistance mutations with highdensity dna probe arrays national disease burden of respiratory viruses detected in children by polymerase chain reaction the cost-effectiveness of nat for hiv, hcv, and hbv in whole-blood donations nested pcr for specific detection and rapid identification of human picornaviruses burden of encephalitisassociated hospitalizations in the united states quality control trial for human immunodeficiency virus type 1 drug resistance testing using clinical samples reveals problems with detecting minority species and interpretation of test results characterization of a novel coronavirus associated with severe acute respiratory syndrome aryldiazomethanes for universal labeling of nucleic acids and analysis on dna chips comparison of a multiplex reverse transcription-pcr-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens real-time pcr in virology clinical virology in real-time children with respiratory disease associated with metapneumovirus in hong kong nat for hbv and anti-hbc testing increase blood safety comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-pcr assay for detection of flaviviruses targeted to a conserved region of the ns5 gene sequences a newly discovered human pneumovirus isolated from young children with respiratory tract disease species differentiation and antibiotic susceptibility testing with dna microarrays linkage between the journal and quality control molecular diagnostics (qcmd) microarray-based detection and genotyping of viral pathogens key: cord-022380-49oti4zg authors: panlilio, adelisa l; gerberding, julie louise title: occupational infectious diseases date: 2009-05-15 journal: textbook of clinical occupational and environmental medicine doi: 10.1016/b978-0-7216-8974-6.50026-9 sha: doc_id: 22380 cord_uid: 49oti4zg nan infections acquired in the work setting represent an eclectic group that is seldom, if ever, considered together as a single category. occupational infections involve several organ systems, respiratory, enteric, and skin infections being particularly common. transmission involves not only casual person-to-person contact, but also a variety of other routes in special work environments. it is thus important to consider what unique features characterize the infectious diseases that can be considered occupational. perhaps one useful way of thinking about these diseases is that, as a group, they tend to be transmitted during work schedules or practices that are systematized. therefore, they can be anticipated, and to the extent that unsafe infectionprone practices can be identified and modified, they can be systematically prevented. another common feature is that many of the occupational infectious diseases can be regarded as behavioral. to the extent that unsafe practices have been defined, and practice policies modified to reduce infection risk, continued transmission often represents failure to follow accepted standards. although certain occupational infections can be prevented by vaccines (e.g., hepatitis b), prevention often depends on simple behavioral changes, such as hand hygiene, use of gloves, and not working while ill. finally, the anthrax cases during the fall of 2001 in the united states demonstrate the possibility of intentional (and criminal) exposure to infectious agents in the workplace, in this instance among those handling mail. 1 these intentional exposures, fortunately, are rare events, but should be considered in assessing sources of exposure for unexpected illnesses. prevention depends primarily on defining risky occupational practices or environments, clearly articulating policies for preventing communicable disease acquisition; removing structural barriers to compliance with policies (e.g., providing soap, hand cleansers, and gloves; allowing time away from work during periods of illness); and promoting healthy practices through behavioral change. because infectious diseases may represent the most common cause of time lost from work, it is important for the clinician concerned with occupational medicine to understand the relationship of specific infections to specific work environments and practices, and to give at least as much attention to prevention as to diagnosis and treatment. occupationally acquired infections have historically been associated with animal exposures and unsanitary work environments. modernization of agrarian techniques and improvement in sanitation have markedly decreased the incidence of these infections in the developed world, though they remain problematic in developing countries. while nearly all infectious diseases could conceivably be transmitted in the workplace, the emphasis here is on those that can be transmitted by casual contact or by specific workrelated exposures, with emphasis on diseases that are most common, most serious, or most readily prevented. healthcare settings pose a unique challenge because of the proximity of infectious patients, susceptible patients, and healthcare personnel. infections transmitted from personnel may have devastating effects on certain groups of patients, particularly the immunosuppressed. likewise, certain infections transmitted to personnel, such as multidrug-resistant tuberculosis, may have serious or even fatal, consequences. table 22 .1 summarizes the microbial etiology, sources, routes of infection, categories of workers at risk, and clinical manifestations of selected infectious diseases that have occupational predilections. a detailed discussion of waterborne microbial diseases is also provided in chapter 54. because the treatment of occupationally acquired infections does not differ from that of infections acquired non-occupationally, 2 the emphasis of this chapter is on the recognition and prevention of these infections. etiologic category, as well as the potential for recurrences, account for the high incidence of the common cold, even among healthy adults. colds are more common in the fall, winter, and early spring, perhaps because of increased crowding among children during the colder seasons. workers are most apt to acquire colds from exposure to young children in the home. secondary cases among coworkers may then develop. adults experience two to four colds each year, although the incidence among adult women exceeds that of men by a small margin, and smokers have a substantially increased risk. the modes of transmission of cold viruses are not entirely elucidated. for rhinoviruses, transmission among experimental subjects occurs most readily by direct handto-hand contact, with a case followed by autoinoculation of the mucous membranes of the eye or nose. such finger-tomucous membrane contact is ubiquitous and unavoidable. other viruses are transmissible by aerosolized droplets. the importance of fomites (such as drinking glasses, telephone receivers, and shared office equipment) as vectors of transmission has not been determined. clinical manifestations typical cold symptoms include nasal congestion, coryza, non-productive cough, sneezing, pharyngitis, and laryngeal irritation. fever is often low grade, or may be absent. viral upper respiratory infections usually resolve within 7-10 days, but longer durations are not uncommon. treatment, prevention, and control treatment for uncomplicated infections is symptomatic. decongestants are more useful in relieving symptoms than are antihistamines. expectorants, saline gargles, and other nonprescription remedies are useful in some cases. antibiotics should not be prescribed for the treatment of colds. colds are difficult to prevent. a policy of work restriction until symptoms improve may prevent the spread of colds but is likely to be impractical (table 22. 2). the cost-benefit analysis of such an approach could be useful, especially in childcare and healthcare settings. hand washing after contact with nasal secretions may be helpful. care should be taken to use tissues when coughing or sneezing and to dispose of soiled tissues after use. epidemiology influenza is a self-limited respiratory illness caused by types a and b influenza virus. epidemics of influenza occur annually in the winter months. adults remain susceptible to the illness despite prior episodes of infection because the antigenic structure of influenza viruses changes frequently, leading to new epidemics. influenza is spread from person to person, primarily by the coughing and sneezing of infected persons or sometimes by direct contact, either with infected persons or a contaminated surface. the disease is easily transmitted, and a single index case may transmit to a large number of susceptible persons in a short period of time. adults and children typically are infectious from 1-2 days before through 5-6 days after the onset of symptoms. clinical syndromes an attack of influenza starts abruptly with fever, malaise, myalgia, and headache. respiratory symptoms mimicking those of the common cold and lower respiratory symptoms including dry cough also are frequent. fever resolves in uncomplicated cases in 48-72 hours, but other symptoms may persist for days to weeks. influenza pneumonia, associated with hypoxemia, cough, and interstitial infiltrates, is not common in healthy adults. elderly patients and those with underlying immunodeficiencies and chronic pulmonary diseases are at high risk for secondary bacterial pneumonias, often caused by streptococcus pneumoniae and less often by haemophilus influenzae and staphylococcus aureus. the diagnosis of influenza frequently is made on the basis of clinical symptoms and signs. however, influenza is very difficult to differentiate from respiratory illnesses caused by other pathogens on the basis of clinical symptoms alone. other pathogens that can cause similar symptoms include, but are not limited to, mycoplasma pneumoniae, adenovirus, respiratory syncytial virus (rsv), rhinovirus, parainfluenza viruses, and legionella species. many pathogens, including influenza, rsv, and parainfluenza, cause outbreaks in a seasonal pattern. laboratory confirmatory tests can be performed to differentiate influenza from other illnesses. appropriate patient samples to collect for laboratory testing can include a nasopharyngeal or throat swab from adults, or nasal wash or nasal aspirates, depending on which rapid test is used. samples should be collected within the first 4 days of illness. rapid influenza tests provide results within 24 hours; viral culture provides results in 3-10 days. most of the rapid tests are more than 70% sensitive for detecting influenza and more than 90% specific. because as many as 30% of samples that would be positive for influenza by viral culture may give a negative rapid test result, negative rapid tests should be followed by viral culture in a sample of the swabs collected. viral culture can also identify other causes of influenza-like illness when influenza is not the cause. serum samples can be tested for influenza antibody to diagnose acute infections. two samples should be collected per person: one sample within the first week of illness and a second sample 2-4 weeks later. if antibody levels increase from the first to the second sample, influenza infection likely occurred. because of the length of time needed for a diagnosis of influenza by serologic testing, other diagnostic testing should be used for rapid detection of possible outbreaks. during community outbreaks, specific virologic or serologic diagnosis is not necessary once the type(s) of influenza virus causing the outbreak have been identified. treatment, prevention, and control persons at high risk for serious morbidity (persons aged 65 and older, persons with chronic underlying diseases) should receive influenza vaccine annually. immunization also is recommended for healthcare personnel and others at risk for transmitting influenza to high-risk patients ( had been associated with reduced work absenteeism and fewer deaths among nursing home patients. 3, 4 most employers do not provide influenza prevention programs for workers outside the healthcare field. 3 amantadine and rimantadine can reduce the duration of uncomplicated influenza a illness when administered within 2 days of onset of illness in otherwise healthy adults. zanamavir and oseltamivir can reduce the duration of uncomplicated influenza a and b illness by approximately 1 day compared with placebo. none of these antiviral agents has been shown to be effective in preventing serious influenza-related complications. to reduce the emergence of antiviral drug-resistant viruses, the duration of therapy should typically be no longer than 5 days. both amantadine and rimantadine are indicated as prophylaxis for influenza a, but not for influenza b infection. oseltamivir has been approved as prophylaxis for influenza a and b. zanamivir has not been approved for prophylaxis, but has been shown to be as effective as oseltamivir in preventing febrile, laboratory-confirmed influenza illness. they are approximately 70-90% effective in preventing illness from influenza a. chemoprophylaxis can be a component of influenza outbreak control programs. 3 epidemiology the incidence of measles (rubeola) has steadily declined in the united states during the last decade and is no longer considered endemic. measles is a major cause of morbidity and mortality worldwide. the majority of cases in the united states in recent years have been imported or secondary cases epidemiologically linked to imported cases. 5 infected persons are highly contagious by the air-borne route during the viral prodrome, and when cough and coryza are prominent, until about 2 days after the rash appears. infection confers lifelong immunity. although most adults born prior to 1957 experienced childhood infection and are no longer susceptible, up to 10% may lack natural immunity. in recent epidemics, cases occurred among unimmunized children, as well as children and young adults who had received a single vaccination with live virus, and among older adults. 5 clinical syndromes measles progresses in several phases. initial virus replication occurs in the respiratory tract and leads to a primary viremic phase, which usually is asymptomatic. release of virus from infected reticuloendothelial cells produces secondary viremia and infection of the entire respiratory system, accompanied by symptoms of coryza, cough, and in some, bronchiolitis or pneumonia. koplik's spots, a bluish-gray enanthem most prominent on the buccal mucosa, precede development of the rash. in a typical case of measles, the rash begins on the face, then progresses to the trunk and distal extremities, and disappears in the same sequence after 5-6 days. treatment, prevention, and control live-attenuated vaccine for prevention of measles became available in the early 1960s. all healthy children should receive the vaccine at age 15 months. because 10% do not respond to a single dose of vaccine, a second dose is now recommended to improve vaccine efficacy. 7 all healthy adults born after 1957 who have not received two doses of live virus vaccine or have not experienced measles also are advised to receive vaccine (table 22. 3). persons who received killed vaccine have a risk of developing atypical measles, and require re-immunization with live virus vaccine. live virus vaccine is contraindicated in infants, pregnant women, and immunosuppressed persons. passive immunization with γ globulin is available for unimmunized persons exposed to infected individuals, but is not routinely recommended for adults. measles rarely can exacerbate tuberculosis and cause a temporary inhibition of delayed hypersensitivity. vaccine administration should be delayed for 1 month after tuberculin testing, and until treatment is under way in persons with active tuberculosis. epidemiology mumps is a viral illness transmitted by the oral or respiratory route during contact with contaminated fomites or aerosolized droplet secretions. mumps is less contagious than measles or rubella but produces significant morbidity, especially among adults. the incubation period ranges from 2 to 3 weeks. virus is detectable for 6 days prior to and 9 days after the appearance of symptoms. most adults are immune to mumps, but 10-20% of unimmunized adults have no serologic evidence of prior infection and are considered susceptible. mumps incidence is now very low in all areas of the united states. the substantial reduction in mumps incidence during the past few years likely reflects the change in the recommendations for use of measles mumps rubella (mmr*) vaccine. 6 clinical syndromes parotitis typically is bilateral, but unilateral disease and involvement of other salivary glands occurs in some persons. localized parotid tenderness and swelling, fever, and painful swallowing suggest the diagnosis. aseptic meningitis is common but benign. encephalitis is a rare but serious manifestation. about 25% of affected postpubescent men develop orchitis, epididymitis, or both, which is bilateral in 15% of cases and may be the sole manifestation of mumps infection. about 50% of cases of mumps orchitis result in testicular atrophy, but neither sterility nor impotence are common sequelae. oophoritis occurs in about 5% of women with mumps. vaccine separated by at least 1 month (i.e., a minimum of 28 days), and administered on or after the first birthday, are recommended for all children and for certain highrisk groups of adolescents and adults. 6 adult men and healthcare personnel with no history of mumps or mumps immunization should be screened for immunity and vaccinated if they are susceptible. immunization is contraindicated in persons with immunosuppression and in pregnant women (table 22. 3). passive immunization with mumps immune globulin decreases the incidence of orchitis and is recommended for mumps in adult men with a single testis. individuals with active mumps should be excluded from work until 9 days after the onset of parotitis to avoid transmission to others in the workplace (table 22. 2). epidemiology fifth disease, also called erythema infectiosum or 'slapped cheek disease', is an infection caused by parvovirus b19. it is a common rash illness that is usually acquired in childhood, but can be an occupational risk for school and childcare personnel. it has been transmitted to personnel in healthcare settings. clinical syndromes symptoms begin with mild fever and symptoms of fatigue. after a few days, the cheeks take on a flushed 'slapped' appearance. there may also be a lacy rash on the trunk, arms, and legs. not all infected persons develop a rash. the child is usually not very ill, and the rash resolves in 7-10 days. most persons who get fifth disease are not very ill and recover without any serious consequences. an adult who is not immune can be infected with parvovirus b19 and either have no symptoms or develop the typical rash of fifth disease, joint pain or swelling, or both. usually, joints on both sides of the body are affected. the joints most frequently affected are the hands, wrists, and knees. the joint pain and swelling usually resolve in a week or two, but they may last several months. about 50% of adults, however, have been previously infected with parvovirus b19, have developed immunity to the virus, and cannot get fifth disease. fifth disease is believed to be spread through direct contact, fomites, or large droplets. the period of infectivity is before the onset of the rash. once the rash appears, a person is no longer contagious. the incubation period is 4-14 days but may be as long as 20 days. treatment, prevention, and control symptomatic treatment for fever, pain, or itching is usually all that is needed for fifth disease. adults with joint pain and swelling may need to rest, restrict their activities, and take anti-inflammatory medications to relieve symptoms. transmission can be prevented by careful attention to hygiene, especially hand washing. no special precautions are necessary. excluding persons with fifth disease from work, childcare centers, or schools is not likely to prevent the spread of the virus, since people are contagious before they develop the rash. epidemiology pertussis, or whooping cough, is an acute infectious disease caused by the bacterium bordetella pertussis. pertussis continues to be an important cause of mortality in the united states. a dramatic decline in the incidence followed the widespread use of whole-cell pertussis vaccines in the mid-1940s. however, since the early 1980s, the reported pertussis incidence has increased cyclically with peaks occurring every 3-4 years. 7, 8 contributing to this increase in incidence is the waning of immunity over time following vaccination, particularly in older age groups. transmission most commonly occurs by contact with respiratory secretions or large aerosol droplets from the respiratory tracts of infected persons and less frequently by contact with freshly contaminated articles of an infected person. analysis of national surveillance data for pertussis during 1997-2000 indicates that pertussis incidence continues to increase in infants too young to receive three doses of pertussis-containing vaccine and in adolescents and adults. 7 clinical syndromes the incubation period of pertussis is commonly 7-10 days. pertussis begins insidiously with non-specific upper respiratory symptoms including coryza, sneezing, low-grade fever, and a mild, occasional cough, similar to the common cold. the cough gradually becomes more severe, and after 1-2 weeks, the second, or paroxysmal stage, begins. characteristically, the patient has paroxysms of numerous, rapid coughs generally with a characteristic high-pitched whoop, commonly followed by vomiting and exhaustion. the patient usually appears normal between attacks. older persons (i.e., adolescents and adults), and those partially protected by the vaccine may become infected with b. pertussis, but usually have milder atypical disease. pertussis in these persons may present as a more persistent cough of greater than 7 days duration, and may be indistinguishable from other upper respiratory infections. inspiratory whoop is uncommon. b. pertussis is estimated to account for up to 7% of cough illnesses per year in older persons. even though the disease may be milder in older persons, these infected persons may transmit the disease to other susceptible persons, including unimmunized or underimmunized infants. the medical management of pertussis cases is primarily supportive, although antibiotics are of some value, with erythromycin being the drug of choice. this therapy eradicates the organism from secretions, thereby decreasing communicability and, if initiated early, may modify the course of the illness. there is no pertussis-containing vaccine (including dtap) currently licensed for persons 7 years of age or older, and vaccination with dtap currently is not recommended after the 7th birthday. vaccine reactions are thought to be more frequent in older age groups, and pertussis-associated morbidity and mortality decrease with increasing age. studies are currently under way to determine if a booster dose of acellular pertussis vaccine administered to older children or adults may reduce the risk of infection with b. pertussis. this may in turn reduce the risk of transmission of b. pertussis to infants and young children who may be incompletely vaccinated. studies among older children, adolescents, and adults examining pertussis disease burden and transmission of disease to infants might guide future policy decisions on the use of acellular pertussis vaccines among persons more than seven years of age. currently, vaccination of children more than 7 years of age, adolescents, and adults is not recommended either routinely or as an outbreak control measure. in the future, licensure of pertussis vaccines for adolescents or adults may lead to new recommendations for the use of vaccines in outbreaks. 9 epidemiology most epidemics of bacterial pneumonia in the workforce are due to community-acquired infections. however, legionellosis is one type of pneumonia which can be transmitted in the workplace. legionella pneumophila is an important cause of both epidemic and endemic adult pneumonia, and it can be associated with outbreaks in the workplace. this organism colonizes aquatic ecosystems and potable water, and it is transmitted to humans by the air-borne route. contaminated air conditioners, humidifiers, and shower heads have been implicated in outbreaks among workers and hospital patients. outbreaks of legionellosis have occurred after persons have breathed mists that come from a water source (e.g., air conditioning cooling towers, whirlpool spas, showers) contaminated with legionella bacteria. persons may be exposed to these mists in homes, workplaces, hospitals, or public places. legionellosis is not passed from person to person. a careful occupational history should be obtained from all adults who present with pneumonia, because occupational exposures cause many otherwise rare pneumonias. public health authorities should be notified if an occupational source is suspected so that an epidemiologic investigation to identify transmission routes and other susceptible individuals may commence. clinical pneumonia syndromes community-acquired bacterial pneumonia usually is exhibited acutely, with fever, chills, productive cough, and often, pleurisy. chest examination demonstrates signs of consolidation that may be confirmed radiologically. sputum examination may aid implementation of empiric therapy by suggesting the etiologic pathogen. blood cultures should be obtained when invasive disease is suspected, and lumbar puncture to evaluate meningeal fluid is indicated when symptoms or signs of meningitis are present. patients with legionnaire's disease usually have fever, chills, and a cough, which may be dry or productive. some patients also have muscle aches, headache, tiredness, loss of appetite, and, occasionally, diarrhea. chest x-rays often show pneumonia but are not pathognomonic. it is difficult to distinguish legionnaire's disease from other types of pneumonia by symptoms alone; other tests are required for diagnosis. the definitive test is culture isolation of the organism in sputum, bronchoalveolar fluid, or pleural fluid. other useful diagnostic tests detect the bacteria in sputum by specialized stains, identify legionella antigens in urine samples, or compare antibody levels to legionella in two blood samples obtained 3-6 weeks apart. the time between the patient's exposure to the bacterium and the onset of illness for legionnaire's disease is 2-10 days. treatment, prevention, and control empiric ambulatory therapy of acute community-acquired bacterial pneumonia, not requiring hospitalization, should include coverage for pneumococcus and h. influenzae, if the patient has a history of chronic obstructive lung disease. amoxicillin, trimethoprim-sulfamethoxazole and cefixime are reasonable choices, unless atypical pneumonia caused by m. pneumoniae or c. pneumoniae is suspected, in which case erythromycin is preferred. erythromycin is the antibiotic currently recommended for treating persons with legionnaire's disease. in severe cases, a second drug, rifampin, may be added. preventing bacterial pneumonia is a difficult challenge. workers at risk for pneumococcal and haemophilus infections should be immunized, although the efficacy of this approach among patients at highest risk is debated (table 22. 3). influenza immunization could eliminate a major risk factor for both primary and secondary bacterial pneumonias. occupational exposures to potential pathogens should be minimized with proper ventilation. prevention of legionellosis is achieved by maintaining an environment that is not conducive to survival or multiplication of legionella. the necessary preventive measures may involve water treatment or modification of air conditioning and ventilation systems. 10 these preventive steps, which may be costly, should be directed at healthcare facilities, and occupational settings where cases have been identified. 11 epidemiology rubella (german measles) virus is transmitted person to person by mucosal exposure to infected droplets of respiratory secretions. since 1969, children in the united states have been routinely immunized against rubella at age 15 months, so that the majority of recognized cases today occur in adults and unimmunized children. since 1992, reported indigenous rubella has continued to occur at a low but relatively constant endemic level with an annual average of less than 200 rubella cases. 7 recent data indicate that the rate of rubella susceptibility and risk for rubella infection are highest among young adults. no large epidemics have occurred since the vaccine was licensed for use in 1969. however, outbreaks continue to occur among groups of susceptible persons who congregate in locations that increase their exposure, and among persons with religious and philosophic beliefs against vaccination. several recent outbreaks have occurred in workplaces where most employees are foreign born, particularly from latin america. 6 reinfection can occur following natural or acquired immunity, but it is usually asymptomatic and only rarely accompanied by viremia. rubella virus is shed from the respiratory tract of infected persons beginning 10 days before the development of rash and for several days after the rash appears. the onset of the rash coincides with the period of maximal contagiousness, and infected persons are not considered infectious for more than 7 days after the rash appears. infected infants shed virus for several months despite the presence of antibody. clinical syndromes adult rubella is often asymptomatic. symptoms occur 12-23 days after exposure. following a prodrome of fever and malaise, adults exhibit a maculopapular rash that begins on the face and extends downward, persists for 3-5 days, and often is accompanied by regional lymphadenopathy of the head and neck, which persists for days to weeks. one-third of adult women may develop arthritis in the fingers, knees, and wrists during the exanthematous phase of illness. children develop hemorrhagic complications more often than adults. in contrast, encephalitis, albeit rare, is more common in adults and is fatal in 20-50% of cases. maternal rubella infection acquired in the first 20 weeks of gestation frequently results in congenital rubella. the earlier in pregnancy rubella occurs, the more severe the fetal consequences. infection in the first trimester results in deafness, congenital heart disease, cataracts or glaucoma, endocrine abnormalities, and mental retardation in up to 60% of newborns. spontaneous abortion also occurs commonly. treatment, prevention, and control immunization of children and susceptible adults with live attenuated rubella virus effectively prevents rubella and accounts for the dramatic decline in the incidence of this disease in the united states. however, many adult women of childbearing age remain susceptible to rubella and require immunization prior to conception to prevent congenital rubella. the hemagglutination-inhibition serologic assay detects natural or acquired immunity. the advisory committee on immunization practices (acip) recommends screening of healthcare personnel who have not been vaccinated, and immunization of susceptible individuals. 7 complications of rubella vaccine occur among adults and include lowgrade fever, symmetric polyarthralgias, distal paresthesias, lymphadenopathy, and rash. vaccine is contraindicated in immunosuppressed persons and pregnant women. pregnancy should be avoided for 3 months after vaccination (table 22. 3). susceptible household contacts of infected adults and children pose a transmission risk in the workplace during the period of virus shedding, beginning about 10 days before the development of rash (about 1 week after exposure) until 7 days after rash appears. therefore, susceptible individuals should not report to work during this time interval (table 22. 2). epidemiology tuberculosis (tb) is caused by mycobacterium tuberculosis and, rarely today, by m. bovis. the incidence of tuberculosis (tb) in the united states declined steadily until the mid-1980s, but then sharply increased, especially in urban areas. the resurgence of tb in the united states in the late 1980s and early 1990s was associated with the emergence of multidrug-resistant tb (mdr-tb) and the hiv/aids epidemic. with this resurgence of tb in the united states came several high-profile nosocomial outbreaks associated with lapses in infection control practices and delays in diagnosis and treatment of persons with infectious tb, as well as the appearance and transmission of mdr-tb strains. since 1992, the declines in the overall number of reported tb cases, including the level of mdr-tb, appear to reflect successful efforts to strengthen tb control following the resurgence of tb and the emergence of mdr-tb. 12 activities emphasizing the first priority of tb control (i.e., promptly identifying persons with tb, initiating appropriate therapy, and ensuring completion of therapy) have been the most important factors in achieving this improvement. such activities reduced community transmission of m. tuberculosis, particularly in areas with a high incidence of aids. improvements in implementation of infection control measures in healthcare settings, concurrent with mobilization of the nation's tb control programs, succeeded in reversing the upsurge in reported cases of tb, and case rates have declined to their lowest levels to date. the threat of mdr-tb is decreasing, and the transmission of tb in healthcare facilities continues to abate due to implementation of infection controls and reductions in community rates of tb. nevertheless, some healthcare personnel are at risk for acquiring tb. pulmonary tb is most commonly transmitted in healthcare settings by inhalation of aerosolized droplet nuclei derived from the respiratory secretions of patients with active respiratory tb. close contact usually is required. most other categories of workers generally are not at risk without close and sustained workplace contact with a person who has active untreated disease. ingestion of unpasteurized milk from cows infected with m. bovis is no longer an important source of tb in most industrialized countries. clinical syndromes primary infection usually is asymptomatic in adults. teenagers and young adults are at higher risk for rapid progression to active disease, usually characterized by apical cavitary disease, than are older adults. primary infection in the elderly usually is exhibited as lower lobe consolidation with hilar adenopathy. primary tuberculosis in persons with advanced hiv infection is commonly symptomatic and progressive. once infection occurs, the organism may disseminate from the lungs to other sites, including the gastrointestinal and genitourinary tracts, and bone. normally, the infection is contained by the host's immune response at this stage. the risk for reactivation is highest in the first year after exposure and declines thereafter. however, aging and stressors such as immunosuppression, intercurrent illness, and chronic malnutrition may increase the risk for reactivation or dissemination of the disease later in life. clinically, reactivation tuberculosis usually is exhibited as upper lobe pulmonary cavitary disease, but virtually any organ system may be involved. treatment, prevention, and control tuberculin skin testing allows determination of prior exposure to tb in immunologically healthy adults, by assessing delayed hypersensitivity to tuberculin antigens using purified protein derivative. the tuberculin skin test (tst) is the only proven method for identifying infection with m. tuberculosis in persons who do not have tb disease. although the available tst antigens are neither 100% sensitive nor specific for detection of infection with m. tuberculosis, no better diagnostic methods have yet been devised. the preferred skin test for diagnosing m. tuberculosis infection is the mantoux test. it is administered by injecting 0.1 ml of 5 tuberculin units (tu) ppd intradermally into the dorsal or volar surface of the forearm. tests should be read 48-72 h after test administration, and the transverse diameter of induration should be recorded in millimeters. there are three cut-off levels recommended for interpretation of the tst results. 14 in hiv-infected persons, any reaction resulting in an induration larger than 5 mm is read as positive. among others, the presence of 15 mm or more of induration always indicates a positive test, 5-10 mm indicates a positive result in persons at risk for tb, and less than 5 mm is negative. a positive tst means an individual has been exposed to tb in the past and is at risk for reactivation. a baseline chest radiogram should be performed on all persons with newly diagnosed tst positivity. if the x-ray study suggests active disease, sputum samples should be obtained, stained for acid-fast bacilli, and cultured for mycobacteria. treatment should be implemented immediately if the index of suspicion is high. public health officials should be notified to institute case management and evaluation of contacts in the home and work environment. if the x-ray study is negative, treatment with isoniazid to suppress or eradicate latent organisms may be recommended, especially in persons younger than age 35 and for those who have recently converted to positive tsts. 13 although bcg vaccine is the most widely administered of all vaccines in the world, and has the highest coverage of any vaccine in the who expanded programme on immunization, it appears to have had little epidemiologic impact on tb. despite its shortcomings, and because of its beneficial effect in children and against leprosy, bcg vaccine likely will remain a component of childhood vaccination strategies in developing countries. however, because of questions about the vaccine's efficacy, and because it induces dermal hypersensitivity to purified protein derivative (ppd) tuberculin in most recipients, bcg has never been recommended for programmatic use in the united states. 14 healthcare providers should follow appropriate infection control procedures, including use of isolation rooms and respiratory protection, when caring for patients with active tuberculosis. 15, 16 varicella/zoster epidemiology varicella virus, the causative agent of chickenpox and zoster, is a highly contagious herpes virus spread by the respiratory route from person to person. the incubation period is about 14 days, and the period of infectivity begins a few days prior to the onset of the rash to about 6 days after the first crop of vesicles appears. immunosuppression usually prolongs the period of infectivity, especially if varicella zoster immune globulin (vzig) has been administered. zoster represents reactivation of varicella virus that is latent in sensory nerve ganglia, and it is not a manifestation of primary infection except in newborns infected in utero. the incidence of zoster increases with age and immunosuppression. susceptible persons in direct contact with zoster lesions risk developing primary varicella. in the prevaccine era, varicella was endemic in the united states, and virtually all persons acquired varicella by adulthood. as a result, the number of cases occurring annually was estimated to approximate the birth cohort, or approximately 4 million per year. this incidence has likely decreased since licensure of the vaccine in 1995. varicella is not a nationally notifiable disease, and surveillance data are limited. 17 clinical syndromes varicella in otherwise healthy children usually is a benign, self-limited disease characterized by low-grade fever and vesicular rash, often preceded by a viral prodrome. varicella vesicles of primary infection appear first on the scalp and trunk and disseminate in crops showing various stages of development over the next 3-4 days. healing results in crusting accompanied by intense pruritus. manifestations of varicella are more severe in adults than in children. about 15% of adults with varicella show radiographic evidence of pulmonary involvement, but this is rarely clinically significant. however, cough, tachypnea, and impaired gas exchange can occur and persist for months after infection. varicella during pregnancy can produce congenital varicella. in its most severe form, this infection can result in mental retardation, blindness, growth retardation, deafness, chorioretinitis, and a peculiar dermatomal lesion of the upper or lower extremity associated with limb atrophy. zoster, the most common manifestation of varicella infection among adults, characteristically produces unilateral vesicular eruptions preceded by pain in one to three dermatomes. disseminated zoster, which is more likely in immunosuppressed patients, probably poses the same risk of infection transmission as primary varicella infection. the major complication of zoster is postherpetic neuralgia, which is especially common in the elderly and may be extremely debilitating. zoster frequently produces cerebrospinal fluid pleocytosis and occasionally encephalitis. immunologically healthy persons may experience recurrences of zoster, usually in the same dermatome as the initial outbreak. zoster is a marker of deteriorating cellmediated immunity among hiv-infected patients, and it may disseminate. treatment, prevention, and control passive immunization with vzig is recommended for immunosuppressed susceptible persons, children with leukemia and other malignancies, and neonates exposed in utero within 5 days before delivery. 18 several antiviral drugs are active against varicella zoster virus, including acyclovir, valacyclovir, famciclovir, and foscarnet. 18 famciclovir and valacyclovir are approved for use only in adults. clinical studies indicate that these drugs may be beneficial if given within 24 hours of onset of rash, resulting in a reduction in the number of days new lesions appeared, in the duration of fever, and in the severity of cutaneous and systemic signs and symptoms. antiviral drugs have not been shown to decrease transmission of varicella, reduce the duration of absence from school, or reduce complications. oral acyclovir can be considered in otherwise healthy adolescents and adults or secondary cases in the household, because of the increased risk of severe illness in these groups. antiviral therapy may also be considered for persons with chronic cutaneous or pulmonary disorders, persons receiving long-term salicylate therapy, and for children receiving short, intermittent or aerosolized courses of corticosteroids. antiviral drugs are not recommended for routine postexposure prophylaxis. systemic steroids in older adults (more than 60 years old) may reduce the incidence and severity of postherpetic neuropathy if started early (within 5 days of skin manifestations). varicella has been difficult to prevent because of the high degree of contagion in households, schools, and healthcare settings. live attenuated virus vaccines have demonstrated efficacy in preventing primary infection, and one was licensed for use in the united states in 1995. routine immunization is now recommended for children less than 18 months of age. 17 varicella vaccination should be given to susceptible adolescents and adults who are at high risk of exposure to varicella. this group includes persons who live or work in environments in which there is a high likelihood of transmission of varicella, such as teachers of young children, residents and staff in institutional settings, and military personnel. varicella vaccination is also recommended for susceptible adolescents and adults who will have close contact with persons at high risk for serious complications of acquired varicella, including healthcare personnel and susceptible family contacts of immunocompromised individuals. the acip recommends that all healthcare personnel be immune to varicella, either from a reliable history of prior varicella infection or vaccination, to reduce the risk of infection and its complications, and to decrease the possibility of transmission of varicella zoster virus to patients (table 22. 3). susceptible adults exposed to children with varicella or with disseminated zoster pose a risk of transmitting varicella to non-immune coworkers, and they should not work until the incubation period is over or, if they become ill, until all lesions are crusted (table 22. 2). dermatomal zoster is not spread efficiently by the air-borne route and otherwise healthy adults afflicted with this illness may be allowed to work if they can avoid touching the lesions and contaminating the work environment. the role of vaccine in the postexposure management of susceptible employees needs to be elucidated. data from the united states and japan in a variety of settings indicate that varicella vaccine is effective in preventing illness or modifying the severity of illness if used within 3 days, and possibly up to 5 days, of exposure. acip recommends vaccine be used in susceptible persons following exposure to varicella. 17 personnel should be excluded from work who have onset of varicella until all lesions have dried and crusted (table 22. 2). following exposure to varicella, personnel who are not known to be immune to varicella (by history or serology) should be excluded from duty beginning on the 10th day after the first exposure until the 21st day after the last exposure (28th day if vzig was given). epidemiology acute gastrointestinal infection follows upper respiratory illness as the next leading category of infectious diseases causing absenteeism among adult workers. a wide array of pathogens, including viruses, bacteria, and protozoa, can result in acute infections of the stomach, small bowel, or colon. a comprehensive discussion of enteric pathogens is provided in chapter 54 (waterborne microbial diseases). most of the etiologic agents are acquired by the fecal-oral route; produce mild, self-limited diseases; and resolve without specific therapy. agents of dysentery (e.g., shigella spp.) often are highly transmissible through low-inoculum exposures. occupational transmission of food-borne or water-borne illnesses occurs; person-to-person transmission has propagated outbreaks of many of these illnesses in healthcare settings, daycare and nursery schools, and institutions where sanitation is poor. instances of such transmission have generally involved food handlers, who are often poorly trained and short-term employees, serving as sources of transmission to others. occupations requiring travel to countries with poor sanitation present a major risk for gastrointestinal infections. poultry workers are frequently exposed to salmonella infections. avoidance of oral contact with sources of fecal contamination is the most important strategy for preventing transmission of pathogens associated with intestinal infections. maintaining good personal hygiene, including careful hand hygiene after using restrooms and before food preparation; proper cooking and storage of foods; and avoidance of contaminated foods and water when traveling are essential prevention strategies. food handlers with diarrheal illnesses should not work until symptoms have resolved, and cure of bacterial infections should be documented by obtaining negative stool cultures more than 48 hours after antimicrobial therapy is completed (table 22. 2). the only vaccines for any of the etiologic agents for acute enteric infections are for typhoid and hepatitis a, which are recommended for personnel in laboratories who frequently work with salmonella typhi or hepatitis a virus (table 22. 3). epidemiology cytomegalovirus (cmv) is a ubiquitous herpes virus transmitted by direct inoculation with infected body fluids (including blood, blood products, respiratory secretions, saliva, and urine) and through sexual contact with infected partners. at least 40% of healthy adults have serologic evidence of prior cmv infection. infection can be acquired perinatally, in utero during maternal primary infection, during birth by passage through infected vaginal secretions, or through ingestion of infected breast milk. cmv is known to be highly transmissible in daycare centers and nursery schools. sexually active adults and recipients of blood products are also at high risk for infection. infants and young children excrete cmv in their urine, saliva, and respiratory secretions for several months after infection. virus is much less readily detected in healthy adults, but intermittent shedding has been documented. like all herpes viruses, cmv remains latent in the host after initial infection. previously infected persons may be reinfected with new strains of cmv. occupational transmission of cmv has been documented in childcare settings, where person-to-person spread through exposure to infected secretions and urine is believed to provide an efficient mode of transmission. up to 50% of seronegative workers in preschool daycare centers have acquired cmv infection in some studies, indicating a potentially serious risk to women of child-bearing age, because of the adverse effects of primary maternal cmv infection on the fetus. at one time, employment in healthcare settings also was believed to pose a high risk for cmv acquisition. however, epidemiologic investigations suggest that most infections in healthcare personnel are acquired sexually, or from exposure to young children in the home, and not from work-related contact. is asymptomatic in healthy persons. a self-limited mononucleosis-like illness occurs in a minority, which may be complicated by hepatitis, pneumonitis, hematologic abnormalities, and myocarditis. immunosuppressed children and adults with primary cmv infection, reactivation, or reinfection may develop severe sequelae. organ transplant recipients, hiv-infected patients, and persons with malignancies have a risk of developing cmv viremia, pneumonia, hepatitis, pancreatitis, enteritis, and retinitis. primary cmv infection at any stage of pregnancy carries a greater risk to the fetus than does recurrent cmv infection during pregnancy. symptoms of congenital cmv infection may be present at birth, and are due to the consequences of active virus replication and resultant end-organ damage. congenital cytomegalic inclusion disease, the most severe form of this entity, includes central nervous system disease, respiratory distress, hepatitis, hepatosplenomegaly, rash, and multi-system failure. infection acquired from exposure to cervical cmv during birth usually is asymptomatic and detected by the onset of virus shedding 4-8 weeks postpartum. with ganciclovir or foscarnet for cmv infection is reserved for immunosuppressed persons at high risk for severe complications. the safety and effectiveness of these agents in preventing congenital cmv have not been established. avoidance of mucosal contact with infected body fluids is the best strategy for preventing cmv transmission. hand washing after contact with secretions and fomites is essential, especially in nurseries and daycare settings. the presence of persons at risk for cmv shedding in the workplace does not pose a hazard to other employees unless direct contact with infected secretions is anticipated. isolation of infected neonates or children is not essential if hand washing is performed after contact with secretions, blood, and urine. pregnant healthcare providers compliant with hand washing protocols can generally safely care for patients with cmv infection. [19] [20] [21] no work restriction is necessary for individuals with cmv infection (table 22 .2). direct exposure to blood and other infected body fluids. children born to infected mothers are at high risk for hbv infection. persons parenterally exposed to blood, including multi-transfused patients, hemophiliacs, dialysis patients, and injection drug users, also are at significant risk. sexual contact with infected partners is another efficient mode of hbv spread. in most industrialized countries, adult infections usually are acquired sexually or by injection drug use. hbv is a relatively hardy virus capable of surviving on environmental surfaces and fomites. transmission in households is well documented and may, in part, be attributable to mucosal contact with fomites contaminated with secretions or blood from infected persons. healthcare personnel and others at risk for occupational blood exposure through percutaneous, mucosal, or dermal routes can acquire hbv infection. the risk associated with accidental needle-stick inoculation of infected blood to susceptible healthcare personnel varies between 5% to 35%, depending on the hepatitis b e antigen (hbeag) status, and hence the viral titer of the source. in up to 50% of occupational infections, a discrete exposure cannot be identified. hepatitis b has an incubation period of 40-180 days. the period of infectivity precedes the development of jaundice by 2-7 weeks and correlates with the presence of hepatitis b surface antigen (hbsag) in the serum; 5-10% of persons with acute (but often clinically silent) infection develop chronic antigenemia. in the united states, up to 1% of adults are carriers of hbv, and provide a reservoir for maintenance of the disease in the population. apparent hepatitis in about one-third of acutely infected adults. clinical hepatitis may be preceded by a prodrome of fever, malaise, urticarial or maculopapular rash, and arthralgias for several days. fever usually resolves before the onset of jaundice. jaundice, dark urine, and scleral icterus usually are present by the time patients seek medical attention. right upper quadrant tenderness, mild hepatic enlargement, and occasionally, splenomegaly are signs that should suggest the diagnosis. the most striking laboratory abnormality is the finding of extreme elevations in the aminotransferase enzymes. alanine aminotransferase (alt) and aspartate aminotransferase (ast) may be elevated to more than 10 times the normal levels, whereas the bilirubin and alkaline phosphatase levels are increased to a much lesser extent. fulminant liver involvement occurs in about 1% of adults and may be complicated by more serious abnormalities, including hypoglycemia, coagulopathy, and hypoalbuminemia. hepatic encephalopathy, hepatorenal syndrome, and bleeding diatheses are life-threatening complications seen in these patients. about 10% of adults with clinically apparent acute hbv infection proceed to chronic hbs-antigenemia, and are at risk for chronic hepatitis, postnecrotic cirrhosis, and primary hepatocellular carcinoma. patients with asymptomatic primary hbv infection are at higher risk for chronic infection than those with symptomatic infection. while chronic persistent hepatitis, a benign illness of little clinical consequence except for the potential for hbv transmission to susceptible individuals, may occur, the major health concern is chronic active hepatitis, which eventually may produce cirrhosis, liver failure, and hepatoma. hepatitis b is differentiated from other causes of hepatitis by serologic assays. 23 a positive hepatitis b surface antigen (hbsag) test identifies patients with current infection and correlates with infectivity during acute and chronic infection. titers of hbsag in the chronic phase of illness may wax and wane and occasionally fall below the limits of laboratory detection, so sequential testing should be performed if chronic hbv is suspected. the presence of hbeag correlates with active virus replication and is a marker of high infectivity and high titer of hbv in the liver and blood. antibody to hepatitis b surface antigen (hbsab) appears when hbsag is cleared and is positive in individuals with immunity after recent or prior infection or immunization. persons with hbsab are not susceptible to acute infection or chronic hepatitis b, except in the very rare case in which reinfection occurs with a strain of hepatitis b against which the normal antibody response does not provide cross-protection. hepatitis b core antibody (hbcab) appears before hbsab and just after hbsag is cleared from the serum, and this is a useful test for diagnosing acute hepatitis b in the window period before hbsab appears. high titers of hbcab persist in chronically infected persons and obviously do not predict immunity from further liver disease. there currently is no treatment for acute hepatitis b. alpha interferon and lamivudine have been licensed for the treatment of persons with chronic hepatitis b. these drugs are effective in up to 40% of cases. hbv infection is largely preventable. inoculation with recombinant vaccines containing hbsag components is safe and highly immunogenic, and appears to confer protection from infection for at least 12 years (table 22. 2). postvaccination testing should be done 1-2 months after completion of the three-dose series to document an appropriate response (i.e., > 10 miu/ml). 23 more than 90% of persons immunized with three properly timed doses (e.g., 0, 1, and 6 months) of vaccine administered intramuscularly in the deltoid region develop protective hbsab levels. factors associated with a lack of response include improper vaccination (improperly stored vaccine, gluteal inoculation, subcutaneous injection), obesity, older age, and smoking. persons who do not respond to the primary vaccine series should receive a second three-dose series or be evaluated for hbsag positivity. since 1982, substantial progress has been made toward eliminating hbv transmission in children and reducing the risk for hbv infection in adults. 24 recommendations of acip have evolved from universal childhood vaccination, to prevention of perinatal hbv transmission, vaccination of adolescents and adults in high-risk groups, and catch-up vaccinations for susceptible children in high-risk populations. 25, 26 the acip vaccination strategies for children and adolescents have been implemented successfully in the united states, and routine immunization of all children is now recommended. the occupational safety and health administration's (osha's) blood-borne pathogen standard mandates provision of vaccine at no cost to all healthcare employees and others at occupational risk for blood exposure. 27 substantial declines in the incidence of acute hepatitis b have occurred among highly vaccinated populations, such as young children and healthcare personnel. vaccine should also be provided to susceptible individuals before sexual maturity, particularly to teenagers in those settings (e.g., inner cities, concentration of poverty) where hbv is highly prevalent, and to all adults at risk for sexual or occupational exposure. preimmunization screening for evidence of prior or persistent infection usually is not cost effective. however, postimmunization testing for antibody response is recommended 1-2 months after the 3 rd dose to detect nonresponders among persons at high risk for exposure. titers of hbsab fall over time and may be undetectable after 5-10 years. the duration of vaccine protection is under investigation. most data suggest that protection persists even when hbsab titers fall below the level of detection, and routine screening and boosting are not recommended. the need for prophylaxis for persons sustaining accidental percutaneous or mucosal exposures to blood should be based on several factors, including the hbsag status of the source, and the hepatitis b vaccination and vaccineresponse status of the exposed person. such exposures usually involve persons for whom hepatitis b vaccination is recommended. any blood or body fluid exposure to an unvaccinated person should lead to initiation of the hepatitis b vaccine series. a summary of prophylaxis recommendations for percutaneous or mucosal exposure to blood according to the hbsag status of the exposure source and the vaccination and vaccine-response status of the exposed person is shown in table 22 .4. 23 when hepatitis b immune globulin (hbig) is indicated, it should be administered as soon as possible after exposure (preferably within 24 hours). the effectiveness of hbig when administered more than 7 days after exposure is unknown. when hepatitis b vaccine is indicated, it § hepatitis b immune globulin; dose is 0.06 ml/kg intramuscularly. ¶ a responder is a person with adequate levels of serum antibody to hbsag (i.e., anti-hbs ≥10 miu/ml). ** a non-responder is a person with inadequate response to vaccination (i.e., serum anti-hbs <10 miu/ml). † † the option of giving one dose of hbig and reinitiating the vaccine series is preferred for non-responders who have not completed a second three-dose vaccine series. for persons who previously completed a second vaccine series but failed to respond, two doses of hbig are preferred. § § antibody to hbsag. should also be administered as soon as possible (preferably within 24 hours) and can be administered simultaneously with hbig at a separate site (vaccine should always be administered in the deltoid muscle). for exposed persons who are in the process of being vaccinated but have not completed the vaccination series, vaccination should be completed as scheduled, and hbig should be added as indicated (table 22 .4). persons exposed to hbsag-positive blood or body fluids who are known not to have responded to a primary vaccine series should receive a single dose of hbig and reinitiate the hepatitis b vaccine series with the first dose of the hepatitis b vaccine as soon as possible after exposure. alternatively, they can receive two doses of hbig, one dose as soon as possible after exposure, and the second dose 1 month later. the option of administering one dose of hbig and reinitiating the vaccine series is preferred for non-responders who did not complete a second three-dose vaccine series. for persons who previously completed a second vaccine series but failed to respond, two doses of hbig are preferred. 23 states. it is estimated that 1.8% of americans have been infected with hcv. hcv-associated end-stage liver disease is the most frequent indication for liver transplantation among us adults. 28 the incubation period for acute hcv infection ranges from 2 to 24 weeks (averaging 6-7 weeks). hcv transmission occurs primarily through exposure to infected blood, such as through injection drug use, blood transfusion, solid organ transplantation from infected donors, unsafe medical practices, occupational exposure to infected blood, and birth to an infected mother (i.e., vertical transmission). hcv may also be acquired through sexual contact, but the importance of this mode of transmission in the united states is not well characterized. hcv is not transmitted efficiently through occupational exposures to blood. healthcare personnel who are parenterally exposed to infected blood through needlestick injuries may acquire hcv infection, but the magnitude of risk (approximately 2 in 100 hcv needlesticks) is less than that associated with hbv exposure. one epidemiologic study indicated that transmission occurred only from hollow-bore needles compared with other sharps. transmission rarely occurs from mucous membrane exposures to blood, and no transmission in hcv has been documented from skin exposures to blood. data are limited on survival of hcv in the environment. in contrast to hbv, the epidemiologic data for hcv suggest that environmental contamination with blood containing hcv is not a significant risk for transmission in the healthcare setting, with the possible exception of the hemodialysis setting where hcv transmission related to environmental contamination and poor infection control practices have been implicated. the risk for transmission from exposure to fluids or tissues other than hcv-infected blood also has not been quantified but is expected to be low. hcv is not known to be transmissible through the airborne route, through casual contact in the workplace, or by fomites. clinical syndromes hepatitis c virus infection produces a spectrum of clinical illness similar to hbv and is indistinguishable from other forms of viral hepatitis based on clinical symptoms alone. serologic tests are necessary to establish a specific diagnosis of hepatitis c. most adults acutely infected with hcv are asymptomatic. after acute infection, 15-25% of persons appear to resolve their infection without sequelae as defined by sustained absence of hcv rna in serum and normalization of alt levels. 28 chronic hcv infection develops in most persons (75-85%), with persistent or fluctuating alt elevations indicating active liver disease developing in 60-70% of chronically infected persons. no clinical or epidemiologic features among patients with acute infection have been found to be predictive of either persistent infection or chronic liver disease. moreover, various alt patterns have been observed in these patients during follow-up, and patients might have prolonged periods (greater than or equal to 12 months) of normal alt activity even though they have histologically confirmed chronic hepatitis. thus, a single alt determination cannot be used to exclude ongoing hepatic injury, and long-term follow-up of patients with hcv infection is required to determine their clinical status and prognosis. the course of chronic liver disease is usually insidious, and progresses slowly without symptoms or physical signs in the majority of patients during the first two or more decades after infection. chronic hepatitis c frequently is not recognized until asymptomatic persons are identified as hcv positive during blood donor screening, or elevated alt levels are detected during routine physical examinations. most studies have reported that cirrhosis develops in 10-20% of persons with chronic hepatitis c over a period of 20-30 years, and hcc in 1-5%, with striking geographic variations in rates of this disease. however, when cirrhosis is established, the rate of development of hcc might be as high as 1-4% per year. longer follow-up studies are needed to assess lifetime consequences of chronic hepatitis c, particularly among those who acquired infection at young ages. although factors predicting severity of liver disease have not been well defined, recent data indicate that increased alcohol intake, being aged greater than 40 years at infection, and being male are associated with more severe liver disease. in particular, among persons with alcoholic liver disease and hcv infection, liver disease progresses more rapidly; among those with cirrhosis, a higher risk for development of hcc exists. in addition, persons who have chronic liver disease are at increased risk for fulminant hepatitis a. screening enzyme immunoassay (eia) and supplemental confirmatory immunoblot tests are licensed and commercially available to detect antibodies to hcv (anti-hcv). anti-hcv may be detected within 5-6 weeks after the onset of infection but a single anti-hcv test cannot distinguish between acute, chronic, or past infection. hcv rna can be detected within 1-2 weeks of exposure to the virus and several weeks before elevations of alt and detection of anti-hcv. testing for anti-hcv by eia is recommended 4-6 months after an exposure to detect infection; testing for hcv rna may be performed 4-6 weeks after exposure if earlier detection of infection is desired. 23 treatment, prevention, and control hcv-positive patients should be evaluated for the presence and severity of chronic liver disease. initial evaluation for presence of disease should include multiple measurements of alt at regular intervals, because alt activity fluctuates in persons with chronic hepatitis c. patients with chronic hepatitis c should be evaluated for severity of their liver disease and for possible treatment. alpha interferon (with or without ribavirin) treatment of hcv appears to prevent hcv replication, decrease hepatic inflammation, and improve symptoms among chronically infected persons. persons with chronic hcv have undergone successful liver transplantation, although recurrences have been documented in this setting. antiviral therapy is recommended for patients with chronic hepatitis c who are at greatest risk for progression to cirrhosis. these persons include anti-hcv-positive patients with persistently elevated alt levels, detectable hcv rna, and a liver biopsy that indicates either portal or bridging fibrosis or at least moderate degrees of inflammation and necrosis. therapy for hepatitis c is a rapidly changing area of clinical practice and consultation with a knowledge specialist (e.g., hepatologist) is recommended. 29 no clinical trials have been conducted to assess postexposure use of antiviral agents (e.g., interferon with or without ribavirin) to prevent hcv infection, and antivirals are not fda approved for this indication. available data suggest that an established infection might need to be present before interferon can be an effective treatment. 23, 29 because there is currently no postexposure prophylaxis (pep) for hcv, the intent of recommendations for postexposure management is to achieve early identification of infection and, if present, referral for evaluation of treatment options. in addition, no guidelines exist for administration of therapy during the acute phase of hcv infection. however, limited data indicate that antiviral therapy might be beneficial when started early in the course of hcv infection. when hcv infection is identified early, the person should be referred for medical management to a specialist knowledgeable in this area. 23 at present, avoidance of parenteral exposure to blood is the only available strategy for preventing hcv infection. epidemiology it is estimated that more than 60 million persons worldwide had been infected by hiv and that 40 million were living with hiv/aids by the end of the year 2000. 30 in the united states, almost 1 million persons are living with hiv. 30 most individuals with hiv infection are active adults employed in the workforce. the primary means of acquiring infection among adults is either through behaviors such as unprotected homosexual or heterosexual intercourse with an infected partner, involving the exchange of body fluids, or injecting drug use involving shared needles and syringes. the virus also is perinatally transmitted to approximately 20-40% of children born to infected mothers, (e.g., vertical transmission). breastfeeding is a bidirectional mode of transmission, to nursing infants of infected mothers and, rarely, to mothers of nursing infants when nipple maceration and biting occur. since 1985, all donated blood in the united states has been screened for hiv infection. the risk of hiv infection due to transfusion of blood products screened by current methods is estimated to be 1 in 1,900,000 units transfused. 32 screening does not completely eliminate the potential for a seronegative but infected unit from a recently infected donor to escape detection. hiv is not transmitted by the air-borne route, by household or workplace contact with infected persons, by exposure to contaminated environmental surfaces, or by insect vectors. the virus is easily inactivated by most common disinfectants, including household bleach (diluted 1:100). commercial sex workers are at the greatest risk of acquiring hiv infection occupationally. the other group of workers at risk for acquiring hiv infection occupationally is healthcare personnel. healthcare providers and other workers in contact with blood or other body fluids who sustain accidental percutaneous or mucosal inoculations with virus-infected material are at risk for infection. the magnitude of risk depends on the severity of exposure, but on the average, about 1 in 300 hiv needlesticks results in infection. the risk for infection following mucosal exposures is estimated to be lower at approximately 0.09%. in the absence of direct exposure, healthcare providers are not at occupational risk for hiv infection. in the united states, through december 2002, there have been 57 cases of occupationally acquired hiv infection reported with an additional 139 possible cases. 32 clinical syndromes the clinical course of hiv infection is variable and changing with the advent of antiretroviral therapy, as well as treatment and prophylaxis for infectious complications. early after infection, within a few weeks to months, an acute febrile illness characterized by malaise, pharyngitis, lymphadenopathy, maculopapular rash, and headache may occur. the frequency of this mononucleosis-like illness has varied widely in reports of seroconverting individuals. at initial presentation of such patients, hiv antibody screening tests (enzyme immunoassay (eia)) may be negative, but viral antigen (p24 antigen) and serologic reactivity to one or more viral components (western blot test) allows the diagnosis to be established at this stage. hiv infection should be suspected in any person with a mononucleosis syndrome lacking a positive heterophil antibody response (monospot test). following initial infection, most persons have generalized asymptomatic lymphadenopathy and appear well. however, laboratory tests document a gradual decline in the number of circulating t-helper lymphocytes (cd4 cells), beginning soon after infection and continuing over the next several years. t-helper cells are essential components of the immune system and mediate aspects of both cellular and humoral immunity. symptoms, signs, and illness suggestive of mild to moderate immunodeficiency appear after about 5 years, when cd4 cells decrease by about 50%, to less than 500 cells/dl. intermittent fever, oral thrush, bacterial pneumonia, enteric infections, and reactivated tb are typically diagnosed at this time. signs suggestive of more rapid deterioration include oral hairy leukoplakia (a wart-like white growth in the oral cavity), shrinking lymphadenopathy, fever, weight loss, and elevated erythrocyte sedimentation rate. when cd4 cell counts fall below 200, serious opportunistic infections can be anticipated. pneumocystis pneumonia (pcp) was the most common index diagnosis in the first 5 years of the epidemic, but the advent of effective pcp prophylaxis has altered the picture. other opportunistic infections and malignancies, including kaposi's sarcoma, lymphoma, disseminated tb, toxoplasmosis, and cryptococcal meningitis, now account for the majority of index hiv diagnoses. with the exception of tb, the infectious complications of hiv infection generally are not transmissible to healthy individuals and pose no risk in the workplace. indeed, the causative organisms are ubiquitous and most adults have already been exposed. opportunistic infections in hivinfected patients usually represent reactivation of dormant organisms when the immune system can no longer keep them inactive. be offered to all patients with symptoms ascribed to hiv infection. 34 recommendations for offering antiretroviral therapy in asymptomatic patients require analysis of many real and potential risks and benefits. information regarding treatment of acute hiv infection from clinical trials is very limited. ongoing clinical trials are addressing the question of the long-term clinical benefit of potent treatment regimens for primary infection. in general, treatment should be offered to individuals with fewer than 350 cd4 t cells/mm or plasma hiv rna levels exceeding 55,000 copies/rnl (by rt-pcr or bdna assay). once the decision has been made to initiate antiretroviral therapy, the goals should be maximal and durable suppression of viral load, restoration and/or preservation of immunologic function, improvement of quality of life, and reduction of hivrelated morbidity and mortality. 33 hiv-infected individuals found to have latent tb infection should be treated with antituberculous therapy to prevent activation of disease. 13 persons at risk for direct contact with blood and other potentially infected materials should receive specific instruction in universal/standard precautions for infection control, as recommended by the centers for disease control and prevention 34, 35 and mandated by the occupational safety and health administration. 27 for most environments outside healthcare settings, common sense and attention to personal hygiene are adequate to protect workers. gloves should be worn to clean up visible sites of blood contamination. environmental surfaces can then be decontaminated with disinfectant solutions or household bleach (diluted 1:100). 34, 36 individuals sustaining accidental parenteral exposures to hiv should be counseled to undergo baseline and followup testing for 6 months after exposure (e.g., 6 weeks, 3 months, and 6 months) to diagnose occupational infection. postexposure chemoprophylaxis with antiretroviral agents has been recommended by the us public health service since 1996 after certain exposures to hiv-infected sources which pose a risk of infection transmission, such as needlesticks, mucous membrane, and non-intact skin exposures (tables 22.5 and 22.6). data from animal models of prophylaxis with these agents suggest that antiviral activity is diminished when treatment is delayed for more than 24 hours. for this reason, immediate reporting and access to chemoprophylaxis is recommended. occupational exposure is a frightening experience. consultation with clinicians knowledgeable about hiv transmission risks who can provide supportive counseling to the worker is essential during the follow-up interval. cdc recommends that occupationally exposed workers refrain from unsafe sexual practices, pregnancy, and blood and organ donation for 6 months after exposure to minimize the risk of transmission. zoonoses are infections that are maintained in nature by transmission between vertebrate animals, and they can be transmitted from other vertebrates to humans or from humans to other vertebrates. zoonotic pathogens can be divided into two major groups: (1) those transmitted primarily among wild animals (e.g., yersinia pestis, rabies), and (2) those transmitted primarily among domestic animals (e.g., sporothrix schenkii, non-typhoid salmonella spp.). other infections not properly classified as zoonoses can result from working directly with animals (e.g., infected wounds resulting from animal bites) or with animal products (e.g., anthrax in carpet weavers). many zoonotic infections present occupational risks, not only to those who work with live or dead vertebrate animals or animal products but also to workers exposed to certain environments contaminated by animals or animal products. thus, workers in veterinary medicine, animal husbandry, and animal research are at risk for acquiring a host of zoonotic infections specific to the type of live animal exposure, just as those involved in healthcare work with humans are at risk for infections acquired from humans. examples of such zoonotic infections include q fever in veterinarians, psittacosis (caused by chlamydia psittaci) in duck farmers, orf (contagious ecthyma) in shepherds, lymphocytic choriomeningitis (e.g., leptospirosis) in laboratory workers who handle rodents, fatal herpes virus simiae infection in primate handlers, and more recently, monkeypox in veterinarians and pet store owners. 37, 38 influenza a (h5nl) (avian flu) infection was shown to have been transmitted from ducks and chickens to poultry workers in hong kong and has become an important source of epidemic infection in various international settings; 39 lyssavirus (related to rabies virus) infections have been transmitted from bats to humans in australia, 41 and a large outbreak of febrile encephalitic and respiratory illnesses among workers who had exposure to pigs was shown to be due to infection with a previously unrecognized paramyxovirus (formerly known as hendra-like virus, now called nipah virus). 41 brucellosis is an example of a zoonotic infection in abattoir workers exposed to live or dead animals or animal products. examples of zoonotic infections acquired by workers exposed to environments harboring or contaminated by contagious animals include leptospirosis in rice field workers, and argentine hemorrhagic fever typically acquired by adult males harvesting corn in cornfields inhabited by rodents, which serve as the reservoir for junin or machupo virus. it is beyond the scope of this chapter to review the large number of zoonoses (about 200 have been described) that could pose a risk to workers in unique jobs that involve contact with various animals or environments. for each type of occupation that involves regular animal contact, it is important to recognize the types of infectious disease risks involved, consider baseline studies and storage of serum for future serologic tests if risks are high, plan preventive measures when possible, and prepare for early diagnosis and treatment of such infections when illness occurs. 42 some of the zoonoses, the occupational groups they affect, and their clinical presentations are included in table 22 .1. infectious diseases continue to emerge, posing threats to the health of workers in numerous settings. a prime example of such a threat is severe acute respiratory syndrome or sars, first identified in early 2003 and responsible for illness and death primarily among exposed healthcare personnel. 43 emerging infectious issues which may prove to be challenges for occupational health include those posed by bioterrorism, biotechnology, 44 and emerging and reemerging infections. these emerging infections emphasize the need for continued vigilance and for careful history taking about occupational exposures when evaluating individuals for illnesses that could possibly be occupationally acquired. timeliness in identification and reporting of cases assists in the accurate estimation of the magnitude of the infectious disease problem and in the development of additional preventive and therapeutic measures. screening of employees for infection with or susceptibility to infectious diseases is an important part of healthcare maintenance, especially when the occupational setting poses a significant risk of transmitting or acquiring infections. screening also is warranted if specific interventions are available to prevent disease transmission among workers. assessment of behaviors, such as smoking, that increase the risk of acquiring infections also is valuable so that employees can be provided educational and other interventions to modify risks. preventing infectious diseases in workers can decrease absenteeism and financial costs associated with disability, sick leave, and health insurance, even if the primary source of infection is non-occupational. attending to these issues at the time of employment obviates the need for ongoing surveillance of many infections and simplifies outbreak investigations by documenting the pre-exposure immune status of contacts. tst screening for active disease identifies those persons who would benefit from prophylaxis (tables 22.5 and 22.6). 14 tb vaccination with bacillus of calmette-guerin (bcg) vaccine, a live attenuated strain of m. bovis, is provided for children and some workers in most european countries, but it is not recommended in the united states because of its unproven efficacy when used in adults and because it induces dermal hypersensitivity to purified protein derivative (ppd) tuberculin in most recipients, impeding the usefulness of tst as a screening tool. persons age 65 years and older, persons with chronic diseases or pulmonary disorders, and healthcare personnel should be offered pneumococcal vaccine and annual influenza vaccine (table 22. 3). 45 rubella immune status should be ascertained and men and women immunized in settings where women of childbearing age are employed (table 22. 3). even though rubella is not often transmitted in the workplace, outbreaks can occur among susceptible individuals and vaccination is an important public health intervention. medical personnel should demonstrate proof of rubella immunity or vaccination prior to patient contact. measles vaccine should be provided to all workers born after 1957 with no documented history of measles who have not received two injections of live virus vaccine (table 22. 3). screening for immunity to varicella and mumps is not routinely recommended, except for healthcare providers and adults with no history of infection with these agents who are exposed to young children. all adults require tetanus immunization. tetanus diphtheria toxoid (td) boosters should be administered every 10 years to adults who have completed primary immunization (table 22. 3). employees with no prior history of tetanus diphtheria immunization or with uncertain histories should receive a series of three primary vaccine injections. 45 similarly, adults with no history of polio immunization should undergo primary immunization with inactivated polio vaccine, especially if they are employed in healthcare settings or when travel to endemic areas is anticipated. persons employed in occupations that pose a risk for parenteral contact with blood and other body fluids should be offered hepatitis b immunization (table 22. 3). healthcare personnel, laboratory workers, animal handlers, first responders, and personal service workers such as barbers, tattooists, and cosmetologists are included in this category. adults with multiple sexual partners also should be encouraged to undergo immunization. serum banking to allow documentation of baseline serostatus is useful for laboratory and healthcare personnel at risk for other bloodborne infections such as hiv or more exotic infections. laboratory workers and animal handlers may be at risk for unusual infectious diseases. q fever, a rickettsial disease transmitted by the air-borne route, is a special risk encountered by handlers of sheep and similar animals. serologic testing for q fever titers prior to occupational exposure is important to document baseline status and to detect seroconversion at follow-up testing. although smallpox vaccine is no longer recommended routinely, genetically engineered vaccines prepared from vaccinia may pose a risk to researchers and clinicians treating patients enrolled in vaccine trials. laboratory workers who handle vaccinia or recombinant vaccinia preparations in culture or in animals should receive vaccinia vaccine. healthcare personnel caring for patients immunized with vaccinia or other orthopoxviruses or tissues and specimens from patients with these infections also should be immunized. a program for smallpox vaccination for selected individuals who may be in the frontline for responding to a bioterrorist attack has recently been initiated in the united states. 46 consultation should be obtained to determine the need for screening, immunization, and testing for other exotic infections. some animal handlers are at risk of acquiring rabies through bites or exposure to infected secretions and tissues. immunization with human diploid cell vaccine (three 1-ml intradermal doses on day 0, 7, and 21 or 28) should be provided to workers at risk for rabies and for persons traveling for more than 1 month to areas where rabies is endemic (table 22. 3). booster injections should be provided every 2 years for those with continuing exposure. surveillance of infectious diseases is conducted to detect increased occurrence of disease so that preventive interventions can be initiated. surveillance can be passive (based on employee health consultations or reports from contractual providers or supervisors) or active (actual monitoring of disease occurrence). active surveillance for infectious diseases is not required in most occupational settings. in work environments where exposure to m. tuberculosis may occur -such as healthcare settings, residential care facilities, shelters, and correctional facilities -active tst surveillance among susceptible individuals is indicated. periodic tsts are especially important in the wake of several recent outbreaks associated with drug-resistant strains of m. tuberculosis in hospitals, adult care settings, and home healthcare settings. 16 skin testing should be performed at least annually in these settings, and perhaps as often as every 6 months, for personnel at high risk for exposure to active tb. surveillance of teachers, travelers to endemic areas, and employees in other institutional settings where close contact with infected individuals is possible also may be warranted, depending on the local prevalence of tb. 14 surveillance for infections among laboratory workers and animal handlers exposed to specific pathogens should be individualized in accordance with standard guidelines for biosafety in microbiologic and biomedical laboratories. 47 maintaining standardized records of reportable infectious diseases is an important component of passive surveillance in the workplace. centralized collection and assessment of these records at regular intervals may allow early detection of outbreaks of occupational infections amenable to specific control interventions. geographic or temporal clusters of cases or clustering among persons with similar attributes or occupational tasks suggest a common source of exposure and infection and warrant investigation. local public health officials and regulatory agencies should be consulted promptly when an outbreak is initially suspected. reporting of occupationally acquired infections permits public health agencies to identify clusters of old and emerging illnesses and ultimately prevent them. these events should be reported as mandated by state and local regulations. 48, 49 return-to-work criteria employees diagnosed with communicable infectious diseases should not return to work until the period of infectivity is past. specific guidelines should be consistent with local public health regulations. some workers, for example food handlers with certain diarrheal illnesses, cannot resume their duties until culture evidence of cure is obtained. employees should be advised of the return-towork policies at the time of employment and when illness is diagnosed. a table for length of work restriction for healthcare personnel can be used to guide return-to-work policies for the workplace (table 22. 2). 19 common sense dictates attention to personal hygiene among all workers. hand washing after using the bathroom and before handling food is essential. the mouth should be covered while sneezing or coughing, and soiled tissues and dressings should be disposed of in trash containers. employers have a responsibility to minimize crowding in the work setting. facilities for hand washing should be available in bathrooms and food preparation areas. proper ventilation also is important. trash should be emptied at regular intervals, and work areas should be clean and free of pests. smoking should be prohibited in common work areas. spills of blood, body fluids, and other potentially infectious substances should be removed with disposable paper towels or other suitable procedures. contaminated areas should then be disinfected with commercial products or with a solution of household bleach (diluted 1:100). 34, 36 infection control in healthcare settings infection control programs in healthcare settings are necessary to prevent transmission of healthcare-related infections to patients and healthcare personnel. the cdc has established a two-tiered system of infection control precautions. 16 the first tier consists of 'standard precautions' which are precautions recommended for delivery of care to all patients regardless of diagnosis or presumed infection status. they are designed to limit exposure to blood or other body substances and include elements such as hand hygiene and use of appropriate protective barriers, e.g., masks, eye protection, and gloves, as needed to prevent direct contact. the second tier of precautions recommended by cdc are 'transmission-based precautions', designed for the management of patients known or suspected to be infected with pathogens whose transmission can be limited by the adoption of additional measures beyond those which are part of standard precautions. they apply to pathogens transmitted by the air-borne or aerosol routes, droplets, and by direct and indirect contact. respiratory precautions are employed for patients with infections communicable by the air-borne route. such patients are housed in private rooms with special ventilation and should wear surgical masks when leaving their rooms. respiratory protection (i.e., n-95 respirators) also are advised for providers in close contact with patients on respiratory precautions. however, the re-emergence of epidemic and mdr-tb has led to a re-emphasis of other fundamentals for prevention of transmission of tuberculosis in healthcare and other settings. early identification of tb allows early indication for therapy, and requires alertness in considering tb in high-risk patients with pulmonary symptoms, especially those with hiv infection. special ventilation measures and respiratory protection are especially important for cough-inducing procedures, such as sputum induction and aerosolized pentamidine administration. healthcare personnel who have the potential for being exposed to m. tuberculosis should be screened on employment and at least annually thereafter by ppd skin testing, comparing previous test results to current results to identify those who have converted to skin test positivity. 15 procedures for disposal of infectious wastes have been developed by the cdc. 36 needles and other sharp objects should be sterilized prior to disposal. liquid and laboratory wastes may be dumped into sewage systems. materials heavily contaminated with bacteria or blood should be placed in special bags that are specifically labeled for infectious waste and should be disposed of according to community standards for such materials. 36 employers have a responsibility to educate employees about infection control. barriers to prevent exposure, including masks, gowns, eye protection, and gloves, should be readily available to workers at risk. hand washing facilities and hand hygiene supplies are essential. where access to sinks or running water is not feasible, alcohol hand rubs or packaged towels containing disinfectants should be provided. impervious containers for the disposal of needles and other sharp objects are essential. 50 such containers should be made available on ambulances and provided to home health aides and other visiting healthcare personnel. personal service workers who use needles, razors, and other sharp objects also should have access to safe disposal units. persons receiving care at home who require injections or other procedures that demand the use of needles also should be provided with impervious disposal containers and instructed in proper disposal methods to protect sanitation workers and others in contact with waste. despite improvements in engineering controls, work practices, and personal protective equipment, laboratory personnel are nevertheless at risk for occupationally acquired infections. laboratory personnel may acquire infection by aerosolization of specimens, mouth pipetting, or percutaneous injury or mucocutaneous contact. methods of infection control applicable to laboratory settings are described in the cdc document entitled 'biosafety in microbiological and biomedical laboratories'. 47 by 1995, it was estimated that 14.5 million children were attending out-of-home daycare in a variety of settings including licensed child daycare centers, regulated daycare homes, and unregulated family daycare homes. 51 many serious infections occur as endemic or microepidemic problems in the daycare setting. these include h. influenzae type b, hepatitis a, cytomegalovirus, parvovirus b 19, and enteric infections (shigella, giardia, rotavirus, clostridium difficile, campylobacter, cryptosporidium, calcivirus, salmonella, enteric adenovirus, astrovirus, and several types of e. coli infections). in addition, the high rate of acute respiratory infections leads to early onset of otitis media, frequent antibiotic use, and emergence of multidrug-resistant enteric pathogens. thus, workers in close contact with children risk exposure to a wide variety of communicable pathogens contained in secretions, urine, and stool. all such personnel, as well as all children in schools and daycare centers, should be screened for immunity to common childhood infections and vaccinated if immunity is not present (table 22. 2). regulation of childcare facilities is essential to reduce risks to children and workers. national standards for infection control in childcare facilities were promulgated in 1990. 52 hand washing facilities and policies are the most important component of disease prevention in school and daycare settings. hands should be washed after contact with mucous membranes and potentially infected body fluids. older children should be instructed in personal hygiene. children with fever or diagnosed infections should be excluded from attending daycare or school until transmission risk is no longer present, and policies for such exclusion should be in place. prompt reporting of disease outbreaks and prompt involvement of public health authorities are essential. employees should be instructed in common-sense first aid procedures for handling wounds, bites, and other situations in which exposure to infected blood or tissues is possible. barrier protection is rarely required in schools, but gloves should generally be available for emergencies requiring first aid. infection with a variety of agents during pregnancy has the potential to cause fetal damage, especially when primary infection occurs. while a number of these infections can be community acquired, the likelihood of exposure to certain of these pathogens can be greater in healthcare settings. infections with as rubella, cmv, and parvovirus are among the infectious agents which may be of special concern to pregnant healthcare personnel. in general, adherence to standard precautions as well as preexposure immunizations when available and appropriate are the best way of preventing the devastating effects of such infections (table 22. 2). 19, 21 immunodeficient workers are at increased risk of devastating infections, particularly with opportunistic agents. the greatest risk for such workers is likely in the healthcare setting where there can be ample opportunity for exposure to these agents. many immunocompromising illnesses would be viewed by the us legal system as disabilities and therefore, individuals with those conditions would be covered under the provisions of the americans with disabilities act of 1990 (see chapter 57.1). 53 such persons should be informed about their risks and furthermore, their employers should make reasonable accommodations to allow their employees to continue to perform their jobs, taking into consideration the provisions of applicable federal, state, and local regulations. occupational infectious diseases encompass a large variety of infections which can involve many organ systems. they include some common infections, such as influenza, that pose a special problem in the workplace because of close interpersonal contact and crowding, and that taken together account for a large proportion of time lost from work. many of these infections are preventable by policies that promote hygiene and provide exclusion from work during periods of contagion. in addition, a variety of less common, but sometimes serious, infections are particularly associated with specific occupations. recognition of the types of infection risk associated with specific occupations can, in most cases, lead to effective, often simple steps for primary prevention, as well as opportunities for early diagnosis and treatment. bioterrorismrelated inhalational anthrax: the first 10 cases reported in the united states principles and practice of infectious diseases centers for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) influenza vaccination in long-term-care hospitals reduces the mortality of elderly patients measles, mumps, and rubella -vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of mumps: recommendations of the advisory committee on immunization practices (acip) changing epidemiology of pertussis in the united states: increasing reported incidence among adolescents and adults guidelines for the control of pertussis outbreaks ashrae guideline 12-2000: minimizing the risk of legionellosis associated with building water systems guideline for prevention of nosocomial pneumonia. the hospital infection control practices advisory committee progressing toward tuberculosis elimination in low-incidence areas of the united states: recommendations of the advisory council for the elimination of tuberculosis centers for disease control and prevention. targeted tuberculin testing and treatment of latent tuberculosis infection the role of bcg vaccine in the prevention and control of tuberculosis in the united states: a joint statement by the advisory council for the elimination of tuberculosis and the advisory committee on immunization practices guidelines for preventing the transmission of tuberculosis in health-care facilities guideline for isolation precautions in hospitals. the hospital infection control practices advisory committee prevention of varicella: update recommendations of the advisory committee on immunization practices (acip) epidemiology and prevention of vaccine-preventable diseases guideline for infection control in healthcare personnel guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force infection control precautions for the pregnant healthcare worker. bailliere's risk and management of bloodborne infections in healthcare workers updated us public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis achievements in public health: hepatitis b vaccination -united states hepatitis b virus: a comprehensive strategy for eliminating transmission in the united states through universal childhood vaccination: recommendation of the immunization practices advisory committee (acip) protection against viral hepatitis: recommendations of the immunization practices advisory committee (acip) occupational safety and health administration, department of labor. 29 cfr part 1910.1030, occupational exposure to bloodborne pathogens; final rule recommendations for prevention and control of hepatitis c virus (hcv) infection and hcv-related chronic disease national institutes of health consensus development conference panel statement. management of hepatitis c an unequal epidemic in an unequal world the risk of transfusion-transmitted viral infections surveillance of healthcare personnel with hiv/aids, as of panel on clinical practices for the treatment of hiv infection. guidelines for the use of antiretroviral agents in hiv-infected adults and adolescents. us department of health and human services recommendations for prevention of hiv transmission in healthcare settings update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis b virus, and other bloodborne pathogens in health-care settings guideline for environmental control in healthcare facilities centers for disease control and prevention. multistate outbreak of monkeypox -illinois risk of influenza a (h5n1) infection among poultry workers, hong kong, 1997-1998 emerging viral diseases: an australian perspective outbreak of hendra-like virus -malaysia and singapore, 1998-1999 risks and prevention of nosocomial transmission of rare zoonotic diseases sars -looking back over the first 100 days occupational standards for the protection of employees in biotechnology centers for disease control and prevention. general recommendations on immunization: recommendations of the advisory committee on immunization practices (acip) and the american academy of family physicians (aafp) recommendations for using smallpox vaccine in a pre-event vaccination program: supplemental recommendations of the advisory committee on immunization practices (acip) and the healthcare infection control practices advisory committee (hicpac) us department of health and human services centers for disease control. case definitions for public health surveillance mandatory reporting of infectious diseases by clinicians. mandatory reporting of occupational diseases by clinicians selecting, evaluating, and using sharps disposal containers. dhhs (niosh) publication no. 97-111 child care arrangements for preschoolers by family characteristics: fall national standards for infection control in out-ofhome child care americans with disabilities act of 1990, 104 stat. 327, 42 u.s.c. sec. 12101 et seq immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infections control practices advisory committee (hicpac) known responder ¶ known non-responder** antibody response unknown hbig § × 1 and initiate hepatitis b vaccine series no treatment hbig x 1 and initiate revaccination or hbig × 2 † † test exposed person for anti-hbs: § § if drug resistance is a concern, obtain expert consultation. initiation of postexposure prophylaxis (pep) should not be delayed pending expert consultation, and, because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. § source of unknown hiv status (e.g., deceased source person with no samples available for hiv testing). ¶ unknown source (e.g., splash from inappropriately disposed blood). ** small volume (i.e., a few drops). † † the designation 'consider pep' indicates that pep is optional and should be based on an individualized decision between the exposed person and the treating clinician. § § if pep is offered and taken, and the source is later determined to be hiv negative, pep should be discontinued. ¶ ¶ large volume (i.e., major blood splash). key: cord-304251-dohglrm1 authors: scully, c; samaranayake, lp title: emerging and changing viral diseases in the new millennium date: 2015-08-06 journal: oral dis doi: 10.1111/odi.12356 sha: doc_id: 304251 cord_uid: dohglrm1 most viral infections encountered in resource‐rich countries are relatively trivial and transient with perhaps fever, malaise, myalgia, rash (exanthema) and sometimes mucosal manifestations (enanthema), including oral in some. however, the apparent benignity may be illusory as some viral infections have unexpected consequences – such as the oncogenicity of some herpesviruses and human papillomaviruses. infections are transmitted from various human or animal vectors, especially by close proximity, and the increasing movements of peoples across the globe, mean that infections hitherto confined largely to the tropics now appear worldwide. global warming also increases the range of movement of vectors such as mosquitoes. thus recent decades have seen a most dramatic change with the emergence globally also of new viral infections – notably human immunodeficiency viruses (hiv) – and the appearance of some other dangerous and sometimes lethal infections formerly seen mainly in, and reported from, resource‐poor areas especially in parts of asia, latin america and africa. this study offers a brief update of the most salient new aspects of the important viral infections, especially those with known orofacial manifestations or other implications for oral health care. professor jens pindborg, in a lecture in the 1970s, spoke of the very few viral infections then known, most of which were regarded as relatively trivial, transient and with few or no serious sequelae and which were largely clinical phenomena with few therapies available (scully, 1979) . indeed, most viral infections encountered in resource-rich countries were and still are, usually relatively trivial and transient with perhaps fever, malaise, myalgia, rash (exanthema) and sometimes mucosal manifestationsan enanthema, and these have been well documented (scully and samaranayake, 1992) . gradually, however, the unexpected consequences of some oral viral infections have emerged and been recognised, not without some surprise (scully, 1983) especially the oncogenicity of some herpesviruses (eglin et al, 1983) and human papillomaviruses (hpvs) which we (eglin et al, 1983; maitland et al, 1987; cox et al, 1993 ) and many others (e.g. lind et al, 1986) have explored, culminating in the appreciation of unanticipated transmission routes for some cancers, such as sexual (scully, 2002) . viruses are increasingly appreciated to cause a wide range of human diseases, ranging from acute self-resolving conditions to acute or chronic fatal diseases. effects that arise long after the primary infection can increase the propensity for chronic conditions (e.g. erythema multiforme) or in some, lead to the development of cancers other than oral (herrington et al, 2015) . in addition, other viruses such as hepatitis c virus (hcv) have been implicated in common but diverse oral lesions such as lichen planus/lichenoid lesions and sicca syndrome (baccaglini et al, 2013) , the latter also sometimes arising after some other viral infections (youinou et al, 2005) although the full aetiopathogenesis of sjogren syndrome remains unclear (kivity et al, 2014) . the possible viral associations in many other oral conditions including periodontitis (vincent-bugnas et al, 2013; ambili et al, 2014; contreras et al, 2014; ly et al, 2014) and several conditions of unknown aetiology, however, remain enigmatic, and associations are not necessarily causal. the recent several decades have also seen a most dramatic change with the emergence globally of new viral infectionsnotably human immunodeficiency viruses (hiv)and the appearance also in resource-rich countries, of some other dangerous and sometimes lethal infections hitherto latent, unrecognised or unappreciated in resource-poor areas. rare lethal and other dangerous viral infections were formerly seen mainly in, and reported from, resourcepoor areas of asia, latin america and africa. pandemics of severe acute respiratory syndrome (sars) and influenza viruses (both h5n1 and h1n1), heralded the surge of zoonotic virus outbreaks. laboratory diagnosis of viral infections is traditionally based on the isolation of the virus, detection of viral nucleic acid or antigens, or serological confirmation (generally presence of igm antibodies), and it is clear that the rate of discovery of new viruses is accelerating, due to a combination of true emergence of new pathogens, increasing awareness and the advance of new mainly molecular biology technologies making rapid detection and characterisation possible (wang, 2011) . various respiratory viruses and ebola virus disease (evd) in particular have served to awaken humanity to the related social inequalities and challenges. this study discusses the importance of emergent new infections and recent findings in relation mainly to those viral infections that may manifest orally or may affect oral health care. fuller descriptions of ebola virus, marburg virus, coronaviruses, nipah virus, noroviruses, enteroviruses, hiv, measles, mumps, respiratory syncytial virus (rsv), influenza, cytomegalovirus (cmv), varicella zoster virus (vzv), epstein-barr virus (ebv), hpvs and kaposi's sarcoma-associated herpesvirus (kshv) are available elsewhere (e.g. herrington et al, 2015) . it is increasingly evident that there is now an amazing range of viral agents, many unidentified, which may be new or newly recognised or old, re-emerging. risk factors for infection include more risk • from greater exposure, • if defences are down, • if agents evade defences, • generally in warm, moist places. emerging viral diseases have arisen mainly because of increasing and changing air travel habits, but other factors that have also increased exposure to the vectors of these infections include conflicts, displacement and migration, as well as changing climate and vector distribution, changing farming/manufacturing, changing lifestyles such as promiscuity and changing clinical practices (mathis et al, 2015) . transmission of respiratory and enteric viruses is highwhile sexually shared infections (ssi) are of low infectivitygenerally requiring the close apposition of infected mucosae, increased when there is epithelial discontinuity. however, with the existence of transnational sexual networks in many countries including usa (hughes, 2001) and europe, with high rates of migration and travel between, for example, amsterdam, barcelona, berlin, london and paris, there have been ssi outbreaks among sexually exploited people of both genders but especially in hiv-positive men. such close encounters, as well as large gatherings of humans, and/or prolonged periods in confined spaces, can enhance transmission of many agents; this has led to the development of a new area of medicine -'mass gathering medicine (mgm)' (memish et al, 2012) . mass gatherings consist of large numbers of people attending an event at a specific site for a finite time. some of the largest mass gatherings are spiritual in nature but other examples include olympics, rock concerts and political rallies. the public health issues associated with the hajj (the annual muslim pilgrimage to mecca, saudi arabia) is the best reported and has international or even intercontinental implications in terms of infection spread. hajj routinely attracts 2.5 million muslims (memish et al, 2012) , and the unavoidable overcrowding raises the risk of respiratory infections. 'hajj cough' is the most frequently reported but influenza (shafi et al, 2008; haworth et al, 2013) and bacterial meningococcal w135 strains16 are more seriousas are severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronavirusesnew viruses that cause severe acute lower respiratory infections (ari), with 10% and 35% mortality rates, respectively, and >50% mortality rates seen in older and immunosuppressed people (gralinski and baric, 2015) . the past decade has also seen the emergence of several other novel viruses that have appeared in resource-rich countriesoften in travelling peopleincluding an h7n9 influenza a virus, a swine-like influenza h3n2 variant virus and a human adenovirus 14p1 (gautret et al, 2014) . a number of zoonotic and vector borne viral diseases have emerged in south-east asia and the western pacific including japanese encephalitis, barmah forest and ross river viruses. australia sees ross river and barmah viruses now appearing regularly. nipah virus causes limited but deadly epidemics in south-east asia. finally, infections by lyssa viruses, kunjin, murray valley and zika viruses are also emerging (bhutta et al, 2014; carson et al, 2015) . in usa, the centers for disease control and prevention (cdc) recognise such threats and have a mission cri-tical (frieden et al, 2015) which focuses on antibiotic resistance and viral infections as shown in box 1. coronaviruses cause illnesses ranging from the common cold to sars. mers-cov has an especially high mortality and has recently spread widely outside the middle east to asia. dr frieden, director of cdc, had stated 'in this interconnected world we live in, we expected mers-cov to make its way to the united states. we have been preparing since 2012 for this possibility (frieden et al, 2015) '. avian influenza a (h5n1) and a (h7n9) viruses have continued to circulate widely in some poultry populations and infect humans sporadically. sporadic human cases of emerging and changing viral diseases c scully and lp samaranayake avian a(h5n6), a(h10n8) and a(h6n1) have also emerged. closure of live poultry markets in china has reduced the risk of a(h7n9) infection (hui and zumla, 2015) . standard infection control precautions are mandatory to prevent cross-transmission from recognised and unrecognised sources of infection. these sources of (potential) infection include blood and other body fluid secretions or excretions (excluding sweat, non-intact skin or mucous membranes) and any equipment or items in the care environment which are likely to become contaminated (booty et al, 2010) . rna viruses, with their high potential for mutation and epidemic spread, are the most common new viral causes of human disease. most emerging viruses are zoonoses; they have jumped from mammals or birds to humans. the annual rate at which novel viruses have been found remains at 2-3 a small number which is an artefact of inadequate surveillance in resource-poor countries, where even established endemic pathogens are often misdiagnosed. indeed, many of the emerging viruses of the future are already infecting humans (rosenberg, 2015) . more awareness, internet communications, advances in diagnostic technology [high-throughput sequencing and specific polymerase chain reaction (pcr) assays] now help assists recognition. promed-mail is one robust and sensitive mechanism for the discovery of emerging disease outbreaks and for rapid dissemination of information to the public health community (madoff and woodall, 2005) . for example, severe fever with thrombocytopenia syndrome (sfts) was recognised as associated with a novel sfts bunyavirus (sftsv) (bao et al, 2011; jiang et al, 2015) using real-time reverse transcription pcr (rt-pcr), viral culture, genetic sequencing, microneutralisation assay (mna) and indirect immunofluorescence assay (ifa). another example is a new arenavirus related to lymphocytic choriomeningitis viruses that was recognised in a cluster of fatal transplant-associated diseases (palacios et al, 2008) . specific pcr assays showed the virus in the kidneys, liver, blood and cerebrospinal fluid of the transplant recipients. awareness of the infections possible and likely, their epidemiology, and a history of travel, contact and relevant vaccinations is crucial to diagnose an infection, but the history is often overlooked. virus infections typically manifest with a fever, but this is neither always present nor measured by the clinician. clinical features are often non-specific and may include malaise, myalgia, rashes and mucosal lesions such as ulceration or bleeding. these are not always major features, not always checked by a clinician, or may often be seen by non-orally trained healthcare workers. examination conditions in the field may be less than ideal; thus, underdiagnoses or misdiagnoses are likely to be quite common. although evd has been recently discussed fully elsewhere (samaranayake et al, 1996 (samaranayake et al, , 2015 scully et al, 2014) , we provide a brief overview account of its oral manifestations below. the three cardinal oral signs and symptoms of evd are gingival bleeding, mucosal lesions (see below) and pain (odynophagia). other important features typical of early and mild forms of disease, which may help oral health care providers suspect evd, include epistaxis, bleeding from injection sites, conjunctivitis and rash. bleeding is very frequent in advanced forms of evd, but it is only relatively frequent in mild or early evd forms; thus, it is likely that ebola-infected patients seeking care may not show such a sign. typically, gingival bleeding is concomitant with other forms of bleeding, particularly epistaxis and bleeding from injection sites. concurrent bleeding at disparate sites is a discriminatory sign of evd. mucosal lesions such as white or red patches, aphthouslike ulceration and greyish exudative lesions may be seen in evd, but these remain to be more accurately defined. odynophagia (discomfort), the consequence of oedema and mucosal lesions, may range from sore throat to severe dysphagia, when mucosal lesions are ulcerated. ebola virus disease is readily transmitted by body fluids, so universal infection control is essential. there is no reliably effective antiviral against this agent yet available. dengue viruses (denvs) cause denguethe most common arthropod-borne viral disease in humansnow seen in more than 100 tropical countries. the word dengue is obtained from swahili phrase ka-dinga pepo meaning 'cramp-like seizure'. it is also called break-bone fever. dengue virus infection can be asymptomatic or a selflimited, acute febrile disease ranging in severity. the classical form is characterised by high fever, headache, abdominal pain, morbilliform rash, myalgia and arthralgia. in a small proportion of cases, the disease progresses to life-threatening dengue haemorrhagic fever, which results in non-infectious bleeding, thrombocytopenia and leakage of plasma, or dengue shock syndrome. there is no reliably effective antiviral against this agent yet available. in the absence of a vaccine and antiviral drugs, the sole control measure is limiting the mosquito vectors. the main mosquitoes, aedes aegypti and aedes albopictus, are potential vectors of numerous arthropod-borne viruses (arboviruses), and have now expanded from the tropics and subtropics, although a. albopictus still plays a relatively minor role compared to a. aegypti in denv transmission. oral manifestations in dengue are rarely reported; gingival bleeding is the most common (lambrechts et al, 2010; mithra et al, 2013; roopashri et al, 2015) . postoperative haemorrhage (dubey et al, 2013) , gingival and lip swelling (pontes et al, 2014) or mucous membrane involvement with no further details (desruelles et al, 1997) have also been noted. neurological sequelae include hypoglossal palsy (jaganathan and raman, 2014) and taste emerging and changing viral diseases c scully and lp samaranayake changes (scully, 2013) . of interest in this context is that saliva has been attempted to be used as a diagnostic fluid for dengue fever (ravi banavar and vidya, 2014) . chikungunya virus (chikv), a togaviridae family alphavirus has emerged as a worldwide threat, causing fever and devitalising arthritis (the name reflects the condition of many of the stricken, 'bent down or become contorted', in the tanzanian makonda language) in a range of countries including many holiday destinations (hasan et al, 2015) . chikungunya virus is a mosquito-borne virus, like dengue transmitted by aedes mosquitoes and has features of fever, headache, rash, nausea, vomiting, myalgia and arthralgia. the presentation differs in adults and childrenthe latter may present with fever, a rash on the face and arms and intra-oral lesions reported as 'koplik spots' (centers for disease c & prevention, 2014). oral manifestations reported also include ulceration (macdonald-ottevanger et al, 2015) and candidiasis (bandyopadhyay and ghosh, 2010) . thus, chikungunya mimics dengue and like dengue, there is no reliably effective antiviral against this agent yet available. indigenous to tropical africa, recent large chikungunya outbreaks have been reported in south-east asia, the indian ocean islands, the caribbean, and the united states and europe (balkans, france, greece, ireland, italy, madeira, the netherlands and spain), mainly in travellers (kumar et al, 2010) . the rapid spread of a. albopictus into europe and the americas coupled with high viraemia in infected travellers returning from endemic areas increases the risk that chikv could establish itself in new endemic regions (thiboutot et al, 2010) . a mutation in chikv (e1-a226v) appears to improve virus survival in a. albopictus and increase its virulence (burt et al, 2012 ). the majority of virus infections of the oral mucosa are due to the herpes group, which are dna viruses. the classical oral manifestations of these virus infections ranging from herpes simplex, herpes zoster to kaposi`s sarcoma, to infections caused by ebv are adequately described elsewhere (balasubramaniam et al, 2014) . the description below refers to recent developments in this regard. herpes simplex virus infections. herpes simplex virus (hsv) infections are increasingly seen in adults, are sometimes caused by hsv-2 and can be an ssi with stomatitis or pharyngitis (looker and garnett, 2005) . herpes labialis recurrences are now recognised to be significantly common in immune defects and to occur where the innate antiviral immune response involving the interferon (ifn-k) promoter is lacking due to polymorphisms within the ifn-k gene (griffiths et al, 2013) . therapy, despite many studies, still involves antivirals such as aciclovir or penciclovir, but hydrocolloid patches may have a place (karlsmark et al, 2008; stoopler and balasubramaniam, 2013) . herpes simplex virus has long been associated with cranial neuropathies and now has also been associated with alzheimer disease (l€ ovheim et al, 2015) . human papillomavirus infections. one of most common ssi in world, hpv can cause cutaneous or mucosal papillomas, common warts (verruca vulgaris), genital warts (condyloma acuminatum) and multifocal epithelial hyperplasia (heck disease). some hpv types (oncogenic or 'high-risk' hpv types such as hpv16) are now implicated in some mouth cancer, especially oropharyngeal cancer (opc). opc incidence has significantly increased, predominantly in economically developed countries (scully et al, 1988; scully, 2002; chaturvedi et al, 2013; brewer and calo, 2015) . hpv is a strong and independent prognostic factor for better survival of opc (ang, 2010; lowy and munger, 2010; o'rorke et al, 2012) , human papillomaviruses infection does not necessarily lead to opc. a study of 1626 males in brazil, mexico, usa; 1 year showed that 0.4% acquired incident oral hpv, 1.7% acquired oral oncogenic hpv infection and 0.6% acquired oral hpv16 infection. new oral oncogenic hpv infections are rare and most are cleared spontaneously within 1 year (kreimer et al, 2013) . the united states food and drug administration (fda) approved the quadrivalent hpv vaccine for girls in 2006 and for boys in 2011 aimed at genital, hpv-related lesions. vaccination has proved to be successful at preventing hpv and associated cervical and other anogenital tumours. hpv vaccines are effective and also against the hpv strains that are most commonly found in the oropharynx (wierzbicka et al, 2014) and have reduced infections (herrero et al, 2013) , but vaccination is yet to be universally recommended for all adolescents. there are two vaccines; both regarded as safe and usually given as a three dose series. • cervarix: recommended for females from 10 to 25 years of age and protects against hpv16 and hpv18. • gardasil: recommended for 11-and 12 year-old girls and also females 13 to 26-year-old. gardasil is also recommended for 9-to 26-year-old males to protect against some genital warts. this vaccine protects against hpv6, hpv11, hpv16 and hpv18. human parvoviruses. the only known parvovirus to infect humans is b19, which is transmitted by droplets, touch and occasionally in blood, and infects rapidly dividing tissues, most commonly the foetus, intestinal epithelium or haematopoietic system. b19 infection in pregnancy is associated with early foetal loss, although the probability of this is low (<10%). b19 also acutely depresses erythrocyte production, which is of little clinical significance, except in patients with other haematological diseases, particularly sickle cell disease, when haemolytic crises may be precipitated. emerging and changing viral diseases parvovirus commonly causes 'fifth disease' (erythema infectiosum; slapped cheek syndrome), a mild illness with a lace-like rash on the face, trunk and extremities, usually in children. papular-purpuric glove-and-sock syndrome is pruritus, oedema and symmetrical erythema, with a 'gloves-and-socks' distribution and oral blisters, erosions and ulcers; 50% of published cases are related to parvovirus b19 infection (segura saint-gerons et al, 2007) . cranial neuropathies have also been reported (soares-fernandes and mar e, 2006) . in many patients (80%) infected with b19, there is also arthropathy, particularly in adults. as the vaccination induced disappearance of rubella, parvovirus is the commonest cause of infection-related transitory arthritis, particularly if it affects the hands. there is no specific therapy available for parvovirus infections. the new century has seen the emergence of several novel viruses that cause respiratory tract infections in humans including sars coronavirus infection mainly in china, mers-cov in saudi arabia and in asia, an h7n9 influenza a virus in eastern china, a swine-like influenza h3n2 variant virus in the usa and a human adenovirus 14p1 also in the usa. mers-cov and h7n9 viruses are still a major worldwide public health concern, but the pathogenesis and mode of transmission of mers-cov and h7n9 influenza a virus are poorly understood and their oral manifestations, if any, ill-defined (gautret et al, 2014) . there are no reliably effective antiviral agents available for these agents. hand, foot and mouth disease. hand, foot and mouth disease (hfmd) is a common childhood illness characterised by fever and vesicular eruptions on hands and feet and in the mouth. complications are rare, but pneumonia, meningitis or encephalitis may occur. hand, foot and mouth disease is not associated with one single agent; it is caused by members of the family picornaviridae in the genus enterovirus; there are over 100 serotypes of enterovirus species a-d, which are the common cause of hfmd and illnesses in infants, such as meningitis and encephalitis . outbreaks of hfmd have been mainly caused by two types of enterovirus a species, coxsackievirus (cv) a16 (cva16), or enterovirus 71 (ev71), and only sporadic cases involve other members of the enterovirus a species have been reported (osterback et al, 2009 ). most cva16associated infections cause only mild symptoms; however, some cva16 infections can lead to severe complications and even death (chen et al, 2014a) . a chinese study of hfmd showed many patients were infected with cva16, fewer with ev71, some were co-infected with cva16 and ev71, and a few were infected with other enteroviruses. upper respiratory tract infection was significantly higher in cva16-associated patients, while neurological complications and hyperglycaemia were significantly higher in ev71-infected patients (liu et al, 2014) . enterovirus infections remain an important public health problem, and other enteroviruses and other agents are emerging as major causative agents of hfmd in some epidemics. serotypes causing severe symptoms such as hfmd including ca16 and ev71 are decreasing, while the proportion of unidentified ev serotypes causing herpangina and viral encephalitis are on the rise . there may be an upward trend in cocirculation of the two pathogens globally and a new role that recombinants play in the emergence of new enterovirus variants. in 2008, a large, hfmd outbreak in fuyang city of anhui province in south-eastern china resulted in a large number of fatalities. phylogenetic analyses of the entire vp1 capsid protein sequence showed isolates belonging to the c4a cluster of the c4 subgenotype, and additionally, genetic recombinations were found between the fuyang hev71 strain and cv-a16, resulting in a recombination virus. in 2008, another nationwide outbreak of hfmd was reported in day care centres and schools in finland (osterback et al, 2009) . from vesicle fluid specimens of hospitalised children, the authors identified the aetiological agent as coxsackievirus a6. enterovirus d68 (ev-d68) appears to cause severe respiratory illness in childrenaffecting most severely those with asthma in the 2014 usa epidemic, and may cause paralyses, including affecting cranial nerves (chen et al, 2014b; foster et al, 2015; maloney et al, 2015) . there is no reliably effective antiviral against these agents yet available. an assay using multilocus pcr and reverse transcription pcr coupled with electrospray ionisation mass spectrometry (rt-pcr/esi-ms), which simultaneously detects and identifies human enterovirus a-d, adenovirus a-f, human herpesvirus 1-8, parvovirus b19 and polyomavirus, detected not only enteroviruses in hfmd, but also herpesviruses, polyomaviruses, adenoviruses and human rhinoviruses in 36% hfmd specimens (chen et al, 2014a) . virus and the orofacial implications of infection have been extensively scrutinised and reported (greenspan, 1998; nokta, 2008; scully, 2014) . hiv infection appeared in the 1920s in the congo from simian origins but was not recognised until the 1980s. the pan epidemic continues to expand with worldwide latest figures of new reported infections (2014) at 2.3 m, a total of over 34 m, and infection in sexually active people now at 1 in 100 (http://www.who.int/hiv/data/en/). worldwide, hiv is mainly heterosexually transmitted and, in the over 50s, there is a threefold increase. africa has a known infected rate >20%, and in eastern europe and central asia, there has been a known increase of 250% in 10 years. uk has among highest rates of new hiv in europe, outstripped by portugal, ukraine, estonia and russia. most hiv infections are in heterosexuals, on vacation but many men having sex with men have hiv. hiv infection is undiagnosed in one in three affected. multidrug-resistant hiv has appeared. the two main viruses, hiv-1 (most common) and hiv-2, infect cells with cd4 surface receptors, mainly t-helper lymphocytes and brain glial cells, and replicate within and damage them, causing hiv disease which may be emerging and changing viral diseases c scully and lp samaranayake symptomless but, over time, ultimately damages cd4+ cells crucial to host defences against fungi, viruses, mycobacteria and parasites, thus causing hiv disease and ultimately producing symptoms (mainly infections and tumours) and the acquired immune deficiency syndrome (aids) and a range of lesions. hiv/aids common manifestations are infections, neoplasms, neurological and autoimmune disorders. infections with viruses, mycobacteria, fungi and parasites, particularly pneumocystis carinii (jiroveci) pneumonia and mucosal candidiasis, are common. loss of weight and wasting ('slim disease') is common. aids is a lethal infection, defined as hiv infection plus 1 or more aids-defining illnesses and a cd4 t lymphocyte count <200 9 10 6 /l. without antiretroviral treatment (art), all eventually develop aids within 5-10 years. candidiasis is universal especially in hiv subtype b strain crf19 infection, but other infections in hiv/ aids depend also upon their environmental exposure; thus, tb is particularly common in people from africa and in urban iv drug users in usa; leishmaniasis is common in persons from around the mediterranean; mycoses such as penicillosis are seen mainly in northern thailand. neoplasms may include virally related kaposi sarcoma, lymphomas or carcinomas (jose et al, 2013; mthethwa et al, 2013; meless et al, 2014; nair et al, 2014) . body fluids such as semen may contain hiv as may saliva, breast milk and blood. hiv transmission is sexual mainly: most new cases are via heterosexual intercourse. hiv can also be transmitted by infected blood or blood products, including plasma or tissues. hiv transmission by contaminated needles and syringes is an important route in injecting drug users. cross-placental transfer is not uncommon. transmission by needlestick ('sharps') injury is an occasional risk for healthcare workers. there is no reliable evidence for hiv transmission by normal social contact or by biting insects. pre-exposure prophylaxis using safe sex practices plus tenofovir and emtricitabine is highly effective to prevent hiv infection. as for treatment, art is typically effective although drugs are costly and often associated with resistance and/ or adverse reactions. immune reconstitution inflammatory syndrome (iris) may follow art and can include exacerbation of some lesions such as tuberculosis, mycobacterium avium complex infections, zoster, hpv, cmv, cryptococcosis and kaposi sarcoma (tsang and samaranayake, 2010) . every effort must be made to avoid needlestick (sharps) injuries, as these could transmit hiv, hepatitis viruses or other infections. in the event of such an injury, speed is of the essence, and where appropriate, counselling and postexposure prophylaxis (pep). current pep for hiv (and other blood-borne agents such as hepatitis b and c) viruses in uk consists of (samaranayake and scully, 2013); • hiv: tenovir, emtricitabine, lopinavir, ritonavir, • hbv: hepatitis immunoglobulin plus hbv vaccine or booster, • hcv: interferon plus ribavirin. mumps. mumps is a common childhood infection caused by the mumps virus (muv), a member of the paramyxoviridae family of enveloped, non-segmented, negative-sense rna viruses. the defining feature of classical mumps is swelling of the parotid gland, but this is not present in all cases and it can also occur in various other disorders. only mumps causes epidemic parotitis. other causes of parotitis include infection by parainfluenza virus types 1 and 3, influenza a virus, coxsackie a virus, echovirus, lymphocytic choriomeningitis virus, human immunodeficiency virus, human t lymphotropic virus 1 and non-infectious causes (e.g. drugs, tumours, immunologic diseases and salivary duct obstruction). mumps virus not only affects salivary glands but also other glands including reproductive glands and pancreas, leading to orchitis or oophoritis (hviid et al, 2008; ternavasio-de la vega et al, 2010) . acute hormonal disturbances are common including decreased testosterone and inhibin b levels with low or normal levels of gonadotropins in 35% and a high incidence of sperm disturbance. importantly, mumps is highly neurotropic, and central nervous system infection ensues in approximately half of cases and may lead to aseptic meningitis, viral encephalitis, and rarely deafness and pancreatitis (rubin et al, 2015) . mumps is vaccine preventable, and one dose of mumps vaccine is about 80% effective against the disease. routine vaccination has proven highly effective in reducing the incidence of mumps and is presently used by most developed countries; however, there have been outbreaks of disease in vaccinated populations (hviid et al, 2008) .since the introduction of the mumps vaccine, the age of appearance of mumps infection has shifted from children to adolescents and young adults, groups with a higher incidence of disease complications and sequelae. in 2005, a large epidemic peaked in the uk, and, in 2006, the american mid-west had several outbreaks. in both countries, the largest proportion of cases was in young adults. in the uk, susceptible cohorts too old to have been vaccinated and too young to have been exposed to natural infections were the primary cause of the mumps epidemic. in the usa, effectiveness and uptake in combination appear not to have been sufficient to obtain herd immunity for mumps in populations such as college students. severe fever with thrombocytopenia syndrome virus (sftsv) infection. an emerging infectious disease, sfts, was identified to be associated with a novel sfts rna bunyavirus (sftsv). transmission of the disease among humans has been described, but clinical impact factors and transmission mechanisms still need further study (bao et al, 2011; li, 2015) . risk factors assessment of the person-to-person transmission revealed that the major exposure factor was blood contact without personal protection equipment. information from this study provided solid references of sfts incubation time, clinical and laboratory parameters related to sfts severity and outcome, and biosafety issues for preventing personto-person transmission or nosocomial infection of sftsv. emerging and changing viral diseases there is no reliably effective antiviral against this agent yet available. viruses are ubiquitous, and they cause a wide range of human diseases, ranging from acute self-resolving conditions to fatal diseases. we continue to witness the emergence of new viruses and infections and there appear to be no signs of abeyance in the march of these elusive enemies. apart from the acute infections with either mild or severe symptoms, a number of new and old virus infections could leave a legacy of secondary illnesses long after the primary infection has subsided. these secondary effects can also increase the propensity for chronic conditions or lead to the development of cancer. the discussion above on emerging viral diseases and old viral diseases appearing in new guises indicates the impact of such infections on the practice of dentistry not only from the perspective of oral manifestations and oral healthcare sciences, diagnosis and management but also from an equally important aspects related to infection control. viruses: are they really culprits for periodontal disease? a critical review human papillomavirus and survival of patients with oropharyngeal cancer urban legends series: lichen planus update on oral herpes virus infections mucocutaneous manifestations of chikungunya fever a family cluster of infections by a newly recognized bunyavirus in eastern china, 2007: further evidence of person-to-person transmission global burden, distribution, and interventions for infectious diseases of poverty standard infection control precautions hpv transmission in adolescent men who have sex with men chikungunya: a re-emerging virus global participation in core data sets for emerging pathogens update on emerging infections: news from the centers for disease control and prevention. notes from the field: chikungunya virus spreads in the americas-caribbean and south america worldwide trends in incidence rates for oral cavity and oropharyngeal cancers detection and identification of viral pathogens in patients with hand, foot, and mouth disease by multilocus pcr, reverse-transcription pcr and electrospray ionization mass spectrometry biology and pathogenesis of cytomegalovirus in periodontal disease human herpes simplex-1 and papillomavirus type 16 homologous dna sequences in normal, potentially malignant and malignant oral mucosa postextraction bleeding following a fever: a case report. oral surg oral med oral pathol oral radiol detection of rna complementary to herpes simplex virus in human oral squamous cell carcinoma enterovirus d68: a clinically important respiratory enterovirus mission: critical -2014 year in review emerging viral respiratory tract infections-environmental risk factors and transmission molecular pathology of emerging coronavirus infections oral manifestations of hiv. hiv insite knowledge base chapter a systematic analysis of host factors reveals a med23-interferon-lambda regulatory axis against herpes simplex virus type 1 replication a comprehensive immunoinformatics and target site study revealed the corner-stone toward chikungunya virus treatment prevention of influenza at hajj: applications for mass gatherings reduced prevalence of oral human papillomavirus (hpv) 4 years after bivalent hpv vaccination in a randomized clinical trial in costa rica viruses and disease: emerging concepts for prevention, diagnosis and treatment the "natasha" trade: transnational sex trafficking emerging respiratory tract viral infections hypoglossal nerve palsy: a rare consequence of dengue fever a cluster of person-toperson transmission cases caused by sfts virus in penglai, china prevalence of oral and systemic manifestations in pediatric hiv cohorts with and without drug therapy randomized clinical study comparing compeed cold sore patch to acyclovir cream 5% in the treatment of herpes simplex labialis infection and autoimmunity in sjogren's syndrome: a clinical study and comprehensive review incidence and clearance of oral human papillomavirus infection in men: the him cohort study oral candidiasis in chikungunya viral fever: a case report consequences of the expanding global distribution of aedes albopictus for dengue virus transmission severe fever with thrombocytopenia syndrome: a newly discovered emerging infectious disease epidemiology of childhood enterovirus infections in hangzhou local immunoreactivity and human papillomavirus (hpv) in oral precancer and cancer lesions co-circulation and genomic recombination of coxsackievirus a16 and enterovirus 71 during a large outbreak of hand, foot, and mouth disease in central china a systematic review of the epidemiology and interaction of herpes simplex virus types 1 and 2 herpes simplex infection and the risk of alzheimer's disease: a nested case-control study prognostic implications of hpv in oropharyngeal cancer altered oral viral ecology in association with periodontal disease the internet and the global monitoring of emerging diseases: lessons from the first 10 years of promed-mail detection of human papillomavirus dna in biopsies of human oral tissue mri findings in children with acute flaccid paralysis and cranial nerve dysfunction occurring during the 2014 enterovirus d68 outbreak emerging and re-emerging infectious threats in the 21st century oral lesions among hivinfected children on antiretroviral treatment in west africa emergence of medicine for mass gatherings: lessons from the hajj oral presentation in dengue hemorrhagic fever: a rare entity the prevalence of hiv associated oral lesions among adults in the era of haart orofacial viral infections-an update for clinicians oral manifestations associated with hiv infection human papillomavirus related head and neck cancer survival: a systematic review and meta-analysis coxsackievirus a6 and hand, foot, and mouth disease a new arenavirus in a cluster of fatal transplant-associated diseases severe oral manifestation of dengue viral infection: a rare clinical description diagnostic efficacy of saliva for dengue -a reality in near future? a piloting initiative clinical and oral implications of dengue fever: a review detecting the emergence of novel, zoonotic viruses pathogenic to humans molecular biology, pathogenesis and pathology of mumps virus needlestick and occupational exposure to infections: a compendium of current guidelines ebola virus infection: an overview viral haemorrhagic fevers with emphasis on ebola virus disease and orodental healthcare fungal and viral infections, skeletal disorders and malignancies. hospital update viruses and cancer: herpesviruses and tumors in the head and neck. a review oral squamous cell carcinoma; from an hypothesis about a virus, to concern about possible sexual transmission persistent metallic taste churchill livingstone: london, uk. oral diseases emerging and changing viral diseases c scully and lp samaranayake clinical virology in oral medicine and dentistry papillomaviruses: the current status in relation to oral disease infection control: ebola aware; ebola beware; ebola healthcare papular purpuric gloves and socks syndrome. presentation of a clinical case hajj: health lessons for mass gatherings isolated velopalatine paralysis associated with parvovirus b19 infection topical and systemic therapies for oral and perioral herpes simplex virus infections mumps orchitis in the post-vaccine era (1967-2009): a singlecenter series of 67 patients and review of clinical outcome and trends chikungunya: a potentially emerging epidemic? immune reconstitution inflammatory syndrome after highly active antiretroviral therapy: a review ebv infection is common in gingival epithelial cells of the periodontium and worsens during chronic periodontitis discovering novel zoonotic viruses hpv vaccination in head and neck hpv-related pathologies viruses contribute to the development of sj€ ogren's syndrome an epidemic analysis of hand, foot, and mouth disease in zunyi we are grateful to dr nihal bandara for the editorial assistance rendered and to the royal college of surgeons of edinburgh for support of professorships (king james iv) for both authors. both authors have equally contributed to the review. key: cord-022633-fr55uod6 authors: nan title: saem abstracts, plenary session date: 2012-04-26 journal: acad emerg med doi: 10.1111/j.1553-2712.2012.01332.x sha: doc_id: 22633 cord_uid: fr55uod6 nan objectives: we sought to determine if the ocp policy resulted in a meaningful and sustained improvement in ed throughput and output metrics. methods: a prospective pre-post experimental study was conducted using administrative data from 15 community and tertiary centers across the province. the study phases consisted of the 8 months from february to september 2010 compared against the same months in 2011. operational data for all centres were collected through the edis tracking systems used in the province. the ocp included 3 main triggers: ed bed occupancy >110%, at least 35% of ed stretchers blocked by patients awaiting inpatient bed or disposition decision, and no stretcher available for high acuity patients. when all criteria were met, selected boarded patients were moved to an inpatient unit (non-traditional care space if no bed available). the primary outcome was ed length of stay (los) for admitted patients. the ed load of boarded patients from 10-11 am was reported the editors of academic emergency medicine (aem) are honored to present these abstracts accepted for presentation at the 2012 annual meeting of the society for academic emergency medicine (saem), may 9 to 12 in chicago, illinois. these abstracts represent countless hours of labor, exciting intellectual discovery, and unending dedication by our specialty's academicians. we are grateful for their consistent enthusiasm, and are privileged to publish these brief summaries of their research. this year, saem received 1172 abstracts for consideration, and accepted 746. each abstract was independently reviewed by up to six dedicated topic experts blinded to the identity of the authors. final determinations for scientific presentation were made by the saem program scientific subcommittee co-chaired by ali s. raja, md, mba, mph and steven b. bird, md, and the saem program committee, chaired by michael l. hochberg, md. their decisions were based on the final review scores and the time and space available at the annual meeting for oral and poster presentations. there were also 125 innovation in emergency medicine education (ieme) abstracts submitted, of which 37 were accepted. the ieme subcommittee was co-chaired by joanna leuck, md and laurie thibodeau, md. we present these abstracts as they were received, with minimal proofreading and copy editing. any questions related to the content of the abstracts should be directed to the authors. presentation numbers precede the abstract titles; these match the listings for the various oral and poster sessions at the annual meeting in chicago, as well as the abstract numbers (not page numbers) shown in the key word and author indexes at the end of this supplement. all authors attested to institutional review board or animal care and use committee approval at the time of abstract submission, when relevant. abstracts marked as ''late-breakers'' are prospective research projects that were still in the process of data collection at the time of the december abstract deadline, but were deemed by the scientific subcommittee to be of exceptional interest. these projects will be completed by the time of the annual meeting; data shown here may be preliminary or interim. on behalf of the editors of aem, the membership of saem, and the leadership of our specialty, we sincerely thank our research colleagues for these contributions, and their continuing efforts to expand our knowledge base and allow us to better treat our patients. david background: two to ten percent of patients evaluated in the emergency departments (ed) present with altered mental status (ams). the prevalence of non-convulsive seizure (ncs) and other electroencephalographic (eeg) abnormalities in this population is not known. this information is needed to make recommendations regarding the routine use of emergent eeg in ams patients. objectives: to identify the prevalence of ncs and other eeg abnormalities in ed patients with ams. methods: an ongoing prospective study at two academic urban ed. inclusion: patients ‡ 13 years old with ams. exclusion: an easily correctable cause of ams (e.g. hypoglycemia, opioid overdose). a 30-minute eeg with the standard 19 electrodes was performed on each subject as soon as possible after presentation (usually within 1 hour). outcome: the rate of eeg abnormalities based on blinded review of all eegs by two boardcertified epileptologists. descriptive statistics are used to report eeg findings. frequencies are reported as percentages with 95% confidence intervals (ci), and inter-rater variability is reported with kappa. results: the interim analysis was performed on 130 consecutive patients (target sample size: 260) enrolled from may to october 2011 (median age: 61, range 13-100, 40% male). eegs for 20 patients were reported uninterpretable by at least one rater (6 by both raters). of the remaining 110, only 30 (27%, 95%ci 20-36%) were normal according to either rater (n = 15 by both). the most common abnormality was background slowing (n = 75, 68%, 95%ci 59-76%) by either rater (n = 47 by both), indicating underlying encephalopathy. ncs was diagnosed in 8 patients (7%, 95%ci, 4-14%) by at least one rater (n = 4 by both), including 6 (5%, 95%ci 2-12%) patients in non-convulsive status epilepticus (ncse). 29 patients (26%,95%ci 19-35%) had interictal epileptiform discharges read by at least one rater (n = 12 by both) indicating cortical irritability and an increased risk of spontaneous seizure. inter-rater reliability for eeg interpretations was modest (kappa: 0.53, 95%ci 0.39-0.67). objectives: to define diagnostic sbi and non-bacterial (non-sbi) biosignatures using rna microarrays in febrile infants presenting to emergency departments (eds). methods: we prospectively collected blood for rna microarray analysis in addition to routine screening tests including white blood cell (wbc) counts, urinalyses, cultures of blood, urine, and cerebrospinal fluid, and viral studies in febrile infants 60 days of age in 22 eds . we defined sbi as bacteremia, urinary tract infection (uti), or bacterial meningitis. we used class comparisons (mann-whitney p < 0.01, benjamini for mtc and 1.25 fold change filter), modular gene analysis, and k-nn algorithms to define and validate sbi and non-sbi biosignatures in a subset of samples. results: 81% (939/1162) of febrile infants were evaluated for sbi. 6.8% (64/939) had sbi (14 (1.5%) bac-teremia, 56 (6.0%) utis, and 4 (0.4%) bacterial meningitis). infants with sbis had higher mean temperatures, and higher wbc, neutrophil, and band counts. we analyzed rna biosignatures on 141 febrile infants: 35 sbis (2 meningitis, 5 bacteremia, 28 uti), 106 non-sbis (49 influenza, 29 enterovirus, 28 undefined viral infections), and 11 healthy controls. class comparisons identified 1,288 differentially expressed genes between sbis and non-sbis. modular analysis revealed overexpression of interferon related genes in non-sbis and inflammation related genes in sbis. 232 genes were differently expressed (p < 0.01) in each of the three non-sbi groups vs sbi group. unsupervised cluster analysis of these 232 genes correctly clustered 91% (128/141) of non-sbis and sbis. k-nn algorithm identified 33 discriminatory genes in training set (30 non-sbis vs 17 sbis) which classified an independent test (76 non-sbis vs 18 sbis) with 87% accuracy. four misclassified sbis had over-expression of interferon-related genes, suggesting viral-bacterial co-infections, which was confirmed in one patient. background: improving maternal, newborn, and child health (mnch) is a leading priority worldwide. however, limited frontline health care capacity is a major barrier to improving mnch in developing countries. objectives: we sought to develop, implement, and evaluate an evidence-based maternal, newborn, and child survival (mncs) package for frontline health workers (fhws). we hypothesized that fhws could be trained and equipped to manage and refer the leading mnch emergencies. methods: setting -south sudan, which suffers from some of the world's worst mnch indices. assessment/intervention -a multi-modal needs assessment was conducted to develop a best-evidence package comprised of targeted trainings, pictorial checklists, and reusable equipment and commodities ( figure 1 ). program implementation utilized a trainingof-trainers model. evalution -1) pre/post knowledge assessments, 2) pre/post objective structured clinical examinations (osces), 3) focus group discussions, and 4) closed-response questionnaires. results: between nov 2010 to oct 2011, 72 local trainers and 708 fhws were trained in 7 of the 10 states in south sudan. knowledge assessments among trainers (n = 57) improved significantly from 62.7% (sd 20.1) to 92.0% (sd 11.8) (p < 0.001). mean scores a maternal osce and a newborn osce pre-training, immediately post-training, and upon 2-3 month follow-up are shown in the table. closed-response questionnaires with 54 fhws revealed high levels of satisfaction, use, and confidence with mncs materials. participants reported an average of 3.0 referrals (range 0-20) to a higher level of care in the 2-3 months since training. furthermore, 78.3% of fhws were more likely to refer patients as a result of the training program. during seven focus group discussions with trained fhws, respondents (n = 41) reported high satisfaction with mncs trainings, commodities, and checklists, with few barriers to implementation or use. conclusion: these findings suggest mncs has led to improvements in south sudanese fhws' knowledge, skills, and referral practices with respect to appropriate management of mnch emergencies. no study has compared various lactate measurements to determine the optimal parameter to target. objectives: to compare the association of blood lactate kinetics with survival in patients with septic shock undergoing early quantitative resuscitation. methods: preplanned analysis of a multicenter edbased rct of early sepsis resuscitation targeting three physiological variables: cvp, map, and either central venous oxygen saturation or lactate clearance. inclusion criteria: suspected infection, two or more sirs criteria, and either sbp <90 mmhg after a fluid bolus or lactate >4 mmol/l. all patients had an initial lactate measured with repeat at two hours. normalization of lactate was defined a lactate decline to <2.0 mmol/l in a patient with an intial lactate ‡2.0. absolute lactate clearance (initial -delayed value), and relative ((absolute clearance)/(initial value)*100) were calculated if the initial lactate was ‡2.0. the outcome was in-hospital survival. receiver operating characteristic curves were constructed and areas under the curve (auc) were calculated. difference in proportions of survival between the two groups at different lactate cutoffs were analyzed using 95% ci and fisher exact tests. results: of 272 included patients, the median initial lactate was 3.1 mmol/l (iqr 1.7, 5.8), and the median absolute and relative lactate clearance were 1 mmol/l (iqr 0.3, 2.5) and 37% (iqr 14, 57 ). an initial lactate >2.0 mmol/l was seen in 187/272 (69%), and 68/187 (36%) patients normalized their lactate. overall sutures on trunk and extremity lacerations that present in the ed. the use of absorbable sutures in the ed setting confers several advantages: patients do not need to return for suture removal which results in a reduction in ed crowding, ed wait times, missed work or school days, and stressful procedures (suture removal) for children. objectives: the primary objective of this study is to compare the cosmetic outcome of trunk and extremity lacerations repaired using absorbable versus nonabsorbable sutures in children and adults. a secondary objective is to compare complication rates between the two groups. methods: eligible patients with lacerations were randomly allocated to have their wounds repaired with vicryl rapide (absorbable) or prolene (nonabsorbable) sutures. at a 10 day follow-up visit the wounds were evaluated for infection and dehiscence. after 3 months, patients were asked to return to have a photograph of the wound taken. two blinded plastic surgeons using a previously validated 100 mm visual analogue scale (vas) rated the cosmetic outcome of each wound. a vas score of 15 mm or greater was considered to be a clinically significant difference. results: of the 100 patients enrolled, 45 have currently completed the study including 19 in the vicryl rapide group and 26 in the prolene group. there were no significant differences in the age, race, sex, length of wound, number of sutures, or layers of repair in the two groups. the observer's mean vas for the vicryl rapide group was 55.76 mm ) and that for the prolene group was 55.9 mm (95%ci 44.77-67.03), resulting in a mean difference of 0.14 mm (95%ci-16.95 to 17.23, p = .98). there were no significant differences in the rates of infection, dehiscence, or keloid formation between the two groups. conclusion: the use of vicryl rapide instead of nonabsorbable sutures for the repair of lacerations on the trunk and extremities should be considered by emergency physicians as it is an alternative that provides a similar cosmetic outcome. objectives: to determine the relationship between infection and time from injury to closure, and the characteristics of lacerations closed before and after 12 hours of injury. methods: over an 18 month period, a prospective multi-center cohort study was conducted at a teaching hospital, trauma center and community hospital. emergency physicians completed a structured data form when treating patients with lacerations. patients were followed to determine whether they had suffered a wound infection requiring treatment and to determine a cosmetic outcome rating. we compared infection rates and clinical characteristics of lacerations with chisquare and t-tests as appropriate. results: there were 2663 patients with lacerations; 2342 had documented times from injury to closure. the mean times from injury to repair for infected and noninfected wounds were 2.4 vs. 3.0 hrs (p = 0.39) with 78% of lacerations treated within 3 hours and 4% (85) treated 12 hours after injury. there were no differences in the infection rates for lacerations closed before (2.9%, 95%ci 2.2-3.7) or after (2.1%, 95%ci 0.4-6.0) 6 hours and before (3.0%, 95% ci 2.3%-3.8%) or after (1.2%, 95% ci 0.03%-6.4%) 12 hours. the patients treated 12 hours after injury tended to be older (41 vs. 34 yrs p = 0.02) and fewer were treated with primary closure (85% vs. 96% p < 0.0001). comparing wounds 12 or more hours after injury with more recent wounds, there was no effect of location on decision to close. wounds closed after 12 hours did not differ from wounds closed before 12 hours with respect to use of prophylactic antibiotics, type of repair, length of laceration, or cosmetic outcome. conclusion: closing older lacerations, even those greater than 12 hours after injury, does not appear to be associated with any increased risk of infection or adverse outcomes. excellent irrigation and decontamination over the last 30 years may have led to this change in outcome. background: deep burns may result in significant scarring leading to aesthetic disfigurement and functional disability. tgf-b is a growth factor that plays a significant role in wound healing and scar formation. objectives: the current study was designed to test the hypothesis that a novel tgf-b antagonist would reduce scar contracture compared with its vehicle in a porcine partial thickness burn model. methods: ninety-six mid-dermal contact burns were created on the backs and flanks of four anesthetized young swine using a 150 gm aluminum bar preheated to 80°celsius for 20 seconds. the burns were randomized to treatment with topical tgf-b antagonist at one of three concentrations (0, 187, and 375 ll) in replicates of 8 in each pig. dressing changes and reapplication of the topical therapy were performed every 2 days for 2 weeks then twice weekly for an additional 2 weeks. burns were photographed and full thickness biopsies were obtained at 5, 7, 9, 14, and 28 days to determine reepithelialization and scar formation grossly and microscopically. a sample of 32 burns in each group had 80% power to detect a 10% difference in percentage scar contracture. results: a total of 32 burns were created in each of the three study groups. burns treated with the high dose tgf-b antagonist healed with less scar contracture than those treated with the low dose and control (52 ± 20%, 63 ± 15%, and 62 ± 14%; anova p = 0.02). additionally, burns treated with the higher, but not the lower dose of tgf-b antagonist healed with significantly fewer full thickness scars than controls (62.5% vs. 100% vs. 93.8% respectively; p < 0.001). there were no infections and no differences in the percentage wound reepithelialization among all study groups at any of the time points. conclusion: treatment of mid-dermal porcine contact burns with the higher dose tgf-b antagonist reduced scar contracture and rate of deep scars compared with the low dose and controls. background: diabetic ketoacidosis (dka) is a common and lethal complication of diabetes. the american diabetes association recommends treating adult patients with a bolus dose of regular insulin followed by a continuous insulin infusion. the ada also suggests a glucose correction rate of 75-100 mg/dl/hr to minimize complications. objectives: compare the effect of bolus dose insulin therapy with insulin infusion to insulin infusion alone on serum glucose, bicarbonate, and ph in the initial treatment of dka. methods: consecutive dka patients were screened in the ed between march '06 and june '10. inclusion criteria were: age >18 years, glucose >350 mg/dl, serum bicarbonate 15 or ketonemia or ketonuria. exclusion criteria were: congestive heart failure, current hemodialysis, pregnancy, or inability to consent. no patient was enrolled more than once. patients were randomized to receive either regular insulin 0.1 units/kg or the same volume of normal saline. patients, medical and research staff were blinded. baseline glucose, electrolytes, and venous blood gases were collected on arrival. bolus insulin or placebo was then administered and all enrolled patients received regular insulin at rate of 0.1 unit/kg/hr, as well as fluid and potassium repletion per the research protocol. glucose, electrolytes, and venous blood gases were drawn hourly for 4 hours. data between two groups were compared using unpaired t-test. results: 99 patients were enrolled, with 30 being excluded. 35 patients received bolus insulin; 34 received placebo. no significant differences were noted in initial glucose, ph, bicarbonate, age, or weight between the two groups. after the first hour, glucose levels in the insulin group decreased by 151 mg/dl compared to 94 mg/dl in the placebo group (p = 0.0391, 95% ci 2.7 to 102.0). changes in mean glucose levels, ph, bicarbonate level, and ag were not statistically different between the two groups for the remainder of the 4 hour study period. there was no difference in the incidence of hypoglycemia in the two groups. conclusion: administering a bolus dose of regular insulin decreased mean glucose levels more than placebo, although only for the first hour. there was no difference in the change in ph, serum bicarbonate or anion gap at any interval. this suggests that bolus dose insulin may not add significant benefit in the emergency management of dka. ihca; 3. return of spontaneous circulation (rsoc). traumatic cardiac arrests were excluded. we recorded baseline demographics, arrest event characteristics, follow-up vitals and laboratory data, and in-hospital mortality. apache ii scores were calculated at the time of rosc, and at 24 hrs, 48 hrs, and 72 hrs. we used simple descriptive statistics to describe the study population. univariate logistic regression was used to predict mortality with apache ii as a continuous predictor variable. discrimination of apache ii scores was assessed using the area under the curve (auc) of the receiver operator characteristic (roc) curve. results: a total of 229 patients were analyzed. the median age was 70 years (iqr: 56-79) and 32% were female. apache ii score was a significant predictor of mortality for both ohca and ihca at baseline and at all follow-up time points (all p < 0.01). discrimination of the score increased over time and achieved very good discrimination after 24 hrs (table, figure) . conclusion: the ability of apache ii score to predict mortality improves over time in the 72 hours following cardiac arrest. these data suggest that after 24 hours, apache ii scoring is a useful severity of illness score in all post-cardiac arrest patients. background: admission hyperglycemia has been described as a mortality risk factor for septic non-diabetics, but the known association of hyperglycemia with hyperlactatemia (a validated mortality risk factor in sepsis) has not previously been accounted for. objectives: to determine whether the association of hyperglycemia with mortality remains significant when adjusted for concurrent hyperlactatemia. methods: this was a post-hoc, nested analysis of a single-center cohort study. providers identified study subjects during their ed encounters; all data were collected from the electronic medical record. patients: nondiabetic adult ed patients with a provider-suspected infection, two or more systemic inflammatory response syndrome criteria, and concurrent lactate and glucose testing in the ed. setting: the ed of an urban teaching hospital; 2007 to 2009. analysis: to evaluate the association of hyperglycemia (glucose >200 mg/dl) with hyperlactatemia (lactate ‡ 4.0 mmol/l), a logistic regression model was created; outcome-hyperlactatemia; primary variable of interest-hyperglycemia. a second model was created to determine if concurrent hyperlactatemia affects hyperglycemia's association with mortality; outcome-28-day mortality; primary risk variablehyperglycemia with an interaction term for concurrent hyperlactatemia. both models were adjusted for demographics, comorbidities, presenting infectious syndrome, and objective evidence of renal, respiratory, hematologic, or cardiovascular dysfunction. results: 1236 ed patients were included; mean age 76 ± 19 years. 133 (9%) subjects were hyperglycemic, 182 (13%) hyperlactatemic, and 225 (16%) died within 28 days of the initial ed visit. after adjustment, hyperglycemia was significantly associated with simultaneous hyperlactatemia (or 3.9, 95%ci 2.48, 5.98). hyperglycemia with concurrent hyperlactatemia was associated with increased mortality risk (or 4.4, 95%ci 2.27, 8.59) , but hyperglycemia in the absence of simultaneous hyperlactatemia was not (or 0.86, 95%ci 0.45, 1.65) . conclusion: in this cohort of septic adult non-diabetic patients, mortality risk did not increase with hyperglycemia unless associated with simultaneous hyperlactatemia. the previously reported association of hyperglycemia with mortality in this population may be due to the association of hyperglycemia with hyperlactatemia. the background: near infrared spectroscopy (sto2) represents a measure of perfusion that provides the treating physician with an assessment of a patient's shock state and response to therapy. it has been shown to correlate with lactate and acid/base status. it is not known if using information from this monitor to guide resuscitation will result in improved patient outcomes. objectives: to compare the resuscitation of patients in shock when the sto2 monitor is or is not being used to guide resuscitation. methods: this was a prospective study of patients undergoing resuscitation in the ed for shock from any cause. during alternating 30 day periods, physicians were blinded to the data from the monitor followed by 30 days in which physicians were able to see the information from the sto2 monitor and were instructed to resuscitate patients to a target sto2 value of 75. adult patients (age>17) with a shock index (si) of >0.9 (si = heart rate/systolic blood pressure) or a blood pressure <80 mmhg systolic who underwent resuscitation were enrolled. patients had a sto 2 monitor placed on the thenar eminence of their least-injured hand. data from the sto 2 monitor were recorded continuously and noted every minute along with blood pressure, heart rate, and oxygen saturation. all treatments were recorded. patients' charts were reviewed to determine the diagnosis, icu-free days in the 28 days after enrollment, inpatient los, and 28-day mortality. data were compared using wilcoxon rank sum and chi-square tests. results: 107 patients were enrolled, 51 during blinded periods and 56 during unblinded periods. the median presenting shock index was 1.24 (range 0.5 to 4.0) for the blinded group and 1.10 (0.5-3.3) for the unblinded group (p = 0.13). the median time in department was 70 minutes (range 22-407) for the blinded and 76 minutes (range 11-275) for the unblinded groups (p = 0.99). the median hospital los was 1 day (range 0-30) for the blinded group, and 2 days (range 0-23) in the unblinded group (p = 0.63). the mean icu-free days was 22 ± 9 for the blinded group and 19 ± 11 for the unblinded group (p = 0.26). among patients where the physician indicated using the sto2 monitor data to guide patient care, the icu-free days were 21.4 ± 9 for the blinded group and 16.3 ± 12 for the blinded group (p = 0.06). background: inducing therapeutic hypothermia (th) using 4°c iv fluids in resuscitated cardiac arrest patients has been shown to be feasible and effective. limited research exists assessing the efficiency of this cooling method. objectives: the objective was to determine an efficient infusion method for keeping fluid close to 4°c upon exiting an iv. it was hypothesized that colder temperatures would be associated with both higher flow rate and insulation of the fluid bag. methods: efficiency was studied by assessing change in fluid temperature (0c) during the infusion, under three laboratory conditions. each condition was performed four times using 1 liter bags of normal saline. fluid was infused into a 1000 ml beaker through 10 gtts tubing. flow rate was controlled using a tubing clamp and in-line transducer with a flowmeter, while temperature was continuously monitored in a side port at the terminal end of the iv tubing using a digital thermometer. the three conditions included infusing chilled fluid at a rate of 40 ml/min, which is equivalent to 30 ml/kg/hr for an 80 kg patient, 105 ml/min, and 105 ml/min using a chilled and insulated pressure bag. descriptive statistics and analysis of variance was performed to assess changes in fluid temperature. results: the average fluid temperatures at time 0 were 3.40 (95% ci 3.12-3.69) (40 ml/min), 3.35 (95% ci 3.25-3.45) (105 ml/min), and 2.92 (95% ci 2.40-3.45) (105 ml/min + insulation). there was no significant difference in starting temperature between groups (p = 0.16). the average fluid temperatures after 100 ml had been infused were 10.02 (95% ci 9.30-10.74) (40 ml/min), 7.35 (95% ci 6.91-7.79) (105 ml/min), and 6.95 (95% ci 6.47-7.43) (105 ml/min + insulation). the higher flow rate groups had significantly lower temperature than the lower flow rate after 100 ml of fluid had been infused (p < 0.001). the average fluid temperatures after 1000 ml had been infused were 16.77 (95% ci 15.96-17.58) (40 ml/min), 11.40 (95% ci 11.18-11.61) (105 ml/min), and 7.75 (95% ci 7.55-7.99) (105 ml/min + insulation). there was a significant difference in temperature between all three groups after 1000 ml of fluid had been infused (p < 0.001). conclusion: in a laboratory setting, the most efficient method of infusing cold fluid appears to be a method that both keeps the bag of fluid insulated and is infused at a faster rate. fluid bolus. patients were categorized by presence of vasoplegic or tissue dysoxic shock. demographics and sequential organ failure assessment (sofa) scores were evaluated between the groups. the primary outcome was in-hospital mortality. data were analyzed using t-tests, chi-squared test, and proportion differences with 95% confidence intervals as appropriate. results: a total of 242 patients were included: 89 patients with vasoplegic shock and 153 with tissue dysoxic shock. there were no significant differences in age (61 vs. 58 years), caucasian race (53% vs. 58%), or male sex (57% vs. 52%) between the dysoxic shock and vasoplegic shock groups, respectively. the group with vasoplegic shock had a lower initial sofa score than did the group with tissue dysoxic shock (5.7 vs. 7.3 points, p = 0.0002). the primary outcome of in-hospital mortality occurred in 8/89 (9%) of patients with vasoplegic shock compared to 40/153 (26%) in the group with tissue dysoxic shock (proportion difference 17%, 95% ci 7-26%, p < 0.0001). conclusion: in this analysis of patients with septic shock, we found a significant difference in in-hospital mortality between patients with vasoplegic versus tissue dysoxic septic shock. these findings suggest a need to consider these differences when designing future studies of septic shock therapies. background: the pre-shock population, ed sepsis patients with tissue hypoperfusion (lactate of 2.0-3.9 mm), commonly deteriorates after admission and requires transfer to critical care. objectives: to determine the physiologic parameters and disease severity indices in the ed pre-shock sepsis population that predict clinical deterioration. we hypothesized that neither initial physiologic parameters nor organ function scores will be predictive. methods: design: retrospective analysis of a prospectively maintained registry of sepsis patients with lactate measurements. setting: an urban, academic medical center. participants: the pre-shock population, defined as adult ed sepsis patients with either elevated lactate (2.0-3.9 mm) or transient hypotension (any sbp <90 mmhg) receiving iv antibiotics and admitted to a medical floor. consecutive patients meeting pre-shock criteria were enrolled over a 1-year period. patients with overt shock in the ed, pregnancy, or acute trauma were excluded. outcome: primary patientcentered outcome of increased organ failure (sequential organ failure assessment [sofa] score increase >1 point, mechanical ventilation, or vasopressor utilization) within 72 hours of admission or in-hospital mortality. results: we identified 248 pre-shock patients from 2649 screened. the primary outcome was met in 54% of the cohort and 44% were transferred to the icu from a medical floor. patients meeting the outcome of increased organ failure had a greater shock index (1.02 vs 0.93, p = 0.042) and heart rate (115 vs 105, p < 0.001) with no difference in initial lactate, age, map, or exposure to hypotension (sbp <100 mmhg). there was no difference in the predisposition, infection, response, and organ dysfunction (piro) score between groups (6.4 vs 5.7, p = 0.052). outcome patients had similar initial levels of organ dysfunction but had higher sofa scores at 24, 48, and 72 hours, a higher icu transfer rate (60 vs 24%, p < 0.001), and increased icu and hospital lengths of stay. conclusion: the pre-shock sepsis population has a high incidence of clinical deterioration, progressive organ failure, and icu transfer. physiologic data in the ed were unable to differentiate the pre-shock sepsis patients who developed increased organ failure. this study supports the need for an objective organ failure assessment in the emergency department to supplement clinical decision-making. background: lipopolysaccharide (lps) has long been recognized to initiate the host inflammatory response to infection with gram negative bacteria (gnb). large clinical trials of potentially very expensive therapies continue to have the objective of reducing circulating lps. previous studies have found varying prevalence of lps in blood of patients with severe sepsis. compared with sepsis trials conducted 20 years ago, the frequency of gnb in culture specimens from emergency department (ed) patients enrolled in clinical trials of severe sepsis has decreased. objectives: test the hypothesis that prior to antibiotic administration, circulating lps can be detected in the plasma of fewer than 10% of ed patients with severe sepsis. methods: secondary analysis of a prospective edbased rct of early quantitative resuscitation for severe sepsis. blood specimens were drawn at the time severe sepsis was recognized, defined as two or more systemic inflammatory criteria and a serum lactate >4 mm or spb<90 mmhg after fluid challenge. blood was drawn in edta prior to antibiotic administration or within the first several hours, immediately centrifuged, and plasma frozen at )80°c. plasma lps was quantified using the limulus amebocyte lysate assay (lal) by a technician blinded to all clinical data. results: 180 patients were enrolled with 140 plasma samples available for testing. median age was 59 ± 17 years, 50% female, with overall mortality of 18%. forty of 140 patients (29%) had any culture specimen positive for gnb including 21 (15%) with blood cultures positive. only five specimens had detectable lps, including two with a gnb-positive culture specimen, and three were lps-positive without gnb in any culture. prevalence of detectable lps was 3.5% (ci: 1.5%-8.1%). the frequency of detectable lps in antibiotic-naive plasma is too low to serve as a useful diagnostic test or therapeutic target in ed patients with severe sepsis. the data raise the question of whether post-antibiotic plasma may have a higher frequency of detectable lps. background: egdt is known to reduce mortality in septic patients. there is no evidence to date that delineates the role of using a risk stratification tool, such as the mortality in emergency department sepsis (meds) score, to determine which subgroups of patients may have a greater benefit with egdt. objectives: our objective was to determine if our egdt protocol differentially affects mortality based on the severity of illness using meds score. methods: this study is a retrospective chart review of 243 patients, conducted at an urban tertiary care center, after implementing an egdt protocol on july 1, 2008 (figure) . this study compares in-hospital mortality, length of stay (los) in icu, and los in ed between the control group (126 patients from 1/1/07-12/31/07) and the postimplementation group (117 patients from 7/1/08-6/ 30/09), using meds score as a risk stratification tool. inclusion criteria: patients who presented to our ed with a suspected infection, and two or more sirs criteria, a map<65 mmhg, a sbp< 90 mmol/l. exclusion criteria: age<18, death on arrival to ed, dnr or dni, emergent surgical intervention, or those with an acute myocardial infarction or chf exacerbation. a two-sample t-test was used to show that the mean age and number of comorbidities was similar between the control and study groups (p = 0.27 and 0.87 respectively). mortality was compared and adjusted for meds score using logistic regression. the odds ratios and predicted probabilities of death are generated using the fitted logistic regression model. ed and icu los were compared using mood's median test. results: when controlling for illness severity using meds score, the relative risk (rr) of death with egdt is about half that of the control group (rr = 0.52, 95% ci [0.278-0.973], p=0.04). also, by applying meds score to risk stratify patients into various groups of illness severity, we found no specific groups where egdt is more efficacious at reducing the predicted probability of death (table 1) . without controlling for meds score, there is a trend in reduction of absolute mortality by 9.7% when egdt is used (control = 30.2%, study = 20.5%, p = 0.086). egdt leads to a 40.3% reduction in the median los in icu (control = 124 hours, study = 74 hours, p = 0.03), without increasing los in ed (control = 6 hours, study = 7 hours, p = 0.50). conclusion: egdt is beneficial in patients with severe sepsis or septic shock, regardless of their meds score. background: in patients experiencing acute coronary syndrome (acs), prompt diagnosis is critical in achieving the best health outcome. while ecg analysis is usually sufficient to diagnose acs in cases of st elevation, acs without st elevation is reliably diagnosed through serial testing of cardiac troponin i (ctni). pointof-care testing (poct) for ctni by venipuncture has been proven a more rapid means to diagnosis than central laboratory testing. implementing fingerstick testing for ctni in place of standard venipuncture methods would allow for faster and easier procurement of patients' ctni levels, as well as increase the likelihood of starting a rapid test for ctni in the prehospital setting, which could allow for even earlier diagnosis of acs. objectives: to determine if fingerstick blood samples yield accurate and reliable troponin measurements compared to conventional venous blood draws using the i-stat poc device. methods: this experimental study was performed in the ed of a quaternary care suburban medical center between june-august 2011. fingerstick blood samples were obtained from adult ed patients for whom standard (venipuncture) poc troponin testing was ordered. the time between fingerstick and standard draws was kept as narrow as possible. ctni assays were performed at the bedside using the i-stat 1 (abbott point of care). results: 94 samples from 87 patients were analyzed by both fingerstick and standard ed poct methods (see table) . four resulted in cartridge error. compared to ''gold standard'' ed poct, fingerstick testing has a positive predictive value of 100%, negative predictive value of 96%, sensitivity of 79%, and specificity of 100%. no significant difference in ctni level was found between the two methods, with a nonparametric intraclass correlation coefficient of 0.994 (95% ci 0.992-0.996, p-value < 0.001). conclusion: whole blood fingerstick ctni testing using the i-stat device is suitable for rapid evaluation of ctni level in prehospital and ed settings. however, results must be interpreted with caution if they are within a narrow territory of the cutoff for normal vs. elevated levels. additional testing on a larger sample would be beneficial. the practicality and clinical benefit of using fingerstick ctni testing in the ems setting must still be assessed. background: adjudication of diagnosis of acute myocardial infarction (ami) in clinical studies typically occurs at each site of subject enrollment (local) or by experts at an independent site (central). from 2000 from -2007 , the troponin (ctn) element of the diagnosis was predicated on the local laboratories, using a mix of the 99th percentile reference ctn and roc-determined cutpoints. in 2007, the universal definition of ami (ud-ami) defined it by the 99th percentile reference alone. objectives: to compare the diagnosis rates of ami as determined by local adjudication vs. central adjudication using udami criteria. methods: retrospective analysis of data from the myeloperoxidase in the diagnosis of acute coronary syndromes (acs) study (midas), an 18-center prospective study with enrollment from 12/19/06 to 9/20/07 of patients with suspected acs presenting to the ed < 8 hours after symptom onset and in whom serial ctn and objective cardiac perfusion testing was planned. adjudication of acs was done by single local principal investigators using clinical data and local ctn cutpoints from 13 different ctn assays, and applying the 2000 definition. central adjudication was done after completion of the midas primary analysis using the same data and local ctn assay, but by experts at three different institutions, using the udami and the manufacturer's 99th percentile ctn cutpoint, and not blinded to local adjudications. discrepant dignoses were resolved by consensus. local vs. central ctn cutpoints differed for six assays, with central cutpoints lower in all. statistics were by chi-square and kappa. results: excluding 11 cases deemed indeterminate by central adjudication, 1096 cases were successfully adjudicated. local adjudication resulted in 104 ami (9.5% of total) and 992 non-ami; central adjudication resulted in 134 (12.2%) ami and 962 non-ami. overall, 44 local diagnoses (4%) were either changed from non-ami to ami or ami to non-ami (p < 0.001). interrater reliability across both methods was found to be kappa = 0.79 (p < 0.001). for acs diagnosis, local adjudication identified 252 acs cases (23%) and 854 non-acs, while central adjudication identified 275 acs (25%) and 831 non-acs. overall, 61 local diagnoses (6%) were either changed from non-acs to acs or acs to non-acs (p < 0 .001). interrater reliability found kappa = 0.85 (p < 0.001). conclusion: central and local adjudication resulted in significantly different rates of ami and acs diagnosis. however, overall agreement of the two methods across these two diagnoses was acceptable. occur four times more often in cocaine users. biomarkers myeloperoxidase (mpo) and c-reactive protein (crp) have potential in the diagnosis of acs. objectives: to evaluate the utility of mpo and crp in the diagnosis of acs in patients presenting to the ed with cocaine-associated chest pain and compare the predictive value to nonusers. we hypothesized that these markers may be more sensitive for acs in nonusers given the underlying pathophysiology of enhanced plaque inflammation. methods: a secondary analysis of a cohort study of enrolled ed patients who received evaluation for acs at an urban, tertiary care hospital. structured data collection at presentation included demographics, chest pain history, lab, and ecg data. subjects included those with self-reported or lab-confirmed cocaine use and chest pain. they were matched to controls based on age, sex, and race. our main outcome was diagnosis of acs at index visit. we determined median mpo and crp values, calculated maximal auc for roc curves, and found cut-points to maximize sensitivity and specificity. data are presented with 95% ci. results: overall, 95 patients in the cocaine positivegroup and 86 patients in the nonusers group had mpo and crp levels measured. patients had a median age of 47 (iqr, (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) , 90% black or african american, and 62% male (p > 0.05 between groups). fifteen patients were diagnosed with acs: 8 patients in the cocaine group and 7 in the nonusers group. comparing cocaine users to nonusers, there was no difference in mpo (median 162 [iqr, ] v 136 ng/ml; p = 0.78) or crp (3 [1] [2] [3] [4] [5] [6] [7] [8] [9] v 5 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] mg/l; p = 0.08). the auc for mpo was 0.65 (95% ci 0.39-0.90) v 0.54 (95% ci 0.19-0.73). the optimal cut-point to maximize sensitivity and specificity was 242 ng/ml which gave a sensitivity of 0.42 and specificity of 0.75. using this cutpoint, 57% v 29% of acs in cocaine users vs the nonusers would be identified. the auc for crp was 0.63 (95% ci 0.39-0.88) in cocaine users vs 0.73 (95% ci 0.52-0.95) in nonusers. the optimal cut point was 11.9 mg/l with a sensitivity of 0.67 and specificity of 0.79. using this cutpoint, 43% v 88% of acs in cocaine users and nonusers would have been identified. conclusion: the diagnostic accuracy of mpo and crp is not different in cocaine users than nonusers and does not appear to have sufficient discriminatory ability in either cohort. results: 18 hrs of moderate pe caused a significant decrease in rv heart function in rats treated with the solvent for bay 41-8543: peak systolic pressure (psp) decreased from 39 ± 1.5 mmhg, control to 16 ± 1.5, pe, +dp/dt decreased from 1192 ± 93 mmhg/sec to 463 ± 77, -dp/dt decreased from )576 ± 60 mmhg/sec to )251 ± 9. treatment of rats with bay 41-8543 significantly improved all three indices of rv heart function (psp 29 ± 2.6, +dp/dt 1109 ± 116, -dp/dt )426 ± 69). 5 hrs of severe pe also caused significant rv dysfunction (psp 25 ± 2, -dp/dt )356 ± 28) and treatment with bay 41-8543 produced protection of rv heart function (psp 34 ± 2, -dp/dt )535 ± 41) similar to the 18 hr moderate pe model. conclusion: experimental pe produced significant rv dysfunction, which was ameliorated by treatment of the animals with the soluble guanylate cyclase stimulator, bay 41-8543. 1 hospital of the university of pennsylvania, philadelphia, pa; 2 cooper university hospital, camden, nj background: patients who present to the ed with symptoms of potential acute coronary syndrome (acs) can be safely discharged home after a negative coronary computerized tomographic angiography (cta). however, the duration of time for which a negative coronary cta can be used to inform decision making when patients have recurrent symptoms is unknown. objectives: we examined patients who received more than one coronary cta for evaluation of acs to determine whether they had disease progression, as defined by crossing the threshold from noncritical (<50% maximal stenosis) to potentially critical disease. methods: we performed a structured comprehensive record search of all coronary ctas performed from 2005 to 2010 at a tertiary care health system. low-tointermediate risk ed patients who received two or more coronary ctas, at least one from an ed evaluation for potential acs, were identified. patients who were revascularized between scans were excluded. we collected demographic data, clinical course, time between scans, and number of ed visits between scans. record review was structured and done by trained abstractors. our main outcome was progression of coronary stenosis between scans, specifically crossing the threshold from noncritical to potentially critical disease. results: overall, 32 patients met study criteria (median age 45, interquartile range [iqr] (37.5-48); 56% female; 88% black). the median time between studies was 27.3 months (iqr, . 22 patients did not have stenosis in any vessel on either coronary cta, two studies showed increasing stenosis of <20%, and the rest showed ''improvement,'' most due to better imaging quality. no patient initially below the 50% threshold subsequently exceeded it (0%; 95% ci, 0-11.0%). patients also had varying numbers of ed visits (median number of visits 5, range 0-23), and numbers of ed visits for potentially cardiac complaints (median 1, range 0-6); 10 were re-admitted for potentially cardiac complaints (for example, chest pain or shortness of breath), and 9 received further provocative cardiac testing, all of which had negative results. conclusion: we did not find clinically significant disease progression within a 2 year time frame in patients who had a negative coronary cta, despite a high number of repeat visits. this suggests that prior negative coronary cta may be able to be used to inform decision making within this time period. 42.7-48.6) compared to non tro ct patients. there was no significant difference in image quality between tro ct images and those of dedicated ct scans in any studies performing this comparison. similarly, there was no significant difference between tro ct and other diagnostic modalities in regards to length of stay or admission rate. when compared to conventional coronary angiography as the gold standard for evaluation of cad, tro ct had the following pooled diagnostic accuracy estimates: sensitivity 0.94 conclusion: tro chest ct is comparable to dedicated pe, coronary, or ad ct in regard to image quality, length of stay, and admission rate and is highly accurate for detecting cad. the utility of tro ct depends on the relative pre-test probabilities of the conditions being assessed and its role is yet to be clearly defined. tro ct, however, involves increased radiation exposure and contrast volume and for this reason clinicians should be selective in its use. background: coronary computed tomographic angiography (ccta) has high sensitivity, specificity, accuracy, and prognostic value for coronary artery disease (cad) and acs. however, how a ccta informs subsequent use of prescription medication is unclear. objectives: to determine if detection of critical or noncritical cad on ccta is associated with initiation of aspirin and statins for patients who presented to the ed with chest pain. we hypothesized that aspirin and statins would be more likely to be prescribed to patients with noncritical disease relative to those without any cad. methods: prospective cohort study of patients who received ccta as part of evaluation of chest pain in the ed or observation unit. patients were contacted and medical records were reviewed to obtain clinical follow-up for up to the year after ccta. the main outcome was new prescription of aspirin or statin. cad severity on ccta was graded as absent, mild (1% to 49%), moderate (50% to 69%), or severe ( ‡70%) stenosis. logistic regression was used to assess the association of stenosis severity to new medication prescription; covariates were determined a priori. results: 859 patients who had ccta performed consented to participate in this study or met waiver of consent for record review only (median age, , 59% female, 71% black). median follow-up time was 333 days, iqr 70-725 days. at baseline, 13% of the total cohort was already prescribed aspirin and 8% on statin medication. two hundred seventy nine (32%) patients were found to have stenosis in at least one vessel. in patients with absent, mild, moderate, and severe cad on ccta, aspirin was initiated in 11%, 34%, 52%, and 55%; statins were initiated in 7%, 22%, 32%, and 53% of patients. after adjustment for age, race, sex, hypertension, diabetes, cholesterol, tobacco use, and admission to the hospital after ccta, higher grades of cad severity were independently associated with greater post-ccta use of aspirin (or 1.9 per grade, 95% ci 1.4-2.2, p < 0.001) and statins (or 1.9, 95% ci 1.5-2.4, p < 0.001). conclusion: greater cad severity on ccta is associated with increased medication prescription for cad. patients with noncritical disease are more likely than patients without any disease to receive aspirin and statins. future studies should examine whether these changes lead to decreased hospitalizations and improved cardiovascular health. background: hess et al. developed a clinical decision rule for patients with acute chest pain consisting of the absence of five predictors: ischemic ecg changes not known to be old, elevated initial or 6-hour troponin level, known coronary disease, ''typical'' pain, and age over 50. patients less than 40 required only a single troponin evaluation. objectives: to test the hypothesis that patients less than 40 years old without these criteria are at <1% risk for major adverse cardiovascular events (mace) including death, ami, pci, and cabg. methods: we performed a secondary analysis of several combined prospective cohort studies that enrolled ed patients who received an evaluation for acs in an urban ed from 1999 to 2009. cocaine users and stemi patients were excluded. structured data collection at presentation included demographics, pain description, history, lab, and ecg data for all studies. hospital course was followed daily. thirty-day follow up was done by telephone. our main outcome was 30-day mace using objective criteria. the secondary outcome was potential change in ed disposition due to application of the rule. descriptive statistics and 95% cis were used. results: of 9289 visits for potential acs, patients had a mean age of 52.4 ± 14.7 yrs; 68% were black and 59% female. there were 638 patients (6.9%) with 30-day cv events (93 dead, 384 ami, 298 pci). sequential removal of patients in order to meet the final rule for patients less than 40 excluded patients based upon: ischemic ecg changes not old (n = 434, 30% mace rate), elevated initial troponin level (n = 237, 60% mace), known coronary disease (n = 1622, 11% mace), ''typical'' pain (n = 3179, 3% mace), and age over 40 (n = 2690, 3.4% mace) leaving 1127 patients less than 40 with 0.8% mace [95% ci, 0.4-1.5%]. of this cohort, 70% were discharged home from the ed by the treating physician without application of this rule. adding a second negative troponin in patients 40-50 years old identified a group of 1139 patients with a 2.0% rate of mace [1.3-3 .0] and a 48% discharge rate. the hess rule appears to identify a cohort of patients at approximately 1% risk of 30-day mace, and may enhance discharge of young patients. however, even without application of this rule, the 70% of young patients at low risk are already being discharged home based upon clinical judgment. background: a clinical decision support system (cdss) incorporates evidence-based medicine into clinical practice, but this technology is underutilized in the ed. a cdss can be integrated directly into an electronic medical record (emr) to improve physician efficiency and ease of use. the christopher study investigators validated a clinical decision rule for patients with suspected pulmonary embolism (pe). the rule stratifies patients using wells' criteria to undergo either d-dimer testing or a ct angiogram (ct). the effect of this decision rule, integrated as a cdss into the emr, on ordering cts has not been studied. objectives: to assess the effect of a mandatory cdss on the ordering of d-dimers and cts for patients with suspected pe. methods: we assessed the number of cts ordered for patients with suspected pe before and after integrating a mandatory cdss in an urban community ed. physicians were educated regarding cdss use prior to implementation. the cdss advised physicians as to whether a negative d-dimer alone excluded pe or if a ct was required based on wells' criteria. the emr required physicians to complete the cdss prior to ordering the ct. however, physicians maintained the ability to order a ct regardless of the cdss recommendation. patients ‡18 years of age presenting to the ed with a chief complaint of chest pain, dyspnea, syncope, or palpitations were included in the data analysis. we compared the proportion of d-dimers and cts ordered during the 8-month periods immediately before and after implementing the cdss. all 27 physicians who worked in the ed during both time periods were included in the analysis. patients with an allergy to intravenous contrast agents, renal insufficiency, or pregnancy were excluded. results were analyzed using a chi-square test. results: a total of 11,931 patients were included in the data analysis (6054 pre-and 5877 post-implementation). cts were ordered for 215 patients (3.6%) in the pre-implementation group and 226 patients (3.8%) in the post-implementation group; p = 0.396. a d-dimer was ordered for 392 patients (6.5%) in the pre-implementation group and 382 patients (6.5%) in the post-implementation group; p = 0.958. in this single-center study, emr integration of a mandatory cdss for evaluation of pe did not significantly alter ordering patterns of cts and d-dimers. identification of patients with low-risk pulmonary emboli suitable for discharge from the emergency department mike zimmer, keith e. kocher university of michigan, ann arbor, mi background: recent data, including a large, multicenter randomized controlled trial, suggest that a low-risk cohort of patients diagnosed with pulmonary embolism (pe) exists who can be safely discharged from the ed for outpatient treatment. objectives: to determine if there is a similar cohort at our institution who have a low rate of complications from pe suitable for outpatient treatment. methods: this was a retrospective chart review at a single academic tertiary referral center with an annual ed volume of 80,000 patients. all adult ed patients who were diagnosed with pe during a 24-month period from 11/1/09 through 10/31/11 were identified. the pulmonary embolism severity index (pesi) score, a previously validated clinical decision rule to risk stratify patients with pe, was calculated. patients with high pesi (>85) were excluded. additional exclusion criteria included patients who were at high risk of complications from initiation of therapeutic anticoagulation and those patients with other clear indications for admission to the hospital. the remaining cohort of patients with low risk pe (pesi £ 85) was included in the final analysis. outcomes were measured at 14 and 90 days after pe diagnosis and included death, major bleeding, and objectively confirmed recurrent venous thromboembolism (vte). results: during the study period, 298 total patients were diagnosed with pe. there were 172 (58%) patients categorized as ''low risk'' (pesi £ 85), with 42 removed because of various pre-defined exclusion criteria. of the remaining 130 (44%) patients suitable for outpatient treatment, 5 patients (3.8%; 95% ci, 0.5% -7.2%) had one or more negative outcomes by 90 days. this included 2 (1.5%; 95% ci, 0% -3.7%) major bleeding events, 2 (1.5%; 95% ci, 0% -3.7%) recurrent vte, and 2 (1.5%; 95% ci, 0% -3.7%) deaths. none of the deaths were attributable to pe or anticoagulation. one patient suffered both a recurrent vte and died within 90 days. both patients who died within 90 days were transitioned to hospice care because of worsening metastatic burden. at 14 days, there was 1 bleeding event (0.8%; 95% ci, 0% -2.3%), no recurrent vte, and no deaths. the average hospital length of stay for these patients was 2.8 days (sd ±1.6). conclusion: over 40% of our patients diagnosed with pe in the ed may have been suitable for outpatient treatment, with 4% suffering a negative outcome within 90 days and 0.8% suffering a negative outcome within 14 days. in addition, the average hospital length of stay for these patients was 2.8 days, which may represent a potential cost savings if these patients had been managed as outpatients. our experience supports previous studies that suggest the safety of outpatient treatment of patients diagnosed with pe in the ed. given the potential savings related to a decreased need for hospitalization, these results have health policy implications and support the feasibility of creating protocols to facilitate this clinical practice change. background: chest x-rays (cxrs) are commonly obtained on ed chest pain patients presenting with suspected acute coronary syndrome (acs). a recently derived clinical decision rule (cdr) determined that patients who have no history of congestive heart failure, have never smoked, and have a normal lung examination do not require a cxr in the ed. objectives: to validate the diagnostic accuracy of the hess cxr cdr for ed chest pain patients with suspected acs. methods: this was a prospective observational study of a convenience sample of chest pain patients over 24 years old with suspected acs who presented to a single urban academic ed. the primary outcome was the ability of the cdr to identify patients with abnormalities on cxr requiring acute ed intervention. data were collected by research associates using the chart and physician interviews. abnormalities on cxr and specific interventions were predetermined, with a positive cxr defined as one with abnormality requiring ed intervention, and a negative cxr defined as either normal or abnormal but not requiring ed intervention. the final radiologist report was used as a reference standard for cxr interpretation. a second radiologist, blinded to the initial radiologist's report, reviewed the cxrs of patients meeting the cdr criteria to calculate inter-observer agreement. patients were followed up by chart review and telephone interview 30 days after presentation. results: between january and august 2011, 178 patients were enrolled, of whom 38 (21%) were excluded and 10 (5.6%) did not receive cxrs in the ed. of the 130 remaining patients, 74 (57%) met the cdr. the cdr identified all patients with a positive cxr (sensitivity = 100%, 95%ci 40-100%). the cdr identified 73 of the 126 patients with a negative cxr (specificity = 58%, 95%ci 49-67%). the positive likelihood ratio was 2.4 (95%ci 1.9-2.9). inter-observer agreement between radiologists was substantial (kappa = 0.63, 95%ci 0.41-0.85). telephone contact was made with 78% of patients and all patient charts were reviewed at 30 days. none had any adverse events related to a background: increasing the threshold to define a positive d-dimer in low-risk patients could reduce unnecessary computed tomographic pulmonary angiography (ctpa) for suspected pe. this strategy might increase rates of missed pe and missed pneumonia, the most common non-thromboembolic finding on ctpa that might not otherwise be diagnosed. objectives: measure the effect of doubling the standard d-dimer threshold for ' 'pe unlikely'' revised geneva (rgs) or wells' scores on the exclusion rate, frequency, and size of missed pe and missed pneumonia. methods: prospective enrollment at four academic us hospitals. inclusion criteria required patients to have at least one symptom or sign and one risk factor for pe, and have 64-channel ctpa completed. pretest probability data were collected in real time and the d-dimer was measured in a central laboratory. criterion standard for pe or pneumonia consisted of cpta interpretation by two independent radiologists combined with necessary treatment plan. subsegmental pe was defined as total vascular obstruction <5%. patients were followed for outcome at 30 days. proportions were compared with 95% cis. results: of 678 patients enrolled, 126 (19%) were pe+ and 93 (14%) had pneumonia. with rgs£6 and standard threshold (<500 ng/ml), d-dimer was negative in 110/678 (16%, 95% ci: 13-19%), and 4/110 were pe+ (posterior probability 3.8%, 95% ci: 1-9.3%). with rgs£6 and a threshold <1000 ng/ml, d-dimer was negative in 208/678 (31%, 27-44%) and 11/208 (5.3%, 2.8-9.3%) were pe+, but 10/11 missed pes were subsegmental, and none had concomitant dvt. the posterior probability for pneumonia among patients with rgs&#8804;6 and d-dimer<500 was 9/110 (8.2%, 4-15%) which compares favorably to the posterior probability of 12/208 (5.4%, 3-10%) observed with rgs& #8804;6 and d-dimer<1000 ng/ml. of the 200 (35%) patients who also had plain film cxr, radiologists found an infiltrate in only 58. use of wells£4 produced similar results as the rgs&#8804;6 for exclusion rate and posterior probability of both pe and pneumonia. conclusion: doubling the threshold for a positive d-dimer with a pe unlikely pretest probability can significantly reduce ctpa scanning with a slightly increased risk of missed isolated subsegmental pe, and no increase in rate of missed pneumonia. background: the limitations of developing world medical infrastructure require that patients are transferred from health clinics only when the patient care needs exceed the level of care at the clinic and the receiving hospital can provide definitive therapy. to determine what type of definitive care service was sought when patients were transferred from a general outpatient clinic operating monday through friday from 8:00 am to 3:00 pm in rural haiti to urban hospitals in port-au-prince. methods: design -prospective observational review of all patients for whom transfer to a hospital was requested or for whom a clinic ambulance was requested to an off-site location to assist with patient care. setting -weekday, daytime only clinic in titanyen, haiti. participants/subjects -consecutive series of all patients for whom transfer to another health care facility or for whom an ambulance was requested during the time period of 11/22/2010 -12/14/2010 and 3/28/2011 -5/13/2011 . results: between 11/22/2010 -12/14/2010 and 3/28/2011 -5/13/2011 patients were identified who needed to be transferred to a higher level of care. sixteen patients (43.2%) presented with medical complaints, 12 (32.4%) were trauma patients, 6 (16.2%) were surgical, and 3 (8.1%) were in the obstetric category. within these categories, 6 patients were pediatric and 4 non-trauma patients required blood transfusion. conclusion: while trauma services are often focused on in rural developing world medicine, the need for obstetric care and blood transfusion constituted six (16.2%) cases in our sample. these patients raise important public health, planning, and policy questions relating to access to prenatal care and the need to better understand transfusion medicine utilization among rural haitian patients with non-trauma related transfusion needs. the data set is limited by sample size and single location of collection. another limitation of understanding the needs is that many patients may not present to the clinic for their health care needs in certain situations if they have knowledge that the resources to provide definitive care are unavailable. background: the practice of emergency medicine in japan has been unique in that emergency physicians are mostly engaged in critical care and trauma with a multi-specialty model. for the last decade with progress in medicine, an aging population with complicated problems, and institution of postgraduate general clinical training, the us model emergency medicine with single-specialty model has been emerging throughout japan. however, the current status is unknown. objectives: the objective of this study was to investigate the current status of implementation of the us model emergency medicine at emergency medicine training institutions accredited by the japanese association for acute medicine (jaam). methods: the er committee of the jaam, the most prestigious professional organization in japanese emergency medicine, conducted the survey by sending questionnaires to 499 accredited emergency medicine training institutions. results: valid responses obtained from 299 facilities were analyzed. us model em was provided in 211 facilities (71% of 299 facilities), either in full time (24 hours a day, seven days a week; 123 facilities) or in part time (less than 24 hours a day; 88 facilities). among these 211 us model facilities, 44% have a number of beds between 251-500. the annual number of ed visits was less than 20,000 in 64%, and 37% have ambulance transfers between 2,001-4,000 per year. the number of emergency physicians was less than 5 in 60% of the facilities. postgraduate general clinical training was offered at us model ed in 199 facilities, and ninety hospitals adopted us model em after 2004, when a 2-year period of postgraduate general clinical training became mandatory for all medical graduates. sixty-four facilities provided a residency program to be a us model emergency physician, and another 9 institutions were planning to establish it. conclusion: us model em has emerged and become commonplace in japan. the background including advance in medicine, aging population, and mandatory postgraduate general clinical training system are considered to be contributing factors. erkan gunay, ersin aksay, ozge duman atilla, nilay zorbalar, savas sezik tepecik research and training hospital, izmir, turkey background: workplace safety and occupational health problems are increasing issues especially in developing countries as a result of the industrial automatisation and technologic improvements. occupational injuries are preventable but they can occasionally cause morbidity and mortality resulting in work day loss and financial problems. hand injuries are one-third of all traumatic injuries and are the most injured parts after occupational accidents. objectives: we aim to evaluate patients with occupational upper extremity injuries for demographic characteristics, injury types, and work day loss. methods: trauma patients over 15 years old admitted to our emergency department with an occupational upper extremity injury were prospectively evaluated from 15.04.2010 to 30.04.2011. patients with one or more of digit, hand, forearm, elbow, humerus, and shoulder injuries were included. exclusion criteria were multitrauma, patient refusal to participate, and insufficient data. patients were followed up from the hospital information system and by phone for work day loss and final diagnosis. results: during the study period there were 570 patients with an occupational upper extremity injury. total of 521 (91.4%) patients were included. patients were 92.1% male, 36.5% between the age 25 to 34, and mean age was calculated 32.9 ± 9.6 years. 43.8% of the patients were from the metal and machinery sector, and primary education was the highest education level for the 74.7% of the patients. most injured parts were fingers with the highest rate for index finger and thumb. crush injury was the most common injury type. 96.3% (n = 502) of the patients were discharged after treatment in the emergency department. tendon injuries, open fractures, and high degree burns were the reasons for admission to clinics. mean work day loss was 12.8 ± 27.2 days and this increases for the patients with laboratory or radiologic studies, consultant evaluation, or admission. the 15-24 age group had a significantly lower work day loss average. conclusion: evaluating occupational injury characteristics and risks is essential for identifying preventive measures and actions. with the guidance of this study preventive actions focusing on high-risk sectors and patients may be the key factor for avoiding occupational injuries and creating safer workplace environments in order to reduce financial and public health problems. background: as emergency medicine (em) gains increased recognition and interest in the international arena, a growing number of training programs for emergency health care workers have been implemented in the developing world through international partnerships. objectives: to evaluate the quality and appropriateness of an internationally implemented emergency physician training program in india. methods: physicians participating in an internationally implemented em training program in india were recruited to participate in a program evaluation. a mixed methods design was used including an online anonymous survey and semi-structured focus groups. the survey assessed the research, clinical, and didactic training provided by the program. demographics and information on past and future career paths were also collected. the focus group discussions centered around program successes and challenges. results: fifty of 59 eligible trainees (85%) participated in the survey. of the respondents, the vast majority were indian; 16% were female, and all were between the ages of 25 and 45 years (mean age 31 years). all but two trainees (96%) intend to practice em as a career. one-third listed a high-income country first for preferred practice location and half listed india first. respondents directly endorsed the program structure and content, and they demonstrated gains in self-rated knowledge and clinical confidence over their years of training. active challenges identified include: (1) insufficient quantity and inconsistent quality of indian faculty, (2) administrative barriers to academic priorities, and (3) persistent threat of brain drain if local opportunities are inadequate. conclusion: implementing an international emergency physician training program with limited existing local capacity is a challenging endeavor. overall, this evaluation supports the appropriateness and quality of this partnership model for em training. one critical challenge is achieving a robust local faculty. early negotiations are recommended to set educational priorities, which includes assuring access to em journals. attrition of graduated trainees to high-income countries due to better compensation or limited in-country opportunities continues to be a threat to long-term local capacity building. background: with an increasing frequency and intensity of manmade and natural disasters, and a corresponding surge in interest in international emergency medicine (iem) and global health (gh), the number of iem and gh fellowships is constantly growing. there are currently 34 iem and gh fellowships, each with a different curriculum. several articles have proposed the establishment of core curriculum elements for fellowship training. to the best of our knowledge, no study has examined whether iem and gh fellows are actually fulfilling these criteria. objectives: this study sought to examine whether current iem and gh fellowships are consistently meeting these core curricula. methods: an electronic survey was administered to current iem and gh fellowship directors, current fellows, and recent graduates of a total of 34 programs. survey respondents stated their amount of exposure to previously published core curriculum components: em system development, humanitarian assistance, disaster response, and public health. a pooled analysis comparing overall responses of fellows to those of program directors was performed using two-sampled t-test. results: response rates were 88% (n = 30) for program directors and 53% (n = 17) for current and recent fellows. programs varied significantly in terms of their emphasis on and exposure to six proposed core curriculum areas: em system development, em education development, humanitarian aid, public health, ems, and disaster management. only 43% of programs reported having exposure to all four core areas. as many as 67% of fellows reported knowing their curriculum only somewhat or not at all prior to starting the program. conclusion: many fellows enter iem and gh fellowships without a clear sense of what they will get from their training. as each fellowship program has different areas of curriculum emphasis, we propose not to enforce any single core curriculum. rather, we suggest the development of a mechanism to allow each fellowship program to present its curriculum in a more transparent manner. this will allow prospective applicants to have a better understanding of the various programs' curricula and areas of emphasis. background: advance warning of probable intensive care unit (icu) admissions could allow the bed placement process to start earlier, decreasing ed length of stay and relieving overcrowding conditions. however, physicians and nurses poorly predict a patient's ultimate disposition from the emergency department at triage. a computerized algorithm can use commonly collected data at triage to accurately identify those who likely will need icu admission. objectives: to evaluate an automated computer algorithm at triage to predict icu admission and 28-day in-hospital mortality. methods: retrospective cohort study at a 55,000 visit/ year level i trauma center/tertiary academic teaching hospital. all patients presenting to the ed between 12/16/2008 and 10/1/2010 were included in the study. the primary outcome measure was icu admission from the emergency department. the secondary outcome measure was 28-day all-cause in-hospital mortality. patients discharged or transferred before 28 days were considered to be alive at 28 days. triage data includes age, sex, acuity (emergency severity index), blood pressure, heart rate, pain scale, respiratory rate, oxygen saturation, temperature, and a nurse's free text assessment. a latent dirichlet allocation algorithm was used to cluster words in triage nurses' free text assessments into 500 topics. the triage assessment for each patient is then represented as a probability distribution over these 500 topics. logistic regression was then used to determine the prediction function. results: a total of 94,973 patients were included in the study. 3.8% were admitted to the icu and 1.3% died within 28 days. these patients were then randomly allocated to train (n = 75,992; 80%) and test (n = 18,981; 20%) data sets. the area under the receiver operating characteristic curve (auc) when predicting icu background: at the 2011 saem annual meeting, we presented the derivation of two hospital admission prediction models adding coded chief complaint (ccc) data from a published algorithm (thompson et al. acad emerg med 2006; 13:774-782) to demographic, ed operational, and acuity (emergency severity index (esi)) data. objectives: we hypothesized that these models would be validated when applied to a separate retrospective cohort, justifying prospective evaluation. methods: we conducted a retrospective, observational validation cohort study of all adult ed visits to a single tertiary care center (census: 49,000/yr) (4/1/09-12/31/10). we downloaded from the center's clinical tracking system demographic (age, sex, race), ed operational (time and day of arrival), esi, and chief complaint data on each visit. we applied the derived ccc hospital admission prediction models (all identified ccc categories and ccc categories with significant odds of admission from multivariable logistic regression in the derivation cohort) to the validation cohort to predict odds of admission and compared to prediction models that consisted of demographic, ed operational, and esi data, adding each category to subsequent models in a stepwise manner. model performance is reported by areaunder-the-curve (auc) data and 95%ci. signs, pain level, triage level, 72-hour return, number of past visits in the previous year, injury, and one of 122 chief complaint codes (representing 90% of all visits in the database). outputs for training included ordering of a complete blood count, basic chemistry (electrolytes, blood urea nitrogen, creatinine), cardiac enzymes, liver function panel, urinalysis, electrocardiogram, x-ray, computed tomography, or ultrasound. once trained, it was used on the nhamcs-ed 2008 database, and predictions were generated. predictions were compared with documented physician orders. outcomes included the percent of total patients who were correctly pre-ordered, sensitivity (the percent of patients who had an order that were correctly predicted), and the percent over-ordered. waiting time for correctly pre-ordered patients was highlighted, to represent a potential reduction in length of stay achieved by preordering. los for patients overordered was highlighted to see if over-ordering may cause an increase in los for those patients. unit cost of the test was also highlighted, as taken from the 2011 medicare fee schedule. physician times. however, during peak ed census times, many patients with completed tests and treatment initiated by triage await discharge by the next assigned physician. objectives: determine if a physician-led discharge disposition (dd) team can reduce the ed length of stay (los) for patients of similar acuity who are ultimately discharged compared to standard physician team assignment. methods: this prospective observational study was performed from 10/2010 to 10/2011 at an urban tertiary referral academic hospital with an annual ed volume of 87,000 visits. only emergency severity index level 3 patients were evaluated. the dd team was scheduled weekdays from 14:00 until 23:00. several ed beds were allocated to this team. the team was comprised of one attending physician and either one nurse and a tech or two nurses. comparisons were made between los for discharged patients originally triaged to the main ed side who were seen by the dd team versus the main side teams. time from triage physician to team physician, team physician to discharge decision time, and patient age were compared by unpaired t-test. differences were studied for number of patients receiving x-rays, ct scan, labs, and medications. results: dd team mean los in hours for discharged patients was shorter at 3.4 (95% ci: 3.3-3.6, n = 1451) compared to 6.4 (95% ci: 6.3-6.5, n = 4601) on the main side, p < 0.01. the mean time from triage physician to dd team physician was 1.4 hours (95% ci: 1.4-1.5, n = 1447) versus to 2.7 hours (95% ci: 2.7-2.8, n = 4568) to main side physician, p < 0.01. the dd team physician mean time to discharge decision was 1.0 hour (95% ci: 1.0-1.1, n = 1432) compared to 2.5 hours (95% ci: 2.4-2.6, n = 4590) for main side physician, p < 0.01. the dd team patients' mean age was 42.6 years (95% ci: 41.9-43.6, n = 1454) compared to main side patients' mean age of 49.1 years (95% ci: 48.5-49.6, n = 4621.) the dd team patients (n = 1454) received fewer x-rays (40% vs. 59%), ct scans (13% vs. 23%), labs (64% vs. 85%), and medications (63% vs. 68%) than main side patients (n = 4621), p < 0.01 for all compared. conclusion: the dd team complements the advanced triage process to further reduce los for patients who do not require extended ed treatment or observation. the dd team was able to work more efficiently because its patients tended to be younger and had fewer lab and imaging tests ordered by the triage physician compared to patients who were later seen on the ed main side. ed objectives: to evaluate the association between ed boarding time and the risk of developing hapu. methods: we conducted a retrospective cohort study using administrative data from an academic medical center with an adult ed with 55,000 annual patient visits. all patients admitted into the hospital through the ed 6/30/2008-2/28/2011 were included. development of hapu was determined using the standardized, national protocol for cms reporting of hapu. ed boarding time was defined as the time between an order for inpatient admission and transport of the patient out of the ed to an in-patient unit. we used a multivariate logistic regression model with development of a hapu as the outcome variable, ed boarding time as the exposure variable, and the following variables as covariates: age, sex, initial braden score, and admission to an intensive care unit (icu) from the ed. the braden score is a scale used to determine a patient's risk for developing a hapu based on known risk factors. a braden score is calculated for each hospitalized patient at the time of admission. we included braden score as a covariate in our model to determine if ed boarding time was a predictor of hapu independent of braden score. results: of 46,704 patients admitted to the hospital through the ed during the study period, 243 developed a hapu during their hospitalization. clinical characteristics are presented in the table. per hour of ed boarding time, the adjusted or of developing a hapu was 1.02 (95% ci 1.01-1.04, p = 0.007). a median of 40 patients per day were admitted through the ed, accumulating 144 hours of ed boarding time per day, with each hour of boarding time increasing the risk of developing a hapu by 2%. conclusion: in this single-center, retrospective study, longer ed boarding time was associated with increased risk of developing a hapu. queried ed and inpatient nurses and compared their opinions toward inpatient boarding. it also assessed their preferred boarding location if they were patients. objectives: this study queried ed and inpatient nurses and compared their opinions toward inpatient boarding. methods: a survey was administered to a convenience sample of ed and ward nurses. it was performed in a 631-bed academic medical center (30,000 admissions/yr) with a 68-bed ed (60,000 visits/yr). nurses were identified as ed or ward and whether they had previously worked in the ed. the nurses were asked if there were any circumstances where admitted patients should be boarded in the ed or inpatient hallways. they were also asked their preferred location if they were admitted as a patient. six clinical scenarios were then presented and their opinions on boarding queried. results: ninety nurses completed the survey; 35 (39%) were current ed nurses (ced), 40 (44%) had previously worked in the ed (ped). for the entire group 46 (52%) believed admitted patients should board in the ed. overall, 52 (58%) were opposed to inpatient boarding, with 20% of ced versus 83% of current ward (cw) nurses (p < 0.0001) and 28% of ped versus 85% of nurses never having worked in the ed (ned) opposed (p < 0.001). if admitted as patients themselves, overall 43 (54%) preferred inpatient boarding, with 82% of ced versus 33% of cw nurses (p < 0.0001) and 74% of ped versus 34% ned nurses (p = 0.0007) preferring inpatient boarding. for the six clinical scenarios, significant differences in opinion regarding inpatient boarding existed in all but two cases: a patient with stable copd but requiring oxygen and an intubated, unstable sepsis patient. conclusion: ward nurses and those who have never worked in the ed are more opposed to inpatient boarding than ed nurses and nurses who have worked previously in the ed. nurses admitted as patients seemed to prefer not being boarded where they work. ed and ward nurses seemed to agree that unstable or potentially unstable patients should remain in the ed. 8 weeks. staff satisfaction was evaluated through pre/ post-shift and study surveys; administrative data (physician initial assessment (pia), length of stay (los), patients leaving without being seen (lwbs) and against medical advice [lama] ) were collected from an electronic, real-time ed information system. data are presented as proportions and medians with interquartile ranges (iqr); bivariable analyses were performed. results: ed physicians and nurses expected the intervention to reduce the los of discharged patients only. pia decreased during the intervention period (68 vs 74 minutes; p < 0.001). no statistically/clinically significant differences were observed in the los; however, there was a significant reduction in the lwbs (4.7% to 3.5% p = 0.003) and lama (0.7% to 0.4% p = 0.028) rates. while there was a reduction of approximately 5 patients seen per physician in the affected ed area, the total number of patients seen on that unit increased by approximately 10 patients/day. overall, compared to days when there was no extra shift, 61% of emergency physicians stated their workload decreased and 73% felt their stress level at work decreased. conclusion: while this study didn't demonstrate a reduction in the overall los, it did reduce pia times and the proportion of lwbs/lama patients. while physicians saw fewer patients during the intervention study period, the overall patient volume increased and satisfaction among ed physicians was rated higher. provider-and hospital-level variation in admission rates and 72-hour return admission rates jameel abualenain 1 , william frohna 2 , robert shesser 1 , ru ding 1 , mark smith 2 , jesse m. pines 1 1 the george washington university, washington, dc; 2 washington hospital center, washington, dc background: decisions for inpatient versus outpatient management of ed patients are the most important and costliest decision made by emergency physicians, but there is little published on the variation in the decision to admit among providers or whether there is a relationship between a provider's admission rate and the proportion of their patients who return within 72 hours of the initial visit and are subsequently admitted (72h-ra). objectives: we explored the variation in provider-level admission rates and 72h-ra rates, and the relationship between the two. methods: a retrospective study using data from three eds with the same information system over varying time periods: washington hospital center (whc) (2008-10), franklin square hospital center (fshc) , and union memorial hospital (umh) . patients were excluded if left without being seen, left against medical advice, fast-track, psychiatric patients, and aged <18 years. physicians with <500 ed encounters or an admission rate <15% were excluded. logistic regression was used to assess the relationship between physician-level 72h-ra and admission rates, adjusting for patient age, sex, race, and hospital. results: 389,120 ed encounters were treated by 90 physicians. mean patient age was 50 years sd 20, 42% male, and 61% black. admission rates differed between hospitals (whc = 40%, umh = 37%, and fshc = 28%), as did the 72h-ra (whc = 0.9%, umh = 0.6%, and fshc = 0.6%). across all hospitals, there was great variation in individual physician admission rates (15.4%-50.0%). the 72h-ra rates were quite low, but demonstrated a similar magnitude of individual variation (0.3%-1.2%). physicians with the highest admission rate quintile had lower odds of 72h-ra (or 0.8 95% ci 0.7-0.9) compared to the lowest admission rate quintile, after adjusting for other factors. no intermediate admission rate quintiles (2nd, 3rd, or 4th) were significantly different from the lowest admission rate quintile with regard to 72h-ra. conclusion: there is more than three-fold variation in individual physician admission rates indicating great variation among physicians in hospital admission rates and 72h-ra. the highest admitters have the lowest 72h-ra; however, evaluating the causes and consequences of such significant variation needs further exploration, particularly in the context of health reform efforts aimed at reducing costs. background: ed scribes have become an effective means to assist emergency physicians (eps) with clinical documentation and improve physician productivity. scribes have been most often utilized in busy community eds and their utility and functional integration into an academic medical center with resident physicians is unknown. objectives: to evaluate resident perceptions of attending physician teaching and interaction after introduction of scribes at an em residency training program, measured through an online survey. residents in this study were not working with the scribes directly, but were interacting indirectly through attending physician use of scribes during ed shifts. methods: an online ten question survey was administered to 31 residents of a midwest academic emergency medicine residency program (pgy1-pgy3 program, 12 annual residents), 8 months after the introduction of scribes into the ed. scribes were introduced as emr documentation support (epic 2010, epic systems inc.) for attending eps while evaluating primary patients and supervising resident physicians. questions investigated em resident demographics and perceptions of scribes (attending physician interaction and teaching, effect on resident learning, willingness to use scribes in the future), using likert scale responses (1 minimal, 9 maximum) and a graduated percentage scale used to quantify relative values, where applicable. data were analyzed using kruskal-wallis and mann-whitney u tests. results: twenty-one of 31 em residents (68%) completed the survey (81% male; 33% pgy1, 29% pgy2, 38% pgy3). four residents had prior experience with scribes. scribes were felt to have no effect on attending eps direct resident interaction time (mean score 4.5, sd 1.2), time spent bedside teaching (4.8, sd 0.9), or quality of teaching (4.9, sd 0.8), as well as no effect on residents' overall learning process (4.6, sd 1.1). however, residents felt positive about utilizing scribes at their future occupation site (6.0, sd 2.7). no response differences were noted for prior experience, training level, or sex. conclusion: when scribes are introduced at an em residency training site, residents of all training levels perceive it as a neutral interaction, when measured in terms of perceived time with attending eps and quality of the teaching when scribes are present. the effect of introduction of an electronic medical record on resident productivity in an academic emergency department shawn london, christopher sala university of connecticut school of medicine, farmington, ct background: there are little available data which describe the effect of implementation of an electronic medical record (emr) on provider productivity in the emergency department, and no studies which, to our knowledge, address this issue pertaining to housestaff in particular. objectives: we seek to quantify the changes in provider productivity pre-and post-emr implementation to support our hypothesis that resident clinical productivity based on patients seen per hour will be negatively affected by emr implementation. methods: the academic emergency department at hartford hospital, the principle clinical site in the university of connecticut emergency medicine residency, sees over 95,000 patients on an annual basis. this environment is unique in that pre-emr, patient tracking and orders were performed electronically using the sunrise system (eclipsys corp) for over 8 years prior to conversion to the allscripts ed emr in october, 2010 for all aspects of ed care. the investigators completed a random sample of days/evening/night/weekend shift productivity to obtain monthly aggregate productivity data (patients seen per hour) by year of training. results: there was an initial 4.2% decrease of in productivity for pgy-3 residents on average from 1.44 patients per hour on average in the three blocks preceding activation of the emr to 1.38 patients seen per hour compared in the subsequent three prior blocks. pgy 3 performance returned to baseline in the subsequent three months to 1.48 patients per hour. there was no change noted in patients seen per hour of pgy-1 and pgy-2 residents. conclusion: while many physicians tend to assume that emrs pose a significant barrier to productivity in the ed, in our academic emergency department, there was no lasting change on resident productivity based on the patients seen per hour metric. the minor decrease which did occur in pgy-3 residents was transient and was not apparent 3 months after the emr was implemented. our experience suggests that decrease in the rate of patients seen per hour in the resident population should not be considered justification to delay or avoid implementation of an emr in the emergency department. emory university, atlanta, ga; 2 children's healthcare of atlanta, atlanta, ga background: variation in physician practice is widely prevalent and highlights an opportunity for quality improvement and cost containment. monitoring resources used in the management of common pediatric emergency department (ed) conditions has been suggested as an ed quality metric. objectives: to determine if providing ed physicians with severity-adjusted data on resource use and outcomes, relative to their peers, can influence practice patterns. methods: data on resource use by physicians were extracted from electronic medical records at a tertiary pediatric ed for four common conditions in mid-acuity (emergency severity index level 3): fever, head injury, respiratory illness, and gastroenteritis. condition-relevant resource use was tracked for lab tests (blood count, chemistry, crp), imaging (chest x-ray, abdominal x-ray, head ct scan, abdominal ct scan), intravenous fluids, parenteral antibiotics, and intravenous ondansetron. outcome measures included admission to hospital and ed length of stay (los); 72-hr return to ed (rr) was used as a balancing measure. scorecards were constructed using box plots to show physicians their practice patterns relative to peers (the figure shows an example of the scorecard for gatroenteritis for one physician, showing resources use rates for iv fluids and labs). blinded scorecards were distributed quarterly for five quarters using rolling-year averages. a pre/post-intervention analysis was performed with sep 1, 2010 as the intervention date. fisher's exact and wilcoxon rank sum tests were used for analysis. results: we analyzed 45,872 patient visits across two hospitals (24,834 pre-and 21,038 post-intervention), comprising 17.6% of the total ed volume during the study period. patients were seen by 100 physicians (mean 462 patients/physician). the table shows overall physician practice in the pre-and post-intervention periods. significant reduction in resource use was seen for abdominal/pelvic ct scans, head ct scan, chest x-rays, iv ondansetron, and admission to hospital. ed los decreased from 129 min to 126 min (p = 0.0003). there was no significant change in 72-hr return rate during the study period (2.2% pre-, 2.0% post-intervention). conclusion: feedback on comprehensive practice patterns including resource use and quality metrics can influence physician practice on commonly used resources in the ed. billboards, via iphone application, twitter, and text messaging. there is a paucity of data describing the accuracy of publically posted ed wait times. objectives: to examine the accuracy of publicly posted wait times of four emergency departments within one hospital system. methods: a prospective analysis of four ed-posted wait times in comparison to the wait times for actual patients. the main hospital system calculated and posted ed wait times every twenty minutes for all four system eds. a consecutive sample of all patients who arrived 24/7 over a 4-week period during july and august 2011 was included. an electronic tracking system identified patient arrival date and the actual incurred wait time. data consisted of the arrival time, actual wait time, hospital census, budgeted hospital census, and the posted ed wait time. for each ed the difference was calculated between the publicly posted ed wait time at the time of patient's arrival and the patient's actual ed wait time. the average wait times and average wait time error between the ed sites were compared using a two-tailed student's t-test. the correlation coefficient between the differences in predicted/ actual wait times was also calculated for each ed. results: there were 8890 wait times within the four eds included in the analysis. the average wait time (in minutes) at each facility was: 64.0 (±62.4) for the main ed, 22.0 (±22.1) for freestanding ed (fed) #1, 25.0 (±25.6) for fed #2, and 10.0 (±12.6) for the small community ed. the average wait time error (in minutes) for each facility was 31(±61.2) for the main ed, 13 (±23.65) for fed #1, 17 (±26.65) for fed #2, and 1 (±11.9) for the community hospital ed. the results from each ed were statistically significant for both average wait time and average wait time error (p < 0.0001). there was a positive correlation between the average wait time and average wait time error, with r-values of 0.84, 0.83, 0.58, and 0.48 for the main ed, fed #1, fed #2, and the small community hospital ed, respectively. each correlation was statistically significant; however, no correlation was found between the number of beds available (budgeted-actual census) and average wait times. conclusion: publically posted ed wait times are accurate for facilities with less than 2000 ed visits per month. they are not accurate for eds with greater than 4000 visits per month. reduction of pre-analytic laboratory errors in the emergency department using an incentive-based system benjamin katz, daniel pauze, karen moldveen albany medical center, albany, ny background: over the last decade, there has been an increased effort to reduce medical errors of all kinds. laboratory errors have a significant effect on patient care, yet they are usually avoidable. several studies suggest that up to 90% of laboratory errors occur during the pre-or post-analytic phase. in other words, errors occur during specimen collection and transport or reporting of results, rather than during laboratory analysis itself. objectives: in an effort to reduce pre-analytic laboratory errors, the ed instituted an incentive-based program for the clerical staff to recognize and prevent specimen labeling errors from reaching the patient. this study sought to demonstrate the benefit of this incentive-based program. methods: this study examined a prospective cohort of ed patients over a three year period in a tertiary care academic ed with annual census of 72,000. as part of a continuing quality improvement process, laboratory specimen labeling errors are screened by clerical staff by reconciling laboratory specimen label with laboratory requisition labels. the number of ''near-misses'' or mismatched specimens captured by each clerk was then blinded to all patient identifiers and was collated by monthly intervals. due to poor performance in 2009, an incentive program was introduced in early 2010 by which the clerk who captured the most mismatched specimens would be awarded a $50 gift card on a quarterly basis. the total number of missed laboratory errors was then recorded on a monthly basis. investigational data were analyzed using bivariate statistics. background: most studies on operational research have been focused in academic medical centers, which typically have larger volumes of patients and are located in urban metropolitan areas. as cms core measures in 2013 begin to compare emergency departments (eds) on treatment time intervals, especially length of stay (los), it is important to explore if any differences exist inherent to patient volume. objectives: the objective of this study is to look at differences in operational metrics based on annual patient census. the hypothesis is that treatment time intervals and operational metrics differ amongst these different categories. methods: the ed benchmarking alliance has collected yearly operational metrics since 2004. as of 2010, there are 499 eds providing data across the united states. eds are stratified by annual volume for comparison in the following categories: <20k, 20-40k, 40-60k, and over 80k. in this study, metrics for eds with <20k visits per year were compared to those of different volumes, averaged from 2004-2010. mean values were compared to <20k visits as a reference point for statistical difference using t-tests to compare means with a p-value < 0.05 considered significant. results: as seen in the table, a greater percentage of high acuity of patients was seen in higher volume eds than in <20k eds. the percentage of patients transferred to another hospital was higher in <20k eds. a higher percentage arrived by ems and a higher percentage were admitted in higher volume eds when compared to <20k visits. in addition, the median los for both discharged and admitted patients and percentage who left before treatment was complete (lbtc) were higher in the higher volume eds. conclusion: lower volume eds have lower acuity when compared to higher volume eds. lower volume eds have shorter median los and left before treatment complete percentages. as cms core measures require hospitals to report these metrics, it will be important to compare them based on volume and not in aggregate. does the addition of a hands-free communication device improve ed interruption times? amy ernst, steven j. weiss, jeffrey a. reitsema university of new mexico, albuquerque, nm background: ed interruptions occur frequently. recently a hands-free communication device (vocera) was added to a cell phone and a pager in our ed. objectives: the purpose of the present study was to determine whether this addition improved interruption times. our hypothesis was that the device would significantly decrease length of time of interruptions. methods: this study was a prospective cohort study of attending ed physician calls and interruptions in a level i trauma center with em residency. interruptions included phone calls, ekg interpretations, pages to resuscitation, and other miscellaneous interruptions (including nursing issues, laboratory, ems, and radiology). we studied a convenience sampling intended to include mostly evening shifts, the busiest ed times. length of time the interruption lasted was recorded. data were collected for a comparison group pre-vocera. three investigators collected data including seven different addendings' interruptions. data were collected on a form, then entered into an excel file. data collectors' agreement was determined during two additional four hour shifts to calculate a kappa statistic. spss was used for data entry and statistical analysis. descriptive statistics were used for univariate data. chi-square and mann whitney u nonparametric test were used for comparisons. results: of the total 511 interruptions, 33% were phone calls, 24% were ekgs to be read, 18% were pages to resuscitation, and 25% miscellaneous. there were no significant differences in types of interruptions pre-vs. post-vocera. pre-vocera we collected 40 hours of data with 65 interruptions with a mean 1.6 per hour. post-vocera, 180 hours of data were collected with 446 interruptions with a mean 2.5 per hour. there was a significant difference in length of time of interruptions with an average of 9 minutes pre-vocera vs. 4 minutes post-vocera (p = 0.012, diff 4.9, 95% ci 1.8-8.1). vocera calls were significantly shorter than non-vocera calls (1 vs 6 minutes, p < 0.001). comparing data collectors for type of interruption during the same 4-hour shift resulted in a kappa (agreement) of 0.73. conclusion: the addition of a hands-free communication device may improve interruptions by shortening call length. '' talk background: analyses of patient flow through the ed typically focus on metrics such as wait time, total length of stay (los), or boarding time. however, little is known about how much interaction a patient has with clinicians after being placed in a room, or what proportion of the in-room visit is also spent ''waiting,'' rather than directly interacting with care providers. objectives: the objective was to assess the proportion of time, relative to the time in a patient care area, that a patient spends actively interacting with providers during an ed visit. methods: a secondary analysis of 29 audiotaped encounters of patients with one of four diagnoses (ankle sprain, back pain, head injury, laceration) was performed. the setting was an urban, academic ed. ed visits of adult patients were recorded from the time of room placement to discharge. audiotapes were edited to remove all downtime and non-patient-provider conversations. los and door-to-doctor times were abstracted from the medical record. the proportion of time the patient spent in direct conversation with providers (''talk-time'') was calculated as the ratio of the edited audio recording time to the time spent in a patient care area (talk-time = [edited audio time/(los -door-to-doctor)]). multiple linear regression controlling for time spent in patient care area, age, and sex was performed. results: the sample was 31% male with a mean age of 37 years. median los: 133 minutes (iqr: 88-169), median door-to-doctor: 42 minutes (iqr: 29-67), median time spent in patient care area: 65 minutes (iqr: 53-106). median time spent in direct conversation with providers was 16 minutes (iqr: 12-18), corresponding to a talk-time percentage of 19.2% (iqr: 14.7-24.6%). there were no significant differences based on diagnosis. regression analysis showed that those spending a longer time in a patient care area had a lower percentage of talk time (b = )0.11, p = 0.002). conclusion: although limited by sample size, these results indicate that approximately 80% of a patients' time in a care area is spent not interacting with providers. while some of the time spent waiting is out of the providers' control (e.g. awaiting imaging studies), this significant ''downtime'' represents an opportunity for both process improvement efforts to decrease downtime as well as the development of innovative patient education efforts to make the best use of the remaining downtime. degradation of emergency department operational data quality during electronic health record implementation michael j. ward, craig froehle, christopher j. lindsell university of cincinnati, cincinnati, oh background: process improvement initiatives targeted at operational efficiency frequently use electronic timestamps to estimate task and process durations. errors in timestamps hamper the use of electronic data to improve a system and may result in inappropriate conclusions about performance. despite the fact that the number of electronic health record (ehr) implementations is expected to increase in the u.s., the magnitude of this ehr-induced error is not well established. objectives: to estimate the change in the magnitude of error in ed electronic timestamps before and after a hospital-wide ehr implementation. methods: time-and-motion observations were conducted in a suburban ed, annual census 35,000, after receiving irb approval. observation was conducted 4 weeks pre-and 4 weeks post-ehr implementation. patients were identified on entering the ed and tracked until exiting. times were recorded to the nearest second using a calibrated stopwatch, and are reported in minutes. electronic data were extracted from the patient-tracking system in use pre-implementation, and from the ehr post-implementation. for comparison of means, independent t-tests were used. chi-square and fisher's t-tests were used for proportions, as appropriate. results: there were 263 observations; 126 before and 137 after implementation. the differences between observed times and timestamps were computed and found to be normally distributed. post-implementation, mean physician seen times along with arrival to bed, bed to physician, and physician to disposition intervals occurred before observation. physician seen timestamps were frequently incorrect and did not improve postimplementation. significant discrepancies (ten minutes or greater) from observed values were identified in timestamps involving disposition decision and exit from the ed. calculating service time intervals resulted in every service interval (except arrival to bed) having at least 15% of the times with significant discrepancies. it is notable that missing values were more frequent post-ehr implementation. conclusion: ehr implementation results in reduced variability of timestamps but reduced accuracy and an increase in missing timestamps. using electronic timestamps for operational efficiency assessment should recognize the magnitude of error, and the compounding of error, when computing service times. background: procedural sedation and analgesia is used in the ed in order to efficiently and humanely perform necessary painful procedures. the opposing physiological effects of ketamine and propofol suggest the potential for synergy, and this has led to interest in their combined use, commonly termed ''ketofol'', to facilitate ed procedural sedation. objectives: to determine if a 1:1 mixture of ketamine and propofol (ketofol) for ed procedural sedation results in a 13% or more absolute reduction in adverse respiratory events compared to propofol alone. methods: participants were randomized to receive either ketofol or propofol in a double-blind fashion according to a weight-based dosing protocol. inclusion criteria were age 14 years or greater, and asa class 1-3 status. the primary outcome was the number and proportion of patients experiencing an adverse respiratory event according to pre-defined criteria (the ''quebec criteria''). secondary outcomes were sedation consistency, sedation efficacy, induction time, sedation time, procedure time, and adverse events. results: a total of 284 patients were enrolled, 142 per group. forty-three (30%) patients experienced an adverse respiratory event in the ketofol group compared to 46 (32%) in the propofol group (difference 2%; 95% ci )9% to 13%; p = 0.798). thirty-eight (27%) patients receiving ketofol and 36 (25%) receiving propofol developed hypoxia, of whom three (2%) ketofol patients and 1 (1%) propofol patient received bag-valve-mask ventilation. sixty-five (46%) patients receiving ketofol and 93 (65%) receiving propofol required repeat medication dosing or lightened to a ramsay sedation score of 4 or less during their procedure (difference 19%; 95% ci 8% to 31%; p = 0.001). procedural agitation occurred in 5 patients (3.5%) receiving ketofol compared to 15 (11%) receiving propofol (difference 7.5%, 95% ci 1% to 14%). recovery agitation requiring treatment occurred in six patients (4%, 95% ci 2.0% to 8.9%) receiving ketofol. other secondary outcomes were similar between the groups. patients and staff were highly satisfied with both agents. conclusion: ketofol for ed procedural sedation does not result in a reduced incidence of adverse respiratory events compared to propofol alone. induction time, efficacy, and sedation time were similar; however, sedation depth appeared to be more consistent with ketofol. with propofol and its safety is well established. however, in 2010 cms enacted guidelines defining propofol as deep sedation and requiring administration by a physician. common edps practice had been one physician performing both the sedation and procedure. edps has proven safe under this one-physician practice. however, the 2010 guidelines mandated separate physicians perform each. objectives: the study hypothesis was that one-physician propofol sedation complication rates are similar to two-physician. methods: before and after, observational study of patients >17 years of age consenting to edps with propofol. edps completed with one physician were compared to those completed with two (separate physicians performing the sedation and the procedure). all data were prospectively collected. the study was completed at an urban level i trauma center. standard monitoring and procedures for edps were followed with physicians blinded to the objectives of this research. the frequency and incremental dosing of medication was left to the discretion of the treating physicians. the study protocol required an ed nurse trained in data collection to be present to record vital signs and assess for any prospectively defined complications. we used chi-square tests to compare the binary outcomes and asa scores across the time periods, and two-sample t-tests to test for differences in age between the two time periods. results: during the 2-year study period we enrolled 481 patients: 252 one-physician edps sedations and 229 3 (-7 to 13) also received bag-valve-mask 3 (2) [0.7 to 6) 1 (1) [0.1 to 4] 1 (-2 to 5) two-physician. all patients meeting inclusion criteria were included in the study. total adverse event rates were 4.4% and 3.1%, respectively (p = 0.450). the most common complications were hypotension and oxygen desaturation, and they respectively showed one-physcian rates of 2.0% and 0.8% and two-physician rates of 1.8% and 0.9% (p = 0.848 and 0.923.) the unsuccessful procedure rates were 4.0% vs 3.9% (p = 0.983). conclusion: this study demonstrated no significant difference in complication rates for propofol edps completed by one physician as compared to two. background: overdose patients are often monitored using pulse oximetry, which may not detect changes in patients on high-flow oxygen. objectives: to determine whether changes in end-tidal carbon dioxide (etco 2 ) detected by capnographic monitoring are associated with clinical interventions due to respiratory depression (crd) in patients undergoing evaluation for a decreased level of consciousness after a presumed drug overdose. methods: this was a prospective, observational study of adult patients undergoing evaluation for a drug overdose in an urban county ed. all patients received supplemental oxygen. patients were continuously monitored by trained research associates. the level of consciousness was recorded using the observer's assessment of alertness/sedation scale (oaa/s). vital signs, pulse oximetry, and oaa/s were monitored and recorded every 15 minutes and at the time of occurrence of any crd. respiratory rate and etco 2 were measured at five second intervals using a capno-stream20 monitor. crd included an increase in supplemental oxygen, the use of bag-valve-mask ventilations, repositioning to improve ventilation, and physical or verbal stimulus to induce respiration, and were performed at the discretion of the treating physicians and nurses. changes from baseline in etco 2 values and waveforms among patients who did or did have a clinical intervention were compared using wilcoxon rank sum tests. results: 100 patients were enrolled in the study (age 35, range 18 to 67, 62% male, median oaas 4, range 1 to 5). suspected overdoses were due to opioids in 34, benzodiazepines in 14, an antipsychotic in 14, and others in 38. the median time of evaluation was 165 minutes (range 20 to 725). crd occurred in 47% of patients, including an increase in o 2 in 38%, repositioning in 14%, and stimulation to induce respiration in 23%. 16% had an o 2 saturation of <93% (median 88, range 73 to 92) and 8% had a loss of etco 2 waveform at some time, all of whom had a crd. the median change in etco 2 from baseline was 5 mmhg, range 1 to 30. among patients with crd it was 14 mmhg, range 10 to 30, and among patients with no crd it was 5 mmhg, range 1 to 13 (p = 0.03). conclusion: the change in etco 2 from baseline was larger in patients who required clinical interventions than in those who did not. in patients on high-flow oxygen, capnographic monitoring may be sensitive to the need for airway support. how reliable are health care providers in reporting changes in etco 2 waveform anas sawas 1 , scott youngquist 1 , troy madsen 1 , matthew ahern 1 , camille broadwater-hollifield 1 , andrew syndergaard 1 , jared phelps 2 , bryson garbett 1 , virgil davis 1 1 university of utah, salt lake city, ut; 2 midwestern university, glendale, az background: etco 2 changes have been used in procedural sedation analgesia (psa) research to evaluate subclinical respiratory depression associated with sedation regiments. objectives: to evaluate the accuracy of bedside clinician reporting of changes in etco 2 . methods: this was a prospective, randomized, singleblind study conducted in ed setting from june 2010 until the present time. this study took place at an academic adult ed of a 405-bed (21 in the ed) and a level i trauma center. subjects were randomized to receive either ketamine-propofol or propofol according to a standardized protocol. loss of etco 2 waveforms for ‡ 15 sec were recorded. following sedation, questionnaires were completed by the sedating physicians. digitally recorded etco 2 waveforms were also reviewed by an independent physician and a trained research assistant (ra). to ensure the reliability of trained research assistants, we compared their analyses with the analyses of an independent physician for the first 41 recordings. the target enrollment was 65 patients in each group (n = 130 total). statistics were calculated using sas statistical software. results: 91 patients were enrolled; 53 (58.2%) are males and 38 (41.8%) are females. mean age was 44.93 ± 17.93 years. most participants did not have major risk factors for apnea or for further complications (86.3% were asa class 1 or 2). etco 2 waveforms were reviewed by 87 (95.6%) sedating physicians and 84 (92.3%) nurses at the bedside. there were 70 (76.9%) etco 2 waveforms recordings, 42 (60.0%) were reviewed by an independent physician and 70 (100%) were reviewed by an ra. a kappa test for agreement between independent physicians and ras was conducted on 41 recordings and there were no discordant pairs (kappa = 1). compared to sedating physicians, the independent physician was more likely to report etco 2 wave losses (or 1.37, 95% ci 1.08-1.73). compared to sedating physicians, ras were more likely to report etco 2 wave losses (or 1.39, 95% ci 1.14-1.70). conclusion: compared to sedating physicians at the bedside, independent physicians and ras were more likely to note etco 2 waveform losses. an independent review of recorded etco 2 waveform changes will be more reliable for future sedation research. background: comprehensive studies evaluating current practices of ed airway management in japan are lacking. many emergency physicians in japan still experience resistance regarding rapid sequence intubation (rsi). objectives: we sought to describe the success and complication rate of rsi with non-rsi. methods: design and setting: we conducted a multicenter prospective observational study using the jean registry of eds at 11 academic and community hospitals in japan during between 2010 and 2011. data fields include ed characteristics, patient and operator demographics, method of airway management, number of attempts, and adverse events. we defined non-rsi as intubation with sedation only, neuromuscular blockade only, and without medication. participants: all patients undergoing emergency intubation in ed were eligible for inclusion. cardiac arrest encounters were excluded from the analysis. primary analysis: we described rsi with non-rsi in terms of success rate on first attempt, within three attempts, and complication rate. we present descriptive data as proportions with 95% confidence intervals (cis). we report odds ratios (or) with 95% ci via chi-square testing. results: the database recorded 2710 intubations (capture rate 98%) and 1670 met the inclusion criteria. rsi was the initial method chosen in 489 (29%) and non-rsi in 1181 (71%). use of rsi varied among institutes from 0% to 79%. success cases of rsi on first and within three attempts are 353 intubations (72%, 95%ci 68%-76%) and 474 intubations (97%, 95%ci 95%-98%), respectively. the success cases of non-rsi on first and within three attempts are 724 intubations (61%, 95%ci 58%-64%) and 1105 intubations (94%, 95%ci 92%-95%). success rates of rsi on first and within three attempts are higher than non-rsi (or 1.64, 95%ci 1.30-2.06 and or 2.14, 95% ci 1.22-3.77, respectively). we recorded 67 complications in rsi (14%) and 165 in non-rsi (14%). there is no significant difference of complication rate between rsi and non-rsi (or 0.98, 95% ci 0.72-1.32). conclusion: in this multi-center prospective study in japan, we demonstrated a high degree of variation in use of rsi for ed intubation. additionally we found that success rate of rsi on first and within three attempts were both higher than non-rsi. this study has the limitation of reporting bias and confounding by indication. (originally submitted as a ''late-breaker.'') methods: this was a prospective, randomized, singleblind study conducted in the ed setting from june 2010 until the present time. this study took place at an academic adult ed of a 405-bed (21 in the ed) and a level i trauma center. subjects were randomized to receive either ketamine-propofol or propofol according to a standardized protocol. etco 2 waveforms were digitally recorded. etco 2 changes were evaluated by the sedating physicians at the bedside. recorded waveforms were reviewed by an independent physician and a trained research assistant (ra). to ensure the reliability of trained ras, we computed a kappa test for agreement between the analysis of independent physicians and ras for the first 41 recordings. a post-hoc analysis of the association between any loss, the number of losses, and total duration of loss of etco 2 waveform and crp was performed. on review we recorded the absence or presence of loss of etco 2 and the total duration in seconds of all lost etco 2 episodes ‡15 seconds. ors were calculated using sas statistical software. results: 91 patients were enrolled; 53 (58.2%) are males and 38 are (41.8%) females. 86.3% participants were asa class 1 or 2. waveforms were reviewed by 87 (95.6%) sedating physicians. there were 70 (76.9%) waveforms recordings, 42 (60.0%) were reviewed by an independent physician and 70 (100%) were reviewed by ras, where there were no discordant pairs (kappa = 1). there were 24 (26.4%) crp events. any loss of etco 2 was associated with a non-significant or of 4.06 (95% ci 0.75-21.9) for crp. however, the duration of etco 2 loss was significantly associated with crp with an or of 1.38 (95% ci 1.08-1.76) for each 30 second interval of lost etco 2 . the number of losses was significantly associated with the outcome (or 1.48, 95% ci 1.15-1.91). conclusion: defining subclinical respiratory depression as present or absent may be less useful than quantitative measurements. this suggests that risk is cumulative over periods of loss of etco 2 , and the duration of loss may be a better marker of sedation depth and risk of complications than classification of any loss. background: ed visits present an opportunity to deliver brief interventions (bis) to reduce violence and alcohol misuse among urban adolescents at risk for future injury. previous analyses demonstrated that a brief intervention resulted in reductions in violence and alcohol consequences up to 6 months. objectives: this paper describes findings examining the efficacy of bis on peer violence and alcohol misuse at 12 months. methods: patients (14-18 yrs) at an ed reporting past year alcohol use and aggression were enrolled in the rct, which included computerized assessment, and randomization to control group or bi delivered by a computer (cbi) or therapist assisted by a computer (tbi). baseline and 12 months included violence (peer aggression, peer victimization, violence related consequences) and alcohol (alcohol misuse, binge drinking, alcohol-related consequences). results: 3338 adolescents were screened (88% participation). of those, 726 screened positive for violence and alcohol use and were randomized; 84% completed 12-month follow-up. as compared to the control group, the tbi group showed significant reductions in peer aggression (p < 0.01) and peer victimization (p < 0.05) at 12 months. bi and control groups did not differ on alcohol-related variables at 12 months. conclusion: evaluation of the saferteens intervention one year following an ed visit provides support for the efficacy of computer-assisted therapist brief intervention for reducing peer violence. violence against ed health care workers: a 9-month experience terry kowalenko 1 , donna gates 2 , gordon gillespie 2 , paul succop 2 1 university of michigan, ann arbor, mi; 2 university of cincinnati, cincinnati, oh background: health care (hc) support occupations have an injury rate nearly 10 times that of the general sector due to assaults, with doctors and nurses nearly 3 times greater. studies have shown that the ed is at greatest risk of such events compared to other hc settings. objectives: to describe the incidence of violence in ed hc workers over 9 months. specific aims were to 1) identify demographic, occupational, and perpetrator factors related to violent events; 2) identify the predictors of acute stress response in victims; and 3) identify predictors of loss of productivity after the event. methods: longitudinal, repeated methods design was used to collect monthly survey data from ed hc workers (w) at six hospitals in two states. surveys assessed the number and type of violent events, and feelings of safety and confidence. victims also completed specific violent event surveys. descriptive statistics and a repeated measure linear regression model were used. results: 213 ed hcws completed 1795 monthly surveys, and 827 violent events were reported. the average per person violent event rate per 9 months was 4.15. 601 events were physical threats (3.01 per person in 9 months). 226 events were assaults (1.13 per person in 9 months). 501 violent event surveys were completed, describing 341 physical threats and 160 assaults with 20% resulting in injuries. 63% of the physical threats and 52% of the assaults were perpetrated by men. comparing occupational groups revealed significant differences between nurses and physicians for all reported events (p = 0.0048), with the greatest difference in physical threats (p = 0.0447). nurses felt less safe than physicians (p = 0.0041). physicians felt more confident than nurses in dealing with the violent patient (p = 0.013). nurses were more likely to experience acute stress than physicians (p < 0.001). acute stress significantly reduced productivity in general (p < 0.001), with a significant negative effect on ''ability to handle/ manage workload'' (p < 0.001) and ''ability to handle/ manage cognitive demands'' (p < 0.05). conclusion: ed hcws are frequent victims of violence perpetrated by visitors and patients. this violence results in injuries, acute stress, and loss of productivity. acute stress has negative consequences on the workers' ability to perform their duties. this has serious potential consequences to the victim as well as the care they provide to their patients. a randomized controlled feasibility trial of vacant lot greening to reduce crime and increase perceptions of safety eugenia c. garvin, charles c. branas perelman school of medicine at the university of pennsylvania, philadelphia, pa background: vacant lots, often filled with trash and overgrown vegetation, have been associated with intentional injuries. a recent quasi-experimental study found a significant decrease in gun crimes around vacant lots that had been greened compared with control lots. objectives: to determine the feasibility of a randomized vacant lot greening intervention, and its effect on police-reported crime and perceptions of safety. methods: for this randomized controlled feasibility trial of vacant lot greening, we partnered with the pennsylvania horticulture society (phs) to perform the greening intervention (cleaning the lots, planting grass and trees, and building a wooden fence around the perimeter). we analyzed police crime data and interviewed people living around the study vacant lots (greened and control) about perceptions of safety before and after greening. results: a total of 5200 sq ft of randomly selected vacant lot space was successfully greened. we used a master database of 54,132 vacant lots to randomly select 50 vacant lot clusters. we viewed each cluster with the phs to determine which were appropriate to send to the city of philadelphia for greening approval. the vacant lot cluster highest on the random list to be approved by the city of philadelphia was designated the intervention site, and the next highest was designated the control site. overall, 29 participants completed baseline interviews, and 21 completed follow-up interviews after 3 months. 59% of participants were male, 97% were black or african american, and 52% had a household income less than $25,000. unadjusted difference-in-differences estimates showed a decrease in gun assaults around greened vacant lots compared to control. regression-adjusted estimates showed that people living around greened vacant lots reported feeling safer after greening compared to those who lived around control vacant lots (p < 0.01). conclusion: conducting a randomized controlled trial of vacant lot greening is feasible. greening may reduce certain gun crimes and make people feel safer. however, larger prospective trials are needed to further investigate this link. screening for violence identifies young adults at risk for return ed visits for injury abigail hankin-wei, brittany meagley, debra houry emory university, atlanta, ga background: homicide is the second leading cause of death among youth ages 15-24. prior studies, in nonhealth care settings, have shown associations between violent injury and risk factors including exposure to community violence, peer behavior, and delinquency. objectives: to assess whether self-reported exposure to violence risk factors can be used to predict future ed visits for injuries. methods: we conducted a prospective cohort study in the ed of a southeastern us level i trauma center. patients aged 15-24 presenting for any chief complaint were included unless they were critically ill, incarcerated, or could not read english. recruitment took place over six months, by a trained research assistant (ra). the ra was present in the ed for 3-5 days per week, with shifts scheduled such that they included weekends and weekdays, over the hours from 8 am-8 pm. patients were offered a $5 gift card for participation. at the time of initial contact in the ed, patients completed a written questionnaire which included validated measures of the following risk factors: a) aggression, b) perceived likelihood of violence, c) recent violent behavior, d) peer behavior, e) community exposure to violence, and f) positive future outlook. at 12 months following the initial ed visit, the participants' medical records were reviewed to identify any subsequent ed visits for injury-related complaints. data were analyzed with chi-square and logistic regression analyses. results: 332 patients were approached, of whom 300 patients consented. participants' average age was 21.1 years, with 57% female, and 86% african american. return visits for injuries were significantly associated with hostile/aggressive feelings (rr 3.7, ci 1.42, 9) , self-reported perceived likelihood of violence (rr 5.16, ci 1.93, 13.78) , recent violent behavior (rr 3.16, ci 1.01, 9.88) , and peer group violence (rr 4.4, ci 1.72, 11.25) . these findings remained significant when controlling for participant sex. conclusion: a brief survey of risk factors for violence is predictive of return visit to the ed for injury. these findings identify a potentially important tool for primary prevention of violent injuries among young adults visiting the ed for both injury and non-injury complaints. background: sepsis is a commonly encountered disease in ed, with high mortality. while several clinical prediction rules (cpr) including meds, sirs, and curb-65 exist to facilitate clinicians in early recognition of risk of mortality for sepsis, most are of suboptimal performance. objectives: to derive a novel cpr for mortality of sepsis utilizing clinically available and objective predictors in ed. methods: we retrospectively reviewed all adult septic patients who visited the ed at a tertiary hospital during the year 2010 with two sets of blood cultures ordered by physicians. basic demographics, ed vital signs, symptoms and signs, underlying illnesses, laboratory findings, microbiological results, and discharge status were collected. multivariate logistic regressions were used to obtain a novel cpr using predictors with <0.1 p-value tested in univariate analyses. the existing cprs were compared with this novel cpr using auc. results: of 8699 included patients, 7.6% died in hospital, 51% had diabetes, 49% were older than 65 years of age, 21% had malignancy, and 16% had positive blood bacterial culture tests. predisposing factors including history of malignancy, liver disease, immunosuppressed status, chronic kidney disease, congestive heart failure, and older than 65 years of age were found to be associated with mortality (all p < 0.05). patients who developed mortality tended to have lower body temperature, narrower pulse pressure, higher percentage of red cell distribution width (rdw) and bandemia, higher blood urea nitrogen (bun), ammonia, and c-reactive protein level, and longer prothrombin time and activated partial thromboplastin time (aptt) (all p < 0.05). the most parsimonious cpr incorporating history of malignancy (or 2.3, 95% ci 1.9-2.7), prolonged aptt (3.0, 2.4-3.8), presence of bandemia (1.7, 1.4-2.0 results: there was poor agreement between the physician's unstructured assessment used in clinical practice and the guidelines put forth by the aha/acc/acep task force. ed physicians were more likely to assess a patient as low risk (42%), while aha guidelines were more likely to classify patients as intermediate (50%) or high (40%) risk. however, when comparing the patient's final acs diagnosis and the relation to the risk assessment value, ed physicians proved better predictors of high-risk patients who in fact had acs, while the aha/acc/acep guidelines proved better at correctly identifying low-risk patients who did not have acs. conclusion: in the ed, physicians are far more efficient at correctly placing patients with underlying acs into a high-risk category, while established criteria may be overly conservative when applied to an acute care population. further research is indicated to look at ed physicians' risk stratification and ensuing patient care to assess for appropriate decision making and ultimate outcomes. compartative conclusion: the amuse score was more specific, but the wells score was more sensitive for acute lower limb dvt in this cohort. there is no significant advantage in using the amuse over the wells score in ed patient with suspected dvt. background: the direct cost of medical care is not accurately reflected in charges or reimbursement. the cost of boarding admitted patients in the ed has been studied in terms of opportunity costs, which are indirect. the actual direct effect on hospital expenses has not been well defined. objectives: we calculate the difference to the hospital in the cost of caring for an admitted patient in the ed and in a non-critical care in-patient unit. methods: time-directed activity-based costing (tdabc) has recently been proposed as a method of determining the actual cost of providing medical services. tdabc was used to calculate the cost per patient bed-hour both in the ed and for an in-patient unit. the costs include nursing, nursing assistants, clerks, attending and resident physicians, supervisory salaries, and equipment maintenance. boarding hours were determined from placement of admission order to transfer to in-patient unit. a convenience sample of 100 consecutive non-critical care admissions was assessed to find the degree of ed physician involvement with boarded patients. results: the overhead cost per patient bed-hour in the ed was $60.80. the equivalent cost per bed-hour inpatient was $23.39, a differential of $37.41. there were 27,618 boarding hours for medical-surgical patients in 2010, a differential of $1,033,189.38 for the year. for the short-stay unit (no residents), the cost per patient hour was $11.36 and the boarding hours were 11,804. this resulted in a differential cost of $583,389.76, a total direct cost to the hospital of $1,616,579.14. review of 100 consecutive admissions showed no orders placed by the ed physician after decision-toadmit. conclusion: concentration of resources in the ed means considerably higher cost per unit of care as compared to an in-patient unit. keeping admitted patients boarding in the ed results in expensive underutilization. this is exclusive of significant opportunity costs of lost revenue from walk-out and diverted patients. this study includes the cost of teaching attendings and residents (ed and in-patient) . in a non-teaching setting, the differential would be less and the cost of boarding would be shared by a fee-for-service ed physician group as well as the hospital. improving identification of frequent emergency department users using a regional health information background: frequent ed users consume a disproportionate amount of health care resources. interventions are being designed to identify such patients and direct them to more appropriate treatment settings. because some frequent users visit more than one ed, a health information exchange (hie) may improve the ability to identify frequent ed users across sites of care. objectives: to demonstrate the extent to which a hie can identify the marginal increase in frequent ed users beyond that which can be detected with data from a single hospital. methods: data from 6/1/10 to 5/31/11 from the new york clinical information exchange (nyclix), a hie in new york city that includes ten hospitals, were analyzed to calculate the number of frequent ed users ( ‡4 visits in 30 days) at each site and across the hie. results: there were 10,555 (1% of total patients) frequent ed users, with 7,518 (71%) of frequent users having all their visits at a single ed, while 3,037 (29%) frequent users were identified only after counting visits to multiple eds (table 1) . site-specific increases varied from 7% to 62% (sd 16.5). frequent ed users accounted for 1% of patients, but for 6% of visits, averaging 9.74 visits per year, versus 1.55 visits per year for all other patients. 28.5% of frequent users visited two or more eds during the study period, compared to 10.6% of all other patients. conclusion: frequent ed users commonly visited multiple nyclix eds during the study period. the use of a hie helped identify many additional frequent users, though the benefits were lower for hospitals not located in the relative vicinity of another nyclix hospital. measures that take a community, rather than a single institution, into account may be more reflective of the care that the patient experiences. indocyanine background: due to their complex nature and high associated morbidity, burn injuries must be handled quickly and efficiently. partial thickness burns are currently treated based upon visual judgment of burn depth by the clinician. however, such judgment is only 67% accurate and not expeditious. laser doppler imaging (ldi) is far more accurate -nearly 96% after 3 days. however, it is too cumbersome for routine clinical use. laser assisted indocyanine green angiography (laicga) has been indicated as an alternative for diagnosing the depth of burn injuries, and possesses greater utility for clinical translation. as the preferred outcome of burn healing is aesthetic, it is of interest to determine if wound contracture can be predicted early in the course of a burn by laic-ga. objectives: determine the utility of early burn analysis using laicga in the prediction of 28-day wound contracture. methods: a prospective animal experiment was performed using six anesthetized pigs, each with 20 standardized wounds. differences in burn depth were created by using a 2.5 · 2.5 cm aluminum bar at three exposure times and temperatures: 70 degrees c for 30 seconds, 80 degrees c for 20 seconds, and 80 degrees c for 30 seconds. we have shown in prior validation experiments that these burn temperatures and times create distinct burn depths. laicga scanning, using lifecell spy elite, took place at 1 hour, 24 hours, 48 hours, 72 hours, and 1 week post burn. imaging was read by a blinded investigator, and perfusion trends were compared with day 28 post-burn contraction outcomes measured using imagej software. biopsies were taken on day 28 to measure scar tissue depth. results: deep burns were characterized by a blue center indicating poor perfusion while more superficial burns were characterized by a yellow-red center indicating perfusion that was close to that of the normal uninjured adjacent skin (see figure) . a linear relationship between contraction outcome and burn perfusion could be discerned as early as 1 hour post burn, peaking in strength at 24-48 hours post-burn. burn intensity could be effectively identified at 24 hours post-burn, although there was no relationship with scar tissue depth. conclusion: pilot data indicate that laicga using lifecell spy has the ability to determine the depth of injury and predict the degree of contraction of deep dermal burns within 1-2 days of injury with greater accuracy than clinical scoring. the objectives: we hypothesize that real-time monitoring of an integrated electronic medical records system and the subsequent firing of a ''sepsis alert'' icon on the electronic ed tracking board results in improved mortality for patients who present to the ed with severe sepsis or septic shock. methods: we retrospectively reviewed our hospital's sepsis registry and included all patients diagnosed with severe sepsis or septic shock presenting to an academic community ed with an annual census of 73,000 visits and who were admitted to a medical icu or stepdown icu bed between june 2009 and october 2011. in may 2010 an algorithm was added to our integrated medical records system that identifies patients with two sirs criteria and evidence of endorgan damage or shock on lab data. when these criteria are met, a ''sepsis alert'' icon (prompt) appears next to that patient's name on the ed tracking board. the system also pages an in-house, specially trained icu nurse who can respond on a prn basis and assist in the patient's management. 18 months of intervention data are compared with 11 months of baseline data. statistical analysis was via z-test for proportions. results: for ed patients with severe sepsis, the preand post-alert mortality was 19 of 125 (15%) and 34 of 378 (9%), respectively (p = 0.084; n = 503). in the septic shock group, the pre-and post-alert mortality was 27 of 92 (29%) and 48 of 172 (28%), respectively (p = 0.977). with ed and inpatient sepsis alerts combined, the severe sepsis subgroup mortality was reduced from 17% to 9% (p = 0.013; n = 622). conclusion: real-time ed ehr screening for severe sepsis and septic shock patients did not improve mortality. a positive trend in the severe sepsis subgroup was noted, and the combined inpatient plus ed data suggests statistical significance may be reached as more patients enter the registry. limitations: retrospective study, potential increased data capture post intervention, and no ''gold standard'' to test the sepsis alert sensitivity and specificity. ) . descriptive statistics were calculated. principal component analysis was used to determine questions with continuous response formats that could be aggregated. aggregated outcomes were regressed onto predictor demographic variables using multiple linear regression. results: 80/100 physicians completed the survey. physicians had a mean of 9.8 ± 9.0 years experience in the ed. 23.8% were female. eight physicians (10%) reported never having used the tool, while 70.8% of users estimated having used it more than five times. 75% of users cited the ''p'' alert on the etb as the most common notification method. most felt the ''p'' alert did not help them identify patients with pneumonia earlier (mean = 2.5 ± 1.2), but found it moderately useful in reminding them to use the tool (3.5 ± 1.3). physicians found the tool helpful in making decisions regarding triage, diagnostic studies, and antibiotic selection for outpatients and inpatients (3.7 ± 1.0, 3.6 ± 1.1, 3.6 ± 1.1, and 4.2 ± 0.9, respectively). they did not feel it negatively affected their ability to perform other tasks (1.6 ± 0.9). using multiple linear regression, neither age, sex, years experience, nor tool use frequency significantly predicted responses to questions about triage and antibiotic selection, technical difficulties, or diagnostic ordering. conclusion: ed physicians perceived the tool to be helpful in managing patients with pneumonia without negatively affecting workflow. perceptions appear consistent across demographic variables and experience. objectives: we seek to examine whether use of the salt device can provide reliable tracheal intubation during ongoing cpr. the dynamic model tested the device with human powered cpr (manual) and with an automated chest compression device (physio control lucas 2). the hypothesis is that the predictable movement of an automated chest compression device will make tracheal intubation easier than the random movement from manual cpr. methods: the project was an experimental controlled trial and took place in the ed at a tertiary referral center in peoria, illinois. this project was an expansion arm of a similarly structured study using traditional laryngoscopy. emergency medicine residents, attending physicians, paramedics, and other acls-trained staff were eligible for participation. in randomized order, each participant attempted intubation on a mannequin using the salt device with no cpr ongoing, during cpr with a manual compression, and during cpr with an automatic chest compression. participants were timed in their attempt and success was determined after each attempt. results: there were 43 participants in the trial. the success rates in the control group and the automated cpr group were both 86% (37/43) and the success rate in the manual cpr group was 79% (34/43 objectives: our primary hypothesis was that in fasting, asymptomatic subjects, larger fluid boluses would lead to proportional aortic velocity changes. our secondary endpoints were to determine inter-and intra-subject variation in aortic velocity measurements. methods: the authors performed a prospective randomized double-blinded trial using healthy volunteers. we measured the velocity time integral (vti) and maximal velocity (vmax) with an estimated 0-20°pulsed wave doppler interrogation of the left ventricular outflow in the apical-5 cardiac window. three physicians reviewed optimal sampling gate position, doppler angle and verified the presence of an aortic closure spike. angle correction technology was not used. subjects with no history of cardiac disease or hypertension fasted for 12 hours and were then randomly assigned to receive a normal saline bolus of 2 ml/kg, 10 ml/kg or 30 ml/kg over 30 minutes. aortic velocity profiles were measured before and after each fluid bolus. results: forty-two subjects were enrolled. mean age was 33 ± 10 (range 24 to 61) and mean body mass index 24.7 ± 3.2 (range 18.7 to 32). mean volume (in ml) for groups receiving 2 ml/kg, 10 ml/kg, and 30 ml/kg were 151, 748, and 2162, respectively. mean baseline vmax (in cm/s) of the 42 subjects was 108.4 ± 12.5 (range 87 to 133). mean baseline vti (in cm) was 23.2 ± 2.8 (range 18.2 to 30.0). pre-and post-fluid mean differences for vmax were )1.7 (± 10.3) and for vti 0.7 (± 2.7). aortic velocity changes in groups receiving 2 ml/kg, 10 ml/kg, and 30 ml/kg were not statistically significant (see table) . heart rate changes were not significant. background: clinicians recognize that septic shock is a highly prevalent, high mortality disease state. evidence supports early ed resuscitation, yet care delivery is often inconsistent and incomplete. the objective of this study was to discover latent critical barriers to successful ed resuscitation of septic shock. objectives: clinicians recognize that septic shock is a highly prevalent, high mortality disease state. evidence supports early ed resuscitation, yet care delivery is often inconsistent and incomplete. the objective of this study was to discover latent critical barriers to successful ed resuscitation of septic shock. methods: we conducted five 90-minute risk-informed in-situ simulations. ed physicians and nurses working in the real clinical environment cared for a standardized patient, introduced into their existing patient workload, with signs and symptoms of septic shock. immediately after case completion clinicians participated in a 30minute debriefing session. transcripts of these sessions were analyzed using grounded theory, a method of qualitative analysis, to identify critical barrier themes. results: fifteen clinicians participated in the debriefing sessions: four attending physicians, five residents, five nurses, and one nurse practitioner. the most prevalent critical barrier themes were: anchoring bias and difficulty with cognitive framework adaptation as the patient progressed to septic shock (n = 26), difficult interactions between the ed and ancillary departments (n = 22), difficulties with physician-nurse commu-nication and teamwork (n = 18), and delays in placing the central venous catheter due to perceptions surrounding equipment availability and the desire to attend to other competing interests in the ed prior to initiation of the procedure (n = 17 and 14). each theme was represented in at least four of the five debriefing sessions. participants reported the in-situ simulations to be a realistic representation of ed sepsis care. conclusion: in-situ simulation and subsequent debriefing provides a method of identifying latent critical areas for improvement in a care process. improvement strategies for ed-based septic shock resuscitation will need to address the difficulties in shock recognition and cognitive framework adaptation, physician and nurse teamwork, and prioritization of team effort. the background: the association between blood glucose level and mortality in critically ill patients is highly debated. several studies have investigated the association between history of diabetes, blood sugar level, and mortality of septic patients; however, no consistent conclusion could be drawn so far. objectives: to investigate the association between diabetes and initial glucose level and in-hospital mortality in patients with suspected sepsis from the ed. methods: we conducted a retrospective cohort study that consisted of all adult septic patients who visited the ed at a tertiary hospital during the year 2010 with two sets of blood cultures ordered by physicians. basic demographics, ed vital signs, symptoms and signs, underlying illnesses, laboratory findings, microbiological results, and discharge status were collected. logistic regressions were used to evaluate the association between risk factors, initial blood sugar level, and history of diabetes and mortality, as well as the effect modification between initial blood sugar level and history of diabetes. results: a total of 4997 patients with available blood sugar levels were included, of whom 48% had diabetes, 46% were older than 65 years of age, and 56% were male. the mortality was 6% (95% ci 5.3-6.7%). patients with a history of diabetes tended to be older, female, and more likely to have chronic kidney disease, lower sepsis severity (meds score), and positive blood culture test results (all p < 0.05). patients with a history of diabetes tended to have lower in-hospital mortality after ed visits with sepsis, controlling for initial blood sugar level (aor 0.72, 95% ci 0.56-0.92, p = 0.01). initial normal blood sugar seemed to be beneficial compared to lower blood sugar level for in-hospital mortality, controlled history of diabetes, sex, severity of sepsis, and age (aor 0.61, 95% ci 0.44-0.84, p = 0.002). the effect modification of diabetes on blood sugar level and mortality, however, was found to be not statistically significant (p = 0.09). conclusion: normal initial blood sugar level in ed and history of diabetes might be protective for mortality of septic patients who visited the ed. further investigation is warranted to determine the mechanism for these effects. methods: this irb-approved retrospective chart review included all patients treated with therapeutic hypothermia after cardiac arrest during 2010 at an urban, academic teaching hospital. every patient undergoing therapeutic hypothermia is treated by neurocritical care specialists. patients were identified by review of neurocritical care consultation logs. clinical data were dually abstracted by trained clinical study assistants using a standardized data dictionary and case report form. medications reviewed during hypothermia were midazolam, lorazepam, propofol, fentanyl, cisatracurium, and vecuronium. results: there were 33 patients in the cohort. median age was 57 (range 28-86 years), 67% were white, 55% were male, and 49% had a history of coronary artery disease. seizures were documented by continuous eeg in 11/33 (33%), and 20/33 (61%) died during hospitalization. most, 30/33 (91%), received fentanyl, 21/33 (64%) received benzodiazepine pharmacotherapy, and 23/33 (70%) received propofol. paralytics were administered to 23/33 (68%) patients, 14/33 (42%) with cisatracurium and 9/33 (27%) with vecuronium. of note, one patient required pentobarbital for seizure management. conclusion: sedation and neuromuscular blockade are common during management of patients undergoing therapeutic hypothermia after cardiac arrest. patients in this cohort often received analgesia with fentanyl, and sedation with a benzodiazepine or propofol. given the frequent use of sedatives and paralytics in survivors of cardiac arrest undergoing hypothermia, future studies should investigate the potential effect of these drugs on prognostication and survival after cardiac arrest. background: the use of therapeutic hypothermia (th) is a burgeoning treatment modality for post-cardiac arrest patients. objectives: we performed a retrospective chart review of patients who underwent post cardiac arrest th at eight different institutions across the united states. our objective was to assess how th is currently being implemented in emergency departments and assess the feasibility of conducting more extensive th research using multi-institution retrospective data. methods: a total of 94 charts with dates from 2008-2011 were sent for review by participating institutions of the peri-resuscitation consortium. of those reviewed, eight charts were excluded for missing data. two independent reviewers performed the review and the results were subsequently compared and discrepancies resolved by a third reviewer. we assessed patient demographics, initial presenting rhythm, time until th initiation, duration of th, cooling methods and temperature reached, survival to hospital discharge, and neurological status on discharge. results: the majority of cases of th had initial cardiac rhythms of asystole or pulseless electrical activity (55.2%), followed by ventricular tachycardia or fibrillation (34.5%), and in 10.3% the inciting cardiac rhythm was unknown. time to initiation of th ranged from 0-783 minutes with a mean time of 99 min (sd 132.5). length of th ranged from 25-2171 minutes with a mean time of 1191 minutes (sd 536). average minimum temperature achieved was 32.5°c, with a range from 27.6-36.7°c (sd 1.5°c). of the charts reviewed, 29 (33.3%) of the patients survived to hospital discharge and 19 (21.8%) were discharged relatively neurologically intact. conclusion: research surrounding cardiac arrest has always been difficult given the time and location span from pre-hospital care to emergency department to intensive care unit. also, as witnessed cardiac arrest events are relatively rare with poor survival outcomes, very large sample sizes are needed to make any meaningful conclusions about th. our varied and inconsistent results show that a multi-center retrospective review is also unlikely to provide useful information. a prospective multi-center trial with a uniform th protocol is needed if we are ever to make any evidence-based conclusions on the utility of th for post-cardiac arrest patients. serum results: mean la was 2.04, sd = 1.45. mean age was 4.5 years old, sd = 5.20. a statistically significant positive correlation was found between la and pulse, respiratory rate (rr), wbc, platelets, and los, while a significant negative correlation was seen with temperature and hco 3 -. when two subjects were dropped as possible outliers with la >10, it resulted in non-significant temperature correlation, but a significant negative correlation with age and bun was revealed. patients in the higher la group were more likely to be admitted (p = 0.0001) and have longer los. of the discharged patients, there was no difference in mean la level between those who returned (n = 25, mean la of 1.88, sd = 0.88) and those who did not (n = 154, mean la of 1.88, sd = 1.35), p = 0.99. furthermore, mean la levels for those with sepsis (n = 138, mean la of 2.18, sd = 1.75) did not differ from those without sepsis (n = 147, mean la of 1.9, sd = 1.08), p = 0.11. conclusion: higher la in pediatric patients presenting to the ed with suspected infection correlated with increased pulse, rr, wbc, platelets, and decreased bun, hco 3 -, and age. la may be predictive of hospitalization, but not of 3-day return rates or pediatric sepsis screening in the ed. background: mandibular fractures are one of the most frequently seen injuries in the trauma setting. in terms of facial trauma, madibular fractures account for 40-62% of all facial bone fractures. prior studies have demonstrated that the use of a tongue blade to screen these patients to determine whether a mandibular fracture is present may be as sensitive as x-ray. one study showed the sensitivity and specificity of the test to be 95.7% and 63.5%, respectively. in the last ten years, high-resolution computed tomography (hct) has replaced panoramic tomography (pt) as the gold standard for imaging of patients with suspected mandibular fractures. this study determines if the tongue blade test (tbt) remains as sensitive a screening tool when compared to the new gold standard of ct. objectives: the purpose of the study was to determine the sensitivity and specificity of the tbt as compared to the new gold standard of radiologic imaging, hct. the question being asked: is the tbt still useful as a screening tool for patients with suspected mandibular fractures when compared to the new gold standard of hct? methods: design: prospective cohort study. setting: an urban tertiary care level i trauma center. subjects: this study took place from 8/1/10 to 8/31/11 in which any person suffering from facial trauma presented. intervention: a tbt was performed by the resident physician and confirmed by the supervising attending physician. ct facial bones were then obtained for the ultimate diagnosis. inter-rater reliability (kappa) was calculated, along with sensitivity, specificity, accuracy, ppv, npv, likelihood ratio (lr) (+), and likelihood ratio (lr) (-) based on a 2 · 2 contingency tables generated. results: over the study period 85 patients were enrolled. inter-rater reliability was kappa = 0.93 (se +0.11). the table demonstrates the outcomes of both the tbt and ct facial bones for mandibular fracture. the following parameters were then calculated based on the contingency table: sensitivity 0.97 (ci 0.81-0.99), specificity 0.72 (ci 0.58-0.83), ppv 0.67 (ci 0.52-0.78), npv 0.97 (ci 0.87-0.99), accuracy 0.81, lr(+) 3.48 ), lr (-) 0.04 (ci 0.01-0.31). conclusion: the tbt is still a useful screening tool to rule out mandibular fractures in patients with facial trauma as compared to the current gold standard of hct. background: appendicitis is the most common surgical emergency occurring in children. the diagnosis of pediatric appendicitis is often difficult and computerized tomography (ct) scanning is utilized frequently. ct, although accurate, is expensive, time-consuming, and exposes children to ionizing radiation. radiologists utilize ultrasound for the diagnosis of appendicitis, but it may be less accurate than ct, and may not incorporate emergency physician (ep) clinical impression regarding degree of risk. objectives: the current study compared ep clinical diagnosis of pediatric appendicitis pre-and post-bedside ultrasonography (bus). methods: children 3-17 years of age were enrolled if their clinical attending physician planned to obtain a consultative ultrasound, ct scan, or surgical consult specific for appendicitis. most children in the study received narcotic analgesia to facilitate bus. subjects were initially graded for likelihood of appendicitis based on research physician-obtained history and physical using a visual analogue scale (vas). immediately subsequent to initial grading, research physicians performed a bus and recorded a second vas impression of appendicitis likelihood. two outcome measures were combined as the gold standard for statistical analysis. the post-operative pathology report served as the gold standard for subjects who underwent appendectomy, while post 2-week telephone follow-up was used for subjects who did not undergo surgery. various specific ultrasound measures used for the diagnosis of appendicitis were assessed as well. results: 29/56 subjects had pathology-proven appendicitis. one subject was pathology-negative post-appendectomy. of the 26 subjects who did not undergo surgery, none had developed appendicitis at the post 2-week telephone follow-up. pre-bus sensitivity was 48% (29-68%) while post-bus sensitivity was 79% (60-92%). both pre-and post-bus specificity was 96% (81-100%). pre-bus lr+ was 13 (2-93), while post-bus lr+ was 21 (3-148). pre-and post-bus lr-were 0.5 and 0.2, respectively. bus changed the diagnosis for 20% of subjects (9-32%). background: there are very little data on the normal distance between the glenoid rim and the posterior aspect of the humeral head in normal and dislocated shoulders. while shoulder x-rays are commonly used to detect shoulder dislocations, they may be inadequate, exacerbate pain in the acquisition of some views, and lead to delay in treatment, compared to bedside ultrasound evaluation. objectives: our objective was to compare the glenoid rim to humeral head distance in normal shoulders and in anteriorly dislocated shoulders. this is the first study proposing to set normal and abnormal limits. methods: subjects were enrolled in this prospective observation study if they had a chief complaint of shoulder pain or injury, and received a shoulder ultrasound as well as a shoulder x-ray. the sonographers were undergraduate students given ten hours of training to perform the shoulder ultrasound. they were blinded to the x-ray interpretation, which was used as the gold standard. we used a posterior-lateral approach, capturing an image with the glenoid rim, the humeral head, as well as the infraspinatus muscle. two parallel lines were applied to the most posterior aspect of the humeral head and the most posterior aspect of the glenoid rim. a line perpendicular to these lines was applied, and the distance measured. in anterior dislocations, a negative measurement was used to denote the fact that the glenoid rim is now posterior to the most posterior aspect of the humeral head. descriptive analysis was applied to estimate the mean and 25th to 75th interquartile range of normal and anteriorly dislocated shoulders. results: eighty subjects were enrolled in this study. there were six shoulder dislocations, however only four were anterior dislocations. the average distance between the posterior glenoid rim and the posterior humeral head in normal shoulders was 8.7 mm, with a 25th to 75th inter-quartile range of 6.7 mm to 11.9 mm. the distance in our four cases of anterior dislocation was )11 mm, with a 25th to 75th interquartile range of )10 mm to )12 mm. conclusion: the distance between the posterior humeral head to posterior glenoid rim may be 7 mm to 12 mm in patients presenting to the ed with shoulder pain but no dislocation. in contrast, this distance in anterior dislocations was greater than )10 mm. shoulder ultrasound may be a useful adjunct to x-ray for diagnosing anterior shoulder dislocations. conclusion: in this retrospective study, the presence of rv strain on focus significantly increases the likelihood of an adverse short term event from pulmonary embolism and its combination with hypotension performs similarly to other prognostic rules. background: burns are expensive and debilitating injuries, compromising both the structural integrity and vascular supply to skin. they exhibit a substantial potential to deteriorate if left untreated. jackson defined three ''zones'' to a burn. while the innermost coagulation zone and the outermost zone of hyperemia display generally predictable healing outcomes, the zone of stasis has been shown to be salvageable via clinical intervention. it has therefore been the focus of most acute therapies for burn injuries. while laser doppler imaging (ldi) -the current gold standard for burn analysis -has been 96% effective at predicting the need for second degree burn excision, its clinical translation is problematic, and there is little information regarding its ability to analyze the salvage of the stasis zone in acute injury. laser assisted indocyanine green dye angiography (laicga) also shows potential to predict such outcomes with greater clinical utility. objectives: to test the ability of ldi and laicga to predict interspace (zone of stasis) survival in a horizontal burn comb model. methods: a prospective animal experiment was performed using four pigs. each pig had a set of six dorsal burns created using a brass ''comb'' -creating four rectangular 10 · 20 mm full thickness burns separated by 5 · 20 mm interspaces. laicga and ldi scanning took place at 1 hour, 24 hours, 48 hours, and 1 week post burn using novadaq spy and moor ldi respectively. imaging was read by a blinded investigator, and perfusion trends were compared with interspace viability and contraction. burn outcomes were read clinically, evaluated via histopathology, and interspace contraction was measured using image j software. results: laicga data showed significant predictive potential for interspace survival. it was 83.3% predictive at 24 hours post burn, 75% predictive 48 hours post burn, and 100% predictive 7 days post burn using a standardized perfusion threshold. ldi imaging failed to predict outcome or contraction trends with any degree of reliability. the pattern of perfusion also appears to be correlated with the presence of significant interspace contraction at 28 days, with an 80% adherence to a power trendline. ventions, 11 isolation, 4 testing, 4 treatment, and 1 ''other'' category intervention were identified. one intervention involving school closures was associated with a 28% decrease in pediatric ed visits for respiratory illness. conclusion: most interventions were not tested in isolation, so the effect of individual interventions was difficult to differentiate. interventions associated with statistically significant decreases in ed crowding were school closures, as well as interventions in all categories studied. further study and standardization of intervention input, process, and outcome measures may assist in identifying the most effective methods of mitigating ed crowding and improving surge capacity during an influenza or other respiratory disease outbreak. communication background: the link between extended shift lengths, sleepiness, and occupational injury or illness has been shown, in other health care populations, to be an important and preventable public health concern but heretofore has not been fully described in emergency medical services (ems objectives: to assess the effect of an ed-based computer screening and referral intervention for ipv victims and to determine what characteristics resulted in a positive change in their safety. we hypothesized that women who were experiencing severe ipv and/or were in contemplation or action stages would be more likely to endorse safety behaviors. methods: we conducted the intervention for female ipv victims at three urban eds using a computer kiosk to deliver targeted education about ipv and violence prevention as well as referrals to local resources. all adult english-speaking non-critically ill women triaged to the ed waiting room were eligible to participate. the validated universal violence prevention screening protocol was used for ipv screening. any who disclosed ipv further responded to validated questionnaires for alcohol and drug abuse, depression, and ipv severity. the women were assigned a baseline stage of change (precontemplation, contemplation, action, or maintenance) based on the urica scale for readiness to change behavior surrounding ipv. participants were contacted at 1 week and 3 months to assess a variety of pre-determined actions such as moving out, to prevent ipv during that period. statistical analysis (chi-square testing) was performed to compare participant characteristics to the stage of change and whether or not they took protective action. results: a total of 1,474 people were screened and 154 disclosed ipv and participated in the full survey. 53.3% of the ipv victims were in the precontemplative stage of change, and 40.3% were in the contemplation stage. 110 women returned at 1 week of follow-up (71.4%), and 63 (40.9%) women returned at 3 months of followup. 55.5% of those who returned at 1 week, and 73% of those who returned at 3 months took protective action against further ipv. there was no association between the various demographic characteristics and whether or not a woman took protective action. conclusion: ed-based kiosk screening and health information delivery is both a feasible and effective method of health information dissemination for women experiencing ipv. stage of change was not associated with actual ipv protective measures. objectives: we present a pilot, head-to-head comparison of x26 and x2 effectiveness in stopping a motivated person. the objective is to determine comparative injury prevention effectiveness of the newer cew. methods: four humans had metal cew probe pairs placed. each volunteer had two probe pairs placed (one pair each on the right and left of the abdomen/inguinal region). superior probes were at the costal margin, 5 inches lateral of midline. inferior probes were vertically inferior at predetermined distances of 6, 9, 12, and 16 inches apart. each volunteer was given the goal of slashing a target 10 feet away with a rubber knife during cew exposure. as a means of motivation, they believed the exposure would continue until they reached the goal (in reality, the exposure was terminated once no further progress was made). each volunteer received one exposure from a x26 and a x2 cew. the exposure order was randomized with a 2-minute rest between them. exposures were recorded on a hi-speed, hi-resolution video. videos were reviewed and scored by six physician, kinesiology, and law officer experts using standardized criteria for effectiveness including degree of upper and lower extremity, and total body incapacitation, and degree of goal achievement. reviews were descriptively compared independently for probe spread distances and between devices. results: there were 8 exposures (4 pairs) for evaluation and no discernible, descriptive reviewer differences in effectiveness between the x26 and the x2 cews when compared. background: the trend towards higher gasoline prices over the past decade in the u.s. has been associated with higher rates of bicycle use for utilitarian trips. this shift towards non-motorized transportation should be encouraged from a physical activity promotion and sustainability perspective. however, gas price induced changes in travel behavior may be associated with higher rates of bicycle-related injury. increased consideration of injury prevention will be a critical component of developing healthy communities that help safely support more active lifestyles. objectives: the purpose of this analysis was to a) describe bicycle-related injuries treated in u.s. emergency departments between 1997 and 2009 and b) investigate the association between gas prices and both the incidence and severity of adult bicycle injuries. we hypothesized that as gas prices increase, adults are more likely to shift away from driving for utilitarian travel toward more economical non-motorized modes of transportation, resulting in increased risk exposure for bicycle injuries. methods: bicycle injury data for adults (16-65 years) were obtained from the national electronic injury surveillance system (neiss) database for emergency department visits between 1997-2009. the relationship between national seasonally adjusted monthly rates of bicycle injuries, obtained by a seasonal decomposition of time series, and average national gasoline prices, reported by the energy information administration, was examined using a linear regression analysis. results: monthly rates of bicycle injuries requiring emergency care among adults increase significantly as gas prices rise (p < 0.0001, see figure) . an additional 1,149 adult injuries (95% ci 963-1,336) can be predicted to occur each month in the u.s. (>13,700 injuries annually) for each $1 rise in average gasoline price. injury severity also increases during periods of high gas prices, with a higher percentage of injuries requiring admission. conclusion: increases in adult bicycle use in response to higher gas prices are accompanied by higher rates of significant bicycle-related injuries. supporting the use of non-motorized transportation will be imperative to address public health concerns such as obesity and climate change; however, resources must also be dedicated to improve bicycle-related injury care and prevention. background: this is a secondary analysis of data collected for a randomized trial of oral steroids in emergency department (ed) musculoskeletal back pain patients. we hypothesized that higher pain scores in the ed would be associated with more days out of work. objectives: to determine the degree to which days out of work for ed back pain patients are correlated with ed pain scores. methods: design: prospective cohort. setting: suburban ed with 80,000 annual visits. participants: patients aged 18-55 years with moderately severe musculoskeletal back pain from a bending or twisting injury £ 2 days before presentation. exclusion criteria included nonmusculoskeletal etiology, direct trauma, motor deficits, and employer-initiated visits. observations: we captured initial and discharge ed visual analog pain scores (vas) on a 0-10 scale. patients were contacted approximately 5 days after discharge and queried about the days out of work. we plotted days out of work versus initial vas, discharge vas, and change in vas and calculated correlation coefficients. using the bonferroni correction because of multiple comparisons, alpha was set at 0.02. results: we analyzed 67 patients for whom complete data were available. the mean age was 40 ± 9 years and 30% were female. the average initial and discharge ed pain scales were 8.0 ± 1.5 and 5.7 ± 2.2, respectively. on follow-up, 88% of patients were back to work and 36% did not lose any days of work. for the plots of the days out of work versus the initial and discharge vas and the change in the vas, the correlation coefficients (r 2 ) were 0.03 (p = 0.17), 0.08 (p = 0.04), and 0.001 (p = 0.87), respectively. conclusion: for ed patients with musculoskeletal back pain, we found no statistically significant correlation between days out of work and ed pain scores. background: conducted electrical weapons (cews) are common law enforcement tools used to subdue and repel violent subjects and, therefore, prevent further injury or violence from occurring in certain situations. the taser x2 is a new generation of cew that has the capability of firing two cartridges in a ''semi-automatic'' mode, and has a different electrical waveform and different output characteristics than older generation technology. there have been no data presented on the human physiologic effects of this new generation cew. objectives: the objective of this study was to evaluate the human physiologic effects of this new cew. methods: this was a prospective, observational study of human subjects. an instructor shot subjects in the abdomen and upper thigh with one cartridge, and subjects received a 10-second exposure from the device. measured variables included: vital signs, continuous spirometry, pre-and post-exposure ecg, intra-exposure echocardiography, venous ph, lactate, potassium, ck, and troponin. results: ten subjects completed the study (median age 31.5, median bmi 29.4, 80% male). there were no important changes in vital signs or in potassium. the median increase in lactate during the exposure was 1.2, range 0.6 to 2.8. the median change in ph was )0.031, range )0.011 to 0.067. no subject had a clinically relevant ecg change, evidence of cardiac capture, or positive troponin up to 24 hours after exposure. the median change in creatine kinase (ck) at 24 hours was 313, range )40 to 3418. there was no evidence of impairment of breathing by spirometry. baseline median minute ventilation was 14.2, which increased to 21.6 during the exposure (p = 0.05), and remained elevated at 21.6 post-exposure (p = 0.01). conclusion: we detected a small increase in lactate and decrease in ph during the exposure, and an increase in ck 24 hours after the exposure. the physiologic effects of the x2 device appear similar to previous reports for ecd devices. use background: public bicycle sharing (bikeshare) programs are becoming increasingly common in the us and around the world. these programs make bicycles easily accessible for hourly rental to the public. there are currently 15 active bikeshare programs in cities in the us, and more than 30 programs are being developed in cities including new york and chicago. despite the importance of helmet use, bikeshare programs do not provide the opportunity to purchase or rent helmets. while the programs encourage helmet use, no helmets are provided at the rental kiosks. objectives: we sought to describe the prevalence of helmet use among adult users of bikeshare programs and users of personal bicycles in two cities with recently introduced bicycle sharing programs (boston, ma and washington, dc). methods: we performed a prospective observational study of bicyclists in boston, ma and washington, dc. trained observers collected data during various times of the day and days of the week. observers recorded the sex of the bicycle operator, type of bicycle, and helmet use. all bicycles that passed a single stationary location in any direction for a period of between 30 and 90 minutes were recorded. data are presented as frequencies of helmet use by sex, type of bicycle (bikeshare or personal), time of the week (weekday or weekend), and city. logistic regression was used to estimate the odds ratio for helmet use controlling for type of bicycle, sex, day of week, and city. results: there were 43 observation periods in two cities at 36 locations. 3,073 bicyclists were observed. there were 562 (18.2%) bicylists riding bikeshare bicycles. overall helmet use was 45.5%, although helmet use varied significantly with sex, day of use, and type of bicycle (see figure) . bikeshare users were helmeted at a lower rate compared to users of personal bicycles (19.2% vs 51.4%). logistic regression, controlling for type of bicycle, sex, day of week, and city demonstrate that bikeshare users had higher odds of riding unhelmeted (or 4.34, 95% ci 3.47-5.50). women had lower odds of riding unhelmeted (or 0.62, 0.52-0.73), while weekend riders were more likely to ride unhelmeted (or 1.32, 1.12-1.55). conclusion: use of bicycle helmets by users of public bikeshare programs is low. as these programs become more popular and prevalent, efforts to increase helmet use among users should increase. background: abusive head trauma (aht) represents one of the most severe forms of traumatic brain injury (tbi) among abused infants with 30% mortality. young adult males account for 75% of the perpetrators. most aht prevention programs are hospital-based and reach a predominantly female audience. there are no published reports of school-based aht prevention programs to date. objectives: 1. to determine whether a high schoolbased aht educational program will improve students' knowledge of aht and parenting skills. 2. to evaluate the feasibility and acceptability of a school-based aht prevention program. methods: this program was based on an inexpensive commercially available program developed by the national center on shaken baby syndrome. the program was modified to include a 60-minute interactive presentation that teaches teenagers about aht, parenting skills, and caring for inconsolable crying infants. the program was administered in three high schools in flint, michigan during spring 2011. student's knowledge was evaluated with a 17-item written test administered pre-intervention, post-intervention, and two months after program completion. program feasibility and acceptability were evaluated through interviews and surveys with flint area school social workers, parent educators, teachers, and administrators. results: in all, 342 high school students (40% male) participated. of these, 317 (92.7%) completed the pretest and post-test with 171 (50%) completing the twomonth follow-up test. the mean pre-intervention, postintervention, and two-month follow-up scores were 53%, 87%, and 90% respectively. from pre-test to posttest, mean score improved 34%, p < 0.001. this improvement was even more profound in young males, whose mean post-test score improved by 38%, p < 0.001. of the 69 participating social workers, parent educators, teachers, and administrators, 97% ranked the program as feasible and acceptable. conclusion: students participating in our program showed an improvement in knowledge of aht and parenting skills which was retained after two months. teachers, social workers, parent educators, and school administrators supported the program. this local pilot program has the potential to be implemented on a larger scale in michigan with the ultimate goal of reducing aht amongst infants. will background: fear of litigation has been shown to affect physician practice patterns, and subsequently influence patient care. the likelihood of medical malpractice litigation has previously been linked with patient and provider characteristics. one common concern is that a patient may exaggerate symptoms in order to obtain monetary payouts; however, this has never been studied. objectives: we hypothesize that patients are willing to exaggerate injuries for cash settlements and that there are predictive patient characteristics including age, sex, income, education level, and previous litigation. methods: this prospective cross-sectional study spanned june 1 to december 1, 2011 in a philadelphian urban tertiary care center. any patient medically stable enough to fill out a survey during study investigator availability was included. two closed-ended paper surveys were administered over the research period. standard descriptive statistics were utilized to report incidence of: patients who desired to file a lawsuit, patients previously having filed lawsuits, and patients willing to exaggerate the truth in a lawsuit for a cash settlement. chi-square analysis was performed to determine the relationship between patient characteristics and willingness to exaggerate injuries for a cash settlement. results: of 126 surveys, 11 were excluded due to incomplete data, leaving 115 for analysis. the mean age was 39 with a standard deviation of 16, and 40% were male. the incidence of patients who had the desire to sue at the time of treatment was 9%. the incidence of patients who had filed a lawsuit in the past was 35%. of those patients, 26% had filed multiple lawsuits. fifteen percent [95% ci 9-23%] of all patients were willing to exaggerate injuries for cash settlement. sex and income were found to be statistically significant predictors of willingness to exaggerate symptoms: 22% of females vs. 4% of males were willing to exaggerate (p = 0.01), and 20% of people with income less than $100,000/yr vs. 0% of those with income over $100,000/ yr were willing to exaggerate (p = 0.03). conclusion: patients at a philadelphian urban tertiary center admit to willingness to exaggerate symptoms for a cash settlement. willingness to exaggerate symptoms is associated with female sex and lower income. background: current data suggest that as many as 50% of patients presenting to the ed with syncope leave the hospital without a defined etiology. prior studies have suggested a prevalence of psychiatric disease as high as 26% in patients with syncope of unknown etiology. objectives: to determine whether psychiatric disease and substance abuse are associated with an increased incidence of syncope of unknown etiology. methods: prospective, observational, cohort study of consecutive ed patients ‡18 presenting with syncope was conducted between 6/03 and7/06. patients were queried in the ed and charts reviewed about a history of psychiatric disease, use of psychiatric medication, substance abuse, and duration. data were analyzed using sas with chi-square and fisher's exact tests. results: we enrolled 519 patients who presented to the ed after syncope, 159 of whom did not have an identifiable etiology for their syncopal event. 36.5% of those without an identifiable etiology were male. 166 (32%) patients had a history of or current psychiatric disease (42% male), and 55 patients (11%) had a history of or current substance abuse (60% male). among males with psychiatric disease, 39% had an unknown etiology of their syncopal event, compared to 22% of males without psychiatric disease (p = 0.009). similarly, among all males with a history of substance abuse, 45% had an unknown etiology, as compared to 24% of males without a history of substance abuse (p = 0.01). a similar trend was not identified in elderly females with psychiatric disease (p = 0.96) or substance abuse (p = 0.19). however, syncope of unknown etiology was more common among both men and women under age 65 with a history of substance abuse (47%) compared to those without a history of substance abuse (27%; p = 0.01). conclusion: our results suggest that psychiatric disease and substance abuse are associated with increased incidence of syncope of unknown etiology. patients evaluated in the ed or even hospitalized with syncope of unknown etiology may benefit from psychiatric screening and possibly detoxification referral. this is particularly true in men. (originally submitted as a ''late-breaker.'') scope background: after discharge from an emergency department (ed), pain management often challenges parents, who significantly under-treat their children's pain. rapid patient turnover and anxiety make education about home pain treatment difficult in the ed. video education standardizes information and circumvents insufficient time and literacy. objectives: to evaluate the effectiveness of a 6-minute instructional video for parents that targets common misconceptions about home pain management. methods: we conducted a randomized, double-blinded clinical trial of parents of children ages 1-18 years who presented with a painful condition, were evaluated, and discharged home in june and july 2011. parents were randomized to a pain management video or an injury prevention control video. primary outcome was the proportion of parents who gave pain medication at home. these data were recorded in a home pain diary and analyzed using a chi-square test. parents' knowledge about pain treatment was tested before, immediately following, and 2 days after intervention. mcnemar's test statistic determined odds that knowledge correlated with the intervention group. results: 100 parents were enrolled: 59 watched the pain education video, and 41 the control video. 72.9% completed follow up, providing information about home pain education use. significantly more parents provided at least one dose of pain medication to their children after watching the educational video: 96% vs. 80% (difference 16%, 95% ci 7.8%, 31.3%). the odds the parent had correct knowledge about pain treatment significantly improved immediately following the educational video for knowledge about pain scores (p = 0.04), the effect of pain on function (p < 0.01), and pain medication misconceptions (p < 0.01). these significant differences in knowledge remained 3 days after the video intervention. the educational video about home pain treatment viewed by parents significantly increased the proportion of children receiving pain medication at home and significantly improved knowledge about at-home pain management. videos are an efficient tool to provide medical advice to parents that improves outcomes for children. methods: this was a prospective, observational study of consecutive admitted cpu patients in a large-volume academic urban ed. cardiology attendings round on all patients and stress test utilization is driven by their recommendation. eligibility criteria include: age>18, aha low/intermediate risk, nondynamic ecgs, and normal initial troponin i. patients >75 and with a history of cad or co-existing active medical problem were excluded. based on prior studies and our estimated cpu census and demographic distribution, we estimated a sample size of 2,242 patients in order to detect a difference in stress utilization of 7% (2-tailed, a = 0.05, b = 0.8). we calculated a timi risk prediction score and a diamond & forrester (d&f) cad likelihood score on each patient. t-tests were used for univariate comparisons of demographics, cardiac comorbidities, and risk scores. logistic regression was used to estimate odds ratios (ors) for receiving testing based on race, controlling for insurance and either timi or d&f score. results: over 18 months, 2,451 patients were enrolled. mean age was 53 ± 12, and 54% (95% ci 52-56) were female. sixty percent (95% ci 58-62) were caucasian, 12% (95% ci 10-13) african american, and 24% (95% ci 23-26) hispanic. mean timi and d&f scores were 0.5 (95% ci 0.5-0.6) and 38% (95% ci 37-39). the overall stress testing rate was 52% (95% ci 50-54). after controlling for insurance status and timi or d&f scores, african american patients had significantly decreased odds of stress testing (or timi 0.67 (95% ci 0.52-0.88), or d&f 0.68 (95% ci 0.51-0.89)). hispanics had significantly decreased odds of stress testing in the model controlling for d&f (or d&f 0.78 (95% ci 0.63-0.98)). conclusion: this study confirms that disparities in the workup of african american patients in the cpu are similar to those found in the general ed and the outpatient setting. further investigation into the specific provider or patient level factors contributing to this bias is necessary. the outcomes for hf and copd were sae 11.6%, 7.8%; death 2.3%, 1.0%. we found univariate associations with sae for these walk test components: too ill to walk (both hf, copd p < 0.0001); highest heart rate ‡110 (hf p = 0.02, copd p = 0.10); lowest sao 2 < 88% (hf p = 0.42, copd p = 0.63); borg score ‡5 (hf p = 0.47, copd p = 0.52); walk test duration £ 1 minute (hf p = 0.07. copd p = 0.22). after adjustment for multiple clinical covariates with logistic regression analyses, we found ''walk test heart rate ‡110'' had an odds ratio of 1.9 for hf patients and ''too ill to start the walk test'' had an odds ratio of 3.5 for copd patients. conclusion: we found the 3-minute walk test to be easy to administer in the ed and that maximum heart rate and inability to start the test were highly associated with adverse events in patients with exacerbations of hf and copd, respectively. we suggest that the 3-minute walk test be routinely incorporated into the assessment of hf and copd patients in order to estimate risk of poor outcomes. the objectives: the objective of this study was to investigate differences in consent rates between patients of different demographic groups who were invited to participate in minimal-risk clinical trials conducted in an academic emergency department. methods: this descriptive study analyzed prospectively collected data of all adult patients who were identified as qualified participants in ongoing minimal risk clinical trials. these trials were selected for this review because they presented minimal factors known to be associated background: increasing rates of patient exposure to computerized tomography (ct) raise questions about appropriateness of utilization, as well as patient awareness of radiation exposure. despite rapid increases in ct utilization and published risks, there is no national standard to employ informed consent prior to radiation exposure from diagnostic ct. use of written informed consent for ct (icct) in our ed has increased patient understanding of the risks, benefits, and alternatives to ct imaging. our team has developed an adjunct video educational module (vem) to further educate ed patients about the ct procedure. objectives: to assess patient knowledge and preferences regarding diagnostic radiation before and after viewing vem. methods: the vem was based on icct currently utilized at our tertiary care ed (census 37,000 patients/ year). icct is written at an 8th grade reading level. this fall, vem/icct materials were presented to a convenience sample of patients in the ed waiting room 9 am-7 pm, monday-sunday. patients who were <18 years of age, critically ill, or with language barrier were excluded. to quantify the educational value of the vem, a six-question pretest was administered to assess baseline understanding of ct imaging. the patients then watched the vem via ipad (macintosh) and reviewed the consent form. an eight-question post-test was then completed by each subject. no phi were collected. pre-and post-test results were analyzed using mcnemar's test for individual questions and a paired t-test for the summed score (sas version 9.2). results: 100 patients consented and completed the survey. the average pre-test score for subjects was poor, 66% correct. review of vem/icct materials increased patient understanding of medical radiation as evidenced by improved post-test score to 79%. mean improvement between tests was 13% (p < 0.0001). 78% of subjects responded that they found the materials helpful, and that they would like to receive icct. conclusion: the addition of a video educational module improved patient knowledge regarding ct imaging and medical radiation as quantified by pre-and posttesting. patients in our study sample reported that they prefer to receive icct. by educating patients about the risks associated with ct imaging, we increase informed, shared decision making -an essential component of patient-centered care. does objectives: we sought to determine the relationship between patients' pain scores and their rate of consent to ed research. we hypothesized that patients with higher pain scores would be less likely to consent to ed research. methods: retrospective observational cohort study of potential research subjects in an urban academic hospital ed with an average annual census of approximately 70,000 visits. subjects were adults older than 18 years with chief complaint of chest pain within the last 12 hours, making them eligible for one of two cardiac biomarker research studies. the studies required only blood draws and did not offer compensation. two reviewers extracted data from research screening logs. patients were grouped according to pain score at triage, pain score at the time of approach, and improvement in pain score (triage score -approach score). the main outcome was consent to research. simple proportions for consent rates by pain score tertiles were calculated. two multivariate logistic regression analyses were performed with consent as outcome and age, race, sex, and triage or approach pain score as predictors. results: overall, 396 potential subjects were approached for consent. patients were 58% caucasian, 49% female, and with an average age of 57 years. six patients did not have pain scores recorded at all and 48 did not have scores documented within 2 hours of approach and were excluded from relevant analyses. overall, 80.1% of patients consented. consent rates by tertiles at triage, at time of approach, and by pain score improvement are shown in tables 1 and 2. after adjusting for age, race, and sex, neither triage (p = 0.75) nor approach (p = 0.65) pain scores predicted consent. conclusion: research enrollment is feasible even in ed patients reporting high levels of pain. patients with modest improvements in pain levels may be more likely to consent. future research should investigate which factors influence patients' decisions to participate in ed research. conclusion: in this multicenter study of children hospitalized with bronchiolitis neither specific viruses nor their viral load predicted the need for cpap or intubation, but young age, low birth weight, presence of apnea, severe retractions, and oxygen saturation <85% did. we also identified that children requiring cpap or intubation were more likely to have mothers who smoked during pregnancy and a rapid respiratory worsening. mechanistic research in these high-risk children may yield important insights for the management of severe bronchiolitis. brigham & women's hospital, boston, ma background: siblings and children who share a home with a physically abused child are thought to be at high risk for abuse. however, rates of injury in these children are unknown. disagreements between medical and child protective services professionals are common and screening is highly variable. objectives: our objective was to measure the rates of occult abusive injuries detected in contacts of abused children using a common screening protocol. methods: this was a multi-center, observational cohort study of 20 child abuse teams who shared a common screening protocol. data were collected between jan 15, 2010 and april 30, 2011 for all children <10 years undergoing evaluation for physical abuse and their contacts. for contacts of abused children, the protocol recommended physical examination for all children <5 years, skeletal survey and physical exam for children <24 months, and physical exam, skeletal survey, and neuroimaging for children <6 months old. results: among 2,825 children evaluated for abuse, 618 met criteria as ''physically abused'' and these had 477 contacts. for each screening modality, screening was completed as recommended by the protocol in approximately 75% of cases. of 134 contacts who met criteria for skeletal survey, new injuries were identified in 16 (12.0%). none of these fractures had associated findings on physical examination. physical examination identified new injuries in 6.2% of eligible contacts. neuroimaging failed to identify new injuries among 25 eligible contacts less than 6 months old. twins were at significantly increased risk of fracture relative to other nontwin contacts (or 20.1). conclusion: these results support routine skeletal survey for contacts of physically abused children <24 months old, regardless of physical examination findings. even for children where no injuries are identified, these results demonstrate that abuse is common among children who share a home with an abused child, and support including contacts in interventions (foster care, safety planning, social support) designed to protect physically abused children. methods: this was a retrospective study evaluating all children presenting to eight paediatric, universityaffiliated eds during one year in 2010-2011. in each setting, information regarding triage and disposition were prospectively registered by clerks in the ed database. anonymized data were retrieved from the ed computerized database of each participating centre. in the absence of a gold standard for triage, hospitalisation, admission to intensive care unit (icu), length of stay in the ed, and proportion of patients who left without being seen by a physician (lwbs) were used as surrogate markers of severity. the primary outcome measure was the association between triage level (from 1 to 5) and hospitalisation. the association between triage level and dichotomous outcomes was evaluated by a chi-square test, while a student's t-test was used to evaluate the association between triage level and length of stay. it was estimated that the evaluation of all children visiting these eds for a one year period would provide a minimum of 1,000 patients in each triage level and at least 10 events for outcomes having a proportion of 1% or more. results: a total of 404,841 children visited the eight eds during the study period. pooled data demonstrated hospitalisation proportions of 59%, 30%, 10%, 2%, and 0.5% for patients triaged at level 1, 2,3, 4, and 5 respectively (p < 0.001). there was also a strong association between triage levels and admission to icu (p < 0.001), the proportion of children who lwbs (p < 0.001), and length of stay (p < 0.001). background: parents frequently leave the emergency department (ed) with incomplete understanding of the diagnosis and plan, but the relationship between comprehension and post-care outcomes has not been well described. objectives: to explore the relationship between comprehension and post-discharge medication safety. methods: we completed a planned secondary analysis of a prospective observational study of the ed discharge process for children aged 2-24 months. after discharge, parents completed a structured interview to assess comprehension of the child's condition, the medical team's advice, and the risk of medication error. limited understanding was defined as a score of 3-5 from 1 (excellent) to 5 (poor). risk of medication error was defined as a plan to use over-the-counter cough/cold medication and/or an incorrect dose of acetaminophen (measured by direct observation at discharge or reported dose at follow-up call). parents identified as at risk received further instructions from their provider. the primary outcome was persistent risk of medication error assessed at phone interview 5-10 days post-discharge. a major barrier to administering analgesics to children is the perceived discomfort of intravenous access. the delivery of intranasal analgesia may be a novel solution to this problem. objectives: we investigated whether the addition of the mucosal atomizer device (mad) as an alternative for fentanyl delivery would improve overall fentanyl administration rates in pediatric patients transported by a large urban ems system. we performed a historical control trial comparing the rate of pediatric fentanyl administration 6 months before and 6 months after the introduction of the mad. study subjects were pediatric trauma patients (age <16 years) transported by a large urban ems agency. the control group was composed of patients treated in the 6 months before introduction of the mad. the experimental group included patients treated in the 6 months after the addition of the mad. two physicians reviewed each chart and determined whether the patient met predetermined criteria for the administration of pain medication. a third reviewer resolved any discrepancies. fentanyl administration rates were measured and compared between the two groups. we used two-sample t-tests and chi-square tests to analyze our data. results: 228 patients were included in the study: 137 patients in the pre-mad group and 91 in the post-mad group. there were no significant differences in the demographic and clinical characteristics of the two groups. 42 (30.4%) patients in the control arm received fentanyl. 34 (37.8%) of patients in the experimental arm received fentanyl with 36% of the patients receiving fentanyl via the intranasal route. the addition of the mad was not associated with a statistically significant increase in analgesic administration. age and mechanism of injury were statistically more predictive of analgesia administration. conclusion: while the addition of the mucosal atomizer device as an alternative delivery method for fentanyl shows a trend towards increased analgesic administration in a prehospital pediatric population, age and mechanism of injury are more predictive in who receives analgesia. further research is necessary to investigate the effect of the mad on pediatric analgesic delivery. methods: this was a prospective study evaluating php-se before (pre) and after (post) a ppp introduction and 13 months later (13-mo). php groups received either ppp review and education or ppp review alone. the ppp included a pain assessment tool. the se tool, developed and piloted by pediatric ems experts, uses a ranked ordinal scale ranging from 'certain i cannot do it' (0) to 'completely certain i can do it' (100) for 10 items: pain assessment (3 items), medication administration (4) and dosing (1) , and reassessment (2). all 10 items and an averaged composite were evaluated for three age groups (adult, child, toddler). paired sample t-tests compared post-and 13-mo scores to pre-ppp scores. results: of 264 phps who completed initial surveys, 146 phps completed 13-mo surveys. 106 (73%) received education and ppp review and 40 (27%) review only. ppp education did not affect php-se (adult p = 0.87, child p = 0.69, toddler p = 0.84). the largest se increase was in pain assessment. this increase persisted for child and toddler groups at 13 months. the immediate increase in composite se scores for all age groups persisted for the toddler group at 13 months. conclusion: increases in composite and pain assessment php-se occur for all age groups immediately after ppp introduction. the increase in pain assessment se persisted at 13 months for pediatric age groups. composite se increase persisted for the toddler age group alone. background: pediatric medications administered in the prehospital setting are given infrequently and dosage may be prone to error. calculation of dose based on known weight or with use of length-based tapes occurs even less frequently and may present a challenge in terms of proper dosing. objectives: to characterize dosing errors based on weight-based calculations in pediatric patients in two similar emergency medical service (ems) systems. methods: we studied the five most commonly administered medications given to pediatric patients weighing 36 kg or less. drugs studied were morphine, midazolam, epinephrine 1:10,000, epinephrine 1:1000, and diphenhydramine. cases from the electronic record were studied for a total of 19 months, from january 2010 to july 2011. each drug was administered via intravenous, intramuscular, or intranasal routes. drugs that were permitted to be titrated were excluded. an error was defined as greater than 25% above or below the recommended mg/kg dosage. results: out of 248,596 total patients, 13,321 were pediatric patients. 7885 had documented weights of <36 kg and 241 patients were given these medications. we excluded 72 patients for weight above the 97%ile or below the 3%ile, or if the weight documentation was missing. of the 169 patients and 187 doses, errors were noted in 53 (28%; 95% ci 22%, 35%). midazolam was the most common drug in errors (29 of 53 doses or 55%; 95% ci 40%, 68%), followed by diphenhydramine (11/53 or 21%; 95% ci 11%, 34%), epinephrine (7/53 or 13%; 95% ci 5%, 25%), and morphine sulfate (6/53 or 11%; 95% ci, 4%, 23%). underdosing was noted in 34 of 53 (64%; 95% ci 50%, 77%) of errors, while excessive dosing was noted in 19 of 53 (36%; 95% ci 23%, 50%). conclusion: weight-based dosing errors in pediatric patients are common. while the clinical consequences of drug dosing errors in these patients are unknown, a considerable amount of inaccuracy occurs. strategies beyond provision of reference materials are needed to prevent pediatric medication errors and reduce the potential for adverse outcomes. drivers background: homelessness affects up to 3.5 million people a year. the homeless present more frequently to eds, their ed visits are four times more likely to occur within 3 days of a prior ed evaluation, and they are admitted up to five times more frequently than others. we evaluated the effect of a street outreach rapid response team (sorrt) on the health care utilization of a homeless population. a nonmedical outreach staff responds to the ed and intensely case manages the patient: arranges primary care follow-up, social services, temporary housing opportunities, and drug/ alcohol rehabilitation services. objectives: we hypothesized that this program would decrease the ed visits and hospital admissions of this cohort of patients. methods: before and after study at an urban teaching hospital from june, 2010-december, 2011 in indianapolis, indiana. upon identification of homeless status, sorrt was immediately notified. eligibility for sorrt enrollment is determined by housing and urban development homeless criteria and the outreach staff attempted to enter all such identified patients into the program. the patients' health care utilization was evaluated in the 6 months prior to program entry as compared to the 6 months after enrollment by prospectively collecting data and a retrospective medical record query for any unreported visits. since the data were highly skewed, we used the nonparametric signed rank test to test for paired differences between periods. results: 22 patients met criteria but two refused participation. the 20-patient cohort had 388 total ed visits (175 pre and 213 post) with a mean of 8.8 (sd 10.1) and median of 6.5 (range 1-44) ed visits in 6 months pre-sorrt as compared to a mean of 10.7 (sd 19.5) and median of 5.0 (0-90) in 6 months post-sorrt (p = 0.815). there were 28 total inpatient admissions pre-intervention and 27 post-intervention, with a mean of 1.4 (sd 2.0) and median of 0.5 (0.7) per patient in the pre-intervention period as compared to 1.4 (sd 1.9) and 1.0 (0-6) in the post-intervention period (p = 0.654). in the pre-sorrt period 50.0% had at least one inpatient admission as compared to 55.0% post-sorrt (p = 1.00). there were no differences in icu days or overall length of stay between the two periods. conclusion: an aggressive case management program beginning immediately with homeless status recognition in the ed has not demonstrated success in decreasing utilization in our population. methods: this was a secondary analysis of a prospective randomized trial that included consenting patients discharged with outpatient antibiotics from an urban county ed with an annual census of 100,000. patients unable to receive text messages or voice-mails were excluded. health literacy was assessed using a validated health literacy assessment, the newest vital sign (nvs). patients were randomized to a discharge instruction modality: 1) standard care, typed and verbal medication and case-specific instructions; 2) standard care plus text-messaged instructions sent to the patient's cell phone; or 3) standard care plus voice-mailed instructions sent to the patient's cell. patients were called at 30 days to determine preference for instruction delivery modality. preference for discharge instruction modality was analyzed using z-tests for proportions. results: 758 patients were included (55% female, median age 30, range 5 months to 71 years); 98 were excluded. 23% had an nvs score of 0-1, 31% 2-3, and 46% 4-6. among the 51.1% of participants reached at 30 days, 26% preferred a modality other than written. there was a difference in the proportion of patients who preferred discharge instructions in written plus another modality (see table) . with the exception of written plus another modality, patient preference was similar across all nvs score groups. conclusion: in this sample of urban ed patients, more than one in four patients prefer non-traditional (text message, voice-mail) modalities of discharge instruction delivery to standard care (written) modality alone. additional research is needed to evaluate the effect of instructional modality on accessibility and patient compliance. figure) . conclusion: cumulative saps ii scoring fails to predict mortality in ohca. the risk scores assigned to age, gcs, and hco 3 independently predict mortality and combined are good mortality predictors. these findings suggest that an alternative severity of illness score should be used in post-cardiac arrest patients. future studies should determine optimal risk scores of saps ii variables in a larger cohort of ohca. objectives: to determine the extent to which cpp recovers to pre-pause levels with 20 seconds of cpr after a 10-second interruption in chest compressions for ecg rhythm analysis. methods: this was a secondary analysis of prospectively collected data from an iacuc-approved protocol. fortytwo yorkshire swine (weighing 25-30 kg) were instrumented under anesthesia. vf was electrically induced. after 12 minutes of untreated vf, cpr was initiated and a standard dose of epinephrine (sde) (0.01 mg/kg) was given. after 2.5 minutes of cpr to circulate the vasopressor, compressions were interrupted for 10 seconds to analyze the ecg rhythm. this was immediately followed by 20 seconds of cpr to restore cpp before the first rs was delivered. if the rs failed, cpr resumed and additional vasopressors (sde, and vasopressin 0.57 mg/kg) were given and the sequence repeated. the cpp was defined as aortic diastolic pressure minus right atrial diastolic pressure. the cpp values were extracted at three time points: immediately after the 2.5 minutes of cpr, following the 10-second pause, and immediately before defibrillation for the first two rs attempts in each animal. eighty-three sets of measurements were logged from 42 animals. descriptive statistics were used to analyze the data. in most cities, the proportion of patients who achieve prehospital return of spontaneous circulation (rosc) is less than 10%. the association between time of day and ohca outcomes in the prehospital setting is unknown. objectives: we sought to determine whether rates of prehospital rosc varied by time of day. we hypothesized that night ohcas would exhibit lower rates of rosc. methods: we performed a retrospective review of cardiac arrest data from a large, urban ems system. included were all ohcas occurring in individuals >18 years of age from 1/1/2008 to 12/31/2010. excluded were traumatic arrests and cases where resuscitation measures were not performed. day was defined as 7:00 am-6:59 pm, while night was 7:00 pm-6:59 am. we examined the association between time of day and paramedic-perceived prehospital rosc in unadjusted and adjusted analyses. variables included age, sex, race, presenting rhythm, aed application by a bystander or first responder, defibrillation, and bystander cpr performance. analyses were performed using chisquare tests and logistic regression. objectives: determine whether a smei helps to improve physician compliance with ihi bundle and reduce patient mortality in ed patients with s&s. methods: we conducted a pre-smei retrospective review of four months of ed patients with s&s to determine baseline pre-smei physician compliance and patient mortality. we designed and completed a smei attended by 25 of 28 ed attending physicians and 28 of 30 ed resuscitation residents. finally, we conducted a twenty-month post-smei prospective study of ongoing physician compliance and patient mortality in ed patients with s&s. results: in the four month pre-smei retrospective review, we identified 23 patients with s&s, with a 61% physician overall compliance and mortality rate of 30%. the average ed physician smei multiple-choice pre-test score was 74%, and showed a significant improvement in the post-test score of 94% (p = 0.0003). additionally, 87% of ed physicians were able to describe three new clinical pearls learned and 85% agreed that the smei would improve compliance. in the twenty months of the post-smei prospective study, we identified 144 patients with s&s, with a 75% physician overall compliance, and mortality rate of 21%. relative physician compliance improved 23% (p = 0.0001) and relative patient mortality was reduced by 32% (p < 0.0001) when comparing pre-and post-smei data. conclusion: our data suggest that a smei improves overall physician compliance with the six hour goals of the ihi bundle and reduces patient mortality in ed patients with s&s. conclusion: using a population-level, longitudinal, and multi-state analysis, the rate of return visits within 3 days is higher than previously reported, with nearly 1 in 12 returning back to the ed. we also provide the first estimation of health care costs for ed revisits. background: the ability of patients to accurately determine their level of urgency is important in planning strategies that divert away from eds. in fact, an understanding of patient self-triage abilities is needed to inform health policies targeting how and where patients access acute care services within the health care system. objectives: to determine the accuracy of a patient's self-assessment of urgency compared against triage nurses. methods: setting: ed patients are assigned a score by trained nurses according to the canadian emergency department triage and acuity scale (ctas). we present a cross-sectional survey of a random patient sample from 12 urban/regional eds conducted during the winters of 2007 and 2009. this previously validated questionnaire, based on the british healthcare commission survey, was distributed according to a modified dillman protocol. exclusion criteria consisted of: age 0-15 years, left prior to being seen/treated, died during ed visit, no contact information, presented with a privacy-sensitive case. alberta health services provided linked non-survey administrative data. results: 21,639 surveys distributed with a response rate of 46%. patients rated health problems as life-threatening (6%), possibly life-threatening (22%), urgent (30%), somewhat urgent (37%), or not urgent (5%). triage nurses assigned the same patients ctas scores of i (<1%), ii (20%), iii (45%), iv (29%) or v (5%). patients self-rated their condition as 3 or 4 points less urgent than the assigned ctas score (<1% of the time), 2 points less urgent (5%), 1 point less urgent (25%), exactly as urgent (38%), 1 point more urgent (24%), 2 points more urgent (7%), or 3 or 4 points more urgent (1%, respectively). among ctas i or ii patients, 54% described their problem as life-threatening/possibly life-threatening, 26% as urgent (risk of permanent damage), 18% as urgent (needed to be seen that day), and 2% as not urgent (wanted to be but did not need to be seen that day). conclusion: the majority of ed patients are generally able to accurately assess the acuity of their problem. encouraging patients with low-urgency conditions to self-triage to lower-acuity sources of care may relieve stress on eds. however, physicians and patients must be aware that a small minority of patients are unable to self-triage safely. when the tourniquet was released, blood spurted from the injured artery as hydrostatic pressure decayed. pressure and flow were recorded in three animals (see table) . the concept was proof-tested in a single fresh frozen human cadaver with perfusion through the femoral artery and hemorrhage from the popliteal artery. the results were qualitatively and quantitatively similar to the swine carcass model. conclusion: a perfused swine carcass can simulate exsanguinating hemorrhage for training purposes and serves as a prototype for a fresh-frozen human cadaver model. additional research and development are required before the model can be widely applied. background: in the pediatric emergency department (ped), clinicians must work together to provide safe and effective care. crisis resource management (crm) principles have been used to improve team performance in high-risk clinical settings, while simulation allows practice and feedback of these behaviors. objectives: to develop a multidisciplinary educational program in a ped using simulation-enhanced teamwork training to standardize communication and behaviors and identify latent safety threats. methods: over 6 months a workgroup of physicians and nurses with experience in team training and simulation developed an educational program for clinical staff of a tertiary ped. goals included: create a didactic curriculum to teach the principles of crm, incorporate principles of crm into simulation-enhanced team training in-situ and center-based exercises, and utilize assessment instruments to evaluate for teamwork, completion of critical actions, and presence of latent safety threats during in-situ sim resuscitations. results: during phase i, 130 clinicians, divided into teams, participated in 90-minute pre-training assessments of pals-based in-situ simulations. in phase ii, staff participated in a 6-hour curriculum reviewing key crm concepts, including team training exercises utilizing simulation and expert debriefing. in phase iii, staff participated in post-training 90 minute teamwork and clinical skills assessments in the ped. in all phases, critical action checklists (cac) were tabulated by simulation educators. in-situ simulations were recorded for later review using the assessment tools. after each simulation, educators facilitated discussion of perceptions of teamwork and identification of systems issues and latent hazards. overall, 54 in-situ simulations were conducted capturing 97% of the physicians and 84% of the nurses. cac data were collected by an observer and compared to video recordings. over 20 significant systems issues, latent hazards, and knowledge deficits were identified. all components of the program were rated highly by 90% of the staff. conclusion: a workgroup of pem, simulation, and team training experts developed a multidisciplinary team training program that used in-situ and centerbased simulation and a refined crm curriculum. unique features of this program include its multidisciplinary focus, the development of a variety of assessment tools, and use of in-situ simulation for evaluation of systems issues and latent hazards. this program was tested in a ped and findings will be used to refine care and develop a sustainment program while addressing issues identified. objectives: our hypothesis is that participants trained on high-fidelity mannequins will perform better than participants trained on low-fidelity mannequins on both the acls written exam and in performance of critical actions during megacode testing. the study was performed in the context of an acls initial provider course for new pgy1 residents at the penn medicine clinical simulation center and involved three training arms: 1) low fidelity (low-fi): torso-rhythm generator; 2) mid-fidelity (mid-fi): laerdal simmanò turned off; and 3) high-fidelity (high-fi): laerdal simmanò turned on. training in each arm of the study followed standard aha protocol. educational outcomes were evaluated by written scores on the acls written examination and expert rater reviews of acls megacode videos performed by trainees during the course. a sample of 54 subjects were randomized to one of the three training arms: low-fi (n = 18), mid-fi (n = 18), or high-fi (n = 18). results: statistical significance across the groups was determined using analysis-of-variance (anova). the three groups had similar written pre-test scores [low-fi 0.4 (0.1), mid-fi 0.5 (0.1), and high-fi 0.4 (0. 2)] and written post-test scores [low-fi 0.9 (0.1), mid-fi 0.9 (0.1), and high-fi 0.8 (0.1)]. similarly, test improvement was not significantly different. after completion of the course, high-fi subjects were more likely to report they felt comfortable in their simulator environment (p = 0.005). low-fi subjects were less likely to perceive a benefit in acls training from high-fi technology (p < 0.001). acls instructors were not rated significantly different by the subjects using the debriefing assessment for simulation in healthcareª (dash) student version except for element 6, where the high-fi group subjects reported lower scores (6.1 vs 6.6 and 6.7 in the other groups, p = 0.046). objectives: we sought to determine if stress associated with the performance of a complex procedural task can be affected by level of medical training. heart rate variability (hrv) is used as a measure of autonomic balance, and therefore an indicator of the level of stress. methods: twenty-one medical students and emergency medicine residents were enrolled. participants performed airway procedures on an airway management trainer. hrv data were collected using a continuous heart rate variability monitoring system. participant hrv was monitored at baseline, during the unassisted first attempt at endotracheal intubation, during supervised practice, and then during a simulated respiratory failure clinical scenario. standard deviation of beat to beat variability (sdnn), very low frequency (vlf), total power (tp), and low frequency (lf) was analyzed to determine the effect of practice and level of training on the level of stress. a cohen's d test was used to determine differences between study groups. results: sdnn data showed that second-year residents were less stressed during all stages than were fourthyear medical students (avg d = 1.12). vlf data showed third-year residents exhibited less sympathetic activity than did first-year residents (avg d = )0.68). the opportunity to practice resulted in less stress for all participants. tp data showed that residents had a greater degree of control over their autonomic nervous system (ans) than did medical students (avg d = 0.85). lf data showed that subjects were more engaged in the task at hand as the level of training increased indicating autonomic balance (avg d = 0.80). conclusion: our hrv data show that stress associated with the performance of a complex procedural task is reduced by increased training. hrv may provide a quantitative measure of physiologic stress during the learning process and thus serve as a marker of when a subject is adequately trained to perform a particular task. objectives: we seek to examine whether intubation during cpr can be done as efficiently as intubation without ongoing cpr. the hypothesis is that the predictable movement of an automated chest compression device will make intubation easier than the random movement from manual cpr. methods: the project was an experimental controlled trial and took place in the emergency department at a tertiary referral center in peoria, illinois. emergency medicine residents, attendings, paramedics, and other acls trained staff were eligible for participation. in randomized order, each participant attempted intubation on a mannequin with no cpr ongoing, during cpr with a human compressor, and during cpr with an automatic chest compression device (physio control lucas 2). participants could use whichever style laryngoscope they felt most comfortable with and they were timed during the three attempts. success was determined after each attempt. results: there were 43 participants in the trial. the success rate in the control group and the automated cpr group were both 88% (38/43) and the success rate in the manual cpr group was 74% (32/43). the differences in success rates were not statistically significant (p = 0.99 and p = 0.83). the automated cpr group had the fastest average time (13.6 sec; p = 0.019). the mean times for intubation with manual cpr and no cpr were not statistically different (17.1 sec, 18.1 sec; p = 0.606). conclusion: the success rate of tracheal intubation with ongoing chest compression was the same as the success rate of intubation without cpr. although intubation with automatic chest compression was faster than during other scenarios, all methods were close to the 10 second timeframe recommended by acls. based on these findings, it may not always be necessary to hold cpr to place a definitive airway; however, further studies will be needed. background: after acute myocardial infarction, vascular remodeling in the peri-infarct area is essential to provide adequate perfusion, prevent additional myocyte loss, and aid in the repair process. we have previously shown that endogenous fibroblast growth factor 2 (fgf2) is essential to the recovery of contractile function and limitation of infarct size after cardiac ischemia-reperfusion (ir) injury. the role of fgf2 in vascular remodeling in this setting is currently unknown. objectives: determine the role of endogenous fgf2 in vascular remodeling in a clinically relevant, closed-chest model of acute myocardial infarction. methods: mice with a targeted ablation of the fgf2 gene (fgf2 knockout) and wild type controls were subjected to a closed-chest model of regional cardiac ir injury. in this model, mice were subjected to 90 minutes of occlusion of the left anterior descending artery followed by reperfusion for either 1 or 7 days. immunofluorescence was performed on multiple histological sections from these hearts to visualize capillaries (endothelium, anti-cd31 antibody), larger vessels (venules and arterioles, antismooth muscle actin antibody), and nuclei (dapi). digital images were captured, and multiple images from each heart were measured for vessel density and vessel size. results: sham-treated fgf2 knockout and wild type mice show no differences in capillary or vessel density suggesting no defect in vessel formation in the absence of endogenous fgf2. when subjected to closed-chest regional cardiac ir injury, fgf2 knockout hearts had normal capillary and vessel number and size in the peri-infarct area after 1 day of reperfusion compared to wild type controls. however, after 7 days, fgf2 knockout hearts showed significantly decreased capillary and vessel number and increased vessel size compared to wild type controls (p < 0.05). conclusion: these data show the necessity of endogenous fgf2 in vascular remodeling in the peri-infarct zone in a clinically relevant animal model of acute myocardial infarction. these findings may suggest a potential role for modulation of fgf2 signaling as a therapeutic intervention to optimize vascular remodeling in the repair process after myocardial infarction. the diagnosis of aortic dissections by ed physicians is rare scott m. alter, barnet eskin, john r. allegra morristown medical center, morristown, nj background: aortic dissection is a rare event. the most common symptom of dissection is chest pain, but chest pain is a frequent emergency department (ed) chief complaint and other diseases that cause chest pain, such as acute coronary syndrome and pulmonary embolism, occur much more frequently. furthermore, 20% of dissections are without chest pain and 6% are painless. for all these reasons, diagnosing dissection can be difficult for the ed physician. we wished to quantify the magnitude of this problem in a large ed database. objectives: our goal was to determine the number of patients diagnosed by ed physicians with aortic dissections compared to total ed patients and to the total number of patients with a chest pain diagnosis. methods: design: retrospective cohort. setting: 33 suburban, urban, and rural new york and new jersey eds with annual visits between 8,000 and 75,000. participants: consecutive patients seen by ed physicians from january 1, 1996 through december 31, 2010. observations: we identified aortic dissections using icd-9 codes and chest pain diagnoses by examining all icd-9 codes used over the period of the study and selecting those with a non-traumatic chest pain diagnosis. we then calculated the number of total ed patients and chest pain patients for every aortic dissection diagnosed by emergency physicians. we determined 95% confidence intervals (cis). results: from a database of 9.5 million ed visits, we identified 782 (0.0082%) aortic dissections, or one for every 12,200 (95% ci 11,400 to 13,100) visits. the mean age of aortic dissection patients was 58 ± 19 years and 57% were female. of the total visits there were 763,000 (8%) with a chest pain diagnosis. thus there is one aortic dissection diagnosis for every 980 (95% ci 910 to 1,050) chest pain diagnoses. conclusion: the diagnosis of aortic dissections by ed physicians is rare. an ed physician seeing 3,000 to 4,000 patients a year would diagnose an aortic dissection approximately once every 3 to 4 years. an aortic dissection would be diagnosed once for approximately every 1,000 ed chest pain patients. patients were excluded if they suffered a cardiac arrest, were transferred from another hospital, or if the ccl was activated for an inpatient or from ems in the field. fp ccl activation was defined as 1) a patient for whom activation was cancelled in the ed and ruled out for mi or 2) a patient who went to catheterization but no culprit vessel was identified and mi was excluded. ecgs for fp patients were classified using standard criteria. demographic data, cardiac biomarkers, and all relevant time intervals were collected according to an on-going quality assurance protocol. results: a total of 506 ccl activations were reviewed, with 68% male, average age 57, and 59% black. there were 210 (42%) true stemis and 86 (17%) fp activations. there were no significant differences between the fp patients who did and did not have catheterization. for those fp patients who had a catheterization (13%), ''door to page'' and ''door to lab'' times were significantly longer than the stemi patients (see table) , but there was substantial overlap. there was no difference in sex or age, but fp patients were more likely to be black (p = 0.02). a total of 82 fp patients had ecgs available for review; findings included anterior elevation with convex (21%) or concave (13%) elevation, st elevation from prior anterior (10%) or inferior (11%) mi, pericarditis (16%), presumed new lbbb (15%), early repolarization (5%), and other (9%). conclusion: false ccl activation occurred in a minority of patients, most of whom had ecg findings warranting emergent catheterization. the rate of false ccl activation appears acceptable. background: atrial fibrillation (af) is the most common cardiac arrhythmia treated in the ed, leading to high rates of hospitalization and resource utilization. dedicated atrial fibrillation clinics offer the possibility of reducing the admission burden for af patients presenting to the ed. while the referral base for these af clinics is growing, it is unclear to what extent these clinics contribute to reducing the number of ed visits and hospitalizations related to af. objectives: to compare the number of ed visits and hospitalizations among discharged ed patients with a primary diagnosis of af who followed up with an af clinic and those who did not. methods: a retrospective cohort study and medical records review including three major tertiary centres in calgary, canada. a sample of 600 patients was taken representing 200 patients referred to the af clinic from the calgary zone eds and compared to 400 matched control ed patients who were referred to other providers for follow-up. the controls were matched for age and sex. inclusion criteria included patients over 18 years of age, discharged during the index visit, and seen by the af clinic between january 1, 2009 and october 25, 2010. exclusion criteria included non-residents and patients hospitalized during the index visit. the number of cardiovascular-related ed visits and hospitalizations was measured. all data are categorical, and were compared using chi-square tests. results: patients in the control and af clinic cohorts were similar for all baseline characteristics except for a higher proportion of first episode patients in the intervention arm. in the six months following the index ed visit, 55 study group patients (27.5%) visited an ed on 95 occasions, and 12 (6%) were hospitalized on 16 occasions. of the control group, 122 patients (30.5%) visited an ed on 193 occasions, and 44 (11%) were hospitalized on 55 occasions. using a chi-square test we found no significant difference in ed visits (p = 0.5063) or hospitalizations (p = 0.0664) between the control and af clinic cohorts. conclusion: based on our results, referral from the ed to an af clinic is not associated with a significant reduction in subsequent cardiovascular related ed visits and hospitalizations. due to the possibility of residual confounding, randomized trials should be performed to evaluate the efficacy of af clinics. reported an income of less than $10,000. there were no significant associations between sex, race, marital status, education level, income, insurance status, and subsequent 30-and-90 day readmission rates. hla score was not found to be significantly related to readmission rates. the mean hla score was 18.9 (sd = 7.87), equivalent to less than 6th grade literacy, meaning these patients may not be able to read prescription labels. for each unit increase in hfkt score, the odds of being readmitted within 30 days decreased by 0.219 (p < 0.001) and for 31-90 days decreased by 0.440 (p < 0.001). for each unit increase in scbs score, the odds of being readmitted within 90 days decreased by 0.949 (p = 0.038). conclusion: health care literacy in our patient population is not associated with readmission, likely related to the low literacy rate of our study population. better hf knowledge and self-care behaviors are associated with lower readmission rates. greater emphasis should be placed on patient education and self-care behaviors regarding hf as a mechanism to decrease readmission rates. comparison of door to balloon times in patients presenting directly or transferred to a regional heart center with stemi jennifer ehlers, adam v. wurstle, luis gruberg, adam j. singer stony brook university, stony brook, ny background: based on the evidence, a door-to-balloon-time (dtbt) of less than 90 minutes is recommended by the aha/acc for patients with stemi. in many regions, patients with stemi are transferred to a regional heart center for percutaneous coronary intervention (pci). objectives: we compared dtbt for patients presenting directly to a regional heart center with those for patients transferred from other regional hospitals. we hypothesized that dtbt would be significantly longer for transferred patients. methods: study design-retrospective medical record review. setting-academic ed at a regional heart center with an annual census of 80,000 that includes a catchment area of 12 hospitals up to 50 miles away. patients-patients with acute stemi identified on ed 12-lead ecg. measures-demographic and clinical data including time from triage to ecg, from ecg to activation of regional catheterization lab, and from initial triage to pci (dtbt , and door to intravascular balloon deployment (d2b). methods: the study was performed in an inner-city academic ed between 1/1/07 and 12/31/10. every patient for whom ed activation of our stemi system occurred was included. all times data from a pre-existing quality assurance database were collected prospectively. patient language was determined retrospectively by chart review. results: there were 132 patients between 1/1/07 and 12/31/10. 21 patients (16%) were deemed too sick or unable to provide history and were excluded, leaving 111 patients for analysis. 85 (77%) spoke english and 26 (23%) did not. in the non-english group, chinese was the most common language, in 22 (20%) background: syncope is a common, potentially highrisk ed presentation. hospitalization for syncope, although common, is rarely of benefit. no populationbased study has examined disparities in regional admission practices for syncope care in the ed. moreover, there are no population-based studies reporting prognostic factors for 7-and 30-day readmission of syncope. objectives: 1) to identify factors associated with admission as well as prognostic factors for 7-and 30-day readmission to these hospitals; 2) to evaluate variability in syncope admission practices across different sizes and types of hospitals. methods: design -multi-center retrospective cohort study using ed administrative data from 101 albertan eds. participants/subjects -patients >17 years of age with syncope (icd10: r55) as a primary or secondary diagnosis from 2007 to june 2011. readmission was defined as return visits to the ed or admission <7 days or 7-30 days after the index visit (including against medical advice and left without being seen during the index visit). outcomes -factors associated with hospital admission at index presentation, and readmission following ed discharge, adjusted using multivariable logistic regression. results: overall, 44521 syncope visits occurred over 4 years. increased age, increased length of stay (los), performance of cxr, transport by ground ambulance, and treatment at a low-volume hospital (non-teaching or non-large urban) were independently associated with index hospitalization. these same factors, as well as hospital admission itself, were associated with 7-day readmission. additionally, increased age, increased los, performance of a head ct, treatment at a low-volume hospital, hospital admission, and female sex were independently associated with 7-30 day readmission. arrival by ground ambulance was associated with a decreased likelihood of both 7-and 7-30 day readmission. conclusion: our data identify variations in practice as well as factors associated with hospitalization and readmission for syncope. the disparity in admission and readmission rates between centers may highlight a gap in quality of care or reflect inappropriate use of resources. further research to compare patient out-comes and quality of patient care among urban and non-urban centers is needed. background: change in dyspnea severity (ds) is a frequently used outcome measure in trials of acute heart failure (ahf). however, there is limited information concerning its validity. objectives: to assess the predictive validity of change in dyspnea severity. methods: this was a secondary analysis of a prospective observational study of a convenience sample of ahf patients presenting with dyspnea to the ed of an academic tertiary referral center with a mixed urban/ suburban catchment area. patients were enrolled weekdays, june through december 2006. patients assessed their ds using a 10-cm visual analog scale at three times: the start of ed treatment (baseline) as well as at 1 and 4 hours after starting ed treatment. the difference between baseline and 1 hour was the 1-hour ds change. the difference between baseline and 4 hours was the 4-hour ds change. two clinical outcome measures were obtained: 1) the number of days hospitalized or dead within 30 days of the index visit (30-day outcome), and 2) the number of days hospitalized or dead within 90 days of the index visit (90-day outcome). results: data on 86 patients were analyzed. the median 30-day outcome variable was 6 days with an interquartile range (iqr) of 3 to 16. the median 90-day outcome variable was 10 days (iqr 4 to 27.5). the median 1-hour ds change was 2.6 cm (iqr 0.3 to 6.7). the median 4-hour ds change was 4.9 cm (iqr 2.2 to 8.2). the 30-day and 90-day mortality rates were 9% and 13% respectively. the spearman rank correlations and 95% confidence intervals are presented in the table below. conclusion: while the point estimates for the correlations were below 0.5, the 95% ci for two of the correlations extended above 0.5. these pilot data support change in ds as a valid outcome measure for ahf when measured over 4 hours. a larger prospective study is needed to obtain a more accurate point estimate of the correlations. background: the majority of volume-quality research has focused on surgical outcomes in the inpatient setting; very few studies have examined the effect of emergency department (ed) case volume on patient outcomes. objectives: to determine whether ed case volume of acute heart failure (ahf) is associated with short-term patient outcomes. methods: we analyzed the 2008 nationwide emergency department sample (neds) and nationwide inpatient sample (nis), the largest, all-payer, ed and inpatient databases in the us. ed visits for ahf were identified with a principal diagnosis of icd-9-cm code 428.xx. eds were categorized into quartiles by ed case volume of ahf. the outcome measures were early inpatient mortality (within the first 2 days of admission), overall inpatient mortality, and hospital length of stay (los). results: there were an estimated 946,000 visits for ahf from approximately 4,700 eds in 2008; 80% were hospitalized. of these, the overall inpatient mortality rate was 3.2%, and the median hospital los was 4 days. early inpatient mortality was lower in the highest-volume eds, compared with the lowest-volume eds (0.8% vs. 2.1%; p < 0.001). similar patterns were observed for overall inpatient mortality (3.0% vs. 4.1%; p < 0.001). in a multivariable analysis adjusting for 37 patient and hospital characteristics, early inpatient mortality remained lower in patients admitted through the highest-volume eds (adjusted odds ratios [or], 0.70; 95% confidence interval [ci], 0.52-0.96), as compared with the lowest-volume eds. there was a trend towards lower overall inpatient mortality in the highest-volume eds; however, this was not statistically significant (adjusted or, 0.92; 95%ci, 0.75-1.14). by contrast, using the nis data including various sources of admissions, a higher case volume of inpatient ahf patients predicted lower overall inpatient mortality (adjusted or, 0.51; 95%ci, 0.40-0.65). the hospital los in patients admitted through the highest-volume eds was slightly longer (adjusted difference, 0.7 day; 95%ci, 0.2-1.2), compared with the lowest-volume eds. conclusion: ed patients who are hospitalized for ahf have an approximately 30% reduced early inpatient mortality if they were admitted from an ed that handles a large volume of ahf cases. the ''practice-makesperfect'' concept may hold in emergency management of ahf. emergency department disposition and charges for heart failure: regional variability alan b. storrow, cathy a. jenkins, sean p. collins, karen p. miller, candace mcnaughton, naftilan allen, benjamin s. heavrin vanderbilt university, nashville, tn background: high inpatient admission rates for ed patients with acute heart failure are felt partially responsible for the large economic burden of this most costly cardiovascular problem. objectives: we examined regional variability in ed disposition decisions and regional variability in total dollars spent on ed services for admitted patients with primary heart failure. methods: the 2007 nationwide emergency department sample (neds) was used to perform a retrospective, cohort analysis of patients with heart failure (icd-9 code of 428.x) listed as the primary ed diagnosis. demographics and disposition percentages (with se) were calculated for the overall sample and by region: northeast, south, midwest, and west. to account for the sample design and to obtain national and regional estimates, a weighted analysis was conducted. results: there were 941,754 weighted ed visits with heart failure listed as the primary diagnosis. overall, over eighty percent were admitted (see table) . fifty-two percent of these patients were female; mean age was 72.7 years (se 0.20). hospitalization rates were higher in the northeast (89.1%) and south (81.2%) than in the midwest (76.0%) and west (74.8%). total monies spent on ed services were highest in the south ($69,078,042) followed by the northeast ($18,233,807), west ($6,360,315) and midwest ($5,899,481) . conclusion: this large retrospective ed cohort suggests a very high national admission rate with significant regional variation in both disposition decisions as well as total monies spent on ed services for patients with a primary diagnosis of heart failure. examining these estimates and variations further may provide strategies to reduce the economic burden of heart failure. background: workplace violence in health care settings is a frequent occurrence. gunfire in hospitals is of particular concern. however, information regarding such workplace violence is limited. accordingly, we characterized u.s. hospital-based shootings from 2000-2010. objectives: to determine extent of hospital-based shootings in the u.s. and involvement of emergency departments. methods: using lexisnexis, google, netscape, pub-med, and sciencedirect, we searched reports for acute care hospital shooting events from january 2000 through december 2010, and those with at least one injured victim were analyzed. results: we identified 140 hospital-related shootings (86 inside the hospital, 54 on hospital grounds), in 39 states, with 216 victims, of whom 98 were perpetrators. in comparison to external shootings, shootings within the hospital have not increased over time (see figure) . perpetrators were from all age groups, including the elderly. most of the events involved a determined shooter: grudge (26%), suicide (19%), ''euthanizing'' an ill relative (15%), and prisoner escape (12%). ambient societal violence (8%) and mentally unstable patients (4%) were comparatively infrequent. the most common injured was the perpetrator (45%). hospital employees comprised only 21% of victims; physician (3%) and nurse (5%) victims were relatively infrequent. the emergency department was the most common site (29%), followed by patient rooms (20%) and the parking lot (20%). in 13% of shootings within hospitals, the weapon was a security officer's gun grabbed by the perpetrator. ''grudge'' motive was the only factor determinative of hospital staff victims (or = 4.34, 95% ci 1.85-10.17). conclusion: although hospital-based shootings are relatively rare, emergency departments are the most likely site. the unpredictable nature of this type of event represents a significant challenge to hospital security and deterrence practices, as most perpetrators proved determined, and many hospital shootings occur outside the building. impact of emergency physician board certification on patient perceptions of ed care quality albert g. sledge iv 1 , carl a. germann 1 , tania d. strout 1 , john southall 2 1 maine medical center, portland, me; 2 mercy hospital, portland, me background: the hospital value-based purchasing program mandated by the affordable care act is the latest example of how patients' perceptions of care will affect the future practice environment of all physicians. the type of training of medical providers in the emergency department (ed) is one possible factor affecting patient perceptions of care. a unique situation in a maine community ed led to the rapid transition from non-emergency medicine (em) residency trained physicians to all em residency trained and american board of emergency medicine (abem) certified providers. objectives: the purpose of this study was to evaluate the effect of the implementation of an all em-trained, abem-certified physician staff on patient perceptions of the quality of care they received in the ed. methods: we retrospectively evaluated press ganey data from surveys returned by patients receiving treatment in a single, rural ed. survey items addressed patient's perceptions of physician courtesy, time spent listening, concern for patient comfort, and informativeness. additional items evaluated overall perceptions of care and the likelihood that the respondent would recommend the ed to another. data were compared for the three years prior to and following implementation of the all trained, certified staff. we used the independent samples t-test to compare mean responses during the two time periods. bonferroni's correction was applied to adjust for multiple comparisons. results: during the study period, 3,039 patients provided surveys for analysis: 1,666 during the pre-certification phase and 1,373 during the post-certification phase. across all six survey items, mean responses increased following transition to the board-certified staff. these improvements were noted to be statistically significant in each case: courtesy p < 0.001, time listening p < 0.001, concern for comfort p < 0.001, informativeness p < 0.001, overall perception of care p < 0.001, and likelihood to recommend p < 0.001. conclusion: data from this community ed suggest that transition from a non-residency trained, abem certified staff to a fully trained and certified model has important implications for patient's perceptions of the care they receive. we observed significant improvement in rating scores provided by patients across all physicianoriented and general ed measures. background: transfer of care from the ed to the inpatient floor is a critical transition when miscommunication places patients at risk. the optimal form and content of handoff between providers has not been defined. in july 2011, ed-to-floor signout for all admissions to the medicine and cardiology floors was changed at our urban, academic, tertiary care hospital. previously, signout was via an unstructured telephone conversation between ed resident and admitting housestaff. the new signout utilizes a web-based ed patient tracking system and includes: 1) a templated description of ed course is completed by the ed resident; 2) when a bed is assigned, an automated page is sent to the admitting housestaff; 3) ed clinical information, including imaging, labs, medications, and nursing interventions (figure) is reviewed by admitting housestaff; 4) if housestaff has specific questions about ed care, a telephone conversation between the ed resident and housestaff occurs; 5) if there are no specific questions, it is indicated electronically and the patient is transferred to the floor. objectives: to describe the effects on patient safety (floor-to-icu transfer in 24 hours) and ed throughput (ed length of stay (los) and time from bed assignment to ed departure) resulting from a change to an electronic, discussion-optional handoff system. conclusion: transition to a system in which signout of admitted patients is accomplished by accepting housestaff review of ed clinical information supplemented by verbal discussion when needed resulted in no significant change in rate of floor-to-icu transfer or ed los and reduced time from bed assignment to ed departure. background: emergency physicians may be biased against patients presenting with nonspecific complaints or those requiring more extensive work-ups. this may result in patients being seen less quickly than those with more straightforward presentations, despite equal triage scores or potential for more dangerous conditions. objectives: the goal of our study was to ascertain which patients, if any, were seen more quickly in the ed based on chief complaint. methods: a retrospective report was generated from the emr for all moderate acuity (esi 3) adult patients who visited the ed from january 2005 through december 2010 at a large urban teaching hospital. the most common complaints were: abdominal pain, alcohol intoxication, back pain, chest pain, cough, dyspnea, dizziness, fall, fever, flank pain, headache, infection, pain (nonspecific), psychiatric evaluation, ''sent by md,'' vaginal bleeding, vomiting, and weakness. non-parametric independent sample tests assessed median time to be seen (ttbs) by a physician for each complaint. differences in the ttbs between genders and based on age were also calculated. chi-square testing compared percentages of patients in the ed per hour to assess for differences in the distribution of arrival times. results: we obtained data from 116,194 patients. patients with a chief complaint of weakness and dizziness waited the longest with a median time of 35 minutes and patients with flank pain waited the shortest with 24 minutes (p < 0.0001) ( figure 1 ). overall, males waited 30 minutes and females waited 32 minutes (p < 0.0001). stratifying by gender and age, younger females between the ages of 18-50 waited significantly longer times when presenting with a chief complaint of abdominal pain (p < 0.0001), chest pain (p < 0.05), or flank pain (p < 0.0001) as compared to males in the same age group ( figure 2 ). there was no difference in the distribution of arrival times for these complaints. conclusion: while the absolute time differences are not large, there is a significant bias toward seeing young male patients more quickly than women or older males despite the lower likelihood of dangerous conditions. triage systems should perhaps take age and gender better into account. patients might benefit from efforts to educate em physicians on the delays and potential quality issues associated with this bias in an attempt to move toward more egalitarian patient selection. background: detailed analysis of emergency department (ed) event data identified the time from completion of emergency physician evaluation (doc done) to the time patients leave the ed as a significant contributor to ed length of stay (los) and boarding at our institution. process flow mapping identified the time from doc done to the time inpatient beds were ordered (bo) as an interval amendable to specific process improvements. objectives: the purpose of this study was to evaluate the effect of ed holding orders for stable adult 3.6 (3.0 -4.1) 7.8 (6.9 -8.7) 15.2 (12.8 -17.6) 4.9 (2.8 -7.0) 17.3 (12.9 -21.7) 6.5 (4.5 -8.5) inpatient medicine (aim) patients on: a) the time to bo and b) ed los. methods: a prospective, observational design was used to evaluate the study questions. data regarding the time to bo and los outcomes were collected before and after implementation of the ed holding orders program. the intervention targeted stable aim patients being admitted to hospitalist, internal medicine, and family medicine services. ed holding orders were placed following the admission discussion with the accepting service and special attention was paid to proper bed type, completion of the emergent work-up and the expected immediate course of the patient's hospital stay. holding orders were of limited duration and expired 4 hours after arrival to the inpatient unit. results: during the 6-month study period, 7321 patients were eligible for the ed holding orders intervention; 6664 (91.0%) were cared for using the standard adult medicine order set and 657 (9.0%) received the intervention. the median time from doc done to bo was significantly shorter for patients in the ed holding orders group, 41 min (iqr 19, 88) vs. 95 min (iqr 53, 154) for the standard adult medicine group, p < 0.001. similarly, the median ed los was significantly shorter for those in the ed holding orders group, 413 min (iqr 331, 540) vs. 456 min (iqr 346, 581) for the standard adult medicine group, p < 0.001. no lapses in patient care were reported in the intervention group. conclusion: in this cohort of ed patients being admitted to an aim service, placing ed holding orders rather than waiting for a traditional inpatient team evaluation and set of admission orders significantly reduced the time from the completion of the ed workup to placement of a bo. as a result, ed los was also significantly shortened. while overall utilization of the intervention was low, it improved with each month. emergency department interruptions in the age of electronic health records matthew albrecht, john shabosky, jonathan de la cruz southern illinois university school of medicine, springfield, il background: interruptions of clinical care in the emergency department (ed) have been correlated with increased medical errors and decreased patient satisfaction. studies have also shown that most interruptions happen during physician documentation. with the advent of the electronic health record and computerized documentation, ed physicians now spend much of their clinical time in front of computers and are more susceptible to interruptions. voice recognition dictation adjuncts to computerized charting boast increased provider efficiency; however, little is known about how data input of computerized documentation affects physician interruptions. objectives: we present here observational interruptions data comparing two separate ed sites, one that uses computerized charting by conventional techniques and one assisted by voice recognition dictation technology. methods: a prospective observational quality initiative was conducted at two teaching hospital eds located less than 1 mile from each other. one site primarily uses conventional computerized charting while the other uses voice recognition dictation computerized charting. four trained observers followed ed physicians for 180 minutes during shifts. the tasks each ed physician performed were noted and logged in 30 second intervals. tasks listed were selected from a predetermined standardized list presented at observer training. tasks were also noted as either completed or placed in queue after a change in task occurred. a total of 4140 minutes were logged. interruptions were noted when a change in task occurred with the previous task being placed in queue. data were then compared between sites. results: ed physicians averaged 5.33 interruptions/ hour with conventional computerized charting compared to 3.47 interruptions/hour with assisted voice recognition dictation (p = 0.0165). conclusion: computerized charting assisted with voice recognition dictation significantly decreased total per hour interruptions when compared to conventional techniques. charting with voice recognition dictation has the potential to decrease interruptions in the ed allowing for more efficient workflow and improved patient care. background: using robot assistants in health care is an emerging strategy to improve efficiency and quality of care while optimizing the use of human work hours. robot prototypes capable of performing vital signs and assisting with ed triage are under development. however, ed users' attitudes toward robot assistants are not well studied. understanding of these attitudes is essential to design user-friendly robots and to prepare eds for the implementation of robot assistants. objectives: to evaluate the attitudes of ed patients and their accompanying family and friends toward the potential use of robot assistants in the ed. methods: we surveyed a convenience sample of adult ed patients and their accompanying adult family members and friends at a single, university-affiliated ed, 9/ 26/11-10/27/11. the survey consisted of eight items from the negative attitudes towards robots scale (normura et al.) modified to address robot use in the ed. response options included a 5-point likert scale. a summary score was calculated by summing the responses for all 8 items, with a potential range of 8 (completely negative attitude) to 40 (completely positive attitude). research assistants gave the written surveys to subjects during their ed visit. internal consistency was assessed using cronbach's alpha. bivariate analyses were performed to evaluate the association between the summary score and the following variables: participant type (patient or visitor), sex, race, time of day, and day of week. results: of 121 potential subjects approached, 113 (93%) completed the survey. participants were 37% patients, 63% family members or friends, 62% women, 79% white, and had a median age of 45.5 years (iqr 18-84). cronbach's alpha was 0.94. the mean summary score was 22.2 (sd = 0.87), indicating subjects were between ''occasionally'' and ''sometimes'' comfortable with the idea of ed robot assistants (see table) . men were more positive toward robot use than women (summary score: 24.6 vs 20.8; p = 0.033). no differences in the summary score were detected based on participant type, race, time of day, or day of week. conclusion: ed users reported significant apprehension about the potential use of robot assistants in the ed. future research is needed to explore how robot designs and strategies to implement ed robots can help alleviate this apprehension. background: emergency department cardioversion (edc) of recent-onset atrial fibrillation or flutter (af) patients is an increasingly common management approach to this arrhythmia. patients who qualify for edc generally have few co-morbidities and are often discharged directly from the ed. this results in a shift towards a sicker population of patients admitted to the hospital with this diagnosis. objectives: to determine whether hospital charges and length of stay (los) profiles are affected by emergency department discharge of af patients. methods: patients receiving treatment at an urban teaching community hospital with a primary diagnosis of atrial fibrillation or flutter were identified through the hospital's billing data base. information collected on each patient included date of service, patient status, length of stay, and total charges. patient status was categorized as inpatient (admitted to the hospital), observation (transferred from the ed to an inpatient bed but placed in an observation status), or ed (discharged directly from the ed). the hospital billing system automatically defaults to a length of stay of 0 for observation patients. ed patients were assigned a length of stay of 0. total hospital charges and mean los were determined for two different models: a standard model (sm) in which patients discharged from the ed were excluded from hospital statistics, and an inclusive model (im) in which discharged ed patients were included in the hospital statistics. statistical analysis was through anova. results: a total of 317 patients were evaluated for af over an 18-month period. of these, 197 (62%) were admitted, 22 (7%) were placed in observation status, and 98 (31%) were discharged from the ed. hospital charges and los in days are summarized in the table. all differences were statistically significant at (p < 0.001). conclusion: emergency department management can lead to a population of af patients discharged directly from the ed. exclusion of these patients from hospital statistics skews performance profiles effectively punishing institutions for progressive care. background: recent health care reform has placed an emphasis on the electronic health record (ehr). with the advent of the ehr it is common to see ed providers spending more time in front of computers documenting and away from patients. finding strategies to decrease provider interaction with computers and increase time with patients may lead to improved patient outcomes and satisfaction. computerized charting adjuncts, such as voice recognition software, have been marketed as ways to improve provider efficiency and patient contact. objectives: we present here observational data comparing two separate ed sites, one where computerized charting is done by conventional techniques and one that is assisted with voice recognition dictation, and their effects on physican charting and patient contact. methods: a prospective observational quality initiative was conducted at two teaching hospitals located less than 1 mile from each other. one site primarily uses conventional computerized charting while the other uses voice recognition dictation. four trained quality assistants observed ed physicians for 180 minutes during shifts. the tasks each physician performed were noted and logged in 30 second intervals. tasks listed were identified from a predetermined standardized list presented at observer training. a total of 4140 minutes were logged. time allocated to charting and that allocated to direct patient care were then compared between sites. results: ed physicians spent 28.6% of their time charting using conventional techniques vs 25.7% using voice recognition dictation (p = 0.4349). time allocated to direct patient care was found to be 22.8% with conventional charting vs 25.1% using dictation (p = 4887). in total, ed physicians using conventional charting techniques spent 668/2340 minutes charting. ed physicians using voice recognition dictation spent 333/1800 minutes dictating and an additional 129.5/1800 minutes reviewing or correcting their dictations. the use of voice recognition assisted dictation rather than conventional techniques did not significantly change the amount of time physicians spent charting or with direct patient care. although voice recognition dictation decreased initial input time of documenting data, a considerable amount of time was required to review and correct these dictations. objectives: for our primary objective, we studied whether emergency department triage temperatures detected fever adequately when compared to a rectal temperature. as secondary objectives, we examined the temperature differences when a rectal temperature was taken within an hour of non-invasive temperature, temperature site (oral, axillary, temporal), and also examined the patients that were initially afebrile but were found to be febrile by rectal temperature. methods: we performed an electronic chart review at our inner city, academic emergency department with an annual census of 110,000 patients. we identified all patients over the age of 18 who received a non-invasive triage temperature and a subsequent rectal temperature while in the ed from january 2002 through february 2011. specific data elements included many aspects of the patient's medical record (e.g. subject demographics, temperature, and source). we analyzed our data with standard descriptive statistics, t-tests for continuous variables, and pearson chi-square tests for proportions. results: a total of 27,130 patients met our inclusion criteria. the mean difference in temperatures between the initial temperature and the rectal temperature was 1.3°f, with 25.9% having higher rectal temperatures ‡2°f, and 5.0% having higher rectal temperatures ‡4°f. the mean temperature difference among the 10,313 patients who an initial noninvasive temperature and a rectal temperature within one hour was 1.4°f. the mean difference among patients that received oral, axillary, and temporal temperatures was 1.2°f, 1.8°f, and 1.2°f respectively. approximately one in five patients (18.1%) were initially afebrile and found to be febrile by rectal temperature, with an average temperature difference of 2.5°f. these patients had a higher rate of admission, and were more likely to be admitted to the intensive care unit. conclusion: there are significant differences between rectal temperatures and non-invasive triage temperatures in this emergency department cohort. in almost one in five patients, fever was missed by triage temperature. background: pediatric emergency department (ped) overcrowding has become a national crisis, and has resulted in delays in treatment, and patients leaving without being seen. increased wait times have also been associated with decreased patient satisfaction. optimizing ped throughput is one means by which to handle the increased demands for services. various strategies have been proposed to increase efficiency and reduce length of stay (los). objectives: to measure the effect of direct bedding, bedside registration, and patient pooling on ped wait times, length of stay, and patient satisfaction. methods: data were extracted from a computerized ed tracking system in an urban tertiary care ped. comparisons were made between metrics for 2010 (23,681 patients) and the 3 months following process change (6,195 patients). during 2010, patients were triaged by one or two nurses, registered, and then sent either to a 14-bed ped or a physically separate 5-bed fast-track unit, where they were seen by a physician. following process change, patients were brought directly to a bed in the 14-bed ped, triaged and registered, then seen by a physician. the fast-track unit was only utilized to accommodate patient surges. results: anticipating improved efficiencies, attending physician coverage was decreased by 9%. after instituting process changes, improvements were noted immediately. although daily patient volume increased by 3%, median time to be seen by a physician decreased by 20%. additionally, median los for discharged patients decreased by 15%, and median time until the decisionto-admit decreased by 10%. press-ganey satisfaction scores during this time increased by greater than 5 mean score points, which was reported to be a statistically significant increase. conclusion: direct bedding, bedside registration, and patient pooling were simple to implement process changes. these changes resulted in more efficient ped throughput, as evidenced by decreased times to be seen by a physician, los for discharged patients, and time until decision-to-admit. additionally, patient satisfaction scores improved, despite decreased attending physician coverage and a 30% decrease in room utilization. ) . during period 1, the ou was managed by the internal medicine department and staffed by primary care physicians and physician assistants. during periods 2 and 3, the ou was managed and staffed by em physicians. data collected included ou patient volume, length of stay (los) for discharged and admitted patients, admission rates, and 30-day readmission rates for discharged patients. cost data collected included direct, indirect, and total cost per patient encounter. data were compared using chi-square and anova analysis followed by multiple pairwise comparisons using the bonferroni method of p-value adjustment. results: see table. the ou patient volume and percent of ed volume was greater in period 3 compared to periods 1 and 2. length of stay, admission rates, 30-day readmission rates, and costs were greater in period 1 compared to periods 2 and 3. conclusion: em physicians provide more cost-effective care for patients in this large ou compared to non-em physicians, resulting in shorter los for admitted and discharged patients, greater rates of patients discharged, and less 30-day readmission rates for discharged patients. this is not affected by an increase in ou volume and shows a trend towards improvement. background: emergency department (ed) crowding continues to be a problem, and new intake models may represent part of the solution. however, little data exist on the sustainability and long-term effects of physician triage and screening on standard ed performance metrics, as most studies are short-term. objectives: we examined the hypothesis that a physician screening program (start) sustainably improves standard ed performance metrics including patient length of stay (los) and patients who left without completing assessment (lwca). we also investigated the number of patients treated and dispositioned by start without using a monitored bed and the median patient door-to-room time. methods: design and setting: this study is a retrospective before-and-after analysis of start in a level i tertiary care urban academic medical center with approximately 90,000 annual patient visits. all adult patients from december 2006 until november 2010 are included, though only a subset was seen in start. start began at our institution in december 2007. observations: our outcome measures were length of stay for ed patients, lwca rates, patients treated and dispositioned by start without using a monitored bed, and door-to-room time. statistics: simple descriptive statistics were used. p-values for los were calculated with wilcoxon test and p-value for lwca was calculated with chi-square. results: table 2 shows median length of stay for ed patients was reduced by 56 minutes/patient (p-value <0.0001) when comparing the most recent year to the year before start. patients who lwca were reduced from 4.8% to 2.9% (p-value <0.0001) during the same time period. we also found that in the first half-year of start, 18% of patients screened in the ed were treated and dispositioned without using a monitored bed and by the end of year 3, this number had grown to 29%. median door-to-room time decreased from 18.4 minutes to 9.9 minutes over the same period of time. conclusion: a start system can provide sustained improvements in ed performance metrics, including a significant reduction in ed los, lwca rate, and doorto-room time. additionally, start can decrease the need for monitored ed beds and thus increase ed capacity. . labs were obtained in 98%, ct in 37%, us in 30%, and consultation in 23%. 18% of the cohort was admitted to the hospital. the most commonly utilized source of translation was a layman (35%). a professional translator was used in 9% and translation service (language line, marty) in 30%. the examiner was fluent in the patient's language in 11%. both the patient and examiner were able to maintain basic communication in 11%. there were 47 patients in the professional/ fluent translation group and 44 patients in the lay translation group. there was no difference in ed los between groups 288 vs 304 min; p = 0.6. there was no difference in the frequency of lab tests, computerized tomography, ultrasound, consultations, or hospital admission. frequencies did not differ by sex or age. conclusion: translation method was not associated with a difference in overall ed los, ancillary test use, or specialist consultation in spanish-speaking patients presenting to the ed for abdominal pain. emergency department patients on warfarin -how often is the visit due to the medication? jim killeen, edward castillo, theodore chan, gary vilke ucsd medical center, san diego, ca background: warfarin has important therapeutic value for many patients, but has been associated with signi-ficant bleeding complications, hypersensitivity reactions, and drug-drug interactions, which can result in patients seeking care in the emergency department (ed). objectives: to determine how often ed patients on warfarin present for care as a result of the medication itself. methods: a multi-center prospective survey study in two academic eds over 6 months. patients who presented to the ed taking warfarin were identified, and ed providers were prospectively queried at the time of disposition regarding whether the visit was the result of a complication or side effect associated with warfarin. data were also collected on patient demographics, chief complaint, triage acuity, vital signs, disposition, ed evaluation time, and length of stay (los). patients identified with a warfarin-related cause for their ed visit were compared with those who were not. statistical analysis was performed using descriptive statistics. results: during the study period, 31,500 patients were cared for by ed staff, of whom 594 were identified as taking warfarin as part of their medication regimen. of these, providers identified 54.7% (325 patients) who presented with a warfarin-related complication as their primary reason for the ed visit. 56.9% (338) each 100 hours of daily boarding is associated with a drop of 1.3 raw score points in both pg metrics. these seemingly small drops in raw scores translate into major changes in rankings on press ganey national percentile scales (a difference of as much as 10 percentile points). our institution commonly has hundreds of hours of daily boarding. it is possible that patient-level measurements of boarding impact would show stronger correlation with individual satisfaction scores, as opposed to the daily aggregate measures we describe here. our research suggests that reducing the burden of boarding on eds will improve patient satisfaction. background: prolonged emergency department (ed) boarding is a key contributor to ed crowding. the effect of output interventions (moving boarders out of the ed into an intermediate area prior to admission or adding additional capacity to an observation unit) has not been well studied. objectives: we studied the effect of a combined observation-transition (ot) unit, consisting of observation beds and an interim holding area for boarding ed patients, on the length of stay (los) for admitted patients, as well as secondary outcomes such as los for discharged patients, and left without being seen rates. methods: we conducted a retrospective review (12 months pre-, 12 months post-design) of an ot unit at an urban teaching ed with 59,000 annual visits (study ed). we compared outcomes to a nearby communitybased ed with 38,000 annual visits in the same health system (control ed) where no capacity interventions were performed. the ot had 17 beds, full monitoring capacity, and was staffed 24 hours per day. the number of beds allocated to transition and observation patients fluctuated throughout the course of the intervention, based on patient demands. all analyses were conducted at the level of the ed-day. wilcoxon rank-sum and analysis of covariance tests were used for comparisons; continuous variables were summarized with medians. results: in unadjusted analyses, median daily los of admitted patients at the study ed was 31 minutes lower in the 12 months after the ot opened, 6.98 to 6.47 hours (p < 0.0001). control site daily los for admitted patients increased 26 minutes from 4.52 to 4.95 hours (p < 0.0001). results were similar after adjusting for other covariates (day of week, ed volume, and triage level). los of discharged patients at study ed decreased by 14 minutes, from 4.1 hours to 3.8 hours (p < 0.001), while the control ed saw no significant changes in discharged patient los (2.6 hours to 2.7 hours, p = 0.06). left without being seen rates did not decrease at either site. conclusion: opening an ot unit was associated with a 30-minute reduction in average daily ed los for admitted patients and discharged patients in the study ed. given the large expense of opening an ot, future studies should compare capacity-dependent (e.g., ot) vs. capacity-independent (e.g, organizational) interventions to reduce ed crowding. fran balamuth, katie hayes, cynthia mollen, monika goyal children's hospital of philadelphia, philadelphia, pa background: lower abdominal pain and genitourinary problems are common chief complaints in adolescent females presenting to emergency departments. pelvic inflammatory disease (pid) is a potentially severe complication of lower genital tract infections, which involves inflammation of the female upper genital tract secondary to ascending stis. pid has been associated with severe sequelae including infertility, ectopic pregnancy, and chronic pelvic pain. we describe the prevalence and microbial patterns of pid in a cohort of adolescent females presenting to an urban emergency department with abdominal or genitourinary complaints. objectives: to describe the prevalence and microbial patterns of pid in a cohort of adolescent patients presenting to an ed with lower abdominal or genitourinary complaints. methods: this is a secondary analysis of a prospective study of females ages 14-19 years presenting to a pediatric ed with lower abdominal or genitourinary complaints. diagnosis of pid was per 2006 cdc guidelines. patients underwent chlamydia trachomatis (ct) and neisseria gonorrhea (gc) testing via urine aptima combo 2 assay and trichomonas vaginalis (tv) testing using the vaginal osom trichomonas rapid test. descriptive statistics were performed using stata 11.0. results: the prevalence of pid in this cohort of 328 patients was 19.5% (95% ci 15.2%, 23.8%), 37.5% (95% ci 25.3%, 49.7%) of whom had positive sexually transmitted infection (sti) testing: 25% (95% ci 14.1%, 35.9%) with ct, 7.8% (95% ci 1.1, 14.6%) with gc, and 12.5% (95% ci 4.2%, 20.8%) with tv. 84.4% (95% ci 75.2, 93.5%) of patients diagnosed with pid received antibiotics consistent with cdc recommendations. patients with lower abdominal pain as their chief complaint were more likely to have pid than patients with genitourinary complaints (or 3.3, 95% ci 1.7, 6.4). conclusion: a substantial number of adolescent females presenting to the emergency department with lower abdominal pain were diagnosed with pid, with microbial patterns similar to those previously reported in largely adult, outpatient samples. furthermore, appropriate treatment for pid was observed in the majority of patients diagnosed with pid. impact background: in resource-poor settings, maternal health care facilities are often underutilized, contributing to high maternal mortality. the effect of ultrasound in these settings on patients, health care providers, and communities is poorly understood. objectives: the purpose of this study was to assess the effect of the introduction of maternal ultrasound in a population not previously exposed to this intervention. methods: an ngo-led program trained nurses at four remote clinics outside koutiala, mali, who performed 8,339 maternal ultrasound scans over three years. our researchers conducted an independent assessment of this program, which involved log book review, sonographer skill assessment, referral follow-up, semi-structured interviews of clinic staff and patients, and focus groups of community members in surrounding villages. analyses included the effect of ultrasound on clinic function, job satisfaction, community utilization of prenatal care and maternity services, alterations in clinical decision making, sonographer skill, and referral frequency. we used qrs nvivo9 to organize qualitative findings, code data, and identify emergent themes, and graphpad software (la jolla, ca) and microsoft excel to tabulate quantitative findings results: -findings that triggered changes in clinical practice were noted in 10.1% of ultrasounds, with a 3.5% referral rate to comprehensive maternity care facilities. -skill retention and job satisfaction for ultrasound providers was high. -the number of patients coming for antenatal care increased, after introduction of ultrasound, in an area where the birth rate has been decreasing. -over time, women traveled from farther distances to access ultrasound and participate in antenatal care. -very high acceptance among staff, patients and community members. -ultrasound was perceived as most useful for finding fetal position, sex, due date, and well-being. -improved confidence in diagnosis and treatment plan for all cohorts. -improved compliance with referral recommendations. -no evidence of gender selection motivation for ultrasound use. conclusion: use of maternal ultrasound in rural and resource-limited settings draws women to an initial antenatal care visit, increases referral, and improves job satisfaction among health care workers. methods: a retrospective database analysis was conducted using the electronic medical record from a single, large academic hospital. ed patients who received a billing diagnosis of ''nausea and vomiting of pregnancy'' or ''hyperemesis gravidarum'' between 1/1/10 and 12/31/10 were selected. a manual chart review was conducted with demographic and treatment variables collected. statistical significance was determined using multiple regression analysis for a primary outcome of return visit to the emergency department for nausea and vomiting of pregnancy. results: 113 patients were identified. the mean age was 27.1 years (sd±5.25), mean gravidity 2.90 (sd±1.94), and mean gestational age 8.78 weeks (sd±3.21). the average length of ed evaluation was 730 min (sd±513). of the 113 patients, 38 (33.6%) had a return ed visit for nausea and vomiting of pregnancy, 17 (15%) were admitted to the hospital, and 49 (43%) were admitted to the ed observation protocol. multiple regression analysis showed that the presence of medical co-morbidity (p = 0.039), patient gravditity (p = 0.016), gestational age (p = 0.038), and admission to the hospital (p = 0.004) had small but significant effects on the primary outcome (return visits to the emergency department). no other variables were found to be predictive of return visits to the ed including admission to the ed observation unit or factors classically thought to be associated with severe forms of nausea and vomiting in pregnancy including ketonuria, electrolyte abnormalities, or vital sign abnormalities. conclusion: nausea and vomiting in pregnancy has a high rate of return ed visits that can be predicted by young patient age, low patient gravidity, early gestational age, and the presence of other comorbidities. these patients may benefit from obstetric consultation and/or optimization of symptom management after discharge in order to prevent recurrent utilization of the ed. prevalence conclusion: there is a high prevalence of ht in adult sa victims. although our study design and data do not allow us to make any inferences regarding causation, this first report of ht ed prevalence suggests the opportunity to clarify this relationship and the potential opportunity to intervene. background: sexually transmitted infections (sti) are a significant public health problem. because of the risks associated with stis including pid, ectopic pregnancy, and infertility the cdc recommends aggressive treatment with antibiotics in any patient with a suspected sti. objectives: to determine the rates of positive gonorrhea and chlamydia (g/c) screening and rates of empiric antibiotic use among patients of an urban academic ed with >55,000 visits in boston, ma. methods: a retrospective study of all patients who had g/c cultures in the ed over 12 months. chi-square was used in data analysis. sensitivity and specificity were also calculated. results: a positive rate of 9/712 (1.2%) was seen for gonorrhea and 26/714 (3.6%) for chlamydia. females had positive rates of 2/602 (0.3%) and 17/603 (2.8%) respectively. males had higher rates of 7/110 (6.4%) (p =< 0.001) and 9/111 (8.1%) (p = 0.006). 284 patients with g/c sent received an alternative diagnosis, the most common being uti (63), ovarian pathology (35), vaginal bleeding (34), and vaginal candidiasis (33); 4 were excluded. this left 426 without definitive diagnosis. of these, 24.2% (87/360) of females were treated empirically with antibiotics for g/c, and a greater percentage of males (66%, 45/66) were treated empirically (p < 0.001). of those empirically treated, 109/132 (82.6%) had negative cultures. meanwhile 9/32 (28.1%) who ultimately had positive cultures were not treated with antibiotics during their ed stay. sensitivity of the provider to predict presence of disease based on decision to give empiric antibiotics was 71.9 (ci 53.0-85.6). specificity was 72.3 (ci 67.6-76.6). conclusion: most patients screened in our ed for g/c did not have positive cultures and 82.6% of those treated empirically were found not to have g/c. while early treatment is important to prevent complications, there are risks associated with antibiotic use such as allergic reaction, c difficile infection, and development of antibiotic resistance. our results suggest that at our institution we may be over-treating for g/c. furthermore, despite high rates of treatment, 28% of patients who ultimately had positive cultures did not receive antibiotics during their ed stay. further research into predictive factors or development of a clinical decision rule may be useful to help determine which patients are best treated empirically with antibiotics for presumed g/c. background: air travel may be associated with unmeasured neurophysiological changes in an injured brain that may affect post-concussion recovery. no study has compared the effect of commercial airtravel on concussion injuries despite rather obvious decreased oxygen tension and increased dehydration effect on acute mtbi. objectives: to determine if air travel within 4-6 hours of concussion is associated with increased recovery time in professional football and hockey players. methods: prospective cohort study of all active-roster national football league and national hockey league players during the 2010-2011 seasons. internet website review of league sties for injury identification of concussive injury and when player returned to play solely for mtbi. team schedules and flight times were also confirmed to include only players who flew immediately following game (within 4-6 hr). multiple injuries were excluded as were players who had injury around all-star break for nhl and scheduled off week in nfl. results: during the 2010-2011 nfl and nhl seasons, 122 (7.2%) and 101 (13.0%) players experienced a concussion (percent of total players), in the respective leagues. of these, 68 nfl players (57%) and 39 nhl players (39%) flew within 6 hours of the incident injury. the mean distance flown was shorter for nfl (850 miles, sd 576 vs. nhl 1060, sd 579) miles and all were in a pressurized cabin. the mean number of games missed for nfl and nhl players who traveled by air immediately after concussion was increased by 29% and 24% (respectively) than for those who did not travel by air nfl: 3.8 (sd 2.2) vs. 2.6 games (sd 1.8) and nhl: 16.2 games (sd 22.0) vs.12.4 (sd 18.6); p < 0.03. conclusion: this is an initial report of an increased rate of recovery in terms of more games missed, for professional athletes flying commercial airlines post-mtbi compared to those that do not subject their recently injured brains to pressurized airflight. the obvious changes of decreased oxygen tension with altitude equivalent of 7,500 feet, decreased humidity with increased dehydration, and duress of travel accompanying pressurized airline cabins all likely increase the concussion penumbra in acute mtbi. early air travel post concussion should be further evaluated and likely postponed 48-72 hr. until initial symptoms subside. background: previous studies have shown better in-hospital stroke time targets for those who arrive by ambulance compared to other modes of transport. however, regional studies report that less than half of stroke patients arrive by ambulance. objectives: our objectives were to describe the proportion of stroke patients who arrive by ambulance nationwide, and to examine regional differences and factors associated with the mode of transport to the emergency department (ed). methods: this is a cross-sectional study of all patients with a primary discharge diagnosis of stroke based on previously validated icd-9 codes abstracted from the national hospital ambulatory medical care survey for 2007-2009. we excluded subjects <18 years of age and those with missing data. the study related survey variables included patient demographics, community characteristics, mode of transport to the hospital, and hospital characteristics. results: 566 patients met inclusion criteria, representing 2,153,234 patient records nationally. of these, 50.4% arrived by ambulance. after adjustment for potential confounders, patients residing in the west and south had lower odds of arriving by ambulance for stroke when compared to northeast (southern region, or 0.45, 95% ci 0.26-0.76, western region, or 0.45, 95% ci 0.25-0.84, midwest region, or 0.56, 95% ci 0.31-1.01). compared to the medicare population, privately insured and self insured had lower odds of arriving by ambulance (or for private insurance 0.48, 95% ci 0.28-0.84 and or for self payers 0.36, 95% ci 0.14-0.93). age, sex, race, urban or rural location of ed, or safety net status were not independently associated with ambulance use. conclusion: patients with stroke arrive by ambulance more frequently in the northeast than in other regions of the us. identifying reasons for this regional difference may be useful in improving ambulance utilization and overall stroke care nationwide. objectives: we sought to determine whether there was a difference in type of stroke presentation based upon race. we further sought to determine whether there is an increase in hemorrhagic strokes among asian patients with limited english proficiency. methods: we performed a retrospective chart review of all stroke patients age 18 and older for 1 year of patients that were diagnosed with cerebral vascular accident (cva) or intracranial hemorrhage (ich). we collected data on patient demographics, and past medical history. we then stratified patients according to race (white, black, latino, asian, and other). we classified strokes as ischemic, intracranial hemorrhage (ich), subarachnoid hemorrhage (sah), subdural hemorrhage (sdh), and other (e.g., bleeding into metatstatic lesions). we used only the index visit. we present the data percentages, medians and interquartile ranges (iqr). we tested the association of the outcome of intracranial hemorrhage against demographic and clinical variables using chi-square and kruskal-wallis tests. we performed a logistic regression model to determine factors related to presentation with an intracranial hemorrhage (ich background: the practice of obtaining laboratory studies and routine ct scan of the brain on every child with a seizure has been called into question in the patient who is alert, interactive, and back to functional baseline. there is still no standard practice for the management of non-febrile seizure patients in the pediatric emergency department (ped). objectives: we sought to determine the proportion of patients in whom clinically significant laboratory studies and ct scans of the brain were obtained in children who presented to the ped with a first or recurrent non-febrile seizure. we hypothesize that the majority of these children do not have clinically significant laboratory or imaging studies. if clinically significant values were found, the history given would warrant further laboratory and imaging assessment despite seizure alone. methods: we performed a retrospective chart review of 93 patients with first-time or recurrent non-febrile seizures at an urban, academic ped between july 2007 to june 2011. exclusion criteria included children who presented to the ped with a fever and age less than 2 months. we looked at specific values that included a complete blood count, basic metabolic panel, and liver function tests, and if the child was on antiepileptics along with a level for a known seizure disorder, and ct scan. abnormal laboratory and ct scan findings were classified as clinically significant or not. results: the median age of our study population is 4 years with male to female ratio of 1.7. 70% of patients had a generalized tonic-clonic seizure. laboratory studies and ct scans were obtained in 87% and 35% of patients, respectively. five patients had clinically significant abnormal labs; however, one had esrd, one developed urosepsis, one had eclampsia, and two others had hyponatremia, which was secondary to diluted formula and trileptal toxicity. three children had an abnormal head ct: two had a vp shunt and one had a chromosomal abnormality with developmental delay. conclusion: the majority of the children analyzed did not have clinically significant laboratory or imaging studies in the setting of a first or recurrent non-febrile seizure. of those with clinically significant results, the patient's history suggested a possible etiology for their seizure presentation and further workup was indicated. background: in patients with a negative ct scan for suspected subarachnoid hemorrhage (sah), ct angiography (cta) has emerged as a controversial alternative diagnostic strategy in place of lumbar puncture (lp). objectives: to determine the diagnostic accuracy for sah and aneurysm of lp alone, cta alone, and lp followed by cta if the lp is positive. methods: we developed a decision and bayesian analysis to evaluate 1) lp, 2) cta, and 3) lp followed by cta if the lp is positive. data were obtained from the literature. the model considers probability of sah (15%), aneurysm (85% if sah), sensitivity and specificity of ct (92.9% and 100% overall), of lp (based on rbc and xanthochromia), and of cta, traumatic tap and its influence on sah detection. analyses considered all patients and those presenting at less than 6 hours or greater than 6 hours from symptom onset by varying the sensitivity and specificity of ct and cta. results: using the reported ranges of ct scan sensitivity and the specificity, the revised likelihood of sah following a negative ct ranged from 0.5-3.7%, and the likelihood of aneurysm ranged from 2.3-5.4%. following any of the diagnostic strategies, the likelihood of missing sah ranged from 0-0.7%. either lp strategy diagnosed 99.8% of sahs versus 83-84% with cta alone because cta only detected sah in the presence of an aneurysm. false positive sah with lp ranged from 8.5-8.8% due to traumatic taps and with cta ranged from 0.2-6.0% due to aneurysms without sah. the positive predictive value for sah ranged from 5.7-30% with lp and from 7.9-63% with cta. for patients presenting within 6 hours of symptom onset, the revised likelihood of sah following a negative ct became 0.53%, and the likelihood of aneurysm ranged from 2.3-2.7%. following any of the diagnostic strategies, the likelihood of missing sah ranged from 0.01-0.095%. either lp strategy diagnosed 99.8% of sah versus 83-84% with cta alone. false positive sah with lp was 8.8% and with cta ranged from 0.2-5.1%. the positive predictive value for sah was 5.7% with lp and from 7.9-63% with cta. cta following a positive lp diagnosed 8.5-24% of aneurysms. conclusion: lp strategies are more sensitive for detecting sah but less specific than cta because of traumatic taps, leading to lower predictive value positives for sah with lp than with cta. either diagnostic strategy results in a low likelihood of missing sah, particularly within 6 hours of symptom onset. background: recent studies support perfusion imaging as a prognostic tool in ischemic stroke, but little data exist regarding its utility in transient ischemic attack (tia). ct perfusion (ctp), which is more available and less costly to perform than mri, has not been well studied. objectives: to characterize ctp findings in tia patients, and identify imaging predictors of outcome. methods: this retrospective cohort study evaluated tia patients at a single ed over 15 months, who had ctp at initial evaluation. a neurologist blinded to ctp findings collected demographic and clinical data. ctp images were analyzed by a neuroradiologist blinded to clinical information. ctp maps were described as qualitatively normal, increased, or decreased in mean transit time (mtt), cerebral blood volume (cbv), and cerebral blood flow (cbf). quantitative analysis involved measurements of average mtt (seconds), cbv (cc/100 g) and cbf (cc/[100g x min]) in standardized regions of interest within each vascular distribution. these were compared with values in the other hemisphere for relative measures of mtt difference, cbv ratio, and cbffratio. mtt difference of ‡2 seconds, rcbv as £0.60, and rcbf as £0.48 were defined as abnormal based on prior studies. clinical outcomes including stroke, tia, or hospitalization during follow-up were determined up to one year following the index event. dichotomous variables were compared using fisher's exact test. logistic regression was used to evaluate the association of ctp abnormalities with outcome in tia patients. results: of 99 patients with validated tia, 53 had ctp done. mean age was 72 ± 12 years, 55% were women, and 64% were caucasian. mean abcd 2 score was 4.7 ± 2.1, and 69% had an abcd 2 ‡ 4. prolonged mtt was the most common abnormality (19, 36%), and 5 (9.4%) had decreased cbv in the same distribution. on quantitative analysis, 23 (43%) had a significant abnormality. four patients (7.5%) had prolonged mtt and decreased cbv in the same territory, while 17 (32%) had mismatched abnormalities. when tested in a multivariate model, no significant associations between mismatch abnormalities on ctp and new stroke, tia, or hospitalizations were observed. conclusion: ctp abnormalities are common in tia patients. although no association between these abnormalities and clinical outcomes was observed in this small study, this needs to be studied further. objectives: we hypothesized that pre-thrombolytic anti-hypertensive treatment (aht) may prolong door to treatment time (dtt). methods: secondary data analysis of consecutive tpatreated patients at 24 randomly selected michigan community hospitals in the instinct trial. dtt among stroke patients who received pre-thrombolytic aht were compared to those who did not receive pre-thrombolytic aht. we then calculated a propensity score for the probability of receiving pre-thrombolytic aht using a logistic regression model with covariates including demographics, stroke risk factors, antiplatelet or beta blocker as home medication, stroke severity (nihss), onset to door time, admission glucose, pretreatment systolic and diastolic blood pressure, ems usage, and location at time of stroke. a paired t-test was then performed to compare the dtt between the propensity-matched groups. a separate generalized estimating equations (gee) approach was also used to estimate the differences between patients receiving pre-thrombolytic aht and those who did not while accounting for within-hospital clustering. results: a total of 557 patients were included in instinct; however, onset, arrival, or treatment times were not able to be determined in 23, leaving 534 patients for this analysis. the unmatched cohort consisted of 95 stroke patients who received pre-thrombolytic aht and 439 stroke patients who did not receive aht from 2007-2010 (table) . in the unmatched cohort, patients who received pre-thrombolytic aht had a longer dtt (mean increase 9 minutes; 95% confidence interval (ci) 2-16 minutes) than patients who did not receive pre-thrombolytic aht. after propensity matching (table) , patients who received pre-thrombolytic aht had a longer dtt (mean increase 10.4 minutes, 95% ci 1.9-18.8) than patients who did not receive pre-thrombolytic aht. this effect persisted and its magnitude was not altered by accounting for clustering within hospitals. conclusion: pre-thrombolytic aht is associated with modest delays in dtt. this represents a feasible target for physician educational interventions and quality improvement initiatives. further research evaluating optimum hypertension management pre-thrombolytic treatment is warranted. post-pds, 7% had only pre-pds, and 9% had both. the most common pds included failure to treat post-treatment hypertension (131, 24%), antiplatelet agent within 24 hours of treatment (61, 11%), pre-treatment blood pressure over 185/110 (39, 7%), anticoagulant agent within 24 hours of treatment (31, 6%), and treatment outside the time window (29, 5%). symptomatic intracranial hemorrhage (sich) was observed in 7.3% of patients with pds and 6.5% of patients without any pd. in-hospital case fatality was 12% with and 10% without a pd. in the fully adjusted model, older age was significantly associated with pre-pds (table) . when post-pds were evaluated with adjustment for pre-pds, age was not associated with pds; however, pre-pds were associated with post-pds. conclusion: older age was associated with increased odds of pre-pds in michigan community hospitals. pre-pds were associated with post-pds. sich and in-hospital case fatality were not associated with pds; however, the low number of such events limited our ability to detect a difference. ct background: mri has become the gold standard for the detection of cerebral ischemia and is a component of multiple imaging enhanced clinical risk prediction rules for the short-term risk of stroke in patients with transient ischemic attack (tia). however, it is not always available in the emergency department (ed) and is often contraindicated. leukoaraiosis (la) is a radiographic term for white matter ischemic changes, and has recently been shown to be independently predictive of disabling stroke. although it is easily detected by both ct and mri, their comparative ability is unknown. objectives: we sought to determine whether leukoaraiosis, when combined with evidence of acute or old infarction as detected by ct, achieved similar sensitivity to mri in patients presenting to the ed with tia. methods: we conducted a retrospective review of consecutive patients diagnosed with tia between june 2009 and july 2011 that underwent both ct and mri as part of routine care within 1 calendar day of presentation to a single, academic ed. ct and mr images were reviewed by a single emergency physician who was blinded to the mr images at the time of ct interpretation. la was graded using the van sweiten scale (vss), a validated grading scale applicable to both ct and mri. anterior and posterior regions were graded independently from 0 to 2. results: 361 patients were diagnosed with tia during the study period. of these, 194 had both ct and mri background: helping others is often a rewarding experience but can also come with a ''cost of caring'' also known as compassion fatigue (cf). cf can be defined as the emotional and physical toll suffered by those helping others in distress. it is affected by three major components: compassion satisfaction (cs), burnout (bo), and traumatic experiences (te). previous literature has recognized an increase in bo related to work hours and stress among resident physicians. objectives: to assess the state of cf among residents with regard to differences in specialty training, hours worked, number of overnights, and demands of child care. we aim to measure associations with the three components of cf (cs, bo, and te). methods: we used the previously validated survey, proqol 5. the survey was sent to the residents after approval from the irb and the program directors. results: a total of 193 responses were received (40% of the 478 surveyed). five were excluded due to incomplete questionnaires. we found that residents who worked more hours per week had significantly higher bo levels (median 25 vs 21, p = 0.038) and higher te (22 vs 19, p = 0.048) than those working less hours. there was no difference in cs (42 vs 40, p = 0.73). eighteen percent of the residents worked a majority of the night shifts. these residents had higher levels of bo background: emergency department (ed) billing includes both facility and professional fees. an algorithm derived from the medical provider's chart generates the latter fee. many private hospitals encourage appropriate documentation by financially incentivizing providers. academic hospitals sometimes lag in this initiative, possibly resulting in less than optimal charting. past attempts to teach proper documentation using our electronic medical record (emr) were difficult in our urban, academic ed of 80 providers (approximately 25 attending physicians, 36 residents, and 20 physician assistants). objectives: we created a tutorial to teach documentation of ed charts, modified the emr to encourage appropriate documentation, and provided feedback from the coding department. this was combined with an incentive structure shared equally amongst all attendings based on increased collections. we hypothesized this instructional intervention would lead to more appropriate billing, improve chart content, decrease medical liability, and increase educational value of charting process. methods: documentation recommendations, divided into two-month phases of 2-3 proposals, were administered to all ed providers by e-mails, lectures, and reminders during sign-out rounds. charts were reviewed by coders who provided individual feedback if specific phase recommendations were not followed. our endpoints included change in total rvu, rvus/ patient, e/m level distribution, and subjective quality of chart improvement. we did not examine effects on procedure codes or facility fees. results: our base average rvu/patient in our ed from 1/1/11-6/30/11 was 2.615 with monthly variability of approximately 2%. implementation of phase one increased average rvu/patient within two weeks to 2.73 (4.4% increase from baseline, p < 0.05). the second aggregate phase implemented 8 weeks later increased average rvu/patient to 3.04 (16.4% increase from baseline, p < 0.05). conclusion: using our teaching methods, chart reviews focused on 2-3 recommendations at a time, and emr adjustments, we were able to better reflect the complexity of care that we deliver every day in our medical charts. future phases will focus on appropriate documentation for procedures, critical care, fast track, and pediatric patients, as well as examining correlations between increase in rvus with charge capture. identifying mentoring ''best practices'' for medical school faculty julie l. welch, teresita bellido, cherri d. hobgood background: mentoring has been identified as an essential component for career success and satisfaction in academic medicine. many institutions and departments struggle with providing both basic and transformative mentoring for their faculty. objectives: we sought to identify and understand the essential practices of successful mentoring programs. methods: multidisciplinary institutional stakeholders in the school of medicine including tenured professors, deans, and faculty acknowledged as successful mentors were identified and participated in focused interviews between mar-nov 2011. the major area of inquiry involved their experiences with mentoring relationships, practices, and structure within the school, department, or division. focused interview data were transcribed and grounded theory analysis was performed. additional data collected by a 2009 institutional mentoring taskforce were examined. key elements and themes were identified and organized for final review. results: results identified the mentoring practices for three categories: 1) general themes for all faculty, 2) specific practices for faculty groups: basic science researchers, clinician researchers, clinician educators, and 3) national examples. additional mentoring strategies that failed were identified. the general themes were quite universal among faculty groups. these included: clarify the best type of mentoring for the mentee, allow the mentee to choose the mentor, establish a panel of mentors with complementary skills, schedule regular meetings, establish a clear mentoring plan with expectations and goals, offer training and resources for both the mentor and mentee at institutional and departmental levels, ensure ongoing mentoring evaluation, create a mechanism to identify and reward mentoring. national practice examples offered critical recommendations to address multi-generational attitudes and faculty diversity in terms of gender, race, and culture. conclusion: mentoring strategies can be identified to serve a diverse faculty in academic medicine. interventions to improve mentoring practices should be targeted at the level of the institution, department, and individual faculty members. it is imperative to adopt results such as these to design effective mentoring programs to enhance the success of emergency medicine faculty seeking robust academic careers. background: women comprise half of the talent pool from which the specialty of emergency medicine draws future leaders, researchers, and educators and yet only 5% of full professors in us emergency medicine are female. both research and interventions are aimed at reducing the gender gap, however, it will take decades for the benefits to be realized which creates a methodological challenge in assessing system's change. current techniques to measure disparities are insensitive to systems change as they are limited to percentages and trends over time. objectives: to determine if the use of relative rate index (rri) better predicts which stage in the system women are not advancing in the academic pipeline than traditional metrics. methods: rri is a method of analysis that assesses the percent of sub-populations in each stage relative to their representation in the stage directly prior. thus, there is a better notion of the advancement given the availability to advance. rri also standardizes data for ease of interpretation. this study was conducted on the total population of academic professors in all departments at yale school of medicine during the academic year of 2010-2011. data were obtained from the yale university provost's office. results: n = 1305. there were a total of 402 full, 429 associate, and 484 assistant professors. males comprised 78%, 59%, and 54% respectively. rri for the department of emergency medicine (dem) is 0.67, 1.93, and 0.78, for full, associate, and assistant professors, respectively while the percentages were 44%, 60%, and 33% respectively. conclusion: relying solely on percentages masks improvements to the system. women are most represented at the associate professor level in dem, highlighting the importance of systems change evidence. specifically, twice as many women are promoted to associate professor rank given the number who exists as assistant professors. within 5 years, the dem should have an equal system as the numbers of associate professors have dramatically increased and will be eligible to promote to full professor. additionally, dem has a better record of retaining and promoting women than other yale departments of medicine at both associate and full professor ranks. objectives: we examine the payer mixes of community non-rehabilitation eds in metropolitan areas by region to identify the proportion of academic and nonacademic eds that could be considered safety net eds. we hypothesize that the proportion of safety net academic eds is greater than that for non-academic eds and is increasing over time. methods: this is an ecological study examining us ed visits from 2006 through 2008. data were obtained from the nationwide emergency department sample (neds). we grouped each ed visit according to the unique hospital-based ed identifier, thus creating a payer mix for each ed. we define a ''safety net ed'' as any ed where the payer mix satisfied any one of the following three conditions: 1) >30% of all ed visits are medicaid patients; 2) >30% of all ed visits are self-pay patients; or 3) >40% of all ed visits are either medicaid or self-pay patients. neds tags each ed with a hospital-based variable to delineate metropolitan/non-metropolitan locations and academic affiliation. we chose to examine a subpopulation of eds tagged as either academic metropolitan or non-academic metropolitan, because the teaching status of non-metropolitan hospitals was not provided. we then measured the proportion of eds that met safety net criteria by academic status and region. results: we examined 2,821, 2,793, and 2,844 weighted metro eds in years 2006-2008, respectively. table 1 presents safety net proportions. the proportions of academic safety net eds increased across the study period. widespread regional variability in safety net proportions existed across all years. the proportions of safety net eds were highest in the south and lowest in the northeast and midwest. table 2 describes these findings for 2008. conclusion: these data suggest that the proportion of safety-net academic eds may be greater than that of non-academic eds, is increasing over time, and is objectives: to examine the effect of ma health reform implementation on ed and hospital utilization before and after health reform, using an approach that relies on differential changes in insurance rates across different areas of the state in order to make causal inferences as to the effect of health reform on ed visits and hospitalizations. our hypothesis was that health care reform (i.e. reducing rates of uninsurance) would result in increased rates of ed use and hospitalizations. methods: we used a novel difference-in-differences approach, with geographic variation (at the zip code level) in the percentage uninsured as our method of identifying changes resulting from health reform, to determine the specific effect of massachusetts' health care reform on ed utilization and hospitalizations. using administrative data available from the massachusetts division of health care finance and policy acute hospital case mix databases, we compared a one-year period before health reform with an identical period after reform. we fit linear regression models at the area-quarter level to estimate the effect of health reform and the changing uninsurance rate (defined as self-pay only) on ed visits and hospitalizations. results: there were 2,562,330 ed visits and 777,357 hospitalizations pre-reform and 2,713,726 ed visits and 787,700 hospitalizations post-reform. the rate of uninsurance decreased from 6.2% to 3.7% in the ed group and from 1.3% to 0.6% in the hospitalization group. a reduction in the rate of the uninsured was associated with a small but statistically significant increase in ed utilization (p = 0.03) and no change in hospitalizations (p = 0.13). conclusion: we find that increasing levels of insurance coverage in massachusetts were associated with small but statistically significant increases in ed visits, but no differences in rates of hospitalizations. these results should aid in planning for anticipated changes that might result from the implementation of health reform nationally. with high levels of co-morbidity when untreated in adolescents. despite broad cdc screening recommendations, many youth do not receive testing when indicated. the pediatric emergency department (ped) is a venue with a high volume of patients potentially in need of sti testing, but assessing risk in the ped is difficult given constraints on time and privacy. we hypothesized that patients visiting a ped would find an audio-enhanced computer-assisted self-interview (acasi) program to establish sti risk easy to use, and would report a preference for the acasi over other methods of disclosing this information. objectives: to assess acceptability, ease of use, and comfort level of an acasi designed to assess adolescents' risk for stis in the ped. methods: we developed a branch-logic questionnaire and acasi system to determine whether patients aged 15-21 visiting the ped need sti testing, regardless of chief complaint. we obtained consent from participants and guardians. patients completed the acasi in private on a laptop. they read a one-page computer introduction describing study details and completed the acasi. patients rated use of the acasi upon completion using five-point likert scales. results: 2030 eligible patients visited the ped during the study period. we approached 873 (43%) and enrolled and analyzed data for 460/873 (53%). the median time to read the introduction and complete the acasi was 8.2 minutes (interquartile range 6.4-11.5 minutes). 90.7% of patients rated the acasi ''very easy'' or ''easy'' to use, 90.6% rated the wording as ''very easy'' or ''easy'' to understand, 60% rated the acasi ''very short'' or ''short'', 60.3% rated the audio as ''very helpful'' or ''helpful,'' 82.9% were ''very comfortable'' or ''comfortable'' with the system confidentiality, and 71.2% said they would prefer a computer interface over in-person interviews or written surveys for collection of this type of information. conclusion: patients rated the computer interface of the acasi as easy and comfortable to use. a median of 8.2 minutes was needed to obtain meaningful clinical information. the acasi is a promising approach to enhance the collection of sensitive information in the ped. the participants were randomized to one of three conditions, bi delivered by a computer (cbi), bi delivered by a therapist assisted by a computer (tbi), or control, and completed 3, 6, and 12 month follow-up. in addition to content on alcohol misuse and peer violence, adolescents reporting dating violence received a tailored module on dating violence. the main outcome for this analysis was frequency of moderate and severe dating victimization and aggression at the baseline assessment and 3, 6, and 12 months post ed visit. results: among eligible adolescents, 55% (n = 397) reported dating violence and were included in these analyses. compared to controls, after controlling for baseline dating victimization, participants in the cbi showed reductions in moderate dating victimization at 3 months (or 0.7; ci 0.51-0.99; p < 0.05, effect size 0.12) and 6 months (or 0.56; ci 0.38-0.83; p < 0.01, effect size 0.18); models examining interaction effects were significant for the cbi on moderate dating victimization at 3 and 6 months. significant interaction effects were found for the tbi on moderate dating victimization at 6 and 12 months and severe dating victimization at 3 months. the computer-based intervention shows promise for delivering content that decreases moderate dating victimization over 6 months. the therapist bi is promising for decreasing moderate dating victimization over 12 months and severe dating victimization over 3 months. ed-based bis delivered on a computer addressing multiple risk behaviors could have important public health effects. figure 1 . the 21-only ordinance was associated with a significant reduction of ar visits. this ordinance was also associated with reduction in underage ar visits, ui student visits, and public intoxication bookings. these data suggest that other cities should consider similar ordinances to prevent unwanted consequences of alcohol. background: prehospital providers perform tracheal intubation in the prehospital environment, and failed attempts are of concern due to the danger of hypoxia and hypotension. some question the appropriateness of intubation in this setting due to the morbidity risk associated with intubation in the field. thus it is important to gain an understanding of the factors that predict the success of prehospital intubation attempts to inform this discussion. objectives: to determine the factors that affect success rates on first attempt of paramedic intubations in a rapid sequence intubation (rsi) capable critical care transport service. methods: we conducted a multivariate logistic analysis on a prospectively collected database of airway management from an air and land critical care transport service that provides scene responses and interfacility transport in the province of ontario. background: motor vehicle collisions (mvcs) are one of the most common types of trauma for which people seek ed care. the vast majority of these patients are discharged home after evaluation. acute psychological distress after trauma causes great suffering and is a known predictor of posttraumatic stress disorder (ptsd) development. however, the incidence and predictors of psychological distress among patients discharged to home from the ed after mvcs have not been reported. objectives: to examine the incidence and predictors of acute psychological distress among individuals seen in the ed after mvcs and discharged to home. methods: we analyzed data from a prospective observational study of adults 18-64 years of age presenting to one of eight ed study sites after mvc between 02/ 2009 and 10/2011. english-speaking patients who were alert and oriented, stable, and without injuries requiring hospital admission were enrolled. patient interview included assessment of patient sociodemographic and psychological characteristics and mvc characteristics. level of psychological distress in the ed was assessed using the 13-item peritraumatic distress inventory (pdi). pdi scores >23 are associated with increased risk of ptsd and were used to define substantial psychological distress. descriptive statistics and logistic regression were performed using stata ic 11.0 (statacorp lp, college station, texas). results: 9339 mvc patients were screened, 1584 were eligible, and 949 were enrolled. 361/949 (38%) participants had substantial psychological distress. after adjusting for crash severity (severity of vehicle damage, vehicle speed), substantial patient distress was predicted by sociodemographic factors, pre-mvc depressive symptoms, and arriving to the ed on a backboard (table) . conclusion: substantial psychological distress is common among individuals discharged from the ed after mvcs and is predicted by patient characteristics separate from mvc severity. a better under standing of the frequency and predictors of substantial psychological distress is an important first step in identifying these patients and developing effective interventions to reduce severe distress in the aftermath of trauma. such interventions have the potential to reduce both immediate patient suffering and the development of persistent psychological sequelae. figure) the predictive characteristics of pets, pesi, and spesi for 30-day mortality in emperor, including auc, negative predictive value, sensitivity, and specificity were calculated. results: the 646 of 1438 patients (44.9%; 95% ci 42.3%-47.5%) classified as pets low had 30-day mortality of 0.5% (95% ci 0.1-1.5%), versus 10.2% (95% ci 8.0%-12.4%) in the pets high group, statistically similar to pesi and spesi. pets is significantly more specific for mortality than the spesi (47.0% v 37.6%; p < 0.0001), classifying far more patients as low-risk while maintaining a sensitivity of 96% (95% ci 88.3%-99.0%), not significantly different from spesi or pesi (p > 0.05). conclusion: with four variables, pets in this derivation cohort is as sensitive for 30-day mortality as the more complicated pesi and spesi, with significantly greater specificity than the spesi for mortality, placing 25% more patients in the low-risk group. external validation is necessary. nicole seleno, jody vogel, michael liao, emily hopkins, richard byyny, ernest moore, craig gravitz, jason haukoos denver health medical center, denver, co background: the sequential organ failure assessment (sofa) score, base excess, and lactate have been shown to be associated with mortality in critically ill trauma patients. the denver emergency department (ed) trauma organ failure (tof) score was recently derived and internally validated to predict multiple organ failure in trauma patients. the relationship between the denver tof score and mortality has not been assessed or compared to other conventional measures of mortality in trauma. objectives: to compare the prognostic accuracies of the denver ed tof score, ed sofa score, and ed base excess and lactate for mortality in a large heterogeneous trauma population. methods: a secondary analysis of data from the denver health trauma registry, a prospectively collected database. consecutive adult trauma patients from 2005 through 2008 were included in the study. data collected included demographics, injury characteristics, prehospital care characteristics, response to injury characteristics, ed diagnostic evaluation and interventions, and in-hospital mortality. the values of the four clinically relevant measures (denver ed tof score, ed sofa score, ed base excess, and ed lactate) were determined within four hours of patient arrival, and prognostic accuracies for in-hospital mortality for the four measures were evaluated with receiver operating characteristic (roc) curves. multiple imputation was used for missing values. results: of the 4,355 patients, the median age was 37 (iqr 26-51) years, median injury severity score was 9 (iqr 4-16), and 81% had blunt mechanisms. thirty-eight percent (1,670 patients) were admitted to the icu with a median icu length of stay of 2.5 (iqr 1-8) days, and 3% (138 patients) died. in the non-survivors, the median values for the four measures were ed sofa 5.0 (iqr 0.0-8.0); denver ed tof 4.0 (iqr 4.0-5.0); ed base excess 7.0 (iqr 8.0-19.0) meq/l; and ed lactate 6.5 (iqr 4.5-11.8) mmol/l. the areas under the roc curves for these measures are demonstrated in the figure. conclusion: the denver ed tof score more accurately predicts in-hospital mortality in trauma patients as compared to the ed sofa score, ed base excess, or ed lactate. the denver ed tof score may help identify patients early who are at risk for mortality, allowing for targeted resuscitation and secondary triage to improve outcomes in these critically ill patients. the background: both animal and human studies suggest that early initiation of therapeutic hypothermia (th) and rapid cooling improve outcomes after cardiac arrest. objectives: the objective was to determine if administration of cold iv fluids in a prehospital setting decreased time-to-target-temperature (tt) with secondary analysis of effects on mortality and neurological outcome. methods: patients resuscitated after out-of-hospital cardiac arrest (oohca) who received an in-hospital post cardiac arrest bundle including th were prospectively enrolled into a quality assurance database from november 2007 to november 2011. on april 1, 2009 a protocol for intra-arrest prehospital cooling with 4°c normal saline on patients experiencing oohca was initiated. we retrospectively compared tt for those receiving prehospital cold fluids and those not receiving cold fluids. tt was defined as 34°c measured via foley thermistor. secondary outcomes included mortality, good neurological outcome defined as cerebral performance category (cpc) score of 1 or 2 at discharge, and effects of pre-rosc cooling. results: there were 132 patients who were included in this analysis with 80 patients receiving prehospital cold iv fluids and 52 who did not. initially, 63% of patients were in vf/vt and 36% asystole/pea. patients receiving prehospital cooling did not have a significant improvement in tt (256 minutes vs 271 minutes, p = 0.64). survival to discharge and good neurologic outcome were not associated with prehospital cooling (54% vs 50%, p = 0.67) and cpc of 1 or 2 in 49% vs 44%, (p = 0.61). initiating cold fluids prior to rosc showed both a nonsignificant decrease in survival (48% vs 56%, p = 0.35) and increase in poor neurologic outcomes (42% vs 50%, p = 0.39). 77% of patients received £ 1l of cooled ivf prior to hospital arrival. patients receiving prehospital cold ivf had a longer time from arrest to hospital arrival (44 vs 34 min, p =< 0.001) in addition to a prolonged rosc to hospital time (20 vs 12 min, p = 0.005). conclusion: at our urban hospital, patients achieving rosc following oohca did not demonstrate faster tt or outcome improvement with prehospital cooling compared to cooling initiated immediately upon ed arrival. further research is needed to assess the utility of prehospital cooling. assessment background: an estimated 10% of emergency department (ed) patients 65 years of age and older have delirium, which is associated with short-and long-term risk of morbidity and mortality. early recognition could result in improved outcomes, but the reliability of delirium recognition in the continuum of emergency care is unknown. objectives: we tested whether delirium can be reliably detected during emergency care of elderly patients by measuring the agreement between prehospital providers, ed physicians, and trained research assistants using the confusion assessment method for the icu (cam-icu) to identify the presence of delirium. our hypothesis was that both ed physicians and prehospital providers would have poor ability to detect elements of delirium in an unstructured setting. methods: prehospital providers and ed physicians completed identical questionnaires regarding their clinical encounter with a convenience sample of elderly (age >65 years) patients who presented via ambulance to two urban, teaching eds over a three-month period. respondents noted the presence or absence of (1) an acute change in mental status, (2) inattention, (3) disorganized thinking, and (4) altered level of consciousness (using the richmond agitation sedation scale). these four components comprise the operational definition of delirium. a research assistant trained in the cam-icu rated each component for the same patients using a standard procedure. we calculated inter-rater reliability (kappa) between prehospital providers, ed physicians, and research assistants for each component. objectives: this study aimed to assess the association between age and ems use while controlling for potential confounders. we hypothesized that this association use would persist after controlling for confounders. methods: a cross-sectional survey study was conducted at an academic medical center's ed. an interview-based survey was administered and included questions regarding demographic and clinical characteristics, mode of ed arrival, health care use, and the perceived illness severity. age was modeled as an ordinal variable (<60, 60-79, and ‡ 80 years). bivariate analyses were used to identify potential confounders and effect measure modifiers and a multivariable logistic regression model was constructed. odds ratios were calculated as measures of effect. results: a total of 1092 subjects were enrolled and had usable data for all covariates, 465 (43%) of whom arrived via ems. the median age of the sample was 60 years and 52% were female. there was a statistically significant linear trend in the proportion of subjects who arrived via ems by age (p < 0.0001). compared to adults aged less than 60 years, the unadjusted odds ratio associating age and ems use was 1.41 (95% ci: background: we previously derived a clinical decision rule (cdr) for chest radiography (cxr) in patients with chest pain and possible acute coronary syndrome (acs) consisting of the absence of three predictors: history of congestive heart failure, history of smoking, and abnormalities on lung auscultation. objectives: to prospectively validate and refine a cdr for cxr in an independent patient population. methods: we prospectively enrolled patients over 24 years of age with a primary complaint of chest pain and possible acs from september 2009 to january 2010 at a tertiary care ed with 73,000 annual patient visits. physicians completed standardized data collection forms before ordering chest radiographs and were thus blinded to cxr findings at the time of data collection. two investigators, blinded to the predictor variables, independently classified cxrs as ''normal,'' ''abnormal not requiring intervention,'' and ''abnormal requiring intervention'' (e.g, heart failure, infiltrates) based on review of the radiology report and the medical record. analyses included descriptive statistics, inter-rater reliability assessment (kappa), and recursive partitioning. results: of 1159 visits for possible acs, mean age (sd) was 60.3 (15.6) and 51% were female. twenty-four percent had a history of acute myocardial infarction, 10% congestive heart failure, and 11% atrial fibrillation. seventy-one (6.1%, 95% ci 4.9-7.7) patients had a radiographic abnormality requiring intervention. ing the likelihood of coronary artery disease (cad) could reduce the need for stress testing or coronary imaging. acyl-coa:cholesterol acyltransferase-2 (acat2) activity has been shown in monkey and murine models to correlate with atherosclerosis. objectives: to determine if a novel cardiac biomarker consisting of plasma cholesteryl ester levels (ce) typically derived from the activity of acat2 is predictive of cad in a clinical model. methods: a single center prospective observational cohort design enrolled a convenience sample of subjects from a tertiary care center with symptoms of acute coronary syndrome undergoing coronary ct angiography or invasive angiography. plasma samples were analyzed for ce composition with mass spectrometry. the primary endpoint was any cad determined at angiography. multivariable logistic regression analyses were used to estimate the relationship between the sum of the plasma concentrations from cholesteryl palmitoleate (16:1) and cholesteryl oleate (18:1) (defined as acat2-ce) and the presence of cad. the added value of acat2-ce to the model was analyzed comparing the c-statistics and integrated discrimination improvement (idi). results: the study cohort was comprised of 113 participants enrolled over 24 months with a mean age 49 (±11.7) years, 59% with cad at angiography. the median plasma concentration of acat2-ce was 938 lm (758, 1099) in patients with cad and 824 lm (683, 998) in patients without cad (p = 0.03) (figure) . when considered with age, sex, and the number of conventional cad risk factors, acat2-ce were associated with a 6.5% increased odds of having cad per 10 lm increase in concentration. the addition of acat2-ce significantly improved the c-statistic (0.89 vs 0.95, p = 0.0035) and idi (0.15, p < 0.001) compared to the reduced model. in the subgroup of low-risk observation unit patients, the ce model had superior discrimination compared to the diamond forrester classification (idi 0.403, p < 0.001). conclusion: plasma levels of acat2-ce, considered in a clinical model, have strong potential to predict a patient's likelihood of having cad. in turn, this could reduce the need for cardiac imaging after the exclusion of mi. further study of acat2-ce as biomarkers in patients with suspected acs is needed. background: outpatient studies have demonstrated a correlation between carotid intima-media thickness (cimt) on ultrasound and coronary artery disease (cad). there are no known published studies that investigate the role of cimt in the ed using cardiac ct or percutaneous cardiac intervention (pci) as a gold standard. objectives: we hypothesized that cimt can predict cardiovascular events and serve as a noninvasive tool in the ed. methods: this was a prospective study of adult patients who presented to the ed and required evaluation for chest pain. the study location was an urban ed with a census of 120,000 annual visits and 24-hour cardiac catheterization. patients who did not have ct or pci or had carotid surgery were excluded from the study. ultrasound cimt measurements of right and left common carotid arteries were taken with a 10mhz linear transducer (zonare, mountain view, ca). anterior, medial, and posterior views of the near and far wall were obtained (12 cimt scores total). images were analyzed by carotid analyzer 5 (mailing imaging application llc, coralville, iowa). patients were classified into two groups based on the results from ct or pci. a subject was classified as having significant cad if there was over 70% occlusion or multi-vessel disease. results: ninety of 102 patients were included in the study; 55.7% were males. mean age was 56.6 ± 13 years. there were 34 (37.8%) subjects with significant cad and 56 (62.2%) with non-significant cad. the mean of all 12 cimt measurements was significantly higher in the cad group than in the non-cad group (0.60 ± 0.20 vs. 0.35 ± 0.23; p < 0.00001). a logistic regression analysis was carried out with significant cad as the event of interest and the following explanatory variables in the model: objectives: to determine the diagnostic yield of routine testing in-hospital or following ed discharge among patients presenting to an ed following syncope. methods: a prospective, observational, cohort study of consecutive ed patients ‡18 years old presenting with syncope was conducted. the four most commonly utilized tests (echocardiography, telemetry, ambulatory electrocardiography monitoring, and cardiac markers) were studied. interobserver agreement as to whether tests results determined the etiology of the syncope was measured using kappa (k) values. results: of 570 patients with syncope, 150 (26%) had echocardiography with 33 (6%) demonstrating a likely etiology of the syncopal event such as critical valvular disease or significantly depressed left ventricular function (k = 0.78). on hospitalization, 349 (61%) patients were placed on telemetry, 19 (3%) of these had worrisome dysrhythmias (k = 0.66). 317 (55%) patients had troponin levels drawn of whom 19 (3%) had positive results (k = 1); 56 (10%) patients were discharged with monitoring with significant findings in only 2 (0.4%) patients (k = 0.65). overall, 73 (8%, 95% ci 7-10%) studies were diagnostic. conclusion: although routine testing is prevalent in ed patients with syncope, the diagnostic yield is relatively low. nevertheless, some testing, particularly echocardiography, may yield critical findings in some cases. current efforts to reduce the cost of medical care by eliminating non-diagnostic medical testing and increasing emphasis on practicing evidence-based medicine argue for more discriminate testing when evaluating syncope. (originally submitted as a ''late-breaker.'') unusual fatigue was reported by 70.7% (severe 29.7%) and insomnia by 47.8% (severe 21.0%). these findings have led to risk management recommendations to consider these symptoms as predictive of acute coronary syndromes (acs) among women visiting the ed. objectives: to document the prevalence of these symptoms among all women visiting an ed. to analyze the potential effect of using these symptoms in the ed diagnostic process for acs. methods: a survey on fatigue and insomnia symptoms was administered to a convenience sample of all adult women visiting an urban academic ed (all arrival modes, acuity levels, all complaints). a sensitivity analysis was performed using published data and expert opinion for inputs. results: we approached 548 women, with 379 enrollments. see table. the top box shows prevalences of prodromal symptoms among all adult female ed patients. the bottom box shows outputs from sensitivity analysis on the diagnostic effect of initiating an acs workup for all female ed patients reporting prodromal symptoms. conclusion: prodromal symptoms of acs are highly prevalent among all adult women visiting the ed in this study. this likely limits their utility in ed settings. while screening or admitting women with prodromal symptoms in the ed would probably increase sensitivity, that increase would be accompanied by a dramatic reduction in specificity. such a reduction in specificity would translate to admitting, observing, or working up somewhere between 29% and 61% of all women visiting the ed, which is prohibitive in terms of personal costs, risks of hospitalization, and financial costs. while these symptoms may or may not have utility in other settings such as primary care, their prevalence, and the implied lack of specificity for acs suggest they will not be clinically useful in the ed. length methods: we examined a cohort of low-risk chest pain patients evaluated in an ed-based ou using prospective and retrospective ou registry data elements. cox proportional hazard modeling was performed to assess the effect of testing modality (stress testing vs. ccta) on the los in the cdu. as ccta is not available on weekends, only subjects presenting on weekdays were included. cox models were stratified on time of patient presentation to the ed, based on four hour blocks beginning at midnight. the primary independent variable was first test modality, either stress imaging (exercise echo, dobutamine echo, stress mri) or ccta. age, sex, and race were included as covariates. the proportional hazards assumption was tested using scaled schoenfield residuals, and the models were graphically examined for outliers and overly influential covariate patterns. test selection was a time varying covariate in the 8am strata, and therefore the interaction with ln (los) was included as a correction term. after correction for multiple comparisons, an alpha of 0.01 was held to be significant. results: over the study period, 841 subjects (of 1,070 in the registry) presented on non-weekend days. the median los was 18.5 hours (iqr 12.4-23.3 hours), 57% were white, and 61% were female. the table shows the number of subjects in each time strata, the number tested, and the number undergoing stress testing vs. ccta. after adjusting all models for age, race, and sex, the hazard ratio (hr) for los is as shown. only those patients presenting between 8am and noon noted a significant improvement in los with ccta use (p < 0.0001). objectives: determine the validity of a managementfocused em osce as a measure of clinical skills by determining the correlation between osce scores and faculty assessment of student performance in the ed. methods: medical students in a fourth year em clerkship were enrolled in the study. on the final day of the clerkship students participated in a five-station em osce. student performance on the osce was evaluated using a task-based evaluation system with 3-4 critical management tasks per case. task performance was evaluated using a three-point system: performed correctly/timely (2), performed incorrectly/late (1), or not performed (0). descriptive anchors were used for performance criteria. communication skills were also graded on a three-point scale. student performance in the ed was based on traditional faculty assessment using our core-competency evaluation instrument. a pearson correlation coefficient was calculated for the relationship between osce score and ed performance score. case item analysis included determination of difficulty and discrimination. the acgme also requires that trainees are evaluated on these 6ccs during their residency. trainee evaluation in the 6ccs are frequently on a subjective rating scale. one of the recognized problems with a subjective scale is the rating stringency of the rater, commonly known as the hawk-dove effect. this has been seen in standardized clinical exam scoring. recent data have shown that score variance can be related to evaluator performance with a negative correlation. higher-scoring physicians were more likely to be a stringent or hawk type rater on the same evaluation. it is unclear if this pattern also occurs in the subjective ratings that are commonly used in assessments of the 6ccs. objectives: comparison of attending physician scores on the acgme 6ccs with attending ratings of residents for a negative correlation or hawk-dove effect. methods: residents are routinely evaluated on the 6ccs with a 1-9 numerical rating scale as part of their training. the evaluation database was retrospectively reviewed. residents anonymously scored attending physicians on the 6ccs with a cross-sectional survey that utilized the same rating scale, anchors, and prompts as the resident evaluations. average scores for and by each attending were calculated and a pearson correlation calculated by core competency and overall. results: in this irb-approved study, a total of 43 attending physicians were scored on the 6ccs with 447 evaluations by residents. attendings evaluated 162 residents with a total of 1,678 evaluations completed over a 5-year period. attending mode score was 9 ranging from 2 to 9; resident scores had a mode of 8 with a range of 1 to 9. there was no correlation between the rated performance of the attendings overall or in each 6ccs and the scores they gave (p = 0.065-0.861). conclusion: hawk-dove effects can be seen in some scoring systems and has the potential to affect trainee evaluation on the acgme core competencies. however, a negative correlation to support a hawk-dove scoring pattern was not found in em resident evaluations by attending physicians. this study is limited by being a single center study and utilizing grouped data to preserve resident anonymity. background: all acgme-accredited residency programs are required to provide competency-based education and evaluation. graduating residents must demonstrate competency in six key areas. multiple studies have outlined strategies for evaluating competency, but data regarding residents' self-assessments of these competencies as they progress through training and beyond is scarce. objectives: using data from longitudinal surveys by the american board of emergency medicine, the primary objective of this study was to evaluate if resident self-assessments of performance in required competencies improve over the course of graduate medical training and in the years following. additionally, resident self-assessment of competency in academic medicine was also analyzed. methods: this is a secondary data analysis of data gathered from two rounds of the abem longitudinal study of emergency medicine residents (1996-98 and 2001-03) and three rounds of the abem longitudinal study of emergency physicians (1999, 2004, 2009 ). in both surveys, physicians were asked to rate a list of 18 items in response to the question, ''what is your current level of competence in each of the following aspects of work in em?'' the rated items were grouped according to the acgme required competencies of patient care, medical knowledge, practice-based learning and improvement, interpersonal and communication skills, and system-based practice. an additional category for academic medicine was also added. results: rankings improved in all categories during residency training. rankings in three of the six categories improved from the weak end of the scale to the strong end of the scale. there is a consistent decline in rankings one year after graduation from residency. the greatest drop is in medical knowledge. mean self-ranking in academic medicine competency is uniformly the lowest ranked category for each year. conclusion: while self-assessment is of uncertain value as an objective assessment, these increasing rankings suggest that emergency medicine residency programs are successful at improving residents' confidence in the required areas. residents do not feel as confident about academic medicine as they do about the acgme required competencies. the uniform decline in rankings the first year after residency is an area worthy of further inquiry. screening medical student rotators from outside institutions improves overall rotation performance shaneen doctor, troy madsen, susan stroud, megan l. fix university of utah, salt lake city, ut background: emergency medicine is a rapidly growing field. many student rotations are limited in their ability to accommodate all students and must limit the number of students they allow per rotation. we hypothesize that pre-screening visiting student rotators will improve overall student performance. objectives: to assess the effect of applicant screening on overall rotation grade and mean end of shift card scores. methods: we initiated a medical student screening process for all visiting students applying to our 4-week elective em rotation starting in 2008. this consisted of reviewing board scores and requiring a letter of intent. students from our home institution were not screened. all end-of-shift evaluation cards and final rotation grades (honors, high pass, pass, fail) from 2004 to 2011 were analyzed. we identified two cohorts: home students (control) and visiting students. we compared pre-intervention (2004) (2005) (2006) (2007) (2008) and postintervention (2008-2011) scores and grades. end of shift performance scores are recorded using a fivepoint scale that assesses indicators such as fund of knowledge, judgment, and follow-through to disposition. mean ranks were compared and p-values were calculated using the armitage test of trend and confirmed using t-tests. results: we identified 162 visiting students (91 pre, 81 post) and 160 home students (90 pre, 80 post). 12 (13.2%) visiting students achieved honors pre-intervention while 31 (38.3%) achieved honors post-intervention (p = 0.000093). no significant difference was seen in home student grades: 28 (31.1%) received honors pre-2008 and 17 (21.3%) received honors post-2008 conclusion: we found that implementation of a screening process for visiting medical students improved overall rotation scores and grades as compared to home students who did not receive screening. screening rotating students may improve the overall quality of applicants and thereby the residency program. background: there are many descriptions in the literature of computer-assisted instruction in medical education, but few studies that compare them to traditional teaching methods. objectives: we sought to compare the suturing skills and confidence of students receiving video preparation before a suturing workshop versus a traditional instructional lecture. methods: 88 first and second year medical students were randomized into two groups. the control group was given a lecture followed by 40 minutes of suturing time. the video group was provided with an online suturing video at home, no lecture, and given 40 minutes of suturing time during the workshop. both groups were asked to rate their confidence before and after the workshop, and their belief in the workshop's effectiveness. each student was also videotaped suturing a pig's foot after the workshop and graded on a previously validated 16-point suturing checklist. 83 videos were scored. results: there was no significant difference between the test scores of the lecture group (m = 11.21, sd = 3.17, n = 42) and the video group (m = 11.27, sd = 2.53, n = 41) using the two-sample independent ttest for equal variances (t(81) = )0.09, p = 0.93). there was a statistically significant difference in the proportion of students scoring correctly for only one point: ''curvature of needle followed'': 25/42 in the lecture group and 35/41 in the video group (chi = 6.92, df = 1, p = 0.008). students in the video group were found to be 2.45 times more likely to have a neutral or favorable feeling of suturing confidence before the workshop (p = 0.067, ci 0.94-6.4) using a proportional odds model. no association was detected between group assignment and level of suturing confidence after the workshop (p = 0.475). there was also no association detected between group assignment and opinion of the suturing workshop (p = 0.681) using a logistic regression odds model. among those students who indicated a lack of confidence before training, there was no detected association (p = 0.967) between group assignment and having an improved confidence using a logistic regression odds model. conclusion: students in the video group and students in the control group achieved similar levels of suturing skill and confidence, and equal belief in the workshop's effectiveness. this study suggests that video instruction could be a reasonable substitute for lectures in procedural education. background: accurate interpretation of the ecg in the emergency department is not only clinically important but also critical to assess medical knowledge competency. with limitations to expansion of formal didactics, educational technology offers an innovative approach to improve the quality of medical education. objectives: the aim of this study was to assess an online multimedia-based ecg training module evaluating st elevation myocardial infarction (stemi) identification among medical students. methods: a convenience sample of fifty-two medical students on their em rotations at an academic medical center with an em residency program was evaluated in a before-after fashion during a 6-month period. one cardiologist and two ed attending physicians independently validated a standardized exam of ten ecgs: four were normal ecgs, three were classic stemis, and three were subtle stemis. the gold standard for diagnosis was confirmed acute coronary thrombus during cardiac catheterization. after evaluating the 10 ecgs, students completed a pre-intervention test wherein they were asked to identify patients who required emergent cardiac catheterization based on the presence or absence of st segment elevation on ecg. students then completed an online interactive multimedia module containing 13 minutes of stemi training based on american heart association/american college of cardiology guidelines on stemi. medical students were asked to complete a post-test of the 10 ecgs after watching online multimedia. objectives: our objective was to quantify the number of pre-verbal pediatric head cts performed at our community hospital that could have been avoided by utilizing the pecarn criteria. methods: we conducted a standardized chart review of all children under the age of 2 who presented to our community hospital and received a head ct between jan 1st, 2010 and dec 31st, 2010. following recommended guidelines for conducting a chart review, we: 1) utilized four blinded chart reviewers, 2) provided specific training, 3) created a standardized data extraction tool, and 4) held periodic meetings to evaluate coding discrepancies. our primary outcome measure was the number of patients who were pecarn negative and received a head ct at our institution. our secondary outcome was to reevaluate the sensitivity and specificity of the pecarn criteria to detect citbi in our cohort. data were analyzed using descriptive statistics and 95% confidence intervals were calculated around proportions using the modified wald method. results: a total of 138 patients under the age of 2 received a head ct at our institution during the study period. 23 patients were excluded from the final analysis because their head cts were not for trauma. the prevalence of a citbi in our cohort was 2.6% (95% ci 0.6%-7.7%) ( (dti) measures disruption of axonal integrity on the basis of anisotropic diffusion properties. findings on dti may relate to the injury, as well as the severity of postconcussion syndrome (pcs) following mtbi. objectives: to examine acute anisotropic diffusion properties based on dti in youth with mtbi relative to orthopedic controls and to examine associations between white matter (wm) integrity and pcs symptoms. methods: interim analysis of a prospective casecontrol cohort involving 12 youth ages 11-16 years with mtbi and 10 orthopedic controls requiring extremity radiographs. data collected in ed included demographics, clinical information, and pcs symptoms measured by the postconcussion symptom scale. within 72 hours of injury, symptoms were re-assessed and a 61-direction, diffusion weighted, spin-echo imaging scan was performed on a 3t philips scanner. dti images were analyzed using tract-based spatial statistics. fractional anisotropy (fa), mean diffusivity (md), axial diffusivity (ad), and radial diffusivity were measured. results: there were no group demographic differences between mtbi cases and controls. presenting symptoms within the mtbi group included gcs = 15 83%, loss of consciousness 33%, amnesia 33%, post-traumatic seizure 8%, headache 83%, vomiting 33%, dizziness 42%, and confusion 42%. pcs symptoms were greater in mtbi cases than in the controls at ed visit (30.1 ± 17.0 vs. 15.5 ± 16.8, p < 0.06) and at the time of scan (19.1 ± 12.9 vs. 5.7 ± 6.5, p < 0.01). the mtbi group displayed decreased fa in cerebellum and increased md and ad in the cerebral wm relative to controls (uncorrected p < 0.05). increased fa in cerebral wm was also observed in mtbi patients but the group difference was not significant. pcs symptoms at the time of the scan were positively correlated with fa and inversely correlated with rd in extensive cerebral wm areas (p < 0.05, uncorrected). in addition, pcs symptoms in mtbi patients were also found to be inversely correlated with md, ad, and rd in cerebellum (p < 0.05). conclusion: dti detected axonal damage in youth with mtbi which correlated with pcs symptoms. dti performed acutely after injury may augment detection of injury and help prediction of those with worse outcomes. background: sports-related concussion among professional, collegiate, and more recently high school athletes has received much attention from the media and medical community. to our knowledge, there is a paucity of research in regard to sports-related concussion in younger athletes. objectives: the aim of this study was to evaluate parental knowledge of concussion in young children who participate in recreational tackle football. methods: parents/legal guardians of children aged 5-15 years enrolled in recreational tackle football were asked to complete an anonymous questionnaire based on the cdc's heads up: concussion in youth sports quiz. parents were asked about their level of agreement in regard to statements that represent definition, symptoms, and treatment of concussion. results: a total of 310 out of 369 parents voluntarily completed the questionnaire (84% response rate). parent and child demographics are listed in table 1 . ninety four percent of parents believed their child had never suffered a concussion. however, when asked to agree or disagree with statements addressing various aspects of concussion, only 13% (n = 41) could correctly identify all seven statements. most did not identify that a concussion is considered a mild traumatic brain injury and can be achieved from something other than a direct blow to the head. race, sex, and zip code had no significant association with correctly answering statements. education (0.24; p < 0.01) and number of years the child played (0.11; p < 0.05) had a small effect. fifty-three percent of parents reported someone had discussed the definition of concussion with them and 58% the symptoms of concussion. see table 2 for source of information to parents. no parent was able to classify all symptoms listed as correctly related or not related to concussion. however, identification of correct concussion definitions correlated with identification of correct symptoms (0.25; p < 0.05). conclusion: while most parents had received some education regarding concussion from a health care provider, important misconceptions remain among parents of young athletes regarding the definition, symptoms, and treatment of concussion. this study highlights the need for health care providers to increase educational efforts among parents of young athletes in regard to concussion. figure 1 ). 2/2 (100%) of patients with baseline liver dysfunction were 25(oh)d deficient and 5/6 (83%) of deaths were patients who had insufficient levels of 25(oh)d. there was an inverse association between 25(oh)d level and tnf-a (p = 0.03; figure 2 ) and il-6 (p = 0.04). background: fever is common in the emergency department (ed), and 90% of those diagnosed with severe sepsis present with fever. despite data suggesting that fever plays an important role in immunity, human data conflict on the effect of antipyretics on clinical outcomes in critically ill adults. objectives: to determine the effect of ed antipyretic administration on 28-day in-hospital mortality in patients with severe sepsis. methods: single-center, retrospective observational cohort study of 171 febrile severe sepsis patients presenting to an urban academic 90,000-visit ed between june 2005 and june 2010. all ed patients meeting the following criteria were included: age ‡ 18, temperature ‡ 38.3°c, suspected infection, and either systolic blood pressure £ 90 mmhg after a 30 ml/kg fluid bolus or lactate of ‡ 4. patients were excluded for a history of cirrhosis or acetaminophen allergy. antipyretics were defined as acetaminophen, ibuprofen, or ketorolac. results: one hundred-thirty five (78.9%) patients were treated with an antipyretic medication (89.4% acetaminophen). intubated patients were less likely to receive antipyretic therapy (51.9% vs. 84.0%, p < 0.01), but the groups were otherwise well matched. patients requiring ed intubation (n = 27) had much higher in-hospital mortality (51.9% vs. 7.6%, p < 0.01). patients given an antipyretic in the ed had lower mortality (11.9% vs. 25.0%, p < 0.05). when multivariable logistic regression was used to account for apache-ii, intubation status, and fever magnitude, antipyretic therapy was not associated with mortality (adjusted or 0.97, 0.31-3.06, p = 0.96). conclusion: although patients treated with antipyretic therapy had lower 28-day in-hospital mortality, antipyretic therapy was not independently associated with mortality in multivariable regression analysis. these findings are hypothesis-generating for future clinical trials, as the role of fever control has been largely unexplored in severe sepsis (grant ul1 rr024992, nih-ncrr). , and caval index )0.09 ± 0.14 (ci )0.14, )0.05) and all were statistically significant. the groups receiving 10 ml/kg and 30 ml/kg had statistically significant changes in caval index; however the 30 ml/kg group had no significant change in mean ivc diameter. one-way anova differences between the means of all groups were not statistically different. conclusion: overall, there were statistically significant differences in mean ivc-us measurements before and after fluid loading, but not between groups. fasting asymptomatic subjects had a wide inter-subject variation in both baseline ivc-us measurements and fluid-related changes. the wide differences within our 30 ml/kg group may limit conclusions regarding proportionality. there were significant differences in performance on ed measures by ownership (p < 0.0001) and region (p = 0.0002). scores on ed process measures were highest at for-profit hospitals (27% above average) and hospitals in the south (5% above average), and lowest at public hospitals (16% below average) and hospitals in the northeast (8% below average). conclusion: there was considerable variation in performance on the ed measures included in the vbp program by hospital ownership and region. ed directors may come under increasing pressure to improve scores in order to reduce potential financial losses under the program. our data provide early information on the types of hospitals with the greatest opportunity for improvement. methods: design/setting -an independent agency mandated by the government collected and analyzed ed patient experience data using a comprehensive, validated multidimensional instrument and a random periodic sampling methodology of all ed patients. a prospective pre-post experimental study design was employed in the eight community and tertiary care hospitals most affected by crowding. two 5.5 month study periods were evaluated (pre: 28/06-12/12/2010; post: 13/12/2010-29/ 05/2011). outcomes -the primary outcome was patient perception of wait times and crowding reported as a composite mean score (0-100) from six survey items with higher scores representing better ratings. the overall rating of care by ed patients (composite score) and other dimensions of care were collected as secondary outcomes. all outcomes were compared using chi-square and two-tailed student's t-tests. results: a total of 3774 surveys were completed in both the pre-ocp and post-ocp study periods representing a response rate of 45%. we compared in-patient mortality from ami for patients who lived in a community with either 2.5 miles or 5 miles of a closure but did not need to travel farther to the nearest ed with those who did not. we used patient-level data from the california office of statewide health and planning development (oshpd) database patient discharge data, and locations of patient residence and hospitals were geo-coded to determine any changes in distance to the nearest ed. we applied a generalized linear mixed effects model framework to estimate a patient's likelihood to die in the hospital of ami as a function of being affected by a neighborhood closure event. results background: fragmentation of care has been recognized as a problem in the us health care system. however, little is known about ed utilization after hospitalization, a potential marker of poor outpatient care coordination after discharge, particularly for common inpatient-based procedures. objectives: to determine the frequency and variability in ed visits after common inpatient procedures, how often they result in readmission, and related payments. methods: using national medicare data for 2005-2007, we examined ed visits within 30 days of hospital discharge after six common inpatient procedures: percutaneous coronary intervention, coronary artery bypass grafting (cabg), elective abdominal aortic aneurysm repair, back surgery, hip fracture repair, and colectomy. we categorized hospitals into risk-adjusted quintiles based on the frequency of ed visits after the index hospitalization. we report visits by primary diagnosis icd-9 codes and rates of readmission. we also assessed payments related to these ed visits. results: overall, the highest quintile of hospitals had 30-day ed visit rates that ranged from a low of 17.8% with an associated 7.3% readmission rate (back surgery) to a high of 27.8% with an associated 13.6% readmission rate (cabg). the most variability was more than 3-fold and found among patients undergoing colectomy in which the worst-performing hospitals saw 24.1% of their patients experienced an ed visit within 30 days while the best-performing hospitals saw 7.4%. average total payments for the 30-day window from initial discharge across all surgical cohorts varied from $18,912 for patients discharged without subsequent ed visit; $20,061for those experiencing an ed visit(s); $38,762 for those readmitted through the ed; and $33,632 for those readmitted from another source. if all patients who did not require readmission also did not incur an ed visit within the 30-day window, this would represent a potential cost savings of $125 million. conclusion: among elderly medicare recipients there was significant variability between hospitals for 30-day ed visits after six common inpatient procedures. the ed visit may be a marker of poor care coordination in the immediate discharge period. this presents an opportunity to improve post-procedure outpatient care coordination which may save costs related to preventable ed visits and subsequent readmissions. objectives: we sought to assess the effect of pharmacist medication review on ed patient care, in particular time from physician order to medication administration for the patient (order-to-med time). methods: we conducted a multi-center, before-after study in two eds (urban academic teaching hospital and suburban community hospital, combined census of 61,000) after implementation of the electronic prospective pharmacy review system (prs). the system allowed a pharmacist to review all ed medication orders electronically at the time of physician order and either approve or alter the order. we studied a 5-month time period before implementation of the system (pre-prs, 7/1/10-11/30/11) and after implementation (post-prs, 7/ 1/11-11/30/11). we collected data on all ed medication orders including dose, route, class, pharmacist review action, time of physician order, and time of medication administration. differences in order-to-medication between the pre-and post-prs study periods were compared using a results: ed metrics that were significantly associated with lbtcs varied across ed patient-volume categories (table) . for eds seeing less than 20k patients annually, the percentage of ems arrivals admitted to the hospital and ed square footage were both weakly associated with lbtcs (p = 0.09). for eds seeing at least 20k-39k patients, median ed length of stay (los), percent of patients admitted to hospital through the ed, percent of ems arrivals admitted to hospital, and percent of pediatric patients were all positively associated, while percent of patients admitted to the hospital was negatively associated with lbtcs. for eds seeing 40k-59k, median los and percent of x-rays performed were positively associated, while percent of ekgs performed was negatively associated with lbtcs. for eds seeing 60k-79k, percent of patients admitted to the hospital through the ed was negatively associated and percent of ekgs performed was positively associated with lbtcs. for eds with volume greater than 80k, none of the selected variables were associated with lbtc. conclusion: ed factors that help explain high lbtc rates differ depending on the size of an ed. interventions attempting to improve lbtc rates by modifying ed structure or process will need to consider baseline ed volume as a potential moderating influence. objectives: our study sought to compare bacterial growth of samples taken from surfaces after use of a common approved quat compound and a virtually non-toxic, commercially available solution containing elemental silver (0.02%), hydrogen peroxide (15%), and peroxyacetic acid (20%) (shp) in a working ed. we hypothesized that, based on controlled laboratory data available, shp compound would be more effective on surfaces in an active urban ed. methods: we cleaned and then sampled three types of surfaces in the ed (suture cart, wooden railing, and the floor) during midday hours one minute after application of tap water, quat, and shp and then again at 24 hours without additional cleaning. conventional environmental surface surveillance rodac media plates were used for growth assessment. images of bacterial growth were quantified at 24 and 48 hours. standard cleaning procedures by hospital staff were maintained per usual. results: shp was superior to control and quat one minute after application on all three surfaces. quat and water had 10x and 40x more bacterial growth than the surface cleaned with shp, respectively. 24 hours later, the shp area produced fewer colonies sampled from the wooden railing: 4x more bacteria for quat, and 5x for water when compared to shp. 24h cultures from the cart and floor had confluent growth and could not be quantified. conclusion: shp outperforms quat in sterilizing surfaces after one minute application. shp may be a superior agent as a non-toxic, non-corrosive, and effective agent for surfaces in the demanding ed setting. further studies should examine sporidical and virucidal properties in a similar environment. objectives: evaluate the effect on patient satisfaction of increasing waiting room times and physician evaluation times. methods: emergency department flow metrics were collected on a daily basis as well as average daily patient satisfaction scores. the data were from july 2010 through february 2011, in a 44,000 census urban hospital. the data were divided into equal intervals. the arrival to room time was divided by 15 minute intervals up to 135 minutes with the last group being greater than 136 minutes. the physician evaluation times were divided into 20 minute intervals, up to 110, the last group greater than 111 with 46 days in the group. data were analyzed using means and standard deviations, and well as anova for comparison between groups. results: the overall satisfaction score for the outpatient emergency visit was higher when the patient was in a room within 15 minutes of arrival (88.4, std deviation 5.9), analysis of variation between the groups had a p = 0.13, for the means of each interval (see table 1 ). the total satisfaction with the visit as well as satisfaction with the provider dropped when the evaluation extended over 110 minutes, but was not statistically significant on anova analysis (see table 2 for means). conclusion: once a patient's time in the waiting room extends beyond 15 minutes, you have lost a significant opportunity for patient satisfaction; once they have been in the waiting room for over 120 minutes, you are also much more likely to receive a poor score. physician evaluation time scores are much more consistent but as longer evaluation times occurred beyond total of 110 minutes we started to see a trend downward in the satisfaction score. results: in all three eds, pain medication rates (both in ed and rx) varied significantly by clinical factors including location of pain, discharge diagnosis, pain level, and acuity. we observed little to no variation in pain medication rates by patient factors such as age, sex, race, insurance, or prior ed visits. the table displays key pain management practices by site and provider. after adjusting for patient and clinical characteristics, significant differences in pain medication rates remained by provider and site (see figure) . conclusion: within this health system, the approach to pain management by both providers and sites is not standardized. investigation of the potential effect of this variability on patient outcomes is warranted. results: all measures showed significant differences, p < 0.01. average pts/h decreased post-cpoe and did not recover post transitional period, 1.92 ± 0.13 vs 1.75 ± 0.11, p < 0.05. rvu/h also decreased post-cpoe and did not recover post transitional period, 5.23 ± 0.37 vs 4.79 ± 0.32 and 4.82 ± 0.33, p < 0.05. charges/h also decreased after cpoe implementation and did not recover after system optimization. there was a sustained significant decrease in charges/h of 4.5% ± 6.5% post cpoe and 3.6% ± 6.4% post optimization, p < 0.05. sub-group analysis for each provider group was also evaluated and showed variability for different providers. conclusion: there was a significant decrease in all productivity metrics four months after the implementation of cpoe. the system did undergo optimization initiated by providers with customization for ease and speed of use. however, productivity measurements did not recover after these changes were implemented. these data show that with the implementation of a cpoe system there is a decrease in productivity that continues even after a transition period and system customization. background: procedural competency is a key component of emergency medicine residency training. residents are required to log procedures to document quantity of procedures and identify potential weaknesses in their training. as emergency medicine evolves, it is likely that the type and number of procedures change over time. also, exposure to certain rare procedures in residency is not guaranteed. objectives: we seek to delineate trends in type and volume of core em procedures over a decade of emergency medicine residents graduating from an accredited four-year training program. methods: deidentified procedure logs from 2003-2011 were analyzed to assess trends in type and quantity of procedures. procedure logs were self-reported by individual residents on a continuous basis during training onto a computer program. average numbers of procedures per resident in each graduating class were noted. statistical analysis was performed using spss and includes a simple linear regression to evaluate for significant changes in number of procedures over time and an independent samples two-tailed t-test of procedures performed before and after the required resident duty hours change. results: a total of 112 procedure logs were analyzed and the frequency of 29 different procedures was evaluated. a significant increase was seen in one procedure, the venous cutdown. significant decreases were seen in 12 procedures including key procedures such as central venous catheters, tube thoracostomy, and procedural sedation. the frequency of five high-stakes/ resuscitative procedures, including thoracotomy and cricothyroidotomy, remained steady but very low (<4 per resident over 4 years). of the remaining 11 procedures, 8 showed a trend toward decreased frequency, while only 5 increased. conclusion: over the past 9 years, em residents in our program have recorded significantly fewer opportunities to perform most procedures. certain procedures in our emergency medicine training program have remained stable but uncommon over the course of nearly a decade. to ensure competency in uncommon procedures, innovative ways to expose residents to these potentially life saving skills must be considered. these may include practice on high-fidelity simulators, increased exposure to procedures on patients during residency (possibly on off-service rotations), or practice in cadaver and animal labs. objectives: to study the effectiveness of a unique educational intervention using didactic and hands-on training in usgpiv. we hypothesized that senior medical students would improve performance and confidence with usgpiv after the simulation training. methods: fourth year medical students were enrolled in an experimental, prospective, before and after study conducted at a university medical school simulation center. baseline skills in participant's usgpiv on simulation vascular phantoms were graded by ultrasound expert faculty using standardized checklists. the primary outcome was time to cannulation, and secondary outcomes were ability to successfully cannulate, number of needle attempts, and needle-tip visualization. subjects then observed a 15-minute presentation on correct performance of usgpiv followed by a 30-minute hands-on practical session using the vascular simulators with a 1:4 to 1:6 ultrasound instructor to student ratio. an expert blinded to the participant's initial performance graded post-educational intervention usgpiv ability. pre-and post-intervention surveys were obtained to evaluate usgpiv confidence, previous experience with ultrasound, peripheral iv access, usg-piv, and satisfaction with the educational format. objectives: this study examines the grade distribution of resident evaluations when the identity of the evaluator was anonymous as compared to when the identity of the evaluator was known to the resident. we hypothesize that there will be no change in the grades assigned to residents. methods: we retrospectively reviewed all faculty evaluations of residents and grades assigned from july 1, 2008 through november 15, 2011. prior to july 1, 2010 the identity of the faculty evaluators was anonymous, while after this date, the identity of the faculty evaluators was made known to the residents. throughout this time period, residents were graded on a five-point scale. each resident evaluation included grades in the six acgme core competencies as well as in select other abilities. specific abilities evaluated varied over the dates analyzed. evaluations of residents were assigned to two groups, based on whether the evaluator was anonymous or made known to the resident. grades were compared between the two groups. results: a total of 10,760 grades were assigned in the anonymous group, with an average grade of 3.90 (95ci 3.88, 3.91). a total of 7,122 grades were assigned in the known group with an average grade of 3.77 (95ci 3.75, 3.79). specific attention was paid to assignment of unsatisfactory grades (1 or 2 on the five-point scale). the anonymous group assigned 355 grades in this category, comprising 3.3% of all grades assigned. the known group assigned 100 grades in this category, comprising 1.4% of all grades assigned. unsatisfactory grades were assigned by the anonymous group 1.9% (95ci 1.5, 2.3) more often. additionally, 5.8% (95ci 3.8, 6.8) fewer exceptional grades (4 or 5 on the five-point scale) were assigned by the anonymous group. conclusion: the average grade assigned was closer to average (3 on a five-point scale) when the identity of the evaluator was made known to the residents. additionally, fewer unsatisfactory and exceptional grades were assigned in this group. this decrease of both unsatisfactory and exceptional grades may make it more difficult for program directors to effectively identify struggling and strong residents respectively. testing to improve knowledge retention from traditional didactic presentations: a pilot study david saloum, amish aghera, brian gillett maimonides medical center, brooklyn, ny background: the acgme requires an average of at least 5 hours of planned educational experiences each week for em residents, which traditionally consists of formal lecture based instruction. however, retention by adult learners is limited when presented material in a lecture format. more effective methods such as small group sessions, simulation, and other active learning modalities are time-and resource-intensive and therefore not practical as a primary method of instruction. thus, the traditional lecture format remains heavily relied upon. efficient strategies to improve the effectiveness of lectures are needed. testing utilized as a learning tool to force immediate recall of lecture material is an example of such a strategy. objectives: to evaluate the effect of immediate postlecture short answer quizzes on em residents' retention of lecture content. methods: in this prospective randomized controlled study, em residents from a community based 3-year training program were randomized into two groups. block randomization provided a similar distribution of postgraduate year training levels and performance on both us-mle and in-training examinations between the two groups. each group received two identical 50-minute lectures on ecg interpretation and aortic disease. one group of residents completed a five-question short answer quiz immediately following each lecture (n = 13), while the other group received the lectures without subsequent quizzes (n = 16). the quizzes were not scored or reviewed with the residents. two weeks later, retention was assessed by testing both groups with a 20-question multiple choice test (mct) derived in equal part from each lecture. mean and median test results were then compared between groups. statistical significance was determined using a paired t-test of median test scores from each group. results: residents who received immediate post-lecture quizzes demonstrated significantly higher mct scores (mean = 57%, median 58%, n = 10) compared to those receiving lectures alone (mean = 48%, median = 50%, n = 15); p = 0.023. conclusion: short answer testing immediately after a traditional didactic lecture improves knowledge retention at a 2-week interval. limitations of the study are that it is a single center study and long term retention was not assessed. background: the task of educating the next generation of physicians is steadily becoming more difficult with the inherent obstacles that exist for faculty educators and the work hour restrictions that students must adhere to. the obstacles make developing curricula that not only cover important topics but also do so in a fashion that helps support and reinforce the clinical experiences very difficult. several areas of medical education are using more asynchronous techniques and self-directed online educational modules to overcome these obstacles. objectives: the aim of this study was to demonstrate that educational information pertaining to core pediatric emergency medicine topics could be as effectively disseminated to medical students via self-directed online educational modules as it could through traditional didactic lectures. methods: this was a prospective study conducted from august 1, 2010 through december 31, 2010. students participating in the emergency medicine rotation at carolinas medical center were enrolled and received education in a total of eight core concepts. the students were divided into two groups which changed on a monthly basis. group 1 was taught four concepts via self-directed online modules and four traditional didactic lectures. group 2 was taught the same core concepts, but in opposite fashion to group 1. each student was given a pre-test, post-test, and survey at the conclusion of the rotation. results: a total of 28 students participated in the study. students, regardless of which group assigned, performed similarly on the pre-test, with no statistical difference among scores. when looking at the summative total scores between online and traditional didactic lectures, there was a trend towards significance for more improvement among those taught online. the student's assessment of the online modules showed that the majority either felt neutral or preferred the online method. the majority thought the depth and length of the modules were perfect. most students thought having access to the online modules was valuable and all but one stated that they would use them again. conclusion: this study demonstrates that self-directed, online educational modules are able to convey important concepts in emergency medicine similar to traditional didactics. it is an effective learning technique that offers several advantages to both the educator and student. background: critical access hospitals (cah) provide crucial emergency care to rural populations that would otherwise be without ready access to health care. data show that many cah do not meet standard adult quality metrics. adults treated at cah often have inferior outcomes to comparable patients cared for at other community-based emergency departments (eds). similar data do not exist for pediatric patients. objectives: as part of a pilot project to improve pediatric emergency care at cah, we sought to determine whether these institutions stock the equipment and medications necessary to treat any ill or injured child who presents to the ed. methods: five north carolina cah volunteered to participate in an intensive educational program targeting pediatric emergency care. at the initial site visit to each hospital, an investigator, in conjunction with the ed nurse manager, completed a 109-item checklist of commonly required ed equipment and medications based on the 2009 acep ''guidelines for care of children in the emergency department''. the list was categorized into monitoring and respiratory equipment, vascular access supplies, fracture and trauma management devices, and specialized kits. if available, adult and pediatric sizes were listed. only hospitals stocking appropriate pediatric sizes of an item were counted as having that item. the pharmaceutical supply list included antibiotics, antidotes, antiemetics, antiepileptics, intubation and respiratory medications, iv fluids, and miscellaneous drugs not otherwise categorized. results: overall, the hospitals reported having 91% of the items listed (range 87-96%). the two greatest deficiencies were fracture devices (range 33-66%), with no hospital stocking infant-sized cervical collars, and antidotes, with no hospital stocking pralidoxime, 1/5 hospitals stocking fomepizole, and 2/5 hospitals stocking pyridoxine and methylene blue. only one of the five institutions had access to prostaglandin e. the hospitals stated cost and rarity of use as the reason for not stocking these medications. conclusion: the ability of cah to care for pediatric patients does not appear to be hampered by a lack of equipment. ready access to infrequently used, but potentially lifesaving, medications is a concern. tertiary care centers preparing to accept these patients should be aware of these potential limitations as transport decisions are made. background: while incision and drainage (i&d) alone has been the mainstay of management of uncomplicated abscesses for decades, some advocate for adjunct antibiotic use, arguing that available trials are underpowered and that antibiotics reduce treatment failures and recurrence. objectives: to investigate the role of antibiotics in addition to i&d in reducing treatment failure as compared to management with i&d alone. methods: we performed a search using medline, embase, web of knowledge, and google scholar databases (with a medical librarian) to include trials and observational studies analyzing the effect of antibiotics in human subjects with skin and soft-tissue abscesses. two investigators independently reviewed all the records. we performed three overlapping meta-analy-ses: 1. only randomized trials comparing antibiotics to placebo on improvement of the abscess during standard follow-up. 2. trials and observational studies comparing appropriate antibiotics to placebo, no antibiotics, or inappropriate antibiotics (as gauged by wound culture) on improvement during standard follow-up. 3. only trials, but broadened outcome to include recurrence or new lesions during a longer follow-up period as treatment failure. we report pooled risk ratios (rr) using a fixed-effects model for our point estimates with shore-adjusted 95% confidence intervals (ci). results: we screened 1,937 records, of which 12 studies fit inclusion criteria, 9 of which were meta-analyzed (5 trials, 4 observational studies) because they reported results that could be pooled. of the 9 studies, 5 enrolled subjects from the ed, 2 from a soft-tissue infection clinic, and 2 from a general hospital without definition of enrollment site. five studies enrolled primarily adults, 3 pediatrics, and 1 without specification of ages. after pooling results for all randomized trials only, the rr = 1.03 (95% ci: 0.97-1.08). exposure being ''appropriate'' antibiotics (using trials and observational studies) resulted in a pooled rr = 1.01 (95% ci: 0.98-1.03). when we broadened our treatment failure criteria to include recurrence or new lesions at longer lengths of follow-up (trials only), we noted a rr = 1.05 (95% ci: 0.97-1.15). conclusion: based on available literature pooled for this analysis, there is no evidence to suggest any benefit from antibiotics in addition to i&d in the treatment of skin and soft tissue abscesses. (originally submitted as a ''late-breaker.'') primary objectives: to compare wound healing and recurrence rates after primary vs. secondary closure of drained abscesses. we hypothesized the percentage of drained ed abscesses that would be completely healed at 7 days would be higher after primary closure. methods: this randomized clinical trial was undertaken in two academic emergency departments. immunocompetent adult patients with simple, localized cutaneous abscesses were randomly assigned to i & d followed by primary or secondary closure. randomization was balanced by center, with an allocation sequence based on a block size of four, generated by a computer random number generator. the primary outcome was percentage of healed wounds seven days after drainage. a sample of 50 patients had 80% power to detect an absolute difference of 40% in healing rates assuming a baseline rate of 25%. all analyses were by intention to treat. results: twenty-seven patients were allocated to primary and 29 to secondary closure, of whom 23 and 27, respectively, were followed to study completion. healing rates at seven days were similar between the primary and secondary closure groups ( we compared 100 consecutive patients each scanned on the 64 or 320 slice ccta in 2010-2011. measures and outcomes-data were prospectively collected using standardized data collection forms required prior to performing ccta. the main outcomes were cumulative radiation doses and volumes of intravenous contrast. data analysis-groups compared with t-, mann whitney u, and chi-square tests. results: the mean age of patients imaged with the 64 and 320 scanners were 49 (sd 10) vs. 51 (13) (p = 0.27). male:female ratios were also similar (57:43 vs. 51:49 respectively, p = 0.40). both mean (p < 0.001) and median (p = 0.006) effective radiation dose were significantly lower with the 320 (6.8 and 6 msv) vs. the 64-slice scanner (12.2 and 10 msv) respectively. prospective gating was successful in 100% of the 320 scans and only in 38% of the 64 scans (p < 0.001). mean iv contrast volumes were also lower for the 320 vs. the 64-slice scanner (74 ± 10 vs. 96 ± 12 ml; p < 0.001). the % non-diagnostic scans was similarly low in both scanners (3% each). there were no differences in use of beta-blockers or nitrates. conclusion: when compared with the 64-slice scanner, the 320-slice scanner reduces the effective radiation doses and iv contrast volumes in ed patients with cp undergoing ccta. need for beta-blockers and nitrates was similar and both scanners achieved excellent diagnostic image quality. background: a few studies have demonstrated that bedside ultrasound measurement of inferior vena cava to aorta (ivc-to-ao) ratio is associated with the level of dehydration in pediatric patients and a proposed cutoff of 0.8 has been suggested, below which a patient is considered dehydrated. objectives: we sought to externally validate the ability of ivc-to-ao ratio to discriminate dehydration and the proposed cutoff of 0.8 in an urban pediatric emergency department (ed). methods: this was a prospective observational study at an urban pediatric ed. we included patients aged 3 to 60 months with clinical suspicion of dehydration by the ed physician and an equal number of control patients with no clinical suspicion of dehydration. we excluded children who were hemodynamically unstable, had chronic malnutrition or failure to thrive, open abdominal wounds, or were unable to provide patient or parental consent. a validated clinical dehydration score (cds) (range 0 to 8) was used to measure initial dehydration status. an experienced sonographer blinded to the cds and not involved in the patient's care measured the ivc-to-ao ratio on the patient prior to any hydration. cds was collapsed into a binary outcome of no dehydration or any level of dehydration (1 or higher). the ability of ivc-to-ao ratio to discriminate dehydration was assessed using area under the receiver operating characteristic curve (auc) and the sensitivity and specificity of ivc-to-ao ratio was calculated for three cutoffs (0.6, 0.8, 1.0). calculation of auc was repeated after adjusting for age and sex. results: 92 patients were enrolled, 39 (42%) of whom had a cds of 1 or higher. median age was 28 (interquartile range 16-39) months, and 53 (58%) were female. the ivcto-ao ratio showed an unadjusted auc of 0.66 (95% ci 0.54-0.77) and adjusted auc of 0.67 (95% ci 0.56-0.79). for a cutoff of 0.6 sensitivity was 26% (95% ci 13%-42%) and specificity 92% (95% ci 82%-98%); for a cutoff of 0.8 sensitivity was 51% (95% ci 35%-68%) and specificity 74% (95% ci 60%-85%); for a cutoff of 1.0 sensitivity was 79% (95% ci 64%-91%) and specificity 40% (95% ci 26%-54%). conclusion: the ability of the ivc-to-ao ratio to discriminate dehydration in young pediatric ed patients was modest and the cutoff of 0.8 was neither sensitive nor specific. background: while early cardiac computed tomographic angiography (ccta) could be more effective to manage emergency department (ed) patients with acute chest pain and intermediate (>4%) risk of acute coronary syndrome (acs) than current management strategies, it also could result in increased testing, cost, and radiation exposure. objectives: the purpose of the study was to determine whether incorporation of ccta early in the ed evaluation process leads to more efficient management and earlier discharge than usual care in patients with acute chest pain at intermediate risk for acs. methods: randomized comparative effectiveness trial enrolling patients between 40-75 years of age without known cad, presenting to the ed with chest pain but without ischemic ecg changes or elevated initial troponin and require further risk stratification for decision making, at nine us sites. patients are being randomized to either ccta as the first diagnostic test or to usual care, which could include no testing or functional testing such as exercise ecg, stress spect, and stress echo following serial biomarkers. test results were provided to physicians but management in neither arm was driven by a study protocol. data on time, diagnostic testing, and cost of index hospitalization, and the following 28 days are being collected. the primary endpoint is length of hospital stay (los). the trial is powered to allow for detection of a difference in los of 10.1 hours between competing strategies with 95% power assuming that 70% of projected los values are true. secondary endpoints are cumulative radiation exposure, and cost of competing strategies. tertiary endpoints are institutional, caregiver, and patient characteristics associated with primary and secondary outcomes. rate of missed acs within 28 days is the safety endpoint. results: as of november 21st, 2011, 880 of 1000 patients have been enrolled (mean age: 54 ± 8, 46.5% female, acs rate 7.55%). the anticipated completion of the last patient visit is 02/28/12 and the database will be locked in early march 2012. we will present the results of the primary, secondary, and some tertiary endpoints for the entire cohort. conclusion: romicat ii will provide rigorous data on whether incorporation of ccta early in the ed evaluation process leads to more efficient management and triage than usual care in patients with acute chest pain at intermediate risk for acs. (originally submitted as a ''late-breaker.'') meta background: many studies have documented higher rates of advanced radiography utilization across u.s. emergency departments (eds) in recent years, with an associated decrease in diagnostic yield (positive tests / total tests). provider-to-provider variability in diagnostic yield has not been well studied, nor have the factors that may explain these differences in clinical practice. objectives: we assessed the physician-level predictors of diagnostic yield using advanced radiography to diagnose pulmonary embolus (pe) in the ed, including demographics and d-dimer ordering rates. methods: we conducted a retrospective chart review of all ed patients who had a ct chest or v/q scan ordered to rule out pe from 1/06 to 12/09 in four hospitals in the medstar health system. attending physicians were included in the study if they had ordered 50 or more scans over the study period. the result of each ct and vq scan was recorded as positive, negative, or indeterminate, and the identity of the ordering physician was also recorded. data on provider sex, residency type (em or other), and year of residency completion were collected. each provider's positive diagnostic yield was calculated, and logistic regression analysis was done to assess correlation between positive scans and provider characteristics. results: during the study period, 15,015 scans (13,571 cts and 1,443 v/qs) were ordered by 93 providers. the physicians were an average of 9.7 years from residency, 36% were female, and 98% were em-trained. diagnostic yield varied significantly among physicians (p < 0.001), and ranged from 0% to 18%. the median diagnostic yield was 5.9% (iqr 3.8%-7.8%). the use of d-dimer by provider also varied significantly from 4% to 48% (p < 0.001). the odds of a positive test were significantly lower among providers less than 10 years out of residency graduation (or 0.80, ci 0.68-0.95) after controlling for provider sex, type of residency training, d-dimer use, and total number of scans ordered. conclusion: we found significant provider variability in diagnostic yield for pe and use of d-dimer in this study population, with 25% of providers having diagnostic yield less than or equal to 3.8%. providers who were more recently graduated from residency appear to have a lower diagnostic yield, suggesting a more conservative approach in this group. background: the literature reports that anticoagulation increases the risk of mortality in patients presenting to emergency departments (ed) with head trauma (ht). it has been suggested that such patients should be treated in a protocolized fashion, including ct within 15 minutes, and anticipatory preparation of ffp before ct results are available. there are significant logistical and financial implications associated with implementation of such a protocol. objectives: our primary objective was to determine the effect of anticoagulant therapy on the risk of intracranial hemorrhage (ich) in elderly patients presenting to our urban community hospital following bunt head injury. methods: this was a retrospective chart review study of ht patients >60 years of age presenting to our ed over a 6-month period. charts reviewed were identified using our electronic medical record via chief complaints and icd-9 codes and cross referencing with written ct logs. research assistants underwent review of at least 25% of their contributing data to validate reliability. we collected information regarding use of warfarin, clopidogrel, and aspirin and ct findings of ich. using univariate logistic regression, we calculated odds ratios (or) for ich with 95% ci. results: we identified 363 elderly ht patients. the mean age of our population was 72, 34 (8.3%) admitted to using anticoagulant therapy, and 23% were on antiplatelet drugs. 14 (3.8%) of the cohort had icb, 3 patients required neurosurgical intervention, and 1 had transfusion of blood products. of the non-anticoagulated patients, 12 (3.6%) were found to have ich, half of those (6) , and mir-223) were measured using real-time quantitative pcr from serum drawn at enrollment. il-6, il-10, and tnf-a were measured using a bio-plex suspension system. baseline characteristics, il-6, il-10, tnf-a and micrornas were compared using one way anova or fisher exact test, as appropriate. correlations between mirnas and sofa scores, il-6, il-10, and tnf-a were determined using spearman's rank. a logistic regression model was constructed using in-hospital mortality as the dependent variable and mirnas as the independent variables of interest. bonferroni adjustments were made for multiple comparisons. results: of 93 patients, 24 were controls, 29 had sepsis, and 40 had septic shock. we found no difference in serum mir-146a or mir-223 between cohorts, and found no association between these micrornas and either inflammatory markers or sofa score. mir-150 demonstrated a significant correlation with sofa score (q = 0.31, p = 0.01), il-10 (q = 0.37, p = 0.001), but not il-6 or tnf-a (p = 0.046, p = 0.59). logistic regression demonstrated mir-150 to be associated with mortality, even after adjusting for sofa score (p = 0.003). conclusion: mir-146a or mir-223 failed to demonstrate any diagnostic or prognostic ability in this cohort. mir-150 was associated with inflammation, increasing severity of illness, and mortality, and may represent a novel prognostic marker for diagnosis and prognosis of sepsis. objectives: to examine the association between emergency physician recognition of sirs and sepsis and subsequent treatment of septic patients. methods: a retrospective cohort study of all-age patient medical records with positive blood cultures drawn in the emergency department from 11/2008-1/ 2009 at a level i trauma center. patient parameters were reviewed including vital signs, mental status, imaging, and laboratory data. criteria for sirs, sepsis, severe sepsis, and septic shock were applied according to established guidelines for pediatrics and adults. these data were compared to physician differential diagnosis documentation. the mann-whitney test was used to compare time to antibiotic administration and total volume of fluid resuscitation between two groups of patients: those with recognized sepsis and those with unrecognized sepsis. results: sirs criteria were present in 233/338 reviewed cases. sepsis criteria were identified in 215/338 cases and considered in the differential diagnosis in 121/215 septic patients. severe sepsis was present in 89/338 cases and septic shock was present in 42/338 cases. the sepsis 6-hour resuscitation bundle was completed in the emergency department in 16 cases of severe sepsis or septic shock. 121 patients who met sepsis criteria and were recognized by the ed physician had a median time to antibiotics of 150 minutes (iqr: 89-282) and a median ivf of 1500 ml (iqr: 500-3000). the 94 patients who met sepsis criteria but went unrecognized in the documentation had a median time to antibiotics of 225 minutes (iqr: 135-355) and median volume of fluid resuscitation of 1000 ml (iqr: . median time to antibiotics and median volume of fluid resuscitation differed significantly between recognized and unrecognized septic patients (p = 0.003 and p = 0.002, respectively). conclusion: emergency physicians correctly identify and treat infection in most cases, but frequently do not document sirs and sepsis. lack of documentation of sepsis in the differential diagnosis is associated with increased time to antibiotic delivery and a smaller total volume of fluid administration, which may explain poor sepsis bundle compliance in the emergency department. background: severe sepsis is a common clinical syndrome with substantial human and financial impact. in 1992 the first consensus definition of sepsis was published. subsequent epidemiologic estimates were collected using administrative data, but ongoing discrepancies in the definition of severe sepsis led to large differences in estimates. objectives: we seek to describe the variations in incidence and mortality of severe sepsis in the us using four methods of database abstraction. methods: using a nationally representative sample, four previously published methods (angus, martin, dombrovskiy, wang) were used to gather cases of severe sepsis over a 6-year period (2004) (2005) (2006) (2007) (2008) (2009) . in addition, the use of new icd-9 sepsis codes was compared to previous methods. our main outcome measure was annual national incidence and in-hospital mortality of severe sepsis. results: the average annual incidence varied by as much as 3.5 fold depending on method used and ranged from 894,013 (300 / 100,000 population) to 3,110,630 (1,031 / 100,000) using the methods of dombrovskiy and wang, respectively. average annual increase in the incidence of severe sepsis was similar (13.0-13.3%) across all methods. total mortality mirrored the increase in incidence over the 6-year period ( background: radiation exposure from medical imaging has been the subject of many major journal articles, as well as the topic of mainstream media. some estimate that one-third of all ct scans are not medically justified. it is important for practitioners ordering these scans to be knowledgeable of currently discussed risks. objectives: to compare the knowledge, opinions, and practice patterns of three groups of providers in regards to cts in the ed. methods: an anonymous electronic survey was sent to all residents, physician assistants, and attending physicians in emergency medicine (em), surgery, and internal medicine (im) at a single academic tertiary care referral level i trauma center with an annual ed volume of over 160,000 visits. the survey was pilot tested and validated. all data were analyzed using the pearson's chi-square test. results: there was a response rate of 32% (220/668). data from surgery respondents were excluded due to a low response rate. in comparison to im, em respondents correctly equated one abdominal ct to between 100 and 500 chest x-rays, reported receiving formal training regarding the risks of radiation from cts, believe that excessive medical imaging is associated with an increased lifetime risk of cancer, and routinely discuss the risks of ct imaging with stable patients more often (see table 1 ). particular patient factors influence whether radiation risks are discussed with patients by 60% in each specialty (see table 2 ). before ordering an abdominal ct in a stable patient, im providers routinely review the patient's medical imaging history less often than em providers surveyed. overall, 67% of respondents felt that ordering an abdominal ct in a stable ed patient is a clinical decision that should be discussed with the patient, but should not require consent. conclusion: compared with im, em practitioners report greater awareness of the risks of radiation from cts and discuss risks with patients more often. they also review patients' imaging history more often and take this, as well as patients' age, into account when ordering cts. these results indicate a need for improved education for both em and im providers in regards to the risks of radiation from ct imaging. background: in nebraska, 80% of emergency departments have annual visits less than 10,000, and the predominance are in rural settings. general practitioners working in rural emergency departments have reported low confidence in several emergency medicine skills. current staffing patterns include using midlevels as the primary provider with non-emergency medicine trained physicians as back-up. lightly-embalmed cadaver labs are used for resident's procedural training. objectives: to describe the effect of a lightlyembalmed cadaver workshop on physician assistants' (pa) reported level of confidence in selected emergency medicine procedures. methods: an emergency medicine procedure lab was offered at the nebraska association of physician assistants annual conference. each lab consisted of a 2-hour hands-on session teaching endotracheal intubation techniques, tube thoracostomy, intraosseous access, and arthrocentesis of the knee, shoulder, ankle, and wrist to pas. irb-approved surveys were distributed pre-lab and a post-lab survey was distributed after lab completion. baseline demographic experience was collected. pre-and post-lab procedural confidence was rated on a six-point likert scale (1-6) with 1 representing no confidence. the wilcoxon signed-rank test was use to calculate p values. results: 26 pas participated in the course. all completed a pre-and post-lab assessment. no pa had done any one procedure more than 5 times in their career. pre-lab modes of confidence level were £3 for each procedure. post-lab modes were >4 for each procedure except arthrocentesis of the ankle and wrist. however, post lab assessments of procedural confidence significantly improved for all procedures with p values <0.05. conclusion: midlevel providers' level of confidence improved for emergent procedures after completion of a procedure lab using lightly-embalmed cadavers. a mobile cadaver lab would be beneficial to train rural providers with minimal experience. background: use of automated external defibrillators (aed) improves survival in out-of-hospital cardiopulmonary arrest (ohca). since 2005, the american heart association has recommended that individuals one year of age or older who sustain ohca have an aed applied. little is known about how often this occurs and what factors are associated with aed use in the pediatric population. objectives: our objective was to describe aed use in the pediatric population and to assess predictors of aed use when compared to adult patients. methods: we conducted a secondary analysis of prospectively collected data from 29 u.s. cities that participate in the cardiac arrest registry to enhance survival (cares). patients were included if they had a documented resuscitation attempt from october 1, 2005 through december 31, 2009 and were ‡1 year old. patients were considered pediatric if they were less than 19 years old. aed use included application by laypersons and first responders. hierarchical multivariable logistic regression analysis was used to estimate the associations between age and aed use. results: there were 19,559 ohcas included in this analysis, of which 239 (1.2%) occurred in pediatric patients. overall aed use in the final sample was 5,517, with 1,751 (8.9%) total survivors. aeds were applied less often in pediatric patients (19.7%, 95% ci: 14.6%-24.7% vs 28.3%, 95% ci: 27.7%-29.0%). within the pediatric population, only 35.4% of patients with a shockable rhythm had an aed used. in all pediatric patients, regardless of presenting rhythm, aed use demonstrated a statistically significant increase in return of spontaneous circulation (aed used 29.8%, 95% ci: 16.2-43.4 vs aed not used 16.8%, 95% ci: 11.4-22.1, p < 0.05), although there was no significant increase in survival to hospital discharge (aed used 12.8%; aed not used 5.2%; p = 0.057). in the adjusted model, pediatric age was independently associated with failure to use an aed (or 0.61, 95% ci: 0.42-0.87) as was female sex (or 0.88, 95% ci: 0.81-0.95). patients who had a public arrest (or 1.35, 95% ci: 1.24-1.46) or one that was witnessed by a bystander (or 1.20. 95%: ci 1.11-1.29) were also predictive of aed use. conclusion: pediatric patients who experience ohca are less likely to have an aed used. continued education of first responders and the lay public to increase aed use in this population is necessary. does implementation of a therapeutic hypothermia protocol improve survival and neurologic outcomes in all comatose survivors of sudden cardiac arrest? ken will, michael nelson, abishek vedavalli, renaud gueret, john bailitz cook county (stroger), chicago, il background: the american heart association (aha) currently recommends therapeutic hypothermia (th) for out of hospital comatose survivors of sudden cardiac arrest (cssca) with an initial rhythm of ventricular fibrillation (vf). based on currently limited data, the aha further recommends that physicians consider th for cssca, from both the out and inpatient settings, with an initial non-vf rhythm. objectives: investigate whether a th protocol improves both survival and neurologic outcomes for cssca, for out and inpatients, with any initial rhythm, in comparison to outcomes previously reported in literature prior to th. methods: we conducted a prospective observational study of cssca between august 2009 and may 2011 whose care included th. the study enrolled eligible consecutive cssca survivors, from both out and inpatient settings with any initial arrest rhythm. primary endpoints included survival to hospital discharge and neurologic outcomes, stratified by sca location, and by initial arrest rhythm. results: overall, of 27 eligible patients, 11 (41%, 95% ci 22-66%) survived to discharge, 7 (26%, 95% ci 9-43%) with at least a good neurologic outcome. twelve were out and 15 were inpatients. among the 12 outpatients, 6 (50%, 95% ci 22-78%) survived to discharge, 5 (41%, 95% ci 13-69%) with at least a good neurologic outcome. among the 15 inpatients, 5 (33%, 95% ci 9-57) survived to discharge, 2 (13%, 95% ci 0-30%) with at least a good neurologic outcome. by initial rhythm, 6 patients had an initial rhythm of vf/t and 21 non-vf/t. among the 6 patients with an initial rhythm of vf/t, 4 (67%, ci 39-100%) survived to discharge, all 4 with at least a good outcome, including 3 out and 1 inpatients. among the 21 patients with an initial rhythm of non-vf/t, 7 (33%, ci 22-53%) survived to discharge, 3 (14%, ci 0-28%) with at least a good neurologic outcome, including 2 out and 1 inpatients. conclusion: our preliminary data initially suggest that local implementation of a th protocol improves survival and neurologic outcomes for cssca, for out and inpatients, with any initial rhythm, in comparison to outcomes previously reported in literature prior to th. subsequent research will include comparison to local historical controls, additional data from other regional th centers, as well as comparison of different cooling methods. protocolized background: therapeutic hypothermia (th) has been shown to improve the neurologic recovery of cardiac arrest patients who experience return of spontaneous circulation (rosc). it remains unclear as to how earlier cooling and treatment optimization influence outcomes. objectives: to evaluate the effects of a protocolized use of early sedation and paralysis on cooling optimization and clinical outcomes in survivors of cardiac arrest. methods: a 3-year (2008-2010), pre-post intervention study of patients with rosc after cardiac arrest treated with th was performed. those patients treated with a standardized order set which lacked a uniform sedation and paralytic order were included in the pre-intervention group, and those with a standardized order set which included a uniform sedation and paralytic order were included in the post-intervention group. patient demographics, initial and discharge glasgow coma scale (gcs) scores, resuscitation details, cooling time variables, severity of illness as measured by the apache ii score, discharge disposition, functional status, and days to death were collected and analyzed using student's t-tests, man-whitney u tests, and the log-rank test. results: 232 patients treated with th after rosc were included, with 107 patients in the pre-intervention group and 125 in the post-intervention group. the average time to goal temperature (33°c) was 227 minutes (pre-intervention) and 168 minutes (post-intervention) (p = 0.001). a 2-hour time target was achieved in 38.6% of the patients (post-intervention) compared to 24.5% in the pre-group (p = 0.029). twenty-eight day mortality was similar between groups (65.4% and 65.3%) though hospital length of stay (10 days pre-and 8 days post-intervention) and discharge gcs (13 preand 14-post-intervention) differed between cohorts. more post-intervention patients were discharged to home (55.8%) compared to 43.2% in the pre-intervention group. conclusion: protocolized use of sedation and paralysis improved time to goal temperature achievement. these improved th time targets were associated with improved neuroprotection, gcs recovery, and disposition outcome. standardized sedation and paralysis appears to be a useful adjunct in induced th. background: ct is increasingly used to assess children with signs and symptoms of acute appendicitis (aa) though concerns regarding long-term risk of exposure to ionizing radiation have generated interest in methods to identify children at low risk. objectives: we sought to derive a clinical decision rule (cdr) of a minimum set of commonly used signs and symptoms from prior studies to predict which children with acute abdominal pain have a low likelihood of aa and compared it to physician clinical impression (pci). methods: we prospectively analyzed 420 subjects aged 2 to 20 years in 11 u.s. emergency departments with abdominal pain plus signs and symptoms suspicious for aa within the prior 72 hours. subjects were assessed by study staff unaware of their diagnosis for 17 clinical attributes drawn from published appendicitis scoring systems and physicians responsible for physical examination estimated the probability of aa based on pci prior to their medical disposition. based on medical record entry rate, frequently used cdr attributes were evaluated using recursive partitioning and logistic regression to select the best minimum set capable of discriminating subjects with and without aa. subjects were followed to determine whether imaging was used and use was tabulated by both pci and the cdr to assess their ability to identify patients who did or did not benefit based on diagnosis. results: this cohort had a 27.3% prevalence (118/431 subjects) of aa. we derived a cdr based on the absence of two out of three of the following attributes: abdominal tenderness, pain migration, and rigidity/ guarding had a sensitivity of 89.8% (95% ci: 83.1-94.1), specificity of 47.6% (95% ci: 42.1-53.1), npv of 92.5% (95% ci: 87.4-95.7), and negative likelihood ratio of 0.21 (95% ci: 0.12-0.37). the pci set at aa <30% pre-test probability had a sensitivity of 94.1% (95% ci: 88.3-97.1), specificity of 49.4% (95% ci: 43.9-54.9), npv of 95.7% (95% ci: 91.3-97.9), and negative likelihood ratio of 0.12 (95% ci: 0.06-0.25). the methods each classified 37% of the patients as low risk for aa. our cdr identified 29.1% (43/148) of low risk subjects who received ct but being aa (-), could have been spared ct, while the pci identified 20.1% (30/149). conclusion: compared to physician clinical impression, our clinical decision rule can identify more children at low risk for appendicitis who could be managed more conservatively with careful observation and avoidance of ct. negative background: abdominal pain is the most common complaint in the ed and appendicitis is the most common indication for emergency surgery. a clinical decision rule (cdr) identifying abdominal pain patients at a low risk for appendicitis could lead to a significant reduction in ct scans and could have a significant public health impact. the alvarado score is one of the most widely applied cdrs for suspected appendicitis, and a low modified alvarado score (less than 4) is sometimes used to rule out acute appendicitis. the modified alvarado score has not been prospectively validated in ed patients with suspected appendicitis. objectives: we sought to prospectively evaluate the negative predictive value of a low modified alvarado score (mas) in ed patients with suspected appendicitis. we hypothesized that a low mas (less than 4) would have a sufficiently high npv (>95%) to rule out acute appendicitis. methods: we enrolled patients greater than or equal to 18 years old who were suspected of having appendicitis (listed as one of the top three diagnosis by the treating physician before ancillary testing) as part of a prospective cohort study in two urban academic eds from august 2009 to april 2010. elements of the mas and the final diagnosis were recorded on a standard data form for each subject. the sensitivity, specificity, negative predictive value (npv), and positive predictive value (ppv) were calculated with 95% ci for a low mas and final diagnosis of appendicitis. background: evaluating children for appendicitis is difficult and strategies have been sought to improve the precision of the diagnosis. computed tomography is now widely used but remains controversial due to the large dose of ionizing radiation and risk of subsequent radiation-induced malignancy. objectives: we sought to identify a biomarker panel for use in ruling out pediatric acute appendicitis as a means of reducing exposure to ionizing radiation. methods: we prospectively enrolled 431 subjects aged 2 to 20 years presenting in 11 u.s. emergency departments with abdominal pain and other signs and symptoms suspicious for acute appendicitis within the prior 72 hours. subjects were assessed by study staff unaware of their diagnosis for 17 clinical attributes drawn from appendicitis scoring systems and blood samples were analyzed for cbc differential and 5 candidate proteins. based on discharge diagnosis or post-surgical pathology, the cohort exhibited a 27.3% prevalence (118/431 subjects) of appendicitis. clinical attributes and biomarker values were evaluated using principal component, recursive partitioning, and logistic regression to select the combination that best discriminated between those subjects with and without disease. mathematical combination of three inflammation-related markers in a panel comprised of myeloid-related protein 8/14 complex (mrp), c-reactive protein (crp), and white blood cell count (wbc) provided optimal discrimination. results: this panel exhibited a sensitivity of 98% (95% ci, 94-100%), a specificity of 48% (95% ci, 42-53%), and a negative predictive value of 99% (95% ci, 95-100%) in this cohort. the observed performance was then verified by testing the panel against a pediatric subset drawn from an independent cohort of all ages enrolled in an earlier study. in this cohort, the panel exhibited a sensitivity of 95% (95% ci, 87-98%), a specificity of 41% (95% ci, 34-50%), and a negative predictive value of 95% (95% ci, 87-98%). conclusion: appyscore is highly predictive of the absence of acute appendicitis in these two cohorts. if these results are confirmed by a prospective evaluation currently underway, the appyscore panel may be useful to classify pediatric patients presenting to the emergency department with signs and symptoms suggestive of, or consistent with, acute appendicitis and thereby sparing many patients ionizing radiation. background: there are no current studies on the tracking of emergency department (ed) patient dispersal when a major ed closes. this study demonstrates a novel way to track where patients sought emergency care following the closure of saint vincent's catholic medical center (svcmc) in manhattan by using de-identified data from a health information exchange, the new york clinical information exchange (nyclix). nyclix matches patients who have visited multiple sites using their demographic information. on april 30, 2010, svcmc officially stopped providing emergency and outpatient services. we report the patterns in which patients from svcmc visited other sites within nyclix. objectives: we hypothesize that patients often seek emergency care based on geography when a hospital closes. methods: a retrospective pre-and post-closure analysis was performed of svcmc patients visiting other hospital sites. the pre-closure study dates were january 1, 2010-march 31, 2010. the post closure study dates were may 1, 2010-july 31, 2010. a svcmc patient was defined as a patient with any svcmc encounter prior to its closure. using de-identified aggregate count data, we calculated the average number of visits per week by svcmc patients at each site (hospital a-h). we ran a paired t-test to compare the pre-and post-closure averages by site. the following specifications were used to write the database queries: of patients who had one or more prior visits to svcmc for each day within the study return the following: a. eid: a unique and meaningless proprietary id generated within the nyclix master patient index (mpi). b. age: thru the age of 89. persons over 90 were listed as ''90 + '' c. ethnicity/race d. type of visit: emergency e. location of visit: specific nyclix site. results: nearby hospitals within 2 miles saw the highest number of increased ed visits after svcmc closed. this increase was seen until about 5 miles. hospitals >5 miles away did not see any significant changes in ed visits. see table. conclusion: when a hospital and its ed close down, patients seem to seek emergency care at the nearest hospital based on geography. other factors may include the patient's primary doctor, availabilities of outpatient specialty clinics, insurance contracts, or preference of ambulance transports. this study is limited by the inclusion of data from only the eight hospitals participating in nyclix at the time of the svcmc closure. upstream methods: data were collected on all ed ems arrivals from the metro calgary (population 1.1 million) area to its three urban adult hospitals. the study phases consisted of the 7 months from february to october 2010 (pre-ocp) compared against the same months in 2011 (post-ocp). data from the ems operational database and the regional emergency department information system (redis) database were linked. the primary analysis examined the change in ems offload delay defined as the time from ems triage arrival until patient transfer to an ed bed. a secondary analysis evaluated variability in ems offload delay between receiving eds. conclusion: implementation of a regional overcapacity protocol to reduce ed crowding was associated with an important reduction in ems offload delay, suggesting that policies that target hospital processes have bearing on ems operations. variability in offload delay improvements is likely due to site-specific issues, and the gains in efficiency correlate inversely with acuity. methods: a pre-post intervention study was conducted in the ed of an adult university teaching hospital in montreal (annual visits = 69 000). the raz unit (intervention), created to offload the acu of the main ed, started operating in january, 2011. using a split flow management strategy, patients were directed to the raz unit based on patient acuity level (ctas code 3 and certain code 2), likelihood to be discharged within 12 hours, and not requiring an ed bed for continued care. data were collected weekdays from 9:00 to 21:00 for 4 months (september -december 2008) (pre-raz) and for 1.5 months (february -march 2011) (post-raz). in the acu of the main ed, research assistants observed and recorded cubicle access time, and nurse and physician assessment times. databases were used to extract socio-demographics, ambulance arrival, triage code, chief complaint, triage and registration time, length of stay, and ed occupancy. background: telephone follow-up after discharge from the ed is useful for treatment and quality assurance purposes. ed follow-up studies frequently do not achieve high (i.e. ‡ 80%) completion rates. objectives: to determine the influence of different factors on the telephone follow-up rate of ed patients. we hypothesized that with a rigorous follow-up system we could achieve a high follow-up rate in a socioeconomically diverse study population. methods: research assistants (ras) prospectively enrolled adult ed patients discharged with a medication prescription between november 15, 2010 and september 9, 2011 from one of three eds affiliated with one health care system: (a) academic level i trauma center, (b) community teaching affiliate, and (c) community hospital. patients unable to provide informed consent, non-english speaking, or previously enrolled were excluded. ras interviewed subjects prior to ed discharge and conducted a telephone follow-up interview 1 week later. follow-up procedures were standardized (e.g. number of calls per day, times to place calls, obtaining alternative numbers) and each subject's follow-up status was monitored and updated daily through a shared, web-based data system. subjects who completed follow-up were mailed a $10 gift card. we examined the influence of patient (age, sex, race, insurance, income, marital status, usual major activity, education, literacy level, health status), clinical (acuity, discharge diagnosis, ed length of stay, site), and procedural factors (number and type of phone numbers received from subjects, offering two gift cards for difficult to reach subjects) on the odds of successful followup using multivariate logistic regression. results: of the 3,940 enrolled, 45% were white, 59% were covered by medicaid or uninsured, and 44% reported an annual household income of <$26,000. 86% completed telephone follow-up with 41% completing on the first attempt. the table displays the factors associated with successful follow-up. in addition to patient demographics and lower acuity, obtaining a cell phone or multiple phone numbers as well as offering two gift cards to a small number of subjects increased the odds of successful follow-up. conclusion: with a rigorous follow-up system and a small monetary incentive, a high telephone follow-up rate is achievable one week after an ed visit. methods: an interrupted time-series design was used to evaluate the study question. data regarding adherence with the following pneumonia core measures were collected pre-and post-implementation of the enhanced decision-support tool: blood cultures prior to antibiotic, antibiotic within 6 hours of arrival, appropriate antibiotic selection, and mean time to antibiotic administration. prescribing clinicians were educated on the use of the decision-support tool at departmental meetings and via direct feedback on their cases. results: during the 33-month study period, complete data were collected for 1185 patients diagnosed with cap: 613 in the pre-implementation phase and 572 post-implementation. the mean time to antibiotic administration decreased by approximately one minute from the pre-to post-implementation phase, a change that was not statistically significant (p = 0.824). the proportion of patients receiving blood cultures prior to antibiotics improved significantly (p < 0.001) as did the proportion of patients receiving antibiotics within 6 hours of ed arrival (p = 0.004). a significant improvement in appropriate antibiotic selection was noted with 100% of patients experiencing appropriate selection in the post-phase, p = 0.0112. use of the available support tool increased throughout the study period, v 2 = 78.13, df = 1, p < 0.0001. all improvements were maintained 15 months following the study intervention. conclusion: in this academic ed, introduction of an enhanced electronic clinical decision support tool significantly improved adherence to cms pneumonia core measures. the proportion of patients receiving blood cultures prior to antibiotics, antibiotics within 6 hours, and appropriate antibiotics all improved significantly after the introduction of an enhanced electronic clinical decision support tool. background: emergency medicine (em) residency graduates need to pass both the written qualifying exam and oral certification exam as the final benchmark to achieve board certification. the purpose of this project is to obtain information about the exam preparation habits of recent em graduates to allow current residents to make informed decisions about their individual preparation for the abem written qualifying and oral certification exams. objectives: the study sought to determine the amount of residency and individual preparation, to determine the extent of the use of various board review products, and to elicit evaluations of the various board review products used for the abem qualifying and certification exams. methods: design: an online survey instrument was used to ask respondents questions about residency preparation and individual preparation habits, as well as the types of board review products used in preparing for the em boards. participants: as greater than 95% of all em graduates are emra members, an online survey was sent to all emra members who have graduated for the past three years. observations: descriptive statistics of types of preparation, types of resources, time, and quantitative and qualitative ratings for the various board preparation products were obtained from respondents. results: a total of 520 respondents spent an average of 9.1 weeks and 15 hours per week preparing for the written qualifying exam and spent an average of 5 weeks and 7.8 hours per week preparing for the oral certification exam. in preparing for the written qualification exam, 90% used a preparation textbook with 16% using more than one textbook and 47% using a board preparation course. in preparing for the oral qualifying exam, 56% used a preparation textbook while 34% used a preparation course. sixty-seven percent of respondents reported that their residency programs had a formalized written qualifying exam preparation curriculum of which 48% was centered on the annual in-training exam. eight-five percent of residency programs had a formalized oral certification exam preparation. respondents reported spending on average $715 preparing for the qualifying exam and $509 for the certification exam. conclusion: em residents spend significant amounts of time and money and make use of a wide range of residency and commercially available resources in preparing for the abem qualifying and certification exams. background: communication and professionalism skills are essential for em residents but are not wellmeasured by selection processes. the multiple mini-interview (mmi) uses multiple, short structured contacts to measure these skills. it predicts medical school success better than the interview and application. its acceptability and utility in em residency selection is unknown. objectives: we theorized that the mmi would provide novel information and be acceptable to participants. methods: 71 interns from three programs in the first month of training completed an eight-station mmi developed to focus on em topics. pre-and post-surveys assessed reactions using five-point scales. mmi scores were compared to application data. results: em grades correlated with mmi performance (f(1.66) = 4:18, p < 0.05) with honors students having higher mmi summary scores. higher third year clerkship grades trended to higher mmi performance means, although not significantly. mmi performance did not correlate with a match desirability rating and did not predict other individual components of the application including usmle step 1 or usmle step 2. participants preferred a traditional interview (mean difference = 1.36, p < 0.0001). a mixed format was preferred over a pure mmi (mean difference = 1.1, p < 0.0001). preference for a mixed format was similar to a traditional interview. mmi performance did not significantly correlate with preference for the mmi; however, there was a trend for higher performance to associate with higher preference (r = 0.15, t(65) = 1.19, n.s.) performance was not associated with preference for a mix of interview methods (r = 0.08, t(65) = 0.63, n.s.). conclusion: while the mmi alone was viewed less favorably than a traditional interview, participants were receptive to a mixed methods interview. the mmi appears to measure skills important in successful completion of an em clerkship and thus likely em residency. future work will determine whether mmi performance correlates with clinical performance during residency. background: the annual american board of emergency medicine (abem) in-training exam is a tool to assess resident progress and knowledge. when the new york-presbyterian (nyp) em residency program started in 2003, the exam was not emphasized and resident performance was lower than expected. a course was implemented to improve residency-wide scores despite previous em literature failing to exhibit improvements with residency-sponsored in-training exam interventions. objectives: to evaluate the effect of a comprehensive, multi-faceted course on residency-wide in-training exam performance. methods: the nyp em residency program, associated with cornell and columbia medical schools, has a 4year format with 10-12 residents per year. an intensive 14-week in-training exam preparation program was instituted outside of the required weekly residency conferences. the program included lectures, pre-tests, high-yield study sheets, and remediation programs. lectures were interactive, utilizing an audience response system, and consisted of 13 core lectures (2-2.5 hours) and three review sessions. residents with previous in-training exam difficulty were counseled on designing their own study programs. the effect on intraining exam scores was measured by comparing each resident's score to the national mean for their postgraduate year (pgy). scores before and after course implementation were evaluated by repeat measures regression modeling. overall residency performance was evaluated by comparing residency average to the national average each year and by tracking abem national written examination pass rates. results: resident performance improved following course implementation. following the course's introduction, the odds of a resident beating the national mean increased by 3.9 (95% ci 1.9-7.3) and the percentage of residents exceeding the national mean for their pgy year increased by 37% (95% ci 23%-52%). following course introduction, the overall residency mean score has outperformed the national exam mean annually and the first-time abem written exam board pass rate has been 100%. conclusion: a multi-faceted in-training exam program centered around a 14-week course markedly improved overall residency performance on the in-training exam. limitations: this was a before and after evaluation as randomizing residents to receive the course was not logistically or ethically feasible. .0 years of practice. among the nonresidency trained, non-boarded em physicians, the percentage of individuals with board actions against them was significantly higher (6.9% vs. 1.9%, 95% ci for difference of 5.0% = 3.1 to 7.5%), but the incidence of actions was not significant (1.3 vs. 3.4 events/ 1000 years of practice, 95% ci for difference of 2.1/ 1000 = )3/1000 to +8/1000), but the power to detect a difference was 30%. conclusion: in this study population, em-trained physicians had significantly fewer total state medical board disciplinary actions against them than non-em trained physicians, but when adjusted for years of practice (incidence), the difference was not significantly different at the 95% confidence level. the study was limited by low power to detect a difference in incidence. objectives: we chose pain documentation as a long term project for quality improvement in our ems system. our objectives were to enhance the quality of pain assessment, to reduce patient suffering and pain through improved pain management, to improve pain assessment documentation, to improve capture of initial and repeat pain scales, and to improve the rate of pain medication. this study addressed the aim of improving pain assessment documentation. methods: this was a quasi-experiment looking at paramedic documentation of the pqrst mnemonic and pain scales. our intervention consisted of mandatory training on the importance and necessity of pain assessment and treatment. in addition to classroom training, we used rapid cycle individual feedback and public posting of pain documentation rates (with unique ids) for individual feedback. the categories of chief complaint studied were abdominal pain, blunt injury, burn, chest pain, headache, non-traumatic body pain, and penetrating injury. we compared the pain documentation rates in the 3 months prior to intervention, the 3 months of intervention, and 3 months post intervention. using repeated-measures anova, we compared rates of paramedic documentation over time. results: our ems system transported 42166 patients during the study period, of whom 15490 were for painful conditions in the defined chief complaint categories. there were 168 paramedics studied, of whom 149 had complete data. documentation increased from 1819 of 5122 painful cases (35.5%) in qtr 1 to 4625 of 5180 painful cases (89.3%) in qtr 3. the trend toward increased rates of pain documentation over the three quarters was strongly significant (p < 0.001). paramedics were significantly more likely to document pain scales and pqrst assessments over the course of the study with the highest rates of documentation compliance in the final 3-month period. conclusion: a focused intervention of education and individual feedback through classroom training, one on one training, and public posting improves paramedic documentation rates of perceived patient pain. background: emergency medical services (ems) systems are vital in the identification, assessment, and treatment of trauma, stroke, myocardial infarction, and sepsis and improving early recognition, resuscitation, and transport to adequate medical facilities. ems personnel provide similar first-line care for patients with syncope, performing critical actions such as initial assessment and treatment as well as gathering key details of the event. objectives: to characterize emergency department patients with syncope receiving initial care by ems and their role as initial providers. methods: we prospectively enrolled patients over 18 years of age who presented with syncope or near syncope to a tertiary care ed with 72,000 annual patient visits from june 2009 to june 2011. we compared patient age, sex, comorbidities, and 30-day cardiopulmonary adverse outcomes (defined as myocardial infarction, pulmonary embolism, significant cardiac arrhythmia, and major cardiovascular procedure) between ems and non-ems patients. descriptive statistics, two-sided ttests, and chi-square testing were used as appropriate. results: of the 669 patients enrolled, 254 (38.0%) arrived by ambulance. the most common complaint in patients transported by ems was fainting (50.4%) or dizziness (45.7%); syncope was reported in 28 (11.0%). compared to non-ems patients, those who arrived by ambulance were older (mean age (sd) 64.5 (18.7), vs. 60.6 (19.5) years, p = 0.012). there were no differences in the proportion of patients with hypertension (20.0% vs 32.0%, p = 0.75), coronary artery disease (8.85% vs 15.3%, p = 0.67), diabetes mellitus (6.5% vs 9.5%, p = 0.57), or congestive heart failure (3.8% vs 6.6%, p = 0.74). sixtynine (10.8%) patients experienced a cardiopulmonary event within 30 days. twenty-eight (4.4%) patients who arrived by ambulance and 41 (6.4%) non-ems patients had a subsequent cardiopulmonary adverse event (rr 1.08, 95%ci 0.68-1.69) within 30 days. the table tabulates interventions provided by ems prior to ed arrival. conclusion: ems providers care for more than one third of ed syncope patients and often perform key interventions. ems systems offer opportunities for advancing diagnosis, treatment, and risk stratification in syncope patients. background: abdominal pain is the most common reason for visiting an emergency department (ed), and abdominopelvic computed tomography (apct) use has increased dramatically over the past decade. despite this, there has been no significant change in rates of admission or diagnosis of surgical conditions. objectives: to assess whether an electronic accountability tool affects apct ordering in ed patients with abdominal or flank pain. we hypothesized that implementation of an accountability tool would decrease apct ordering in these patients. methods: before and after study design using an electronic medical record at an urban academic ed from jul-nov 2011, with the electronic accountability tool implemented in oct 2011 for any apct order. inclusion criteria: age >= 18 years, non-pregnant, and chief complaint or triage pain location of abdominal or flank pain. starting oct 17 th , 2011, resident attempts to order apct triggered an electronic accountability tool which only allowed the order to proceed if approved by the ed attending physician. the attending was prompted to enter the primary and secondary diagnoses indicating apct, agreement with need for ct and, if no agreement, who was requesting this ct (admitting or consulting physician), and their pretest probability (0-100) of the primary diagnosis. patients were placed into two groups: those who presented prior to (pre) and after (post) the deployment of the accountability tool. background: there has been a paradigm shift in the diagnostic work-up for suspected appendicitis. edbased staged protocols call for the use of ultrasound prior to ct scanning because of its lack of radiation, and the morbidity related to contrast. a barrier to implementation is the lack of 24/7 availability of ultrasound. objectives: to evaluate the impact of the implementation of ed performed appendix ultrasounds (apus) on ct utilization in the staged workup for appendicitis in the emergency department. methods: we performed a quasi-experimental, before/ after study. we compared data from the first 8 months of 2009, before the availability of ed performed apus, with the same interval in 2011 after introduction of ed apus. we excluded patients who had appendectomies for reasons other than appendicitis or had been diagnosed prior to arrival. no patient identifiers were included in the analysis and the study was approved by the hospital irb. we report the following descriptive statistics (percentages, sensitivities, and absolute utilization changes conclusion: implementation of an ed apus in the staging work up of appendicitis was associated with a significant reduction in overall ct utilization in the ed. objectives: this study aims to evaluate ed patients' knowledge of radiation exposure from ct and mri scans as well as the long-term risk of developing cancer. we hypothesize that ed patients will have a poor understanding of the risks, and will not know the difference between ct and mri. methods: design -this was a cross-sectional survey study of adult, english-speaking patients at two eds from 6/13/11-8/13/11. setting -one location was a tertiary care center with an annual ed census of 45,000 patient visits and the other was a community hospital with annual ed census of 35,000 patient visits. obser-vations -the survey consisted of six questions evaluating patients' understanding of radiation exposure from ct and mri as well as long-term consequences of radiation exposure. patients were then asked their age, sex, race, highest level of education, annual household income, and whether they considered themselves health care professionals. results: there were 500 participants in this study, 315 (of 5,589 total) from the academic center and 185 (of 4,988 total) from the community hospital during the study period. overall, only 10% (95% ci 7-12%) of participants understood the radiation risks associated with ct scanning. 60% (95% ci 56-65%) of patients believed that an abdominal ct had the same or less radiation as a chest x-ray. 25% (95% ci 21-29%) believed that there was an increased risk of developing cancer from repeated abdominal cts. only 22% (95% ci 19-26%) of patients knew that mri scans had less radiation than ct. 44% (95% ci 39-49%) either didn't know or believed that repeated mris were associated with an increased risk of developing cancer. higher educational level, household income, and identification as a health care professional all were associated with correct responses, but even within these groups, a majority gave incorrect responses. conclusion: in general, ed patients do not understand the radiation risks associated with advanced imaging modalities. we need to educate these patients so that they can make informed decisions about their own health care. background: homelessness has been associated with many poor health outcomes and frequent ed utilization. it has been shown that frequent use of the ed in any given year is not a strong predictor of subsequent use. identifying a group of patients who are chronic high users of the ed could help guide intervention. objectives: the purpose of this study is to identify if homelessness is associated with chronic ed utilization. methods: a retrospective chart review was accomplished looking at the records of the 100 most frequently seen patients in the ed for each year from 2005-2010 at a large, urban academic hospital with an annual volume of 55,000. patients' visit dates, chief complaints, dispositions, and housing status were reviewed. homelessness was defined by self-report at registration. patients were categorized according to their ed utilization with those seen >4 times in at least three of the five years of the study identified as chronic high utilizers; and those who visited the ed >20 times in at least three of the five years of the study were identified as chronic ultra-high utilizers. descriptive statistics with confidence intervals were calculated, and comparisons were made using non-parametric tests. results: during the 5-year study period, 189,371 unique patients were seen, of whom 0.7% patients were homeless. 335 patients were identified as frequent users. there were patients who presented on the top 100 utilizer lists from multiple years. 67 (20%, 95%ci 16-25) patients were identified as homeless. 148 patients were seen >4 times in at least three of the 5 years and 23 (16%, 11-22) were homeless. 12 patients were seen >20 times in at least three of the 5 years and 5 (41%, 19-68) were homeless. our facility has a 40% admission rate; however, non homeless chronic ultra-high utilizers had admission rates of 24% and homeless chronic ultra-high utilizers were admitted 14%. conclusion: chronic ultra-high utilizers of our ed are disproportionately homeless and present with lower severity of illness. these patients may prove to be a cost-effective group to house or otherwise involve with aggressive case management. the debate over homeless housing programs and case management solutions can be sharpened by better defining the groups who would most benefit and who represent the greatest potential saving for the health system. background: the prevalence of obese patients presenting to our emergency department (ed) is 38%: obese patients present in disproportionate number compared to the general population (us rate = 27%). in spite of this, there is a disconnect in patients' perceptions of weight and health: many patients underestimate their weight and report a key barrier to weight loss is patient-provider communications; such discussions have proven to be highly effective in smoking, drug, and alcohol cessation, an important initial step toward promoting wellness. information about patient provider communication is essential for designing and implementing emergency department (ed) based interventions to help increase patient awareness about weightrelated medical issues and provide counseling for weight reduction. objectives: we assessed patients' perceptions about obesity as disease and patient communication with their providers through two questions: do you believe your present weight is damaging to your health? has a doctor or other health professional every told you that you are overweight? methods: a descriptive cross-sectional study was performed in an academic tertiary care ed. a randomized sample of patients (every fifth) presenting to the ed (n = 453) was enrolled. pregnant patients, patients who were medically unstable, cognitively impaired, or who were unable or unwilling to provide informed consent were excluded. percentages of ''yes'' and ''no'' are reported for each question based on patient bmi, ethnicity, sex, and the number of comorbid conditions. regression analysis was used to determine differences in responses between subgroups. results: among overweight/obese, white/black patients, 42.5% do not feel their weight is damaging to their health and 54.7% reported they have not been told by a doctor they are overweight. of individuals who have been told by a doctor they were overweight, 23.2% still believe their present weight is not damaging to their health. of individuals who have not been told by a doctor they were overweight, 41.5% believe their present weight is damaging to their health. differences in race and age were not found. p values <0.05 for all results. conclusion: our data point toward a disconnect regarding patients' perceptions of health and weight. timely education about the burden of obesity may lead to a decrease in its overall prevalence. (originally submitted as a ''late-breaker.'') objectives: to examine the attitudes and expectations of patients admitted for inpatient care following an emergency department visit. methods: a descriptive study was done by surveying a voluntary sample of adult patients (n = 210) admitted to the hospital from the emergency department in one urban teaching hospital in the midwest. a short, ninequestion survey was developed to assess patient attitudes and expectations towards hiv testing, consent, and requirements. analyses consisted of descriptive statistics, correlations, and chi-square analyses. results: the majority of patients report that hiv testing should be a routine part of health care screening (82.4%) and that the hospital should routinely test admitted patients for hiv (78.6%). despite these overall positive attitudes towards hiv testing, the data also suggest that patients have strong attitudes towards consent requirements with 80% acknowledging that hiv testing requires special consent and 72% reporting that separate consent should be required. the data also showed a statistically significant difference in the proportion of patients who believed that hiv testing is a part of routine health care screening by race (v2 = 6.825, df = 1, p = .009). conclusion: patients attitudes and expectations towards routine hiv testing are consistent with the cdc recommendations. emergency departments are an ideal setting to initiate hiv testing and the findings suggest that patients expect hospital policies outline procedures for obtaining consent and screening all patients who are admitted to the hospital from the ed. results: the analysis revealed a ''hot spot'', a cluster of 833 counties (24.5%) with high ca rates adjacent to counties with high ca rates, located across the southeastern us (p < 0.001). within these counties, the average ca rate was 14% higher than the national average. a ''cool spot'', a cluster of 548 counties (16.1%) with low rates, was located across the midwest (p < 0.001). in this cool spot the average ca rate was 12% lower than the national average. figures 1 and 2 show us adjusted rates and spatial autocorrelation of ca deaths, respectively. conclusion: we identify geographic disparities in ca mortality and describe the cardiac arrest belt in the southeastern us. a limitation of this analysis was the use of icd-10 codes to identify cardiac arrest deaths; however, no other national data exist. an improved understanding of the drivers of this variability is essential to targeted prevention and treatment strategies, especially given the recent emphasis on development of cardiac resuscitation centers and cardiac arrest systems of care. an understanding of the relation between population density, cardiac arrest count, and cardiac arrest rate will be essential to the design of an optimized cardiac arrest system. we defined ed utilization during the past 12 months as non-users (0 visits), infrequent users (1-3 visits), frequent users (4-9 visits), and super-frequent users ( ‡10 visits). we compared demographic data, socioeconomic status, health conditions, and access to care between these ed utilization groups. results: overall, super-frequent use was reported by 0.4% of u.s. adults, frequent use by 2%, and infrequent ed use by 19%. higher ed utilization was associated with increased self-reported fair to poor health (55% for super-frequent, 48% for frequent, 22% for infrequent, 10% for non-ed users). frequent ed users were also more likely to be impoverished, with 31% of superfrequent, 25% of frequent, 13% of infrequent, and 9% of non-ed users reporting a poverty-income ratio <1. adults with higher ed utilization were more likely to report the ed as the place they usually go when sick (10% for super-frequent, 6% for frequent, 2% for infrequent, 0.5% for non-ed users). they also reported greater outpatient resource utilization, with 73% of super-frequent, 48% of frequent, 25% of infrequent, and 10% of non-ed users reporting ‡10 outpatient visits/year. frequent ed users were also more likely than non-ed users to be covered by medicaid (34% for super-frequent, 26% for frequent, 12% for infrequent, 5% for non-ed users). conclusion: frequent ed users were a vulnerable population with lower socioeconomic status, poor overall health, and high outpatient resource utilization. interventions designed to divert frequent users from the ed should also focus on chronic disease management and access to outpatient services, rather than focusing solely on limiting ed utilization. objectives: we explored factors associated with specialty provider willingness to provide urgent appointments to children insured by medicaid/chip. methods: as part of a mixed method study of child access to specialty care by insurance status, we conducted semi-structured qualitative interviews with a purposive sample of 26 specialists and 14 primary care physicians (pcps) in cook county, il. interviews were conducted from april to september 2009, until theme saturation was reached. resultant transcripts and notes were entered into atlas.ti and analyzed using an iterative coding process to identify patterns of responses in the data, ensure reliability, examine discrepancies, and achieve consensus through content analysis. results: themes that emerged indicate that pcps face considerable barriers getting publicly insured patients into specialty care and use the ed to facilitate this process. ''if i send them to the emergency room, i'm bypassing a number of problems. i'm fully aware that i'm crowding the emergency room.'' specialty physicians reported that decisions to refuse or limit the number of patients with medicaid/chip are due to economic strain or direct pressure from their institutions ''in the last budget revision, we were [told], 'you are losing money, so you need to improve your patient mix'''. in specialty practices with limited medicaid/chip appointment slots, factors associated with appointment success included: high acuity or complexity, personal request from or an informal economic relationship with the pcp, geography, and patient hardship. ''if it's a really desperate situation and they can't find anybody else, i will make an exception''. specialists also acknowledged that ''patients who can't get an appointment go to the er and then i am obligated to see them if they're in the system.'' conclusion: these exploratory findings suggest that a critical linkage exists between hospital eds and affiliated specialty clinics. as health systems restructure, there is an opportunity for eds to play a more explicit role in improving care coordination and access to specialty care. albert amini, erynne a. faucett, john m. watt, richard amini, john c. sakles, asad e. patanwala university of arizona, tucson, az background: trauma patients commonly receive etomidate and rocuronium for rapid sequence intubation (rsi) in the ed. due to the long duration of action of rocuronium and short duration of action of etomidate, these patients require prompt initiation of sedatives after rsi. this prevents the potential of patient awareness under pharmacological paralysis, which could be a terrifying experience. objectives: the purpose of this study was to evaluate the effect of the presence of a pharmacist during traumatic resuscitations in the ed on the initiation of sedatives and analgesics after rsi. we hypothesized that pharmacists would decrease the time to provision of sedation and analgesia. methods: this was an observational, retrospective cohort study conducted in a tertiary, academic ed that is a level i trauma center. consecutive adult trauma patients who received rocuronium in the ed for rsi were included during two time periods: 07/01/07 to 07/ 30/08 (pre-phase -no pharmacy services in the ed) and 07/01/09 to 06/30/11 (post-phase -pharmacy services in the ed). since the pharmacist could not respond to all traumas in the post-phase, this was further categorized based on whether the pharmacist was present or absent at the trauma resuscitation. data collected included patient demographics, baseline injury data, and medications used. the median time from rsi to initiation of sedatives and analgesics was compared between the pre-phase group (group 1), post-phase pharmacist absent group (group 2), and post-phase pharmacist present group (group 3) using the kruskal-wallis test. results: a total of 200 patients were included in the study (group 1 = 100, group 2 = 70, and group 3 = 30). median age was 35, 48.5, and 54.5 years in groups 1, 2, and 3, respectively (p = 0.005). there were no other differences between groups with regard to demographics, mechanism of injury, presence of traumatic brain injury, glasgow coma scale score, vital signs, ed length of stay, or mortality. median time between rsi and post-intubation sedative use was 13, 15, and 6 minutes in groups 1, 2 and 3, respectively (p < 0.001). median time between rsi and post-intubation analgesia use was 80, 16, and 10 minutes in groups 1, 2, and 3, respectively (p < 0.001). the presence of a pharmacist during trauma resuscitations decreases time to provision of sedation and analgesia after rsi. background: outpatient antibiotics are frequently prescribed from the ed, and limited health literacy may affect compliance with recommended treatments. objectives: among patients stratified by health literacy level, multimodality discharge instructions will improve compliance with outpatient antibiotic therapy and follow-up recommendations. methods: this was a prospective randomized trial that included consenting patients discharged with outpatient antibiotics from an urban county ed with an annual census of 100,000. patients unable to receive text messages or voicemails were excluded. health literacy was assessed using a validated health literacy assessment, the newest vital sign (nvs). patients were randomized to a discharge instruction modality: 1) usual care, typed and verbal medication and case-specific instructions; 2) usual care plus text messaged instructions sent to the patient's cell phone; or 3) usual care plus voicemailed instructions sent to the patient's cell phone. antibiotic pick-up was verified with the patient's pharmacy at 72 hours. patients were called at 30 days to determine antibiotic compliance. z-tests were used to compare 72-hour antibiotic pickup and patient-reported compliance across instructional modality and nvs score groups. results: 758 patients were included (55% female, median age 30, range 5 months to 71 years); 98 were excluded. 23% had an nvs score of 0-1, 31% 2-3, and 46% 4-6. the proportion of prescriptions filled at 72 hours varied significantly across nvs score groups; self-reported medication compliance at 30 days revealed no difference across different instructional modalities nor nvs scores (table 1) . conclusion: in this sample of urban ed patients, 72hour prescription pickup varied significantly by validated health literacy score, but not by instruction delivery modality. in this sample, patients with lower health literacy are at risk of not filling their outpatient antibiotics in a timely fashion. has been developed, validated, and utilized to study the processes of care involved in successful care transitions from inpatient to outpatient settings, but has not been utilized in the ed. objectives: we hypothesized that the ctm-3 could be successfully implemented in the ed without differential item difficulty by age, sex, education, or race; and would be associated with measures of quality of care and likelihood of following physician recommendations. methods: a descriptive study design based on exit surveys was used to measure ctm-3 scores and likelihood of following treatment recommendations. surveys were administered to a daily cross-sectional sample of all patients leaving the ed between 7a-12a by research assistants in an urban academic ed setting for 3 weeks in november 2011. we report means and standard deviations, and analysis of variance to identify differences in ctm-3 scores for those who planned and did not plan to follow ed recommendations. results: 750 surveys were completed; patients were 43 ± 19 years old, 58% black, 61% female, 56% with at least some college education, and 38% were admitted. average ctm-3 score was 87.1 ± 21.6 (range 0-100). scores were not associated with sex (p = 0.57), race (p = 0.19), or education level (p = 0.25). lower ctm scores were associated with increasing age (p = 0.03), patient perceptions that the ed team was less likely to use words that they understood, listen carefully to them, inspire their confidence and trust, or encourage them to ask questions (all p < 0.01). those who reported they were ''very likely'' to follow ed treatment had an average score of 89 ± 21, while those who were ''unlikely'' or ''very unlikely'' to follow ed treatment plans had an average score 47 ± 28 (p = 0.00). conclusion: the ctm-3 performs well in the ed and exhibited only differential item difficulty by age; there was no significant difference by race, sex, or education level. furthermore, it is highly associated with likelihood of following physician recommendations. future studies will focus on ctm-3 scores ability to discriminate between patients who did or did not experience a subsequent ed visit or rehospitalization. age and race were found to be significant predictors of the race pathway. regression of the data by race revealed blacks (or 1.9: ci 1.3-2.6; p < 0.0002), hispanics (or 3.0: ci 1.3-2.6; p = 0.0001), and asians (or 2.3: ci 1.1-4.9; p = 0.03), were more likely to enter the race cohort than were whites; however, much of this discrepancy is accounted for by age. the mean age of minority patients was 62 years, while white patients were older at 71 years (p = 0.002). conclusion: in a diverse demographic population we found that racial minorities were presenting at younger ages for chest pain and were more likely to receive cardiac testing at bedside than their white counterparts; and hence, were selected to a lower level of care (nonmonitored unit background: expanding insurance coverage is designed to improve access to primary care and reduce use of emergency services. whether expanding coverage achieves this is of paramount importance as the united states prepares for the affordable care act. objectives: we examined ed and outpatient department use after the state children's health insurance program (schip) coverage expansion, focusing on adolescents (a major target group for schip) versus young adults (not targeted). we hypothesized that coverage would increase use of outpatient services and emergency department services would decrease. methods: using the national ambulatory medical care survey and the national hospital ambulatory medical care survey, we analyzed years 1992-1996 as baseline and then compared use patterns in 1999-2009 after schip launch. primary outcomes were populationadjusted annual visits to ed versus non-emergency outpatient settings. interrupted time-series were performed on use rates to ed and outpatient departments between adolescents (11-18 years old) and young adults (19-29 years old) in the pre-schip and schip periods. outpatient-to-ed ratios were calculated and compared across time periods. results: the mean number of outpatient adolescent visits increased by 299 visits per 1000 persons (95% ci, 140-457), while there was no statistically significant increase in young adult outpatient visits across time periods. there was no statistically significant change in the mean number of adolescent ed visits across time periods, while young adult ed use increased by 48 visits per 1000 persons (95% ci, 24-73). the adolescent outpatient-to-ed ratio increased by 1.0 (95% ci, 0.49-1.6), while the young adults ratio decreased by 0.53 across time periods (95% ci, )0.90 to )0.16). conclusion: since schip, adolescent non-ed outpatient visits increased while ed visits remained unchanged. in comparison to young adults, expanding insurance coverage to adolescents improved access to health care services and suggests a shift to non-ed settings. as an observational study we are unable to control for secular trends during this time period. also as an ecological study we are unable to examine individual variation. expanding insurance through the affordable care act of 2010 will likely increase use of outpatient services but may not decrease emergency department volumes. background: cancer patients are receiving a greater proportion of their care on an outpatient basis. the effect of this change in oncology care patterns on ed utilization is poorly understood. objectives: to examine the characteristics of ed utilization by adult cancer patients. methods: between july 2007 and march 2009, all new adult cancer patients referred to a tertiary care cancer centre were recruited into a study examining psychological distress. these patients were followed prospectively until september 2011. the collected data were linked to administrative data from three tertiary care eds. variables evaluated in this study included basic we have previously shown that reducing non-value-added activities through the application of the lean process improvement methodology improves patient satisfaction, physician productivity and emergency department length of stay. objectives: in this investigation, we tested the hypothesis that non-value-added activities reduce physician job satisfaction. methods: to test this hypothesis, we conducted timemotion studies on attending emergency physicians working in an academic setting and categorized their activities into value-added (time in room with patient, time discussing cases and educating medical learners, time in room with patient and learner), necessary non-valueadded activities (charting, sign out, looking up labs), and unnecessary non-value-added activities (looking for things, looking for people, on the phone). the physicians were then surveyed using a 10-point likert scale to determine their relative satisfaction with each of the individual tasks (1 worst part of day, 10 best part of day). results: physicians spent 46% of their shift performing value-added work, 38% of their shift performing necessary non-value-added activities, and 16% of their shift performing unnecessary non-value-added activities (waste). weighted physician satisfaction (satisfaction x [percent time spent performing the activity / percent time engaged in activity category]) was highest when the physician was performing value-added work (8.75) compared to performing either necessary non-valueadded work (3.35) or waste (2.61). conclusion: the attending physicians we studied spent the majority of their time performing non-value-added activities, which were associated with lower satisfaction. application of process improvement techniques such as lean, which focus on reducing non-value-added work, may improve emergency physician job satisfaction. background: rocuronium and succinylcholine are the most commonly used paralytics for rapid sequence intubation (rsi) in the ed. after rsi, patients need sustained sedation while they are mechanically ventilated. however, the longer duration of action of rocuronium may influence subsequent sedation dosing, while the patient is therapeutically paralyzed. objectives: we hypothesized that patients who receive rocuronium would be more likely to receive lower doses of post-rsi sedation compared to patients who receive succinylcholine. methods: this was an observational, retrospective cohort study conducted in a tertiary, academic ed. consecutive adult patients, who received rsi using etomidate for induction of sedation between 07/01/09 to 06/30/10, were included. patients were then categorized based on whether they received rocuronium or succinylcholine for paralysis. the dosing of post-rsi sedative infusions was compared at 0, 30, 60, and 120 minutes after initiation between the two groups using the wilcoxon rank-sum test. results: a total of 254 patients were included in the final analysis (rocuronium = 127, succinylcholine = 127). mean age was 52 and 47 years in the rocuronium and succinylcholine groups, respectively (p = 0.04). there were no other baseline differences between groups with regard to demographics, reason for intubation, stroke, traumatic brain injury, glasgow coma scale score, pain scores, or vital signs. in the overall cohort, 90.2% (n = 229) of patients were given a sedative infusion or bolus in the ed. most patients were initiated on propofol (n = 169) or midazolam (n = 49) infusions. median propofol infusion rates at 0, 30, 60, and 120 minutes were 20, 20, 27.5, and 30 mcg/kg/min in the rocuronium group and 20, 40, 45, and 45 mcg/kg/ min in succinylcholine group, respectively. the difference was statistically significant at 30 (p < 0.001) and 60 (p = 0.003) minutes. median midazolam infusion rates at 0, 30, 60, and 120 minutes were 2, 2, 2, and 3 mg/hour in the rocuronium group and 2, 3, 4, and 4.5 mg/hour in succinylcholine group, respectively. the difference was statistically significant at 60 (p = 0.003) and 120 (p = 0.04) minutes. conclusion: patients who receive rocuronium are more likely to receive lower doses of sedative infusions post-rsi due to sustained therapeutic paralysis. this may put them at risk for being awake under paralysis. what is the impact of the implementation of an there was a difference in presenting pain (p < 0.001), stress (p < 0.001), and anxiety (p < 0.001) among patients that received an opioid in the ed. there was a difference in presenting pain (p < 0.001) for patients discharged with an opioid prescription, but not for stress (p = 0.32) or anxiety (p = 0.90). conclusion: patient-reported pain, stress, and anxiety are higher among patients who received an opiate in the ed than in those who did not, but only pain is higher among patients who received a discharge prescription for an opioid. methods: this was a prospective, randomized crossover study on the use of gvl and dl by incoming pediatric interns prior to advanced life support training. at the start of the study, the interns received a didactic session and expert modeling of the use of both devices for intubation. two scenarios were used: (1) normal intubation with a standard airway and (2) difficult intubation with tongue edema and pharyngeal swelling. interns then intubated laerdal simbaby in each scenario with both gvl and dl for a total of four randomized intubation scenarios. primary outcomes included time to successful intubation and the rate of successful intubation. the interns also rated their satisfaction with the devices using a visual analog scale (0-10) and chose their preferred device for their next intubation. results: 29 interns were included in this study. in the normal airway scenario, there were no differences in the mean time for intubation with gvl or dl (62.9 ± 24.1 vs 61.8 ± 26.2 seconds, p = ns) or the number of interns who performed successful intubation (23 vs 22, p = ns). in the difficult airway scenario, the interns took longer to intubate with gvl than dl (92.3 ± 26.6 vs 59.9 ± 22.7 seconds, p = 0.008), but there were no differences in the number of successful intubations (17 vs 19, p = ns). interns rated their satisfaction higher for gvl than dl (7.3 ± 1.8 vs 6.5 ± 1.5, p = 0.05) and gvl was chosen as the preferred device for their next intubation by a majority of the interns (19/29, 66%). conclusion: for novice clinicians, gvl does not improve the time to intubation or intubation success objectives: to determine the time to intubation, the number of attempts, and the occurrence of hypoxia, in patients intubated with a c-mac device versus those intubated using a standard laryngoscope. methods: randomized controlled trial using exception from informed consent that included patients undergoing endotracheal intubation with a standard laryngoscope at an urban level i trauma center. eligible patients were randomized to undergo intubation using the c-mac or standard laryngoscopy. standard laryngoscopy was performed using a c-mac device laryngoscope with the video output obstructed to ensure equivalent laryngoscope blades in the two groups. data were collected by a trained research assistant at the patient's bedside and video review by the investigators. the number of attempts made, the initial and lowest oxygen saturation (spo 2 ), and the total time until the intubation was successful was recorded. hypoxia was defined as an oxygen saturation <93%. data were compared with wilcoxon rank sum and chi-square tests. results: thirty-eight patients were enrolled, 20 (70% male, median age 58, range 28 to 86, median spo 2 97%, range 79 to 100) in the standard laryngoscopy group and 18 (67% male, median age 58, range 19 to 73, median spo 2 96.5%, range 78 to 100) in the c-mac group. the median number of attempts for standard laryngoscopy was 1, range 1 to 3, and for c-mac was 1, range 1 to 2 (p = 0.43). the median time to intubation for the standard laryngoscopy group was 54 seconds (range 7 to 89) and for the c-mac group was 41 seconds (range 4 to 101)(p = 0.05). hypoxia was detected in 5/20 (20%) in the standard laryngoscopy group and 1/18 (6%) in the c-mac group (p = 0.15). the median decrease in oxygen saturation during the attempt was 5.4% (range 0% to 31%) for the standard laryngoscopy group and 2.3% (range 0% to 16%) for the c-mac group. conclusion: we did not detect a difference in number of attempts, the occurrence of hypoxia, or the diagnosis of aspiration pneumonia between standard laryngoscopy and the c-mac. the time to successful intubation was shorter for patients intubated with the c-mac. the c-mac device appears to be superior to standard laryngoscopy for emergent endotracheal intubation. (originally submitted as a ''late-breaker.'') the background: aspiration pneumonia is a complication of endotracheal intubation that may be related to the difficulty of the airway procedure. objectives: to determine the association of the device used, the time to intubation, the number of attempts to intubate, and the occurrence of hypoxia with the subsequent development of aspiration pneumonia. methods: this was a prospective observational study of patients undergoing endotracheal intubation by emergency physicians at an urban level i trauma center conducted from 7/1/2010 until 11/1/2011. the device used on the initial attempt to intubate was at the discretion of the treating physician. data were collected by a trained research assistant at the patient's bedside. the device used, the number of attempts made to intubate, the lowest oxygen saturation during the attempt, and the total time until intubation was successfully accomplished were recorded. patient's medical records were reviewed for the subsequent diagnosis of aspiration pneumonia. hypoxia was defined as an oxygen saturation <93%. data were analyzed using multinomial logistic regression and odds ratios (or). results: 654 patients were enrolled; 141 (22%) subsequently developed aspiration pneumonia. 328 were intubated with a standard laryngoscope (sl), 277 using the c-mac, 26 with an intubating laryngeal mask, and 23 with nasotracheal intubation (ni) (or 0.87, 95% ci = 0.70-1.06). comparison of individual devices versus sl did not show an association by device type. the median number of attempts for patients with aspiration pneumonia was 1, range 1 to 3, and for those without was 1, range 1 to 9 (or 0.78, 95%ci = 0.43-1.38). the median time to intubation for patients who developed aspiration pneumonia was 55 seconds (range 4 to 756) and for those who did not was 54 seconds (range 4 to 721)(or 1.00, 95%ci = 0.99-1.00). hypoxia during intubation was detected in 53/141 (38%) in the aspiration pneumonia group and 175/513 (34%) in the no aspiration pneumonia group (or 1.06, 95% ci = 0.65-1.72). conclusion: there was not an association between the device used, the number of attempts, the time to intubation, or the occurrence of hypoxia during the intubation, and the subsequent occurrence of aspiration pneumonia. background: japanese census data estimate that 35 million, or nearly 29% of the overall population, will be over age 65 by the year 2020. similar trends are apparent throughout the developed world. although increased patient age affects airway management, comprehensive information in emergency airway management for the elderly is lacking. objectives: we sought to characterize emergency department (ed) airway management for the elderly in japan including success rate, and major adverse events using a large multi-center registry. methods: design and setting: we conducted a multicenter prospective observational study using the japanese emergency airway network (jean) registry of eds at 11 academic and community hospitals in japan between 2010 and 2011 inclusive. data fields included ed characteristics, patient and operator demographics, methods of airway management, number of attempts, success rate, and adverse events. participants: patient inclusion criteria were all adult patients who underwent emergent tracheal intubation in the ed. primary analysis: patients were divided to into two groups defined as follows: 18 to 64 years old and over 65 years old. we describe primary success rates and major adverse events using simple descriptive statistics. categorical data are reported as proportions and 95% confidence intervals (cis). results: the database recorded 2710 patients (capture rate 98%) and 2623 met the inclusion criteria. of 2623 patients, 1104 patients were 18 to 64 years old (62%) and 1519 were over 65 years old (38%). the older group had a significantly higher success rate at first attempt intubation (1074/1519; 70.7%, 95% ci 68.8-72.6%) compared with the younger group (710/1104; 64.3%, 95% ci 61.9-66.7%). the older group had similar major adverse event rates (112/1519; 7.4%, 95% ci 6.3-8.5%) compared with the younger group (83/1104; 7.5%, 95% ci 6.2-8.8%). (see table 1) background: the degree to which a patient's report of pain is associated with changes in blood pressure, heart rate, and respiratory rate is not known. objectives: to determine to what degree a standardized painful stimulus effects a change in systolic blood pressure (sbp), diastolic blood pressure (dbp), heart rate (hr), or respiratory rate (rr), and compare changes in vital signs between patients based on pain severity. methods: prospective observational study of healthy human volunteers. subjects had their sbp, dbp, hr, and rr measured prior to pain exposure, immediately after, and 10 minutes after. pain exposure consisted of subjects placing their hand in a bath of 0 degree water for 45 seconds. the bath was divided into two sections; the larger half was the reservoir of cooled water monitored to be 0 degrees, the other half filled from constant overflow over the divider. water drained from this section into the cooling unit and was then pumped up into the base of the reservoir through a diffusion grid. subjects completed a 100 mm visual analog scale (vas) representing their perceived pain during the exposure and graded their pain as minimal, moderate or severe. data were compared using 95% confidence intervals. results: 90 subjects were enrolled, mean pain vas 40 mm, range 0 to 77, 49 reported mild pain, 41 moderate pain, and 0 severe pain. the percent change from baseline in vital signs during the exposure and 10 minutes after are presented in the table. conclusion: there was a wide variety in reported pain among subjects exposed to a standard painful stimulus. there was a larger change in heart rate during the exposure among subjects who described a standardized painful exposure as moderate than in those who described it as severe. the small observed changes in blood pressure and respiratory rate seen during the exposure did not differ by pain report or persist after 10 minutes. background: vital signs are often used to validate intensity of pain. however, few studies have looked at the capacity of vital signs to estimate pain intensity, particularly in patients with a diagnosis that a majority of physicians would agree produce significant pain in the ed. objectives: to determine the association between pain intensity and vital signs in consecutive ed patients and in a sub-group of patients with diagnosis known to cause significant pain. methods: we performed a post-hoc analysis of prospectively acquired data in a cohort study done in an urban teaching hospital with computerized triage and nurses records. we included all consecutive ed adult patients ( ‡16 years old), who had any level of pain intensity measured during triage, from march 2008 to november 2010. the primary outcome was the mean heart rate, systolic and diastolic blood pressure for every pain intensity level from 1 to 10 on a verbal numerical scale. our secondary outcomes where the same but limited to patients with the following diagnosis: fracture, dislocation, and renal colic. we performed descriptive statistics, one-way and two-way anovas when appropriate. results: during our study period, 42,947 patients ‡16 years old where triaged with a pain intensity of at least 1/10 and 3939 had a diagnosis known to cause significant pain. 56.5% of patients were female, with a mean pain intensity of 6.8/10, mean age of 47.9 years (±19.3), and 22.3% were ‡65 years old. there was a statistically significant difference (p < 0.05) in mean heart rate, systolic and diastolic blood pressure for each level of pain intensity, ex: difference between 1/10 and 10/10 for mean heart rate was 3.9 beats per minutes, for systolic pressure was 4.0 mmhg and for diastolic 4.5 mmhg. results are similar for painful diagnosis: difference for mean heart rate was 0.3 beats per minutes, for systolic pressure was 6.5 mmhg and diastolic 8.8 mmhg. however, these differences are not clinically significant. conclusion: although our study is a post hoc analysis, pain intensity, heart rate, systolic and diastolic pressures during triage are usually reliable data and a prospective study would likely produce the same result. these vital signs cannot be used to estimate or validate pain intensity in the emergency department. 8% had a positive urine drug screen. logistic multivariate regressions analyses revealed the following factors to be significantly associated with the risk of having an abnormal head ct: association with seizure (p = 0.0072); length of time of loss of consciousness, ranging from none to 0-30 min to >30 min (p = 0.0013); alteration of consciousness (p = 0.00009); post-traumatic amnesia (p = 0.0132); alcohol intake prior to injury (p = 0,0003); and initial ed gcs (p = 0.0255). conclusion: in an emergency department cohort of patients with traumatic brain injury, symptoms including loss of or alteration in consciousness, seizure, post traumatic amnesia, and alcohol intake appear to be significantly associated with abnormal findings on head ct. these clinical findings on presentation may be useful in helping triage head injury patients in a busy emergency department, and can further define the need for urgent or emergent imaging in patients without clearly apparent injuries. background: the etiology of neurogenic shock is classically attributed to diminished peripheral vascular resistance (pvr) secondary to loss of sympathetic outflow to the peripheral vasculature. however, the sympathetic nervous system also controls other key elements of the cardiovascular system such as the heart and capacitance vessels and disruptions in their function could complicate the hemodynamic presentation. objectives: we sought to systematically examine the hemodynamic profiles of a series of trauma patients with neurogenic shock. methods: consecutive trauma patients with documented spinal cord injury complicated by clinical shock were enrolled. hemodynamic data including systolic and diastolic blood pressure, heart rate (hr), impedance-derived cardiac output, pre-ejection period (pep), left ventricular ejection time (lvet), and calculated systemic pvr were collected in the ed. data were normalized for body surface area and a validated integrated computer model of human physiology (guyton model) was used to analyze and categorize the hemodynamic profiles based on etiology of the hypotension using a systems analysis. correlation between markers of sympathetic outflow (hr, pep, lvet) and shock etiology category was examined. results: of 9 patients with traumatic neurogenic shock, the etiology of shock was decrease in pvr in 4 (45%; 95% ci 19 to 73%), loss of vascular capacitance in 3 (33%; 12 to 65%), and mixed peripheral resistance and capacitance responsible in 2 (22%; 6 to 55%). the markers of sympathetic outflow had no correlation to any of the elements in the patients' hemodynamic profiles. conclusion: neurogenic shock is often considered to have a specific well-characterized pathophysiology. results from this study suggest that neurogenic shock can have multiple mechanistic etiologies and represents a spectrum of hemodynamic profiles. this understanding is important for the treatment decisions made in the management of these patients. -year (2008-2010) , pre-post intervention study of trauma patients requiring massive blood transfusion was performed. we divided the population into two cohorts: a pre-protocol group (pre) which included trauma patients receiving mbt not aided by a protocol, and a post-protocol group (post) who underwent mbt via the mbtp. patient demographics, 24hour blood component totals, timing of blood component delivery, trauma injury severity score (iss), initial glasgow coma scale (gcs) score, trauma mechanism, and patient mortality data were collected and analyzed using fisher's exact tests, student's t-tests, and mann-whitney u tests. results: fifty-two patients were included for study. median times to delivery of first products were reduced for prbcs (4 minutes), ffp (16 minutes), and platelets (33 minutes) between the pre and post cohorts. median time to delivery of any subsequent blood product was significantly reduced (10 minutes) in the post cohort (p = 0.024). the median number of blood products delivered was increased by 5.5 units for prbcs, 4 units for ffp, 0.5 units for platelets, and 1 unit for cryoprecipitate after implementation of mbtp. the percentage of patients receiving higher blood product ratios (>3:1) was reduced between the pre and post cohorts for prbc to ffp (25% reduction) and prbc to platelet ratio groups (7 % reduction). despite improved transfusion timing and ratios, we found no significant difference in mortality (p = 0.129) between pre and post cohorts when we adjusted for injury severity. conclusion: protocolized delivery of massive blood transfusion might reduce time to product availability and delivery, though it is unclear how this affects patient mortality in all us trauma centers. background: burns are common injuries that can result in significant scarring leading to poor function and disfigurement. unlike mechanical injuries, burns often progress both in depth and size over the first few days after injury, possibly due to inflammation and oxidative stress. a major gap in the field of burns is the lack of an effective therapy that reduces burn injury progression. objectives: since mesenchymal stem cells (msc) have been shown to improve healing in several injury models, we hypothesized that species-specific msc would reduce injury progression in a rat comb burn model. methods: using a 150 gm brass comb preheated to 100 degrees celsius, we created four rectangular burns, separated by three unburned interspaces on both sides of the backs of male sprague-dawley rats (300 g). the interspaces represented the ischemic zones surround-ing the central necrotic core. left untreated, most of these interspaces become necrotic. in an attempt to reduce burn injury progression, 20 rats were randomized to tail vein injections of 1 ml rat-specific msc 10 6 cells/ml (n = 10) or normal saline (n = 10) 60 minutes after injury. tracking of the stem cells was attempted by injecting several rats with quantum dot-labeled msc. results: by four days post-injury, all of the interspaces in the control rats (54/54, 100%) became necrotic while in the experimental group, 29/48 (60%) of the interspaces became necrotic (fisher's exact test; p < 0.001). at 7 days, the percentage of the unburned interspaces that became necrotic in the msc treated group was significantly less than in the control group (80% vs. 100%, p < 0.0001). we were unable to identify any quantum dot labeled msc in the injured skin. no adverse reactions or wound infections were noted in rats injected with msc. conclusion: intravenous injection of rat msc reduced burn injury progression in a rat comb burn model. although basic demographics of bicyclists in accidents have been described, there is a paucity of data describing the street surface involved in accidents, and whether designated bicycle roadways offer protection. this lack of information limits informed attempts to change infrastructure in a way that will decrease morbidity and/or mortality of cyclists. objectives: to identify road surface types involved in pedal cyclist injuries and determine the relationship between injury severity and the use of designated bicycle roadways (dbr) versus non-designated roadways (ndr). we hypothesized that more severe injuries would happen at intersections regardless of dbr versus ndr. methods: this retrospective cohort study reviewed the trauma database from a level i trauma center in tucson, az. we identified all bicyclists in the database injured in accidents involving a motor vehicle from january 1, 2009 1, through december 31, 2009 . the patients were then linked to a local government database that documents location (latitude/longitude) and direction of travel of the cyclist. seventy-eight total incidents were identified and categorized as occurring on a dbr versus ndr and occurring at an intersection versus not at an intersection. results: only one patient who arrived at the trauma center died. fifty-one of the accidents (65%) occurred on dbrs; 63% of accidents occurring on dbrs took place in intersections. conversely, 63% of accidents on ndrs occurred outside of intersections. the odds of an injury occurring at an intersection versus not at an intersection were 2.9 times higher (95% ci: 1.0-8.5) for dbrs compared to ndrs. the odds of a trauma being severe (admitted) versus not severe (discharged home) were 2.7 times higher (95% ci: 0.9-8.7) when a collision occurred not at an intersection versus at an intersection. conclusion: contrary to our hypothesis, in this study group severe injuries were more likely outside of an intersection. however, intersections on dbrs were identified as problematic as cyclists on a dbr were more likely to be injured in an intersection. future city planning could target improved cyclist safety in intersections. background: minor thoracic injury (mti) is frequent and a significant proportion will still have moderate to severe pain at 90 days. there is a lack of risk factors to orient specific treatment at ed discharge. objectives: to determine risk factors of having pain ( ‡3/10, on a numerical intensity pain score from 0 to 10) at 90 days in a population of minor thoracic injury patients discharged from the ed. methods: a prospective multi-center cohort study was conducted in four canadian eds, from november 2006 to january 2010. all consecutive patients, 16 years and older, with mti (with or without rib fracture), a normal chest x-ray, and discharged from the ed were eligible. a standardized clinical and radiological evaluation was done at 1 and 2 weeks. standardized phone interviews were done at 30 and 90 days. pain evaluation occurred at five time points (ed visit, 1 and 2 weeks, 30 and 90 days). using a pain trajectory model (sas), we planned to identify groups with different pain evolution at 90 days. the final model was based on the importance of difference in pain evolution, confidence intervals, and number of patients in each group. to judge the adequacy of the final model, we examined whether the posteriori probabilities (i.e., a participant's probability of belonging to a certain trajectory group) averaged at least 70% for each trajectory group. then using logistic multinomial regression and the low risk group of having pain as the control group, we identified significant predictors of patients in the moderate and high risk groups having pain at 90 days. results: in our cohort of 1,057 patients, 1,025 had an evaluation at 90 days. we identified three groups at low (34%), moderate (50.6%), and high risk (15.4%) of having pain ‡3/10 at 90 days. using risk factor identified by univariate analysis, we created a model to identify patients at risk containing the following predictors: age ‡ 30 years old, women, current smoker, two or more rib fractures, complaint of dyspnea, and saturation <95% at initial visit. posteriori probabilities for low, moderate, and high risk were 76%, 74%, and 88%. conclusion: to our knowledge, this is the first study to identify potential risk factor for having pain at 90 days after minor thoracic injury. these risk factors should be validated in a prospective study to guide specific treatment plan. the use of ultrasound to evaluate traumatic optic neuropathy benjamin burt, lisa montgomery, cynthia garza meissner, sanja plavsic-kupesic, nadah zafar ttuhsc -paul l foster school of medicine, el paso, tx background: whenever head trauma occurs, there is the possibility for a patient to have an optic nerve injury. the current method to evaluate optical nerve swelling is to look for proptosis. however, by the time proptosis presents, significant damage has already occurred. therefore, there is a need to establish a method to evaluate nerve injury prior to the development of proptosis. objectives: fundamental to understanding the pathophysiology of optic nerve injury and repair is an understanding of the optic nerve's temporal response to trauma including blood flow changes and vascular reactivity. the aim of our study was to assess the dependability and reproducibility of ultrasound techniques to sequence optic nerve healing and monitor the vascular response of the ophthalmic artery following an optic nerve crush. methods: the rat's orbit was imaged prior to and following a direct injury to the optic nerve, at 72 hours and at 28 days. 3d, 2d, and color doppler techniques were used to detect blood flow and the course of the ophthalmic artery and vein, to evaluate the course and diameter of the optic nerve, and to assess the extent of optic nerve trauma and swelling. the parameters used to evaluate healing over time were pulsatility and resistance indices of the ophthalmic artery. results: we have established baseline ultrasound measurements of the optic nerve diameter, normal resistance and pulsatility indices of the ophthalmic artery, and morphological assessment of the optic nerve in a rat model. longitudinal assessment of 2d and 3d ultrasound parameters were used to evaluate vascular response of the ophthalmic artery to optic nerve crush injury. we have developed a rat model system to study traumatic optic nerve injury. the main advantages of ultrasound are low cost, non-invasiveness, lack of ionizing radiation, and the potential to perform longitudinal studies. our preliminary data indicate that 2d and 3d color doppler ultrasound may be used for the evaluation of ophthalmic artery and total orbital perfusion following trauma. once baseline ultrasound and doppler measurements are defined there is the opportunity to translate the rat model to evaluate patients with head trauma who are at risk for optic nerve swelling and to assess the usefulness of treatment interventions. background: alcoholism is a chronic disease that affects an estimated 17.6 million american adults. a common presentation to the emergency department (ed) is a trauma patient with altered sensorium who is presumed to be alcohol intoxicated by the physicians based on their olfactory sense. often ed physicians may leave patients suspected of alcohol intoxication aside until the effects wear off, potentially missing major trauma as the source of confusion or disorientation. this practice often results in delays in diagnosing acute potentially life-threatening injuries in the patients with presumed alcohol intoxication. objectives: this study will determine the accuracy of physicians' olfactory sense for diagnosing alcohol intoxication. methods: patients suspected of major trauma in the ed underwent an evaluation by the examining physician for the odor of alcohol as well as other signs of intoxication. each patient had determination of blood alcohol level. alcohol intoxication was defined as a serum ethanol level ‡80 mg/dl. data were reported as means with 95% confidence intervals (95% ci) or proportions with inter-quartile ranges (iqr 25%-75%). results: one hundred and fifty one patients (70% males) were enrolled in the study, median age 45 years (iqr 33-56). the median score for glasgow coma scale was 15. the level of training of examining physician was a median of pgy 4 (iqr pgy 3 -attending). prevalence of alcohol intoxication was 43% (95% ci: 35% to 51%). operating characteristics: physician assessment of alcohol intoxication, sensitivity 84% (95% ci: 73% to 92%), specificity 87% (95% ci: 78% to 93%), positive likelihood ratio 6.6 (95% ci: 3.8 to 11.6), negative likelihood ratio 0.18 (95% ci: 0.1 to 0.3), and accuracy 86% (95% ci: 80% to 91%). patients who were falsely suspected of being intoxicated were 7.3% (95% ci: 4% to 13%). conclusion: although the physicians had a high degree of accuracy in identifying patients with alcohol intoxication based on their olfactory sense, they still falsely overestimated intoxication in a significant number of non-intoxicated trauma patients. the background: optimal methods for education and assessment in emergency and critical care ultrasound training for residents are not known. methods of assessment often rely on surrogate endpoints which do not assess the ability of the learner to perform the imaging and integrate the imaging into diagnostic and therapeutic decisions. we designed an educational strategy that combines asynchronous learning to teach imaging skills and interpretation with a standardized assessment tool using a novel ultrasound simulator to assess the learner's ability to acquire and interpret images in the setting of a standardized patient scenario. objectives: to assess the ability of emergency medicine and surgical residents to integrate and apply information and skills acquired in an asynchronous learning environment in order to identify pathology and prioritize relevant diagnoses using an advanced cardiac ultrasound simulator. methods: 12 em r2 residents and 12 r2 surgical residents completed an online focused training program in cardiac ultrasonography (iccu elearning, https:// www.caeiccu.com/lms). this consisted of approximately 14 hours of intensive training in cardiac ultrasound. residents were then given cases with a patient scenario that lacked significant details that would suggest a specific diagnosis. the resident was then given a list of 17 possible diagnoses and asked to rank the top five diagnoses in order of most likely to least likely. each resident (blinded to the pathology displayed by the simulator) then imaged using an ultrasound simulator. after imaging, the residents were given the same list of potential diagnoses, and asked to rank them again from 1-5. results: overall, residents ranked the correct diagnosis in the top five significantly more times post-ultrasound than pre-ultrasound. additionally, the residents made the correct diagnosis significantly more times postultrasound than pre-ultrasound. similar patterns occur for congestive heart failure, pericardial effusion with tamponade, and pleural effusion. there was no significant difference pre-and post-ultrasound for pulmonary embolism and anterior infarction. conclusion: an asynchronous online learning program significantly improves the ability of emergency medicine and surgical residents to correctly prioritize the correct diagnosis after imaging with a standardized pathology imaging simulator. mark favot, jacob manteuffel, david amponsah henry ford hospital, detroit, mi background: em clerkships are often the only opportunity medical students have to spend a significant amount of time caring for patients in the ed. it is imperative that students gain exposure to as many of the various fields within em as possible during this time. if the exposure of medical students to ultrasound is left to the discretion of the supervising physicians, we feel that many students would complete an em clerkship with limited skills and knowledge in ultrasound. the majority of medical students receive no formal training in ultrasound during medical school and we believe that the em clerkship is an excellent opportunity to fill this educational gap. objectives: evaluate the usefulness and effectiveness of a focused ultrasound curriculum for medical students in an em clerkship at a large, urban, academic medical center. methods: prospective cohort study of fourth year medical students doing an em clerkship. as part of the clerkship requirements, the students have a portion of the curriculum dedicated to the fast exam and ultrasound-guided vascular access. at the end of the month they take a written test, and 1 month later they are given a survey via e-mail regarding their ultrasound experience. em residents also completed the test to serve as a comparison group. all data analysis was done using sas 9.2. scores were integers ranging between 0 and 10. descriptive statistics are given as count, mean, standard deviation, median, minimum, and maximum for each group. due to non-gaussian nature of the data and small group sizes, a wilcoxon two-sample test was used to compare the distributions of scores between the groups. results: in the table, the distribution of scores was compared between the residents (controls) and the students (subjects). the mean and median scores of the student group were higher than those of the resident group. the difference in scores between the two groups was statistically significant (p = 0.021). conclusion: our data reveal that after completing an em clerkship with time devoted to learning ultrasound for the fast exam and vascular access, fourth year medical students are able to perform better than em residents on a written test. what remains to be determined is if their skills in image acquisition and in performance of ultrasound-guided vascular access procedures also exceed those of em residents. results: there were 106 respondents (total response rate 24.71%). compared to non-em students, students pursuing em (8 students, 7.55%) were more drawn to their specialty for work hour control (p < 0.0009) and shorter residency length (p < 0.0338). em students were less likely than non-em students to be drawn to their chosen specialty for future academic opportunities (p < 0.0085). em students formed their mentorships by referral significantly more than non-em students (p < 0.0399), though there was no statistical difference in quality of existing mentorships amongst students. of the 93 students not currently and never formerly interested in em, the most common response (25.8%) for why they did not choose em was the lack of a strong mentor in the field. conclusion: the results confirmed previous findings of lifestyle factors drawing students to em. future academic opportunities were less likely to draw students to em than students pursuing other specialties. lack of mentorship in the field was the most common reason given for why students did not consider em. given the lack of direct em exposure until late in the curriculum of most medical schools, mentorship may be particularly important for em and future study should focus on this area. background: misdiagnosis is a major public health problem. dizziness leads to 10 million visits annually in the us, including 2.6 million to the emergency department (ed). despite extensive ed workups, diagnostic accuracy remains poor, with at least 35% of strokes missed in those presenting with dizziness. ed physicians need and want support, particularly in the best method for diagnosis. strong evidence now indicates the bedside oculomotor exam is the best method of differentiating central from peripheral causes of dizziness. objectives: after a vertigo day that includes instruction in head impulse testing, emergency medicine residents will feel comfortable discharging a patient with signs of vestibular neuritis and a positive head impulse test without ordering a ct scan. methods: post graduate year 1-4 emergency medicine residents participated in a four hour vertigo day. we developed a mixed cognitive and systems intervention with three components: an online game that began and ended the day, a didactic taught by dr. newman-toker, and a series of small group exercises. the small group sessions included the following: a question and answer session with the lecturer; vertigo special tests (cerebellar assessment, dix hall-pike, epley maneuver); a head impulse hands-on tutorial using a mannequin; and a video lecture on other tests useful in vertigo evaluation (nystagmus, test of skew, vestibulocular reflex, ataxia). results: thirty emergency medicine residents were studied. before and after the intervention the residents were given a survey in which one question asked ''in a patient with acute vestibular syndrome and a history and exam compatible with vestibular neuritis, i would be willing to discharge the patient without neuroimaging based on an abnormal head impulse test result that i elicited''. resident answers were based on a sevenpoint likert scale from strongly agree to strongly disagree. twenty-five residents completed both surveys. of the seven residents who changed their responses pre to post,a significant proportion (100%) changed their answer from disagree/neutral to agree after a 4hour vertigo day (mcnemar's test, p value = 0.0082). conclusion: in this single-center study, teaching headimpulse testing as part of a vertigo day increases resident comfort with discharging a patient with vestibular neuritis without a ct scan. background: previous studies have been inconsistent in determining the effect of increased ed census on resident workload and productivity. we examined resident workload and productivity after the closure of a large urban ed near our facility, which resulted in a rapid 21% increase in our census. objectives: we hypothesized that the closure of a nearby hospital closure with a resulting influx of ed patients to our facility would not change resident productivity. methods: this computer-assisted retrospective study compared new patient workups per hour and patient load before and after the closure of a large nearby hospital. specifically, new patient workups per hour and the 4 pm patient census per resident were examined for a one-year period in the calendar year prior to the closing and also for one year after the closing. we did not include the four month period surrounding the closure in order to determine the long-term overall effect. background: emergency medicine residents use simulation for training due to multiple factors including the acuity of certain situations they are faced with, and the rarity of others. current training on highfidelity mannequin simulators is often critiqued by residents over the physical exam findings present, specifically the auscultatory findings. this detracts from the realism of the training, and may also lead a resident down a different diagnostic or therapeutic pathway. wireless remote programmed stethoscopes represent a new tool for simulation education which allows any sound to be wirelessly transmitted to a stethoscope receiver. objectives: our goal was to determine if a wireless remote programmed stethoscope was a useful adjunct in simulation-based cases using a high-fidelity mannequin. our hypothesis was that this would represent a useful adjunct in simulation education of emergency medicine residents. methods: starting june 2011, pgy1-3 emergency medicine residents were assessed in two simulation-based cases using pre-determined scoring anchors. an experimental randomized crossover design was used in which each resident performed a simulation case with and without a remote programmed stethoscope on a highfidelity mannequin. scoring anchors and surveys were used to collect data with differences of means calculated. results: fourteen residents participated in the study. residents noted most realistic physical exam findings associated with the case with the adjunct in 13/14 (93%) and that their preference was for the use of the adjunct in 13/14 (93%). based off of a five-point likert scale, with 5 being the most realistic, the adjunct-associated case averaged 4.4 as compared to 3.0 without (difference of means 1.4, p = 0.00017). average scores of residents with the adjunct were 2.5/3 with the use of the adjunct and 2.3/3 without (difference of means 0.2, p = 0.076). average total times were 28:49 with the adjunct as compared to 30:02 without. conclusion: a wireless remote programmed stethoscope is a useful adjunct in simulation training of emergency medicine residents. residents noted physical exam findings to be more realistic, preferred its use, and had approached significant improvement of scores when using the adjunct. background: prior studies predict an ongoing shortage of emergency physicians to staff the nation's eds, especially in rural areas. to address this, em organizations have discussed broadening access to acgme or aoa accredited em residency programs to physicians who previously trained in another specialty and focusing on physicians already practicing in rural areas. objectives: to investigate whether em program directors (pds) from allopathic and osteopathic residency programs would be willing to accept applicants previously trained in other specialties and whether this willingness is modified by applicants' current practice in rural areas. methods: a five-question web-based survey was sent to 200 u.s. em pds asking questions about their policies on accepting residents with past training and from rural practices. questions included whether a pd would accept a resident with prior training in other specialties, how many years from this training would the applicant be still a competitive candidate and if a physician was practicing in a rural region would the likelihood of acceptance to the program be improved. different characteristics of the residency programs were recorded including length of program, years in existence, size, type, and location of program. we compared responses by program characteristics using chi-square test. results: of the 96 (48%) pds responding to date, a large majority (87%) reported they do accept applicants with previous residency training, although directors of osteopathic programs were less likely to accept these applicants (56% vs 94% for allopathic; p < 0.001). overall, 28% of pds reported no limit on the length of time from prior training to when they are accepted at an em program. 73% reported it is very or possibly realistic they would accept a candidate who had completed training and was board certified in another specialty. a majority of all respondents (61%) felt a physician practicing in a rural setting might be viewed as a more favorable candidate, even if the resident would only be in the program for 2 years after receiving training credit. directors of newer programs (<5 years of existence) were more likely to view these candidates favorably than older programs (91% vs 53%; p = 0.02). conclusion: there appear to be many em residency programs that would at least review the application and consider accepting a candidate who trained in another specialty. a qualitative assessment of emergency medicine self-reported strengths todd guth university of colorado, aurora, co background: self-reflection has been touted as a useful way to assess the acgme core competencies. objectives: the purpose of this study is to gain insight into resident physician professional development through analysis of self-perceived strengths. a secondary purpose is to discover potential topics for selfreflective narrative essays relating to the acgme core competencies. methods: design: a small qualitative study was performed to explore the self-reported strengths of emergency medicine (em) residents in a single four-year residency. participants: all 54 residents regardless of year of training were also asked to report their selfperceived strengths. observations: residents were asked: ''what do you feel are your greatest strengths as a resident? provide a quick description.'' the author and another reviewer identified themes from within each year of residency with abraham maslow's conscious competence conceptual framework in mind. occurrences of each theme were counted by the reviewers and organized according to frequency. once the top ten themes for each year of residency were identified and exemplar quotes identified, the two reviewers identified trends. inter-rater agreements were calculated. results: representing unconscious incompetency, the first trend was the reported presence of ''enthusiasm and a positive attitude'' from residents early in their training that decreases further along in training. additionally, a ''willingness and motivation to improve and learn'' was reported as a strength throughout all the years of training but most frequently reported in the first two years of residency. entering into conscious incompetence, the second trend identified was ''recognition of limitations and openness to constructive feedback'' that was mentioned frequently in the second and third years of residency. demonstrating conscious competence, the third trend identified was the increase in identification of the strengths of ''educational leadership, teamwork skills and communication, and departmental patient flow and efficiency'' in the later years of residency. conclusion: self-reported strengths has helped to identify both themes within each year of residency and trends among the years of residency that can serve as areas to explore in self-reflective narratives relating to the acgme core competencies. training. pofu can also be used to assess the acgme core competency of practice-based learning. the exact form or frequency of pofu assessment among various em residencies, however, is not currently known. objectives: we aimed to survey em residencies across the country to determine how they fulfill the pofu requirement and whether certain program structure variables were associated with different pofu systems. we hypothesized that implementation of pofu systems among em residencies would be highly variable. methods: in this irb-approved study, all program directors of acgme allopathic em residencies were invited to complete a 10-question survey on their current approaches to pofu. respondents were asked to describe their current pofu system's characteristics and rate its ease of use, effectiveness, and efficiency. data were collected using surveymonkey(tm) and reported using descriptive statistics. results: of 158 residencies surveyed, 81 (51%) submitted complete data. 77.5% were completed by program directors and over three-fourths (76.1%) of em residencies require monthly completion of pofus. the mean total pofus required per year was 78 (95% ci 58-98), with a median of 64 and a range of 2-400. almost 2/3 (63%) of residencies use an electronic pofu system. most (84%) 4-year em residencies use an electronic pofu system, compared with half (54%) of 3-year residencies (difference 30%, p = 0.025, 95% ci 5.1%-47.2%). seven commercially available electronic programs are used by 71% of the residencies, while 29% use a customized product. most respondents (88%) rated their pofu system as easy to use, but less than half (49%) felt it was an effective learning tool or an efficient one (45%). onethird (34%) would use a different pofu system if available, and almost half (44%) would be interested in using a multi-residency pofu system. conclusion: em residency programs use many different strategies to fulfill the rrc requirement for pofu. the number of required pofus and the method of documentation vary considerably. about two-thirds of respondents use an electronic pofu system. less than half feel that pofu logs are an effective or efficient learning tool. background: certification of procedural competency is requisite to graduate medical education. however, little is known regarding which platforms are best suited for competency assessment. simulators offer several advantages as an assessment modality, but evidence is lacking regarding their use in this domain. furthermore, perception of an assessment environment has important influence on the quality of learning outcomes, and procedural skill assessment is ideally conducted on a platform accepted by the learner. objectives: to ascertain if a simulator performs as well as an unembalmed cadaver with regard to residents' perception of their ability to demonstrate procedural competency during ultrasound (us) guided internal jugular vein (ij) catheterization. methods: in this cross-sectional study at an urban community hospital during july of 2011, 15 residents in their second or third year of training from a 3-year em residency program performed us guided catheterizations of the ij on both an unembalmed cadaver and a simulator manufactured by blue phantom. after the procedure, residents completed an anonymous survey ascertaining how adequately each platform permitted their demonstration of proficiency on predefined procedural steps. answers were provided on a likert scale of 1 to 10, with 1 being poor and 10 being excellent. p values < 0.10 were considered educationally significant. results: the median overall rating of the simulator (s) to serve as an assessment platform was similar to that of the cadaver (c) with scores of 8.0 and 8.3 respectively, p = 0.89. median ratings for permitting the demonstration of specific procedural steps were as follows: conclusion: senior em residents positively rate the blue phantom simulator as an assessment platform and similarly to that of a cadaver with regard to permitting their demonstration of procedural competency for us guided ij catheterization, but did prefer the cadaver to a greater degree when identifying and guiding the needle into the ij. methods: in fall 2011, wcmc and wcmc-q students taking the course completed a 20 question pre-and post-test. wcmc-q students also completed a postcourse single-station objective structured clinical examination (osce) that evaluated their ability to identify and perform eight actions critical for a first responder in an emergency situation (table 1) . results: on both campuses, mean post-test scores were significantly higher than mean pre-test scores (p £ 0.001). on the pre-test, mean wcmc student scores were significantly higher than for wcmc-q students (p = 0.02); however, no difference was found in mean post-test scores (p = 0.895). there was no association between the scores on the osce (mean = 7.01, sd = 1.00) and the post-test (p = 0.683) even after adjusting for a possible evaluators' effect (table 2) . clinical skills course was effective in enhancing student knowledge in both qatar and new york as evidenced by the significant improvement in scores from the pre-to post-tests. the course was able to bring wcmc-q student scores and presumably knowledge up to the same level as wcmc students. students performed well on the osce, suggesting that the course was able to teach them the critical actions required of a first responder. the lack of association between the post-test and osce scores suggests that student knowledge does not independently predict ability to learn and demonstrate critical actions required of a first responder. future studies will evaluate whether the course affects the students' clinical practice. assess breathing 3 assess circulation 4 call ems 5 call ems and assess abcs prior to other interventions 6 immobilize 7 localize and control bleeding 8 splint fractured extremity and skills specific to wilderness medicine by incorporating simulated medical scenarios into a day-long adventure race. this event has gained acceptance nationally in wilderness medical circles as an excellent way to appreciate the challenges of wilderness medicine, however its effectiveness as a teaching tool has not yet been verified. objectives: the objective of this study was to determine if improvement in simulated clinical and didactic performance can be demonstrated by teams participating in a typical medwar event. methods: we developed a complex clinical scenario and written exam to test the basic tenets that are reinforced through the medwar curriculum. teams were administered the test and scored on a standardized scenario immediately before and after the 2011 midwest medwar race. teams were not given feedback on their pre-race performance. scenario performance was based on the number of critical actions correctly performed in the appropriate time frame. data from the scenario and written exams were analyzed using a standard paired difference t-test. results: a total of 31 teams participated in both the pre-and post-event scenarios. the teams' pre-race scenario performance was 71.0% (sd = 17.0, n = 31) of critical actions met compared to a post-race performance of 89.7 % (sd = 11.4, n = 31). the mean improvement was 18.7% (sd = 18.7, n = 31, 95% ci 12. 1, 25. 3) with a significant paired two-tailed t-test (p £ 0.01). a total of 95 individual subjects took the written pre-and posttests. the written scores averaged pre-race 84.5% (sd = 12.5, n = 95) and post-race 88.7% (sd = 11.5, n = 95). the mean improvement was 4.2% (sd = 11.7, n = 95, ci )7.5, 15.9), with a significant paired twotailed t-test (p £ 0.01). conclusion: medwar participants demonstrated a significant improvement in both written exam scores and the management of a simulated complex wilderness medical scenario. this strongly suggests that medwar is an effective teaching platform for both wilderness medicine knowledge and skills. palliative methods: ed residents and faculty of an urban, tertiary care, level i trauma center were asked to complete an anonymous survey (6/2010-10/2011). participants ranked 22 statements on a five-point likert scale (1 = strongly disagree-5 = strongly agree). statements covered four main domains of barriers related to: 1) education/training, 2) communication, 3) ed environment; 4) personal beliefs. respondents were also asked if they would call pc consult for 15 ed clinical scenarios (based on established triggers). results: 30/45 (67%) eligible participants completed the survey (23 residents, 7 faculty), average age was 31 years, 52% (15/29) male, and 58% (15/26) caucasian. respondents identified two major barriers to ed-pc provision: lack of 24 hour availability of pc team (mean score 4.4) and lack of access to complete medical records (4.2). listed domain barriers included: communication-related issues (mean 3.3) like access to family or primary providers, ed environment (2.8) for example chaotic setting with time-constraints, education/training (2.7) related to pain/pc, and personal beliefs regarding end-of-life (2.5). all respondents agreed that they would call pc consult for a 'hospice patient in respiratory distress', and a majority (73%) would consult pc for 'massive intracranial hemorrhage, traumatic arrest, and metastatic cancer'. however, traditional in-patient triggers like frequent re-admits for organ failure issues (dementia, congestive heart failure, and obstructive pulmonary disease exacerbations) were infrequently (10%) chosen for pc consult. conclusion: to enhance pc provision in the ed setting, two main ed physician perceived barriers will likely need to be addressed: lack of access to medical records and lack of 24-7 availability of pc team. ed physicians may not use the same criteria to initiate pc consults as compared to the traditionally established inpatient pc consult trigger models. percent of charts with an mse by ait prior to resident evaluation (a measure of reduced diagnostic uncertainty and decision-making), (4) ed volume. results: there were no educationally significant differences in productivity or acuity between the pre-ait and post-ait groups. mse was recorded in the chart prior to resident evaluation in 10.9% of cases. ed volume rose by 9.0% between periods. conclusion: ait did not affect productivity or acuity of patients seen by em2s. while some volume was directed away from residents by ait (patients treated-andreleased by ait only), overall volume increased and made up the difference. this is similar to previously reported rankings that program directors gave to the same criteria. although medical students agreed with program directors on the importance of most aspects of the nrmp application areas of discordance included higher medical student ranking for extracurricular activities and a lower relative ranking for aoa status than program directors. this can have implications for medical student mentoring and advising in the future. background: emergency care of older adults requires specialized knowledge of their unique physiology, atypical presentations, and care transitions. older adults often require distinctive assessment, treatment and disposition. emergency medicine (em) residents should develop expertise and efficiency in geriatric care. older adults represent over 25% of most emergency department (ed) volumes. yet many em residencies lack curricula or assessment tools for competent geriatric care. the geriatric emergency medicine competencies (gemc) are high-impact geriatric topics developed to help residencies meet this demand. objectives: to examine the effect of a brief gemc educational intervention on em resident knowledge. methods: a validated 29-question didactic test was administered at six em residencies before and after a gemc focused lecture delivered summer and fall of 2009. scores were analyzed as individual questions and in defined topic domains using a paired student's t-test. results: a total of 301 exams were included. the testing of didactic knowledge before and after the gemc educational intervention had high internal reliability (87.9%). the intervention significantly improved scores in all domains (table 1) . graded increase in geriatric knowledge occurred by pgy year with the greatest improvement seen at the pgy 3 level (table 2) . conclusion: even a brief gemc intervention had a significant effect on em resident knowledge of critical geriatric topics. a formal gemc curriculum should be considered in training em residents for the demands of an ageing population. the overall procedure experience of this incoming class was limited. most r1s had never received formal education in time management, conflict of interest management, or safe patient trade-off. the majority lacked confidence in their acute and chronic pain management skills. these entry level residents lacked foundational skill levels in many knowledge areas and procedures important to the practice of em. ideally medical school curricular offerings should address these gaps; in the interim, residency curricula should incorporate some or all of these components essential to physician practice and patient safety. background: the american heart association and international liaison committee on resuscitation recommend patients with return of spontaneous circulation following cardiac arrest undergo post-resuscitation therapeutic hypothermia. in post-cardiac arrest patients presenting with a rhythm of vf/vt, therapeutic hypothermia has been shown to reduce neurologic sequelae and decrease overall mortality. objectives: to explore clinical practice regarding the use of therapeutic hypothermia and compare survival outcomes in post-cardiac arrest patients. a secondary outcome was to assess whether the initial presenting cardiac arrest rhythm (ventricular fibrillation/ventricular tachycardia (vf/vt) versus pulseless electrical activity (pea) or asystole) was associated with differences in outcomes. methods: a retrospective medical record review was conducted for all adult ( ‡18 years) post-cardiac arrest patients admitted to the icu of an academic tertiary care centre (annual ed census 150,000) from 2006-2007. data were extracted using a standardized data collection tool by trained research personnel. results: 200 patients were enrolled. mean (sd) age was 66 (16) and 56.5% were male. of 58 (29.0%) patients treated with hypothermia, 27 (46.6%) presented with an initial rhythm of vf/vt and 31 (53.4%) presented with pea or asystole. nine (33.3%) patients with vf/vt were treated with therapeutic hypothermia and discharged from hospital compared to 2 (6.4%) patients with pea or asystole (d 26.9%; 95% ci: 6.4%, 46.3%). of 142 patients not treated with hypothermia, 37 (26.1%) presented with vf/vt, 93 (65.5%) presented with pea or asystole, and 12 (8.4%) initial rhythms were unknown. fifteen (40.5%) patients with vf/vt, not treated with hypothermia, were discharged from hospital compared to 13 (13.9%) patients with pea or asystole (d 26.6%; 95% ci: 10.0%, 43.5%). regardless of initial presenting rhythm or initiation of therapeutic hypothermia, 37 (88.1%) discharged patients had good neurological function as assessed by the cerebral performance category (cpc score 1-2). conclusion: although recommended, post-cardiac arrest therapeutic hypothermia was not routinely used. patients with vf/vt and treated with hypothermia had better outcomes than those with pea or asystole. further research is needed to assess whether cooling patients with presenting rhtyhms of pea or asystole is warranted. racial background: chronic obstructive pulmonary disease (copd) is a major public health problem in many countries.the course of the disease is characterised by episodes, known as acute exacerbations (ae), when symptoms of cough, sputum production, and breathlessness become much worse. the standard prehospital management of patients suffering from an aecopd includes oxygen therapy, nebulised bronchodilators, and corticosteroids. high flow oxygen is used routinely in prehospital areas for breathless patients with copd. there is little high quality evidence on the benefits or potential dangers in this setting but audits have shown increased mortality, acidosis, and hypercarbia in patients with aecopd treated with high flow oxygen. objectives: to compare standard high flow oxygen treatment with titrated oxygen treatment for patients with an aecopd in the prehospital setting. methods: cluster randomized controlled parallel group trial comparing high flow oxygen treatment with titrated oxygen treatment in the prehospital setting. in an intention to treat analysis (n = 405), the risk of death was significantly lower in the titrated oxygen arm compared with the high flow oxygen arm for all patients and for the subgroup of patients with confirmed copd (n = 214). overall mortality was 9% (21 deaths) in the high flow oxygen arm compared with 4% (7 deaths) in the titrated oxygen arm; mortality in the subgroup with confirmed copd was 9% (11 deaths) in the high flow arm compared with 2% (2 deaths) in the titrated oxygen arm. titrated oxygen treatment reduced mortality compared with high flow oxygen by 58% for all patients (p = 0.02) and by 78% for the patients with confirmed chronic obstructive pulmonary disease (p = 0.04). patients with copd who received titrated oxygen according to the protocol were significantly less likely to have respiratory acidosis or hypercapnia than were patients who received high flow oxygen. conclusion: titrated oxygen treatment significantly reduced mortality, hypercapnia, and respiratory acidosis compared with high flow oxygen in aecopd. these results provide strong evidence to recommend the routine use of titrated oxygen treatment in patients with breathlessness and a history or clinical likelihood of copd in the prehospital setting. (originally submitted as a ''late-breaker.'') trial registration australian new zealand clinical trials register actrn12609000236291. background: toxic particulates and gases found in ambulance exhaust are associated with acute and chronic health risks. the presence of such materials in areas proximate to ed ambulance parking bays, where emergency services' vehicles are often left running, is potentially of significant concern to ed patients and staff. objectives: investigators aimed to determine whether the presence of ambulances correlated with ambient particulate matter concentrations and toxic gas levels at the study site ed. methods: the ambulance exhaust toxicity in healthcare-related exposure and risk [aether] program conducted a prospective observational study at an academic urban ed / level i trauma center. environmental ambient gas was sampled over a continuous five-week period from september to october 2011. two sampling locations in the public triage area (public patient dropoff area without ambulances) and three sampling locations in the ambulance triage area were randomized for 24-hour monitoring windows with a temporal resolution of 2 minutes to obtain 7 days of non-contiguous data for each location. concentrations of particulate matter less than 2.5 microns in aerodynamic size (pm2.5), oxygen, hydrogen sulfide (h 2 s), and carbon monoxide (co) as well as lower explosive limit for methane (lel) were monitored with professionally calibrated devices. ambulance traffic was recorded through offline review of 24/7 security video footage of the site's ambulance bays. results: 4,118 measurements at the public triage nurse desk space revealed pm2.5 concentrations with a mean of 21.32 ± 27.01 lg/m 3 (median 15.95 lg/m 3 ; maximum 1,152.58 lg/m 3 ). 4,867 ambulance triage nurse desk space pm2.5 concentrations recorded a mean of 60.45 ± 53.38 lg/m 3 (p < 0.0001, unpaired t test; median 43.37 lg/m 3 ; maximum 580.78 lg/m 3 ). oxygen levels remained steady throughout the study period; co, h 2 s, and lel were not detected. ambulance activity levels had the highest correlations with pm2.5 concentrations at the ambulance triage foyer (r = 0.47) and desk area (r = 0.42) where patients wait and ed staff work 8-12 hr shifts. conclusion: ed spaces proximate to ambulance parking bays had higher levels of pm2.5 than areas without ambulance traffic. concentrations of ambient particulate matter in acute care environments may pose a significant health threat to patients and staff. an ems ''pit crew'' model improves ekg and stemi recognition times in simulated prehospital chest pain patients sara y. baker 1 , salvatore silvestri 1 , christopher d. vu 1 , george a. ralls 1 , christopher l. hunter 1 , zack weagraff 2 , linda papa 1 1 orlando regional medical center, orlando, fl; 2 florida state university college of medicine, orlando, fl background: prehospital teams must minimize time to ekg acquisition and stemi recognition to reduce overall time from first medical contact to reperfusion. auto-racing ''pit crews'' model rapid task completion by pre-assigning roles to team members. objectives: we compared time-to-completion of key tasks during chest pain evaluation in ems teams with and without pre-assigned roles. we hypothesized that ems teams using the ''pit crew'' model would improve time to recognition and treatment of stemi patients. methods: a randomized, controlled trial of paramedic students was conducted over 2 months at orlando medical institute, a state-approved paramedic training center. we compared a standard ems chest pain management algorithm (control) with a pre-assigned tasks (''pit crew'') algorithm (intervention) in the evaluation of simulated chest pain patients. students were randomized into groups of three; intervention and control groups did not interact after randomization. all students reviewed basic prehospital chest pain management and either the standard or pre-assigned tasks algorithm. groups encountered three simulated patients. laerdal simmanò software was used track completion of tasks: taking vital signs, iv access, ekg acquisition and interpretation, asa administration, hospital stemi notification, and total time on scene. results: we conducted 54 simulated-patient encounters (30 control / 24 intervention encounters). mean time-to-completion of each task was compared in the control and intervention groups respectively. time to obtain vital signs was 4:18 vs. 2:21 min (p = 0.001); time to asa administration was 3:54 vs 2:00 min (p < 0.001); time to ekg acquisition was 5:39 vs 3:42 min (p < 0.001); time to ekg interpretation was 6:43 vs 4:21 min (p < 0.001); time to iv access was 5:42 vs 4:45 min (p = 0.05); time to stemi notification was 7:19 vs 4:26 min (p < 0.001); and time to scene completion was 9:02 vs 5:27 min (p < 0.001). conclusion: paramedic student teams with pre-assigned roles (the ''pit crew'' model) were faster to obtain vital signs, administer asa, acquire and interpret the ekg, stemi notification, and overall time on scene during simulated patient encounters. further study with experienced ems teams in actual patient encounters is necessary to confirm the relevance of these findings. background: use of automated external defibrillators (aed) has remained low in the u.s. understanding the effect of neighborhoods on the probability of having an aed used in the setting of a public arrest may provide important insights for future placement of aeds. objectives: to determine associations between the racial and income composition of neighborhoods (as defined by u.s. census tracts), individual arrest characteristics, and whether bystanders or first responders initiate aed use. methods: cohort study using surveillance data prospectively submitted by emergency medical services systems and hospitals from 29 u.s. sites to the cardiac arrest registry to enhance survival between october 1, 2005 and december 31, 2009 . neighborhoods were defined as high-income vs. low-income based on the median household income being above or below $50,000 and as white or black if >90% of the census tract was of one race. neighborhoods without a predominant racial composition were defined as integrated. arrests that occurred within a public location (excluding medical facilities and airports) were eligible for inclusion. hierarchical multi-level modeling, using stata v11.0, was used to determine the association between individual and census tract characteristics on whether an aed was used. results: of 2,769 eligible cases, an aed was used in 1336 arrests (48.2%) by a first responder (n = 1,127, 40.8%) or bystander (n = 209, 7.5%). patients whose arrest was witnessed (odds ratio [or] 1.26; 95% confidence interval [ci] 1.06-1.50) were more likely to have an aed used (table) . when compared to high-income white neighborhoods, arrest victims in low-income black neighborhoods were least likely to have an aed used (or 0.54; 95% ci 0.33-0.87). arrest victims in lowincome white (or 0.57; 95% ci 0.32-1.02) and lowincome integrated (or 0.70; 95% ci 0.51-0.96) were also less likely to have an aed used. conclusion: arrest victims in black and low-income neighborhoods are least likely to have an aed used by a layperson or first responder. future research is needed to better understand the reasons for low rates of aed use for cardiac arrests in these neighborhoods. the impact of an educational intervention on the pre-shock pause interval among patients experiencing an out-of-hospital cardiac arrest jonathan studnek 1 , eric hawkins 1 , steven vandeventer 2 1 carolinas medical center, charlotte, nc; 2 mecklenburg ems agency, charlotte, nc background: pre-shock pause duration has been associated with survival to hospital discharge (std) among patients experiencing out-of-hospital cardiac arrest (oohca) resuscitation. recent research has demonstrated that for every 5-second increase in this interval there is an 18% decrease in std. objectives: determine if a decrease in the pre-shock pause interval for patients experiencing oohca could be realized after implementation of an educational intervention. methods: this was a retrospective analysis of data obtained from a single als urban ems system from 1/1/2010 to 12/31/10 and 8/1/11 to 11/6/2011. in august 2011, an educational intervention was designed and delivered to approximately 150 paramedics emphasizing the importance of reducing the time off chest during cpr. specifically, the time period just prior to defibrillation was emphasized by having rescuers count every 20th compression and pre-charge the defibrillator on the 180th compression. in order to determine if this change resulted in process improvement, 12 months of data were assessed before and 3 months after the educational intervention. pre-shock pause was the outcome variable and was defined as the time period after compressions ceased until a shock was delivered. this interval was measured by a cpr feedback device connected to the defibrillator. inclusion criteria were adult patients who required at least one defibrillation and had the cpr feedback device connected during the defibrillation attempt. analysis was descriptive utilizing means and 95% ci as well as wilcoxon rank sum test to assess difference between the two time periods. results: in the pre-intervention period there were 117 patients who received 211 defibrillations compared to 30 patients receiving 71 defibrillations in the post-intervention phase. the mean duration of the pre-shock pause pre-intervention was 35 seconds (95% ci 20-50) while the post-intervention duration was 9 seconds (95% ci 7-12). the difference in pre-shock pause duration was statistically significant with p < 0.001. conclusion: these data indicate that after a simple educational intervention emphasizing decreasing time off chest prior to defibrillation the pre-shock pause duration decreased. future research must describe the sustainability of this intervention as well as the effects this process measure may have on outcomes such as survival to hospital discharge. background: the broselow tape (bt) has been used as a tool for estimating medication dosing in the emergency setting. the obesity trend has demonstrated a tendency towards insufficient pediatric weight estimations from the bt, and thus potential under-dosing of resuscitation medications. objectives: this study compared drug dosing based on the bt with dosing from a novel electronic tool (et) that accounts for provider estimation of body habitus. methods: data were obtained from a prospective convenience sample of children ages 1 to 8 years arriving to a pediatric emergency department. a clinician performed an assessment of body habitus (average/underweight, overweight, or obese), blinded to the patient's actual weight and parental weight estimate. parental estimate of weight and measured length and weight were collected. epinephrine dosing was calculated from the measured weight, the bt measurement, as well as from a smart-phone tool based on the measured length and clinician's estimate of body habitus, and a modified tool (mt) incorporating the parent estimate of habitus. the wilcoxson rank-sum test was used to compare median percent differences in dosing. results: one hundred children (mean age 3 years) were analyzed; 47% were overweight or obese. clinicians correctly identified children as overweight/obese 23% of time (ci 0.12-0.38). adding parent estimate of weight improved this to a sensitivity of 74% (ci 0.59-0.86). the median difference between the weight-based epinephrine dose and bt dose was 11%. for the et the median difference from the weight-based dose was 7% (p = 0.05 compared to the bt), and for the mt was 1.7% (p < 0.01 compared to the bt). when a clinically significant difference was defined as ±10% of the actual dose, bt was within that range 40% of the time, et was within range 56% of the time (p = 0.02), and mt was within range 64% of the time ( background: in most out-of-hospital cardiac arrest (ohca) events, a call to 9-1-1 is the first action by bystanders. accurate diagnosis of cardiac arrest by the call taker depends on the caller's verbal description. if cardiac arrest is not suspected, then no telephone cpr instructions will be given. objectives: we measured the effect of a change in the ems call taker question sequence on the accuracy of diagnosis of cardiac arrest by 9-1-1 call takers. methods: we retrospectively reviewed the cardiac arrest registry to enhance survival (cares) dataset for january 1, 2009 through june 30, 2011 from a city, population 750,000, with a longstanding telephone cpr program (apco). we included ohca cases of any age who were in arrest prior to the arrival of ems and for whom resuscitation was attempted. in early 2010, 9-1-1 call takers were taught to follow a revised telephone script that emphasized focused questions, assertive control of the caller, and provision of hands-only cpr instructions. the medical director personally explained the reasons for the changes, emphasizing the importance of assertive control of the caller and the comparative safety of chest compressions in patients not in cardiac arrest. beginning in 2010, call recordings were reviewed regularly with feedback to the call taker by the 9-1-1 center leadership. the main outcome measure was sensitivity of the 9-1-1 call taker in diagnosing cardiac arrest. bystander cpr was reported by ems crews attending the event. we compared 2009 with 2010 and 2011 using the v 2 test and odds ratios (or). results: there were 504 ohca cases in 2009, 457 cases in 2010, and 287 in the first half of 2011 (68/100,000 population). the mean age was 57 ± 21 years, and 27% of the events were witnessed. before the revision, 40% of ohca cases were identified by 9-1-1 dispatchers; and after the revised questioning sequence, 74% were identified (or 4.3, 95% ci 3.2-5.6). the false positive rate changed little (from 56/month to 72/month). the mean time to question callers was unchanged (53 vs 51 seconds). bystander cpr was performed in 37.3% of events in 2009, 39.2% in 2010, and 49.1% of events in 2011 (p < 0.001). conclusion: emphasis on scripted assessment improved sensitivity without loss of specificity in identifying ohca. with repeated feedback, it translated to an increase in victims receiving bystander cpr. in an out-of hospital cardiac arrest population confirmed by autopsy salvatore silvestri, christopher hunter, george ralls, linda papa orlando regional medical center, orlando, fl background: quantitative end-tidal carbon dioxide (etco 2 ) measurements (capnography) have consistently been shown to be more sensitive than qualitative (colorimetric) ones, and the reliability of capnography for assessing airway placement in low perfusion states has sometimes been questioned in the literature. objectives: this study examined the rate of capnographic waveform presence of an intubated out-of-hospital cardiac arrest cohort and its correlation to endotracheal tube location confirmed by autopsy. our hypothesis is that capnography is 100% accurate in determining endotracheal tube location, even in low perfusion states. methods: this cross-sectional study reviewed a detailed prehospital cardiac arrest database that regularly records information using the utstein style. in addition, the ems department quality manager routinely logs the presence of an alveolar (four-phase) capnographic waveform in this database. the study population included all cardiac arrest patients from january 1, 2009 through december 31, 2009 managed by a single ems agency in orange county, florida. patients were included if they had endotracheal intubation performed, had capnographic measurement obtained, failed to regain return of spontaneous circulation (rosc), and had an autopsy performed. the main outcome was the correlation of the presence of an alveolar waveform and the location of the ett at autopsy. results: during the study period, 921 cardiac arrests were recorded. of these, 263 had an advanced airway placed (ett or laryngeal tube airway), and no rosc. of the 263 advanced airway cases, 73 were managed with an ett. autopsies were performed on 30 of these patients and resulted in our study cohort. the location of the ett at autopsy was recorded on all 30 of these cases. capnographic waveforms were recorded in the field in all 30 of these study patients, and 100% of the tubes were located within the trachea at autopsy. the sensitivity of capnography in determining proper endotracheal tube location was 100% in this study. conclusion: in our study, the presence of a capnographic waveform was 100% reliable in confirming proper placement of endotracheal tubes placed in outof-hospital patients with poor perfusion states. results: over 60 variables were presented to the 34 ems medical directors responding (100% survey population captured). among the myriad of responses, 14 (42%) initiate cardiopulmonary resuscitation (cpr) at 30 compressions to 2 ventilations consistent with il-cor/aha guidelines. seven (21%) initiate continuous chest compressions from the start of cpr with no pause and interposed ventilations. nine (26%) begin chest compressions only during the first 2-3 minutes, with either passive oxygenation by oxygen mask (six; 18%) or no oxygen (three; 9%). airway management following non-invasive oxygenation and ventilation by primary endotracheal intubation occurs in 12 systems (35%), while six (18%) use supraglottic devices. fourteen (42%) allow paramedics to decide between endotracheal and supraglottic device placement. thirty systems (88%) utilize continuous waveform capnography. the initial approach to non-ems witnessed ventricular fibrillation is chest compression prior to first defibrillation in 30 systems (88%). eighteen systems (52%) escalate defibrillation energy settings, with four systems (12%) utilizing dual sequential defibrillation. twenty (59%) initiate therapeutic hypothermia in the field. conclusion: wide variability in ca care standards exists in america's largest urban ems systems in mid-2011, with many current practices promoting more continuity in chest compressions than specified in the 2010 ilcor/aha guidelines. endotracheal intubation, a past mainstay of ca airway management, is deemphasized in many systems. immediate defibrillation of non-ems witnessed ventricular fibrillation is uncommon. objectives: determine the out-of-hospital cardiac arrest survival in this area of puerto rico using the utstein method. methods: prospective observational cohort study of adult patients presenting with an out-of-hospital cardiac arrest to the upr hospital ed. study endpoints will be survival and neurologically intact survival at hospital discharge, 6 months, and 12 months. results: a total of 144 consecutive cardiac arrest events were analyzed for a period of 2 years. one-hundred fifteen events met criteria for primary cardiac etiology (79.86%). the average age for this group was 68.47 years. there were 45 female (39.13%) and 70 male (60.86%) participants. the average time to start cpr was 14.60 minutes. transportation to the ed was 71.3% by ems and 25.22% by private vehicle. a total of 68 events were witnessed (59.13%). the survival rate to hospital admission was 23.66%. the overall cardiac arrest survival was 9.30% and overall neurologically intact survival was 4.30%. neurologically intact survival at 6 and 12 months was 2.15%. the rate of bystander cpr in our population was 16.13% with a survival rate of 6.66%. conclusion: survival from out-of-hospital cardiac arrest in the area served by the upr hospital is low but comparable to other cities in the us as reported by the cdc cardiac arrest registry to enhance survival (cares). this low survival rate might be due to low bystander cpr rate and prolonged time to start cpr. background: hyperventilation has been directly correlated with increased mortality for out-of-hospital cpr. ems providers may hyperventilate patients at levels above national bls guidelines. real-time feedback devices, such as ventilation timers, have been shown to improve cpr ventilation rates towards bls standards. it remains unclear if the combination of a ventilation timer and pre-simulation instruction would influence overall ventilation rates and potentially reduce undesired hyperventilation. objectives: this study measured ventilation rates of standard cpr (and pre-instruction on effects of hyperventilation) compared to cpr with the use of a commercial ventilation timer (and pre-instruction on effects of hyperventilation). we propose that use of a ventilation timer, measuring and displaying to ems providers real-time ventilations delivered, will have no difference in ventilation rates when comparing these groups. methods: this prospective study placed ems providers into four groups: two controls measuring ventilation rates before (1a) and after instruction (1b) on the deleterious effects of hyperventilation, and a concurrent intervention pair with before (2a) and after instruction (2b), with the second pair measuring ventilation rates with a ventilation timer that provides immediate feedback on respirations given. ventilation rates were measured for a 60-second period after one minute of simulated cpr using mannequins. the control set without instruction (1a, n = 12) averaged 14.21 breaths (95% ci = 10.31-18.11) and with instruction (1b, n = 13) averaged 20.23 breaths (95% ci = 16.16-24.30). the intervention set without instruction (2a, n = 11) averaged 13.04 breaths (95% ci = 9.29-16.78) and with instruction (2b, n = 13) averaged 11.77 breaths (95% ci = 8.02-15.51). there was a significant improvement (p = 0.016) in ventilation rates with use of a ventilation timer (control group versus intervention group regardless of pre-instruction). there was no statistically significant difference between groups with respect to instruction alone (p = 0.223). conclusion: the use of a ventilation timer significantly reduced overall ventilation rates, providing care closer to bls guidelines. the addition of pre-simulation instruction added no significant benefit to reducing hyperventilation. background: in 2010, the american heart association (aha) recommended a compression rate of (roc) 100/ min and a depth of compressions (doc) at least 2 inches for effective cpr. as an educational tool for lay rescuers, the aha as adopted the catch phrase ''push hard, push fast''. objectives: in this irb-exempt study, we sought to determine if persons without formal cpr training could perform non-ventilated cpr as well as those who have been trained in the past or those currently certified. methods: a convenience sample of patrons of the new york state fair was asked to perform 2 minutes of hands-only cpr on a prestan pp-am-100m adult cpr manikin. these devices provide visual indicators of acceptable rate and depth of compressions. each subject was video recorded on a dell latitude 620 laptop computer with a logitech quick cam using logitech quick cam 8.4.6 for windows software. results: a total of 175 volunteers (74 male, 102 female) aged 16-68 years participated: 52 were never certified (nc) in cpr, 73 were previously certified (pc), and 50 were currently certified (cc). there was no difference in age across the groups. the cc group had a higher proportion of females (chi-square = 9.71, p < 0.008). cc volunteers sustained roc and doc for an average of 57.1 seconds as compared to an average of 18.5 seconds (pc) and 2.3 seconds (nc) respectively. (f = 27.8, p < 0.001). the cc maintained roc of closer to 100/ min (mean 111.6/min) when compared to the pc (mean 85.3/min) and nc (mean 86.0/min) groups (f = 14.7, p < 0.001). a higher proportion of volunteers of the cc group were able to perform adequate doc (chi-square = 11.2, p < 0.004), and hand placement (chisquare = 19.21, p < 0.001) when compared to the other two groups. conclusion: compared to the target roc and doc, none of the groups did well and only 14 subjects met target roc/doc. increased out-of-hospital cardiac arrest survivability due to lay rescuer intervention is only assured if cpr is effectively administered. the effect and benefit of maintaining formal cpr training and certification is clear. background: more than 300,000 out-of-hospital cardiac arrests (ohcas) occur annually in the united states (us). automated external defibrillators (aeds) are life-saving devices in public locations that can significantly improve survival. an estimated 1 million aeds have been sold in the us; however, little is known about whether locations of aeds match oh-cas. these data could help determine optimal placement of future aeds and targeted cpr/aed training to improve survival. objectives: we hypothesized that the majority (>50%) of aeds are not located in close proximity (200 feet) to the occurrence of cardiac arrests in a major metropolitan city. methods: this was a retrospective review of prospectively collected cardiac arrest data from philadelphia ems from january 1, 2008 until december 31, 2010. included were ohcas of presumed cardiac etiology in individuals 12 years of age or older. excluded were oh-cas of presumed traumatic etiology, cases where resuscitation was terminated at the scene, and those dead on arrival. aed locations in philadelphia were obtained from myheartmap, a database of installed and wallmounted aeds in pennsylvania. we used gis mapping software to visualize where ohcas occurred relative to where aeds were located and to determine the radius of ohcas to aeds. arrests within a 200, 400, and 600 foot radius of aeds were identified using the attribute location selection option in arcgis. the lengths of radii were estimated based on the average time it would take for a person to walk to and from an aed (200 feet2 minutes; 400 feet4 minutes; 600 feet6 minutes). results: we mapped 3,483 ohcas and 2,314 aeds in philadelphia county. ohcas occurred in males (55%; 1916/3483) and the mean age was 65.4 years. ventricular fibrillation occurred in 19% (662/3483). aeds were primarily located in schools/universities (30%), office buildings (22%), and residential buildings (4%). aeds were not identified within 200 feet in 93% (3,239) of ohcas, within 400 feet of 90% (3,135) of ohcas, and within 600 feet in 79% (2,752) of ohcas. the figure (large black circles) illustrates aed/ohca within 200 feet on the left and 600 feet on the right. conclusion: aeds were rarely close to the locations of ohcas, which may be a contributor to low cardiac arrest survival rates. innovative models to match aed availability with ohcas should be explored. (originally submitted as a ''late-breaker.'') potential background: early and frequent epinephrine administration is advocated by acls; however, epinephrine research has been conducted primarily with standard cpr (std). active compression-decompression cpr with an impedance threshold device (acd-cpr + itd) has become the standard of care for out of hospital cardiac arrest in our area. the hemodynamic effects of iv epinephrine under this technique are not known. objectives: to determine the hemodynamic effects of iv epinephrine in a swine model undergoing acd-cpr+itd. methods: six female swine (32 ± 1kg) were anesthetized, intubated, and mechanically ventilated. intracranial, thoracic aorta, and right atrial pressures were recorded via indwelling catheters. carotid blood flow (cbf) was recorded via doppler. etc0 2 , sp0 2 , and ekg were monitored. ventricular fibrillation was induced and went untreated for 6 minutes. three minutes each of standard cpr (std), std-cpr+itd, and acd-cpr+itd was preformed. at minute 9 of the resuscitation, 40 lg/kg of iv epinephrine was administered and acd-cpr+itd was continued for 1 minute. statistical analysis was performed with a paired t-test. results: aortic pressure and calculated cerebral and carotid perfusion pressures increased from std < std+itd < acd-cpr+itd (p £ 0.001). epinepherine administered during acd-cpr+itd signficantly increased mean aortic (29 ± 5vs42 ± 12, p = 0.01), cerebral (12 ± 5 vs 22 ± 10, p = 0.01), and coronary perfusion pressures (8 ± 7 vs 17 ± 4, p = 0.02); however, mean cbf and etco 2 decreased (respectively 29 ± 15 vs 14 ± 7.0, p = 0.03; 20 ± 7 vs 18 ± 6, p = 0.04). conclusion: the administration of epinepherine during acd-cpr+itd signficantly increased markers of macrocirculation, while significantly decreasing etco 2 , a proxy for organ perfusion. while the calculated cerebral perfusion pressures increased, the directly measured cbf decreased. this calls into question the ability of calculated perfusion pressures to accurately reflect blood flow and oxygen delivery to end organs. hypoxia background: during cardiac arrest most patients are placed on 100% oxygen with assisted ventilations. after return of spontaneous circulation (rosc), 100% oxygen is typically continued for an extended time. animal data suggest that immediate post-arrest titration of oxygen by pulse oximetry produces better neurocognitive/ histologic outcomes. recent human data suggest that arterial hyperoxia is associated with worse outcomes. objectives: to assess the relationship between hypoxia, normoxia, and hyperoxia post-arrest and outcomes in post-cardiac arrest patients treated with therapeutic hypothermia. methods: we conducted a retrospective chart review of 190 post-arrest patients admitted to an academic medical center between january, 2000 and december, 2007 who had arterial blood gases (abg) drawn after rosc. demographic variables were analyzed using anova and chi-square tests as appropriate. unadjusted logistic regression analyses were performed to assess the relationship between hypoxia (pao 2 < 60 mmhg), normoxia (60-300 mmhg), hyperoxia (>300 mmhg), and mortality. results: on first abg (190 patients), 37 (19.5%) were hypoxic, 92 (48.4%) normoxic, and 61 (32.1%) hyperoxic. the average age of the cohort was 62.8 years (no difference for hypoxic, normoxic, and hyperoxic patients). overall mortality was 70.5% (134/190). there were no significant differences between initial heart rate, systolic blood pressure, sex, race, or pre-arrest functional status. in-hospital mortality was significantly higher when the first abg demonstrated hypoxia (94.6%; 35/ 37) than for normoxia (68.5%; 63/92) or hyperoxia (59%; 36/61). in unadjusted logistic regression analysis of first pao 2 values, hyperoxia was not associated with increased mortality (or 0.7; 95% ci 0.3-1.4) but hypoxia was associated with increased mortality (or 6.1; 95% ci 1.4-27.5). conclusion: hypoxia but not hyperoxia on first abg was associated with mortality in a cohort of post-arrest patients. background: there are over 330,000 deaths due to cardiac arrest per year in the us. the aha recommends monitoring the quality of cpr primarily through the use of end tidal co 2 (etco 2 ). the level of etco 2 is significantly dependant on minute ventilation and altered by pressor and bicarbonate use. cerebral oximetry (cereox) uses near infrared spectroscopy to non-invasively measure oxygen saturation of the frontal lobes of the brain. cereox has been correlated with cerebral blood flow and jugular vein bulb saturations. objectives: the objective of this study is to compare the simultaneous measurement of etco 2 and cereox to investigate which monitoring method provides the best measure of cpr quality as defined by return of spontaneous circulation (rosc). methods: a prospective cohort of a convenient sample of patients using out-of-hospital and ed cardiac arrest from two large eds. patients were monitored simultaneously by etco 2 and cereox during cpr. patient demographics and arrest data were collected using the utstein criteria. all patients were monitored throughout the resuscitation efforts. rosc was defined as a palpable pulse and a measurable blood pressure for a minimum of thirty minutes. results: twenty two patients were enrolled with complete data sets; 27% of the subjects had rosc. average down time of rosc subjects was 12 minutes (sd ± 14.6) and 31 minutes (sd ± 17.8) for subjects without rosc. the inability to obtain a value of 30 either for etco 2 or cereox was 50% and 75% specific with an 80% and 100% npv respectively for predicting lack of rosc. obtaining a value of 30 either for etco 2 or cereox was 66% and 100% sensitive, respectively in identifying rosc. subjects with rosc had sustained values above 30 for 1.25 mins on cereox and 4.9 mins on etco 2 prior to rosc. the increase in values over a three minute period prior to rosc was 13.5 on cereox and 1.3 on etco 2 . conclusion: the inability to obtain a value of 30 on either the etco 2 or cereox strongly predicted lack of rosc. cereox provides a larger magnitude and closer temporal increase prior to rosc than etco 2 . attaining a value of 30 on cereox was more predictive of rosc than etco 2. an discrepancies due to communicating information to multiple listeners in a short amount of time. this creates a communication barrier not always apparent to practitioners. we examine the perceptions of ems and ed personnel on the transfer of care and its correlation to missing patient data. objectives: evaluate provider perception of information transfer by ems and ed personnel and compare this to an external observer's objective assessment. methods: this is a retrospective quality improvement program at an academic level i trauma center. transfers of medical and trauma patients from ems to ed personnel were attended by trained external observers, research associates (ra). ra recorded the data communicated: name, age, past medical history (pmh), allergies, medications, events, active problems, vital signs (vs), level of consciousness (loc), iv access, and treatments given. then, ems and ed staff rated their perception of transfer on a 1-10 rating scale. results: ra evaluated 448 patient transfers (268 medical and 180 trauma). transfer time did not differ, 4.05 minutes for medical (95% ci: 3.77-4.32), 3.92 minutes for trauma patients (95% ci: 3.53-4.31)(p = 0.57). missing data between the two groups also did not differ, except loc and treatment were missed more in medical transfers, while pmh was missed more in the trauma transfers. comparing the transfers with all vs present (67%, 300/448) and all vs missing (12%, 55/ 448), with all vs missing, there was no difference in perception of transfer for ems (9.6/10 vs present vs 9.4/10 vs absent) or ed staff (9.5/10 vs present, 9.4/10 vs absent). when all vital signs were missing, ra rated 69.1% of transfers as poor, whereas when all vs were present 80.8% of transfers were considered good. conclusion: ems and ed staff felt transfers of care were professional, teams were attentive, and had similar amounts of interruptions for both medical and trauma cases. their perception of transfer of care was similar even when key information was missing, although external observers rated a significant amount of transfers poorly. thus, ems and ed staffs were not able to evaluate their own performance in a transfer of care and external observers were found to be better evaluators of transfers of care. swati singh, john brown, prasanthi ramanujam ucsf, san francisco, ca background: ems transports a large number of psychiatric emergencies to emergency departments (ed) across the us. research on paramedic education related to behavioral emergencies is sparse, but based on expert opinion we know that gaps in paramedic knowledge and training exist. in our system, paramedics triage patients to medical, detoxification, and purely psychiatric destinations, so a paramedic's understanding of these emergencies directly affects the flow of patients in our eds. objectives: our objectives were to understand the gaps in current training and develop a targeted curriculum for field providers with a long term goal of appropriately recognizing and triaging subjects to the ed. methods: data were collected using a survey that was distributed during a paramedic association meeting in october 2011. subjects were excluded if they did not complete the survey. survey questions addressed demographics of paramedics, frequency of various psychiatric emergencies and their confidence in managing these emergencies. data were collated, analyzed, and presented as descriptive statistics. results: forty-nine surveys were distributed with a response rate of 82% (n = 40/49). of the respondents, 70% (n = 28) were male and 68% (n = 27) had at least five years experience. mood, thought, and cognitive disorders were the most frequently encountered presentations and 65% (n = 26) of respondents came across psychiatric emergencies multiple times a week. many respondents did not feel confident managing agitated delirium (n = 16, 40%), acute psychosis (n = 17, 43%), and intimate partner or elder abuse (n = 14, 35%). a third to a half of the respondents felt they have little or no training in chemical sedation (n = 18, 45%), verbal de-escalation (n = 14, 35%), and triaging patients (n = 21, 53%). conclusion: we identified a need for a revised curriculum on management of psychiatric emergencies. future steps will focus on development of a curriculum and change in knowledge after implementation of this curriculum. background: prehospital endotracheal intubation has long been a cornerstone of resuscitative efforts for critically ill or injured patients. paramedic airway management training will need to be modified due to the 2011 acc/aha guidelines to ensure maintenance of competency in overall management of airway emergencies. how best to modify the training of paramedics requires an understanding of current experience. objectives: the purpose of this report is to characterize the airway management expertise of experienced and non-experienced paramedics in a single ems system. methods: we retrospectively reviewed all prehospital intubations from an urban/suburban ambulance service (professional ambulance, inc.) over a five-year period (january 01, 2006 to december 31, 2010). characteristics of airway management by paramedics with 0-5 years of experience (group 1) were compared to those with greater than 5 years of experience (group 2). airway management was guided by massachusetts statewide treatment protocols governing direct laryngoscopy and all adjunctive approaches. attempts are characterized by laryngoscope blade passing the lips. difficult and failed airways were managed with extraglottic devices (egd) or needle cricothyroidotomy. we reviewed patient characteristics, intubation methods, rescue techniques, and adverse events. results: 150 patients required airway management: 120 (80%) were performed by group 1 and 30 (20%) were performed by group 2. group 1 was both faster to intubate (1.39 vs 1.83 attempts, p = 0.0035) and less likely to use a rescue device (19.1% vs 50.0%, p = 0.0009). both are equally likely to go directly to a rescue device (10% vs 10%, p = 1.0). all patients were successfully oxygenated and ventilated with either an endotracheal tube or egd. no surgical airways were performed and no patients died as a result of a failed airway. conclusion: while intubation success rates of paramedics with less than and greater than five years of experience are similar, less experienced paramedics use fewer attempts and are less likely to use a rescue device. both recognize difficult airways and go directly to rescue devices equally. this highlights difficulties faced maintaining competence. education requirements must be evaluated and redesigned to allow paramedics to maintain competence and emphasize airway management according to the latest resuscitation guidelines. how well do ems 9-1-1 protocols predict ed utilization for pediatric patients? stephanie j. fessler 1 , harold k. simon 1 , daniel a. hirsh 1 , michael colman 2 1 emory university, atlanta, ga; 2 grady health systems, atlanta, ga background: the use of emergency medical services (ems) for low-acuity pediatric problems has been well documented. however, it is unclear how accurately general ems dispatch protocols predict the subsequent ed utilization for these patients. objectives: to determine the ed resource utilization rate of pediatric patients categorized as low acuity by 9-1-1 dispatch protocols and then subsequently transferred to a children's hospital. methods: all transports for pediatric patients from the scene by a large urban general ems provider that were prioritized as low acuity by initial 9-1-1 dispatch protocols were identified. protocols were based on the national academy of medical priority dispatch system, v12. starting on jan 1, 2010, 100 consecutive cases of patients transported to three pediatric emergency departments (ped) of a large tertiary care pediatric health care system were reviewed. demographics, ped visit characteristics, resource utilization, and disposition were recorded. those patients who received meds other than po antipyretics, had labs other than a strep test, a radiology study, a procedure, or were not discharged home were categorized into the significant ed resource utilization group. results: 93% of the patients were african american and either had public insurance or self-pay (86%, 13% respectively). the median age was 11 months (4d-13yr). 54% were female. none of these low-acuity patients were upgraded by ems operators en route. upon arrival to the ped, 45% of transported patients were classified into the significant utilization group. six of the 100 total patients were admitted, including a 2 y/o requiring emergent intubation, an 8 m/o old with a broken cvl, a 6 y/o with sickle cell pain crisis, and a 2 y/o with altered mental status. the remainder of the significant resource utilization group consisted of children needing procedures, anti-emetics, narcotic pain control, labs, and xrays. conclusion: in this general ems 9-1-1 system, dispatch protocols for pediatric patients classified as low priority did poorly in predicting subsequent ed utilization with 45% requiring significant resources. further, ems operators did not recognize a critical child who needed emergent intervention. opportunity exists to refine general ems 9-1-1 protocols for children in order to more accurately define an ems priority status that better correlates with ultimate needs and resource utilization. the objectives: determine if there is an association between a patient's impression of the overall quality of care and his or her satisfaction with provided pain management. it was hypothesized that satisfaction with pain management would be significantly associated with a patient's impression of the overall quality of care. methods: this was a retrospective review of patient satisfaction survey data initially collected by an urban als ems agency from 1/1/2007 to 8/1/2010. participants were randomly selected from all patients transported proportional to their paramedic defined acuity; categorized as low, medium, or high with a goal of 100 interviews per month. the proportions of patients sampled from each acuity level were 25% low, 50% medium, and 25% high. patients were excluded if there was no telephone number recorded in the prehospital patient record or they were pronounced dead on scene. all satisfaction questions used a five-point likert scale with ratings from excellent to poor that were dichotomized for analysis as excellent or other. the outcome variable of interest was the patient's perception of the overall quality of care. the main independent variable had patients rate the staff who treated them at the scene on their helping to control or reduce their pain. demographic variables were assessed for potential confounding. results: there were 2,759 patients with complete data for the outcome and main independent variable with 45.0% male respondents and an average age of 54.1 (sd = 22.7). overall quality of care was rated excellent by 66.0% of patients while 59.1% rated their pain management as excellent. of patients who rated their pain management as excellent, 87.9% rated overall quality of care as excellent while only 34.2% of patients rated overall quality excellent if pain management was not excellent. when controlling for potential confounding variables, those patients who perceived their pain management to be excellent were 13.9 (95% ci 11.5-16.9) times more likely to rate their overall quality of care as excellent compared to those with non-excellent perceived pain management. conclusion: patients' perceptions of the overall quality of care were significantly associated with their perceptions of pain management. objectives: the purpose of this study is to determine whether ground-based paramedics could be taught and retain the skills necessary to successfully perform a cricothyrotomy. methods: this retrospective study was performed in a suburban county with a population of 160,000 and 21,000 ems calls per year. participants were groundbased paramedics in a local ems system who were taught wire-guided cricothyrotomy as part of a standardized paramedic educational update program. as part of the educational program, paramedics were taught wire-guided cricothyrotomy on a simulation model previously developed to train emergency medicine residents. after viewing an instructional video, the participants were allowed to practice using a 16step checklist. not all of these 16 steps were automatic failures. each paramedic was individually supervised performing a cricothyrotomy on the simulator until successful; a minimum of five simulations was required. retention was assessed using the same 16-step checklist during annual skills testing, after a minimum of 6 weeks to a maximum of 3 months posttraining. results: a total of 55 paramedics completed both the initial training and reassessment during the time period studied. during the initial training phase, 100% (55 of 55) of the paramedics were successful in performing all 16 steps of the wire-guided cricothyrotomy. during the retention phase 87.3% (48 of 55) retained the skills necessary to successfully perform the wire-guided cricothyrotomy. of the 16-step checklist, most steps were performed successfully by all the paramedics or missed by only 1 of the 55 paramedics. step #8, which involved removing the needle prior to advancing the airway device over the guidewire, was missed by 34.5% (19 of 55) of the participants. step #8 was not an automatic failure since most participants immediately self-corrected and completed the procedure successfully. conclusion: paramedics can be taught and can retain the skills necessary to successfully perform a wireguided cricothyrotomy on a simulator. future research is necessary to determine if paramedics can successfully transfer these skills to real patients. helicopter emergency medical services in background: netcare911 is one of the largest private providers of emergency air medical care in south africa. each hems (helicopter emergency medical service) crew is manned by a physician-paramedic team and is dispatched based on specific medical criteria, time to definitive care, and need for physician expertise. objectives: to describe the characteristics of net-care911 air medical evacuations in gauteng province and to analyze the role of physicians in patient care and effect on call times. methods: all patients transported by a netcare911 helicopter over a one year period from january -december 2008 were enrolled in the study. injury classifications, demographics, procedures, scene and flight times were collected retrospectively from run sheets. data were described by medians and interquartile intervals. results: a total of 386 patients were transported on 384 flights originating from the netcare911 gauteng helicopter base. ninety-two percent were traumarelated, with 74% resulting from motor vehicle accidents. physician expertise was listed 30% of the time as the indication for air medical response. a total of 105 advanced procedures were performed by physicians on 93 patients, including paralytic-assisted intubations, chest tube placement, and cardiac pacing. the median total call time was 46 minutes with 10 minutes spent on scene, compared with 54 and 24 minutes when advanced procedures were performed by hems (p < 0.001). conclusion: trauma accounts for an overwhelming majority of patients requiring emergency air medical transportation. advanced medical procedures were performed by physicians in nearly a quarter of the patients. there were significant differences in call times when advanced procedures were performed by hems. objectives: we sought to evaluate the level of awareness and adoption of the off-line protocol guidelines by utah ems agencies. methods: we surveyed all ems agencies in utah 18 months after protocol guideline release. medical directors, ems captains, or training coordinators completed a short phone survey regarding their knowledge of the emsc protocol guidelines, and whether their agency had adopted them. in particular, participants were asked about the pain protocol guideline and their management of pediatric pain. results: of the 186 agencies, 182 participated in the survey (98%). of those participating, 15 agencies (8%) were excluded from the analysis: 4 (2%) who only treat adults and 11 (6%) who do not participate in electronic data entry. of the remaining 171 agencies (94%), 155 (91%) were familiar with the utah emsc protocol guidelines; 116 agencies (68%) have either partially or fully adopted the protocol guidelines. 132 agencies (77%) were familiar with the pain treatment protocol guideline; 29 (17%) had adopted it; 34 (21%) planned to either partially or fully adopt the protocol. overall, 84 agencies (49%) had offline protocols allowing the administration of narcotics to children. of those, 49 (58%) had intranasal fentanyl as an available medication and delivery route. of the 84 agencies with offline protocols for pain, 77 (83%) reported familiarity with the emsc pain protocol guideline. conclusion: the creation and dissemination of statewide emsc protocol guidelines results in widespread awareness (91%) and to date 68% of agencies have adopted them. future investigation into factors associated with protocol adoption should be explored. background: intranasal (in) naloxone is safe and effective for the treatment of opioid overdose. while it has been extensively studied in the out-of-hospital environment in the hands of paramedics and lay people, we are unaware of any studies evaluating the safety and efficacy of in naloxone administration by bls providers. in recent years in naloxone has been added to the bls armamentarium; however, most services/states require an als unit be dispatched and attempt an intercept if in naloxone is administered by the bls providers. objectives: the purpose of this study is to evaluate the safety and effectiveness of bls-administered in naloxone in an urban environment. methods: retrospective cohort review as part of the ongoing qa process of all patients who had in naloxone administration by bls providers. the study was part of a special projects waiver by massachusetts oems from february 2011 through november 2011 in a busy urban tiered ems system in the metro-boston area. exclusion criteria: cardiac arrest. demographic information was collected, as well as vital signs, number of naloxone doses by bls, patient response to bls naloxone administration (clinical improvement in mental status and/or respiratory status), als intercept. descriptive statistics and confidence intervals are reported using microsoft excel and spss 17.0. results: fifty-six cases of bls-administered in naloxone were identified, and 2 were excluded as cardiac arrests. the included cases had a mean age of 38.8 years ±13.5 (range 16-82), and 74% (ci 60-85) were male. of the 54 included cases, 76% (ci 62-87) of patients responded to bls administration of naloxone. of the responders, 17% (ci 7-32) required two doses. there were 10 protocol violations representing 19% (ci 9.2-31.4) of the total administrations, however in 100% of these 10 protocol violations the patients had a positive response to the administration of in naloxone. seven of the protocol violations were patients who required a second 2 mg dose of naloxone. eleven cases did not have an als intercept; only 1 of these 11 patients did not respond to bls administration of naloxone. there were no identified adverse events. conclusion: bls providers safely and successfuly administered in naloxone achieving a response rate consistent with studies of als providers' administration of in naloxone. given the success rate of bls providers, it may be feasible for bls to manage responders without the aid of an als intercept. background: an estimated 20% of patients arriving by ambulance to the ed are in moderate to severe pain. however, the management of pain in the prehospital setting has been shown to be inadequate, and untreated pain may have negative consequences for patients. objectives: to determine if focused education on pediatric pain management and implementation of a pain management protocol improved the prehospital assessment and treatment of pain in adult patients. specifically, this study aimed to determine if documentation of pain scores and administration of morphine by ems personnel improved. methods: this was a retrospective before and after study conducted by reviewing a county-wide prehospital patient care database. the study population included all adult patients transported by ems between 01 february 2006 and 28 february 2010 with a working assessment of trauma or burn. ems patient care records were searched for documentation of pain scores and morphine administration 2 years before and 2 years after an intensive pediatric focused pain management education program and implementation of a pain management protocol. frequencies and 95% cis were determined for all patients meeting the inclusion criteria in the before and after time period and chisquare was used to compare frequencies between time periods. a secondary analysis was conducted using only subjects documented as meeting the protocol's treatment guidelines. results: 7,999 (10%) of 77,122 adult patients transported by ems during the study period met the inclusion criteria: 4,357 in the before and 3,642 in the after period. subject demographics were similar between the two periods. documentation of pain score did not change between the time periods ( background: there is a presumption that ambulance response times affect patient outcome. we sought to determine if shorter response times really make a difference in hospital outcomes. objectives: to determine if ambulance response time makes a difference in the outcomes of patients transported for two major trauma (motor vehicle crash injuries, penetrating trauma) and two major medical (difficulty breathing and chest pain complaints) emergencies. methods: this study was conducted in a metropolitan ems system serving a population total of 800,000 including urban and rural areas. cases were included if the private ems service was the first medical provider on scene, the case was priority 1, and the patient was 13 years and older. a 12-month time period was used for the data evaluation. four diagnoses were examined: motor vehicle crash injuries, penetrating trauma, difficulty breathing, and chest pain complaints. ambulance response times were assessed for each of the four different complaints. the patients' initial vital signs were assessed and the number of vital signs out of range was recorded. a sampling of all cases which went to the single major trauma center was selected for evaluation of hospital outcome. using this hospital sample, number of vital signs out of range were assessed as a surrogate marker indicating severity of hospital outcome. correlation coefficients were used to evaluate interactions between independent and outcome variables. results: of the 2164 cases we reviewed over the 12month period, we found that the ems service responded significantly faster to trauma complaints at 4.53 minutes (n = 254) than medical complaints at 5.92 minutes (n = 1910) . in the hospital sample of 587 cases, number of vital signs out of range were positively correlated with hospital days (r = 0.11), admits (r = 0.12), icu admits (r = 0.10), and deaths (r = 0.09), but not response times (r = (-)0.08). in the entire sample, there was no correlation between vital signs out of range and response times for any diagnosis (see figure) . conclusion: conclusions: based on our hospital sample which showed that number of vital signs out of range was a surrogate marker of worse hospital outcomes, we find that hospital outcomes are not related to initial response times. adverse effects following prehospital use of ketamine by paramedics eric ardeel baylor college of medicine, houston, tx background: ketamine is widely used across specialties as a dissociative agent to achieve sedation and analgesia. emergency medical services (ems) use ketamine to facilitate intubation and pain control, as well as to sedate acutely agitated patients. published studies of ems ketamine practice and effects are scarce. objectives: describe the incidence of adverse effects occurring after ketamine administration by paramedics treating under a single prehospital protocol. methods: a retrospective analysis was conducted of 98 consecutive patients receiving prehospital ketamine from paramedics in the suburban/rural ems system of montgomery county hospital district, texas between august 1, 2010 and october 25, 2011. ketamine administration indications were: need for rapid control of violent/agitated patients requiring treatment and transport; sedation and analgesia after trauma; facilitation of intubation and mechanical ventilation. ketamine administration contraindications were: equivalent ends achieved by less invasive means; hypertensive crisis; angina; signs of significantly elevated intracranial pressure; anticipated inability to support or control airway. all patients were included, regardless of indication for ketamine administration. data were abstracted from electronic patient care records and available continuous physiologic monitoring data, and analyzed for the presence of adverse effects as defined a priori in ''clinical practice guidelines for emergency department ketamine dissociative sedation: 2011 update.'' results: no patients were identified as experiencing adverse effects as defined by the referenced literature. ketamine was utilized most often for patients with the following nemsis provider's primary impression: 25 (26%) altered level of consciousness, 23 (23%) behavioral/psychiatric, 20 (20%) traumatic injury. overall, combativeness was associated with 64 (65%) patients. the mean age was 41 years (range 3-94 years) and 50 (51%) were male. the mean ketamine dose was 150 mg (range 25-500 mg) and twenty-four (24%) patients received multiple administrations. conclusion: in this patient population, our data indicate that prehospital ketamine use by ems paramedics, across all indications for administration, was safe. further study of ketamine's utility in ems is warranted. an background: rigorous evaluation of the effect of implementing nationally vetted evidence-based guidelines (ebgs) has been notoriously difficult in ems. specifically, human subjects issues and the health insurance portability and accountability act (hipaa) present major challenges to linking ems data with distal outcomes. objectives: to develop a model that addresses the human subjects and hipaa issues involved with evaluating the effect of implementing the traumatic brain injury (tbi) ebgs in a statewide ems system. methods: the excellence in prehospital injury care (epic) project is an nih-funded evaluation of the effect of implementing the ems tbi guidelines throughout arizona (ninds-1r01ns071049-01a1). to accomplish this, a partnership was developed between the arizona department of health services (adhs), the university of arizona, and more than 100 ems agencies that serve approximately 85% of the state's population. results: ebg implementation: implementation follows all routine regulatory processes for making changes in ems protocols. in arizona, the entire project must be carried out under the authority of the adhs director. evaluation: a before-after system design is used (randomization is not acceptable). hipaa: as an adhsapproved public health initiative, epic is exempt from hipaa, allowing sharing of protected health information between participating entities. for epic, the state attorney general provided official verification of hi-paa exemption, thus allowing direct linkage of ems and hospital data. irb: once epic was officially deemed a public health initiative, the university irb process was engaged. as an officially sanctioned public health project, epic was determined to not be human subjects research. this allows the project to implement and evaluate the effect of this initiative without requiring individual informed consent. conclusion: by utilizing an ems-public health-university partnership, the ethical and regulatory challenges related to evaluating implementation of new ebgs can be successfully overcome. the integration of the department of health, the attorney general, and the university irb can properly protect citizens while permitting efficient implementation and rigorous evaluation of the effect of ebgs. this novel approach may be useful as a model for evaluation of implementing ems ebgs in other states and large counties. (20.6%-58.1% by age) were transported to non-trauma centers. the most common reasons cited by ems for hospital selection were: patient preference (50.6%), closest facility (20.7%), and specialty center (15.2%). patient preference increased with age (p for trend 0.0001) and paralleled under-triage ( figure 1 ). iss ‡ 16 patients transported to non-trauma hospitals by patient request had lower unadjusted mortality (3.8%, 95%ci 1.9-5.8) than similar patients transported to trauma centers (11.8%, 95%ci 10.7-12.8) or transported for other reasons (12.6%, 95%ci 11.4-13.7) (figure 2) . under-triage appears to be influenced by patient preference and age. self-selection for transport to non-trauma centers may result in under-triaged patients with inherently better prognosis than triagepositive patients. background: only 25% of all out-of-hospital cardiac arrest (ohca) patients receive bystander cpr (cardiopulmonary resuscitation). the neighborhood in which an ohca occurs has significant influence on the likelihood of receiving bystander cpr. objectives: to utilize geographic information systems to identify ''high-risk'' neighborhoods, defined as census tracts with high incidence of ohca and low cpr prevalence. methods: design: secondary analysis of the cardiac arrest registry to enhance survival (cares) dataset for denver county, colorado. population: all consecutive adults (>18 years old) with ohca due to cardiac etiology from january 1, 2009 through december 31, 2010. data analysis: analyses were conducted in arc-gis. three spatial statistical methods were used: local morans i (lmi), getis-ord gi*(gi*), and spatial empirical bayes (seb) adjusted rates. census tracts with high incidence of ohca, as identified by all three spatial statistical methods, were then overlain with low bystander cpr census tracts, which were identified in at least two out of three statistical methods (lmi, gi*, or the lowest quartile of bystander cpr prevalence). overlapping census tracts identified with both high ohca incidence and low cpr prevalence were designated as ''highrisk''. results: a total of 728 arrests in 142 census tracts occurred during the study period, with 595 arrests included in final sample. events were excluded if they were unable to be geocoded (n = 41), outside denver county (n = 8), or occurred in a jail (n = 3), hospital/ physician's office (n = 7), or nursing home (n = 74). for high ohca incidence: lmi identified 29 census tracts, gi* identified 45 census tracts, and the seb method identified 28 census tracts. twenty-five census tracts were identified by all three methods. for low bystander cpr prevalence: lmi identified 9 census tracts, gi* identified 16 census tracts, and 101 census tracts were identified as being in the lowest quartile of cpr prevalence. twenty-four census tracts were identified by two of the three methods. two census tracts were identified as high-risk having both high ohca incidence and low cpr prevalence (figure) . high-risk census tract demographics as compared to denver county are shown in the table. conclusion: the two high-risk census tracts, comprised of minority and low-income populations, appear to be possible sites for targeted community-based cpr interventions. objectives: we sought to assess the accuracy and correlation of geographic information system (gis) derived transport time compared to actual ems transport time in ohca patients. methods: prospective, observational cohort analysis of ohca patients in vancouver, b.c., one of the sites of the resuscitation outcomes consortium (roc). a random sample from all of the ohca cases from 12/05 through 05/07 was selected for analysis from one site of the roc epistry. using gis, ems transport time was derived from reported latitude/longitude coordinates of the ohca event to the actual receiving hospital. this was calculated via the actual network distance using arcgis. this gis-derived time was then compared to the actual ems transport time (in minutes) using the wilcoxon signed rank test. scatter plot analysis of actual vs. gis times were created to evaluate the relationship between actual and calculated time. a linear regression model predicting actual ems transport time from the derived gis-time was also developed in order to examine the potential relationship between the two variables. differences in the relationship were also investigated based on time of the day to reflect varying traffic conditions. results: 641 cases were randomly selected for analysis. the median actual transport time was significantly longer than the median gis derived transport time (7.08 minutes vs. 5.50 minutes). scatter plot analysis did not reveal any significant correlation between actual and gis-based time. additionally, there was poor approximation of gis-based time and actual ems time (r 2 = 0.20) with no evidence of a significant linear relationship between the two. the poorest correlation of time was observed during the morning hours (07:00-09:00; r 2 = 0.02) while the strongest correlation was during the overnight hours (00:00-07:00; r 2 = 0.26). conclusion: gis derived time does not appear to correlate well with actual ems transport time of ohca patients. efforts should be made to accurately obtain actual ems transport times for ohca patients. objectives: we first sought to describe the incidence of ohca presenting to the ed. we then sought to determine the association between hospital characteristics and survival to hospital admission. methods: we identified patients with diagnoses of cardiac arrest or ventricular fibrillation (icd-9 427.5 or 427.41) in the 2007 nationwide emergency department sample, a nationally representative estimate of all ed admissions in the us. eds reporting ‡1 patient with ohca were included. our primary outcome was survival to hospital admission. we examined variability in hospital survival rate and also classified hospitals into high or low performers based on median survival rate. we used this dichotomous hospital level outcome to examine factors associated with survival to admission including hospital and patient demographics, ed volume, cardiac arrest volume, and cardiac catheterization availability. all unadjusted and adjusted analyses were performed using weighted statistics and logistic regressions. results: of the 966 hospitals, 949 (98.2%) were included. in total, 44,782 cases of cardiac arrest were identified, representing an estimated 203,331 cases nationally. overall ed ohca survival to hospital admission was 23.5% (iqr 0.1%, 29.4%) in adjusted analyses, increased survival to admission was seen in hospitals with teaching status (or 2.7, 95% ci 1.7-4.4, p < 0.001), annual ed visits ‡10,000 (or 3.9, 95% ci 2.5-6.1, p < 0.001), and pci capability (or 9.1, 95% ci 1.2-68.2, p = 0.032). in separate adjusted analyses including teaching status and pci capabilities, hospitals with >40 annual cardiac arrest cases (or 3.0, 95% ci 2.2-4.2, p < 0.001) were also shown to have improved survival (figure) . conclusion: ed volume, cardiac arrest volume, and pci capability were associated with improved survival to hospital admission in patients presenting to the ed after ohca. an improved understanding of the contribution of ed care to ohca survival may be useful in guiding the regionalization of cardiac arrest care. background: prior investigations have demonstrated regional differences in out-of-hospital cardiac arrest (ohca) outcomes, but none have evaluated survival variability by hospital within a single major us city. objectives: we hypothesized that 30-day survival from ohca would vary considerably among one city's receiving hospitals. methods: we performed a retrospective review of prospectively collected cardiac arrest data from a large, urban ems system. our population included all ohcas with a recorded social security number (which we used to determine 30-day survival through the social security death index) that were transported to a hospital between 1/1/2008 and 12/31/2010. we excluded traumatic arrests, pediatric arrests, and hospitals receiving less than 10 ohcas with social security numbers over the three-year study period. we examined the associa-tion between receiving hospital and 30-day survival. additional variables examined included: level i trauma center status, teaching hospital status, ohca volume, and whether post-arrest therapeutic hypothermia (th) protocols were in place in 2008. statistics were performed using chi-square tests and logistic regression. results: our study population comprised 550 arrest cases delivered to 18 unique hospitals with an overall 30-day survival of 14.4%. mean age was 69.0 (sd 16.2) years. males comprised 54.2% of the cohort; 53.3% of victims were black. thirty-day survival varied significantly among the hospitals, ranging from 4.8% to 35.0% (chi-square 32.3, p = 0.014). ohcas delivered to level i trauma centers were significantly more likely to survive (19.5% vs. 12.7%, p = 0.05), as were those delivered to hospitals known to offer post-arrest th (19.2% vs. 11.8%, p = 0.018). hospital teaching status and ohca volume were not associated with survival. conclusion: there was significant variability in ohca survival by hospital. patients were significantly more likely to survive if transported to a level i trauma center or hospital with post-arrest th protocols, suggesting a potential role for regionalization of ohca care. limiting our population to ohcas with recorded social security numbers reduced our power and may have introduced selection bias. further work will include survival data on the complete set of ohcas transported to hospitals during the three-year study period. background: traumatic brain injury is a leading cause of death and disability. previous studies suggest that prehospital intubation in patients with tbi may be associated with mortality. limited data exist comparing prehospital (ph) nasotracheal (nt), prehospital orotracheal (ot), and ed ot intubation and mortality following tbi. objectives: to estimate the associations between ph nt, ph ot, and ed ot intubation and in-hospital mortality in patients with moderate to severe tbi, with hypotheses that ph nt and ph ot intubation would be associated with increased mortality when compared to ed ot or no intubation. methods: an analysis using the denver health trauma registry, a prospectively collected database. consecutive adult trauma patients from 1995-2008 with moderate to severe tbi defined as head abbreviated injury scale (ais) scores of 2-5. structured chart abstraction by blinded physicians was used to collect demographics, injury and prehospital care characteristics, intubation status and timing, in-hospital mortality and survival time, and neurologic function at discharge. poor neurologic function was defined as cerebral performance category score of 3-5. multivariable logistic regression and survival analyses were performed, using multiple imputation for missing data. results: of the 3,517 patients, the median age was 38 (iqr 27-51) years. the median ph gcs was 14 (iqr 6-15), median injury severity score was 20 (iqr 13-29), and median head ais was 4 (iqr 3-5). ph nt occurred in 15.8%, ph ot in 9.5%, and ed ot in 17.4%, while mortality occurred in 17.5%. the 24-, 48-, and 72-hour survival analyses are outlined in the table. survival curves for ph nt, ph ot, and ed ot are demonstrated in the figure (p < 0.001) . conclusion: prehospital intubation in patients with moderate to severe tbi is associated with increased mortality. contrary to our initial hypothesis, there was also a significant association between ed intubation and mortality. these associations persisted despite survival time, and while adjusting for injury severity. background: sbdp150 is a breakdown product of the cytoskeletal protein alpha-ii-spectrin found in neurons and has been detected in severe tbi. objectives: this study examined whether early serum levels of sbdp150 could distinguish: 1) mild tbi from three control groups; 2) those with and without traumatic intracranial lesions on ct (+ct vs -ct); and 3) those having a neurosurgical intervention (+nsg vs -nsg) in mild and moderate tbi (mmtbi). methods: this prospective cohort study enrolled adult patients presenting to two level i trauma centers following mmtbi with blunt head trauma with loss of consciousness, amnesia, or disorientation and a gcs 9-15. control groups included uninjured controls and trauma controls presenting to the ed with orthopedic injuries or an mvc without tbi. mild tbi was defined as gcs 15 and moderate tbi as having a gcs <15. blood samples were obtained in all patients within 4 hours of injury and measured by elisa for sbdp150 (ng/ml). the main outcomes were: 1) the ability of sbdp150 to distinguish mild tbi from three control groups; 2) to distinguish +ct from -ct and; 3) to distinguish +nsg from -nsg. data were expressed as means with 95%ci, and performance was tested by roc curves (auc and 95%ci). results: there were 275 patients enrolled: 54 tbi patients (42 gcs 15, 12 gcs 9-14), 23 trauma controls (16 mvc controls and 7 orthopedic controls), and 198 uninjured controls. the mean age of tbi patients was 39 years (range 19-70) with 63% males. fourteen (14%) had a +ct and 9% had +nsg. mean serum sbdp150 levels were 0.764 (95%ci 0.561-0.968) in normal controls, 1.035 (0.091-2.291) in orthopedic controls, 1.209 (0.236-2.181 ) in mvc controls, 2.764 (1.700-3.827 ) in mild tbi with gcs 15, and 5.227 (0.837-9.617) in tbi with gcs 9-14 (p < 0.001). the auc for distinguishing mild tbi from both controls was 0.83 (95%ci 0.68-0.99). mean sbdp150 levels in patients with -ct versus +ct were 2.170 (1.340-3.000) and 6.797 (2.227-11.368) respectively (p < 0.001) with auc = 0.78 (95%ci 0.61-0.95). mean sbdp150 levels in patients with -nsg versus +nsg were 2.492 (1.391-3.593) and 6.867 (3.891-9.843) respectively (p < 0.001) with auc = 0.88 (95%ci 0.77-0.98). conclusion: serum sbdp150 levels were detectable in serum acutely after injury and were associated with measures of injury severity including ct lesions and neurosurgical intervention. further study is required to validate these findings before clinical application. utility of platelet background: pre-injury use of anti-platelet agents (e.g., clopidogrel and aspirin) is a risk factor for increased morbidity and mortality in patients with traumatic intracranial hemorrhage (tich). some investigators have recommended platelet transfusion to reverse the anti-platelet effects in tich. objectives: this evidence-based medicine review examines the evidence regarding the effect of platelet transfusion in emergency department (ed) patients with pre-injury anti-platelet use and tich on patientoriented outcomes. methods: the medline, embase, cochrane library, and other databases were searched. studies were selected for inclusion if they compared platelet transfusion to no platelet transfusion in the treatment of adult ed patients with pre-injury anti-platelet use and tich, and reported rates of mortality, neurocognitive function, or adverse effects as outcomes. we assessed the quality of the included studies using ''grading of recommendations assessment, development and evaluation'' (grade) criteria. categorical data are presented as percentages with 95% confidence interval (ci). relative risks (rr) are reported when clinically significant. results: five retrospective, registry-based studies were identified, which enrolled 635 patients cumulatively. based on standard criteria, three studies were of ''low'' quality evidence and two studies had ''very low'' qualities. one study reported higher in-hospital mortality in patients with platelet transfusion (ohm et al), another showed a lower mortality rate in patients receiving platelet transfusion (wong et al). three studies did not show any statistical difference in comparing mortality rates between the groups (table) . no studies reported intermediate-or long-term neurocognitive outcomes or adverse events. conclusion: five retrospective registry studies with suboptimal methodologies provide inadequate evidence to support the routine use of platelet transfusion in adult ed patients with pre-injury anti-platelet use and tich. abnormal levels of end-tidal carbon dioxide (etco 2 ) are associated with severity of injury in mild and moderate traumatic brain injury (mmtbi) linda papa 1 , artur pawlowicz 2 , carolina braga 1 , suzanne peterson 1 , salvatore silvestri 1 1 orlando regional medical center, orlando, fl; 2 university of central florida, orlando, fl background: capnography is a fast, non-invasive technique that is easily administered and accurately measures exhaled etco 2 concentration. etco 2 levels respond to changes in ventilation, perfusion, and metabolic state, all of which may be altered following tbi. objectives: this study examined the relationship between etco 2 levels and severity of tbi as measured by clinical indicators including glasgow coma scale (gcs) score, computerized tomography (ct) findings, requirement of neurosurgical intervention, and levels of a serum biomarkers of glial damage. methods: this prospective cohort study enrolled adult patients presenting to a level i trauma center following a mmtbi defined by blunt head trauma followed by loss of consciousness, amnesia, or disorientation and a gcs 9-15. etco 2 measurements were recorded from the prehospital and emergency department records and compared to indicators of tbi severity. results: of the 46 patients enrolled, 21 (46%) had a normal etco 2 level and 25 (54%) had an abnormal etco 2 level. the mean age of enrolled patients was 40 (range 19-70) and 32 (70%) were male. mechanisms of injury included motor vehicle collision in 19 (41%), motor cycle collision in 9 (20%), fall in 8 (17%), bicycle/ pedestrian struck in 8 (17%), and other in 2 (4%). eight (17%) patients had a gcs 9-12 and 38 (83%) had a gcs 13-15. of the 11 (24%) patients with intracranial lesions on ct, 10 (91%) had an abnormal etco 2 level (p = 0.006). of the 5 (11%) patients who required a neurosurgical intervention, 100% had an abnormal etco 2 level (p = 0.05). levels of a biomarker indicative of astrogliosis were significantly higher in those with abnormal etco 2 compared to those with a normal etco 2 (p = 0.026). conclusion: abnormal levels of etco 2 were significantly associated with clinical measures of brain injury severity. further research with a larger sample of mmtbi patients will be required to better understand and validate these findings. background: acetaminophen (apap) poisoning is the most frequent cause of acute hepatic failure in the us. toxicity requires bioactivation of apap to toxic metabolites, primarily via cyp2e1. children are less susceptible to apap toxicity; one current theory is that children's conjugative pathway (sulfonation) is more active. liquid apap preparations contain propylene glycol (pg), a common excipient that inhibits apap bioactivation and reduces hepatocellular injury in vitro and in rodents. cyp2e1 inhibition may decrease toxicity in children, who tend to ingest liquid apap preparations, and suggests a potential novel therapy. objectives: to compare phase i (toxic) and phase ii (conjugative) metabolism of liquid versus solid prepara-tions of apap. we hypothesize that ingestion of a liquid apap preparation results in decreased production of toxic metabolites relative to a solid preparation, likely due to the presence of pg in the liquid preparations. methods: design-pharmacokinetic cross-over study. setting-university hospital clinical research center. subjects-adults ages 18-40 taking no chronic medications. interventions-subjects were randomized to receive a 15 mg/kg dose of a commercially available solid or liquid apap preparation. after a washout period of greater than 1 week, subjects received the same dose of apap in the alternate preparation. apap, apap-glucuronide and apap-sulfate (phase 2 metabolites), apap-cysteinate and apap-mercapturate (phase 1 metabolites) were analyzed via lc/ms in plasma over 8 hours. peak concentrations and measured auc were compared using paired-sample t-tests. plasma pg levels were measured. results: fifteen subjects completed the protocol. peak concentrations and aucs of the cyp2e1 derived toxic metabolites were significantly lower following ingestion of the liquid preparation (table, figure) . the glucuronide and sulfate metabolites were not different. pg was present following ingestion of liquid but not solid preparations. conclusion: ingestion of liquid relative to solid preparations in therapeutic doses results in decreased plasma levels of toxic apap metabolites. this may be due to inhibition of cyp2e1 by pg, and may explain the decreased susceptibility in children. a less hepatotoxic formulation of apap can potentially be developed if co-formulated with a cyp2e1 inhibitor. background: pressure immobilization bandages have been shown to delay mortality for up to 8 hours after coral snake envenomation, providing an inexpensive and effective treatment when antivenin is not readily available. however, long-term efficacy has not been established. objectives: determine if pressure immobilization bandages, consisting of an ace wrap and splint, can delay morbidity and mortality from coral snake envenomation, even in the absence of antivenin therapy. methods: institutional animal care and use committee approval was obtained. this was a randomized, observational pilot study using a porcine model. ten pigs (17.3 kg to 25.6 kg) were sedated and intubated for 5 hours. pigs were injected subcutaneously in the left distal foreleg with 10 mg of lyophilized m. fulvius venom resuspended in water, to a depth of 3 mm. pigs were randomly assigned to either a control group (no compression bandage and splint) or a treatment group (compression bandage and splint) approximately 1 minute after envenomation. pigs were monitored daily for 21 days for signs of respiratory depression, decreased oxygen saturations, and paresis/paralysis. in case of respiratory depression, pigs were euthanized and time to death recorded. chi-square was used to compare rates of survival up to 21 days and a kaplan-meier survival curve constructed. results: average survival time of control animals was 412 ± 90 minutes compared to 12,642 ± 7,132 minutes for treated animals. significantly more pigs in the treatment group survived to 24 hours than in the control group (p = 0.03). two of the treatment pigs survived to the endpoint of 21 days, but showed necrosis of the distal lower extremity. conclusion: long-term survival after coral snake envenomation is possible in the absence of antivenin with the use of pressure immobilization bandages. the applied pressure of the bandage is critical to allowing survival without secondary consequences (i.e. necrosis) of envenomation. future studies should be designed to accurately monitor the pressures applied. background: patients exposed to organophosphate (op) compounds demonstrate a central apnea. the kölliker-fuse nuclei (kf) are cholinergic nuclei in the brainstem involved in central respiratory control. objectives: we hypothesize that exposure of the kf is both necessary and sufficient for op-induced central apnea. methods: anesthetized and spontaneously breathing wistar rats (n = 24) were exposed to a lethal dose of dichlorvos using three experimental models. experiment 1 (n = 8) involved systemic op poisoning using subcutaneous (sq) dichlorvos (100 mg/kg or 3x ld50). experiment 2 (n = 8) involved isolated poisoning of the kf using stereotactic microinjections of dichlorvos (625 micrograms in 50 microliters) into the kf. experiment 3 (n = 8) involved systemic op poisoning with isolated protection of the kf using sq dichlorvos (100 mg/kg) and stereotactic microinjections of organophosphatase a (opda), an enzyme that degrades dichlorvos. respiratory and cardiovascular parameters were recorded continuously. histological verification of injection site was performed using kmno4 injections. animals were followed post-poisoning for 1 hour or death. betweengroup comparisons were performed using a repeated measured anova or student's t-test where appropriate. results: animals poisoned with sq dichlorvos demonstrated respiratory depression starting 5.1 min post exposure, progressing to apnea 15.9 min post exposure. there was no difference in respiratory depression between animals with sq dichlorvos and those with dichlorvos microinjected into the kf. despite differences in amount of dichlorvos (100 mg/kg vs 1.8 mg/kg) and method of exposure (sq vs cns microinjection), 10 min following dichlorvos both groups (sq vs microinjection respectively) demonstrated a similar percent decrease in respiratory rate (51.5 vs 72.2, p = 0.14), minute ventilation ( background: patients sustaining rattlesnake envenomation often develop thrombocytopenia, the etiology of which is not clear. laboratory studies have demonstrated that venom from several species, including the mojave rattlesnake (crotalus scutulatus scutulatus), can inhibit platelet aggregation. in humans, administration of crotaline fab antivenom (av) has been shown to result in transient improvement of platelet levels; however, it is not known whether platelet aggregation also improves after av administration. objectives: to determine the effect of c. scutulatus venom on platelet aggregation in vitro in the presence and absence of crotaline fab antivenom. methods: blood was obtained from four healthy male adult volunteers not currently using aspirin, nsaids, or other platelet-inhibiting agents. c. scutulatus venom from a single snake with known type b (hemorrhagic) activity was obtained from the national natural toxins research center. measurement of platelet aggregation by an aggregometer was performed using five standard concentrations of epinephrine (a known platelet aggregator) on platelet-rich plasma over time, and a mean area under the curve (auc) was calculated. five different sample groups were measured: 1) blood alone; 2) blood + c. scutulatus venom (0.3 mg/ml); 3) blood + crotaline fab av (100 mg/ml); 4) blood + venom + av (100 mg/ ml); 5) blood + venom + av (4 mg/ml). standard errors of the mean (sem) were calculated for each group. results: antivenom administration by itself did not significantly affect platelet aggregation compared to baseline (103.8 ± 3.4%, p = 0.47). administration of venom decreased platelet aggregation (72.0 ± 8.5%, p < 0.05). concentrated av administration in the presence of venom normalized platelet aggregation (101.4 ± 6.8%) and in the presence of diluted av significantly increased aggregation (133.9 ± 9.0%); p < 0.05 for both groups when compared to the venom-only group. to control for the effects of the venom and av, each was run independently in platelet-rich plasma without epinephrine; neither was found to significantly alter platelet aggregation. conclusion: crotaline fab av improved platelet aggregation in an in vitro model of platelet dysfunction induced by venom from c. scutulatus. the mechanism of action remains unclear but may involve inhibition of venom binding to platelets or a direct action of the antivenom on platelets. background: routine use of both breathalyzers and hand sanitizers is common across emergency depart-ments. the most common hand sanitizer on the market, purell, contains 62% ethyl alcohol and a lesser amount of isopropyl alcohol. previous investigations have documented that risk is low to the health care worker who applies frequent hand sanitizers to themselves. however, it is unknown whether this alcohol mixture causes false readings on a breathalyzer machine being used to determine alcohol levels on others. objectives: to determine the effect on the measurement of breathalyzer readings in individuals who have not consumed alcohol after hand sanitizer is applied to the experimenter holding a breathalyzer machine. methods: after obtaining informed consent, a breathalyzer reading was obtained in participants who had not consumed any alcohol in the last 24 hours. three different experiments were performed with 25 different participants in each. in experiment 1, two pumps of hand sanitizer were applied to the experimenter. without allowing the sanitizer to dry, the experimenter then measured the breathalyzer reading of the participant. in experiment 2, one pump of sanitizer was applied to the experimenter. measurements of the participant were taken without allowing the sanitizer to dry. in experiment 3, one pump of sanitizer was placed on the experimenter and rubbed until dry according to the manufacturer's recommendations. readings were recorded and analyzed using paired t-tests. results: the initial breathalyzer reading for all participants was 0. after two pumps of hand sanitizer were applied without drying (experiment 1), breathalyzers ranged from 0.02 to 0.17, with a mean above the legalintoxication limit of 0.11 (t(24) = )15.3, p < 0.001). after one pump of hand sanitizer was applied without drying (experiment 2), breathalyzers ranged from 0.02 to 0.11, with a mean of 0.06 (t(24) = )14.1, p < 0.001). after one pump of hand sanitizer was applied according to manufacturer's directions (experiment 3), breathalyzers ranged from 0.0 to 0.02 with a mean of 0.01 (t(24) = )5.1, p < 0.001). conclusion: use of hand sanitizer according to the manufacturer's recommendations results in a small but significant increase in breathalyzer readings. however, the improper and overuse of common hand sanitizer elevates routine breathalyzer readings, and can mimic intoxication in individuals who have not consumed alcohol. stephanie carreiro, jared blum, francesca beaudoin, gregory jay, jason hack objectives: the primary aim of this study is to determine if pretreatment with ile affects the hemodynamic response to epinephrine in a rat model. hemodynamic response was measured by a change in heart rate (hr) and mean arterial pressure (map). we hypothesized that ile would limit the rise in map and hr that typically follow epinephrine administration. methods: twenty male sprague dawley rats (approximately 7-8 weeks of age) were sedated with isoflurane and pretreated with a 15 ml/kg bolus of ile or normal saline, followed by a 15 mcg/kg dose of epinephrine intravenously. intra-arterial blood pressure and hr were monitored continuously until both returned to baseline (biopaq). a multifactorial analysis of variance (manova) was performed to assess the difference in map and hr between the two groups. standardized t-tests were then used to compare the peak change in map, time to peak map, and time to return to baseline map in the two groups. results: overall, a significant difference was found between the two groups in map (p = 0.01) but not in hr (p = 0.34). there was a significant difference (p = 0.023) in time to peak map in the ile group (54 sec, 95% ci 44-64) versus the saline group (40 sec, 95% ci 32-48) and a significant difference (p = 0.004) in time to return to baseline map in ile group (171 sec, 95% ci 148-194) versus the saline group (130 sec, 95% ci 113-147). there was no significant difference (p = 0.28) in the peak change in map of the ile group (75.4, mmhg, 95% ci 66-85) versus the saline group (69.9 mmhg, 95% ci 64-76). conclusion: our data show that in this rat model ile pretreatment leads to a significant difference in map response to epinephrine, but no difference in hr response. ile delayed the peak effect and prolonged the duration of effect on map but did not alter the peak increase in map. this suggests that the use of ile may delay the time to peak effect of epinephrine if the drugs are administered concomitantly to the same patient. further research is needed to explore the mechanism of this interaction. rasch analysis of the agitation severity scale when used with emergency department acute psychiatry patients tania d. strout, michael r. baumann maine medical center, portland, me background: agitation is a frequently observed and problematic phenomenon in mental health patients being treated in the emergency setting. the agitation severity scale (agss), a reliable and valid instrument, was developed using classical test theory to measure agitation in acute psychiatry patients. objectives: the aim of this study was to analyze the agss according to the rasch measurement model and use the results to determine whether improvements to the instrument could be made. methods: this prospective, observational study was irb-approved. 270 adult ed patients with psychiatric chief complaints and dsm-iv-tr diagnoses were observed using the agss. the rasch rating scale model was employed to evaluate the 17 items comprising the agss using winsteps statistical software. unidimensionality, item fit, response category performance, person and item separation reliability, and hierarchical ordering of items were all examined. a principle components analysis (pca) of the rasch residuals was also performed. results: variable maps revealed that all of the agss items were used to some degree and that the items were ordered in a way that makes clinical sense. several duplicative items, indicating the same degree of agitation, were identified. item (5.19) and person (2.01) separation statistics were adequate, indicating appropriate spread of items and subjects along the agitation continuum and providing support for the instrument's reliability. keymaps indicated that the agss items are functioning as intended. analysis of fit demonstrated no extreme misfitting items. pca of the rasch residuals revealed a small amount of residual variance, but provided support for the agss as being unidimensional, measuring the single construct of agitation. the results of this rasch analysis support the agss as a psychometrically robust instrument for use with acute psychiatry patients in the emergency setting. several duplicative items were identified that may be eliminated and re-evaluated in future research; this would result in a shorter, more clinically useful scale. in addition, a gap in items for patients with lower levels of agitation was identified. generation of additional items intended to measure low levels of agitation could improve clinician's ability to differentiate between these patients. background: attempted suicide is one of the strongest clinical predictors of subsequent suicide and occurs up to 20 times more frequently than completed suicide. as a result, suicide prevention has become a central focus of mental health policy. in order to improve current treatment and intervention strategies for those presenting with suicide attempt and self-injury in the emergency department (ed), it is necessary to have a better understanding of the types of patients who present to the ed with these complaints. objectives: to describe the epidemiology of ed visits for attempted suicide and self-inflicted injury over a 16year period. methods: data were obtained from the national hospital ambulatory medical care survey (nhamcs). all visits for attempted suicide and self-inflicted injury (e950-e959) during 1993-2008 were included. trend analyses were conducted using stata's nptrend (a nonparametric test for trends that is an extension of the wilcoxon rank-sum test) and regression analyses. a two-tailed p < 0.05 was considered statistically significant. results: over the 16-year period, there were an average of 420,000 annual ed visits for attempted suicide and self-inflicted injury (1.50 [95% confidence interval (ci) 1.33-1.67] visits per 1,000 us population). the overall mean patient age was 31 years, with visits most common among ages 15-19 (3.70; 95%ci 3.11-4.30). the average annual number of ed visits for suicide attempt and self-inflicted injury more than doubled from 244,000 in 1993-1996 to 538,000 in 2005-2008. during the same timeframe, ed visits for these injuries per 1,000 us population almost doubled for males (0.84 to 1.62), females (1.04 to 1.96), whites (0.94 to 1.82), and blacks (1.14 to 2.10). no temporal differences were found for method of injury or ed disposition; there was, however, a significant decrease in visits determined by the physician to be urgent/emergent from 95% in 1993 to 70% in 2008. conclusion: ed visit volume for attempted suicide and self-inflicted injury has increased over the past two decades in all major demographic groups. awareness of these longitudinal trends may assist efforts to increase research on suicide prevention. in addition, this information may be used to inform current suicide and self-injury related ed interventions and treatment programs. benjamin l. bregman, janice c. blanchard, alyssa levin-scherz george washington university, washington, dc background: the emergency department (ed) has increasingly become a health care access point for individuals with mental health needs. recent studies have found that rates of major depression disorder (mdd) diagnosed in eds are far above the national average. we conducted a study assessing whether individuals with frequent ed visits had higher rates of mdd than those with fewer ed visits in order to help guide screening and treatment of depressed individuals encountered in the ed. objectives: this study evaluated potential risk factors associated with mdd. we hypothesized that patients who are frequent ed visitors will have higher rates of mdd. methods: this was a single center, prospective, crosssectional study. we used a convenience sample of noncritically ill, english speaking adult patients presenting with non-psychiatric complaints to an urban academic ed over 6 months in 2011. we oversampled patients presenting with ‡3 visits over the previous 364 days. subjects were surveyed about their demographic and other health and health care characteristics and were screened with the phq 9, a nine-item questionnaire that is a validated, reliable predictor of mdd. we conducted bivariate (chi-square) and multivariate analysis controlling for demographic characteristics using sta-ta v. 10.0. our principal dependent variable of interest was a positive depression screen (phq 9 score ‡10). our principal independent variable of interest was ‡3 visits over the previous 364 days. results: our response rate was 90.7% with a final sample size of 1012. of our total sample, 313 (30.9%) had three or greater visits within the prior 364 days. one hundred (32%) frequent visitors had a positive phq 9 mdd screen as compared to 142 (20.3%) of subjects with fewer than three visits (p < 0.0001). in our multivariate analysis, the odds for having three or more visits for subjects who had a positive depression screen was 1.42 (1.03, 1.97). of subjects with three or more visits with a positive depression screen, only 116 (37%) were actively being treated for mdd at the time of their visit. conclusion: our study found a high prevalence of untreated depression among frequent users of the ed. eds should consider routinely screening patients who are frequent consumers for mdd. in addition, further studies should evaluate the effect of early treatment and follow up for mdd on overall utilization of ed services. access to psychiatric care among patients with depression presenting to the emergency department janice c. blanchard, benjamin l. bregman, dana rosenfarb, qasem al jabr, eun kim george washington university, washington, dc background: literature suggests that there is a high rate of major depressive disorder (mdd) in emergency department (ed) users. however, access to outpatient mental health services is often limited due to lack of providers. as a result, many persons with mdd who are not in active treatment may be more likely to utilize the ed as compared to those who are currently undergoing outpatient treatment. objectives: our study evaluated utilization rates and demographic characteristics associated with patients with a prior diagnosis of mdd not in active treatment. we hypothesized that patients who present to the ed with untreated mdd will have more frequent ed visits. methods: this was a single center, prospective, crosssectional study. we used a convenience sample of noncritically ill, english speaking adult patients presenting with non-psychiatric complaints to an urban academic ed over 6 months in 2011. subjects were surveyed about their demographic and other health and health care characteristics and were screened with the phq 9, a nine-item questionnaire that is a validated, reliable predictor of mdd. we conducted bivariate (chi-square) and multivariate analysis controlling for demographic characteristics using stata v. 10.0. our principal dependent variable of interest was a positive depression screen (phq 9 ‡ 10). our analysis focused on the subset of patients with a prior diagnosis of mdd with a positive screen for mdd during their ed visit. results: our response rate was 90.7% with a final sample size of 1012. 243 (24.0%) patients screened positive for mdd with a phq 9 score ‡10. of the 243 patients with a positive depression screen, 55.1% reported a prior history of treatment for mdd (n = 134). of these patients, only 57.6% were currently actively receiving treatment. hispanics who screened positive for depression with a history of mdd were less likely to actively be undergoing treatment as compared to non-hispanics (22.2% versus 46.9%, p = 0.041). patients with incomes less than $20,000 were more likely to actively be receiving treatment as opposed to higher incomes (76.3% versus 42.7% p = 0.003). conclusion: patients presenting to our ed with untreated mdd are more likely to be hispanic and less likely to be low income. the emergency department may offer opportunities to provide antidepressant treatment for patients who screen positive for depression but who are not currently receiving treatment. evaluation of a two-question screening tool (phq-2) for detecting depression in emergency department patients jeffrey p. smith, benjamin bregman, janice blanchard, nasser hashim, mary pat mckay george washington university, washington, dc background: the literature suggests there is a high rate of undiagnosed depression in ed patients and that early intervention can reduce overall morbidity and health care costs. there are several well validated screening tools for depression including the nine-item patient health questionnaire (phq-9). a tool using a two-question subset, the phq-2, has been shown to be an easily administered, reasonably sensitive screening tool for depression in primary care settings. objectives: to determine the sensitivity and specificity of the phq-2 in detecting major depressive disorders (mdd) among adult ed patients presenting to an urban teaching hospital. we hypothesize that the phq-2 is a rapid, effective screening tool for depression in a general ed population. methods: cross sectional survey of a convenience sample of 1012 adult, non-critically ill, english speaking patients with medical and not psychiatric complaints presenting to the ed between 9am and 11pm weekdays. patients were screened for mdd with the phq-9. we used spss v19.0 to analyze the specificity, sensitivity, positive predictive value (ppv), negative predictive value (npv), and kappa of phq-2 scores of 2 and 3 (out of possible total score of 6) compared to a validated cut-off score of 10 or higher of 27 points on the phq-9. the two questions on the phq-2 are: ''over the last two weeks, how often have you had little interest in doing things? how often have you felt down, depressed or hopeless?'' responses are scored from 0-3 based on ''never'',''several days'', ''more than half'', ''nearly every day''. results: 1012 subjects of 1116 approached agreed to participate (90.7% response rate), and 975 (96.3%) completed the phq-9. the phq-9 identified 225 (23.1%) subjects with mdd. table 1 outlines the percent of subjects who were positive and the sensitivity, specificity, positive, and negative predictive values and kappa for each cut-off on the phq-2. conclusion: the phq-2 is a sensitive and specific screening tool for mdd in the ed setting. moreover, the phq-2 is closely correlated with the phq-9, especially if a score of 3 or greater is used. given the simplicity and ease of using a two-item questionnaire and the high rates of undiagnosed depression in the ed, including this brief, self-administered screening tool to ed patients may allow for early awareness of possible mdd and appropriate evaluation and referral. patients. however, much of this self-harm behavior is not discovered clinically and very little is known about the prevalence and predictors of current ed screening practices. attention to this issue is increasing due to the joint commission's patient safety goal 15, which focuses on identification of suicide risk in patients. objectives: to describe the prevalence and predictors of screening for self-harm and of presence of current self-harm in eds. methods: data were obtained from the nimh-funded emergency department safety assessment and followup evaluation (ed-safe). eight u.s. eds reviewed charts in real time for 35-40 hours a week between 8/ 2010 and 11/2011. all patients presenting during enrollment shifts were characterized as to whether a selfharm screening had been performed by ed clinicians. a subset of patients with a positive screening was asked about the presence of self-harm ideation, attempts, or both by trained research staff. we used multivariable logistic regression to identify predictors of screening and of current self-harm. data were clustered by site. in each model we examined day and time of presentation, age < 65 years, sex, race, and ethnicity. results: of the 92,154 patients presenting during research shift, 24,240 (26%) were screened for self-harm. screening rates varied among sites and ranged from 4% to 32%, with one outlier at 93%. of those screened, 2,471 (10%) had current self-harm. among those with selfharm approached by study personnel (n = 1,037), 916 (88%) had thoughts of self-harm (suicidal or non-suicidal), 806 (78%) had thoughts of suicide, 444 (43%) had self-harm behavior, and 316 (31%) had suicide attempt(s) over the preceding week. predictors of being screened were: age < 65 years, male sex, weekend presentation, and night shift presentation (table) . among those screened, predictors of current self-harm were: age < 65 years, white race, and night shift presentation. conclusion: screening for self-harm is uncommon in ed settings, though practices vary dramatically by site. patients presenting at night and on weekends are more likely to be screened, as are those under age 65 and males. current self-harm is more common among those presenting on night shift, those under age 65, and whites. results: there were 1328 out-of-hospital records reviewed, and hospital discharge data were available in 1120 non-cardiac arrest patients. of the 1120 patients, 1084 (96.8%) patients survived to hospital discharge and 36 (3.2%) died during hospitalization. the mean age of those transported was 54 years (sd20), 612 (55%) were male, 128 (11%) were trauma-related, and 112 (10%) were admitted to the icu. average systolic blood pressure (sbp), pulse (p), respiratory rate (rr), oxygen saturation (o 2 sat), and end-tidal carbon dioxide (etco 2 ) were sbp = 141 (sd29), p = 95 (sd25), rr = 24 (sd9), o 2 sat = 95% (sd8), and etco 2 = 34 (sd10 conclusion: of all the initial vital signs recorded in the out-of-hospital setting, etco 2 was the most predictive of mortality. these findings suggest that pre-hospital etco 2 is a useful clinical tool for determining severity of illness and appropriate triage. background: the prehospital use of continuous positive airway pressure (cpap) ventilation is a relatively new management for acute cardiogenic pulmonary edema (acpe) and there is little high quality evidence on the benefits or potential dangers in this setting. objectives: the aim of this study was to determine whether patients in severe respiratory distress treated with cpap in the prehospital setting have a lower mortality than those treated with usual care. methods: randomized, controlled trial comparing usual care versus cpap (whisperflowò) in a prehospital setting, for adults experiencing severe respiratory distress, with falling respiratory efforts, due to a presumed acpe. patients were randomised to receive either usual care, including conventional medications (nitrates, furosemide, and oxygen) plus bag-valve-mask ventilation, versus conventional medications plus cpap. the primary outcome was prehospital or in-hospital mortality. secondary outcomes were need for tracheal intubation, length of hospital stay, change in vital signs, and arterial blood gas results. we calculated relative risk with 95% cis. results: fifty patients were enrolled with mean age 79ae8 (sd 11ae9), male 56ae0%, mortality 20ae0%. the risk of death was significantly reduced in the cpap arm with mortality 34ae6% (9 deaths) in the usual care arm compared to 4ae2% (1 death) in the cpap arm (rr, 0ae12; 95% ci 0ae02 to 0ae88; p = 0ae04). patients who received cpap were significantly less likely to have respiratory acidosis (mean difference in ph 0ae09; 95% ci 0ae01 to 0ae16; p = 0ae02; n = 24) than patients receiving usual care. the length of hospital stay was significantly less in the patients who received cpap (mean difference 2ae3 days; 95% ci )0ae01 to 4ae6, p = 0ae05). conclusion: we found that cpap significantly reduced mortality, respiratory acidosis, and length of hospital stay for patients in severe respiratory distress caused by acpe. this study shows the use of cpap for acpe improves patient outcomes in the prehospital setting. (originally submitted as a ''late-breaker.'') trial reg. anzctr actrn12609000410257; funding fisher and paykal suppliers of the whisperflowò cpap device. background: because emergency service utilization continues to climb, validated methods to safely identify and triage low-acuity patients to either alternate care destinations or a complaint-appropriate level of ems response is of keen interest to ems systems and potentially payers. though the literature generally supports the medical priority dispatch system (mpds) as a tool to predict low-acuity patients by various standards, correlation with initial patient physiologic data and patient age is novel. objectives: to determine whether the six mpds priority determinants for protocol 26 (sick person) can be used to predict initial ems patient acuity assessment or severity of an aggregate physiologic score. our longterm goal is to determine whether mpds priority can be used to predict patient acuity and potentially send only a first responder to do an in-person assessment to confirm this acuity, while reserving als transport resources for higher acuity patients. methods: calls dispatched through the wichita-sedgwick county 9-1-1 center between july 20, 2009 and october 1, 2011 using mpds protocol 26 (sick person) were linked to the ems patient care record for all patients 14 and older. the six mpds priority determinants were evaluated for correlation with initial ems acuity code, initial vital signs, rapid acute physiology score (raps), or patient age. the ems acuity code scores patients from low to severe acuity, based on initial ems assessment. results: there were 9370 calls dispatched using protocol 26 for those 14 years of age and older during the period, representing approximately 13% of all ems calls. there is a significant difference in the first encounter vital signs among different mpds priority levels. based on the logistic regression model, the mpds priority code alone had a sensitivity of 68% and specificity of 55% for identifying low-acuity patients with ems acuity score as the standard. the area under the curve (auc) for roc is 0.62 for mpds priority codes alone, while addition of age increases this value to 0.69. if we use the raps score as the standard to the mpds priority code, auc is 0.528. if we include both mpds and age in the model, the auc is 0.533. conclusion: in our system, mpds priority codes on protocol 26 (sick person) alone, or with age or raps score, are not useful either as predictors of patient acuity on ems arrival or to reconfigure system response or patient destination protocols. alternate ambulance destination program c. nee-kofi mould-millman 1 , tim mcmahan 2 , michael colman 2 , leon h. haley 1 , arthur h. yancey 1 1 emory university, atlanta, ga; 2 grady ems, atlanta, ga background: low-acuity patients calling 9-1-1 are known to utilize a large proportion of ems and ed resources. the national association of ems physicians and acep jointly support ems alternate destination programs (adps) in which low-acuity patients are allocated alternative resources non-emergently. analysis of one year's adp data from our ems system revealed that only 4.5% of eligible patients were transported to alternate destinations (ambulatory clinics). reasons for this low success rate need investigation. objectives: to survey emts and discover the most frequent reasons given by them for transportation of eligible patients to eds instead of to clinics. methods: this study was conducted within a large, urban, hospital-based ems system. upon conducting an adp for 12 months, a paper-based survey was created and pre-tested. all medics with any adp-eligible patient contact were included. emts were asked about personal, patient, and system related factors contributing to ed transport during the last 3 months of the adp. qualitative data were coded, collated, and descriptively reported. results: sixty-three respondents (26 emt-intermediates and 37 emt-paramedics) completed the survey, representing 79% of eligible emts. thirty-one emts (49%) responded that they did not attempt to recruit eligible patients into the adp in the last 3 program months. of those emts, 25 (81%) attributed their motive to multiple, prior, failed recruitment attempts. the 32 emts who actively recruited adp patients were asked reasons given by patients for clinic transport refusals: 19 (60%) cited that patients reported no prior experience of care at the participating clinics, and 23 (72%) reported patients had a strong preference for care in an ed. regarding system-related factors contributing to non-clinic transport, 24 of the 32 emts (75%) reported that clinic-consenting patients were denied clinic visits, mostly because of non-availability of same-day clinic appointments. conclusion: respondents indicated that poor emt enrollment of eligible patients, lack of available clinic time slots, and patient preference for ed care were among the most frequent reasons contributing to the low success rate of the adp. this information can be used to enhance the success of this, and potentially other adp programs, through modifications to adp operations and improved patient education. the effect of a standardized offline pain treatment protocol in the prehospital setting on pediatric pain treatment brent kaziny 1 , maija holsti 1 , nanette dudley 1 , peter taillac 1 , hsin-yi weng 1 , kathleen adelgais 2 1 university of utah, school of medicine, salt lake city, ut; 2 university of colorado, school of medicine, aurora, co background: pain is often under treated in children. barriers include need for iv access, fear of delayed transport, and possible complications. protocols to treat pain in the prehospital setting improve rates of pain treatment in adults. the utah ems for children (emsc) program developed offline pediatric protocol guidelines for ems providers, including one protocol that allows intranasal analgesia delivery to children in the prehospital setting. objectives: to compare the proportion of pediatric patients receiving analgesia for orthopedic injury by prehospital providers before and after implementation of an offline pediatric pain treatment protocol. methods: we conducted a retrospective study of patients entered into the utah prehospital on-line active reporting information system (polaris, a database of statewide ems cases) both before and after initiation of the pain protocol. patients were included if they were age 3-17 years, with a gcs of 14-15, an isolated extremity injury, and were transported by an ems agency that had adopted the protocol. pain treatment was compared for 2 years before and 18 months after protocol implementation with a wash-out period of 12 months for agency training. the difference in treatment proportions between the two groups was analyzed and 95% cis were calculated. results: during the two study periods, 1155 patients met inclusion criteria. patient demographics are outlined in the table. 93/501 (18.6%) patients were treated for pain before compared to 174/654 (26.6%) patients treated after the pain protocol was implemented; a difference of 8.0% (95% ci: 3.2%-12.8%). patients were more likely to receive pain medication if they had a pain score documented (or: 1.16; 95% ci: 1.09-1.22) and if they were treated after the implementation of a pain protocol (or: 1.27; 95% ci: 1.00-1.62). factors not associated with the treatment of pain include age, sex, and mechanism of injury. conclusion: the creation and adoption of statewide emsc pediatric offline protocol guideline for pain management is associated with a significant increase in use of analgesia for pediatric patients in the prehospital setting. background: evidence-based guidelines are needed to determine the appropriate use of air medical transport, as few criteria currently used predict the need for air transport to a trauma center. we previously developed a clinical decision rule (cdr) to predict mortality in injured, helicopter-transported patients. objectives: this study is a prospective validation of the cdr in a new population. methods: a prospective, observational cohort analysis of injured patients ( ‡16 y.o.) transported by helicopter from the scene to one of two level i trauma centers. variables analyzed included patient demographics, diagnoses, and clinical outcomes (in-hospital mortality, emergent surgery w/in 24 hrs, blood transfusion w/in 24 hrs, icu admit greater than 24 hrs, combined outcome of all). prehospital variables were prospectively obtained from air medical providers at the time of transport and included past medical history, mechanism of injury, and clinical factors. descriptive statistics compared those with and without the outcomes of interest. the previous cdr (age ‡ 45, gcs £ 13, sbp < 90, flail chest) was prospectively applied to the new population to determine its accuracy and discriminatory ability. results: 416 patients were transported from october 2010-august 2011. the majority of patients were male (59%), white (79%), with an injury occurring in a rural location (60%). most injuries were blunt (95%) with a median iss of 9. overall mortality was 5%. the most common reasons for air transport were: mvc with high risk mechanism (17%), gcs £ 13 (16%), loc >5 minutes (16%), and mvc >20 mph (14%). of these, only gcs £ 13 was significantly associated with any of the clinical outcomes. when applying the cdr, the model had a sensitivity of 100% (81.2%-100%), a specificity of 51.2% (50.6%-51.6%), a npv of 100% (98.1%-100%), and a ppv of 9.9% (8.0%-9.9%) for mortality. the area under the curve for this model was 0.92, suggesting excellent discriminatory ability. conclusion: the air transport decision rule in this study performed with high sensitivity and acceptable specificity in this validation cohort. further external validation in other systems and with ground transported patients are needed in order to improve decision making for the use of helicopter transport of injured patients. background: acute non-variceal upper gastrointestinal (gi) bleeding is a common indication for hospital admission. to appropriately risk-stratify such patients, endoscopy is recommended within 24 hours. given the possibility to safely manage patients as outpatients after endoscopy, risk stratification as part of an emergency department (ed) observation unit (ou) protocol is proposed. objectives: our objective was to determine the ability of an ou upper gi bleeding protocol to identify a lowrisk population, and to expeditiously obtain endoscopy and disposition patients. we also identified rates of outcomes including changes in hemoglobin, abnormal endoscopy findings, admission, and revisits. background: acute uncomplicated pyelonephritis (pyelo) requires no imaging but a ct flank pain protocol (ctfpp) may be ordered to determine if patients with pyelo and flank pain also have an obstructing stone. the prevalence of kidney stone and the characteristics predictive of kidney stone in pyelo patients is unknown. objectives: to determine elements on presentation that predict ureteral stone, as well as prevalence of stone and interventions in patients undergoing ct for pyelo. methods: retrospective study of patients at an academic ed who received a ctfpp scan between 8/05 and 4/09. 5497 ctfpps were identified and 1899 randomly selected for review. pyelo was defined as: positive urine dip for infection and >5 wbc/hpf on formal urinalysis in addition to flank pain/cva tenderness, chills, fever, nausea, or vomiting. patients were excluded for age < 18 y.o., renal disease, pregnancy, urological anomaly, or recent trauma. clinical data (178 elements) were gathered blinded to ct findings; ct results were abstracted separately and blinded to clinical elements. ct findings of hydronephrosis and hyrdroureter (hydro) were used as a proxy for hydro that could be determined by ultrasound prior to ct. patients were categorized into three groups: ureteral stone, no significant findings, and intervention or follow-up required. classification and regression tree analysis was used to determine which variables could identify ureteral stone in this population of pyelo patients. results: out of the 1899 patients, 105 (7.0%) met criteria for pyelo; subjects had a mean age of 39 ± 15.9 and 82% (n = 87) were female. ct revealed 31 (29%, 95% ci = 0.22-0.39) symptomatic stones, and 72 (68%, 95% ci = 0.59-0.76) exams with no significant findings. two patients needed intervention/ follow-up (1%, 95% ci = 0.0052-0.0667), one for perinephric hemorrhage and the other for pancreatitis. hydro was predictive for ureteral stone with an or = 18.4 (95% ci = 6.4-52, p < 0.0001). eleven (35%) ureteral stone patients were admitted and 9 (8%) of them had procedures. of these patients, 100% had ct signs of obstruction, 8 (88%) had hydronephrosis, and 1 (11%) had hydroureter. conclusion: hydronephrosis was predictive of ureteral stone and in-house procedures. prospective study is needed to determine whether ct scan is warranted in patients with pyelonephritis but without hydronephrosis or hydroureter. curative objectives: the specific aim of this analysis was to describe characteristics of patients presenting to the emergency department (ed) at their index diagnosis, and to determine whether emergency presentation precludes treatment with curative intent. methods: we performed a retrospective cohort analysis on a prospectively maintained institutional tumor registry to identify patients diagnosed with crc from 2008-2010. emrs were reviewed to identify which patients presented to the ed with acute symptoms of crc as the initial sign of their illness. the primary outcome variable was treatment plan (curative vs. palliative). secondary outcome variables included demographics, tumor type and location. descriptive statistics were conducted for major variables. chi-squre and fisher's exact tests were used to detect the association between categorical variables. two-sample t-test was used to identify the association between continuous and categorical variables. results: between jan 1 2008 and dec 31 2010, 376 patients were identified at our institution with crc. 214 (57%) were male and 162 (43%) were female, with mean age 60.6; sd: 13.3. thirty-three patients (8.8%) initially presented to the ed, of whom 5 (15.5%) received palliation. of 339 patients who initially presented elsewhere, 69 (20.5%) received palliation. acute ed presentation with crc symptoms did not preclude treatment with curative intent (p = 0.47). patients who presented emergently were more likely to be female (64% vs male 41%; p = 0.01) and older (65 vs. 60; p = 0.02). there was no statistically significant relationship between age, sex, tumor location, or type and treatment approach. conclusion: patients with crc may present to the ed with acute symptoms, which ultimately leads to the diagnosis. emergent presentation of crc does not preclude patients from receiving therapy with curative intent. cannabinoid (or 2.93, , and white blood cell (wbc) count ‡14,000/mm 3 (or 11.35, 95% ci 3.42-37.72). conclusion: age ‡65 years is not associated with need for admission from an ed observation unit. older adults can successfully be cared for in these units. initial temperature, respiratory rate, and pulse were not predictive of admission, but extremely elevated blood pressure was predictive. other relevant predictor variables included comorbidities and elevated wbc count. advanced age should not be a disqualifying criterion for disposition to an ed observation unit. older adult fallers in the emergency department luna ragsdale, cathleen colon-emeric duke university, durham, nc background: approximately 1/3 of community-dwelling older adults experience a fall each year, and 2.2 million are treated in u.s. emergency departments (ed) annually. the ed offers a potential location for identification of high-risk individuals and initiation of fall-prevention services that may decrease both fall rates and resource utilization. objectives: the goal of this study was to: 1) validate an approach to identifying older adults presenting with falls to the ed using administrative data; and 2) characterize the older adult who falls and presents to the ed and determine the rate of repeat ed visits, both fall-related and all visits, after an index fall-related visit. methods: we identified all older adults presenting to either of the two hospitals serving durham county residents during a six month period. manual chart review was completed for all encounters with icd9 codes that may be fall-related. charts were reviewed 12 months prior and 12 months post index visit. descriptive statistics were used to describe the cohort. results: a total of 4452 older adults were evaluated in the ed during this time period; 1714 (55.7%) had an icd9 code for a potentially fall-related injury. of these, record review identified 534 (12%) with a fall from standing height or less. of the fallers, 65.9% of the patients were discharged, 31% were admitted, and 3% were admitted under observation. of those who fell, 38.2% had an ed visit within the previous year. approximately 1/3 (33.3%) of these were fall related. over half (53.4%) of the patients who fell returned to the ed within one year of their index visit. a large proportion (44.4%) of the return visits was fall-related. follow-up with a primary care provider or specialist was recommended in 46% of the patients who were discharged. overall mortality rate for fallers over the year following the index visit was 18%. conclusion: greater than fifty percent of fallers will return to the ed after an index fall, with a large proportion of the visits related to a fall. a large number of these fallers are discharged home with less than fifty percent having recommended follow-up. the ed represents an important location to identify high-risk older adults to prevent subsequent injuries and resource utilization. objectives: we studied whether falls from a standing position resulted in an increased risk for intracranial or cervical injury verses falling from a seated or lying position. methods: this is a prospective observational study of patients over the age of 65 who presented with a chief complaint of fall to a tertiary care teaching facility. patients were eligible for the study if they were over age 65, were considered to be at baseline mental status, and were not triaged to the trauma bay. at presentation, a questionnaire was filled out by the treating physician regarding mechanism and position of fall, with responses chosen from a closed list of possibilities. radiographic imaging was obtained at the discretion of the treating physician. charts of enrolled patients were subsequently reviewed to determine imaging results, repeat studies done, or recurrent visits. all patients were called in follow-up at 30 days to assess for delayed complications related to the fall. data were entered into a standardized collection sheet by trained abstractors. data were analyzed with fisher's exact test and descriptive statistics. this study was reviewed and approved by the institutional review board. results: two-hundred sixty two patients were enrolled during the study period. one-hundred ninety eight of these had fallen from standing and 64 fell from either sitting or lying positions. the mean age for patients was 84 (sd 7.9) for those who fell from standing and 84 (sd 8.4) for those who fell from sitting or lying. there were 6 patients with injuries who fell from standing: three with subdural hematomas, one with a cerebral contusion, one with an osteophyte fracture at c6, and one with an occipital condyle fracture with a chip fracture of c1. there were 2 patients with injuries who fell from a seated or lying position: one with a traumatic subarachnoid hemorrhage and one with a type ii dens fracture. the overall rate of traumatic intracranial or cervical injury in elders who fell was 3%. no patients required surgical intervention. there was no difference in rate of injury between elders who fell from standing versus those who fell from sitting or lying (p = 1). (table) . conclusion: both instruments identify the majority of patients as high-risk which will not be helpful in allocating scarce resources. neither the isar nor the trst can distinguish geriatric ed patients at high or low risk for 1or 3-month adverse outcomes. these prognostic instruments are not more accurate in dementia or lower literacy subsets. future instruments will need to incorporate different domains related to short-term adverse outcomes. background: for older adults, both inpatient and outpatient care involves not only the patient and physician, but often a family member or informal caregiver. they can assist in medical decision making and in performing the patient's activities of daily living. to date, multiple outpatient studies have examined the positive roles family members play during the physician visit. however, there is very limited information on the involvement of the caregiver in the ed and their relationship with the health outcomes of the patient. objectives: to assess whether the presence of a caregiver influences the overall satisfaction, disposition, and outpatient follow-up of elderly patients. we performed a three-step inquiry of patients over 65 years old who arrived to the upenn ed. patients and care partners were initially given a questionnaire to understand basic demographic data. at the end of the ed stay, patients were given a satisfaction survey and followed through 30 days to assess time to disposition, whether the patient was admitted or discharged, outpatient follow-up, and ed revisit rates. chi-square and t-tests were used to examine the strength of differences in the elderly patients' sociodemographics, self-rated health, receiving aid with their instrumental activities of daily living, and number of health problems by accompaniment status. multivariate regression models were constructed to examine whether the presence or absence of caregivers affected satisfaction, disposition, and follow-up. results: overall satisfaction was higher among patients who had caregivers (2.4 points), among patients who felt they were respected by their physician (3.8 points), and had lower lengths of stay (2 hours). patients with caregivers were also more likely to be discharged home (or 2.4) and to follow-up with their regular physician (or 2.1). there was no evidence to suggest caregivers affected the overall rates of revisits back to an ed. conclusion: for older adults, medical care involves not only the patient and physician, but often a family member or an informal care companion. these results demonstrate the positive influence of caregivers on the patients they accompany, and emergency physicians should define ways to engage these caregivers during their ed stay. this will also allow caregivers to participate when needed and can help to facilitate transitions across care settings. background: shared decision making has been shown to improve patient satisfaction and clinical outcomes for chronic disease management. given the presence of individual variations in the effectiveness and side effects of commonly used analgesics in older adults, shared decision making might also improve clinical outcomes in this setting. objectives: we sought to characterize shared decision making regarding the selection of an outpatient analgesic for older ed patients with acute musculoskeletal pain and to examine associations with outcomes. methods: we conducted a prospective observational study with consecutive enrollment of patients age 65 or older discharged from the ed following evaluation for moderate or severe musculoskeletal pain. two essential components of shared decision making, 1) information provided to the patient and 2) patient participation in the decision, were assessed via patient interview at one week using four-level likert scales. results: of 233 eligible patients, 110 were reached by phone and 87 completed the survey. only 25% (21/87) of patients reported receiving 'a lot' of information about the analgesic, and only 21% (18/87) reported participating 'a lot' in the selection of the analgesic. there were trends towards white patients (p = 0.06) and patients with higher educational attainment (p = 0.07) reporting more participation in the decision. after adjusting for sex, race, education, and initial pain severity, patients who reported receiving 'a lot' of information were more likely to report optimal satisfaction with the analgesic than those receiving less information (78% vs. 47%, p < 0.05). after the same adjustments, patients who reported participating 'a lot' in the decision were also more likely to report optimal satisfaction with the analgesic (82% vs. 47%, p < 0.05) and greater reductions in pain scores (mean reduction in pain 4.6 vs. 2.7, p < 0.05) at one week than those who participated less. background: quality of life (qol) measurements have become increasingly important in outcomes-based research and cost-utility analyses. dementia is a prevalent, often unrecognized, geriatric syndrome that may limit the accuracy of patient self-report in a subset of patients. the relationship between caregiver and geriatric patient qol in the emergency department (ed) is not well understood. objectives: to qualify the relationship between caregiver and geriatric patient qol ratings in ed patients with and without cognitive dysfunction. methods: this was a prospective, consecutive patient, cross-sectional study over two months at one urban academic medical center. trained research assistants screened for cognitive dysfunction using the short blessed test and evaluated health impairment using the quality of life-alzheimer's disease (qol-ad) test. when available in the ed, caregivers were asked to independently complete the qol-ad. consenting subjects were non-critically ill, english-speaking, community-dwelling adults over 65 years of age. responses were compared using wilcoxon signed ranks test to assess the relationships between patient and caregiver scores from the qol-ad stratified by normal or abnormal cognitive screening results. significance was defined by p < 0.05. results: patient qol ratings were obtained from 108 patient-caregiver pairs. patients were 51% female, 52% african-american, with a mean age of 76-years, and 58% had abnormal cognitive screening tests. compared with caregivers, cognitively normal patients had no significant qol assessment differences except for questions of energy level and overall mood. on the other hand, cognitively impaired patients differed significantly on questions of energy level and ability to perform household chores with a trend towards significant differences for living setting (p = 0.097) and financial situation (p = 0.057). in each category, the differences reflected a caregiver underestimation of quality compared with the patient's self-rating. conclusion: discrepancies between qol domains and total scores for patients with cognitive dysfunction and their caregivers highlights the importance of identifying cognitive dysfunction in ed-based outcomes research and cost-utility analyses. further research is needed to quantify the clinical importance of the patient-and caregiver-assessed quality of life. background: age is often a predictor for increased morbidity and mortality. however, it is unclear whether old age is a predictor of adverse outcome in syncope. objectives: to determine whether old age is an independent predictor of adverse outcome in patients presenting to the emergency department following a syncopal episode. methods: a prospective observational study was conducted from june 2003 to july 2006 enrolling consecutive adult ed patients (>18 years) presenting with syncope. syncope was defined as an episode of transient loss of consciousness. adverse outcome or critical intervention were defined as gastrointestinal bleeding or other hemorrhage, myocardial infarction/percutaneous coronary intervention, dysrhythmia, alteration in antidysrhythmics, pacemaker/defibrillator placement, sepsis, stroke, death, pulmonary embolus, or carotid stenosis. outcomes were identified by chart review and 30-day follow-up phone calls. results: of 575 patients who met inclusion criteria, an adverse event occurred in 24% of patients. overall, 35% of patients with risk factors had adverse outcomes compared to 1.6% of patients with no risk factors. in particular, 28/127 (22%; 95% ci 16-30%) of patients <65 with risk factors had adverse outcomes, while 85/196 (43%; 95% ci 36-50%) of the elderly with risk factors had adverse outcomes. in contrast, among young people 2/196 (1%; 95% ci 0.04-3.8%) of patients without risk factors had adverse outcomes while 2/56 (3.6%; 95% ci 0.28-13%) of patients ‡65 without risk factors had adverse outcomes. conclusion: although the elderly are at greater risk for adverse outcomes in syncope, age ‡ 65 or older alone does not appear to be a predictor of adverse outcome following a syncopal event. based on these data, it should be safe to discharge home from the ed patients with syncope, but without risk factors, regardless of age. (originally submitted as a ''late-breaker.'') antibiotics background: adherence to national guidelines for hiv and syphilis screening in eds is not routine. in our ed, hiv and syphilis screening rates among patients tested for gonorrhea and chlamydia (gc/ct) have been reported to be 45% and 30%, respectively. objectives: to determine the effect of a sexually transmitted infection (sti) laboratory order set on hiv and syphilis screening among ed patients tested for gc/ct. we hypothesized that a sti order set would increase screening rates by at least 30%. methods: a 6-month, quasi-experimental study in an urban ed comparing hiv and syphilis screening rates of gc/ct-tested patients before (control phase) and after the implementation of a sti laboratory order set (intervention phase). the order set linked blood-based rapid hiv and syphilis screening with gc/ct testing. consecutive patients completing gc/ct testing were included. the primary outcome was the absolute difference in hiv and syphilis screening rates among gc/ ct-tested patients between phases. we estimated that 550 subjects per phase were needed to provide 90% power (p-value of £0.05) to detect an absolute difference in screening rates of 10%, assuming a baseline hiv screening rate of 45%. results: the ed census was 42,461. characteristics of patients tested for gc/ct were similar between phases: the mean age was 33 years (sd = 12) and most were female (65%), black (49%), hispanic (30%), and unmarried (84% services have recommended the use of immunization programs against influenza disease within hospitals since the 1980s. the emergency department (ed) being the ''safety net'' for most non-insured people is an ideal setting to intervene and provide primary prevention from influenza. objectives: the purpose of this study is to assess whether a pharmacist-based influenza immunization program is feasible in the ed, and successful in increasing the percentage of adult patients receiving the influenza vaccine. methods: implementation of pharmacist-based immunization program was developed in coordination with ed physicians and nursing staff in 2010. the nursing staff, using an embedded electronic questionnaire within their triage activity, screened patients for eligibility for the influenza vaccine. the pharmacist using an electronic alert system within the electronic medical record identified patients who we deemed eligible and if agreed the pharmacist vaccinated the patient. patients who refused to be vaccinated were surveyed to ascertain their perception concerning immunization offered by a pharmacist in the ed. feasibility and safety data for vaccinating patient in the ed were recorded. results: 149 patients were approached and enrolled into the study. of the 149, 41% agreed to receive the influenza vaccine from a pharmacist in the ed. the median screening time was 5 minutes and median vaccination time was 3 minutes for a total of 8 minutes from screening time to vaccination time. 74% were willing to receive the influenza vaccine from a pharmacist, and 78% were willing to receive the vaccine in the ed. the main reason given for refusing to receive the influenza vaccine was ''patient does not feel at risk of getting the disease''; only 14.6% stated they were vaccinated recently. conclusion: a pharmacist-based influenza immunization program is feasible in the ed, and has the potential to successfully increase the percentage of adult patients receiving the vaccine. 1.4 ± 0.1, p < 0.05). ed visits by hiv-infected patients also had longer lengths of ed stay (317 ± 26.0 minutes vs. 222.5 ± 5.6 minutes, p < 0.05) and were more likely to be admitted (29% vs. 15%, p < 0.05), than their non-hiv infected counterparts. conclusion: although ed visits by hiv-infected individuals in the u.s. are relatively infrequent, they occur at rates higher than the general population, and consume significantly more ed resources than the general population. the background: the influence of wound age on the risk of infection in simple lacerations repaired in the emergency department (ed) has not been well studied. it has traditionally been taught that there is a ''golden period'' beyond which lacerations are at higher risk of infection and therefore should not be closed primarily. the proposed cutoff for this golden period has been highly variable (3-24 hours in surgical textbooks). objectives: to answer the following research question: are wounds closed via primary repair after the golden period at increased risk for infection? methods: we searched medline, embase, and other databases as well as bibliographies of relevant articles. we included studies that enrolled ed patients with lacerations repaired by primary closure. exclusion: 1. intentional delayed primary repair or secondary closure, 2. wounds requiring intra-operative repair, skin graft, drains, or extensive debridement, and 3. grossly contaminated or infected at presentation. we compared the outcome of wound infection in two groups of early versus delayed presentations (based on the cut-offs selected by the original articles). we used ''grading of recommendations assessment, development and evaluation'' (grade) criteria to assess the quality of the included trials. frequencies are presented as percentages with 95% confidence intervals. relative risk (rr) of infection is reported when clinically significant. results: 418 studies were identified. four trials enrolling 3724 patients in aggregate met our inclusion/exclusion criteria. two studies used a 6-hour cut-off and the other two used a 12-hour cut-off for defining delayed wounds. the overall quality of evidence was low. the infection rate in the wounds that presented with delay ranged from 1.4% to 32%. one study with the smallest sample size (morgan et al), which only enrolled lacerations to the hand and forearm, showed higher rates of infection in patients with delayed wounds (table). the infection rates in delayed wound groups in the remaining three studies were not significantly different from the early wounds. conclusion: the evidence does not support the existence of a golden period, nor does it support the role of wound age on infection rate in simple lacerations. background: although clinical studies in children have shown that temperature elevation is an independent and significant predictor of bacteremia in children, the relationship in adults is largely unknown or equivocal. objectives: review the incidence of positive blood cultures on critically ill adult septic patients presenting to an emergency department (ed) and determine the association of initial temperature with bacteremia. methods: july 2008 to july 2010 retrospective chart review on all patients admitted from the ed to an urban community hospital with sepsis and subsequently expiring within 4 days of admission. fever was defined as a temperature ‡38°c. sirs criteria were defined as: 1) temperature ‡38°c or £36°c, 2) heart rate ‡90 beats/ minute, 3) respiratory rate ‡20 or mechanical ventilation, 4) wbc ‡ 12,000/mm 3 or <4,000 or bands ‡10%. objectives: we examined the utility of limited genetic sequencing of bacterial isolates using multilocus sequence typing (mlst) to discriminate between known pathogenic blood culture isolates of s. epidermidis and isolates recovered from skin. methods: ten blood culture isolates from patients meeting the centers for disease control and prevention (cdc) criteria for clinically significant s. epidermidis bacteremia and ten isolates from the skin of healthy volunteers were studied. mlst was performed by sequencing 400 bp regions of seven genes (arc, aroe, gtr, muts, pyr, tpia, and yqil) . genetic variability at these sites was compared to an international database (www.sepidermidis.mlst.net) and each strain was then categorized into a genotype on the basis of known genetic variation. the ability of the gene sequences to correctly classify strains was quantified using the support vector machine function in the statistical package r. 1,000 bootstrap resamples were performed to generate confidence bounds around the accuracy estimates. results: between-strain variability was considerable, with yqil being most variable (6 alleles) and tpia being least (1 allele). the muts gene, responsible for dna repair in s. epidermidis, showed almost complete separation between pathogenic and commensal strains. when the seven genes were used in a joint model, they correctly predicted bacterial strain type with 90% accuracy (iqr 85, 95%). conclusion: multilocus sequence typing shows excellent early promise as a means of distinguishing contaminant versus truly pathogenic isolates of s. epidermidis from clinical samples. near-term future goals will involve developing more rapid means of sequencing and enrolling a larger cohort to verify assay performance. conference are presented by influenza scenario in table 1 and background: antiviral medications are recommended for patients with influenza who are hospitalized or at high risk for complications. however, timely diagnosis of influenza in the ed remains challenging. influenza rapid antigen tests have short turn-around times, making them potentially useful in the ed setting, but their sensitivities may be too low to assist with treatment decisions. objectives: to evaluate the test characteristics of the binaxnow influenza a&b rapid antigen test (rat) in ed patients. methods: we prospectively enrolled a systematic sample of patients of all ages presenting to two eds with acute respiratory symptoms or fever during three consecutive influenza seasons (2008) (2009) (2010) (2011) . research personnel collected nasal and throat swabs, which were combined and tested for influenza with rt-pcr using cdc-provided primers and probes. ed clinicians independently decided whether to obtain a rat during clinical care. rats were performed in the clinical laboratory using the binaxnow influenza a&b test on nasal swabs collected by ed staff. the study cohort included subjects who underwent both a research pcr and clinical rat. rat test characteristics were evaluated using pcr as the criterion standard with stratified sub-analyses for age group and influenza subtype (pandemic h1n1 (ph1n1), non-pandemic influenza a, influenza b). results: 561 subjects were enrolled; 131 subjects were pcr positive for influenza (76 ph1n1, 20 non-pandemic influenza a, and 35 influenza b). for all age groups, rat sensitivities were low and specificities were high ( hiv infection with cd4 < 200; and among nursing home residents, inability to independently perform activities of daily living. sources for bacterial cultures included blood, sputum (adults only), bronchoalveolar lavage (bal), tracheal aspirate, and pleural fluid. only sputum specimens with a bartlett score ‡1+ were considered adequate for culturing. results: among 461 children enrolled, 7 (2%) had s. aureus cultured from ‡1 specimen, including 5 with methicillin-resistant s. aureus (mrsa) and 2 with methicillin-susceptible s. aureus (mssa). specimens positive for s. aureus included 3 pleural fluid, 2 blood, 2 tracheal aspirates, and 1 bal. two children with s. aureus had evidence of co-infection: 1 influenza a, and 1 streptococcus pneumoniae. among 673 adults enrolled, 17 (3%) grew s. aureus from ‡1 specimen, including 9 with mrsa and 8 with mssa. specimens positive for s. aureus included 5 blood, 11 sputum, and 3 bal. five adults with s. aureus had evidence of co-infections: 2 coronavirus, 1 respiratory syncytial virus, 1 s. pneumoniae, and 1 pseudomonas aeruginosa. presenting clinical characteristics and outcomes of subjects with staphylococcal cap are summarized in tables 1-2. conclusion: these preliminary findings suggest s. aureus is an uncommon cause of cap. although the small number of staphylococcal cases limits conclusions that can be drawn, in our analysis staphylococcal cap appears to be associated with co-infections, pleural effusions, and severe disease. future work will focus on continued enrollment and developing clinical prediction models to aid in diagnosing staphylococcal cap in the ed. background: emergency care has been a neglected public health challenge in sub-saharan africa. the goal of global emergency care collaborative (gecc) is to develop a sustainable model for emergency care delivery in low-resource settings. gecc is developing a training program for emergency care practitioners (ecps). objectives: to analyze the first 500 patient visits at karoli lwanga ''nyakibale'' hospital ed in rural uganda to determine the knowledge and skills needed in training ecps. methods: a descriptive cross-sectional analysis of the first 500 consecutive patient visits in the ed's patient care log was reviewed by an unblinded abstractor. data on demographics, procedures, laboratory testing, bedside ultrasounds (us) performed, radiographs (xrs) ordered, and diagnoses were collated. all authors discussed uncertainties and formed a consensus. descriptive statistics were performed. results: of the first 500 patient visits, procedures were performed in 367 (73.4%) patients, including 244 (48.8%) who had ivs placed, 47 (9.4%) who received wound care, and 42 (8.4%) who received sutures. complex procedures, such as procedural sedations, lumbar punctures, orthopedic reductions, nerve blocks, and tube thoracostomies, occurred in 49 (9.8%) patients. laboratory testing, xrs, and uss were performed in 188,(37.6%), 99 (19.8%), and 45 (7%) patients, respectively. infectious diseases were diagnosed in 217 (43.4%) patients; 78 (15.6 %) with malaria and 57 (11.4%) with pneumonia. traumatic injuries were present in 140 (28%) patients; 77 (15.4%) needing wound care and 31 (6.2%) with fractures. gastrointestinal and neurological diagnoses affected 58 (11.6%) and 27 (5.4%) patients, respectively. conclusion: ecps providing emergency care in sub-saharan africa will be required to treat a wide variety of patient complaints and effectively use laboratory testing, xrs, and uss. this demands training in a broad range of clinical, diagnostic, and procedural skills, specifically in infectious disease and trauma, the two most prevalent conditions seen in this rural sub-saharan africa ed. assessment of point-of-care ultrasound in tanzania background: current chinese ems is faced with many challenges due to a lack of systematic planning, national standards in training, and standardized protocols for prehospital patient evaluation and management. objectives: to estimate the frequency with which prehospital care providers perform critical actions for selected chief complaints in a county-level ems system in hunan province, china. methods: in collaboration with xiangya hospital (xyh), central south university in hunan, china, we collected data pertaining to prehospital evaluation of patients on ems dispatches from a ''1-2-0'' call center over a 2-month period. this call center services an area of just under 5000 km 2 with a total population of 1.36 million. each ems team consists of a driver, a nurse, and a physician. this was a cross-sectional study where a single trained observer accompanied ems teams on transports of patients with a chief complaint of chest pain, dyspnea, trauma, or altered mental status. in this convenience sample, data were collected daily between 8 am and 6 pm. critical actions were pre-determined by a panel of emergency medicine faculty from xyh and the university of maryland school of medicine. simple statistical analysis was performed to determine the frequency of critical actions performed by ems providers. results: during the study period, 1170 patients were transported, 452 of whom met the inclusion criteria. 218 (48.2%) evaluations were observed directly for critical actions. the table shows the frequency of critical actions performed by chief complaint. none of the patients with chest pain received an ecg even though the equipment was available. rapid glucose was checked in only 2.1% of patients presenting with altered mental status. a lung exam was performed in 22.7% of patients with dyspnea, and the respiratory rate was measured in 9.1%. among patients transported for trauma, blood pressure, and heart rate were only measured in 1% and 4.1%, respectively. conclusion: in this observation study of prehospital patient assessments in a county-level ems system, critical actions were performed infrequently for the chief complaints of interest. performance frequencies for critical actions ranged from 0 to 22.7%, depending on the chief complaint. standardized prehospital patient care protocols should be established in china and further training is needed to optimize patient assessment. trends little is known about the comparative effectiveness of noninvasive ventilation (niv) versus invasive mechanical ventilation (imv) in chronic obstructive pulmonary disease (copd) patients with acute respiratory failure. objectives: to characterize the use of niv and imv in copd patients presenting to the emergency department (ed) with acute respiratory failure and to compare the effectiveness of niv vs. imv. methods: we analyzed the 2006-2008 nationwide emergency department sample (neds), the largest, all-payer, us ed and inpatient database. ed visits for copd with acute respiratory failure were identified with a combination of copd exacerbation and respiratory failure icd-9-cm codes. patients were divided into three treatment groups: niv use, imv use, and combined use of niv and imv. the outcome measures were inpatient mortality, hospital length of stay (los), hospital charges, and complications. propensity score analysis was performed using 42 patient and hospital characteristics and selected interaction terms. results: there were an estimated 101,000 visits annually for copd exacerbation and respiratory failure from approximately 4,700 eds. ninety-six percent were admitted to the hospital. of these, niv use increased slightly from 14% in 2006 to 16% in 2008 (p = 0.049), while imv use decreased from 28% in 2006 to 19% in 2008 (p < 0.001); the combined use remained stable (4%). inpatient mortality decreased from 10% in 2006 to 7% in 2008 (p < 0.001). niv use varied widely between hospitals, ranging from 0% to 100% with median of 11%. in a propensity score analysis, niv use (compared to imv) significantly reduced inpatient mortality (risk ratio 0.57; 95% confidence interval [ci] 0.48-0.56), shortened hospital los (difference )3 days; 95%ci )4 to )3), and reduced hospital charges 044; 855) . niv use was associated with a lower rate of iatrogenic pneumothorax compared with imv use (0.04% vs. 0.6%, p < 0.001). an instrumental analysis confirmed the benefits of niv use, with a 5% reduction in inpatient mortality in the niv-preferring hospitals. conclusion: niv use is increasing in us hospitals for copd with acute respiratory failure; however, its adoption remains low and varies widely between hospitals. niv appears to be more effective and safer than imv in the real-world setting. background: dyspnea is a common ed complaint with a broad differential diagnosis and disease-specific treatment. bronchospasm alters capnographic waveforms, but the effect of other causes of dyspnea on waveform morphology is unclear. objectives: we evaluated the utility of capnographic waveforms in distinguishing dyspnea caused by reactive airway disease (rad) from non-rad in adult ed patients. methods: this was a prospective, observational, pilot study of a convenience sample of adult patients presenting to the ed with dyspnea. waveforms, demographics, past medical history, and visit data were collected. waveforms were independently interpreted by two blinded reviewers. when the interpreters disagreed, the waveform was re-reviewed by both reviewers and an agreement was reached. treating physician diagnosis was considered the criterion standard. descriptive statistics were used to characterize the study population. diagnostic test characteristics and inter-rater reliability are given. results: fifty subjects were enrolled. median age was 52 years (range 21-82), 50% were female, 34% were caucasian. 29/50 (58%) had a history of asthma or chronic obstructive pulmonary disease. rad was diagnosed by the treating physician in 19/50 (38%) and 32/50 (64%) had received treatment for dyspnea prior to waveform acquisition. the interpreters agreed on waveform analysis in 47/50 (94%) cases (kappa = 0.88). test characteristics for presence of acute rad, including 95%ci, were: overall accuracy 70% (55.2%-81.7%), sensitivity 69% (43.5%-86.4%), specificity 71% (51.8%-85.1%), positive predictive value 59% (36.7%-78.5%), negative predictive value 79% (58.5%-91.0%), positive likelihood ratio 2.25 (1.36-3.72) , negative likelihood ratio 0.42 (0.23-0.74). conclusion: inter-rater agreement is high for capnographic waveform interpretation, and shows promise for helping to distinguish between dyspnea caused by rad and dyspnea from other causes in the ed. treatments received prior to waveform acquisition may affect agreement between waveform interpretation and physician diagnosis, affecting the observed test characteristics. asthma background: asthma and chronic obstructive pulmonary disease (copd) patients who present to the emergency department (ed) usually lack adequate ambulatory disease control. while evidence-based care in the ed is now well defined, there is limited inform-ation regarding the pharmacologic or non-pharmacologic needs of these patients at discharge. objectives: this study evaluated patients' needs with regard to the ambulatory management of their respiratory conditions after ed treatment and discharge. methods: over 6 months, 94 adult patients with acute asthma or copd, presenting to a tertiary care alberta hospital ed and discharged after being treated for exacerbations, were enrolled. using results from standardized in-person questionnaires, charts were reviewed by respiratory researchers to identify care gaps. results: overall, 58 asthmatic and 36 copd patients were enrolled. more patients with asthma required education on spacer devices (52% vs 31%). few asthma (9%) and no copd patients had written action plans; asthma patients were more likely to need adherence counseling (53% vs 36%) for preventer medications. more patients with asthma required influenza vaccination (72% vs 39%; p = 0.003); pneumococcal immunization was low (36%) in copd patients. only 22% of asthmatics reported ever being referred to an asthma education program and 19% of the copd patients reported ever being referred to pulmonary rehabilitation. at ed presentation, 28% of the asthmatics required the addition of inhaled corticosteroids (ics) and 16% required the addition of ics/long acting beta-agonist (ics/laba) combination agents. on the other hand, 36% of copd patients required the addition of long-acting anticholinergics while most (83%) were receiving preventer medications. finally, 31% of copd and 29% of asthma patients who smoked required smoking cessation counseling. conclusion: overall, we identified various care gaps for patients presenting to the ed with asthma and copd. there is an urgent need for high-quality research on interventions to reduce these gaps. methods: this is an interim, sub-analysis of an interventional, double-blinded study performed in an academic urban-based adult ed. subjects with acute exacerbation of asthma with fev1 < 50% predicted within 30 minutes following initiation of ''standard care'' (including a minimum of 5 mg nebulized albuterol, 0.5 mg nebulized ipratropium, and 50 mg corticosteroid) who consented to be in a trial were included. all treatment was administered by emergency physicians unaware of the study objectives. patients were randomly assigned to treatment with placebo or an intravenous beta agonist. all subjects had fev1 and ds obtained at baseline, 1, 2, and 3 hours after treatment. fev1 was measured using a bedside nspire spirometer, and ds was calculated using a modified borg dyspnea score. results: thirty-eight patients were included for analysis. spearman's rho test (rho) was used to measure correlations between fev1 and ds at 1, 2, and 3 hours post study entry and subsequent hospitalization. rho is negative for fev1 (higher fev1 correlates to lower rate of hospitalization) and positive for ds (higher ds correlates to higher rate of hospitalization). at each time point, ds were more highly correlated to hospitalization than were fev1 (see table) . conclusion: dyspnea score at 1, 2, and 3 hours were significantly correlated with hospital admission, whereas fev1 was not. in this set of subjects with moderate to severe asthma exacerbations, a standardized subjective tool was superior to fev1 for predicting subsequent hospitalization. methods: this is an interim, subgroup analysis of a prospective, interventional, double-blind study performed in an academic urban ed. subjects who were consented for this trial presented with acute asthma exacerbations with fev1 £ 50% predicted within 30 minutes following initiation of ''standard care'' (includes a minimum of 2.5 mg nebulized albuterol, 0.5 mg nebulized ipratropium, and 50 mg of a corticosteroid). ed physicians who were unaware of the study objectives administered all treatments. subjects were randomized in a 1:1 ratio to either placebo or investigational intravenous beta agonist arms. blood was obtained at 1 and 1.25 hours after the start of the hour long infusion. blood was centrifuged and serum stored at )80°c, and then shipped on dry ice for albuterol and lactate measurements at a central lab. the treatment lactate and d lactate were correlated with 1 hr serum albuterol concentrations and hospital admission using partial pearson correlations to adjust for ds. results: 38 subjects were enrolled to date, 20 with complete data. the mean baseline serum lactate level was 18.1 mg/dl (sd ± 8.6). this increased to 32.7 mg/ dl (sd ± 15.0) at 1.25 hrs. the mean 1 hr ds was 3.85 (sd ± 2.0). the correlations between treatment lactate, d lactate, 1 hr serum albuterol concentrations (r, s and total) and admission to hospital are shown (see table) . both treatment and d lactate were highly conrrelated with total serum albuterol, r albuterol, and s albuterol. there was no correlation between treatment lactate or d lactate and hospital admission. conclusion: lactate and d lactate concentrations correlate with albuterol concentrations in patients presenting had asthma. fifty one percent were <21 years old and 54% were female. we found a decline of 27% (95% ci: 23%-30%, p < 0.0001; r 2 = 0.73, p < 0.0001) in the overall yearly asthma visits to total ed visits from 1996 to 2010. when we analyzed sex and age groups separately, we found no statistically significant changes for females or for males <21 years old (r 2 £ 0.016, p ‡ 0.65). for females and males >21 years old, yearly asthma visits to total ed visits from 1996 to 2010 decreased 39% (95% ci: 33%-43%, p < 0.0001; r 2 = 0.90, p < 0.0001) and 20% (95% ci: 14%-26%, p < 0.0001; r 2 = 0.80, p < 0.0001), respectively. conclusion: we found an overall decrease in yearly asthma visits to total ed visits from 1996 to 2010. we speculate that this decrease is due to greater corticosteroid use despite the increasing prevalence of asthma. it is unclear why this decrease was seen in adults and not in children and why it was greater for adult females than males. objectives: our objectives were to describe the use of a unique data collection system that leveraged emr technology and to compare its data entry error rate to traditional paper data collection. methods: this is a retrospective review of data collection methods during the first 12 months of a multicenter study of ed, anti-coagulated, head injury patients. on-shift ed physicians at five centers enrolled eligible patients and prospectively completed a data form. enrolling ed physicians had the option of completing a one-page paper data form or an electronic ''dotphrase'' (dp) data form. our hospital system uses an epicòbased emr. a feature of this system is the ability to use dps to assist in medical information entry. a dp is a preset template that may be inserted into the emr when the physician types a period followed by a code phrase (in this case ''.ichstudy''). once the study dp was inserted at the bottom of the electronic ed note, it prompted enrolling physicians to answer study questions. investigators then extracted data directly from the emr. our primary outcomes of interest were the prevalence of dp data form use and rates of data entry errors. results: from 7/2009 through 8/2010, 883 patients were enrolled. dp data forms were used in 288 (32.6%; 95% ci 29.5, 35.7%) cases and paper data forms in 595 (67.4%; 95% ci 64.3, 70.5%). the prevalence of dp data form use at the respective study centers was 11%, 16%, 18%, 31%, and 85%. sixty-six (43.7 %; 95% ci 35.8, 51.6%) of 151 physicians enrolling patients used dp data entry at least once. using multivariate analysis, we found no significant association between physician age, sex, or tenure and dp use. data entry errors were more likely on paper forms (234/595, 39.3%; 95% ci 35.4, 43.3%) than dp data forms (19/288, 6.6%; 95% ci 3.7, 9.5%), difference in error rates 32.7% (95% ci 27.9, 37.6%, p < 0.001). conclusion: dp data collection is a feasible means of study data collection. dp data forms maintain all study data within the secure emr environment obviating the need to maintain and collect paper data forms. this innovation was embraced by many of our emergency physicians. we found lower data entry error rates with dp data forms compared to paper forms. background: inadequate randomization, allocation concealment, and blinding can inflate effect sizes in both human and animal studies. these methodological limitations might in part explain some of the discrepancy between promising results in animal models and non-significant results in human trials. whereas blinding is not always possible, in clinical or animal studies, true randomization with allocation concealment is always possible, and may be as important in minimizing bias. objectives: to determine the frequency with which published emergency medicine (em) animal research studies report randomization, specific randomization methods, allocation concealment, and blinding of interventions and measurements, and to estimate whether these have changed over time. methods: all em animal research publications from 1/ 2000 through 12/2009 in ann emerg med and acad emerg med were reviewed by two trained investigators for a statement regarding randomization, and specific descriptions of randomization methods, allocation concealment, blinding of intervention, and blinding of measurements, when possible. raw initial agreement was calculated and differences were settled by consensus. the first (period 1 = 2000-2004) and second (period 2 = 2005-2009) 5-year periods were compared with 95% confidence intervals. results: of 117 em animal research studies, 109 were appropriate for review because they involved intervention in at least two groups. blinding of interventions and measurements were not considered possible in 37% and 3%, respectively. significant differences between period 1 and 2 were absent, although there was a trend towards less blinding of interventions and more blinding of measurements. raw agreement was 91%. conclusion: although randomization is mentioned in the majority of studies, allocation concealment and blinding remain underutilized in em animal research. we did not compare outcomes between blinded and non-blinded, randomized and non-randomized studies, because of small sample size. this review fails to demonstrate significant improvement over time in these methodological limitations in em animal research publications. journals might consider requiring authors to explicitly describe their randomization, allocation, and blinding methods. background: cluster randomized trials (crts) are increasingly utilized to evaluate quality improvement interventions aimed at health care providers. in trials testing ed interventions, migration of eps between hospitals is an important concern, as contamination may affect both internal and external validity. objectives: we hypothesized geographically isolating emergency departments would prevent migratory contamination in a crt designed to increase ed delivery of tpa in stroke (the instinct trial). methods: instinct was a prospective, cluster-randomized, controlled trial. twenty-four michigan community hospitals were randomly selected in matched pairs for study. following selection of a single hospital, all hospitals within 15 miles were excluded from the sample pool. individual emergency physicians staffing each site were identified at baseline (2007) and 18 months later. contamination was defined at the cluster level, with substantial contamination defined a priori as >10% of eps affected. non-adherence, total crossover (contamination + non-adherence), migration distance and characteristics were determined. results: 307 emergency physicians were identified at all sites. overall, 7 (2.3%) changed study sites. one moved between control sites, leaving 6 (2.0%) total crossovers. of these, 2 (0.7%) moved from intervention to control (contamination) and 4 (1.3%) moved from control to intervention (non-adherence). contamination was observed in 2 of 24 sites, with 17% and 9% contamination of the total site ep workforce at follow-up, respectively. two of 6 crossovers occurred between hospitals within the same health system. average migration distance was 42 miles for all eps in the study and 35 miles for eps moving from intervention to control sites. conclusion: the mobile nature of emergency physicians should be considered in the design of quality improvement crts. use of a 15-mile exclusion zone in hospital selection for this crt was associated with very low levels of substantial cluster contamination (1 of 24) and total crossover. assignment of hospitals from a single health system to a single study group and/or an exclusion zone of 45 miles would have further reduced crossovers. increased reporting of contamination in cluster randomized controlled trials is encouraged to clarify thresholds and facilitate crt design. objectives: an extension of the lr, the average absolute likelihood ratio (aalr), was developed to assess the average change in the odds of disease that can be expected from a test, or series of tests, and an example of its use to diagnose wide qrs complex tachycardia (wct) is provided. methods: results from two retrospective multicenter case series were used to assess the utility of qrs duration and axis to assess for ventricular tachycardia (vt) in patients with undifferentiated regular sustained wct. serial patients with heart rate (hr) >120 beats per minute and qrs duration >120 milliseconds (msec) were included. the final tachydysrhythmia diagnosis was determined by a number of methods independent of the ecg. the aalr is defined as: aalr = 1/n total [r (n i *lr i ) (for lr > 1) + r (n k /lr k ) (for lr < 1)], where lr i and lr k are the interval lrs, and n i and n k are the number of patients with test results within the corresponding intervals. roc curves were constructed, and interval lrs and aalrs were calculated for the qrs duration and axis tests individually, and when applied together. confidence intervals were bootstrapped with 10,000 replications using the r boot package. results: 187 patients were included: 95 with supraventricular tachycardia (svt) and 92 with vt. optimal qrs intervals (msec) for distinguishing vt from svt were: qrs £ 130, 130 < qrs < 160, and qrs ‡ 160. qrs axis results were dichotomized to upward right axis (181-270 degrees) or not ()89 to 180 degrees). results are listed in the table. conclusion: application of the qrs interval and axis tests together for patients with wide qrs complex tachycardia changes the odds of ventricular tachycardia, on average, by a factor of 3.5 (95% ci 2.4 to 6.2), and this is mildly improved over the qrs duration test alone. both a strength and weakness of the aalr is its dependence on the pretest probability of disease. the aalr may be helpful for clinicians and researchers to evaluate and compare diagnostic testing approaches, particularly when strategies with serial non-independent tests are considered. consultation for adults with metastatic solid tumors at an urban, academic ed located within a tertiary care referral center. field notes were grouped into barrier categories and then quantified when possible. patient demographics for those who did and did not enroll were extracted from the medical record and quantified. patients who did not meet inclusion criteria for the study (e.g., cognitive impairment) were excluded from the analysis. results: attempts were made to enroll 42 eligible patients in the study, and 23 were successfully enrolled (55% enrollment rate). barriers to enrollment were deduced from the field notes and placed into the following categories from most to least common: patient refusal (6); diagnostic uncertainty regarding cancer stage (4); severity of symptoms preclude participation (4); patient unaware of illness or stage (3); and family refusal (2). conclusion: patients, families, and diagnostic uncertainty are barriers to enrolling ed patients with advanced illness in clinical trials. it is unclear whether these barriers are generalizable to other study sites and disease processes other than cancer. objectives: the purpose of this study was to evaluate the use of a high-fidelity mannequin bedside simulation scenario followed by a debriefing session as a tool to improve medical student knowledge of palliative care techniques. methods: third year medical students participating in a 12-week simulation curriculum during a surgery/ emergency medicine/anesthesia clerkship were eligible for the study. all students were administered a pretest to evaluate their baseline knowledge of palliative care and randomized to a control or intervention group. during week 3 or 4, students in the intervention group participated in and observed two end-of-life scenarios. following the scenarios, a faculty debriefer trained in palliative care addressed critical actions in each scenario. during week 10, all students received a posttest to evaluate for improvement in knowledge. the pre-test and post-test consisted of 12 questions addressing prognostication, symptom control, and the medicare hospice benefit. students were de-identified and pre-and post-tests were graded by a blinded scorer. results: from jan-dec 2011, 70 students were included in the study and 5 were excluded due to incomplete data. the mean score on the pre-test for the intervention group was 3.16, and for the control group was 3.45 (p = 0.90 the results indicate that educators identify the most important scenarios as protocol-based simulations. respondents also suggested that scenarios of very common emergency department presentations bear a great deal of importance. emergency medicine educators assign priority to simulations involving professionalism and communication. finally, many respondents noted that they use simulation to teach the presentation and management of rare or less frequent, but important disease processes. the identification of these scenarios would suggest that educators find simulation useful for filling in ''gaps'' in resident education. background: prescription drug misuse is a growing problem among adolescent and young adult populations. objectives: to determine factors associated with past year prescription drug misuse defined as using prescription sedatives, stimulants, or opioids to get high, taking them when they were prescribed to someone else or taking more than was prescribed among patients seeking care in an academic ed. methods: adolescents and young adults (14-20) presenting for ed care at a large, academic teaching hospital were approached to complete a computerized screening questionnaire regarding demographics, prescription drug misuse, illicit drug use, alcohol use, and violence in the past 12 months. logistic regression was used to predict past year prescription drug misuse. results: over the study time period, there were 2156 participants (86% response rate) of whom 300 (13.9%) endorsed past year prescription drug misuse. specifically, rates of past year misuse for opioids was 8.7%, sedatives was 5.4%, and stimulants was 8.0%. significant overlap exists among classes with over 40% misusing more than one class of medications. in the multivariate analysis significant predictors of past year prescription drug misuse included female gender (or conclusion: approximately one in seven adolescents or young adults seeking ed care have misused prescription drugs in the past year. while opioids are the most common drug misused, significant overlap exists among this population. given the correlation of prescription drug misuse with the use and misuse of other substances (i.e. alcohol, cough medicine, marijuana) more research is needed to further understand these relationships and inform interventions. additionally, future research should focus on understanding the differences in demographics and risk factors associated with misuse of each separate class of prescription drugs. prospective 10 objectives: this study aims to examine the association of depression with high ed utilization in patients with non-specific abdominal pain. methods: this single-center, prospective, cross-sectional study was conducted in an urban academic ed located in washington, dc as part of a larger study to evaluate the interaction between depression and frequency of ed visits and chronic pain. as part of this study, we screened patients using the phq-9, a nineitem questionnaire that is a validated, reliable predictor of major depressive disorder. we analyzed the subset of respondents with a non-specific abdominal pain diagnosis (icd-9 code of 789.xx). our principal outcome of interest was the rate of a positive depression screen in patients with non-specific abdominal pain. we analyzed the prevalence of a positive depression screen among this group and also conducted a chi-square analysis to compare high ed use among abdominal pain patients with a positive depression screen versus those without a positive depression screen. we defined high ed utilization as >3 visits in a 364-day period prior to the enrollment visit. background: numerous studies have found high rates of co-morbid mental illness and chronic pain in emergent care settings. one psychiatric diagnosis frequently associated with chronic pain is major depressive disorder (mdd). objectives: we conducted a study to characterize the relationship between mdd and chronic pain in the emergency department (ed) population. we hypothesized that patients who present to the ed with selfreported chronic pain will have higher rates of mdd. methods: this was a single-center, prospective, crosssectional study. we used a convenience sample of noncritically ill, english speaking adult patients presenting with non-psychiatric complaints to an urban academic ed over 6 months in 2011. we oversampled patients presenting with pain-related complaints (musculoskeletal pain or headache). subjects were surveyed about their demographic and other health and health care characteristics and were screened with the phq 9, a nine-item questionnaire that is a validated, reliable predictor of mdd. we conducted bivariate (chi-square) and multivariate analysis controlling for demographic characteristics (race, income, sex, age) using stata v. 10.0. our principal dependent variable of interest was a positive depression screen (phq 9 score ‡10). our principal independent variable of interest was the presence of self-reported chronic pain (greater than 3 months). results: of 77 patients enrolled, 2 did not meet all inclusion criteria. 50 had two or more assessments for comparison. their average age was 39 (range 21-59), 70% were male, and 74% were in police custody. 38% used methadone alone; 16% heroin alone; 4% oxycodone alone; and the rest used multiple opioids. the average dose of im methadone was 10.3 mg (range 5-20 mg); all but 3 patients received 10 mg. the mean cows score before receiving im methadone was 11.19 (range 3-23), compared to 4.83 (range 0-20) 30 minutes after methadone (p < 0.001; mean difference = )6.36; 95% ci = )4.57 to )8.15). the mean wss before and after methadone was )1.54 (range )1 to )2) and )0.755 (range )2 to 2), respectively (p < 0.001; 95% ci = )1.0 to )0.57). the mean physician-assessed wss was significantly lower than the patient's own assessment by 0.78 (p < 0.001). adverse events included an asthmatic patient with bronchospasm whose oxygen saturation decreased from 95% to 88% after receiving methadone, a patient whose oxygen saturation decreased from 95% to 93%, and two patients whose amss decreased from )1 to )2 (indicating moderate sedation). background: as the us population ages, the coexistence of copd and acute coronary syndrome (acs) is expected to be more frequent. very few studies have examined the effect of copd on outcomes in acs patients, and, to our knowledge, there has been no report on biomarkers that possibly mediate between copd and long-term acs patient outcomes. objectives: to determine the effect of copd on longterm outcomes in patients presenting to the emergency department (ed) with acs and to identify prognostic inflammatory biomarkers. methods: we performed a prospective cohort study enrolling acs patients from a single large tertiary center. hospitalized patients aged 18 years or older with acs were interviewed and their blood samples were obtained. seven inflammatory biomarkers were measured, including interleukin-6 (il-6), c-reactive protein (crp), tumor necrosis factor-alpha (tnf-alpha), vascular cell adhesion molecule (vcam), e-selectin, lipoprotein-a (lp-a), and monocyte chemoattractant protein-1 (mcp-1). the diagnoses of acs and copd were verified by medical record review. annual telephone follow-up was conducted to assess health status and major adverse cardiovascular events (mace) outcomes, a composite endpoint including myocardial infarction, revascularization procedure, stroke, and death. background: aortic dissection (ad) is an uncommon life-threatening condition requiring prompt diagnosis and management. thirty-eight percent of cases are missed upon initial evaluation. the cornerstone of accurate diagnosis hinges on maintaining a high index of clinical suspicion for the various patterns of presentation. quality documentation that reflects consideration for ad in the history, exam, and radiographic interpretation is essential for both securing the diagnosis and for protecting the clinician in missed cases. objectives: we sought to evaluate the quality of documentation in patients presenting to the emergency department with subsequently diagnosed acute ad. methods: irb-approved, structured, retrospective review of consecutive patients with newly diagnosed non-traumatic ad from 2004 to 2010. inclusion criteria: new ad diagnosis via ed. exclusion criteria: ad diagnosed at another facility; chronic, traumatic, or iatrogenic ad. trained/monitored abstractors used a standardized data tool to review ed and hospital medical records. descriptive statistics were calculated as appropriate. inter-rater reliability was measured. our primary performance measure was the prevalence of a composite of all three key historical elements (1. any back pain, 2. neurologic symptoms including syncope, and 3. sudden onset of pain.) in the attending emergency physician's documentation. secondary outcomes included documentation of: ad risk factors, pain quality, back pain at multiple locations, presence/absence of pulse symmetry, mediastinal widening on chest radiograph, and migratory nature of the pain. results: 65/203 met our inclusion/exclusion criteria. the mean age was 58.4 years; 65% were male, 23 (35.4%) were stanford a. 32 (60%) presented with a chief complaint of chest pain. primary outcome measure: 6/65 (9.2%; 95%ci = 3.5,19.0) documented the presence/ absence of all three key historical elements. [back pain = 42/65; 64.6% (51.8, 76.1); neuro symptoms = 39/ 65; 60% (47.1, 72.0); sudden onset = 12/65; 18.5% (9.9, 30.0).] limitations: small number of confirmed ad cases. conclusion: in our cohort, emergency physician documentation of key historical, physical exam, and radiographic clues of ad is suboptimal. although our ed miss rate is lower than that which has been reported by previous authors, there is an opportunity to improve documentation of these pivotal elements at our institution. objectives: this study assessed the opinions of iem and gh fellowship program directors, in addition to recent and current fellows regarding streamlining the application process and timeline in an attempt to implement change and improve this process for program directors and fellows alike. methods: a total of 34 current iem and gh fellowship programs were found through an internet search. an electronic survey was administered to current iem and gh fellowship directors, current fellows, and recent graduates of these 34 programs. results: response rates were 88% (n = 30) for program directors and 53% (n = 17) for current and recent fellows. the great majority of current and recent fellows (77%) and program directors (83%) support transitioning to a common application service. similarly, 88% of current and recent fellows and 83% of program directors support instituting a uniform deadline date for applications. however, only 47% of recent/current fellows and 33% of program directors would support a formalized match process like nrmp. conclusion: the majority of fellows and program directors support streamlining the application for all iem and gh fellowship programs. this could improve the application process for both fellows and program directors, and ensure the best fit for the candidates and for the fellowship programs. in order to establish effective emergency care in rural sub-saharan africa, the unique practice demographics and patient dispositions must be understood. objectives: the objectives of this study are to determine the demographics of the first 500 patients seen at nyakibale hospital's ed and assess the feasibility of treating patients in a rural district hospital ed in sub-saharan africa. methods: a descriptive cross-sectional analysis of the first 500 consecutive patient visits in the ed's patient care log was reviewed by an unblinded abstractor. data collected included age, sex, condition upon discharge, and disposition. all authors discussed uncertainties and formed a consensus. descriptive statistics were performed. results: of the first 500 patient visits, 254 (50.8%) occurred when the outpatient clinic was open. there were 275 (55%) male visits. the average age was 25.2 years (sd ± 22.2). pediatric visits accounted for 218 (43.6%) patients, and 132 (26.4%) visits were for children under five years old. only one patient expired in the ed, and 401 (80.2%) were in good condition after treatment, as subjectively defined by the ed physicians. one person was transferred to another hospital. after treatment, 180 (36%) patients were discharged home. of those admitted to an inpatient ward, 126 (25.2%) patients were admitted to medical wards, 97 (19.4%) to pediatrics, and 60 (12%) to surgical. only six (1.2 %) patients went directly to the operating theatre. conclusion: this consecutive sample of patient visits from a novel rural district hospital ed in sub-saharan africa included a broad demographic range. after treatment, most patients were judged to be in ''good condition'', and over one third of patients could be discharged after ed management. this sample suggests that it is possible to treat patients in an ed in rural sub-saharan africa, even in cases where surgical backup and transfers to higher level of care are limited or unavailable. background: communication failures in clinical handoffs have been identified as a major preventable cause of patient harm. in italy, advanced prehospital care is provided predominantly by physicians who work on ambulances in teams with either nurses or basic rescuers. the hand-offs from prehospital physicians to hospital emergency physicians (eps) is especially susceptible to error with serious consequences. there are no studies in italy evaluating the communication at this transition in patient care. studying this, however, requires a tool that measures the quality of this communication. objectives: the purpose of this study is to develop and validate a tool for the evaluation of communication during the clinical handoff from prehospital to emergency physicians in critically ill patients. methods: several previously validated tools for evaluating communication in hand-offs were identified through a literature search. these were reviewed by a focus group consisting of eps, nurses, and rescuers, who then adapted and translated the australian isbar (identification, situation, background, assessment, recommendation), the tool most relevant to local practice. the italian isbar tool consists of the following elements: patient and provider identification; patient's chief complaint; patient's past medical history, medications, and allergies; prehospital clinical assessment (primary survey, illness severity, vital signs, diagnosis); treatment initiated and anticipated treatment plan. we conducted and video-taped the hand-offs of care from the prehospital physicians to the eps in 12 pediatric critical care simulations. four physician raters were trained in the italian isbar tool and used it to independently assess communication in each simulation. to assess agreement we calculated the proportion of agreement among raters for each isbar question, fleiss' kappas for each simulation, as well as mean agreement and mean kappas with standard deviations. results: there was 100% agreement among the four physicians on 70% of the items. the mean level of agreement was 91% (sd 0.15). the overall mean kappa was 0.67 (sd 0.10). conclusion: the standardized tool resulted in good agreement by physician raters. this validated tool may be helpful in studying and improving hand-offs in the prehospital to emergency department setting. objectives: we hypothesized that residents who were provided with vps prior to hfs would perform more thoroughly and efficiently than residents who had not been exposed to the online simulation. methods: we randomized a group of 30 residents from an academic, pgy 1-4 emergency medicine program to complete an online vps case, either prior to (vps group, n = 14 residents) or after (n = 16) their hfs case. the vps group had access to the online case (which reviewed asthma management) 3 days prior to the hfs session. all residents individually participated in their regularly scheduled hfs and were blinded to the content of the case -a patient in moderate asthma exacerbation. the authors developed a dichotomous checklist consisting of 33 items recorded as done/not done along with time completed. a two sample proportion test was used to evaluate differences in the individual items completed between groups. a wilcoxon rank sum test was used to determine the differences in overall and subcategory performance between the two groups. median time to completion was analyzed using the log-rank test. results: the vps group had better overall checklist performance than the control group (p-value 0.046). in addition, the vps group was more thorough in obtaining an hpi (p-value 0.009). specific actions (related to asthma management) were performed better by the vps group: inquiring about last/prior ed visits (0.038), total number of hospitalizations in the prior year (0.029), prior intubations (0.001), and obtaining peak flow measurements (0.030). overall there was no difference in time to event completion between the two groups. conclusion: we found that when hfs is primed with educational modalities such as vps there was an improvement in performance by trainees. however, the improved completeness of the vps group may have served as a barrier to efficiency, inhibiting our ability to identify a statistical significant efficiency overall. vps may aid in priming the learners and maximize the efficiency of training using high-fidelity simulations. training using an animal model helped develop residents' skills and confidence in performing ptv. retention was found to be good at 2 months post-training. this study underscores the need for hands-on training in rare but critical procedures in emergency medicine. methods: in this cross-sectional study at an urban community hospital, 15 residents in their second or third year of training from a 3-year em residency program performed us-guided catheterizations of the ij on a simulator manufactured by blue phantom. two board-certified em physicians observed for the completion of pre-defined procedural steps using a checklist and rated the residents' overall performance of the procedure. overall performance ratings were provided on a likert scale of 1 to 10, with 1 being poor and 10 being excellent. residents were given credit for performing a procedural step if at least one rater marked its completion. agreement between raters was calculated using intraclass correlation coefficients for domain and summary scores. the same protocol was then repeated on an unembalmed cadaver using two different board-certified em physician raters. criterion validity of the residents' proficiency on the simulator was evaluated by comparing their median overall performance rating on the simulator to that on the cadaver and by comparing the proportion of residents completing each procedural step between modalities with descriptive statistics. results: em residents' overall performance rating on the simulator was 7.4 (95% ci: 6.0 to 8.8) and on the cadaver was 6.1 (95% ci: 4.7 to 7.5). the results for each procedural step are summarized in the attached figure. inter-rater agreement was high for assessments on both the simulator and cadaver with overall kappa scores of 0.89 and 0.96 respectively. background: the environment in the emergency department (ed) is chaotic. physicians must learn how to multi-task effectively and manage interruptions. noise becomes an inherent byproduct of this environment. previous studies in the surgical and anesthesiology literature examined the effect of noise levels and cognitive interruptions on resident performance during simulated procedures; however, the effect of noise distraction on resident performance during an ed procedure has not yet been studied. objectives: our aim was to prospectively determine the effects of various levels of noise distraction on the time to successful intubation of a high-fidelity simulator. methods: a total of 45 emergency medicine, emergency medicine/internal medicine, and emergency medicine/family medicine residents were studied in a background noise environments of less than 50 decibels (noise level 1), 60-70 decibels (noise level 2), and of greater than 70 decibels (noise level 3). noise levels were standardized by a dosimeter (ex tech instruments, heavy duty 600). each resident was randomized to the order in which he or she was exposed to the various noise levels and had a total of 2 minutes to complete each of the intubation attempts, which were performed in succession. time, in seconds, to successful intubation was measured in each of these scenarios with the start time defined as the time the resident picked up the storz c-mac video laryngoscope blade and the finish time defined as the time the tube passed through the vocal cords as visualized by an observer on the storz c-mac video screen. analytic methods included analysis of variance, student's t-test, and pearson's chi-square. results: no significant differences were found between time to intubation and noise level nor did the order of noise level exposure affect the time to intubation (see table) . there were no significant differences in success rate between the three noise levels (p = 0.178). a significant difference in time to intubation was found between the residents' second and third intubation attempts with decreased time to intubation for the third attempt (p = 0.001). conclusion: noise level did not have an effect on time to intubation or intubation success rate. time to intubation decreased between the second and third intubations regardless of noise level. background: growing use of the emergency department (ed) is cited as a cause of rising health care costs and a target of health care reform. eds provide approximately one quarter of all acute care outpatient visits in the us. eds are a diagnostic center and a portal for rapid inpatient admission. the changing role of eds in hospital admissions has not been described. objectives: to compare if admission through the ed has increased compared to direct hospital admission. we hypothesized that the use of the ed as the admitting portal increased for all frequently admitted conditions. methods: we analyzed the nationwide inpatient sample (nis), the largest us all-payer inpatient care database, from 1993-2006. nis contains data from approximately 8 million hospital stays each year, and is weighted to produce national estimates. we used an interactive, webbased data tool (hcupnet) to query the nis. clinical classification software (ccs) was used to group discharge diagnoses into clinically meaningful categories. we calculated the number of annual admissions and proportion admitted from the ed for the 20 most frequently admitted conditions. we excluded ccs codes that are rarely admitted through the ed (<10%) as well as obstetbackground: the optimal dose of opioids for patients in acute pain is not well defined, although 0.1 mg/kg of iv morphine is commonly recommended. patient-controlled analgesia (pca) provides an opportunity to assess the adequacy of this recommendation as use of the pca pump is a behavioral indication of insufficient analgesia. objectives: to assess the need for additional analgesia following a 0.1 mg/kg dose of iv morphine by measuring additional self-dosing via a pca pump. methods: a three-arm randomized controlled trial was performed in an urban ed with 75,000 annual adult visits. a convenience sample of ed patients ages 18 to 65 with abdominal pain of <7 days duration requiring iv opioids was enrolled between 4/2009 and 6/2010. all patients received an initial dose of 0.1 mg/kg iv morphine. patients in the pca arms could request additional doses of 1 mg or 1.5 mg iv morphine by pressing a button attached to the pump with a 6-minute lock-out period. for this analysis, data from both pca arms were combined. software on the pump recorded times when the patient pressed the button (activation) and when he/she received a dose of morphine (successful activation). results: 137 patients were enrolled in the pca arms. median baseline nrs pain score was 9. mean amount of supplementary morphine self-administered over the 2 hour study period subsequent to the loading dose was 5.7 mg and 6.7 mg for the 1 and 1.5 mg pca groups respectively. 124 patients activated the pump at least once (91%, 95% ci: 84 to 94%). figure 1 shows the frequency distribution of the number of times the pump was activated. of those who activated the pump, the median number of activations per person was 5 (iqr: 3 to 12). there were 1124 activations of the pump. 60% of activations were successful (followed by administration of morphine), while 40% were unsuccessful as they occurred during the 6-minute lock-out periods. 19% of the activations occurred in the first 30 minutes, 29% in the second 30 minutes, 25% in the third 30 minutes, and 27% in the last 30 minutes after the initial loading dose. conclusion: almost all patients requested supplementary doses of pca morphine, half of whom activated the pump five times or more over a course of 2 hours. this frequency of pca activations suggests that the commonly recommended dose of 0.1 mg/kg morphine may constitute initial oligoanalgesia in most patients. marie-pier desjardins, benoit bailey, fanny alie-cusson, serge gouin, jocelyn gravel chu sainte-justine, montreal, qc, canada background: administration of corticosteroid at triage has been suggested to decrease the time to corticosteroid administration in the ed. objectives: to compare the time between arrival and corticosteroid administration in patients treated with an asthma pathway (ap) or with standard management (sm) in a pediatric ed. methods: chart review of children aged 1 to 17 years diagnosed with asthma, bronchospasm, or reactive airways disease seen in the ed of a tertiary care pediatric hospital. for a one year period, 20% of all visits were randomly selected for review. from these, we reviewed patients who were eligible to be treated with the ap ( ‡18 months with previous history of asthma and no other pulmonary condition) and who had received at least one inhaled bronchodilator treatment. charts were evaluated by a data abstractor blinded to the study hypothesis using a standardized datasheet. various variables were evaluated such as age, respiratory rate and 0 2 saturation at triage, type of physician who saw patient first, treatment prior to visit, in ed, and at discharge, time between arrival and corticosteroid administration, and length of stay (los background: return visits comprise 3.5% of pediatric emergency department (ped) visits, at a cost of >$500 million/year nationally. these visits are typically triaged with higher acuity and admission rates and raise concern for lapses in quality of care and patient education during the first visit. objectives: the aim of this qualitative study was to describe parents' reasons for return visits to the ped. methods: we prospectively recruited a convenience sample of parents of patients under the age of 18 years who returned to the ped within 72 hours of their previous visit. we excluded patients who were instructed to return, had previously left without being seen, arrived without a parent, were wards of the state, or did not speak english. after obtaining consent, the principal investigator (ce) conducted confidential, in-person, tape-recorded interviews with parents during ped return visits. parents answered 12 open-ended questions and 9 closed-ended questions using a five-point likert scale. responses to open-ended questions were analyzed using thematic analysis techniques. the scaled responses were grouped into three categories of agree, disagree, or neutral. results: from the 49 closed-ended responses, 86% of parents agreed that their children were getting sicker, and 92% agreed that their children were not getting better. 80% agreed that they were unsure how to treat the illness, however only 41% agreed they did not feel figure 1 : frequency distribution of number of pca activations comfortable taking care of the illness. only 29% agreed that the medical condition and/or the instructions were not clearly explained in the first visit. some common themes from the open-ended questions included worsening or lack of improvement of symptoms. many parents reported having unanswered questions about the cause of the illness and hoped to find out the cause during the return visit. conclusion: most parents brought their children back to the ped because they believed the symptoms had worsened or were not improving. although a large proportion of parents believed that the medical condition was clearly explained at the first visit, many parents still had unanswered questions about the cause of their child's illness. while worsening symptoms seemed to drive most return visits, it is possible that some visits related to failure to improve might be prevented during the first ped visit through a more detailed discussion of disease prognosis and expected time to recover. pediatric background: experience indicates that it is difficult to effectively quell many parents' anxiety toward pediatric fevers, making this a common emergency department (ed) complaint. the question remains as to whether athome treatment has any effect on the course of emergency department treatment or length of stay in this population. objectives: to determine whether anti-pyretic treatment prior to arrival in the emergency department affects the evaluation or emergency department length of stay of febrile pediatric patients. methods: a convenience sample of children, ages 0-12 years, who presented to a tertiary care ed with chief complaint of fever were enrolled. parents were asked to participate in an eight-question survey. questions related to demographic information, pre-treatment of the fever, contact with primary care providers prior to ed arrival, and immunization status. upon admission or discharge, investigators recorded information regarding length of stay, laboratory tests and imaging ordered, and medications given. results: eighty-one patients were enrolled in the study. seventy-six percent of the patients were pre-treated with some form of anti-pyretic by the caregiver prior to ed arrival. there was no significant effect of pre-treatment on whether laboratory tests or medications were ordered in the ed or whether the patient was admitted or discharged. the length of ed stay was found to be significantly shorter among those who received anti-pyretics prior to arrival (184 ± 11 vs. 247 ± 36 minutes; p = 0.03). conclusion: among febrile children, those who receive anti-pyretics prior to their ed visit had statistically significant shorter length of stays. this also supports implementation of triage or nursing protocols to administer an anti-pyretic as soon as possible in the hope of decreasing ed throughput times. background: during the past two decades, the prevalence of overweight (bmi percentile >95) in children has more than doubled, reaching epidemic proportions both nationally and globally. the public health burden is enormous given the increased risk of adult obesity as well as the adverse consequences on cardiovascular, metabolic, and psychological health. despite the overwhelming prevalence, the effect of obesity on emergency care has received little attention. objectives: the goal of this study is to determine the relation of weight on reported emergency department visits in children from a nationally representative sample. methods: weight (as reported by parents) and height along with frequency of and reason for emergency department (ed) use in the last 12 months were obtained from children aged 10-17 y (n = 46,707) in the cross-sectional, telephone-administered, national survey of children's health (nsch). bmi percentiles were calculated using sex-specific bmi for age growth charts from the cdc (2000). children were categorized as: underweight (bmi percentile£5), normal weight (>5 to <85), at-risk for overweight (85 to <95), and overweight ( ‡95). prevalence of ed use was estimated and compared across bmi percentile categories using chisquare analysis and multivariable logistic regression. taylor-series expansion was used for variance estimation of the complex survey design. results: the prevalence of at least one ed use in the past 12 months increased with increasing bmi percentiles (figure 1, p < 0.001). additionally, overweight children were more likely to have more than one visit. overweight children were also less likely to report an injury, poisoning, or accident as the reason for ed visit compared to other bmi categories (47, 55, 59, 54% in overweight, at-risk, normal, and underweight respectively, p < 0.05). conclusion: as rates of childhood obesity continue to grow in the u.s., we can expect greater demands on the ed. this will likely translate into an increased emphasis on the care of chronic conditions rather than injuries and accidents in the pediatric ed setting. results: mean pediatric satisfaction score was 84.1 (sd 3.9) compared with 81.4 (3.2) for adult patients (p < 0.001); monthly sample sizes ranged from 14-74 and from 30-125 for the two populations, respectively. both populations showed an increase in satisfaction after opening of the ped-ed. for both populations there was no significant trend in patient satisfaction from the beginning of the study period to the opening of the ped-ed, but after the opening the models of the populations differed. the pediatric satisfaction model was an interrupted two-slope model, with an immediate jump of 3.5 points in november and an increase of 0.2 points per month thereafter. in contrast, adult satisfaction scores did not show a jump but increased linearly (two slope model) after 11/2011 at a rate of 0.3 per month. prior to the opening of the ped-ed, mean monthly pediatric and adult satisfaction scores were 81.5 (2.4) and 79.5 (2.8), respectively (difference 2.0 95% ci 0.1-3.8, p = 0.04). after the opening the mean scores were 86.8 (3.1) and 83.2 (2.4), respectively (difference 3.6, 95% ci 2.1-5.0, p < 0.001). conclusion: opening of a dedicated ped-ed was associated with a significant increase in patient satisfaction scores both for children and adults. patient satisfaction for children, as compared to adults, was higher before and after opening a ped-ed. the background: there are racial disparities in outcomes among injured children. in particular, black race appears to be an independent predictor of mortality. objectives: to evaluate disparities among ed visits for unintentional injuries among children ages 0-9. methods: five years of data (2004) (2005) (2006) (2007) (2008) from the national hospital ambulatory cares survey were combined. inclusion criteria were defined as unintentional injury visits (e-code 800.0 to 869.9 or 888.0 to 929.9) and age 0-9 years. visit rates per 100 population (defined by the us census) were calculated by race and age group. weighted multivariate logistic regression analysis was performed to describe associations between race and specific outcome variables and related covariates. primary statistical analyses were performed using sas version 9.1.3. results: 21,524,000 of 585,294,000 weighted ed visits met our inclusion criteria (3.7%). per 100 persons, black children had 1.5 times as many ed visits for unintentional injuries as whites (table) . there were no racial differences in the sex ratio (1.4 boy visits: 1 girl), proportion of visits by age, ed disposition, immediacy with which they needed to be seen, whether or not they were evaluated by an attending physician, metropolitan vs. rural hospital, admission length of stay, mode of transportation for ed arrival, number of procedures, diagnostic services, or ed medications. background: sudden cardiac arrests in schools are infrequent, but emotionally charged events. little data exist that describes aed use in these events. objectives: the purpose of our study was to 1) describe characteristics and outcomes of school cardiac arrests (ca), and 2) assess the feasibility of conducting bystander interviews to describe the events surrounding school ca. methods: we performed a telephone survey of bystanders to ca occurring in k-12 schools in communities participating in the cardiac arrest registry to enhance survival (cares) database. the study period was from 8/2005-12/2010 and continued in one community through 2011. utstein style data and outcomes were collected from the cares database. a structured telephone interview of a bystander or administrative personnel was conducted for each ca. a descriptive summary was used to assess for the presence of an aed, provision of bystander cpr (bcpr), and information regarding aed deployment, training, and use and perceived barriers to aed use. descriptive data are reported. results: during the study period there were 30,603 ca identified at cares communities, of which 73 were identified as educational institutions. of these, 46 (0.15%) events were at k-12 schools with 21 (45.7%) being high schools. of the 46 arrests, a minority were children (15 (32.6%) < age 19), most (32, 84.8%) were witnessed, a majority (36, 76.1%) received bcpr, and 26 (56.5%) were initially in ventricular fibrillation (vf). most arrests 28/40 (70%) occurred during the school day (7a-5p). overall, 14 (30.4%) survived to hospital discharge. interviews were completed for 29 of 46 (63.0%) k-12 events. eighteen schools had an aed on site. most schools (84.2%) with aeds reported that they had a training program and personnel identified for its use. an aed was applied in 10 of 18 patients, and of these 8 were in vf and 4 survived to hospital discharge. multiple reasons for aed non-use (n = 8) were identified. conclusion: cardiac arrests in schools are rare events; most patients are adults and received bcpr. aed use was infrequent, even when available, but resulted in excellent (4/10) survival. further work is needed to understand aed non-use. post-event interviews are feasible and provide useful information regarding cardiac arrest care. physician background: gastroenteritis is a common childhood disease accounting for 1-2 million annual pediatric emergency visits. current literature supports the use of anti-emetics reporting improved oral re-hydration, cessation of vomiting, and reduced need for iv re-hydration. however, there remains concern that using these agents may mask alternative diagnoses. objectives: to assess outcomes associated with use of a discharge action plan using ed-dispensed ondansetron at home in the treatment of gastroenteritis. methods: a prospective, controlled, observational trial of patients presenting to an urban pediatric emergency department (census 22,400) over a 12-month period for acute gastroenteritis. fifty patients received ondansetron in the ed. twenty-nine patients were enrolled in the pediatric emergency department discharge action plan (ped-dap) where ondansetron for home use was dispensed by the treating clinician. twenty-one patients were controls. control patients did not receive home ondansetron. ped-dap patients were given instructions to administer the ondansetron for ongoing symptoms any time 6 hours post ed discharge. all patients were followed by phone at 7-14 days to assess for the following: time of emesis resolution, alternative diagnoses, unscheduled visits, and adverse events. results: all 50 patients were followed by phone. 24/29 ped-dap patients received home ondansetron. 21/29 patients had resolution of emesis in the ed. 7/29 had resolution of their emesis between time of discharge and 24 hours. 1/29 of ped-dap patients reported emesis after 24 hours from ed discharge. five patients reported an unscheduled visit. all five return visits returned to the ed (1/5 returned for emesis, 4/5 for diarrhea). 17/21 controls reported resolution of symptoms within the ed. 2/21 of controls had resolution between time of discharge and 24 hours. 1/21 of the control patients had resolution with between 24 and 48 hours post discharge. 1/21 had an unscheduled appointment with the pmd at 72 hours post-discharge for ongoing fever and nausea. in follow-up there were no alternative diagnoses identified. the effect of the ped-dap on resolution of emesis between discharge and 24 hours appears to be statistically significant (p value < 0.04). conclusion: ondansetron given in schedule with a discharge action plan appears to provide a modest benefit in resolution of symptoms relative to a control population. objectives: to determine the repeatability coefficient of a 100 mm vas in children aged 8 to 17 years in different circumstances: assessments done either at 3 or 1 minute interval, when asked to recall their score or to reproduce it. methods: a prospective cohort study was conducted using a convenience sample of patients aged 8 to 17 years presenting to a pediatric ed. patients were asked to indicate, on a 100 mm paper vas, how much they liked a variety of food with four different sets of three questions: (set 1) questions at 3 minute interval with no specific instruction other than how to complete the vas and no access to previous scores, (set 2) same format as set 1 except for questions at 1 minute interval, (set 3) same as set 1 except patients were asked to remember their answers, and (set 4) same as set 1 except patients were shown their previous answers. for each set, the repeatability coefficient of the vas was determined according to the bland-altman method for measuring agreement using repeated measures: 1.96 x ö 2 x s w where s w is the within-subject standard deviation by anova. the sample size required to estimate s w to 10% of the fraction value as recommended was 96 patients if we obtained three measurements for each patient. results: a total of 100 patients aged 12.1 ± 2.4 years were enrolled. the repeatability coefficient for the questions asked at 3 minute intervals was 12 mm, and 8 mm when asked at 1 minute interval. when asked to remember their previous answers or to reproduce them, the repeatability coefficient for the questions was 7 mm and 6 mm, respectively. conclusion: the condition of the assessments (variation in intervals or patients asked to remember or to reproduce their previous answers) influence the testretest reliability of the vas. depending on circumstances, the theoretical test-retest reliability in children aged 8 to 17 years varies from 6 to 12 mm on a 100 mm paper vas. background: skull radiographs are a useful tool in the evaluation of pediatric head trauma patients. however, there is no consensus on the ideal number of views that should be obtained as part of a standard skull series in the evaluation of pediatric head trauma patients. objectives: to compare the sensitivity and specificity of a two-and four-film x-ray series in the diagnosis of skull fracture in children, when interpreted by pediatric emergency medicine physicians. methods: a prospective, crossover experimental study was performed in a tertiary care pediatric hospital. the skull radiographs of 100 children were reviewed. these were composed of the 50 most recent cases of skull fracture for which a four-film radiography series was available at the primary setting and 50 controls, matched for age. two modules, containing a random sequence of two-and four-film series of each child, were constructed in order to have all children evaluated twice (once with two films and once with four films). board-certified or -eligible pediatric emergency physicians evaluated both modules two to four weeks apart. the interpretation of the four-film series by a radiologist, or when available, the findings on ct scan, served as the gold standard. accuracy of interpretation was evaluated for each patient. the sensitivity and specificity of the two-film versus the four-film skull xray series, in the identification of fracture, were compared. this was a non-inferiority cross-over study evaluating the null hypothesis that a series with two views would have a sensitivity (specificity) that is inferior by no more than 0.055 compared to a series with four views. a total of 50 controls and 50 cases were needed to establish non-inferiority of the two-film series versus the four-film series, with a power of 80% and a significance level of 5%. results: ten pediatric emergency physicians participated in the study. for each radiological series, the proportion of accurate interpretation varied between 0.20 to 1.00. the four-film series was found to be more sensitive in the detection of skull fracture than a two-film series (difference: 0.084, 95%ci 0.030 to 0.139). however, there was no difference in the specificity (difference: 0.004, 95%ci )0.024 to 0.033). conclusion: for children sustaining a head trauma, a four-film skull radiography series is more sensitive than a two-film series, when interpreted by pediatric emergency physicians. the objectives: we developed a free online video-based instrument to identify knowledge and clinical reasoning deficits of medical students and residents for pediatric respiratory emergencies. we hypothesized that it would be a feasible and valid method of differentiating educational needs of different levels of learners. methods: this was an observational study of a free, web-based needs assessment instrument that was tested on 44 third and fourth year medical students (ms3-4) and 29 pediatric and emergency medicine residents (r1-3). the instrument uses youtube video triggers of children in respiratory distress. a series of cased-based questions then prompts learners to distinguish between upper and lower airway obstruction, classify disease severity, and manage uncomplicated croup and bronchiolitis. face validity of the instrument was established by piloting and revision among a group of experienced educators and small groups of targeted learners. final scores were compared across groups using t-tests to determine the ability of the instrument to differentiate between different levels of learners (concurrent validity). cronbach's alpha was calculated as a measure of internal consistency. results: response rates were 19% among medical students and 43% among residents. the instrument was able to differentiate between junior (ms3, ms4, and r1) and senior (r2, r3) learners for both overall mean score (61% vs.78%, p < 0.01) and mean video portion score (74 vs. 84%, p = 0.02). table 1 compares results of several management questions between junior and senior learners. cronbach's alpha for the test questions was 0.47. conclusion: this free online video-based needs assessment instrument is feasible to implement and able to identify knowledge gaps in trainees' recognition and management of pediatric respiratory emergencies. it demonstrates a significant performance difference between the junior and senior learners, preliminary evidence of concurrent validity, and identifies target groups of trainees for educational interventions. future revisions will aim to improve internal consistency. results: the survey response rate was 87% (60/69). among responding programs, 40 (67%) reside within a children's hospital (vs. general ed); 51 (85%) are designated level i pediatric trauma centers. forty-three (72%) programs accept 1-2 pem fellows per year; 53 (88%) provided at least some eus training to fellows, and 42 (70%) offer a formal eus rotation. on average this training has existed for 3 ± 1 years and the mean duration of eus rotations is 4 ± 2 weeks. twenty-eight (67%) programs with eus rotations provide fellow training in both a general ed and a pediatric ed. there were no hospital or program level factors associated with having a structured training program for pem fellows. conclusion: as of 2011, the majority of pem fellowship programs provide eus training to their fellows, with a structured rotation being offered by most of these programs. background: ed visits are an opportunity for clinicians to identify children with poor asthma control and intervene. children with asthma who use eds are more likely than other children to have poor control, not be using controller medications, and have less access to traditional sources of primary care. one significant barrier to ed-based interventions is recognizing which children have uncontrolled asthma. objectives: to determine whether the pacci, a 12item parent-administered questionnaire, can help ed clinicians better recognize patients with the most uncontrolled asthma and differentiate between intermittent and persistent asthma. methods: this was a randomized controlled trial performed at an urban pediatric ed. parents were asked to answer questions about their child's asthma including drug adherence and history of exacerbations, as well as answer demographic questions. using a convenience sample of children 1-18 years presenting with an asthma exacerbation, attending physicians in the study were asked to complete an assessment of asthma control. physicians were randomized to receive a completed pacci (intervention) or not (control group). using an intent-to-treat approach, clinicians' ability to accurately identify 1) four categories of control used by the national heart, lung, and blood institute (nhlbi) asthma guidelines, 2) intermittent vs. persistent level asthma, and 3) controlled / mildly uncontrolled vs. moderate/severely uncontrolled asthma were compared for both groups using chi-square analysis. results: between january and august 2011, 57 patients were enrolled. there were no statistically significant differences between the intervention and control groups for child's sex, age, race and parents' education. conclusion: the pacci improves ed clinicians' ability to categorize children's asthma control according to nhlbi guidelines, and the ability to determine when a child's control has been worsening. ed clinicians may use the pacci to identify those children in greatest need for intervention, to guide prescription of controller medications, and communicate with primary care providers about those children failing to meet the goals of asthma therapy. figure) . fewer than half of physicians reported the parent of a 2-year-old being discharged from their ed following an mvc-related visit would receive either child passenger safety information or referrals (table) . conclusion: emergency physician report of child passenger safety resource availability is associated with trauma center designation. even when resources are available, referrals from the ed are infrequent. efforts to increase referrals to community child passenger safety resources must extend to the community ed settings where the majority of children receive injury care. background: pediatric subspecialists are often difficult to access following ed care especially for patients living far from providers. telemedicine (tm) can potentially eliminate barriers to access related to distance, and cost. objectives: to evaluate the overall resource savings and access that a tm program brings to patients and families. methods: this study took place at a large, tertiary care regional pediatric health care system. data were collected from 1/2011-10/2011. metrics included travel distance saved (round trip between tm presenting sites and the location of the receiving sites), time savings, direct cost savings (based on $0.55/mile) and potential work and school days saved. indirect costs were calculated as travel hrs saved/encounter (based on an average speed of 55 miles/hr). demographics and services provided were included. results: 690 tm consults were completed by 13 separate pediatric subspecialty services. most patients were school aged (86% >/= 5yrs old objectives: to analyze test characteristics of the pathway and its effects on ed length of stay, imaging rates, and admission rate before versus after implementation. methods: children ages 3-18 presenting to one academic pediatric ed with suspicion for appendicitis from october 2010 -august 2011 were prospectively enrolled to a pathway using previously validated lowand high-risk scoring systems. the attending physician recorded his or her suspicion of appendicitis and then used one of two scoring systems incorporating history, physical exam, and cbc. low-risk patients were to be discharged or observed in the ed. high-risk patients were to be admitted to pediatric surgery. those meeting neither low-nor high-risk criteria were evaluated in the ed by pediatric surgery, with imaging at their discretion. chart review and telephone follow-up were conducted two weeks after the visit. charts of a random sample of patients with diagnoses of acute appendicitis or chief complaint of abdominal pain and undergoing a workup for appendicitis in the eight months before and after institution of the pathway were retrospectively reviewed by one or two trained abstractors. results: appendicitis was diagnosed in 65 of 178 patients prospectively enrolled to the pathway (37%). mean age was 9.6 years. of those with appendicitis, 63 were not low-risk (sensitivity 96.9%, specificity 48.7%). the high-risk criteria had a sensitivity of 73.8% and specificity of 77.0%. a priori attending physician assessment of low risk had a sensitivity of 100% and specificity of 49.6%. a priori assessment of high risk had a sensitivity of 58.5% and specificity of 90.2%. we reviewed 232 visits prior to the pathway and 290 after. mean ed length of stay was similar (256 minutes before versus 257 after). ct was used in 12.1% of visits before and 7.3% after (p = 0.07). use of ultrasound increased (44.8% before versus 55.9% after, p < 0.02). admission rates were not significantly different (48.3% before versus 42.7% after, p = 0.2). conclusion: the low-risk criteria had good sensitivity in ruling out appendicitis and can be used to guide physician judgment. institution of this pathway was not associated with significant changes in length of stay, utilization of ct, or admission rate in an academic pediatric ed. computer-delivered alcohol and driver safety behavior screening and intervention program initiated during an emergency department visit mary k. murphy 1 , lucia l. smith 2 , anton palma 2 , david w. lounsbury 2 , polly e. bijur 2 , paul chambers 2 1 yale university, new haven, ct; 2 albert einstein college of medicine, bronx, ny background: alcohol use is involved in 32 percent of all fatal motor vehicle crashes and recent estimates show that at least 448,000 people were injured due to distracted driving last year. patients who visit the emergency department (ed) are not routinely screened for driver safety behavior; however, large numbers of patients are treated in the ed every day creating an opportunity for screening and intervention on important public health behaviors. objectives: to evaluate patient acceptance and response to a computer-based traffic safety educational intervention during an ed visit and one month follow-up. methods: design. pre /post educational intervention. setting. large urban academic ed serving over 100,000 patients annually. participants. medically stable adult ed patients. intervention. patients completed a self-administered, computer-based program that queried patients on alcohol use and risky driving behaviors (texting, talking, and other forms of distracted driving). the computer provided patients with educational information on the dangers of these behaviors and collected data on patient satisfaction with the program. staff called patients one month post ed visit for a repeat query. results: 150 patients participated; average age 39 (21-70), 58% hispanic, 52% male. 96% of patients reported the program was easy to use and were comfortable receiving this education via computer during their ed visit. self-reported driver safety behaviors pre, post intervention (% change): driving while talking on the phone 45%,16% ()29%, p = 0.001), aggressive driving 44%,15% ()29%, p = 0.001), texting while driving 28%,9% ()19%, p = 0.001), driving while drowsy 18%,4% ()14%, p = 0.002), drinking in excess of nih safe drinking guidelines15%,%7 ()8%, p = 0.039), drinking and driving 10%,1% ()9%, p = 0.006). conclusion: we found a high prevalence of selfreported risky driving behaviors in our ed population. at 1 month follow-up, patients reported a significant decrease in these behaviors. overall patients were very satisfied receiving educational information about these behaviors via computer during their ed visit. this study indicates that a low-intensity, computer-based educational intervention during an ed visit may be a useful approach to educate patients about safe driving behaviors and promote behavior change. prevalence of depression among emergency department visitors with chronic illness janice c. blanchard, benjamin l. bregman, jeffrey smith, mohammad salimian, qasem al jabr george washington university, washington, dc background: persons with chronic illnesses have been shown to have higher rates of depression than the general population. the effect of depression on frequent emergency department (ed) use among this population has not been studied. objectives: this study evaluated the prevalence of major depressive disorder (mdd) among persons presenting with depression to the george washington university ed. we hypothesized that patients with chronic illnesses would be more likely to have mdd than those without. methods: this was a single center, prospective, crosssectional study. we used a convenience sample of noncritically ill, english-speaking adult patients presenting with non-psychiatric complaints to an urban academic ed over 6 months in 2011. subjects were screened with the phq 9, a nine-item questionnaire that is a validated, reliable predictor of mdd. we also queried respondents about demographic characteristics as well as the presence of at least one chronic disease (heart disease, hypertension, asthma, diabetes, hiv, cancer, kidney disease, or cerebrovascular disease). we evaluated the association between mdd and chronic illnesses with both bivariate analysis and multivariate logistic regression controlling for demographic characteristics (age, race, sex, income, and insurance coverage). results: our response rate was 90.7% with a final sample size of 1012. of our total sample, 525 (51.9%) had at least one of the chronic illnesses defined above. of this group, 162 (30.9%) screened positive for mdd as compared to 82 (16.6%) of the group without chronic illnesses (p < 0.0001). in multivariate analysis, persons with chronic illnesses had an odds ratio for a positive depression screen of 1.80 (1.31, 2.50) as compared to persons without illness. among the subset of persons with chronic illnesses (n = 525), 46.9% had ‡3 visits in the prior 364 days as compared to 34.4% of persons with chronic illnesses without mdd (p = 0.007). conclusion: our study found a high prevalence of untreated mdd among persons with chronic illnesses who present to the ed. depression is associated with more frequent emergency department use among this population. initial blood alcohol level aids ciwa in predicting admission for alcohol withdrawal craig hullett, douglas rappaport, mary teeple, daniel butler, arthur sanders university of arizona, tucson, az background: assessment of alcohol withdrawal symptoms is difficult in the emergency department. the clinical institute withdrawal assessment (ciwa) is commonly used, but other factors may also be important predictors of withdrawal symptom severity. objectives: the purpose of this study is to determine whether ciwa score at presentation to triage was predictive of later admission to the hospital. methods: a retrospective study of patients presenting to an acute alcohol and drug detoxification hospital was performed from july 2010 through january 2011. patients were excluded if other drug withdrawal was present in addition to alcohol. initial assessment included age, sex, vital signs, and blood alcohol level (bal) in addition to hourly ciwa score. admission is indicated for a ciwa score of 10 or higher. data were analyzed by selecting all patients not immediately admitted at initial presentation. logistic regression using wald's criteria for stepwise inclusion was used to determine the utility of the initially gathered ciwa, bal, longest sobriety, liver cirrhosis, and vital signs in predicting subsequent admission. results: there were 123 patients who fit the inclusion criteria, with 9 admitted for treatment at initial intake and another 27 admitted during the following 10 hours. logistic regression indicated that presenting bal was a strong predictor (p = 0.01) of admission for treatment after initial presentation, as was presenting ciwa (p = 0.03). thus, presenting bal provided a substantial addition above initial ciwa in predicting later admission. no other variables added significantly to the prediction of later admission. to determine the interaction between presenting bal and ciwa scores, we ran a repeated measures analysis of the first five ciwa scores (from presentation to 4 hours later), using bal split into low (bal < 0.10) and high (bal > 0.10) groups (see figure) . their interaction was significant, f (1, 93) = 11.86, p < 0.001, g 2 = 0.11. those presenting with higher initial bal had suppressed ciwa scores that rose precipitously as the alcohol cleared. those with low presenting bal showed a decline in ciwa over time conclusion: initial assessment using the common assessment tool ciwa is aided significantly by bal assessment. patients with higher presenting bal are at higher risk for progression to serious alcohol withdrawal symptom. objectives: to describe patient and visitor characteristics and perspectives on the role of visitors in the ed and determine the effect of visitors on ed and hospital outcome measures. methods: this cross-sectional study was done in an 81,000-visit urban ed, and data were attempted to be collected from all patients over a consecutive 96-hour period from august 25 to 28, 2011. trained data collectors were assigned to the ed continuously for the study period. patients assigned to a rapid care section of the ed (24%) were excluded. a visitor was defined as a person other than a health care provider (hcp) or hospital staff present in a patient's room at any time. patient perspectives on visitors were assessed in the following domains: transportation, emotional support, physical care, communication, and advocating for the patient. ed and hospital outcome measures pertaining to ed length of stay (los) and charges, hospital admission rate, hospital los and charges were obtained from patient medical records and hospital billing. data analyses included frequencies, student's t-tests for continuous variables, and chi-square tests of association for categorical variables. all tests for significance were two-sided. objectives: to examine the effect of sunday alcohol availability on ethanol-related visits and alcohol withdrawal visits to the ed. methods: study design was a retrospective beforeafter study using electronically archived hospital data at an urban, safety net hospital. all adult non-prisoner ed visits from 1/1/2005 to 12/31/2009 were analyzed. an ethanol-related ed visit was defined by icd-9 codes related to alcohol (291.x, 303.x, 305.0, 980.0 ). an alcohol withdrawal visit was defined by icd-9 codes of delirium tremens (291.0), alcohol psychosis with hallucination (291.3), and ethanol withdrawal (291.81). we generated a ratio of ethanol-related ed visits to total ed visits (ethanol/total) and ratio of alcohol withdrawal ed visits to total ed visits (withdrawal/total). a day was redefined as 8 am to 8 am. the ratios were averaged within the four seasons to account for seasonal variations. data from summer 2008 were dropped as it spanned the law change. we stratified data into sunday and non-sunday days prior to analysis to isolate the effects of the law change. we used multivariable linear regression to estimate the association of the ratio with the law change while adjusting for time and the seasons. each ratio was modeled separately. the interaction between time and the law change was assessed using p < 0.05. results: during the study there were a total of 212,189 ed visits including 12,042 (6% of total) ethanol-related visits and 5,496 (3% of total) alcohol withdrawal visits. unadjusted ratios in seasonal blocks are plotted in the figure with associated 95% ci and best fit regression line for before and after law change, respectively. after adjusting for time and season in the multivariable linear regression, we found no significant association of either ethanol/total or withdrawal/total with the law change. this remained true for both sunday and non-sunday data. all interactions assessed were not significant. conclusion: the change in colorado law to allow the sale of full-strength alcoholic beverages on sundays did not significantly affect ethanol-related or alcohol withdrawal ed visits. background: olanzapine is a second-generation antipsychotic (sga) with actions at the serotonin/histamine receptors. post-marketing reports and a case report have documented dangerous lowering of blood pressure when this antipsychotic is paired with benzodiazepines, but a recent small study found no bigger decreases in blood pressure compared to another antipsychotic like haloperidol. decreases in oxygen saturations, however, were larger when olanzapine was combined with benzodiazepines in alcohol-intoxicated patients. it is unclear whether these vital sign changes are associated with the intramuscular (im) route only. objectives: the assessment of vital signs following administration of either oral (po) or im olanzapine, either with or without benzodiazepines (benzos) and with or without concurrent alcohol intoxication. methods: this is a structured retrospective chart review of all patients who received olanzapine in an academic medical center ed from 2004-2010 who had vital signs documented both before medication administration and within four hours afterwards. vital signs were calculated as pre-dose minus lowest post-dose vital sign within 4 hours, and were analyzed in an anova with route (im/po), benzo use (+/)), and alcohol use (+/)) as factors. significance level was set to <0.05. results: there were 482 patients who received olanzapine over the study period. a total of 275 patients (225 po, 50 im) met inclusion criteria. systolic blood pressures decreased across all groups as patients reduced their agitation. neither the route of administration, concurrent use of benzos, nor the use of alcohol were associated with significant changes in systolic bp (p = ns for all comparisons; see figure 1 ). decreases in oxygen saturations, however, were significantly larger for alcoholintoxicated patients who subsequently received im olanzapine + benzos compared to other groups (route: p < 0.001; alcohol: p < 0.01; route x alcohol: p < 0.001; route x benzos x alcohol: p < 0.05; see figure 2 ). conclusion: alcohol and benzos are not associated with significant decreases in blood pressure after po olanzapine, but im olanzapine + benzos is associated with potentially significant oxygen desaturations in patients who are intoxicated. intoxicated patients may have differential effects with the use of im sgas such as olanzapine when combined with benzos, and should be studied separately in drug trials. patients with a psychiatric diagnosis rasha buhumaid, jessica riley, janice blanchard george washington university, washington, dc background: literature suggests that frequent emergency department (ed) use is common among persons with a mental health diagnosis. few studies have documented risk factors associated with increased utilization among this population. objectives: to understand demographic characteristics of frequent users of the emergency department and describe characteristics associated with their visits. it was hypothesized that frequent visitors would have a higher rate of medical comorbidities than infrequent visitors. methods: this was a retrospective study of patients presenting to an urban, academic emergency department in 2009. a cohort of all patients with a mental health-related final icd-9 coded diagnosis (axis i or axis ii) was extracted from the electronic medical record. using a standard abstraction form, a medical chart review collected information about medical comorbidities, substance abuse, race, age, sex, and insurance coverage, as well as diagnosis, disposition, and time of each visit. results: our sample consisted of 109 frequent users ( ‡4 visits in a 365 day period) and 442 infrequent users (£3 visits in a 365 day period). frequent users were more likely to be male (68% vs. 54.5% p = 0.01), black (86% vs. 59% p < 0.0001), and had a higher average number of comorbid conditions (2.0, 95%ci 1.73,2.26) as compared to infrequent users (1.0, 95%ci 0.90,1.10). a higher percentage of visits in the infrequent user group occurred during the day (49% vs. 38.3% p < 0.0001) while a higher number of visits in the frequent users occurred after midnight (24.3% vs. 16.6% p = 0.0003). visits in the frequent user group were less likely to be for a psychiatric complaint (34.3% vs. 81.2%) and less likely to result in a psychiatric admission (18.3% versus 56.7%) as compared to the infrequent user group (p < 0.0001). conclusion: our data indicate that among patients with psychiatric diagnoses, those who make frequent ed visits have a higher rate of comorbid conditions than infrequent visitors. despite their increased use of the ed, frequent visitors have a significantly lower psychiatric admission rate. many of the visits by frequent users are for non-psychiatric complaints and may reflect poor access to outpatient medical and mental health services. emergency departments should consider interventions to help address social and medical issues among mental health patients who frequently use ed services. background: the world health organization estimates that one million people die annually by suicide. in the u.s., suicide is the fourth leading cause of death between the ages of 10 and 65. many of these patients are seen in ed, while outpatient visits for depression are also high. no recent analysis has compared these groups in the recent years. objectives: to determine if there is a relationship between the incidence of suicidal and depressed patients presenting to emergency departments and the incidence of depressed patients presenting to outpatient clinics from 2002-2008. the secondary objective is to analyze trends in suicidal patients in the ed. methods: we used nhamcs (national hospital ambulatory medical care survey) and namcs (national ambulatory medical care survey), national surveys completed by the centers for disease control, which provide a sampling of emergency department and outpatient visits respectively. for both groups, we used mental-health-related icd-9-cm, e codes and reasons for visit. we compared suicidal and depressed patients who presented to the ed, to those who presented to outpatient clinics. our subgroup analyses included age, sex, race/ethnicity, method of payment, regional variation, and urban verses rural distribution. results: ed visits for depression (1.14%) and suicide attempts (0.49%) remained stable over the years, with no significant linear trend. however, office visits for depression significantly decreased from 3.14% of visits in 2002 to 2.65% of visits in 2008. non-latino whites had a higher percentage of ed visits for depression (1.25%) and suicide attempt (0.57%) (p < 0.0001), and a higher percentage of office visits for depression than all other groups. among patients age 50-69 years, ed visits for suicide attempt significantly increased from 0.12% in 2002 to 0.44% in 2008. homeless patients had a higher percent of ed visits for depression (6.5%) and suicide attempt ( background: for potentially high-risk ed patients with psychiatric complaints, efficient ed throughput is key to delivering high-quality care and minimizing time spent in an unsecured waiting room. objectives: we hypothesized that adding a physician in triage would improve ed throughput for psychiatric patients. we evaluated the relationship between the presence of an ed triage physician and waiting room (wr) time, time to first physician order, time to ed bed assignment, and time spent in an ed bed. methods: the study was conducted from 11/2009-2/ 2011 at an academic ed with 55000 annual visits and a dedicated on-site emergency psychiatric unit. we performed a pre/post retrospective observational cohort study using administrative data, including weekend visits from noon-10pm, 8 months pre and post addition of weekend triage physicians. after adjusting for patient age, sex, insurance status, emergency severity index score, mode of arrival, ed occupancy rate, wr count, boarding count, and average wr los, multiple linear regression evaluated the relationship between the presence of a triage physician and four ed throughput outcomes: time spent in the wr, time to first order, time spent in an ed bed, and the total ed los. results: 565 visits met inclusion criteria, 280 in the 8 months before and 285 in the 8 months after physicians were assigned to triage on weekends. table 1 reports demographic data; multivariate analysis results are found in table 2 . the presence of a triage physician was associated with an 8 (95% ci 0.6-15.2) minute increase in wr time and no associated change in time to first order, time spent in an ed bed, or in the overall ed los. conclusion: use of triage physicians has been reported to decrease the time patients spend in an ed bed and improve ed throughput. however, for patients with psychiatric complaints, our analysis revealed a slight increase in wr time without evident change in the time to first order, time spent in an ed bed, or total ed los. improvements in ed throughput for psychiatric patients will likely require system-level changes, such as reducing ed boarding and improving lab efficiency to speed the process of medical clearance and reduce time spent in the unsecured wr. these findings may not be generalizable to eds without a dedicated ed psychiatric unit with full-time social workers to assist with disposition. initial assessment included ciwa scoring, repeated hourly, as well as other variables (see table 1 ). treatment and admission to the inpatient hospital was indicated for a ciwa score of 10 or higher. statistical analysis was performed utilizing repeated measures general linear modeling for ciwa scores and anova for all other variables. results: there were 123 patients who fit the inclusion criteria, with 9 admitted for treatment at initial intake and another 27 admitted during the following 10 hours. the table below compares the three most prevalent ethnic populations seen at our hospital. native americans presented at a significantly younger age (p < 0.05) than the other two ethnicities. initial ciwa scores taken on admission were significantly lower in the native american group than the other two groups (p < 0.05) and at 1 hour a difference existed but failed to reach significance. repeated measures analysis indicate that ciwa scores progressed in a u-shaped curvilinear fashion (see figure 1 ) conclusion: initial assessment utilizing ciwa scores appears to be affected by ethnicity. care must be taken when assessing and making decisions on a single initial ciwa score. further research is needed in this area as our numbers are small and differences might be seen in subsequent scoring. in addition, our study consists of primarily male patients and does not include african-american patients. background: age is a risk factor for adverse outcomes in trauma, yet evidence supporting the use of specific age cut-points to identify seriously injured patients for field triage is limited. objectives: to evaluate under-triage by age, empirically examine the association between age and serious injury for field triage, and assess the potential effect of mandatory age criteria. methods: this was a retrospective cohort study of injured children and adults transported by 48 ems agencies to 105 hospitals in 6 regions of the western u.s. from 2006-2008. hospital records were probabilistically linked to ems records using trauma registries, emergency department data, and state discharge databases. serious injury was defined as an injury severity score (iss) ‡16 (the primary outcome). we assessed under-triage (triage-negative patients with iss ‡16) by age decile, different mandatory age criteria, and used multivariable logistic regression models to test the association (linear and non-linear) between age and iss ‡ 16, adjusted for important confounders. results: 260,027 injured patients were evaluated and transported by ems over the 3-year period. under-triage increased markedly for patients over 60 years, reaching 58% for those over 90 years ( figure 1 ). mandatory age triage criteria decreased under-triage, while substantially increasing over-triage: one iss ‡ 16 patient identified for every 65 additional patients triaged to major trauma centers. among patients not identified by other criteria, age had a strong non-linear association with iss ‡ 16 (p < 0.01); the probability of serious injury steadily increased after 30 years, becoming more notable after 60 years ( figure 2 ). conclusion: under-triage in trauma increases in patients over 60 years, which may be reduced with mandatory age criteria at the expense of system efficiency. among patients not identified by other criteria, serious injury steadily increased after 30 years, though there was no age at which risk abruptly increased. background: although limited resuscitation with hemoglobin-based oxygen carriers (hbocs) improves survival in several polytrauma models, including those of traumatic brain injury (tbi) with uncontrolled hemorrhage (uh) via liver injury, their use remains controversial. objectives: we examine the effect of hboc resuscitation in a swine polytrauma model with uh by aortic tear +/) tbi. we hypothesize that limited resuscitation with hboc would offer no survival benefit and would have similar effects in a model of uh via aortic tear +/) tbi. methods: anesthetized swine subjected to uh inflicted via aortic tear +/) fluid percussion tbi underwent equivalent limited resuscitation with hboc, lr, or hboc+nitroglycerin (ntg) (vasoattenuated hboc) and were observed for 6 hours. comparisons were between tbi and no-tbi groups with adjustment for resuscitation fluid type using two-way anova with interaction and tukey kramer adjustment for individual comparisons. results: there was no independent effect of tbi on survival time after adjustment for fluid type (anova, tbi term p = 0.59) and there was no interaction between tbi and resuscitation fluid type (anova interaction term p = 0.12). there was a significant independent effect of fluid type on survival time (anova p = 0.005 background: intracranial hemorrhage (ich) after a head trauma is a problem frequently encountered in the ed. an elevated inr is recognized as a risk of bleeding. however, in a patient with an inr in normal range, a level associated with a lower risk of ich is not known. objectives: the aim of this study was to identify an inr threshold that could predict a decreased risk of an ich after a head trauma in patients with a normal inr. it is hypothesized that there is a threshold at which the likelihood of bleeding decreases significantly. methods: we did a study using data from a registry of patients with mild to severe head trauma (n = 3356) evaluated in a level i trauma center in canada between march 2008 and february 2011. all the patients with a documented scan interpreted by a radiologist and a normal inr, defined as a value less then 1.6, were included. we determined the correlation between inr value binned by 0.1 and the proportion of patients with an ich. threshold was defined by consensus as an abrupt change of more than 10% in the percentage of patients with ich. univariate frequency distribution was tested with pearson's chisquare test. logistic regression analysis was then used to study the effects of inr on ich with the following confounding factors: age, sex, and intake of warfarin, clopidogrel, or aspirin. results are presented with 95% confidence intervals. results: 751 patients met the inclusion criteria. the mean age was 55.3 years ± 29.9 and 65% were men. 267 patients (35.6%) had an ich on brain scan. we found a significantly lower risk of ich at a threshold of inr less than 1.0 (p < 0.001, univariate or = 0.37, 95%ci 0.25-0.54) and a strong correlation between the risk of bleeding for every increase of the inr (r 2 = 0.8987). in fact, after adjustment for confounding variables, every 0.1 inr increase was associated with an increased risk of having an ich (or 1.50; 95% ci 1.31-1.72). conclusion: we were able to demonstrate an inr threshold under which the probability of ich was significantly lower. we also found a strong association between the risk of bleeding and the increase in inr within a normal range, suggesting that clinicians should not be falsely reassured by a normal inr. our results are limited by the fact that this is a retrospective study and a small proportion of traumatic brain injured patients in our database had no scan or inr at their ed visit. a prospective cohort study would be needed to confirm our results. background: increasingly, patients with tbi are being seen and managed in the emergency neurology setting. knowing which early signs are associated with prognosis can be helpful in directing the acute management. objectives: to determine whether any factors early in the course of head trauma are associated with shortterm outcomes including inpatient admission, in-hospital mortality, and return to the hospital within 30 days. methods: this irb-approved study is a retrospective review of patients head injury presenting to our tertiary care academic medical center during a 9-month period. the dataset was created using redcap, a data management solution hosted by our medical school's center for translational science institute. results: the median age of the cohort (n = 500) was 26, iqr = 15-48yrs, with 62% being male. 84% had a gcs of 13-15 (mild tbi), 3% 9-13 (moderate tbi), and 13% gcs < 8 (severe tbi). 39% of patients were admitted to the hospital. the median length of hospital stay was 2 days, with an iqr of 1-5 days. of those admitted, 53% had an icu stay as well. the median icu los was also 2 days, with an iqr of 1-6days. twenty nine (6%) patients died during their hospital stay. lower gcs was predictive of inpatient admission (p = 0.0003) as well as icu days (p < 0.0001). significant predictors of re-admission to the hospital within 30 days included hypotension (p = 0.002) upon initial presentation. the prehospital and ed gcs scores were not statistically significant. significant predictors of in-hospital death in a model controlling for age included bradycardia (p = 0.0042), hyperglycemia (p = 0.0040), and lower gcs (p = 0.0003). the incidence of bradycardia (hr < 60) was 4.4%. conclusion: early hypotension, hyperglycemia, and bradycardia along with lower initial gcs are associated with significantly higher likelihood of hospital admission, including icu admission, as well as intrahospital death and re-admission. background: over 23,000 people per day require treatment for ankle sprains, resulting in lost workdays and training for athletes. platelet rich plasma (prp) is an autologous concentration of platelets which, when injected into the site of injury, is thought to improve healing by promoting inflammation through growth factor and cytokine release. studies to date have shown mixed results, with few randomized or placebo-controlled trials. the lower extremity functional scale (lefs) is a previously validated objective measure of lower extremity function. objectives: is prp helpful in acute ankle sprains in the the emergency department? methods: prospective, randomized, double-blinded, placebo-controlled trial. patients with severe ankle sprains and negative x-rays were randomized to trial or placebo. severe was defined as marked swelling and ecchymosis and inability to bear weight. both groups had 50 cc of blood drawn. trial group blood was centrifuged with a magellan autologous platelet separator (arteriocyte, cleveland) to yield 3-4 cc of prp. prp along with 0.5 cc of 1% lidocaine and 0.5 cc of 0.25% bupivicaine was injected at the point of maximum tenderness by a blinded physician under ultrasound guidance. control group blood was discarded and participants were injected in a similar fashion substituting sterile 0.9% saline for prp. both groups had visual analog scale (vas) pain scores and lefs on days 0, 3, 8, and 30. all participants had a posterior splint and were made non weight bearing for 3 days after which they were reexamined, had their splint removed, and were asked to bear weight as tolerated. participants were instructed not to use nsaids during the trial. results: 1156 patients were screened and 37 were enrolled. four withdrew before prp injection was complete. eighteen were randomized to prp and 15 to placebo. see tables for results. vas and lefs are presented as means with sd in parentheses. demographics were not statistically different between groups. conclusion: in this small study, prp did not appear to offer benefit in either pain control or healing. both groups had improvement in their pain and functionality and did not differ significantly during the study period. limitations include small study size and large number of participant refusals. methods: a structured chart review of all icd-9 radius fracture coded charts spanning march 18, 2010 to july 17, 2011 was conducted. specific variable data were collected and categorized as follows: age, moi, body mass index, and fracture location. the charts were reviewed by two medical students, with 10% of the charts reviewed by both students to confirm inter-rater reliability. frequencies and inter-quartile ranges were determined. comparisons were made with fisher's exact test and multiple logistic regression. results: 187 charts met inclusion criteria. 46 charts were excluded due to one of the following reasons: no fracture or no x-ray (14), isolated ulnar fracture (19), or undocumented or penetrating moi (13). of the analyzed patients (n = 141), distal radius fractures were most common (66%), followed by proximal (32%) and midshaft (2%). chart reviewers were found to be reliable (j = 1). age and moi were significantly associated with fracture location (see table) . ages 18-54 and bike accidents were more strongly associated with proximal radius fractures (odds ratio: 12 [2-94] and 5 [2-13], respectively). conclusion: patients presenting to our inner city ed with a radius fracture are more likely to have a distal fracture. adults 18-54 and bike accidents had a significantly higher incidence of proximal fractures than other ages or mois. background: trauma centers use guidelines to determine the need for a trauma surgeon in the ed on patient arrival. a decision rule from loma linda university that includes penetrating injury and tachycardia was developed to predict which pediatric trauma patients require emergent intervention, and thus are most likely to benefit from surgical presence in the ed. objectives: our goal was to validate the loma linda rule (llr) in a heterogeneous pediatric trauma population and to compare it to the american college of surgeons' major resuscitation criteria (mrc). we hypothesized that the llr would be more sensitive than the mrc for identifying the need for emergent operative or procedural intervention. methods: we performed a secondary analysis of prospectively collected trauma registry data from two urban level i pediatric trauma centers with a combined annual census of approximately 115,000 visits. consecutive patients <15 years old with blunt or penetrating trauma from 1993 through 2010 were included. patient demographics, injury severity scores (iss), times of ed arrival and surgical intervention, and all variables of both rules were obtained. the outcome (emergent operative intervention within 1 hour of ed arrival or ed cricothyroidotomy or thoracotomy) was confirmed by trained, blinded abstractors. sensitivities, specificities, and 95% confidence intervals (cis) were calculated for both rules. results: 8,079 patients were included with a median age of 5.9 years and a median iss of 9. emergent intervention was required in 51 patients (0.6%). the llr had a sensitivity ranging from 59.4%-59.7% (95% ci: 25.9%-93.5%) and specificity ranging from 49.5%-86.5% (95% ci: 21.6%-82.1%) between both institutions. the mrc had a sensitivity ranging from 73.6%-81.6% (95% ci: 54.7%-95.1%) and specificity ranging from 69.4%-84.7% (95% ci: 54.7%-90.1%) between institutions. conclusion: emergent intervention is rare in pediatric trauma patients. the mrc was more sensitive for predicting the need for emergent intervention than the llr. neither set of criteria was sufficiently accurate to recommend their routine use for pediatric trauma patients. droperidol for sedation of acute behavioural disturbance leonie a. calver 1 , colin page 2 , michael downes 3 , betty chan 4 , geoffrey k. isbister 1 1 calvary mater newcastle and university of newcastle, newcastle, australia; 2 princess alexandra hospital, brisbane, australia; 3 calvary mater newcastle, newcastle, australia; 4 prince of wales hospital, sydney, australia background: acute behavioural disturbance (abd) is a common occurrence in the emergency department (ed) and is a risk to staff and patients. there remains little consensus on the most effective drug for sedation of violent and aggressive patients. prior to the food and drug administration's black box warning, droperidol was commonly used and was considered safe and effective. objectives: this study aimed to investigate the effectiveness of parenteral droperidol for sedation of abd. methods: as part of a prospective observational study, a standardised protocol using droperidol for the seda-acute and delayed behavioral deficits were demonstrated in this rat model of co toxicity, which parallels the neurocognitive deficit pattern observed in humans (see figure) . similar to prior studies, pathologic analysis of brain tissue demonstrated the highest percentage of necrotic cells in the cortex, pyramidal cells, and cerebellum. the collected data are summarized in the table. we have developed an animal model of severe co toxicity evidenced by behavioral deficits and neuronal necrosis. future efforts will compare neurologic outcomes in severely co poisoned rats treated with hypothermia and 100% inspired o2 versus hbo to normothermic controls treated with 100% inspired o2. increasing in popularity, attracting more than 70,000 annual participants worldwide. prior studies have consistently documented renal function impairment, but only after race completion. the incidence of renal injury during these multi-day ultramarathons is currently unknown. this is the first prospective cohort study to evaluate the incidence of acute kidney injury (aki) in runners during a multi-day ultramarathon foot race. objectives: to assess the effect of inter-stage recovery versus cumulative damage on resulting renal function during a multi-day ultramarathon. methods: demographic and biochemical data gathered via phlebotomy and analyzed by istatò (abbott, nj) were collected at the start and finish of day 1 (25 miles), 3 (75 miles), and 5 (140 miles) during racing the planet'sò150-mile, 7-day self-supported desert ultramarathons. pre-established rifle criteria using creatinine (cr) and glomerular filtration rate (gfr) defined aki as ''no injury'' (cr <1.5x normal, decrease of gfr <25%), ''risk'' (cr 1.5x normal, decrease of gfr by 25-49%), and ''injury'' (cr 2x normal, decrease of gfr by 50-75%). results: thirty racers (76% male) with a mean (+/) sd) age of 39 + /-10 years were studied during the 2008 sahara (n = 7, 23.3%), 2008 gobi (n = 10, 33%), and 2009 namibia (n = 13, 43.3%) events. the average decrease in gfr from day 1 start to day 1 finish was 28 + /-25 (p < 0.001, 95% ci 18.5-37.6); day 1 start to day 3 finish was 29.6 + /-20.1 (p < 0.001, 95% ci 18.4-40.7); and day 1 start to day 5 finish was 30.9 ± 17.5 (p < 0.001, 95% ci 20.8-41). runners categorized as risk and injury for aki after stage 1 was 44.8 % and 10%; after stage 3 was 67% and 13%, and after stage 5 was 57.1% and 7.1% conclusion: the majority of participants developed significant levels of renal impairment despite recovery intervals. given the changes in renal function, potentially harmful non-steroidal anti-inflammatory drugs should be minimized to prevent exacerbating acute kidney injury. background: more than 10% of the elderly abuse prescription drugs, and emergency medicine providers frequently struggle to identify features of opioid addiction in this population. the prescription drug use questionnaire (pduqp) is a validated, 42-item, patient-administered tool developed to help health care providers better identify problematic opioid use, or dependence, in patients who receive opioids for the treatment of chronic pain. objectives: to identify the prevalence of prescription drug misuse features in elderly ed patients. methods: this cross-sectional, observational study was conducted between 07/2011 and 08/2011 in the ed of an urban, university-affiliated community hospi-tal that serves a large geriatric population. all patients aged 65 to 89 inclusive were eligible, and were recruited on a convenience basis. exclusion criteria included known dementia, and critical illness. outcomes of interest included self-reported history of prior prescription opioid use, substance abuse history, aberrant medication-taking behaviors, and pduqp results. results: one hundred patients were approached for participation. two were excluded for inability to read english, three were receiving analgesia for metastatic cancer, 28 had never taken a prescription opioid, and seven refused to participate beyond pre-screening. sixty patients completed the study (see table 1 ). of those, 13.3% reported four or more visits within 12 months; chronic pain was reported by 56.7%; debilitating pain by 55.9%; prior pain management referral by 18.3%; and storing opioids for future use by 30%. seventeen patients reported current prescription opioid use, and were administered the pduqp (see figure) . in this population, 47.1% thought their pain was not adequately being treated; 41.2% reported having to increase the amount of pain medication they were taking over the prior 6 months; 35.3% saved up future pain medication; 11.8% had doctors refuse to give them pain medication for fear that the patient would abuse the prescription opioids; and 29.4% reported having a previous drug or alcohol problem. conclusion: screening instruments, such as the pduqp, facilitate identification of geriatric patients with features of opioid misuse. a high proportion of patients in this study save opioids for further use. interventions for safe medication disposal may decrease access to opioids and subsequent morbidity. age extremes, male sex, and several chronic health conditions were associated with increased odds of heat stroke, hospital admission, and death in the ed by a factor of 2-3. chronic hematologic disease (e.g. anemia) was associated with a 10-12 fold increase in adjusted odds of each of these outcomes. conclusion: hri imposes a substantial public health burden, and a wider range of chronic conditions confer susceptibility than previously thought. males, older adults, and patients with chronic conditions, particularly anemia, are likely to have more severe hri, be admitted, or die in the ed. background: carbon monoxide (co) poisoning is a remarkable cause of death worldwide. co, produced by the incomplete combustion of hydrocarbons, has many toxic effects on especially the heart and brain. co binds strongly to cytochrome oxidase, hemoglobin, and myoglobin causing hypoxia of organs and issues. co converts hemoglobin to carboxyhemoglobin and makes transport of oxygen through the body impossible and causes severe hypoxia. objectives: the aim of this study is to investigate the levels of s100b and neuron specific enolase (nse) measured both during admittance and at the sixth hour of hyperbaric and normobaric oxygen therapy carried out on patients with a diagnosis of co poisoning. methods: the study is designed as a prospective observational laboratory study. forty patients were enrolled in the study: 20 underwent normobaric oxygen therapy (nbot) and the other 20 underwent hyperbaric oxygen therapy (hbot). levels of s100b and nse were measured both during admittance and at the sixth hour of admittance of all patients. demographic data, clinical characteristics, and outcome measures were recorded. all data were statistically analyzed. results: in both treatment groups, mean levels of nse after therapy were significantly lower than admittance levels. although levels of nse measured before and 6 hours after treatment in hbot group were high, the difference between groups was not statistically significant (p > 0.05). in both treatment groups, mean levels of s100b after therapy were significantly lower than admittance levels; likewise nse. although levels of s100b measured before and 6 hours after treatment in hbot group were high, the difference between groups was not statistically significant (p > 0.05). additionally, while levels of s100b measured after treatment in the hbot group were lower compared to the nbot group, the difference between groups was also not statistically significant (p > 0.05). conclusion: levels of s100b and nse as evidence for brain injury elevation in case of co poisoining and decrease by therapy according to our study as well as previous studies. decrease in levels of s100b is more significant. according to our results, s100b and nse may be useful markers in case of co poisoning; however, we did not meet any data providing more value in determining hbot indications and determining levels of cohb in the management of patients with a diagnosis of co poisoining. neurons objectives: this study was conducted to determine if neurons in the dmh, and its neighbor the paraventricular hypothalamus (pvn), were likewise involved in mdma-mediated neuroendocrine responses, and if serotonin 1a receptors (5-ht1a) play a role in this regional response. methods: in both experiments, male sprague dawley rats (n = 5-12/group) were implanted with bilateral cannulas targeting specific regions of the brain, i.v. catheters for drug delivery, and i.a. catheters for blood withdrawal. experiments were conducted in raturn cages, which allow blood withdrawal and drug administration in free moving animals while recording their locomotion. in the first experiment, rats were microinjected into the dmh, the pvn, or a region between, with the gabaa agonist muscimol (80 pmol/100nl/side) or pbs (100nl) and 5 min later were injected with either mdma (7.5 mg/kg i.v.) or an equal volume of saline. blood was withdrawn prior to microinjections and 15 minutes after mdma for ria measurement of plasma acth. locomotion was recorded throughout the experiment. in a separate experiment of identical design, either the 5-ht1a antagonist way 100635 (way, 5 nmol/100 nl/side) or saline was microinjected followed by i.v. injection of mdma or saline. in both experiments, increases in acth and distance traveled were compared between groups using an anova analysis. results: when compared to controls, microinjections of muscimol into the dmh, pvn, or the area in between attenuated plasma increases in acth and locomotion evoked by mdma. when microinjected into the dmh or pvn, way had no effect on acth, but when injected into the region of the dmh it significantly increased locomotion. background: poor hand-offs between physicians when admitting patients have been shown to be a major source of medical errors. objectives: we propose that training in a standardized admissions protocol by emergency medicine (em) to internal medicine (im) residents would improve the quality of and quantity of communication of vital patient information. methods: em and im residents at a large academic center developed an evidence-based admission handover protocol termed the '7ps' (table 1) . em and im residents received '7ps' protocol training. im residents recorded prospectively how well each of the seven ps were communicated during each admission pre-and post-intervention. im residents also assessed the overall quality of the handover using a likert scale. the primary outcome was the change in the number of 'ps' conveyed by the em resident to the accepting im resident. data were collected for six weeks before and then for six weeks starting two weeks after the educational intervention. results: there were 78 observations recorded in the preintervention (control) group and 48 observations in the post-intervention group. for each of the seven 'ps' the percentage of observation where all of the information was communicated is shown in table 2 . the communication of 'ps' increased following the intervention. this rise was statistically significant for patient information and pending tests. in the control group the mean of total communicated ps was 5 and in the intervention group, the mean increased to 6 (p < 0.005). the quality of the handover communication had a mean rating of 3.9 in the control group and 4.3 in the intervention group (p < 0.05). conclusion: this educational intervention in a cohort of em and im residents improved the quality and quantity of vital information communicated during patient handovers. the intervention was statistically significant for patient information transfer and tests pending. the results are limited by study size. based on our preliminary data, an agreed-upon handover protocol with training improved the amount and quality of communication during patients' hospital admission on simple items that were likely had been taken for granted as routinely transmitted. we recruited a convenience sample of residents and students rotating in the pediatric emergency department. a two-sided form had the same seven clinical decisions on each side: whether to perform blood, urine, spinal fluid tests, imaging, iv fluids, antibiotics, or a consult. the rating choices were: definitely not, probably not, probably would, and definitely would. trainees rated each decision after seeing a patient, but before presenting to the preceptor, who, after evaluating the patient, rated the same seven decisions on the second side of the form. the preceptor also indicated the most relevant decision (mrd) for that patient. we examined the validity of the technique using hypothesis testing; we posited that residents would have a higher degree of concordance with the preceptor than would medical students. this was tested using dichotomized analyses (accuracy, kappa) and roc curves with the preceptor decision as the gold standard. results: thirty-one students completed 130 forms (median 4 forms; iqr 2,6) and 23 residents completed 206 (6; iqr 3,12). preceptors included 24 attending physicians and 3 fellows (9; iqr 4, 21). students were concordant with preceptors in 70% (k = 0.38) of mrd while residents agreed in 79.6% (p = 0.045), k = 0.59. roc analysis revealed significant differences between students and residents in the auc for the mrd (0.84 vs 0.72; p = 0.03). conclusion: this measure of trainee-preceptor concordance requires further research but may eventually allow for assessment of trainee clinical decision-making. it also has the pedagogical advantage of promoting independent trainee decision-making. background: basic life support (bls) and advanced cardiac life support (acls) are integral parts of emergency cardiac care. this training is usually reserved in most institutions for residents and faculty. the argument can be made to introduce bls and acls training earlier in the medical student curriculum to enhance acquisition of these skills. objectives: the goal of the survey was to characterize the perceptions and needs of graduating medical students in regards to bls and acls training. methods: this was a survey-based study of graduating fourth year medical students at a u.s. medical school. the students were surveyed before voluntarily participating in a student-led acls course in march of their final year. the surveys were distributed before starting the training course. both bls and acls training, comfort levels, and perceptions were assessed in the survey. results: of the 182 students in the graduating class, 152 participated in the training class with 109 (72%) completing the survey. 50% of students entered medical school without any prior training and 49% started clinics without training. 83.5% of students reported witnessing an average of 3.0 in-hospital cardiac arrests during training (range of 0-20). overall, students rated their preparedness 2.0 (sd 1.0) for adult resuscitations on a 1-5 likert scale with 1 being the unprepared. 98% and 92% of students believe that bls and acls should be included in the medical student curriculum respectively with a preference for teaching before starting clerkships. 36% of students avoided participating in resuscitations due to lack of training. of those, 95% said they would have participated had they been trained. conclusion: to our knowledge, this is one of the first studies to address the perceptions and needs for bls and acls training in u.s. medical schools. students feel that bls and acls training is needed in their curriculum and would possibly enhance perceived comfort levels and willingness to participate in resuscitations. background: professionalism is one of six core competency requirements of the acgme, yet defining and teaching its principles remains a challenge. the ''social contract'' between physician and community is clearly central to professionalism so determining the patient's understanding of the physician's role in the relationship is important. because specialization has created more narrowly focused and often quite different interactions in different medical environments, the patient concept of professionalism in different settings may vary as well. objectives: we hoped to determine if patients have different conceptions of professionalism when considering physicians in different clinical environments. methods: patients were surveyed in the waiting room of an emergency department, an outpatient internal medicine clinic, and a pre-operative/anesthesia clinic. the survey contained 18 examples of attributes, derived from the american board of internal medicine's eight characteristics of professionalism. participants were asked to rate, on a 10-point scale, the importance that a physician possess each attribute. an anova analysis was used to compare the sites for each question. results: of 604 who took the survey, 200 were in the emergency department, 202 were in the medicine clinic, and 202 were in the pre-operative clinic. females comprised 56% of the study group and the average age was 49 with a range from 18 to 94. there was a significant difference on the attribute of ''providing a portion of work for those who cannot pay;'' this was rated higher in the emergency department (p = 0.003). there was near-significance (p = 0.05) on the attribute of ''being able to make difficult decisions under pressure,'' which was rated higher in the pre-op clinic. there was no difference for any of the other questions. the top four professional attributes at each clinical site were the same -''honesty,'' ''excellence in communication and listening,'' ''taking full responsibility for mistakes,'' and ''technical competence/ skill;'' the bottom two were ''being an active leader in the community'' and ''patient concerns should come before a doctor's family commitments.'' conclusion: very few differences between clinical sites were found when surveying patient perception of the important elements of medical professionalism. this may suggests a core set of values desired by patients for physicians across specialties. emergency medicine faculty knowledge of and confidence in giving feedback on the acgme core competencies todd guth, jeff druck, jason hoppe, britney anderson university of colorado, aurora, co background: the acgme mandates that residency programs assess residents based upon six core competencies. although the core competencies have been in place for a number of years, many faculty are not familiar with the intricacies of the competencies and have difficulty giving competency-specific feedback to residents. objectives: the purpose of the study is to determine the extent to which emergency medicine (em) faculty can identify the acgme core competencies correctly and to determine faculty confidence with giving general feedback and core competency focused feedback to em residents. methods: design and participants: at a single department of em, a survey of twenty-eight faculty members, their knowledge of the acgme core competencies, and their confidence in providing feedback to residents was conducted. confidence levels in giving feedback were scored on a likert scale from 1 to 5. observations: descriptive statistics of faculty confidence in giving feedback, identification of professional areas of interest, and identification of the acgme core competencies were determined. mann-whitney u tests were used to make comparisons between groups of faculty given the small sample size of the respondents. results: there was a 100% response rate of the 28 faculty members surveyed. eight faculty members identified themselves as primarily focused on education. although those faculty members identifying themselves as focused on education scored higher than non-education focused faculty for all type of feedback (general feedback, constructive feedback, negative feedback), there was only a statistical difference in confidence levels 4.57 versus 2.65 (p < 0.002) for acgme core competency specific feedback when compared to noneducation focused faculty. while education focused faculty correctly identified all six of acgme core competencies 94% of the time, not one of the non-education focused faculty identified all six of the core competencies correctly. non-education focused faculty only correctly identified three or more competencies 25% of the time. conclusion: if residency programs are to assess residents using the six acgme core competencies, additional faculty development specific to the core competencies will be needed to train all faculty on the core competencies and on how to give core competency specific feedback to em residents. there is no clear consensus as to the most effective tool to measure resident competency in emergency ultrasound. objectives: to determine the relationship between the number of scans and scores on image recognition, image acquisition, and cognitive skills as measured by an objective structured clinical exam (osce) and written exam. secondarily, to determine whether image acquisition, image recognition, and cognitive knowledge require separate evaluation methodologies. methods: this was a prospective observational study in an urban level i ed with a 3-year acgme-accredited residency program. all residents underwent an ultrasound introductory course and a one-month ultrasound rotation during their first and second years. each resident received a written exam and osce to assess psychomotor and cognitive skills. the osce had two components: (1) recognition of 22 images, and (2) acquisition of images. a registered diagnostic medical sonographer (rdms)-certified physician observed each bedside examination. a pre-existing residency ultrasound database was used to collect data about number of scans. pearson correlation coefficients were calculated for number of scans, written exam score, image recognition, and image acquisition scores on the osce. results: twenty-nine residents were enrolled from march 2010 to february 2011 who performed an average of 247 scans (range 118-617). there was no significant correlation between number of scans and written exam scores. an analysis of the number of scans and the ocse found a moderate correlation with image acquisition (r = 0.42, p = 0.029) and image recognition (r = 0.61, p = <0.01)). pearson correlation analysis between the image acquisition score and image recognition score found that there was no correlation (r = 0.175, p = 0.383). there was a moderate correlation with image acquisition scores to written scores (r = 0.541, p = 0.025) and image recognition scores to written scores (r = 0.596, p = 0.019). conclusion: the number of scans does not correlate with written tests but has a moderate correlation with image acquisition and image recognition. this suggests that resident education should include cognitive instruction in addition to scan numbers. we conclude that multiple methods are necessary to examine resident ultrasound competency. background: although emergency physicians must often make rapid decisions that incorporate their interpretation of an ecg, there is no evidence-based description of ecg interpretation competencies for emergency medicine (em) trainees. the first step in defining these competencies is to develop a prioritized list of ecg findings relevant to em contexts. objectives: the purpose of this study was to categorize the importance of various ecg diagnoses and/or findings for the em trainee. methods: we developed an extensive list of potentially important ecg diagnoses identified through a detailed review of the cardiology and em literature. we then conducted a three-round delphi expert opinion-soliciting process where participants used a five-point likert scale to rate the importance of each diagnosis for em trainees. consensus was defined as a minimum of 75 percent agreement on any particular diagnosis at the second round or later. in the absence of consensus, stability was defined as a shift of 20 percent or less after successive rounds. results: twenty-two em experts participated in the delphi process, sixteen (72%) of whom completed the process. of those, fifteen were experts from eleven different em training programs across canada and one was a recognized expert in em electrocardiography. overall, 77 diagnoses reached consensus, 42 achieved stability, and one diagnosis achieved neither consensus nor stability. out of 120 potentially important ecg diagnoses, 53 (43%) were considered ''must know'' diagnoses, 62 (51%) ''should know'' diagnoses, and 7 (6%) ''nice to know'' diagnoses. conclusion: we have categorized ecg diagnoses within an em training context, knowledge of which may allow clinical em teachers to establish educational priorities. this categorization will also facilitate the development of an educational framework to establish em trainee competency in ecg interpretation. ''rolling refreshers background: cardiac arrest survival rates are low despite advances in cardiopulmonary resuscitation. high quality cpr has been shown to impart greater cardiac arrest survival; however, retention of basic cpr skills by health care providers has been shown to be poor. objectives: to evaluate practitioner acceptance of an in-service cpr skills refresher program, and to assess for operator response to real-time feedback during refreshers. methods: we prospectively evaluated a ''rolling refresher'' in-service program at an academic medical center. this program is a proctored cpr practice session using a mannequin and cpr-sensing defibrillator that provides real-time cpr quality feedback. subjects were basic life support-trained providers who were engaged in clinical care at the time of enrollment. subjects were asked to perform two minutes of chest compressions (ccs) using the feedback system. ccs could be terminated when the subject had completed approximately 30 seconds of compressions with <3 corrective prompts. a survey was then completed by to obtain feedback regarding the perceived efficacy of this training model. cpr quality was then evaluated using custom analysis software to determine the percent of cc adequacy in 30-second intervals. results: enrollment included 88 subjects from the emergency department and critical care units (55 nurses, 17 physicians, 16 students and allied health professionals). all participants completed a survey and 61 cpr performance data logs were obtained. positive impressions of the in-service program were registered by 81% (71/88) and 74% (65/88) reported a self-perceived improvement in skills confidence. eighty-three percent (73/88) of respondents felt comfortable performing this refresher during a clinical shift. thirtynine percent (24/61) of episodes exhibited adequate cc performance with approximately 30 seconds of cc. of the remaining 37 episodes, 71.1 ± 29.2% of cc were adequate in the first 30 seconds with 80.1 ± 28.6% of cc adequate during the last 30 second interval (p = 0.1847). of these 37 individuals, 30 improved or had no change in their cpr skills, and 7 individuals skills declined during cc performance (p = 0.007). conclusion: implementation of a bedside cpr skill refresher program is feasible and is well received by hospital staff. real time cpr feedback improved upon cpr skill performance during the in-service session. teaching emergency medicine skills: is a self-directed, independent, online curriculum the way of the future? tighe crombie, jason r. frank, stephen noseworthy, richard gerein, a. curtis lee university of ottawa, ottawa, on, canada background: procedural competence is critical to emergency medicine, but the ideal instructional method to acquire these skills is not clear. previous studies have demonstrated that online tutorials have the potential to be as effective as didactic sessions at teaching specific procedural skills. objectives: we studied whether a novel online curriculum teaching pediatric intraosseus (io) line insertion to novice learners is as effective as a traditional classroom curriculum in imparting procedural competence. methods: we conducted a randomized controlled educational trial of two methods of teaching io skills. preclinical medical students with no past io experience completed a written test and were randomized to either an online or classroom curriculum. the online group (og) were given password-protected access to a website and instructed to spend 30 minutes with the material while the didactic group (dg) attended a lecture of similar duration. participants then attended a 30-minute unsupervised manikin practice session on a separate day without any further instruction. a videotaped objective structured clinical examination (osce) and post-course written test were completed immediately following this practice session. finally, participants were crossed over into the alternate curriculum and were asked to complete a satisfaction survey that compared the two curricula. results were compared with a paired t-test for written scores and an independent t-test for osce scores. results: sixteen students completed the study. pre-course test scores of the two groups were not significantly different prior to accessing their respective curricula (mean scores of 32% for og and 34% for dg, respectively; p > 0.05). post-course written scores were also not significantly different (both with means of 76%; p > 0.05); however, for the post-treatment osce scores, the og group scored significantly higher than the dg group (mean scores of 92.6% and 88.1%; t(14) = 1.76, p < 0.05.) conclusion: this novel online curriculum was superior to a traditional didactic approach to teaching pediatric io line insertion. novice learners assigned to a selfdirected online curriculum were able to perform an emergency procedural skill to a high level of performance. em educators should consider adopting online teaching of procedural skills. background: applicants to em residency programs obtain information largely from the internet. curricular information is available from a program's website (pw) or the saem residency directory (sd). we hypothesize that there is variation between these key sources. objectives: to identify discrepancies between each pw and sd. to describe components of pgy1-3 em residency programs' curricula as advertised on the internet. methods: pgy1-3 residencies were identified through the sd. data were abstracted from individual sd and pw pages identifying pre-determined elements of interest regarding rotations in icu, pediatrics, inpatient (medicine, pediatrics, general surgery), electives, orthopedics, toxicology, and anesthesia. agreement between the sd and pw was calculated using a cohen's unweighted kappa calculation. curricula posted on pws were considered the gold standard for the programs' current curricula. results: a total of 117 pgy1-3 programs were identified through the sd and confirmed on the pw. ninetyone of 117 programs (78%) had complete curricular information on both sites. only these programs were included in the kappa analysis for sd and pw comparisons. of programs with complete listings, 66 of 91 programs (73%) had at least one discrepancy. the agreement of information between pw and sd revealed a kappa value of 0.26 (95% ci 0.19-0.33). analysis of pw revealed that pgy1-3 programs have an average of 4.15 (range, 2-9), 3.1 (range, 1-6), 1.7 (range, 0-4), and 1.0 (range, 0-4) blocks of icu, pediatrics, elective, and inpatient, respectively. common but not rrc-mandated rotations in orthopedics, toxicology, and anesthesiology are present in 77, 80, and 93 percent of programs, respectively. conclusion: publicly accessible curricular information through the sd and pw for pgy1-3 em programs only has fair agreement (using commonly accepted kappa value guides). applicants may be confused by the variability of data and draw inaccurate conclusions about program curricula. from the gravid uterus and improves cardiac output; however, this theory has never been proven. objectives: we set out to determine the difference in inferior vena cava (ivc) filling when third trimester patients were placed in supine, llt, and right lateral tilt (rlt) positions using ivc ultrasound. methods: healthy pregnant women in their third trimester presenting to the labor and delivery suite were enrolled. patients were placed in three different positions (supine, rlt, and llt) and ivc maximum (max) and minimum (min) measurements were obtained using the intercostal window in short axis approximately two centimeters below the entry of the hepatic veins. ivc collapse index (ci) was calculated for each measurement using the formula (max-min)/max. in addition, blood pressure, heart rate, and fetal heart rate were monitored. patients stayed in each position for at least 3 minutes prior to taking measurements. we compared ivc measurements using a one-way analysis of variance for repeated measures. results: twenty patients were enrolled. the average age was 25 years (sd 5.7) with a mean estimated gestational age of 39.5 weeks (sd 1.4). there were no significant differences seen in ivc filling in each of the positions (see table 1 ). in addition, there were no differences in hemodynamic parameters between positions.ten (50%) patients had the largest ivc measurement in the llt position, 7 (35%) patients in the rlt position, and 3 (15%) in the supine position. conclusion: there were no significant differences in ivc filling between patient positions. for some third trimester patients llt may not be the optimal position for ivc filling. background: although the acgme and rrc require competency assessment in ed bedside ultrasound (us), there are no standardized assessment tools for us training in em. objectives: using published us guidelines, we developed four observed structured competency evalua-tions (osce) for four common em us exams: fast, aortic, cardiac, and pelvic. inter-rater reliability was calculated for overall performance and for the individual components of each osce. methods: this prospective observational study derived four osces that evaluated overall study competency, image quality for each required view, technical factors (probe placement, orientation, angle, gain, and depth), and identification of key anatomic structures. em residents with varying levels of training completed an osce under direct observation of two em-trained us experts. each expert was blinded to the other's assessment. overall study competency and image quality of each required views were rated on a five-point scale (1poor, 2-fair, 3-adequate, 4-good, 5-excellent), with explicit definitions for each rating. each study had technical factors (correct/incorrect) and anatomic structures (identified/not identified) assessed as binary variables. data were analyzed using cohen's and weighted k, descriptive statistics, and 95% ci. results: a total of 185 us exams were observed, including 33 fast, 53 cardiac, 53 aorta, and 46 pelvic. total assessments included 185 ratings of overall study competency, 691 ratings of required view image quality, 2998 ratings of technical factors, and 2978 ratings of anatomic structures. inter-rater assessment of overall study competency showed excellent agreement, raw agreement 0.84 (0.77, 0.89), weighted k 0.87 (0.82, 0.91). ratings of required view image quality showed excellent agreement: raw agreement 0.75 (0.72, 0.79), weighted k 0.82 (0.79, 0.84). inter-rater assessment of technical factors showed substantial agreement: raw agreement 0.96 (0.95, 0.97), cohen's k 0.78 (0.74, 0.82). ratings of identification of anatomic structures showed substantial agreement: raw agreement 0.86 (0.85, 0.88), cohen's k 0.64 (0.60, 0.67). conclusion: inter-rater reliability is substantial to excellent using the derived ultrasound osces to rate em resident competency in fast, aortic, cardiac, and pelvic ultrasound. validation of this tool is ongoing. a objectives: the objective of this study was to identify which transducer orientation, longitudinal or transverse, is the best method of imaging the axillary vein with ultrasound, as defined by successful placement in the vein with one needle stick, no redirections, and no complications. methods: emergency medicine resident and attending physicians at an academic medical center were asked to cannulate the axillary vein in a torso phantom model. the participants were randomized to start with either the longitudinal or transverse approach and completed both sequentially, after viewing a teaching presentation. participants completed pre-and post-attempt questionnaires. measurements of each attempt were taken regarding time to completion, success, skin punctures, needle redirections, and complications. we compared proportions using a normal binomial approximation and continuous data using the t-distribution, as appropriate. a sample size of 57 was chosen based on the following assumptions: power, 0.8; significance, 0.05; effect size, 50% versus 75%. results: fifty-seven operators with a median experience of 85 prior ultrasounds (26 to 120 iqr) participated. first-attempt success frequency was 39/57 (0.69) for the longitudinal method and 21/57 (0.37) for the transverse method (difference 0.32, 95% ci 0.12-0.51); this difference was similar regardless of operator experience. the longitudinal method had fewer redirections (mean difference 1.8, 95% ci 0.8-2.8) and skin punctures (mean difference 0.3, 95% ci )2 to 0.18). arterial puncture occurred in 2/57 longitudinal attempts and 7/ 57 transverse attempts, with no pleural punctures in either group. among successful attempts, the time spent was 24 seconds less for longitudinal method (95% ci 3-45). though 93% of participants had more experience with the transverse method prior to the training session, 58% indicated after the session that they preferred the longitudinal method. methods: a prospective single-center study was conducted to assess the compressibility of the basilic vein with ultrasound. healthy study participants were recruited. the compressibility was assessed at baseline, and then further assessed with one proximal tourniquet, two tourniquets (one distal and one proximal), and a proximal blood pressure cuff inflated to 150 mmhg. compressibility was defined as the vessel's resistance to collapse to external pressure and rated as completely compressible, moderately compressible, or mildly compressible after mild pressure was applied with the ultrasound probe. results: one-hundred patients were recruited into the study. ninety-eight subjects were found to have a completely compressible basilic vein at baseline. when one tourniquet and two tourniquets were applied 64 and 58 participants, respectively, continued to have completely compressible veins. a fisher's exact test comparing one versus two tourniquets revealed no difference between these two techniques (p = 0.46). only two participants continued to have completely compressible veins following application of the blood pressure cuff. the compressibility of this group was found to be statistically significant by fisher's exact test compared to both tourniquet groups (p < 0.0001). furthermore, 24 participants with the blood pressure cuff applied were found to have moderately compressible veins and 72 participants were found to have mildly compressible veins. conclusion: tourniquets and blood pressure cuffs can both decrease the compressibility of peripheral veins. while there was no difference identified between using one and two tourniquets, utilization of a blood pressure cuff was significantly more effective to decrease compressibility. the findings of this study may be utilized in the emergency department when attempting to obtain peripheral venous access, specifically supporting the use of blood pressure cuffs to decrease compressibility. background: electroencephalography (eeg) is an underused test that can provide valuable information in the evaluation of emergency department (ed) patients with altered mental status (ams). in ams patients with nonconvulsive seizure (ncs), eeg is necessary to make the diagnosis and to initiate proper treatment. yet, most cases of ncs are diagnosed >24 h after ed presentation. obstacles to routine use of eeg in the ed include space limitations, absence of 24/7 availability of eeg technologists and interpreters, and the electrically hostile ed environment. a novel miniature portable wireless device (microeeg) is designed to overcome these obstacles. objectives: to examine the diagnostic utility of micro-eeg in identifying eeg abnormalities in ed patients with ams. methods: an ongoing prospective study conducted at two academic urban eds. inclusion: patients ‡13 years old with ams. exclusion: an easily correctable cause of ams (e.g. hypoglycemia, opioid overdose). three 30-minute eegs were obtained in random order from each subject beginning within one hour of presentation: 1) a standard eeg, 2) a microeeg obtained simultaneously with conventional cup electrodes using a signal splitter, and 3) a microeeg using an electrocap. outcome: operative characteristics of micro-eeg in identifying any eeg abnormality. all eegs were interpreted in a blinded fashion by two board-certified epileptologists. within each reader-patient pairing, the accuracy of eegs 2 and 3 were each assessed relative to eeg 1. sensitivity, specificity, and likelihood ratios (lr) are reported for microeeg by standard electrodes and electrocap (eegs 2 and 3). inter-rater variability for eeg interpretations is reported with kappa. results: the interim analysis was performed on 130 consecutive patients (target sample size: 260) enrolled from may to october 2011 (median age: 61, range: 13-100, 40% male). overall, 82% (95% confidence interval [ci], 76-88%) of interpretations were abnormal (based on eeg1). kappa values representing the agreement of neurologists in interpretation of eeg 1-3 were 0.54 (0.36-0.73), 0.57 (0.39-0.75), and 0.55 (0.37-0.74), respectively. conclusion: the diagnostic accuracy and concordance of microeeg are comparable to those of standard eeg but the unique ed-friendly characteristics of the device could help overcome the existing barriers for more frequent use of eeg in the ed. (originally submitted as a ''late-breaker.'') a background: patients who use an ed for acute migraine are characterized by higher migraine disability scores, lower socio-economic status, and are unlikely to have used a migraine-specific medication prior to ed presentation. objectives: to determine if a comprehensive migraine intervention, delivered just prior to ed discharge, could improve migraine impact scores one month after the ed visit. methods: this was a randomized controlled trial of a comprehensive migraine intervention versus typical care among patients who presented to an ed for management of acute migraine. at the time of discharge, for patients randomized to comprehensive care, we reinforced their diagnosis, shared a migraine education presentation from the national library of medicine, provided them with six tablets of sumatriptan 100 mg and 14 tablets of naproxen 500 mg, and if they wished, provided them with an expedited free appointment to our institution's headache clinic. patients randomized to typical care received the care their attending emergency physician felt was appropriate. the primary outcome was a between-group comparison of the hit6 score, a validated headache assessment instrument, one month after ed discharge. secondary outcomes included an assessment of satisfaction with headache care and frequency of use of migraine-specific medication within that one month period. the outcome assessor was blinded to assignment. results: over a 19 month period, 50 migraine patients were enrolled. one month follow-up was successfully obtained in 92% of patients. baseline characteristics were comparable. one month hit6 scores in the two groups were nearly identical (59 vs 56, 95%ci for difference of 3: )5, 11), as was dissatisfaction with overall headache care (17% versus 18%, 95%ci for difference of 1%: )22, 24%). not surprisingly, patients randomized to the comprehensive intervention were more likely to be using triptans or migraine-preventive therapy (43% versus 0%, 95%ci for difference of 43%: 20, 63%) one month later. conclusion: a comprehensive migraine intervention, when compared to typical care, did not improve hit6 scores one month after ed discharge. future work is needed to define a migraine intervention that is practical and useful in an ed. background: lumbar puncture (lp) is the standard of care for excluding non-traumatic subarachnoid hemorrhage (sah), and is usually performed following head ct (hct). however, in the setting of a non-diagnostic hct, lp demonstrates a low overall diagnostic yield for sah (<1% positive rate). objectives: to describe a series of ed patients diagnosed with sah by lp following a non-diagnostic hct, and, when compared to a set of matched controls, determine if clinical variables can reliably identify these ''ct-negative/lp-positive'' patients. methods: retrospective case-control chart review of ed patients in an integrated health system between the years 2000-2011 (estimated 5-6 million visits among 18 eds). patients with a final diagnosis of non-traumatic sah were screened for case inclusion, defined as an initial hct without sah by final radiologist interpretation and a lp with >5 red blood cells/mm 3 , along with either 1) xanthochromic cerebrospinal fluid, 2) angiographic evidence of cerebral aneurysm or arteriovenous malformation, or 3) head imaging showing sah within 48 hours following lp. control patients were randomly selected among ed patients diagnosed with headache following a negative sah evaluation with hct and lp. controls were matched to cases by year and presenting ed in a 3:1 ratio. stepwise logistic regression and classification and regression tree analysis (cart) were employed to identify predictive variables. inter-rater reliability (kappa) was determined by independent chart review. results: fifty-five cases were identified. all cases were hunt-hess grade 1 or 2. demographics are shown in table 1 . thirty-four cases (62%) had angiographic evidence of sah. five variables were identified that positively predicted sah following a normal hct with 98% sensitivity (95% ci, 90-100%) and 25% specificity (95% ci, 19-32%): age > 50 years, neck pain or stiffness, onset of headache with exertion, vomiting with headache, or loss of consciousness at headache onset. kappa values for selected variables ranged from 0.75-1.0 (18% sample). the c-statistic (auc) and hosmer-lemeshow test p-value for the logistic regression model are 0.87 and 0.74, respectively (table 2) . conclusion: several clinical variables can help safely limit the amount of invasive testing for sah following a non-diagnostic hct. prospective validation of this model is needed prior to practice implementation. background: post-thrombolysis intracerebral hemorrhage (ich) is associated with poor outcomes. previous investigations have attempted to determine the relationship between pre-existing anti-platelet (ap) use and the safety of intravenous thrombolysis, but have been limited by low event rates thus decreasing the precision of estimates. objectives: our objective was to determine whether pre-existing ap therapy increases the risk of ich following thrombolysis. methods: consecutive cases of ed-treated thrombolysis patients were identified using multiple methods, including active and passive surveillance. retrospective data were collected from four hospitals from 1996-2005, and 24 distinct hospitals from 2007-2010 as part of a cluster randomized trial. the same chart abstraction tool was used during both time periods and data were subjected to numerous quality control checks. hemorrhages were classified using a pre-specified methodology: ich was defined as presence of hemorrhage in radiographic interpretations of follow up imaging (primary outcome). symptomatic ich (secondary outcome) was defined as radiographic ich with associated clinical worsening. a multivariable logistic regression model was constructed to adjust for clinical factors previously identified to be related to postthrombolysis ich. as there were fewer sich events, the multivariable model was constructed similarly, except that variables divided into quartiles in the primary analysis were dichotomized at the median. results: there were 830 patients included, with 47% having documented pre-existing ap treatment. the mean age was 69 years, the cohort was 53% male, and the median nihss was 12. the unadjusted proportion of patients with any ich was 15.1% without ap and 19.3% with ap (difference 4.2%, 95% ci )1.2% to 9.6%); for sich this was 6.1% without ap and 9% with ap (difference 3.1%, 95%ci )1 to 6.7%). no significant association between pre-existing ap treatment with radiographic or symptomatic ich was observed (table) . conclusion: we did not find that ap treatment was associated with post-thrombolysis ich or sich in this cohort of community treated patients. pre-existing tobacco use, younger age, and lower severity were associated with lower odds of sich. an association between ap therapy and sich may still exist -further research with larger sample sizes is warranted in order to detect smaller effect sizes. background: post-cardiac arrest therapeutic hypothermia (th) improves survival and neurologic outcome after cardiac arrest, but the parameters required for optimal neuroprotection remain uncertain. our laboratory recently reported that 48-hour th was superior to 24-hour th in protecting hippocampal ca1 pyramidal neurons after asphyxial cardiac arrest in rats. cerebellar purkinje cells are also highly sensitive to ischemic injury caused by cardiac arrest, but the effect of th on this neuron population has not been previously studied. objectives: we examined the effect of post-cardiac arrest th onset time and duration on purkinje neuron survival in cerebella collected during our previous study. methods: adult male long evans rats were subjected to 10-minute asphyxial cardiac arrest followed by cpr. rats that achieved return of spontaneous circulation (rosc) were block randomized to normothermia (37.0 deg c) or th (33.0 deg c) initiated 0, 1, 4, or 8 hours after rosc and maintained for 24 or 48 hours (n = 21 per group). sham injured rats underwent anesthesia and instrumentation only. seven days post-cardiac arrest or sham injury, rats were euthanized and brain tissue was processed for histology. surviving purkinje cells with normal morphology were quantified in the primary fissure in nissl stained sagittal sections of the cerebellar vermis. purkinje cell density was calculated for each rat, and group means were compared by anova with bonferroni analysis. results: purkinje cell density averaged (+/) sd) 35.9 (2.4) cells/mm in sham-injured rats. neuronal survival in normothermic post-cardiac arrest rats was significantly reduced compared to sham (10.7% (5.0%)). overall, th resulted in significant neuroprotection compared to normothermia (38.9% (15.7%) of sham). purkinje cell density with 24-hour duration th was 35.0% (11.2%) of sham and 48-hour duration th was 43.3% (15.6%), both significantly improved from sham (p = 0.245 between durations). th initiated 0, 1, 4, and 8 hours post-rosc provided similar benefit: 44.6% (21.6%), 33.2% (8.1%), 36.6% (12.9%), and 41.1% (9.3%) of sham, respectively. conclusion: overall, these results indicate that postcardiac arrest th protects cerebellar purkinje cells with a broad therapeutic window. our results underscore the importance of considering multiple brain regions when optimizing the neuroprotective effect of post-cardiac arrest th. the effect of compressor-administered defibrillation on peri-shock pauses in a simulated cardiac arrest scenario joshua glick, evan leibner, thomas terndrup penn state hershey medical center, hershey, pa background: longer pauses in chest compressions during cardiac arrest are associated with a decreased probability of successful defibrillation and patient survival. having multiple personnel share the tasks of performing chest compressions and shock delivery can lead to communication complications that may prolong time spent off the chest. objectives: the purpose of this study was to determine whether compressor-administered defibrillation led to a decrease in pre-shock and peri-shock pauses as compared to bystander-administered defibrillation in a simulated in-hospital cardiac arrest scenario. we hypothesized that combining the responsibilities of shock delivery and chest-compression performance may lower no-flow periods. methods: this was a randomized, controlled study measuring pauses in chest compressions for defibrillation in a simulated cardiac arrest. medical students and ed personnel with current cpr certification were surveyed for participation between july 2011 and october 2011. participants were randomized to either a control (facilitator-administered shock) or variable (participantadministered shock) group. all participants completed one minute of chest compressions on a mannequin in a shockable rhythm prior to initiation of prompt and safe defibrillation. pauses for defibrillation were measured and compared in both study groups. results: out of 200 total enrollments, the data from 197 defibrillations were analyzed. subject-initiated defibrillation resulted in a significantly lower pre-shock handsoff time (0.57 s; 95% ci: 0.47-0.67) compared to facilitator-initiated defibrillation (1.49 s; 95% ci: 1.35-1.64). furthermore, subject-initiated defibrillation resulted in a significantly lower peri-shock hands-off time (2.77 s; 95% ci: 2.58-2.95) compared to facilitator-initiated defibrillation (4.25 s; 95% ci: 4.08-4.43). conclusion: assigning the responsibility for shock delivery to the provider performing compressions encourages continuous compressions throughout the charging period and decreases total time spent off the chest. this modification may also decrease the risk of accidental shock and improve patient survival. however, as this was a simulation-based study, clinical implementation is necessary to further evaluate these potential benefits. objectives: to determine the sensitivity and specificity of peripheral venous oxygen (po 2 ) to predict abnormal central venous oxygen saturation in septic shock patients in the ed. methods: secondary analysis of an ed-based randomized controlled trial of early sepsis resuscitation targeting three physiological variables: cvp, map, and either scvo 2 or lactate clearance. inclusion criteria: suspected infection, two or more sirs criteria, and either systolic blood pressure <90 mmhg after a fluid bolus or lactate >4 mm. peripheral venous po 2 was measured prior to enrollment as part of routine care, and scvo 2 was measured as part of the protocol. we analyzed for agreement between venous po 2 and scvo 2 using spearman's rank. sensitivity and specificity to predict an abnormal scvo 2 (<70%) were calculated for each incremental value of po 2 . results: a total of 175 were analyzed. median po 2 was 43 mmhg (iqr 32, 55). median initial scvo 2 was 79% (iqr 70, 88). thirty-nine patients (23%) had an initial scvo 2 < 70%. spearman's rank demonstrated fair correlation between initial po 2 and scvo 2 (q = 0.26). a cutoff of venous po 2 < 57 was 90% sensitive and 20% specific for detecting an initial scvo 2 < 70%. twenty-seven patients (20%) demonstrated an initial po 2 of >56. conclusion: in ed septic shock patients, venous po 2 demonstrated only fair correlation with scvo 2, though a cutoff value of 56 was sensitive for predicting an abnormal scvo 2 . twenty percent of patients demonstrated an initial value above the cutoff, potentially representing a group in whom scvo 2 measurement could be avoided. future studies aiming to decrease central line utilization could consider the use of peripheral o 2 measurements in these patients. sessions. ninety-two percent were rns, median clinical experience was 11-15 years, and 56% were from an intensive care unit. provider confidence increased significantly with a single session despite the highly experienced sample (figure 1 ). there was a trend for further increased confidence with an additional session and the increased confidence was maintained for at least 3-6 months given the normal sensitivity analysis. conclusion: high fidelity simulation significantly increases provider confidence even among experienced providers. this study was limited by its small sample size and recent changes in acls guidelines. background: recent data suggest alarming delays and deviations in major components of pediatric resuscitation during simulated scenarios by pediatric housestaff. objectives: to identify the most common errors of pediatric residents during multiple simulated pediatric resuscitation scenarios. methods: a retrospective observational study conducted in an academic tertiary care hospital. pediatric residents (pgy1 and pgy3) were videotaped performing a series of five pediatric resuscitation scenarios using a high-fidelity simulator (simbaby, laerdal): pulseless non-shockable arrest, pulseless shockable arrest, dysrhythmia, respiratory arrest, and shock. the primary outcome was the presence of significant errors prospectively defined using a validated scoring instrument designed to assess sequence, timing, and quality of specific actions during resuscitations based on the 2005 aha pals guidelines. residents' clinical performances were measured by a single video reviewer. the primary analysis was the proportion of errors for each critical task for each scenario. we estimated that the evaluation of each resident would provide a confidence interval less than 0.20 for the proportion of errors. results: twenty-four of 25 residents completed the study. across all scenarios, pulse check was delayed by more than 30 seconds in 56% (95%ci: 46%-66%). for non-shockable arrest, cpr was started more than 30 seconds after recognizing arrest in 21% (95%ci 7-42%) and inappropriate defibrillation was performed in 29% (95%ci 13-51%). for shockable arrest, participants failed to identify the rhythm in 58% (95%ci 37-78%), cpr was not performed in 25% (95%ci 10-47%), while defibrillation was delayed by more than 90 seconds in 33% (95%ci 16-51%) and not performed in one case. for shock, participants failed to ask for a dextrose check in 71% (95%ci 51-86%), and it was delayed by more than 60 seconds for all others. conclusion: the most common error across all scenarios was delay in pulse check. delays in starting cpr and inappropriate defibrillation were common errors in non-shockable arrests, while failure to identify rhythm, cpr omission, and delaying defibrillation were noted for shockable arrests. for shock, omission of rapid dextrose check was the most common error, while delaying the test when ordered was also significant. future training in pediatric resuscitation should target these errors. background: many scoring instruments have been described to measure clinical performance during resuscitation; however, the validity of these tools has yet to be proven in pediatric resuscitation. objectives: to determine the external validity of published scoring instruments to evaluate clinical performance during simulated pediatric resuscitations using pals algorithms and to determine if inter-rater reliability could be assessed. methods: this was a prospective quasi-experimental design performed in a simulation lab of a pediatric tertiary care facility. participants were residents from a single pediatric program distinct from where the instrument was originally developed. a total of 13 pgy1s and 11 pgy3s were videotaped during five simulated pediatric resuscitation scenarios. pediatric emergency physicians rated resident performances before and after a pals course using standardized scoring. each video recording was viewed and scored by two raters blinded to one another. a priori, it was determined that, for the scoring instrument to be valid, participants should improve their scores after participating in the pals course. differences in means between pre-pals and post-pals and pgy1 and pgy3 were compared using an anova test. to investigate differences in the scores of the two groups over the five scenarios, a two-factor anova was used. reliability was assessed by calculating an interclass correlation coefficient for each scenario. results: following the pals course, scores improved by 8.6% (3.8 to 13.3), 15.7% (8.6 to 22.7), 6.3% ()1.8 to 14.3), 18.2% (9.3 to 27), and 4.1% ()3.0 to 11.2) for the pulseless non-shockable arrest, pulseless shockable arrest, dysrhythmia, respiratory, and shock scenarios respectively. there were no differences in scores between pgy1s and pgy3s before and after the pals course. there was an excellent reliability for each scoring instrument with iccs varying between 0.85 and 0.98. conclusion: the scoring instrument was able to demonstrate significant improvements in scores following a pals course for pgy1 and pgy3 pediatric residents for the pulseless non-shockable arrest, pulseless shockable, and respiratory arrest scenarios only. however, it was unable to discriminate between pgy1s and pgy3s both before and after the pals course for any scenarios. the scoring instrument showed excellent inter-reliability for all scenarios. a background: medical simulation is a common and frequently studied component of emergency medicine (em) residency curricula. its utility in the context of em medical student clerkships is not well defined. objectives: the objective was to measure the effect of simulation instruction on medical students' em clerkship oral exam performance. we hypothesized that students randomized to the simulation group would score higher. we predicted that simulation instruction would promote better clinical reasoning skills and knowledge expression. methods: this was a randomized observational study conducted from 7/2009 to 5/2010. participants were fourth year medical students in their em clerkship. students were randomly assigned on their first day to one of two groups. the study group received simulation instruction in place of one of the lectures, while the control group was assigned to the standard curriculum. the standard clerkship curriculum includes lectures, case studies, procedure labs, and clinical shifts without simulation. at the end of the clerkship, all students participated in written and oral exams. graders were not blinded to group allocation. grades were assigned based on a pre-defined set of criteria. the final course composite score was computed based on clinical evaluations and the results of both written and oral exams. oral exam scores between the groups were compared using a two-sample t-test. we used the spearman rank correlation to measure the association between group assignment and the overall course grade. the study was approved by our institutional irb. results: sixty-one students participated in the study and were randomly assigned to one of two groups. twenty-nine (47.5%) were assigned to simulation and the remaining 32 (52.5%) students were assigned to the standard curriculum. students assigned to the simulation group scored 5.34% (95% ci 2.78-7.91%) higher on the oral exam than the non-simulation group. additionally, simulation was associated with a higher final course grade (p < 0.05). limitations of this pilot study include lack of blinding and interexaminer variability. conclusion: simulation training as part of an em clerkship is associated with higher oral exam scores and higher overall course grade compared to the standard curriculum. the results from this pilot study are encouraging and support a larger, more rigorous study. initial approaches to common complaints are taught using a standard curriculum of lecture and small group case-based discussion. we added a simulation exercise to the traditional altered mental status (ams) curriculum with the hypothesis that this would positively affect student knowledge, attitudes, and level of clinical confidence caring for patients with ams. methods: ams simulation sessions were conducted in june 2010 and 2011; student participation was voluntary. the simulation exercises included two ams cases using a full-body simulator and a faculty debriefing after each case. both students who did and did not participate in the simulations completed a written post-test and a survey related to confidence in their approach to ams. results: 154 students completed the post-test and survey. 65 (42%) attended the simulation session. 48 (31%) attended all three sessions. 58 (38%) participated in the lecture and small group. 15 (10%) did not attend any session. post-test scores were higher in students who attended the simulations versus those who did not: 7 (iqr, 6-8) vs. 6 (iqr, 4-7); p < 0.001. students who attended the simulations felt more confident about assessing an ams patient (58% vs. 42%; p = 0.05), articulating a differential diagnosis (66% vs. 47%; p = 0.03), and knowing initial diagnostic tests (74% vs. 53%; p = 0.01) and initial interventions (79% vs. 56%; p = 0.003) for an ams patient. students who attended the simulations were more likely to rate the overall ams curriculum as useful (94% vs. 61%; p < 0.001). conclusion: addition of a simulation session to a standard ams curriculum had a positive effect on student performance on a knowledge-based exam and increased confidence in clinical approach. the study's major limitations were that student participation in the simulation exercise was voluntary and that effect on applied skills was not measured. future research will determine whether simulation is effective for other chief complaints and if it improves actual clinical performance. background: the acgme has defined six core competencies for residents including ''professionalism'' and ''interpersonal and communication skills.'' integral to these two competencies is empathy. prior studies suggest that self-reported empathy declines during medical training; no reported study has yet integrated simulation into the evaluation of empathy in medical training. objectives: to determine if there is a relation between level of training and empathy in patient interactions as rated during simulation. methods: this is a prospective observational study at a tertiary care center comparing participants at four different levels of training: first (ms1) and third year (ms3) medical students, incoming em interns (pgy1), and em senior residents (pgy3/4). trainees participated in two simulation scenarios (ectopic pregnancy and status asthmaticus) in which they were responsible for clinical management (cm) and patient interactions (pi). this was the first simulation exposure during an established simulation curriculum for ms1, ms3, and pgy1. two independent raters reviewed videotaped simulation scenarios using checklists of critical actions for clinical management (cm: 0-11 points) and patient interactions (pi: 0-17 points). inter-rater reliability was assessed by intra-class correlation coefficients (iccs objectives: we explored attitudes and beliefs about the handoff, using qualitative methods, from a diverse group of stakeholders within the ems community. we also characterized perceptions of barriers to high-quality handoffs and identified strategies for optimizing this process. methods: we conducted seven focus groups at three separate gatherings of ems professionals (one local, two national) in 2010/2011. snowball sampling was used to recruit 48 participants with diverse professional, experiential, geographic, and demographic characteristics. focus groups, lasting 60-90 minutes, were moderated by investigators trained in qualitative methods, using an interview guide to elicit conversation. recordings of each group were transcribed. three reviewers analyzed the text in a multi-stage iterative process to code the data, describe the main categories, and identify unifying themes. results: participants included emts, paramedics, physicians, and nurses. clinical experience ranged from 4 months to 36 years. recurrent thematic domains when discussing attitudes and beliefs were: perceptions of respect and competence, professionalism, teamwork, value assigned to the process, and professional duty. modifiers of these domains were: hierarchy, skill/training level, severity/type of patient illness, and system/ regulatory factors. strategies to improving barriers to the handoff included: fostering familiarity and personal connections between ems and ed staff, encouraging two-way conversations, feedback, and direct interactions between ems providers and ed physicians, and optimizing ways for ems providers to share subjective impressions (beyond standardized data elements) with hospital-based care teams. conclusion: ems professionals assign high value to the ed handoff. variations in patient acuity, familiarity with other handoff participants, and perceptions of respect and professionalism appear to influence the perceived quality of this transition. regulatory strategies to standardize the contents of the handoff may not alone overcome barriers to this process. miology, public health) then developed an approach to assign ems records to one of 20 symptom-based illness categories (gastrointestinal illness, respiratory, etc). ems encounter records were characterized into these illness categories using a novel text analytic program. event alerts were identified across the state and local regions in illness categories using either change detection from baseline with (cusum) analysis (three standard deviations) and a novel text-proportion (tap) analysis approach (sas institute, cary, nc). results: 2.4 million ems encounter records over a 2year period were analyzed. the initial analysis focused upon gastrointestinal illness (gi) given the potential relationship of gi distress to infectious outbreaks, food contamination and intentional poisonings (ricin). after accounting for seasonality, a significant gi event was detected in feb 2010 (see red circle on graph). this event coincided with a confirmed norovirus outbreak. the use of cusum approach (yellow circle on graph) detected the alert event on jan 24, 2010. the novel tap approach on a regional basis detected the alert on dec 6, 2009. conclusion: ems has the advantage of being an early point of contact with patients and providing information on the location of insult or injury. surveillance based on ems information system data can detect emergent outbreaks of illness of interest to public health. a novel text proportion analytic technique shows promise as an early event detection method. assessing chronic stress in the emergency medical services elizabeth a. donnelly 1 , jill chonody 2 1 university of windsor, windsor, on, canada; 2 university of south australia, adelaide, australia background: attention has been paid to the effect of critical incident stress in the emergency medical services (ems); however, less attention has been given to the effect of chronic stress (e.g., conflict with administration or colleagues, risk of injury, fatigue, interference in non-work activities) in ems. a number of extant instruments assess for workplace stress; however, none address the idiosyncratic aspects of work in ems. objectives: the purpose of this study was to validate an instrument, adapted from mccreary and thompson (2006) , that assesses levels of both organizational and operational work-related chronic stress in ems personnel. methods: to validate this instrument, a cross-sectional, observational web-based survey was used. the instrument was distributed to a systematic probability sample of emts and paramedics (n = 12,000). the survey also included the perceived stress scale (cohen, 1983) to assess for convergent construct validity. results: the survey attained a 13.6% usable response rate (n = 1633); respondent characteristics were consistent across demographic characteristics with other studies of emts and paramedics. the sample was split in order to allow for exploratory and confirmatory fac-tor analyses (n = 847/n = 786). in the exploratory factor analysis, principal axis factoring with an oblique rotation revealed a two-factor, 34-item solution (kmo = 0.943, v 2 = 23344.38, df = 561, p £.001). confirmatory factor analysis suggested a more parsimonious, two-factor, 20-item solution (v 2 = 632.67, df = 168, p £ 0.001, rmsea = 0.06, cfi = 0.92, tli = 0.91, srmr = 0.04). the factors demonstrated good internal reliability (operational stress a = 0.877, organizational stress a = 0.868). both factors were significantly correlated (p £ 0.01) with the hypothesized convergent validity measure. conclusion: theory and empirical research indicate that exposure to chronic workplace stress may play an important part in the development of psychological distress, including burnout, depression, and posttraumatic stress disorder (ptsd). workplace stress and stress reactions may potentially interfere with job performance. as no extant measure assesses for chronic workplace stress in ems, the validation of this chronic stress measure enhances the tools ems leaders and researchers have in assessing the health and well-being of ems providers. effect of naltrexone background: survivors of sarin and other organophosphate poisoning can develop delayed encephalopathy that is not prevented by standard antidotal therapy with atropine and pralidoxime. a rat model of poisoning with the sarin analogue diisoprophylfluorophosphate (dfp) demonstrated impairment of spatial memory despite antidotal therapy with atropine and pralidoxime. additional antidotes are needed after acute poisonings that will prevent the development of encephalopathy. objectives: to determine the efficacy of naltrexone in preventing delayed encephalopathy after poisoning with the sarin analogue dfp in a rat model. the hypothesis is that naltrexone would improve performance on spatial memory after acute dfp poisoning. the sarin analogue dfp was used because it has similar toxicity to sarin while being less dangerous to handle. methods: a randomized controlled experiment at a university animal research laboratory of the effects of naltrexone on spatial memory after dfp poisoning was conducted. long evans rats weighing 250-275 grams were randomized to dfp group (n = 4, rats received a single intraperitoneal (ip) injection of dfp 5 mg/kg) or dfp+naltrexone group (n = 5, rats received a single ip injection of dfp (5 mg/kg) followed by naltrexone 5 mg/kg/day). after injection, rats were monitored for signs and symptoms of cholinesterase toxicity. if toxicity developed, antidotal therapy was initiated with atro-background: one of the primary goals of management of patients presenting with known or suspected acetaminophen (apap) ingestion is to identify the risk for apap-induced hepatotoxicity. current practice is to measure apap level at a minimum of 4 hours post ingestion and plot this value on the rumack-matthew nomogram. one retrospective study of apap levels drawn less than 4 hours post-ingestion found a level less than 100 mcg/ml to be sufficient to exclude toxic ingestion. objectives: the aim of this study was to prospectively determine the negative predictive value (npv) for toxicity of an apap level of less than 100 mcg/ml obtained less than 4 hours post-ingestion. methods: this was a multicenter prospective cohort study of patients presenting to one of five tertiary care hospitals that are part of the toxicology investigator's consortium (toxic). eligible patients presented to the emergency department less than 4 hours after known or suspected ingestion and had the initial apap level obtained at greater than 1 but less than 4 hours post ingestion. a second apap level was obtained at 4 hours or more post-ingestion and plotted on the rumack-matthew nomogram to determine risk of toxicity. the outcome of interest was the npv of an initial apap level less than 100 mcg/ml. a power analysis based on an alpha = 0.05 and power of 0.80 yielded the requirement of 71 subjects. results: data were collected on 171 patients over a 30month period from may 2009 to nov 2011. patients excluded from npv analysis consisted of: initial apap level greater than 100 mcg/ml (31), negligible apap level on both the initial and confirmatory apap level (31), initial apap level drawn less than one hour after ingestion (15), or an unknown time of ingestion (1). ninety-three patients met the eligibility criteria. two patients (2.2%) with an initial apap level less than 100 mcg/ml (54 mcg/ml at 90 min, 38 mcg/ml at 84 min) were determined to be at risk for toxicity based on oh s330 2012 saem annual meeting abstracts implementation of an emergency department sign-out checklist improves patient hand-offs at change of shift nicole m ma computer-assisted self-interviews improve testing for chlamydia and gonorrhea in the pediatric emergency department is the australian triage system a better indicator of psychiatric patients' needs for intervention than the ena emergency severity index triage system? patients were given an initial dose of 10 mg droperidol intramuscularly followed by an additional dose of 10 mg after 15 min if required. inclusion criteria were patients requiring physical restraint and parenteral sedation. the primary outcome was the time to sedation. secondary outcomes were the proportion of patients requiring additional sedation within the first hour, over-sedation measured as -3 on the sedation assessment tool, and respiratory compromise measured as oxygen saturation <90%. results: droperidol was administered to 424 patients and 370 of these had sedation scores documented. presentations included 56% with alcohol intoxication. dose ranged from 2.5 mg to 30 mg, median 10 mg (interquartile range conclusion: droperidol is effective for rapid sedation for abd and rarely causes over-sedation serum creatinine (scr) is widely used to predict risk; however, gfr is a better assessment of kidney function. objectives: to compare the ability of gfr and scr to predict the development of cin among ed patients receiving cects. we hypothesized that gfr would be the best available predictor of cin. methods: this was a retrospective chart review of ed patients ‡18 years old who had a chest or abdomen/pelvis cect between 06/01/11 and 07/31/11. baseline and follow-up scr levels were recorded. patients with initial scr >1.6 mg/dl were excluded, as per hospital radiology department protocol. cin was defined as a scr increase of either 25%, 0.5 mg/dl, or a gfr decrease of 25% within 72 hours of contrast exposure. gfr was calculated using the ckd epi and mdrd formulae, and analyzed in original units and categorized form (<60, ‡60) with each additional unit decrease in ckd epi, subjects were 3% more likely to develop cin (or = 1.03) (p < 0.0281). additionally, subjects with ckd epi <60 were 3.20 (or) times more likely to have cin than subjects with ckd epi ‡60 in original units, ckd epi (p < 0.0001) and mdrd (p < 0.0016) both had a significantly higher auc than scr. conclusion: age, as an independent variable, is the best predictor of cin, when compared with scr and gfr. due to a small number of cases with cin, the confidence intervals associated with the odds ratios are wide. future research should focus on patient risk stratification and establishing ed interventions to prevent cin. 694 a rat model of carbon monoxide induced neurotoxicity heather ellsworth non-traumatic subarachnoid hemorrhage diagnosed by lumbar puncture following non-diagnostic head ct: a retrospective case-control study and decision a dass score of >14 has been previously defined as an indicator of increased stress levels. multivariable logistic regression was utilized to identify demographic and work-life characteristics significantly associated with stress. results: 53.6% of individuals responded to the survey (34,340/64,032) and prevalence of stress was estimated at 5.9%. the following work-life characteristics were associated with stress: certification level, work experience, and service type. the odds of stress in paramedics was 32% higher when compared to emt-basics (or = 1.32, 95% ci = 1.23-1.42). when compared to £2 years of experience 28-2.18) were more likely to be stressed. ems professionals working in county (or = 1 ci = 1.07-1.51) and private services (or = 1 56) were more likely than those working in fire-based services to be stressed. the following demographic characteristics were associated with stress: general health and smoking status finally, former smokers (or = 1.34, 95% ci = 1.17-1.54) and current smokers (or = 1.37, 95% ci = 1.18-1.59) were more likely to be stressed than non-smokers literature suggests this is within the range of stress among nurses, and lower than physicians. while the current study was able to identify demographic and work-life characteristics associated with stress, the long-term effects are largely unknown methods: design: prospective randomized controlled trial. subjects: female sus scrofa swine weighing 45-55kg were infused with amitriptyline 0.5 mg/kg/minute until the map fell to 60% of baseline values. subjects were then randomized to experimental group (ife 7 ml/kg followed by an infusion of 0.25 ml/kg/minute) or control group (sb 2 meq/kg plus equal volume of normal saline). interventions: we measured continuous heart rate (hr), sbp, map, cardiac output (co), systemic vascular resistance (svr), and venous oxygen saturation (svo 2 ). laboratory values monitored included ph, pco 2 , bicarbonate, lactate, and amitriptyline levels. descriptive statistics including means, standard deviations, standard errors of measurement, and confidence limits were calculated. results: of 14 swine, seven each were allocated to ife and sb groups. there was no difference at baseline for each group regarding hr, sbp, map, co, svr, or svo 2 . ife and sb groups required similar mean amounts of tca to reach hypotension one ife and two sb pigs survived. conclusion: in this interim data analysis of amitriptyline-induced hypotensive swine, we found no difference in mitigating hypotension between ife and sb lipid rescue 911: a survey of poison center medical directors regarding intravenous fat emulsion therapy michael r. christian 1 , erin m. pallasch cook county hospital (stroger), chicago, il 745 reliability of non-toxic acetaminophen concentrations obtained less than 4 hours after ingestion evaluating age in the field triage of injured background: hiv screening in eds is advocated to achieve the goal of comprehensive population screening. yet, hiv testing in the ed is sometimes thwarted by a patient's condition (e.g. intoxication) or environmental factors (e.g. other care activities). whether it is possible to test these patients at a later time is unknown. objectives: we aimed to determine if ed patients who were initially unable to receive an hiv testing offer might be tested in the ed at a later time. we hypothesized that factors preventing testing are transient and that there are subsequent opportunities to repeat testing offers. methods: we reviewed medical records for patients presenting to an urban, academic ed who were approached consecutively to offer hiv testing during randomly selected periods from january 2008 to january 2009. patients for whom the initial attempted offer could not be completed were reviewed in detail with standardized abstraction forms, duplicate abstraction, and third-party discrepancy adjudication. primary outcomes included repeat hiv testing offers during that ed visit, and whether a testing offer might eventually have been possible either during the initial visit or at a later visit within 6 months. outcomes are described as proportions with confidence intervals. results: of 824 patients approached, initial testing offers could not be completed for 120 (15%). these 120 were 62% male, 52% white, and had a median age of 41 (18-64). a repeat offer of testing during the initial visit would have been possible for 99/120 (83%), and 52/99 (53%) were actually offered testing on repeat approach. of the 21 for whom a testing offer would not have been possible on the initial visit, 14 (67%) had at least one additional visit within 6 months, and 11/14 (79%) could have been offered testing on at least one visit. overall, a repeat testing offer would have been possible for 110/120 (93%, 95% ci 85-96%). conclusion: factors preventing an initial offer of hiv testing in the ed are generally transient. opportunities for repeat approach during initial or later ed encounters suggest that, given sufficient resources, the ed could succeed in comprehensively screening the population presenting for care. ed screening personnel who are initially unable to offer testing should repeat their attempt. hiv adopt an ''opt-out'' rapid hiv screening model in order to identify hiv infected patients. previous studies nationwide have shown acceptance rates for hiv screening of 20-90% in emergency departments. however, it is unknown how acceptance rates will vary in a culturally and ethnically diverse urban emergency department.objectives: to determine the characteristics of patients who accept or refuse ''opt-out'' hiv screening in an urban emergency department.methods: a self-administered, anonymous survey is administered to ed patients who are 18 to 64 years of age. the questionnaire is administered in english, russian, mandarin, and spanish. questions include demographic characteristics, hiv risk factors, perception of hiv risk, and acceptance of rapid hiv screening in the emergency department. results: to date 145 patients responded to our survey. of the 145, 102 (70.3%) did not accept an hiv test (group 1) in their current ed visit and 43 (29.7%) accepted an hiv test (group 2). the major two reasons given for opting out (i.e., group 1) was ''i do not feel that i am at risk'' (59.8%) and ''i have been tested for hiv before'' (25.5%). there was no difference between the groups in regards to sex (p = 0.737), age (p = 0.351), religious affiliation (p = 0.750), marital status (p = 0.331), language spoken at home (p = 0.211), and whether they had been hiv tested before (73.2% in group 1 and 59.4% in group 2; p = 0.123). however, there was a statistically significant difference with regards to educational level and income. more patients in group 1 (69.0%) and 46.1% in group 2 had less than a college level education (p < 0.05). similarly, more patients in group 1 (58.3%) and only 34.8% in group 2 had an annual household income of £$25,000 (p < 0.05). conclusion: in a culturally and ethnically diverse urban emergency department, patients with a lower socioeconomic status and educational level tend to opt out of hiv screening test offered in the ed. no significant difference in acceptance of ed hiv testing was found to date based on primary language spoken at home or religious affiliation background: antimicrobial resistance is a problem that affects all emergency departments. objectives: our goal was to examine all urinary pathogens and their resistance patterns from urine cultures collected in the emergency department (ed).methods: this study was performed at an urban/suburban community-teaching hospital with an annual volume of 40,000 visits. using electronic records, all cases of urine cultures received in 2009 were reviewed for data including type of bacteria, antibiotic resistance, and health care exposure (hcx). hcx was defined as no prior hospitalization within the previous six months, hospitalization within the previous three months, hospitalization within the previous six months, nursing home resident (nh), and presence of an indwelling urinary catheter (uc). an investigator abstracted all data with a second re-abstracting a random 5% for kappa statistics between 0.697 and 1.00. group background: approximately 12-20% of patients treated with epinephrine for anaphylaxis receive a second dose but the risk factors associated with repeat epinephrine use remain poorly defined. objectives: to determine whether obesity is a risk factor for requiring 2 + epinephrine doses for patients who present to the emergency department (ed) with anaphylaxis due to food allergy or stinging insect hypersensitivity. methods: we performed a retrospective chart review at four tertiary care hospitals that care for adults and children in new england between the following time periods: massachusetts general hospital (1/1/01-12/31/ 06), brigham and women's hospital (1/1/01-12/31/06), children's hospital boston (1/1/01-12/31/06), hasbro children's hospital (1/1/04-12/31/09). we reviewed the medical records of all patients presenting to the ed for food allergy or stinging insect hypersensitivity using icd9cm codes. we focused on anthropomorphic data and number of epinephrine treatments given before and during the ed visit. among children, calculated bmis were classified according to cdc growth indicators as underweight, healthy, overweight, or obese. all patients who presented on or after their 18th birthday were considered adults.background: transitions of care are ubiquitous in the emergency department (ed) and inevitably introduce the opportunity for errors. despite recommendations in the literature, few emergency medicine (em) residency programs provide formal training or standard process for patient hand-offs. checklists have been shown to be effective quality improvement measures in inpatient settings and may be a feasible method to improve ed hand-offs. objectives: to determine if the use of a sign-out checklist improves the accuracy and efficiency of resident sign-out in the ed as measured by reduced omission of key information, communication behaviors, and time to sign-out each patient. methods: a prospective study of first-and second-year em and non-em residents rotating in the ed at an urban academic medical center with an annual ed volume of 55,000. trained clinical research assistants observed resident sign-out during shift change over a two-week period and completed a 15-point binary observable behavior data collection tool to indicate whether or not key components of sign-out occurred. time to sign out each patient was recorded. we then created and implemented a computerized sign-out checklist consisting of key elements that should be addressed during transitions of care, and instructed residents to use this during hand-offs. a two-week post-intervention observation phase was conducted using the same data collection tool. proportions, means, and non-parametric comparison tests were calculated using stata. results: one hundred fifteen sign-outs were observed prior to checklist implementation and 72 after; one sign-out was excluded for incompleteness. significant improvements were seen in four of the measured signout components: inclusion of history of present illness increased by 18% (p < 0.001), likely diagnosis increased by 17% (p = 0.015), disposition status increased by 18% (p < 0.01), and patient/care team awareness of plan increased by 19% (p < 0.01). (figure 1 ) time data for 108 sign-outs pre-implementation and 72 post-implementation were available. seven sign-outs were excluded for incompleteness or spurious values. mean length of sign out was 83s (95% ci 65 to 100) and 71.7s (95% ci 52 to 92) per patient. conclusion: implementation of a checklist improved the transfer of information but did not affect the overall length of time for the sign-out. the objectives: to determine risk factors associated with adult patients presenting to the ed with cellulitis who fail initial antibiotic therapy and require a change of antibiotics or admission to hospital. methods: this was a prospective cohort study of patients ‡18 years presenting with cellulitis to one of two tertiary care eds (combined annual census 120,000). patients were excluded if they had been treated with antibiotics for the cellulitis prior to presenting to the ed, if they were admitted to hospital, or had an abscess only. trained research personnel administered a questionnaire at the initial ed visit with telephone follow-up 2 weeks later. patient characteristics were summarized using descriptive statistics and 95% confidence intervals (cis) were estimated using standard equations. backwards stepwise multivariable logistic regression models determined predictor variables independently associated with treatment failure (failed initial antibiotic therapy and required a change of antibiotics or admission to hospital). results: 598 patients were enrolled, 47 were excluded, and 53 were lost to follow-up. the mean (sd) age was 53.1 (18.4) and 56.4% were male. 497 (99.8%) patients were given antibiotics in the ed. 185 (37.2%) were given oral, 231 (46.5%) were given iv, and 81 (16.3%) patients were given both oral and iv antibiotics. 102 (20.5%) patients had a treatment failure. fever (temp >38°c) at triage (or: 4.1, 95% ci: 1.5, 10.7), leg ulcers (or: 3.1, 95% ci: 1.4, 6.6), edema or lymphedema (or: 2.5, 95% ci: 1.4, 4.5), and prior cellulitis in the same area (or: 1.8, 95% ci: 1.1, 2.9) were independently associated with treatment failure. conclusion: this analysis found four risk factors associated with treatment failure in patients presenting to the ed with cellulitis. these risk factors should be considered when initiating empiric outpatient antibiotic therapy for patients with uncomplicated cellulitis. use background: children presenting for care to a pediatric emergency department (ped) commonly require intravenous catheter (iv) placement. prior studies report that the average number of sticks to successfully place an iv in children is 2.4. successfully placing an iv requires identification of appropriate venous access targets. the veinviewer visionò (vvv) assists with iv placement by projecting a map of subcutaneous veins on the surface of the skin using near infrared light. objectives: to compare the effectiveness of the vvv versus standard approaches: sight (s) and sight plus palpation (s+p) for identifying peripheral veins for intravenous catheter placement in children treated in a ped. methods: experienced pediatric emergency nurses and physicians identified peripheral venous access targets appropriate for intravenous cannulation of a cross-sectional convenience sample of english speaking children aged 2-17 years presenting for treatment of sub-critical injury or illness whose parents provided consent. the clinicians marked the veins with different colored washable marker and counted them on the dorsum of the hand and in the antecubital fossa using the three approaches: s, s+p, and vvv. a trained research assistant photographed each site for independent counting after each marking and recorded demographics and bmi. counts were validated using independent photographic analyses. data were entered into sas 9.2 and analyzed using paired t-tests. results: 146 patients completed the study. clinicians were able to identify significantly more veins on the dorsum of the hand using vvv than s alone or s+p, 3.26 (p < 0.0001, ci 2.89-3.64) and 2.31 (p < 0.0001, ci 1.97-2.65), respectively, as well as significantly more veins in the antecubital fossa using vvv than s alone or s+p, 2.62 (p < 0.0001, ci 2.29-2.96) and 1.93 (p < 0.0001, ci 1.62-2.42), respectively. the differences in numbers of veins identified remained significant at p < 0.05 level across all ages, races, and bmis of children and across clinicians and validating independent photographic analyses. conclusion: experienced emergency nurses and physicians were able to identify significantly more venous access targets appropriate for intravenous cannulation in the dorsum of the hand and antecubital fossa of children presenting for treatment in a ped using vvv than the standard approaches of sight or sight plus palpation. an background: mental health emergencies have increased over the past two decades, and contribute to the ongoing rise in u.s. ed visit volumes. although data are limited, there is a general perception that the availability of in-person psychiatric consultation in the ed and of inpatient psychiatric beds is inadequate. objectives: to examine the availability of in-person psychiatry consultation in a heterogeneous sample of u.s. eds, and typical delays in transfer of ed patients to an inpatient psychiatric bed. methods: during 2009-2011, we mailed a survey to all ed directors in a convenience sample of nine us states (ar, co, ga, hi, ma, mn, or, vt, and wy). all sites were asked: ''are psychiatric consults available in-person to the ed?'' (yes/no), with affirmative respondents asked about the typical delay. sites also were asked about typical ed boarding time between a request for patient transfer and actual patient departure from the ed to an inpatient psychiatric bed. ed characteristics included rural/urban location, visit volume (visits/hour), admission rate, ed staffing, and the proportion of patients without insurance. data analysis used chi-square tests and multivariable logistic regression. results: surveys were collected from 495 (91%) of the 541 eds, with >80% response rate in every state. overall, only 30% responded that psychiatric consults were available in-person to the ed. in multivariable logistic regression, ed characteristics independently associated with lack of in-person psychiatric consultation were: location within specific states (eg, ar, ga), rural location, lower visit volume, and lower admission rate. among the subset of eds with psychiatric consults available, 48% reported a typical wait time of at least 1 hour. overall, 54% of eds reported that the typical time from request to actual patient transfer to an inpatient psychiatric bed was >6 hours, and 47% reported a maximum time in past year of >1 day (median 3 days, iqr 2-4). in a multivariable model, location in ma and higher visit volume were associated with greater odds of a maximum wait time of >1 day. conclusion: among 495 surveyed eds in nine states, only 30% have in-person psychiatric consultants available. moreover, approximately half of eds report boarding times of >6 h from request for transfer to actual departure to an inpatient psychiatric bed.background: many emergency departments (ed) in the united states use a five tiered triage protocol that has a limited evaluation of psychiatric patients. the australian triage scale (ats), a psychiatric triage system, has been used throughout australia and new zealand since the early 1990s. objectives: the objective of the study is to compare the current triage system, emergency nurses association (ena) esi 5-tier, to the ats for the evaluation of the psychiatric patients presenting to the ed. methods: a convenience sample of patients, 18 years of age and older, presenting with psychiatric complaints at triage were given the ena triage assessment by the triage nurse. a second triage assessment, performed by a research fellow, included all observed and reported elements using the ats protocol, a self-assessment survey and an agitation assessment using the richmond agitation sedation scale (rass). the study was performed at an inner city level i trauma center with 60,000 visits per year. the ed was a catchment facility for the police department for psychiatric patients in the area. patients were excluded if they were unstable, unable to communicate, or had a non-psychiatric complaint. results were analyzed in spss v16. the analysis of data used frequencies, descriptive and anova. results: a total of 100 patients were enrolled in the study: 72% were african american, 14% caucasian, 13% hispanic, 1% asian, and 1% indian; 63% of subjects enrolled were male. the patients' level of agitation using rass showed 59% were alert and calm, 22% were restless and anxious, 6% were agitated, and 5% combative, violent, or dangerous to self. the only significant correlation found was among the ats and several self assessment questions: ''i feel agitated on a 0 to 10 scale'' (p = 0.031) and ''i feel violent on a 0 to 10 scale'' (p = 0.001). there were no significant correlations found among the ena triage, rass scores, and throughput times. conclusion: the ats test was more sensitive to the patient declaring that he or she was agitated or felt violent. this shows that this system might be a more useful system in determining the severity of need of psychiatric patients presenting to the ed. variations background: hemoglobin-based oxygen carriers (hbocs) have been evaluated for small-volume resuscitation of hemorrhagic shock due to their oxygen carrying capability, but have found limited utility due to vasoactive side-effects from nitric oxide (no) scavenging. objectives: to define an optimal hboc dosing strategy and evaluate the effect of an added no donor, we use a prehospital swine polytrauma model to compare the effect of low-vs. moderate-volume hboc resuscitation with and without nitroglycerin (ntg) co-infusion as an no donor. we hypothesize that survival time will improve with moderate resuscitation and that an no donor will add additional benefit. methods: survival time was compared in groups (n = 7) of anesthetized swine subjected to simultaneous traumatic brain injury and uncontrolled hemorrhagic shock by aortic tear. animals received one of three different resuscitation fluids: lactated ringers (lr), hboc, or vasoattenuated hboc with ntg co-infusion. for comparison, these fluids were given in a severely limited fashion (sl) as one bolus every 30 minutes up to four total, or a moderately limited fashion (ml) as one bolus every 15 minutes up to seven total, to maintain mean arterial pressure ‡60 mmhg. comparison of resuscitation regimen and fluid type on survival time was made using two-way anova with interaction and tukey kramer adjustment for individual comparisons. results: there was a significant interaction between fluid regimen and resuscitation fluid type (anova, p = 0.011) indicating that the response to sl or ml resuscitation was fluid type-dependent. within the lr and hboc+ntg groups, survival time (mean, 95%ci) was longer for sl, 323.5 min ( injuries are common and result from many different mechanisms of injury (moi). knowing common fracture locations may help in diagnosis and treatment, especially in patients presenting with distracting injuries that may mask the pain of a radius fracture.objectives: we set out to determine the incidence of radius fracture locations among patients presenting to an urban emergency department (ed).background: carbon monoxide (co) is the leading cause of poisoning morbidity and mortality in the united states. standard treatment includes supplemental oxygen and supportive care. the utility of hyperbaric oxygen (hbo) therapy has been challenged by a recent cochrane review. hypothermia may mitigate delayed neurotoxic effects after co poisoning as it is effective in cardiac arrest patients with similar neuropathology. objectives: to develop a rat model of acute and delayed severe co toxicity as measured by behavioral deficits and cell necrosis in post-sacrifice brain tissue.methods: a total of 28 rats were used for model development; variable concentrations of co and exposure times were compared to achieve severe toxicity. for the protocol, six senescent long evans rats were exposed to 2,000 ppm of co for 20 minutes then 1,500 ppm for 160 minutes, followed by three successive dives at 30,000 ppm with an endpoint of apnea or seizure; there was a brief interlude between dives for recovery. a modified katz assessment tool was used to assess behavior at baseline and 2 hours, 1 day, and 1, 2, 3, 4, 5, and 6 weeks post-exposure. following this, the brains were transcardially fixed with formalin, and 5 lm sagittal slices were embedded in paraffin and stained with hematoxylin and eosin. a pathologist quantified the percentage of necrotic cells in the cortex, hippocampus (pyramidal cells), caudoputamen, cerebellum (purkinje cells), dentate gyrus, and thalamus of each brain to the nearest 10% from 10 randomly selected high power fields (400x background: there remains controversy about the cardiotoxic effects of droperidol, and in particular the risk of qt prolongation and torsades des pointes (tdp).objectives: this study aimed to investigate the cardiac and haemodynamic effects of high-dose parenteral droperidol for sedation of acute behavioural disturbance (abd) in the emergency department (ed). methods: a standardised intramuscular (im) protocol for the sedation of ed patients with abd was instituted as part of a prospective observational safety study in four regional and metropolitan eds. patients with abd were given an initial dose of 10 mg droperidol followed by an additional dose of 10 mg after 15 min if required. inclusion criteria were patients requiring physical restraint and parenteral sedation. the primary outcome was the proportion of patients who have a prolonged qt interval on ecg. the qt interval was plotted against the heart rate (hr) on the qt nomogram to determine if the qt was abnormal. secondary outcomes were frequency of hypotension and cardiac arrhythmias. results: ecgs were available from 273 of 424 patients with abd given droperidol. the median dose was 10 mg (iqr 10-15 mg; range: 5 to 30 mg). the median age was 33 years (rnge: 16 to 92) and 163 were males (60%). a total of four (1%) qt-hr pairs were above the ''at-risk'' line on the qt nomogram. transient hypotension occurred in 8 (3%), and no arrhythmias were detected.conclusion: droperidol appears to be safe when used for rapid sedation in the dose range of 5 to 30 mg. it rarely causes hypotension or qt prolongation. blood background: soldiers and law enforcement agents are repeatedly exposed to blast events in the course of carrying out their duties during training and combat operations. little data exist on the effect of this exposure on the physiological function of the human body. both military and law enforcement dynamic entry personnel, ''breachers'', began expressing sensitivity to the risk of injury as a result of multiple blast exposures. breachers apply explosives as a means of gaining access to barricaded or hardened structures. these specialists can be exposed to as many as a dozen lead-encased charges per day during training exercises.objectives: this observational study was performed by the breacher injury consortium to determine the effect of short-term exposure to blasts by breachers on whole blood lead levels (blls) and zinc protoporphyrin levels (zppls). methods: two 2-week basic breaching training classes were conducted by the united states marine corps' weapons training battalion dynamic entry school. each class included 14 students and up to three instructors, with six non-breaching marines serving as a control group. to evaluate for lead exposure, venous blood samples were acquired from study participants on the weekend prior and following training in the first training class, whereas the second training class had an additional level performed mid-training. blls and zppls were measured in a whole-blood sample using the furnace atomic absorption method and hematofuorimeter method, respectively. results: analysis of these blast injury data indicated students demonstrated significantly increased blls post-explosion (mean = 7 mcg/dl, sd 2.42, p < 0.001) compared to pre-training (mean = 3 mcg/dl, sd 1.60) and control subjects (mean = 3 mcg/dl, sd 2.73, p < 0.001). instructors also demonstrated significantly increased blls post explosion (mean = 6 mcg/dl, sd 1.95, p < 0.02) compared to pre-training (mean = 3 mcg/ dl, sd 1.14) and control subjects (mean = 3 mcg/dl, sd 2.73, p < 0.001). student and instructor zppls were not significantly different in post-training compared to pretraining or control groups. conclusion: the observation from this study that breachers are at risk of mild increases in blls support the need for further investigation into the role of lead following repeated blast exposure with munitions encased in lead. direct observation of the background: notification of a patient's death to family members represents a challenging and stressful task for emergency physicians. complex communication skills such as those required for breaking bad news (bbn) are conventionally taught with small-group and other interactive learning formats. we developed a de novo multi-media web-based learning (wbl) module of curriculum content for a standardized patient interaction (spi) for senior medical students during their emergency medicine rotation.objectives: we proposed that use of an asynchronous wbl module would result in students' skill acquisition for breaking bad news. methods: we tracked module utilization and performance on the spi to determine whether students accessed the materials and if they were able to demonstrate proficiency in its application. performance on the spi was assessed utilizing a bbn-specific content instrument developed from the griev_ing mnemonic as well as a previously validated instrument for assessing communication skills.results: three hundred seventy-two students were enrolled in the bbn curriculum. there was a 92% completion rate of the wbl module despite students being given the option to utilize review articles alone for preparation. students interacted with the activities within the module as evidenced by a mean number of mouse clicks of 42.1 (sd 21.6). overall spi scores were 94.5%, (sd 4.4) with content checklist scores of 92.8% (sd 5.7) and interpersonal communication scores 97.9% (sd 4.7). five students had failing content scores (<75%) on the spi and had a mean number of clicks of 30.8 (sd 28.2), which is not significantly lower than those passing (p = 0.21). students in the first year of wbl deployment completed self-confidence assessments which showed significant increases in confidence (2.86 tobackground: pelvis ultrasonography (us) is a useful bedside tool for the evaluation of women with suspected pelvic pathology. while pelvic us is often performed by the radiology department, it often lacks clinical correlation and takes more time than bedside us in the ed. this was a prospective observational study comparing the ed length of stay (los) of patients receiving ed us versus those receiving radiology us. objectives: the primary objective was to measure the difference in ed los. the secondary objectives were to 1) assess the role of pregnancy status, ob/gyn consult in the ed, and disposition, in influencing the ed los; and 2) to assess the safety of ed us by looking at patient return to the ed within 2 weeks and whether that led to an alternative diagnosis.methods: subjects were women over 13 years old presenting with a gi or gu complaint, and who received either an ed or radiology us. a t-test was used for the primary objective, and linear regression to test the secondary objective. odds ratios were performed to assess for interaction between these factors and type of ultrasound. subgroup analyses were performed if significant interaction was detected. results: forty-eight patients received an ed us and 85 patients received a radiology us. subjects receiving an ed us spent 162 minutes less in the ed (p < 0.001). in multivariate analysis, even when controlling for pregnancy status, ob/gyn consult, and disposition, patients who received an ed us had a los reduction of 108 minutes (p < 0.05). in odds ratio analysis, patients who were pregnant were 11 times more likely to have received an ed us (p < 0.05). patients who received an ob/gyn consult in the ed were five times more likely to receive a radiology us (p < 0.05). there was no association between type of us and disposition. in subgroup analyses, pregnant and non-pregnant patients who received an ed us still had a los reduction of 140 minutes (p < 0.01) and 112 minutes (p < 0.05), respectively. sample sizes were inadequate for subgroup analysis for subjects who had ob/gyn consults. in patients who did not receive an ob/gyn consult, those who received an ed us had a los reduction of 139 minutes (p < 0.001). finally, 10% of subjects returned within two weeks, but none led to an alternative diagnosis. conclusion: even when controlling for disposition, ob/gyn consultation, and pregnancy status, patients who received an ed us had a statistically and clinically significant reduction in their ed los. in addition, ed us is safe and accurate. background: although early surface cooling of burns reduces pain and depth of injury, there are concerns that cooling of large burns may result in hypothermia and worse outcomes. in contrast, controlled mild hypothermia improves outcomes after cardiac arrest and traumatic burn injury. objectives: the authors hypothesized that controlled mild hypothermia would prolong survival in a fluidresuscitated rat model of large scald burns. methods: forty sprague-dawley rats (250-300 g) were anesthetized with 40 mg/kg intramuscular ketamine and 5 mg/kg xylazine, with supplemental inhalational isoflurane as needed. a single full-thickness scald burn covering 40% of the total body surface area was created per rat using a mason-walker template placed in boiling water (100 deg c) for a period of 10 seconds. the rats were randomized to hypothermia (n = 20) and nonhypothermia (n = 20). core body temperature was continuously monitored with a rectal temperature probe. hypothermia was induced through intraperitoneal injection of cooled (4 deg c) saline. the core temperature was reduced by 2 deg c and maintained for a period of 2 hours, applying an ice or heat pack when necessary. the rats were then rewarmed back to baseline temperature. in the control group, room temperature saline was injected into the intraperitoneal cavity and core temperature was maintained using a heating pad as needed. the rats were monitored until death or for a period of 7 days, whichever was greater. the primary outcome was death. the difference in survival was determined using a kaplan-meier analysis or log rank test. results: the mean core temperatures were 32.5 deg c for the hypothermic group and 35.6 deg c for the normothermic group. the mean survival times were 124 hours for the hypothermic group (95% confidence interval [ci] = 98 to 150) and 100 hours for the normothermic group (95% ci = 68 to 132). the seven-day survival rates in the hypothermic and non-hypothermic groups were 67% and 53%. these differences were not significant, p = 0.33 for both comparisons. conclusion: induction of brief mild hypothermia increases but does not significantly prolong survival in a resuscitated rat model of large scald burns. serum objectives: we sought to determine levels of serum mtdna in ed patients with sepsis compared to controls and the association between mtdna and both inflammation and severity of illness among patients with sepsis. methods: prospective observational study of patients presenting to one of three large, urban, tertiary care eds. inclusion criteria: 1) septic shock: suspected infection, two or more systemic inflammatory response (sirs) criteria, and systolic blood pressure (sbp) <90 mmhg despite a fluid bolus; 2) sepsis: suspected infection, two or more sirs criteria, and sbp >90 mmhg; and 3) control: ed patients without suspected infection, no sirs criteria, and sbp >90 mmhg. three mtdnas (cox-iii, cytochrome b, and nadh) were measured using real-time quantitative pcr from serum drawn at enrollment. il-6 and il-10 were measured using a bio-plex suspension array system. baseline characteristics, il-6, il-10, and mtdnas were compared using one way anova or fisher exact test, as appropriate. correlations between mtdnas and il-6/il-10 were determined using spearman's rank. linear regression models were constructed using sofa score as the dependent variable, and each mtdna as the variable of interest in an independent model. a bonferroni adjustment was made for multiple comparisons.results: of 93 patients, 24 were controls, 29 had sepsis, and 40 had septic shock. we found no significant difference in any serum mtdnas among the cohorts (p = 0.14 to 0.30). all mtdnas showed a small but significant negative correlation with il-6 and il-10 (q = )0.24 to )0.35). among patients with sepsis or septic shock (n = 69), we found a small but significant negative association between mtdna and sofa score, most clearly with cytochrome b (p = 0.001). conclusion: we found no difference in serum mtdnas between patients with sepsis, septic shock, and controls. serum mtdnas were negatively associated with inflammation and severity of illness, suggesting that as opposed to trauma, serum mtdna does not significantly contribute to the pathophysiology of the sepsis syndromes. methods: we consecutively enrolled ed patients ‡18 years of age who met anaphylaxis diagnostic criteria from april 2008 to july 2011 at a tertiary center with 72,000 annual visits. we collected data on antihypertensive medications, suspected causes, signs and symptoms, ed management, and disposition. markers of severe anaphylaxis were defined as 1) intubation, 2) hospitalization (icu or floor), and 3) signs and symptoms involving ‡3 organ systems. antihypertensive medications evaluated included beta-blockers, angiotensin converting enzyme (ace) inhibitors, and calcium channel blockers (ccb). we conducted univariate and multivariate analyses to measure the association between antihypertensive medications and markers of severe anaphylaxis. because previous studies demonstrated an association between age and the suspected cause of the reaction with anaphylaxis severity, we adjusted for these known confounders in multivariate analyses. we report associations as odds ratios (ors) and corresponding 95% cis with p-values. results: among 302 patients with anaphylaxis, median age (iqr) was 44 (31-58) and 204 (67.5%) were female. eight (2.7%) patients were intubated, 57 (19%) required hospitalization, and 139 (46%) had ‡3 system involvement. forty-nine (16%) were on beta-blockers, 34 (11%) on ace inhibitors, and 22 (7.3%) on ccb. in univariate analysis, ace inhibitors were associated with intubation and ‡3 system involvement and ccb were associated with hospital admission. in multivariate analysis, after adjusting for age and suspected cause, ace inhibitors remained associated with hospital admission and beta-blockers remained associated with both hospital admission and ‡3 system involvement. conclusion: in ed patients, beta-blocker and ace inhibitor use may predict increased anaphylaxis severity independent of age and suspected cause of the anaphylactic reaction. background: advanced cardiac life support (acls) resuscitation requires rapid assessment and intervention. some skills like patient assessment, quality cpr, defibrillation, and medication administration require provider confidence to be performed quickly and correctly. it is unclear, however, whether high-fidelity simulation can improve confidence with a multidisciplinary group of providers with high levels of clinical experience. objectives: the purpose of the study was to test the hypothesis that providers undergoing high-fidelity simulation of cardiopulmonary arrest scenarios will express greater confidence. methods: this was a prospective cohort study conducted at an urban level i trauma center from january to october 2011 with a convenience sample of registered (rn) and license practical nurses, nurse practitioners, resident physicians, and physician assistants who agreed to participate in 2/4 high-fidelity simulation (laerdal 3g) sessions of cardiopulmonary arrest scenarios about 3 months apart. demographics were recorded. providers completed a validated preand post-test five-point likert scale confidence measurement tool before and after each session that ranged from not at all confident (1) to very confident (5) in recognizing signs and symptoms of, appropriately intervening in, and evaluating intervention effectiveness in cardiac and respiratory arrests. descriptive statistics, paired t-tests, and anova were used for data analysis. sensitivity testing evaluated subjects who completed their second session at 6 months rather than 3 months. results: sixty-five subjects completed consent, 39 completed one session, and 23 completed at least two background: prehospital studies have focused on the effect of health care provider gender on patient satisfaction. we know of no study that has assessed patient satisfication with patient and prehospital provider gender. some studies have shown higher patient satisfaction rates when cared for by a female health care provider.objectives: to determine the effect of ems provider gender on patient satisfaction with prehospital care. methods: a convenience sampling of all adult patients brought in to our ed, an urban level i trauma center by ambulance. a trained research associate (ra) stationed at triage conducted a survey using press ganey ems patient satisfaction questions. there were thirteen questions evaluating prehospital provider skills such as driving, courtesy, listening, medical care, and communication. each skill was assigned a point value between one and five; the higher the value the better the skill was performed. the patient's ambulance care report was copied for additional data extraction.results: a total of 225 surveys were done. average patient age was 71, and 54% were female. scores for all questions totaled 65 (mean 62.63 ± 5.1). prehospital providers pairings were: male-male (n = 141), male-female (n = 71), and female-female (n = 13). there were no statistically significant differences in scores between our pairings (mean scores for male:male 19.3, male:female 19.1, and female:female 19.2; p = 0.73). we found nonstatistical differences in satisfaction scores based on the gender of the emt in the back of the ambulance: males had a mean score of 62.7 and females had a mean score of 62.6 (p = 0.91). we examined gender concordance by comparing gender of the patient to the gender of the prehospital provider and found that male-male had a mean score of 62.8, female-female 62.2, and when the patient and prehospital provider gender did not match, 62.5 (p = 0.71). conclusion: we found no effect of gender difference on patient satisfaction with prehospital care. we also found that overall, patients are very satisfied with their prehospital care. objectives: we set out to determine the sensitivity and specificity of eps in determining the presence of recently ingested tablets or tablet fragments.methods: this was a prospective volunteer study at an academic emergency department. healthy volunteers were enrolled and kept npo for 6 hours prior to tablet ingestion. over 10 minutes subjects ingested 800 ml of water and 30 tablets. ultrasounds video clips were performed prior to any tablet ingestion, after drinking 200 ml of water, after 10 tablets, after 20 tablets, after 30 tablets, and 60 minutes after the final tablet ingestion yielding six clips per volunteer. all video clips were randomized and shown to three eps who were fellowship-trained in emergency ultrasound. eps recorded the presence or absence of tablets.results: ten volunteers underwent the pill ingestion protocol and sixty clips were collected. results for all cases and each rater are reported in the table. overall there was moderate agreement between raters (kappa = 0.42). sub-group analysis of 10, 20, or 30 pills did not show any significant improvement in sensitivity and specificity.conclusion: ultrasound has moderate specificity but poor sensitivity for identification of tablet ingestion. these results imply that point-of-care ultrasound has limited utility in diagnosing large tablet ingestion. background: intravenous fat emulsion (ife) therapy is a novel treatment that has been used to reverse the acute toxicity of some xenobiotics with varied success. us poison control centers (pcc) are recommending this therapy for clinical use, but data regarding these recommendations are lacking.objectives: to determine how us pcc have incorporated ife as a treatment strategy for poisoning. methods: a closed-format multiple-choice survey instrument was developed, piloted, revised, and then sent electronically to every medical director of an accredited us pcc using surveymonkey in march 2011; addresses were obtained from the aapcc listserv, participation was voluntary and remained anonymous; three reminder invitations were sent during the study period. data were analyzed using descriptive statistics.results: forty-five of 57 (79%) pcc medical directors completed the survey. all 45 respondents felt that ife therapy played a role in the acute overdose setting. thirty (67%) pcc have a protocol for ife therapy: 29 (97%) recommend an initial bolus of 1.5 ml/kg of a 20% lipid emulsion, 28 (93%) pcc recommend an infusion of lipids, and 27/28 pcc recommend an initial infusion rate of 0.25 ml/kg of a 20% lipid emulsion. thirty-three (73%) felt that ife had no clinically significant side effects at a bolus dose of 1.5 ml/kg (20% emulsion). forty-four directors (98%) felt that the ''lipid sink'' mechanism contributed to the clinical effects of ife therapy, but 26 (58%) felt that there was a yet undiscovered mechanism that likely contributed as well. in a scenario with cardiac arrest due to a single xenobiotic, directors stated that their center would always or often recommend ife after overdose of bupivicaine (43; 96%), verapamil (36; 80%), amitriptyline (31; 69%), or an unknown xenobiotic (12; 27%). in a scenario with significant hemodynamic instability due to a single xenobiotic, directors stated that their pcc would always or often recommend ife after overdose of bupivicaine (40; 89%), verapamil (28; 62%), amitriptyline (25; 56%), or an unknown xenobiotic (8; 18%).conclusion: ife therapy is being recommended by us pcc. protocols and dosing regimens are nearly uniform. most directors feel that ife is safe but are more likely to recommend ife in patients with cardiac arrest than in patients with severe hemodynamic compromise. further research is warranted. levels drawn at 4 hours or more (240 mcg/ml at 5 hours, 198 mcg ⁄ ml at 4 hours, respectively). npv for toxic ingestion of an initial apap level less than 100 mcg/ml was 97.8% (95% ci 92.3-99.7%).conclusion: an apap level of less than 100 mcg/ml drawn less than 4 hours after ingestion had a high npv for excluding toxic ingestion. however, the authors would not recommend reliance on levels obtained under 4 hours to exclude toxicity as the potential for up to 6.7% false negative results is considered unacceptable. background: genetic variations in the mu-opioid receptor gene (oprm1) mediate individual differences in response to pain and addiction.objectives: to study whether the common a118g (rs1799971) mu-opioid receptor single nucleotide polymorphism (snp) or the alternative splicing snp of oprm1 (rs2075572) was associated with overdose severity, we assessed allele frequencies of each including associations with clinical severity in patients presenting to the emergency department (ed) with acute drug overdose. methods: in an observational cohort study at an urban teaching hospital, we evaluated consecutive adult ed patients presenting with suspected acute drug overdose over a 12-month period for whom discarded blood samples were available for analysis. specimens were linked with clinical variables (demographics, urine toxicology screens, clinical outcomes) then de-identified prior to genetic snp analysis. in-hospital severe outcomes were defined as either respiratory arrest (ra, defined by mechanical ventilation) or cardiac arrest (ca, defined by loss of pulse). blinded taqman genotyping (applied biosystems) of the snps were performed after standard dna purification (qiagen) and whole genome amplification (qiagen repli-g). the plink 1.07 genetic association analysis program was used to verify snp data quality, test for departure from hardy-weinberg equilibrium, and test individual snps for statistical association. results: we evaluated 178 patients (37% female, mean age 41.2) who overall suffered 13 ras and 3 cas (of whom 2 died). urine toxicology was positive in 33%, of which there were positives for 32 benzodiazepines, 26 cocaine, 21 opiates, 13 methadone, and 6 barbiturates. all genotypes examined conformed to hardy-weinberg equilibrium. the 118g allele was associated with 2.5fold increased odds of ca/ra (or 2.5, p < 0.05). the rs2075572 mutant allele was not associated with ca/ ra. conclusion: these data suggest that the 118g mutant allele of the oprm1 gene is associated with worse clinical severity in patients with acute drug overdose. the findings add to the growing body of evidence linking the a118g snp with clinical outcome and raise the question as to whether the a118g snp may be a potential target for personalized medical prescribing practices with regard to behavioral/physiologic overdose vulnerability. key: cord-314098-1i6c0l3e authors: hayward, joshua a; tachedjian, mary; cui, jie; cheng, adam z; johnson, adam; baker, michelle l; harris, reuben s; wang, lin-fa; tachedjian, gilda title: differential evolution of antiretroviral restriction factors in pteropid bats as revealed by apobec3 gene complexity date: 2018-03-29 journal: mol biol evol doi: 10.1093/molbev/msy048 sha: doc_id: 314098 cord_uid: 1i6c0l3e bats have attracted attention in recent years as important reservoirs of viruses deadly to humans and other mammals. these infections are typically nonpathogenic in bats raising questions about innate immune differences that might exist between bats and other mammals. the apobec3 gene family encodes antiviral dna cytosine deaminases with important roles in the suppression of diverse viruses and genomic parasites. here, we characterize pteropid apobec3 genes and show that species within the genus pteropus possess the largest and most diverse array of apobec3 genes identified in any mammal reported to date. several bat apobec3 proteins are antiviral as demonstrated by restriction of retroviral infectivity using hiv-1 as a model, and recombinant a3z1 subtypes possess strong dna deaminase activity. these genes represent the first group of antiviral restriction factors identified in bats with extensive diversification relative to homologues in other mammals. bats are reservoirs of emerging viruses that are highly pathogenic to other mammals including humans. in recent years, viruses such as ebola, marburg, hendra, nipah, sosuga, and severe acute respiratory syndrome coronavirus (sars-cov) have crossed species barriers from their natural hosts into humans (calisher et al. 2006; smith and wang 2013; amman et al. 2015; hayman 2016) . intriguingly, experimental infections have demonstrated that specific bat species can be infected with ebola, marburg, sars-cov, hendra, and nipah viruses without the development of clinical symptoms (swanepoel et al. 1996; williamson et al. 1998; middleton et al. 2007; watanabe et al. 2010; jones et al. 2015) . this has led to efforts to uncover differences that might exist between the antiviral strategies of bats and other mammals zhang et al. 2013; zhou et al. 2016) . based upon studies of endogenous retroviruses, bats are an important source of mammalian retroviruses and many cross-species transmissions of retroviruses from bats to other mammals have been documented (hayward et al. 2013; cui et al. 2015) . a number of antiviral restriction factors are known to be capable of inhibiting the normal replication of retroviruses; among the most well studied of these are the members of the apobec3 (a3) protein family. a3 proteins are capable of restricting the replication of several virus families including retroviruses, hepadnaviruses, and parvoviruses, in addition to retrotransposons which are genomic parasites (chen et al. 2006; muckenfuss et al. 2006; renard et al. 2010) . a3 proteins are dna cytosine deaminases capable of hypermutating viral genomes and sterically hindering nascent dna synthesis (sheehy et al. 2002; lecossier et al. 2003; refsland and harris 2013) . all a3 proteins contain one or two zinccoordinating cytosine deaminase domains (z-domains) which are required for deaminase activity (chiu and greene 2008) . a3 proteins are divided into three phylogenetic subgroups, z1, z2, and z3, which are distinguished by the conservation of amino acid residues within the z-domain (larue et al. 2009; refsland and harris 2013) . we hypothesized that, given the prolonged history of coexistence between bats and their retroviruses, differences exist relative to other mammals in the antiviral genes responsible for retroviral restriction. pteropid bats are species within the family pteropodidae, and are implicated as reservoirs of emerging human viral pathogens (hayman 2016) . here, we report assemblies of the a3 loci in the closely related pteropids, pteropus vampyrus and pteropus alecto, which harbor nipah and hendra viruses, respectively (halpin et al. 2000; rahman et al. 2010) . several bat genomes (fang et al. 2015) have been published in recent years; however, no available assembly has resolved the a3 gene locus. this failure is likely due to the presence of many repetitive sequences, generated by extensive gene duplication and recombination, which are known to hinder genome assembly (zhang et al. 2013; fang et al. 2015) . in this study, pteropid a3 loci were generated through the step-wise extension of cdna-mapped gene scaffolds followed by remapping of sequence read archives, supported experimentally by an a3 expression analysis of bat spleen tissue. the functionality of p. alecto a3 subtypes were investigated through analysis of their ability to deaminate dna, and restrict the infectivity of a model retrovirus. the role of a3mediated restriction of ancient bat retroviruses was assessed using a hypermutation analysis of endogenous retroviruses within the genome of p. vampyrus. pteropid bats express a diverse range of apobec3 gene products pteropid a3 gene expression was initially determined by searching for a3 homologues in the p. alecto transcriptome database through a tblastn analysis using the human a3 proteins a3a, a3c, and a3h, representing the three zinccoordinating cytosine deaminase motif (z-domain) subtypes a3z1, a3z2, and a3z3, as search queries. a3 expression was subsequently assessed through analysis of cdna (complementary dna) generated from p. alecto spleen tissue, which harbors multiple immune cell types known to express a3 genes in humans (refsland et al. 2010 ). this analysis revealed 20 distinct a3 mrna transcripts derived from seven a3z1 genes, five a3z2 genes, and a single a3z3 gene (supplementary table s1, supplementary material online). pairwise comparison of these a3 transcripts (supplementary fig. s1 , supplementary material online) reveal a wide range of sequence identities between pteropid a3 genes, indicating considerable diversity among homologs: a3z1 genes range from 86% to 97% identity, whereas a3z2 genes range from 54% to 99% identity. in addition to the expected a3z1, a3z2, and a3z3 subtypes, several a3 amino acid sequences contained z-domains distinct from the canonical domains as defined by a specific combination of key conserved residues (larue et al. 2009 ). this group, designated a3z2b, carried both the a3z2 domain-specific tryptophan-phenylalanine (wf) residues and the a3z1 domain-specific arginine-adjacent isoleucine (ri) motif ( fig. 1a ). of the 20 unique transcripts, 19 harbored a single z-domain and one contained two z-domains, an nterminal a3z2b domain paired with a c-terminal a3z3 domain. these results indicate that pteropid bats express a more diverse repertoire of a3 mrna than reported to date for other mammals ( fig. 1c ). the generation of the pteropid a3 loci was accomplished in a step-wise manner outlined in supplementary fig. s2 , supplementary material online. the a3 gene locus in eutherian mammals is located between the cbx6 and cbx7 genes (larue et al. 2008) . blastn searches using the p. alecto a3 cdna products as queries revealed that publicly accessible bat genomes of p. alecto and p. vampyrus did not contain assembled a3 gene loci located within the boundaries of cbx6 and cbx7. however, the bat a3 cdna could be mapped, albeit across numerous discontiguous scaffolds, to the p. vampyrus genome accessible through the ensembl database. the a3 locus contains many large, repetitive sequence elements that result in the failure of automated assembly. this problem was overcome by matching exons derived from sequenced cdna, allowing the informed pairing of discontiguous scaffolds. this was not possible using the p. alecto genome as it did not contain sufficient assembled scaffolds to which a3 cdna products could be mapped. it was reasonable to map p. alecto cdna products against the p. vampyrus genome as these species are very closely related, with a recent divergence of $4.4 ma and 97-99% genomic nucleotide sequence identity (almeida et al. 2014 ). the p. vampyrus a3 locus was then generated through a step-wise extension of preexisting p. vampyrus gene scaffolds to join the gaps between exon-paired scaffolds, followed by remapping of the sequence read archives (sra) to validate and generate the 190 kb p. vampyrus a3 locus. the p. alecto a3 locus was generated by mapping p. alecto reads against the p. vampyrus a3 locus and was found to be slightly shorter at approximately 188 kb as a result of multiple short 3-20 nt insertions and deletions. these indels occurred in the a3 loci since the divergence of p. alecto and p. vampyrus, likely as a result of nonhomologous recombination events, the frequency of which is known to be increased in regions of the genome that contain sections of repetitive dna (metzenberg et al. 1991) . within the p. alecto a3 locus 18 gene coding regions were identified including 12 a3z1, one prototypical a3z2 (herein described as a3z2a), four a3z2b, and one a3z3 ( fig. 1b) . the organization and number of the a3 genes was found to be identical between p. vampyrus and p. alecto. neural network promoter prediction revealed promoter elements immediately upstream of each gene. interferon-stimulated response element (isre) transcription factor binding sites for interferon regulatory factors and nf-jb were also identified in each promoter region (supplementary data s1, supplementary material online). these data indicate that each pteropid a3 gene is most likely capable of being transcribed and that their expression is potentially upregulated as part of the antiviral interferon response. differential evolution of antiretroviral restriction factors in pteropid bats . doi:10.1093/molbev/msy048 mbe all p. alecto a3 cdna sequences were successfully mapped against the assembled p. alecto loci, revealing various alternative splicing schemes that allow many of the a3 genes to express numerous transcripts (supplementary fig. s3 and table s1, supplementary material online). a comparison of the a3 loci of p. vampyrus and p. alecto against the a3 loci of other mammals ( fig. 1c ) reveals that pteropid bats possess the largest a3 locus yet reported, containing 18 putative a3 analysis of the phylogenetic relationships between the a3 proteins of p. alecto and other mammals ( fig. 2 ) confirms the expected phylogenetic positions of the a3z1, a3z2a, and a3z3 proteins, and reveals that the a3z2b proteins, containing both z1-and z2-defining sequence motifs, phylogenetically cluster within the broader a3z2 clade. notably, pteropid a3z2a and a3z2b are positioned a greater sequence distance from each other than from the a3z2 proteins of distantly related mammals, revealing that pteropid bats possess four phylogenetically distinct a3 z-domain subtypes, two of which appear to be derivatives of the z2 clade. we tested the activity of the bat a3 proteins for which expression was verified (supplementary table s1, supplementary material online) by assessing their ability to cause mutations in e. coli though a rifampicin mutagenesis assay, utilizing human a3b and a3g as positive controls because they are known to be expressed and enzymatically active in e. coli (holden et al. 2008; shi et al. 2015) . mutations in the rpob gene increase with mutagenic stress mediated by a3 (harris et al. 2002; shi et al. 2015) . mutational frequencies above background levels were only observed for a3z1 proteins, and we found that four bat a3z1s showed a 10-fold or greater increase in mutation frequency ( fig. 3a ). in particular, a3z1c isomer b showed over 1000-fold increase above empty vector and 100-fold above a3z1c isomer a. to determine if the difference in mutation frequency correlated with differences in mutation signatures, we sequenced a pcr-amplified portion of the rpob gene known to harbor hotspots for rifampicin resistance. bat a3 mutation signatures were compared against human a3b and a3g ( fig. 3b ), which have dinucleotide sequence preferences for 5 0 -tc and 5 0 -cc, respectively. we found that bat a3z1c isomer a was highly specific for causing mutation at a known 5 0 -tcg hotspot at nucleotide position 1585, which is also the preferred target site for a3b (shi et al. 2015) . in contrast, a3z1c isomer b targeted sites at position 1565, 1585, and 1592, which have 5 0 -tct, 5 0 -tcg, and 5 0 -tcc contexts, respectively. decreased specificity may account for the high mutation frequency observed in the rifampicin mutagenesis assays. these results show that bat a3 proteins are functionally active and can mutate cytosines in a diverse range of dinucleotide contexts. the antiviral activity of bat a3 proteins was determined by assessing the capacity of representatives of each bat a3 subtype to restrict hiv-1 infectivity in target cells. hiv-1 encodes the anti-a3 counter-measure, vif, a protein that causes the proteolytic degradation of a3 through the recruitment of the cellular e3 ubiquitin ligase (marin et al. 2003; sheehy et al. 2003; kane et al. 2015) . hiv-1 stocks were generated by cotransfection of 293 t producer cells with bat a3 and an hiv-1 variant that does not express vif. hiv-1 particles were harvested from the cell culture supernatant, and virion production was determined by quantifying virion-associated reverse transcriptase (rt) activity. hiv-1 infectivity was assessed by inoculating human tzm-bl target cells, which express luciferase in the presence of hiv-1 infection, with virus normalized to equivalent levels of virion-associated rt activity. four bat a3 proteins demonstrated a dose-dependent inhibition of hiv-1 infectivity ( fig. 4) . the sole double z-domain bat a3 protein, a3z2bd-z3, produced the largest reduction in hiv-1 infectivity, which was comparable to inhibition by the human a3 protein, a3g. these data indicate that bat a3 proteins are functional restriction factors. fig. 2. mammalian apobec3 phylogeny. the phylogenetic relationships of mammalian a3 proteins were inferred using the maximum likelihood method and jtt þ g model with 1,000 bootstrap replicates. a3 subgroups z1, z2, and z3 are paralogs, which have expanded independently. bootstrap values for nodes represented in >40% of trees are shown. a3z1, a3z2/z2a, a3z2b, and a3z3 clades are shown in green, orange, pink, and blue, respectively. the scale represents the number of amino acid substitutions per site. differential evolution of antiretroviral restriction factors in pteropid bats . doi:10.1093/molbev/msy048 mbe ancient bat retroviruses were hypermutated by apobec3 a3 activity results in c to u lesions in the negative (cdna) strand, which lead to positive (genomic) strand g to a mutations in endogenous retroviruses (ervs) within host genomes (perez-caballero et al. 2008) . a3 proteins also exhibit local sequence-context bias in the nucleobases they deaminate. the presence of strand-biased, context-specific a3 mutagenic signatures in ervs constitutes unambiguous evidence of a past encounter with one or more cellular a3 enzymes (lee et al. 2008) . to assess hypermutation of pteropid ervs, we identified a group of closely related ervs from each of the betaretrovirus and gammaretrovirus genera present in the genome of p. vampyrus. we interrogated the p. vampyrus genome since the p. alecto genome assembly does not contain sufficient contiguous endogenous retroviral sequences to conduct this analysis. a previously reported group of bat endogenous betaretroviruses, sub-classified as group 7 betaretroviruses, was selected and a group of bat endogenous gammaretroviruses (herein described as group 1 gammaretroviruses) was identified in the manner described previously (hayward et al. 2013 ). phylogenies were generated using the maximum likelihood method and an ancestral reconstruction was performed by extrapolation of the common ancestor of each group (see supplementary fig. s4 , supplementary material online). mutations in each erv were identified by comparison against the group's common ancestor. strand-biased g to a hypermutation and contextspecific gg and ga dinucleotide preferences were identified in both groups ( fig. 5 ). these results are consistent with the hypothesis that ancient bat retroviruses were subjected to a3-mediated hypermutation, supporting the expected role of bat a3 proteins as antiviral restriction factors. the divergence times of the bat a3z1 genes were estimated using a molecular clock dating analysis that indicates these genes arose through duplication events beginning approximately 27 ma (supplementary fig. s5 , supplementary material online). this study was undertaken to address the question of differential antiviral gene evolution in bats, and more specifically, whether important differences exist in a major class of genes that control and restrict retroviruses and genomic retroelements. we demonstrated that bats possess 18 a3 genes, 13 of which are transcriptionally active in spleen tissue, with several proving active in a heterologous e. coli base mutation assay or mbe an hiv-1 restriction assay. in addition to the expected a3z1, a3z2, and a3z3 phylogenetic subtypes, we identified an additional division within the bat a3z2 clade in which we designated a3z2a as the prototypical a3z2 subtype, and a3z2b, as a subdivision that possesses a combination of invariant amino residues key to the amino acid character of both the a3z1 and a3z2 subtypes. representative a3 loci have previously been delineated for every eutherian order with the exceptions of chiroptera (bats) and all orders within the clade afrotheria (e.g., elephants, tenrecs, dugongs, and hyraxes) (nakano et al. 2017) . with the caveat that the a3 loci of the vast majority of mammalian species remain to be resolved, the pteropid bat species studied here are shown to possess the largest and most phylogenetically diverse antiviral a3 gene repertoire of any mammal reported to date. assessment of the functionality of bat a3 proteins through a rifampicin mutagenesis assay revealed several a3z1 proteins as potent mediators of cytosine deamination with a3z1 isomer b demonstrating a mutational frequency greater than human a3b and a3g. although some a3z1 proteins, and a3 proteins of other subtypes were not found to be catalytically active in this assay, numerous factors could account for inactivity within e. coli, such as protein misfolding or missing cellular cofactors and localization requirements. the capacity of bat a3 proteins to restrict retroviral infection was observed through the use of hiv-1 as a model target for a3 functionality. four bat a3 proteins representing single and double z-domain proteins of the z2a, z2b, and z3 subtypes were observed to be capable of inhibiting hiv-1 infectivity in a dose-dependent manner. the bat a3z1 proteins found to be mutagenic were not found to be capable of restricting hiv-1 infectivity. a possible explanation for this is that not all bat a3 proteins are capable of restricting this specific virus, as is the case for human a3 proteins such as a3b and a3c which inhibit siv but not hiv, whereas a3f and a3g are potent inhibitors of hiv (sheehy et al. 2003; yu et al. 2004; zheng et al. 2004) . although many endogenous retroviruses are present in bat genomes (cui et al. 2012; hayward et al. 2013) , no exogenous bat retroviruses have yet been reported. to address our expectation that bat retroviruses could be subjected to restriction by bat a3 proteins we performed a hypermutation analysis of the endogenized retroviruses present in the genome of p. vampyrus and found evidence of strand-biased, context-dependent, cytosine deamination of both beta-and gammaretroviruses. these results implicate bat a3 proteins as the mediators of g to a hypermutation in bat retroviruses. the large number of a3z1 genes present in the pteropid a3 locus is surprising given the scarcity of this subtype among other mammals (larue et al. 2009 ). the presence of 12 a3z1 genes in pteropid bats indicates an evolutionary advantage for pteropid bats in the maintenance and expansion of this subtype. the most comparable apobec3 protein in humans is a3a, the sole apobec3 single-domain z1 protein. a3b and a3g are double-domain proteins that have a z1 paired with a z2a domain (fig. 1c ). endogenous a3a is cytoplasmically fig. 4 . antiviral activity of a3 proteins. vif-deficient hiv-1 was generated in the presence of a3 proteins. human 293 t cells were transfected with 400 ng of the hivdvif expression plasmid piiibdvif and variable amounts (25-600 ng) of human or bat a3 expression plasmids. tzm-bl indicator cells were inoculated with 293 t cell culture supernatants normalized for viral particles by virion-associated rt activity. infectivity relative to hivdvif alone was measured by quantifying luciferase activity in tzm-bl lysates. hiv, human immunodeficiency virus type 1; ha3g, human apobec3g; z1c iso a, pteropus alecto apobec3z1c isomer a; z1c iso b, pteropus alecto apobec3z1c isomer b; z2a iso a, pteropus alecto apobec3z2a isomer a; z2ba iso c, p. alecto apobec3z2ba isomer c; z3 iso a, p. alecto apobec3z3 isomer a; z2bd-z3, p. alecto apobec3z2bd-z3. error bars represent the standard error of the mean from three independent assays. differential evolution of antiretroviral restriction factors in pteropid bats . doi:10.1093/molbev/msy048 mbe located (land et al. 2013 ) but accesses the nucleus during telophase, the final stage of mitosis ). it has restrictive activity against retrotransposons (chen et al. 2006; koito and ikeda 2013) and viruses including hiv-1, adenoassociated virus, and human papillomavirus (chen et al. 2006; berger et al. 2011; warren et al. 2015) . in humans, evidence suggests that a3z1 domain-containing proteins may represent a double-edged sword, as potent inhibitors of genomic retrotransposons and a possible threat to the cell's own genomic sequence integrity (larue et al. 2008; roberts et al. 2013; taylor et al. 2013; burns et al. 2015; green et al. 2016 ). one possible reason for the observed expansion of a3z1 genes in bats may be related to the evolution of flight, which is hypothesized to be an important link to the physiological conditions allowing bats to be sources of high viral diversity (o'shea et al. 2014 ). increased metabolic capacity has been linked to the evolution of sustained flight in bats (shen et al. 2010) , and the by-products of oxidative metabolism include reactive oxygen species that are known to damage cellular dna (cooke et al. 2003) . the most notable genetic adaptations involved in the development of flight in bats have been found to exist in the genes responsible for the detection and repair of cellular dna damage (zhang et al. 2013; zhou et al. 2016) . it is possible that enhanced tolerance to dna damage would allow for the expansion of genes such as a3 whose products are known to cause such damage. many studies have indicated that a3 enzymes restrict endogenous transposable elements such as line-1 (long interspersed nuclear element 1) and alu (muckenfuss et al. 2006; stenglein and harris 2006; kinomoto et al. 2007; wissing et al. 2011 ). interestingly, line-1 elements became extinct in the pteropid bat lineage approximately 24 ma (cantrell et al. 2008) . preliminary dating of the a3 gene expansions (supplementary fig. s5 , supplementary material online) indicate that fig. 5 . hypermutation analysis of endogenized retroviruses reveals that ancient bat retroviruses were subjected to restriction by a3 proteins. (a) comparison of guanine to adenine (g to a) versus cytosine to thymidine (c to t) mutations on the coding (þ) strand of endogenous retroviruses reveals strand-biased cytosine deamination. (b) subsequent analysis of the dinucleotide context of the g to a mutations in the coding strand reveals that the gg to ag and ga to aa mutations typical of human a3g are the most common mutations in the endogenous retroviruses of bats. statistical significance was calculated using fisher's exact test. *p < 0.05; **p < 0.01; ***p < 0.001. hayward et al. . doi:10.1093/molbev/msy048 mbe timing of the initial expansion of the bat a3z1 subtype may coincide with this event and further investigation of a possible connection between pteropid a3 genes and the line-1 extinction may be warranted. this study represents the first report of the differential evolution of an important antiviral gene family in bats relative to other mammals. the full account and explanation of the ability of bats to act as such effective hosts and transmitters of zoonotic viral pathogens will likely consist of many complex and interlinking factors beyond simple metrics such as gene numbers and amino acid sequence characteristics. ongoing studies of this expanded gene family should further demystify the strategies and mechanisms utilized by bats that result in an impressive tolerance for viral infections. approval for the use of bat tissues was granted by the commonwealth scientific and industrial research organization's australian animal health laboratory animal ethics committee (protocol aec1281). transcriptome data sets of p. alecto were generated from pooled total rna from mitogen-stimulated spleen, lymph node, white blood cells, and unstimulated bone-marrow and thymus from one pregnant female and one adult male, and the unstimulated thymus of a juvenile male as described previously (papenfuss et al. 2012 ensembl: enst00000348946] . the human a3 transcripts were translated into protein sequences using the clc genomics workbench 8.0 (https://www.qiagenbioinformatics.com/; last accessed april 3, 2018). to identify transcripts of interest the tblastn function of clc genomics workbench was used with the following parameters: blosum62 matrix, word size ¼ 3, e-values < 1 â 10 à12 , gap costs of existence 11, extension 1, with no filtering of regions of low complexity. transcripts were confirmed as a3 homologues by initial identification of, in their translated protein sequences, the presence of the conserved z-domain, hx 1 ex 21-27 s/twspcx 2-4 c (where "x" indicates a nonconserved position [conticello et al. 2005; larue et al. 2009 ]), and subsequent identification of the a3 subtype-specific conserved motifs described in detail in larue et al. (2008, 2009 ). to confirm a3 gene product expression, polymerase chain reaction (pcr) amplification assays were performed using cdna generated from p. alecto spleen tissue and various combinations of forward and reverse primers designed using the a3 transcript predictions identified in the transcriptome analysis. p. alecto trapping, tissue collection and rna extraction was performed as reported previously (cowled et al. 2011 ) except rnalater (ambion) preserved spleen from four male adult bats was pooled prior to tissue homogenization followed by total rna extraction with the qiagen rneasy mini kit with on-column removal of genomic dna with dnase i. total rna was reverse transcribed into cdna with the qiagen omniscript reverse transcriptase according to the manufacturer's protocol except that the reaction contained 100 ng/ll total rna, 1 lm oligo(dt) 18 (qiagen), and 10 lm random hexamers (promega). all pcr amplification assays were performed using the roche faststart high fidelity pcr system (cat # 04738292001) utilizing an annealing temperature gradient of 54-64 c in 2 c increments. each reaction was made up to a total of 20 ll, containing 1 unit of polymerase, 2 ng of total cdna, and 8 pmol of each primer. all other parameters for pcr amplification were performed according to the manufacturer's recommended protocol. pcr reaction products were separated by agarose gel electrophoresis, using a 1% (w/v) agarose gel run at 120 v for 25 min. all dna bands were excised from the gel and purified using the qiagen qiaquick gel extraction kit (cat # 28706) according to the manufacturer's protocol. the gel extracted pcr products were cloned into the pcr2.1 plasmid vector using the invitrogen topo ta cloning kit (cat # 450641) according to the manufacturer's protocol and transformed into escherichia coli top10 (invitrogen). the nucleotide sequence of clones were determined by sanger sequencing using the m13f (5 0 -gtaaaacgacggccag-3 0 ) and m13r (5 0 -caggaaacagctatgac-3 0 ) sequencing primers. to identify the gene scaffolds representing fragments of the a3 gene locus in the p. vampyrus genome, a blastn analysis was performed using the a3 gene products amplified in the cdna analysis as queries. the p. vampyrus genome was downloaded from ensembl and all blastn searches were performed locally using clc genomics workbench with the following parameters: match score 2, mismatch cost à3, word size ¼ 5, e-values < 1 â 10 à5 , gap costs of existence 5, extension 2, with no filtering of regions of low complexity. to extend the sequence of each scaffold, 150 nt at both the 5 0 and 3 0 termini was used as the query sequence for a blastn search against the p. vampyrus illumina whole genome sequencing (wgs) project sra [sra: srp047390] using the ncbi blastn suite (http://blast.ncbi.nlm.nih.gov/blast.cgi; last accessed april 3, 2018) with the following parameters: program ¼ megablast, maximum target sequences ¼ 500, e-values < 1 â 10 à20 , word size ¼ 28, maximum matches in a query range ¼ 13, match score ¼ 1, mismatch cost ¼ 2, gap costs ¼ linear, with no filtering or masking. all hits with a differential evolution of antiretroviral restriction factors in pteropid bats . doi:10.1093/molbev/msy048 mbe percentage identity of 97% or higher were downloaded and assembled using the clc genomics workbench assemble sequences tool into a contiguous consensus sequence using the following parameters: minimum aligned read length ¼ 20, alignment stringency ¼high, conflicts ¼ vote (a, c, g, t). consensus sequences were aligned with the original query to confirm that the assembly represented a terminally extended match of the original query sequence; they were subsequently used as a new query in an otherwise identical blastn search against the same sra. this process was iteratively repeated until all scaffolds could be extended out far enough that their sequences overlapped with another scaffold. the extended and overlapping scaffolds were joined end to end to create a contiguous reference sequence construct representing the p. vampyrus a3 gene locus. to validate the draft a3 locus reference sequence, all p. vampyrus wgs sra were downloaded from ncbi and locally converted into paired fastq data sets using the ncbi sra toolkit 2.4.3 (http://www.ncbi.nlm.nih.gov/traces/sra/sra.cgi; last accessed april 3, 2018). each data set was subjected to a quality control check using the fastqc program 0.11.3 (babraham bioinformatics, cambridge). paired fastq data sets were imported into clc genomics workbench using the illumina import function and the clc genomics workbench next generation sequencing (ngs) toolkit was used for all following tasks. all data sets were trimmed on the basis of quality using the default parameters. data sets with short paired-read distances were assessed for the presence of overlapping pairs and these were merged where appropriate using the default parameters. the p. vampyrus data set included long paired reads with insert sizes of 2-20 kb which are helpful for the resolution of repetitive gene regions. validation of the p. vampyrus a3 locus was performed by mapping all reads to the reference sequence construct using the following parameters: linear gap cost, length fraction ¼ 0.9, similarity fraction 0.9, with automatic detection of paired read distances. other parameters were left in their default settings. potential gene coding regions were identified by mapping p. alecto a3 cdna sequences against the locus through a local blastn analysis using clc genomics workbench with default settings. promoter elements were predicted using the online neural network promoter prediction 2.2 tool (reese 2001) (http://www.fruitfly.org/seq_tools/promoter.html; last accessed april 3, 2018) using the default parameters. isre were predicted using the online promo transcription factor binding site prediction tool (farr e et al. 2003) (http://alggen. lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirdb=tf_ 8.3 ; last accessed april 3, 2018) using the parameters: species: mammalia, factors: mammalia. the identities of the putative bat a3 gene products were confirmed by phylogenetic analysis. all reference sequences were downloaded from ensembl (https://www.ensembl.org/; last accessed april 3, 2018) and aligned with p. alecto sequences using muscle (edgar 2004 ). regions of low conservation and uncertain alignment were removed using the gblocks program (talavera and castresana 2007) . phylogenetic relationships were inferred using the maximum likelihood (ml) method available in the mega6 program (tamura et al. 2013) , employing nearest-neighbor interchange and branch swapping, and the best-fit model of amino acid substitution was found to be jtt þ g (tamura et al. 2013) . to determine the robustness of each node 1,000 bootstrap replications were incorporated. bat a3 cdnas were pcr amplified from parental vectors and subcloned into pet24(þ) vectors (emd biosciences). plasmids were transformed into calcium competent c43 (de3) strain e. coli (gift from dr. do-hyung kim) and grown in lb plates with kanamycin. single colonies were selected and grown in lb with kanamycin for 28 h to ensure stationary phase growth. thereafter, cultures were spread on either rifampicin plates or serially diluted in m9 salt media and spread on kanamycin plates. colony counts were obtained after 18 h. rpob mutation frequency was obtained by dividing the number of viable colonies on rifampicin plates by colonies on kanamycin plates and correcting for the dilution factor. a portion of the rpob gene was pcr amplified using primers: forward 5 0 -ttggcgaaatggcggaaaacc and reverse 5 0 -caccgacggataccacctgctg. pcr products were enzymatically purified using exonuclease i and rsap (neb). purified fragments were sequenced using the forward primer (genewiz, nj). human embryonic kidney cells (293 t cells) and human epithelial cervical adenocarcinoma cells modified to express constitutively high levels of hiv-1 receptors and coreceptors (tzm-bl cells), were utilized in the inhibition of infectivity assay. the tzm-bl cells were obtained through the nih aids research and reference reagent program (wei et al. 2002) . all cell cultures were maintained at 37 c, 5% co 2 in dulbecco's modified eagle's medium (thermo) supplemented with heat-inactivated fetal bovine serum (100 ml/l; invitrogen), glutamine (292 lg/ml; invitrogen), and the antibiotics penicillin (100 units/ml; invitrogen) and streptomycin (100 units/ml; invitrogen). the antiviral activity of bat a3 was assessed by examining the capacity to restrict hiv-1 infectivity. bat a3 cdnas were pcr amplified from parental vectors and subcloned into pcdna3.1 mammalian expression vectors (thermo fisher, ma). 293 t cells were cotransfected with different quantities of bat a3 or human a3g (phu-a3gha; [mariani et al. 2003 ]) expression plasmids (25-600 ng) and 400 ng of piiibdvif (simon et al. 1995) , which generates hiv-1 virions in the absence of vif. untransfected cells and cells transfected only with piiibdvif were used as controls. transfected cell cultures were incubated at 37 c, 5% co 2 for 48 h, and then virion-containing supernatants were collected and clarified by centrifugation at 200 â g for 5 min. virus production was determined by quantifying virion-associated rt activity, as previously described (wapling et al. 2005) . tzm-bl cells hayward et al. . doi:10.1093/molbev/msy048 mbe were inoculated with rt normalized supernatant and incubated at 37 c, 5% co 2 for 48 h. hiv-1 infection of tzm-bl cells was assessed through quantitation of luciferase activity in cell lysates as previously described (tyssen et al. 2010 ). the impact of cytosine deamination on the genomes of ancient bat retroviruses was assessed by hypermutation analysis (lee et al. 2008) . the genomes of all ervs used in the analysis were extracted from the p. vampyrus genome. multiple outgroups were tested to confirm that the position of the ancestral node remained consistent. for the betaretroviruses, we . the most closely related betaretrovirus and gammaretrovirus to our erv groups were jsrv and mmlv, respectively, and were employed as outgroups for the phylogenies. sequence alignments and phylogenies were conducted as described for the apobec3 phylogenetic analysis, and the best-fit model of nucleotide substitution was found to be t92 þ g for both groups of retroviruses. ancestral sequences were computationally extrapolated using the mega6 program (tamura et al. 2013) using the mutations accumulated in each erv since integration by estimating the ancestral state of each node in the maximum likelihood phylogenetic tree. the state is chosen to be the one that maximizes the probability of the given sequence data. mutational biases were then evaluated by analyzing an alignment of the ancestral sequence and the ervs (supplementary data s2 and s3, supplementary material online) using the online hypermut tool (http://www.hiv. lanl.gov/content/sequence/hypermut/hypermut.html; last accessed april 3, 2018) (rose and korber 2000) with context enforced on the ancestral reference sequence. the hypermut tool incorporates fisher's exact test to determine statistical significance. to estimate the time of the expansion of a3z1 genes in bat genome, molecular dating was performed using the bayesian evolutionary method as implemented in beast v1.8.2 (drummond et al. 2012) . all reference nucleotide sequences were aligned using muscle and adjusted manually. a recombination test was then performed using the rdp, geneconv and maxchi methods implemented in rdp3 (martin et al. 2010) . after removing recombinant sequences, all sequences were submitted to beast for estimation of the divergence time. fossil node calibrations were taken from published literature (kumar et al. 2017 ma. bayesian phylogeny was inferred using the gtr þ g substitution model, the yule speciation tree prior and lognormal relaxed clock (uncorrelated) prior. the beast processes were run for ten million generations until all relevant parameters converged [effective sample size (ess)>200], with 10% of the bayesian markov chain monte carlo (mcmc) chains discarded as burn-in. supplementary data are available at molecular biology and evolution online. each flying fox on its own branch: a phylogenetic tree for pteropus and related genera (chiroptera: pteropodidae) a recently discovered pathogenic paramyxovirus, sosuga virus, is present in rousettus aegyptiacus fruit bats at multiple locations in uganda apobec3a is a specific inhibitor of the early phases of hiv-1 infection in myeloid cells apobec3b: pathological consequences of an innate immune dna mutator bats: important reservoir hosts of emerging viruses loss of line-1 activity in the megabats apobec3a is a potent inhibitor of adenoassociated virus and retrotransposons the apobec3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements evolution of the aid/apobec family of polynucleotide (deoxy) cytidine deaminases oxidative dna damage: mechanisms, mutation, and disease molecular characterisation of toll-like receptors in the black flying fox pteropus alecto identification of diverse groups of endogenous gammaretroviruses in mega and microbats bats and rodents shape mammalian retroviral phylogeny bayesian phylogenetics with beauti and the beast 1.7 muscle: multiple sequence alignment with high accuracy and high throughput bgd: a database of bat genomes identification of patterns in biological sequences at the alggen server: promo and malgen apobec3a damages the cellular genome during dna replication isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus rna editing enzyme apobec1 and some of its homologs can act as dna mutators bats as viral reservoirs identification of diverse full-length endogenous betaretroviruses in megabats and microbats crystal structure of the anti-viral apobec3g catalytic domain and functional implications experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera lineagespecific viral hijacking of non-canonical e3 ubiquitin ligase cofactors in the evolution of vif anti-apobec3 activity all apobec3 family proteins differentially inhibit line-1 retrotransposition intrinsic immunity against retrotransposons by apobec cytidine deaminases. front microbiol timetree: a resource for timelines, timetrees, and divergence times subcellular localization of the apobec3 proteins during mitosis and implications for genomic dna deamination endogenous apobec3a dna cytosine deaminase is cytoplasmic and nongenotoxic the artiodactyl apobec3 innate immune repertoire shows evidence for a multi-functional domain organization that existed in the ancestor of placental mammals guidelines for naming nonprimate apobec3 genes and proteins hypermutation of hiv-1 dna in the absence of the vif protein hypermutation of an ancient human retrovirus by apobec3g species-specific exclusion of apobec3g from hiv-1 virions by vif hiv-1 vif protein binds the editing enzyme apobec3g and induces its degradation rdp3: a flexible and fast computer program for analyzing recombination homology requirements for unequal crossing over in humans experimental nipah virus infection in pteropid bats (pteropus poliocephalus) apobec3 proteins inhibit human line-1 retrotransposition a conflict of interest: the evolutionary arms race between mammalian apobec3 and lentiviral vif bat flight and zoonotic viruses the immune gene repertoire of an important viral reservoir, the australian black flying fox evidence for restriction of ancient primate gammaretroviruses by apobec3 but not trim5a proteins application of a time-delay neural network to promoter annotation in the drosophila melanogaster genome the apobec3 family of retroelement restriction factors quantitative profiling of the full apobec3 mrna repertoire in lymphocytes and tissues: implications for hiv-1 restriction apobec1 and apobec3 cytidine deaminases as restriction factors for hepadnaviral genomes in non-humans in vivo an apobec cytidine deaminase mutagenesis pattern is widespread in human cancers detecting hypermutations in viral sequences with an emphasis on g!a hypermutation isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein the antiretroviral enzyme apobec3g is degraded by the proteasome in response to hiv-1 vif adaptive evolution of energy metabolism genes and the origin of flight in bats crystal structure of the dna deaminase apobec3b catalytic domain complementation of vif-defective human immunodeficiency virus type 1 by primate, but not nonprimate, lentivirus vif genes bats and their virome: an important source of emerging viruses capable of infecting humans apobec3b and apobec3f inhibit l1 retrotransposition by a dna deamination-independent mechanism experimental inoculation of plants and animals with ebola virus improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments mega6: molecular evolutionary genetics analysis version 6.0 dna deaminases induce break-associated mutation showers with implication of apobec3b and 3a in breast cancer kataegis structure activity relationship of dendrimer microbicides with dual action antiviral activity mutations that abrogate human immunodeficiency virus type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer apobec3a functions as a restriction factor of human papillomavirus bat coronaviruses and experimental infection of bats, the philippines emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (t-20) monotherapy transmission studies of hendra virus (equine morbilli-virus) in fruit bats, horses and cats endogenous apobec3b restricts line-1 retrotransposition in transformed cells and human embryonic stem cells apobec3b and apobec3c are potent inhibitors of simian immunodeficiency virus replication comparative analysis of bat genomes provides insight into the evolution of flight and immunity human apobec3f is another host factor that blocks human immunodeficiency virus type 1 replication contraction of the type i ifn locus and unusual constitutive expression of ifn-a in bats key: cord-002774-tpqsjjet authors: nan title: section ii: poster sessions date: 2017-12-01 journal: j urban health doi: 10.1093/jurban/jti137 sha: doc_id: 2774 cord_uid: tpqsjjet nan food and nutrition programs in large urban areas have not traditionally followed a systems approach towards mitigating food related health issues, and instead have relied upon specific issue interventions char deal with downstream indicators of illness and disease. in june of 2004, the san francisco food alliance, a group of city agencies, community based organizations and residents, initiated a collahorarive indicator project called rhe san francisco food and agriculture assessment. in order to attend to root causes of food related illnesses and diseases, the purpose of the assessment is to provide a holistic, systemic view of san francisco\'s food system with a focus on three main areas that have a profound affect on urban public health: food assistance, urban agriculture, and food retailing. using participatory, consensus methods, the san francisco food alliance jointly developed a sec of indicators to assess the state of the local food system and co set benchmarks for future analysis. members collected data from various city and stare departments as well as community based organizations. through the use of geographic information systems software, a series of maps were created to illustrate the assets and limitations within the food system in different neighborhoods and throughout the city as a whole. this participatory assessment process illustrates how to more effectively attend to structural food systems issues in large urban areas by ( t) focusing on prevention rather than crisis management, (2) emphasizing collaboration to ensure institutional and structural changes, and (3) aptly translating data into meaningful community driven prevention activities. to ~xplore the strategies to overcome barriers to population sample, we examined the data from three rapid surveys conducted at los angeles county (lac). the surveys were community-based partic· 1patory surveys utilizing a modified two-stage cluster survey method. the field modifications of the method resulted in better design effect than conventional cluster sample survey (design effect dose to that if the survey was done as simple random sample survey of the same size). the surveys were con· ducte~ among parents of hispanic and african american children in lac. geographic area was selected and d1.v1ded int.o small c~usters. in the first stage, 30 clusters were selected with probability proportionate to estimated size of children from the census data. these clusters were enumerated to identify and develop a list of households with eligible children from where a random sample was withdrawn. data collectmn for consented respondents involved 10-15 minutes in-home interview and abstraction of infor· ma~ion from vaccine record card. the survey staff had implemented community outreach activities designed to fost~r an~ maintain community trust and cooperation. the successful strategies included: developing re.lat1on .w.1th local community organizations; recruitment of community personnel and pro· vide them with training to conduct the enumeration and interview; teaming the trained community introduction: though much research has been done on the health and social benefits of pet ownership for many groups, there have been no explorations of what pet ownership can mean to adults who are marginalized, living on fixed incomes or on the street in canada. we are a community group of researchers from downtown toronto. made up of front line staff and community members, we believe that community research is important so that our concerns, visions, views and values are presented by us. we also believe that research can and should lead to social change. method: using qualitative and exploratory methods, we have investigated how pet ownership enriched and challenged the lives of homeless and transitionally housed people. our research team photographed and conducted one-on-one interviews with 11 pet owners who have experienced home· lessncss and live on fixed incomes. we had community participation in the research through a partnership with the fred vicror centre camera club. many of the fred victor centre camera club members have experienced homelessness and being marginalized because of poverty. the members of the dub took the photos and assisted in developing the photos. they also participated in the presenta· tion of our project. results: we found that pet ownership brings important health and social benefits to our partici· pants. in one of the most poignant statements, one participant said that pet ownership " ... stops you from being invisible." another commented that "well, he taught me to slow down, cut down the heavy drugs .. " we also found that pet ownership brings challenges that can at times be difficult when one is liv· ing on a fixed income. we found that the most difficult thing for most of the pet owners was finding affordable vet care for their animals. conclusion: as a group, we decided that research should only be done if we try to make some cha.nges about what we have learned. we continue the project through exploring means of affecting social change--for example, ~eti.tions and informing others about the result of our project. we would like to present our ~mdmgs and experience with community-based participatory action resea.rch m an oral. presentarton at yo~r conference in october. our presentation will include com· mumty representation ~f. both front-hne staff and people with lived experience of marginalization and homdessness. if this is not accepted as an oral presentation, we are willing to present the project m poster format. introduction the concept of a healthy city was adopted by the world health organization some time ago and it includes strong support for local involvement in problem solving and implementation of solutions. while aimed at improving social, economic or environmental conditions in a given community, more significantly the process is considered to be a building block for poliq reform and larger scale 'hange, i.e. "acting locally while thinking globally." neighbourhood planning can he the entry point for citizens to hegin engaging with neighbours on issues of the greater common good. methods: this presentation will outline how two community driven projects have unfolded to address air pollution. the first was an uphill push to create bike lanes where car lanes previously existed and the second is an ongoing, multi-sectoral round table focused on pollution and planning. both dt•monstrate the importance of having support with the process and a health focus. borrowing from traditions of "technical aid"• and community development the health promoter /planner has incorporated a range of "determinants of health" into neighbourhood planning discussions. as in most urban conditions the physical environment is linked to a range of health stressors such as social isolation, crowding, noise, lack of open space /recreation, mobility and safety. however typical planning processes do not hring in a health perspective. health as a focus for neighbourhood planning is a powerful starti_ng point when discussing transportation planning or changing land-uses. by raising awareness on determmants of health, citizens can begin to better understand how to engage in a process and affect change. often local level politics are involved and citizens witness policy change in action. the environmental liaison committee and the dundas east hike lanes project resulted from local level initiatives aimed at finding solutions to air pollution -a priority identified hy the community. srchc supported the process with facilitation and technical aid. _the processs had tangible results that ultimately improve living conditions and health. •tn the united kmgdom plannm in the 60's established "technical aid" offices much like our present day legal aid system to provide professional support and advocacy for communities undergoing change. p2-15 (c) integrating community based research: the experience of street health, a community service agency i.aura cowan and jacqueline wood street health began offering services to homeless men and women in east downtown ~oronto in 1986. nursing stations at drop-in centres and shelters were fo~lowed by hiv/aids prevent10~, harm reduction and mental health outreach, hepatitis c support, sleeping bag exchange, and personal tdennfication replacement and storage programs. as street health's progi;ams expanded, so to~ did the agency:s recognition that more nee~ed t~ be done to. address the underl~ing causes of, th~ soct~l and economic exclusion experienced by its clients. knowing t.h~t. a~voca~y ts. helped by . evtd~nce , street he.alt~ embarked on a community-based research (cbr) initiative to 1dent1fy commumty-dnven research priorities within the homeless and underhoused population. methods: five focus groups were conducted with 46 homeless people, asking participants to identify positive and negative forces in their lives, and which topics were important to take action on and learn more about. findings were validated through a validation meeting with participants. results: participants identified several important positive and negative forces in their lives. key positive forces included caring and respectful service delivery, hopefulness and peer networks. key negative forces included lack of access to adequate housing and income security, poor service delivery and negative perceptions of homeless people. five topics for future research emerged from the process, focusing on funding to address homelessness and housing; use of community services for homeless people; the daily survival needs of homeless people and barriers to transitioning out of homelessness; new approaches to service delivery that foster empowerment; and policy makers' understanding of poverty and homelessness. conclusions: although participants expressed numerous issues and provided much valuable insight, definitive research ideas and action areas were not clearly identified by participants. however, engagement in a cbr process led to some important lessons and benefits for street health. we learned that the community involvement of homeless people and front-line staff is critical to ensuring relevance and validity for a research project; that existing strong relationships with community parmers are essential to the successful implementation of a project involving marginalized groups; and that an action approach focusing on positive change can make research relevant to directly affected people and community agency staff. street health benefited from using a cbr approach, as the research process facilitated capacity building among staff and within the organization as a whole. p2-16 (c) a collaborative process to achieve access to primary health care for black women and women of colour: a model of community based participartory research notisha massaquoi, charmaine williams, amoaba gooden, and tulika agerwal in the current healthcare environment, a significant number of black women and women of color face barriers to accessing effective, high quality services. research has identified several issues that contribute to decreased access to primary health care for this population however racism has emerged as an overarching determinant of health and healthcare access. this is further amplified by simultaneous membership in multiple groups that experience discrimination and barriers to healthcare for example those affected by sexism, homelessness, poverty, homophobia and heterosexism, disability and hiv infection. the collaborative process to achieve access to primary health care for black women and women of colour project was developed with the university of toronto faculty of social work and five community partners using a collaborative methodology to address a pressing need within the community ro increase access to primary health care for black women and women of colour. women's health in women's hands community health centre, sistering, parkdale community health centre, rexdale community health centre and planed parenthood of toronto developed this community-based participatory-action research project to collaboratively barriers affecting these women, and to develop a model of care that will increase their access to health services. this framework was developed using a process which ensured that community members from the target population and service providers working in multiculrural clinical settings, were a part of the research process. they were given the opportunity to shape the course of action, from the design of the project to the evaluation and dissemination phase. empowerment is a goal of the participatory action process, therefore, the research process has deliberately prioritized _ro enabling women to increase control over their health and well-being. in this session, the presenters will explore community-based participatory research and how such a model can be useful for understanding and contextualizing the experiences of black women and women of colour. they will address. the development and use of community parmerships, design and implementation of the research prorect, challenges encountered, lessons learnt and action outcomes. they will examine how the results from a collaborative community-based research project can be used as an action strategy to poster sessions v61 address che social determinants of women health. finally the session will provide tools for service providers and researchers to explore ways to increase partnerships and to integrate strategies to meet the needs of che target population who face multiple barriers to accessing services. lynn scruby and rachel rapaport beck the purpose of this project was ro bring traditionally disenfranchised winnipeg and surrounding area women into decision-making roles. the researchers have built upon the relationships and information gachered from a pilot project and enhanced the role of input from participants on their policy prioriries. the project is guided by an advisory committee consisting of program providers and community representatives, as well as the researchers. participants included program users at four family resource cencres, two in winnipeg and two located rurally, where they participated in focus groups. the participants answered a series of questions relating to their contact with government services and then provided inpuc as to their perceptions for needed changes within government policy. following data analysis, the researchers will return to the four centres to share the information and continue che discussion on methods for advocating for change. recommendations for program planning and policy development and implementation will be discussed and have relevance to all participants in the research program. women's health vera lefranc, louise hara, denise darrell, sonya boyce, and colleen reid women's experiences with paid and unpaid work, and with the formal and informal economies, have shifted over the last 20 years. in british columbia, women's employability is affected by government legislation, federal and provincial policy changes, and local practices. two years ago we formed the coalition ior women's economic advancement to explore ways of dealing with women's worsening economic situations. since the formation of the coalition we have discussed the need for research into women's employabilicy and how women were coping and surviving. we also identified how the need to document the nature of women's employability and reliance on the informal economy bore significanc mechodological and ethical challenges. inherent in our approach is a social model of women's health that recognizes health as containing social, economic, and environmental determinants. we aim to examine the social contexc of women's healch by exploring and legitimizing women's own experiences, challenging medical dominance in understandings of health, and explaining women's health in terms of their subordination and marginalizacion. through using a feminist action research (far) methodology we will explore the relationship between women's employability and health in 4 communities that represent bricish columbia's social, economic, cultural/ethnic, and geographic diversities: skidegate, fort st . .john, lumhy, and surrey. over the course of our 2 year project, in each community we will establish and work with advisory committees, hire and train local researchers, conduct far (including a range of qualitative methods), and support action and advocacy. since the selected communities are diverse, the ways that the research unfolds will 1·ary between communities. expected outcomes, such as the provision of a written report and resources, the establishment of a website for networking among the communities, and a video do.:umentary, are aimed at supporting the research participants, coalition members, and advisory conuniuces in their action efforts. p2t 9 (c) health & housing: assessing the impact of transitional housing for people living with hiv i aids currently, there is a dearth of available literature which examines supporrive housing for phas in the canadian context. using qualitative, one-on-one interviews we investigace the impact of transitional housing for phaswho have lived in the up to nine month long hastings program. our post<'r pr<·senta-t1on will highlight research findings, as well as an examination of transitional housing and th<· imp;kt it has on the everyday lives of phas in canada. this research is one of two ground breaking undertakings within the province of ontario in which fife house is involved. p2-20 (c) eating our way to justice: widening grassroots approaches to food security, the stop community food centre as a working model charles l.evkoe food hanks in north america have come co play a central role as the widespread response to growing rates of hunger. originally thought to be a short term-solution, over the last 25 years, they have v62 poster sessions be · · · 1· d wi'thi'n society by filling the gaps in the social safety net while relieving govemcome mst1tut1ona 1ze . . . t f the ir responsibilities. dependent on corporate donations and sngmauzmg to users, food banks men so th' . ·11 i i . are incapable of addressing the structural cause~ of ~u~ger. 1s pres~ntation w1 e~~ ore a ternanve approaches to addressing urban food security while bmldmg more sustamabl.e c~mmumt1es. i:nrough the f t h st p community food centre, a toronto-based grassroots orgamzanon, a model is presented case 0 e 0 h'l k' b 'id · b that both responds to the emergency food needs of communities w 1 e wor mg to. u1 ~ sustama le and just food system. termed, the community food centre model. (cfc), ~he s~op is worki?g to widen its approach to issues of food insecurity by combining respectful ~1rect service wit~ com~~mty ~evelop ment, social justice and environmental sustainability. through this approach, various critical discourses around hunger converge with different strategic and varied implications for a~ion. as a plac~-based organization, the stop is rooted within a geographical space and connected directly to a neighbourhood. through working to increase access to healthy food, it is active in maintaining people's dignity, building a strong and democratic community and educating for social change. connected to coalitions and alliances, the stop is also active in organizing across scales in connection with the global food justice movement. inner city shelter vicky stergiopoulos, carolyn dewa, katherine rouleau, shawn yoder, and lorne tugg introduction: in the city of toronto there are more than 32,000 hostel users each year, many with mental health and addiction issues. although shelters have responded in various ways to the health needs of their clients, evidence on the effectiveness of programs delivering mental health services to the home· less in canada has been scant. the objective of this community based research was to provide a forma· tive evaluation of a multi-agency collaborative care team providing comprehensive care for high needs clients at toronto's largest shelter for homeless men. methods: a logic model provided the framework for analysis. a chart review of 56 clients referred over a nine month period was completed. demographic data were collected, and process and outcome indicators were identified for which data was obtained and analyzed. the two main outcome measures were mental status and housing status 6 months after referral to the program. improvement or lack of improvement in mental status was established by chart review and team consensus. housing outcomes were determined by chart review and the hostel databases. results: of the clients referred 75% were single and 98% were unemployed. forty four percent had a psychiatric hospitalization within the previous two years. the prevalence of severe and persistent men· tal illness, alcohol and substance use disorders were 60%, 26% and 37% respectively. six months after referral to the program 37% of clients had improved mental status and 41 % were housed. logistic regression controlling for the number of general practitioner and psychiatrist visits, presence of person· ality or substance use disorder and treatment non adherence identified two variables significantly associ· ated mental status improvement: the number of psychiatric visits (or, 1.92; 95% ci, 1.29-2.84) and treatment non adherence (or, 0.086; 95% ci, 0.01-0.78). the same two variables were associated with housing outcomes. history of forensic involvement, the presence of a personality or substance use disorder and the number of visits with a family physician were not significantly associated with either outcome. conclusions: despite the limitations in sample sire and study design, this study can yield useful informa· tion to program planners. our results suggest that strategies to improve treatment adherence and access to mental health specialists can improve outcomes for this population. although within primary care teams the appropriate collaborative care model for this population remains to be established, access to psychiatric follow up, in addition to psychiatric assessment services, may be an important component of a successful program. mount sinai hospital (msh) has become one of the pre-eminent hospitals in the world by contributing to the development of innovative approaches to effective health care and disease prevention. recently, the hospital has dedicated resources towards the development of a strategy aimed at enhancing the hospital's integration with its community partners. this approach will better serve the hospital in the current health care environment where local health integration networks have been struck to enhance and support local capacity to plan, coordinate and integrate service delivery. msh has had early success with developing partnerships. these alliances have been linked to programs serving key target populations with _estabhshe~. points of access to msh. recognizing the need to build upon these achievements to remain compe~mve, the hospital has developed a community integration strategy. at the forefront of this strategy is c.a.r.e (community advisory reference engine): the hospital's compendium of poster sessions v63 community partners. as a single point of access to community partner information, c.a.r.e. is more than a database. c.a.r.e. serves as the foundation for community-focused forecasting and a vehicle for inter and intra-organizational knowledge transfer. information gleaned from the catalog of community parmers can be used to prepare strategic, long-term partnership plans aimed at ensuring that a comprehensive array of services can be provided to the hospital community. c.a.r.e. also houses a permanent record of the hospital's alliances. this prevents administrative duplication and facilitates the formation of new alliances that best serve both the patient and the hospital. c.a.r.e. is not a stand-alone tool and is most powerful when combined with other aspects of the hospital's community integration strategy. it iscxpected that data from the hospital's community advisory committees and performance measurement department will also be stored alongside stakeholder details. this information can then be used to drive discussions at senior management and the board, ensuring congruence between stakeholder, patient and hospital objectives. the patient stands to benefit from this strategy. the unique, distinct point of reference to a wide array of community services provides case managers and discharge planners with the information they need to connect patients with appropriate community services. creating these linkages enhances the patient's capacity to convalesce in their homes or places of residence and fosters long-term connections to neighborhood supports. these connections can be used to assist with identifying patients' ongoing health care needs and potentially prevent readmission to hospital. introduction: recruiting high-risk drug users and sex workers for hiv-prevention research has often been hampered by limited access to hard to reach, socially stigmatized individuals. our recruitment effom have deployed ethnographic methodology to identify and target risk pockets. in particular, ethnographers have modeled their research on a street-outreach model, walking around with hiv-prevention materials and engaging in informal and structured conversations with local residents, and service providers, as well as self-identified drug users and sex workers. while such a methodology identifies people who feel comfortable engaging with outreach workers, it risks missing key connections with those who occupy the margins of even this marginal culture. methods: ethnographers formed a women's laundry group at a laundromat that had a central role as community switchboard and had previously functioned as a party location for the target population. the new manager helped the ethnographers invite women at high risk for hiv back into the space, this time as customers. during weekly laundry sessions, women initiated discussions about hiv-prevention, sexual health, and eventually, the vaccine research for which the center would be recruiting women. ra.its: the benefits of the group included reintroducing women to a familiar locale, this time as customers rather than unwelcome intruders; creating a span of time (wash and dry) to discuss issues important to me women and to gather data for future recruitment efforts; creating a location to meet women encountered during more traditional outreach research; establishing the site as a place for potential retention efforts; and supporting a local business. women who participated in the group completed a necessary household task while learning information that they could then bring back to the community, empowering them to be experts on hiv-prevention and vaccine research. some of these women now assist recruitment efforts. the challenges included keeping the group women-only, especially after lunch was provided, keeping the membership of the group focused on women at risk for hiv, and keeping the women in the group while they did their laundry. conclusion: public health educators and researchers can benefit from identifying alternate congregation sites within risk pockets to provide a comfortable space to discuss hiv prevention issues with high-risk community members. in our presentation, we will describe the context necessary for similar research, document the method's pitfalls and successes, and argue that the laundry group constitutes an ethical, respectful, community-based method for recruitment in an hiv-prevention vaccine trial. p3-0t (c) upgrading inner city infrastructure and services for improved environmental hygiene and health: a case of mirzapur in u.p. india madhusree mazumdar in urgency for agricultural and industrial progress to promote economic d.evelopment follo_wing independence, the government of india had neglected health promotion and given less emphasis on infrastructure to promote public health for enhanced human pro uct1v_ity. ong wit r~p1 m astrucrure development, which has become essential if citie~ are to. act ~s harbmger.s of econ~nuc ~owth, especially after the adoption of the economic liberalization policy, importance _is a_lso ~emg g1ve.n to foster environmental hygiene for preventive healthcare. the world health orga~1sat10~ is also trj:'1!1g to help the government to build a lobby at the local level for the purpose by off~rmg to mrroduce_ its heal.thy city concept to improve public health conditions, so as to reduce th_e disease burden. this pape~ 1s a report of the efforts being made towards such a goal: the paper descr~bes ~ c~se study ?f ~ small city of india called mirzapur, located on the banks of the nver ganga, a ma1or lifeline of india, m the eastern part of the state of uttar pradesh, where action for improvement began by building better sanitation and environmental infrastructure as per the ganga action plan, but continued with an effort to promote pre· ventive healthcare for overall social development through community participation in and around the city. asthma physician visits in toronto, canada tara burra, rahim moineddin, mohammad agha, and richard glazier introduction: air pollution and socio-economic status are both known to be associated with asthma in concentrated urban settings but little is known about the relationship between these factors. this study investigates socio-economic variation in ambulatory physician consultations for asthma and assesses possible effect modification of socio-economic status on the association between physician visits and ambient air pollution levels for children aged 1 to 17 and adults aged 18 to 64 in toronto, canada between 1992 and 2001. methods: generalized additive models were used to estimate the adjusted relative risk of asthma physician visits associated with an interquartile range increase in sulphur dioxide, nitrogen dioxide, pm2.5, and ozone, respectively. results: a consistent socio-economic gradient in the number of physician visits was observed among children and adults and both sexes. positive associations between ambient concentrations of sul· phur dioxide, nitrogen dioxide and pm2.5 and physician visits were observed across age and sex strata, whereas the associations with ozone were negative. the relative risk estimates for the low socio-«onomic group were not significantly greater than those for the high socio-economic group. conclusions: these findings suggest that increased ambulatory physician visits represent another component of the public health impact of exposure to urban air pollution. further, these results did not identify an age, sex, or socio-economic subgroup in which the association between physician visits and air pollution was significantly stronger than in any other population subgroup. eco-life-center (ela) in albania supports a holistic approach to justice, recognizing the environ· mental justice, social justice and economic justice depend upon and support each other. low income cit· izens and minorities suffer disproportionately from environmental hazards in the workplace, at home, and in their communities. inadequate laws, lax enforcement of existing environmental regulations, and ~ea.k penalties for infractions undermine environmental protection. in the last decade, the environmental 1ust1ce m~ve~ent in tirana metropolis has provided a framework for identifying and exposing the links ~tween irrational development practices, disproportionate siting of toxic facilities, economic depres· s1on, and a diminished quality of life in low-income communities and communities of color. the envi· ~onmental justice agenda has always been rooted in economic, racial, and social justice. tirana and the issues su.rroun~ing brow~fields redevelopment are crucial points of advocacy and activism for creating ~ubstantia~ social chan~~ m low-income communities and communities of color. we engaging intensively m prevcnnng co'.'1mumnes, especially low income or minority communities, from being coerced by gov· ernmenta~ age_nc1es or companies into siting hazardous materials, or accepting environmentally hazard· ous_ practices m order to create jobs. although environmental regulations do now exist to address the environmental, health, and social impacts of undesirable land uses, these regulations are difficult to poster sessions v65 enforce because many of these sites have been toxic-ridden for many years and investigation and cleanup of these sites can be expensive. removing health risks must be the main priority of all brown fields action plans. environmental health hazards are disproportionately concentrated in low-income communities of color. policy requirements and enforcement mechanisms to safeguard environmental health should be strengthened for all brownfields projects located in these communities. if sites are potentially endangering the health of the community, all efforts should be made for site remediation to be carried out to the highest cleanup standards possible towards the removal of this risk. the assurance of the health of the community should take precedence over any other benefits, economic or otherwise, expected to result from brownfields redevelopment. it's important to require from companies to observe a "good neighbor" policy that includes on-site visitations by a community watchdog committee, and the appointment of a neighborhood environmentalist to their board of directors in accordance with the environmental principles. vancouver 1976-2001 michael buzzelli, jason su, and nhu le this is the second paper of research programme concerned with the geographical patterning of environmental and population health at the urban neighbourhood scale. based on the vancouver metropolitan region, the aim is to better understand the role of neighbourhoods as epidemiological spaces where environmental and social characteristics combine as health processes and outcomes at the community and individual levels. this paper builds a cohort of commensurate neighbourhoods across all six censuses periods from 1976 to 2001, assembles neighbourhood air pollution data (several criterion/health effects pollutants), and providing an analysis to demonstrate how air pollution systematically and consistently maps onto neighbourhood socioeconomic markers, in this case low education and lone-parent families. we conclude with a discussion of how the neighbourhood cohort can be further developed to address emergent priorities in the population and environmental health literatures, namely the need for temporally matched data, a lifecourse approach, and analyses that control for spatial scale effects. solid waste management and environment in mumbai (india) by uttam jakoji sonkamble and bairam paswan abstract: mumbi is the individual financial capital of india. the population of greater mumbai is 3,326,837 and 437 sq. km. area. the density of population 21, 190 per sq. km. the dayto-day administration and rendering of public services within gr. mumbai is provided by the brihan mumbai mahanagar palika (mumbai corporation of gr. mumbai) that is a body of 221 elected councilors on a 5-year team. mumcial corporation provides varies conservancy services such as street sweeping, collection of solid waste, removal and transportation, disposal of solid waste, disposal of dead bodies of animals, construction, maintance and cleaning of urinals and public sanitary conveniences. the solid waste becoming complicated due to increase in unplanned urbanization and industrialization, the environment has deteriorated significantly due to inter, intra and international migration stream to mumbai. the volume of inter state migration to mumbai is considerably high i.e. 20.89 lakh and international migrant 0.77 lakh have migrated to mumbai. present paper gives the view on solid waste management and its implications to environment and health. pollution from a wide varity of emission, such as from automobiles and industrial activities, has reached critical level in mumbai, causing respiratory, ocular, water born diseases and other health problems. sources of generation of waste are -household waste, commercial waste, institutional waste, street sweeping, silt removed from drain/nallah/cleanings. disposal of solid waste in gr. mumbai done under 1 incineration 2. processing to produce organic manure. 3. vermi-composting 4. landfill the study shows that the quantity of waste disposal of through processing and conversion to organic ~anure is about 200-240 m.t. per day. the processing is done by a private agency m/s excel industries ltd. who had set up a plant at the chincholi dumping ground in western mumbai for this purpose. the corporation is also disposal a plant of its waste mainly market waste through the environment friendly, natural pro-ces~ known as vermi-composing about 100 m.t. of market waste is disposed of in this manner at the various sites. there are four land fill sites are available and 95 percent of the waste matter generated m mumbai is disposed of through landfill. continuous flow of migrant and increa~e in slum population is a complex barrier in the solid waste management whenever community pamc1pat1on work strongly than only we can achieved eco-friendly environment in mumbai. persons exposed to residential craffic have elevated races of respiratory morbidity an~ ~ortality. since poverty is an important determinant of ill-health, some h~ve argued that t~es~ assoc1at1ons may relate to che lower socioeconomic status of those living along ma1or roads. our ob1ect1ve was to evaluate the association between traffic intensity at home and hospital admissions for respiratory diagnoses among montreal residents older than 60 years. morning peak traffic estimates from the emmej2 montreal traffic model (motrem98) were used as an indicator of exposure to road traffic outside the homes of those hospitalised. the influence of socioeconomic status on the relationship between traffic intensity and hospital admissions for respiratory diagnoses was explored through assessment of confounding by lodging value, expressed as the dollar average over road segments. this indicator of socioeconomic status, as calculated from the montreal property assessment database, is available at a finer geographic scale than socioeconomic information accessible from the canadian census. there was an inverse relationship between traffic intensity estimates and lodging values for those hospitalised (rho -0.23, p 3160 vehicles during che 3 hour morning peak), even after adjustment for lodging value (crude or 1.35, cl95% 1.22-1.49; adjusted or 1.13, cl95% 1.02-1.25). in montreal, elderly persons living along major roads are at higher risk of being hospitalised for respiratory illnesses, which appears not simply to reflect the fact that those living along major roads are at relative economic disadvantage. the paper argues that human beings ought to be at the centre of the concern for sustainable development. while acknowledging the importance of protecting natural resources and the ecosystem in order to secure long term global sustainability, the paper maintain that the proper starting point in the quest for urban sustainability in africa is the 'brown agenda' to improve che living and working environment of che people, especially che urban poor who face a more immediate environmental threat to their health and well-being. as the un-habitat has rightly observed, it is absolutely essential "to ensure that all people have a sufficient stake in the present to motivate them to take part in the struggle to secure the future for humanity.~ the human development approach calls for rethinking and broadening the narrow technical focus of conventional town planning and urban management in order to incorporate the emerging new ideas and principles of urban health and sustainability. i will examine how cities in sub-saharan africa have developed over the last fifty years; the extent to which government policies and programmes have facilitated or constrained urban growth, and the strategies needed to achieve better functioning, safer and more inclusive cities. in this regard i will explore insights from the united nations conferences of the 1990s, especially local agenda 21 of the rio summit, and the istanbul declaration/habitat agenda, paying particular attention to the principles of enablement, decentralization and partnership canvassed by these movements. also, i will consider the contributions of the various global initiatives especially the cities alliance for cities without slums sponsored by the world bank and other partners; che sustainable cities programme, the global campaigns for good governance and for secure tenure canvassed by unhabit at, the healthy cities programme promoted by who, and so on. the concluding section will reflect on the future of the african city; what form it will take, and how to bring about the changes needed to make the cities healthier, more productive and equitable, and better able to meet people's needs. heather jones-otazo, john clarke, donald cole, and miriam diamond urban areas, as centers of population and resource consumption, have elevated emissions and concentrations of a wide range of chemical contaminants. we have developed a modeling framework in which we first ~stimate the emissions and transport of contaminants in a city and second, use these estimates along with measured contaminant concentrations in food, to estimate the potential health risk posed by these che.micals. the latter is accomplished using risk assessment. we applied our modeling framework to consider two groups of chemical contaminants, polycyclic aromatic hydrocarbons (pah) a.nd the flame re~ardants polybrominated diphenyl ethers (pbde). pah originate from vehicles and stationary combustion sources. ~veral pah are potent carcinogens and some compounds also cause noncancer effects. pbdes are additive flame retardants used in polyurethane foams (e.g., car seats, furniture) 105fer sessions v67 and cl~ equipm~nt (e.g., compute~~· televisio~s). two out of three pbdes formulations are being voluntarily phased by mdustry due to rmng levels m human tissues and their world-wide distribution. pbdes have been .related to adv.erse neurological, developmental and reproductive effects in laboratory ijlimals. we apphed our modelmg framework to the city of toronto where we considered the southcattral area of 21 by 21 km that has a population of 1.3 million. for pah, local vehicle traffic and area sources contribute at least half of total pah in toronto. local contributions to pbdes range from 57-85%, depending on the assumptions made. air concentrations of both compounds are about 10 times higher downtown than 80 km north of toronto. although measured pah concentrations in food date to the 1980s, we estimate that the greatest exposure and contribution to lifetime cancer risk comes from ingestion of infant formula, which is consistent with toxicological evidence. the next greatest exposure and cancer risk are attributable to eating animal products (e.g. milk, eggs, fish). breathing downtown air contributes an additional 10 percent to one's lifetime cancer risk. eating vegetables from a home garden localed downtown contributes negligibly to exposure and risk. for pbdes, the greatest lifetime exposure comes through breast milk (we did not have data for infant formula), followed by ingestion of dust by the toddler and infant. these results suggest strategies to mitigate exposure and health risk. p4-01 (a) immigration and socioeconomic inequalities in cervical cancer screening in toronto, canada aisha lofters, rahim moineddin, maria creatore, mohammad agha, and richard glazier llltroduction: pap smears are recommended for cervical cancer screening from the onset of sexual activity to age 69. socioeconomic and ethnoracial gradients in self-reported cervical cancer screening have been documented in north america but there have been few direct measures of pap smear use among immigrants or other socially disadvantaged groups. our purpose was to investigate whether immigration and socioeconomic factors are related to cervical cancer screening in toronto, canada. methods: pap smears were identified using fee codes and laboratory codes in ontario physician service claims (ohip) for three years starting in 1999 for women age 18-41 and 42-66. all women with any health system contact during the three years were used as the denominator. social and economic factors were derived from the 2001 canadian census for census tracts and divided into quintiles of roughly equal population. recent registrants, over 80% of whom are expected to be recent immigrants to canada, were identified as women who first registered for health coverage in ontario after january 1, 1993. results: among 397,967 women age 18-41 and 328,885 women age 42-66, 55.3% and 55.5%, rtspcctively, had pap smears within three years. low income, low education, recent immigration, visible minority and non-english language were all associated with lower rates (least advantaged quintile:most advantaged quintile rate ratios were 0.84, 0.90, 0.81, 0.85, 0.83, respectively, p < 0.05 for all). similar gradients were found in both age groups. recent registrants comprised 22.5% of women and had mm;h lower pap smear rates than non-recent registrants (37.2 % versus 63. 7% for women age 18-41 and 35.9% versus 58.2% for women age 42-66). conclnsions: pap smear rates in toronto fall well below those dictated by evidence-based practice. at the area level, immigration, visible minority, language and socioeconomic characteristics are associated with pap smear rates. recent registrants, representing a largely immigrant group, have particularly low rates. efforts to improve coverage of cervical cancer screening need to be directed to all ~omen, their providers and the health system but with special emphasis on women who recently arrived m ontario and those with social and economic disadvantage. challeges faced: a) most of the resources are now being ~pent in ~reventing the sprea.d of hiv/ aids and maintaining the lives of those already affected. b) skilled medical ~rs~nal are dymg under· mining the capacity to provide the required health care services. ~) th.e comphcat1o~s of hiv/aids has complicated the treatment of other diseases e.g. tbs d) the ep1dem1c has led. to mcrease number of h n requiring care and support. this has further stretched the resources available for health care. orp a s d db . . i methods used on our research: 1. a simple community survey con ucte y our orgamzat1on vo · unteers in three urban centres members of the community, workers and health care prov~ders were interviewed ... 2. meeting/discussions were organized in hospitals, commun.ity centre a~d with government officials ... 3. written questionnaires to health workers, doctors and pohcy makers m th.e health sectors. lessors learning: • the biggest-health bigger-go towards hiv/aids prevention • aids are spreading faster in those families which are poor and without education. •women are the most affected. •all health facilities are usually overcrowded with hiv/aids patients. actions needed:• community education oh how to prevent the spread of hiv/aids • hiv/aids testing need to be encouraged to detect early infections for proper medical cover. • people to eat healthy • people should avoid drugs. implications of our research: community members and civic society-introduction of home based care programs to take care of the sick who cannot get a space in the overcrowded public hospitals. prl-v a te sector private sector has established programs to support and care for the staff already affected. government provision of support to care-givers, in terms of resources and finances. training more health workers. introduction: australian prisons contain in excess of 23,000 prisoners. as in most other western countries, reliance on 'deprivation of liberty' is increasing. prisoner numbers are increasing at 7% per annum; incarceration of women has doubled in the last ten years. the impacts on the community are great -4% of children have a parent in custody before their 16th birthday. for aboriginal communities, the harm is greater -aboriginal and/or torres strait islanders are incarcerated at a rate ten times higher than other australians. 25% of their children have a parent in custody before their 16th birthday. australian prisons operate under state and territory jurisdictions, there being no federal prison system. eight independent health systems, supporting the eight custodial systems, have evolved. this variability provides an unique opportunity to assess the capacity of these health providers in addressing the very high service needs of prisoners. results: five models of health service provision are identified -four of which operate in one form or another in australia: • provided by the custodial authority (queensland and western australia)• pro· vided by the health ministry through a secondary agent (south australia, the australian capital territory and tasmania) • provided through tendered contract by a private organization (victoria and northern territory) • provided by an independent health authority (new south wales) • (provided by medics as an integral component of the custodial enterprise) since 1991 the model of the independent health authority has developed in new south wales. the health needs of the prisoner population have been quantified, and attempts are being made to quantify specific health risks /benefits of incarceration. specific enquiry has been conducted into prisoner attitudes to their health care, including issues such as client information confidentiality and access to health services. specific reference will be made to: • two inmate health surveys • two inmate access surveys, and • two service demand studies. conclusions: the model of care provision, with legislative, ethical, funding and operational independence would seem provide the best opportunity to define and then respond to the health needs of prisoners. this model is being adopted in the united kingdom. better health outcomes in this high-risk group, could translate into healthier families and their communities. p4-04 (a) lnregrated ethnic-specific health care systems: their development and role in increasing access to and quality of care for marginalized ethnic minorities joshua yang introduction: changing demographics in urban areas globally have resulted in urban health systems that are racially and ethnically homogenous relative to the patient populations they aim to serve. the resultant disparities in access to and quality of health care experienced by ethnic minority groups have been addressed by short-term, instirutional level strategies. noticeably absent, however, have been structural approaches to reducing culturally-rooted disparities in health care. the development of ethnic-specific h~alth car~ systems i~ a structural, long-term approach to reducing barriers to quality health care for eth· me mmonty populations. methods: this work is based on a qualitative study on the health care experiences of san francisco chinatown in the united states, an ethnic community with a model ethnic-specific health care infrastrucrure. using snowball sampling, interviews were conducted with key stakeholders and archival research was conducted to trace and model the developmental process that led to the current ethnic-specific health care system available to the chinese in san francisco. grounded theory was the methodology ijltd to analysis of qualitative data. the result of the study is four-stage developmental model of ethnic-specific health infrastrueture development that emerged from the data. the first stage of development is the creation of the human capital resources needed for an ethnic-specific health infrastructure, with emphasis on a bilingual and bicultural health care workforce. the second stage is the effective organization of health care resources for maximal access by constituents. the third is the strengthening and stability of those institutional forms through increased organizational capacity. integration of the ethnic-specific health care system into the mainstream health care infrastructure is the final stage of development for an ethnic-specific infrastructure. conclusion: integrated ethnic-specific health care systems are an effective, long-term strategy to address the linguistic and cultural barriers that are being faced by the spectrum of ethnic populations in urban areas, acting as culturally appropriate points of access to the mainstream health care system. the model presented is a roadmap to empower ethnic communities to act on the constraints of their health and political environments to improve their health care experiences. at a policy level, ethnic-specific health care organizations are an effective long-term strategy to increase access to care and improve qualiiy of care for marginalized ethnic groups. each stage of the model serves as a target area for policy interventions to address the access and care issues faced by culturally and linguistically diverse populations. users in baltimore md: 1989-2004 noya galai, gregory lucas, peter o'driscoll, david celentano, david vlahov, gregory kirk, and shruti mehta introduction: frequent use of emergency rooms (er) and hospitalizations among injection drug users (idus) has been reported and has often been attributed to lack of access to primary health care. however, there is little longitudinal data which examine health care utilization over individual drug use careers. we examined factors associated with hospitalizations, er and outpatient (op) visits among idus over 14 years of follow-up. methods: idus were recruited through community outreach into the aids link to lntravenous experience (alive) study and followed semi-annually. 2,551 who had at least 2 follow-up visits were included in this analysis. outcomes were self-reported episodes of hospitalizations and er/op visits in the prior six months. poisson regression was used accounting for intra-person correlation with generalized estimation equations. hits: at enrollment, 73% were male, 95% were african-american, 33% were hiv positive, median age was 35 years, and median duration of drug use was 15 years. over a total of 37,512 visits, mean individual rates of utilization were 11 per 100 person years (py) for hospitalizations and 123 per 100 py for er/op visits. adjusting for age and duration of drug use, factors significantly associated with higher rates of hospitalization included hiv infection (relative incidence [ri(, 1.4), female gender (ri, 1.2), homelessness (ri, 1.6), as well as not being employed, injecting at least daily, snorting heroin, havmg a regular source of health care, having health insurance and being in methadone mainte.nance treatment (mmt). similar associations were observed for er/op visits except for mmt which was not associated with er/op visits. additional factors associated with lower er/op visits were use of alcohol, crack, injecting at least daily and trading sex for drugs. 10% of the cohort accounted for 45% of total er/op visits, while 11 % of the cohort never reported an op visit during follow-up. . . . lgbt) populations. we hypothesized that prov1dmg .appomtments .for p~t1~nts w1thm 24 hours would ensure timely care, increase patient satisfaction, and improve practice eff1c1ency. further, we anticipated that the greatest change would occur amongst our homeless patients.. . methods: we tested an experimental introduction of advanced access scheduling (usmg a 24 hour rule) in the primary care medical clinic. we tracked variables inclu~ing waiting ti~e fo~ next available appointment; number of patients seen; and no-show rates, for an eight week penod pnor to and post introduction of the new scheduling system. both patient and provider satisfaction were assessed using a brief survey (2 questions rated on a 5-pt scale). results and conclusion: preliminary analyses demonstrated shorter waiting times for appointments across the clinic, decreased no-show rates, and increased clinic capacity. introduction of the advanced access scheduling also increased both patient and provider satisfaction. the new scheduling was initiated in july 2005. quantitative analyses to measure initial and sustained changes, and to look at differential responses across populations within our clinic, are currently underway. introduction: there are three recognized approaches to linking socio-economic factors and health: use of census data, gis-based measures of accessibility/availability, and resident self-reported opinion on neighborhood conditions. this research project is primarily concerned with residents' views about their neighborhoods, identifying problems, and proposing policy changes to address them. the other two techniques will be used in future research to build a more comprehensive image of neighborhood depri· vation and health. methods: a telephone survey of 658 london, ontario residents is currently being conducted to assess: a) community resource availability, quality, access and use, b) participation in neighborhood activities, c) perceived quality of neighborhood, d) neighborhood problems, and e) neighborhood cohesion. the survey instrument is composed of indices and scales previously validated and adapted to reflect london specifically. thirty city planning districts are used to define neighborhoods. the sample size for each neighborhood reflects the size of the planning district. responses will be compared within and across neighborhoods. data will be linked with census information to study variation across socio-eco· nomic and demographic groups. linear and gis-based methods will be used for analysis. preliminary results: the survey follows a qualitative study providing a first look at how experts involved in community resource planning and administration and city residents perceive the availability, accessibility, and quality of community resources linked to neighborhood health and wellbeing, and what are the most immediate needs that should be addressed. key-informant interviews and focus groups were used. the survey was pre-tested to ensure that the language and content reflects real experiences of city residents. the qualitative research confirmed our hypothesis that planning districts are an acceptable surrogate for neighborhood, and that the language and content of the survey is appropriate for imple· mentation in london. scales and indices showed good to excellent reliability and validity during the pre· test (cronbach's alpha from 0.57-0.96). preliminary results of the survey will be detailed at the conference. conclusions: this study will help assess where community resources are lacking or need improve· ment, thus contributing to a more effective allocation of public funds. it is also hypothesized that engaged neighborhoods with a well-developed sense of community are more likely to respond to health programs and interventions. it is hoped that this study will allow london residents to better understand the needs and problems of their neighborhoods and provide a research foundation to support local understandmg of community improvement with the goal of promoting healthy neighborhoods. p4-08 (a) hiv positive in new york city and no outpatient care: who and why? hannah wolfe and victoria sharp introduction: there are approximately 1 million hiv positive individuals living in the united sta!es. about. 50% of these know their hiy status and are enrolled in outpatient care. of the remaining 50 yo, approx~mately half do not know their status; the other group frequently know their status but are not enrolled m any .sys~em of outpatient care. this group primarily accesses care through emergency departments. when md1cated, they are admitted to hospitals, receive acute care services and then, upon poster sessions v71 di5'harge, disappear from the health care system until a new crisis occurs, when they return to the emergency department. as a large urban hiv center, caring for over 3000 individuals with hiv we have an active inpatient service ".'ith appr~xi~.ately 1800 discharges annually. we decided to survey our inpatients to better charactenze those md1v1duals who were not enrolled in any system of outpatient care. results: 18% of inpatients were not enrolled in regular outpatient care: 2% at roosevelt hospital and 35% at st.luke\'s hospital. substance abuse and homelessness were highly prevalent in the cohort of patients not enrolled in regular outpatient care. 84% of patients not in care (vs. 33% of those in care) were deemed in need of substance use treatment by the inpatient social worker. 74% of those not in care were homeless (vs. 15% of those in care.) patients not in care did not differ significantly from those in me in terms of age, race, or gender. patients not in care were asked "why not:" the two most frequent responses were: "i haven't really been sick before" and "i'd rather not think about my health. conclusions: this study suggests that there is an opportunity to engage these patients during their stay on the inpatient units and attempt to enroll them in outpatient care. simple referral to an hiv clinic is insufficient, particularly given the burden of homelessness and substance use in this population. efforts are currently underway to design an intervention to focus efforts on this group of patients. p4.q9 (a) healthcare availability and accessibility in an urban area: the case of ibadan city, nigeria in oder to cater for the healthcare need of the populace, for many years after nigeria's politicl independence, empphasis was laid on the construction of teaching, general, and specialist hospital all of which were located in the urban centres. the realisation of the inadequacies of this approach in adequately meeting the healthcare needs of the people made the country to change and adopt the primary health care (phc) system in 1986. the primary health care system which is in line with the alma ata declaration of of 1978, wsa aimed at making health care available to as many people as possible on the basis of of equity and social justice. thus, close to two decades, nigeria has operated primary health care system as a strategy for providing health care for rural and urban dwellers. this study focusing on urban area, examimes the availabilty and accessibility of health care in one of nigeria's urban centre, ibadan city to be specific. this is done within the contest of the country's national heath policy of which pimary health care is the main thrust. the study also offers necessary suggestion for policy consideration. in spite of the accessibility to services provided by educated and trained midwifes in many parts of fars province (iran) there are still some deliveries conducted by untrained traditional birth attendants in rural parts of the province. as a result, a considerable proportion of deliveries are conducted under a higher risk due to unauthorised and uneducated attendants. this study has conducted to reveal the pro· portion of deliveries with un-authorized attendants and some spatial and social factors affecting the selection of delivery attendants. method: this study using a case control design compared some potentially effective parameters indud· ing: spatial, social and educational factors of mothers with deliveries attended by traditional midwifes (n=244) with those assisted by educated and trained midwifes (n=258). the mothers interviewed in our study were selected from rural areas using a cluster sampling method considering each village as a cluster. results: more than 11 % of deliveries in the rural area were assisted by traditional midwifes. there are significant direct relationship between asking a traditional birth attendant for delivery and mother age, the number of previous deliveries and distance to a health facility provided for delivery. significant inverse relationships were found between mother's education and ability to use a vehicle to get to the facilities. conclusion: despite the accessibility of mothers to educated birth attendants and health facilities (according to the government health standards), some mothers still tend to ask traditional birth attendants for help. this is partly because of unrealistic definition of accessibility. the other considerable point is the preference of the traditional attendants for older and less educated mothers showing the necessity of changing theirs knowledge and attitude to understand the risks of deliveries attended by traditional and un-educated midwifes. p4-12 (a) identification and optimization of service patterns provided by assertive community treatment teams in a major urban setting: preliminary findings &om toronto, canada jonathan weyman, peter gozdyra, margaret gehrs, daniela sota, and richard glazier objective: assertive community treatment (act) teams are financed by the ontario ministry of health and long-term care (mohltc) and are mandated to provide treatment, rehabilitation and support services in the community to people with severe and persistent mental illness. there are 13 such teams located in various regions across the city of toronto conducting home visits 1-5 times per week to each of their approximately 80 respective clients. each team consists of multidisciplinary health professionals who assist clients to identify their needs, establish goals and work toward them. due to complex referral patterns, the need for service continuity and the locations of supportive housing, clients of any one team are often found scattered across the city which increases home visit travel times and decreases efficiency of service provision. this project examines the locations of clients in relation to the home bases of all 13 act teams and identifies options for overcoming the geographical challenges which arise in a large urban setting. methods: using geographic information systems (gis) we geocoded all client and act agency addresses and depicted them on location maps. at a later stage using spatial methods of network analysis we plan to calculate average travel rimes for each act team, propose optimization of catchment areas and assess potential travel time savings. resnlts: initial results show a substantial scattering of clients from several act teams and substan· rial overlap of visit travel routes for most teams. conclusions: reallocation of catchment areas and optimization of act teams' travel patterns should lead to substantial savings in travel times, increased service efficiency and better utilization of resourc_~· ~e l'<!tenri~i modifications to catchment areas must be conducted with appropriate attention to specific clients servtce needs, service continuity and applicable regulations by the mohl tc. venice lido is a popular island within and outside italy because it hosts the international cinema festival yearly. only few people remember the island for its hospital, which, in the fifties, was one of the most important hospitals in italy and in europe, being a modern center for wind-, sun-and thermal-therapy. since the sixties, its fame became to drop away, since its structure was no longer adequate for the evolving medicine science. at the end of the seventies all the wards were moved into a new big building in the same area. at present the hospital is made up by about forty 'pavilions' (mostly partially used or empty) and a big building, disseminated in a 10 hectares area, on the sea front, on the 500 m long beach. the aim of my research is to suggest a realistic solution, sustainable on the functional use of the area according to basic local needs and economically realizable. i used a philological and multi-disciplinary method, more in detail: -from historical documents, i found what was the very first call of the property and how it adapted to the needs of the demographic development and the sanitary supply over time; -by contacting some operators from different fields, i realized which functions were suitable to the buildings in existence, to the population's needs and to the present sanitary supply. moreover the planning idea considers: -the debate going on between local people who strongly want to preserve the property, and die ones who want to renew it by different new opportunities; and -the regional sanitary politics, aiming at rationalizing the regional hospitals dislocation, and the budget needs. this plan analyzes and proposes some new alternative solutions to satisfy different needs. the different activities will be dealt out and the environmental fall will be limited as much as possible. after having analyzed its basic call, considered its environmental peculiarities and its position, verified the emerging needs of the local population, the plan proposes 5 different activities: the hospital; the social-rehabilitation center; the elderly house; the thermal center; the residential center; and other activities. such a plan wants to share in the solution of a basic problem by giving architectonic and functional dignity to an historical completely degraded area and by starting again a social-economical broken off process, by bringing in venice lido some fresh necessary elements for its so wished new throw. p4-14 (c) dilemma of free health care in spokane, wa david bunting introduction: the paper reports on the activities of project access spokane [pas], a physician sponsored initiative to provide uncompensated health care to low income, uninsured residents of spokane county wa. program providers included all county hospitals, over 600 physicians, other medical professionals, and specialized clinics. each prescription requires only a $4 co-payment. sponsored and administered by the county medical society, pas is funded by local and national foundations, private corporations, municipalities, civic organizations and individuals. method: the paper is based on a first year assessment report funded by pas, using hcfa [health care financing administration] insurance claim data documenting utilization rates, disease categories and donated charge information and patient cares [centralized applications, referrals and enrollment status) data. results: while essentially free for enrollees, the program involved real costs. an administrative organization to refer over 700 patients to any of over 800 providers had to be developed and staffed, using both paid and unpaid personnel. methods to identify potential patients and veri.fy eligibility. had to ~devised. patient appointments and transportation had to be scheduled and monitored. pu~hc relanons, provider solicitations and fundraising activities were continuous. information requirements regarding program operation were also significant. data reporting the volume. ~nd value of dona~ed medical services were necessary to demonstrate accomplishments and attr~ct addmon~l support. providers required assurances their contributions were both significant and eqmtable. funding sources sought evidence that their support had important consequences. however, generating this ~~ta proved difficult. many providers failed to submit appropriate insurance claims forms because .of a~dmonal donated effort and administrative cost, thereby not only reducing their own apparent conmbut1ons but also the overall significance of the program. conclusions: during its initial year, the estimated cost to deliver free health care was ~bout 5500 per enrollee. the lesson is that without an elaborate administrative structure and record keeping s.yst~~· efforts to provide free health care probably will fail. eligible enrollees c~n not be ~parated from in~hgi ble ones, care will not be accessed or delivered in an efficient manner, neither fundmg nor ~upport "". 1 11 be offered without evidence of tangible benefits, and providers will withdraw if they perceive mequ1table tteannent. v74 p4-1s (c) mobility in prostitution and the impact of health therese van der helm and henk sulman poster sessions introduction: many of the estimated 8.000 commer~ial ~ex wor~ers (c~w)in a'.'1~terdam ~~me from developing countries and eastern europe. to gain ins~ght mto their workmg and_ hvmg condinons the intermediary project (ip) at the municipal health serv1~e _contacts these women via ?utreach work on regular basis. since the new brothel law in october 2000 1s 1~troduc~d, ~sw ~rom outside the eu and who have no dutch residence permit are not allowed to work m prost1tut10n. smee then, many of these csw therefore have gone "underground." the consequences of the new law in the netherlands will be discussed with regard to the accessibility of csw and their risk for stl/hiv acquisition and unplanned pregnancies. methods: topics during outreach work are the interventions to increase the knowledge of safe sex and birth control for csw. written information is handed out in relevant languages and with addresses of health and social services. in conversations with the women, it appeared that many are not aware of these services. to provide easier access to health care for women, staff of the ip carries out free sti control csw working places, in windows brothels and sex houses. also, women are offered vaccination against hepatitis b (hbv). results: from january 2004 till july 2005 we approached 900 csw for first contact. one third was dutch; others were from developing countries, other eu countries and eastern europe. 350 sti consul· rations were done among non-iv drug using sex workers, of whom 50 % was dutch. of these, 2 % was diagnosed with syphilis, 1 % with gonorrhea, and 9% with chlamydia. of 234 women tested for hiv, none were infected. due to the high turnover of csw in .brothels, most wnmen were tested only once. among 845 csw tested for hbv -of whom one third is dutch -22 % had antibodies for hbv, 12 were carrier of hbv, most of whom came from hbv endemic areas. conclusions: sti control and hbv vaccination in brothels is an important support in health care. our findings suggest that csw do not play an important role in the transmission of sti. many csw are highly mobile and often not aware of the existing health and social services. continuous outreach is important to remain in contact with this mobile population. regulations in the new brothel law should not hamper contacts with (illegal) csw. heart disease (cho) is projected to become the leading cause of death. studies conducted in europe and the united states have proven a higher rate of cho in south asians who have migrated from south asia, most notably india, pakistan and bangladesh. approximately 50% of all heart attacks among south asian men occur under the age of 55; 25% of them occur under the age of 40-unheard of in any other population. associated risk factors such as diabetes, high blood pressure and cholesterol are also com· mon in this group. the population of south asians residing in nyc has more than doubled over the past decade to over 300,000, the majority being young, working-class and with limited health care access. many south asian men are employed as taxi drivers (4 out of 10 nyc taxi drivers are from south asial, working daily12 hour plus shifts. methods: acknowledging that this vulnerable, at-risk group was unavailable for outreach through conventional venues, three of the hospitals of the new york city health and hospital corporation, (elmhurst, queens and bellevue) conducted a one day outreach effort at nyc's jfk international air· ~rt's taxi holdin~ ar~a, ~here over one thousand taxis regularly assemble. bringing an outreach event directly to the taxi dnvers workplace, a tent was set up where cardiovascular-related health screenings, in~luding bl~ pressure, sugar, cholesterol, body mass index (bmi), were provided. results were shared with and explamed to the drivers and each participant received a south asian health education brochure. a total of 84 taxi drivers who identified as south asian were involved. rau/ts: participants were all male, ranging from 20 to 61 years; mean age, 41.5. screenings revealed 57% hypertensive; 46% had cholesterols over 200; 26% blood glucose over 160; 91 % had a bmi greater than 25. conc~on: a unique outreach activity, adapting to logistical, occupational limitations, is pre· se~ted. on-site f~back and explanation of results, reiterating and reinforcing the concern that south asia~. men are at mcreased danger for early coronary heart disease, and the dissemination of a culturally sensmve and r~levant brochure, may heighten awareness of prevailing cardiac risk factors in this immi· grant community. p4-17 (c) the community-hospital integration program framework: community-hospital panoenhips to improve the population's health john stevenson, richard blickstead, ann-marie marcolin, and sandi kendal v75 introduction: st. josephs health centre is a catholic community teaching hospital located in the heart of southwest toronto. st. joseph\'s was founded from a community-expressed need for hospital services, and since then has stayed committed to fostering a healthy community through collaboration and parmership. st. joseph's has developed an innovative model, the community-hospital integration program (chip), to strengthen st. joseph's partnerships with community stakeholders, and facilitates the building of links between community needs, service provision, and public policy outcomes to improve the health and wellness of the diverse neighbourhoods they together serve. methods: development of the chip framework st. joseph's health centre developed the chip framework through a multidisciplinary approach that included extensive international literature reviews, consultations, and key informant interviews with community service agencies and academics. to ensure active community voice, st. joseph's has hosted a number of community consultations including a community outreach forum attended by over ninety representatives from 68 community partners. results: the chip framework the chip framework aims to improve the health and wellness of the urban communities served by st. josephs health centre through four intersecting pillars: • raising community voices provides an infrastructure and process that supports community stakeholder input into health care service planning, decision-making, and delivery by the hospital and across the continuum of care; • sharing reciprocal capacity promotes healthy communities through the sharing of our intellectual and physical capacity with our community partners; • cultivating integration initiatives facilitates vertical, horizontal, and intersectoral integration initiatives in support of community-identified needs and gaps; and • facilitating healthy exchange develops best practices in community integration through community-based research, and facilitates community voice in informing public policy. the chip framework drives the complex inter-relationships between community-hospital engagement, reciprocal capacity-building, integration initiatives, and community-based research and evaluation, to create an interconnected network of health care services. the fluidity and simplicity of this framework, and its dedication to balancing community needs and hospital mandate, posits the st. josephs health centre's chip model as an integral component in building healthy and thriving communities. p4-18 (c) home based care promotion: improving acess to quality services and livelihood in the face of aids home based care is a service provided to persons affected/living with hiv/aids, a menu of care/support services at home/community. it is a prominent alternative approach. in uganda, hospital bed ocupacny is high, patient health care worker ratio deteriorating. empowering communities offers solutions to the problems; adressing cost, effectiveness of care. hiv/aids demands changes in attitudes, behaviour, approaches which is enhanced in home/community settings. for example art requires high levels of adherecnce, which medical workers have vety little opportunity to enforce. it also demands other support mechanisims in terms of nutrition, personal displine, etc which work best in stigma free environments. hence the relevance of home based care. this paper highlights the gaps that call for increased action in terms of home based care, examples of whre it has worked key challenges and recommendations for the future. p4-19 (c) health care for one ... health care for alli katharina kovacs burns introduction: at the surface, it appears that every person in canada _has. ~ccess to a publicly funded health care system. those living in urban centers should have the best ava1l~b1hty, chmce, and access to a variety of health care services because of the distribution of health care services, fac1lmes, and health professionals in concentrated in urban centers. this is not true for everyone -people with low income or who are homeless experience the challenges of social exclusion and being marginalized: there are many factors at play making it difficult for the latter group of people to access health care. service they need,.when they need them. health care is not as accessible or inclusive as intended. the quesuon to be explore.cl 1~: if health care is available and accessible to one person, what makes it not available to all people? the ~1gmfic~nce of having marginalized people accessing health care and other services includes e~hanced quality of hfe and decreased risks associated with being homeless, and cost savings in ion~ ter~ w1rh acute health care. methodology: community-based participatory research is applied m a case study on health care access and challenges experienced by low income and homeless people in one urban centre, edmonton, edmonton, and their challenges. the data is being gathered and ana_lyzed usmg basic m1x~d methods. results: the results will focus on how services can be more mtegrated and accessible to all people with low income and who are homeless. there will also be discussion about specific perceptions of not only the service providers but also of people with low income an~ who are homeless~ and vario~s decision makers. the results will be compared to the literature regardmg health care services access mother urban centers in canada and elsewhere. conclusions: recommendations will need to address social exclusion and other factors which can make health care and other services more available to all people in the community. the implications for health care will also be discussed. additional information will be gathered in the next phases of the study to develop a community services access model. p4-20 (c) availability and access exemplified: a case study beth hayhoe and ruth ewert introduction: access to health care in canada requires either the correct documentation or money. street youth have neither. in addition, health services in canada are designed by adults for adults. youth in general are often wary of having trust in adults with respect to their health concerns and frequently feel misunderstood. street youth are even more mistrustful of adults and public organizations, making their contact with health care professionals minimal. this combined with their risky behaviours and dangerous environment creates a population at risk for numerous health issues but with no place to have them investi· gated. at our non-government, not for profit health service designed specifically to provide a broad range of health care to street youth, professional services are provided almost entirely by volunteers. methods: using a retrospective analysis of the 11 years of data gathered from this organization and reviewing the initial reasons gathered from youth about their needs, the success of the health centre was examined. results: all the things youth had listed as being important in a health centre have been implemented, and even more things have been added to improve the breadth and quality of services offered. the use of the health centre has increased by more than six times since its opening in 1994. in addition, tens of thousands of visits have been paid to the health centre, costing the government and taxpayers nothing. as far as we know there is nothing else in canada that offers so many diverse and innovative services to youth in need. conclusion: it is clear from this case, that health care targeted at the needs of specific disadvan· taged populations can successfully provide them with appropriate health care. a model of accessible health care for marginalized populations can easily be developed for high impact and low cost from this successful case. toronto is one of the most ethno-racially diverse cities in the world of the estimated 2,529,280 toronto residents, 24% (597,218) live in scarborough population growth in scarborough is faster than toronto. scarborough's population increased by 6% from 1996 to 2001 while toronto increased only by 4%. toronto's population is projected to increase by 10% (252 000) in 2011 while scarbor· ough's will increase by 12%. (census canada 2001) low income, pove;ty, seniors, hidden homeless, new~omers to canada, the uninsured, are just a few of the issues concerning health care in our community. health care needs in scarborough are greatly impacted by diversity of ethnic and racial groups, poverty and settlement issues. this paper will share how the scarborough hospital (tsh) an urb~n communir_y hospital cares f~~ it~ community. tsh recognizes: •the changing community• b_arriers t~ accessing care • lnequalmes m health care the hospital in caring for its community pro· v1~es services that '!'e~e cu~turally, racially, and linguistically sensitive to our community. the paper will share the hospitals unique program on access and equity. the paper will share the needs in scar· borough and tsh's response to these needs: working in partnership with the local health committee, i:ne scarborough homeless co.mminee, the scarborough network of immigrant services organiza· nons a~d others. 1:'1e paper will also share the many initiatives this urban community hospital has taken viz. community and home programs in service areas such as mental health, haemodialysis day care, t~~ home oxyg~n program, the palliative at-home program, and above all support for the volunteer clinic for the uninsured. j"'"1tllu:lion: in canada's universal health care system it is generally assumed that everyone has equal access to health care. however, some immigrant groups in canada have historically been uninswed. these groups include failed refugee claimants, those overstaying their visas, and new immigrants in transition who are waiting to become eligible for health insurance. some estimates have suggested that at the present time there may be as many as one hundred thousand undocumented and thus uninsured immigrants in toronto alone. understandably, information on the characteristics of this marginalized population is very limited, since undocumented immigrants tend to have little contact with formal institutions. knowledge of the demographic characteristics of this population may aid in the development of health and social programs to better identify and meet the needs of undocumented immigrants in toronto. we conducted a review of all undocumented, uninsured patients, 15 years of age or older at access alliance multicultural community health centre (aamchc) between 1994 and 2004. demographic information such as age, gender, preferred language, length of time living in canada, selfreported education level and household income were recorded. rmdts: 72% of all adult patients at access alliance were undocumented and uninsured. 80% were under40years of age (with a median age of 30 years), 72% were women and only 19% spoke english. this group lived in canada for an average period of 2.2 years before they were first seen at aamchc. 74% of undocumented clients reported having completed at least a secondary or post-secondary education. 81% had self-reported household incomes of less than 20,000$/year, while 97% of households reported earning less than $30,000/year. the mean income per person in an average household was 425$/month. conchuions: uninsured individuals at aamchc were predominantly young females with limited knowledge of the english language. despite having attained a high level of education most were living well below the poverty line. recognition of these characteristics may assist in the development of health and social programs that are better adapted to meet the needs of undocumented immigrants. p4-23 (c) sharing expertise: a role for the hospital lactation consultant in the community. badrgrmuul: st. michael's hospital is a tertiary care hospital that serves a large inner city population in downtown toronto. in order to provide care to this population, the hospital has developed a number of unique outreach roles. one such role was developed to work with pregnant and breastfeeding women living in regent park, a social housing complex located near the hospital. the population of regent park includes new immigrants, refugees and the socially disadvantaged. approach: the outreach lactation consultant provides care, as part of the canada prenatal nutrition program, in the community prenatally, in the hospital during their inpatient stay and postnatally when they return to the community. the continuity of care enhances the probability of long term successful breastfeeding. aside from the health benefits of breastmilk, breastfeeding is especially important for women who are financially needy and must depend on food banks for formula. . . lason learned: aher two years of partnership, the relationship between the an-hospital ~stpar tum experience for breastfeeding mothers and the community postpartum support, the breastfeeding rate ~thin this community is high. the professional support and visibility of the 1:8ctation consultant, both m the hospital and in the community, contributes to the support of breastfee~m~.. . in light of ongoing funding constraints, this ha1son between hospital and community could be at risk and discontinued. in light of the benefits to the mother/baby dyad, the lactation consultant as a community partner in the canada prenatal nutrition program should be safeguarded. . lldpodiu:tion: although the importance of adequate health care in pregna~cy is well recognised, ethnic disparities in utilization of prenatal care still exist in many western coun~~es. a fun~amental factor in these disparities may be language proficiency, as it influences women's ab1hty. to ob~m and understand health information, to find the way in the health care system and to communicate with health care v78 poster sessions providers. we investigated the role of language proficiency in use and knowledge of folic acid as aspect of prenatal care in an urban multi-ethnic pregnancy c?hort. . . design: prospective cohort study. setting and parnc1pants: amsterdam ~regnant women attending obstetric care providers for their first antenatal visit (n= 8050). country of birth defined ethnicity: the netherlands, surinam, antilles, turkey, morocco, ghana, other non-~est~m (nw) and other western (w) country. main outcome measures: knowledge about and use of.fohc a~d (fa) supplements in pregnancy, and determinants of these in diffe~~t ethnic ~~ups. deterrrunants mcl~ded age, ed~ tion, parity, pregnancy intention and dutch prof1c1ency. stansncs: ~2 to co~p~re e~c groups, stranfied logistic regression (forward stepwise method) to explore determmants "'.'thm ethmc groups .. use of fa supplements was significantly lower among ghanaian, moroccan, turkish and other nw women (21%to41 %) than among dutch (86%) or other w women (78%). use amongsurinames and antillean women was intermediate (51%and60%). ethnic differences in fa knowledge were similar. knowledge was the strongest determinant of use in all ethnic groups, with odds ratios (ors) ranging from 10.9 to 42.0. language proficiency was the strongest determi~a~t of kno~ledge in ethnic groups with a mother tongue different from dutch (ors good vs. low proficiency ranging from 3.2 ro 16.0). educational level had a modifying role, as shown by an interaction effect between education and language proficiency in the nw group. here, the odds of having fa knowledge given a high education and good dutch proficiency was 20 times the odds given a good proficiency but low education. conclusions: appropriate periconceptional use of folic acid supplements in non-western ethnic groups is low, reflecting an absence of knowledge that is largely determined by the inability to speak and understand the language of the habitual country. tailored interventions using communication channels most likely to address (pregnant) women from ethnic minority groups are necessary. apan from specific interventions, our results are in support of strong incentives on language education despite current political debate. introduction: recent research suggests living in an economically disadvantaged neighborhood is asso· ciated with decreased likelihood of undergoing mammography and increased risk of late-stage breast can· cer diagnosis. long distances and travel times to facilities offering low-or no-cost mammography may be imponant barriers to adherence to mammography screening recommendations for residents of economically disadvantaged neighborhoods. the purpose of this study was to examine whether facilities providing low-and no-cost screening mammography were less spatially accessible in low-income neighborhoods in chicago, and the extent to which the relationship between neighborhood income and the spatial accessibility of facilities varied by the proponion of african-american residents in the neighborhood. methods: the sample consisted of 343 chicago neighborhoods. for each neighborhood, we con· structed three measures of the spatial accessibility of facilities: street network distance to the nearest facility, public transportation travel time to the nearest facility, and shortest automobile travel time to a facility. using 2000 decennial census data, we characterized the neighborhoods according to proportion of residents with incomes below the poverty line, proportion of residents in each of four raciavethnic groups (african-american, latino, white, and other), and population density. we used ordinary least squares (ols) and spatial exogenous lag regression to examine relationships. model 1 estimated the relationship between neighborhood poverty and the spatial accessibility of facilities, adjusting for raciaveth· nic composition and population density. model 2 added a multiplicative interaction term between neighborhood poverty and african-american. restllts: we identified deven facilities that provided low-or no-cost screening mammography to chicago residents in 2004. we found that the distance and travel times via automobile and public trans· portation to facilities generally decreased as neighborhood poveny increased. however, we also found that the ~egative associations between neighborhood poverty and two of the spatial accessibility mea· sures -distance and public transportation travel time -were less strong in neighborhoods with the highest proponions of african-american residents. among neighborhoods in the highest tertile for poveny, th~ mean distance and mean public transportation travel time to facilities were over twice as long in neighborhoods in the highest tertile than in those in the lowest tenile of african-american residents. . ~ persistent socioeconomic and racial/ethnic disparities in breast cancer stage at diagno-s11 ~nd su~1val suggest that ensuring an equitable distribution of affordable mammography is a worth-w~e policy .goal. the ~~dy sugges~ that ~ture investigations should consider both neighborhood soc1oecono1d1c charactensbes and rac1avethmc composition when examining the spatial distribution of health resources. , 2004) . epidemiological data suggest tha~ hiv prevalence among msm is over 1 % in some metropolitan cities, such .as beijing (ibid.). due to widespread homophobia in chinese society, however, this population remains invisible within current health/social services. lack of research on sexuality, specifically homosexuality, has impaired our understanding of health and health practices of chinese msm. focusing on the illness experiences of chinese msm with hiv/aids, this paper explores the socio-cultural impacts of hiv/ aids on this group. the data used for this paper were collected through semi-constructed in-depth face-toiace interviews with 21 adult plwhas (including 11 msm) in beijing, china. with the permission of the participants, the interviews were audio-taped or recorded in notes. the transcribed interviews and interview notes were analyzed by using n-vivo, a software program for qualitative data analysis. this phenomenological study aimed to understand the experiences chinese plwhas (including msm) from their own perspectives. results: it is found that the illness experiences of chinese msm with hiv/aids are profoundly shaped by the socio-cultural meanings of homosexuality, which are further complicated by the dominant discourses on hiv/aids in china. at a macro level, homophobia in the larger society has decreased the capability of this group to respond to this epidemic in a more efficient way. at a meso level, aids-phobia within the chinese msm circuits has prevented this group from openly confronting this disease and offering support to those who are already affected by it. at a micro level, however, these hiv-infected/ affected msm have tried their best to locate spaces to live and to construct hope for their future lives despite various barriers within family, community, and the larger society. this study suggests that facilitating chinese msm's response to the aids crisis urgently requires bridging awareness, commitment, knowledge and resources both within and without this group. it also illustrates the importance of understanding homosexuality in the local context of hiv i aids, which will be helpful for developing culturally sensitive hiv/aids programs for this population on the community, national and international levels. introduction: having a regular family doctor (rfd) is known to be positively correlated with a good medical follow up, including access to preventive and/or chronic care. inversely, a lack of primary care may lead to high rates of avoidable hospitalizations, especially among poor people and/or in underserved neighbourhoods. in such a matter, a recent comparative study showed that paris was better ranged than other cities, such as nyc, tokyo or london. nevertheless, despite a quasi universal health insurance and a high density of general practitioners, a noticeable fraction of the paris population does not have any regular physician and we aimed to understand who they are and for what reasons rhey don't have any rfd. mdbods: cross sectional population survey among a random sample of households in 2 ~nderpriv ileged neighbourhoods in paris inner city, performed in 2003, using a face-to-face quest10nna1re collecr-1ng more than 400 social and health characteristics. ruults: a quarter (26.3 %) of rhe study popularion did not have any rfd. this proporrion was iignificantly higher among male (or= 2.00, 95%ci = [ 1.41-2.781), younger (e.g. or 18 -29/> .s l _= 4._oo, 95"1.ci = (2.13-7.69)), and/or unemployed (or =2.01, 95%ci = ij .21-3.3411_ people. in multtvanate analysis, after a full adjustment on gender, age, health status, health insura~ce, income, occupat10n and tducation level, we observed significant associations between having no rfd and: ~arrtal and_ pare~t hood status (e.g. or single no kids/in couple+kids = 2.12, 95%ci = ( 1.26-3.59()~ quality of relattonsh1ps with neighbours (or bad/good= 3 .82, 95%ci = [ 1.84-7.94)), and length of residence m the neighbourhood (with a dose/effect statistical relationship). . co11clusion: gender, age, employment status, mariral and parenthood stat~s as well as ~e1gh bourhood anchorage seem to be major predictors of having a rfd, even when um.versa! health i~sur ance has reduced most of financial barriers. in urban contexts, where residential migrattons and single lift (or family ruptures) are frequent, specific information may be conducted to encourage people to ket rfd. :tu~y tries to assess the health effects and costs and also analyse the availability and accessibility to health care for poor. . methods: data for this study was collected by a survey on 300 households of the local community living near the factories and 100 households where radiation hazard w~s n?~ present. ~~art from mor· bidity status and health expenditure, data was collected ~n access, a~ail~b1.hty and eff1c1ency of healrh care. a discriminant analysis was done to identify the vanables that d1scnmmate between the study and control group households in terms of health care pattern. a contingent valuation survey was also undertaken among the study group to find out the factors affecting their willingness to pay for health insurance and was analysed using logit model. results: the health costs and indebtedness in families of the study group was high as compared to control group households and this was mainly due to high health expenditure. the discriminant analysis showed that expenditure incurred by private hospital inpatient and outpatient expenditure were significant variables, which discriminated between the two types of households. the logit analysis showed !hat variables like indebtedness of households, better health care and presence of radiation induced illnesses were significant factors influencing willingness to pay for health insurance. the study showed that study group households were dependent on private sector to get better health care and there were problems with access and availability at the public sector. conclusion: the study found out that the quality of life of the local community is poor due to health effects of radiation and the burden of radiation induced illnesses are so high for them. there is an urgent need for government intervention in this matter. there is also a need for the public sector to be efficient to cater to the needs of the poor. a health insurance or other forms of support to these households will improve the quality for health care services, better and fast access to health care facilities and reduces the financial burden of the local fishing community. the prevalence of substance abuse is an increasing problem among low-income urban women in puerto rico. latina access to treatment may play an important role in remission from substance abuse. little is known, however, about latinas' access to drug treatment. further, the role of social capital in substance abuse treatment utilization is unknown. this study examines the relative roles of social capital and other factors in obtaining substance abuse treatment, in a three-wave longitudinal study of women ages 18-35 living in high-risk urban areas of puerto rico, the inner city latina drug using study (icldus). social capital is measured at the individual level and includes variables from social support and networks, familism, physical environment, and religion instruments of the icdus. the study also elucidates the role of treatment received during the study in bringing about changes in social capital. the theoretical framework used in exploring the utilization of substance abuse treatment is the social support approach to social capital. the research addresses three main questions: ( t) does social capital predict parti~ipating in treatment programs? (2) does participation in drug treatment programs increase social capital?, and (3) is there a significant difference among treatment modalities in affecting change in ~ial capital? the findings revealed no significant association between levels of social capital and gettmg treatment. also, women who received drug treatment did not increase their levels of social capital. the findings, however, revealed a number of significant predictors of social capital and receiving drug ~buse treatment. predictors of social capital at wave iii include employment status, total monthly mcoi:rie, and baseline social capital. predictors of receiving drug abuse treatment include perception of physical health and total amount of money spent on drugs. other different variables were associated to treatment receipt prior to the icldus study. no significant difference in changes of social capital was found among users of different treatment modalities. this research represents an initial attempt to elucidate the two-way relationship between social capital and substance abuse treatment. more work is necessary to unden~nd. ~e role of political forces that promote social inequalities in creating drug abuse problems and ava1lab1hty of treatment; the relationship between the benefits provided by current treatment poster sessions v81 sctrings and treatment-seeking behaviors; the paths of recovery; and the efficacy and effectiveness of the trtaanent. and alejandro jadad health professionals in urban centres must meet the challenge of providing equitable care to a population with diverse needs and abilities to access and use available services. within the canadian health care system, providers are time-pressured and ill-equipped to deal with patients who face barriers of poverty, literacy, language, culture and social isolation. directing patients to needed supportive care services is even more difficult than providing them with appropriate technical care. a large proportion of the population do not have equitable access to services and face major problems navigating complex systems. new approaches are needed to bridge across diverse populations and reach out to underserved patients most in need. the objective of this project was to develop an innovative program to help underserved cancer patients access, understand and use needed health and social services. it implemented and evaluated, a pilot intervention employing trained 'personal health coaches' to assist underserved patients from a variety of ethno-linguistic, socio economic and educational backgrounds to meet their supportive cancer care needs. the intervention was tested with a group of 46 underserved cancer patients at the princess margaret hospital, toronto. personal coaches helped patients identify needs, access information, and use supportive care services. triangulation was used to compare and contrast multiple sources of quantitative and qualitative evaluation data provided by patients, personal health coaches, and health care providers to assess needs, barriers and the effectiveness of the coach program. many patients faced multiple barriers and had complex unmet needs. barriers of poverty and language were the easiest to detect. a formal, systematic method to identify and meet supportive care needs was not in place at the hospital. however, when patients were referred to the program, an overwhelming majority of participants were highly satisfied with the intervention. the service also appeared to have important implications for improved technical health care by ensuring attendance at appointments, arranging transportation and translation services, encouraging adherence to therapy and mitigating financial hardship -using existing community services. this intervention identified a new approach that was effective in helping very needy patients navigate health and social services systems. such programs hold potential to improve both emotional and physical health out· comes. since assistance from a coach at the right time can prevent crises, it can create efficiencies in the health system. the successful use of individuals who were not licensed health professionals for this purpose has implications for health manpower planning. needle exchange programs (neps) have been distributing harm reduction materials in toronto since 1990. counterfit harm reduction program is a small project operated out of a community health centre in south-east toronto. the project is operated by a single full-time coordinator, one pan-rime mobile outreach worker and two peers who work a few hours each week. all of counterfit's staff, peers, and volunteers identify themselves as active illicit drug users. yet the program dis~rib utes more needles and safer crack using kits and serves more illicit drug u~rs t~an the comb1~e~ number of all neps in toronto. this presentation will discuss the reasons behind this success, .s~1f1cally the extended hours of operation, delivery models, and the inclusion of an. extremely marg1~ahzed community in all aspects of program design, implementation and eva.luat1?n. ~ounterfit was recently evaluated by drs. peggy milson and carol strike, two leading ep1dem1olog1st and researchers in the hiv and nep fields in toronto and below are some of their findings: "the program has experienced considerable success in delivering a high quality, accessible and well-used program .... the pro· gram has allowed (service users) to become active participants in providing. services to others and has resulted in true community development in the best sense. " ... counterf1t has ~~n verr succe~sful attracting and retaining clients, developing an effective peer-based model an.d assisting chen~s ~1th a vast range of issues .... the program has become a model for harm red~ctmn progr~ms withm the province of ontario and beyond." in june 2004, the association of ?ntano co~mumty heal~~ <:en· ires recognized counterfit's acheivements with the excellence m community health initiatives award. in kenya, health outcomes and the performance of government health service~ have det~riorated since the late 1980s, trends which coincide with a period of severe resource constramts necessitated by macro-economic stabilization measures after the extreme neo-liberalism of the 1980s. when the govern· ment withdrew from direct service provision as reform trends and donor advocacy suggested, how does it perform its new indirect role of managing relations with new direct health services providers in terms of regulating, enabling, and managing relations with these health services providers? in this paper therefore, we seek to investigate how healthcare access and availability in the slums of nairobi has been impacted upon by the government's withdrawal from direct health care provision. the methodology involved col· leering primary data by conducting field visits to 8 health institutions located in the slum areas of kibera and korogocho in nairobi. purposive random sampling was utilized in this study because this sampling technique allowed the researcher(s) to select those health care seekers and providers who had the required information with respect to the objectives of the study. in-depth interviews using a semi-structured ques· tionnaire were administered ro key informants in health care institutions. this sought to explore ways in which the government and the private sector had responded and addressed in practical terms to new demands of health care provision following the structural adjustment programmes of the 1990s. this was complemented by secondary literature review of publications and records of key governmental, bilateral and multilateral development partners in nairobi. the study notes a number of weaknesses especially of kenya's ministry of health to perform its expected roles such as managing user fee revenue and financial sustainability of health insurance systems. this changing face of health services provision in kenya there· fore creates a complex situation, which demands greater understanding of the roles of competition and choice, regulatory structures and models of financing in shaving the evolution of health services. we rec· ommend that the introduction of user fees, decentralization of service provision and contracting-out of non-clinical to private and voluntary agencies require a new management culture, and new and clear insri· tutional relationships. experience with private sector involvement in health projects underlines the need not only for innovative financial structures to deal with a multitude of contractual, political, market and risks, but also building credible structures to ensure that health services projects are environmentally responsive, socially sensitive, economically viable, and politically feasible. purpose: the purpose of this study is to examine the status of mammography screening utilization and its predictors among muslim women living in southern california. methods: we conducted a cross-sectional study that included 202 women aged ::!: 40 years. we col· leered data using a questionnaire in the primary language of the subjects. the questionnaire included questions on demography; practices of breast self-examination (bse) and clinical breast examination (cbe); utilization of mammography; and family history of breast cancer. bivariate and multiple logistic regression analyses were performed to estimate the odds ratios of mammography use as a function of demographic and other predictor variables. . results: among the 202 women, 78% were married, 68% were 40-50 years old, and 20% had family h1story of breast cancer. thirty-two percent of the participating women never practiced bse and 32% had not undergone cbe during the past two years. the data indicated that 46% of the women did not have mammography in the last two years. logistic regression analysis showed that age (0r=5.1, 95% confi· dcnc~ interval (cl)=l.8-14.2), having clinical breast examination (0r=24.9, 95% cl=8.4-73.7), and practtce of self-breast examination (0r=2.6, 95% cl= 1.1-6.2), were strong predictors of mammography use . . conclusions: the data point to the need for intervention targeting muslim women to inform and motivate th.cm a~ut practices for early detection of breast cancer and screening. further studies are needed to investigate the factors associated with low utilization of mammography among muslim women population in california. we conducted a review of the scientific literature and° government documents to describe ditnational health care program "barrio adentro" (inside the neighborhood). we also conducted qualiurivt interviews with members of the local health committees in urban settings to descrihe the comm unity participation component of the program. rtsmlts: until recently, the venezuelan public health system was characterized by a lack or limited access w health care (70% of the population) and long waiting lists that amounted to denial of service. moit than half of the mds worked in the five wealthiest metropolitan areas of the country. jn the spring oi2003, a pilot program hired 50 cuban mds to live in the slums of caracas to provide health care to piople who had previously been marginalized from social programs. the program underwent a massive expansion and in only two years 20,000 cuban and 6,500 venezuelan health care providers were working acmss the country. they provide a daily average of 20-40 medical consultations and home visits, c1lly out neighborhood rounds, and deliver health prevention initiatives, including immunization programs. they also provide generic medicines at no cost to patients, which treat 80% of presenting ill-ij!m, barrio adentro aims to build 8,000 clinics (primary care), 1,200 diagnostic and rehabilitation ctnrres (secondary care), and upgrade the current hospital infrastructure (tertiary care). local health committees survey the community to identify needs and organize a variety of lobby groups to improve dit material conditions of the community. last year, barrio adentro conducted 3.5 times the medical visits conducted by the ministry of health. the philosophy of care follows an integrated approach where btalrh is related to housing, education, employment, sports, environment, and food security. conclusions: barrio adentro is a unique collaboration between low-middle income countries to provide health care to people who have been traditionally excluded from social programs. this program shows that it is possible to develop an effective international collaboration based on participatory democracy. low-income americans are at the greatest risk of being uninsured and often face multiple health concerns. this evaluation of the neighborhood health initiative (nh!), an organization which uses multiple programmatic approaches to meet the multiple health needs of clients, reflected the program's many activities and the clients' many service needs. nh! serves low-income, underserved, and hard-to-reach residents in the des moines enterprise community. multiple approaches (fourth-generation evaluation, grounded theory, strengths-and needs-based) and methods (staff and client interviews, concept mapping, observations, qualitative and quantitative analysis) were used to achieve that reflection. results indicate good targeting of residents in the 50314 zip code and positive findings in the way of health insurance coverage and reported unmet health needs of clients. program activities were found to match client nttds, validating the organization\'s assessment of clients. important components of nhi were the staff composition and that the organization had become part of both the formal and informal networks. nhi 1 1 positioned as a link between the target population and local health and social sc:rvice agencies, working to connect residents with services and information as well as aid local agencies in reaching this underserved population. p4-36 (c) welfare: definition by new york city maribeth gregory for an individual who resides in new york city, to obtain health insurance under the medicaid policy one must fall under certain criteria .. (new york city's welfare programs 2003) if the individual _is on ssi or earns equal to or less than $934 per month, he is entitled to receive no more than $5,600 m resources. a family the size of two would need to earn less than $942 per month to qualify for no greater than ss,650 worth of medicaid benefits. a family of three would qualify for $5,650 is they earned less than $942 per month and so on. introduction: the vancouver gay communiry has a significant number of asian descendan!l. because of their double minority status of being gay and asian, many asian men who have sex with men (msm) are struggling with unique issues. dealing with racism in both mainstream society and the gay communiry, cultural differences, traditional family relations, and language challenges can be some of their everyday srruggles. however, culturally, sexually, and linguistically specific services for asian msm are very limited. a lack of availability and accessibiliry of culturally appropriate sexual health services isolates asian msm from mainstream society, the gay community, and their own cultural communities, deprives them of self-esteem, and endangers their sexual well-being. this research focuses on the qualita· tive narrative voices of asian msm who express their issues related to their sexualiry and the challenges of asking for help. by listening to their voices, practitioners can get ideas of what we are missing and how we need to intervene in order to reach asian msm and ensure their sexual health. methods: since many asian msm are very discreet, it is crucial to build up trust relationships between the researcher and asian msm in order to collect qualitative data. for this reason, a community based participatory research model was adopted by forming a six week discussion group for asian msm. in each group session, the researcher tape recorded the discussion, observed interactions among the participants, and analyzed the data by focusing on participants' personal thoughts, experiences, and emotions for given discussion topics. ra11lts: many asian msm share challenges such as coping with a language barrier, cultural differ· ences for interpreting issues and problems, and westerncentrism when they approach existing sexual health services. moreover, because of their fear of being disclosed in their small ethnic communities, a lot of asian msm feel insecure about seeking sexual health services when their issues are related to their sexual orientation. conclflsion: sexual health services should contain multilingual and multicultural capacities to meet minority clients' needs. for asian msm, outreach may be a more effective way to provide them with accessible sexual health services since many asian msm are closeted and are therefore reluctant to approach the services. building a communiry for asian msm is also a significant step toward including them in healthcare services. a communiry-based panicipatory approach can help to build a community for asian msm since it creates a rrust relationship between a worker and clients. p4-38 (c) identifying key techniques to sustain interpretation services for assisting newcomers isolated by linguistic and cultural barriers from accessing health services s. gopi krishna lntrodaetion: the greater toronto area (gta) is home to many newcomer immigrants and other vulnerable groups who can't access health resources due to linguistic, cultural and systemic barriers. linguistic and cultural issues are of special concern to suburbs like scarborough, which is home to thousands of newcomer immigrants and refugees lacking fluency in english. multilingual community ~nterpreter. service~ (mcis) is a non-profit social service organization mandated to provide high quality mterpretanon services. to help newcomers access health services, mcis partnered with the scarborough network of immigrant serving organizations (sniso) to recruit and train volunteer interpreters to accompany clienrs lacking fluency in english and interpret for them to access health services at various locati?ns, incl~~ing communiry ~c:-lth centres/social service agencies and hospitals. the model envisioned agencies recruin~ and mcis ~.mm.g and creating an online database of pooled interpreter resources. this da.tabase, acces&1bl~ to all pama~~g ?rganization is to be maintained through administrative/member · ship fees to. be ~1d by each parnapanng organization. this paper analyzes the results of the project, defines and identifies suc:cases before providing a detailed analysis for the reasons for the success . . methods:. this ~per~ q~ntitative (i.e. client numben) and qualitative analysis (i.e. results of key •~ormant m~rv1ews with semce ~sers and interpreters) to analyze the project development, training and 1mplementanon phases of the project. it then identifies the successes and failures through the afore· mentioned analysis. poster sessions vss resljts: the results of the analysis can be summarized as: • the program saw modest success both ia l?lllls of numbers of clients served as well as sustainability at various locations, except in the hospital iririog. o the success of the program rests strongly on the commitment of not just the volunteer interprmr, but on service users acknowledgments through providing transponation allowance, small honororia, letter of reference etc. • the hospital sustained the program better at the hospital due to the iolume and nature of the need, as well as innate capacity for managing and acknowledging volunceers. collc/llsion: it is possible to facilitate and sustain vulnerable newcomer immigrants access to health !ul'ices through the training and commitment of an interpreter volunteer core. acknowledging volunteer commitment is key to the sustenance of the project. this finding is important to immigration and health policy given the significant numbers of newcomer immigrants arriving in canada's urban communities. nity program was established in 1993 to provide support to people dying at home, especially those who were waiting for admission to the resi<lential hospice. with the advent of haart and corresponding challenges for people living with hiv/aids in the community, the community program has developed a unique case management approach. this model features an interdisciplinary team providing a clientdrivcn service. the case manager provides continuity of care to clients in a variety of settings both within and outside of the health care system. a case study is used to demonstrate how formal and informal networks of support facilitate efficient and appropriate resource utilization to promote client health. this case study demonstrates the value of consistent coordination with a client who has complex physical, spiritual, substance use and mental health needs. the authors will show how they liaised with the housing authority, police, social services as well as the family physician, clinic staff and a hospital emergtncy room to ensure this client did not fall between the cracks. tarek hussain recognition of the importance of community involvement and sectoral cooperation in health and !ocial services, formalized in alma-ata declaration in 1978, was reinforced at riga meeting held at the mid·point between alma-ata and the year 2000. then at the 20th anniversary of alma-ata declaration, ir was declared that 'primary health care is everybody's business'. health does not exist in isolation. health cannot be defined only as an outcome of 'medical care' but also as one of 'social action'; the responsibility of disease prevention and health promotion rests not only on governments, but also with don·govemmentorgan izations, the community, and individuals. the major accomplishment to date may be that rhere is growing realization in the health sector that community involvement is more than a political right-it is absolute necessary. lessons have learned during the implementation of disease-specific l'cltical programmes, such as cdd, ari, and epi; first, integrated and holistic approaches to childcare art needed, and second, the need for community involvement has become evident. imc! is a broad inte-gra1ed strategy with an overall objective contributing to reducing child morbidity and mortality in developing countries. and one of the imci components, 'community !mci', which is now broadening ~s an entry point for child-focused community development initiatives. community jmcl/c~1ld health is ~n mtcgrated approach to the promotion of key family and community practices that have impact on: child growth and development, disease prevention, home care for sick, malnourished child~en, a.nd care-seekmg behavior and compliance with advice and treatment. the presentation addres~es, m brief, the ~evel opinent of community !mci, operational aspect of community/im cl, key strategies and tools ava1la~le for implementation of c/imci. the paper draws some successful programme examples on working logether for imc! with the community in various countries which had substantial benefns on programme outcomes. p4-4 t (c) healthy child screening: an innovative service initiative ann-marie marcolin and joyce allen . introduction: with mounting attention for creative and integrated models of c~re .that are populallon and community focused, the healthy child screening initiative achiev.es t~ese pnncipl~s and more! . • parkdale high park rotary (sponsorship) the heal~h ~h1ld scree?1?g is a ~opl~-centred model of care. agency and professional sector-specific silos are ehmmated, ~rov1dmg f~~1hes wit~ one-sto~ access to primary prevention services in a local school gym! children at nsk are rece1vmg early mtervent10n and appropriate referrals to the right care provider, at the right time a~d in t~e right place. further, with a complimentary focus on prevention, the program serves to keep at nsk children healthy. healthy child screening results: the service model is developed around four distinct phases: 1) planned outreach; 2) coordinated intake and registration; 3) interdisciplinary screening; and 4) targeted referral. since the fall 2003 and through six collaborative healthy child screenings 158 children ranging in age from 2 to 6 have benefited from this unique service model of care. conclusion: the presentation will provide practical information on the development and implementation of the model. there will also be a focus on lessons learned in building community service provider-hospital relationships. according to statistics canada (2001) 49% of the toronto population was born outside of canada and 21 % were new immigrants. immigrants often arrive in canada in superior health compared to the canadian born population, but they lose this health advantage over time. changes in immigrant health status over time may be attributed to a variety of factors including barriers to accessing and receiving care as well as limitations in the mainstream health service organizations and their approaches to health care delivery (hyman, 2001 ) . for example, immigrant women are less likely to receive pap tests, which places them at a greater risk for developing cervical cancer. similarly, immigrant women receive less mammography screening than their canadian counterparts. the immigrant women's health centre mobile health unit (mhu) was created as an alternative care delivery model to address the need for increased accessibility as well as culturally and linguistically appropriate reproductive health care. toronto western hospital and the immigrant women's health centre have formed a collaborative partnership which incorporates a primary care nurse practitioner and lay ethnocultural counselors providing care on the mhu. currently, a qualitative ethnonursing study is underway to understand the unique experiences of women using the mhu for their reproductive health care as well as the perspectives of the mhu health care providers. based on a literature review and preliminary findings from provider and participant interviews, the proposed presentation will address the themes of health status for immigrant and refugee women, accessibility issues as well strategies to improve their reproductive health care. we will discuss benefits as well as limitations to health care provision using this alternative service delivery model. michael carden, brian edlin, andrew talal, elizabeth getter, and marla shu lntrod.ction: to date most active drug users have not had access to treatment for hepatitis c virus (hcv) infection and substantial barriers continue to exist that interfere with treatment availability for this population. methods: active illicit drug users interested in pursuing hcv care and treatment were recruited in partnership w~th co~munity-based organizations (cbo's) in new york city. baseline data were collected on quahty of hfe, substance use patterns, depression, health care utilization, hcv health beliefs and other relev.ant variables. medical evaluations were conducted, including liver biopsy when indicated, an~ treatment ts ~ffered when no contraindications are present. participants also receive psychiatric evaluahons to determme the appropriateness of interferon treatment. renlts: fifteen individuals have been recruited to date and received an initial medical evaluation. the mean age was 40 years and _the average age of initial heroin or cocaine use was 16.8. eight (53%) ha~ been homeless at least once m the last 6 months and s (33%) were homeless for most or all of this penod. fourteen (93%) had a history of injection drug use with the mean age at first injection being 21 111s1frsewons v87 ,an. elrven persc.!ns (79%) reported inje~~ng .heroin or cocaine within the last 30 days while the .wing four (21 l'o) reported regular non-m1ect1on use of cocaine and street-obtained benzodiazepines. sijiy seven percent of the sample (n= 10) reported ever being referred to hcv treatment while 2 ( t 3 % ) ftponeddiattreann~nt had been offered by~ ~e.dical provider. none had ever received a liver biopsy or araanent. most believed that hcv was a s1gmf1cant threat to their health (80%) and that there was a pm)dchance they would eventually die if their hepatitis c was not treated (67%). though 7 individuals 147%1 believed that there was no cure for hcv, 13 (87%) reported that they would initiate treatment if i was recommended by their doctor. thirteen (87%) had a current psychiatric diagnosis including «pmsion, anxiety disorder, schizophrenia, schizoaffective disorder, and borderline and antisocial persooaliry disorders. among eight individuals who had the beck depression inventory administered, the mean score was 29.75. attendance rates were 83% (83/100) at scheduled medical and psychiatric 1ppointments and 50% (2/4) at liver biopsy. <andtuions: active drug users, despite experiencing multiple psycho-social problems, can be mgaged in hcv care using a multidisciplinary approach, but require intensive follow-up support. hcv aunnent of active drug users should not be dismissed. n-44 (c) antiretroviral therapy in mv infected infants: when to initiate therapy an african experience kingsley okonkwo, ademola adeyemo, israel sokeye, and nwaife umeike /""°""ction: as technology for early diagnosis of human immunodeficiency virus [hiv] improves, m to commence highly active antiretroviral therapy [haarn is yet to be fully evaluated. this prospective study describes our initial experience with early haart in hiv infected nigerian infants and rqions the outcome. we also analyzed the socioeconomic implications of early haart therapy in infants in the african setting. method: hiv infected infants were started on haart before 6 months and followed up at 4 mkly intervals.we monitored baseline and 3 monthly cd4 . conclusion: initial results suggest early haart before 6 months resulted. in improv~d out~ome in african children. treatment appears to be as effective as in developed counmes. in. a~nca as. m most developing countries with limited resources it is possible and effective to use haart m 1~~ants if p~oper llloniroring facilities are available. however the occurrence of long-term drug related tox1c1ty and mk of emergence of resistant viral strains create need for further evaluation of haart in infants. background: most risk factors for cardiovascular disease (cvd) can be mitigated through ~th clinical interventions and lifestyle change. we used data from a telephone survey of new york city (nyc) adults to characterize health care access and healthy behavior among those with three or more cvd risk factors. methods: the study population included respondents to the nyc community health survey 2002 (n= 9,674) self-reporting~ 3 of the following: smoking, diabetes, high cholesterol level, high blood pres· sure, body mass index >25, and age >45 (males) or >55 (females) (n=2,439). results: based on self-report, an estimated 1.447,000 (24%) of nyc adults have~ 3 or more cvd risk factors. this population is 51 % male, 47% white, 25% black, and 53% with s 12 years of education. most report good access to health care, indicated by having health insurance (95%), regular doctor (89%), their blood pressure checked within last 6 months (91 %), and their choles· terol checked within the past year (90% ). only 29% reported getting at least 20 minutes of exercise ~ 4 times per week and only 9% eating ~ 5 servings of fruits and vegetables the previous day. among current smokers, 59% attempted to quit in past 12 months, but only 32% used medication or counseling. implications: these data suggest that most nyc adults known to be at high risk for cvd have access to regular health care, but most do not engage in healthy lifestyle or, if they smoke, attempt effective quit strategies. more clinic-based and population-level interventions are needed to support lifestyle change among those at high risk of cvd. introduction: recently, much interest has been directed at "obesogenic" (obesity-promoting) (swinburn, egger & raza, 1998) built environments, and at geographic information systems (gis) as a tool for their exploration. a major geographical concept is accessibility, or the ease of moving from an origin to a destination point, which has been recently explored in several health promotion-related stud· ies. there are several methods of calculating accessibility to an urban feature, each with its own strengths, drawbacks and level of precision that can be applied to various health promotion research issues. the purpose of this paper is to describe, compare and contrast four common methods of calculating accessibility to urban amenities in terms of their utility to obesity-related health promotion research. practical and conceptual issues surrounding these methods are introduced and discussed with the intent of providing health promotion researchers with information useful for selecting the most appropria1e accessibility method for their research goal~ ~ethod: this paper describes methodological insights from two studies, both of which assessed the neighbourhood-level accessibility of fast-food establishments in edmonton, canada -one which used a relatively simple coverage method and one which used a more complex minimum cos1 method. res.its: both methods of calculating accessibility revealed similar patterns of high and low access to fast-food outlets. however, a major drawback of both methods is that they assume the characteristics of the a~e~ities and of the populations using them are all the same, and are static. the gravity potential method is introduced as an alternative, since it is ·capable of factoring in measures of quality and choice. a n~mber of conceptual and pr~ctical iss~es, illustrated by the example of situational influences on food choice, make the use of the gravity potential model unwieldy for health promotion research into sociallydetermined conditions such as obesity. co.nclusions: i~ ~ommended that geographical approaches be used in partnership with, or as a foun~ation for, ~admonal exploratory methodologies such as group interviews or other forms of commumty consultation that are more inclusive and representative of the populations of interest. qilhl in los angeles county ,,..ia shaheen, richard casey, fernando cardenas, holman arthurs, and richard baker ~the retinomax autorefractor has been used for vision screening of preschool age childien. ir bas been suggested to be used and test school age children but not been validated in this age poup. ob;taiw: to compare the results of retinomax autorefractor with findings from a comprehensive i!' examination using wet retinoscopy for refractive error. mllhods: children 5-12 years old recruited from elementary schools at los angeles county were iaml with snellen's chart and the retinomax autorefractor and bad comprehensive eye examination with dilation. the proportion of children with abnormal eye examination as well as diesensitiviry and specificity of the screening tools using retinomax autorefractor alone and in combinalion wirh snellen's chart. results of the 258 children enrolled in the study (average age= 8.5± 2.1 years; age range, 5-12 years), 6?% had abnormal eye examination using retinoscopy with dilation. for the lerinomax, the sensitivity was 85% (95% confidence interval [ci] 78%-90%), and the specificity was 31% (95% ci, 22%-41 o/o). simultaneous testing using snellen's chart and retinomax resulted in gain in 111sitiviry (94%, 95% cl= 89, 97), and loss in specificity (28%, 95% cl= 19%-38%). the study showed that screening school age children with retinomax autorefractor could identify most cases with abnormal vision but would be associated with many false-positive results. simuhaneous resting using snellen's chart and retinomax maximize the case finding but with very low specificiry. mdhotjs: a language-stratified, random sample of 2366 members of the college of family physicians of canada received a confidential survey. the questionnaire collected data on socio-demographic characteristics, medical training, practice type, setting and hcv-related care practices. the self-adminisratd questionnaire was also made available to participants for completion on the internet. batdti: response proportion was 33%. median age was 41 years (47% female) and the proporlionoffrench questionnaires was 26%. approximately 88% had completed family medicine residency lllining in canada; median year of training completion was 1995. sixty-seven percent, 38% and 29% work in private offices/clinics, community hospitals and emergency departments, respectively. regarding ~practices, 94% had ever requested a hcv test and 87% of physicians had screened for hcv iafrction in rhe past 12 months· median number of tests was 10. while 17% reported having no hcv-uaed patients in their practic~, 44% had 1-5 hcv-infected patients. regarding the level of hcv care provided, 4.3% provide ongoing advanced hcv care including treatment and dose monitoring for ctmduions: in this sample of canadian family physicians, most had pro~ided hcv screening. to •least one patient in the past year. less than half had 1-5 hcv-infected patients and 41 % provide ~:relared care the role of socio-demographic factors, medical training as wel_i as hcv ca~e percep-lldas 10 rhe provision of appropriate hcv screening will be examined and described at the time of the canference. '4-50 (c) healthcare services: the context of nepal meen poudyal chhetri """1tl.ction healthcare service is related with the human rights and fundamental righ~ of the ci~ ciaaiuntry. however, the growing demand foi health care services, quality heal~care service, accessib1b~ id die mass population and paucity of funds are the different but interrelated issues to .be ~ddressed. m nepat.1n view of this context, public health sector in nepal is among other sectors, which is struggling -.i for scarce resources. . . . nepal, the problems in the field of healthcare servic~s do not bnut ~o the. paucity of faads and resources only, but there are other problems like: rural -urban imbalance, regional unbalance, poster sessio~ f the ll ·m1 ·ted resources poor healthcare services, inequity and inaccessibility of the poor management o , . poor people of the rural, remote and hilly areas for the healthcare services and so on.. . . . · . i f ct the best resource allocation is the one that max1m1zes t e sum o m ivi ua s u11 · ea t services. n a , · h d' ·b · · · h . ·t effi.ciency and efficient management are correlated. it might be t e re istn utmn of mes. ence, equi y, . . . . 1 . income or redistribution of services. moreover, maximizanon of available resources, qua tty healthcare services and efficient management of them are the very important and necessary tools and techmques to meet the growing demand and quality healthcare services in nepal. p4-51 (a) an jn-depth analysis of medical detox clients to assist in evidence based decision making xin li, huiying sun, ajay puri, david marsh, and aslam anis introduction: problematic substance use represents an ever-increasing public health challenge. in the vancouver coastal health (vch) region, there are more than 100,000 individuals having some probability of drug or alcohol dependence. to accommodate this potential demand for addiction related services, vch provides various services and treatment, including four levels of withdrawal management services (wms). clients seeking wms are screened and referred to appropriate services through a central telephone intake service (access i). the present study seeks to rigorously evaluate one of the services, vancouver detox, a medically monitored 24-bed residential detox facility, and its clients. doing so will allow decision makers to utilize evidence based decision-making in order to improve the accessibility and efficiency of wms, and therefore, the health of these clients. methods: we extract one-year data (october 1, 2003 -september 30, 2004 from an efficient and comprehensive database. the occupancy rate of the detox centre along with the clients' wait time for service and length of stay (los) are calculated. in addition, the effect of seasonality on these variables and the impact of the once per month welfare check issuance on the occupancy rate are also evaluated. results: among the 2411 clients (median age 40, 65% male) who were referred by access! to vancouver detox over the one-year period, 1448 were admitted. the majority (81 %) of those who are not admitted are either lost to follow up (i.e., clients not having a fixed address or telephone) or declined service at time of callback. the median wait time was 1 day [q3-ql: 3-1], the median los was 5 days iq3-qt: 6-3], and the average bed occupancy rate was 83%. however, during the threeday welfare check issue period the occupancy rate was lower compared to the other days of the year 175% vs. 84%, p conclusion: our analysis indicates that there was a relatively short wait time at vancouver detox, however 40% of the potential clients were not served. in addition, the occupancy rate declined during the welfare check issuance period and during the summer. this suggests that accessibility and efficiency at vancouver detox could be improved by specifically addressing these factors. background: intimate partner violence (ipv) is associated with acute and chronic physical and men· tal health outcomes for women resulting in greater use of health services. yet, a vast literature attests to cultural variations in perceptions of health and help-seeking behaviour. fewer studies have examined differences in perceptions of ipv among women from ethnocultural communities. the recognition, definition, and understanding of ipv, as well as the language used to describe these experiences, may be different in these communities. as such, a woman's response, including whether or not to disclose or seek help, may vary according to her understanding of the problem. methods: this pilot study explores the influence of cultural factors on perceptions of and responses to ipv among canadian born and immigrant young women. in-depth focus group interviews were con· ducted with women, aged 18 to 24 years, living in toronto. open-ended and semi-structured interview questions were designed to elicit information regarding how young women socially construct jpv and where they would go to receive help. interviews were transcribed, then read and independently coded by the research team. codes were compared and disagreements resolved. qualitative software qsr n6 was used to assist with data management. . ruu~ts_: res~nses_abo~t what constitutes ipv were similar across the study groups. when considering specific ab.us1ve ~1tuanons and types of relationships, participants held fairly relativistic views about ipv, especially with regard to help-seeking behaviour. cultural differences in beliefs about normaive m;ile/femal~ relations. familial.roles, and customs governing acceptable behaviours influenced partictpants perceptions about what 1n1ght be helpful to abused women. interview data highlight the social l05ter srnfons v91 11111 suucrural _impact these factors ha:e on you?g women and provide details regarding the dynamics of cibnocultur~ m~uences on help-~eekmg behav10ur: t~e ro~e of such factors such as gender inequality within rtlaoo?sh1ps and t_he ~erce1ved degree of ~oc1al 1solat1on and support nerworks are highlighted. collc~ the~ findmgs unde~score the _1mporta_nc_e of understanding cultural variations in percrprions of ipv ~ relanon to ~elp-seekmg beha~1':'ur. th1s_mformation is critical for health professionals iodiey may connnue developmg culturally sensmve practices, including screening guidelines and protorol s. ln addition, _this study demonstr~tes that focus group interviews are valuable for engaging young romen in discussions about ipv, helpmg them to 'name' their experiences, and consider sources of help when warranted. p4-s3 (a) health problems and health care use of young drug users in amsterdam .wieke krol, evelien van geffen, angela buchholz, esther welp, erik van ameijden, and maria prins /11trod11ction: recent advances in health care and drug treatment have improved the health of populations with special social and health care needs, such as drug users. however, still a substantial number dots not have access to the type of services required to improve their health status. in the netherlands, tspccially young adult drug users (yad) whose primary drug is cocaine might have limited access to drugrreatment services. in this study we examined the history and current use of (drug associated) treatmmt services, the determinants for loss of contact, and the current health care needs in the young drug mm amsterdam study (yodam). methods: yodam started in 2000 and is embedded in the amsterdam cohort study among drug mm. data were derived from y ad aged < 30 years who had used cocaine, heroin, ampheramines and i or methadone at least 3 days a week during the 2 months prior to enrolment. res11lts:of 195 yao, median age was 27 years (range: 18-30 years), 72% was male and 83% had 1dutch nationality at enrolment. nearly all participants (97%) reported a history of contact with drug llt.lnnent services (methadone maintenance, rehabilitation clinics and judicial treatment), mental health car? (ambulant mental care and psychiatric hospital) or general treatment services (day-care, night-care, hdp for living arrangements, work and finance). however, only 61 % reported contact in the past six l!xlllths. this figure was similar in the first and second follow-up visit. among y ad who reported no current contact with the health care system, 87% would like to have contact with general treatment serl' icts. among participants who have never had contact with drug treatment services, 67% used primarily cocaine compared with 22% and 8% among those who reported past or current contact, respectively. saied on the addiction severity index, 70% reported at least one mental health problem in the past 30 days, but only 11 % had current contact with mental health services. concl11sion: results from this study among young adult drug users show that despite a high contact rm with health care providers, the health care system seems to lose contact with yao. since 87% indicatt the need of general treatment services, especially for arranging house and living conditions, health m services that effectively integrate general health care with drug treatment services and mental health care might be more successful to keep contact with young cocaine users. mtthods: respondents included adults aged 18 and over who met dsm-iv diagn?snc criteria for an anxiety or depressive disorder in the past 12 months. we performed two sets of logisnc regressmns. thtdichotomous dependent variables for each of the regressions indicated whether rhe respondenr_vis-ud a psychiatrist, psychologist, family physician or social worker in the _past_ 12 months. no relationship for income. there was no significant interaction between educatmn an mco~:· r: ::or respondents with at least a high school education to seek help ~rom any of the four servic p were almost twice that for respondents who had not completed high school. th . d ec of analyses found che associacion becween educacion and use of md-provided care e secon s · · be d · · ·f· ly 1 ·n che low income group for non-md care, the assoc1anon cween e ucatlon and was s1gm icant on -· . . . . use of social workers was significant in both income groups, but significant only for use of psychologists in che high-income group. . . . conclusion: we found differences in healch service use by education level. ind1v1duals who have nor compleced high school appeared co use less mental he~lt~ servi~es provided ~y psyc~iatrists, psycholo· gists, family physicians and social workers. we found limited e.v1dence _suggesting the influence of educa· tion on service use varies according to income and type of service provider. results suggesc there may be a need to develop and evaluate progr~ms.designe~ to deliver targeted services to consumers who have noc completed high school. further quahtanve studies about the expen· ence of individuals with low education are needed to clarify whether education's relationship with ser· vice use is provider or consumer driven, and to disentangle the interrelated influences of income and education. system for homeless, hiv-infected patients in nyc? nancy sahler, chinazo cunningham, and kathryn anastos introduction: racial/ethnic disparities in access to health care have been consistently documented. one potential reason for disparities is that the cultural distance between minority patients and their providers discourage chese patients from seeking and continuing care. many institucions have incorporated cultural compecency craining and culturally sensicive models of health care delivery, hoping co encourage better relacionships becween patients and providers, more posicive views about the health care system, and, ulcimacely, improved health outcomes for minority patients. the current scudy tests whether cultural distance between physicians and patients, measured by racial discordance, predicts poorer patient attitudes about their providers and the health care system in a severely disadvantaged hiv-infected population in new york city that typically reports inconsistent patterns of health care. methods: we collected data from 396 unscably housed black and latino/a people with hiv who reported having a regular health care provider. we asked them to report on their attitudes about their provider and the health care system using validated instruments. subjects were categorized as being racially "concordant" or "discordant" with their providers, and attitudes of these two groups were compared. results: the sample consisted of 256 (65%) black and 140 (35%) latino/a people, who reported having 80 (20%) black physicians, 49 (12%) latino/a physicians, 167 (42%) white physicians, and 100 (25%) physicians of another/unknown race/ethnicity. overall, 260 (75%) subjects had physicians of a different race/ethnicity than their own. racial discordance did not predict negative attitudes about rela· tionship with providers: the mean rating of a i-item trust in provider scale (lo=high and o=low) was 8.0 for both concordant and discordant groups, and the mean score in 13-icem relationship with provider scale (4=high and !=low) was 3.5 for both groups. however discordance was significantly associated with distrust in che health care syscem: che mean score on a 7-icem scale (5=high discrust and l=low distrust) was 3.4 for discordant group and 3.0 for che concordant group (t= 2.66, p= 0.008). we further explored these patterns separacely in black and lacino/a subgroups, and using different strategies ro conceptualize racial/ethic discordance. conclusions: in this sample of unscably housed black and latino/a people who receive hiv care in new york city, having a physician from the same racial/ethnic background may be less important for developing a positive doctor-patient relationship than for helping the patients to dispel fear and distrust about the health care system as a whole. we discuss the policy implications of these findings. ilene hyman and samuel noh . .abstract objectiw: this study examines patterns of mental healthcare utilization among ethiopian 1mm1grants living in toronto. methods: a probability sample of 342 ethiopian adults ( 18 years and older) completed structured face-to-face interviews. variables ... define, especially who are non-health care providers. plan of analysis. results: approximately 5% of respondents received memal health services from mainstream healthcare providers and 8% consulted non-healthcare professionals. of those who sought mental health services from mainstream healthcare providers, 3.1 % saw family physicians, 2.1 % visited a psychiatrist. and 0.6% consulted other healthcare providers. compared with males, a significantly higher proportion 1gsfer sessions v93 ri ftlnales consulted non-healthcare_ professionals for emotional or mental health problems (p< 0.01 ). tlbile ethiopian's overall use of mamstream healthcare services for emotional problems (5%) did not prlydiffer from the rate (6%) of the general population of ontario, only a small proportion ( 12.5%) rjerhiopians with mental health needs used services from mainstream healthcare providers. of these, !oj% received family physicians' services, 4.3 % visited a psychiatrist, and 2.2% consulted other healthll/c providers. our data also suggested that ethiopian immigrants were more likely to consult tradioooal healers than health professionals for emotional or mental health problems ( 18.8% vs. 12.5% ). our bivariate analyses found the number of somatic symptoms and stressful life events to be associated with an increased use of medical services and the presence of a mental disorder to be associated with a dfcreased use of medical services for emotional problems. however, using multivariate methods, only die number of somatic symptoms remained significantly associated with use of medical services for emooonal problems. diu#ssion: study findings suggest that there is a need for ethnic-specific and culturally-appropriate mrcrvention programs to help ethiopian immigrants and refugees with mental health needs. since there ~a strong association between somatic symptoms and the use family physicians' services, there appears robe a critical role for community-based family physicians to detect potential mental health problems among their ethiopian patients, and to provide appropriate treatment and/or referral. the authors acknowledge the centre of excellence for research in immigration and settlement (ceris) in toronto and canadian heritage who provided funding for the study. we also acknowledge linn clark whose editorial work has improved significantly the quality of this manuscript. we want to thank all the participants of the study, and the ethiopian community leaders without whose honest contributions the present study would have not been possible. this paper addresses the impact of the rationalization of health-care services on the clinical decision-making of emergency physicians in two urban hospital emergency departments in atlantic canada. using the combined strategies of observational analysis and in-depth interviewing, this study provides a qualitative understanding of how physicians and, by extension, patients are impacred by the increasing ancmpts to make health-care both more efficient and cost-effective. such attempts have resulted in significantly compromised access to primary care within the community. as a consequence, patients are, out of necessity, inappropriately relying upon emergency departments for primary care services as well as access to specialty services. within the hospital, rationalization has resulted in bed closures and severely rmricted access to in-patient services. emergency physicians and their patients are in a tenuous position having many needs but few resources. furthermore, in response to demands for greater accountability, physicians have also adopted rationality in the form of evidence-based medicine. ultimately, ho~ever, rationality whether imposed upon, or adopted by, the profession significantly undermines physu.:1ans' ability to make decisions in the best interests of their patients. johnjasek, gretchen van wye, and bonnie kerker introduction: hispanics comprise an increasing proportion of th.e new york city (nyc) populanon !currently about 25%). like males in the general population, h1spamc males (hm) have a lower prrval,nce of healthcare utilization than females. however, they face additional access barriers such as bnguage differences and high rates of uninsurance. they also bear a heavy burden of health problems lllehasobesity and hiv/aids. this paper examines patterns of healthcare access and ut1hzat1on by hm compared to other nyc adults and identifies key areas for intervention. . . . 148 9 01 5 8 9 01 and older are significantly lower than the nhm popu anon . 10 v. . 10, p<.05), though hi\' screening and immunizations are comparable between the two groups. conclusion: findings suggest that hm have less access t? healthcare than hf or nhm. hown1r, hm ble to obtain certain discrete medical services as easily as other groups, perhapsdueto!rtor are a hm. i i . subsidized programs. for other services, utilization among 1s ower. mprovmg acc~tocareinthis group will help ensure routine, quality care, which can lead to a greater use of prevennve services iii! thus bener health outcomes. introduction: cancer registry is considered as one of the most important issues in cancer epidemiology and prevention. bias or under-reporting of cancer cases can affect the accuracy of the results of epidemiological studies and control programs. the aim of this study was to assess the reliability of the regional cancer report in a relatively small province (yasuj) with almost all facilities needed for c3llcll diagnosis and treatment. methods: finding the total number of cancer cases we reviewed records of all patients diagnoicd with cancer (icd 140-239) and registered in any hospital or pathology centre from1999 until 2001 i n yasuj and all (5) surrounding provinces. results: of 504 patients who were originally residents of yasui province, 43.7% wereaccoulll!d for yasuj province. the proportion varies according to the type of cancer, for exarnplecancetsofdiglstive system, skin and breast were more frequently reported by yasuj's health facilities whereas cancmoi blood, brain and bone were mostly reported by neighbouring provinces. the remaining cases (56.3%1 were diagnosed, treated and recorded by neighbouring provinces as their incident cases. this is partly because of the fact that patients seek medical services from other provinces as they believed that the facil. ities are offered by more experienced and higher quality stuffs and their relative's or temporary acooiii' modation addresses were reported as their place of residence. conclusion: measuring the spatial incidence of cancer according to the location of report ortht current address affected the spatial statistics of cancer. to correct this problem recording the permanm! address of diagnosed cases is important. p4-60 (c). providing primary healthcare to a disadvantaged population at a university-run commumty healthcare facility tracey rickards the. c:ommuni~y .h~alth ~linic (chc) is a university sponsored nurse-managed primary bealthwt (p~c:l clime. the clm1c is an innovative model of healthcare delivery in canada that has integrated tht principles of phc ser · · h' . vices wit ma community development framework. it serves to provide access to phc services for members of th · · illi · dru is ii be . . e community, particularly the poor and those who use or gs, we mg a service-learning facil'ty f d · · · · · · d rionll h . . .,m.:. · t · . meet c ient nee s. chmc nursing and social work staff and srudents r·--· ipa em various phc activities and h .l.hont" less i . f . outreac services in the local shelters and on the streels to'"" popu auon o fredericton as well th chc · model iii fosterin an on oi : . • e promotes and supports a harm reduction . · local d!or an~ h ng ~art:ersh1p with aids new brunswick and their needle exchange program, w1tha ing condoms and :xu:t h:~~~e e~aint~nance therapy clients, and with the clie~ts themselves ~_r; benefits of receiving health f ucation, a place to shower, and a small clothing and food oai~· care rom a nurse p · · d d · --""~'i"· are evidenced in th r research that involved needsaans mvo ves clients, staff, and students. to date the chc has unacn-1 · sessment/enviro i . d ; •• '""""1ll eva uanon. the clinic has also e . d nmenta scan, cost-benefit analysis, an on-go...,,1"".'i'~ facility and compassionate lea x~mme the model of care delivery' focusing on nursing roles wi~ cj rmng among students. finally, the clinic strives to share the resu•p v95 . -arch with the community in which it provides service by distributing a bi-monthly newsletter, and plllicipating in in-services and educational sessions in a variety of situations. the plan for the future is coolinued research and the use of evidence-based practice in order to guide the staff in choosing how much n~ primary healthcare services to marginalized populations will be provided. n-61 (c) tuming up the volume: marginalized women's health concerns tckla hendrickson and betty jane richmond bdrotbu:tion: the marginalization of urban women due to socio-economic status and other determinants negatively affects their health and that of their families. this undermines the overall vitaliry of urban communities. for example, regarding access to primary health care, women of lower economic surus and education levels are less likely to be screened for breast and cervical cancer. what is not as widely reported is how marginalized urban women in ontario understand and articulate their lack of access to health care, how they attribute this, and the solutions that they offer. this paper reports on the rnults of the ontario women's health network (owhn) focus group project highlighting urban women's concerns and suggestions regarding access to health care. it also raises larger issues about urban health, dual-purpose focus group design, community-based research and health planning processes. mdhods: focus group methodology was used to facilitate a total of 30 discussions with 55 urban and 54 rural women across ontario from 2003 to 2005. the women were invited to participate by local women's and health agencies and represented a range of ages, incomes, and access issues. discussions focussed on women's current health concerns, access to health care, and information needs. results were analyzed using grounded theory. the focus groups departed from traditional focus group research goals and had two purposes: 1) data collection and dissemination (representation of women's voices), and 2) fostering closer social ties between women, local agencies, and owhn. the paper provides a discussion and rationale for a dual approach. rax/ts: the results confirm current research on women's health access in women's own voices: urban women report difficulty finding responsive doctors, accessing helpful information such as visual aids in doctors' offices, and prohibitive prescription costs, in contrast with rural women's key concern of finding a family doctor. the research suggests that women's health focus groups can address access issues by helping women to network and initiate collective solutions. the study shows that marginalized urban women are articulate about their health conctrns and those of their families, often understanding them in larger socio-economic frameworks; howtver, women need greater access to primary care and women-friendly information in more languages and in places that they go for other purposes. it is crucial that urban health planning processes consult directly with women as key health care managers, and turn up the volume on marginalized women's voices. women: an evaluation of awareness, attitudes and beliefs introduction: nigeria has one of the highest rates of human immunodeficiency virus ihivi seroprrvalence in the world. as in most developing countries vertical transmission from mother to child account for most hiv infection in nigerian children. the purpose of this study was ro. determine the awareness, attitudes and beliefs of pregnant nigerian women towards voluntary counseling and testing ivct! for hiv. mnbod: a pre-tested questionnaire was used to survey a cross section '.>f.240 pregnant women ~t 2 (lrlleral antenatal clinics in awka, nigeria. data was reviewed based on willingness to ~c~ept or re1ect vct and the reasons for disapproval. knowledge of hiv infection, routes of hiv transm1ssmn and ant1rnroviral therapy iart) was evaluated. hsults: 72% of the women had good knowledge of hiv, i 5% had fair knowledge while 1.1% had poor knowledge of hiv infection.48% of the women were not aware of the association of hreast milk feeding and transmission of hiv to their babies. majority of the women 87% approved v~t while 13% disapproved vct, 93% of those who approved said it was because vct could ~educe risk of rransmission of hiv to their babies. all respondents, 100% who accepted vc.i ~ere willing to be tnted if results are kept confidential only 23% accepted to be tested if vc.t results w.111 be s~ared w1.th pinner and relatives 31 % attributed their refusal to the effect it may have on their marriage whale 69 '-gave the social 'and cultural stigmatization associated with hiv infection for their r~fusal.s 9 % wall accept vct if they will be tested at the same time with their partners.81 ~0 of ~omen wall pref~r to breast feed even if they tested positive to hiv. women with a higher education diploma were 3 times v96 more likely to accept vct. knowledge of art for hiv infected pregnant women as a means of pre. vention of maternal to child transmission [pmtct) was generally poor, 37% of respondents wm aware of art in pregnancy. conclusion: the acceptance of vct by pregnant women seems to depend on their understanding that vct has proven benefits for their unborn child. socio-cultur al factors such as stigmatizationof hiv positive individuals appears to be the maj_or impedi~ent towards widespread acceptanee of ycr in nigeria. involvemen t of male partners may 1mpro~e attitudes t~wa~ds vct:the developmentofm novative health education strategies is essential to provide women with mformanon as regards the benefits of vct and other means of pmtct. p4-63 (c) ethnic health care advisors in information centers on health care and welfare in four districts of amsterdam arlette hesselink, karien stronks, and arnoud verhoeff introduction : in amsterdam, migrants report a "worse actual health and a lower use of health care services than the native dutch population. this difference might be partly caused by problems migrants have with the dutch language and health care and welfare system. to support migrants finding their way through this system, in four districts in amsterdam information centers on health care and welfare were developed in which ethnic health care advisors were employed. their main task is to provide infor· mation to individuals or groups in order to bridge the gap between migrants and health care providers. methods: the implementat ion of the centers is evaluated using a process evaluation in order to give inside in the factors hampering and promoting the implementat ion. information is gathered using reports, attending meetings of local steering groups, and by semi-structu red interviews with persons (in)directly involved in the implementat ion of the centers. in addition, all individual and groupcontaets of the health care advisors are registered extensively. results: since 2003 four information centers, employing 12 ethnic health care advisors, are implemented. the ethnicity of the health care advisors corresponds to the main migrant groups in the different districts (e.g. moroccan, turkeys, surinamese and african). depending on the local steering groups, the focus of the activities of the health care advisors in the centers varies. in total, around 2000 individual and 225 group educational sessions have been registered since the start. most participants were positive about the individual and group sessions. the number of clients and type of questions asked depend highly on the location of the centers (e.g. as part of a welfare centre or as part of a housing corporation). in all districts implementa tion was hampered by lack of ongoing commitment of parties involved (e.g. health care providers, migrant organization s) and lack of integration with existing health care and welfare facilities. discussion: the migrant health advisors seem to have an important role in providing information on health and welfare to migrant clients, and therefore contribute in bridging the gap between migrants and professionals in health care and welfare. however, the lack of integration of the centers with the existing health care and welfare facilities in the different districts hampers further implementation . therefore, in most districts the information centres will be closed down as independent facilicities in the near future, and efforts are made to better connect the position of migrant health advisor in existing facilities. the 2005 who report ranks the philippines as ninth among 22 countries with a high tb prevalence. about a fourth of the country's population is infected, with majority of cases coming from the lower socioeconomic segments of the community. metro manila is not only the economic and political capital of the philippines but also the site of major universities and educational institutions. initial interviews with the school's clinicians have established the need to come up with treatment guidelines and protocols for students and personnel when tb is diagnosed. these cases are often identified during annual physical examinations as part of the school's requirements. in many instances, students and personnel diagnosed with tb are referred to private physicians where they are often lost to follow-up and may have failure of treatment due to un monitored self-administered therapy. this practice ignores the school clinic's great potential as a tb treatment partner. through its single practice network (spn) initiative, the philippine tuberculosis initiatives for the private sector (philippine tips), has established a model wherein school clinics serve as satellite treatment partners of larger clinics in the delivery of the directly observed treatment, short course (dots) protocol. this "treatment at the source" allows school-based patients to get their free government-suppl ied tb medicines from the clinic each day. it also cancels out the difficulty in accessing medicines through the old model where the patient has to go to the larger clinic outside his/her school to get treatment. the model also enables the clinic to monitor the treatment progress of the student and assumes more responsibility over their health. this experience illustrates how social justice in health could be achieved from means other than fund generation. the harnessing of existing health service providers in urban communities through standardized models of treatment delivery increases the probability of treatment success, not only for tb but for other conditions as well. p4-66 (c) voices for vulnerable populations: communalities across cbpr using qualitative methods martha ann carey, aja lesh, jo-ellen asbury, and mickey smith introduction: providing an opportunity to include, in all stages of health studies, the perspectives and experiences of vulnerable and marginalized populations is increasingly being recognized as a necessary component in uncovering new solutions to issues in health care. qualitative methods, especially focus groups, have been used to understand the perspectives and needs of community members and clinical staff in the development of program theory, process evaluation and refinement of interventions, and for understanding and interpreting results. however, little guidance is available for the optimal use of such information. methods: this presentation will draw on diverse experiences with children and their families in an asthma program in california, a preschool latino population in southern california, a small city afterschool prevention program for children in ohio, hiv/aids military personnel across all branches of the service in the united states, and methadone clinic clients in the south bronx in new york city. focus groups were used to elicit information from community members who would not usually have input into problem definitions and solutions. using a fairly common approach, thematic analysis as adapted from grounded theory, was used to identify concerns in each study. next we looked across these studies, in a meta-synthesis approach, to examine communalities in what was learned and in how information was used in program development and refinement. results: while the purposes and populations were diverse, and the type of concerns and the reporting of results varied, the conceptual framework that guided the planning and implementation of each study was similar, which led to a similar data analysis approach. we will briefly present the results of each study, and in more depth we will describe the communalities and how they were generated. conclusions: while some useful guidance for planning future studies of community based research was gained by looking across these diverse studies, it would be useful to pursue a broader examination of the range of populations and purposes to more fully develop guidance. background: the majority of studies examining the relationship between residential environments and cardiovascular disease have used census derived measures of neighborhood ses. there is a need to identify specific features of neighborhoods relevant to cardiovascular disease risk. we aim to 1) develop methods· data on neighborhood conditions were collected from a telephone survey of s,988 fesi· dents in balth:.ore, md; forsyth county, nc; and new york, ny. a sample of 120 of the i.ni~~l l'elpondents was re-interviewed 2-3 weeks after the initial interview t~ measure the tes~-~etest rebab1~1ty of ~e neighborhood scales. information was collected across seven ~e1ghborho~ cond1~ons (aesth~~ ~uah~, walking environment, availability of healthy foods, safety, violence, social cohesion, and acnvmes with neighbors). neighborhoods were defined as census tracts or homogen~us census tra~ clusters. ~sycho metric properties.of the neighborhood scales were accessed by ca~cu~~.ng chronba~h s alpha~ (mtemal consistency) and intraclass correlation coefficients (test-r~test reliabilmes) .. pear~n s .corre~anons were calculated to test for associations between indicators of neighborhood ses (tncludmg d1mens1ons of race/ ethnic composition, family structure, housing, area crowding, residential stability, education, employment, occupation, and income/wealth) and our seven neighborhood scales. . chronbach's alphas ranged from .73 (walking environment) to .83 (violence). intraclass correlations ranged from .60 (waling environment) to .88 (safety) and wer~ high~~~ .7~ for ~urout of the seven neighborhood dimensions. our neighborhood scales (excluding achv1hes with neighbors) were consistently correlated with commonly used census derived indicators of neighborhood ses. the results suggest that neighborhood attributes can be reliably measured. further development of such scales will improve our understanding of neighborhood conditions and their importance to health. childhood to young adulthood in a national u.s. sample jen jen chang lntrodfldion: prior studies indicate higher risk of substance use in children of depressed mothers, but no prior studies have followed up the offspring from childhood into adulthood to obtain more precise estimates of risk. this study aimed to examine the association between early exposure to maternal depl'elsive symptoms (mds) and offspring substance use across time in childhood, adolescence, and young adulthood. methods: data were obtained from the national longitudinal survey of youth. the study sample includes 4,898 mother-child/young adult dyads interviewed biennially between 1992 and 2002 with children aged 4 to 16 years old at baseline. data were gathered using a computer-assisted personal interview method. mds were measured in 1992 using the center for epidemiologic studies depression scale. offspring substance use was assessed biennially between 1994 and 2002. logistic and passion regression models with generalized estimation equation approach was used for parameter estimates to account for possible correlations among repeated measures in a longitudinal study. rnlllta: most mothers in the study sample were whites (42%), urban residents (79%), had a mean age of 31 years with at least a high school degree (82%). the mean child age at baseline was 9 years old. offspring cigarette and alcohol use increased monotonically across childhood, adolescence, and young adulthood. differential risk of substance use by gender was observed. early exposure to mds was associated with increased risk of cigarette (adjusted odds ratio (aor) = 1.52, 95% confidence interval (0): 1.12, 2.08) and marijuana use (aor = 1.46, 95% ci: 1.02, 2.08), but not with alcohol use across childhood, adolescence, and young adulthood, controlling for a child's characteristics, socioeconomic status, ~ligiosity, maternal drug use, and father's involvement. among the covariates, higher levels of father's mvolvement condluion: results from this study confirm previous suggestions that maternal depressive symptoms are associated with adverse child development. findings from the present study on early life experi-e~ce have the potential to inform valuable prevention programs for problem substance use before disturbances become severe and therefore, typically, much more difficult to ameliorate effectively. the ~act (~r-city men~ health study predicting filv/aids, club and other drug transi-b~) study 15 a multi-level study aimed at determining the association between features of the urban enyjronment mental health, drug use, and risky sexual behaviors. the study is randomly sampling foster sessions v99 neighborhood residents and assessing the relations between characteristics of 36 ethnographically defined urban neighborhoods and the health outcomes of interest. a limitation of existing systematic methods for evaluating the physical and social environments of urban neighborhoods is that they are expensive and time-consuming, therefore limiting the number of times such assessments can be conducted. this is particularly problematic for multi-year studies, where neighborhoods may change as a result of seasonality, gentrification, municipal projects, immigration and the like. therefore, we developed a simpler neighborhood assessment scale that systematically assessed the physical and social environment of urban neighborhoods. the impact neighborhood evaluation scale was developed based on existing and validated instruments, including the new york city housing and vacancy survey which is performed by the u.s. census bureau, and the nyc mayor's office of operations scorecard cleanliness program, and modified through pilot testing and cognitive testing with neighborhood residents. aspects of the physical environment assessed in the scale included physical decay, vacancy and construction, municipal investment and green space. aspects of the social environment measured include social disorder, social trust, affluence and formal and informal street economy. the scale assesses features of the neighborhood environment that are determined by personal (e.g., presence of dog feces), community (e.g., presence of a community garden), and municipal (e.g., street cleanliness) factors. the scale is administered systematically block-by-block in a neighborhood. trained research staff start at the northeast corner of an intersection and walk around the blocks in a clockwise direction. staff complete the scale for each street of the block, only evaluating the right side of the street. thus for each block, three or more assessments are completed. we are in the process of assessing psychometric properties of the instrument, including inter-rater reliability and internal consistency, and determining the minimum number of blocks or street segments that need to be assessed in order to provide an accurate estimate of the neighborhood environment. these data will be presented at the conference. obj«tive: to describe and analyze the perceptions of longterm injection drug users (idus) about their initiation into injecting. toronto. purposive sampling was used to seek out an ethnoculturally diverse sample of idus of both genders and from all areas of the city, through recruitment from harm reduction services and from referral by other study participants. interviews asked about drug use history including first use and first injecting, as well as questions about health issues, service utilization and needs. thematic analysis was used to examine initiation of drug use and of injection. results: two conditions appeared necessary for initiation of injection. one was a developed conception of drugs and their (desirable) effects, as suggested by the work of becker for marijuana. thus virtually all panicipants had used drugs by other routes prior to injecting, and had developed expectations about effects they considered pleasureable or beneficial. the second condition was a group and social context in which such use arose. no participants perceived their initiation to injecting as involving peer pressure. rather they suggested that they sought out peers with a similar social situation and interest in using drugs. observing injection by others often served as a means to initiate injection. injection served symbolic purposes for some participants, enhancing their status in their group and marking a transition to a different social world. concl111ion: better understanding of social and contextual factors motivating drug users who initiate injection can assist in prevention efforts. 10 ma!onty of them had higher educational level (57%-highschool or higher).about 20.2 yo adffiltted to have history of alcohol & another 12.4% had history of smoking. only 3.2% people were on hrt & 3.1 % were receiving steroid. majority of them (81.2) did not have history of osteoporosis. 13.6% have difficulty in ambulating. only 8.8% had family history of osteoporosis. bmd measurements as me~sured by dual xray absorptiometry (dexa) were used for the analysis. bmd results were compare~ w1~ rbc folate & serum vitamin b12 levels. no statistical significance found between bmd & serum v1taffiln b12 level but high levels of folate level is associated with normal bmd in bivariate and multivariate analysis. conclusion: in the studied elderly population, there was no relationship between bmd and vitamin b12; but there was a significant association between folate levels & bmd. introduction: adolescence is a critical period for identity formation. western studies have investigated the relationship of identity to adolescent well-being. special emphasis has been placed on the influence of ethnic identity on health, especially among forced migrants in different foreign countries. methodology: this study asses by the means of an open ended question identity categorization among youth in three economically disadvantaged urban communities in beirut, the capital of lebanon. these three communities have different histories of displacement and different socio-demographic makeup. however, they share a history of displacement due to war. results and conclusion: the results indicated that nationality was the major category of identification in all three communities followed by origin and religion. however, the percentages that self-identify by particular identity categories were significantly different among youth in the three communities, perhaps reflecting different context in which they have grown up. mechanical heart valve replacement amanda hu, chi-ming chow, diem dao, lee errett, and mary keith introduction: patients with mechanical heart valves must follow lifelong warfarin therapy. war· farin, however, is a difficult drug to take because it has a narrow therapeutic window with potential seri· ous side effects. successful anticoagulation therapy is dependent upon the patient's knowledge of this drug; however, little is known regarding the determinants of such knowledge. the purpose of this study was to determine the influence of socioeconomic status on patients' knowledge of warfarin therapy. methods: a telephone survey was conducted among 100 patients 3 to 6 months following mechan· ical heart valve replacement. a previously validated 20-item questionnaire was used to measure the patient's knowledge of warfarin, its side effects, and vitamin k food sources. demographic information, socioeconomic status data, and medical education information were also collected. results: sixty-one percent of participants had scores indicative of insufficient knowledge of warfarin therapy (score :s; 80%). age was negatively related to warfarin knowledge scores (r= 0.27, p = 0.007). in univariate analysis, patients with family incomes greater than $25,000, who had greater. than a grade 8 education and who were employed or self employed had significantly higher warfarm knowledge scores (p= 0.007, p= 0.002 and p= 0.001 respectively). gender, ethnicity, and warfar~n therapy prior to surgery were not related to warfarin knowledge scores. furthermore, none of t~e. m-hospital tea~hing practices significantly influenced warfarin knowledge scores. however, panic1~ants who _rece1v~d post discharge co~unity counseling had significantly higher knowledge scores tn comp~r1son with those who did not (p= 0.001 ). multivariate regression analysis revealed that und~r~tandmg the ~oncept of 1?ternational normalized ratio (inr), knowing the acronym, age and receiving ~ommum1!' counseling after discharge were the strongest predictors of warfarin kn~wledge. s~1oeconom1c status was not an important predictor of knowledge scores on the multivanate analysis. poster sessions v101 ~the majority of patients at our institution have insufficient knowledge of warfarin therapy.post-discharge counseling, not socioeconomic status, was found to be an important predictor of warfarin knowledge. since improved knowledge has been associated with improved compliance and control, our findings support the need to develop a comprehensive post-discharge education program or, at least, to ensure that patients have access to a community counselor to compliment the in-hospital educatiop program. brenda stade, tony barozzino, lorna bartholomew, and michael sgro lnttotl#ction: due to the paucity of prospective studies conducted and the inconsistency of results, the effects of prenatal cocaine exposure on functional abilities during childhood remain unclear. unlike the diagnosis of fetal alcohol spectrum disorder, a presentation of prenatal cocaine exposure and developmental and cognitive disabilities does not meet the criteria for specialized services. implications for public policy and services are substantial. objective: to describe the characteristics of children exposed to cocaine during gestation who present to an inner city specialty clinic. mnbods: prospective cohort research design. sample and setting: children ages 5 to 15 years old, referred to an inner city prenatal substance exposure clinic since november, 2003. data collection: data on consecutive children seen in the clinic were collected over an 18 month period. instrument: a thirteen (13) page intake and diagnostic form, and a detailed physical examination were used to collect data on prenatal substance history, school history, behavioral problems, neuro-psychological profile, growth and physical health of each of the participants. data analysis: content analysis of the data obtained was conducted. results: twenty children aged 6 to 14 years (mean= 9.8 years) participated in the study. all participants had a significant history of cocaine exposure and none had maternal history or laboratory (urine, meconium or hair) exposure to alcohol or other substances. none met the criteria of fetal alcohol spectrum disorder. all were greater than the tenth percentile on height, weight, and head circumference, and were physically healthy. twelve of the children had iqs at the 19th percentile or less. for all of the children, keeping up with age appropriate peers was an ongoing challenge because of problems in attention, motivation, motor control, sensory integration and expressive language. seventy-four percent of participants had significant behavioral and/or psychological problems including aggressiveness, hyperactivity, lying, poor peer relationships, extreme anxiety, phobias, and poor self-esteem. conclusion: pilot study results demonstrated that children prenatally exposed to cocaine have significant learning, behavioural, and social problems. further research focusing on the characteristics of children prenatally exposed to cocaine has the potential for changing policy and improving services for this population. methods: trained interviewers conducted anonymous quantitative surveys with a random sample (n= 148) of female detainees upon providing informed consent. the survey focused on: sociodemographic background; health status; housing and neighborhood stability and social resource availability upon release. results: participants were 70% african-american, 16% white, 9% mixed race and 5% native american. participants' median age was 37, the reported median income was <s2,000 year, and 53% reported receiving a high school diploma or equivalent. women reported a number of health conditions rypical of underserved populations, including asthma (42%), sexually transmitted infections (39%), high blood pressure (19%), hcv (14%), diabetes (5%) and hiv (4%). nearly half (46%) did not anticipate having a place to stay for at least 30 days upon release. factors significantly associated housing stability upon release were: high family support score (adjusted odds ratio [aor) 6.15; 95% confidence interval (95% cl) 1.76, 21.61), high neighborhood stability score (aor 4.41; 95% cl 1.25, 15.52), wanting a commercial sex worker (csw) support group (aor 0.25; 95% cl 0.10, 0.62); and identifying as bisexual (aor 0.24; 95% cl 0.07, 0.75). women desired employment (95%), housing (84%), education (77%), drug treattnent (74%), help with custody of children (31 %), childcare (28%), and domestic violence suppon (20%) services. conclusions: female detainees represent one of urban centers' most marginalized pcjpwatioos. short periods of detention represent a unique opportunity for _interven?~ns bri~ co~s and public health institutions. service providers should collaborate ~1th local ~ails to .p~de a con1111uwnof care for female detainees upon release that includes transportation , housing, residential drug treallllcnt, employment, education, family reunification, childcare and domestic viol~nce su~rt. ~pecial attenlion is warranted to lesbian and bisexual women and csws, who may be especially margmalized &om family and service-based support networks. introduction: "health care and ethnic minority immigrants" examines how health care policy dil· ferences between canada and the united states impact the health-related hardships, access and use of preventative care, and health outcomes of urban ethnic minority immigrants in both countries. methods: the findings of this paper build on the results of my qualitative comparative study of hotel workers in vancouver, bc and seattle, wa that revealed greater health related hardships for similarly matched employees in seattle compared to vancouver. in this paper, i examine the generalizability of these findings at the cross-national level for similar ethnic minority immigrant groups. i compare the impact of health care policy differences on specific groups that have emigrated to cities in both canada and the united states. these include immigrants from china, viemam, philippines, west indies, africa, sri lanka, pakistan, and latin america. comparative statistical analysis -utilizing rel· evant data from statistics canada and the u.s. census -reveal the extent to which health policy differ· ences help explain how current health policy in the united states disadvantages urban ethnic minority immigrants. results: many ethnic minority immigrants are a part of the working poor, a group which disproportionately lacks health insurance coverage in the united states. their position in the labour market makes them most vulnerable to exclusion from health care services. the data reveals how current u.s. health policy creates barriers for recently arrived ethnic minority immigrants to access health insurance and care. it remains difficult to ascribe health outcome differences directly to policy differences because of the challenge in disentangling the complex interacting interventing variables, including income inequality, that determine health outcomes. yer suggestive evidence suggests that health policy in the united states is harming the health of urban ethnic minority immigrants. the current health policy regime in the united states harms the health access, care, and outcomes of urban ethnic minority immigrants. comparing the fortunes of similar immigrants in cana· dian and u.s. cities reveals these patterns of disadvantage and some of their consequences. these barriers are an understudied factor in the sociological literature on "segmented assimilation" and social exclu· sion. the paper ends with policy recommendatio ns to improve access to health care among ethnic minor· ity immigrants in both countries, with the goal of reducing health related hardships, and improving health outcomes. mass index deyu pan, richard baker, and keith norris badtgrou~ over the past 3 decades, there has been dramatic increase in prevalence of obesity in the us population. however, the relationship between obesity and the prevalence of cardiovascular dis· ease (cv~) risk factors in different race/ethnic groups over time is not well known. . ik~rgn: _we use 2 cross-sectional , nationally representative surveys: national health and nutri· t10nal examination survey (nhanes iii 1988 -1994 (n= 14 029) and nhanes 1999 nhanes -2002 , including white, black, and hispa~ic races who were no~preg~ant, aged 20 to 74 years. res11lt1: the preval~nce of high cholesterol level (~ 240 mg/dl), untreated high blood pressure (2: l40/90 mm hg) an~ diabetes were calculated according to bmi group (lean, <25; overweight, 25-29; and obese, 2: 30) for different race/ethnicity groups. over the approximately 10 years period (from 1988l ~94 ~o [1999] [2000] [2001] [2002] , reducrion in 3 cvd risk factors in non-hispanic white has been observed. for mmonty race/~hnicity groups, black and hispanic, there had been increases in prevalence in high blood fa pressure and diabetes. the lean group experienced larger increase in prevalence of those cvd risk ctors. . fo~~ the prevalence in cvd risk factors over time had reduced in most of the bmi ca1egoes r t e w ~~e population. however, for black and hispanic, prevalence of 2 of 3 cvd risk factors ave generauy uncreased, especially in overweight and obese groups. methods: strategies identified to achieve these goals include: developing meaningful neighbourhood and district boundaries for comparing health information, providing free access to neighbourhood health profiles (including relevant data at the smallest level for which valid rates could be calculated); mapping relationships of inequality at a variety of nested levels; providing a range of formats (maps, tables) to meet the needs of users; providing technical support; seeking user input to advance and improve the site; and conducting workshops to foster access and use of data for decision-making , advocacy and collaboration. an evaluation strategy is being developed to assess the efforts and impact of these strategies. a preliminary assessment of the results of the first six months of activity will be completed in october 2005. results: significant differences in health are shown at all the geographic levels indicating success in developing the boundaries. results tabulated for the first six months use of the site include: site visits; media use; reference/acknow ledgement of the site in other documents; number of contacts through presentations and workshops; feedback from users identifying strengths and limitations; and, response from health decision makers. members of the partnership are identifying additional tools and services to further support actions that reduce health inequalities. the assessment will be used to develop more specific goals, targets and monitoring mechanisms. conclusions: our experience with paper-based health profiles indicates that they have been used extensively for program planning and advocacy. the greater availability of information in a publiclyavailable web-based format is expected to translate into even greater usefulness and wider application. the web site has had considerable usage to date, extensive media coverage. site users and workshop participants have provided positive feedback and suggestions for next steps. the six month review will be available in october 2005. in india, more than 4 and a half people are hiv-positive and half a million have aids. india currently has the largest number of positive people in the world, oumumbering south africa and accounting for nearly one tenth of the global hiv/aids prevalence. the world bank predicts that by 2015, there will be 35 million hiv/aids cases in india. the central challenge facing hiv prevention efforts in south asia today is learning how to respond to the societal determinants of vulnerability to hiv. hiv/aids is an issue largely engulfed by social stigma. stigma and discrimination are often considered the foremost barriers to effective poster sess10ns v104 prevention and care initiatives. stigma can make a person afraid of disclosing their status~ ~yone el se, and may make them feel depressed or alone. stigma can .also le~d to poor adherence to medicanon, ~ likelihood to access health and social services, and less desire to hve. hiv+ men and women may beafraidtotd! their co-workers that they have hiv for fear of their reactions or for fear of losing their jobs. in a study 1 conducted of people living with hiv/aids in delhi, india ov~r. 2003-~, many respo~ts shared their experiences of discrimination in their families, in their communmes, an~ m health ~e settmgs. these findings will be shared in the workshop, and will be compa~ed to m~ ex~nences workmg as an ~ co~l~ at the center for aids prevention studies in san francisco, cahforma and congreso de lannos unidos m philadelphia, pennsylvania. the workshop will include an overview of hiv/aids in urban contexts in tht united states (los angeles, san francisco, new york, philadelphia) and south asia (delhi, hyderabad, chennai). differences in methods for outreach, prevention and treatment efforts between devdoped world and developing world contexts will be discussed. a large portion of the workshop will be centered on discussion. participants will engage in an exercise to categorize their ideas of stigma. the workshop will also include a powerpoint presentation on the quantitative data gathered from the survey. the number of homeless and runaway youth is increasing in toronto, which is currently home to 3 out of 4 youth in the gt a who live alone, and it is the preferred locale for the street-involved and homeless youth. the city's youth population is expected to grow by nearly 20% by 2011, the school dropout rate is rising, and there are is a paucity of empirical data on what works with these at-risk youth. over a two-year period, (2002) (2003) (2004) , youthlink-inner city, conducted an evaluation of the impact of a harm reduction program aimed at reducing street youth's risk of contracting/infecting others with the hep c virus, which has both short-term and long-term deleterious effects. demographic data from 102 street-youth, along with resources/services used, employment methods, type and frequency of drug use, health status, police contacts, and their views of program impact were analyzed using a standardized sur· vey. additionally, focus groups with youth, agency staff and community key informants were conducted, as well as in-depth interviews with three youth, over a sixth-month period. this paper details study results, which both inform both practice and future study about what works, why it works, and how best to work with these vulnerable youth. sho~ed .that the wasteland in this area was contaminated by as cd zn pb cu simultaneously. the con-ta~matton of as ~d was quit significant. the contamination problems and environmental issues in this region are very. serious. the awareness on environmental and health issues was investigated by spot survey and analysis. overall awareness among the residents in the region is very poor. the measures must be taken to assu~e t~e .sustainable economy development by local and central gorvernments. keywords nandan guangx1; mmmg area; environmental awareness. concern like, epileptic fits, vision problems, earache, cough of more than 3 weeks and urinary problems. ninety-five subjects did not take bath regularly and 42 were exposed to sex. the health problems identified are only of a particular point of time and it may be difficult to interpret the depth of "iceberg" of the street children's world also draw the health-illness continuum. periodic interaction and follow-up is very difficult as they generally disappear from the research setting due to their frequent mobility for survival measures. as per the prediction of the iceberg theory, only some problems may have been identified, larger problems in respect to their health may not have been identified. in spite of various limitations, this attempt had proven that, it is possible to enter the street children world, explore the iceberg of illnesses, and establish data of health-illness continuum through an effective nursing agency. it is recommended to deworm these children, provide nutritional education, establish "condom corners" and "street children help line" in the city and urban slum areas and undertake action research on their health. malnutrition is a serious problem in developing countries like bangladesh. malnutrition causes a great deal of child suffering. malnutrition is one of the major causes of morbidity and mortality among the child. the aim of present study was to investigate the nutritional status and its relation to socioeconomic conditions of urban disadvantaged child of under five years age. nutritional status was determined by anthropoemetric measurement(heig ht, weight, mid-arm circumference etc.). determination of total protein and serum albumin were done for biochemical examination. haematological examination was done by blood haemoglobin profile estimation by cynmethhaemoglob in method and determination of haematocrit value or packed cell volume (pcv). stool examination for ova of hook worm, round worm, trichuris species and other species were also done. relevant information on socioeconomic characteristics was recorded by interviewing the house hold head using pretested questionnaire. our study demonstrated that nutritional status of the children are positively correlated with family income, parents level of formal education and negatively related to the family size. the effect of family income on the nutritional status, haematological and biochemical indices of urban disadvantaged child under five will be discussed in detail which will help us to determine proper way of utilization of health care facilities exists in developing countries. introduction: with the increase in morbidity and reduction in mortality and with the introduction of highly active antiretroviral therapy (haart), there is increasing focus on the determinants of healthrelated quality of life (hrqol) in hiv-infection. the focus on the present study is to examine the relationship between social support, depression, and hiv disease severity, and the extent to which these variables impact on hrqol in adult men with hiv-infection. methods: as part of a larger ongoing natural history study focusing on the neurobehavioural complications of hiv and aids, we administered questionnaires to assess depression (beck depression inventory), social support, and hrqol (mos-hiv) to 366 adult men with hiv-infection. a structured interview was used to collect health information and data on hiv disease severity. physical and mental health summary scores (phs and mhs) were derived through principal components analysis. participants were grouped according to level of social support: "well supported" (n= 180), "moderately supported" (n=90), "little or ineffective support" (n=96). analysis of variance was conducted to assess the effects of three levels of perceived social support, depression status (present vs absent), and hiv disease severity (aids vs non-aids) on mental and physical health dimensions of hrqol. potential interaction effects were also evaluated. results: social support was found to have a significant effect on both mhs and phs. presence of depression was found to have a significant effect on mhs but not phs. hiv disease severity (presence of aids diagnosis) was found to have a significant effect on phs but not mhs. manov a analyses revealed no significant interactions effects. conclusions and implications: level of social support, presence of depression and hiv disease severity were shown to have independent effects on hrqol. feeling "well supported" appears to have significant benefits for mental and physical health. biological indicators of hiv disease appear to have a selective impact on physical health while presence of depression is related to significant reductions in mental health. the development and evaluation of behavioural interventions to improve perceived social support network and depression will likely result in significant improvements in health outcomes of adults with hiv and aids. introduction: ongoing medical advances have greatly enhanced the longevity of penons living with aids (plwa). however, for certain subgroups living with hiv/aids, including, members of minority groups, women and the economically underprivilege d, aids outcomes have not changed so favorably. one obvious factor to consider when explaining disparities in aids outcomes is the environ· ment within which an individual resides. few studies of aids outcomes have focused on how commu· nity setting influences the effectiveness of care delivery. the principle research questions were: 1) what is the relationship between the locations of areas with concentrations of pl wa and the distribution of both primary and ancillary service providers? 2) how has the widespread availability of haart (after 1996) impacted aids outcomes at the community level? 3) how do aids mortality and fatality rates vary when related community characteristics, such as household income, ethnic mix, and geographic access to primary and ancillary hiv/aids services are considered? we examine this issue across residential communities in the los angeles area. methods: los angeles county's comprehensive aids service providers database (compiled and maintained by aids project los angeles) was geo-referenced and distances between weighted mean population centers and the nearest hiv/aids service providers were calculated to provide a measure of access which was then merged with aids diagnosis and mortality data along with relevant socioeconomic and population indicators and for analysis using bivariate and multivariate statistics at the zip code level. results: there is a growing disparity in hiv/aids outcomes between low-income minority areas and affluent non-minority areas in the post-haart era (p = 0.002). ancillary aids services such as case management and counseling and testing are located near the places where low income hiv/aids service consumers are likely to live (p =.000). in spite of having more access to ancillary services low income areas continue to be associated with smaller decreases post-haart aids mortality rates (p=0.301). conclusions: given the increasing disparities in aids outcomes between lower and upper income communities in the post -haart era, more specific research into the capacities of aids services providers and the efficiency of their interventions is required. although it is clear that ancillary services are operating in the areas of greatest need, their effectiveness remains limited. understanding the reasons for t~is, be they lack of adequate resources for inner city service providers, failure to reach target popula· tmns or other causes, requires additional research. in 20?3, y.out~ who were able to speak either french or english and had been absent from their parent's/ c~regivers ~es.1dence for at least three consecutive nights took part in the survey which consisted of inter· viewer-adm1ms tered question~aires. p.arti~i~an~s were recruited from drop in centres in 7 cities across <?-nada.y~uth self-report as either using in1ect1on or non-injection drugs. statistical analyses were car· r1ed out using sas version 8. the 'home' is a contested site for many women living in urban areas. women living in poverty and using illicit drugs are largely excluded from participation in dominant 'home-base' gender roles of wife and mother because of economic disadvantage. they thus 'make home' in ways that are specific to their everyday experiences. while research has established links between housing, health and drug use, this literature does not tend to consider the meanings that home may have within the lives of women who lack access to adequate tenured living spaces. this presentation reports the findings of my master's thesis work. using grounded theory methodology within a poststructuralist feminist conceptual framework, i conducted in-depth, open-ended interviews with 11 women living in the toronto area. the women were recruited from within the community through posters at different social service agencies (e.g., drop-in centres, community health centres). they were asked to describe their housing experiences and what home meant to them over the course of their lives. through the data analysis process, a substantive theory of 'making home' for women living in poverty and using illicit drugs emerged. the central concept of making home is a continual process of learning about and interacting with the environment. the process of making home entails different subprocesses: finding home (knowing what is available and accessible, and accessing home), adaptation, and leaving home. the different meanings of home that women developed from their experiences and knowledge emerged as critical components for the process of making home. the women i interviewed were very mobile and experienced constant change with regards to home. home was not considered only in relation to physical living space. home could be made within a relationship or with regards to a possession. while dominant meanings of home relation to tenured living spaces are generally positive, my research indicated that home could also be a negative context. the presentation will describe my research process and findings as well as the theoretical and policy implications of my results. in the western democracies, social citizenship bundles the right to social provision (defined as a share of the social good, not necessarily proportionate to the market value of a citizen's contribution) with participation in the paid labour force. for this reason, social citizenship rights, which usually involve some kind of redistributive measures, are a key mechanism by which states ensure economic equality, or at least, economic equality of opportunity, but are also a mechanism of social exclusion. social rights are inscribed around a single kind of relationship, that which exists between a male family breadwinner and the paid labor market; those who make economic contributions that fall outside this relationship -such as unpaid care-work, or, work that occurs in shadow or underground economiesare to a greater or lesser degree barred from accessing citizen-based social provisions. furthermore, social provision that occurs outside of the breadwinner/market-eco nomy dyad is very often meanstested and is accompanied by regulatory state apparatuses (this includes a range of social services, from welfare payments, to child-care subsidies, to disability pensions). social citizenship is thus stratified; the legal relationship that is at the heart of social citizenship acts as a form of social closure against claims for a share of the social good made by those who are 'lesser' citizens than others. of particular interest in this paper is how citizenship inequalities translate into inequalities in the social provisions that emerge from economic participation: the right to safety on the job, protection from employer harassment, protection from the vagaries of the market as well the right to adequate, comprehensive, and appropriate health services. drawing on mixed methods, longitudinal data from adult sex workers (n= 170) located in two research sites with different social welfare regimes -one in canada and one in the u.s. -we examine the consequences of working in a "shadow" or "underground" economy on various facets of health and access to health services, taking into consideration the mediating role that citizenship constructs and social welfare regimes play in accessing other key social and economic determinants of health. methods: the study population included methadone patients (re)admitted at the mhs in the period from 1/1/1996 to 31/1/2002, born in the netherlands, morocco, surinam, dutch antilles or turkye, and officially registered in the population register of amsterdam. the population register was used to ascer· rain the vital status. causes of death were provided by the central bureau of statistics. mortality was categorised as hiv related, directly drug related (overdose) and other.mortality. results: the methadone patients had the following characteristics: 38 years of age at entry; 81% male; 37% ethnic minority; 37% ever injected. in total, 9558 personyear (py) of observation time and 173 deaths (18% hiv related, 14% overdose; 57% other) were observed. hence, the crude mortality rate was 18/1000 py (95% ci 16-21/1000 py). the percentage of (ever) injectors was higher among the native dutch than among the ethnic minorities, 51 % and 14 % respectively. higher rates were observed among native dutch drug users compared to those belonging to an ethnic minority (age adjusted hazard ratio (hr) 1.9 (95% ci 1.4-2.7). the age adjusted hr of (ever) injecting drug users versus non injecting users was 3.0 (95% cl 2.2-4.1 ). after additional adjustment for route of administration a non significant difference between the dutch and ethnic minorities remained (hr 1.3, 95% cl 0.9-1.9, p-value 0.151. conclusion: the mortality rate among the heroin users in amsterdam is strongly affected by the high prevalence of non-injecting drug use. moreover, this study confirms that differences in mortality between the native dutch and the ethnic minorities is related to differences in route of administration. the ongoing reduction of injecting drug use (due tot selctieve mortality, and switching route ofadministration and popularity of crack cocaine) may lead to a further reduction of mortality rates among heroin users in amsterdam. interventions preventing the initiation of injecting should be encouraged. . introduction: food insecurity is an inequality that is central in the lives of many low-income cana· d1ans. people lack food security when regular access to nutritious food is limited or variable due to high prices'. low income, lack of transportation or inadequate food distribution, or they become disadvan· taged m other w~ys through the acquisition of food. a group comprising academics, health profession· als, and commumty workers who have experience in food insecurity, developed a pilot study in ottawa that sought to und~rstand the lived experience of food insecurity. the study also used the united states department of agriculture (usda) community food security module. methods: a series of group meetings determined the best research approaches; questionnaires and consent forms were developed by a working group. twenty-three self-identified food insecure respon· dents were interviewed. the data were analysed using frequency distributions· the food security module was coded according to the usda coding guide, and open-ended responses w~re coded using a grounded approach. renlts: preliminary results from our pilot study have given the research team cause for considerable concern. out of 14 households without children, 11 experienced food insecurity with hunger; 5 ~ere at the severe l~vel. out of 9 households with children, 7 experienced food insecurity with hunger. ~•rst person reflecnons commonly featured feelings of depression low self-esteem and despair. participants' account tha d . an 1 cu ty gettmg to the store, severely limited putting knowledge and skills mto practice. l'oster sessions v109 conclusion: based on these early findings, our research team is looking to challenge what appear to be assumptions about poverty and food insecurity and for best ways to use this information. some policy approaches and interventions that encourage people to eat better and exercise more, may be missing the mark for low-income individuals, and may serve to maintain food insecurity. future research will build on this pilot. with methods augmentation, and a comprehensive knowledge dissemination plan, we hope co contribute to research and policy frameworks at all levels. ps-25 (a) mental health and the corrections system: population-based analyses in urban, semi-urban, and rural settings julian somers, robert watts, and michelle patterson introduction: according to recent epidemiological research, rates of mental disorders vary considerably across countries and across regions within countries. nevertheless, rates of mental disorders (including substance use disorders) are consistently higher in populations involved in the corrections sys-1em. courts have long recognized the heavy burden of mental illness within their purview, and have developed innovative programs to better manage the needs of such individuals. the majority of these innovations (e.g., specialized courts) exist in urban settings. however, few studies have examined the degree of variability in rates of different mental disorders between courts in urban and other settings (e.g., semi-urban, rural). variability between courts in different settings, if present, holds implications for needs-based resource planning. the present study was conducted as part of a long-term inter-ministerial collaboration in british columbia (bc). analyses were conducted on linked administrative data regarding population health and correctional services. a database was constructed including all individuals sentenced in bc in a single year (n=49,142). corrections records were matched to records of health services utilization (hospital and outpatient services) for mental disorders and substance use disorders in the years preceding sentencing. services for mental health and substance-related problems were aggregated into three groups: severe mental illness (smi); less-severe mental illness (lsmi); alcohol/other drug (ad). courts were grouped, based on their annual volumes, inro three categories: "urban"; "semi-urban" and "rural." rates of service use for mental disorders were compared between groups for 1-year and 5-years preceding sentencing. results: there were significant differences in the rates of mental disorders in courts of different size (urban, semi-urban, rural) . however, differences between the rates of disorders within each of these groups exceeded the discrepancies between groups. comparatively high and low rates of disorders were observed in courts of each size. moreover, the courts with the highest rates of certain disorders (e.g., ad) did not have the highest rates of other types of mental disorders (e.g., smi). conclusions: rates of mental disorders differed significantly among the annual populations sentenced across courts in bc. urban courts (which process comparatively large numbers of people) have implemented services to address the needs of the mentally ill. the present analyses strongly suggest that program development should be closely linked to the demonstration of need. the results caution that a uniform approach to court programs for the mentally ill is likely to be inefficient, over-supplying services in some settings and underestimating need in others. results: public settings used for injection are characterized by highly unhygienic conditions, the presence of unsafely disposed syringes, limited availability of sterile syringes and a lack of sterile water for injecting. a number of unsafe injection practices observed were common among public injectors. the presence of police officers nearby and the potential of assault by street predators fostered rushed injecting among those consuming drugs in public spaces. the ecological features of these environments encourage unhygienic and unsafe injecting practices, heighten risk for overdose and the transmission of blood borne viruses. introduction: approximately 13% of women experience depression in the first weeks or months after giving birth. although it has been argued that postpartum depression (ppd) is a culture-bound p~ nomenon, experienced only in western, industrialized countries, recent international research studies have confirmed that the prevalence of ppd is similar around the world. however, little is known about variables that may influence the experience of ppd in women from diverse cultures and immigrant women. research is needed to identify the role culture may play in the presentation of ppd (i.e., types of symptoms most commonly reported), how women from various racial, ethnic and cultural backgrounds perceive and attribute their symptoms of ppd, and finally, how culture can be appropriately addressed in ppd treatment programs. method: to examine the role of culture in ppd, semi-structured interviews and self-report question· naires were administered to clients of the women's health centre of st. joseph's health centre, tor· onto. participants were identified as either first generation canadians (relative newcomen) (n=8) or second generation canadian (parents were immigrants to canada) (n=6). data were collected qd these two groups of women based on: severity of depression, symptom presentation, perceived role of social supports and culturally-specific traditions, and barriers and responses to treatment. the interview data were analyzed using qualitative methods (thematic content analysis), and the following themes were identified: 1) stress about passing on their culture to their children, 2) lack of social support and feelings of isolation, 3) perceived importance or lack of importance of cultural based tra· ditions, 4) conflicts with family members. about cultural traditions and beliefs, 5) mental health stigma within ethnic communities and beyond, 6) race-and sex-based discrimination, and 7) language barriers. conclrllion: identifying the role culture plays in the presentation of ppd, and how women from diverse backgrounds perceive and attribute their symptoms of ppd, is critical in order to establishcultur· ally appropriate guidelines for treatment options. furthermore, information gathered from this study can be used to develop policies and resources for delivering culturally competent care for newcomer wo~ who are dealing with ppd as well as the issues around acculturation (i.e., language barriers, racism). llus is of particular importance for urban health centers which provide postpartum care to diverse groups of women, and particularly recent immigrants or newcomers. ps-~8 (al. contaminated 'therapeutic landscape': perceptions of the aamjiwnaang fint nation keyln smith and isaac luginaah . in~: this. paper pres;ents the findings of an ongoing study among the aamjiwnaang f!rst nanon. aam11wnaang is located m the heart of samia's 'chemical valley', surrounded by cherrucal ~(ants such as esso, imperial oil, shell, suncor energy, and canada's largest hazardous waste disposal sate. safety kleen. this fint nation community is located within the st. clair river •area of concem'. as destgna~ by health canada for the population of samia and the surrounding region. although this comm_umty, by way of. its proximity to the numerous chemical plants is already exposed to high levels pollu~on, recent che?ucal dumping in ~mjiwnaang, as well as a failed proposal for another ethanol plant m the community, only helped to mtensify community concerns about potential health impacts of exposure. research on the cultural beliefs and traditions of first nation communities has established that th~ ~isting rel~tionship between first nations people and 'mother earth' is not only physical, but also_ spmtual. in this study we explore how the complex relationship between the aamjiwnaang first naaon and 'mother earth' be ha · · · i .. may c ngmg 111 a contammated 'therapeutic' landscape. the study a so ~lores h?w aam11wnaang residents are coping with these changes and with the probable conramina· boa of their community. ra!jts: aamjiwnaang residents acknowledge that they are living in a contaminated environment and are being impacted by emissions from the surrounding 'chemical valley' especially for future generations, and have expressed concerns that members of the community and mother earth are 'sick' as a result of this exposure. while moving from that 'place' has been discussed, residents have strong personal and generational connections to the land and insist however, that aamjiwnaang is their 'home' and will remain that way despite growing community concerns about the impact from industry. the contribution of this study lies in the direct involvement of community leadership in designing and conducting this research and distributing the results to further local policy development regarding community concerns on the reserve. background: truck driver are at very high risk for drug abuse because the factor contributing to drug abuse among driver are multiple. it is important to gain an understanding of the key factors that contributing to drug abuse among truck drivers. methods: seventy-truck driver aged 25-35 years were conveniently selected from the population. all the participants were male. an interview schedule was developed in the light of literature data and data were collected by personal interview with informed consent in selected urban area of eastern part of nepal. results: a considerable percentage of truck driver have less knowledge about drug abuse, its effect on body and their complication. majority of the participants (90%) explained that lack of education, financial crisis were leading factors of drug abuse. the average numbers of respondents were believed that lack of meaning in life, marital disharmony. friendship with drug abuser, stress, tiredness, modernization of life pattern and freedom from the home pressure were the influencing risk factors to drug abuse. conchuion: this study reveals those truck drivers are at risk in urban area for developing drugs abuse, mostly influenced by social and environmental factors. hence, these groups should undergo certain educational programme to prevent not only drug abuse, but also prevent transmission of infectious disease among these groups. introduction: food insecurity is an inequality that is central in the lives of many low-income canadians. our group of university researchers, health professionals, and community developers conducted a study in ottawa with community workers who work with people identified as food insecure. the purpose of the study was to understand the nature of their work, and their perspectives, perceptions and experiences of the causes and consequences of food insecurity. (a parallel study was conducted with food insecure participants). methods: sampling was through local knowledge of organizations involved in the provision of food to community members. written permission for the researchers to contact workers in confidence was obtained from each organization. thirteen in-depth interviews were conducted. the interviews consisted of a number of open-ended questions. the data were collated, and analysed using a grounded approach. results: workers interviewed represented organizations that offered diverse programming and methods of food distribution intended to address the needs of the community. the provision of food was seen as a necessary response to address hunger that arose for the majority from poverty caused by low income, or levels of government income support and safety nets that were insufficient to support food requirements. workers reported that food requirements varied according to different. ethnic backgrounds, ages of recipients and family configurations, and with food preferences, and d'.etary (health) requirements. workers' observations of the effects of insufficient food on adults and children such as depression, low energy and non-participation or social disengagement matched those of food insecure respondents. food insecurity was cited as "another srressor among the mountain of problems." overall, workers presented a picture of a silent, stigmatized, poor population including children, and a lack of advocacy to address the issues. be tha th · ·ty the results suggest that workers and organizations, like the commuruty mem rs t ey msecun • · · · · f ilab'l' f food · t d ·n thei·r ab1'l1'ty to address food insecurity. l1m1tanons m terms o ava 11ty o , serve, are restnc e 1 . . funding, and donations appeared to set the para?1eters of pr~g:am r.espon~ to commuruty food msecurity. workers were concerned with program des1g": and a~m1mstranon that m general addr~d hunger on an emergency short-term basis, although food msecunty was long-ter_m for ~any. sustainable solutions that include knowledge translation strategies, and health and social pohcy responses were suggested and will be explored in future research that hopes to build on this pilot. background: socioeconomic deprivation as well as low levels of soc!al s.uppoi:r have .been associated with poor outcomes following cardiac surgery. pre-operative depression m ~anents with coron~ry artery disease awaiting cabg ranges from 27-47% and is an independent predictor of post-operanve mortality. since st. michael's hospital has a large inner city population, this study was designed to determine the both the prevalence of depressive symptomatology (ds) in patients awaiting cabg as well as its relationship with socioeconomic status (ses) and social support. methods: consecutive patients referred for isolated cabg were invited to complete the centre for epidemiological studies depression scale (ces-d) as well as a social support scale and a life stressors inventory. a ces-d score of 2: 16 represents mild ds and a score of 2:27 represents moderate to severe ds. participants also recorded information on functional status, perceived progression of heart failure, demographic and ses variables. all participants with ces-d scores 2: 16 were considered to have ds. results: of the 75 patients (75%) who returned the survey, 22 (29.3%) of participants had a ces-d score 2: 16 with 8 (10.7%) of those having scores suggestive of severe depression. females and those with ongoing medical concerns tended to have more ds (p= 0.06 and p= 0.07 respectively). age was not related to ds. perceived worsening of symptoms and increased ccs angina class were significantly related to the presence of ds (p= 0.03 and p= 0.03 respectively) but not the number of diseased heart vessels. satisfaction with personal relationships including having someone to confide in (p=0.01), feeling lonely (p= 0.04) and not having a partner (p= 0.007) were significantly related to ds. being very upset by a recent breakup in the immediate family was also significantly related to ds (p= 0.02). having greater than a grade 9 education was the only ses variable related to ds (p=0.06). multivariate logistic regression suggested that not being partnered and having low education were the most significant predictors of ds. conclnsion: pre-operative depression is present in at least one quarter of patients referred for cabg. since ds is related to poor outcomes following cabg, single patients with few social supports and low education should be considered at high risk for ds. these findings support the need for the development of supportive interventions in patients at risk in order to decrease post-operative morbidity. this study examines the relationship between childhood sexual abuse, sexual assault during adolescence, and hiv-risk-related behaviours in a sample of 919 homeless youths. over the past decade, there has been increasing research devoted to the impact of childhood sexual abuse. some studies have suggested that the experience of childhood sexual abuse is associated with substance use and abuse, at· risk sexual behaviour and subsequent higher rates of hiv infection (ompad et al., 2005; cinq-mars et al., 2003; brown et al., 1997) . other studies have found that childhood abuse, especially sexual abuse, is c_o~n among stree~ youth (noell et al. 1999 ). this study compares sexually abused males and females livtng on the street ~th non-sexually abused street youth, with regards to unsafe sexual behaviours and e~ures to potmnal t:dv sources (e.g., tattooing, body piercing and injection drug use). the hypothe-sis oft~ ~nt study is that the prevalence of hn-risk-related behaviours will be higher among street youth vtctuns of sexual abuse than those who have not been sexually abused. the sample includes 919 youths aged 13 to 25 who met specific criteria's for itinerancy (649 male and 270 female, with a mean ~of 19.4 y· and a standard deviation of 2.9). they were recruited through five organisations working ~th~ you~ and were~ of an ongoing cohort study. all youth completed a 45 to 60 minute ques· ~onnaue on their sexual behaviours, use of drugs and alcohol and other hiv risk behaviours. oral spec· unens for hiv testing were. also collected. a total of 357 (38.9%) youth reported at least one incident of ~i •~use/assault (1~1 girls (67.0% of all the girls) and 176 boys (27.2% of all the boys]). preliminary descnpnve analyses pomt to sexually abused/assaulted youth showing the highest prevalence of sexual poster sessions v113 at-risk behaviours. for example, 43.3% of sexual abuse/assault victims have engaged in prostitution in their lives compared with 13.4% in the non-sexually abused/assaulted group. in addition, 42.6% of sexual abuse/assault victims reported having injected drugs, compared with 32.0% in the non-sexually abused/assaulted group. more statistical analyses will bring other significant factors to light, especially when we analyze separately those victim of childhood sexual abuse as compare to those victim of sexual assault after childhood. elucidating the risk factors for getting involved in hiv-risk-related behaviours is important for the development of comprehensive and appropriate prevention and treatment intervention strategies. introduction: new immigrants and refugees to canada frequently emigrate from regions of the world where intestinal parasitic infections with worms are endemic. of note, some of these parasites area capable of surviving for years to decades within a given host and can have life threatening consequences many years after initial infection. thus, early diagnosis and treatment of intestinal parasitic worms is considered important in high risk immigrant populations, however there remains a lack of consensus regarding the best method of accomplishing this. diagnosing worm infections through stool examinations is costly and has limited sensitivity, while presumptive treatment of all immigrants, although advocated by some, is expensive and results in healthy individuals being unnecessarily treated. herein, we examine the utility of a hematologic marker (i.e. peripheral blood eosinophilia) as a screening instrument to identify parasitic worm infections in new immigrants to toronto. we identified 192 adults, 15 years of age or older, who were screened for intestinal parasitic infections by stool examination as part of a recently developed screening protocol at a downtown toronto community health centre. we examined the prevalence of infections with worms and subsequently evaluated the impact of several demographic factors on the presence of worm infections. we also determined the sensitivity and specificity of peripheral blood eosinophilia (defined as greater than 500 eosinophils per ml) as a marker for intestinal parasitic worm infections. results: overall, 21 % of the 192 immigrants tested had evidence of at least one worm infection on a single stool examination, while 4% of such individuals bad infections with multiple worms concurrently. age did not appear to be associated with the presence of worms, however 29% of males had evidence of worm infections compared with 16% of females (p= 0.03). immigrants from asia, sub-saharan africa, and central america had the highest burden of overall infection. the sensitivity and specificity of peripheral blood eosinophilia for worm infections was 24% and 92% respectively. conclusions: one in five recent immigrants presenting to a community health centre in downtown toronto had evidence of at least one intestinal parasitic worm infection. the highest burden of illness was in men and in those emigrating from less developed regions of the world. peripheral blood eosinophilia is a poor screening test for worm infections however its presence is highly suggestive of the existence of worms in new immigrants. more than a decade ago, the journal of american college health recommended that a national study be conducted to measure the health status of african american college students, 'both health disparities and [their] positive healthful behaviors' (fennell, 1994) . it was later decided that when exploring the level of health risk behaviors for african american students, gender is an important variable and should be included in the research (fennell, 1997) . at historically black college and universities (hbcus) college health researchers must tackle gender disparities through highly-effective health promotion and disease prevention programs. the purpose of this review is to examine the literature that addresses the overarching health behaviors (i.e. risky sexual behavior, poor diet, smoking, physical inactivity, etc.) of african american men who attend hbcus. despite the various social, cultural, and academic benefits of african american students who attend hbcus, the number of research studies that adequately address their health and health behaviors is limited. studies indicate that african american men who attend hbcus are actively engaged in poor health behaviors. compared to females at hbcus, african american men are more likely to behave in ways that could result in injuries and the use tobacco, alcohol, and ~ther dru~. the city of toronto has recently proposed to conduct a street count of the homeless. like proposals that have come before, this most recent proposal is controversial among many homeless advocates and service providers because it is perceived as intrusive, likely to undercount the target population, and unnecessary for addressing the problem. advocates of the count view it as necessary to gauge the scope of the problem and advise city leaders about how to most effectively direct resources ro.the various homeless services. this paper first reviews the history of efforts to count homeless populat10ns m toronto, describing the motivations and arguments of people on both sides of the issue. second, the paper reviews the methodological complexities of counting homeless populations, describing approaches that use shelter-counts, administrative data and street observations. those interested in counts for toronto and elsewhere can benefit from this review of approaches that have been proposed and carried out in other municipalities. third, the paper proposes a new community-base d strategy that joins the skills of methodologists, local experts, and service providers to ensure a fair and accurate count. the method of systematic social observation avoids some of the problems of early counts by utilizing a repeated identification approach to minimize undercount bias. the method also has the advantage of involving and compensating homeless individuals for assisting with the street count estimates. the estimation approach can be implemented on a rolling basis throughout the year. drug transitions) is a multi-level study aimed at examining the associations between features of the urban environment and mental health, drug use, and risky sexual behaviors. research participants are being recruited in 36 new york city (nyc) neighborhoods. an essential first step in the implementation of this study was to delineate boundaries for each target neighborhood that could then be used to establish sampling frames and as key units of analytic interest. although many neighborhood studies rely on pre-established definitions (e.g. those used for the census or administrative purposes), such definitions do not always reflect contemporary settlement patterns. we felt that street level observations were a neces· sary component of the definition process. methods: the procedures used to delineate neighborhoods were: (1) development of census block maps of targeted areas; (2) review of land use maps and census tract data to ensure, prior to going into the field, tha.t particular blocks are residential and likely to have sufficient population for recruitment purposes; and (3) field vislts and observation in each of the targeted areas. field observations focused on: housing characteristics, including housing type and condition; characteristics of commercial areas, such as likely customer base (e.g. local or no~, socio-economic status and ethnicity of customers); pedestrian volume (including con· gregauons of people m parks and outside stores or residences); obstructions to pedestrian traffic (e.g. ma1or thoroughfares, multi-block industry or institutions); and maintenance and use of open spaces. results: in making the final decisions regarding neighborhood boundaries and the inclusion or exclusion of particular. blocks, we considered a broad range of factors including evidence of homogeneity an~ heterogeneity (socmeconomic, ethnic, housing type, environmental) across blocks within and blocks ad1acent to a nei.ghborhood, and across the 36 sample neighborhoods; the potential for efficient recruit· ment of appropriate particip.ants (i.e. sufficient local pedestrian traffic); and boundaries used in reporting census data (s? that population data can be used in our analysis). . altho~gh utilization of field observations for neighborhood definitions is relatively time consuming (ap~~ox1mately 2 hours for a 12 block area, with more diverse neighborhoods taking even longer), we annc1p~t~ that it will substantially improve the quality and consistency of our data gath· ~rmg ~nd analyse~. spec1f1c e?'amples from neighborhoods defined through this process will be given dur· m~ this presentatmn. we will also discuss the merits and limitations of characterizing neighborhoods usmg street level observations. has been no consideration for how social networks are associated with sse. the objective of this study was to identify personal and social network characteristics associated with being a recipient of sse. in this cross-sectional study, idus who injected in the past 6 months were recruited from syringe exchange programs in montreal, canada, between april 2004 and january 2005. information on each participant and on the persons with whom contact had occurred in the past month were elicited using a structured, interviewer-administe red questionnaire. participants provided detailed demographic and drug use information on up to 5 idu members with whom drugs or injecting materials were shared. using logistic regression, personal and network characteristics were examined in relation to receipt of sterile syringes. res.its: of 277 idus, 39% reported receiving sterile syringes in the past month from another idu. the sample mean age was 33 years (range 18-55), 73% male, 91 % caucasian, and 85% cocaine injectors. in multivariate analyses adjusted for age and gender, recipients of sterile syringes were more likely than non-recipients to share drugs after preparation (0r=2.39(1.06-5.42 )), to require help or to have helped inject another idu (or= 3.48(1.60-7.60)), to have asked someone to get sterile syringes from a syringe exchange program (or=2.54[1.20-5.40 )), to lend syringes (or=2.44[1.16-5.16) ) and to use drug preparation water that was previously used by another iou (0r=2.46(1.42-4.28) ) during the past 6 months. recipients also had a higher rate of change (p=0.01) of idus in their drug injecting network during the past month. with respect to social networks, 45% ( 171/348) of idu network members were providers of sterile syringes and were more likely than non-providers to inject everyday (or= 2.50( 1.51-4.15)). when stratified by their role as a sexual partner or as someone who provides support (e.g. friend, family member), further risk behaviours were observed in relation to sharing injecting materials with the participant. conclusion: recipients of sterile syringes and their idu network members had several high-risk behaviours for infection with bloodborne diseases. sse may afford an opportunity for the dissemination of other salutary behaviours such as information sharing on safe injecting practices between recipients and distributors. furthermore, drug injecting networks may be an avenue to reach idus who may not otherwise be exposed to preventive measures. there are numerous sources of stigma and labelling of mental and physical illness. first, the sociocultural dimension to stigma and labelling clearly influences patients, families, health care professionals and society's perception and treatment of illness. this creates multiple challenges related to illness identification and recovery, particularly in culturally diverse societies. second, the media is a major source of stigma and stereotyping with regard to a variety of mental and physical illnesses. despite increased public knowledge of many illnesses, studies have shown that stigma has intensified over the years in certain cases. a third element of stigma and labels appears with the use of diagnosis as a solution to human problems with the result that difficult to treat patients are repudiated and social issues are unaddressed. this presentation will serve to outline the development of stigma, various expressions of stigma, and consequences of stigma. it will provide some practical recommendations for the reduction of stigma related to mental and physical illness. purpose: to assess the knowledge, attitude, and practice about immunization for parent of 2-3 years old hispanic and african american children in los angeles county (lac). methods: we conducted two cluster surveys in lac. the sample was selected with probability proportionate to estimated size at the first stage and simple random sample of children at the second stage. data were collected through interview. we absuacted vaccine coverage data from immunization record card (irc) at home or from clinic records. the participation was 81 % in hispanics and 83% in african americans populations. irc was not available at home for 26% of african american and 16% of hispanic children. results: all parents perceived their children got all necessary vaccine~. fa~orable attitudes to.w~rd immunization were reported by 82 % of african american and 90% of h1s?amc parents. '.he m~1ority of the parents (90% african american and 95% hispanic) agreed that vaccines prevent senous d1se~ses among children. regarding vaccine safety, significantly more hispanic parents (91 %) than african american parents (75%) mentioned that vaccines are safe. introduction: cervical cancer has been shown to be preventable by papanicolau tests in heterosexual women; however, the utility of papanicolau test in lesbians is controversial. cervical cancer is caused by the human papillomavirus (hpv) which is transmitted by sexual _intercourse; howev_er, its' transmission by homosexual intercourse is also controversial. the goal of this study was to review the evidence for papanicolau tests in lesbians. methods: a pub med, ovid ( 1996 ovid ( to 2005 , and cochrane library search was performed using the mesh headings "homosexuality , female," "vaginal smears," and "papillomavirus, human." in pub med, the clinical queries feature was used with "diagnosis" and "broad, sensitive" filters. a google advanced search of the internet was conducted with the terms "lesbian," "cervical cancer," and "pap rest." the url was limited to ".ca," ".edu," or ".gov." results: the search yielded 8 original articles and 4 reviews, but no cochrane reviews. none of the publications were canadian. 111 website hits were found and selected websites were reviewed. studies showed that lesbians have fewer papanicolau tests than heterosexual women; however, many reported risk factors for cervical cancer, including multiple past or current sexual partners (male and female), early age of first coitus, history of sexually transmitted diseases, and cigarette smoking. hpv has been detected in 13-30% of lesbians and 6-19% of lesbians who have never had sex with men. case studies of abnormal papanicolau tests in lesbians who have never had sex with men have also been reponed. sexual transmission of hpv among lesbians can be explained by several factors: 1) 53-99% of lesbians have had sex with men and 21-30% of lesbians continue to have sex with men. 2) hpv may be transmitted by sexual behaviours among women, like digital-vaginal sex, digital-anal sex, oral sex, and the use of insertive sex toys. 3) hpv transmission requires only skin-to-skin contact. genital hpv has been identified on human fingers. some professional organizations, like the society of obstetricians and gynecologists of canada and the american cancer society, recognize the need for papanicolau tests in lesbians. other organizations, like the canadian cancer society, do not have official policy statements. the current ontario cervical screening program does not specifically address lesbians. conclusion: although the data was limited, most studies recommended that lesbians should receive papanicolau tests. the frequency of these tests was controversial. policy makers and professional organizations should address the needs of this special population and make specific recommendations . monique chaaya, ahia sibai, hiam chemaitelly, and zana el roueiheb . literature concerning the mental and physical effect of work at an old age is contradictory. in addinon, urban environments are physically and socially harsh with reduced potential for income generation and employment and increased potential for depression. the present paper is an attempt to study how do work, or lack of work, link to the experience of depression among older adults in underprivileged lebanese urban communities. the data comes from a cross-sectional survey, where 740 elderly residing in 3 underprivileged communities in beirut, including a palestinian refugee camp, were successfully inter-v1ewed through face-to-face interviews. logistic regression was performed to assess the effect of work on depression ad_justing for many other covariates. data showed that 17.5% of people aged 60 years and more were still workmg. there was a highly protective effect of work on depression in elderly males (or= 0.342, 95% cl= 0.128-0.909). the current study is the first of its kind in the arab region and more work shou_ld be _don_e in this area. it is important for the lebanese government to consider elderly rights for social mclus10n m terms of employment opportunities. amam nuru-jeter, nancy adler, and burton singer . l~u~on:_ the socioeconomic gradient is perhaps the most persistent and significant finding in social epidem1ological research. the insistent nature of the ses effect is evidenced by its significance after acc?untmg for ~umerous confounds. the significance and magnitude of the effect, however, varies by ::•al group. evidence_ o_f the relative effect of race and ses is inconclusive. further, less attention has f ~ ptid to the specific interactions of race and ses than to examinations of which is a better predictor 0 d ~ th. ~sues are further complicated by the explanatory complexity of the various measures of ses anl . owht ey relate to the health of various social groups. understanding the specific nature of these re a11ons ips 1s necessary for guiding he ith d · i 1· · h a an soc1a po mes and programs aimed at improving t e poster sessions v117 health of our most disadvantaged groups, and therefore population health, overall. this study examines the synergistic effects of ses with both race and gender in predicting group differences in mortality. methods: analyses use data from the national longitudinal mortality survey (n= 559, 715) for the years 1979, 1980, and 1981-85; and were linked to the national death index for deaths occurring through 1985. results were confirmed using data from the national mortality followback survey for people who died in 1986 (n= 13,491) and the 1986 national health interview survey (denominator). we used correlation analysis and the standardized mortality ratio to examine ses (education and income), race, and gender as predictors of group differences in changes in mortality. results: for the total population, analyses support the income gradient showing significant declines in mortality ratios from low to high income along the income strata. education shows a significant drop in mortality with completion of high school and completion of college, exhibiting a threshold rather than a gradient effect. compared to whites, blacks receive diminishing health returns for each subsequent level of income and education; with whites closely mirroring the total population in ses thresholds. similarly, compared to men, women do not experience the same health gains at comparable ses levels. for education, black men and women only show significant mortality declines upon completion of high school. the ses gradient operates differently among race and gender subgroups. threshold effects suggest resource gains in life opportunities at particular milestones along the ses gradient. further, ses matters less for blacks and women compared to whites and men suggesting differing implications for health and social policy aimed at reducing disparities. objective: we assessed the impact of diabetes in a large puerto rican community of chicago by measuring the prevalence of diagnosed diabetes and calculating the diabetes mortality rate. methods: we analyzed data from a comprehensive health survey conducted in randomly selected households in community areas. questions on diagnosed diabetes and selected risk factors were asked. in addition, mortality data were analyzed in order to calculate the age-adjusted diabetes mortality rate. when possible, rates were compared to those found in other studies. results: the diabetes prevalence located in this community (20.8%: 95% cl= 10.1 %-38.0%) is one of the highest ever reported for puerto ricans. for instance, it is more than three times higher than the prevalence for the us (6.1 %) and twice the prevalence for puerto ricans in new york ( 11.3%) and puerto rico (9.3%-9.6%). diagnosed diabetes was found to be significantly associated with obesity (p=0.023). the prevalence was particularly high among older people, females, those horn in the us, and those with a family history. the diabetes mortality rate (67.6 per 100,000 population) was more than twice the rate for all of chicago (31.2) and the us (25.4). conclusions: understanding why the diabetes and mortality rates for puerto ricans in this commu· nity are so much higher than those of other communities is imperative. collaboration between researchers, service providers and community members can help address the issues of diabetes education, early screening and diagnosis and effective treatment needed in this community. introduction: sars is a respiratory illness that is spread from person to person through dose contact. this infectious disease outbreak was unusual due to the high rate of infection among health care workers. the aim of the study is to explore the demographic and attitudinal determinants of psychological distress due to sars among health care workers of a toronto hospital. methods: a total of 997 adult participants completed questionnaires co assess psychological distress (impact of event scale -jes) and attitudes to sars. of these, 845 participants completed the questionnaire during the sars i outbreak, and 152 participants completed the questionnaire during the sars ii outbreak. participants also provided information on demographic variables, including occupation and exposure to sars. principal components analysis was used to derive eight attitudinal scales: fear: system, protection, prevention, job stress, stigma, instrumental coping, and psychological copm~. l111ear regression analyses were applied to evaluate the associations of the demographic and amtudmal variables, as well as time, with psychological distress. . . regression analyses showed that time and higher scores on fear, 1ob stress, stigma'. and instrumental coping were associated with a higher total ies score: (r2 =.30). contrary to expectations, none of the demographic variables was associated with total ies score. conclusion: the presence of psychological distress in health care workers is indicative of intrusive or avoidant responses to thoughts, feelings, and memories associated with the sars outbreak. this study shows that ( 1) fear for one's health and the he~lth of.others, (2) ex~erien~in~ job stress, (3)_tbe per· ception of stigma, (4) usage of instrumental copmg skills, ~n~ (5) t1m_e s1grufi~an~ly c~nmb~te to increased psychological distress in health care workers. potential mterventmns during mfecttous diseaie outbreaks in health care settings should be targeted to minimize the impact of these factors. purpose: the purpose of this study was to expand knowledge regarding feeding practices, know!· edge and nutritional beliefs of mothers currently residing in the dominican republic (dr). the rising incidence of obesity in children is a major national health concern and obesity in first and second-generation immigrant children also continues to rise. we know that parents (particularly mothers) shape children's early diet patterns and that immigrant mothers experience additional challenges related to acculturation and assimilation into a new culture, however there is a paucity of literature concerning the effect of immigrant adaptation to american culture on the nutrition of children. in addition, new immigrant knowledge about the causes of obesity and the resulting health implications for childhood obesity has not been assessed and reported. this qualitative study used focus group methodology to discuss views and practices related to the introduction of food for infants and feeding practices for young children, along with a discussion on knowledge and beliefs related to the causes and health implications of child· hood obesity. ten dominican mothers' of young children (birth to 6 years old) who reside in a small town in the western frontier area of the dr were participants in the focus group. results from this srudy will be compared to results from an identical study that will be conducted in fall 2005 with new immi· grant mothers from the dr to the boston area. we will compare feeding practices, beliefs and knowledge about nutrition for young children between these two groups. methods: using participatory principles, a focus group design was used to interview a group of mothers in the dr. the investigator along with a bilingual translator conducted the focus group. the session was tape recorded and is currently in the process of being transcribed and translated. transcribed data will be systematically analyzed themes will be identified. a qualitative data analysis software will be used to organize and code the data according to the specific aims of the study. supporting quotations will be selected to substrate each theme. lmpli~tions for urban health practice: an ultimate goal of this study is to lay a foundation for understanding the process of acculturation on feeding practices of mothers when they immigrate to the us. understanding beliefs, knowledge and feeding practices that mothers use with young children will help m ~he ~evelopment of culturally and linguistically appropriate educational strategies and materials for use m primary prevention of pediatric obesity. in canada more than seventy-five percent of immigrants choose to stay in three major urban centers of toronto, montreal and vancouver. approximately fifty percent of the immigrants eventually set· tie m ~~e greater tor<_>nto area. there is mounting evidence that the increasing immigrant population has a_ sigmfic~nt health disadvantage over canadian-born residents. this health disadvantage manifests particularly m the ma1"ority of 1"mm1"gr t h h d be · · h . . . . an s w o a en m canada for longer than ten years. this group as ~n associ~te~ with higher risk of chronic disease such as cardiovascular diseases. this disparity twccb n ma1onty of the immigrant population and the canadian-born population is of great importance to ur an health providers d" · i i · b as isproporttonate y arge immigrant population has settled in the ma1or ur an centers. generally the health stat f · · · · · · h h been . us 0 most 1mm1grants 1s dynamic. recent 1mm1grants w o av_e ant •;ffca~ada _for less ~han ~en years are known to have a health advantage known as 'healthy imm1• ~ants r::r · ~:s eff~ 1~ defined by the observed superior health of both male and female recent immiimmigrant participation in canadian society particularly the labour market. a new explanation of the loss of 'healthy immigrant effect' is given with the help of additional factors. lt appears that the effects of social exclusion from the labour market leading to social inequalities first experienced by recent immigrant has been responsible for the loss of healthy immigrant effect. this loss results in the subsequent health disadvantage observed in the older immigrant population. a study on patients perspectives regarding tuberculosis treatment by s.j.chander, community health cell, bangalore, india. introduction: the national tuberculosis control programme was in place over three decades; still tuberculosis control remains a challenge unmet. every day about 1440 people die of tuberculosis in india. tuberculosis affects the poor more and the poor seek help from more than one place due to various reasons. this adversely affects the treatment outcome and the patient's pocket. many tuberculosis patients become non-adherence to treatment due to many reasons. the goal of the study was to understand the patient's perspective regarding tuberculois treatment provided by the bangalore city corporation. (bmc) under the rntcp (revised national tuberculosis control programme) using dots (directly observed treatment, short course) approach. bmc were identified. the information was collected using an in-depth interview technique. they were both male and female aged between 4-70 years suffering from pulmonary and extra pulmonary tuberculosis. all patients were from the poor socio economic background. results: most patients who first sought help from private practitioners were not diagnosed and treated correctly. they sought help form them as they were easily accessible and available but they. most patients sought help later than four weeks as they lacked awareness. a few of patients sought help from traditional healers and magicians, as it did not help they turned to allopathic practitioners. the patients interviewed were inadequately informed about various aspect of the disease due to fear of stigma. the patient's family members were generally supportive during the treatment period there was no report of negative attitude of neighbours who were aware of tuberculosis patients instead sympathetic attitude was reported. there exists many myth and misconception associated with marriage and sexual relationship while one suffers from tuberculosis. patients who visited referral hospitals reported that money was demanded for providing services. most patients had to borrow money for treatment. patients want health centres to be clean and be opened on time. they don't like the staff shouting at them to cover their mouth while coughing. conclusion: community education would lead to seek help early and to take preventive measures. adequate patient education would remove all myth and conception and help the patients adhere to treatment. since tb thrives among the poor, poverty eradiation measures need to be given more emphasis. mere treatment approach would not help control tuberculosis. lntrod#ction: the main causative factor in cervical cancer is the presence of oncogenic human papillomavitus (hpv). several factors have been identified in the acquisition of hpv infection and cervical cancer and include early coitarche, large number of lifetime sexual partners, tobacco smoking, poor diet, and concomitant sexually transmitted diseases. it is known that street youth are at much higher risk for these factors and are therefore at higher risk of acquiring hpv infection and cervical cancer. thus, we endeavoured to determine the prevalence of oncogenic hpv infection, and pap test abnormalities, in street youth. ~tbods: this quantitative study uses data collected from a non governmental, not for profit dropin centre for street youth in canada. over one hundred females between the ages of sixteen and twentyfour were enrolled in the study. of these females, all underwent pap testing about those with a previous history of an abnormal pap test, or an abnormal-appearing cervix on clinical examination, underwent hpv-deoxyribonucleic (dna) testing with the digene hybrid capture ii. results: data analysis is underway. the following results will be presented: 1) number of positive hpv-dna results, 2) pap test results in this group, 3) recommended follow-up. . the results of this study will provide information about the prevalence of oncogemc hpv-dna infection and pap test abnormalities in a population of street youth. the practice implic~ tions related to our research include the potential for improved gynecologic care of street youth. in addition, our recommendations on the usefulness of hpv testing in this population will be addressed. methods: a health promotion and disease prevention tool was developed over a period of several years to meet the health needs of recent immigrants and refugees seen at access alliance multicultural community health centre (aamchc), an inner city community health centre in downtown toronto. this instrument was derived from the anecdotal experience of health care providers, a review of medical literature, and con· sultations with experts in migration health. herein we present the individual components of this instrument, aimed at promoting health and preventing disease in new immigrants and refugees to toronto. results: the health promotion and disease prevention tool for immigrants focuses on three primary health related areas: 1) globally important infectious diseases including tuberculosis (tb), hiv/aids, syphilis, viral hepatitis, intestinal parasites, and vaccine preventable diseases (vpd), 2) cancers caused by infectious diseases or those endemic to developing regions of the world, and 3) mental illnesses includiog those developing among survivors of torture. the health needs of new immigrants and refugees are complex, heterogeneous, and ohen reflect conditions found in the immigrant's country of origin. ideally, the management of all new immigrants should be adapted to their experiences prior to migration, however the scale and complexity of this strategy prohibits its general use by healthcare providers in industrialized countries. an immigrant specific disease prevention instrument could help quickly identify and potentially prevent the spread of dangerous infectious diseases, detect cancers at earlier stages of development, and inform health care providers and decision makers about the most effective and efficient strategies to prevent serious illness in new immigrants and refugees. lntrodmction: as poverty continues to grip pakistan, the number of urban street children grows and has now reached alarming proportions, demanding far greater action than presently offered. urbanization, natural catastrophe, drought, disease, war and internal conflict, economic breakdown causing unemployment, and homelessness have forced families and children in search of a "better life," often putting children at risk of abuse and exploitation. objectives: to reduce drug use on the streets in particular injectable drug use and to prevent the transmission of stds/hiv/aids among vulnerable youth. methodology: baseline study and situation assessment of health problems particularly hiv and stds among street children of quetta, pakistan. the program launched a peer education program, including: awareness o_f self and body protection focusing on child sexual abuse, stds/hiv/aids , life skills, gender and sexual rights awareness, preventive health measures, and care at work. it also opened care and counseling center for these working and street children ar.d handed these centers over to local communities. relationships among aids-related knowledge and bt:liefs and sexual behavior of young adults were determined. rea.sons for unsafe sex included: misconception about disease etiology, conflicting cultural values, risk demal, partner pressur~, trust and partner significance, accusation of promiscuity, lack of community endorsement of protecnve measures, and barriers to condom access. in addition socio-economic pressure, physiological issues, poor community participation and anitudes and low ~ducation level limited the effectiveness of existing aids prevention education. according to 'the baseline study the male children are ex~ to ~owledge of safe sex through peers, hakims, and blue films. working children found sexual mfor~anon through older children and their teachers (ustad). recommendation s: it was found that working children are highly vulnerable to stds/hiv/aids, as they lack protective meas":res in sexual abuse and are unaware of safe sexual practices. conclusion: non-fatal overdose was a common occurrence for idu in vancouver, and was associated with several factors considered including crystal methamphetamine use. these findings indicate a need for structural interventions that seek to modify the social and contextual risks for overdose, increased access to treatment programs, and trials of novel interventions such as take-home naloxone programs. background: injection drug users (idus) are at elevated risk for involvement in the criminal justice system due to possession of illicit drugs and participation in drug sales or markets. the criminalization of drug use may produce significant social, economic and health consequences for urban poor drug users. injection-related risks have also been associated with criminal justice involvement or risk of such involvement. previous research has identified racial differences in drug-related arrests and incarceration in the general population. we assess whether criminal justice system involvement differs by race/ethnicity among a community sample of idus. we analyzed data collected from idus (n = 1,084) who were recruited in san francisco, and interviewed and tested for hiv. criminal justice system involvement was measured by arrest, incarceration, drug felony, and loss/denial of social services associated with the possession of a drug felony. multivariate analyses compared measures of criminal justice involvement and race/ethnicity after adjusting for socio-demographic and drug-use behaviors including drug preference, years of injection drug use, injection frequency, age, housing status, and gender. the six-month prevalence of arrest was highest for whites (32%), compared to african americans (25%) and latinos (27% ), in addition to the mean number of weeks spent in jail in the past 6 months (7.0 vs. 5.8 and 4.2 weeks). these differences did not remain statistically significant in multivariate analyses. latinos reported the highest prevalence of a lifetime drug felony conviction (48%) and mean years of lifetime incarceration in prison (13.3 years), compared to african americans (48%, 10.7 years) and whites (34%, 6.9 years). being african american was independently associated with having a felony conviction and years of incarceration in prison as compared to whites. the history of involvement in the criminal justice system is widespread in this sample. when looking at racial/ethnic differences over a lifetime including total years of incarceration and drug felony conviction, the involvement of african americans in the criminal justice system is higher as compared to whites. more rigorous examination of these data and others on how criminal justice involvement varies by race, as well as the implications for the health and well-being of idus, is warranted. homelessness is a major social concern that has great im~act on th~se living.in urban commu?ities. metro manila, the capital of the philippines is a highly urbanized ar~ w.1t~ the h1gh~st concentration of urban poor population-an estimated 752,229 families or 3,005,857 md1v1duals. this exploratory study v122 is the first definitive study done in manila that explores the needs and concerns of street dwdlent\omc. less. it aims to establish the demographic profile, lifestyle patterns and needs of the streetdwdlersindit six districts city of manila to establish a database for planning health and other related interventions. based on protocol-guid ed field interviews of 462 street dwellers, the data is useful as a template for ref!!. ence in analyzing urban homelessness in asian developing country contexts. results of the study show that generally, the state of homelessness reflects a feeling of discontent, disenfranchisem ent and pow!!· lessness that contribute to their difficulty in getting out of the streets. the perceived problems andlar dangers in living on the streets are generally associated with their exposure to extreme weather condirioll! and their status of being vagrants making them prone to harassment by the police. the health needs of the street dweller respondents established in this study indicate that the existing health related servias for the homeless poor is ineffective. the street dweller respondents have little or no access to social and health services, if any. some respondents claimed that although they were able to get service from heallh centers or government hospitals, the medicines required for treatment are not usually free and are beyond their means. this group of homeless people needs well-planned interventions to hdp them improve their current situations and support their daily living. the expressed social needs of the sucet dweller respondents were significantly concentrated on the economic aspect, which is, having a perma· nent source of income to afford food, shelter, clothing and education. these reflect the street dweller' s need for personal upliftment and safety. in short, most of their expressed need is a combination of socioeconomic resources that would provide long-term options that are better than the choice of living on the streets. the suggested interventions based on the findings will be discussed. . methods: idu~ aged i 8 and older who injected drugs within the prior month were recruited in 2005 usmg rds which relies on referral networks to generate unbiased prevalence estimates. a diverse and mon· vated g~o~p of idu "seeds." were given three uniquely coded coupons and encouraged to refer up to three other ehgibl~ idu~, for which they received $5 usd per recruit. all subjects provided informed consent, an anonymous 1~t erv1ew and a venous blood sample for serologic testing of hiv, hcv and syphilis anti~!· results. a total of 213 idus were recruited in tijuana and 206 in juarez, of whom the maion!)' were .male < 9 .l.4% and 92.2%) and median age was 34. melhotls: using the data from a multi-site survey on health and well being of a random sample of older chinese in seven canadian cities, this paper examined the effects of size of the chinese community and the health status of the aging chinese. the sample (n=2,272) consisted of aging chinese aged 55 years and older. physical and mental status of the participants was measured by a chinese version medical outcome study short form sf-36. one-way analysis of variance and post-hoc scheffe test were used to test the differences in health status between the participants residing in cities representing three different sizes of the chinese community. regression analysis was also used to examine the contribution of size of the chinese community to physical and mental health status. rmdts: in general, aging chinese who resided in cities with a smaller chinese population were healthier than those who resided in cities with a larger chinese population. the size of the chinese community was significant in predicting both physical and mental health status of the participants. the findings also indicated the potential underlying effects of the variations in country of origin, access barriers, and socio-economic status of the aging chinese in communities with different chinese population size. the study concluded that size of an ethnic community affected the health status of the aging population from the same ethnic community. the intra-group diversity within the aging chinese identified in this study helped to demonstrate the different socio-cultural and structural challenges facing the aging population in different urban settings. urban health and demographic surveillance system, which is implemented by the african population & health research center (aphrc) in two slum settlements of nairobi city. this study focuses on common child illnesses including diarrhea, fever, cough, common cold and malaria, as well as on curative health care service utilization. measures of ses were created using information collected at the household level. other variables of interest included are maternal demographic and cultural factors, and child characteristics. statistical methods appropriate for clustered data were used to identify correlates of child morbidity. preliminary ratdts: morbidity was reported for 1,087 (16.1 %) out of 6,756 children accounting for a total of 2,691 illness episodes. cough, diarrhoea, runny nose/common cold, abdominal pains, malaria and fever made up the top six forms of morbidity. the only factors that had a significant associ· ation with morbidity were the child's age, ethnicity and type of toilet facility. however, all measures of socioeconomic status (mother's education, socioeconomic status, and mother's work status) had a significant effect on seeking outside care. age of child, severity of illness, type of illness and survival of father and mother were also significantly associated with seeking health care outside home. the results of this study have highlighted the need to address environmental conditions, basic amenities, and livelihood circumstances to improve child health in poor communities. the fact that socioeconomic indicators did not have a significant effect on prevalence of morbidity but were significant for health seeking behavior, indicate that while economic resources may have limited effect in preventing child illnesses when children are living in poor environmental conditions, being enlightened and having greater economic resources would mitigate the impact of the poor environmental conditions and reduce child mortality through better treatment of sick children. inequality in human life chances is about the most visible character of the third world urban space. f.conomic variability and social efficiency have often been fingered to justify such inequalities. within this separation households exist that share similar characteristics and are found to inhabit a given spatial unit of the 'city. the residential geography of cities in the third world is thus characterized by native areas whose core is made up of deteriorated slum property, poor living conditions and a decayed environment; features which personify deprivation in its unimaginable ma~t~de. there are .eviden~es that these conditions are manifested through disturbingly high levels of morbidity and mortality. ban · h h d-and a host of other factors (corrupt10n, msens1t1ve leaders 1p, poor ur 1ty on t e one an , . · 1 f · 1 · · th t ) that suggest cracks in the levels and adherence to the prmc1p es o socta 1usnce. ese governance, e c . . . . . ps £factors combine to reinforce the impacts of depnvat10n and perpetuate these unpacts. by 1den· grou o . ·1 "id . . bothh tifying health problems that are caused or driven by either matena _or soc1a e~nvanon or , t e paper concludes that deprivation need not be accepted as a way. of hfe a~d a deliberate effon must be made to stem the tide of the on going levels of abject poverty m the third world. to the extent that income related poverty is about the most important of all ramifications of po~erty, efforts n_iu_st include fiscal empowerment of the poor in deprived areas like the inner c~ty. this will ~p~ove ~he willingness of such people to use facilities of care because they are able to effectively demand 1t, smce m real sense there is no such thing as free medical services. ). there were 322 men with hiv-infection included in the present study (mean age and education of 41.8 (sd=8.4) and 13.9 (sd=2.7), respectively). a series of multiple regressions were used to examine the unique contributions of symptom burden (depression, cognitive, pain, fatigue), neuropsychologic al impairment (psychomotor efficiency), demographics (age and education) and hiv disease (cdc-93 staging) on iirs total score and jirs subscores: ( 1) activities of daily living (work, recreation, diet, health, finances); (2) psychosocial functioning (e.g., self-expression, community involvement); and (3) intimacy (sex life and relationship with partner). resnlts: total iirs score (r 2 "0.43) was associated with aids diagnosis (ii= 0.11, p <0.01) and symptoms of pain (ii= -0.14, p < 0.01 ), fatigue (ji = -0.34, p < 0.001) and cognitive difficulties (p =0.30, p < 0.001 ). for the three dimensions of the iirs, multiple regression results revealed: ( 1) activities of daily living (r2=0.42) were associated with aids diagnosis (ii =0.17, p < 0.01) and symptoms of pain <p =-0j6, p < 0.01 ), fatigue (ji = -0.31, p < 0.001) and cognitive difficulties (ji =0.32, p < 0.001 ); (2) psychosocial functioning (r2=0.31) was associated with was associated with symptoms of fatigue (ii= -0.25, p <0.001) and cognitive difficulties <ii =0.19, p <0.001); and (3) intimacy (r2=0.17) was related to was associated with symptoms of fatigue (ij = -0.17, p < 0.01) and cognitive difficulties (ii= 0.19, p < 0.001 ). methods: the sif evaluation methodology involves a comprehensive database located at the sif, a randomly selected p~o.spective coh~rt of sif users, and two pre-existing external control cohorts. . . ~ts: in addmon to reporting process data and descriptions of the sif users, we report on data indicating that the establishment of the sif has been independently associated with reductions in public ~':°g ~ (p <?.001), and syringe sharing (aor =0.3[95%ci: 0.1 _ 0.7); p =0.013) and other unsafe m1ecnon pract1_ces_ (p ~ 0.010), and increased uptake of detox services (log-rank p =0.017). as well, we report on da~ md1cat1ng that the sif has not prompted adverse changes in community drug use patterns. the vancouver sif has been well accepted by the target population, and while adve~ events s~c~ as overdoses have occurred, these events have been successfully managed. externally compiled data indicate that the sif has been associated with substantial declines in public disorder !'oster sessions v125 associated with injection drug use, syringe sharing, and increased uptake of detox services. as well, the establishment of the sif has not exacerbated community drug use patterns. ongoing evaluation activities wiu involve assessing the impact of the sif on a variety of outcomes, including infectious disease transmission, fatal overdose, and utilization of health and social services. city, brazil, 1996 -2002 maria cristina almeida, waleska caiaffa, renato assum;iio, and fernando proietti dengue cases were described according to time, space, intensity, age, gender, onset of symptoms, residence address and census tract. spatial distribution of two groups (children and women over 64 years old and men aged 20-59) were compared, under assumption that they have distinct behaviors regarding their displacement around the city. analysis included local moran index, ripley\\'s k function and recurrence of census areas over time. about 99,559 cases were identified in seven epidemic waves. concentration of cases in small areas, followed by a spread either in space or time suggested endemic patterns. regarding age-gender groups, distinct patterns suggested that residence might not be a good proxy in determining the local of infection for men aged 20-59. dengue is shifting to endemic pattern in this city and transmission may not be sole related to home environment, suggesting that additional spatial information must be couected aiming efficient control measures. murukan kandamuthan and subodh kandamuthan lnl7uduction: illness or disablement of a child may affect the economic functioning of a family as it alters the employment pattern and earnings of parents this paper tries to assess the health status and the quality of life of disabled children, and how generally, severe disablement in a child affected their fami-lies\' finances, and to quantify any such effects the problems faced by children in their home and to provide information relevant to the formation of criteria for new or increased cash support. methods: a case-control study was done in the thiruvananthapuram district, capital city of kerala by selecting a random sample of 300 families with severely disabled children below 15 years and a random sample of 300 normal children matched for age and occupation of the parents. comparative and subjective data were collected. money spent on children\'s medical care, loss of earnings of the parents, and other economic burden to the family due to the disability of the child in the family. a discriminant analysis along with univariate analysis was done to assess the factors that mostly differentiate the disabled and normal children. the mean expenditure of the families with a disabled child was rs. 852/-per month, which is significantly higher than the corresponding expenditure of rs. 389/-per month of families with normal child, (t= 16.86, p <.00001). of the disabled children, 81 % were not getting any social security payments and 90% had no special concessions for medical and other educational purposes. the analysis of income and expenditure pattern indicated that financial impact of disablement in a child on the family is significant. the discriminant model results revealed that medical expenditure was a significant variable that differentiated the disabled and normal child. the study found that the health status and quality of life of the disabled children were far from satisfactory. this in fact affected the economic status of the disabled children. in addition to reduced earnings, there are extra costs for disabled children for travel, domestic help, medical care, and health care expenditures (hospital care, physician services, dentistry, drugs and others) for disabled individuals. a strong case can be made for their long-term care by improving the financial support to such families possibly through the introduction of an allowance specially to compensate for the earnings lost by parents due to the disability of their children. widows in india are considered disadvantage group of population. because they are characterized by pangs of separation from spouse, and consequently they go through mental agony, social ~s?lation, and economic dislocation. in addition to these, poverty, landlessness, homelessness, malnutr1non etc. endure serious deterioration of their health. according to census 2001, there are 44 million women are widowed and one fourth live in urban areas. there is a noticeable increase of urban widows from the previous census (almost half). the paper examines the type of disability and chronic ailment borne among elderly widows (60 years and above). fifty second round of national sample survey aske~ the individuals three sets of questions regarding their health: their status of health, whether they are physically poster sessions v126 immobile, and whether or not they have had any specific chronic illnesses. although health infrastruc-1 1 ·1 ble 1 ·n urban setting in india as compared to rural counterpart, prevalence of tures are arge y ava1 a . . . . . h · ·11 · h h"gher 1 "n urban areas analysis shows 1omt problems m old age qmte common c romc 1 ness is muc 1 • . . and nearly 44 percent elderly widows are suffering from this ail'.11ent in urban areas. one-fifth of widows are suffering from high/low bp. heart disease, diabetes and urinary problems are much ~ore pre~alent in urban areas though disabilities is comparatively lower in urban areas. however, the d~fference is not statistically significant. disability with respect to vision is more pronounced and one third_ of the total respondents have problems regarding eyesight. one fifths ofdderly widows are _ha.rd of hearing. though disability increases with age, similar trend is not observed with respect to chr~mc illness. the percepnon of self-health among elderly becomes poorer as their age increases. the perceived health status need not be always corrected to objective indicators of health. it is noted that around 27 per~en~ of those perceived their health as "excellenr" or "very good" are found to be suffering from chrome disease of1omts and 17 percent have visual disability. to conclude an elderly widow living in urban areas have similar health problems as she jives in rural areas. this can be easily explained, as they were not made aware to seek treatment from health facility. in addition, the study shows that the economic conditions are deterrent factors for their illness. it is essential that intervention should be taken up improving their economic conditions so that wellbeing of this most vulnerable group of the society can be taken care of. heart failure is a disease that affects nearly 15 million people world wide. the united states alone accounts for one third of this population. blacks are stricken with heart failure twice as often as whites. men are more likely to develop chf than women, however since the majority of the chf population are seniors, there are actually more women than men. congestive heart failure (chf) has become a pandemic. as the baby boomer generation ages, the number of chf cases will rise dramatically. (young, j. & mills, r.,2004). after the age of 45, each proceeding decade doubles the incidence level of chf. the average person has a 1 in 5 chance of developing heart failure within their lifespan according to the framingham study (nih.gov). individuals aging 65 or older compose over half of the entire documented heart failure population. in this age group, chf is the leading cause of hospitalization. many patients are continuously readmitted. it is estimated that the chf population will increase by one million within the next 25 years. mortality was at 50% for a two year period and 70% for five years (young, j. & mills, r.,2004 ). data from: aha as technology evolves, the understanding of the nature of heart failure has become clearer. in the beginning of the 20th century heart failure was diagnosed as a condition that presented with edema (swelling) in the extremities caused by water retention. treatment in the beginning of the past cenrury was limited to abdominal drainage and diuretics. the discovery of the heart's insufficient pumping ability in heart failure patients lead to new surgeries and devices such as heart transplants and ventricular assist devices (bridge until transplant is available). ventricular hypertrophy often occurs before the development of heart failure. hypertrophy is the term given for cardiac remodeling. cardiac remodeling also includes chamber dilation (young, j. & mills, r.,2004) . chf financial burden the us healthcare financing administration has ranked chf as the highest cost illness in the diagnose-related area. direct costs related to chf in the us for 2004 were nearly $26 billion. these costs included hospital admissions, physician fees, home health aids and medications. indirect costs were over $2 billion in 2004. loss of jobs or death due to chf was the criteria for this category (young, j. & mills, r., 2004). . introduction: the injection drug user quality of life scale (iduqol) is a relatively new scale ~es1gned to capture the unique and individual circumstances that determine quality of life among injection drug users (idus). the 20 life areas comprising the instrument were based on the research literature an~ ~ocus group discussions with idus. the purpose of the present study was to evaluate the content validity of t.h~ iduqol using judgmental methods based on subject matter experts' (smes) ratings. content vah~1ty refers to the de~ee to which elements of an assessment tool are representative of the construct of interest and appropnate for a given population. methods: data were obtained from six smes, from the field of idu research and practice, who ~ere. asked t.o evaluate various aspects of the iduqol, including the title of the instrument, administraon instruchons, clarity of the names and descriptions of each life area, response format, scoring instruc-no~s and examples, record form and whether each life area should be included in the instrument. sme ratings were made using either 3 or 4 point scales. sm es were also given the opportunity to comment on poster sessions v127 each section and to suggest whether important life areas had been overlooked, were redundant, not relevant, or in need of revision. (cvi) and the ~v.erage ~eviation index (ad) were used to evaluate smes' agreement on ratings of the content vahd1ty vanables. both measures provided support for the content validity of various aspects of the iduqol, although results from the stricter ad index noted some areas of disagreement, including the description of particular life areas (e.g., being useful, drugs, sex), the inclusion of some life areas (e.g., drug treatment, education, independence and free choice), and the ease of the scoring instructions. the findings from this study provide (a) content validity evidence to support the use of the iduqol as well as (b) recommendations to suengthen both the instrument (e.g., improved descriptions for some items) and the accompanying administration and scoring manual (e.g., an easier scoring template). changes made to the instrument and its manual make the iduqol even easier for researchers, practitioners, and program evaluators to use as a way of assessing, and tracking changes over time or as a result of interventions in, quality of life in injection drug users. introduction: strict adherence to prescribed regimens is required to derive maximal benefit from highly active antireuoviral therapy (haart) in persons living with hiv/aids (phas). adherence is a key determinant of the degree and durability of viral suppression. adherence is also essential in reducing the spread of hiv and ensuring that today's treatments remain effective for as long as possible. the objective of this study is to conduct a systematic review of the research literature on the effectiveness of education and patient support strategies for improving adherence to haart. methods: a systematic search of the core databases was performed from january 1996 until may 2005. randomized controlled trials examining the effectiveness of education and patient support interventions to improve the adherence to haart were considered for inclusion. only those studies that measured adherence at a minimum of six weeks were included. study selection, quality assessments, and data abstraction were performed independently by two reviewers. results: study heterogeneity with respect to differing populations, interventions, outcomes, and length of follow-up did not allow for meta-analysis. we included 19 studies involving 2, 159 ph as. sample sizes ranged from 22 to 367. study duration ranged from a single session to a variable number of sessions delivered over one year. many of the interventions involved the provision of an educational component to improve adherence and often addressed strategies to overcome barriers to adherence and the development of problem-solving skills. they ranged from simple interventions (e.g., the provision of reminder devices) to complex interventions (e.g., cognitive-behavioural therapy delivered by licensed psychologists). eleven of the 19 studies demonstrated a statistically significant advantage associated with the described adherence intervention. the advantage associated with adherence outcomes does not seem to translate into the more clinically relevant outcome of viral suppression. only 4 out of 12 studies that reported virological or immunological results found a significant effect associated with the intervention. the studies had several methodological shortcomings. conclusions: there is a need for standardization and methodological rigour in the conduct of adherence trials. some interventions may have a significant impact on improving adherence. further research is required before any specific adherence improving strategy can confidently be incorporated into standard clinical practice. focus groups were used to collect data from a purposive sample of treatmentexperienced participants. focus group data were analyzed using a grou~ded t~eory appr~ach. i:ne themes identified from the interviews were used to identify the effects of ant1rerrov1rals on quality of hfe. the content of the mos-hiv was appraised against the themes identified from our analysis. participants also completed the mos-hiv survey and were asked whether the survey covered all important aspects of quality of life on antiretroviral medications. results: five key informant interviews and five focus groups with 38 participants were used to identify effects on quality of life that are not directly ~aptu~ed by_ the mos-hiv health survey. our~~ showed that our participants described quality of hfe as mvol~mg_ a set of ~rsonal trad~ffs ~stay alive. in most cases, worsened quality of life was traded-off for gams 1~ longevlty from ~~canon use. these eff~'ts or tradeoffs included: ( 1) downstream consequences of side effects and toxlanes; (2) loss of lrufe. pende~t decision-making privileges, (3) having to choose drugs over ~areer; (~)burden of medication-taking responsibilities; and (5) living life under a pretense and havmg to hide-the net effect of the tradeoffs, or "prices" to pay led to what was described as "longevity with hn without hope and future~. conclusion: our study highlights five major themes summarizing the impact of haart on quality on life, and suggests that currently used scales for measuring quality of life do not a~atd~ cap~e these findings and the associated consequences. the conceptual framework for measuring quality of bfe in hiv should be reconsidered in order to better capture the important effects of the medications on quality of life. this may involve widening the boundaries of the definition of health-related quality oflife to include aspects such as finances, employment, and housing. we should further consider the development of a haart adverse effect specific instrument, using a broad definition of adverse effect in order to capture all types of adverse impacts of hiv treatments on quality of life. introduction: measles (rubeola) is the most contagious vaccine preventable disease of humans. while cases of measles are uncommon in canada today, the disease continues to be a leading cause of morbidity and death in the developing world. as a result of human migration, measles cases still occur in canada, primari~ among immigrants, returning travelers, and other susceptible groups. canada's national advisory council on immunizations (naci) recommends that immigrants without records of measles immunization be considered for vac:cination since these individuals may not have been appropriately vaccinated in their native country. on rhe other hand, natural immunity to measles in immigrants is likely to be high given the high incidence of disease in developing parts of rhe world. thus it remains unclear if the most efficient strategy to achieve high levels of measles immunity in immigrants should entail initial serologic screening followed by vaccination of susceptible individuals or preemptive vaccination of all persons lacking immunization records. understanding the epidemiology of measles immunity in immigrant populations could help guide such decisions. . methods: we identified 527 adults, 15 years of age or older, who were screened for serologtc immunity to measles as part of a recently developed screening protocol at a downtown toronto community health centre. we subsequently determined if these individuals had any immunization records from their native country or from within canada. we then examined the relationship between several demographic factors and immunity to measles. results: 93% of immigrants had no prior immunization records. overall, 93% of the 527 immigrants rested for immunity to measles were found to be immune; 88% of those under the age of 20, 93% of those between 20 and 39 years of age, 99% of those between 40 and 59 years of age, and 100% of those 60 years of age or older. gender, education status, household income, and immigration status were nor ~ssoc1ated wirh immunity to measles. immunity to measles was lowest among immigrants from eastern europe (8~%), whil~ immigrants from other regions of the globe had greater than 90% immunity. c~lusrons: immigrants and refugees appear to have reasonably high levels of immunity to mea· sics, possibly related to natural infection wirh the measles virus. immigrants from eastern europe appear to have slightly. lower l~vel~ of immunity to the measles virus. universal screening for immunity to meales or preemptive vac:cmauon may both be inefficient public health strategies, however further research is needed to corroborate these findings. nancy r~ss, stephane tremblay, saeeda khan, daniel crouse, mark tremblay, and jean-marie berrhelor o~ve: the speed of the rise in obesity in places like canada suggests that rather than a shift in the gcncnc_ com~sirion of the population, the root of the obesity pandemic is an environment that suprrs ~besity. this paper examines the influence of neighbourhood and metropolitan characteristics. bour~lts:. while accounting. for individual socio-demographics and behaviours, residents of neighs with a large proportmn of poorly educated individuals had higher bmis than those living in poster sessions v129 neighbourhoods with more highly educated individuals (p <.01 ). residing in a neighbourhood with a high proportion of recent immigrants was associated with lower bmi for men (p<.01), but not women. neighbourhood dwelling density, a proxy for walkability, was not associated with bm! for either sex. metropolitan sprawl was associated with higher male bmi (p=.02) but the effect was negligible for women (p=.09). bmi was significantly lower for women (p<.01) living in a city in the province of quebec, even after accounting for the influence of individual and neighbourhood covariates. conclusions: bm! was strongly patterned by individual-level social position in urban canada. the incremental influence on bmi of neighbourhood related to the neighbourhood's social conditions and not to their physical form. metropolitan sprawl was associated with higher bm! for men, a group with longer car-dependent commutes than women. the findings suggest that both individuals and their environments (both social and physical) influence the distribution of bmi in urban populations. introduction: urban environments have been linked to a range of human health issues. in singapore, prevalence of end stage renal disease (esrd) is predicted to rise (2633 in 1999 to 6000 in 2010 , kidney international, vol.67, 2005 . the clinical and economic burden of esrd has promoted development of strategies aimed at preventing the development and progression of chronic kidney disease. these include population based screening programs such as the national kidney foundation, sin-gapore\'s (nkfs) "partnership for prevention."the program, aims to reduce incidence of esrd through comprehensive intervention and screening for early detection of urinary abnormalities, blood pressure (bp), and random blood glucose (rbg). objective of this study is to examine gender, race and age wise prevalence of elevated bp, rbg and proteinuria. methodology: this current analysis includes 575,438 subjects, age 18 to 86 years, who underwent screening during september 1999 and december 2004. participants were asked to give demographic information and the history of diseases. tests were given to get clinical information such as proteinuria, blood glucose, sbp, and dbp. blood pressure abnormalities were defined according to ]nc vi criteria. proteinuria was defined as the presence of 1 plus or greater protein (equivalent to> 30 mg/di) on dipstick analysis. results: there were 296, 116 (51.5%) males. racial distribution was chinese (78.8% ), malay (8.8% ), indians (8. 7%) and others (3. 7% ).among participants, who were apparently "healthy" (asymptomatic and without history of dm, ht, or kd), gender and race wise % prevalence of elevated (bp> 140/90), rbg (> 140 mg/di) and positive urine dipstick for protein was as follows male: (20.5;6.9; 3.5) female:(13.6;5.0;3. 2) chinese:( 17.1;6.0;3) malay: (19.4;7.3;5.6) indian:( 15.9;7.5;3.0) others: (15.4;4.5;2.9) total:(l 7.1, 6.1,3.2). percentage of participants with more than one abnormality were as follows. those with bp> 140/90mmhg, 14% also had rbg> 140mg/dl and 6.4% had proteinuria> i. those with rbg> 140mgldl, 11 % also had proteinuria> 1 and 35% had bp> 140/90mmhg. those with proteinuria> 1, 18% also had rbg> 140mg/dl, and 38% had bp> 140/90mmhg. conclusion: we conclude that sub clinical abnormalities in urinalysis, bp and rbg readings are prevalent across all genders and racial groups in the adult population. the overlap of abnormalities, point towards the high risk for esrd as well as cardiovascular disease. this indicates the urgent need for population based programs aimed at creating awareness, and initiatives to control and retard progression of disease. introduction: various theories have been proposed that link differential psychological vulnerability to health outcomes, including developmental theories about attachment, separation, and the formation of psychopathology. research in the area of psychosomatic medicine suggests an association between attachment style and physical illness, with stress as a mediator. there are two main hypotheses explored in the present study: ( t) that individuals living with hiv who were upsychologically vulne~able" at study entry would be more likely to experience symptoms of depression, anxiety and phys1ca! illness over. the course of the 9-month study period; and (2) life stressors and social support would mediate the relat10nship between psychological vulnerability and the psychological ~nd physical outcomes. . (rsles), state-trait anxiety inventory (stai), beck depr~ssi~n lnvento~ (bdi), and~ _21-item pbys~i symptoms inventory. we characterized participants as havmg psychological vulnerability and low resilience" as scoring above 35 on the raas (insecure attachment) or above 120 on the das (negative expectations about oneself). . . . . . " . . ,, . results: at baseline, 55% of parnc1pants were classified as havmg low resilience. focusmg on anxiety, the average cumulative stai score of the low-resilience group was significandy hi~e~ than that of the high-resilience group ( 18.45 sd= 10.6 versus 9.57 sd= 8.6; f(l,80)= 16.74, p <.001). similar results were obtained for bdi and physical symptoms (f( 1,80)= 14.65, p<.001 and f( 1,80)= 5.50, p<.05, respec· tively). after controlling for resilience, the effects of variance in life stres".°rs averaged over time wa~ a_sig· nificant predictor of depressive and physical symptoms, but not of anxiety. ho~e_ver, these assooan~s became non-significant when four participants with high values were removed. s1id1larly, after controlling for resilience, the effects of variance in social support averaged over time became insignificant. conclusion: not only did "low resilience" predict poor psychological and physical outcomes, it was also predictive of life events and social support; that is, individuals who were low in resilience were more likely to experience more life events and poorer social support than individuals who were resilient. for individuals with vulnerability to physical, psychological, and social outcomes, there is need to develop and test interventions to improve health outcomes in this group. rajat kapoor, ruby gupta, and jugal kishore introduction: young people in india represent almost one-fourth of the total population. they face significant risks related to sexual and reproductive health. many lack the information and skills neces· sary to make informed sexual and reproductive health choices. objective: to study the level of awareness about contraceptives among youth residing in urban and rural areas of delhi. method: a sample of 211 youths was selected from barwala (rural; n= 112) and balmiki basti (urban slums; n= 99) the field practice areas of the department of community medicine, maulana azad medical college, in delhi. a pre-tested questionnaire was used to collect the information. when/(calen· dar time), by 2, fisher exact and t were appliedxwhom (authors?). statistical tests such as as appropriate. result: nearly 9 out of 10 (89.1 %) youth had heard of at least one type of contraceptive and majority (81.5%) had heard about condoms. however, awareness regarding usage of contraceptives was as low as 9.4% for terminal methods to 39.3% for condom. condom was the best technique before and after marriage and also after childbirth. the difference in rural and urban groups was statistically signif· icant (p=.0001, give confidence interval too, if you provide the exact p value). youth knew that contra· ceptives were easily available (81 %), mainly at dispensary (68.7%) and chemist shops (65.4%). only 6.6% knew about emergency contraception. only advantage of contraceptives cited was population con· trol (42.6%); however, 3.8% believed that they could also control hiv transmission. awareness of side effects was poor among both the groups but the differences were statistically significant for pills (p=0.003). media was the main source of information (65%). majority of youth was willing to discuss a~ut contraceptive with their spouse (83.4%), but not with others. 51.2% youth believed that people in their age group use contraceptives. 35% of youth accepted that they had used contraceptives at least once. 81 % felt 2 children in family is appropriate, but only 59.7% believed in 3 year spacing. . conclusion: awareness about contraceptives is vital for youth to protect their sexual and reproduc· tive health .. knowledge about terminal methods, emergency contraception, and side effects of various contraceptives need to be strengthened in mass media and contraceptive awareness campaigns. mdbot:ls: 740 elderly aged 60+ were interviewed in 3 poor communities in beirut the capital of f:ebanon, ~e of which is a palestinia~. refugee camp. depression was assessed using the i 5-item geriat· nc depressi~n score (~l?s-15). specific q~estions relating to the 3 aspects of religiosity were asked as well as questions perta1rung to demographic, psychosocial and health-related variables. results: depression was prevalent in 24% of the interviewed elderly with the highest proportion being in the palestinian refugee camp (31 %). mosque attendance significantly reduced the odds of being depressed only for the palestinian respondents. depression was further associated, in particular communities, with low satisfaction with income, functional disability, and illness during last year. condiuion: religious practice, which was only related to depression among the refugee population, is discussed as more of an indicator of social cohesion, solidarity than an aspect of religiosity. furthermore, it has been suggested that minority groups rely on religious stratagems to cope with their pain more than other groups. implications of findings are discussed with particular relevance to the populations studied. nearly thirty percent of india's population lives in urban areas. the outcome of urbanization has resulted in rapid growth of urban slums. in a mega-city chennai, the slum populations (25.6 percent) face greater health hazards due to overcrowding, poor sanitation, lack of access to safe drinking water and environmental pollution. amongst the slum population the health of women and children are most neglected, resulting in burden of both communicable and non-communicable diseases. the focus of the paper is to present the epidemiology profile of children (below 14 years) in slums of chennai, their health status, hygiene and nutritional factors, the social response to health, the trends in child health and urbanization over a decade, the health accessibility factors, the role of gender in health care and assessment impact of health education to children. the available data prove that child health in slums is worse than rural areas. though the slum population is decreasing there is a need to explore the program intervention and carry out surveys for collecting data on some specific health implications of the slum children. objective: during the summer of 2003 there was a heat wave in central europe, producing an excess number of deaths in many countries including spain. the city of barcelona was one of the places in spain where temperatures often surpassed the excess heat threshold related with an increase in mortality. the objective of the study was to determine whether the excess of mortality which occurred in barcelona was dependent on age, gender or educational level, important but often neglected dimensions of heat wave-related studies. methods: barcelona, the second largest city in spain (1,582,738 inhabitants in 2003) , is located on the north eastern coast. we included all deaths of residents of barcelona older than 20 years that occurred in the city during the months of june, july and august of 2003 and also during the same months during the 5 preceding years. all the analyses were performed for each sex separately. the daily number of deaths in the year 2003 was compared with the mean daily number of deaths for the period 1998-2002 for each educational level. poisson regression models were fitted to obtain the rr of death in 2003 with respect to the period 1998-2002 for each educational level and age group. results: the excess of mortality during that summer was more important for women than for men and among older ages. although the increase was observed in all educational groups, in some age-groups the increase was larger for people with less than primary education. for example, for women in the group aged 65-74, the rr of dying for 2003 compared to 1998-2002 for women with no education was 1.30 (95%ci: 1.04-1-63) and for women with primary education or higher was 1.19 (95%ci: 0.90-1.56). when we consider the number of excess deaths, for total mortality (>=20 years) the excess numbers were higher for those with no education ( 17 5. 7 for women and 46. 7 for men) and those with less than primary education (112.5 for women and 11-2 for men) than those with more than primary edm:ation (75.0 for women and -10.3 for men). conclusion: age, gender and educational level were important in the 2003 barcelona heat wave. it is necessary to implement response plans to reduce heat morbidity and mortality. policies should he addressed to all population but also focusing particularly to the oldest population of low educational level. introduction: recently there has been much public discourse on homelessness and its imp~ct on health. measures have intensified to get people off the street into permanent housing. for maximum v132 poster sessions success it is important to first determine the needs of those to be housed. their views on housing and support requirements have to be considered, as th~y ar~ the ones affected. as few res.earch studies mclude the perspectives of homeless people themselves, httle is known on ho~ they e~penence the 1mpacrs on their health and what kinds of supports they believe they need to obtain housing and stay housed. the purpose of this study was to add the perspectives of homeless people to the discourse, based in the assumption that they are the experts on their own situations and needs. housing is seen as a major deter· minant of health. the research questions were: what are the effects of homelessness on health? what kind of supports are needed for homeless people to get off the street? both questions sought the views of homeless individuals on these issues. methods: this study is qualitative, descriptive, exploratory. semi-structured interviews were conducted with homeless persons on street corners, in parks and drop-ins. subsequently a thematic analysis was carried out on the data. results: the findings show that individuals' experiences of homelessness deeply affect their health. apart from physical impacts all talked about how their emotional health and self-esteem are affected. the system itself, rather than being useful, was often perceived as disabling and dehumanizing, resulting in hopelessness and resignation to life on the street. neither welfare nor minimum wage jobs are sufficient to live and pay rent. educational upgrading and job training, rather than enforced idleness, are desired by most initially. in general, the longer persons were homeless, the more they fell into patterned cycles of shelter /street life, temporary employment /unemployment, sometimes addictions and often unsuccessful housing episodes. conclusions: participants believe that resources should be put into training and education for acquisition of job skills and confidence to avoid homelessness or minimize its duration. to afford housing low-income people and welfare recipients need subsidies. early interventions, 'housing first', more humane and efficient processes for negotiating the welfare system, respectful treatment by service providers and some extra financial support in crisis initially, were suggested as helpful for avoiding homelessness altogether or helping most homeless people to leave the street. this study is a national homelessness initiative funded analysis examining the experiences and perceptions of street youth vis-a-vis their health/wellness status. through in-depth interviews with 140 street youth in halifax, montreal, toronto, calgary, ottawa and vancouver, this paper explores healthy and not-so healthy practices of young people living on the street. qualitative interviews with 45 health/ social service providers complement the analysis. more specifically, the investigation uncovers how street youth understand health and wellness; how they define good and bad health; and their experiences in accessing diverse health services. findings suggest that living on the street impacts physical, emotional and spiritual well·being, leading to cycles of despair, anger and helplessness. the majority of street youth services act as "brokers" for young people who desire health care services yet refuse to approach formal heal~h care organizational structures. as such, this study also provides case examples of promising youth services across canada who are emerging as critical spaces for street youth to heal from the ravages of ~treet cultur~. as young people increasingly make up a substantial proportion of the homeless population in canada, it becomes urgent to explore the multiple ways in which we can support them to regain a sense of wellbeing and "citizenship." p5-77 (c) health and livelihood implications of marginalization of slum dwellers in provision of water and sanitation services in nairobi city elizabeth kimani, eliya zulu, and chi-chi undie . ~ntrodfldion: un-habitat estimates that 70% of urban residents in kenya live in slums; yet due to their illegal status, they are not provided with basic services such as water sanitation and health care. ~nseque~tly, the services are provided by vendors who typically provide' poor services at exorbitant prices .. this paper investigates how the inequality in provision of basic services affects health and livelihood circumstances of the poor residents of nairobi slums . . methods: this study uses qualitative and quantitative data collected through the ongoing longitudmal .health and demographic study conducted by the african population and health research center m slum communities in n ·rob" w d · · · · ai 1. e use escnpnve analytical and qualitative techmques to assess h~w concerns relating to water supply and environmental sanitation services rank among the c~mmumty's general and health needs/concerns, and how this context affect their health and livelihood circumstances. results: water (32%) and sanitation (20%) were the most commonly reported health needs and also key among general needs (after unemployment) among slum dwellers. water and sanitation services are mainly provided by exploitative vendors who operate without any regulatory mechanism and charge exorbitantly for their poor services. for instance slum residents pay about 8 times more for water than non-slum households. water supply is irregular and residents often go for a week without water; prices are hiked and hygiene is compromised during such shortages. most houses do not have toilets and residents have to use commercial toilets or adopt unorthodox means such as disposing of their excreta in the nearby bushes or plastic bags that they throw in the open. as a direct result of the poor environmental conditions and inaccessible health services, slum residents are not only sicker, they are also less likely to utilise health services and consequently, more likely to die than non-slum residents. for instance, the prevalence of diarrhoea among children in the slums was 31 % compared to 13 % in nairobi as a whole and 17% in rural areas, while under-five mortality rates were 151/1000, 62/1000 and 113/1000 respectively. the results demonstrate the need for change in governments' policies that deprive the rapidly expanding urban poor population of basic services and regulatory mechanisms that would protect them from exploitation. the poor environmental sanitation and lack of basic services compound slum residents' poverty since they pay much more for the relatively poor services than their non-slum counterparts, and also increase their vulnerability to infectious diseases and mortality. since 1991 iepas've been working in harm reduction becoming the pioneer in latin america that brought this methodology for brazil. nowadays the main goal is to expand this strategy in the region and strive to change the drug policy in brazil. in this way harm reduction: health and citizenship program work in two areas to promote the citizenship of !du and for people living with hiv/aids offering law assistance for this population and outreach work for needle exchange to reduce damages and dissemination of hiv/aids/hepatit is. the methodology used in outreach work is peer education, needle exchange, condoms and folders distribution to reduce damages and the dissemination of diseases like hiv/aids/hepatitis besides counseling to search for basic health and rights are activities in this program. law attendance for the target population at iepas headquarters every week in order to provide law assistance that includes only supply people with correct law information or file a lawsuit. presentations in harm reduction and drug policy to expand these subjects for police chiefs and governmental in the last year attended 150 !du and 403 nidu reached and 26.364 needles and syringes exchanged. in law assistance 740 (420 people living with aids, 247 drug users, 43 inject drug users, 30 were not in profile) people attended. 492 lawsuits filed 218 lawsuits in current activity. broadcasting of the harm reduction strategies by the press helps to move the public opinion, gather supporters and diminish controversies regarding such actions. a majority number of police officer doesn't know the existence of this policy. it's still polemic discuss this subject in this part of population. women remain one of the most under seviced segments of the nigerian populationand a focus on their health and other needs is of special importance.the singular focus of the nigerian family welfare program is mostly on demographic targets by seeking to increase contraceptive prevalence.this has meant the neglect of many areas of of women's reproductive health. reproductive health is affected by a variety of socio-cultural and biological factors on on e hand and the quality of the service delivery system and its responsiveness on the other.a woman's based approach is one which responds to the needs of the adult woman and adolescent girls in a culturally sensitive manner.women's unequal access to resources including health care is well known in nigeria in which stark gender disparities are a reality .maternal health activities are unbalanced,focusi ng on immunisation and provision of iron and folic acid,rather than on sustained care of women or on the detection and referral of high risk cases. a cross-sectional study of a municipal government -owned hospitalfrom each of the 6 geo-political regions in igeria was carried out (atotal of 6 ce~ters) .. as _part ~f t~e re.search, the h~spital records were uesd as a background in addition to a 3-week mtens1ve mvesuganon m the obstemc and gynecology departments. . . . : little is known for example of the extent of gynecological morbtdtty among women; the little known suggest that teh majority suffer from one or more reproductive tr~ct infect~ons. although abortion is widespread, it continues to be performed under ilegal and unsafe condmons. with the growing v134 poster sessions hiv pandemic, while high riskgroups such ascomn;iercial sex workers and their clients have been studied, little has been accomplished in the large populat10ns, and particularly among women, regardmgstd an hiv education. . . conclusions: programs of various governmentalor non-governmental agen,c1es to mvolve strategies to broaden the narrow focus of services, and more importan~, to put wo~en s reproducnve health services and information needs in the forefront are urgently required. there is a n~d to reonent commuication and education activities to incorprate a wider interpretation of reproducnve health, to focus on the varying information needs of women, men, and youth and to the media most suitable to convey information to these diverse groups on reproductive health. introduction: it is estimated that there are 250-300 youths living on the streets, on their own with the assistance of social services or in poverty with a parent in ottawa. this population is under-serviced in many areas including health care. many of these adolescents are uncomfortable or unable to access the health care system through conventional methods and have been treated in walk-in clinics and emergency rooms without ongoing follow up. in march 2004, the ontario government provided the ct lamont institute with a grant to open an interdisciplinary and teaching medical/dental clinic for street youth in a drop-in center in downtown ottawa. bringing 5 community organizations together to provide primary medical care and dental hygiene to the streetyouths of ottawa ages 12-20, it is staffed by a family physician, family medicine residents, a nurse practitioner, 2 public health nurses, a dental hygienist, dental hygiene students and a chiropodist who link to social services already provided at the centre including housing, life skills programs and counselling. project objectives: 1. to improve the health of high risk youth by providing accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. 2. to model and teach interdisciplinary adolescent care to undergraduate medical students, family medicine residents and dental hygiene students. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative:a) semi-structured interviews b) focus groups with youth quantitative:a) electronic medical records for 12 months b) records (budget, photos, project information). results: in progress-results from first 12 months available in august 2005. early results suggest that locating the clinic in a safe and familiar environment is a key factor in attracting the over 130 youths the clinic has seen to date. other findings include the prevalence of preventative interventions including vaccinations, std testing and prenatal care. the poster presentation will present these and other impacts that the clinic has had on the health of the youth in the first year of the study. conclusions: 1) the clinic has improved the health of ottawa streetyouth and will continue beyond the initial pilot project phase. 2) this project demonstrates that with strong community partnerships, it is possible meet make healthcare more accessible for urban youth. right to health care campaign by s.j.chander, community health cell, bangalore, india. introduction: the people's health movement in india launched a campaign known as 'right to health care' during the silver jubilee year of the alma ata declaration of 'health for all' by 2000 ad in collahoration with the national human rights commission (nhrc). the aim of the campaign was to establish the 'right to health care' as a basic human right and to address structural deficiencies in the pubic health care system and unregulated private sector . . methods: as part of the campaign a public hearing was organized in a slum in bangalore. former chairman of the nhrc chaired the hearing panel, consisting of a senior health official and other eminent people in the city. detailed documentation of individual case studies on 'denial of access to health care' in different parts of the city was carried out using a specific format. the focus was on cases where denial of health services has led to loss of life, physical damage or severe financial losses to the patient. results: _fourte_en people, except one who had accessed a private clinic, presented their testimonies of their experiences m accessing the public health care services in government health centres. all the people, e_xcept_ one person who spontaneously shared her testimony, were identified by the organizations worki_ng with the slum dwellers. corruption and ill treatment were the main issues of concern to the people. five of the fourteen testimonies presented resulted in death due to negligence. the public health cen· n:s not only demand money for the supposedly free services but also ill-treats them with verbal abuse. five of these fourteen case studies were presented before the national human right commission. the poster sessions v135 nhrc has asked the government health officials to look into the cases that were presented and to rectify the anomalies in the system. as a result of the public hearing held in the slum, the nhrc identified urban health as one of key areas for focus during the national public hearing. cond#sion: a campaign is necessary to check the corrupted public health care system and a covetous private health care system. it helps people to understand the structure and functioning of public health care system and to assert their right to assess heath care. the public hearings or people's tribunals held during the campaign are an instrument in making the public health system accountable. ps-82 (a) violence among women who inject drugs nadia fairbairn, jo-anne stoltz, evan wood, kathy li, julio montaner, and thomas kerr background/object ives: violence is a major cause of morbidity and mortality among women living in urban settings. though it is widely recognized that violence is endemic to inner-city illicit drug markets, little is known about violence experienced by women injection drug users (!du). therefore, the present analyses were conducted to evaluate the prevalence of, and characteristics associated with, experiencing violence among a cohort of female idu in vancouver. methods: we evaluated factors associated with violence among female participants enrolled in the vancouver injection drug user study (vidus) using univariate analyses. we also examined self-reported relationships with the perpetrator of the attack and the nature of the violent attack. results: of the 346 active iou followed between december 1, 2003 and may 6, 2005, 73 (21.1 %) had experienced violence during the last six months. variables positively associated with experiencing violence included: homelessness (or= 3.46, 95% ci: 1.66-7.21, p < 0.01), public injecting (or= 3.45, 95% ci: 1.43 -8.35, p < 0.01 ), frequent crack use (or= 2.99, 95% ci: 1. 72 -5.17, p < 0.01 ), recent incarceration (or =2.81, 95% cl: 1.38 -5.72, p < 0.01), receiving help injecting (or =2.77, 95% cl: 1.54-5.00, p < 0.01 ), shooting gallery attendance (or =2.46, 95% ci: 1.22 -4.93, p < 0.01 ), sex trade work (or =2.30, 95% cl: 1.35 -3.93, p < 0.01 ), frequent heroin injection (or= 1.96, 95% cl: 1.13 -3.40, p < 0.02), and residence in the downtown eastside (odds ratio [or] = 1.85, 95% ci: 1.09 -3.13, p < 0.02). variables negatively associated with experiencing violence included: being married or common-law (or =0.47. 95% ci: 0.25 -0.87, p < 0.02) and being in methadone treatment (or =0.53, 95% ci: 0.31 -0.91, p < 0.02). the most common perpetrators of the attack were acquaintances (48.0%), strangers (27.4%), police (9.6%), or dealers (8.2%). attacks were most frequently in the form of beatings (65.8%), robberies (21.9%), and assault with a weapon (13.7%). conclusion: violence was a common experience among women !du in this cohort. being the victim of violence was associated with various factors, including homelessness and public injecting. these findings indicate the need for targeted prevention and support services, such as supportive housing programs and safer injection facilities, for women iou. introduction: although research on determinants of tobacco use among arab youth has been carried out at several ecologic levels, such research has included conceptual models and has compared the two different types of tobacco that are most commonly used among the lebanese youth, namely cigarette and argileh. this study uses the ecological model to investigate differences between the genders as related to the determinants of both cigarette and argileh use among youth. methodology: quantitative data was collected from youth in economically disadvantaged urban communities in beirut, the capital of lebanon. results: the results indicated that there are differences by gender at a variety of ecological levels of influence on smoking behavior. for cigarettes, gender differences were found in knowledge, peer, family, and community influences. for argileh, gender differences were found at the peer, family, and community l.evels. the differential prevalence of cigarette and argileh smoking between boys and girls 1s therefore understandable and partially explained by the variation in the interpersonal and community envi.ronment which surrounds them. interventions therefore need to be tailored to the specific needs of boys and girls. introduction: the objective of this study was to assess the relationship between parents' employment status and children' health among professional immigrant families in vancouver. our target communmes v136 poster sessions included immigrants from five ethnicity groups: south korean, indian, chine~e, ~ussian, and irani~ with professional degrees (i.e., mds, lawyers, engineers, ma?~ger~, and uru~ers1ty professors) w11h no relevant job to their professions and those who had been hvmg m the studied area at least for 36 months. methodology: the participants were recruited by collaboration from three local community agencies and were interviewed individually during the fall of 2004. ra#lts: totally, 109 complete interviews were analyzed: 33 from south-east asia, 59 from south asia, 17 from russia and other eastern europe. overall, 14.5% were employed, 38.5% were underemployed, 46% indicated they were unemployed. overall, 58.5% were not satisfied with their current job. russians and other eastern europeans were most likely satisfied with their current job, while south-east asians were most satisfied from their life in canada. about 53% indicated that their spouses were not satisfied with their life in canada, while 55% believed that their children are very satisfied from their life in canada. in addition, around 30% said they were not satisfied from their family relationship in canada. while most of the responders ranked their own and their spouses' health status as either poor or very poor, jut 3% indicated that their first child's health was very poor. in most cases they ranked their children's health as excellent or very good. the results of this pilot study show that there is a need to create culturally specific child health and behavioral scales when conducting research in immigrant communities. for instance, in many asian cultures, it is customary for a parent either to praise their children profusely, or to condemn them. this cultural practice, called "saving face," can affect research results, as it might have affected the present study. necessary steps, therefore, are needed to revise the current standard health and behavioral scales for further studies by developing a new scale that is more relevant and culturally sensitive to the targeted immigrant families. metboda: database: 2003 national health survey (ministry of health www.msc.es). two thousand interviews were performed among madrid population (0.04% of the whole); 593 corresponded to older adults (0.04% of the 1. 7 million aged 50 years and over). study sample constitutes 95.3% (565 out of 593) of those older adults, who live in urban areas. demographic structure (by age and gender) of this population in relation to health services use (medical consultations, dentist visits, emergence services, hospitalisation) was studied using general linear model univariate procedure. a p0.005), while age was associated with emergence services use (26% of the population: 21 %, 28% and 45% of each age group) and hos~italisation (17% .oft~~ population: 13%, 20% and 31%, of each age ~oup) (p0.005) was fou~d with respect to dennst v1s1ts (18% vs 20%), medical consultations (29% vs 36%), and emergence services use (26% vs 26%), while an association (p= 0.005) was found according to hospitalisation (20% vs 16%). age. an~ g~der interaction effect on health services use was not found (p> 0.005), but a trend towards bosp1tal1sanon (p=0.04) could be considered. concl.uions: demographic structure of urban older adults is associated with two of the four health se~ices use studi~. a relation.ship ber_ween age. and hospital services use (emergence units and hospitalisanon), but not with ~ut-hosp1tal sei:vices (medical and dentist consultations), was found. in addition ro age, gender also contnbutes to explam hospitalisation. . sexua experiences. we exammed the prevalence expenences 10 relation to ethnic origin and other sociodemographic variables as wc1i as y1j7 die relation between unwanted sexual experiences, depression and agreuion. we did so for boys and prts separately. mdhods: data on unwanted sexual expcric:nces, depressive symptoms (ce.s-d), aggrc:uion (bohi-di and sociodemographic facron were collected by self-report quescionnairc:s administettd to 35 31 students in the: 2nd grade (aged 12-16) of secondary schools in amsterdam, the netherlands. data on the nature ol unwanted sexual experiences were collected during penonal interviews by trained schoolnursn. ltaijtj: overall prevalences of unwanted sexual experiences for boys and girls were 6.5% and 5.7% respectively. unwanted sexual experiences were more often ttported by turkish ( 17.1 %), moroc· an (10.4%) and surinamese/anrillian boys (7.4%) than by dutch boys (2.2%). moroccan and turkish girls, however, reported fewer unwanted sexual experiences (respectively 2.3 and 2.7%) than durch girls did (6.9%). depressive symptoms(or=4.6, cl=3.1-7.0) covert agression (0r•4.9, cl•3.2-7.7) and cmrt aggression (or= 2.6, cl• 1.6-4.4) were more common in girls with an unwanted sexual experi· met. boys with an unwanted sexual experience reported more depressive symptoms (or= 2.2; cl• i . .l· 3.9) and oven agression (or= 1.5, cl= 1.0-2.4) . of the reported unwanted sexual experiences rnpec· timy 17.5% and 73.5% were confirmed by male and female adolescents during a personal interview. cond11sion: we ..:an conclude that the prevalence of unwanted sexual experiences among turkish and moroccan boys is disturbing. it is possible that unwanted sexual experiences are more reported hy boys who belong to a religion or culture where the virginity of girls is a maner of family honour and talking about sexuality is taboo. more boys than girls did not confirm their initial disdosurc of an lllwalltc:d sexual experience. the low rates of disclosure among boys suggcsu a necd to educ.:atc hcahh care providen and others who work with migrant boys in the recognition and repomng of 1exu.il ... iction. viramin a aupplc:tmntation i1 at .h'yo, 1till far from tafl'eted 100%. feedinit pracn~:n panku· lerty for new born earn demand lot of educatton ernpha111 a• cxdu11ve hrealt fecdtnit for dnared rcnoj of 6 months was observtd in only 6.s% of childrrn thoulh colckturm w.11 givm 1n rn% of mwly horn ct.ildrm. the proportion of children hclow-2 waz (malnounshrdl .con" a• h!jh •• 42.6% anj "rt'i· acimy tc.. 11 compared to 1998 data. mother's ~alth: from all is 10 womm in ttprod~uvr •ill' poup, 83% were married and among marned w~ .\9% only w\"rt' u1mic wmr cnntr.-:cruve mt1h· odl 44% were married bdorc thc •ar of 18 yean and 27% had thnr ftnc prcicnancy hcftitt dlt' •icr nf 21 yean. the lt'f'vicn are not uutfactory or they arc adequate but nae unh1ed opumally. of thote' l'h mothen who had deliverrd in last one year, 80% had nailed 11ntmaral eum1nat1on 11 ira" oncc, .~o-... bad matt rhan four ttmn and ma1ortty had 1heir tetanus toxotd tnin,"t1or"'" nlht "'"'"· ljn1r11ned rn· win ronductrd 12.4% dchvcnn and 26% had home deh\'t'oc'i. ~md~: the tervtcn unbud or u111led are !tu than dnarame. the wr· l'kft provided are inadequate and on dechm reprcwnttng a looun1t ~p of h11hnto good coytti\#' ol wr· ncn. l!.ckground chanpng pnoriry cannoc be ruled out u °"" of thc coatnbutory bc10f. ps-ii ia) dcpn:wioa aad anuccy ia mip'mu ia awccr._ many de wn, witco tui~bmjer. jack dekker, aart·jan lttkman, wim gonmc:n. and amoud verhoeff ~ a dutch commumry-bucd icudy thawed 12-moarh•·prc:yalm«i al 17 .44'1. kw anx1· ay daorden and 13. 7% foi' dqrasion m anmttdam. nm .. 11p1tficantly hlllhn than dwwhrft .. dw ~thew diffamca m pttyalcnca att probably rdarcd to tlk' largr populanoa of napaan 111 ..\mturdam. <turkish 5%, moroccan 9%, and surinam 10%1. lndttd. ic'wt'll ltudla hatt ~ ~ high prevalena rata among migrant poupi in rbe ncdwrlands. howc'ytt au ttlluchn iuffenod from wry low raporne rara, or med screnung talel thac lack a ~y yabdarcd ~ in order to study the prevalence of depression and anxiety, and the (barriers to) use of mental health care among the different ethnic groups the following study was recently conducted: . the study consisted of a two-step approach. the first step was mcl~ded m the ~ h ith survey of 2004 (ahs), and consisted of three screening scales for depression and aruaety (klo, g::q-12 and mhl-5). in this survey all respondents were asked_ permission for~ second app~ 1:' 18t consisted of a structured interview containing the cidi for aruaety and depression, and questtollllalres on health care seeking, amongst others. all respondents who gave permission for a second approachwere invited by jetter for a suuctured interview with multilingual interviewers at home at a preset date and time in the following week. . in total, 439 dutch, 317 turkish, 322 moroccan and 124 surinam respondents took part in the ahs and gave permission for a second approach. in the second step, fo~ 21~ tur~h, 184m~roc can 87 surinamese and 320 dutch respondents, information from the extensive interview was available (res~nse rates between 60 and 70% ). since the data collection was completed only recently (june 2005), prevalence estimates can be presented at the conference for the first time. we have shown that with this approach it is feasible to achieve an acceptable response rate in a study on mental health among migrants. therefore we expect that this study will provide reli· able estimates of depression and anxiety in the turkish, moroccan and surinam migrants in amstw!3~· for the first time. in addition, insight into mechanisms underlying the differences between and within ethnic groups and into barriers to mental health care will provide pretexts for the improvement of pre· vention and mental health care for migrant groups. this study aimed to determine spatial patterns of mortality and morbidity of five major health problems in an urban environment: homicides, pregnancy among adolescents ((<20 years old), asthma hospitalization among young children(< 5 years old) and two mosquito-borne diseasesdengue and visceral leishmaniasis, during 1999-2003. all events were obtained through the city health database and, subsequently, geoproccsscd using the address of residence and the geographical and administrative division of the municipality, composed by 80 unit of planning (up), which in tum were formed, each one, by census tract units. two research questions were investigated: are there spatial patterns to the events dis· tribution, and moreover, are these patterns overlapping across space? we use thematic maps, index of comparative mortality/morbidity by up and the overlapped rank of the 20th worse up rates for each event. a spatial pattern of high rates of homicides, proportion of young mothers and hospitalization of asthma were overlapping in areas social and economically disadvantaged. for mosquito-borne diseases, high rates with great dispersion were found in unprivileged areas in contrast with very low rares among privileged ones. these results pointed toward a coexistence of heavier burden of diseases for those living in areas of the city where misery, poverty, lack of political public health may be modulating social health problems. a possible environmental intervention in one mosquito•bome disease might be playing a role in the occurrence of other. although with limitations, this study may provide useful information for a joint urban planning under the public perspective, articulated for use in health impact assessment. jalil safaei bdrod#aion: the association of socioeconomic status (ses), usually measured by income, and health status is an established result in bcalth studies. the poor and low income individuals have lower health sta· tus then_ those with higher incomes. in general. such finding has been usually obtained from sample surveys on speafic populations. a problem with many sample survey$ is that their results are not comparable aaoss ~ndaries as they may use different sampling methods, ask different questions, categorize the answers differently, or define groups differently. moreover, the sample data need to be standardized using the demographic: katurea of a ~ population. this study, however, uses the national population health survey (nphs) cycle 3 public use mictodata files which is based on a common survey template ·~~three~ area in canada, namely montreal, toronto, and vancouver. it also utili7.es the built-m stanclatdizaaon of the nphs data which accounts for its complex survey design. . ~:the stu~ usc:s two well-known measures of health inequalitythe relative index of meqaality and die concentranon indexto capture the extent of income related health inequality among poster sessions v139 urban female and male populations in each of the three metropolitan areas. personal income is classified into ten groups by the nphs with "no income" as the first group and "more than $60,000" as the last. health status is measured by three variables -having a chronic condition (chc), self assessed health (sah), and health utility index (hui) -using appropriate indices. alternative formulations are used to calculate the standard deviations of the estimated or calculated measures. the findings of the study suggest that the measured health inequality depends on the health measure used. for chc and hui the measured inequality indices do indicate poorer health for lower income individulas, however, they are not statistically insignificant. health inequalities are more pronounced when health is measured by sah. the results do not support any systematic ranking of the three metropolitan areas in terms of health inequalities. they do not reveal any systematic pattern of inequality between men and women, either. conclusions: despite the use of highly aggregated nphs data, income related health disparities are observed in the three metropolitan areas in canada. this is more the case with self perceived (sah) health. such disparities are preventable and call for broader health policies that address poverty and low income among other social determinants of health. introduction: the analysis of health indicators under the spatial perspective is configured as an important instrument in the detection of intra-urban differentials. this study aimed to examine the spatial distribution of the births occurred in belo horizonte, in 2001, analyzing the presence of spatial clusters of health indicators for newborns (rn) and their mothers, using data from the information system on live birth database (sinasc). method: for each covered area of the basic units of health (ubs), we calculated the proportion of: adolescent mothers, mothers with less than 8 years of schooling, first pregnancy, mothers with four or more pregnancies, dead babies born on previous pregnancies, stillbirths, cesarean section, less than four prenatal care attendance, moderate and severe hypoxemia in the 1st and 5th minutes of life, low or very low birth weight. we used empiric bayesian methods for smoothing the estimates. for spatial analysis, the indicators obtained from the global moran (i) index and local indicators of spatial association (lisa) were used. maps were built to allow the visualization of the spatial clusters. in all analysis, significance statistics was considered p~ 0.05. results: a total of 36, 12 7 births of residents in belo horizonte were registered in 200 i. the estimated bayesian proportions for the entire municipality was close to the one observed for all variables. analysis using lisa showed the presence of relevant spatial clusters for adolescent and low educated mothers, dead babies form previous pregnancies, cesarean section, low attendance to the prenatal care especially in area with low socio-demographic characteristics. three areas presented consistent clusters, with important spatial auto-correlation for almost all studied indicators. conclusion: the used methodology was configured as a great instrument of detection risk areas where clustering occurs. it can easily be incorporated in any surveillance system as a mechanism for controlling events related to births in the municipality. moreover, it can be applied to discriminate target areas for prompt public health interventions, such as improving health services access and the consolidation of better obstetric practices. introduction: gentrification, the displacement of low income and non-majority people out of longtime neighborhoods and residences is a global phenomenon. it has been identified as occurring in both developed and less developed countries and as inequality persists or increases, many communities are vulnerable to disruption and loss. while there may be some benefits to gentrification, these benefits are less likely to impact those who have been forced to move out of a gentrifying community or those who remain but economically stressed. . . . . this paper lays out a theoretical framework for 1dent1fymg and ~ddressm~. the_ health consequences of gentrification on residents living in a community prior .to o.r durmg gent~1~1cat.10n. by drawing on the literature of housing, sociology, health and urban plannmg, 1t places genmf1catt~n and neighborhood change into the context of other forces affecting th~ .hea.lth of vulnerable populations. it then proposes solutions to prevent or mitigate the impacts of genmf1cat1on. results: four main categories of consequences potentiallr re~ult from ~entr~fica~~n: !'°°r housing· related issues, spatial effects, mental health impacts and contr1bunons to racial ~spanttes m heal~. the housing issues include increased susceptibility to asthma, lead exposure, allergiesl~topy an~. acetdents/ injuries. spatial effects include reduced access to health ~are, reduce~ access to 1obslttadinonal food sources, decreased physical activity, and increased obesity. the __ pnmary mental health effects_ :ire increased stress increased risk of depression and disruption of trad1t1onal support networks. in addinon to the above he~lth issues, gentrification has the potential to increase racial disparities in health by reduc· ing access to long term and multi-culturally experienced health care provide~s. conclusions: public health practice provides a framework for addressmg the health consequences of gentrification. in this context primary prevention would involve preventing gentrification in the first place along with a renewed commitment to helping distressed communities a~~ im_proving the quality ~f life for long term residents. the next layer of meeting the challenges of gentrification would be to assjst long rime residents to stay in a community and thus potentially allowing them to participate in the bene~ts of gentrification (such as better public services). only if all these efforts fail -and these other prevennon strategies must be a priority, public policy should then work to help people and instirutions move to other communities through grants and the provision of alternative safe affordable housing and neighborhoods. sarah romans, marsha cohen, and tonia forte lnh'odlletion: there has been sustained interest in urban rural (ur) differences in mental health over the last 50 years of epidemiological research, with most studies reporting higher urban rates of mor· bidity, particularly when psychotic disorders have been studied. most recently, in britain, urban rates of non-psychotic disorder were greater than rural and semi-rural rates, both before and after the more adverse circumstances of urban dwellers were considered (paykel et al 2003) . surprisingly, there were no ur differences in help seeking. in canada, wang (2004) found greater urban rates for major depression only after he controlled for the confounding effects of race, immigration, employment and marital status, a result reminiscent of work from the us (blazer et 1985) . rural participants were less likely to have sought help for their mental health problems. in this study, we sought to examine urban/rural differences in rates of depression and help seeking in canada. method; data came from the 2000 canadian community health survey 1.2, a community survey conducted by statistics canada of people aged 15 and older, with the total sample size of 36,984 respon· dents, 77% response. analyses used weighted data; differences were assessed by chi-squared. ra.its: bivariate cross-tabulations showed a modest increase in 12 month depression rates for urban (5.4%) over rural (4.3%, p= 0.03) dwellers; there were no ur differences when the sample was examined separately by gender and age group ( 15-29, 30-44, 45-69) . in general, more urban (10.6%) than rural people (8. 7%, p= 0.002) had sought mental health treatment in the past year. there was no ur difference in helping seeking amongst those with depression (u 58.9%, r 50.1 % ns). amongst the whole population, helping seeking was gendered with more urban men accessing help (7.4%) than rural men (5.4%, p=0.004); this did not apply for women (13.7% vs 12.1% p=0.1). ~ons: the urban-rural demographic continues to generate intriguing findings, even recently when internal migration is common and people can seek out the environment most conducive to their health, ~th physical and mental. people with depression are a disadvantaged, frequently stigmatized group, with ~r quality of life a_nd often impaired function. unlike many other factors associated wi~h urban or rural hfe per se, depression can be treated. the low help seeking rates is a cause for concern, m both the ur~n and_ rural populations. it behooves researchers, policy makers and service planners to ensutt effective services are available for both humane and economic reasons. methods: using united states 2000 census income data, we assigned the 12 manhattan community districts to two major socioeconomic groups, manhattan i and ii. manhattan i is composed of community districts with average household incomes above the median for new york city; manhattan ii is those below. with public new york city department of health 1999 death records and 2000 us census data, we calculated a "standard" new york city population and estimated mortality rate distributions for manhattan i and ii. we then compared these age-adjusted actual mortality distributions with a standard t-test. results: we explored premature deaths using several cut-off points (before the ages of 65, 70, and 75), and with subcomparisons for males and females. every comparison showed the wealthier area, manhattan i, to have significantly lower premature mortality rates than the lower income manhattan ii. further, we compared age-adjusted mortality rates among the city's five boroughs, and found no significant differences based on local geography alone. conclusions: these findings suggest that in manhattan, characterized by its extremes of wealth and poverty, health status, as measured by premature mortality, does vary significantly with the local income level. in manhattan, the relative average income by community district varies as much as 4: 1. this ratio is greater than that found in the other cities we have studied: for example, the range among tokyo's kus is half that in manhattan, or about 2: 1. paris has shown the longest life expectancy and changing premature mortality rates. from our preliminary work, we expect to find less variation in premature mortality rates in comparable cities. this study will continue to explore the relationship of premature mortality rates and life expectancy to income levels and different health systems among wcp cities with a view to measuring health policy options. introduction: according to the world health organization (who), smoking-related illness is currently the world's second major cause of death, currently responsible for one out of ten adult deaths worldwide. the who's framework convention on tobacco control (fctc) aims to reduce tobaccorelated disease and deaths by changing national public policies. mexico, which, according to the who, had smoking prevalences of 32% among adults in 1998 and 23% among health care providers (hcps) in 1997, ratified the fctc in 2004. objective: to examine knowledge of and attitudes towards cigarette smoking and smoking cessation among hcps, and clinical practices regarding smoking cessation. methods: in june 2005, a convenience sample of hcps from one public clinic in urban oaxaca, mexico, part of mexico's national network of public health care clinics, were interviewed in spanish using a verbally administered questionnaire that was standard among all participants. during primary care outpatient visits, hcps were observed treating patients to assess the relative importance of smoking cessation as a health care priority. results: of the 18 hcps interviewed, including ten physicians, three nurses, and five medical students, 67% reported smoking regularly. all hcps reported assessing patients' history of smoking, knowledge of the health risks associated with smoking, and feeling qualified to discuss these risks with patients. all hcps reported counseling patients who currently smoked to quit. all hcps reported recommending nicotine replacement therapy (eg., patch and/or gum) and/or behavioral therapy (eg., psychologist, support groups) to patients who smoked. all hcps reported that there was no government funding for smoking cessation, and that all costs associated with smoking cessation were patients' out-of-pocket expenses. observation of hcp interaction with patients revealed that hcps always inquired about smoking history with new patients and rarely inquired about smoking history with returning patients. hcps were not observed counseling patients who reported current smoking on smoking cessation methods and its benefits. observation of the clinical environment suggested a focus on preventative child health (eg., immunization, water-borne illnesses), family planning, and chronic diseases (eg., diabetes, hypertension). conclusions: while the mexican government has made smoking cessation a public health priority and hcps report addressing smoking cessation, observation of hcps suggests: 1) smoking status is assessed only in new patients; and 2) smoking cessation methods are not addresse~. observ_atio~ of hcps and the clinical environment in a public clinic in oaxaca suggests that smoking cessation 1s of lower priority than other heath care issues. p6-04 (a) urban agriculture and food and nutrition security in kampala, uganda fiona yeudall, renee sebastian, abdelrahman lubowa, selahadin ibrahim, and donald cole introduction: urban agriculture is an important livelihood strategy contributing to household food security by increasing access and availability to food in urban settings (koc et al., 1999) . the purpose of poster sessions v142 rhis study was to examine relationships between child nutritional securiry outcomes, household food security, and urban agriculture activiries in kampala, uga~da. . . questionnaires assessing socio-demographic, farmmg and household food secunry (hfsi characreristics were administered to 270 households. food diversiry was ~lculate~ from ~e number of foods consumed over 24 hours for one child (2-5 years) per household. heigh~ weight,. nud-upper·ann· circumference (muac) and tricep skinfolds (tsf) were measured for the mdex child. z-sc.ores for height-for·age (haz), weighr-for-age (waz) and body mass index (zbmi) were ~~ulated usmg cen· rres for disease control and prevenrion reference data. z-scores for body composition measures were calculated using nhanes i and ii data for african americans. the lms method was used to c~.t for skewed z·score indices. all dara was checked for normaliry and univariable and backward mulnvanablc linear regression analysis conducted. ruwlts: household food securiry was significantly associated with wealth (b= 0. 71, p< 'a acre were more dependenr on assets for hfs. although sex of head of household was ~ot related to j-:ifs, it modi· fied relationships in rhar female headed households had greater food securtry when fai:mmg less than 1,4 acre compared ro male headed households, and vice versa. hfs was significantly associated with food diversiry (s=0.15, p). urbanization, especially in developing countries, has substantially increased the vulnerability of rhe mass of low-income urban dwellers. the livelihoods and quality of life for many of the poor espe· cially in larin america, asia, and africa, have deteriorated significantly over the years. urban areas are increasingly unable to provide for their populations, resulting in poverry, massive unemploymenr, job cuts, poor housing, lack of or poor public services, and a compromised health status. botswana, a country rhat lies in the sourhern parr of africa, has had its share of urban problems such as rhe mush· rooming of squarters and low-income urban sertlements. despite efforts to address the substandard living conditions in low-income urban areas, these problems have continued to grow. these living con· diuons are porenrially stressful to rhe residenrs and likely affects their health. the primary aim of the 11udy wa1 to examine the complex relationship between communiry-level stressors and interveners and health-related quality of life among residents of low-income neighborhoods in francistown, botswana. u1i"" a croa1-icctional quantitative design (both descriptive and explanatory) and using primarily cloae-mded interview• with a random sample of 388 residents, this study examined the role of chrome life 1trnson and environmental quality on overall health status qualiry of life) and the physical, psy· chological and level of independence domains of health. the major hypothesis of the study was that community-levrl stre1sor1 would influence health-related quality of life and that social capital would moderate these relationships. findings indicate that neighborhood quality is a powerful predictor of healrh 1tatu1 rhan socioeconomic status and individual life stressors. social capital was also found to he a 11111ificant positive predictor of healrh and also moderator of structural factors. social capital moderated the effects of low environmental quality on level of indrpendence and on physical health outcomn, but nor on psychological and global health outcomes. these findings suggest that as the environment get1 betttr. stresson are reduced, hence promoting berter health outcomes. the study ends with implication• for social justice, public health and social work practice, and research, focusing mostly on the ~ole of social capital and the environmental quality in predicting health outcomes. spt· cafically • anenhon should be focused on political and civic society's commitment for social justice and poveny alleviation, ~uction of the threat of insecuriry and violence; cultivating social capital and good governance; and improving the health and social environments, especially housing and environ· mental 1aiutanon to name a few. urban and other uprisings by non-elites that lead to transitions, can disrupt sexual and injection "risk" networks that transmit infectious diseases by changing mixing patterns. they can also weaken urban social networks, their associated protective norms and their informal social control mechanisms which can lead to increased sexual and drug risk behaviors and violence (fullilove 2004; wallace & wallace1998, aral 2002 , friedman & reid 2000 hankins et al 2002) . for example, the 1979 revolution and transition to theocratic rule, and subsequent urban and national conflicts, have been followed by many urban youth in iran engaging in clandestine high-risk sexual and drug behaviors. in palestine, conflicts and restrictions on movement have led to increased fatalities from chronic diseases due to limited access to hospitals and modern medical facilities (union of palestinian medical relief committees); and heroin use and violence-related blood exposure have also increased. social movements "from below" that are rooted both in urban social network dynamics and in underlying patterns of injustice can have both protective and risky effects. for example, the social movements that led to the collapse of the ussr and its dependent governments in other countries-with subsequent increases in sex trade, alcoholism, drug use, tuberculosis, hiv, stis, and mortality in many localities-were based originally on such city-based movements in eastern europe. their impacts on health were mediated by urban social dynamics and structures. some urban social movements have improved health by prevent· ing the spread of hiv, hepatitis and other diseases. examples include movements of (a) gays and lesbians and (b) drug users. these have been rooted in social networks that overlap with risk networks. theories of urban health should include social conflict as a core concept. mechanisms that generate conflicts and the pathways by which conflicts affect health should be a major part of urban health research agendas. p6-07 (c) demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital (lasuth) chest clinic john bako, adewale akeredolu, and wale alabi tuberculosis has become a resurgent public health problem in recent times in the world. because resources are limited, control programmes frequently must target population at greatest risk. the purpose of this study was to study the demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital. a total of 76 patients who have been both radiologically and bacteriologically confirmed tuberculosis patients were used for this study between january and march. 57 (75%) were males, while 19 (25%) were females. notable risk factors among patients with tuberculosis were overcrowding (46.1 % ), homelessness ( 11.8%) cigarette smoking (22.4% ), alcoholism (30.3%), non vaccination (25%), secondary contact (36.8%) and poor knowledge (92. l 'x,). man· agement history patterns among the patients were herbs ( 13.2 'yo), orthodox medicine ( .h . .l'x.). intervention targeting early-identified groups may be an effective way to reduce the incidence of tuber· culosis. such intervention should focus on health education, modification of life style and improvement of standard of living. p6-08 (c) profiles in urban health in 9 cities of the americas in the countries of the americas, there is an increasing migration of national and international populations to urban centers. this presentation will focus on some of the issues created by this influx of populations, the ways that 9 cities in 8 countries are dealing with them, and the efforts to promote local participation and solutions in management and decision-making. the methodology used to collect this information has been to draw upon and analyze information produced by the mayor's and ministry of health and development offices in each of the cities under consideration. an analysis of the impact of urbanization on health and health determinants will be presented. the information covers issues such as health status by geographic areas within the cities, highlighting issues such as marginality, barriers and physical, economic and cultural constructs and inequities in the delivery of and access to essential services (such as health, education, water, and basic sanitation). issues of governance and citizen participation will be highlighted to provide insight in~o the balance of power and mechanisms for decision-making at the local level. part of the analysis will show the relationship between democratic processes and social participation. public ~olicies will be reviewed to indicate those that are most beneficial in promoting health and overcoming barriers to it, as well as ways of capitalizing on local assets and resources. final~y, evidence of effectiveness of various strategies will be indicated. the presentation will conclude wuh suggesuons for future strategic directions to improve the quality of life and the promotion of health in large urban centers of the americas. introduction: much of the illicit drug in mexico is concentrated in northern border areas. the 2000 mile border between the u.s. and mexico is also characterized by migration; the border crossing between tijuana, baja california, mexico and san diego, california, u.s.a. is rep~rt~dl~ the busiest land border crossing in the world. we attempt to describe the migration scene among m1ect1on drug users (idus) m tijuana and investigate service needs. methods: migration trends were investigated in a cross-sectional study conducted from february to june 2005 among idus in tijuana. enrollment criteria included informed consent, being 18 years or older, and having injected drugs within the prior month. subjects were recruited by respondent-dnven sampling and were administered a quantitative survey and serology for hiv, hcv, and syphilis. logistic regression was used to compare idus who had migrated to the u.s. versus those who had not. results: of 222 idus enrolled, 91 % were male and median age and age at first injection were 35 and 20, respectively. drug combinations injected most frequently in the past 6 months were heroin (35%1 and crystal methamphetamine mixed with heroin (53%). of those enrolled, 212 responded to quesnons on migration and drug treatment and were included in this analysis. although 76% had resided in tijuana for at least 5 years, 70% of users were born outside of baja california. working outside of mexico was common, with 38% working abroad in the last 10 years (94% in u.s.), and 17% in the past year. in the last 6 months, 10% of idus had crossed the border to the u.s., with 57% crossing at least once per month. those working outside of mexico in the past year were less likely to have ever received substance abuse treatment, [29% vs. 53%, or 0.38, ] and were marginally less likely to have received drug treatment in the past 6 months [6% vs. 18 %, or 0.28, 95% ci (0.06-1.2)]. drug use patterns, age and gender did not differ between migrants and non-migrants, although migrants were more likely to report being in need of substance abuse treatment (68% vs. 57%, respectively). conclusions: migration is common among idus in tijuana. maintaining drug treatment regimens and determining how to best target education and services to a highly mobile population pose challenges to officials on both sides of the border. these data underscore the need to develop coordinated binational prevention and treatment efforts. introduction: despite the expanding literature on the important role public policy plays in influencing the broader determinants of the public\'s health, profound differences exist among jurisdictions in rhe attention placed upon such activities. we take an international perspective on health by examining rhe d?minant public _health paradigms of canada, usa, uk, and sweden and exploring how these paradigms shape public health practice. _method: we carefully analyze governmental and public health agencies documents to discern ~he dommant para~igms driving public health approaches and practices. we also consider the unique paht1· cal and economic contexts within each nation and consider how these contexts drive public health pahcy and approaches. . . ~u~: the canadian and usa public health communities -with some exceptions --focus upon m~ividuahzed approaches to risk management. in contrast, the uk and swedish public health scenes are oriented toward broader approaches to health determinants. we find that the extent to which govern· ments, public health agencies and public health workers concern themselves with public policy approaches £<_> ~ddress ~ro.ad~r .determinants of health depends upon the particular health parad'.~ adhered. ~o withm each 1urisd1ctton. and whether a paradigm is adopted depends upon the ideologi~a and pol~ncal context of each nation. nations such as sweden that have a long tradition of public policies promonng social jus~ce an~ equity are naturally receptive to evolving population health concepts. '[he usa represen~ a ~bey en~ro~~t where such is~ues are clear!~ subordinate. ., our findings mdicate that there 1s a strong political component that influences pubh ~ealth a~proaches and practi~ within the jurisdictions examined. the implications are that those seek· m~ to raise the broader detennmants of the public's health should work in coalition to raise these issues with non-health organizations and age · ca d d th · badrgrollnd: in developed countries, social inequalities in health have endured or even worsened comparatively throughout different social groups since the 1990s. in france, a country where access to medical and surgical care is theoretically affordable for everyone, health inequalities are among the high· est in western europe. in developing countries, health and access to care have remained critical issues. in madagascar, poverty has even increased in recent years, since the country wenr through political crisis and structural adjustment policies. objectives. we aimed to estimate and compare the impact of socio· economic status but also psychosocial characteristics (social integration, health beliefs, expectations and representation, and psychological characteristics) on the risk of having forgone healthcare in these 2 dif· fercnt contexts. methods: population surveys conducted among random samples of households in some under· served paris neighbourhoods (n= 889) and in the whole antananarivo city (n= 2807) in 2003, using a common individual questionnaire in french and malagasy. reslllts: as expected, the impact of socioeconomic status is stronger in antananarivo than in paris. but, after making adjustments for numerous individual socio-economic and health characteristics, we observed in both cities a higher (and statically significant) occurrence of reponed forgone healthcare among people who have experienced childhood and/or adulthood difficulties (with relative risks up to 2 and 3.s respectively in paris and antananarivo) and who complained about unhealthy living conditions. in paris, it is also correlated with a lack of trust in health services. coneluions: aside from purely financial hurdles, other individual factors play a role in the non-use of healthcare services. health insurance or free healthcare seems to be necessary hut not sufficienr to achieve an equitable access to care. therefore, health policies must not only focus on the reduction of the financial barriers to healthcare, but also must be supplemented by programmes (e.g. outreach care ser· vices, health education, health promotion programmes) and discretionary local policies tailored to the needs of those with poor health concern .. acknowledgments. this project was supported by the mal>io project and the national institute of statistics (instat) in madagascar, and hy the development research institute (ird) and the avenir programme of the national institute of health and medical research (inserm) in france. for the cities of developing countries, poverty is often described in terms of the living standard~ of slum populations, and there is good reason to believe that the health risks facing these populations are even greater, in some instances, than those facing rural villagers. yet much remains to be learned ahour the connections between urban poverty and health. it is not known what percentage of all urban poor live in slums, that is, in communities of concentrated poverty; neither is it known what proportion of slum residents are, in fact, poor. funhermore, no quantitative accounting is yet available that would sep· arare the health risks of slum life into those due to a househoid•s own poverty and those stemminic from poveny in the surrounding neighborhood. if urban health interventions are to be effectively targeted in developing countries, substantial progress must be made in addressing these cenrral issues. this paper examines poverty and children's health and survival using two large surveys, one a demographic and health survey fielded in urban egypt (with an oversampling of slums) and the other a survey of the slums of allahabad, india. using multivariate statistical methods. we find, in both settings: ( 11 substan· rial evidence of living standards heterogeneity within the slums; (21 strong evidence indicating that household-level poverty is an imponant influence on health; and (3) staristically significant (though less strong) evidence that with household living standards held constant, neighborhood levels of poverty adversely affect health. the paper doses with a discussion of the implications of these findings for the targeting of health and poverty program interventions. p6-13 (a) urban environment and the changing epidemiological surfacr. the cardiovascular ~ &om dorin, nigeria the emergence of cardiovascular diseases had been explained through the concomitants o_f the demographic transition wherein the prevalent causes of morbidity and monality ~hangr pr~mmant infectious diseases to diseases of lifestyle or chronic disease (see deck, 1979) . a ma1or frustration m the v146 poster sessions case of cvd is its multifactural nature. it is acknowledged that the environment, however defined is the d · f · t' b tween agents and hosts such that chronic disease pathogenesis also reqmre a me 1an o mterac ion e . spatio-temporal coincidence of these two parties. what is not clear is which among ~ever~( potennal fac· · h b pace exacerbate cvd risk more· and to what extent does the ep1dem1olog1cal trans1· tors m t e ur an s ' . . . . tion h othesis relevant in the explanation of urban disease outlook even the developmg cities like nigeri~: thesis paper explorer these within a traditional city in nigeria. . . . the data for the study were obtained from two tertiary level hospitals m the metropolis for 10 years (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) . the data contain reported cases of cvd in the two facilities for the period. adopting a series of parametric and non-parametric statistics, we draw inferences between the observed cases of cvds and various demographic and locational variables of the patients. findings: about 28% of rhe cases occurred in 3 years (1997) (1998) (1999) coinciding with the last year of military rule with great instability. 55.3% occurred among male. 78.8% also occurred among people aged 31-70 years. these are groups who are also likely to engage in most stressful life patterns. ~e study also shows that 63% of all cases occurred in the frontier wards with minor city areas also havmg their •fair' share. our result conformed with many empirical observation on the elusive nature of causation of cvd. this multifactoral nature had precluded the production of a map of hypertension that would be consistent with ideas of spatial prediction. cvd -cardiovascular diseases. mumbai is the commercial capital of india. as the hub of a rapidly transiting economy, mumbai provides an interesting case study into the health of urban populations in a developing country. with high-rise multimillion-dollar construction projects and crowded slums next to each other, mumbai presents a con· trast in development. there are a host of hi-tech hospitals which provide high quality care to the many who can afford it (including many westerners eager to jump the queue in their healthcare systems-'medical tour· ism'), at the same time there is a overcrowded and strained public healthcare system for those who cannot afford to pay. voluntary organizations are engaged in service provision as well as advocacy. the paper will outline role of the voluntary sector in the context of the development of the healthcare system in mumbai. mumbai has distinct upper, middle and lower economic classes, and the health needs and problems of all three have similarities and differences. these will be showcased, and the response of the healthcare system to these will be documented. a rising hiv prevalence rate, among the highest in india, is a challenge to the mumbai public healthcare system. the role of the voluntary sector in service provision, advocacy, and empowerment of local populations with regards to urban health has been paramount. the emergence of the voluntary sector as a major player in the puzzle of urban mumbai health, and it being visualized as voices of civil society or communiry representatives has advantages as well as pitfalls. this paper will be a unique attempt at examining urban health in india as a complex web of players. the influence of everyday socio·polirical-cultural and economic reality of the urban mumbai population will be a cross cutting theme in the analysis. the paper will thus help in filling a critical void in this context. the paper will thus map out issues of social justice, gender, equiry, effect of environment, through the lens of the role of the voluntary sector to construct a quilt of the realiry of healthcare in mumbai. the successes and failures of a long tradi· tion of the active advocacy and participation of the voluntary sector in trying to achieve social justice in the urban mumbai community will be analyzed. this will help in a better understanding of global urban health, and m how the voluntary sector/ngos fir into the larger picture. ba~und: o~er. half _of n~irobi's 2.5 million inhabitants live in illegal informal settlements that compose 5 yo of the city s res1dent1al land area. the majority of slum residents lack access to proper san· iranon and a clean and adequate water supply. this research was designed to gain a clearer understand· mg of what kappr · · · h f . . opnate samtanon means or the urban poor, to determine the linkages between gender, hvehhoods, and access to water and sanitation, and to assess the ability of community sanitation blocks to meet water and sanitation needs in urban areas. m~tbojs_: _a household survey, gender specific focus groups and key informant interviews were conducted m maih saba, a peri-urban informal settlement. qualitative and quantitative research tools were u~ to asses~ the impact and effectiveness of community sanitation blocks in two informal settlements. results ropna e samtarmn me u es not only safe and clean latrines, but also provision ° adequate drainage and access to water supply for cleaning of clothes and homes. safety and cleanliness poster sessions v147 were priorities for women in latrines. levels of poverty within the informal settlements were identified and access to water and sanitation services improved with increased income. environmental health problems related to inadequate water and sanitation remain a problem for all residents. community sanitation blocks have improved the overall local environment and usage is far greater than envisioned in the design phase. women and children use the blocks less than men. this is a result of financial, social, and safety constraints. the results highlight the importance a need to expand participatory approaches for the design of water and sanitation interventions for the urban poor. plans need to recognize "appropriate sanitation" goes beyond provision of latrines and gender and socioeconomic differences must be taken into account. lessons and resources from pilot projects must be learned from, shared and leveraged so that solutions can be scaled up. underlying all the challenges facing improving water and sanitation for the urban poor are issues of land tenure. p6-16 (c) integrating tqm (total quality management), good governance and social mobilization principles in health promotion leadership training programmes for new urban settings in 12 countries/ areas: the prolead experience susan mercado, faren abdelaziz, and dorjursen bayarsaikhan introduction: globalization and urbanization have resulted in "new urban settings" characterized by a radical process of change with positive and negative effects, increased inequities, greater environmental impacts, expanding metropolitan areas and fast-growing slums and vulnerable populations. the key role of municipal health governance in mitigating and modulating these processes cannot be overemphasized. new and more effective ways of working with a wide variety of stakeholders is an underpinning theme for good governance in new urban settings. in relation to this, organizing and sustaining infrastructure and financing to promote health in cities through better governance is of paramount importance. there is a wealth of information on how health promotion can be enhanced in cities. despite this, appropriate capacity building programmes to enable municipal players to effectively respond to the challenges and impacts on health of globalization, urbanization and increasing inequity in new urban settings are deficient. the who kobe centre, (funded by the kobe group( and in collaboration with 3 regional offices (emro, searo, wpro) with initial support from the japan voluntary contribution, developed a health promotion leadership training programme called "prolead" that focuses on new and autonomous structures and sustainable financing for health promotion in the context of new urban settings. methodology: country and/or city-level teams from 12 areas, (china, fiji, india, japan, lebanon, malaysia, mongolia, oman, philippines, republic of korea, tonga and viet nam) worked on projects to advance health promotion infrastructure and financing in their areas over a 9 month period. tools were provided to integrate principles of total quality management, good governance and social mobili1.ation. results: six countries/areas have commenced projects on earmarking of tobacco and alcohol taxes for health, moblization of sports and arts organizations, integration of health promotion and social health insurance, organizational reforms, training in advocacy and lobbying, private sector and corporate mobilization and community mobilization. results from the other six areas will be reported in 01..;obcr. conclusions: total quality management, good governance and social mobilization principles and skills are useful and relevant for helping municipal teams focus on strategic interventions to address complex and overwhelming determinants of health at the municipal level. the prolead training programmes hopes to inform other processes for building health promotion leadership capacity for new urban settings. the impact of city living and urbanization on the health of citizens in developing countries has received increasing attention in recent years. urban areas contribute largely to national economies. however, rapid and unplanned urban growth is often associated with poverty, environmental degradation and population demands that outstrip service capacity which conditions place human health at risk. local and national governments as well as multi national organizations are all grappling with the challenges of urbanization. with limited data and information available, urban health characteristics, including the types, quantities, locations and sources in kampala, are largely unknown. moreover, there is n? basis for assessing the impact of the resultant initiatives to improve health ~onditions amo~g ~o":1":1um ties settled in unplanned areas. since urban areas are more than the aggregation ?f ~?pie w~th md_1v1dual risk factors and health care needs, this paper argues that factors beyond the md1v1dual, mcludmg the poster sessions v148 · i d h · i · ment and systems of health and social services are determinants of the health soc1a an p ys1ca environ . of urban populations. however, as part of an ongoing study? ~s pape~ .addresses the basic concerns of urban health in kampala city. while applying the "urban hvmg conditions and the urban heal~ pen· alty" frameworks, this paper use aggregated urban health d~ta t~ explore the role of place an~ 111st1tu· tions in shaping health and well-being of the population m kampala by understanding how characteristics of the urban environment and specific features of the city are causally related to health of invisible and forgotten urban poor population: results i~dica~e that a .range o~ urb~n he~l~h hazards m the city of kampala include substandard housing, crowdmg, mdoor air poll.ut1on, msuff1c1ent a~d con· taminated water, inadequate sanitation and solid waste management services, vector borne .diseases, industrial waste increased motor vehicle traffic among others. the impact of these on the envtronment and community.health are mutually reinforcing. arising out of the withdra"'.al of city pl~nning systems and service delivery systems or just planning failure, thousands of people part1cularl~ low-mc~me groups have been pushed to the most undesirable sections of the city where they are faced with ~ va_r1ety ~f envj· ronmental insults. the number of initiatives to improve urban health is, however, growing mvolvjng the interaction of many sectors (health, environment, housing, energy, transportation and urban planning) and stakeholders (local government, non governmental organizations, aid donors and local community groups). key words: urban health governance, health risks, kampala. introduction: the viability of urban communities is dependent upon reliable and affordable mass transit. in particular, subway systems play an especially important role in the mass transit network, since they provide service to vast numbers of ridersseven of the 95 subway systems worldwide report over one billion passenger rides each year. surprisingly, given the large number of people potentially affected, very little is known about the health and safety hazards that could affect both passengers and transit workers; these include physical (e.g., noise, vibration, accidents, electrified sources, temperature extremes), biological (e.g., transmission of infectious diseases, either through person-to-person spread or vector-borne, for example, through rodents), chemical (e.g., exposure to toxic and irritant chemicals and metals, gas emissions, fumes), electro-magnetic radiation, and psychosocial (e.g., violence, workstress). more recently, we need to consider the threat of terrorism, which could take the form of a mass casualty event (e.g., resulting from conventional incendiary devices), radiological attack (e.g., "dirty bomb"), chemical terrorist attack (e.g., sarin gas), or bioterrorist attack (e.g., weapons grade anthrax). given the large number of riders and workers potentially at risk, the public health implications are considerable. methods: to assess the hazards associated with subways, a structured review of the (english) litera· ture was conducted. ruults: based on our review, non-violent crime, followed by accidents, and violent crimes are most prevalent. compared to all other forms of mass transit, subways present greater health and safety risks. however, the rate of subway associated fatalities is much lower than the fatality rate associated with automobile travel (0.15 vs. 0.87 per 100 million passenger miles), and cities with high subway ridership rates have a 36% lower per capita rate of transportation related fatalities than low ridership cities (7.5 versus 11.7 annual deaths per 100,000 residents). available data also suggest that subway noise levels and levels of air pollutants may exceed recommended levels. . ~: there is a paucity of published research examining the health and safety hazards associated with subways. most of the available data came from government agencies, who rely on passively reported data. research is warranted on this topic for a number of reasons, not only to address important knowled~ gaps, but also because the population at potential risk is large. importantly, from an urban perspecnve, the benefits of mass transit are optimized by high ridership ratesand these could be adversely affu:ted by unsafe conditions and health and safety concerns. veena joshi, jeremy lim. and benjamin chua ~ ~rban health issues have moved beyond infectious diseases and now centre largely on chrome diseases. diabetes is one of the most prevalent non-communicable diseases globally. 9 % of adult ¥151 benefit in providing splash pads in more parks. given the high temperature and humidity of london summers, this is an important aspect and asset of parks. interviewed parents claimed to visit city parks anywhere between 1 to 6 days per week. corrduion: given that the vast majority of canadian children are insufficiently active to gain health benefits, identifying effective qualities of local parks, that may support and foster physical activity is essential. strategies to promote activity within children's environments are an important health initiative. the results from this study have implications for city planners and policy makers; parents' opinions of, and use of city parks provides feedback as to the state current local parks, and modifications that should be made for new ones being developed. this study may also provide important feedback for health promoters trying to advocate for physical activity among children. introdt1clion: a rapidly increasing proportion of urban dwellers in africa live below the poverty line in overcrowded slums characterized by uncollected garbage, unsafe water, and deficient sanitation and overflowing sewers. this growth of urban poverty challenges the commonly held assumption that urban populations enjoy better health than their rural counterparts. the objectives of this study are (i) to compare the vaccination status, and morbidity and mortality outcomes among children in the slums of nairobi with rural kenya, and (ii) to examine the factors associated with poor child health in the slums. we use data from demographic and health survey representative of all slum settlements in nairobi city carried out in 2000 by the african population & health research center. a total of 3,256 women aged 15-49 from 4,564 households were interviewed. our sample consists of 1,210 children aged 0-35 months. the comparison data are from the 1998 kenya demographic and health survey. the outcomes of interest include child vaccination status, morbidity (diarrhea, fever and cough) and mortality, all dichotomized. socioeconomic, environmental, demographic, and behavioral factors, as well as child and mother characteristics, are included in the multivariate analyses. multilevel logistic regression models are used. l'nlimin11ry rest1lts: about 32 % of children in the slums had diarrhea in the two weeks prior to the survey, compared to 16% of rural children. these disparities between the urban poor anj the rural residents are also observed for fever (64% against 42%), cough (46% versus 20%), infant mortality (91/ 1000 against 76/1000), and complete vaccination (48% against 64%). preliminary multivariate results indicate that health service utilization and maternal education have the strongest predictive power on child morbidity and mortality in the slums, and that household wealth has only minor, statistically insignificant effects. conclruion: the superiority of health of urban children, compared with their rural counterparts, masks significant disparities within urban areas. compared to rural residents, children of slum dwellers in nairobi are sicker, are less likely to utilize health services when sick, and stand greater risk to die. our results suggest policies and programs contributing to the attainment of the millennium development goal on child health should pay particular attention to the urban poor. the insignificance of socioeconomic status suggests that poor health outcomes in these communities are compounded by poor environmental sanitation and behavioral factors that could partly be improved through female education and behavior change communication. introduction: historic trade city surat with its industrial and political peace has remained a center of attraction for people from all the comers of india resulting in to pop.ulatio~ explosio~ a~d stressed social and service infrastructure. the topography,dimate and demographic profile of the city 1s threat to the healthy environment. aim of this analysis is to review the impact of managemt'nt reform on health indicators. method: this paper is an analysis of the changing profile of population, sanitary infr~s~rucrure, local self government management and public health service reform, secondary health stat1st1cs data, health indicator and process monitoring of 25 years. . . health of entire city and challenge to the management system. plague outbr~ak (1994) was the turning point in the history of civic service management including p~blic ~e~lth service management. ~ocal self government management system was revitalized by reg~lar_ field v1s1ts o~ al~ cadre~, _decentraltzanon of power and responsibility, equity, regular vigilant momtormg, commumcanon facility, ream_approach and people participation. reform in public health service management was throu_gh stan~~rd1zed intervention protocol, innovative intervention, public private partnership, community part1c1panon, academic and service institute collaboration and research. sanitation service coverage have reached nearer to universal. area covered by safe water supply reached to 98%(2004) from 40% (1991) and underground drainage to 97% (2004) from 17% ( 1991) the overhauling of the system have reflected on health indicators of vector and water born disease. malaria spr declined to 1.23 (2004) from 23.06'yo(!991) and diarrhea case report declined to 1963(2004) from 3431 (2004). except dengue fever in 2002 no major disease outbreaks are reported after 1991. city is recipient of international/national awards/ranking for these achievements. the health department have developed an evidence and experience based intervention and monitoring system and protocol for routine as well as disaster situation. the health service and management structure of surat city have emerged as an urban health model for the country. introduction: the center for healthy communities (chc) in the department of family and com· munity medicine at the medical college of wisconsin developed a pilot project to: 1) assess the know· ledge, attitudes, and behaviors of female milwaukee public housing residents related to breast cancer; 21 develop culturally and literacy appropriate education and screening modules; 3) implement the developed modules; 4) evaluate the modules; and 5) provide follow-up services. using a community-based participatory research model the chc worked collaboratively with on-site nurse case management to meet these objectives. methods: a "breast health kick off event" was held at four separate milwaukee public housing sites for elderly and disabled adults. female residents were invited to complete a 21-item breast health survey, designed to accommodate various literacy levels. responses were anonymous and voluntary. the survey asked women about their previous physical exams for breast health, and then presented a series of state· ments about breast cancer to determine any existing myths. the final part gathered information about personal risk for breast cancer, the highest level of education completed, and whether the respondents h;td ever used hormone replacement therapy and/or consumed alcohol. responses were collected for descriptive analysis. results: a total of 45 surveys (representing 18% of the total female population in the four sites) were completed and analyzed. 89% reported that they had a physical exam in the previous rwo years. 96% of respondents indicated they never had been diagnosed with breast cancer. 85% reported having had a mammogram and 87% having had a clinical breast exam. those that never had a mammogram reported a fear of what the provider would discover or there were not any current breast problems ro warrant an exam. 80% agreed that finding breast cancer early could lower the chance of dying of cancer. over 92% reported that mammograms were helpful in finding cancer. however, 27% believed that hav· ing a mammogram actually prevents breast cancer. 14% indicated that mammograms actually cause cancer and 16% reported that a woman should get a mammogram only if there is breast cancer in her family. conclusion: this survey indicates that current information about the importance of mammograms and clinical breast exams is reaching traditionally underserved women. yet there are still critical oppor· tunities to provide valuable education on breast health. this pilot study can serve as a tool for shaping future studies of health education messages for underserved populations. located in a yourh serv· ~ng agency m downtow~ ottawa, the clinic brings together community partners to provide primary medical care. and dent~i hygiene t? the street youths of ottawa aged 12-20. the primary goal of the project is to provide accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. these efforts respond to the pre-existing body of evidence suggesting that the principle barrier in accessing such care for these youths are feelings of intimidation and vulnerability in the face of a complex healthcare system. the bruyere fhn satellite clinic is located in the basement of a downtown drop-in and brings together a family medicine physician and her residents, a dental hygienist and her 2nd year students, a nurse practitioner, a chiropodist and 2 public health nurses to provide primary care. the clinic has been extremely busy and well received by the youth. this workshop will demonstrate how five community organizations have come together to meet the needs of high risk youths in ottawa. this presentation will showcase the development of the clinic from its inception through its first year including reaction of the youths, partnerships and lessons learned. it will also focus on its sustainability without continued funding. we hope to have developed a model of service delivery that could be reproduced and sustained in other large cities with faculties of medicine. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative-a) semi structured interviews with providers & partners b)focus groups with youth quantitative a)electronic medical records for 12 months records (budget, photos, project information). results: 1) successfully built and opened a medicaudental clinic which will celebrate its 1 year anniversary in august. 2) over 140 youths have been seen, and we have had over 300 visits. conclusion: 1) the clinic will continue to operate beyond the 18 month project funding. 2) the health of high risk youth in ottawa will continue to improve due to increased access to medical services. p7-11 (a) health services -for the citizens of bangalore -past, present and future savita sathyagala, girish rao, thandavamurthy shetty, and subhash chandra bangalore city, the capital of karnataka with 6.5 million is the 6th most populous city in india; supporting 30% of the urban population of karnataka, it is considered as one of the fastest growing cities in india. known as the 'silicon valley of india', bangalore is nearly 500 years old. bangalore city corporation (bmp), is a local self government and has the statutory commitment to provide to the citizens of bangalore: good roads, sanitation, street lighting, safe drinking water apart from other social obligations, cultural development and poverty alleviation activities. providing preventive and promotive heahh services is also a specific component. the objective of this study was to review the planning process with respect to health care services in the period since india independence; the specific research questions being what has been the strategies adopted by the city planners to address to the growing needs of the population amidst the background of the different strategies adopted by the country as a whole. three broad rime ranges have been considered for analysis: the 1950s, 1970s and the 1990s. the salient results are: major area of focus has been on the maternal and child care with activities ranging from day-care to in-patient-care; though the number of institutions have grown from 5 to the current day 79, their distribution has been far from satisfactory; obtaining support from the india population projects 3 and 8 major upgradarions have been undertaken in terms of infrastructure; over the years, in addition to the dispensaries of modern system of medicine, local traditional systems have also been initiated; the city has partnered with the healthy cities campaign with mixed success; disease surveillance, addressing the problems related to the emerging non-communicable diseases including mental health and road traffic injuries are still in its infancy. isolated attempts have been made to address the risks groups of elderly care and adolescent care. what stands out remarkably amongst the cities achievements is its ability to elicit participation from ngos, cbos and neighbourhood groups. however, the harnessing of this ability into the health sector cannot be said totally successful. the moot question in all the above observed development are: has the city rationally addressed it planning needs? the progress made so far can be considered as stuttered. the analysis and its presentation would identify the key posirive elements in the growth of banglore city and spell a framework for the new public health. introduction: anaemia associated with pregnancy is a major public health problem all over the world. different studies in different parts of india shown prevalence of anaemia between 60-90%. anaemia remains a serious health problem in pregnancy despite of strong action taken by the government of india through national programmes. in the present study we identified th~ social beha~iors, responsible for low compliance of if a tablets consumption in pregnancy at community level and intervention was given with new modified behaviors on trial bases. . in vadodara urban. 60 anganwadies out of 289 were selected from the list by random sampling for tips (trials of improved practices) study. . . participants: 266 pregnant women (132, intervention group+ 134, control. group) registered m the above 60 anganwadies. study was conducted in to three phases: phase: 1. formative research and baseline survey (frbs). data was collected from all 266 pregnant women to identify behaviors that are responsible for low compliance of ifa tablets. both qualitative and quantitative data were collected. haemoglobin was estimated of all pregnant women by haemo-cue. phase: 2. phase of tips. behaviors were identified both social & clinical for low compliance of ifa tablets consumption in pregnancy from frbs and against those, modified behaviors were proposed to pregnant women in the intervention group on trial bases by health education. trial period of 6 weeks was given for trial of new behaviors to pregnant women in the interven· tion group. phase: 3. in this phase, feedbacks on behaviors tried or not tried were taken from pregnant women in intervention group. haemoglobin estimation was carried out again in all 266 pregnant women. at the end of the study, messages were formulated on the bases of feedbacks from the pregnant women. results: all pregnant women in the intervention group had given positive feedback on new modified behaviors after intervention. mean haemoglobin concentration was higher in intervention group (10.04±0.11 gm%) than control group (9.60±0.14 gm%). ifa tablets compliance was improved in intervention group (95.6%) than control group (78.6%). conclusion: all pregnant women got benefits after trial of new modified behaviors in the intervention group. messages were formulated from the new modified behaviors, which can be used for longterm strategies for anaemia control in the community. introduction: in order to develop a comprehensive mch handbook for pregnant women and to assess its effect among them, a pilot study was carried out at the maternal and child health training institute (mchti), in dhaka, bangladesh. methods: from mchti a sample of 600 pregnant women was selected and all subjects were women who were attending the first visit of their current pregnancy by using a random sampling method. of the 600 subjects, 240 women were given the mch handbook as case and 360 women were not given the handbook as control. data on pre and post intervention of the handbook from the 240 cases and 360 controls were taken from data recording forms between the 1st of november 2002 and 31st of october, 2003 and data was analysed by using a multilevel analysis approach. this was a hospital-based action (case-control) research, and was applied in order to measure the outcome of pre and post intervention following the introduction of the handbook. data was used to assess the effects of utilisation of the handbook on women's knowledge, practice and utilisation of mch services. results: this study showed that the change of knowledge about antenatal care visits was 77.1% among case mothers. knowledge of danger signs improved 49.2 %, breast feeding results 31.5%, vaccination 32.0% and family planning results improved 60.3% among case. results showed some positive changes in women's attitudes among case mothers and study showed the change of practice in antenatal care visits was .u.5% in the case. other notable changes were: change of practice in case mother's tetanus toxoid (ti), 55.2%; and family planning 41.2%. in addition, handbook assessment study indicated that most women brought the handbook on subsequent visits (83.3%), the handbook was highly utilised (i.e. it was read by 84.2%, filled-in by 76.1 %, and was used as a health education tool by 80.4%). most women kept the handbook (99.5%) and found it highly useful (78.0%) with a high client satisfaction rate of 88.0%. conclusion: pregnant women in the case group had higher knowledge, better practices, and higher utilisation of mch services than mothers in the control groups who used alternative health cards. if the handbook is developed with a focus on utilising a problem-oriented approach and involving the recomendations .of end~users, it is anticipated that the mch handbook will contribute significantly to ensuring the quahry of hfe of women and their children in bangladesh. after several meetmgs to identify the needs of the community, a faso clinic was opened at ncfs. health care professionals from smh joined with developmental and social service workers from ncfs to implement the faso diagnostic process and to provide culturally appropriate after-care. the clinic is unique in that its focus is the high risk urban aboriginal population of toronto. it accepts referrals of not only children and youth, but also of adults. lessons learned: response to the faso clinic at native child and family services has been overwhelming. aboriginal children with f asd are receiving timely diagnosis and interventions. aboriginal youth and adults who have been struggling with poveny, substance abuse, and homelessness are more willing to enter the ncfs centre for diagnosis and treatment. aboriginal infants prenatally exposed to alcohol born at st. michael's hospital or referred by other centres have access to the developmental programs located in both of the partnering agencies. the presentation will describe the clinic's development, and will detail the outcomes described, including interventions unique to the aboriginal culture. p7-15 (c) seeds, soil, and stories: an exploration of community gardening in southeast toronto carolin taran, sarah wakefield, jennifer reynolds, and fiona yeudall introduction: community gardens are increasingly seen as a mechanism for improving nutrition and increasing food security in urban neighbourhoods, but the evidence available to support these claims is limited. in order to begin to address this gap in a way that is respectful of community knowledge and needs, the urban gardening research opportunities workgroup (ugrow) project explored the benefits and potential risks of community gardening in southeast toronto. the project used a community-based research (cbr) model to assess community gardens as a means of improving local health. the research process included interviews, focus groups, and participant observation (documented in field notes). we also directly engaged the community in the research process, through co-learning activities and community events which allowed participants to express their views and comment on emerging results. most of the research was conducted by a community-based research associate, herself a community gardener. key results were derived from these various sources through line-by-line coding of interview transcripts and field note review, an interactive and iterative process which involved both academic and community partners. results: these various data sources all suggest that enhanced health and access to fresh produce are important components of the gardening experience. they also highlight the central importance of empowering and community-building aspects of gardening to gardeners. community gardens were thought to play a role in developing friendships and social support, sharing food and other resources, appreciating cultural diversity, learning together, enhancing local place attachment and stewardship, and mobilizing to solve local problems (both inside and outside the garden). potential challenges to community gardens as a mechanism for communiry development include bureaucratic resistance to gardens, insecure land tenure and access, concerns about soil contamination, and a lack of awareness and under· standing by community members and decision-makers of all kinds. conclusion: the results highlight many health and broader social benefits experienced by commu· nity gardeners. they also point to the need for greater support for community gardening programs, par· ticularly ongoing the ongoing provision of resources and education programs to support gardens in their many roles. this research project is supported by the wellesley central health corporation and the centre for urban health initiatives, a cihr funded centre for research development hased at the univer· sity of toronto. p7-16 (c) developing resiliency in children living in disadvantaged neighbourhoods sarah farrell, lorna weigand, and wayne hammond the traditional idea of targeting risk reduction by focusing on the development of eff~ctive coping strategies and educational programs has merit in light of the research reportmg_ that_ ~10lupl.e forms of problem behaviour consistently appear to be predicted by increasing exposure to 1den_uf1able risk factors. as a result, many of the disadvantaged child and youth studies have focused on trymg to better _unde.r· stand the multiple risk factors that increase the likelihood of the development of at nsk behaviour m ch1ldren/youth and the potential implications for prevention. this in turn has led t_o. the conclus1on that community and health programs need to focus on risk reduction by helpm~ md1v1duals develop more effective coping strategies and a better understanding of the limitations of cenam pathologies, problematic v156 poster sessions coping behaviours and risk factors potentially inheren~ in high needs co~unities. ~owever, another ai:ea of research has proposed that preventative interventions should cons1de~ .~rotecnve fa~ors alo~~ with reducing risk factors. as opposed to just emphasizing problems, vulnerab1ht1es, and deficits, a res1liencybased perspective holds the belief that children, youth and their families. have strengths, reso~ce.s and the ability to cope with significant adversity in ways that are not only effective, but tend to result m mcreased ability to constructively respond to future adversity. with this in mind, a participatory research project sponsored by the united way of greater toronto was initiated to evaluate and determine the resiliency profiles of children 8 -12 years (n = 500) of recent immigrant families living in significantly disadvantaged communities in the toronto area. the presentation will provide an overview of the identified protective factors (both intrinsic and extrinsic) and resiliency profiles in an aggregated format as well as a summary of how the children and their parents interpreted and explained these strength-based results. as part of the focus groups, current community programs and services were examined by the participants as to what might be best practices for supporting the development and maintaining of resiliency in children, families and communities. it was proposed that the community model of assessing resiliency and protective factors as well as proposed best strength-based practice could serve as a guide for all in the community sector who provide services and programs to those in disadvantaged neighbourhoods. p7-17 (c) naloxone by prescription in san francisco, ca and new york, ny emalie huriaux the harm reduction coalition's overdose project works to reduce the number of fatal overdoses to zero. located in new york, ny and san francisco, ca, the overdose project provides overdose education for social service providers, single-room occupancy hotel (sro) residents, and syringe exchange participants. the project also conducts an innovative naloxone prescription program, providing naloxone, an opiate antagonist traditionally administered by paramedics to temporarily reverse the effects of opiate overdose, to injection drug users (idus). we will describe how naloxone distribution became a reality in new york and san francisco, how the project works, and our results. the naloxone prescription program utilizes multiple models to reach idus, including sro-and street-based trainings, and office-based trainings at syringe exchange sites. trainings include information on overdose prevention, recognition, and response. a clinician conducts a medical intake with participants and provides them with pre-filled units of naloxone. in new york, funding was initially provided by tides foundation. new york city council provides current funding. new york department of mental health and hygiene provides program oversight. while the new york project was initiated in june 2004, over half the trainings have been since march 2005. in san francisco, california endowment, tides foundation, and san francisco department of public health (sfdph) provide funding. in addition, sfdph purchases naloxone and provides clinicians who conduct medical intakes with participants. trainings have been conducted since november 2003. to date, nearly 1000 individuals have been trained and provided with naloxone. approximately 130 of them have returned for refills and reported that they used naloxone to reverse an opiate-related overdose. limited episodes of adverse effects have been reported, including vomiting, seizure, and "loss of friendship." in new york, 400 individuals have been trained and provided with naloxone. over 30 overdose reversals have been reported. over half of the participants in new york have been trained in the south bronx, the area of new york with the highest rate of overdose fatalities. in san francisco, 570 individuals have been trained and provided with naloxone. over 96 overdose reversals have been reported. the majority of the participants in san francisco have been trained in the tenderloin, 6th street corridor, and mission, areas with the highest rates of overdose fatalities. the experience of the overdose project in both cities indicates that providing idus low-threshold access to naloxone and overdose information is a cost-effective, efficient, and safe intervention to prevent accidental death in this population. p7-18 (c) successful strategies to regulate nuisance liquor stores using community mobilization, law enforcement, city council, merchants and researchers tahra goraya presenta~ion _will discuss ~uccessful environmental and public policy strategies employed in one southen:1 cahf?rmna commumty to remedy problems associated with nuisance liquor stores. participants ~111 be given tools to understand the importance of utilizing various substance abuse prevention str~tegi~ to change local policies and the importance of involving various sectors in the community to a~_1st with and advocate for community-wide policy changes. recent policy successes from the commultles of pa~ad~na and altad~na will highlight the collaborative process by which the community mobilized resulnng m several ordmances, how local law enforcement was given more authority to monitor poster sessions v157 nonconforming liquor stores, how collaborative efforts with liquor store owners helped to remove high alcohol content alcohol products from their establishments and how a community-based organiz,uion worked with local legislators to introduce statewide legislation regarding the regulation of nuisance liquor outlets. p7-19 (c) "dialogue on sex and life": a reliable health promotion tool among street-involved youth beth hayhoe and tracey methven introduction: street involved youth are a marginalized population that participate in extremely risky behaviours and have multiple health issues. unfortunately, because of previous abuses and negative experiences, they also have an extreme distrust of the adults who could help them. in 1999, toronto public health granted funding to a non governmental, nor for profit drop-in centre for street youth aged 16-24, to educate them about how to decrease rhe risk of acquiring hiv. since then the funding has been renewed yearly and the program has evolved as needed in order to target the maximum number of youth and provide them with vital information in a candid and enjoyable atmosphere. methods: using a retrospective analysis of the six years of data gathered from the "dialogue on sex and life" program, the researchers examined the number of youth involved, the kinds of things discussed, and the number of youth trained as peer leaders. also reviewed, was written feedback from the weekly logs, and anecdotal outcomes noted by the facilitators and other staff in the organization. results: over the five year period of this program, many of youth have participated in one hour sessions of candid discussion regarding a wide range of topics including sexual health, drug use, harm reduction, relationship issues, parenting, street culture, safety and life skills. many were new youth who had not participated in the program before and were often new to the street. some of the youth were given specific training regarding facilitation skills, sexual anatomy and physiology, birth control, sexually transmitted infections, hiv, substance use/abuse, harm reduction, relationships and discussion of their next steps/future plans following completion of the training. feedback has been overwhelmingly positive and stories of life changing decisions have been reported. conclusion: clearly, this program is a successful tool to reach street involved youth who may otherwise be wary of adults and their beliefs. based on data from the evaluation, recommendations have been made to public health to expand the funding and the training for peer leaders in order ro target between 100-200 new youth per year, increase the total numbers of youth reached and to increase the level of knowledge among the peer leaders. p7-20 (c) access to identification and services jane kali replacing identification has become increasingly more complex as rhe government identification issuing offices introduce new requirements rhar create significant barriers for homeless people to replace their id. new forms of identification have also been introduced that art' not accessible to homekss peoplt-(e.g. the permanent resident card). ar rhe same time, many service providers continue to require identifi· cation ro access supports such as income, housing, food, health care, employment and employmt·nt training programs. street health, as well as a number of other agencies and community health centres, h,1, been assisting with identification replacement for homeless peoplt· for a number of years. the rnrrt·nr challenges inherent within new replacement requirements, as well as the introduction of new forn1' of identification, have resulted in further barriers homeless people encounter when rrring to access t:ssential services. street health has been highlighting these issues to government identification issuing offices, as well as policy makers, in an effort to ensure rhar people who are homeless and marginalized have ac'ess to needed essential services. bandar is a somali word for •·a safe place." the bandar research project is the product of the regent park community health centre. the research looks ar the increasing number of somali and afri· can men in the homeless and precariously house population in the inner city core of down~own toronto. in the first phase of the pilot project, a needs assessment was conducted to 1dennfy barners and issues faced by rhe somali and other african men who are homeless and have add1cr10ns issues. th_e second phase of rhe research project was to identify long rerm resources and service delivery mechamsms that v158 poster sessions would enhance the abiity of this population to better access detox, treatment, and post treatment ser· vices. the final phase of the project was to facilitate the development of a conceptual model of seamless continual services and supports from the streets to detox to treatment to long term rehabilitation to housing. "between the pestle and mortar" -safe place. p7-22 (c) successful methods for studying transient populations while improving public health beth hayhoe, ruth ewert, eileen mcmahon, and dan jang introduction: street youth are a group that do not regularly access healthcare because of their mis· trust of adults. when they do access health care, it is usually for issues severe enough for hospitalization or for episodic care in community clinics. health promotion and illness prevention is rarely a part of their thinking. thus, standard public health measures implemented in a more stable population do not work in this group. for example, pap tests, which have dearly been shown to decrease prevalence of cer· vical cancer, are rarely done and when they are, rarely followed up. methods to meet the health care needs and increase the health of this population are frequently being sought. methods: a drop-in centre for street youth in canada has participated in several studies investigating sexual health in both men and women. we required the sponsoring agencies to pay the youth for their rime, even though the testing they were undergoing was necessary according to public health stan· dards. we surmised that this would increase both initial participation and return. results: many results requiring intervention have been detected. given the transient nature of this population, return rates have been encouraging so far. conclusion: it seems evident that even a small incentive for this population increases participation in needed health examinations and studies. it is possible that matching the initial and follow-up incentives would increase the return rate even further. the fact that the youth were recruited on site, and not from any external advertising, indicates that studies done where youth trust the staff, are more likely to be successful. the presentation will share the results of the "empowering stroke prevention project" which incor· porated self-help mutual aids strategies as a health promotion methodology. the presentation will include project's theoretical basis, methodology, outcomes and evaluation results. self-help methodology has proven successful in consumer involvement and behaviour modification in "at risk," "marginalized" settings. self-help is a process of learning with and from each other which provides participants oppor· tunities for support in dealing with a problem, issue, condition or need. self-help groups are mechanisms for the participants to investigate existing solutions and discover alternatives, empowering themselves in this process. learning dynamic in self-help groups is similar to that of cooperative learning and peertraining, has proven successful, effective and efficient (haller et al, 2000) . the mutual support provided by participation in these groups is documented as contributory factor in the improved health of those involved. cognizant of the above theoretical basis, in 2004 the self-help resource centre initiated the "empowering stroke prevention project." the project was implemented after the input from 32 health organizations, a scan of more than 300 resources and an in-depth analysis of 52 risk-factor-specific stroke prevention materials indicated the need for such a program. the project objectives were:• to develop a holistic and empowering health promotion model for stroke prevention that incorporates selfhelp and peer support strategies. • to develop educational materials that place modifiable risk factors and lifestyle information in a relevant context that validates project participants' life experiences and perspectives.• to educate members of at-risk communities about the modifiable risk factors associated with stroke, and promote healthy living. to achieve the above, a diverse group of community members were engaged as "co-editors" in the development of stroke prevention education materials which reflected and validated their life experiences. these community members received training to become lay health promoters (trained volunteer peer facilitators). in collaboration with local health organizations, these trained lay health promoters were then supported in organizing their own community-based stroke prevention activities. in addition, an educational booklet written in plain language, entitled healthy ways to prevent stroke: a guide for you, and a companion guide called healthy ways to pre· vent stroke: a facilitator's guide were produced. the presentation will include the results of a tw<>tiered evaluation of the program methodology, educational materials and the use of the materials beyond the life of the project. this poster presentation will focus on the development and structure of an innovative street outreach service that assists individuals who struggle mental illness/addictions and are experiencing homelessness. the mental health/outreach team at public health and community services (phcs) of hamilton, ontario assists individuals in reconnecting with health and social services. each worker brings to the ream his or her own skills-set, rendering it extremely effective at addressing the multidimensional and complex needs of clients. using a capacity building framework, each ream member is employed under a service contract between public health and community services and a local grassroots agency. there are public health nurses (phn), two of whom run a street health centre and one of canada's oldest and most successful needle exchange programs, mental health workers, housing specialists, a harm reduction worker, youth workers, and a united church minister, to name a few. a community advisory board, composed of consumers and professionals, advises the program quarterly. the program is featured on raising the roors 'shared learnings on homelessness' website at www.sharedlearnings.ca. through our poster presentation participants will learn how to create effective partnerships between government and grassroots agencies using a capacity building model that builds on existing programs. this study aims to assess the effects of broadcasting a series of documentary and drama videos, intended to provide information about the bc healthguide program in farsi, on the awareness about and the patterns of the service usage among farsi-speaking communities in the greater vancouver area. the major goals of the present study were twofold; ( 1) to compare two methods of communications (direct vs. indirect messages) on the attitudes and perceptions of the viewers regarding the credibility of messengers and the relevance of the information provided in the videos, and (2) to compare and contrast the impact of providing health information (i.e., the produced videos) via local tvs with the same materials when presented in group sessions (using vcr) on participants' attitudes and perceptions cowards the bc healrhguide services. results: through a telephone survey, 545 farsi-speaking adults were interviewed in november and december 2004. the preliminary findings show that 53% of the participants had seen the aired videos, from which, 51 % watched at least one of the 'drama' clips, 8% watched only 'documentary' clip, and 41% watched both types of video. in addition, 27% of the respondents claimed that they were aware about the program before watching the aired videos, while 73% said they leaned about the services only after watching the videos. from this group, 14% said they called the bchg for their own or their "hildren's health problems in the past month. 86% also indicated that they would use the services in the future whenever it would be needed. 48% considered the videos as "very good" and thought they rnuld deliver relevant messages and 21 % expressed their wish to increase the variety of subjects (produ\:e more videos) and increase the frequency of video dips. conclusion: the results of this study will assist public health specialists in bc who want to choose the best medium for disseminating information and apply communication interventions in multi\:ultural communities. introduction: many theorists and practitioners in community-based research (cbr) and knowledge transfer (kt) strongly advocate for involvement of potential users of research in the development of research projects, yet few examples of such involvement exist for urban workplace health interventions. we describe the process of developing a collaborative research program. methods: four different sets of stakeholders were identified as potential contributors to and users of the research: workplace health policy makers, employers, trade unions, and health and safety associations. representatives of these stakeholders formed an advisory committee which met quarterly. over the 13 month research development period, an additional 21 meetings were held between resc:ar~h~rs and stakeholders. in keeping with participant observation approaches, field notes of group and md1v1~ ual meetings were kept by the two co-authors. emails and telephone calls were also documented. qu~h tative approaches to textual analysis were used, with particular attention paid to collaborattve v160 poster sessions relationships established (as per cbr), indicators of stakeholders' knowledge utilization (as per kt), and transformations of the proposed research (as per cbr). results: despite initial strong differences of opinion both among stakeho~ders .an~ between stakeholders and researchers, goodwill was noted among all involved. acts of rec~proc1ty included mu.rual sharing of assessment tools, guidance on data utilization to stakeho~der orga~1zat10ns, and suggestions on workplace recruitment to researchers. stakeholders demonstrated mcreases m concep~ual. un~erstand ing of workplace health e.g. they more commonly discussed more complex,. psychosocial md1cators of organizational health. stakeholders made instrumental use of shared materials based on research e.g. adapting their consulting model to more sophisticated dat~ analysis. sta~ehol?~rs recogni_zed the strategic use of their alliance with researchers e.g., transformational leadership trainmg as a~ inducement to improve health and safety among small service franchises. stakeholders helped re-define the research questions, dramatically changed the method of recruitment from researcher cold call to stakeholderbased recruitment, and strongly influenced pilot research designs. owing a great deal to the elaborate joint development process, the four collaboratively developed pilot project submissions which were all successfully funded. conclusion: the intensive process of collaborative development of a research program among stakeholders and researchers was not a smooth process and was time consuming. nevertheless, the result of the collaborative process was a set of projects that were more responsive to stakeholder needs, more feasible for implementation, and more broadly applicable to relevant workplace health problems. introduction: environmental groups, municipal public health authorities and, increasingly, the general public are advocating for reductions in pesticide use in urban areas, primarily because of concern around potential adverse health impacts in vulnerable populations. however, limited evidence of the relative merits of different intervention strategies in different contexts exists. in a pilot research project, we sought to explore the options for evaluating pesticide reduction interventions across ontario municipalities. methods: the project team and a multi-stakeholder project advisory committee (pac), generated a list of potential key informants (kl) and an open ended interview guide. thirteen ki from municipal government, industry, health care, and environmental organizations completed face to face or telephone interviews lasting 30-40 minutes. in a parallel process, a workshop involving similar representatives and health researchers was held to discuss the role of pesticide exposure monitoring. minutes from pac meetings, field notes taken during ki interviews, and workshop proceedings were synthesized to generate potential evaluation methods and indicators. results: current evaluation activities were limited but all kls supported greater evaluation effons beginning with fuller indicator monitoring. indicators of education and outreach services were imponant for industry representatives changing applicator practices as well as most public health units and environmental organizations. lndictors based on bylaw enforcement were only applicable in the two cities with bylaws, though changing attitudes toward legal approaches were being assessed in many communities. the public health rapid risk factor surveillance system could use historical baseline data to assess changes in community behaviour through reported pesticide uses and practices, though it had limited penetration in immigrant communities not comfortable in english. pesticide sales (economic) data were only available in regional aggregates not useful for city specific change documentation. testing for watercourse or environmental contamination might be helpful, but it is sporadic and expensive. human exposure monitoring was fraught with ethical issues, floor effects from low levels of exposure, and prohibitive costs. clinical episodes of pesticide exposure reported to the regional poison centre (all ages) or the mother risk program (pregnant or breastfeeding women) are likely substantial underestimates that would be need to be supplemented with sentinel practice surveillance. focus on special clinical populations e.g., multiple chemical sensitivity would require additional data collection efforts . . conc~ons: broad support for evaluation and multiple indicators were proposed, though con-s~raints associate~ with access, coverage, sensitivity and feasibility were all raised, demonstrating the difficulty of evaluating such urban primary prevention initiatives. interventionists. an important aim of the youth monitor is to learn more about the health development of children and adolescents and the factors that can influence this development. special attention is paid to emo· tional and behavioural problems. the youth monitor identifies high-risk groups and factors that are associated with health problems. at various stages, the youth monitor chancrs the course of life of a child. the sources of informa· tion and methods of research are different for each age group. the results arc used to generate various kinds of repons: for children and young persons, parents, schools, neighbourhoods, boroughs and the municipality of rotterdam and its environs. any problems can be spotted early, at borough and neigh· bourhood level, based on the type of school or among the young persons and children themselves. together with schools, parents, youngsters and various organisations in the area, the municipal health service aims to really address these problems. on request, an overview is offered of potentially suitable interventions. the authors will present the philosophy, working method, preliminary effects and future developments of this instrument, which serves as the backbone for the rotterdam local youth policy. social workers to be leaders in response to aging urban populations: the practicum partnership program sarah sisco, alissa yarkony, and patricia volland 1"'"1tliu:tion: across the us, 77.5% of those over 65 live in urban areas. these aging urban popu· lations, including the baby boomers, have already begun encounter a range of heahh and mental hcahh conditions. to compound these effects, health and social service delivery fluciuates in cities, whit:h arc increasingly diverse both in their recipients and their systems. common to other disciplines (medicine, nursing, psychology, etc.) the social work profession faces a shortage of workers who are well-equipped to navigate the many systems, services, and requisite care that this vast population requires. in the next two decades, it is projected that nearly 70,000 social workers will be required to provide suppon to our older urban populations. social workers must be prepared to be aging-savvy leaders in their field, whether they specialize in gerontology or work across the life span. mllhotu: in 2000, a study conducted at the new york academy of medicine d<>1:umcntcd the need for improved synchroniciry in two aspects of social work education, classroom instruction and the field experience. with suppon from the john a. hanford foundation, our team created a pilot proj~"t entitled the practicum pannership program (ppp) in 11 master's level schools of social work, to improvt" aginr exposure in field and classroom content through use of the following: i) community-university partnrr· ships, 2) increased, diverse student field rotations, ll infusion of competcn1."}'·drivm coursework, 41 enhancement of field instructors' roles, and 5) concentrated student recruitment. we conductt"d a prr· and post-test survey into students' knowledge, skills. and satisfaction. icarlja: surveys of over 400 graduates and field inltnk."tors rcflected increased numlk-n of .1rrm:y· univmity panncrships, as well as in students placed in aging agencin for field placements. there wa1 11 marked increase in student commitments to an aging specialization. onr year por.t·gradu:nion rcvealrd that 93% of those surveyed were gainfully employed, with 80% employed in the field of aginic. by com· bining curricular enhancement with real-world experiences the ppp instilled a broad exposurr for llu· dents who worked with aging populations in multiple urban settings. coltdtuion: increased exposure to a range of levels of practicr, including clinical, policy/ajvocaq, and community-based can potentially improve service delivery for older adulh who live in elfin, and potentially improve national policy. the hanford foundation has now elected to 1uppon cxpantion of the ppp to 60 schools nationwide (urban and rural) to complement other domntic initiatives to cnhalk"c" holistic services for older adults across the aging spectrum. bodrgnn.ntl: we arc a team of rcscarcbcn and community panncn working tcj8c(her to develop an in"itepth understanding of the mental health needs of homeless youth ~ages 16 to 24) (using qualiutivc and quantitative methods 8' panicipatory rncarch methods). it is readily apparmt that '-neless youth cxpcricnce a range of mental health problems. for youth living on the street, menul illnew may be either a major risk factor for homelessnal or may frequently emcsge in response to coping with rhe multitudinous stressors associated with homclcslllcsi including exposure to violence, prasutt to pamaplte in v162 poster sessions survival sex and/or drug use. the most frequent psychiatric diagnoses amongst the homeless gencrally include: depression, anxiety and psychosis. . . . the ultimate ob1ective of the pr~am of rei:e~ is to ~evelop a plan for intervention to meet the mental health needs of street youth. prior t_o pl~nnmg mtervenbons, .it is necessary to undertake a comprehensive assessment ~f mental health needs m this ~lnerable populanon. thus, the immediate objective of this research study is to undertake a comprehensive assessment of men· tal health needs. . . melbotlology: a mixed methodology triangulating qualitative, participatory acnon and quantitative methods will capture the data related to mental health needs of homeless youth. a purposive sample of approximately 60-80 subjecrs. ages 16 to 24, is currently being ~ted ~participate from the commu.nity agencies covenant house, evergreen centre fo~ srrc;et youth, turning p?1?t and street ~ serv~. youth living on the street or in short -term residennal programs for a mmimum of 1 month pnor to their participation; ages 16 to 24 and able to give infonned consent will be invited to participate in the study. o..tcomes: the expected outcome of this initial survey will be an increased understanding of mental health needs of street youth that will be used to develop effective interventions. it is anticipated that results from this study will contribute to the development of mental health policy, as well as future programs that are relevant to the mental health needs of street youth. note: it is anticipated that preliminary quantitative data (25 subjects) and qualitative data will be available for the conference. the authors intend to present the identification of the research focus, the formation of our community-based team, relevance for policy, as well as preliminary results. p7-31 (a) the need for developing a firm health policy for urban informal worken: the case of despite their critical role in producing food for urban in kenya, urban farmers have largely been ignored by government planners and policymakers. their activity is at best dismissed as peripheral eveo, inappropriate retention of peasant culture in cities and at worst illegal and often some-times criminal· ized. urban agriculture is also condemned for its presumed negative health impact. a myth that contin· ues despite proof to the contrary is that malarial mosquitoes breed in maize grown in east african towns. however, potential health risks are insignificant compared with the benefits of urban food production. recent studies too rightly do point to the commercial value of food produced in the urban area while underscoring the importance of urban farming as a survival strategy among the urban poor, especially women-headed households. since the millennium declaration, health has emerged as one of the most serious casualties consequent on the poverty, social exclusion, marginalisation and lack of sustain· able development in africa. hiv/aids epidemic poses an unprecedented challenge, while malaria, tuber· culosis, communicable diseases of childhood all add to the untenable burden. malnutrition underpins much ill-health and is linked to more than 50 per cent of all childhood deaths. kenya's urban poor people ~ace ~ h~ge burde~ of preventable and treatable health problems, measured by any social and bi~ medical md1cator, which not only cause unnecessary death and suffering, but also undermine econonuc development and damage the country's social fabric. the burden is in spite of the availability of suitable tools and re:c=hnology for prevention and treatment and is largely rooted in poverty and in weak healah •rstems. this pa~ therefore challenges development planners who perceive a dichotomy instead of con· tmuum between informal and formal urban wage earners in so far as access to health services is con· cemed. it i~ this gap that calls for a need to developing and building sustainable health systems among the urban mformal ~wellers. we recommend a focus on an urban health policy that can build and strengthen the capacity of urban dwellers to access health services that is cost-effective and sustainable. such ~ health poli<=>: must strive for equity for the urban poor, displaced or marginalized; mobilise and effect1~ely use sufficient sustainable resources in order to build secure health systems and services. special anenti_on. should ~ afforded hiv/aids in view of the unprecedented challenge that this epidemic poses to africa s economic and social development and to health services on the continent. methods: a review of the literature led us to construct three simple models and a composite model of exposure to traffic. the data were collected with the help of a daily diary of travel activities using a sample of cyclists who went to or come back from work or study. to calculate the distance, the length of journey, and the number of intersections crossed by a cyclist different geographic information systems (gis) were operated. statistical analysis was used to determine the significance between a measure of exposure on the one hand, and the sociodemographic characteristics of the panicipants or their geographic location on the other hand. restlltj: our results indicate that cyclists were significantly exposed to road accidents, no matter of where they live or what are their sociodemographic characteristics. we also stress the point that the fact of having been involved in a road accident was significantly related to the helmet use, but did not reduce the propensity of the cyclists to expose themselves to the road hazards. condlllion: the efforts of the various authorities as regards road safety should not be directed towards the reduction of the exposure of the vulnerable users, but rather towards the reduction of the dangers to which they could face. keywords: cyclist, daily diary of activities, measures of exposure to traffic, island of montreal. p7-33 (a) intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods olumuyiwa akinbamijo intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods abstract urbanization panicularly in nigerian cities, ponends unprecedented crises of grave dimensions. from physical and demographic viewpoints, city growth rates are staggering coupled with gross inabilities to cope with the consequences. environmental and social ills associated with unguarded rapid urbanization characterize nigerian cities and threaten urban existence. this paper repons the findings of a recent study of the relationship between environmental health across inrraurban residential communities of akure, south west nigeria. it discuses the typical urbanization process of nigerian cities and its dynamic spatial-temporal characteristics. physical and socio-demographic attributes as well as the levels and effectiveness of urban infrastructural services are examined across the core residential districts and the elite residential layouts in the town. the incidence rate of cenain environmentally induced tropical diseases across residential neighborhoods and communes is examined. salient environmental variables that are germane to health procurement in the residential districts, incidence of diseases and diseases parasitology, diseases prevention and control were studied. field data were subjected to analysis ranging from the univariate and bivariate analysis. inferential statistics using the chi-square test were done to establish the truthfulness of the guiding hypothesis. given the above, the study affirms that there is strong independence in the studied communities, between the environment and incidence of diseases hence health of residents of the town. this assertion, tested statistically at the district levels revealed that residents of the core districts have very strong independence between the environment and incidences of diseases. the strength of this relationship however thins out towards the city peripheral districts. the study therefore concludes that since most of the city dwellers live in urban deprivation, urban health sensitive policies must be evolved. this is to cater for the urban dwellers who occupy fringe peripheral sites where the extension of facilities often times are illegally done. urban infrastructural facilities and services need be provided as a matter of public good for which there is no exclusive consumption or access even for the poorest of the urban poor. many suffer from low-self esteem, shame and guilt about their drug use. in addition, they often lack suppon or encounter opposition from their panners, family and friends in seeking treatment. these personal barriers are compounded by fragmented addiction, prenatal and social care services, inflexible intake systems and poor communication among sectors. the experience of accessing adequate care between services can be overwhelming and too demanding. the toronto centre for substance use in pregnancy (t-cup) is a unique program developed to minimize barriers by providing kone-stop" comprehensive healthcare. t-cup is a primary care based program located in the department of family medicine at st. joseph\'s health centre, a community teaching hospital in toronto. the interdisciplinary staff provides prenatal and addiction services, case management, as well as care of newborns affected by substance use. regular care plan meetings are held between t-cup, labour and delivery nurses and social workers in the y164 poster sessions maternity and child care program. t-cup also connects "'.omen with. inpatient treatment programs and community agencies such as breaking the cycle, an on-site counselmg group for pregnant substance users. · f · d d h ith method: retrospective chart review, qualitative patient ~ans action stu ~· an ea care provider surveys are used to determine outcomes. primary outcomes mclude changes m maternal su~tance use, psychosocial status and obstetrical complications (e.g. pre-rupture of membrane, pre-eclampsia, placen· ral abruption and hemorrhage). neonatal measures ~~nsisted of .bir~h pa_rame~ers, length of h~spital st.ay and complications (e.g. feral distress, meconium stammg, resuscitation, 1aund1ce, hypoglycemia, seventy of withdrawal and treatment length). chart review consisted of all t-cup patients who met clinical cri· reria for alcohol or drug dependence and received prenatal and intra-partum care at st. joseph's from october 2003 to june 2005. participants in the qualitative study included former and current t-cup patients. provider surveys were distributed on-site and to a local community hospital. raulb: preliminary evaluation has demonstrated positive results. treatment retention and satisfaction rates were high, maternal substance use was markedly reduced and neonatal outcomes have shown to be above those reported in literature. conclusion: this comprehensive, primary care model has shown to be optimal in the management of substance use in pregnancy and for improving neonatal outcomes. future research will focus on how this inexpensive program can be replicated in other health care settings. t-cup may prove to be the optimal model for providing care to pregnant substance users in canada. lntrod11ction: cigarette smoking is one of the most serious health problems in taiwan. the prevalence of smoking in 2002 is 48.1 % in males 5.9% in females aged 18 years and older. although the government of taiwan passed a tobacco hazards control act in 1997, it has not been strongly enforced in many places. therefore, community residents have often reported exposure of second hand smoke. the purpose of the study was to establish a device to build up more smoke-free environments in the city of tainan. methods: unique from traditional intervention studies, the study used a healthy city approach to help build up smoke-free environments. the major concept of the approach is to build up a healthy city platform, including organizing a steering committee, setting up policies and indicators, creating intersectoral collaboration, and increasing community participation. first, more than 80 enthusiastic researchers, experts, governmental officers, city counselors and community leaders in tainan were invited in the healthy city committee. second, smoke-free policies, indicators for smoke-free environments, and mechanisms for inter-departmen· tal inspections were set up. third, community volunteers were recruited and trained for persuading related stakeholders. lastly, both penalties and rewards were used for help build up the environments. raults: aher two-year (2003 aher two-year ( -2005 execution of the project, the results qualitatively showed that smoke-free environments in tainan were widely accepted and established, including smoke-free schools, smoke-free workpla~es, smoke-free households, smoke-free internet shops, and smoke-free restaurants. smoke~s were. effectively educated not to smoke in public places. community residents including adults and children m the smoke-free communities clearly understand the adverse effects of environmental tobacco smoke and actively participated anti-smoking activities. conclruions: healthy city platform is effective to conquer the barrier of limited anti-smoking rc:sources. nor. only can it enlar:ge community actions for anti-smoking campaigns, but also it can provide par_merships for collaboratjon. by establishing related policies and indicators the effects of smoke· free environments can be susta1·ned a d th · · · ' · n e progression can be monitored m a commuruty. these issues are used ~· oi::c it~ goals, weuha identifies issues that put people's health at risk. presently, team com~u:c: ran ee~tion !earns. (iats) that design integrative solutions ~tesj'°~ g om six to fifteen members. methods in order to establish wo-poster sessions v165 projects for weuha, the following approach was undertaken: i. a project-polling template was created and sent to all members of the alliance for their input. each member was asked to identify thdr top two population groups, and to suggest a project on which to focus over a 12-18 month period for each identified population. 2. there was a 47% response to the poll and the top three population groups were identified. data from the toronto community health profile database were utilized to contextualize the information supplied for these populations. a presentation was made to the steering committee and three population-based projects were selected, leaders identified and iats formed. three population-based projects: the population-based projects and health care issues identified are: newcomer prenatal uninsured women; this project will address the challenges faced by providers to a growing number of non-insured prenatal women seeking care. a service model where the barrier of "catchments" is removed to allow enhanced access and improved and co-ordinated service delivery will be pilot-tested. children/obesity/diab etes: using a health promotion model this team will focus on screening, intervention, and promoting healthy lifestyles (physical activity and nutrition) for families as well as for overweight and obese children. seniors health promotion and circle of discharge: this team will develop an early intervention model to assist seniors/family unit/caregivers in accessing information and receiving treatment/care in the community. the circle of discharge initiative will address ways of utilizing community supports to keep seniors in the community and minimize readmissions to acute care facilities. results/expected outcomes: coordinated and enhanced service delivery to identified populations, leading to improved access, improved quality of life, and health care for these targeted populations. introduction: basic human rights are often denied to high-risk populations and people living with hiv/aids. their rights to work and social security, health, privacy, non discrimination, liberty and freedom of movement, marriage and having a family have been compromised due to their sero-positive status and risk of being positive. the spread of hiv/aids has been accelerating due to the lack of general human rights among vulnerable groups. to formulate and implement effective responses needs dialogue and to prevent the epidemic to go underground barriers like stigma need to be overcome. objective: how to reduce the situation of stigma, discrimination and human rights violations experienced by people living with hiv/aids and those who are vulnerable to hiv/aids. methodology and findings: consultation meetings were strm.-rured around presentations, field visits, community meetings and group work to formulate recommendations on how govt and ngos/cbos should move forward based on objective. pakistan being a low prevalence country, the whole sense of compl;u:enc.:y that individuals are not subject to situations of vulnerable to hiv is the major threat to an explosion in th•· epidemic, therefore urgent measures are needed to integrate human rights issues from the very start of the response. the protection and promotion of human rights in an integral component of ;tll responses to the hiv/aids epidemic. it has been recognized that the response to hiv/aios must he multi sectoral and multi faceted, with each group contributing its particular expertise. for this to occur along with other knowlcdg<" more information is required in human rights abuses related to hiv/ aids in a particular scenario. the ~·on sultarion meetings on hiv/aids and human rights were an exemplary effort to achieve the same ohj<..:tivc. recommendations: the need for a comprehensive, integrated and a multi-sectoral appro;u.:h in addressing the issue of hiv/aids was highlighted. the need social, cultural and religious asp•·ct' to he: prominently addressed were identified. it was thought imperative measures even in low prevalence countries. education has a key role to play, there is a need for a code of ethics for media people and h<"alth care providers and violations should be closely monitored and follow up action taken. p7-38 (c) how can community-based funding programs contribute to building community capacity and how can we measure this elusive goal? mary frances maclellan-wright, brenda cantin, mary jane buchanan, and tammy simpson community capacity building is recognized by the public health agency of canada (phac) as an important strategy for improving the overall health of communities by enabling communities to addre~s priority issues such as social and economic determinants of health. in 2004/2005 phac.:, alberta/nwf region's population health fund (phf) supported 12 community-based projects to build community capacity on or across the determinants of health. specifically, this included creating accessible and sup· portive social and physical environments as well as creating tools and processes necessary for healthy policy development and implementation. the objective of this presentation is to highlight how the community capacity building tool, developed by phac ab/nwf region, can demonstrate gains in v166 poster sessions · · the course of a pror· ect and be used as a reflective tool for project planning and community capacity over . . . . i · a art of their reporting requirements, 12 pro1ect sites completed the community caparny eva uanon. s p . . th t i ii i'd d . building tool at the beginning and end of their ~ne-year prorect. e oo ~o ects va 1 an reliable data in the context of community-based health prorects. developed through a vigorous ~nd collabora11ve research process, the tool uses plain languag~ to expl~re nine key f~atures o~ commuruty cap~city with 35 't ch with a section for contextual information, 26 of which also mdude a four-pomt raong 1 ems, ea f fu d · scale. results show an increase in community capacity over the course o the nde prorects. pre and post aggregate data from the one-year projects measure~ statistic.ally si~n~ficant changes for 17 of the 26 scaled items. projects identified key areas of commumty capacity bmldmg that needed strengthemng, such as increasing participation, particularly among people with low incomes; engaging community members in identifying root causes; and linking with community groups. in completing the tool, projects examined root causes of the social and economic determinants of health, thereby exploring social justice issues related to the health of their community. results of the tool also served as a reflec· cion on the process of community capacity building; that is, how the project outcomes were achieved. projects also reported that the tool helped identify gaps and future directions, and was useful as a project planning, needs assessment and evaluation tool. community capacity building is a strategy that can be measured. the community capacity building tool provides a practical means to demonstrate gains in community capacity building. strengthening the elements of community capacity building through community-based funding can serve as building blocks for addressing other community issues. needs of marginalized crack users lorraine barnaby, victoria okazawa, barb panter, alan simpson, and bo yee thom background: the safer crack use coalition of toronto (scuc) was formed in 2000 in response to the growing concern for the health and well-being of marginalized crack users. a central concern was the alarm· ing hepatitis c rate ( 40%) amongst crack smokers and the lack of connection to prevention and health ser· vices. scuc is an innovative grassroots coalition comprised of front-line workers, crack users, researcher! and advocates. despite opposition and without funding, scuc has grown into the largest crack specific harm reduction coalition in canada and developed a nationally recognized sarer crack kit distribution program (involving 16 community-based agencies that provide outreach to users). the success of our coalition derives from our dedication to the issue and from the involvement of those directly affected by crack use. setting: scuc's primary service region is greater toronto, a diverse, large urban centre. much ofour work is done in areas where homeless people, sex trade workers and drug users tend to congregate. recently, scuc has reached out to regional and national stakeholders to provide leadership and education. mandate: our mandate is to advocate for marginalized crack users and support the devdopmentof a com.p.rehensive harm reduction model that addresses the health and social needs facing crack users; and to fac1htare the exchange of information between crack users, service providers, researchers, and policy developers across canada. owrview: the proposed workshop will provide participants with an overview of the devdopment of scuc, our current projects (including research, education, direct intervention and consultation), our challenge~ and s~ccesses and the role of community development and advocacy within the coalition. pre-senter~ will consist of community members who have personal crack use experience and front-line work· ers-, sc.uc conducted a community-based research project (toronto crack users perspectives, 2005) , in w~ich 1 s focus groups with marginalized crack users across toronto were conducted. participants iden· t1f1ed health and social issues affecti h b · · · d " red . . ng t em, arrsers to needed services, personal strategies, an oue recommendations for improved services. presenters will share the methodology, results and recommen· datmns resulting from the research project. conc/usio": research, field observations and consultations with stakeholders have shown that cradck shmoke~s are at an. increased risk for sexually transmitted infections hiv/aids hepatitis c, tb an ot er serious health issues health · ff, · ' ' · · . · issues a ectmg crack users are due to high risk behavmurs, socio· economic factors, such as homeless d. · · · · d · 1 . 1 . ness, 1scrsmmat1on, unemployment, violence incarceraoons, an soc1a 1so at1on, and a lack of comprehe · h i h · ' ns1ve ea t and social services targeting crack users. · · sinct · s, owever arge remains a gross underesurnaoon. poster sessions v167 these are hospital-based reports and many known cases go unreported. however teh case, young age at first intercourse, inconsistent condom use and multiple partnersplace adolescents at high risks for a diverse array of stls, including hiv. about 19% of female nigerian secondary school students report initiating sexual intercourse before age 13 years. 39% of nigerian female secondary school students report not using a condom the last time they had sexual intercourse. more than 60% of urban nigerian teens report inconsistent condom use. methods: 371 adolescents were studied, ages 12 to 19, from benin city in edo state. the models used were mother-daughter(119), mother -son(99), father -son (87), and father-daughter(66). the effect of parent-child sexual communicationat baseline on child\'s report of sexual behavior, 6 to 12 months later were studied. greater amounts of sexual risk communication were asociated with markedly fewer episodes of unprotected sexual intercourse, reduced number of sexual partners and fewer episodes of unprotected sexual intercourse. results: this study proved that parents can exert more influence on the sexual knowledge attitudes and practise of their adolescent children through desired practises or rolemodeling, reiterating their values and appropriate monitoring of the adolescents\' behavior. they also stand to provide information about sexuality and various sexual topics. parental-child sexual communication has been found to be particularly influential and has been associated with later onset of sexual initiation among adolescents, less sexual activity, more responsible sexual attitudes including greater condom use, self efficacy and lower self -reported incidence of stis. conclusions: parents need to be trained to relate more effectively with their children/wards about issues related to sex and sexuality. family -based programs to reduce sexual risk-taking need to be developed. there is also the need to carry out cross-ethnicaland cross-cultural studies to identify how parent-child influences on adolescent sexual risk behavior may vary in different regions or countries, especially inthis era of the hiv pandemic. introduction: public health interventions to identify and eliminate health disparities require evidence-based policy and adequate model specification, which includes individuals within a socioecological context, and requires the integration of biosociomedical information. multiple public and private data sources need to be linked to apportion variation in health disparities ro individual risk factors, the health delivery system, and the geosocial environment. multilevel mapping of health disparities furthers the development of evidence-based interventions through the growth of the public health information network (phin-cdc) by linking clinical and population health data. clinical encounter data, administrative hospital data, population socioenvironmental data, and local health policy were examined in a three-level geocoded multilevel model to establish a tracking system for health disparities. nj has a long established political tradition of "home rule" based in 566 elected municipal governments, which are responsible for the well-being of their populations. municipalities are contained within counties as defined by the us census, and health data are linked mostly at the municipality level. marika schwandt community organizers from the ontario coaliti~n again~t pove~, .along ":ith ~edical practitioners who have endorsed the campaign and have been mvolved m prescnbmg special diet needs for ow and odsp recipients, will discuss the raise the rates campaign. the organizati~n has used a special diet needs supplement as a political tool, meeting the urgent needs o.f .poor ~ople m toront~ while raising the issues of poverty as a primary determinant of health and nutrtnous diet as a preventative health mea· sure. health professionals carry the responsibility to ensure that they use all means available to them to improve the health of the individuals that they serve, and to prevent future disease and health conditions. most health practitioners know that those on social assistance are not able to afford nutritious foods or even sufficient amounts of food, but many are not aware of the extra dietary funds that are available aher consideration by a health practitioner. responsible nurse practitioners and physicians cannot, in good conscience, ignore the special needs diet supplement that is available to all recipients of welfare and disabiliry (ow and odsp). a number of toronto physicians have taken the position that all clients can justifiably benefit from vitamins, organic foods and high fiber diets as a preventative health measure. we know that income is one of the greatest predictors of poor health. the special needs diet is a health promotion intervention which will prevent numerous future health conditions, including chronic conditions such as cardiovascular disease, cancer, diabetes and osteoporosis. many communiry health centres and other providers have chosen to hold clinics to allow many patients to get signed up for the supplement at one time. initiated by the ontario coalition against poverty, these clinics have brought together commu· niry organizers, community health centers, health practitioners, and individuals, who believe that poverty is the primary determinant of poor health. we believe that rates must be increased to address the health problems of all people on social assistance, kids, elders, people with hiv/aids -everyone. even in the context of understaffing, it could be considered a priority activity that has potentially important health promotion benefits. many clients can be processed in a two hour clinic. most providers find it a very interesting, rewarding undertaking. in 2004 the ontario coalition for social justice found that a toronto family with two adults and two kids receives $14,316. this is $21,115 below the poverty line. p7-43 (c) the health of street youth compared to similar aged youth beth hayhoe and ruth ewert . lntrod~on: street youth are at an age normally associated with good health, but due to their risky ~hav1ours and th~ conditions in which they live, they experience health conditions unlike their peer~ an more stable env1r~nments. in addition, the majority of street youth have experienced significant physical, sexual ~nd em.ot1onal abuse as younger children, directly impacting many of the choices they make around their physical and emotional health. we examined how different their health really is. . , methodl: using a retrospective analysis of the 11 years of data gathered from yonge street mis· 510~ 5 • evergreen health centre, the top 10 conditions of youth were examined and compared with national tren~s for similar aged youth. based on knowledge of the risk factors present in the group, rea· sons for the difference were examined. d' ~its: street youth experience more illness than other youth their age and their illnesses can bt . irect t ·~kc~ to the. conditions in which they live. long-term impacts of abus~ contribute to such signif· ~~nt t e t 0 d~slpl air that youth may voluntarily engage in behaviours or lack of self care in the hope at t cir 1ve~ w1 perhaps come to a quicker end. concl11non: although it has ion b k h th' dy clearly shows 3 d'fi . h g ee~ no~n t at poverty negatively affects health, ~siu be used to make ; erence m t .e health of this particular marginalized population. the infonnanon can relates to th . ecommendatio.ns around public policy that affects children and youth, especially as it e1r access to appropriate health care and follow up. p7-44 (cl why do urban children · b gt . tarek hussain 10 an adesh die: how to save our children? the traditional belief that urban child alid. a recent study (dhs d fr 17 r~n are better off than rural children might be no longer v urban migrants are highata th om h c~untn~s i demonstrates that the child survival prospects of rural· er an t ose m their r j · · ·grants. in bangladesh, currently 30 million 0 ~r~ 0~1gm and lower than those of urban non-idi million. health of the urban 1 ~ p~e are hvmg m urban area and by the year 2025, it would be so the popu at1on 1s a key a eals that urban poor have the worse h 1 h . concern. recent study on the urban poor rev ea t situation than the nation as a whole. this study shows that infant poster sessions v169 mortality among the urban poor as 120 per thousand, which are above the rural and national level estimates. the mortality levels of the dhaka poor are well above those of the rest of the city's population but much of the difference in death rates is explained by the experience of children, especially infants. analyzing demographic surveillance data from a large zone of the city containing all sectors of the population, research showed that the one-fifth of the households with the least possessions exhibited u5 child mortality almost three times as high as that recorded by the rest of the population. why children die in bangladesh? because their parents are too poor to provide them with enough food, clean water and other basic needs to help them avoid infection and recover from illness. researchers believed that girls are more at risk than boys, as mothers regularly feed boys first. this reflects the different value placed on girls and boys, as well as resources which may not stretch far enough to provide for everyone. many studies show that housing conditions such as household construction materials and access to safe drinking water and hygienic toilet facilities are the most critical determinants of child survival in urban areas of developing countries. the present situation stressed on the need for renewed emphasis on maternal and child healthcare and child nutrition programs. mapping path for progress to save our children would need be done strategically. we have the policies on hand, we have the means, to change the world so that every child will survive and has the opportunity to develop himself fully as a healthy human being. we need the political will--courage and determination to make that a reality. p7-45 (c) sherbourne health centre: innovation in healthcare for the transgendered community james read introduction: sherbourne health centre (shc), a primary health care centre located in downtown toronto, was established to address health service gaps in the local community. its mission is to reduce barriers to health by working with the people of its diverse urban communities to promote wellness and provide innovative primary health services. in addition to the local communities there are three populations of focus: the lesbian, gay, bisexual, transgendered and transexual communities (lgbtt); people who are homeless or underhoused; and newcomers to canada. shc is dedicated to providing health services in an interdisciplinary manner and its health providers include nurses, a nurse practitioner, mental health counsellors, health promoters, client-resource workers, and physicians. in january 2003 shc began offering medical care. among the challenges faced was how to provide responsive, respectful services to the trans community. providers had considerable expertise in the area of counselling and community work, but little in the area of hormone therapy -a key health service for those who want to transition from one gender to another. method: in preparing to offer community-based health care to the trans community it was clear that shc was being welcomed but also being watched with a critical eye. trans people have traditionally experienced significant barriers in accessing medical care. to respond to this challenge a working group of members of the trans community and health providers was created to develop an overall approach to care and specific protocols for hormone therapy. the group met over a one year period and their work culminated in the development of medical protocols for the provision of hormone therapy to trans individuals. results: shc is currently providing health care to 281 registered clients who identify as trans individuals (march 31 2005) through primary care and mental health programs. in an audit of shc medical charts (january 2003 to september 2004) 55 female-to-male (ftm) and 82 male-to-female (mtf) clients were identified. less than half of the ftm group and just over two-thirds of the mtf group presented specifically for the provision of hormones. based on this chart audit and ongoing experience shc continues to update and refine these protocols to ensure delivery of quality care. conclusion: this program is an example of innovative community-based health delivery to a population who have traditionally faced barriers. shc services also include counselling, health promotion, outreach and education. p7-46 (c) healthy cities for canadian women: a national consultation sandra kerr, kimberly walker, and gail lush on march 4 2005, the national network on environment and women's health held a pan-canadian consultation to identify opportunities for health research, policy change, and action. this consultation also worked to facilitate information sharing and networking between canadian women working as urban planners, policy makers, researchers, and service workers on issues pertaining to the health of women living in canadian cities. methods: for this research project, participants included front-line service workers, policy workers, researchers, and advocates from coast to coast, including francophone women, women with disabilities, racialized women, and other marginalized groups. the following key areas were selected as topics for du.bnes i1 alto kading .:auk of end·sugr ieaal clileue ia singapore, accounting for more than so% of new can singapore (nkfs) to embark on a prevention program (pp) 10 empo~r d1ahc 1j1u1f dieir condition bttter, emphasizing education and disease sdf·managemen1 lkilla a. essennal camponenn of good glycaemic control. we sought 10 explore the effects of a 1pecialijed edu.:a11on pro· pun od glycacmic conuol, as indicated by, serum hba ic values budine serum hba ic values were determined before un<k-rgmng the education progr ;am, which couisted of comprehensive dietary advice, ideal weight goals, and improving lipid profiln. serum hbaic values were obtained ar 3,6,9 months later to determine impact of the program. analy11• wu done uling paired !·tests significant reduction in hba le levels from 0 ro 9 month• was observed in females, chinese race, older age group(> so yean). ohew-ibmi ~ 27.nwm2, wai11 hip ratio> l),up to primary and above secondary level education and those having om urine iclt showed that increasing hbalc levels (9) had increasing urmary protein (38.± 117; .18 ±i ih so± 136) and crearinine (s2.s ± 64 7s ± 71; ioi± 7s) levels fbg rnults showed that the management nf d1abetn m the nkfs preven· tion programme is effec;rive. results also indicated 1har hba le leve11 have a linnr trend wnh unnary protein and creatinine which are imponant determinants of renal diseate tal family-focused cinical palbway1 promoce politivc outcollln for ua inner city canu allicy ipmai jerrnjm1 care llctivirits in preparation for an infanr'' dilchargr honlr, and art m1endnl lo improve effi.:k'fl.:tn of c.are. 11lere i11 paucity of tttran:h, and inconsi1trncy of rnulta on 1ht-•m!*-1 of f1m1ly·fc"-'uw d1nm 1a: to determinr whrthrr implrmentation of family.focuted c:pt 1n 1 ntnn.tt.tl unit w"n mg an inner city 1;ommunity drcl't'aki leftarh of lf•y (i.osi and rromclll'i family uo•fkllon and rt.1j1 nest for dikhargr. md6odt: family-focuk"d cpi 1041 data wm coll«ted for all infant• horn btrwttn 29 and 36 wft"k• 1t"lal111mi atr who wrtt .1dm111ed to the ntonatal unit lmgdl of -.y 111. 9 n. 14.8 day'o p c o.osi ind pma .11 d•mr., ho.nr 137.3 t 1.3 n. 36.4 ± i. i wb, p < o.os) wett n«01fiamly f.lfrt 1n the pre.(]' poup. ~11.fxtmon icofn for famihn wrre high. and families noctd thc:y wnr mott prepued to ah thrar t..lby "'-· thett was .a cosi uving of s 1,814 (cdn) per patient d1teharpd home 1n the pmi-cp poap c.-pated 10 the p"''lfoup· cortclaion· lmplrmrnr.rion of family·foanrd c:p. in a nrona1.1i umt tc"fyidi an 1nnn an com· muniry decre.ned length of'"'" mft with a high dcgrft of family uujamon, and wrre coll~nt at least 35% percent of the kathmandu population lives in slum like conditions with poor access to basic health services. in these disadvantaged areas, a large proportion of children do not receive treatment due to inaccessibility to medical services. in these areas, diarrhea, pneumonia, and measles, are the key determinants of infant mortality. protein energy malnutrition and vitamin a deficiency persists and communicable diseases are compounded by the emergence of diseases like hiv/aids. while the health challenges for disadvantaged populations in kathmandu are substantial, the city has also experienced various forms of innovative and effective community development health programs. for example, there are community primary health centers established by the kathmandu municipality to deliver essential health services to targeted communities. these centers not only provide equal access to health services to the people through an effective management system but also educate them hy organizing health related awareness programs. this program is considered one of the most effective urban health programs. the paper/presentation this paper will review large, innovative, and effective urhan health programs that are operating in kathmandu. most of these programs are currently run by international and national ngos a) early detection of emerging diseases in urban settings through syndromic surveillance: 911 data pilot study kate bassil of community resources, and without adequate follow-up. in november 2003 shelter pr.oviders ~et with hospital social workers and ccac to strike a working group to address some of th~ issues by mcre.asing knowledge among hospital staff of issues surrounding homelessness, and to build a stro?g workmg relationship between both systems in hamilton. to date the hswg has conducted four w~lkmg to~ of downtown shelters for hospital staff and local politicians. recently the hswg launched its ·~ool.k1t for staff working with patients who are homeless', which contains community resources and gu1dehnes to help with effective discharge plans. a scpi proposal has been submitted to incre~se the capacity of the hswg to address education gaps and opportunities with both shelters and hospitals around homelessness and healthcare. the purpose of this poster presentation is to share hamilton's experience and learnings with communities who are experiencing similar issues. it will provide for intera~tion around shared experiences and a chance to network with practitioners across canada re: best practices. introduction and objectives: canadians view health as the biggest priority for the federal government, where health policies are often based on models that rely on abstract definitions of health that provide little assistance in the policy and analytical arena. the main objectives of this paper are to provide a functional definition of health, to create a didactic model for devising policies and determining forms of intervention, to aid health professionals and analysts to strategize and prioritize policy objectives via cost benefit analysis, and to prompt readers to view health in terms of capacity measures as opposed to status measures. this paper provides a different perspective on health, which can be applied to various applications of health such as strategies of aid and poverty reduction, and measuring the health of an individual/ community/country. this paper aims to discuss theoretical, conceptual, methodological, and applied implications associated with different health policies and strategies, which can be extended to urban communities. essentially, our paper touches on the following two main themes of this conference: •health status of disadvantaged populations; and •interventions to improve the health of urban communities.methodology: we initially surveyed other models on this topic, and extrapolated key aspects into our conceptual framework. we then devised a theoretical framework that parallels simple theories of physkal energy, where health is viewed in terms of personal/societal health capacities and effort components.after establishing a theoretical model, we constructed a graphical representation of our model using selfrated health status and life expectancy measures. ultimately, we formulated a new definition of health, and a rudimentary method of conducting cost benefit analysis on policy initiatives. we end the paper with an application example discussing the issues surrounding the introduction of a seniors program.results: this paper provides both a conceptual and theoretical model that outlines how one can go about conducting a cost-benefit analysis when implementing a program. it also devises a new definition and model for health barred on our concept of individual and societal capacities. by devising a definition for health that links with a conceptual and theoretical framework, strategies can be more logically constructed where the repercussions on the general population are minimized. equally important, our model also sets itself up nicely for future microsimulation modeling and analysis.implications: this research enhances one's ability to conduct community-based cost-benefit analysis, and acts as a pedagogical tool when identifying which strategies provide the best outcome. p7-06 (a) good playgrounds are hard to find: parents' perceptions of neighbourhood parks patricia tucker, martin holmes, jennifer irwin, and jason gilliland introduction: neighbourhood opportunities, including public parks and physical activity or sports fields hav~ been. iden.tified as correlates to physical activity among youth. increasingly, physical activity among children 1s bemg acknowledged as a vital component of children's lives as it is a modifiable determinant of childh~d obesity. children's use of parks is mainly under the influence of parents; therefore, the purpose of this study was to assess parents' perspectives of city parks, using london ontario as a case study.m~~: this qualitative study targeted a heterogeneous sample of parents of children using local parks w1thm london. parents with children using the parks were asked for 5 minutes of their time and if willing, a s.hort interview was conducted. the interview guide asked parents for their opinion 'of city parks, particularly the one they were currently using. a sample size of 50 parents is expected by the end of the summer.results: preliminary findings are identifying parents concern with the current jack of shade in local parks. most parents have identified this as a limitation of existing parks, and when asked what would make the parks better, parents agree that shade is vital. additionally, some parents are recognizing the v170 poster sessions focused discussions during the consultation: 1. women in _poverty 2. women with disability 3. immi· grant and racialized women 4. the built and _physica_l environment. . . . . r its· participants voiced the need for integration of the following issues withm the research and policy :::na; t) the intersectional nature of urban women's health i~sues wh~ch reflects the reality of women's complex lives 2) the multisectional aspect of urban wo_m~n s health, 1ss~es, which reflects the diversity within women's lives 3) the interse~roral _dynamics within _womens hves and urban health issues. these concepts span multiple sectors -mdudmg health, educat10n, and economics -when leveraging community, research, and policy support, and engaging all levels of government.policy jmplicatiom: jn order to work towards health equity for women, plans for gender equity must be incorporated nationally and internationally within urban development initiatives: • reintroduce "women" and "gender" as distinct sectors for research, analysis, advocacy, and action. •integrate the multisectional, intersectional, and intersecroral aspects of women's lives within the framework of research and policy development, as well as in the development of action strategies. • develop a strategic framework to house the consultation priorities for future health research and policy development (for example, advocacy, relationship building, evidence-based policy-relevant research, priority initiatives}.note: research conducted by nnewh has been made possible through a financial contribution from health canada. the views expressed herein do not necessarily represent the views of health canada.p7-47 (c) drugs, culture and disadvantaged populations leticia folgar and cecilia rado lntroducci6n: a partir de un proyecto de reducci6n de daiios en una comunidad urbana en situ· aci6n de extrema vulnerahilidad surge la reflexion sobre el lugar prioritario de los elementos sociocuhurales en el acceso a los servicios de salud de diferentes colectivos urbanos. las "formas de hacer, pensar y sentir" orientan las acciones y delimitan las posibilidades que tienen los individuos de definir que algo es o no problema, asf como tambien los mecanismos de pedido de ayuda. el analisis permanenre del campo de "las culturas cotidianas" de los llamados "usuarios de drogas" aporta a la comprension de la complejidad del tema en sus escenarios reales, y colabora en los diseiios contextualizados de politicas y propuestas socio-sanitarias de intervenci6n, tornandolas mas efectivas.mitodos: esta experiencia de investigaci6n-acci6n que utiliza el merodo emografico identifica elementos socio estructurales, patrones de consumo y profundiza en los elementos socio-simb61icos que estructuran los discursos de los usuarios, caracterizandolos y diferenciandolos en tanto constitutivos de identidades socia les que condicionan la implementaci6n del programas de reduccion de daiios.resultados: los resultados que presentaremos dan cuenta de las caracteristicas diferenciadas v relaciones particulares ~ntre los consumidores de drogas en este contexto espedfico. a partir de este e~tudio de caso se mtentara co1?1enzar a responder preguntas que entendemos significativas a la hora de pensar intcrvcnciones a la med1da de poblaciones que comparten ciertas caracteristicas socio-culturales. (cuales serian las .motivaciones para el cambio en estas comunidades?, cque elementos comunitarios nos ayudan a i:nnstnur dema~~a? • cque tenemos para aprender de las "soluciones" que ellos mismos encuenrran a los usos problemat1cos? methods: our study was conducted by a team of two researchers at three different sites. the mapping consisted of filling in a chart of observable neighbourhood features such as graffiti, litter, and boarded housing, and the presence or absence of each feature was noted for each city block. qualitative observations were also recorded throughout the process. researchers analyzed the compiled quantitative and qualitative neighbourhood data and then analyzed the process of data collection itself.results: this study reveals the need for further research into the effects of physical environments on individual health and sense of well-being, and perception of investment in neighbourhoods. the process reveals that perceptions of health and safety are not easily quantified. we make specific recommendations about the mapping methodology including the importance of considering how factors such as researcher social location may impact the experience of neighbourhoods and how similar neighbourhood characteristics are experienced differently in various spaces. further, we discuss some of the practical considerations around the mapping exercise such as recording of findings, time of day, temperature, and researcher safety.conclusion: this study revealed the importance of exploring conceptions of health and well-being beyond basic physical wellness. it suggests the importance of considering one's environment and one's own perception of health, safety, and well-being in determining health. this conclusion suggests that attention needs to be paid to the connection between the workplace and the external environment it is situated in. the individual's workday experience does not start and stop at the front door of their workplace, but rather extends into the neighbourhood and environment around them. our procedural observations and recommendations will allow other researchers interested in the effect of urban environments on health to consider using this innovative methodology. introduction: responding ro protests against poor medical attention for sexually assaulted women and deplorable conviction rates for sex offenders, in the late 1970s, the ontario government established what would become over 30 hospital-based sexual assault care and treatment centres (sactcs) across the province. these centres, staffed around the clock with specially trained heath care providers, have become the centralized locations for the simultaneous health care treatment of and forensic evidence collection from sexually assaulted women for the purpose of facilitating positive social and legal outcomes. since the introduction of these centres, very little evaluative research has been conducted to determine the impact of this intervention. the purpose of our study was to investigate it from the perspectives of sexually assaulted women who have undergone forensic medical examinations at these centres.method: women were referred to our study by sactc coordinators across ontario. we developed an interview schedule composed of both closed and open-ended questions. twenty-two women were interviewed, face-to-face. these interviews were approximately one-to-two hours in length, and were transcribed verbatim. to date, 19 have been analyzed for key themes.results: preliminary findings indicate that most women interviewed were canadian born (79'yo), and ranged in age from 17 to 46 years. a substantial proportion self-identified as a visible minority ( 37'x.). approximately half were single or never married (47%) and living with a spouse or family of origin (53%). most were either students or not employed (68%). two-thirds (68%) had completed high school and onethird (37%) was from a lower socio-economic stratum. almost two-fifths (37%) of women perceived the medical forensic examination as revictimizing citing, for example, the internal examination and having blood drawn. the other two-thirds (63%) indicated that it was an empowering experience, as it gave them a sense of control at a time when they described feeling otherwise powerless. most (68%) women stated that they had presented to a centre due to health care concerns and were very satisfied ( 84 % ) with their experiences and interactions with staff. almost all (89%) women felt supported and understood.conclusions: this research has important implications for clinical practice and for appropriately addressing the needs of sexually assaulted women. what is apparent is that continued high-quality medical attention administered in the milieu of specialized hospital-based services is essential. at the same time, we would suggest that some forensic evidence collection procedures warrant reevaluation. the study will take an experiential, approach by chroruclmg the impa~ of the transition f m the streets to stabilization in a managed alcohol program through the techruque of narrative i~:uiry. in keeping with the shift in thinking in the mental health fie!~ ~his stu~y is based on a paradigm of recovery rather than one of pathology. the "inner views of part1c1pants hves as they portray their worlds, experiences and observations" will be presented (charm~z, 1991, ~· 38~)-"i?e p~ of the study is to: identify barriers to recovery. it will explore the exj?cnence of ~n~t1zanon pnor to entry into the program; and following entry will: explore the meanmg ~nd defirutto~s of r~overy ~~d the impact of the new environment and highlight what supports were instrumental m movmg pan1apants along the recovery paradigm.p7-st (a) treating the "untreatable": the politics of public health in vancouver's inner city introdudion: this paper explores the everyday practices of therapeutic programs in the treadnent of hiv in vancouver's inner city. as anthropologists have shown elsewhere, therapeutic programs do not siinply treat physical ailments but they shape, regulate and manage social lives. in vancouver's inner city, there are few therapeutic options available for the treatment of 1-ilv. public health initiatives in the inner city have instead largely focused on prevention and harm reduction strategies such as needle exchange programs, safe injection sites, and safer-sex education. epidemiological reports suggest that less than a quarter of those living with hiv in the downtown eastside (dtes) are taking antiretroviral therapies raising critical questions regarding the therapeutic economy of antiretrovirals and rights to health care for the urban poor.methods: this paper is drawn from ethnographic fieldwork in vancouver's otes neighborhood focusing on therapeutic programs for hiv treatment among "hard-to-reach" populations. the research includes participant-observation at inner city health clinics specializing in the treatment of hiv; semi· structured interviews with hiv positive participants, health care professionals providing hiv treatment, and administraton working in the field of inner city public health; and, lastly, observation at public meetings and conferences surrounding hiv treatment.r.awlts: hiv prevention and treatment is a central concern in the lives of many residents living in the inner city -although it is just one of many health priorities afflicting the community. concerns about drug resistance, cost of antiretrovirals, and illicit drug use means that hiv therapy for most is characterized by the daily observation of their medicine ingestion at health clinics or pharmacies. daily observed treatment (dot) is increasingly being adopted as a strategy in the therapeutic management of "untreatable" populations. dot programs demand a particular type of subject -one who is "compliant" to the rules and regimes of public health. over emphasis on "risky practices," "chaotic lives," and "~dh~rence" preve~ts the public health system from meaningful engagement with the health of the marginalized who continue to suffer from multiple and serious health conditions and who continue to experience considerable disparities in health.~ the ~ffec~s of hiv in the inner city are compounded by poverty, laclc of safe and affordable houamg, vanous 1llegal underground economies increased rates of violence and outbreaks of ~~~·~ly tr~nsmitted infections, hepatitis, and tuberculosi: but this research suggests 'that public health uunauves aimed at reducing health disparities may be failing the most vulnerable and marginal of citiztl1s. margaret malone 1~ vi~lence that occurs in families and in intimate relationships is a significant urban, ~unity, and pu~hc health problem. it has major consequences and far-reaching effects for women, ~~--renho, you~ sen1on, and families. violence also has significant effects for those who provide and ukllc w receive health care violence · · i · · . all lasses, · is a soc1a act mvolvmg a senous abuse of power. it crosses : ' : ' ~ 11 s;nden, ag~ ~ti~, cultures, sexualities, abilities, and religions. societal responseshali ra y oc:used on identificatton, crisis intervention and services for families and individuajs.promoten are only "-"--:-g to add h · ' · i in intimate relationshi with"-~"'.". ress t e issues of violence against women and vjoence lenga to consider i~ m families. in thi_s p~per, i analyze issues, propose strategies, and note c~· cannot be full -...l'-~ whork towards erad1canng violence, while arguing that social justice and equity y -.1ucvcu w en thett are people wh mnhod: critical social theory, an analysis that addresses culturally and ethnically diverse communities, together with a population health promotion perspective frame this analysis. social determinants of health are used to highlight the extent of the problem of violence and the social and health care costs.the ottawa charter is integrated to focus on strategies for developing personal skills, strengthening community action, creating supportive environments, devdoping healthy public policies, and re-orientating health and social services. attention is directed to approaches for working with individuals, families, groups, communities, populations, and society.ratdts: this analysis demonstrates that a comprehensive interdisciplinary, multisectoral, and multifaceted approach within an overall health promoting perspective helps to focus on the relevant issues, aitical analysis, and strategies required for action. it also illuminates a number of interacting, intersecting, and interconnecting factors related to violence. attention, which is often focused on individuals who are blamed for the problem of violence, is redirected to the expertise of non-health professionals and to community-based solutions. the challenge for health promoters working in the area of violence in families and in intimate relationships is to work to empower ourselves and the communities with whom we work to create health-promoting urban environments. social justice, equiry, and emancipatory possibilities are positioned in relation to recommendations for future community-based participatory research, pedagogical practices for health care practitioners, and policy development in relation to violence and urban health. the mid-main community health center, located in vancouver british columbia (bc), has a diverse patient base reflecting various cultures, languages, abilities, and socio-economic statuses. due to these differences, some mid-main patients experience greater digital divide barriers in accessing computers and reputable, government produced consumer health information (chi) websites, such as the bc healthguide and canadian health network. inequitable access is problematic because patient empowerment is the basis of many government produced chi websites. an internet terminal was introduced at mid-main in the summer of 2005, as part of an action research project to attempt to bridge the digital divide and make government produced chi resources useful to a broad array of patients. multi-level interventions in co-operation with patients, with the clinic and eventually government ministries were envisioned to meet this goal. the idea of implementing multi-level interventions was adopted to counter the tendency in interactive design to implement a universal solution for the 'ideal' end-user [ 1 ), which discounts diversity. to design and execute the interventions, various action-oriented and ethnographic methods were employed before and during the implementation of the internet terminal. upon the introduction of the internet terminal, participant observation and interviews were conducted using a motion capture software program to record a digital video and audio track of patients' internet sessions. this research provided insight into the spectrum of patients' capacities to use technology to fulfil their health information needs and become empowered. at the mid-main clinic it is noteworthy that the most significant intervention to enhance the usefulness of chi websites for patients appeared to be a human rather than a technological presence. as demonstrated in other ethnographic research of community internet access, technical support and capacity building is a significant component of empowerment (2). the mid-main wired waiting room project indicates that medical practitioners, medical administrators, and human intermediaries remain integral to making chi websites useful to patients and their potential empowerment. (1) over the past 5 years the environmental yo~th alliance has been of~ering a.youth as~t. mappin~ program which trains young people in community research and evaluation. wh1~st the positive expenenc~ and relationships that have developed over this time attest to the success of this program, no evaluations has yet been undertaken to find out what works for t.he youth, what ~ould be changed, and what long term outcomes this approach offers for the youth, their local community, and urban governance. these topics will be shared and discussed to help other community disorganizing and uncials governments build better, youth-driven structures in the places they live.p7-55 (a) the world trade center health registry: a unique resource for urban health researchers deborah walker, lorna thorpe, mark farfel, erin gregg, and robert brackbill introduction: the world trade center health registry (wfchr) was developed as a public health response to document and evaluate the long-term physical and mental health effects of the 9/11 disaster on a large, diverse population. over 71,000 people completed a wfchr enrollment baseline survey, creating the largest u.s. health registry. while studies have begun to characterize 9/11 bealth impacts, questions on long-term impacts remain that require additional studies involving carefully selected populations, long-term follow-up and appropriate physical exams and laboratory tests. wtchr provides an exposed population directory valuable for such studies with features that make ita unique resource: (a) a large diverse population of residents, school children/staff, people in lower man· hattan on 9/11 including occupants of damaged/destroyed buildings, and rescue/recovery/cleanup work· ers; (b) consent by 91 % of enrollees to receive information about 9/11-related health studies; (c) represenration of many groups not well-studied by other researchers; (c) email addresses of 62% of enrollees; (d) 30% of enrollees recruited from lists with denominator estimates; and (e) available com· parison data for nyc residents. wfchr strives to maintain up-to-date contact information for all enrollees, an interested pool of potential study participants. follow-up surveys are planned.methods: to promote the wtchr as a public health resource, guidelines for external researcher.; were developed and posted on (www.wtcregistry.org) which include a short application form, a twopage proposal and documentation of irb approval. proposals are limited to medical, public health, or other scientific research. researchers can request de-identified baseline data or have dohmh send information about their studies to selected wfchr enrollees via mail or email. applications are scored by the wtchr review committee, comprised of representatives from dohmh, the agency for toxic subst~nces and disease registry, and wtchr's scientific, community and labor advisory committees. a data file users manual will be available in early fall 2005.~suits: three external applications have been approved in 2005, including one &om a non-u.s. ~esearcher, all requesting information to be sent to selected wtchr enrollees. the one completed mail· mg~~ wtchr enrollee~ (o 3,700 wfc tower evacuees) generated a positive survey response rate. three additional researchers mtend to submit applications in 2005. wfchr encourages collaborations between researchers and labor and community leaders.conclusion: studies involving wtchr enrollees will provide vital information about the long· term health consequences of 9/11. wtchr-related research can inform communities, researcher.;, policy makers, health care providers and public health officials examining and reacting to 9111 and other disasters. t .,. dp'"f'osed: thi is presentation will discuss the findings of attitudes toward the repeat male client iden· 1 ie as su1e1 a and substance us'n p · · · · i · 'd . . -1 g. articipants will learn about some identified effective strategies or service prov1 ers to assist this group of i · f men are oft · d bl men. n emergency care settings, studies show that this group 0 en viewe as pro emaric patient d i r for mental health p bl h h 5 an are more ikely to be discharged without an assessmen 200!) ea 1 1 rofr ems t. an or er, more cooperative patients (forster and wu 2002· hickey er al., · r y resu ts om this study suggest th · · ' ' l · d tel' mining how best to h 1 . d 1 at negative amtudes towards patients, difficu nes e · as well pathways l_e_ p patientsblan ~ck of conrinuity of care influence pathways to mental health care. • uc\:ome pro emat1c when p ti k · che system. m a ems present repeatedly and become "get stuc id methods: semi-structured intervie d . · (n=5), ed nurses (n=5) other ed ;s were con ucted with male ed patients (n=25), ed phys1oans ' sta (n= 7) and family physicians (n= 7). patients also completed a poster sessions v175 diagnostic interview. interviews were tape-recorded, transcribed verbatim and managed using n6. transcripts were coded using an iterative process and memos prepared capture emergent themes. ethics approval was obtained and all participants signed a detailed informed consent form.introduction: urban settings are particularly susceptible to the emergence and rapid spread of nt•w or rare diseases. the emergence of infectious diseases such as sars and increasing concerns over the next influenza pandemic has heightened interest in developing and using a surveillance systt·m which detects emerging public health problems early. syndromic surveillance systems, which use data b,1scd on symptoms rather than disease, offer substantial potential for this by providing near-real-rime data which are linked to an automated warning system. in toronto, we are piloting syndromic data from the 911 · emergency medical services (ems) database to examine how this information can be used on an ongoing basis for the early detection of syndromes including heat-related illness (hri), and influenza-like-illness (ill). this presentation will provide an outline of the planned desi_gn of this system and proposed evaluation. for one year, 911 call codes which reflect heat-related illness or influenza-like-illness will be selected and searched for daily using software with a multifactorial algorithm. calls will he stratified by call code, extracted from the 911-ems database and transferred electronically to toronto public health. the data will be analyzed for clusters and aberrations from the expected with the realtime outbreak and disease surveillance (rods) system, a computer-based public health tool for the early detection of disease outbreaks. this 911-ems surveillance system will be assessed in terms of its specificity and sensitivity through comparisons with the well-established tracking systems already in place for hr! and ill. others sources of data including paramedic ambulance call reports of signs and . this study will introduce complementary data sources t~ the ed ch1e~ complamt an~ o~~rthe-counter pharmacy sales syndromic surveillance data currently bemg evaluated m ~ther ontar~o cltles. . syndromic surveillance is a unique approach to proactive(~ dete~tmg early c.hangesm the health status of urban communities. the proposed study aims to provide evidence of differential effectiveness through investigating the use of 911-ems call data as a source of syndromic surveillance information for hr! and ili in toronto. introduction: there is strong evidence that primary care interventions, including screening, brief advi<:e, treatment referral and pharmacotherapy are effective in reducing morbidity and mortality caused by substance abuse. yet physicians are poor at intervening with substance users, in part because of lack of time, training and support. this study examines the hypothesis that shared care in addictions will result in decreased substance use and improved health status of patients, as well as increased use of primary care interventions by primary care practitioners (pcps). methods: the addiction medicine service (ams) at st. joseph's health centre's family medicine department is in the process of being transformed from its current structure as a traditional consult service into a shared care model called addiction shared care (asc). the program will have three components: education, office systems and clinical shared care. as opposed to a traditional consult service, the patient will be booked with both a primary care liaison worker (pcl) and addictions physician. patients referred from community physicians, the emergency department and inpatient medical and psychiatric wards will be recruited for the study as well as pcps from the surrounding community. the target sample size is 100-150 physicians and a similar number of patients. after initial consult, patient will be recruited into the study with their consent. the shared-case model underlines the interaction and collaboration with the patient's main pcp. asc will provide them with telephone consults, advice, support and re-assessment for their patients, as well as educational sessions and materials such as newsletters and informational kits.results: the impact of this transition on our patient care and on pcp's satisfaction with the asc model is currently being evaluated through a grant provided by the ministry of health & long term care. a retrospective chart review will be conducted using information on the patient's substance use, er/clinic visits, and their health/mood status. pcp satisfaction with the program will be measured through surveys and focus groups. our cost-effectiveness analysis will calculate the overall cost of the program per patient..conclusion: this low-cost service holds promise to serve as an optimal model and strategy to improve outcomes and reduce health care utilization in addict patients. the inner city public health project introduction the inner city public health project (icphp) was desi.gned to explore new an~ innovative ways to reach marginalized inner city populations that par-t1c1pate m high health-nsk beha~1ors. much of this population struggles with poverty, addictions, mental illness and homelessness, creatmg barriers to accessing health services and receiving follow-up. this pro1ect was de~igned to evaluat~ .~e success of offering clinics in the community for testing and followup of communicable diseases uuhzmg an aboriginal outreach worker to build relationships with individuals and agencies. v1n(demographics~ history ~f testi~g ~nd immunization and participation in various health-risk behaviors), records of tesnng and 1mmumzat1ons, and mterviews with partner agency and project staff after one year.. results: t~e chr ~as i~strumental in building relationships with individuals and partner agencies ' .° the c~mmun_1~ re_sultmg m req~ests for on-site outreach clinics from many of the agencies. the increase m parnc1pat10n, the chr mvolvement in the community, and the positive feedback from the agen? staff de~onstrated that.the project was successfully creating partnerships and becoming increasingly integrated m the community. data collected from clients at the initial visit indicated that the project was reaching its target populations and highlighted the unique health needs of clients, the large unmet need for health services and the barriers that exist to accessing those services. ~usion: the outreach clinics were successful at providing services to target populations of high health-nsk groups and had great support from the community agencies. the role of the chr was critical to the success of the project and proved the value of this category of health care worker in an urban aboriginal population. the unmet health needs of this disadvantaged population support the need for more dedicated resources with an emphasis on reducing access barriers. building a caring community old strathcona's whyte avenue, a district in edmonton, brought concern about increases in the population of panhandlers, street people and homeless persons to the attention of all levels of government. the issue was not only the problems of homelessness and related issues, but feelings of insecurity and disempowerment by the neighbourhood residents and businesses. their concerns were acknowledged, and civic support was offered, but it was up to the community itself to solve the problem. within a year of those meetings, an adult outreach worker program was created. the outreach worker, meets people in their own environments, including river valley camps. she provides wrap-around services rooted in harm reduction and health promotion principles. her relationship-based practice establishes the trust for helping clients with appropriate housing, physical and/or mental health issues, who have little or no income and family support to transition from homelessness. the program is an excellent example of collaboration that has been established with the businesses, community residents, community associations, churches, municiple services, and inner-city agencies such as boyle street community services. statistics are tracked using the canadian outcomes research institute homes database, and feedback from participants, including people who are street involved. this includes an annual general meeting for community and people who are homeless. the program's holistic approach to serving the homeless population has been integral, both in creating community awareness and equipping residents and businesses to effectively interact with people who are homeless. through this community development work, the outreach worker engages old strathcona in meeting the financial and material needs of the marginalized community. the success of this program has been surprising -the fact is that homeless people's lives are being changed; one person at a time and the community has been changed in how they view and treat those without homes. over two years, the program has successfully connected with approximately seventy-five individuals who call old strathcona home, but are homeless. thirty-six individuals are now in homes, while numerous others have been assisted in obtaining a healthier and safer lifestyles by becoming connected with other social/health agencies. the program highlights the roots of homelessness, barriers to change and requirements for success. it has been a thriving program and a model that works by showing how a caring community has rallied together to achieve prosperous outcomes. the spn has created models of tb service delivery to be used m part~ers~1p with phannaceunca compa-. · · -. t' ns cooperatives and health maintenance orgamzanons (hmos). for example, the mes, c1v1c orgamza 10 , . · b tb d' · spn has established a system with pharmaceutical companies that help patients to uy me 1cmes at a special discounted rate. this scheme also allows patients to get a free one-_month's worth of~ dru_g supply if they purchase the first 5 months of their regimen. the sy_s~e~s were ~es1gned to be cm~pattble with existing policies for recording and documentation of the ph1hppme national tuberculosis program (ntp). aside from that, stakeholders were also encouraged to be dots-enabled through the use of m~nual~ and on-line training courses. the spn initiative offers an alternative in easing the burden of tb sc:rv1ce delivery from rhe public sector through the harnessing of existing private-sector (dsos). the learnmgs from the spn experience would benefit groups from other locales that _work no~ only on ~ but other health concerns as well. the spn experience showcases how well-coordinated private sector involvements help promote social justice in health delivery in urban communities.p7-63 (c) young people in control; doing it safe. the safe sex comedy juan walter and pepijn v. empelen introduction: high prevalence of chlamydia and gonorrhoea have been reported among migrants youth in amsterdam, originating from the dutch antilles, suriname and sub-sahara africa. in addition, these groups also have high rates of teenage-pregnancy (stuart, 2002) and abortions (rademakers 1995), indicating unsafe sexual behaviour of these young people. young people (aged 12 -30) from the so· called urban scene (young trendsetters in r&b/hip hop music and lifestyle) in amsterdam have been approached by the municipal health service (mhs) to collaborate on a safe sex project. their input was to use comedy as vehicle to get the message a cross. for the mhs this collaboration was a valuable opportunity to reach a hard-to-reach group.mdhods: first we conducted a need assessment by means of a online survey to assess basic knowledge and to similtaneously examine issues of interest concerning sex, sexuality, safer sex and the opposite sex. second, a small literature study was conducted about elements and essential conditions for succesful entertainment & education (e&e) (bouman 1999), with as most important condition to ensure that the message is realistic (buckingham & bragg, 2003) . third a program plan was developed aiming at enhancing the stl/hiv and sexuality knowledge of the young people and addressing communication and educational skills, by means of drama. subsequently a safe sex comedy show was developed, with as main topics: being in love, sexuality, empowerment, stigma, sti, hivand safer sex. the messages where carried by a mix of video presentation, stand up comedy, spoken word, rap and dance.results: there have been two safe sex comedy shows. the attendance was good; the group was divers' with an age range between 14 and 50 year, with the majority being younger than 25 year. more women than men attended the show. the story lines were considered realistic and most of the audients recognised the situations displayed. eighty percent of the audients found the show entertaining and 60% found it edm:arional. from this 60%, one third considers the information as new. almost all respondents pointed our that they would promote this show to their friends.con.clusion: the s.h<_>w reached the hard-to-reach group of young people out of the urban scene and was cons1d_ered entert~mmg, educational and realistic. in addition, the program was able in addressing important issues, and impacted on the percieved personal risk of acquiring an sti when not using condoms, as well as on basic knowledge about stl's. introduction: modernity has contributed mightily to the marginality of adults who live with mental illnesses and the subsequent denial of opportunities to them. limited access to social, vocational, educational, and residential opportunities exacerbates their disenfranchisement, strengthening the stigma that has been associated with mental illness in western society, and resulting in the denial of their basic human rights and their exclusion from active participation in civil society. the clubhouse approach tn recovery has led to the reduction of both marginality and stigma in every locale in which it has been implemented judiciously. its elucidation via the prism of social justice principles will lead to a deeper appreciation of its efficacy and relevance to an array of settings. methods: a review of the literature on social justice and mental health was conducted to determine core principles and relationships between the concepts. in particular, fondacaro and weinberg's (2002) conceptualization of social justice in community psychology suggests the desirability of the clubhouse approach in community mental health practice. a review of clubhouse philosophy and practice has led to the inescapable conclusion that there is a strong connection between clubhouse philosophy-which represents a unique approach co recovery--and social justice principles. placing this highly effective model of community mental health practice within the context of these principles is long overdue. via textual analysis, we will glean the principles of social justice inherent in the rich trove of clubhouse literature, particularly the international standards of clubhouse development.results: fondacarao and weinberg highlight three primary social justice themes within their community psychology framework: prevention and health promotion; empowerment, and a critical pnsp<"<·tive. utilizing the prescriptive principles that inform every detail of clubhouse development and th<" movement toward recovery for individuals at a fully-realized clubhouse, this presentation asserts that both clubhouse philosophy and practice embody these social justice themes, promote human rights, and empower clubhouse members, individuals who live with mental illnesses, to achieve a level of wdl-heing and productivity previously unimagined.conclusion: a social justice framework is critical to and enhances an understanding of the clubhouse model. this model creates inclusive communities that lead to opportunities for full partic1pil!ion 111 civil society of a previously marginalized group. the implication is that clubhouses that an· based on the international standards for clubhouse programs offer an effective intervention strategy to guarantee the human rights of a sizable, worthy, and earnest group of citizens. to a drastic increase in school enrollment from 5.9 million in 2002 to 7.5 million in 200.s. however, while gross enrollment rates increased to 104°/., in the whole country after the introduction of fpe, it remained conspicuously low at 62% in the capital city, nairobi. nairobi city's enrollment rate is lower .than thatof all regions in the country except the nomadic north-eastern province. !h.e.d1sadvantage of children bas_ed in the capital city was also noted in uganda after the introduction of fpe m the late 1990s_-many_ education experts in kenya attribute the city's poor performance to the high propornon of children hvmg m slums, which are grossly underserved as far as social services are concerned. this paper ~xammes the impact of fpe and explores reasons for poor enrollment in informal settlements m na1rob1 city. methods: the study utilizes quantitative and qualitative schooling data from the longitudinal health and demographic study being implemented by the african population and health research center in two informal settlements in nairobi. descriptive statistics are used to depict trends in enrollment rates for children aged 5-19 years in slum settlements for the period 2000-2005. results: the results show that school enrollment has surprisingly steadily declined for children aged 15-19 while it increased marginally for those aged 6-14. the number of new enrollments (among those aged 5 years) did not change much between 2001 and 2004 while it declined consistently among those aged 6-9 since 2002. these results show that the underlying reasons for poor school attendance in poverty-stricken populations go far beyond the lack of school fees. indeed, the results show that lack of finances (for uniform, transportation, and scholastic materials) has continued to be a key barrier to schooling for many children in slums. furthermore, slum children have not benefited from fpe because they mostly attend informal schools since they do not have access to government schools where the policy is being implemented.conclusion: the results show the need for equity considerations in the design and implementation of the fpe program in kenya. without paying particular attention to the schooling needs of the urban poor children, the millennium development goal aimed at achieving universal primary education will remain but a pipe dream for the rapidly increasing number of children living in poor urban neighborhoods.ps-04 (c) programing for hiv/aids in the urban workplace: issues and insights joseph kamoga hiv/aids has had a major effect on the workforce. according to !lo 35million persons who are engaged in some form of production are affectefd by hiv/aids. the working class mises out on programs that take place in communities, yet in a number of jobs, there are high risks to hiv infection. working persons sopend much of their active life time in workplaces and that is where they start getting involved in risky behaviour putting entire families at risk. and when they are infected with hiv, working people face high levels of seclusion, stigmatisation and some miss out on benefits especially in countries where there are no strong workplace programs. adressing hiv/aids in the workplace is key for sucessfull responses. this paper presents a case for workplace programing; the needs, issues and recommendations especially for urban places in developing countries where the private sector workers face major challenges. key: cord-313729-mydyc68y authors: mcdiarmid, melissa a. title: hazards of the health care sector: looking beyond infectious disease date: 2014-11-25 journal: ann glob health doi: 10.1016/j.aogh.2014.08.001 sha: doc_id: 313729 cord_uid: mydyc68y background: possessing every hazard class, the health care sector poses significant health threats to its workforce in both high-resource settings and lowand middle-income countries (lmics). objectives: the aim of this paper was to examine the applicability of the classical hierarchy of hazard control technologies in resource-constrained health care settings. methods: using a biologic and chemical hazard example, the hazard control hierarchy was applied for risk mitigation. findings: even when resource constraints force a reordered selection of hazard control elements, risk reduction can be achieved across a variety of hazard classes. conclusion: for lmics with limited resources, the hazard control hierarchy can be effectively employed, although the selection of methods may be reordered, to achieve significant hazard control. such prevention strategies can thereby strengthen and sustain a critical pillar of the health system, its workforce. it is counter-intuitive that the health care industry, whose mission is the care of the sick, is itself a "high-hazard" industry for the workers it employs. this industry sector consistently demonstrates poor workforce injury and illness statistics, among the highest in the united states 1 and in the european union (eu), about 30% higher than the average work-related accident rate. 2 in both the united states and the eu, about 10% of all workers are employed in the health care sector. this workforce is overwhelmingly female, even in some low-and middle-income countries (lmics), with about 70% of the total being women workers. 3 with such a large portion of the global workforce being employed in this high-hazard sector and with forecasts for the increasing need for health workers in the future, the magnitude of the health threat is considerable and demands address. a significant array of hazards is posed by the sector, with biologic agents and infectious diseases the most widely recognized. indeed, such "health careeacquired" infections in patients, other unintended errors, such as medication overdoses, and the known side effects of hazardous treatments have recently spawned the highly visible "patient safety movement." 4 less apparent, however, has been the risk to health that those same hazards and this same environment impose on the men and women who work there. 5 although preventing exposure to infectious agents and musculoskeletal injuries resulting from patient lifting have been the primary focus of employee safety programs, the chemical hazards in health care have been more slowly recognized. these include novel agents, some of which are unique to health care such as sterilants, germicidal agents, and pharmaceuticals including the highly toxic anticancer drugs. many of these drugs are themselves cancer-causing or toxic to human reproduction and have been the subject of environmental monitoring campaigns in recent years, showing widespread work-area contamination. 6 in the context of this highly complex and hazardous work environment, particular challenges arise in pursuing protections for health care workers in this unique employment sector. biases within the health care industry and the safety and health community itself collude to limit both the awareness of hazards that do exist and the successful application of classical approaches used to assure safe jobs. 7 this occurs for several reasons. because health care is a nontraditional employment setting, imagined by the public to be clean and safe, hazard awareness often is lacking. also, due to its unique mission of caring for the sick, self-preservation behaviors, which normally aid in protecting workers, are suspended in a culture of self-less commitment to patient care. there is an erroneous "either/or" mentality historically present that sometimes forces a false choice to be made by a worker between providing good care or protecting oneself. importantly, these threats to caregiver health have been named as critical factors in the us nursing shortage according to the american nurses association (ana), which published in a recent study that health and safety of the work environment impacts nurses' decision to stay in the profession. 8 internationally as well, conditions of work and health threats have been found to contribute to the current global shortage of health workers. 9 in a recent document from the world health organization (who), "monitoring the building blocks of health systems," the health workforce is described as one of the essential 6 pillars of a strong and sustainable health system. 10 although enlarging capacity through skills building and training is emphasized to bolster the health workforce, also discussed in the prevention of workforce shortages is mitigation of "losses caused by death, retirement, career change or out-migration." clearly, failing to address health threats in the work environment will be a barrier to retaining and sustaining caregiver ranks, which in turn, threaten the delivery of health care globally. workers in the health care sector, which possesses every hazard class, may encounter health threats both common to other workers, such as those related to large facility operations and maintenance, including asbestos, heavy metals, and solvents, and those hazards unique to the provision of care to ill patients. a number of excellent reviews of hazard management in the healthcare sector have been published 5,11-13 and the reader is directed to these resources. however, several overarching hazards require specific address, due both to their strategic threat to the global health workforce and because they are eminently preventable. biologic hazards, airborne, and bloodborne pathogen exposure biologic hazards are encountered in all health care settings and include airborne and bloodborne pathogens. certainly the best-known airborne hazard is tuberculosis (tb), but other agents are also acquired by the airborne route, such as measles and severe acute respiratory syndrome and most recently, middle east respiratory syndrome. critical elements of an airborne hazard prevention plan are the early identification and isolation of patients as well as administrative and work practice controls to minimize exposure and disease transmission. 14 in the long arc of the unfolding of the hiv pandemic, one little-known development has been the unintended effect experienced by health care workers in some settings in southern africa. due to the endemicity of hiv infection in these regions, including among health care workers, and the often fatal collusion of tb infection in the already hiv infected, there have been alarmingly high tb infection rates and losses of life among nurses and other health workers. 15 often, the typical tb prevention and treatment services afforded hiv patients may not be sought by hiv-infected health workers, due to the stigma they would experience with public knowledge of their hiv condition, which can occur when they line up for tb preventive treatment at the same clinics where they worked, for example. because of this, the morbidity and mortality among workers was substantial, even as many of the countries in the region were already hobbled by a health worker shortage. against this backdrop, in 2011, a new initiative was launched jointly by the who, international labor organization (ilo), and unaids to protect health workers. joint who-ilo-unaids policy guidelines were issued for improving health worker access to hiv and tb prevention, treatment, care, and support 16 to curb this alarming, preventable loss of life among health workers. this initiative promotes worker education regarding tb exposure risk and urges that prevention and treatment services be provided at points of care, while maintaining the privacy of health workers. such occupational health services currently are not widely available in these affected areas, but could be provided by building upon some existing clinical and infection control resources. as well, awareness must be brought to local and regional health ministers that threats to the health care workforce also threaten the viability of health systems with the loss of caregivers to preventable disease and death. indeed, the who identifies the health care workforce as 1 of 6 essential pillars of its health system strengthening initiative. 10 bloodborne pathogens, which include viruses capable of causing hepatitis or hiv infections continue to threaten health workers in both high-resource areas and in lmics. 17 in developing countries, 40% to 65% of hepatitis b (hbv) and c virus (hcv) infections in health care workers were attributed to percutaneous occupational exposure. in industrialized countries, such infections rates are lower, with 8% to 27% of infections for hcv attributable to occupation and around 10% for hbv. these lower rates are due to immunization and post-exposure prophylaxis (pep). the range of hiv infections related to occupational exposure is estimated at 0.5% to 11%. 18 the likelihood of infection occurring after a percutaneous exposure has been observed to occur in a specific order with hbv infections (18%-30%) > hcv (1.8%) > hiv infections (0.3%) displaying generally a 10-fold difference in infection likelihood between each of these agents. [18] [19] [20] [21] health workers are at risk for exposure to bloodborne pathogens while performing routine duties involving the use of "sharps" such as injection needles and from unsafe sharps disposal. the who has many resource guides to protect health workers from exposure to bloodborne viruses, the elements of which include: 1) the use of "universal or standard" precautions-a system of work practices and behaviors that minimizes exposure such as prohibition of manual needle recapping after use and safe sharps disposal; 2) availability of hepatitis b immunization for health workers; 3) use of personal protective equipment (ppe) and apparel such as use of gloves; and 4) post-exposure management (including prophylaxis, where appropriate) counseling, and support. 22 although health threat interventions in lmics have largely targeted infectious disease risks, the who recently has enlarged its focus to include chronic disease prevention. this is in acknowledgment that nearly 80% of noncommunicable disease deaths-29 million annually-occur in lmics. 23 chronic disease includes heart and respiratory disease, cancer, and diabetes. therefore, widening the focus of attention given to occupational health threats related to the treatment of chronic disease must become a part of a comprehensive safety and health plan for the sector. "exposure to potentially hazardous chemicals is a fact of life for health care workers," according to stellman in her overview article on chemical hazards in health care. 24 examples include laboratory reagents and chemicals required in diagnostic or therapeutic procedures. pharmaceuticals are of increasing concerning, especially the hazardous anticancer chemotherapy drugs, which are highly toxic and require vigilance in their use and handling. the term hazardous drug was first applied to most anticancer and some other limited classes of drugs by the american society of health-system pharmacists 25 and was adopted by the occupational safety and health administration 26 and the national institute for occupational safety and health 27 in their publications promoting safe handling practices. drugs are classified as hazardous if studies indicate that exposures to them have the potential for causing cancer in animals or humans, or if they cause developmental or reproductive toxicity, or other organ-system damage. most hazardous drugs are those used to treat cancer but also include hiv therapies and other antiviral agents. 27 occupational exposures to hazardous drugs can lead to acute effects such as skin disorders, allergic reactions, and hair loss; and chronic effects, including adverse reproductive events and possibly cancer. 6 in the pan american health organization (paho) monograph, "safe handling of hazardous chemotherapy drugs in low resource settings," 28 a safer approach to handling these highly toxic, but lifesaving drugs is described when resource limitations might mitigate against "state-of-the-art" practices, but the hazard, nonetheless, requires address. because these drugs require extensive manual manipulation during formulation (compounding) of the patient dose, there is opportunity for worker exposure. a heavy reliance on worker training, ppe use, and scrupulous work practices must be applied to minimize worker exposure. the paho document provides a detailed approach to safe handling of hazardous drugs in resource-constrained settings. in its 2004 document "recommendations for protecting healthcare workers' health," international commission on occupational health called for a "systematic occupational risk prevention program" for health care workers to include training regarding work risks and the provision of protective measures, as an integral part of an administrative process addressing health care quality. 29 the basic occupational health approach to minimizing exposure to any workplace hazard uses a combination of protective industrial hygiene control methods that are applied in a specified order or hierarchy. this approach has achieved success across many industrial settings. 30 in most cases, the elements of this hierarchy can be applied to the health care setting (fig. 1) . the hierarchy of hazard control technologies relies on engineering controls, such as a biological safety cabinet, a glove box, or other type of hazard barrier or containment, as the first technology applied. however, engineering solutions often are the most costly type of hazard control. in resource-limited settings where engineering controls are unaffordable or otherwise not feasible, scrupulous use of work practices that minimize aerosol and dust generation along with administrative controls that limit personnel access to areas where hazards are encountered can minimize exposure. figure 1 displays this upside-down hierarchy approach. importantly, the lack of resources for the more costly elements of a hazard control approach does not relieve the facility from responsibility from applying some of the control measures that are available to make the work setting the most safe it can be, even in the setting of resource constraints. the hierarchy of hazard control technologies relies first on engineering controls, as just described. for the airborne hazard example of tb, early identification and isolation of potentially infectious patients, in a negative pressure room, is the ideal "engineering" intervention to minimize exposure to airborne mycobacterium. however, in settings where such negative pressure isolation rooms are not available, other administrative and work practice controls of the airborne hazard can be applied, such as placing the infectious patient in a single room away from others. in some locations, a cough-inducing procedure, such as obtaining a sputum specimen, may occur outside, away from other patients. in situations where "engineered" sharps with safety features are not available, careful work practice controls, such as refraining from manual recapping of needles, can mitigate needle-stick injuries. for preparation of hazardous, anticancer drugs when the engineering control containment of a biologic safety cabinet is not available, applying a work practice of preparing drugs in low-traffic and clean preparation areas, and controlling personnel access to this area can minimize the number of workers potentially exposed to fugitive drug aerosol, as described in the paho document. 28 although only 2 hazard types (biologic and chemical) are discussed in detail here, the "hierarchy of hazard control" approach can effectively be employed to limit exposure to other hazard classes and sources of risk to health workers including musculoskeletal risks of patient lifting 31,32 and workplace violence. 33 in both well-resource settings and in lmics, the health care workforce is threatened daily with harm from exposure to agents encountered in this unique and complex workplace. even here, however, the classical hierarchy of hazard control technologies can still be effectively applied to mitigate risk. importantly, the selection of hazard control methods may be reordered in low-resource settings to provide some, albeit not ideal, hazard control. as lmics enlarge their prevention services beyond infectious disease control, other health care hazards related to chronic disease care, such as cancer will require programmatic address by occupational health staff to protect and retain the vital health workforce, which is a fundamental pillar of all health systems. healthcare illness and injury statistics gender in health workforce to err is human, building a safer health system niosh (national institute for occupational safety and health/cdc) a nora report: state of the sector: healthcare and social assistance preventing occupational exposures to antineoplastic drugs in health care settings chemical hazards in health care: high hazard, high risk, but low protection american nurses associations (ana) working together for health. the world health report monitoring the building blocks of health systems: a handbook of indicators and their measurement strategies healthwise work improvement in health services trainers' guide health and safety of workers in the health sector: a manual for managers and administrators health workers. health worker occupational health guidelines for preventing the transmission of mycobacterium tuberculosis in health care settings tb in health care workers in the joint who-ilo-unaids policy guidelines on improving health worker's access to hiv and tb prevention, treatment, care and support services: a guidance note preventing needlestick injuries among healthcare workers: a who-icn collaboration sharps injuries: global burden of disease from sharps injuries to health-care workers. geneva: world health organization update on the subject of epidemiology of blood-transmitted occupational infections a caseecontrol study of hiv seroconversion in health care workers after percutaneous exposure joint who/ilo guidelines on post exposure prophylaxis (pep) to prevent hiv infection aide mã©moire for a strategy to protect health workers from infection with bloodborne viruses preventing chronic disease: a vital investment chemcial in the health care environment. geneva: international labor organization ashp technical assistance bulletin on handling cytotoxic and hazardous drugs osha instruction ted (training and education directive), 1.15 directorate of technical support: controlling occupational exposure to hazardous drugs niosh alert: preventing occupational exposures to antineoplastic and other hazardous drugs in health care settings international commission on occupational health (icoh) and the international social security association (ssa). recommendations for protection health care workers' health patty's industrial hygiene and toxicology safe lifting programs at long-term care facilities and their impact on workers' compensation costs sit-stand powered mechanical lifts in long-term care and resident quality indicators national institute for occupational safety and health (niosh). violence occupational hazards in hospitals key: cord-263645-wupre5uj authors: morgan, brittany s; forte, jordan e; hargrove, amanda e title: insights into the development of chemical probes for rna date: 2018-09-19 journal: nucleic acids res doi: 10.1093/nar/gky718 sha: doc_id: 263645 cord_uid: wupre5uj over the past decade, the rna revolution has revealed thousands of non-coding rnas that are essential for cellular regulation and are misregulated in disease. while the development of methods and tools to study these rnas has been challenging, the power and promise of small molecule chemical probes is increasingly recognized. to harness existing knowledge, we compiled a list of 116 ligands with reported activity against rna targets in biological systems (r-bind). in this survey, we examine the rna targets, design and discovery strategies, and chemical probe characterization techniques of these ligands. we discuss the applicability of current tools to identify and evaluate rna-targeted chemical probes, suggest criteria to assess the quality of rna chemical probes and targets, and propose areas where new tools are particularly needed. we anticipate that this knowledge will expedite the discovery of rna-targeted ligands and the next phase of the rna revolution. the field of rna biology has exploded in recent years with the discovery of non-coding rnas (ncrnas) that regulate essential processes in all living organisms (1) . these processes include transcription, translation, and evasion in bacteria and archaea (1, 2) as well as replication, persistence, and cellular transformation in viruses (3) . within the human genome, protein-coding genes are vastly outnumbered by regulatory ncrnas that can influence a wide range of cellular functions (2, 4) . many of these ncrnas are dysregulated in and implicated as drivers of various human diseases, including metastatic cancers and neurologi-cal and neuromuscular disorders (2, 5, 6) . this 'rna revolution' is radically changing our understanding of the role rna plays in fundamental biology and is rapidly driving scientific innovation. methods and tools to structurally and functionally characterize rnas at the molecular level, however, are more difficult and/or lacking as compared to those for proteins (7) (8) (9) (10) . one important example is the development of chemical probes, which has greatly progressed the study of proteins and related diseases (11, 12) but has been challenging for non-ribosomal rnas. this powerful chemical tool requires small molecules with well-defined biological activity, cell permeability, and selectivity to accurately and reliably probe specific mechanistic and phenotypic questions (11, 12) . given the potential advantages of small molecule chemical probes over biological approaches (e.g. sirnas, asos and crispr-cas) (13, 14) and the power of using both approaches in tandem (12) , the development of rnatargeted chemical probes has the potential to greatly benefit both chemists and biologists interested in rna. while ligands that bind non-ribosomal rna in vitro have been reported for decades, the development of chemical probes with evidence of specific small molecule:rna engagement in cell or animal models has dramatically increased in the last four years. recent studies report several drug-like small molecules that target a range of rnas in animal models, including riboswitches (15) , mirnas, (16, 17) splice sites (18) , and mature mrnas (19) , at least one of which is currently in clinical trials (nct02268552). multivalent ligands have been reported that target r(cug) exp repeats of myotonic dystrophy type 1 (dm1) in d. melanogaster (20) and mouse models (21) . these recent successes confirm that selective rna targeting is achievable in biological systems; however, the limited examples over years of effort highlight the challenges associated with selectively probing rna. recently, we compiled the rna-targeted bioactive ligand database (r-bind), which comprised organic small molecules that target non-ribosomal rnas and show activity in cell culture or animal models (22) . for this survey, the compilation was updated to include chemical probe discoveries through may 2017 for a total of 116 chemical probes (see supplementary material, r-bind 1-1.xls). aminoglycosides are excluded due to established non-specific binding behavior (23) (24) (25) as well as peptides and oligonucleotides due to distinctive medicinal chemistry properties (26, 27) . the chemical probes were divided into two classes: monovalent, traditional drug-like small molecules (sm) and multivalent ligands (mv) with alkyl, aryl, or peptidyl linkers between multiple binding moieties. previous work compared the physicochemical, structural, and spatial properties of the small molecules to fdaapproved drugs as well as rna-binding ligands without reported biological activity (22) . this analysis revealed several key differences between these libraries that can in turn be used to bias small molecules toward biological rna targeting. in addition to that work, the curation of this collection allowed us to gain insights into: (i) the rna elements targeted; (ii) the design and discovery strategies utilized and (iii) the in cellulo characterization of these chemical probes. herein, we discuss these insights, highlight unique examples, and consider the need to establish standards for cellbased selectivity. we conclude by proposing future directions that utilize our current and prospective chemical biology toolbox to expedite the discovery of chemical probes for rna. the 116 chemical probes targeted 33 distinct rna elements, including those from bacterial, fungal, human, or viral systems [ figure 1 ]. although some overlap between targets was observed, the small molecules probed a wider range of rnas in cell culture than the multivalent ligands [ figure 1a ]. the most common small molecule target was the hiv-1 trans-activation response element (tar) rna, a well-studied and frequently screened rna that binds to the viral protein tat (23) . disruption of this interaction reduces viral production and represents an alternative strategy against hiv. some of the first rna-targeted chemical probes were developed for tar rna, including a tetraaminoquinozaline (28) and a 6-aminoquinolone (29, 30) with an ec 50 value of 16 m and an ic 50 value of 0.85 m, respectively, in chronically infected hiv cell models. on the other hand, only one bioactive small molecule was identified for a fungal target, specifically the candida albicans lsu group 1 ribozyme (31). this essential ribozyme is a desirable antifungal target as it leads to failed ribosomal assembly when mutated and is absent in the human genome. further, 10/75 small molecule chemical probes demonstrated efficacy in animal models, targeting seven unique rna elements in bacterial and human systems. one recent example (19) targeted the g-quadruplex structure located in the 5 -untranslated region (utr) of human vascular endothelial growth factor (hvegf) mrna, an angiogenic growth factor involved in tumor progression. in a breast cancer mouse model, the small molecule showed antitumor efficacy similar to that of doxorubicin but with fewer indications of side effects. multivalent chemical probes targeted fewer distinct rna elements, with 27/41 unique ligands targeting nucleotide repeat expansions [ figure 1b ]. there are several advantages to targeting these rna repeats: (i) long repeat stretches are typically not present elsewhere in the human genome; (ii) nuclear localization minimizes competition with ribosomal rna and (iii) targetable motifs are separated by a specific distance (32) . another target of interest was the heat shock response element of the 32 factor mrna in e. coli. this rna element contains a rare, perfectly paired three-way junction that can be stabilized by symmetrical triptycenebased molecules, forming a distinct shape-selective fit (33) . this stabilization resulted in an ∼60% reduction in translation of an 32 -gfp fusion protein and could potentially lead to antimicrobial activity (34) . in addition, 7/41 chemical probes showed efficacy in animal models, targeting two rna elements: r(cug) exp repeats and pri-mirna-96. pri-mirna-96 is an oncogenic rna that suppresses the translation of a pro-apoptotic protein, foxo1. in a mouse model of triple negative breast cancer, a modular ligand designed to target the drosha processing site on the rna led to a statistically significant reduction in tumor size and to changes in rna and protein levels consistent with the proposed mode of action (17) . notably, examination of this list further exposes the rna-driven processes and diseases that still lack functional chemical probes. ideal rna targets have defined functional sites and/or clear phenotypes while also being of high abundance, and several untargeted rnas, ranging from archaeal ncrna to oncogenic lncr-nas, meet these criteria. many of the monovalent ligands were discovered through traditional screening methods. approximately one-third of the rna:ligand interactions were identified by each of the following approaches: focused-screening (fcs), highthroughput screening (hts), and hts followed by lead optimization (hts-lo) [ figure 2a ]. in this survey, fcs is defined by the use of biased libraries, which are typically based on prior knowledge of a particular chemotype binding to an rna element. in contrast to fcs libraries, molecules specifically designed to explore structureactivity relationships were classified as lead optimization (lo). the starting points for several of the fcs libraries and/or other small molecule identification strategies included rna-binding natural products, chemical similarity searching, and scaffold-based synthesis [ figure 2b -d]. we caution that the relative success of these various approaches cannot be evaluated since failed attempts are not typically documented in the literature. hit rates are one of the benchmarks used to assess the efficiency of a screen. we note prior to the discussion that comparisons across studies should be interpreted with caution as the definition of a small molecule lead, the specific assays used in primary screens, the controls utilized in the assay, and the number of false-positives and -negatives can be highly variable and are not always reported. of the 41 rna:small molecule interactions discovered through hts and fcs, 20 had reported hit rates, which were compared by screening approach, primary screen, and primary library [ figure 3 ]. the higher hit rates found in some fcs approaches provide compelling evidence that fcs is efficient for rna targets, as it is known to be for protein targets (35, 36) . moving forward, characterization of additional rna tertiary structures (8, 9, 37, 38) and the identification of novel rna-binding chemotypes (8, 39, 40) will expedite the fcs approach for discovering biologically active rna-targeted ligands. while hit rates varied widely within each type of primary screen and primary library, these com-parisons support the potential of many distinct paths toward rna ligand discovery. specific aspects of library and screen design are discussed below. small molecules discovered by hts were typically from large libraries (n > 50 000 small molecules), including three corporate libraries and the nih small molecule repository [ table 1 ]. there were select examples of smaller libraries as well: fda-approved drugs (n = 1120) and ucla academic library (n = 1692). importantly, some of these reports explicitly stated that libraries were filtered to yield small molecules with favorable medicinal chemistry propers-amino acid conjugates a screening approaches ties prior to screening. while successful in protein-targeted drug discovery (41), only one report identified a bioactive ligand from a fragment-based library (commercial library) (42) . once optimized, the scaffold yielded four additional molecules that targeted the influenza a rna promoter with ic 50 values ranging from 34 to 44 m in a cell-based luciferase assay (43) . similarly, only two reports yielded bioactive small molecules from natural productbased libraries (synthetic library and academic library) (19, 44) . both of these screens contained fewer than 150 small molecules, yet identified ligands that bind and modulate g-quadruplex structures located in the 5 -utr of two distinct mrnas. it is promising that small molecules were discovered from a variety of hts libraries, suggesting that biologically active, rna-binding ligands can be found in a subset of current small molecule chemical space (22) . further validation and exploration of this space could lead to greater efficiency and success in identifying bioactive leads as well as rna-privileged chemotypes. as expected, fcs used smaller libraries, typically containing fewer than 150 small molecules [ table 1 ]. the largest fcs library (n = 320, academic library) was designed through a chemical similarity search of the bis-benzimidazole and similar cores, which have shown preferences for 1 × 1 nucleotide internal loops (45) . in addition, this library was filtered for favorable medicinal chemistry properties, and the screen resulted in three leads that: (i) bound r(cug) exp in vitro; (ii) led to a statistically significant decrease in ctnt mini-gene exon inclusion in cells and (iii) were selective for a mini-gene with 960 r(cug) repeats compared to a mini-gene without the repeats. fcs also encompassed rna structure-guided design, which included two studies utilizing molecular modeling to identify ligands structurally similar to guanine for the xpt-pbux riboswitch (46, 47) . in another structure-guided approach, a small library of p-terphenylene-based ligands was designed to mimic an ␣-helix of rev, a protein-binding partner of the hiv-1 rev response element (rre) (48) . leads were selected by docking, with one ligand disrupting the rev-rre interaction in vitro (ic 50 = 6.8 m), inhibiting hiv-1 replication (ic 50 values of 3.4-5.9 m), and exhibiting on-target effects via a rre-luciferase reporter assay (ic 50 values of 10-21 m). further, we did not identify successful reports of biologically active ligands from small molecule libraries biased to general rnabinding. recently, chemical companies have designed such focused libraries (chemdiv, nucleic acid ligands: http://www.chemdiv.com/nucleic-acid-ligands/; otava chemicals, rna targeted library: http: //www.otavachemicals.com/products/targeted-librariesand-focused-libraries/rna-binding); however, the success of these libraries is yet to be reported. to date, successful fcs strategies have utilized knowledge of the rna structure and/or a small molecule binder(s), neither of which is known for many therapeutically-relevant rnas. the primary screening assay for each small molecule was categorized as computational, in vitro, or cell-based [ figure 4a ]. this list contained a wide range of primary screening assays, with limited examples of the same assay being used for multiple targets. the majority of the chemical probes were discovered by in vitro primary screening assays (n = 28) with fewer in cellulo or silico examples. of those in vitro primary screens, 15 were rna:protein displacement assays [ figure 4b ]. these included fluorescence-based assays (förster resonance energy transfer (fret) and fluorescence anisotropy) and radiolabel-based methods (mobility shift, scintillation proximity, and filtration assays). one rather unique assay utilized a molecular beacon approach to probe for stabilization of stem loop 3 (sl3), a presumptive structural switch located in the -packing domain in hiv-1 that is destabilized by binding of the gag protein prior to packaging of the virus (49) . in this assay, the 5 -and 3 -terminal ends of the sl3 rna were labeled with a tet fluorophore and a blackhole quencher (bhq1), respectively. in the presence of gag protein, the rna construct became single stranded and the fluorescence was 'turned on'. when a small molecule stabilized the folded hairpin form of sl3 rna, the gag-promoted rna destabilization was reduced and the fluorescence was quenched. the researchers screened a modest sized library (>2500 small molecules) and discovered a ligand that reduced viral production similar to models with a mutated -packing domain (p24 50 = 11.3 m). the remaining in vitro assays consisted of two activitybased screens and various rna binding assays [figure (v) competition dialysis and (vi) indicator displacement. we note that a lack of correlation in small molecule activity between in vitro rna binding and rna:protein displacement assays has sometimes been reported, (16, 51, 52) highlighting the importance of multiple assays and/or choosing the most relevant assay for a particular system. we also note that, as in all screens, a lack of correlation can be observed between in vitro activity and cell culture activity. these differences can be attributed to many factors, including small molecule uptake, localization, and metabolism, specificity or off-target effects, and target availability due to binding of other macromolecules or metabolites. nonetheless, we emphasize the success of the in vitro assays mentioned here in developing rna bioactives and the valuable insights gained from other ligands discovered by in vitro rna assays without reported biological activity. cell-based screens were the second most common primary screening assay for hts or fcs approaches [ figure 4a ] and often the preferred screen for bacterial and viral rna targets. the exception was a splicing assay of human serotonin receptor 2c (htr2c) mrna where a green fluorescent protein (gfp) reporter was used to evaluate the inclusion or exclusion of a particular exon (53) . bacterial and viral rna cell-based studies also utilized reporter systems such as gfp or lacz gene as well as more traditional phenotypic screens, such as growth inhibition or cell death. in one particular example, ∼57 000 ligands were screened in a growth inhibition assay against escherichia coli with and without supplementation of riboflavin (15). this differential supplementation allowed researchers to specifically probe the riboflavin pathway and confirm the fmn riboswitch was targeted by ribocil-b. in addition, one report measured enzyme activity and antigen production in addition to cell death measurements to assess antiviral activity against hiv-1 (30). given these successes, cell-based assays likely offer unforeseen promises as primary screening assays to discover rna-targeted probes. high-throughput and focused computational screens were used to identify four small molecules [ figure 4a ]. two small molecules were identified by docking against experimentally determined structures of hiv-1 tar (54) and rre rna (48) . in another example, small molecules were modeled into an x-ray diffraction structure of the xpt-pbue guanine riboswitch aptamer after the native ligand was removed (46) . criteria such as geometrical constraints, hydrogen bonding patterns, and molecule planarity were used to assess the 'fit' of the ligand, leading to the selection of two small molecules, one of which had antimicrobial activity against 9 of the 15 gram-positive bacteria species tested and was selective for species with the guaa gene under riboswitch control. the fourth example utilized a computationally-predicted 3d structure of the severe acute respiratory syndrome coronavirus (sars-cov) pseudoknot (55) . a library of 80 000 small molecules was docked against the predicted structure, and the 58 highest scoring molecules were tested in an in vitro activity-based assay. the screen resulted in a biologically active ligand with an ic 50 value of 0.45 m in cell-based models. additional advances in computational structural prediction and rna:ligand docking will undoubtedly lead to improved computational primary screens and thus more efficient experimental screens (56, 57) . in addition to the primary screens, a database of known rna motif:small molecule interactions, inforna (39) , was utilized to identify seven small molecules. the database was generated using a library versus library approach named 2-dimensional combinatorial screening (2dcs). in this method, small molecules are immobilized onto a microarray slide and then incubated with libraries of labeled, randomized rna secondary structures. the bound rnas are excised, sequenced, and assigned a fitness score using structure-activity relationships through sequencing (starts). fitness scores reflect the affinity and selectivity of a given rna motif:small molecule interaction and are represented on a numerical scale, where a higher score represents greater selectivity. to utilize the inforna database, a computational or experimental secondary structure of rna is input, the 2dcs data is searched and lead molecules are proposed. this strategy identified bioactive ligands for five targets: (i) mapt pre-mrna, 1-nucleotide bulge; (58) (ii) pre-mirna-96, 1 × 1 internal loop (59); (iii) pre-mirna-18a, 1-nucleotide bulge (60); (iv) pre-mirna-210, 1 × 1 internal loop (16) and (v) pre-mirna-544, 1 × 1 internal loop (61) . other examples of ligands not identified from a primary screening assay included the selection and characterization of four metabolite analogs for riboswitch inhibition (62) (63) (64) (65) . in contrast to small molecules, most multivalent ligands were developed through rational design based on the secondary structure of the rna target [ figure 5a ]. generally, development began by the identification of monovalent ligands that bound to a particular secondary structure motif(s) by screening, literature search, or using inforna [see section 'other methods of small molecule discovery']. monomers were covalently linked by selecting and optimizing appropriate sized spacers. for several multivalent ligands, the design was inspired by a crystal structure of r(cug) sequences, leading to ligands targeting r(cug) exp in drosophila melanogaster models (66) . this approach linked acridine and a triaminotriazine unit, the latter of which was proposed to recognize the non-optimal base pairing of u-u mismatches by janus-wedge hydrogen bonding. stacking of the two units was expected to decrease nonspecific intercalative binding. this early design was optimized to yield bisamidinium conjugates that mitigated the glossy and rough eye phenotype observed in a dm1 transgenic drosophila melanogaster model (67) . a different approach utilized hoechst 33258, which had been previously reported to bind a 5 cug/3 guc internal loop (68) . hoechst 33258 was modified to contain an azide handle and then covalently linked to a peptoid backbone via click chemistry (21) . after extensive linker optimization, multivalent ligands were identified that improved r(cug) exprelated splicing defects in a mouse model of dm1. one notable exception to the aforementioned design strategies is the use of dynamic combinatorial chemistry (dcc) [ figure 5b ] (69) . several multivalent ligands were derived from a library of resin-bound, cysteine-containing monomers, which were allowed to incubate with the rna of interest, probing thousands of multivalent ligand combinations by forming covalent yet reversible disulfide linkages. the binders with highest affinity were thus enriched and then isolated, characterized, and validated for rnabinding. after replacing the disulfide linkage with more sta-ble bioisosteres, the method yielded bioactive ligands for two rnas of known structure: dm1 r(cug) exp , where statistically significant improvements in splicing were observed in mouse models (70) , and hiv-1 frameshift-stimulating rna, (71, 72) where in one example the decrease in viral infectivity (ec 50 values of 3.9 and 26 m) correlated to frame-shifting activity (>50% at 50 m) in cell culture (73) . a powerful advantage of dcc is that multivalent ligands can be constructed without knowledge of the rna structure, including larger and complex tertiary folds. in general, both rational design and dcc yield multivalent probes with significantly increased affinity and specificity for rna targets relative to small molecules. while achieving high potency in biological systems with larger molecules may require more development than with traditional small molecules, the examples identified support the possibility of success. evaluating target engagement, off-target effects, potency/appropriate concentration, and other criteria is critical to understanding the quality of a chemical probe and thus any experimental conclusions (11, 12, 74, 75) . when curating the collection of chemical probes, strict benchmarks related to these criteria could not be included due to the lack of consistency within the field. in the next paragraphs, the characterization techniques utilized for rna-targeted chemical probes will be described for each biological system and notable examples highlighted. one of the most common validation experiments in bacterial and fungal systems was serial passage (15, 31, 46, 62, 65, 76, 77) . in this technique, ligand resistant mutants were grown in the presence of compound and mutations were mapped by whole-genome sequencing. in addition to confirming target engagement, the results revealed off-target effects and unexpected modes of action. further, select examples performed the serial passage experiments in multiple bacterial strains and measured binding affinity to the mutants in vitro, which provided added confidence in target engagement (15, 31, 77) . in select cases where serial passage experiments did not yield mutated isolates, the ligands were tested against mutants with established variations in structure or activity (76, 65) . another powerful strategy for assessing target engagement in riboswitches was phenotype rescue by addition of the native ligand (15, 46) . lastly, several of the targeted rna elements regulated the expression or translation of specific genes, which was assessed by measuring the quantity of the rna or protein, respectively. one study went beyond measuring the expected transcripts and performed a transcriptomic microarray analysis of genes involved in many different cellular processes (46) . the observed repression was consistent with riboswitch inhibition, although addition of the native ligand failed to rescue the expression of several genes, indicating a potential cellular stress response. this strategy and other genome-wide analyses can provide compelling evidence of target engagement, though it must be noted that target specificity does not always lead to biological specificity (12) . compared to the other systems, chemical probes targeting viral rnas were less characterized in terms of target engagement and specific activity. a select few studies validated probe activity by testing mutant versions of the virus (49, 78) or closely related native viruses (30, 73) . notably, one report validated probe activity with a second research group to ensure reproducibility of the biological effect (71) . rnaspecific reporter systems were occasionally used to confirm on-target effects, including fusion-induced gene stimulation (79) , heterologous tethering (54) , and a viral protein reporter (48) . a noteworthy example of assessing off-target effects was the use of rna-seq at increasing ligand concentrations in the absence of the target rna (72) . the experiment in hek293t cells revealed that 53 of the 17 822 transcripts assayed had statistically significant alterations at two concentrations, potentially reflecting a cellular stress response. it is also intriguing that the most biologically potent ligand in this study was the least selective analog in an in vitro trna competition assay. this observation underlies potential differences in cellular activity versus in vitro binding selectivity and thus the importance of progressing multiple ligands to biological assays. there are several noteworthy examples of probing ligand promiscuity and/or selectivity in human systems. multiple studies utilized genome-wide analyses to globally assess changes in mirna, (16, 17, 59, 60, 80) mrna, (18, 50) or splicing (18, 21, 47, (81) (82) (83) (84) (85) ) levels compared to the target rna or process. toward a more direct assessment, chemical cross-linking and isolation by pull down (chem-clip) was utilized to investigate proximity-based engagement of rna (86) . in this method, the ligand is appended with a nucleic-acid reactive module (e.g. chlorambucil) and a biotin purification tag. after incubating cells with the modified ligand, the cells are lysed, the ligand is captured by streptavidin beads, and the bound targets are characterized by qrt-pcr or rna-seq. this strategy as well as competitive chem-clip (86) were used to characterize onand off-target engagement of ligands that modulate repeat expansions (45, 84, 85, 87) and mirnas (16, 17, 60) . in another study, dual luciferase reporter assays were utilized to compare ligand binding to four g-quadruplex structures, including the rna of interest and three other regulatory rnas (44) . this study was one of few examples in which selectivity within a target family was assessed in cell culture. various controls were also used to evaluate on-target effects. for example, the impacts of chemical probes have been analyzed following sirna knockdown of foxo1 mrna (59) and following inhibition of the mtor signaling pathway modulated by mirna-544 (61) . another example overexpressed mirna-210 and assessed the effect of the chemical probe on the phenotype (16) . likewise, a ligand targeting r(cug) exp was tested with the rna under conditional expression (88) . another notable example used rna immunoprecipitation (rip) to detect rna-binding at increasing concentrations of ligand and identified a dosedependent response (19) . for precipitation, a g-quadruplex specific antibody, bg4, was utilized and the complex was characterized by two complementary methods: dot blotting and qrt-pcr. another important control, particularly for human systems, was to replicate on-target effects in at least two cell lines, though this control was performed for only a limited number of chemical probes (44, 53, 47, 81, 84) . for many years, rna has been labeled as 'undruggable' or 'impossible to probe selectively'; however, the reports described herein demonstrate the substantial progress that has been achieved in the past four years. this includes the development of chemical probes for 33 unique rna elements, though the number of 'targetable' rna elements is certain to vastly exceed this list (5, 6, 23, 89) . those in archaea, for example, are unrepresented in reports of rnatargeted chemical probes, despite the established importance of small regulatory rnas in archaea metabolism, morphology, and adaption to extreme conditions (90, 91) . furthermore, there are several rnas implicated in diseases for which novel treatment strategies are needed, including insect-borne viruses (23) , genetic disorders (92) , and metastatic cancers (5,6) as well as bacterial targets amid the antibiotic-resistance crisis (89, 93) . there are also many opportunities for rna-targeting in fungal systems (94) , especially as fungal infections are experiencing a rise in cases and a therapeutic plateau (95) . fundamentally, the development of chemical probes will allow for the rapid and reversible interrogation of novel and complex rna biology in ways not attainable by knockdown and genetic approaches (74) . while the potential benefits of selective rna targeting are staggering, approaches toward chemical probe development must thoughtfully consider a number of variables, including transcript abundance and tissue-specific expression of the rna target. by total mass and number of molecules, rrna and trna account for greater than 90% of total cellular rna in humans (96) , and thus mrnas and other ncrnas exist in a much lower abundance (1, 96, 97) . even within these low abundance transcripts, copy numbers can vary widely within and across cell types with reports of mrna levels spanning four orders of magnitude and some ncrnas averaging less than one copy per cell (1, 97, 98) . a direct impact of rna expression levels was recently reported, in which the authors proposed that ligand occupancy of the mirna target was driven by the relative abundance of structurally similar rna elements (16) . rna with well-defined function and that are highly expressed thus represent low-hanging fruit within the field (6, 99) . the selection of a library and primary screening strategy is also critical for the success of chemical probe discovery. for rna targets with known structure and/or ligands, the use of focused screening libraries (35, 36) has proven to be an efficient strategy, though this approach generally limits the chemical diversity of the library. advances such as the identification of more rna-privileged scaffolds (39) and biologically relevant rna chemical space (22, 100) will facilitate the discovery of chemical probes for additional rna targets. these efforts could be expedited by additional fragment-or natural product-based screening to access vast chemical space with fewer ligands (41) and to probe known biologically relevant chemical space, respectively (101) . additional high-throughput screens could likewise discover novel chemotypes, though the expense may exceed the resources available in academia, where focused approaches are more attainable. continued progress in the development and refinement of computational tools will also aid in expanding the boundaries of focused screening and structurebased design; (56, 57) although the latter will also depend upon advancements for accurately determining atomic resolution structures (8, 9, 37, 38) . in addition, in vitro or cellular activity-based assays that probe well-studied rna functions (e.g. splicing, translation, or processing) may be more practical starting points to identify chemical probes. this includes screening strategies described herein as well as others recently developed (102, 103) . finally, numerous opportunities exist to build upon the established multivalent targeting strategies discussed above, particularly the application of dynamic combinatorial chemistry to rnas of unknown structure. when using a chemical probe in a biological system, the quality and specificity of the probe must be well characterized to draw accurate and meaningful conclusions, as recently highlighted by several preeminent chemical biologists (11, 12, 74, 75) . evaluation of the chemical probes revealed a diverse spectrum of characterization techniques with few ligands meeting the traditional criteria for robust chemical probes, and not all of these gaps can be attributed to a lack of relevant tools. for example, characterization inconsistencies include incomplete reports of cytotoxicity and a lack of attention to cell permeability and localization. in addition, many in vitro assays are accessible to help establish the potency and selectivity of a probe in multiple experiments, and these assays should include evaluation against both specifically mutated targets and a number of other structured rnas. any cell-based observations should be reproducible in multiple cell lines and validated in the absence of the target by utilizing sirna or crispr-cas technologies. further, on-target effects should be established by using a number of spatial-based experiments (12) . these include the biochemical methods described herein (e.g. chem-clip or rip) and novel applications of other technologies such as in-cell chemical probing (104, 105) to observe changes in rna secondary structure upon ligand binding or photoaffinity labeling (106) to assess target engagement under temporal control. if plausible, serial passage experiments followed by deep sequencing should also be performed, even in human systems (107) , to identify ligandescaping mutations to confirm target engagement and/or off-target effects. lastly, it is critical to use an inactive analog and an active analog from a different chemical class, if feasible, to draw conclusions regarding the targeted biology. moving toward these standards will be crucial for the rna-targeting field to avoid the scientific pollution that has plagued many others (11, 12, 74) . for comparison, we note that over a decade ago chemical probe discovery for another 'undruggable' target, protein:protein interactions, was in its infancy with only 19 known small molecule inhibitors (108) . however, novel advances in screening approaches and design strategies led to the rapid discovery of thousands of protein:protein interaction inhibitors with several entering the clinic and most also breaking the rules of 'drug-like chemical space' (109) . in the next five years, we anticipate that the innovations described herein and the ones yet to be discovered will lead to a similar surge in reports of rna-based chemical probes and therapeutics. we also expect that novel and well-characterized chemical probes will allow rna biologists to uncover many more exciting and unanticipated roles for rna, propelling us into the next phase of the rna revolution. supplementary data are available at nar online. the noncoding rna revolution-trashing old rules to forge new ones the rise of regulatory rna viral noncoding rnas: more surprises the encode project consortium (2007) identification and analysis of functional elements in 1% of the human genome by the encode pilot project non-coding rnas as drug targets the therapeutic targeting of long noncoding rna linking rna biology to lncrnas synthetic receptors for oligonucleotides and nucleic acids biochemical methods to investigate lncrna and the influence of lncrna:protein complexes on chromatin opportunities and challenges in rna structural modeling and design the impact of chemical probes in drug discovery: a pharmaceutical industry perspective choose and use your chemical probe wisely to explore cancer biology comparison of small molecules and oligonucleotides that target a toxic, non-coding rna overcoming cellular barriers for rna therapeutics selective small-molecule inhibition of an rna structural element small molecule inhibition of microrna-210 reprograms an oncogenic hypoxic circuit design of a small molecule against an oncogenic noncoding rna smn2 splice modulators enhance u1-pre-mrna association and rescue sma mice discovery of small molecules for repressing cap-independent translation of human vascular endothelial growth factor (hvegf) as novel antitumor agents rationally designed small molecules that target both the dna and rna causing myotonic dystrophy type 1 features of modularly assembled compounds that impart bioactivity against an rna target discovery of key physicochemical, structural, and spatial properties of rna-targeted bioactive ligands small molecules targeting viral rna targeting rna with small molecules chemical and functional diversity of small molecule ligands for rna the medicinal chemistry of peptides the medicinal chemistry of therapeutic oligonucleotides inhibitors of protein-rna complexation that target the rna: specific recognition of human immunodeficiency virus type 1 tar rna by small organic molecules new anti-human immunodeficiency virus type 1 6-aminoquinolones: mechanism of action ) 6-aminoquinolones as new potential anti-hiv agents pentamidine inhibition of group i intron splicing in candida albicans correlates with growth inhibition rational design of chemical genetic probes of rna function and lead therapeutics targeting repeating transcripts recognition of nucleic acid junctions using triptycene-based molecules modulation of the e. coli rpoh temperature sensor with triptycene-based small molecules recent developments in focused library design: targeting gene-families the design and application of target-focused compound libraries recent advances in developing small molecules targeting rna insights into rna structure and function from genome-wide studies inforna 2.0: a platform for the sequence-based design of small molecules targeting structured rnas identifying the preferred rna motifs and chemotypes that interact by probing millions of combinations twenty years on: the impact of fragments on drug discovery a novel small-molecule binds to the influenza a virus rna promoter and inhibits viral replication targeting influenza a virus rna promoter for up-regulating the translation of antiamyloidogenic secretase, a disintegrin and metalloproteinase 10 (adam10), by binding to the g-quadruplex-forming sequence in the 5 untranslated region (utr) of its mrna studying a drug-like, rna-focused small molecule library identifies compounds that inhibit rna toxicity in myotonic dystrophy novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways chemical correction of pre-mrna splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(cag)-containing transcripts structure-based design of an rna-binding p-terphenylene scaffold that inhibits hiv-1 rev protein function targeting rna-protein interactions within the human immunodeficiency virus type 1 lifecycle a small molecule that represses translation of g-quadruplex-containing mrna thermodynamic studies of a series of homologous hiv-1 tar rna ligands reveal that loose binders are stronger tat competitors than tight ones development of small molecules with a noncanonical binding mode to hiv-1 trans activation response (tar) rna pyrvinium pamoate changes alternative splicing of the serotonin receptor 2c by influencing its rna structure structure-based computational database screening, in vitro assay, and nmr assessment of compounds that target tar rna identification of rna pseudoknot-binding ligand that inhibits the -1 ribosomal frameshifting of sars-coronavirus by structure-based virtual screening structure based approaches for targeting non-coding rnas with small molecules structure-based discovery of small molecules binding to rna bottom-up design of small molecules that stimulate exon 10 skipping in mutant mapt pre-mrna sequence-based design of bioactive small molecules that target precursor micrornas defining rna-small molecule affinity landscapes enables design of a small molecule inhibitor of an oncogenic noncoding rna biogenesis disrupts adaptive responses to hypoxia by modulating atm-mtor signaling novel riboswitch-binding flavin analog that protects mice against clostridium difficile infection without inhibiting cecal flora roseoflavin is a natural antibacterial compound that binds to fmn riboswitches and regulates gene expression structure-guided mutational analysis of gene regulation by the bacillus subtilis pbue adenine-responsive riboswitch in a cellular context thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine a simple ligand that selectively targets cug trinucleotide repeats and inhibits mbnl protein binding targeting toxic rnas that cause myotonic dystrophy type 1 (dm1) with a bisamidinium inhibitor specific binding of hoechst 33258 to site 1 thymidylate synthase mrna dynamic combinatorial selection of molecules capable of inhibiting the (cug) repeat rna-mbnl1 interaction in vitro: discovery of lead compounds targeting myotonic dystrophy (dm1) from dynamic combinatorial 'hit' to lead: in vitro and in vivo activity of compounds targeting the pathogenic rnas that cause myotonic dystrophy n-methylation as a strategy for enhancing the affinity and selectivity of rna-binding peptides: application to the hiv-1 frameshift-stimulating rna hiv-1 frameshift rna-targeted triazoles inhibit propagation of replication-competent and multi-drug-resistant hiv in human cells high-affinity recognition of hiv-1 frameshift-stimulating rna alters frameshifting in vitro and interferes with hiv-1 infectivity the promise and peril of chemical probes target identification and mechanism of action in chemical biology and drug discovery design and antimicrobial action of purine analogues that bind guanine riboswitches dual-targeting small-molecule inhibitors of the staphylococcus aureus fmn riboswitch disrupt riboflavin homeostasis in an infectious setting conformational inhibition of the hepatitis c virus internal ribosome entry site rna inhibition of hiv-1 tat-tar interaction by diphenylfuran derivatives: effects of the terminal basic side chains small molecule inhibition of mir-544 biogenesis disrupts adaptive responses to hypoxia by modulating atm-mtor signaling design of a bioactive small molecule that targets the myotonic dystrophy type 1 rna via an rna motif-ligand database and chemical similarity searching induction and reversal of myotonic dystrophy type 1 pre-mrna splicing defects by small molecules targeting the r(cgg) repeats that cause fxtas with modularly assembled small molecules and oligonucleotides small molecule recognition and tools to study modulation of r(cgg)(exp) in fragile x-associated tremor ataxia syndrome precise small-molecule recognition of a toxic cug rna repeat expansion approaches to validate and manipulate rna targets with small molecules in cells discovery of a biomarker and lead small molecules to target r(ggggcc)-associated defects in c9ftd/als lomofungin and dilomofungin: inhibitors of mbnl1-cug rna binding with distinct cellular effects non-coding rnas as antibiotic targets small regulatory rnas in archaea small rnas in bacteria and archaea: who they are, what they do, and how they do it rna structures as mediators of neurological diseases and as drug targets antibacterial drug discovery in the resistance era fungal rna biology the antifungal pipeline: a reality check non-coding rna: what is functional and what is junk? front specificity and nonspecificity in rna-protein interactions an abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data micrornas and other non-coding rnas as targets for anticancer drug development enhancements of screening collections to address areas of unmet medical need: an industry perspective the re-emergence of natural products for drug discovery in the genomics era development and implementation of an hts-compatible assay for the discovery of selective small-molecule ligands for pre-micrornas rna fluorescence in situ hybridization for high-content screening progress and challenges for chemical probing of rna structure inside living cells detection of rna-protein interactions in living cells with shape photoaffinity labeling in target-and binding-site identification using transcriptome sequencing to identify mechanisms of drug action and resistance emerging classes of protein-protein interaction inhibitors and new tools for their development small molecules, big targets: drug discovery faces the protein-protein interaction challenge a small molecule that targets r(cgg)(exp) and improves defects in fragile x-associated tremor ataxia syndrome development of pharmacophore models for small molecules targeting rna: application to the rna repeat expansion in myotonic dystrophy type 1 identification of biologically active, hiv tar rna-binding small molecules using small molecule microarrays importance of ribosomal frameshifting for human immunodeficiency virus type 1 particle assembly and replication bioavailable inhibitors of hiv-1 rna biogenesis identified through a rev-based screen a modular approach to synthetic rna binders of the hepatitis c virus internal ribosome entry site artificial nucleobase-amino acid conjugates: a new class of tar rna binding agents we thank the members of the hargrove lab for stimulating discussion and input. key: cord-302784-jkjdglns authors: alotaibi, badriah; bieh, kingsley; yassin, yara; mushi, abdulaziz; maashi, fuad; awam, amnah; mohamed, gamal; hassan, amir; yezli, saber title: management of hospitalized drug sensitive pulmonary tuberculosis patients during the hajj mass gathering: a cross sectional study date: 2019-07-13 journal: travel med infect dis doi: 10.1016/j.tmaid.2019.07.007 sha: doc_id: 302784 cord_uid: jkjdglns background: to document the management of drug-sensitive tb patients during the hajj and assess compliance with the saudi tb management guidelines. method: the study was conducted in hospitals in makkah during the 2016 and 2017 hajj seasons. structured questionnaire was used to collect data on relevant indices on tb management and a scoring system was developed to assess compliance with guidelines. results: data was collected from 31 tb cases, 65.4% (17/26) were saudi residents. sputum culture was the only diagnostic test applied in 67.7% (21/31) of patients. most (96.8%, 30/31) confirmed tb cases were isolated, but only 12.9% (4/28) were tested for hiv and merely 37% (10/27) received the recommended four 1st-line anti-tb drugs. guideline compliance scores were highest for infection prevention and control and surveillance (9.6/10) and identifying tb suspects (7.2/10). the least scores were obtained for treating tb (5.0/10) and diagnosing tb (3.0/10). conclusions: healthcare providers training and supervision are paramount to improve their knowledge and skill and ensure their compliance with existing tb management guidelines. however, there may be a need for the introduction of an international policy/guideline for tb control and management during mass gatherings such as the hajj to guide providers’ choices and facilitate monitoring. tuberculosis (tb) remains a global public health problem with significant morbidity and mortality. in 2017, the world health organisation (who) estimated that 10 million people developed active tb causing up to 1.6 million deaths [1] . the who's end-tb strategy aims to reduce the overall number of tb deaths by 95% and the tb incidence rate by 90% in 2035 compared with the 2015 baseline incidence and mortality figures [2] . key to achieving these targets is the early diagnosis and appropriate management of tb cases worldwide according to national and international guidelines [3] [4] [5] [6] . this includes in the context of mass gatherings such as the annual hajj in makkah, kingdom of saudi arabia (ksa), where over 2 million pilgrims, many originating from tb endemic areas, congregate in crowded settings and worship under conditions that increase the risk of tb transmission [7, 8] . in the context of the hajj, respiratory tract infection including those caused by viruses have been researched in detail [9] . however, while tb cases have been reported during the event [10, 11] , tb management approaches during this unique event remained largely undocumented, and it is unknown whether these are consistent with the ksa and international tb management guidelines. hospitals in makkah serve both hajj pilgrims as well as local residents and the hajj workforce during the mass gathering free of charge. the additional stress on these health facilities during hajj may compromise the services provided for all patients during the event, including those diagnosed with tb. this study documents the management of drug-sensitive tb patients during hajj and explores the compliance of healthcare providers with the ksa tb management guidelines in the ministry of health (moh) hospitals in makkah during the mass gathering. this cross sectional study took place in makkah, saudi arabia, and https://doi.org/10.1016/j.tmaid.2019.07.007 received 28 january 2019; received in revised form 12 june 2019; accepted 10 july 2019 included 13 hospitals comprising those serving pilgrims in hajj holy sites. the study was conducted during the hajj lunar month (1st-30th dulhija) during the 2016 and 2017 hajj seasons, corresponding to 2nd sep-1st oct 2016 and 22nd aug-21st sep 2017, respectively. all hospitalized adults (> 18 years old) diagnosed with drug-sensitive pulmonary tb (ptb) during the study period were enrolled in the study if they consented. these patients are referred to in the manuscript as "suspected tb patients" until the time they were confirmed to have tb by the healthcare facility there were admitted to. the management of tb patients was documented using a specifically designed data collection form which included patients' demographics data, underlying health conditions and tb risk factors as well as clinical data including various aspects of tb management such as patients' screening, infection prevention and control (ipc), tb diagnosis and treatment and case notification and outcome. data was collected by the study team from patient's records, attending physician and through interviews with patients. all analyses were done using spss 22.0 (spss inc., chicago, usa) and sas 9.4 (sas institute inc., nc, usa) software program. variables were characterized using frequencies and mean for the respective categorical and continuous variables. a scoring system was developed, having identified four key themes from the questionnaire and literature review which were relevant to tb management in hajj. the themes were 1) identifying tb suspect, 2) ipc and surveillance, 3) diagnosing tb and 4) treating drug-sensitive tb. for each theme, relevant indicators were identified from the questionnaire (tables 2-5) . a score of 1 was assigned for each indicator/variable that was consistent with the 2014 ksa tb management guideline [6] (latest version during study period) and 0 for inconsistency with the guideline. the indicator subscore (x) for guideline consistency was obtained as follows: x = number of cases consistent with guidelines (c)/ total number eligible of cases (d) * 10 the eligible cases summed the consistent and inconsistent responses with tb guideline and strictly excluded missing data and unknown responses. the guideline consistency score for each theme was obtained by calculating the mean of the indicator sub-scores for each theme. the study was approved by the king fahad medical city ethics committee and the institutional review board (irb log: 16-329e) and conducted in accordance with the ethics committee's guidelines. characteristics of the study population are presented in table 1 . for the two-year period, 31 confirmed drug-sensitive ptb patients were recruited for the study. the mean age of the study population was 52 years (sd = 18.1 years, range 21-83 years). most (80.6%, 25/31) were males and over half were over 50 years old (60.7%, 17/28), with only primary or no formal education (55.1%, 16/29). the tb patients were nationals of 10 countries but the majority (65.4%, 17/26) had been residing in ksa for at least one year (table 1) . over one third of the cases (37.5%, 9/24) did not complete their hajj rituals while the status of 50% (12/24) of the cases was not known. three confirmed tb cases (12.5%, 3/24) did complete their hajj rituals in individual ambulances. no mortality was recorded among the tb patients at the time when the study ended with half (14/28) having been discharged (table 1) . in relation to tb risk factors, only 6.6% (2/30) of respondents reported prior travel to high tb burden countries (pakistan and indonesia) and 27.6% (8/29) stated that they had performed hajj or umrah within the past 1 year. the majority (75%, 6/8) of the previous hajj or umrah pilgrims were saudi residents. a sizable proportion of tb cases declared that they had smoked or were current smokers of tobacco products (44.8%, 13/29) or had chronic diseases (63.0%, 17/27) especially diabetes and hypertension (table 1) . upon registration, most (76.6%, 23/30) suspected tb patients were admitted to an isolation room or ward. otherwise, the patients were either admitted into the emergency room (er [6.7%, 2/30]), icu (6.7%, 2/30), neurosurgery ward (3.3%, 1/30), orthopedic ward (3.3%, 1/30) or general ward (3.3%, 1/30). while a proportion (48.4%, 15/ 31) of suspected tb patients was separated from other patients during registration, the triaging status of 45.2% (14/31) of suspected tb patients was not reported ( table 2 ). the proportion of suspected tb patients put in isolation while waiting diagnosis was 77.4% (24/31) and rose to 96.8% (30/31) once drug-susceptible tb was confirmed. only 1 (3.2%) confirmed tb patient was managed in the er. generally, most (93.1%, 27/29) confirmed tb patients spent less than 1 day in the health facilities before they were isolated. in all cases, appropriate symptoms were sought from tb suspects, in particular, cough ≥2 weeks (64.5%, 20/31) and fever with chills/night sweat (67.7%, 21/31). the history of contact with active tb cases was obtained from 45.2% (14/31) of suspected tb cases, although the status of this variable was unknown in a further 41.9% (13/31) of the cases (table 3 ). in general, 44.8% (13/29) of the tb suspects were questioned about other tb risk factors. specifically, their country of residence, hiv status and potential occupational exposure to tb (table 3) . furthermore, in the majority of cases (67.7%, 21/31) providers used recommended screening test for active tb (chest x-ray) in case management. in terms of diagnostic period, 50% (5/10) of tb suspected patients within known health facilities visits status had > 2 visits to health facilities before appropriate screening/diagnostic tests were ordered (table 4 ). however, the period between patient registration/arrival in health facilities and order of tb screening/diagnostic test(s) was ≤12 h in 77.4% (24/31) of cases. similarly, in most cases (83.4%, 25/30), the period between ordering screening/diagnostic test(s) and confirmation of tb diagnosis was ≥2 days. in general, 62.5% (15/24) of cases were diagnosed within 2 days of arrival/registration to healthcare facilities. sputum culture was the diagnostic test utilized in the majority (67.7%, 21/31) of cases (table 4 ). xpert mtb/rif assay was not utilized for tb diagnosis. in one instance, none of the recommended diagnostic tests were ordered for the tb suspected patient. only 12.9% (4/25) of suspected/confirmed tb cases were screened for hiv. in over half of cases (58.1%, 18/31), the tb suspected patients were questioned about their tb history, although for a further 25.5% (8/31), response to this variable was unknown ( based on the duration of hajj season (around one month), the possibility of monitoring treatment completion is unfeasible in a hajj study. thus, appropriate post-hajj referral and provision of drugs to last during the referral period were proxies for estimating possible continuity of care. in most cases it was not known whether confirmed tb patients were referred for further treatment after completion of hajj (77.3%, 17/22) or were given enough anti-tb drugs to last until they arrive in their country of residence (61.6%, 11/18). in 76.7% (23/30) of cases, the confirmed tb cases were reported to the ksa moh preventive medicine department. the reporting status of the rest of the cases (23.3%, 7/30) was unknown. a proportion (44.8%, 13/29) of the confirmed tb cases was reported to the appropriate country medical missions' office. this excludes the 48.3% (14/29) of cases with unknown medical missions' reporting status. among the latter, 57.1% (8/14) were saudi residents. the confirmed tb case status of two residents of pakistan and myanmar were not reported to their respective country medical mission's office. the tb management guidelines compliance scores across the 4 identified themes are presented in table 6 . out of a maximum possible score of 10, the overall guideline compliance score was highest for the themes ipc and surveillance (9.6) and identifying tb suspects (7.2). the least scores were obtained for the themes treating tb (5.0) and diagnosing tb (3.0). some notable variations in theme's sub-scores were observed. for instance, while the overall score for the identifying tb suspects theme was high, a low score was documented for obtaining history of tb risk factors from patients. inversely, while the overall score for treating tb was average, high score was seen in relation to not starting tb treatment for patient before tb diagnosis. this study exemplifies the compliance of tertiary healthcare providers with the saudi national guidelines for tb management during the hajj. the result showed high level of compliance with the assessed tb management guidelines indices for systematic screening of tb suspects as well as ipc and surveillance, but low compliance scores were obtained for prompt tb diagnosis and use of standardized treatment regimen for drug-susceptible tb. most tb cases in the current study were males and over half were above 50 years old with primary or no formal education. this is in accordance with global and hajj-related data and established risk factors for tb [1, 7, 10, 12] . however, the prevalence of coexisting chronic diseases (63%, 17/27) among tb patients, especially diabetes, was higher than that reported internationally as well as previous studies among hajj pilgrims with tb [7, [12] [13] [14] . the presence of chronic diseases, increases the risk of tb disease, predisposes to severe illness and complicates tb treatment [1, 12] . as such, the management of comorbidities is now a key focus of the integrated, patient-centered care and prevention strategy of global tb control [2] . around 28% (8/29) of the tb cases reported being current smokers and a similar proportion (5/18) indicated that they did smoke in the past. this is higher than that reported in another study among hajj pilgrims with tb (13.3%) [7] but lower than figures from some international reports [15, 16] . other risk factors for tb such as visit to, or residence in high-burden countries and occupational exposure were uncommon among tb patients in the study and so was previous hajj or umrah performance. while the latter events are not an established risk factor for tb transmission, hajj is a risk of tb infection and both clinically-recognized or undiagnosed active tb have been reported at the pilgrimage [7, 10, 17] . the majority (65.4%, 17/26) of tb patients in this study were ksa residents. this may be explained by the fact that the study included both pilgrims and non-pilgrims and that healthcare facilities in makkah provide healthcare to pilgrims and non-pilgrims during the hajj. although saudi arabia is not a high tb burden country, tb incidence in the country show significant regional variation with the makkah region showing much higher tb incidence rates than the rest of the country and rising trend [18, 19] . in addition to the hosting of the hajj and umrah mass gatherings, this high tb incidence may also be related to the fact that around 40% of the makkah region population are non-saudis, many originate from and frequently visit high tb burden countries [18, 19] . regardless, it is evident that in addition to strategies to control imported active tb [7] , interventions to prevent transmission during hajj from locals and internal pilgrims with tb should also be developed and implemented. generally, home-based care for tb is preferred to methods of care that are based on strict hospitalization. in the ksa context, medical or mental instability and residence in congregate settings are among factors that may warrant hospitalization [6] . in this study, most of the suspected and confirmed tb patients were admitted and isolated for tb management. to the best of our knowledge, there is no standardized global protocol guiding the choice of suitable models of care-whether home based or hospitalized care-during international mass gatherings. however, considering the potential of tb transmission in such crowded settings, the constant mobility of pilgrims and challenges in verifiable or stable residences for pilgrims during hajj, hospitalization, although undesirable, seems a logical and practical choice for tb management during the mass gathering. ipc in healthcare settings is one of the key strategies for tb control [20] . however, implementation of ipc recommendations seems to be inadequate with several studies reporting poor tb infection control measures in health facilities [21] [22] [23] [24] . further, many hcws are practicing without adequate infection control training and often lack knowledge on tb infection control strategies and guidelines [24, 25] . in the current study, we report high compliance with the aspects of tb ipc [26] . early detection through systematic screening of tb suspects is key to improving tb case detection. the who recommends that persons with signs and symptoms consistent with tb should be evaluated for tb to ensure prompt diagnosis and treatment [3, 5] . similarly, the saudi tb guideline recommends that healthcare workers (hcws) should be knowledgeable about tb symptoms to facilitate the efficient identification of tb suspects for diagnosis and treatment [6] . in the current study, providers utilized presenting symptoms to correctly identify suspected tb patients in all cases. cough and fever with chills/night sweat were the most frequent symptoms among patients. this finding corroborates existing evidence that identifies cough as the most common symptom of ptb [3, 27] . further, in majority of cases (67.7%, 21/31), chest x-ray, a recommended screening tool for active tb, was conducted for the tb suspects. chest x-ray is particularly more sensitive for tb screening after a positive symptom screening [27] . however, we also found that less than half of the tb suspected cases were questioned about tb risk factors. adequate knowledge of tb symptoms and risk factors among providers are prerequisites for correct and prompt identification of suspected tb patients for screening and diagnosis [5, 6] . in view of the significant use of both symptom-based and radiological screening methods in this study, a total guideline compliance score of 7.2 out of 10 was obtained for the prompt identification and screening of tb suspects theme for tb management. delayed diagnosis of tb can enhance the transmission of infection, worsen the disease and increase the risk of death [28, 29] . in the current study, half of the tb cases had more than 2 visits to healthcare facilities before tb screening/diagnosis tests were ordered for the patients. while studies from other settings reported similar findings [30, 31] , our results are concerning, as delays in diagnosing tb during hajj may lead to significant transmission given the crowded setting during the event. similarly, sputum culture (which takes at least 2-3 weeks to produce results) was the only recommended diagnostic test applied in about 70% (21/31) of cases. the application of sputum culture as the singular diagnostic test is not consistent with approved standards for tb diagnosis [6] . as 75% (18/24) of suspected cases were confirmed to have tb by the third day of arrival in the health facility, it appears that providers relied on screening tests, such as chest x-ray, for the confirmation of tb diagnosis. this practice is inconsistent with both national and international guidelines; chest radiography is only recommended for screening purposes. the 2014 ksa tb guidelines recommended the use of xpert mtb/ rif as an initial tb diagnostic test on a conditional basis [6] . as such, the latter was not included in the scoring criteria for this study. nonetheless, xpert mtb/rif, which could detect tb and mdr-tb by proxy in the same day [32] , was not applied for tb diagnosis in this study. although available in a number of saudi hospitals and reference labs, the roll out of xpert mtb/rif has been slow and its use for pointof-care testing is limited [7, 33] . access to same day diagnosis of tb could prove valuable in a highly mobile hajj population where followup visits to the same health facility may not be guaranteed and where delays in diagnosis may increase the risk of transmission in such crowded settings. as such, ksa authorities should consider the provision of tb molecular testing capability in health facilities within the hajj areas to facilitate rapid (same-day) diagnosis of tb during the mass gatherings. due to the synergistic relationship between hiv and tb, it is recommended that all tb patients should be screened for hiv [5] . yet, only a fraction of tb patients were questioned about their hiv status (20.7%, 6/29) or tested for hiv (12.9%, 4/28) in this study. this is much lower than what is reported globally [1] . as a low prevalence setting, knowledge of hiv among healthcare workers is low in saudi arabia [34] . yet, hiv could be a more frequent comorbidity among pilgrims who arrive with active tb from areas with high hiv disease prevalence [1] . more so, a missed or delayed hiv diagnosis in a tb patient stalls the commencement of appropriate treatment and results in poor outcomes for the patient, community and health system [35] . therefore, healthcare providers in ksa ought to be trained and guided to conduct screening for hiv and other comorbidities in all suspected tb patients irrespective of their nationality. in general, because of delays in diagnosis, infrequency of hiv testing and failure to utilize the appropriate diagnostic tests for suspect tb patients, the combined score for the tb diagnosis theme was 3 out of a maximum of 10, the lowest score of all tb management themes in the current study. treatment of tb in ksa is free of charge for pilgrims and other patients and both the ksa and who guidelines for tb management recommend the use of four 1st-line anti-tb drugs in the treatment of drug-susceptible tb [5, 6] . the guideline compliance score for tb treatment in this study was average; partly because 63% (17/27) of tb patients received fewer than four 1st-line anti-tb drugs. in general, inappropriate treatment of tb is common worldwide. in a systematic review that included 37 studies from 22 countries, inappropriate treatment regimens were prescribed in 67% of the studies and the percentage of patients on inappropriate regimens varied between 0.4% and 100% [36] . poor knowledge of national and international tb management guidelines contributes to inappropriate prescription of anti-tb drugs by healthcare providers, and the use of inappropriate regimen drives the occurrence of relapse and the emergence of drugresistant tb [37, 38] . both the who and ksa tb guidelines recommend that all patients with ptb being treated with the 1st-line regimen should have their sputum samples tested by the end of the 2nd, 5th and 6th month of treatment [6, 39] . in the current study, all confirmed tb cases with known notification status were reported to the saudi health authorities. however, it is unknown whether the continuum of care was maintained for tb cases who were international pilgrims and who had to return to their home countries soon after the pilgrimage (before the end of the treatment period). any travel-related treatment interruptions could breed treatment relapse and drug-resistance and propagate community spread of tb. both national and international tb guidelines fall short of providing guidance on tb control at international mass gatherings, including procedures for ensuring access to care and support services during travel. thus, the development and dissemination of a multinational hajj and umrah and/or mass gatherings-specific tb management protocols are needed. these protocols should also include pathways for the safe transfer across borders and follow up of tb patients involved in mass gatherings. the current study is among the foremost surveys of tb management at international mass gatherings. while the small number of cases and high proportion of unknown responses for some variables constituted limitations, the tb management indices obtained was a fair representation of the compliance of providers with national and international tb guidelines in moh hospitals during the hajj. the findings provides a basis for the review of existing practices across settingsprivate and public sector vs national and foreign health facilities-and serves as a reference for the development of appropriate guideline and protocol for tb management at the hajj and umrah, as well as other settings with similar health system resources and population dynamics hosting recurrent international mass gatherings. in the short term, availability of rapid molecular diagnostic techniques for tb as we all improving hcws' knowledge regarding tb management guidelines and monitoring compliance are needed to ensure tb patients are management appropriately during hajj and that tb transmission is prevented. no conflicts of interest to declare. none to declare. the study was approved by the king fahad medical city ethics committee and the institutional review board (irb log: 16-329e) and conducted in accordance with the ethics committee's guidelines. all participants gave verbal consent before enrolment. world health organization. gear up to end tb: introducing the end tb strategy. world health organization world health organization. early detection of tuberculosis: an overview of approaches, guidelines and tools world health organization. guidelines for the treatment of drug-susceptible tuberculosis and patient care compendium of who guidelines and associated standards: ensuring optimum delivery of the cascade of care for patients with tuberculosis. world health organization saudi ministry of health. basics of tuberculosis control in saudi arabia. public health agency ntcp, ministry of health, kingdom of saudi arabia undiagnosed active pulmonary tuberculosis among pilgrims during the 2015 hajj mass gathering: a prospective cross-sectional study tuberculosis infection during hajj pilgrimage. the risk to pilgrims and their communities a systematic review of emerging respiratory viruses at the hajj and possible coinfection with streptococcus pneumoniae tuberculosis is the commonest cause of pneumonia requiring hospitalization during hajj (pilgrimage to makkah) clinical and temporal patterns of severe pneumonia causing critical illness during hajj tuberculosis comorbidity with communicable and non-communicable diseases: integrating health services and control efforts tuberculosis non-communicable disease comorbidity and multimorbidity in public primary care patients in south africa comorbidities in pulmonary tuberculosis cases in puducherry and tamil nadu, india: opportunities for intervention high prevalence of smoking among patients with suspected tuberculosis in south africa prevalence of smoking and its impact on treatment outcomes in newly diagnosed pulmonary tuberculosis patients: a hospital-based prospective study high risk of mycobacterium tuberculosis infection during the hajj pilgrimage tuberculosis incidence trends in saudi arabia over 20 years: 1991-2010 tuberculosis in saudi arabia: prevalence and antimicrobial resistance world health organization. who policy on tb infection control in health-care facilities, congregate settings and households a national infection control evaluation of drug-resistant tuberculosis hospitals in south africa the status of tuberculosis infection control measures in health care facilities rendering joint tb/ hiv services in "german leprosy and tuberculosis relief association" supported states in nigeria infection control and the burden of tuberculosis infection and disease in health care workers in china: a cross-sectional study assessment of knowledge and practice of health workers towards tuberculosis infection control and associated factors in public health facilities of addis ababa, ethiopia: a cross-sectional study updates on knowledge, attitude and preventive practices on tuberculosis among healthcare workers a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of world health organization. systematic screening for active tuberculosis: principles and recommendations. world health organization the relationship between delayed or incomplete treatment and all-cause mortality in patients with tuberculosis delayed tuberculosis diagnosis and tuberculosis transmission missed opportunities to diagnose tuberculosis are common among hospitalized patients and patients seen in emergency departments time delays in diagnosis of pulmonary tuberculosis: a systematic review of literature roadmap for rolling out xpert mtb/rif for rapid diagnosis of tb and mdr-tb evaluation of genexpert mtb/rif for detection of mycobacterium tuberculosis complex and rpo b gene in respiratory and non-respiratory clinical specimens at a tertiary care teaching hospital in saudi arabia knowledge and attitudes of doctors toward people living with hiv saudi arabia early versus delayed antiretroviral therapy for hiv and tuberculosis co-infected patients: a systematic review and meta-analysis of randomized controlled trials prevalence of inappropriate tuberculosis treatment regimens: a systematic review knowledge of tuberculosis-treatment prescription of health workers: a systematic review multidrug resistance after inappropriate tuberculosis treatment: a meta-analysis world health organization. treatment of tuberculosis: guidelines. geneva: world health organization key: cord-022888-dnsdg04n authors: nan title: poster sessions date: 2009-08-19 journal: eur j immunol doi: 10.1002/eji.200990224 sha: doc_id: 22888 cord_uid: dnsdg04n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx3, the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx3 facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th1-oriented adaptive response. actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx3, which does not show direct activity on fungal cells. finally, ptx3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx3-mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx3 amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx3-opsonised conidia, cd11b activation, internalization, recruitment to the phagocytic cup and cd11b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd11b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx3, fcgriia/cd32 mediates inside-out activation of cd11b and consequently phagocytosis of c3b-opsonised conidia. in vivo phagocytosis experiments performed with c1q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx3) and the cellular arm (fcgrs, cr3). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx3 binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx3-/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx3. we found high concentration of ptx3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( x 2 ng/ml). the presence of ptx3 in human colostrum seems to be due to the secretion of ptx3 by human mammary gland since we report the production of ptx3 by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx3 production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx3 supplying by breast feeding: (i) soluble ptx3 in colostrum (ii) hmc that can secrete ptx3 upon stimulation in the specific tissue, (iii) an increase of ptx3 production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx3 may participate to the beneficial role of breast feeding on the newborn health. a. m. baru 1 , j. stephani 2 , h. wagner 2 , t. sparwasser 1 1 twincore, institute for infection immunology, hannover, germany, 2 technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of 10 receptors in humans (tlr 1-10) and 12 in mice (tlr 1-9, 11-13). as transmembrane receptors, tlrs are expressed on the cell surface (tlr 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (tlr 3, 7, 8 and 9) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about 5 years later to this, toll-like receptor 9 (tlr9) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr9 (hutlr9) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr9 (mutlr9) is also expressed on conventional dendritic cells (cdcs). consequently, tlr9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr9 (henceforth called as hut9 mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr9 knock-out background. we expect that hut9-mutlr -/mice mimic the human specific expression pattern of tlr9, i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr9. by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr9 ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c1q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. the main inducers of il-12p40 are microbial components activating toll-like receptors with the magnitude of il-12p40 induction depending on the specific tlr engaged. differential induction of il-12p40 upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il-12p40 promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il-12p40 promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. acetylation of h4 showed a specific distribution pattern and occured mainly in regulatory elements within the il-12p40 promoter, whereas acetylation of h3 took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue 4 on h3 turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue 1 , w. ellmeier 1 1 medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi 1 , m. buxadé 1 , j. minguillón 1 , r. berga 1 , j. aramburu 1 , c. lópez-rodríguez 1 1 universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat5 is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat5 participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat5 is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat5 could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat5 is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat5 is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat5. our work indicates that nfat5 is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr3, tlr4, tlr7 and tlr8 and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p50 and p65 nfxb subunits. interestingly, we observed that tlr3 stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr4, tlr7 and tlr8 stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl-2 expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr3, tlr4, tlr7 and tlr8. finally, in vivo tlr7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr4, tlr7 or tlr8 triggering can directly favor tumor development whereas tlr3 signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms-354825) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd8 + and cd4 + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr 4 ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd4 + and cd8 + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd4 + and cd8 + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: 1. innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr 4 stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . 2. adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni 1 , r. oatuni 1 , m. collini 2 , m. caccia 2 , p. castagnoli 3 , g. chirico 2 , f. granucci 1 1 university of milano-bicocca, biotechnology and bioscience, milan, italy, 2 university of milano-bicocca, physics, milan, italy, 3 singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin-2 (il-2) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il-2 production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca2+/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca2+ influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr4 engagement, depending instead exclusively on cd14. we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase-2 (cox-2) that, by generating prostaglandins (pgs), such as pge2, regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd14. given the essential involvement of cd14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd14 represents a major step towards the development of potential treatments with modes of action involving interference with cd14 functions. we have examined the interaction of cd55, a 80-kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd14. human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd14 antibody coupled to biotin visualised by qd-525-streptavidin and anti-cd55 antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd55 and cd14, suggesting a new functional role of cd55 as a member of a multimeric lps receptor complex. l. lundvall 1 , r.r. schumann 1 1 charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase-1 induction leads to an increased release of mature il-1b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d39) was established for assessing il-1b induction during this disease. the murine raw 264.7 cell line, the human astroglial u-87 mg and the murine microglial cell-line bv-2 were stimulated with the tlr4 ligand lps, the tlr2 ligand pam 2 cys, the nod2 ligand mdp, or the nod1 ligand c12-ie-dap, and, as control, atp alone or in combination. we assessed il-1b by elisa and caspase-1 and pro-il-1b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il-1b induction pathway. s. pneumoniae (d39) infected mice showed a significant increase in il-1b release after 24 hours. in vitro, an increase in il-1b levels after costimulation with lps or pam 2 cys, and mdp or c12-ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il-1b by tlr-and nlr-ligands was observed in raw cells, bv-2 cells, u-87 cells and primary astrocytes. active caspase-1 (p10) was induced by mdp or c12-ie-dap, corresponding with high il-1b responses when lps or pam 2 cys was added. sirna experiments show that a knock-down of nod2 leads to a diminished il-1b release after lps-and mdp-stimulation. the precursor forms of il-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il-1b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il-1b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il-1b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg 1 1 toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd3 and cd28 activation model system -tlr4 presence on cd4+ cells is found in mouse t cells, human t cells and jurkat cell lines. following cd3/cd28 activation for 48 hours we have identified a small but distinct populationof tlr4+ cells. further characterization indicates these cells to be cd4+cd25+ cells. further characterization of the expression and functional acitvity of the tlr4+ t cells indicates co-expression of tlr4 with md-2 indicating a functional tlr4 receptor. in addition lps activiation did not lead to upregulation of tlr4 expression in t-cells. the data indicate that tcr activation leads to tlr4 expression. the expression appears to be associated with cd4+cd25+ cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann 1 , d. kaul 1 , c. krüger 1 , f. zipp 1 , r. nitsch 2 , s. lehnardt 1 1 charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, 2 charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr7 plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr7 (and human tlr8) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr7 is expressed in these cells. incubation of microglia with all three of the above mentioned tlr7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il1-b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr7 knock out (ko) microglia by realbecause neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin-1 is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin-1-transfected plb985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd11b labeling. concerning apoptosis, increased coronin-1 expression in dmf-differentiated plb985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin-1 significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or bid truncation suggesting that coronin-1 interfered with mitochondria-related events. to validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin-1 immunolabeling. we concluded that coronin-1 could constitute a potential target in resolving inflammation. p.-n. hsu 1 1 national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw264.7 macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p38 map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf-6 sirna and traf-6 decoy peptide, indicating this pathway is traf-6 dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b7-h1 and b7-dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il-6 was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il-2 and ifn-g which could not be overcome by restimulation with acd3. this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd4 t cells into t helper 17 (th17) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th17 differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th17 cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il-1 receptor-related protein st2 could be implicated in lps tolerance. the natural ligand of st2 was recently identified as interleukin-33 (il-33), a new member of the il-1 family. in this study, we investigated whether il-33 triggering of st2 was able to induce lps desensitization of mouse macrophages. we found that il-33 actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il-33 enhancing effect of lps response is mediated by the st2 receptor since it is not found in st2 ko mice. the biochemical consequences of il-33 pretreatment of mouse macrophages were investigated. our results show that il-33 increases the expression of the lps receptor components myeloid differentiation protein-2 (md2), cd14 and tlr-4 and the myeloid differentiation factor 88 (myd88) adaptor molecule. in addition, il-33 pretreatment of macrophages enhances the cytokine response to tlr-2 but not to tlr-3 ligands. thus, il-33 treatment preferentially affects the myd88-dependent pathway activated by the tlr. c. klotz 1 , b. lenz 1 , r. lucius 1 , s. hartmann 1 1 humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., 2008) . the cystatin effect was blocked by the application of anti-il-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin-10 (il-10). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il-10 in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il-10 production after avcystatin stimulation. application of specific inhibitors revealed that the il-10 induction was p38 and erk dependent, and inhibitor titration indicated a higher sensitivity towards p38. western blotting experiments confirmed the phosphorylation of p38 and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il-10 is also regulated by the phosphatidylinositol-3-kinase (pi3k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il-10 and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose-7-phosphate (s-7p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose-5p and xylulose-5p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p38/jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s-7p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts 1 , y. morias 1 , p. de baetselier 1 , a. beschin 1 1 vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m1) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m1 in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd88, ifng, il-10, ccr2 and nf-kb to the development and/or recruitment of pathogenic m1 in the liver was investigated using knock-out mice or by delivering il-10 in infected mice. results: we established that cd11b+ly6c+cd11c+ tnf and inos producing dcs (tip-dcs) represent the major m1 liver subpopulation. tip-dcs differentiated in an ifng/myd88-dependent manner from cd11b+ly6c+ inflammatory monocytes in the liver of infected mice. ccr2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr2 ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il-10 in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p50 was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p50 and il-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m1, in particular tip-dcs. the inflammatory activity of liver m1 is controlled by il-10 and/or p50 nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov 1 , j. driesen 1 , z. abdullah 2 , a. niño castro 1 , t. chakraborty 3 , m. krönke 4 , o. utermöhlen 4 , c. wickenhauser 5 , j.l. schultze 1 1 limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, 2 institute of molecular medicine and experimental immunology, bonn, germany, 3 institute of medical microbiology, university of giessen, giessen, germany, 4 institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, 5 institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and cd25 and production of prostaglandin e2 (pge 2 ) and interleukin 10. all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd25, expressed by regulatory myeloid cells, acts as an il-2 scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge 2 or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd25 + ido + cox-2 + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin 12 (il-12) and il-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il-12 and il-18 or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il-12 and il-18. in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il-12 and il-18. therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel 1 , m. rodriguez gomez 2 , m. niedermeier 2 , y. talke 2 , n. göbel 2 , k. schmidbauer 2 , m. mack 2 1 unversity hospital regensburg, internal medicine ii, regensburg, germany, 2 university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il-4 and il-6. activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp-1 cells infected with m. bovis bcg, the avirulent m. tuberculosis h37ra strain and the virulent m. tuberculosis h37rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva 1 , l.d. miteva 1 , s.a. stanilova 1 1 trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il-23 is a heterodimeric cytokine composed of a p19 subunit associated with the il-12/23p40 subunit. like il-12, il-23 is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il-12p40-related cytokines controls the appearance of normal th 1 and pathological th 17 mediated immune responses. in this study, we examined the dynamics of inducible il-12p40 and il-23p19 mrna expression and protein production in purified human monocytes and how jnk and p38 mapks inhibitors influenced il-12p40 and il-23 production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il-23p19 gene expression was higher than those observed for il-12p40 gene at all time-points. the level of il-23p19 mrna increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for c3bgp and 22.7 fold for lps). c3bgp and lps triggered il-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. the higher level of il-12p40 gene expression was detected at 6 th h in lps compared to c3bgp stimulated monocytes (8.1 vs. 4.9 fold). however, il-12p40 and il-23 protein production was increased in the highest level after c3bgp stimulation. the inhibition of p38 led to the statistical significant augmentation of c3bgp stimulated il-12p40 production. the inhibition of the same map kinase enhanced lps stimulated il-12p40 production without significant difference. the inhibition of jnk and p38 mapks significantly decreased c3bgp stimulated il-23 production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c3bgp and lps to induce the expression of il-12p40 and il-23p19 mrnas in purified human monocytes. we showed that inhibition of p38 mapk down regulated il-23 and upregulated il-12p40 production in stimulated monocytes. we concluded that in human monocytes p38 map kinase activation has an opposite effect on the il-12p40 and il-23p19 expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd83, cd86 and cd40, and secrete il-12, thus acquiring the ability to induce proliferation and th1 polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd1c, bdca-3, and cd16. by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd16+ subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd16+ dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd16+ dc, including their survival and their ability to produce il-12p70. besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il-6, il-8 and mcp-1 involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg 1 , s. wolke 2 , a. brakhage 2 , m. gunzer 1 1 otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, 2 hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in 2004 nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to 3 hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor 7 (irf7), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr9. in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr9 to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens 1 , s. vander beken 1 , p. bogaert 1 , j. grooten 1 1 ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th1-driven immunological counterpart of the th2-driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd45 bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th1-and th2-driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd45.1 + ram remained constant in cell number for the first 2 days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker 1 , a. stojanovic 1 , a. cerwenka 1 1 german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr-1 + cd11b + f4/80 + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas 1 , f. marchesi 1 , m. fabbri 1 , s. schiarea 2 , c. chiabrando 2 , a. mantovani 1,3 , p. allavena 1 1 istituto clinico humanitas, rozzano, italy, 2 istituto mario negri, milano, italy, 3 università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m1 and m2. m1 macrophages act as a first line of defence against pathogens whereas m2 cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd16, cd68 and low levels of mhc class ii. tc-macro produced il-10, il-6, tnf but not il-12, even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl2, cxcl8, ccl17 and cxcl10. the transcriptional profile of tc-macro revealed that several genes in line with an m2 polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g 100.000 kda). il-3 and il-6 were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl-2h3 rat mast cell line. we demonstrate that gr nuclear translocation begins within 5 minutes and completes after 30 minutes in dx treated rbl-2h3 cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within 5 minutes as non-genomic. studying gc-caused apoptosis, rbl-2h3 cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl-2h3 cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. 5 minutes of dx treatment inhibited ca 2+ -signaling in antigen stimulated rbl-2h3 cells in the concentration range of 100nm -10mm. moreover, 5 minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl-2h3 cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box 1 (hmgb1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . extracellular hmgb1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb1 exerts antibacterial functions in human adenoid and testis (2) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity (3). we asked whether hmgb1 from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for 40 or 120 minutes with 25 nm phorbol ester (pma), 100 ng/ml interleukin 8 (il-8), or 100 ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h2a-histone h2b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb1 is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb1 localizes on nets. we hypothesize that net-bound hmgb1 might exert a direct antimicrobial function, or that nets might concentrate hmgb1 locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation (37°c, 3 h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr-1 and anti-vegfr-2 antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il-18. altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd1 molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd1a and cholesterol-dependent lipid rafts impact on cd1a surface expression and cd1a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd1-restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd1 group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd1a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd1 molecules in multiple sclerosis. we first analyzed cd1 expression on monocytes from 16 ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd1a was not expressed on ms patient monocytes, while the other members of the cd1 family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd1 molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd1 molecules in this context. c. ohnmacht 1 , d. voehringer 1 1 ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th2 cells. these results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il-1beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il-6 and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il-6 and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant 143038). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, mph1), and during resolution of inflammation (type-2 macrophages, mph2). methods: mph1 / 2 were generated by culturing cd14 + human monocytes for 6 days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph1 / 2 of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph1 / 2 were stimulated with lps and secreted il-6, il-8, il-10, il-12p40 and tnf were quantified by elisa. results: mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas mph1 are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph1 and mph2 which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il-6, il-12 and tnf production is suppressed while il-10 secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp70 in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). intracellular ros and hsp70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (invitrogen) and anti-hsp70 antibody (brm-22, sigma), respectively. intracellular level of hsp70 was also estimated in neutrophils after heat shock (hs) performed at 43°c for 10 min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at 1-15 min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for 1 min at 42°c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp70 (hsp70 basal ) and ros level, both intracellular and extracellular. at the same time increased hsp70 level immediately after hs (hsp70 (0 min)) correlated negatively with intracellular ros (initial and after hs). the hsp70 increase value (hsp70 (0 min) -hsp70 basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp70 increase was registered in 60 min after hs (hsp70 (60 min) -hsp70 basal ). thus it was found that within this age group the alteration in hsp70 induced by hs in neutrophils but not basal hsp70 itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant #3303. d. goyeneche-patiño 1 , z. orinska 1 , f. mirghomizadeh 1 , s. bulfone-paus 1 1 forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd8 + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type 1 ifn. two novel genes, receptor transporter protein (rtp4) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp4 and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd88 and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd8 -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during 8 and 48 h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp4 and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine 2000. stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp4 in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd88 knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr 3 and 9, as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp4 and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd88 and trif and the pathways that they represent are not relevant in the expression of rtp4 and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano 1 , e. erba 2 , r. frapolli 2 , m. d'incalci 2 , a. anselmo 1 , s. pesce 1 , p. allavena 1 , a. mantovani 1, 3 1 humanitas clinical institute, rozzano, italy, 2 mario negri institute, milan, italy, 3 institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et-743) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl2, cxcl8 and the inflammatory protein pentraxin3 (ptx3) either at transcriptional and protein level, especially after tnfa/il1b stimulation. down-regulation of ccl2, cxcl8, ptx3 and also of il6 and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin-4 (il-4) is a key cytokine of the t helper 2 cell response. il-4 has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il-4 production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il-4 producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day 3-6 after birth, a small population of il-4 producing cells was observed in the absence of any stimuli. the il-4 + population was not detectable at 6 or 12 weeks of age. other cytokine producing cells (ifn-g, il-10) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il-4 + cell population. phenotyping of the neonatal il-4 + cells showed that they were ige + /mhcii -/cd4cells. the occurrence of cd4 + il-4 producing cells after pma stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il-4 at day 5 after birth. neonatal pbmc collected before colostrum uptake did not produce il-4 in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il-4 responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il-4 response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel 1 , d. voehringer 1 1 ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat6-dependent manner when stimulated with the th2 cytokines il-4 or il-13. alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat6-dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat6-deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat6 expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat6 expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat6-deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il-4 exposed macrophages from wild-type and stat6-deficient mice. candidate genes will be expressed using retroviral transfections of stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. the following human malignant cell lines were tested: a549, ht29, hct116, sw60, mcf7, mda-mb231, jurkat and hl60. results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m1, and the tams isolated from the malignant effusions similar to m2. among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms4a4a and ms4a6a: highest trascriptional level after 18h of stimulation with 10-6m dexamethasone. ms4a murine genes are differently expressed respect to the human counterpart and only the homologs of ms4a6a (ms4a6b, 6c and 6d) have a similar regulation. finally, egfp-tagged ms4a4a, ms4a6a, and ms4a7 expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms4a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms4a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (mcp-1)/ccl2 and the gpcr ligand fmlp or the tlr4 agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il-1b or ifn-g, significantly and dose-dependently synergized with ccl2 in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin-8/cxcl8, but not of the ccl2 receptor ccr2 in monocytic cells. in turn, the induced cxcl8 synergized with ccl2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr1/cxcr2, because cxcl8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl2 intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, 2004) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for 20 min at 37°c with gentle shaking, followed by spinning down of pmns for 5 min, at 4°c and 500g. the supernatant was sedimented at 15000g for 10 min, 4°c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd56 has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd56 low cd33+ blood monocytes (mo) in healthy individuals and the increase in cd16+cd56+ blood mo in patients with inflammation has been reported. the functional activity of human cd56+ blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd56 expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd33 and activation markers such as hla-dr and trem-1. percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (p x 0.0001) in the total number of circulating cd56 low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd33 and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd56+ blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd33+ blood mo expressed increased levels of cd56 on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem-1+) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd56 on cd14 high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m1-and m2-polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m1 and m2 macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m1 type and were cd14 negative but cd80 and mhcii positive and produced high levels of il-8, tnfalpha and il-6 following lps stimulation. culture in the presence of mcsf resulted in induction of the m2 phenotype. these cells were cd14 positive with intermediate expression of cd80 and mhcii expression and produced high levels of il-10, il-6 and il-8 following lps stimulation. interestingly, already baseline il-8 production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m1-and m2-polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr4 ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma (50 ng/ml) treatment for 48h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl60 cells expressed antigen receptors cd14, tlr2, tlr4 and cd68 , and displayed enhanced phagocytic activity and production of ros. expression of cd13, cd33 and cd15 was also maintained however the cells were hla-dr and cd1a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl60 cells to differentiate into macrophages in response to pma. hl60 may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta 1 , n. ferrer 2 , a. gómez 2 , j. gonzalo 2 , a. arbués 2 , a. anel 1 , c. martín 2 , j. pardo 1 , apoptosis, immunity and cancer 1 university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, 2 university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so2, a m. tuberculosis phop mutant strain that was shown (perez et al 2001) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al 2006) . in the present study, we compare the time course and phenotype of cell death induced by so2, bcg and wild type m. tuberculosis on the murine macrophage cell line j774 and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type 1 receptor for vip (vipr1) gene is highly conserved through species and, in humans, is highly polymorphic. vipr1 has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from 53 blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr1 in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr1 after 3, 6, 9 and 12 h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g 50 %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from 53 donors that had been typed for the relevant vipr1 snps. the down-regulation of vipr1 correlates with the presence of a t at rs896 mapping in the 3'-end of the gene (p= 0.004). the vipr1 protein level was decreased about 40 % in monocytes of 3 subjects typed as t/t at rs896 whereas 3 subjects typed as c/c at rs896 maintained a high level of expression after 9h of lps treatment. the data show that different haplotypes of the vipr1 gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska 1 , t. vavrochova 1 , d. filipp 1 , immunobiology 1 institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd14 in the early mouse embryo (me). facs analysis of cell suspension prepared from 10.5 day me showed that about 0.7-1 % of cells were positive for cd11b. these cells exclusively were also positive for cd14, tlr2, and cd45 antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e7,5), embryo-derived mhcii + macrophages start to appear in the embryo around day 13. multicolor facs analysis of cd11b, cd45, cd14, f4/80, tlr2, tlr4, c-kit and mhcii surface markers revealed differential expression of tlr2 and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd11b + tlr2 + cells isolated from the e10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il12/il23-dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several ifng pathway genes, other candidate genes are being investigated for the 2 other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp1. we did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: tlr2 is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il-10 cytokine. using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to 70-80 % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, 2009. hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp70 have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp70 on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp70 were used. exogenous hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp70 on their surface. results demonstrating effect of exogenous hsp70 on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp70 on amplitude of respiratory burst. the cells expressing surface hsp70 impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp70 might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp70 to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd14/cd86 and cd14/cd163 double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd14/86 subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd14/163 subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m2 polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević 1 , i. pilipović 1 , s. stanojević 1 , k. mitić 1 , k. radojević 1 , v. pešić 2 , g. leposavić 1,2 1 institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, 2 faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h 2 o 2 ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both no and h 2 o 2 production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h 2 o 2 production. an increase in h 2 o 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h 2 o 2 production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd14 ++ cd16 -('classical') and cd14 + cd16 + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd14 + cd16 + monocytes display higher tlr2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd14 ++ cd16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr2 stimulation. methods: cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for cd14 and cd16 and intracellular il-12p70 and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il-12p70 (% positive cells and geomfi) was significantly higher for cd14 + cd16 + cells than for cd14 ++ cd16cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd14 ++ cd16cells were positive for tnf to a significantly higher extent than the cd14 + cd16 + cells. in particular the tnf response to tlr2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd14 ++ cd16monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. after 2 months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf1 gene concerns the well-known gt deletion in the second exon of ncf1 gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after cci. levels of tnf-a, il-1b, il-6, il-10 and il-1 receptor antagonist were analyzed in brain tissue using real time rt-pcr at 4 and 72 hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. data were analyzed with the mann-whitney u test and a two-tailed p x 0 .05 was considered significant. all cytokines were highly up-regulated 4 hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il-10 were significantly lower in the ubiquitin treated animals, whereas the levels of il-6 and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight (38) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while 18 were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p=0.02), tpp (p=0.00), osi (p = 0.00), mda (p = 0.00) were significantly raised but taa (p = 0.01) was significantly reduced in ptb patients compared with controls. the levels of mda (p = 0.04), neopterin (p=0.00) and tpp (p=0.00) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p=0.00), mda (p=0.00), neopterin (p=0.02), osi (p=0.00) were significantly reduced while taa (p=0.01) was significantly raised in c+m after 4 weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after 4 weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas 1 , e.m. cunha 1 , m.j. oliveira 1 1 icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap-70 is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap-70 regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., 2006; sivalenka and jessberger, 2004) . swap-70-/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., 2002; sivalenka and jessberger, 2004; sivalenka et al., 2008) . crucial regulators of these processes are members of the rho family of small gtpases such as rac1 and rhoa. swap-70 interacts with rac1 in vitro and preferentially binds the active gtp-bound rac1. swap-70 supports the increase of active rac1 in vitro by a yet to be defined mechanism (shinohara et al., 2002) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap-70 and rac1. it was found that fulllength swap-70 preferentially binds to constitutively active rac1 (rac1q61l) but not to its dominant negative form (rac1t17n). binding assays with swap-70 truncated mutants showed interaction of swap-70's n-terminus with gtpgs rac1 or rac1 depleted of guanine nucleotide, whereas swap-70 central or c-terminal regions do not bind to any form of rac1. preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire swap-70 sequence, mapped the rac1 binding site near the n-terminus of swap-70. full-length swap-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap-70 with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap-70. v. c. barbosa 1 , c. d. polli 1 , m.c. roque-barreira 1 , m.c. jamur 1 , c. oliver 1 , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl-2h3 cells were sensitized with ige anti-tnp and stimulated with dnp 48 -hsa or artinm. artinm binding to rbl-2h3 cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp-1 and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp-1 release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand 1 (psgl-1), b1 and b7-integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd44 (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il-1b, il-18 and tnf-a. herpes simplex virus 1 (hsv-1) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il-18 and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv1. methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) (10ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv-1 for 18 h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot 2.0 software. results: in the first itraq experiment over 300 human proteins were identified in the hsv1 infected cell supernatants. from these proteins 119 had at least 3 fold increase after poly(i:c) + hsv1 infection compared to the uninfected cells. hsv1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of 2.7 fold increase in the protein amount in the poly(i:c) + hsv1 infected cell supernatant and a 2.6 fold increase in the hsv1 infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand 10, il-6, tnf-a induced protein 6, complement factor b, galectin-1 and mxa. at present, further experiments are on-going for more detailed analysis of the hsv1 infected macrophage secretome. h. p. prakash 1,2 1 german cancer research centre, translational immunology, heidelberg, germany, 2 max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein-1 (ciap-1) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal3, laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal3 or rpmi and monocytes (1x10 5 ) were added into each insert. when necessary, hrgal3 was pre-incubated with 10mm lactose or sacarose. mcp-1 (100ng/ml) was used as positive control. we observed that hrgal-3 binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal-3, we observed that the association between these glycoproteins and hrgal-3 resulted in a 60 % increase in the number of migrating cells. both n-and c-terminal domains of hrgal-3 are involved in the association between laminin or fibronectin and hrgal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal-3 induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for 3 weeks in the presence of bmp4,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd14+cd45+ cells. the sorted cd14+cd45+ cells were further cultured for 7 -10 days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd14, m-csf receptor cd115 and the scavenger-receptor cd36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd14 and cd16 (fcgammariii) : the cd14lowcd16-and cd14+ cd16+. trancscriptional, phenotypic and functional assays suggest the alternative (m2) polarization of cd14+cd16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m2 -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi 1 , p. kropf 1 1 imperial college london, immunology department, london, united kingdom the balance between t helper (th) 1 and th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th1 or th2 responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd4 + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg9vd2 t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f1-atpase) is expressed on many cell types and is a possible specific ligand for the vg9vd2 tcr. the present study aims at understanding the role of f1-atpase in antigen regognition. using video microscopy calcium imaging in single vg9vd2 t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f1-atpase. purified f1-atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg9vd2 cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f1-atpase. thus the f1-atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg9vd2 t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f1-atpase. by using purified f1-atpase and peptides derived from vg9vd2 tcr sequences, interaction sites between f1-atpase and tcr were identified on both ligands. based on these findings a generalized model for vg9vd2 t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg2d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg2d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg2d activation in vivo. by transiently overexpressing the nkg2d ligand rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma5vdelta1 gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg2d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg2d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg2d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae1 induction. the primary systemic th2 response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg2d receptor suggest that different ones may play unique roles. a novel nkg2d-ligand, h60c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae-1 induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg2d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd1d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th1 (ifn-g, tnf) and th2 (il-4, il-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type 1 diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd4 -nk1.1 -inkt cells producing high levels of the pro-inflammatory cytokine il-17 has been identified (inkt17 cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt17 cells in type 1 diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il-17 as compared to c57bl/6 mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt17 cells present in nod mice were mainly cd4 -nk1.1 -, express the ror-g transcription factor and il-23 receptor, both molecules being usually associated with th17 commitment. we are currently analyzing, using co-transfer experiments, whether these inkt17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in nod mice. j. s. dodd 1 , r. muir 1 , s.s. affendi 1 , p.j. openshaw 1 1 imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd1d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd1d-deficient mice with poor nkt cell responses have inefficient induction of cd8 t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th2 immunity (increasing il-5 and il-10), promoting pulmonary eosinophilia and ablating cd8 t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd8 t cell activity (as measured by cd25 expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d4) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d7) weight loss by a cd8 t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd4 + ab t cells, are regarded as immature or t h 2 biased. vg9 + vd2 + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg9 + vd2 + t cells towards these activators. because il-23 is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg9 + vd2 + t cells. herein, we observed that zoledronate induced neonatal vg9 + vd2 + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h 1-like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h 2-like cytokines such as il-4 and il-5 were not induced. addition of il-23 to zoledronate selectively costimulated ifn-g production from neonatal vg9 + vd2 + t cells. furthermore, zoledronate/il-23 treatment resulted in neonatal vg9 + vd2 + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il-23 (il-23r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il-23 resulted in a further increase of t-bet expression in neonatal vg9 + vd2 + t cells. these changes in the expression of il-23r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il-23 treatment. of note, in contrast to adult peripheral blood vg9 + vd2 + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg9 + vd2 + t cells. altogether, these observations show that neonatal vg9 + vd2 + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il-23 is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e3 ligases such as hhv8 encoded k3, k5 or mhv68 encoded mk3. these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e3 ligases manipulate them. we discovered that both the cellular march and viral e3 ligases ubiquitinate cd1 molecules. however, whereas viral molecules inhibit cd1-antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd1 molecules. in contrast mhc class ii was only targeted by cellular and not by viral e3 ligases. furthermore cd1 molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e3 ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd2 negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht29 colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd2 neg clones in vivo, we generated hypodermal ht29 tumors in immunodeficient mice. concomitant injections of vd2 neg clones, in contrast to vd2 + cells, prevented the development of ht29 tumors. vd2 neg clones expressed chemokine c-c motif receptor 3 (ccr3) and migrated in vitro in response to chemokines secreted by ht29 cells, among which were the ccr3 ligands macrophage inflammatory protein (mip)-1d and monocyte chemoattractant protein (mcp)-4. more importantly, a systemic intraperitoneal (i. p.) treatment with vd2 neg clones delayed the growth of ht29 subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr3 antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd2 neg clones were not able to inhibit the growth of a431 hypodermal tumors. our findings suggest that cmv-specific vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht29 cells expressing the luciferase and realized orthotopic injection of ht29-luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint1, currently the only known determinant of a canonical iel compartment, that is selectively required for vg5vd1 + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint1 expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint1 function; for example, we demonstrate that the constitutive expression of wild-type skint1 fully restores detc development in a skint1 mutant mouse, but does not rescue normal detc function. thus, skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd1d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd19 + cd21 hi cd23 lo cd11c -) and splenic conventional dendritic cells (cdcs) (cd11c hi cd8a +/-cd11b +/-b220 -) from wt and cd1d -/mice as apcs for nkt cells from va14-ja18 transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd1d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd1d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il-4 by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il-4 producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h 2 response, whereas cdc-primed nkt cells rather favor a t h 1 response. objectives: il-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il-18 give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd1d -/-) mice. we set out to investigate the activated b cells in il-18 injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il-18 (2 mg) for 10 days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. results: mice injected with il-18 developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il-18 was increased in inkt cell deficient (cd1d -/-) mice, in contrast to published data. an increased response to il-18 was also observed in ja281 -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il-18 induced antibody responses. further characterization of the recruitment of b cells in il-18 injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il-18 induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd1d, are prone to autoantibody production and often involved in early immune responses. the il-18 induced antibody response in mzb deficient (cd19 -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il-18 induced antibody responses. we conclude that the role for inkt cells in il-18 induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c57bl/6 mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha 18-/-and cd1d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il-4. moreover, ifn-g production required the presentation of ehlppg by cd1d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il-12. similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd16 in cmv-infected individuals. cd16 is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that 71.9 ± 15.9 % of gamma-delta t cells from 21 cmv-infected ktr expressed cd16, when compared with only 19.8 ± 16.7% in 11 non cmv-infected ktr (p x 0.0005). similarly, 51.9 ± 25.7 % of gamma-delta t cells from 13 cmv-seropositive blood donors expressed cd16 compared to 27.1 ± 25.1 % in 15 cmv-seronegative donors (p x 0.01). cd16+ gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd16-dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il-12 and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd16 mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd107a on cd16+ gamma-delta t cell lines. cd16 is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd16+ gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a431 skin carcinoma cell line pre-incubated either with rituximab (anti-cd20) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd16-dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k5mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il-6, ifn-g, tnf-a, osm, ccl2, cxcl8, cxcl10, and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd40, cd86, hla-dr, and dc-sign, and lost cd14, ccr2, ccr5, and cxcr4. addition of further microbial stimuli (lps, peptidoglycan) induced ccr7 and enabled these inflammatory dcs to trigger antigenspecific cd4 + effector ab t cells expressing ifn-g and/or il-17. importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg9/vd2 t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg9/vd2 t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg9/vd2 t cells stimulated with hmb-pp in the presence of il-21 express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il-21 regulate expression of the b cell attracting chemokine cxcl13/bca-1, its receptor cxcr5, and co-stimulatory molecules involved in b cell help. purified peripheral vg9/vd2 t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to 4 days with and without hmb-pp, in the absence or presence of il-2 or il-21, or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl13 protein were detected in co-culture supernatants only when both il-21 and hmb-pp were provided, implying an il-21-dependent and tcr-dependent expression. vg9/vd2 t cells were confirmed as producers of cxcl13 by flow cytometry and immunofluorescence. under the same conditions, activated vg9/vd2 t cells expressed cd27, cd28, cd40l, cd70, icos and ox40. in contrast, neither cxcr5 nor ccr7 changed markedly by il-21 stimulation of peripheral vg9/vd2 t cells. conclusion: our findings confirm on the protein level that stimulation of vg9/vd2 t cells with hmb-pp and il-21 induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto 1 , m. emoto 1 1 gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)-12. although downmodulation of surface t cell receptor (tcr)/nkr-p1c (nk1.1) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il-12 stimulation. to determine whether failure to detect inkt cells after il-12 stimulation is caused by dissociation/internalization of tcr and/or nkr-p1c or by block of de-novo synthesis of these molecules, and to examine the role of il-12 in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p1c by inkt cells after stimulation with a-galcer or il-12, and influence of il-12 neutralization on down-modulation of stcr/snkr-p1c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il-12 neutralization. whereas s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of il-12, s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p1c remained unaffected by a-galcer or il-12 treatment, despite the down-modulation of ctcr and/or cnkr-p1c protein expression. in contrast, ctcr + cnkr-p1c + stcr -snkr-p1c -inkt cells and cnkr-p1c + snkr-p1c -inkt cells were detectable after in-vitro stimulation with a-galcer and il-12, respectively. our results indicate that tcr and nkr-p1c expression by inkt cells is differentially regulated by signaling through tcr and il-12r. they also suggest that il-12 participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p1c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg9-gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region 3 d1 (cdr3d1) and cdr3d2 repertoire, with a striking enrichment in a specific germline-encoded cdr3d1 sequence. differentiated gd t cells and the enriched cdr3d1 sequence were detected as early as after 21 weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd1-jd1 and vgi-jg1.3/2.3 rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd2-jd1 and vg9-jg1.2 selection was seen in pb tcrgd cells. detailed analysis of the cdr3 motifs revealed selection determinants in both vg9-jg1.2 (canonical length and cdr3 motif) and vd2-jd1 (minimal cdr3 length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚4) and tcrgd cb cells (6-7) to tcrgd pb cells (˚10 or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type 1 diabetes (t1d). we have previously shown that transgenic expression of a cd1d-restricted, va3.2-vb9 tcr in nod mice lead to an increase in cd1d-restricted type ii nkt cells (24abnkt cells), and prevention of the development of t1d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by 24abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc2.5 cd4+ t cells, in the presence or absence of selected cells from 24abnkt cell transgenic mice. results: in 24ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, 24abnkt cells expressed a high level of cxcr3 and a low level of ccr7 and cd62l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd4+ bdc2.5 t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with bdc2.5 cd4+ cells resulted in the prevention of diabetes development. the protection from disease required a minor cd4+ subset of 24ab+ nkt cells, but was independent of cd25+ t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd1d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs44, induces in vitro inkt cell expansion and ifng and il-4 secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th2 profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il-10 with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr1 cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th17 immune response is characterized by the secretion of il-17a and il-17f. the il-17 locus encodes the highly conserved il-17a and il-17f cytokines that are syntenic in 44kb distance to each other. besides cd4 + th17 and nkt cells, approximately 50 % of the il17a producers are gd t-cells. like cd4 + th17 cells, il-17 producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il-17 production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th17 cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il-17 or ifn-g. combined with the well known classification of il-17 producing gd t-cells along the markers cd27 and cd122, our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th17-type immune responses in vivo. in this context, the potential redundancy of il-17a and il-17f may complicate the analysis. so far, most studies were carried out with il-17a single-deficient or il-17f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il-17a and il-17f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg9vgd2 t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b1 and il-15 differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd2 tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp3) and, similarly to ab tregs, suppress the proliferation of anti-cd3/anti-cd28 stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd2 tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il-17 was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd11b in the liver following a-galcer treatment, numbers of cells lacking cd11b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma9/vdelta2 t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma9/vdelta2 t cells expansion upon stimulation with zoledronic acid (za) and interleukin-2 (il-2). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in 43 patients (49 %) (responders, r), whereas 44 patients (51 %) were non-responders (nr). vgamma9/vdelta2 t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt2, whereas temra of r patients had an higher expression of the costimulatory molecule nkg2d. the proliferative response of vgamma9/vdelta2 t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, 82 % of r patients were m, whereas 77 % of um patients were nr (p x 0.001). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c57bl/6 mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il-12 p40 (clone r2-10f6; atcc) or anti-cd-1d (clone 1b1) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at 18 hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il-12p40, a component of both il-12 and il-23; pretreatment with anti-cd1d mab had no effect. il-12rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il-12 was not a critical factor. this suggestion is supported by the increased expression of il-23p19 and il-12p40 (but not il-12p35) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il-23 production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb1) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing il-17. this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd27-defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine 123. cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to 40 % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, 40-60 % of gd 27+ cells were positive for pgp activity, although their gd 27counterparts remained largely negative (p x 0.01). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd27. as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma9vdelta2 t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma9vdelta2 t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma9vdelta2 t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma9vdelta2 t cells from healthy donors were stimulated with different compounds (zol, ipp) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma9vdelta2 t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma9vdelta2 t cells. moreover, mcp-2 depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma9vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma9vdelta2 tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma9vdelta2 t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg9vd2 t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg9vd2 t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg9vd2t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg9vd2 t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg9vd2 t cells and their effects on the viability of glioma cells, we expanded in vitro vg9vd2t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg9vd2t cell lines to kill three different glioma cell lines (t70, u251, u373) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg9vd2t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg9vd2 t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg9vd2 t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg9vd2 t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd1d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e7-expressing transplanted skin is dependent on interactions between populations of cd1d-expressing cd11c+/f480+ myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e7 graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il-2 and a-galcer. expression of cd1a, cd1d and the costimulatory molecules cd86 and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il-4) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd1d expression on the cell surface in comparison with control cells. in contrast cd1a expression was decreased. cd86 and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd1d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd1a, cd1b and cd1c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il-4 producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd1d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, 0.5-10 % of circulating lymphocytes express a vg9vd2 t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg9-enriched genes compared to conventional mhc-restricted cd8 + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf4 & crtam in gd and ab t cells respectively. because igsf4 binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd8 + ab t cells express crtam, and that engagement of igsf4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf4/crtam. we therefore sought to answer: 1. what is the function of igsf4/crtam on gd t cells? 2. how is the igsf4-crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf4 enrichment on resting gd t cells, with expression also detected on˚10 % of ab t cells. the properties of those cells are being examined. however, igsf4 generally correlates with markers of activation/antigen experience such as cd45ro. thus, igsf4 cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg9 + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf4 and crtam within 48 hours. however, engagement of igsf4 by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg2d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf4 interactions are sufficient for cd8 t cells to kill gd t cells and/or vice versa. instead, crtam-igsf4 interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd3z is a 16 kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately 3-6 % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd2 tcr variable segment associated with the vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg9vd2 t cells without antigen presentation. in vitro stimulated vg9vd2 t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg9vd2 t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg9vd2 t cell anergy observed in hiv+ patients. to this aim, cd3z expression and ifn-g production by vg9vd2 t cells from hiv+ and hiv-subjects were analyzed. we show that vg9vd2 t cells from hiv-infected patients expressed lower level of cd3z compared with healthy donors. a direct correlation between cd3z expression and ifn-g production capability by vg9vd2 t cell was found. however, pkc activation by pma is able to restore cd3z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg9vd2 t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner 1 , m. swamy 1 , s.l. clarke 1 , a. hayday 1 1 king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd8aa or cd8 -cd4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. the cells are initially activated by plate-coated acd3 antibody and a cytokine cocktail and maintained further in medium containing low levels of il-2. after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg2d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il-6) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after 6 hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd94 in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg2 molecules in heterodimer with cd94, we screened the presence of these receptors on t cell subsets in bd. the expression of nkg2 a/c/d molecules on gd and cd8+ t cells were analyzed in 17 active and 9 inactive patients with bd and 21 healthy controls. expression of nkg2 molecules was evaluated on cd8+, gd t and cd56+ nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls (4.4 vs. 2.8 %, p=0.001). in addition to the increase of gd t cells, increased expression of activating nkg2c molecules was also observed on gd t cells (20 % vs. 11 %, p= 0.01). nkg2a expression on gd t cells was found to be higher than nkg2c expression in patients and controls; but nkg2a expression on the t cells was not statistically different in both groups (33.5 vs. 40 %). nkg2d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd8+ cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th1 and th2 cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg9/vd2+ lymphocytes. we have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg9/vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two vg9/vd2+ resistant ("non-target") cell lines (under 20 % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs 697 (non-target), and validated the results by rt-qpcr quantification. results: we identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp1, ifitm1 and prame, for example, are up-regulated, whereas cd274 and clec2d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg9/vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd1d, in the rat. mice and rats have very similar cd1d and inkt tcr genes, with the exception of the va14 gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd1d revealed a very similar pattern of cd1d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il-4 production was 10-100 fold lower in the best responder rat strain (f344) compared to mouse (c57/bl6). since nkrp1a (rat homologue of mouse nk1.1) and tcr are not appropriate markers for rat inkt, cd1d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd1d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd1d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd1d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd1d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g9d2 t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g9d2 t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g9d2 t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g9d2 t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp-1. a significant (p x 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of pbmcs cultured with picostim+il-2 in comparison with pbmcs cultured with il-2 or media alone. picostim+il-2 caused significant expansion and activation of gd t cells following culture of pbmcs for 10-14 days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers 100-fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il-2, reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma9/vdelta2 (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than 5 % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: 1) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; 2) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); 3) to establish whether the same issues could be solved using a simplified protocol of dc generation. 1) dc were generated from cd14 + cells of healthy donors/mm patients; immaturedc on day 6 were induced to fully mature by incubation for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za. after 7 days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; 2) idc generated from cd14 + cells of hla-a*0201 + healthy donors/patients were pulsed with sv-peptide and stimulated for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za; after 2 rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd8 + t cells was determined by svpentamers staining; 3) the same experiments were performed both with dc generated following a standard protocol and a 48h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [6-3 h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd4 + cd25 + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp3 negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd1 subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg2d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd8+ have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n=8) and inactive (bdna) (n=20), versus healthy controls (hc) (n=30) and patients with recurrent oral ulcerations (ru) (n=14). to determine gd cytokine profile and surface markers treg-related in bd (n=9) and hc (n=11). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta1, vdelta2, cd8alpha, cd8beta, nkg2d, nkg2a and cd103. -intracellular expression of ctla-4, and foxp3. -intracellular expression of il-2, il-4, ifngamma, il-10 and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta2+ cells were significantly increased in ru. vdelta1+ and gdcd8+ lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg2d was slightly increased in gd from bda. -nkg2a expression by gdcd8+ was not different in bd versus hc. -most of gdcd8+ presented cd8alpha-alpha homodimers in bd and hc and were negative for cd103, foxp3 and ctla-4. gdcd8+ and gdcd8-subsets were (in bd and hc): -high ifngamma-producers without differences. -low il-2-producers: il2+ cells were lower in gdcd8+ than in gdcd8-. -low il-10-producers: il10+ cells were lower in gdcd8+ than in gdcd8-. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd8+ than in gdcd8--very low producers of il4 in most of cases. the hallmark in bd was the increase of gdcd8/vdelta1+. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg2a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd8+, except a lower percentage of il-2+ cells than in the gdcd8-subset. gdcd8+ from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd56 in humans. a subgroup, the invariant nkt cells (inkt), expresses the va24vb11 tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd3 + cd56 + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with ifn-g (50 ng/ml), anti-cd3 (25 ng/ml) and il-2 (500 ui/ml). these cells were cultured for 21 days and the expanded cd3 + cd56 + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd3 + c56 + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x 0,05 was considered significant. results: we could significantly expand cord blood cd3 + cd56 + nkt cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). table 1 shows the percentage (mean±sd,n=10) of phenotypic markers in cd3 + cd56 + cells at baseline (day 0) and after 21 days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb4 and vb8 families (p x 0,001). conclusion: our results show that cord blood-derived nkt cells are mainly cd8 + and cd94 + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb4 and vb8 families in these cells. l. marischen 1 , d. wesch 1 , p. rosenstiel 2 , a. till 2 , d. kabelitz 1 1 institute of immunology, kiel, germany, 2 institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod2. peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod2 gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp1 19 and pvama1 proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day 5 culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd69 + and cd25 + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day 3 after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd107a + (lysosomal associated membrane proteins: lamp-1) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th1 and th2 cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th1-like and th2-like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-cd3 and monoclonal antibody specific for the cdr3 loop of the invariant tcr a chain of inkt cells (clone:6b11). furthermore, after pma/ionomycin stimulation for 4 hours, intracellular ifng and il-4 cytokines were detected in cd4+cd8-, cd4-cd8-(dn), cd4-cd8+ and cd4+cd8+ subsets of inkt cells by five colour flow cytometry in patients with ad (n=10) and healthy controls (n=10). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x 0.01) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x 0.01). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r=0.726 and p x 0.001) and healthy controls (r=0.693 and p x 0.001). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il-4 level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x 0.05). the frequency, the number of inkt cells and the cytokine producing capacity of the cd4/cd8 inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il-4 that promotes the th2 differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells (1.8 %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd4 + nkt and cd4 -cd8 -nkt cells. nk1.1 + nkt cells may release large amounts of il-2, il-4, ifn-g and il-10 after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il-2 had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il-2. there was a significantly increase in the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd69 molecule existed in 68.95 % of the cells from large-scale selection. the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. under a light microscope at x400 magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. the differentiation pathway of the seb-activating cd8 + and tcrvb8 + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd8 + nkt cell and tcrvb8 + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl3 which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo-1am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h2begfp reporter mice. stimulation with anti-gd clone gl3 or anti-cd3 clone 2c11 elicited activation of gd t cells suggesting that tcr gd and cd3 molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl3 in vitro produced ccl4, ifng and tnfa. therefore, we were interested whether the ccl4 production of gd iiel influenced the homing of ccr5 cells such as lamina propria (lp) cd4 + foxp3 + cells (tregs). to test this, wt mice were i. p. treated with gl3 mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr5 + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr5 + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr5 expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il-4 production in g4 reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il-4 gene. in the absence of any manipulation gfp was expressed from the il-4 locus in populations of immature thymic nkt cells (predominantly cd4+cd44lotcrhi cells on a balb/c background, and cd4+cd44lonk1.1-on a c57bl/6 background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il-4 production was induced predominantly in cd4+ nkt cell subsets of the liver and spleen, and after i. n. administration, in cd4+ nkt cells of the airways. spontaneous and a-galcer-induced expression from the il-4 locus occurred in the absence of stat6 signalling, and did not require initial exposure to il-4 protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin-17 (il-17) plays an important role in neutrophil recruitment. herein, we investigated the role of il-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c57bl/6 (wt) and il-17 receptor gene-deficient (il-17r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. the ability of il-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il-17r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il-17 induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il-17r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il-17receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il-17 also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin-1 receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il-1r associated kinases to activate nfxb and p38 mitogen-activated protein kinase. the il-33-induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il-33 in different mast cell subsets. methods: different mast cells subsets (hmc-1, human cbmcs and murine bmmcs) were stimulated with il-33. the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk1/2, pakt, pnfxb, p38 and pjnk). additionally, we studied the signal transduction of il-33 in il-33r transfected hek293t cells. results: we found, that a tir family member, il-33r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il-33-induced cytokine production depends on c-kit transactivation. il-33r and il-1r accessory protein (il-1racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il-1r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il-33-induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il-33 in mast cells in absence of iger activation. (1) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease-1 (ho-1) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho-1 expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap1 small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap1 activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. rna interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in rap1 activation and that the oscillations in the formation of rap1-gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c3g (rap1gef) and spa1 (rap1gap): preliminary results suggest that spa-1 has probably a role in the early activation peak. since this oscillatory chemokine-induced rap1 activation is present on other myeloid cell lines (hl60, 32d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different sh3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)-3 and il-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il-3 and il-5 receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il-3 , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn 328 are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: (1) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna-146 expression is induced by activation of the toll-like/interleukin-1 receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna-146a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (casp8/3). in line with this, mirna-146a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna-146a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna-146a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna-146a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il-15, il-7r a-chain and il-15r a -chain genes in hiv disease progression. methods: we studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (ltnp). we analyzed 2 snps in the il-15 gene, 5 snps in the il-7r gene and 3 snps in the il-15r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il-15r aa 182 and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il-7r and il-15r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd4 and cd8 t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art1, and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art1 in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art1 transcripts by rt-pcr analysis in human blood leukocytes. soluble art1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek293 cells with art1 and tnf resulted in modification of tnf at at least 2 distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r32, a site that has previously been implicated in binding to tnfr2. binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit225 than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art1 resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik 1 , t.v.poroshina 1 1 institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and 11 control men were assessed for slpi, tnf-alpha, il-8 and free tgf-b1 in ejaculate by elisas using stat-fax 303 plus. a 3 to 5 day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p x 0.001) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p x 0.05). the il-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p x 0.05) in seminal fluid. free tgf-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b1 concentrations were 36.2 ± 6.2 pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il-8 and free tgf-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi 1 , e. a. elgaaeid 1 1 faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th1 or th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod2/card15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod2/card15 gene in cd. we performed a cases /controls study upon 75 cd patients and 90 healthy controls. this study suggests that in northen tunisian population, 3020insc mutation in nod2/card15 gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il-10 polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il-10 cytokine genetic profile in patients with ibd. we examined the contribution of il-10 gene promoter polymorphisms (-627 and -1117) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card15/nod2 gene mutations in cd presentation and location. in tunisian population, the 3020insc insertion in nod2/card15 gene is a marker of susceptibility to cd, while the a allele at position -627 in the il-10 promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il-10 gene polymorphisms and card15/nod2 gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il-2-dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il-2-driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il-2 molecule by ganglioside. but gangliosides apparently can also form complexes with il-2r; such complexes influence on the signal transduction through il-2r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il-2r subunits. in our work we use il-2-dependent cytotoxic t-cell murine line ctll-2. two different approaches for study of possible interaction types between exogenous gangliosides and il-2r subunits were applied: antibody staining of il-2r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 i-dcp-gm1 followed by immunoprecipitation of il-2r subunits. the fluorescence intensity of the antibody-labeled il-2r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside gm1, and by 10.7 % after incubation with gm2. labeling of the cells with antibodies to the il-2r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm1 (2.8 %) or with gm2 (5.9 %). to determine the mode of this impressive masking influence of ganglioside gm1, photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm1 with subunits of il-2r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il-2r, but not for il-2r a-subunit. these results demonstrate that exogenous ganglioside gm1 can interact with a-and bsubunits of il-2r in different modes. interaction of il-2r b-subunit with ganglioside gm1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa 1 , j. bukur 1 , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak2 and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak2. results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other ifn-g signal transduction components like stat1 and jak1 were not affected. jak2 blockade by two jak2-specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak2 leading to a lack of jak2 expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak2 inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of 506 controls of the spanish population. methods: two il18 polymorphisms located on the il-18 promoter region, snps -607 a/c (rs1946518) and -137 g/c (rs187238) were genotyped using taqman snp genotyping assays. the functional il-18 gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il-18 -607 p=0,318. il18 -137 p=0,740) but may contribute to disease onset and aggressiveness: the genotype il-18 -607 cc genotype, which is associated with higher il-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), t (p=0,001), m (p=0,012) and stage (p=0,002). the influence of the il-18 -103 gg genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), t (p=0,026) and stage (p=0,011). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il-18 polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il-18 gene (il-18 -607 and -137) may be associated with an worse prognosis of rcc. high levels of il-18 production can play an important role in grow, invasion and metastasis of renal cancer. interleukin-18 (il-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il-18 is its ability to induce the production of ifn-gamma in presence of il-12. moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l0. then it seems that il-18 has a crucial role in immunity against brucella infection. since the expression of il-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il-18 gene polymorph isms and brucellosis. methods: a total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il-18 polymorphisms at position -137 g/+113t/+127c (correlated with high production of il-18), codon 35/3c and -656g/-607c (correlated with higher production of il-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). discussion: as data revealed genotypes that have correlation with higher production of il-18 are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il-18 at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor 2 (ifngr2) mouse mutant. we have generated a mouse line carrying a conditional ifngr2 allele using the 2 loxp 2-flp recognition target (frt) approach. a targeting vector with 2 loxp sites flanking exons 4-6 and 2 frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat1 following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat1 activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd4 cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr2 on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez 1 , r. carretero 2 , p. saenz-lopez 2 , j. cantón 2 , j. carretero 2 , f. ruiz-cabello 2 , l.m. torres 1 , cts-143 1 hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, 2 hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and 130 normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca)12 allele of ifgn is significant more frequent in healthy control than in cin patient (p=0,030) in contrast homozygosis for (ca)12 allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). conclusions: our study suggest that ifng (ca)12 allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin-15 (il-15), a th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il-15 single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il-15 (c267t, g367a, c13687a and a14035t) polymorph isms using pcr-rflp. results: at position 267, the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p=0.025 and p=0.042, respectively). at position 13687, the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p=0.050, p=0.015). discussion: as shown the frequency of cc genotype and c allele at position 267 were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position 13687 is lower in patients than the healthy controls and the frequency of c allele at position 13687 is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf-7 and skbr-3, as well as their ability to respond to several cytokines and to the g-1 gpr30 agonist. the mcf-7 and skbr-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il-2ra, il-2rg, il-3/il-5/gm-csfr, il-4/il-13r, il-5ra, il-6ra, il-6r (gp130), il-7ra, il-10r, tnfr i, tnfr ii, ifngra, cxcr1, cxcr2, cxcr3, cxcr4, cxcr5. cells were incubated with il-10, ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr30 agonist. cytosolic ca 2+ concentrations [ca 2+ ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr-3 cells were found positive for a larger number of receptors than the mcf-7 cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf-7 cells with il-10 reduced the calcium response to g-1, while pretreatment with ifn-g and tnf-a potentiated the calcium response to g-1. tnf-b had no effect on mcf-7 g-1 stimulation. no direct effect on basal [ca 2+ ] i stimulation could be noticed when administering the cytokines alone. in skbr-3 cells, pretreatment with il-10 or tnf-b had no effect on basal [ca 2+ ] i and did not significantly alter the calcium response to g-1, while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g-1. pretreatment with tnf-a produced calcium oscillations and reduced the response to g-1. conclusions: mcf-7 and skbr-3 cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr30 stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd8a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd8a -pdcs and cdcs were infected with mcmv, whereas after 24h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd8 t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag1-deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag1 and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd8 t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg 1668, respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd8a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt3-l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f4/80 + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd8 t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr7 and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd11c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd8 t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd4 t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd4 t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd8 t cells in response to an adova infection due to an impaired cd4 t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than 500 passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il-12. to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp33 and vacv-gp33 expressing the gp33 epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p14 t cell receptor recognizing the gp33 epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p14ifnar -/mice. upon adoptive co-transfer of p14ifnar -/and p14ifnar wt/wt t cells and subsequent challenge with mva-gp33, a massive expansion of p14ifnar wt/wt t cells was observed, whereas p14ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp33 induced a rather similar expansion of ifnar competent and ifnar deficient p14 t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h5n1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h5n1 induced an exceptionally nf-kb dependent antiviral response. irf3 is essential part of this interferon-response of human endothelia. furthermore, we identified hmga1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h5n1. finally, nfatc4 was found to be a transcriptional regulator for specifically h5n1-induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h5n1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns1, delta-ns1 influenza virus (a recombinant influenza virus lacking the ns1 gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered t cell priming in vivo. a direct comparison of delta-ns1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns1 protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns1 protein. thus, we propose that the virally encoded ns1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin 18 is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin 18 is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of 14-3-3 target proteins. thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections. i. wessels 1 , d. fleischer 1 , l. rink 1 , p. uciechowski 1 1 institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il-1b expression is not elucidated, yet. it is known that the il-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il-1b when stimulated. b-cells which are il-1b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il-1b promoter and the impact of methylation on il-1b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl-60 cells were differentiated into monocytic cells after dihydroxyvitamine d 3 treatment. the monocytic phenotype was confirmed by flow cytometry. the il-1b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with 5-aza-2-deoxycytodine (aza) and changes in il-1b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl-60 cells, differentiated cells displayed upregulation of cd14 antigen and acquired the ability to express il-1b. by comparing the accessibilities of il-1b promoter we detected that the il-1b promoter was not accessible in undifferentiated hl-60 cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl-60 cells, demonstrating that the chromatin remodeling of the il-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il-1b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il-1b expression were found. our data indicate that the il-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg 1 , g. wetzel 1 , h. arnold 1 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p65 family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il-1b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp2, 3, 4, 5 and 6 mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd88 was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. (2)). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd8 + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. 2008 aug 1;181 (3)). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. 2008 dec; 82 (24) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il-1b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory 4t1 breast cancer cells over expressing il-1b (4t1/il-1b) tumor cells, but rarely in untransfected 4t1 cells. secretion of il-1b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg2d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il-4ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used 130 healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array 5.0 having p x 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of il-6, il-8, il-10, tnf-alfa and il1-ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed 150 snps with p x 1,0*10 -4 . to identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. a combined analysis revealed 10 snps with p x 9,1*10 -5 .these results are not genome wide significant. the 10 snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find 10 nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp60 on adipocytes. for the first time we could show a hsp60-stimulated release of the proinflammatory cytokines il-6, cxcl1 and mcp-1 in a time-and concentration-dependent manner from murine 3t3-l1 adipocytes. analyses of hsp60-signalling in these adipocytes revealed that members of the mapk-family (erk1/2, p38) and the transcription factor nfkb are involved in hsp60-mediated induction of the mediators il-6, cxcl1 and mcp-1. binding-studies with fluorescence-labelled hsp60 demonstrated that the interaction of hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mm. binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of hsp60-binding. further analyses to characterize hsp60-receptor structures on adipocytes revealed the presence of toll-like receptor (tlr)4 on adipocytes. tlr4 has been found to be expressed on macrophages and to interact with hsp60, therefore suggesting tlr4 as a potential receptor candidate for hsp60 on adipocytes. in order to identify the responsible binding-epitope of hsp60 we investigated the effect of specific antibodies directed against different epitopes of the hsp60-molecule. incubation with antibodies directed against the n-terminus of hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the hsp60-binding to adipocytes (47-80 %) indicating that the n-terminal region of hsp60 is involved in receptor binding. our experiments demonstrate that hsp60 stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf3. we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf3 knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with 3 % of fetal calf serum at 37°c and 5 % co2. bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd10, cd29, cd73, cd21 and stro-1 and were negative for cd45. intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost 100 % of msc. interleukin-2, ifn-gamma and tnf (th1 cytokines) increased msc contractility, whereas il-10 (a th2 cytokine) decreased msc contractility. by immunofluorescence, we observed that il-2, ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il-10 decreased that incorporation. our results suggest that th1 and th2 cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca 2+ -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as 40 nm and 400 nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents (10 mm) at the 24-h culture interval reached the values of approximately 3 ng/ml and 2 ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of 2-6 h in rat pecs. the 24-h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p38 and erk1/2. it was not suppressed by the calcium chelating agents bapta-am and tmb-8. the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr 305/07/0061. pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin1 substrates and targeting sites. recent data show that pin1 interacts with apo-bec3g (a3g). the pin1/a3g interaction results in a reduced a3g expression and a diminished a3g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin1 gene (-842 g/c and -667 t/c) modulate pin1 expression; in particular, the -842 gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, 28; 69-74, 2007) . the -842 c/g and -667 t/c polymorphisms in the promoter of pin1 gene as well as pin1 protein levels were analyzed in 30 exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; 40 hiv-infected patients (hiv) and 40 healthy controls (hc). the genotype and allele distributions of the -842 snp was skewed in esn (genotype: p= 0.008; allele: p= 0.013). in particular esn showed a significantly lower frequency of the -842 gg genotype compared to hiv and hc (p=0.017 and p=0.019, respectively) and consequently a lower g allele frequency (p=0.026 and p=0.028, respectively). no significant differences were found for the -667 snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, 9-(r)[2-(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. secretion of cytokines was determined after the 5-h culture by elisa. production of no was assayed at the interval of 24 h using griess reagent. approximately 300 compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately 30 compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip-1a and cytokines tnf-a and il-10. although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n 6 -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mm. the remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant 1m0508. host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and 10 million units of ifn-a treatment three times a week for 6 months was initiated. before and after treatment: percentages of the il-2 and ifn-g in cd4+ t cells were assessed to determine intracellular t helper cell 1 (th1) type cytokine expression. similarly, percentages of intracellular il-2 and ifn-g were detected to verify cytotoxic t cell 1 (tc1) type cytokine expression in cd8+ t cells. percentage of th2 and tc2 type cytokine expression, (il-4 and il-13) were determined in cd4+ and cd8+ t cells, respectively. six (50 %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th2 cells with respect to healthy controls before treatment. tc percentages, both tc1 and tc2, were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il-4 expression was higher and the percentages of th1 type cells were significantly low. il-13 expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency (29,8 %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt 1 , f. juengerkes 1 , b. schumak 1 , g. gielen 1 , j. kalff 2 , p. knolle 1 , b. holzmann 3 , a. limmer 1 1 university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, 2 university hospital bonn, department of surgery, bonn, germany, 3 department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il-12, the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh 1 1 rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il-17 and il-23 in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, 45 h. pylori-infected du patients (23 patients were positive for anti-caga antibody and 22 patients were negative for anti-caga antibody), 30 h. pylori-infected as carriers (15 subjects were positive for anti-caga antibody and 15 subjects were negative for anti-caga antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il-17 and il-23 was measured by elisa method. the mean serum levels of il-17 in total du patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total as subjects (5.19 pg/ml ± 3.75, p x 0.001) and healthy control group (3.55 pg/ml ± 3.76, p x 0.0001). in du group, it was found that the mean serum levels of il-17 in subjects with positive test for anti-caga (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-caga (7.65 pg/ml ± 4.74; p x 0.05). the mean serum levels of il-23 in du (8.66 pg/ml ± 8.41) and as groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, p x 0.02 and p x 0.03, respectively). however, no significant difference was observed for mean serum levels of il-23 between du and as groups. moreover, in both du and as groups the mean serum levels of il-23 was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il-17 and il-23 in h. pylori-infected subjects as compared with control group. in du group the expression of il-17 influenced by the bacterial caga factor. a. aral 1,2 , a. atak 1 1 gazi university faculty of medicine, department of immunology, ankara, turkey, 2 gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il-6 levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il-6 elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il-6 levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the 1st week and started to decrease at the 3rd week. il-6 reached to its peak at the 3rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic 1 , m. jurisic 2 1 university of kragujevac, school of medicine, kragujevac, serbia, 2 university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd3, cd20 and cd68. tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x 0.05) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. 1) drawings blood from 20 patients and 20 healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. 2) detection of the gene expression of prolactin and tlr-2 in cd14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr-2 and peripheral prolactin expression in cd14+ monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u937 culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table-2 ). when tb household contacts and healthy controls were compared, cfp10 and esat6 seemed to be more useful than tst in tb contacts for displaying ltb (table-2) . although cfp10 spot numbers were much more than esat6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: 0,069)( table-3 ). both esat6 and cfp10 spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip-10, il-6 and il-10. notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type 1 ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/1/68 (h3n2) (hk) differs from its putative avian precursor by 7 amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk-5aa-i62r, n81d, k92n, s193n, g144a, human (2-6); rhk-r2-l226q, s228g and rhk-7aa-i62r, n81d, k92n, s193n, g144a, l226q, s228g, avian (2-3)). among these variants, the double mutant rhk-r2 and the seven mutant (rhk-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l226q and s228g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip-10 and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r2 and rhk-7aa as compared to rhk and rhk-5aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il-8, shedded adhesion molecules (cd25, vcam-1, icam-1), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of 98 newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il-10, and neutrophilic cd64 levels, procalcitonin and il-10 were measured by elisa technique while, neutrophilic cd64 by single colour flowcytometric technique. of these 49 "infected" infants, 16 had positive blood culture (subgroup ia: culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another 49 newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il-10, cd64, and crp. il-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/ml. for pct, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/ml. neutrophilic cd64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il-10 and neutrophilic cd64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and npv 86 %. the combination of il-10 and crp had high sensitivity and moderate specificity, 93 % and 79 %, respectively. conclusions: il-10 and neutrophilic cd64 levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp3, naip5 or ipaf and the adaptor asc are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of il-1b and il-18. recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi3k, vps34 and can be inhibited with the pi3k inhibitors wortmannin and 3 methyladenine (3ma). in contrast, activation of akt, via class i pi3k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il-1b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with 3ma, wortmannin or an akt inhibitor. supernatants were analysed for il-1b by elisa. results: 3ma enhanced il-1b secretion by bmdc treated with the tlr3 and tlr4 ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il-1b secretion was greatly reduced in bmdc from nalp3 -/mice compared to wild type c57/bl6 controls. treatment with the akt inhibitor had no effect on lps-induced il-1b secretion by bmdc. tlr-dependent secretion of il-1a was also enhanced by treatment with 3ma. conclusions: these data demonstrate that il-1b secretion by bmdc in response to treatment with pi3k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp3 inflammasome. this response is limited to tlr3 and tlr4 agonists. inhibition of akt had no effect on lps-induced il-1b production, suggesting that the effect of wortmannin and 3ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi3k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (s1p) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the s1p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s1p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s1p, and later analyzed by flow cytometry. a pharmacological analysis of the s1p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam-1 expression was increased following lps and s1p concomitant stimulation in both venous and arterial cells, suggesting that tlr4 and s1p receptors cooperate in the expression of icam-1. conversely, no cooperation was observed when tlr2 ligands were used. in order to elucidate which s1p receptor subtype was involved in the increase of icam-1 expression, we used a pharmacological approach with s1p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam-1 after lps and s1p challenge was significantly reduced by blocking s1p receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s1p receptors 2 and 3, which suggest that s1p receptor 1 might be involved in the effect. conclusions: altogether these data demonstrate that tlr4 and s1p receptors can interact to increase adhesion molecules such as icam-1 in human endothelial cells, and the s1p receptor subtype involved in the effect differs between arterial and venous cells. 15 with ssc without pah) and a pool of 12 sera of healthy controls (hc) were tested. results: in 1 dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in 2 dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. twenty one protein spots were recognized by more than 80 % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam-1 and vcam-1 were assesed by an enzyme immunoassay (elisa). results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). reference value for icam-1 was 130-299 ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients (40 %) having increasd levels of icam-1. high icam-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). mean values were found 735,5 ng/ml and 400 ng/ml accordingly. those findings verify the positive correlation between icam-1 and inflammatory diseases and tissue damage but not for vcam-1. colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: 16 cases of ulcerative colitis (urc), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. immunohistoobjectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). samples of outlying veined blood were extracted to the 24 hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd18 in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in 3 degrees: degree 0. without expression. degree 1. weak; degree 2. moderate; degree 3. intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. the placebo group: mixed pattern, degree 1-2. group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree 2-3. the percentage of cells that expressed cd 18 was greater in the group study (40 mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd18 answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam10 and adam17. we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam17. these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam17 activity in the tissue. in the presence of the adam17 inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam17 as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf-460/ vegf+405 is associated with rcc risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). on the other hand, the polymorphism vegf -2578 a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. therefore, pten is one of the main antagonists of the pi3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il-6 as well as il-8 levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il-6 as well as il-12 and il-23 mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th-17 t cells, we measured th-17 cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il-17 and a strong reduction of il-22 mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il-1ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il-10. based on our previous observation that support a direct role of il-10-activated stat3 in the enhancement of il-1ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il-10. quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il-1ra promoter. crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after il-10 addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il-1ra promoter. chip assays showed that the pol ii recruitment to the il-1ra promoter induced by lps is significantly increased by il-10, further strengthening the concept that the rapid enhancement of lpsinduced il-1ra gene expression by il-10 initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat3 immunoprecipitated dna showed statistically significant levels of stat3 binding to the il-1ra promoter only in cells stimulated with lps in the presence of il-10. surprisingly, anti-p65 and anti-p50 chip assays revealed enrichment of both p65 and p50 recruitment to the il-1ra promoter when il-10 was added to lps-stimulated cells, suggesting that il-10 enhances the recruitment of nf-kb to the il-1ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il-10-treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il-1ra promoter site is dependent on il-10-activated stat3, since it is greatly reduced when stat3 activation by il-10 is impaired. the molecular mechanism through which il-10-activated stat3 promotes the recruitment of nf-kb to the il-1ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mmcp-2). we examined the mechanisms underlying the repression of mmcp-2 gene expression. our data show that the "repressor" effects of il-15 on mmcp-2 promoter activity are still operating on the mmcp-2 591 bp long minimal promoter. moreover, il-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy1. furthermore, chromatin immunoprecipitation revealed that il-15 promoted specific reciprocal recruitment of c/ebpb but not yy1 to the mmcp-2 promoter. finally, il-15 deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp-2. thus, we proposed that the expression of mmcp-2 and possibly other immunoregulatory genes may be regulated by il-15 through epigenetic modification and by balancing the content and binding of c/ ebpb and yy1 in mast cells. i. nagy 1 , k. filkor 1 , a. vörös 1 , l. kemény 2 , a. szász 1 1 bzaka, baygen, szeged, hungary, 2 university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir-203, mir-146a and mir-155, which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir-203 expression; in contrast, pgn re-stimulation had no further effect on mir-146a and mir-155 expression. next, we investigated the correlation between the expression of mir-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (socs-3). although the gene-expression profile of neither p63 nor socs-3 changed, we found that the expression of mir-203 reversibly correlates with both p63 and socs-3 protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir-203 prior to pgn-treatment completely abolished both p63 and socs-3 down-regulation, revealing the involvement of mir-203 in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir-203 expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb1+/-had a lower incidence and burden of benign papillomas when compared to tgfb1+/+ animals. however, more scc developed in the tgfb1+/-mice. after acute and chronic promotion, tgfb1+/-skin showed a reduced proliferative response with no increase in epidermal tgfb1 or nuclear p-smad2 compared to tgfb1+/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb1 gene dosage. further, pharmacological inhibition of alk5 suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb1 are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb1+/-skin, tgfb1+/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb1+/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb1+/+ but not tgfb1+/-keratinocytes, indicating that tgfb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb1 +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of 132 patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp-1) and fibroblast (mrc-5) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of 132 ra patients and 82 controls the levels of survivin correlated to urokinase (upa) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that 30/132 ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc-5 and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type b (itpkb) phosphorylates inositol (1, 4, 5) trisphosphate (ins(1,4,5)p 3 ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca 2+ responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h1 and h2 receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of il-1b by thp-1 cells after a 24h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il-1b secretion. tissue transglutaminase (tg2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg2 in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg2 expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg2 can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg2 not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg2 inhibitor, in a transgenic mouse model cf and in the taz10 transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz10 tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg2 in generating inflammation in two very different pathologies. this work underlines the critical role of tg2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge 2 stimulated a-dc. preliminary results indicate no accumulation of hif-1alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif-1alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p38 but not erk1/2 or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il-12 and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt5a -but not wnt3a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp130 in te. after infection with t. gondii, mice lacking neuronal gp130 (synapsin-cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. death of synapsin-cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp130 expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il-1b, il-6 and il-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il-4, il-10 and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb1 further may generate more effective therapies. as tnfa mrna 3' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb1 suppresses tnfa protein production by upregulating the rnabinding protein fxr1, which can bind to tnfa mrna and inhibit translation. methods: using raw 264.7 cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb1, we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 3'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb1 and luciferase expression was quantified. cells treated with lps and tgfb1 were also examined for fxr1 expression using pcr and western blot. following fxr1 knockdown using sirna, the influence of tgfb1 and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb1. using the luciferase-tnf-3'utr vector we show that tgfb1 targets the 3'utr of tnfa. furthermore, tgfb1 and il-10 both upregulate fxr1 mrna and protein; and treatment with tgfb1 and lps can synergistically upregulate mrna expression, more than tgfb1 alone. following sirna inhibition of fxr1, tgfb1 can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp-1 and mmp-3 secretion from saecs, nhbes and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. interleukin-17 augmented comtb-stimulated up-regulation of mmp-1 and mmp-3 secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin-17 down-regulated mmp-9 secretion from saecs by 50 %. interleukin-22 up-regulated mmp-1 and mmp-3 secretion from fibroblasts but not from saecs. timp1 secretion from saecs was enhanced by interleukin-17 but there was no effect of interleukin-22. mmp up-regulation by interleukin-17 and comtb was inhibited by the pi3kinase inhibitor ly294002 and on western analysis akt (protein kinase b) was phosphorylated at 30 minutes. chemical inhibition of the p110d isoform of pi3kinase with ic87114 abrogated the il-17 and comtb driven secretion of mmp-3 from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome 10) accentuated mmp-3 secretion. these inhibitory effects were confirmed with sirna. mmp-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. in summary, interleukin-17 and interleukin-22 drive transcription dependent mmp-1 and mmp-3 secretion from airway epithelial cells and fibroblasts. interleukin-17 also increases timp but down-regulates mmp-9 gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi3kinase pathway is central in interleukin-17 driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto 1 , l.s. moreira 2 , e. gonzalez-rey 2 , m. delgado 1 1 institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, 2 university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within 15 years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper 1 response. cholesterol metabolism is regulated by factors such as pparg1 (proliferator activated receptor g), srb1 (a class b scavenger receptor), cd36 or abca1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th1 immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg1, srb1, cd36, and abca1. we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to 60 % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il-6 in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il-6 and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il-6, tnf-a, il-1b, il-8 and il-12, and the anti-inflammatory cytokine il-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il-23 was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il-6 and il-10, and in some cases, of il-23. the maximal plasma levels of il-6 and il-10 were found at 24 hours and of il-23 between 48-72 hours. modest plasma levels of il-8 were also observed, with maximal production at various time points (4-24 hours). by contrast, production of tnf-a, il-1b and il-12 did not occur to a significant extent, while production of il-17 occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il-6, il-8, il-23 and il-10 also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il-6, il-10, il-8 and il-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving 165 patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il-6, activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il-33 is a novel il-1 cytokine family member that is expressed as an intracellular precursor (pro-il-33) and is thought to be cleaved by caspase-1 to yield a mature bioactive form of the molecule (mat-il-33). to date however, evidence of cell-associated proteolytic processing and caspase-1 dependent secretion of mat-il-33 has not been reported. here we show that pro-il-33 but not mat-il-33 is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il-33 to the il-33r and also il-33r-dependent bioactivity of pro-il-33 on mast cells. we propose a previously unrecognized role for pro-il-33 as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il-23 p19 mrna expression in myeloid cells, and markedly increase secretion of il-23, but not il-12, by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il-23 promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop10. chromatin immunoprecipitation (chip) assays using anti-chop10 and isotype control mab were performed using nuclear lysates from u937 cells and il-23 promoter dna measured by qpcr. chop10 binding on the il-23 promoter was detected following stimulation of u937 cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il-23 promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop10 in il-23 gene transcription, u937 cells expressing shrna's specific for chop10 or non-specific gene target were tested for their ability to express il-23 following tlr and er stress stimulation. u937 expressing three independent shrna targets for chop10 exhibited significant reductions in il-23p19 mrna (up to 87 % reduction of the response to lps+tp) compared to u937 expressing a control shrna. chop10 shrna expression did not affect the expression of other lpsresponsive genes, including il-1, il-8, ccl3 and sod2. to identify if er stress induction of il-23 mediated by chop10 expression plays a role in a more physiological setting, we examined the role of chop10 in the induction of il-23p19 gene expression following chlamydia trachomatis (ct) infection. infection of u937 cells with live but not g-irradiated ct induced expression of er stress response genes, including chop10. u937 infected with live ct exhibited increased il-23p19 mrna expression compared to u937 infected with nonviable bacteria. chop10 silencing significantly reduced the ability of live ct to induce il-23p19mrna, confirming the important role of chop10 in this response. these data suggest that er stress induction of chop10 could contribute significantly to the pathogenesis of diseases in which il-23 plays an major role, through induction of il-17 and il-22 producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd45b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd45b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd45b maintained a sustained activation of p38 kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd5 and cd69 were elevated on gadd45b-deficient thymocytes. thus, we provide evidence that gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo 1 1 institut pasteur, paris, france, 2 monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic il-7 niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il-7 promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il7 transcripts (il-7 hi cells). il-7 hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl19, ccl25, cxcl12) and cytokines (il15) that are critical for normal thymopoiesis. in the adult thymus, il-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il-7 levels. conversely, the frequency of il-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il-7-expression by tecs. > together, our temporal-spatial analysis of il-7-expressing cells in the thymus suggests that thymic il-7 levels are dynamically regulated under distinct physiological conditions. this novel il-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il-7 expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l5 and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl2-tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl2 and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl2-tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl2-tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg2 plasma cells and serum igg2 levels were˚5-10 fold increased. importantly, serum from young slc-tg;em-bcl2-tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in 2/3 and 6/6 cases, respectively. these values were increased when compared with control groups: 1/6 in fcgriib -/and 0/6 in bcl2-tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd27 + cd135 + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd117 hi cells, representing multipotent progenitors, or cd127 + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb2 and ephb3 expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b1 and/or ephrin b2, the ligands of ephb2 and ephb3 receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb1 or ephrinb2 genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb1/b2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb1 and ephrinb2 in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink 1 , d. vanhecke 1 1 university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op9-dl1 cell culture system (1) . using this in vitro assay we obtain large numbers of human cycd3 + and cd4 + cd8 + double positive thymocytes starting from umbilical cord blood (ucb) derived cd34 + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il-7 and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd34 + cells upon notch signaling is cd7 followed by cd127. t cell specification is accompanied by the induction of cd1a and loss of cd34 on cd7 + cd127 + cells. these cd34 -cd1a + cd7 + cells become dependent on continuous il7 and notch signaling for sustained survival and further differentiation into cd4 + cd8ab + dp thymocytes. we found that flt3l is not essential for the differentiation of cd4 + cd8 + human thymocytes but that addition of exogenous flt3l in the co-cultures increases the number of cd34 + precursors and consequently result in higher yields of developing cd4 + cd8ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd3 + cd4 + cd8ab + dp subset in this in vitro assay suggesting that op9 stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn3 to dn4 stage, where bdnf and its receptor p75 are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga 1 , c. lópez-rodríguez 1 1 pompeu fabra university, barcelona, spain nfat5 is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat5 are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat5 participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat5 deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd8 + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat5-null mice are unable to mount cd4+-and cd8 + -immune responses. data from our laboratory indicate that nfat5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat5 during the development of t lymphocytes, we developed mouse models that delete nfat5 at early (lck-cre + /nfat5 flox/flox ) or late (cd4-cre + /nfat5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat5 is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat5 at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p53-/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p53 has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic t-cell tumors. s. myrczek 1 , r. pardi 2 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, 2 vita-salute san raffaele university school of medicine, milano, italy jab1 is the catalytic subunit of the highly conserved cop9 signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab1 regulates the activity of ap1 transciption factors. to date jab1 is thought to be essential for every cell type as jab1 knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab1. to investigate the function of jab1 in b cells we established a mouse strain deficient for jab1 selectively in b cells. mice with floxed alleles of jab1 kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb1-locus (m. reth, freiburg). ablation of jab1 expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b1 and b2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl2 under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab1-deficient, bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab1 in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab1 when apoptosis is prevented. t. nitta 1 , s. murata 2 , k. tanaka 3 , y. takahama 1 1 university of tokushima, tokushima, japan, 2 university of tokyo, tokyo, japan, 3 rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd8 t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd8 t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd8 t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th1 and th2 antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd4+ t cells or functional deficits that selectively interfere with th1 or germinal centre responses. in this talk i will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn1 c-kit high (etp), dn2 and dn3 thymocyte populations was hybridised to affymetrix mouse 430a-2.0 genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as acvr1, bmpr1, fzd7, chemokine receptors cx3cr1, cxcr6 and integrins a2, a4, a9, ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata-3, tcf-1, notch-1, rag-1, rag-2 and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn2 stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn3 population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd8aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e14 thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day 18 of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd34 + cd1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd34 + cd1precursors with recombinant wnt3a (100 ng/ml) or with licl (10 mm) for 12hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab (8e7) under conditions of phosphatase activity inhibition. wnt3a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il-15 and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st2 cells suplemented with il-7 and flt3l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt3a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt3a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il-7 and flt3l and modify the transcription factor profiles of cd34 + cd1thymocytes mainly increasing hes-1 and id3 expression levels. human th17 clones and circulating th17 cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th1 and th2 clones or circulating th1oriented t cells, respectively. accordingly, human th17 cells exhibited lower expression of clusterin, and higher bcl-2 expression and reduced apoptosis in the presence of tgf-beta, in comparison with th1 cells. umbilical cord blood naï ve cd161(+)cd4(+) t cells, which contain the precursors of human th17 cells, differentiated into il-17a-producing cells only in response to il-1beta plus il-23, even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd161(+) t cells but it increased the relative proportions of cd161(+) t cells differentiating into th17 cells in response to il-1beta plus il-23, whereas under the same conditions it inhibited both t-bet expression and th1 development. these data suggest that tgf-beta is not critical for the differentiation of human th17 cells, but indirectly favors their expansion because th17 cells are poorly susceptible to its suppressive effects. m. irla 1 , w. reith 1 1 university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd4(+) or cd8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd4(+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd40 on mtecs by cd40l induced on the positively selected cd4(+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd4(+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu 1 , i. ravens 1 , g. bernhardt 1 1 hannover medical school, institute of immunology, hannover, germany cd155 is originally identified as human poliovirus receptor (pvr) and as rodent tage4, which is overexpressed in rodent colon carcinoma. cd155 is also known as necl-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd155 is overexpressed in transformed cells and promotes the cell cycle. thus, cd155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd4 and cd8; the dn subset is further subdivided into four stages (dn1-4) by differential expression of cd44 and cd25. thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd4+ sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd8 lineage. here we show that the frequency of terminally matured cd8+ sp cells but not that of cd4+ sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd155, a selective deficiency of mature cd8+ sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd155 is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd8+ sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd8+ t cells. in cd155 deficient animals, a shift in the tcr repertoire displayed by the pool of cd8+ sp cells was found demonstrating that cd155 is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. 2005 , 202, 1599 . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e9 (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd71 + ter119 + erythroid and cd45 + cd11b + myeloid cells are simultaneously cycling (s/g2/m) in the post-gastrulation mouse embryo (e10-12). the peak of lymphohematopoietic cell proliferation occurs at e13 in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd40-cd154 interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd40, we hypothesised a role for cd40-cd154 interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd40 expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd154 message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd154-/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd154 on reconstitution of b cell numbers following depletion. we show that cd40 is expressed by pro-b cells, and these cells proliferate in response to cd40 signalling in vitro. pcr identified a source of cd154, negative for cd3eta, in the bm of wt mice showing this cd154 is not provided by activated re-circulating t cells. we have shown that when cd154 -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn1 (cd4 -/cd8 -/cd44 + /cd25 -) to the dp (cd4 + /cd8 + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn1 stage on. in the bone marrow we found yfp + /b220 + and yfp + /b220populations. thus these pta expression analyses show closely similar pattern to those observed with hucd25 preta-reporter transgenic mice (gounari f. et al. 2002 , martin et al. 2003 . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. 3, 489-496 (2002 ) martin c. h. et al., nat. immunol. 4, 866-873 (2003 . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz 1 , g. klein 1 1 zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm-332 which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x 70kda.mmp-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm-332. in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp-19. by western blotting the zymogen and the activated form of mmp-19 can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm-332 and mmp-19 can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp-19 which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp-19, timp-2 and timp-3, are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd4/cd8 expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd4+cd8+ double positive (dp) tcrab low cells entering selection, and their cd4+cd8+ dp tcrab-immediate precedents followed by underrepresentation of the selected cd4+cd8+ dp tcrab high and the most mature cd4-cd8+ and, particularly, cd4+cd8-single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd4-cd8-double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd4+cd8-/cd4-cd8+ sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a 1 -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a 1 -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd4/ cd8/tcrab), in adult wistar rats subjected to 14-day-long treatment with a 1 -ar blocker urapidil (0.20mg/kg body weight/day s. c.). the a 1 -ar immunoreactivity was found in both thymocytes (mainly less mature cd3and cd3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed1-postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd4/ cd8/tcrab expression were observed. these changes comprised of an increase in the percentage of cd4+8+ tcrabthymocytes, which was accompanied by the reduction in that of cd4+8+ tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd4+8-tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd4-8+ tcrab high was reduced. in addition, the percentage of cd4+ t regulatory and cd161+tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a 1 -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c57bl/6 mice infected with m. avium (10 6 cfus, iv) were sacrificed at different time points after infection (5, 16 and 25 weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il-10) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at 16 weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at 24 weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il-10 in this organ. in the spleen, ifn-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm29935 and ai067906 from nih and grant i-823 from the robert a. welch foundation to wtg, and by grants hl067256 and hl61897 from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. 2001) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c57bl/6 x balb/c)f1 mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f1 mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski 1 , s. lang 1 , m. stein 1 , t. winkler 1 1 friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h3ac) or methylation of h3 on lysine 4 (h3k4me2/3). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h3ac and h3k4me2/3) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar #3) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 5'rlm race at the ivar #3 element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo2 locus, is thought to be accessible only during the double-negative (dn) 1 and 2 thymocyte stages based on mrna expression, implying that translocations between lmo2 and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx1 (hox11), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed 1) to evaluate lmo2 and tlx1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for lmo2 or tlx1 translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo2 accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo2 and tlx1 loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn1 and dn2 development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn1, dn2, dn3 and immature single positive (isp) stages for both lmo2 and tlx1. conclusion: our findings show that both the lmo2 and tlx1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn1 and dn2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e10-12), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag2 -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd4 cell depletion. however, cd8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd8 t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd8 cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd8 t cells has important implications for vaccine development against neonatal infections. (2008) showed that irf8 knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf8 as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf8 in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf8 were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs17444416. conclusions: although recent findings indicated that irf8 function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf8 that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez 1 , t. boehm 1 1 max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l1 expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd4 and cd8 t cells from tuberculosis patients highly express pd-1 when compared to healthy uninfected individuals. in addition, analysis of pd-1 expression in lung biopsies from tuberculosis patients revealed that pd-1 is expressed on cd4 and cd8 t cells confined to lung granulomatous lesions. finally, blocking of the pd-1/pd-l1 axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd-1/pd-l1 pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p100 to p52. among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly 1 wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th17 and th1 cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd4 + t cells, nfatc1 and c2 are predominantly expressed. nfatc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc1/a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc1/c. as demonstrated by y2h screen and co-ips, nfatc1/c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc1/c -but not the unsumoylated nfatc1/a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc1 target gene interleukin-2. other lymphokines like ifng and il13 are reversely regulated. interestingly, ntreg cells which do not express il2 exerted only nfatc1/c, but no nfatc1/a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc1 function. therefore, especially ntreg cells and anergized cd4 + t cells might be regulated by the long sumoylatable isoform nfatc1/c. lnk/sh2b3 and aps/sh2b2, two members of the lnk/sh2b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b-1 cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il-7 signalling in pre-b cells overexpression of lnk dramatically inhibits il-7-dependent growth demostrating that lnk negatively regulates il-7 pathways. furthermore, we showed that il-7 stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e3 ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav1. to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd3 complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd3 complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd4 + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh2 or sh3 domains known to mediate protein-protein interactions are key players in these processes. sly2 (sh3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a sh3 and a sam domain as well as a bipartite nls. sly2 belongs to a family of three molecules: sly1, sly2 and sash1.in humans, the sly2 gene is located on chromosome 21, in mice on chromosome 16. sly2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly2 we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin3-associated polypeptide p30 (sap30) as a putative interaction partner of sly2. sap30 is a conserved member of the sin3a-hdac corepressor complex that contains histone deacetylase 1 (hdac1) and histone deacetylase 2 (hdac2) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293t cells. in addition, we could show a direct interaction between sly2 and hdac1. to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly2 increases the activity of hdac1 in whole cell lysates and, more precisely, in nuclear extracts of 293t cells. the interaction of sly2 with sap30 and hdac1 indicates a transcriptional function of this protein. within the sin3a-hdac corepressor complex sly2 might act as a switch for the activity of hdac1. cd46-cyt1 and cyt2 are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd19 (b cell marker) fused to the transmembrane and intracellular domain of cd46-cyt1 or cyt2 in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt1 coengagement controlled il-10 secretion, while cyt2 coligation inhibited ifng production. moreover, our preliminary data suggest that cd46-cyt2 inhibits the phosphorylation of several molecules known to be activated by cd46 stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd46 cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh2 domain and three sh3 domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut78 cells with gst fusion proteins containing full length nck, the three sh3 domains or the individual sh3 and sh2 domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp76 and cd3epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam68) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip55 once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g4, results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g4) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g4) degranulation as measured by cd107a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi3k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the vps10p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla-4 (sctla-4) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla-4 in immune responses. using a specific anti-human soluble ctla-4 monoclonal antibody, jmw-3b3 that selectively binds the soluble isoform but not membrane bound ctla-4, or cleaved fragments of it, we demonstrate that sctla-4 plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla-4, secreting increased amounts of cytokines including interferon-g, il-17 and tnf-a, but lower amounts of il-10. soluble ctla-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla-4 induced secretion of the immunoregulatory cytokine il-10 by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sctla-4. it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla-4 it is crucial to t cell inhibition. membrane-bound ctla-4 exists as a homo-dimer on t cells but sctla-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b7 ligands on antigen presenting cells. a third important observation from this study is that sctla-4 exists both in serum and culture supernatants as a natural 46kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il-10 dependent regulation is most critical, boosting sctla-4 secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin-1/efhd-2, in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin-1/efhd2 is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin-1/efhd-2 in the immature murine b cell line wehi231 enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin-1/efhd-2 impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g1 cell cycle arrest. to understand how swiprosin-1/efhd2 enhances pro-apoptotic bcr signals, we analyzed whether swiprosin-1/efhd2 is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin-1/efhd2 enhanced bcr-induced calcium flux in wehi231 cells, whereas shrna-mediated down-regulation of swiprosin-1/ efhd2 impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin-1/efhd2 interacts with phospholipase cg2 (plcg2) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg2 and swiprosin-1/efhd2 was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin-1/efhd2 silenced wehi231 cells with swiprosin-1/efhd-2 was inhibited by the syk inhibitor bay 61-3606. in analogy, swiprosin-1/efhd2 regulated syk activity positively. moreover, swiprosin-1/efhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg2 and of syk tyrosine residue 352, which is involved in syk activation. finally, reconstitution of swiprosin-1/efhd2 knock-down cells with swiprosin-1/efhd2 mutants revealed that the n-terminal putative sh3-binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi231 cells. interestingly, swiprosin-1/efhd2 re-expression in swiprosin-1/efhd2-silenced cells induced already in unstimulated cells raft partitioning of syk, plcg2 and the bcr, which was reversed after 2 min of bcr stimulation. in summary, swiprosin-1/efhd2 is an accelerator of proximal bcr signalling and acts through syk and plcg2 by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e3 ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc1 tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd28 co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e3 ligase dead mice can spontaneously reject tc1 tumors. conclusion: cbl-b e3 ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e3 ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e3 ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd8 t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd8 + cd45ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip10 and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-cd3/cd28 antibodies. gene expression patterns were compared for cells stimulated with anti-cd3/cd28 beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip10, itac). the enhanced expression of granzyme-b, ifn-g, trail and ip10 were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd3-coated p815 cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal-3 to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd3 + foxp3 + and cd4 + cd25 high t cells. moreover, t cell receptor activation with combined anti-cd3 and anti-cd28 stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk-506, but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv-1 infection leads to immune dysfunction owing to a successive loss of the cd4 + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv-1 virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd4 and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk1/2. this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk1/2 directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap-1, nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk1/2 is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal 1 , t. brdicka 1 , v. horejsi 1 1 institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd45 are key regulators of src-family kinases in leukocytes. while cd45 is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh2 domain of csk binds phosphotyrosine 317 of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd25 and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd69 upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd25-csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd8 t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd8 t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie-2kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd8 t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd8 t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd8 t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd8 t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa-1 integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git2 in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi 1 , s. parusso 1 , b. frossi 1 , g. gri 1 , c. pucillo 1 1 university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il-6 -/-mcs and anti-il-6 receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il-6 derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd40 on naï ve b cells and the interaction of cd40 on b cell surface and cd40l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd40l e cd40 on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves 1 , n. bercovici 1 , a. caignard 1 1 inserm u567, paris, france inhibitory killer ig-like receptors (kir2dl1-2/3) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd8 + t cell subsets. these receptors suppress cd8+ t cell activation through recruitment of the src homology 2 domain-containing protein tyrosine phosphatase 1 (shp-1). to further analyse the yet largely unclear role of inhibitory kir receptors on cd4+t cells, kir2dl1 transfectants were obtained from a cd4 + t cell line and primary cells. the transfection of cd4 + t cells with kir2dl1 dramatically increased the t cell receptor (tcr)-induced production of il-2 independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir2dl1-itim phosphorylation, shp-2 recruitment, zap-70 and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp-2 and p-pkc-v but not of shp-1. in contrast, the kir2dl1/hla-cw4 interaction led to a strong synaptic accumulation of kir2dl1 and the recruitment of shp-1/2, inhibiting tcr-induced il-2 production. kir2dl1 may induce two opposite signaling outputs in cd4 + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir2dl1 receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg1 is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg1 antibody during these conditions. the observed igg1 switching behaviour mimics that of b cells responding to lps and il-4, but is mediated by a different, stat6-independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana 1 , c. schwindling 1 , m. pasche 2 , c. junker 1 , c. kummerow 1 , u. becherer 2 , e.c. schwarz 1 , j. rettig 2 , m. hoth 1 1 saarland university, biophysics, homburg, germany, 2 saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai1 channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than 150 nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd4 + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd4 + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. under tolerogenic conditions (ova alone), cd4 + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, 4-1bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd4 + t cell expansion and contraction kinetics in the early phase of the t cell response (days 1-6). in the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic cd4 + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il-2, ifn-g, and il-17a, and only few ova-specific foxp3 + regulatory t cells ( x 10 %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd4 + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells (30 %) was substantially increased. on day 14, both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells (50 %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd4 + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il-17a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd8 t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd8 tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by cd8 tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd8 tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd8 tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog 35-55 protein. our results provide direct evidence that mc contribute to cd8-specific priming in eae and show that the tc proliferation failure is specific for cd8 tc from mog 35-55 -immunized w/w sh mice. the role of mc-cd8 tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd8 tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd8 tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd8 tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd28 engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd28 costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd3complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla-4, but not via any conventional motifs in this region. overexpression of trim augments ctla-4 surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla-4 localisation, mainly restricted to the tgn. ctla-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla-4 trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla-4 expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla-4 transport to the cell surface. it is imperative to reveal the mechanisms by which ctla-4 is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd1 molecules, nkt cells are activated and release cytokines, including ifn-g, il-2 and il-4. nkt cells are efficiently recruited to the liver via cxcr6-dependent chemotaxis toward cxcl16 and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd8 t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd8 t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd8 t cell tolerisation via interaction with lsec. to this end we analysed cd1d expression on lsec and their ability to activate nkt cells by presentation of the cd1d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd1d, as agalcerpresenting-lsec were capable to induce tnf-a, il-2, il-4 and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd54 and b7-h1 on lsec. as naï ve cd8 t cell tolerisation by lsec critically depends on b7-h1, we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg9, jg1.2, and vd2 gene products. they recognize nonpeptide antigens like (e)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd-1. one of the inhibitory receptors, pd-1, is a member of cd28/ctla-4 family and contains a single ig v-like domain in its extracellular region. pd-1 can bind to two b7 homologue molecules, pd-l1 and pd-l2. it has been reported that interaction of pd-1 with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd-1 is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus il-2 to obtain gd t cells. pd-l1+ and pd-l1-human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l1 mabs, the pd-l1 extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd-1 in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd-1/pd-l1 interaction. results: gd t cells expressed pd-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l1. we first examined whether or not the engagement of pd-1 receptor could modulate the cytotoxic activity of gd t cells. pd-l1-expressing tumor cells tempered cytotoxic activity of pd-1+ gd t cells, and cytokine production such as tnf-a was down-regulated by pd-1 engagement. in addition, inclusion of anti-pd-l1 mab reversed cytotoxic activity and cytokine production when pd-l1-expressing tumor cells were challenged by pd-1-expressing gd t cells. conclusion: pd-1 delivers inhibitory signals in gd t cells upon engagement with pd-l1. peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il-2 production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e3 ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd4 + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova 323-339 peptide)-specific t cells from do11.10 tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e3 ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e3 ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler 1 , p. aichele 1 1 immh, university freiburg, immunology, freiburg, germany interleukin 12 (il-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il-12 has a direct influence on cd 8 + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal 1) and costimulation (signal 2). we analysed direct il-12 signaling to cd8 t -12 signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd8 t cells lacking il-12 signaling failed to up-regulate klrg1 and to down-regulate cd127 in the context of listeria but not viral infections. thus direct il-12 signaling to cd8 t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd8 + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd8 + t cells are tightly controlled in their effector functions by cd152 (ctla-4). we demonstrate that signals induced by cd152 reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd8 + t cells. for this novel function cd152 specifically represses the transcription factor eomes, but not t-bet. a cd152 mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd152-mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd8 + t cells differentiated in the absence of cd152 signaling could be demonstrated in vivo. the novel insights that cd152-mediated signal transduction in vivo indeed alters cd8 + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd8 + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p2 receptors. p2x 1-7 receptors open to non-selective ion channels, whereas p2y1, 2, 4, 6, 11-14 are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p2 receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr9 but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca(2+)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek 1 , a. brouckova 1 , d. filipp 1 1 institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih3t3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y505flck. comparative 2-d gel analyses followed by ms/maldi identified rack1 as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih3t3 cells of ha-tagged rack1 with either a wild type lck or constitutively active y505flck revealed a significantly enhanced complex formation between y505flck and rack1 compared to that of wtlck. ectopic expression of y505flck with its domain-inactivating mutations showed that lck-rack1 interaction depends on functional sh2, sh3 and the c-terminal tail sequence of lck. lck-rack1 interaction is readily detectable also in primary cd4 + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack1 with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack1 in the context of lck translocation to lr is further strengthen by the observation that rack1 is associated with elements of cytoskeleton. these results are the first to characterize rack1 as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (il-21) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd19+cd20+cd27-cd38-igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th2 response to alumova inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to igg1. the plasma cells up-regulate cxcr4, but not cxcr3, a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th1-associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚5%), igg2a (˚30 %), igg2b (˚30 %) or igg1 (˚30 %). in addition to cxcr4, some 70 % of these plasma cells strongly express cxcr3. the induction of cxcr3 in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg2a switching during th1 responses. this is functionally significant for oti-dependent cxcr3 expression, as well as induction of switching to igg2a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg2b. t-bet is known to be induced in b cells exposed to ifng or tlr9 stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd88. objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd272) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd4+ t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd4+ t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla-4 pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa-1 and cd2 in the psmac of untransformed human t-cells. in marked contrast, tcr/cd3 accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl-1 regulates interclonal t cell competition during acute and chronic immune activation. we found p53-independent noxa gene induction and mcl-1 downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl-1, which was delayed in noxa -/cells. using ot-1 cells and altered peptide ligands we observed that the level of mcl-1 downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl-1 -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np366) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen 9 (ebag9) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag9 confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-adaptin in t cells. both interactions suggested an involvement of ebag9 in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag9-/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag9-deficient ctls. these data imply a role for ebag9 in regulating the formation of mature ctl granules and identify ebag9 as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag9 defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd8 t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd80/86 and il-12. in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd3/acd28 or pma/ionomycin could not significantly activate cd4 and cd8 t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca 2+ influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena 1 , s. carrasco 1 , i. merida 1 1 centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp1-ras-erk signal intensity is critical to determine the final cell outcome. rasgrp1 is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. 1. analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. 2. develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd8 cells expressing gfp-c1 domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c1 domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h4, cd278), a cd28-like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th2 biased responses and high production of the anti-inflammatory cytokine il-10, and was essential to the development of germinal centres. however, icos can also help in the il-21-dependent differentiation of inflammatory th17 cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd28 a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p85 subunit of class ia pi-3 kinases. these can complex with one of the three 110 kda catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate pip 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi3k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi3 kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi3-k regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) pi3-kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p85a g p53a g g p85b and p110a g p110d g g p110b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd3-induced secretion of il-10 and il-4. during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap450, a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap450 construct that acts as a dominant negative, or sirna knockdown of endogenous akap450 expression in t cells prevents the correct organization of cd3z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa-1 localization was also analyzed to assess p-smac architecture and, interestingly, confocal 3d reconstruction revealed that lfa-1 ring was not clear in the akap450-disrupted cells. moreover, akap450 was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap450 decrease the phosphorylation of molecules such as lat, plcg1 and pkcv. these defective activation events as reflected in a reduction of il-2 production. together, our results underscore a key role for akap450 in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin85/cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh3 domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca2+ mobilization and cell activation involving the pi3k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc1d10c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr 1) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as 2) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. 3) we then studied the phenotype of carabin kd (shrna expressing) a20 b cells after bcr stimulation. 1) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t1 tot2 b cells and to follicular mature b cells. 2) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. 3) bcr simulation, but not lps stimulation, of carabin kd a20 b cells shows an acceleration of ras target erk1/2 phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk1/2 pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor 3 (tlr3) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd4 + t cells express tlr3 and respond to the well characterized synthetic tlr3 ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd4 + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr3. tlr3 stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd4 + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor 3 (irf3) as revealed by realtime-rcr analysis of ifn b and irf7, whose transcription depends on the activity of irf3. combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr3 signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd4 + t cells. this study was supported by dfg spp 1110 "innate immunity" (ka 502/8-3). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd3e cytoplasmic domain (cd3e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd3e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd3e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd3e cd and a fluorescent membrane dye (r18). with this assay, we show that the cd3e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd3e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd3e cd to lipid bicells. membrane binding by the cd3e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia 1 , y. ge 1 , u. quitsch 1 , p. beckhove 1 1 german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd4+ t cell help is believed to contribute to optimal cd8+ memory expansion via cd40l on cd4+ t cells binding cd40 on dendritic cells. however, a few reports suggest that cd40l-cd40 engagement may mediate direct cell-cell contacts between cd4+ and cd8+ t cells. in this study, we investigated the importance of cd4-cd8 co-operation and cd40l-cd40 interactions for t em proliferation. methods: we isolated human cd4+ and cd8+ t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd4+ and cd8+ populations were activated in vitro using anti-cd3/cd28 beads. proliferation was measured by [ 3 h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd40 or cd40l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd4+ or cd8+ t em cells, demonstrating that optimal t em expansion requires direct cd4-cd8 interactions. surprisingly, not only cd8+ but also cd4+ t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd4+ t em proliferation depends on signals from cd8+ t em cells. activation induced the expression of cd40 on both populations and cd40l on subsets of cd4+ and cd8+ t cells. blocking of cd40l on cd8+ t em cells impaired significantly cd4+ t em proliferation, which confirms that the improved expansive potential of cd4+ t em cells in mixed populations depends on cd40l co-stimulation by the cd8 t em subset. conclusions: our data demonstrate for the first time that activated cd8+ t em cells deliver help to the cd4+ t em subset via cd40l-cd40 signalling and may play an important role for cd4+ t em expansion upon stimulation. the t cell surface glycoprotein cd5, a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd5 localization upon t cell:apc conjugation. we have questioned which domains of cd5 mediate the localization within the is, and for this we have expressed cd5 mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd5 mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd5 by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd5 depends on sequences within the cytoplasmic domain, as a cd5 deletion mutant lacking most of the cytoplasmic tail, cd5.k384 stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd5 translocation was mapped within amino acids glu 418 and his 449 since the cd5.h449 stop mutant, just short of 22 aa is still able to translocate to the is, whereas cd5.e418 stop , that lacks 2 important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu 418 -his 449 ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna-2 is missing, but lmp-1 is still expressed. using a hl derived cell line, we have shown that the cytokine il-4 can induce lmp-1 expression in vitro and can replace ebna-2. we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is 2, 3 or 4. a high affinity stat6 binding site is spaced by 4 nucleotides. we found three potential stat binding sites in the lmp-1 promoter, which we named lrs, tr and edl1. they were spaced by 4, 3 and 2 nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il-4-treated or non-treated kmh2-ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat6 binding site, or lrs-stat6. a stat6 complex binding to the gl-epsilon promoter and lrs-stat6 was induced by il-4. the specificity of the stat6 complex was shown by supershift experiments with anti-stat6, but not anti-stat5 antibodies. when gl-epsilon or lrs-stat6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat6 contains a functional stat6 binding site. oligonucleotides, corresponding to lrs in which the stat6 site had been mutated, could not compete for stat6 binding. interestingly, the unlabeled lrs-tr with 3 nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by 2 nucleotides (lrs-edl1), it could not compete. thus, expression the transforming protein lmp-1 can be induced directly by the t cell derived cytokine il-4 in a stat6 dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp-1 and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd8 t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji 2007 (ji . 179:2250 . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd4 t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd4+ t cells primarily affect the proportion of cd4+ t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd40, cd80 and cd86 on parameters of cd4+ t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd40l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd40 leads to an enhancement of ig and cytokine production. the current dogma postulates that these 2 signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd40l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd40l (rmcd40l) can increase proliferation induced by tlr3 (poly ic) and tlr4 (lps) agonists. by contrast, we never observed any synergy between rmcd40l and tlr1/2 (pam3csk4) or tlr2/6 (pam2csk4) agonists. to go further in the study of cd40l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd40l, named mini-cd40ls, based on a c 3 -symmetry core holding cd40-binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd40ls bind to immobilized human cd40 and compete with the binding of cd40l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd40l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd40l, mini-cd40ls synergize tlr4 (lps), tlr3 (poly ic) and tlr7/8 (r848) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd40ls and tlr 1/2 (pam3csk4), tlr 2/6 (pam2csk4) and tlr9 (odn 2395) agonists. synergy between cd40l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd40l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd8 + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd8 + t cell tolerance. we have defined a phenotypic profile for cd8 + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd25 and cd69, and high levels of expression of cd62l and ly6c. whereas, cd8 + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd25 and cd69, and loss of cd62l and ly6c expression. ly6c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly6c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly6c, expressed on naïve cd8 + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd8 + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to 60 days after infection. results: daily injection of paraoxon induced g 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. mice exposed to paraoxon exhibited g 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th1 and th17 cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide 3-kinases (pi3k) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi3k are divided into two types: class ia p85/p110 heterodimers, which are activated by tyr kinases, and the class ib p110g (p110gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p110g deletion affects tcr-induced t cell stimulation. mice lacking p110g show a partial defect in t cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd4+/cd8+ t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi3kg-specific inhibitor causes a reduction in the number of cd4+ memory t cells that mediate renal injury. similarly, pi3kg deletion in p65 pi3k transgenic mice also reduces the numbers of cd4+ memory t cells. there is therefore evidence that pi3kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi3kg regulates this process is not well understood. we studied the specific role of p110g in t cell activation. methods: we studied whether the tcr activates p110g and the consequences of interfering with p110g expression or function on t cell activation. results: we found that after tcr engagement, p110g interacts with and forms a complex with ga q/11 , lck and zap70. tcr stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p110g controls rac1 activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p110g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p110g-/-t cells. our observations clarify the activation mechanism and mode of action of p110g in the control of t cell activation, confirming a crucial role for p110g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a4b2 and a7, expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c57bl/6j mice. they were stained with fluorescently labeled igm-, cd40-or cd23specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by 3 h-thymidine incorporation upon stimulation with anti-cd40 and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a7 nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a4 or b2 nachr subunits inhibited binding of igm-and cd23-specific antibodies but facilitated that of cd40-specific antibody. in contrast, antibody against a7 subunit prevented binding of anti-cd40 but not of anti-igm or anti-cd23 suggesting that a7 nachrs are located close to cd40, while a4b2 ones are close to bcr/cd23. consequently, anti-cd40-induced b lymphocyte proliferation was increased by mla much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd40-mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd4 + t h -lymphocytes to antigen presenting cells or of cd8 + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim1 and trpc3), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about 96 %. down-regulation of stim1 was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd4 + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, 2008) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein 70 (hsp70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd8 + cytotoxic t-lymphocyte (ctl) and cd4 + t-helper cell (th1) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k562 cells as well as targetindependent cytotoxicity. cd4 + cells exhibited a strong increase in proliferation after stimulation with hsp70, with rates of up to 29 %. in the presence of target cells, a 35-fold up-regulation of granzyme b mrna was observed after stimulation of cd4 + t-helper cells with hsp70 in combination with il-7, -12 and -15. the target cell-independent secretion of granzyme b by cd4 + cells was greatly augmented after stimulation with hsp70 plus il-2 or il-7, -12 and -15. in this study, we have shown that hsp70 is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd4 + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd3 + and cd8 + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp70 on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd40, which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd150 receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk1 overexpression in a model system was achieved by transfection. pjnk1/2 expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd150 on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk2, but not jnk1 activation. cd40 ligation on primary tonsillar b cells also resulted in jnk2 activation. however, bcr crosslinking did not affect the level of jnk1/2 phosphorylation. cd150-mediated jnk2 activation was independent from sh2d1a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd150 and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase 1 (hpk1) was associated with cd150 in primary b cells as well as in b cell lines. cd150-hpk1 association was independent from cd150 tyrosine phosphorylation and sh2d1a expression. overexpression of hpk1 in a model system significantly enhanced cd150mediated jnk2 phosphorylation. it is known that tnf family receptors such as cd30, cd40, rank trigger survival signals in hrs cells. we observed the expression of pjnk1/2 in hrs cells of primary classical hl. cd150 could be involved in sustained jnk2 activation in primary hrs cells, and this may reflect the role of cd150 receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk2 is activated via cd150 in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk1 is involved in cd150-mediated jnk2 activation. objectives: cd5 has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd5 upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd5 are involved in c-cbl phosphorylation and association. methods: el4 thymoma cell line was stably transfected with wild-type human cd5 or hcd5 cytoplasmic tail mutants: cd5.k384stop (maintaining only a pseudo itim); cd5.h449stop (lacking the distal s and y in the carboxy-terminal region); cd5. ¿ e418-l444stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y700 in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd3 in combination or not with anti-human cd5 biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py700) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x 0.05). in murine thymocytes, co-crosslinking of cd3 with cd5 induces an increase in c-cbl phosphorylation compared to cd3 alone. analysis of the el-4 transfectants showed that mutants cd5.k384stop and cd5.h449stop lost the ability to costimulate cd3-mediated phosphorylation of c-cbl. in contrast, cd5. ¿ e418-l444stop mutant, was able to efficiently costimulate cd3-mediated c-cbl phosphorylation, similarly to the hcd5wt. our results indicate that the absence of the pseudo itam in cd5 does not interfere with c-cbl phosphorylation in response to cd3 plus cd5 crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd5 appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd5 cytosplasmic tail, but rather, may indirectly associate with cd5 through the interaction with other sh2-sh3 domain-containing molecules, that may be recruited to cd5 through its carboxy-terminal region. l. kolly 1 , s. narayan 1 , j. tschopp 2 , a. so 1 , n. busso 1 1 chuv, rheumatology, lausanne, switzerland, 2 unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent il-1b and il-18 production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd4 + and cd8 + t cells were activated in vitro through anti-cd3 stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd3 ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd3 activated asc -/-t cells predominantly displayed a more th 2 phenotype, producing more il-10 (199 vs. 692 pg/ml; asc +/+ vs. asc -/-t cells respectively; p=0.0074) and less ifn-g (15,831 vs. 6 ,921 pg/ml; asc +/+ vs. asc -/-t cells respectively; p = 0.0021). when asc +/+ and asc -/-t cells were purified into cd4 + and cd8 + t cell fractions and activated individually using anti-cd3, no inhibition in proliferation was observed amongst activated asc -/-cd4 + and cd8 + t cells. interestingly, the activated asc -/-cd4 + t cell fraction produced significantly more il-10 when compared to activated asc -/-cd8 + t cells and asc +/+ cd4 + and cd8 + t cells (asc -/-cd4 + t cells = 380 pg/ml il-10; asc -/-cd8 + t cells = undetectable il-10; asc +/+ cd4 + t cells = 11 pg/ml il-10; asc +/+ cd8 + t cells = undetectable il-10). cd4 + and cd8 + t cell mixing experiments revealed that asc -/-cd4 + t cells are able to inhibit the proliferative ability of asc -/-cd8 + t cells, asc +/+ cd4 + and cd8 + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd4 + t cells. collectively, these results demonstrate that the absence of asc drives cd4 + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd4 + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge 1 and pge 2 suppress some t-cell functions including proliferation, activation and cytokine production. pge 2 signals through four types of gpcrs called the ep receptors. at low concentrations, pge 2 is believed to be necessary for t cell function, whereas at higher concentrations, pge 2 inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge 2 and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge 2 diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd4 + t cells with ep receptor antagonists was found to impair cell surface expression of cd71, cd69, cd25 and ox40 but not cd44. suppression of t cell proliferation by pge 2 has already been widely studied. however, blocking ep receptors in cd4 + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd4 + t cells to the chemokine sdf-1b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd44 + cd4 + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd4 + t cells. our results also suggest that considering pge2-mediated camp signaling in cd4 + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd4 + human t-cells was examined. oxidation affects several ca 2+ signalling pathways by altering the activity of ip 3 receptors, trp channels and store operated ca 2+ channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca 2+ signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd147 (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd147 knockout mouse possess enhanced mixed lymphocyte reactions and cd147 monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd147 signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd147 in jurkat t cells augments the secretion of the t cell growth-factor interleukin-2 (il-2) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il-2 promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd147, we identified the immunomodulatory sub-domain of cd147. supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd40 and il4 (simulates t cell-dependent activation). microarray assays identified about 104 mirnas in unstimulated b cells. 35 of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor-4 (irf-4). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf-4 transcripts differs from that observed for irf-4 protein abundance after b cell stimulation. further analysis identified the irf-4 transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk592 and the dfg forschergruppe for832. objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on 11 different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey 1 , f. hauck 2 , i. berberich 1 , f. berberich-siebelt 2 , gk 520 -immunomodulation 1 institute for virology and immunobiology, university of wuerzburg, würzburg, germany, 2 university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd4+ t lymphocytes, the transcription factor is predominantly expressed in t helper 2 (th2) compared to t helper 1 (th1) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g1 phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp-1 is expressed in differentiated effector t cells where it is higher in th2 than th1 cells. the regulation of the blimp1 expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm1 promoter and activates blimp-1 expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp-1 in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp-1 in b cells using cre recombinase. moreover we found a new putative blimp-1 isoform lacking exon 2. currently, we analyze the expression of c/ebpb and blimp-1 in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening 53 cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd21low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd4 + t cells but not transitional b cells or cd8 + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (mip-1a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy2 revealed constitutively high background levels in cd21low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas 1 , v. courtois 1 , k. de luca 1 , r. sodoyer 1 1 sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il-2, il-4 or il-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il-2, il-4 and il-6. furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a-136-0033-01835). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen-4 (ctla-4) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla-4 during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il3) in the tissue and the released eggs and first stage larvae (l1) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd3 and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla-4 during the infection, a neutralysing antibody (a-ctla-4; 4f10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with s. ratti il3. the in vivo neutralisation of ctla-4-signalling by applying a-ctla-4 during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th2 cytokines, such as il-4 and il-5 and a reduction of the proinflammatory cytokines ifn-g and il-17. the investigation of the humoral response showed a remarked increase of the igg1-titer in the serum during secondary infection in mice that had been treated with a-ctla-4 during primary infection. furthermore, the blockade of ctla-4 resulted in a diminished worm burden as indicated by reduced release of l1 in the faeces. these results suggest that the blockade of ctla-4 during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg1 during secondary infection also reflects the induction of a potent th2 response. objectives: cd36 is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd36 is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd36 on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd36 expression. cd36 knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd36 ko compared to heterozygous mice. since reduced levels of cd36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd36 ko mice. after one injection of apoptotic cells, cd36 ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd36 on b-cells are involved in setting this threshold. conclusion: our data suggest that cd36 is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd152 acts as a major check-point of immune responses, but the mechanism by which cd152 controls peripheral t cell responses is unknown. the consequences of cd152 signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd152 in th1 cells reduced migration towards ccl4, cxcl12 and ccl19. crosslinking of cd152 together with cd3 and cd28 stimulation on activated th1 cells increased expression of the chemokine receptors ccr5 and ccr7, which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd152 expression and cd152-mediated migration-enhancing signals. importantly, migration of cd152 positive th1 lymphocytes in in vivo experiments increased, as compared to cd152 negative counterparts, showing that indeed cd152 orchestrates specific migration of selected th1 cells to sites of inflammation and antigenic challenge in vivo. these data show that cd152 signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd152 adds to the already significant role of cd152 in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd152 signaling on the longevity of cd28 null t cells. using a sensitive staining method for cd152, we show that human cd4 + cd28 null and cd8 + cd28 null t cells rapidly express surface cd152. serological inactivation of cd152 using specific fab fragments or blockade of cd152 ligands using ctla4ig in cd4 + cd28 null and cd8 + cd28 null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd152 crosslinking on activated cd28 null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd152 is mediated by pi3'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd152 act directly on activated cd28 null t lymphocytes and, due to its exclusive expression as a receptor for cd80/cd86 on cd28 null t cells, prevention of cd152 mediated signaling is likely a major target mechanism taking place during therapy with ctla4ig. objectives: cd45 is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd45 -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd148 (ptprj, dep-1) which acts as a positive regulator of sfk in cd45 -/-b cells and macrophages and can compensate for cd45 deficiency in these cells. indeed, combined deficiency of cd45 and cd148 in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd148 and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd148 -/mice were reported so far. however, in human t cells the role of cd148 may be different since naive human t cells express cd148 at a level comparable to b cells. using cd45 -/-/cd148 -/human t cell line (jurkat-derived js-7 cells) we tested the ability of cd148 to complement cd45 deficiency in t cells. we used retroviral transduction to express human cd45 or cd148 in js-7 cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd148 to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js-7 cells. expression of wild type cd45 or cd148 in js-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd69 upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js-7 cells expressing cd148 when compared to control cells. finally, cd148 substrate trapping mutant expressed in js-7 cells interacted with lck in vivo suggesting that lck is a direct substrate of cd148 in js-7 cells. the results suggest a level of redundancy between cd45 and cd148 in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam1.6) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr 505 and tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native 2d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca2+ release-activated ca2+) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa-1 prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al.2006) we describe a non-viral vector based approach (vockerodt et al. 2008) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in 9 independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd40 signaling cascade was conducted. after activating this particular signaling cascade (cd40) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. 2005 ). a rat thymic epithelial cell (tec) line (r-tnc.1) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc.1 cell line in vitro. it was found that a number of adhesion molecules, such as cd2, cd4, cd8, cd11a, cd18, cd54, cd90 was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc.1 line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il-2 activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc.1 cells. we found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mab to cd2 and cd8 molecules (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells). early adhesion was inhibited by mab to cd90, abtcr, mhc class i molecule (ifn-g stimulated r-tnc.1 cells) and cd4 molecule (ifn-g unstimulated r-tnc.1 cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc.1 cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mab to cd2, cd4, cd8, cd90 molecule (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc.1 cells). our results also suggest the involvement of cd11a/cd18 dependent -cd54 independent pathway in adhesion and cd11a/cd18 dependent -cd54 dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin-27 (il-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il-27 favours naï ve cd4 t cell differentiation into th1 cells to the detriment of th17 or th2 differentiation. the il-27 receptor (il-27r) is a heterodimer composed of tccr, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. il-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd8 t cells, but the potential impact of il-27 on human cd8 t cells has not been elucidated. our goal is to investigate the impact of il-27 on human cd8 t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il-27r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd8 than cd4 t cells expressing the complete surface il-27r (gp130+tccr). however, we detected high amounts of intracellular tccr in both, cd4 and cd8 t cells, but only polyclonal activation (anti-cd3) of cd8 t cells led to an actual increase of il-27r surface expression. purified cd8 t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il-27 activated stat1 and stat3 signalling with rapid kinetics in both cd8 and cd4 t cells, indicating the capacity of il-27 to signal through these molecules. addition of il-27 to anti-cd3 activated cd8 t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il-27 on human cd8 t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd-1/pd-l1 pathway is associated with production of the immunoregulatory cytokine il-10, the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l1 and pd1, as well as myelin basic protein (mbp)-stimulated il-10 production, pakt inhibition, and apoptosis (annexin v), in 50 ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); 24 had a diagnosis of stable disease (sms). results showed that: 1) pd-l1 -expressing cd14+ and cd19+ cells are reduced in ams compared to sms individuals (p=0.04); and 2) pd1 expression is increased in cd4+ t cells of sms individuals and is comparable on cd8+ t cells of ams and sms patients. this is associated with a significant decrease in il-10 production by mbp-stimulated cd14+ and cd19+ cells of ams patients (p=0.03). additionally, cd8+ anexin v+ (av+) cells were diminished and cd8+ pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd4+av+ and cd4+ pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l1/pd-1 pathway seen in ams patients result in a reduced mbp-specific il-10 production by cd14+ and cd19+ cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd8+ t. the pd1/pdl1 pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti-4-1bb in cd8 cells. this difference could be due to down regulation of cd28 by activated lymphocytes and possible preferential response of cd8 cells to anti-4-1bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin7 (stx7) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx7. rna interference technique was also used to down regulate stx7 expression in primary human ctls. results: we identified stx7 in ctls by pcr and immunocytochemistry. stx7 accumulates at the is after ctl/target cell contact. when stx7 function was blocked by overexpression of a dominant negative stx7 mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx7 is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the 20 years history of mouse t h 1 and t h 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h 0, t h 1 or t h 2 phenotypes, based on their cytokine production (il-2, ifng or il-4). a comparative analysis of t-bet, ifng and il-4 mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca 2+response, membrane raft expression/organization, k + -and ca 2+ -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h 1 g t h 0 g t h 2, although the membrane cholesterol level (detected with anti-cholesterol ab, ac8) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h 1 cells upon activation than in t h 2 cells. t h 1 cells produced a more sustained calcium response with higher amplitude than t h 2 cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav1.2 and kv1.3 ion channels, major functional determinants of the sustained calcium influx. t h 2 cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd19-dynabeads. cd19 + isolated cells were facs sorted into cd19 + cd27naïve b or cd19 + cd27 + memory b cells. dna synthesis was measured by 3h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd19 + cd27naï ve b cells, of which bmp-6 and -7 were most efficient (40 % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd19 + cd27 + memory b cells by 40 -50 %. to induce differentiation, both naï ve and memory b cells were stimulated with cd40l and il-21. this increased the production of igm, iga and igg 10 -100-fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad1/5/8 in cd19 + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd40l/il-21-induced production of igs in mature human b cells. s. gutenberger 1 , k. warnatz 1 1 university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (erk 1/2). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd86 upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of 20 hd and 25 cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk1/2. to increase the signal intracellular phosphatases were inhibited by h2o2. as markers of activation and initiation of proliferation, cd86 and ki67 expression were measured after 2 days of in vitro stimulation. k. theil 1 , p. aichele 1 1 immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd8 t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p14 cd8 t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b6-recipient mice and compared their expansion with wild-type (wt) p14 t cells after viral infection. we could demonstrate a severe impairment in the capacity of p14 t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p14 t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd8 t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p14 t cells into h8 mice. h8 mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp33-41. therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h8 mice are infected with lcmv8.7. this is a lcmv variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the p14 t cells. s. frischbutter 1 , r. baumgrass 1 1 deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap1 which are important for expression of cytokines such as il-2, ifng and il17. it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl-10 was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl-10 phosphorylation but rather an inhibition of bcl-10 dephosphorylation. furthermore, calcineurin and bcl-10 co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl-10/malt1 signaling complex and dephosphorylates bcl-10 and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop 1 , j. lamoureux 1 , c. fortin 1 1 université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: 25 healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla-4 has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla-4 expression was higher after stimulation in t cells of elderly. there was differences between cd4 and cd8 t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd95l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh3 and ww domain proteins. since fasl surface expression is regulated by adam10-mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl2a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh3 domain proteins that potentially interact with the riped fasl prd, we used a sh3 domain phage display library containing all 288 sh3 domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conseoptimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b7 family members on antigen-presenting cells with cd28 on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd28 co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound12, with classical gcs regarding their suppressive effect on cd28-costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd3 and anti-cd28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd28-costimulated human memory/effector cd4+ t cells by compound12 than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound12 was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound12 and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound12 than prednisolone. when evaluating possible mechanism for the increased activity of compound12 in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound12 which was not prevented by the partial gc receptor antagonist, ru-486, in vitro. moreover, in vivo we observed less induction of il-1beta and tnf-alpha by pre-treatment with compound12 than with prednisolone. our data indicate that the non-steroidal segra, compound12, may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b6 mice elicits robust cd8+ t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il-2+ cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa 224 -specific cells also express tnfa, but only about half of the ifng+ np 366 -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd8+ t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd8+ t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova 257-264 peptide shows a close correlation with division in vivo. early after antigen encounter (0-2 divisions) the vast majority of cells express only tnfa. after 3-4 divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd4+ t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd8+ t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd8+ t cells, expansion plays a very important role in the regulation of cd8+ t cell effector function. in addition to its chemo-attractant function, sdf-1a (stromal-cell derived factor-1a, cxcl12) has been described to costimulate cd4 + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd4 + t cells was increased by immobilized sdf-1a to a level similar to that obtained with the costimulatory molecule cd28. as visualized by real time confocal microscopy, t cells entering in contact with sdf-1a formed a tether and displayed an active scanning activity. since sdf-1a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd3 mabs, it is conceivable that the sdf-1a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf-1a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf-1a in the context of cd4 + t activation by antigen-presenting cells secreting sdf-1a. this study should help us to better define how sdf-1a modulates cd4 + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd4 + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd3 and anti-cd28 antibodies and cape for 2 days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by 3h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il-5 production and lymphoproliferation in cd4 + t cells stimulated by anti-cd3/cd28. cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd4 t cells express a variety of molecules on their surface, such as mhc-class ii, cd80, cd86, cd70, whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd4 t cells might induce t cell proliferation and differentiation from cd4 resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd4 t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd4 memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd25, cd30, and cd69. analysis of the cytokine profile of these cells revealed the differentiation of il-10-and ifn-g-double-producing cells in response to contact with th1 effector cells, and il-4-producing cells in response to contact with th2 effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd3/cd28 stimulation. whereas neutralization of ifn-g or il-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd70, cd80, and cd86 could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. conclusion: interaction of cd4 memory t cells with activated t cells resulted in the production of the cytokines il-4, il-10, and ifn-g. given the immunomodulatory capacity of il-4 and il-10, these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir 1 , s. thompson 1 , j.j. murphy 1 1 king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il-2 and il-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp36l1 by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp36l1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl1 differentiation was observed to be associated with downregulation of zfp36l1 protein. in an attempt to determine whether zfp36l1 downregulation was directly linked to bcl1 differentiation, a zfp36l1 shrna expressing lentivirus (psicor) was employed to knockdown zfp36l1 expression. this reagent downregulated zfp36l1 expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp36l1 shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp36l1 downregulation in promoting bcl1 plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of 32 patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all 32 isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd95 (fas, apo-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd3/cd28-triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo1 mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd95l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il-2 and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk1/2 phosphorylation, the expression of various activation markers, the il-2 production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb415) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb1,3galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd4+ and cd8+ t cells. in addition, mitogenic stimulus increase 3-fold the all binding to cd4+ t cells. previous studies in human pbmc showed that all binds to human cd4+ t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd4+ t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd3-dependent activation of purified cd4+ t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd25 and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd25 expression, but enhanced the anti-cd3-dependent proliferation and cd25 expression of purified cd4+ t cells. analisis of calcium influx showed that all enhanced anti-cd3 dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd4+ t cells by up-regulating t cell activation mediated by anti-cd3 stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in214609) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd3 complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd95 receptor. it has been previously demonstrated that cd95 ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp-76, slap-130 and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il-1r, il-2r, il-3r, il-7r, il-12r, il-18r, cd27, cd28, cdw137( 4-1bb), cd 95(fas) and cd178 (fasl) on hpbls in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. 1. the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) the expressions of membrane immune molecules before cultured. 2) expressions of the membrane molecules on hpbls during culturing. 5% fbs rpmi 1640 group (1640 group), il-2 group, pha group... (1) mtt assay. (2) proliferative times and growth curves of hpbls... 1. during cultured in vitro, there are expression changes of the il-1rs (il-1ra, il-2ra, il-2rg, il-3r, il-7r, il-12r, il-18r), co-stimulatory molecules (cd27, cd28, 4-1bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in 1640 group, il-2 group and pha group, but the rests are different. 2. our data also suggest that the hpbls cultured in cd3mcab+cd28mcab+il2+il1a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. 3. celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun 1 , r. tauber 1 1 zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the ras/rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-jun n-terminal kinase (1), which leads to an enhanced binding of l-selectin to soluble ligands (3). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues (4). here we show that the protein phosphatase 2a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp2a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp2a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer 1 , e. glasmacher 1 1 helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago1-4 genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm1 and rck or expression of dominant-negative gw182. interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd4 + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd8 + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd8 + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd8 + t-cell activation and differentiation. naïve cd62l hi cd44 lo cd8 + t cells were sorted and stimulated by anti-cd3 and anti-cd28 antibodies. results: firstly, we show that the activation and differentiation of cd8 + t cells require il-2 provided by activated cd4 + t cells at the initial priming stage after stimulation. secondly, this critical il-2 signal is delivered through il2rbg of cd8 + cells and is independent of il-2ra. besides promoting cell proliferation, il-2 stimulation increases the amount of ifng and granzyme b produced by cd8 + t cells. conclusion: therefore, our studies demonstrate that a full cd8 + t-cell response is elicited by a critical temporal function of il-2 released from cd4 + t cells, providing mechanistic insights into the regulation of cd8 + t cell activation and differentiation. most antigenic peptides recognized by cd8 t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a3168-176 is a tumor antigenic peptide presented by hla-a1 and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a3168-176 by tumor cells. by electroporating hla-a1 cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart-1/melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart-1/melan-a 26-35 specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart-1/melan-a 26-35 ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart-1 expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart-1/melan-a 26-35 epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart-1/melan-a 26-35 ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd8 + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd4 t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev 207: 293-313, 2005) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h2-m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity 11: 453-462, 1999) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. furthermore, also reduction of tap1 and tap2 transcription was observed contrasting to the elevated levels of erp57 and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their ifng-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about 50% of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a*0201) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a*0201 binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a*0201 from tapasin, but only ttp-ha dissociated hla-a*0201 from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl-721.220-a2 showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a*0201 from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a*0201. m. basler 1,2 , c. lauer 2 , m. groettrup 1,2 1 biotechnology institute thurgau, kreuzlingen, switzerland, 2 university of constance, division of immunology, department of biology, konstanz, germany two lmp7-dependent antigens have been described that relied on the 'structural presence' of lmp7 in the proteasome but not on the activity of lmp7. here we have investigated processing of the h-2d b -restricted uty 246-254 epitope of the male minor antigen uty reported to be lmp7-dependent. using splenocytes from lmp7 -/-, lmp2 -/and mecl-1 -/mice we found that the uty 246-254 epitope requires lmp7 and lmp2 but not mecl-1. curiously, a selective lmp2 inhibitor did not interfere with uty 246-254 presentation. objective: we investigated why the deletion but not the inhibition of lmp2 interferes with uty 246-254 presentation. we hypothesized that the 'structural' requirement for lmp2 is based on replacement of the caspase-like activity of b1 in the proteasome. methods: it was determined if t1a mutants of lmp2 and/or b1 can rescue the uty 246-254 epitope. we used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. finally we determined by mass spectrometry if the uty 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by lmp2-deficient and proficient immunoproteasomes in vitro. results: we found that t1a mutants of lmp2 and b1 rescue presentation of uty [246] [247] [248] [249] [250] [251] [252] [253] [254] . also inhibition of cells with a b1-selective inhibitor preserves uty [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. an aspartate in position 7 of the uty 246-254 sequence wmhhnmdli is preferentially used as a cleavage site by lmp2-deficient but not half as frequently by lmp2-proficient immunoproteasomes. the generation of the uty 246-254 epitope relies on the replacement of the caspase-like activity of b1 by lmp2 because the b1 activity destroys the uty 246-254 epitope. this is the first example for the 'structural' requirement of lmp2 for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp2 and perhaps also lmp7 and mecl-1 exert their function in antigen processing. a. linnemann 1 , a. musiol 2 , r. lindner 1 1 hannover medical school, cell biology, hannover, germany, 2 hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf6) were overexpressed in 3t3 fibroblasts. we show that antibody-mediated oligomerization of mhc i in 3t3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf6: endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf6-regulated to a novel, arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv40 virus and since sv40 binding triggers mhc i oligomerization, this novel pathway may be involved in sv40 uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of 57 rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab3b, 3c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab3b and rab3c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab3b or 3c positive vesicles. furthermore, the rab3b, 3c positive compartment were colocalizd with a fraction of rab27a at a juxtaposition of phagosomes. together these data demonstrate that rab3b and rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over 90 % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd8+ t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf2a and bglf5 have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf1, has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens 2009). this represents a novel function for a virally-encoded gpcr. bilf1 is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf1 displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf1 is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf1 can hinder the recognition of virally-infected cells by cytotoxic cd8+ t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg1 is transcribed in thymus from both male and female individuals. finally, we detected the semg1 mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). deficiency in beta5i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta5i-deficient cells could be restored by treatment with d3t, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr2, 4 and 5, which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd4 + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd4 + t cells. the presence of exogenous tlr3 and tlr8 ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james 1 , i. bailey 1 , t. elliott 1 1 university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct26 colon carcinoma. this response is long lasting and mediated by both cd4 and cd8 t cells. importantly, the treg depleted ct26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a20, c26, bcl1, renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw11, which is h2-d d restricted. analysis of the generation of gsw11 in ct26 revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct26 cells substantially increased the amount of gsw11 present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct26 and immunised balb/c mice. greater than 80 % of mice injected with eraap kd ct26 were found to reject the tumour. analysis of t cell responses revealed the presence of gsw11-specific t cells, however, these responses were small (0.5-1 %). this compared to a much larger response to ct26 (˚5 %). preliminary results indicate that the majority of the t cell responses (non-gsw11-specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [1] . the di-allelic hla-a2 restricted minor histocompatibility antigen ha-1 locus codes for the highly immunogenic ha-1 his and the non-immunogenic ha-1 arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha-1 arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for ha-1 his and less than 5/ cell for ha-1 arg [2] . as ha-1 his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha-1 his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha-1 mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha-1 allelic peptides, we investigated the impact of the ha-1 his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha-1 his and ha-1 arg peptides were performed. moreover, the crystal structures of hla-a2 in complex with either ha-1 his , ha-1 arg or a ha-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the hla-a2/ha-1 peptide complexes. our results exclude a role for antigen processing in preventing ha-1 arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha-1 arg allele in the hla-a2 peptide binding groove [3] . they provide a rationale for the lack of ha-1 arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd8 t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd8 t cell response. on the other hand, immunosubunit expression is not essential for development of cd8 t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp7 (ib5) and mecl-1 (ib2) develop a variety of autoimmune responses, including a latent form of t1d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd8 but not cd4 t cells. a cd8 t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd8 t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd8 t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, 162 autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr3 and dr4. analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type 1 (hsv-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd4 + and cd8 + t cells in hsv-1 immunity have yet to be clearly elucidated. to better understand the role of hsv-1-specific cd4 + t cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein d of hsv-1. following flank infection, gd-specific cd4 + t cells were first detected in the draining brachial and axillary lymph nodes (ln) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gd-specific cd4 + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day 6 pi and peaked at day 9. while hsv-specific t cells were first observed in the draining ln at day 5 pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd4 + t cells was observed up to 23 days post-infection in the brachial ln. ex vivo analyses suggest that only cd11c + cells were involved in gd antigen presentation at days 2, 5 and 15 post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd8a + dcs can present the gd antigen to cd4 + t cells at day 2 pi, whereas by day 5 pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap1 and erap2 unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap1/erap2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp57. upon assembly of the heavy chain with b 2 m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp57. both crt and erp57 are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp57 to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp57 or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp57 must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp57 point mutant that fails to bind to cnx or crt was just as effective as wild type erp57 in normalizing rates of disulfide formation. we conclude that erp57 does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp57 point mutant, class i heavy chains, crt and the tapasin-erp57 disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp57 and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp57 have no effect on plc stability or class i surface expression, suggesting that erp57 plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb1*0405/dqb1*0401 haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr*0405 transgenic ab0 nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd4+ and cd8+ t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr*0405 transgenic mice (cd4+ t cell competent) develop aip even unprovoked, similar to ab0 nod mice (cd4+ t cell deficient), while hla-dr*0401, hla-dq8 or hla-dr*0405/dq8 (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr*0405 fails to protect from aip, likely due to defects in the induction of cd4+ regulatory t cells. our results identify hla-dr*0405 as a prominent risk factor for aip on the hla-drb1*0405/dqb1*0401 haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan 1 , c. britten 1 , h. overkleeft 2 , g. van der marel 2 , k. melief 1 , d. filippov 2 , f. ossendorp 1 1 leiden university medical center, section tumorimmunology, leiden, netherlands, 2 leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd8 and cd4 t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi 1,2 , x. hu 1,2 , a. kawanatachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping 8-mer and 10-mer epitopes (rypltfgwcf (nef138-10) and rypltfgw (nef138-8)) were found to be presented by hla-a*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y2f, y2w, t5c, f6l, w8r, f10r, f10y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef138-10 (wild type) and its four mostly common immune escape mutants (y2f, t5c, y2f&t5c, f6l), and also nef138-8 (wild type). we found that there was little difference between the nef138-10 (wild type) and nef138-10 (y2f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y2 or f2. interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (t5c) and nef138-10 (y2f&t5c), which may affect the binding of peptide and the recognition of t cell receptor. for nef138-10 (f6l), the side chain of l6 was more flexible compared to the nef138-10 (wild type). alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of f6 was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd8+ and cd4+ t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a2 restricted cmvpp65 derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. this pattern was seen for 5/9 epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to 2-5 log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b7 restricted cmvpp65 rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t2 cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd8+ t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp110, and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor-5 (fgf-5) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf-5 spliced peptide by cells transfected with mutant constructs encoding fgf-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b*4402, are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b*4402 and b*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us3 protein. to address this hypothesis, us3 was stably coexpressed in b lymphoblastoid cell lines expressing hla-b*4402 or hla-b*4405. in the presence of us3, the surface expression of hla-b*4402 was substantially reduced whereas hla-b*4405 expression was relatively unaffected. although us3 was able to form complexes with both hla class i allotypes, only hla-b*4402 was retained intracellularly in an immature form whereas hla-b*4405 was transported to the cell surface. accordingly, in the presence of us3, hla-b*4405, but not hla-b*4402, constitutively presented a hla-b44 restricted alloantigen to reporter t cells, suggesting that us3 binds hla-b*4405 without interfering with peptide loading. us3 has been reported by others to bind the plc but surprisingly we have not detected such us3-plc complexes in our system. rather, in the presence of us3 we identified a pool of class i molecules distinct from the plc and only present in us3 expressing cells, implying that us3 may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa 1 , b. seliger 1 1 martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il12p70. since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for 24h with the gold standard of maturation (tnfa, il1b, il6 and pge2) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase-3 (lap3) had a more than 20-fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap1 and erap2 and of the immunoproteasome subunits lmp2 and lmp10 was enhanced between 2 and 5-fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr4 ligand mpla in comparison to the tlr-3 and 7/8 ligand polyi:c and r848. these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd4 + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il-4 induced surface expression of mature mhc class ii molecules and cd86. when ifn-g/il-4 primed pcmc were used as antigen presenting cells for cd4 + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd69 up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b15-23 to specific cd8+ t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd8 + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th1 immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd8+ t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd8+ tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd8+ t cell clones, faure, 2009, eur j immunol 39 (2): 380-90). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd8+ t cells after their in vitro differentiation into dc, 6 days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv-1 genome contains arfs within gag, pol and env genes encoding for a panel of hla-b*07 restricted epitopes. qprsdthvf (q9vf/5d) is one such epitope but its parental epitope qprsnthvf (q9vf/5n) has a significant higher frequency among hiv-1 isolates. strikingly, q9vf/5d-or q9vf/5n-specific ctls recognize apcs infected with hiv strains encoding for q9vf/5d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad8 ) encoding for q9vf/5n do not activate ctl responses raising the possibility that q9vf/5n epitope is not presented by infected cells. we asked whether introducing mutations within q9vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q9vf/5n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q9vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q9vf mhc-i presentation. we performed in vitro proteasomal digestions of 28mer peptides encompassing q9vf/5d or q9vf/5n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q9vf/5n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q9vf/5n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q9vf/5d or q9vf/5n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q9vf/5d peptide. we cloned and sequenced hiv-1 genomes from the three donors. surprisingly, out of 20 hiv proviral genomes isolated from pbmcs of q9vf/5d reactive donors, we could not find any virus bearing the q9vf/5d sequence. the isolated hiv sequences either encoded for q9vf/5n or had a stop codon within the epitope. in contrast, viruses encoding for q9vf/5d were isolated from pbmcs of the q9vf/ 5d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv-1 seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch 1 , y. wang 1 , d.c. tscharke 1 1 the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd8 + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd8 + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd8 + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h-2 bxd f 1 mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f 1 progeny, with some peptides being almost equally immunogenic in f 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in f 1 mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f 1 mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f 1 mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd8 + t cells with a restricted vb usage in f 1 mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr2 agonist, s-[2,3-bispalmitoyiloxy-(2r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp-2), as an adjuvant for cross-priming against cellular and soluble antigens. malp-2 has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd8 + and cd8 -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr2/6 results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap1) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to 90 % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, 50 and 10 % as compared to control rma cells, were injected s. c. in the flank of c57bl/6 syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to 45 days after injection. all mice injected with rma clone with a 50 % level of eraap expression developed a tumor and died within 23 days after injection. surprisingly, any animal injected with rma clone with a 10 % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd4 + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd4+ t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor (207 bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd20 therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor (2.5 bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg4 levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb1*0701-restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with 20-mer synthetic fviii peptides to stain epitope-specific cd4+ cells.cd4+ t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a2 domain: fviii 405-424 , fviii 421-440 and fviii 653-672 , as well as the c1 domain peptide fviii 2093-2112 , but not any c2 domain peptides. the c1 domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost.85:123-33, 2005 ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni 1 , s. albini 1 , m.z. limongi 1 , l. cifaldi 1 , d. fruci 1 1 ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p50 nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap1 and erap2, we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap1, erap2 and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap1 and erap2. this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p65 nf-kb subunit, we demonstrated that nf-kb is involved in erap1 and erap2 expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap1 and erap2 and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap1, erap2 and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch 1 , s. tenzer 1 , h. schild 1 , e. von stebut-borschitz 1 1 uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c57bl/6 mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th1 immunity, whereas ifng secretion of both cd4 + th1 and cd8 + tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il-12-dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd8 + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd8 + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm7 (lmp7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c57bl/6 mice. in addition, ex vivo co-cultures with infected dc from either lmp7 -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc1 cell ifng secretion and the dc restimulatory capacity of cd8 + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd8 + t cells, isolated from low dose infected c57bl/6 wildtype or lmp7 -/mice, were not detected. in summary, our data indicate that despite the fact that cd8 responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd8 + t cells against l. major is independent of the lmp7 subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a20, generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd4 + t cell epitope (84-103) from the g1 domain of aggrecan 10,000 times more efficiently than non-specific b cells and over 100 times more efficiently than the macrophage line j774. however, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by j774. treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b27 is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b*2705, peptide loading is relatively independent of this chaperone. we analyzed the effect of b27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b27 subtypes. the association of b27 heavy chain with tapasin was analyzed in c1r cells transfected with hla-b27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta-1, which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me1 monoclonal antibody that recognizes b27properly folded b27/peptide complexes. the formation of fully assembled b27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc10, which recognizes mhc class i free heavy chains (hc), or with me1. maturation was analyzed by pulse-chase analysis, immunoprecipitation with me1 and treatment with endoglycosidase h (endo h). hla-b27 polymorphic positions other than 116, both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b27 subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd4 + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd8 + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c57bl/6 mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately 15 % of vacv global presentation in the context of h-2 b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd8+ and cd8-dc, which differ in their mhci antigen presentation capacities. cd8+ dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd8-dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd8+ and cd8-dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd8+ and cd8-dc was determined. surprisingly, cd8+ dc exhibit rapid surface mhci turnover compared to cd8-dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd8+ and cd8-dc were stabilized and no longer underwent rapid turnover. this suggests that cd8+ and cd8-dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd8+ and cd8-dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h-2kb loaded with the ova-derived peptide, siinfekl. cd8+ and cd8-dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd8+ dc, but not the cd8-dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd8+, and not cd8-dc. this data further validates the role for cd8+ dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp-1 cytosol using gelatinase b/mmp-9 as a model enzyme. in the first dimension, ion exchange chromatography separated the thp-1 proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp-9. in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp-1 cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp-9-cleaved or intact thp-1 cytosol. proliferation was assessed by measuring incorporated 3 hthymidine. results: 100-200 mmp-9 candidate substrates were isolated, of which 69 were identified, revealing many novel mmp-9 (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp-9 decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e3-19k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue 56 in hla-a11 and residue 93 in e3-19k are highly critical for association of both proteins. this defines a putative interaction surface between e3-19k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e3-19k and the class i antigen presentation pathway. moreover, because soluble e3-19k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap1 and tap2. tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap1 and tap2 were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap1 on tap2 was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap1 mutants suggested that the lack of tap1 protein expression is associated with a strong reduction of tap2 protein levels, which could be restored by tap1 gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap1 on tap2 different shrna plasmids specifically targeting tap1 or tap2, respectively, were stably transfected into constitutively tap1 and tap2 expressing hacat keratinocytes and colo794 melanoma cells. in both cell types the shrna-mediated tap1 and tap2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap1 caused not only an almost complete loss of tap1, but also a strong decrease of tap2 protein expression. in contrast, the tap2 knock down exhibited no influence on the tap1 expression. specific inhibition of the proteasome prevented the degradation of tap2 in the tap1 knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap1 on tap2 protein expression is not restricted to rare tap1 mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of uv. blister roofs were then collected before and 1, 4 and 10 days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd14 + cd36 + and of a macrophagic cd14 -cd36 + cell subsets that emerge 1 and 4 days post exposition, respectively. more importantly whereas classical cd1a hi cd207 + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd1a low cd207and cd1a low cd207 + that emerged 1 and 4 days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd86 and hladr. finally, 10 days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey 1 , e. reeves 1 , t. elliott 1 , e. james 1 1 university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd4+ cd25+ regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct26, stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct26-derived cross-protective antigen, gsw11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. this protein has previously been shown to encode ct26-specific cd8 and cd4 antigens, implicating it as a 'hot-spot' for ct26 tumour antigens. interestingly, we have identified a truncated version of gp70 which may be responsible for generation of gsw11. expression studies have revealed increased gp70 expression in ct26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw11) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p4, p6 and p9 tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp2 molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp85-99, bound to hla-dp2 with high affinity (10-30 nm). binding studies of mbp85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f91a, f92a and k93a) were affected, and only by a 20-50 fold reduction in affinity for hla-dp2. the observation that none of the substitutions resulted in a complete loss of binding to hla-dp2 indicates that 1) hla-dp2 binding to peptides does not depend on a large hydrophobic residue accomodated in p1, or 2) mbp85-99 can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp2 molecule binds the immunodominant epitope in ms, mbp85-99, possibly in more than one register. additional studies are required to resolve the hla-dp2 peptide binding properties, and to determine whether expression of hla-dp2 affects the disease course in ms patients. results: depletion of mncs for cd14+ cells abrogated the tg-induced cytokine production and proliferation of cd4+ t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g 98 % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd4+ t cell proliferation was significantly reduced in cd14/cd19-depleted mnc cultures, as compared to cultures depleted for cd14+ monocytes alone. the same applied to the production of il-2, il-6 and tnf-a. production of ifn-g and il-10 was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa 1,2 , p. pala 1 , g. miiro 3 , j. todd 3 , p. kaleebu 1,2 , n. imami 2 , f. gotch 2,3 1 mrc uganda, basic science, entebbe, uganda, 2 imperial college, london, united kingdom, 3 mrc uganda, entebbe, uganda background: hiv-1-specific t-cell responses are preserved in hiv-1 infected individuals with non-progressing hiv-1 disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv-1 for g 8 years, maintaining cd4+ t-cell counts g 500, and with minimal cd4+ decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: 17 art naï ve hiv-1 infected patients from the entebbe cohort in uganda were recruited and stratified by cd4+ t-cell count, cd4+ decline slopes, and time of enrolment, into 2 groups -10 ltnp and 7 rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd4 t-cell count, and ifn-g, il-2 and il-4 elispot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). peptides were based on consensus sequences of gag and nef from hiv-1 clades a1, a2 and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il-4 responses were significantly higher in the ltnp than in rp (p=0.02, ifn-g responses to gaga2 pool 1; p=0.04, il-4 responses to gaga1 pool 2). il-2 responses were low and not significantly different between ltnp and rp. there was a positive correlation between il-4 responses to gaga1 pool 2 and cd4 t-cell counts (r 2 =0.502, p=0.04), but no correlation between either il-4 or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv-1 specific responses were higher in ltnps than in rps confirming previous results. non-specific il-4 responses were high possibly reflecting baseline th2 responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her2/neuand her2/neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using 7-aad for dna analysis and specific antibodies directed against the proliferation marker ki-67, the m-phase specific phistone h3 as well as for the h-2l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her2/neuversus her2/neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g 0 /g 1 -, s-and g 2 /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g 0 /g 1 -phase, which continuously raised and peaked within the s-phase. however, h-2l d surface antigen expression was not altered in her2/neucells during cell cycle progression. in contrast to her2/neufibroblasts the her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h-2l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd207) and ccr6 were cultured from cd34+ stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd34+ derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il-4-dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mø). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to 45 min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mø than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mø have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, 1999) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, 1992, 1999) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x 0,0001), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, 2252 patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for 2207 of them. for 538 of these patients hla-drb1 data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p=0.02). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb-1a preparations, whereas no differences were shown for ifnb-1b. an association between hla-drb1*11 and nab-negativity was detected (p=0.028). the known associations between hla-drb1*15 and csf-positive ms and hla-drb1*04 and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb-1a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb-1b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart-1 and gp100 more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni 1 , s. lorenzi 1 , f. mattei 1 , f. spadaro 1 , l. gabriele 1 1 istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd8 t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd8a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd8 t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd8 t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg7 thymoma line, we show that type i ifn promote the ability of cd8a + dc to capture apoptotic eg7 cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd8a + dc and the persistence of apoptotic eg7-cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg7-derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd8a + dc. as a result, eg7-loaded dc become competent at inducing ot-i cd8 t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd8a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. (2). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd8+ dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam3csk4, and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd8+ dc but not cd8-dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm1, which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a549 human lung cancer cell line. methods: dm1 induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a549 cells. we examined the morphological change using optical microscope. and gfp-lc3 punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm1 was showed high cytotoxicity on various cancer cell line, especially a549 cells. dm1 treated a549 cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc3 and beclin-1 protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc3 was increased concentration and time-dependently. beclin-1 also was increased. and then, we examined gfp-lc3 punctation on a549/ gfp-lc3 stable cells using confocal. a549 cells were formed gfp punctuation after dm 1 treatment. to confirm these data, we measured cell death ratio using 3-methyladenine (3-ma), an autophagocytosis inhibitor. after 3-ma treatment, dm1 induced cell death was decreased comparing with dm1 treatment. conclusion: dm1 did not induce apoptotic cell death. but dm1 showed several characteristics of autophagic cell death. taken together, dm1 induced autophagocytosis on a549 cells. these finding indicated that therapeutic potential of dm1 by triggering autophagic cell death. s. b. rasmussen 1 , s. r. paludan 1 1 university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr9 recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv-1 infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv-1, we found that tlr9 dependent ifn regulatory factor 3 activation was abrogated. in addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr9 dependent recognition. in the ongoing project we are examining how hsv-1 triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko 1 , i. berberich 1 , gk 520 -immunomodulation 1 universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a1 and its human homologue bfl-1 are anti-apoptotic members of the bcl-2 family. a1 is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a1. unless the c-terminal ends of other bcl-2 proteins the tail of a1 does not function as a strong membrane anchor. nevertheless, the last amino acids of a1 are important for the protein. in fact, the c-terminus of a1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a1 undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl-2 factor bimel results in increased stability of a1. this is due to reduced ubiquitination of a1 after binding of bimel. we conclude that the cterminus of a1/bfl-1 serves as a docking site for e3 ubiquitin ligase(s) that control the stability of a1 by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e3 ubiquitin ligase that interacted with a1 in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs-1 in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs-1 is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs-1 significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs-1 was manifest not only by the suppression of jaks acting upstream of p38 mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak 1/2 activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs1 which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs-1 overexpressing cells. the results strongly suggest that socs-1 acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl12/sdf-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl12 effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u-87, and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p53 and fas-l expression was carried out by western-blot. results: cxcl12 induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p53 pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p53-ser46 levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after 24h of tnf-a treatment. conclusions: cxcl12 induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p53function/levels. s. y. demiroglu 1 , r. dressel 1 1 universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp70 does not protect against cell death mediated by ctl. acute overexpression of hsp70 can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res 63: 8212) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp70 on granzyme b-induced apoptosis. methods: hsp70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp70 induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. results: hsp70 did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp70 in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g1 peak measurements (p=0.01). in contrast, a partial protection of ge-tet cells was observed after acute hsp70 overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp70 does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood 102:1788) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk1034 and dr394/2-3. the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr1 and tnfr2, both belonging to the tnf receptor superfamily. tnfr1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr2-associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr2, resulting in depletion of traf2 from the cytoplasm. here we confirm that tnfr2 induced depletion of cytosolic traf2 by the use of tnfr1-and tnfr2-specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf2 in the alternative nfkb pathway, we show that tnfr2 induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr2 and membrane tnf. j. c. morales 1 , d. ruiz-magaña 1 , d. carranza 1 , c. ruiz-ruiz 1 1 universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase-8. in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r2 and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp36l1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp36l1 may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl-2 protein is an important cell survival protein at different stages of b cell development. bcl-2 mrna also contains are regions that could possibly be targeted by zfp36l1 protein. in the present study, we have tested the ability of zfp36l1 protein to bind to a bcl-2 mrna are probe and to degrade bcl-2 mrna. recombinant bacterially expressed zfp36l1 protein was shown to bind specifically to a bcl-2 are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp36l1 also provided evidence that endogenous zfp36l1 in b cells could bind to the bcl-2 are. in order to examine whether zfp36l1 binding to bcl-2 are resulted in bcl-2 mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp36l1 knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl-2 mrna was expressed in both wild-type and knockout mefs. the half-life of bcl-2 mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp36l1 does play a role in degradation of bcl-2 mrna. overall, our data are consistent with a role for zfp36l1 in degradation of bcl-2 mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp1. since lmp1 is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp1 function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi3-kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te1) and lymphocytic (nc5) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes1 or tes2 (transforming effectors site) derived from the c-terminal intracellular part of lmp1, in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp1's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase-8. using this nc5 tumorigenic model in scid mice, we showed that induction of the dn lmp1-tes2 prevent development of tumours and mouse death. these dominant negative derived from lmp1 could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs1 and socs3 differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38mapk activities, while socs3 promoted apoptosis with an increase in p38 activities. in contrast, both socs1 and socs3 display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs1 or socs3 induced a slight decrease in g1 or s phase cells and a prominent increase in g2/m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g1/s transition and g2/m arrest induced by socs1 or socs3. socs1 promoted dna damage-induced p21 induction and g2/m cyclin b expression, while socs3 induced decrease in g1 cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein 90 (hsp90) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp90 has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp90 can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp90 is released during necrotic cell death and proinflammatory effects of extracellular hsp90 have been observed. thus, we asked whether apoptozing cells cleave hsp90 during apoptosis or how hsp90 is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp90 protein content. we observed that hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp90 (hsp90 alpha and hsp90 beta) we could show that the 55 kda fragment is formed after degradation of the alpha isoform of hsp90. further, we were able to show, that hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp90 and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp90 was inhibited or if exogenous hsp90 was added. these results demonstrate that cleavage of hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc3-i is processed to lc3-ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf-7 gfp-lc3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl-2/beclin-1 control of autophagy. recently, jnk was shown to be necessary for beclin-1 upregulation, and jnk-mediated phosphorylation of bcl-2 is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf-7 cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol 3-angelate (pep005), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep005 provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb4 turned their response to pep005 from an increased to decreased rate of apoptosis. furthermore, our data show that pep005 inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl-1 and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p75, trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch11 and analysed by flow cytometry. expression of the caspase 8 inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch11 treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor 1 (tnf-r1), and tumor necrosis factor (tnf)-a receptor 2 (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd95 independent of its ligand fasl/cd95l. fas/cd95 is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl2-family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo 201, sw1116, lovo, caco-2, ht-29) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki-67, pcna, p53, bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl-2, bax, caspase 3, 6, 9, nfkb, parp1/89kda, tnfr1/cd120a and cd95/fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl-2, bax, nfkb, parp1 was significantly lower and expression of caspases, tnfr1 as well as percentage of cd95 cells significantly higher as compared with control group. caspase 3 expression was significantly higher as compared with nfkb, parp1 and tnfr1. in comparison to the standard nutrition preoperative immunonutrition increased bcl-2 and nfkb expressions and decreased caspases and parp1 expressions. in addition, we found a significant down-regulation of bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x 0.01). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x 0.007). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either la1 (10 9 cfu/day) or placebo, during 57 days prior to uv (2 × 1.5 med). blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p53). while a similar decrease of lc for both groups was observed on day 1 after uv exposure compared to placebo, la-1 group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd1a low cd207 -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il-10 and a tendency to inhibit il-6 was observed in la-1 group compared to placebo. on day 4, la-1 group presented a greater recruitment of early lc precursors and a trend to increase cd1a low cd207 + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il-6, tnfa, il-8, and il-10) was observed in la1 group compared to placebo. we show that la1 limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la1 supplementation for photoprotection at a lower dose (1 log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around e11.5. previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn1 in a nude (foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn1. if true, it should be possible to target this cell type by use of foxn1-promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either 19 or 82 glutamine residues under the control of foxn1 promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei 1,2 , y. toriumi 3 , k. kimura 4 1 european university viadrina, frankfurt (oder), germany, 2 shimane institute of health science, izumo, japan, 3 shimane university faculty of medicine, department of pediatrics, izumo, japan, 4 japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp2). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r=0.83, p x 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r=0.77, p x 0.05) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd4 (r=0.96, p=0.0001). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd4, within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd4 + cd25 + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox40l expressed on mcs and the constitutive expression of ox40 (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox40l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca 2+ responses and degranulation. the cross-talk between t reg cells and mcs through ox40-ox40l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th1 or th2 effector cells and the induction of antigen specific foxp3+ regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th2 cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th2 and treg subset, gata3 and foxp3 respectively, counter regulate each other (mantel y et al., 2008) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp3+ tregs and high gata3+ th2 cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost 100 million allergic patients are sensitized to the major birch pollen allergen bet v 1, which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v 1 recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v 1 molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g 90 %) of patients' ige binding to bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (p2) and aa 74-104 (p6) of bet v 1. cross-reactive ige epitopes between bet v 1 and related pollen allergens and plant food allergens involved primarily p2. p2 and p6 are not adjacent peptides in the bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of bet v 1. as determined by surface plasmon resonance, monoclonal antibody mab2 specific for p2 and mab12 specific for p6 showed high affinity, i. e., dissociation constants, k(d) = 8.35e-11 m and k(d) = 1.05e-9 m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p2 and anti-p6 antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v 1 allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th1 cells as well as tc1 cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th2 immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th1/tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd4 + and cd8 + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd4 (gk1.5) or anti-cd8 (53.6.72) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr-1 monoclonal antibody rb6-8c5 or monoclonal antibody nimp-r14. one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd8 + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd4 + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd8 + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd8 + t cells, cd4 + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd4 + or cd8 + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th1 and tc1 cells operative in the elicitation of airway inflammation. conclusions: robust type 1 immune responses, although highly effective in the counter-regulation of local th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h292. the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p 1, we were able to show that the major allergen of phleum pratense, phl p 1, although sharing molecular similarities with der p 1, does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p 1. chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il-6 and il-8 from nci-h292 cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p 1. in contrast to hdm extract grass pollen allergens induce the release of mcp-1 from respiratory epithelial cells, as well as moderate levels of il-12. detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr22) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, 3 distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): 7.2±5.7) and of il-13 (mean±sd: 1157±909 pg/ml), and reduced il-10 (mean±sd of il-10-spot forming units/2x10 5 cells (sfu): 912±510), compared to healthy subjects (3.4±2.7 si, p x 0.05; 355±396 pg/ml, p x 0.05; 1272±623 sfu, for proliferation, il-13 and il-10, respectively); children with non-ige mediated cma had a significant reduction of il-13 (192±362 pg/ml), compared to ige patients (p x 0.0004) and an increased, although not statistically significant, production of ifn-g (33.7±54.6 sfu) compared to control (9.5±9.5 sfu) and to ige-allergic patients (0.6±0.8 sfu). finally, tolerant patients showed reduced il-13 (636±1048 pg/ ml, p x 0.05) and proliferation (3.9±3.5 si), compared to acute ige-cma children. interestingly, the high level of il-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il-4 was undetectable in all patients even blocking the il4-receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th2-like response with il-13 and proliferation is dominant in ige-mediated cma patients; by contrast a th1-skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang 1 , b. chua 1 , f.c. lew 1 , a. ho 1 , k. wong 1 , k.l. wong 1 , d. m. kemeny 1 1 national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd4 th2 t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd8 t cells inhibits the induction of the th2 response. here we have investigated the effect of cd8 t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd8 t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of 3 airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd8 + t cells (36.7 %±4.1 % to 17.6 %±2.7 %). when ifn-gamma -/-ot-i cd8 t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for cd8 t cell ifn-gamma. cd11c + cd103 + cd11blung dcs from cd8 transferred mice secreted higher levels (500pg/ml) of il-12p70 following ex-vivo stimulation as compared with animals that were not given cd8 t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd8 t cells can subsequently divert the local lung environment to one that favors th1 immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in 8 % of patients with hiv infection. this reaction is strongly associated with hla-b*5701 and appears to be mediated by cd8+ t cells producing inflammatory cytokines. we show that cd8+t cell responses can be primed in vitro, in normal blood donors who are hla-b*5701+, but not in non-b*5701+ donors. cd8 t cells, but not cd4 t cells, are expanded by abacavir pulsed autologous apc over a13-day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b*5701. similar responses were detected in abacavirhypersensitive hlab*5701+ patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b*5701 since they were undetectable using apc expressing closely related hla-b57 or b58 allotypes. responses to apc expressing mutants of hla-b*5701 demonstrated a crucial role for residue 116. isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b*5701 triggering cd8 t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b*5701 may be relevant to the role of other disease-associated class i allotypes such as hla-b27 and b51. a. jenckel 1 , s. bulfone-paus 1 , n. föger 1 1 research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin1a (coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro1a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro1a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro1a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro1a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro1a during mast cell activation. cell fractionation experiments demonstrated that the association of coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro1a phosphorylation. a functional correlation between coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro1a. thus, coro1a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin1a (coro1a) deficient mice have demonstrated that coro1a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin1b (coro1b) is a closely related homolog of coro1a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro1b deficient mice and crossed them with coro1a deficient mice to obtain coro1a/coro1b double deficient mice. analysis of t lymphocytes from coro1a/coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro1a/coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro1a/1b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro1a/1b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn1 (also known as hacs1 or sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology 3 (sh3) domain and a predicted bipartite nuclear localization signal. the samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn1 in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn1 in all tested hematopoietic cell types. the highest expression level of samsn1 mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd4+ and cd8+ t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly1 (hacs2) and sly3 (sash1) -were expressed only at a very low level in mast cells. the high level of samsn1 mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn1 deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn1 deficient mast cells. in additional studies we are now investigating the effects of samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., 2008). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg1 abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st8siai-v; st6galnac i-iv, st3gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg1 abs. results: we observed that the expression of st3gal i, iii and v coding genes was similar in both hybridomas, while the st3gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg1. in addiction, the expression levels of st8sia and st6galnac genes in the hybridoma producer of anaphylactic igg1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg1. conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg1 abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h 2 cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after 2 times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il-5 and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf1 mediates the differentiation into th1 cells by activating multiple genes which are independently crucial for the development of naive t cells into th1 cells. because irf1 is expressed in many different tissues, it can be considered as a master switch factor for th1 cell differentiation. methods: here, we tested mice deficient in irf1 in the murine acute asthma model to evaluate its importance in this th2 cell-mediated disease. the protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype cd8+ t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf1 deficient mice with the help of adoptively transferred cd8+ t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by 3.5 fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. 1) were also significantly higher in irf1 deficient mice. conclusion: interferon regulatory factor 1 plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd8+ t cells. mechanistically, a potential counterregulation of asthma by th1 cells is not available in irf1 knockout mice. together with our previous report that irf1 represents a susceptibility gene for allergy in the human, our data highlight irf1 as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c57bl/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for 24hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk1-receptor and ppt1 mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd11c expressing apc substets characterized by cd4 and cd8 expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th-1 cytokine profile and increased levels of il-2 mrna. further the number of cd25 + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: 1) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase3, 2) asthma and ee are prototypic th2 diseases in which the cytokines il-4 and il-13 play a principal role through the activation of the transcription factor stat6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate stat6 by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat6, we analyzed its effect in the activation of stat6 by il-4 and il-13. we found that treatment of cells with ppis inhibited the ability of il-4 and il-13 to signal stat6 activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th2 mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied 12 asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd4 and cd25 high (treg)), that might inhibit the development of a th2 response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for 48 hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd69 or cd25 at 48 hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h4r. the level of intracellular ccl2 production in human lc was reduced after stimulation with h4r agonists and basal production could be restored when h4r was blocked with the specific antagonist jnj7777120. moreover histamine and a h4r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h4r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h4r in allergic skin diseases and encourage further exploration of the h4r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g1p3, an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g1p3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g1p3 which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd20 antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th2 to chronic th1-predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g 5,000; g 6,000; g 19,000 mg/dl, respectively). all patients underwent prior treatment with omalizumab for 6 months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up 1 year), ige levels decreased from 5,512 to 1,500 mg/dl. the other two patients are in the 3 and 1-months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd20 antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) 1 and th2 cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either 1 %dncb, 25 %tma or to vehicle alone for 0.5-6h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il-2 (mean=131pg/ml, n=12, p x 0.001), il-17 (mean=69pg/ml, n=12, p x 0.001) and il-1a (683pg/ml, n=12, p x 0.001) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant il-2 or of the known regulator of lc migration; il-1b. interleukin-1b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p x 0.001). in contrast, il-2 or control injections were without effect. however, il-2 administration caused an increase in cutaneous il-17 production (347pg/ml, n=12, p x 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). in addition, systemic treatment with anti-il-2 antibody failed to impact on lc migration provoked by dncb (33 % reduction; n=6, p x 0.001). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il-1a (987pg/ml, n=10, p x 0.001) and il-17 (248pg/ml, n=6, p x 0.01) expression was reduced to baseline levels by anti-il-2 treatment. these data demonstrate that il-2 is not involved in the regulation of lc migration, unlike il-1b and tnf-a. however, il-2 is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il-2 may influence lc and dermal dc maturation, via the expression of il-1a and il-17. allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey 2005), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p38-mapk-pathway related protein (p38prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p38 mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct-2005 -018681, www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, 15 % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il-1 receptor antagonist (il-1ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il-1b and of il-1-like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il-1-related genes by real-time pcr on mrna from blood cd14+ monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il-18, il-18bp, and caspase-1, both before and after therapy with kineret. il-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il-1b, tnfa, il-12) nor anti-inflammatory cytokines (il-10, tgfb) were detected. as expected, il-1ra was only detectable after therapy. il-18 was detectable in schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pm) and decreased after therapy (13.2 pm). the circulating il-18 inhibitor il-18bp was lower than in controls and not affected by therapy. thus, free il-18 levels were increased in schnitzler's patients as compared to controls (17.6 pm vs. 7.2 pm in controls) and decreased after therapy (9.9 pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il-1-related cytokines (il-1b, il-18), nor of the il-1/il-18-converting enzyme caspase-1 in blood monocytes. however, the high circulating levels of il-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around 20 % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr3 signal via myd88 to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd88 signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c57bl/6 myd88-deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg1-titers in ova-treated myd88-/-mice compared to wildtype mice, although the nacl-treated myd88-/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = 4) or from non-infested contra-lateral sites. expression of mip-1a, igf-1, mcp-1 and ip-10 genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd3, cd4, cd8, cd21, mhc class ii, and p46 than that of holsteins. lymphocytes expressing wc1 and cd21 antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x 0.05). infested skin of nelores contained significantly more message for mip-1a, igf-1, mcp-1 and ip-10 than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th2 response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd3+ t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il-1 plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa-1 in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam-1/lfa-1 binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th1 than a th2 local response pattern by virtue of il-1 secretion and icam-1/lfa-1 interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th2 like micromilieu in acute towards a th1 dominated milieu in chronic lesions. the genotyping of ccl4l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi07/0329). psoriasis is an inflammatory dermatosis with 2 % prevalence among caucasians. hla-cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in -308 and -238 positions. strong association was found between polymorphisms in the -238 region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b27, and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. hla-a -b -c -dr -dq alleles and tnf-238 and -308 snp were differentiated by pcr/ssp. analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. in the male group, the mean age at disease onset was 16,3 years. severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw*06 allele was positive in 10 cases (47,62 %), tnf 238 g/a genotype was found in 5 (23,8 %) and tnf 308 g/a in 4 (19,1 %). in the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. cw*06 allele was positive in 26 cases (54,2 %), tnf 238 g/a genotype was presented in 16 (33,3%) and tnf 308 g/a in 8 (16,6 %). severe disease was seen in male patients. there was no difference in frequency of cw*06 allele between male and female groups, but there was a tendency of significant difference in tnf 238 g/a genotype. we found that c57bl/6 mice were more susceptible than balb/c and dba/2 mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il-6 and mrp8/14, among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that 1)the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. 2) in particular, in many cases it is proved that there is a personal but also a family history of atopy. 3) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd86 we could observe dose dependent upregulation of programmed death ligand 1 (pdl-1) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a 60 year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since 35 years. in march 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters (120 mg) every second day. in april 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the auricle. the histological examination showed an hhv-8 positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th1 type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs1, socs3 and foxp3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for socs1, socs3 ve foxp3 mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd3, cd4, cd25, foxp3, cd4 + cd25 high , cd4 + foxp3 + . expression of socs3 and foxp-3 mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs1 was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd25, foxp3, cd4+cd25 high , cd4 + foxp3 + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs1, socs3 and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs1 and socs3 molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing fcs (exposed), and 43 healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than 0.05 was considered significant.neopterin level was significantly elevated in exposed (3,2±0,8ng/ml) compared to controls (1,8±1,3ng/ml; p=0,0001). moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls (81,9±22,7au vs 64,3±16,7au p=0,0001) and to silicosis patients (69,6±20,0au p=0,002). in contrast, igmcic was significantly elevated in silicosis than in exposed (70,2±18,8au vs 62,1±24,2au; p=0,03).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p=0,001 and 0,02 respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a*01, b*08, drb1*03 (these of good prognosis) *12/*13/*14/*15) or tbc (drb1*02/*05/*14/*16 and associations with dqa1*02/*03/*05) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a*01, b*08 and drb1*03 (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a*01, drb1*14. as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd4 + , cd25 ++ , foxp3 + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein 60 (hhsp60). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd4 + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il-17, il-6, il-8, tgf-b and ifn-g production prevailed, pointing to a th17/th1 weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp60. importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th17/th1 cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp60 might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices (1) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a 384 well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c3-complement, myeloid related protein 14 and two new proteins, integrin-ß4 and fibrinogen. data from more than 100 patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least 3 mice per group with distinct cytokine pattern: th1 (c57bl/10, c57bl/6) and th2 (balb/c). muscular injury was performed by injection of bupivacaine. both th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd3 bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at 4 dpi. balb/c mice showed increased collagen expression and decrease of mmp-9 activity associated with more mrna for tgf-b1. this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th1 cytokines) or myonecrosis and collagen deposition (th2 cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß1-adrenergic receptor (anti-ß1ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß1ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß1ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß1ec ii -specific rat monoclonal antibody (clone 13f6), and showed by elisa that it binds to the linearized ß1ec ii peptide. additionally, we confirmed with flow cytometry that 13f6 also binds the ß1ec ii in its native conformation, i. e. directly labeled circular ß1ec ii (dyl649-ß1ec ii ) peptide. moreover, we demonstrated activation of the ß1-adrenoreceptor by 13f6 using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß1ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß1ec ii -specific peptide-based therapy, by intravenously applying a circular ß1ec ii peptide in the dcm lewis rat model to neutralize the anti-ß1ec ii antibodies. while the peptide therapy strongly reduced the anti-ß1ec ii titers in the serum by up to 80 % and consecutively lead to clinical remission, elispot assays for the detection of ß1ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß1ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß1ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b3 or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca 614-629 epitope elicits autoreactive cd4 + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca 614-629 peptide. in a first step, hybridoma cells were generated by fusing bw5147 tcra -cd8lymphoma cells with myhca 614-629 -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle 1 , a. schwarting 1 , p.r. galle 1 1 university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase 3 (pr3), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr3-positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il-8. pr3-mrna from the murine cancer cell line wehi-274 was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr3-promoter in the second intron of the mouse pr3 gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr3 transcript and its promoter indicates that the murine pr3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th1 mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd1d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th2 cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n=13) or pbs (n=11) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr4 activation, in the presence/ absence of och. subsequently we transferred 1.5x10 6 mdcs (n=11) or och-primed mdcs (n=11) (3 times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a 70.6 % reduction in plaque size compared to mice treated with mdcs (p x 0.05). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p x 0.05) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with och-primed dcs (p x 0.05). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il-10. discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th2 phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c57bl/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c57bl/6 and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr3 regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg2c were isolated only from fcgriib deficient mice, but not from c57bl/6 control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box 1 protein (hmgb1), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb1. we investigated if hmgb1-containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb1 remains associated with ncs released from late apoptotic cells in vitro. hmgb1-ncs complexes were detected also in the blood of patients with sle. hmgb1 containing ncs from apoptotic cells induced secretion of il-b, il-6, il-10, and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd88 and toll-like receptor 2. neither hmgb1-free ncs from living cells nor from apoptotic hmgb1-or hmgb1/2-deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb1 activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il-10 release by macrophages. immunizations with hmgb1-containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr2-dependent manner. in conclusion, hmgb1 in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher 1 , k.p. hoefig 1 , e. kremmer 1 , v. heissmeyer 1 stretches and helps in the selection of the correct splicing borders. a allele of (r61h) creates a strong binding site for a splicing enhancer protein srp40 according to bioinformatics. our findings indicate that, the putative branch point, r61h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank1. finally, we believe that bank1 delta 2 protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta 2 protein may contribute to the observed reduction in sle susceptibility. s. beermann 1 , r. seifert 1 , d. neumann 1 1 hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h 1 receptor (h 1 r), h 2 r, h 3 r, and h 4 r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd3 antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or 4-methylhistamine, a h 4 r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and 4-methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c57bl/6 mice induced by either a-cd3 antibodies or cpg-odn. this histamine effect was completely inhibited by the h 2 r-specific antagonist famotidine, while h 4 r-, h 1 r-, and h 3 /h 4 r-selective antagonists had no or only moderate effects. interestingly, the h 4 r-selective reagent jnj7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd3 induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h 4 r, jnj7777120 may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h 2 r and, to a much lesser extend, the h 4 r. using this assay system, cells obtained from control c57bl/6 mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of 20 ng/ml resolvin e1(rve1) on the release of tgfb, il-6 and il-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (pma) (1nm), and the combination of pma and ionomycine (3 mg/ml) for 72 hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = 0.010) and sjögren's (p=0.017) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve1 significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p=0.041) or pma+ionomycin (p=0.021), however rve1 was ineffective at reducing tgfb release in the sle and ss patients. rve1 caused a non-significant decrease in il-6 release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il-17 was not significantly modified by rve1 in any of the groups tested. the release of tgfb by 20 ng/ml of rve1 can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve1 tested, il-6 and il-17 release were not significantly affected in healthy or autoimmune patients. omega-3 fatty acid derived rve1 may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis 1 , a.k. tsirogianni 1 , m. herrmann 2 , h. m. moutsopoulos 1 , m.n. manoussakis 1 1 university of athens, dpt pathophysiology, athens, greece, 2 university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included 29 with primary ss (american-european criteria 2002) and 14 with sle (acr criteria 1997). age-and sex-matched healthy blood donors to the ss and sle groups (13 donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd68-staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p=0.0001). in ss patients, defective mb-phagocytosis involved only monocytes (p x 0.0001) and significantly correlated with the presence of extraglandular manifestations (p=0.02). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x 0.0001) and of ss (p=0.001). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: 30 healthy persons have been included in our study and 71 sle patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b 2glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in 36,6 %, aphl of igg class -in 45,1 % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x 2 =6,4; p x 0,02). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x 2 =3,9; p x 0,05). the increased levels of anti-ada were revealed in 11 of 18 women with hnp, and the combination of anti-ada and aphl (9/18) was found out more often than isolated anti-ada (2/18, x 2 =6,5; p x 0,02) or isolated aphl (3/18, x 2 =4,5; p x 0,05). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc1 and mdc2, and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc1, mdc2 and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from 31 pss patients fulfilling the american european consensus group criteria (aecc) and 28 gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p=0,0002) and mdc2 (p x 0,0001) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd5+ b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin 21 (il-21) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd5+ b cells and increased il-21 levels. we therefore hypothesized that il-21 may have similar biological effects on cd5+ b cells in autoimmune diseases. here we demonstrate that the amount of il-21 in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to 14 % of cd5+ b cells in sle individuals expressed grb. in-vitro experiments revealed that il-21 was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor 9. importantly, il-21 significantly decreased the cd5+/cd5-b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd5+ b cell death. these results suggest that il-21-induced grb may play a regulatory role for cd5+ b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il-21 and grb levels in sle and showing that il-21 reduces the cd5+/ cd5-b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il-21 may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il-21 in sle and other autoimmune diseases. r. de palma 1 , e. d'aiuto 1 , s. vettori 1 , g. abbate 1 , g. valentini 1 1 second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd4+ t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il-1beta, il-6 and il-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed 169 igg antibodies from single facs purified cd19+cd27+cd38+cd138+ bone marrow plasma cells of 3 patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran 1 , e. moazemi godarzi 1 , e. kamali sarvestani 1 , e. aflaki 1 1 shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin 6 (il-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il-6 gene at positions -572 g/c and -174 g/c have been described that are key regulators of il-6 gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il-6 gene polymorphism at -174 position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the -572 position significantly differed in sle patients and controls. at this position gg genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p x 0.014). the frequency of -572 g allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x 0.04). at -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. in conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to sle and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained 161 patients with ibd (89 with cd, 41 with uc, 31 with gluten sensitive enteropathy, gse) and 33 healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of 7 asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd4-ve cd8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd4(+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling-1 (rgs-1) protein, we have now found substantial over-expression (10-100 fold) of rgs-1 in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs-1 decreases primary t cell responses to cxcl12 and ccl19, strongly implying that it may regulate t cell localisation. thus, rgs-1 may be a novel target for modulating t cells in ibd, consistent with which snps in rgs-1 have been associated with both coeliac disease and type 1 diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c57bl/6j mice were divided in control (c) and experimental (e) groups. c1-c3 received peanut protein immunization, animals of the control groups c4 were sham immunized, and control group c5 received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a 30 day peanut containing cd, the experimental group was divided into 5 groups (e1-e5). ova feeding began 7 days prior cd (e1) on day 0 (e2), 7 (e3), 14 (e4) and 21 (e5) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e1) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive il1-b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il1-b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (dss) in the drinking water for 8 days to wildtype mice. when ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl 1 , n. schneiderhan-marra 1 , m. blum 2 , g. stein 2 , m. schmolz 2 , t. joos 1 1 nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, 2 edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [1] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h 1-associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h 2-like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il-17 production and t h 17 polarization and decreases recruitment of cd4 + cd25 + foxp3 + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd11c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd11c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h 17 and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen 1,2 , g. van assche 3 , p. rutgeerts 3 , a. liston 1 , j. l. ceuppens 2 1 k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, 2 k. u. leuven, experimental immunology lab, leuven, belgium, 3 k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp-1 allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced 3 types of colitis dss (th1/th17), tnbs (th1) and oxazolone (th2) in hp ko mice. neutralizing anti-il-17 mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th17/th1 cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. 1) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; 2) in dss but not in oxa colitis mice, il-17, ifn-g, tgf-b and il-6 were significantly increased (p x 0.01, dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x 0.05, ko vs wt). in tnbs colitis, we found elevated il-12 and ifn-g (p x 0.01, tnbs vs control). although not significant, il-17 was also somewhat upregulated; 3) in dss colitis we observed that il-23 enhanced differentiated th17 cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il-6 and il-21, more mln-t cells from hpko mice differentiated into th17 cells; 4) anti-il-17 mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il-17 showed reduced il-6, il-17 and ifn-g in both mln and lp (p x 0.05, anti-il-17 vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il-17, supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann 1 , g. talabér 1 , g. süt" o 2 , p. németh 1 , t. berki 1 1 university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, 2 university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that 2.2 million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd4+cd25hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th1 and th2 cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd4+ and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd4+ inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells (74.5 ± 3.5 %, mean ± sem), while cd4+ inkt cells ratio was moderate (25.4 ± 3.5 %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: 38.0 ± 7.1 %; cd: 34.0 ± 5.2 %, mean ± sem), while the percentage of cd4+ inkt cells was elevated (uc: 62.0 ± 7.1 %; cd: 65.9 ± 5.2 %, mean ± sem) in both disease groups. proportions of foxp3+ treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b1 gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b1 adenovirus (tgf-b1-exo). the t cell inhibitory function of tgf-b1-exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b1-exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b1-exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd4 + foxp3 + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b1-exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b1-exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd4 + foxp3 + regulatory t cells (treg) revealed that the relative numbers of cd4 + foxp3 + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b1 gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd4 + foxp3 + treg. tgf-b1-exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd8+ t-cells was similar in the four vaccinees as observed by ifng, il-2 production-profiles. however, the killing capacity of expanded cd8+ t-cells was distinct as observed by the competence to kill ns3-peptide presenting transfectants in vitro. as depicted in figure 1 , cd8+ t-cells cells from both vac1 (cleared ) and vac2 (chronic) produced il-2 and ifng after stimulation with ns3-peptide59. however, specific killing of the peptide loaded transfectants was only observed in vac 1, who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure 1 ] killing of ns3 peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd8+ t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd8 t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns3 from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr4-expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns3 might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns3 or eda-ns3 under the control of cmv promoter were prepared. recombinant ns3 and the fusion protein eda-ns3 were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd40 agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns3 rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns3. results: immunization of mice with the plasmids expressing eda-ns3, but not ns3 alone, induced strong t cell responses against the main hla-a2 restricted cytotoxic t cell determinants from ns3. the recombinant eda-ns3 fusion protein, but not ns3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp-1 monocyte cell line. immunization of hhd mice with eda-ns3 fusion protein induced both cd4+ and cd8+ t cell responses against ns3 and, when immunized with poly(i:c) and anti-cd40 antibodies, was able to down-regulate the intrahepatic expression of hcv-ns3 rna. the recombinant eda-ns3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even 25 years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e2 protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd207+cd1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd8+ responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to 3 years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd1a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd8+ t cells by mature dcs transduced with melan-a-recombinant human coronavirus 229e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin 2 (il2) and interleukin 15 (il15) will be involved, and fms-like tyrosin kinase 3 ligand (flt3l) will be expressed to modulate dendritic cells. il2 is known to enhance early t cell expansion and limits t cell overshoot, whereaes il15 guarantees survival of high affinity t cells during memory phase. on the other hand flt3l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik 1 , ag waisman 1 uniklinik mainz, 1. med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd8 t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il-10 production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd8 t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response 7 days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il-10 produced by b cells by conditional deletion of the il-10 gene in these cells. we found that b cell secreted il-10 has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb490 to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for 10 days in the presence of il-2 and il-7 with exposure to overlapping peptide pools of latent ebna-1 and lytic bzlf-1 antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd3, cd4, cd8, ccr7, cd45ra, il-2, tnfa, ifng and cd107a. the majority of ex vivo ebv-reactive cd4+ t cells as well as ebna-1-reactive cd8+ t cells were il-2 and tnfa-producing memory cells, the later being more frequent in bone marrow (cd4+, median, ebna-1: bm 0.69 %;pb 0.12 %; bzlf-1: bm 0.37 %;pb 0.01 %, p=0.039). after in vitro expansion a major subset of ebv-specific cd4+ and cd8+t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd4+ and cd8+ t cells retained, however, a tnfa single, tnfa/il-2 or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd4+ and cd8+ t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr7/cd45ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il-2 delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd4 + and cd8 + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd154 or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd4 + and cd8 + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd8 + and cd4 + t cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to 29-90 % in the t cell lines and the absolute numbers of both hexon-specific cd4+ and cd8+ t cells were 2 to 3 log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd8 + and cd4 + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype 5 (ad5) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad5 vaccine vectors expressing whv core protein was first examinated in c57bl6 mice. groups of mice were immunized with a dna prime-ad5 boost regimen or with dna and ad5 alone. ad5 was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd8+ t cell responses against peptide pools 1 and 3 in spleens of dna and dna-ad5 immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd8+ t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad5 very weak cd8+ t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad5 level of antibodies did not change. those antibodies were only igg2a subclass what indicates th1 t helper type of response. ad5-immunized mice had over 3-fold lower level of anti-whc: both igg2a and igg1 subtypes were detected. the weak response induced by ad5 may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m2e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal 2007) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m2e serum ab titers against peptide and native m2e. this correlated with a large number of m2-specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h1n1), which contains a variant m2e-sequence different in 3 amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x31 (h3n2) and the highly pathogenic pr/8 (h1n1) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m2e as a potential "universal" influenza vaccine. this research is supported by a nih t32 fellowship ca09171-32, the nih grant ai 46457 and a grant from the commonwealth of pa. l. yu 1 1 zhejiang university, zhangzhou, china interleukin-18 (il-18) is a cytokine produced by stimulated mononuclear macrophage system. in this report, 18-day-old chicken embryos were vaccined with the plasmid dna (pci-chil-18) encoding chicken interleukin-18 and the copy numbers of chil-18 in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil-18 could accelerate high concentrations of chil-18 in nonage peripheral blood, accelerate high expression of chil-18 in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil-18 could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil-18 (p x 0.05). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd8 cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on 12 volunteers and a phase i study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd8 cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv-1 neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) 120 and the transmembrane protein gp41. two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (v3) is recognized by the human monoclonal antibody 447-52d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies 4e10 and 2f5. in order to elicit anti-hiv-1 neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp120-v3 region or the gp41-mper. the vlps are based on the acyltransferase component (e2 chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e2 chain self-assembles into a 24 nm protein scaffold resembling a vlp and that contains 60 copies of e2. efficient display and refolding of the v3 and mper regions in e2 vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for e2. the priming and boosting with a combination of vlps and specific hiv-1 envelope dna were used to immunize mice and rabbits. results: the v3-e2 and mper-e2 vlps were purified as stable 60mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of 447-52d, 4e10 and 2f5 antibodies to hiv-e2 monomers was confirmed by western blot. we obtained high titers of hiv-1 gp140-specific antibodies in mice immunized with a combination of vlps plus dna (hiv-1 sf162 gp160). these antibodies generated a low (20 %-27 %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e2 vlps plus dna elicited a low titer of hiv-1 gp140 specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e2 vlps are able to display antigenic determinants of hiv and to induce high titers of hiv-1-specific antibodies. the e2 vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h5n1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h5n1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h5n1 whole virus wild-type vaccine, could induce an immune response and protect mice against h5n1 influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h5n1 antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h5n1 virus. the protective efficacy of the trivalent vaccine derived mainly from the h1n1 component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h5n1 virus, suggesting that antibodies are the main contributor to protection. h1n1 specific serum did not inhibit neuraminidase activity of h5n1 virus suggesting that protection was not mediated by neuraminidase n1-specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h5n1 whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h5n1 vaccine enhanced anti-h5n1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h5n1 vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h5n1 candidate vaccine. [1] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of 335 children in the first grade of primary school in the area of central and west macedonia. there were 234 greek and 101 foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following 1) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%. 2) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). 3) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type 1 diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om-85, a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd28 -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om-85. cytokine secretion, activation and proliferation of b cells and foxp3+ tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr2, tlr4 or the myd88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om-85. two synthetic tlr4 agonists used as adjuvant in human (om-174-dp and om-197-mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om-85-induced protection of diabetes required natural tregs, as nod-cd28 -/mice were not protected. remarkably, om-85 activated b cells and not t cells, promoting their proliferation and il-10 secretion, two phenomena that were tlr4-and myd88-dependent. om-174-dp and om-197-mp-ac two synthetic murine tlr4 agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd4 + cd25 + foxp3 + t cells and the proliferation of il-10 secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr4 signaling in the protective effect of om-85 on development of diabetes and show that two other tlr4 agonists induce proliferation of b cells and their secretion of il-10 as well as stimulation of regulatory cd4 + cd25 + foxp3 + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit 1 , a. legat 1 , s. thomas 1 , m. van mechelen 2 , p. hermand 2 , m. goldman 1 1 institute for medical immunology/université libre de bruxelles, gosselies, belgium, 2 glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide 4-phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor 4 (tlr4). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd14 (scd14) for its tlr4 ligand activity, we investigated the involvement of both forms of cd14 in the responses elicited by crx-527, a prototypical agp. first, we found that crx-527 efficiently induces nf-kb and irf3 activation in hek cells transfected with tlr4 and md-2 genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd14. likewise, crx-527 efficiently induces the synthesis of nf-kb and irf-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd14 prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd14 respond to crx-527 but not to lps in serum-free medium. the addition of the soluble form of cd14 did not modify the levels of il12 and tnf produced by crx-527 stimulated dc but increased the levels of interferon-b (ifn-b). when scd14 was added to hek cells expressing tlr4/md-2, nf-kb activity was not modified but irf3 activity was increased in a dose-dependent manner in response to crx-527. we will further compare the responses induced by crx-527 in wild-type and cd14 deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h-2d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled 14 and 28 days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin-15 gene polymorph isms (c267t, g367a, c13687a and a14035t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype infg+/cd69-, p=0.02) and activated (subtype infg+/cd69+, p=0.028) cbmc, producing ifn-g, as compared with newborns from urban mothers (n=79) exposure to pets and the risk of allergic symptoms during the first 2 years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc09/16 to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6) tb has the highest diagnostic value in children n 4 years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp-10 antigen is more sensitive for detection of tb-specific t cells compared to esat-6 antigen. 4. in children with tst 5-14 mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr2 and tlr4 in the development of spontaneous lupus disease by creating tlr2 or tlr4 deficient c57bl/6 lpr/lpr mice. methods: tlr2 and tlr4 deficient lupus prone mice have been generated by crossing c57/bl6-tlr2 -/-or c57/bl6-tlr4 -/-mice with c57/bl6 lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr2 or tlr4 deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr2 or tlr4 in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr4 deficient mice suggesting an important role of tlr4 in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr4 also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr4 pc14/13 expression of full length mcl-1 and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl-1, an anti-apoptotic protein. a splice variant of mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl-1 (mcl-1l) and its splice variant (mcl-1s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl-1. method: neutrophils were isolated from children (diagnosed x 17 years) with jsle (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, jsle serum alone or with jsle serum plus 20pg/ml gm-csf. quantitative real time pcr was used to assess mcl-1l and mcl-1s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl-1s to mcl-1l was also higher in jsle patients compared to controls (p x 0.05). the addition of gm-csf to jsle serum was associated with an increase in mcl-1l (1.66 ± 0.31) and a decrease in mcl-1s (2.56 ± 1.1) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl-1s, and less anti-apoptotic full length mcl-1 cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd4 + cyld ex7/8 t cells were transferred into rag1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex7/8 cd4 + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex7/8 mice are capable to control inflammatory responses. for this purpose cd4 + cd25 + cells were co-transferred with naïve wt cd4 + t cells into rag1 -/-recipients. interestingly, rag1 -/-recipients of cyld ex7/8 t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc17/16 the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a*03 in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb1*13, *01 and *0103 alleles, and a decreased allele frequency of drb1*15 in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb1*0103 is associated with the development of severe disease and positive association of cd with drb3*0301 and drb1*13. indeed, drb1*15 was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb1*07 frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb1*04 in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb1*15 were strongly increased with respect to cd and controls. however, the frequency of drb1*04 was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb1*15 is associated with uc in european, north american, japanese and korean populations methods: a total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 x 5 years of age, 12 5-10 years of age and 2 g 10 years of age) and 23 girls (10 x 5 years of age, 6 5-10 years of age and 7 g 10 years of age). pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). the lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis g 20.000 in 9 cases (17,6 %). the most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and 3) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il-13ra2, previously believed to be a decoy for il-13 only, is able to transmit a signal via il-13. our results support this and may suggest that il-13/ il-13ra2 signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il-4ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il-13 or il-13ra2 and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd4 + cd25 -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il-6 and il-1b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd4 + cd25 -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi-98, l. acidophilus ncfm tm and b. bifidum bi-504) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi-98 strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il-6 or il-1b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 1 1 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed 21 pediatric cd patients (13 active, 8 remission), 24 pediatric uc patients (17 active, 7 remission), and 37 age-matched non-ibd controls. nkg2d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x 0.05 was considered significant. results: nkg2d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd3+cd56+ nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg2d ligands on circulating monocytes is also observed. the dramatic increase of nkg2d+ lymphocytes, and the strong upregulation of nkg2d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg2d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc17/26 peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp3+ t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd4+ t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp7 knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than 200 million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec-205 antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa 1-191) or hcv-ns3 (aa 1027-1218) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns3 and core we successfully optimized culture and protein purification conditions. briefly, ns3 was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec-205 to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec-205/ hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd01/2 mva-nef vaccination induces polyfunctional cd4 t-cells and increases the proliferative capacity of cd8 t-cells in hiv-1 infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv-1 protein nef (mva-nef) was safe in hiv-1 infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd4 t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il-2 and mip-1b, the expression of the activation marker cd154 and the differentiation marker cd45ra in nef-specific cd4 and cd8 t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il-2 and mip-1b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd4 t-cell response and a significant increase of polyfunctional nef-specific cd4 t-cells, simultaneously expressing ifn-g, il-2 and cd154. using the standard ics no increase of nef-specific cd8 t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd8 t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il-2 and cd154 expressing cd4 t-cells and the increase of proliferating cd8 t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd4 t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd8 t-cells in hiv-1 infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv-1 mediated membrane fusion and p24-detected replication. db81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with 25mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd4 + and cd8 + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns4 (1677-1756 aa) and rns5a (2061-2302 aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/2j mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns4 protein. immunization with rns5a-immunomax conjugate and rns5 in cfa (1.4 mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns5a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns5a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd01/21 degree of cross-genotype reactivity of hcv-specific cd8 t cells directed against ns3 the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd8 t cell response targeting hcv genotype 1 (gt1) and genotype 3 (gt3) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: 53 subjects (17 with gt1, 22 with gt3 and 14 anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns3 from gt1 or gt3. individual reactive peptides and the degree of cross-reactivity between the gt1 and gt3 variants were determined by ics. complete ns3 is sequenced from all viremic patients pd01/22 anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes 1, 4, 6 or 9 suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. decreased splenic viral load after ifna1 treatment correlated with an expansion of activated fv-specific cd8 + t cells and nk cells in the spleen, whereas in ifna4-and ifna9-treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna2, 5 and 11 are under investigation pd01/23 elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd8 t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd8 t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd8 t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a*0201-or k b -restricted cd8 t cell responses by these dna vaccines differed. k b /ova 257-264 -and k b /s 190-197 -specific cd8 t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd4 + and cd8 + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of 5 peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, 3 hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd8 + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd8 + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il-12-exposed cd8 + t cells showed at least five-fold increase of survival rate in vivo than il-2-exposed cd8 + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd8 + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il-12-exposed cd8 + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd8 + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il-12 and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x 0.01) . there was a significant elevation of t ada and ada1 activities in iga deficient patients as compared to healthy individuals (p x 0.01) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd01/61 hiv-1 sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after 2-6 weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in 12 out of 17 patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, 10-day-old ross 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/79 strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/79 and h120 strains, via the drinking water at 10 days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd4 t cells from 25 patients with active tb and 28 patients with successfully treated tb were analysed for simultaneous expression of ifng and il2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with ppd. moreover, cells were stimulated with esat-6 and cfp-10 receiver operator characteristics analysis revealed that a percentage of ifng /il2 dual positive cells x 56 % served as an accurate marker for active tb patients (specificity 100 %, sensitivity 65 %), while frequencies g 56 % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd03/7 necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in 1993 and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, 12 out of 14 children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently 6.12 to 100 000 inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd03/8 novel analogues of thalidomide inhibit cd80 expression and production of tnf-a, il-12, ifn-g, cxcl-9 this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta1-dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta1r7k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd80 and cd86. these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il-1a and il-1b, while il-6 levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il-1b production. using bmdc from nlrp3 -/-mice we examined il-1b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il-1b production is dependent on nlrp3. collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho-1 expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from 20 % in wild type (hmox1 +/+ ) mice to 87 % in ho 1 deficient (hmox1 -/-) mice. hmox1 -/-but not hmox1 +/+ mice developed end-stage multiorgan failure. mortality of hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h 2 o 2 or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb1. similarly, circulating and cytoplasmic hmgb1 was increased in hmox1 -/-relative to hmox1 +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b-1,2-linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and 2-4 weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were 7.7 and 9.4 iu/ml and those of dtwp-pasteur were 8.2 and 8.6 iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were 30.2 eu/ml for dtwp-local and 47.9 eu/ml for dtwp-pasteur vaccines, respectively (p x 0.001). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd05/18 united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b:2 , a gram-negative coccobacillus. jrmt12, an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 cfu of jrmt12. a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b:2 on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for 1 h before adding concanavalin-a (cona) pd06 -vaccination and immunotherapy against parasitic diseases pd06/1 evaluation of simian adenoviral vector adch63 expressing msp-1 as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype 5 (adhu5) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch63 is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp-1 and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm128 while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho-1 expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox1 locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho-1 is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp63 expression was confirmed by sds-page and elisa using monoclonal antibody against gp63. discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a5-180recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a5-180recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at -20°c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and 2 seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r=0.216, p=0.641) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il-17 on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il-17 to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il-17 and il-17-related inhibition of cfu-e growth were dependent on p38 mapk activity, since the p38 mapk inhibitor, sb203580, markedly downregulated il-17-induced activation of nos and reversed the growth inhibitory effects of il-17 on cfu-e. the in vivo stimulating effect of il-17 on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il-17 effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il-17 acts on bone marrow cells and also revealed a link between the il-17, no and hematopoiesis. further studies on il-17-mediated induction of both inos and enos methods: a total of 1785 blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of 68 samples (3,8 %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x 100 iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had 100 iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 iu/l anti-hbs in all the blood units, which also goes for our study pd14/20 neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen 1,2 1 national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa-1a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa-1a iggs we monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. in 11 of them all was diagnosed for the first time. 1 subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. 2005, nature 435, 452-8). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 3'utr of icos, in which a 47bp sequence, containing a possible mir-101 binding site, was sufficient (yu et al. 2007, nature, 450, 299-303). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. 2000, j biol chem 275, 33655-62). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia-1-and ago2-deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r01 ai 42269. we have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw 264.7 macrophages. in this report, we investigated the effect of 6-hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin-8 (il-8), monocyte chemotactic protein-1 (mcp-1) in ht-29 human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed 6-hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated 6-hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. 6-hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p65. in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of 6-hma significantly reduced the mrna levels of il-8 and mcp-1 stimulated by tumor necrosis factor-a (tnf-a) in the ht-29 cells. pretreatment of 6-hma also significantly blocked the ixba degradation and nf-xb p65 nuclear translocation stimulated by tnf-a in the ht-29 cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of 6-hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, 6-hma could be new potential therapeutic agent for inflammatory bowel disease.cd4 serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd4 igg immune response in hiv-1-exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd4 domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human cd4, in the absence of the target cells expressing the co-receptor ccr5. the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (ccr5 expressing) cells. thus, in the absence of ccr5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv-1 infections. a conventional protocol for the generation of monoclonal antibodies was used. db-81 (igg1, x), one of the anti-cd4 antibodies obtained, recognized preferentially cd4 complexed to gp120, as compared to cd4 alone, not competed for the gp120 binding site on cd4 and was specific for the second extracellular domain of cd4. g. röder 1 , l. geironson 2 , a. darabi 3 , m. harndahl 1 , c. schafer-nielsen 4 , k. skjödt 5 , s. buus 1 , k. paulsson 2 1 copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, 2 lund university, immunology bmc d14, lund, sweden, 3 lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, 4 schafer-n, copenhagen, denmark, 5 department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first 87 n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a*0201. to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn 1-87 /80 , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn /80 was demonstrated to be located on tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first 87 n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd8+ t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region 301-498 of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b*2705-restricted np-flu epitope (aa 383-391) was replaced by hla-b27 or hla-a2-restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard 51 cr release assay and through the ifn-g production. results: using hla-b27 or hla-a2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a2 molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a2 restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b27 as a remarkable hla-class i molecule that, differently from hla-a2, can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b27 molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv1. consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p17-gag and p24 region was determined in vitro. 25mer peptides were digested with i20s and c20 proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv-1 p17-and p24-gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers 1 , g. koster 1 , o. landsverk 1 , f. skjeldal 1 , o. bakke 1 1 university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi3 kinases and thus ptdins(3)p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier 1 , l. visvabharathy 1 , j. fu 1 1 university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e3-19k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e3-19k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type 2 e3-19k and class i molecules.results: we showed that e3-19k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a11 molecules, with the mature form being more tightly associated. we also provided evidence that e3-19k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e3-19k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e3-19k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il-12, il-6, tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands 2 and 4, but also with the t-celldependent stimulus cd40l (cd154) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd40, cd83, and cd86 was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p38 and erk-1/2. strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd4+ t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th2 (il-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th1 (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th1 / th2 balance and to identify potential t cell target antigens (ags), we analyzed circulating cd4+ t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il-4 expression in peripheral blood cd3+cd4+ lymphocytes were measured in 9 ccs patients and 7 healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il-4/ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il-4 and lower ifn-g intracellular expression were detected in cd4+ t cells from css patients, as compared to hs (il-4: 1.3±0.3 % vs. 0.50±0.21 %, p x 0.05; ifn-g: 14.2±4.5 % vs. 27.0±4.8 %, p x 0.025). similar results were obtained by elisa (il-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p x 0.05; ifn-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p x 0.05). elispot counts of hexavalent vaccine-stimulated cd4+ cells were positive for il-4 in 4/5 (80 %) css patients and in 5/5 (100 %) hs, and for ifn-g in 2/9 (22 %) css patients and 7/7 (100 %) hs. mpo stimulation determined significant ifn-g release in 5/8 (62 %) css patients, but not in hs (0/5) no il-4 response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd4+ cells from css patients show a th2-polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd4+ t cells, and responses towards it are instead th1-polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd4+ t cells in css. a. voigt 1 , e. opitz 1 , k. savvatis 2 , k. klingel 3 , k. stangl 1 , u. kuckelkorn 1 , p.-m. kloetzel 1 1 charité -universitätsmedizin berlin campus mitte, berlin, germany, 2 charite -campus benjamin franklin, berlin, germany, 3 universität tübingen, tübingen, germanymurine models of coxsackievirus b3 (cvb3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp2 of the stress-induced ip, were infected with 1x10e5 pfu cvb3 nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp7 as early as day 4 p. i. in lmp2-deficient mice. however, lmp2-deficiency was linked to less severe myocarditis at day 8 p. i. (he stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. lmp2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p x 0.001). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x 0.05), there was no difference in lmp2-deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb3 infection in lmp2-deficient mice. likewise, diastolic function was preserved in lmp2-deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp2-immunosubunit function in regulatory processes of viral replication. absence of lmp2 confers host protection in enterovirus myocarditis. h. w. liao 1 , j. xu 1 , j.q. huang 1 1 sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype 2 (den-2) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr1 and tnfr1-2 were constantly very low whereas trailr2-4 decreased after den-2 infection. fasl was expressed at similar levels on huvecs throughout den-2 infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by den-2 infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for 7 days into wis rat paw web tissue in saline containing 7.5x10 7 cells. group ii. s.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day 8.they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p x 0.05). immunohistochemical pictures showed increase in percentage of ox62 (migrating dendritic cell), mhc ii, his48 (granulocytes), ox7 (stem cells) and cd54 (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed1 (macrophages) and ox62 cells. popliteal lymph nodes contained evidently less of ox62, his48 and mhcii cells than in group i (p x 0.05). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega-3 fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption. the results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega-3 fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising 1 % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e2f2 mutation, and other affecting b cell apoptosis control, such as bcl-2 over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c57bl/6 (b6) genetic background. e2f2-/-bcl-2tg were obtained backcrossing e2f2-/-and e2f2+/+hbcl-2tg mice. e2f2-/-bcl-2tg, e2f2-/-, e2f2+/+hbcl-2tg and control mice (e2f2+/+) were followed up to 18mo-old. serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h3]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl-2tg in b lymphocytes of e2f2-/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e2f2-/-bcl-2tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e2f2-/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e2f2 or the overexpression of a bcl-2 tg in the b6 genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor 9 (tlr9) abolishes the generation of antinucleosome igg2a and igg2b autoantibodies but exacerbates lupus disease. however, the tlr9-dependent tolerance mechanism is unknown. here we show that loss of tlr9 in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th1 and th17 t cells. transfer of a synthesized monoclonal polyreactive igm to tlr9 deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr9-dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n=115) (vitali et al. 2002) . ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g 1 (fs+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x 0.05). interleukin (il) 17, il-1ra, il-1beta, il-12p40, il-15, macrophage inflammatory protein (mip) 1alpha, mip-1beta, eotaxin, interferon (ifn) alpha, and il-4 levels were significantly increased in the gc+ patients (n=27) compared with the gc-patients (n=70). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il-4 found in these patients (gao et al. 2006) . increased titers of th17-associated cytokines, il-17, il-1beta and the il-23 subunit il-12p40, may indicate a higher activity of these cells in gc+ patients (nguyen et al. 2008) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il-6. the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg2. production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g 50 %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd88-dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd88-dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/-1 baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il-8. objectives: increased levels of il-18, an innate and inflammatory cytokine of the il-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il-18 and other genes of the il-1/il-1r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il-18 and il-18bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il-18 are higher than in controls. serum il-18 correlates with disease activity and decreases upon remission. monocyte expression of the receptor il-18rb is increased and correlates with disease severity, while expression of tir8/sigirr (a down-regulatory receptor of the il-1r/il-18r family) is reduced. in mrl lpr/lpr mice, expression of il-18, caspase-1 and il-18rb genes precedes disease onset in lymph-nodes. in other organs, changes in il-1-related genes (il-33 and tigirr-1 up-regulation, tir8/sigirr down-regulation) occur after disease onset. free il-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il-18 levels correlate with autoimmune lupus both in mice and humans. free il-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il-18rb is a marker of pathology, suggesting increased il-18-dependent activation. both in mouse organs and human monocytes, tir8/sigirr expression decreases with disease, suggesting impaired control of il-18r activation. thus, il-18 may be involved in autoimmune lupus pathology, and il-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f4 (amino acid (aa) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f4 was recognized by all patients. fragment f1 (aa 5-30) was recognized by 9 of 34 dcssc patients. fragment f8 (aa 350-400) was recognized by 4 of 8 sle patients. analysis of clinical data revealed a significant difference between the f1 negative and f1 positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f1 and f8 could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd4 + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd4 + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril-2. by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd4 + t cells enriched in tregs were co-cultured with activated cd4 + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd4 + t cells anergy was also evaluated based on cbl-b, grail and foxp3 mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd4 + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd4 + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b-1 cells through targeting p110d by shrnas strategy. methods: we used the drugs, ly294002 and wortmannin, pan-specific inhibitors against pi3ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. we then introduced either pan-specific inhibitors against all pi3ks or p110d-targeting shrnas into an sle-prone animal model, nzb/w f1 mice, for therapeutic purposes. the results suggested that pi3ks are not only important for the development of b-1 cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p110d. either pan-specific inhibitors against pi3ks or p110d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f1 mice. one inhibitor, ly294002, and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi3ks are critical for the maintenance of b-1 cell populations might shed light on future treating other diseases associated with b-1 cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool 1 , m. alarcón-riquelme 1 , s. kozyrev 1 1 institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank1 gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs10516487, r61h). we identified that bank 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank1 isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs17266594), which is in complete ld with r61h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia1, which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a 57-year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti-2gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow 1 1 cnrs 9021, strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below 2. b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd19 surface protein for all patients. this cd19 lower expression is associated with cd45 lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf2 expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf2 lacking an exon in the c2-domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf2 in t-cells. smurf2 expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf2 expression was upregulated by tgf-beta stimulation in t-cells and smurf2 was markedly upregulated in tumor infiltrating cd4+ lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day 42, smurf2 transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf2 showed an upregulation of the tgfbrii and an activation of smad2 and 3 as compared to wild-type t-lymphocytes, which were previously described as typical smurf2 targets for degradation. in addition the transfection of smurf2 and a caga-luc plasmid into cos-cells for smad3-promotor studies yielded the same effect as shown by upregulation of the smad3 activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf2 splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf2 has beneficial effects on mucosal inflammation and tumor development. smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat3 has important functions in cytokine signalling in a variety of tissues. however, the role of stat3 in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat3 activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat3 activation in dss colitis is dependent on il-22 rather than il-6. il-22 was secreted by colonic cd11c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat3 activity were highly susceptible to experimental colitis, indicating that epithelial stat3 regulates gut homeostasis. stat3 iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il-22 and epithelial stat3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat3 activation regulates immune homeostasis in the gut by promoting il-22-dependent mucosal wound healing. stat3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd40 and tlr4 expression at day 3 and day 7 after infection. mycobacterial infection did not result in differential tlr2 expression as compared to uninfected cells. cd40 is involved in stimulating th1 pro-inflammatory responses, although map may interfere with cd40 signalling (1) . tlr4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd40 and tlr4 receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd8 t cells by exogenous antigen leads to colitis. methods: eight million naïve cd8 + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h2-kb, were transferred i. v. into b6 mice. at day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day 2 after the injection of ot-i cells. the phenotype of effector cells was evaluated at day 5 by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd8, the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd8 + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd8 + t cells. our study demonstrates that the local activation of antigen-specific cd8 t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b6 mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x 2 and t-test programmes. results: in 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. on the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: 1) helicobacter pylori infection is relatively common in the general population (30,5%).2) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova 1 , t. ulmannova 1 , k. stechova 1 , k. tesarova-flajsmanova 1 , v. stavikova 1 , j. nevoral 1 1 charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il-4, il-6, il-10, il-17, il-23, ifn-gamma, tgf-beta1, egf and eotaxin. man-whitney u test was used for statistical analysis, p x 0.05 was considered statistically significant.results: il-10 as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). higher concentrations of il-4 and lower concentration of tgf-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. 8310-5. background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il-8 (murine homologs kc and mip-2) and its receptor cxcr2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. anti-kc antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. results: facs analysis revealed 80 % neutrophil purity, 35 % of which were cxcr2 + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, 4 h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model (1.5 % dss in drinking water for 1 week, followed by pure drinking water for 1 week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, 2 % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell 2004), treatment of commensal-depleted mice with the tlr4 ligand lps in drinking water protected against the lethality of 2 % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. at 2 % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by 1.5 % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il-13 production which is an important pathological factor. neutralizing il-4 or il-13 prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th2 cytokines in colitis remain undefined, we used mice deficient in il-4/il-13 or the key receptor through which they signal, il-4ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il-4ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il-4/il-13 double deficient mice which were protected from colitis. removing il-13 production from il-4ra -/mice, by using il-4ra/il-13 double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il-13 mediates susceptibility in an il-4ra independent manor. recent evidence pc17/33 introduction: the activation of cd4 + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th1-like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th1 cytokines, such as il-5,-6 and il-13. however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc2 was found in uc and cd colonic tissue compared to control specimen. transmitted to the th2-mediated oxazolone-induced colitis model, nfatc2-production is significantly increased in both diseases, too. nfatc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc2 deficient mice compared to control mice, which can be observed by tunel assays, caspase3 and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl-2 and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il-6, ifn-gamma, il-13 and il-17 by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc2 regulates il-23/il-17 in an indirect way. last, administration of il-6 blocked the protective effects of the nfatc2 deficiency in experimental colitis, suggesting that nfatc through il-6 signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc2 in colitis by controlling mucosal t cell activation in an il-6 dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd4cd45rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd4cd45rb high t cells also regulatory cd4cd45rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at 3 different time points during colitis progression. after 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. microrna was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from 11 micrornas that demonstrated an induction during the development of disease we selected 4 micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd4cd45rb high transfer model. objective: the purpose of this clinical trial (id: nct00287677 of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in 3 groups: group a (n=8) receiving haart+ gh (for 6 months) + tt+hva/b vaccines (at month 6 post gh adminsitration); group b (n= 6) receiving haart+gh but not vaccines; and group c (hiv control group, n=7) with haart+vaccines (at month 6) but without gh. all patients are followed up 6 months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after 24 weeks of administaring hormone the absolute numbers of cd4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (mean and sem; p x 0.0025). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p x 0.02). viral load remained undetectable in all patients. despite the increase in cd4 counts the percentage of recent thymic emigrants (as assessed by the expression of cd31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv (80,000-160,000), hepatitis c virus (hcv; 2.3-4.7 million) and hepatitis b virus (hbv; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv-1 infected patients with g 350 cd4 + t-cell counts and plasma hiv-rna levels of x 1.7 log 10 copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from 4 timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g 50 and when the responses were g 2 times the standard deviation above the mean of replicate negative controls (mock electroporated dc). 16/17 patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in 4/16 patients before and 11/16 patients after vaccination. for rev, 7/16 patients showed a pre-existing rev specific response and 14/16 patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus 71 (ev71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev71 is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev71 inactivated virus. methods: mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after 0, 2, and 4 weeks. blood samples were collected at 0, 21, 35, and 45 days. the total serum anti-ev71 igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il-5 from splenocytes was also measured. results: immunization with ev71/ps-g showed that the anti-ev71 igg levels were significantly increased compared with ev71 alone or ev71/cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev71/ps-g group compared to ev71 alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev71/ps-g-immunized mice compared to those of ev71 or ev71/cfa/ifa-immunized mice, indicating a th1 cells response elicited by heat-inactivated ev71 vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd8+ t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd8+ t-cell responses against hcv-ns3, studying induction of il-2, ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il-2 and ifng production by cd8+ t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv-1-recognizing cd8 + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv-1 replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd8 + t cells by electroporation, and chose tcrs which were able to recognize the hla-a2 restricted hivpol-peptide iv9, or the hivgag-peptide sl9. results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il-2, tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd25, after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd4 + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd8 + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd8 + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt3l pretreated balb/c mice were incubated for 3 hrs with hcv ns5-coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g 80 % dcs and was used for immunization. dc expression of the maturation markers cd40, cd80 and cd86 was determined before and after ingestion of ns5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns5 expressing sp2/0 cells). results: in immunized animals, the ctl activity was increased 3-fold compared to mock immunized mice. accordingly, tumor challenge with ns5 expressing tumor cells showed a significant reduction in tumor growth. the number of cd4 + ifn-g + cells was increased g 3-fold and the number of cd4 + il-2 + increased g 5fold in the dc-ns5-bead immunization group. these results paralleled the proliferative response of splenocytes to ns5 protein obtained from immunized animals with the most significant response in the cd4+ population of dc-ns5-bead immunized animals. the use of ns5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th1 biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec-205 induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec-205 antibody can be developed. we screened the anti-dec-205 sequence computationally for putative hla dr4-restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec-205 antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec-205 as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih 1r21ai078800 a live oral vaccine based on human adenovirus (ad)4 has proved safe and effective in us military recruits for nearly 50 years. in these experiments, we have investigated whether replication-deficient ad4 can be an efficient potential vaccine carrier for oral vaccination. ad5 vectors were used throughout to provide a benchmark for efficacy. we generated novel ad4 and ad5 vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad4 than ad5. ad5 routinely infected and provided transgene expression in˚10 % of human cd4+ and cd8+ t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to 25 -30 % of cd8+ t cells present, showing that ad5 infected a surprisingly large proportion of t cells. in comparison, ad4 provided egfp expression in x 2 % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad4 and ad5 vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad4 and ad5 induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. interestingly, when delivered together ad4 and ad5 vectors targeted the same subset of cells. together, these data show that ad4 is a viable alternative to ad5-based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; g 300 in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the 667 mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, 2005; gros et al, 2008; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd8 + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp60) of rhdv at two different locations: 1) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp-2), and 2) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp-306). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd8 + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp-2) was more immunogenic than insertion at position 306 for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp-2 at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr-2 ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd8+ t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd8+ t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np 147-155 specific cd8+ t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr-2 targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l929cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during 7 days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd2+, cd4+, cd8+, cd16+ t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns4 and ns5a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h5n1 influenza virus inhibits the ifn-g synthesis (mibayashi et al., 2007) and causes a decrease in cd4 + and cd8 + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., 2000) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a 10 amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions 129-131 (tat'kov c. i. et al., 2000) . deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 iu once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast rna preparation (5 mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., 1983) . results: when deltaferon was administered in doses of 2-20*10 4 iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of 2*10 4 iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon (2*10 4 iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd8 responses (tetramer+ hiv-1 gag p24 cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd8 responses after pla-p24 immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p24 were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas 1 , c. prego 2 , s. vicente 2 , m.j. alonso 2 , a. gonzález-fernández 1 1 university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, 2 university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to balb/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease (100 miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigenbackground: infection with human immunodeficiency virus type 1 (hiv-1) is characterized by dysfunction of hiv-1-specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from hiv-1 and the tlr3 ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p24 microcapsules containing p24 and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for 10 days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p24 peptides. intracellular staining of interferon-gamma (ifn-g), interleukin-2 (il-2) and cd107a after p24 stimulation was also performed. results: mddc from hiv(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p24 containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il-2, upon p24 peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd16b between our model cytokine il-2 and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p30gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il-2 dependent proliferation assays of il-2 variants. results: murine il-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il-2 dependent cell line ht-2. we found that the membrane anchors comprising one and four ig-like domains (il-2::1iggpi and il-2::4iggpi) resulted in severely reduced vlp production by 293 producer cells and despite of an increased targeting of il-2::4iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il-2 fused to the minimal gpi-anchor acceptor sequence of hcd16b (il-2::gpi). il-2::2iggpi, however, showed comparable particle production and biological activity in vitro when compared to il-2::gpi. furthermore, il-2 fused to 2ig::gpi showed an increased capacity to co-stimulate primary p14 tcr transgenic t-cells specific for lcmv-gp 33-41 in the context of h2-d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. 2ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il-2::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f1816-b13 of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant 812079 & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild 1 , interdisciplinary transplant laboratory 1 charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with 6 different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from 2,5 month to 16 days. t cell lines are composed of cd8 and cd4 cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker 1 , m. assenmacher 1 , a. richter 1 1 miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp65-specific cd4 + and cd8 + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd45ro -cd25 -) cd4 + and cd8 + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp65 peptide pool and cd3-depleted autologous pbmc as feeder cells in the presence of il-2, il-7, and il-15. already 9-13 days after primary activation pp65 495-503 /a2-tetramer + cd8 + t cells were detectable for 3 hla-a2 + blood donors. to analyze cd8 + t cells having other specificities than for the peptide pp65 495-503 as well as probably primed cd4 + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to 3.9% of the cd8 + t cells and up to 3.8% of the cd4 + t cells after restimulation with pp65 peptide pool, but not with either irrelevant ie-1 peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd154 and cd137 only if restimulated with the pp65 peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il-2, indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd4 + and cd8 + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf 1 , k. mozdzanowska 1 , l. otvos 2 , j. erikson 1 1 the wistar institute, philadelphia, united states, 2 temple university, philadelphia, united statesthe influenza virus a matrix protein 2 ectodomain (m2e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m2e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine 2003; virology journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-m2e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd8 + t cells, have been considered. clinical data confirm a crucial role for antiviral cd8 + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a*0201 and hla-b*0702 previously. considering other frequent hla alleles cmv specific cd8 + t cells were monitored longitudinally in 30 hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd8 + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd8 + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a*2402/pp65 (341-349) (7.6x10 6 cells/l) and hla-b*3501/pp65 (123-131) (4.4x10 6 cells/l) specific cd8+ t cells are significantly lower than those for hla-a*0101/pp50 (245-253) (17.2x10 6 cells/l) and hla-a*0201/pp65 (495-503) (13.2x10 6 cells/l) specific cd8 + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. (2,5 -50 mcg) . no adverse effects were indicated during trials (up to 25 month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of vichrepol.no differences were detected in cd4+ and cd8+ t cell counts and cd4+/cd8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp70 or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp70 or gp70 alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb6f1 hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb6f1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd4 + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd4 + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf01 exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf01 did not show any increase in their levels of ifn-gamma. conclusion: caf01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from 13 children aged 1-2 years old (6 boys and 7 girls) -group 1, and 11 children (6 boys and 5 girls) 6-7 years old -group 2 were isolated on a gradient of density before vaccination ( or revaccination) with priorix, 1 week, and 2 months after and incubated with cfse. then 2 million/ ml pbl were incubated in rpmi-1640 supplemented with 10 % fcs (the negative control), at presence of 5 mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % cî 2 at 37°c within 7 day. intensity of a fluorescence estimated on fl1 by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control 90 % pbl in both groups did not enter mitosis. in the positive control 90 % of cells have passed one and more mitoses. in group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. in group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. in a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. production of the interleukin (il) 6, ifn-g, tnf-a, il-4, il-1 was more informative than il-5, il-7, il-8, il-12. measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp120 cd4-binding site (cd4bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv-1 neutralizing polyclonal iggs directed against the cd4bs. the mabs were shown to react with an anti-cd4bs human neutralizing mab (b12), to elicit antibodies that recognize the gp120 molecule and an anti-hiv-1 neutralizing response in rabbits, confirming them as cd4bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd4bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd4bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp120 titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b12 in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p1 and p2) reacted in elisa only with the cd4bs-directed igg fraction. the clones were also recognized in elisa by b12. p1 and p2-immunized rabbit sera showed a strong anti-gp120 titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb2 strain glycoproteins. in particular, 3/5 rabbits in the p1 group and 1/5 in the p2 group showed an 80 % hiv neutralization at dilutions ranging from 1:20 to 1:150. the immunoenzymatic assay used, allowed to detect a p1 and p2 reactivity in hiv-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines (17d and 17dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a 23-year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following 17d-204 vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd16, cd32 and cd64) along with increased levels of nkt-cells (cd3 + cd16 +/-cd56 +/-/cd3 + ratio) and activated t-cells (cd4 + and cd8 + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il-6, il-17, il-4, il-5 and il-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il-4 + ), cd8 + t-cells (il-4 + and il-5 + ) and b-lymphocytes (tnf-a + , il-4 + and il-10 + ). the analysis of cd4 + t-cells revealed a complex profile with increased frequency of il-12 + and ifn-g + and decreased percentage of tnf-a + , il-4 + and il-5 + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester 1 , h.-g. rammensee 1 , s. stevanović 1 1 eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd8 t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd8 t cell epitopes for the frequent mhc class i alleles a*01, a*02, and a*24 from the proteins pii (hexon), pviii, and e1a of adv strains ad2 and ad5 by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a 12-day recall stimulation of pbmcs from at least 16 healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd8 t cells. we could identify 27 new peptides eliciting ifn-g responses, several of which were confirmed as novel cd8 t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle (5 ara criteria) was diagnosed in a 40-year-old african female patient with hiv-1 (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd4 t-cell count x 200 cells/mm 3 . initiation of 500 mg mmf bid was associated with biological and clinical remission of sle and cd4 t-cell increase. no opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to art. hiv-1-specific cd4 and cd8 t-cell responses were analyzed after 18 months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il-2 production following stimulation with a panel of 192 hiv-1-derived optimal epitopes (9/10-mers) covering various hiv regions and a pool of 105 hiv-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. all peptides are derived from hiv-1 consensus strain iiib. results: highly polyfunctional hiv-1 specific cd4 and cd8 t-cell responses against gag were detected. 11 epitope-specific cd8 t-cell responses were identified: except for one response restricted by hla a*23 and another one by hla cw*07, all the others were restricted by hla-b alleles and mostly by b*58 (n = 5). seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il-2+) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv-1 specific cd4 and cd8 t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv-1 t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of 10 6 dl50/ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the 7 th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt-46767, cofaa and cgpi-20090305. . state of vaccine-induced measles immunity was determined by means of elisa in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in 4-6 years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with mt conversion. in 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with mt conversion in 1-3 years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic mt igg level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il-17 producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein 65 (dnahsp65). methods: balb/c female mice were infected by intra-tracheal route with 10 5 h37rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. each dose consisted of 100 micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd4+, cd8+ and gamma-delta t cells from lungs were determined 10 and 50 days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x 0.05 were considered significant (t test). results: at day 10 after the end of the immunotherapy, dnahsp65 treated mice exhibit increased numbers of absolute cd8+ and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il-17 producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp65 treated mice showed more ifn-gamma producing cells in both cd8+ and cd4+ cell populations. at day 50 after the end of the therapy, the main observation in mice which received dnahsp65 treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd4+ and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd8+ cells, together with a more frequency of gamma-delta t cells producing il-17. finally, the immunotherapeutic effects of dnahsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd8+ cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il-17, are the main effects of dnahsp65 immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il-17 and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as 60 loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput (96-well format), requires only 100 ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that 5 candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls 71 % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all 5 m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards 4 of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to 100 % in pb, 75 % in rx and 93 % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd40l, a co-stimulatory molecule preferentially expressed on activated cd4+ t cells, is the ligand of cd40. cd40-cd40l interaction induces the production of il-12 and the initiation of a th1-type immune response. several studies show that cd40l is required for the activation of macrophages and the maturation of dcs. moreover, cd40l enhances the capacity of cd8+ t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd40l. we have constructed the recombinant bcg strain expressing cd40l (rbcg2) by electroporation of bcg with pgfm11/signalsequenceag85b-cd40lec and an another recombinant strain with empty vector pgfm11 (rbcg1) as a control. the expression of cd40l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd40l 2 weeks after vaccination but not at 4 and 8 weeks. rbcg2 seems to be more protective against paratuberculosis than rbcg1, but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd40l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il-1a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x 0.05) of tnfa, ifng and il-1a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x 0.05) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd40 mab and ag85a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd40 binding on cd40 transfected l929 fibroblasts. the conjugates were tested in vivo in wild type and cd4 + cell-depleted mice for the induction of specific anti-ag85a serum antibodies. splenocytes were challenged ex vivo with ag85a and were examined for their ability to produce th1-related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il-2. we developed a method to successfully crosslink anti-cd40 mab to ag85a. serum antibodies against ag85a were detected after immunisation with this conjugate vaccine in both wild type and cd4 + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag85a alone. production of two other th1-related cytokines, tnfa and il2, was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd40 conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd4 + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer 1 , r. burger 2 1 robert koch-institute, cellular immunology, berlin, germany, 2 robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd4-positive and cd8-positive t-lymphocytes. cd8-positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd8+ t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for 5 days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to 30 % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd4+ t-lymphocytes in vivo. similarly, the cd4-positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd4+ and the cd8+ subpopulation depended on the presence of apc. stimulation oft cd8+ cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd8-positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target 6 (esat-6) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, 83 iranian and afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of esat-6 and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of 4 spot forming unit ( g 20 spots per million), we found detectable response to esat-6 in almost 80 % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable esat-6 specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th1 immune response, against esat-6, in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal 1 , p.k. upadhyay 1 1 national institute of immunology, pdc-1, delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl 2 solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c57bl/6 mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: 2.4 % alginate, 0.012 % pva concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % pvp (poly vinyl pyrollidone). there was 6 fold increase in proliferation index of spleenocytes and releases 600 pico-gram of interferon gamma after 4 week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by 10 % increase in cd86 and 10.5 % in ccr7 expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b100 is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b100s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b100s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b100s. by cell cycle analysis we determined that b100s is able to induce apoptosis in up to 80 % of jurkat and molt-4 t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b100s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b100s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that b100s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b100s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b100s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd40, cd80 and cd86, and of the inflammatory cytokines il-6, il-23 and mcp-1. using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd4 t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th2 and th1/th17 skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd4 t cell responses. microcapsule based vaccination resulted in a marked induction of il-17 secreting th17 cells, without inducing strong th1 (cfa) or th2 (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg1, igg2c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd4 + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd80, cd86 and cd40 and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il-6 and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr4-dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr4-defective c3h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il-1b secretion by dc, through its effects on caspase-1 processing, which is also tlr4-independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr4 signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc2461 (st11) or lactobacillus rhamnosus ncc4007 (lpr), each at 5x10 8 cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week 1 to week 7 of life. fresh feces were collected at 3, 5 and 7 weeks of life for determination of iga levels (assessed by elisa). pups were bled 2 and 4 weeks after immunization for determination of measles-specific igg1 and igg2a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st11-fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from 200 kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at 3:1 ration and washed 3 times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw264.7 by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc6/propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that 15 up to 24 euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd3+ cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on 35 th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il-5 and ige serum levels were increased on animals from group i when compared to control.the enhancement on th2 immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd8+ ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd4+t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg2a and igg2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren 1,2 , n. almqvist 2 , a. lönnqvist 1 , s. östman 1 , c. rask 1 , e. telemo 2 , a.e. wold 1 1 university of göteborg, department of clinical bacteriology, göteborg, sweden, 2 university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. examination of gut sections from sea treated donor mice revealed increased density of cd8a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp21, proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna 1826, a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp21, or groel together with cpg-dna 1826 reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp21 together with cpg-dna 1826. the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp21 microspheres induced protective ifng-secreting cd4 + t-cells and raised pulmonary pmp21-specific iga levels in vivo. also, pmp21 microspheres caused lower il-6 serum levels upon administration than the injection of pmp21 together with cpg-dna 1826, indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in 44 cases of s. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised 44 iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen 2006).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter 2007) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st59 strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st59 strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in 15 and 7 ds children, respectively. samples were taken before and 3-4 weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg 1 -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at 4 years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg 1 , igg 2 and igg 4 ). at 9 years, ds children had lower postvaccination geometric mean igg 4 anti-tt-titers only. post-vaccination igg 1 -anti-tt avidity levels were decreased in 8/15 and 4/7 ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. 2 kda; 30, 6 kda; 23, 9 kda; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kda and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of 100 families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an 8-month period. sera were obtained from healthy subjects from the same community (2 months to 88 years of age). the highest incidence of salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. the lowest incidence, 10/1000, was observed in the population aged 10 to 59. whereas serum from individuals ranging 15-70 years of age showed maximum igg1, igg3 and iga anti-s. typhimurium titres, children less than 5 years-old did not show detectable igg1 and igg3 titres and had weak igg2, iga and igm antibody levels; only their igg4 levels were comparable to those detected in adults. moreover, the levels of igg2 and igg3 antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková 1 , s. bystrický 1 1 institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o135 using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd19, expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age 18-55 years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg1, igg2 and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present 5 years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after 5 to 7 days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg2 response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers 1:3200). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones (1:800 vs. 1:3200). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv 0032-06 and vega 2/7029/27 grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked d-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs (250g-350g) were treated orally with 50 mg respivax five consecutive days. after the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. the populations of cd8, cd4 and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd8 positive cells, which number reached maximum at the end of the second week. on day 14 b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd4 and cd8 positive cells. on days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta 1 , s. majumdar 1 1 bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein-10 (ip-10) and interleukin-8 (il-8) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (inos2) expression and was linked to the mapk signaling pathway via antagonistic regulation of p38mapk and erk1/2. further, ip-10 was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th1 cytokines and no. thus this study strongly demonstrates that ip-10, like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th1/th2 cytokines mediated by cd8+ t cells and activating cd4+ t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x 0.05) following immunization and after challenge with leishmania major. il-4 values were increased in all groups, but there was no statistical difference between the groups(p g 0.05) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x 0.05) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x 0.05) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x 0.05). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at 3 week intervals. three weeks after booster injection, 5×10 5 stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg2a/igg1was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type 1 immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin 10 (il-10), and cellular expression of cd4 and cd25. results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd4+ lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with 1,600 infective n. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg1 and igg2a). this was independent of level of me supply. feeding regime did not affect levels of the type-2 cytokines il-4 and il-13. conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp63 is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp63 of l.major strain mrho/ir/75/er. methods: l.major promastigotes were grown in rpmi1640 supplemented with 10 % fcs. l.major rna extraction and cdna synthesis were carried out. gp63 gene segment was amplified by specific primers and cloned into ptz57r to construct ptz57r/gp63. the presence of gp63 into ptz57r was confirmed by pcr. then, ptz57r/gp63 was sent to determine the sequence of its nucleotides. after that the gp63 gene segment was sub-cloned into pet32 a (+) expression vector and transformed into e.coli bl21 (de3) plyss and gp63 protein was expressed in presence of 1mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, 2007) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. subjects received virosomal formulations containing 50 mg of ama 49-c1 (pev301t), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (pe)-peptide conjugate and 10 ug of uk39 (pev302t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days 0, and 90. results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma9vdelta2 t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il-10 responses, to epitopes on the dominant rbc autoantigen, anion exchanger-1 (ae-1, or band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla-4 + regulatory t cells or soluble forms of ctla-4, may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =-0.81) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd8 + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b 1 (r medium =-0.74) and il-10 (r medium =-0.65) in isolated splenic hscs ex vivo. analysis of effector cd4 + t cells in spleen showed decrease of t h 1 cells quantity and simultaneous t h 2 cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd4 + cd25 + ctla-4 + -and cd4 + cd25 -il-10 + il-4regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h 1, t h 2 and regulatory t cells were correlated (r th1 =-0.74, r th2 =0.86 and r th3 =0.80 and r tr1 =0.68) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th3 =0.84 and r tr1 =0.76) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b1-and il-10-produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p38, jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il-17, as compared to non-infected controls. the infection also altered the effect of il-17 on mapk activation by preventing its stimulating effect on p38 mapk. moreover, in s.obvelata-infected animals il-17 markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il-17 induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r 2 g 0.98. the analytical range was from 1 g/l to 70 g/l. the coefficient of variation (cv) was x 10 % for [mc] n 1 g/dl, and x 20 % for [mc] x 1 g/dl. this procedure was successfully implemented to quantify the mc in 1100 serum samples between march 2008 and february, 2009. among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: 1. we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. 2. the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. moreover, cd34 + cd38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b 1 , il-10, pge 2 and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd40-ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd40l is cleaved to soluble (s)cd40l. we sought to examine the levels of scd40l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded 5 atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd40l -along with other products -were assayed by quantitative elisa. levels of il8, cd62p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd40l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l . to examine if scd40l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il-6 secretion. the il-6 concentration was consistently below 5-10 pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il-6. the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il-6 production over control (p x 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l (p x 0.05). pre-incubation of b cells with cd40-blocking antibodies substantially abrogated il-6 secretion, unlike isotype-matched control. the partial blocking of cd40 binding on cd40 + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd40l (under investigation) and indicates a sustained role for pc-derived scd40l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd40l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule-1 (dnam-1) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from 30 aml patients before specific anti-leukemia therapy and 47 healthy donors. all results were analyzed statistically using spss version 15. results: aml patients under 65 years showed a significant reduction in the expression of dnam-1, nkp30 and nkp46 compared with age-matched controls. both healthy individuals and aml patients older than 65 years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp44 expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam-1 on nk cells and its ligand cd112 on aml blasts has been found in aml patients under 65 years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam-1 expression on aml patients and confirm previous reports showing a significant decrease of nkp30 and nkp46 on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel 1 , e. gorska 1 , u. demkow 1 , m. wasik 1 1 medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved 10 children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 m. analysis of: p-gp surface expression, p-gp function (rhodamine 123 test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was 14,52 %±11,32. after 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine 123 efflux, which characterized p-gp activity, was 2,62 %±3,15. after 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). average phi in lymphoblasts was 7,78±0,19. acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . rest of lymphoblasts showed alkalization (phi -8,00).the percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-v test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd49d/cd29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd29 on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd38 and anti-cd29 (clon huts21) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures (18 hours). results: cd29 active form was expressed in the majority of normal and tumoral bm pc from healthy subjects (67.3±6.6, n=9) and mm patients in the early stages of the disease (32.6±7.5, n=17). in these cells, huts21 epitope was clearly upregulated by mn 2+ . in contrast, circulating pc were almost all huts21 negative, and levels did not significantly augment when these cells were exposed to mn 2+ . moreover, not only pb but also bm malignant cells from pcl patients were also huts21 negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index (2.35±0.9 brdu+ cells, n=3) in comparison to pc from mm patients in the first stages of disease (1.3±0.2 brdu+ cells, n=5). these results suggest that the active form of cd29 must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either 10 % fetal bovine serum (fbs) with and without fgf2,or 10 % human platelet lysate (hpl), 5 %hpl, (10 % fbs + 10 % hpl), (10 % fbs + 5 % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day 18). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p53 dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: 1) ten cases with fa diagnosed on the basis of dna breakage analysis, 2) ten cases with aaa, and 3) ten normal control cases. the presence of p53 dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p53 dna was demonstrated in bone marrow of 90 % of children with fa, compared to 10 % in children with aaa (p x 0.001), while, no p53 dna was seen in normal control. a positive correlation between dna breakage and presence of p53 dna was seen in bone marrow from fa (p x 0.02, r0.81). the presence of p53 tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord-265146-j0n3a4m6 authors: hsieh, ying-hen title: ascertaining the 2004–2006 hiv type 1 crf07_bc outbreak among injecting drug users in taiwan date: 2013-02-12 journal: int j infect dis doi: 10.1016/j.ijid.2013.01.002 sha: doc_id: 265146 cord_uid: j0n3a4m6 objective: to ascertain the explosive 2004–2006 outbreak of hiv-1 crf07_bc among intravenous drug users (idu) in taiwan, which more than doubled the total number of reported hiv cases in less than 3 years, resulting in a 45-fold increase in cumulative idu/hiv cases and a 40-fold increase in previously seldom-reported female idu/hiv cases. methods: a mathematical model was utilized to fit the monthly case data, in order to estimate the turning points (peak incidence) and the reproduction number r of the outbreak. furthermore, correlation analysis was carried out to assess the correlation between infections among the male and female idus. results: model fit revealed a two-wave epidemic during april 2004–march 2007. the larger second wave started shortly after may 2005 and peaked in october 2005 before gradually subsiding. r was estimated to be 3.15 (3.14–3.16) and 27.21 (26.73–28.05) for the two respective waves. the time series of monthly differences in male and female case data were found to be most significantly correlated at lag 0 (i.e., r > 0.7) with r = 0.906 and 0.804, respectively in each direction. the granger causality test indicated that the male time series caused the corresponding female time series with a lag of 2 months or less. conclusions: the modeling results revealed the presence of a small first wave in 2004, before an explosion of cases after may 2005. furthermore, a harm reduction program implemented in august 2005 contributed to the downturn in the epidemic after october. correlation results also suggest that the upsurge in male hiv cases led to the subsequent drastic surge in female cases. in taiwan, where active hiv/aids surveillance has been in place since 1996, with 2.1-2.5 million annual screening tests and free antiretroviral therapy (art) introduced since 1997, [1] [2] [3] [4] during this time-period, an outbreak of hiv-1 crf07_bc infections among idus, including many previously seldom-seen female idu hiv-infected cases, resulted in more reported cases than all reported hiv cases among all risk groups combined in the previous 20 years since 1984, when the first aids case was reported. it has been speculated that the source of this outbreak was a drugtrafficking route to taiwan from yunnan province via southeast china, guangxi province, and hong kong, [6] [7] [8] from where a substantial amount of heroin was being smuggled into taiwan. moreover, five idus from southern taiwan were diagnosed as the country's first hiv-1-seropositive cases infected with crf07_bc in 2002. 2 it has been reported that the percentage of persons receiving a diagnosis of aids within 12 months of diagnosis of hiv infection dropped suddenly from 20% in 2003 to 8.3% in 2005, during the time when most of the newly diagnosed cases came from the idu population, which implies that the detected idu cases were in the early stage of hiv infection. 4 it has also been reported 1 that 96% of objective: to ascertain the explosive 2004-2006 outbreak of hiv-1 crf07_bc among intravenous drug users (idu) in taiwan, which more than doubled the total number of reported hiv cases in less than 3 years, resulting in a 45-fold increase in cumulative idu/hiv cases and a 40-fold increase in previously seldom-reported female idu/hiv cases. methods: a mathematical model was utilized to fit the monthly case data, in order to estimate the turning points (peak incidence) and the reproduction number r of the outbreak. furthermore, correlation analysis was carried out to assess the correlation between infections among the male and female idus. results: model fit revealed a two-wave epidemic during april 2004-march 2007. the larger second wave started shortly after may 2005 and peaked in october 2005 before gradually subsiding. r was estimated to be 3.15 (3.14-3.16) and 27.21 (26.73-28 .05) for the two respective waves. the time series of monthly differences in male and female case data were found to be most significantly correlated at lag 0 (i.e., r > 0.7) with r = 0.906 and 0.804, respectively in each direction. the granger causality test indicated that the male time series caused the corresponding female time series with a lag of 2 months or less. conclusions: the modeling results revealed the presence of a small first wave in 2004, before an explosion of cases after may 2005. furthermore, a harm reduction program implemented in august 2005 contributed to the downturn in the epidemic after october. correlation results also suggest that the upsurge in male hiv cases led to the subsequent drastic surge in female cases. ß 2013 international society for infectious diseases. published by elsevier ltd. all rights reserved. hiv cases infected through idu diagnosed in 2003-2004 were infected with hiv subtype crf07_bc, which is totally different from the previously common subtype b and subtype crf01_ae in taiwan. as is typical of underreporting of hiv/aids among many hardto-count high-risk populations for hiv/aids, 9, 10 there is a significant discrepancy between reported and estimated hiv/aids cases in the idu group because of the difficulty reaching idus. in taiwan, where needle-sharing or apparatus-sharing behaviors have been found to be common among idus, the majority of newly diagnosed hiv/idus were detected through mandatory inmate screening upon entry to correctional facilities. however, mandatory hiv screening of persons under police custody due to violation of the narcotics control act since late 2004 could also have partially contributed to the sharp increase in detection. in response to the outbreak in idus, a harm reduction program, which involved a needle-syringe program (nsp) and substitution treatment, was implemented by the taiwanese government in august 2005 as an intervention to the rapidly increasing hiv epidemic since 2004. 4 there has been a steady drop in hiv incidence among idus in recent years, down from being the major mode of hiv transmission in taiwan in [2004] [2005] [2006] to below that of homosexual and heterosexual transmissions since 2007, but still significantly higher than its pre-2003 level. a recent molecular epidemiology study after the crf07_bc outbreak in taiwan concluded that while the percentage of crf07_bc among all hiv infections decreased from 2007 to 2009, the percentage of subtype b actually increased. 11 however, many questions remain regarding this sudden outbreak among the idu population. in this study the reported hiv/idu case data and a simple mathematical model, the richards model, [12] [13] [14] were used to investigate the temporal progression of this epidemic among idus in taiwan. in particular, the total case data are fit to the model, as well as the male and female case datasets separately, in order to ascertain the epidemic. correlation analysis was performed in an attempt to determine the relationship between the male and female idus. the data used here were extracted from the monthly reported hiv case data between april 2004 and march 2007, for a total of 36 months, made available by the taiwan centers for disease control and prevention (tcdc) on the tcdc website. 5 the data are provided for each risk group/factor and gender. however, the hiv/ idu case data by gender are only available after august 2004, the fifth month of the dataset. in what follows, for ease of illustration in the tables and figures, the months are numbered, namely, april 2004 is month 1 and march 2007 is month 36. the richards model, a logistic-type mathematical model was used in this study. the explicit solution of the richards model is of the form: where c(t) is the cumulative number of deaths at week t and the prime ' 0 ' denotes the time rate of change. k is the final outbreak size over a single wave of outbreak, r is the per capita growth rate of the cumulative case number, a is the exponent of deviation of the cumulative case curve, and t i is the turning point of the epidemic (which signifies the moment of upturn or downturn for the increase in the cumulative case number). the basic premise of the richards model is that the incidence curve of a single wave of infections consists of a single peak of high incidence, resulting in an s-shaped cumulative case curve and a single turning point (or the inflection point of the cumulative case curve) of the outbreak. this turning point t i , which is defined as the point in time at which the rate of accumulation changes from increasing to decreasing, or vice versa, can easily be pinpointed via the richards model. when more than one wave of infection occurs, a variation of the s-shaped richards model is proposed, 15 which makes the distinction between two types of turning points. other than the first turning point ending the initial exponential growth of the cumulative case number, a second type of turning point is present in a multi-wave epidemic where the growth rate of the cumulative case number begins to increase again, signifying the beginning of the next wave. for further illustrations, the readers are referred to hsieh and cheng 15 and hsieh and chen, 16 in which the incidence curves for the 2003 great toronto area severe acute respiratory syndrome (sars) and the 2007 taiwan dengue outbreaks containing two peaks (or two turning points of the first type) and one valley (or a turning point of second type) are investigated. for the computation of the basic reproduction number r 0 , the formula r 0 = exp (rt) was used, where t is the generation interval of the disease or the average interval from onset of one individual to the onset of his/her contacts. it has been shown mathematically 17 that, given the growth rate r, the expression r 0 = exp (rt) provides an upper bound for the basic reproduction number, regardless of the assumed distribution of the generation interval. we noted that in this instance, the estimate obtained is not the basic reproduction number, but the effective reproduction number r, since in taiwan hiv is endemic among the idu population and multiple intervention measures have already been in place for some years. the model parameters of epidemiological importance are k, r, and the turning point t i of the epidemic. the cumulative death data can be fitted to the richards model to obtain estimates of these model parameters, using any standard software with a least-squares approximation tool, e.g., sas, matlab, etc. more applications of the richards model on other infectious disease outbreaks such as dengue can also be found in hsieh and chen 16 and hsieh and stefan. 18 we first examined whether the times series were 'stationary', in order to avoid spurious regression, which could possibly result in a biased and inconsistent estimator. 19 a stationary time series means that its statistical characteristics do not change in time. in the event of a non-stationary time series, it can be suitably transformed to achieve stationarity. the augmented dickey-fuller (adf) test 19 was employed to verify if the random variables were indeed stationary series. three equations were used to test if the series process has a non-stationary character: 1. without drift and trend terms: 3. both drift and trend terms: the null hypothesis is non-stationary or unit root, i.e. to determine the correlation between the epidemic among the male and female idus through their time series of monthly case numbers, a distributed lag model (dlm) was employed to describe the relationship between the male and female time series. a dlm is a regression model that includes current and lagged values of one or more explanatory variables. this model allows the determination of what the effects are for a change in a time series. the resulting correlation coefficient, r, is a useful measure of linear strength between two random variables. the mathematical formula for computing r is: where n is the number of pairs of data. the value of r is such that à1 r + 1. the '+' and 'à' signs are used for positive linear correlations and negative linear correlations, respectively. if there is no linear correlation or a weak linear correlation, r is close to 0. a value near zero means that there is a random, nonlinear relationship between the two variables. 'jrj 0.4' means 'low correlation', '0.4 < jrj 0.7' means 'moderate correlation', and 'jrj > 0.7' means 'high correlation'. in other words, a correlation greater than 0.7 is generally described as strong, whereas a correlation less than 0.4 is generally described as weak. the granger approach 20 is used to ascertain how much of the current values of time series y can be explained by past values or some lagged values of time series y. the commonly used software eviews was developed originally by economists for use in economics applications, but can also be useful in other statistical applications. eviews version 5.0 (http://www.eviews.com/) was used to analyze the data. in general, it is better to use more lags rather than fewer lags, since the theory is couched in terms of the relevance of all past information. it is advisable to pick a lag length, l, which corresponds to the reasonable beliefs about the longest time over which one of the variables could help to predict the other. eviews performs bivariate regressions of the form: for all possible pairs of series in the group. the reported f-statistics are the wald statistics for the joint hypothesis: the null hypothesis is that series x does not granger-cause series y in the first regression and that y does not granger-cause x in the second regression. the monthly time series data of reported hiv cases for male idus, female idus, and all idus in taiwan were fit to the richards model as in figure 1 and table 1 the effective reproduction number r was computed for each wave. however, it is unclear what the generational interval is for hiv. it has been proposed that the rates of partner change for homosexuals and heterosexuals tend to be of the order of 1 year, 21 but it is unclear what the rate of needle-syringe sharing is among idus, [22] [23] [24] although it is most likely of shorter length than that of sexual transmission of hiv. due to the lack of a reliable estimate for the hiv generation time among idus in the literature, a generational interval of 5 or 6 months was assumed, based on an estimate of doubling time for aids cases among idus in the northeastern usa early in the epidemic. 22 it should be noted that the main purpose for estimating r in this study was to compare the transmissibility estimated using different datasets and to ascertain the relative temporal change in transmissibility that occurred in each wave of infection during the course of the epidemic. to table 2 , where the time series of monthly reported cases and the difference of two successive monthly reported cases were found to be stationary for both males and females. note that we use 'male' and 'female' to denote the respective time series of monthly reported male and female hiv cases, and '4male' and '4female' to denote the respective time series of differences of monthly male and female hiv cases. next, the correlation between the monthly reported case data (male and female) and monthly differences in case data (4male and 4female) were analyzed. the correlation coefficient r is a useful measure of the linear strength between two random variables. applying a univariate model of y = b 0 + bx t à lag + e t for 'x causes y' (or x ! y) with lag, male ! female and 4male ! 4female were found to be most significantly correlated (i.e., r > 0.7) with r = 0.906 and 0.804 with lag 0, respectively. the correlation plots for the correlation are given in figure 2 , which indicates the most significant correlations between the male reported cases and female reported cases, as well as their differences, are consistently at lag 0 (in red). the granger causality test was subsequently carried out between the time series of male and female and between the time series of 4male and 4female for time lags up to 5 months. test results, also given in table 3 with the causal direction for each pair indicated with an arrow, indicate that both male time series caused the corresponding female time series after a lag of 2 months. the timelines of the epidemic for all cases, as well as for the male and female case numbers, are illustrated in figure 3 , indicating good agreement among estimates of temporal progression of the epidemic using the three datasets, and pinpointing may 2005 as the month that separated the two waves obtained from all three datasets. it is further concluded that the turning points for the first of these two waves differ slightly at 16 17 .05), respectively, for the total, male, and female case data (see table 1 (18 + 4.19) , for all cases, males, and females, respectively. it is interesting to note that in a recent study, 25 phylogenetic tree analysis of 451 hiv-infected inmates with an idu history in taiwan was employed to demonstrate that there were two waves of hiv-1 crf07_bc infection from mainland china to taiwan. although no timeline of the two waves was available from the molecular study, it does corroborate our modeling results. the estimates for r indicate that the transmissibility of hiv among the idu population increased from the first wave to the second wave according to all three datasets, culminating in a peak around october 2005, when a downward trend ensued. it is interesting to note that the harm reduction program that was implemented by the taiwanese government in august 2005 could have impacted the downturn in case numbers after october, as revealed by our modeling results. however, only a trial harm reduction program, which included a needle-syringe program (nsp) and substitution treatment in four counties, was established at that time. after 1 year of the pilot study, data indicated that the hiv incidence in cities with an nsp decreased from 13.9 to 13.3 per 100 000 persons compared to an incidence increase from 11.5 to 15.3 per 100 000 persons in cities without an nsp. 26 subsequently, the harm reduction program was expanded to the whole of taiwan in july 2006. therefore, the country-wide downturn in case numbers after october 2005 may only be partially attributable to the harm reduction program. r for females was higher than that of males in the first wave, but lower in the second wave and ending earlier in june 2006, perhaps reflecting the more rapid initial increase in female incidence in the early stages of the epidemic. however, this drastic upsurge in female hiv-infected idus was relatively more difficult to sustain in the second wave, as indicated by the shorter length of this wave when compared to that of the males. therefore the epidemic impacted female idus more drastically initially, but the overall magnitude of infection was still less than that of the males, which was the group most affected by the epidemic. correlation analysis of the time series of male and female cases indicates that the outbreak among female idus was most likely driven by the infections among male idus, who were far more numerous at the beginning of the epidemic. in the granger causality test, the lag length corresponds to the longest time over which one of the variables could help to predict the other. therefore, the minimum p-value at a lag of 2 months for both the time series of case numbers and the first differences of the time series suggests that the strongest causality relationship, of male infections causing female infections, was within 2 months, a rather immediate relationship that led to the sudden upsurge in reported female hiv/idu cases shortly after the upsurge of the male idu cases in early 2004. it also indicates that there is likely some needle-sharing or apparatus-sharing among and between male and female idus, although one cannot rule out the possibility of sexual contact between the male and female idus, since one study 25 in taiwan found that 99.1% of the hiv-infected idus participating in the study were heterosexual. unfortunately no data on any relationships that might exist between the reported cases were available. the study further demonstrates how quickly a disease traditionally endemic in a male population can develop into an epidemic in the female population given appropriate circumstances; hence transmission across gender is an important aspect of disease surveillance. in summary, the abrupt outbreak among idus in taiwan in 2004-2007, which was caused by the recently introduced crf07_bc recombinant, led to two waves of infection with increasing transmissibility as measured by the effective reproduction number r during each wave, suggesting the presence of a small first wave in 2004 before the explosion of cases after may 2005. this further demonstrates the future potential of real-time modeling and analysis of disease data 27 as part of a disease surveillance system, which could conceivably detect and alert the authorities of a possible herald wave before the arrival of a major outbreak. the waves ended by march 2007, which could be attributable to a timely and effective harm reduction program implemented in august 2005; this has been essential in preventing further occurrences of wide-spread infections among idus in taiwan since 2007. furthermore, the infections among male idus led to the epidemic among the female idus, a population that had previously been mostly devoid of hiv infections. the limitation of this modeling study arises mainly from the nature of the hiv surveillance data, which typically consists of a longer period of time due to the long hiv incubation period, and hence is highly dependent on temporal changes in testing, reporting, and interventions over the years. in this study, the data that were used spanned a period of roughly 3 years, during which time the explosive outbreak among idus emerged; this subsequently led to interventions that included wider testing of idus in taiwan and the harm reduction program. moreover, while the simple mathematical model that was employed was able to reveal the temporal progression, culmination, and conclusion of the outbreak, it was unable to further pinpoint the exact impact of these intervention measures on the outbreak, which would require much more detailed data on the reported cases as well as a far more complicated mathematical model. hiv-1 crf07_bc infections, injecting drug users hiv-1 in taiwan molecular epidemiology of hiv-1 infection and full-length genomic analysis of circulating recombinant form 07_bc strains from injecting drug users in taiwan the changing epidemiology of prevalent diagnosed hiv infections in taiwan disease control and prevention. hiv/aids database timeline of the hiv epidemic among the idu population in taiwan diseases%20&%20prevention/issues%20of%20hiv-aids/statistics%20of%20hiv-aids/download%20hiv-aids%20data.htm a recent outbreak of human immunodeficiency virus type 1 infection in southern china was initiated by two highly homogeneous, geographically separated strains, circulating recombinant form ae and a novel bc recombinant asia and africa: on different trajectories? the lack of epidemiological link between the hiv type 1 infections in hong kong and mainland china empirical bayes approach to estimate the number of hiv-infected individuals in hidden and elusive populations ascertaining hiv underreporting in low prevalence countries using the approximate ratio of underreporting surveillance of hiv type 1 recent infection and molecular epidemiology among different risk behaviors between 2007 and 2009 after the hiv type 1 crf07_bc outbreak in taiwan a flexible growth function for empirical use severe acute respiratory syndrome epidemic in asia real-time forecast of multi-wave epidemic outbreaks turning points, reproduction number, and impact of climatological events on multi-wave dengue outbreaks how generation intervals shape the relationship between growth rates and reproductive numbers intervention measures, turning point, and reproduction number for dengue testing for unit roots in autoregressive moving average models of unknown order investigating causal relations by econometric models and crossspectral methods epidemiological parameters of hiv transmission needles that kill: modeling human immunodeficiency virus transmission via shared drug injection equipment in shooting galleries rm. drugs, sex and hiv: a mathematical model for new york city population dynamics of hiv-1 inferred from gene sequences molecular epidemiology of hiv-1 subtype b, crf01_ae, and crf07_bc infection among injection drug users in taiwan oral presentation epidemic modeling in real time: 2009 novel a (h1n1) influenza outbreak in canada yhh is supported by grants nsc 100-2314-b-039-028-my3 and 100-2115-m-039-002 from the national science council of taiwan. the author is grateful to the reviewers for their constructive and insightful comments, which significantly improved this manuscript.conflict of interest: no competing interest declared. key: cord-316273-vo6j8zb0 authors: cosset, françois-loic; lavillette, dimitri title: cell entry of enveloped viruses date: 2011-02-08 journal: adv genet doi: 10.1016/b978-0-12-380860-8.00004-5 sha: doc_id: 316273 cord_uid: vo6j8zb0 enveloped viruses penetrate their cell targets following the merging of their membrane with that of the cell. this fusion process is catalyzed by one or several viral glycoproteins incorporated on the membrane of the virus. these envelope glycoproteins (envgp) evolved in order to combine two features. first, they acquired a domain to bind to a specific cellular protein, named “receptor.” second, they developed, with the help of cellular proteins, a function of finely controlled fusion to optimize the replication and preserve the integrity of the cell, specific to the genus of the virus. following the activation of the envgp either by binding to their receptors and/or sometimes the acid ph of the endosomes, many changes of conformation permit ultimately the action of a specific hydrophobic domain, the fusion peptide, which destabilizes the cell membrane and leads to the opening of the lipidic membrane. the comprehension of these mechanisms is essential to develop medicines of the therapeutic class of entry inhibitor like enfuvirtide (fuzeon) against human immunodeficiency virus (hiv). in this chapter, we will summarize the different envelope glycoprotein structures that viruses develop to achieve membrane fusion and the entry of the virus. we will describe the different entry pathways and cellular proteins that viruses have subverted to allow infection of the cell and the receptors that are used. finally, we will illustrate more precisely the recent discoveries that have been made within the field of the entry process, with a focus on the use of pseudoparticles. these pseudoparticles are suitable for high-throughput screenings that help in the development of natural or artificial inhibitors as new therapeutics of the class of entry inhibitors. enveloped viruses have a core incorporating the genetic material of the virus surrounded by a lipidic membrane acquired from the cells they bud from. their genetic material enters target cells following the merging of their membrane with the membrane of the cells at either the plasma membrane or the membrane of another internal compartment. the process of membrane merging is called membrane fusion, which preserves the integrity of the cell membrane. the fusion of two separate lipid bilayers in a nonaqueous environment first requires that they come into close contact. this is followed by an intermediate stage characterized by the merger of only the closest contacting monolayer, a process called hemifusion. third, the fully completed fusion results in whole bilayer merging, followed by the opening of the pore. to achieve this process, the envelope glycoproteins (envgp) have evolved in order to combine two features. on the one hand, they acquired a domain to bind to a specific cellular protein, named "receptor." on the other hand, they developed in a different manner, according to the genus of the virus, a function of fusion that allows the destabilization of the membrane and the opening of a pore through which the genetic material will enter the cell. despite three different classes of fusion protein having been described so far, three common main steps are described for achieving the pre-to postconformational changes. the first one, after envgp activation upon receptor binding or acidification of the endosomal compartment, exposes the fusion peptide that is projected toward the top of the glycoprotein, allowing the initial interaction with the target membrane. the second one is the folding back of the c-terminal region onto a trimeric n-terminal region that leads to the formation of a postfusion protein structure. the final and third step also requires further refolding of the membrane proximal and transmembrane regions in order to obtain a full-length postfusion structure where both membrane anchors (fusion peptide and tm domains) are present in the same membrane. the viral glycoprotein-induced fusion must be controlled to allow the virus to leave the cell, to prevent the aggregation of the viruses, and to release genetic material next to a compartment that will permit the continuity of the infectious cycle. this regulation is executed principally by the inactivation or the masking of the fusion machinery that directly disorganizes the lipid bilayer. on one hand, most of these envgp are synthesized as a precursor that requires a cleavage by a cellular protease (like furin) and prepares the molecules for the subsequent necessary changes of conformation for the fusion process. on the other hand, the activation of the conformational changes is induced by interaction with the receptor and/or the action of the ph (that protonates some amino acids), modifying the interactions of the envgp and their structure. in addition, some proteases or other enzymes are necessary to achieve some complementary priming of the envgp to make it competent for fusion (such as cathepsin b and l). two types of fusion mechanisms can occur, namely, ph independent and ph dependent. in the first case, the recognition between virus and receptor directly triggers conformational changes in the envgp that leads to the direct fusion between the two membranes (viral and plasma) and to the liberation of the viral genetic material. this activation of envgp at neutral ph allows the fusion in vitro and in vivo of envgp-expressing cells with receptor-expressing cells. this fusion leads to the merging of cell cytoplasms and to the generation of multinucleated cells called syncytia. in the second case, for ph-dependent fusion, the interaction between the envgp and the receptor leads to the obligatory endocytosis of the virus-receptor complex, and the acidification of endosomes triggers conformational changes in the envgp. for the ph-dependent virus, such a fusion can be reproduced in cell culture in vitro or in a liposomevirus fusion assay in the tube after decreasing the ph, but cannot occur in vivo. research during the last few years has greatly advanced our understanding of the cell surface receptors for viruses and has provided many surprising insights. these advances were achieved largely by identification and molecular cloning of the cell surface or cytoplasmic proteins that have been subverted for use as viral receptors or cofactor, and by parallel advances in studies of the viral envgp that bind to the receptors. another key area of new insights concerns the physical-chemical process of viral adsorption and of pulling the virus closely onto the cellular membrane. indeed, adsorption is a severely limiting step in infections of cultured cells, and the initial attachment often does not involve the receptors that ultimately mediate infections (andreadis et al., 2000; guibinga et al., 2002; pizzato et al., 1999 pizzato et al., , 2001 ugolini et al., 1999) . thus, we need to distinguish cell surface molecules such as heparan sulfate proteoglycans, dc-sign, or integrins that can enhance infections by concentrating retroviruses onto cells (bounou et al., 2002; geijtenbeek et al., 2000; jinno-oue et al., 2001; mondor et al., 1998; pohlmann et al., 2001; saphire et al., 2001) from authentic receptors that induce conformational changes in envgp that are a prerequisite for fusion of the viral and cellular membranes. in contrast to other cell surface components such as lectins or proteoglycans that influence infections indirectly by enhancing virus adsorption onto specific cells, the true receptors induce conformational changes in the viral envgp that are essential for infection. however, it appears that more and more intracellular proteins have roles in controlling viral host ranges, and the proteins involved in traffic of intracellular vesicles like endosomes, play a critical role in entry. therefore, proteins from pathways specifically characterized might be considered as a cofactor as their role is not to mediate direct contact with envgp but is crucial for entry. in addition, some viruses are able to use more than one endocytic pathway and the cellular proteins involved thus direct the virus to the entry door beneficial for the virus under a particular condition. pseudoparticles are retroviridae cores which incorporate heterogeneous envelope protein from a different virus, possibly of a different family. their entry process mimics precisely that of the wild-type viruses from which the envelope glycoprotein was derived. they represent a useful tool to study molecular processes of envelope viruses as they are very flexible, allowing the analysis of numerous mutants. moreover, they allow a precise measurement of the infectivity that depends on the envelope glycoprotein for the entry step and they allow the establishment of virus-liposome fusion assays. we will first introduce envelope architecture and structure of the viral fusion machineries that have been developed to achieve the fusion and entry steps. despite many differences of structure, they share a common refolding process that activates different fusion domains found in most fusion proteins. we will then illustrate the different fusion regulation processes that have been developed by viruses to lead to functional virions, both in terms of cleavage activation of fusion proteins during exit and in terms of activation during binding and endocytosis. despite some differences between distinct players, some common principles can be proposed for all fusion processes. we will illustrate the molecular details characterizing the maturation of the different fusion proteins, defined by the following three characteristics: the cleavage of an envelope protein precursor, the presence and triggering of the exposition of a fusion peptide, and an association as a trimeric complex association in its active fusion conformation. the progression of these structural rearrangements slows down the kinetic barrier between hemifusion and fusion-pore formation. in a second part, we will present the different entry pathways and cell proteins that are used by viruses to infect cells. viruses have emerged as valuable tools for the study of endocytic mechanisms. these properties have been crucial for the development of pseudoparticles for their use in terms of vector for gene therapy and for their use in terms of tool for receptor cloning, transgenesis, or transduction. finally, we will give examples of strategies that have been developed in vivo and in vitro to inhibit the entry step of the enveloped viruses which have led, in the case of hiv, to the development of inhibitors that are used in the clinic. a. membrane fusion according to the stalk-pore model envgp are responsible for bringing the membranes closer, triggering the link and the destabilization of the outer leaflet, the merging of the whole membrane, and the opening of a pore through the cell and virus membrane to allow the core to enter the cytoplasm of the cell. the hypothesis of the pore model in viral membrane fusion mechanism is supported by experimental results. the first evidence for a hemifusion intermediate was achieved by studying influenza virus entry that occurs after the hemagglutinin glycoprotein binding to the host cell. the substitution of the hemagglutinin transmembrane domain by a glycosylphosphatidylinositol (gpi) revealed the importance of the transmembrane region for the fusion pore opening and expansion. hemifusion structures are connections between outer leaflets of apposed membranes, whereas the inner leaflets remain distinct. this is a transient structure that either dissociates or gives rise to the fusion pore (chernomordik and kozlov, 2008) . interestingly, the helix breaker residues within the tm domain are critical for the fusogenicity of different retroviral env, such as hiv (owens et al., 1994) and mo-mlv (taylor and sanders, 1999) , being important for both the hemifusion and pore opening step of the fusion process. in addition, hemifusion intermediate has been detected in the case of hiv env-mediated fusion (munoz-barroso et al., 1998) by using peptide inhibitors that target a prefusion or prehairpin structure such as hiv-1 gp41 t-20. once the pore is formed, it allows a connection between two compartments initially separated by the apposed membranes. the ability of the membrane to hemifuse and develop a fusion pore has been found to depend on the lipid microdomain composition, for example, cholesterol (chernomordik and kozlov, 2003) . indeed, a potential lipid dependence of virus entry processes was first deduced from experiments on influenza virus, implying a role for detergent-resistant lipidic microdomain (takeda et al., 2003) . for retroviruses, the tm palmitoylations which contribute to the env localization in detergent-resistant lipidic microdomain domains (li et al., 2002) indirectly influence the fusion process (ochsenbauer-jambor et al., 2001) . as an alternative to the lipidic pore hypothesis, a direct fusion mechanism has also been proposed. the fusion pore is a full proteic channel-like structure dependent only on the transmembrane domains of the glycoproteins. in this model, the pore is opened by the joining of two hemipores located on each membrane kozlov, 2005, 2008) . after fusion pore opening and enlargement (melikyan et al., 2005) , the genetic material enters the cytoplasm of the cell, instigating the virus cell cycle. the fusion of the viruses that enter directly at membrane plasmic level (as with the paramyxoviruses and most retroviruses) is triggered by the activation of the viral envelope protein at neutral ph. in this case, only the binding of the virus to its receptor activates the fusogenic potential of the complexes of envgp. this mechanism is ph-independent. in the case of retroviruses, the binding of the surface subunit (su) to its receptor induces conformational changes not only in itself but also in the tm with which it interacts, thus inducing fusion. this activation at neutral ph permits the envelope glycoprotein of ph-independent viruses, in certain experimental conditions or in vivo, to induce the fusion between the cells expressing the envelope glycoprotein and the cells expressing the receptor (harrison, 2008; kielian and rey, 2006; weissenhorn et al., 2007) . this intercellular fusion, by merging their plasma membranes, places the cell cytoplasms in continuity, and one or more cells become giant multinucleated cells named syncytia. in contrast, the fusion of fig. 4 .1 most other viruses depends strictly on their internalization into one of the numerous endocytic pathways such as the clathrin-dependent, clathrin-independent, and caveolae-independent, as well as the macropinocytosis (vaccinia virus) (mercer and helenius, 2008) or the phagocytose (as recently described for equine herpesvirus 1 virus (frampton et al., 2007) and the hiv virus (trujillo et al., 2007) , although in this case not resulting in a productive infection; marsh and helenius, 2006; sieczkarski and whittaker, 2002; figure 4 .1). as described so far, the enveloped viruses that use these itineraries have fusion reactions that require exposure to a moderately acid ph in the different endocytosis vesicles (ph-dependent viruses). classical examples of viruses that fuse at low ph with the membrane of endosomes or artificial membranes in the test tube include the influenza orthomyxovirus, the dengue or the tick-borne encephalitis (tbev) flaviviruses, and the semliki forest alphavirus (sfv) (harrison, 2008; kielian and rey, 2006; roche et al., 2008; weissenhorn et al., 2007) . interestingly, an intermediate mechanism has been described for two retroviruses, asls and jsrv. the proposed mechanism of aslv virion entry occurs in two steps involving a receptor-priming step that induces env conformational changes, thus allowing the env to become sensitive to the action of acid ph (mothes et al., 2000) . this hybrid mechanism does not lead to cell-cell fusion in vivo. jsrv also uses receptor priming for fusion activation of jsrv env at a low ph, but the mechanism differs slightly to aslv, requiring dynaminassociated endocytosis (bertrand et al., 2008) . so far, many different endocytosis pathways have been described (marsh and helenius, 2006; mercer and helenius, 2009; mercer et al., 2010b) as being used by both ph-dependent and ph-independent viruses. however, reinvestigations of these entry pathways are clearly needed for many ph-independent viruses that were originally thought not to rely on endocytosis. for example, nipah paramyxoviruses induce fusion between cells at neutral ph and were considered as a ph-independent virus not reliant on endocytosis for entry. however, recently, it was proposed that nipah viruses (nivs) use macropinocytosis for entry (pernet et al., 2009) . it should be noted that viruses that use a phindependent mechanism of activation of envgp may still enter the cell by endocytosis without any imperative requirement for acidification activation of envgp in the endosomes. clathrin-dependent endocytosis is the best-characterized pathway of entry and is the major itinerary of entry for ph-dependent viruses. this process is initiated by the formation of the characteristic invaginations of membrane, known as clathrin-coated pits (ccp) (fig. 4.1) . the ccp assembly takes place on the internal face of the plasma membrane following a signal induced by the activation of a receptor. this clathrin-dependent method of internalization is used by the semliki forest and sindbis alphaviruses, the rubella rubivirus, and the viruses exploit different endocytosis pathways to enter host cells. multiple mechanisms have been defined as pinocytic, that is, they are involved in the uptake of fluids, solutes, and small particles. these include clathrin-mediated, macropinocytosis, caveolar/raftmediated mechanisms, as well as several novel mechanisms. large particles are taken up by phagocytosis, a process restricted to a few cell types. though poorly documented, the entry of some viruses can be mediated by numerous cargoes which can be endocytosed by mechanisms that are independent of the clathrin coat protein and the fission gtpase, dynamin. these pathways include rhoa-(il-2 pathway), arf6-, and cdc42-regulated pathway (geec pathway). after binding of the particles to the cell surface entry receptor, genetic material is delivered into the cytoplasm of the cell via a specific endocytosis pathway. recently, macropinocytosis has emerged as an important entry pathway for viruses (1). the entry of most ph-dependent viruses is mediated by the use of clathrine-coated endocytic vesicles (2). other viruses penetrate host cells via the formation of vesicles covered by caveola molecule (3). using this pathway, the viral particles are targeted to neutral ph caveosomes or to early endosomes with moderately acid ph. alternately, the virus can use non-clathrin, non-caveolin-dependent pathways, both dynamin-dependent or independent (4, 5, 6). these pathways are differentiated by the implication of different gtpases (rhoa, cdc42, or arf6) and their different compositions (cholesterol, flotillin, tem, etc). hantaan hantavirus, for example (detulleo and kirchhausen, 1998; helenius et al., 1980; jin et al., 2002; kee et al., 2004) . macropinocytosis is another endocytosis pathway utilized by viruses belonging to vaccinia, adeno, picorna, and other virus families. macropinocytosis is an endocytic mechanism normally involved in fluid uptake induced by growth factors, phorbol ester. however, the binding of virus particles can also activate signaling pathways that trigger actinmediated membrane ruffling and blebbing. this is followed by the formation of large vacuoles (macropinosomes) at the plasma membrane, internalization of virus particles, and penetration by the viruses or their capsids into the cytosol through the limiting membrane of the macropinosomes (mayor and pagano, 2007; mercer and helenius, 2009) . a variety of evidences suggest that macropinocytosis can be a mechanism for hiv-1 and epstein-barr herpes virus entry in some primary cells or cell lines (marechal et al., 2001; miller and hutt-fletcher, 1992) . based on the more defined criteria of macropinocytosis (mercer and helenius, 2009) , these conclusions are probably premature and may depend on the experimental conditions that use very concentrated viruses which may influence the utilization of less specific pathways. it should be noted that different strains of the same virus can elicit dramatically different responses in host cells during entry, and different macropinocytic mechanisms are possible in the same cell line through subtle differences in the activating ligand (mercer et al., 2010a ). an additional endocytosis pathway, whilst badly defined, is known as a clathrin-and caveola-independent pathway. the dependence on different gtpase and cargo proteins (dynamin, rho, cdc42, arf6, etc.) defines this endocytosis pathway. several viruses are using this clathrin-and caveolaindependent pathway, including the influenza orthomyxovirus (as a second possible pathway), the sars-cov coronavirus, the lymphocytic choriomeningitis arenavirus (lcmv), and picornaviruses (madshus et al., 1987; matlin et al., 1981; wang et al., 2008) . finally, other vesicles (characterized by caveolin or caveosome, with a high concentration in cholesterol and sphingolipids) are clathrin-independent but dynamin-dependent and do not have acid compartments before their merging with early endosomes (mayor and pagano, 2007) . the nonenveloped sv40 virus and some human enteroviruses are the prototypes of the ph-independent viruses that use this endocytosis pathway. so far, only two enveloped viruses have been described to use this pathway, the newcastle disease paramyxovirus (ndv) (cantin et al., 2007) and ebola virus (empig and goldsmith, 2002; sanchez, 2007) . most viruses have been shown to use only one entry pathway; however, there are more and more recent examples that indicate that some viruses use multiple endocytosis pathways to enter their target cells (influenza, hiv). for example, ebola viruses are known to enter cells by clathrin-mediated endocytosis (sanchez, 2007) , but lipid raft-associated, caveolin-mediated endocytosis has also been proposed as a mechanism of ebola virus uptake (empig and goldsmith, 2002) . in terms of the activation process of the membrane fusion and the need for particular microdomains, it is not always clear why the viruses are using so many different pathways. it is possible that the viruses that evolved to fuse intracellularly have a selective advantage to release their genome to specific intracellular sites that will allow the rapid and efficient establishment of the infectious cycle. one selective advantage could also be the requirement for particular lipid microdomains, like those enriched in cholesterol. indeed, it was shown that contrary to the class ii flavivirus dengue virus and the yellow fever virus, the e1 fusion protein from semliki forest virus (sfv) binds cholesterol which explains its dependence for cholesterol and compartment containing cholesterol (umashankar et al., 2008) . for other viruses, the dependence on cholesterol is linked to bulk effects on membrane fluidity and the maintenance of particular microdomains where receptors are located and where they have some particular diffusion. in addition to these dichotomies of ph-independent or ph-dependent fusion process, of plasma or endosomes membrane fusion, the mechanisms of activation of the fusion proteins of the different enveloped viruses are very diverse. some reactions of fusion are triggered by the interaction of one envelope glycoprotein with a unique receptor. one example is the interaction of the envelope glycoprotein su-tm of the -retrovirus with the multitransmembrane receptor (table 4 .1). in another case, one unique envelope glycoprotein can interact with several receptors. for example, the fusion of hiv-1 is triggered by the sequential interaction of the su gp120 with the cd4 receptor, followed by interaction with a coreceptor such as ccr5 or cxcr4, chimiokine receptors of seven transmembrane domains (hunter, 1997) . previously, it was believed that binding to receptors directly triggered a series of conformational changes in the viral envgp culminating in fusion of the viral and cellular membranes. however, new evidence suggests that -retroviral association with receptors triggers an obligatory cooperative interaction or cross-talk between envgp on the viral surface for hiv and mlv (tailor et al., 2003) . if this intermediate step is prevented, infection fails. conversely, in several circumstances, this cross-talk can be induced in the absence of a cell surface receptor for the virus, in which case, infection can proceed efficiently. this new evidence strongly implies that the role of cell surface receptors in infections of -retroviruses (and perhaps of other enveloped animal viruses) is more complex and interesting than was previously imagined. in all these cases, the envgp have a double function attachment to the receptor and an exclusive role in fusion in the same protein. however, for other viruses, these two functions are filled by different proteins. the e2 protein of the glut-1 neuropilin 1 hs immune recognition (1 tmd) chimiokine receptor (7 tmd) na-pi cotransporter (10 tmd) na-pi cotransporter (10 tmd) na-pi cotransporter (10 tmd) g-protein coupled receptor (10 tmd) cotransport na-a.a. neutres (7 tmd sfv alphavirus binds the receptor that permits the endocytose of the virus in a compartment where the acid ph will activate the e1 protein that will induce the fusion (table 4 .1). for most paramyxoviruses (including newcastle parainfluenza virus, human parainfluenza virus type 3 (hpiv-3), mumps virus, bovine rsv (brsv), ovine rsv (orsv), and human rsv (hrsv)), the hypothesis is that the binding of hn or g/sh to their receptor induced not only conformational changes in the receptor but also in the f protein with which it interacts. these changes make f competent for the membrane fusion, with the exception of the f protein of the simian parainfluenza virus 5 (sv5), the brsv, orsv, and hrsv that do not strictly require hn or g/sh to induce the fusion (though the fusion is greatly increased by hn or g/sh, respectively). this complexity in the distribution of the functions is still more evident in, for example, the alphaherpesvirus (hsv-1, hsv-2) (rey, 2006) . in this case, the binding of the gd glycoprotein to one of its three receptors (hvem for herpesvirus entry molecule mediator, or nectin 1, or a specific heparan sulfate) activates a complex of gb trimers associated to gl and gh proteins, all the three being essential to the fusion (gianni et al., 2006; table 4 .1). in the same way as for the hepatitis c virus, the e1e2 heterodimer induces entry following the recognition of at least four receptors, cd81, srb1, claudin, and ocludine (zeisel et al., 2009) . another interesting variation of activation of fusion is the artificial reverse process. in some cases, it was described that some exosomes or pseudoparticle-incorporating receptors were able to enter cells expressing envelope. for example, the multitransmembrane receptor mcat-1 from ectropic murine leukemia virus (mlv) or the tva receptor for avian sarcoma-leukosis virus (aslv-a) can be used to infect cells that express their respective retroviral envgp (balliet and bates, 1998) . similarly, the incorporation of both cd4 and one other coreceptor of human immunodeficiency virus (hiv), ccr5 (endres et al., 1997) or cxcr4 (endres et al., 1997; mebatsion et al., 1997; schnell et al., 1997) , allows the production of viral particles infecting cells that express simian immunodeficiency virus (siv) or hiv envelope glycoprotein. interestingly, it was shown that nef, an accessory protein of human immunodeficiency virus type 1 (hiv-1) that enhances the infectivity of progeny virions when expressed in virus-producing cells, significantly enhanced the infectivity of cd4-chemokine receptor pseudotypes for cells expressing hiv-1 env. surprisingly, nef also increased the infectivity of hiv-1 particles pseudotyped with tva, even though nef had no effect if the ph-dependent env protein of aslv-a was used for pseudotyping (pizzato et al., 2008) . this process indicates that the difference of superficial tension between the cell (weak because the radius of the cell is big) and the virus (strong as the radius of the virus is small) is not crucial for the development of an oriented fusion process with envgp on the one side and receptor on the other. having said that, the cell is not an empty bubble, and this efficacy of the fusion process regardless of which membrane harbors the receptor or the envgp may reflect the adaptation of the virus to optimize the membrane merging independently of the superficial tension. cells are not completely round, and superficial tension depends on the localization where the virus binds. moreover, the plasma membrane has different lipid compositions and interacts with the cytoskeleton which will attenuate or increase the superficial tension at certain localizations. d. structural classification: class i, class ii, and class iii fusion proteins glycoproteins from enveloped viruses have evolved to combine two main features. first, they have the capacity to bind with a specific cellular receptor and second, they include a fusion domain (peptide fusion and transmembrane domain) that can be activated to mediate the merging (fusion) of viral and cellular membranes. three different classes of viral fusion proteins have been identified to date based on key structural features at pre-and postfusion stages. many studies have demonstrated that the structural transition from a pre-to a postfusion conformation leads to a stable hairpin conformation. this includes class i fusion proteins, characterized by trimers of hairpins containing a central alpha-helical coiled-coil structure, and class ii fusion proteins, characterized by trimers of hairpins composed of beta structures. a third class of fusion proteins has been described recently, that also forms trimers of hairpins by combining the two structural elements alpha-helix and beta-sheet structures (roche et al., 2006 (roche et al., , 2007 . the synthesis and the conformation of these classes i, ii, or iii fusion proteins are different. class i viral fusion proteins of diverse virus families, including retroviridae, filoviridae, orthomyxoviridae, paramyxoviridae, and coronaviridae, differ greatly in size and amino acid sequence, but their membrane-anchored domains share common structural features that are essential for membrane fusion, including two heptad repeats (called hr-1 and hr-2), preceded by a hydrophobic fusion peptide. class i membrane-fusion reaction is mediated by the refolding of the fusion protein to a highly stable rod-like structure with a central trimeric -helical coiled coil. such coiled-coil structures are emblematic of class i proteins, and physical demonstration or computer prediction of such a structure is frequently used to help define a fusion protein as belonging to class i. the envelope glycoprotein of a retrovirus is generated by the cleavage of its precursor in one su and one transmembrane subunit containing an anchoring sequence to the membrane (hunter, 1997) . this maturation, essential to the process of fusion, frees a hydrophobic sequence to the n terminal extremity of the tm, named fusion peptide, as it is supposed to insert into the target membrane and initiates the fusion. initially, the fusion peptide is masked inside the trimer of envgp, a form competent for the fusion (fig. 4.2a ). in general, the cleavage of the fusion proteins is mandatory for most class i proteins to render them competent for fusion (earp et al., 2005; harrison, 2005) . exceptions are the envelope glycoprotein from ebola or sars-cov viruses, classified as class i on the grounds of the three-dimensional structure of one of their fragments, which are not cleaved but yet are functional. though an nterminal fusion peptide is predicted in the potential transmembrane subunit (localized by analogy to homologous viruses), their functionality can be explained also by the presence of an internal fusion peptide (ebola). another explanation of their functionality is their requirement for the l or b cathepsins that cleave the envgp during endocytosis (see below). the number of complexes on the surface of viruses harboring class i fusion proteins is very variable. it seems that lentiviruses have only 10-20 su-tm trimers, whereas the coronaviruses have hundreds of trimers coating the surface, giving them their characteristic morphology of "crown" which is the origin of the name of this family (tables 4.1 and 4.2). the process of assemblage and biosynthesis of class virus ii is very different to the class i proteins (kielian and rey, 2006) (fig. 4.2b) . during biosynthesis, the alphavirus (e1) and flavivirus (e) fusion proteins fold cotranslationally with a companion or regulatory protein, termed p62 (or pe2) for alphaviruses and prm for flaviviruses . this heterodimeric interaction is important for the correct folding and transport of the fusion protein. both p62 and prm are cleaved by the cellular protease furin late in the secretory pathway, in a maturation reaction that is a crucial regulatory step for subsequent virus fusion (salminen et al., 1992; stadler et al., 1997; wengler, 1989; zhang et al., 2003a) . though this cleavage in the envelope glycoprotein complex is important, contrary to class i fusion protein, it is not crucial, since mutated alphavirus and flavivirus for which the regulating proteins are not cleaved have only a decreased infectivity (salminen et al., 1992; zhang et al., 2003b) . in the case of the sfv alphavirus, the fusion process induced by the uncleaved fusion proteins can be a trigger after treatment of the virus at ph5 or less, rather than the normal fusion threshold ph 6 of the wildtype virus (salminen et al., 1992; zhang et al., 2003a) . this variation in the threshold of ph of the fusion is necessary for the dissociation of the heterodimer. the class ii fusion proteins are either homodimers or heterodimers (e homodimer for the flaviviruses or e2-e1 heterodimer for alphaviruses) that form an envelope netting the viral membrane (lescar et al., 2001; rey et al., 1995) . the internal fusion peptide is masked at the interface of the dimers at the extremity of a long beta sheet. one important difference between these two groups of viruses is the budding site . in alphaviruses, the p62-e1 complex is transported to the plasma membrane, and the heterodimer interaction is maintained after p62 processing. new virions bud at the plasma membrane, in a process that is driven by lateral contacts between e2 and e1 heterodimers (e2 being the mature companion protein) to induce the required curvature of the lipid bilayer, in combination with interactions of the cytosolic tail of e2 with the nucleocapsid. budding results in the formation of icosahedral-enveloped particles of triangulation t ¼ 4, containing 80 trimeric e2/e1 spikes. by contrast, flavivirus particles bud into the endoplasmic reticulum as immature virions formed by 60 trimers of prm-e. the immature particles have an organization similar to that of mature alphaviruses, with each trimer forming a of the endosomal compartment, exposes the fusion peptide that is projected toward the top of the glycoprotein, allowing the initial interaction with the target membrane. the second step is the folding back of the c-terminal region onto a trimeric n-terminal region that leads to the formation of a postfusion protein structure. the third and final step also requires further refolding of the membrane proximal and transmembrane regions in order to obtain a full-length postfusion structure where both membrane anchors (fusion peptide and tm domains) are present in the same membrane. three different classes of fusion have been identified so far based on common structural motives. (a) the class i fusion proteins are characterized by trimers of hairpins containing a central alpha-helical coiled-coil structure. for retroviruses, receptor binding induces the movement of the su, allowing a loop-to-helix transition of a polypeptide segment of tm that was previously buried underneath the su heads, projecting the fusion peptide 100 å toward the target membrane, where it inserts irreversibly. this occurs by a "spring-loaded" mechanism. the hr2 c-terminal end (green) of the long tm -helix jackknifes back, reversing the direction of the viral-membrane-proximal segment of tm, which then interacts in an antiparallel fashion with the groove formed by the n-terminal hr1 (blue) trimeric coiled coil. the final postfusion conformation of tm is, therefore, a highly stable rod with the tm and fusion-peptide segments together at the same end of the molecule, a structure termed a "trimer of hairpins" or helix buddle (hb). (b) class ii fusion proteins are characterized by trimers of hairpins composed of beta structures. the red, yellow, and blue parts of each subunit correspond, respectively, to domains i, ii, and iii of the ectodomain. the fusion loop is at the tip of domain ii. monomeric transition between the prefusion dimer and the trimeric-extended intermediate is shown. after exposure to the low ph of the endosomes, domains i and ii swing outward, while domain iii and the stem remain oriented against the membrane roughly similar to the prefusion state. the fusion loop, at the top of the diagram, interacts with the target bilayer. domains i and ii associate into the trimeric core of the postfusion conformation, and domain iii must then zip back along the trimer core, thus reorientating the domain iii. (c) a third class of fusion proteins has been described recently, which also forms trimers of hairpins by combining the two structural elements alphahelix and beta-sheet structures. class iii fusion proteins are composed of five domains that give rise to a molecular architecture very distinct from any reported class i or class ii fusion proteins. interestingly, the ectodomain of g has been crystallized in its pre and postfusion (low-ph) state. during the conformational change that occurs upon low ph exposure, the domains of g radically change their position and orientation as a result of rearrangements that occur in the linker regions. domain i (yellow), carrying the fusion loops, and the transmembrane domain move 16 nm from one end of the molecule to the opposite ). only domain iii (blue) undergoes significant refolding with extension of the central helix f. to complete the process, the c-terminal helices of domain iv (red) insert into crevices formed by two other protomers in the postfusion form, reminiscent of the structural changes observed during refolding events of class i fusion proteins with hb formation. spike in which prm covers the fusion protein e. the newly formed virions are then transported to the external milieu through the exocytic pathway. processing of prm generates the mature m protein with a short ( 40 residues) ectodomain (kuhn et al., 2002; zhang et al., 2003b) . presumably because of the removal of a large portion of the prm ectodomain, the flavivirus surface dramatically reorganizes after processing to give 90 e-e homodimers arranged with icosahedral symmetry. the mature flavivirus particles display a smooth, spikeless surface, with e dimers ordered in a characteristic "herring bone" pattern. there is no correlation between these criteria for class i and class iii fusion proteins, but all the viruses harboring class ii fusion proteins are ph-dependent. finally, the four class iii fusion proteins resolved (vsv-g, gb of hsv1 and ebv and baculovirus gp64) in spite of their homology of trimeric structure have some major functional differences arising from their difference of biosynthesis. g protein is the only class iii fusion protein whose prefusion structure is known. the proteins of class fusion iii have a combination of the two structural elements, alpha helix like class i and beta sheet like class ii heldwein et al., 2006; kadlec et al., 2008; roche et al., 2006 roche et al., , 2007 (fig. 4.2c) . the trimers are maintained by interaction between central alpha helixes, but each domain of fusion exposes two buckles of internal fusion peptide placed at the extremity of a long beta sheet. the g protein is the only protein responsible for the binding and the entry of the vsv. the rhabdovirus vsv possesses 1200 molecules of the g protein on its surface and forms 400 trimers. in contrast, the hsv-1 virus incorporates 12 different envgp on its surface, of which 4 are essential for the entry step (gd, gb, and gh/gl) (turner et al., 1998) . during their biosynthesis, neither the g protein of vsv nor gb of hsv-1 are cleaved, and they present internal fusion peptides. however, whereas the g is functional by itself, the gb envelope glycoprotein alone is not sufficient to induce the entry of the virus or the membrane fusion. nevertheless, currently, no precise interaction between gd, gb, and gh/gl has been identified, even though the gd ectodomain has been shown to allow the entry of engineered hsv-1 virus particles that lack gd (that is gd-null mutants; cocchi et al., 2004) . gp64 is the major component of the viral envelope, and the sole fusogenic proteins that are triggered to induce the fusion in the low ph environment of endosomes for baculovirus. interestingly, the distinguishing feature between g and gp64 and any other fusion protein is that they can undergo a reversible conformational change, unlike class i and most class ii fusion proteins, for which the postfusion conformation is thermodynamically more stable at all ph values, and the conformational rearrangement is effectively irreversible. vsv or gp64 exposure to low ph inactivates the virus, but the fusion activity can be fully recovered when the ph is raised. it has been proposed that the reversibility of the conformational change allows g to avoid unspecific activation during transport through the acidic golgi vesicles. as seen in the previous sections, though there is much described variation in the manner of activation of the fusion proteins and additional mechanisms await discovery, only three classes of fusion proteins have been defined so far (weissenhorn et al., 2007) . as previously described, based on the main structural organization of their envgp, the viruses are now assigned to the class i, ii, or iii. in parallel, based on the activation mechanism of the fusion protein, viruses have been classified as ph-dependent or -independent. interestingly, the relationship between the mechanism of activation of the fusion and the class of protein of fusion is not correlated. the hiv or influenza envgp are both class i and yet they have a process of activation that is ph-independent and -dependent, respectively, for the entry of the virus. similarly, the class iii fusion proteins enclose the herpes virus simplex 1 (hsv-1) gb protein and the rhabdovirus vsv-g protein that are ph-independent and ph-dependent, respectively, for their activation. so far, all the class ii fusion proteins have been phdependent for their activation. although there are notable differences between the activation processes, the structural motives used, and the initial oligomeric states of the fusion proteins (the native trimeric conformation of class i and iii proteins in opposition to the homo-or heterodimers of the class ii fusion proteins), the common features of the final structures obtained after fusion seem to suggest some generic mechanism of conformational changes common to all envgp of enveloped viruses (see fig. 4 .2). first, the activation of the fusion protein following the interaction with the cellular receptor(s), coupled or not to the exposure to the acid environment of the endosomes, exposes the fusion peptide that is projected toward the top of the glycoprotein, allowing the initial interaction with the cellular target membrane. for class i fusion proteins, the proposed model indicates that the transition of conformation requires the transformation of a part of the molecule in alpha helix and the association of this in three helixes bundle (named the "coiled coil"; weissenhorn et al., 2007) . for retroviruses, this movement allows a loopto-helix transition of a polypeptide segment of tm that was previously buried underneath the su heads, projecting the fusion peptide 100 å toward the target membrane, where it inserts irreversibly. in the case of class i fusion proteins like retroviruses, this occurs by a "spring-loaded" mechanism. this initial change is proposed to result in a "prehairpin intermediate," an extended structure that is anchored both in the target membrane by the fusion peptide and in the virus membrane by the tm transmembrane segment. for the class ii fusion proteins, the projection of the fusion peptide requires the dissociation of the hetero-or homodimers and modifications in the "hinge region" unstructured before conformational changes (stiasny and heinz, 2006) . for the class iii (roche et al., 2008) , similarly to the fusion proteins of class i, the exhibition of the fusion peptide requires a rearrangement of the domains mediated by modification of the central helixes that do remain parallels. second, the folding back of the region including the fusion peptide onto a trimeric c-terminal region leads to the formation of a postfusion protein structure with the outer regions zipped up against an inner trimeric core. interestingly, it has been described that all the class i, ii, and iii peptides can inhibit this step by competing with the interaction of envgp-specific domain for the formation of this structure. third, the final steps require further refolding of the juxtamembrane and transmembrane regions to obtain a stable postfusion structure with the fusion peptides and the transmembrane domains at the same extremity of a stable stem of a protein complex anchored in the target membrane. this structure brings the two membranes proximal and provides free energy to overcome the barrier of membrane merging (melikyan, 2008) . membrane fusion occurs, which leads to pore formation and release of the viral genome into the cytoplasm. the refolding of class ii fusion proteins generates trimers from monomeric intermediate. the existence of monomeric intermediates for class i and iii is not well known. however, if the steps that led to exposition of the fusion peptide and its interaction with the membrane targets maintain a three-order symmetry, the refolding of the c-terminal region requires the destruction of the trimer at least to the juxtamembrane region. moreover, from the differences observed between the amino acids involved in the interface of the reconstituted resolved trimers in their pre-and postfusion conformations, it is probable that the conformational changes are going through a monomeric intermediate for the class iii g envelope glycoprotein of vsv and the class i f protein of paramyxovirus. on the contrary, the interface of the class i ha2 subunit of the ha envelope glycoprotein trimer is very similar between the pre-and postfusion conformation, shedding doubt on the existence of monomeric intermediates. in contrast, when fusion is initiated at low ph, the dimers of the class ii fusion proteins are broken, freeing monomers that reassociate in trimers. finally, precise structural information of the native metastable conformation (prefusion) and the final stable conformation (postfusion) is available only for a limited number of viruses (for envelope glycoprotein of influenza virus, sfv, tbev, vsv, and the parainfluenza 5 f protein). the structural conversion of the native metastable conformation in a final stable conformation is not precisely known and is highly speculative for most viruses, and the envelope glycoprotein domains implied in these molecular rearrangements are littlereferenced. clearly, more studies are necessary to identify the intermediaries of envelope glycoprotein conformations. these intermediates would identify the domains that interact during the conformational changes which will highlight ways to generate inhibitory peptides. f. domain organizations/fusion domains 1. acquisition of fusion competence: priming by cleavage during virus production, the host cell is basically preserved, since the expression of fusogenic competent glycoproteins is highly controlled for most viruses. however, for some viruses, the envgp induce a cytopathic effect that leads to the generation of multinucleated cells, called syncytia, which are induced by the fusion of cells that express the envelope glycoprotein and receptors. abundant glycoprotein at the surface of the cell could induce cellular death by syncytia formation, toxicity via receptor interaction, or immune recognition. for these reasons, the localization and the amount of the oligomerized envelope glycoprotein at the host cellular surface are highly modulated by fine trafficking and sequestration mechanisms. the receptor interference mechanism (either by saturation of binding site on receptor by envelope glycoprotein or by internalization of receptor by different viral proteins, as it is achieved by some retroviruses; hunter, 1997) can also limit the amount of receptors available for fusion between infected cells. the control of the cleavage of the envgp to free the fusion peptide is also a regulation process of the fusion protein fusogenicity (labonte and seidah, 2008) . finally, envgp fusion competency may be a late event that occurs during virus budding, as described for mulv retroviruses (rein et al., 1994) . proteolytic priming is a common method of controlling the activation of membrane fusion mediated by viral glycoproteins. the members of the proprotein convertase (pc) family play a central role in the processing and/or activation of various protein precursors involved in many physiological processes and various pathologies such as neurodegenerative pathology, cancer bacterial toxins activation, and viral infections. the proteolysis of these precursors that occurs at basic residues within the general motif (k/r)-(x)-(k/r) is mediated by the proprotein convertases pc1/3, pc2, furin, pace4, pc4, pc5 (also called pc6), and pc7 (also called pc8, lpc, or spc7), whereas the proteolysis of precursors within hydrophobic residues performed by the convertase s1p/ski-1 and the convertase narc-1/pcsk9 seems to prefer to cleave at a lvfaqsip motif (lahlil et al., 2009 ). the seven pcs have different, albeit partly, overlapping expression patterns and subcellular localization. they have conserved aminotermini with highest homology in the subtilisin-like catalytic domain. data on various infectious viruses revealed that the cleavage of their envelope glycoprotein precursors by one or more pcs is a required step for the acquisition of the infectious capacity of viral particles. indeed, various studies have demonstrated the capacity of the pcs to correctly cleave a variety of viral surface glycoproteins. these include the hiv-1 gp160 (decroly et al., 1996) and surface glycoproteins of hong kong, ebola virus, the severe acute respiratory syndrome coronavirus and chikungunya virus (basak et al., 2001; bergeron et al., 2005; ozden et al., 2008) . in parallel, other studies revealed that the inhibition of processing of these viral surface glycoproteins by the pc inhibitors such as dec-r-v-k-r-cmk completely abrogated the virus-induced cellular cytopathicity. the surface glycoproteins of other viruses, particularly the hemorrhagic fever viruses (arenaviridae family), such as lassa (basak et al., 2002; lenz et al., 2001) , crimean congo hemorrhagic fever (vincent et al., 2003) , and lymphocytic choriomeningitis (beyer et al., 2003) , were shown to be cleaved by the convertase ski-1 that cleaves at hydrophobic residues. similarly, blocking of ski-1 activity by a specific inhibitor has also shown to affect the processing and the stability of the glycoproteins of these viruses (pullikotil et al., 2004) . for all highly pathogenic avian influenza (hpai) viruses of subtypes h5 and h7 known to date, the cleavage of ha occurs at the c-terminal r residue in the consensus multibasic motifs, such as r-x-k/r-r with r at position p4 and k-k/r-k/t-r with k at p4, and leads to systemic infection. early studies demonstrated that the ubiquitously expressed furin and proprotein convertases (pcs 5 and 6) are activating proteases for hpai viruses (basak et al., 2001; stieneke-grober et al., 1992) . recently, ubiquitous type ii transmembrane serine proteases, mspl and its splice variant tmprss13, have been proposed as novel candidates for proteases processing ha proteins of hpai (okumura et al., 2010) . is it interesting to note that viral receptors can also be modified by proprotein convertase. indeed, pcsk9 impedes hepatitis c virus infection in vitro, modulates liver cd81 expression, and enhances the degradation of the low-density lipoprotein receptor (ldlr) (labonte et al., 2009) , bestowing on the proprotein convertase an additional role in controlling the fusogenicity of the envelope glycoprotein. the exhibition and insertion of a hydrophobic fragment of 10-30 residues in the membrane, named "fusion peptide" or "fusion loop" is a crucial step of the fusion process (epand, 2003) . the fusion peptides in an n-terminal position (such as for the retrovirus or the influenza virus) is liberated for most viruses after envelope glycoprotein cleavage, and it can insert into the external layer of the membrane in an oblique manner, whereas the fusion loop (for the class ii and iii viruses) remains probably more superficial (see table 4 .3). the fusion peptide of the class i and ii proteins is initially buried in the envelope glycoprotein trimer or dimer, respectively. for the class iii, the fusion loop is present outside of the structure, most likely because the fusion peptide of class iii fusion proteins is weakly hydrophobic and probably requires a cooperation between several loops to be functional and efficient. the simple picture of a viral fusion protein acting on cell and viral membranes by means of only two restricted segments, that is to say, the fusion peptide and the transmembrane domain, is too simplistic. instead, a more complex concerted action of different membranotropic segments of the fusion proteins is necessary. more conformational changes are required to achieve a complete fusion of the two lipid bilayers. as described previously, the class i-iii fusion proteins roughly share common refolding processes and formation of intermediates. several regions of the fusion protein complex indirectly aid the fusion process, as for example, the "stem" regions (see below). contrary to the relatively simple and canonical organization of the fusion peptides for the influenza virus or the flavivirus e protein, the recently resolved structures of the gb glycoprotein of the simplex herpes type 1 virus (heldwein et al., 2006) and g protein (roche et al., 2006 (roche et al., , 2007 of the vesicular stomatitis virus (vsv) indicated a bipartite fusion peptide composed of two hydrophobic loops, each loop being relatively nonpolar or very weakly hydrophobic (which rarely leads to the identification of a fusion peptide by fusion peptide prediction based on hydrophobic domain identification). these differences in the organization of the fusion peptides suggest differences of action of the fusion proteins, notably in the number required. according to experiments using neutralization assays, it seems that one hiv envelope glycoprotein is capable of inducing membrane fusion. however, it has been shown that the induced fusion by ha requires a collaboration of several complexes of envelope glycoprotein (8-9 trimers). in the same way, different networks of class ii fusion proteins have been proposed, such as the hexagonal organization observed on the surface of liposomes or an association of five trimers in a structure similar to a volcano based on structure predictions (stiasny and heinz, 2006) . concerning the fusion protein of class iii, a hexagonal structure has been proposed for rabies virus envelope glycoprotein and recently for vsv-g according to a modeling using its structure. the requirement for multiple fusion peptides may be compared to the number of receptors required. the assembly of a complex containing several receptors may be a prerequisite for the membrane fusion steps that require multiple envgp molecules to cooperatively participate in this process. for example, in the case of hiv-1, the presence of more than one cd4 in contact with the virus enhances the infectivity dramatically and reduces the concentration of coreceptors needed for infection (platt et al., 1998) . further investigation of this system has implied that a critical complex containing approximately four to six coreceptors is a requirement for infection, although it is not known whether this complex performs a transient role and then disperses or is maintained throughout the membrane fusion process (kuhmann et al., 2000) . despite some uncertainties, several lines of evidence have suggested that three to six hemagglutin trimers may cooperatively participate in the influenza a virusmediated membrane fusion reaction (blumenthal et al., 1996; boulay et al., 1988) and that multiple envelope glycoprotein trimers are required for rabies virus-mediated membrane fusion (roche and gaudin, 2002) . see table 4 .3. the cytoplasmic tails of envelope viruses harbor different motifs that are responsible for its trafficking and are variable in length. it is surprising to see that the cytoplasmic tails of fusion proteins are not exchangeable. for example, when the hcv e1 and e2 cytoplasmic tails or the f cytoplasmic tails of hrsv (human respiratory syncytial virus) are substituted for that of vsv-g envelope glycoprotein (or cd4), the fusogenicity of these envelopes in cell-cell fusion assays and virus-cell assay (infection) is destroyed (buonocore et al., 2002; oomens et al., 2006) . similarly, when the ha glycoprotein is anchored by a gpi, the entry process is stopped at the hemifusion step (kemble et al., 1994; markosyan et al., 2000) . the cellular localizations (e.g., cholesterol-rich microdomains) are sometimes modified, and biochemical modifications (glycosylation, oligomerization) can affect the properties of the vsv-g envgp (kemble et al., 1994) . nevertheless, in their initial context, these cytoplasmic tails allow fusion. for some viruses, regulation of fusion is mediated by the cleavage of the cytoplasmic tail. for -retrovirus, in addition to the su-tm cleavage, a significant fraction of virion-associated tm is further processed by the viral protease removing the c-terminal 16 amino acids of the cytoplasmic domain, the r peptide. -retrovirus virions assemble and bud from infected cells as immature particles that must undergo an additional proteolytic maturation to become infectious (green et al., 1981; lavillette et al., 1998 lavillette et al., , 2002 rein et al., 1994) . this maturation concerns the viral protease-dependent cleavage of the so-called peptide r at the c-terminus of the cytoplasmic tail. the r peptide inhibits fusion, and different hypothesis have been proposed. first, the r peptide contains an y-x-x-internalization motif, and the removal of this motif following the cleavage of the r peptide might result in a higher amount of envelope at the surface membrane and thus more fusion (song et al., 2003) . second, following the r peptide cleavage, the remaining cyt tail forms a membrane-embedded amphiphilic alpha-helix domain that destabilizes the membrane (rozenberg-adler et al., 2008; zhao et al., 1998) . third, it has been proposed that as the r peptide contains a palmitoylation, its removal induces the close trimerization of the cyt tail and drastic conformational changes in the ectodomain of env (aguilar et al., 2003) which might influence env fusogeneity by destabilizing su-tm complexes. these conformational changes are necessary for the isomerization of the su-tm disulfide mlv env (loving et al., 2008) . this r peptide cleavage is the last step leading to a fusion competent infectious mlv retrovirus, but this final modification does not exist in lentiviruses which harbor a long cytoplasmic tail. however, truncations of the long cytoplasmic domains of lentiviral env proteins occur under certain culture conditions (chakrabarti et al., 1989) and increase env fusogenicity in a similar way to mutated truncated versions of siv, hiv-1, and hiv-2 envs (mulligan et al., 1992; spies et al., 1994; wilk et al., 1992) . this regulation is a hallmark of adaptation of endogenous retroviruses, as this cleavage is fulfilled by a cellular protease that activates the endogenous envgp herv-w in relevant tissues involved in placenta development. it is interesting to note that for most viruses (and not only the -retrovirus), the truncation of the cytoplasmic tail increases the fusogenicity of the envgp, as is seen in paramyxoviruses (moll et al., 2002) . in many cases, this truncation seems to increase the cell surface expression (since cellular trafficking signals in the cytoplasmic tails are modified), which may explain the increase of fusogenicity. for certain other env, the truncation leads to conformational changes in the ectodomain which lowers the activation threshold for fusion, resulting in enhanced env fusion activity and kinetics (aguilar et al., 2003; cote et al., 2008; spies et al., 1994; zhao et al., 1998) . finally, the cleavage of the cytoplasmic tail sometimes allows, at least partially, a cell-cell fusion at neutral ph (ph-independent), although the entry of the virus remains ph-dependent. the notion of phdependence, seems in this case, to be due to a specific conformation or a particular density of envelope glycoprotein . numerous studies on several viruses have highlighted the critical role of the pretransmembrane sequence (ptm), also called the membrane proximale region or the juxtamembrane domain (jmd), which is rich in aromatic amino acids. the class i fusion glycoproteins of coronavirus, lentivirus (hiv, siv, fiv), ebola virus (munozbarroso et al., 1999; saez-cirion et al., 2003) , and many other viruses contain this short jmd region in the ectodomain between the end of hr-2 and the beginning of the transmembrane (tm) domain . the class ii (sfv, dengue, tebv) and class iii (vsv and the herpes virus) fusion proteins also possess these regions, although they are less rich in tryptophan than the class i jmd (jeetendra et al., 2002 (jeetendra et al., , 2003 roche et al., 2008) . although the jmds of fusion proteins of enveloped viruses are rich in aromatic amino acids, the number, spacing, and sequence of the aromatic amino acids are quite variable; however, the function remains the same. these jmds contribute to the conformational changes that occur during membrane fusion, interact with membranes, induce membrane destabilization, and/or facilitate membrane fusion (munoz-barroso et al., 1999) . therefore, the jmd of viral fusion proteins is a potential target for viral inhibitors. entry inhibitors that target the jmd of class i fusion proteins include monoclonal antibodies that bind the jmd of gp41 to the fiv and to the hiv (lorizate et al., 2006; purtscher et al., 1994) . some peptides that mimic the jmd have been designed for envgp of fiv (giannecchini et al., 2004) , hiv (moreno et al., 2006) , ebola virus (saez-cirion et al., 2003) , and the sars virus (howard et al., 2008) and inhibit viral entry. this strategy has been also broadly used against many other paramyxoviruses such as the sendai virus (joshi et al., 1998; rapaport et al., 1995) , the newcastle disease virus (young et al., 1999) , the human parainfluenza type 3 (hpiv-3) (yao and compans, 1996) , the respiratory syncytial virus (rsv), and the measles virus (mv) (lambert et al., 1996) . in the same way, peptides that mimic the jmd from class ii and iii fusion proteins have also been developed against infection by dengue virus (hrobowski et al., 2005) and cmv (english et al., 2006; lopper and compton, 2004) . in the case of hcv, some juxtamembrane domains have been proposed in e1 and e2 (drummer and poumbourios, 2004; drummer et al., 2007) . high-throughput screening (hts) of peptides derived from e1 and e2 sequences has identified inhibitory peptides close to the transmembrane domain of e2, though they are not among the most inhibitory (cheng et al., 2008) . however, these strategies suffer from certain limitations. the derived peptides of these jmd often have the capacity to oligomerize, which inactivates them, and some stratagems need to be implemented to make the peptides more bioreactive against viruses. moreover, these peptides that prevent the correct conformational change of the envelope glycoprotein, must act in a certain window of time and in a particular compartment compatible with their active structure. indeed, the acid ph of some compartment is not compatible with the bioactive structure of the peptide. for the sars-cov virus, the peptide is able to inhibit the entry of the virus in the presence of protease at the cellular surface, but the peptide has little effect on the entry of the virus by endocytosis or on the activation by cathepsin l in the acid conditions of the endosome (ujike et al., 2008) . in the case of influenza, it has not been possible to develop such inhibitory peptides targeting the juxtamembrane domain. reasons suggested for this include the entry through acid compartments which modify protein structures, the entry by different pathways of endocytose, and the rapidity of the conformation changes of ha. some modifications can be made to these peptides to increase their efficiency for viruses that fuse at the plasma membrane. for three paramyxoviruses, hpiv-3, a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses hendra virus (hev) and niv, which cause lethal central nervous system (cns) diseases, the addition of cholesterol to a paramyxovirus hrc-derived peptide (derived from the heptad repeat immediately preceding the transmembrane domain) increased antiviral potency by 2 log units (porotto et al., 2010) . this enhanced activity is the result of the targeting of the peptide to the plasma membrane. the cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the f protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses. the definition of a receptor is very complicated and has limitations. it is sometimes difficult to distinguish between "simple" receptors that mediate adsorption or binding and that may not even initiate conformational changes, and "critical" receptors for fusion which, upon binding, will generate the conformational changes that will allow the exposure of the fusion peptide and lead to membrane fusion. some cellular molecules are also involved in the localization or trafficking of viral receptors and these are important cofactors of entry. another ambiguity is the role of enzymatic activities. some are necessary to process the fusion protein inside producer cells, or inside the endosomes of target cells, and they are necessary to activate the potential of the envgp for membrane fusion. while it is inaccurate to consider them as "receptors", they are certainly critical cofactors. all viruses likely bind at least weakly to multiple cell surface components such as heparan sulfate proteoglycans, dc-sign, integrins, or glycolipids (bounou et al., 2002; cantin et al., 1997; fortin et al., 1997; jinno-oue et al., 2001; mondor et al., 1998; saphire et al., 1999 saphire et al., , 2001 . although such binding substances probably do not induce conformational changes in envgp that are necessary for membrane fusion, they can enhance viral adsorption and substantially increase efficiencies of infections, thus contributing to pathogenesis (alvarez et al., 2002; bounou et al., 2002; geijtenbeek et al., 2000; jinno-oue et al., 2001; saphire et al., 2001) . because such binding proteins contribute to infections, it can be difficult to unambiguously distinguish them from receptors that directly mediate the membrane fusion process, especially for viruses that bind to their authentic receptors only weakly (e.g., for retroviruses, in the cases of felv-t or polytropic mulvs; anderson et al., 2000; marin et al., 1999; temin, 1988) . we emphasize this because pathogenic variants of different animal viruses have often been associated with abilities to bind to apparently novel cell surface components, and it has sometimes been inferred that the viruses have switched their receptor specificities. in these instances, it has generally not been established that the cell surface binding components are receptors that directly mediate infections. in the case of ebola, for example, the receptor(s) that mediates its entry has yet to be definitively identified. c-type lectins such as dc-sign and dc-signr are thought to serve as adherence factors for ebola or marburg virus (marzi et al., 2006; matsuno et al., 2010) . other plasma membrane-associated proteins have been implicated in ebov uptake, including folate receptor alpha and the tyrosine kinase receptor axl (chan et al., 2001; shimojima et al., 2006 shimojima et al., , 2007 sinn et al., 2003) , but the physical interaction of ebov gp and these proteins has not been demonstrated, and cells that do not express these proteins are permissive for ebov gp-mediated virion uptake. some previous studies have implicated the actin cytoskeleton in ebov entry, where agents such as cytochalasin d and swinholide a that impair microfilament function, inhibited gp-mediated entry (yonezawa et al., 2005) . similarly, vsv was shown to bind ubiquitously to cells via phosphatidylserine (ps) (schlegel et al., 1983) . however, a more recent study reports that ps is not a receptor for vsv, as no correlation was found between cell surface ps levels and vsv infection, and annexin v, which specifically binds ps, did not inhibit infection of vsv (coil and miller, 2004) . therefore, the cell surface receptors for vsv have not been identified, but it is generally thought that binding via the g-protein is rather unspecific and involves negative charges on the plasma membrane (carneiro et al., 2006; coil and miller, 2005) . adsorption of viruses onto cultured cells from the medium is usually a very slow and inefficient process, principally because of the slow rates of their diffusion into contact with the cell surfaces (allison and valentine, 1960; andreadis et al., 2000) . in general, the rate of contact cannot be significantly enhanced by mixing or stirring, because the boundary layer of the relatively stationary fluid that surrounds walls or other large objects (e.g., cells) in flowing liquids is substantial compared with the rate of virus diffusion, hence stirring does not increase the concentration of virus surrounding this boundary zone (allison and valentine, 1960) . in the case of retroviruses, it has become especially clear that adsorption is a severely limiting step in infection of cultured cells. in classic studies in which virus samples were incubated with cells for several hours before washing with fresh medium and subsequently detecting the foci of infection, it was estimated that only 1/1000 or fewer of the virions in the medium were infectious. in contrast to previous interpretations, studies suggest that this low infectivity-to-virion ratio is principally caused by the inefficiency of adsorption (andreadis et al., 2000) . accordingly, serial incubation of a virus-containing medium for 2-h periods with sequential cell cultures results in the same titers in each of the cultures after correction for spontaneous viral decay (kabat et al., 1994) . furthermore, centrifuging the virus down onto the cultured cells (i.e., spinoculation) often increases retroviral titers by 1-2 log orders of magnitude (bahnson et al., 1995) . studies aiming to count retrovirions adsorbed onto cell surfaces by confocal immunofluoresence microscopy or by quantitative pcr methods (marechal et al., 2001; pizzato et al., 1999 pizzato et al., , 2001 have demonstrated that receptors for viral entry are irrelevant for initial adsorption of retrovirions onto surfaces of most cells (pizzato et al., 1999 (pizzato et al., , 2001 . on the contrary, the initial steps of virus attachment seem to more critically depend on accessory cellular binding substances, such as heparin sulfates, integrins, or lectins, including dc-sign (bounou et al., 2002; guibinga et al., 2002; jinno-oue et al., 2001; mondor et al., 1998; saphire et al., 2001) . by forming multivalent weak reversible bonds with such abundant cell surface components, a virus would become efficiently bound in a manner that would allow it to "graze" until it makes appropriate contact with a true receptor (haywood, 1994; park et al., 2000) . it is well known that for several viruses (rubella togavirus, bvdv pestivirus, newcastle disease paramyxovirus, hiv lentivirus, mouse hepatitis coronavirus (mhv)), rearrangements of the thiol content and of the disulfide bridges, induced by thioredoxine or protein disulfide isomerase (pdi), are essential to induce some big conformational changes necessary for the membrane fusion (fenouillet et al., 2007) . interestingly, the mlv and htlv retroviruses developed an "internal" oxydoreduction activity by adapting a catalytic motif involved in disulfure bridge isomerization. the two su and tm subunits can be linked in either a covalent or noncovalent manner. for hiv-1, the existence of the soluble gp120 protein indicates a noncovalent link between su and tm (kowalski et al., 1987) . however, for most other retroviruses, a covalent link was described at one stage. in all cases, with the exception of mmtv and jsrv, a disulfide bond between the su and the tm is formed between the highly conserved cx6cc motif of the tm and the cxxc of the su (pinter et al., 1997; schulz et al., 1992; sitbon et al., 1991) . this cxxc motif is extremely rare in cellular proteins and is similar to a motif found in the catalytic site of enzymes involved in thiol isomerization, like pdi or thioredoxin (pinter et al., 1997; sanders, 2000) . this motif in the su has been shown to be part of an autocatalytic isomerization function of su to destroy the initial bond between su and tm generated during env synthesis and create an intra-su bond inside the cxxc motif wallin et al., 2004) . this disulfide bond isomerization is crucial for the fusogenicity of -retrovirus (mlv) (fenouillet et al., 2007) . some viruses use the protease activity of particular cellular enzymes localized in the endosomes or at the cellular surface for activating their envgp. the ebola filovirus (for which the gp1-gp2 envelope glycoprotein is cleaved by the furine in the producer cells) (chandran et al., 2005; schornberg et al., 2006) , the hev (pager and dutch, 2005) , niv (diederich et al., 2009) , or the sars-cov and mhv-2 coronaviruses (for which the s spikes is not cleaved in the producer cells) (huang et al., 2006; qiu et al., 2006; simmons et al., 2005) require the cysteins lysosomal proteases, the l or b cathepsine (catl or catb) for their entry process. after virus uptake following angiotensin-converting enzyme 2 receptor binding, cathepsin l-mediated proteolysis induces conformational changes in the sars-cov s glycoprotein to trigger the endosomal membrane fusion process (simmons et al., 2005) . likewise, cleavage of the ebola glycoprotein by catl cleavage removes a glycosylated glycan cap and mucin-like domain (muc domain) and exposes the conserved core residues implicated in receptor binding (chandran et al., 2005; hood et al., 2010; lee et al., 2008; schornberg et al., 2006) . entry of this virus is ph-dependent and associated with the cleavage of gp by proteases, including catl and/or catb, in the endosome or cell membrane, which is required for entry into the host cell. however, the precise role of the cleavage of ebola envelope glycoprotein, which is already cleaved intracellularly during its exit, is uncertain. the cleavage of the gp to a stable form of 18 kda of gp1 may increase binding, suggesting that the cleavage facilitates the interaction with a cellular receptor (dube et al., 2009; kaletsky et al., 2007) . another possibility is that the catb cleavage is required to facilitate the triggering of viral membrane fusion by destabilizing the prefusion conformation of ebola envelope glycoprotein (wong et al., 2010) . the cathepsins comprise a family of lysosomal protease enzymes whose primary function (i.e., protein degradation) plays a critical role in normal cellular homeostasis. cathepsin l is one of the 11 members of human lysosomal cysteine proteases (i.e., b, c, f, h, k, l, o, s, v, w, and x) that fall in the c1 family (papain family) of the ca clan (rossi et al., 2004) . these enzymes were traditionally linked to nonspecific proteolytic activity within lysosomes. more recently, cathepsin l has been implicated in regulatory events relating to cancer, diabetes, immunological responses, degradation of the articular cartilage matrix, and other pathological processes (vasiljeva et al., 2007) , including osteoporosis, rheumatoid arthritis, and tumor metastasis (palermo and joyce, 2008) . interestingly, several host cell proteases appear to be able to prime fusion activation in the case of sars-cov, including cathepsin l, trypsin, factor xa, thermolysin, plasmin, tmprss11a, and elastase. the proteolytic cleavage events in sars-cov s that lead to membrane fusion occur both at the s1/s2 boundary and adjacent to a fusion peptide in the s2 domain (belouzard et al., 2009) . elastasemediated activation of sars-cov was originally reported by taguchi and coworkers, and it has been proposed that elastase may have important implications for viral pathogenesis (matsuyama et al., 2005) . elastase is known to be secreted by neutrophils as part of an inflammatory response to a viral infection and is also produced by opportunistic bacteria (e.g., pseudomonas aeruginosa) that can colonize virally infected respiratory tissue. as such, it has been considered that elastasemediated activation of sars-cov might be an important factor in the severe pneumonia seen in sars-cov-infected patients (belouzard et al., 2010) . in conclusion, in order to better classify the receptors, the receptors must be differentiated according to their precise function: those that permit a nonspecific adsorption, such as the glycosaminoglycans (used by dengue, tbe, hsv-1), the type c lectin receptors, such as l-sign, dc-sign, the hmgl, the lsectin, and the asialoglycoprotein receptor (used by hcv, ebola, marburg); those that permit a specific adsorption receptor (binding receptor) allowing the sorting (toward particular endosomes and intracytoplasmic compartment) and the initial conformational changes (such as the cd4 to hiv, the integrins or the laminin receptor for the dengue, sindbis, or lassa viruses); and finally, those that permit the exposition of the domains implicated in the destabilization of the membrane (like the fusion peptide) and are the latest receptors to act (such as the hiv coreceptors). therefore, studying the kinetics of the conformation changes of the envgp and the kinetics of action and utilization of the receptors is essential to accurately categorize the receptors (nonspecific adsorption receptor, receptor of binding, receptor of fusion). all the cellular proteins that allow the exposition, localization, and the trafficking of these receptors and/or endosomes should be considered as cofactors. with all the sirna screens that are coming out, the list of such cofactors is dramatically increasing. the use of different receptors often correlates with the need of a virus to overcome barriers existing in the cell type or tissue that they infect. one wellstudied example is the binding of coxsackievirus b to decay-accelerating factor (daf) in the apical surface of epithelial cells, and subsequently to the coxsackievirus and adenovirus receptor (car), which is localized in the tight junction region. daf helps to bring the virus to the tight junctions, and car induces a conformational change and promotes endocytosis (coyne and bergelson, 2006) . almost all animal viruses use receptors exclusively containing a single tm sequence (see table 4 .1). in striking contrast, cell surface receptors for -retroviruses have multiple transmembrane (tm) sequences, compatible with their identification in known instances as transporters for important solutes. similarly, hepatitis c virus, in addition to ldl receptor, exclusively uses multitransmembrane receptors:the scavenger receptor class b type 1 (srb1 with two transmembrane domains), the tetraspanin receptors cd81 and claudins (4 tms), and another tight junction protein occludin-1 (4 transmembrane domains; . another surprise is that some viruses, including many -retroviruses, use not just one receptor but pairs of closely related receptors as alternatives (tailor et al., 2003) . this appears to have enhanced viral survival by severely limiting the likelihood of host escape mutations. all of the receptors used by -retroviruses contain hypervariable regions that are often heavily glycosylated and that control the viral host range properties, consistent with the idea that these sequences are battlegrounds of virus-host coevolution. however, in contrast to previous assumptions, it is probable that -retroviruses have adapted to recognize conserved sites that are important for the receptor's natural function and that the hypervariable sequences have been elaborated by the hosts as defense bulwarks surrounding the conserved viral attachment sites. the fact that all virus groups have been severely limited throughout evolution in the types of receptors they can employ, may initially appear inconsistent with evidence that some viruses can switch their receptor specificities with apparent ease. this has been most dramatically suggested by shifts of influenza a viruses between animal reservoirs, which involve single amino acid changes in the viral hemagglutinin, enabling recognition of different sialic acid structures (e.g., n-acetyl or n-glycolyl neuraminic acids in 2,6 or 2,3 linkages to galactose) that predominate in the different host species (baranowski et al., 2001; gambaryan et al., 2005; skehel and wiley, 2000) . similarly, slight changes in specificity for receptors accompanied the emergence, in 1978, of the canine parvovirus (parker et al., 2001) . however, these are small shifts in receptor specificities rather than global jumps to dissimilar receptors. similar slight shifts are involved in the change from the ccr5 to cxcr4 coreceptor usage during aids progression (scarlatti et al., 1997) . small shifts in usages of highly similar receptors have also been reported during cell culture selections of subgroup b, d, and e avian leukosis viruses that all use polymorphic variants of the same tvb receptor (taplitz and coffin, 1997) and during cell culture selections of hiv-1 variants (platt et al., 1998) . therefore, despite the rarity of receptor repertoire expansions throughout millions of years of retrovirus evolution, limited switches can occur within single infected animals. for example, there is an evolution of coreceptor usage in hiv-1/aids, some in vivo adaptations of ecotropic mulvs and formation of polytropic mulvs, and some evolution of altered receptor usages in domestic cats infected with felv-a (tailor et al., 2003) . several viruses have been reported to use multiple alternative receptors or even alternative pathways for infection of cells. for example, mv isolates appear to be capable of using cd46 or slam, which both contain single tm domains (baranowski et al., 2001; oldstone et al., 1999; tatsuo et al., 2000) . complex viruses such as herpesviruses that contain several distinct envgp are also typically able to bind to several cell surface components (baranowski et al., 2001; borza and hutt-fletcher, 2002) . the foot-and-mouth disease picornavirus (fmdv) may also use multiple receptors, including heparan sulfates and integrins, and may, in addition, be able to invade cells via immunoglobulin fc receptors when the virus is coated with antibodies (baranowski et al., 2001; mason et al., 1994) . this alternative entry route is also used by the dengue flavivirus, which may explain the extremely strong pathogenesis that occurs when it reinfects previously exposed individuals (baranowski et al., 2001) . in the case of fmdv, it has not been established whether heparin sulfate is a true receptor that directly mediates infection or merely a binding factor that influences infection indirectly by enhancing virus adsorption. hiv-1 infections are also strongly stimulated by accessory cell surface binding components including heparan sulfates, glycolipids, and dc-sign (bounou et al., 2002; geijtenbeek et al., 2000; pohlmann et al., 2001) . similarly, a paralysis-inducing neurotropic variant of friend mulv binds more strongly than the parental virus to heparan sulfate, and thereby becomes more infectious for brain capillary endothelial cells while still remaining dependent on the cat1 receptor (jinno-oue et al., 2001) . these examples illustrate how changes in affinities for accessory binding substances can dramatically alter cellular tropisms and pathogenesis of viruses, and why it has often been difficult to distinguish such accessory binding factors from true receptors or coreceptors that are essential for infections. on the basis of these considerations, we believe that the available evidence strongly supports our proposal that all virus groups have been severely constrained in the types of receptors they can employ for infection of cells. however, some viruses have evolved several pathways for infection, and viruses such as hiv-1 have evolved distinct sites in a single su glycoprotein for recognition of dissimilar receptors and coreceptors. the complexity of envgp is variable. an example of a very simple fusion protein is the fast proteins of orthoreovirus that do not belong, however, to the family of the enveloped virus (barry et al., 2010; shmulevitz and duncan, 2000) . the orthoreovirus genus includes some no-enveloped viruses that cause the cell-cell fusion of infected cells. this fusion activity is due to the small no-structural membrane viral proteins named fast proteins (fusion-associated small transmembrane protein), in size ranging between 10 and 15 kda. the fast protein ectodomains are very small, with extreme cases of only 20 residues, and contain hydrophobic short regions with/without acid properties, and a myristoylation site. though their small size challenges their ability to form a hairpin structure, these fast proteins expressed alone are sufficient to induce the membrane fusion (barry et al., 2010; shmulevitz and duncan, 2000) . surprisingly, they are able to traffic as far as the plasma membrane without inducing intracellular disorder. indeed, by opposition, the expression of retroviral tm or orthomyxovirus ha2 alone does not lead to cell surface membrane expression, and these subunits are blocked intracellularly. the fusion induced by the fast protein is very broad, which questions their use of a particular protein receptor. thus, some fusion proteins are simple and possess solely the fusion function, and others comprise several domains in addition to the domain involved in fusion. the function of some of these domains has remained more or less independent from the fusion domain, and sometimes, they can be naturally separated in different proteins (i.e., h and f from paramyxoviruses) or experimentally on different fragments, as explain below. such is the case of the -retroviruses for which the function of binding to the receptor can be separated from the function of fusion. this line of inquiry was initiated by the studies of bae et al. (1997) relating to a conserved phq motif that occurs near the amino-terminal ends of su glycoproteins in all -retroviruses. mutation of this phq motif blocked membrane fusion but had no effect on receptor attachment. subsequently, we discovered that noninfectious -retrovirions lacking this histidine could be transactivated by addition of a soluble su or an amino-terminal fragment of su, called the receptor-binding domain (rbd), to the cultured cells (lavillette et al., 2000) . interestingly, studies by overbaugh and coworkers have demonstrated that similar transactivation processes can occur in natural infections by -retroviruses. specifically, they found that infections by the immunosuppressive felv-t virus, which has a pro in place of his in its phq motif, require transactivation either by a soluble felv-b-related su glycoprotein termed felix that is endogenously expressed in cat t cells or by an felv-b su glycoprotein . this activating process parallels that of herpes simplex virus for the transmission of a fusogenic signal among the envgp of the herpes simplex virus on receptor binding by glycoprotein gd. the soluble gd ectodomain has been shown to allow entry of engineered hsv-1 virus particles that lack gd (i.e., gd-null mutants; cocchi et al., 2004) . the evidence reviewed provides very strong support for the hypothesis that attachment of viruses to their receptors initiates a pathway that obligatorily contains intermediate steps. these intermediate steps very likely include viral association with multiple receptors, cooperative conformational changes within env glycoprotein, and cross-talk between env on the viral surfaces. in the case of the retroviruses, this evidence suggests that virus binding to receptors does not directly induce irreversible structural changes in su-tm complexes as was previously believed. rather, it implies that the binding to receptors induces su-su interactions that are prerequisites for later steps in a highly coordinated membrane fusion pathway. we anticipate that similar intermediate steps are likely to be involved in infections by other groups of retroviruses and perhaps in infections by other membrane-enveloped viruses. other viruses can be activated by soluble receptors that, by interacting with the envelope glycoprotein, induce some of the conformational changes necessary to trigger fusion. this example indicates that binding and fusion steps can be separated and can take place at different locations. in these cases, mhv, avian retrovirus aslv, and the herpes simplex 1 (hsv-1) can infect some cells that do not harbor binding receptors provided that the soluble form is present in the infection media (the soluble ceacam1 receptor for mhvr, the soluble tva receptor for aslv, and the soluble nectin-1 for hsv-1; kwon et al., 2006; matsuyama and taguchi, 2002; mothes et al., 2000) . vectors derived from retroviruses such as lentiviruses and oncoretroviruses are probably among the most suitable tools to achieve long-term gene transfer, since they allow stable integration of a transgene and its propagation in daughter cells. lentiviral vectors should be the preferred gene-delivery vehicles over vectors derived from oncoretroviruses (mlv) since, in contrast to the latter, they can transduce nonproliferating target cells. moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer approaches in vivo (frecha et al., 2008b) . to achieve such in vivo gene transfer, innovative approaches have been developed to upgrade lentiviral vectors for tissue or cell targeting and which have potential for in vivo gene delivery. one strategy is to develop vectors that harbor envgp with selective tropisms. vectors derived from retroviruses offer particularly flexible properties in gene transfer applications, given the numerous possible associations of various viral surface glycoproteins (determining cell tropism) with viral cores. selective tropisms were achieved by taking advantage of the natural tropisms of envgp from other membrane-enveloped viruses. for instance, the use of surface glycoproteins derived from viruses that cause lung infection and infect via the airway epithelia, such as ebola virus or influenza virus, may prove useful for gene therapy of the human airway (kobinger et al., 2001) . exclusive transduction of retinal pigmented epithelium could be achieved following subretinal inoculations of some vector pseudotypes in rat eyes (duisit et al., 2002) . high transgene expression was detected in dermal fibroblasts transduced with vsv-g-, eboz-, or mulv-pseudotyped hiv vector and effectively targeted quiescent epidermal stem cells which underwent terminal differentiation resulting in transgene expression in their progenies (hachiya et al., 2007) . importantly, several viral envgp target lentiviral vector to the cns such as rabies (wong et al., 2004) , mokola (watson et al., 2002; wong et al., 2004) , lymphocytic choriomeningitis virus envelope (lcmv) (miletic et al., 2004; stein et al., 2005) , or ross river (kang et al., 2002) viral envgp that even permit transduction of specific cell types within the cns. likewise, screening of a large panel of pseudotyped vectors established the superiority of the gibbon ape leukemia virus (galv) stitz et al., 2000) and the cat endogenous retroviral-modified glycoproteins (rd114) for transduction of progenitor and differentiated hematopoietic cells. recently, a new lv carrying the mv envgp on its surface was able to overcome vector restrictions in both quiescent t and b cells (frecha et al., 2008a (frecha et al., , 2009 ). importantly, naive as well as memory t and b cells were efficiently transduced, while no apparent activation, cell-cycle entry, or phenotypic switching were detected, opening the door to a multitude of gene therapy and immunotherapy applications. vectors derived from hiv pseudotyped with sendai virus fusion protein f (kowolik and yee, 2002) or e1e2 from hepatitis c virus (bartosch et al., 2003) , and such vectors are able to transduce human hepatoma cells and primary human hepatocytes efficiently, although they are unable to enter nonliver cells. the gp64 glycoprotein from baculovirus autographa californica multinuclear polyhedrosis virus pseudotyped fiv efficiently and also showed excellent hepatocyte tropism (kang et al., 2005) . the screening of cdna libraries has emerged as a powerful tool to identify and clone viral entry receptors. it is an alternative to the use of human/chinese hamster radiation hybrid panels of cells. in order to clone an unknown receptor by complementation screens, cdna from a cell permissive to infection by a certain virus is introduced into a nonpermissive cell. as some recessive cell lines are poorly transfectable, the use of pseudoparticles provides a good tool to transduce the cdna library for this cloning strategy. a retroviral cdna library approach, involving transfer and expression of cdnas from highly infectable cells to nonpermissive cells, has been used to clone and identify the mulv polytropic x-receptor tailor et al., 1999a; yang et al., 1999) , the rd114 asct2 receptor tailor et al., 1999b) , felv-c flvcr1 receptor from human and domestic cat cdna libraries (quigley et al., 2000; tailor et al., 1999c) , and two closely related human proteins, phur-a1 and phur-a2, that function as receptors for perv-a (ericsson et al., 2003) . briefly, when a cell line that is not susceptible to a particular pseudotype retrovirus vector harboring an envelope for which the receptor is not known, a cdna library from a cell line highly susceptible to transduction, was constructed by cloning the cdna into a retroviral expression vector. afterward, the cdna retroviral expression library was transduced into nonsusceptible cells by infection at a relatively low multiplicity of infection so that the majority of infectants would contain single-copy provirus inserts. the library-containing cells were then screened for susceptibility to pseudotype vector transduction through selection of drug-resistant cells after exposure to the vector carrying a resistance gene. of drug-resistant clones obtained from the primary screen and using pcr primers specific for vector sequences, cdna products from clones with conferred susceptibility were identified after nested pcr was performed on dna extracted from reinfectable clones. however, for many viruses, initial attempts using a retroviral cdna library were unsuccessful due to an inherent background of nonspecific infection with pseudoparticles. in fact, no cell line was completely nonpermissive to even "no envelope" pseudoparticles bearing no viral envelope proteins, indicating the existence of nonspecific uptake mechanisms. in the screens, this resulted in a high background of drug-resistant colonies, independent of glycoproteinmediated cell entry. thus, unless the entry factor cdna was highly represented in the library, a single round of transduction/challenge would not suffice. to deal with the high background observed in screens, methods that would allow multiple rounds of selection and enrichment have been developed. recently, the use of a cyclic packaging rescue (cpr) system using non-self-inactivating vectors has been shown to increase the efficiency of receptor cloning with powerful iterative screening methods (evans et al., 2007; . most retroviral vectors commonly used for gene-delivery applications are self-inactivating vectors that contain deletions in the long terminal repeat (ltr) elements. no packaging competent retroviral rna transcripts are generated from such integrated proviruses; instead, transgene expression is driven by an internal nonretroviral promoter. in contrast, if the cdna library is constructed in a provirus that retains the complete ltr elements, the retroviral promoter is active in transduced cells and a full-length viral rna is expressed. expression of the packaging components, gag-pol and vesicular stomatitis virus vsv-g envelope (that will efficiently infect recessive cell lines), in these cells allows packaging of the rna into pseudoparticles capable of transducing naive cells. this approach, termed cpr (bhattacharya et al., 2002; koh et al., 2002) , allows retrieval of the library after selection has been performed, followed by transduction of a naive cell population, concluded by a new round of selection. this process can be repeated sequentially for an unlimited number of selection/ enrichment steps. for an additional level of selection, different challenge virus genomes can be used, each encoding a different drug-selectable marker (e.g., puromycin (puror) or zeocin (zeor) resistance), in self-inactivating retroviruses. thus, after challenge and selection of a library-transduced population with one pseudoparticle-packaged selection cassette (e.g., puror), the population can be pooled and rechallenged with the second-selectable pseudovirus (e.g., zeor). then, during cpr, only the full-length retroviral transcripts from the non-selfinactivating provirus that encodes the library but not the self-inactivating challenge virus genomes are repackaged and transferred to the naive cell population. this enables researchers to perform multiple rounds of selection, thereby overcoming the background of nonspecific pseudoparticle uptake. moreover, using this scheme, underrepresented cdna will be enriched. once the final round of selection of pseudotype-susceptible cells has been achieved, genomic dna can be prepared from selected clones and used as a template in a pcr across the provirus cdna-cloning site. the nonpermissive target cell line for the cdna screen adheres to several stringent criteria. as stated above, the primary requirement is that (1) to minimize nonspecific background, cell lines with minimal uptake of pseudoparticle of interest and "no envelope" pseudoparticles were preferred. (2) to ensure that nonpermissiveness was a phenotype due to the lack of a pseudoparticlespecific entry factor(s) rather than poor infection by pseudotypes in general, chosen cell lines should be highly permissive to an unrelated pseudoparticle (vsvgpp, rhabdovirus) infection. in addition, this also ensures that the target cell line would be easily transduced with the cdna library. (3) for selection, candidate cell lines also had to be susceptible to the desired drug selections. (4) to perform multiple rounds of screening involving cpr, the ideal cell line had to demonstrate this method well and be highly transfectable. (5) finally, to facilitate the screen, the chosen cell line needed to be relatively fast growing and clone efficiently. to gain insights into virus entry, it is necessary to examine several inhibitors of pathway-mediated endocytosis in terms of their role in blocking infection mediated by pseudotypes with different envgp. an advantage of pseudoparticles is that the entry process of pathogens of bsl4 can be studied in bsl2 conditions. for example, to gain insights into ebola virus entry, inhibitors against different endocytosis pathways have been examined for their ability to block infection mediated by hiv pseudotyped with the ebola envgp (bhattacharyya et al., 2010) . the use of control pseudoparticles (e.g., pseudotyped with vesicular stomatitis virus envgp (vsv g)) can be used as controls to assess cell viability and specificity of inhibition. inhibition of clathrin function traditionally relied on three principal approaches: drugs that inhibit acidification of endosomes (such as bafa1, a specific, nonreversible endosomal proton pump inhibitor), as well as commonly used lysosomotropic agents (such as ammonium chloride (nh 4 cl) and chloroquine); potassium depletion; and finally, treatment of cells with brefeldin a (bfa) or chlorpromazine (sieczkarski and whittaker, 2002) . as such, these drugs have multiple effects on cell function, and their use to inhibit virus infection should be treated with some caution. thanks to pseudotypes, some other studies have implicated the actin cytoskeleton in ebola virus entry, where agents such as cytochalasin d and swinholide a that impair microfilament function were shown to inhibit envgp-mediated entry (yonezawa et al., 2005) . ebola enters cells through a low-ph-dependent, endocytosis-mediated process. a large body of evidence indicates that ebola viruses enter cells by clathrin-mediated endocytosis (sanchez, 2007) , but lipid raftassociated, caveolin-mediated endocytosis has also been proposed as an alternative mechanism of ebola virus uptake (empig and goldsmith, 2002) . low-ph events lead to cathepsin-dependent cleavage of ebola virus envgp that is required for productive uptake of the virus (chandran et al., 2005; kaletsky et al., 2007; schornberg et al., 2006) . other low-ph-dependent events have been postulated to be required as well (schornberg et al., 2006) . furthermore, ebola virus likely uses a rho-mediated pathway, as is seen in vsv virus, suggesting that this may be a route of entry utilized by many different viruses (quinn et al., 2009 ). more recently, proteins interfering with endocytosis, such as the use of dominant-negative eps15, or rna interference (rnai) have been developed, and such approaches target the different pathways with higher specificity (mercer and helenius, 2009; mercer et al., 2010b) . the rnai approach allows researchers to perform screens to identify previously unrecognized host factors that are required for viral replication. rnai screens rely on either short interfering rnas (sirnas) or short hairpin rnas (shrnas) to knock down the function of a particular gene in a cell. researchers can then infect the cells with specific viruses and monitor levels of viral replication. if viral replication is reduced, then the knocked-down gene might be necessary for the virus to replicate itself or function within the host cell. some small-scale screens have been developed using wild-type viruses (kolokoltsov et al., 2007) or pseudoparticles (trotard et al., 2009) . pseudoparticles can be exploited to focus a sirna screen specifically on the entry step of a virus. indeed, only the entry steps are governed by the envgp, whereas all the uncoating, integration, and expression steps of the transgene depend on retroviral proteins. having said that, this suffers an inherent drawback in that some steps are dependent on retroviral proteins and therefore limits the scope of the screen. one limitation to the sirna screen is specificity and cell toxicity, which can be overcome by the use of pseudotypes. indeed, a sirna screen can be done with different pseudotypes in parallel. if, contrary to the pseudotypes of interest, some pseudotypes are not affected by sirna, this indicates that the sirna specifically inhibits the virus of interest and, moreover, that the sirna is not toxic. to better characterize the entry pathway of the hepatitis c virus, a small interfering rna library dedicated to membrane trafficking and remodeling was screened in the context of the huh-7.5.1 liver cell line cells infected by hcv pseudoparticles (hcvpp) (trotard et al., 2009) . results showed that the downregulation of different factors implicated in clathrin-mediated endocytosis inhibit hcvpp cell infection. in addition, knockdown of the phosphatidylinositol 4-kinase type iii-alpha (pi4kiiialpha) prevented infection by hcvpp, and the presence of pi4kiiibeta in the host cells influenced their susceptibility to hcvpp infection. this library screening using pseudoparticles identified two kinases, pi4kiiialpha and beta, as being involved in the hcv life cycle. these results have been confirmed in published works of the identification of cellular factors required for the hcv life cycle using sirna libraries screening either over 4500 drugable genes (ng et al., 2007; vaillancourt et al., 2009) , 140 cellular membrane-trafficking genes coller et al., 2009) , kinome (supekova et al., 2008) , or the entire genome (li et al., 2009; tai et al., 2009 ). these works are much more time-and cost-consuming, and even though pseudoparticles may appear to be an overly simple model to study and identify host factors necessary for viral infection, they are also powerful and flexible tools that have led to major discoveries, as these examples have shown. recently, however, in order to identify host factors necessary for viral infections, researchers are turning to genome-wide genetic screens. indeed, the sequencing of the human genome and the emergence of technologies that allow the silencing of individual genes in cells one by one using sirna, when combined with the use of genome-wide sirna libraries and automated hts methodology, allows molecular information to be gained about virtually every critical intracellular event occurring during virus infection. within the last few years, there have been several publications describing such genome-wide sirna screens that have been applied to virus infections in tissue-cultured cells. rnai screens have now been preformed for several viruses, including hiv konig et al., 2008; yeung et al., 2009; zhou et al., 2008) , west nile virus (krishnan et al., 2008) , influenza (karlas et al., 2010; konig et al., 2008) , and, in drosophila cells, dengue (sessions et al., 2009) and vaccinia virus (moser et al., 2010) . from the hundreds of cellular factors that have been implicated in the outcome of infection, some were already known from other studies, but the majority are entirely novel, and the validation and analysis of data is ongoing in many laboratories. however, a poor overlap between results from different screens published on the same viruses has been observed. the differences are probably linked not only to technical variations but also to the inherent risk of false positives through off-target effects and false negatives due to inefficient silencing. cell toxicity is a common complication, as well as cell-type specificity. following-up such screens with pseudoparticle studies is useful to validate the cell proteins that are involved in the entry step only and facilitate the analysis, compared to the use of a replication competent virus. resistance to individual antivirals is likely to develop for most specific therapeutics targeting particular viral proteins, thus making therapy consisting of a combination of drugs targeting different stages of the viral life cycle highly desirable. the entry process represents another series of potential targets for therapeutic intervention. it has not been extensively explored due to highthroughput experimental limitations for some viruses that require biosafety level 3 or 4 or for the viruses with no robust infection system. pseudoparticle infection systems, utilizing different reporter genes or proteins (i.e., firefly luciferase or gfp), can be developed for hts of small molecule libraries, peptide libraries, or neutralizing antibodies for their entry inhibitor properties. in order to facilitate the screen, assay performance can be improved by modifying the properties of both the parental host cell line and the pseudovirus. for example, cells with improved pseudoparticle entry and cell spreading can be selected. pseudoparticles can be improved by using envgp that increase infectivity either by selecting a specific variant or by expressing a human codon-optimized envelope glycoprotein sequence. finally, the pseudoviruses can be engineered to express a human codon-optimized reporter to improve the sensitivity of the assay. using these modifications, hts can be developed using 96-or 384-well plates. compounds found to inhibit pseudoparticle infection can then be counterscreened against pseudoparticles containing other variants to characterize the cross-reactivity of the molecules in a virus family. moreover, molecules can then be counterscreened against pseudoparticles harboring envgp from other virus families to evaluate the specificity of the inhibitor or its large spectrum. a wide variety of entry inhibitors, namely, peptides, chemical compounds, or antibodies, exist. they can interfere with the different steps of the entry process, such as the binding to receptors or the conformational changes of the fusion proteins, by acting on the envelope protein itself, on the acidification of the endosomes, if necessary, or on the endocytosis. for hiv, most marketed drugs for treating aids are inhibitors of hiv-1 reverse transcriptase or protease enzymes, but new targets include the integrase enzyme, cell-surface interactions, membrane fusion, and also virus particle maturation and assembly (kaushik-basu et al., 2008) . entry inhibition entails preventing hiv-1 breaching the cell, either as a strategy to prevent infection altogether or to curtail infection of new cells in an hiv-positive individual. several strategies have proved effective in hiv-1 entry inhibition either in vitro (using pseudoparticles or replication competent viruses) or in vivo: binding to viral surface proteins gp120 and gp41, binding to human cell surface receptor cd4, and binding to human cell surface coreceptors, ccr5 and cxcr4 (leonard and roy, 2006; liu et al., 2007) . in particular, the synthesized peptide t-20 is believed to act by binding to gp41 (wild et al., 1993 (wild et al., , 1994 and is currently in clinical use. yet, several more recent studies have revealed that t-20 does not block the six-helix bundle prehairpin formation (liu et al., 2005) . another peptide, c37, derived from the c-terminus of gp41 (and nearly identical to the widely reported c-peptide c34; liu et al., 2005; root et al., 2001) , is also described as having strong anti-hiv entry activity due to the tight binding of the gp41 n-terminal helices. maraviroc is a small antiretroviral compound known to be a ccr5 antagonist, which blocks r5-tropic hiv entry into cd4 cells (hunt and romanelli, 2009) . several studies have established that synergy can occasionally be observed when two fusion inhibitors are combined in a viral assay or in vitro fusion assay. for example, some synergy is observed in the combination of ccr5 antibodies with t-20 (ji et al., 2007; murga et al., 2006) , the combination of small molecular antagonists of coreceptors with t-20 (tremblay et al., 2000 , the combination of small molecular antagonists of ccr5 with ccr5 antibodies (ji et al., 2007; murga et al., 2006) , and the combination of small molecular antagonists of ccr5 with chemokines (murga et al., 2006; tremblay et al., 2002) . in the case of hcv, entry into hepatocytes is a multistep process, involving at least four cellular receptors, leading to virion endocytosis and fusion of the viral and cellular membranes. unlike the hcv replication process, these steps have not been thoroughly exploited as targets for antiviral intervention. recently, with the development of hcvpp and the jfh1 infectious molecular clone, it has become possible to test drugs against entry. in vitro, proof-of-concept studies for inhibiting the hcv entry process have been demonstrated using cyanovirin-n that targets the n-linked glycans of the viral envelope proteins and prevents e2-cd81 interaction (helle et al., 2006) , neutralizing antibodies directed against the hcv e1 and e2 proteins (broering et al., 2009; habersetzer et al., 1998; keck et al., 2008; law et al., 2008; owsianka et al., 2005; perotti et al., 2008; schofield et al., 2005; yu et al., 2004) , antibodies against cellular receptors cd81 ( bartosch et al., 2003; cormier et al., 2004; lavillette et al., 2005; molina et al., 2008) , sr-bi (catanese et al., 2007; zeisel et al., 2007) , claudin-1 (krieger et al., 2010) , and agents that block endosomal acidification (bartosch et al., 2003; hsu et al., 2003; koutsoudakis et al., 2006; lavillette et al., 2005) . in vivo studies using humanized trimera mice model or human liver-u-pa-scid mice have also demonstrated prophylactic efficacy of monoclonal anti-e2 (eren et al., 2006) and anti-cd81 antibodies (meuleman et al., 2008) , respectively. some broad-spectrum antiviral drugs have also been tested (pecheur et al., 2007) , but more recent studies have used the hcvpp system in order to undertake a screening campaign that led to the discovery of a class of small molecule hcvspecific inhibitors consisting of several structurally related compounds defined by a common triazine core (baldick et al., 2010) . inhibition of entry was confirmed by using time-of-addition experiments to demonstrate that inhibitor activity is confined to the first 3 h of infection, with inhibition occurring postattachment and closely linked to the inhibition kinetics of the endosomal acidification inhibitor bafilomycin. pseudoparticles can also be used to identify the mode of action of molecules identified using a replication competent virus in a cell-based screening system involving multiple rounds of infection in a 96-well format. after analysis of a publicly available library of 446 clinically approved drugs, the impact of 33 identified compounds on viral entry was tested using hcvpp infection to recapitulate hcv particle adsorption, internalization, and viral envelopemediated fusion (gastaminza et al., 2010) . many of the candidates were lysosomotropic compounds that inhibited hcv entry with differential efficacy. both pseudotype (larson et al., 2008) and infectious virus screening (bolken et al., 2006) identified broadly active arenavirus entry inhibitors. isolation and mapping of resistant viruses, as well as chimeras between sensitive and resistant strains, mapped the target of activity to the gp2 subunit of the g envelope protein complex, specifically to the interface between the c-terminal stem and tmd domains. mechanistic studies showed that these arenavirus inhibitors prevented low-ph-induced fusion by blocking reorganization between the gp2 stem with n-terminal domains of the g protein complex (york et al., 2008) . another alternative for developing entry inhibitor compounds is to target endosomal protease necessary for entry of certain viruses. inhibitors of cathepsin l block viral entry of severe acute respiratory syndrome coronavirus (sars-cov) and ebola virus and impair conversion of hev glycoprotein into the mature, active form (chandran et al., 2005; pager and dutch, 2005; simmons et al., 2005) . with respect to the development of antiviral agents, inhibitors of human cathepsin l are not subject to resistance arising from rapid mutations of the viral genome (shah et al., 2010) , making cathepsin l an attractive target for drug development. hts for cathepsin l inhibitors identified a novel thiocarbazate compound exhibiting potent inhibition against cathepsin l myers et al., 2008; shah et al., 2008) . this compound prevented 293t cell infection by the ebola and sars-cov pseudotypes, respectively. in addition, the thiocarbazate inhibited in vitro propagation of malaria parasite plasmodium falciparum and inhibited leishmania major . finally, another natural entry inhibitor is provided by monoclonal neutralizing antibodies. many monoclonal antibodies can be isolated from immortalized b-cells recovered from patients or from mice hybridomas following immunization. the antibodies can be selected by elisa assay using soluble envelope proteins or pseudoparticles. the neutralizing potential of these antibodies can be easily screened using pseudoparticles with high-throughput infection assays, and the inhibitory effect can be verified with replicating viruses. in the case of hcv, using an antibody antigen-binding fragment phagedisplay library generated from a donor chronically infected with hcv, of 115 clones showing specific binding to hcv e2 glycoprotein, 5 monoclonal antibodies presented neutralizing activities against cell-culture hcv (hcvcc), jfh-1 virus, and a panel of hcvpp displaying e1-e2 from diverse genotypes (law et al., 2008) . overall, neutralizing antibodies can inhibit the different steps of the entry process from binding to membrane fusion by targeting the domains involved in this step or by limiting the conformational changes of the envelope complex. interestingly, one study has recently highlighted the feasibility of targeting short-lived envelope glycoprotein intermediates for inhibition of membrane fusion using monoclonal antibodies (york et al., 2010) . this action is very similar to the peptides against hiv membrane fusion on the market. such strategies to effectively target fusion peptide function in the endosome may lead to the discovery of novel classes of antiviral agents, and screens using pseudoparticles will provide an easily wielded system to identify such infrequent antibodies. all viruses have developed varied mechanisms to reach the same goal. they vary greatly in structure, but all seem to have a common mechanism of action, in which a ligand-triggered large-scale conformational change in the fusion protein is coupled to apposition and merger of the two bilayers. in spite of the different mechanisms to activate the fusion peptide, fusion proteins are distributed into three classes based on their structural homologies. future experiments must aim to elucidate the molecular mechanisms and the dynamics of the conformational changes driving virus entry. this will require the development of new approaches to study the rapid conformational changes of a small number of membrane interacting protein molecules and domains. a more realistic goal is the determination of all the structures of proteins that mediate the entry of all human viruses, either at a prebinding or postfusion stage. another challenge will be the identification of the cognate cellular receptors. identification of all the cellular receptors for human viruses would be an important contribution to our understanding of virus tropism and pathogenesis. once known, the role of receptors in entry, as a specific or nonspecific binding factor, or as receptors needed for conformational changes, or for routing the virus to the right compartment, will have to be established. moreover, for most viruses, the envgp appear to function in an autonomous manner and can permit fusion without a requirement for receptors at acidic ph. thus, the role of varied receptors remains enigmatic for many viruses. it is difficult to predict the roles of a receptor in fusion, sorting and routing the virus toward a particular favorable compartment in the pursuit of the infectious cycle based upon its family and its structure. the activation process of envgp and the postbinding events are early steps that are crucial to understand, as they could provide targets for the development of therapeutics. the better understanding of the envelopereceptor interaction also raises hopes for the possibility of designing entry machines that can deliver genes and other molecules to any cell of choice. recently, the use of whole genome sirna screen has become more and more widely used for different viruses in order to identified factors important for entry. it should drastically increase our knowledge of the factors necessary for entry of viruses. similarly, many high-throughput interactome studies will identify cellular proteins interacting with the different viral components. altogether, the comparison of all these high-throughput screens should help us to identify cellular proteins and pathways common to different viruses which may help, with rational structure/mechanism-based design of entry inhibitors, to develop inhibitors that cross-react with different pathogens. in a similar fashion, the design of vaccine immunogens that are capable of eliciting potent, broadly neutralizing antibodies of known epitopes, is expected to contribute toward the development of vaccines. in parallel, the development of panels of human monoclonal antibodies against every entry-related protein from all pathogenic human viruses could accelerate our understanding of entry mechanisms and help to fight viral diseases. if research continues at the present pace, most of these goals could be accomplished within the next few decades. cytoplasmic tail of moloney murine leukemia virus envelope protein influences the conformation of the extracellular domain: implications for mechanism of action of the r peptide virus particle adsorption. ii. adsorption of vaccinia and fowl plague viruses to cells in suspension c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans identification of a cellular cofactor required for infection by feline leukemia virus toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection class iii viral membrane fusion proteins class iii viral membrane fusion proteins functional dissection of the moloney murine leukemia virus envelope protein gp70 centrifugal enhancement of retroviral mediated gene transfer a novel small molecule inhibitor of hepatitis c virus entry efficient infection mediated by viral receptors incorporated into retroviral particles evolution of cell recognition by viruses features of a spatially constrained cystine loop in the p10 fast protein ectodomain define a new class of viral fusion peptides infectious hepatitis c virus pseudo-particles containing functional e1-e2 envelope protein complexes implication of the proprotein convertases furin, pc5 and pc7 in the cleavage of surface glycoproteins of hong kong, ebola and respiratory syncytial viruses: a comparative analysis with fluorogenic peptides a rapid fluorometric assay for the proteolytic activity of ski-1/s1p based on the surface glycoprotein of the hemorrhagic fever lassa virus a human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: possible role in g protein-coupled signal transduction molecular docking of cathepsin l inhibitors in the binding site of papain activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the s2 domain roles for endocytic trafficking and phosphatidylinositol 4-kinase iii alpha in hepatitis c virus replication implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus jaagsiekte sheep retrovirus utilizes a ph-dependent endocytosis pathway for entry endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase ski-1/s1p identification of gene function by cyclical packaging rescue of retroviral cdna libraries ebola virus uses clathrin-mediated endocytosis as an entry pathway dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events identification and characterization of potent small molecule inhibitor of hemorrhagic fever new world arenaviruses alternate replication in b cells and epithelial cells switches tropism of epstein-barr virus posttranslational oligomerization and cooperative acid activation of mixed influenza hemagglutinin trimers presence of host icam-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and cd4(+)-tcell depletion in human lymphoid tissue, a major site of replication in vivo identification of host proteins required for hiv infection through a functional genomic screen identification and characterization of broadly neutralizing human monoclonal antibodies directed against the e2 envelope glycoprotein of hepatitis c virus characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis c virus glycoproteins the presence of host-derived hla-dr1 on human immunodeficiency virus type 1 increases viral infectivity newcastle disease virus may enter cells by caveolae-mediated endocytosis probing the interaction between vesicular stomatitis virus and phosphatidylserine high-avidity monoclonal antibodies against the human scavenger class b type i receptor efficiently block hepatitis c virus infection in the presence of high-density lipoprotein the cytoplasmic domain of simian immunodeficiency virus transmembrane protein modulates infectivity folate receptor-alpha is a cofactor for cellular entry by marburg and ebola viruses endosomal proteolysis of the ebola virus glycoprotein is necessary for infection a virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis c virus infection in vitro protein-lipid interplay in fusion and fission of biological membranes membrane hemifusion: crossing a chasm in two leaps mechanics of membrane fusion the soluble ectodomain of herpes simplex virus gd contains a membraneproximal pro-fusion domain and suffices to mediate virus entry phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus enhancement of enveloped virus entry by phosphatidylserine rna interference and single particle tracking analysis of hepatitis c virus endocytosis cd81 is an entry coreceptor for hepatitis c virus fusogenicity of jaagsiekte sheep retrovirus envelope protein is dependent on low ph and is enhanced by cytoplasmic tail truncations virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions identification of the paired basic convertases implicated in hiv gp160 processing based on in vitro assays and expression in cd4(+) cell lines the clathrin endocytic pathway in viral infection nipah virus fusion protein: influence of cleavage site mutations on the cleavability by cathepsin l, trypsin and furin hepatitis c virus glycoprotein e2 contains a membrane-proximal heptad repeat sequence that is essential for e1e2 glycoprotein heterodimerization and viral entry mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis c virus glycoprotein e1 the primed ebolavirus glycoprotein (19-kilodalton gp1,2): sequence and residues critical for host cell binding five recombinant simian immunodeficiency virus pseudotypes lead to exclusive transduction of retinal pigmented epithelium in rat the many mechanisms of viral membrane fusion proteins association of the caveola vesicular system with cellular entry by filoviruses targeting of hiv-and siv-infected cells by cd4-chemokine receptor pseudotypes rational development of beta-peptide inhibitors of human cytomegalovirus entry fusion peptides and the mechanism of viral fusion preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis c virus (hcv): a potential treatment to prevent hcv reinfection in liver transplant patients identification of receptors for pig endogenous retrovirus claudin-1 is a hepatitis c virus coreceptor required for a late step in entry cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus host-derived icam-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity equine herpesvirus 1 enters cells by two different pathways, and infection requires the activation of the cellular kinase rock1 efficient and stable transduction of resting b lymphocytes and primary chronic lymphocyte leukemia cells using measles virus gp displaying lentiviral vectors stable transduction of quiescent t cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins strategies for targeting lentiviral vectors receptor specificity of influenza viruses from birds and mammals: new data on involvement of the inner fragments of the carbohydrate chain budding of alphaviruses unbiased probing of the entire hepatitis c virus life cycle identifies clinical compounds that target multiple aspects of the infection dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances transinfection of t cells the membrane-proximal tryptophan-rich region in the transmembrane glycoprotein ectodomain of feline immunodeficiency virus is important for cell entry the herpesvirus glycoproteins b and h. l are sequentially recruited to the receptor-bound gd to effect membrane fusion at virus entry sequence-specific antibodies show that maturation of moloney leukemia virus envelope polyprotein involves removal of a cooh-terminal peptide cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and vsv-g pseudotyped mlv-derived retrovirus vectors to target cells characterization of human monoclonal antibodies specific to the hepatitis c virus glycoprotein e2 with in vitro binding neutralization properties gene transfer in human skin with different pseudotyped hiv-based vectors mechanism of membrane fusion by viral envelope proteins viral membrane fusion virus receptors: binding, adhesion strengthening, and changes in viral structure crystal structure of glycoprotein b from herpes simplex virus 1 on the entry of semliki forest virus into bhk-21 cells cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity aromatic amino acids in the juxtamembrane domain of severe acute respiratory syndrome coronavirus spike glycoprotein are important for receptor-dependent virus entry and cell-cell fusion peptide inhibitors of dengue virus and west nile virus infectivity hepatitis c virus glycoproteins mediate ph-dependent cell entry of pseudotyped retroviral particles sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells maraviroc, a ccr5 coreceptor antagonist that blocks entry of human immunodeficiency virus type 1 viral entry and receptors. in "retroviruses the membrane-proximal domain of vesicular stomatitis virus g protein functions as a membrane fusion potentiator and can induce hemifusion the membraneproximal region of vesicular stomatitis virus glycoprotein g ectodomain is critical for fusion and virus infectivity ccr5 small-molecule antagonists and monoclonal antibodies exert potent synergistic antiviral effects by cobinding to the receptor hantaan virus enters cells by clathrin-dependent receptor-mediated endocytosis a unique heparin-binding domain in the envelope protein of the neuropathogenic pvc-211 murine leukemia virus may contribute to its brain capillary endothelial cell tropism a core trimer of the paramyxovirus fusion protein: parallels to influenza virus hemagglutinin and hiv-1 gp41 differences in cd4 dependence for infectivity of laboratory-adapted and primary patient isolates of human immunodeficiency virus type 1 the postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity in vivo gene transfer using a nonprimate lentiviral vector pseudotyped with ross river virus glycoproteins persistent expression of factor viii in vivo following nonprimate lentiviral gene transfer genome-wide rnai screen identifies human host factors crucial for influenza virus replication peptide inhibition of hiv-1: current status and future potential therapeutic control of hepatitis c virus: the role of neutralizing monoclonal antibodies effects of endocytosis inhibitory drugs on rubella virus entry into veroe6 cells lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion virus membrane-fusion proteins: more than one way to make a hairpin filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo novel retroviral vectors to facilitate expression screens in mammalian cells small interfering rna profiling reveals key role of clathrin-mediated endocytosis and early endosome formation for infection by respiratory syncytial virus global analysis of host-pathogen interactions that regulate early-stage hiv-1 replication characterization of the early steps of hepatitis c virus infection by using luciferase reporter viruses functional regions of the envelope glycoprotein of human immunodeficiency virus type 1 preferential transduction of human hepatocytes with lentiviral vectors pseudotyped by sendai virus f protein inhibition of hepatitis c virus infection by anti-claudin-1 antibodies is mediated by neutralization of e2-cd81-claudin-1 associations rna interference screen for human genes associated with west nile virus infection cooperation of multiple ccr5 coreceptors is required for infections by human immunodeficiency virus type 1 structure of dengue virus: implications for flavivirus organization, maturation, and fusion soluble v domain of nectin-1/hvec enables entry of herpes simplex virus type 1 (hsv-1) into hsv-resistant cells by binding to viral glycoprotein d pcsk9 impedes hepatitis c virus infection in vitro and modulates liver cd81 expression emerging viruses: risk of pandemic the potential anti-tumorigenic and anti-metastatic side of the proprotein convertases inhibitors peptides from conserved regions of paramyxovirus fusion (f) proteins are potent inhibitors of viral fusion identification of a broad-spectrum arenavirus entry inhibitor a prolinerich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes activation of a cell entry pathway common to type c mammalian retroviruses by soluble envelope fragments the envelope glycoprotein of human endogenous retrovirus type w uses a divergent family of amino acid transporters/cell surface receptors characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis c virus broadly neutralizing antibodies protect against hepatitis c virus quasispecies challenge structure of the ebola virus glycoprotein bound to an antibody from a human survivor the lassa virus glycoprotein precursor gp-c is proteolytically processed by subtilase ski-1/s1p the hiv entry inhibitors revisited the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph palmitoylation of the murine leukemia virus envelope protein is critical for lipid raft association and surface expression intersubunit disulfide isomerization controls membrane fusion of human t-cell leukemia virus env a genomewide genetic screen for host factors required for hepatitis c virus propagation different from the hiv fusion inhibitor c34, the anti-hiv drug fuzeon (t-20) inhibits hiv-1 entry by targeting multiple sites in gp41 and gp120 hiv entry inhibitors targeting gp41: from polypeptides to smallmolecule compounds coiled-coil domains in glycoproteins b and h are involved in human cytomegalovirus membrane fusion recognition and blocking of hiv-1 gp41 pre-transmembrane sequence by monoclonal 4e10 antibody in a raft-like membrane environment r-peptide cleavage potentiates fusion-controlling isomerization of the intersubunit disulfide in moloney murine leukemia virus env effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of hep 2 cells by picornaviruses human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis polymorphisms of the cell surface receptor control mouse susceptibilities to xenotropic and polytropic leukemia viruses the lipid-anchored ectodomain of influenza virus hemagglutinin (gpi-ha) is capable of inducing nonenlarging fusion pores virus entry: open sesame the signal peptide of the ebolavirus glycoprotein influences interaction with the cellular lectins dc-sign and dc-signr rgd sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement pathway infectious entry pathway of influenza virus in a canine kidney cell line different potential of c-type lectin-mediated entry between marburg virus strains receptor-induced conformational changes of murine coronavirus spike protein protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection pathways of clathrin-independent endocytosis a cxcr4/cd4 pseudotype rhabdovirus that selectively infects hiv-1 envelope protein-expressing cells common principles and intermediates of viral protein-mediated fusion: the hiv-1 paradigm imaging individual retroviral fusion events: from hemifusion to pore formation and growth vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells virus entry by macropinocytosis vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry virus entry by endocytosis anti-cd81 antibodies can prevent a hepatitis c virus infection in vivo selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycoproteins epstein-barr virus enters b cells and epithelial cells by different routes serum-derived hepatitis c virus infection of primary human hepatocytes is tetraspanin cd81 dependent importance of the cytoplasmic tails of the measles virus glycoproteins for fusogenic activity and the generation of recombinant measles viruses human immunodeficiency virus type 1 attachment to hela cd4 cells is cd4 independent and gp120 dependent and requires cell surface heparans the membranotropic regions of the endo and ecto domains of hiv gp41 envelope glycoprotein a kinome rnai screen identified ampk as promoting poxvirus entry through the control of actin dynamics retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein cytoplasmic domain truncation enhances fusion activity by the exterior glycoprotein complex of human immunodeficiency virus type 2 in selected cell types dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41 role of the membraneproximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion potent antiviral synergy between monoclonal antibody and small-molecule ccr5 inhibitors of human immunodeficiency virus type 1 design, synthesis, and evaluation of inhibitors of cathepsin l: exploiting a unique thiocarbazate chemotype identification of host genes involved in hepatitis c virus replication by small interfering rna technology palmitoylation of the rous sarcoma virus transmembrane glycoprotein is required for protein stability and virus infectivity novel type ii transmembrane serine proteases, mspl and tmprss13, proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication measles virus infection in a transgenic model: virus-induced immunosuppression and central nervous system disease the cytoplasmic tail of the human respiratory syncytial virus f protein plays critical roles in cellular localization of the f protein and infectious progeny production mutations in the membrane-spanning domain of the human immunodeficiency virus envelope glycoprotein that affect fusion activity monoclonal antibody ap33 defines a broadly neutralizing epitope on the hepatitis c virus e2 envelope glycoprotein inhibition of chikungunya virus infection in cultured human muscle cells by furin inhibitors: impairment of the maturation of the e2 surface glycoprotein cathepsin l is involved in proteolytic processing of the hendra virus fusion protein cysteine cathepsin proteases as pharmacological targets in cancer cell surface heparan sulfate proteoglycans: selective regulators of ligand-receptor encounters canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells biochemical mechanism of hepatitis c virus inhibition by the broadspectrum antiviral arbidol nipah virus entry can occur by macropinocytosis identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis c virus e2 protein localization of the labile disulfide bond between su and tm of the murine leukemia virus envelope protein complex to a highly conserved cwlc motif in su that resembles the active-site sequence of thiol-disulfide exchange enzymes initial binding of murine leukemia virus particles to cells does not require specific env-receptor interaction evidence for nonspecific adsorption of targeted retrovirus vector particles to cells nef can enhance the infectivity of receptorpseudotyped human immunodeficiency virus type 1 particles effects of ccr5 and cd4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1 human occludin is a hepatitis c virus entry factor required for infection of mouse cells towards a small animal model for hepatitis c dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans viral entry inhibitors targeted to the membrane site of action development of protein-based inhibitors of the proprotein of convertase ski-1/s1p: processing of srebp-2, atf6, and a viral glycoprotein a broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1 endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry cloning of the cellular receptor for feline leukemia virus subgroup c (felv-c), a retrovirus that induces red cell aplasia rho gtpases modulate entry of ebola virus and vesicular stomatitis virus pseudotyped vectors a synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of sendai virus-cell fusion: an emerging similarity with functional domains of other viruses the rd114/simian type d retrovirus receptor is a neutral amino acid transporter function of the cytoplasmic domain of a retroviral transmembrane protein: p15e-p2e cleavage activates the membrane fusion capability of the murine leukemia virus env protein molecular gymnastics at the herpesvirus surface the envelope glycoprotein from tick-borne encephalitis virus at 2 a resolution characterization of the equilibrium between the native and fusion-inactive conformation of rabies virus glycoprotein indicates that the fusion complex is made of several trimers crystal structure of the low-ph form of the vesicular stomatitis virus glycoprotein g structure of the prefusion form of the vesicular stomatitis virus glycoprotein g structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited protein design of an hiv-1 entry inhibitor comprehensive search for cysteine cathepsins in the human genome membrane-proximal cytoplasmic domain of moloney murine leukemia virus envelope tail facilitates fusion pre-transmembrane sequence of ebola glycoprotein. interfacial hydrophobicity distribution and interaction with membranes membrane fusion process of semliki forest virus. ii: cleavage-dependent reorganization of the spike protein complex controls virus entry a conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity analysis of filovirus entry into vero e6 cells, using inhibitors of endocytosis, endosomal acidification, structural integrity, and cathepsin (b and l) activity sulfhydryl involvement in fusion mechanisms lentiviral vectors pseudotyped with a modified rd114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and cd34+ cells derived from human and nonhuman primates host cyclophilin a mediates hiv-1 attachment to target cells via heparans syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages in vivo evolution of hiv-1 co-receptor usage and sensitivity to chemokine-mediated suppression inhibition of vsv binding and infectivity by phosphatidylserine: is phosphatidylserine a vsv-binding site? construction of a novel virus that targets hiv-1-infected cells and controls hiv-1 infection human monoclonal antibodies that react with the e2 glycoprotein of hepatitis c virus and possess neutralizing activity role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein conserved structural features in the interaction between retroviral surface and transmembrane glycoproteins? discovery of insect and human dengue virus host factors kinetic characterization and molecular docking of a novel, potent, and selective slow-binding inhibitor of human cathepsin l a small-molecule oxocarbazate inhibitor of human cathepsin l blocks severe acute respiratory syndrome and ebola pseudotype virus infection into human embryonic kidney 293t cells tyro3 family-mediated cell entry of ebola and marburg viruses the mechanism of axl-mediated ebola virus infection a new class of fusion-associated small transmembrane (fast) proteins encoded by the non-enveloped fusogenic reoviruses dissecting virus entry via endocytosis inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha substitution of leucine for isoleucine in a sequence highly conserved among retroviral envelope surface glycoproteins attenuates the lytic effect of the friend murine leukemia virus receptor binding and membrane fusion in virus entry: the influenza hemagglutinin a tyrosine motif in the cytoplasmic domain of masonpfizer monkey virus is essential for the incorporation of glycoprotein into virions truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain proteolytic activation of tick-borne encephalitis virus by furin the lymphocytic choriomeningitis virus envelope glycoprotein targets lentiviral gene transfer vector to neural progenitors in the murine brain flavivirus membrane fusion influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease lentiviral vectors pseudotyped with envelope glycoproteins derived from gibbon ape leukemia virus and murine leukemia virus 10a1 identification of human kinases involved in hepatitis c virus replication by small interference rna library screening a functional genomic screen identifies cellular cofactors of hepatitis c virus replication cloning and characterization of a cell surface receptor for xenotropic and polytropic murine leukemia viruses a sodium-dependent neutralamino-acid transporter mediates infections of feline and baboon endogenous retroviruses and simian type d retroviruses a putative cell surface receptor for anemiainducing feline leukemia virus subgroup c is a member of a transporter superfamily cell surface receptors for gammaretroviruses influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion selection of an avian retrovirus mutant with extended receptor usage virus entry is a major determinant of cell tropism of edmonston and wildtype strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins the role of the membrane-spanning domain sequence in glycoprotein-mediated membrane fusion mechanisms of cell killing/cytopathic effects by nonhuman retroviruses strong in vitro synergy between the fusion inhibitor t-20 and the cxcr4 blocker amd-3100 anti-human immunodeficiency virus interactions of sch-c (sch 351125), a ccr5 antagonist, with other antiretroviral agents in vitro kinases required in hepatitis c virus entry and replication highlighted by small interference rna screening noninfectious entry of hiv-1 into peripheral and brain macrophages mediated by the mannose receptor glycoproteins gb, gd, and ghgl of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a cos cell transfection system hiv-1 attachment: another look heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway differential cholesterol binding by class ii fusion proteins determines membrane fusion properties identification of a lipid kinase as a host factor involved in hepatitis c virus rna replication emerging roles of cysteine cathepsins in disease and their potential as drug targets crimean-congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase ski-1 isomerization of the intersubunit disulphide-bond in env controls retrovirus fusion sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with vsv, ebola, mokola, lcmv, or mulv envelope proteins virus membrane fusion cell-associated west nile flavivirus is covered with e+pre-m protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release a synthetic peptide from hiv-1 gp41 is a potent inhibitor of virus-mediated cell-cell fusion peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection retained in vitro infectivity and cytopathogenicity of hiv-1 despite truncation of the c-terminal tail of the env gene product transduction patterns of pseudotyped lentiviral vectors in the nervous system a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry receptors for polytropic and xenotropic mouse leukaemia viruses encoded by a single gene at rmc1 peptides corresponding to the heptad repeat sequence of human parainfluenza virus fusion protein are potent inhibitors of virus infection a genome-wide short hairpin rna screening of jurkat t-cells for human proteins contributing to productive hiv-1 replication studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha ph-induced activation of arenavirus membrane fusion is antagonized by small-molecule inhibitors an antibody directed against the fusion peptide of junin virus envelope glycoprotein gpc inhibits ph-induced membrane fusion interaction of peptides with sequences from the newcastle disease virus fusion protein heptad repeat regions neutralizing antibodies to hepatitis c virus (hcv) in immune globulins derived from anti-hcv-positive plasma scavenger receptor class b type i is a key host factor for hepatitis c virus infection required for an entry step closely linked to cd81 hepatitis c virus entry: molecular mechanisms and targets for antiviral therapy furin processing and proteolytic activation of semliki forest virus structures of immature flavivirus particles functional domains in the retroviral transmembrane protein genome-scale rnai screen for host factors required for hiv replication our research is supported by inserm, ens lyon, cnrs, and ucb lyon-i and by grants from the european union (lshb-ct-2004-005246 "compuvac"), the european research council (erc advanced grant to flc no. 233130 "hepcent"), the agence nationale de recherches sur le sida et les hepatites virales (anrs), and the finovi foundation. we thank sarah kabani for helpful comments on this chapter. we apologize to the many authors of important original contributions that were not directly cited. lastly, we thank the past and present members of our laboratory for their many experimental and intellectual contributions. key: cord-314331-7k0oym5i authors: menza, timothy w.; garai, jillian; ferrer, joshua; hecht, jen title: rapid uptake of home-based hiv self-testing during social distancing for sars-cov2 infection in oregon date: 2020-06-27 journal: aids behav doi: 10.1007/s10461-020-02959-2 sha: doc_id: 314331 cord_uid: 7k0oym5i we implemented a pilot home hiv self-testing program one week after a stay-home order for sars-cov2 was enacted in oregon. we advertised the program on a geospatial networking app and community partner websites targeting men who have sex with men; nine percent of web visits resulted in an order. over 70% of the kits initially allotted to the program were ordered in the first 24 h of launch. one-third of participants had never tested for hiv. we found enthusiasm for discreet, free, home-based testing and uncovered an unmet need for hiv testing as clinical and outreach programs shuttered in oregon. men who have sex with men (msm) continue to comprise the greatest proportion of new hiv infections in the united states and in oregon [1] . hiv testing is a critical intervention toward ending the hiv epidemic among msm. testing is the first step to status-neutral hiv prevention comprised of pre-and post-exposure prophylaxis for those who test hiv-negative; anti-retroviral treatment and viral suppression for those who test positive; and, testing and treatment for sexually transmitted infections (stis) for all [2] . however, msm may not test for hiv with a frequency commensurate with their risk [3] . for example, in 2017, 31% of sexually active msm residing in the portland, oregon metro area had not tested in the prior year [4] . barriers to hiv testing include the stigma of requesting hiv testing; lack of knowledge of testing services; the low density of accessible hiv prevention services; concerns around confidentiality and trust; high perceived cost and low perceived benefit of testing; experiences of racism, homophobia, and other biases within healthcare settings; and, lack of provider cultural competence and comfort working with diverse racial, ethnic, sexual and gender identities [5] [6] [7] . for young, gender diverse, black and latinx msm, these barriers may be further amplified. the implementation of sars-cov2 mitigation strategies, while necessary to slow the spread of sars-cov2, present an additional barrier to hiv testing access. hiv testing in both clinical and non-clinical outreach settings has decreased [8] . while social distancing guidelines recommend against new sexual partnerships, it is implausible to expect complete cessation of sexual contact with non-household members. as a way to maintain social and sexual connectedness during this time, the use of geospatial networking applications (henceforth referred to as "apps") may be increasing [8] . public health agencies must find alternative ways to provide hiv testing while social distancing measures are in effect. apps represent an opportunity to provide that service [9] . in fact, the american men's internet survey (amis) found that 22% of msm who use apps had never tested for hiv [9] . in addition, 77% of msm who use apps wanted social or sexual networking apps to add a feature that would allow them to order an hiv home test from the app. among men who had never tested for hiv, 83% desired this feature. in a recent randomized trial among msm, access to home hiv self-testing increased the frequency of hiv testing and identified more new hiv infections compared to the control group, and, through participant distribution of tests to social and sexual contacts, diagnosed hiv in participants' social and sexual networks [10] . home-based hiv testing, thus, has the potential to increase engagement in status-neutral hiv prevention. in the present study, we present oregon's experience implementing a statewide pilot home hiv self-testing program. in 2016, less than 5% of insured oregonians had tested for hiv and less than 40% of all oregonians reported that they were ever tested for hiv [11] . given these low testing rates, increasing hiv testing became a key priority in our plan to end new hiv infections in oregon. barriers to hiv testing vary widely across oregon's urban and rural geographies. therefore, we sought to employ a webbased hiv self-testing program advertised on apps and other digital media designed to be accessible across oregon. we learned of the development of a new national program, with a strong connection to apps, that was intended to be implemented at the jurisdictional level. oregon was the first state to participate in the pilot of this program. in partnership with building healthy online communities (bhoc), on march 31, 2020, one week after a stay-home order was issued in oregon [12] , we launched a pilot of a free, mail-order, home hiv testing program targeted to msm. we advertised the program website, takemehome. co, through banner advertisements on grindr, a geospatial sexual networking app, and posts on community hiv prevention partner facebook and instagram pages and websites. the conversion rates (orders/visit) for the first 223 orders were high: 7% for grindr (152/2116), 15% (42/284) for direct search, 11% (8/74) for facebook, and 25% (21/84) for community hiv prevention partner websites. the overall conversion rate was 9% (223/2559). eligible participants were ≥ 18 years old and had not tested for hiv in the prior year. participants could order up to two oraquick hiv tests (orasure technologies, bethlehem, pa) at one time to share with a sexual or social network member or save for testing at a later date. we collected age, race/ethnicity, sex assigned at birth, current gender identity, number of sexual partners in the prior year, and timing of most recent hiv test at the time of order placement. participants could provide short written feedback at the time of order placement or through a follow-up email. in this analysis, we present descriptive statistics of demographic characteristics, risk, and testing information and summarize participant feedback. we used stata 16.1 (statacorp, college station, tx) for all analyses. within 24 h of the launch, we received orders for 109 (73%) of the 150 kits initially allotted to the program. we subsequently increased our kit allotment by an additional 324 tests and allowed only one kit per order. from march 31 through may 31, 233 individuals assigned male at birth ordered 248 kits. fifty-one (22%) of 233 participants lived in rural or frontier zip codes. from march 31 through april 3, the data system did not capture the participant-reported demographic, risk, and testing information at the time of order placement. the error in data capture was fixed immediately after its discovery, but we were unable to retrieve the data that was not recorded properly. therefore, we present these measures for the latter 149 (64%) of 233 participants who ordered kits from april 4 through may 31 ( table 1) one-third had never tested for hiv and only 2% had ever used a home test in the past. there were key themes to the feedback we received (table 1) . first, participants expressed appreciation for free testing. second, they reported that the home-testing program was easy, convenient, and discreet. third, the testing felt safe. for one participant, that safety was avoiding the embarrassment and nervousness of going to a healthcare facility for testing. for another, it was using the home-testing program instead of going to a healthcare facility where they might risk exposure to sars-cov2. finally, up-to-date hiv testing results were important to two participants who reported being sexually active during the stay-home order. we implemented a pilot home-based hiv testing program to increase access to hiv testing separate from the sars-cov2 pandemic; however, its launch coincided with rising sars-cov2 cases and the stay-home order in oregon. we observed rapid uptake of this hiv self-testing service, thus revealing enthusiasm for discreet, free, home-based testing and uncovering an unmet need for hiv testing as clinical and outreach programs shuttered. one-third of program participants had never tested before, a proportion greater than that found in a prior survey of msm who use apps and in a trial of home hiv self-testing [9, 10] . for almost all, participation in the pilot program was their first introduction to home hiv self-testing. program participants represented key demographics of msm affected by hiv in oregon [1, 4] : a quarter of our sample was less than 25 years of age; 34% were people of color (25% of oregon's population is comprised of people of color) with 17% identifying as latinx (13% of the oregon population is latinx), 4% identifying as black (2% of the oregon population is black) and 3% identifying as native american (1.2% of the oregon population is native american); and, 25% had more than three sexual partners in the prior year. a goal of our statewide home testing program was to reach msm outside of oregon's urban areas; over one-fifth of program participants lived in rural/frontier areas with poorer access to hiv testing [11] . our pilot testing program was advertised on, and most orders originated from, a single app. diversification of the apps and websites on which we advertise self-testing may allow us to reach more young, gender diverse, black, indigenous, latinx, and rural msm in addition to more sexually active msm. based on the feedback provided by participants, home self-testing appears to ameliorate several barriers to accessing an hiv test. first, testing was free to avoid concerns related to cost and insurance coverage. however, public health agencies must find a way to sustain access to free testing, particularly at a time when std/hiv programs may be underfunded as health departments dedicate substantial resources to responding to the sars-cov2 epidemic. options include prioritizing grant and state/county general funds for home testing programs; billing insurance for hiv tests for those with insurance while continuing to provide never tested for hiv 51 (34) ever tested at home 3 (2) select client feedback "thanks for the free test" "very easy and convenient" "easy to use and feel safe" "i've always been too embarrassed and nervous to go in and get a test. you guys made it so easy and private" "i like how discreet it was and the fact you don't have to wait in a waiting room just do it from the comfort of your own home" "as a person who is nervous to go into the hospital right now, i appreciate this service so much" "i've just really opened myself back up with this covid thing going on, really happy about this, i can keep my profile updated" "being sexually active with the current pandemic, this means a lot to me" free testing for uninsured participants; and/or, allowing participants to donate tests to another participant (a pay-itforward model). as federal funds account for a substantial source of state and local health department funding for hiv prevention, funders should allow for maximum flexibility in how dollars can be spent for home testing initiatives. restrictive caps on use of funds and excessively burdensome data reporting requirements should be avoided. second, testing was "easy" and "convenient." it did not require participants to take a day off from work or put other responsibilities on hold for an appointment at a healthcare setting. third, proximity to local hiv testing resources is less crucial, particularly in rural and frontier areas, when a test can be delivered to one's door. fourth, testing can be performed in the safety and privacy of one's home without fear of judgment or discrimination based on race, ethnicity, sexual and gender identity, and sexual practices. in fact, healthcare providers may judge sexual activity with non-household members quite harshly during stay-home orders. finally, home hiv testing allowed participants to test without risking exposure to sars-cov2 infection in a healthcare setting. while our data are limited in that they represent a single state experience and, due to technical issues, 64% of the individuals who ordered kits, this pilot program suggests that at-home hiv testing can address existing gaps and barriers in testing in many areas of the united states both in the presence and absence of social distancing measures. in the coming months, we plan to expand the sexual health services offered by our home self-testing program to include the ability to order multiple hiv tests for distribution to social and sexual contacts and the option for comprehensive sti testing, hepatitis c screening, and labs for pre-exposure prophylaxis. estimated hiv incidence and prevalence in the united states redefining prevention and care: a status-neutral approach to hiv recommendations for hiv screening of gay, bisexual, and other men who have sex with men-united states hiv infection risk, prevention, and testing behaviors among men who have sex with men-national hiv behavioral surveillance, 23 u.s. cities barriers to hiv testing within a sample of spanish-speaking latinx gay, bisexual, and other men who have sex with men: implications for hiv prevention and care understanding structural barriers to accessing hiv testing and prevention services among black men who have sex with men (bmsm) in the united states barriers to hiv testing among young men who have sex with men (msm): experiences from clark county characterizing the impact of covid-19 on men who have sex with men across the united states in national trends in sexual behavior, substance use and hiv testing among united states men who have sex with men recruited online effect of internet-distributed hiv selftests on hiv diagnosis and behavioral outcomes in men who have sex with men: a randomized clinical trial conference on retroviruses and opportunistic infections (croi) state of oregon executive order 20-12 author contributions twm conceived the study, had full access to the data, analyzed and interpreted the data, and wrote the manuscript. jh acquired the data and had full access to the data. jg and jh contributed to the interpretation of the data. jf assisted with study conception. all authors revised the manuscript for critical intellectual content, agree to be accountable for the integrity of the work, and approved the final manuscript.funding this work was supported by the oregon health authority. conflict of interest there are no conflicts of interest to declare. key: cord-015324-y44sfr0c authors: nan title: scientific programme date: 2007-09-01 journal: pediatr nephrol doi: 10.1007/s00467-007-0558-3 sha: doc_id: 15324 cord_uid: y44sfr0c nan the kidney plays an important role in ion homeostasis in the human body. several hereditary disorders characterized by perturbations of renal magnesium reabsorption leading to hypomagnesemia were described over the past 50 years. only recently, mutations in renal ion channels and transporters have been identified in several of these diseases, following positional cloning strategies in families with these disorders. muations in the slc12a3 gene have been identified in patients with gitelman syndrome, an autosomal recessive disorders characterized by hypokalemia, hypomagnesemia and hypocalciuria. slc12a3 encodes the thiazide-sensitive na + , cl-cotransporter (ncc) in the distal convoluted tubule (dct), the nephron site of active renal magnesium reabsorption. the hypomagnesemia in gitelman syndrome has been shown to be secondary to the primary defect in ncc. mutations in the fxyd2 gene have been found in patients with autosomal dominant renal hypomagnesemia associated with hypocalciuria (idh). fxyd2 encodes the gamma-subunit of the na + , k + -atpase, which is expressed primarily in the kidney with the highest expression levels in dct and tal. hypomagnesemia in patients with idh can be severe (<0.40 mm) and cause generalized convulsions. the molecular mechanism for renal mg 2+ loss in this autosomal dominant type of primary hypomagnesemia remains to be elucidated. in patients with autosomal recessive hypomagnesemia with hypercalciuria and nephrocalcinosis (fhhnc) mutations in the cldn16 gene, and very recently also in the cldn19 gene, have been identified. these genes encode claudin16 and claudin19 respectively, which are essential components of the paracellular pathway for magnesium at the level of the thick ascending limb of henle's loop. mutations in trpm6, encoding the epithelial mg 2+ channel trpm6 in the apical membrane of dct cells, have been identified in patients with mg 2+ wasting and secondary hypocalcemia (hsh). this autosomal recessive disease is due to defective intestinal and renal mg 2+ (re)absorption, and affected individuals show neurologic symptoms of hypomagnesemic hypocalcemia, including seizures and muscle spasms during infancy. in my lecture, i will give an update on the proteins involved in magnesium reabsorption and the emerging pathophysiological understanding of hereditary disorders in which renal handling of this divalent ion is disturbed. transcriptional control of genes that regulate tissue patterning and the cell cycle is fundamental to normal renal development. the mammalian kidney arises from reciprocal inductive tissue interactions between the ureteric bud and the metanephric mesenchyme. these interactions result in growth and branching of the ureteric bud and its daughter collecting ducts, a process termed, renal branching morphogenesis, and formation of nephrogenic progenitors and mature nephrons. expression and activity of transcription factors in ureteric and metanephric mesenchyme cells determines the patterning of ureteric bud and metanephric mesenchyme derived tissue elements. this lecture will highlight transcription factors critical to renal branching morphogenesis and nephrogenesis, with particular emphasis on those factors that are mutated in humans with renal malformation. the regulation of transcription factor activity will be discussed in the context of growth factor signaling pathways downstream of wnts, bone morphogenetic proteins and sonic hedgehog and pathways such as notch that control cell differentiation. the complexity of transcriptional signaling will be highlighted in the normal and malformed kidney at the level of transcriptional factor interactions and target gene promoters that can be targeted by mutiple signaling pathways. t. benzing diseases of the glomerular filter of the kidney are a leading cause of end-stage renal failure. recent studies have emphasized the critical role of the slit diaphragm of podocytes for the size-selective filtration barrier of the kidney and revealed novel aspects of the mechanisms leading to proteinuria, both in inherited and acquired diseases. several critical structural protein components of the slit diaphragm have been identified. recently, it has been shown that slit diaphragm proteins, in addition to their structural functions, participate in common signaling pathways. this talk will focus on what is known about the importance of the podocyte for the function of the glomerular filter of the kidney. it will provide a snapshot of our current understanding of the signaling properties of slit diaphragm proteins and project a framework for further studies necessary to delineate the function and dynamics of the slit diaphragm protein complex and the pathogenesis of nephrotic syndrome. long-term survival of children with end-stage renal disease (esrd) has increased in the last 20 years but the mortality rate remains high. cardiovascular disease accounts for 40 to 50% of all deaths, infectious disease about 20%. a prolonged period of dialysis versus having a renal graft and persistent hypertension are mortality risk factors. the prevalence of all sorts of morbidities is high among those who have reached adulthood. nearly 50% of all these patients suffer from left ventricular hypertrophy and life threatening vascular changes; nearly one third has clinical signs of metabolic bone disease. this accounts for both dialysis and transplanted patients. the chance of getting cancer is 10 times increased as compared to the general population; skin cancer and non-hodgkin lymphomas are most reported. a long period of dialysis at childhood is associated with impairment of both cognitive and educational attainment in adulthood. yet, despite all these negative outcomes, the health perception of young adults with childhood onset esrd is good. unemployment under young adults on chronic dialysis with pediatric onset of disease is higher than among healthy age related peers, but much lower than among dialysis patients of the same age with adult onset of disease. an impaired social development in childhood is associated with an impaired mental health perception at adult age. research and therapy in children with esrd should focus not only on prevention of graft failure but also on prevention of co-morbidity, especially cardiovascular disease, life threatening infections and malignancies. early transplantation, more extended forms of frequent hemodialysis in those who can not be transplanted, a more rigorous treatment of hypertension, avoidance of calcium-containing phosphate binders, reduction of the chronic inflammatory state, and tailor made anti-rejection therapy after transplantation may all be targets to improve outcome in future patients. psychosocial care should be more focused on attainment of independency and preparation for adult life, i. e. schooling and job career. applying regenerative medicine to combat organ shortage laboratory of regenerative nephrology, edmond and lili safra children's hospital, sheba medical center, tel hashomer, sackler school of medicine, tel aviv university, tel aviv, israel organ transplantation has been one of the major medical advances of the past 30 years; however, it is becoming increasingly apparent that the supply of organs is limited and will not improve with current medical practice. regenerative medicine is focused on the development of cells and tissues for the purpose of restoring function through transplantation. when facing late stages of esrd, which necessitate whole kidney transplantation, organogenesis represents an alternative to combat organ shortage. indeed, previous data pinpoints a window of time in human and pig kidney organogenesis that may be optimal for transplantation into mature recipients. "window" transplants are defined by their remarkable ability to grow, differentiate and undergo vascularization, achieving successful organogenesis of urine-producing miniature kidneys with no evidence of trans-differentiation into non-renal cell types, lack of tumorigenicity and reduced immunogenicity compared to adult counterparts. in contrast, when dealing with earlier stages of esrd or acute renal disease, which allow for individual cell replacement, both bonemarrow derived and kidney specific stem cells, might be applicable. accordingly, we show in proof-of-principle experiments that a novel population of multi-potential stem cells derived from the adult murine kidney are capable of re-populating ischemically injured tubular cells, while hematopoietic stem cells (cd34+cd133+) or hemangioblastic progenitor cells can give rise to peritubular endothelial cells. thus, different types of biological materials (embryonic kidney, adult kidney, adult bone marrow) offer new and powerful strategies for future tissue development and regeneration in renal medicine. k. tory, é. kis, a. szabó, g. reusz 1st department of pediatrics, semmelweis university, budapest, hungary it is well known that chronic renal failure significantly increases the risk of cardiovascular mortality in adults. transplantation decreases this risk; however, it still exceeds that of the normal population. as the number of children on renal replacement therapy (including transplantation) has significantly increased in recent years, there has been a growing amount of evidence regarding the increased cardiovascular vulnerability of this specific patient group. one important factor of cardiovascular mortality in adults is the dysfunction of the autonomic nervous system, previously designated as autonomic neuropathy. in a series of studies using the conventional ewing tests as well as heart rate variability (hrv) monitoring we were able to show altered sympathovagal balance in children on peritoneal-and hemodialysis, that was at least partly reversible following renal transplantation. according to our data sympathetic overactivity plays a major role in the sympathovagal disequilibrium observed. correspondingly, beta-adrenergic blockade improves the decreased hrv in the young. early, accelerated arteriosclerosis is another important, recently recognized consequence of crf in children. arterial calcification leads to increased arterial stiffness deteriorating the cushioning function of the aorta. arterial stiffness can be assessed by the reproducible and non-invasive measurement of the pulse wave velocity (pwv). in adults, it was found to be a strong, independent predictor of cardiovascular mortality. hypertension, hyperphosphatemia/elevated serum calciumphosphate product and vitamin d deficiency are the principal factors associated with pwv. recently, pwv was also shown to be increased in children on dialysis. however, the difference to the normal population may only be apparent if a control group matched for body dimensions is used, because of the dependence of pwv on body dimensions and uremic children's significant growth deficit. furthermore, increased pwv could also be shown in children following renal transplantation (tx) indicating the persistence of these lesions even following successful tx. the data on the autonomic nervous system and arterial dysfunction in young patients point to the necessity of early prevention in order to avoid the cardiovascular complications of crf. this study was supported by grants otka-t046155-f048842-f042563 and ett 435/2006. nitric oxide (no) production is reduced in ckd due to decreased renal and widespread decreases in endothelial no production. possible causes of no deficiency are: 1). substrate (l-arginine) limitation; 2). increased levels of circulating endogenous inhibitors of nitric oxide synthase (particularly asymmetric dimethylarginine [adma]); 3). decreased nos protein abundance/activity. decreased l-arginine availability in ckd is likely due to perturbed renal biosynthesis secondary to damage to the renal parenchyma. in addition, inhibition of transport of larginine into endothelial cells and shunting of l-arginine into other metabolic pathways (e. g. arginase) will also decrease availability. elevated plasma and tissue levels of adma in ckd are functions of reduced renal excretion (minor), reduced catabolism by dimethylarginine dimethylamino-hydrolase (ddah) and possibly increased protein methylation. an increase in adma has emerged as a major independent risk factor in many forms of cardiovascular disease as well as end-stage renal disease (and probably ckd). decreased renal nos protein abundance and activity have been reported by us in multiple models of ckd in the rat. the neuronal (nos 1) isoform is always reduced in the presence of injury, and is preserved in settings where there is protection from damage (female; wistar furth rat vs. sprague dawley). generalized and nnos specific inhibition accelerates underlying renal injury and in vulnerable animals will cause injury in the absence of underlying renal disease. we also find that relaxin (rlx) mediated prevention and repair of damaged kidneys requires an intact no system in order to function. in summary, no deficiency can cause cardiovascular and renal disease, and ckd results in no deficiency, contributing to a vicious cycle that promotes progression. an intact no system is required for rlxmediated repair of kidney damage. no deficiency in ckd is likely due to many causes: decreased arginine synthesis/availability and transport; increased endogenous adma functioning as competitive inhibitor; decreased enzyme abundance and activity will all lead to reduction in no generation. in addition, the oxidative stress of ckd will further reduce nos activity, switch the nos to become oxidant generators and will scavenge no to form the harmful peroxynitrite. selected glomerulopathies due to single gene defects such as finnish type nephropathy, diffuse mesangial sclerosis alport/thin membrane disease and inherited lipid disorders and will be discussed. finnish nephropathy is due to mutations of nephrin, a major structural component of the slit diaphragm. over 50 nephrin mutations are known associated with variably absent slit diaphragms, originally thought to be specific for constitutional nephrin mutations. however, sporadic minimal change disease and membranous glomerulonephritis may have absent slit diaphragms suggesting that nephrin may participate in the nephrotic syndrome in nonhereditary diseases. dms is one cause of congenital nephrotic syndrome, characterized by podocyte proliferationis shared by wt1 (denys-drash syndrome), laminin α2 (pierson syndrome) and galloway-mowat syndrome mutations. alport nephritis is a paradigm in which only a subset of mutations may predict disease severity (col ivα5)). heterozygous alport is similar to thin membrane disease (tmd), but 25% of tmd harbor col (iv) α3 mutations and a subset has col ivα2. nail patella syndrome due to heterozygous loss of lmx1b gene which regulates col ivα3-5 expression during kidney development. in classic nail-patella pathology fibrillar collagen bundles within the gbm are identical in severe and mild disease. thus the mutation does not correlate with prognosis. a mutation overlap in factor h gene in lipid disorders such as dense deposit disease (ddd) and lcat underscores glomerular pathologies that ranges from classic ribbon-like deposits in ddd, to lucent gbm deposits characteristic of lcat, or subendothelial lipid pools in lipoprotein glomerulopathy. the importance of genetics underscoring the pathology is amply demonstrated but mutations may not determine prognosis. background genes, post-translational modification and or protein/protein interactions in podocytes or the gbm may act as phenotypic modifiers. acute renal failure (arf) has many different definitions. rifle criteria distinguish "risk, injury, failure, loss and end-stage renal disease" features of this event. classic forms include pathophysiological cases of renal hypoperfusion and direct parenchymal injury, as well as postrenal anatomical obstruction. microvascular mechanism is in general the effect of disturbed balance between vasoconstriction (in response to endothelin, angiotensin ii, thromboxane a2, leukotrienes and increased sympathetic nerve activity) and vasodilatation (in response to nitric oxide, pge2 and bradykinin). endothelial and vascular smooth muscles cells may undergo structural damage and increased leukocytes-endothelial adhesion is a cause of inflammation. cytoskeletal breakdown, loss of polarity, apoptosis, necrosis, desquamation of viable and necrotic cells with tubular obstruction are underlying mechanisms of acute tubular necrosis. ischemic and direct tubular injury dominate as causes of arf, however specific epidemiology is strictly age-related. sepsis and major surgeryrelated events are the most common causes of arf in hospitalized patients. data on genetic background of certain genes polymorphisms and susceptibility to specific risk factors in newborns and infants are conflicting. several prophylactic and therapeutic techniques are available, however not of universal value. appropriate fluid management is crucial in ischemic arf, classic hemolyticuremic syndrome and rhabdomyolysis, however fluid overload is the one of major predictors of poor outcome in children admitted to icu. neither "renal dose" of dopamine, nor loop diuretics change the outcome. involvement of extrarenal organs worsens the overall outcome, which is the poorest in patients with multi-organ failure. early introduced renal replacement therapy is one of the key modalities in icu-treated patients. continuous techniques are of major value. in specific cases, such as hepato-renal syndrome, albumin ("liver") dialysis serves as an effective bridge to liver transplantation. hyperostosis-hyperphosphatemia syndrome (hhs) is a rare recessively inherited disease, manifested by persistent severe hyperphosphatemia and self-remitting episodic bone pain with radiologic findings of periosteal reaction and cortical hyperostosis. hyperphosphatemia in this patient population is not counter-balanced by pth or vitamin d, posing a mirror image of two hypophosphatemic states which result from increased activity of fibroblast growth factor 23 (fgf23). the two hypophosphatemic disorders which result from enhanced urinary phosphate leak are: dominantly inherited hypophosphatemic rickets and tumor induced osteomalacia. this observation was the impetus to study the role of fgf23 in hhs. affected individuals were found to have low levels of the full length, biologically active fgf23, but markedly augmented amounts of the cleaved inactive fragments. patients were found to be homozygous for a mutation in the galnt3 gene encoding a peptide involved in mucin-type o-glycosylation. decreasing the expression of the galnt3 gene by rna interference resulted in augmenting processing of the intact fgf23. our research indicates that the primary defect in hhs is a state of underglycosylation of fgf-23 resulting from reduced expression of ppgantase-t3, due to mutations in galnt3, and leading to augmented processing at the cleavage site. these changes in fgf-23 would abolish its phosphaturic effect and lead to severe persistent hyperphosphatemia. this study provides the pathogenetic mechanism of the first mucin-type o-glycosylation defect identified. our observation lends further credence to the primary and essential role of fgf23 in phosphate homeostasis through a pth-independent pathway. this was substantiated most recently by several reports which showed that mutations in the fgf23 gene are responsible for familial hyperphosphatemic tumoral calcinosis (fhtc) . a further study showed that hhs and familial hyperphosphatemic tumoral calcinosis are allelic disorders with a founder mutation in our region. immune responses govern the outcome of many forms of chronic kidney disease. gene therapy offers the potential to modify immune genes to improve outcome. we will discuss the potential use of gene transduction as a therapy for chronic kidney disease. background: chronic proteinuric renal disease is a major cause of end stage renal disease in man. adriamycin nephropathy is a murine model of chronic proteinuric renal disease where initial chemical injury is followed by immune and structural changes that mimic human disease. foxp3 is a gene specifically expressed by regulatory t (treg) cells. forced expression of the gene foxp3 causes transduced t cells to develop a regulatory phenotype. we hypothesised that foxp3 transduced regulatory t cells could protect against renal injury in adriamycin nephropathy. methods: the retroviral vectors expressing foxp3 and green fluorescent protein (gfp) (foxp3/migr) and gfp alone (migr) were transfected into package cell lines ecopack2-293, which produced the two retroviruses. cd4+ t cells were isolated from spleen of balb/c mice and stimulated by anti-cd3 mab and il-2 for 24 hours and then were infected with either retrovirus. expression was confirmed and phenotypic and in vitro functional assays demonstrated a regulatory phenotype. one week after infection, the gfp+ positive cells were sorted. foxp3 and control vector (migr) transduced t cells were administered to adriamycin (adr)-induced progressive renal nephropathy in mice. results: adoptive transfer of the foxp3 transduced t cells protected against renal injury. urinary protein excretion was reduced; there was less renal injury as measured by glomerulosclerosis, and interstitial infiltrates. serum creatinine, glomerular sclerosis and tubulointerstitial alterations were significantly lower in adr-foxp3 group, compared to those without treatment (adr) and treated with control vector (migr) transduced group (adr+migr). the foxp3 transduced cd4 t cells also showed suppressive activity in vitro. we conclude that foxp3 induced t reg cells may have a therapeutic role in protecting against immune injury and disease progression in chronic proteinuric renal disease. the italkid project is a prospective, population-based registry that was started in 1990. prevalent and incident cases of chronic renal insufficiency (cri) in children and adolescents were identified throughout italy (total population base: 16.8 million children). the inclusion criteria were: i) creatinine clearance (ccr): <90ml/min/1.73m 2 bsa; and ii) an age of <20 years at the time of registration. as to december 31st, 2006 a total of 2026 patients had been registered. the incidence of cri was estimated 12.1 cases per million and the (point) prevalence 74.7 per million of the age-related population (marp). the probability of kidney survival at 20 years of age was significantly different depending on the ccr at study entry, being 63% in patients with mild renal insufficiency (ccr 51-75 ml/min), 30% in those with moderate renal insufficiency (ccr 25-50 ml/min) and 3% in those with severe renal insufficiency (ccr <25 ml/min). the patients with normal (<0.2) and low (0.2-1.0) upr/ucr compared to those with mild (>1.0), showed a significantly slower decline in ccr (deltaccr +0.2±3.62 and -0.6±3.67 vs -3.76±5.64 ml/min/yr) and a higher kidney survival (96.7 and 94.1 vs 44.9%). the incidence of renal replacement therapy (rrt) was 7.3/year/100 patients and the casefatality rate on conservative treatment 1.41%. patients showed a significantly different slope of ccr before pubertal growth spurt as compared to after: -0.31±4.02 ml/min/1.73mq/yrs and -3.1±5.04. a non-linear pattern of decline in the probability of kidney survival, with a steep decrease during pubertal and early post-pubertal age was observed: the overall probability of rrt at age 10 was estimated 9.4%, while 51.8% of the patients will eventually required rrt before the 2nd decade of life was over. suggesting that pubertal development triggers the progression of cri in children. treatment with angiotensin converting enzyme inhibitors did not significantly modify the naturally progressive course of hypodysplastic nephropathy. ambulatory blood pressure monitoring (abpm) is a relatively new technique of blood pressure assessment in children and adolescents that offers several distinct advantages over traditional methods of blood pressure measurement, including the ability to detect white coat and masked hypertension, as well as the ability to assess nocturnal blood pressure. the role of abpm in the diagnostic evaluation of pediatric patients with elevated blood pressure is well-established, and recent surveys have demonstrated that it is used quite often by pediatric nephrologists and other practitioners during the initial evaluation of elevated blood pressure. it is less well-established, however, what role abpm might play in hypertensive children and adolescents once hypertension has been diagnosed and treatment initiated. studies in adults have demonstrated that abpm can assess whether patients on antihypertensive medications have achieved goal blood pressure, or whether their hypertension has progressed in severity. abpm can be used to follow nocturnal blood pressure in patients with chronic kidney disease (ckd) and diabetes, facilitating early identification of non-dipping, which is a known risk factor for progression of ckd or development of diabetic nephropathy, respectively. abpm can also be incorporated into clinical trials of antihypertensive medications to help assess their efficacy and safety. while additional data supporting these applications of abpm to pediatric hypertension clearly need to be generated from well-designed clinical trials, we propose that there is ample justification to utilize abpm just as frequently after the diagnosis of hypertension as before. childhood obesity is increasing globally in epidemic proportions and affects children in both industrialized and non-industrialized nations. in the last 30 years the percentage of overweight or obese children has increased from 5% to almost 30%. if current trends continue, as many as 50% of children may be overweight or obese by the year 2010. obesity predisposes to development of the metabolic syndrome, which is defined in children as the presence of three or more of the following features: abdominal adiposity (bmi >95%ile), serum triglycerides >95%ile, serum hdl cholesterol <5% ile, fasting blood glucose >110 mg/dl, and/or hypertension (systolic or diastolic blood pressure >95%ile adjusted for age, gender and height). the metabolic syndrome occurs in about 5% of children, but in 30-50% of overweight children. the presence of the metabolic syndrome increases the risk for cardiovascular disease and chronic kidney disease almost ten-fold in adults. adipose tissue does not just store fat, but also has important endocrine and immune functions mediated through adipocytokines, including leptin, adiponectin, resistin, apelin and visfatin, and classical cytokines such as tnf-a and il-6. increased leptin, decreased adiponectin and increased inflammatory cytokines, which occur in obesity, are known to induce vascular endothelial dysfunction and increase blood pressure. increase in adipose tissue also leads to infiltration by monocytes, macrophages and lymphocytes, which are stimulated to produce additional cytokines that may contribute to the systemic inflammation associated with obesity and vascular inflammation associated with hypertension. screening for hypertension and metabolic syndrome in obese children is critical to allow early identification of the metabolic syndrome and aggressive early intervention to reduce the risks for progression to cardiovascular disease and chronic kidney disease in later life. therapeutic strategies must include lifestyle changes of weight loss, healthier diet and regular physical exercise as well as treatment of hypertension, hyperlipidemia or hyperglycemia. blood pressure (bp) regulation is affected by numerous physiologic, biochemical, genetic and environmental factors. it has been suggested that exogenous conditions affecting intra-uterine growth and development may pre-program individuals for hypertension, metabolic abnormalities and cardiovascular morbidity in later life. animal data and human autopsy findings support a link between intrauterine growth, nephron endowment and postnatal hypertension. conceptually, it appears increasingly unclear whether the association of birth weight and bp in later life is mediated by intrauterine growth retardation as suggested by various animal models, whether prematurity per se affects bp programming independent of the fetus, nutritional status, or whether postnatal circumstances statistically linked to low birth weight affect this relationship. we have designed a study to evaluate the relationship between gestational age, birth weight and bp abnormalities by applying abpm in a group of children born preterm with and without intrauterine growth retardation and a local control group of children born at term with appropriate weight. this study represents the first systematic assessment of 24-hour cardiovascular regulation in children and adolescents born preterm. our findings indicate that a fraction of these preterm born subjects has a selective nocturnal increase in systolic bp, resulting in an elevated prevalence of non-dipping. our analysis suggests that intrauterine growth retardation, rather than prematurity per se, is the major effector of the early cardiovascular abnormalities observed in preterm children. moreover, we have found that nocturnal systolic bp was closely linked to heart rate, pointing to a possible role of sympathetic hyperactivation. while little data is available regarding a link between low birth weight and/or prematurity and sympathoadrenal function in adult humans, a role of the sympathetic nervous system in the programming of adult hypertension has been consistently demonstrated in various animal models of fetal growth retardation. in conclusion, we detected subtle abnormalities of circadian bp regulation in those preterm born children who suffered from intrauterine growth retardation and this may reflect sympathetic hyperactivation. the intrinsic tendency of kidney disorders with reduced nephron mass to progress and the quest for renoprotective strategies are an ongoing focus of renal research. in adults, hypertension is not only a marker but also a major driving force of renal failure progression. renin-angiotensin system blockers (ace inhibitors and at1 receptor blockers) are preferred antihypertensive drugs in ckd due to their specific renoprotective effects beyond blood pressure (bp) control, mediated by their antiproteinuric, antiinflammatory and antifibrotic properties. it is less clear whether bp reduction to low-normal values has an additional salutory effect on gfr preservation. children suffer from a markedly different spectrum of renal diseases than adults, with a preponderance of hypo/dysplastic kidney malformations. hypertension and proteinuria are common but usually moderate. to elucidate the renoprotective efficacy of ace inhibition and strict bp control in children, the escape trial was launched by a consortium of 33 european pediatric nephrologists. a total of 400 children and adolescents with stage ii-iv ckd received a fixed dose of ramipril and were randomized to intensified (<50th percentile) or conventional bp control (50th-95th pct). 24 h-bp, proteinuria and gfr were monitored over a 5-year period. 24 h mean arterial pressure (map) was reduced by ramipril from 89 to 83 mm hg (i. e. from 1.5 to 0 sds) on average. subsequently, mean map was lowered further to 79 mm hg at 24 months in the intensified arm, and maintained around 83 mm hg (0.2 sds) in the conventional control arm (p<0.0001). on average 1.1 antihypertensive drugs were added to ramipril in the intensified and 0.4 in the conventional treatment arm (p=0.0001). treatment tolerability was excellent in both arms, with less than 3% dropouts due to side effects. in summary, ramipril was effective and well tolerated in children with ckd. it is possible and safe to target low-normal bp in children. final results regarding renal survival will be available in summer 2007 and presented at the ipna meeting. nephronophthisis and joubert syndrome: converging on convergent extension? nephronophthisis (nphp), an autosomal recessive cystic kidney disease, leads to terminal kidney failure in adolescence. nphp may be associated with retinitis pigmentosa (rp) in senior-loken syndrome (slsn) or with cerebellar vermis aplasia in joubert syndrome (jbts). we have identified by positional cloning 7 genes as mutated in nphp. the gene product of nphp1, nephrocystin-1, acts in focal adhesion signaling. by positional cloning we detected mutations in the nphp2/inversin gene as causing infantile nphp (type 2) with association of situs inversus or rp. we demonstrated expression of nphp1, 2 in primary cilia of renal epithelial cells, supporting a new unifying theory for the pathogenesis of cystic kidney diseases (watnick et al. nature genet 34: 355, 2003) , stating that the products of all genes mutated in cystic kidney disease in humans, mice, or zebrafish are expressed in primary cilia, basal bodies, or centrosomes of renal epithelial cells (hildebrandt et al. nature rev genet 6: 928, 2005) . identification of nphp3 mutations in nphp type 3 also revealed the cause of the renal cystic disease mouse model "pcy", for which treatment has been demonstrated. positional cloning of nphp4 led to the demonstration that its gene product, nephrocystin-4, is conserved in c. elegans and expressed together with nephrocystin-1 in ciliated head and tail neurons of the nematode. the role of primary cilia function for retinal-renal syndromes was confirmed by identification of the novel nphp5 gene. recently, several signaling pathways have been implicated in the downstream signaling pathways that connect ciliary/basal body function to the renal cystic phenotype. these include the wnt signaling/planar cell polarity pathways, and the hedgehog signaling pathway. we implicated the planar cell polarity signaling pathway in nphp by positional cloning of mutations in the gene nphp6/cep290 as causing joubert syndrome. abrogation of nphp6 function in zebrafish caused planar cell polarity (convergent extension) defects and recapitulated the human phenotype of joubert syndrome. further gene identification in nphp will result in the definition of functional networks of primary cilia, centrosomes, and planar cell polarity as they pertain to the pathogenic mechanisms of nphpassociated syndromes and other cystic kidney diseases. hnf1b is a transcription factor that is expressed in bile ducts, intestine, pancreas and renal epithelia. germ-line inactivation of the mouse hnf1β/tcf2 gene is embryonic lethal (e7.5) due to defective differentiation of the extra-embryonic visceral endoderm. hnf1β is later expressed in endoderm derivatives and in the mesonephros. to understand the function of hnf1b at later stages of development and during organogenesis, we applied a conditional inactivation based on a (cre-loxp) strategy. with the use of a cre recombinase that is expressed in renal collecting ducts and henle loops (ksp-cre) we found that hnf1b inactivation leads to polycystic kidney disease (pkd). this is reminiscent of the renal phenotype of patients carrying heterozygous mutations in tcf2/hnf1β. these patients suffer from maturity onset diabetes of the young type 5 (mody5) and at the same time from renal cysts (renal cysts and diabetes or rcad). this cystic phenotype is linked to the defective expression of pkhd1 and pkd2, two genes mutated in pkd patients. the cellular and molecular mechanisms underlying pkd are still poorly understood. it was recently speculated that planar-cell-polarity signalling (wnt) could be at the basis of cyst formation. maturation of nephrons during development is accompanied by a considerable lengthening of tubules. this process involves an intense proliferative phase without any increase in tubule diameter. interestingly, we discovered that the progeny of consecutive cell divisions are adjacent one another and oriented in parallel to the axis of tubules. this suggested that upon lengthening, cells divide according the tubule axis. in addition, 3d image reconstructions revealed that oriented cell division is due to the alignment of the mitotic spindle with the axis of the tubule. we hypothesized that oriented cell division could play an essential role in preventing tubular dilation during the massive proliferative phase that accompanies tubular elongation. indeed, our results indicated that both in ksp-cre-kidney-specific-hnf1β-deficient pups and in the pck rats, lacking the expression of pkhd1, the mitotic alignments were highly distorted. our results indicate that hnf1β plays a crucial role in activating the expression of genes involved in the control of tubular size maintenance during tubular elongation. emerging evidence suggests that the intravenous injection of bone marrow-derived cells improves renal function after acute tubular injury. examination of human transplant biopsies of female kidneys that had been transplanted into male recipients have shown the presence of tubular cells that co-express the y chromosome and epithelial markers. however, controversy exists as to whether the protective effect is due to engraftment of the cells in the injured tubule or an endocrine/paracrine effect of the injected cells. our studies demonstrate that intravenous infusion of whole bone marrow from male mice into female recipients results in the appearance of significant numbers of y chromosome + cells in the kidney interstitium, and rare y chromosome + cells in the tubules. the majority of the interstitial cells express leukocyte markers such as cd45. in addition, we have found that i. v. or i. p. injection of bone marrow stromal cells (msc, adherent non-hematopoietic marrow cells) into mice reduced the severity of cisplatin-or ischemia-induced aki. examination of these kidneys demonstrates that mscs enhance tubular cell proliferation and decrease tubular apoptosis after injury. examination of multiple tissue sections at 1 or 7 days after injury failed to reveal any examples of y chromosome cells within the tubules, and only rare examples of y chromosome cells within the renal interstitium. furthermore, exposure to conditioned medium from cultured mscs (msc-cm) significantly diminished cisplatin-induced death of cultured proximal tubule cells in vitro, while i. p. administration of msc-cm in the mouse markedly diminished the rise in bun associated with cisplatin injection. thus our data suggests that hematopoietic cells and their derivatives from adult bone marrow enter the kidney in response to injury where they are primarily localized to the interstitial space as inflammatory cells. in rare instances, these cells may differentiate into, or fuse with, tubular epithelial cells. in contrast, bone marrow mscs fail to enter the kidney in significant numbers, but can protect the endogenous tubular cells from toxic injury by secreting a factor or factors that limit apoptosis and enhance proliferation. renal hypodysplasia (rhd) is characterized by a reduced nephron number, small kidney size and disorganized renal tissue. a hereditary basis has been established for a subset of affected patients, suggesting a major role of developmental genes involved in early kidney organogenesis. gene mutations with dominant inheritance causing rhd, urinary tract anomalies and defined extrarenal symptoms have been identified in tcf2 (renal cysts and diabetes syndrome (rcad)), pax2 (renal-coloboma syndrome (rcs)), eya1 and six1 (branchio-oto-renal syndrome (bor)) for the most frequent of these syndromes. a recent study on a cohort of 100 patients with rhd and consecutive renal insufficiency demonstrated that 16% of them had mutations in one gene encoding for a transcription factor. the majority of mutations were identified in tcf2 (hnf1β) (especially in the subset with kidney cysts) and pax2. this study demonstrates that subtle extrarenal symptoms in syndromal rhd easily can be missed. genetic testing in children with rhd should be preceded by a thorough clinical evaluation for extrarenal symptoms, including eye, ear, and metabolic anomalies. the presence of these anomalies increases the likelihood of detecting a specific genetic abnormality. in addition, mutations in genes that are usually associated with syndromes can occur in patients with isolated rhd. the ret receptor, its ligand gdnf and the co-receptor gfra1 play a pivotal role during early nephrogenesis and enteric nervous development. in humans, activating ret mutations cause multiple endocrine neoplasia, whereas inactivating mutations lead to hirschsprung disease. while ret deficiency also causes renal hypodysplasia (rhd) in the mouse model, genetic abnormalities in ret have not been characterized in human isolated renal malformations to date. the ret mutations, y971f and s649l, reportedly predisposing to medullary thyroid carcinoma (mtc) were found in one and six patients respectively in the same rhd cohort. none of the patients or their carrier relatives had clinical evidence of mtc at the time of the study. our findings suggest that ret mutations predispose to both mtc and rhd, with a low penetrance for either disorder. interestingly, a gdnf mutation was found in addition to a ret mutation and to an eya1 mutation in a patient with the branchio-oto-renal syndrome suggesting an oligogenic inheritance. mutations in clcn5, the gene encoding the chloride/proton exchanger clc-5, underlie most forms of dent's disease, an x-linked nephrolithiasis syndrome that is always associated with low molecular weight proteinuria. clc-5 belongs to the clc gene family of chloride channels and transporters and, in addition to other sites, is expressed in apical endosomes of the proximal tubule. in these vesicles, it co-localizes with the proton pump and with endocytosed proteins. previously thought to be a clchannel, it is now known to mediate electrogenic cl -/h + -exchange. this notion remains compatible with a role in supporting the acidification of endosomes by providing an electrical shunt for the h + -atpase. to clarify the pathogenesis of dent's disease, we created a knock-out mouse model. it displayed low molecular weight proteinuria due to a largely reduced endocytosis by proximal tubular cells (fluid-phase and receptor-mediated). the acidification of renal cortical endosomes was reduced in the ko. the failure of the pt to endocytose pth led to an increased luminal, but not systemic concentration of this hormone. this, in turn, resulted in hyperphosphaturia due to a retrieval of the phosphate transporter napi-2a from the pt plasma membrane. nephrolithiasis can be explained by altered renal handling of pth and vitamind, all of which are secondary to the impaired endocytosis. there is an increased prevalence of nephrolithiasis in individuals with obesity, type ii diabetes, and the metabolic syndrome. in particular, uric acid constitutes a much higher percentage of stones in these patients compared to the general stone-forming population. we strived to examine the pathophysiologic connection between the metabolic syndrome and uric acid stones. in the vast majority of cases, the principal abnormality in uric acid stones is not hyperuricosuria but an excessively low urinary ph. uric acid nephrolithiasis is in fact a disease of 'urinary acidification' although there is no systemic metabolic acidosis in the classical sense because acid-base balance is achieved and no excessive acid is accumulated. however, the excretion of protons using low pk closed buffers rather than the high pk open buffer ammonia dictates a low urinary ph. since protonated uric acid has a sparingly low solubility compared to ionized urate, low urine ph promotes uric acid precipitation. thus uric acid nephrolithiasis is an innocent bystander of low urinary ph. the link between the metabolic syndrome and urinary ph as a continuous variable is explored by epidemiologic, human metabolic, and laboratory studies. in population-based studies in stoneformers, low urinary ph is associated with higher body weight. urinary ph is also lower with increasing number of features of the metabolic syndrome (waist circumference, high triglycerides, low high density lipoproteins, hypetension, hyperglycemia) and within each of the features, the severity of each parameter is inversely proportional to urinary ph. in metabolic studies in humans, low urinary ph is associated with low peripheral insulin sensitivity. when studied as in-patients on identical metabolic diets, patients with type ii diabetes and uric acid stone-formers have high net acid generation than normal volunteers. this alone lowers urinary ph although the reason for the elevated acid load is not clear at present. in addition to high acid generation, uric acid stone formers tend to use buffers other than ammonia to buffer protons in the urinary resulting in unduly acidic urine ph. when challenged with an acute acid load, the ammonium excretion response is markedly blunted in uric acid stone-formers. a similar urinary abnormally of acidic urine ph and underutilization of ammonia is seen in the zucker diabetic fatty (zdf) rat compared to their lean counterpart. these animals have peripheral insulin resistance, elevated serum free fatty acid and have significant steatosis in their kidneys. one abnormality in the kidney is reduced expression and activity of the na + /h + exchanger nhe3 which is the major transporter that excretes ammonium into the urine and its activity is stimulated by insulin. causality is supported by the fact that treatment of the animals with a thiazolidinedione partially reversed the fat infiltration, urinary acidification abnormality and reduced expressed of nhe3. the direct effect of fat on the renal proximal tubule was tested in cultured cells. provision of fatty acids beyond the oxidation capacity of the tubule leads to dose-dependent impairment of nhe3, generalized dysfunction and eventually cell death. a sub-cytotoxic dose of free fatty acid did not affect baseline nhe3 activity and expression but reduced the ability of insulin to activate nhe3. in summary, the metabolic syndrome is associated with increase acid generation that is independent of diet. this increased acid load is effectively excreted by the kidney. failure to maximally utilize the ammonium buffer system results in lower urinary ph and titration of urate to its insoluble form as uric acid and results in uric acid nephrolithiasis. part of the pathogenic mechanism maybe lipotoxicity from fat infiltration of organs. uric acid nephrolithiasis is an "innocent bystander" which is a sentinel of a more generalized alteration in acid generation and excretion. genetic hypercalciuria recurrent kidney stone production is one of the most common diseases of the bipedal human condition, occurring in up to 10% of the population in western societies. unlike four-footed animals who likely perish in the wild from a selection disadvantage from a painful calculus, human beings continue to function, and have the ability to express the biologic defects repetitively that result in a stone. idiopathic hypercalciuria, where a specific gene defect has not yet been established, is a shrinking subpopulation of recurrent calcium stone formers, as specific mutations and functional polymorphisms of genes intimately or distantly related to calcium homeostasis arise from the sequencing of the human genome applied to clinical stone disease. other postulated mechanisms can be sought for in this manner as well, and may await larger population-based studies. the familial nature of nephrolithiasis is clear and robust for most calcium-based stone formers, and may have a gender-specificity for that inheritance. the role of environment in its expression remains controversial. the systemic nature of genetic hypercalciuria may be seen through its associated effects on skeletal health and biology. between 25-33% of children with genetic hypercalciuria have abnormally low bone density measured by dual-energy absorptiometry that cannot be explained by correction for height or body mass. further, normalization of urinary calcium excretion with a variety of therapies is associated with improvements in bone density values over time. a proposal for re-classifying hypercalciuria pathogenetically will be presented, and linked to observational data across the world for successful therapeutic approaches. a call for a stone registry and more careful determination of etiology will be sought through the ipna congress. the systems that regulate blood pressure are plastic during development and can be permanently reset. experiments in animals show that it is surprisingly easy to produce lifelong changes in blood pressure by minor manipulations of the mother's diet before and during pregnancy. this phenomenon has been referred to as "programming". epidemiological and animal studies show that programmed effects operate within the normal range of growth and development, and influence the risk of hypertension, coronary heart disease and stroke in later life. a clinical study of 2003 people aged 62 years from the helsinki birth cohort showed that two different paths of growth preceded the development of hypertension. people already diagnosed as having hypertension had small body size a birth and low weight gain from birth to two years but grew rapidly after two. at age eleven years their body size was around the average. as adults they tended to be obese and insulin resistant. a second group of people had not been diagnosed but their blood pressures were classified as hypertensive under current definitions. they were short at birth, had low weight gain from birth to two years and remained small after two. at age eleven years they were short and thin. as adults they tended to be overweight and have atherogenic lipid profiles. the first path of growth is similar to that which leads to coronary heart disease in this cohort. the second path is that which leads to stroke. this paper will present data on the maternal and placental influences through which these paths originate. nephron number is a key feature of the conceptual paradigm positing that cardiovascular and metabolic diseases that arise during childhood and adulthood have their origin in events that occur during fetal life. in mammals, nephrons are derived from a subset of cells resident within the metanephric blastema. blastemal cells participate in reciprocal inductive tissue interactions with the ureteric bud. these interactions induce the ureteric bud to grow and branch. in turn, ureteric bud branches induce discrete populations of the metanephric blastema to undergo successive transitions resulting in the formation of mature nephrons. a distinct population of metanephric blastema cells in the stroma modulates branching morphogenesis and nephrogenesis. identification of genes that control both branching morphogenesis and nephrogenesis is providing insight into the molecular pathways that could be targeted by environmental, nutritional and hormonal factors that control fetal programming. this lecture will highlight the morphologic and cellular events critical to renal branching morphogenesis and nephrogenesis, and the gene networks that regulate or counter-regulate these events. these gene networks will then be considered in the light of non-genetic factors that modulate their activities. it is now accepted that early life environment can modulate adult phenotype, including the blood pressure. the likely primary mechanism is epigenetic modulation of gene expression during a sensitive period of fetal maturation, but the pathogenesis of the later development of hypertension is unclear. participation by extrarenal factors such as central regulation and peripheral vascular function has been evoked; however, a strong body of experimental evidence suggests that the "setpoint" for renal regulation of na balance and extracellular volume is altered. because both humans and many experimental models with prenatally programmed hypertension appear to have a decrease in the total number of nephrons, impaired filtration of na has been hypothesized to be an important pathogenetic factor. more recent evidence has suggested that postnatal inflammation and accumulation of reactive oxygen species in the renal interstitium may contribute to the genesis of hypertension by upregulating distal nephron na transport. furthermore, the role of renal vascular function remains to be determined. the different mechanisms are not mutually exclusive; it is conceivable that both a prenatal "priming" and a postnatal "second hit" are required for hypertension to become manifest. j. ingelfinger epidemiological studies published in the late 1980s by barker and his group -and since replicated in many populations -provide evidence of an inverse relationship between birthweight and risk of cardiovascular disease, hypertension, and renal dysfunction in adult life. both clinical studies and animal models have been used to investigate mechanisms underlying these observations [as cited in recent reviews. the concept that changes in the intrauterine milieu affect the growing fetus resulting in alterations in physiology and general health in later life has been termed perinatal programming or developmental origins of health and disease [dohad] . yet, despite a large and burgeoning literature about this phenomenon and its relationship to cardiovascular and renal disease, involved mechanisms remain elusive. maternal malnutrition or exposure to various medications or substances leads to an adverse in utero environment that may impair nephrogenesis, evident in experimental animal studies as well as in clinical reports in humans. nephron deficit at birth persists throughout life, creating "low glomerular endowment, " an important risk factor for hypertension and esrd in adulthood. for a number of years it has been hypothesized that nephron number may strongly influence blood pressure as well as susceptibility to renal disease in later life. recently clinicopathologic observations suggest that a relationship more directly. renal morphogenesis involves complex events in which many genes interact in the formation of the final kidney. when the normal pattern of nephrogenesis is interrupted, renal abnormalities may ensue. during renal development, two major events -ureteric bud (ub) branching and mesenchymal-to-epithelial transformation -greatly impact the outcome of renal morphogenesis. renal malformation accounts for approximately 40 percent of childhood renal failure and represents the end result of failure of fundamental embryonic processes in ub and metanephric mesenchyme (mm) lineages. this presentation will review the data concerning renal responses to perinatal challenges as these occur and later evolve during childhood. we will consider the implications and the data available concerning screening, follow-up and management of at-risk persons. generation of oxidized lipoproteins in obstructive nephropathy -atherogenic or fibrogenic? children's hospital and regional medical center, division of pediatric nephrology, seattle, united states chronic kidney disease, regardless of etiology, is characterized by a relentless progression of fibrosis that gradually destroys the normal renal architecture, particularly in the tubulointerstitium. obstructive uropathy accounts for approximately 35 percent of pediatric patients with chronic kidney disease (ckd) and end-stage renal disease (esrd). after post-natal relief of obstruction, the optimal treatment of children with obstructive uropathies remains unknown and for many the progression of ckd to esrd is inevitable. therapeutic options are limited by an incomplete understanding of fibrogenic pathways in the kidney. the major renal fibrotic pathways identified thus far can be broadly classified into those involved in transforming growth factor beta (tgf-b) activation, macrophage-mediated inflammation, angiogenesis, and extracellular matrix production/degradation. it is becoming increasing clear that key molecules have multiple roles in several of these pathways triggering fibroinflammatory events targeting specific cell types. oxidative stress represents an intersection of many of these fibroinflammatory pathways leading to cellular activation and tissue injury. oxidized lipoproteins accumulate in the circulation and renal interstitium in both experimental models and patients with ckd and esrd. many parallels have been drawn between atherogenesis and the pathogenetic mechanisms of progressive kidney destruction by fibrosis. although ckd is clearly associated with an increased cardiovascular risk, it is not clear whether oxidized lipoproteins amplify fibrogenic pathways in the kidney. research in our laboratory suggests that hypercholesterolemia increases injury severity in obstructed kidneys. scavenger receptors mediate the cellular effects of oxidized lipoproteins during atherosclerosis and activate both inflammatory and oxidative pathways. dietary and antioxidant therapies have clear benefits in animal models but limited efficacy in patients. our studies suggest that blocking key scavenger receptors leads to a significant attenuation of both oxidative and pro-inflammatory pathways during chronic injury by obstruction in hypercholesterolemic mice. interventions targeting scavenger receptor signaling may represent an alternative strategy to attenuate both the progression of ckd and cardiovascular disease. the resolution of injury and promotion of renal repair comprises a delicate balance between cell death and destruction of tissue architecture in relation to cell differentiation, maturation and extracellular matrix (ecm) remodeling. although many studies have focused on the cellular and molecular events leading to the development of renal fibrosis, less is understood about the process of renal repair and regeneration. this is despite the fact that the kidney has a significant capacity for regeneration and cellular replacement following acute damage. the present study describes the structural, functional, and expression profile analysis of endogenous renal repair and the regenerative potential of the kidney following reversal of ureteral obstruction (r-uuo) in the mouse.10 days after unilateral ureteral obstruction there is renal tubular cell loss, activation of an inflammatory cascade leading to widespread cortical interstitial fibrosis and the loss of normal medullary architecture. following 2 to 6 weeks after r-uuo there was marked tubular repair and regeneration of medullary components, ecm remodeling and decreased inflammatory cell infiltration. the structural repair observed at 6 weeks post-release of ureteral obstruction was associated with a 50-86% recovery of the glomerular filtration rate (gfr). expression profile analysis was performed to visualize patterns of gene expression that were differentially expressed in the repaired and remodeling areas following r-uuo. we are also interested in the regulation of cellular recovery and the processes involved in epithelial cell re-differentiation in regenerating tubules following injury. tubular epithelial cell cilia may play potential roles in directing the orientation of cell division and epithelial differentiation during the endogenous remodeling process. our results suggest that renal cilium lengthening may be an important factor in the response to injury and subsequent recovery of renal function. these studies propose that a lengthening of renal epithelial cilia increases their sensitivity to flow and reduces damaging epithelial dedifferentiation in the injured renal tubules. a better understanding of the key events involved in endogenous renal repair and remodeling may open the way to new interventions based on their manipulations aimed at acceleration of renal regeneration and prevention of scarring. soren nielsen has kindly agreed to present this topic, however was not able to submit an abstract. the final regulation of urinary k excretion in the fully differentiated kidney is accomplished in the distal nephron, including the cortical collecting duct (ccd), where cell k passively diffuses into the urine through apical k selective channels. the prevalence of the sk/romk channel and its high p o at the resting membrane potential has led to the belief that this channel mediates baseline k secretion. less easily detected is the bk channel which is characterized by a low p o at the physiologic resting membrane potential and [ca 2+ ] i . bk channels are activated by membrane depolarization, elevation of [ca 2+ ] i , and/or membrane stretch, and can be selectively blocked by iberiotoxin (ibx). we have reported that flow-stimulated net k secretion (jk) in the adult rabbit ccd is (i) blocked by ibx and (ii) associated with increases in net na absorption (jna) and [ca 2+ ] i , leading us to conclude that bk channels mediate this process. recent studies have examined the acute and chronic regulation of bk channel-mediated flow-stimulated jk. we reported that flowstimulated jk requires an increase in [ca 2+ ] i due to luminal ca 2+ entry and er ca 2+ release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. channel expression is regulated long term during postnatal development and by dietary k intake. specifically, an increase in tubular fluid flow rate fails to elicit an increase in jk in the rabbit ccd until the 5 th wk of postnatal life, coincident with appearance of immunodetectable bk channels, whereas flow-induced increases in jna and [ca 2+ ] i in 2-wk-old ccds are "mature". a role for the bk channel in renal k adaptation has been suggested by the observation that dietary k loading leads to an increase in abundance of bk message in microdissected ccds with redistribution of immunodetectable channel proteins from an intracellular pool to the apical membrane. additionally, ccds isolated from k loaded animals demonstrate enhanced flow-stimulated jk compared to tubules from control fed animals. in sum, emerging evidence suggests that the bk channel plays a prominent role in distal k secretion in response to increases in urinary flow rate and dietary k intake. the late developmental appearance of this channel is compatible with the need of the growing animal to retain k early in postnatal life. recent advances in molecular genetics of hereditary hypomagnesemia substantiated the role of a variety of genes in human magnesium transport. this knowledge on underlying genetic defects helps to distinguish different clinical subtypes and gives insight into molecular components involved in magnesium transport. during the last four decades, numerous reports concerning inherited magnesium losing disorders have been published and their distinctive phenotypic features have been discussed. phenotypic characterization of affected individuals and experimental studies of appropriate animal models have contributed to a growing knowledge of renal magnesium transport mechanisms. the identification of the affected nephron segments, the different modes of inheritance and the observation of additional characteristic symptoms promoted a classification into different subtypes of inherited magnesium losing disorders. in general, primary magnesium wasting disorders are relatively rare. the prevalence of the more frequent entities, for example gitelman syndrome, has been estimated to be approximately 1: 50.000. for most of the other disease entities, relatively few cases have been reported in the literature. depending on the genotype, the clinical course is sometimes mild or even asymtomatic. therefore, the disease prevalence might be underestimated for some of these syndromes. magnesium transport has been intensively studied in humans and various animal models leading to accepted concepts underlying the pathophysiology of the different forms of hypomagnesemia. however, the electrophysiological characterization of magnesium pathways has been complicated by unintentional simultaneous measurement of other cations so that the molecular correlates mediating mammalian magnesium transport components remained undefined. a different approach to study components of magnesium transport arises from genetic analysis of families affected with magnesium wasting diseases. linkage studies enabled the localization of several genes involved in hereditary hypomagnesemia and in the last decade, a number of genes have been identified by positional cloning. these genes have provided first insight into mammalian magnesium transport molecules. distal renal tubular acidosis may be inherited as an autosomal dominant or recessive trait. mutations in three genes -slc4a1, atp6v1b1 and atp6v0a4 -are associated with the various forms of disease, and give rise to a wide spectrum of clinical severity. in general, dominant mutations do not affect ion transport function per se and do not affect hearing, whereas recessive disease is characterized by loss of function and deafness. some unusual functional consequences of mutations in ae1 and a4 proteins are mistargeting and loss of protein-protein interaction respectively, and these will be discussed. the heart in pediatric nephrotic syndrome y. frishberg shaare zedek medical center, pediatric nephrology, jerusalem, israel in recent years, the molecular bases of several conditions which lead to steroid-resistant nephrotic syndrome (srns) have been identified. the common denominator shared by these clinical entities is that they all result from structural defects in the glomerular barrier, thus explaining their unresponsiveness to immunosuppressants. for instance, the congenital ns of the finnish type is caused by mutations in the nphs1 gene encoding nephrin. a recessive form of srns was found to result from mutations in nphs2 encoding podocin which is specifically expressed in podocytes. we have previously shown that a founder mutation in nphs2 (r138x) is the prevalent cause of hereditary srns (55% of tested are homozygotes) among arab children in israel. interestingly, we noted that a number of patients who are homozygous for the r138x mutation in podocin have a co-existing cardiac disorder. only a few case reports described an association between srns and cardiac defects. we questioned whether the glomerular-barrier disorders, which have been considered to be kidney-specific, have implications on other organ-systems. thus, we systematically reviewed the cardiac status of these srns patients at the time of diagnosis while they had normal blood pressure and preserved renal function (31). cardiac anomalies were detected in 89% of children, the most common of which were cardiac hypertrophy and pulmonary stenosis. analyzing two control groups enabled us to conclude that cardiac disorders in homozygotes for mutations in nphs2 cannot be attributed to an association by chance or to a state of persistent ns. because human podocin mrna is expressed in fetal heart, we hypothesize that it may have a role in normal cardiac development and this will be an issue of further investigation. this is the first study showing a role for podocin in extra-renal tissues and therefore recommends early cardiac evaluation for timely medical management. cvd is the world-wide biggest obstacle to long-term survival of children and adolescents with ckd. mortality from cvd is excessively high on dialysis and continues to be a threat after renal transplantation. early diagnosis of the individual risk for cvd would enable preventive measures on an individual basis. however, prospective studies with hard end points -cardiac events -are difficult if not impossible to conduct in children and adolescents. on the other hand, the known high morbidity and mortality in elderly dialysis patients may largely result from pre-existing comorbidity (advanced atherosclerosis has been demonstrated in this age group at initiation of dialysis). for this reason, investigations in patients with childhood onset ckd may provide a diagnostic window to study the pathogenesis of cardiac and vascular changes in subjects without comorbidities. several studies using non-invasive measurements of surrogate markers for cvd have demonstrated a pattern of early systemic cvd, including changes in intima-media thickness (imt) of conduit arteries (aorta, a. carotis) and muscular arteries (a. femoralis), altered function of peripheral resistance arteries (venous occlusion plethysmography) and abnormalities in the heart (echocardiography). patients show a significant decrease in post-ischemic vascular reactivity with evidence of vascular stiffness and frequently have extraosseous calcifications often involving coronary arteries and heart valves. importantly, these studies have found little evidence for correlations with classical risk factors (except hypertension), but with abnormalities of calcium and phosphorus metabolism and their therapy, including the intake of active vitamin d preparations and calcium-containing phosphate binders. thus, patients with childhood-onset ckd are at high risk to develop systemic cardiovascular changes, which may represent a new disease originating from the survival of ckd, a previously deadly disease, and interventions for the prevention of renal osteodystrophy. this therapeutic challenge needs to be addressed with high priority to enable long-term survival of children with ckd. while their mere existence is still questioned by some investigators, lipid rafts have recently gained a large amount of attention because of their apparent involvement in various cellular processes, including signaling, membrane trafficking, polarization, and endo-as well as exocytosis of proteins as well as pathogens. membrane rafts have been defined as small (10-200 nm) heterogeneous, highly dynamic, sterol-and sphingolipid-enriched domains that compartmentalize cellular processes and that can sometimes be stabilized into larger platforms by protein-protein or proteinlipid interactions. rafts are of special interest for pediatric nephrologists for two reasons: 1. they play a critical role in immune cell activation, especially in the formation of the immunological synapse (is), and thus in many important disease processes affecting our patients, including -but not limited to -transplant rejection. beyond their participation in is formation, rafts also facilitate signaling through other immune cell receptors, such as the interleukin-2 receptors, where they may ascertain cytokine selectivity and specificity. 2. equally important, rafts serve as essential site for proper interactions between nephrin and podocin, thus establishing the integrity of the glomerular filter. general aspects of raft biology as well as their role in immune cell signaling will be reviewed in more depth in this presentation. raft involvement in glomerular filter formation and especially genetic aspects relevant to disturbances of this involvement and of the associated integrity of the filter, as seen in hereditary nephrotic syndromes, are discussed in subsequent presentations in this symposium. reports of familial forms of fsgs date back to 1956, with the observation of an autosomal recessive disease primarily within the finnish population. it is characterized by massive proteinuria in utero, with up to 20 to 30 grams of protein loss per day. nphs1 encodes a gene product termed nephrin that localizes to lipid rafts within the slit diaphragm of the podocyte. steroid-resistant nephrotic syndrome (srns) is an autosomal recessive nephrotic syndrome and manifests between 3 months and 5 years of age, rapid progression to esrd, and with few cases of recurrence after renal transplantation. the gene product is podocin (nphs2), located on 1q25-31. podocin most likely functions in the structural organization of the slit diaphragm and regulation of its filtration function. it has been shown to interact in vivo with both nephrin and cd2-associated protein (cd2ap), a cytoplasmic binding partner of nephrin. mutations in the alpha-actinin 4 gene (actn4), localized to chromosome 19q13 have been associated with autosomal dominant fsgs, characterized by adult onset disease of variable severity and rate of progression to esrd. fractions of the mutant protein have been shown to form large aggregates within podocytes ultimately compromising the function of the normal actin cytoskeleton, both through its abnormal function and toxic accumulation. recently, a disease-causing mutation for hereditary fsgs has been localized to chromosome 11q 21-22, and identified as the transient receptor potential cation channel, subfamily c, member 6 (trpc6). the missense mutation causes a highly conserved proline in the first ankyrin repeat of trpc6 to become a glutamine at position 112 (p112q). the trpc6 p112q mutation causes increased and prolonged calcium transients in transfected cells. the mutant channel also significantly enhances cation signals triggered by at1 receptor activation. biotinylation and immunostaining studies reveal that the mutation also appears to cause mislocalization of the ion channel to the cell surface. whereas previously reported mutations such as nphs1, nphs2 and actn4 have emphasized the importance of cytoskeletal and structural proteins in glomerular diseases, trpc6 related fsgs suggests an additional mechanism for renal disease pathogenesis. knowledge of trpc6 mediated calcium entry into cells may offer unique insights into therapeutic options for glomerular diseases. t. huber university hospital, department of nephrology, freiburg, germany the sense of touch relies on the ability of specialized sensory cells to convert mechanical stimulation into ionic currents. mechanoreceptor cells respond to external force by opening ion channels. recent findings highlight now a potential role for the mechanosensitive ion channel trpc6 at the glomerular filtration barrier. trpc6 localizes to the slit diaphragm and mutations of trpc6 cause familial glomerular disease. mutations of the phb-domain protein podocin are the most common cause of hereditary nephrotic syndrome and we demonstrate that podocin and mec-2, the closest homologue of podocin in caenorhabditis elegans, bind cholesterol to regulate the activity of associated ion channel complexes: deg/enac channels for mec-2 and trpc channels for podocin. both the mec-2-dependent activation of mechanosensation in c. elegans and the podocin-mediated activation of trpc6 channels requires cholesterol. our data suggest that the recruitment of cholesterol by podocin and mec-2 to ion channels plays an important role in regulating their activity. these findings promote the concept that podocin, similar to the function of mec-2, may be part of a mechanosensitive protein complex at the slit diaphragm of podocytes. isidro salusky has kindly agreed to present this topic, however was not able to submit an abstract is there a role for bisphosphonates in pediatric bone disease? f. santos hospital universitario central de asturias, universidad de oviedo, pediatria, oviedo, spain bisphosphonates are being increasingly and successfully utilized to prevent bone fractures and treat bone pain in children with severe osteoporosis from different origins. a largest experience has been accumulated with the administration of cycles of intravenous pamidronate in children with osteogenesis imperfecta. in addition, to the bone resorption inhibition mediated by their effects on osteoclasts, bisphosphonates given in large doses inhibit normal and ectopic mineralization. thus, bisphosphonates have also been used in children with hypercalcemia and in the treatment of calcinosis and heterotopic ossification, bisphosphonates have been administered to renal transplanted adults to prevent or treat bone loss induced by chronic administration of glucocorticoids and might also be useful in the management of urolithiasis in selected hypercalciuric patients. the potential clinical utilization of bisphosphonates in the prevention and treatment of vascular calcification in patients with chronic renal failure is now being explored, although no data on children in this clinical setting are available. a number of questions as to the precise clinical indications to start bisphosphonates' administration, the type of bisphosphonate to be used, the duration of the treatment, the best way to monitor its effectiveness and the risk of longterm toxic effects remain to be answered. water is the solvent in which all metabolic reactions occur. body water moves between compartments with diverse compositions that are separated by semi-permeable lipid membranes. water exchanges also occur between maternal and fetal blood separated by several layers of tissue, the so-called placental membranes. the fetus appears to be dependent on placental flow and perfusion pressure for the bulk of his water requirements, and the prostanoids play a significant role in the control of ureteroplacental and umbilicoplacental blood flows. osmotic and hydrostatic forces control placental water flux. the amniotic fluid (af) appears early during gestation and its volume increases rapidly. the net af volume turnover approximates 95% per day. major sources of af production are represented by fetal lungs secretions, and by fetal urine. the af is initially isotonic, and becomes hypotonic when significant amounts of dilute urine are produced by the fetus. disposal of water is effected by fetal swallowing of af and by the intramembranous (im) pathway, that is the route of absorption between the fetal circulation and the amniotic cavity. this route appears to play an important role in the overall regulation of af volume and composition. the fact that water crosses the im pathway in excess of solutes suggests a role for aquaporin water channels in allowing this transport. circumstantial evidence indicates that the im water flow is regulated by aquaporin 1. homeostatic changes in placental permeability could thus be up or down-regulated by the number of aquaporin water channels in the membrane. systemic lupus (sle) is a multigenic and multifactorial disease, characterized by b lymphocytes polyclonal activation with decreased tolerance, autoantibodies production and immune complexes formation. dna was initially thought to be the mayor auto-antigen, however, it is not immunogen and injection of dna-anti dna does not induce lupus nephritis. the complex of dna and histones (nulceosome) is provided with a positive electrical charge which favours the binding to heparansulphates in the gbm. in sle high levels of nucleosomes are present in circulation due to accelerated lymphocytes apoptosis or defective removal of apoptotic cells. lupus nephritis (ln) is consequent to deposition of immune complexes, activation of lymphomononuclear cells and reactivity of renal cells. the who classification of lupus nephritis has been recently reviewed by the isn/rps. the new classification takes into account a distinction between forms without endocapillary hypercellularity (mesangial or subepithelial deposits) and others with endocapillary hypercellularity (involving less or more than 50% with segmental or global distribution). prognosis and treatment of ln need a flexible therapy, tailored on histological picture and clinical data. steroids must be given at high doses for induction therapy but have drawbacks of heavy morbidity and mortality. the addition of immunosuppressive drugs improves the therapeutic index. the nih protocol used cyclophosphamide (cyc) pulses in severe ln (monthly pulse for 6 months followed by quarterly pulses for 1 year). more recent studies, comparing low with high doses cyc pulses, failed to prove a significantly different effect. for maintenance therapy of ln, mycofenolate mofetil may be a good alternative to cya or aza. rituximab, a chymeric monoclonal antibody anti cd20, which selectively and profoundly depletes b lymphocytes, has provided very interesting results in sle with poor response to classical therapy or in relapse. the possibility of rotating agents with different side effects may allow to lower the doses of steroids, to reduce drug-specific morbidity, and to improve the compliance of patients. efforts should be done to minimize steroids and cyc which are very effective but are the main responsible of invalidating and even lifethreatening complications. for half a century now, physicians have tried to classify vasculitic syndromes. classification of vasculitides is required to put in perspective the pathogenesis and therapeutic advances and to provide a uniform language, given the variation in the epidemiology of these diseases. the "american college of rheumatology" criteria have been used for classification and the chapel hill consensus criteria for definition purposes in children as well however they are based totally on adult criteria. taking into account the differences in children and the new developments in medicine a group of pediatricians have aimed to revise the classification of vasculitic syndromes encountered in children. the consensus group consisted of a multinational panel of experts who were pediatricians, pediatric rheumatologists and pediatric nephrologists. the delphi and nominal group techniques were used. this project has provided classification criteria for henoch-schönlein purpura, childhood polyarteritis nodosa, wegener granulomatosis and takayasu arteritis. this was an important task since appropriate classification criteria for vasculitis in children has been missing for far too long. we hope that the international and multispecialist composition of the expert group involved will facilitate the applicability of this classification for most vasculitic diseases in children seen around the world and will meet the needs of pediatricians. these criteria are now to be validated using a large registry for childhood vasculitides. anti-neutrophil cytoplasm antibodies (anca) are well established as a diagnostic marker for small vessel vasculitis, including wegener's granulomatosis and microscopic polyangiitis. there is increasing evidence that anca are directly involved in the pathogenesis of vascular inflammation in these disorders. in clinical studies, there is a clear relationship between levels of anca and the activity and extent of disease, and anca positivity confers an increased risk of relapse. rising titres of anca predict relapse, and two studies have shown that pre-emptive treatment of those with rising titres can prevent relapse. of great interest is the recent report of a newborn child who developed glomerulonephritis and lung haemorrhage after transplacental transfer of anca. in vitro experiments have shown that anca can activate cytokine primed neutrophils to release oxygen radicals, enzymes and inflammatory cytokines. this is achieved through both direct f(ab) 2 binding and fc receptor engagement. in co-culture, anca can induce neutrophil mediated killing of endothelial cells. in flow chamber studies, anca can induce neutrophil adhesion and transmigration across endothelial cell monolayers. more recently, anca have been shown to be pathogenic in experimental models of disease. high titre anti-mpo antibodies induced by immunisation of mpo deficient mice can transfer glomerulonephritis and vasculitis to naive recipients. immunisation of rats with mpo induces anti-mpo autoantibodies which lead to crescentic glomerulonephritis. intravital microscopy in this model confirms that anca can induce leukocyte transmigration and microvascular haemorrhage in vivo. however, many questions remain unanswered. some patients may have high levels of anca without disease activity, and others may have typical disease without detectable anca. it seems likely that t cells may be involved, not only in providing help for anca synthesis, but also in mediating tissue damage. it is also possible that an additional inflammatory stimulus, for example an infection is required, to enhance the inflammatory effects of anca. greater understanding of the role of anca in vascular inflammation will hopefully lead to safer and more effective approaches to treatment. hacettepe university, faculty of medicine, department of pediatric nephrology, ankara, turkey the therapy of vasculitic syndromes poses a problem for the caring pediatrician. the treatment of anca associated vasculitides will be presented to cover microscopic polyangiitis, wegener granulomatosis and churg strauss syndrome. treatment of all anca-associated diseases are similar. steroids and cyclophosphamide are the mainstay of induction treatment. in severe patients with kidney involvement steroids can be given in the form of intravenous methylprednisolone for 1-3 days (15-30 mg/kg/d, max.1 gr), followed by daily oral corticosteroids (1.5 mg/kg/d, max.60 mg/d). cyclophosphamide may be given at 2mg/kg/d p. o. or monthly iv pulses 0.75gr/sqm for at least 6 months. for maintenance treatment there are again many different regimens for oral corticosteroids. along with corticosteroids for maintenance regimen there are a number of protocols suggesting the continuation of cyclophosphamide. the cycazarem study has shown that the replacement of cyclophosphamide with azathioprine at 3 months was also as effective for disease control. methotrexate has also been shown to be an alternative for maintenance treatment. treatment of the childhood polyarteritis nodosa (pan) with systemic disease is similar to that of anca-related vasculitides. there are a number of non-anca associated vasculitides in childhood. the most frequent in childhood are henoch-schönlein purpura (hsp), kawasaki disease (kd) and takayasu arteritis (ta). the treatment of hsp is usually symptomatic. however, for severe kidney involvement with extracapillary proliferation and rapidly progressive disease severe immunosuppressive treatment is indicated. triple treatment with steroids, cyclophoshamide and dipyridamole have been given in various series. for kd intravenous immunoglobulin at a dose of 2g/kg still remains the first choice of treatment along with salicylates. for ta therapy depends on the extent of vessel invovlement: severe disease necessitates steroids and cyclophosphamide whereas for less intensive vessel involvement methotrexate and steroids may suffice. treatment period depends on the actiivty of the disease. lb. zimmerhackl haemolytic uraemic syndrome (hus) is the most common cause of acute renal failure in children. the syndrome is defined by the triad of microangiopathic haemolytic anaemia, thrombocytopenia and acute renal failure (creatinine over the 97 th percentile). world wide hus is increasing. in a german/austrian multicenter study we follow 628 children with hus occurring in the years 1997 to 2002.5 year follow-up data are now available www. hus-online. at. from this study the following results are obvious. hus affects predominantly children of kindergarten age. the median age at onset is 2,9 years. the majority of hus is of infectious origin. shigatoxin (stx) producing escherichia coli (stec, ehec) are present in over 80% of patients. the predominant shigatoxin type is type ii. hus is classified into two clinical subgroups. "typical" hus usually occurs after a prodrome of diarrhoea (d+hus), and "atypical" hus (ahus), which is not associated with diarrhoea (d-hus). the majority of d+hus worldwide is caused by ehec type o157: h7, which is transmitted to humans via different routes. however non-o157 groups are emerging and are predominant in europe. transmission of disease in elder patients is predominantly through food poisoning and direct contact to farm animals. in infants and small children direct transmission from human to human seems to be more likely. currently there are no specific therapies preventing the disease course. anti-shigatoxin antibodies are being tested by several companies. if this may prevent hus is open to study. otherwise the therapy at present is symptomatic. parenteral volume expansion before hus in patients with positive stx or ehec stool culture may counteract the effect of thrombotic process before development of hus and attenuate renal injury. use of antibiotics, antimotility agents, narcotics and non-steroidal anti-inflammatory drugs should be avoided during the acute phase in particular during the prodromal phase. from our own study the prevention is best done by preventing primary ehec infection. however, patients with severe course and long term sequelae should be screened for genetic abnormalities in the complement system. if auto antibodies against complement proteins or the vwf play an relevant role is under discussion. patients under one year of age at onset have a significant worse outcome and should be kept under surveillance. patients below 3 month of age are very likely to have an inborn error of complement or vwf and should be tested specifically. the european registry on hus and related disorders may help to determine these abnormalities: www. haemolytic-uraemic-syndrome. org. in order to improve long term outcome of these patients, increased awareness and an european (international?) task force is mandatory. in adults with chronic kidney disease, protein-energy malnutrition and inflammation are risk factors for death and accelerated cardiovascular disease. the "malnutrition-inflammation-cachexia syndrome" (mics); anorexia, increased basal metabolic rate, and loss of lean body mass is associated with low serum albumin, decreased protein intake, elevated c-reactive protein, and low serum cholesterol levels in adults. in children, mics manifests as growth retardation. clinical research focusing on the evolution of mics in pediatric ckd to understand its causes and consequences and how nutritional interventions alter its course may be the key to improving survival in ckd. baseline cross sectional data from the ongoing chronic kidney disease in children study, of children (n=335) aged 1-16yrs (mean=11.1yrs) with estimated gfr 30±90 ml/min/1.73 m 2 (mean iohexol gfr 38 ml/min/1.73 m 2 ) shows substantial growth retardation in ckd with median height percentile=23. greater height deficits are seen at lower gfr's. symptoms of decreased appetite and nausea are reported by 35% and 47% of those with gfr <30 respectively. mean ldl cholesterol is lowest in those with gfr <30 (97 vs 123mg/dl in gfr 30±40 ml/min/1.73 m 2 group). serum albumin declines as serum creatinine increases (r=-0.22). unlike previous reports in adult ckd, increases in crp were not associated with lower gfr at baseline. further exploration of the mics in pediatric ckd will be presented. the causes of growth failure in pediatric patients with chronic kidney disease (ckd) are multifactorial. it is an open question as to which factors play a key role in diminishing physical growth. it is also unclear which mechanisms may become pace makers for the therapeutic improvement of growth. furthermore, growth failure in children with ckd affects total body height, body proportions and composition as well as organ development. growth failure may also lead to disproportion which can only be identified by detailed anthropometric measurements. we were able to demonstrate that ckd patients have a specific age-dependent pattern of growth and distinct changes in segmental growth (trunk, arm and leg length) from birth to adolescence. leg growth in relation to other parameters of linear growth showed the most dynamic growth changes and emerged as the best indicator of growth in children with ckd. trunk growth had little synchronicity with leg growth. furthermore, we found that anthropometric measurements can be used as a diagnostic tool in distinguishing different sub-groups of ckd patients, for instance, children with syndromic ckd. in the heterogeneous group of patients with focal and segmental glomerulosclerosis, patients with schimke's disease were found to have a dramatically decreased sitting height/leg length ratio. as the disturbance in growth of ckd patients is a marker of the severity of the disease and of the quality of renal care, the annual analysis of growth failure from early childhood to adolescence should be used as a landmark information of the quality of medical care and as a helpful tool in differential diagnosis and of specific courses of ckd in sub-groups of patients. for example, patients with congenital or acquired renal diseases, dialyzed or transplanted patients and in gender differences. in addition, anthropometric measurements are able to identify specific growth patterns in children with ckd, which should be considered in the assessment of treatment efficacy such as in rhgh therapy. b. tönshoff, l. weber, b. höcker university children's hospital, 1st department of pediatrics, heidelberg, germany it is currently under debate whether steroid avoidance or late steroid withdrawal provides the best overall risk-to-benefit ratio in pediatric renal transplantation. late steroid withdrawal has the advantage over steroid avoidance that immunological high-risk patients and those with unstable graft function can easily be identified beforehand and be excluded from steroid-free immunosuppression. in order to further validate this approach, we performed a prospective randomized open-label multicenter trial in 41 low-risk pediatric renal transplant recipients (12 f, 29 m; mean age 10.1 yrs; range, 3.4 to 17.8) on csa (target trough level 100-200 ng/ml), mmf (1200 mg/m 2 per day) and methylprednisolone (3) (4) mg/m 2 per day), who were randomly assigned >1 year posttransplant to continue steroids or to withdraw over a period of 3 months. an interim analysis was performed at a mean observation period of 29 mo after study entry; 31 patients had been followed for at least 15 mo. there were 3 drop-outs (1 reversible acute rejection episode, 1 switch to sirolimus and 1 to tacrolimus). transplant function as assessed by calculated ccr remained stable in both groups, no graft was lost. prepubertal children off steroids gained relative height from baseline -1.5±0.5 sds to -0.9±0.6 sds after 2 yrs, while patients on steroids lost relative height (-1.1±0 .6 sds at baseline, -1.6±0.7 sds after 2 yrs); a comparable pattern was observed in pubertal patients. the standardized body mass in patients off steroids declined from 0.75±1.01 sds at baseline to 0.31±1.17 after 2 yrs (p<0.05), while it tended to increase in patients on steroids (baseline, 0.40±1.35 sds; after 2 yrs, 0.56±1.39). the rate of adverse events, mainly infections, was comparable in both groups. patients off steroids required less frequently antihypertensive medication (62%) than patients on steroids (93%). a significant reduction of serum cholesterol (by 20%) and triglycerides (by 13%) in response to steroid withdrawal was observed. conclusions: this interim analysis indicates that late steroid withdrawal in selected pediatric renal transplant recipients on csa and mmf is safe, allows catch-up growth and ameliorates cardiovascular risk factors. at the ipna meeting, full 12 month outcome data of all patients will be available. u. frei, j. noeldeke renal transplantation faces two major challenges: the organ shortage resulting in extended waiting times and an aging population resulting in increased death with a functioning graft. the eurotransplant senior program (esp) allocates kidneys within a narrow geographic area from donors aged >65 years to recipients >65 years regardless of hla. this analysis investigates the impact of the esp on waiting time, graft and patient survival. the esp group (n=1406, old to old) was compared to two groups allocated via the eurotransplant kidney allocation system (etkas) with either similar donor age [old to any (o/a), donor age >65, n=446] or recipient age [any to old, (a/o), recipient age 60-64, n=1687]. all patients were transplanted between 1999 and 2004. since initiation of the esp (1999), availability of elderly donors doubled and waiting time for esp patients decreased. local allocation led to shorter cold ischemia time (11.9 vs. >17.0 hours, p<0.001) and less delayed graft function (dgf, esp 29.7% vs o/a 36.2%, p=0.047) but 5-10% higher acute rejection rates. importantly, graft and patient survival were not negatively affected by the esp allocation scheme. the esp age matching of elderly donors and recipients is an effective allocation system for organs from elderly donors. the effect of age matching on the duration of the waiting time a. rahmel, m. slot, j. smits eurotransplant international, leiden, the netherlands in eurotransplant the proportion of children, i. e. patients aged under the age of 16 at time of registration, with end-stage renal disease (esrd) on the renal waiting list amounts to only 1.2%. despite this small proportion the eurotransplant kidney allocation system (etkas) specially addresses pediatric transplant candidates. in order to increase their chances of receiving a transplant in time children are assigned via etkas extra points for hla matching and receive an age bonus. in order to evaluate this allocation policy, the chances for receiving a kidney for patients in different age groups were evaluated. for the cohort of patients registered in the year 1999, a 5 year followup was obtained. for patients aged under 6 at time of registration [n=134] 47% and 81% received a kidney from a post-mortem donor, within 1 year and 5 years after listing. for children aged 6 to 10 [n=108] these rates were at 1 and 5 years after listing 31% and 67%, and for children between age 11 and 16 [n=192] 33% and 72% respectively. adult patients were less likely to receive an organ: 11% and 42% of patients aged between 16 and 64 were transplanted within 1 and 5 years after listing. senior esrd patients can benefit from the eurotransplant senior program (esp), their chances for a transplant were 28% and 61% within 1 and 5 years, respectively. the life span of a renal allograft is limited in time. in the eurotransplant experience 80% of the post-mortem kidneys used for transplantation in children failed within 20 years, compared to 70% for the living donor renal transplants (rtx). donor age is an important factor associated with long term renal allograft function. age matching between donor and recipient is hampered by the low number of pediatric donors. in the period 2000-2005, 78 heart beating kidney donors aged under 1 were reported and used for rtx, 69 donors were between 2 and 5 years of age, and 319 donors were between 6 and 16 years old. in the same period 9981 adult donors were reported and used for transplantation. in the period 2000-2005, 18% of the pediatric donor kidneys ultimately served a pediatric recipient. to improve the allocation of pediatric donors to pediatric recipients, et is in the process of implementing a rule giving pediatric recipients priority in case of pediatric donors. gender and sex hormones are playing a central role in the incidence and progression of different renal diseases. vascular tone, endothelial function and immune response are influenced by gender. in renal transplantation ischemic injury is always present. females are more resistant to postischemic renal failure than males. following ischemia/reperfusion injury renal vascular resistance decreases, allowing fast restitution of blood flow and oxygen supply in females. additionally, stabilization and preservation of tubular function following ischemia is achieved by the protection of na+/k+ atp-ase and heat shock protein activity contributing to the observed gender differences. posttransplant immune reactions also differ between genders. female gender predisposes to more severe acute rejection following kidney transplantation, partly due to differences in the effectivity of immunosuppressants used. ineffective immunosuppression in females, as well as different cytotoxicity profile of the drugs is contributing to more prone immune reactions shortly after transplantation resulting in more severe acute rejection. in contrast, similar to the slower deterioration of other renal diseases, progression of chronic graft failure is less pronounced in females in experimental and clinical settings. estradiol decreases profibrotic processes, preserves endothelial function and modifies influx of immune cells into the graft. the fine balance between alloantigen dependent immune reactions and alloantigen independent factors and its impact on longterm graft function is modified by gender. new evidences supporting the significance of sexual dimorphism following kidney transplantation may present the base of gender modified therapeutic approaches in the future. iga nephropathy: aetiology, incidence, and geographic distribution iga nephropathy (igan) is characterized by mesangial deposits of iga, proliferation of mesangial cells and expansion of matrix. the accumulation of iga and complement fractions within glomeruli was initially ascribed to deposition of iga immune complexes (igaic) due to mucosal immune response with predominant iga synthesis. this hypothesis has offered an explanation of the relationship between infections of the upper airways and gross hematuria. high levels of igaic have been detected in 30-70% of patients, mostly of polymeric iga1, and polymeric iga1 are detectable in renal deposits. this observation is consistent with hypothesis of either bone marrow or mucosal origin. however, no specific viral or alimentary antigens have been found in mesangial deposits, and the qualitative properties of polymeric iga1 have rather become of interest, particularly the glycosylation pattern of iga1. human iga1 is o-glycosylated with carbohydrate chains of n-acetyl galactosamine (galnac) and galactose (gal) which may be covered with sialic acid (neu5ac). patients with igan exhibit circulating iga1 with reduced gal and/or neu5ac and increased exposure of n-galnac. such aberrantly glycosylated iga can circulate in monomeric form or participate in the formation of autoaggregates/true immune complexes. it likely escapes clearance by hepatic receptors and has a preferential renal deposition by virtue of enhanced reactivity with the mesangial matrix components. the role of a triggering event has not ruled out. in recent reports, antigens of bacterial origin and the secretory tract component have been found in renal deposits, making scientists reconsidering again the role of bacterial infections. tonsils recurrent infections may provide a suitable aetiology for igan, and the effect of tonsillectomy on the long term outcome of igan is under evaluation. the prevalence of igan varies in different areas, due to ethnic and environmental factors, being particularly frequent in mediterranean europe, northern europe, asia and australia. it is still debated whether differencies in frequency, clinical features and disease progression among patients with igan from different countries are actually due to uneven distribution of this diseases or these discrepancies are due to the different criteria for performing renal biopsy. definition. igan is characterized by the presence of dominant (or codominant) mesangial deposits of iga on immunofluorescence microscopy, frequently with c3 and sometimes with igg or igm. classification. the glomeruli display a broad spectrum of histologic changes, related in part to the differences in the indication for the biopsy of the referring nephrologist. a number of classification systems have been used to describe the histologic manifestations of igan. two will be referred to here: the system of m. haas (ajkd 29:829-842, 1997 ): 1) minimal histologic lesion -the glomeruli exhibit no more than a minimal increase in mesangial cellularity, without segmental sclerosis or crescents. 2) fsgs-like -the glomeruli display focal segmental sclerosis in a pattern resembling primary focal segmental glomerulosclerosis, with at most a minimal increase in mesangial cellularity and no crescents. 3) focal proliferative glomerulonephritis (gn) -50% or fewer of the glomeruli are hypercellular. the increase in cellularity may be limited to mesangial areas, or there may be obstruction of glomerular capillaries by proliferated endocapillary cells. crescents may be present. 4) diffuse proliferative gn -more than 50% of glomeruli are hypercellular. the hypercellularity may be segmental or global, and crescents may be present. 5) advanced chronic gn -40% or more of the glomeruli are globally sclerotic, and/or there is 40% or more tubular atrophy or loss in the cortex. the system of s. emancipator (heptinstall's pathology of the kidney, lippincott-raven, philadelphia, 1998, pp 479-512) : a -normal/minimal glomerular lesions b -focal mesangial proliferation c -diffuse mesangial proliferation d1 -focal segmental endocapillary proliferation superimposed on mesangial proliferation d2 -focal segmental endocapillary proliferation alone e -diffuse endocapillary proliferation f -diffuse extracapillary proliferation (crescents in >70%), with or without endocapillary proliferation g -glomerulosclerosis (>70% of the glomeruli are sclerotic) h -unclassifiable or combined lesions diffuse proliferative gn (c) and focal proliferative gn (d1) are the major patterns of expression of glomerular injury. indicators of a poor outcome. these include a high proportion of glomeruli with crescents, numerous sclerotic glomeruli, interstitial fibrosis and tubular atrophy, extension of the iga deposits into the peripheral glomerular capillary walls, and hyaline arteriolosclerosis. differential diagnosis. mesangial iga deposits are present in henoch-schönlein nephropathy, and may be present in lupus nephritis, chronic liver disease, coeliac sprue, certain dermatologic diseases, and some rheumatologic diseases. iga nephropathy [igan] is defined by the presence of mesangial iga, but otherwise the histopathological and clinical features are very variable. we do not yet know sufficient about pathogenic mechanisms to understand whether igan is a single disease. although traditionally called an 'immune complex' disease there is no direct evidence that mesangial iga deposition in igan occurs through classical antigen-antibody interactions. mesangial cells do carry receptors for iga, which may play a key role in iga deposition and subsequent injury, but these are not yet fully characterised. mesangial iga in igan is polymeric iga1. there is no evidence of mucosal immune system overactivity, indeed there seems to be underproduction and abnormal t cell control of mucosal iga production, along with overproduction by the marrow of iga1 iga1 has a hinge region peptide structure which is a site for o-glycosylation. in igan circulating and mesangial iga1 both have abnormal o-glycosylation at the hinge region. the same defect is seen in henoch-schönlein purpura only when there is renal involvement. the glycosylation defect is not due to abnormal peptide structure of the hinge; the possibility that there is reduced activity of the key enzyme b1, 3 galactosyltransferase in b cells or plasma cells has not yet been confirmed. alternatively the glycosylation changes may reflect a shift in the production of mucosal type of iga1 to the marrow. while iga1 deposition is caused by disease mechanisms specific to igan, subsequent inflammatory and fibrotic events are probably driven by mechanisms common to other chronic glomerular disease. in some patients iga1 deposition is not followed by inflammation, in others inflammation resolves without fibrosis. cytokine and growth factor production by mesangial cells is a sufficient explanation for glomerular inflammation and fibrosis. however little is yet understood of any genetic or environmental influences which protect some patients from progressive renal injury. our recent controlled trials by the japanese pediatric iga nephropathy treatment study group demonstrated that treatment of children with severe iga nephropathy with prednisolone, azathioprine, heparin/warfarin, and dipyridamole for 2 years early in the course of the diseaseprevents immunological renal injury and progression of the disease. the majority of patients with iga nephropathy in our series are diagnosed early in the course of the disease, and the asymptomatic period before the discovery of urinary abnormalities is short. early diagnosis and early treatment is very important in iga nephropathy. 1. combined therapy for severe childhood iga nephropathy (j am soc nephrol 10: [101] [102] [103] [104] [105] [106] [107] [108] [109] 1999) . seventy-eight children with newly diagnosed iga nephropathy showing diffuse mesangial proliferation were randomly assigned to receive either the combined therapy of prednisolone, azathioprine, heparin-warfarin, and dipyridamole for two years (group 1) or the combination of heparin-warfarin and dipyridamole for two years (group 2). urinary protein excretion was significantly reduced in group 1 patients, but remained unchanged in group 2 patients. the percentage of glomeruli showing sclerosis was unchanged in group 1 patients, but significantly increased in group 2 patients. 2. steroid treatment for severe childhood iga nephropathy (clin j am soc nephrol, 1:511-517, 2006) . in this study we have compared the effects of prednisolone, azathioprine, warfarin, and dipyridamole (combination) with those of prednisolone alone in 80 children with newly diagnosed iga nephropathy showing diffuse mesangial proliferation. patients were randomly assigned to receive either the combination or prednisolone alone for two years. the primary endpoint was the disappearance of proteinuria, defined as urinary protein excretion <0.1 g/m 2 /day. thirty-nine of the 40 patients receiving the combination and 39 of the 40 receiving prednisolone completed the trial. thirty-six of the 39 patients (92.3%) receiving the combination and 29 of the 39 (74.4%) receiving prednisolone reached the primary endpoint by the two-year follow-up point (p=0.007 log-rank). the percentage of sclerosed glomeruli was unchanged in the patients receiving the combination, but increased in the prednisolone group (p=0.0003). the frequency of side-effects was similar in the two groups. long-term administration of recombinant human erythropoietin (epo) has become the most common way of treating anemia in chronic renal disease. standard amounts per unit body weight have been recommended for the initial dose. however, several authors have noted that the dose per unit body weight needed for a given response is higher in younger children than in older children or adolescents and that it is increasing with decreasing body weight. furthermore, for a given absolute dose of epo the outcome was investigated in adult hemodialysis patients, but no dependence was found in 54 patients weighing 45-86 kg. a similar model to the hemoglobin-time data of 8 children aged 8-15 years treated with epo for renal anemia did not find an impact of body weight on response when it was modelled in terms of absolute dose. in a similar analysis to the hemoglobin-time data 52 children and adolescents aged 5-20 years were analyzed, in order to more definitively answer the question if, for given absolute doses the hematopoetic response to epo in children depends on body weight. neither the dose response parameter e max and ed 50 showed dependence on body weight. the median hemoglobin response to a standard dose was similar to that reported for adults. it can be concluded that younger and smaller children need relatively more epo than older children. doses for children should be specified as absolute amounts rather than amounts per unit body weight. references: scigalla p. effect of recombinant human erythropoietin treatment on renal anemia and body growth of children with end-stage renal disease. in: baldamus ca, scigalla p, wieczorek l, et al, editors erythropoetic agents are the mainstay of treatment for renal anemia. although there are several different marketed forms of erythropoetin, they are not substantially different in their ability to stimulate erythropoesis. however, darbepoetin, which differs in one amino acid, and has additional glycosylation sites, has a longer half-life and therefore a presumed longer duration of action. this provides a theoretical advantage, suggesting that less frequent injections will be required. clinical experience and studies have confirmed that both erythropoetin (epo) and darbepoetin (dar) are effective to maintain hb values within the recommended range. also, though there is some experience with epo injected every couple of weeks, the overall evidence suggests that dar has a longer duration of action, and injections are required less frequently. for many children, particularly the pre-dialysis population, anemia is successfully controlled with dar injected every 3-4 weeks. however, this apparent advantage of dar is somewhat reduced because of the increased pain reported with dar injections compared to epo. both epo and dar have been used successfully in children of all ages. with epo, the doses required to maintain hb values appear to be increased in infants compared to older children; this may not be the case with dar, but there is much less experience with use of dar in infants. also, the administration of dar in infants is hindered by the need to inject portion of a unidose pre-filled syringe, which may introduce inaccuracies in dosing, is wasteful, and is not user-friendly. the side effect profile for each product is similar, and specifically each has been associated with development of hypertension, which is most likely with higher hb values. pure red cell aplasia has also been reported with each product. thrombocytosis has been reported with dar use. overall, there is little to recommend one product over the other. the need for less frequent injections may favor dar for use in most children beyond infancy, whereas it is easier to administer epo accurately in infants. how much iron is needed and how much is toxic? iron deficiency is the primary reason for ineffective erythropoiesis in patients with chronic kidney disease (ckd) who receive an erythropoiesis stimulating agent (esa). reasons for iron deficiency include inadequate dietary intake, blood loss from the gastrointestinal tract, frequent blood tests, loss of blood in the extracorporeal circuit of hemodialysis (hd), as well as the hepcidin related impairment of the intestinal absorption of iron and its release from the reticuloendothelial system. supplementation of iron can be by either the oral or intravenous (iv) routes, although the national kidney foundation-kidney disease outcomes quality initiative (k/doqi) strongly recommends the preferential use of iv iron in children and adult patients receiving hd. the k/doqi recommended target iron indices for all children on dialysis are a serum ferritin >100ng/ml and a transferrin saturation (tsat) >20%. while the serum ferritin should not regularly be >500 ng/ml, some such patients will achieve a higher hemoglobin value following an iv course of iron therapy. of the iv iron agents, the non-dextran products appear to be safest, and pediatric dosing recommendations exist for ferric gluconate and are currently being studied for iron sucrose. differences do exist for the clinical (e. g. proteinuria) and subclinical (e. g. oxidative injury) toxicities associated with the currently available iv iron products and should be taken into consideration when prescribed. until recently the major physiological function of erythropoietin (epo) was thought to be the induction of erythropoiesis. however, a growing body of evidence indicates that epo has tissueprotective properties and prevents ischemia induced tissue damage in several organs including the kidney. a main target of epo´s action is the endothelium, and one of the pivotal intracellular pathways mediating the beneficial effects of epo is the activation of akt, i. e. serine/threonine protein kinase b. as a result, akt phosphorylates the proapoptotic factor bad, which in turn causes inhibition of programmed cell death (apoptosis). moreover, experimental studies revelead that epo is a potent regulator of endotheial progenitor cell (epc) proliferation and differentiation. collectively, these data support the hypothesis that epo is a key molecule in the process of endothelial (vascular) repair and neoangiogenesis. treatment with rhuepo or analogues could therefore open new therapeutic strategies in regenerative cardiovascular medicine. introduction: africa as a continent is besieged by many health challenges including malaria, hiv (upwards of 60% of paediatric admissions), tuberculosis and malnutrition with an infant mortality rate (imr) ranging from 62(southern africa) to 132(mozambique). good health facilities are on the whole not available except in the extreme north and south of the continent with doctors/100 000 population in south africa 69 but in kenya only 13 and even lower in other parts of africa. renal disease: renal disease in adults is an unknown quantity with no information being available regarding children's renal disease, where many babies do not even have access to antenatal ultrasounds. situation in south africa: in reality, there are only 2 centres (cape town and johannesburg/ pretoria) doing paediatric dialysis and transplantation in significant numbers with small numbers interspersed in the rest of the country. this raises numerous issues: -accessibility for children to renal care -retaining minimum standards of care for children -both dialysis and transplantation -combined with adult units -central government funding in form of tertiary services grant for paediatric renal care -children not first priority on any transplant program in terms of organ allocation -private facilities vs state facilities -ethical decisions dialysis facilities: peritoneal dialysis (pd) first line therapy for acute renal failure, as can be performed in any setting with minimal equipment and expertise. in the setting at rxh, we have a greater than 60% survival in infants and children dialysed using pd even in a sophisticated intensive care setting. haemodialysis and chronic dialysis may not be easily available in most settings and realistically need transfer to one of the major centres. transplantation: again limited to only few centres, with at least 30% living related donations from family members. initial good 1 year (90%) and 5 year (80%) graft survival but results then deteriorating as patients enter their adolescent years often with little support and transfer to adult units in their early teens. controversial issues here include access to one kidney transplant only, no chronic dialysis if not suitable for transplant and transplantation in hiv positive recipients. conclusions: adequate resource allocation is required for paediatric renal care especially where resources are limited. immunosuppression remains the cornerstone of successful transplantation. in developing countries transplantation is mostly from living donors and transplant is thus a once in a lifetime chance. in this backdrop immunosuppression is a challenge as several issues have to be overcome. namely, non-availability of newer drugs, high costs and paucity of drug monitoring facilities. in most countries immunosuppression is based on a triple drug regimen of cyclosporin (cya), prednisolone (pred) and azathioprine (aza). in the last few years mmf and tacrolimus (tac) has been initiated in a few centres. several tailoring strategies have been employed taking into consideration costs, drug availability, tissue typing facilities and drug monitoring. firstly in hla identical transplants which constitute 15-20% of the total, cya based regimen are used. marked reduction to 1-2 mg/kg at 6 months and complete withdrawal at one year in rejection free transplants is safe with continuing of aza and pred. secondly several centres selectively use tac and mmf in cases with early rejection or transplants with >3 mismatches. it is possible to switch to cya or aza after 6 months on individual basis. thirdly induction protocol with atg for 3 days in transplants with >4 mismatches and in retransplants is cost effective. cost considerations have increased the use of generic calcinurin inhibitors with market share of 20-100% in different countries. co administration of p450 competitors has reduced doses of cya by 50-60% with considerable cost reductions. siut has been running a living related transplant program for more than 20 years with dialysis and follow-up of recipients and donors. keeping in mind economic constraints a model of government public partnership was developed which provides all facilities including drugs free of cost. we adopted a number of tailoring strategies e. g. strict monitoring, tailoring by hla match and donor age and use of biological agents in risk groups. in first 10 years we used parent drugs, however increasing costs necessitated use of generics after establishing bioequivalence in controlled trials. graft survival rates were maintained at 92% and 85% at 1 and 5 years. in conclusion, immunosuppression in developing countries require besides newer drugs, monitoring facilities, affordable costs and regular follow-up as transplantation for majority is once in a lifetime chance and failure equates with death. occurrence of infections following kidney transplantation is a major reason for hospitalization, and an important cause for renal dysfunction & mortality in developing countries. more than 50% renal transplant recipients get serious infections, with a 20-40% risk of mortality. infections in the first month after transplantation are similar to those in surgical patients; opportunistic pathogens and cmv predominate between 1-6 months; and tuberculosis (tb) after 6 months. the risk of tb in patients on maintenance dialysis & following transplantation is between 10-20%. the onset of tb is usually within 12 months of transplantation. clinical syndromes include pleuropulmonary tb, followed by disseminated tb, pyrexia of unknown origin and lymph node disease. demonstration of m. tuberculosis, on microscopy or culture might not be possible. nonrifampicin based treatment regimens (inh, pyrazinamide, ethambutol, fluoroquinolone for 3 months, followed by hef for 9 months) are used. inh prophylaxis (6-9 months) is advised in patients with tuberculin positivity or contact with active tuberculosis. cmv disease results in considerable morbidity & mortality; its timing is influenced by donor/recipient serological combination, state prior to transplantation, use of antilymphocyte induction therapy or preventive strategies. cmv disease is characterized by a non-specific febrile illness; features of enterocolitis, pneumonia, hepatitis, myocarditis, esophagitis, chorioretinitis & bone marrow involvement are variable; disseminated disease is rare. cmv disease exacerbates the net immunosuppression, increasing the risk for opportunistic infections. patients with cmv disease are also at risk for acute rejection and chronic allograft injury, atherosclerosis & vascular injury. diagnosis of cmv disease is based on a combination of viral serology, shell vial cultures, pp65 antigen assay and pcr. the cost of prophylaxis and treatment is a major limitation to the use of ganciclovir or valganciclovir. other infections in transplant recipients include malaria, p. carinii and fungi (cryptococcosis, candidiasis, mucormycosis, aspergillosis). infections are an important cause of morbidity & mortality in renal transplant recipients. their management continues to be challenging due to difficulties in diagnosis, unsatisfactory follow-up and cost of medications. ethical issues of renal replacement therapies n. orta, p. zibaoui, e. lara university of carabobo/insalud, pediatric nephrology, valencia, venezuela important ethical issues in pediatric nephrology (pn): genetic and molecular techniques, prenatal therapies for urinary anomalies, treatment of children with chronic renal disease (esrd). ecosonography can detect nephrourological anomalies and studies of amniotic fluid give information on chromosome alterations and renal function. particular situations, can lead to dilemmas related to pregnancy interruption and neonatal dialysis and transplantation (dt) possibilities. renal insufficiency (ri) presents at any age, and peritoneal, hemodialysis or hemofiltration could be applied. patients without structural abnormalities, ri secondary to toxics or isquemic nephropathies have better prognosis. these procedures are complicated and costly and increases, and may have secondary effects with ethical implications. treatment by dialysis developed in the 60's and inclusion of children was not considered. this has changed with advances in dt. since 1970's dt programs were setup for children, but received criticisms and considered unethical, but were continued because of familiar and humanitarian demands and today it is clear that children benefit with that. scientific societies recommend: 1. children who receive dialysis must meet the following criteria: -diagnosis of esrd, legal authorization, possibility of transplantation, acceptable quality of life; may not be rejected for economic, social or psychological reasons, nor gender, age, race or mental conditions. biopsies are the current 'gold standard' for monitoring transplant patients, but it has been shown that even mild rejection episodes, based on pathology grading, can have poor outcomes and even biopsies with normal early pathology can progress rapidly with chronic allograft injury. there is considerable inter-observer variability of biopsy pathology readings that adds an additional confounder to this method of analysis. identification of non-invasive biomarkers in blood or other fluids would allow for the possible elimination of frequent biopsies. hence, the identification of diagnostic and predictive non-invasive genomic markers will be a worthy tool to aid in the clinical monitoring of transplant patients. the use of dna microarrays as a hypothesis generation tool for determining gene expression differences across thousands of genes in a data set is increasing. recently, the use of microarrays has been applied to the transplant field and holds great promise for unraveling the mechanisms at play in various transplant processes and for identifying new tissue specific and non-invasive biomarkers predictive of clinical outcomes. as microarrays produce large amounts of data, bioinformatics tools are being developed to determine gene expression patterns. gene clustering and class prediction tools aid in the discovery of molecular signatures in different disease processes while literature mining, gene family analysis and pathway analysis help in understanding the biological relevance of these signatures. initial studies in acute rejection and graft dysfunction have produced possible markers for risk stratification of the rejection event and have suggested underlying mechanisms at play, that may now allow us to test novel drugs for treating specific acute rejection episodes. the most exciting application of microarrays lies in our ability to predict clinical outcomes by non-invasive serial monitoring, eliminate the requirement of transplant biopsies and individualize patient management by accurately predicting the patient's sensitivity to immunosuppression-sufficient to suppress the allo-response, yet insufficient to abrogate the innate response. responsible array data handling and accessible reporting will open new doors for transplant researchers through increased use of computers and collaborations towards these kinds of novel insights and treatment options for transplant patients in the future. this talk focuses on dna microarrays, their application to transplantation, and discusses some of their limitations and recent applications, as well as some key research studies where dna microarrays are applied to understanding the molecular differences in acute transplant rejection that segregate and likely control the differences in rejection treatment responsiveness as well as decline in graft function, as well as gaining insights into the different biological processes that govern these differences. the molecular pathogenesis of vur is not well understood. uroplakins (ups) are expressed in the urothelium and are developmentally regulated by rab27b and tbx genes. up ii and iiia-null mice exhibit a primary (1 o ) vur phenotype. using microarrays and rt-pcr, we analyzed gene expression in surgically-discarded ureteric tissue from children undergoing reimplantation for 1 o reflux, compared with adult living-related transplant donors as normal controls, as well as children with 2 o reflux (megaureter, duplicated ureters, and posterior urethral valves) as age-matched disease controls. we also studied urine protein profiles using 2-dimensional electrophoresis (2d-page) followed by time-of-flight mass spectroscopy (q-tof-ms). rt-pcr showed partial expression of ureteric upia, upib, upii and upiiia genes in patients with 1 o reflux. protein screening using western immunobloting confirmed that some uroplakins were undetectable. real-time rt-pcr revealed that ureteric up ia, up ib, upii, and up iiia gene expression decreased significantly, whereas upiiib gene expression increased at least 2-fold compared to controls. compared with patients with 2 o vur, the decrease of upiiia expression in those with 1 o reflux was highly statistically significant (p<0.00001). the expression of rab27b and tbx genes also decreased significantly in patients with 1 o and 2 o reflux as well. total urinary protein concentration increased by 6-fold in the 1 o vur patients without detectable renal scarring on dmsa scans; and increased further in patients with renal scarring, 13-fold for 1 o vur and 11-fold for 2 o vur patients. proteomic analyses of proteinuria revealed an overall increase of protein with mw>60.4 kda in the pi range 4.0 to 7.0 in patients. q-tof ms identified one predominant urinary protein of ~ 105 kda as sera-transferrin, which was confirmed by elisa quantitation. we hypothesize that the abnormal developmental expression pattern of the up, rab27b and tbx genes in vur patients results in abnormal urothelial functions, which lead to leakage and/or secretion of proteins into the urine, and that this occurs in the absence of scarring from urinary infections. further identification of these protein biomarkers may lead to non-invasive diagnostic tests for vur. biomarker discovery in acute kidney injury p. devarajan cincinnati children's hospital medical center, department of pediatric nephrology and hypertension, cincinnati, united states acute kidney injury (aki), previously referred to as acute renal failure (arf), represents a common and persistent problem in clinical medicine. despite significant improvements in therapeutics, the mortality and morbidity associated with aki remain high. a major reason for this is the lack of early markers for aki, akin to troponins in acute myocardial disease, and hence an unacceptable delay in initiating therapy. fortunately, the application of innovative technologies such as functional genomics and proteomics to human and animal models of aki has recently uncovered several novel genes and gene products that are emerging as biomarkers. the most promising of these are chronicled in this symposium. these include a plasma panel (ngal and cystatin c) and a urine panel (ngal, . since they represent sequentially expressed biomarkers, it is likely that the aki panels will be useful for timing the initial insult and assessing the duration of aki. based on the differential expression of the biomarkers, it is also likely that the aki panels will distinguish between the various types and etiologies of aki. however, they have hitherto been tested only in small studies and in a limited number of clinical situations. it will be important in future studies to validate the sensitivity and specificity of these biomarker panels in clinical samples from large cohorts and from multiple clinical situations. such studies will be markedly facilitated by the availability of commercial tools for the reliable and reproducible measurement of biomarkers across different laboratories. a. watson children and young people's kidney unit, notthingham university hospitals, nottingham, united kingdom the achievement of adequate chronic peritoneal dialysis in children requires close attention to clinical, dietetic and psychosocial aspects of care. successful dialysis is dependent upon excellent peritoneal access and swan neck coil catheters with downward facing exit sites are increasingly favoured with many placed laparoscopically. automated peritoneal dialysis is employed in most developed countries but adolescents may be given the choice of capd which may be the only modality available in developing countries. training and support by committed nursing staff are of paramount importance as is regular review by a paediatric renal dietitian. growth parameters should be regularly monitored and supplemental enteral feeding introduced early. many infants are nasogastrically fed, but the preferred route for supplemental enteral feeding is via a gastrostomy which can be placed at the same time as the pd catheter. dialysis fill volumes should be assessed in terms of body surface area and intra-abdominal pressure measurements. there can be discrepancies between urea and creatinine clearances and adequate dialysis includes combining clinical parameters with regular growth measurements as well as biochemical data including phosphate, calcium and parathyroid levels. peritoneal function tests are useful in monitoring progress but the greatest challenges are in sustaining long-term dialysis, particularly in infants where transplantation is likely to be delayed. support for the families with a respite care 'package' may delay or prevent burnout and possibly reduce peritonitis rates. conventonal pd solutions are acidic, contain unphysiological concentrations of lactate and glucose and highly toxic glucose degradation products (gdp), which are substrates for advanced glycation end product (age) formation. repeated administration induces epithelial to mesenchymal cell transition, loss of the mesothel cell layer, progressive submesothelial fibrosis and angiogenesis, ultimately leading to ultrafiltration failure. meanwhile pd solutions with an improved toxicity profile are available. multi chamber pd solutions separate glucose from the buffer at a very low ph, which largely prevents gdp formation. after mixture ph is close to normal. they are equally effective with regard to solute-and water transport, reduce inflow pain and systemic age load. dialysate effluent markers indicate increased mesothelial cell mass, reduced peritoneal inflammation and reduced angiogenesis. animal studies demonstrate preservation of pd membrane morphology and function, respective human biopsy data however are pending. a european paediatric multi centre trial accomplished recently elucidates the impact of the lactate and bicarbonate buffer. a randomized cross over trial in adult patients points to an improved preservation of residual renal function; a korean registry suggests improved technique and patient survival. icodextrin solutions reduce glucose and gdp exposure, improve extracellular fluid status, left ventricular mass and stabilize membrane function in anuric adult capd patients and should be beneficial in children, too. amino acid based solutions achieve similar clearance and ultrafiltration rates, reduce glucose and gdp load and allow for a phosphate free amino acid supply. the nutritional benefit however is questionable. other, thus far experimental strategies include addition of locally active compounds to pd fluids such as gdp scavengers, inhibitors of age formation and antifibrotic agents. surface active phospholipids may increase peritoneal contact area and ultrafiltration. gene therapy appears highly promising but is still far from being clinically applied. in summary, there is substantial evidence for increased biocompatibility of the recently introduced multi chamber and icodextrin based pd solutions. they should improved long term morbidity and mortality of pd patients; this however still needs to be proven. the impact of the ippr on the treatment of peritonitis b. a. warady children's mercy hospital and clinics, pediatrics nephrology, kansas city, united states the international pediatric peritonitis registry (ippr) is an initiative that was established to evaluate the safety and efficacy of largely opinion based peritonitis treatment guidelines in which empiric antibiotic therapy (1 st generation cephalosporin and ceftazidime or a glycopeptide and ceftazidime) was stratified by disease severity. forty-seven centers from 14 countries contributed data on 392 children and 501 peritonitis episodes treated in accordance with the guidelines. culturenegative peritonitis accounted for 31% of all episodes, with a marked regional variability in the incidence of this disorder, as well as in the peritonitis causative organisms. overall, 89% of cases achieved full functional recovery, a portion following relapsing peritonitis (9%). in-vitro evaluation revealed only 69% sensitivity of gram-positive organisms to a 1 st generation cephalosporin (eastern europe > north america) and 80% sensitivity of gram-negative organisms to ceftazidime. in contrast, 94% of gram-positive organisms and 93% of gram-negative organisms were sensitive to the combination of either a 1 st generation cephalosporin or an aminoglycoside. whereas the risk of empiric treatment failure was associated with the presence of a gram-negative infection (p=0.0004), neither the risk factors assumed by the guidelines nor the choice of empiric therapy were predictive of the final functional outcome of the peritonitis episodes. the data collected by the ippr will serve as an important source of evidence to be incorporated into revised pediatric peritonitis treatment guidelines. ch. aufricht medical university, pediatrics, vienna, austria peritoneal dialysis (pd) is a safe, cost effective and widely used form of renal replacement therapy in patients with end stage renal failure. however, up to a third of patients on pd will suffer from technical failure during their course. identification of the patients at highest risk would be of high clinical relevance. cytotoxicity of pd fluids due to low ph, hyperosmolarity, and/or high concentrations of lactate, glucose and its degradation products causes mesothelial cell injury that ranges from minor cellular dysfunction to overt (apo)necrosis. the same physicochemical properties of pdf that cause such cellular injury also induce pathways leading to repair and recovery. this so-called cellular stress response results in a switch of the cellular machinery from routine procedures towards reaction against stressors. the complex machinery of the cellular stress responses not only counteract direct toxic injury caused by pdf but also attenuate inflammation or other potentially deleterious cellular processes. infectious, uremic and toxic injuries might converge in cellular inflammation. whereas inflammatory processes triggered by infection protect the peritoneal cavity against invading microorganism, chronic sterile smoldering 'cytotoxic' inflammation may result in aberrant healing processes and peritoneal fibrosis. recently, polymorphisms of many proteins involved in relevant cellular responses have been described and related to altered resistance and/or susceptibility to pathogenetic processes with potential influence in pd. in this review, we will focus on key effectors of stress responses, of cellular inflammation and of fibrogenesis such as heat shock proteins (hsp), cytokines (il-6), chemokines (il-8) toll-like receptors (tlr) and growth factors. given the recently shown role of these 'players' for the interplay between mesothelial injury, inflammation and cytoprotection, these polymorphisms will likely be relevant for mesothelial cell damage during pd. taken together, the ability to understand -and ultimately modify -the risk profile of a given patient will be essential for tailoring individual pd therapies. the pathologic diagnosis of chronic allograft nephropathy (can) was introduced in 1993 by the banff classification system for renal allograft injury. it originally included at least four entities that it was acknowledged could not always be distinguished by biopsy: 1) chronic rejection; 2) chronic calcineurin inhibitor (cni) toxicity; 3) hypertensive vascular disease; 4) chronic infection and/or reflux (solez, ki 1993 44: 411) . over the ensuing decade, the definition of can has expanded and fluctuated such that some pathologists have come to use the term to mean a specific pathologic entity comprising "…progressive graft dysfunction accompanied by chronic interstitial fibrosis, tubular atrophy, vascular occlusive changes and glomerulosclerosis. " (nankivell, nejm 2003 349: 2326 , while others have suggested can should be used to describe all causes of renal allograft dysfunction involving fibrosis. the resulting confusion over terminology has in some ways hindered the growing awareness of the multiple discrete, diagnosable and often treatable diseases capable of causing chronic graft injury. these diseases include: 1) chronic (primarily antibody-mediated) rejection; 2) de novo and recurrent glomerular disease; 3) calcineurin inhibitor toxicity; 4) interstitial fibrosis and tubular atrophy without evidence of any specific etiology. to this list should be added other recognizable causes of late graft dysfunction such as polyoma virus infection and hypertension. in the new banff 2005 classification can will no longer be a diagnostic category (colvin, world transplant congress, 2006) . instead, the term sclerosis will be used to describe: "interstitial fibrosis/tubular atrophy, not otherwise specified, " or "if/ta nos. " the increasing use of protocol biopsies in clinically stable patients has dramatically increased awareness of the ongoing pathological changes almost every renal allograft appears to be undergoing almost from the first moments of engraftment. techniques are now available in most centers to reliably recognize the presence of chronic antibody-mediated rejection. these include: 1) typical morphological findings: lamination of glomerular basement membranes, arterial intimal fibrosis, interstitial fibrosis/tubular atrophy; 2) c4d staining in peritubular capillaries or glomeruli; 3) the presence of circulating donorspecific antibodies (takemoto ajt 2004 4: 1033 . treatment of late, chronic antibody-mediated rejection is in its infancy and may include use of anti-b cell antibody (rituximab), ivig, and/or plasmapheresis. calcineurin inhibitor toxicity can be more reliably identified by focusing on blood vessels in the allograft, primarily the location of hyaline deposits in the arterioles. nodular, peripheral hyalinosis is found almost exclusively in cni toxicity, whereas sub-endothelial and transmural hyaline deposits are non-specific and can be seen in hypertension, aging and diabetic nephropathy. there is increasing concern that recent gains in short-term renal allograft survival and reductions in acute rejection rates have not resulted in improved long-term graft survival. while this is likely due to multiple factors, including infections and malignancies associated with over immunosuppression, unrecognized (sub-clinical) acute cellular rejection (moreso ajt 2006 6: 747), and noncompliance in some patients, much evidence points to the primary role of cni toxicity in many if not the majority of chronically failing allografts maintained on cni's. experience with conversion from cni to sirolimus in adult patients has been reviewed in a recent editorial (betard nephrol dial transplant 2006 21: editorial comments) . in 10 studies involving nearly 400 patients, between 54% and 80% of patients with late allograft dysfunction failed to respond favorably to cni withdrawal and replacement with sirolimus. acute rejection episodes were rare, occurring in only six patients. common adverse effects included dyslipidemia, anemia and proteinuria. in one study, 32 of 50 patients developed proteinuria after conversion, 18 in the nephrotic range. pediatric experience with cni withdrawal has been limited (kerecuk, pediatr nephrol 2005 20: 1630 falger, pediatr transplant 2006 10: 565, hocker, pediatr transplant 2006 . our recent experience at stanford with cni withdrawal in 17 patients was not favorable, with 41% of patients experiencing an associated acute rejection episode (weintraub, wtc 2006) . prior history of acute rejection significantly increased the relative risk of acute rejection after cni withdrawal (rr=1.8), and proteinuria was common. patients with advanced chronic graft dysfunction were at increased risk for graft loss. in summary, the use of the term can to describe all causes of chronically failing renal allografts is to be discouraged in favor of a search for specific etiologies whenever possible. antibody-mediated rejection and cni toxicity are major causes of late allograft dysfunction. protocol biopsies are helpful in identifying patients with treatable causes of late allograft dysfunction. cni withdrawal/avoidance may be successful, but optimum patient selection criteria and withdrawal/replacement strategies have not been determined. pediatric experience to date with cni withdrawal has been limited, but it appears that late withdrawal after cni injury and graft dysfunction have become well established may be associated with inferior outcomes. prospective trials in pediatric patients are needed to address these issues. which immunosuppression in pediatric transplantation? p. hoyer university children's hospital, essen, germany recent results in pediatric renal transplantation have reached one year graft survival rates better than 90%. current immunosuppressive drugs should be classified according to their interference with the immunesynapsis as signal 1, signal 2 and signal 3 blocking agents. the variety of immunosuppressive drugs does allow more treatment combinations than the potential number of large scale studies in children. the definition of current unmet needs should guide employment of drugs. calcineurin inhibitors (cni) are still the basis of immunosuppression. individual risk profile leads to preferences for cyclosporine or tacrolimus. while in adults cni reduction seems to be the major goal, the search for steroid sparing or avoidance protocols has attracted major interest in pediatric transplantation. growth and body configuration as well as cardiovascular risk factors should be in the focus of research. antibody induction protocols, mainly with il-2 receptor antibodies, are increasingly popular, but efficacy is less clear than concluded from adult studies and might depend on initial combination therapy. mmf (cellcept®) or mpa (myfortic®) are effective drugs with cni sparing potential. early adequate dosing seems to be of greater importance than the choice of the cni, but the price to pay may be an increase in infectious complications. mtor inhibitors are promising in avoiding nephrotoxic side-effects; specific side effects on male gonadal function and possible interference with growth should be considered before any recommendation can be given. fty 720 would have been of especial interest for children because of maintaining viral infectious response, but phase-2 studies are on withhold. the vision of tolerance has stimulated research on regulatory t-cells; i. e. cd4 cd25+t-reg cells and the mastergene foxp3. the impact of lymphocyte depletion induction protocols with campath on operational tolerance needs further studies. newer drugs in development are isa 242, a cyclosporine analog without nephrotoxicity; a modified release form of tacrolimus which might improve compliance; the phosphokinase inhibitor aeb071 with the potential to avoid cnis; the jak3 inhibitor cyp 690, 550 which might interfere with signal 3 (il2 and il15-receptor); and lea29y (belatacept) with blocks costimulatory signals. according to the new european drug legislation some of these drugs will be subject for mandatory testing in pediatric patients to get marketing authorisation. the future might be a more tailored immunosuppression according to the induvidual needs of the patient. immunosuppression minimization strategies, under the umbrella of a newer generation of more powerful induction and maintenance immunosuppressants, are being increasingly applied to pediatric organ transplantation, with the greatest emphasis on minimization of steroids and calcineurin inhibitor agents. safe elimination of these steroids carry unprecedented advantages for reducing patient morbidity and chronic graft injury, but may also result in unanticipated changes in immunological homeostasis and drug pharmacokinetics, heralding closer surveillance for posttransplant infections and alterations in drug bioavailability and dosing, as well as break-through immunologic responses. in single center studies, pediatric renal transplantation appears safe without steroids. daclizumab first dose doubling and extended use for 6 months replaces steroids effectively without evidence of over-immunosuppression, and may be the pivotal causative for the reduced acute rejection seen in the face of steroid avoidance. this pilot protocol has been tested in a prospective, multicenter randomized us and canadian study. b. maecker, c. klein department of pediatric hematology/oncology, hannover medical school, hannover, germany posttransplant lymphoproliferative disorders are severe complications of immunosuppressive therapy after solid organ transplantation causing significant morbidity and mortality. to define prognostic factors, we have analyzed 55 pediatric solid organ graft recipients (kidney, liver, heart/lung) that were reported to the german ped-ptld registry. ptld was diagnosed at a median time of 29 months post organ transplantation with sigifcantly shorter lagtime in liver versus heart or renal graft recipients. the five-year overall and event-free survival was 68% and 59%, respectively. stage iv disease with bone marrow and/or cns involvement was independently associated with poor survival. no differences in outcome were observed between early and late onset ptld, monomorphic or polymorphic ptld, and ebv-positive or ebv-negative ptld, respectively. patients with burkitt-like ptld and c-myc translocations had very short survival. these factors should be important to consider for future prospective interdisciplinary trials that are urgently needed to define rational treatment strategies. cystinosis is an autosomal recessive lysosomal storage disorder affecting children and adults all over the world. in cystinosis cystine is trapped in the lysosomal compartment due to a defect in its egress transport protein cystinosin encoded by the gene ctns. by mechanisms still not fully understood this leads to tubular and glomerular kidney and other organ failures (e. g., thyroid, muscles) if left untreated. kidney involvement is prominent from shortly after birth on as renal tubular fanconi syndrome with the clinical consequences of failure to thrive and rickets. cystinosis is the single most common cause of renal fanconi syndrome in childhood. therefore, any recognition of renal glucosuria, generalized aminoaciduria, phosphaturia, small molecular weight proteinuria, polyuria, and metabolic acidosis (due to renal bicarbonate loss) should lead to prompt consideration of cystinosis as possible cause. this can be done utilizing biochemical analytical methods measuring the cystine content of polymorphonuclear leucocytes. corneal cystine crystals, seen in slit lamp examination, are pathognomic as well, but may not be visible before 16 months of age. left undiagnosed and untreated, patients will develop in addition to the existing tubular insufficiencies pronounced glomerular kidney failure often already present at diagnosis and inevitably leading to end stage renal failure typically at the end of the first decade of life. kidney failure in cystinosis presents differently from other forms of glomerular kidney failure because of the overlap of tubular and glomerular insufficiencies. kidney transplantation will be curative with respect to kidney function. specific treatment with cysteamine to lower the intralysosomal cystine content apparently is as important as before transplantation to prevent or attenuate other organ failures and therefore has to be continued after kidney transplantation. cysteamine treatment has been introduced in the 1970s and has been approved in the 1990s. early diagnosis and diligent treatment is able to prevent or ameliorate major organ complications. unfortunately, until now no newborn screening exists due to the biochemistry and cell biology involved. the high prevalence of a european founder mutation, i. e., a 57 kb deletion in the ctns gene, makes molecular based methods not feasible. lowe syndrome and dent's disease: two ends of a spectrum d. böckenhauer great ormond street hospital for children nhs trust, nephrology, london, united kingdom lowe syndrome and dent's disease are x-linked disorders; the former is a systemic disorder characterized by cataracts, mental retardation and proximal tubulopathy whilst the latter was originally described as an isolated kidney disorder with tubular proteinuria, hypercalciuria/ nephrocalcinosis and progressive renal impairment. mutations in ocrl1 underlie lowe syndrome, whereas the majority of cases with dent's disease are caused by mutations in clcn5. recently, mutations in ocrl1 have been identified in a subgroup of patients with dent's disease (also called dent-2). it is unclear, why some patients with ocrl1 mutations get dent's disease, while others develop lowe syndrome. however, careful clinical observation has revealed that dent-2 patients have evidence of systemic involvement. moreover, the degree of severity of symptoms in lowe syndrome is highly variable. thus, mutations in ocrl1 can cause a spectrum of symptoms, from tubular proteinuria and hypercalciuria to the full manifestations of lowe syndrome. here, we will review the clinical phenotype of the disorders, what is known about their pathophysiology and discuss genotype/phenotype correlation. fabry disease, the second most prevalent lysosomal storage disorder after gaucher disease, is an xlinked inborn error of the glycosphingolipid metabolic pathway and affects approximately 1:44,000 live births. mutations in the gene encoding alpha-galactosidase a, lysosomal hydrolase, lead to systemic glycosphingolipid deposition, resulting in profound dysfunction of neurological, renal, cardiac, and cerebrovascular systems. the initial phase begins in childhood or adolescence and is characterized by neuropathic pain, angiokeratomas, and ocular deposits. the later phase is distinguished by progressive cardiac, cerebral, and renal involvement, leading to multi-organ dysfunction and death. few patients have historically survived past their mid 50s. although renal involvement usually becomes prominent in adulthood, adolescents may develop proteinuria and decreased glomerular filtration rate. timely diagnosis is critical, given that the enzyme replacement therapy likely delays progression of the serious complications of fabry disease and may have potentially preventive benefits. specifically, there is growing evidence that initiation of enzyme therapy slows progression of chronic kidney disease (ckd); it remains unknown whether enzyme therapy in the currently employed doses can prevent ckd. pediatricians have a particularly important role in making the diagnosis, since they are likely to be the first providers to encounter fabry patients and thus initiate therapy before irreversible tissue injury develops. nephrologists should consider the diagnosis of fabry disease when a patients present with ckd with nephrotic or subnephrotic proteinuria, often with skin lesions (but these may be limited in distribution), episodic extremity pain, and/or psychiatric problems. further research is required to determine the efficacy of enzyme replacement therapy to prevent organ damage in children, to identify optimal therapeutic doses and schedules, and to define efficacy of additional treatments for fabry kidney disease, include angiotensin converting enzyme inhibitors and angiotensin receptor blockers. w. van't hoff great ormond street hospital for children nhs trust, pediatric nephrology, london, united kingdom children with a complete deficiency of methylmalonyl coa mutase develop both renal tubular and glomerular dysfunction. the renal tubular dysfunction is characterised by renal tubular acidosis, defective urinary concentration and hyporeninaemic hypoaldosteronism. a chronic interstitial nephritis also develops leading to long-term glomerular renal damage. chronic kidney disaes is not evident on routine testing as muscle mass is reduced and protein intake is markedly restricted. formal measurement of gfr using chromium 51 edta showed that 7 of 12 such mma patients have a gfr <60 mls/min/1.73 m 2 ), and by 12 years, 7 of 9 patients had a gfr <40. most mma patients with ckd do not have excess proteinuria nor hypertension. although vit b12 responsive mma patients in general have a more favourable outcome, late onset renal complications have been seen. in addition to ckd, mma patients can develop cardiomyopathy, pancreatitis, gut dysmotility and chronic or acute encephalopathy. management is based on a protein-restricted, high calorie diet, allopurinol to control hyperuricaemia, supplements of carnitine and metronidazole therapy to reduce production of propionate. haemodialysis has successfully cleared plasma mma and improved the metabolic and nutritional status. liver transplantation has been performed in a number of younger children, as enzyme replacement therapy, but is associated with a significant morbidity and mortality (including late neurological deaths). renal transplantation has been reported in a small number of older patients but it is not yet clear how good the graft outcome will be. combined liver-kidney transplantation has been undertaken again in small numbers, with a high morbidity and significant mortality although there are a very few remarkable survivors. so far, there are only centre specific experiences and unfortunately reviewing the literature will not give a full picture of long-term outcome and complications, due to the bias of the reported results towards favourable outcomes. there is a clear need to share data on the management of these rare patients in order to better understand the best approach. estimations of the extent of hiv disease in sub-saharan africa are that 26 million people are currently infected of which the total number of children is 2.1 million. worldwide it is thought that 2.3 million children are infected so it can be seen that the majority reside in africa. in south africa, we have a minimum of 250 000 hiv infected children aged less than 14yrs of age with only 15 000 on highly active anti-retroviral therapy (haart) at present. currently 40-60% of all paediatric admissions to hospital may be hiv related. hiv associated renal disease, is not yet well documented among children -especially from africa -but now with the growing availability of haart, this information has become more important for appropriate management. hiv renal disease presents in many forms, including including hiv-assoc nephropathy (hivan) and hiv immune complex kidney disease (hivick) amongst others. questions have now been raised as to whether a screening program should be introduced as haart, despite side effects and drug interactions, has revolutionised management if hiv infection detected early enough. transplantation which was thought previously to be an absolute contraindication and potential waste of valuable resource is now possible, provided there is maintenance of haart, hiv viral load is undetectable for >3 mths and cd4 >200cells/mul. there remains concern about feasibility in children with increased rejection, reduction of calcineurin inhibitors and infections such as recurrence of hepatitis c post-transplant. despite these concerns, studies in adherent adults have clearly shown that adult patients with hiv infection do better following transplantation than on dialysis. of concern, are some studies, showing that certain black patients may have a genetic predisposition to hiv infection. overall, transplantation can be successful in these patients provided the hiv disease is under control. however, hiv disease -including renal disease -remains a major problem in sub-saharan africa due to limited resources and lack of availability of drugs, where other health priorities may prevail. background. previous studies suggest that hiv-1 induces dysfunction and/or injury of endothelial cells, leading to the systemic release of fibroblast growth factor -2 (fgf-2). to date, the role that circulating fgf-2 may play in the pathogenesis of childhood hivan is not clearly understood. objective. here we sought to determine the potential role of circulating fgf-2 in the pathogenesis of hivan using wild type (wt) and hiv-tg 26 mice. methods and results. to determine whether circulating fgf-2 induced ultrastructural changes in renal glomerular endothelial cells and podocytes, we injected human recombinant fgf-2 daily for 10 days into fvbn wt and hiv-tg 26 mice (n=4 in each group). by electron microscopy we found that fgf-2 induced endothelial swelling and mild fusion of the foot processes in wt mice. these lesions were more severe in hiv-tg 26 mice, which showed enlargement of podocytes, protein reabsorption droplets, significant fusion of the foot processes, and collapsing glomerulopathy. subsequently, to confirm these findings in a different experimental model system, we used recombinant adenovirus carrying a secreted form of human fgf-2 (ad-fgf-2). four to five weeks old hiv-tg 26 mice without evidence of abnormal protenuria (urine protein/creatinine ratio (up/uc) <10), and their wild type littermates were injected with 5x10 8 plaque forming units (pfu) per mouse of ad-fgf-2 or control ad-lacz vectors through the retro-orbital (r. o.) venous plexus (n=6 per group). all mice were followed for three weeks. all hiv-tg 26 mice injected with rad-fgf-2 developed heavy proteinuria (up/uc >40). in addition, 50% showed elevated bun levels (>25 mg/dl) and renal histological lesions typical of hivan. in contrast, wt mice, developed transient and moderate proteinuria (up/uc <40), without renal failure or permanent renal damage. the number of animals with proteinuria in hiv-tg 26 mice injected with ad-lacz group was consistent with the natural history of the renal disease progression. control wt mice injected with ad-fgf-2 developed mild proteinuria (40%) that returned to normal values14-21 days after the injection. fgf-2 induced microcystic tubular dilatation and recruitment of mononuclear cells both in wt type and hiv-tg 26 mice, although the changes were more significant in the later group. conclusion. taken together, these results suggest that the accumulation of fgf-2 in the circulation of hiv-tg 26 mice induces glomerular endothelial and podocyte injury leading to the development of heavy proteinuria, renal failure, and the typical renal histological features of hivan. we conclude that the elevated levels of fgf-2 in the circulation of hiv-infected children may be a significant risk factor for the development and/or progression of hivan. human immunodeficiency virus associated nephropathy (hivan) is the third leading cause of kidney failure in young african american adults in the united states. the prevalence and natural history of the disease in children is not well documented. we have examined our experience in 315 children, aged 2 months to 22 years (mean age 9.4±0.3 years) with predominantly perinataly acquired hiv-1 infection from their mothers. since 1998, 286 of these children have been evaluated routinely for evidence of renal disease with quantitative assessment of proteinuria by random urine protein to creatinine ratios (upr/cr). renal functional studies included serum creatinine with estimation of glomerular filtration rate (egfr), urinalyses, renal scintigraphy, ultrasound and renal biopsy when possible. the patients were divided according to their level of proteinuria. those with nephrotic range proteinuria (upr/cr>1.0) were designated as hiv-ns (n=32; 11%). those with persistent intermediate range proteinuria (upr/cr: 0.2<1.0) were designated as hiv-pp (n=62; 22%). those with no proteinuria (upr/cr<0.2) were designated as having no nephropathy (hiv-non; n=192; 67%). the great majority of patients were treated with highly active antiretroviral therapy (haart), although those with hiv-ns had less time on treatment when their proteinuria was discovered. moreover, the virulence of the hiv infection as measured by viral load (vl) was significantly greater in those patients with proteinuria. also, viral load correlated positively with degree of proteinuria (r=0.5; p<0.0001). mortality as shown by kaplan-meier survival curves was significantly greater in the hiv-ns group as compared to the pp and non groups. renal failure occurred only in the group with nephrotic range proteinuria. during the past 25 years we have dialyzed 27 children with hiv infection in our end stage renal disease (esrd) program for a total of 660 patient dialysis months. since 1999, all patients are managed on hemodialysis due to the high occurrence of fungal peritonitis in our initial experience. survival has improved remarkably during the past 10 years with a median survival of 35 months (range 4 to 125 months). the next stage is to begin renal transplantation in those patients with adequate control of the hiv infection. aim: to determine the spectrum of severe renal disease in our hiv infected children; excluding severe sepsis and septic shock syndromes. indications for their referral, renal biopsy and histology relating to outcome. methods: retrospective analysis of all children referred to the paediatric renal service from the general wards and hiv clinic from january 1996 to december 2005. analysis includes age of presentation, sex, nutritional status, symptoms, renal function, histology, associated diseases including infections and follow-up. results: total of 60 children with a mean age of 6.6±0.7 years with a male: female ratio of 1:1.3. there was an overlap of symptoms but the commonest presenting symptom was haematuria in 50%, severe urinary tract infection and pyelonephritis in 23%, anasarca with or without any nephrosis in 33%, acute renal failure in 20%, chronic renal failure in 5%, severe electrolyte disturbances in 5%. renal biopsy was performed in 52 (86.7%) children. histogically 50% had immune complex disease (icd) of which 65.1% had lymphoid interstitial pneumonitis (lip).11.5% had fsgs of which one had lip but also had icd, 11.5% had severe interstitial nephritis, associated infectious changes in 11.5% and atn in 5.8%. one patient each had minimal change, mpgn, abdominal kaposi sarcoma and kidney/bladder stones. 33.3% had or were treated for pulmonary tuberculosis. mean follow-up was 12±12.9 months. death occurred in 16.7% (10). all patients with fsgs with some renal dysfunction and severe sepsis demised. conclusion: despite the high burden of hiv disease, severe renal complications have a low prevalence. good correlation of icd and lip (65.1%) but prognosis is good. pulmonary tuberculosis and infection is the main complications resulting in high mortality in nephrotic syndrome with fsgs. hiv associated nephropathy (hivan) is one of the most common causes of renal failure in hiv seropositive african americans. in the usa, hivan has become the third leading cause of end stage renal disease (esrd) in african americans over the age of 20. while the introduction of haart has decreased both the mortality and infectious complications of hiv infection, the incidence of hivan has reached a plateau and has not decreased. with reduced mortality, the prevalence of seropositive patients in the esrd program continues to increase dramatically. the histopathological findings of hivan include focal segmental glomerulosclerosis of the collapsing variant combined with microcystic tubule dilatation. the most common other diagnosis is mesangioproliferative glomerulonephritis associated with hepatitis c infection, a common comorbid condition. typical features of hivan include renal enlargement and echogenicity by ultrasound analysis. microscopic findings include coexistent microcystic tubule dilatation and glomerular involvement. usually there is mild to moderate tubulointerstitial inflammation, interstitial edema, and fibrosis as well. glomerulosclerosis is usually focal and segmental with collapse of the glomerular tuft and hypertrophy of visceral epithelial cells. hivan is caused by renal epithelial infection by hiv-1 in a susceptible host. clearly there are genetic factors, based on the racial predilection of this disease. patients with hivan are 5.4 times more likely to have a relative with renal failure. in susceptible individuals, hiv infection induces renal epithelial proliferation and apoptosis. the kidney represents a tissue-specific compartment in which hiv-1 can replicate in a previously unrecognized reservoir. while hivan is not currently considered to be an aids-defining condition, patients with hivan should be treated with haart. in some instances, haart has completely reversed the disease process, although it does not rid the kidney of virus. thus, in patients with hivan, the kidney is a true reservoir for replication competent hiv. whether this occurs in other forms of renal disease associated with hiv infection or in patients without renal disease remains to be determined. prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. renal sympathetic nerves directly innervate renal tubules and blood vessels and plays a role in the regulation of glomerular filtration rate and renal sodium excretion. we examined if renal nerves play an a role in mediating the hypertension in prenatal programming. pregnant sprague-dawley rats were injected daily with intraperitoneal dexamethasone between 15 th and 18 th day of gestation. renal norepinephrine concentration was measured at 3 weeks of age. renal denervation was preformed at age 6 weeks of age in control and prenatal dexamethasone treated rats and blood pressure was measured at age of 8 weeks. renal norepinephrine concentration was 338±25 ng/gr in controls and 591±82 ng/gr in the group that received prenatal dexamethasone (p<0.05). systolic blood pressure at 8 weeks of age was 124±1 mmhg in sham operated controls, 122±5 mmhg denervated controls (p=ns). blood pressure was elevated to 133±1 mmhg in dexamethasone treated sham operated group (p<0.05), but was normal at 115±1 mmhg in the dexamethasone treated denervation group. in conclusion, prenatal dexamthasone results in elevated renal norepinephrine levels. bilateral renal denervation normalized the systolic blood pressure in rats that received prenatal dexamethasone. these data are consistent with the renal nerve playing an important role in mediating the hypertension in prenatal programming by dexamethasone. objective: evaluation of clinical outcomes in surgically treated children with renovascular hypertension (rvh). methods: 35 rvh patients treated surgically at a single centre between 1979 and 2005 were retrospectively reviewed: 63% were male, 0.4-17.9 (median 7.6) years of age, with systolic blood pressure (sbp) 141 mmhg (105-300 mmhg). results: bilateral renal artery stenosis was present in 53%, midaortic syndrome(mas) in 40%, intrarenal disease in 49% and coexisting cerebral disease in 24% of patients. surgical procedures (n=47) included: a) nephrectomy (n=18), b) autologous surgery for both aortic reconstruction (n=1) and renal revascularisation (n=15) (renalartery reimplantation (n=4), renal bypass (n=9) and autotransplantation (n=2)) and c) synthetic graft interposition for renal revascularisation (n=6), aortic reconstruction (n=6) or both (n=1). the majority (92%) of patients who received synthetic grafts had vascular anatomy too complex for autologous surgery. technical failure leading to secondary nephrectomy occurred in 3 patients. postoperative complications were haemorrhage (n=5), septicaemia (n=4), and chylous ascites (n=1). there were no operative deaths. patients from the uk were followed up for 5.1 (0.05-16) years. sbp post-surgery improved (116 mmhg, range 90-160 mmhg, p<0.0001). outcomes were normal sbp without treatment (52%), improved (34%) or unchanged sbp (14%). reduction of sbp led to loss of contralateral kidney in 1 patient. 10 children required re-interventions (15 angioplasties and 6 surgical procedures) for progressive disease (n=6), narrowing of the synthetic graft (n=4) and re-stenosis of the autologous bypass (n=2). conclusion: rvh is a progressive disease of extensive nature. surgery benefited 86% of children when performed in conjunction with conservative therapy and, if indicated, interventional radiology. h. wong 1 unlike adults with predominant primary hypertension (htn), the majority of children diagnosed with htn traditionally suffered from secondary forms of htn. however, substantial changes have occurred to the demographics of pediatric population over the past 20 years. in addition, our definition, awareness and understanding of children with hypertension has also changed. we therefore retrospectively reviewed the current causes of htn in children followed in a single tertiary care pediatric nephrology referral center between january 2003 and december 2006. patients who were either diagnosed with or treated for htn at the time of their last visit where included. gender, height, weight, age at the time of diagnosis, causer of htn and casual blood pressure were recorded. out of 1554 patients, 399 (25.7%) were diagnosed with htn. the majority were males (n=249, 62%). median was 10 years (1 month-18 years) age at time of diagnosis and 13.4 years (1.7 months-19 years) at time of the last follow-up. secondary htn was the most common cause of pediatric htn (n=329, 82%) followed by primary (n=51, 13%) and then white coat (n=19, 5%). renal htn was the most common cause of secondary htn (307/329, 93%). for patients referred initially for assessment of htn (n=137), primary (n=43, 31%) and secondary (n=41, 30%) were the two most common diagnosis followed by normotension (n=38, 28%) and white coat htn (n=15, 10.9%). twenty two (5.5%) patients were diagnosed before the age of 1 year. out of 23 renal transplant patients, 18 had htn (78%). renal htn remains the most common cause of pediatric htn overall and continues to represent a large portion of children referred for htn. all children suspected of having htn should continue to have thorough investigations of renal disease to identify the underlying cause. m. sinha 1 , c. booth 1 , j. simpson 2 , n. dalton 3 , s. qureshi 2 , c. reid 1 , s. rigden 1 1 evelina childrens hospital, guys and st. thomas's nhs foundation trust, department of pediatric nephrology, london, united kingdom 2 evelina childrens hospital, guys and st. thomas's nhs foundation trust, department of pediatrics cardiology, london, united kingdom 3 king's college london, department of medicine, london, united kingdom background: hypertension (ht) is a frequent complication in paediatric tx patients. the evolution of end organ damage and its relationship to ht in these patients is not well described. aim: to study the predictive value of an abnormal 24-hour abp profile for end organ damage. methods: patients underwent simultaneous casual blood pressure (cbp), abpm, echocardiogram (lvh if lvmi >38g/m 2.7 ) and ecg assessment, with measurement of several biochemical cardiovascular risk markers. cbp data for 18-months prior to study date was analysed as time averaged z-score. results: we present initial results of a 5-year prospective study. 22 patients (16 male) aged 12.7y±3.4 (mean±sd) and 5.9y±2.6 since tx were studied. on the day of study all patients had normal cbp. 73% had lvh of whom 65% had abnormalities on abpm. overall, 55% patients had both abnormal abpm and lvh. 45% had other findings: 10% abnormal abpm but no lvh; 10% normal abpm but no lvh; 25% normal abpm and lvh. data were analysed for differences between 2 groups of patients, with and without lvh. bp alone was significantly associated with increased lvmi. time averaged cbp was normal in all patients although differed significantly between the 2 groups: systolic bp z-score [mean (ci)] 0.27 (0.57, -0.03) with lvh; -.31 (-0.09, -0.54) without lvh. there were significant differences by abpm criteria relating to several components of the abpm profile including mean arterial pressure, systolic and diastolic bp load. no difference was found between the groups for hb, ca*po4 product, ipth and cgfr. conclusions: we have found the majority of our renal transplant patients have normal cbp but have abnormal abpm in association with lvh. this suggests better control of hypertension should be achieved in patients who have abnormal abp profiles and lvh. a new group of patients with normal abpm but lvh has also been identified. background: obesity is an independent risk factor for renal failure. therefore, we compared the body composition of pediatric nephrology patients with the general child population over two decades. methods: 6, 575 patients with a mean age of 9.5±4.2 years were studied. in 4, 595 patients (70.0%), sufficient data were available to analyze body composition. body composition was measured as body mass index (bmi) z-score because of the age dependency, calculated on the basis of data from the national (usa) center for health statistics (2000) . results: enuresis (24.88%), hematuria (16.45%), recurrent urinary tract infections (11.98%) and proteinuria (11.17%) were the most common diagnoses. the bmi z-score of the pediatric nephrology patients increased significantly from 0.33±0.47 in 1985-1991 to 0.72±1.06 in 1992-1999 and 1.27±1.54 in 2000-2006. while the rate of this increase was not statistically different from that seen in the normal population, they consistently demonstrated a significantly higher bmi z-score (average +0.545) over time. nephrotic edema, non-nephrotic proteinuria and hypertension were not confounding factors. conclusions: patients seen in our pediatric nephrology service over two decades had a higher bmi than the average child population. this implies that these patients are at even greater risk for development of chronic kidney disease later in life. we recommend therapeutic intervention to address this potentially modifiable risk factor. objective: b cell dysregulation is believed to be involved in the development of childhood-onset systemic lupus erythematosus (sle). there is limited evidence regarding efficacy and safety of interventions targeting b cell in children. in our study we evaluated efficacy and safety of b lymphocyte depletion therapy. methods: data of 20 children (15% male) with sle aged 14.1 years (6.1-16.2) treated with rituximab in a single centre were retrospectively reviewed. biochemical parameters were evaluated before and after treatment, and the normalisation of the parameters was assessed as a primary outcome. results: prior to rituximab therapy all patients received extensive immunosuppressive agents. indications for rituximab therapy were chronic (n=13) or acute illness, in either relapsing sle (n=3) or first presentation (n=4). rituximab 750 mg/m 2 was intravenously administered twice within a 2-week period in combination with cyclophosphamide. patients were followed up for 1.5 years (0.1-3.6) . no serious side effects were seen, except for viral infections such as herpes zoster (n=5). all patients with high creatinine (n=5; 226±99 mmol/l) prior to rituximab showed a decline within 2 months (mo), achieving a stable level by 8 mo (69±14 mmol/l, p<0.03). all patients with hypoalbuminaemia (n=12; 28.6±4.2 g/l) improved (5 mo: 36.1±6.4 g/l, p<0.001). low c3 level (0.40±0.12 g/l) as seen in 12 patients prior to treatment resulted in an increase up to 5 mo (0.77±0.24 g/l, p<0.002). a decline of previously high anti-dsdna (n=13; 162±163 iu/ml) was observed in all patients (5 mo: 56±62 iu/ml, p<0.02). conclusion: rituximab is safe and effective when used in combination with standard immunosuppressive agents. further prospective studies are essential to evaluate the longterm safety of the drug. outcome of severe henoch-schönlein purpura nephritis treated with longterm immunosuppression m. shenoy, m. bradbury, l. lewis, n. webb royal manchester children's hospital, department of nephrology, manchester, united kingdom aims: to look at the long-term outcome of all children with severe henoch-schönlein purpura nephritis (hsn) treated with long term immunosuppression in a single centre over a ten year period. patients and methods: retrospective review of the records of 27 children (19 male) with iskdc grade 3b, 4, 5 and 6 hsn managed at our institution from 01/01/92 to 31/12/01 results: the mean age at presentation was 9.8 years (range 3.6-15.8 years). the median estimated glomerular filtration rate (egfr) at presentation was 91 ml/min/1.73 m 2 (iqr 51. 5-96.6 ) and urine protein: creatinine ratio (up: uc) was 556 mg/mmol (iqr 292-1363). the indication for biopsy was nephrotic syndrome in 9, nephrotic range proteinuria in 7, sub-nephrotic range proteinuria in 3, acute nephritis in 6 and nephritic-nephrotic syndrome in 2. a total of 25 patients were treated with weaning dose of steroids, of whom 22 were also commenced on long-term azathioprine (mean duration 8.9 months). 18 of these children received an 8-12 week course of oral cyclophosphamide (2-3 mg/kg/day) prior to azathioprine therapy (2-3 mg/kg/day). outcome: after a mean follow-up period of 7 years, 10 (37%) have made a complete recovery, 11 (40.7%) have persistent proteinuria but normal egfr, 2 (7.4%) have persistent proteinuria and are on anti-hypertensive therapy with normal egfr and 4 (14.8%) have progressed to esrd. older age at presentation was the only independent risk factor for poor outcome (12.8 years vs. 8.9 years, p=0.005). conclusions: despite treatment with cyclophosphamide, long-term steroids and azathioprine, a majority of children with hsn grade -3b on initial biopsy have persistent renal abnormalities on long term follow-up. only older age at presentation was associated with poor outcome. background: resent advances in podocyte biology indicated that the main cause of the heavy proteinuria in nephrotic syndrome (ns) is a dysfunction of slit diaphragm. on the other hand, the classical charge selective barrier is not likely to have a place in slit diaphragm. therefore we reevaluated the charge selective barrier function in ns and chronic glomerulonephritis using recently established charge selectivity index (csi; takahashi et al, pediatr res 59: 336, 2006) in comparison with dent disease. patients and methods: csi is a clearance ratio of igg (stokes einstein radius 49-60, pi 4.5-9.0) and iga (stokes einstein radius 61, pi 3. 5-5.5) . the assay of serum and urinary igg and iga was performed using laser nepherometry and enzyme immuno-assay. in order to evaluate the csi of normal glomerular filtrate, we measured the csi of dent disease. the urine of dent disease is considered to be a concentrate of filtered protein from normal glomerulus, without having a process of tubular protein reabsorption. thirty eight patients with podocyte diseases (focal and segmental glomerulosclerosis 4, finnish type congenital nephrotic syndrome 1, steroid sensitive nephrotic syndrome 33), 75 patients with chronic glomerulonephritis (iga nephritis 41, henoch-schönlein purpura nephritis 21, mesangiocapillary glomerulonephritis 5, alport syndrome 8) and 8 patients with dent disease, were analyzed. results and conclusion: csi (mean±sd) of podocyte disesses, chronic glomerulonephritis and dent disease was 1.12±0.25, 0, 42±0.31 and 0.18±0.04 respectively. the results apparently indicated that the charge selective barrier of gcw is working strongly in normal glomerulus, less strongly in podocyte diseases and not working in chronic glomerulonephritis. objectives: nphs2 mutations have been reported in familial and sporadic srns. we investigated the prevalence of nphs2 mutations among south-east asian chinese and their association with clinical outcomes. methods: genomic dna from 29 patients with primary sporadic srns (mean age at onset 5.4±4.0 years, range 0.58-12.0 years) and 45 cord blood controls were screened on all 8 exons and exonintron boundaries using direct sequencing. results: a missense heterozygous 871c>t mutation in exon 7 was identified in only one patient. polymorphisms 954t>c and1038a>g in exon 8 were detected in both groups. in the patient group, the genotypic frequency of tt, tc, cc at position 954 was 0.24, 0.55 and 0.21, and aa, ag and gg at position 1038 was 0.86, 0.14 and 0 respectively, consistent with hardy-weinberg expectations. there was no significant difference in allele frequencies between patients and controls. using binary logistic regression analysis, individual polymorphisms did not appear as informative predictors of poor clinical outcome, defined as persistent proteinuria or renal failure (p>0.05). on analyzing composite genotypes, carriers of at least a copy of the 954c allele were significantly associated with poor outcome (p=0.047, or=12.7, 95% ci: 1.03-157), while heterozygotes for 1038a>g were associated with good outcome (p=0.048, or=0.06, 95% ci: 0.004±0.979), suggesting possible interactions between the polymorphic sites. further analysis showed that the genotypic combination of 954tc/cc with 1038aa was more likely to have a poor clinical outcome. no significant linkage disequilibrium was detected between the two polymorphisms. conclusion: the concomitant occurrence of at least a copy of the 954c allele and the 1038aa genotype may be associated with poor clinical prognosis in srns but larger studies are needed to confirm these findings. renal hypodysplasia (rhd) is characterized by a reduced kidney size and/or maldevelopment of the renal tissue following disturbed organogenesis. numerous deletion mouse models of developmental genes have been established presenting with anomalies of the kidneys resembling rhd, among these the knock-out of bmp4 and six2. here, we report on the first human mutations in bmp4 and six2 identified in children with rhd, among these three different mutations in bmp4 in five unrelated patients (ser91cys, thr116ser, asn150lys) and three different mutations in six2 also in five unrelated individuals (leu43phe, pro241leu, asp276asn). overexpression assays in zebrafish demonstrated that ventralization and dorsalization caused by bmp4 and six2 overexpression, respectively, could be diminished after overexpression of mutant constructs expressing the human mutations identified. morpholino knock-down of zebrafish bmp4 and six2.1 reveals specific roles of these genes for pronephric development, affecting the expression of wilms tumor-1 (wt1) and glomerular development. rna analysis of cos7 and hek293 transfected with different bmp4 constructs showed a lower level of mrna abundance in bmp4 mutants, indicating a possible negative feedback of the mutants on their own mrna expression and/or stability. nonreducing western analysis revealed that s91c-bmp4 forms alternative protein complexes as compared to wildtype-bmp4, due to the formation of extra disulfide bonds. these studies implicate six2 and bmp4 as important players in the development of the renal system, and suggest that defects in these proteins could affect kidney development at multiple stages leading to the congenital defects observed in rhd patients. apoptosis is important in normal renal development in which regulation of cell numbers is critical. studies have shown that apoptosis occurs in non-cystic tubules in pre-uremic pkd kidneys, thus providing the mechanism whereby expansion of cysts is accompanied by loss of normal nephrons. to date, it is unclear which initiating factors (intrinsic, through mitochondrial damage or extrinsic, through ligand activation of death receptors) are responsible for apoptosis in pkd and whether they are the same in ad-and arpkd. aim: to elucidate the sequence of activation of intracellular apoptotic pathway(s) in both ad-and arpkd. methods: proteins were extracted from tissues of human ad-and arpkd kidneys and normal adult (nhk) and fetal kidneys (hfk). quantitative western immunoblot analysis was carried out for 7 markers of intrinsic and extrinsic apoptosis. immunohistochemical staining for these markers was performed on tissue sections of the same kidneys. results: markers of extrinsic apoptotic pathways, caspases 8 and 10, were significantly increased in ar-and in adpkd tissue by comparison to nhk and hfk tissues. these increases were seen in early stages of adpkd and were more pronounced later in the disease. for the intrinsic pathway, caspase 9 and bid were increased in hfk tissue, but unchanged in ad-and arpkd tissue compared to nhk tissue. staining for caspase 9 was found in hfk, but was absent in all other kidney tissues. staining for caspase 8 was seen in early adpkd and endstage (es) adpkd as well as in es arpkd. caspase 3 was identified as the main executing caspase of fetal development and adpkd, but wasn't seen in arpkd, where caspase 7 predominated. conclusion: induction of extrinsic pathways of apoptosis predominates in pkd and occurs early in the disease process in adpkd, but only later in arpkd. intrinsic apoptosis predominates during development. intrauterine growth restriction (iugr) is a risk factor for an aggravated course of renal diseases in later life. the influence of postnatal factors, eg. accelerated catch-up growth, is not well understood. we therefore analysed the influence of postnatal nutrition after iugr on the developement of later renal inflammation and fibrosis in the rat. iugr was induced by low protein diet (8% vs. 20%) in pregnant wistar dams. litter size was reduced to 6 or 10 male animals in iugr (lp6, lp10) and control animals (np6, lp10), respectively. animals were sacrificed on day 70. mean arterial blood pressure was similar in all four groups. lp6 -(31.7±6.4 ml/h/100 g) and np6 animals (37.68±16.6 ml/h/100 g) showed reduction of endogen creatinine clearance by 50% (vs. np10and lp10) (p<0.001). renal mrna expression of il6 (5, 7 x), tgfβ1 (1, 5 x) , endothelin 1 (2, 7x) und osteopontin (2, 3 x) was significantly higher in lp6 than in np6. lp6 showed the highest glomerulosclerosis-score ((0.39±0.07) (vs. np6 (0.1±0.07), lp10 (0.09±0.02) and, np10 (0.03±0.02)) (p<0.01). as marker of extra cellular matrix expansion glomerular collagen-iv deposition was significantly higher in lp6 (17.8±6.3%) (vs. np6 (14.3±4.0%), lp10 (7.4±4.0%) and np10 (7.2±1.9%)) (p<0.01). postnatal nutrition modifies the consequences of iugr in the kidney. increased postnatal nutrition of the individual animal is associated with aggravated renal inflammation and fibrosis after iugr. unilateral renal agenesis (ura): how intensively do we need to investigate and follow-up? s. rhodes, a. watson nottingham university hospitals, children and young people's kidney unit, nottingham, united kingdom a single kidney is one of the commonest urinary tract abnormalities in the general population. concerns remain about long-term outcomes with a reduced nephron mass and hence intensity of investigations and duration of follow-up. we identified 52 cases with ura (ectopic and pelvic kidneys excluded) from our nephrourology database between 1985 and 2006. 38 (73%) occurred in males and 25 (48%) had left ura. 27 (52%) were detected antenatally with 12/27 (44%) recognised in the last 2 years. median age of detection for postnatal ura was 7 mths (range 0.25-179 mths) with uti (29%) as the main indication for ultrasound scan (uss). 60% patients were classified as simple ura with no associated abnormality and 40% as complex with problems such as vesicoureteric reflux (vur)(21%), hydronephrosis or scarring. all cases reviewed had uss which showed compensatory hypertrophy in 25/52 (48%). 29/52 (56%) had dmsa scan and 24/52 (46%) micturating cystogram. of those antenatally detected single kidneys with normal initial uss none had vur, scarring, hypertension or proteinuria. these patients were discharged from follow-up at the median age of 6 mths (range 1-122 mths). all complex cases have continued under follow-up. conclusions: our data suggest that the incidence of antenatally detected ura may be increasing. investigations need to be individualised depending upon the initial uss. the value of routine dmsa and mcug in simple cases is questioned. most of these patients can be discharged after adequate documentation of compensatory hypertrophy of the normal kidney, absence of proteinuria, normal blood pressure and renal function. autosomal dominant pdk (adpkd) in childhood j. crocker, p. wornell, p. acott iwk health centre/dalhousie univeristy, division of nephrology, halifax, canada autosomal dominant polycystic kidney disease (adpkd) is the most common genetic kidney disease with 1 in 6 adults progressing to end-stage renal disease (esrd). a program for intervention in childhood at presentation with adpkd was developed and instituted in 1992. our long-term objective is to modify clinical parameters that may contribute to risk of development of esrd in adulthood. seventy children with adpkd (average age 11.47 yr) were followed, of which 10 (14%) had nephromegaly +/-cysts on antenatal ultrasound. modifiable risk factors were common including hypertension (22%), hyperlipidemia (54%), and proteinuria (7%). ace inhibitors were first line therapy for proteinuria and/or hypertension. ace inhibitors may modify cyst progression so they have been made available to non-hypertensive children as well. most patients with hyperlipidemia responded to dietary intervention with one patient developing gallstones. in the past 5 years we have focused on renal calculi, as this is a known risk for a subgroup of adults with poor prognosis. we noted 14 patients (20%) have glycinuria, which is a precursor to oxaluria. four patients have passed calculi with two of these being diagnosed by genetic linkage analysis for adpkd without radiographic cysts present. cerebral vascular studies of 14 patients with severe headaches revealed two with vascular structural anomalies. all adolescent females received counseling regarding appropriate contraception and their pregnancy risks of hepatic cyst progression. an early intervention program targeting modifiable risk factors of adpkd patients during childhood and adolescence may modify adult renal failure risk in this population. background: techniques of chronic infant dialysis have evolved during the past 2 decades but morbidity and mortality are not well described. methods: a retrospective review was performed on 45 infants -18 months of age with end stage renal disease (esrd) treated with maintenance dialysis during the past 23 years. the experience was divided into era 1 (1983 era 1 ( -1994 (n=23) and era 2 (1995 era 2 ( -2006 . dialysis modality, morbidity, and long term survival were assessed and compared between the 2 eras. results: all patients were begun on peritoneal dialysis (pd). there were 22 males and 23 females. age at initiation of dialysis was 4, 5 months. the predominant diagnoses were dysplasia/obstructive uropathy (n=24), autosomal recessive polycystic kidney disease (arpkd) (n=10), congenital nephrotic syndrome (cns) (n=7), other (n=4). overall survival is 38% (17/45) with current age of survivors ranging from 5 months to 23 years. mortality between era 1=70% and era 2=55% was not significantly different. fifteen (33%) survived to receive a kidney transplant. overall median survival is 3.1 years (era 1=3.5 years; era 2=3.1 years; p=0.97). conclusions: although long term survival is possible in infants with esrd, mortality and morbidity remain high. improved technologies for automated pd should address the needs of the infant <6 kilograms. joubert syndrome and related disorders (jsrd) are a group of autosomal recessive conditions characterized by a complex neuroradiological malformation resembling a molar tooth on imaging. clinically, jsrd are characterized by overlapping phenotypes presenting neurological signs and variable involvement of other organs such as kidneys (mainly nephronophthisis -nph), retina and liver. seventy-nine italian jsrd patients from 63 unrelated families were recruited in pediatric nephrology and neurology centers. medical records and brain mri were reviewed. patients without chronic renal failure (crf) underwent measurement of their glomerular and tubular function, a ddavp urine concentration test and a renal ultrasound. retinal examination was performed in most patients. seven patients younger then 18 years had normal renal function but were not fully tested. of the remaining 72 cases, 44 had no evidence of renal disease, 16 had developed crf mostly in their second decade of life, and 9 were younger then 11 years of age and had urinary concentration defects, of whom 5 also had hyperechogenic kidneys by renal ultrasound. when comparing this latter group with age-matched jsrd patients, no other tubular function abnormality was detected. in 13 multiplex pedigrees, no discrepancies in renal involvement between affected family members was observed. retinal involvement was significantly associated with renal disease. conclusions. evidence of interstitial renal disease was observed in one third of italian patients with jsrd. renal involvement should always be suspected in these patients, particularly if they have evidence of retinal dysplasia or other family members with symptoms related to nph. these patients should be assessed with a urine concentration test. we examined the course of 49 children with 56 primary obstructive megaureters (pom) treated in our hospital from 1994-2006. pom occurred more often in boys (71%, p<0.01) and on the left side (67%, p<0.05). 43 pom (77%) were only treated conservatively. four underwent immediate surgery following the first mag3 renography. one of these was nephrectomized after unsuccessful urinary diversion and persistent renal hypertension. of the 52 kidneys primarily managed conservatively, 9 needed a ureterocystoneostomy later on due to increasing obstruction. surgical correction of pom required a mean of 15 days of hospitalization (incl. temporary urinary diversion, removal of catheters and treatment of complications). one child required surgical revision of early post-operative ureteric stenosis. urinary tract infections (uti) were a common complication (n=66, mean 1.3 per patient). as utis occurred mainly in infants, hospital admission was commonly required (45%). only 14 children never acquired a uti (29%). eight patients had a poor outcome as defined by partial kidney function <40% on the affected side (n=6), atrophy on final ultrasound (n=3) or nephrectomy (n=1). the most potent predictor of reduced unilateral kidney function was a small kidney present from birth. secondary kidney growth failure occurred only in one case. the initial degree of obstruction on renography, but not the degree of hydronephrosis or size of megaureter predicted outcome. also, prenatal diagnosis of pom, surgical treatment and the occurrence of utis showed no association with outcome. in summary, the long-term prognosis of pom appears favorable. adverse outcomes were more closely related to congenital kidney size deficit than to the degree of obstruction. surgical interventions and the high uti incidence led to significant hospitalization times. r. bhimma, m. adhikari, k. asharam university of kwazulu-natal, maternal and child health, durban, south africa background: steroid resistant (sr) forms of ns have a poorer outcome in blacks compared to other racial groups. methods: 223 children with srns 1-16 years old were analysed retrospectively for the period 1976-2004. treatment schedules included oral cyclophosphamide with prednisone only (n=90); prednisone on alternate days with methylprednisolone and oral cyclophosphamide (n=117); oral prednisone on alternate days, 3 doses of intravenous methylprednisolone on alternate days and monthly doses of intravenous cyclophosphamide (n=10) or cyclosporine adjusted to a trough level of 150-200 mg/ml (n=6). we compared the clinical, biochemical characteristics and outcome of these children using different forms of therapies. results: 183 (82.1%) underwent renal biopsy.84 (45.9%) were indian and 99 (54.1%) were black. 66 (36.1%) had minimal change ns, 66 (36.1%) had focal segmental glomerulosclerosis (fsgs), 15 (8.2%) a proliferative form of ns, and 36 (19.7%) had other forms of ns. 58/84 (69.0%) indian children biopsied were in complete remission and 29/40 (72.5%) with mcd who were treated with oral cyclophosphamide and prednisone only achieved complete remission. 32/40 (80%) indian children not biopsied who received only oral prednisone and cyclophosphamide achieved complete remission. 20/99 (20.2%) black children who were biopsied achieved complete remission. none of the eight black children who were not biopsied given only oral cyclophosphamide and prednisone achieved complete remission. conclusion: since 80% of indian children with srns responded to a trial of oral cyclophosphamide and prednisone but none of the black children did, we propose the use of oral cyclophosphamide therapy in non black children before embarking on renal biopsy. mj. kemper, c. moeller, n. rink, m. van husen, de. müller-wiefel university of hamburg, pediatric nephrology, hamburg, germany introduction: ssns is often complicated by a refractory clinical course with frequent relapses and steroid dependency despite aggressive alternative immunosuppressive regimens. since recent immunological findings suggest an alteration of not only t-but also b-cell immunity in ssns (kemper 2003 (kemper , 2004 we hypothesized that that immunological targeting of b-cells with antibodies directed against cd20 (rituximab) is able to maintain remission (rm) in treatment refractory patients with ssns methods: a total of six patients with complicated courses of ssns had severe steroid-dependency and toxicity. all patients had previously been treated with cyclophosphamide, two patients relapsed on maintenance therapy with cyclosporine, one relapsed on additional levamisole and one on mmf. treatment was initiated at a median age of 13 (range 11-15.6) years and rtx was given at steroid induced rm at a dose of 4x 375 mg/m 2 bsa within 4 weeks. results: a complete b-cell depletion was induced for at least 5 months in all patients. rm could be maintained in all patients and steroid treatment could be discontinued after a median of 2.5 months (range 1-4.6) . also csa could be discontinued in the two patients on maintenance treatment. at current follow-up of 11.5 months (range 5-23.5) the two patients after csa discontinuation relapsed after 8.7 and 12.7 months, respectively, but responded after steroid induced rm-to a second course of rtx. all other patients remained in remission off treatment so far. no significant clinical side effects were noted. conclusion: in summary and conclusion, rtx seems to be a therapeutic option in complicated ssns. long-term follow-up and future prospective studies are necessary to further define the role of rtx in the treatment of refractory steroid sensitive nephrotic syndrome. efficient control of secondary hyperparathyroidism can be achieved by calcimimetics. they increase calcium sensing receptor (car) sensitivity to extracellular calcium. in r-568-treated uremic rats, proteinuria has been significantly reduced by calcimimetics. thus, we examined the potential direct effect of r-568 on podocytes. the car was expressed in cultured immortalized podocytes (quantative rtpcr, western blot) and in podocytes obtained from healthy and subtotally nephrectomized rats (immunohistochemistry). glomerular car abundance was increased by uremia and r-568. the car colocalized with the plasma membrane and intracellular filaments in cultured podocytes, but not with the caveolin 1-and 2-rich membrane fractions. we then studied the effect of r-568 on the mitogen-activated protein kinase (mapk) family. jnk was not activated. p38 showed a biphasic response pattern, whereas erk1/2 (and further downstream, p90 ribosomal s6 kinase) showed dose-(1-50 nmol/l) and time-dependent phosphorylation, resulting in the activation of the transcription factor camp response element binding protein (creb). creb phosphorylation induced bcl-xl expression and bad phosphorylation, both of which have prosurvival activity. specificity was confirmed by addition of mek 1/2 inhibitor u0126, which completely blocked r-568-induced creb phosphorylation. facs analysis revealed a significant, 50% decrease in puromycin-induced apoptosis in podocytes treated with r-568 for 48, 60 and 72 hrs. in conclusion, calcimimetics induced prosurvival gene expression in podocytes via mapk, protecting them from apoptosis. calcimimetics may have a direct renoprotective action beyond control of hyperparathyroidism in chronic kidney disease patients. we report our experience with plasma therapy in a family of three sisters from whom two are homozygous twins (patients b and c), presenting a missense mutation of the exon 23 of the human complement factor h gene (hf-1). the factor h concentration has always remained normal and no systemic complement activation has ever been detected. in a ten years period, the followings have been observed: 1/ no use of plasma exchange lead to immediate esrf of native kidneys (patient a); 2) conventional plasma therapy (10 sessions at presentation followed by repeated 10 ml/kg plasma infusions when relapse of hemolysis and thrombopenia) could not impede esrf (patient b); 3) the use of intensive pe at presentation (daily pe (40 ml/kg ffp until plasma creatinine normalization followed by one pe/2w indefinitely) lead to a normal gfr 5 years after presentation despite 2 relapses (patient c); 4/ no prophylactic pe for tx lead to immediate hus relapse and transplant loss (patient 1); 5) prophylactic pe allowed a successful tx in patient b (pl. creat 110 mcmol/l after 5 years); 6) early and intensive pe allowed complete normalization of several hus relapses after tx (patient b). conclusions: in fh mutation-related atypical hus, 1) intensive and indefinitely prolonged pe can allow a normal function of native kidney at long term; 2) prophylactic pe allows successful kidney tx; 3) early and intensive pe allow reversion of hus relapse after tx. the success of plasma therapy is bound to the followings: a/ the use of pe and not plasma infusion. b/ the prolongation of daily pe after normalization of hemolysis parameters, and further prophylactic pe. c/ the use of pe prophylaxis from before tx. d/ a minimum pes frequency of one/week in case of prophylactic treatment for tx. e/ immediate intensification of pe frequency in case of relapse. objectives of study: heparan sulfates (hss) are highly polyanionic sugar chains in the glomerular basement membrane (gbm) and have been reported to play an important role in the chargeselective permeability of the kidney. alterations in hs expression have been reported in a number of renal pathologies. in this study, we evaluated if degradation of hs in the gbm resulted in proteinuria in rats, using a controlled in vivo approach. methods: heparinase iii and neuraminidase were injected i. v. in 2-month old wistar rats at t=0 hr and t=8 hr, and kidneys were removed at t=24 hr. urine samples were taken at various time points. cryosections were stained for hs using specific antibodies, and for hs stubs (generated by heparinase). in addition, hs was evaluated at the electron microscopical level. neuraminic acid expression was analysed by peanut agglutinin lectin. the hs content in urinary samples was evaluated by agarose gel electrophoresis. urinary neuraminic acid was studied by an enzymatic colorimetric assay. presence of urinary albumin (proteinuria) was investigated by sds-page and by a competition elisa. results: injection with heparinase iii resulted in an almost complete absence of glomerular hs staining. cupromeronic blue staining was also greatly reduced in the gbm, further indicating that hs was largely degraded. staining for the hs proteoglycan core protein agrin was unaltered. in the urine a strong increase in hs was found, already at the first point in time of urine collection (2 hr after the first injection). however, no urinary albumin or other proteins could be detected at any point in time analysed. injection of rats with neuraminidase resulted in a major increase of albumin in the urine. conclusion: in conclusion, removal of hs from the gbm does not result in acute albuminuria, whereas removal of neuraminic acids does. introduction and methods: 192 children undergoing deceased (156) or living (36) donor kidney transplantation were randomized to receive tacrolimus, azathioprine, steroids and two doses of basiliximab (tas+b) or tacrolimus, azathioprine and steroids(tas). previously reported six month follow-up biopsy-proven acute rejection rates were 19.2% (tas+b) and 20.4% (tas). patient survival was 100% and graft survival 95% in both arms (am j transplant 2006; 6: 1666-72) . in this investigator-driven follow-up study individual outcome data were submitted annually to the coordinating centre. results: two year follow-up data were obtained for 144 (87.8%) of the 164 who completed the six month study. there was one death in the tas group occurring at month 20. there were 5 graft losses in the tas+b arm and 9 in the tas arm. all 5 graft losses in the tas+b arm occurred within the first 6 months. kaplan-meier estimates of 2 year graft survival were94.9% for tas+b and 89.5% for tas (p=0.28 breslow generalised wilcoxon test). episodes of biopsy proven acute rejection occurred in 23 in tas+b and 26 in tas, kaplan-meier estimates of freedom from rejection being 76.1% and 69.6% respectively (p=0.49). renal function did not differ significantly between the two arms; the median (iqr) plasma creatinine levels were 90 (74-113) in tas+b and 96 (77-115) in tas (p=0.94). similarly there was no evidence of a difference in either systolic or diastolic blood pressure between the two arms. there was one case of b cell ptld in the tas+b arm at 11 months in addition to two cases in the tas arm previously reported in the 6 month study. conclusions: the addition of basiliximab to a regimen of tacrolimus, azathioprine and steroids does not appear to result in an improvement in either acute rejection rate or graft survival at two year follow-up. further data collection is ongoing. chronic renal dysfunction is a major complication after heart transplantation (htx). the pathophysiology is not yet fully understood but is thought to be in part due to calcineurin inhibitor (cni) toxicity, and reducing cni exposure has become one of the main strategies aimed at ameliorating renal outcome in htx recipients. we previously reported a significant improvement of renal function in 14 htx children with biopsy-proven cni nephrotoxicity and chronic renal failure, 1 year after the reduction of cni dosage with a concomitant replacement of azathioprine by mycophenolate mofetil. we ought to determine whether this improvement was persistent after 3 years of follow-up, despite the histological lesions of chronic cni nephrotoxicity. gfr evaluated by annual inulin clearance had improved from a mean of 46.5±9.6 ml/min/1.73 m 2 (range 34-60) at time of the switch to 77.6±6.0 ml/min/1.73 m 2 (range 54-102) (p=0.019) one year after (67% improvement), and remained stable at 76.4±18.2 ml/min/1.73 m 2 (range 50-102) (p=0.76) 3 years after the switch. maximal urinary osmolality followed the same increase profile from 559±103 mosm/kg before the switch to 762±122 at one year and 736±154 after 3 years (p=0.64). meanwhile, the occurrence of serious adverse events such as infectious episodes, acute rejection and chronic allograft dysfunction as assessed by clinical examination, echocardiography and endomyocardial biopsies were not different from a control group of patients whose treatment was unchanged. no malignancy was observed in either group. in conclusion, reduction of cni dosage and replacement of azathioprine by mycophenolate mofetil lead to a safe and long-lasting improvement of renal function in children with heart transplants and cni-induced nephropathy. previous studies have demonstrated reduced bone mineral density (bmd) in some patients with idiopathic hypercalciuria (ih). reduced bmd during childhood may impact adult peak bone mass. bisphosphonates employed in adults with ih and reduced bmd resulted in conflicting outcomes. we evaluated the effects of oral bisphosphonate, alendronate (ale), in 3 patients with persistent ih and reduced bmd. patients presented at ages 6, 12, 13 yr, with hematuria/dysuria, 2 had recurrent urolithiasis, and all had ih (uca >4 mg/kg/24 hr). despite maximal traditional rx with low na/high k diet, thiazides, k-citrate, ih persisted (5.6-12 mg/kg/24 hr). at ages 10, 16 and 14.5 yr, dxa showed reduced bmd, and ale 10, 40 and 70 mg/once weekly was given for 15, 7 and 18 months, respectively. after 6 months of ale, uca decreased significantly compared to baseline (mean±sd, 3.0±1.9 vs. 6.3±1.8 mg/kg/24 hr, p<0.05). compared to baseline, bmd z scores 1 year after starting ale improved at the spine (-1.1±0.4 vs. -2.3±0.6, p<0.05) and hip (-1.9±0.3 vs. -2.3±0.2) . the decline in uca during ale rx correlated with the increased bmd z scores in the spine (r=-0.98, p=0.01) and hip (r=-0.93, p<0.05). height z scores, serum creatinine, ca, p, electrolytes and pth remained normal throughout the rx period and no further hematuria or new stones occured. in summary, ale normalized uca previously resistant to traditional rx, eliminated urinary symptoms and improved reduced bmd in children with ih. the use of a single oral weekly dose appears adequate and safe. larger prospective studies are needed to confirm these preliminary results. in addition to its classical role in the regulation of calcium (ca) and phosphate (po 4 ) homeostasis, vitamin d has important immunomodulatory and anti-inflammatory effects, that in turn can influence atherosclerotic vascular disease. we studied the impact of vitamin d levels and inflammation on vascular structure and function in children on dialysis. 59 children (age 13.4±4.1 yrs) on dialysis (mean duration 1.1±0.9 yrs) were studied. all children received 1α-hydroxyvitamin d 3 (alfacalcidol) . cumulative data on ca, po 4 and pth, doses of po 4binders and alfacalcidol were recorded.1, 25-dihydroxyvitamin d 3 (vit d) and high-sensitivity crp (hs-crp) levels were measured by 125 i radioimmunoassay and elisa respectively. all children had carotid intima-media thickness (cimt), pulse-wave velocity and cardiac ct for coronary calcification. 23 (39%) children had vit d deficiency (levels<40 pmol/l), 25 (42%) hadnormal levels (40±150 pmol/l) and 11 (18%) had high levels (>150 pmol/l). vit d positively correlated with serum ca, caxpo 4 and alkaline phosphatase. both cimt and calcification showed a bimodal distributionpatients with vit d levels <40 or >150 pmol/l were significantly more likely to have calcification or raised imt than those with vit d levels in the normal range. hs-crp levels independently predicted cardiac calcification (p=0.02) but not cimt. there was a strong inverse correlation between vit d levels and hs-crp (p<0.0001, r=-0.69). patients with vitamin d levels<40 pmol/l and hs-crp levels>10 mg/l had 6-fold greater calcification scores than subjects below these cutoffs. in conclusion, vit d deficiency is common despite treatment in children on dialysis. vit d may be an important mediator of vascular damage both through its hypercalcaemic and anti-inflammatory actions. severe growth failure remains one of the challenging problems in the care of children suffering from chronic renal failure (crf). although, rhgh has been proven to increase final adult height in prepubertal crf patients only limited data on its efficacy in the pubertal age-range are available. in addition, the impact of the underlying renal disease and the mode of renal replacement therapy on final height in these patients remain unclear. we report on final height data of 240 (47 female) severely growth-retarded crf patients (standardized height <-2 sds; database: kigs medical outcomes, pfizer). mean age at start of rhgh therapy was 13.7±3.0 years, standardized height was -3.6±1.2 sds and duration rhgh therapy was 4.6±2.5 years (range 1.0 to 14.2 years). at baseline 45% of the patients were on conservative treatment, 28% were on dialysis and 27% had a functioning renal allograft. in the whole study population mean standardized height was increased in the first treatment year and at attainment of final adult height by +0.4 sds and +1.2 sds, respectively (each p<0.0001 vs. baseline). prepubertal children aged less than 12 years at start of rhgh therapy showed the best growth response (+1.5 sds). in pubertal patients mean increase in standardized height was +1.2 sds, whereas growth response in patients with delayed onset of puberty (>+2 sd) was significantly lower (+0.8 sds; p=0.039 vs. other groups). the duration of rhgh therapy was positively associated with cumulative height gain. growth response was significantly lower in patients on long-term dialysis and in patients with nephropathic cystinosis. conclusion: rhgh therapy in severely growth retarded prepubertal and pubertal crf patients results in an increased final adult height. growth response is diminished in crf patients with markedly delayed onset of puberty. tightly regulated rankl/opg system is essential for normal bone remodelling. however, the exact roles of those osteogenic markers in uremic bone disease have yet to be defined. we therefore assessed the potential relationship of the rankl/opg system in bone biopsy proven secondary hyperparathyroidism (hpt) in dialyzed children. methods: 40 patients aged 13±1years were on ccpd for 14±4 months. s-ca, p, alk p'tase, pth, opg and rankl levels were measured. bone biopsies were obtained after double tetracycline labeling and none of the patients were treated with vitamin d for four weeks prior to biopsy. results: s-ca levels were 9.2±0.7 mg/dl, p: 6.1±1.5 mg/dl, alk p-tase: 401±297 u/l, pth: 898±415 pg/ml. bone biopsy findings revealed high turnover bone disease in 31 patients, 9 patients had normal bone formation rate (bfr). mean opg and rankl levels were 4.46±1.46 and 0.66±0.81 pmol/l, respectively (reference control=opg: 1.8; rankl: 0.42). opg correlated with bfr (r=0.473, p<0.01) and adjusted apposition rate (r=0.42, p<0.01), while inversely correlated with osteoid maturation time (omt) (r=-0.34, p<0.03) and mineralization lag time (r=-0.45, p<0.01). rankl/opg ratio was negatively correlated with mineral apposition rate (r=-0.35, p<0.03) and positively with omt (r=0.31, p<0.05). pth was correlated with bfr (r=0.40, p<0.01) and resorption area (r=0, 54, p<0.01), but not with any mineralization markers. there were no correlations between pth and opg or rankl. conclusion: opg, in contrast to rankl, exerts a dual effect on the skeleton by promoting mineralization and increasing bfr. these osteogenic markers might be of benefit in characterizing the turnover, mineralization and volume of the skeletal lesions of secondary hpt as recently recommended by kdigo. "sevcan bakkaloglu was supported by tübitak (scientific and technological research council of turkey). this study was supported by usphs grants dk-35423, dk-67563 and mo1-rr00865. " pth values are widely used to guide therapy for renal osteodystrophy in patients treated with maintenance dialysis yet target values are based on cross-sectional studies and discrepancies between bone formation rates (bfr) and pth have been described during intermittent calcitriol rx. thus, we evaluated the relationship between serum biochemical parameters and bone turnover during treatment with intermittent vitamin d sterols. 60 patients aged 13±1 yrs with biochemical and bone biopsy (bbx) proven 2 nd hpt received 8 months of vitamin d (1, 25d 3 or 1d 2 ) given in twice weekly oral dosing and phosphate binders. dose of vitamin d was titrated upwards monthly to maintain 1 st pth-ima(nichols r ) levels between 300-400 pg/ml. bbx was then repeated. s-ca, p, alk p-tase and pth by 1 st and 2 nd pth-imas were obtained monthly throughout therapy. baseline values were: p: 6.2±0.1 mg/dl, ca: 9.2±0.1 mg/dl, alk p-tase: 397±36 iu/l, 1 st pth-ima 940±55 pg/ml, 2 nd pth-ima: 510±41 pg/ml. final values were: p: 5.5±0.1mg/dl, ca: 9.4±0.1 mg/dl, alk p-tase: 324±35 iu/l, 1 st pth-ima: 565±43 pg/ml, and 2 nd pth-ima: 280±27. bone formation rate (bfr/bs) decreased from 114±8 to 55±5 um 2 /mm 2 /d (nl: 10-73.4); 72% achieved normal bone turnover. 2 nd pth-ima values were 40-50% lower than 1 st pth-ima levels; there was no difference in predictive capability of the two assays. patients achieving normal bfr/bs had 1 st pth-ima values of 508±44 pg/ml. the sensitivity, specificity and ppv of a 1 st pth-ima range of 300-600 pg/ml for normal bfr/bs were 70% (95% ci: 50-86%), 60% (26-88%), and 83% (61-95%) respectively. during therapy with intermittent vitamin d sterols, maintaining pth levels higher than currently recommended results in normal bfr/bs and prevents adynamic bone. maternal diabetes will induce abroad array of congenital malformation. consistently with these hypotheses, we observed the defects of renal development of diabetic pregnancy from late phase of embryogenesis to postnatal period in animal model (c57bl/6j mice). histological analysis of phenotypes revealed decreased glomerular numbers, glomerular hypertrophy and hypercellularity, as well as renal tubular detachment in sequential late phase of kidney development in the offspring of diabetic female mice. tunel assay showed signficinatly increased cell apoptosis in fetal kidneys of hyperglycemic group. rt-pcr and fish study of kidneys of fetal and newborn mice revealed that gdnf and early growth response alpha (egr-α) are two of crucial genes inhibited in hyperglycemic ambience. in human, we presumed that children of gestational hyperglycemic mothers have the same defects of renal development as our animal model similarly, thus we measured and compared echogram of kidney/liver echoenic ratio in 76 children born to gestational hyperglycemic mothers and 240 health children. interestingly, our human ultrasonic study indicated that after age of 6 months, the diabetic children had increased echogenic ratio of kidney/liver than the healthy age matched children (p<0.01). we also found that 26.3% of diabetic children had nonobstructive hydronephrosis. this simple sonographic procedure may provide a permissive was for clinicians to obtain the basis of long-lasting follow-up of these high-risk children as early as possible. keywords: diabetic embryopathy, renal development, glial cell line-derived neurotrophic factor, early growth response alpha, renal sonogram. genetic inactivation of spry 1 in mice results in increased number of ub branches and expanded gdnf, c-ret and wnt-11 expression domains (basson et al., dev. cell, 2005) , indicating that spry1 is a negative regulator of the gdnf-ret-wnt11 pathway. ang ii induced ub branching morphogenesis, partly via stimulation of egfr tyrosine kinase activity (yosypiv et al. jasn, 2006) . we tested the hypothesis that ang ii stimulates the gdnf-ret pathway via repression of spry1. cd1 mice metanephroi were dissected on embryonic (e) day e12.5, grown on filters in the presence or absence of ang ii (10 -5 m, n=5/group) for 24 hours and subjected to whole-mount ish with digoexigenin-labelled spry1, c-ret, wnt-11 and gdnf crna probes. spry1 mrna was expressed in ub branches and in condensing mesenchyme. c-ret and wnt-11 were expressed in ub tips, and gdnf-in the metanephron mesenchyme. ang ii downregulated spry1 expression in the ub. in contrast, c-ret and wnt-11 were induced by ang ii in the ub tip cells. gdnf expression in the mesenchyme was also upregulated by ang ii. in addition, ang ii stimulated ub tip cell proliferation, as determined by in vivo brdu in corporation (28.5±2.4 vs.9.7±1.2; p<0.001) compared to control. these findings suggest a model in which ang ii-mediated inhibition of spry1 gene expression releases. ret tyrosine kinase activity leading to upregulation of c-ret and its downstream target gene, wnt-11. enhanced wnt-11 expression, in turn, induces gdnf in the adjacent mesenchyme. this causes focal bursts of ub tip cell proliferation and branching. these results support the hypothesis that abnormal collecting system development in angiotensinogen, renin, ace or at1-deficient mice is at least partly due to aberrant regulation of the ub branching morphogenesis program. objectives of study: nephrogenesis requires a fine balance of many factors that can be disturbed by intrauterine growth restriction (iugr), leading to a low nephron endowment. our previous studies have shown that offspring born to mothers supplied low protein diets during pregnancy have fewer glomeruli than normal at birth in sd rats. the aim of this study was to identify the possible pathogenesis of abnormal nephrogenesis and decreased glomerular number in iugr by comparative proteomic approach. methods: iugr was induced in sd rats by isocaloric protein restriction in pregnant dams. kidney proteins were obtained from 6 neonatal normal rats (control group) and 8 iugr rats (iugr group) respectively. a series methods including 2-de, silver staining, mass spectrometry and database searching were used. the 2-de test was repeated three times in each group. results: after silver staining, the 2-de image analysis detected average 730±58 spots in iugr group and 711±73 spots in control group. the average matched rate was 86% and 81% respectively. the differential proteomic expression analysis found eleven protein spots were expressed only in iugr group and one in control group. seven protein spots were up-regulated more than 5 folds and two down-regulated more than 5 folds in iugr group compared with those in control group. these 21 protein spots were preliminarily identified, which were structural constituents of cytoskeleton including vimentin, cytokeratin 10, perlecan and b-actin, transcriptional factors including splicing factor, rho gdp dissociation inhibitor alpha and cell division proteinkinase 2, enzymes including retinal dehydrogenase1, transketolase and so on. conclusions: data from this study may provide, at least partly, valuable experimental evidence of proteins involved in the pathogenesis of abnormal nephrogenesis and decreased glomerular number in iugr. the aim of the study is to describe the natural history of tcf2/hepatocyte nuclear factor-1 beta linked disease in children. prenatal and postnatal renal evolution and extrarenal manifestations of 33 patients were reported. thirty one had prenatal diagnosis of developmental nephropathy: bilateral foetal hyperechogenic kidneys or a hyperechogenic kidney with a controlateral multicystic dysplastic kidney. 64% had cysts. the mean prenatal renal length was normal (0, 29 sd) . intrauterine growth was normal with median birth weight of 3.1 kg (range 1.6-3.6) and 30% were small for gestational age. after a mean follow-up of 79 months, the renal growth was impaired with a mean renal size of -0.78 sd. patients with a solitary functioning kidney showed no compensatory hypertrophy. thirty one patients developed bilateral nephropathy and 2 isolated unilateral multicystic kidney. twenty eight patients had cysts, mainly cortical bilateral microcysts. the mean gfr was 73.19±30.43 ml/min/sc. sixteen patients had stage 1 chronic kidney disease (ckd), four were classified as stage 2, eleven as stage 3 or 4 ckd and two were diagnosed with end stage renal failure. annual decline of the glomerular filtration rate was 3.4 ml/min. extrarenal manifestations included 3 patients with diabetes, one with exocrine pancreatic insufficiency, one with pancreatic hypoplasia without clinical symptoms and 4 with cholestasis. we found a complete heterozygous deletion of the tcf2 gene in 20 patients and nine different point mutations. three patients had the g76c mutation, 2 with unilateral mcdk and 1 with bilateral severe hypoplasia leading to early renal dysfunction. tcf2 gene anomalies are associated with low renal function decline but in some patients end stage renal failure appeared early in life. long term follow-up is needed to determine the incidence of extrarenal symptoms, particularly diabetes. objectives: although it has been observed more than one hundred years ago that the urinary tract is very resistant to infection, its antimicrobial mechanisms have not yet been systematically characterized. we sought to elucidate the expression and relevance of the antimicrobial peptide cathelicidin in this area. methods: in order to investigate the expression of cathelicidin in health, urine samples from healthy children, pieces of healthy renal tissue, and cell cultures were analyzed. to evaluate the expression of cathelicidin during infection, urine samples from children with urinary tract infection, a mouse model of ascendant pyelonephritis and cell culture experiments were employed. the relevance of cathelicidin production was tested by bacterial challenge of cathelicidin-deficient and neutropenic mice. in addition, we studied the in vitro effects of cathelicidin on the growth and multicellular behavior of bacteria as well as we tested sensitivity of clinical e. coli strains to the peptide. results: cathelicidin is expressed by epithelial cells of healthy urinary tract and excreted into urine in low concentrations. during urinary tract infection, the secretion of cathelicidin by epithelial cells significantly increases within minutes. invading neutrophils are the source of the second wave of cathelicidin. epithelial cathelicidin protects the urinary tract against bacterial infection while neutrophil-derived cathelicidin influences the severity of infection. cathelicidin displays multiple effects on bacteria. low concentrations of cathelicidin inhibit multicellular behavior, e. g. biofilm formation of e. coli, and high peptide concentrations kill bacteria. bacteria resistant to cathelicidin have an increased ability to invade the urinary tract. conclusion: the antimicrobial peptide cathelicidin is a key factor of the mucosal immunity of the urinary tract. the predictive value of procalcitonine (pct) plasma level for renal scarring after an acute pyelonephritis (apn) is still debated. during apn, the bacterial lipopolysaccharide of the membranes induce the release of tnf, il1 and il6. these cytokines lead to inflammation syndrome and fibrosis sequellae resulting from cell apoptosis induced by nitric oxide (no) release that is catalysed by no-synthase. the aim of this work was to clarify the physiological role of pct in renal parenchyma apoptosis and fibrosis caused by apn. we conducted a prospective study in children with a first apn episode (fever>38.5c°, crp>20 mg/l, monomicrobial urine positive culture>10.5 fcu/ml). we excluded patients with any concomitant infection, renal dysplasia or obstructive uropathy or grade 4-5 vur 133 children were enrolled (age 37.2 m, median 17 m). on admission, pct, crp and phospholipase a2 (pla2) were quantified in serum. scintigraphy with 99m tc-dmsa was performed on day 4 and 9 months later in case of initial abnormalities. fisher's test was performed and statistical significance was defined as p<0.05. results: on day 4, 107 (79%) presented renal parenchyma alterations, at 9 m 57 underwent control scan and 17 (28%) had renal scars. pla2 and pct levels were correlated with early dmsa lesion but not with renal scars at 9m. paradoxically, initial pct level was significantly lower in the presence of renal scars (4.19 vs 7.59 ng/ml, p<0.01). significant pct increase was observed in favourable progress (recovery 7.55 vs aggravation 3.34 ng/ml, p<0.01) and no difference between recovery and improvement evolution. these results suggest the protective effect of pct against apoptosis resulting from down-regulation of nitric oxide release; so high pct values could be interpreted with caution in apn. acute pyelonephritis (apn) may lead to renal scarring with later risk of hypertension and renal insufficiency. the aims of this study were to analyse prospectively renal scars progression with time and their impact on renal growth. methods: 52 patients (pts), aged from 0 to 18 years who presented scars on dmsa at 6 months after apn were included. in these children a second dmsa was done after 3 years. evolution of scars was analyzed following pre-established criteria independently by 3 observers. scar progression was classified as follows: 0=no change, 1=partial improvement, 3=total resolution. in addition a renal ultrasound was repeated to assess kidney growth using z-score. results: 104 renal units (52 pts: 32 f, 22 m) were studied. the incidence of vesicoureteral reflux (vur) was 34.6% (18/52). per renal units, vur were observed in 27/104 (26%); (51% vur grade (g) i, ii and 49% g iii, iv). there were 91 scars observed 6 month after apn which evolution over 3 years was as follow: 0=26% (24/91), 1=65% (59/91) and 2=9% (8/91). incidence of vur was higher in children presenting 3 scars; 5/7 (71%) against 12/45 (26%) in those with 1 or 2 scars. to identify factors interfering with renal growth, we analyzed potential confounding variables using robust linear regression. z-score was worsened with increased number of scars observed at 6 month after apn p<0.001) and improved with disappearance of vur (0.89 ci 0.001-1.7; p<0.05). conclusion: between 6 month and three years after apn, 82% of scars improved. in our population, the number of scars secondary to pna was the most important factor affecting renal growth. the second prognosis criterion was the correction of vur. prevention of pna and rapid treatment to avoid kidney scars seems to be the essential aim to preserve optimal kidney growth. v. smolkin 1, 2 , r. halevy 1, 2 , w. sakran 1, 4 , y. kennes 3 , a. koren 1, 4 1 ha'emek medical center, pediatric b, afula, israel 2 ha'emek medical center, pediatric nephrology unit, afula, israel 3 ha'emek medical center, bacteriology and microbiology laboratory, afula, israel 4 the ruth and baruch rappaport school of medicine, haifa, israel febrile urinary tract infection (uti) is a relatively common infection disease in childhood. children consider at risk for uti commonly receive prophylactic antibiotics to prevent recurrence of this urinary tract disease because of the risk of kidney scarring, which may lead to complications such as hypertension or end-stage renal disease. compliance with antibiotic prophylaxis after uti was assessed in 69 children, using a parent questionnaire and a urine test for antibacterial substances. thirty six children (1 st group) received prophylactic treatment during the period of 4 months (range 3-7months) and the other 34 patients (2 nd group) received antibiotic prophylaxis for a longer duration (12 months, range 11-16 months). in the first group of patients, 35 (98%) of parents reported giving the antibiotics every day in comparison with 30 (88%) in the 2 nd group. twenty nine (81%) of urine tests were positive for antibacterial substances in the 1 st group but only 16 (47%) in the second group. in the 1 st group of patients the difference in recurrent infection between regular takers and non-takers was statistically highly significant. no significant difference was found in recurrent uti rate in takers and non-takers in the 2 nd group. failure to understand the reason for prophylaxis and forgetfulness was found to be a main reason for non-compliance. imaging studies evaluating the kidneys and urinary tract are performed routinely after a febrile uti. evidence of their value in changing management or affecting long term outcome is limited. as part of a multicentre, rct (iris 1) evaluating different antibiotic regimes in the treatment of first febrile urinary tract infections, we undertook as a secondary objective, an evaluation of the diagnostic protocol. the imaging modalities (ultrasound, dmsa scintigraphy within 10 days and voiding cystogram within 1-2 months) were assessed for their ability to predict long term parenchymal damage. 337 children of 2 years of age or less at the time of investigation and who had normal renal function and antenatal ultrasound, were considered suitable for analysis of the diagnostic course. us was performed in 336 children with 288 (86%) normal, minor changes were noted in the remainder, apart from 1 case requiring a change in management in the form of a pyeloplasty for pelvi-ureteric obstruction. the cystourethrogram was performed in 323 of the children with 65 (20%) demonstrating vur. the cystogram was a poor predictor of long term damage, being positive for reflux in only 17 of the 39 children who subsequently developed scarring. an acute dmsa scan performed in all children, exhibited findings consistent with acute pyelonephritis in 212 (63%). a repeat dmsa scan at 12 months in those with evidence of acute pyelonephritis demonstrated a scarring rate of 24%. the data did not demonstrate the clinical efficacy of routinely performing scintigraphy in the acute phase or a cystourethrogram. our recommendations for a reasonable diagnostic work up in small children with their first episode of uti are 1) performance of a scintigram 6 months following the acute infective episode, and 2) close surveillance to identify eventual recidivists. introduction: focal segmental glomerulosclerosis (fsgs) has a high recurrence rate after renal transplantation (rtx). recurrence can lead to tx loss. disease recurrence (dr) seems to be influenced by genetic background. methods: 77 patients with childhood onset of biopsy proven fsgs who were transplanted were evaluated. genetic investigations of the nphs2 gene were done in 46 patients (59.7%). results: mean age at diagnosis was 6.06 years. first renal transplantation was performed at age of 12.3 years.18 patients received a living related (lrd), 55 a deceased donor (dd) graft. 4 patients data under investigation. 31 p (40.3%) recurred after a mean time of 6, 3 years, 5 p with lrd (27.8%) , 25 p with a dd (45.5%). 23 patients (29.9%) lost their graft after 6, 8 years, 15 patients due to recurrence. a second rtx was performed in 16 patients, 11 with dd, 2 with lrd, 3 patients data under investigation, with 71% dr. a third transplantation was performed in 7 patients and a fourth in one patient. screening for nphs2 mutations revealed 10 patients with mutation in the nphs2 gene, 9 homozygot, 1 heterozygot. all patients with a homozygote nphs2 mutation did not recur and only the one patient with a heterozygous mutation (1/10) recurred compared to 17/36 without a mutation (p<0.05). conclusion: living related transplantation was advantageous to decreased donor transplantation. patients with nphs2 mutations had a lower risk of recurrence after transplantation compared to patients without mutations. patients with fsgs should be screened for nphs2 mutations. objective: prospective ebv surveillance post tansplantation reduces incidence of acute rejection and risks of post transplant lymphoproliferative disorders (ptld). methods: prospective screening of ebv by polymerase chain reaction (pcr) and serology in all patients transplanted between 2003 -2006 . results: 27 patients transplanted between may 2003 and september 2006 . 22 were cadaveric and 5 live-related transplants. recepient ebv serology status was known but not on donors. basiliximab was used for induction and maintenance comprised of tacrolimus/azathioprine (aza) until 2004 and mycophenolate (mmf)/tacrolimus from 2004. all received corticosteroids. 17 had mmf/tacrolimus, 10 aza/tacrolimus. ebv screened within first week post transplant, weekly for a month, monthly for 6 months. when ebv pcr lv >5.0, mmf/aza was withdrawn, tacrolimus levels kept between 6-8 ng/ml. pcr/ serology is checked 2 weekly for 3 months, monthly for 6, until lv falls <5.0 and vca igg becomes positive. the mmf/aza is reintroduced. patients with ebv pcr lv <5.0 are kept on full immunosuppression while viral load and vca igg monitored. 17 ebv viraemia detected following prospective screening.1 was re-activation. 9 were symptomatic. onset varied 1 to 21.7 months post transplant (mean 4.3 months). 10 were on mmf/tacrolimus 7 received aza/tacrolimus. 12 had pcr lv>5.0 consequently maintained on tacrolimus an alternate day steroids for period of time. cmv patient and donor status was known and antiviral prophylaxis given to nonimmune. the use of antivirals made no impact on viral load. conclusion: no patient experienced acute rejection or ptld despite modification of therapy with our prospective screening programme. antivirals have no role in protecting or reducing viral load in already infected patients. graft attrition rate is highest in the first three months after renal transplantation. we analysed data in a recent cohort from all dutch centres for pediatric renal transplantation to determine incidence and causes of such early graft failure. methods: data from all pediatric renal transplants performed between 01-01-98 and 01-01-06 were analysed retrospectively. a common immunosuppressive protocol was used in all centres. thrombosis prophylaxis was given according to centre policy. early failure was determined as graft failure within 3 months after transplantation. prevalence of possible risk factors of graft failure identified by literature search were extracted from patient charts. data were analysed with univariate and multivariate logistic regression. results: the cohort consisted of 228 transplants. age of the recipients was 3-18 yrs (mean 10.9). there were 19 early graft failures (8.2%). major causes of graft failure were thrombosis (5.3%) and are(1.8%). univariate analysis identified an association of early failure with complications during surgery (or 10.0, p=0.00), cold ischemia time (or 1.1, p=0.026), side of graft donation (or 2.8, p=0.046) and diuresis in the first hour after transplantation (or 0.3, p=0.026). multivariate regression analysis showed that complications during surgery were associated with the risk of early failure (or 6.4, p=0.002), as were side of graft donation (or 5.7, p=0.015) and diuresis in the first hour after transplantation (or 0.3, p=0.038) . only the occurrence of complications during surgery was significantly associated with early graft failure due to thrombosis (or 13.6, p=0.006). conclusion: thrombosis is the major cause of early graft failure after renal transplantation in dutch children, especially after surgical complications. prospective studies are needed to determine whether early failure can be decreased by more rigorous prophylaxis. background: interleukin (il)-1 is a major contributor to inflammation and cell death during ischemia-reperfusion (ir) injury and its deleterious effects are mediated mainly by nuclear factor-κb (nf-κb) activation. receptor binding and signalling of il-1 can be blocked by the il-1 receptor antagonist (il-1ra). the aim of our study was to characterize the effects of il-1ra administration on inflammation, apoptosis and infiltration in renal ir injury. methods: renal ischemia was induced in lewis rats (n=7/group) by clamping of the left renal artery for 45 min followed by reperfusion of 24 h or five days respectively, when kidney were removed for histological and molecular analysis. results: treatment with il-1ra ameliorated ischemic renal tissue injury and inflammatory infiltration. futhermore, the number of apoptotic tubular cells was lower in il-1ra treated animals 24 h after ischemia, which was paralleled by a bax/bcl-2 mrna ratio towards anti-apoptotic effect. il-1ra reduced the expression of monocyte chemoattractant protein-1 (mcp-1) mrna 24 h and 5 days and that of intracellular adhesion molecule-1 (icam-1) expression 24 h after reperfusion in the kidney. conclusions: our results indicate that il-1ra treatment ameliorates renal ir injury and this protective effect might be mediated by reduced induction of nf-κb mediated mcp-1, icam-1 and the decreased ratio between bax and bcl-2 mrna expression. k. satomura, y. santo osaka medical center for maternal and child health, pediatric nephrology and metabolism, izumi, japan many studies have reported that angiotensin-converting enzyme inhibitor (acei) and angiotensin receptor blocker (arb) show renoprotective effects in adult patients with acquired renal diseases. however, these effects have not been studied in children with renal hypoplasia or dysplasia (rhd). in this study, we explored the renoprotective effects of these drugs in children with rhd. patients and methods: participants of the study were patients with rhd aged less than 15 years. a follow-up for more than one year was conducted in our outpatient clinic. the patients had chronic renal failure, and had not received acei or arb in the past. the change of glomerular filtration rate (gfr) per year, urinary protein/creatinine (up/cr) ratio, serum potassium levels, and blood pressure before the treatment with trandolapril or candesartan were compared with those of one year after the treatment. the estimated gfr was calculated by the schwartz formula. wilcoxon rank sum test was applied to evaluate the statistical significance. results: eleven patients were eligible for this study. the mean age of the patients was 8.4±2.6 years and the estimated gfr was 57.3±9.0 ml/min/1.73 m 2 when trandolapril or candesartan was administered. the change in the estimated gfr for a year significantly improved after the treatment with trandolapril or candesartan compared with the pre-treatment period (-6.1±5.4 vs. 1.9±5.9 ml/min/1.73 m 2 /year, p=0.003). the up/cr ratio significantly decreased at one year after the treatment compared with that before the treatment (0.67±0.62 vs. 0.49±0.53, p=0.041). the blood pressure and serum potassium level showed no significant changes. conclusion: these data suggest that treatment with acel or arb has beneficial effects on the renal function in children with mild to moderate renal failure due to rhd. objective of the study: postischaemic arf is influenced by sex hormones. dehydroepiandrosterone (dhea) pretreatment diminishes postischemic injury. previously we demonstrated that after renal ischaemia-reperfusion (i-r) injury the expression and activity of na, k-atpase (nka) is impaired in male rats. here we tested the impact of dhea on postischaemic survival, renal damage, mrna and protein expression of nka. methods: left renal pedicles of dhea (4.0 mg/kg/day) and propylene glycol, as vehicle (pg) treated male rats (g dhea and g pg , respectively) were clamped for 55 min followed by 2 (t 2 ) and 24 (t 24 ) hours of reperfusion. survival rate, histological damage, serum creatinine (cn) and urea nitrogen (bun) were investigated. the mrna expression and protein level of nka α1 and β1 subunit were also determined. sham operated animals served as control. results: dhea treatment was associated with better postischemic survival (p<0.05 g dhea vs. g pg ). postischaemic cn and bun were higher and renal histology showed more rapid progression vs. controls, however there was no difference between g dhea and g pg groups. mrna expression of nka α1 subunit was higher in g dhea vs. g pg at every time points, however it was decreased in i-r groups vs. controls (p<0.05). similar changes were observed in nka α1 protein level (p<0.05, g dhea vs. g pg in controls, t 2 , t 24 ). mrna and protein expression of α1 subunit were different only at t 24 (p<0.05, g dhea vs. g pg ). conclusions: our study indicates that nka is more protected from the detrimental effects of i-r injury in dhea treated animals, which might be a contributing factor of improved postischaemic renal function and could help to preserve cell and organ homeostasis even in male animals. the work was supported by otka f048842-t37578, bolyai and semmelweis grants. objective of study: recently bnp has been identified as a useful cardiac marker for risk stratification in adults. whether bnp has a similar diagnostic potential in pediatric population with ckd has to be established. the aim of the study was to assess the value of bnp to predict the cardiac dysfunction in children with ckd. methods: to test this suggestion, the relationship between plasma concentrations of bnp, echocardiographic parameters and cardiovascular risk factors (lipid profile, hypertension, secondary hyperparathyreoidism) has been evaluated. a cohort group consisted of 46 children and young adults (35 pts mean age x=16.12±5.14 years; 11 controls mean age x=14.73±5.04years). out of 35 studied pts 24 children were with ckd stage 4 and 5 k/doqi (12 pts ckd stage 4; 12 dialyzed pts) and 11 transplanted subjects with stable graft function (gfr=1.17±0.7 ml/s/1.73 m 2 ). results: no association was found between bnp and gfr and/or s-creat (p=0.12; p=0.1; p=0.44). the highest bnp plasma levels have been found in dialyzed children (620.77 pg/ml; resp. 75.98 pg/ml in tx; resp. 51, 85 pg/ml in ckd stage 4 group) as compared to controls (35, 25 pg/ml p<0,001). bnp was independently related to left ventricular mass (r=0.84; p<0.001) as well as to ejection fraction (r=-0.6; p<0.001) and preload (r=0.78; p<0.001). significant correlation between bnp and hypertension (r=0.608; p<0.001) has been observed. more over, bnp correlated with ipth (r=0.37; p<0, 05) and diastolic dysfunction (r=0.61; p<0, 001). plasma bnp concentrations did not significantly differ before/after dialysis procedure (p=0.07). conclusions: bnp is a sensitive marker for cardiac dysfunction in ckd children. however, prospective studies in larger group of pts are needed to confirm our preliminary data. we have reviewed the data of 20 patients with nephropathic cystinosis (nc) followed in our department since 1987. overall, 2 patients have died during the follow-up period. the mean follow-up was 17,6±6,7 yrs (range 6, 8) . 13/20 patients have initiated dialysis and 10/20 received a cadaveric renal transplant. univariate analysis showed that the time to reach a serum creatinine value of 2 mg/dl was significantly associated with the patient's age at the last follow-up (o. r. 1, 3/yr; p<0.001), the age at the beginning of cysteamine (mea) treatment (o. r. 1, 3/yr; p=0.006), the dose of mea (o. r. 0, 99/g, p=0.035) and the use of ace-inhibitors (o. r. 0, 15; p=0.014). by multivariate analysis, only the period of treatment (or 1, 24, p=0.037) and therapy with ace-inhibitors (or 0, 16, p=0.041) were significantly associated with better outcome. similar results were obtained when assessing the time to dialysis. there was a significant correlation between the dose of mea with intra-leucocytic cystine levels (ilc). by univariate analysis, the dose of mea expressed in mg/m 2 correlated more with the outcome then the dose expressed in mg/kg. statural growth was significantly associated by multiple correlation analysis only with growth hormone (gh) treatment (p<0.05). altogether, these results indicate that the renal prognosis of nc has been improving over the past two decades. this improvement may not be solely attributable to the introduction of mea for the treatment of nc. our data suggest that the overall management of these patients has improved, including more efficient symptomatic treatment of the fanconi syndrome and the use of medications such as ace inhibitors and gh. background: the chronic kidney disease in children (ckid) cohort study, targeted to enroll 540 children aged 1-16 yrs with chronic kidney disease (ckd) by estimated gfr (egfr schwartz) of 30-90 ml/min/1.73 m 2 aims to assess risk factors for kidney disease progression in children. objective: to describe the characteristics of the ckid cohort at study entry. design/methods: at the ckid baseline visit, gfr is measured by plasma disappearance of iohexol. children undergo physical exam, standardized bp measurements, blood and urine testing and parent and child interview. results: as of 01/16/07, 335 children had completed the baseline visit, median age 11.1 yrs, 71% caucasian, 16% african american, 62% male and 15% hispanic ethnicity. median age-adjusted height and weight percentiles were 23% and 42%, respectively. underlying cause of ckd was urologic, cystic or hereditary in 68%, 21% had acquired glomerular disease. median egfr at study entry was 54.6 ml/min/1.73 m 2 , 40% higher than the median iohexol-based gfr 38.5 ml/min/1.73 m 2 . 48% of participants reported a history of hypertension, 35% anemia, 11% seizures, and 7% depression. symptoms of headache, nausea, loss of appetite and weakness steadily increased in prevalence with lower gfr. assessment of family history in parents and grandparents revealed: high blood pressure 81%, high cholesterol 66%, kidney disease 29%, dialysis 12%, and kidney transplant 6%. conclusions: children with stage iii ckd have significant height deficits, hypertension, anemia, and seizure disorders. cross-sectionally, increasing non-specific symptoms are associated with lower gfr. collection of plasma, serum and genetic material will facilitate understanding of novel risk factors and biologic mechanisms underlying kidney disease progression and associated morbidities. aim: increased glomerular filtration rate (gfr) has been implicated in the development of diabetic nephropathy. large normal interindividual variations of gfr hamper the diagnosis of renal hemodynamic alterations. we examined renal functional reserve (rfr) in children with insulindependent diabetes mellitus (iddm) to assess if hyperfiltration occurs. methods: the renal hemodynamic response following dopamine infusion was examined in 51 iddm children (7.7±3.6 y) with a mean duration of diabetes of 6.2 years and compared to 34 controls. results: mean baseline gfr in diabetic children did not differ from control population (130.7±22.96 vs. 124.8±25 ml/min/1.73 m 2 ), whereas renal plasma flow was significantly lower (463.7±103.9 vs. 587.2±105ml/min/1.73 m 2 , p<0.001) and filtration fraction was increased (29±8 vs. 21±2%, p<0.001), compared to controls. the mean rfr was lower (p<0.001) than in control subjects (-0, 77±23 vs. 21±8 ml/min/1.73 m 2 ). conclusion: the observation that rfr is reduced or absent suggests that all children are in a state of glomerular hyperfiltration with increased intraglomerular pressure regardless of the baseline normal values of gfr. while measurements of rfr may be helpful in diagnosing the presence of hemodynamic changes, the relevance to development of diabetic nephropathy remains unknown. only 30-40% develops diabetic nephropathy, suggesting that glomerular hyperfiltration is only a concomitant risk factor. lp diet induces ischemic renal injury involving epithelial cells from osom. here, we tested whether hsp70 would stabilize renal na+k+atpase attachment to the cytoeskeleton during recovery from lp. after weaning, rats (n=8), were fed for 14 days with a lp diet (8%), then were recovered by means of a normal protein diet (24%rp) each group had an age-matched control group (24%, np). tissues from cortex and osom were homogeneized in buffer plus 0.1% triton x-100. protein levels were measured by western blot. in vitro coincubation of solublen on cytoeskeletal and insoluble cytoskeletal-associated fractions in the presence or absence of anti-hsp70 antibody was performed. interaction between proteins was determined by coimmunoprecipitation. increased na+k+atpase dissociation was shown in soluble fraction from osom as a result of lp diet vs. np (196.5±1.1 vs. 151±1.3, p<0.05). meanwhile, decreased hsp70 levels in the same fraction was shown (lp: 179.3±10.5 vs. np 224.7±1.85, p<0.05). translocation of hsp70 to the cytoeskeletal injured fraction associated with stabilization of na+k+atpase was shown in osom from lp, after in vitro coincubation of the cytoskeletal fraction of lp and non cytoskeletal fraction of rp. these effects were abolished by the addition of anti-hsp70 antibody. coimmunoprecipitation showed that the amount of hsp70 coprecipitating with na+k+atpase increased in lp osom, in rp results were similar to control. in cortex, absence of significant differences was shown in the na+k+atpase and hsp70 expression at in vivo and in vitro experiments among groups. in lp and rp cortex tissues, interaction of both proteins was similar to control. our results allow us to suggest that hsp70 has a critical protective role in the integrity of the cytoskeletal anchorage of na+k+atpase during recovery from ischemic lp injury in osom. tubular reabsorption of mg +2 occurs predominantly by paracellular flux in the thick ascending limb of the loop of henle. it is mediated by the, tight junction, mg +2 channel protein, paracellin-1, which is encoded by the gene pcln-1. cyclosporin a (csa), a widely used immunomodulatory drug, decreases tubular mg +2 reabsorption leading to urinary mg +2 wasting and hypomagnesemia. the exact molecular mechanisms of this modulation are unknown. in this study we examined the effect of csa on the paracellin-1 gene in renal cell lines and mouse kidney. the effect of csa on the pcln-1 gene promoter was examined. a plasmid, containing the human pcln-1 (hpcln-1) promoter (2.5kb) upstream to a luciferase reporter gene, was transfected into opossum kidney (ok) and human embryonic kidney (hek293) cells prior to exposure to 5 micrograms/ml csa for 24 hours. mice received csa (15 mg/kg/day) for up to 5 days. at 24 h intervals blood/urine mg +2 and ca +2 levels were determined and kidneys harvested for paracellin-1 mrna quantitation (real-timepcr) as well as protein quantification (western blot) and visualization (immunofluorescence). csa decreased hpcln-1 promoter activity by 25%-50% in transfected ok and hek293 cells, compared to control cells. csa-treated animals displayed hypomagnesemia with increasing urine mg +2 and ca +2 levels paralleled by a 20-30% decrease in pcln-1 mrna after 3 and 5 days of csa exposure. a similar effect on paracellin-1 protein expression inthe renal tubule was evident in response to csa treatment. csa may influence paracellin-1-mediated mg +2 transport at the transcriptional level. involvement of the calcineurin-dependent tonebp pathway or the calcineurin-independent map kinase pathway in this response is subject to future research. this effect of csa on the paracellin-1 gene may play a role in the renal magnesium wasting and hypomagnesemia induced by csa. it is well established that proteinuria induces tubular injury, which is closely associated with progressive decline of renal function. megalin has an important physiological role in the reabsorption of a variety of low molecular weight proteins along the proximal tubule. in this study, we examined whether megalin mediates tubular injury secondary to glomerular diseases. we utilized megalin knockout (ko) mice, in which 60% of proximal tubular cells have mosaic nullmutation in the megalin gene. to induce glomerular injury, these were mated with a transgenic line, nep25, in which selective podocyte injury can be induced by injection of immunotoxin, lmb2. megalin ko/nep25 mice were injected with lmb2 (0.625 ng/g bw) and analyzed 2 weeks later. they showed moderate proteinuria (upro/cr=80, vs 12.5 before lmb2) and mild tubular injury, which were comparable to those observed in nep25 mice with intact megalin gene injected with the same dose of lmb2. in the latter, megalin was still detectable in injured tubules. within each megalin ko/nep25 mouse, we compared megalin-intact (+) vs deficient (-) proximal tubular cells by immunostaining. the majority of megalin+ cells showed enhanced staining for albumin. in contrast, most megalin-cells were not stained for albumin. aquaporin-1, which is highly expressed in normal proximal tubules, was diminished in 39.7% of megalin+ cells, whereas it was diminished in only 11.3% of megalin-cells. some proximal tubular cells expressed mcp-1. in that, 69% of mcp-1 expressing cells were megalin+. the results suggested that megalin plays an important role in the reabsorption of massively filtered proteins in glomerular diseases, thereby contributing to proximal tubular cell injury. objectives of study: the hallmark of nephropathic cystinosis is lysosomal cystine accumulation, primarily leading to fanconi syndrome. although all tissues haveelevated cystine levels, it is not known why the kidney is first affected. it is postulated that decreased atp production in cystinosis results in defective proximal tubular reabsorption, a process driven by na, k-atpase. to study this hypothesis, we have monitored atp levels and viability in conditionally immortalized proximal tubular cells (ptc) of cystinosis and healthy controls. methods: urinary sediment of cystinotic patients and healthy controls was suspended ins supplemented culture medium. primary cultures were transfected with sv40 ts a58 t antigen allowing proliferation at 33°c and maturation at 37°c. to confirm the proximal origin of the cells 1) expression of aquaporin-1 (aqp1) and dipeptidyl-peptidase iv (dpp-iv) was demonstrated using immunoblot technique and 2) morphology was determined using em. ptc were matured at 37°c for 0-10 days, followed by cystine measurement using hplc and atp determination using luciferase assay. results are expressed as nmol/mg protein. results: colonies of cystinotic and control cell lines (n=2) with cobblestone morphology developed after 2 weeks and expressed aqp1 and dpp-iv, confirming their proximal origin. in cystinotic ptc, cystine levels increased from 2.0 at 33°c to 7.5 after 10 days at 37°c compared to 0.3 in controls. intracellular atp decreased in cystinotic ptc during 10 days from 8.8to 1.6, while atp levels in controls remained stable (range 9.7-13.7). atp levels inversely correlated with cystine accumulation in cystinotic ptc (r=-0.95, p=0.005). conclusion: decreased atp levels in conditionally immortalized ptc during 10 days maturation suggest that alterations in atp levels are involved in tubular dysfunction in cystinotic patients. aquaporin ( 13 aqps have been identified to date. aqp1 conveys 50% of the peritoneal water transport in pd. cell migration and angiogenesis are altered in aqp1 ko mice; erythrocyte pco 2 depends on aqp1. human peritoneal mesothelial cells (hpmc) from non-uremic patients and a human mesothelial cell line (met-5a) were incubated with conventional (c-pdf), icodextrin (ico), bicarbonate (b-pdf) and lactate based double chamber pd solution (l-pdf). mrna was measured by real time rtpcr, and protein by western blot and immunocytochemistry. hpmc and met-5a express aqp1, 3, 9 and 11. incubation of met-5a and hpmc with c-pdf, ico and l-pdf did not change aqp1 and 3 expression compared to medium. in contrast, incubation with b-pdf increased aqp1 and 3 mrna and protein in both hpmc (24 h aqp1 mrna: 262±21, aqp3: 3226±634%) and met-5a (aqp1: 1860±1180, aqp3: 2025±1624%). the effect on aqp1 was in part explained by differences in ph, the effect on aqp3 was largely independent of ph. addition of 34 mmol/l of bicarbonate to l-pdf increased aqp1 and 3 mrna (l-pdf+bic: aqp1: 983±110, aqp3: 326±26%). aqp9 expression was suppressed by all three pd fluids (b-pdf: 25±2.4, l-pdf: 16±9.6, c-pdf 75%: 26±11%), aqp11 was up regulated by b-pdf (377±37%). b-pdf improved hpmc migration. we for the first time demonstrate the expression of four different aqps in peritoneal mesothelial cells. they are markedly regulated by pd-solutions. the upregulation of aqp1, 3, and 11 by bicarbonate based pdf may have important implications on long term peritoneal membrane function, wound healing and neoangiogenesis. the regulation of cx3cl1 (fractalkine) by thromboxane a2 in inflammation there is marked leukocyte infiltration into the damaged tissue. chemokines recruit and direct leukocytes to the injured site. the chemokine cx3cl1 is up-regulated in renal inflammation such as glomerulonephritis and allograft rejection. thromboxane a2 (txa2), a potent vasoconstrictor in the kidney, is also pro-fibrotic. txa2 is up-regulated early in inflammation and its effects are mediated by binding to the tp receptor. the aim of this study was to examine the effect of txa2 on cx3cl1 expression. we previously showed that cx3cl1 recycles between the cell surface and an internal compartment. cell surface cx3cl1 is cleaved by the enzyme, tace, to yield the soluble species of the chemokine. we generated human ecv-304 cells stably expressing cx3cl1 and treated cells with a tp agonist. levels of cx3cl1 were quantitated by western blotting. cx3cl1 decreased by 30-60 min after tp stimulation, but recovered by 4h. using flow cytometry, we found that cell surface levels of cx3cl1 decreased by 30 min, but began to recover before levels of total cx3cl1 were replenished. to verify if early recovery of cx3cl1 was due to redistribution from the internal to the plasma membrane compartment, we used fluorescence recovery after photobleaching (frap). after 60 min tp stimulation decreased endocytosis of cx3cl1 was seen. we postulated that loss of cell surface cx3cl1 is due to cleavage by tace. accordingly, a tace inhibitor prevented early loss of cx3cl1 after tp stimulation. in summary, txa2 induces rapid proteolytic shedding of cell surface cx3cl1 by tace, but later increases expression of cx3cl1 at the cell surface by inhibiting internalization of the chemokine. this could release the soluble chemotactic protein from the cell adhesive transmembrane protein, promoting leukocyte recruitment initially, whilst promoting leukocyte adhesion later in the time course. the response to steroid therapy is used to characterize idiopathic nephritic syndrome (ins) as steroid sensitive (ssns) or steroid resistant (srns). ssns pathogenesis has been associated with activation of t-cells and srns has been associated with activation of tgfb1 and progression to chronic kidney disease. our objective was to determine differences in the urinary excretion of inflammatory cytokines; opn, icam1 and tgfb1, between ssns (n=12), srns (n=12), and controls (ctr, n=12) in an attempt to identify specific biomarkers of steroid sensitivity or lack thereof. for this purpose, we used a protein array high-throughput technique and confirmed the findings by elisa. in addition, we performed immunohistochemistry (ihc) for the above cytokines on renal biopsy samples. mean age, gender, race, body mass index, and estimated glomerular filtration rate were not statistically different among three groups. there were no statistically significant differences between ssns and srns in regard to the presence of hypertension, ace treatment, and renal histology (p=0.99, 0.22, and 0.99, respectively) . urinary excretion of icam1, opn, and tgfb1 were statistically significantly higher in ins subjects vs. ctr. urinary icam1 and opn were higher in ssns than in srns (p=0.005 and p=0.0002, respectively). however, the urinary excretion for tgfb1 was similar in ssns and srns. the ihc failed to reveal differences in renal tissue expression of the studied cytokines. there was no correlation between urine and kidney tissue expression. in summary, our preliminary data suggest that urinary opn and icam1 may serve as biomarkers of ssns. this assumption needs to be tested prospectively. methods: this is an interim report of this single center study on proteinuria in hiv positive children. there are 250 children aged 0-18yrs. registered in our hiv clinic. all children attending the outpatient clinic or seen on admission are studied. questionnaires included bio and clinical data. blood pressure was measured in all children. a clean catch or bag urine is obtained from all and urine biochemistry done on an aliquot. patients found to have proteinuria 1+ or above are referred to the nephrology unit. their urine is quantitated (timed urine collection), renal function, renal ultrasound and cd4 count estimated. anf, le cells, fbc including platelet count and genotype will be assayed. a renal biopsy will be performed for nephrotic range proteinuria. result: eighty children positive for hiv have been studied. there were 41 males and 39 females giving a ratio of 1.1: 1. their mean and median ages were 3.28yrs and 3.25 years respectively. all had normal blood pressures, renal ultrasound, and renal function. fifteen of the 80 children (18.8%) had 30-100mg/dl proteinuria; 19 (23.8%) haematuria. their mean cd4 + cell count was 316.9/mm 3 , range 180-1267. there was no significant difference between low cd4 + count and the presence of proteinuria (p>0.3). seventy seven of the patients had vertical transmission of hiv; two acquired the infection from blood transfusion; one from scarification marks. conclusion: there is a high prevalence of proteinuria among children with hiv infection in our region. hiv positive children should be screened and intervention instituted to avert or delay the onset of chronic renal failure. carotid intima media thickness (cimt) and brachial artery flow mediated dilatation (fmd) are novel indices of subclinical atherosclerosis. we studied these indices in children with nephrotic syndrome (ns) using high resolution ultrasonography (hrus). 52 children with ns (42 ssns;10 srns) of 24-207 months duration and 50 normal sibling controls underwent cimt and fmd determination using a 7.5 mhz us probe. routine biochemistry, lipid profile and hscrp were carried out to look into associations with cimt and fmd. statistical analysis was carried out using epiinfo 6.0. cases and controls were similar in age, sex, growth and egfr. mean maximum cimt (+-95% ci) (in mm) was higher in the ns group (0.435 +-0.014; range 0.333-0.570) vis-a-vis controls (0.398+-0.091; range: 0.345-0.490) (p<0.01). srns and ssns cases were similar (0.441+-0.037 vs 0.434+-0.015 mm, p=0.36). nephrotic children showed significantly lower fmd (10.56+-4.16% vs 17.39+-3.94%, p=0.02). cimt and fmd varied with frequency of relapses in last 12 months (0.415+0.035mm, 10.27+-5.68% (no relapses); 0.449+-0.019 mm, 7.57+-5.39% (>3 relapses). children with ns had mean hscrp of 5.25+-0.57 mg/l. on univariate analysis, cimt correlated with disease profile at onset (r=0.33, p<0.01), fmd (r=-0.23, p=0.02), male sex (r=0.23, p=0.02), bmi (r=0.23, p=0.02), cyclosporine exposure (r=0.5, p<0.01), total cholesterol (tc) (r=0.34, p<0.01), ldl (r=0.33, p<0.01), vldl (r=0.29, p<0.05), triglycerides (r=0.33, p<0.05), atherogenic index (ai) (r=0.33, p<0.01) and creatinine (r=0.25, p=0.03). fmd correlated with systolic blood pressure, age, bmi, weight, total cholesterol, vldl and triglycerides (p<0.05). to conclude, hrus can detect early atherosclerosis using fmd and cimt as the indices in children with nephrotic syndrome with disease duration of 20 years and more. a ten-year old girl, who presented with biopsy proven acute severe changes of type 1 membranoproliferative nephritis (mpgn1) appears to have shown complete resolution of her disorder after nine months mycophenolate moffetil (mmf) (cellcept -roche) added to relatively rapidly reducing steroids. she presented aged nine years with incidental finding of significant proteinuria and upper tract haematuria when presenting with abdominal pain. blood pressure, chemical renal function and haematology were normal. c3 and total haemolytic complement were at the lower limit of normal. renal biopsy showed light microscopy (lm) changes of markedly enlarged glomeruli with significantly increased mesangial cellularity and matrix, together with 'double contours' of basement membrane. inmmunofluorescence (if) was strongly positive for igg and c3. electron microscopy (em) showed mesangial cell interposition into the basement membranes. she was treated with iv pulse methylprednisolone, followed by high dose oral prednisolone (pnl). after one month, mmf 500mg bd was added as pnl was tailed to alternate day therapy. pnl was reduced more quickly than usual due to difficulty with side effects and excellent clinical response. within three months of mmf, proteinuria and haematuria had disappeared. blood pressure and renal function have remained normal throughout treatment. after nine months, while on 0.5mg/kg alternate day pnl and 500mg bd mmf, repeat biopsy showed normal sized glomeruli on lm with no significant changes, no positive staining on if and normal em without mesangial interposition. this case suggests that mmf may be a key drug in management of mpgn1. g. stringini, e. malagnino, f. emma bambino gesu children's hospital and research institute, department of nephrology and urology, rome, italy serum creatinine values may vary considerably between normal subjects. these variations are in part secondary to differences in muscle mass, body composition and creatinine tubular secretion. in addition, low nephron number, which is influenced by genetic and intrauterine factors, is associated with increased incidence of arterial hypertension, late-onset proteinuria and chronic renal failure. despite compensatory glomerular hypertrophy, autopsy data indicates that kidneys with fewer glomeruli tend to be significantly smaller. on these bases, we have hypothesized that a significant part of the variance of normal serum creatinine levels is related directly to individual differences in renal size. to test this hypothesis, we have reviewed the data of 1746 renal ultrasound examinations performed at our institution between 1991 and 2006. patients were selected if they had conditions that should not influence renal size (low tract uti, enuresis, bladder instability, idiopathic hypercalciuria), if they had normal gfr and an ambulatory blood pressure measurement. analyses were performed after expressing renal length, serum creatinine levels and blood pressure values as standard deviation scores (sds) corrected for gender, age, height and weight, by multiple nonlinear regression analysis. a significant negative correlation was found between serum creatinine levels and renal length (p<0.001). when renal length measurements were grouped in quartiles, serum creatinine sds was on average 0.37 sds higher in the 1st quartile (small kidneys), in comparison with the 4th quartile (large kidneys). no correlation was found between kidney length and single measurements of ambulatory blood pressure. these data indicate that renal size is accountable for a significant part of the variance in serum creatinine levels that is observed in the normal pediatric population. henoch-schönlein purpura (hsp) is the commonest small vessel vasculitis of childhood. henoch-schönlein purpura nephritis (hspn) is the major determinant of long term prognosis in hsp. the objective of this randomised controlled trial was to determine the effect of early prednisolone treatment on the development and progression of nephropathy in children with hsp. methods: children under 18 years of age, with a diagnosis of hsp, presenting to secondary care centres in england and wales were randomly assigned to receive either placebo or prednisolone (2mg/kg/day (max 80mg) for 7 days, followed by 1mg/kg/day for 7days (max 40mg)). patients, parents, paediatricians and investigators were 'blinded' to assignment of treatment or placebo. the primary outcome measure was the determination of the presence or absence of proteinuria, defined as urine protein: creatinine ratio (up: uc) >20mg/mmol, 12 months after initial presentation in treated and untreated patients. results: 353 children with hsp were randomised. 181 patients were assigned to receive prednisolone (group a) and 172 patients were assigned to receive placebo (group b). 36 patients in group a and 27 patients in group b did not complete the study. there was no difference in the incidence of proteinuria at 12 months, in patients receiving prednisolone (19/145) compared with those receiving placebo (15/145) or=1.32, 95% ci 0.59-2.94, p=0.49. conclusions: this is largest prospective randomised placebo-controlled trial of the role steroids in hsp in the literature to date. this study provides compelling evidence of absence of a beneficial effect of early treatment with prednisolone in the development of nephropathy 12 months after disease onset in children with hsp. while quality of life (qol) is generally assumed to be poor on dialysis and to improve markedly after kidney transplantation, systematic assessments of qol in children have not been performed to date. to date, we have examined general and health-related qol in 35 children and adolescents with esrd aged 4-16 years treated by hemodialysis (hd; n=7), peritoneal dialysis (pd, n=8) or renal transplantation (tx, n=20), using a well-established instrument (kindl) comprising an ageadapted set of questionnaires (kindl). the obtained measures of overall and item-specific qol were transformed to standard deviation scores (sds). general qol was close to normal and did not differ significantly between hd (-0.37±1.58 sds), pd (-0.20±1.04) and tx (-0.12±0.95). psychological well-being was better in children on pd (0.54±1.04) than after tx (-0.53±1.30, p=0.05) and on hd (-0.98±1.73, p=0.06). physical wellbeing did not differ between treatment groups (hd: -0.39±1.19, pd: 0.10±1.21, tx: (-0.16±1.06). qol related to family life tended to be compromised in dialyzed (-0.87±1.58) vs. tx children (-0.11±0.88, p=0.07) . social interaction with friends was considered moderately impaired by all patient groups (hd: -0.89±1.92, pd: -1.15±1.26, tx -0.82±1.44). self-esteem was close to normal in all groups (hd: -0.3±0.85, pd: -0.27±1.35, tx 0.21±.0). in summary, our preliminary results suggest that subjective qol is remarkably good in children with esrd, with surprisingly small differences between treatment modalities. tx appears to be perceived beneficial with respect to the integrity of family life but provides poorer psychological qol than pd, possibly due to the constant concern with graft loss. background: gfr is determined in the ckid cohort study by plasma disappearance of iohexol (igfr). updated serum creatinine (scr)-based formulas (sgfr=k ht/scr) are needed for bedside clinical use. objective: derive formulas based on enzymatic scr to estimate gfr in the ckid study in children aged 1-16 yrs with sgfr of 30-90 ml/min/1.73 m 2 . methods: from 253 children, height (ht, meters), igfr, and scr & bun (mg/dl) by bayer advia 2400 were obtained. gfr was estimated from regression models: a(ht/scr) b (35/bun) c where a is gfr if ht=scr and bun=35; b and c are exponents. results: 60% (n=152) were male; median age=11, scr=1.4, bun=31 and igfr=38. formulas ranged from an updated schwartz formula (a=40.4, b=1, c=0; r 2 =0.66), to sex-specific (a=38.8, b=0.98, c=0 in girls, and a=40.5, b=0.79, c=0 in boys), which yielded r 2 =0.69, to the most complex (a=37.7, b=0.86, c=0.16 in girls, and a=39.7, b=0.69, c=0.16 in boys), which yielded r 2 =0.71, higher (p<0.001) than the r 2 of the updated schwartz formula. by random selection of 127 of 253 subjects in 100 independent trials, we assessed the predictive ability of each formula on the other 126 observations. sex-specific formulas produced unbiased results and increasing precision with increasing complexity. the updated schwartz underestimated igfr in boys by 3% (p<0.001) and overestimated igfr in girls by 4% (p<0.001). conclusions: scr, ht, sex, and bun together provided unbiased estimates of igfr and explained 71% of its variability in children with igfr of 14 to 76 ml/min/1.73 m 2 . extrapolation to ht/scr of 3 (normal body habitus and kidney function) and bun of 10 yielded predictions of 118 ml/min/1.73 m 2 for gfr in girls and 104 boys. including cystatin c and broadening the gfr range to include normal levels are data being collected by ckid to extend the formulas and provide wider clinical utility. f. hussain, m. mallik, a. watson nottingham university hospitals, children and young people's kidney unit, nottingham, united kingdom considerable variation exists between units in terms of techniques used for renal biopsy. the bapn agreed an audit using published standards and a questionnaire was sent to all 13 uk paediatric nephrology centres. 11 agreed to a prospective audit between 1/1/05 and31/12/05. the survey revealed information leaflets are sent pre-biopsy in 5 (45%) centres with only one using play preparation. 6 (55%) routinely perform biopsies as daycase procedures (dc); 6 (55%) use general anaesthetic (ga). realtime ultrasound is the favoured method in 8 (73%) of centres. biopsies are performed by nephrologists only in 4 (36%), nephrologists with radiologists in 5 (45%) and radiology alone in 2 (19%). of 531 biopsies (352 native), 164 (31%) were performed as a dc with 262 (49%) being done under ga. the mean age of patients was 11yrs (range 0.8±18.9yrs). the standard for the number of passes of native kidneys (<3 in 80%) was achieved in 86.4% with no significant difference between grade of operator or nephrology/radiology speciality. standard number of passes in transplanted kidneys (<2 in 80%) was achieved in 73.4%. adequate tissue was obtained for diagnosis in 97.5% (standard >95%). the significant complication rate (macroscopic haematuria and/or delay indischarge) was higher than the set standard of <5% at 7.3%. there was no significant difference in complication rates whether the biopsy was performed as a dc or inpatient procedure (p=0.73) or whether ga or sedation was used (p=0.8). conclusions: the survey highlights significant variation in practice with limited use of preparation materials and dc. the results may enable individual units to reflect upon their techniques and complication rates and has stimulated constructive debate about indications and training issues. objective: to characterize bp in children enrolled in the ckid study, an observational cohort study of children 1-16y old with schwartz estimated gfr of 30-90 ml/min/1.73 m 2 . design/methods: 3 bp's were obtained using an aneroid sphygmomanometer. gfr was measured by iohexol disappearance. bp was classified according to the 4th report on bp in children. hypertension (htn) was defined as bp-95th percentile (uncontrolled), or as self-reported htn plus current treatment with antihtn meds. pre-htn was defined as bp 90th-95th percentiles. results: 284 children (mean age 11±4y; 60% male) were studied. mean gfr was 41±14 ml/min/1.73 m 2 and mean ckd duration was 7±4y. for sbp, 139/280 (49.6%) subjects had htn of these, 59 subjects (21%) had uncontrolled htn. 14 additional subjects (5%) had pre-htn. of subjects with systolic htn, only 57% had measured sbp<90th percentile, and among the 73 subjects with measured sbp>90th percentile, only 32 (44%) were taking antihtn meds. for dbp, 136/280 subjects (48.6%) had htn of these, 53 subjects (19%) had uncontrolled htn. 30 subjects (11%) had pre-htn. of subjects with diastolic htn, just 52% had measured dbp <90th percentile, and among the 83 subjects with measured dbp>90th percentile, only 35 (42%) were taking antihtn meds. among children with gfr<50 ml/min/1.73 m 2 , the prevalence of systolic or diastolic htn appeared to increase with decreasing gfr. conclusions: a significant proportion of children enrolled in the ckid study suffer from elevated bp. nearly 40% of children with ckd had measured bp>90th percentile, and more than 50% of these children were not receiving antihypertensives, indicating that htn in pediatric ckd is frequently under-or even untreated. long-term follow-up of the ckid cohort should reveal the effect of elevated bp on ckd progression. background: we recently demonstrated elevated excretion of the putative urinary biomarkers tgf-β and et1 in a large cohort of children with ckd. tgf-β excretion was highest in kidney disorders associated with marked tubulointerstitial fibrosis, such as obstructive uropathy, nephronophthisis and pkd. aim: to investigate the course and predictive value of urinary tgf-β and et1 excretion in children with ckd undergoing ace inhibition (acei). methods: to date, 165 patients have been followed for 2 years and 91 patients for 3 years in a prospective, interventional trial investigating the nephroprotective efficacy of acei w/out intensified blood pressure control. results: average bp was reduced to the mid normal range. proteinuria initially decreased by 50%, but gradually reincreased to baseline levels within 3 years. urinary tgf-β excretion, which was initially elevated 3-fold above healthy controls, continuously decreased during treatment from 32.6±31.5 ng/g creatinine to 27.4±20.7, 20.6±44.0, and 18.4±20.5 after 1, 2, and 3 years, respectively (p<0.001). in contrast, et1 excretion increased by 100% within the first 2 years of follow-up. neither baseline nor the change in tgf-β and et1 excretion during treatment predicted the short-or long-term antihypertensive, antiproteinuric response to acei or ckd progression rate. conclusions: the marked reduction of urinary tgf-β excretion probably reflects the antifibrotic effect of acei but, at least over 3 yrs of observation, does not predict the early and late course of proteinuria and ckd progression. hence, urinary tgf-β appears to be a biomarker of tubulointerstitial disease activity rather than of global disease progression. the surprising increase of et1 excretion may reflect the induction of compensatory hemodynamic mechanisms during acei. cardiovascular complications are the most important cause of death in pediatric patients with endstage renal disease. therefore early diagnosis and treatment are very important. brain natriuretic peptide (bnp) is released in response to volume overload or conditions that cause ventricular stretch. the aim of the study was to investigate whether bnp can be used for early diagnosis of cardiac complications in pediatric patients with esrd. twenty-four patients on peritoneal dialysis (mean age 13.3±4.4 years), 21 patients on hemodialysis (mean age 15.0±3.6 years) and 39 sex and age matched healthy children (mean age 12.4±3.0 years) were included the study. plasma bnp levels were significantly higher in the patient group than those in the control group (590.4±765.8 vs 159.0±23.7 pg/ml, respectively, p<0.05), but there was no significant difference between hemodialysis and peritoneal dialysis patients. in patients with hypertension, bnp levels, left ventricular systolic and diastolic diameters were significantly higher than those in the patients without hypertension. in patients with higher crp levels, bnp levels were significantly higher than those in the patients with low crp levels. bnp levels had a positive correlation with left ventricular mass index (r=0.32, p=0.04) and a negative correlation with ejection fraction (r=-0.59, p<0.001) and shortening fraction (r=-0.55, p<0.001) significantly. there was no significant relation between bnp levels and anemia, dialysis duration, and dialysis modality. in conclusion, high plasma bnp level is significantly correlated with dilated left ventricle and it may be useful as a biochemical marker for identification of pediatric dialysis patients with cardiac dysfunction. we observed a high prevalence of left ventricular hypertrophy (lvh) and impaired lv contractility in children with stage ii-iii chronic kidney disease (ckd) (jasn 2006 , jasn 2007 . in a prospective, open-label assessment in 84 children receiving ace inhibition (ramipril 6 mg/m 2 /d) with or without additional antihypertensive medication, we evaluated by echocardiography lv mass (lvm), geometry and myocardial mechanics at baseline and after 12 (n=65) or 24 mos (n=55) of treatment. lvh was defined by lvm index>38 g/m 2 and concentric geometry by relative wall thickness>0.375 (95 th normal percentile). lv systolic function was assessed at the midwall level by circumferential shortening (ms). normalized 24 h mean arterial blood pressure (bp) was reduced from 1.3±1.4 at baseline to 0.0±1.4 and -0.5±1.0 sds after 12 and 24 mos respectively. lvmi was reduced significantly after 12 (from 34.0±8.4 to 31.6±8.0 g/m 2.7 , p<0.02) and 24 mos (from 34.4±7.6 to 31.9±9.7, p<0.05). of those patients presenting with lvh at baseline, lvm regressed to the normal range in 10/19 (53%) after 12 mos and in 10/18 (55%) after 24 mos. the prevalence of concentric lv geometry remained unchanged (baseline: 8%, 12mos: 9%, 24mos: 7%). age and afterload-corrected myocardial function increased from 93±15% to 100±13% at 24mos (p<0.005). the changes in lvm and myocardial performance were independent of the randomized bp target and gfr. change in lvm was correlated with change in hemoglobin level (r=0.30, p<0.05) and change in myocardial function with change in bp level (r=0.39, p<0.05). in conclusion, fixed-dose ace inhibition and tight bp control induce regression of established lvh in the majority of children with stage ii-iii ckd. this is associated with a normalization of myocardial contractility. objectives of the study: periods with insufficient erythropoietic activity may occur during the erythropoietin (epo) treatment in chronic haemodialysis (hd). we determined the effects of a short-term suspension of epo therapy on various oxidative stress parameters during a 12-week follow-up in hd patients. methods: the antecedent epoetin beta (eb) treatment was suspended for 10 days. after that, 9 patients received eb two times a week and 7 patients received darbepoetin alfa (da) once weekly. concentrations of whole blood oxidized and reduced glutathione (gssg, gsh) and various haematological parameters were determined weekly. erythrocyte malondialdehyde (e-mda) and the activities of erythrocyte superoxide dismutase, catalase and glutathione peroxidase were determined at weeks 0, 4 and 12. results: the ratio gssg/gsh was increased in both groups after continuation of the suspended epo therapy (p<0.05 and p<0.01 week 1 vs. the baseline in the da and eb group, respectively) and also at week 9 (p<0.01 and p<0.001 vs. the baseline in the da and eb patients, respectively). the activities of the antioxidant enzymes were increased at week 4 in both groups (p<0.05 vs. the baseline for da and p<0.001 vs. the baseline for eb), and returned to their week 0 levels by week 12. the e-mda level decreased in both groups (p<0.05 week 12 vs. the baseline for da and p<0.05 weeks 4 and 12 vs. the baseline for eb). conclusions: a short-term suspension of epo therapy caused characteristic time-dependent changes in the oxidative stress. the ratio gssg/gsh increased at weeks 1 and 9. activities of the antioxidant enzymes were elevated at week 4, resulting in an improvement in lipid peroxidation. these results might have implications in certain conditions with transient alterations in the erythropoietic activity in hd patients. rrf has been associated with better nutritional status both in adults and children on peritoneal dialysis and in adults on chronic hd. there are no data on the influence of rrf on nutrition in children on chronic hd.179 three-days dietary reports and simultaneous urea kinetic monitoring of 30 children, adolescents and young adults on chronic hd (age: 4.3-24.5 years) were retrospectively analyzed. protein catabolic rate, an index of nutrition adequacy, was normalized by body weight (npcr). in patients with rrf, total kt/v (kt/vtot) was calculated summing hd kt/v(kt/vhd) and rrf (evaluated by residual urea clearance ku-). in all patients, npcr and dietary protein intake (ndpi) were significantly correlated (p<0.0001). kt/vtot was correlated with npcr(p<0.0001) while correlation between kt/vhd and npcr was not significant (p: 0.11). in patients with rrf, ku resulted significantly associated with npcr (p<0.0001) while kt/vhd was not (p: 0.10). npcr was significantly higher in patients with rrf (1.46±0.41 vs 1.03±0.33 g/kg/day; p<0.0001). patients on recombinant growth hormone (rhgh) treatment showed npcr higher than those without rhgh (1.34±0.41 vs 1.01±0.39 g/kg/day; p<0.0001). however, in a multiple regression model including age, the use of rhgh, rrf, kt/vtot and kt/vhd, npcr resulted significantly associated only with rrf (b: 0.128; p<0.0001). inconclusion: in children, adolescents and young adults on chronic hd treatment, rrf is associated with better nutrition. rrf positively affects nutrition independently from hd efficiency and rhgh effects. possible hypothesis are a more selective (although decreased) depuration and the positive influence of water excretion maintenance on food intake. more efforts have to be made in order to maintain rrf in children on chronic hd. during the past three years, six women have undergone chronic haemodialysis during pregnancy at the pediatric renal unit of the adelaide women's and children's hospital. five have delivered normal infants at between 33 and 38 weeks gestation; one is presently at 28 weeks gestation, with an apparently normal fetus. four were already receiving chronic haemodialysis at the time of conception; the others began dialysis at 20 weeks gestation. the protocol includes six days per week dialysis for at least two hours per treatment. a number of practical and emotional issues have arisen, with major potential psychosocial hazards, including: unplanned pregnancy due to ignorance of fertility; precipitation onto the dialysis program; acceptance of increased dialysis time; concerns regarding effects of drugs and dialysis/kidney failure on the fetus; the likelihood of prematurity; the perceived difficulties of motherhood while on a chronic dialysis program; loss of income, social networks and independence. the program has been successful due to the cooperative approach from the multidisciplinary team consisting of nephrologists, obstetricians, obstetric physicians dialysis nurses, midwives, dieticians, physiotherapists, psychiatrists and social workers. the social worker has provided counselling and coordinated transport and assistance with housework, childminding and other day to day tasks. although the program has had overall success, there have been a number of pitfalls worth discussing, including those arising from the complex interactions between the members of the various disciplines, and those involved in maintaining the balance of clinical and psychosocial needs of the women. (median 17.5) , weight from 4.5 to 15.4 kgbw (median 9). the vascular access was a central catheter (kt) in 12 children, an arteriovenous fistula (avf) in 6, avf and kt alternatively in 14 patients. duration of hd ranged from 1 to 63 m (median 14.5).35 dual lumen tunnelled cuffed kt were inserted in 26 children through the internal jugular vein [ijv]), surgically in 8, percutaneously in 27. all kt have had an immediate good function. nine kt were exchanged over a guide wire for dysfunction. kt infections with positive blood culture were successfully treated in 8 cases. duration of kt ranged from 1 to 20 m (median 5). at the end of the follow-up, 7 kt were still in function, 6 removed for a mature avf, 11 after renal transplantation (rt), 1 for improved gfr and 1 failed. patency of the venous network after withdrawal of kt was assessed in 16 children (doppler 3, mri 13) and showed normal patency in 6, ijv thrombosis in 5, brachiocephalic thrombosis in 3 and stenosis in 2. thirty avf were created in 20 children, distal in 18 (60%). immediate patency was obtained in all cases except 1. the median blood flow ranged from 350 to 1800 ml/min/m 2 (median 750). following the primary surgery, 41 repeated surgical procedures included a superficialization of the vein in 10, refection for stenosis or thrombosis in 9, reduction of overflow in 3, 2 nd avf creation in 10 and ligation after rt in 9. percutaneous transluminal angioplasty was performed in 5 children. duration of patency ranged from 1 to 168 m (median 36.5). in conclusion, hd is feasible in small children. nevertheless, kt are associated with risk of venous occlusion and obtention of a reliable avf frequently need several interventions, altogether leading to a limitation of hd indications in young children. 1994-2005, 60 (19%) were <2 years of age. the underlying disorders were congenital nephropathies in 70% (malformations 33%, hereditary 37%) and acquired diseases in 30%. living related donation (ld) was performed in 59% and preemptive tx in 33%. immunosuppressive (is) protocol varied considerably between the countries and over time.1-year graft survival (gs) was 96% in ld and 87% in grafts from deceased donors (dd). gs improved significantly for dd grafts with time. the number of acute rejections (ar) during the first year posttx was significantly lower in ld recipients, in tac-treated children and during the second half of the study period. this improvement over time was also seen in separate analysis of cya-treated children. the proportion of rejection-free patients increased in all countries. median height sds at tx was -1.8 (-8.3 to +1.7)(boys -1.9, girls -1.6). height sds increased to -1.5 at 3 years posttx. conclusions: gs results were excellent and the frequency of ar low, especially in children with ld grafts and tac treatment. interesting differences between the countries concerning donor source, preemptive tx, is and use of protocol biopsies were found. n. marcun varda, a. gregoric maribor teaching hospital, department of pediatrics, maribor, slovenia objectives: essential hypertension (eh), identifiable in children, is associated with cardiovascular (cv) diseases in adulthood. the aim of our study was to evaluate the presence of some traditional and non-traditional cv risk factors in our children and young adults with eh in the search for additional cv risk. the prevalence of metabolic syndrome (ms) was also investigated. methods: a total of 104 children and young adults, diagnosed with eh, were included in our study. they were compared with a control group of 50 healthy children, matched for sex and age, with regards to specific aspects in the history, body mass index (bmi), waist: hip ratio, full blood count, crp, serum cholesterol, hdl cholesterol, ldl cholesterol, triglycerides, uric acid, glucose, insulin, fibrinogen, homocysteine, apolipoprotein a1, apolipoprotein b and lipoprotein (a). in addition, the prevalence of ms was calculated. ms was defined as having three or more ms components according to the national cholesterol education program's adult treatment panel iii criteria, tailored for children. results: the differences in values of bmi, crp, platelets, triglycerides, uric acid, apolipoprotein a1, apolipoprotein b and homocysteine between the hypertensive patients and the controls were statistically significant. in all hypertensives positive family history of hypertension in the first or second generation was revealed. overweight (bmi >90 th percentile for age and sex) was identified in 40%, obesity (bmi >97 th percentile) in 24%, abnormal glucose homeostasis in 23%, high serum triglycerides in 42% and low hdl in 48% of the hypertensives. ms was present in 48% of these children and in 6% of our controls. conclusion: we demonstrated that children and young adults with eh differ from the population of healthy children in some specific cv risk factors, and are therefore at an increased cv risk. background: rtd is a rare autosomal recessive disease of differentiation of fetal kidneys with poorly developed proximal tubules. fetal and neonatal findings include oligohydramnios, preterm birth and neonatal death due to pulmonary hypoplasia, anuria and hypotension. mutations in genes in the renin-angiotensin system (ras), encoding for angiotensinogen, renin, angiotensin converting enzyme and angiotensin ii receptor type 1, have been revealed. we report the first rtd patients surviving the neonatal period and still being alive. both patients had affected siblings demised in utero or neonatally. case 1: oligohydramnios, birth at 38 weeks, 2850 grams. neonatal course: pulmonary hypoplasia, pneumothorax, hypotension and anuria with ventilation (4 weeks) and dialysis (2 days) . current findings at 15 years: creatinine 200 umol/l, normal blood pressure. genetic and functional analysis: homozygous mutation of angiotensinogen gene (1124g-a); very low angiotensinogen concentration (21.4 ng/ml; normal 1024-275), absent plasma renin activity (normal 1.5-3.0 ng/ml/h) despite very high active renin (199 pg/ml; normal 22-11). case 2: oligohydramnios, birth at 35 weeks, 2520 grams. neonatal course: mild respiratory distress, hypotension and anuria with oxygen (1 day) and dialysis (5 months) . current findings at 10 years: renal transplantation at age 4 years, good graft function. genetic and functional (as neonate) analysis: homozygous mutation of renin gene (1124g-a); very low active renin (<2.5 pg/ml; normal 24-850). conclusions: rtd is caused by inactivating mutations in genes encoding the ras resulting in chronic low perfusion pressure of the fetal kidney. genetic and functional analysis of ras contributes to diagnosis of rtd. this observation extends the rtd phenotype from a uniformly fatal to a more favourable disease. objectives of study: to evaluate the relationship between serum uric acid (ua), new onset essential hypertension (eh) and endothelial dysfunction (ed) in the youth. methods: 29 subjects with abpm proved new onset eh (aged 19, 9±3, 5 years, bmi 28, 9±3, 5 kg/m 2 ), 36 overweight/obese normotensive youth (oo) matched for age and bmi, and 73 age matched healthy normotensive controls (nt) with normal weight were enrolled. ua, total cholesterol, hdl, ldl, triglycerides and creatinine were analyzed in blood, glomerular filtration rate (gfr) was calculated according to schwartz formula and endothelial function was assessed using flow-mediated dilation (fmd, %). results: new onset eh was associated with overweight/obesity in 90% of subjects. serum ua levels were significantly higher in eh than oo (381±92 vs.336±72 umol/l, respectively, p<0, 05), in eh than nt (381±92 vs.277±62 umol/l, respectively, p<0, 0001), and in oo than nt (336±72 vs.277±62 umol/l, respectively, p<0, 0001). total cholesterol, hdl, ldl and triglycerides were significantly higher in eh and oo compared with nt (p<0, 0001). no significant differences were found in lipidograms between eh and oo. serum creatinine and gfr did not differ significantly between the groups. fmd was significantly lower in eh comparing with oo and nt (5,49±4,83 vs.12,75±3,47 vs. 14,55±5,32%, respectively, p<0, 0001) and in oo comparing with nt (12,75±3,47 vs.14, 55±5,32%, respectively, p<0, 05). conclusions: new onset eh in the youth is associated with overweight/obesity, higher serum ua and ed. ua may play a causal role in the pathogenesis of eh and commonly with eh and proatherogenic serum profile contributes to ed in overweight/obese hypertensive youth. objective of study: fibromuscular dysplasia (fmd) is a systemic arteriopathy of the small and medium sized vessels. it is the first cause of renal arterial stenosis (ras) in childhood. the aim of this study was to describe the natural history of fmd in children. we analysed all the data of 12 children with fmd. results: mean age at diagnosis was 4 years 9 months old. hbp was discovered fortuitously in 5 cases, after symptoms of malignant hypertension in 7. in all cases bp values corresponded to severe hypertension (mean bp of 190/105 mmhg). extra renal localizations were found in half of the patient, and the most frequent pathological arteries were the superior mesenteric and the carotida. the mean number of arterial stenosis at diagnosis was 2 per patient. seven patients had a primary fmd with familial history for 1 patient. in the others fmd were associated with polymalformatif syndromes: reported by grange (2), moya-moya disease (2), neurofibromatosis type i (1). after a median follow-up time of 81 months the number and severity of stenosis increase at 6 arteries per patient. these lession, although to a lesser extend, were already observed as soon as 30 months of follow-up. percutaneous transluminal renal artery angioplasty were proposed in 6 cases when the bp was uncontrolled indeed a multi antihypertensive therapy with severe renal stenosis (>80%) or when renal growth was impaired. prognosis is severe with cardiologic complications (10/12) and 1 death. conclusions: intimal form of fmd accounts for the majority of multivisceral fmd. at diagnosis several hypertension is almost present. vascular ultrasonography imaging techniques is usefull in follow-up. a conservative treatment has to be privilegied. this disease is evolutive with increase of pathological arteries number, aggravation of stenosis degree and sometimes renal function impairment. objective: the effect of over weight on blood pressure elevation (bp) is more frequently present in childhood. several studies have demonstrated the efficacy of angiotensin-converting enzyme inhibitors (acei) therapy in obese hypertensive adults, but data on children are very limited. methods: 110 obese (bmi: z score >2.5 sd) primary hypertensive (systolic ordiastolic blood pressure >95th) children (aged 6-7 years) were enrolled to this single centre prospective study. patients received ramipril (0.1-0.15 mg/bwkg/day) once daily. office and ambulatory bloodpressure measurements and serum biochemical analysis were performed at start and after 1 and 6 months of treatment. 36 patients (23,7%) hadimpaired glucose tolerance (igt) on oral glucose tolerance test (ogtt). results: 94 (85,5%) patients completed the six months ramipril therapy. reduction in 24-hour mean arterial pressure (map) was 4, 95 mmhg (-1, 02sd) after 1 month and 10, 14 mmhg (-2,05 sd) 6 months treatment respectively. bp was reduced with equal efficacy during day-and night time. 3/110 (2, 7%) patients were lost during the follow-up. 15 (15,9%) patients with high uric acid levels also were treated with allopurinol. eleven patients (10%) received second antihypertensive medication because of the blood pressure remained uncontrolled. (7 pts metoprolol, 4 pts amlodipine) 8 (7,2%) patients suffered recurrent cough, but otherside effect has not observed. the serum glucose and insulin levels havenot changed significantly during the follow-up. conclusion: oncea day given ramipril significantly decreases the blood pressure inobese hypertensive children. it is effective, tolerable and safe bloodpressure lowering monotherapy in childhood. further studies and longer follow-up are necessary to prove its long term beneficial effect in the childhood metabolic syndrome combined with hypertension. hw. zhang, j. ding, f. wang, yf. wang peking university first hospital, department of pediatric nephrology, beijing, people's republic of china objectives of study: females with x-linked alport syndrome (xlas) have variable phenotypes, from microscopic hematuria to chronic renal failure, which can not be clarified solely by mutation features of col4a5 gene. x-inactivation has been suspected to be one of the responsible reasonsfor this phenomenon, but no definite correlation has been demonstratedso far. in order to confirm whether the phenotypes of xlas femalescorrelate with x-inactivation, we analyzed the xinactivation patternsin peripheral blood cells in 36 xlas females and in skin fibroblasts in12 xlas females. methods: the x-inactivation analysis was performed using hpaiipredigestion of dna followed by polymerase chain reaction (pcr) of thehighly polymorphic cag repeat of the androgen receptor (ar) gene. results: results showed that the average x-inactivation levels of the mutant allele decreased while the degree of proteinuria increased, there was anegative correlation between the degree of proteinuria and thex-inactivation ratios of the mutant allele in blood cells (r=-0.474, p=0.006). however, there was no correlation between the degree of proteinuria and the x-inactivation ratios of the mutant allele in skin fibroblasts (r=-0.131, p=0.701). though 7 of 12 patients (58.33%) had the similar x-inactivation ratios in both blood cells and skin fibroblasts, there was also no correlation between the x-inactivation ratios of the mutant allele in skin fibroblasts and that in peripheral blood cells (r=0.180, p=0.575). conclusion: we concluded that the x-inactivation ratios in blood cells correlated with the degree of proteinuria, which might explain partially the diverse phenotypes in female xlas patients. more studies, including post-transcription regulation, environmental factors, and so on, are still needed. objective: to report frasier syndrome (fs) with wt1 mutation and abnormal expression of podocyte molecules which is the first case in mainland china. methods: peripheral blood cells were analyzed for chromosome karyotype and wt1 gene mutation. the ratio of +kts/-kts isoforms was quantified with genescan and genescan software. expressions of podocyte molecules were detected by immunohistochemical staining. result: the patient presented with steroid-resistant fsgs and male pseudohermaphroditism. the wt1 ivs 9 +5 g>a mutation was found in one allele in the proband, but not in her parents. the ratio of +kts/-kts was 0.67 in the proband, and was 1.35 and 1.42 in her mother and father, respectively. podocyte molecules expression altered in normal-and abnormal-appearing glomeruli. wt1 expression showed diffuse nuclear staining with less obvious speckles compared with that in controls. wt1 (antibody against c-terminal) displayed strong, normal, faint and negative stained podocyte nuclei within the same glomerulus. the staining intensity of wt1 (antibody against the n-terminal) was very faint. conclusion: taking clinical data, pathology, karyotype analysis and genetic testing together, we diagnosed the first case of fs in mainland china, which prompts there might be more cases underdiagnosed. wt1 displayed diffuse nuclear staining with less visible speckles compared to controls, supporting the view of the differential nuclear localization of kts isoforms. our study further confirmed that wt1 mutation resulted in abnormal expression of podocyte molecules in glomeruli of fs, though we did not know whether this phenomenon directly or indirectly resulted from loss of wt1 regulation. dent disease is an x-linked disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, calcium nephrolithiasis and the development of renal insufficiency after the middle age. so far, two genes responsible for the development of dent disease have been identified, i. e., clcn5 and ocrl1. ocrl1 was originally identified as a causative gene for lowe syndrome. in japan, igarashi et al. described for the first time that idiopathic tubular proteinuria in japan is identical to dent disease by mutational analyses. most of japanese patients with dent disease are identified by annual urinary screening as chanced proteinuria and several clinical phenotypes, such as incidence of nephrocalcinosis and long term outcome of renal function remains to be elucidated. furthermore, identification of ocrl1 as a second causative gene for dent disease has made the understanding of dent disease more complex. here we report 90 patients with dent disease phenotype with the results of genetic analyses and clinical features. out of 90 patients in 90 different families, 58 mutations (64%) in clcn5 were identified. in the 32 patients with no clcn5 mutations, genetic analysis in ocrl1 is ongoing, and at the moment 4 different mutations in ocrl1 ((i127stop, r301c, r476w and r318h) were identified. the remaining patients are now beeing investigated. among the patients in whom clcn5 mutations were detected, hypercalciuria are not always present. in several elderly patients, mild renal function impairment is present. there are similar clinical phenotypes between patients with clcn5 or ocrl1 mutations, but serum levels of ldh and ck are likely to be higher in those with ocrl1 mutations. in summary, we will present genotype and phenotype heterogeneity in japanese dent disease, and will discuss the clinical spectrum of dent disease. a. gulati 1 , s. sethi 1 , j. lunardi 2 , m. kabra 1 , n. gupta 1 , p. hari 1 , a. bagga 1 1 all india institute of medical sciences, 1st department of pediatrics, new delhi, india 2 hôpital de la tronche, genetics, grenoble cedex. france, france objectives: to study the clinical features and genetic basis of patients with lowe syndrome and identify female carriers. methods. case records of 6 patients with lowe syndrome presenting to this hospital, between 3-7 yr old, were reviewed. the clinical features were recorded and glomerular and tubular functions assessed. a detailed genetic analysis was performed on dna extracted from peripheral blood of patients and their mothers, which involved sequencing of all 23 exons of the ocrl1 gene. results: all patients showed failure to thrive, bilateral congenital cataracts, refractory rickets, delayed motor and language milestones and proximal renal tubular acidosis with fanconi syndrome. the mean schwartz gfr was 90 ml/min/1.73 sq. m. genetic analysis showed distinct mutations of ocrl1 gene in all patients studied. we report 4 new mutations having identified the variants c.779 a>g and c.853 g>t in exon 10, the variant c.1183_1184 ins t in exon 12 and c.2351-2 a>g in intron 22 in 4 independent patients. we also found 2 previously described mutations involving the region c.2309_2310 del tg and c.2377 c>t in exon 21. four of 6 mothers were heterozygous carriers. all genotypically proven carriers showed characteristic lenticular opacities. conclusion: the identification of mutations in the ocrl1 gene provides confirmation of the diagnosis of lowe syndrome. the new mutations described in north indian children expand the range of mutations that give rise to this condition. these observations have important implications for molecular diagnosis and genetic counseling in families with lowe syndrome. juvenile nephronophthisis (nph), an autosomal recessive nephropathy, is the most common genetic cause of chronic renal failure in childhood. in 10-15% of known cases, nph is associated with joubert syndrome (js), a neurological disorder described in patients with cerebellar ataxia, mental retardation, hypotonia and respiratory dysregulation. mutations in three genes, ahi1, nphp1 and nphp6 have been identified in patients with nph and js. as nphp1 mutations usually cause isolated nephronophthisis, the factors which predispose to the development of neurological symptoms in some patients are poorly understood. to determine such genetic factors and to assess the mutation rate of ahi1, nphp1 and nphp6 in nph and js, a cohort of 30 families with nph and at least one js-related neurological symptom was screened for mutations in these genes. thirteen (43%) and 8 (27%) unrelated patients were homozygous or compound heterozygous for nphp1 and nphp6 mutations, respectively. in 4 patients (13%) without nphp1, nphp6 or ahi mutations, mutations in a novel gene (nphp8/cors7) encoding a ciliary protein have been identified. interestingly, 7 of the 13 patients with nphp1 mutations carried either a heterozygous truncating mutation in nphp6 (1 patient), a heterozygous missense mutation in ahi1 (1) or the ahi1 variant r830w (5) . the variant r830w affects an amino acid conserved in vertebrates and predicted to be 'possibly damaging' by polyphen software. in conclusion, nphp1, nphp6 or nphp8 mutations can be found in 83% of patients with nph and js in our cohort. our finding that half of all patients with nphp1 mutations carry a mutation or a damaging variant in nphp6 or in ahi1 strongly supports the notion that epistatic effects provided by these genes contribute to the appearance of neurological symptoms in patients with nphp1 mutations. molecular cytogenetic techniques such as array-based cgh have been instrumental in the identification of microimbalances associated with syndromic phenotypes. we investigated 10 patients with unclear syndromic nephropathies (e. g. urinary tract malformations, focal segmental glomerulosclerosis, and persistent hematuria/proteinuria) and additional clinical features, such as mental retardation, heart defects or growth abnormalities. array-cgh analysis was performed with a whole-genome array with 8000 large insert clones providing an average resolution of <0.5mb. results: in one 16-year old female patient presenting with microhematuria, proteinuria, mental retardation including severe speech impairment, senso-neuronal hearing loss, and recalcitrant focal epilepsy, we detected a microdeletion in chromosomal bands xq22.3-q23. this deletion was verified by fish, found to be uniallelic, 2.2-3.7mb in size, and not to be inherited from the mother. electron microscopy of kidney biopsy showed splitting of the lamina densa and a thin basal membrane, which is diagnostic for alport syndrome. high-resolution cranial magnetic resonance imaging including white fibre tracking revealed a severe neuronal migration disorder with subcortical band heterotopia (double cortex syndrome), i. e. a second band of cortical neurons within the white matter below the true cortex. conclusions: in 10 patients with unclear syndromic nephropathies, we identified a female with a contiguous gene syndrome at xq22.3-q23. the microdeletion includes the x-linked alport syndrome gene col4a5 and the lisx gene associated with subcortical heterotopia, mental retardation and epilepsy. thus, the phenotype observed in our female patient combines features of the amme-complex (alport syndrome, mental retardation, midface hypoplasia, elliptocytosis) with x-linked lissencephaly. (29 f, 29 m; 17.80±6 .50 yrs, hemoglobin 9.67±1.08 gr/dl, ferritin 2827±1895 mg/dl) were enrolled. lipid profile, acute phase proteins (apps) were measured. renal tubular functions, plasma vegf level, urinary nag/cre ratio were determined. abpm and imt measurements were performed. the results were compared with healthy controls. results: mean 24-hour, day and night systolic-blood pressure (sbp), diastolic-bp (dbp) and mean arterial pressure values were comparable to that of control group. dipping in dbp was less in tm (19.03% vs 23.73%; p<0.05). the ratio of patients with less than 10% dipping in sbp (non-dippers) was higher in tm (51.9% vs 35.0%, p<0.05). mean plasma vegf level was 30.16±22.95 pg/ml [2.69-112.25] in tm, was within normal range (<10 pg/ml) in controls. apps were normal. imt of common carotid artery (cca) was 0.455±0.050 mm in tm group and 0.273±0.039 mm in controls, (p<0.01); imt of internal carotid artery (ica) was 0.344±0.037 mm in tm group and 0.203±0.041 mm in controls (p<0.01). positive correlations were found between vegf and microalbuminuria, b2-microglobulin and homosistein; between nag/cre and microalbuminuria; between cca-imt and age, and a negative correlation between cca-imt and ferritin. conclusions: renal tubular damage, abnormalities in abpm, increase in vegf and increase in cca-imt and ica-imt occur when the patients are asymptomatic and routine laboratory test are normal. optimal hemoglobin levels and deferoxamine therapy do not prevent the development of renal and vascular damage. l. sylvestre 1 , e. santos 1 , p. granzotto 2 , e. siqueira 2 , l. moreira 2 , l. rispoli 2 , n. mendes 2 , r. meneses 1 1 hospital pequeno principe, pediatric nephrology, curitiba, brazil 2 centro universitario positivo unicenp, curitiba, brazil introduction: hypertension is frequently underdiagnosed in children. early diagnosis and a planned follow-up is helpful in detecting and preventing the harmful long-term consequences of hypertension. therefore, we created a specific outpatient clinic to have a better follow-up of these patients. material and methods: we reviewed the files of patients from the outpatient clinic of hypertension, from the dept. of pediatric nephrology in hospital pequeno principe, curitiba, brazil, followed for more than 3 months, from march 2005 to december 2006. we analyzed demographic and anthropometric data, diagnosis, staging of hypertension, presence of target-organ damage and treatment. results: 72 patients were eligible, 40 boys, mean age at the first visit 6.3 years old, mean follow-up of 11 months. mean bmi=18.92 (44% overweight and obese). secondary hypertension was present in 47% of the cases, predominantly due to parenchymal renal disease; essential hypertension associated to overweight and obesity in 19 patients (27%) and there was no established diagnosis yet in other 19 patients. fifty-five patiens performed at least one abpm of 24 hours, 46 showed hypertension. twelve from 69 patients showed left ventricular hypertrophy and 18 from 61 patients had abnormalities of the retinal vasculature associated to hypertension. the most frequent drugs used to treat hypertension were ace inhibitors (49) and calcium channel blockers (47). conclusion: our data are in accordance that secondary hypertension is frequent in children, mostly associated to renal disease. furthermore, we could detect a large number of obese hypertensive children and adolescents. target-organ abnormalities are not as frequent as in adults but need to be monitored. intrauterine growth retardation (iugr) is characterized by low nephron number with or without reduced kidney size. leptin, an important hormone in the regulation of body fat massand weight, is decreased in fetuses with iugr. in the present study, weexamined the effects of leptin in a metanephric mesenchymal cell line ms7. ms7 is generated from the metanephroi of embryonic day 11.5 homozygous mouse transgenic for h-2kb-tsa. incubation of ms7 withleptin 1, 10, 50, 100 or 500 ng/ml for 24 h did not affect either [ 3 h]-thymidine incorporation or cell number. on the other hand, [ 3 h]-leucine incorporation was significantly increased by leptin in a dose dependent manner (120±9%, 123±2%, 130±3%, and 131±3% of control by 1, 10, 50, and500 ng/ml, respectively). protein/dna was also increased 1.7-fold by leptin 10 ng/ml. leptin 10 ng/ml activated both erk and p38. the levels of phosphorylated-erk (p-erk) and phosphorylated-p38 (p-p38) , as assessed by western blot analysis, started to increase at 10 min, peaked at 30 min (1.6-and 1.7-fold increase respectively) remaining elevated at 1, 2, and 6 h, and began to decrease at 12 h returning to the baseline level at 24 h. the levels of p-erk and p-p38 at 30 min were increased by leptin in a dose dependent manner (1.2-, 1.6-, 1.6-, and 1.7-fold increase by 1, 10, 100, and 500 ng/ml respectively). the levels of total erk and p38 remained unchanged. increase in [ 3 h]-leucine incorporation stimulated by leptin 10 ng/ml was completely inhibited by coincubation with a mek inhibitor pd98059 5 μm or a p38 inhibitor sb203580 5 μm. these results demonstrate that leptin induces hypertrophy of metanephric mesenchymal cells via erk and p38. the hypertrophic effect of leptin may play a role in normal renal development and may explain reduced kidney size in a hypoleptinemic state, iugr. objectives of study: to investigate the mechanism of nephron deficit in rat model of intrauterine growth retardation (iugr). methods: a rat model of iugr was built by maternal low-protein (6%) dietthroughout pregnancy. newborn male pups were chose as our studyobjects. the proliferation and apoptosis in kidney was showed by ki-67 detection and tunel method. expression of wt1, bcl-2, bax, and p53 mrnas in renal tissue were examined by real-time pcr, and expression of wt1 and bcl-2 gene products in renal tissue were examined by immunohistochemistry and western blot. the final number of glomeruli was determined at 2 weeks of age when nephrogenesis has finished. results: at 2 weeks postnatally, iugr offspring had fewer glomeruli per kidney than those in controls (p<0.001). in iugr newborns, tunel positive cells were more numerous in the nephrogenic zone. renalwt1 and bcl-2 mrna levels were significantly reduced in newborn iugrpups, and the bcl-2 mrna/bax mrna ratio was also decreased, but therewas no change in the expression of p53 mrna. in iugr newborns, the wt1and bcl-2 protein expressions were significantly decreased, and the irimmunostaining were also suppressed in the nephrogenic zone. conclusions: these results suggest that reduction of nephron number in iugr rat may be associated with enhanced apoptosis in kidneydevelopment. decreased wt1 and bcl-2 expressions and reduction of the bcl-2/bax ratio may contribute to the molecular mechanisms behind these findings. objective of the study: the aim of this study was to identify risk factors for urinarytract infection (uti) during follow-up of children with isolatedantenatal hydronephrosis. methods: between 1999 and 2006, 192 patients were diagnosed with isolated fetal renal pelvicdilatation (rpd) and were prospectively followed. after initialclinical and imaging evaluation, us scans, clinical examination, andlaboratory reviews were scheduled at 6-month intervals. the event ofinterest was time until occurrence of first episode of uti. cox's regression model was applied to identify variables that wereindependently associated with the uti. results: a total of 192patients were included in the analysis (140 boys and 52 girls) themedian fetal rpd was 10 mm (iq range, 7.8±14) and 95 patients (49.5%) presented bilateral rpd. seventyeight (41%) infants presented urinarytract anomaly. the most frequent detected uropathy was upjo (55), followed by primary vur (16), and megaureter (7). median follow-up timewas 24 months (iq range, 12-39 months). during follow-up, uti occurred in 27 (14%) of the 192 children. the incidencerate of uti was 5 episodes per 1000 person-month. the incidence rate of uti has decreased from 7.2 episodes per 1000 person-month in the first year of life to 3.3 in the second year, and to 1.4 after the third year. by survival analysis, the risk of uti for the whole series was estimated in 8.5% at 12 months, 14% at 24 months, and 21% at 36 months of age. after adjustment, two variables were independent predictors of uti during follow-up: female gender (rr=2.2, 95% ci, 1.04-4.8, p=0.03) and presence of uropathy (rr=4.6, 95% ci, 1.8-11.4, p=0.001). conclusion: according to findings, in a cohort of antenatal hydronephrosis, girls with vur or urinary tract obstructionhad a higher risk of uti during follow-up. objective of the study: to identify predictive factors of resolution of fetal renalpelvic dilatation (rpd) in a cohort of medically managed children. methods: between 1999 and 2006, 192 patients were diagnosed with isolated rpd and were prospectively followed. of 192 infants, 165 (86%) were clinically managed. after initial clinical and imaging evaluation, us scans, clinical examination, and laboratory reviews were scheduled at 6-month intervals. the event of interest was rpd resolution, regardedas renal pelvis <5 mm on two consecutive renal sonograms. cox's regression model was applied to identify variables that were independently associated with the event. results: a total of 165 patients were included in the analysis and uropathy was diagnosed in 51(31%) infants. median follow-up time was 22 months (interquartile range, 12 to 37 months). during follow-up, 60 (36%) patients presented rpd resolution. by survival analysis, the estimate of rpd resolution for the whole series was 23% at 12 months, 39% at 24 months, and 42% at36 months of age. the median time for rpd resolution was estimated at 49 months (95% ci, 36-62). in the survival analysis, three variables were found to be significantly associated with resolution of rpd during follow-up: mild fetal rpd, grades 1-2 (sfu grading system), and presence of uropathy. after adjustment, only absence of uropathy remained as an independent predictor of rpd resolution (rr=3.6, 95% ci, 1.7-7.4, p<0.001). conclusion: according to these findings, it was estimated that rpd resolution occurs in about half of the patients without uropathy at 2 years of age. objective: to study clinical and pathological characteristics of antineutrophil cytoplasmic autoantibody (anca)-positive glomerulonephritis(gn). methods: clinical data of thirteen patients during 5 years, with anca-positive gn were analyzed. results: of patients with anca-positive gn with an average age of 8.8±2.5 (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) years, 11 patients were female and 2 were male. the average course was 2.2±1.9 months. 8 cases onset between december and february. there was 3, 5, 2 cases whose chief complaint on admission was anaemia, swollen and hematuria respectively. the main clinical symptoms were: anaemia (100%), hematuria/proteinuria (100%), renal functional lesion (100%), edema (76.9%), oliguresis (38.5%), hypertension (61.5%), arthralgia (30.8%), rash (15.4%), abdominal pain (46.2%), fever (7.7%). laboratory tests: bun, scr and esr were high while hemoglobin was low in all patients. mpo-anca was positive in 13 cases c3 was normal in 4 cases and low in 9 cases. pathology features: all glomeruli are affected and show different degree of segment and glomus sclerosis. there was different degree of capsula glomeruli thickening. glomerulus capillary loop was necrotic, nuclear leukocytes and cell debris could be seen. inflammatory cell interstitial infiltration with lymphocyte and plasmocyte. endotheliocyte of small vessels of interstitial was swelling and vessel wall was edema, necrosis and glassy degeneration. immunofluorescence showed no or small immune complex deposition and showed pauci-immune gn. conclusion: onset of patients with anca-positive gn was hiding and there were nonspecific clinicalsymptoms in the early stage that reduced late diagnoses until esrd. female patients was predominant. renal pathology showed segment necrotic nephritis interstitial inflammation and polyangitis. the disease had unfavourable prognosis. background: renal function maturation isn't achieved at birth. vlbw infants exposed to intensive care have an increased risk of developing renal function impairment. moreover, we showed that ibu was associated with a significant impairment in renal function at one week of life in vlbw infants. objective: to evaluate renal function development in infants treated with ibu for pda closure as compared to infants not exposed to ibu, during the first month of life. methods: multicentric prospective cohort study of 27 to 31 weeks gestation (ga) infants exposed or not exposed to ibu within the first 5 days of life. infants presenting with renal impairment at birth, urinary tract malformation, or contraindication to ibu were excluded. infants exposed to ibu were paired to controls according to ga, centre and crib score. creatinine clearance (ml/min/1.73 m 2 ) was measured for glomerular filtration rate evaluation. fractional excretion of sodium (fena%), micro-albuminuria (mg/l) and alpha1-microglobinuria/creatininuria (1/ucr: mg/mmol) were also measured once a week up to 1 month of life results: 120 infants were studied, half exposed to ibu. birth weight was 1100±278 g (mean±sd), and ga 28±1 wks. results are presented in the table: *p=0.02;**=0.01;***<0.01. j. rouillard lafond, mp. morin, c. girardin centre hospitalier universitaire de sherbrooke, département de pédiatrie, sherbrooke, canada objectives of study: many studies have focused on the negative effects of low birth weight, especially caused by intra-uterine growth restriction, on adult renal function. however, few have addressed the impact of extreme prematurity (<28 weeks) on renal function of these children during their later childhood and adolescence. the aim of the present study is to estimate the renal outcome of this population, by determining blood pressure, glomerular filtration rate, fractional excretion of sodium and microalbuminuria in 24 children (11 girls and 13 boys) aged 7 to 18 years old (mean age 12) born between 24-28 weeks of gestation (mean: 27 weeks). methods: during one encounter, height, weight and blood pressure were measured for each subject. blood tests were conducted to quantify creatinine, electrolytes and cystatin c. microalbumine, creatinine and electrolytes were dosed in one micturition. pertinent risk factors of renal damage in their perinatal history were noted. results: renal insufficiency, defined by a clearance of less than 90 ml/min/1,73 m 2 , was present in 3 subjects (22%) when estimated by schwartz formula (84,67-86,20 ml/min/1,73 m 2 ) and in 1 (4%) when estimated by cystatin c (47,4 ml/min/1,73 m 2 ). furthermore, 5 (20%) children presented an elevated fractional excretion of sodium (1, 19-1, 56%) . finally, 8 children (33%) presented microalbuminuria, with albumin/creatinine ratio greater than 2,0 mg/mol (2,04-17,25 mg/mol) . those children presented more episodes of hypotension during neonatal period (p=0,028) and have a tendency to have had neonatal asphyxia more than the others (p=0,065). conclusions: these results suggest that children born extremely preterm may present renal insufficiency and sign of tubulopathy as early as adolescence, with microalbuminuria possibly announcing upcoming glomerulopathy. organogenesis isregulated by epithelial-mesenchymal interactions that take place in theembryonic kidney between the metanephric mesenchyme (mm) and theepithelium of the ureteric bud (ub). the mm expresses signals thatregulate ureteric branching while the ub signaling leads to inductionof nephrogenesis. as a response to the ub signals the mm cellscondense, aggregate, epithelialize and undergo simple morphogenesis togenerate segmented nephrons during kidney organogenesis in connectionto ureteric bud branching. we reported earlier that mutagenesis of fgf8 function from the whole embryonic mesoderm leads to kidney failure. activation of wnt-4 gene expression encoding another essential signal for nephrogenesis also depends on fgf8 function (perantoni et al., 2005) . given the important role of fgf8 in kidney development we targeted fgf8 roles in urogenital system (ugs) development with an ugs specific cre line pax 8 cre. pax8 credeletion was expected to recombine the floxed genome in the nephronprecursors, ureteric bud and the wolffian duct derivatives. in crossesbetween fgf8 c/c and fgf8 n/+ ; pax8 cre mouse lines fgf8 gene was deleted successfully. as a result the whole ugswas affected. the kidney was severely reduced in size. newborn nullmice were born alive but died within 24 hours likely due to kidneyfailure. in the deficient kidney the organization of theproximal-intermediate segments of the developing nephron was disturbed. marker analysis with in situ hybridization was consistentwith serious defects in nephrogenesis. we conclude that fgf8 functionis involved in the control of wolffian duct development andsegmentation of the assembling nephron. the zellwegerspectrum disorders (zsds) are characterized by a generalized loss of peroxisomal functions caused by deficient peroxisomal assembly. clinical presentation and survival are heterogeneous. although most peroxisomal enzymes are unstable in the cytosol of peroxisomedeficient cells of zsd patients, a few enzymes remain stable among which alanine: glyoxylate aminotransferase (agt). its deficiency causes primary hyperoxaluria type 1 (ph1, mim 259900), aninborn error of glyoxylate metabolism characterized by hyperoxaluria, nephrocalcinosis, and renal insufficiency. despite the normal level of agtactivity in zsd patients, hyperoxaluria has been reported in several zsdpatients. we aimed to determine the prevalence of hyperoxaluria in zsds and tofind clinically relevant clues that correlate with the urinary oxalate load. methods: we reviewed medical charts of 31 dutch zsd patients with prolonged survival (>one year). results: urinary oxalate excretion was assessed in 23 and glycolate in 22 patients. hyperoxaluriawas present in 19 (83%), and hyperglycolic aciduria in 14 (64%). renal involvement with urolithiasis and nephrocalcinosis was present in five of whichone developed end-stage renal disease. the presence of hyperoxaluria, potentially leading to severe renal involvement, was statistically significant correlated with the severity of neurological dysfunction. discussion: zsd patients should be screened by urinalysis for hyperoxaluria and renal ultrasound for nephrocalcinosis in order to take timely measures to preventrenal insufficiency. menkes disease is a very rare x-linked recessivedisorder of copper metabolism. the frequency is 1: 250.000 live births. mutations in the atp7a gene are described, which encodes for aintracellular copper-binding membrane protein. pathogenetically a defect in copper absorption is responsible for inadequate synthesis of copper requiringenzymes and causes multisystemic manifestations. the clinical picture ischaracterized by early neurodegenerative symptoms like muscular hypotony andcerebral seizures. the patients also present with the so called kinky hair, hyperelastic skin, and anomalies of the kidneys and the urinary tract. wereport on a patient of non consanguineous parents of german origin. theprenatal ultrasound did not show any malformations. birth at 35 weeks ofgestation. postnatally a softening of the cranial bones and a vesicoureteralreflux iv° with dilatation of the renal pelvis combined with a subpelvicureteral stenosis had been observed, which were operated at the 2 nd month of life. at the age of 3 months the patient presented with seizuresand abnormal hair structure. within the diagnostic course menkes diseasehas been suspected and the nonsense mutation parg980x has been identified inthe atp7a gene. the mother is not a conductor, a mutation in thegermline cannot be excluded. conclusion: the combination of urinary tract malformations and neurodegenerative symptoms should let you think of the very rare menkes disease. introduction: primary hyperoxaluria type 1 (ph1) is an inborn error of glyoxylate metabolism due to the deficient activity of the hepatic peroxisomal enzyme agt (alanine: glyoxylate aminotransferase). it leads to excessive endogenous oxalate production. patients develop urolithiasis and renal insufficiency. the contribution of specific precursors in the pathway leading to endogenous oxalate synthesis is not known. this is warranted to design appropriate treatments. we aimed to test the contribution of different precursors to oxalate synthesis. methods: wild type mouse hepatocytes were incubated with different potential precursors of glyoxylate, either in the presence or absence of alanine. in the absence of alanine flux through agt is deficient thereby mimicking agt deficiency. similar experiments were also performed in hepatocytes from agtdeficient mice. results: oxalate production was found to be highest with glyoxylate as substrate in the absence of alanine, whereas oxalate production was lower with glycolate, hydroxypyruvate, glycine, fructose, and ethylene glycol. the results obtained in wild-type hepatocytes incubated in the absence of alanine were comparable to those obtained in hepatocytes from agt-deficient mice. upon addition of alanine to wild-type mouse hepatocytes, however, resulted in 30% lower rates of oxalate production, in contrast to hepatocytes from agt deficient mice. discussion: hepatocytes derived from agt deficient mice represent a good model to study the contribution of different precursors to oxalate production in ph1. s. grisaru 1 , c. geary-joo 2 , f. snider 2 , j. cross 2 1 university of calgary, department of pediatrics, calgary, canada 2 university of calgary, department of biochemistry and molecular biology, calgary, canada gcm1 (glial cell missing), is a transcription factor necessary for the formation of placental syncytiotrophoblast in mice. gcm1 mutant mice die before nephrogenesis at embryonic day (e) 10. during early murine development gcm1 expression is limited to the placenta. however, immediately after birth, gcm1 is increasingly expressed in the kidney in proximal tubular cells in the outer medulla. we recently reported successful rescue the gcm1 null phenotype using a tetraploid aggregation approach (jasn vol.17, 2006) . since our previous report, further analysis of the aggregation products confirmed only 3 homozygous mutants (2 males and a female) obtained from six hundred and twenty five transferred aggregate embryos resulting in 121 live pups. abnormal cortico-medullary patterning was demonstrated by histology analysis of adult gcm1 null mice kidneys. this abnormality was further defined by immunohystochemical detection of known nephron segment-specific markers (aquaporin-7, aquaporin-2 and tamm-horsfall protein) in gcm1 null kidney sections. to define the expression of gcm1 in human kidneys, commercially available anti-human gcm1 polyclonal antibodies were used to detect gcm1 protein in tissue sections of newborn kidneys obtained from autopsies. gcm1 was detected by immunohystochemistry in the renal cortex in tubular structures with cells having a brush border suggestive of proximal tubules. gcm1 signal was not detected in the renal medulla. conclusion: in humans, gcm1 is expressed in renal proximal tubules at birth whereas in adult mice its mutation is associated with abnormal renal cortico-medullary ultrastructure. this effect may represent a primary role for gcm1 in late renal development and patterning, or structural changes occurring postnatally secondary to alterations of tubular physiology caused by gcm1 inactivation. objectives: lupus nephritis (ln) in singapore children treated with cyclophosphamide and/or azathioprine has a poor prognosis with a reported 10-year renal survival of 59%. this study examined the long-term outcome of children with lupus nephritis using a new protocol comprising pulse intravenous methylprednisolone, mmf ± cyclosporine. method: twenty-one children with ln (age range at start of treatment 3.7-14.8 years) who were treated between the years 1995 to 2007 were included in this retrospective study. mmf dose was 1200 mg/m 2 /day. mean duration of follow-up was 3.6±2.0 (range 1.3-8.6) years. treatment outcome was defined by systemic lupus erythematosus disease activity index (sledai), renal function, proteinuria and serologic markers. effect of steroids on growth was assessed by height standard deviation score (htsds). statistical analysis was performed using wilcoxon signed rank test. results: at presentation, 72% had nephritic-nephrotic syndrome, 14% had nephrotic syndrome, while 14% had renal failure requiring dialysis. renal biopsy classification (who) was ii in 19%, iii in 24%, iv in 33%, and v in 24%. comparing pre-mmf treatment and current follow-up parameters respectively, sledai (17.4±8. objectives of study: to understand the effects of response gene to complement 32 (rgc-32) in tgf-β1 induced epithelial-mesenchymal transition (emt) on human renal proximal tubular epithelial cells (hptecs). methods: constructed rgc-32 expression plasmids and rgc-32 sirna hairpin plasmids, transient transfected them into hptecs in vitro, and then treated hptecs with tgf-β1 (5ng/ml) or vehicle alone for indicated time (0, 30min, 2h, 8h, 24 h) . rt-pcr and western blot were used to determine the expression of a-sma, ecm (col-i, fn1). the mrna expressions of e-cadherin and sm22a were detected by rt-pcr. results: (1) the promoting effects of rgc-32 on emt. instead of stimulation with tgf-β1, the hptecs, those overexpressed rgc-32 gene, de novo obtain the ability to produce markers of myofibroblast phenotype (a-sma, col-i and fn) and sm22a gene, as well as lost the capability of expressing e-cadherin gene. (2) the eliminating effects of rgc-32 sirna on emt that induced by tgf-β1. after stimulation of tgf-β1 for 24 hours, the expression of a-sma, col-i, and fn as well as sm22a gene in hptecs, those rgc-32 genes were interfered with rgc-32 sirna, were significantly decreased than that in controls. conclusions: rgc-32 was an important regulator for tgf-β1 and its downstream signalling smad proteins on emt. background: low birth weight is associated with a low nephron endowment. this may predispose to hyperfiltration and cascading proteinuria particularly if obesity develops. our report relates to an emerging population of children with proteinuric kidney disease in our multiethnic community. methods: forty-two obese children (mean age 14±5 years) with proteinuric kidney disease (kd) were studied. twenty-four were of normal birth weight (nbw>3000 grams) and 18 were of low birth weight (lbw<1200 grams). there was a female (24/42=57%) and an ethnic predominance (23 african, 16 hispanic). degree of proteinuria was determined by the random urine protein (pr) and albumin (alb) to creatinine (cr) ratios (upr/cr and ualb/cr). renal function (egfr) was estimated from the schwartz formula. body mass index was used as a measure of obesity (>95% centile). insulin resistance was measured by the homeostatic model assessment (homa). kidney tissue was obtained in 28 of the patients for pathology and histomorphometry. results: average bmi was 96±4% tile. fasting insulin and homa scores were not significantly different in the obese nbw versus obese lbw children. renal biopsy specimens revealed focal glomerulosclerosis (fsgs) in the majority of patients (23/28=83%). progression to end stage kidney disease was significantly greater in lbw compared to nbw children with a median renal survival of 11 years, p<0.01. glomerulomegaly as measured by glomerular diameter was similar in obese patients and significantly greater than non-obese controls with fsgs. conclusions: obesity appears to be a confounding factor in the development of glomerulosclerosis and progression of kidney disease in children. low birth weight and concomitant low nephron endowment may contribute to disease progression, especially in those of african and hispanic descent. objective: to determine long term outcome and prognostic factors of iga nephropathy in a large single center cohort of pediatric patients. patients and methods: we have reviewed the medical charts of 79 patients with biopsy proven igan that have been followed at our institution from 1983 to 2004, with a minimal follow-up of 2 years. follow-up data, including proteinuria >500 mg/24 h or the need for ace-is therapy, chronic renal failure (crf) and hypertension were analysed after 2 years and 5 years of follow-up. data from 60 patients with follow-up longer than 5 years were also available (mean follow-up 9.9 years, range 5-24 years). 61% of patients received therapy (cyclophosphamide in 10 patients and/or steroid±ace-is in the remaining). clinical features at the onset, histology class (lee and haas) and treatment during the first 2 years were analysed by multivariate analysis against the above mentioned dependet variables. results: the average follow-up was 7.85 years (range 1-24 years). presentation symptoms included macroscopic hematuria in 76% of patients. at the end of follow-up, renal function was normal in 94% patients, 3 patients have reached end-stage renal disease and 2 had chronic renal failure. proteinuria or the need for aceis at 2 years was significantly associated with the age of onset (or: 1.30 [1.09-1.56] ) and proteinuria at the onset (or: 1.86 [1.05-3.32] ). crf was significantly associated with familial igan (or>10). hypertension at onset was significantly associated with persistent hypertension during follow-up (or: 8.82 [1.55-50] . conclusion: taken together, these data indicate that the overall prognosis of igan is good during childhood and that the worst prognostic factor for development of crf is familial igan. overall, histological classification had a poor correlation with the outcome of the disease. this study was designed to compare three urinary protein expert systems for profiling proteinuria (pu) in children with kidney diseases. freshly voided urine was collected from 61 children with glomerular diseases, 19 children with tubular diseases and 25 healthy children aged 3-16 years. 23 out of 80 children with renal disease had a glomerular filtration rate (gfr) <90 ml/min/1.73 m 2 . the urinary protein expert systems were 1. albumin/total protein ratio (apr), 2. alpha-1microglobulin/alpha-1-microglobulin + albumin algorithm (aaa), and 3. the complex upes algorhithm (using serum creatinine, urinary total protein, alpha-1-microglobulin, albumin, igg, alpha-2-macroglobulin and dipsticks). apr correctly identified glomerular pu in 47 of 61 (77%) children with glomerular diseases, tubular pu in 16 of 19 (84%) children with tubular diseases and normal pu in 23 of 25 (92%) healthy children. aaa correctly identified glomerular pu in all 61 (100%) children, tubular pu in 18 of 19 (95%) children, and all 25 healthy children were characterized as having no pathological pu. upes differentiated the type of pu in children with glomerular diseases into glomerular (50/61 patients) and mixed glomerulo-tubular (6/61 patients). tubular pu in children with partial or complete renal fanconi syndrome was identified in 16/19 patients and described as mixed glomerulo-tubular pu in 3/19 patients. mixed glomerulo-tubular pu was only found in children with ckd stages 2-5 of glomerular and tubular diseases. in conclusion, urinary protein expert systems may be used to distinguish between glomerular and tubular pu. the aaa algorithm had the highest reliability when compared with the two other expert systems and the accuracy was not negatively influenced by a decrease of gfr. however, upes provided additional information on mixed glomerulo-tubular pu in patients with a low gfr. background: the three lmw proteins cystatin c (cys), β2-microglobulin (β2-m) and β-trace protein (β-tp) are useful markers of gfr. cys is particularly well suited for the detection of incipient renal failure. however, corticosteroid medication has been shown to stimulate cys production. aim of the study: analysis of the effect of corticosteroid therapy on the correlation between gfr and the three lmw markers. patients: 119 patients (47 f, 72 m; median age 10.9 years, range 0.2 to 18.9) with malignant (n=47) or nephrological (n=72) diseases underwent a single-shot inulin clearance. the respective lmw proteins were measured by particle-enhanced immuno-nephelometry. 27 children received corticosteroids (prednisone or dexamethasone) in a mean dosage of 28 mg/m 2 /d of prednisone equivalent (pred/bsa). multiple linear regression analysis was performed between the lmw markers as dependent and both gfr and steroid-dose as independent variables. results: mean gfr was 77.2±27 ml/min/1.73 m 2 , mean cys 1.08±0.5 mg/l, β2-m 2.17±1.33 mg/l and β-tp 0.97±0.57 mg/l. cys was highly correlated with the reciprocal of gfr (p<0.0001) but not with corticosteroid-dose (p=0.138), whereas both β2-m and β-tp were highly correlated (p<0.0001) with both the reciprocal of gfr and the reciprocal of pred/bsa. discussion: using gold-standard gfr measurements, we cannot confirm earlier reports indicating an increase in serum cys during corticosteroid medication. by contrast, steroids significantly lowered both β2-m and β-tp serum concentrations. we conclude that at least in patients with mild renal insufficiency cys -unlike β2-m and β-tp -appropriately reflects gfr also during steroid therapy. this further supports the concept of cys being a superior marker of incipient renal failure. objective: to repeatedly follow kidney function since onset of type1diabetes and evaluate whether gfr can predict development of micro-or macroalbuminuria or end stage renal disease. design: observational cohort study. methods: since 1978, all diabetic patients undergo renal function tests every 2nd to 3rd year from onset. 363 children, 204 boys, have done 1640 clearance studies. 81 healthy children and young adults, 5-30 years of age, served as controls. gfr was evaluated by clearance of inulin during water diuresis and continuous infusion. results: gfr during the first 15 years after onset of diabetes was significantly higher than that of controls (mean 130-142 vs. 116 ml/min/1.73 m 2 ). at onset, 2 and 5 years after, boys had significantly higher gfr than girls (mean 135, 148, 146 vs. 128, 134, 133 ml/min per 1.73 m 2 ) but after 8 years no differences were found between sexes. the occurrence of microalbuminuria, albuminuria during the first 15-20 years was analysed and mean of gfr of 0, 2 and 5 years, 2 and 5 years, 2, 5 and 8 years and all those gfrs separately were compared between patients still normoalbuminuric, microalbuminuric and macroalbuminuric after 10 and 20 years resp. no significant differences were found between the groups. moreover the change in gfr from 0 to 5, 2 to 5 and 2 to 10 and 5 to 10 years were also compared between the groups and no significant differences were found.8 young adults reached end stage renal disease (esrd) after 15-29 (median 20) years and comparing their gfrs during the first 5-8 years with those still normoalbuminuric after 20 years, no significant difference was found. conclusions: hyperfiltration is found in children with type 1 diabetes during the first 12-15 years from onset and hyperfiltration was equally seen during the first 5-8 years in those children who in the future developed normo-, micro-, albuminuria and/or esrd. background: hiv associated nephropathy (hivan) remains an important entity despite the use of highly active anti-retroviral therapy (haart). our objectives were to determine the prevalence and severity of renal manifestations in a cohort of hiv infected children during the haart era. methods: a retrospective analysis was conducted on 286 children infected with hiv. renal assessments included quantitation of proteinuria, radiologic abnormalities, and renal function. persistent proteinuria (pp) was defined by urine protein to creatinine ratio (upr/cr) >0.2 detected on at least two measurements 1 month apart. renal sonography and mag 3 renal scintigraphy were categorized according to the presence of bilateral increased echogenicity and/or nephromegaly, and cortical retention and/or diffuse parenchymal disease, respectively. hivan was considered in those children that had pp associated with any radiological abnormalities. results: of the 286 children, 98.6% were perinatally infected. eighty-five (29.7%) had pp. of these, 46 had pp alone, while 39 (13.6%) developed hivan. the mean age of onset of hivan was 9.4±0.8 years. overall mortality at the time of analysis was 3.8% and it was highest in those with hivan. viral load (vl) >100, 000 copies was significantly associated with hivan. creatinine clearance was significantly decreased in patients with hivan. conclusions: the prevalence of pp in our population of perinatally infected children remains high (29.7%), with at least half of them showing evidence of hivan. persistently high vl (>100, 000 copies) was associated with the presence of hivan. a spectrum of renal related disorders is a frequent occurrence in hiv infected children and should be sought with periodic urinalysis, quantitation of proteinuria and renal function, and imaging and/or histopathological studies. mmf has shown to be effective in adult ln, whereas only anectodical data are reported in childhood. we evaluated mmf in 15 children with ln, 11 f/4 m, mean age: 12.4±3.9 yrs, proteinuria >3 g/day, decreased c3 and increased anti-dsdna serum levels, normal renal function. renal biopsies, before mmf, showed the following classes (weening) : iv in 8 cases, iii in 1, ii in 5, vi in 1. before mmf: 2 patients have received i. v. cyp; 2 more received aza and csa but were in flare-up of disease; the remaining 11 were newly diagnosed patients. each patient received three i. v. metilprednisolone pulses and thereafter mmf (plus oral prednisone(p): 1 mg/kg/day) was administered (mean dose: 29±7.7 mg/kg/day; through level: 3.6±1.2 μg/ml). outcome was monitored by sledai score, renal function, proteinuria. in 11 children p was tapered and, after 4.6±2.3 month mean time, stopped; 4 children were receiving p (0.3 mg/kg/day). the mean followup is 35±16 months. sustained clinical remission was observed: proteinuria was absent in all, in 10 patients an increase of serum c3 and c4 and a decrease of anti-dsdna levels was seen. significant steroid sparing effect was obtained: hypercorticysm dramatically improved. of the 15 patients 8 achieved 2 years of mmf treatment and in them, at this time, a serial second renal biopsy was performed: histopathological activity indices reduced (8.76±2.55 vs. 5.3±6.97, p<0.01), whereas chronicity indices did not change (3.47±1.56 vs. 3.3±3.31). no haematological and/or gastrointestinal side effects were observed. our pilot study suggests that mmf represents a good alternative to traditional therapy in the treatment of sle in children, and in controlling disease activity and as steroid sparing agent without significant side effects over the entire period of therapy. mmf has shown to be effective, during treatment, in mantaining remission of childhood sd and csad ns, but few data are available on the mmf long term effectiveness after drug stopping. we report the results of two years mmf treatment in 33 children with sd and csad ns. the characteristics of sd and csad groups were, respectively: 19 patients, 11 boys, mean age 6.7 yrs vs. 14 pts, 11 boys, mean age: 11.6 yrs (p<0.005); first episode ns mean age: 3.4 yrs vs. 6.3 yrs; ns mean duration: 2.9 yrs vs. 7.4 yrs (p<0.001); mean steroid therapy duration: 2.2 yrs vs. mean csa therapy duration: 3.2 yrs; histologic features: fsgs 2, mc 17 vs. fsgs 9, mc 5. in both groups mmf was started after remission was achieved with prednisone administered at the last relapse. mmf treatment: lenght: 24 months; mean dose: 27.8±4, 1 mg/kg/day; plasma through level: 3.6±1.2 mcg/ml; non responder: patient presenting a ns relapse during mmf. in sd group 17(83%) and in csad 12 (85%) subjects were responders. in 14 patients of sd group and 10 patients of csad group p could be withdrawn over a mean period of 11.3 months, so that ns remission was sustained just by mmf; remaining patients were receiving p at a mean dose of 0.3 mg/kg/a. d. two years mmf treatment was accomplished in 26/33 (78%) patients. at 24.3±5 months mean followup since mmf withdrawal, 6 (23%) patients (4 of sd and 2 of csad group) relapsed after 9.3 months (3.2-12.1) mean time no haematological or gastrointestinal side effects were observed. our results demonstrate that two years mmf treatment in sd and csad ns children is effective not only in maintaining remission during therapy, but also in achieving persistent remission after withdrawal of drug in a significant rate (>70%) of patients; the side effects and the rate of mmf dependence are negligible with respect to those of steroids and csa. information on long-term renal function following treatment for wilms tumor (wt) are relatively scanty. previous studies reported a worrying late development of microalbuminuria (uma), hypertension (hpt) and even reduction in glomerular filtration rate (gfr). the aim of the present study was to evaluate the long-term renal outcome in a cohort of patients who underwent uninephrectomy for wt. glomerular function (as creatinine clearance by cockroft-gault formula) was calculated and uma (as uma/ucr ratio) as well as urinary b 2 microglobulin excretion were detected. 24-hours ambulatory blood pressure monitoring was also recorded. fifteen patients (11 f) with a median age at wt diagnosis of 4.4 yrs (range 1.8-16.6) were studied. the median follow-up was 13.3 yrs (10.2±19.3). eight patients had been classified as wt stage 1 and 7 as wt stage ii. all patients had been treated with unilateral total nephrectomy and chemotherapy. two of the children had also been addressed to radiotherapy. the primary disease did not recur in any of the patients. the median age at time of investigation was 18, 3 yrs (range: 12, 7-30, 4). none of them had a gfr below normal limit (mean gfr was 109.8±26.8 ml/min/1.73 m 2 ). urinary b 2 microglobulin excretion was normal (mean ub 2 /ucr: 0.5±0.3) in all of the patients. the mean uma/ucr ratio, was 0.13±0, 1 with only 1 patient exhibiting higher then normal values (uma/ucr ratio: 5, 3). the 24 hr blood pressure was normal in all patients with a mean systolic and diastolic blood pressure sds of -0.5±0.7 and -0.1±0.8, respectively. we conclude that as far as renal function, unilateral total nephrectomy combined with chemotherapy for low-stages wt can be look at as a safe treatment although it might be wise to monitor renal function at 5-year interval. classicaly, patients are divided into monosymptmatic enuresis (mne) and non-monosymptomatic enuresis, however, there is upcoming evidence that this subtyping might be artificial. the aim was to register the characteristics of nocturnal diuresis-rate and bladder volume in both subtypes. methods: retrospective analysis of 1000 consecutive patient-files, age 6-18, primary consulting for enuresis in a tertiary center. registration of incontinentia diurna (id) and maximal functional bladder volume (vmax), 24 hours urine-collections in 4 day and 4 nighttime-collections with uosmol and diuresis-volume (dv) and-rate. patients are divided into a mne and nmne-group. results: 1) vmax is significantly lower than bladder volume for age, 2) nocturnal polyuria is only present in 1/3 patients, 3) nocturnal diuresis is >vmax, 4) there is a significant linear correlation between nd and the nocturnal/daytime-diuresis-ratio, indicating fluid intake dependency. 5) there is a negative correlation between nd and urinary osmolality. 6) but the positive correlation between total nocturnal osmotic excretion and nd is much stronger. this unexpected observation cannot be explained by the classical primary vasopressin-theory. conclusion: our data show (almost) no statistical difference between the mne and nme-groups, suggesting a continuum instead of 2 separate identities. both groups have a significant low vmax and nocturnal polyuria. the observation of the extremely strong correlation of nocturnal polyuria with the high osmotic excretion and high 24 h urine-production suggests that nocturnal diuresis-rate is highly fluid-and nutrition-dependent, and therefore more attention should be given to this part of the urotherapy. a. deguchtenaere, a. raes, j. dehoorne, r. mauel, e. vanlaecke, p. hoebeke, j. vande walle there is increasing evidence that a subgroup of patients with nocturnal polyuria may have an abnormal circadian rhythm of tubular sodium which may result in vasopressin resistance. the pathogenesis of this phenomenon remains to be elucidated. however if the increased sodiumexcretion overnight results in the ddavp-resistance, decrease of the sodium-excretion-overnight may respond in subsequent ddavp response. aim of the study: retrospective study on the circadian rhytm of diuresis-rate and osmotic excretion in basal condition and subsequent during introduction of ddavp, diet and furosemide. results + discussion: 1) baseline-values show significant lower uosmol and higher diuresis-rate overnight compared to controls. striking is the >40% part of electrolytes to explain the high osmotic excretion. 2) introduction of ddavp results in a normalization of nocturnal uosmol, but despite a significant decrease of uosmol overnight, nocturnal polyuria persists. 3) protein-and sodium-restriction results only in slight differences, but of course we do not have data on the compliance. 4) furosemide in the morning results in a significant increase of daytime diuresis, osmotic and sodium-excretion, but as compensation decreased nighttime diuresis, osmotic and sodiumexcretion. 5) in 8/10 cases the antidiuretic effect results in an anti-enuretic effect. conclusion: this pilot study clearly demonstrates that introduction of early morning furosemide results in significantly lower nocturnal diuresis. because the urinary osmolality remains high, this correlates with decreased nocturnal osmotic excretion associated with increased osmotic excretion (sodium) during daytime. background: the elasticity of the vessel walls decreases with age, this process is dramatically speeded up by uremia. as an early indicator of arteriosclerosis pulse wave velocity (pwv) increases along with arterial stiffness. aim: to establish normal values for pwv in healthy children; to compare it with children on dialysis. patients, methods: pwv was measured with a pulsepen device in 116 healthy children and young adults (age range 6-23 years) as well as in 10 uremic children (14,4±4 years) (crf) treated by hemodialysis (n=7) or peritoneal dialysis (n=3). two control groups of 10-10 childrens were formed using the database of healthy children: one matched for age (a-c) and one adjusted for height and weight (h/w-c). blood pressure, heart rate, serum calcium (ca), phosphate (p) , and parathyroid hormone levels were also determined. results: a significant linear correlation was found between pwv and age (r=0, 60), height (r=0, 49), weight (r=0, 43), (p<0, 01), systolic blood pressure (r=0, 38) and heart rate (r=-0, 24) (p<0, 05). crf patients were smaller by 15, 3 cm than a-c (p<0, 05), and younger than h/w-c by 4, 5 years (p<0, 01). pwv in crf (5, 68±0, 96 m/s) did not differ significantly from a-c (5, 04±1, 18), however it was elevated in comparison to h/w-c (4, 51±0, 55 p<0, 01). serum p, caxp and pth was increased in crf (p<0, 001) conclusion pwv is higher in children with crf as a sign of increased arterial stiffness. controls matched for height and weight should be used in states of severe growth retardation. a number of established risk factors potentially responsible for arterial dysfunction are present in crf. ngal has been identified as an early marker of acute renal failure (arf). sepsis in very low birth weight (vlbw) infants is associated with arf more often than recognized. the aim of this study is to determine whether ngal represents a marker of renal impairment in vlbw infants affected by sepsis. samples of urine of 36 vlbw infants were prospectively collected for weekly measurement of ngal. after evaluation of the clinical course, 2 groups were identified: group sepsis includes 14 infants affected with 16 septic events associated with some degree of renal impairment and group normal includes 22 uncomplicated vlbw infants. a mouse model of sepsis was created in 11 neonatal mice by intra-peritoneal introduction of salmonella lipopolysaccharide. kidneys were harvested 24 hours after the challenge, and ngal mrna was quantified by real time pcr. ngal values of the normal group did not differ with gestational age or post-natal age of the neonates. the upper bound of the 95th percentile confidence interval was 40 ng/ml. the median value of ngal in the sepsis group at 7 days before the septic event was 75 ng/ml and during sepsis was 150 ng/ml: these values were not significantly different, but both were significantly higher (p<0.001) than the median of the normal group (10 ng/ml). once sepsis had been treated, the median value of urinary ngal was similar to normal (10 ng/ml p=0.17). changes in urine ngal concentration paralleled changes in serum creatinine. sepsis induced animal models showed a dramatic increase of ngal mrna in kidney tubules that paralleled acute renal failure. these neonatal animal and human data suggest that ngal may be an early marker of renal impairment in septic vlbw babies. further investigation is necessary to more exactly define the temporal relationship between the onset of sepsis/arf and rise in urine ngal concentration. we report 4 cases of acute renal failure (arf) associated with orellanus syndrome, a cortinarius mushroom poisoning. grand-father, mother, father and son presented with arf 1 week after gastrointestinal symptoms and 2 weeks after repetitive ingestion of wild mushrooms. critically arf was observed for the 11 year-old boy: anuria, severe metabolic disorders (hyponatremia 124 mmol/l, hyperkaliemia 9 mmol/l, serum creatinine 1137 mmol/l, blood urea 54 mmol/l). renal biopsy was performed for the grand-father, father and son (day 6, 9 and 8 respectively after presentation) and showed similar lesions: severe tubulointerstitial nephritis (tin) with tubular necrosis and interstitial fibrosis. renal replacement therapy was necessary for the father and the son. the mother recovered completely in two weeks without dialysis while renal function improved slowly for the two men. the boy is still hemodialyzed 5 months later. his main problem is uncontrolled hypertension. the diagnosis was confirmed 3 weeks later since fungal spores of cortinarius orellanoides were observed in the contaminated meal by light microscopy. the severity of the disease seems to be related to the toxin quantity: the 40kg boy ate mushrooms as much as his father and grand-father, the mother ate much less. cortinarius spp poisoning is an exceptional paediatric cause of arf. gastrointestinal disorders are the main symptoms of the initial phase of the poisoning appearing 3 days after the mushrooms ingestion. the renal phase is delayed (median 8.5 days) characterized by arf secondary to tin. the toxin effects are dose-related, explaining the severity of the boy's symptoms. the prognosis is severe with 60% of end stage renal failure. currently no treatment is available. although rare, mushroom poisoning should be considered in the differential diagnoses of arf with tin. l. mendels, ah. bouts, j-c. davin, j. groothoff emma children's hospital/academic medical center, pediatric nephrology, amsterdam, the netherlands background: inchronic dialysis, tertiary hyperparathyroidism (th) is clinically revealed by persistent combined high parathyroid hormone (pth), normalor high total serum calcium (tca) and normal phosphate levels. sincethe introduction of bicarbonate containing dialysate in peritoneal dialysis (pd), we have observed combined high tca and pth level sunusually early after onset of dialysis therapy. in most of the secases, ionized calcium (ica) levels were low. we aimed to investigate the extent of this discrepancy and its association with the mode of therapy. methods: serum ica, tca, pth, bicarbonate and capillary ph were assessed over 2 years in 10 pediatric pd, 11 hemodialysis (hd) and in 9 transplanted (tx) patients. associations between tca, pth and ica were analyzed. results: comparedto tx patients, we found in pd and hd patients a lower mean ica/tcaratio (both p<0.0001), an increased mean tca-ica (p<0.001 and p <0.01, respectively), and a higher number of combined normal/increased tca and decreased ica and increased pth values (44.7% and 32.3%, respectively for pd and hd, vs.0% for tx). alow ica/tca was associated with a high capillary ph (r=-0.51; p<0.0001), a high venous bicarbonate (r=-0.34, p=0.001) and a lowage (r=0.21, p=0.02). conclusions: ica levels are warranted for monitoring calcium phosphate homeostasis in dialysis patients, especially in young pd patients. the use of only tca levels might lead to an inadequate treatment with vitamin d, and henceinduce the development of autonomous hyperparathyroidism in these patients. preterminal renal failure (prf)and end-stage renal disease in children and adolescents are associatedwith an increased risk of atherosclerosis and cardiovascular disease. oxidative stress is one of the pathogenetic factors that could possiblybe influenced by therapeutic interventions. we investigated biomarkers of oxidative stress in 12 children (median age 9.6 years) with prf (median gfr 43 ml/min/1.73 m 2 , range 15-86) andin 11 children (median age 11.9 years) under peritoneal dialysis (pd)and 13 healthy age-matched controls (c). plasma samples wereinvestigated for malondialdehyd (hplc) and carbonyl groups in proteins(elisa) as biomarkers for oxidative stress as well as the plasmaantioxidative substances vitamin c (photometric), vitamin e (hplc), ubichinols (hplc), sulfhydryl groups in proteins(photometric), erythrocyte resistance to radicals and the total radicaltrapping antioxidant capacity (trap). in both patient groups prf and pdwe found a depletion of sulfydryl groups and ubichinol-10 and a reducedresistance of erythrocytes to radicals. malondialydehyd (p<0.05) andcarbonyl groups (p<0.01) were elevated in the pd group compared tocontrols. conclusion: from these studies we concludethat in children under peritoneal dialysis biomarkers of oxidativestress are elevated. moreover antioxidative defenses in preterminalrenal failure as well as under peritoneal dialysis are impaired. significant acute renal failure due to non-steroidal anti-inflammatory drugs: inpatient setting in united states non-steroidal anti-inflammatory drugs (nsaid) are freely available over-the counter. many children routinely use them without medical supervision. fourteen inpatients mean age of 15.0±2.84 years (4 males, 10 females), were referred to nephrology for acute renal failure. based on history, biochemistry, imaging and urinalysis the diagnosis of acute renal failure due to nsaid was made. all patients admitted to taking ibuprofen and six also consumed naproxen. the exact doses of either could not be scientifically determined as none were prescribed by a physician. none of the patients had underlying renal diseases at the time of admission. nine patients had proteinuria and 12 had hematuria (including one with gross hematuria). one patient had nephrotic syndrome but resolved spontaneously without steroids and has remained in remission for 2 years. two patients required dialysis. only one of the dialyzed patient required steroid therapy for recovery of renal function. all data are expressed as mean±sd. the mean duration of hospitalization was 6±4.3 days. the mean serum creatinine at the peak of renal failure was 3.97±4.74 mg/dl (range 1.2-16.6). all patients recovered renal function with normalization of serum creatinine to 0.72±0.15 mg/dl (range 0.5-1, p<0.01). however, the duration from onset to normalization of serum creatinine was 48±63 days; indicating many patients had abnormal renal function for aprolonged period. in conclusion, nsaids pose significant risk of renal failure forsignificant duration and as an entity may be under recognized. objectives: treatment with growth hormone (gh) improves growth retardation of chronic renal failure. cdna microarrays were used to investigate gh-induced modifications in gene expression in the growth plate of uremic young rats, the organ where longitudinal growth takes place. methods: rna was extracted from the tibial growth plate from two groups (n=10) of young rats: uremic (nx) and uremic treated with 3.3 mg/kg/day of intraperitoneal gh for one week (nxgh). after reverse transcription, agilent technology was used to analyze differential gene expression by microarrays containing 21, 000 rat probes (four hibridizations were performed). most expressed genes were detected using linear models and bayesian methods. to confirm gene expression changes shown by the chips, some genes known to play a physiological role in growth plate metabolism were analyzed by real-time quantitative polimerase chain reaction (qpcr). the ribosomal protein l4 (rpl4) expression did not show changes in the array and was used as the housekeeping gene. results. gh modified the expression of 224 genes, 195 being upregulated and 29 down-regulated. the assay was validated by the qpcr results, which confirmed the sense of expression modification found in the arrays for insulin like growth factor i (down), insulin like growth factor ii (up), collagen 5 alpha 1 (down) and proteoglican type 2 (up) . conclusions: this study shows for the first time the profile of growth plate gene expression modifications caused by gh treatment in experimental uremia. the further analysis of selected individual genes, whose expression is differentially modified by gh will contribute to explain the mechanism of the stimulating effect of gh on growth in chronic renal failure. objectives of study: children with chronic renal failure have an increased risk of cardiovascular disease. this is associated with endothelial dysfunction, a key pathophysiological factor in atherosclerotic disease. circulating endothelial progenitor cells (epcs) have the potential to repair endothelial damage and promote angiogenesis. in adults, the number of epc in peripheral blood correlates with endothelial function and reduced epc levels are associated with a higher incidence of cardiovascular events. we aimed to investigate if children on long-term hemodialysis (hd) therapy have reduced epc levels. methods: we quantified circulating epc in 12 pediatric hd patients before a midweek hd session and 11 healthy age-matched controls. epc are a subfraction of the haematopoeietic stem cells (hsc) expressing both hsc-marker cd34 and the vegf-receptor-2 kdr. using flow cytometry, epcs were identified as cd34+kdr+ cells and quantified relative to the number of granulocytes in the sample. results: the number of epcs in the peripheral blood was significantly reduced in hd patients (12.0±1.9 vs 29.0±7.3/10 5 granulocytes, 59% reduction; p=0.03). the total number of circulating hsc also tended to be lower in hd patients (71.2±7.3 vs 112.8±23.7/10 5 granulocytes, 39% reduction; p=0.09). conclusion: the number of circulating epcs is significantly reduced in children on long term hd. reduced epc levels may contribute to endothelial dysfunction and accelerated atherosclerosis in children on long term hd. future studies are needed to identify the cause of this deficiency and to evaluate if increasing epc levels provides therapeutic benefit. objectives: darbepoetin alfa (aranesp ® ) is a novel erythropoiesis stimulating protein that has been shown in adult trials to have safety and tolerability equivalent to recombinant human erythropoietin. however, to date there is only limited published data on the use of aranesp inpaediatric patients. the objective of this study was to determine the safety and efficacy of darbepoetin in children with chronic and endstage kidney disease. methods: from 2003 to 2006, 30 children with either chronic or end stage kidney disease were enrolled in a prospective observational study. the initialdose of darbepoetin was 0.45 mcg/kg weekly (either iv or sc) and subsequent dose was titrated to achieve haemoglobin (hb) between 110 and 130 g/dl. results: data analysis to date includes 22 patients (16 male : 6 female) whose agesranged from 1 month to 17 years (mean 9 years). hb improved significantly with darbepoetin treatment from mean 84 g/dl (range 64-107) at start of treatment to 115 g/dl (range 81-147, p<0.001) at completion. the mean starting dose was 0.55 mcg/kg/week (range 0.4-0.9) which was not significantly different to the dose atthe end of the study (0.57 mc/kg/week, range 0.5-2.5). however, there was a significant change in the frequency of administration, with 85% commencing on weekly treatment, but only 25% still on weekly treatment at the end of the study (p<0.001). the most common treatment interval in stable patients was fortnightly (40%) but a significant number tolerated even longer intervals (25% dosed every 3 weeks or longer). injection pain was common, but there were no other significant adverse events. conclusions: darbepoetin alfa is a safe and effective therapy for anaemia associated with kidney disease. the majority of children will maintain satisfactory haemoglobin at a dosing interval of every 2 weeks orgreater. high prevalence (43%) of left ventricular hypertrophy (lvh) and impaired systolic myocardial function in children with mild-to-moderate chronic renal failure (crf) were observed in previous studies (jasn2006, jasn2007). in adult patients with uncomplicated arterial hypertension, lv mass (lvm) exceeding compensatory value for body size and cardiac workload (inappropriate or ilvm) is associated with poor prognosis, independently of lvh. we tested in crf children if increased lvm compensates or exceeds the expected values for individual cardiac load and if ilvm is associated with impaired cardiac function. complete anthropometrics, biochemical profile and doppler echocardiograms were obtained in 24 children (age 11.5±5.2 yrs; gfr 48.7±30.3 ml/min/1.73 m 2 ). ilvm was defined above 109% of the value predicted for individual body size, gender, and stroke work and lvh was defined as lvm/m 2.7 >38 g/m 2.7 . 9 patients showed ilvm. children with ilvm had higher mean age and lower heart rate as compared to patients with appropriate lvm (both p<0.05), without differences in blood pressure, bmi and gfr. after controlling for differences in age, gender distribution and presence of lvh, patients with ilvm showed similar cardiac geometry and diastolic function parameters compared to children with compensatory lvm (p=ns). in contrast, presence of ilvm was associated with lower lv ejection fraction (53.5±3.7% vs 63.0±3.6%) and lower midwall fractional shortening (15.9±1.8% vs 18.6±1.4%)compared to children with compensatory lvm (both p<0.01), indicating impaired lv chamber performance and reduced systolic myocardial function. in conclusion, in 37.5% of children with mild-to-moderate crf, lvm is inappropriately increased for individual cardiac workload and body size. presence of ilvm is associated with reduced systolic function, independently of age, gender and presence of lvh. pediatric nephrology centers were enrolled. age groups of the patients were as follows: 41.9% newborn, 19.7% 2-12 months, 23.7% 13 months -10 years, 14.7% 11-18 years. underlying diseases were prematurity (30.1%), malignancy (10.7), congenital heart diseases (chd, 16.0), urologic disorders (7.3%). low fluid intake was noticed in 35% of cases.50.4% of cases developed arf after they have been hospitalized. time to diagnose arf was longer in the surgery department (4.32±5.01 days) compared to pediatrics (1.12±3.33 days), p<0.05. thirty-nine percent of patients were on mechanical ventilation (mv) before the diagnosis of arf, an additional 6.2% needed mvl after the diagnosis of arf. arf was prerenal, intrinsic and obstructive in 37%, 61% and 2% respectively. hemodialysis and peritoneal dialysis was performed in 9.1% and 21.9% of cases. mortality was 31.8%; and it was secondary to non-arf related causes in 79.5% of cases; presence of mv, intrinsic arf, prematurity, chd, malignancy and being in intensive care unit were poor prognostic factors. conclusion: our nationwide data suggest that nephrologist, intensivist and pediatrician should focus on risk groups to prevent and to diagnose arf earlier. appropriate fluid intake and earlier consultation to a nephrologist are simple but may be effective measures to prevent arf.. hemolytic uremic syndrome is characterized by the triad of hemolytic anemia, acute renal failure, and thrombocytopenia. recent studies have shown that shiga-toxins (st) may stimulate apoptotic cell death in renal tubular cells, but the underlying molecular mechanisms remain to be elucidated. in the present study, confluent llc-pk1 cells were exposed to st and cell death was studied with morphological and biological assay. in llc-pk1 cells st was found to induce apoptotic cell death in a dose-and time-dependent manner. the expression of calpain and bax were significantly up regulated by st, while the expression of bcl-2 was down regulated. cell death was completely inhibited by a specific calpain inhibitor, but not by a broad caspase inhibitor, zvad-fmk, implicating a caspase-independent pathway via calpain. moreover, we found that serum factors could trigger a survival signal against st-induced cell death through pi3k/akt pathway. in conclusion, activation of calpain mediates st-induced renal proximal tubular cell death, and the expression of bcl-2 and bax were oppositely altered. stimulation of pi3k/akt signalling protects cells against death. verocytotoxin (vt)-producing e. coli (vtec) infection represents the main cause of hemolytic uremic syndrome (hus) in children. a nationwide surveillance system of hus was introduced in italy in may 1988 to follow the trend of vtec infections. for each patient, epidemiological and clinical information was collected by a standardized questionnaire. laboratory diagnosis of vtec infection was based on the detection of vtec and free vt in stools and of antibodies to the lipopolysaccharide (lps) of serogroups, o26, o103, o111, o145, and o157 in the sera. the immuno-detection of vt on circulating neutrophils was also performed on some patients. as of december 2005, 481 cases have been notified, accounting for a mean annual incidence of 0.31x100,000 in the 0-14 age group with a significant difference among the regions (from 0.15 to 1.1); median age of patients: 23 months, 53% males. most cases (53%) occured in summer from june to september. seventy-nine per cent of the cases had prodromal symptoms such as bloody diarrhea (26%) and non-bloody diarrhea (51%). five patients (1.0%) died from the disease. stools and/or sera were collected from 380 cases. evidence of vtec infection was observed in 255 cases (67%). the vtec serogroups most commonly detected were o157 (37% of the vtec-positive patients), followed by o26, o145, o111 and o103. the number of cases associated with non-o157 infections increased over time: from 1997 the o26-associated cases are the most frequent. during the surveillance-period 4 epidemic clusters have been registered: 1992-lombardia, 1995-veneto, 1997 and 2005 campania. the role of vesico-ureteral reflux (vur) as a predisposition for acquired renal scarring with urinary tract infections (uti) has been questioned in recent years. few studies have investigated baseline factors associated with chronic nephropathy in severe reflux. we aimed to evaluate dmsa scans in children having any degree of primary vur associated with uti in order to identify variables that are predictors of the presence and/or development of renal scar. data of patients with proven uti who have primary vur were evaluated retrospectively. patients and renal units were classified as scar (+) and scar (-) by dmsa results. the following parameters were assessed with respect to their relation to presence of renal scarring: sex, age at diagnosis, grade (g) of reflux, number of subsequent utis (on new renal scars). there were 138 patients (m/f: 53/85, median age 42 months) and 212 refluxing units. variables increasing the likelihood of scar detection were: male gender (32/53 vs. 35/85, p=0.043; or 2.2), >26 months of age (for girls only; 31/59 vs. 4/26, p=0.003; or 6.1), g iv-v reflux (or 9.1 vs. g i-iii reflux and 23.3 vs. no reflux). all boys having g iv-v reflux and girls over 26 months of age having g iv-v reflux had very high rate of scarring compared to the rest (21/23 vs. 46/115, p=0.0001, or 15.8 and 14/16 vs. 53/122, p=0.002, or 9.1, respectively). however, variables increasing the likelihood of new/progressive scar development were only the presence of previous scar (or 8.3) and uti number >1 (or 3.0). neither uti number nor new/progressive scar development was affected by vur grade. in conclusion, the most predictive variables for the presence of renal scarring among children presenting with a uti were male gender, age (being >26 months; only for females) and grades iv-v reflux, while new/progressive scar development was associated with presence of previous scar and uti number. d. hothi 1 , e. harvey 1 , c. goia 2 , d. geary 1 1 hospital for sick children, department of pediatric nephrology, toronto, canada 2 hospital for sick children, educational, toronto, canada introduction: adequate ultrafiltration (uf) is necessary for good health in dialysis dependent patients. however uf can be hindered by development of intradialytic symptoms and hypotension. objectives: to determine whether sodium ramping, uf profiles and mannitol could improve uf without increasing intradialytic morbidity in children. method: a standardized hd practice was instituted in our unit. we prospectively analysed 506 dialysis treatments from 11 chronic patients with routine scheduled hd, 4hrs x3-4/wk. results: uf volumes between 3.2 to 9.7% of the dry weight were achieved. mannitol reduced the risk of developing intradialytic symptoms by 64% (p<0.05) without altering the risk of hypotension, with a mean uf volume of 6.2% of the dry weight. a linear sodium ramp (148-138mmol/l) increased the odds of intradialytic symptoms (p=0.10) and hypotension (p<0.05), with no difference in the mean uf volume. all uf profiles increased the risk of intradialytic symptoms but the effect was not statistically significant except with profile 2 (stepwise reduction of uf during procedure). achievement of dry weight was least likely with uf profile 2 (p<0.05); there was no statistical difference in the mean uf volume between them all. conclusion: uf volumes higher than the traditional recommendations of 5% of the dry weight can be achieved in children. the use of mannitol increased the uf volumes and reduced symptoms without increased hypotensive episodes. objective: innate immunity and urinary tract response play a central role int he development of urinary tract infection (uti), in which heat shock protein (hsp)70 and toll-like receptor 4 have a key position. patients and methods: hspa1b a(1267)g and tlr4 a(896)g genotypes were determined using allele-specific polymerase chain reaction in 103 patients treated with recurrent urinary infection. allelic prevalence was related to reference values of 235 healthy controls. clinical data were also reviewed and statistically evaluated. results: hspa1b (1267)gg genotype and hspa1b (1267)g allele occurred more frequently in uti patients versus controls (p=0.0001) and both were associated with a higher risk of renal scarring (p=0.012 and 0.049, respectively). tlr4 (896)ag genotype and tlr4 (896) g allele had also higher prevalence among uti patients than controls (p=0.031 and 0.041, respectively). the combination of carrying ag genotype at both sites meant the greatest risk for uti (p=0.05). conclusion: our data indicate an association between the carrier status of hspa1b (1267)g and tlr4 (896) one of the major goal of hemodialysis adequacy is to achieve the fixed endsession body weight, so called dry weight, in order to limit overhydration and thereby cardiovascular risks. the prescribedultrafiltration, can induce hypotensive episodes and thereby limit thedry weight achievement. on line equipments offer the assessment of theblood volume (bv) and its relative variation. the bv is derived fromdirect measurement of the hematocrite. weroutinely use such an equipment (fresenius a 2008 c) over all thesessions since february 2002, conducting to a clinical experience of 7800 bvm curves. these registered curves could be related to thehemodialysis prescription parameters (uf, nad, td, kt/v) to the dryweight, the blood pressure and to the clinical dialytic symptoms. thisexperience conducts us to define the normal bv curve over a sessionand its variations. starting dialysis induces an acute initial (5 to 30 min) decrease of bv, 5 to 8%, mostly asymptomatic: extra corporeal circuit filling; this initialdecrease is a sign of normality. there after the normal bv curve should be flat; uf rate/amount being compensated by the plasma refilling rate: iso osmotic dialysis, no symptoms, no cramps, no hypotension, no vomiting. incase of no bv decrease over the dialysis session, the patient isoverloaded: reduce his dry weight. in case of a decrease of the bvhigher than 10%, there is a hypotensive risk: uf rate/amount, dryweight, sodium dialysate, sodium temperature and kt/v urea (cellular water shift) should be individually adapted conducting to arefilling curve. the effectiveness on the bv of these individual changein dialysis prescription can be directly, on line attested by the bvmcurve: plasma refilling capacity test. the bv changes reactivity is rapid, 5 to 15 min. renalscarring following acute pyelonephritis (apn) in children is a frequentcomplication which may impair renal growth. its pathophysiology includes host response, bacterial virulence, associated malformationand/or renal dysplasia. we prospectively studied virulence factors of e. coli isolates from children with a first episode of apn (fever >38.5 c°, crp >20 mg/l, monomicrobial e. coli positive culture >10*5 cfu/ml). we excluded patients with anyconcomitant infection, renal dysplasia or obstructive uropathy (us examination, renal length <2 sd) or grade 4-5 vur. renal scarring was evaluated by dmsa scan performed 6 to 9 months after apn. 383 patients were included in a multicentre prospective randomized study comparing short vs long i. v. treatment with ceftriaxone as a first line antibiotic treatment [in press]; 161 out of them fulfilled criteria for virulencestudy. six virulence genes were investigated by multiplex pcr (pap, sfa, afa adhesine genes; cnf1, hly toxin genes, aeraerobactin gene) and the k1 capsular antigen was researched by latextest. we identified 21 distinctive virulence profiles; 99% of e. coli strains had one or more virulence factors; 64% expressed the aer genewith at least one adhesin or one cytotoxic factor. renal scars occurredin 18% of cases and low grade (1-2-3) vur was detected in 46%. statistical analysis did not show any correlation between the presenceof scars and e. coli virulence pattern. in addition, scarring was not correlated with the antibiotoc regimen but was correlated with grade 3 vur (p 0.002). most e. colistrains associated with apn in children show several virulence factors, mainly adhesins and cytotoxins, but their profile was not correlated with renal scarring. background: acute lobar nephronia (aln), a severe renal parenchymal inflammatory disease, ranging between acute pyelonephritis (apn) and frank abscess formation, has been diagnosed with increasing frequency due to the advancement of non-invasive diagnostic modalities and the development of systematic diagnostic schemes. e. coli is the most common bacterialpathogen isolated from the urine samples of aln patients and the associated percentage is significantly higher than those among the patients with first time urinary tract infections. this prospective study was conducted to elucidate and differentiate the bacterialvirulence factors associated with aln and apn in pediatric patients. methods: patients included in the present study were those suspected of anupper uti and underwent a systematic scheme of ultrasonographic, ct and tc99m-dmsa evaluation for the differential diagnosis of aln andapn. exclusion criteria were any evidence of underlying diseases orurinary anatomical anomalies except vur. the e. coli isolates from the urine samples of patients were screened with pcr analysis for various urovirulence genes. pulsed-field gelelectrophoresis was used to analyze the genetic association of theisolates. results: a total of 88 patients were enroled. forty-six patients were diagnosed as aln, while the other 42 cases were apn. diverse genotypes were found among the e. coli isolates in either group. among the pathogenetic determinants examined, multivariate logistic regress analysis indicating that a papg ii allele was the only significant urovirulence factor associated with aln (p<0.005; odds ratio, 17.16). conclusions: while no specific genetic lineage was identified among the e. coli isolates studied, a papgii gene was found strongly associated with the cause of aln among pediatric patients without underlying disease other than vur. 0-<21 yr during 1996-2005 were sent to leading paediatric nephrologists of 13 asian countries/regions. those having national renal registry were to use the registry data. results: data from 11 countries/regions were returned (incl. 3 national registries), namely china, hong kong sar, india, indonesia, japan, malaysia, pakistan, philippines, singapore, south korea & thailand. a total of 2275 esrd patients were reported: 772 on peritoneal dialysis (pd), 678 on haemodialysis (hd), & 825 transplant (tx) of 34%, 30% & 36% respectively. chronic pd: capd and automated pd (apd) were the main modes of pd at ratio of 2.6 to 1. only 3 countries had apd morethan capd. peritonitis rate ranged from 1 episode in 3 to 50 patient-months, and seemed less common in those having more apd. chronic hd: hd comprised of 47% chronic dialysis, mostly adolescents. a-v fistula wasused in 76%, and permanent catheter 15% for vascular access. background: current data suggest the role of chronic inflammation and lipid disorders in atherogenesis. the aim of the study was to evaluate established and new markers of atherosclerosis in apd and hd pediatric patients and to assess whether the method of dialysis has an impact on those factors. methods: soluble(s) e-selectin, il-4 and il-12 concentrations were evaluated by elisa in sera of 18 apd patients on, 9 hd patients and 15 controls. hscrp levels were assessed by nephelometry. the lipid profile (total cholesterol (chol), hdl-chol, ldl-chol, triglycerides (tgl)) was also estimated. results: se-selectin concentrations in dialyzed patients were higher than in controls (apd p<0.0001; hd p<0.0001) and in apd were increased vs. hd (p<0.05). there were no differences in median values of il-4, il-12 and hscrp between examined groups. chol levels were increased only in apd vs. controls (p<0.05). hdl-chol concentrations were decreased in all dialyzed patients when compared to controls (apd p<0.0001; hd p<0.01), without difference between apd and hd. ldl-chol in apd and hd were higher than in controls (apd p<0.0001; hd p<0.05), but failed to differentiate between two dialysis modalities. tgl levels behaved in the same way. conclusions: the elevated se-selectin concentrations in all patients show the role of endothelium in atherogenesis in ckd children. thus, the se-selectin augmentation may serve as an early marker of endothelial activation, appearing prior to inflammation (unchanged hscrp, il-4). increased seselectin and cholesterol levels in apd patients prove that children on peritoneal dialysis are more prone to atherosclerosis than those on hemodialysis. background: end-stage renal disease (esrd) is associated with an increased risk of cardiovascular morbidity and mortality. according to recent data, arterial stiffness measured by aortic pulse wave velocity (pwv) and augmentation index (aix) is a strong independent predictor of cardiovascular mortality in adult esrd patients. few studies have been reported regarding arterial stiffness in the paediatric renal population. methods: aortic pwv and aix (difference between the first and the second systolic peaks on the aortic pressure waveform divided by the pulse pressure) were determined in 14 haemodialysis children (10 boys; age 12±4 years) by applanation tonometry using a sphygmocor device. seven of the hd patients (5 boys; age 12±3.5 years) received a renal transplant (tx) and were restudied (6±2 months post tx). the immunosuppressive regimen included basiliximab induction, cyclosporine, mycophenolate mofetil, and steroids. results: in the hd population, aortic pwv (6.3±1.3 m/s) was correlated with age (p=0.019), weight (p=0.036), height (p=0.0036) and systolic blood pressure (p=0.0038). in the transplanted cohort aix decreased in six children out of seven after transplantation (7.57±9.45% on hd versus -6.57±19.7% after tx). no significant change was observed for aortic pwv (6.37±1.18 m/s on hd versus 6.86±1.87 m/s after tx). objective: to study the pathogenic role of host and escherichia coli virulence factors in the development of e. coli febrile urinary tract infection (uti) in children with acute cystitis (ac), acute pyelonephritis (apn) and renal scar. materials and methods: isolates recovered from 125 children consecutively admitted to the hospital with e. coli febrile uti that diagnosed as ac (n=36) or apn (n=89) were retrospectively enrolled into this study. virulence genes of e. coli, that included papg genes (classes i-iii), aer, cnf1, fimh, hlya, afa, sfa/foc, iha, usp, irone and ompt, were detected by polymerase chain reaction analysis. results: young age (=<5 months), male sex were more frequently associated host factors for patients with apn, but old age (>5 months) and female sex were more frequently associated with renal scar formation. after multilogistic regression analysis, with regard to e. coli virulence factors, the papg class ii gene might play a more important role in the development of e. coli apn. however, iha was significantly higher in young children with acute pyelonephritis. afterwards, age, gender, duration of fever before admission, and crp level were considered as potential confounders for the further multivariate analyses, specifically estimating the relative risks of e. coli genotypes to the incidence of acute pyelonephritis and renal scar by age group and the existence of vesicoureteral reflux. odds ratios with 95% confidence intervals for each variable were utilized to estimate the relative risk of acute pyelonephritis and renal scar. in addition, there were no differences between young children and old children, if we excluded the factor of vesicoureteral reflux (vur). conclusion: both host and e. coli virulence factors contribute to the development of febrile uti, apn and renal scar. since 2/06 we have started an acute peritoneal dialysis (duration 1-61 days) in 8 infants (age 7 days to 9 months) with the use of the baxter acute set for children under constant warming of the complete dialysate inflow tract to 37 °c (barkey system). as dialysate solutions we chose a glucose/bicarbonate/lactate solution (physioneal 40) in all patients and in 4 patients a mixture of this solution with a 1.1% amino acid solution (nutrineal) in a 3:1 ratio. the body weight before the renal insufficiency was 1.3 to 5.2 kg. in 5 patients the renal failure followed cardiothoracic surgery. the handling of the system was easy. because of obstruction by omentum and fibinous layers, respectively, the dialysis catheter had to be cleared surgically in 2 patients. body temperature could be kept constant and in the normal range, even with low body weight and intensive dialysis (as measured by dialysate volume per kg body weight and day). in the infants dialyzed with the glucose/amino acid-mixture a decreased loss of albumin through the dialysate (as measured by the necessary intravenous albumin substitution), a better glucose homeostasis (less episodes of hyperglycemia and less need for insulin infusions) as well as a better acid-base control (less episodes of metabolic alkalosis) could be found. the detected tendency to a better homeostasis (concerning body temperature, serum albumin, blood glucose and acid-base) with this dialysis system and the used dialysate solutions could help to increase the survival chances of infants with renal failure. this will be evaluated prospectively. objectives: varicella-zoster-virus (vzv) infection can cause significant morbidity and mortality in the immunocompromised patient. since there is no clear correlation between antibody titers and protection monitoring after vzv vaccination is unclear. patients and methods: serum samples of nineteen pediatric transplant recipients were investigated for vzv igg antibody titers and avidity (elisa test). a relative avidity index (rai) <40% showed evidence for low-avidity antibodies, an rai >60% high-avidity antibodies, borderline avidity inbetween. the control group consisted of 27 healthy children. 22 had suffered from varicella infection after wild-virus contact, 5 had undergone varicella vaccination. as there was no difference between diseased and vaccinated controls both subsets were treated as one group. results: median vzv igg antibody titers were 560 u/ml (range 100-2600) for transplanted children and 790 u/ml (range 50-2300) for control subjects (n. s.). median rai was 83% for transplant patients and 90% for controls (p=0,04). there was no correlation between rai and antibody titers in either group. rai increased significantly with time after vaccination or infection in both groups. despite protective antibody titers after vaccination and an rai of 68% one transplant recipient showed a moderate vzv infection that required antiviral treatment. postinfectious course showed an increase of rai up to nearly 100%. conclusion: vzv igg antibody avidity might be a pathbreaking parameter regarding decision making for a second vaccination before transplantation or preemptive treatment in a transplanted patient in case of exposure. in japan, almost half of children with esrd received renal transplantation and more than 90% of those were living kidney transplants from their parents. burden of esrd during childhood frequently causes psychosocial problems in their families, including parental relationship problems. we investigated how parental divorce or death affected the choice of renal transplantation in children. patients and methods: in 68 children younger than 20 years old who started renal replacement therapy in our hospital, percentages of the children losing a parent or parents by divorce or death were investigated. we compared renal transplantation rates and percentages of fathers as kidney donors between those living with both parents and those without. results: in observation periods (4.2±3.3 years), 43 (63%) received renal transplants from 40 living and 3 deceased donors. nineteen children (28%) lost a parent or parents by divorce (n=16) or death (n=3). in all divorced families, mothers got parental authority. ten parents divorced during ckd period, 2 after start of dialysis, and 4 after transplant. of 49 children living with both parents, 31 (63%) transplanted with kidneys from 9 fathers, 20 mothers, and 2 deceased donors. of 16 children whose parents divorced, 11 (69%) transplanted from 4 fathers (2 just before and 2 after their divorce), 5 mothers and 2 other family members. however, in 3 children at least one parent died, only 1 (33%) transplanted from a deceased donor. conclusion: a high parental divorce rate from ckd period was observed in children with esrd, suggesting burden of the disease on their families. renal transplantation was preferred even in divorced families and divorced fathers still were willing to donate kidneys to their children. objective of study: to determine the importance of gastrointestinal evaluation in pre-transplantation phase in pediatrics with end stage renal disease (esrd). methods: twenty four children with esrd (13 female, 11 male) mean age 14.7 (±3.4) years on maintenance hemodialysis were included in this study. upper gastrointestinal endoscopies were performed and four gastric antral and duodenal biopsy specimens were obtained for urease test and histological study for all patients. serum gastrin levels were measured in all patients, too. a control group was chosen to compare the rate of h. pylori infection using student's t. test. results: gastrointestinal symptoms were present in 16 (67%) of 24 patients. seventeen (71%) patients had abnormal upper gastrointestinal endoscopic findings. h. pylori was detected in 66% of patients and 20% in control group (p<0.001). in symptomatic patients 75% had abnormal endoscopic findings and 63% had positive urease test for h. pylori infection. while, in asymptomatic cases these rates were 30% and 75%, respectively. seventy one percent of patients with gastrointestinal lesions and 50% of patients with normal endoscopic examination were infected. high serum gastrin levels in infected and non-infected patients were detected in 75% and 12.5%, respectively (p<0.001). conclusion: we demonstrated a significant number of patients with peptic ulcer diseases and h. pylori infection and secondary hypergastrinemia. this study showed that, clinical symptoms are not a reliable predictor of gastrointestinal problems. our results emphasize the importance of periodic, and also pre-transplant gastrointestinal evaluation in these patients to find out their problem and manage appropriately. key words: renal failure, hemodialysis, peptic ulcer disease, helicobacter pylori, hypergastrinemia. background: preservation of a stable allograft function in children following renal transplantation (rtx) depends on various factors including genetic variability. the gene of the angiotensin i converting enzyme (ace) has been shown to influence allograft function. we therefore analysed various polymorphisms of the renin-angiotensin system in 91 children following rtx and 32 kidney donors and associated genotypes with loss of renal function. patients and methods: 91 children and adolescents (60 male, 31 female, mean age at transplantation 9.7±5.2 years) with stable renal function and observation period exceeding 6 months were included. mean follow-up time was 5.4 years (0.5 to 16 years). dna was extracted from all recipients and 32 donors and genotyped using rflp. the following polymorphisms were studied: renin 18 g/a; ace i/d; angiotensinogen (agt) 235 met/thr and angiotensin ii receptor type-1 (at1r) 1166 a/c. the slope of glomerular filtration rate (gfr) was determined by linear regression analysis and correlated with the genotype. results: allelic frequencies were not different from healthy controls. genotypes of renin, agt and at1r showed no significant association with the slope of gfr but patients homozygous for the ace-d-allele had a significantly steeper decline of gfr when compared to homozygous carriers of the ace-i-allele (slope dd: -4.3±0.8 vs. ii: -1.3±1.1; p=0.035). the dd-genotype was also present in 11 out of 32 donors and in four cases a dd-recipient received a kidney from a dd-donor. those four patients showed a more pronounced decline of gfr (-5.2±0.5; p=0.002 ). in addition, dd-recipients had a significantly increased systolic and diastolic blood pressure before rtx. conclusions: the dd-genotype is associated with a faster, non-immunological loss of graft function which has to be evaluated in prospective studies. year large single-center review b. warshaw, l. hymes, l. greenbaum, s. amaral from1995-2006, 207 children received renal transplants at emory university/children's healthcare of atlanta of whom 29 (14%) had nephroticsyndrome (ns) as their primary diagnosis (27 fsgs, 2 minimal change). all children received calcineurin-based immunosuppression. thirteen children (45%) developed recurrent ns within the first week post-transplant and received plasmapheresis (pp) 3-5 times weekly. nine (70%) had complete resolution of proteinuria. two who responded to pp suffered graft loss fromlate recurrences of ns at 7 years. ns did not resolve with pp in 4 children who suffered either delayed function (1) or early cessationof function (3); each of the latter lost their grafts within the first 3 months. 16 patients did not have recurrences of ns. comparison of these 2 groups showed significantly increased riskfor recurrence with younger patient age (p<0.006), interval <3 years from onset of ns to esrd (p<0.02), and living donor source (7/9=78% ld vs 6/20=30% dd; p<0.02). no differences were seenfor hla match, donor age, gender, or african american race. actuarial graft survival for children with recurrence was 70% at1 year and 60% at 2 years vs. 100% & 92% for patients without recurrence. conclusions: ns recurred in 45% of children with ns as their primary cause of esrd. risk factors for recurrence included younger age, interval <3 years from onset of ns to esrd, and living donors. most recurrences responded to pp (70%); failure to respond was associated with delayed or early cessation ofrenal function & early graft loss. the incidence of recurrence was strikingly high (78%) among living donor recipients, suggesting a need to explore prophylactic strategies such as preemptive pp in this group. methods: a single-centre retrospective case-controlled study including 24 children <3 yr (g1) and 24 matched kidney tx recipients older than 3 yr ofage (g2). patients were matched for donor type and tx period. kaplan-meier method was used for patient and graft survival. results: 23 tx were performed using deceased donors in both groups. median recipient age, weight and height at tx in g1 were 1.5 [0.6-2.7] yr, 9.1 [5.6-11.1] kg and 74.8 [64.5-87.0] cm, respectively, while median age in g2 was 11.2 [3.3-17.2] yr. hypoplasia-dysplasia wasmore frequent in g1 (42 vs 21%). median hla-dr mismatch, time ondialysis and number of blood transfusion before tx were not different. in g1, kidneys were placed intraperitoneally and most of vascular anastomoses were done on distal aorta (83%) and inferior vena cava (88%). median follow-up was 8.7 [0.02-15.9] yr and 6.5 [0.3-15 .6] yr ing1 and g2, respectively. patient survival was 94% at 5 yr and 88% at 10yr in g1; 3 patients died in g1 (2 ptld, 1 recurrence of primary disease), none in g2. fiveyear graft survival was 79% in g1 and 77% in g2. acuterejection episodes occurred in 46% in g1 and in 67% in g2 (p 0.15 ). chronic rejection led to 3 late graft losses in g2 (5.2 to 6.9 yrafter tx) vs 0 in g1. renal function did not differ between the 2 groups during the first 5 yr post tx. the average height gain was better in g1 at 5 yr post tx (+1.3 sds vs. -0.1). primary disease recurrence was observed in 4 cases (1 in g1) causing graft lossin 3 cases. two arterial thromboses were observed in g1 causing 1 earlygraft-loss. conclusion: the outcome after cadaveric kidney transplantation is as good in children under 3 yr of age at transplantation as in older recipients in our experience. c. garcia, v. bittencourt, d. malheiros, a. tumelero, j. antonello, a. oliveira, v. garcia cancer is an increasingly recognized problem associated with immunosuppression. recent reports, however, suggest that sirolimus (srl) has anti-cancer properties that could address this problem. aim: to report a retrospective analysis of preliminary results of 6 patients who received srl because of post-transplant de novo malignancies in a consecutive cohort of 348 pediatric kidney recipients. patient and methods: we retrospectively evaluated the efficacy and safety of srl in 6 pediatric renal transplantation recipients, who were 12±7 years when converted to srl. the 6 post-transplant de novo malignancies were: gall bladder and hepatic leiomyoma (n=1), wilms tumor in the native kidney (n=2), ptld (n=2) and hpv-associated neoplasia. the immunossupressive regimen at the malignancy diagnosis was tacrolimus/cyclosporin, mmf/azathioprin and prednisone. all were converted to double immunossupression with sirolimus at a standard dose of 2mg/day (tl5-10ng/ml) and prednisone. patients with ptld were also treated with rituximab, and the patients with wilms tumor received chemotherapy. mean follow-up after srl conversion is 35±25 months. results: all patients were maintained with srl and pred without rejection, with good renal function and no cancer recurrence (follow-up 6 to 75 months). the patients with wilms tumor are still on chemotherapy. follow-up control after srl conversion in the 6 patients: conclusion: although the intraperitoneal group had characteristics associated with increased surgical risk (they tended to be younger and smaller, with a higher incidence of aortic anastomoses and a higher incidence of multiple vessels), surgical complications were significantly lower than expected in the extraperitoneal group 7% v. 33%. objective of study: proteinuria is a frequent complication in adult patients after renal transplantation (r-tx) and is associated with poor graft survival. in children, there are no studies focusing primarily on proteinuria after r-tx. the aim of this study was to investigate the prevalence of proteinuria in children after r-tx and to evaluate changes of proteinuria during a 2-yr study on intensified antihypertensive therapy. methods: protein excretion was measured in 24-hr urine and proteinuria defined as >96 mg/m 2 /day. proteinuria was investigated at baseline, 1 and 2 years after intensifying of the antihypertensive therapy in children with uncontrolled hypertension at baseline (i. e. additional antihypertensive drugs given to children with blood pressure >95. pc). 33 children (13.7±4.3 yrs) out of 45 from our center fulfilled inclusion criterias (>6 months after r-tx, no acute rejection (ar) in the last 3 months, no recurrent fsgs). results: the prevalence of proteinuria was 82% at baseline, 76% after 1 year (ns) and it decreased to 52% after 2 years (p<0.05). the mean protein excretion was 256±303 mg/m 2 /day at baseline, 224±208 after 1 year (ns) and it decreased to 134±88 after 2 years (p<0.01). mean number of antihypertensive drugs increased from 2.1±0.9 drugs/patient to 2.7±0.8 after 2 years (p<0.01). mean nighttime bp decreased significantly after 2 years. the number of patients on ace-inhibitors increased from 19% at baseline to 39% after 2 years (p<0.05). conclusions: this is the first study on proteinuria in children after r-tx. it showed that proteinuria is a frequent finding in transplanted children and that intensified long-term antihypertensive treatment using ace-inhibitors can decrease not only bp but also proteinuria in these patients. allograftrejection involves t cell activation and proliferation and multipleinflammatory components. monocyte chemotactic peptide-1 (mcp-1) is achemoattractant and activating factor for monocytes. interleukin-6 (il-6) may contribute to monocyte recruitment, results intubulointerstitial damage. cytotoxic lymphocytes induce target cell death by ligation of the fas-fasligand. allelic polymorphisms in recipient genes coding for them reported to beassociated with variations of outcome in renal transplantation. the aim was to investigate impact of mcp-1 -2518 a/g and il6-174 g/c, fas-670 a/g polymorphisms on acute allograft rejection (ar). there were 20 males and 27 females, 19±4.6 years meanly; 21 of the graftscame from living-related donors, 26 were from cadavers. the controlgroup consisted of 150 unrelated, healthy individuals with similar ageand sex. ar group was composed by 17 patients experienced at least one ar episode within the first 6 months of transplantation. the non ar group was comprised by 30 kidney transplant patients without ar. there was no significant difference between renal transplant patients and healthy controls in genotype distribution of allelic frequencies of il-6, fas and mcp-1 polymorphisms. while il-6 and fas gene polymorphisms had no effect on the incidence of ar episodes, there was significant association with mcp-1. the distribution of the genotypes for mcp-1 -2518 a/g in ar group were aa/ag/gg 23, 6%, 64, 7%, 11, 7% respectively. the distribution of the genotypes for mcp-1 -2518 a/g was aa/ag/gg 60%, 30%, 10% in nonar group respectively. the carriage of g allele at -2518 position of mcp-1 gene has a significant association with ar (or: 4, 87, 95%, ci: 1, 27-18, 5). the il-6 and fas gene polymorphisms had no effect on the incidence of ar. mcp-1 -2518 g allele carriage increases the ar risk in turkish renal transplant patients. the high recurrence rate of focal segmental glomerulosclerosis (fsgs) in transplanted kidney recipients suggests the hypothesis that such patients have a circulating factor that changes glomerular capillary permeability. serum from patients with fsgs increases glomerular permeability to albumin, and this permeability factor was partially identified as a protein. the removal of this protein by plasmapheresis (pp) decreases proteinuria. object: the aim of this paper is to provide data about the therapeutic effect of pp in fsgs children with recurrence in the transplanted kidney. methods and results: twenty eight pediatric kidney transplant recipients had fsgs as cause of renal failure from 1990 to 2007 in our center, confirmed by biopsy pre-transplant. seventeen of these (60.7%) had a recurrence (proteinuria >1 g/m 2 per day associated with hypoalbuminemia). the mean age was 12±4.3 years, 85, 7% were caucasians and 67, 8% were performed with living donor. since 2001, patients who presented fsgs recurrence were treated with 10 cycles of pp (3 cycles/weekly), initiated immediately post-recurrence (n=11). immunosuppression comprised of cyclosporin in high doses (c 2 levels of 1700-1800 ng/ml) or tacrolimus (tl=10 mg/dl), mycophenolate sodium or mofetil (until 1997 azathioprine was used) and prednisone. among patients who received pp (n=11), 7 (63.6%) achieved a complete remission. there were no cases of remission among those six patients who were not treated with pp. those who achieved remission after pp had no recurrence. the patients treated with pp had infectious complications: one patient had cytomegalovirus disease and two patients had varicella. conclusion: pp appears to be effective in treating recurrent fsgs following kidney transplantation. it should be started as soon as possible. because the calcineurin inhibitors (cni) cyclosporin a (csa) and tacrolimus (tac) are drugs with a narrow therapeutic index, individualization of cni dosage by therapeutic drug monitoring is indisputable. however, the optimal strategy for monitoring cni therapy is currently under debate. dosing of cnis according to the molecular effect of the drug on its target cells could optimize immunosuppressive therapy with cnis. for this purpose, we developed a reliable, precise, and robust whole blood assay based on the measurement of the expression of three nfat-regulated genes (il-2, ifng and gm-csf) in pma/ionomycin-stimulated lymphocytes before and 1.5 (tac, c 1.5 ) or 2 hrs (csa, c 2 ) after oral drug intake. the inhibition of genes in this assay is independent from other commonly used immunosuppressive drugs and reflects calcineurin inhibition expressed as residual nfat activity. in a pilot study, 40 patients (mean age 14 yrs, mean time period posttransplant 42 mo.) were analyzed. in csa-treated patients (n=12), a mean c 2 concentration of 380 ng/ml (range, 180-900 ng/ml) corresponded to a mean residual nfat-activity of 32% (range, 3-115%) ; the correlation between individual residual nfat-activity and c 2 was moderate (r=0, 59, p<0.05). at a csa-c 2 of 400 ng/ml, the residual nfat-activity varied between 8% and 25%. in tactreated patients (n=28), a mean c 1.5 concentration of 18 ng/ml (range, 6-54 ng/ml) corresponded to a mean residual nfat-activity of 40% (range, 7-70%); the correlation between individual residual nfat-activity and c 1.5 was also only moderate (r=0, 60, p<0.05). conclusion: these data indicate that there is a considerable inter-patient variability of residual nfat-activity at a given maximal cni blood concentration. ongoing studies are validating this assay regarding clinical outcome criteria of immunosuppression such as acute rejection and infections. hus due to antibodies against factor h (husafhab) is a very rare disease for which a limited experience in its management is available. we present a case of husafhab who was transplanted but lost her graft after only 4 mos. in aug 2002 a 5-mos old child was admitted for a d-hus which did not go into remission and required chronic peritoneal dialysis. no fh and mcp gene mutation were detected nor adamst-13 activity was decreased. afhab were not searched at that time. in apr. 06 the child underwent cadaver renal transplant (rtx). the immunosuppressive regimen was basiliximab-prednisone-cyclosporine-mycophenolate mofetil. fifteen days after rtx, following surgery for urinoma, the child exhibited an hus relapse with thrombocytopenia, haemolytic anemia and increased serum creatinine (scr). high afhab were detected (2400 au/ml). four plasma exchanges (pe) were performed over 2 wks with a drop of afhab to control level (196 au/ml) and a remission of hus. until the end of aug, afhab remained low (<400 au/ml) and the child was well (scr range: 0.6-1.2 mg/dl). in early aug, the patient was shifted to tacrolimus for severe hypertrichosis with a fluctuation of its plasma level (lower recorded value: 1.4 ng/ml) signs of acute rejection developed and 4 metilprednisolone (mtp) pulses brought scr to baseline level. in oct. 06, following an urti, the child presented with severe proteinuria (upr/ucr: 15, 6) without other clear signs of hus recurrence. since afhab were increased (849 au/ml), pe was restarted but an acute hemorrhagic complication (hemotorax) occurred and pe had to be interrupted. septicemia followed and the child became anuric. the renal biopsy performed 3 days later showed signs of glomerular and vascular thrombotic microangiopathy. renal function did not recover despite mtp pulses and 4 pe. in nov the child was back to pd and in dec the graft was removed. background & aims: recently, the role of nitric oxide (no) in the pathogenesis of idiopathic nephrotic syndrome (ins) has been intensively investigated. however, its rapid turnover has made us impossible to investigate the quantity and the source. as we have developed a novel method for quantitative analysis for no by a new fluorescent indicator, 4, 5-diaminofluorescein (daf-2), its amount produced by both t and b lymphocytes in ins was studied. methods: five children with steroid-sensitive ins (mean age: 4.0 y) were included in this study, together with 7 children with other renal diseases (mean age: 5.0 y) such as chronic glumerulonephritis and alport syndrome with significant amount of proteinuria, and 10 healthy adults (mean age: 28.0 y) for the control. no production from cd3+ cell and cd19+ cell was investigated by a flow cytometry using daf-2. results were expressed as mean fluorescence intensity and were compared among the groups. results: the amount of no produced by cd3+ cell and cd19+ cell in children with ins was significantly greater than those in children with other renal diseases and healthy adults (cd3+ cell: 57.5±6.8 [mean±sd], 27.6±7.0, and 21.7±2.1, respectively; cd19+ cell: 35.7±3.5, 24.9±3.9, and 18.9±2.3, respectively, p<0.05). additionally, both cd3+ cell and cd19+ cell during nephrotic relapse produced more no than in nephrotic remission (p<0.05). discussion: patients with relapsing ins showed increased production of no by both t and b lymphocyte. these findings indicate that no plays some role in the pathogenesis of ins and suggest that an abnormal immune system may exist not only in t but also in b lymphocytes. a. bagga, a. sinha, s. menon, p. hari aim: the treatment of patients with srns is challenging. based on suggestions that b-lymphocytes are crucial in the pathogenesis of nephrotic syndrome, we examined the efficacy of rituximab (rtx) in patients with srns refractory to standard therapies. methods: six patients (4 with initial, 2 late resistance), 3-16 yr old, were included; biopsy showed minimal change & focal segmental glomerulosclerosis in 3 each. all had previously received iv high-dose steroids, alkylating agents & calcineurin inhibitors (cni) for 2-14 yr with periods of partial (pr; urine 1-2+) or complete remission (cr; urine trace/negative). all now had srns refractory to 6-months treatment with cni. rtx (375 mg/sq. m) was infused iv every week for 4 weeks. therapy with cni and/or alternate-day prednisolone, & cotrimoxazole prophylaxis was continued. patients were monitored for proteinuria and renal functions. results: at a median interval of 4 wk following the last rtx dose, cr was seen in 4 & pr in 2 patients. remission was sustained in 4 patients, despite tapering doses of steroids & cni. one case had relapse of nephrotic syndrome 6-months later, which responded to steroid treatment. at median follow-up of 28 wk, cr, pr and recurrence of nephrotic proteinuria (3-4+) were seen in 3, 2 and 1 patients respectively. mean urine albumin-to-creatinine ratio was 8.9 at baseline and 0.9 at followup; respective blood levels of albumin were 1.4 and 3.7 g/dl & cholesterol 481 and 221 mg/dl (all p<0.01, anova); difference in leukocyte counts and levels of igg were not significant and none had serious infections. conclusions: this is the first report on the efficiacy of rtx in sustaining remission in patients with srns. therapy with this agent appears promising for difficult srns, with a better risk/benefit profile than other medications. quantitative or functional deficiency of factor h results in uncontrolled complement activation and is an important cause of familial hemolytic uremic syndrome (ahus). factor h-related proteins (fhr) constitute a protein family which share structural and most likely functional similarities to factor h. we here describe complete deficiency of fhr-1/-3 as novel cause of ahus. factor h and fhr-1/-3 were quantified by elisa and were further analyzed by western blot using specific antibodies. complement activation was determined by measuring c3 and c4. serial hgb (g/dl), platelet, creatinine (mg/dl), and ldh (u/l) were measured. a 12 year old girl presented with a 5 day history of lethargy, pallor, vomiting and hypertension. hgb was 6 g/dl, platelets 40x10 9 /l and creatinine was 6.8 mg/dl. initial therapy consisted of packed red cell and platelet transfusion, followed by steroid therapy. representation occurred 7 weeks later with hypertension, edema, persistent anemia thrombocytopenia and renal dysfunction (creatinine 4.5 mg/dl). renal biopsy demonstrated features of chronic thrombotic microangiopathy. low c3 levels indicated activation of the complement system. while western blot analysis showed normal factor h level, fhr-1/-3 was absent. by repetitive plasma infusion and plasmapheresis, the cycle of hemolysis and thrombocytopenia could be disrupted. chronic periodical plasma infusion q 14 days resulted in regression of renal impairment to a degree (current baseline creatinine 1.5 mg/dl). in conclusion, fhr-1/-3 are thought to have co-factor activity and play a role in complement activation in ahus. deficiency of fhr-1/-3 may lead to a subclinical form of ahus such that patients may initially present with features of chronic renal failure. however, factor replacement therapy may lead to regression of renal impairment. s. choudhry objectives of the study: the aim of the study is to investigate the long term prognosis ofsevere childhood iga nephropathy after 2 years combined therapy. patients: we examined 39 patients who had entered thejapanese pediatric iga nephropathy treatment study group between 1990and 1994, had been treated with 2 years combined therapy (combinationof prednisolone, azathioprine, dipyridamole and heparin/warfarin) andhad been followed for more than 3 years after the therapy. significantproteinuria is defined as more than 0.2g/m 2 /day. results: mean age at onset was 11.4 years (4-16 years), proteinuria at diagnosis was 1.11±0.84 g/m 2 /day, proteinuria after the combined therapy was 0.17±0.22g/m 2 /day and mean follow-up period after the combined therapy was 7.2 years (3.2-9.9 years). proteinuria improved in all patients during thecombined therapy (p<0.0001). the final prognoses were as follows: 21 patients (54%) showed no proteinuria, 15 (38%) showed proteinuria withnormal renal function and 3 (8%) proteinuria with decreased renalfunction. we compared the clinical and pathological parameters betweenpatients with proteinuria (n=18) and those without proteinuria (n=21) at the last observation. period between disease onset and start of treatment, glomeruli showing crescents before the combined therapy (%) and glomeruli showing pathological changes after the combined therapy (%) were the significant risk factors for the proteinuria at the last observation. logistic multivariable analysis revealed that glomeruli showing pathological changes after the combined therapy (%) was theonly independent risk factor for the proteinuria at the last observation. conclusions: pathological activity of nephritis at the end of the combined therapy might correlate well with the final proteinuria and the long term prognosis. s. arun, a. bagga, s. bhatnagar, p. hari, s. menon, s. saini aim: relapses in ssns of ten follow minor infections and are associated with perturbed t-cell function. based on data that zn supplements modulate t-cell function and reduce risk of infections, we examined its efficacy in reducing relapses in patients with ssns. methods: in this double blind rct, 81 consecutive patients with ssns 1-16 yr old, stratified into frequent (fr=52) and infrequent (ifr=29) relapsers, were randomized to 12-months therapy with zn (10 mg daily) or placebo. patients with fr also received long-term, alternate day prednisone. relapse and infection rates were monitored monthly. blood levels of zn, sil2r, il4 and interferon (ifn) were measured at baseline, relapse andend of study. results: patientsin the zn (n=40) and placebo (n=41) groups had similar baseline clinical & laboratory features. the former showed 20% lower frequency of relapses (difference in means -0.2; 95% ci -0.7, 0.3) with trend towards reduction from 6-months onward. a higher proportion (44.7%) of patients in the zn group had sustained remission compared toplacebo (27.5%). reduction in relapse rates was higher in fr receiving zn vs. placebo (-0.3; ci -0.9, 0.3); respective sustained remission was seen in 46% and 16% patients (relative risk 0.5; ci 0.3, 0.9; p 0.02). no differences were found in infection rates, and levels of zn, sil2r & il4; levels of ifn were higher in those receiving zn compared to placebo (p=0.02). conclusions: zn supplementation for 1-yr was effective in maintaining remission and reducing relapse rates. the effect was mediated not by reducing infections but perhaps through effect on th1 cytokines. while zn therapy appears promising, these results need confirmation in patients with frequent relapses and perhaps at a higher dose. background: n-glycosylation process in the endoplasmic reticulum (er) is tightly regulated and orchestrated by many factors such as chaperones and energy systems. we showed that adequate nglycosylation is crucial for nephrin to assemble to the plasma membrane (jasn, 2002) . additionally we recently demonstrated that the er stress evoked by glucose starvation induces hypoglycosylated nephrin retained in the er, which is rescued by dexamethasone (dex) (ki, 2006) and immunosuppresant: mizoribine (mzr) (submitting). in the present study, we tested whether other er stress inducer, hypoxia, could also interfere the alteration of nephrin n-glycosylation system and whether dex and immunosuppressants rescue its defective process. methods: nephrin-expressing cell line was cultured either in 21% o 2 or 1% o 2 for 24 hours in the presence or absence of dex, mzr and cyclosporin a (csa), followed by western blot analysis with nephrin, cytochrome c. intracellular atp concentrations were measured by the highperformance liquid chromatography. protein expression of cyclophilin d (cyp-d), a component of mitochondrial permeability transition pore (mptp), was tested in the samples from human glomeruli and cultured podocyte. results: hypoxia induced hypoglycosylated nephrin. csa, but not dex and mzr, inhibited the formation of this hypoglycosylated nephrin and rescued mature form. csa inhibited the increase of cytochrome c in the cytoplasm caused by hypoxia. in addition, csa partially rescued the decrease of intracellular atp. cyp-d was distinctly observed in human glomeruli and located in podocytes. conclusion: csa inhibit the formation of hypoxia-induced hypoglycosylated nephrin through protecting the mptp opening via selectively binding to cyp-d in the mitochondria, resulting in a recovery of atp. csa may exert direct action on the alteration of nephrin biogenesis induced by the er stress. background and objectives: in two previous randomized controlled trials (rcts) we showed that treatment of severe childhood iga nephropathy (iga-n) with diffuse mesangial proliferation using prednisolone, azathioprine, heparin-warfarin, and dipyridamole reduced immunologic renal injury and prevented any increase of sclerosed glomeruli. in one of the two rcts we also showed that treatment with prednisolone alone did not prevent a further increase of sclerosed glomeruli. accordingly, the immunosuppressant is considered to play an important role in the combination therapy. often however, we were unable to complete the azathioprine regimen due to its severe side effects. therefore we considered that a different, but effective immunosuppressant would be worth trying. mizoribine, like azathioprine, is an antimetabolite that exerts its immunosuppressant effect by inhibiting lymphocyte proliferation. design, setting, participants, and measurements: in this pilot study, we administered mizoribine instead of azathioprine as part of the combination therapy for treatment of 23 children with severe iga-n and evaluated the efficacy and safety of the regimen. results: eighteen patients reached the primary endpoint (urinary protein/creatinine ratio <0.2) during the two-year treatment period. the cumulative disappearance rate of proteinuria determined by the kaplan-meier method was 80.4%. mean urinary protein excretion was reduced from 1.86 g/m 2 /day to 0.20 g/m 2 /day (p<0.0001). after treatment, the mean percentage of glomeruli showing sclerosis was unchanged in comparison with that before treatment. no patients required a change of treatment due to side effects. the efficacy and safety of the mizoribine combination seems to be acceptable for treatment of children with severe iga-n. objectives of study: alport syndrome is a hereditary renal disease which is normally diagnosed by either histopathological studies or a genetical analysis. we attempted to diagnose alport syndrome by means of immunofluorescence staining of cultured cells with collagen type 4 alpha chains obtained from voided human urine specimens. methods: the cells were cultured from the voided human urine of 1 patient with x-linked alport syndrome, 1 patient with sporadic alport syndrome, and 1 patient with autosomal-dominant alport syndrome. for comparison purposes, controls cells were cultured from the voided human urine of 1 patient with iga nephropathy, 1 patient with fsgs, and 1 patient with purpura nephritis. the cultured cells were stained by immunofluorescence techniques with collagen type 4 alpha 1, 2, 3, 4, and 5 chains. results: in the cultured cells of the controls staining for collagen type 4 alpha 1 and 2 chains were observed both in the extracellular matrix and in the cytoplasm while for the staining of collagen type 4 alpha 3, 4, and 5 chains were observed in the cytoplasm. in cultured cells of xlinked and sporadic alport syndrome staining of collagen type 4 alpha 5 chain was lost or attenuated. in the cultured cells of autosomal-dominant alport syndrome the staining patterns of collagen type 4 alpha 3, 4, and 5 chains were observed. the staining patterns of the collagen type 4 alpha 5 chain in cultured cells of alport syndrome correlated with that observed in renal biopsies obtained from alport syndrome patients as reported previously. hence, the staining of cultured cells with the collagen type 4 alpha 5 chain obtained from voided human urine may therefore be a potentially useful means of diagnosing alport syndrome in a non-invasive manner. the aim of our study: was to examine zeta chain expression in cd4+, cd8+ t cells and nk cells from ins children in active phase of the disease and in remission and to assess the effect of 24 h and 72 h anti-cd3 + ril-2 stimulation on zeta chain expression. we enrolled 11 ins children in relapse before initiating the therapy, 12 ins children in complete remission of proteinuria on prednisone for 2-4 weeks (2mg/kgbw/d) and 15 age-matched controls. zeta expression was determined by flow cytometry as mean fluorescence intensity (mfi). results: cd4+cells: mfi in relapse was higher than in remission and in controls. anti-cd3 + ril-2 stimulation had no impact on zeta expression in acute phase and in cotrols, but increased mfi values after 72h in pts with remission. cd8+cells: zeta expression stayed unchanged irrespective of the examined group or antibody stimulation. nk cells: there were no differences between mfi values before stimulation and in relapse after stimulation. in remission and in controls antibody stimulation decreased zeta expression. in controls, mfi values for nk cells were higher than those for cd4+ and cd8+ populations, but this preponderance disappeared after 72h stimulation. in remission, that nk cell predominance vanished after 24 h stimulation. in relapse, mfi values in all cells were comparable and stimulation had no impact on lymphocyte proportions. urinary protein, creatinine, and scd80 were measured. scd80 was measured using a elisa kit. scd80 was expressed as ng/g of urine creatinine. data were analyzed using anova and spearman rank correlation (src) tests. 1) scd80 urinary concentrations in patients with mlns in relapse were higher than patients in remission, healthy controls and other patients with proteinuria (anova p 0.0001) but similar to those seen in sle. 2) in patients with mlns relapse, scd80 concentration did not correlate with urine protein/creatinine ratio (src, r: 0.1719, p: 0.297) 3) in 3 patients with mlns, serial urine scd80 concentrations were measured over a period a time. scd80 was increased at the time of relapse and decreased toward normal range weeks before patients went into remission. conclusion: urinary scd80 is elevated in mlns patients in relapse. this increased urinary concentration is not due to the proteinuria, since scd80 was not elevated in other patients with glomerulopathies and proteinuria. upregulation of podocyte scd80 during relapse may play a role in the pathogenesis of proteinuria in mlns. aim: patients with mpgn type ii/dense deposit disease (ddd) and atypical haemolytic uremic syndrome (ahus) secondary to defective complement control are found to have a high prevalence of y402h polymorphism of factor h gene (cfh), which is linked to age-related macular degeneration (amd). however, no studies have looked into an ocular phenotype of children with complement based renal diseases, yet. methods: in two pediatric nephrology centres all patients with mpgn ii/ddd and ahus were identified and screened prospectively for the presence of y402h polymorphism and drusen maculopathy. all patients have been on immunomodulatory therapy at the time of screening. results: four children were identified (male:female=3:1) with mpgn ii/ddd and ahus. there were two children in either group. the median (range) age at examination was 8.6 years (3.5 to 13.3 years). of the four patients, all were found to have the y402h polymorphism. none of the patients had evidence of drusen at the time of examination. two patients with mpgn ii/ddd and one patient with ahus had additional factor h mutations, which were thought to be responsible for the renal disease. conclusion: in our study of children with mpgn ii/ddd and ahus we found a 100% prevalence of the y402h polymorphism of factor h gene, which puts these patients at higher risk for drusen maculopathy. therefore, all children with either mpgn ii/ddd or ahus should be screened for y402h polymorphism; and if found positive have regular follow-up ophthalmologic examination. in the future, should y402h be proven to be of functional significance with respect to complement control, there would be a role for plasma infusion or replacement of purified factor h for the treatment of both the renal and the ocular phenotype. we previously demonstrated that angiotensin-(1-7) is an endogenous ligand for the g-protein coupled receptor mas. the aim of this study was to evaluate the role of angiotensin-(1-7) mas receptor in kidney structure and function by using transgenic mice with genetic deletion of the receptor mas. mas -/-(knockout) mice were compared to mas +/+ (wild type) mice regarding renal function parameters and kidney histology. the animals were housed in metabolic cages to obtain 24-hour urine samples for measuring urinary volume, osmolality and microalbuminuria. at the end of the experiment, mas -/-and mas +/+ mice were sacrificed by decapitation to harvest the kidneys for histological analysis and immunofluorescence of collagen types iii and iv. the quantification of collagen expression was determined by confocal microscopy (zeiss lsm510). urinary volume was significantly reduced in mas -/-mice as compared to mas +/+ animals (1.32±0.49 vs. 1.80±0.83 ml/24 hs in mas +/+ mice, p<0.05). this change was associated with an increase in urinary osmolality (3846±796 vs. 3219±840 mosm/kg in mas +/+ mice, p<0.05) and albumin excretion (0.23±0.16 vs. 0.04±0.02 mg/24 hs in mas +/+ mice, p<0.05). the histological analysis showed a significant reduction in the glomerular diameter in mas -/-mice as compared to mas +/+ animals (51.84±0.73 vs. 58.25±0.79 μm in mas +/+ mice, p<0.05), where as any significant change was observed in tubular diameter. the immunofluorescence of mas -/-kidneys revealed a marked increase of the expression of collagen types iii and iv in periglomerular region as well as in external and internal medulla. collagen iv was also augmented in mesangial area of mas -/-mice. these results suggest that the receptor mas is critical for the regulation of renal structure and function. a circadian rhythm in calciuria is evident with a peak after 18:00 h, a lower excretion overnight and a nadir in the early morning. during the peak period both ca/creat ratio (figure) and calcium output frequently surpass the reference values of 0.21mg/mg and 4 mg/kg/day respectively. conclusion: a significant circadian variation in calciuria is evident in normal school age children, with peaks surpassing commonly used reference values for hypercalciuria. this peak excretion is independent from urine output, glomerular filtration and intake. the average 24 h excretion of 4 mg/kg is rarely surpassed despite this peaks. the management of children with secondary hyperparathyroidism is complicated and should start early in the course of renal insufficiency. in spite of an optimal management hyperparathyroidism is sometimes uncontrolled and calcimimetics like cinacalcet hydrochloride which directly stimulates calcium sensing receptors and potently suppress pth secretion are an alternative to parathyroidectomy. they are very promising agents, but paediatric experience is lacking. a 11 years old girl with a bardet biedl syndrome without medical care came last year with end stage renal failure. thanks to daily hemodialysis, serum phosphate level was kept within normal limits and phospho-calcic product remains below 4.4 as recommended. in spite of this treatment, serum pth level increased to 1297 ng/l. this toxic hyperparathyroidism with optimal monitoring of serum calcium, phosphate, vitamin d levels led us to start calcimimetics. a 1,5 mg/kg cinacalcet dose was administrated and induced a decline of pth level to 684 ng/l one month later. a 11 years old boy with recessive polycystosis and chronic renal failure was treated with calciumbased phosphate binders and sevelamer hydrochloride but with a poor compliance and a free diet which led him to hyperparathyroidism (pth to 813 ng/l). in addition, a parathyroid gland hyperplasia with two adenoma was individualized. start of calcimimetics treatment (less than 1mg/kg) led to a rapid decline in pth level from 813 ng/l to 457 ng/l. the two adenoma decreased in size. in case of hyperparathyroidism, with close monitoring of serum calcium, phosphate, alkaline phosphatase and vitamin d levels, calcimimetics are an excellent alternative to surgery, with safe use and low dose in these cases. preliminary favourable experience has been reported in children but paediatric experience is lacking. side effects result from chronic administration of steroids. the aims of this study are to analyze graft function and metabolic effects of low dose and withdrawal of steroid therapy. this is a single center pilot, one arm and prospective study. methylprednisone (mp) was decreased to 0.07±0.03 mg/kg/day (low dose) after 4 months. we studied changes in graft function, height velocity, lipid profile, body composition and bone mass after 1 year of low dose mp and after a 2 nd year following mp withdrawal. patients received daclizumab induction; tacrolimus and mycophenolate mofetil. the inclusion criteria was 1 st living related graft and pra <10%; 16 patients were enrolled and total follow-up was 2.5 years. age at transplantation was 3.1-21 years, 5 females, 9 prepubertal, 7 postpubertal patients. patients and graft survival were 100%, acute rejection (ar) occurred after 14 months of mp withdrawal in 2/9 (22%) prepubertal patients (banff 97, 1b). in prepubertal patients, at the moment of steroid withdrawal and 1 year later creatinine clearance was 99.7±15.2 and 82.9±3.9 ml/min/1.73 m 2 , p<0.001; height velocity 6.9±2.5 and 7.4±2.9 cm/year, p: ns, with a height increment of 0.31±0.1 sds, p<0.05 during the last year of follow-up. graft function did not change in postpubertal patients. in all patients at the moment of steroid withdrawal and 1 year later lipid profile was normal; fat body mass 10.0±6.7 and 9.2±5.5 kg, p: ns; lean body mass 29.3±11.4 and 31.7±12.8 kg, p<0.02; total skeleton bmd -1.0±0.6 and -0.7±0.8 sds, p: ns and lumbar spine bmd -1.3±1.04 and -0.86±0.86 sds, p<0.05. this study demonstrates that low dose and steroid withdrawal allow catch up growth, normal lipid profile with no fat accumulation. steroid withdrawal prevents bone loss with increment of lean body mass, but with a concerning rate of ar and graft function deterioration in prepubertal patients. children with x-linked hypophosphatemic rickets (xlh) usually present with progressive disproportionate stunting. we therefore conducted a 3 year randomized controlled trial on theeffect of rhgh therapy on body anthropometry (28 parameter) in 16prepubertal children with xlh (each 8 patients in rhghandcontrol-group). age at baseline was 7.3±2.0 yrs (mean±sd), height was -3.2±0.8 sd, calcitriol dosage was 23 ng/kg x day, and phosphate dosagewas 55 mg/kg x day (each p>0.05 rhgh-vs. control-group). results: within the whole study population longitudinal bodydimensions were more affected than transversal body dimensions aswell as circumferences (extremities/trunk). leg length (-3.7±0.9 sd; p<0.0001) was the most impaired longitudinal parameterexplaining 90% of the overall variability of total body height, whereassitting height (-1.7±0.8 sd) was the best preserved longitudinalparameter. longitudinal body dimensions were significantly increased after 12 months rhgh-treatment (height +0.72±0.18 sd; sitting height +0.61±0.39 sd, leg length +0.57±0.55 sd; arm length +0.74±0.59 sd; each p<0.001). in contrast, progressive stunting was noted in control patients (height -0.16±0.42 sd; sitting height -0.01±0.24 sd; leg length -0.1±0.56 sd; arm length -0.01±0.26 sd; each p<0.001 vs. rhgh-group). rhgh treated patients showed a significant increase in all transversal body dimensions (each p<0.001), as well as a decrease in skin folds (range -0.2 up-to -0.9 sd; each p<0.001). one year rhgh treatment in severely growth retardedprepubertal children with xlh leads to improvement of longitudinal bodydimensions, harmonization of body (i. e. transversal body dimensions & body proportions), and decrease of body fat. thalassemia is an hemolytic anemia characterized by decreased production of β globin. the chronic hemolysis requires frequent bloodtransfusions that caused hemosiderosis, including iron deposition in the kidney. removal of excess iron via iron chelators, the most commonof which is desferal produce iron excretion in urine and stool. few studies have been published, the studied patients, mostly adults, exhibited proteinuria, aminoaciduria, low urine osmolarity and excess secretion of the proximal tubular function markers, n-acetyl-d-glucoseaminidase (nag) and β2 microglobulin. the iron accumulation may causes lipid oxidative peroxidation and this oxidative process cause's tissue damage. in this study we examined 40 thalassemia major patients treated with desferal. the degree of hemosiderosis was determined by measuring serumiron and ferritin. 12 children without iron metabolism disorders orrenal disease serve as controls. the renal and tubular function was analyzed. no differences in creatinine clearance, blood hco3, na and k fe, tmp/gfr was found. serum uric acid was equal in the two groups but itsurine excretion was significantly higher in the thalassemic group probably due to persistent hemolysis. the nag level and its ratio to creatinine were significantly higher compared to the control group. the results of our work showed that most of the thalassemic patients have sub clinical disturbances in tubular function and high nag in theurine. these results raise the question of treating thalassemic patients with oral chelators which removes iron from cells preventing the oxidative process; moreover, and add antioxidants which may prevent the deposition of iron in tissues. in light of these findings, it would be advisable to routinely check glomerular and tubular function as part ofthe follow-up in these patients. objectives of study and methods: little information is available on the long-term follow-up in patients with biallelic mutations in the two genes slc12a1 and kcnj1, encoding the bumetamidesensitive na-k-2cl cotransporter and the in wardly rectifying renal potassium channel, respectively. we evaluated the long-term follow-up (>7 years) in 12 patients with these two forms of bartter syndrome. the slc12a1 and kcnj1 genes were screened by dhplc and sequencing techniques. results: the long-term follow-up period was 7 to 22 years, median 11, after diagnosis, in 8patients with mutations in slc12a1 (4 homozygotes, 4 compound heterozygotes) and 4 patients with mutations in kcnj1 (2 homozygotes, 2 compound heterozygotes) genes. medical treatment with indo methacin at last follow-up control including supplementation with potassium in 7patients and medical treatment with indomethacin in 10 patients (meandose 1.1 mg/kg/day). in two patients (one with slc12a1 and another one with kcnj1 mutations) growth hormone (gh) deficiency was detected with specific tests. both patients were treated with recombinant human gh. at the end of follow-up, body height was <3° percentile for agein 2 of the 12 patients, 1 of whom with gh deficiency and body weightwas <3° percentile for age in 3 patients (none of them with gh decifiency conclusions: these data demonstrate that some patients with biallelic mutations inthe slc12a1 and kcnj1 genes tend to present slight impaired glomerular kidney function after a median follow-up of 11 years and that growth retardation and gh deficiency are often present in these patients. the cytosolic c-terminus of nhe3 does not mediate its apical localization or baseline activity -implications for blood pressure and ph homeostasis the epithelial sodium proton exchanger, nhe3, is expressed in the apical membrane and subapical endosomes of the proximal tubule. apical localization is fundamental to proximal tubular na+, water and hco3-absorption, a process necessary for the maintenance of plasma ph and blood pressure. moreover the redistribution of nhe3 between these compartments alters its activity. for example, acute hypertension leads to an increased endomembrane accumulation of nhe3 and pressure natriuresis. nhe3 is composed of a n-terminus with 12 transmembrane helices and a cytosolic c-terminus. the former is necessary for sodium-proton exchange while the latter regulates activity. the goal of our studies was to evaluate the contribution of the cytosolic c-terminus to apical localization and therefore to nhe3 function. to this end we generated a series of nhe3 constructs with sequential deletions in the cytosolic c-terminus. all constructs contained an extracellular epitope tag to facilitate the delineation between cell surface and endomembrane nhe3. stable renal epithelial cell lines were generated expressing each construct. surprisingly, even when the entire c-terminus was deleted nhe3 was still detectable at the apical membrane. we proceeded to evaluate the activity of the truncated exchangers and found they retained activity. finally, we assayed the attachment of nhe3 to the actin cytoskeleton (a process thought to be mediated by the c-terminus), by measuring their solubility in the weak detergent triton x-100 and by measuring their mobility in the plane of the plasma membrane. both assays confirmed that exchangers lacking the c-terminus retained their association with the plasma membrane. in conclusion, the cytosolic c-terminus of nhe3 is not necessary for apical localization nor baseline nhe activity. further studies will be needed to confirm its role in specific regulatory processes. background: ibu is used as a safer alternative to indomethacine for pda treatment. however, safety/efficacy balance has not been extensively studied. animal and a few clinical studies suggested that ibu might also have significant side effects. objective: to evaluate renal function at one week of life in infants treated with ibu as compared to infants not exposed to ibu, within the first days of life. methods: multicentric prospective cohort study of 27 to 31 weeks gestation (ga) infants exposed or not exposed to ibu. infants presenting with renal impairment at birth, urinary tract malformation, or contraindication to ibu treatment were excluded. infants exposed to ibu were paired to controls according to ga, centre and crib score. creatinine clearance (ml/min/1.73 m 2 ) was measured for glomerular function evaluation; fractional excretion of sodium (fena) and 1microglobinuria/creatininuria (1/ucr) for tubular function evaluation. results: 120 infants were studied: 60 exposed to ibu for pda closure with 83.3% efficiency and 60 controls. birth weight was 1100±278 g (mean+sd), and ga 28.2±1.2 wks. glomerular filtration rate (gfr) significantly decreased on day 7 in ibu exposed infants. noteworthy, diuresis remained in the normal range, but was significantly lower after ibu treatment (given on day 2) as compared to controls. antiresorptives, particularly bisphosphonates (bp), offer a promising treatment inpediatric bone disease. bp have been successfully used to treat childrenwith osteogenesis imperfecta (oi), secondary osteoporosis, fibrousdysplasia, hypercalcemia. concerns exist regarding bone mineralizationand bone growth and transient adverse reactions. according to available literature, bp were successfully used in ~1000 children, intravenous pamidronate (pm) being the most frequently used one (>500 children, mostly with oi), followed by alendronate (al) (~300 patients with oi, secondary osteoporosis, hypercalcemia), risedronate, etidronate, zoledronate, clodronate, olpadronate. oi treatment with bp resulted in an increase in bmd, improved growth, relief from pain, mobility improvement, improved quality of life and a drop in fracture rate there are sparse data comparing oral and i. v. bp. oral and i. v. bp seem to have a similar effect in children with oi. daily al therapy seems to be safe and effective in children with oi, and daily or weekly administration of al led to increase in bmd in patients with secondary osteoporosis. there is scarcity of randomized, double blind, placebo-controlled or active comparator trials with bp in children. currently, double-blind, placebo-controlled study with risedronate andactive comparator trial with intravenous zoledronate in children with oi are planned. bp therapy should be used in context of a well-runclinical program with specialist knowledge in the management of pediatric metabolic bone disorders. other emerging antiresorptive agents include denosumab, glucacon-likepeptide-2 and calcitonin tablets. these drugs are tested in phase iii trials in adults. their applicability to children is likely to be discussed in the coming years. the current kdigo recommendations on classification or renal osteodystrophy recommend assessment of three areas of bone histology: turnover, mineralization, and volume. while lesions of bone turnover are prevalent in children treated with dialysis, little is know about the prevalence of mineralization defects and their response to therapy with vitamin d sterols and phosphate binders. we evaluated the skeletal mineralization (osteoid thickness (o. th.) and osteoid maturation time (omt)) in 207 patients ages 13±1 years treated with maintenance dialysis who were not receiving vitamin d sterol therapy. serum biochemical markers were: ca: 9.2±0.1 mg/dl, p: 6.2±0.2 mg/dl, pth: 660±40 pg/ml, alk p'tase: 390±21 iu/dl. 53% of patients with high turnover bone disease (95% ci: 43-62%) and 29% (18-41%) of patients with normal or adynamic bone had abnormal skeletal mineralization as reflected by both a prolonged omt and widened o. th. subsequently, a subset of 103 patients underwent treatment with calcitriol and calcium carbonate. while serum pth levels decreased by 36% (p<0.05) and bone formation rate decreased by 41% (p<0.05) with therapy, there was no change in o. th or omt from baseline. indeed, 67% (55-77%) of patients had abnormal mineralization after therapy. we conclude that mineralization defects are prevalent in children treated with dialysis and are not affected by current therapeutic options. further studies are needed to determine the pathophysiology and optimal treatment of these defects. introduction: clara cell secretory protein (cc16) is a protein synthesized primarily by non-ciliated bronchiolar epithelial cells, the clara cells. like other low-molecular size proteins, plasma cc16 is rapidly eliminated by glomerular filtration and reabsorbed by proximal tubular cells. this protein has been rarely studied in the paediatric age. the sensitivity of cc16 in urine was compared to that of b 2-microglobulin (b 2m) and n-acetyl-b-d-glucosaminidase (nag conclusions: cc16 is a good marker of the proximal tubular function. cc16 is related better with the urinary b 2m elimination, since both are low-molecular size proteins that are reabsorbed by the proximal tubule. sd. kim, bs. cho kyunghee university hospital, pediatrics, seoul, south korea background: many studies have demonstrated that arb prevents renal progression in patients with glomerular nephritis or diabetic nephropathy by inhibition of hmc proliferation and reduce of extracellular matrix expansion and glomerulosclerotic changes. however, the molecular effects of arb in cultured hmc have not been completely defined. we investigated differential gene expression by arb treatment on cultured hmc according to time sequence using cdna chip (affymetrix). methods: mc was grown in dmem with 10% fbs and then arb was treated on cultured mc. rnas of hmc at different time points (4, 8 , and 24 h after arb treatment and no treatment) were compared using affymetrix cdna chip. to validate the patterns of gene expression analyzed by the microarrays, some genes were selected and semi-quantitative rt-pcr was performed. results: among genes, humoral immune response, cytokine activity, il-8 receptor binding, chemotaxis, cell cycle arrest, and morphogenesis associated genes were down-regulated for 4, 8, and 24 h after arb treatment. they also showed different clustering according to their changing patterns. conclusion: the present study demonstrates profile of gene expression as time goes by after arb treatment on proliferation of human mc. gene expression by arb treatment on cultured hmc showed sequential changes. our results showed that chemotaxis and immune response associated genes were suppressed by arb treatment on hmc. further evaluation of individual genes will be conducted to elucidate molecular mechanism. podocin is the major component of the slit diaphragm, the site responsible for size and charge selectivity of filtration. mutations in the nphs2 gene, which encodes podocin can lead to steroidresistant nephrotic syndrome (srns). in this work we studied whether genetic changes of podocin might predispose to the development of sporadic non-familial srns in childhood. we screened for podocin mutations 50 children (26m/24f; mean age 11.9±0.7 years) with sporadic srns and 50 healthy controls. renal biopsy in srns patients revealed mesangial proliferative glomerulonephritis (n=21), focal segmental glomerulosclerosis (fsgs; n=13), membranoproliferative glomerulonephritis (n=13) and membranous nephropathy (n=3). the mean age of onset of srns was 8.8±0.7 years. all 8 exons of podocin gene of patients with srns and controls were amplified and direct dna sequencing was performed. there is one novel nonsense heterozygous mutation of c.328 g>t p.87glu>stop was identified in the 1 st exon of nphs2 gene in srns child with fsgs who has renal transplantation at the age of 2 years without recurrence of fsgs in her graft. we found one kind of single nucleotide polymorphism c.872+7a>g in the 7 th exon of nphs2 gene in 6% (3/50) srns patients (2 with fsgs; 1 with membranoproliferative glomerulonephritis) and 10% (5/50) children in controls. allele frequency of this polymorphism of the nphs2 gene in srns patients and controls was not different significantly. our results indicate that mutation-detected rate in the nphs2 gene in russian children with sporadic srns was 2%. the low mutation-detected rate and identified polymorphism c.872+7a>g in nphs2 gene unlikely predispose to the development of sporadic srns in russian children. further determining the slit diaphragm genetic profile of children with sporadic srns is warranted in order to improve disease classification and tailoring of treatment. for diagnose of essential hypertension is necessary except all causes of secondary hypertension (sh). case: family history of 17-year-old female patient was unremarkable for hypertension, incl. renal diseases. during last 6 months she had often headaches with intermittent vertigo. she was not anywhere examined. at admission on intensive care unit patient had headache, her pulse rate was 130/min, respiratory rate 12/min, bp (right arm) was 220/180 mmhg and the lower extremity bp 225/188 mmhg. other physical examination was normal, incl neurological exam. fundoscopy of the eyes did not show any evidence of hypertensive retinopathy. cardiac ultrasound showed mild left ventricular hypertrophy. abnormal laboratory parameters (without medication) were: hypokalemia (3.0 mmol/l), metabolic alkalosis (ph 7.48, hco 3 26.3 mmol/l), low urinary output of sodium (22 mmol) and chloride (<18 mmol), high urinary output of potassium (34 mmol). renal function tests were normal and no abnormalities were detected by renal ultrasonography, incl. doppler exam of renal blood flow. by large hormonal analyse were detected high plasma renin activity (pra; 12.8 ng/ml/hod; n.r. 0.5-1.9) and very mild increased serum aldosterone (ald; 0.63 nmol/l; n.r. 0-0.60). during the ct renal angiography was found solid mass in the upper part of the left kidney. a partial left nephrectomy was performed and histological exam revealed juxtaglomerular cell tumor (so-called reninoma, re). after the resection we stopped subsequently patients antihypertensive therapy. in a 3-month follow-up our girl was normotens. re is a very rare cause of sh. re typically present during adolescence or young adulthood. this cause of severe sh should be considered along renal artery stenosis in adolescents presenting with secondary hyperaldosteronism or in causes with renin-secreting tumor of non-renal origin. we report our preliminary molecular findings in twelve turkish cystinosis patients. the patients were 3 to 22 years; male/female ratio was 7: 5. all presented initially with severe failure to thrive, polyuria and polydipsia. cystinosis was diagnosed at age 1 month to 6 years. six of the patients reached end stage renal failure at ages ranging from 6.5 to 15 years necessitating renal replacement therapy; 3 are currently on hemodialysis, one is on capd, and two were transplanted. while three of remaining 6 have renal fanconi syndrome with proteinuria, 3 had kidney failure in varying degrees. molecular analyses involve an initial multiplex pcr, checking for the presence or absence of 57 kb founder deletion, and subsequent sequencing of the 9 coding exons of ctns. interestingly, none of the 12 nephropathic cystinosis patients carried the 57 kb deletion. instead, one patient had a new homozygous 10 kb deletion of exons 4 to 9 of ctns. one patient was homozygous for a known 4 bp deletion in exon 3, i.e., c.357del gact. two patients were homozygous for new missense mutations in exons 7 and 9, i.e., c.451g>a (r151g) and c.518a>g (y173c), respectively. the most common mutation in our turkish patients was a new exonic splice site mutation in exon 9, i.e., c.681g>a (e227e). of 24 alleles studied, 7 carried this mutation, which is expected to disrupt proper splicing. two patients were compound heterozygous for one of the above-mentioned mutations and a known missense mutation in exon 12, i.e., c.1015g>a (g339r). in two patients, we could not find any disease-causing mutation; in two patients, we could find only one disease-causing mutation. in summary, cystinosis patients of turkish ancestry show ctns mutations different from those of western european patients. these findings will be of relevance for molecular-based screening and diagnostic methods for cystinosis. introduction: the association of hypertension with bell's palsy in adults has been reported from 4-11%, but there is few data in children. the presence of bell's palsy in children requires a complete evaluation for hypertension, solid tumors, leukemia, neurofibroma, and trauma. hypertensive children usually present with clinical manifestations of underlying disease, but with substantial elevation, symptoms of hypertension develop. although headache, dizziness, blurred vision and seizure are common neurologic symptoms of hypertension, but facial paralysis is not a wellrecognized presenting feature of hypertension in children. case report: this paper describes two severely hypertensive children who referred to children's hospital of tabriz with periferal hemifacial nerve paresis and initial diagnosis of bell's palsy. case 1: a 12 years old girl who admitted with blood pressure of 230/120 mmhg on admission. renal ultrasound study revealed left small size kidney and renal scintigraphy followed by angiography confirmed renovascular hypertension. case 2: a 5 years old boy with blood pressure of 180/100 mmhg on admission. sonography of kidneys was normal except for a small size (4 mm) stone in left kidney. renal scintigraphy and angiography were normal. all other evaluations for etiology of hypertension including cardiac and endocrine investigations were negative. treatment of hypertension with antihypertensive drugs resulted in complete recovery of facial paresis in both cases within 3-4 months. conclusion: unilateral peripheral facial nerve paresis is a rare presentation of hypertension in children. unawareness of this presentation may result in delay in diagnosis of hypertension which may increase further with steroid therapy for bell's palsy. to uncover the frequency and the spectrum of nphs2 mutations in egyptian children with non familial steroid-resistant nephrotic syndrome (srns), 16 patients were screened by pcr-singlestrand confirmation polymorphism analysis of nphs2 gene followed by direct sequencing. nphs2 mutations were evident in 5 patients (31.3%) who were bearing five novel mutations including two frame shift mutations (713-714insg and 132-133insg), two missense mutations (556a>c and 647t>a) and a silent mutation (408a>t). there were no phenotypic or histological characteristics of patients bearing nphs2 mutations, apart from the earlier onset of the disease, compared to those who were not bearing mutations. in conclusion, nphs2 mutations are prevalent in egyptian children with non-familial srns and this may in part explain the less favorable prognosis reported in these patients. , which is an inherited systemic disease of connective tissue primarily affecting the skin, retina, and cardiovascular system, also leads to rvh. these symptoms of pxe are usually apparent in adulthood and rarely observed in children. here, we describe a very rare pediatric case of rvh caused by pxe. case report: a 6-year-old boy was noticed to have severe hypertension (183/128 mmhg) when he was admitted to our hospital for an operation for exotropia. he had no previous or family history of pxe or hypertension. laboratory examination showed that plasma renin (517.0 pg/ml) and aldosterone (71.3 ng/dl) concentrations were markedly elevated. his systolic and diastolic blood pressure (bp) decreased 12.7% and 22.2% from baseline after administration of captopril, respectively. a computerized tomographic scan of his abdomen showed multiple calcified vessels in kidneys and spleen. yellowish papules on the bilateral axillary regions and inguinal area were detected. characteristic histological changes of pxe such as elastic fiber mineralization and calcification were noticed in the biopsy of affected skin lesions. conclusion: pxe is characterized clinically by high heterogeneity in the age of onset and extent and severity of organ system involvement. although its symptoms usually appear in the second or third decade of life, gastrointestinal bleeding and acute myocardial infarction have been reported in childhood. we should also consider pxe as one of the causes of rvh in children, because its prognosis depends largely on the extent of extracutaneous organ involvement. the clcnkb gene is rarely reported as having a large deletion mutation, but all cases reported previously were large homozygous deletions and a large heterozygous deletion is impossible to detect by direct sequencing. patients and methods: this report concerns a genetic analysis of 5 japanese patients with type iii bs. to identify the mutations in the clcnkb gene, we used polymerase chain reaction (pcr) and direct sequencing to investigate all exons and exon-intron boundaries in the patients and their family members. to detect large heterozygous deletion mutations of the clcnkb gene, we conducted semi-quantitative pcr amplification using capillary electrophoresis. results: four mutations were identified, comprising 1 novel 2 bp deletion mutation (c.1334_1335delct), an entire heterozygous deletion and a heterozygous deletion mutation of exon 1 and 2 of the clcnkb gene. the nonsense mutation w610x in the clcnkb gene was detected in all patients (2 of them were homozygous and 3 were heterozygous) and this finding indicates that this nonsense mutation is likely to constitute a founder effect in japan. two patients had large heterozygous deletion of the clcnkb gene, which was proved by semi-quantitative pcr amplification. conclusions: capillary electrophoresis is a new method and extremely useful for detecting large heterozygous deletions, and should be employed to examine type iii bs cases in whom only a heterozygous mutation has been detected by direct sequencing. obesity defined as body mass index (bmi) above 95 th percentile for age and gender is regarded as a risk factor for cardiovascular (cv) target organ damage (tod) in children and adults with ph. however, when using percentile-based definition of obesity one may miss children at risk of cv complications. the aim of the study was to compare sensitivity of traditional definition of obesity (bmi>95 th pc) and bmi thresholds (tbmi) for cv complications described by katzmarzyk et al. (2004) majority of children with idiopathic nephrotic syndrome (frequent relapsers, steroid dependent and steroid resistant) require adjunctive therapy. the response to cyclophosphamide (cp) in these children is variable and difficult to predict. there may be an effect of polymorphic expression of gst on the remission with cp. in this study, we have tried to evaluate the correlation of gst polymorphism and response to cp therapy in these children we studied gst polymorphism in 73 consecutive children (54 males, 19 females) with steroid sensitive (n=44) and steroid resistant (n=29) nephrotic syndrome, receiving cp therapy. we evaluated the inter-relationships between gstm1, gstt1, and gstp1 genotypes and correlated it with the response to cp therapy in these children. the mean age of onset was 5.2±4.4 years. out of 73 children, 67% children responded to cyclophosphamide therapy. the null genotype of gstm1 and gstt1 was observed in 39.7% and 64.4% respectively while val105 genotype of gstp1 was seen in 22% children. there was no significant correlation among the various individual genotypic combinations. however, a trend was seen towards remission in children with a combination of gstp1 polymorphism with gstt1 null genotype (p=0.08) or combination of gstp1 polymorphism with gstm1 wild type genotype (p=0.0885) another important finding in our study was that there was a significantly higher frequency of val105 allele of gstp1 in responders to cyclophosphamide as compared to nonresponders (p=0.01). the results indicate that the presence of the val105 allele correlates with the response to cyclophosphamide in these children. gst gene polymorphism may be a significant therapeutic tool in the management of children with idiopathic nephrotic syndrome receiving therapy. objectives of study: primary vesicoureteral reflux (vur) is a common pediatric disease that may lead to reflux nephropathy and end-stage renal disease. the renin-angiotensin system (ras) was proposed to be associated with primary vur. the objective of this study was to investigate whether the gene polymorphisms of the ras are involved in primary vur and correlation with the severity of vur in taiwanese children. methods: we studied the angiotensin ii type 2 receptor (at2r) c3123a gene polymorphism for association with susceptibility to primary vur and disease severity in 100 vur children and 60 healthy controls. fifty four of the 100 vur patients had low-grade vur (grade i-iii) and 46 had high-grade vur (grade iv and v). to analyze the polymorphisms in the c3123a of at2r gene, the snp genotyping assay were performed. the genotypic frequency and allele frequency for four ras genes were analyzed to detect the correlation between the patients with mild, severe vur, and healthy control. results: we found that the c3123a of at2r gene was associated with the development and severity of vur. significantly higher c and lower a allele frequencies were presented in vur patients (c allele 0.67 and a allele 0.33) compared with controls (c allele 0.56 and a allele 0.44, p<0.005). the similar results were observed in both mild (c allele 0.73 and a allele 0.27) and severe (c allele 0.62 and a allele 0.38) vur compared with controls (p<0.005). the cc genotype was higher in vur patients compared with controls (p<0.005). conclusions: at2r c3123a gene polymorphism was associated with the development and the severity of vur in taiwanese children. it raises the possibility to utilize the genotype of at2r as a risk factor to evaluate the development and severity of primary vur. results: stone analysis led to diagnosis of cystinuria in 12 patients (1% of all), but only 5 were children (1.4% of the paediatric stone patients). age at diagnosis in three patients was 17-30 years and in four 30-49 years. time from first manifestation (pain 7x, gross haematuria 3x, urinary retention 2x, uti 4x) until diagnosis was 0-2 years in seven, 5-10 in three and even 34 and 39 years in two. renal function was impaired in 1 patient (ckd stage ii). the urinary cystine test was positive in all 9 patients with cystine stones examined, but was negative in 2 family members thus excluding cystinuria in them. family history of urinary stones was positive in two; the brother of one had died of renal failure due to bilateral urolithiasis. conclusions: delays in diagnosis of cystinuria in armenia are unacceptable. production of tablets containing nickel/dithionite which is non-toxic in contrast to brand test (cyanide/nitroprusside) is planned and could allow screening of all patients with stones of unknown origin. background: classically, childhood hypertension has always been recognized as secondary and frequently demanded an exhaustive etiological investigation. however, in the last two decades, many studies have been shown that pediatric patients also present primary hypertension and, sometimes, the adult disease probably begins during childhood. the aim of this study was to analyze a cohort of patients followed-up at a single tertiary unit (belo horizonte, brazil). methods: in this retrospective cohort study, the records of 220 diagnosed with arterial and followed at our unit between 1994 and 2004 were analized. a data base was used for statistical analysis. results: of 220 patients, 53.2% were male and 46.8% female. the distribution of patients according to the age at diagnosis was: 0-1.99 years, 3(1.4%), 2 yr-6.99 yr, 51 (23.2%), 7 yr-11.99 yr, 55 (25%), and 12 yr, 111 (50.4%). the causes for hypertension were: renal diseases in 172 (78%), primary hypertension in 33 (15%), renovascular disease in 9 (4%) and ohers in 6 patients (3%). the comparison between primary hypertensive subjects and patients with renal diseases showed that, although blood pressure was similar at admission, a better control was achieved in the first group (p<0.05). the frequency of overweight and/or obesity was higher in primary than in secondary hypertension ( nephrnophthisis (nphp) is a very rare cause of crf in korea. identification of five genes mutated in nphp subtypes 1-5 has linked the pathogenesis of nphp. ten percent of affected individuals have retinitis pigmentosa, constituting the senior-löken syndrome. we experienced juvenile-onset crf with leber's amaurosis in two sibilings. case 1: a 10-year-old boy presented with pale appearance. he had severe renal impairment and visual disturbance, but no symptom of polyuria and polydipsia. his past annual school screening urinalysis was normal. his growth and development were normal except opthalmological findings and pallor. case 2: his 14-years-old sister also had a visual disturbance and was found to have crf. there was no specific problem during perinatal period. opthalmologic findings were similar to her brother's. nphp1 and 5 on chromosome 2q13 was analyzed by direct sequencing. sequencing of mitochondrial dna-mbol digestion for 14484 mutation was analyzed by pcr-sequencing and rflp. electroretinogram revealed a decreased in amplitude of a and b waves. on the kidney biopsy, some of glomeruli were globally sclerotic. remaining showed no cellular proliferation or capillary wall thickening. interstitium was diffusely fibrotic and in which were scattered lymphocytes. most tubules were preserved well but a few dilated tubules were intermingled. no positive reaction for immunoglobulines or complements in if. by em, tubular membranes showed thickening, splitting and disintegration. nphp1 and 5 genes were not identified. there was no transition mutation at mitochondrial dna nt.11778, 3480, 14484, 15257. late-onset senior-löken syndrome were diagnosed in these cases even though there were no polyuria and polydipsia of initial symptoms of nphp and nphp1 and 5 were not identified. we need to identify other known or novel mutation genes. nail-patella syndrome (nps) is an autosomal dominant disease characterized by classic tetrad of dysplastic nails, absent or hypoplastic patellae, elbow dysplasia, and iliac horns. some patients manifest nephropathy and adult-onset glaucoma. nps is associated with mutations in the lmxib gene. there is marked inter-and intrafamilial variability in the phenotypes. in this study, phenotype-genotype correlation was analyzed in 7 unrelated korean children with nps. the probands were 3 boys and 4 girls. they manifested dysplastic nails (7/7, 100%), absent or hypoplastic patellae (6/7, 86%), elbow dysplasia (4/7, 57%), iliac horns (3/7, 43%) and nephropathy (2/7, 29%) . four missense mutations/2 in the lim-b domain (h114q and l127p) and 2 in the homeodomain (r200q and a213p)/and 1 frame-shifting deletion (c,680dela) were identified in the lmxib gene.r200q and a2123p are known to be common mutations, and r200q was detected in 3 patients in this study. autosomal dominant inheritance was identified in 3 patients by phenotype and genotype analysis of the family members and in 2 patients by phenotype analysis only. remaining 1 patient had de novo r200q mutation. one patient and her mother with r200q mutation developed nephrotic syndrome, which progressed to end-stage renal disease (esrd). another patient with h114q mutation had asymptomatic proteinuria with microscopic hematuria, and her father had esrd. galucoma was not detected in any patients or family members affected. there were inter-and intrafamilial variability of the phenotypes, but no genotype-phenotype correlation was identified. this is the first study to characterize mutations in the lmx1b gene in korean patients with nps. r200q is a common mutation in korean, also. the mechanism underlying the phenotypic variations and predisposing factors to the development nephropathy remain unknown. the pathological mechanism which would be responsible for higher production of digitalis similar compounds in essential arterial hypertension (ah), would be a genetically defect kidney, which causes difficulty in na + excretion. higher intake of salts can be an etiological factor inessential ah only patients with inherited abnormalities of thetransport mechanisms in the cell membrane.the objective of the studywas to establish the incidence and type of ah and frequency of genetic factor in the investigated population of 3000 school children (age 7-16years). in children with essential and borderline ah we evaluated the activity of erythrocyte membrane na+, k+-atpase in the presence ofvarying atp concentrations. atp, adp and amp levels and lipid peroxides (as tbars) in the erythrocytes and tbars in plasma were measured. our data have shown that the prevalence of ah is lowest in 7-8 years oldchildren, while it is the highest in 15-16 years oldchildren. essential ah was established in 11 (0.36%) and borderline in 17 (0.56%) children. genetic factor were found in 54.5% of children with essential ah and in 60.4% of children with borderline ah. but, statistical analyses of the first and second degree relatives of children with essential and borderline ah suggested normal prevalence of hypertension. atp, adp and amp levels, as well as, lipid peroxides were not significantly altered compared to healthy children. kinetic profileof erythrocyte plasma membrane na+, k+-atpase activity revealed the presence of noncompetitive (allosteric) inhibition of enzyme activityin the children with essential and borderline ah. the differences in the kinetic properties of na+, k+-atpase between two investigated groups of children suggested that this dynamic model could be used as potential biological marker for early diagnosis and differentiation of these two type of ah. majority of these patients were obese and with increasing body weight nighttime hypertension became prominent in these patients. objectives: it has been shown that weight gain is directly related to increase inblood pressure. the objective of this study was to analyze the frequency of overweight/obesity in children/adolescents with primary hypertension (ph) and the relationship between the overweight/obesityand the stage of hypertension (h). methods: we analyzed the data of 113 patients aged 10-17.5 years (m 65; f 48) diagnosed with primary hypertension. overweight/obesity was defined asperc. of bmi -85/95 c. the stage of hypertension was determined according to the 4 th report on the diagnosis, evaluation,and treatment of high blood pressure in children and adolescents of the nhlbi working group. results: out of 113 pts, prehypertension was found in 10 pts (8.9%), stage i h in 30 (26.5%) and stage ii h in 73 pts (64.6%). stage ii h was more frequentin boys (69% vs. 58% in girls). combined systolic/diastolic h was found most frequently, in 58 pts (51.3%), isolated elevation in systolic blood pressure (bp) was found in 47 pts (41.6%), while only 8 pts (7.1%) had isolated elevation in diastolic bp. sixty-one pts (53.98%) were found overweight/obese; of which 33 (54.1%) above 95c bmi. theobese children were predominantly found to have stage ii h: 84.8% ofthe obese children. none of the obese children had prehypertension. contrary to the general perception, boys were found more frequently overweight/obese (67.2% boys vs. 32.8% girls). conclusion: our data shows that stage ii ph is not rare also in theteen-age group. frequently it is accompanied with overweight/obesity, especially in boys. we can conclude that the roots of ph in the adultage are already set from the childhood. our efforts should focus onearly diagnosis of ph and include prevention of obesity as the possible contributing factor for the development of ph. objectives of study: alport syndrome (as) is a progressive renal disease characterized by hematuria, progressive renal failure, high-tone sensorineural hearingloss and ocular lesions. xlinked dominant alport syndrome (xlas) isthe major inheritance form, accounting for almost 80% of the cases, caused by mutations in col4a5 genes. there are no good cures available for as at present, but there are some patients who requestfor prenatal diagnosis and genetic counciling. this study performed thefirst prenatal diagnosis in chinese as family. methods: the entire coding sequence of col4a5 mrna of peripheral blood lymphocytes was amplified using nest pcr to screen mutations in a chinesexlas family. mutation analysis of the fetus was performed on both cdna-based level and dna-based level of amniocytes. fetus sex was determined by pcr amplification of sry as well as karyotypes analysis. maternal cells contamination was excluded by linkage analysis. results: mutation screen showed that there was a g to a substitution at position 4271 in exon 46 of col4a5 gene (c.g4271a), it was subsequently confirmed at genomic dna level by pcr amplification of exon 46 of col4a5gene. the mutation was identified in the index case, family members aswell as the pregnant lady. the prenatal diagnosis showed that fetus didnot carry the same mutation as the mother detected by both cdna-basedand dna-based mutation analysis. pcr amplification product of sryas well as karyotypes analysis revealed a male fetus. linkage analysis of the three x chromosome markers showed that there was no contamination of maternal cells in amniocytes. conclusion: after mutation identification of col4a5gene in a large chinese as family, a successfully prenatal diagnosis was performed in one of the female members in this family based on both cdna as well as genomic dna level of amniocytes. objectives of study: alport syndrome (as) is a clinical and genetic heterogenousdisease. autosomal recessive as (aras) is caused by mutations in col4a3 and col4a4 genes, accounting for 10% of the cases. thin glomerular basement membrane nephropathy (tbmn), is also caused by mutations in col4a3 and col4a4genes, inherited as an autosomal dominant trait. differentiation between early stages of aras and tbmn may be difficult in clinic, itdepends on gene mutation analysis of col4a3 or col4a4. methods: the whole entire coding regions of col4a3 and col4a4 mrna of peripheral blood lymphocytes were analyzed using reverse transcription polymerase chain reaction (rt-pcr) and direct sequencing to screen mutations in three chinese aras families, the corresponding exon with flanking intronic sequence was further amplified to confirm the abnormality. results: both the entire coding regions of col4a3 and col4a4 mrna were successfully amplified and completely sequenced by seven overlapping pcr products, respectively. results showed that there were 10 variations of col4a3 and 4 variations of col4a4, respectively. among the 10 variations of col4a3,three were snp reported previously, four were new snp, one nonsense mutation, one small insertions and one splicing mutation, the latter three were the pathogenic mutations for the three families, respectively. all the four variations of col4a4 were snp reported previously. we concluded that rt-pcr and direct sequencing using peripheral blood lymphocytes rna is a practical, sensitive and feasible approach for analysis col4a3 and col4a4 gene variants in autosomal recessive alport syndrome patients, which offers a useful, inexpensive and timesaving approach for systematic gene analysis in patients with aras. objectives of study: gitelman syndrome (gs) is an autosomal recessive tubular disorder characterized by metabolic alkalosis, hypokalemia, hypomagnesemia and hypocalciuria. the majority of gs patients carry inactivating mutations of the slc12a3 gene encoding the sodiumchloride cotransporter located in the distal convoluted tubule. this study aimed to detect mutations in a chinese family with gs. methods: two brothers presenting muscle weakness, hypokalemic metabolic alkalosis, hypomagnesemia and hypocalciuria were clinically diagnosed as gitelman syndrome. the two brothers, as well as their healthy brother and parents, were detected for mutations in slc12a3 gene by direct pcr for each exon and then sequencing on genomic dna extracted from peripheral blood cells. results: the two patients were found to have the same compound heterozygous mutations (c.917c>t and ivs 14-8t>c) in the slc12a3 gene. the two mutations were also detected in paternal and maternal genomic dna, respectively. the unaffected brother had one mutation (c.917c>t) only. conclusion: a novel compound heterozygous mutation on the slc12a3 gene was revealed in a chinese family with gs. the objective: to determine the clinical value of ambulatory blood pressure monitoring (abpm) in pediatric kidney disease. methods: 83 patients with common kidney diseases aged from 5-16 yrs were enrolled. 24-hour abp were performed by welch allyn abpm6100. the number of cases whose ccr>90mmol/l/1.73 m 2 , ccr 60-89 mmol/l/1.73 m 2 , ccr 30-59 mmol/l/1.73 m 2 , ccr 15-29 mmol/l/1.73 m 2 and ccr<15 mmol/l/1.73 m 2 is 64, 4, 5, 1 and 9, respectively. seven patients only took fosinoprilto control blood pressure did abpm again more than one weeks after therapy. 1141 healthy children performed in german in 1997 was used asthe normal data. casual bp was measured by sphygmomanometer. 23 children took echocardiography to calculate left ventricular mass index (lvmi). results: the incidence of nocturnal hypertension was significantly higher than that of diurnal hypertension (p<0.001).theincidence of non-dipper in 83 children with kidney disease was 68.7%. nocturnal dipping rate in the patients was significantly lower than that in the healthy children (p<0.01). the incidence of masked hypertension and white coat hypertension inchildren with kidney disease were 3.6% and 9.1% respectively. nocturnal systolic and diastolic dipping rate of the children with lupus nephritis and acute glomerulonephritis were lower than those of the children with henoch-schönlein purpura nephritis (p<0.05). lvmi hadpositive correlation with 24-hour diastolic blood pressure load (r=0.414, p=0.036), but it had no correlation with casual bp. after taking fosipril, 24-h, diurnal and nocturnal average abp decreased significantly (p<0.05). nocturnal dipping rate increased, but did not reach statistical difference (p>0.05). conclusions: abpm was an effective tool for diagnosis and management of hypertension in childhood with kidney disease. identification of 6 novel mutations in the col4a5 gene of japanese patients with x-linked alport syndrome background: alport syndrome consists of nephritis, often progressing to renalfailure, and sensorineural hearing loss. x-linked phenotype (omim#301050) is the result of mutation in the gene for the alpha-5 chain of collagen iv (col4a5). objectives of study: to find mutations causing x-linked alport syndrome in japanese patients. patients and methods: diagnosis criteria of alport syndrome were proteinuria, familial history of renal failure andchanges of gbm in electron microscopy. to identify the mutations in the col4a5 gene. results: for the time being we finished genetic analysis of 8 patients with suspected x-linked alport syndrome, 6 males and 2 females. we identified mutations in 7 of 8 patients; 6 of them were novel mutations. they comprise: 2 nonsense mutations, 2 missense mutations and 3 mutations of splicing acceptor site resulting in exon skipping or truncation, and establishing of premature stop codon. in previous reports of x-linked alport syndrome, mutation detection rate was around 60%. the present study provides a detection rate of 87.5%, although our number of examined cases is limited. conclusions: direct sequencing of rt-pcr and pcr products is efficient method of finding mutations in col4a5 gene. we found mutations in high percentage of investigated patients. objectives of study: to make a genetic diagnosis of juvenile nephronophthisis for a chinese boy. methods: analyze the presentation, family history, laboratory investigations, and histological features of a patient suspected of juvenile nephronophthisis, make a clinical diagnosis. to confirm the diagnosis on genetic level by pcr amplification of satellite markers located within the known homologous deletion of nphp1,including del-2, del-9, del-16, del-5-(5)2, del-10 and markers outside the deletions (ranbp11/12 and d2s1896)as control. results: the boy was nine years old, he was admitted becauseof renal failure for 6 months. this case manifested by school age, negligible onset, anemia, polyuria, renal failure at 9 years old,without hypertension and abnormal urine analysis. his sister presented with anemia at 1 year old, and died of uremia at 6 years old.laboratory investigations showed no proteinuria and hematuria. thekidney size is normal, and the cortical medullary boundary is obscure.the histological features consist of the disintegration of tubularbasement membrane, the atrophy and dilation of renal tubules, theinterstitial cell infiltration and fibrosis. he was suspected asjuvenile nephronophthisis. by pcr amplification, we found the missingof the satellite markers of del-2, del-9, del-5-(5)2 and del-10, which indicate that there is the common large homologous deletion (250kb) in the child's nphp1. conclusions: nephronophthisis is the major hereditary cause ofchronic renal failure. the boy was suspected of nephronophthisis because of chronic renal failure, family history, normal kidney sizeand hitological features. according to the age of renal failure, he was suspected as juvenile nephronophthisis. the diagnosis was confirmed by gene analysis. it is the first juvenile nephronophthisis case for chinese confirmed by gene analysis. cutaneous small-vessel vasculitis is a rare condition in children and is commonly associated with a wide spectrum of systemic inflammatory conditions, malignancies, infections or drug hypersensitivities. enalapril is anangiotensin-converting enzyme inhibitor, commonly used for the treatment of hypertension. cutaneous vasculitis due to enalapril has very rarely been reported and only with adults. we report a case of an 8-yea-old boy presented with a pruritic eruptionover his lower legs that started 6 hours after he had initiated treatment with enalapril at a dose of 0.5 mg/kg/daily. he had a history of hypertension due to a shrieked right kidney since infancy and he wason treatment with nifedipine and propranolol for the last three years. this medication was discontinued because it was ineffective, the day before initiation of enalapril. clinical examination revealed palpable purpuric lesions involving lower legs and ankles. he was otherwise well. a skin biopsy of the lesions showed leucocytoclastic vasculitis of small vessels. abnormal investigations were elevated c-reactive protein, esr and leucocytosis. further laboratory tests including creatinine, liver function tests, urineanalysis, antinuclear antibody, cryoglobulins, viral serology, complement levels, were normal or negative. enalapril was discontinued and he was started oral prednizolone and ceterizine over the next ten days. the skin eruption regressed rapidly. cutaneous vasculitis induced by enalapril is a rare adverse effect and it is essential a prompt recognition and withdraw of the suspicious drug. objectives of study: barttin, the gene product of the bsnd gene involved in bartter's syndrome with sensorineural deafness, is an essential b-subunit for clc-ka and clc-kb chloride channels, and it is expressed in the kidney as well as in the inner ear. one patient affected by deafness and renal bartter phenotype without bsnd mutations has been previously reported with simultaneous mutations in both clcnkb (responsible for classic bartter syndrome) and clcnka genes. we report here on a new case of a 6-months-old boy presenting such a disease. a severe polyhydramnios was detected during the pregnancy. he presented also polyuria, growth retardation, nephrocalcinosis and sensorineural deafness. laboratory studies revealed metabolic alkalosis (plasma hco3 47,7 mmol/l), hypokalemia (2,4 mmol/l), hypercalcuria (cau/cru 1,6 mg/mg) and elevated plasma renin (320 pg/ml). the aim of the study was to identify the cause of the severe renal salt wasting and sensorineural deafness in this patient. methods: dna sequencing analysis was performed on the bsnd, clcnkb, and clcnka genes. results: no mutation was detected in the bsnd gene. we identified a reciprocal partial homozygous deletion of exons 1-6 for the clcnkb gene and theloss of exons 7-19 for the clcnka gene. conclusions: the disruption of both clc-ka and clc-kb chloride channels leads to a syndrome clinically not distinguishable from bsnd, characterized by severe salt wasting and deafness. this digenic disorder is due to simultaneous mutations on the two genes clcnka and clcnkb respectively. the tight topology of the highly homologous clcnka genemight predispose to an unequal crossing over leading to partial orcomplete deletions of the clcnkb gene. we hypothesize that thischimaeric resulting gene interferes with the correct function of both the channels clc-ka and clc-kb, and leads to a bsnd-like phenotype. familiary hypomagnesemia with hypercalciuria and nephrocalcinosis (fhhnc) is a nautosomal recessive tubular disorder that is frequently associated with progressive renal failure. mutations in cldn16 which encodes the renal tight junction protein paracellin-1 were identified as the underlying genetic defect. in this work we present a gipsy family with fhhnc in which the mother and daughter both showed homozygous mutations in the cldn16 gene. a three-year-old girl was investigated after acute pyelonephritis and was found to have medullary nephrocalcinosis, hypomagnesemia (0,5 mmol/l), hypercalciuria (10,5 mg/kg/d), hypermagnesuria (femg 15%) and incomplete distal renal tubular acidosis. her renal function was normal. her mother demonstrated also bilateral medullary nephrocalcinosis, hypomagnesemia (0.2 mmol/l) with high femg (86%), moderate renal failure (creatinine 234 umol/l) and high ipth levels (398 pg/ml). as a child she had afebrile seizures, but serum mg levelwas not measured. index patient's uncle had history of recurrent passage of calculi in childhood, bilateral medullary nephrocalcinosis, normocalcemic hypercalciuria but his serum mg was not determined. he developed obstructive uropathy due to impaction of the calculus in urethra and eventually progressed to terminal uremia and later was transplanted. mutational analysis confirmed that the index case, motherand uncle were homozygous for the common mutation in the cldn16gene (leu 151 phe), while the father was heterozygous. we present a new family from macedonia with fhhnc with unusual presentation over two generation. mutational analysis confirmed also the diagnosis of fhhnc in the uncle although as a child he was considered to have idiopathic hypercalciuria. thus, serum mg should be routinely checked in children with nephrolithiasis/nephrocalcinosis. m. pan ' czyk-tomaszewska, j. s ' ladowska, j. so l /tyski, m. roszkowska-blaim arterial hypertension is one of the complications of reflux nephropathy. the aim of the study was to assess the risk factors of arterial hypertension in children with primary vesicoureteric reflux (vur). we studied 150 children aged from 4 to18 years mean 9±3.3, 82 with unilateral and 68 with bilateral vur. in all children ambulatory blood pressure monitoring (abpm) and 99m tc dmsa renal scanning (dmsa) were performed and z score body mass index (z-score bmi) were calculated. the following criteria were examined as predictive risk factors: age, gender, age at diagnosis andat resolution of vur, period between diagnosis and resolution of vur,grade of vur, type of vur (unilateral or bilateral), treatment modality (medical, surgical), z-score bmi, grade of dmsa and birth weight. results: i grade of dmsa scan was diagnosed in 27 children, iigrade in 85, iii grade in 31 and iv in 7 patients. hypertension (hp) was diagnosed in 20 children (13%). the mean value of z-score bmi in children with hypertension was significantly higher in comparison withchildren with normal blood pressure (0.043±0.21 and 0.74±0.86). the multivariate discriminate analysis showed that zscore bmi and grades of dmsa scars were significant risk factors for developing arterial hypertension in children with vur. both parameters had the same influence on development of hypertension (standardized coefficient of discriminate analysis 0,748 and 0,736 respectively). conclusion: the development of hypertension in children with vur is associated with higher bmi and higher grades of renal scars in dmsa scan. grange syndrome comprises arterial stenoses with hypertension, brachy syndactyly, bone fragility, learning disability, and cardiac defects and it has been reported in the members of two families up to now. we report here a 14-year-old boy with hypertension, multiple arterial stenoses, microcephaly and brachysyndactyly. severe hypertension (200/120 mmhg) was detected on his routine control for acute rheumatic carditis and he was hospitalised for investigation. he had no complaints. his parents were first degree relatives. his physical examination revealed microcephaly, bilateral brachysyndactyly between the forth and the fifth fingers of hands and the second and the third fingers of feet. complete blood count, electrolytes, renal, thyroid, and hepatic functions and acute phase reactants were all in normal limits. echocardiography showed aortic and mitral insufficiency and left ventricular hypertrophy. renal and cerebral magnetic resonance angiographies demonstrated stenoses at bilateral renal, internal carotid, superior cerebral and posterior cerebellar arteries. association of hypertension with microcephaly and finger abnormalities and multiple arterial stenosis suggested grange syndrome. chromosomal analysis showed 46 xy karyotype. hypertension persisted despite triple antihypertensive agents and percutaneous renal angioplasty was performed leading to a reduction of antihypertensive medication. with this case report we wanted to remind the recognition of this extremely rare syndrome in a hypertensive child with multiple morphological abnormalities and percutaneous angioplasty as a treatment modality in cases with persistent severe hypertension. hypertension is usually indicative of an underlying disease process in children. the combination of severe hypertension with hyponatremia is called hyponatremic hypertensive syndrome (hhs). in this report we present a case of hhs. the boy was referred to our hospital at the age of 7 years old with generalized tonic clonic convulsion. his past history was insignificant and positive findins of the patients were as follows: hypertension (170/115 mm/hg), agressive behaviors after convulsive period, metabolic alkalozis (blood gases, ph: 7,51, hco 3 : 24,8, pco 2 : 27,3), hypocloremia (87 meq/l), hypokalemia (3 meq/l), hyponatremia (123 meq/l), low urine density, polyuria and high serum aldosterone levels (1000 pg/ml, range: 20-240). the serum renin, cortisol, tyroid and antidiuretic hormones were within normal range. abdominal color doppler ultrasound were revealed double renal arteries in both kindneys. cranial and abdominal tomography examinations and cebrospinal-fluid examination were normal. the diagnosis of hhs was suggested based on these clinical and laboratory findins and therapy of nifedipine was started. the arterial pressure improved with this therapy. in hss due to renal ischemia, activation of renin angiotensin system which induce pressure natriuresis and thus, thence hyponatremia. we think that presence of renal arterial anomaly might be a cause of an ischemic condition and activation of renin-angiotensin-aldosterone system in our patient. also, activation of aldosterone could enhance with hyponatremia and it could be suppressed of renin secretion in our patient. [hadtstein et al. hypertension 2004] . the aim of this study was to analyze whether children with renal scars have altered rhythms of mean arterial pressure (map) and heart rate (hr). study design: ambulatory 24-hour blood pressure profiles of 61 untreated patients with renal scars associated with recurrent urinary tract infections and vur were investigated and compared to 938 healthy controls. results: pre-pubertal, but not pubertal patients with renal scars displayed a number of significant differences to controls. before puberty, the mesors of map and hr were significantly elevated, and the peak-trough difference of the 24 h curve was blunted by 7.5 mm hg for map and 7.8 bpm for hr. the amplitudes of 24 h and 8h map rhythms were blunted by 2.0 and 1.5 mmhg, and those of 24 h and 12h hr rhythms by 1.9 and 1.2 bpm (all p<0.05). pubertal patients did not display any abnormalities of bp or hr rhythms. we did not find any correlations of the degree of renal scarring, bmi or gfr with the abnormalities in cardiovascular rhythms. conclusion: in summary, pre-pubertal children with renal scarring due to vur show blunted circadian and ultradian rhythms of bp and hr. this phenomenon may reflect subtle alterations of autonomous nervous signaling in children with damaged kidneys; it remains to be addressed whether such abnormalities constitute an independent cardiovascular risk factor. mutations in the eya1 gene are associated with bor/bo syndrome and rarely with an isolated renal phenotype. we present a family from macedonia displaying a novel eya1 gene mutation. a six-year-old female was found to have bilateral grade iii vesicoureteral reflux (vur) after an episode of acute pyelonephritis. mutational analysis detected a novel eya1 gene mutation [an inframe 3bp deletion (c.4_6delttg), resulting in l2del of eya1a]. she had no clinical stigmata for bor/bo syndrome. the paternal grandmother died due to end stage renal failure. all first grade relatives underwent detailed physical and ophtalmological examination, audiometry, ultrasound screening of the urinary tract and mutational analysis of the eya1 gene. the father and older sister were mutation carriers and both had normal ultrasound findings. the physical examination did not reveal abnormalities suggestive for bor/bo syndrome, except the sister had esotropia/ hypermetropia and horizontal nystagmus. the male sibling was found to have mild hydronephrosis on the left side. he was not a mutation carrier and cystographic study was not performed at that time. on repeat ultrasound examination he demonstrated dilatation of the right prevesical ureter. the cystography revealed presence of right sided vur grade iii. in conclusion: we present two siblings with vur; although eya1 gene mutation was found in one of them, it can not be incriminated for the renal phenotype in this family, as co-segregation of the mutation with the vur does not occur in all affected members of the family. decision to perform cystographic study should be still based on clinical grounds. associations asymmetric dimethylarginine (adma) is a novel predictor of cardiovascular (cv) outcomes in adults. aim: to evaluate adma in children with hypertension. subjects: 23 children (4 girls/19 boys; median age: 15 yrs) with primary (n=10) and secondary (n=13) hypertension. methods: adma levels were analyzed by elisa method. in study subjects antropometrical data, ambulatory blood pressure monitoring (abpm), left ventricular mass index (lvmi), carotid artery intimamedia thickness (cimt), 24-hour urinary albumin excretion (uae) and serum biochemical markers (lipids, uric acid) were also evaluated. results: adma concentration was positively correlated to serum uric acid (r=0.42, p<0.05) and uae (r=0.45, p<0.05). when analyzed in boys separately, more apparent correlation was found for uae (r=0.57) and a tendency for adma to be associated with lvmi r=0.44, p=0.059). interestingly, when analysis restricted to older children (above the median age), adma correlated to lvmi more significantly (r=0.65, p<0.05). no relation was found of adma to cimt, lipids, and abpm parameters. moreover, patients with left ventricular hypertrophy (43%) and microalbuminuria (55%) had a tendency for higher adma values compared to those without these abnormalities (2.5±0.83 vs 1.74±1.1 μmol/l, p=0.081 and 2.39±1.23 vs 1.66±0.74 μmol/l, p=0.13, respectively). when data analyzed on the basis of adma values, patients in the top quartile showed the worst profile. when compared to the lowest quartile, they had higher lvmi (p<0.05), higher uae (p<0.05; inter-quartile comparison p=0.012), and higher uric acid concentration (p=0.052). conclusions: the associations of adma with known factors of cv damage found in this study provide evidence that adma is closely linked to the early cv dysfunction in hypertensive children. nephrogenic diabetes insipidus (ndi) is a disease characterized by unresponsiveness of distal tubule and collecting duct to vasopressin. although ndi usually presents with polydipsia and polyuria, most infants do not have any of these symptoms and presentation is with features of dehydration like fever, constipation, vomiting, failure to thrive and developmental delay. so, the diagnosis is usually delayed until hypernatremia is noted. almost all infants go through a period of hypogammaglobulinemia at approximately the 5th-6th months of age. at this time, the serum ig g level reaches its lowest point, and many normal infants begin to experience recurrent respiratory tract infections. the clinical findings of ndi and transient hypogammaglobulinemi of infancy (thi) may be seen at the same period. here, we report a five-month old boy with ndi. on his history, he was admitted because of recurrent fever attacks and was diagnosed as thi when he was three-months-old. on his follow-up, hypernatremic dehydration was detected and he was diagnosed as ndi. at the time of diagnosis, he had intracranial calcification secondary to delayed diagnosis of ndi. in infants with ndi, recurrent fever attacks due to dehydration may occur and incorrectly lead to think as an infectious disease. we think that this report can be an important warning for clinicians. we analyzed nphs1, nphs2 and neph1 gene in 8 japanese cns patients independent of the patients in previous report (k.i. 2005) from different japanese group which suggested that the mutation of nphs1 was not a major cause of cns in japanese patients. we extracted genomic dna from leukocytes and analyzed all exons and exon-intron boundaries for nphs1, nphs2 and neph1 using polymerase chain reaction and direct sequencing after informed consent had been obtained. the study was approved by the ethics committees of okayama university medical school. results: we found compound heterozygous mutations of nphs1 in 4 patients and homozygous mutations in 2 patients. one of these 2 homozygous mutations was already reported in paper from europe and was not found in more than 50 healthy japanese. another one cause defection of trafficking to the membrane as we reported before (k.i.2000 (k.i. , ajkd 2002 . the other 2 patients have only heterozygous mutation in nphs1 that healthy parent also has. we could find any mutations in neither nphs2 nor neph1 in the 2 patients with heterozygous mutation. one of these 2 patients has mild form of cns. another one has neither expression of nephrin nor podocin protein on kidney tissue by immunohistochemistry. interestingly, 4 patients out of 8 have the same mutation in nphs1 as nt2515(delc). parents who have this mutation heterozygously were from neighboring prefectures. all mutations including this mutation but one have never been reported out of japan which was isolated from continent. all amino acids substituted by missense mutations which seem to be causative were preserved among mouse, rat and human. conclusions: our studies clearly demonstrated that nphs1 is a major cause of cns even in japanese patients. moreover, nt2515(delc) is a common mutation in japanese cns patients like fin major or minor. long-term survival of childhoodonset ckd is mainly limited by the manifestation of cardiovasculardisease (cvd). the development of early stages of cvd can be assessedby non invasive methods, e.g. flow mediated vasodilation (fmd) ofperipherial arteries and measurement of intima media thickness (imt) of the common carotid artery. wetherefore performed fmd and imt investigations in 24 children and adolescents (mean age 15.8±5.1 years) suffering from ckd (mean gfr 44±33 ml/min/1.73 m 2 ) on conservative treatment (n=11), dialysis treatment (5 hd, 1 pd) and after renal transplantation (n=7), and in 24 sex-and age-matched healthy controls. ckdpatients showed significantly decreased fmd (5.2±3.1% vs 9.8±2.3%, p<0.001), where as imt did not significantly differ between patients and controls (0.48±0.06mm vs 0.46±0.08 mm, p=0.13). within the ckd group the presence of arterial hypertension tended to be associated with increased imt (0.48±0.006 vs 0.45±0.05 mm, p=0.05). in contrast, duration of ckd, mode of renal replacement therapy and degreeof renal dysfunction was not significantly associated with fmd and imt findings. conclusion: children and adolescents suffering from ckd show decreased arterial elasticity irrespective of the mode of renal replacement therapy, the rebycontributing to increased long-term cardiovascular morbidity and mortality. the genetic researches have shown the connection between the essential hypertension and angiotensinogen gene. the researches of preeclampsia also showed the connection with angiotensinogen gene. according to established connection of angiotensinogen gene with essential hypertension and also preeclampsia the aim of our research was to determine by the help of m235t, g-6a anda-20c polymorphisms of angiotensinogen gene whether there is a genetic disposition for essential hypertension in those children whose mothers had preeclampsia. at the same time we wanted to establish whether the polymorphisms of angiotensinogen gene can be connected with preeclampsia in women in our population. two groups of children were studied: children of mothers who had preeclampsia and children of mothers without hypertensive disease in the pregnancy. we also studied two groups of mothers: mothers who had preeclampsia and mothers without hypertensive disease in the pregnancy. m235t, g-6a and a-20c polymorphisms of angiotensinogen gene were performed using the pcr method. in investigating the differences between the two groups of children no statistically significant differences were found in m235t,g-6a and a-20c polymorphisms of angiotensinogen gene. in investigating the differences between two groups of mothers no statistically significant differences were found in genotype distribution. the results of our study failed to confirm that with help of m235t, g-6aand a-20c polymorphisms of angiotensinogen gene we might establish genetic disposition for essential hypertension in those children whose mothers had preeclampsia. we did not confirm the association between m235t, g-6a and a-20c polymorphism of angiotensinogen gene and preeclampsia either. (11) previously treated also with cyclosporine. before start of fosinopril treatment all had proteinuria 0.7-4.2 g/24 h and gfr >60 ml/1.73 m 2 /min. all active steroid and immunosuppressive treatment were discontinued. eleven children also were treated with calcium channel blockers to control hypertension. after twelve months of fosinopril (0.3-0.4 mg/kg) treatment a dna analysis for ace-gene polymorphism was performed. three patients carried ii, 10-id and 4-dd gene polymorphism of ace gene. no significant differencein proteinuria at start of fosinopril treatment between all polymorphism types was observed. all 3 patients with ii polymorphism had a good antiproteinuric effect of fosinopril with more than 4-fold decrease in proteinuria in comparison with just 2,3 fold decrease in id polymorphism and no response in dd. renal function remained stable in all children except of 2 with id and 2 with dd gene polymorphism who demonstrated decrease in gfr. we suggest that ii polymorphism of ace gene may be associated with better antiproteinuric effect of acei while dd predicts poor response. wider study is required to confirm genetic predisposition for ace blockade efficacy in proteinuric diseases. introduction: dent disease is x-linked recessive proximal tubulopathy, due to mutations in the clcn5 gene. it is characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and progresive renal failure. aim: case report of patient with short stature and proximal tubulopathy where dent disease is determined by gene analysis. method: seven-year-old boy is refered after endocrinological exemination where abdominal ultrasound showed nephrocalcinosis. there are anamnestic data neither of oedema, macrohaematuria, nor poliuria or hypertension. there are also no data of chronic renal failure in the family. we describe detailed diagnostic procedures performed in the boy and his family. results: we determined: proteinuria (1,8 g/day), elevated urinary excretion of beta-2microglobuline, microscopic hemathuria, hypercalciuria (8-10 mg/kg/day), nephrocalcinosis, decreased tubular reabsorption phosphate (65-72%). values of grow hormone, parathormone on thyroid hormone are normal. except hypercalciuria, which is registered in patient's mother, all other analysis performedin family members are betwen referent values. diagnosis is finalized by mutation analysis, which has showed s244l substitution on cncl5. mutation carrier is mother with normal fenotype. conclusion: dent disease is rare x-linked nephrocalcinosis. definitive diagnosis ofthis proximal tubulopathy which leads to progressive renal demage, is not possible without evidence of gene mutation in renal chlor channel. introduction: the segmental renal infarction is a rare disease, allthough it is more frequent in children than in adults and can be clinically veryrelevant. objective: we describe the clinical course of two children with idiopaticsegmental renal infarction who suffered severe arterial hypertension. cases description: the typical clinical picture of this disease, as seen in our twopatients, is characterized by metabolic alkalosis, hyponatremia, hypokaliemia, hyper-reninemia, hyperaldosteronism and renal salt lossfrom the contralateral enlarged kidney. hypertension was diagnosedduring the study of haematuria in the first case and due to ahypertensive emergency in the second case. the ethiology was found tobe renovascular in both patients, involving the occlusion of small renal arteries causing segmental renal infarction. our first patient was treated with partial nephrectomy, and the second patient wastreated with antihypertensive medication given the impossibility ofremoving the renal infarcted area. conclusions: in the clinical picture, the sodium depletion with increased urinary volume of the contralateral healthy kidney can be explained by the goldblatt model. the gold standard test for the diagnosis is theselective renal arteriography, which is the most sensible and specifictest for diagnosing renovascular hypertension secondary to a segmental renal infarction because it helps identifiing the segmentary artery ofthe infarcted area, this being the source of increased focal renin production. the definitive treatment is the surgical segmentectomy. if segmentectomy is not feasible because of the localization of the infracted area as inour second patient, a medical treatment is needed. conclusion: although an inverse association between birth weight/blood pressure has been suggested in several large studies, interpretation is still controversial. in our study, we have found only a weak inverse correlation between birth weight and pad (abpm): no statistical significance has been found among other variables, although graphs show a trend of low birth weight children towards hypertension. probably a long term follow-up is necessary. he took longer to have improvement, but his calcemia, phosphatemia and alkalinephosphatase are normal. the coding region of the vdr was amplified by pcr and directly sequenced. results: three mutations (two novels) were identified. two patients had anovel mutation in exon 7, changing the amino acid glutamine to glutamicacid at position 259 (q259e). one patient had a novel mutation in exon 8 changing glycine to valine at position 319 (g319v). those mutations are in the ligand binding domain and belong to the patients with better control of the disease. one had mutation in exon 3, in the dna binding domain, changing arginine to a stop codon at position 73 (r73x) and it was from the patient with worse response to the treatment. conclusions: two novel mutations are presented in the vitamin d receptor and it could be possible to do a correlation between the clinical evolution and the localization of the mutations. we aimed to evalute the effects of intrauterine growth retardation (iugr) on blood pressure (bp) in small for gestational age (sga) children. 23 sga children (6f,17m) aged 9.4±3.9 y (5 preterm and 18 term) and 13 (8f, 5m) agematched control with appropriate for ga (aga) were included in the study. 24-hr ambulatory blood pressure monitoring (abpm)was performed using an oscillometric device (mobilograph). 95th centile limit was set according to published data sets, nocturnal threshold was 10% less than awake limits. bp was classified as normal if the mean sbp is <95th percentile and sbp load is >25%. nocturnal bp dip for sbp and dbp were evaluated. in 2 sga and in 1 aga case, office sbp were above 95th%, all dbp values were normal in both groups. according to the mean of 52 abpm records, 2 systolic,1 diastolic ht were determined in sga group, and 3 systolic, no diastolic ht were in aga group. there were 9 sga and 5 aga children whose sbp load over 25%, and only 5 of them remarked as hypertensive according to mean sbp values. 5 children considered as hypertensive had significantly higher sbp loads than those that were not hypertensive(38% vs 10%, p<0.05). abpm records were not different between preterm and term sgas. sga group had lower nocturnal bpdip for sbp and dbp in comparison to aga group(13.5% vs 14.5% for sbp,15.4%vs 18.1%for dbp, p: 0.62). although it was not statistically significant, the frequency of non-dipper children was higher in sga group (22.7%) than those in aga group (8.3%). except the lower nocturnal bp dip and higher sbp loads in sga group which may be a marker for further hypertension, no clear association between being sga and hypertension could be found in our study population. pres is a rare clinico-radiological entity associated with acute hypertension, renal insufficiency and immunosuppressive therapy. it is typically reversible but cases with irreversibility had rarely been reported. we report 2 contrasting cases of pres associated with renal disease, one with full recovery and the other subsequently had epilepsy. first patient had steroid sensitive nephrotic syndrome with acute hypertension, fluid overload and acute renal failure while the second patient had severe flare of lupus nephritis with malignant hypertension, renal failure and thrombotic thrombocytopenia. both had acute onset of drowsiness, blindness and seizures and mri findings of subcortical oedema in the parietal-occipital-temporal regions. while the second patient had full recovery, the first patient developed temporal epilepsy and repeat mri showed mesial temporal sclerosis. pres is attributed to capillary leak from functional cerebral vascular changes of hypertension, rather than infarction. prolonged vascular disruption may lead to ischemia resulting in neurological sequelae and chronic epilepsy. using mri, pres can be readily diagnosed and normalisation of blood pressure is imperative in patients at high risk of pres. efforts have been done to describe the significance of various genetic polymorphisms (snps) in acute renal failure (arf). available reports do not investigate the predictive value of snps in disease forecasting, because so many interacting factors influence arf that classical statistical methods become unstable. high-dimensional nonparametric methods such as random forest technique overcome this problem and help to identify each clinical and genetic factor that possibly contributes to disease. we tested the classification value of basic clinical data available at birth and 19 snps in arf of 111 preterm infants. we determined the relative importance score (ri) of each parameter in classification. low birth weight and gestational age had the highest ri; just few snps had medium ri, while the majority of snps had small ri. then we created variable-patterns and searched for pattern with the highest accuracy of classification. for each complication, the accuracy was 0.71 solely basic clinical data were considered. if snps were incorporated, the accuracy of classification for arf was not improved. in contrast with previous observations these findings suggest that the snps do not provide additional information about arf-risk of the general preterm population. conclusions: srbd in children with elevated blood pressure is higher than that of the general pediatric population. the prevalence appears highest in patients with pre-hypertension and may be higher in patients with essential and secondary hypertension than in patients with white-coat hypertension. the renal phenotype in lowe syndrome is similar to dent's disease a 22-month-old girl presented with heart failure due to severe hypertension (200/120). doppler ultrasonography (us) revealed a right renal artery stenosis. biological findings showed normal serum creatinine (scr) (35 mmol/l), hyponatremia (131 mmol/l) and hypokalemia (3 mmol/l). aldosterone plasma level was increased as were epinephrine (429 pg/ml n<185) and norepinephrine (1230 pg/ml n<450) plasma levels measured 3 times. renal arteriography confirmed the presence of a nearly obstructive right renal artery stenosis with a normal left kidney. pta failed to improve signifcantly the renal artery flow. in the following hours after pta, her scr level increased, up to 160 mmol/l. doppler us showed an unchanged right kidney but 3 hypoperfused area in the left kidney confirmed by the tc 99 -dmsa scan. both aspects were strongly in favor of focal ischemic events. secondarly, her renal function improved but hypertension remained difficult to control autotransplantation of the right kidney was then done, but unfortunately, thrombosis of the right renal artery occured in the following hours. six months later, her plasma creatinine level is of 55 mmol/l. her cardiac function is normal but she has to remain on antihypertensive drugs. what exactly happened during arteriography in her left kidney, in order to provoke such ischemic injury, knowing that the left renal artery was not catheterized during arteriography and pta? one hypothesis is that arterial vasospasms occured, explained by a high vasospastic predisposition due to enhaced cathecolamines secretion. such increased has already been drescribed in patients with renal artery stenosis. this case raised the question of preparing patients in this condition with efficient antispasm therapy agents before radiological vascular investigations are done. rtd is a cause of oligohydramnios leading to potter's sequence and neonatal anuria. it is characterized by absent or hypoplasic proximal tubules. these lesions can be secondary to non genetic conditions involving renal hypoperfusion. genetic forms of rtd have autosomal recessive transmission. mutations of genes encoding for the renin-angiotensin system have been reported. evolution is most often severe with in utero or neonatal death. case report: this boy was born at 38 weeks of gestation with oligohydramnios, arthrogryposis, large fontanels with poor calvarian ossification. he was referred to intensive care unit for respiratory distress, low blood pressure (bp) unresponsive to vasopressors and anuria (plasma creatinine 370 μmol/l at day 3). renal ultrasound (us) showed enlarged kidneys with bilateral cortical hyperechogenicity. weaning from ventilation and vasoactive drugs was possible at day 7. glomerular filtration recovered (plasma creatinine 45 μmol/l at day 26). at age 10, the child is well with normal growth and a mild chronic renal insufficiency (creatinine clearance 75 ml/min/1,73 m 2 ). bp is in the low normal ranges with plasma renin x40 over the upper range of normal values and normal plasma aldosterone. on renal us, kidney volume normalized with persistent cortical hyperechogenicity. genetic study revealed compound heterozygous mutation in the gene encoding angiotensin converting enzyme. one is a nonsense mutation (r1209p), the other is a truncating mutation (k601fsx640). it is possible that the first mutation is less deleterious than the other and this could partly explain the unusually favourable evolution of this rtd. in conclusion, this is a rare report of a child with autosomal recessive rtd surviving after neonatal period. further functional studies are needed to explain this unusual phenotype. data on hbp in children are very limited, and it is unclear how much it is specific in pediatric patients. the objective of our study was to determine the reproducibility of hbp in children and adolescents. automatic omron 705-cp devices, which have been validated for use in children and adolescents, were provided to all families. hypertensive children measured blood pressure (bp) every minutes, three times consecutively in the morning or early afternoon, and three times in the evening during 13 days. the reproducibility of hbp was quantified using repeated measures analysis of variance test and the sd of differences between average hbp values of consecutive days. confidence interval (ci) was calculated. we studied 41 patients (14 girls, 27 boys); mean age was 12,1 year, range 4-18 years. there were 78 measurements of systolic blood pressure (sbp) and diastolic blood pressure (dbp). in the morning or early afternoon the mean sbp and dbp were 110,9 mmhg (sd 10, 65) (95% ci 107, 5-114, 3 mmhg) and 64, 4 mmhg (sd 9, 08) (95% ci 61, 4-67, 3 mmhg), respectively. in the evening the mean sbp and dbp was 111,6 mmhg (sd 10,14) (95% ci 108,3-114,8 mmhg) and 65,0 mmhg (sd 8,83) (95% ci 62,2-67,9 mmhg). the mean average difference between the daily measurements of sbp and dbp were analyzed with each period and were not statistically significant (p>0.05). on the basis of these results, we conclude that self-monitoring of blood pressure at home in children has good reproducibility and is not influenced by white coat effect. case report: a 3-year-old caucasian girl presented with history of severe polyuria and polydipsia of few months duration. past history was significant for head injury and possible seizure activity at age 1 yr. there was no history of headaches, vomiting, or visual problems. family history negative for di. physical examination was essentially normal. basal plasma and urine osmolality, and plasma vasopressin were 294 and 96 mosm/kg, and <0.5 pg/ml, respectively. at the conclusion of water deprivation test these readings were 309 and 98 mosm/kg, and 31.3 pg/ml, respectively. however, urine osmolality increased to 493 mosm/kg after desmopressin injection, confirming the diagnosis of cdi. mri scan revealed absence of the bright spot on t 1 weighted images. genetic analysis detected avp gene mutation a19t, where the normal alanine at amino acid position 19 is changed to threonine. elevated levels of plasma vasopressin after water deprivation were misleading and intriguing. we believe that elevated plasma vasopressin, although immunoreactive was devoid of biological activity. conclusion: molecular genetic evaluation should be performed in all children with cdi without an identifiable cause, even in the absence of a positive family history of cdi. mutations of the nphs2 gene encoding podocin lead to autosomal recessive corticoresistant nephrotic syndrome, focal and segmental glomerulosclerosis (fsgs), and early end-stage renal disease (esrd). in mice, podocin inactivation by homologous recombination results in diffuse mesangial and glomerular sclerosis, esrd and death within the first 5 weeks of life. this early demise precludes extensive study and elucidation of the molecular pathways engaged by podocin in the mature kidney. we have, therefore, generated a novel mouse model in which a tamoxifeninducible creer-loxp system allows for conditional inactivation in a temporal fashion in podocytes. following tamoxifen administration in nphs2 flox/-, cre+ mice, cre expression was noted in 88% of glomeruli and in 60-70% of podocytes. two weeks after cre induction, podocin expression is decreased in podocytes correlating with the appearance of selective albuminuria at 10-16 days. this progresses to massive, nonselective proteinuria by 4 weeks, along with high blood pressure and impaired renal function. optical microscopy of kidneys showed no lesion at 1 week and fsgs progressively developed by 4 weeks along with tubular lesions. however electron microscopy revealed a partial foot process effacement at 1 week with no significant albuminuria that extends by 2 weeks along with abnormalities of the basement membrane and development of albuminuria. no mesangial or vascular lesion has been noted, differentiating it from our previous podocin null model in which renal development may be affected by podocin loss or nephrotic syndrome. this model will allow a better understanding of the mechanisms underlying the development of nephrotic syndrome and the role of podocin in the mature kidney, and will be crucial to test new therapeutic approaches. glomerulonephritis is a group of diseases with complex etiology, pathogenesis, morphological features and clinical course. the renin-angiotensin and coagulation systems genes are important group of candidate genes involved in pathogenesis of chronic renal diseases. the purpose of our study was to analyze the association of genetic polymorphisms of these genes with glomerular kidney diseases. the study population consisted of 181 patients with immunological glomerular kidney diseases and 19 patients with renal failure with glomerulonephritis as primary disease. the control group consisted of 80 healthy subjects. by means of the polymerase chain reaction (pcr) the following polymorphisms were evaluated: insertion/deletion (i/d) polymorphism in intron 16 of the angiotensin-converting enzyme gene (ace), 4g/5g polymorphism of the plasminogen activator inhibitor-1 (pai-1). no significant association was found between the ace and pai-1 allele and genotype frequencies and the disease. more progressing of glomerulonephritis current was marked at patients with simultaneous has dd genotype of ace gene and 4g/4g gene pai-1(c 2 =9,1; p=0,008). our results suggest that ace i/d and pai-1 4g/5g polymorphism is an important modifying gene in the progression of glomerulonephritis. captopril objectives: secondary causes of hypertension such as renovascular hypertension are more abundant in children unlike adult population. the objective of this retrospective study is to assess the use of captopril renography (cr), which provides a non-invasive approach in the differential diagnosis of hypertension. patients and methods: clinical, radiological and scintigraphic data of a total of 64 patients were analyzed. there were 22 girls and 42 boys (mean age: 13±3 years). none of the patients had parenchymal renal disease or reduced renal function. all patients were orally hydrated before scintigraphic study. cr was performed 1 hour after orally captopril administration and iv furosemide injection was done simultaneously with 1-5 mci tc99m-mag3. when post-captopril study was normal, baseline scintigraphy was not performed. computed tomography angiography (cta) was performed in 11 and gadolinium-enhanced magnetic resonance angiography (mra) was performed in 8 children in addition to routine renal doppler ultrasonography (dus). results: nineteen out of 64 patients had other comorbid diseases as follows: obesity n=10, previosly-diagnosed fibomuscular dysplasia n=3, n=2 neurofibromatosis, n=2 demyelinating diseases, n=1 bartter and n=1 turner syndrome. cr was normal in 48 patients. in 8 children abnormal cr findings in correlation with radiological methods were reported. correlation with radiological methods could not be done due to suboptimal technique in 7 patients, of whom dus in 5 obese children could not be interpreted. in the other 1 patient, although cr was abnormal, radiological methods could not confirm scintigraphy. conclusions: captopril renography is a useful and simple-to-perform imaging modality in children suspected of having renovascular hypertension. takayasu's arteritis (ta) is a chronic, inflammatory, large-vessel vasculitis affecting the aorta and its main branches, which causes stenosis, occlusion, rarely aneurysm and distal ischemia. the disease is most common in young women, it is rare in inviduals before the age of 16 years. clinical presentation may be heterogeneous. in this report, we present a pediatric patient with ta who had hypertension as the sole manifestation of multipl critical arterial involvement but no other symptoms. a 14-year-old boy was admitted with hypertension. the acute-phase reactants were moderately elevated with an erythrocyte sedimentation rate 70 mm/h, and a c-reactive protein value of 26 mg/l. serologic tests including ana, anti-ds dna, c and panca, complement c 3 , and c 4 were negative and other laboratory data were normal. mr angiography showed multiple severe stenosis or occlusions of the thoracic and abdominal aorta together with bilateral renal arteries, and saccular aneurysm in the abdominal aorta. immunosuppressive treatment including pulse steroid and methotrexate was prescribed. the patient underwent angioplasty of bilateral renal arteries and suprarenal aorta, and a stent was placed in the right renal artery. ta should be kept in mind in the differential diagnosis of hypertension in children, even if they do not have other associated symptoms of the disease. human urotensin ii is the most potent vasoconstrictor which circulates in the plasma of healthy individuals. it was suggested that it has an endocrine role in sodium handling and even in metabolic syndrome. the aim of this study was to investigate the role of u-ii in obese adolescents with hypertension. fourteen obese adolescents with essential hypertension (group 1) were compared with thirteen age-and sex-matched obese adolescents with white-coat hypertension (group 2). they underwent twenty-four hour abpm and echocardiographic investigation, complete physical examination, including adiposity indexes. plasma and urinary levels of u-ii were measured by ria. obese adolescents in group 1 have significantly higher blood pressure measurements than those in group 2 confirmed by abpm. there was no significant difference in left ventricular mass index between two groups. no significant difference was found in plasma u-ii concentrations (pg/ml) between group 1 and group 2 (35.23±4.90 and 35.98±5.74, respectively), whereas mean urinary u-ii level (pg/mg urinary creatinine) was significantly higher in group 2 than that of group 1 (28.80±6.83, 44.44±16.78, respectively). considering the renal synthesis and vasoactive role of u-ii, these results suggest that u-ii may have a role in adolescents with white-coat hypertension. distal renal tubular acidosis (drta) and deafness is a rare autosomal recessive disease characterized by severe metabolic acidosis in childhood and inappropriately high urinary ph along with sensorineural hearing loss. the disease is caused by defects in the atp6v1b1 gene. the aim of the present study was to determine the molecular basis for drta with deafness in eight patients from four families (a-d) in israel. molecular testing was done by sequencing the coding exons of the atp6v1b1 gene in one affected child from each family. a population screen was performed for mutations found in family a. the results yielded a different mutation in the atp6v1b1 gene, as follows: families a and d: missense mutation, 1037c>g (p346r), in addition to a single nucleotide polymorphism (2t>c) in the first codon; family b: insertion mutation, 1155-1156insc (i386fs); family c: a novel nonsense mutation, 340 c>t (r114x). in conclusion, the phenotype of drta and deafness concurs with mutations in the atp6v1b1 gene. in the present study, four families of different origins with the same phenotype had three different genotypes, indicating that there is no single common mutation in israel. these findings have implications for genetic counseling during pregnancy and testing of families. the fact that all the patients that were examined have harbor mutations in the atp6v1b1 gene, pointed for the specific clinical phenotype. making correct and early clinical diagnosis is a fundamental step in finding the molecular basis of this rare disorder. delta f508 is the most common cystic fibrosis (cf) mutation worldwide. the prevalence is approximately 70% in caucasian and 14-55% in asian while f311l is demonstrated in only 0.2%. homogenous delta f508 mutation is recognized as the most common genotype however there are small numbers of cf patients having delta f508/f311l. in the present study we report a 2 year-old thai boy, originated from north india, presented with recurrent episodes of febrile illness, hyponatremia, hypokalemia and metabolic alkalosis since 4 months of age. he was transferred to our hospital for further investigation. blood chemistry revealed serum electrolytes: sodium 122, potassium 3.69, chloride 79.7, bicarbonate 33.8 meq/l, and urine electrolytes: sodium<10, potassium 45.7, chloride<10 meq/l. after intravenous fluid administration, hyponatremia and metabolic alkalosis improved. dna sequencing analysis of his blood demonstrates compound heterogenous mutation for delta f508 and f311l in cftr gene. t to g transversion at nucleotide 2694 and g to a transversion at nucleotide 4521 are found without altering amino acid encoding gene. in conclusion, we report a rare case of cf with delta f508/f311l genotype presented with recurrent hyponatremia and metabolic alkalosis. awareness of electrolyte abnormalities during febrile illness, proper genetic counseling and long-term follow-up are necessary in this patient. objectives of study: about 10% of families with congenital nephrogenic diabetes insipidus (ndi) have mutations in the aquaporin 2 gene (aqp2), which codes for the vasopressin-sensitive water channel. only seven aqp2 mutations are known to cause a dominant form of the disease. in this study we performed genetic and clinical studies in a 5 generation family with autosomal dominant inheritance of a partial di phenotype. methods and results: the proband (man, 37 yrs old) was initially diagnosed with partial central diabetes insipidus due to antidiuretic effect of ddavp. his daughter (1.3 yrs old) also showed polyuria and polydipsia which was responsive to high doses of intranasal ddavp. the pedigree was consistent with an autosomal dominant inheritance pattern. sequence analysis of the entire coding region of the avp gene and aqp2 gene was performed in the proband and his affected daughter and in 2 unaffected family members. the avp gene was normal in all subjects. in the two affected patients but not in the healthy subjects we identified a novel missense mutation in one allele of exon 4 of the aqp2 gene (c.760c>t) which predicts a substitution in the c-terminal part of the aqp2 protein (p.r254w). conclusion: partial ndi can be caused by heterozygosity for a p.r254w substitution of the aqp2 water channel. the substitution is likely to significantly alter the c-terminal tail of aquaporin 2 which is important for proper trafficking to the apical plasma membrane. the preservation of some residual antidiuretic function indicates that some aqp2 tetramers are processed correctly. the study further illustrates the importance of molecular diagnostic tools in establishing a correct differential diagnosis in familial cases of di. a background and aim: dent's disease is a renal tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. mutations in the clcn5 gene encoding the chloride/proton exchange transporter clc-5 cause this disease. we have described an alu insertion in clcn5 that leads to exon 11 skipping in the patient's mrna. in this study, we investigated the consequence of this mutation on the function of two putative exonic splicing enhancers (eses) in exon 11 and the role of alu 5'-end sequences on splicing inhibition. methods: minigenes were constructed by inserting exon 11 and exon 11-alu with their intronic flanking sequences into the exon trap vector. artificial mutations were introduced by site-directed mutagenesis. plasmids were transfected into cos7 cells by electroporation. pre-mrna splicing analysis was performed by rt-pcr. the ese finder web resource was used to predict the mutagenesis effects on the eses. results: alteration of one of the putative eses, the predicted binding site for srp40, induced exon 11 skipping. restoration of this site within the alu sequence promoted the incorporation of exon 11-alu in the mrna. however, restoration of both eses leaving the alu sequence intact resulted in exon skipping. furthermore, substitution of the eses for the first seven nucleotides of the alu element, and insertion of this sequence next to the eses increased exon skipping. deletion or modification of the alu 5'-end enhanced exon 11 inclusion in the mrna. in the patient carrying the clcn5 alu insertion, dent's diseases is caused by disruption of an ese and by inclusion of splicing inhibitory sequences leading to exon skipping. this work was supported by grant pi042620 from fondo de investigación sanitaria. factor h-associated hemolytic uremic syndrome (fhd-hus) is a rare disease with incomplete penetrance. it is not clear why and how carriers of the same mutation will eventually develop or not develop the disease and this uncertainty is sometimes responsible for anxiety in the carriers badly affecting their quality of life (particularly of parents with children carrying the mutation). in the attempt of estimating disease risk in subjects with factor h gene mutation, the 6 families of fhd-hus patients (all of which had a documented mutation on the scr20) currently being treated at our center, were screened to identify the carriers after having been informed on the purpose of the analysis. twenty-eight subjects (age range 0-89 yrs), out of 55 analyzed, revealed heterozygous mutations in the carboxy-terminal region of fh1 gene (51%). among subjects carrying the mutation, 9 (32%) had exhibited the disease at a mean age of 12 years (range 0.2-32), 6 of which (67%) before age 10, whereas the remaining 19 subjects (mean age of 53 yr -range 11-89) were healthy at time of the study. the figure shows the probability of carriers to be hus-free by age (no event occurred after age 32 yrs). in the meantime that predisposing factor are identified, the presented survival curve (fig.1) can be an useful tool to estimate the residual risk of hus in individual carriers according to their specific age. cerebello-oculo-renal syndrome (cors) (also called joubert syndrome (js) type b) and meckel (mks) syndrome belong to the cilliopathy group of developmental autosomal recessive disorders associated with primary cilium dysfunction. nephronophthisis (nphp), the most frequent genetic cause of renal failure in children and young adults, is associated with retinal degeneration and cerebellar vermis aplasia in cors. mks is characterizd by renal cystic dysplasia, central nervous system malformations and hepatic developmental defects. these syndromes are genetically heterogenous: mutations in ahi1 and nphp1 can cause cors and mutations in mks1 can lead to mks, while mutations in mks3 and nphp6 are found in both syndromes. using snp mapping, we identified missense and truncating mutations in a novel genee, nphp8, in both cors and mks. interestingly, all sequence changes were either nonsense or frameshift mutations in fetuses with mks whereas patients with cors had either only one or no truncating mutation. we then show that inactivation of nphp8 mouse orthologue recapitulates the cerebral and renal defects of cors/mks. we further demonstrate that nphp8 protein co-localizes at the basal body/centrosomes with nephrocystin-6 and nephrocystin-4, the protein products of both nphp6 and nphp4, known disease genes for nphp. in addition, missense mutations of nphp8 protein found in cors patients diminishes its interaction with nephrocystin-4. taken together. our findings demonstrate that mutations of this novel ciliary gene can cause the multiorgan syndromes of either cors or mks, which therefore represent a continuum of the same underlying disorder. severe antenatal bartter's syndrome ga 25 + 4, bw: 750 g). both had classical signs of antenatal bs including very poor thrive, polyuria (5-10 ml/kg b.w./h) and responded to indomethacin treatment. direct sequencing was performed on pcr-amplified genomic dna. the entire coding region of both genes was analyzed. the kcnj1 gene was normal but both children were homozygous for a single base substitution (39671t>a) in exon 12 of the slc12a1 gene predicting a premature stop codon (y538x). the parents of both children and two siblings were heterozygous for the same mutation. by restriction enzyme analysis, the 39671t>a substitution was absent among 100 chromosomes from healthy subjects. conclusion: two children with severe antenatal bs were identified and the clinical phenotype was characterized. we identified in both children a novel mutation causing a premature stop codon of the bumetanide-sensitive-sodium-potassium-chloride co-transporter (nkcc2). despite large potassium supplements and indomethacin treatment, both children show persistent polyuria, vomiting, and insufficient thrive. background: mutations of nphs1 encoding the transmembrane slit diaphragm protein nephrin cause congenital nephrotic syndrome of the finnish type, characterized by massive proteinuria and the development of nephrotic syndrome before 3 months of age. about 70 different nphs1 mutations have been described. a large number of these are missense mutations resulting in single amino acid replacement mostly located in the extracellular domain. some nphs1 mutations have been associated with mild phenotypes responding to angiotensin-converting enzyme and prostaglandin synthesis inhibition. patient history: we report on a son of consanguineous turkish parents with a novel nphs1 alteration and steroid-resistant congenital nephrotic syndrome. after normal pregnancy and delivery at 37 weeks of gestational age the boy presented with edema and proteinuria at the age of 2 months. he responded to angiotensin converting enzyme inhibitor treatment reducing his proteinuria from a maximum of 66 g/g creatinine to 10 g/g creatinine and allowing him to develop normally until the age of 7 months when cardiovascular insufficiency in the course of a hyperpyretic infection led to hypoxic encephalopathy. genetic testing revealed a homozygous in-frame 3-bp insertion in exon 11 of nphs1. both parents were found to be heterozygous. no mutations were found in nphs2 or in exons 8 and 9 of wt1. the alteration results in an insertion of a glycine in an extracellular immunoglobulin domain of nephrin. the pathogenicity of this alteration is unclear. nephrin may be misfolded and mislocalized as in some missense mutations of the extracellular domain. alternatively the alteration might lead to an altered extracellular homo-and heterodimerization of nephrin. functional studies are currently underway to determine the effect of this novel alteration in nphs1. objectives of study: interleukin 1 receptor antagonist (il-1ra) gene polymorphism has been found to affect disease susceptibility and activity in several inflammatory diseases. we investigated the association between il1ra gene polymorphism and childhood nephrotic syndrome (ns) in a turkish population. methods: we analyzed the genetic polymorphism of il-1ra gene in 91 patients with childhood ns and 100 healthy controls by using pcr. five alleles of the il-1ra gene were identified and designated as il-1ra*1, il-1ra*2, il-1ra*3, il-1ra*4, and il-1ra*5, according to the variable number of tandem repeats in intron 2. results: in the ns group, the allele frequencies of il-1ra*1, il-1ra*2, il-1ra*3, il-1ra*4, and il-1ra*5 were 74.7, 13. 2, 4.4, 2.2, and 5 .5% compared with 58%, 28%, 1%, 0%, and 12% in the controls. a high allele frequency of il-1ra*1(x 2 =5.28, p=0.021) and a lower allele frequency of il-1ra*2 (x 2 =6.31, p=0.012) were found in childhood ns. the other polymorphisms were not significantly different from normal controls. conclusions: a high allele frequency of the il-1ra*1 allele may affect the disease susceptibility in childhood ns. objectives of study: congenital anomalies of the urinary tract (caut) are common in humans, and the incidence is increasing with recent advances in prenatal ultrasonographic examinations. interstitial fibrosis, which correlates with infiltration of inflammatory cells is common finding in the kidney with long-term ureteral obstruction. up-regulation of monocyte chemoattractant protein-1 (mcp-1), may be a common regulatory pathway involved in the progressive renal damage with any etiologies leading to interstitial fibrosis. mcp-1 gene polymorphism -2518 a/g had been suggested to influence circulating mcp-1 levels and gene expression thus, might be one of the genetic markers for progression of renal damage. our aim was to investigate the frequency of mcp-1 genotypes and allele -2518g in patients with caut. objectives of study: congenital urological anomalies are well recognized as causing renal dysfunction. on the other hand, patients without congenital anomalies but with urinary bladder dysfunction (bd) could also develop renal parenchymal damage. it has been reported that angiotensin converting enzyme (ace) i/d polymorphism was a risk factor for renal parenchymal damage in certain renal diseases including vesicoureteral reflux. the aim was to determine i/d polymorphism as a potential risk factor for renal parenchymal damage in patients with congenital obstructive uropathy and to compare them to patients born with normal anatomy and innervation of bladder, sphincter and pelvic floor but which could develop upper urinary tract abnormalities. objectives od study: to determine the frequency of pyelocaliceal dilation (pcd) in asymptomatic infants and its connection with presence of other urinary tract anomalies (uta) and urinary tract infection (uti). methods: ultrasonographic screening (us) of urinary tract (ut) was performed on unselected population of 1000 healthy infants ranging between 7 days and 6 months of age (511 boys and 489 girls). kidneys were divided into two groups according to grade of pcd. group i consisted of kidneys with pcd, whose anterio-posterior diameter was 5-10 mm, while patients from the group ii had a-p diameter wider than 10 mm.patients were followed up (ranging 5-58 months) by serial ultrasound examinations and with other methods for presence of uti and uta if necessary. results: in examined population 2,2% had uta, and 8,4% had uti. pcd was found in 74 infants (7,4%). in the group i, consisted of 63 infants (35 males and 28 females) pcd increased during the time in 3,2% infants, remained unchanged in 11,1%, decreased in 14,3%, and disappeared in 71,4% patients. uta was found in 4 (6,3%) infants, and uti in 9 (14,3%) infants. in the group ii, consisted of 11 male infants, pcd has not changed in 5 (45,4%) infants, decreased in 2 (18,2%), while dilation disappeared in 4 (36,4%) patients. uta was found in 6 (54,5%), and uti in 4 (36,4%) infants. conclusions: us of ut is useful and valuable method for detecting pcd. our results indicate that mild pcd in infants increases the risk for uta approximately 3 times and uti 2 times, while severe pcd raises the risk for uta approximately 24 times, and uti 4 times. early diagnosis and early treatment of uta and alertness for possible uti should be the final goal of the kidney damage prevention. therefore we recommend that us of ut should be done in all children in the first months of life. in order to determine factors involved in kidney development, the spatial and temporal expression pattern of intermediate filaments (cytokeratins, vimentin) , epithelial growth factor (egf) and transforming growth factor alpha (tgf-α), was investigated in developing kidneys (mesonephros and metanephros) of 5-9 week human embryos. immunohistochemistry for detecting specific antibodies was used on paraffin sections. in the 5-9 week human mesonephros, vimentin was moderately expressed in all mesonephric structures, while cytokeratins were seen only in the mesonephric nephrons. moderate to strong expression to egf and tgf-α detected in all mesonephric structures, decreased with advancing development. in the 5-6-week metanephros, vimentin was mildly expressed in all metanephric structures. later on, its expression increased in collectin system and interstitium. in the 5 th developmental week, first to appear were cytokeratins 8 and 19 in the ureteric bud and ampullae, while from the 8 th week onward, both cytokeratins showed increasing expression in the collecting system and nehrons. at early stages, egf and tgfá showed moderate to strong reactivity in the collecting system, the metanephric mesenchyme and cups. from the 7 th week onward, expression of both factors decreased in differentiating nephrons. expression of all investigated developmental factors was in line with subsequent mesonephric degeneration. expression pattern of intermediate filaments in the metanephros might be associated with mesenchimal to epithelial transformation of developing nephrons. some mutations of cytokeratins are lethal, while others might lead to some multifactorial disorders. egf and tgf-α expression patterns of the metanephros indicate their role in induction, proliferation and growth of metanephric structures. their disturbed expression might cause reduction in kidney growth. we have demonstrated that renin-angiotensin system (ras) and mitogen-activated protein kinase (mapk) family constribute to the renal development. growing evidences indicate that aldosterone, a final element or ras, is an independent and powerful mediator of various renal disease. purpose of this study was to evaluate the role of endogenous aldosterone in renal development including cell proliferation and apoptosis, and the expression of mapk family. newborn rat pups were treated with spironolactone (200 mg/kg/d) in olive oil or vehicle for 7 d. to identify cellular changes, kidneys were examined for proliferating cell nuclear antigen (pcna) by immunohistochemical (ihc) stain, and apoptotic cells by tunel stain. immunoblot, ihc stain and rt-pcr for mapks, phospho-mapks, and p53 gene were performed. spironolactone treatment resulted in decreased body weight, decreased pcna-positive proliferating cells and increased tunel-positive apoptotic cells, especially renal cortical epithelial cells (p<0.05). in the spironolactone-treated group, c-jun n terminal kinase (jnk)-2 and phospho-jnk-2 protein expressions were significantly increased, whereas extracellular signal -regulated kinase (erk)-2 and p38 protein expressions were sigificantly decreased compared with the control group (p<0.05) in immunoblot and ihc stain. expressions of erk-1, phospho-erk-1 and 2, and p53 sere not changed by spironolactone treatment. in rt-pcr, erk-2 and p38 mrna expressions were significantly increased in the spironolactone-treated group (p<0.05). we conclude that aldosterone inhibition in the developing rat kidney decreases cellular proliferation and increases apoptosis, and modulates the expressions of jnk-2, erk-2 and p38. mapk family expression may be implicated to differentially participate in aldosterone-related intracellular signaling pathways in the developing kidney. objectives: early detection of anomalies of the kidney and urinary tract (ut) helps to prevent complications but is hampered in moldova by diagnostic and logistic problems. to assess the extent of late diagnosis we studied the clinical data of all children referred to us for suspected ut infection (uti) in 2004/5 and found to have renal or ut anomalies. methods: 92 children (27 males; age 3 months-15 years) found to have anomalies of the kidney and ut and treated conservatively were studied (newborns and infants <3 months were seen elsewhere). work-up included ultrasonography in all, voiding cysto-urethrography (vcug) in 40%, urography in 57% and scintigraphy in 3% of patients. results: reasons for referral were febrile uti (38%); abdominal pain (31%), diarrhea and vomiting (16%), enuresis (9%) and dysuria (6%). uti was confirmed by urine culture in two thirds of cases. age at diagnosis of renal or ut anomaly was <1 year in 9%, 1-3 years in 6%, 3-7 years in 7%, 7-10 years in 30% and >10 years in 48%. renal hypo/dysplasia was found in 14, solitary kidney in 10 and a horseshoe kidney in 9 children. anomalies of the ut included hydronephrosis due to ureteral obstruction (13 up and 11 uv-obstruction), vesico-ureteral reflux (8), duplex systems (17) and bladder anomalies or mild infravesical obstruction (10). serum creatinine was in the normal range in all children. urolithiasis was found in 2 patients. conclusions: anomalies of the kidney and ut were diagnosed in nearly half beyond the age of 10 years, thus with considerably delay. based on the results of this study a new strategy of renal ultrasound screening in newborns including prenatal diagnosis and of closer co-operation with referring physicians has been implemented. objective of the study: the purpose of this study was to report the outcome of infants with antenatal hydronephrosis. methods: between may 1999 and june 2006, all patients diagnosed with isolated fetal renal pelvic dilatation (rpd) were prospectively followed at asingle tertiary renal unit. inclusion criteria were presence of rpd equal to or greater than 5 mm on prenatal ultrasound after 28 weeks gestation, at least six-months of follow-up, and at least two postnatalus scans. the events of interest were presence of uropathy, rpd resolution, urinary tract infection (uti), and hypertension. rpd was classified as mild (5-9.9 mm), moderate (10-14.9 mm) and severe (>15 mm objective of the study: the aim of this study was to compare the accuracy of ultrasound renal parameters to discriminate between significant uropathy and idiopathic renal pelvis dilatation. methods: 193 neonates who were found to have isolated fetal renal pelvis dilatation (rpd) underwent systematic investigation and were prospectively followed. a us scan was performed after the first week of life and all infants underwent vcug. neonates with rpd larger than 10 mm were examined by renal scintigraphy. receiver-operating characteristic (roc) plots were constructed to determine the accuracy of three indexes: fetal rpd, postnatal rpd, and sfu grading system. results: a total of 193 infants were included in the analysis. ninety-five fetuses (49%) presented bilateral renal pelvis dilatation. seventy-nine infants (41%) presented urinary tract anomaly, corresponding to 95 renal units. the most frequent detected uropathy was ureteropelvic junction obstruction (61), followed by primary vesicoureteral reflux (26), and megaureter (8) conclusions: our data support the view that fetal and postnatal us renal parameters are useful markers to identify infants with clinically significant uropathies. there was no significant difference in performance among the indexes. objective of the study: the aim of this study was to evaluate the diagnostic accuracy of ultrasound (us) renal parameters to identify vur in infants withisolated antenatal hydronephrosis. methods: 193 neonates who were found to have isolated fetal renalpelvis dilatation (rpd) underwent systematic investigation and wereprospectively followed. a us scan was performed after the first week of life and all infants underwent vcug. neonates with rpd larger than 10mm were examined by renal scintigraphy. receiver-operating characteristic (roc) plots were constructed to determine the accuracy of three indexes: fetal rpd, postnatal rpd, and sfu grading system. results: a total of 193 infants were included in the analysis. seventeen infants (8.8%) presented vur, corresponding to 26 renal units. to discriminate between renal units with and without vur areaunder the curve (auc) was estimated by the roc curve, which was 0.52 (95% ci=0.47-0.57), 0.58 (95% ci, 0.53-0.63), and 0.55 (95% ci, 0.50-0.60) for fetal rpd, postnatal rpd, and sfu grading system, respectively. there was no statistically significant difference between the indexes. the optimal threshold for fetal rpd was 7 mm with a sensitivity of 69% (95% ci, 48-86) and specificity of 45% (95% ci, 40-51), for postnatal rpd the respective figures were: 54% (95% ci, 33-73) and 72% (95% ci, 67-77) for the cut-off of 8.8 mm. conclusions: our data shown that the magnitude of rpd is a poor predictor of presence of primary vur. there was no significant difference in performance among the indexes. objetive: we investigated the prevalence of renal calcification in children with autosomal ressecive polycystic kidney disease (arpkd) and studied the metabolic changes thats could cause this complication. methods: 9 patients with arpkd. 6 girls/3 boys; range age 2 m to 15 y. criteria inclusion presence typical imaging findings: enlarged kidney and diffusely increased renal echogenicity and poorly defined renal margins on sonography; suggestive imaging features with positive results renal or liver biopsy, results 7 patients, 5 girls and 2 boys, range of age 9-15 y, had ct scan renal clacification bilaterally. without renal clacifications <1 year. 7 children renal calcifications, 7 sistemic hypertension, 4 portal hypertension and gastrointestinal bleeding and one renal colic. renal insufficiency 6 patients, it was mild in 3 (gfr >50 ml/min/1,73 mt 2 ), moderate in 2 (gfr 30-50), and severe in one (gfr<30). all with renal insufficiency had distal tubular acidosis. hypocitraturia urine 7 patients. urinary calcium, uric acid, oxalates, and cystines normal in all. tuberoussclerosis (tsc) -an autosomal dominant inherited genetic disorder -is characterized by development of hamartomatous growths in many organs.two causative genes, tsc1 (chromosomal locus 9q34) and tsc2 (chromosomal locus 16p13.3) have been identified. tsc2 gene is adjacent to pkd1, the major gene for autosomal dominant polycystic kidney disease (adpkd) on chromosom 16p13 and contiguous germline deletion of both genes results in severe polycystic kidney disease phenotype at birth. at 6 months of age bilateral abdominal masses were occasionally palpatedin a previously healthy girl. ultrasonography demonstrated enlarged (approximately 15 cm) kidneys with multiple large cysts resembling those seen in adpkd. renal function and blood pressure was normal. suspicion of tuberous sclerosis was raised due to numerous hypopigmented cutaneous macules on the trunk and extremities. echocardiography demonstrated 2 rhabdomyomas in the left ventricle with no hemodynamic significance. an isolated juxtapapillary astrocytoma was found in the left eye. her psychomotor development was normal with no history of seizures. cerebral magnetic resonance imaging revealed multiple subependymal nodules with noobstruction of the cerebral fluid pathways. by multiplex ligation-dependent probe amplification (mlpa) a large dna deletion was identified spanning from tsc2 exon 10 to pkd1 exon 46 permitting the diagnosis of tsc2-pkd1 contiguous gene syndrome. cardiac rhabdomyoma and cutaneous manifestations were found in her father as well but no renal changes. at 3 years of age the girl is doing fine with ace blockers against hypertension. renal function is still normal. the size of the cardiac rhabdomyomas is diminishing while the cerebral and ocular hamartomasare unchanged. this additional report focuses tsc in an infant presenting with polycystic kidneys and cutaneous lesions. improvements in ultrasound technology and the appropriate timing of antenatal ultrasound has led to refined prenatal diagnosis and enhanced accuracy of diagnosis of fetal renal anomalies and makes it possible to treat obstructive and/or refluxing uropathies before the onset of clinical symptoms.a retrospective review of 165 patients; 44 girls (26,7%) and 121 boys (73,3%) admitted to our clinic between january 1999-december 2006 with antenatal urinary anomalies were investigated to determine the urinary tract anomalies, and the follow-up results are presented. routine prophylaxis was started at admision and the imaging studies were performed. the mean gestational age at detection was 28,4±5,87 weeks. the mean age of admittance was 32,95±29,63 days and the average follow-up period was 19,15±26,05 months. antenatal ultrasonoghrapy examination showed anomalies in 220/330 renal units. of these 220 antenatally observed renal units, 216 had postnatal urinary tract anomalies. of these 216 postnatally observed renal units, 235 urinary tract anomalies were detected (multiple urinary tract anomalies in 48 patients) ( table 1) . fifty-two (31,5%) children had 59 surgical interventions such as; ureteroneocystostomy, pyeloplasty, nephroureterectomy, puv resection. eighty-one (49%) of our patients had urinary tract infection during follow-up and renal scar was detected in 58 (35%) patients. acute renal failure developed in 6 patients and chronic renal failure developed in 3 patients and three patients died. we conclude that all infants with fetal urinary abnormalities should be evaluated, so that we can recognize and treat congenital anomalies that may affect renal function or cause urinay tract infection, renal scarring. the majority of patients with fetal urinary anomalies can be managed safely with close conservative follow-up. fetal urinary tract obstruction at 50 days gestation (e50) produces small kidneys with cysts, whereas obstruction at 60 days (e60) generates large kidneys with cysts. in the present study, we investigated the mechanism for the generation of small kidneys by urinary tract obstruction at an earlier gestational age, e50. fetal lambs underwent urethral and urachal ligation at e50 (n=17) or e60 (n=39). fetal lambs were delivered by c-section 5, 7, 20 days after obstruction, or at term (145 days gestation). unoperated kidneys of e50, e60, e80, and at term served as controls. the percentage cystic area of kidneys obstructed at e50 and e60 was not different 5 days after obstruction (17±5% vs 17±6%). after 7 days, however the percentage of cystic area became larger in kidneys obstructed at e50 (40±17% vs 23±5%), and was significantly larger 20 days after obstruction (51±18% vs 17±17%). proliferating cells, detected by pcna staining, were found in cysts and tubules of obstructed kidneys increasing toward term, and were more abundant in kidneys obstructed at e60. on the other hand, pcna-positive cells in the nephrogenic zone were reduced in obstructed kidneys. the decrease was more prominent in kidneys obstructed at e50. apoptotic cells, identified by tunel staining, were detected in the inner medulla of obstructed kidneys equally in kidneys obstructed at e50 and e60 during the fetal stage. at term, tunel-positive cells were rarely present in normal kidneys or kidneys obstructed at e50, but were found abundantly in the interstitium and occasionally in cysts and tubules of kidneys obstructed at e60. in conclusion, urinary tract obstruction at an earlier gestational age produces small kidneys by inhibition of mesenchymal cell proliferation, which may be due to compression by cysts. a. results: detrusor hyperreactivity, most commonly in i and iv or v grade was found in 47.9% of vur children. the maximum detrusor pressure was above 70 cm h 2 0. detrusor-external dyssymetry was found in 17% of children, most frequently in grade i and iv or v grades, and detrusor-internal dyssymetry was recognized in 19.4% of children with vur, most frequently in grade i and iv or v. in 12.5% of children with i-iii grade of vur cystometric capacity was reduced but 1 child with v grade had increased capacity of the urinary bladder. glomerular filtration according to schwartz equation was normal and independent of vur grade. decrease in osmolality below 800mosm/kg h 2 0 in nocturnal urine was only detected in the group of children with iv and v grade of vur,. there was no correlation between detrusor tension and osmolality of urine and glomerular filtration rate. conclusions: 1). dysfunction of the lower urinary tract, with detrusor hyperactivity was detected, as the most frequent dysfunction in 74% of children below 2 year old with i-v degree of vur, 2). the maximun detrusor pressure in the voiding phase was highest in grade i and iv iv-v reflux children. hypomelanosis of ito was first described as a disorder characterized by unusual unilateral or bilateral macular hypopigmented whorls, streaks and patches. subsequently, neurologic, skeletal and ocular involvement were described. kidney involvement has only been exceptionally reported. herein, we describe a case of a male infant with hypomelanosis of ito and renal involvement. the patient was born at 40 weeks of gestation by cesarian delivery. the ultrasound scan at 34 weeks of gestation revealed bilateral enlargement of kidneys, decreased corticomedullary differentiation and cysts located in the cortical and subcapsular regions. these findings were confirmed at two months of life by ct scan. skin examination showed hypopigmented linear and round diffuse lesions located on the right leg and arms. the ophthalmological examination showed anterior capsular and posterior subcapsular cataract of the left eye. as previously reported in other cases of hypomelanosis of ito, the patient presented a transient leucocytosis (max 30.000/mm 2 ) during the first 8 months of life.the renal biopsy showed a classic picture of glomerulocystic kidney disease, whereas the skin biopsy confirmed the clinical diagnosis of hypomelanosis of ito.three other cases of kidney disease in hypomelanosis of ito have been reported. of these, one case presented abnormalities of the glomerular basement membrane, and one case presented with polycistic kidney disease. the third case had renal cystic dysplasia with a histological picture containing glomerular cysts. alltogether these reports suggest that genes involved in hypomelanosis of ito are important for normal renal development and may be implicated in cystogenesis, when mutated. here we report on a male newborn (birth weight 3070 g, length 51 cm) who presented with progressive edemas, oliguria and failure to thrive during the first week of live. on clinical examination he showed bilateral microcoria and decreased muscle tone. laboratory work-up revealed large proteinuria (28 g/g creatinine), hypoalbuminemia (13 g/l) and renal failure ( objectives. to investigate the incidence, nature, and management of associated genitourinary malformations in children with multicystic dysplastic kidney (mcdk). methods. in this retrospective study, we analyzed the medical records and imaging studies of 24 children with mcdk. in 20 children (83%) anomalies of the urinary tract were suspected prenatally in ultrasound studies. in the remaining 4 children the diagnosis of mcdk was made postnatally. results. the male/female sex ratio was 8: 16. the left side was involved in 11(46%) children. voiding cystourethrography was done in 14 (58%) children, the isotopic 99m tc-dmsa scan of the kidney in 15 (63%). urogential anomalies were present in 10 (42%) children. among them, contralateral urologic anomalies were found in 3 patients (vesicoureteral reflux in 2 and hydronephrosis in 1), and ipsilateral in 8 (vesicoureteral reflux in 1, ureterocele in 3, and hydroureter in 2). genital abnormalities such as uterine didelphys and hydrocele were found in 2 children. fourteen (58%) patients underwent follow-up examinations with ultrasonography (mean follow-up 4.15 years, range 7 months to 12 years). compensatory hypertrophy of the contalateral kidney was found in most children and decreased size of ipsilateral dysplastic kidney was found in 9 out of 14 children with follow-up. no cases of hypertension or tumor developed during the follow-up. conclusion. ultrasound can be used safely to diagnose unilateral mcdks and associated genitourinary malformation. although the risk of hypertension and development of malignancy is low, follow-up evaluation of contralateral renal function and genitalia will be needed. in cases of hydronephrosis and/or urinary tract infection, voiding cystourethrography is necessary and possibility of association with genital anomalies should be considered until the puberty. a. background: ectopic ureter is a rare anomaly. its incidence is at least four times higher in females and also more than 80% of the ectopic ureters drain duplicated systems in females. the most common presenting symptoms of an ectopic ureter are urinary tract infection and incontinence. diagnosis is often delayed and may remain undiagnosed until adulthood. case report: a 3-month-old girl was admitted to our hospital with the complaints of fever and discomfort. her mother recognized intermittent dribbling of urine while changing her napkin. physical examination revealed fever (38.3 °c), diaper dermatitis and intermittent dribbling of urine. urinalysis revealed leukocytouria, acute phase reactants were elevated (crp: 1.45, esr: 27) and renal function tests were normal. the patient was hospitalized with the diagnosis of acute pyelonephritis. tc99m dimercaptosuccinic acid (tc-dmsa) scan revealed an increase in the size of the right kidney and decreased uptake in the upper half of the same kidney. ultrasound was performed with the suspicion of an ectopic ureter and it showed right duplicated kidney with marked dilatation of the upper collecting system. the ureter was also dilated in its whole length and ended ectopically distal to the bladder. contrast-enhanced magnetic resonance urography (mru) demonstrated right obstructed duplicated system with vaginal ectopic insertion of upper pole ureter. discussion: this case was presented to underscore the role of careful physical examination in the diagnosis of this rare anomaly that is by paying attention to the complaints of the family. ultrasound is the initial, important diagnostic modality in these patients especially if done by experienced radiologists. the diagnosis can be confirmed with mru by depicting the exact insertion of the ectopic ureter. objectives of study: to evaluate the occurrence and severity of vesicoureteral reflux (vur) in young infants with a history of mild prenatal hydronephrosis. the usefulness of voiding urosonography (vus) in the diagnosis of vur was also evaluated. methods: forty seven infants (31 males, 16 females) with a history of mild prenatal hydronephrosis, diagnosed between 21 st to 30 th week of gestation, were enrolled in the study. postnatal ultrasound was performed within the first month of life. voiding cystourethrography (vcug) and at the same time, contrast-enhanced harmonic vus was performed at the age of 1.5-2.5 months. results: the prenatal ultrasound revealed an anterior-posterior pelvic diameter of 5-7 mm in 33 fetuses and 7-10 mm in 14. postanatal ultrasound showed an anterior-posterior pelvic diameter of 5-7 mm in 34 infants and 7-10 mm in 13. vur was found in 9 of 47 (19.1%) infants (grade i: 2, ii: 4, iii: 3). the vur was detected by both vcug and vus in 4 of 9 children, only by vcug in 2 and only by vus in 3 of 9 infants. the vur that was missed by vcug was more severe (grade ii and iii), compared with this one missed by vus (grade i). conclusions: even though prenatal hydronephrosis was associated with a quite important occurrence of vur, this was of mild or moderate severity. comparison between the two imaging modalities showed that the vur missed by vus were with no clinical significance (grade i), whereas the vur missed by vcug were more severe. although further study is needed, vus could be an alternative method, mainly in girls, in whom the imaging of the urethra is not necessary, thus avoiding the radiation exposure. early treatment with indomethacin in a neonate with a bartter syndromea case report neonatal bs is a rare genetic disorder characterized by sodium, potassium and chloride urinary wasting, hypokalemic metabolic alkalosis with hyperreninemia and hyperaldosteronism in the absence of hypertension and high level of urinary prostaglandins. indomethacin therapy is controversial because of toxicity for gut and kidney. a premature boy of unrelated couple was born by cesarean section at 28 weeks because of early rupture of membranes and fetal distress. birth weight was 900 g, length 36 cm and apgar score 2/6/8. pregnancy was complicated by severe polyhydramnios. baby was mechanically ventilated for first 12 hours and treated with antibiotics because of suspected sepsis. marked polyuria and dehydration were present from 1st week. metabolic parameters revealed hypokalemia, hyponatremia and hypochloremic metabolic alkalosis. serum creatinine was slightly elevated and gfr in normal ranges. blood pressure was normal with raised plasma renin activity and aldosterone level. sodium excretion via urine and level of renal and systemic prostaglandins were increased. nephrocalcinosis was detected on us from 2 week. additonally to electrolyte supplementation indomethacin was started on the 18th day at a dose of 1 mg/kg/day. in first 2 months child experienced 4 septic episodes, candida pyelonephritis and was operated because of bowel obstruction. dna analysis of affected child found 2 new mutations in romk gene: p185l and q289x in herited from parents who carried one mutation each. at 18 months height and weight are at 3 and head circumference at 50 percentile with slightly retarded psychomotoric development. he continues on 1.2 mg/kg/day indomethacin therapy. blood electrolyte profile is normal without supplements. us shows no progression of nephrocalcinosis. early treatment with indomethacin may prevent life-threatening complications and reduce the development of nephrocalcinosis. nimuselide, a cox2 inhibitor is widely used in india for relief of pain and fever. we describe 6 cases of fetal renal abuse leading to neonatal renal failure due to maternal ingestion of nimuselide in the third trimester of pregnancy. results: all 6 patients were diagnosed as having renal failure in the first few days of life.there were 5 boys and one girl.. all the mothers had normal pregnancies except for oligohydramnios that was detected during the last 2 months of pregnancy in all cases.4 children presented with anuria from birth. the remaining two had non-oliguric renal failure with metabolic acidosis as the presenting feature in one and poor feeding and lethargy in the other. usg revealed normal sized kidneys. one patient also showed increased echogenicity. 3 out of the 4 anuric children underwent peritoneal dialysis for a period varying from 3 to 4 weeks without recovery of renal failure. the remaining 3 were managed conservatively. two of these patients are now in chronic renal failure at ages 2 and 5 years. only one patient recovered completely after 5 days of anuria. all the mothers had taken nimuselide in the last trimester for periods varying from few days to several weeks for relief of pain or fever. some of them had taken multiple courses of short duration. the mother of the child who recovered completely had taken nimuselide in the last 5 days before delivery. renal biopsy done in one baby revealed renal tubular dysgenesis. conclusion: nimuselide intake in the last trimester of pregnancy can be associated with oligohydramnios and neonatal renal failure that can be irreversible. renal tubular dysgenesis may be the underlying pathology. objectives of study: pkd is the most common inherited renal disease. aim of ours study was to analyse clinical and laboratory features of the different types pkd. patients and methods: we described the clinical presentation of 47 children with pkd (26 boys, 21 girls) diagnosed in pediatric nethrology department between 1989 and 2007. the patient's age range was from 3 months till 18 years. we retrospectively studied the family histories, clinical and biochemical data (physical examination, level of arterial blood pressure, blood and urine creatinine levels, serum levels of urea, glomerular filtration rate), ultrasonography, scintigraphy. results: the analysis of the family histories revealed adpkd in 27 patients (14 boys, 13 girls), arpkd in 6 (2 boys, 4 girls) and nondifferential pkd in 14 (10 boys, 4 girls) children. pkd diagnosed by antenatal ultrasound in 6 cases (2 adpkd, 4 arpkd). the mean follow-up adpkd were 4,4 year (range 1 -13 years), arpkd -5,3 year (range 1 -14,5 years). conclusion. patients with arpkd demonstrated the early beginning of an arterial hypertension and progressing chronic renal failure. one girl with neonatal form of arpkd died. chronic renal failure developed in 9 (19,1%) cases of pkd. objectives: this prospective study was to answer the question on the need of long-term follow-up and correlation of renal functions with the age at surgery and grade of o.u. prior to surgery in patients after surgery of obstructive uropathy (o.u.). methods: selected biochemical markers of glomerular and tubular functions and ultrasonographic findings in 62 patients (40 boys) who underwent surgery due to uni-or bilateral o.u. of grade iii. and iv. (age at surgery <12 months) in 1994-1997 were examined at mean age of 6.3±0.9 years. the results were compared to 32 healthy controls and/or to reference values according to age. consequently, patients were devided into groups according to the age at surgery (0-3 months, 4-6 months, 7-12 months) and grade of o.u. prior to surgery. results: serum concentration of cystatin c (s-cysc) was significantly higher in patients when compared to control group (p<0.001). while s-creatinine was within reference interval in all patients, s-cysc was increased in 63.3% when compared to reference interval. decreased tubular resorption and concentration ability was found in 67% and 26% patients, respectively. non-specific aminoaciduria was detected in 42.9% patients. on ultrasound, 66.7% kidneys after surgery had residual dilatation of renal pelvis. the differences in renal functions in patients according to their age at first surgery were not significant except for u-nag activity with significant negative linear trend with higher age at surgery. the grade of o.u. prior to surgery did not have significant influence on renal functions. conclusions: mild tubular dysfunction and slightly reduced gfr in the part of patients make longterm nephrologic follow-up reasonable. our results support the trend of postponing early postnatal surgical intervention in patients with positive ultrasound screening of o.u. and normocreatininemia. objectives of study: to evaluate, during almost five years of follow-up, the changes among preoperative and postoperative renal function in 46 infants (36m/10f) with prenatal severe hydronephrosis (hn) and upj obstruction. methods: upj obstruction was diagnosed by a mag3 renal scan, performed at 4-6 weeks of age to establish baseline differential renal function; surgery was made if there was evidence of obstruction injury, and/or progression of hn and/or symptoms. the group was re-imaged 3 months after surgery, after 6 months, 1 year and then annually. results: initial differential renal function was moderate in 41.6% of males and in 30% of females and good in 58.4% and in 70% respectively. also,75% of kidneys required surgery because of declining function, with mean differential renal function in the affected kidney of 32% that improved to 44% already at 3 months after pyeloplasty. there was no significant functional improvement, in the kidneys that underwent correction because of increased hn or symptoms and final renal function was >40%. after pyeloplasty t was >30 min. in 21% and 20-30 min. in 79% of cases (p<0.05) there was no statistically significant correlation between initial grade of hn and initial renal function. surgical treatment was performed between 9-16 months of age and there was no significant difference in postoperative results, ascribed to patient age at surgery. ma values, greater than controls at diagnosis, reduced at 18 months after surgery in all, but 16% of children. during the follow-up, the mean ccr and bp values were in normal range for age in all children. conclusions: our findings showed improvement also in kidney with preoperative uptake less of 40%. this may be to explain in according to an inverse correlation between degree of renal dysplasia and gestational age. objective of study: is to determine the postnatal course and follow-up of children with fetal hydronephrosis. methods: in 6 years period (2000) (2001) (2002) (2003) (2004) (2005) (2006) we followed 72 infants with antenataly detected hydronephrosis. all infants were submitted to ultrasonographic examination of kidneys and bladder. if indicated, the isotope renography, micturating cysto-urethrography, i.v.urography and mr were performed. results: the diagnosis of hydronephrosis was established during 15 -39 th weeks of gestation by obstetritian. first postnalat ultrasound investigation was performed during neonatal period in most children (69%). in 48 (66,7%) infants we diagnosed idiopathic hydronephrosis, in 12 (16,6%) vesicouretheral reflux (vur) grade ii-iv, in 7 (9,7%) hypofunction of one kidney, in 2 (2,8%) ureteropelvic junction obstruction (upjo), in 1 (1,4%) ren duplex and ureterocele, in 1 (1,4%) ampular pelvis and in 1 (1,4%) afunction of one kidney. after 6-12 months we found normal ultrasound in 35 (48,6%) children. the ultasound results were stable in 24 (33,3%) children and in 9 (12,5%) there was progression of hydronephrosis. four (5,5%) infants underwent immediate surgery. conclusions: in a group of 72 infants with antenataly detected hydronephrosis the diagnosis was confirmed postnataly in 89% infants. more than 50% of infants required long term follow-up. in 5,5% the immediate surgey was required. this data support the need for antenatal detection of hydronephrosis. in the same period we followed up 207 infants with urologic abnormalities which were not detected antenataly.the fetal ultrasound is reliable screening method in detection of urologic abnormalities. the considerable number of anomalies which were not detected antenataly are the result of insuffitient use of fetal ultrasound investigation. hemolytic uremic syndrome (hus) is defined by acute renal failure, microangiopathic hemolytic anemia, and thrombopenia. perinatal asphyxia (pa) may cause renal failure after birth and is often associated to disseminated intravascular coagulopathy (dic) with platelet consumption. however, no biological investigation permits to distinguish clearly neonatal hus from dic. we report three neonates with renal failure due to different degrees of pa. they presented biological features compatible with hus such as fragmentocytes (3%), thrombopenia (<50,000/mm3), anemia (<8 g/dl). serum creatinine on day 5 was 293, 152, 372 mmol/l respectively, requiring peritoneal dialysis in one patient. haptoglobin was undetectable for all three patients. factor h and i were in the lower normal range; components of the complement system (c3, c4) and adamt13 activity were decreased. two patients received daily fresh frozen plasma infusions over the first 4 weeks. renal function improved in two patients until day 30; one patient has chronic renal failure. all other parameters suggestive for hus were normal on day 12, 30, and 60 respectively. no severe neurological consequences were noted for either of them. pa may be responsible for multiorgan damage via ischemic lesions. ischemia may result in endothelial cell injury, the crucial event for the development of thrombotic microangiopathy. we hypothesize that endothelial cell damage concomitant with pa may lead to a vicious circle resulting in consumption of platelets and plasma factors involved in hemostasis and/or fibrinolysis. early use of fresh frozen plasma may correct these alterations. renal biopsies might have been useful but are technically difficult in newborns. in conclusion, pa and neonatal hus are difficult to distinguish and endothelial cell damage may be a common pathyphysiological aspect and might requirespecific treatments. a. medynska, m. nalesniak, k. kilis-pstrusinska, d. zwoliñska congenital posterior urethral valves (puv) are the most common cause of lower urinary tract obstruction in male neonates. we aimed to review our experience with puv children (24 boys) in respect to retrospective analysis of the clinical course of disease. we analyzed: ultrasound during pregnancy, age of disease onset, clinical symptoms at admittance to hospital, outcome. average lenght of follow-up was 7,3 years, varying between 2 month and 20 years.obstruction of urinary tract was suspected by prenatal ultrasound in 9 patients. the initial presenting symptoms were as follows: urinary tract infection 7 boys, failure to thrive 3 patients, increased abdominal circumference -2, abdominal pain 1, enuresis -3, acute renal failure 1, and chronic renal failure 14 children. in association with puv renal dysplasia/hypoplasia in 4 patients, undescended testis 3, bladder trabeculation 4 were found. the diagnosis of puv was confirmed by voiding cystourethrograms and/or cystoscopy. primary vesicouretral reflux was documented in 7 pts. hydronephrosis and/or megaureter were observed the most often in 14 boys. the diagnosis of puv was established in 10 pts at the age of less 1 month, in 6 pts between 2 -12 months, and in 8 between 1-15 years. surgery was performed in 7 pts in neonates period including primary valve surgical ablation and/or cutaneostomy vesicostomy. chronic renal failure was diagnosed in 7 boys in first year of live. 4 of them progressed to end-stage renal disease. globally during the follow-up 7 pts developed end-stage renal disease. 4 pts have done a graft. only 5 boys survive without progression to chronic renal failure. the presentation of puv is variable and currently antenatal detection is the most common mode. outcome boys with puv is poor. patients need nephrologic assesment from birth. background: furosemide is among the 10 most frequently used drugs in the neonatal unit but few studies analyze the beneficial effect and complications in this patient group. objectives of the study: to analyze the therapeutic clinical effect and to document side effects of furosemide therapy in extremely preterm infants born <28 weeks gestational age (ga). methods: twelve infants born <28 weeks ga were prospectively included during the fall 2006. the following parameters were documented prior, during and after furosemide administration: clinical status, serum/urine electrolytes, creatinine, albumin, blood gases and furosemide exposure. ultrasound of the kidneys and a wrist radiograph were performed at 6-8 weeks to rule out osteopenia/rickets and nephrocalcinosis respectively. no statistical analysis were done due to the small study size. results: general oedema, respiratory rate, apneic spells and oxygen supplementation decreased. arterial/venous pco 2 decreased and partial oxygen saturation increased indicating improved lung function. hco 3 increased and ph decreased. urinary excretion of sodium, potassium, chloride and calcium increased while phosphate excretion decreased. serum sodium and chloride decreased and potassium increased initially. six infants had electrolyte disturbances and metabolic alkalosis. one infant died during the study period. in the remaining 11 infants, 3 of 5 had worsening of their patent ductus arteriosis, 3 had osteopenia or rickets and 1 had nephrocalcinosis. the total side effect score was increased in the infants with the highest furosemide exposure. conclusions: this small study suggests that furosemide is beneficial in extremely preterm infants born <28 weeks ga and that the associated side effects correlate to the total drug exposure. we recommend caution for long term administration of furosemide. conclusion: although children in our study suffered significant neonatal hie resulting in arf, glomerular and tubular function recovered sufficiently to cope with increasing body mass and metabolic needs. unlike reported studies, we did not find any significant evidence of cri in the survivors of neonatal hie and arf. one with marginal microalbuminuria will need further observation. c was born after a monozygotic monoamniotic twin pregnancy (gestational age=34 weeks). she presented with twin-twin transfusion syndrome with hypotrophy (birth weight=2020g, other twin=2950 g), severe hypovolemia, anemia and acute renal failure. she required a blood transfusion, mechanical ventilation (5 days), and peritoneal dialysis (10 days). she recovered without bronchodysplasia but with chronic renal failure (creatininemia at 2 months=90 μmol/l). the following months were uneventful but, as usual in tts, she exhibited growth retardation and slight mental delay compared to her twin. she reached terminal renal failure at 12 years of age and was successfully kidney transplanted. after age of 6 years, she presented with increasingly frequent and severe pulmonary infections predominantly in lower lobes of both lungs. after extensive explorations, the diagnosis of bilateral bronchiectasis was made. the search for classical aetiologies of such pathology was negative. the symmetric aspect and the absence of other etiologies lead to the consideration of bronchiectasis as a congenital pathology. numerous publications demonstrate the role of the renin-angiotensin system (ras) in renal and cerebral damages in tts. authors demonstrate the role or ras in development of vasculature in fetus but also of cartilage and muscle in different organs including lung. associations between tts and lung pathologies need to be further investigated. urofacial ( the urofacial (ochoa) syndrome (ufs) is a rare disease that occurs in both sexes and is more frequent when the parents are closely related. it has both urinary and facial abnormalities. ufs is a rare autosomal recessive disorder and a potential gene has been mapped to chromosome 10q23-q24. they present a bladder voiding dysfunction due to impaired neural communication between the bladder and the spinal cord, resulting in incomplete emptying of the bladder. this usually results in enuresis, urinary tract infection, hydronephrosis and in some severely affected patients, end-stage renal disease developed. the facial abnormality is a characteristic expression that, when these patients smile, their facial musculature inverts and they appear as if they are crying. we report a 15year-old girl who has inverted facial appearance, voiding dysfunction and vezicoureteral reflux. she had constipation and did the intermittan uretral cathaterization for five years. after a detailed evaluation, she was diagnosed as ochoa syndrome due to inverted facial expression. we report this case, because early diagnosis of ufs is very important for early assessment and management of urinary problems to prevent development of chronic renal failure. we think that only a smile can give a strong high light for this unusual 'inverted' facial expression and patients can be screened earlier for severe voiding dysfunction. tar syndrome is a congenital malformation syndrome characterized by bilateral absence of the radii and a thrombocytopenia. the known urinary anomalies with tar are duplex ureter, dilatation of renal pelvis, horseshoe kidney, and functional problems like vesicouretheral reflux and pyelonephritis. case: nine years old patient with tar syndrome was admitted with a complaint of bright red urine repeated three times. the microscopic hematuria without dysmorphism of erythrocytes accompanied with no proteinuria were determined in repeated urine sample microscopy. iga, ana, anti dna serology were negative. urine culture was clean. stone formation (6x7 mm) in the upper pole of the right kidney was established by the abdominal ultrasonography. postoperative chemical analysis of the stone, revealed that it was consisted of oxalate monohydrate and dihydrate. but the patient discontinued his follow-ups afterwards for a year. in this period, he had macroscopic hematuria attack once. when he applied for the second time, he reported macroscopic hematuria. cystoscopy was done for etiology. many tortuous and engorged vessels were seen by this evaluation in the bladder mucosa ( figure 1 ). there was no active bleeding point. result: in this report, kidney stone and telangiectasia found co-incidentally in the bladder of a patient with tar syndrome during the examination of hematuria are discussed as there is no case report demonstrating nephrolithiasis and telangiectasia in tar patients in the literature. figure 1 : many tortuous and engorged vessels in the cystoscopy. antenatal hydronephrosis (anh) is one of the common fetal abnormalities detected on ultrasound (us). the long-term renal prognosis for infants with mild to moderate postnatal hydronephrosis (hn) is unclear and controversial. a systematic review of the published literature was performed to determine an evidence based approach in infants with antenatally diagnosed hn and to identify those at risk of significant post natal nephro-uropathy (pnu). key questions were identified. does anh predict renal tract pathology in neonates? what is the value of prophylactic antibiotics in infants with anh? can postnatal us diagnose significant pnu? when is the optimal time to screen infants postnatally? which imaging modalities are necessary to diagnose the cause of the hn? how many neonates with anh would need to be investigated to prevent one case of esrf/crf? a search strategy was formulated. out of 362 titles only seven studies met the validity criteria for inclusion. the results indicate that antenatal us is a valid screening test for pnu (sensitivity 16%, specificity 98%), but is not a predictor of pnu. the detection rate of hn by us is the same whether it is done early or late in neonates. two normal ultrasounds over a minimum of one month are required to screen for pnu. infants with a renal ap diameter over 7mm are at risk of a significant pnu and should be investigated further. us is not a substitute for cystourethrogram or dynamic isotope studies to determine the cause of hn. none of the studies addressed the role of prophylactic antibiotics. there was insufficient data to calculate the total number of cases that would need to be investigated to prevent one unfavourable outcome (esrf/crf). high quality population based cohort studies with long term follow-up into adulthood are required to determine the optimum postnatal management of mild to moderate hn in infants with anh. jeune asphyxiating thoracic dysplasia: a case report jeune asphyxiating thoracic dysplasia (jatd) is one of the congenital hepatorenal fibrocystic syndromes. it is characterized by renal, hepatic, pancreatic abnormalities with associated skeletal abnormalities including a long and narrow thorax, metaphyseal irregularities, and shortness of the ribs and long bones. this report describes a pediatric patient with jatd developed end-stage renal failure. a 7-year-old boy was admitted with complaint of vomiting and pallor. he had dysmorphic appearance including trigonocephaly, short stature, long thorax and short limbs and fingers, polydactyly and fascial dysmorphism. laboratory findings revealed severe anemia (hb 3.8 g/dl), high bun and creatinine levels (111 mg/dl and 7.9 mg/dl respectively) and normal liver tests. abdominal usg showed a severe intrahepatic biliary tract dilatation and intraparenchymal cysts in liver, pancreas and kidneys. mr pancreatocholangiography was consistent with caroli's disease. jatd should be considered when caroli's disease exists with skeletal abnormalities. follow-up antenatal hydronephrosis: one centre experience the most common renal abnormality detected antenatally is hydronephrosis (1% to 5% of all pregnancies). in this paper, we report follow-up our patients with antenatal hydronephrosis (ah) between 2001 and 2006. diagnosis of hydronephrosis was made >5 mm of antero-posterior diameter (ap) of the renal pelvis. ah was detected in 74 patients on antenatal ultrasound examination. of the 74 patients with ah, 46 (62.1%) were found to have hydronephrosis postnatally and 34 (37.8%) postnatal scans were normal. in 17 of these patients (36.9%) had bilateral and 29 (63.1%) of these patients had unilateral hydronephrosis. in these patients with ah, uretero-pelvic junction obstruction (upj) 28 (60.8%), reflux 12 (26.2%), uretero-vesical junction obstruction 3 (6.5%), posterior urethral valves 2 (4.3%) and mega-ureter 1 (2.2%) were identified. in follow-up period, 2 (16.6%) patients with reflux, 19 (41.3) patients with upj were treated with surgery (p<0.05). in conclusion, upj was most important cause of ah and most of them were treated with surgery. hyponatremia and renal tubular acidosis in severe vesicoureteral refluxa case report sahlgrenska academy, department of pediatric, gothenburg, sweden case report: 1st child to healthy parents without heredity for kidney or metabolic disease. antenatal bilateral hydronephrosis. postnatal examination showed bilateral vur grade v, normal urethra. antibiotic prophylaxis was started, no urinary tract infection (uti) occurred. normal s-creat and s-na + at 2 weeks of age. the electrolytes remained normal during the next 3 months. normal growth. the boy was thirsty with high urine volumes. at 3 month of age feeding problems began with retarded weight gain. no vomiting or diarrhoea. at admission to the hospital the child was dehydrated, s-na + 120 mmol/l, s-cl -99 mmol/l, s-ph 7.11, bicarbonate 11 mmol/l, s-creat 36 μmol/l. crp 8 mg/l, urine culture negative. u-na + 10 mmol/l, u-ph 6.0, u-osmolality 214 mosm/kg. the anion gap was normal and there was no lactacidosis. the s-aldosterone was elevated 53,1 nmol/l, s-cortisone normal. treatment included intravenous rehydration, na + supplement and oral bicarbonate. blood chemistry normalised and the child's general condition improved rapidly. conclusions. children with severe reflux are at high risk for uti but may also develop impaired tubular function with diabetes insipidus, renal tubular acidosis and hyponatremia. the mechanisms include down-regulation of vasopressin receptors and impaired distal tubular function. close clinical monitoring of these children with regular blood chemistry and weight controls is important. purpose: we aim to prospectively study the natural history of minimal to severe grades of antenatal hydronephrosis (anhn) in our local chinese population and correlate the renal pelvic diameter (rpd) with the outcome. patients and methods: cases of anhn were prospectively followed up along a predefined protocol using us, mag3 and mcu in all. obstruction (pujo or vujo) was defined as a need for surgery based on symptoms and deteriorating function, not on mag3 drainage time. results: 174 neonates were followed up for minimum of 5 years. eighty percent had normal or minimal hn on postnatal scans (rpd 5-9 mm), 11.4% had mild (10-14mm) or moderate (15-19) hn and 8.5% severe hn (>20 mm). seventy-eight percent infants had benign course; 6.9% had partial obstruction which improved; 11.5% had vesicoureteric reflux (vur) one of which required surgery. another 5.7% required surgery for obstruction. the roc curve for obstruction requiring surgery showed optimal cut-off point of 18.5 mm (sensitivity 100%, specificity 99.2%). prolonged diuretic t1/2 was not predictive of surgery. severity of hn did not correlate with presence or grade of vur. fifteen patients developed uti despite antibiotic prophylaxis and 7 had focal scars, all occurred in association with high grade vur or obstruction. the prognosis of infants with minimal or mild anhn is good. however rpd is poorly correlated with vur. the chances of obstructive lesions requiring surgery are high when the rpd is above 18 mm. in those with high grade reflux or obstruction, urinary tract infection may lead to renal scars. objectives of study: cystinosis is a rare disease presented initially with renal fanconi syndrome, and renal glomerular failure develops later in childhood. without cysteamine treatment, patients affected with cystinosis uniformly died during childhood in the absence of renal replacement therapy (rrt). cysteamine is not available here and in some other areas of the world. the aim of this paper is to describe a beneficial effect of acacia gum in a patient with cystinosis and chronic renal failure. method: 9 years old girl with cystinosis presented with symptomatic uremia as she didn't receive cysteamine. serum creatinine 7.4 mg/dl, blood urea 200 mg/dl. the girl was hospitalized and vomiting controlled with intravenous fluid and pyridoxine. chronic dialysis was not available for her and the parents refused treatment with intermittent acute peritoneal dialysis. the girl was treated with a new therapeutic regimen (therapy2006;3 (2): 321) combining the traditional conservative management of crf (dietary and pharmacologic) with addition of acacia gum (ag) 25 g/day as an urea lowering agent aiming at improving her condition without dialysis. results: treatment was associated with amelioration of the uremic symptoms and improved general well being. after 2 weeks of treatment, serum creatinine 1.9 mg/dl, blood urea 69 mg/dl. during 4 months of follow-up she continued in experiencing improved well being and urea levels was kept below 70 mg/dl without dialysis. conclusion: it was possible to improve the health of patient with cystinosis despite the nonavailability of cysteamine and the appropriate rrt. objectives of study: the pattern of renal tubular disorder (rtds) has been infrequently reported in the literature, and the pattern of rtds in iraqi and arab children is not known. methods: from june 2000 to august 2006, it was possible to evaluate 40 children with suspected rtd to determine the type of their tubular defect. there was evidence of rtd in only 35 patients; 22 males (63%) and 13 females (37%). their ages at referral ranged between 8 months and 14 years (mean 4.8 years). in 4 patients with oculo-cerebro-renal syndrome, there was no evidence of rtd and one patient had hyperoxaluria which not a rtd. results: seven types of rtds were identified. the three most common disorders were: idiopathic hypercalciuria (37%), cystinosis (21%) and renal tubular acidosis rta (21%). four of the patients with rta have proximal rta, and four have distal rta. four of the patients with hypercalciuria have also significant hyperoxaluria >3 mg/kg/day. conclusion: the pattern of rtds in iraqi children differs from the previous studies: in germany the three most frequent disorders were cystinosis, xlhr, and idiopathic hypercalciuria. objectives of study: few literatures reported the incidence of ocular abnormalities in chronic renal failure (crf). the aim of this paper is to determine the incidence of ocular abnormalities in childhood crf. methods: from january 1993 to december 2005, 80 patients with crf (at the university hospital in al kadhimiyia) were examined to determine the presence of ocular abnormalitites. fifty patients were males (62.5%) and 30 (37%) were female. their age at referral ranged from 2 months to 18 years (mean 9 year). they were followed for a period ranged from 5 days to f years. results: corneal cystine crystals were the most common ocular abnormalities associated with childhood crf observed in 6 patients with nephropathic cystinosis (7.5%). congenital cataract & glaucoma were observed in 3 patients (3.75%) with oculo-cerebro-renal syndrome (ocrs). congenital cataract & chorioretinal hypoplasia were present in 1 patient with ocrs. hypertensive retinopathy occurred in 2 patients. acquired cataracts occurred in one patient with hinman syndrome in association with hypocalcaemia and non-compliance with calcium and onealphacalciferol supplementation. retinitis pigmentosa in one patient with laurence moon biedl syndrome. bilateral optic atrophy in one patient with familial nephropathy associated with club feet. proptosis in one patient with membranoproliferative glomerulonephritis. conclusion: ocular abnormalities are relatively common in childhood crf occurring in approximately 19%. objective: hypocalcaemia has been reported as a complication of phototherapy especially in neonates. we studied the relation between serum calcium level and urine calcium to creatinine ratio in neonates under phototherapy. method: 50 icteric newborns (30 males and 20 females) treated by phototherapy entered into study by non accidental sampling. the consent was taken from parents on admission. all were breastfed healthy newborns. weight was checked and serum samples for calcium and bilirubin and urine aliquots for calcium, creatinine and osmoloality were sent on arrival (group i), after 48 hour of starting (group ii) and 24 hour after discontinuing phototherapy (group iii). hypercalciuria was defined by uca/ucr >0.8, hypocalcemia was defined by serum calcium <8mg/dl in the term and <7 mg/dl in the premature. chi2, anova, wilcoxon rank test and spearman were used to compare frequency, means, median and correlation. p<0.05 was considered significant. two groups were designed, pateints whose therapy were finished at least 12 months (group 1) and those either on therapy or less than 12 months passed from the last protocol of cytostat (group 2). demographic data, cumulative dosages of anticancer drugs, history of other nephrotoxic agents, nephrectomy, radiotherapy and acute renal failure were recorded. we used ctc2 (1999) to evaluate renal function. chi2 and mann whitney u test and biniary logistic regression were used to compare percentage, scoring and correlation respectively. p value less than 0.05 was considered significant. result: 58 out of 115 patients were in group 1 and 57 ones were in group 2. the mean of age was 11.86 years (±5.3 sd). the median (range) of therapy and termination was 36 months (1-156 months) and 1 month (0-12 month) in group 2 and 36 month (24-240) and 51 months (15-120 months) in group 1 respectively. the percentage of reversible renal failure, proteinuria, abnormal serum calcium and magnesium, metabolic acidosis and urinary concentration defect was higher in group 2. (table 1) these differences were statistically significant (p<0.05). we found no correlation between ctc score and dosage of drugs, age, sex, history of radiotherapy or nephrotoxic agents (p>0.05). conclusion: mild to moderate tubular dysfunction has been observed in survivors of leukemia. routine follow-up of renal functions is recommended. v. tramma, k. giourtzis, v. fotoulaki, k. nousia-arvanitaki aristotle university, 4th pediatric clinic, thessaloniki, greece although cftr is expressed in the kidney, patients with cystic fibrosis (cf) have not been reported to have major renal abnormalities with the exception of urolithiasis. the aim of this study was to determine renal function and the potential risk factors for renal stone formation in cf patients older than 10 years of age. the findings of metabolic evaluation of 4 cf patients having confirmed urolithiasis (mean age: 22,37±5,78) were compared with those of 27 cf patients without urolithiasis (mean age: 24,25±4,03) and those of 10 healthy volunteers (mean age: 22,13±4,98). evaluation included plasma sodium (na), potassium (k), chloride, bun, creatinine, uric acid, calcium (ca), phosphorus (p), magnesium (mg) and parathormone (pth). twenty-four hour urine collection for creatinine, uric acid oxalates, ca, mg, k + , na + and microalbuminuria was also performed. glomerular filtration rate (gfr) was calculated and fresh urine samples were examined for the presence of crystals, erythrocytes, glycosuria and microorganisms. patients with cf and urolithiasis showed significantly increased values of bun (p: 0.012), pth (p: 0.018) and gfr (p: 0.003), very low urine mg levels (p: 0.014) and microalbuminuria (p: 0.034) as compared to cf patients without urolithiasis. there was no correlation of urolithiasis with hypercalciuria and hyperoxaluria. furthermore, all cf patients showed significantly increased pth levels (p: 0.032), very low urine mg levels (p: 0.024) and microalbuminuria (p: 0.024) as compared to healthy volunteers. conclusions: renal dysfunction was demonstrated in older cf patients, probably, secondary to the primary defect of renal chloride channels. extracellular volume regulators, such as hormones, may also be implied. urolithiasis may be the result of renal dysfunction. conclusions: the morbidity of hsp had obviously increased in recent years. the familial cases, the initial symptoms of no palpable purpura at onset and the cerebral, pulmonary, cardiac and pancreatic involvement should be paid attention. objectives of study: systemic lupus erythematosus (sle) is an autoimmune disease affecting multiple organs and tissues including central nerve system, cardiovascular system and kidney. although etiologic mechanisms of sle are incompletely known, overproduction of immunoglobulin g autoantibody may contribute to onset of this disease. while still incompletely understood, the etiology of systematic sle is considered to involve both genetic and environmental factors. we encountered two boys with severe sle from unrelated families and analyzed polymorphisms of the gene that encodes cytotoxic t-lymphocyte associated (ctla)-4, a protein important in t-cell activation and immune tolerance. abnormal function of the gene may participate in causation of autoimmune disease including sle, resulting in production of immunoglobulin against various self-antigens. case report: in family 1, a boy showed serious cardiovascular complications associated with heart failure while his mother also had clinically active sle including nephritis. a boy in family 2 developed severe renal complications and peripheral vasculitis accompanied by disseminated petechiae in the lower extremities; his paternal grandfather had died from fibrinous pneumonia caused by sle. results: analysis of the ctla-4 gene indicated that the boy in family 1 and his mother possess a gg genotype in ctla-4 exon 1 at +49 together with a 106-bp fragment length of the 3' untranslated region (utr) in exon 4. the boy in family 2 also showed gg at +49. no association with disease activity was found for polymorphism of the promoter region in exon 1 at -318 in either family. conclusions: disorders of the ctla-4 gene, especially a gg genotype in exon 1 at +49 and/or 106bp fragment length of the 3'utr in exon 4, may be involved in early development of sle in japanese children such as the boys described here. this disorder is transmitted mainly as x-linked trait, and is caused by mutations in the col4a5 gene encoding α5 chain of type iv collagen. in some families, x-linked as is associated with diffuse leiomyomatosis. we present clinical, pathologic and molecular-genetic findings in japanese family with this inheritance mode of as in association with leiomyomatosis. case report and results: as was diagnosed in a one-year-old boy with recurrent aspiration pneumonia caused by esophageal stenosis from leiomyomatosis. he had macroscopic hematuria and bilateral cataracts. diagnosis was confirmed by electron microscopy coupled with type iv collagen chain subtype staining in a renal biopsy specimen. thickening and irregular contours of the glomerular basement membrane (gbm) and splitting of the lamina densa were evident by electron microscopy. immunofluorescent staining for type iv collagen chains failed to show staining for α3 (iv), α4 (iv), or α5 (iv) in the gbm, associated with lack of α5 (iv) and α6 (iv) staining in the bm of bowman's capsule. his mother, who exhibited esophageal leiomyomatosis and is heterozygous for as, showed a discontinuous staining pattern for α5 (iv) along the epidermal bm. genetic analysis in the boy revealed the deletion of the first two exon of col4a6 together with deletion of the 5' end of col4a5. conclusion: identification of an as patient during infancy is extremely rare. clinical manifestations, including macrohematuria, cataracts and leiomyomatosis caused by the large deletion involved col4a5 to col4a6, led to early presentation with as. functional voiding disorders of the bladder occur in the absence of any anatomic/neurological abnormality and present with wetting. invasive urodynamic studies are discomforting, not easily available in emerging countries and costly. this study aims to validate non-invasive urodynamics. children below 12 years, with possible voiding disorders evaluated prospectively. non-invasive evaluation included history, examination, frequency volume charting, ultrasonography, urinalysis and renal functions. micturating cystourethrogram was carried out for children with urinary tract infection. all children underwent invasive urodynamic studies (uds) and the significance of association of the parameters of non-invasive assessment with invasive urodynamics was determined. the chi square test was used for statistical analysis using the epi 6 software. 34 children underwent invasive uds. the commonest abnormality was detrusor instability (di) in 21 (61.7%). dysynergic voiding (dv) noted in 6 (17.6%), lazy bladder in 2 and an occult neurogenic bladder in 2. the study was normal in 3. repressing the disease progression may be 1.5 mg/kg/day or more background: henoch-schoenlein purpura is classified into the small vessel vasculitis. there may be no reliable indicator of the disease activity. steroid treatment (1 mg/kg/day of prednisolone) has been thought of as a means with which alleviate abdominal pain. however, this dose seemed to be not effective to intervene the disease progression to nephritis. objectives: forty-three japanese children with henoch-schoenlein purpura were enrolled in this study. fibrinogen degradation product e-fraction (fdp-e) value was measured once or twice a week in the patients. coagulation factor xiii was simultaneously measured in 36 of the patients in the early phase of illness. with an aim to alleviate abdominal pain, 0.7-2.0 mg/kg/day of prednisolone had been administered to 15 of the patients. results: at presentation, only 16 of the 36 patients had low factor xiii activity. on the contrary, 33 of them had elevated serum fdp-e value. longitudinal fdp-e measurement revealed that patients whose fdp-e value normalized within the second weeks of illness had minimal risk of nephritis. in this group, 2 of 28 had microhematuria. in the other patients group with prolonged (after fourteen days of illness) elevation of fdp-e values, 8 of 15 had nephritis. furthermore, 7 of 8 had proteinuria after three months of illness. these 8 patients who had received prednisolone therapy with less than 1.5 mg/kg/day in the early phase of illness. the other 7 patients with 1.5 mg/kg/day or more prednisolone therapy had no nephritis. summary: the disease activity of hsp and hspn might reflect the duration of elevated serum fdp-e. more than 1.5mg/kg/day of prednisolone may repress the disease progression to nephrits. background: all major organs are involved more or less in thalassemia and most of them have been studied thoroughly in previous literature. renal system involvement has not been scrupulously scrutinized yet. method: in a randomized prospective study, renal findings of 58 children and young adults, aged 3-24 years, with thalassemia major (group 1) were compared to other 50 cases of thalassemia intermedia (group 2). blood urea nitrogen, serum creatinine, uric acid, calcium, phosphate, urinalysis, and sonographic findings were evaluated. results: mean age was 17±3.5 years in group 1 and 18±3.0 in group 2. mean serum ferritin level was 3503±201 ng/ml in group 1 vs. 871±81.8 ng/ml in thalassemia intermedia group (p<0.05). 92% of subjects of group 2 had received or was on hydroxyurea at the time of evaluation. serum uric acid was significantly higher in patients with thalassemia intermedia ( conclusion: significant renal involvement is not a frequent complication in children and young adults suffering from thalassemia. hyperuricemia and microscopic hematuria is more common in thalassemia intermedia than thalassemia major. case: a 13-year-old female patient was born at term (weight 3750 g, lenght 51 cm) and for aspiration of amniotic fluid required resuscitation and mechanical ventilation for 17 days. perinatal hypoxia was a cause of her acute renal failure but dialysis was not needed. patient's follow-up during next 12 yrs showed mild form of chronic renal failure (crf) without hypertension: serum creatinine (scr; range during follow-up [rdf] 95-112 μmol/l), glomerular filtration rate (gfr; rdf 66-73.8 ml/min/1,73 m 2 ) and p (rdf 30-180 mg/24 h). at the age 13yrs we performed renal biopsy (rb) because girl's p and scr increased (358 mg/24 h, 139 μmol/l, respectively) and gfr decreased (51 ml/min/1.73m 2 ). rb showed c1q-nephropathy (c1qn) with focal glomerular sclerosis and hyalinosis. c1qn is a rare disease and as a first diagnostic step is differentiation against lupus nephritis. progression of c1qn to crf is infrequent. probably two renal diseases were a cause of crf in our patient -hypoxic renal damage during neonatal period and c1qn. objective: renal dysfunction has been reported in survivors of neoplastic disease. early diagnosis of renal damage may decrease the morbidity in those with partial or complete remission. we studied the frequency of nephrotoxicity in pediatric patients whose therapy were completed. method: 108 pediatric cancer patients (44 f, 64 m) who were at least one year off therapy, enrolled in a prospective cross sectional study from 2003 to 2005 in oncology department of ali asgar children hospital. demographic data, cumulative dosages of anticancer drugs, history of other nephrotoxic agents, nephrectomy, radiotherapy were recorded. fasting blood and urine samples were collected to calculate fractional excretion of mg, ca, p, upr/ucr, clcr, urine osmolality and blood gas analysis. result: the mean of age was 12.9 years. 80 out of 108 patients had lymphoproliferative malignancies (group 1) and 28 had solid tumors (group 2). the mean of therapy was 35.44 month. treatment was discontinued for 52.48 month in average. the median of blood ca, p, mg, bicarbonate,and cr were 9.3 mg/dl, 3.6 mg/dl, 2.1 mg/dl, 22.65 meq/l and 0.6 mg/dl, respectively. the median of fractional excretion of ca was 0.81% this rate was 7.47% for p excretion and 2.4% for mg excretion. clcr was 135.3 ml/min/m 2 in median. the medians of urine osmolality was 850 mosmol/kg/h 2 o. the median of urine protein to urine creatinine ratio was 0.07 mg/mg. these values were not different between two groups (p>0.05) but urine concentration was defective in solid tumors group (p=0.001). mild to moderate nephrotoxicity was seen in 52.8% of cases. using binary logistic regression we found no correlated factor (p>0.05). conclusion: mild to moderate tubular dysfunction has been observed in survivors of chemotherapy. routine follow-up of renal functions are recommended. the study is to discuss the treatment of hemolytic uremic syndrome (hus) after acute stage. methods: there were 13 children who lived through acute stage of hus then continued treating. besides angiotensin converting enzyme inhibitors (acei) and early restriction of protein intake, the study was to use therapeutic schedule according to clinical classification, response to prednisone and pathological manifestation, which referred to clinical classification diagnosis and treatment of child with glomerulus disease (the program) established by nephrology group in pediatric branch of chinese medical association (cma). results: after 5 months to 12 years follow-up 4 mild type children maintained the normal blood pressure and renal function and urine examination, except for 1 recurrence. in 9 gravis type children 6 maintained the normal blood pressure and renal function and urine examination, another 3 children who manifested as durative abnormality of urine examination developed into end stage renal failure (esrd) and died in the 5th, 8th and 13th month at last. another 4 gravis type children untreated after stage of hus died in the 27th day to 48th day of the course. conclusion: it could improve the prognosis of children after acute stage of hus evidently to use therapeutic schedule according to clinical classification, pathological manifestation. objective: to find the prevalence of hematuria in patients with thalassemia major. methods: of total 1000 patients with thalassemia major under regular blood transfusion, 500 cases were randomly selected. history was reviewed and physical examination was done. urinalysis was performed in all the patients. in those with hematuria (5 or more rbc/hpf) or suspicious to hematuria (3-4 rbc/hpf) second urinalysis was done at the next transfusion time. more investigations were done in those with persistent hematuria. results: the patients had age range of 6 months to 32 years and male to female ratio was 1.05. hematuria was detected in 20 (4%) and suspicious in 33 patients (6.6%). sixty four percent of the patients with hematuria were female and it was persistent in urinalysis in 90% of cases. in 81% of the patients with hematuria, blood transfusion was started before the end of first year. in those with hematuria or suspicious to hematuria, 4% had sterile pyuria and 16% had proteinuria (these figures were 2.1% and 1.4% respectively, in those without hematuria). hypertension was not detected, but 2 patients had secondary diabetes mellitus. conclusion: hematuria is not uncommon in patients with thalassemia major and is more prevalent in girls and in those with early transfusion. background: it has been widely recognized that cyclosporine a (cya) is a useful immunosuppressive drug in renal transplantation. although it has been also accepted that cya is an effective drug for pediatric nephrotic syndrome in the past two decades, the effective serum concentrations are not revealed. the functional roles of cya has been reported that cya inhibits the production of interleukin 2 (il-2) in vivo and in vitro. aim: in this study, we investigated the correlation of serum concentrations of cya levels with il-2 levels in pediatric nephrotic syndrome cases. methods: seven children (6 boys and 1 girl, mean age 9.5±4.24 years) with minimal change nephritic syndrome were enrolled in this investigation. cya (mean dose, 3.49±0.88 mg/kg/day) was administrated in two divided doses before meal with or without administration of predonisolone. blood samples were collected just before, 1, 2, 3 and 4 hours after administration of cya. the serum concentrations of cya and il-2 were measured immediately. results: the peak blood concentrations of serum cya were observed at 1 hour after administration. the concentrations of serum il-2 levels reduced at 1 hour after administration of cya, and kept the same levels during 4 hours afterwards. the serum concentrations of cya which inhibited more than 80% of the serum concentrations of il-2 required 500 ng hr/ml. conclusions: we confirmed that cya inhibited the production of il-2 in children with nephrotic syndrome. these findings suggest that the necessary serum concentrations of cya to maintain the sufficient suppressive rate were more than 500 ng hr/ml in pediatric nephrotic syndrome. this study aimed to evaluate the circulating angiotensins in female adolescents with type 1 diabetes (dm1) and to compare with the results obtained in healthy age-matched adolescents to disclose possible changes in plasma peptide concentrations that could be related to microalbuminuria and metabolic control. patients were divided as female adolescents with dm1 (n=25) and adolescent age-matched controls (n=18). diabetic patients were evaluated at our endocrinology center and healthy adolescents were selected from our primary care unit. plasma levels of angiotensin (ang) i, ang ii and ang-(1-7) were determined by radioimmunoassay. glycohemoglobin and microalbuminuria were also measured. results were expressed as medians or means and standard deviation. kruskal wallis was used for median comparisons and t-test for means. the level of significance was p<0.05. adolescent dm1 patients exhibited high levels of glycohemoglobin (9.5±0.4%). microalbuminuria was detected in 5 (20%) patients with a disease duration of 8.4±0.7 years. angiotensin concentrations were significantly increased in dm1 patients (p<0.05 compared to controls) and ang-(1-7) levels were 4-fold higher than control values. on the other hand, the levels of ang-(1-7) in microalbuminuric patients were significantly lower than in non-microalbuminuric diabetic adolescents (p<0.05). the comparisons between ratios of ang-(1-7)/ang i and of ang ii/ang i suggested a predominance of ang ii formation rather than ang-(1-7) in microalbuminuric diabetics when compared to normoalbuminuric patients. our results showed an overall increase of angiotensins in a young female diabetic population, and further suggested a pathophysiological role for angiotensin-(1-7) in dm1. the pediatric nephrologist is often faced with the difficulty of determining adequate iron supplementation in children with chronic kidney disease (ckd). soluble transferrin receptor (stfr) and the stfr to log(ferritin) ratio (stfr-f index) have been proposed as markers of iron deficiency (id) independent of inflammation; however, their relationship with c-reactive protein (crp) and their age dependency have not been established. we therefore embarked on a prospective study of 436 healthy children undergoing minor surgery to determine reference ranges for stfr (dade behring n-latex stfr analyser, dade behring bn prospec) and stfr-f index. we studied the relationship between crp and ferritin, transferrin, stfr and stfr-f index. we also compared the relationship between mean corpuscular volume (mcv) and ferritin, stfr and stfr-f index in 205 children. results: for ages 0. .0001, 0.0050) with mcv, which we used as a marker of id in the absence of a non-invasive gold-standard; however, only stfr-f index, but not ferritin, transferrin and stfr, was independent of crp. this study shows that ferritin, transferrin, and stfr, but not stfr-index, are dependent on acute phase reactions. it is therefore hypothesized that stfr-f index provides a more useful marker for monitoring the iron status in ckd patients. conclusions: low osmolality is a crucial factor to facilitate water absorption at least in the rat small intestine, while the absorption of sodium may be influenced by the concentration of sodium and glucose. (definition iccs). standard treatment consisted of general advice on voiding and drinking habits, alarm treatment and occasionally vasopressin. constipation was diagnosed on clinical considerations (history, stool charts, physical examination, occasionally x-ray). all patients received general instructions according to bowel habits. laxatives were prescribed when the patient was diagnosed as constipated. treatment goal was daily bowel movements. treatment results were evaluated 3 months and 2 years after discharge. results: 151 patients were included. mean follow-up was 2.2 yrs. overall success rate (full response) was 80.8% (3 months) and 72.6% (2 years). laxative use: 11.2% (n=17) of the patients received laxatives, 88% (n=133) did not. in 1 patient information was lost. there was no significant difference in success rate between the laxative group compared with the non-laxative group (p=0.14, chi-square). treatment modality: 6.0% (n=9) received general advice only without laxatives, all but one had a full response. 90.1% (n=136) were treated with advice and alarm, 16 (10.6%) of them received laxatives. response to alarm treatment was 82.5%. no significant difference in success rate between the laxative and non-laxative group (p=0.13, chi-square). 1 patient was dry after vasopressin and laxatives. conclusion: the majority of patients with mse can achieve nocturnal continence without laxatives. constipation treatment with laxatives may be supportive, but is not essential in the treatment of mse. aim: hypocalcemic tetany is a known complication of plasmapheresis. we studied the changes in ionised and total calcium, and magnesium concentrations during plasmapheresis, with and without supplementing the replacement fluid with calcium and magnesium. methods: plasmapheresis was carried out by using 4.5% human albumin solution (has) with or without supplements for the first 85% of the exchange, and fresh frozen plasma (ffp) for the last 15%. we measured ionised and total calcium and total magnesium at the beginning and end of the has, and after 15 minutes of ffp infusion. results: we undertook 31 pairs of plasmapheresis runs with and without supplements in 11 children who had a variety of renal conditions. during the exchange with unsupplemented has, the total calcium fell from 2.02 to 1.70 mmol/l (ci 9.9-20.3, p<0.000), the ionised calcium fell from 1. have raised significant problems in minor surgeries. but the developmental mechanism of ponv is not clear until now. previously, we have experienced a case with ihn and ponv who showed extremely high plasma antidiuretic hormone (adh) level at the onset of ihn and that elevated adh level induced by minor surgery was supposed a trigger of ihn and ponv. in this study we investigated various values including plasma adh in cases taking kidney biopsy in order to clarify the mechanism of ihn and ponv. methods: fifteen patients taking percutaneous kidney biopsy were study subjects (mean age 10.5 years). plasma adh, serum electrolytes and osmolality were measured before and 4-5 hours after kidney biopsy. urine samples were collected to measure electrolytes and osmolality. results: high plasma adh level (19.9±8.33 pg/ml) was observed in 9 out of 15 subjects (60%). serum sodium level dropped significantly in these cases. six of 9 cases showed ponv, we divided all 15 cases into 2 groups: ponv group and non-ponv group. the result was that plasma adh level was significantly high in ponv group. conclusion: our study make it clear that elevated plasma adh level is frequently seen in children taking kidney biopsy and suggest that hydration with hypotonic saline solution after surgery is inappropriate because of the risk of developing ihn. it also become clear that high plasma adh level might lead to ponv as the same mechanism seen in motion sickness. it is suggested that adh secretion by stress after minor surgery is associated with not only ihn but also the onset mechanism of ponv. polyarteritis nodosa (pan) occurs more commonly in patients with familial mediterranean fever (fmf) and visceral hematomas are seen in almost half of the patients. we report here a 14 year-old girl with pan and fmf presented with multiple visceral hematomas. the patient was on colchicine therapy for four years because of fmf but uncompliant to therapy. she addmitted with the complaints of fever, malaise, abdominal pain and artralgia lasting for two months. she was pale and extremely cachectic with atrophic muscles of extremities. she had fever, hypertension, hepatosplenomegaly and arthritis. she had anemia with normal renal and hepatic function tests, albumin levels, and electrolytes. multiple hypoechogenic mass lesions were detected on liver and bilateral kidneys on ultrasonography and computerised tomography and diagnosed to be hematomas by laparascopic examination. urinalysis, hematological tests for bleeding and blood marrow examinations were normal. bacterial cultures and serological tests did not reveal any infectious agent. serum complement levels were normal with negative antinuclear antibody, anti-dna, p and c antineutrophil cytoplasmic antibodies. renal angiography showed multiple aneurysms in bilateral renal arteries leading to the diagnosis of pan. she was successfully treated with intravenous pulse methylprednisolone followed by oral perdnisolone and oral cyclophosphamide together with colchicine and antihypertensive agents. she has been followed up for four years without any complaints and normal laboratory and radiological findings except multiple scar formations on kidneys on dmsa-renal scanning. results: the stone-free rate was 86% after one eswl session. the above rate increased to 92% and 96% after the second and the third session respectively. regarding surgical treatment with pcnl, the overall stone-free rate was 93%. in 3 children initially treated with pcnl, an eswl session was performed later successfully, for residual calculi. open surgical removal was required in 9 children with structural anomalies. the patients with staghorn calculi underwent nephrolithotomy combined with eswl in cases of residual fragments. 12 patients underwent ureterscopic procedures to address ureteral stones and complete fragment removal was obtained. no major sideeffect were observed, during the above procedures. conclusions: it seems that the advances in instrument technology provide a variety of safe and effective methods in the management of paediatric urolithiasis. the incidence of open surgery has thus fallen. minimally invasive methods must form the first choice of treatment, while open surgery should be undertaken mainly in cases of coexisting congenital abnormalities. in all children the following parameters were estimated: a) timetable of ne, b) feeling/volume chart (frequency and biggest quantity of urination=functional capacity of bladder), c) ultrasound of urinary tract (size of kidneys, bladder capacity, bladder wall thickness, postvoiding residual urine) and d) urodynamic parameters (uroflowmetry and water cytometry). the ud bladder parameters were then correlated with the us and voiding diary findings. results: all children had sufficient data registered to allow reliable analysis. ud studies showed that children with mild pne had normal urodynamics findings, us parameters and voiding diary findings as well. ud studies reveal a relatively high incidence of instability in children with moderate and mainly in those with serious ne. conclusions: in children with pne, urodynamics did not have a significant additional value compared to baseline diagnostics and it should be avoided. on the contrary, findings from urodynamic studies in children with serious ne show that it has a useful role in this type of enuresis evaluation and management. objectives of study: hyperlipidemia, especially when started during early childhood will increase the risk of atherosclerosis. it is also a major risk factor for allograft nephropathy and post-transplant hyperlipidemia, so its diagnosis and treatment is highly suggested. in this study we have evaluated the effect of hemodialysis on the lipid profile of children with end stage renal disease (esrd). methods: twenty-two children with esrd who were on maintenance hemodialysis in shiraz pediatric hemodialysis unit were studied. they were asked not to take greater than 30% of their total daily calories as fat at least 1 month before sampling. after a 12-hour overnight fasting and before starting dialysis, blood samples were taken for lipid profiles. for each patient with total cholesterol, tg or ldl-c levels more than 95th percentile for age and gender or hdl-c level less than 35 mg/dl, was defined as dyslipidemia. results: nineteen out of 22 children (86.4%) had abnormal lipid profiles. atherogenic factor of tg/hdl-c ratio more than 5 as a major risk factor for cardiovascular disease was in 86%. conclusion: dyslipidemia is common in hemodialysed children. so, hemodialysis set-up change and antilipimic medication, and replacement of l-carnitinine is recommended for correction of dyslipidemia in this group of patients. objectives of study: bipolar renal length measurement is an integral part of the assessment of urinary tract in childhood, and is routinely performed on ultrasonography and renal scintigraphy investigation. correlation between kidney size measurement on ultrasonography and dimercaptosuccinic acid (dmsa) scintigraphy is not well recognized. the purpose of this study was to comparison renal size measured by dmsa scintigraphy and ultrasonography to find if there is acceptable agreement between renal lengths by these two methods. methods: as cross sectional retrospective study, 90 patients enrol in this study and their dmsa scan results and kidney ultrasonography reports compared. the agreement between renal size measured by two methods for left and right kidneys were evaluated separately using bland-altman plot. pearson's correlation coefficient was used to examine their correlation. statistical significance was calculated by paired-student t-test. the same tests were used to for kidneys with normal dmsa scans. results: correlation coefficient showed close correlation between kidney length measured by ultrasound and dmsa scan, but there were significant differences between two methods (paired t-test, p<0.0001). comparison between renal size measured by ultrasonography and dmsa scan using the methods of bland & altman plot in all patients and the group with normal kidneys showed a systematic bias of about +6.5 mm for left and +6.3 mm for right kidneys. conclusion: despite of close correlation between dmsa scan and ultrasonography for kidney length measurement; kidney size is overestimated by about 10 percent in dmsa scan study and this matter must be considered in practice. medical treatment of cystinuria is often considered disappointing. patients undergo frequent surgery which is often followed by early relapse. the aim of our study was to prospectively evaluate, in a paediatric population, the efficacy of a conservative medical approach for long-term treatment of cystinuria, to prevent the formation of new renal stones and reduce the number and dimension of pre-existing stones. twenty-one stone former cystinuric patients were treated with a combined approach which included cystine-binding drugs. free and bound urine cystine levels were routinely measured every four months. drug dosage was adjusted in order to maintain a steady free urine cystine level below 100 mmol/mmol creatinine (a three fold increase of normal level). in the 19 patients who completed the study, renal stone episodes were reduced from 0.28 to 0.03 episodes/year, and in several patients the number and dimension of pre-existing renal stones were reduced. during the entire follow-up, percutaneous lithotripsy to remove an obstructive stone was required in only one subject. no relapse was observed 12 months after treatment. the dosage required to achieve target levels was very closely correlated to patient body weight: older children required a lower dose to achieve target levels. in conclusion: medical management of cystinuria is feasible. the treatment must be personalised, at least in pediatric age. the amount of required drug is strictly depending from body size. it is mandatory to obtain a low free urine cystine level before any invasive procedure to reduce the risk of early relapse. objective: to study the pathophysiology of nutcracker syndrome (ns) and to assess the role of the upright position imaging and superior mesenteric artery (sma) angle measurement in the diagnosis. methods: doppler us findings in 23 children with ns and in 26 healthy control subjects were compared. the mesenteric angle, peak velocity (pv) and anteroposterior (ap) diameter of the left renal vein (lrv) at the hilar and aortomesenteric portions were measured in both the supine and upright positions. the means ±sd of the sma angle, ap diameter and pv ratio between the two portions were calculated and cut-off levels for the diagnosis of ns were established. results: the diameter and pv ratios were significantly different between the patient and control groups both in the supine and upright positions (p<0.001). differences (d) between the supine and upright positions were also significant for the diameter of the lrv at the aortomesenteric portion, diameter ratio and sma angle in both groups. upright position imaging revealed comparatively narrower sma angles and more pronounced entrapment findings in patients with ns. the sma angle measurement had a sensitivity of 69.6% and a specificity of 61.5% in the supine position and 87.0% and 76.9% in the upright position when the cut-off values were set to less than 41° and 21°, respectively. the upright position has significant effects on the lrv hemodynamics and angle of sma both in patients and healthy subjects. sma angle measurement may be a useful adjunct parameter in the diagnosis of ns. ). in addition, a statistically higher rate of pathological abnormalities on renal biopsy was noted in the group with microscopic hematuria combined with proteinuria and also in cases with more severe hematuria. conclusions: school urinary mass screening has greatly contributed to diagnosing chronic renal diseases. continuous medical observation is required when abnormal urinalysis is observed, and a more aggressive medical approach such as renal biopsy should also be performed if necessary. this study compared the outcome of children with proliferative ln (who class iii/iv) using a new protocol comprising pulse intravenous methylprednisolone, mmf +/-cyclosporine, with standard prednisolone and cyclophosphamide/azathioprine. method: twenty-three children with proliferative ln (age range at diagnosis 3.7-18.6 years) who were followed up for 6.9-4.5 (range 1.3-21.0) years, were included in this retrospective study. group i (n=11) received prednisolone with cyclophosphamide and/or azathioprine. group ii (n=12) received the combined mmf protocol with mmf dose of 1200 mg/m 2 /day. poor outcome was defined as death or chronic renal failure. survival analysis was performed using the log-rank test. effect of treatment on growth at last follow-up was assessed using height standard deviation score (htsds). differences between the groups were analyzed using the mann-whitney and fisher's exact test. results: at last follow-up, significantly more group i compared to group ii patients had higher serological activity as defined by low serum complement c3 (50% vs 0% respectively, p=0.014). in addition, 8-year actuarial survival was higher in group ii (100%) compared to group i (61%). all the group ii patients achieved complete remission of proteinuria compared to group i (0.01±0.06 vs 0.79±1.0 g/d/1.73 m 2 respectively, p=0.002). group ii patients also had lower htsds on long-term follow-up compared to group i (-0.26±1.05 vs -1.96±1.73 respectively, p=0.025). conclusion: combination immunosuppressive protocol involving mmf +/-cyclosporine resulted in better renal outcome in children with proliferative ln without compromising on growth. this regimen allowed steroid tapering to alternate day dosing without increasing lupus activity. background: a full dose of corticosteroid is required to induce complete remission (cr) in steroidsensitive nephrotic syndrome (ssns), unless it is possible to taper and discontinue along with the course after cr. however, the mechanism of this change in steroid sensitivity remains unknown. p-glycoprotein (pgp) has a function to eliminate given corticosteroids from cytoplasm, which results in inducing corticosteroid resistance. therefore, we analyzed a drug delivery perspective using the real-time polymerase chain reaction (pcr) of multiple drug-resistant gene 1 (mdr1; encoding pgp) messenger rna (mrna) expression. patients and methods: fourteen patients with steroid-sensitive nephrotic syndrome (ssns; male/ female: 14/0, age: 1-23 years old; mean 10.4) were enrolled in this study. mdr1 mrna gene expression of peripheral blood nucleated cells (pbnc), before and after cr (a total of nineteen sets of blood samples), were quantified using real-time pcr and then carried for analysis. results: the mdr1 mrna levels before cr were variable in each patient. however, there was an apparent decrease in the mdr1 gene expression of pbnc after cr (p<0.003). the results suggest that pgp may play a role in the ability to taper corticosteroids after cr in ssns. : 6 week prednisone 60 mg/m 2 /day + 6 weeks 40 mg/m 2 every other day). all other patients (b) received daily prednisone 1.5-2 mg/kg/day for 8 weeks and 30-50% of initial dose for 1 week, followed by alternate day steroids (41 week) with tapering by 2.5 mg every 6-8 weeks down to 2.5-5 mg. "frequent relapses" (less than 2 months after discontinuation of initial steroid therapy or of first relapse therapy) were treated with chlorambucil 0.15-0.2 mg/kg/day for 8 weeks and half of this dose for 6-10 months. results: seven patients (with long treatment) were lost to follow-up and 48 were studied. six of 11 (55%) of a had a relapse 4.6±1.7 months after the end of initial therapy; 3 became infrequent and 3 frequent relapsers. 19 of 37 (51%) of b relapsed 7.8±1.3 months after the end of initial therapy; 14 became infrequent and 5 frequent relapsers. frequent relapsers (27% in a and 14% in b) were treated with chlorambucil and all but one achieved long remission (>1 year). conclusions: first relapse occurred later after onset of ssns in patients with long (50 weeks) as compared to short (12 weeks) initial steroid therapy but the time interval between the end of initial therapy and the first relapse and the proportion of relapsers were similar. longer initial therapy may result in a lower number of frequent relapsers. nearly all patients had long remissions periods that were, however, achieved at the expense of early administration of chlorambucil. conclusions: the medium age of pts with metabolic stones was found to be higher than the medium age of pts with infectious stones. the familial occurrence of kidney stones was found to be important 46%. the ultrasonographic examination is the most important one. the stones composed by calcium oxalate and calcium phosphate were found to have the highest percentage. metabolic abnormalities were found in 75% of patients and hypercalciuria was the most common disorder. hypocitraturia is considered to be a risk factor the calcium stones. in an attempt to explore the new treatment for the childhood-onset intractable steroid-dependent nephrotic syndrome (sdns), we have recently performed the treatment with high-dose mizoribine (mzr), the inhibitor of inosine monophosphate dehydrogenase, and suplatast tosilate dimethylsulfonium std), a selective th2 cytokine inhibitor, which were both made in japan. mzr has been commonly used for the treatment of frequently relapsing sdns in japan at a dose of 3-4 mg/kg/day (maximally 150 mg/day) divided into two doses (kidney int 58: 317,2000). we used high-dose mzr (mean: 10.1 mg/kg/day) once before morning meal for 9 adolescent patients with frequently relapsing sdns who had been treated with long-term cyclosporine (csa) resulted in moderate to severe csa nephropathy. with this treatment for 2 years, seven out of 9 patients weaned off csa and experienced less relapses without apparent adverse effects by high-dose mzr. std is a both il-4 and il-5 inhibitor and commonly used for childhood asthma. we used std at a dose which is for the treatment of asthma for 13 children with sdns (mean 9.2 years) without previous csa treatment. after one year follow-up with std treatment, the relapse rate of nephrosis was decreased from 1.54±1.20 to 0.15±0.56 per year (p=0.0008 by wilcoxon signed-ranks test), where as the dosage of orally given predonisolone was also decreased from 0.19±0.21 to 0.04±0.05 mg/kg/day (p=0.0187 by wilcoxon signed-ranks test). objectives of study: to evaluate the efficacy and safety of long-term cya treatment for pediatric sle patients. pediatric sle patients in their teens suffer from many relapses and severe side effects caused by steroid and cyclophosphamide. there have hardly been any reports on the long-term cya treatment in children. methods: we retrospectively compiled 6 cases of childhood-onset sle female patients (mean: 12.1 years) admitted to our department from 1995 to 2004. the initial treatment was methylprednisolone pulse therapy followed by prednisolone (psl). at the onset, 4 patients had class 2 lupus nephritis and 2 showed class 3. after several relapses, cya was added and used for 20 to 68 months. the dose of cya ranged from 2.8 to 4.9 mg/kg/day, and the target trough level from 50 to 80 ng/ml. results: under this low-dose cya treatment, 5 patients had no relapse while 1 had a relapse after 49 months. in all patients, psl was reduced to alternate day treatment (mean: 10.8 mg/2 days), and 3 patients under 14 years gained the target height. of all patients, 5 developed hypertrichosis, 1 gingival hyperplasia, 1 transient elevation of s-cr with acute gastro-enteritis. although 1 case had elevated s-cr after 20 months of cya treatment, it returned to normal level within 3 months after the cessation of cya. five patients had second biopsy after 2 years, and 2 showed mild tubulointerstitial (t-i) changes. two had third biopsy after 5 years and both showed mild t-i changes only. the presence of t-i changes had no relation to s-cr, u-beta2 mg and u-nag. conclusion: low-dose cya treatment might be an effective and safe second line treatment for sle patients with many relapses in teens. it is also important to perform renal biopsy periodically to detect cya-induced renal damage which hardly shows any abnormalities in blood or urinary tests. 3 hypersensitivity to inulin is rare; two cases of food allergy and some cases of allergy after inulin infusion have been reported. an 11-year-old boy suffering from severe iga nephropathy (igan) is reported with both anaphylactic reaction and concomitant relapse of his nephropathy due to inulin infusion, used for measuring gfr 2 years after first symptoms. pruritus, sibilants and cough were observed during a first renal function test. prick and intradermal tests were negative for inulin. the patient presented with pallor and asthenia during a second inulin infusion performed under dexchlorpheniramin, leading to immediate infusion stop. he was read mitted because of fatigue and nausea; acute renal failure was diagnosed 4 days after inulin infusion. a drug-induced acute interstitial nephritis was first suspected. however, due to the presence of macroscopic hematuria and proteinuria, a renal biopsy was performed and showed acute proliferative relapse of igan. few data are available about inulin-induced hypersensitivity. chandra described anaphylaxis and cardiorespiratory arrest immediately after administration of sinistrin. a retrospective study of all recorded cases of hypersensitivity associated with renal function tests was performed by our pharmacovigilance unit. 6,328 tests using inulin clearance were realized both in adults and children; 31 patients experienced side effects which were divided into 3 groups: respiratory symptoms, rash and general signs. most side effects were minor and no life threatening complication occurred. the underlying mechanism of inulin hypersensitivity is not well known. although 55% of patients with inulin-associated hypersensitivity underwent a first renal function test, we can speculate that presensitization with food inulin may occur, sometimes leading to severe problems such as in our patient with iga-mediated immunological dysregulation. . we have previously demonstrated a composite heterozygous nphs2 mutation of both v165x and r168h in a chinese patient with srns. however, it is not clear the molecular mechanisms of mutant podocinlead to proteinuria. some evidences proved the possible interaction between podocin and trpc6. this study explored the effects of mutant podocin on the free cytosolic ca2+ and apoptosis of podocyte in order to clarify the possible causative mechanism of mutant podocin. methods: 1. the pdsred2 n1-wild/mutant podocin was constructed by using site-directed mutagenesis. 2. mouse podocyte clone was cultured and transfected with pdsred2 n1-wild/mutant podocin. 3. free cytosolic ca2+ was measured using the fluorescent indicator, fluo 3-am. results: the low level of free cytosolic ca2+ was detected in normal podocyte and the transfected podocytes with r168h mutant podocin. the v165x mutant podocin increased the free cytosolic ca2+more evidently than the over-express podocin in transfected podocytes. podocyte apoptosis were not detected in the blank-vector (just pcdna 3.0) transfected podocytes and normal podocyte. the v165x and r168h mutant podocin increased the podocyte apoptosis more evidently than the over-express podocin in transfected podocytes. conclusions: the v165x mutant podocin might induce podocyte apoptosis via the increment of free cytosolic ca2+. however, whether the increment of free cytosolic ca2+ is induced by trpc6 and the involved signal pathway should be further investigated. y. xing, q. fan, j. ding objectives of study: podocytes slit diaphragm (sd) associated molecules (nephrin, podocin and cd2ap) play a critical role on maintaining the integrity of glomerular filter. vegf is produced by podocyte, and acts on endothelium and podocyte itself. but, it is not clear whether there are some relationships between vegf and sd associated molecules. our study detected the expression of sd associated molecules and vegf in adriamycin (adr) nephrotic rats. methods: the adr rat was established by adr injection. distributions of sd molecules and vegf were detected by immunochemistry. the mrna and protein of sd molecules and vegf was examined by real-time pcr and western, respectively. nephrin phosphorylation were detected by immunoprecipitation. results: distribution of nephrin, podocin and cd2ap changed evidently, and the staining intensity of vegf decreased evidently. nephrin mrna increased at day 7, and returned to the normal at day 14 and 28; podocin and cd2ap mrna constantly increased from day 3 until day 28. the protein of nephrin increased at day 7 until day 28; podocin was dramatically upregulated at day 7, and thereafter recovered again, but was downregulated at day 28; cd2ap prominently increased at day 14 and day 28. tyrosine phosphorylation of nephrin was decreased evidently at day 28, and vegf mrna did not show significantly changes at any time points observed. however, vegf protein reduced significantly from the 7 th day, and also reduced evidently at days 14 and days 28. conclusion: the abnormality of nephrin, podocin and cd2ap may be one of the mechanisms that lead to proteinuria in adr-induced nephrotic rats. the occurrence of proteinuria in adr rats may be also associated with the reduced vegf protein, which may be related with the reduction of nephrin phosphorylation. these results suggested there may be some relationship between vegf and sd molecules. the objectives of study: mutations in genes encoding structural proteins of slit diaphragm can lead to nephritic syndrome. just recently, another gene trpc6 mutation was identified in autosomal dominant fsgs. trpc6 encodes ion channel protein trpc6, whose expression has not been clarified completely in kidney. this study aims to explore the expression and distribution of trpc6 in normal human, mouse and rat renal tissue and the mouse podocyte clone (mpc5). methods: distributions of trpc6 in normal human, mouse and rat renal tissue and cultured podocyte was observed with immunochemistry staining. the mrna expression of trpc1, 2, 3, 4, 5 and 6 was detected by using rt-pcr. the protein expression of trpc6 in human, mouse and rat renal cortex and differentiated mpc5 was detected with western. results: trpc6 showed weak staining in glomeruli and strong in renal tubules and vessels in human kidney, however, strong in glomeruli and was mainly distributed along the capillary loops and mesangium in mouse and rat kidney. the staining of trpc6 was observed in differentiated mpc5, which is distributed evenly on the cell membrane. the specific pcr band of trpc1, 2, 3, 4, 5 and 6 was detected in mouse kidney and differentiated mpc5. the sequence of the amplified pcr products is same as that published in genebank. the specific 106kda protein band of trpc6 was detected in normal human, mouse renal cortex and differentiated mpc5. conclusion: the expression of trpc6 was verified in normal human, mouse and rat kidneys and in differentiated mpc5. these results will benefit for further screening the possible mutation of trpc6 in acquired nephrotic syndrome, and investigating the relationship between trpc6 and the proteinuria-related podocyte molecules. methods: twenty children with kd (13 boys and 7 girls, aged from 3 to 78 months) were enrolled in our study. kidney sizes (including kidney length and kidney volume) were measured during acute stage in these patients. twenty age-and sex-matched healthy children and 15 febrile children served as healthy controls and fever controls. left kidney length and age were used for correlation analysis and analysis of covariance. results: kidney lengths in patients with kd were significantly larger than those of healthy children (p<0.001). the mean sd score of kidney length was 2.54±0.97 for these patients (p<0.001, vs -0.23±0.82 in normal control). kidney volume analysis yields the similar result (51.83±17.93cm 3 vs 35.62±11.24cm 3 , p=0.001). there was no kidney enlargement in the fever controls. up to 70% of the children with kd have absolute nephromegaly (>mean+2sd). this incidence is as frequent as that of lymphadenopathy and extremities change, the 2 diagnostic criteria of kd. conclusion: these results confirm the presence of large kidneys in the children with kd and also provide another useful indicator for kd diagnosis if the diagnostic criteria is not yet well established. during renal inflammation macrophages infiltrate the renal parenchyma, and their number correlates with the intensity of inflammation. macrophage migration inhibitory factor (mif) was described originally to be a product of t-cells and macrophages. mif plays an important role in renal tissue injury. to our knowledge, the studies that assessed the role of macrophages in acute renal infection were few and the role of mif was not evaluated. the aim of this study was to assess mif in uti and compare the urinary excretion of mif in pyelonephritis, cyctitis and also control group to find a non-invasive and sensitive method to differentiate them. in this prospective case-control study 31 pediatric patients with uti (25 patients with acute pyelonephritis, 8 patients with acute cystitis) and 40 healthy children were recruited. urine mif concentration was quantitated by elisa and corrected for urine creatinine. the mean ratios of urine mif/cr were calculated as 66.14 (sem=23.78) pg/μmol creatinine in acute pyelonephritis, 1.58 (sem=0.59) in acute cystitis and 1.85 (sem=0.35) in healthy individuals. urine mif/cr ratio was significantly higher in pyelonephritis than the ones in acute cystitis (p=0.0001) and control (p=0.0001). roc analysis was demonstrated that urine mif/cr ratio could considered potentially useful index to detect acute pyelonephritis [p=0.0001, area under curve (auc)=0.959]. the optimal cut-point of 5.39 pg/μmol creatinine for urine mif/cr ratio could potentially separates acute pyelonephritis patients from healthy individuals (sensitivity and specificity of 92% and 92.5%, respectively). the underlying histopathological characteristics in biopsied renal diseases are of great importance in determining the long-term prognosis and provides useful information in clinical practice. ethnicity seems to play a critical role in the epidemiology of biopsied renal diseases the aim of this study is to provide data of clinical manifestations of biopsy-proven native renal diseases in iranian children. in this retrospective study, 476 iranian children who were diagnosed as renal disease between 1980 and january 2003, were evaluated. diffuse and focal mesangial proliferative glomerulonephritis was present in 31.5% of all biopsies performed. mpgn, fsgs and mcd were observed in 9.2%, 10.2% and 13.4% the most common clinical syndrome at any age is nephrotic syndrome (50.4%), followed by nephritic syndrome (13.8%), nephrotic-nephritic syndrome (13.4%), recurrent macroscopic hematuria (7.3%), asymptomatic urine abnormalities (aua) (7.7%) and azotemia was seen in 6.7% of patients. mesangial proliferatiove gn (18/70=25.7%), poststreptococal gn (15/70=21.4%) are the most frequent pathologies with acute nephritic syndrome presentation. the most frequent causes of aua were mesangial proliferative gn and hsp. thrombotic microangiopathy (hus) was the most prevalent cause of arf. inthis study, chronic tubulo interstitial nephritis (33.3%) and alport (13.3%) were the most common causes of crf presentation in our patients. in conclusion, mpgn remains the most common histopathological subtype in children with renal biopsied disease. the incidence of fsgs continues to be high in iranian children. the aim of this study is to assess postnatal kidney volume development and to compare the intrauterine and extrauterine kidney growth curves in premature infants. one hundred neonates were enrolled in this study. all infants had their kidney volumes measured by renal ultrasound examination. group ga consisted of 44 neonates whom were evaluated within 48 hours after birth, and their gestational ages were used in the analysis. group ca included 56 premature infants born before 34 weeks of gestation and was evaluated 14-96 days after birth, and their conceptional ages were used in the analysis. left kidney volume, body weight, body height and age were used in the correlation analysis. kidney volumes in group ca infants were significantly larger before 31 weeks of age, but smaller after 31 weeks of age than those of group ga infants (p=0.001). there was a significantly better growth in body weight (p=0.001) and body height (p<0.001) in group ga infants. however, a larger kidney volume was noted in group ca infants with the same body weight (p<0.001). conclusion: chart of postnatal growth of normal kidney volume before 40 weeks of conceptional age in premature infants is presented. our data suggests that intrauterine growth may have a regulatory influence on kidney growth, and the reduced kidney volume in the premature infants may start from the early extrauterine period. objectives of study: to illuminate the role of prohibitin (phb), a tumor suppressor which inhibit cell proliferation by repressing e2f-mediated transcription, in tubulointerstitial fibrosis (tif). methods: renal biopsy specimens were obtained from 48 children with primary glomerulonephritis. phb and α-sma proteins expression were detected by immunohistochemistry. subcellular location of phb in nrk-49f was detected by confocal microscope. changes of phb protein and mrna expression in cells upon tgf-β1 stimulation were detected. after transfected with phb plasmid, cell cycle and α-sma protein and mrna expression in cells treated with or without tgf-β1 were detected. results: phb protein was found at normal renal tissues, with a positive distribution in interstitial cells and tubular epithelial cells. phb was down-regulated in tissues with tif and negatively correlated with tif degrees (p<0.05). phb is majority located at cytoplasm as well as at nucleus in nrk-49f. phb protein and mrna expression in cells were decreased when treated with tgf-β1, and the effects were both time-dependent and dose-dependent. extraneous phb inhibited cells proliferation induced by tgf-β1, and phb over-expressing cells failed to enter the cell cycle compared with non-transfected cells (p<0.01). α-sma was not expressed in control cells while de novo expression of α-sma in cells upon tgf-β1 stimulation was increased. overexpression of phb did not affect basic α-sma expression but dramatically repressed tgf-β1-initiated α-sma expression (p<0.01). conclusions: extraneous phb suppresses renal interstitial fibroblasts proliferation and cell phenotypic change induced by tgf-β1, which indicates phb as a potential target to halt tif progression. results: the prevalence of urine abnormalities of first screening was over 5.00%, and that of the second screening was about 1.00%. the prevalence was different with various methods. the specificity of method b was higher than method a. testing two urine samples for each child had higher specificity. the direct cost of method a and b was -1.70 and -2.90 rmb, respectively. for screening twice, the corresponding cost was no more than -1.90 and -3.00 rmb, respectively. using method a to screen twice for each child was convenient and economical, which also reduced the false positive rate effectively. the prevalence of urine abnormalities of junior highschool children was significantly higher than that of elementary school-children in xh and the peak point was seen at the point of 12 years old. however, there was no significant difference between children in ja and yp. more than 10 months of follow-up diagnosed 2 cases of iga nephropathy. conclusions: urine abnormalities of school-children could be detected through urine screening at school. for shanghai, method a with screening twice was convenient, economical, and could reduce the false positive rate effectively. objectives: angiopoietin-like3 protein (angptl3) is involved in lipid metabolism and angiogenesis. the present study was to examine angptl3 expression in human kidneys with proteiuria, in adramycin rats (adr), and in puromycin induced podocyte damage. methods: immunohistochemistry was performed on kidney biopsies from children with mcd, mn, fsgs, tbmn. in adr, angptl3 expression was determined by quantitative real-time rt-pcr in glomeruli and tubuli dissected from frozen section of kidneys with laser microdissection system. in mpc5, a conditionally immortalized mouse podocyte cell line in vitro, angptl3, perlecan and agrin were detected through real-time pcr with the induction of puromycin. detachment assay was performed in podocytes tranfected by angptl3-pcdna3.1. results: in human kidneys, co-labeling showed angptl3 expressed in the cytosol of wt1 positive cells. quantitative computerized analysis showed that angptl3 in glomeruli in mcd and mn were significantly higher than that of tbmn, fsgs respectively (p<0.05). in adr, angptl3 in glomeruli increased significantly at 21 st or 28 th day (p<0.05) after adriamycin injection compared with control. and the expression of angptl3 in glomeruli was correlated with 24 h urinary protein (r=0.81, p<0.05). in mpc5 both protein and mrna expression of angptl3 on podocytes were up-regulated with the induction of puromycin. in podocytes transfected by angptl3-pcdna3.1 the expression of perlecan or agrin increased significantly compared with control (p<0.05). the attachment ratio was shown 95.7%±3.3% 24 hs after puromycin treatment on podocytes transfected by angptl3 compared with 38.6%±4.7% on normal podocytes, and 27.4%±3.5% on untransfected podocytes. conclusions: angptl3 is predominantly expressed in podocytes which could be involved in podocyte damage and the development of proteiuria. (1), iv (1) and iii (1), respectively. only one patient had microhematuria. we found that 5 of them had a very low c3 serum levels. clq and c4 deposits were all strong positive in 6 renal tissues. our findings suggest that biopsy should be strongly considered in this patient population. the significant renal involvement (class iii, iv, or v ln) could be found in sle patients with very lower proteinuria with or without hematuria. patients in bfb group received computer-assisted biofeedback program while those in ddavp group took minin. both therapies were carried out for 1 month and then 3 months follow-up was taken. parameters of follow-up included enuresis diary-urine flow rate and aqp2 in urine. results: 50 pne patients were recruited (26 boys, and 24 girls), whose mean age was (8.4±0.9) years. at the end of treatment and three months later, total effective rates in bfb group were significantly higher than those in ddavp group. uroflowmetry findings showed that in bfb group maximum flow rate, voided volume and ratio of coordinative detrusor-sphincter contraction increased after treatment. ratio of normal flow curve increased at second follow-up (p<0.05). in ddavp group voided volume and voiding time decreased after treatment. ratio of normal flow curve and coordinative detrusor-sphincter contraction had no change after treatment. two bands of aqp2 (29 000 and 43 000) were detected in the morning urine. density of patients bands was significantly lower than that of the controls. density of 29 000 bands in ddavp group after treatment were significantly higher than that before, but there was no difference between datas before and after treatment in bfb group. conclusions: bfb and ddavp are both effective therapies for pne in children. bfb is helpful in correcting voiding dysfunction and ddavp can increase aqp2 protein in the urine. with higher effective rate within four month, bfb is strongly recommended. objective: to describe the clinical course of non-parasitic chyluria in a thai pediatric case. this is the first report in children. results: the 7-year-old boy presented milky urine lasting for one year. urine tests showed heavy proteinuria (protein to creatinine ratio 9.1 mg/mg), lipiduria (triglyceride 29 mg/dl). the proof of a pronounced hypertriglyceriduria led to the diagnosis of chyluria. his renal function was normal. numerous red cells and lymphocytes were observed in the urine, and postprandial cystoscopy revealed milky cloudy urine emanating from right ureteral orifices. retrograde pyelography demonstrated pyelolymphatic backflow. serum immunoglobulin g4 for wuchereria bancrofti and circulating filarial antigen in the peripheral blood were negative. chest x-ray, abdomen computed tomography and intravenous urography did not demonstrate abnormal mass or malformation. proteinuria and lipiduria ceased before sixth week of a medium-chain triglyceride-rich diet. there was no recurrent chyluria after 12 weeks of mct-rich diet were completed. conclusion: in non-parasitic chyluria with unknown etiology, the low-fat diet with mct supplementation alone is effective. the prognosis is excellent. there was a significant improvement of waz comparing data at admission and at the end of follow-up (p<0.001). there was also a significant improvement of whz comparing data at admission and at the end of follow-up (p=0.02). only 9 (2.3%) patients presented with a whz less than -2.00 at the end of the follow-up. conclusion: children with primary vur presented an improvement in somatic growth with medical management. objective: the aim of this study is to investigate the clinical practical value of using doppler ultrasound to detect renal blood flow in renal parenchymatous diseases of children. methods: the renal arteries, segmental arteries and interlobar arteries were detected by doppler ultrasound. the parameters were peak systolic velocity (vmax), minimum velocity in diastole period (vmin), vmax/vmin (s/d), resistive index (ri) and pulsatility index (pi). there were 30 cases of healthy children, 20 cases of acute poststreptococcal glomerulonephrits, 20 cases of primary nephrotic syndrome and 15 cases of chronic renal failure. results: the doppler renal blood flow in normal school children was high velocity and low resistant type. 15 typical cases of acute nephritis with edema and oliguria appeared low velocity and high resistant type, ri, pi and s/d of all renal arteries were significantly increased, vmin are significantly decreased (p<0.05). after 2 to 3weeks all parameters returned to normal. during edema period and convalescence, the renal blood flow of primary nephrotic syndrome is low resistant type, ri, pi and s/d of segmental arteries and interlobararteries were significantly decreased (p<0.01). the feature of low circulating blood capacity was not alleviated even though edema was vanished and urine output was increased. the doppler in chronic renal failure was high resistant and low velocity type. ri, pi and s/d were all significantly increased, vmin were significantly decreased (p<0.01). when ri was great than 0.8, the extent of damage in kidney function was serious and the prognosis was bad. conclusion: renal blood flow provided a new non-invasive method for clinic diagnosis and evaluation of the prognosis in children renal parenchymatous diseases. we concluded that the dms is an important cause of congenital nephrotic syndrome. the outcome of our patients was poor and most of our patients died before 5 years old. objectives: the antiphospholipid syndrome is defined by the association of arterial/venous thromboses or obstetrical fetal loss with the presence of antiphospholipid antibody. this syndrome may be primary or secondary, particularly in association with systemic lupus erythematous. this study is to examine the frequency of anticardiolipin antibodies and the association between anticardiolipin antibodies with some symptoms in children with lupus nephritis. methodology: twenty-five children with lupus nephritis from 03/2006 to 11/2006 in department of nephrology, children's hospital o 1 were included in the study. we find the relationship between anticardiolipin antibodies with hematologic and renal involvement. results: anticardiolipin antibodies was positive in 11 patients (44%), 6 for anticardiolipin igm antibody (24%), 7 for anticardiolipin igg antibody (28%). there was a positive correlation between the presence of anticardiolipin antibodies and thrombocytopenia. in 11 patients with positive anticardiolipin antibodies, 3 patients had mta on renal biopsy. conclusion: anticardiolipin antibodies are associated with thrombocytopenia and mta. aim: the methodologies for quantitating urinary calcium excretion have not been standardized. the aim of this study was to compare urinary calcium/osmolality (uca/osm) ratio with calcium/creatinine (uca/cr) ratio and to assess the correlation of both ratios with daily urinary calcium excretion for the diagnosis of hypercalciuria in children. patients and methods: 364 children aged 6-14 years (mean 9.4±2.2 years) were included in the study. they were randomly selected from previous study's larger patient population. non-fasting, second morning urine samples were collected from all children. children were divided into two main groups: 1) 180 children with uca/cr <0.21(mg/mg) and 2) 184 children with uca/cr <0.21. 24-hour urine samples were collected from the second group, who were further divided into two subgroups: 2a) 113 children with daily calcium excretion <4 mg/kg/day and 2b) 46 hypercalciuric children (daily calcium excretion >4 mg/kg/day). results: mean uca/osm ratio was significantly lower in the first (1) group than the second (2) group (0.11±0.07 vs 0.31±0.14 mg/l/mosm/kg, p<0.05); but there was no difference between 2a and 2b subgroups. the correlations of both uca/osm and uca/cr ratios with 24-hour calcium excretion were poor (r=0.27 for both). conclusion: uca/osm ratio correlated with spot uca/cr ratio. but its superiority on uca/cr ratio in the diagnosis of hypercalciuria could not be shown. interestingly, values of 24-hour calcium excretion as a definite diagnosis test of hypercalciuria; did not correlate mathematically with those ratios of hypercalciuric or non-hypercalciuric children. using uca/osm ratio as a screening test would not separate hypercalciuric children. background: microalbuminuria is a biomarker of renal damage. the presence of microalbuminuria in patients with a solitary kidney has been described, but the pathophysiology leading to its occurrence is poorly understood. it is postulated that microalbuminuria is the early result of hyperfiltration. methods: we concomitantly measured inulin clearance, filtration fraction (ff) and microalbuminuria in children with a single kidney. correlation between the occurrence of microalbuminuria and a high filtration fraction was done. microalbuminuria was defined as an albumin/creatinine ratio (acr) >3 g/mol for boys and girls. normal filtration fraction was defined as <26%, and normal inulin clearance as >90 ml/min x 1.73 m 2 . during the same study, we also measured microalbuminuria in children with severe grade iii to v vesico-ureteral reflux (vur). results: 27 children with a single kidney were evaluated. 18 patients (67%) had a normal ff, and only one (6%) in that group had an abnormal acr. 9 patients (33%) had elevated ff, and 5(56%) had an abnormal acr. the presence of an abnormal acr was highly correlated with an abnormal ff (p=0.008). the mean gfr between the groups with normal or abnormal microalbuminuria did not differ significantly (94±31 ml/min x 1.73 m 2 vs. 83±16 ml/min x 1.73 m 2 , respectively). there was no significant association between microalbuminuria and a high ff in patients with severe reflux (p=0.3). discussion: we found the presence of microalbuminuria to be significantly associated with an elevated ff in children with a single kidney. this finding goes in line with the pathophysiology of a reduced nephron mass, leading to hyperfiltration, and ultimately to glomerular sclerosis. the benefit of renin-angiotensin-aldosterone blockade in these patients remains to be proven. chyluria is the excretion of chyle from the urinary tract and indicates the presence of an abnormal communication between intestinal lymphatics and the urinary tract. it can be of parasitic or nonparasitic etiology. southern brazil is not an endemic region for filariasis. aim: report a case of a 14-yrs caucasian adolescent girl referred to our out-patient clinic. history: 3 yrs before, she started to pass milky urine with white clots. no edema. normal blood pressure. she was investigated in another hospital and underwent a renal biopsy, that was normal. a diagnosis of nephrotic syndrome was made. she was treated initially with steroids and after changed to cyclosporin, lisinopril and simvastatin. conclusion: chyluria, although a rare conditione specially in children and adolescent in nonendemic areas, should beconsidered in the differential diagnosis of nephrotic syndrome. macroscopic examination of the urine, that is milky and cloudy, is simple and very helpful. also, triglycerides are found only in the urine of patients with chyluria. these simple tests will avoid unnecessary treatment, which is not without side effects. low-density lipoprotein apheresis (ldl-a) has been tried in the treatment of patients with steroidimmunosupression resistant nephrotic syndrome (ns) due to focal segmental glomerulosclerosis (fsgs). we would like to report a child case study of fsgs with ns and renal insufficiency due to mitochondrial abnormality treated by ldl-a and to clarify the therapeutic effects of this treatment. a 12-year-old boy was referred to our hospital with complaints of heavy proteinuria and edema. a routine examination revealed proteinuria of 7.0 g/day, serum albumin (alb) of 1.9 g/dl and creatinine clearance (ccr) of 58.5 ml/min. renal biopsy specimen showed fsgs and perceptive deaf nass was recognized, necessitating a hearing aid. the a3243g point mutation in mitochondrial gene was detected by using genomic dna isolated from peripheral blood leukocytes and by the molecular analysis using an allele-specific polymerase chain reaction (pcr). oral prednisone (2 mg/kg/day for eight weeks), intravenous methyl-prednisone pulse therapy (1.0 g/day,three times a week on the consecutive days for three weeks) and oral cyclophosphamide (75 mg/day for eight weeks) were not effective to reduce proteinuria. a protocol of ldl-a was designed for treatment twice-a-week for four weeks and then once-a-week for six weeks. following treatment by ldl-a, serum total cholesterol and ldl were markedly changed form 271 to 118 mg/dl and from 198 to 91 mg/dl, respectively. a small but significant increase in alb from 1.9 to 2.9 g/dl and a remarkable decrease in proteinuria from 7.0 to 1.4 g/day were also successfully obtained. conversely, no marked changes in ccr were detected. the results of the present study indicate that a rapid decrease in proteinuria and an excellent increase in alb by ldl-a provide a possible therapy for drug-resistant ns due to fsgs with mitochondrial abnormality. glomerular filtration rate (gfr) can be estimated in children by various formulas based on body height and serum creatinine (s cr ) measurements such as the schwartz formula (egfr sch =kxbh/s cr ). we evaluate the performance of egfr sch in estimating gfr in a pediatric cohort when compared to 125 i-iothalamate clearance (igfr), used as the reference standard for measuring gfr. between 1996 and 2006, we obtained 265 igfr and egfr sch on 171 subjects. for subjects who had more than one igfr, the first measurement was used for analysis. mean age was 13±6 (range 1-20, 56% age=13), 55% male. mean s cr was 1.2 mg/dl (median 0.8), mean igfr 76±38 ml/min/1.73 m 2 and mean egfr sch 116±59 ml/min/1.73 m 2 . figure 1 shows a scatter plot of the data with a line representing perfect agreement. figure 2 shows a residual plot comparing the difference between estimated and measured gfr to egfr sch . pearson r correlation between the two variables was 0.87 (ln scale). accuracy of egfr sch within 20% and 50% was 20% and 50%, respectively. the median difference between igfr and egfr sch was 40.1 ml/min/1.73 m 2 (median % difference 50%). for igfr >60, 30-59, 15-29 and <15 ml/min/1.73 m 2 , egfr sch overestimated gfr by 46%, 66%, 48% and 44%, respectively. however, the median difference between igfr and egfr sch for the same groups was 39, 18, 8 and 4 ml/min/1.73 m 2 , respectively. in conclusion, agreement between egfr sch and igfr is poor. egfr sch overestimates igfr at all levels of gfr, but bias of egfr sch vs. igfr increases progressively with higher gfr levels. in clinical instances when an accurate estimation of gfr is critical for patient management, the use of egfr sch should be reconsidered. until a more applicable estimation equation is developed, isotope measurement of gfr remains the ideal method to determine gfr in this population. background: immunosuppressive therapies other than corticosteroids, potentially associated with serious adverse effects, are urgently required for children with frequently relapsing nephrotic syndrome (frns). this study evaluated the efficacy and safety of long-term treatment with a moderate dose of cyclosporine (cya) in children with frns. methods: in this prospective, open-label multicenter trial, patients, from 2 to 16 years old, were randomly divided into two groups. for the first 6 months, both groups received cya (sandimmune) in a dose that maintained the whole-blood trough level between 80 to 100 ng/ml. during the next 18 months, the dose of cya was adjusted to maintain a trough level between 60 and 80 ng/ml in group a, while group b received a fixed dose of 2.5 mg/kg per day of cya. the primary end point was the rate of sustained remission. results: at 24 months, the rate of sustained remission was 52% in group a (n=24 patients), as compared with only 15% in group b (n=20) (p=0.006). the hazard ratio for relapse was 0.36 (95% ci, 0.17 to 0.77) in group a as compared with group b (hazard ratio=1.0). at 24 months, the rate of progression (to frns)-free survival was 78% in group a and 56% in group b (p=0.12). mild arteriolar hyalinosis of the kidney was found in 4 (19.0%) of 21 patients in group a and 1 (5.6%) of 18 in group b; no patient had striped interstitial fibrosis or tubular atrophy. conclusion: cya given for 2 years in a dose producing a trough level between 80 and 100 ng/ml for the first 6 months, followed by a trough level between 60 and 80 ng/ml for the next 18 months is an effective and relatively safe treatment for children with frns. with this regimen, about 50% of patients are expected to remain relapse-free during 2 years of treatment, without the most critical adverse effect of cya, i.e., interstitial changes of the kidney. renal stone disease has been regarded as an uncommon problem in children especially in the first year of life. we evaluated clinical findings and metabolic examination of 49 children with urinary tract stone presenting in the first year of life. there were 25 boys (51%) and 24 girls (49%), the mean age of admittance was 6,04±3,15 months. the average follow-up period was 30,91±24,65 months. urolithiasis was diagnosed during evaluation for uti and incidentally. positive family history for urolithiasis was reported in 22 (44,8%) patients. in 28/38 (73,7%) patients urinary metabolic examination was not normal (table 1 ). in 48 of 49 patients (98%), stones were located in kidneys which was bilateral in 26 (52%) patients and one patient had passing stone which had never seen in ultrasonographic examination. stones were examined in 3 subgroups. in 39 (80%) patients stones were measured 5 mm or smaller (group 1), in 9 patients (18%) they were between 5-9,9 mm (group 2) and in only 1 patient the stone (cystin) was larger than 10 mm (group 3). stones measuring 5 mm and larger were found highly associated (in 7 of 10 children, 70%) with abnormal ultrasonographic findings mainly hydronephrosis. in group 1, stones disappeared spontaneously in 12/23 (52%) patients. urinary tract infections (uti) were present in 28 (57%) patients. one fourth of cases had associated genitourinary tract abnormalities mainly vesicoureteral reflux in 9 (18%) patients. we conclude that the presenting symptoms of urolithiasis in the first year of life show a wide spectrum so that high index of suspicion is important for early detection. stones measuring 5 mm and smaller may have great chance to disappear. we also emphasize the importance of screening for uti in patients with urolithiasis under 1 year of age. background: long term complications of glycogen storage diseases (gsds) include delayed puberty, hepatic adenoma and renal disease. in the present study we aimed to detect renal involvement in children with glycogen storage disease and to determine the most accurate laboratory test to be the gold standard for early detection of this renal dysfunction. methods: twenty-seven children known to have gsd were included in this study. fifteen healthy age-and sex-matched children were also included as controls. routine urine analysis, urinary β2 microglobulin and microalbumin were done for all patients and controls. renal function tests, serum electrolytes, alkaline phosphatase, urinary calcium, blood and urine ph, urinary and plasma aminogram, in addition to calculation of glomerular filtration rate (gfr), bone x-ray to detect rachitic manifestations and abdominal ultrasound to measure renal size were done for all patients. results: twenty-one patients had one or more renal abnormality. the most common was increased urinary β2 microglobulin (15/21) followed by abnormal gfr whether low or high (8/21) and microalbuminuria (6/21). sonographically there was nephrocalcinosis in one case and renal stone in another one. the auroc curve for β2 microglobulin was 0.86, (p=0.01) and 0.7 for urinary microalbumin/creatinine ratio (p=0.15). the best cutoff level to predict renal abnormality for urinary β2 microglobulin was 0.22 mg/l with 70% sensitivity and 100% specificity and the best cutoff value for urinary microalbumin/creatinine ratio was 4.5 with 86% sensitivity and 50% specificity. in conclusion: renal abnormalities are common in patients with gsd. urinary β2 microglobulin can be considered the gold standard for early detection of renal dysfunction in these patients. the aim of this study was to investigate the role of neutrophil activation, protein oxidation and ceruloplasmin in the pathogenesis of hsp, which has been not investigated previously. serum activities of myeloperoxidase (mpo) and arylesterase (aryl) and levels of free thiol, ceruloplasmin (clp) and total oxidant status (tos) were measured in 29 children with hsp (16 boys, 13 girls; mean age 9.3±2.7 years) at the onset of the disease and during remission in comparison with 30 matched healthy subjects. patients at active stage had significantly higher mpo activity (391±277 vs. 155±154 u/l, p<0.001), higher clp (832±120 vs. 682±114 mg/dl, p<0.001) and tos values (20.7±11.8 vs. 7.5±2.8 μmol h 2 o 2 /l, p<0.001) than controls. patients had significantly lower aryl activity (158000±39000 vs. 187000±46000 u/l, p<0.001) and lower free thiol levels (234±48 vs. 279±26 μmol/l, p<0.001) than controls. there were 17 patients with gis involvement, 14 with joint and 13 with renal involvement. no significant differences were found in the oxidant stress markers between patients with or without organ involvement (p>0.05). significantly positive correlations were found between tos and mpo (r=0.437, p=0.018), and tos and clp (r=0.409, p=0.028) at the disease onset; while a negative correlation was found between mpo and thiol (r=-0.597, p=0.001) during remission. in conclusion, protein oxidation and neutrophil activation may play important roles in the pathogenesis of hsp. gastrointestinal system, joint and/or renal involvements were not together with different magnitude of oxidant stress. further studies are required to identify oxidizing substances and to develop therapeutic strategies to reduce oxidant stress in hsp. 1, 21) . if the first remission occurred after 8 days, the median time to relapse after discontinuation of steroid therapy was significantly lower than in children with shorter remission time (0.5 vs 3.0 months; p<0,0001). in conclusion children who fail to achieve a prompt remission after a first episode of ns are more likely to have frns or sdns. these retrospective data provide the rationale for individualizing the initial steroid treatment of mcns according to the time to obtain a remission. a prospective study is needed to validate this approach. the aim of this study was to determine the influence of osmolality of the first morning urine (ofmu) to efficacy of the desmopressin therapy in enuretic (pne) children and to compare the values of ofmu in enuretic and non-enuretic children. methods: we investigated ofmu in group of 50 children with pne and in group of 36 control non-enuretic children. pne group was divided into subgroup i (ofmu <600 mosm/kg h 2 o) and subgroup ii (ofmu >600 mosm/kg h 2 o). additionally, we measured ofmu 1 months after the initiation of desmopressin therapy and recorded the number of wet nights. regarding the number of wet nights we divided pne group to 3 subgroups: subgroup a (<5 wet nights/month), subgroup b (5-10 wet nights/month), and subgroup c (>10 wet nights/month). results: the statistically significant difference between control group and pne group regarding ofmu was not found (p=0.612). all 10 children from subgroup i had <5 wet nights/month during desmopressin therapy. 14 children from subgroup ii had <5 wet nights/month, and 26 had >5 wet nights/month during desmopressin therapy. the difference between those two groups was statistically significant (x 2 =13.3, p=0.0003). in children from subgroup a the difference between ofmu-s during and before treatment was 413 mosm/kg h 2 o, in children from subgroup b it was 261 mosm/kg h 2 o and in children from subgroup c it was 117,5 mosm/kg h 2 o. there was the statistically significant difference among those subgroups. conclusion: children with pne had usually similar ofmu like non-enuretic children. low ofmu is a good prognostic factor for desmopressin therapy of pne, especially in patients whose ofmu is <600 mosm/kg h 2 o. children with bigger difference of ofmu before and during therapy had better response to desmopressin therapy. we can conclude that ofmu can help in choosing the appropriate therapy for pne in children. yh. ng 1 , kl. chan 2 1 kk women's and children's hospital, pediatric nephrology, singapore, singapore 2 singapore general hospital, neonatology, singapore, singapore aim: to evaluate the clinical course and outcome of primary vesicoureteric reflux (vur) in patients with antenatal hydronephrosis in a neonatal unit. method: a prospective observational study of neonates with antenatal hydronephrosis born between january 1991 and december 2000 in the neonatal unit of singapore general hospital. neonates with significant hydronephrosis postnatally underwent micturating cystourethrography (mcu). records were reviewed with regards to the clinical course and outcome of primary vur. results: of 280 neonates with antenatal hydronephrosis, 139 (49%) had significant hydronephrosis postnatally and underwent mcu. 12.9% (18/139) were diagnosed with primary vur at median age of 7 weeks. there were more male (n=11) than female infants with primary vur. 10 (56%) infants had bilateral vur. 18% (n=5) of the renal refluxing units (rru) had low grade vur and 82% had high grade vur with the majority (57%) being grade iii vur. repeat mcu for 15 rru at 2 years showed that 60% (n=9) had spontaneous resolution of vur, 27% had improved vur grade and 13% had similar vur grades as before. 2 infants develop vur in the contralateral kidney which was previously normal. 14 infants (23 rru) underwent dmsa with renal scarring noted in 4 infants. all 4 infants were noted to have renal scarring without a history of urinary tract infection (uti). interestingly, 2 male siblings were found to have grade iii vur with renal scarring with subsequent spontaneous resolution. none of the study subjects underwent surgery. median age of follow-up was 3.8 years (range 0.3-12.5 years). conclusion: unlike neonates with vur detected after uti, infants with primary vur were predominantly male, had higher grade of vur with spontaneous resolution in the majority. early diagnosis of primary vur may provide the opportunity for reduced incidence of reflux nephropathy. t. neveus 1 , g. läckgren 1 , j. wahlberg 2 , n. wahlin 1 1 uppsala university children's hospital, uppsala, sweden 2 uppsala university hospital, department of transplantation surgery, uppsala, sweden objectives and methods: loin pain hematuria syndrome is a rare entity consisting of recurrent macroscopic hematuria with debilitating loin pain. it has only been described in adults, etiology is unclear and treatment is controversial but the therapy with best recorded success is to remove the kidney and reposition it in the pelvis. our objective was to show that the condition exists in childhood as well. results: aj, a previously healthy 9-year-old girl, was admitted because of recurring cystitis-like symptoms with microscopic hematuria but without bacteriuria. ultrasound and urography were normal. the episodes continued during the following years with increasing hematuria, now macroscopic, and increasing loin pain that was somewhat exercise-dependent. a renal ct scan was normal, as was cystoscopy, urography and ultrasound, but during cystoureteroscopy dilated vessels were noted in the mucosa of the right renal pelvis. antegrade pyelography, high resolution renal ct angiography, invasive renal angiography, mag3 renogram were all normal, gfr 111. nephrological evaluation, including kidney biopsy and coagulation tests were also normal and during cystoscopy blood could be seen emerging from the right ureteral orifice. by this time the patient was 15 years old and was dependent on opioids in order to be able to go to school. after long discussions with the nephrologist, urologists, pain specialist and transplantation surgeon, the family opted for autotransplantation as a last resort. this was performed january 2006 and the girl became almost momentarily pain-free. nowadays she does not need any analgesics, but after prolonged exercise (like several days of horseback riding) she may experience slight pain in the left loin and/or hematuria. conclusions: idiopathic loin pain hematuria syndrome exists in childhood and may possibly be treated with renal autotransplantation. j. van der deure, a. ockhuijsen, m. sondaar deventer ziekenhuis, 1st department of pediatrics, deventer, the netherlands objective: enuresis is a common pediatric problem. psychosocial factors (psf) influence the results of enuresis treatment in children. aim: to determine the short and long term effects of psf on enuresis treatment in a general pediatric population. methods: we reviewed the data of our enuresis patients treated from 2002-2004. relevant contributing psf were categorized. initial follow-up was at 3 months after training. a written questionnaire was sent 2 years after training. treatment success was defined as >90% improvement in dry nights. results: 211 pts were included. in 65 pts (30.8%) contributing psf were recognised. categorized problems: family related n=28 (43%), behavioural problems n=28 (43%), motivation/support n=22 (33.8%), learning disabilities n=21 (32.3%). overall success rate was 75.3% at 3 months and 65.3% at 2 yrs (overall resp quest 63.7%, psf 60.6%, 1 psf 51%, >1 psf 73%). success rate in the psf group was 49.2% (1 psf 61.5%, >1 psf 30.7%) at 3 months and 43.2% (1 psf 45%, >1 psf 42%) at 2 yrs. statistics: success rate in the group with psf is significantly lower as compared to no psf at 3 months (p<0.001 (chi-square), or 9.3, 95% ci 4.2-20.2) and at 2 yrs (p=0.0017 (chi-square), or 3.7, 95% ci 1.6-8.1) success rate at 3 months is significantly lower in pts with >1 psf, compared with 1 psf. (p=0.011 (chi square), or 4.6, 95% ci 1. 5-13.9) . no significance could be demonstrated at 2 yrs (p=0.85, chi-square) but this may be due to the variety in response rates. t. papalia, r. greco, r. bonofiglio hospital annunziata, nephrology, cosenza, italy actually a new litholitic therapy includes the phytotherapy agents as phyllantus niruri (pn), a plant used for years in brazil to treat urinary stones. in this work we estimate the effect of pn intake (uristone 2 gr/die) in 15 children (9 m/6 f, 9±5 years old) with urolithiasis. the pn has been administered for short term (from 1 to 3 months) in 13 children wih caox urinary stones and for 6 months in 2 with struvite stones. besides all children treated with dietary intervention: high fluid intake, sodium restriction, normal calcium intake and a diet low in animal protein. urinary and plasma analysis, body weight, map, ph, creatinine clearance, urinary excretion of mg and citrate were determined at baseline, 1 month and at the end of the study. the patients were studied by renal ultrasonography at baseline, 1, 3, 6 months. nobody of them had been undergone extracorporal shock wave lithotripsy. there were no differences in the mean values of urinary and plasma parameters before and after pn intake, except for a significant reduction in the mean urinary calcium in 8 hypercalciuric pts (6±1,3 vs 3,5±1 mg/kg/die). in this follow-up n°10 patients showed a faster stone clearance after a regular intake of pn and the others showed a smaller stone diameter. previous reports showed pn has a potent inhibitory effect on caox crystal adhesion and/or endocytosis by renal tubular cells and inhibitory effect on crystal growth, which might be related to the higher incorporation of gags into the calculi. our results suggest pn appears to represent a nontoxic and a low cost alternative for the prevention and treatment of stone disease, especially in the children. further studies are necessary to validate these preliminary findings. d. weitzel, c. schäfer, k. hohenfellner, u. pfeffer, m. neukirch german clinic for diagnostic, pediatrics, wiesbaden, germany objective: does sonographic quantification of the renal parenchyma allow estimation of isotopic renal function? method: sonographic kidney images of 88 patients (age 1 to 195 months; mean 47) were measured retrospectively. in all images of both kidneys taken from dorsal the volume on the base of length, width and depth was calculated. the parenchymal area (pa) in the longitudinal and cross section was calculated by planimetry. the distribution of renal function via mag3 was compared with sonographic values as volume and pa of each kidney in relation to whole kidney volume and pa respectively. patients with reduced global kidney function and time space of more than 3 months between isotopic study and sonography were excluded. results: interrater variability regarding planimetry of pa in longitudinal section (from dorsal taken images) was as good as measurement of kidney length (correlation coefficient (k)=0,968-0,977 and 0,97-0,99 respectively). all sonographic parameters correlated significantly with the isotopic parameters of renal function. the latter correlated best with the pa in longitudinal section (from dorsal taken images) k 0,939. the combination with planimetry in cross section did not improve correlation (k 0,94). difference of the proportional pa of the left kidneys (in correlation to whole kidney pa) in comparison to isotopic proportional renal function lead to mean difference of -0,3% with a standard deviation 6,7%. if only kidneys with split function of 45-55% the mean difference of proportional pa was -0,6% and the standard deviation 3,6%. conclusion: the distribution of total pa of both kidneys correlates significantly with the distribution of renal function (left and right) in isotopic studies. if sonographic planimetry might change the indication for isotopic studies in respect of renal function needs to be proofed in prospective studies. background: childhood incontinence is a common important urologic problem. especially daytime incontinence is often neglected by the parents until it turns out to be a significant clinical problem. the aim of this study was to evaluate the clinical characteristics of the patients with incontinence that were followed in our nephrology clinic. study design: patients were followed between the dates of 01.01. 2004-31.12.2006 and they were admitted solely due to incontinence or with concomitant urinary tract infections were enrolled. results: the study comprised 99 patients (38 m, 61 f; mean age 9.78±3.06 years). fourty-two patients had only nocturnal enuresis (ne) (29 primary, 13 secondary). twelve patients had daytime incontinence (di) (4 primary, 8 secondary) and 45 had both ne and di (25 primary, 13 secondary and 7 both primary ne and secondary di). all, except two (neurogenic bladder), had functional incontinence. twelve patients had additional fecal incontinence and 8 had constipation. sixty-two percent of the patients had one or recurrent urinary tract infections (uti) in their past history, 16% had accompanying vesicoureteral reflux and 4% had urinary stones. ultrasound revealed unilateral or bilateral dilatation in 20% and other anomalies in 5% of the patients. nineteen patients had abnormal dimercaptosuccinic acid scintigraphy findings. timed voiding schedule and double voiding were recommended to all patients with daytime incontinence, 43% of the patients received anticholinergic treatment and 50% received antimicrobial prophylaxis. discussion: overall, approximately 2/3 of our patients had associated uti and 1/5 had abnormal dmsa findings. therefore every patient with uti should be questioned about urinary incontinence and be treated carefully if present. the aim of the study was to determine early parameters of ultrasound and dmsa scanning diagnostics of reflux nephropathy (rn) in children. we examined 150 children with rn and vesicoureteric reflux (vur). all children were comparable on gender and age. all patients underwent color doppler ultrasound (cdus), x-ray and dmsa scan. they were divided into two groups: 1) children with unilateral rn a according to classification of smellie j. et all, 1975 (n=75) ; 2) children with vur without renal damage (n=75). we established that data of cdus (diastolic velocities (vd) 5,94±0,99 mm/sec, systolic velocities (vs) 22,3±5,74 mm/sec, resistive indices (ri) 0,63±0,06, pulsatility indices (pi) 0,76±0,19), dmsa scanning (time of the maximal accumulation 11,8±0,91 sec, maximal activity 189,3±20,4 sob/sec, mean velocities of accumulation 29,1±1,9 mm/sec, the contribution to the common accumulation 45,3±3,5%) are characteristic for patients with rn a. data of cdus (vd 10,7±1,68 mm/sec, vs 23,9±1,7 mm/sec, ri 0,63±0,02, pi 1,08±0,24), dmsa scanning (time of the maximal accumulation 7,5±0,86 sec, maximal activity 81,5±11,9 sob/sec, mean velocities of accumulation 7,72±1,07 mm/sec, the contribution to the common accumulation 34,27±3,03%) are characteristic for patients with vur without renal scars. the ranges of cdus and dmsa scanning were significantly different between children from comparing groups (p<0,05). our result suggest that data of cdus (vd, vs, ri, pi), dmsa scanning (time of the maximal accumulation, maximal activity, mean velocities of accumulation, the contribution to the common accumulation) can be used to early diagnostics of scarring in children with vur. the purpose of this study was to determine normal reference values for urinary uric acid/creatinine ratios in healthy turkish children. in this study, random urine specimens from 1306 children (662 boys, 644 girls) aged 1 month to 15 years were analyzed for uric acid and creatinine, and urinary uric acid/creatinine ratios were determined from each sample. uric acid/creatinine ratios were the highest in children aged 1-6 months and showed a significant decrease with age (p<0.05). uric acid/creatinine ratios were not significantly different between the sexes except 12-15 years. girls between 12-15 years had higher urinary uric acid/creatinine ratios when compared with boys (p<0.05). there was no correlation between urinary uric acid/creatinine ratios and protein intake. our results show that urinary uric acid/creatinine ratio changes with age. when assessing the urinary uric acid/creatinine ratio, a child's age should be considered. we provided normal reference values of urinary uric acid/creatinine ratio for using in our region. the aim of the study was to investigate microbiological marker of activity of uti. e. coli and s. aureus p 209 were isolated from urine of 180 children with uti. the children were divided into 4 groups: 1. with pyelonephritis in the acute period (n=45); 2. with pyelonephritis in the period of remission (n=45); 3. with cystitis in the acute period (n=45); 4. with cystitis in the period of remission (n=45) 20 healthy children consists the group of control. definition of bactericidal activity of urine (bau) was carried out by our original method. the essence of the method consisted in measuring of the optical density (od) of the bacteria after their contact with urine (experience) and isotonic solution of nacl (control) after 30 minutes of endurance in meat peptone mediums with 37 c during 3-5 hours. bau was calculated under the formula: bau (%)=(odc-ode)/odc*100%, (odc -control group, ode -experience group). we established that the level of bau did not correlate with urine ph (r=0,2), osmolality (r=0,1), lysozymuria (r=0,2), lysinuria (r=-0,2). we established that the low level of bau was marked in children of control group (10-40%). the patients in active period of uti had high level of bau (>70%). the parameters of bau didn't depend from the level of uti (pyelonephritis or cystitis). the level of bau reduced in the period of remission of uti. we established that the level of bau correlated with bacteriuria (r=0,8), leucocyturia (r=0,5). the level of bau didn't depend from the degree of urine dissemination (r=-0,3). so, the level of bau is correlated with laboratory parameters of uti and can be used as new additional microbiological marker of diagnostics of activity of uti. the evolution of the alport syndrome in brazilian children vesicoureteric reflux (vur) is common in children with urinary tract infections (uti). if vur coexisting with uti there is a high risk of progression to end-stage renal disease (esrd). the correct diagnosis is important. we observed 136 children (104 girls and 32 boys) aged 1 mo to 12 yrs at the time they have been diagnosed as having vur. the follow-up period was 1 mo up to 11 yrs after the diagnosis. all 22 children with vur grade 5 have been operated. after antireflux operation incidence of uti dramatically decreased even this cannot prevent progressive kidney damage in some patients. children with less severe vur have been put on prophylaxis. controlled mcu was performed usually after 1 year later. if vur disappeared medication have been stopped. vur grade 2-4 had a tendency of resolution under conservative treatment in 25.5% of the patients. in 16 children associated urinary tract malformations were found: duplicated system, dysplastic kidney, kidney agenesia, dystopic kidney, urethral stenosis and bladder outlet obstruction. in 2 patients nonfunctioning kidney have been found. dysfunctional voiding was common finding. blood pressure and physical development have been controlled. kidney size, function and scar formation have been followed by dmsa scan. we observed kidney growth at 6 mos intervals. during the follow-up period 3 infants have had reversible renal insufficiency. one patient with bilateral vur grade 5 went into renal failure at the age of 9 yrs. conclusions: vur is still one of the most common leading causes to esrd in childhood. even the existing controversy concerning treatment modalities it is obvious that low grade vur does not need operative treatment. it is indicated in high grade vur to prevent repeated and severe uti but it cannot preserve progression of the disease because of high incidence of coexisting kidney dysplasia. results: from forty children with nephrolitiasis, 28 (70%) were boys and 12 (30%) were girls. the mean age was 80.5±57.5 months. the youngest age was 4 months old. the most common clinical presentation was abdominal discomfort 22 (55%), followed by uti 21 (52%), microscopic hematuria 13 (32.5%), macroscopic hematuria 12 (30%), spontaneous urinary calculi 9 (22%), flank pain 8 (20%). nine of 40 children were presenting with chronic renal failure (crf). statistical analysis showed that age had correlation with the present of crf in children with nephrolitiasis (p=0.007). the clinical presentations of nephrolitiasis were varied. abdominal discomfort and uti were the major signs and symptoms. there was correlation that age may influence the present of crf in children with nephrolitiasis. objective: renal involvement is one of the most frequent and serious manifestations of sle. we analyzed the treatment and renal outcome of patients with lupus nephritis. methods: seventy-seven identified patients were retrospectively analyzed from jan. 1996 to dec. 2005 . the outcome was divided as complete remission (24-hour proteinuria <0.5g, plasma creatinine level normal and sledai <5), partial remission (abnormal renal damage index improved >50%, 24-hour proteinuria >0.5g, sledai <10) and no response, respectively. results: fifty-four patients were biopsy proven ln (70%). fifty-seven patients followed up more than 6 month. all the eleven patients with class i or ii achieved remission, using prednisolone together with either hcq, or tripterygium or mmf or cyclophophamide (ctx). in forty-three patients with class iii or iv or v, they were given prednisolone together with mmf or ctx. we found that remission was in 18 cases, part remission 17 cases and no response in 8 cases. associations between methylenetetrahydrafolate reductase (mthfr) c677t polymorphisms and several vascular diseases have been reported. this is a clinical study designed to investigate the possible effects of (mthfr) c677t polymorphisms on the developement of henoch-schönlein purpura (hsp), renal involvement, and clinical course. fourty-one patients with hsp (25 m/16 f) mean age (7,8±2,9 years) were included in the study. eighteen of the patients had renal involvement. the control group consisted of 50 healthy children. blood samples were obtained for mthfr c677t transition, homocysteine, folic acid and vitamine b12 in the patients and controls. the genotype frequencies (cc/ct/tt) of mthfr in the hsp group were 0,56/0,39/0,12 and 0,58/0,38/0,04 in the control group, respectively (ns). the genotype frequencies (cc/ct/tt) were 0,39/0,39/0,22 in the patients without renal involvement and 0,78/0,22/0,00 in those with renal involvement, respectively (ns). homocysteine levels were 10,6±6,8 in the hsp patients and 12,9±4,5 μmol/l in controls (ns). vitamine b12 levels were 320,6±157,5 pg/ml in the hsp patients and 302,6±209,6 pg/ml in the control group (ns). folic acid levels were 5,7±3,0 in the hsp and 4,0±1,6 ng/ml in the control group (p<0,002). no significant relationship was present with the mthfr genotype and plasma homocysteine, folic acid and vitamine b12 levels. no association with mthfr gene polymorphism and homocysteine plasma levels could be detected in patients with hsp and hsp nephritis. although mthfr gene polymorphisms have been found to be associated with several vascular diseases, the results of this study indicate that other mechanisms should be operative in the developement of hsp and hsp nephritis. we report the symptoms, signs and laboratory values at onset and during 6 month-follow-up of hsp in a prospective study of 220 children (99 girls, 121 boys) with mean age of 7.1 years (1.6-16.7 y). the first sign of hsp was purpura in 152 (70%), oedema or other joint symptoms in 121 (55%), abdominal pain in 49 (22%) and melena in 3 (1%) patients. petechiae appeared on the average 5 days after the first symptoms (1-22 d) if purpura was not the first sign (n=67). 78% of the cases were diagnosed between september and march. the mean delay from the first symptoms to the diagnosis was 7 days (0-65d our results based on an unselected and prospective patient material demonstrate that renal symptoms in hsp children develop early, are common and should be followed up at least 6 months. the kidney is a metabolically active organ, so any alteration in kidney function might affect nutrient utilization. objective: analyze the nitrogen balance (nb) as a marker for adequate food consumption in children with chronic renal insufficiency (cri). material and methods: 60 patients (12 boys, 48 girls) diagnosed and managed in the nephrology and nutrition departments. they were placed in two groups depending on age: group a: 20 patients aged 5±2.5 years, follow-up 4±1.1 years, glomerular filtration rate (gfr) estimated by cr-edta: 38±20 ml/min/1.73 m 2 ; group b: 40 patients aged 12.5±3 years, follow-up 10.9±3.5 years, gfr estimated by cr-edta: 37±17 ml/min/1.73 m 2 results: group a had a worse weight and height evolution: weight: -0.95±0.15 sd in group a vs 0.15±2 sd in group b; height -1.29±0.29 sd vs -0.34±0.23 sd (respectively). group a showed a significant increase in tnf blood levels (p<0.03) that was inversely related with weight and height. bn was significantly greater in group a (1.59±1.93 gr/day) than in group b (-2.6±4gr/day) (p<0.01) and this was related with higher calorie (p<0.003) and protein (p<0.004) intakes. there was no difference in alimentary nutritional breakdown. nb improved significantly over the follow-up (p<0.01). there was no relation between nb and gfr. there was a significant increase in triglyceride levels and significantly lower blood urea levels in group a (p<0.04). we conclude that nitrogen balance depends on protein and calorie consumption and is independent of the severity of renal affectation. we present a case of a 6 months old boy, who was delivered to our intensive care unit because of high fever, acute renal failure and dilatation of the urinary tract. his hemoculture and urinary culture were positive for e. coli and a severe urosepsis was diagnosed. his urethral catheterization was unsuccessful, so a suprapubic puncture was performed to relieve the urinary system and provide sufficient urinary flow. after the urinary sepsis was cured with adequate board spectrum antibiotics, a voiding cystourethrography (vcug) was performed to reveal the suspected anatomical abnormality, vesicoureteral reflux (vur) respectively. meanwhile a continuously growing nodular tumor of the penis was observed. the vcug showed the signs of urethral stenosis, but posterior urethral valve and vur was excluded. the doppler ultrasound found a solid vascularised tumor, 2x2.5 cm in its diameter. during the surgical operation the tumor couldn't been totally removed, as it was infiltrating the surrounding tissues and the urethra. the histopathological examination of the biopsy specimen confirmed a juvenile xanthogranuloma (jxg). this is an uncommon, benign, non-langerhans cell histiocytosis, primarily seen in infancy as a solitary cutaneous lesion, predominantly in males. systemic form of the disease is rarely seen. usually it resolves spontaneously without any further treatment, but the differentiation from a malignant neoplasm is essential. according to the authors' search, this is the 2nd reported case in the literature and the first pediatric report of the jxg of the penis. urine examination using x-ray diffractometry background and goal: x-ray powder diffraction analysis is widely used in chemistry and pharmacology and for other industrial purposes. in medical science it is used for analyzing kidney stones and investigating retained crystals in tissue sections. in the department of mineralogy and petrology we investigated urine samples, at first diagnostically to detect urinary amino acids, glucose and compounds and, secondly, to detect calcium oxalate hydrate, which can be employed for early detection of renal tubular injury when no significant differences in renal function values exist. materials and methods: after sedimentation and dehydration, authors investigated more than 100 urine samples of children using x-ray diffractometry. results: 12 of them were glucosuric due to diabetes mellitus; in these cases glucose could be detected in each of their urine samples. in 5 cases different amino acids due to aminoacidopathy were detected. the urine samples of 8 children -kidney stone problems in history -were examined, in one case struvit, in the other cases ca oxalate crystals were identified. also, 30 samples of 10 children were examined, 24 hours after at least 2 hours long anesthesia, ca oxalate hydrate appeared in their urine referring to renal tubular injury due to inhalational anesthetic agents. in 10 cases urine samples of children treated in the intensive care unit were analyzed, in 50% ca oxalate crystals could be detected. in 40 cases healthy children's urine were investigated, as control ones. conclusion: x-ray diffractometry, as a highly sensitive method can be used efficiently in clinical measurements. further investigations are needed in order to determine its place in clinical trials. authors emphasize the importance of collaboration of different sciences, as well. drug intake in the background of sudden death? microalbuminuria was significantly more in patients more than 10 years of age as compared to younger patients on bivariate analysis (p=0.001). on logistic regression analysis, though microalbuminuria was more in patients more than 10 years of age, it was not statistically significant. the association between microalbuminuria and urinary specific gravity levels of <1.010 was statistically significant (p=0.001), similar results were seen on logistic regression analysis. there was no correlation between microalbuminuria and hospitalizations, crises, previous blood transfusions, hemoglobin electrophoresis and serum creatinine levels. conclusion: identification of risk factors for microalbuminuria may allow earlier intervention to prevent renal complications in patients with ssd. in developing countries at primary health care level urinary specific gravity should be done routinely in patients with ssd to identify cases at risk of microalbuminuria. m. bak, e. serdaroglu, y. bicilioglu aim: the aim of the present randomized-controlled study was to compare desmopressin (dp), alarm and combined treatments in nocturnal enuresis. the study included 101 children (67 boys and 34 girls) with nocturnal enuresis. the mean age was 10.7±2.4 years (ranged 5-16 years) and the mean wet-nights was 14.9±6.1 day per month before treatment. the patients were followed for one month before treatment and randomized to dp (33 patients), alarm (34 patients) and combined (34 patients) treatment groups. the dp group was received 40 μg orally one-hour before sleep and alarm group was used wet-stop bedwetting alarm device after education of parents. the patients were followed 6 months in treatment period and 2 months after discontinuation of treatments. results: wet-nights per months was significantly reduced between before treatment and last month of treatment in dp (14.5±5.7 to 4.8±6.5, p<0.001), alarm (14.1±5.9 to 2.9±4.1, p<0.001) and combined treatment (16.2±6.9 to 1.9±2.5, p<0.001) groups. treatment success (>50% decreasing in wet-nights) and complete response (100% dry) rates was 79%, 91%, 97% and 30%, 27%, 35% in dp, alarm and combined treatment groups respectively. the more rapidly decreasing in wetnights was observed between dp used and only alarm treatment group but this effect disappeared after 3 months. relapse rates was 67%, 11% and 22% in dp, alarm and combined treatment groups respectively between successfully treated patients (p=0.002). conclusion: alarm treatment is the best intervention with low relapse rates and no potential adverse effect in nocturnal enuresis. dp group has higher relpse rate but adding to dp may achieve more rapid decreasing in wet nights especially in patients and parents expecting rapid result. aim: it is often difficult to collect urine from infants. use of specifically designed urine collection pads gives reliable results for routine biochemistry tests in adult urine. their use for routine and metabolic tests in paediatric urine has not been investigated. the aim of this study was to evaluate whether the pads give reliable results for routine and metabolic biochemistry tests in paediatric urine. methods: urine collected by bag or clean-catch from infants <2y without metabolic disorders was divided into two aliquots, one of which was added to a collection pad, incubated for 15min at 37°c, then recovered by aspiration. routine and metabolic analyses were performed on pad/non-pad aliquots. additionally, selected metabolic analyses were performed on pad/non-pad urine from patients with diagnosed inborn errors and urine spiked to simulate metabolic disorders. for quantitative analyses, pad/non-pad results were compared using bland-altman bias plots, passing and bablok regression and paired t-tests. results: routine tests (urea, electrolytes, creatinine, osmolality, calcium: creatinine, phosphate: creatinine, magnesium: creatinine, urate: creatinine, n=32) showed close concordance with no clinically significant pad/non-pad differences. in infants without metabolic disorders, aminoacids (n=10), organic acids (n=12) and mucopolysaccharides (n=8); and in patients with metabolic disorders -phenylketonuria (n=1), mucopolysaccharidoses ii (n=2) and iii (n=1), inborn errors of organic acid metabolism (n=6) and cystinuria (n=3), all showed excellent pad/non-pad concordance. sugar chromatography in urine spiked with glucose/galactose/fructose showed identical staining intensity in pad/non-pad samples. conclusion: urine collection pads give reliable results for a wide range of routine and metabolic tests in paediatric urine. a post-translational modification of arginine residues in proteins and subsequent proteolysis result in release of symmetric dimethylarginine (sdma). sdma is considered an end product of metabolism, excreted primarily by the kidney. several previous studies have reported a significant relationship between glomerular filtration rate (gfr) and plasma sdma. to determine the potential value of sdma in the assessment of gfr in children we have measured sdma in samples taken during routine measurement of glomerular filtration rate (gfr) using plasma clearance of inutest. 257 patients (144 male) requiring routine gfr measurement were studied. median (range) age 11.8y (1.6-20.2) , height (ht) 145cm (82-188), and surface area 1.3 m 2 (0.5-2.6). gfr was measured using the plasma clearance of inutest, and plasma sdma and creatinine (pcr) by liquid chromatography stable isotope dilution electrospray mass spectrometry-mass spectrometry method. estimated gfr (egfr) was calculated from the formula 31 x ht (cm)/pcr (μmol/l). median gfr was 80 ml/min/1.73 m 2 (6-217), plasma sdma 0.530 μmol/l (0.266-4.460 ), pcr 54.5 μmol/l (12.7-777), and egfr 82 ml/min/1.73 m 2 (7-212). as expected both plasma sdma and pcr increased with a decline in gfr. compared to gfr the correlation with 1/sdma, r=0.83 (p<0.001) was better than for 1/creatinine, r=0.75 and similar to that for egfr, r=0.86. comparing sdma with inutest gfr for detection of gfr <90 ml/min/1.73 m 2 the area under the roc curve was 0.87 (p<0.001). the equivalent areas for pcr and egfr were 0.84 and 0.89, respectively. in conclusion plasma sdma is an endogenous marker of gfr in children and is superior to pcr because it appears to be independent of body size. since the calculation of egfr requires accurate measurement of height, plasma sdma may provide a practical alternative for assessment of gfr in children. thrombotic microangiopathy (tma) consists of thrombocytopenia, microangiopathic hemolysis, and thrombi in the microvasculature of vital organs. broad categories of causes of tma include infectious (verotoxin-induced hus), hematologic (ttp), complement based (atypical hus), immune-mediated (sle, anti-phospholipid antibody syndrome), and drug-induced (cyclosporine). thorough investigation is required to detremine the underlying etiology in order to provide specific therapy and information about prognosis. a 14 year girl presented with short stature and was found to have hypertension, proteinuria, renal failure (creatinine 350 umol/l), and anemia (hgb 72 g/l). platelets were normal. she also had spondylometaphyseal dysplasia, scoliosis, lymphopenia, mild pulmonary hypertension and aortic stenosis. on pulmonary function testing, her dlco was decreased. chest ct demonstrated small micronodules. a ventilation-perfusion scan was normal. renal biopsy showed features of tma. she was treated with dialysis and underwent renal transplantation one year later. there has been no disease recurrence 6 months post-transplant. genetic and immunologic workups were negative, including anti-cardiolipin antibodies. a thrombophilia workup was negative apart from a heterozygous mutation in mthfr. c3 levels were mildly reduced. anti-neutrophil antibodies were negative. complement system studies, including factor h, and smarcal1 analysis for schimke immuno-osseous dysplasia are underway. although the descriptive diagnosis of tma can be applied here, the underlying pathophysiologic diagnosis has still to be defined. it is of particular importance that further efforts be made to identify the etiology given the potential risk of disease recurrence in the renal graft. background: long term outcome of renal functions after liver transplantation (lt) in wd is not studied yet. aim: the aim of this study was to determine the long term outcome of renal functions in children receiving lt for wd. patients and methods: renal functions were examined in 9 (f/m: 2/7) liver transplanted patients for wd before and long after lt and compared with renal functions of 9 patients (f/m: 5/4) with lt for a hepatic disease other than wd. the mean age of subjects was 12.1±3.2 years in the patients group and 9.8±4.1 years in the controls. the mean duration of follow-up was at least 2 years. glomerular and tubular functions were assessed using the conventional equations for measured creatinine clearance (gfr), tubular phosphate reabsorption (tpr), daily protein and calcium excretion in both groups. results: mean gfr before lt was 81.7±32 ml/min/1.73 m 2 in the study group and 146.3±37.6 ml/min/1.73 m 2 in the controls (p=0.007). the mean tpr before lt was found to be 67.9%±18.2% in the study group and 88%±9.6% in the controls (p=0.03). daily protein excretion rate before lt was found to be high in both groups, as well as urinary calcium excretion. an increase in gfr was observed in the study group after lt (p> 0.05), while it was slightly decreased in the controls (p>0.05). tpr increased significantly in the study group after lt (84.8%±9.5%) (p=0.04) and although it was found to be significantly lower in the study group than the controls before lt, in the long term follow-up the difference between the groups was disappeared (p=0.59). conclusion: tubular dysfunction is frequent in patients with wd. liver transplantation for hepatic failure secondary to wd is a lifesaving procedure. it corrects the underlying hepatic defect as well as renal defects and leads to long-term survival. rather conflicting results are available regarding the neurocognitive development of children with ckd, due to small sample sizes, cross-sectional study designs, differing methodological approaches and historical trends in patient selection for renal replacement therapy. we prospectively examined in a standardized, multi-center effort 59 children with ckd aged 0.8-15 years. children were treated either conservatively (n=30, gfr <25 ml/min/1.73 m 2 ) or by dialysis (hd: n=7, pd: n=22). a sub-sample of 18 children underwent repeated testing after 12 -14 months. bayley and snijders-oomen developmental tests were performed and the measured values normalized to standard deviation scores (sds). general cognitive development averaged at -1.02 (±1.48) sds. 42% of the patients scored <-1.5 sds, i.e. below the 10 th percentile of the normal population. no significant differences were observed between pre-dialysis (-0.87+1.57) and dialyzed patients (-1.16+1.40). impaired neurocognitive function was marked in infants (-1.63+1.31 sds), whereas school children showed a distribution similar to healthy children (0.43+1.42 sds, p<0.005). the global neurocognitive sds remained unchanged in the longitudinal sample (t 1 =-0.97±1.57; t 2 =-0.91±1.71). in summary, our preliminary results demonstrate a high prevalence of neurocognitive impairment in infants with ckd. we assume that this finding reflects the improved survival of children with complex disorders affecting not only the kidney but also brain development. the poor performance of this age group highlights the importance of close neurocognitive follow-up and early developmental interventions. objectives of study: to make a diagnosis of a girl with kidney subcapsular hydrops, abnormal urinanalysis and hypertension. methods: physical examination and laboratory investigations were analyzed. results: a 5 years old girl was admitted because of kidney subcapsular hydrops, proteinuria without edema, and hypertension (130/94 mmhg) for 50 days. no trauma or familial hypertension history were provided. no hydrops was found before the age of 1 year. urinary rbcs were 5-7/hp and protein was 77 mg/kg/24 hr. the serum albumin was normal. ultrasound examination revealed normal sized kidneys, increased echogenicity in both kidneys, and subcapsular hydrops on the upper pole of the right kidney connected with an old renal fissure. ucg and fundus examinations were normal. gfr of the right kidney was slightly decreased as compared to the left (65 ml/min vs. 67 ml/min, by dtpa scan). by puncture of hydrops, yellow clear fluid was drained, the analysis showed similar composition to that of original urine, so subcapsular urinoma was diagnosed. urine collection from two kidneys separately was performed by cystoscopy; nonselective proteinuria of 1+ was found in urine from the right and 2+ from the left kidney. analysis revealed urea 36.8 mmol/l, potassium 6.92 mmol/l, creatinine 0.42 mmol/l in the right kidney urine compared to urea 77.2 mmol/l, potassium 11.19 mmol/l, and creatinine 1.29 mmol/l in the left, which suggested that the right kidney function was compromised. according to proteinuria from both kidneys with microscopic hematuria, without edema and hypoalbuminemia, glomerulonephritis was diagnosed. the girl was diagnosed with glomerulonephritis and subcapsular urinoma. it was a rare case because of their co-incidence. reasons for the hypertension, if caused by the glomerulonephritis or the pressure by subcapsular urinoma, as well as reasons for subcapsular urinoma need to be clarified during the follow-up. the aim of this study was to detect factors that could interfere with the results of des treatment. methods: fifty-six patients 5.9 to 15.2 years old with des without improvement by previous therapies were randomly distributed into two voiding training programs: group 1 (g1): 26 patients submitted to 24 kegel exercises training sessions for three months; group 2 (g2): 30 patients submitted to 16 biofeedback sessions over a two month period. both groups adhered to a voiding and drinking schedule, received adequate toilet posture instructions and were reinforced through the maintenance of voiding diaries. clinical evaluation was carried out before each programs initiation and 1, 6, and 12 months after end of the program. all patients were submitted to renal and dynamic ultrasound before and 6 months after each program's conclusion. the following variables were analyzed: gender, age at diagnosis, treatment group type, vesico-ureteric reflux, constipation, urinary tract infection, asymptomatic bacteriuria, bladder wall thickening and post void residual (pvr) urine. the logistic regression model was applied to identify independent variables associated with response to treatment. results: urinary continence was improved after completion of either training program. success in diurnal urinary incontinence varied from 72.7 to 80% in g1 and from 65.2 to 89.4% in g2. success in nocturnal urinary incontinence varied from 66.7 to 84.2% in g1 and from 65.4 to 86.9% in g2. in multivariate analysis three variables remained independently associated with bad response to treatment: constipation with soiling, bladder wall thickening and pvr urine (p<0.05). conclusion: studies using multivariate regression analysis to identify predictors of response to behavioral therapy are important for the development of selection criteria for prescribing these therapies to children. we report the case of a four-year-old child born to consanguineous parents, who presented first at two months of age with respiratory failure. during the admission he developed panyctopenia, hypertension and nephrotic range proteinuria. the metabolic workup revealed methylmalonic academia and aciduria along with homosystinuria; highly suggestive of cobalamin deficiency. the renal biopsy showed chronic thrombotic microangiopathy (tma). the muscle biopsy showed the presence of nemaline rods on electron microscopy. cobalamin c deficiency was confirmed by genetic analysis as the patient was homozygous for the mutation c547/548. at the age of one, he was noted to be visually inattentive and developmentally delayed. on ophthalmology examination there was evidence of bilateral maculopathy and dysfunction of rods and cons on electroretinogram. he subsequently went on to develop bilateral bovine maculopathy. since his initial presentation he has been maintained on hydroxycobalamin, betaine and folate; with no further relapse of proteinuria or panyctopenia. however, despite adequate doses of hydroxycobalamin, the maculopathy progressed. to our knowledge, this is the first report of a child with both tma and bovine maculopathy. the aim of the study was to assess the rate of vesicoureteral reflux (vur) in patients with lower urinary tract dysfunction (lutd) of nonneurogenic origin. dysfunctional voiding may result in lower urinary tract symptoms in children and is commonly associated with urinary tract infections and vur. we investigated 111 patients with voiding dysfunction during last three years: 31 boys and 80 girls, a mean age of 8.2 years, with a mean follow-up of 19 months, all with normal renal function. mean pre and post treatment symptom scores were 23.6 and 7.6 respectively. vur was detected in 21% of the children with unstable bladders and 17.5% of the children with stable bladders as detected by video urodynamic investigation, 22% of the children with urinary tract infection (uti) on admission, and 18% of the children with no history of uti on admission. the co-existence of vur in our group with voiding dysfunction was 19.8% (22 patients). all patients with vur had low grade reflux (grades i-iii) and 3 of them bilateral, the remaining 19 unilateral. renal us was performed in 55 patients and revealed hydronephrosis in 13 patients, while the remainder showed no abnormalities. vur was found to be present in 30% of the children with abnormal us findings, while only 2% of the children with normal us findings were affected. furthermore, the patients that had spontaneous resolution of reflux showed a markedly greater improvement in symptom scores. a significant portion of patients with lutd (19.8%) have low grade vur. in detection of vur in patients with lutd, hydronephrosis is a good indicator of the presence of reflux, while utis and urodynamic findings were not found to be significant indicators. the overall spontaneous resolution rates of vur in patients with lutd and stable bladder following treatment was found to be 22.7% and 14.2%, respectively. 200-250g) were grouped: group 1 (n: 5) was the sham group. group 2, 3 and 4 (n: 7 for each) received 50 mg/kg twice daily ptx intraperitoneally (i.p.), 100 mg/kg/day gen i.p. and both ptx and gen at the same dosages for 8 consecutive days, respectively. the rats were weighed at the beginning and than weekly during the study. after the last dose 24-hour urines were collected. then, rats were sacrificed and blood samples were obtained from the abdominal aorta. bun, serum creatinine (scr), creatinine clearance (ccr), renal superoxide dysmutase (sod), catalase (cat) and thiobarbituric acid reactive substances (tbars) levels were determined. all the parameters were compared between the groups. results: body weights were not different between the groups either at the beginning or during the study. bun levels were significantly higher in group 3 than the other groups (p<0.01). scr and ccr levels were similar between the groups 3 and 4, but the levels were higher than those of groups 1 and 2 (p<0.01). sod and tbars levels were similar between all groups. the levels of tubular cell apoptosis and caspase-3 expression were significantly higher in group 3 than the other groups (p<0.05). conclusion: we may conclude that ptx administration significantly reduced the apoptosis in gentoxicity, but we could not demonstrate any evidence of ptx-related reduced oxidative stress. the lack of evidence for the widespread use of antimuscarinics and holding exercises in mne prompted us to design a randomized controlled trial comparing interventions in four groups of children with mne: placebo (a) or oxybutynin chloride (b) in combination with a daily regimen of standardized fluid intake and holding exercises or as monotherapy (c) and (d). a fifth group, to be treated with alarm only, was planned as control. randomization was stratified for participating hospital, sex, age, tanner stage, family history of mne, previous treatment, and bladder capacity class, being the largest from either the maximum voided volume (mvv) from a 48 h frequency volume chart or the volume after 4 baseline holding exercises (hev). after 12 weeks intervention with holding exercise (a and b) , hev had increased, both with oxybutynin (208±84 (sd) ml to 311±121 (sd) ml, p<0.001) and placebo (216±82 (sd) ml to 259±90 (sd) ml, p<0.05). without holding exercises, only oxybutynin (d) increased hev (223±79 (sd) ml to 274±111 (sd) ml (p<0.05). mvv increased also in groups a and b (holding exercises), not c and d. cure rate (less than 1 wet night in the last for 4 weeks) was low: a 1/29, b 3/30, c 0/30 and d 4/30. control group with alarm had 22/30 cure. cure was not related to hev, mvv or delta hev and mvv. this questions the relevance of increasing bladder capacity in mne. background: sickle cell disease (scd) is an inherited disorder of beta-globulin synthesis of haemoglobin, resulting in a tendency for haemoglobin polymerisation and consequent vasoocclusion, tissue hypoxia, and ensuing organ damage. the kidney is a particularly sensitive to hypoxia and renal failure is a major cause of morbidity and mortality in scd. children with scd commonly hyperfiltrate, the glomerular filtration rate (gfr) then typically falls back towards normal in adulthood. routine estimation of gfr in children is primarily derived from the height and plasma creatinine (pcr) measurements using k a constant dependent on the creatinine analytical method. this formula has reduced accuracy in children with a gfr >80ml/min/1.73 m 2 . it has never been validated in hyperfiltration or children with scd. recently, new gfr markers have been proposed including symmetrical dimethylarginine (sdma) that may be independent of body size. in this pilot study we tested the hypothesis that estimated gfr and/or sdma allow a reliable estimation of gfr in scd. methods: 13 hbss patients, age range 10-20yrs (mean age 15yrs) attending the evelina children's hospital were studied. the patients were on regular blood transfusions for stroke management and undertook a formal gfr measurement using plasma clearance of inutest. the plasma samples were also used for the measurement of pcr and sdma by stable isotope dilution mass spectrometry. egfr was calculated using k=35. results: inutest gfr ranged from 70-175 ml/min/1.73 m 2 . there was significant inverse correlation between sdma and inutest gfr (p<0.01)). there was no significant correlation between either pcr or egfr and inutest gfr. conclusions: this early data suggests that sdma might prove valuable in monitoring gfr in children with scd. introduction: the aim of this study was to identify the risk factors for renal scarring in children with lower urinary tract dysfunction (lutd) by using data available at the time of patient admission to the interdisciplinary management program of lutd at hospital das clínicas. material and methods: medical records of 120 patients were assessed retrospectively concerning gender, presence of vesicoureteric reflux (vur), bladder capacity, detrusor overactivity, residual urine, urinary tract infection (uti), asymptomatic bacteriuria, constipation, detrusor-sphincteruncodination (dsu), high detrusor pressure at maximal cystometric capacity, low compliance, thickness and trabeculation of the bladder wall. renal scarring was diagnosed by dmsa scan. statistical analysis was performed by univariate and multivariate analysis. a p value <0.05, 95% confidence interval was considered significant. results: renal scarring was detected in 38 patients (31%). abnormal bladder capacity, detrusor overactivity, residual urine, asymptomatic bacteriuria, constipation, dsu, high detrusor pressure and low compliance were not associated with renal sccaring. vur, uti, decreased bladder capacity, urinary residue, trabeculated and thick bladder wall were associated with scarring at univariate analysis. multivariate analysis showed vur (p<0.0001) and female gender (p=0.05) as independent risk factors for renal scarring. thickness of the bladder wall was a marginal risk factor (p=0.07). conclusion: urodynamic parameters didn´t predict renal damage in this study. uti was not a risk factor for renal scarring; however, it was associated with vur (p=0.03). although vur was the main risk factor in our analysis, renal scarring was probably due to multifatorial causes as vur was associated with itu. results: comparison of test a and b demonstrates that a mild fluid load 1 h before the administration of ddavp nasal spray (2 puffs) results in 3 major significant differences.1). maximal concentrating capacity (cc) is reached later than 1h after administration (p<0.05). in 8/46% cc at sleeping time is <70% of max, thereby resulting in a persistent np in the first hour of the night. 2) uosmol is significantly (23%) lower in the overnight collection (u4), correlating with a higher diuresis-rate (16%)3). the duration of the ddavp effect is shorter leading to an increase in diuresis-rate and to a decrease in the u osmolality in 24/46 children in the urine-collection between 4-7h in the morning. this proves that in up to 50% of the patients the ddavp-effect does not cover the full night. conclusion: test b demonstrate sthat fluid intake prior to the ddavp-administration influences significantly the antidiuretic effect of ddavp (onset, maximum and duration). this might explain the partial response, suggestive for insufficient pd (and pk) effect of the spray. test a proves that fluid restriction 1 h before ddavp administration, significantly reduces the nocturnal diuresisvolume. m. schmidts, c. schnakenburg, k. häffner, c. jacobi, k. schwab, m. pohl university hospital, center for pediatrics and adolescent medicine, freiburg, germany background: enteropathic hemolytic uremic syndrome (d+hus) is responsible for 90% of all hus cases. in addition to the nephrological and neurological complications, pancreatic damage resulting in diabetes mellitus is also possible and appears to be associated with more severe cases and elevated mortality. case report: a 3-year-old girl suffering from d+hus with severe colitis, acute renal failure, dyskinesia, dysarthria and agitation provoked by shiga-like toxin positive ehec infection in july, 2006. the severe affection was characterized by partial thalamic infarction, prolonged leukocytosis (max. 51 g/l) and the necessity of 4 weeks of dialysis. 6 days after the disease onset, hyperglycemia (max. 530 mg/dl glucose) was noted. c-peptide was found to be low indicating reduced insulin secretion. 6 months after hus the patient continues to be insulin dependent. clinically apparent exocrine pancreatic insufficiency has resolved spontaneously. gfr had recovered to 28 ml/h/1,73 m 2 , but then kidney function deteriorated and dialysis had to be resumed after 5 months. kidney histology at this time showed severe nephron loss compatible with chronic changes after hus and ruled out other unrelated kidney disease. conclusion: in addition to the kidney damage, chronic pancreatic damage can occur in hus. therefore blood glucose levels should be monitored in all hus patients. it is tempting to speculate that patients after childhood hus might be at risk for diabetes mellitus later in life. objective: childhood nephrotic syndrome (ns) is characterised by a relapsing course resulting in significant corticosteroid burden or prescription of cytotoxic immunosupressive therapy. this randomised controlled study was carried out over 3 years at a single centre in sri lanka to compare the efficacy and safety in children with steroid dependant ns treated with intravenous cyclophosphamide or intravenous vincristine therapy. methods: thirty-nine sequential children with steroid dependant ns with evidence of steroid toxicity were randomly allocated to receive either intravenous cyclophosphamide (500 mg/m 2 monthly for 6 months) or vincristine (1.5 mg/m 2 weekly 4 doses followed by 4 doses monthly). both groups received an identical tapering regimen of oral prednisolone for 6 months. all children were reviewed on monthly basis for one year focusing on recurrence of proteinuria and side effects of therapy. finding of +++ proteinuria for 3 consecutive days was diagnostic of relapse. results: there were 18 (11m: 7f) children in the cyclophosphamide group (mean age 6.4 years) and 21 (15m: 6f) in the vincristine group (mean age 7.2 years). during one year of follow-up 6/18 (33%) in the cyclophosphamide group suffered a relapse while 13/21 (62%) suffered a relapse in the vincristine group. p=0.03 (comparison of 2 proportions using standard error. ci 0.105 to 0.49). no serious adverse effects were encountered in either group. conclusion: in steroid dependant ns, intravenous cyclophosphamide therapy is superior to intravenous vincristine therapy in maintaining sustained remission. we present the clinical and biochemical features of a patient with antenatal bartter syndrome who was found to have a novel romk mutation. the patient presented antenatally with severe polyhydramnios. polyuria, hyponatraemia and hyperkalaemia were evident soon after birth. she had marked hypercalciuria and developed medullary nephrocalcinosis in early infancy. failure to thrive was evident from 12 months. hypokalaemia was a late feature, developing gradually from 18 months. serum chloride levels were consistently 95-100 mmol/l whilst urinary chloride levels were consistently <30 mmol/l, only reaching higher levels after treatment was commenced. alkalosis was not present and the patient did demonstrate some response to furosemide implying some functional capability of na-k-2cl. renin and aldosterone levels were persistently elevated. treatment with indomethacin, nacl and kcl produced a good clinical response. mutational analysis revealed compound heterozygous mutations in kcnj1, the gene encoding romk. both mutations, m357t and l359r are in the terminus of the protein thought to have a role in channel gating. similarities and differences from the classically described presenting features of antenatal bartter syndrome highlight the clinical heterogeneity in this condition. this relates to the different identified kcnj1 mutations which are likely to affect romk function in different ways. the m357t mutation has been described, but past electrophysiological studies in sf9 cells transfected with the m357t mutant have not identified any differences in k + conductivity from wild type romk. given that the mutation appears to be clinically relevant in this case, further functional studies are indicated. l359r is to our knowledge a novel mutation. expression of these mutations in oocytes is now planned to enable evaluation their effects on channel function and regulation. a 10-year-old boy was admitted because of nephrotic syndrome. renal biopsy, performed after 4 weeks of prednisone (60 mg/m 2 od) + 3 methylprednisolone pulses (15 mg/kg each), showed focal segmental glomerulosclerosis. prednisone was tapered (to 30 mg/m 2 od) and enalapril was introduced, without any significant improvement. two weeks later, the patient had transient hypoperfusive acute renal failure which required acei discontinuation. due to the persistence of proteinuria, cyclosporine was started (5mg/kg/day). proteinuria gradually decreased and ceased within the subsequent two weeks. in the meantime the boy had started to complain of low-grade fever without other symptoms and with normal physical examination. laboratory tests only showed leucocytosis (wbc 22 x 10 3 /ml) and increased c-reactive protein (65 mg/l). a chest x-ray revealed an upper mediastinal enlargement and a total body ct scan confirmed the presence of several enlarged mediastinal lymph nodes, whose biopsy led to the diagnosis of hodgkin disease (hd) nodular sclerosis subtype, cd30+; stage was iia. cyclosporine and the residual steroid treatment were discontinued and the patient was given six cycles of abvd (doxorubicin, bleomycin, vinblastine, dacarbazine), followed by radiation therapy. presently, 18 months after stopping chemotherapy, both hd and nephrotic syndrome are still in remission. once again, this case points out both that fsg can be secondary to a lymphoproliferative disease and that chemotherapy for hd might have been effective to keep fsgs in long-lasting remission. several treatment methods including increased fluid intake and dietary modification, medical therapies such as potassium citrate and use of extracorporeal shockwave lithotripsy (eswl) and finally surgery methods are used for treatment of urolithiasis. the aim of this study was to evaluate the etiological and clinical characteristics and level of response to medical therapy with polycitra in children with urolithiasis. one hundred thirty-four patients had urolithiasis of which 109 cases followed thetreatment instructions and fulfilled the inclusion criteria for this study. struvite stone were excluded from the study. all other patients who had an initial ultrasonography showing stone inurinary tract were treated with polycitra-k (potassium citrate 220 grams, citric acid 66.8 grams in 1 liter of distilled water) irrespective of the cause of the stone. at the end, complete resolution or passage or a decrease in the size of stone in later sonography was defined as response to treatment. hypercalciuria and hyperuricosuria were found to be etiological factor in 25% and 19% of patients respectively. the stone analysis was found that 50% of them were ca-oxalate. stone disease was more common between the age of 1-5 years. the most common complaint was hematuria (20%). eswl was performed in 42% of patients who did not respond to polycitra and had surgically active stones. calcium-oxalate stones were the most frequent stone which responded to polycitra. the response rate in girls and boys was equal and in different age groups the response rate was almost equal. methods: mmcs were expanded in culture and immunocytochemistry was used to characterize the cells. after gentamicin-induced atn, fluorescently-labeled cells were transplanted and traced in kidney tissues by fluorescence microscopy. kidney pathology was studied by hematoxylin-eosin staining, apoptosis was examined by the tunel assay, and ki-67 and bcl-2 expression were examined by immunohistochemistry and reverse transcription-polymerase chain reaction, respectively. results: (1) mmcs (rimm-18 cells) were successfully expanded in culture. the phenotype of the cultured cells were vimentin-positive and kreatin-negative by immunocytochemistry. (2) in the mmcs-treated group: the mortality rate decreased; renal function clearly improved; damage to the cell-treated kidneys was reduced and histopathologic lesion scores were lower; proliferation of renal tubular epithelial cells was improved; the apoptosis of renal tubular epithelial cells was reeuced; and the expression of bc1-2 mrna and protein was upregulated. the subcapsular transplantation of mmcs could ameliorate renal function and repair kidney injury. atn is the most common reason for arf, and there is still an absence of effective therapies. this study was done to observe the effect of mobilizing bone marrow cells with stem cell stimulating factor (scf) and gm-csf on recovery from gentamicin-induced acute tubular necrosis (atn) in rats. atn was included in male sprague-dawley rats with five daily high dose intraperitoneal injections of gentamicin. subcutaneous injections of scf and gm-csf were administered simultaneously and these cytokines was observed at day 2, 5, 10, 17, 24 and 31. leukocyte numbers, percent venous blood cd34+ cells, mortality rate, and concentration of the urine proteins, urine nag, bun, scr, and ccr, histopathogic lesion scores were determined. twelve hours after bone marrow ablation (bma) by lethal x-ray radiation, gentamicin-induced spf atn rats were given five daily injections of scf and gm-csf. bun, scr, and histopathogic lesion scores were evaluated at day 2, 5 and 10. the effects and mechanism of scf and gm-csf on atn was observed. the number of leukocytes and the cd34+ cell percentage increased significantly in atn rats between 2 and 10 days after scf and gm-csf injection. in addition, mortality rates dropped, the peak value of renal function increased, renal function were rapid ameliorated and histopathologic lesions were reduced. there was no significant effect on atn rats after bma. this study demonstrates that scf and gm-csf effectively mobilized bone marrow stem cells in atn rats. rapidly improving renal function and decreasing mortality rate. these results suggest that bone marrow stem cell mobilization may be an effective therapy for atn. key words: acute tubular necrosis, bone marrow stem cells, stem cell factor, granulocytemacrophage colony stimulating factor, irradiation. objective of the study: the response to recombinant human erythropoietin (rhuepo), 50 unit/kg twice weekly was studied prospectively in 35 children and adolescents with end stage renal failure who were either transfusion dependent or had hematocrits (hct) <25%. methods: rhuepo was given to 22 haemodialysis (hd) patients and 13 patients on conservative treatment, with mean age (10.84±4.08) years, 25 males and 10 females with mean hct (26.75±4.70). blood pressure, haematocrit, iron-indices, serum potassium, calcium, phosphorus, alkaline phosphatase, urea nitrogen and intact parathyroid hormone (ipth) were monitored serially. results: serum aluminum was measured randomly in 6 patients, results were normal ranging from 12-22 ug/l. when serum ferritin was <100ng/ml during therapy, they received iron supplementation. according to the response, patients were divided into 2 groups, the non-responders group with hct<27, mean age (9.97±3.55) years, 13 males and 6 females with mean ipth (669.9±461.77 pg/ml) and group of responders with hct >27 with mean age (11.66±4.32 years),12 males and 4 females with mean ipth ( increase of fatty acids such as nonesterified fatty acid (nefa) and triglyceride (tg) with oxidative stress and production of cytokine/chemokine occured during cisplatin (cp) toxicity. statin (3hydroxy-3-mthylglutaryl coenzyme a reductase inhibitors) have been postulated to have pleiotrophic effects. we examined whether statin, pravastatin, would ameliorate renal damage induced by cp. male wistar rats (weight 180-200 g) fed standard chow were divided into 3 groups: control-rats received tap water alone for 19 days; cp treatment-rats that received cp (10 mg/kg, i.v.) on the 14 th day of the study; cp+pravastatin treatment-rats that received pravastatin (6 mg/kg/day) in their drinking water for 19 days and cp injection as indicated in the preceeding group. blood and urine samples were collected and the kidney were removed 5 days after cp treatment. urinary excretions of protein and 8-hydroxy-deoxyguanosine (8-ohdg), serum levels of creatinine and fatty acids were measured. histology was evaluated by light microscopy with immunohistochemistry for pentosidine, n-carboxymethyllysine (cml), and heme oxygenase (ho)-1. expression of ho-1 mrna in the kidney was also measured. pravastatin decreased urinary excretions of protein and 8-ohdg and ameliorated renal function diminishing areas of tubular damage and positive staining of pentosidine and cml compared with those of cp treatment alone. however, positive staining area and mrna expression of ho-1 were not significantly changed by pravastatin treatment. although pravastatin did not influence serum levels of total and low-density lipoprotein cholesterol, serum tg level decreased and was equivalent to that of control group. these findings suggest that pravastatin treatment partially protects against cp-induced nephrotoxicity in rats, through its antioxidant as well as lipid-lowering effect. n. bresolin, v. fernandes, f. carvalho, j. goes, l. araujo, m. simon, g. gamborgi, m. zanin hospital infantil joana de gusmo, nephrology pediatric department, florianópolis, brazil objectives: the objective is a report case of acute renal failure (arf) in a child contact with lonomia obliqua caterpillar in southern brazil. the accidents with lonomia oblique has increased in south of brazil in recent years. this increase can be related with reduction of caterpillar natural predators and deflorestation. the venom of lonomia caterpillar provokes hemorrhagic syndrome resembling disseminated intravascular coagulation (dic) and hemolytic anemia. arf could be an important complication of hemoglobinuria that has been recently described in adults. methods: a case report. results: the present is a first case described of arf in a child after contact with lonomia obliqua caterpillar. conclusions: the arf can occur in any age, however, the contributing factor to the development of arf remains obscure. there are 3 mechanisms pointed out: fibrin deposition in glomerular microcirculation, ischemia in hemorrhagc shock with hypotension and venom direct action. the present article related a case of lonomia obliqua accident in a child who revealed coagulation disorder, hemolytic anemia, arf and always, she was hemodynamic stable. the treatment included antilonom serum, urine alkalinization, hyperhydration and peritoneal dialysis for four days. she was treated and followed per 1 year when she recovered her normal renal function. introduction: secondary hyperparathyroidism develops in chronic kidney disease as a consequence of impaired phosphate, calcium and vitamin d homeostasis. the treatment includes phosphate binders and vitamin d analogues, but sometimes ineffective. we report two cases of refractory secondary hyperparathyroidism treated successfully with cinacalcet. case 1: a 21-month-old boy with end stage renal disease (esrd) due to posterior urethral valve started on peritoneal dialysis at 12 months of age. he developed secondary hyperparathyroidism with serum parathyroid hormone (pth) level reaching 585 pg/ml. serum calcium had been in the range of 6.7 to 9.6mg/dl and serum phosphorus in the range of 1.7 to 10.7 mg/dl. despite treatment with phosphate binders and vitamin d analogues, pth levels kept increasing to 897 pg/ml. x-ray showed the cupping and fraying of the distal ends of radius and ulna. we started cinacalcet 10 mg daily and increased the dosage up to 30 mg daily. eight months later, pth level decreased to 154 pg/ml and bone changes resolved. case 2: a 12-year-old boy with esrd due to primary hyperoxaluria started on peritoneal dialysis at 10 years of age. he developed secondary hyperparathyroidism with serum pth level of 641 pg/ml. serum calcium had been in the range of 7.9 to 9.7 mg/dl and serum phosphorus in the range of 5.9 to 11.1 mg/dl. despite treatment with phosphate binders and vitamin d analogues, pth level kept increasing to 1457 pg/ml. x-ray showed the distal radius and ulnar fracture of left arm and right femur fracture. we started cinacalcet 30 mg daily. five months later, pth levels decreased to 151 pg/ml. conclusion: cinacalcet is effective in the secondary hyperparathyroidism resistant to phosphate binders and vitamin d analogues in children with chronic renal failure. rota virus (r) is a common pathogen as the cause of gastroenteritis in childhood. we experienced some cases with r infection who had the renal failure induced by uroammoniac calculi as well as dehydration. we examined the clinical feature and laboratory findings of the cases with viral acute gastroenteritis in r and non-rota virus (nr) groups for 2005-2006. with rapid diagnosis test, we checked the patients that needed the hospitarization medical treatment from newborn to five-years old. in the 42 cases of r group and 23 cases of nr group, we campared the clinical findings, blood chemistry test and urinalysis of r groups in acute and convalescent a tage with those of nr group. r group had significantly (p<0.05) lower in ph (vein blood gas test) and higher in blood uric acid (bua) compared with those of nr group. these findings suggest that the elevation of bua and acidosis in r group may induce the formation of renal calculus resulting in postrenal failure. our patient is white male 9 y/o. at 4 year of age, he was dx. with acute lymphocytic leukemia and was induced to remission on pog 9404; he presented tumor lysis syndrome during induction, acute renal failure with no recovery in renal function and rrt was started (ccpd) and continue on the same therapy. during the last years, he presented chronic pancreatitis, ards, sepsis. he had episodes of major vessel thrombosis probably due to central lines (several femoral lines and vascath placed in his thorax) and l-asparaginase. our patient did receive 3 years of chemotherapy and radiation; and he is on complete remission. on 11/2,005, for pre-kidney transplant evaluation, a mri test was requested using gadolinium 1 ml/kg for evaluating his vascular system (abdomen and pelvis) with inconclusive results. another mri test was requested 3 months later using gadolinium 0.3 ml/kg. 8 weeks later, his mother noticed brawny hyperpigmented, shiny, tightly bound-down, indurate plaques of his bilateral lower extremities, (more significantly on his lateral and posterior calves, and in his anterior and lateral thighs) a punch skin biopsy showed increasing number of fibroblastic cells and widened space between septa in the deep reticularis dermis compatible with nfd. nfd is a fibrosing disorder identified among patients with renal disease with cutaneous finding of skin thickening in extremities and trunk, very similar to those of systemic sclerosis. etiology, pathogenesis and clinical course remain unknown. the majority of cases have been reported in dialysis or kt patients and gadolinium has been identified as a trigger of nfd. in our case, there appears to be a link between the use of gadolinium and the developed of nfd. background: chronic renal failure (crf) is an independent cardiovascular risk factor. changes in calcium-phosphate homeostasis not only affect the quality of bones but also constitute the biochemical base for vascular calcification. aim: to find a method better describing factors of calcification using routine laboratory examinations and computed evaluation of complex equilibria. methods, patients: data of 12 crf and of 24 transplanted (tx) children were compared to a healthy control group of 15. tx children's parameters were taken before and 12 and 48 month following transplantation. three different strategies were used to analyse factors of calcification: ca x p product, ca-p activity value and the concentration of cahpo 4 . the ca x p product and the ca-p activity value were not informative, because they didn't represent the direction of change in the complex chemical equilibrium. the cahpo 4 concentration was increased in the crf and the tx group (before transplantation) (0.380±0.173 mmol/l) compared to the healthy control group (0.260±0.060 mmol/l) (p<0.01). after 12 and 48 months to transplantation the cahpo 4 concentration was in the normal range in the tx group. a negative correlation was found between the concentration of ica 2+ (ionic calcium) and pth (parathyroid hormone) in the dialysed children (r=0.720), but not in the transplanted group (r=0.122). conclusion: according our findings the force driving calcification is better represented by the concentration of cahpo 4 , the base compound of all primary calcification, than by measuring ca and p separately. follow-up study is needed to establish the predictive value of determination of cahpo 4 . this study was supported by grants otka-t046155 and ett 435/2006, the national office for research and technology (nkth) and szentágothai j. knowledge centre. introduction: acute renal failure is a rare complication of nephrotic syndrome and its cause is still unknown. several investigators reported that the most important factor for renal failure was acute tubular necrosis (atn); however, some cases did not have laboratory findings of tubular dysfunction paradoxically. patients and methods: we reviewed 11 cases of nephrotic syndrome with acute renal failure (nsarf) since 1990 at metropolitan kiyose children's hospital. seven of 11 cases had calcium deposition in the distal tubules predominantly. we analyzed the clinicopathological findings in these 7 cases. results: the pathological diagnoses of these 7 cases were as follows: 4 minor glomerular abnormalities, 2 mesangial proliferative glomerulonephritis (without iga deposition), and 1 focal segmental glomerulosclerosis. interstitial nephritis was not documented in any case. the common characteristics of these 7 cases were calcium deposits in the distal tubules. some cases had focal simplification of tubular epithelium. all cases were an initial episode of nephrotic syndrome, and 6 cases were steroid resistant. almost all cases had hypertension. in addition, the range of urinary β2 microglobulin and fena were 94 to 2980 and 0.1 to 0.33%, respectively. discussion: in these 7 cases, there was enhanced na reabsorption and urinary β2 microglobulin was only mildly elevated at the time of renal failure. these findings were not consistent with atn. additionally, pathological finding of severe tubular damages was not found on biopsy. contrarily, tubular obstructions due to ca depositions were consistent with these clinicopathological findings. conclusion: our findings suggest that tubular obstruction due to calcium deposition plays an important role in the etiology of nsarf except for atn. background: natriuretic factor was found in urine of chronic uremia for more than 30 years. n-terminal pro-b-type natriuretic peptide (prontbnp) is postulated to be the natriuretic factor owing to its elevation in chronic kidney disease (ckd). however, salt wasting and non-saltwasting chronic kidney disease haven't been partitioned as different entities before. this study is designed to clarify whether prontbnp is different in salt wasting and non-salt wasting ckd and if it is the main causative factor of natriuresis. methods: 17 patients with salt wasting ckd, which are mainly composed of congenital renal structure abnormalities, and 11 patients with non-salt wasting ckd, which are mainly composed of glomerulonephritis, were collected. all of them are non-dialysis-dependent and without heart failure. serum prontbnp in these two groups and 28 normal controls were sampled. fractional excretion of sodium (fena) and estimated clearance of creatinine (ccr) were also checked. results: prontbnp was elevated in salt wasting group compared to normal controls (p=0.012). moreover, prontbnp was even significantly higher in non-salt wasting group than in salt wasting one (p<0.0001). in salt wasting ckd, prontbnp and fena were correlated (r=0.7, p=0.017), but the same result was not observed in non-salt wasting group. prontbnp was significantly correlated to ccr and age using multiple regression analysis (p<0.05). conclusion: prontbnp elevation was different between salt wasting and non-salt wasting ckd. volume expansion explained the difference and prontbnp elevation in salt wasting ckd resulted from other mechanisms. since salt wasting ckd, in which the influence of volume expansion was eliminated, had a good correlation between prontbnp and fena, prontbnp may be one of the major contributing natriuretic factor in chronic uremia. background: cidofovir is a new antiviral drug that has a broad spectrum of activity against a number of dna viruses. several observations reported its efficacy as topical treatment of resistant warts. we herein report a systemic complication of acute renal failure associated with such topical therapy. casereport: girl aged 17, received a renal transplant in 1999 and 2001 for end-stage renal failure secondary to haemolytic uremic syndrome. her initial immunosuppressive regimen consisted of prednisone, tacrolimusand mycophenolate. five years post transplant she developed chronic allograft nephropathy with mild renal impairment. as a consequence of long standing immunosuppression she also developed invalidating, widespread periungueal warts. a well conducted, aggressive, conventional management of her warts including reduction of immunosuppression failed to improve the symptoms. she subsequently received an intralesional injection of a 75 mg/ml cidofovir solution. forty eight hours after the treatment she developed local swelling, inflammation and pain along with acute alteration of renal function. although cidofovir seric dosages performed on day 4, 5, 7 and 8 postinjection were negative we attributed this acute renal failure to a potential nephrotoxic side effect of cidofovir. renal biopsy showed no evidence of rejection but mild alterations of the tubes compatible with tubulo-interstitial nephritis. spontaneous recovery of renal function occurred within 2 months. conclusion: we describe an acute renal failure as a consequence of topical cidofovir treatment of warts in the context of renal transplant with mild preexisting renal failure. topical administration of cidofovir needs as carefull use and monitoring as its parenteral administration, especially in patients with altered renal function. the hemolytic uremic syndrome (hus) is a disease characterized by microangiopathic hemolytic anemia and variable organ impairment with a predominat feature of renal failure in children. the aim of this study was to investigate the presence of plasma lipid peroxidation products in the acute phase of hus. we have analyzed the levels of lipid peroxidation, determined fluorometrically as thiobarbituric acid-reactive substances (tbars), in the plasma of 8 patients (aged 15-48 months with a median of 23) diagnosed with the shiga toxin-associated form of the disease. in all cases, tbars determinations were performed at hospital admission, during treatment and upon discharge. tbars values averaged 1.27±0.14 and 1.12±0.20 mm sem at admission and discharge respectively (reference value <0.5 mm). of the patients examined, 4 presented conserved diuresis during the course of the syndrome, while the other 4 were anuric and required dialysis. maximum tbars values resulted significantly higher (p<0.05 by anova followed by newman-keuls test) in the dialysis-requiring patients as compared to those that had conserved diuresis (2.66±0.48 vs 1.58±0.39 mm respectively) we also investigated a possible correlation between tbars and plasma creatinine, urea, lactate dehidrogenase (ldh) and platelet count. positive and highly significant correlations (r=0.45 and 0.47, p<0.01) were observed when tbars values were plotted as a function of plasma creatinine and urea values respectively. no significant correlations were detected for tbars and plasma ldh values or platelet count. thus, patients affected by shiga toxin-associated hus exhibit increased levels of oxidative stress. also, there is a positive correlation between the magnitude of oxidative stress and the severity of renal failure. c. soares, j. diniz, e. lima, g. oliveira, c. camargos, b. sousa, e. oliveira objective of the study: the purpose of this retrospective cohort study was to report the clinical course of children and adolescents with chronic kidney disease (ckd) enrolled in a pre-dialysis program. methods: the records of 108 patients with ckd admitted to an interdisciplinary pre-dialysis program from 1990 to 2006 were retrospectively analyzed. the program consisted of conservative management of children and adolescents with ckd and was conducted by an interdisciplinary team including pediatric nephrologists, pediatricians, nurses, psychologists, nutritionists, and social workers. the patients were admitted with a glomerular filtration rate (gfr) equal to or below 75% of the value expected for their age according to normal reference data. demographic, clinical and laboratory data at entry and at the end of the follow-up were analyzed. renal survival analysis was performed using the kaplan-meier method. results: the median age at admission was 8.8 years (interquartile range: 2.6-13.2 yr). the most frequent primary renal disease was congenital urological anomaly in 50 patients (46%), following by glomerular diseases in 25 (26%), cystic diseases in 19 (18%), and others in 14 children (13%). at admission, 8 patients (7%) had ckd stage 2, 60 (56%) had ckd stage 3, and 40 (37%) presented ckd stage 4. median follow-up time was 5.3 years (iq range, 2.6-7.8). at the end of the follow-up, 12 children presented ckd stage 1 or 2 (13%), 20 patients were in stage 3 (18.5%), 16 in stage 4 (15%), and 58 children were in stage 5 (54%). it was estimated by survival analysis that the probability of ckd stage 5 was 24% at 3 years, 37% at 5 years, and 62%at 10 years after admission to the pre-dialysis program. conclusion: the long-term overall renal survival is relatively poor in a cohort of children and adolescents with ckd. objective: characterize the association between proteinuria and gfr in a cross-sectional study of children with mild to moderate chronic kidney disease (ckd). methods: first morning urine protein to creatinine ratios (up/c) and gfr (measured by iohexol blood-disappearance) for 260 children enrolled in the ckid cohort study were examined using loglinear regression of proteinuria and gfr, adjusted for age, sex, body surface area (bsa), and ckd cause. conclusions: in children with mild to moderate ckd, there was a log-linear relation between proteinuria and gfr. the prospective cohort design of ckid will assess the risk of gfr decline associated with level of proteinuria in children with glomerular vs non-gn causes of ckd. objective: to study the clinical characteristics of chronic renal failure (crf) in children. the clinical data of 81 children with crf in the last fifteen years were retrospectively analyzed. results: the first main causes of crf in children were still glomerular disease, and congenital deformities of urinary system and hereditary renal disorders were the second main causes. the initial age of children with crf which were caused by congenital deformities of urinary system and hereditary renal disorders were relatively younger than children with crf which were caused by glomerular disease. the symptoms of some crf in children were not typical. conclusions: glomerular disease were still the first main causes of crf in children. urine screening test, early renal function examination and kidney b-mode ultrasonic were attribute to early diagnosis and proper management of crf in children. the aim of the study was the evaluation of influence of clinical and biochemical parameters upon degree of impairment of cardiac function in dialysed children. methodology: 16 chronically dialysed (6 hd, 10 pd) children participated in the study (10 m, 6 f), aged 5-18,5 yrs (x=12,2±3.8 yrs). echocardiography examinations were carried out with a hp 5500 device. diastolic and systolic left ventricular (lv) dimension, ejection fraction (ef) and lv mass index (lvmi) were evaluated. mean values of estimated parameters in 6 months preceeding echo were calculated. results: on the basis of echo examinations 3 groups were singled out: a (n=3) of normal heart function, b (n=3) of impaired systolic and diastolic heart function and c (n=10) of normal systolic and impaired diastolic heart function. no differences between groups according age, bmi, dialysis and renal insufficiency duration were found. in group of children with severe cardiac lesion (b group) a higher lv mass (a vs b vs c: 74,7 vs 119,9 vs 73,5 g/m) and statistically significant lower ejection fraction (68,1 vs. 33,7 vs. 65,9%) were ascertained. these children were anuric (996 vs. 0 vs. 1112 ml/d), their systolic (102,1 vs. 118,4 vs. 117,9) and diastolic (64,4 vs. 84,8 vs. 77,9) blood pressure were significantly higher, so was the number or hipotensive medications (0,33 vs. 1,72 vs. 1,44) . bioimpedance analysis showed overhydration in group b (tbw% 63 vs 67 vs 62.5; ecw/icw 0.67 vs 0.76 vs 0.7). conclusions: the vast majority of chronically dialysed children demonstrate an impairment of cardiac function mainly of diastolic parameters. anemia, malnutrition, hypervolemia, anuria and hypertension stand for a significant risk factors of cardiac impairment in dialysed children. we report here the clinical findings and prognosis of 10 patients (5 girls, 5 boys) with infantile oxalosis diagnosed between june 1990 and december 2006 in order to remind this rare autosomal recessive metabolic disorder as the potential cause of acute renal failure in infancy. the mean age of the patients was 4.7 months (range: 2.5-10 months). all patients had the complaint of vomiting since birth, 3 had irritability, and 2 had convulsions. seven of them had parental consanguinity (70%) and family histories revealed urolithiasis in four patients and infantile deaths in one. two patients were cousins without any other family history. all patients presented with anemia (hemoglobin: 4.5-9.6 g/dl), renal failure (creatinine levels: 3.2-10.2 mg/dl) and metabolic acidosis. abdominal x-rays showed bilateral nephrocalcinosis, renal ultrasonographies revealed normal sized kidneys with diffuse and intensive hyperechogenity and loss of corticomedullary differentiation in all patients. crystal deposition was demonstrated in fundoscopic examination of 5 patients, bone marrow aspiration of 2 patients, and renal tissues of 8 patients that underwent renal biopsy. besides supportive therapy, peritoneal dialysis was performed on 7 patients, but only one patient accepted to continue the therapy. four patients died within few months and 4 were lost to follow-up, probably died. two patients have been followed up for 3 months one on continuous ambulatory peritoneal dialysis. in conclusion, this rare disease with fatal outcome should be remembered and investigated in infants with renal failure and bilateral nephrocalcinosis since early combined renal and liver transplantation may be life saving if it can be performed. acute renal failure following halothane anesthesia in young child? case report we present here the case of renal failure and hepatocellular lysis that developed 24 h after the exposure of halothane anesthesia during eye examination. previously a healthy 7 months old boy had a cardiac arrest, during the above mentioned diagnostic procedure, therefore cpr was applied which all happened in another hospital. after a few hours he was transferred to icu at our hospital, consciousness, hemodinamic stabile. in the following 24 hours he developed acute non-oliguric renal failure (maximal level of urea 29 mmol/l, creatinine 259 mmol/l), as well as hepatocellular lysis (alt 1879 u/l, ast 2157 u/l). plasmaferesis and continuous venovenous hemodiafiltration were immediately applied followed by conservative measures. the level of serum transaminase returned to the normal values within a week and the level of creatinine and urea within two weeks. fully recovered boy was released home. toxic effects of halothane anesthesia in children are reported in only few cases in the literature. objective of study: many cases of chronic aristolochic acid nephropathy (caan) in adults had been reported, but it had rarely been described in children. we reported 3 children with caan to investigate its clinical and pathological characteristics. methods: three cases were studied retrospectively and relevant literatures were reviewed. results: three children received traditional chinese herb medicine containing aristolochic acid 4 to 8 months for different basal diseases including chronic aggressive hepatitis b, secondary hydrocephalus and purpura nephritis and suffered from caan from 2 mouths to 6 years after they began receiving the chinese medication. the main manifestations were renal failure of various degree accompanied with proximal and distal tubular dysfunctions. two of them began with anemia and suffered from serious renal failure. the onset of another case was glucosuria with renal function impaired mildly, and she presented secondary fanconi's syndrome simultaneously. their pathological characteristics were diffuse paucicellular interstitial fibrosis and marked tubular atrophy with mild glomerular impairment. after withdrawal of the chinese herb medicine, renal failure in two patients attenuated progressively. the clinical features of caan in children are progressive renal failure and renal tubular disfunction. its pathological characteristics are diffuse paucicellular interstitial fibrosis and marked tubular atrophy. therefore, we emphasize the recognition of the nephrotoxicity of traditional chinese herb medicine and prevent caan from happening in children. objective of study: recombinant human erythropoietin (rhuepo) treatment may cause pure red cell aplasia (prca), but it had been rarely described in children. in order to acquaint ourselves with this disease, we reported a boy with chronic renal failure developed prca following rhuepo treatment. methods: one case was studied retrospectively and relevant literature was reviewed. results: a 10-year-old boy suffered from chronic renal failure combining with renal anemia and received rhuepo treatment. two weeks later, his hemoglobin (hb) increased from 57 to 90g/l, and maintained 90 to 94g/l for 6 weeks. subsequently his hb declined suddenly at a rate of 1g/l/day, despite rhuepo increased in dose. his reticulocyte count reduced to 0.4 10 9 /l without other cytopenias in peripheral blood. a series of laboratory examinations were performed, including bone marrow cell smear and culture to confirm his prca. because various of other factors such as parvovirus etc. induced prca was excluded, we considered the boy's prca was due to rhuepo treatment, although we didn't detect anti-epo antibodies for lacking of the reagent. the boy received erythrocyte suspension transfusion to correct his anemia and was waiting for renal transplant in the following period. conclusions: during the treatment of rhuepo, sudden and rapid reducing of hb might be a hint of rhuepo induced prca. the diagnosis should be based on the clinical presentation, the absence of red cell precursors in bone marrow and the detection of anti-epo antibodies. renal transplant and immunosuppressive therapy might be effective. m. zaniew, b. mroziñski, a. warzywoda, e. stefaniak, a. siwiñska, j. zachwieja among ambulatory blood pressure monitoring (abpm) parameters, pulse pressure (pp) provide an information on cardiovascular (cv) status. recently, a novel parameter i.e. ambulatory arterial stiffness index (aasi) has been proposed. aim: to investigate pp and aasi in relation to left ventricular (lv) geometry and carotid intima-media thickness (cimt) in children with chronic renal failure (crf). subjects: 24 children (6 f/18 m; median age: 15 yrs) with crf treated conservatively (n=8) and with dialysis (hd; n=7/pd; n=9). methods: we studied demographic data, echocardiography data [left ventricular mass (lvm), left ventricular mass index (lvmi), interventricular septum (ivs), left ventricular posterior wall (lvpw), left ventricular end diastolic diameter (lvedd), relative wall thickness (rwt)], cimt and abpm. from abpm, we calculated pp [difference between systolic blood pressure (sbp) and diastolic blood pressure (dbp)] and plotted dbp against sbp (regression slope). aasi was defined as 1 minus this regression slope. results: a positive correlation was found between aasi and pp (r=0.35, p<0.05). aasi was correlated to lvedd (r=0.39, p<0.05) and rwt (r=-0.45, p<0.01). pp was related to: weight (r=0.47, p<0.01), body surface area (r=0.41, p<0.05), body mass index (r=0.43, p<0.05), and lvm (r=0.58, p<0.001), lvmi (r=0.54, p<0.01), lvpw (r=0.35, p<0.05), lvedd (r=0.68, p<0.001), rwt (r=-0.35, p<0.05). neither aasi nor pp was associated to cimt. children with lv hypertrophy had higher pp than without this alteration (p<0.05). when data analyzed across quartiles of pp, children in the upper quartile showed higher lvm (inter-group comparisons p<0.001), lvmi (p<0.01), lvpw (p<0.01) and lvedd (p<0.001). conclusions: pp is a stronger predictor than aasi of lv geometry in children with crf.. acute renal failure in newborn period may be caused by prenatal, natal and postnatal factors. among them, obstructive lesions of the genital tract (e.g. imperforate hymen and vaginal atresia) are very rare. children with hydrometrocolpos due to distal vaginal atresia may present with severe obstructive uropathy and its consequences. hydrometrocolpos is the result of vaginal obstruction and can become an emergency in newborn period. acute renal failure associated with distal vaginal atresia appears to be rare, with only one report in the paediatric literature. here we report a 27-dayold infant with a hydrometrocolpos causing life-threatening renal failure. percutaneous drainage did result in dramatically improved clinical and laboratory findings of the patient. objectives of study: sympathetic overactivity is currently considered as an important mechanism of both development and progression of chronic renal failure. however, this statement was mostly based on the researches in which the participants were adult patients with terminal renal failure. little information is available on autonomic changes in pediatric patients with mild renal insufficiency. our aim was to determine whether there is sympathetic activation in children with chronic pyelonephritis in a stage of mild renal insufficiency. methods: 47 patients met the inclusive criteria were selected and assigned into two groups according to the creatinine clearance. group i had 26 patients with creatinine clearance between 60 and 90 ml/min/1.73 m 2 while group ii have 21 patients with normal creatinine clearance. baseline of age (from 10-17 years old), gender and diagnosis between the 2 groups are comparable. time domain analysis of heart rate variability in short-term recordings of 2 minutes was performed in both groups. as well vain questionnaire for assessment of autonomic state was performed in all participants with their parents help. results: the outcomes of heart rate variability analysis showed sympathetic overactivity of 73.0% patients in group i vs 30.8% in group ii, and the difference is statistically significant (t=2.497, p<0.05). significant difference was also found in results of vain questionnaire: 69.2% of patients in group i were estimated as "sympathetic", while only 33.3% in group ii (t=2.32, p<0.05). conclusions: based on the consistent findings of the two methods used in this study, we conclude that sympathetic overactivity may be found in children with even mild renal insufficiency, and it should be considered as an early event in the development of pediatric renal failure. the aim of this study was to describe the prevalence of myocardiopathy in pediatric patients with different stages of ckd (stages 2 to 5).methodology: inclusion criteria -gfr <75 ml/1.73 m 2 , ckd treatment >6 months, age <19 years old. echocardiograms were performed using standard techniques. left ventricular mass (lvm) was measured by two-dimensional directed m-mode echocardiography according to the american society of echocardiography criteria. lvm index was assessed and when >51g/m 2.7 it was considered severe hypertrophy. the relative wall thickness (rwt) was measured to assess the lv geometric pattern. age, high blood pressure (hbp), anemia, time and type of treatment were compared to the echocardiographic findings. results: we evaluated 53 patients, mean age 8 years old, 41% on pre-dialysis, 36% on hemodialysis (hd) and 23% on peritoneal dialysis (pd). patients on hd were evaluated in the interdyalitic period. twenty-seven patients (51%) had myocardiopathy, clvh in 14 (25%), elvh in 7 (14.5%) and cr in 6 (11%). severe hypertrophy ocurred in 20 pacients (37.7%). there was no significant difference in relation to age and high blood pressure. patients with clvh were on hd for longer time and had a lower hematocrite when compared to the patients without clvh (14±3 vs 4±2 months; p<0.01) and (33±1 vs 27±1; p<0.001) respectively. there was a significant correlation between hematocrite and left ventricular mass (r 2 =0.28). conclusion: we observed a high prevalence of myocardiopathy in this study population. the risk factors associated to clvh were anemia and time on hd. larger and prospective studies are needed to analyze the impact of myocardiopathy in the cardiovascular mortality in children as well as the results of interventions applied to correct these risk factors. [0] [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . urological problems such as vesicoureteral reflux (18, 5%), obstructive uropathy (10,7%) and neurogenic bladder (15,1%) were the leading underlying conditions causing cri with a total of 46,3%. while majority of the patients were having gfr levels less than 15 (33,5%) or between 25 to 50 ml/min/1,73 m 2 (31,2%), gfr level was between 50-75 in only 16.5%of the patients. patients with pth levels between 100-300 and more than 300 pg/ml comprised the majority (32,4 and 40% respectively). the gfr levels correlated positively with hemoglobin/hematocrit and calcium levels and negatively with phosphorus and pth levels (p<0.05). renal replacement therapies were initiated in 33,6% of the patients. the most striking result of this study is the predominance of vur or related urological problems that are relatively preventable as the underlying causes of cri in children. early diagnosis and appropriate medical or surgical management of these conditions should be emphasized together with achieving broader coverage of the pediatric cri population in terms of supportive treatment. twin-twin transfusion syndrome (ttts) complicates up to 15% of monochorionic twin pregnancies. shunting of blood between twins through common placental blood vessels can cause growth restriction, oligohydramnios and anaemia in the donor twin. renal impairment in the donor twin has been reported as a transient phenomenon with full recovery. we describe a case series of three children with chronic renal failure due to ttts. all 3 cases were monochorionic diamniotic twins. in cases 1 and 2, growth discrepancy was noted on prenatal scanning. in the case 3 anomaly scans were normal. gestation at birth ranged between 31 and 36 weeks. in all cases there was a significant birth weight discrepancy between twins. post-natal ultrasound appearances were variable: case 1 had normal scans within the first month but echogenic kidneys at 3 months; case 2 had initial increased echogenicity but features consistent with cortical necrosis at 4 weeks; case 3 had small kidneys with no cortico-medullary differentiation. all 3 children became dialysis dependent within the first year of life. age of commencement ranged from 4 days to 7 months. case 3 was successfully transplanted at 3 years. case 2 had an unsuccessful transplant at 2 years and remains on dialysis at 4 years. case 1 remains on dialysis at 2 years. cases 2 and 3 are now at mainstream school with only mild learning difficulties; case 1 has some gross motor delay but other wise normal development. with advances in neonatal intensive care and improved survival of low birth weight babies, this presentation may become increasingly common. even severe renal involvement can occur in the absence of other significant hypoperfusion injury. the management of survivors of ttts with established renal failure may present unique challenges and opportunities. introduction: endothelial heparan sulfate proteoglycans (hspg) play an important role in various biological processes in the renal glomeruli. it is involved in the inflammatory process by acting as a ligand for l-selectin. furthermore, leukocytes are able to interact with chemokines bound to hspg (examples are il-8, rantes and mcp-1). this will lead to activation of the integrins on leukocytes and result in more stable leukocyte-endothelial wall adhesion. in this work, we have studied the effect of a subtoxic dose of shiga-like toxin (stx) 1 and 2 on the hspg and the possible implications on the pathogenesis of the hemolytic uremic syndrome (hus). methods: to study this effect, primary human umbilical venous endothelial cells (huvec) and primary human glomerular microvascular endothelial cells (gmvec) were incubated for 24 hours with a subtoxic dose of stx1 or 2. then, cells were treated with the enzyme heparinase 3 (which cleaves off hspg). non-treated cells were used as controls. after treatment with the enzyme, the endothelial cells were exposed to flowing human leukocytes in a parallel plate flow chamber. the amount of adherent leukocytes was calculated. -: not treated with heparinase 3, +: treated with heparinase 3 conclusion: stx1 and 2 cause an upregulation of the amount of adhering leukocytes on both endothelial cell types. treatment of the endothelial cells with heparinase 3 decreases markedly this upregulation. the effect can not be detected without stx-incubation. this can be explained by a possible upregulation of hspg on endothelial cells by stx, resulting in a higher level of bound chemokines or an increased binding of released chemokines to hspg. this will lead to an increased adhesion of leukocytes and will induce a more severe inflammatory process in the renal endothelium. this effect will contribute to the devastating pathogenesis of the hus. c. soares, j. diniz, e. lima, g. oliveira, c. camargos, b. sousa, m. almeida, e. oliveira objective of thestudy: the purpose of this study was to identify clinical, nutritional and laboratory factors associated with the rate of progression of chronic kidney disease (ckd) among the children and adolescents admitted to a pre-dialysis interdisciplinary program. methods: one hundred eight children and adolescents aged 2 months to 19 years with ckd in conservative management were prospectively followed up from 1990 to 2006. renal survival was analyzed by the kaplan-meier method and cox's regression model. a multivariate model was developed from the admission until occurrence of ckd stage 5 (glomerular filtration rate <15 ml/min). the following data were obtained at admission: gender, race, age at admission, weight z score, renal primary disease, blood pressure, and laboratory data (serum creatinine, serum urea, glomerular filtration rate, 24 hr urinary protein excretion, hematuria). results: median follow-up time was 5.3 years (iq range, 2.6-7.8) and 58 patients (54%) progressed to ckd stage 5. in the univariate analysis the following variables were significantly associated with the event: non-white race (p=0.04), age >8 years (p=0.01), ckd stage at baseline (p=0.01), renal primary disease (p<0.001), severe proteinuria (p<0.001). after adjustment, 3 variables remained as independent predictors of ckd stage 5: severe proteinuria (rr=2.1, 95% ci=1.4±3.1), glomerular disease as renal primary disease (rr=1.5, 95% ci=1.2±1.0), and ckd stage 4 at baseline (rr=2.3, 95% ci, 1.4±3.7). conclusion: the combination of three factors -severe proteinuria, glomerular disease, and ckd stage 4 at admission -was an independent indicator of adverse outcome in children and adolescents in a pre-dialysis interdisciplinary program. background: allogenic hematopoetic stem cell transplantation (allo-hsct) has gained world wide acceptance as a treatment for various diseases. renal dysfunction is one of the major complications that influences transplant related mortality (trm) rates following hsct. in this prospective study, we aimed to investigate the effect of allo-hsct on renal function in children. methods: renal ultrasonography, dmsa scintigraphy, analysis of renal and tubular function tests were performed before, shortly and 1-year after hsct. acute renal toxicity (art) was classified according to bearman criteria. grade 1=increase in creatinine up to twice the baseline; grade 2=increase in creatinine above twice baseline; grade 3=increase in creatinine above twice baseline and need for dialysis. chronic renal insufficiency (cri) was defined as gfr<70 ml/min/1.73 m 2 and failure (crf) as need for dialysis. results: between april 1999 and june 2006, 57 children (median age: 10.0 years) underwent 58 allo-hsct because of hematologic disorders (malignancy, 19; hemoglobinopathy, 28; aplastic anemia, 10). all patients except one received nontbi conditioning regimen. six patients (10.3%) were died because of trm but none of these patients had primary art. during the first 100 days, 10 patients (17.2%) had grade 2 art (csa nephrotoxicity in 7, vod in 3 patients). grade 3 art was defined in 5 patients (8.6%) (vod in 3, sepsis in 1, csa nephrotoxicity in 1 patient). eleven patients had structural renal abnormalities before hsct, 3 of them persisted and 3 new patients had pathologic results one year after hsct. in long term period, 5 (8.6%) of 37 evaluable children had cri and no patient had crf requiring dialysis. conclusion: renal dysfunction was found to be a frequent complication after allo-hsct in children. therefore, renal functions should be followed carefully in these patients. .26 years and at the end of follow-up, and compared between the three groups. there were no significant differences between groups in so far as gender, underlying disease, age at diagnosis, proteinuria, hypertension, hyperparathyroidism, use of ace inhibitors or renal size. erithropoietin use was significantly higher (p<0.001) in group 1. gfr improved in all three groups during their first two years of life. the cut-off point on the roc curve indicating worse gfr long term evolution was 50 ml/min/1.73 m 2 at two years of life (sensitivity 85%, specificity 88%). g. zilleruelo, am. onder, j. chandar, o. nwobi, c. abitbol background: catheter-related bacteremia (crb) is a common complication of tunneled-cuffed hemodialysis catheters. our objective was to investigate the effectiveness of tissue-plasminogen activator-tobramycin locks (tpa-abl) in preventing crb in pediatric patients. methods: a retrospective analysis of 52 pediatric hemodialysis patients was performed. patients with >10 crb/1000 catheter days (cd) were designated as high risk. those with <10 crb/1000 cd were of average risk. three eras of 6 consecutive months were studied. in era 1, high risk patients were detected. during era 2, the high-risk group was placed on 3 times weekly prophylactic tpa-abl. in era 3 the high-risk group was given once weekly tpa-abl. results: there was a significant decrease in the rate of crb with prophylactic tpa-abl which was more pronounced when given three times/week. conclusions: the use of prophylactic tpa-abl 3 times weekly significantly reduces the rate of crb in patients at high risk. prophylaxis once weekly may be less effective than thrice a week. l. was born after a normal pregnancy without a special personal or familial history, was seen at the age of 4 years, after a 6 days long ordinary oro-pharyngeal viral infection treated by symptomatic treatments. he presented with inflammation of the left cheek with slight fever (38°) and a sole purpuric lesion of the left leg (1cm) and some petechiae on the thorax. blood count showed haemolytic anemia (haemoglobin=10 g/dl, schizocyte=2%, increased ldh), 23000 white cells (90% neutrophils) and 123000 platelets. no germs were found in hemoculture, lumbar punction, stools or urines cultures. creatininemia was 95 μmol/l. in the following hours, despite immediate antibiotics administration and without any shock sign, several purpuric extensive necrotic lesions appeared, renal insufficiency increased (creatininemia=175 μmol/l), platelets count decreased to 46000 and markers of diffuse intravascular coagulation dramatically increased. in the following days, proteinuria, macroscopic hematuria and hypertension appeared. after 3 days on anticoagulation therapy, renal function remained stable while anemia, thrombopenia and coagulation disorders persisted. coagulation factor tests demonstrated a heterozygote deficiency of factor v leiden (also found in the father) and an acquired protein s deficiency secondary to streptococcal infection. after protein s infusion and plasma exchanges, his state improved and necrotic lesions ceased. this initial hematologic and renal presentation could have suggest a hus but the large purpuric lesions remain unusual in such pathology. a in children on chronic peritoneal dialysis malnutrition is being diagnosed with an objective combined nutritional score (abn score) based on anthropometry and bioimpedance analysis indices (nephrol dial transpl 2006; 21: 1946-51) . aim of this study was to investigate the prevalence of malnutrition and the main factors associated with it in children with ckd 2-4. we planned a cross-sectional study of 77 children with ckd 2-4, mean age 8.9±5.8 years, who underwent controls in our institution between september 1 and december 31, 2006. the data of abn score, age, primary renal disease, standard blood and urinary tests, and estimated gfr (schwartz formula) were collected.the prevalence of malnutrition (abn score <10.33) in the whole population was 23%. the abn score was positively correlated with age, height sds, serum hemoglobin, total protein and albumin (p value from <0.0001 to <0.05), while a negative correlation was found with serum phosphate and proteinuria (p<0.005). patients with steroid-resistant nephrotic syndrome had lower abn score values than those with other primary renal diseases (9.61±0.85 vs 11.67±1.93; p<0.05). the percentage of children with malnutrition in the different stages of ckd increased in parallel with the decrease of gfr, from 10% in the ckd 2 group to 29.2% in the ckd 4 group. in conclusion, the prevalence of malnutrition in children with ckd 2-4 is not negligible. low hemoglobin, total protein and albumin levels and high serum phosphate and urinary protein excretion, particularly in small children with growth impairment, strongly suggest the need for an in depth nutritional assessment, in order to diagnose malnutrition and treat it accordingly. e.c. developed atypical hus at 6 months of age. a heterozygous factor h gene mutation was found. despite plasma-exchange treatment numerous relapses led the child to ckd stage 4: creatinine clearance (ccr) 15.02 ml/min/1.73 sqm according to schwartz formula. at 4 yrs we started a programme of fresh frozen plasma (ffp) infusions, 10-15 ml/kg bw. the child was poliuric and hypertensive, notwithstanding the treatment with ramipril, amlodipine, clonidine and furosemide. in the first 6 months he received ffp every 10 days. mean ccr during this period was 16.8±1.7 ml/min/1.73 sqm. haptoglobin (hap) was still <20 mg/dl in 10/15 tests (66.7%), ldh was increased (543.9±87.5 u/l), hemoglobin and platelet count were always in the normal range. the treatment schedule was then changed to ffp every 7 days for the next 4 months. during this period, ccr was significantly higher (23.8±2.4; p<0.0001) and ldh significantly lower than in the first 6 months (445.5±31.7; p<0.005); haptoglobin was always >20 mg/dl. no differences between the two periods were found for hemoglobin, platelet count, proteinuria, microalbuminuria and blood pressure values. both in the first and the second period, ccr was negatively correlated with ldh (r 2 0.40, p<0.0005) and with the bioimpedance analysis (bia) measure of resistance, which is an index of body hydration. in conclusion: 1. the only signs of disease activity in atypical hus can be minor abnormalities in laboratory tests, such as increased ldh and decreased haptoglobin levels; 2. ffp infusions are useful in maintaining hus remission and preserving kidney function, provided that the treatment schedule is optimized; 3. bia is useful in monitoring hydration status of children with ckd, especially those with poliuria and under ace-inhibitors, as it allows for the correct interpretation of serum cr values. analgesic-antipyretic agents are commonly used medications worldwide for the treatment of pain and fever in children. acute renal failure is commonly seen in adults after treatment with analgesicantipyretic agents. this complication has rarely been reported in children. two patients, ages 12 and 16 years were admitted with a diagnosis of acute, non-oliguric renal failure. one had symptoms of upper respiratory tract infection, and the other had been suffering from vomiting and abdominal pain. appropriate evaluations including detailed history especially the history of drug ingestion, physical examination, and measurement of serum creatinine concentrations were performed in the emergency department. both patient had ingested analgesic-antipyretic agents (mefenamic acid, and paracetamol) before the onset of acute renal failure. none of the patients had previous history of renal disease or concomitant treatment with other drugs. none had oliguria or anuria, dehydration, abnormal serum electrolyte concentrations, or evidence of glomerulonephritis. microscopic hematuria and leukocyturia were found in one patient. serum creatinine was 1.1 and 2.29 mg/dl at presentation. both of them recovered completely within a week. we emphasize that clinicians must be aware of renal toxicity of analgesic-antipyretic drugs with low doses. with the increasing use of over-the-counter analgesic-antipyretic agents, this association may become more prevalent. cardiovascular disease is a main cause of morbidity and mortallty in patients with chronic kidney disease (ckd). the pathophysiology of cardiovascular disease (cvd) in ckd remains uncertain but nowadays sympathetic hyperactivity is recognized as an important mechanism involved. this observational and transversal study of 40 patients from five to 21 years old, submitted to dialysis or at least four months after kidney transplantation (ktx), without signs of transplantations rejections, with definite ckd and creatinine clearance of 50 ml/min or less. the subject (median age=14 years; 62.5% female) were classified accordingly with treatment modality: conservative (n=7), peritoneal dialysis (capd) (n=5), hemodyalisis (n=13) and renal transplantation (n=15) submitted to 123l-metaiodobenzilguanidine (123l-mibg) planar and tomography scintigraphy and heart rate variability (hrv). comparisons among groups were made using anova and the association between variables was assessed using spearman's correlation coefficients and bonferroni correction was used during multiple comparisons testes. a p value <0.05 was considered significant. hemodialysis patients presented increased cardiac washout (p=0.002), heterogeneous pattern of 123l-mibg distribution (p=0.036) and lower values of low frequency component of hrv (p=0.040). capd subjects had reduced lung washout (p=0.030). the cardiac washout had positive correlation with pth and negatives correlation with creatinine clearance. there was a significant negatives association between the rr interval in low frequency (lf) and cardiac washout. the uremic cardiac disautonomia might be characterized by decreased low frequency component of hrv and increased 123l-mibg washout and heterogeneous distribution pattern by left ventricular walls; these abnormalities subsided after ktx. d+hus is the main cause of acute renal failure in children. extrarenal manifestations are associated in more than 30% of the cases. hus causes toxin mediated endothelial cell damage, resulting in thrombotic microangiopathy and intraluminal thrombosis of small vessels, with subsequent tissue ischemia and necrosis of involved organs. a 3-year-old boy has been admitted for d+hus associated with escherichia coli o145 diarrhea. he presented with renal failure and hypertension requiring hemodialysis for 28 days, hemolytic anemia (5g/dl, schizocytes 6%) requiring 7 blood transfusions, severe thrombopenia (10 g/l) and hyperleucocytosis (39600/mm 3 ). severe hemorragic colitis with duodenitis required prolonged parenteral nutrition. at 6 days after onset, the child presented with confusion, slurred speech followed by loss of consciousness associated with major hyperglycemia (107 mmol/l) and elevated corrected natremia (155 mmol/l) without ketosis requiring transfer in intensive care unit (icu). continuous hemofiltration associated with insulin therapy (0.05 ui/kg) was then established with slow decrease of natremia and serum glucose within 72 hours. neurologic condition rapidly improved. serum amylase and lipase were normal. insulinemia at the same time as the highest hyperglycemia was low (1,7 ui/ml), and search for human insulin, islet cell and glutamic acid decarboxylase antibodies were negative. insulin therapy could be discontinued within 15 days. at the last follow-up, 6 months later, neurologic examination, serum glucose and glomerular filtration rate were normal. in conclusion: 1/ hyperglycemic hyperosmolar coma is a severe complication yet never reported in d+hus; 2/ continuous hemofiltration with constant monitoring of biologic parameters could avoid permanent lesions due to rapid correction of this major metabolic unbalance. chronic renal failure (crf) can interfere with the regulation and time dependent secretion of multiple hormones. adrenocortical function in children with crf is examined in a few studies with a limited number of patients, and the results are controversial. in this study our aim is to evaluate adrenocortical function in basal and stimulated conditions, and to determine the relationship with the glomerular filtration rate (gfr), etiology and duration of crf in a larger group of patients. sixty-one patients with crf (28 f, 33 m; mean age 12.6±3.2 years) were studied. the patients were grouped according to etiology [structural abnormalities (n: 33), glomerulonephritis (n: 11), others (n: 11)], duration of crf [0-36 (n: 25) , 36-72 (n: 22) and >72 months (14)] and gfr [ 25-75 (n: 24), 10-25 (n: 19), <10 ml/min/1.73 m 2 (n: 18)]. cortisol levels were measured at 08: 00 a.m. (basal cortisol) and 11: 00 p.m. low dose acth test (0.5 micg/m 2 synacthen iv) was performed. delta cortisol was calculated as peak cortisol minus basal cortisol during the acth test. diurnal rhythm is accepted to be preserved if 08: 00 a.m. cortisol/11:00 p.m. cortisol is greater than two. basal cortisol levels and peak cortisol response to low dose acth is similar in all groups. median levels of delta cortisol found to be higher in the gfr<10 ml/min/1.73 m 2 group; p=0.06. diurnal rhythm seems to be more preserved in the gfr 25-75 ml/min/1.73 m 2 group (%68) compared to gfr<10 group (%44); p=0.612. no correlation was found between the basal, peak and delta cortisol, gfr and duration of crf. our preliminary results have shown that there is no difference in the basal and peak cortisol levels between all groups. this is the first study in children showing that adrenocortical function is preserved in groups with gfr levels between 10-75 ml/min/1.73 m 2 . objective: this study was we evaluated bk virus and jc virus in urinary after renal transplantation. methods: because these polyoma viruses are excreted in urine, these 12 patients (7 females and 6 males, 16.1±6.7 years old) was analyzed by polymerase chain reaction. all patients were living donor renal transplantation from a parent. results: two patients have detected bk virus in urine. as the type of immunosuppressive treatment, one had tacrolimus and mycophenolate mofetil, and one more had methylprednisolone and tacrolimus. seventeen percent of the patients had quantifiable bk virus-dna in urine. thirty-three percent of the patients had quantifiable jc virus-dna in urine. there was non significant relationship between these polyoma viruses in urine and the type of immunosuppressive treatment. no patients developed interstitial nephritis during the study. conclusions: the activation of bk virus and jc virus does not seem to be related to the type of immunosuppressive treatment. the pathogenetic role of polyoma virus infection in renal transplantation recipients further researches are needed. background: urinary tract infection (uti) due to neurogenic bladder, secondary to large spina bifida as myelomeningocele, is well known, but the association of small occult spina bifida (sbo), having normal physical examination, with that of infection has not been reported. we studied the frequency of spina bifida occulta in children with urinary tract infection. method: the kub of voiding cystouretrography in 104 patients (72 f, 31 m) with average of age 6 year (±3.5 sd), who referred to radiology department of ali asgar children hospital for uti, were observed for sbo. all patients had normal physical examination. chi2 were used to find the frequency of the level of sbo and the differences respectively. p<0.05 was considered significant. result: 80 out of 104 patients had sbo. the order of frequency of the level of sbo was s1 (51%), l5 (14,4%), s1s2 (6,7%), l5s1 (4,8%), and l5s1s2 (1%). 22 patients had vesicoureteral reflux (bilateral in 8 cases, 10 at left and 3 at right side). the severity of vur was 76% grade (g) ii, 14,35% g iii, 9.1% g iv, and 4.8% g i. spina bifida occulta was detected in 19 out of 23 patients with vur. this difference was not statistically significant (p=0.43): conclusion: the high frequency of lumbosacral sbo in the patients with urinary complaint may imply some lower urinary tract dysfunction in these patients although we need a control study in normal children. we evaluated interleukin-6 and interleukin-8 levels in the urine of 33 children with renal scarring, with vesicoureteral reflux (23/33, group a 1 ) and without vesicoureteral reflux (10/33, group a 2 ) and in the urine of 7 healthy children (group b). none of the children had urinary tract infection for the last ten months. interleukin-6 and interleukin-8 were determined using a commercially available human enzyme-linked immunosorbent assay kit. results: urinary interleukin-8 levels were below the lower detection limit in all samples. interleukin-6 levels were detectable in all urine samples of children with renal scarring and below the detection limits in the urine samples of healthy children. there were no statistically significant differences between urinary interleukin-6 levels in children with and without vesicoureteral reflux. there is a relationship between the grade of renal scars, the time past from their development and the urinary levels of il-6. the introduction of mycophenolate mophetil (mmf) was an important advance in immunosuppressive therapy but its use is associated with gastrointestinal intolerability (gi) requiring dose reductions or drug interruptions. enteric coated mycophenolate sodium (ec-msp) was designed to improve mycophenolic acid (mpa)-related gi by delaying the release of mpa until reaching the small intestine. at present, its immunosuppressive activity in pediatric renal transplant with gi has not been clarified. we studied trough levels of active metabolite mpa (c 12 ), changes in kidney function, mixed lymphocyte culture, cytotoxic antibodies as well as cytotoxic response before and after 3 months of the conversion from mmf to ec-mps in 8 renal transplant recipients with gi. in the immunosuppressive protocol used: mmf, tacrolimus and corticosteroid, mmf 444±46 mg/m 2 /day was replaced by ec-mpa 318±34 mg/m 2 /day. after 3 months of treatment with ec-mpa, the incidence of gi decrease to 12.5%. the levels of mpa were about 50% higher on ec-mps (6.9±1.1 ug/ml) compared to mmf administration (4.2±0.9 ug/ml). serum creatinine, creatinine clearance and urinary protein excretion did not change (1.3±0.3 to 1.4±0.3 mg/dl, 71.7±10.3 to 70.2±10.4 ml/min/1.73 m 2 and 0.08±0.06 to 0.12±0.06 gr/24 hr, respectively). also, during ec-mpa, proliferative response and cytotoxic antibodies showed no significant change. the release of il-10 was striking augmented with mmf or ec-mps therapy, but interferon-γ and tnf were low under both treatments. our data indicate that conversion from mmf to ec-mps leads to an improvement in gi without altering key elements of immunosuppression. however, the monitoring of mpa before and after conversion should be appropriated to the optimization of therapy efficacy. re. munarriz, ju. arakaki, ce. munarriz pediatric clinic, pediatric nephrology, buenos aires, peru objective: to describe the results of a program of chronic ambulatory peritoneal dialysis (capd) in children in peru by means of conventional indicators. methods: 61 children (52, 45% male) were included in a longitudinal descriptive study. the average age was 10, 4±4,02 years (rank of 1, [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . 42% were from lima (the capital city) and 58% came from other cities of the country. primary glomerulonephritis (46, 1%) was the main cause of the renal insufficiency. we evaluated the program from 2001 to 2006. results: the average weekly kt/v was 2,8±1,13. the average dose of erythropoietin (epo) was 98 ui/kg/week. 60% of the patients had average annual albumin of more than 3,5 gr./dl. the average annual protein catabolism index (icp) was 1,0±0,37 gr/kg/d. the weight/age, height/age, weight/height z scores at the beginning and at the end were 2.1/1.3, -2.095/1.3, -2.9/2, 4 -3.4/2.2, 1.9/7.0-0.8/2.1, respectively. the average hematocrit was 26.5±7.6%. the rate of peritonitis adjusted for the period was 0, 62 episodes/patients-year (1 episode every 19 months) and the mortality for the same period were 14%. conclusions: the indicators evaluated in our patients are according to the results of the international literature. is repeated urine culture essential after antibiotic therapy in uti in thai children? background: in 1999, the american academy of pediatrics recommended for uti treatment, that urine culture be repeated only in children <2 years with fever >48 hours, since the chance of a positive urine culture in other children is very low. objective: to evaluate the cost-effectiveness of repeated urine culture after antibiotic therapy in childhood uti. patients and methods: a retrospective review of the records of children diagnosed with uti in songklanagarind hospital from jan 1-dec 31, 2004. results: there were 479 patients total, of which 30 were excluded due to no repeated urine culture. 449 patients with 533 uti episodes were analyzed (245 boys and 204 girls). 241 (53.7%), 71 (15.8%) and 137 (30.5%) patients aged <1, 1-2 and >2 years respectively. 49 (9.2%) had a repeated urine culture with significant growth. multivariate analysis showed that age <1 year, etiologic agents psuedomonas aeroginosa or enterococci spp., fever >72 hours and kub anomalies were the most significant risk factors for positive repeated culture, while vur and recurrent uti episodes were not significant risk factors. if we consider that a child who has at least one of the above risk factors should have a repeat urine culture, then 351 cases (65.9%) will require repeating urine culture and 182x3.7=$us 672.4 will be saved and 5 (10.2%) positive repeated urine culture will be missed. conclusion: our study in a group of thai children indicates that repeated urine culture after antibiotic therapy is still recommended. our aim was to find out if there are any signs of renal scarring and reduced renal function without recurrent uti in patients with obstructive pyelonephritis. there were investigated 18 children (4-12 y) with 1-4 years passed after diagnosis of obstructive pyelonephritis. these patients were investigated during 2 years long period without uti. all the children had a treatment with nitrofurantoin during season viral infections. we investigated excretion with urine of the collagen product (hydroxyproline) and activity of lisosomal enzymes (b n-acetilglutamase, elastase, pseudocholiesterase). as a control group, 20 healthy children of the same age were investigated. our result demonstrates the decrease of the level in urine of collagen product and lisosomal enzymes and normalization of tubular and glomerular functions during 2 years remission of pyelonephritis. prevention of recurrent uti and maintenance of the remission of pyelonephritis leads to the decrease of sclerosing processes on kidney. regular dialysis induces insecurity and special psychological problems associated with staff-, machine-and artificial material-dependence. the more severe the child's physical problems are the more probable is developing of emotional disorders, a sense of loneliness and an exaggerated dependence on the parents and staff. a psychological and social study of 19 children on regular dialysis was performed. there were 8 girls and 11 boys, aged 5-18 (mean: 11) ys. two children (10%) are exclusively treated with haemodialysis, 11 (58%) are on peritoneal dialysis and in 6 (32%) both methods interchanged. somatic concomitant disorders are present in 5 (26%) of children. among psychological disorders, an inclination to unsociability, autoaggression or aggression towards the immediate environment has developed in 4 children (21%), 6 (32%) do not like talking about disease while 9 (47%) are communicative and sociable. psychosocial characteristics showed: emotional difficulties (anxiety, mild depression) in 32%, feeling of being physically different from peers (short stature, less physical ability) in 42%, lowered self-esteem and social isolation as a result of school absenteeism in 26%. overprotection of parents was presented in 58%. different psychological changes were present in some children. of the 19 children 17 are of school age: 3 (18%) attend special schools and the remaining 14 follow regular education programs. with the help from their teachers, children on dialysis can master regular school programs, in spite of the time spent on dialysis. a good and continuing co-operation of dialysis staff and sick children and their parents as well as a more intensive co-operation with psychologists and teachers are necessary to reduce psychological disorders and promote a better adaptation to the life of their healthy coevals. we report a 15-year-old girl with syndrome frasier who developed b cell lymphoma within nine month after live related kidney transplantation. in induction therapy we applied atg up to 5 days, mycophenolate mofetil and cyclosporine. routine abdominal ultrasound revealed multifocal hypoechogenic changes in liver and spleen. computed tomography showed diffuse focuses of changed liver tissue in length up to 4.5 cm, precontrast density of 30-40 hu and postcontrast of 50-65 hu. in spleen there were three similar changes (up to 2.3 cm). after surgical biopsy of liver, patohistological examination confirmed diffuse large cell b lymphoma (cd 20 positive, moderate risk). pcr ebv was positive. we immediately started with ganciclovir intra venously and decreased cyclosporin and mmf (within two weeks) up to completely exclusion from therapy. in the same time we increased prednisolon on 15 mg daily. after 7 weeks from beginning ganciclovir she treated with rituximab, one dose of 500 mg every week (five weeks). repeated abdominal ultrasound and two controls computed tomography showed markedly regression of tumorous lesions. after 6.5 months of last rituximab doses scan showed normal spleen and liver findings. all lesions were resolved. in two occasions she developed severe leucopenia without any infection complication. she still has got moderate lymphopenia due to continual ganciclovir therapy. during this period (11 months) of illness course she treated with ganciclovir intravenously due to repeated ebv reactivation (positive pcr). despite enormous reduction in immunosupressive therapy renal function remain stabile without episodes of acute rejection. 5-20.5 years) . the most of them, 92% (23 patients) received graft from live related donor. 84% of patients had preoperative dialysis and 14% were preemptive. the mean waiting period for transplantation was 30 months (range 0 to 120 months). congenital anomalies of kidney and urinary tract were the most common underlying diseases (13 patients), then nephrotyc syndrome (4 patients), hereditary nephritis (3 patients), polycystic kidney disease (2 patients) and others. daclizumab was the most commonly used as immunosuppressive induction agent and the maintenance immunosuppressive therapy consisted of azathioprine or mycophenolate mofetil (mmf), cyclosporine or tacrolimus and prednisone. two patients had immediate postoperative surgical complication and graft loss due to thrombosis of a. renalis and donor tubular necrosis respectively. three patients had delayed graft function; two of them underwent cadaveric transplantation. one patient had recurrence of primary kidney disease only few days after transplantation and graft loss after one year. other patient lost the graft after 2 years due to noncompliance and chronic cellular rejection. after one year of follow-up graft survival rate is 86% and patient survival rate 100%. at the end of follow-up (mean range 29.21 months), 18 patients had normal, 3 patients slightly decreased renal graft function. catch-up growth was seen during the first year after transplantation from mean height sds of -1.65 to 1.25. the mean goal of our further intention is improvement of cadaver renal transplantation. j. lee, y. shim, s. lee objectives; although the variable risk factors of urinary tract infection (uti) in children such as virulence factors of the pathogenic bacteria and vesicoureteral reflux are already well known, the role of normal flora clononizing the intestinal tract and genitourinary tract is not fully understood. the change in colony forming units (cfu) of lactobacillus in chidren with uti is primarily investigated to explore the role of lactobacillus, one of the human normal flora, in development of uti. methods; lactobacillus was cultured from stool, urine, and periurethral or vaginal discharge of febrile infants with uti. those with confirmed uti by the suprapubic urine culture were classified to uti group (n=-60), and those with negative urine culture and confirmed viral illness were classified to control group (n=31). lactobacillus was anaerobically cultured in dico rogosa sl agar (becton, dickinson and company, usa) for 48 hours at 37°c. results: the cfu of lactobacillus in stool was correlated with those in periurethra, vagina and urine (p<0.05). the cfu of lactobacillus in stool was significantly lower in uti group than in control group (19,286±30,920 vs 16,969,129±89,717,956, p<0.05) . the cfu of lactobacillus in periurethra or vagina was significantly lower in uti group than in control group (2,788±5,365 vs 43,038±179,089, p<0.05). the cfu of lactobacillus in urine was significantly lower in uti group than in control group (173±469 vs 2,087±5,681, p<0.05). the cfu was distributed mostly at low level in uti group, which was significantly different from that in control group (p<0.05) conclusions; the cfu of lactobacillus in stool, periurethra, vagina and urine is low in infants with uti. it is suggested that lactobacillus has a role in the development of uti. pediatric small bowel transplantations are associated with pronounced electrolyte inbalances in the posttransplant period. we investigated the sources of possible electrolyte losses. our hypothesis was that electrolyte inbalances after intestinal transplantation might be augmented by fk 506 (tacrolimus) mediated renal toxicity rather than short bowel syndrome alone. we retrospectively reviewed eleven living-related small bowel transplantations between october 2002 and december 2006. the data collected included frequent serum and urine electrolyte profiles, renal function parameters, enterostoma electrolytes, and fk 506 levels in the postoperative period up until either discharge or graft loss. we analyzed pearson's correlations between fk 506 levels, serum electrolytes, renal function parameters, and also fractional excretions (fe). in order to investigate possible delayed nephrotoxic effects of fk 506, we correlated all values of the same day as well as with fk 506 levels of 24, 48, and 72 hours earlier. furthermore, we analyzed our data stratified by fk 506 dosing ranges. our results show decreased gfr and prominent increase of renal sodium, phosphorus, and magnesium losses along with rising fk 506 levels, suggesting this pathway as a major contributor to their imbalances. furthermore, fk 506 levels were associated with serum calcium and phosphorus decline, though urinary calcium excretion was not significantly changed. signs of renal toxicity are apparent within the first 24 hours of fk 506 challenge. our data suggest that fk 506 mediated nephrotoxiticy is a significant contributor to electrolyte imbalances observed after small bowel transplantation. objectives of study: urinary tract infections (uti) are common in children and c-reactive protein (crp) in serum is often used to evaluate the severity of the renal inflammation. recently it was demonstrated that crp can be produced locally in the kidney. we therefore measured the crp concentration in both serum (s-crp) and urine (u-crp) from children with uti to evaluate the extent of local production and the usefulness of measuring u-crp for diagnosis of inflammatory kidney disease. methods: 56 children (31 boys) with uti were studied (median age 0.8 years, range 12 days-5 years). 43 children had fever 38.5 °c or more. the uti was caused by e.coli in 50 patients. all children were examined within a few days of diagnosis by dmsa scan for evaluation of renal function. as controls were used 14 children with respiratory infection (pneumonia in most) and elevated s-crp in whom uti was ruled out by negative urine culture. u-crp was measured in a commercial hscrp elisa. normal value was <6 mg/l results: in the uti patients, the median s-crp was 126 mg/l (range 0-440) and u-crp 144 mg/l (range 0-937). there was a significant correlation between the s-crp and u-crp concentrations (<0.001). dmsa scans were abnormal in 37 (66%) uti patients. the proportion of abnormal scans increased significantly with the crp concentration in both serum (<0.01) and urine (<0.05). all control patients had increased s-crp concentrations (median 157 mg/l, range 45-243) but the median u-crp was 2.5 mg/l (range 0-73). the u-crp in the patients with uti was significantly higher than in the controls with other infections (<0.05). conclusions: our data strengthens the concept that crp can be produced locally in the kidney during uti. the usefulness of measuring u-crp to evaluate inflammatory kidney disease needs to be tested further. s. paunova, a. kucherenko, i. smirnov, g. serova, i. donin, l. revenkova, n. goltsova in order to reveal the role of cytokines in renal tissue damage in infants with urinary tract infection (uti) 42 patients aged from 0 to 6 months were examined. in all of them inflammatory markers (esr, crp, leukocyte count), urine concentration of tumor necrosis factor-α (utnf-α) and transforming growth factor-β (utgf-β) standardized to urinary creatinine concentrations were evaluated. two groups of patients were determined: 1) with uti and normal urodynamics (n=33), 2) with uti and urodynamic disorders (vur, hydronephrosis) (n=9). 12 healthy infants were used as controls. as a result, all the patients demonstrated significantly elevated utnf-α, utgf-β creatinine ratio in comparison with controls (p<0,05) with no difference between groups. but 16 children with normal urodynamics (from the 1 st group) presented with severe uti (1 st -a) showed urine tnf-α tgf-β creatinine ratio in 1,5 times higher than other patients of 1 st gr and close to 2 nd gr. data (1 st gr.-utnf-α/ucr=9,32±1,82, utgf-β/ucr=1231,65±133,36, 1 st gr.-utnf-α/ucr=6,89±0,98, utgf-β/ucr=782,7±87,77; 2 nd gr -utnf-α/ucr=12,58±4,25, utgf-β/ucr=1130,58±280,45, p1,2-1<0,05) . to conclude, tnf-α and tgf-β are responsive for renal tissue impairement in infants with uti. elevated tnf-α tgf-β creatinine ratio in a part of infants with normal urodynamics suggests that renal damage begins early in infection mostly due to inflammation. one may suspect a predisposition of that subgroup of patients to fibrogenic process and subsequent scar formation. prompt diagnosis and localization of pyelonephritis are of great importance in treatment and prognosis of the patients. the urinary excretion of enzymes, in particular n-acetyl-beta-dglucosaminidase (nag), is considered a relatively simple and non-invasive method in the detection of renal tubular function under various conditions such as pyelonephritis. this study was performed to determine the diagnostic value of urinary nag in acute pyelonephritis and to compare it with other indices traditionally used for this purpose in children. this is a quasi experimental study conducted from april 2005 to may 2006 on children with pyelonephritis. diagnosis of pyelonephritis has been based on standard criteria. seventy-two patients between 1 month and 15 years were recruited. fresh random urine samples were obtained on the admission time and at 48 th hour of treatment and were tested for nag and creatinine. there was a significant difference in pre and post-treatment urinary nag/creat ratio (p<0.000) and the sensitivity and specificity of urinary nag in diagnosis of pyelonephritis were 72% and 78% respectively. there was a significant correlation between urinary nag level and kidney ultrasonography results. patients whose ultrasonography showed hydronephrosis, had the highest level of urinary nag and patients who showed urinary stone in their ultrasonography had the lowest level of urinary nag. in addition, there was a reverse correlation between urine culture results and urinary nag level; patients who had negative urine culture had higher level of urinary nag in comparison with patients with positive urine culture. we conclude that urinary nag is elevated in children with pyelonephritis especially in the presence of urinary tract abnormality and the absence of renal stone. type-1 hyperoxaluria (ph-1) is an autosomal recessive disorder caused by the impaired activity of the hepatic peroxisomal alanine-glyoxilate aminotransferase. the disease leads to end stage renal disease (esrd) and combined liver-kidney transplantation (clkt) is required. we report 3 cases diagnosed with ph-1 who received clkt. case-1: she had attacks of dark urine without any pain and renal stones were determined on sonography when she was 2.5 years old. she was diagnosed with ph-1 and received peritoneal dialysis (pd) at the end of the first year and cadaveric clkt was performed when she was 9 yearsold. at the age of 16, she had chronic allograft nephropathy and began to have hemodialysis (hd). recently, 1.5 year after hd, cadaveric renal transplantation (tx) was performed. she is well after the second tx. case-2: he was admitted with polyuria, polydypsia, stomachache and renal stones were determined on sonographic examination. he had esrd and pd was started when he was 7 years old. at the age of 10, cklt was performed from his mother. his liver and renal functional tests are well 8 months after cklt. case-3: he was evaluated because of having an older brother diagnosed with ph-1 when he was 4.5 years old. he had no complain, but sonography showed multiple calculi. within 5 years he experienced flank ache, hematuria attacks and anuric phases due to obstruction and esrd appeared and he received hd and clkt was performed from his full-match sister at the age of 9.5. he is doing well 5 years after tx. here, we present the favourable clinical outcomes of our patients with cklt to indicate the validity of this treatment of choice in ph-1. tenascin (tn) is a glycoprotein component of extracellular matrix (ecm) which is not present in the normal kidney tissue. tissue plasminogen activator inhibitor-1 (pai-1) regulates fibrinolysis and the plasmin mediated matrix metalloproteinase activation and it is also not expressed in the normal kidney. recent studies focus on the pathogenesis of advanced renal diseases. in this study we evaluated tn and pai-1 staining in the renal tissues of pyelonephritic rats using immunohistochemistry (ihc) as to understand if these markers may be served as histological parameters of pyelonephritis like fibrosis, tubularatrophy or vascular changes. seven rats were injected 0.1 ml solution containing e. coli atcc 25922 10 10 cfu/ml into left renal medullae. seven rats were designed as sham group and were given 0.9% nacl. rats were sacrificed 7 days after injections. renal tissues were studied histopathologically by hematoxylin and eosinand scored for the parameters of pyelonephritis. tn and pai-1 expressions were studied semiquantatively by ihc by tenascin novocastra (ncc-tenas-c) and pai-1 (h-300) santa cruz biotechnology. both tn and pai-1 expressions increased in the pyelonephritic groups. we observed acorrelation between tenascin and fibrosis, vascular changes and tubular atrophy and pai-1 expression showed a correlation with only fibrosis. we conclude that increase in renal tn and pai-1 expression shown in experimental pyelonephritis are the responsible factors for the fibrotic changes of persistent renal damage. introduction: urinary beta 2 microglobulin (β2mg) urinary excretion is a good index of proximal tubular cell dysfunction. objective: to determine β2mg excretion significance in determining the outcome in respect to scar and renal insufficiency. patients and methods: urinary β2mg and creatinine (cr) was measured in 83 urine samples of whom 53 proceed to do dmsa renal scan at the time of admission and 6 months later to detect scar. β2mg was measured using radioimmunoassay method using β2mg 96-test kit (radim company). twenty children had various grades of renal scars. results were compared with ratios of 19 children with low uptake and 14 normal scanning and 30 normal children. student t test, anova, and unpaired t-test was used for analysis and differences considered significant if p<0.05. results: the mean urinary β2mg/cr was significantly higher in the scarring group (5.23±10.6) than in the normal group (0.19±0.2) and in low uptake group (0.49±0.86) (p<0.05). patients with grade iii had higher values (14.69±15.82) than grades i (0.36±0.35) and ii (3.37±5.20) (p<0.05). patients without renal scar had β2mg/cr ratio below 0.46 microgram/mg. the mean β2mg/cr was higher in patients with vur grades 4 and 5 (8.93±12.01) than patients with vur grades 1 to 3 (0.81±3.04) (p=0.02). maximum β2mg/cr was detected in patients with grade 4 vur (33.3) and the minimum was zero in non-refluxing patients and normal children. two patients with high grade vur and the highest levels of β2mg/cr ratio (33.3 and 26) progressed to renal failure in 2 years time, the first patient was treated by hemodialysis and the second underwent renal transplantation. conclusion: measurement of urinary β2mg is useful in the early detection of tubular damage in patients with renal scars. introduction: chronic allograft nephropathy refer to the progressive decline of renal function seen in some renal transplant recipients in association with alloantigen-dependent and alloantigenindependen factors. hyperlipidemia is a risk factor for chronic allograft nephropathy in adult pts, where no data exist in pediatric transplant population. methods: in this cross sectional study, 62 patients (32 can/30 non-can) that aged 5-18 yr and 9-93 mo (mean: 48 mo) after transplantation, were evaluated for lipid profile and renal function tests. results: hyperlipidemia has high prevalence in our patients both pre and posttransplantation and hypercholesterolemia and increased ldl had significant correlation with chronic allograft nephropathy (p=0.009) and p=0.025 respectively. conclusion: in pediatric patient as in adults hyperlipidemia and particularly hypercholesterolemia has significant correlation with chronic allograft nephropathy and as adults may need specific therapy. results: pre-transplantation renal replacement therapy time ranged from 0 to 132 months. eleven children underwent pre-emptiverenal transplantation. 43/72 transplants were from living related donors. donor age ranged from 0 days to 74 years. 9 grafts were from donors <1 year of age and 5 of these grafts were transplanted en-block. hla mismatches ranged from 0 to 5 antigens. primary disease was: focal segmental glomerulosclerosis in 9, rapidly progressive glomeluronephritis in 6, reflux nephropathy in 23, nephronophthisis in 9, iga nephropathy in 1, congenital nephrotic syndrome in 2, dysplasia-hypoplasia in 11, idiopathic membranous glomerulonephritis in 1, henoch-schönlein purpura in 1, hemolytic-uremic syndrome in 1, nephroblastoma in 1, polycystic kidney disease in 1 and of unknown origin in 4 children. patient survival at five years was 97%. allograft survival with living related transplants at one, two and five years was 95%, 95% and 82% respectively and with cadaveric transplants at the same periods was 86%, 86% and 86% respectively (p<0.05). regarding en-block grafts, they functioned immediately and satisfactory and presented excellent graft function 10 years later. most kidneys were lost due to acute or chronic rejection (n=6). other causes were renal artery thrombosis (n=3), infections (n=2), withdrawal of immunosupressive regime (n=1). conclusions: results of this single center series of pediatric renal transplants are encouraging from the standpoint of patient and allograft survival. conclusion: in infants with hn, the incidence of uti was higher especially in those with ou, hn of higher sfu grade or hun. the antibiotic prophylaxis may be recommended during 1 year after birth in infant with hn because the incidence of uti was high in these period. results: the underlying diseases were: sepsis with mods (26.8%), septic shock (11.3%), severe intoxication (15.5%), trauma with sirs (11.3%), drowning (2.8%), abdominal compartment syndrom (2.8%) and inborn metabolic disorders (2.8%). 43 children (61%) had acute renal failure, 28 (39%) patients met non-renal crrt criteria. cvvh was performed in 39 (55%) children, cvvhdf in 32 (45%) children. crrt duration was 6 to 216 hours (median 177.6 hours). dynamics of blood urea, creatinine, c-reactive proteine (crp) and white blood cells (wbc) were evaluated. significant decline (p<0.001) of creatinine along the treatment with cvvh as well as during cvvhdf was observed. blood urea levels showed significant decrease only in children treated with cvvhdf (p<0.05). significant decrease of wbc and crp was observed only using cvvh. 37 children from the study group survived (overall mortality 47.9%). in non-survivors was time from crrt initiation to its termination compared to time interval from crrt initiation to the death of children with 3-4 organs failure significant (p<0.05) where as in non-survivors with 1-2 failured organs it was not. conclusion: cvvh is efficient at removing urea and creatinine as well as inflammatory mediators (crp, wbc). cvvhdf is more potent to blood urea elimination. authors suggest preferring cvvh to cvvhdf in critically ill children to affect basic inflammatory parameters. to analyse hd and pd prescription (pr) adopted in chronic dialysis children, were viewed data of 147 pd regimens in 91 patients (0.5-18years) and 100 hd regimes in 74 patients (age 1.8-18 years) treated in 12 pediatric centres in 2005. pd patients were on automated pd: -nightly intermittent pd (nipd; 89 pr): 9.6±1.3 (8-13) hrs; 13.3±5.5 (6-27) cycles/night; dwell volume (dv) 889±232 (465-1400) ml/m 2 bsa; -tidal pd (42 pr): 9.7±1.9 (8-18) hrs; 16.9±5.6 (9-31) cycles/night; dv 1008±182 (600-1200) ml/m 2 ; tidal volume 63.8±9.5 (50-85)%; -continuous cycling pd (ccpd;16 pr): 10.3±1.6 (8-14) hrs; 15.6±5.6 (7-27) cycles/night; dv 1017±162 (600-1280) ml/m 2 ; daytime dv 62±18 (50-100)% of night dv. in 27% of pr dialysis fluid (df) glucose concentration was >1.36%, and in 12% buffer was bicarbonate. df of daytime dwell was 1.36% glucose (13 pr) or icodextrin (12 pr). patients with residual diuresis were 63.5% of those on nipd, 56% on tidal pd, and 6.2% on ccpd. hd was performed as bicarbonate hd (79%), hemodial filtration (15%) and acetate-free biofiltration(6%). patients received 3 sessions/week in 66% of cases, 4/week in 23%, and 2/week in 9% of cases; 2 oxalosis patients were on daily hd. session duration was 3 hrs in 44 pr, 4 hrs in 36, and 3.5 hrs in17. dialyser membrane was: polysulfone (38%); hemophane (19%); polyamide s (16%); cellulose acetate (9%); polyacrylonitrile (5%); cellulosediacetate (4%); cellulose triacetate (4%); polymethylmetacrylate (4%); polyether/carbonate (1%). ratio between dialyser surface area and patient bsa was 1.05±0.19 (0.70-1.43) and was 1-1.20 in 31%, 0.8-1 in 23%, >1.20 in 12%, and <0.8 in 7% of cases. isoniazid (inh) is widely used in most prophylactic and therapeutic anti-tuberculosis regimens because of its effectiveness and low cost. acute intoxication by isoniazid is known to cause symptoms of seizures, metabolic acidosis, coma, and even death. we present a case of acute isoniazid poisoning in a seven years old patient who ingested 7 tablets (2100 mg) of isoniazid. she was admitted unconscious, with ventilatory insufficiency and convulsions. renal and liver function tests were in normal ranges. she was intubated and mechanically ventilated. despite parenteral midazolamand pyridoxine (vitamine b6) treatments convulsions went on. then hemodialysis was performed and after hemodialysis convulsions and ventilatory insufficiency were disappeared and the patient was conscious and she was extubated. hemodialysis may be an effective treatment alternative for the patients who doesn't respond pyridoxine treatment. the aim of this study is to analyse children under two years of age, with their first febrile urinary tract infection (uti), identifying bacteriological etiology, antimicrobial resistance, urologicalabnormalities and renal damage. this is a prospective study including 92 children (63% girls) with their first febrile uti. mean age was 6 months (3-10), 27 (29%) patients were younger than 3 months (62% of them were boys). urine was obtained by suprapubic aspiration (65.2%) or transurethral catheterization (34.8%). 37% had positive nitrite on urinalysis and 95.6% had leukocyturia. they were submitted to ultrasonography (usg), dmsa scan (within 4 months) and voiding cystourethrography (vcug). the most frequent microorganism found in urine culture was escherichia coli, (94.6%). in this study high bacterial resistance to antimicrobials was observed in relation to the following antibiotics: ampicillin (57.6%), first generation cephalosporyn (32.6%), sulfamethoxazole/ trimethopin (43.5%). resistance to second generation cephalosporyn, aminoglycoside, nitrofurantoin and nalidixic acid was lower than 3.5%. renal ultrasound was abnormal in 43.5% of the infants. vesico-ureteral reflux (vur) was observed in 34.7%, although only 7.6% had vur grade iii or more. the dmsa scan showed that 23 (25%) patients had renal parenchymal damage. fourteen of these (60.9%) had normal esr. there were 8 (8.7%) reinfections within a 6 months period, even under prophylactic treatment, and the presence of vur grade iii or more was the only one with a significant relationship. conclusion: there were high levels of bacterial resistance to frequent used antimicrobials. this finding points toward a need for reviewing criteria of choosing initial blind therapy. investigation with dmsa scan is important in the detection of renal parenchymal scars, irrespective of the reflux grade. purpose: urinary tract infection (uti) has a risk of renal damage and is associated with vur. vur is investigated only by vcu. however, vcu is an invasive, painful study and many patients hesitate to be taken the study. we studied the correlation of vur in vcu and defect of dmsa scan and investigated the possibility of substitution of vcu by dmsa scan. material and methods: from 1999 to 2006, the medical records were searched for children admitted to cheongju st.mary's hospital with the first uti who had been evaluated with both dmsa scan and vcu within 1 months of the infection. the value of several clinical signs, laboratory findings, the resultsof dmsa scan and vcu were investigated. bacteriuria was defined as 100,000 or greater colony-forming units in urine from a bag, midstream or catheter sample. results: there were 54 patients underwent both studied at the first uti. mean age of the patients was 13 months old. the male patients were 38 (70%). the vur was found in 19 of the 54 patients (35%), grade i-ii in 5 and grade iii-v in 14 patients. there was no significant correlation with the presence of vur in sex, fever duration, blood white cell count and the level of serum creactive protein (crp). but the patients with vur grade iii-v were significantly older than the patients with grade i-ii reflux or without vur. there were abnormal dmsa scan findings in 22 of 54 (41%). of these patients, 11 were without vur, 1 with vur grade i-ii and 10 with vur grade iii-v. the abnormal dmsa scan was correlated with the presence and severity of vur. but vur was found in 25% of patients with normal dmsa scan. conclusions: abnormal dmsa scan is correlated with the presence of vur, so the patients with abnormal dmsa scan require vcu. in order to prevent missing a quarter of patients with vur shown normal dmsa, vcu should be recommended in all children with first febrile uti. cuba is the largest of the carribean islands with its 11,5 millions inhabitants. cuba is considered as a developing country and is classified in the group of: "lower middle income countries and territories". despite low financial resources, cuba has succeeded to develop an efficacious health care system with comparable results to those of west europe and usa (who data) ccl: 1) hd is the most prominent form of pediatric dialysis (automated night pd is in progress); 2) the no of transplantations is relatively low because of no participation to an international transplantation network; 3) high no of pediatric nephrologists; 4) high quality of patients care. background: inborn errors of metabolism in neonates are often characterised by hyperammonaemic coma within the first days of life and require prompt diagnosis and specific treatment such as toxin removal and nutritional support. cvvhd seems to be the optimal modality for extracorporal ammonium detoxification, however, little experience with small numbers of children has been accumulated. patients and methods: from 1996 to 2007, 21 patients with hyperammonaemia (16 male, 5 female) were admitted for dialysis treatment: 18 neonates (mean age 3.8±2.4 days, range 1-10 days) with a mean birth weight of 3430±1023g and 3 infants (mean age 5.6±4.6 years, range 3 to 11 years). in 14 neonates and 2 infants we inserted venous double lumen shaldon catheters (predominately femoral or jugular vein) for cvvhd treatment while 4 neonates and 1 infant underwent capd treatment. results: plasma ammonia levels (range 508-7267 μg/dl before dialysis and 27-3317 μg/dl after dialysis) were reduced by 50% within 4.5±2.7 h by cvvhd and within 18.7±9.2 h by capd (p<0.05). total dialysis time was 35.9±25.7h for cvvhd patients. no major mechanical complications were observed with cvvhd and stable blood flows (10-40 ml/min) and dialysate flows (1000-3000 ml/m 2 /h) were achieved. due to severity of underlying disease, 2 out of 14 neonates (14%) undergoing cvvhd died on day 2-3 while 2 out of 4 neonatal capd patients (50%) died on day 3 and one infant patient died after 498 days of capd treatment. twelve out of 14 neonates (86%) survived with no or moderate mental retardation. conclusions: cvvhd is an effective modality to eliminate plasma ammonia within few hours. however, vascular access and blood flows are critical restrictions. mental retardation has to be evaluated in larger scale studies. r. vilalta, e. lara, a. madrid, s. chocron, j. nieto hospital materno-infantil vall de hebron, nefrologia pediatrica, barcelona, spain background: transplant nephropathy is the main cause of renal failure in kidney transplanted children. until this situation is proved by biopsy, sometimes the progressive raise of creatinine leads to raise the anticalcineurinic (cni) drugs with added nephrotoxicity. sirolimus (sir) plus an anticalcineurinicin less dose and mycophenolate (mmf) could offer in kidney-transplanted children an immunosuppressive regime with less toxicity and even an improvement of renal function. methods and patients: 12 paediatric kidney-transplanted patients developed biopsy-proved chronic allograft nephropathy (age 6-19 y, mean 8) a follow-up post-transplant of 6 y and 7 exhibit also tubular involvement and acute cni toxicity. sir was added in all patients as a rescue therapy at 0.08 mg/kg/d. results: after a follow-up period of 18 months, creatinine level diminished (p<0.04) in 8 patients (4 in group tac, 3 in group cya, with no significant differences). creatinine level did not show a significant change in the other 4 patients (2 group tac, 3 group cya, basal creatinine 2.8 mg/dl. serum cholesterol changed from 230±30 mg/100 ml to 223±2 (ns) and serum tryglicerides from 110±32 mg/100 ml to 121±24 (ns). proteinuria also did not show changes (24±8 to 22±4 mg/m 2 /h (ns). conclusions: a poly-drug approach with less dose of anticalcineurinic added to the antiproliferative effect of sirolimus and the inhibition of purine synthesis based on mycophenolate mofetil could suppose an improvement of the renal function in children graft nephropathy an even in the graft survival. steroids have been a cornerstone in renal transplant immunosuppression. several strategies have been used to minimize their side effects. new immunosuppressive drugs have allowed steroid withdrawal or total avoidance. aim: to evaluate a new protocol with steroid-free maintenance immunosuppression in pediatric renal transplant. patients and methods: a prospective, non-randomized study in 46 consecutive first renal allograft recipients, followed-up to 24 months. patients received prednisone the first 6 days, two-dose basiliximab induction and maintenance therapy with tacrolimus (tac) and mycophenolate mofetil (mmf). no steroids were given after 6 d posttransplant. controls were 20 historical-matched steroid-based children receiving basiliximab, tac and mmf. all patients gave informed consent. anthropometric, biochemical variables, acute rejection and cmv infection were compared using student-t test and regression analysis. results: a better growth pattern was seen in steroid-free maintenance group reaching a normal growth at 24 months. gfr and serum glucose were similar in both groups. total cholesterol levels were significantly lower in the study group. the incidence of acute rejection was 4.3% in steroidfree maintenance vs 8.6% in steroid-based group, no differences in cmv infection and blood pressure were observed. hematocrit levels were lower during the first months after transplant in the steroid-free group, increased after 6 months post-transplant. patient and graft survival was 98% at two-yr post transplant in the two groups. conclusions: this steroid-free maintenance immunosuppressive protocol was efficacious, safe, with a lower incidence of acute rejection, not increased risk of infection, preserving optimal growth, renal function and reducing cardiovascular risk factors. objectives of study: to evaluate the lipid profile and its possible implications in children with end stage renal disease (esrd) or renal transplantation. methods: 19 children (11 boys, 8 girls) aged from 3.5 to 18 years, 9 on peritoneal dialysis (group i) and 10 with renal transplantation (group ii) were studied. in all children were examined: serum creatinine, total cholesterol, triglycerides, high density lipoproteins (hdl) and low density lipoproteins (ldl). a cardiac and liver ultrasonography were also performed. the body mass index (bmi) and blood pressure were evaluated in all children. 13/19 children received also a triplex carotid study. the median values of blood creatinine, cholesterol, triglycerides, hdl, ldl as well as the number of children with bmi over 95 th percentile in both groups were shown in table i . all children had normal findings in triplex carotid study. cardiac ultrasonography was abnormal in 1 child of group i and in 2 children of group ii. only 1 child presented lipoid invasion in liver ultrasound. 3/9 children of group i and 6/10 children of group ii presented hypertension, well controlled, with antihypertensive therapy. conclusions: frequent evaluation of lipid profile is recommended in all children with esrd or renal transplantation independently their bmi. in our study, children with renal transplantation presented better lipid profile compared with children on peritoneal dialysis. group i with <18 months (n=14; 7,8±4,9 months) and group ii >18 months (n=7; 36,8±19,9 months). results: serum albumin, serum lipids values and the distribution of the categories of the peritoneal membrane did not present significant differences between the groups. hypertension, renal residual function (p=0,005), the renal urea kt/v (p=0,007) and the weekly renal ccr (p=0,005) were significantly higher in group i, while the weekly peritoneal ccr (p=0,025) and the total weekly ccr (p=0,037) were significantly higher in group ii. catch-up was not observed in any group. control of the cholesterolemia, trigliceridemia and albuminemia were maintained with the dialysis time in both groups. the goals of adequacy of the doqi for kt/v and ccr were gotten respectively in 85,71% and 64,29% of the group i and in 71,43% and 42,86% of the group ii. the longer time in dialysis was associated with the lowest values of renal residual function, renal kt/v and renal ccr. the capacity of transport of the membrane was similar in both groups. objectives of study: to explore the characterize of peritoneal transport in chinese children with chronic peritoneal dialysis. methords: pet was carried out 10 times for six children (mean ages 9.3±4.4, aged from 2 to 14 years) who were maintained by capd, and the infusion volume of dialysate was 1195±597 ml (1100ml/m 2 ). the peritoneal solution transport rate was evaluated by the standards of twardowski's and ppdsc's criteria. results: in our study, the initial pet was performed at 38.7±15.6 days following initiation of pd, the 4-hours of peritoneal creatinine clearance (4h-d/p) and glucose absorption (4h-d/d0) was 0.85±0.24 and 0.34±0.19, respectively. according to the standards of twardowski's and ppdsc 's criteria, the peritoneal transport categories were divided into high transport (h) (6/10), high average transport (ha) (1/10), low average (la) (3/10) for peritoneal solution transport, and h (3/10), ha (4/10), la (1/10), low transport (2/10) for glucose absorption. no low transport type of solution was uesd in our patients. the coincidence rate of peritoneal creatinine and glucose transport types were 100% and 90% between the twardowski's and ppdsc's criteria, respectively. the different changes of peritoneal transport type were found in two patients with continuous pet. the value of 4h-d/p increased after peritonitis episodes. our results showed that the pet in 70% of capd children fall into high and high average transport categories elevated by ppdsc's and adult standards. the peritoneal solute clearance was adequacy in the children, but net water ultrafiltration was lower. standard pediatric pet and its criterie are consistent with the adult criteria. the capability of peritoneal solute transport increased after peritonitis episodes. verapamil (vp) is known to alter cyclosporine (csa) bioavailability. the impact on immunoregulators (il-2, tgf-β 1 , and tgf-β 2 ) in allograft recipients remains unresolved. a prospective open study to examine the impact of vp on peripheral blood cell mrna encoding il-2, tgf-β 1 , and tgf-β 2 and serum il-2, tgf-β 1 , tgf-β 2 protein levels was performed. parental written informed consent was obtained in all cases. children with stable renal allograft function (<6 months), and receiving immunosuppression (csa, pdn, either with aza or mmf) were included. in the first visit, a clinical examination, two-point (2 and 12h) csa pharmacokinetic profile, serum creatinine, serum for il-2, tgf-β 1 , and tgf-β 2 protein levels (by elisa) were obtained; peripheral mononuclear cells were collected for measurement of transcripts for 18s rrna (house keeping gene) and mrna for il-2, tgf-β 1 , and tgf-β 2 (by real time quantitative pcr assay). after the visit one, patients were either withdrawn of vp (if the subject was already receiving vp) or started on vp 2mg/kg/day (if the subject was not receiving vp). two weeks after, a repeat clinical evaluation and blood collection, as in the first visit, were performed. 21 pediatric recipients of renal allografts were included (17 were from ld, mean post-transplant time 4.8 years, mean csa dose 3.8 mg/kg/day). the c2h and calculated auc 0-12h were significantly higher in those receiving vp, but there was no difference in csa trough levels. protein and mrna levels of il-2 tgf-β 1 , and tgf-β 2 were not different. were previously seen by a nephrologist. logistic regression was performed on anemia (hgb<11.0 g/dl) and showed relative risk in blacks was 3.74 vs. whites. relative risk in those who did not receive epo was 2.77 vs. those who did. of 19 black patients, 16 were anemic and 9 previously seen by a nephrologist. of 55 white patients, 32 were anemic and 16 previously seen by a nephrologist. in summary, blacks and patients not receiving epo at the time of dialysis initation were more likely to be anemic. despite being seen previously by a nephrologist, nearly 60% of patients were anemic when starting dialysis. further analysis is needed to determine causality to improve anemia control in incident dialysis patients. 2 of the avf were in whites with 1 in a black patient. the avg was in a black patient, with a cvc distribution of 20 whites and 11 blacks. 21 patients with cvc had been previously followed by a nephrologist and of these 12 had been followed for >6 months. in summary, incident pediatric hemodialysis patients are primarily having cvc as initial access type. with 56.7% having been previously seen by a nephrologist and 57% of these for greater than 6 months, the reasons behind not having an avf or avg as primary access need to be explored and improved upon. this high incident cvc use is consistent with data reported in the united states, but not with other european and asian countries. an effort to have a permanent avf or avg in incident pediatric hemodialysis patients needs to be made by the patient's nephrologist. to find the preventive measures for recurrent uti in infants with first febrile uti and normal urinary tract (ut), the incidence of recurrent uti and its risk factors were investigated. method: from june, 2002 to june, 2005 under 12 months of age (-6 mon: 152, 6-12 mon: 38), who were diagnosed as the first febrile uti and proved to have normal ut, were enrolled to the retrospective study. for all infants with nonretractile prepuce, topical application of hydrocortisone for 2-4 weeks and physiotherapy was recommended. during the following 1 year, the incidence of recurrent uti and the well-known risk factors such as female, young age, phimosis, vaginal reflux, and initial 99mtc-dmsa(+) pyelonephritis were evaluated. result: the incidence of recurrent uti in infants with normal ut was 21.1% and recurrent uti episode was 0.23/patient-year. the recurrent incidence in male infants was 22.8%, which was not significantly different from 12.5% in female infants (p=0.193). the recurrent incidence in younger infants was significantly higher than in older infants [-6 mon: 25.0%, 6-12 mon: 5.3%, p=0.008]. this age-related difference was significant in male infants [-6 mon: 25.8%, 6-12 mon: 7.7%, p=0.045], but not in female infants (p=0.169). in infants with persistent nonretractile prepuces, recurrent uti developed in 34.3%, which was higher than 15.6% in infants with retractile prepuces (p=0.041). the presence of the vaginal reflux (p=0.087) or initial 99mtc-dmsa(+) pyelonephritis (p=0.041) showed no significant difference in the incidence of recurrent uti. conclusion: in uti infants with normal ut, younger infants under 6 months of age and nonretractile prepuces of male infants were the risk factors for recurrent uti. objective: vascular endothelial growth factor (vegf) appears to play a central role in the process leading to peritoneal angionesis and increased level of vegf may conrtibute to high peritoneal small-solute transport rate (ptsr) in continuous ambulatory peritoneal dialysis (capd) patients in adult. vegf-c is related to lymphogenesis, but its role in peritoneal solute transport rate is not known. in this study, we evaluated possible relationship between dialysate vegf and vegf-c levels and pstr in children. method: twelve children with no apparent inflammation process or disease, who had been on capd, were enrolled. standard peritoneal equilibration test (pet) was done to evaluate pstr. d/pcreat and d/d 0 gluc were calcualted at 4hr of pet. overnight dialysate levels of vegf and vegf-c were measured using commercial elisa kit. correlation between dialysatevegf (or vefg-c) and d/pcreat (d/d 0 glu) was analyzed. results: mean peritoneal dialysis duration was 9.25±7.18 months. mean overnight dialysate vegf and vegf-c level were 44.74±30.49 pg/ml and 119.6±78.26 pg/ml, respectively. a significant correlation was noted between the dialysate vegf-c and vegf level (r=0.809, p=0.001). dialysate vegf level had negative correlation with d/d 0 glu of 4hr pet (r=-0.691, p=0.009). vegf-c had no correlation with d/d 0 glu or d/p creat. conclusion: there was significant relationship between dialysate vegf and vegf-c levels in children and significant correlation was also noted between dialysate vegf and ptsr. it seems that vegf contribute to high ptsr also in children on capd. m. feldkötter, l. stapenhorst, b. beck, u. bangen, b. hoppe we currently use sirolimus as a second line medication in transplanted patients with a distinct nephrotoxicity of calcineurin-inhibitors. as our short term experiences were not as positive as expected, we performed a short term meta-analysis in our renal transplant recipients under sirolimus treatment: we give an account of seven kidney transplant patients who were either directly started or were switched to a medication with sirolimus during september 2004 to february 2006. the reasons for this action taken were calcineurin-inhibitor side effects like severe arteriolopathy with lossof gfr, atypical haemolytic-uraemic-syndrome, seizures after the first dosages of cya and a tacrolimus induced exanthema. in four of seven patients switched to sirolimus we observed severe side effects, exaggerating those of the calcineurin-inhibitor and hence, in three patients the latter treatment was installed again. findings were distinct proteinuria in two patients, hyperlipidemia in three patients, wound healing disorders and, most strikingly, treatment resistant severe pancytopenia in one patient and severe interstitial pulmonary fibrosis in another patient, both with amelioration after termination of the medication, but still the need of oxygen therapy in the latter patient. in addition we noticed a slightly faster reduction of the gfr calculated with the schwartz formula in five patients compared to the previous immunosuppressive regimen. based on these findings we strongly feel that a more critical discussion of each case is necessary before changing the immunosuppressive medication. also, the question arises on whether sirolimus can really be valued as an equivalent alternative to a calcineurin-inhibitor based immunosuppressive regimen in pediatric kidney transplantation. y. kovalski, r. cleper, i. krause, m. davidovits schneider children's medical center of israel, nephrology and dialysis unit, petah tiqwa, israel background: despite significant technical improvements, haemodialysis in infants with end-stage renal disease (esrd) is still associated with significant morbidity and mortality. methods: the files of patients weighing less than 15 kg with esrd who were treated with haemodialysis at our institute between 1995 and 2005 were reviewed for background and treatment characteristics, morbidity and outcome. results: the study group included 11 patients aged 7-75 months (mean 34.2 months) weighing 7.2-14.9 kg (mean 10.9 kg). mean duration of dialysis was 11.3 months. vascular access posed the major problem. ten patients were dialysed through a central venous cuffed catheter and one through an arteriovenous fistula. an average of three different vascular accesses was required per patient (range 1-9). mechanical difficulties were the most common cause of central line removal (56.5%), followed by infections (15.6%). major complications causing significant morbidity were intradialytic haemodynamic instability, hyperkalemia, coagulation within the dialysis set, anaemia, hypertension, inadequate fluid removal and recurrent hospitalisations. analysis of outcome revealed that 8 patients underwent successful transplantation, one returned for haemodialysis after 4.5 years due to graft failure, and 2 died. conclusion: haemodialysis is a suitable option for low-weight paediatric patients with esrd awaiting transplantation, when performed in highly qualified centers. the importance of antibiotic prophylaxis in management of vur vesicoureteral reflux (vur) cause urinary tract infection (uti) and renal scarring is a common condition in children. the detection and treatment of vur before renal scarring is vital. recently, optimal management of low grade vur is controversial. the aim was to explore the kidney outcome in a cohort of patients with vur. the patients were divided into five subgroups according to vur grades. all of them were treated with low dose prophylactic antibiotics until the age of 5 years. urine culture was repeated monthly. background: anemia is a common complication in patients on hemodialysis. treatment of anemia with recombinant human erythropoietin (rhuepo) may lead to iron deficiency. intravenous sodium ferric gluconate complex (sfgc) therapy improves iron stores. objectives of study: aim of our study was to assess effects of maintenance sodium ferric gluconate therapy in pediatric patients on hemodialysis on mean hemoglobin (hb), hematocrit (hct), transferrin saturation (tsat), serum ferritin and rhuepo dose, as well as safety of therapy with sfgc. methods: intravenous sfgc therapy was administered for 3 months in mean dose of 1.2 mg/kg/week to eight pediatric patients on hemodialysis. patients were from 2 to 16 years old (4 males and 4 females, aged 11.5±5 years). all patients were prescribed rhuepo before start of study. results: sfgc therapy was successful in maintenance of mean hb (increased from 10.5 to 11.3 g/dl), mean hct (improved from 31% to 33%), mean tsat (from 23 to 30%) and mean ferritin level (from 592 to 765 ng/ml). high ferritin levels in two patients were due to inflammatory disease rather than the sign of iron overload. the mean weekly rhuepo dose decreased from 4380 to 3500 iu. no significant adverse event due to intravenous sfgc therapy occurred. conclusions: intravenous maintenance sfgc use in pediatric patients on hemodialysis was safe and successful in maintenance of iron indices, thus allowing reduced use of rhuepo. the the viral hepatitis b still remains a serious problem, especially actual in patients with end-stage renal disease (esrd) on renal replacement therapy (rrt). the high frequency of hbv infection transmission in hemodialysis units and immunodeficiency modify hepatitis clinical course and outcomes and worsening vaccination results and renal graft survival. we have analyzed the results and influence on transmission of hb -infection of hepatitis b vaccination in 28 children aged from 6 to 17 years with esrd on chronic hemodialysis. majority of children were boys (53.5%) older than 11 years (85.7%). an assessment of hbs-ag has been conducted prior and during the vaccination (engerixb) by scheme 0-1-6 months. after first vaccination hbv infection was detected there after in 10.7% of children, after second vaccination in 7.1%. in all patients (11) which have received three tours of vaccination, an active immune response was developed. thus, vaccination against viral hepatitis b is effective and prevents hbv infection in children with end stage renal disease on chronic hemodialysis. renal transplantation (tx) represents the best treatment for the patient with crf. scientific advance has been able to optimize the immunosuppressive treatment however the adherence to treatment has been not maintained. aims: to identify the factors that influence in non-adherent behavior with the purpose of designing effective educational strategies. methods: the qualitative focus was carried out through patients and tutors interviews. the quantitative aspect applies for epidemic variables, time post-tx, percentages and frequency of the sentences coming from the analysis of the interviews. nurse, psychologist and a social worker were incorporated with the purpose of elaborating an instrument based on seven questions related to the transplantation, risk and/or loss of the graft; besides the events happened as consequence of this, allowing that interviewed manifested with freedom their opinions. the interview was recorded in a microcassette and later transcribed. analysis was determined by categories containing the answers of each question granting the agreement sentences according to the frequency which was repeated in each interview. informed consent was obtained. results: 150 tx (1989 -2006 ; 15 non-adherent, 80% of them were interviewed. mean age: 9.7 ys. loss the graft: 50%, time post-tx: 37.7 months, dd: 67% ld: 33%. the lack of supervision in the taking of medications, numbers/schedules medications, family conflicts and the poor communication with the parents/medical team seem to be the main factors for non-adherence. conclusion: it is necessary to modify the pattern of the patient's attention transplanted under the pattern of chronic suffering that allows the sick person's and their family active incorporation to the process in an integral way to the multidisciplinary group. infantile results: 9 patients (4 females, 5 males) <15 kg, 6/9-<10kg at pd start were treated. they consisted 20% of our center's pd patients (43 pts). age at pd start: 21.6±16 months (median 17), 8/9 pts <24 mo. pd therapy duration: 4-21 mo (median 6), 3 pts >12 mo. esrf cause: congenital nephrotic syndrome 4 pts, dysplastic kidneys 4, cortical necrosis 1. 6 pts were fed by gastrostomy, 4 pts received gh (growth hormone). 5/9 pts had hypertension (ht) treated with >2 drugs and 1-4 cv events. pd type: 5/9 cycler-assisted, 1/9 capd (continuous ambulatory pd), 3/9 both. pd adequacy targets (kt/v>2.5) were reached in 8/9. peritonitis: 0.22 episodes/patient-month, 3 pts had >3 episodes. 6/9 pts had >2 pd catheters and 5/9 >5 pd-related surgery. outcome: 6/9-kidney transplantation, 3/9 switched to hd for infections or uncontrolled ht. height ±sds median 0, weight ±sds median +1.1. conclusions: small infants with esrf can be successfully treated by pd despite high rate of infectious, cv and surgical complications. pd therapy main target is optimal growth towards kidney transplantation. hyperlipidemia is a well recognised complication of renal transplantation. it is a fairly common problem in the paediatric renal transplant population. its prevalence ranges from 16% to 72% though the mechanism is not clear. steroids, calcineurin inhibitors and rapamycin are the main culprits in inducing hyperlipidemia, which is a potential risk factor for cardiovascular heart disease and graft dysfunction. long term effects of these immunosuppressive drugs in children have not been adequately studied. of the calcineurin inhibitors cyclosporine (csa) was found to induce hyperlipidemia compared to tacrolimus (tac). post-ransplant hyperlipidemia is well described in adults; the same cannot be said in children. in adults, post-transplant hyperlipidemia increases risk of cardiovascular disease 3 to 4 fold. screening and management of hyperlipidemia has therefore become an important part of current long term management of transplant patients. there is a limited data on prevalence of hyperlipidemia in renal transplant in children and even more so locally here in south africa. most of the known studies have been conducted in the first world, there was therefore need to determine prevalence locally. this information would ultimately assist in the overall management of our renal transplant recipients. majority of the patients had normal lipid profile. 25% of the patients had high cholesterol levels, while 25% of the patients had high tg levels. 4/10 (40%) of the patients on csa had hypercholesterolemia compared to only 1/11 (9%) on tac (p=0.15). 2/10 (20%) of the patients on csa had high tg compared to 3/11 (28%) on tac (p=1.0). the study concluded that the prevalence of hypercholesterolemia and hypertriglyceridemia in renal transplant pts is high, comparable to other studies and that there is a tendency towards having more lipid abnormalities in transplant pts on csa. grade 3 vur in 24 (39%) and . the incidence of abnormal findings was significantly higher in children with uti and vur than in those with uti without vur (88.5% vs 67%; p<0.05). in children with no vur, grades 1-2 vur, grade 3vur and grades 4-5 vur, renal scarring rates were 67%, 70%, 90% and 88%, respectively. the patients with higher grades vur tended to have more than 3 scars on their dmsa scans (p<0.01). our findings suggest that renal scarring resulting from uti is mostly related to vur, but sometimes is caused by the infection itself. we can conclude that vcu is essential for diagnosis of vur, but 99mtc-dmsa scan shouldn't be avoid in the management of children with uti. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] years. seventythree (97.3%) of them were on triple immunosuppressive therapy, one on double therapy, and one didn't use any medication. overall, 9 subjects (12%) had at least one episode of uti. twenty-four episodes of urinary tract infection occurred in 9 children: 7 episodes in two girls with neurogenic bladder (ngb), 9 episodes in two boys with posterior urethral valve (puv), four episodes in an obese girl with laurence-moon-biedl syndrome, 2 episode of uti in 2 girl with unknown primary renal disease and 2 episodes in 2 girl, one with polycystic kidney disease and one nephronophthisis. conclusion: uti following kidney transplantation was more common in children with known lower urinary tract abnormalities. key words: urinary tract infection, kidney transplantation. background: in japan, the severe shortage of cadaveric kidneys led clinicians to attempt performing abo-incompatible living kidney transplantation (tx). some reports demonstrate successful results of the combination of plasmapheresis (pp), strict immunosuppression and splenectomy. however, we should concern about a great risk of surgical invasion and postoperative serious infection in younger patients who underwent splenectomy. recently, a few reports suggested anti-cd20 monoclonal antibody (rituximab) can be an alternative to splenectomy. patient and method: a 9 years old boy with bilateral hypoplastic kidneys had been treated with peritoneal dialysis for 3 years. since his blood type o was incompatible with paternal blood type a, we arranged to perform tx using pp and rituximab without splenectomy. a single pp was performed on days -10, -7, and -3 to reduce anti-a antibody (ab) titer. rituximab was administered in a single dose of 375 mg/m 2 on day -6. basiliximab, tacrolimus, mycophenolate mofetil and methylprednisolone were used for immunosuppression. results: before tx, the anti-a ab titer was reduced from 1:32 to 1:8, and cd19 level was suppressed from 25% to 3%. tx was performed without splenectomy and he had excellent initial graft function. we observed anti-a ab titer rose up to 1:128 on day +5, but it decreased spontaneously to 1:16. there were no side effects and severe infections during the perioperative period, but the neutropenia treated by gcsf appeared 3 months after rituximab administration. the protocol biopsies were performed in 1 month and 6 months, which revealed no signs of rejection. conclusions: abo incompatible tx using pp and rituximab without splenectomy can be a therapeutic option in children to avoid a invasive surgery and infectious risks. further establishment of optimal protocol for children are necessary to obtain excellent outcomes safely. 1.70.2, 1.320.4, and 75.65.5, 66.415.1, in hivan, and od, p=0.06, p=0 .08, respectively. serum p levels were 6.71. 1, 4.60.9, and cap were 54.517.0, and 39.68.7, in hivan and od, p=0.044, p=0. the aim of this study is to identify the outcome of the physically or socially handicapped children with end stage renal disease (esrd) receiving chronic peritoneal dialysis (cpd). among 95 patients commenced on cpd, 11 handicapped children with esrd receiving cpd were identified in our unit during the period between november 1995 and february 2007. age at cpd initiation ranged from 5.5 to 15.5 years (median age: 11; 6 girls, 5 boys). underlying diseases were neuropathic bladder and vesicoureteral reflux (in 3 patients), chronic pyelonephritis (in 3 patients), vesicoureteral reflux (in 2 patients), amyloidosis (in 2 patients), and alport syndrome (in 1 patient). causes of handicapped status against cpd were inadequacies of indoor resources (in 3 patients), cerebral palsy (in 2 patients), down syndrome (in 1 patient), inadequate psychosocial status (in 1 patient), surgically corrected rectovesicale fistula and ectopic anus (in 1 patient), blindness (in 1 patient), ventriculoperitoneal shunt and paraplegia (in 1 patient), colostomy (in 1 patient). all catheters were implanted percutaneously by the same pediatric nephrologist. median duration of dialysis was 24 (range 3-124) months. during a total of 443 dialysis months, 27 episodes of peritonitis (1 episode/16.4 patient-month), 1 episode of exit-site infection, and 1 episode of tunnel infection occurred in 8 of 11 children. except for an inguinal hernia in 1 patient, we did not observe any mechanical complication related to catheter. cpd was terminated in 6 children (death in 3, renal transplantation in 2, switch to hemodialysis in 1). before initiation of renal replacement therapy, some negative baseline factors may not be really contraindications for cpd. the socioeconomic and geographic factors greatly influence the prevalence and outcome of renal disease in children. the subspecialty of pediatric nephrology in sudan was established few years ago and the facilities for the management of renal problems are limited. the aim of this study is to review the demographic profile, complications and outcome of capd after nineteen months of treatment. all children who underwent capd from june 2005 to january 2007 were studied. there were 22 children (12 males), the mean age was 11 years (range from 11 months to 16 years). the majority of children has undetermined cause of esrd (11 children). the most common complication was peritonitis (peritonitis rate was 1/8.3 patient/months. 8 patients had refractory peritonitis that necessitate catheter removal, exit-site infection was documented in 4 children and catheter block in 4 children. there were 12 drop out of the program 4 were due to deaths, 7 changed modality and one family withdraw treatment. in conclusion this analysis has stimulated improvement in nurses training and supervision as well as attempts to improve catheter survival and microbiology monitoring. the -5) , looking for symptoms of hypovolemia (cramps, abdominal pain or headache) during or at the end of hd treatment. bioimpedance measurements were performed at the end of each session according to the 50 khz tetrapolar technique; resistance and reactance values were plotted on the age and gender specific 50 th , 75 th and 95 th percentiles of the vector distribution in the healthy population (reference tolerance ellipses) as a resistance-reactance graph. hypovolemia (hv) was indicated by a vector shifted to the upper pole, out of the 95% tolerance ellipse; normovolemia (nv) by a vector inside the 95° ellipse. patients complained of one or more of the above-mentioned symptoms in 26% of hd sessions, while biva suggested hv in 47.9% of the sessions. symptoms were significantly more common (p<0.05) in sessions with hv (20/57 cases; positive predictive value 35.1%) than in those with nv (11/62 cases; negative predictive value 82.3%). biva suggested hv in 20/31 sessions with symptoms (sensitivity 64.5%) and nv in 51/88 sessions without symptoms (specificity 58%). no significant differences in the accuracy of biva were found between patients either younger vs older than 18 years, or with height sds <-2 vs >-2, or taking vs not taking antihypertensive drugs. in conclusion, biva can be useful in assessing dry weight in children and young adults on hd: since patients with a vector shifted to the upper pole, out of the reference 95% tolerance ellipse, are at high risk of hypovolemia during the next hd treatment, the increase of the dry weight is then indicated chronic antibody-mediated rejection can occur as a de novo complication in renal allograft recipients and is associated with c4d deposition in peritubular capillaries in the renal graft and positive circulating anti-hla antibodies, although the sensitivity and specificity of positive c4d staining for chronic humoral rejection requires further study. renal outcome appears to be worse in c4d positive patients. current treatment strategies to manage c4d-positive chronic humoral rejection are poorly defined. various protocols with enlarged doses of tacrolimus, mycophenolate mofetil, plasmapheresis, ivig and rituximab have been reported in adult patients. we investigated four pediatric patients (mean age 13.6 yrs; range 10 to 17 yrs) after renal transplantation that developed c4d positive chronic rejection. in 3 of 4 patients, maintenance immunosuppression with calcineurin inhibitors had previously been minimized because of severe toxicity. in 3 of 4 patients, an elevated anti hla class ii antibody titre could be detected; donorspecific antibodies were positive in 2 patients. all patients experienced a progressive deterioration of graft function. treatment with repeated intravenous immunoglobulin (ivig) (1 g/kg body weight per week over four consecutive weeks) followed by a single dose of rituximab (375 mg/m 2 ) was therefore initiated. three of four patients showed an improvement of graft function with a mean increase of gfr by 25%. one patient with advanced chronic transplant nephropathy lost his graft after 6 months. this pilot study demonstrates that the combination of high-dose ivig and rituximab can stabilize or improve transplant function in chronic antibody-mediated rejection without major side effects. the use of ivig and rituximab appears to reduce the active immunologic process, but larger trials are needed to support these observations. cardiovascular diseases are some of the most important causes of morbidity and mortality in children with end stage renal disease (esrd). chronic inflammation has been suggested to be a risk factor for cardiovascular diseases. the aim of this study was to investigate the relation between crp and cardiac changes in children on hemodialysis. this study was conducted on 60 patients (30 patients were hypertensive & 30 were normotensive), 39 males (65%) and 21 females (35%) on regular hemodialysis due to esrd. their ages ranged from 7 to 16 years (mean 14.93±3.0). sixty age-and sex-matched controls were also included. significantly higher velocity of circumferential fibre shortening (vcfs), tei index, interventricular septum thickness in diastole (ivsd), left ventricular mass index (lvmi) and isovolumetric relaxation time (ivrt) and significantly lower e/a ratio were found in all patients as well as in hypertensive & in normotensive groups as compared to the controls. significantly higher ivsd and lvmi were found in hypertensive patients than normotensive patients. significantly higher high sensitivity crp (hs-crp) & crp latex were found in all patients as well as in hypertensive & in normotensive groups when compared to the controls. crp was significantly higher in both study groups with cns symptoms and cardiac symptoms in comparison to those without. it was also significantly higher in patients with increased lvmi & than in those with abnormal e/a ratio. hdlc showed a significantly direct negative effect on crp. s. ca 2+ , se. p and ca x p had a significantly direct positive effect on it. we can conclude that the cardiac affection in children with esrd appears in the form of lv hypertrophy with early diastolic affection. crp could be correlated to these changes and to cns symptoms and cardiac symptoms in these patients. is. lim, hs. lee, dw. kim, wh. choi chung-ang university, pediatrics, seoul, south korea purpose: urinary tract infections are common clinical problems occurring in infants and pediatric patient groups, most frequently caused by uropathogenic e. coli. urinary pathogens almost always infect the host through ascension from the rectum, vagina to the urethra and bladder. recurrent urinary tract infection is a disorder involving repeated or prolonged bacterial infection of the bladder or lower urinary tract. in this study, we examined the substitusion effect of probiotics in the high risk group of recurrent urinary tract infection. objectives & methods: patients diagnosed as recurrent urinary tract infection were administered probiotics for six months, and urine cultures were checked during the period. probiotics in this study were selected among the products commercially saled in korea, namely lactobacillus acidophillus, bacillus subtilis, and bifidobacterium infantis. single blind study was done for selection of probiotics for patients. result: the separated bacteria from the urinary tracts of the patients were the same as administered probiotics in some patients. conclusion: in recurrent urinary tract infection, there seemed to be a substitution effect of probiotics for uropathogenic bacteria, and it is reasonable to administer probiotics for long period in the high risk group of recurrent urinary tract infection. renal insufficiency therapy in children: quality assessment and improvement: the rich q study objectives: outcome studies in children on chronic renal replacement therapy (crrt) have revealed a 30-time increased mortality and 40% co-morbidity in adult survivors. information on the quality of care of treatment centers and on the impact of advised quality indicators on outcomes in children are lacking. no data exist on the impact on these outcomes of the different treatment modalities, peritoneal dialysis, hemodialysis & transplantation either. until now, no structural corporation and consensus on general guidelines with respect to crrt exist between the 9 dutch (nl) and belgium (b) centers for pediatric crrt. aim of the study: 1. assessment of the current quality of treatment crrt in children (qt) in nl & b and of the effect of recurrent peer review of the achieved outcomes on the qt. 2. the assessment of the effect of different treatment modalities on outcomes. 3. the creation of a format for multicenter trials. methods: all prevalent patients on chronic dialysis aged <19 years at onset of the study & all incident patients during the study period with onset of crrt<19 years of age, from b & nl will be included. treatment characteristics and quality indicators of crrt with respect to physical and psychosocial outcomes will be collected of all patients. operational data collection and management will be performed by members of the dutch institute for quality care in dialysis patients (hans mak institute). each 6 months, all data will be revealed and actively discussed by representatives of all centers (peer review). the effect of registration and peer review on the qt will be analyzed after 2 & 4 years. comparison will be made between the effects of cumulative periods of different rrt models on outcomes. the study will be performed between august 2007 & 2011. on estimation, 200 patients will be analyzed. renal renal transplantation in patients with lower urinary tract dysfunction (lutd) of different origin is a challenging issue in field of pediatric transplantation. we report our single centre experience to evaluate the patient and graft survival as well as risks of the surgery and immunosupressive therapy. among 70 pediatric transplant patients 11 patients had severe lower urinary tract dysfunction. videourodynamic test was performed in all patients preoperatively and postoperatively. the cause of urological disorders was secondary to neurogenic bladder (n: 5) and valve bladder (n: 6). clean intermittent cathatetization (cic) was needed in 6 patients to empty the bladder. pretransplant augmentation ileocystoplasty was created in four patients and gastrocystoplasty in one patient to achieve low-pressure reservoir with adequate capacity. three of the patients received kidneys from cadaveric and 9 of them from living donors. the mean age at transplantation was 15±4.7 years. the median duration of transplantation was 18 months (range 1-49 months). at their last visit median creatinin levels were 0.95 mg/dl (0.8-2.4) . three patients had recurrent symptomatic urinary tract infections who had augmented bladder and on cic. one of them had creatinine levels of 2.4 mg/dl. one patient with ileocystoplasty who developed urinary leak and ureteral stricture in early postoperative period was treated by antegrade j stent. severe lutd reserves high risks for graft kidney. however our data suggests that renal transplantation is safe and effective treatment modality if the underlying urologic disease properly managed during the whole course of transplantation period. since surgery and follow-up of these patients is more complicated, patient compliance and experience of transplantation team will have significant impact on the outcome. r. meneses, l. sylvestre, j. sousa, d. ribeiro hospital pequeno principe, pediatric nephrology, curitiba, brazil introduction: in july 2000, we started a systematic evaluation program of each patient on chronic pd. the aim of this study was to analyze the long-term outcome of children on pd program. material and methods: we evaluated all the patients on pd between july 2000 and may 2006, who performed 3 complete protocols, with a minimal interval of 6 months between them, consisting of: anthropometric measurements, blood pressure and cardiological status, standardized laboratorial evaluations, pet test, clearance and kt/v, measurement of the intra-peritoneal pressure (ipp), occurrence of infections, hernias or constipation and need to change the catheter. we then compared all the evaluations using the graphpad prism software, a p value <0.05 was considered significant. results: 36 out of 74 patients were eligible, mean age 8±5 years old at the first evaluation, 61% boys, primary renal disease: 45% uropathies, 28% glomerulopathies, 8% tubulopathies and 19% other causes. there was an improvement on bmi and weight/height z-scores and worsening of height/age z-score, but none was significant. there was also no significant decrease in residual renal function (p=0.51), adequacy parameters remained stable: clearance (p=0.84) and kt/v (p=0.58). most patients were converted from capd to ccpd and nipd, and some had to increase daytime dwells (p=0.0098).constipation and the number of infections improved but not significantly. laboratorial evaluations, peritoneal membrane characteristics, ipp, need to change the catheter and occurrence of hernias did not change over the time. conclusion: a long-term maintenance of children in peritoneal dialysis program is possible, but reaching a satisfactory clinical condition is a great challenge. several points need to be checked for planning a better adequacy and survival of dialysis technique in children waiting for a graft. a rigorous follow-up protocol seems helpful in precocity of prescription strategy modifications. we observed a stable long-term outcome observing these adequacy tools. outcome the recurrence of primary disease in transplants is a well-known problem. we report our single centre experience to assess the frequency of the recurrence of primary glomerulonephritis in children after renal transplantation. medical reports after 1990 of 12 children with primary glomerular disease were evaluated. the 13 grafts were nine from living related and four from cadaveric donors. eight of them were diagnosed as fsgs, 2 of them mpgn and 2 of them pan. the mean age was 15.5±5.4 years. however the median transplantation duration was 47 months, one of the fsgs patient had hyperacute rejection. five years later she had second graft with the serum creatinine 0.7 mg/dl at 7th year of second transplantation. and all recipients were immunosuppressed with either cyclosporin a or tacrolimus, azothioprine or mmf and steroid based regimens. mutational analysis was available in two patients, they had homozygous podocin mutations. post transplant recurrence of fsgs was confirmed in one patient. glomerular tip lesion was the only histologic abnormality on graft biopsy. he has treated with plasmapheresis with no improvement of proteinuria. two of the fsgs patients had thromboses after transplantation. one of them had cardiac thrombosis with heterozygote mthfr mutation and one of them had renal artery thrombosis and loss of graft with prothrombin 20210a mutation. both of them have had additional risk factors for thrombosis. they have all functioning grafts except one. we have not observed any recurrence in patients with pan and mpgn. although the number of our patients quite small, renal and patient survival seems to be more favourable in our experience but we strongly recommend the evaluation of all risk factors of thrombosis and give appropriate anticoagulation. skin involvement in factor h deficiency (fhd) associated to hemolytic uremic syndrome (hus) has never been reported. we describe the case of a young adult on regular hemodialysis (hd) for fhd-hus who developed microangiopatic skin lesions and was successfully treated with plasma exchange (pe). the patient developed end stage renal disease secondary to fhd-hus (scr20) in 2004, when she was 32. after one year of hd she complained of severe night pain in the perimalleolar areas, followed by skin lesions which evolved into superficial ulcers (fig1). in august 2005, due to the worsening of the skin lesions, the patient started hd and pe (2 litres of fresh frozen plasma per session twice a wk) based on the hypothesis that skin lesions were expression of thrombotic microangiopathy. after 7 wks of pe there was a skin improvement (fig. 2) and a pain relief. pe was discontinued. 3 wks later she started to complain of the usual pain in the right foot. pe program combined to hd was restarted and the symptoms ceased again. pe was gradually discontinued and she was addressed to regular plasma infusion of 0.5 litre per wk. so far, after 18 months the pain and the skin lesions did not show up again. m. belingheri, s. cristino, p. basile, v. bianchi, a. leoni, s. testa, l. ghio, a. edefonti, g. ardissino ospedale maggiore policlinico irccs, mangiagalli e regina elena, pediatric nephrology, milan, italy background: in rapidly growing children on hemodialysis (hd), the determination of dry weight still remains troublesome. bioimpedance analysis (bia) is potentially helpful in quantifying the fluid to be removed but its specific role, in routine clinical practice, is not yet clearly set. the aim of the present study was to test the feasibility of prescribing ultrafiltration (uf) exclusively based on bia parameters. methods: differences in body weight, resitance (rx) and reactance (x-c) between pre-and post-hd were calculated in order to derive the equivalence between uf and bia parameters in a 3 years old girl over a period of 16 months. for 21 consecutive hd sessions, uf was prescribed exclusively based on the derived uf-bia equivalence. this period was compared with 21 hd sessions where uf was prescribed by the conventional approach. results. xc correlated with ultrafiltration better (r: .75) than rx (r: .64). bia-based compared with weight-based uf prescription showed a significantly lower number of hd sessions complicated by hypotension (19% vs. 50%), need of fluid reinfusion (5% vs. 50%) and a better quality of the hd sessions (86% vs. 37%). conclusion. prescription of uf solely based on xc is feasible and provides a better outcome compared to the conventional modality of uf prescription. we believe that this approach could be useful for any patients with low tolerance to uf or with problems in setting the correct dry weight. aim: the aim of the present prospective study was to determine the incidence of urinary tract infection (uti) and related abnormalities in children ages between 0-2 years. material and methods: all children between 0-2 years old whose admitting to first step health offices (routine controls and immunization) was screened for uti with urine dipstick test after education of minimum two persons from first step health offices according to protocol with two tertiary child care center and health directorate of izmir province in turkey between july 2005 and july 2006. all patients with urine dipstick test abnormalities were referred to tertiary child care centers for evaluation. urine microscopic evaluation and urine cultures and other further investigations were performed in tertiary care centers after obtaining urine with urinary catheterization. results: 14.918 children (55% boys) were screened with urine dipstick test. the children's mean age was 11.7±5.6 mo (median 10 mo). screening test was found normal in 12.527 (84%) children. 962 of 2391 (40%) of referred child were admitted to tertiary care centers and evaluated for uti. uti was demonstrated in 419 children (2.8% of screened and 44% of evaluated children's). uti incidence was found 4.1% in girls and 1.7% in boys. urinary tract abnormalities were found in 24 children (0.2% of screened and 5.7% of evaluated children's). the most common urinary abnormality was vesicoureteral reflux (17 patients). conclusion: the uti incidence was 2.8% in children ages between 0-2 years, uti more common in girls than boys in this age group and only small group of children has urinary tract abnormality which is determined with routine urine screening. knowledgement: thank you for this opportunity to health directorate of izmir province. we describe ds post-peldrt in 2 children with no known neurologic problems and discuss potential predisposing factors. a 12.2 kg girl with renal dysplasia was started on a calcineurin inhibitor (cni) one week pre-t and when her blood urea nitrogen (bun) was 110 mg/dl. on admission for t, the bun had increased to 146, and her serum sodium (na) was 142 mmol/l. post-t, she remained intubated and paralyzed to permit generous volume supplementation, including 1: 1 replacement of her vigorous urine output (uop), initially with 0.45% nacl in water. five hours post-t, her bun was 17 and her na 126. after modification of uop replacement, her na normalized. on the morning of post-t day 1, paralysis was discontinued, but she did not awaken and had sluggish pupillary reactions. computed tomography of the head (cth) revealed diffuse cerebral edema, and brain death occurred. a 39 kg adoloescent with polycystic kidneys was started on a cni 4 days before peldrt. his bun and na then were 76 and 142, respectively, and had not changed on the day of t. post-t, the patient was immediately extubated. with uop replacements as described above, his bun and na decreased from 74 to 12 and 143 to 124, respectively, over 24 hours despite adjustments in the na content of his intravenous fluids based on urine na levels. the patient then had a 10-second tonic-clonic seizure, followed by a 5-hour post-ictal state. cth was negative, and the patient recovered completely. we conclude that ds, caused by a rapid decrease in serum bun and thus osmolality, may complicate peldrt in settings even with older pediatric recipients or without excessive elevation of pre-t bun. other factors contributing to this ds may include relatively mild hyponatremia and cni effects on both pre-t uremia and seizure threshold. results: 89 patients, predominantly males, ages between 5 months and 21 years old. the mean incidence of peritonitis was 0.1 episodes/patient months. fifty-seven patients (64%) had at least one episode of peritonitis.there were 145 peritonitis, 85% percent from all episodes began at home, 35% caused by gram negatives, 38% by gram positives, 8% by fungus, 19% had a negative culture and in less than 1% it was not performed. the mean treatment time was 19 days, 45% had a good response to initial empiric antibiotics (cefazoline and amicacine). the interval between the beginning of dialysis and the first peritonitis episode varied from 1 to 1462 days, occurring in the first 6 months in 50% of the patients. successful treatment occurred in 75% of the cases, 18% were transfered to hemodialysis, 7% had a consecutive peritonitis episode, and 1 patient died due to mesenteric artery trombosis. conclusion: peritonitis occurred early in our patients. even though most of them have a good initial response, there is still a great amount that have complications leading to technique failure. continuous education for the patients and health team, aiming early diagnosis and treatment, are useful to preserve the technique and decrease morbidity and mortality associated to peritonitis. d. davis, j. emancipator, x. zhu, c. rosen objective: to assess for sd in p chronic kidney disease (ckd) patients before and after rtx. methods: we assessed 4 symptom (sx) domains of sleep disorders: 1) sleep-disordered breathing (sdb); 2) insufficient sleep (is) (shortened sleep time or nap); 3) excessive daytime sleepiness (eds); and 4) restless leg syndrome (rls) using a set of standardized questionnaires in 51 patients with ckd (age 5-18 yrs) including non-d non-tx (ndntx) (n=19), d (n=11), rtx (at least 3 months post-tx) (n=21), and 15 age-matched sibling controls (c) without known ckd. the presence of an overall sd was defined by positive responses in any of the 4 sx domains. results: mean age (se) ranged from 11.9 (3.7) to 14.7 (3.8) in the 4 pt groups (p>0.05) without significant differences in gender, race, or congenital ckd. estimated mean gfr (ml/min/1.73m 2 ) (se) was significantly higher in the rtx group [75.8 (22.4) methods: in a prospective design, 64 renal transplanted children, who had renal transplantation at least 3 months before, at namazee hospital, were enrolled in our study. immunosuppressive regimen consisted of cyclosporine and prednisolone plus mycofenolat mofetil or azathropin. data regarding gfr, serum creatinine, electrolytes, lipids and c 0 and c 2 levels was collected at beginning, in one-month, and five-month intervals. cyclosporine was adjusted to 100-250 ng/ml based on c 0 level. patients were divided into two c 0 (<100 and >100 ng/ml) and two c 2 (<800 and >800 ng/ml) subgroups. discussion: similar creatinine levels, drug dosage, and complications of c 0 and c 2 subgroups may be due to dependence of renal function to several factors other than cyclosporine dosage. regarding coefficient of variation, c 2 was more accurate and reliable than c 0 level. as there was no significant difference in mean c 0 and c 2 levels, and renal function at beginning and the end of the study, there seems to be no need to check c 2 levels after renal transplantation. purpose: preparation is necessary in order to effectively meet the critical needs of the post-operative pediatric kidney transplant patient upon their arrival to the icu following transplantation. the increasing number of children requiring liver transplantation services has made it evident that it is important to have guidelines in place for their initial and often specialized post-operative care. methods: the main goal is to provide the child with appropriate post-operative care and to recognize and quickly address complications. therefore the icu nurse will: · monitor the patient continually and conduct full assessments a minimum of 1 time/hour (airway, breathing, ventilation, perfusion, neurological status, etc) . · observe the incision for signs of bleeding, evisceration, and dehiscence. · treat post-operative pain. · update family with findings, etc. · see that appropriate post-operative studies (ultrasound, laboratory studies, etc) are completed. outcomes: nurses in the icu monitor the pediatric post-operative kidney transplant patients very closely as outlined. this allows for quick recognition of problems and immediate intervention. it is the practice of these nurses to be fully aware of the patient's status as well as any changes that might be problematic. conclusions: nurses are prepared to care for pediatric kidney transplant patients and very carefully follow established guidelines for assessment. following guidelines for assessing and caring for pediatric kidney transplant patients upon admission to the icu has proven to be affective in allowing nurses to quickly recognize complications and notify the appropriate clinician. background: uremia is an independent cardiovascular risk factor. transplantation increases life expectations of patients with crf, however there is still an increased risk of accelerated arteriosclerosis. the pulse wave velocity (pwv) is a non-invasive marker of arterial distensibility, it increases along with arterial stiffness, as an early indicator of arteriosclerosis. aim: to evaluate pwv values of transplanted (tx) children. patients, methods: pwv was measured with a pulsepen in 21 tx (age 14,3±3,0 years). two control groups were formed using a database of 116 healthy children (6-23 years): one matched for age (a) and one adjusted for height and weight (h/w). blood pressure, heart rate, serum calcium (ca), phosphate (p) , and pth were also determined before transplantation and at the time of the pwv measurement. results: tx patients were smaller by 15,3 cm (p<0,05) than a and younger by 2,9 years than h/w (p<0,01). pwv in tx (5,5±0,7 m/s) did not differ significantly from a (5,1±0,9) , however it was elevated in comparison to h/w (4,6±0,6 p<0,01). serum p, caxp and pth was increased before transplantation, all the values returned into the normal range except for creatinine (106±50 micromol/l) at the time of the study. there was no correlation between pwv and the actual values of ca, p and pth. conclusion: pwv is higher in transplanted children as a sign of increased arterial stiffness. controls matched for height and weight should be used in states of severe growth failure. although a number of established risk factors potentially responsible for arterial dysfunction were present before transplantation, they were normal at the time of the study. the long lasting effect of uremia before transplantation could be in part responsible for the increased pwv in children after transplantation. supported otka-t046155-fo48842-f042563 and ett 435/2006. d. derakhshan 1 , h. jalaeian 2 , a. derakhshan department of pediatric nephrology, shiraz, iran 2 shiraz organ transplantation center, nemazee hospital, shiraz, iran backgrounds: bartter syndrome is an inherited recessive autosomal tubulopathy characterized by hypochloremia, hypokalemia, metabolic alkalosis associated with potassium renal leakage, and normal blood pressure despite increased plasma renin activity. patients with this syndrome may have proteinuria or hematuria, but most of them have normal gfrs. here we report on a child with bartter syndrome who developed esrd (end stage renal disease) and underwent successful cadaveric kidney transplantation. case presentation: a 7-year-old girl presented to the pediatrics nephrologist with failure to thrive, severe hypokalemia, hypochloremia, metablolic alkalosis, and normal blood pressure and the diagnosis of bartter syndrome was considered for her. however, due to poor compliance, she did not receive any medications, did not give consent for kidney biopsy and did not attend her opd follow-up visits for about 8 years, when she developed esrd and went on chronic hemodialysis (3/weeks) . her little sibling also was diagnosed to be suffering from bartter syndrome at this time. after 10 months, she received a cadaveric renal allograft. afterwards, her kidney function, serum electrolytes, and growth have improved dramatically. discussion: in this case, we postulate that long-term hypokalemia due to bartter syndrome led to chronic interstitial nephritis and renal dysfunction. successful renal transplantation, even after the onset of esrd, for severe clinically bartter syndrome results in correction of metabolic abnormalities and excellent graft function. we propose that bartter syndrome should be considered as a possible cause of esrd and an indication for early renal transplantation, a procedure that results in a cure for the underlying disease and significant improvements in patient's quality of life. h. jalaeian 1 , a. derakhshan 2 , d. derakhshan 2 , m. fallahzadeh 2 , z. bazargani 3 , m. basiratnia 4 1 shiraz organ transplantation center, nemazee hospital, shiraz, nemazee hospital, shiraz, iran 3 fasa university of medicine, pediatrics, fasa, iran 4 shiraz university of medical sciences, pediatric nephrology, shiraz, iran background: obesity is a major issue in the end stage renal disease population. while studies evaluating the effect of obesity on transplant outcomes in adults have yielded varying results, this issue is even still more controversial in children. methods: in a cross-sectional design, 71 pediatric recipients, aged 4-19 at transplantation and with normal graft function for at least 7 months after transplantation, were evaluated. we grouped the data with regard to the body mass index (bmi) percentiles as group i (bmi >95th), group ii (bmi <95th), group iii (bmi >85th), group iv (bmi <85th). we compared the clinical and laboratory findings between groups i and ii and between groups iii and iv. obesity was defined as bmi >95th and being overweight was defined as bmi >85th. results: there were 71 children (45 males, 26 females) with mean age at time of transplantation of 12.6±3.5 years (range, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] , and mean follow-up of 4.0±2.4 years. 12.7% of children were overweight and 5.6% were obese. no difference was found regarding age, height, duration of pretransplantation dialysis, or age at transplantation between groups i and ii and between groups iii and iv. (p>0.05). further more, no difference was found in regard to serum creatinine, bun, glomerular filtration rate, and 1-year graft survival rates among obese and/or overweight and other children. no correlation was found between bmi and gfr (p>0.05). conclusion: obese and overweight recipients can have excellent graft function and survival rates that are comparable to their non-obese counterparts. denying patients access to renal transplantation on the basis of obesity per se does not appear to be justified. d. derakhshan 1 , h. jalaeian 2 , a. derakhshan 1 , b. sabet 2 , m. fallahzadeh nemazee hospital, shiraz, iran 2 shiraz organ transplantation center, nemazee hospital, shiraz, iran introduction: tb is an important cause of morbidity and mortality in renal transplant recipients, especially in developing countries. this study was done to identify the incidence of tuberculin test positivity before transplantation as well as the influence on outcome of graft function and patient survival in children who receive renal allografts. methods: all 196 children with esrd who received a renal allograft between 1990 and 2006 were evaluated. as a routine pre-operative measure, a tb test was administered, using ppd. the ppdpositive recipients were compared with ppd-negative subjects, regarding age, gender, graft function, graft outcomes and patient survival rates. patients were divided into <5 mm versus >5 mm induration. results: the mean age of recipients was 14.03±3.20 years (range, 4-18) with a male/female ratio of 1.26:1. the majority of children were on chronic dialysis with mean duration on dialysis of 12.90±12.96 months. the tuberculin test was positive in 14.3% of children; all of them received isoniazide prophylaxis on diagnosis of latent tuberculosis. overall, the 1-year, 2-year, and 3-year survival rates were 94.47±0.02%, 92.44±0.025%, and 88.86±0.03%. three year survival rate was not different among ppd positive or ppd negative individuals. (90.00±0.09 vs. 93±0.03%; p>0.05) in addition, no difference was found for 1-year or 2-year graft survival rates (p>0.05). also serial serum creatinine levels at 1-month, 6-month, 1-year, 3-year, and 5year interval after transplantation was not statistically different (p>0.05). conclusions: detection of latent tuberculosis infection is an important step in the control of tuberculosis among asymptomatic pediatric kidney transplant. with proper management, latent tb does not affect transplantation outcome among children. h. xu, q. shen, ss. ruan, yl. bi, yq. lu, x. wang children's hospital of fudan university, department of nephrology, shanghai, people's republic of china objectives of study: we have started the first pediatric renal transplantation project in children hospital in china from 2004. survival of patients and grafts for the 5 patients are 100%. the clinical features were analysed and specific problems related to drugs and infections were reviewed. methods: 5 children (9~16 years old) underwent rtx. the duration of follow-up was 4 months to 32 months (average of 19.8 months). results: all the 5 patients were on automated peritoneal dialysis prior to rtx. the transplanted kidneys came from cadaveric donors (one were a 6-year-old brain-dead boy). 4 patients received il-2 receptor antibodies as induction therapy and the other one with alg due to high level of population reactive antibody. after the rtx, all the 5 patients were on triple immunosuppressive treatment (prednisolone, mmf, fk506 or csa) . no patient developed postoperative complication and delayed graft function occured. during the follow-up, 1 case suffered from calcineurin inhibitor renal toxicity and changed to rapamycin treatment, 1 from acute respiratory distress syndrome due to infection and 2 from elevated liver enzymes owing to drugs. one had acute rejection at 2 months after the operation. a severe anemia appeared on him after the rejection recovered. the cause of the anemia was found by the positive of serum anti-parvovirus b19 igm and completely recovered from the ivig treatment. at latest follow-up, the mean serum creatinine level of the 5 patients was 84.4±36.5 umol/l and egfr was 118.0±38.8 ml/min/1.73 m 2 . some patients received a support from "shanghai child renal failure trust fund". conclusions: the improvement of surgical technique, adequate dialysis prior to rtx, rational use of medicine, financial support and regular follow-up are all important for improving the outcome. h. bunker-wiersma 1,3 , j-c. davin the area under the curve (auc) of cyclosporine is strongly related to the efficacy and toxicity of the drug and its variability is mainly determined by the absorption phase. close therapeutic drug monitoring (tdm) is warranted to optimise therapy, using more appropriate methods to estimate the auc, than trough concentration measurement. from july 2004 we started auc guided monitoring of cyclosporine therapy based on two concentration measurements, c0 and c2. the results of this method are reported. methods: all paediatric renal transplant recipients treated with cyclosporine were included in the analysis. bayesian, model based estimation of auc values was performed at each out-patient visit or during hospital admission. calculated pharmacokinetic parameters, treatment efficacy data and side effects were collected over the 18 month period after introduction. target auc values were derived from previous studies in adult patients: 5400 ng/h/ml in the first three months after transplantation and 3250 ng/h/ml after three months. results: 15 early or stable post renal transplant patients were evaluated, divided in two groups (group i: <3 months after transplantation; group ii: >3 months after transplantation). in patients with trough concentrations below the therapeutic range, more than 50% auc's were in the therapeutic range in both groups. conclusion: auc guided monitoring of cyclosporine after kidney transplantation in children using c0 and c2 is practically feasible and presents the major advantage that c2 has not to be determined precisely 2 hours after csa administration. it is more closely related to the total drug exposure as compared with trough concentration monitoring and isolated c2. this method may facilitate the use of lower doses of cyclosporine and by this way limitside-effects. objectives: there is no satisfying data about reproductive functions after kidney transplantation in adolescence who have end stage renal disease (esrd) during childhood. we analysed the reproductive functions of kidney transplanted male adolescences. patients and methods: nine patients who followed between 1995-2006 were enrolled in the study. except one preemptively transplanted patient, all were on hemodialysis/peritoneal dialysis before transplantation. mean dialysis duration was 21 (9-54) months. their ages ranged between 10-17 years (mean 13.8) at transplantation. at the urologic examination, their mean age was 19 years. all patients had normal renal functions. results: all patients had normal testicular volume, libido and erectil functions. except one all patients had normal serum levels of lh, fsh, total and free testosterone. seven of the 9 patients semens were available for analysis. 3/7 patients had normal sperm parameters. transplantation had been performed before adolescence period in these 3 patients. one of these patients had been treated with intensive cyclophosphamide before. oligospermia was detected in 2, defective morphology in 3, low sperm motility in 6/7 patients. conclusion: although adult transplanted patients mostly have normal semen profiles; male children with end stage renal failure would not have normal spermatogenesis at the adolescence period; even after successful renal transplantation. in our study only 3 patients had normal semen profile, even hormon levels were normal. renal transplantation age seemed to be more crucial than the duration of esrd, of primary diagnosis or previous cyclophosphamide usage. r. vilalta, j. nieto, e. lara, a. madrid, s. chocron hospital materno-infantil vall de hebron, department of pediatric nephrology, barcelona, spain background: inhibition of il-2 receptors by basiliximab is irreversible and extended in time (mean 30 days). basiliximab (anti cd25 receptor) is used usually in the induction regime in our first cadaveric-donor kidney transplants. its re-use when chronic allograft nephropathy (can) develops could be useful. however some concern could exist related to possible adverse reactions (anaphylactic shock) linked to re-exposure to this drug because is an heterologous protein. less adverse reactions as lymphokine release syndrome has been described with the use of other monoclonal antibodies as the anti-cd20 receptor rituximab. objective: to describe our experience in the treatment with basiliximab of seven children with banf ii can. patients: seven children (2-16 years old (means 8.2 y.), 5 boys, 2 girls) showed biopsy-proved banf ii can. its period post-transplant ranged from 2 to 5 years (mean 4.1 y.) and their creatininine level from 2 to 4 mg/100ml (mean 3.2). all of them had been received at the transplant time basiliximab, tacrolimus or cyclosporine, mycophenolate and tapered steroids to reach 0.2 mg/kg/day in the third month post-transplant. when can developed, sirolimus was used in two patients, but was withdrawn due to increase of proteinuria. results: one dose of basiliximab (20 mg/1.73 m 2 ) was administered after 3 steriod pulses (10 mg/kg/day) in all patients. their basal immunosuppression was not changed. plasma creatinine diminished by 30% in four patients in the second week post-treatment and this improvement was sustained in two patients after one year follow-up. proteinuria did not change in any patient. in the course of this treatment no adverse reactions were observed. conclusion: 1) use of basiliximab in can is safe and possibly useful. 2) exposure to different monoclonal antibodies in paediatric kidney transplantation could be usual in the future; in the induction time, in the treatment of humoral rejection if exists (rituximab) and in the treatment of can. 3) it is necessary to establish that exposure and re-exposure to different antibodies is safe and without major adverse effects as our limited experience supports. r. vilalta, e. lara, a. madrid, s. chocron, j. nieto hospital materno-infantil vall de hebron, department of pediatric nephrology, barcelona, spain background: there are limited knowledge of kinetics and pharmacodynamic effect of sirolimus in paediatric renal transplantatation. provided that sirolimus is effective and safe in combination with tacrolimus and mycophenolate (mmf), the initial dose needed, the evolution of blood levels and the steady state should be studied in order to optimise its clinical use. objective: to establish a possible correlation between dose/level ratio of rapamune and other parameters as age, gender or puberal state. patients and methods: between 2000 and 2006, 19 paediatric patients (11 girls, 8 boys) received a cadaveric kidney transplant. age ranged from 4 to 16 years (mean 8 y.), and all of them received mmf and steroids. sirolimus were used from 0.02 to 0.1 mg/kg/day, to obtain levels between 6 to 12 ng/ml. results: dose/level ratio obtained allowed us to describe three types of patients: an infant-type i dose-level patient (age 4-8 y), a prepuberal type ii (age 8-12y) and an adult-type iii dose-level patient (age 12-16y). type i needed sirolimus between 0.08 and 0.1 mg/kg/day (sd±0.01), type ii between 0.04 and 0.8 mg/kg/day (sd±0.015) and type iii between 0.02 and 0.04 mg/kg/day (sd±0.018) to obtain all of them a constant blood levels between 6 and 12 ng/ml. the same positive correlation was obtained regarding the puberal status. no correlation were observed regarding the gender. introduction: developing of diabetes mellitus after renal transplantation is one of the determining factor in the survival of the patient and the graft. in present study we assessed the carbohydrate metabolism status of ntx. methods: we analyzed 45 patients' data about recently developed carbohydrate metabolism failure after ntx. children underwent ntx between 1990-2005 were investigated. thirty-nine children (16 girls/23 boys) underwent ogtt, who had no ptdm. we analyzed the incidence of ptdm/igt, the combination of immunsuppressive therapy, the number of transplants, the proportion of cadaver/living donor, hcv, blood pressure, lipid metabolism, bmi, graft function parameters and the time since ntx. results: ptdm developed in 6 children (13%). four of 6 patients required insulin therapy. we diagnosed igt in 7 of 39 with ogtt investigated patients (16%). all ptdm/igt patient got tacrolimus and continous steroid therapy. the dose of steroid was 6.5 mg/day in the ptdm/igt group vs. 5.2 mg/day no ptdm/igt (p<0.05). during ogtt the trough level of tacrolimus was higher in the ptdm/igt group 14.3 ng/ml vs. 10.1 ng/ml (p<0.05). in the other parameters we did not find any significant differences between ptd/igt and no ptdm/igt patients. discussion: the most important reasons in the development of ptdm and igt after transplant are steroid therapy and higher tacrolimus trough level. in transplant children we recommend the regular fasting glucose and ogtt examimation, the reduction of steroid and tacrolimus in case of stable graft function. otka f-042563, otka-t046155, ett 435/2006 , ett 184/2003 the introduction: measurement of plasma bnp is a novel noninvasive approach in the assessment of cardiovascular status. in our study we investigated the role of bnp in the monitoring of cardiovascular status of children with crf or renal transplant (ntx). methods: we examined 32 children with crf (n=17, 11 boys/6 girls, age: 12,1 year (3,5-20)) or ntx (n=15, 9 boys/6 girls, age: 16, 25) ). patients underwent echocardiographic investigations (ivs, lvedd, lvesd, pw and fs) and their bnp levels were measured (age matched normal values were used). other cardiovascular risk factors, such as hgb, htk, ca, p, creatinine and blood pressure were also evaluated. a correlation between bnp and echocardiographic results was calculated. results: the values of lvesd, fs and bnp levels of renal transplant patients were significantly better than those of crf patients (p<0,05). the other parameters did not show significant differences. bnp levels were significantly higher in all age groups of crf patients as compared to the normal levels. in younger ntx patients this value was within normal limits. in older ntx patients, and in those that had their transplants a long time ago we measured higher bnp levels, which correlated significantly with graft function as well (p<0,05). bnp showed a significant positive correlation with lvesd and a significant negative correlation with fs only in crf patients. the elevated bnp levels showed the worsening of cardiac function even when the echocardiographic parameters were still normal. the hgb, htk, ca, p and creatinine values were significantly better in ntx patients and showed no correlation with bnp. summary: bnp is an early, easily usable marker in diagnosing and following decreased cardiac function of both crf patients and after ntx. otka f-042563, otka-t046155, ett 435/2006 , ett 184/2003 c. garcia 1 , v. bittencourt 1 , s. vitola 3 , e. didone 3 , e. guerra 3 , f. pires 3 , a. tumelero 1 , d. malheiros 1 , v. garcia department of pediatric nephrology, porto alegre, brazil 2 complexo hospitalar santa casa, department of nephrology and kidney transplantation, porto alegre, brazil 3 complexo hospitalar santa casa, department of surgery, porto alegre, brazil the objective is relate the results of 348 consecutive kidney transplants carried out in children in a single center. patients and methods: analysis of kidney transplants performed in patients less than 18 years old, carried out from may 1977 to december 2006. results: 348 kidney transplants were performed. 48% of the patients were female, 86% were caucasian and 14% were african-brazilian. the mean age at the transplant was 11.3±4.5 years. the most frequent etiology of renal failure was vesico-ureteral reflux/obstructive uropathy (35%), followed by glomerulopathy (26%). the donor was deceased in 34% and living related in 66% (parents 82%). the initial immunosuppression was cya+aza+pred in 38.9%, cya+mmf+pred in 6,9%, tac+aza+pred in 8.6%, tac+mf+pred in 23.9%, tac + mf without pred in 8.9%. sirolimus was employed initially in 4 cases. induction with okt3/atg occurred in 5 patients and 157 received anti-il2 receptor antibody. the 110 graft losses during 29 years of follow-up were secondary to chronic allograft nephropathy in 58 (51%), vascular thrombosis in 6 (5.2%), acute rejection in 13 (11.2%), recurrence of original disease in 14 (12.1%). there were 27 transplants in 21 patients with focal segmental glomerulosclerosis, 12 (57.1) had a recurrence after transplant. eight were treated with plasmapheresis and 75% obtained a total remission. the survival of graft in the first, fifth and tenth year was: 90%, 72% and 59% respectively. the graft survival in the 5th year according the immunosuppression was 41% using azathioprin and prednisone, 72% with cya/aza or mmf and 78% with tac/aza or mmf. the patient survival in the first, fifth and tenth year was: 95%, 93% and 85% respectively, infection was the main cause of death. j. feber 1 , p. geier 1 , b. chaudry 1 , h. wong 1 , g. filler 2 1 children's hospital of eastern ontario, division of pediatric nephrology, ottawa, canada 2 london health science center, departments of pediatrics, london, ontario, canada successful pediatric renal transplantation (tx) should fully correct the metabolic abnormalities of end-stage renal failure. however, ckd may persist because of only half of the normal nephron endowment and other factors (ischemia, nephrotoxocity etc). height, bmi and blood pressure (bp) z-scores, cystatin c-gfr, hemoglobin (hb), serum pth, hco 3 , cholesterol, mycophenolic acid (mpa) and sirolimus (sir) levels were analyzed retrospectively in 21 tx recipients (10 males, age 8.5±5.8 years) at 4 months post tx (t1) and at 3.26±2.21 years (median 2.4) post tx (t2). data are expressed as mean±sd. height z-scores remained significantly lower than controls (t1: -0.90±1.17; t2: -0.75±1.23, ns), growth failure occurred in 19% of pts at t1 and 26% of pts at t2. bp z-score did not change from t1 to t2, but hypertension was diagnosed in 66% pts at t1 and 79% pts at t2. gfr (ml/min/1.73 m 2 ) was 71.9±18.6 at t1 and 70.9±23.7 at t2 (ns), mean decline of gfr was 3.9±1.1%/year. hb z-score remained below normal at -1.55±2.04 at t1 and -1.10±1.27 at t2 (ns) and anemia was diagnosed in 62% and 63% of pts at t1 and t2 respectively, despite trough levels of both mpa (2.77±1.43 mg/ml, 11 pts) and sir (7.73±2.55 mg/ml, 10 pts) that would be considered adequate. hypercholesterolemia was detected in 23.8% pts at t1 and 42% pts at t2, whereas only 9.5% of pts at t1 and 16.8% of pts at t2 were labeled as obese. bone disease was diagnosed in 28.5% pts at t1 and 15.8% pts at t2. we observed suboptimal growth, hypertension, hypercholesterolemia, bone disease and persistent anemia in a significant proportion of tx children despite iron supplementation, adequate mpa and sir levels and good kidney function. these ckd complications require careful monitoring and intervention. a. al midani 1 , g. koffman 2 , j. john 2 , s. stephen 2 , s. suzanne 2 , r. lord 2 1 royal free hospital, transplantation, london, united kingdom 2 great ormond street hospital for children nhs trust, transplantation, london, united kingdom objectives: to document factors predisposing towards surgical complications over 7 years in a single pediatric renal transplant centre. methods: we retrospectively analysed 179 consecutive renal transplants between jan 2000 and dec 2006. patients were divided into group 1, without complications, and group 2, with complications. we compared variables previously identified as risks for surgical complications between the two groups: live/deceased donor, donor and recipient age, gender and weight, side of organ donation, cold ischaemia time, single/multiple vessels, intraperitoneal/extraperitoneal approach, anastomosis to aorta/iliac vessels, thrombosis prophylaxis (changed from heparin to aspirin in oct 2000). results: 141/179 (81%) were complication free; 19% patients developed one or more surgical complication: wound infection 2/179 (1.1%), wound dehiscence 3 (1.7%), prolonged ileus 1 (0.5%), lymphocoele 7 (3.9%). 7 patients were re-explored: 5 (2.8%) for bleeding, 2 (1.1%) for graft repositioning. we observed 2 (1.1%) cases of renal artery stenosis. overall, 5 (2.8%) graft loss occurred secondary to thrombosis, 13% (3/23) prior to changing our prophylaxis from heparin to aspirin (1.2% on aspirin). urological complications occurred in 7 (3.9%): 3 ureteric leaks and 4 ureteric stenoses. the variables between group 1 and group 2 were as follows: under 20 kg: 19% v 35%, less than 5 yrs old: 12% v 30%, intraperitoneal approach: 13% v 37%, anastomosis onto the aorta: 18% v 43%, no aspirin prophylaxis: 9% v 24%, other variables were the same in both groups. conclusions: 19% of patients developed surgical complications. a higher rate of surgical complications was seen in recipients under 5, using the intraperitoneal approach onto the aorta. the introduction of aspirin prophylaxis reduced graft loss due to thrombosis from 13% to 1.2%. other variables did not affect the complication rate. m. medeiros 1 , v. sharma 2 , r. ding 2 , s. valverde 1 , am. hernández 1 , p. garcía 1 , y. fuentes 1 , m. suthanthiran 2 1 hospital infantil de mexico federico gomez, departamento de nefrologia, mexico, mexico 2 weill cornell medical college, immunogenetics and transplantation center, new york, ny, united states the forkhead transcription factor foxp3 is highly expressed in cd4+cd25+ regulatory cells (tregs). the foxp3+cd25+cd4+ cells play a central role in immune tolerance and tgf-β 1 is reported to induce foxp3 expression in vitro. whether there is an in-vivo association between foxp3 and tgf-β 1 is not known. we investigated the hypothesis that there is a positive association between foxp3 and tgf-β 1 in children with stable renal graft function. parental written informed consent was obtained before enrollment in all cases. 24 children with stable renal allograft function for a minimum of 12 months were studied. a complete clinical examination was performed; peripheral mononuclear cells were collected for measurement of transcripts for foxp3, tgf-β 1 , tgf-β 2 , and 18s rrna (house keeping gene) using by real time quantitative pcr assay. correlation between transcript levels was performed using pearson r. results: 24 pediatric recipients of renal allografts were studied. tgf-β 1 and foxp3 were highly expressed in peripheral blood mononuclear cells, and there was a highly significant and positive correlation between levels of mrna for foxp3 and tgf-β 1 (r=0.811, p<0.0001)), whereas no significant correlation was found between tgf-β 1 vs. tgf-β 2 (and tgf-β 2 vs. foxp3). conclusion: foxp3 expression in vivo is strongly correlated with tgf-β 1 expression in peripheral mononuclear cells of stable renal transplant recipients. introduction: studies suggest that pre-emptive lamivudine therapy improves survival in hbv renal transplants. however, long-term outcome is not well established. method: four chinese adolescents with chronic hbv infection were transplanted. they were put on cyclosporin a, mycophenolate mofetil and prednisolone. prophylactic lamivudine was given just before transplantation and was continued afterwards. hbv status and liver enzymes were monitored serially. results: four patients were transplanted at the age of 15.5±3.0 (13.9-19.9) yrs old. they were followed up for 74.7±10 (61-85) months and no mortality was reported. alanine transaminase (alt) was only transiently elevated in the first 2 months post-transplant in all cases and became normal afterwards. there was no hepatitis flare and liver function was normal at the last follow-up. hbeag and hbv dna were positive in 1 patient before transplantation and remained positive at the latest follow-up. mutation in the ymdd motif of the hbv genome was detected in the same patient and undeterminable in the other three due to low virus load. this patient remained clinically stable with normal liver function except there was a rise of viral load from baseline. all grafts were functioning and there was one late acute cellular rejection which responded to treatment and there was no hepatitis flare. latest mean serum creatinine was 141±63 (56-208) umol/l. conclusion: ymdd mutation and resistance to lamivudine treatment may happen but appear to have little clinical significance. our long-term results showed that renal transplant seems feasible and safe in this population up to 7 yrs follow-up. there are no studies in mexican children (mx) . the aim of the study was to determine tacrolimus pharmacokinetics (pk) in mexican renal transplant children and compare it wih reported pk in aa and ca. methods: a seven point pharmacokinetic profile (0, 0.5, 1, 2, 4, 8 and 12h) was performed in ten children receiving tacrolimus as part of the immunosuppressive therapy, mean age was 13.9±2.19 years, mean post transplant time 9.8±35 months. c0 and cmax were obtained directly from experimental points, the auc and t1/2 was obtained with a non-compartmental model using winnonlin version 3.0. results: in cyclosporine (csa) is widely used for immunsuppression in transplant recipients and for treatment of srns. however, patients can develop csa associated cutaneous side effects, e. g. hypertrichosis, skin cancer, and viral warts due to human papillomavirus infection. here we report on a 23-yearold boy suffering from a microdeletion syndrome (18 q-) and srns (histology: minimal change nephropathy) starting at the age of 20 years. since the initial combination therapy with corticosteroids and cyclophosphamide was associated with severe side effects (sepsis, leukopenia) and did not lead to sustained remission csa therapy was initiated. csa-treatment resulted in rapid clinical remission. however, after 12 months the patient developed viral warts (hands, trunk and head), although csa trough levels were kept below 100 μg/l. therefore, immunosuppression was switched to mmf (2x500 mg/day) resulting in sustained remission of srns and rapid disappearance of viral warts within 4 months. conclusion: conversion to mmf may be a usefull treatment strategy in srns showing csa associated side effects like viral warts. 2003-2006. 5 were live related (lrd) and the remaining cadaveric (cad). 6 were pre-emptive(pet). all received basiliximab induction 2 hrs prior to surgery. in addition, induction immunosuppression consisted of tacrolimus and methylprednisolone. in all but 2 patients, basiliximab was re-administered at day 4. 2 patients, aged 11 and 6 yrs (one cad and other lrd respectively) developed acute noncardiogenic pulmonary oedema 6-48 hrs after transplantation. both children had renal dysplasia as primary cause of renal failure. both required delayed ventilation and were ventilated for 4 to 6 days respectively. there was a rapid rise in c reactive protein in both patients. both grafts had primary function, but the cad transplant subsequently developed acute tubular necrosis, and was eventually lost within 3 weeks due to thrombotic micro angiopathy and severe acute antibody mediated rejection despite immunosuppression with sirolimus, mycophenylate, steroids and plasma exchange therapy. conclusion: we report a rare but serious side effect of basiliximab. to our knowledge, this is the first report of basiliximab induced non-cardiogenic oedema so early post transplantation and in such young children. early recognition and aggressive appropriate supportive therapy is vital for patient and where possible, graft survival. 3 (17); cmv seroconversion (4); seizures (4); hypertension (9), uti (6), adverse reaction to basiliximab (2), delayed graft function (4), acute rejections (2), chronic allograft nephropathy (1). 17 patients had well matched kidneys (3 or less mismatches), 11 were poorly matched. 2 grafts were lost from latter group, both were cad, 1 had acute tubulointerstitial nephritis and tacrolimus toxicity and the other thrombotic microangiopathy and eventually acute antibody mediated rejection. chronic allograft nephropathy (can) is a complex phenomenon caused by underlying kidney disease and superimposed by environmental and genetic factors. we investigated the association of polymorphism in the genee nos with the can. nitricoxide is synthesized from l-arginine in vascular endothelial cells by nitric oxide synthase. endothelial nitric oxide plays an important role in endothelial dysfunction and involved in the inflammation. the gene encoding enos maps to chromosome 7q35 7q36.7. a missense variant of the enos gene in exon 7 shows a transversion of g to t at nucleotide position 894 (g894t) that results in a replacement of glu by asp at amino acid residue 298 (glu298asp). the aim was to investigate the association between can and g894t polymorphism of the endothelial nitric oxide synthase gene. the g894t mutation at exon 7 of the endothelial nitric oxide synthase gene, enos gene polymorphism, was analyzed in 61 turkish children with renal transplantation. the g894t polymorphism of the endothelial nitric oxide synthase gene was determined by polymerase chain reaction and restriction fragment length polymorphism. were grouped according to stages of chronic kidney disease (ckd) as estimated by the calculated glomerular filtration rate (gfr, schwartz formula). measurements of structural and functional surrogates for cardiovascular disease (cvd) included intima-media thickness (imt) of the common carotid artery (cca), pulse wave velocity (pwv) and augmentation index (aix). aix and pwv reflect the degree of arterial stiffness and were calculated from pulse wave recordings at the arteria carotis and arteria femoralis (sphygmocor device). results: patients and healthy control subjects had a mean age of 14 years. imt was not significantly different in patients and controls. significant differences were found in the aix, which was increased by 50%: the mean aix was in healthy subjects was -28,2% and in transplanted subjects -14,4%. pwv was increased by 15% (4,74 m/s vs. 5,43 m/s). both aix and pwv increased in parallel with the degree of renal impairment (stages of ckd). table 1 . discussion: weight gain post transplant is multifactorial, like cultural, psychological and associated to steroids. weight gain was observed in general, patients with overweight or in risk of overweight didn't loose weight postransplant even they were aware of the consequences. introduction: tacrolimus is metabolized by cytochrome p450 3a and has a narrow therapeutic range. we report a kinetic interaction between tacrolimus and amlodipine, a potent cytochrome p450 inhibitor, resulting in anuric acute renal failure. case report: a 14-year-old male renal transplant recipient received amlodipine, a calcium channel blocker as antihypertensive treatment while he was on tacrolimus (0.1 mg/kg per day). he presented first with diarrhea and developed subsequently, dizziness and fatigue, related to acute anuric renal failure, requiring hemodialysis for 20 days. tacrolimus trough levels were in the desired therapeutic range (3-6 ng/ml) until recently. three days after introduction of amlodipine, tacrolimus trough levels increased to a toxic level of 38.8 ng/ml. after discontinuation of amlodipine, tacrolimus levels returned to the normal range in seven days and renal function recovered progressively. no polymorphisms in the expression of cyp3a and p-glycoprotein were detected. discussion: tacrolimus is known to be a substrate of p-glycoprotein, responsible for drug secretion into the intestinal lumen and metabolized by enterocytic cyp3a. amlodipine is a competitive inhibitor of cyp3a. as no abnormalities of cyp3a and p-glycoprotein were found, we suspect that drug interaction due to competitive inhibition of tacrolimus metabolism by amlodipine was responsible for these toxic effects. concomitant diarrhea might have played an additional role for increased tacrolimus serum levels, presumably in relation to diarrhea associated dysfunction of enterocytic cyp3a and p-glycoprotein. conclusions: amlodipine and diarrhea increase tacrolimus blood concentration by inhibiting its metabolism. amlodipine should not be used in patients on tacrolimus. careful monitoring of tacrolimus blood levels is recommended in case of concomitant diarrhea. urinary tract infection (uti) remains a significant cause of infectious complications in renal transplant recipients. the aim of the study was to determine the frequency of uti following renal transplantation in our center. the records of 34 patients (f/m: 14/20) who underwent renal transplantation were evaluated retrospectively. among them 12 patients (f/m: 9/3) were found to have at least one episode of uti during follow-up. the records were examined for the age, sex, primary disease, and duration of chronic renal failure, donors, posttransplant follow-up and recurrence of uti. biochemical analysis of blood for renal functions, complete blood count, creactive protein and sonographic examination of patients were also recorded and results were compared with renal transplanted patient who did not develop any episode of uti (group 2). the mean age of the group 1 was 14.6±4.7 years, while it was 14.5±3 years in the group 2. mean duration of post-transplant follow-up was 2.8±1.7 years for group 1 and 2.4±1.7 for group 2. four patients (33.3%) in group 1 and 7 patients (33.3%) in group 2 had vesicoureteral reflux (p>0.05). five patients had single uti while 7 patients had more than one uti. though we did not find any difference between girls and boys in terms of presence of vesicoureteral reflux, frequency of uti in girls was found to be significantly higher than in boys (p=0.003). ultrasonographic examination of patients during uti in group 1 revealed pyelonephritis in 3 and hydronephrosis in 2 patients. the most frequent microorganism causing uti was e. coli. age, donor source and etiology of chronic renal failure did not influence the incidence of urinary tract infection. our data suggests that urinary tract infection remains a frequent but mostly benign complication in the pediatric transplant population, especially in female gender. the growing population of transplanted patients requires the consideration of the potential side effects of the different treatment regimens. experience of the last decade with calcineurin and nucleoside reverse transcriptase inhibitors revealed important renal side effects. we describe a 15 years old girl who was known to have liver failure related to wilson's disease (wd). she had orthotopic liver transplantation from her mother 3 years ago and treatment with tacrolimus and mmf was initiated. although she was known to have proximal renal tubular acidosis secondary to wd, renal tubular functions were found to be normal within the three months of transplantation. two years after the transplantation lamivudine was initiated because of de novo hepatitis b infection in transplanted liver. a couple of months later she developed renal fanconi syndrome with metabolic acidosis, hypophosphatemia, glycosuria and aminoaciduria. she needed high doses of sodium bicarbonate and phosphate supplementation. tacrolimus was suspected to be the cause of late post transplant renal acidosis and was replaced by sirolimus. however, 3 months later, at the 6 th month of lamivudine treatment, she was hospitalized because of metabolic acidosis, mild hyperglycemia and inability to walk. electromyographic examination revealed myopathic changes while liver biopsy was normal with a normal tissue copper level. renal biopsy showed findings of karyomegalic nephropathy which could be the result of the action of antimitotic agents. we suspect that our patient's tubular dysfunction, myopathy and hyperglycemia may have resulted from mitochondrial dysfunction which is triggered by tacrolimus and augmented by lamivudine. however, randomized and prospective studies with large groups of patients are needed for definite results about mithocondrial side effects of these drugs. recombinant factor viia (rfviia, novoseven) is a new hemostatic agent that was initially indicated in hemophiliac patients. recently it has been used successfully for the treatment of bleeding in patients with thrombocytopenia, and acquired and congenital platelet dysfunction. epstein syndrome, also known as alport-like syndrome, is a rare autosomal dominant disease characterized by proteinuria, chronic renal failure, hearing loss, and thrombocytopenia with giant platelets. our group previously reported functional alterations of giant platelets of boy with epstein syndrome, who rapidly progressed to end stage renal disease during adolescence. the first nonheartbeating kidney transplantation at age 17 was failed because of the severe postoperativebleeding irresponsible for traditional therapy (packed red cells, platelets, and fresh frozen plasma), result in immediate graft failure and the need for transplant nephrectomy. the second kidney transplantation was 4 years later, after a single intravenous bolus injection 70 μg/kg body weight rfviia, which was repeated one and 12 hours after the surgery. rfviia successfully controlled the bleeding in the peri-and postoperative phase and no side effect and thrombotic complication occurred and his graft function is still stable after 4 years. recombinant factor viia may have a potential role in the treatment of phenotypic bleeding associated with chronic kidney disease. cyclosporin a (csa) and mycophenolic acid (mpa) have a wide interindividual variability in their pharmacokinetics (pk). among others, intestinal p-glycoprotein (p-gp) expression and cyp3a4 activity have been held to be responsible for that variability. in adult kidney transplant (rtx) patients, an influence of these gene polymorphisms has not been shown; however, there are no data in pediatric patients. we reasoned that such an influence might be masked in adults by confounding environmental factors accumulating over the decades of life. we therefore investigated a possible influence of gene polymorphisms of p-gp and cyp3a4 on defined dose-adjusted pk-parameters in 70 children with rtx (age 11.6; range, 3.2-17.4 yrs). pk parameters (auc, c2) were assessed 1, 3, 12, and 24 weeks after rtx. real-time, rapid-cycle pcr methods were used for genotyping. the allele frequencies for the mdr1 c3435t allele (expression and in vivo activity of p-gp) of 62% and for the cyp3a4-v allele of 3% were comparable to those reported for caucasian populations. dose-adjusted pk parameters of csa and mpa were not significantly different in patients with and without the cyp3a4-v allele or patients with different mdr1 c3435t genotypes. along with that finding, neither of the polymorphisms investigated into was associated with renal function or the incidence of acute rejection episodes. we studied how the il-2r β-chain becomes enriched in lipid rafts of activated human t cells, isolated by ficoll gradient and sheep red cell rosetting, and how its tyrosine phosphorylation, which requires its heterodimerization with the common cytokine r β-chain (βc), occurs there. imunoblots (ibs) of sucrose gradient fractions of cell lysates obtained during a 72-hour activation with phytohemeagglutinine (pha) showed the gradual, largely selective, translocation of the β-chain into rafts. as dimerization or lipid modification can be mechanisms underlying raft enrichment, we assessed lysates of pha-activated cells in ibs under non-reducing versus reducing and crosslinking conditions but did not see evidence of β-chain dimerization. however, exposure to cycloheximide to interfere with post-translational acylation, or to the palmitic acid analogue 2-bromohexadecanoic acid substantially diminished raft enrichment of the il-2r β-chain. we next performed ibs of il-2r β-chainand βc-immunoprecipitates from raft and non-raft fractions of activated t cells before and after il-2 treatment. we found that il-2 exposure triggers the translocation of small amounts of βc, accompanied by il-2r β-chains, into rafts, resulting in its heterodimerization with the il-2r β-chain and their tyrosine phosphorylation. all of these processes were attenuated in the presence of the il-2r β-chain-blocking antibody daclizumab. we conclude that the raft enrichment of the il-2r β-chain requires palmitoylation and provides the focal point for the formation of the highaffinity il-2r via il-2r β-chain-mediated "chaperoning" of few βcs into these domains, establishing novel raft-dependent mechanisms underlying cytokine r specificity and selectivity in human t cells. iga nephropathy (igan) is an immunecomplex disease resulting from a defect in mucosal iga response. food antigens have been implicated in the pathogenesis. gut permeability to antigenic substances is immature at birth and its maturation is delayed by early administration of antigenic foods while breast feeding accelerates this process. we aimed to evaluate if exposure to antigenic foods in early life is associated with a predisposition for igan in childhood. three groups including children with igan (group 1, n=33), primary non-iga glomerulopathies (group 2. n=25) and healthy controls (group 3, n=40) were formed. their parents filled a questionnaire regarding the age at diagnosis, gestation time, birth weight, feeding by breast milk, formula, cow's milk and complementary foods. all groups were similar for age, sex, gestation period, birth weight and the rate and duration of breast feeding. in addition, the rate of formula feeding was also similar in all groups. however, cow's milk consumption rate was higher in group 1 and 2 than in group3. introduction of formula was earlier in groups 1 and 2 than in group 3. in addition, the children in group 1 were younger than the other groups at the onset of feeding by cow's milk and weaning. roc curves predicted 3.5, 3.75 and 5.5 months as the best cut-off age values for formula feeding, cow's milk feeding and weaning for predicting the presence of igan, respectively. ors for igan with respect to these cutoff levels were 28 (95% ci: 4-189), 5.7 (95% ci: 1.9-17.2) and 10.5 (95% ci: 3.9-28.0), respectively. the results of this preliminary study indicate that early introduction of antigenic foods might increase the risk of future primary igan. results: they were 8 males and 2 females, with a male: female ratio of 4: 1. their ages ranged from 5 months to 15 years (mean 6.8 years), with a peak age of 5-9 years. the common presenting complaints were generalised oedema (60%); oliguria (50%) and hypertension (50% we report nine patients (three males) with mesangiocapillary glomerulonephritis (mcgn) from a single paediatric nephrology centre. the average age at presentation was 12.0 years (range 9.2 to 13.4). all had nephrotic syndrome. seven had mcgn type 1 and two had mcgn type ii. six of seven tested had positive c3 nephritic factor. three patients responded well to steroids and ace inhibitors and received no further therapy. five had a good response initially but relapsed when steroids were tapered and one patient had a poor response to steroids. these six patients received calcineurin inhibitor (ci) therapy. four responded well with resolution of proteinuria. one patient relapsed when tacrolimus was withdrawn after 23 months of therapy but proteinuria resolved after re-introduction of therapy. two patients had a poor response to ci therapy. one remains stable but with heavy proteinuria. the second patient (with mcgn type ii) initially had a complete remission of proteinuria on steroids but relapsed 30 months after presentation while on prednisolone 5 mg on alternate days. repeat biopsy showed 65% crescents. treatment with pulsed intravenous steroids, cyclosporin and mycophenolate mofetil was ineffective and she progressed rapidly to end stage renal failure. we conclude that ci treatment might be useful in mesangiocapillary glomerulonephritis. prospective, randomised controled trials are required to determine their place in the management of this disease. iga nephropathy (igan) is caused by a primary defect in mucosal iga response leading to increased antigenic stimuli reaching to bone marrow. enteric flora is important for mucosal and systemic immunity, and probiotics regulate specific and innate immunity by maintaining microbial balance in the gut. saccharomyces boulardii (s.boulardii), a probiotic, increases intestinal siga production, protects enteric infections and also prevents atopic and immunoinflammatory diseases. we aimed to evaluate the effect of s.boulardii on experimental igan, induced by oral polio virus vaccine (opv) administration to the mice. four groups of male balb/c mice (n=7 for each) were formed. groups i and ii were immunized enterally by opv at the onset, 2 nd and 4 th weeks. group ii was also given s.boulardii in drinking water throughout the study. group iii was given only s.boulardii, while group iv received no treatment. two weeks after the last opv dose, all the animals were sacrificed to obtain their kidneys for histopathological evaluation and all four groups were compared with respect to the severity of histopathological changes. while there was mild to moderate mesengial proliferation and widening, tubular atrophy, interstitial inflammation and fibrosis in group i, no remarkable histological changes in the other groups were noted. immunofluorescence microscopy revealed universal deposition of iga and some c3 in group i, while there was no iga or c3 deposition in the other groups. electronmicroscopy revealed mesengial proliferation along with matrix expansion, focal basement membrane thickening and electron-dense deposits in the mesengial area in only opv group and the other groups were normal. in conclusion, enteral s.boulardii administration prevented experimental igan development in mice. the aim was to assess the correlation of renal histopathological findings with clinical diagnosis in order to recognize the pattern of kidney diseases in our pediatric population. methods: a total of 95 renal biopsies performed on children who presented to the surgical kidney hospital in damascus during a period of 3 years were retrospectively reviewed. results: nephrotic syndrome alone accounted for 52% of all cases, followed by hematuria in 21%, mild to moderate renal impairment including allograft dysfunction in 15%, nephritic syndrome in 10%, and hsp in 2%. the most common histologic lesion was mcd in (29%). fsgs was the second most common lesion (13%) followed by mesangial gn (11%), mpgn (9%), post-infectious gn (5%), iga nephropathy (4%), membranous gn (3%), cns of finnish type (2%), alport syndrome (2%), interstitial nephritis (2%), nephronophthisis (2%), hsp (2%), acute rejection (2%), chronic rejection (2%), nephrocalcinosis (1%), crescentic gn of undetermined origin (1%), and lastly, 5% were completely within normal limits. familial and inherited diseases were encountered in 15%. histopathologic diagnosis was mostly useful in nephrotic cases. while in hematuria cases, the usefulness of the histologic findings in terms of therapeutic and/or prognostic point of view was definitely less. one of the reason for that in our series is perhaps because we still do not have facilities to perform electron microscopic evaluation of the renal tissue. however, controversy about the usefulness of renal biopsy in such cases is still there. conclusion: this study provides an important data on the pattern of pediatric renal diseases in our center and highlights the usefulness histologic findings in guiding the therapeutic plan especially for nephrotic children. aim: the aim of this study was to determine the efficacy of tacrolimus in the management of sr fsgs in children. study design: this was a prospective study of 20 children with sr fsgs treated with tacrolimus (0.2-0.4 mg/kg per day in 2 divided doses over 12 hours adjusted to a trough level between 7-15ng/ml) for 12 months in combination with low dose steroids. other therapies included angiotensin converting enzyme inhibitors, folic acid, multivitamins and lipid lowering agents. results: the mean age at study entry was 11.1 years (range 5.6-16.8). the mean duration of nephrotic syndrome before initiation of tacrolimus therapy was 4.7 years (range 2.1-7.6). at the end of the treatment period 8 (40%) children were in complete remission, 9 (45%) children were in partial remission and 3 (15%) failed to respond. the average period of follow-up following cessation of tacrolimus treatment was 27.5 months (range 13.7-43.7). at last hospital follow-up 5 (25%) of children were in complete remission, 10 (50%) in partial remission and 2 (10%) in relapse. 3 children demised from dialysis related complications following cessation of tacrolimus treatment. adverse events included sepsis (2), nausea (2) diarrhea (2), anaemia (4) and worsening of hypertension (4) . conclusion: tacrolimus is a safe and effective treatment for sr fsgs. however, like cyclosporine some children tend to relapse following cessation of treatment. it has been rarely reported in association with graves-disease. now we present a previously healthy 6-year-old japanese girl who had proteinuria due to stage i mn and graves disease. patient: she was found to have 2+ proteinuria and a goiter at her school medical examination simultaneously. serum free thyroxine was 4.98 ng/dl (normal range 0.95~1.74), thyroid-stimulating hormone (tsh) less than 0.003 microu/ml (0.34~3.88), anti-microsomal antibody 1600t (~100), anti-thyroglobulin antibody 400t (~100), and tsh-receptor antibody 84% (~10) consistent with graves' disease. the electron microscopy finding of her renal biopsy specimen showed the presence of electoron-dense deposits located in the subepitherial and intramembranous spaces. with immunofluorescence microscopy, the bright granular staining of igg along the gromerular capillary wall was found. these findings were characteristic of mn. objectives of study: to investigate whether graves disease caused mn in this patient. methods: we examined the presence of thyroid microsome and thyrogrobulin in glomeruli by immunofluorescence study using anti-thyroid microsomal antibody and anti-thyrogrobulin antibody. result: glomerular granular staining of thyroid microsomal antigens was demonstrated corresponding to igg granular deposits, but that of thyrogulobulin was absent. conclusion: mn in this patient is presumed to be caused by immunecomplexes mediated by thyroid microsomal antigens. objective: to explore the role of oxidative stress reaction on the injury of glomerular podocyte slit diaphragm molecular barrier. methods: thirty-two male spraque-dawley (sd) rats were randomly divided into control, low dose (3.0 mg/kg), nephrotic (7.5 mg/kg), overdose (10.0 mg/kg) groups by the dosage of adriamycin (adr) injection.the levels of malondialdehyde (mda) glutathione peroxidase (gsh-px), hydroxy radical ( . oh) and superoxide dismutase (sod) in renal cortex were measured; the expression of podocin was measured with immunohistochemistry. results: (1) compared with control group, the levels of mda in renal cortex and 24-hour urinary protein were increased, the levels of sod in renal cortex was decreased in adr-treated groups, especially in nephrotic group (p<0.05). (2) in control group, podocin staining was a sable linearlike pattern along the capillary loops of glomerulus; in nephrotic group, podocin staining was a light tan discontinuous punctiform or short linear-like pattern along the capillary loops of glomerulus. compared with control group, the score of podocin immunohistochemical staining was decreased in adr groups, especially in nephrotic group (p<0.05). (3) there were some significant negative correlations between the score of podocin immunohistochemical staining and the levels of mda in renal cortex. there were some significant positive correlations between the score of podocin immunohistochemical staining and the levels of sod in renal cortex. conclusion: (1) there was close relationship between podocin and the development of proteinuria. (2) there were significant correlations between the reduction of podocin in glomerular podocyte slit diaphragm and oxidative stress reaction, especially lipid peroxidation. lupus nephritis (ln) remains an important problem in patients with sle. to evaluate the clinical course, histopathology and the efficacy and safety of high-dose pulse cyclophosphamide (ctx) in children with ln. retrospectively, 25 children with ln were studied; all patients underwent renal biopsy and were followed up for at least 3 years. the clinical and serologic data at the time of renal biopsy were recorded. five of them were excluded because of short period of follow-up or defective laboratory data. based on renal biopsy (who classification for ln), 20 patients were treated with the following regimens: one patient (class i) with low-dose prednisolone (pred), 7 (class ii, iii) with high-dose of pred, 12 (class iv) with high-dose pred and 13 received intermittent intravenous (iv) ctx pulses (monthly for 6 months, then every 3 months) followed by mycophenolate mofetil (mmf) as maintenance therapy. there were 13 girls and 7 boys. the mean age at the time of diagnosis of sle was 10.2 years. eighteen patients were more than 8 years old. sixty percent of the patients were presented as nephritic-nephrotic syndrome. there was 1 with class i, 5 with class ii, 2 with class iii, 12 with class iv and none with class v based on biopsy. eighty-five percent of cases went in remission, one was hemodialytic and 2 died due to renal failure and cns involvement. among 12 cases with class iv, 11 responded to pred and iv ctx pulses. there was no evidence of side effects. it seems that iv pulse ctx does induce remission of clinical and renal disease in the majority of early diagnosed children with severe ln. furthermore, it appears that mmf is an appropriate drug for maintenance therapy. however, this study was based on a small number of subjects. further studies to confirm the long-term efficacy and safety of ctx pulse therapy on larger numbers of patients are needed. forming group i were compared with 25 children on short course steroid therapy (iskdc regime) (group ii). children were examined for steroid side-effects and underwent blood tests, ophthalmologic evaluation and radiological examination. results: though remission was achieved in <4 weeks by 84% in group ii against 60% in group i, the total dose received (25-50 mg/kg) was lower in group i (44% vs 20%). forty-six % had 1-2 relapses, 44% had 3-6 relapses and 6% had 6-10 relapses. the proportion of children having >2 relapses was much higher in group ii (60% versus 40%). mean relapse/patient/ year was 1.6 (sd 0.5) in group i against 3.1 (sd 1.6) in group ii. delayed bone age (44%), radiological evidence of osteoporosis (42%). cushingoid facies (28%), posterior subcapsular cataract (16%), decreased growth velocity (14%) and hypertension (12%) were the side effects and were almost equally distributed in the two groups. more patients from group ii received a higher cumulative dose/ kg/year of >150 mg (76%versus 56% in group i) and these had higher risk for hypertension, delayed bone age and osteoporosis. conclusion: alternate day, prolonged therapy (soyka regime) compared to short course, daily therapy (iskdc) resulted in lower cumulative dose to the patient. acute side effects and severity of infections were less. mean relapse/patient/year were lower. group ii patients receiving higher cumulative dose had osteoporosis and delayed bone age. objetive: to assess urinary protein excretion decrease in patients with primary srns treated with ec-mps methods: cohort of 13 patients, mean age: 9 years with primary srns. inclusion criteria: primary sncr with of focal segmental glomerulosclerosis (fsgs). exclusion criteria: glomerular filtration rate (gfr) 30% than baseline level, leukopenia, absence of decrease of proteinuria excretion by month 6 of treatment with ec-mps. mean time after the initial diagnosis of ns until the introduction of ec-mps was calculated in patients who decreased urinary protein excretion below nephrotic range and in non-responsive patients and the glomerular damage, interstitial fibrosis and tubular atrophy were classified as: absent, mild, moderate, severe (0 to 3, risk grade>6). ec-mps dosing: 450-700 mg/m 2 /day. complete response was considered a reduction in the urinary protein excretion lower than or equal to 4mg/m 2 /hour; partial response: urinary protein excretion ranging from 4 to 40 mg/m 2 /hour and absence of response: urinary protein excretion in the nephrotic range. laboratory monthly assessments: serum creatinine, urea, hemoglobin and blood cell counts, lipids, serum proteins, amilase, 24 hours urinary protein excretion. . no significant differences in the frequency of both alleles were observed among patients with different grades of hypertension or proteinuria. in conclusion, drb1* 03011, and possibly 1105 alleles confer susceptibility to psagn. however the severity of the disease is not determined by these two alleles. methods: fifty children who diagnosed as biopsy-proven fsgs were studied retrospectively by medical records. response to treatment and pathologic slides, we compared normal renal function group (n=28) and decreased renal function group (n=22), assessed the factors affecting renal survival and progression to renal failure. results: the mean age at onset was 8 1/12 years, gender ratio m: f was 2.3: 1 and the mean duration of follow-up was 7 1/12 years. the overall renal survival rate was 34% at 5 years, 8% at 10 years. five-year survival rate was 74% in normal renal function group, but 27% in decreased renal functin group. between the two groups, there were no significant differences in age at onset, gender ratio, amount of proteinuria, incidence of hematuria, hypertension and mesangial hypercellularity. decreased renal function group showed higher serum creatinine level, poor response to treatment, higher percent of glomeruli with sclerosis, moderate to severe tubulointerstitial change and vascular change (p<0.05). the prognostic factors of renal survival rate were same as above (p<0.05). there were no significant factor has shown relations with the progression rate to renal failure. conclusion: we reviewed the factors affecting long-term outcome of fsgs. serum creatinine level, steroid responsiveness and the degree of glomerulosclerosis were significant prognostic factors. but, age at onset, gender, amount of proteinuria, incidence of hematuria and hypertension were not considered as a prognostic factor. a background: several studies have suggested that cyclosporine (csa) and methylprednisolone pulse therapy (mpt) may be effective for idiopathic steroid-resistant nephrotic syndrome (isrns) in children; however, the optimal regimen has yet to be established. the present study evaluated the efficacy and safety of 2 years' treatment with csa (neoral) combined with mpt and prednisolone (psl) in such patients. methods: in this prospective study, children with biopsy-proved isrns were enrolled. all patients received csa and psl (1 mg/kg every other day). patients who had focal segmental glomerular sclerosis (fsgs) additionally received mpt (methylprednisolone at a dose of 30 mg/kg per day for 3 consecutive days at weeks 1, 2, 5, 9, and 13) . the dose of csa was adjusted to maintain a trough level from 120 to 150 ng/ml for the initial 3 months, followed by 80 to 100 ng/ml for months 4 to 12, and 60 to 80 ng/ml for months 13 to 24. results: twenty-six patients were enrolled. their mean age was 4.5±3.9 years. two-year follow-up was completed in 22 patients. histological examination at study entry revealed minimal changes in 16 patients, diffuse mesangial proliferation in 3, and fsgs in 7. at the end of the therapy, 19 patients had complete remission, including 6 who had occasional relapses of steroid-sensitive nephrotic syndrome, and 1 had partial remission; the remission rate was 90.8%. nephrosis persisted in 1 patient. disease progressed to end stage renal failure in 1 patient. serial renal biopsy at the end of the study showed mild signs of csa-related renal toxicity, including tubulointerstitial fibrosis in 2 (10.5%) of 19 patients. conclusion: combination therapy with csa, mpt, and psl for 2 years was clearly effective and produced a high remission rate without serious csa-related renal toxicity in children with isrns, in contrast to previous reports. objectives: patients with hemolytic uremic syndrome who do not require dialysis in acute stage usually have a good prognosis. however the spectrum of renal compromise is wide. we believed non-anuric patients with higher creatinine values in acute stage could have different evolution when compared to patients with lower values. aims: 1) to analyze the outcome after a 5 year and 2) to determine if peak serum creatinine values in acute stage would be a prognostic marker. methods: 130 patients, aged 13.8 months at hemolytic uremic syndrome, were analyzed. they were classified into 4 groups: group i, complete recovery; group ii had 2 subgroups: iia, microalbuminuria, and iib, proteinuria and/or high blood pressure, both with normal renal function; group iii, chronic renal failure; and group iv, end stage renal disease. peak creatinine value was definided as the highest value of at least 2 determinations in acute stage. these data were available in 57 patients and they were divided in those with creatinine equal to or higher than 1,5 mg/dl (26 patients) and those with lower values (31 patients). the relationship between creatinine and final outcome was analyzed. we applied fisher's test. results: after a mean follow-up of 12 years, 83 patients were in group i, 27 in group iia, 15 in group iib (6 with hypertension, 5 with proteinuria and 4 with both) and 5 in group iii. eight out of 26 patients (30%) with creatinine equal to or higher than 1.5 mg/dl in acute stage and 1 out of 31 (3.2%) with lower values were in groups iib and iii in the last visit (p=0.007). conclusions: 1) after 12 years, 15% developed proteinuria, high blood pressure or chronic renal failure and 21% microalbuminuria. 2) peak creatinine values in acute stage were a prognostic indicator. objective of study: the aim of this study was to assess the serum concentration of hs-crp in children with nephrotic syndrome (ns) treated with prednisone and cyclosporine a (cya). methods: patients were divided into 3 groups: i -20 ns children (4-14 years) in relapse, examined twice: a -before treatment and b -after proteinuria regression (a 3-4 week course of prednisone therapy), ii -20 children with steroid-dependent or steroid-resistant ns, treated with cya, also examined twice: d -before treatment with cya, e -6 months after therapy. control group (c) consisted of 20 healthy children. serum hs-crp level was determined using a nephelometric method with behring nephelometer 100 analyzer, dade behring. results: it was shown that median hs-crp concentration was the highest in children with relapsing steroid-sensitive ns before treatment (ia). after proteinuria regression (ib), the hs-crp level decreased and did not differ from healthy controls (c) (p>0.05). in group ii, before cya administration (iid) the level of hs-crp was normal, but increased after 6 months of treatment (iie) up to a level six times higher that of the control group (p<0,01). conclusions: in children with steroid-sensitive nephrotic syndrome in relapse, the serum hs-crp level is increased but returns to normal values after a 3-4 week glucocorticoid treatment course. in children chronically treated with cya due to ns, serum hs-crp level increases significantly during the therapy. slit diaphragm connecting adjacent foot processes of podocyte is the final barrier of glomerular capillary wall to prevent proteinuria. both podocytes and neuronal cells are terminally differential cells and they share many common features. nurexin is a presynaptic adhesion molecule that plays a role in synaptic differentiation, and they have been understood to be specifically expressed in neuronal tissue. we found that neurexins are expressed not only in neuronal cells but also in several organs including kidney. our immunofluorescence study shows that neurexins are restrictedly expressed in glomeruli in kidney. dual-labeling immunofluorescence studies show that neurexins are localized close to a cd2ap. we also detected some portions of neurexin staining are coincident with that of rab3a, a synaptic vesicle associated molecule. we found that a single splice variant of neurexin-1 is expressed in glomeruli. the staining intensity of neurexin in the glomeruli clearly reduced and their staining pattern shift to more discontinuous patchy pattern in puromycin aminonucleoside nephropathy and anti-nephrin antibody induced nephropathy. the alteration of neurexin in these models was detected more clearly and rapidly than nephrin. we confirmed that neurexin is expressed in podocyte also in human kidney section. these observations suggest that neurexin is involved in the development of proteinuria and that neurexin could be an early diagnostic marker of podocyte injury. to further elucidate the clinical relevance of t-cell abnormality in minimal change nephrotic syndrome (mcns), and to predict the consequences of mcns, we studied t-cell receptor (tcr) diversity by analizing cdr3 size distribution and the frequency of v repertoire usage. thirty-six pediatric patients with mcns were enrolled. eighteen were frequent relapsers and/or steroiddependent (fr/sd) and 18 were non-frequent relapsers (nfr). the study was performed to analyze serial changes of tcr v repertoires in the two groups of patients. frequencies of v repertoire usage were determined by flowcytometry, and tcr cdr3 length distribution was analyzed by genescan. in nfr patients, abnormalities in the distribution of 21 v repertoires were few in both cd4 + and cd8 + t cells. in fr/sd patients the patterns were normal in cd4 + t cells, while selected v repertoires were significantly increased in cd8 + t cells in some patients. furthermore, tcr diversity was significantly reduced in both cd4 + and cd8 + t cells in fr/sd patients as shown by marked skewing of cdr3 size distributions. it is noteworthy that in some fr/sd patients the initially abnormal tcr diversity improved as the clinical symptoms improved such that they became nfr over the years. analysis of tcr diversity may delineate the subgroup of patients with fr/sd and provide a rationale for early intervention with immunosuppressive therapy for these patients. background: transforming growth factor-β is known to play a role in the interaction between metabolic and homodynamic factors in mediating accumulation of extracellular matrix in the diabetic nephropathy. tgf-β1 gene polymorphism was associated with circulating tgf-β levels and influenced the pathogenesis of fibrotic diseases including diabetic nephropathy. in this study, we examined the relationship between tgf-β1 gene codon 10 polymorphism and type 2 diabetic nephropathy with more than 10-year history of disease. methods: we conducted a case-control study, which enrolled 325 type 2 diabetes. a total of 176 patients with diabetic nephropathy were compared with 149 patients without diabetic nephropathy. tgf-β1 codon 10 genotyping was determined using polymerase chain reaction with sequence specific primers method. results: distribution of tgf-β1 codon 10 genotype in the patients either with nephropathy or without nephropathy is confined to hardy-weinberg equilibrium. methods: nzb/w f1 female mice were distributed into three experimental groups (n=5 per group) according to age: 3, 4.5 and 6 months. at specific time-point for each group, 24-hour urine and blood samples were collected to determine proteinuria, osmolality and creatinine levels. after sacrifice, kidneys were removed to measure chemokines and cytokines levels by elisa (pg/100 mg of tissue). results: urinary flux was significantly lower at 4.5 than at 3 and 6 months. a significant reduction in creatinine clearance and an increase in proteinuria were detected at 4.5 and 6 months when compared to 3 months. no significantly changes were observed in serum and urinary osmolality. regarding inflammation, mcp-1 significantly increased at 4.5 (262.7±50.5) and remained elevated at 6 months (334.6±87.7) when compared to 3 months values (206.6±37.9). kc was also higher at 6 than at 3 months ( background: nephrotic syndrome (ns) is related to immunological factors and renal inflammatory mechanisms. many studies showed that inflammatory mediators, especially interleukin-8 (il-8) and monocyte chemoattractant protein-1 (mcp-1), have a role in kidney injury. changes in their urine concentration were found in lupus nephritis and iga nephropathy. thus, the aims of this study were to evaluate il-8 and mcp-1 in serum and urine samples of pediatric patients with primary ns and to verify the relation between these measurements and protein excretion. methods: patients were divided according to current 24-hour proteinuria into two groups: lower than 200 mg/24 hours (group 1, n=11) and higher than 200 mg/24 hours (group 2, n=14). blood and 24-hour urine samples were collected and stored at -80 °c. il-8 and mcp-1 were measured by elisa standard methods. results: blood il-8 and mcp-1 did not differ between the groups. urinary il-8 levels (pg/mg of creatinine) were significantly elevated in patients of group 2 when compared to group 1 (51.06±9.67 vs. 20.48±5.24, p<0.05). although group 2 also exhibited higher values of urinary mcp-1 (223.3±66.64 pg/mg creatinine) than group 1 (151.1±31.64 pg/mg creatinine), they did reach statistical difference. conclusion: the inflammatory process in ns seems to be a local phenomenon, since blood levels of these chemokines were similar in all groups. moreover, our findings showed a relation between il-8 and the presence of proteinuria and suggested a role for this local inflammatory mediator in disease activity. the characteristics of iga nephropathy detected in school urinary screening iga nephropathy (igan) is one of common types of glomerulonephritis in children. however, progression to esrd in patients with igan is not as rare as originally thought. in korea, school urinary screening (sus) program, an useful tool to find out abnormal urinary findings, initiated in 1998. igan was the most common histopathological change in children with isolated hematuria and/or proteinuira in sus. we studied to clarify the clinical and pathologic characteristics of iga nephropathy detected by sus in korea. we investigated 35 patients (symptomatic group=14 vs sus group=21) had been diagnosed with igan following renal biopsy at the yeungnam univ. hospital between may 1998 and may 2004. these patients were analyzed clinical nature, laboratory data and histopathologic findings (haas classification in lm) and progress, retrospectively. their mean age were 10.3±2.5 and 9.5±3.6, respectively, at the time of kidney biopsy. gross hematuria and edema apt to be common in symptomatic group. there were no significant difference in serum iga level, estimated ccr, 24-hours protein amount, light microscopic class and electron microscopic findings between two groups. mesangial iga deposition was significantly more intense in symptomatic group with gross hematuria. in addition to iga deposition in capillary and immune dense deposition in intramembrane is significantly common in symptomatic group with nephrotic range proteinuria. however, progression to chronic renal failure was not noted in both groups during 32.2±28.8 and 24.8±11.2 months respectively. also there were no difference in outcome according to treatment modalities. a longer follow-up period is needed to obtain more information on progression of igan with nephritic range proteinuria by disclosing iga deposition in capillary and immunedense deposition in intramembrane. outcome of srns is uncertain and especially patients unresponsive to treatment have a high risk to develop esrd. prognosis has improved with the introduction of csa, however but long-term follow-up data are scarce and response to csa in patients with genetic forms of srns is uncertain. we report on 25 patients with srns, that was diagnosed at a median age of 3.6 (range 0.5-11.2) years. treatment with csa was initiated on concomitant prednisone therapy, however steroids were discontinued after due course in all patients. median follow-up is 9.5 (0.3-19.1) years. 17 patients had fsgs on renal histology and 12 patients had mcns. complete remission (cr) was defined as reduction of proteinuria <100 mg/m 2 /d. partial remission (pr) was defined as reduction of proteinuria of at least 50% and cessation of edema with serum albumine levels >25g/l. results: 12 patients (6 fsgs, 6 mcns) reached cr with csa treatment. in 8 of these (5 fsgs, 3 mcns) csa could be tapered and discontinued successfully after a median time of 2 (0.5-6.1) years. eight patients showed (7 fsgs) a pr while 5 patients (3 fsgs) showed no repsonse (nr). of 6 patients going into esrd 5 had podocin mutations (pm). only one patient with pm showed partial remission on csa. our data indicate a positive effect of csa treatment in srns, especially in sporadic cases. in patients with cr tapering and even discontinuation of csa is possible. prognosis of srns has improved with csa treatment. objective: we planned to investigate the effects of rsv on the proteinuria and glomerular structure of rats and to explore the role of rsv in the pathogenesis of minimal change nephrotic syndrome. methods: sd rats were inoculated with 6 10 2 , 10 4 , and 10 6 pfu rsv respectively, and sacrificed on days 4, 8, 14, 28 and 60 postinoculation (rsv 4 , rsv 8 , rsv 14 , rsv 28 , rsv 60 ). renal histology was observed by light microscopy and electron microscopy. meanwhile, the proteinuria and serum parameters were measured. the rsv rna and rsv titer were determined by in situ hybridization and plaque assay respectively. immune complex deposits were detected by immunofluorescence microscopy. results: after inoculation, the urinary protein excretion was increased, especially in 6 10 6 pfu rsv 14, 28, 60 (p<0.05). the serum albumin of 6 10 6 pfu rsv 14, 28, and different-titer rsv 60 decreased, but no significant differences in cholesterol, urea nitrogen and creatinine were found among all. slight hypercellularity in minority glomeruli and swelling of partial tubular epithelial cells were observed in rsv 4, 8 of different-titer, whereas a relief of the above changes and no abnormalities were detected in rsv 14 and rsv 28 respectively under a light microscopy. extensive foot process effacement was observed in 6 10 6 pfu rsv 14, 28, 60 under an electron microscope. rsv rna signal and rsv titer of renal and pulmonary tissues, depending on the dose of inoculum, reached their climax on day 8 postinoculation, especially in 6 10 6 pfu rsv 8 . no immune complex deposits were detected in the renal tissues. conclusion: our study reports for the first time that rsv can lead to nephropathy in rats on day 14-60 postinoculation, especially in 6 10 6 pfu rsv-inoculated rats, which may be a new exploration of the pathogenesis of mcns. objectives of study: podocytes play an important role in maintaining the glomerular filtration barrier structure and functions, which associates with several podocyte-specific proteins. our previous study indicated the antiproteinuric effects of dexamethasone were achieved by changes the expression of certain podocyte molecules in vivo. the extracellular signal-regulated kinases regulates a wide range of cellular processes. the aim of the present study is to analyze the potential effects of dexamethasone on podocytes in vitro and to investigate its associated signal transduction pathway. methods: immortalized mouse podocyte clone was divided into three groups: the dexamethasonetreated group (dex-group), the dexamethasone with puromycin aminonucleoside-treated group (dex-pangroup), and control. in dex-group, cultured podocytes were treated with dexamethasone, while in dex-pan group, cells were treated with dexamethasone for 30min first, then added puromycin for different time periods. changes of the protein expression of podocyte-specific proteins and phosphorylation of erk were analyzed by western blotting analysis. result: compared with the control group, in dex-group, the expression of nephrin, podocin, cd2ap, α-actinin4 proteins and vegf protein started to elevate at 24 hr, while neph1 showed no obviously change. erk signaling pathways was activated. increased phosphorylation of erk was marked but transient, which increased from 2 min to 15 min, then decreased. at 30 min the level of phosphorylation of erk returned to the baseline. the phosphorylation of erk level was significant raised in dex-pan group, and lasted to 60 min. conclusion: dexamethasone alters the expression of certain podocyte-specific proteins not only in vivo but also in vitro, and in part, through the activation of the erk signal pathway. kh. kim minimal lesion in the renal biopsy of idiopathic nephrotic syndrome (ns) patients (pts) generally predicts a benign course. fsgs lesions in nspts, on the other hand, are associated with increased risk of steroid resistance and progression to renal failure. fsgs may be observed in follow-up biopsies despite being initially undetectable. whether this represents sampling error or the true natural history of this condition, earlier markers of steroid resistance and poorer prognosis could prove helpful. renal morphometric analyses was performed in 13 ns pts, ages 2 to 19 years, initially diagnosed with minimal lesion, with (n=8) or without(n=5) subsequent progression to fsgs or end stage renal disease (esrd), along with 5 controls ages 3 to 16 years. the age of patients in progressive and non-progressive group and controls were similar. gbm width in patients with minimal lesion (368±52 nm) who subsequently progressed, was significantly greater than that in non-progressive group (290±35 nm, p<0.02). gbm width in both groups was not significantly different compared with the controls (321±49 nm). average foot process width was increased in patients both with progression and non-progression as compared with controls, but there was no significant difference between progressive and nonprogressive group. the length density of podocyte detachment per gbm surface was not significantly different between progressive, non-progressive groups and control groups. there were no significant differences in the mean glomerular volume and cortical interstitial volume fraction between progressive and non progressive group. in conclusion, gbm width may help to differentiate between progressive and non-progressive groups in minimal lesion nephropathy. this may be a pathogenetic clue and needs further study. objective: our study is to investigate the correlation between clinical features and pathological characteristics on henoch-schönlein purpura nephritis (hspn) and the therapeutic methods. methods: fifteen boys and five girls aged 7-15 years (median 10.5 years) with hspn were analyzed retrospectively. the clinical characteristics, laboratory data, pathological findings and therapeutic methods were investigated. results: the patients with isolated hematuria and isolated proteinuria, the pathologic patterns were lighter then gradeii b ; eight patients with hematuria and proteinuria and seven patients with nephrotic syndrome, the pathologic patterns of injury is more severe then gradeii b , whose pathologic patterns injury exceeded gradeii b is 77.8%. twelve patients with nephritic-range proteinuria (>50mg/kg.d) and nephrotic syndrome received corticosteroids, cyclophosphamide, heparin and dipyridamole treatment. nine of twelve patients received intravenous pulse methylprednisolone (mp) and pulse cyclophosphamide (ctx). fourteen of twenty patients obtained stable remission after 3 months to 5 years, and five of twenty patients have asymptomatic microscopic hematuria, another one only has minimal proteinuria. conclusions: if the clinical features showed nephritic-range proteinuria or nephrotic syndrome, the renal pathologic changed markedly as well. for patients whose pathologic exceeded grade iii b or have renal tubule and interstitial damage, our study suggests that mp pulse therapy have satisfied curative effect. materials and methods: in experiment 1, higa mice, c57bl/6 mice and balb/c mice were inoculated intravenously with live cb4 and inactivated cb4 once a month from 1 to 12 mo of age. in experiment 2, higa mice, c57bl/6 mice and balb/c mice were inoculated intravenously with live cb4 and inactivated cb4 once at 6 wk of age. mice in the control group were inoculated with vehicle. the kidneys were extirpated from 5 mice of each group killed with time after inoculation for histological evaluation. experiment 3: we examined prostaglandin (pg) synthetic activity of cultured human mesangial cells. results: the scores for mesangial iga deposition, pcna-positive and matrix scores at 20 weeks were higher in higa mice with live cb4 than in higa mice with inactivated cb4 or without cb4. on em examination, swelling and detachment of endothelial cells from 24 hours after inoculation and increase of serum ifn-gamma concentration were found in mice with live cb4. the scores for mesangial iga and igg depositions, pcna-positive and matrix scores at 30 weeks were more frequently found in balb/c mice with live cb4. production of pge2 and txb2 significantly increased in cultured human mesangial cells damaged by live cb4. conclusion: these results suggest that cb4 provokes exacerbation of renal pathological findings in higa mice via endothelial injury and ifn-gamma production, and may play important roles in igalike glomerulonephritis pathogenesis. rapidly progressive glomerulonephritis (rpgn) is a rare occurrence in alport syndrome (as). this report describes the case of a 10-year-old malewith as who developed rpgn and considers the cause of rpgn in this patient. he had a history of persistent hematuria and proteinuria since birth. at the age of 2 years, he was diagnosed with as based on a renal biopsy. he developed nephrotic syndrome at the age of 5 years. before administration of cyclosporine (cya), repeated renal biopsy was performed at the age of 7 years. the biopsy specimen showed pathologic lesions characteristic of as without crescents. using immunofluorescence (if) staining, the expression of alfa-5 chains of collagen iv was found to be absent in the glomeruli. therefore, cya was administered for eight months. although he recovered from nephrotic syndrome, the effect of cya was limited. after the cessation of cya, his renal function slowly exacerbated. at the age of 9 years, the administration of angiotensin receptor blocker was started. no subsequent anti-proteinuric effect was noted and his renal function improved to cr 0.79 mg/dl. however, by the age of 10 years, he showed macrohematuria and acute deterioration in renal function to cr 9.38 mg/dl. subsequently, he underwent a third renal biopsy. on light microscopy, the biopsyspecimen showed diffuse cellular crescentic glomerulonephritis. if findings indicated pauci-immune type and electron micrography showed a few subepithelial deposits. a serological study revealed negative results for mpo-anca, pr3-anca, and anti-glomerular basement membrane antibody. despite immediate treatment with pulses of methylprednisolone, cyclophosphamide, and plasma exchange, he progressed to end stage kidney disease. the patient reported here presents either a super imposition of rpgn upon a preexisting case of as or a new morphologic and clinical presentation for as. the wt1 gene encodes a zinc finger transcription factor involved in kidney and gonadal development. mutations of the wt1 gene have been shown to cause denys-drash syndrome (dds) and frasier syndrome (fs). the association of early onset nephrotic syndrome progressing to renal insufficiency, xy pseudohermaphrodism and wilms' tumor characterizes dds. renal biopsy shows diffuse mesangial sclerosis (dms). fs is also characterized by xy pseudohermaphrodism and nephropathy, but patients have delayed kidney failure characterized histologically by fsgs. this report examines three girls with nephrotic syndrome related to mutations in the wt1 gene, but with normal female karyotype and development. two girls with early-onset steroid-resistant nephrotic syndrome presented classical wt1 mutations coding for an amino acid change (d396n) and (r394w) at exon 9 that is typical of dds. both children were phenotypically and genotypically females. they developed end stage renal failure within 7 years. one girl had a wilms' tumor on the right kidney. the third child was identified with heavy proteinuria at 7 years of age. laboratory investigations revealed a protein level of 7.2 g/dl (6-8 g/dl), albumin 3.7 g/dl (3.9-5.3 g/dl). proteinuria worsened to 4 g/24 h and she failed to respond to prednisone. renal biopsy demonstrated fsgs in 20% of glomeruli. the splice site mutation ivs9+4 g >a, known to be associated with fs, was found in this patient. karyotype was 46, xx and she had normal uterus and adnexae on ultrasound. angiotensin-converting enzyme inhibitors were prescribed but she still has heavy proteinuria without renal failure. the classical clinical presentation of dds and fs is out dated. pediatric nephrologists need to consider the possibility of these genetic syndromes in evaluation of females with steroid-resistant nephrotic syndrome. we aimed to compare the effects of cyclosporine (csa) or mmf with or without combination of vitamin a,d,e or n-acetyl-l-cysteine (nac). the study included 64 rats in eight groups: control, nephrotic syndrome without treatment, treatment with csa, mmf, vitamin a,d,e, combination of csa and vit ade, combination of mmf and vit ade and combination of nac and vit ade. all rats except the control group were given adriamycin. blood samples were drawn and 24-h urine were collected on day 1 and at weeks 5 and 16. at the 5 th week, 24-h urinary protein excretions in the treatment groups were higher and serum albumin levels were lower than that of 0 th week and control groups (p>0.05). at the 16 th week, urinary protein excretions in group csa&ade was lower than of the groups of csa, mmf, mmf&ade, ade and nac&ade with non-significance (p=0. 42, p=0.94, p=0.72, p=0.82, p=0.53) . serum albumin in group mmf&ade and group ade were significantly higher than of control group (p=0.015, p=0.01). serum albumin in group ade was significantly higher than that of groups of csa and csa&ade (p=0.05, p=0.045). serum triglyceride in group mmf&ade was significantly lower than that of groups csa and csa&ade. serum creatinine in group mmf&ade was lower than that of the groups of csa, csa&ade, mmf, ade and nac&ade and was significantly lower than that of control group (p=0.004). serum creatinine in group csa was significantly higher than of groups of csa&ade, mmf, mmf&ade (p=0.004, p=0.001, p=0.001, respectively). in group nas+ade, total oxidant was significantly higher and total anti-oxidant was significantly lower than other treatment groups (p<0.05). we showed that the better effect on proteinuria, serum triglyceride and albumin and lower serum creatinine by adding vitamin a, d, e in the treatment of experimental nephrotic syndrome with csa or mmf. conclusion: ht is associated with known risk factor of progression of biopsy-proven gn such as s-cr or proteinuria. the crb is an important tool for studies focusing on the epidemiology of gn in the czech republic and serves as a basis for cooperation in this field. (4), lupus-nephritis (3), iga-nephropathy (2), minimal change disease and membranous nephropathy (by 1) were observed. the distinction of morphological variants of the fsgs was started recently (last 6 months). among the patients with fsgs, which were biopsied during this short period (4), all have had a tip-lesion. these patients were started cyclosporine a 150 mg/m 2 /day with complete (3) and partial (1) remission achievement after 1-3 months. thus, the focal and segmental glomerulosclerosis is the most frequent cause of the nephrotic syndrome in children. the focal and segmental glomerulosclerosis with tip-lesion is characterized by favourable course and good response to therapy with cyclosporine a during the short-time period. objectives of study: molecules of monocyte chemoattractant protein-1 (mcp-1), β-catenin and cytokeratin19 (ck19) express increased in cellular crescent, which is a severe pathological change in renal diseases. however, it is unknown whether these molecules in urine correlate to the number or extent of cellular crescents. methods: urinary molecules mentioned above were detected in 20 healthy subjects and 124 patients with renal diseases by elisa. the expressions of these molecules and macrophage (cd68 positive) in 87 biopsy specimens were also investigated. results: significant higher levels of these molecules in urine were demonstrated in all patients with renal diseases compared with healthy subjects (p<0.01), but the highest level in patients of the cellular crescent group. the significant correlations were revealed between these molecules in urine and the index of cellular crescents (r=0.75, r=0.21, r=0.63, p<0.01), between these molecules in glomeruli and the index of cellular crescents (r=0.58, r=0.66, r=0.52, p<0.01), and between these molecules in glomeruli and in urine (r=0.62, r=0.50, r=0.20, p<0.01). conclusions: this study suggested that the detection of urinary mcp-1, β-catenin and ck19 might become potential biomarkers for clinical diagnosing cellular crescent lesions and assessing cellular crescent extent in renal diseases. this study aims to evaluate the benefits and harms of levamisole in steroid dependent (sd) and frequent-relapsing (fr) nephrotic children. material and methods: a total of 24 steroid-sensitive nephrotic children were recruited prospectively from january 2004 to december 2006. 5 females and 19 males, 11 (48%) fr, 12 (52%) sd. mean age at the beginning of levamisole was 5,7 years. twelve didn't receive any alternative drug before, 12 received cyclophosphamide. renal biopsy was perfomed in 5 patients, 4 had minimal change disease (mc), one patient had mc at the first biopsy and fsgs in the second. the patients were divided in two groups according to their steroid response: sd and fr. levamisole was started at a dose of 2,5 mg/kg/48 h as a second line drug in order to try to prolong periods of remission. clinical and laboratorial controls were performed monthly. patients were evaluated as: total responders: when steroid withdrawal was possible, partial responders: when steroid reduction was possible, non-responders: no steroid modification in 3 months. the responders used levamisole for one year. results: the response of frequent-relapsing patients was: 63% of total response, 18% of partial response and 18% of non-responders. steroid-dependent patients had 50% of total response, 25% partial and 25% non-responsers. only one patient developed leukopenia. now 21% (5) are out of levamisole and in remission, 50% (12) are still using levamisole(alone or low-dose of prednisone), 21% (5) are using cyclosporin, one used cyclophosphamide and one is using low-dose prednisone. conclusion: levamisole is a promising alternative drug for nephrotic syndrome. the major advantage of levamisole is its steroid-sparing effect with minimal toxicity. conclusions: hypertension and renal insufficiency were less frequently seen in chinese children with fsgs, isolated hematuria as unique clinic presentation was common in fsgs. all pathological variants had tubular-interstitial lesions, but vascular lesions were rarely seen. most fsgs children with nephrotic syndrome were sensitive to steroid at initial stage, and easy to develop frequent relapse gradually, immunosuppressive agent may be helpful to elevate remission rate. the aim of the study was to assess whether the serum index iga/c3 can be a usefull marker of activity igan and hsn in children. twenty children with igan (mean age 10.0±3.6 years) and 33 children with hsn (mean age 9.53±4.17 years) were retrospectively analysed. in all children urine analysis, gfr were checked and the levels of serum iga and c3 were measured before therapy, serum iga/c3 was than calculated. at the onset of illness, igan and hsn was diagnosed based on the renal biopsy in mean time 1.06±1.37 years. changes in light microscopy were graded i-v according to the classification of who (cwho). all biopsies were scored for activity and chronicity index (ai max score 9, ci max score 12) (pediatr.nephrol.1989,3,248 heparan sulfate proteoglycans are present both as structural components of the gbm and as modifiers of growth factor signaling on the cell surface. in mcns the presence of certain hs epitopes inthe gbm is decreased. hsulf1 and hsulf2 are recently identified endosulfatases, that can remodel the sulfation pattern of hs and thereby control the availability and presentation of factors such as fgf and hgf to their high affinity receptors. sulfatase activity requires posttranslational modification by formylglycine-generating enzyme or sulfatase modifying factor 1 (fge/sumf1) that is counteracted by its paralogue pfge/sumf2. we demonstrated that podocyte, endothelial and tubular epithelial cell lines express mrna for sulf1, sulf2, sumf1 and sumf2. we investigated the in vivo distribution patterns of these enzymes in human kidney specimens by in situ hybridization. in histologically normal kidneys (n=5) expression of hsulf1 mrna was largely restricted to peritubular and glomerular endothelial cells, where as hsulf2 mrna was weakly expressed in all glomerular resident cell types. expression of sumf1 and sumf2 mrna was present in a minority of mesangial cells and podocytes, as well as in avariable number of glomerular and peritubular endothelial cells and invascular smooth muscle cells. in mcns (n=5), the glomerular expression patterns of hsulf1, sumf1 and sumf2 mrna did not differ significantly from that in controls. in contrast, hsulf2 mrna expression was increased in podocytes. increased hsulf2 expression by podocytes is a novel factor to be considered in the pathogenesis of mcns. the onset and duration of ins, histopathological changes in renal biopsy, results of corticosteroid therapy and proteinuria selectivity reflecting the alteration of glomerular capillary wall were analysed. materials and methods: 80 children with ins aged 2-18, followed up 12 to 36 month, and 20 healthy children were studied. in all patients 1 stage of ckd were diagnosed. ins children were divided in two groups regarding to serum cystatin c levels as a marker of gfr: group i (n=37)children with unchanging gfr, ii (n=43) -patients with impairment renal function over the study period. serum total protein, albumin, cholesterol, cystatin c, creatinine and immunoglobulin igg, and urinary protein, albumin, igg and creatinine were measured. results: serum cystatin c levels were higher in both groups of ins patients compared to healthy children (gr. i: 0.8±0.11, gr. ii: 1.19±0.42 vs 0.7±0.1 mg/l; p<0.01). in group ii amount of 24-hour proteinuria was significantly higher than in group i (2.93±2.27 vs 3.76±2.79 g/l; p<.001), however other biochemical parameters (including igg excretion, albuminuria, selectivity index) were not different. in group ii higher age of onset was found. in this group mesangial proliferation and focal segmental glomerulosclerosis more often were observed. conclusions: the age of onset, histopathological diagnosis and proteinuria can be considered as important markers of ckd progression in children with ins. probably, longer follow-up of children with ins is necessary to find other prognostic factors. k. kilis-pstrusinska, k. fornalczyk, d. zwoliñska cyclosporine a (csa) has been used as a therapeutic option for steroid-dependent and steroidresistant nephrotic syndrome (ns). the aim of the study was to assess the effects of long term csa treatment in children with ns. methods: we performed retrospective study to evaluate safety and efficacy of csa therapy in 44 children with ns (20 girls and 24 boys), aged 2-16 years. results: before introducing csa, in 30 patients steroid-dependent ns and in 14 -steroid-resistant ns were observed. 28 children presented symptoms of steroid toxicity. pre-treatment renal biopsy was performed in 42 patients (2 children without biopsy because of renal agenesia). minimal change disease was diagnosed in 18 (43%) children, focal segmental glomerulosclerosis (fsgs) in 9 (21%), mesangial glomerulonephritis (gn) in 8 (19%) and membranoproliferative gn in 7 (16%) cases. all children were taking csa (target blood trough levels 50-150 ng/ml) more than 6 months, mean 24 months (range 7-72). complete remission was achieved in 32 (73%) children, partial in 9 (20%). in 17 (39%) patients csa treatment was continued with mild dose of steroids. 3 patients (7%) did not respond to the therapy and one of them end stage renal failure developed. following side effects have been observed: hyperuricuria (20% of patients), hyperuricaemia (9%), hypomagnesaemia (7%), hypertension (11%), hypertrichosis (9%), gingival hyperplasia (5%), hepatotoxicity (2%), gastritis and ileitis symptoms (2%). in 15 patients control renal biopsy was performed after 24-36 months of csa therapy. in 4 patients progression to fsgs was seen. only in one case histological findings of csa nephrotoxicity occurred. conclusion: long-term csa treatment in children with steroid-dependent and steroid-resistant nephrotic syndrome can be consider as an effective and safe therapy. introduction: membranous nephritis represents a rare disease in childhood with an incidence of 0.8 to 6% in renal biopsy specimens among various types of glomerulonephritis. although in many cases the disease is considered as idiopathic the association of membranous nephritis and infectious agents it is also well known. aim: we report a case of a 19 months old baby girl of asian origin, presented with macroscopic haematuria of glomerular origin, proteinuria, 1-3 gr/24 hours, hyaline and granular casts, maculopapular rash on the legs and microcephaly. methods: renal function tests in terms of plasma urea and creatinine were normal. the renal biopsy showed membranous nephritis. tests for infectious diseases (torch screen) showed a primary cmv infection. this diagnosis was based on the presence of high level cmv specific igm antibodies, increased igg antibodies and low avidity of cmv specific igg antibodies. a real-time-pcr of the renal biopsy specimen was positive for cmv as well, confirming this virus as the causative agent of membranous nephritis in the presented case. treatment with gancyclovire per os was introduced and it was most impressive since proteinuria disappeared in the following two to three weeks. two years after the diagnosis the child remains well and asymptomatic. in summary: to our knowledge, among various infectious agents, there is no case of congenital or secondary nephropathy described so far due to cmv infection in children. objective of study: pulse methylprednisolone therapy (pmt) has been shown effective in proteinuric renal diseases. but its exact effect in children with steroid-sensitive relapsing nephrotic syndrome still has no definite conclusion. to evaluate the effect and adverse effects of pmt, we performed a retrospective study. methods: there were 11 cases of steroid-sensitive relapsing nephritic syndrome received pmt. they all had been treated with oral prednisone in similar condition previously. a self control design was used to compare the effect of pmt and oral prednisone. results: the average age was 7.5±3.1 years. ten cases attained complete remission after the first course of pmt, and the average duration of pmt until remission was 7.4±4.4 days. one case attained complete remission after the second course of pmt. compared with the effect of oral prednisone previously, seven cases attained complete remission more rapidly. paired-samples t-test was performed to compare the effects of pmt and oral prednisone in six cases with very similar state, however, the difference between them was not significant (p=0.082). during the treatment of pmt, adverse effects were found in 6 cases. conclusion: compared with oral prednisone, the superiority of pmt had not been definitely confirmed, and adverse effects might appear during the treatment. therefore we should be very strict with administering pmt in children with steroid-sensitive nephrotic syndrome. the study we performed was retrospective, so it was necessary to design a prospective clinical randomized controlled trial to further evaluate its effect. objective of study: pulse methylprednisolone therapy (pmt) has been shown effective in proteinuric renal diseases. but the exact effect of pmt in children with steroid sensitive nephrotic syndrome (ssrn) still has no definite conclusion. to evaluate the effect and adverse effects of pmt, we performed a prospective study. methods: a prospective clinical randomized controlled trial was conducted to compare the effect and adverse effects of pmt with oral prednisone (op) in children with ssrn. thirty-one children were enrolled with only 23 suitable for evaluation including 9 patients in the pmt group and 14 in op group. results: there was no significant difference between both groups in complete remission rate. the average durations of therapy until remission were 4.9±1.5 days in pmt group and 9.6±6.3 days in op group, respectively. complete remission was more rapidly attained in pmt group (p=0.036; <0.05). during the treatment and the following 3 months, no significant difference of adverse effects was found between both groups. there was no significant difference between both groups in relapse rate during 3 months follow-up. conclusion: compared with oral prednisone, pmt could induce complete remission more rapidly and did not company with more adverse effects in children with ssrn. pmt had no effect on the reduction of relapse rate in the following 3 months after administration. outcome of childhood henoch-schönlein purpura nephritis with nephroticrange proteinuria in a single center objective: the majority of children with henoch-schönlein purpura nephritis (hsn) only with hematuria and/or mild proteinuria have good chances for recovery. however, it is still unclear how we should treat hsn with persistent massive proteinuria. the aim of this study is to evaluate the outcome of childhood hsn with nephrotic-range proteinuria treated with angiotensin converting enzyme inhibitor (acei) and corticosteroids. methods: 109 patients with henoch-schönlein purpura (58 boys, 51 girls) ranging from 0.5 to 13.8 (6.3±2.7) years old at onset visited our hospital between april 1997 and december 2006. thirty seven (33.9%) developed hsn. mean age of 21 (13 boys, 8 girls) hsn patients with nephroticrange proteinuria (>40 mg/h/m 2 ) at the time of diagnosis was 8.3±2.9 years old. two developed nephrotic syndrome, but none had renal insufficiency at onset. one patient suffered moderate proteinuria and renal dysfunction 8 years after the onset. renal biopsies were done in 16 cases and showed 1 for grade i, 4 for grade ii and 10 for grade iii respectively (classification by international study of kidney diseases in children). results: eleven of the 21 patients received acei and no patients were treated with immunosuppressive agents except for corticosteroids and methylprednisolone pulses. after a mean follow-up of 3.8 years, 11 patients showed complete remission, 9 had mild proteinuria or microscopic hematuria, only one postpartum patient presented with nephrotic-range proteinuria, and none developed renal insufficiency. conclusions: our results suggested that the short-term outcome of childhood hsn with nephroticrange proteinuria but not renal insufficiency was relatively favorable. thus, the degree of proteinuria is not a sole prognostic factor for childhood hsn. therefore, the indication for corticosteroid therapy should be clarified. recent observations determined a r1160x specific exon 27 mutation in the gene encoding nephrin (nphs1) which evidenced an unexpectedly mild finnish-type congenital nephrotic syndrome (cns) phenotype. the long-term follow-up of patients with this mutation is actually known only in a few patients. we reported the long term follow-up of a girl originary of sicily (italy) presenting such a mutation. the first 3 years of her life were previously reported (s.guez et al pediatr nephrol 1998). in brief the child was pre-term born from related parents and she presented proteinuria (3-6 g/day) from birth. renal biopsy was consistent with the diagnosis of cns and molecular evaluation demonstrated a homozygous exon 27 r1160x nonsense mutation in the nphs1 gene. in the first 2 years she was treated by human albumin without a regression of proteinuria that decreased up to 0.3-0.5 g/day with enalapril treatment (0.75 mg/kg/day); clearance of creatinine at 3 years of age ranged between 49 and 73 ml/min/1.73 mq. during the following 10 years (at the last control the girl was 13 year old) she had continued enalapril treatment (0.20 mg/kg/day). creatinine clearance was 80 ml/min/1.73 mq/day, serum total protein 66 g/l; serum albumin 40 g/l and proteinuria ranged between 1.5-4.0 g/day. both height and weight were at 50 th for age and pubertal stage was normal; blood pressure was 90/60 mmhg. in conclusion, this is the first reported long-term follow-up in an italian patient affected by finnishtype cns with the specific r1160x nonsense mutation in the nphs1 gene: even if proteinuria persisted there was no worsening in gfr. serum total protein, albumin and growth were normal confirming the milder phenotype in comparison with the typical finnish forms. long-term enalapril treatment may also have contributed to the prognosis in this specific form of congenital nephrosis. atypical hemolytic uremic syndrome (ahus) frequently results in end-stage renal failure and can be lethal in many cases. recently, it has been recognized that many cases of ahus are associated with a defective control of the complement activation cascade. more than sixty different mutations in complement factor h gene (cfh) have been reported so far. guidelines for treatment modalities are yet to be established although plasma infusions and exchanges are often advocated. we describe a patient who presented at 7 months of age with ahus (anemia, thrombopenia, acute renal failure requiring hemodialysis) associated with heterozygous combined de novo complement factor h mutations (s1191l and v1197a) on the same allele. laboratory investigations showed normal levels of complement c4, c3 and factor h. during the first episode, daily plasma exchanges (pe) using cryosupernatant (cr) as replacement fluid resulted in a resolution of hemolysis and complete normalization of renal function. two ahus recurrences were successfully treated with daily pe and subsequently pe were weaned to twice weekly. one year after the first episode, pe were stopped and pi regimen (30 ml/kg twice weekly and weekly thereafter) was started. at the present time, the patient has been receiving weekly pi (30 ml/kg) for one year. transitory falls in haptoglobin levels or platelet counts are observed periodically and successfully treated by intensification of pi (30 ml/kg) twice weekly for one or two weeks. renal function remains normal.although our observation demonstrates the effectiveness and good tolerance of large volumes of pi (30 ml/kg), long-term efficacy of such a therapy remains to be evaluated. because of the possibility of secondary failure of plasma therapy, it is important to investigate alternative approaches such as combined liver-kidney transplantation. common variable immunodeficiency (cvid) is characterized by reduced serum immunoglobulin levels and recurrent bacterial infections. there have been only 3 previous case reports of renal granulomas in cvid and only one of them was associated with immune complex glomerulonephritis. we present a case of renal granuloma and glomerulopathy in a patient with cvid. a 17-year-old girl, with a past history of uveitis, presented with cardiac tamponnade and bilateral pleural effusions. investigations revealed nephrotic syndrome (serum albumin of 23 (normal 41-54) g/l and proteinuria >3g/l), microscopic hematuria and reduced serum igg levels (4.49 g/l, normal 5.29-15.21 g/l). lupus nephritis and serositis were first suspected and corticosteroids were initiated. no serum anti-nuclear or anti-dna antibodies, nor complement activation were detected. a renal biopsy was performed and showed global glomerular endocapillary proliferation. intratubular calcium deposits were present. in the interstitium, a noncaseating epithelioid granuloma was found. immunofluorescence studies showed significant mesangio-parietal igm deposits with few c3 and c1q. as igm-immune complex glomerulonephritis has been reported in sarcoidosis, this diagnosis was highly suspected. further investigation revealed normal lung parenchyma and mediastinum, normal serum angiotensinconverting enzyme, 1.25 oh 2 d 3 , calcemia and calciuria. the nephrotic syndrome gradually improved, but serum igg levels remained persistently low (2.73 g/l). the diagnosis of cvid was confirmed and iv immunoglobulins were initiated. this is the first report of concomitant renal granuloma and igm-immune complex (without igg) glomerulonephritis in a cvid patient. in summary, cvid must be included in the differential diagnosis of renal granuloma and should be differentiated from sarcoidosis to ensure appropriate therapy. o. nwobi, c. abitbol, j. chandar, w. seeherunvong, g. zilleruelo background: rituximab, an anti-cd 20 antibody, has been proposed as therapy for refractory systemic lupus erythematosus (sle), although its use in children remains anecdotal. we present our initial longterm experience on the safety and efficacy of rituximab for treatment of sle in children. methods: 17 pediatric patients with sle refractory to standard treatment protocols were treated with rituximab for 2-4 doses (375 mg/m 2 ). all had proliferative nephritis and systemic manifestations of vasculitis. clinical disease activity was scored using the sle-disease activity index (sledai). proteinuria is reported as the urine protein to creatinine ratio (upr/cr). patients have been followed an average of 2.3±i.2 years. results: b cell depletion occurred within 1 month and remained suppressed for up to 3 months. clinical course, renal function and proteinuria improved in the majority of patients as summarized below: objective: to assess efficiency and safety of treating crns patients with srl. subjects: 5 patients, mean age 11.6 years old (6 yrs-18 yrs) (3 females) affected by crns. inclusion criteria: histological diagnosis of focal and segmental glomerulosclerosis, glomerular filtration rate (gfr) above 60ml/min/1.73 m 2 , negative plasma pregnancy test, signed child and parents informed consent. exclusion criteria: secondary nephrotic syndrome, white blood cells (wbc) count below 4000/mm 3 , chronic hepatopathy, coagulopathy, tumoral or infection processes. discontinuation criteria: sustained decrease in gfr by more than 30% of the initial rate, decrease of the wbc count to less than 4000/mm 3 , occurrence of lymphoprolipherative and tumoral processes, severe infection, alterations in coagulation parameters, positive pregnancy test or inflammatory process or lack of changes in proteinuria or its increase after four-month treatment. methods: srl dose and dosage: dose of 1 to 2 mg/m 2 /day (up to 5 mg/day) administered once a day. expected dosage range: 5 to 10 ng/ml (hplc-uv method). treatment duration: 12 months. results: 3 patients showed nephrotic syndrome remission; average srl dose: 2 mg/m 2 /day; average srl dosage: 5.75 ng/ml (range 5.3-6.3). average proteinuria prior to treatment: 157 mg/m 2 /hour (range 80-263); average proteinuria after treatment: 3 mg/m 2 /hour (range 2.5-4); average time of remission: 2 months and 20 days, none of 5 patients showed adverse events that would have lead to the treatment discontinuation. if these data are representative of the universe of patients we are dealing with, the confidence interval (p=0.95) of the percentage of patients with not proteinuria after treatment is going to be between 45% and 75% if 40 patients are assessed. conclusions: srl caused crns remission in patients who were refractory to traditional immunosuppressive treatments. background: retinoic acid-inducible gene-i (rig-i) may play an important role on inflammatory and immune processes by regulating the expression of various genes, and has been reported to be expressed in various inflammatory diseases. we studied the expression of rig-i in human lupus nephritis and evaluated the correlation between its expression and the histological activity of the renal disease in these cases. methods: expression of rig-i in the glomeruli was assessed by indirect immunofluorescence method; frozen sections of ten kidney tissue specimens obtained from eight patients with lupus nephritis were stained with a monoclonal antibody for rig-i. kidney tissue specimens from eight patients with minimal-change (mc) disease were used as controls. results: expression of rig-i was detectable as granular immunofluorescence in a mesangial and capillary distribution in all the patients with lupus nephritis, but was absent or only trace-like in the patients with mc disease. the glomerular immunoreactivity for rig-i was correlated with the histological activity and severity of the renal disease in the patients with lupus nephritis. conclusions: rig-i expression occurs at levels detectable by indirect immunofluorescence and may be potentially useful as a parameter reflecting the renal histological activity in cases of lupus nephritis. cyclophosphamide pulse therapy for crescentic, proliferative iga nephropathy in children iga nephropathy is one of the most common glomerular kidney diseases in europe, up to 25% of affected adults need dialysis after 10 years. therapy of crescentic, proliferative iga nephropathy in children is not well examined. between 01/2000 and 12/2006 seventeen children (main age 10 years (6-18), male/female=10/7) with biopsy-proven mesangioproliferative glomerulonephritis as manifestation of iga nephropathy were enrolled. nine patients (male/female=7/2) had severe clinical manifestations (renal failure, nephrotic proteinuria or cerebral vasculitis) with extracapillary glomerular proliferations (crescents). this children were treated by intravenous methylprednisolone pulses (400/200/200/100 mg/m 2 body surface area over 4 days) followed by monthly intravenous cyclophosphamide (cph) pulses (6x500 mg/m 2 body surface area) with gradually tapered oral prednisolon (4-10 mg/m 2 body surface area/48 h over 6 months) and aceinhibitor (enalapril 0.05-0.2 mg/kg/d). after 6 months of treatment a significant reduction of proteinuria (2.9±0.4 to 0.24±0.08 g/m 2 /d; p<0.0001) and improvement of kidney function (gfr 117±20.5 vs. 174±7 ml/min/1.73 m 2 , p<0.05) was observed. no notable adverse events were recorded. six patients had a second renal biopsy after completing cph-therapy; only 1 crescent was found in 245 examined glomeruli (initial findings: 48 crescents in 231 glomeruli). after follow-up of 37 (8-75) months all children have unaltered renal function, further episodes of macrohematuria or gross proteinuria did not occur. intravenous cph pulse therapy seems to be an effective therapeutic option in paediatric patients suffering from crescentic iga nephropathy. eye involvement in children with primary fsgs distinct eye abnormalities have been described in children with nephrotic syndrome, particularly in diffuse mesangial sclerosis (pierson syndrome). the aim of the study is to investigate whether there are any associated ocular anomalies in childhood primary focal segmental glomerulosclerosis (fsgs). demographic characteristics, age at onset, drug therapy and duration of the disease were defined in 30 patients (14 girls,16 boys, mean age 10.1±3.9 years) with biopsy proven fsgs. standard steroid therapy was prescribed to the patients. a detailed ophtalmological examination was performed in all patients. the median age at diagnosis was 6 years (1-16 years). mean followup time was 36 months (4-116 months). eleven patients (36.6%) reached to chronic renal failure during follow-up period. overall, 15 patients (50%) showed various eye abnormalities. nuclear opacity was found inone child and posterior subcapsular opacities probably due to corticosteroid therapy were present in two cases. two patients had myopic astigmatism and two had exotrophia. importantly, 4 patients had anisometropic ambliyopia, 3 had mittendorf spots and 1 had pigmentary changes in macula, which had never been described in the literature. mutational analysis for nephrin, podosin, wt1 and lamb2 genes are still going on. we don't know whether particular mutations are related to particular eye findings like pierson syndrome, yet. however our findings emphasize that ophtalmological evaluation should be performed in all patients with primary fsgs at the time of diagnosis. regardless of the underlying disease, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the highly specialized interdigitating foot processes. to investigate whether high glucose and advanced glycosylation endproduct (age) induce podocyte phenotypical changes, including quantitative and distributional changes of zo-1 protein, we cultured rat glomerular epithelial cells (gepc) under 1) normal glucose (5mm,=control) or 2) high glucose (30 mm) or 3) age-added or 4) high glucose plus age-added conditions. high glucose plus age-added condition could increase the permeability of monolayered gepcs and induce ultrastructural separation between confluent gepcs. zo-1 moved from peripheral cytoplasm to inner actin filaments complexes by both age-added and/or high glucose condition in cutured gepc by confocal imaging. high glucose plus age-added condition also decreased zo-1 protein amount and its mrna expression to statistically significant level compared to normal glucose or osmotic control conditions. we could also confirm the activation of pkb/akt signaling pathway in gepc by age, high glucose and insulin in pi3k-dependent manner. in addition, an inhibition of pi3 kinase by ly294002 was able to prevent quantitative and distributional changes of zo-1 protein induced by high glucose and age. these findings suggest that both high glucose-and age-added condition induce the cytoplasmic translocation and suppresses the production of zo-1 at transcriptional level and these changes may be mediated by pi3k/akt signaling. this pathway could explain the role of zo-1 in the phenotypic changes of podocytes in diabetic conditions. henoch-schönlein purpura (hsp) is common in the pediatric population, while wegener's granulomatosis (wg) is rare. although both diseases are classified into the vasculitis syndrome, their clinical symptoms, the treatment and the prognosis are considerably different. the classic clinical triad of wg consists of upper and lower airway disease, renal involvement and small vessel vasculitis. we present a rare childhood case in whom hsp-like symptoms were developed prior to wg symptoms. a 13-year-old girl developed ankle joint pain and swelling and purpura on bilateral legs and hands in the absence of abdominal pain. she was diagnosed as hsp and treated with oral prednisolone (psl) therapy. although high dose psl temporarily rescued these symptoms, purpura and joint swelling and pain reappeared in parallel with a reduction of psl dose. at this moment, because microscopic hematuria, mild proteinuria and hoarseness were also noticed for the first time, serum proteinase-3 antineutrophil cytoplasmic antibody (pr-3anca) was evaluated and detected to be high. moreover renal pathology showed necrotizing pauci-immune glomerulonephritis, leading to the final diagnosis as wg. after methylprednisolone pulse therapy followed by oral psl combined with cyclophosphamide, her clinical symptoms with wg were resolved together with the reduction of serum pr-3anca titer. taken together we emphasize that pr-3anca should be evaluated even in the patients who only develop hsp symptoms. background: short-term efficacy of steroid therapy for pediatric patients with iga nephropathy (igan) has been reported. however, there are a number of cases in whom their igan recurs after stopping the steroid therapy. recent japanese study indicated that tonsillectomy had a significant impact on renal outcome. also, another japanese study showed mizoribine was effective for igan in children. in this study, we examined the effects of a treatment regimen consisting of tonsillectomy and steroid pulse therapy followed by mizoribine (tx) for chronic relapsing igan in children. method: ten cases who showed chronic relapsing igan were included as the subjects (mean age at onset: 12.0 yrs, mean age at initiation of tx: 19.4 yrs). they were divided into two group, a normal renal function group (group a, n=6) and an impaired renal function group (group b, n=4). the changes in hematuria, proteinuria, renal function, and adverse events were prospectively examined for more than 12 months. result: a negative of hematuria was observed in 50% of group a and 75% of group b. a negative of proteinuria was seen in 33% of group a. in addition, in group b, deterioration of renal function was not observed during the observation periods. there were no serious adverse events associated with this treatment regimen. conclusion: a treatment regimen consisting of tonsillectomy and steroid pulse therapy followed by mizoribine treatment seems to be effective to control of acute inflammation coexisting with chronic glomerular lesions, and can be a valuable addition to therapeutic options for treating patients with chronic relapsing igan in children. objective of study: to present a case of silent stenosing ureteritis in a boy with henoch-schönlein purpura (hsp). case report: a 4-year-old boy was admitted with a typical hsp. urine findings were normal on admission. a gastrointestinal bleeding on the 4 th day of illness suggested corticosteroid treatment. the ultrasound on the 4 th and 7 th day revealed a normal urinary tract. on the 10 th day he developed non-painful macroscopic haematuria, followed by microscopic haematuria and proteinuria, which reached the nephrotic range on the 11 th day. thus, the boy was treated with methylprednisolone pulses and cyclophosphamide for 8 weeks. renal biopsy was not performed because of his parent's refusal. microscopic haematuria and proteinuria gradually subsided, with complete disappearance at the 5 th month. during this time he was asymptomatic, with no episodes of macroscopic haematuria or colicky flank pain. at the 8 th month of illness a new ultrasound revealed a major left hydronephrosis. computer tomographic urography showed a complete ureteropelvic junction obstruction. the 99mtc-dmsa scan revealed a 5% relative function ipsilaterally. a left pyelostomy was performed. during the next four months after draining, the urine volume of the affected kidney was 0.2-0.3 ml/kgr/hour, the creatinine clearance 2-3 ml/min/1.73 m 2 and the 99mtc-dmsa scan showed a 4% relative function. based on the above findings a nephrectomy was decided. conclusions: although rare, stenosing ureteritis should be considered in hsp. the typical clinical presentation with haematuria in association with colicky flank pain may not always occur, as in the present case, or may be confused with the symptoms of hsp itself. thus, the repetition of an ultrasound during the process of the disease may be necessary, in order for this complication to be diagnosed and treated early, preventing serious renal outcome. mitochondrial diseases are either due to sporadic or inherited mutations mainly in mitochondrial dna located genes. with regard to renal manifestations, tubular dysfunctions are common; however, the existence of solitary glomerulopathy has recently become apparent. in such case, the pathomechansim of the glomerular proteinuria is still obscure. wepresent a 12 year-old girl who was found to have asymptomatic proteinuria (up/cr1.0) in the absence of hematuria, azotemia, tubular dysfunctions, lacking any neurological manifestations. her family history showed maternal inheritance with mild proteinuria of grandmother and renal insufficiency of young uncle. light microscopy revealed 7 glomeruli of normal appearance and 3 of global sclerosis. electron microscopy showed swollen mitochondria in podocyte of normal appearance glomerulus. pointmutation rate of mitochondrial dna, a3243g, was detected as less than 1% in grandmother, 6% in mother and 39% in the patient examined byperipheral blood cells. the proteinuria completely disappeared 3 months after treatment with combined therapy of arb and acei. to determine the responsible molecule for the pathomechanism of proteinria, immunostaining followed by conforcal microscopy with slit diaphragm associated molecules (sdm) (nephrin, podocin), gbm associated molecules (type iv collagen alpha chains, laminin isoforms, perlecan) and podocalyxin was studied and compared to the controls. interestingly distinct decrease of expression with sdm was observed even in normal appearance glomerulus of the patient. taken together, a3243g mutation itself may lead to depletion of atp and/or increase of free radicals in podocyte, which predominantly affect the biogenesis of sdm, result in pathological glomerular proteinuria. the mechanism of antiproteinuric effect of arb/acei therapy should be evaluated by serial biopsy specimen. objectives: to report the effectiveness of pulse cyclophosphamide induction therapy in children with diffuse proliferative lupus nephritis. to identify predictors for unresponsiveness to the treatment. methods: thai children under 15 years of age with biopsy-proven diffuse proliferative lupus nephritis who were admitted to chiang mai universityhospital between 2001and 2006 were retrospectively studied. responsiveness to treatment, defined as urinary protein to creatinine ratio of less than 0.3, was assessed at the end of induction period. the clinical characteristics and laboratory data including gender, age at diagnosis of sle, duration of disease before treatment, hypertension, clinical nephrotic syndrome, amount of proteinuria, serum creatinine, creatinine clearance, serum c 3 level and presence of crescentic formation in renal biopsy were compared between the two groups who responded and did not respond to the treatment. results: a total of 27 patients (89% female) with the mean age at diagnosis of sle of 10.3±2.6 year were studied. nineteen patients (70%) achieved remission at the end of induction therapy. there were no significant differences in all parameters studied between responsive and nonresponsive groups. despite the indifference in the amount of proteinuria, the proportion of patients with nephrotic-range proteinuria was higher in unresponsive group. conclusions: pulsecyclophosphamide is an effective regimen for induction therapy in children with diffuse proliferative glomerulonephritis. although no definite predictor was detected in this study, higher proportion of patients with nephrotic-range proteinuria in the unresponsive group wasnoted. born small for gestational age, but not early postnatal weight gain aggravates the course of idiopathic nephrotic syndrome in children clinical and animal studies have shown a higher risk of an aggravated course of renal disease in childhood after birth small for gestational age (sga). fast catch-up growth after sga seems to support the development of later disease. in a retrospective analysis of 62 cases with idiopathic nephrotic syndrome treated between 1994 and 2004 in a university centrefor paediatric nephrology we identified 6 children as sga and 56 asappropriate for gestational age (aga). we related the course of disease to birth weight and catch-up growth. median age of manifestation in sga was 6.4 (1.9-15.2) years vs. 3.7 (1.2-15.33 ) years in aga children. in all sga children renal biopsy was performed, while only 55% of the aga children underwent renal biopsy showing nodifference in renal histology. in the sga group, 66% patients developed steroid resistance (vs. 21% aga, p<0.05). the number of relapses was not different. 83% sga children needed antihypertensive treatment inthe course of the disease compared to 39% of aga children. catch-up weight gain between birth and 24 months of age did not influence the course of disease. in conclusion we could find evidence for an aggravated course of idiopathic nephrotic syndrome in former sga children, but weight gainduring the first two years of life did not influence the course of disease. the mechanisms of perinatal programming in later renal disease need further investigation. wilson's disease(wd) is a disorder of copper metabolism that affects numerous organsystems including kidneys. besides renal tubular dysfunction as a result of excessive storage of copper, renal manifestations due to therapeutic complications can also develope specially with d-penicillamin. in this study we investigated the frequency and spectrum of renal manifestations during d-penicillamin therapy in wd. of 62 patients receiving d-penicillamin for wd, 4 patients(6.4%) (3 boys, 1 girl) developed findings of glomerulopathy within 1 month to 1 year after initiation of therapy and all were histologically diagnosed to have membranoproliferative glomerulonephritis (mpgn). the patients were between 6-11 years old, and they had normal urinalysis and renal function tests in their first presentations. two siblings developed hematuria and proteinuria below nephrotic range while the other two developed nephrotic syndrome. one of these latter patientsalso had acute renal failure needing temporary peritoneal dialysis. three patients had low complement (c) 3 levels, 2 had antinuclearantibody (ana) positivity, two had c-antineutrophil cytoplasmic antibody (anca) and one p-anca positivity. d-penicillamin therapy replaced by zinc sulphate in all patients. all renal findings improvedin patients within 3-6 months with normal renal functions and complement levels, and negative ana, p and c-anca tests. after 2 years all were clinically in remission of mpgn confirming the role of d-penicillamin in development of renal disease. objectives of the study: the use of tacrolimus in steroid-resistant (sr) focal segmental glomerulosclerosis (fsgs) has been reported in single and small series. the aim of this report is to exhibit experienceon the management of children with sr fsgs in whom tacrolimus had been started on due to the therapy resistance. methods: fk506 combined low-dose oral steroid was started on three male patients (3, 8, 14-yearold) with sr fsgs who had been following for three years. all of them had failed various cyclosporin a, cyclophosphamide and steroid regimens prior to treatment with fk 506. the application was 0.1 mg/kg/day in two divided doses over 12 h adjusted to a trough blood level between 5 and 10 ng/ml for 12 months. other therapies included angiotensin-converting enzyme inhibitors, vitamin d and calcium analogues, and lipid-lowering agents. results: a reduction in proteinuria to normal levels was noted between 2-4 weeks following the initiation. the remission was achieved overall during the treatment. the relapse was recorded following cessation of tacrolimus in 2-4 weeks. the drug was generally well tolerated with no sideeffects and adverse reactions. the ratio of infectious events did not differ from the former regimens. conclusion: tacrolimus may be effective in controlling the proteinuria of patients with srfsgs during the therapy. there is a trend of relapse following cessation of treatment. the duration for drug uptake is a topic of debate. further study in larger population is warranted. introduction: histological features of focal segmental glomerulosclerosis are found in 75% of pediatric patients with steroid resistant nephrotic syndrome. upto 50% (between 13-78%) children with fsgs progress to esrd. objective: to study the clinical course of childhood fsgs and determine the possible predictive factors of chronic kidney disease. method: case records of children who had biopsy proven fsgs and had presented to the sindh institute of urology and transplantation between 1995-2005, were retrieved. clinical and laboratory parameters at baseline, response to steroids and cyclosporine, development of crf (as defined by a gfr of <60 ml/min/1.73 m 2 ), and histopathological details were analysed. result: a cohort of 59 children with a mean age of 8.5±6.5 years and a m: f ratio of 3: 1 was identified. after a mean follow-up time of 5 years, 21/59 (35.5%) developed crf. on univariate analysis, male sex (90% vs 65%, p-0.04), >6 years age at onset (62% vs. 32%, p-0.02), hypertension (57% vs. 26%, p-0.05) and microhematuria at presentation (62% vs. 25%, p-0.007) were significantly associated with risk of developing crf. steroid resistant course (90% vs. 53%, p=0.003) was more prevalent in those who developed crf. the crf group was also more likely to have an elevated creatinine at baseline (47.6% vs. 5%, p<0.05). moderate tubular atrophy and a high percentage of segmentally and globally sclerosed glomeruli were found in those who developed crf. patients progressing to crf were more likely to have a partial response to cyclosporine (86% vs. 33%, p=0.02) conclusion: factors such as age, microhematuria, hypertension, elevated baseline creatinine, steroid resistance, tubular atrophy, percent global sclerosis and partial response to cyclosporine are likely predictive of progression to chronic kidney disease in children with fsgs presenting to our center. chronic glomerular nephropathies in children are marked by an often unfavourable evolution, so that the establishing of a prognosis at the time of the diagnosis is both a professional and a moral duty for the pediatric nephrologists. purpose: the estimation of the current practice renal survival prognosis in children with chronic glomerular nephropathies, by using clinical and laboratory elements in different histological forms of primitive chronic glomerulonephritis (cgn), with a minimum period of observation of one year. we analyzed parameters that may intervene in the duration of renal survival: type of cgn, age at the debut of the illness, histological scores of activity and chronicity, the presence of tubular atrophylesions and that of interstitial fibrosis, renal failure (rf) installment time, in cases with normal renal function at the beginning, the time until the initiation of dialysis in cases with esrf, respectively. the statistic analysis of data has been carried outwith epi soft (fishcer test). the results have been as follows: unfavourable evolution has been taken into consideration in the cases which have presented fixed nitric retention or which required the initiation of dialysis. the initiation of dialysis was necessary in 19 cases (76%), out of which 11 (44%) having associated between 4 and 6 of the considered risk factors. if the histological type (sfgs, dgs, mpgn) is added to the obtained score, the accuracy of the estimation increases to 89%. in conclusion: the usage of prognosis scores composed of current elements of diagnosis that have proven to have statistical significance, as far as the renal survival prognosis is concerned, may allow the invoking of a medium-term prognosis in the evolution of children with cgn. introduction: the srns can lead to a progressive deterioration of the renal function and therefore needs an aggressive therapy. one of the alternatives of treatment is the association described by mendoza et al which consists of prolonged use of methylprednisolone with cyclophosphamide (cp), which has a remission rate of 66%. the objective of this study is to evaluate retrospectively the clinical evolution of 15 children with srns treated with mendoza's protocol. method: between 1993 and 2005, 15 children, 8 male and 7 female, with srns were subjected to a renal biopsy and later treated with mendoza's protocol. cp was used in children that had not responded to pulses of methylprednisolone and presented a relapse. all of the patients received supplemental calcium. the clinical evaluation included stature, weight, ophthalmic fundus examination, proteinuria in urine recollection of 24 hours and/or protein to creatinine ratio and blood chemistry. results: eleven patients (73%) had fsgs. nine (60%) presented complete remission, two (13%) had partial remission, four (26%) did not respond to treatment, 3 of which evolved to terminal kidney disease. nine patients received cp. of the complications secondary to treatment with steroids, 80% had a linear growth suppression and an increase in their bmi and 1 patient presented cataracts with visual impairment. conclusion: the prolonged treatment with boluses of methylprednisolone and cyclophosphamide is a good alternative in patients with srns. the prolonged use of high doses of steroids can cause linear growth suppression and other adverse reactions, so it is advisable torealize a genetic study in all of the patients with srns to be able to exclude the patients that have a genetic mutation and so avoid unnecessary treatment. objectives: to study the clinicopathological profile and outcome of lupus nephritis (ln) in indian children. methods: clinical and histopathological features and outcome of children with ln was retrospectively reviewed. patients were included if they fulfilled the acr criteria for the diagnosis of sle and had either of persistent proteinuria, active urinary sediments or renal dysfunction. outcome was analyzed at 3 years and at last follow-up. results: ofthe 53 children studied, 13 were boys. the mean age (sd) at diagnosis was 9.8±2.3 (median 9.8, range 5-14.4) yr; 23 children were youngerthan 10 years of age. the mean age at presentation and renal biopsy was 10.8±2.2 (median 10.4, range 6.5-15) years. the mean duration of follow-up was 3.1±2.9 (median 2.5, range 0.2-10.3 years) years. 41.2% patients were followed for more than 3 years. commonest clinical manifestations were fever (77%), hypertension (58%) and malar rash (56%). 35 and 28% of patients presented with nephrotic and nephritic syndrome respectively before the diagnosis of sle. the commonest pathology was class iv nephritis (41%) followed by class ii (17%). hypertension, hematuria, nephrotic syndrome and decreased egfr were significantly associated with class iv ln. at last follow-up 31.5, 15.7 and 0% patients were in ckd stage ii, iii, and iv respectively. the patient survival rate at 3 year and at last follow-up was 100 and 94% respectively, while no patient developed esrd at 3 years. infections were seen in 30% cases that resulted indeath in 2 patients; 1 died of hepatic encephalopathy. conclusion: sle nephritis has a varied presentation and high morbidity. a significant proportion of patients developed infections during the course of disease. clinical, pathological features and outcome in our study do not differ markedly from those in most pediatric series. background: atypical hus has a frequently relapsing course and a poor renal prognosis. low c3 plasma concentration suggests alternate complement pathway regulatory abnormalities: factor h (fh), factor i (fi) and membrane cofactor protein (mcp). case report: we report an 11 year old girl with atypical hus due to acquired fh deficiency caused by anti-fh antibodies (abs). hus was diagnosed on the basis of acute renal failure, microangiopathic hemolyticanemia and thrombocytopenia. past history: recurrent fever with culturenegative pharyngitis (suspected pfapa syndrome). decreased c3 (64 mg%, normal>80) with normal c4. adamts 13 activity was depressed, no cleaving protease abs. hemodialys (hd) and plasma exchange (pex) were started 12 days later. severe urticaria and angoiedema during pex was treated with methylprednisolone and chlorpheniramine. hematological and renal improvement were observed after the 3 rd pex session. 4 relapses occurred in 3 months: 1 -controlled by pex; 2 -(with suspected pfapa) with single dose corticosteroids, 3 -with prednisone therapy, 4terminated with single dose ivig 200 mg/kg. elevated anti fh ab titer-1471 au with decreased fh functional activity were found in pre-pex plasma. fh, fi, factor b and c3 antigenic levelsnormal. hematological remission and renal function improvement without hus relapse ensued while tapering corticosteroids. two years after presentation, onprednisone 5 mg/day anti fh titer dropped significantly with restoration of functional fh activity, gfr ~75ml/min/1.73 m 2 . conclusions: in cases of atypical hus, active search for anti fh ab iscrucial for implementing specific and effective therapy: plasma exchange, iv ig and steroids for improving course and longterm prognosis. research centre for child health rams, department of pediatric nephrology, moscow, russian federation 25 children aged 1,5-16 years with idiopathic biopsy-proven steroid-resistant focal and segmentary glomerulosclerosis (fsgs) were treated with cyclosporine a (csa) 3,2-7,6 mg/kg as initial dosage, oral prednisolone 1,5 mg/kg every other day tapered to the 12-th month and methylprednisolone pulses (mp) 20-30 mg/kg every other day for the first 2-4 weeks in 17 of patients. serum creatinine level was controlled once a month. after 6 months of csa treatment complete or partial remission of proteinuria was in 15 (60%) of children, no effect in 10 (40%). serum creatinine level increased in 14% on an average in the group remission of proteinuria. in the group of non responded patients the creatinine elevation was significant same -15%. after 1 year of csa treatment complete or partial remission observed in 18 (72%), no effect in 7 (28%). elevation of serum creatinine level in children with remission was 15,5% (without significant difference compared to 6 month's csatreatment). increasing of the creatinine level more than 30% in two patients leaded to double tapering csa dose resulted in normalization of serum creatinine level. in the group of csa non responders the significant increasing of serum creatinine level (35%) was revealed (compared to 6 month's csa treatment). in 3 cases the elevation ofcreatinine level was more than 50% and these patients turned into esrd eventually. in all of non-responders csa was discontinued. we concluded that serum creatinine level in csa respondrers was stable without significant elevation during the 1 year of treatment. csa therapy for 1 year without any effect influenced on renal function decreasing. clinical objective: to describe the clinical course and outcome of pediatric patients with cgd treated with iv mpt (30 mg/kg x 3 doses monthly for 12 months, then 1 dose monthly for the next 6 months) methods: patients' medical records were reviewed. pre, post-treatment and follow-up 24 hr urineprotein, creatinine and gfr were compared using paired t-test; and proteinuria, hematuria and blood pressure using mcnemar's chi square. outcome measures were analyzed usingmean ± sd and frequency distribution. results: 30 patients were included, 13 male, 17 female. mean age at disease onset is 7.972±4.298 years. mean duration of follow-up is 23.733±18.714 months. 70% achieved complete remission after a mean of 1.8 cycles, 17% partialremission after a mean of 2.8 cycles and 13% were treatment failures. mean relapse rate is 0.700±1.208 on treatment and 0.733±1.596 at follow-up. renal survival rate is 93%. 24 hour urine protein and the proportion of patients with proteinuria, hematuria and hypertension significantly decreasedafter treatment and remained stable at follow-up. serum cr/gfr were also stable pre, posttreatment and at follow-up. no serious side effects were noted. conclusion: this protocol induced a high and early remission rate (70% after 1.8 cycles)among the patients. majority demonstrated stable renal function and blood pressure over time. relapse rates were low and treatment is generally safe. recommendation: the protocol can be offered to oral-steroid or alkylating agent-resistant patients with satisfactory remission rates. objectives of study: nestin, an intermediate filament protein which has a role in regulating cellular cytoskeletal structure, is restrictedly expressed in the podocytes of human kidneys. in the present study nestin expression was investigated in biopsy specimens of children with focal segmental glomerulosclerosis (fsgs). methods: 36 kidney biopsy specimens taken from children with diagnosis fsgs were investigated. diagnosis was performed on light microscopy, immunofluorescence microscopy, taking into account clinical data. for immunomorphology monoclonal anti-nestin antibody from mouse (sc-23927, clone 2c1.3a11, santa cruz biotechnology) diluted 1: 100, was applied on cryostat or paraffin sections using labeled streptavidinebiotin (lsab+ dako) method. visualization was performed by dako aec substrate. 10 kidney biopsies of patients without nephrotic syndrome, mainly with mesangioproliferative gn were used for control staining. results: the mean age at the time of biopsy was 10.2±4.9 years, and all patients had nephrotic syndrome. half of them revealed some focal tubulointerstitial changes: tubular atrophy, slight interstitial fibrosis and lympho-monocytic infiltration. in two cases mutation of wt1 gene and in one case mutation of nphs2 gene was detected. four cases had familial character of fsgs. nestin expression was variably present in different cases of fsgs. decreased expression was detected in glomeruli with segmental mesangial sclerosis and capsular adhesions. conclusion: fsgs revealed heterogenity concerning nestin expression. nestin expression was diminished in affected glomeruli. background: renal effects of altered ob/ob-r pathway may contribute to obesity, and diabetesassociated proteinuria. in the kidney ob/ob-r stimulates collagen type i and iv synthesis and upregulates tgfβ1 and tgfβ2 receptors. objective: to determine ob/ob-r and its downstream (jak/stat/socs) pathway expression in nephrotic syndrome (ns) and fsgs. design/methods: microarray analysis of kidneys of 2-week (2w) and 4-month (4m) old transgenic mice (tg) and controls (ctr) was performed, and confirmed by quantitative pcr. kidney sections were analyzed by immunohistochemistry (ihc) and western blot analysis (wb). urinary ob/ob-r of children with ns classified as steroid sensitive (ssns) or steroid resistant (srns), were measured by elisa. results: ob, ob-r, and jak1,2,3 mrna expressions were not statistically different at 2w and 4m between ctr and tg. stat3 and socs mrna were increased 2.04-fold (sem ±0.52), and 17.38fold (sem±8.6) at 4m in tg, p=0.04 and p=0.01 respectively. ihc and wb (p=0.65) of kidney sections showed no significant difference between the 2 groups. we examined 12 ctr, 10 ssns, and 11 srns patients with comparable bmi, age, race and gender. urinary protein to creatinine ratio in ssns and srns was 0.18 (sem±0.06) and 3.12(sem±1.03) respectively, p=0.03. urinary ob (p=0.1), ob-r (p=0.09), and tgfβ1 (p=0.12) were not statistically significantly different between the 3 groups. conclusions: ob/ob-r was not upregulated in tg at the onset of proteinuria and fsgs. however, advanced fsgs (4m tg) showed significant activation of socs3, an ob/ob-r negative regulatory pathway. pediatric patients with early srns had no significant increase in urine ob/ob-r. this data suggests the role for ob/ob-r regulatory pathways in the development of advanced fsgs. primary fsgs presents clinically with steroid-resistant (srns) or steroid-dependent (sdns) nephrotic syndrome or proteinuria. the data shows, that 43% patients with fsgs progress to esrd in 15 years follow-up. objectives: the aim of the study was to analyze the long-term outcome of patients with primary fsgs diagnosed in kidney biopsy. material: the study group consisted of 113 children (54 males, 58 females) followed from 1982. all patients were treated with immunosupression and renoprotection. the clinical data were analyzed after follow-up lasting for mean 10±6,1 years (0,5-25 years) . 85 children presented with nephrotic syndrome (66 srns and 17 sdns) and 28 with proteinuria. the age ranged from 0,5-16 years mean 5,9±4,5 at the time of diagnosis. more then 1 kidney biopsy was performed in 55 children -in 29 progression from mcd (n=18) and mes (n=11) to fsgs were observed. results: the clinical remission was observed in 73/113 patients (64%) and was not correlated with initial proteinuria (mean 11,3±9). in 20/113 patients crf was observed, 15 of them progressed to esrd (12 were successfully transplanted). among patients, who had fsgs as their initial glomerular lesion (n=84), the percentage of glomerular sclerosis was significantly higher in a group in which remission was not obtained after long-term follow-up (31,7 vs. 17%, p<0,001). in 9 out of 49 patients with follow-up over 10 years progression to esrd was observed (18,4%), 10/49 were transmitted to adult centers with persistent proteinuria. conclusions: immunosuppression and renoprotection in patients with fsgs can prevent the progression of crf. extensive glomerular sclerosis is a predictor of unfavourable outcome. further clinical and genetical studies are needed to establish the effective therapy modalities. mycophenolate and who had previously undergone a renal biopsy, recieved 600 mg twice daily (maximum 1 gram twice daily) during six months. prednisone was concurrently prescribed at at dosage of 1 mg/kg/every other day, during 8 weeeks and 0.5 mg/kg/every other day during the subsequent 8 weeeks. results are expressed as mean±sd. results: seven chidren, 5 boys and two girls were enrolled. oncet of their ns was at age 6.2±4, 16 yr (range 2-13 yr) and mmf was initiated at 9.4±4.4 yr (4.5-15.5 yr) . six were sr and one sd. two patients had previously recieved cyclosporine, two patients cyclophosphamide and one chlorambucil. renal histology displayed: focal segmental glomerulosclerosis (n=3), minimal change (n=2), mesangial proliferation (n=1) and membranous glomerulonephritis (n=1). at the end of the follow-up: three patients were in partial remission, two were in complete remission and two had no response to mmf. initial and final serum creatinine concentration 0.53±0.08 vs 0.57±0.09 mg/dl), estimated gfr (124.2 vs. 111±37 ml/min/1.73 m 2 ) and serum albumin ( idiopathic nephrotic syndrome is the most frequent glomerular disease in childhood. most patients are steroid responsive but half of them relapse and often become steroid-dependent. they are exposed to long term steroid complications on the one hand and relapses due to insufficient disease control on the other hand. our aim is to determine predictive risk factors for high degree steroid dependence. in france, steroid-resistance is defined as persistent proteinuria after one month of daily oral prednisone (60 mg/m 2 ) and 3 pulses of methylprednisolone (mpn) (1 g/1.73 m 2 ). we included 27 steroid responsive children with disease onset between 2000 and 2006. the mean age at diagnosis was 4.6 years (range 1.5-16). all patients initially received prednisone 60 mg/m 2 per day. the following parameters were analysed: age at onset, gender, days to remission with initial steroid therapy, mpn pulses, numbers of relapses, steroid dependency, immunosuppressive drugs. twenty of the 27 patients were steroid-dependent; among the 20 steroid dependent patients, 11 received mpn pulses. 90% of those patients (10/11) were treated by cyclosporine during follow-up. on the other hand, only 10% (1/9) of the patients who did not receive mpn required cyclosporinebased therapy during follow-up (chi-square test, p=0.0018). interestingly, there was no correlation between treatment days until remission during the initial prednisone course and the risk for later steroid dependence. conclusion: the need for mpn pulses, but not the time interval until remission helps to predict steroid dependence. patients, necessitating mpn pulses to obtain remission are at risk to require cyclosporine for disease control. by identifying these children, we could eventually 1/ avoid multiple relapses by earlier use of adequate immunosuppression and 2/ avoid side effects related to long term high dose steroid therapy. membranous nephropathy (mn) with antitubular basement membrane antibodies is a rare condition. relapse of tubular dysfunction in renal transplant recipients has been published in one case, but relapse of mn in the renal graft has not yet been reported. a 3-year old boy presented first with steroid resistant nephrotic syndrome associated to tubular dysfunction. renal biopsy revealed mn associated to interstitial fibrosis and granular deposits of iga, igg, and c3 along the tubular basement membrane. indirect immunofluorescence (if) revealed circulating anti-tubular basement membrane antibodies. he received a renal allograft at the age of 6 years. an acute rejection episode on day 21 required three steroid pulses and okt-3. renal biopsy revealed the presence of interstitial and vascular rejection (banff iii) and relapse of tubular basement membrane deposits. renal function normalized within 10 days and remained stable (gfr estimated by schwarz formula=103 ml/min per 1.73). proteinuria remained negative and urinalysis normal over 5 years under the immunosuppressive regimen including fk, azt, and prednisone. at the age of 11 years, proteinuria increased progressively over 6 weeks reaching 2.8 g/day, whereas serum creatinine remained stable. renal biopsy revealed the presence of granular deposits along the glomerular basement membrane suggesting a late relapse of mn in the transplant. rituximab therapy (day 0, 15, 30, and 60) followed by switch from azathioprine (aza) to mmf resulted in a complete biological remission with negative proteinuria. indirect if revealed progressive decrease of antitubular basement ab level during rituximab treatment and a negative signal was obtained 3 months after the switch from aza to mmf. this is the first report of glomerular relapse of mn with anti tubular basement antibodies in a renal transplant recipient. nephrotic children are at risk for severe pneumococcal infections. the best moment for antipneumococcal vaccination is controversially discussed. we investigated the serologic response after pneumo23 vaccination in 25 children (10 girls) with nephrotic proteinuria and hypoalbuminemia, immediately after initialisation of prednisone therapy at 60 mg/m 2 (group 1) and in 16 children after tapering down of prednisone to <0.5 mg/kg eod (group 2). there was no difference in both groups concerning antibody (ab) response, relapse frequency, or number of steroid dependent forms. in group 1, pneumo23 ab levels at presentation (m0) were 1.20±0.22 (mean±se) . at m1 antibody levels increased 9-fold to 9.28±1.35 (p<0.0001). serum levels at m3 were 7.58±1.67. one year after vaccination ab levels (4.4±1.78) decreased compared to m1 (p<0.01), but remained increased compared to m0 (p<0.01). there was no increased delay until remission in both groups compared to a retrospective control group. severe hypoalbuminemia (<10 g/l) at the time of vaccination was not related to a lower serological response on m1. during relapses, antibody levels decreased significantly compared to levels before relapse (p<0.01), but increased again once remission was obtained. even during relapses, ab levels remained higher (>3-fold) than pre-vaccination levels. conclusion: nephrotic children on high dose glucocorticoid therapy respond to anti-pneumococcal vaccination and their ab levels remain elevated during relapses. vaccination at disease onset may be beneficial as those patients with relapses during the tapering down of steroids already have increased anti pneumo23 ab at the time of relapse. crescentic glomerulonephritis with isolated c3 deposits associated to complement abnormalities: a new entity? introduction: several progressive renal diseases present proteinuria, as a result of glomerular and tubulointerstitial injuries. thus, some studies prove that proteinuria is an important predict factor for progression of renal failure. angiotensin converting enzyme inhibitors (acei) are efficient in reducing proteinuria and preserve renal function in patients with diabetic or non-diabetic nephropathy. the purpose of this study was to evaluate the efficacy and security of acei in children. material and methods: the acei (enalapril) was used in normotensive and hypertensive patients with chronic renal disease, with microalbuminuria/proteinuria. results: we studied 28 patients (13 girls), for at least 6 months, mean age was 10.4±3.5 years (1 month to 17 years). 5/28 patients had glomerulopathy, 13/28 chronic pyelonephritis, 4/28 systemic disease, 2/28 renal hypoplasia/ dysplasia, 3/28 cystic renal disease and 1/28 arterial hypertension. the mean dose of enalapril was 0,25mg/kg/d (0, 04 to 0, 38) and it was used during 36,6 months (mean). we observed a normalization of proteinuria/microalbuminuria in 53, 5% of cases. in seven patients, the drug was discontinued due to: 3/7 irregular use, 2/7 vertigo, 2/7 hypercalemia and acute renal failure recovered after withdrawn the drug. during the use of enalapril we did not observe significant difference in potassium or creatinine serum levels, as well as blood pressure measurements. conclusions: the use of enalapril in pediatric patients with renal disease and proteinuria/ microalbuminuria showed security and efficient. therefore, we suggest it as a antiproteinuric and renoprotective agent in children. a. filleron, al. adra-delenne, l. ichay, f. dalla-vale, h. valette, d. morin chu montpellier, pediatric nephrology, montpellier, france in order to evaluate the long-term efficacy of oral cyclophosphamide (cp) in children with sdns, the outcome of 33 patients (11 girls) treated in our unit for steroid sensitive ns were studied retrospectively. median age at diagnosis was 3.8 years (range 1.5 to 13). initially, they received oral prednisone (p) : 60 mg/m 2 /d for 4 weeks and p dosage was then tapered for the next 14 weeks (totale dose p: 3390 mg/kg). relapses of proteinuria were treated with p (60 mg/m 2 /day) and, in case of steroid dependency (sd), p was maintained on an alternate day regimen. one single child had no relapse, while 8 had a relapse rate of less than 1/year. the remaining 24 frequently relapsed and 14 received oral cp -2 mg/kg/day for 12 weeks, totale dose 168 mg/kgbecause of their high relapse rate with steroid toxicity. median duration of p treatment was 3.7 years (range 0.75 to 10) before cp was given. in one case cp had to be stopped because of hemorragic cystitis. follow-up after cp treatment was, at least, 2 years. p was stopped in the following 6 months after cp in 10/13 children, but has to be continued in the remaining 3 because of early relapse of proteinuria. among those 13 children, only one had no more relapse 4 years after cp. in 12 children, relapse of proteinuria occured 11.5±7 months after cp. in those patients, 8/12 had to receive another steroid sparing drug such as cyclosporine or mycophenolate mofetil (mmf) because of recurrence of sd. in our experience, cp treatment in ns with steroid toxicity is associated with a significant change in the relapse rate in only 5/13 children and in the remaining 8, improvement was transient. our results suggest that alternative treatment, such as mmf, has to be evaluated as first-line steroidsparing agent in those patients with sdns and steroid toxicity. interleukin the aim of our investigation is to compare the concentration of total ige, specific ige, interleukin-4 (il-4) and gamma-interferon (gamma-ifn) in serum of 27 children with initial and relapsed steroid-sensitive mcns and of 26 children with atopic dermatitis at the age from 1 to 16 years. the concentration of total ige was measured by immunoenzymatic method and il-4, gamma-ifn by immunoassay technique using monoclonal antibodies. the result showed that 64% of 26 children with atopic dermatitis had the increased concentration of total ige; specific ige was increased to alimentary allergens-84,6%, household-69,2%, inhaled-26,9%. the concentracion of il-4 was 34,4±9,4 pg/ml, of gamma-ifn was 102±11,3 pg/ml. the result showed that 9% of 27 children with mcns had increased concentration of total ige; specific ige was increased to alimentary allergens-96,2%, household-77,8%, inhaled-29,6%. the concentration of il-4 was 22,31±3,8 pg/ml, of gamma-ifn was 91,97±9,3 pg/ml. according to our investigation, the concentration of il-4 and gamma-ifn in children with mcns were not significantly different then in children with atopic dermatitis. conclusion: the fact that there were not significant differences in serum total ige and specific ige, il-4 and gamma-ifn in children with mcns and atopic dermatitis gives us a reason to suppose that these diseases have identic mechanisms of pathogenesis with ige reaction i-type with activation of t-limfocyte. to clarify the pathogenesis of mcns, comprehensive studies for these cells would be worthwhile. there are several lines of evidence that the slit diaphragm (sd) not only serves as a structural framework for filtration barrier but also has an essential role as a signaling platform. nephrin is tyrosine phosphrylated by src-family tyrosine kinase, fyn. phosphorylated nephrin recruits nck to sd, and regulates assembly of actin filament. the crucial roles of tyrosine phosphorylation in podocyte is also indicated by renal malfunction observed in fyn knockout mice. neph1 has a longer cytoplasmic domain and a larger number of tyrosine residues in its cytoplasmic region than nephrin. but knowledge about tyrosine phosphorylation of neph1 is limited. here we characterize neph1 as a substrate of fyn. fyn interacted with and phosphorylated cytoplasmic domain of neph1 in vitro and in cultured cells. peptide mass fingerprinting of neph1 cytoplasmic domain phosphorylated by fyn in vitro identified at least five tyrosine phosphorylation sites. site-directed mutagenesis confirmed that these tyrosine residues were indeed phosphorylated in cultured cells. in pull-down analysis with neph1 from rat glomerular lysate, neph1 specifically bound to an adaptor protein grb2 and a tyrosine kinase csk in a phosphorylation-dependent manner. coimmunoprecipitation experiments revealed phosphorylation of y637 and y638 were crucial in neph1-grb2 binding. furthermore, tyrosine phosphorylated neph1 suppressed erk activation elicited by fyn, and also inhibited fyn-induced ap-1 transcriptional activation. these inhibitory effects required the intact binding motif of the grb2 sh2 domain, and both y637f and y638f mutants failed to inhibit erk activation. these results indicate that fyn orchestrates a wide spectrum of protein-protein interactions at sd by phosphorylating neph1 as well as nephrin, and neph1 modulates downstream signaling by phosphorylation-dependent association with adapter proteins. celiac disease (cd) is a common disorder in southern europe and has a protean clinical presentation. hla class ii aplotypes dq2 and/or dq8 are present in 99% of cd patients and in 30% of the normal population. the observation of three patients with both cd and nephritic syndrome (ns) prompted us to study hla class ii aplotypes in our patients with ns. in all children with ns admitted to our unit we determined the presence of dq2/dr3, dq2/dr7, dq2/dr4 e dq8/dr4 aplotypes and anti-transglutamidase antibodies (ab-httg). hla typing was done by dna extraction and pcr amplification and electrophoresis in agarose; ab-httg determination was made by elisa. as control groups we examined 27 children with cd and 70 first degree relatives (of theirs). in so far we have studied 40 children with ns (27 males, 13 females, age ranged 3-18 years); 34 are steroid sensitive (ssns), 6 steroid resistant (srns). a renal biopsy was done in 10 and showed minimal lesions in 3, focal and segmental sclerosis in 2, membranoproliferative gn in 2, membranous gn in 2 and iga deposition in 1. corticosteroids or other immunosuppressant were administered in 27 when blood was drown. dq2 and/or dq8 aplotypes were present in 33 out of 40 patients (82.5%), in 29 out of 34 ssns (85.3%), in 43 out of 70 cd relatives (61%) and in all cd patients. dq2/dr3 combination was present in a smaller percentage of ns compared to control groups. ab-httg were detected in one patient out of 26 (3.8%). purpose: to investigate activity of antithrombin and a protein c at 57 children with mcns: at 39 active period (proteinuria more 1 g/m 2 /d; hypoalbuminemia <25 g/l), at 46 in incomplete remission (the third day of absence of proteinuria, hypoalbuminemia <35 g/l), at 11 in proof remission. methods: activity of natural anticoagulants in blood was defined by a clotting method with use of reactants "roche" and "behring". results: activity of antithrombin in blood in the active period of disease sharply decreased (60,6±3,9%, p<0,0001), and already in the period of incomplete remission came back to norm (96,2±2,6%), characteristic for the period of full remission (96,3±4,8%). activity of a protein c in blood in the active period of mcns was high (154,1±7,5%, p<0,001), during incomplete remission decreased (127,1±7,5%, p<0,001), in the period of proof remission was in norm (94,2±5,7%, p<0,001). at children with mcns dependence of decrease in activity antithrombin from weight hypoalbuminemia (r=0,5, p<0,005), hyperfibrinogenemia (r=-0,6, p<0,005), hypercholesterolemia (r=-0,5, p<0,01) and hyper-lipoproteinemia (r=-0,5, p<0,03) is established. authentic distinctions of factor of the attitude of activity of protein c/ activity of antithrombin depending on the period (the active period -2,5, incomplete remission -1,3, proof remission -0,98) are received. conclusion: at children with mcns changes in system of natural anticoagulants: decrease in activity antithrombin below 80% and increase of factor of a parity of anticoagulants (more than 1,0) testify to hypercoagulation and risk of thrombosis. varicella objectives: the pattern of steroid responsiveness of nephrotic syndrome may change during the course of the disease in children with steroid sensitive nephrotic syndrome (ssns) and/or in different populations. patients and results: a prospective cohort study was conducted in 22 centers. patients who were initially diagnosed as ssns in 2001 and followed for five years were included. standard questionary forms from 268 children(149 boys) with a mean age of 4.3 years (22 months-16 years) at presentation were submitted for entry to data coordinating center. 165/268 patients who showed initial steroid sensitivity with a follow-up period of at least one year (1-5 years) were included in the study. seventy three (44.5%) children remained in sustained remission at 1 year; nine patients showed steroid resistance. 67/165 patients were followed for 5 years, whose clinical course were sustained remission in 46 (70%) and steroid resistance in 4(6%). steroid response rate from 1 to 5 years remained stable (93-95%). eight children out of totally 13 patients who were steroid sensitive initially, became steroid resistant in the first year. the remainder showed steroid resistance at the 2 nd year (2), at 4 th year (1) or at 5 th year (2) . renal biopsy was performed in 19 children who developed steroid dependency or steroid resistance. nine patients revealed fsgs, 8 minimal change disease, 1 mesangioproliferative gn, 1 membranoproliferative gn, one igm nephropathy. only two patients who had minimal change nephropathy in initial biopsy progressed to fsgs after 2 and 5 years. conclusion: steroid response rate was between 93-95% and steroid resistance was 4-6% in 5 years follow-up. secondary steroid resistance within the first year of presentation seemed to be predictive for their subsequent courses. the need of biopsy was not high. ssns seemed still as a relatively benign condition in our population. the aim of this study was to asses the changes in coagulation/fibrinolysis system in chronic renal disease (crd) by measuring plasma levels of von willebrand factor (vwf) and plasminogen activator inhibitor -1 (pai-1). we studied 74 children (5-16 years old) with nephrotic syndrome (ns): minimal change disease (n=14), focal segmental glomerulosclerosis (n=17), mesangioproliferative glomerulonephritis (n=24), membranoproliferative glomerulonephritis (n=7). relapse of the disease was observed in 34 patients. 15 healthy age matched children served as controls. serum levels of pai-1: ag and vwf were measured by elisa. results. pai-1 and vwf levels were elevated in all morphological forms of ns in relapse and remission compared with controls (p<0,01) except the mcd remission in which they were the same as controls (p>0,05). the highest levels of pai-1 and vwf were discovered in relapse of proliferative forms (mespgn, 70±31 ng/ml and 5,0±1,7 me/ml, respectively; mpgn, 100,05±61,1 ng/ml and 3,14±0,65 me/ml, respectively) compared with nonproliferative (mcd 52,98±24,03 ng/ml and 1,81±0,54 me/ml, respectively; fsgs 52,27±20,39 ng/ml and 2,42±0,84 me/ml, respectively, p<0,001). conclusion. these data suggest activation of coagulation/fibrinolysis system in relapse of ns and the absence of normalization in the remission phase. our results confirmed that more severe fibrin formation via activation of intraglomerular coagulation and fibrin accumulation is characteristic for mpgn, likely by deficiency of the fibrinolysis system. introduction. several recent case reports suggest that rituximab (rtx) could be an effective treatment for idiopathic nephrotic syndrome. in a retrospective study, data were collected from 11 patients (mean age: 12.6 years) treated with rtx for steroid dependent nephrotic syndrome (mean duration of the disease: 112 months). four of 11 were treated during a remission period. eight of 11 were treated in association with one or several other immunosuppressive (is) treatments (prednisone, anticalcineurin, mycophenolate mofetil). rtx efficacy was admitted when the previous is treatment was withdrawn or significantly tapered-off, or when the proteinuria disappeared with no other change than rtx treatment. a complete b-cell depletion was confirmed in all patients when assessed (9/11) even when rtx was infused during a period with nephrotic proteinuria. rtx was considered to be effective in 6 cases especially when given in association with other immunosuppressive treatment during a period with remission of proteinuria (4/4 success, follow-up 3 to 10 months). conversely rtx failed to induce remission among patients who were treated during a proteinuric period with no other immunosuppressive drug (3/3 failures). finally rtx was considered to be effective among 2 of 4 patients treated in association with other is drugs during a proteinuric period (follow-up 3 and 31 months). there was no significant side effect during rtx infusion. delayed side effects were observed for 2 patients: 1 case of neutropenia and pneumocystis pneumonia and 1 case of hypogammaglobulinemia. conclusion. rtx is an effective treatment in a subset of patients with severe steroid dependent nephrotic syndrome. further prospective data are necessary to determine if rtx could become an alternative to other immunosuppressive drugs in patients with toxic side effects. infections are leading causes of death in lupus patients. disseminated histoplasmosis has been commonly documented in immunocompromised patients including lupus patients. we report a case of lethal cerebral histoplasmosis in a child originating from french guyana. lupus disease was revealed by typical malar rash. she developed a class ii lupus nephritis treated with prednisone and azathioprine. then she developed restrictive lung disease, recurrent arthritis and pericarditis. later on, nephrotic syndrome revealed a class iii lupus nephritis treated with methylprednisolone pulses and mycophenolate mofetil. four years following the onset of the disease, she was admitted because of febrile seizures and five months later for a febrile coma. repeated lumbar punctures displayed hypercellularity with depressed levels of glucose and elevated protein concentrations but sterile cultures. according to the presence of high titers of lupic specific antibodies and cerebral mri suggesting vasculitis, neurological flare of lupus was considered and immunosuppressive treatment was increased (methylprednisolone and cyclophosphamide pulses, plasma exchanges). a repeated lumbar puncture evidenced presence of histoplasma capsulatum. despite antifungic treatment the child died. our report emphazises the difficulty to discriminate opportunistic infections from the wide spectrum of lupus clinical features. symptoms of infection may mimic those of lupus, or conversely, may be masked by immunosuppressive drugs. infection screening should take in account clinical feature as well as endemic context. our report is the first case of isolated cerebral histoplasmosis in a child with systemic lupus. renal manifestations of mitochondrial cytopathies have been described, but nephrotic syndrome with respiratory chain disorders (rc) was described extremely rarely in infancy. we report a 9 months-old boy with a mitochondrial cytopathy preceded by 2 months history of steroid-resistant nephrotic syndrome. on admission his clinical condition was deteriorating rapidly with gross oedema, ascites, hypertension and oliguria. fundoscopic examination revealed salt-pepper sign which was thought to be consistent with intrauterine infection (iui) at that time. however, serologic and microbiologic investigation of iui was inconclusive. a sensorineural hearing loss was found to associate his findings. podocin mutation was negative. a percutaneous renal biopsy was undertaken and revealed diffuse mesengial sclerosis. a significant decrease in mitochondria was observed on electron microscopic examination. the child progressed to end stage renal failure and was successfully managed by peritoneal dialysis. during his follow-up a fine tremor was observed in his hands and cranial mri revealed demyelinisation in left thalamus and occipital lobe. steroid resistant nephritic syndrome, sensorineural hearing loss, ocular and neurologic findings has led us to be suspicious about mitochondrial cytopathy and muscle biopsy was done. though muscle biopsy was normal, the results of biochemical analysis showed a deficiency of the respiratory chain complex iv (cytochrome c oxidase) (rc iv). the clinical phenotype and the deficiency of respiratory complex iv thought to be compatible with deficiency of the cytochrome c oxidase deficiency protein cox10. nephrotic syndrome with rc disorder were described extremely rarely in infancy. based on these observations, we suggest that rc disorders should be considered in patients with early onset nephritic syndrome. human parvovirus b19 (hpvb19) was identified as the cause of a self-limited childhood febrile illness with rash, namely erythema infectiosum. most of hpv-b19 infections are usually mild or asymptomatic, but in some cases infection is associated with serious systemic complications. renal involvement in patients with hpvb19 infection was discussed in recent, mostly anecdotal, case reports. the majority of these reports were described in adults, whereas only a few cases of childhood were defined whom presented with mesangiocapillary proliferative glomerulonephritis, fsgs or tubulointerstitial nephritis. a literature search revealed no cases of acute endocapillary proliferative glomerulonephritis in childhood. a 10-year-old girl was admitted with fever, cough, maculopapuler rash, hemoptysis, dark-colored urine, multiple lymphadenopathies, hepatosplenomegaly. she developed acute nephritic syndrome during the course of this complex clinical features. laboratory data showed proteinuria, hematuria, hypocomplementemia, the presence of igm and igg antibodies to hpvb19 and positive reaction of serum hpvb19 dna using a polymerase chain reaction. renal biopsy showed acute endocapillary proliferative glomerulonephritis with coarse granular c3 depositions in a "starry sky pattern" which is more peculiar to poststreptococcal glomerulonephritis. electron microscopy revealed subendothelial and hump-shaped subepithelial dens deposits. there was no evidence of either a mycobacterial or a streptococcal infection and the diagnosis of goodpasture syndrome and connective tissue disorders were excluded by clinical and laboratory investigations. based on the literature review, this case represents, to our knowledge, the first time that a direct relationship between parvovirus infection and acute endocapillary proliferative glomerulonephritis has been demostrated in a child. objective: the purpose of this retrospective cohort study was to report the clinical course of children and adolescents with focal segmental glomerulosclerosis (fsgs) according to steroid response. methods: the records of 88 patients with biopsy-proven fsgs admitted between 1972 and 2005 were retrospectively reviewed. demographic, clinical and laboratory data at entry and at the end of the follow-up were analyzed. the patients were classified according to the initial prednisone response into two groups: group 1 (g1): response (complete or partial remission) (n=63) and group 2 (g2): non-response (prednisoneresistant) (n=25). renal survival analysis was performed using the kaplan-meier method. results: the median age at admission was 4.55 years (iq range: 0.99±12.84 yr) in g1 and 6.86 years (iq range: 1.08±15.55 yr) in g2. seventeen patients (27%) of g1, and 17 patients (68%) of g2 presented with hematuria at admission, and 31 (49%) children of g1 and 13 (52%) of g2 presented blood pressure levels above the 95th percentile. g2 presented a higher 24 h proteinuria (4.87 mg/24 h) at admission when compared to g1 (3.17 mg/24 h, p=0.019). median follow-up time was 8.72 years in g1 and 3.96 years in g2. the renal survival rate was 98% at 5 years and 92% at 12 years in g1, 69% at 5 years and 26% at 13 years in g2. conclusion: progressive renal insufficiency was more frequent in patients with fsgs who have initial resistance to prednisone therapy. objectives of the study: adults with chronic kidney disease (ckd) show impaired immune status. in this study, the profile of mononuclear cell subsets was related to the age and actual gfr in children and compared to healthy controls. methods: the expression of lymphocyte surface antigens was evaluated on peripheral blood (pb) mononuclear cells using three-color flow cytometry in 45 children with ckd (stage2-5) on conservative treatment. we analyzed absolute and relative numbers of total leukocytes, total lymphocytes and subsets: cd19+, cd3+, cd3+cd4+, cd3+cd8+, cd3-cd16/56+, cd3+hla-dr+, cd3+cd25+, cd69+, cd3+αβ+, cd3+γδ+, cd45ra+, cd45ra+cd4+, cd45ra+cd8+, cd45ro+, cd45ro+cd4+, cd45ro+cd8+, cd4+cd25+. results: in younger ckd children (below 10 years) absolute numbers of cd3+, cd3+cd8+, cd3+cd25+, αβ+t, γδ+t cells and cd4/cd8 ratio was higher, the percentage of cd3+cd8+, cd45ra+cd4+, cd45ro+cd4+, cd45ro+cd8+, αβ+t cells and the absolute number of cd45ro+cd8+ cells was lower than in the oldest group. in children with the lowest gfr (below 15 ml/min) the percentage of cd3+, cd19+ was lower, the absolute number of cd3+cd25+, cd69+, and the percentage of nk-cells, cd3+cd25+, cd69+, cd45ro+cd8+ cells was elevated as compared to ckd stage 2 group. the absolute number of cd8+, cd3+cd25+, cd45ra+cd8+, αβ+t, γδ+t cells and percentage of total lymphocytes, cd19+, cd3+cd25+, cd69+ was lower in ckd children than in controls. conclusion: impaired immune status is observed already in early stages of ckd. progressive disturbances in pb lymphocytes percentage mostly in the naive and memory t cells and the shift in the cd4/cd8 balance were found in pre-dialysis children with ckd. with progressive loss of renal function, we observed an increased expression of activation markers on t cells such as cd25 or cd69. introduction: relapse of steroid resistant nephrotic syndrome (srns) after renal graft occurs iñ 30% of the pediatric patients. medical management is based on increased immunosuppression with the use of iv cya and plasma exchanges (pe). however, this strategy fails in ~30% of the treated patients. new immunosuppressive agents may improve the outcome of relapsing srns post transplant. case report: a 15-year-old boy with srns reached esrd and received a cadaveric kidney transplant after two years on hemodialysis. the immunosuppressive regime was cya, mmf and steroids. seven days post transplant gross proteinuria (4 g/day) reoccurred. iv cya was administered over two months (blood level: ~450 ng/ml) associated to pefloxacine and pe (n=15), and followed by oral cyclophosphamide (cyp), resulting in partial disease control. cyp was discontinued due to haematological toxicity after one month. proteinuria increased again from 1 to 3 g/day within 3 months, despite high dose oral cya (10 mg/kg/day) and mmf. etanercept (a tnf blocking agent) was introduced at a dose of 25 mg twice weekly (combined with three steroid pulses) and maintained over two months: proteinuria decreased to 0.4 g/day over 20 days. etanercept was discontinued followed by a relapse of the ns and re-introduced eight months later, with, again a significant decrease of proteinuria to a baseline level of 0.5 g/day. conclusion: treatment with anti-tnf agents in nephrotic children has been reported once in a boy with high degree steroid dependent ns; a spontaneous decrease of disease activity over time cannot be excluded in this patient and might jeopardize data interpretation. our case is the first report of successful antiftreatment despite a constantly high activity of the nephrotic syndrome, demonstrated by relapse after discontinuation of etanercept while the patient was on post transplant immunosuppression. fournier´s gangrene (fg) is defined as a specific, quick and progressive form of synergic necrotizing fasciitis of multi-bacterial origin that affects perineum muscular fascia, genital region and surrounding areas with skin gangrene due to thrombosis of subcutaneous blood vessels. it describes the clinical case in a male preschooler of four years of age with idiopathic nephrotic syndrome (ns) that subsequent presented fg of the scrotum. broad-spectrum antibiotics, intravenous albumin and surgical cleaning of the scrotal necrotic tissues were indicated. pseudomona aeruginosa was isolated from necrotic tissue cultures. the later evolution was satisfactory with healing of the affected area and remission of the ns subsequent to the steroidal treatment. fg is an uncommon in children, rapidly progressive infection of the genital, perineal and perianal regions. it is characterized by a synergistic necrotizing fasciitis leading to thrombotic occlusion of small subcutaneous vessels and development of gangrene. until now few cases have been report fg in children, and still less associate to the kidney diseases. et all (1999) described a 10-year-old boy presenting with steroid resistant ns developed fg of the scrotum so that to our knowledge, this patient seems be the second case reported in medical literature with both pathologies. high index of suspicion, prompt diagnosis, broad spectrum antibiotics followed by wide debridement is the key to successful treatment. objectives of study: to evaluate a long term experience on iga nephropathy (igan) presenting in childhood and investigate clinical and histological factors that may act as early markers of renal disease progression. methods: retrospective review of data from children and adolescents with biopsy proven igan in the last 16 years. demographic and clinical data at presentation and severity of renal histological involvement were recorded and related to renal dysfunction markers identified at the last review. results: twenty-five patients were studied (19m/6f) with median age at onset of 11 (7-18) and follow-up of 6 (1-16) years. on presentation recurrent macroscopic hematuria was present in 19 patients, microscopic hematuria (mh) in 6, proteinuria in 11 (1 nephrotic), hypertension in 10 and transient acute renal failure in 1. renal histology findings were focal mesangioproliferative in 17, focal proliferative in 3, diffuse proliferative in 3 and focal sclerosing glomerulonephritis in 2. six patients showed tubulointersticial and extraglomerular vascular lesions (tevl) with glomerular crescents in 2. on follow-up, 7 patients remitted (2 spontaneously, 5 with ace inhibitors). of the remaining, 14 were kept on ace inhibitors due to proteinuria (7), hypertension (1) or both (6). one patient (with focal glomerulosclerosis and tevl) developed esrd within a year after diagnosis, despite treatment. at last review, 6 patients presented progressive renal disease with a mean decrease of 6 ml/min/1,73 m 2 in gfr per year. these (5m/1f) showed mainly mh and proteinuria at onset and tevl. conclusions: early renal function impairment in childhood igan can occur and may be associated with mh and proteinuria at presentation and with focal glomerulosclerosis and tevl on renal histology. proteinuria persistence in a number of patients emphasizes the need for long term followup into adulthood. adhesion molecules, il-12+p40 and cd23+cd19+ and cd25+cd4+ lymphocyte subsets in childhood nephrotic syndrome background: parathyroid hormone (pth) can modulate t cell activation and proliferation through as yet incompletely identified mechanisms. since the pth receptor (pthr) is a g protein-coupled receptor and thus a candidate for association with lipid rafts, and since pth has been shown to alter membrane phospholipid metabolism, we explored the relationship of the pthr with lipid rafts in human t cells. methods and results: we found by flow cytometry that neither physiologic nor pathologically elevated concentrations of pth affect the up-regulation of the raft marker gm-1 or of the partially raft-associated activation marker cd25 in purified t cells stimulated with phytohemagglutinin (pha). moreover, we detected the pthr exclusively in non-raft fractions of these cells after sucrose gradient separation. conclusions: these data indicate that in human t cells, the pthr does not associate with lipid rafts and that pth does not modulate these domains. accordingly, other mechanisms underlying the actions of pth on human t cells need to be sought. the direction and magnitude of potassium transport in nephron segments depend on the sitespecific distribution of transporters in tubule cell membranes. potassium depletion has been demonstrated to be associated with altered sodium reabsorption in renal tubule segments. we examined whe her potassium transporters protein expression is associated with altered abundance of major renal na + transporters, that may contribute to the development of hypokalemia in lp. after weaning rats (n=8) were fed 14 days with lp diet (8%), then they were recovered with a normal protein diet (24%, rp), each group had a control group (24%, np). we examined the changes in the abundance of the na + /h + exchanger (nhe3), na + k + atpase, na + k + 2clcotransporter (bsc-1), na + clcotransporter (tsc), epithelial sodium channel (enac) subunits and romk in kidneys of lp, np, rp rats. controls were normalized to 1. results reduced clcreat (ml/min) in lp vsnp (0.6±0.2 vs 1.6±0.2), hypokalemia (3.6±0.1 vs 5.1±0.2 meq/l) and increased fe k+ (44.2±0.5 vs 29.9±0.3%) were demonstrated. immunoblotting revealed that the abundance of nhe3 in cortex was severely decreased. the amount of bsc-1 (1.9±0.07, p<0.05) and tsc (1.4±0.15, p<0.05) protein levels were enhanced in the inner stripe (isom) and outer stripe of the outer medulla (osom), respectively. romk protein levels were increased in lp (1.23±0.04, p<0.05), the protein abundance of the enac subunits α, β and γ was increased near 1.25 fold each in response to lp. na + k + atpase protein levels showed no differences in cortex and osom. after rp, na + transporters expression returned to control values. conclusion: increased expression of bsc-1, tsc, enac subunits and romk, contributing to distal potassium secretion was shown in hypokalemia from lp. a role of aldosterone may be suggested. v. belostotsky 1 , mz. mughal 2 , j. berry 3 , n. webb 1 1 royal manchester children's hospital, pediatric nephrology, manchester, united kingdom 2 st. mary's hospital, pediatrics, manchester, united kingdom 3 manchester royal infirmary, vitamin d laboratory, manchester, united kingdom aims: to describe the prevalence of vitamin d deficiency in south asian and white uk children with renal disease. to establish how decreased levels of vitamin d affect pth in patients with a normal gfr. methods: 143 children aged 1-18 years were enrolled in the study: 99 were of white uk, 38 of s asian and 6 of other ethnic origin. 18 were on dialysis, 18 had chronic renal failure, 46 had various renal disorders with normal gfr (>80 ml/min/1.73 m 2 ), 61 had a transplant (42 with anormal gfr). blood samples were collected to establish the levels of 25-vitamin d (25-ohd); pth; creatinine. 25-ohd concentration <10 ng/ml was defined as vitamin d deficiency; levels between 10-20 ng/ml as vitamin d insufficiency. serum pth of 0.8-3.9 pmol/l was defined as normal. results: the prevalence of vitamin d deficiency/insufficiency was higher in s asian (87%) than white (36%) children (p<0.0001). ofthe 88 (28 s asian, 56 white and 4 other) children with normal gfr17/28 s asian and 28/56 white children had pth concentrations >3.9 pmol/l. of these 12/17 s asian, 11/28 white children had low levels of 25-ohd (p=0.04). of 19 transplant patients with reduced gfr, 11 of 14 with a high pth had low 25-ohd levels, compared with 1 of 5 with a normal pth (p=0.04). conclusions: many s asian children attending our renal clinic are vitamin d deficient/insufficient and the prevalence of this problem is significantly higher than that in the white population. high pth values in the setting of a normal gfr can often be explained by vitamin d deficiency and should result in serum 25-ohd levels being measured. nephronophthisisis a rare recessive autosomal disease which may be either limited to progressive chronic tubulointerstitial nephritis or associated with extrarenal involvement (eye, liver, central nervous system, etc.); mutations/deletions have been found in at least 6 nphp genes. fibrous dysplasia is a benign skeletal lesion due to an activating mutation inthe gene that encodes the α subunit of stimulatory g protein and occurs after fertilization in somatic cells; it involves one or several bones and may be part of mccune albright syndrome. we report on a boy with fibrous dysplasia of bone diagnosed at 3 yrs of age, who underwent protocol renal function tests at 7 yrs of age in the follow-up of pamidronate treatment. inulin clearance was 75 ml/min/1.73 m 2 and potassium reabsorption rate was 55.2% where as there was neither urinary phosphate wasting nor hypercalciuria. serum magnesium was decreased (0.74 mmol/l) without reabsorption abnormality and serumuric acid progressively increased with age. in addition, due to increasing microalbuminuria, a treatment with acei was started at 11 yrs of age. renal ultrasonography at 11 yrs of age showed hyperechoic reduced-sized kidneys with few microcysts. a renal biopsy (light and electron microscopy) was performed at 11 yrs of age, which showed nephronophthisis-like lesions, i.e., diffuse interstitial fibrosis and focal thickening of tubular membrane basement. dna analysis revealed no nphp1 gene deletion but is still under investigation.nephronophthisis has been reported in association with skeletal involvement (coneshapedepiphyses) and fibrous dysplasia with hypophosphatemic rickets or fanconi syndrome. however no association between fibrous dysplasia of bone and nephronophthisis-like lesions has been described and may be a new picture of the nephronophtisis/medullary cystic kidney disease complex. m. bald, m. holder, h. leichter olgahospital, pediatric nephrology, stuttgart, germany puumalaviruses belong to the group of hanta viruses and are transmitted by inhalation of aerosolized particles of the red bank vole (cletriomonysglareolus) which is endemic in the alb-danube region of southern germany. infections with puumala virus were first described as "nephropathia epidemica" in scandinavia with the clinical symptoms of fever, thrombocytopenia and acute renal failure. over the last seven years three boys with acute renal failure were admitted to our hospital after vacationing in the region endemic for puumalavirus. all three presented with high fever, influenza like symptoms aswell as pronounced abdominal or flank pain. they showed a decreased gfr (14, 47 and 71 ml/min/1.73 m 2 , respectively) with hematuria and proteinuria. cbc revealed no leucocytosis or anemia, but thrombocytopenia in 2 of the 3 patients. they had no oliguria, but 2 patients had marked polyuria in the recovery phase of renal function. arenal biopsy in the boy with the most severe presentation showed diffuse tubular damage. puumala virus infections were confirmed in all patients by serological tests, and renal function normalized within 2-3 weeks. nephropathia epidemica due to puumala virus infections have to be included in the differential diagnosis of acute renal failure in patients from endemic regions. severe abdominal or flank pain are common symtoms in these patients; renal failure is transient and the general prognosis is good. aims: the objective of this study is to determine the relationship of urinary calcium excretion (uca) with sodium and protein intake in a pediatric population of families with low income. methods: 109 children, 59 f and 50m, ages 8 to 15 years from families with income <$100/month were studied. protein intake was estimated with a 7-day dietary record. a nonfasting urine sample was collected for dipstick, calcium, creatinine, sodium, potassium, urea and uric acid. urinarycalcium/creatinine (ca/cr), sodium/potassium (na/k), uricacid/creatinine (au/cr) and urea/creatinine (u/cr) ratios were calculated. children with a urinary ca/cr >0.20 mg/mg, were submitted to a 15 day period of high sodium foods restriction after which a second urine sample was collected. results: mean (x) and standard deviation (sd) for ca/cr, na/k, au/cr and u/cr ratios were: 0.09±0.05 mg/mg, 5.68±5.41 meq/meq, 0.5±0.24 mg/mg and 16.12±5.3 mg/mg respectively. the 95th percentiles for ca/cr, na/k, au/cr and u/cr were 0.19, 13.07, 0.96 and 26.5 respectively. x±ds for protein intake was 1.27±0.27 g/kg/day. the incidence of hypercalciuria was 6.4% in the initial urine sample and 2.7% in the second. correlation was significant between ca/cr ratio and na/k ratio (r: 0.5, p<0.05), acu/cr ratio (r: 0.38, p<0.05) and u/cr (r: 0.30, p<0.05), not significant between ca/cr ratio and protein intake. conclusions: the incidence of hypercalciuria in this serie is lower than previously reported values in venezuela for the general pediatric population and decreases further when sodium intake is controlled. although no correlation was found between uca and protein intake, we could speculate that protein intake near to the daily recommended requirements of 1 g/kg/day, could be a possible reason for the lower incidence of hypercalciuria in this population. 16-year old girl presented with rapid onset of muscular weakness and a short history of severe dysphagia, dysphonia nad significant wasting. on examination, she was dystrophic (bmi 15,7) and had signs of myopathy. laboratory findings confirmed myopathy (cpk 106,4 ukat/l, ast 2,86 ukat/l, myoglobin 1582 ug/l). there was striking hypokalemia (s-k 1,8 mmol/l) suggesting hypokalemic paralysis. diagnosis of distal renal tubularacidosis (drta) was based on confirmation of hyperchloremicmetabolic acidosis, severe hypokalemia, high urinary ph and positive value of urinary anion gap (s-cl 120 mmol/l, ph 7,31, be 10, urinary ph 7,5). there was evidence of other signs of renal tubular impairment (urinary beta-2-microglobulin 213 mg/l, glomerulo-tubular proteinuria 1,01 g/24 h). autoimmune tests (high positive rheumatoid factor, anf, ena ss-a/ro, ss-b/la, high circulating immunocomplexes) and low values of sialometric measurements (7,5 ml/2x15 minutes) revealed primary sjogren´s syndrome as the underlying cause of drta. the renal biopsy confirmed chronic tubulo-interstitial nephritis compatible with this diagnosis. full recovery of muscle weakness and laboratory findings of hypokalemia and acidois followed potassium and alkali replacement. corticosteroids were administered with subsequent addition of cyclosporine a because of disease activity. conclusion: primary sjogren´s syndrome is a rare diagnosis in childhood and adolescence and should be considered in patients presenting with hypokalemic paralysis due to drta. m. caletti, h. lejarraga, s. caíno, a. jiménez introduction: ndi is a chronic, genetic disease caused by an inability to effectively conserve urinary water, due to a lack of response of distal renaltubule to antidiuretic hormone. the main symptom is polyuria, polydipsia and growth impairment. objective: to evaluate long term growth in height and weight of 16 children with ndi. patients and methods: sixteen patients with ndi attending hospital for a median period of 11.5 years (range 3.4/16.2 yrs) were studied. treatment consisted of indometacine, hydroclorotiazide and amiloride (iha). height and weight was measured with standardized anthropometric techniques. z scores(sds) for all measurements were calculated according to national standards. results: all children responded favourably to treatment. mean birth weight sds was not different from zero; mean height and weight at diagnosis was ±2.31 and ±2.66 sds respectively, and at the end of follow-up was ±1.30 and ±0.98 respectively. the majority of patients´s growth curves evolved below the 50 th centile. ten out of 16 children experienced some catch up in height (mean height gain: 1.38 sds (r: -0.50/2.42)). mean weight gain during follow-up was 1.74 sds (r: 0.86/4.24) . mean gain in body mass index was 1.72 sds (range 0.5/4.8). in the two patients who attained adult height, adolescent growth spurt was normal, and final height was within normal limits for standards and for parental height. correlation coefficient between gain in height andage at diagnosis was ±0.58. conclusion: although mean height at follow-up was below the expected normal value, combined therapy with iha is compatible with some catch up growth in height and weight. the lower the initial height, the greater the height gain. adherence to treatment is essential for normal growth in children with ndi. body growth of children with steroid-responsive idiopathic nephrotic syndrome m. noer, i. irwanto, n. sumiarso, m. chalim soetomo hospital, school of medicine airlangga university, department of child health, surabaya, indonesia objectives of study: the present study was designed to evaluate the statural growth of children with steroid-responsive idiopathic nephrotic syndrome, attending the pediatric nephrology unit department of child health, school of medicine airlangga university, soetomo hospital, surabaya, indonesia, with a minimum follow-up of 2 years. methods: anthropometrice valuation included weight, height, and growth velocity expressed as mean and standard deviation scores (sds), relative to the normal population (nchs/cdc 2000). these indices were analyzed at admission and then every 6 months of follow-up. all patients were treated with prednisone, according to indonesian consensus of management of idiopathic nephrotic syndrome in children. results: of 157 children (105 boys and 52 girls), 43 patients (34 boys) aged 26/12 years to 16 years (mean 7.05 years) were analyzed. initial mean height and z score (height for age) were 108.41±15.2 cm and -1.85±1.41, respectively. mean height and z score (height for age) of the last follow-up were 121.88±15.1 cm and -1.34±1.5, respectively. mean growth velocity were 6.25±2.05 cm/year where 3 boys (6.9%) had growth velocity less than 4 cm/year. total cumulative dose of steroid during 2 years of follow-up were 4283.90±2863.35 mg or 233.76±153.73 mg/kgbw. conclusions: the cumulative dose of steroid up to 233.76 mg/kg body weight in children with nephrotic syndrome during 2 years of treatment did not influence their growth velocity. background: recently it has been reported in adult patients (pts) that deterioration of renal function was associated with the lost of nocturnal blood pressure (bp) dip and enhanced urinary sodium (una) and protein (uprt) excretion during night. objectives of study: to investigate the circadian rhythms of bp, una and uprt in children with chronic kidney disease stage i (ckd i). methods: in 34 pts (15 boys) aged 11.2±4.3 years with ckd i (chronic glomerulopathy confirmed by renal biopsy in 85% pts), 24 hour bp was monitored during daytime (d) and nighttime (n) and urinary samples for uprt, urinary creatinine (ucr), and una, were collected for both periods. results: serum creatinine-based gfr was 127±22 ml/min/1.73 m 2 , uprt ranged from 21 to 5455 (median 141) mg/24 h, and una from 27 to 267 (median 102) mmol/24 h. in general we found a highly significant nocturnal decrease in systolic bp (from 116 to 108 mmhg), diastolic bp (71 to 62 mmhg), mean arterial pressure (87 to 78 mmhg), heart rate (91 to 75/min) urinary output (uo), una and uprt. the regression equations were as follows: uod (ml/m 2 /h)=32+1.6xuon (ml/m 2 /h); unad (mmol/l)=73.5+0.14xunan (mmol/l); uprtd (mg/m 2 /h)=5.4+1.83xuprtn (mg/m 2 /h) and urinary osmolality (us) d=13+0.65xusn. nocturnal decrease of uo correlated with nocturnal decrease of ucr and uprt, and nocturnal decrease of uprt correlated with nocturnal decrease of uo. more than half of the patients were classified as non dipper. they differ significantly from dipper only in night/day changes of us. conclusion: night/day changes of uo, una and uprt in pts with ckd i may be calculated from the given regression equations. these changes are not correlated with nocturnal bp decrease. non dippers have greater nocturnal change of us compared to dippers. follow-up of these parameters will clarify their importance in progression of ckd. autosomal autosomal dominant proximal renal tubular acidosis (prta) it is described l. g. brenes (1977) at seven members of one family. we diagnosed seven members of the afghani family with prta: mothers and 6 children (5 girls, 1 boy from 2,5 till 16 years) with hyperchloremic metabolic acidosis. pedigree analysis suggested an autosomal dominant inheritance pattern. observable patients did not have ricket and nephrocalcinosis. deafness and ocular abnormalis are absent. the plasma hco 3 concentration is decrease in the range of 14,9 to 19,0 mm/l, minimal urine ph is <5,5. parameters of blood creatinin and glomerular filtration rate were normal. urine calcium excretion was normal. therapy strategy of prta at observable patients provides high dozes of citrates/bicarbonates 10-15 mmol/kg per 24 h. all india institute of medical sciences, division of nephrology, department of pediatrics methods: retrospective case-search. data of previously reported prospective trial (n=19) was also included. results: all except 6 patients had previously been treated with both levamisole and cyclophosphamide. forty-two cases qualified for the study and were administered mmf for a mean duration of 14.3 months (95% ci, 12.0, 16.6/patient in the first 6 months of treatment and 0.7 episodes (n=35) in next six months of mmf treatment (p<0.0001), an average reduction of 62% (95% ci, 49.1, 75.0) from the pre-mmf phase. nine (21.4%) patients had no relapses while on mmf therapy we present sibling cases of as with heavy proteinuria at early childhood. a boy (3 year-old) and a girl (2 yearold) were diagnosed as x-linked as. since a boy developed persistent heavy proteinuria (up/cr 2.9) with macrohematuria andresistant to arb/acei therapy, we treated him with cyclosporina (csa) that could lead to complete remission. to investigate pathomechanism of proteinuria, we tested immunostaining for slit diaphragm associated molecules (nephrin, podocin), gbmassociated molecules (laminin, perlecan, agrin) and podocalyxin using frozen sections from his firstand second biopsy and girl's one (up/cr 0.4). in the specimens from boy's first biopsy and girl's one, light microscopy revealed mild mesangial proliferation and no differences ofthe expression with perlecan, agrin and podocalyxin compared with controls. however, when determined laminin isoforms, fetal type laminin (alpha2beta1gamma1) wasdistinctly observed in the gbm, whereas that was localized only inmesangium with controls. interestingly when compared mature laminin isoform (alpha5beta2gamma1), beta2 chain was specifically less expressed in the gbm. however there were no differences of expression of these molecules in the specimens between pre-and post-treatmentwith csa. in boy's second biopsy, 50% glomeruli were detected to becollapsed. together with the recent report showing that laminin beta2 mutation causes congenital nephrotic syndrome factor v leiden mutation and steroid resistant membranous glomerulonephritis: a case report m. buyukcelik 1 , m. karakok turkey renal compications of d-penicillamin therapy in wilson's disease sami ulus children's hospital, department of pediatric nephrology sami ulus children's hospital, department of pediatrics sami ulus children's hospital hass classification), treatment and outcome. thirty-nine patients; 29 boys (74.4%) and 10 girls (25.6%) time of the last examination (median 60 months, min<1 year) after the admission as a long term follow-up. clinically, group i; while microscopic hematuria was detected in 6 patients, 19 patients had repeated attacks of hematuria, 4 had isolated mild proteinuria/hematuria, group ii; 6 patients had nephritic, 1 had nephrotic syndrome and 3 had both. biopsy grades in the 39 patients: 55%, 60% had grade i, 10.4%, 10% had grade ii, 31%, 20% had grade iii, 6%, 10% had grade iv in group i and ii, respectively. group i and ii patients recovered completely (no hematuria and proteinuria) 34.4%, 44.8% as well as 20%, 50%, short-term, longterm, respectively. while recovery rates in fish-oil and/or ace-inh treatment group was 34.4% and 48.2%, in corticosteroids group, it was 25%, 50% short-term, long-term,respectively. no patients who received immunosuppressive treatment had improved. however, 3 (7.6%) patients would suffer from esrd. initial presentation, severity of renal involvement and type of the treatment were not found to have a prognostic value (p>0.05). in children, igan is characterized by extreme pathogenetic, clinical and histological polymorphisms radojevic 1 1 university of belgrade, faculty of medicine, institute of pathology, belgrade, serbia 2 institute of mother and child health of serbia, belgrade, serbia 3 university children's hospital, department of nephrology, belgrade, serbia celiac disease hla aplotype in children with nephrotic syndrome s i (if) or mcp or with anti-cfh autoantibodies. varicella hasn't been described as a triggering event of ahus. we report two cases of ahus associated with complement dysfunction revealed after varicella infection. case 1. a five year-old boy presented with non post-diarrheal hus, 17 days after varicella. serum creatinine was 300 μmol/l, hemoglobin 6.5 g/dl, schizocytes 17%, platelets 19 g/l. glomerular filtration rate normalized within 14 days. search for shiga toxin-producing e coli in the stools and serum anti-lipopolysaccharides antibodies were negative. plasma c3, cfh and if levels were normal. no mutations of cfh and if were found. mcp cell-surface expression was decreased and a c30f mutation in mcp exon 2 was demonstrated. case 2. a four year-old girl had ahus 5 days after varicella at that time, complement system study showed normal c3 level (677 mg/l, normal 660 to 1260 mg/l), normal cfh level (81%, normal 70-130%), but the presence of anti-cfh autoantibodies. no mutations of cfh, if or mcp were found. in conclusion, these 2 cases outline that varicella can be the triggering event of ahus associated with complement dysregulation about 450 (62.5%) children had relapses after initial remission. various infections were responsible for relapsed with in 200/450 (44.44%) who had relapses. about 120 children (32.69%) relapsed with out cause where as poor compliance was observed in 47 (12.8%). overall infection and relapse rate was 1.4 and 1.00/pt/yr respectively. among 367 children with infections, most common types of infections were acute respiratory infections (ari), diarrhea and uti seen in 200 (54.49%), 82 (22.34%) and 30 (8.17%) of cases respectively. other types of infections like malaria, peritonitis, skin infection and pulmonary tuberculosis were seen serum pai-1 and tgf-beta 1 levels in profliferative forms of glomerulonephritis in children russian federation 2 research centre for child health rams, department of pathology, moscow, russian federation we aimed to investigate serum levels of plasminogen activator inhibitor -1 (pai-1) and transforming growth factor-beta 1 (tgf-beta 1 ) in children with proliferative forms of glomerulonephritis (gn). 51 children were examined (5-16 years old) with gn (steroidresistent ns, n=27; steroidsensitive ns, n=11, isolated haematuria, n=13) and 15 healthy age matched controls. mesangioproliferative gn (mespgn) was detected in 39 patients, membranoproliferative gn (mpgn) in 12 cases. serum levels of pai-1: ag and tgf-beta 1 were measured by elisa. results. the highest levels of pai-1: ag and tgf-beta 1 were observed in relapse of mpgn: 93,51±71,63 ng/ml and 16 these results confirm prosclerotic effects of pai-1 and tgf-beta 1 via increased fibrin deposits and extracellular matrix accumulation in the renal tissue and promotion of disease progression rituximab treatment for idiopathic nephrotic syndrome: a retrospective study of 11 cases v. guigonis 1 , a. dallocchio 1 , m. dehennault urinary and serum annexin v levels in children with steroid sensitive and steroid-resistant nephrotic syndrome hôpital robert debré-aphp, pediatric intensive care unit indonesia this study was aimed to evaluate the efficacy of pulse dose of cyclophosphamide in children with srns admitted in child health department faculty of medicine university of indonesia/cipto mangunkusumo hospital jakarta between 2001-2006. 6-month period, one child died, and the rest (5 children) did not complete the regimen. five out of 7 children who finished the treatment had remission, while 2 others were still experiencing heavy proteinuria. remission was achieved in various time, 3 children were in remission after the first dose, in 2 children it was achieved at the third and sixth dose. in further follow-up time; one child remained in remission, one child had relapse when still receiving cpa, 2 children got relapse one month after stopping cpa, and one child had relapse after 6 months ceasing cpa. nausea and vomiting were found in 2 children indonesia this study is to evaluate the anthropometric measurements of children with nephrotic syndrome methods: a descriptive retrospective study at child health department, cipto mangunkusumo hospital, jakarta. data were collected from medical records of nephrotic syndrome 9%) irns; body height and body weight of <p3 was found in none of frns/sdns, 9/32 (28,1%) srns, and 20/101 (19,8%) irns; bmi >p95 was found in 4/45 (8,9%) frns/sdns, 3/32 (9,4%) srns, 4/101 (3,9%) irns. conclusion: the percentage of children with frns/dsns and srns with body height <p3 il-12) during active stage (as: urinary protein >40 mg/m 2 per hour, serum albumin <2.0 g/dl) and remission stage (rs) in ss-mcns. a total of 48 patients with ss-mcns (29 of 48 patients with as and 19 of 48 patients with rs) and 19 healthy children were recruited for studies. the mean±sd of serum il-12, se-selectin and sicam, levels were significantly higher in patients with as than in patients with rs (277±188.4/157.2±119 pg/ml; 132.1±67.9/88.3±40.5 ng/ml and 168.3±120.3/82.2±27.1 ng/ml respectively, p<0.05). in spite of, higher levels of il-12, se-selectin and sicam in patients with as than controls, difference was not statistically important. the percentage of cd3+cd23+ lymphocyte subsets were statistically grater in patients with as severe proximal renal tubular acidosis in pearson syndrome birth weight was 4000 g. pallor was initially noted during the neonatal period and referred to our hospital with anorexia, vomiting, diarrhea, weakness, and increased pallor at 8 wk of age. on the physical examination she was pale and the other systems were unremarkable. investigation showed hypoplastic anemia, and bone marrow examination showed cytoplasmic vacuolization of both myeloid and erythroid precursors, and maturation arrest of granulopoesis. family history was negative for hematological disease. the diagnosis ps was considered on the basis of early severe refractory anemia associated with vacuolization of bone marrow precursor cells and ring sideroblasts. treatment was started consisting of vitamine b6 and folic acid. she was followed with growth retardation, moderate anemia and leucopenia up to age 4.5. at that age, the girl was readmitted with severe vomiting and dehydration. on admission, she had moderate metabolic acidosis, hypokalemia, high plasma lactate, and hypophosphatemia. further investigations showed tubuler proteinuria, glucosuria, aminoaciduria, and defective bicarbonate reabsorbtion in the proximal tubule. she developed refractory metabolic acidosis resulting in a cardiac and respiratory failure and death. to confirm the diagnosis of ps, molecular studies were performed 4977 bp common deletion was found medial calcification in intact human arteries from children with chronic kidney disease is associated with apoptosis and osteogenic differentiation -clinical and laboratory correlations r using intact human vessels we studied the phenotypic changes in medium sized arteries ex vivo and in vitro after exposure to ca and po 4 . arteries were retrieved during insertion of pd catheters or at transplantation from 34 children: dialysis (n=20), ckd stage iv (n=8) and compared with mesenteric vessels from 6 controls. vessel rings were incubated with graded concentrations of ca and po 4 upto 21 days. calcium and alkaline phosphate (alk) were measured by cresolphtalein complexone and colorimetry. immunohistochemistry for bone marker proteins, inhibitors of calcification and apoptosis were performed. laboratory findings were related to patient's clinical and biochemical parameters, carotid intimamedia thickness (cimt) and coronary calcification. vessels from ckd or dialysis patients had increased baseline vessel wall ca compared with controls (p=0.001). when exposed in vitro to ca and/or po 4 , dialysis vessels showed greater calcification than those from controls or ckd patients (p<0.0001). in the presence of elevated po 4 even a small increase in ca increased calcification (p<0.001). calcification was associated with apoptosis (tunel +) and could be inhibited using apoptosis inhibitor zvad. alk in ckd and dialysis vessels and along with upregulation of bone-markers suggests an osteogenic conversion of vsmcs using our unique in-vitro model, we have shown for the first time that vascular damage induced by elevated ca-po 4 as well as factors specific to dialysis primes vessels for rapid progression of medial calcification altered expression of major renal na + and k + transporters c. ruete 2 , p. vallés 1,2 1 university of cuyo, department of pathology turkey the effect of corticosteroid therapy on bone metabolism in nephrotic syndrome was examined. sixty-nine patients with idiopathic nephrotic syndrome (age: 7.0±2,5 years) and 21 healthy controls (age: 6.4±3,5 years) were divided into 3 groups: group 1: patients who were on remission but still receiving steroid therapy, group 2: patients who were on remission and free of steroids within the last year and group 3: patients with active nephrotic syndrome and receiving steroid therapy. serum total calcium, ionized calcium, phosphorus, alkaline phosphatase, magnesium, parathyroid hormone, 25(oh)d, serum cystatin c, urine protein, urine creatinine and urine cystatin c levels measured in all patients including the control group. in addition, lumbar spine bone mineral density z scores were measured in the patient group objectives of study: we evaluated the clinical, laboratory and urinary tract echosonographic findings in patients with ah and rh. methods: there was prospective clinical study, included 30 patients (mean age 6.6±2.5) with ih (normocalcemic, normophosphatemic, with 24 hours urinary calcium excretion greater than 4 mg/kg/day) all analyzed parameters (dysuria, positive family history of urolithiasis, microscopic hematuria, urinary tract microlithiasis) showed low sensitivity and specificity, and none of the parameters could be considered reliable in differentiating ah versus rh. average value of una/cr was greater in patients with rh conclusion: none of the analyzed clinical parameters, laboratory and echosonographic parameters except values of urinary excretion after calcium deprivated diet could be considered reliable in differentiating ah versus rh. patients with rh have higher level of 24 hours urinary calcium excretion than patients with ah. patients with rh have significantly greater excretion of urinary sodium compared with patients with ah idiopathic hypercalciuria (ih) is defined as hypercalciuria with no detectable cause. low bmd with increased fracture rate and tendency to short stature has been reported in ih patients. we aimed to perform calcaneus qus in children with ih and relate to u-ca, body height and number of prevalent fractures (fx). 11 children (8 girls, 3 boys; patient. body height was recorded and qus was measured on both heels with cuba clinical. the 24-h u-caexcretion (u-ca/24 h) was assessed and calculated in mmol/kg/24 h. results were expressed as z-scores ±sd. czech anthropometric parameters from a 1991 survey and previously obtained qus values of the healthy czech pediatric population served as reference data. qus results were also calculated as height adjusted values with the use of heightmatched standards. u-ca/24 h was matched to healthy european paediatric population values we found no correlations between fx and bua (either age-related orheight-adjusted) or fx and vos (age-related or height-adjusted). neither were there any correlations between u-ca and fx, or u-ca/24 hand bua or vos, respectively. in conclusion, children with ih had normal height, normal values of bua and low vos 40 (fc) 491 (p) 652 (p) 268 (mr) 23 (fc) 126 (fc) 194 (op) 523 (p) alpay h. 399 (p) 854 (p) amann k. 37 (fc), 810 (p), 818 (p) amanullah f. 521 (p) 169 (op) amaro a. 77 (op) 164 (op) amore a. 48 (sy) 831 (p) anarat a. 277 (fc) 131 (mr) 322 (p) 191 (op) 213 (fc) 75 (op) 345 (p) 391 (p) bael a. 192 (op) 308 (p), 543 (p), 600 (p) 32 (fc) 129 (fc) 658 (p) balat a. 349 (p) 444 (p) 19 (fc) bayazit ak. 372 (p) 906 (p) 708 (p), 709 (p), 710 (p) 338 (p) 356 (p) 671 (p) 805 (p) bensman a. 162 (op) 153 (sa), 384 (p) 425 (p) 118 (fc) bereczki cs. 66 (op) 214 (fc) 93 (op), 285 (fc) 197 (op) 12 (sy) bi yl. 484 (p) 23 (fc) 370 (p) 171 (op) 841 (p) biyikli n. 399 (p) 398 (p) 227 (fc) bocanegra v. 224 (fc) 122 (fc) 352 (p) boh m. 134 (fc) bökenkamp a. 92 (op) 271 (fc) 22 (fc) 177 (op) 65 (op) 92 (op) bourrier t. 425 (p) bouts ah. 103 (op), 117 (fc) 77 (op) 903 (p) bubic-filipi lj 368 (p) 168 (op), 271 (fc) 78 (op) 486 (p) 389 (p) caropreso m. 95 (op) 277 (fc), 371 (p) 40 (fc) 459 (p), 460 (p) chartapisak w. 817 (p) 530 (p) 693 (p) chaves a. 77 (op) 394 (p) chemodanova m. 911 (p) 559 (p) chen j. 76 (op) 724 (p) 488 (p) 539 (p) 654 (p) 160 (op) 396 (p) 645 (p), 650 (p), 836 (p) clermont mj 737 (p) 211 (fc), 472 (p) 55 (sy) codoceo a. 523 (p) coelho g. 77 (op) 49 (sy), 221 (fc) 48 (sy) 704 (p) 214 (fc) 211 (fc) 846 (p) 206 (fc) 456 (p) coutinho s. 868 (p) coviello d. 333 (p) 223 (fc) craig j. 108 (op), 556 (p) cransberg k. 214 (fc) 765 (p) 425 (p) cristino s. 672 (p) 39 (fc) 532 (p) cross j. 86 (op) cruz mr 22 (fc) 534 (p) 117 (fc), 558 (p), 630 (p) 55 (sy) deanfield j. 127 (fc) 845 (p) decena-galvez a. 601 (p) 124 (fc) 571 (p) 415 (p) deguchtenaere a. 98 (op) 859 (p) 679 (p) 303 (sa), 373 (p) 381 (p) delucchi a. 633 (p) 821 (p) 338 (p) 439 (p) 485 (p) 653 (p) 284 (fc), 599 (p) 20 (fc) dinda a. 756 (p) 325 (p) 695 (p) 607 (p) dittrich k. 37 (fc) 28 (fc) 307 (p), 445 (p) dötsch j. 37 (fc), 810 (p), 818 (p) dragon-durey ma 388 (p) 34 (fc) 576 (p) 25 (fc) 833 (p) 594 (p) 512 (p) 660 (p) dursun i. 344 (p) 344 (p) 500 (p) edefonti a. 380 (p) 374 (p) eke f. 265 (fc), 718 (p) 322 (p) 220 (fc), 269 (fc), 333 (p), 403 (p) 32 (fc) 338 (p) 706 (p) 384 (p) evim m. 430 (p) f faerch m. 378 (p) fallahzadeh mh. 448 (p) 473 (p) 248 (sy) feig d. 353 (p) 681 (p) 644 (p) fella a. 96 (op) feneberg r. 176 (op) 77 (op) 862 (p) filler g. 25 (fc) 849 (p) 104 (op) fischbach m. 157 (op) sy) fitoz s. 408 (p) 117 (fc) 798 (p) 470 (p) fujita h. 75 (op) 441 (p) fujita t. 769 (p) 443 (p) fukuhara d. 816 (p) 171 (op) 843 (p) 803 (p) 171 (op) 189 (op) garnier a. 365 (p) 762 (p) 155 (op) 251 (sy) geary-joo c 21 (fc) 32 (fc) 177 (op) 141 (sy) 32 (fc) 605 (p) gross ml. 116 (fc) 610 (p) 331 (p) 136 (fc) 205 (fc) 533 (p) 821 (p) guo w. 76 (op) 746 (p) 70 (op) 344 (p) haberal m. 537 (p) 42 (fc) 72 (op), 128 (fc) 271 (fc), 538 (p) 285 (fc) 266 (fc), 747 (p) 266 (fc) 851 (p) 268 (mr) 344 (p) 470 (p) 183 (op) 164 (op) hernández am. 638 (p) 75 (op) 183 (op) 28 (fc) 190 (op), 271 (fc) 814 (p) hou jr. 315 (p) 334 (p) 168 (op) 315 (p) 204 (fc) 29 (fc) 305 (p), 306 (p) iharada a 186 (op) 185 (op) 677 (p) 267 (fc), 283 (fc) 618 (p) 664 (p) 49 (sy) 793 (p) kathiravelu a. 25 (fc) 100 (op) 154 (op) 104 (op), 115 (fc) 385 (p) 665 (p) 765 (p) 734 (p) 749 (p) 643 (p) 18 (mr) 334 (p) 69 (op) 471 (p) kondo y. 29 (fc) 647 (p), 891 (p) kosuljandic-vukic d. 503 (p) 205 (fc) kovalski y. 645 (p) 190 (op) 815 (p) 312 (p) 611 (p) kru èic d. 910 (p) krylova-olefirenko a. 825 (p) 616 (p) 582 (p) 746 (p) 260 (sy) 118 (fc) 340 (p) 544 (p) kurayama r. 185 (op) 599 (p) 266 (fc) lalatta f. 177 (op) 515 (p) 257 (sy), 354 (p) 160 (op) 432 (p) lau yw 77 (op) 77 (op) 559 (p) llanas b. 71 (op), 136 (fc), 859 (p) llewellyn-edwards a. 270 (fc) 284 (fc) 348 (p) 263 (fc) luis-yanes m 354 (p) 589 (p) 293 (sy) maeda a. 566 (p) maekawa k. 602 (p) 830 (p) 453 (p) 477 (p) 841 (p) 410 (p) 323 (p) mak r. 49 (sy) 95 (op) 171 (op) 625 (p) 20 (fc), 26 (fc) 226 (fc) 457 (p) 254 (sy) 20 (fc) 893 (p) 638 (p) 578 (p) medynska a. 421 (p) 350 (p) mehls o. 128 (fc) 855 (p) 277 (fc), 426 (p) 74 (op) 179 (op) 174 (op) 115 (fc), 758 (p) 420 (p) 23 (fc) 368 (p) molnár-varga m. 525 (p) 227 (fc) 279 (fc) 487 (p) montini g. 209 (fc) 774 (p) 523 (p) 74 (op) 587 (p) 569 (p) morimoto t. 187 (op) 364 (p) 71 (op) 345 (p) morita t. 563 (p) 392 (p) mortazavi f. 309 (p) 283 (fc) moscaritolo e. 502 (p) 728 (p) mosig d. 493 (p) 191 (op) 226 (fc) 498 (p) moxey-mims m. 221 (fc) 158 (op) 521 (p) mudun a. 652 (p) 116 (fc) mughal mz. 895 (p) 828 (p) müller t. 165 (op) müller v. 218 (fc), 244 (sy) müller-esterl w. 745 (p) müller-wiefel de. 23 (fc) 49 (sy), 221 (fc) 174 (op) fc) 185 (op) 31 (fc) 504 (p) nghia h. 490 (p) niaudet p. 113 (mr) 372 (p) 312 (p) nuzzi f. 95 (op), 96 (op), 541 (p) nwobi o. 592 (p) 171 (op) 77 (op) 393 (p) 75 (op) 338 (p) 257 (sy) 567 (p) ostalska-nowicka d. 522 (p) 621 (p) otukesh h. 477 (p) 322 (p) 710 (p) 543 (p) ozen a. 498 (p) 230 (sy), 308 (p) 380 (p) 876 (p) paik kh. 643 (p) 853 (p) pan'czyk-tomaszewska m. 337 (p) 459 (p) 35 (fc) paripovic d. 611 (p), 612 (p), 647 (p) 207 (fc) pasqualini t. 194 (op) fc) pastori a. 463 (p) pászka d. 100 (op) 95 (op) 334 (p) pereira a. 77 (op) 129 (fc) 275 (fc), 574 (p) 576 (p) fc) 110 (op), 344 (p) raes a. 98 (op), 99 (op), 223 (fc) 218 (fc) 535 (p) rigden s. 22 (fc) 209 (fc) ring e. 346 (p) rink n. 104 (op) 74 (op) ristoska-bojkovska n 271 (fc), 538 (p) roszkowska-blaim m. 337 (p) 576 (p) 894 (p) ruffo gb. 914 (p) ruhlmann a. 193 (op) ruiter j. 85 (op) rumeau r. 206 (fc) rusai k. 215 (fc) rusnak f. 608 (p) rüther u. 381 (p) rutjes n. 785 (p) rybarova s. 772 (p) rychlik i. 776 (p) ryckewaert-dhalluin a 477 (p) sabasiñska a. 401 (p) 684 (p) sabolic-avramovska v. 348 (p) 721 (p) 162 (op) safouh h. 499 (p) saha a. 530 (p) sahin h. 512 (p) 184 (op) saint-cyr c. 805 (p) saito a. 450 (p) 29 (fc) sakalli h. 537 (p), 708 (p), 866 (p) sakalli ercoban h. 426 (p) 13 (sy) 868 (p) salusky i. 129 (fc) 74 (op) 717 (p), 720 (p) sarkissian a. 316 (p) 805 (p) 217 (fc) 414 (p) 215 (fc) 154 (op) sayili a. 590 (p) schäfer b. 691 (p) 32 (fc), 34 (fc), 42 (fc), 51 (sy), 116 (fc), 228 (fc), 271 (fc), 276 (fc) 215 (fc) schmidt-gayk h. 281 (mr) schmidts m. 549 (p) schmitt cp. 116 (fc) 195 (op) 549 (p) 272 (fc) schneider-maunoury s. 381 (p) 549 (p) 49 (sy), 221 (fc) 23 (fc) 180 (op) 869 (p) seeherunvong w. 40 (fc) 887 (p) 185 (op) 468 (p) semama d 76 (op) 748 (p) 470 (p) 656 (p), 657 (p) shin yh 382 (p) 564 (p) 22 (fc) 372 (p) 416 (p), 519 (p), 652 (p) siwiñska a. 343 (p) 276 (fc) 154 (op) 717 (p) spasojevic b. 612 (p), 647 (p), 910 (p) spasojevic-dimitrijeva b stanic m. 386 (p), 891 (p), 910 (p) stankovic a. 386 (p) 644 (p) 125 (mr) 41 (fc) 74 (op) 18 (mr) 361 (p) 869 (p) 711 (p) 711 (p) 772 (p) 869 (p) szteblich a. 188 (op) t 32 (fc) 679 (p) taha g. 499 (p) 532 (p) taheri derakhsh n. 436 (p) 602 (p) 814 (p) 31 (fc), 790 (p), 807 (p) tan ph 654 (p) 654 (p) 31 (fc) 422 (p) 177 (op) 536 (p) 524 (p) 882 (p) tizard e. 270 (fc) 738 (p) toenshoff b. 120 (fc) 432 (p) tönshoff b. 42 (fc) 731 (p) 160 (op) 218 (fc) 20 (fc) 171 (op) 66 (op) 652 (p) 154 (op) 219 (fc) urdaneta-carruyo e. 551 (p), 872 (p) urdaneta-contreras a 277 (fc) 572 (p) 587 (p) valverde s. 638 (p) 713 (p) van den heuvel l. 118 (fc) 511 (p), 529 (p) van't hoff w. 297 (sy), 348 (p) 486 (p) 213 (fc), 700 (p) 11 (sy) 55 (sy) 272 (fc), 275 (fc) 738 (p), 791 (p), 792 (p) waters a. 181 (op) 38 (fc), 121 (fc), 273 (fc) 268 (mr) 72 (op) 117 (fc) 129 (fc) 72 (op) 227 (fc), 904 (p) 33 (fc) wingen a. 168 (op) 142 (sy) 219 (fc) 49 (sy), 221 (fc) 21 (fc), 693 (p) 264 (fc) 13 (sy) 473 (p) 135 (fc) 322 (p) 798 (p) 816 (p) 79 (op), 784 (p) 29 (fc) 775 (p) yap hk. 31 (fc) 795 (p) yata n. 497 (p) 174 (op) 340 (p) 801 (p) yilmaz a. 519 (p) 174 (op) 67 (op) 439 (p) 579 (p) 794 (p) zhou jh 40 (fc) 165 (op), 211 (fc), 233 (sy) zingg-schenk a 858 (p) 209 (fc) zurowska a. 32 (fc) 161 (op) the il6 (-174g/c) polymorphism is associated with initial peritoneal transport status in children commencing chronic pd acknowledge: this study was from iran university of medical science. atypical hemolytic uremic syndrome: an unsolved case of complement dysregulation 788 (p) aim: we have shown that rats overexpressing il-13 gene developed a mcn with proteinuria, hypoalbuminemia and hypercholesterolemia. this study aimed to determine the role of il-13 on hypercholesterolemia in this model. methods: recombinant rat il-13 gene in a mammalian expression vector was transfected into quadriceps of wistar rats by in-vivo electroporation. serum il-13, albumin, cholesterol, creatinine and urine albumin were measured serially. after sacrifice on day 70, hepatic gene expression of hmg-coa reductase (hmg-coar), acat2, cholesterol-7a-hydroxylase (ch7ah), ldl-receptor (ldlr) and il-13 receptor subunits were determined by rt-pcr. results: compared to control rats (n=17), il-13-transfected rats (n=41) showed significant albuminuria (0.36±0.37 vs 3.45±0.89 mg/day, p<0.001), hypoalbuminemia and hypercholesterolemia (1.72±0.05 vs 3.39±0.34 mmol/l, p<0.001) at day 70. serially, this rise in serum cholesterol preceded the increase in proteinuria and fall in serum albumin. serum cholesterol correlated significantly with il-13 levels (r=0.64, p<0.001). in 14 of the il-13-transfected rats with serum cholesterol>3.10 mmol/l, hepatic gene expression (mean±sem) was significantly upregulated compared to controls for hmg-coar (0.25±0.08 vs 0.54±0.05, p=0.02), acat2 (0.26±0.03 vs 0.43±0.04, p=0.009), ch7ah (0.50±0.12 vs 1.08±0.10, p=0.004) ldlr (0.10±0.01 vs 0.15±0.02, p=0.02) and il-13ra2 (0.003±0.002 vs 0.080±0.015, p<0.001). conclusion: the increased cholesterol synthesis in il-13-induced mcn was associated with increased hepatic gene expression of hmg-coar and acat2, which are important enzymes for cholesterol synthesis. associated increased hepatic gene expression of ch7ah and ldlr involved in cholesterol metabolism could be a negative feedback response. e. bordador, f. anacleto philippine general hospital, pediatric nephrology, manila, philippines general objective: to formulate a clinical scoring system that will predict the presence of glomerular crescents in patients with severe nephritis. specific objectives: (1) to describe the profile of children with acute nephrotic-nephritic syndrome (2) to correlate clinical parameters with renal histopathology. methods: this is a retrospective study. twenty six charts from the philippine general hospital, admitted between january 2002 -march 2006 were retrieved. included in the study were children with acute nephritic -nephritic syndrome who underwent kidney biopsy. excluded were patients with small/contracted kidneys on renal ultrasound. statistical tool used were univariate analysis, pearson's product moment method and chi -square test. results: profile variables of the subjects were analyzed. afterwhich, each clinical parameter was tested using univariate analysis, if significantly correlated with the renal biopsy result using pearson's product moment method at critical value=0.388 at p<0.05. out of 11 parameters, only 3 parameters were noted to be significant, these were serum creatinine, blood urea nitrogen and glomerular filtration rate. further analysis was made by separately treating male from female population using a chi-square test with critical value x 2 (1,.05) =3.28. the test identified another three clinical features among female which were significantly associated with renal biopsy results, these were blood pressure, anemia and hematuria. from these significant parameters associated with renal biopsy results, a clinical scoring system was then conceptualized in order to identify patients with high probability of crescents on renal biopsy. conclusion: cresent maybe use as an accurate screening tool to predict presence of glomerular crescent in patients with severe nephritis. heterogeneous effect of acei therapy in children with proteinuric nephropathies b. kranz, s. diepenbruck, u. vester, r. buescher, a. wingen, p. hoyer university of duisburg-essen, pediatric nephrology, essen, germany background: chronic proteinuric nephropathies are at high risk of developing progressive renal insufficiency. the renin-angiotensin-aldosteron-system blockade is a well documented strategy to reduce proteinuria in adult patients. the efficacy and risks of renal-protective therapy with angiotensin-converting-enzymeinhibitors (acei) in children with proteinuric kidney diseases is of concern. method: in this retrospective study the efficacy of acei as antiproteinuric therapy in children with chronic proteinuric nephropathies is evaluated. patients: sixty-three children (mean age 10.7±3.5 years) have been treated with acei for a mean of 2.7±2.3 years (range 0.1±10.6 years) because of proteinuria (hus n=10, alport syndrome n=22, psh n=13, others n=18). results: proteinuria in all patients declined from a median of 1.5 g/d to 0.9 g/d after 2 years (p=0.01). there was a drop out of 5 patients because of end stage renal failure during the followup. one-third of patients showed a continuous reduction in proteinuria from 3.1 g/d to 0.4 g/d (median) within 3 years while a second third had a transientimprovement followed by a reincreasing proteinuria later. patientswith hus showed a good response (decrease of proteinuria from 2.8 g/d to 0.4 g/d) in contrast to patients with alport syndrome who developed increased proteinuria (0.8 g/d to 3.0 g/d) despite of acei and patients with psh who had no change in proteinuria.interpretation: it has to be discussed whether biological compensation mechanisms may bypass the ace inhibition in single patients and whether the underlying illness may predict the efficacy of acei.aims: to determine the clinicolaboratory renal manifestations; glomerular, extra-glomerular histopathologic lesions; renal tubular dysfunction (rtd) frequency, and outcome of a short-term renal follow-up in nigerian children with systemic lupus erythematosus (sle). methods: a non-randomized prospective study of consecutive cases of childhood-onset sle with nephropathy. baseline/follow-up clinicolaboratory data were collected. each patient was followedup for 12 months. results: seven of 11 children studied were girls (f:m, 1.75). median age at diagnosis was 11.0 years. median diagnosis time interval (1.9 years) and median time of renal disease onset (1.0 year) were similar. hypertension, nephrotic syndrome, and acute renal failure occurred in 45.5%, 54.5% and 63.7% of the patients, respectively. the glomerular lesions were non-proliferative lupus nephritis (ln) in 9.0% (class ii ln); focal (class iii ln) and diffuse (class iv ln) proliferative ln (pln) in 27.0% and 64.0%, respectively. tubulointerstitial nephritis (tin, 91.0%), and rtd (64.0%) were common. arf (p=0.033) and rtd (p=0.015) were significantly associated with severe tin. complete renal remission rate at end-point was 71.4%. relapse and renal survival rates were 14.3% and 86.0%, respectively. rtd was persistent in 43.0%. conclusion: renal function disorders, diffuse pln, and extra-glomerular lesions were frequent. significant association of arf and rtd with severe tin in this series suggests the need for early renal tubular function (rtf) assessment in our sle patients. deranged rtf may be marker of severe tin in sle warranting early confirmatory renal biopsy and aggressive interventional treatment.we report two cases of children with crescentic glomerulonephritis (gn) associated to isolated c3 deposits. patient 1. a 9 year old girl was admitted for macroscopic hematuria, nephrotic range proteinuria (proteinuria/creatininuria=1000 mg/mmol) and renal failure (serum creatinine 600 μmol/l). anca and other antibodies were negative. c3, c4 and ch50 activity were normal but c3nef was detected. the patient was treated by prednisolone and cyclophosphamide pulses followed by oral corticotherapy. renal function normalized, but proteinuria persisted (proteinuria/creatininuria=500 mg/mmol). a relapse occurred ten months later. corticotherapy and cyclophosphamide pulses were reinitiated and were successful, followed by mmf maintenance therapy. patient 2. a 4 year old girl was admitted after viral infection for macroscopic hematuria and fever. proteinuria was of nephrotic range (proteinuria/creatininuria=708 mg/l). the search for autoimmunity was negative. c3nef was detected but c3, c4 and ch50 activity were normal. serum creatinine increased to 369 μmol/l. after three pulses of prednisolone followed by oral prednisone, renal function normalized. histological examination of the two renal biopsies revealed endo-and extracapillary gn with numerous granulocytes in the capillary lumen. cellular crescents involved 90% of the glomeruli. immunofluorescence demonstrated isolated c3 deposits in the mesangium and along the glomerular basement membrane (humps). c1q, igg, iga and igm staining were negative. background: the risk of end stage renal disease (esrd) is low in unilateral wilms tumor, although patients with wagr or associated genitourinary anomalies are at higher risk. esrd is attributed mostly to hyperfiltration in the remnant kidney. immune complex glomerulonephritis (icg) has not been previously reported in wilms tumor patients. objectives: to report 2 cases of icg in unilateral wilms tumor. methods: retrospective chart review. patient #1 a boy with cryptorchidism, diagnosed with unilateral wilms tumor at 3 y of age. patient #2 a girl with wagr and unilateral wilms tumor diagnosed at 2 y of age. both had chemotherapy after tumor resection. results: patient #1: within 2 mo of tumor resection, a proteinuria of 3.8 g/24 hrs and rising creatinine were noted. renal biopsy was consistent with icg. within 5 mo of surgery the patient developed esrd requiring chronic hemodialysis. patient #2: within 4 y of tumor resection, the patient developed progressive, asymptomatic proteinuria up to 2 g/24 hrs. the renal biopsy revealed changes typical for icg. the patient was treated initially with enalapril and prednisone. due to no response, mycophenolate mofetil (mmf) was added and prednisone was weaned. after 4 mo of therapy, her proteinuria improved to 0.5 g/24 hrs. her serum creatinine continued to be normal at 0.6 mg/dl, with calculated gfr of 113 ml/min/1.73 m 2 . conclusions: this is the first report of icg in patients with unilateral wilms tumor with rapid progression to esrd in the first patient, but successful response to mmf in the second patient. despite low risk for progressive proteinuria in wilms tumor patients, it is prudent to monitor these patients for proteinuria and perform a renal biopsy if proteinuria is progressive. mmf therapy may be attempted to decrease proteinuria and to delay the onset of esrd.aim of the study: to present our first results of rhgh treatment mainly in children on hemodialysis. patients and methods: sixteen children, aged 4.5-17.1 years (mean age 11.25±3.57) with height below -2.0 standard deviation score (sds) for age or height velocity below -2.0 sds for age, were selected to receive rhgh therapy at our nephrology and hemodialysis department. most of them were on hemodialysis (14 children) with mean spent time 2.88±2.68 years (0-9 years) before an initiation of rhgh therapy. one half of patients were prepubertal (8 children) and second half were in early puberty (testicular volume between 4 and 8 ml for boys and breast development b2 or b3 in girls). all received 28-30 iu/ml per week by daily subcutaneous injection for 6 months to 3 years. the year before rhgh therapy served as a control period. results: during the first year of treatment, mean height velocity in hemodialysis patients increased from 2.25 cm/year to 6.59 cm/year (p<0.0001) and in the second year was 5.25 cm/year (p=0.004). the mean height sds in hemodialysis children did not improved significantly during the first year of rhgh treatment (from -3.01 sds to -2.77 sds, p=0.063). neither weight, nor the body mass index varied compared with the pretreatment period. no change was observed in glucose, total proteins, albumins, cholesterol and triglycerides levels. the mean increment in bone age was 1.1 years. pubertal status had no influence on response to rhgh. conclusions: therapy with rhgh improved height velocity in children with esrd. no significant side effects were observed in 16 children during the 23.5 treatment years. two patients developed secondary hyperparathyroidism but the relationship with rhgh remains uncertain. in our treatment group rhgh therapy was safe. a. waters 1 , a. trautmann 2 , p. zipfel 3 , e. harvey 1 , ch. licht 1 1 hospital for sick children, department of pediatric nephrology, toronto, canada 2 university children's hospital, department of pediatric nephrology, heidelberg, germany 3 atypical hemolytic uremic syndrome (ahus) is characterized by the absence of a diarrheal prodrome, the tendency to relapse and a poor outcome. functional and quantitative deficiency of complement regulatory proteins or inhibiting autoantibodies result in uncontrolled complement activation, which eventually causes ahus. -we report a case of ahus with complement dysregulation associated with a progressive refractory response to plasma infusions. factor h and factor h-related proteins (fhr) were quantified by elisa and were further analyzed by western blot. complement activation was determined by measuring c3. serial hgb (g/dl), platelet, creatinine (mg/dl), ldh (u/l) were measured. initial presentation was at 7 months of age with thrombocytopenia, hemolytic anemia and increased creatinine. ahus was suspected as e coli infection was ruled out. disease remission followed several plasma exchanges. monthly plasma infusion maintained remission. therapy intervals exceeding 1 month promoted relapses. nine years later, the relapse interval decreased and over the subsequent 3 years, thrombocytopenia persisted as plasma infusion requirements increased. a concomitant decline in renal function (creatinine 1.6 mg/dl) occurred with the development of persistent proteinuria and hypertension. at 13 years of age, she deteriorated acutely with hypocomplementemia and thrombopenia. quantitative factor h was normal. autoantibodies to platelets and factor h were negative. intravenous immunoglobulin combined with oral steroids resulted in normalization of platelet count. complement dysregulation is associated with ahus. hereditary defects can be treated with factor replacement therapy. refractory responses may subsequently arise due to the development of autoantibodies against complement regulatory proteins. complement dysregulation requires further analysis in our patient. objectives: angiopoietin-like 3 protein (angptl3) is involved in lipid metabolism and angiogenesis. the present study was to examine angptl3 expression in human kidneys with proteiuria, in adramycin rats (adr), and in puromycin induced podocyte damage. methods: immunohistochemistry was performed on kidney biopsies from children with mcd, mn, fsgs, tbmn. in adr, angptl3 expression was determined by quantitative real-time pcr in glomeruli and tubuli dissected from frozen section of kidneys with laser microdissection system. in mpc5, a conditionally immortalized mouse podocyte cell line in vitro, angptl3, perlecan and agrin werer detected through real-time pcr with the induction of puromycin. detachment assay was performed in podocytes tranfected by angptl3-pcdna3.1. results: in human kidneys, co-labeling showed angptl3 expressed in the cytosol of wt1 positive cells. quantitative computerized analysis showed that angptl3 in glomeruli in mcd and mn were significantly higher than that of tbmn, fsgs respectively (p<0.05). in adr, angptl3 in glomeruli increased significantly at 21 st or 28 th day (p<0.05) after adriamycin injection compared with control. and the expression of angptl3 in glomeruli was correlated with 24 h urinary protein (r=0.81, p<0.05). in mpc5 both protein and mrna expression of angptl3 on podocytes were up-regulated with the induction of puromycin. in podocytes transfected by angptl3-pcdna3.1 the expression of perlecan or agrin increased significantly compared with control (p<0.05). the attachment ratio was shown 95.7%±3.3% 24 hs after puromycin treatment on podocytes transfected by angptl3 compared with 38.6%±4.7% on normal podocytes, and 27.4%±3.5% on untransfected podocytes. conclusions: angptl3 is predominantly expressed in podocytes which could be involved in podocyte damage and the development of proteiuria. purpose: we present 2 cases of a previously undescribed pattern of membranoproliferative glomerulonephritis, in children with neuroblastoma on chemotherapy. the pattern of injury shows unusual focal capillary loop proteinaceous deposits possibly related to toxic chemotherapeutic drugs. the resultant hypertension and renal impairment made bone marrow transplant a challenging prospect. method: case series results: case 1: this boy with stage 4 neuroblastoma, developed severe renal impairment and hypertension during treatment. thus there were difficulties in administration of chemotherapy and he required early surgery. at tumor resection a nephrectomy was necessary. he received an autologous stem cell transplant. he became unwell at transplant and required haemofiltration. he made a good renal recovery and did not relapse. case 2: he was diagnosed at 2 months with stage 4s neuroblastoma. he completed treatment but later relapsed. during treatment his gfr reduced and he developed severe hypertension. this lead to restrictions in chemotherapy. renal biopsy was carried out at tumor resection. at bone marrow transplant he was very unwell and required haemofiltration. he has chronic hypertension and low gfr. light microscopy findings in both -global diffuse membranoproliferative pattern of injury -large numbers of proteinaceous resorption droplets -features of a protein deposition disease electron microscopy findings common to both -large number of differently sized protein droplets in the endothelial cells -no obvious immunecomplexes/deposits -protein deposition disease with a membranoproiferative pattern of injury conclusion: both cases showed deposits in the kidneys which may be tumor protein in origin and the resultant glomerulonephritis, hypertension and renal impairment lead to challenges in transplant. the long term consequences are yet to be revealed. methods: establish the cultured glomerular mesangial cells of rat in vitro, 3~10 generations of cells were used in the experiment after identification. the experiment included five groups: ctrl, lps, high, middle and low dose fos groups. gmc proliferation were detected by mtt. the changes of ln, fn and col protein secretion were detected by the elisa. the changes of lnbeta 2 mrna expression were detected by semi-quantitative real-time pcr. results: (1) fos can inhibit the effect of proliferation induced by lps. (2) mesangial cells can secrete some ecm protein in normal culturing medium, ecm protein secreted by mesangial cells was significantly higher in lps group than that in ctrl group (p<0.01), while ecm protein was significantly lower in all fos groups than that in lps group (p<0.01). (3) lnbeta 2 mrna expression was significantly higher in lps group than that in ctrl group, while that in fos group was significantly lower than in lps group. conclusions: lps can induce the increase of secretion of the ecm, including ln, fn, col fos can inhibit the secrection of ecm in gmc as dose-dependent manner at the mrna and protein levels. the conclusion supplies the theoretical evidence for the renal protection of fos. h. hong, w. na, y. li, w. qiang guangzhou first municipal people's hospital, department of pediatrics, guangzhou, people's republic of china objective: to observe the effects of fosinopril (fos), the new generation angiotensin-converting enzyme inhibitor (acei), on protein and mrna expression of tgf-β1 of rat glomerular mesangial cell (gmc) induced by lps; to demonstrate the preventive mechanism against glomerular sclerosis by applying fos. methods: culture rat gmc in classic way. the cultured cell were divided into 3 groups, namely (1) control group, (2) lps group, (3) lps+fos group. detect tgf-β1 concentration in gmc supernatant fluid by elisa; estimate tgf-β1 mrna expression by semiquantitative real-time rt pcr. results: lps group is obviously higher than control groups in tgf-β1 secretion and mrna expression, while lps+fos group drops distinctively in tgf-β1 secretion and mrna expression compared with lps group. conclusions: fos has obvious inhibited on tgf-β1 expression of rat gmc both at protein level and mrna level, which reveals that it might be an important mechanism by fos on restraining the development of glomerulosclerosis. r. kahn 1 , n. akbari 1 , j. wieslander 2 , w. müller-esterl 3 , a. christensson 4 , k. westman 4 , t. hellmark 4 , d. karpman 1 1 lund university, pediatrics, lund, sweden 2 wieslab ab, lund, sweden 3 institute of biochemistry ii, frankfurt, germany 4 lund university, nephrology, lund, sweden vasculitis is an inflammation with neutrophil influx in and around blood vessels. patients may have elevated plasma levels of neutrophil-derived proteinase 3 and anti-neutrophil cytoplasmic antibodies (anca) directed to proteinase 3, suggested to be involved in the pathogenesis of disease. we have previously shown that the kallikrein-kinin system (kks) is activated in vasculitis. in vivo the kks is activated on endothelial cells and neutrophils when high-molecular-weight kininogen (hk) is cleaved by kallikrein thus liberating bradykinin. bradykinin is a potent mediator of inflammation. in the present study we investigated if neutrophil-derived proteases, and proteinase 3 in particular, could induce activation of the kks and bradykinin release. purified neutrophils from ten vasculitis patients (3 adults, 7 children) and thirteen controls were treated with triton-x to induce lysis. proteinase 3 was immunoadsorbed from the neutrophil extracts. bradykinin and proteinase 3 levels were measured by elisa. hk proteolysis was detected by immunoblotting. proteinase 3 incubated with purified hk induced physiological breakdown of hk and bradykinin release. this was inhibited by preincubation of proteinase 3 with anti-proteinase 3. triton-x treated neutrophil extracts from both patients and controls induced hk proteolysis and bradykinin release whereas the neutrophil extracts from which proteinase 3 had been immunoadsorbed did not. levels of proteinase 3 in the neutrophil extracts from patients and controls did not differ. these findings suggest that neutrophil derived proteinase 3 can proteolyse hk in a physiological manner thus liberating bradykinin, thereby initiating kallikrein-independent activation of the kks. introduction: this is a prospective study to evaluate the safety and efficacy of tacrolimus in 22 consecutive children with steroid-resistant nephrotic syndrome (srns). methods: all of them were subjected to kidney biopsy. tacrolimus was given in dose of 0.15-2.0 mg/kg/day in two divided doses to attain trought levels of 5.0-10.0 ng/l. these patients were followed-up every 2 weekly initially for the first month, followed by monthly visits. urine spot protein creatinine estimation was done at each visit. besides blood glucose, serum creatinine, urea, electrolytes, albumin, and complete blood count were done once a month. results: the mean age of onset was 8.6±5.9 yrs. of the 22 children, 9 had mcd, 11 had fsgs and another 2 had dmh on histopathology. tacolimus had to be withdrawn in 3 children: of the rest 19 children who received adequate therapy, complete remission was seen in 16 (76%) children, 2 (11%) attained partial remission and 1 was non responsive. the mean time to achieve remission was 59.9+45.8 days and the mean dose of tac was 0.18+0.07mg/kg. the mean urine spot protein/creatinine ratios were significantly lower (0.64±1.09 vs 14.5±23, p=0.01) and mean serum albumin significantly greater (3.73±0.71 vs 2.45±0.52, p=0.001) as compared to those prior to tac. of the 16 children who attained complete remission, 2 patients are off steroids and tac and in sustained remission, while the rest 14 are still on tac therapy. conclusions: this is the largest study so far on the safety and efficacy of tacrolimus therapy in children with srns. we conclude that tacrolimus is a useful therapeutic alternative in children with srns who are unresponsive to cyclophosphamide and cyclosporine. objectives: to describe hiv infected paediatric patients from our centre with pathology proven renal disease. methods: retrospective review of biopsy data base and case notes of patients with hiv referred with renal problems. results: 14 patients were identified who had biopsy confirmed renal disease. the mean age of the patients was 5,7 yrs (range: 6 months to 16 years). twelve of the patients were african and two were of mixed race. renal pathology was divided into three groups: 1) hiv associated nephropathy (hivan): five patients. 2) mesangioproliferative nephropathy: 4 patients 3) other: acute pyelonephritis in 1, mesangial proliferation plus interstitial nephritis in 1, renal tuberculosis in 2, hiv immune complex disease (hivick) in one. conclusion: there is a high degree of variability of renal pathology in children with hiv and renal disease which upholds the need accurate diagnosis when confronted with these patients. background: acute poststreptococcal glomerulonephritis (apsgn) is the most common glomerular disease of children in our country. it has not been studied well in this region yet. here, we report our experience with psgn in a tertiary referral center during a five-year period. method: hospital records of all 137 children who had been admitted from mar. 2001 to mar. 2006 to nemazee hospital with diagnosis of acute glomerulonephritis (agn) were reviewed. all demographic, clinical, paraclinical data and consumed medications were obtained. results: among 137 children diagnosed as agn, 122 (89%) had apsgn. other 15 (11%) children had mpgn (n=4), mespgn (n=4), iga nephropathy (n=2), lupus nephritis (n=2), rpgn (n=2), and fsgs (n=1). mean age in children with apsgn was 8±3.5 (range, years. 117 children (96%) developed apsgn following a sore throat or upper respiratory infection while 5 (4%) cases developed after impetigo. ninety-five (78%) patients developed apsgn during the cold seasons of the year. periorbital edema was found in119 children (97.5%), hypertension in 92 (75%), gross hematuria in 88 (72%), oliguria in 45 (37%), generalized edema in 23(19%), azotemia (bun>20) in 98 (80%), and nephrotic range proteinuria in 30 (24.5%). aso titer was high in 103 (84%). low c 3 was detected in 105 (86%) and low c 4 in 16 (13%). dilutional anemia in 63 (51.5%), hyponatremia in 33 (27%), and hyperkalemia in 17 (14%) children among whom, 3 required hemodialysis. regarding medications, 29 patients had received only furosemide, 73 cases took furosemide and nifidipine and for 10 patients furosemide+nifidipine+another antihypertensive medication was prescribed. conclusion: acute psgn is the most common type of glomerulonephritis in this region. it follows sore throat in the majority of cases. it usually has an uneventful course. y. guo, zh. wang, x. liu west china second university hospital, sichuan university, department of pediatrics, chengdu, people's republic of china objective: we planned to explore the mechanism of glomerular basement membrane (gbm) damaged by rsv, through investigating the effects of lmwh on proteinuria and glomerular structure of rsv nephropathy. methods: sd rats were inoculated with 6 10 6 pfu rsv and lmwh 400 iu/kg. group a: rsv was given in the first 3 days, and lmwh was given for the following 11 days; group b: the mixture of rsv and lmwh was given in the first 3 days, then lmwh was given for other 11 days; group c: lmwh was continuously given throughout 14 days and on the 4 th -6 th day rsv was also given; group rsv and the control were respectively inoculated by rsv and dmem for 3 days. renal histology, urinary protein excretion and serum parameters were observed. rsv rna in renal and pulmonary tissue was determined by in situ hybridization. results: there was no significant increase in urinary protein excretion of the lmwh-treated groups (a 7.4053±4.057, b 7.101±1.833, c 9.209±1.625, mg/24 h) compared with the control, but that of group rsv (32.041±3.844 mg/24 h) gradually increased after rsv inoculation. there was just a decrease in albumin (15.060±1.335g/l) and an increase in urea nitrogen (12.9203±3.932μmol/l) of group rsv only. no change of the glomeruli detected in all lmwh-treated groups, while congestion and swelling in glomeruli of group rsv were observed significantly. glomerular microstructures of the lmwh-treated groups were almost normal, while extensive foot process effacement was observed in group rsv. rsv rna signal expressed weaker in the lmwh-treated groups than in group rsv. conclusion: rsv damages hs on gbm by electrostatic interaction. lmwh, as the analog of hs, charged with anion, competes with hs to combine with rsv to keep gbm from being destroyed, and then reduce the proteinuria. s. zhai, zh. wang west china second university hospital, sichuan university, department of pediatrics, chengdu, people's republic of china objective: the study is to explore the relevance among gags, hpa and ela in the steroid responsive nephrotic syndrome (srns). methods: (1) 55 children with srns were selected, including the active (n=20), the convalescent (n=20), the remissive (n=15). 19 purpuric nephritis and 15 healthy children were served as the control.(2) using the improved whiteman process detected the urinary gags. ela activities in plasma were determined by the amount of 4-nitroaniline released per unit time. immunocytochemistry and image analysis method were used to detect the expression of hpa of peripheral blood leukocyte. results: 1. gags of the active were the highest (4.1051±1.1722) of all (p<0.001). in contrast with the healthy, the active and the convalescent (2.5666±0.6826, p<0.001) were significant difference, but the remissive no difference (1.6588±0.3103, p>0.05). 2. all of ns showed higher level of hpa than the healthy (p<0.05). comparing with the healthy, hpa was significant difference both in the active (iod 54786.2137±28714.0671, p<0.001) and in the convalescent (iod 15708.6270±4036.2843, p<0.001),but no difference in the remissive (iod 4181.0056±878.3909, p>0.05). by contrasting the active and the purpuric, their difference of hpa was no statistic significance (p>0.05). 3. all of ns showed higher levers of ela than the healthy (p<0.05). the healthy was strikingly different, contrasting with the active (77.4304±17.6366) and the convalescent (53.6803±7.4168, p<0.001), but no difference with the remissive (38.6552±9.1556, p>0.05). 4.there was a significant correlation among the urinary protein, urinary gags, hpa and ela with simple linear regression analysis. conclusion: in the srns, proteinuria may be resulted by the spallation of hpa and ela for gags on gbm. (4), combination withhaematuria, hypertension and/or renal insufficiency (23), extrarenalsymptoms (6), and familial mediterranean fever (fmf, 40) . of the 85 non-nephrotic patients, 26 had extrarenal symptoms, 15 were nephritic,9 had rapidly progressive glomerulonephritis (gn), 6 renal failure and 29 isolated urinary abnormalities. biopsy samples were evaluated by light microscopy in yerevan and zurich and by electron microscopy (except for amyloidosis) and immunohistochemistry (last 28) in zurich. results: the most common histological lesion was renal amyloidosis (25%), followed by focal segmental glomerulosclerosis (fsgs, 9%), lupus nephritis (8%),systemic vasculitis/hus and minimal change disease (mc, 7.5% each),mesangioproliferative gn/iga-nephropathy and membranous nephropathy(mn, 7% each), hereditary nephritis and membrano-proliferative gn typei (6% each), acute postinfectious gn (apgn, 4%), and dense deposit disease (ddd, 3%). the miscellaneous group includes,apart from interstitial nephritis (1%), unclassified or inadequate biopsies and specimens with mostly sclerosed glomeruli(9%). the majority of patients with amyloidosis of fmf, fsgs/mc andmn were nephrotic, but 18% of patients with amyloidosis had non-nephrotic proteinuria. conclusions: several glomerular lesions were considerably more frequent than in other studies, particularly amyloidosis of fmf, mn and lupus nephritis, and to lesser extent membranoproliferative gn type 1 and ddd. apgn is underrepresented because less than 1% of all patients (>700) had a biopsy. this study would not have been possible without international collaboration. henoch-schönlein purpura in children: an epidemiological study amongst dutch pediatricians on incidence and diagnostic criteria j. aalberse 1,2 , k. dolman 2 , g. ramnath 3 , r. rodrigues pereira 4 , j-c. davin the aim of the present study on the incidence of henoch-schönlein purpura (hsp) in dutch children is not only to give some insight in the epidemiology of hsp in the netherlands but also to record the diagnostic criteria used by dutch pediatricians and to evaluate the accuracy of the latter using the presence of iga in the skin when biopsies are available. methods: in 2004, all dutch pediatricians received monthly a card asking to mention new diagnosed hsp. pediatricians reporting one or more new patients with hsp were sent a list of questions concerning symptoms, blood and urine parameters, skin biopsy, diagnostic criteria and follow-up duration needed. results: two hundred and thirty-two patients from 0-18 of age (6.1/10 5 ) were reported as having started hsp in 2004. twenty nine % presented with renal symptoms. in accordance with the classification criteria of the american collegeof rheumatology (acr), eighty percent of pediatricians consider that isolated purpura (without hematological abnormalities) is sufficient to allow the diagnosis of hsp in children. from the 17 skin biopsies performed, only 9 (53%) presented with iga deposits. the follow-up duration considered as necessary was longer in case of renal symptoms at presentation. however, 45% of patients without renal symptoms would be followed for more than one year. conclusion: considering the recent (2006) eular/pres endorsed consensus criteria for the classification of childhood vasculitides, hsp should have been diagnosed in only 160 of the 179 patients of our study. the use of isolated non-thrombocytopenic purpura as only criterian to diagnose hsp in children might therefore lead to over diagnosis and unnecessary follow-up. noteworthy, the eular/pres criteria remain to be validated by a prospective study. the clinical presentation and response to therapy of childhood pan in johannesburg, south africa. method: retrospective record review of twelve children with a clinical diagnosis of pan treated between 1993 and 2005. results: there was unequal number of males and females; average age at presentation was 6.8±0.83-13.9 years, all were black children. eight children had more than 3 acr criteria and 11 sufficient clinical criteria (eular/pres consensus criteria). musculoskeletal and cardiac diseases were the commonest finding at presentation (75%), cutaneous, hypertension (58%), renal and gastrointestinal disease (50%), central nervous system disease (42%) and constitutional features (33%). two children had bone involvementwith periosteal reactions on plain x-ray. angiographic abnormalities were found in 8 (67%), and 9 (75%) had positive histology (skin/renal biopsies). tuberculosis was diagnosed in 6 (50%), and 6 had positive streptococcal titers. all patients were ana and hepatitis b negative, but there were five patients anca positive (2=p-anca, 2=c-anca, 1=both) (42%). the crp was elevated in 8/12 (67%), esr also in 8/12, while 58% had both elevated. all patients received oral glucocorticoids, 6 methylprednisolone (1-3 pulses-500 mg/m 2 ), 7 ivi cyclophosphamide (1-6 pulses, 500 mg/m 2 ), 10 oral azathioprine, and 1 required i. conclusions: children with pan in johannesburg present at a younger age with multi-organ disease. they require aggressive therapy with both glucocorticoids and cytotoxic therapy to ensure good outcomes. objective of the study: in order to evaluate the predictive factors of chronic kidney disease (ckd), the records of 47 children with biopsy-proven mesangioproliferative nephrotic syndrome (mpns) admitted between 1972 and 2005 were retrospectively reviewed. methods: renal survival was analyzed by the kaplan-meier method and cox's regression model. two multivariate models were developed: (1) from the onset of symptoms to the occurrence of ckd and (2) from the time of renal biopsy to ckd. the following data were obtained at admission an dat the time of renal biopsy: gender, race, age at the onset ofnephrotic syndrome symptoms, age at admission, blood pressure, laboratory data (serum creatinine, serum urea, glomerular filtration rate, 24-hr urinary protein excretion, hematuria). patients were classified according to the response to the initial course of prednisone: (1) a complete response was defined as a proteinuria <0.3g/day; (2) a partial response was defined as urinary protein excretion of <1 g/day and >0.3g/day, and (3) no response was defined as urinary protein excretion of >1 g/day. results: median follow-up time was 8 years (iq range, 3.9±13.7) and 12 patients (26%) progressed to ckd. at baseline, after adjustment 2 variables remained as independent predictors of ckd: creatinine >1.0 mg/dl (rr=3.8, ci 95%=1.01±11.3) and non-response to steroids (rr=10.7, ci 95%=2.2±51.2). at the time of renal biopsy, after adjustment 2 variables remained as independent predictors of ckd: age>7.5 yr (rr=3.8, ci 95%=1.0±14.3) and creatinine >1.2 mg/dl (rr=3.3, ci 95%=0.97±11.2). conclusion: serum renal function at baseline and initial response to prednisone were strong predictors of progression to ckd in our cohort of children with mesangioproliferative nephrotic syndrome. methods: an illustrative case history of a boy with sle and vhd in the absence of antiphospholipid antibodies was described. results: a 14-year-old boy with a history of sle for about five years was admitted to our hospital due to intermitted arthralgia, facial erythema, increased serum creatinine and oliguresis in october 2006. he had been treated irregularly with prednisone and immunosuppressants. however,the disease was not always controlled well. during thishospitalization, the problems of hypertension, renal failure and anemia had been resolved and maintained, but the problem of intolerance to increased circulation volume was obvious, and three times echocardiography (ecc) showed moderate to severe mitral valve insufficiency. reviewed his history, suspected mitral leaflets vegetations was revealed by ecc four months after onset, he complained of chest pain, chest distress and breathholding several times without causes since eighteen months after onset and manifested with heart failure once, and ecc showed moderate to severe mitral valve regurgitation four months ago and twenty days ago respectively. ruling out the possibility of infective endocarditis, rheumatic heart disease and congenital heart diseases, vhd were considered secondary to sle. since the vhd becomes hemodynamically significant, valve surgery is needed. conclusions: vhd is generally asymptomatic and omitted easily, so routine cardiac evaluation of children with sle using electrocardiography, echocardiography and chest x-ray is recommended to early detect and treat cardiac abnormalities, which may lead to better survival. objective: we have previously reported that deleted in esophageal cancer 1 (dec1), a potent tumour suppressor gene, was specifically upregulated in cd4+ cells of patients with relapse of mcns, using differential display rt-pcr. this study aimed to further characterize the potential function of dec1 in mcns. methods: semi-quantitative rt-pcr was used to verify the dec1 gene expression in children with relapse and remission of mcns. jurkat cells were transfected with plasmid containing dec1 gene or vector alone. gene expression was regulated by tet-on/off system. the effect of dec1 on jurkat cell proliferation was assessed by 3 h-thymidine incorporation. cell cycle analysis was performed following propidium iodide staining. protein localization was determined by immunofluorescent staining with anti-dec1 antibody. results: we confirmed that dec1 gene expression was significantly increased in 15 children with mcns in relapse (0.93±0.17), as compared to remission (0.44±0.13, p<0.002), 10 normal controls (0.59±0.06, p<0.01), 7 patient controls with viral infections (0.58±0.03, p<0.03) and 7 nephrotic patients with other forms of glomerulonephritis (0.48±0.10, p<0.03). in dec1-transfected jurkat cells, cell proliferation was inhibited by 47.2%, compared with vector control. cell cycle analysis indicated that dec1 arrested jurkat cell cycle progression by blocking its entry into the g2/m phase. immunofluorescent staining with anti-dec1 antibody suggested that dec1 was a cytoplasmic protein, which was in agreement with psort. conclusion: dec1 gene expression was significantly upregulated in children with relapse of mcns. our results showed that dec1 acts as a t-cell proliferation suppressor and arrests cell cycle progression, and thus may be important in mediating the number or function of cd4+ cells during relapse of mcns. objective of study: the aim of the study was to assess plasma and urine concentrations of vascular endothelial growth factor (vegf) in nephrotic syndrome children (ns) depending on the total dose of glucocorticoids (gc) and the percentage of lymphocytes with glucocorticoid receptor expression (cd3/gcr). methods: we examined 51 children (2-15 years) , allocated to three groups: group i: 13 children with the first ns onset, group ii: 13 children with ns relapse, group c: 25 healthy children. the ns patients were examined: a: before treatment and b: 4-5 weeks after prednisone administration at a dose of 60 mg/m 2 /24 h. plasma and urinary vegf levels were determined using the immunoenzymatic elisa method. flow cytometry was applied to assess cd3/gcr expression. results: higher plasma and urinary vegf concentrations were noted in ns children before treatment (a), as compared to control subjects (c). following prednisone therapy (b), vegf level was reduced but it was still higher than in the control group. positive correlation was observed between vegf and protein in the urine (group i r=0.660, p<0.05, group ii r=0.818, p<0.01) and a weak positive correlation between vegf in plasma and urine (group i r=0.531, p<0.05, group ii r=0.581, p<0.05). cd3/gcr expression was lower in group ii. in both groups, the correlation between plasma vegf and cd3/gcr was positive (p<0.05). conclusions: 1. plasma and urinary vegf levels increase during nephrotic syndrome onset. 2. glucocorticoid treatment reduces plasma and urinary vegf levels in ns children. objective of study: the aim of the work was to determine the expression of p-glycoprotein (p-gp) on peripherial lymphocytes (cd3) in children with steroid-dependent nephrotic syndrome (sdns) during cyclosporine a (cya) and ace-inhibitor (ace-i) treatment. methods: the study group (i) consisted of 20 children with sdns aged 5-18 years, with a subsequent proteinuria relapse at the time of prednisone dose reduction. all ns children were examined three times: a: at proteinuria relapse, before cya treatment, b: after 3 months, c: after 12 months of cya administration. control group (ii) consisted of 20 healthy children. cd3/p-gp was measured using a flow cytometry assay. serum cya level was assessed by means of the immunofluorescence method. results: the expression of cd3/p-gp in ns relapse, prior to cya+ace-i administration was much higher (median 9.15%, range 1.50-13.50%) when compared to healthy controls (1.20% range 0.30-5.70%). the absolute number of cd3/p-gp in this examination was almost 5 times higher when compare to healthy controls (p<0.01). after 3 months of cya+ace-i therapy the expression of cd3/p-gp decreased dramatically and was similar to the controls. similar results were obtained after 12-months of treatment. when analysing the correlation between cd3/p-gp and serum cya concentration a strong negative correlation was found in both examinations. the correlation was stronger in group ib (during the treatment with higher cya doses): (r=-0.624, p<0.01) than in ic (after reduction the cya dose: (r=-0.464, p<0.01) conclusions: the results of our studies indicate that cya in sdns inhibits the expression of p-gp. cya is an alternative therapy that may lead to optimization of glucocorticoid doses, thus reducing the risk that goes along with treatment. backround: hemolytic uremic syndrome is considered as the main cause of acute renal failure in childhood. it is characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. the epidemiology of the disease varies between counties. aim of the study: to describe demographic and epidemiologic aspects of hus, presented in a 25 years period at the biggest children's hospital in the country. material-methods: 27 patients aged 11 days -12 years, mean age 4.8 years, (20 boys, 7 girls) were diagnosed with the syndrome. of them 5, 18.5%, presented as d+ cases, 11 with preceeded respiratory system symptoms and 11 with no preceeded symptoms. treatment consisted of fresh frozen plasma (ffp) and whole blood transfusions in all cases. peritoneal dialysis was necessary in 7 cases, haemodialysis in 2, while plasmapheresis was performed in 3 cases. three children, all boys, suffered from recurrent type of syndrome. a girl required treatment with peritoneal dialysis for 4 months followed by recovery of renal function. one-five years follow-up: one baby 8 months old died, 4 children received renal transplantation, in 5 renal function remained mildly reduced (gfr 50-80 ml/min/1.73 m 2 ) and 6 suffered of hypertension. the rest 11 recovered fully. summary: hus represents a rather minor public health problem with low mortality rate in greece. methods: three children with pauci-immune ln were retrospectively reviewed. results: the reported cases fulfilled the american rheumatism association criteria for sle. at admission, all had a 1-month history of glomerulopathy without intensive therapy. case 1 presented with nephrotic proteinuria, macroscopic hematuria and progressively deterioration of renal function. laboratory data included a positive antineutrophil cytoplasmic antibodies (anca) on myeloperoxidase immunoassay. she was positive for lupus anticoagulant (la) but negative for anti-cardiolipin (acl) and anti-beta-2 glycoprotein i (β 2 gpi) antibody. renal biopsy showed pauciimmune necrotizing and crescentic ln. case 2 manifested with proteinuria, microhematuria and hypertension. anca was negative. case 3 had nephrotic proteinuria, microhematuria and hypertension. the case had positive la, acl and anti-β 2 gpi antibody. anca was negative. the biopsy findings of both case 2 and case 3 indicated pauci-immune mesangial proliferative ln. electronic microscopy revealed segmental and diffuse foot process effacement respectively. all three ln remitted following the treatment of steroid and immunosuppressive agents. conclusions: these atypical ln were considered to be associated with the distinct pathogenesis, rather than immune complex-mediated glomerular injuries. we supposed the possibility of ancaassociated necrotizing and crescentic glomerulonephritis for the first case. for the others, primary or secondary podocytes lesions might alter the glomerular permeability. the nephrotic syndrome is a clinical picture that characterized by proteinuria, hypoproteinemia, oedema and hyperlipidemia. although the primer nephrotic syndrome is the most common type of nephrosis in children, it may also develop during the course of infections including hepatitis b and c virus infections, whereas hepatitis a virus infection related nephrotic sendrome is very rare in children. in this paper, we present a case of who had suspected diagnosis hepatitis a virus infection related nephrotic syndrome. the boy at the age of 3 years was referred to our hospital due to vomiting, abdominal distension and oliguria. physical examinations were revealed palpebral and tibial oedema, hepatomegaly, decreased breath sounds and mat percussion on right hemithorax. icter were not detected. laboratory examination were showed proteinuria, hyperlipidemia, hypoalbuminemia, high alt and ast levels with normal levels of bilirubin, alkaline phosphatase, complement c3/c4, urea and creatinine. in viral serologic examinations, hepatitis b and c related antibody were found negative, but anti-hav igm was found positive. chest radiography and ultrasonography examination were revealed right pleural effusion and abdominal ultrasonography examination was revealed ascites. based on these findings, we thought that the nephrotic syndrome could be developed due to anicteric hepatitis a virus infection in our patient. low dose steroid treatment was started (prednisolon 1 mg/kg/day, 1 month). edema was improved, negative urine protein was seen, and liver functions were not deteriorated during this therapy in our patient. hepatitis a virus may be cause of nephrotic syndrome by forming of immune-complexes. in conclusion, although hepatitis a virus infection is rare cause of nephrotic syndrome in children, it should be investigated as a seconder casuse. membranoproliferative glomerulonephritis (mpgn) is a rare, chronic glomerulonephritis, and its prognostic factors and outcome are not known very well in childhood. in this retrospective study, we reviewed the clinical, laboratory and histopathological features of children with primary mpgn. thirty-three children (18 boys, 15 girls) presented at a mean age of 11.0 years (range 2-16 years). they were followed for a median of 44 months (range 12-92 months). the clinical presentation in children was nephritic-nephrotic syndrome in 15 children (45.5%), nephritic syndrome in 14 (42.4%) and nephrotic syndrome in 4 (12.1%). nine patients had renal failure at presentation. at the end of study, 25 patients (75.8%) had complete remission and 8 patients had abnormal findings (4 proteinuria, 2 microscopic hematuria, 1 hypertension, 1 esrd). all of patients were treated with steroid. eight patients were given another immunosuppressant drug (cyclophosphamide 7, azathioprine 1) in addition to steroid therapy. the interval between appearance of symptoms and admission, durations of microscopic hematuria and proteinuria, and systolic blood pressure at presentation were higher in children with abnormal findings when compared with children with complete remission. there were no significant differences in clinical presentation type or histopathological features between children with abnormal findings and children with complete remission. our results showed that delayed detection and treatment, uncontrolled hypertension and unresponsiveness to steroid in early period were poor prognosis predictors. we did not determine any correlation between histopathological findings and outcome. we need further investigations including larger patient population. partial lipodystrophy (pl) is a rare condition of unknown etiology, with childhood onset. it is characterized by progressive loss of subcutaneous fat of face, neck, trunk and upper extremities together with c3 hypocomplementemia. usually, patients do not have clinically evident renal disease or abnormalities until they have had the disease for 8 or more years. but, the case we reported here, firstly presented with membranoproliferative glomerulonephritis (mpgn), and during follow-up pl was observed. a six years old girl was presented with sore throat, vomiting and loss of appetite for the last fifteen days. her development was normal and the child was asymptomatic till 6 years of age. the parents were relatives of the third degree. there was no history of similar cases in the family. the physical examination of the patient was normal. urinalysis revealed hematuria and pyuria. proteinuria was not present. renal function tests were normal. laboratory examination revealed low serum complement c3 but normal serum c4. the lipid profile was also normal. patient was followed with the diagnosis of poststreptococcic glomerulonephritis. because of persistent low complement c3 and nephrotic range proteinuria renal biopsy was performed at the 3 rd month of follow-up. renal histology revealed mpgn consistent with type 2. c3 nephritic factor was negative. at the same time it was observed that the face took on a cadaverous look with prominent malar bones, chin and zygoma because of the loss of buccal fat. according to these findings, partial form of lipodistrophy, which is frequently associated with mpgn, was considered in the patient. during two years of follow-up she had two recurrences of nephrotic range proteinuria and she is now in remission with prednisolone therapy. as far as we know this is the first example of pl which is developed during follow-up of mpgn. central nervous system abnormalities in children with acute post-streptococcal glomerulonephritis (apsgn) are rare and are considered to be secondary to acute severe hypertension, electrolyte imbalances and uraemia. cerebral vasculitis associated with acute post-streptococcal glomerulonephritis has been rarely reported in pediatric literature. a 13-year-old girl with a severe headache, vomiting, edema and macroscopic heamaturia is presented. she had history of upper respiratory infection before two weeks. so, the patient was diagnosed as apsgn. on admission, she was normotensive and biocemically well balanced. two hour later, she experienced a grand mal seizure. mri examination of brain showed not only multiple areas of increased density in white and gray matter, and cerebellum but also subarachnoid bleeding, consistent with vasculitis. during follow-up, abducens nerve palsy was detected. histopathological features on renal biopsy specimen, an elevated antistreptolysin level and fallen c3 complement level were compatible with apsgn. all clinical and laboratory abnormalities improved with steroid therapy (pulse and oral methylprednisolon). in conclusion, children with apsgn may present central nervous system abnormalities without hypertension, uraemia and electrolyte disturbances. results: hsp was diagnosed in 105 patients, median age 6 years old. all had the skin manifestations, 49.5% abdominal pain and 41% arthritis. 45 patients developed hsp nephritis (42.9%), mean presentation time was 4.5 months after phs diagnosis. renal biopsy was performed in 14 patients, and the most common histopathological finding was hsp nephritis grade iii a. age of onset older than 10 years was statistically significant for nephritis development (chi square < 0.05). chronic renal insufficiency incidence was 0.95%. conclusions: the main complication of hsp is nephritis. follow-up should include evaluation by a pediatric nephrologist. age of onset older than 10 years is an important risk factor for hsp nephritis. objectives of study: acquired abnormalities of coagulation and fibrinolysis in nephrotic syndrome have been implicated in the pathogenesis of deep vein and arterial thrombosis. resistance to activated protein c due to amutation in the gene for factor v, the commonest inherited risk factor for venous thrombosis, could contribute to risk of both arterial and deep vein in patients with nephrotic syndrome. we report an arterial thrombosis in a young girl with idiopathic membranousglomerulonephritis (mgn) and factor v leiden mutation. case report: a 14 year-old girl was admitted to our hospital with swelling of both legs and cyanosis of her toes. her mother had history of abortion in the second trimester of gestation, and parents were second-degree relatives. physical examination showed peripheral edema in both extremities and peripheral cyanosis in toes. laboratory study showed the nephrotic range proteinuria. serum albumin was 2.2 g/dl, and cholesterol was 380 mg/dl. creatinine, electrolytes, prothrombin/partial-thromboplastin times, c 3 , c 4, fibrinogen, protein s, c, antithrombin iii and homosistein levels were within normal limits. ana, anti-dsdna, p-anca, and c-anca, antiphospholipid igg/igm, anticardiolipin igg/igm were all negative. genetic study showed the heterozygote mutation of factor v leiden (fv/g1691a). in arteriography, there was complete occlusion on the posterior tibiaartery of left leg, occlusion of mid portion of right femoral artery and the posterior tibia artery of right leg. renal biopsy findings werefelt to be compatible with mgn. conclusions: we postulate that patients with concurrent nephrotic syndrome and factor v leiden mutation may have an increased risk of thrombosis. screening for factor v leiden mutation may be indicated in patients with idiopathic nephrotic syndrome. introduction: hus (d+) is the 2nd cause of chronic renal failure (crf) in children in argentina. proteinuria is the main predictor of progression to crf. patients with renal failure, when treated with restricted proteinin take show slower progression to end-stage of rcf. proteinuria appeared in patients with hus sequelae subject to an overload of protein intake. objective: to evaluate the effect of a controlled protein intake on proteinuria in patients with renal disease due to hus and normal renal function (>80 ml/min/1.73 m 2 ). patients and methods: within a multicenter, randomized, double blind, controlled trial, carried out inorder to study the effect of iace on the development of renal disease*, proteinuria before and after a controlled diet was measured in 102 patients with proteinuria due to hus and normal renal function. protein intake was indicated according to rda for age. protein intake was estimated with a questionnaire on former 72 hours, and with 24 hrs urea excretion. proteinuria was measured in 24 hours urine collection at the beginning of the study and at 15, 30 and 60 days after onset of the study. results: median age at onset was 16.5 months (r: 7.0-85.0); mean of length of follow-up after hus onset was 48.0 months (r: 4.0-155). in sixty five (63,7%) patients, proteinuria reduced to normal values; in the remaining 37 (36,3%) there was no change. median of initial and final proteinuria in the 65 children whose proteinuria became normalised, was 9.83 mg/k/day (ds 3.71) and 2.44 (ds1.34) respectively <0.0005. conclusion: controlled protein diet (rda) normalizes proteinuria in patients with significant proteinuria secondary to hus and normal renalfunction. * a trial financed by roemmers laboratory, argentina. differential diagnosis of hematuria is the significant task of any nephrologist while most common reasons for hematuria demand histopathological diagnosis. iga-nephropathy is the progressive glomerular disease which is known to be a common reason for hematuria. the diagnosis ofiganephropathy is often missed because of variability of clinical presentations, long-term asymptomatic course and therefore waiting tactics in prescribing renal biopsy. the aim of the study was to evaluate the incidence of iga-nephropathy and to define its characteristic clinical and morphological features in children in belarus. we present results of immunohistochemical staining for iga mesangial deposition of 65 kidney biopsy preparations. the mean age of the patients enrolled in the study was 11,77±0,66 years. at the moment of biopsy patients clinically presented isolated hematuria (36%), hematuria with proteinuria (29%), nephrotic syndrom (12%), nephrotic syndrom with hematuria and hypertension (5%), isolated proteinuria (5%) and others in less than 2% each. iga mesangial deposition was detected using immuno histochemical staining with polyclonal rabbit anti-human iga. we found diffuse mesangial deposition of iga in 18 patients (28% of all). two of them had nonnephrotic isolated proteinuria, one nephrotic syndrom. vast majority of patients were hematuric (either isolated or combined with proteinuria). iga nephropathy incidence among hematuric patients was 38%. in histopathological examination we found mild segmental mesangial hypercellularity in 14 patients, mild to moderate global and segmental mesangial proliferation in 3 (in two of this patients we also found cellular and fibrous crescents in less than 30% glomeruli). one patient had focal-segmental glomerulosclerosis secondary to diffuse mesangial proliferative glomerulonephritis and clinically presented heavy proteinuria. we analyzed nphs2 mutations in chilean pediatric patients with srns due to fsgs, diffuse mesangial sclerosis (dms) and minimal change disease (mcd). nphs2 mutation analysis was performed in 31 patients (sporadic cases n=27, familial cases n=4). nphs2 exons 5 and 7 were amplified by pcr and subjected to automatic sequencing or enzyme restriction analysis using clai (c.686g>a) and hin6i (c.851c>t) respectively. patients median age at disease onset was 24 months (range , 55% of which were male. histological diagnosis were fsgs (n=26), dms (n=2) and mcd (n=3). ten of 31 patients (32.3%) had mutations in nphs2 (9/26 fsgs, 0/2 dms, 1/3 mcd). eight of 10 patients bearing mutations were sporadic cases. seven patients were compound heterozygous for r229q and a284v. patients with mutations were significantly older than those without mutations (median age 24 versus 84 months, p<0.01). resistance to cyclosporin was observed in 6 of 7 cases with mutations and 11 of 18 cases without mutations (ns). we studied the frequency of r229q and a284v in 60 healthy volunteers with similar ethnic background. only one control individual was heterozygous for r229q. no a284v mutation was detected. our study demonstrated that nphs2 mutations are a major cause of srns in chilean pediatric patients. since most of the srns patients bearing nphs2 mutations were cyclosporin resistant, it is advisable to perform nphs2 mutation screening before starting immunosuppressives. in this study we aimed to investigate the long-term prognosis of henoch-schönlein nephritis (hsn) in childhood. between 1991 and 2003, 156 patients with hsn were investigated retrospectively. there were 86 males and 70 females with a mean age of 9.6 years. they were graded according to the degree of renal involvement. grade 1: isolated microscopic hematuria (n: 31); grade 2: hematuria and mild proteinuria (n: 60); grade 3: acute nephritic syndrome (n: 4); grade 4: nephrotic syndrome/hematuria (n: 18); grade 5: acute nephritic and nephrotic syndrome (n: 43). renal biopsy was performed on 43 patients in grade 4 and 5. twenty patients had extensive crescent formation (>50%) on renal biopsy, and were given triple therapy [iv pulse methylprednisolone (mpz) 30 mg/kg/d for 3 days, followed by oral prednisolone (op), oral cyclophosphamide-2mg/kg/day for 2-3 months and dipyridamole]. the other 23 patients with <50% crescent formation were given mpz followed by op and dipyridamole. the patients in grade 3 and 4 were given op and dipyridamole. grade 1 and 2 were not given any immunosupressive agent. during the follow-up period (mean 30±3.5; range 12-96 months), 23 patients in grade 1, 38 patients in grade 2, 2 patients in grade 3, 8 patients in grade 4 and 21 patients in grade 5 showed complete remission (59%). of the 5 patients with extensive fibrosis on renal biopsy, 2 had persistent nephropathy (1%) and 3 developed endstage renal failure (2%). the remaining 59 patients showed near-complete recovery with minimal urinary abnormalities (38%). in conclusion, although initial presentation of renal involvement determines the prognosis in hsn, intensive treatment with triple therapy appears to be effective in severe renal disease especially if started before the development of fibrotic changes in crescents and tubulointerstitial tissue. aim: the bacterial infection in nephrotic syndrome (ns) is still the big problem and is one of the leading death causes in ho chi minh city, vietnam. we did this study with aims to overview this complication in children with ns. methods: our study population consisted of 132 children with nephrotic syndrome during one-year prospective cohort. twenty seven out of 132 patients who developed severe bacterial infection including pneumonia, peritonitis, urinary tract infection, bacteremia or cellulitis were recorded. results: from june 2004 to june 2005, there were 132 children with nephrotic syndrome recruited to the study. the severe bacterial infection stood at 20.5% (n=27). in 27 these children, 14 children were first ns, 13 children were relapsing ns. the percentages of pneumonia, urinary tract infection, cellular infection and peritonitis were 12.9% (n=17), 4.5% (n=6), 3% (n=4) and 2.3% (n=3), respectively. no patients with bacteremia were recorded. in 6 patients with uti, e.coli wasin 3 patients, proteus in 2 and enterococus in 1. ns children with uti were asymptomatic. ns children with peritonitis had typically clinical manifestations of fever, abdominal pain, tenderness. one out of four patients with peritonitis cultured streptococcus pneumoniae inperitoneal fluid. some factors associated with severe bacterial infection were increase in weight (12.7 vs 9.4%, p=0.02), very low serum albumin (10.3 vs 13 g/l; p=0.003), rise in serum a 2 globulin level (39.5% vs 34.6%; p=0.005) and hyperfibrinogenemia (5.8 vs 5.3 g/l; p=0.02). conclusions: bacterial infection has still been a common problem in children with nsin ho chi minh vietnam. it is important to investigate and manage this complication early to reduce mortality rate. primary nephrotic syndrome (ns) is the most common glomerular disease in children which mainly responds to corticosteroids. however, >70% of children experience a relapse with recurrent episodes. the aim was investigation of outcome in ns patients, retrospectively. clinical, histological data and treatment responses of children presenting with ns from 1996 to 2006 were reviewed. all patients were treated with steroid treatment firstly and classified as steroid sensitive ns (ssns) and steroid resistant ns (srns) according to clinical or laboratory responses. there were 91 patients, 52 male and 39 female, had followed-up for 64.17±54.79 months. age at onset ranged from 9-186 (median 34) months. seven patients were admitted with hematuria and ns (5 mpgn, 2 mgn) that excluded from this study. 60 patients were treated by only classical steroid treatment. thus, 60 (71.4%) patients were ssns and classical steroid therapy was successful in this group.14 of all ssns patients were evaluated with biopsy (6 fsgs, 8 dmp) because of frequently recurrent ns. in srns group (24 patients) with one cytotoxic agent, complete and stable remission was induced in 13 patients (6 in fsgs, 5 in mlh, 2 in dmp) while 6 patients (6 in fsgs) who responded to more than one cytotoxic agents had partial remission with symptomatic relief. five children (5 in fsgs) were refractory to all cytotoxic therapies. cyclophosphamide (cp) was used as the first cytotoxic agent in 18 patients and induced complete remission in 11 (61.1%). the patients who relapsed following cp and patients who failed to respond were treated with further cytotoxic therapies such as cyclosporine a (cs) or mmf. in 6 patients, cs was used as the first cytotoxic agent and induced remission in 2 patients (33.3%). steroid must be the initial drug in childhood ns. cp could be used successfully as an immunosuppressive agent in srns patients. aim: annexin v has a molecular weight of 32-35 kda and has been reported to possess anticoagulant activity, inhibition of phospholipase a2, regulation of membrane transport, proliferation and signal transduction. it is reported that urinary annexin v concentration may be an indicator of apoptosis and acute renal injury related to the urinary protein level. the aim of this study is to define the role of urinary annexin v, serum annexin v concentrations as new prognostic tools and follow criteria in children with steroid sensitive and resistant ns. methods: annexin v concentrations were measured in serum and 24 hour urine samples in 23 steroid sensitive nephrotic syndrome (ns) patients in both relaps and remission periods (group 1 and 2 respectively) and in 22 steroid-resistant ns (group 3) and sex and age matched 22 healthy controls (group 4). total protein, albumin, cholesterol concentrations and 24 hour urinary excretion of protein and creatinine were measured in all groups. results: in steroid resistant ns group (group 3), median of urinary annexin v/creatinine ratio was significantly higher than all the other groups (5048.8 ng/g creatinine (min-max: 1272.5-40498.4) vs 2839.5 ng/g creatinine (min-max: 1131.0-15835.4) in group 1 (p: 0.06); 2500.0 ng/g creatinine (min-max: 1424.2-11055.6) in group 2 (p: 0.028), and 2018.3 ng/g creatinine (min-max: 1225.9-6887.4) in group 4 (p: 0.028)). serum annexin v concentration was significantly higher in group 3 (median 16.6 ng/ml) than in group 4 (median 8.2). no significant correlation was found between urinary protein excretion and urinary annexin v/creatinine ratio. conclusion: remarkably increased urinary annexin v/creatinine ratio could be used as a determinant factor in children with steroid resistant ns, and it may be a prognostic factor in these children. v. baudouin 1 , f. bernaudin 2 , a. garnier 1 , t. kwon 1 , m. peuchmaur 3 , g. deschenes 1 , c. loirat 1 1 hôpital robert debré-aphp, service de néphrologie pédiatrique, paris, france 2 chic, service de pédiatrie, créteil, france 3 cgvhd is the most common late complication of hsct. clinical manifestations mimic lupus disease but renal involvement is unusual. a 4 year-old boy underwent hsct from an hla-identical sibling donor for sickle cell disease. he received myeloablative therapy (cyclophosphamide, busulfan, antithymocyte globulin). prophylaxis of acute gvh consisted in prednisone and mmf until month 4. moderate skin and mucosa cgvh appeared at month 6 so that prednisone and mmf were restarted. at month 9 prednisone was stopped and mmf decreased to half dose. at 1 year, while clinical features of cgvh intensified, fortuitous diagnosis of ns was done. glomerular filtration rate and blood pressure were normal. biopsy showed membranous glomerulonephritis and mesangial hypercellularity. immunofluorescence confirmed granular immune deposits of igg and c3treatment consisted in prednisone (1.5 mg/kg/d for 1 month, progressively tapered to 0.5 mg/kg/e.o.d at month 4) and cyclosporine (5 mg/kg/d, trough levels 40-50 ng/ml). proteinuria decreased to <0.5g/l in 2 months. cyclosporine was progressively stopped between month 6 and 12 without relapse of proteinuria. ns associated with cgvhd had been reported in about 60 patients (5 patients <18 yr-old). retrospective studies estimate occurrence around 1% of patients with cgvhd. it was secondary to membranous glomerulonephritis in 67% of cases, minimal changes disease in 21%. strengthening of immunosuppression led to complete remission in 90% of patients with minimal changes disease and 27% of those with membranous glomerulonephritis (60% partial remission). usual treatment consisted in prednisone (87%) and cyclosporine (51%). this manifestation of cgvhd is probably underestimated and can occur at the same time or later than other clinical features. early detection of proteinuria in patients with cgvhd is recommended to adapt immunosuppressive treatment. objectives: to see if cyclosporine a (csa) is safe and effective in reducing proteinuria in children with the iga nephropathy (igan) or the henoch-schoenlein purpura nephritis (hspn). methods: the biopsy proven 34 patients (19 with igan, 15 with hspn) who showed increased proteinuria (>1+) for longer than 4 months were included. the blood level of csa, serum chemistries, urine analysis and complete blood cell counts were carried out every other month along with the physical exams. results: csa was given at an amount of 4.41.0 mg/kg/day for 10.13.3 months in average. complete remission of proteinuria in 27 (79.4%) and partial remission in 2 (5.9%) were achieved by csa treatment. five (14.7%) non-responders were discontinued for csa treatment in the middle of the trial. the ration of urine protein to creatinine was initially 3.95.0 and reduced gradually with time to 0.81.6, 0.30.7, 0.50.9 at 4, 8, 12 months after csa treatment, respectively. twenty eight patients showed hypertrichosis, three experienced transient elevation of serum creatinine, and two complained difficulties in taking the medication due to severe nausea. for 28.3 months after completion of the csa treatment, 9 patients redeveloped proteinuria and had to receive the 2 nd csa trial. no clear difference was observed in the pharmacokinetic profiles of csa attributable to the non-response or recurrence. follow-up renal biopsies were carried out in 11 patients after completion of the csa therapy and no csa toxicity was found. there was no alteration of linear growth pattern. conclusions: this study has a limitation of lacking the control group but the csa treatment is assumed to be very effective and a safe method to attain the remission of proteinuria in pediatric patients with the igan or hspn. j. madrigal 1 , e. fernandez 2 , p. noguera 2 , p. carranza 3 1 the aim of this retrospective study was : 1) to correlate the histopathological diagnosis with steroid response,persistent hematuria, hypertension and or abnormal renal function tests (gfr) 2) to evaluate the response of the patients with srns and sdns to the oral cytotoxic drugs , in a period of 10 consecutive years. 3) to correlate the response of this group of patients to the oral cytotoxic drugs with their histopathological diagnosis 4) to observe the incidence of fsgs in costarrican children during a period of 10 years. 5) based on these observations, reevaluate the indications of the kidney biopsy in our patients . we reviewed all the clinical records of patients with the diagnosis of ns and in whom a kidney biopsy have been done. patients with incomplete data were excluded. two consecutive reviews were made: the first included all patients diagnosed between january 1993 and december 1997 (group a) and the second, between january 1998 and december 2002 (group b). a total of 81 medical records were analyzed; 20 patients were excluded, and the remaining 61 were studied. results: all patients had been referred for edema or new onset nephrotic syndrome before treatment had been initiated. in our patients the steroid response was also the most important factor to predict the histological diagnosis and the response to the treatment with cytotoxics. the presence of hematuria and abnormal serum creatinine at the time of diagnosis were a predictive factor for the steroid response but not for the histological diagnosis. arterial hypertension achieved statistical significance only between mcns and fsgs but it was useful as a predictive factor for the hystological. in our group of patients with srns treated with cytotoxics, 55.5% with mcns responded, versus 37.8% of the patients with dmgn p<0,05. a. guersoni, v. mello, v. benini, s. laranjo children with srns are exposed to prolonged and high doses of steroid therapy and other immunosupressants that can lead to a variety of serious side effects. these include statural growth impairment, obesity, osteoporosis, cataract, hypertricosis and psychological disturbances.we carried out a prospective single-center study to evaluate the efficacy of mycophenolate in 20 children with steroid-resistant disease, 7 female and 13 male, aged 11.5±4.2 years. histological findings were: minimal change disease (mcd) in 8 children, focal segmental glomerulosclerosis (fsgs) in 11 and membranous nephropathy in one. all patients had been treated with at least one course of cyclophosphamide, metil-prednisolone in 15, while 13 had also been treated with cyclosporine before mmf. the initial dose of mmf was 600 mg/m 2 per day, together with a minimal reduction dose of corticosteroids associated with angiotensin ii receptor blockers (arbs) and sinvastatin.three patients went into complete remission, 15 into partial remission and 2 showed no remission. partial remission was described as loss of edema and improvement in symptoms, despite persistence of significant but improved proteinuria, that was classified either as moderate or low proteinuria according to the level. side effects were: diarrhea (n=1), neutropenia (n=2), infectious disease (n=1). mmf is an important new therapeutic option when associated with angiotensin ii receptor blockers (arbs) and sinvastatin for srns with mcd or fsgs, providing improvement in edema and symptoms despite persistence of proteinuria, with no compromise of physical appearance or risk of nephrotoxicity. background: primary focal segmental glomerulosclerosis (fsgs) that is resistant to steroids and other immunosuppressive agents has a guarded long-term prognosis. patients who fail to respond to current treatment may benefit from therapies that inhibit renal fibrosis and retard progressive loss of kidney function. objective: the font study (novel therapies in resistant fsgs) is a phase i clinical trial designed to test the safety, tolerability, and pharmacokinetics (pk) of novel agents that reduce renal fibrosis in patients with resistant fsgs. methods: patients, age 2-40 yr and egfr >40 ml/min/1.73 m 2 , with resistant fsgs who fail treatment in the nih supported trial evaluating cyclosporine vs. dexamethasone plus mycophenolate mofetil or who are screen failures due to prior exposure to these drugs are eligible. patients are assigned to receive rosiglitazone 3 mg/m 2 per day po or adalimumab 24 mg/m 2 every other wk sc. the treatment phase is 16 wk and patients undergo an initial (wk 0) and steady state (wk 16) pk assessment. results: 24 patients have been screened and 16 have been randomized 8 to each test therapy. 6 patients have completed rosiglitazone therapy and 2 have completed adalimumab. four serious adverse events have occurred in patients receiving rosiglitazone, none related to the study drug. fatigue (37%), gastrointestinal complaints (37%), and headache (25%) were the most common adverse events in patients given rosiglitazone. the outcomes in the adalimumab group have not been analyzed. conclusion: these preliminary results indicate the feasibility of performing phase i assessments of novel agents that can target renal fibrosis in patients with resistant fsgs. the findings will be used to design phase ii randomized clinical trials in this cohort of patients at high risk of progression to end stage renal disease. supported by grant niddk r2170341. object: to study the effect of fty720 on glomerulosclerosis and expression of cell cycle regulatory proteins in subtotal nephrectomized rats. methods: 24 rat were divided into sham-operation group, glomerulosclerosis model group and fty720 treated glomerulosclerosis group, 8 rats in each groups. the rats in later two groups were subjected to 5/6 nephrectomy. after operation, the treat group was fed with fty720 for 12 weeks. the expression of collagen, fibronectin, and cycline, p21, p27 were determined by immunohistochemistry methods. results: after treatment with fty720, up began to decrease from 4w after operation, significantly lower than in model group (p<0.01). the model group showed higher level of scr from 8w, which was much higher than in control group (p<0.05). in fty720 treated group, scr level were much lower than in model group. fty720 could obviously inhibit the expressionof col-and fn in glomeruli and attenuate the extent of glomerulosclerosis. moreover, fty720 could upregulate glomerular expression of p21 and downregulate glomerular expression of p27 and cycline. the expression levels of p21 and cycline were significantly lower in treatment group than in model group (p<0.05), but still higher than in control group (p<0.05). p27 expression in glomeruli was stronger in treatment group than in model group (p<0.05), and lower than in normal group but without significant statistic difference. conclusion: fty720 can diminish urine protein excretion and prevent glomerulosclerosis in subtotal nephrectomized rats. this protective effect is presumed to be associated with its role in downregulation of cycline expression and upregulation of p27 expression in glomerular cells, and inhibition of extracellular matrix accumulation in glomeruli. the authors illustrate severe side effect of steroid therapy ulcerative gastroduodenitis-and rare complication (multiple cerebral thrombo-embolism) in the case of a 10 year old girl with steroid resistent nephrosis. in childhood occurence of thrombosis in steroid sensitive nephrosis syndrome is 1.5%, while it comes up to 3.8% in steroid resistant cases. on admittance she presented the classic symptoms of nephrotic syndrome. one month after the initiation of steroid therapy haematemesis, melaena occurred, and after appropriate therapy was cured. due to the progression of the nephrotic syndrome, steroid shot and later immunesuppressive therapy was started. eight weeks after onset she became unconscious for a shortwhile and had a transient episode of right hemiparesis. at the same time ct and mri disclosed bilateral parieto occipital ischemic territorial vascluar lesions, with relative sparing of the cortical ribbbon. following icu observation her state rapidly improved and after a two week period she became free of symptoms. renal biopsy disclosed the pattern of minimal change nephrosis with diffuse mesangial hypercellularity and a slight amount of igm positivity. immunological evaluation-with the capacitiy to reveal systemic immunological diseases remained negative. having been put on an evidence based protocol the patient's present nephrological state was unremarkable, with proteinuria less then 1g/day. in the present work the authors discuss the factors predisposing to thrombo-embolism with special emphasis on the possible preventive measures and therapy. igg autoantibodies to c1q (antic1q) have been reported to play a pathogenic role in immunecomplex mediated diseases (sle, apsgn, membranoproliferative gn, etc). the occurrence of antic1q in adult patients with sle has been shown to correlate with disease activity and some immunological parameters (hypocomplementemia, anti-dsdna) and may be useful in the early diagnosis of lupus nephritis (ln) or even as a predictor of renal flares. the presence of antic1q in children with apsgn was associated with more severe disease manifestations and a lack of spontaneous recovery. associations between antic1q, c4 and c3 complement levels and disease manifestations in 129 children with gn were investigated and compared with healthy controls. antic1q were measured by elisa and c3 and c4 by immunoturbidimetry, respectively. 29 of 129 patients with gn were positive for antic1q compared to 0/40 healthy controls. antic1q were associated with active ln and hypocomplementemia: 10/15 patients with sle were found to be antic1q-positive. nine of these 10 had active renal disease at the time of blood sampling compared to 1/5 being antic1q-negative. 7/10 antic1q-positive patients had low c3 level and 4/10 had low c4 level. in children with apsgn, 13/38 were positive for antic1q. antic1q positive patients had significantly higher proteinuria, more often hypertension and c3-hypocomplementemia. all 5 patients in which apsgn did not resolve spontaneously were antic1q-positive. antic1q were associated with active nephritis and hypocomplementemia in patients with sle. in children with apsgn antic1q-positive patients have more severe disease and stronger c3-hypocomplementemia then those being antic1q-negative. m. zahrane 1 , l. fawaz 2 , l. nesseim 3 1 cairo university hospital, pediatric nephrology, cairo, egypt 2 cairo university hospital, pediatrics, cairo, egypt 3 cairo university hospital, cell pathology, cairo, egypt objective of the study: growth hormone (gh) and insulin-like growth factors are essential for normal growth and development. chronic renal failure (crf) results in major changes in the circulating growth hormone/insulin-like growth factor (igf) system. our aim is to study clinical and laboratory parameters of growth and osteodystrophy including igf1 and igfbp2 as part of the somatotropic hormone axis in egyptian children suffering from crf on conservative therapy. methods: 62 egyptian children (47 boys and 15 girls) with a mean age of 9.7y (0.47 to 21.12y) suffering from crf on conservative therapy and 21 controls were included in the study. ht, wt and tsf were measured and followed up for a period of 6 months. at the end of the follow-up period serum for igf1 and igfbp2, renal function, electrolytes, ca, p and alkaline phosphatase and acid base balance were measured and an x-ray of the left hand and wrist was done to determine their bone age by tanner and whitehouse. results: our study shows that children suffering from crf in egypt on conservative therapy have growth retardation with a mean ht of 3.7 sds, a mean wt of -2.24 sds. tsf mean was -1.3 sds. on the average the patients had a delay of 2.95y (±2.0) in their bone age. their height was retarded more than their bone age with a height age/bone age of 0.8 (±0.18). alkaline phosphatase as a markers of renal osteodystrophy is significantly correlated to the height, height age, bone age and to the ph. the mean igf1sds (-0.6±1.8) did not differ from that of controls while the mean igfbp2sds (2.4±4.6) was significantly higher in patients with crf than in controls. height and weight were significantly correlated to igf1 but not igfbp2. there is a significant correlation between igfbp2 level and the glomerular filtration rate. conclusions: the imbalance between normal insulin-like growth factor-i (igf-i) and markedly increased igfbp2 plasma levels plays a pathogenic role for growth retardation in children with chronic renal failure. the lower the gfr the higher the igfbp2 level. the latters inhibitory action may provide hope for improving growth in cases of crf by reducing the level of igfbp2 or displacing igf1 from it. s. sultana 1 , h. rahman 2 , m. hossain 2 1 bangladesh medical college, pediatric department, dhaka, uttara, bangladesh 2 bangabandu sheikh mujib medical university, pediatric nephrology, dhaka, bangladesh objective: to find out the impact of different etiology of chronic renal failure on growth in children. methods: this prospective study was carried out in the department of pediatrics, bangabandhu sheikh mujib medical university (bsmmu), dhaka, bangladesh, from october 2001 to october 2003. fifty children of both sexes under 15 years of age with clinical and biochemical evidence of chronic renal failure (crf) with creatinine clearance (ccr) of <75 ml/min/1.73 m 2 were included in the study. on the basis of underlying causes of crf, the children were divided into congenital (n=30) and acquired (n=20) groups. all patient's height and weight were measured. radiographs of hands, digits, ankle and knee joints, lumbar spine & skull were obtained to evaluate the presence of renal osteodystophy (rod) and for assessment of bone age. serum intact parathormone (ipth) level was also assayed in all patients. growth parameters and presence of radiographic and biochemical features were evaluated in two groups. results: crf children due to congenital anomalies had stunting and wasting in 23 (76.7%) and 20 (66.7%) cases respectively and the difference between two groups of crf patients was highly significant (p<0.001). alkaline phosphatase (467.70±218.55 u/l) and ipth (91.43±33.42 pg/ml) were also significantly higher than acquired group (p<0.001 and p<0.05 respectively). radiographic features of rod were present in 15 (50%) cases in congenital group in comparison to 4 (20%) in acquired group and the growth zone lesion was the commonest type of rod in congenital group (66.7%). conclusion: all efforts should be made to diagnose the presence of crf as early as possible, especially in infants and in children with early onset crf who seem to lose growth potential. introduction: in patients with thalassemia major the most important cause of morbidity and mortality is organ failure due to iron deposits, desferioxamine toxicity and anemia. this study was designed to define renal abnormalities associated with thalassemia and to find early marker (markers) of renal dysfunction. patients & method: 39 thlassemic children (18 female and 21 male) with mean age of 11.8±2.3 yr. were studied. all of them were received desforioxamine. 22 age and sex matched healthy children were involved in the study. blood and timed urine sample were obtained for hematologic and biochemical tests. the results were compared between case and control group. results: mean value of bun, serum creatinine, creatinine clearance, serum electrolytes, urine osmolality, fractional excretion of sodium and potassium were not statistically different between two groups. level of urinary nag (n-acetyle-beta-d-glycosaminidase) was significantly higher in patients than in controls (p: 0.001). there was a positive relation between urinary nag and duration of disease (p: 0.04). the was no statistically significant relation between urinary nag and serum ferritin. tubular function was not altered by hypertransfusion. conclusion: proximal tubular dysfunction is found in thalassemic patients. measuring urinary nag can guide the physician to find the early tubular abnormality in patients without frank renal dysfunction. severity of the abnormalities is correlated with the duration of disease. the present study aimed to investigate the effects of isolated ma and ma associated with mild renal function impairment on fracture healing in rats. ma was induced by chronic ingestion of 2% ammonium chloride solution as the unique source of liquid and renal dysfunction was produced by unilateral nephrectomy. thirty male holtzman rats (200-260g) were divided into six groups: control group (c,n=5) non-operated rats receiving tap water, acidotic group (ac,n=5) non-operated rats ingesting 2% ammonium chloride; sham water (s,n=5) sham-operated animals receiving tap water; sham acidotic (sac,n=5) sham-operated rats ingesting 2% ammonium chloride; nephrectomy water (n,n=5) nephrectomized rats ingesting tap water; and nephrectomy acidotic (nac,n=5) nephrectomized rats ingesting 2% ammonium chloride. after one week, blood samples were obtained to measure ph and gases, and a fracture of the right tibia was manually produced. four weeks later, fracture healing was evaluated by radiological and histological parameters. blood ph and gases, serum electrolytes and creatinine were also determined. data were compared by anova followed by newman keuls or fisher's exact test. fracture healing in nac, ac, sac animals was significantly altered as compared to c group. there was an additive effect of metabolic acidosis and unilateral nephrectomy in fracture healing process as shown by the comparison of sac and ac rats using radiological and histomorphometrical parameters. there was no difference between electrolytes and creatinine levels in all groups at the end of the experiment. this study showed a higher frequency of delayed fracture healing and nonunion in the presence of ma, which is worsened by unilateral nephrectomy. our data indicated an important interaction between bone and kidney in acid base homeostasis. introduction: dent disease, an x-linked recessive tubulopathy, is historically characterized by lowmolecular weight proteinuria, hypercalciuria, nephrocalcinosis/lithiasis and slowly progressive renal failure. most cases are caused by mutations in the clcn5 gene (dent disease 1, omim #300009), some patients with dent like phenotype have defects in the lowe syndrome gene ocrl1 (dent disease 2, omim #300555). patients: 10 male patients from 7 families with urinary findings resembling dent disease are reported. in 7 patients, mutations in the clcn5 gene were found, in 3 patients ocrl1 mutations. all children have increased values of urinary alpha-1-microglobuline, but also unselective glomerular proteinuria. 6/10 have mild hypercalciuria, 4/10 demonstrate mild renal insufficiency. amost all patients have increased echogenicity of renal parenchyma, but mild medullary hyperechogenicity is found only in 2 of 10 patients. metabolic acidosis or renal phosphate wasting is not found. interestingly, 5 of 10 children have increased values of creatinine kinase of unknown origin, clinically asymptomatic and independant of clcn5 or ocrl1 mutations. conclusion: the phenotype and genotype of dent disease is very heterogeneous. diagnostic criteria of dent disease and of lowe syndrome should be discussed. e. sahpazova, d. kuzmanovska, l. spirevska, n. ristoska bojkovska pediatric clinic, nephrology, skopje, macedoniathe nutritional condition of 35 children (22 males and 13 females) mean age 8.18±4.04 (range 1-16 years) with moderate renal failure have been followed for three year. glomerular filtration rate (gfr) was measured by creatinine clearance calculated with schwartz formula and was ranged from 13.59 to 75 ml/min per 1.73 m 2 . the nutritional condition was determined by anthropometrics and nutritional measurements. the patients were divided in four groups depending of their protein intake, primary disease, ages and glomerular filtration rate. all patients were following an -ad libidum -diet. nutritional intake was determined by minimum of two 3-day prospective dietary diaries. 46% of children received significantly lower protein intake. the mean protein intake (% of who recommendation) determined by dietaries of patients with -sub-optimal intake -was 94.79% vs. 175.45% in patients with -adequate protein intake -(p<0.05). all patients have a calorie intake of at least 80% of the who recommendations. the relative distribution of calories was 11.22% from proteins, 57.66% from carbohydrates and 31.12% from lipids. nitrogen balance in 15 patients was positive and correlated most significantly with increasing energy intake (r=0.58). average values for height, weight, triceps skin fold, mid-arm muscle circumference, and body mass index were within 2 sd of the mean of the normal population. the protein intake, primary disease and age of the children did not have any effect on growth and development. only patients with more advanced renal disease showed small score for height and growth velocity. key words: chronic renal failure; uremic children; nutritional status; nutritional intake; u. aslanova 1 , t. morimoto 1 , e. farajov 2 , n. kumagai 1 , n. sugawara 1 , a. ohsaga 3 , y. maruyama 3 , s. tsuchiya 1 , s. takahashi 4 , y. kondo 2 1 tohoku university, pediatrics, sendai, japan 2 tohoku university, medical informatics, sendai, japan 3 tohoku university, physiology, sendai, japan 4 nihon university school of medicine, pediatrics, tokyo, japan the extracellular calcium-sensing receptor (casr) located in either luminal or basolateral cell membranes of various types of renal tubules including proximal tubules, henle's loop and collecting ducts has been thought to play a fundamental role in electrolyte metabolism. to further identify the physiological roles of the casr, we examined the effects of ca 2+ and calcimimetics neomycin (neo), gentamicin and gadolinium chloride gd 3+ on the intracellular ph (phi) of in vitro microperfused mouse medullary thick ascending limb (mtal) cells of henle's loop, by loading the cells with fluorescent ph indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and measuring the ratio of fluorescence emission at 530 nm after exciting the dye at 490 and 440 nm. in a steady-state condition in hepes-buffered solution, the phi in the mtals was 7.29±0.04 (n=9). a concentration of 200 micromol/l neo in the basolateral side decreased the phi after 1 min by -0.13±0.02 (n=34, p<0.0001). the other calcimimetics showed similar effects on phi, whereas none of these calcimimetics in the lumen affected phi. na + removal or the inhibition of na + and proton transport with amiloride, bumetanide, or bafilomycin did not eliminate the effect of neo on phi. on the other hand, clremoval clearly eliminated the neo-induced phi decrease (-0.06±0.01 vs -0.00±0.05 in clremoval, n=4, p<0.003). thus, we have demonstrated for the first time that the casr is involved in the regulation of the phi in the mtal and requires clto exert its effect. background: paediatric nephrologists are often consulted for atypical rickets of renal or non-renal origin. the comeback of vitamin d deficient (classical) rickets in armenia and elsewhere is not only a public health problem but also a new diagnostic challenge for nephrologists. the aim is to analyse all paediatric patients seen in 2006 with bone deformities and suspected rickets at the arabkir hospital in yerevan. patients and methods: patients with bone deformities came spontaneously or were referred by one of us (gk). routine serum chemistry was done in yerevan. further investigations, if needed, and urine chemistry were performed in zurich. patients with rickets due to renal insufficiency were excluded. results: in 2006 we have seen 22 patients (12 males) with rachitic bone deformities aged 17-46 months (mean 32±9.4) at diagnosis. of these, 8 patients had florid vitamin d deficient rickets and 9 had sequelae of rickets but were radiologically and biochemically cured. these 17 children with classical rickets had to be distinguished from 5 patients with other forms of rickets: x-linked hypophosphataemia (xlh; 2), vitamin d dependant rickets type1 (1), and renal fanconi syndrome (2) due to fanconi-bickel syndrome and idiopathic. conclusions: (i) the rising number of vitamin d deficient rickets is of concern and due to neglected prophylaxis, (ii) children with classical rickets came late and were in the same age range as patients with atypical rickets, (iii) hence, and because of the larger number of rachitic children, an increased awareness of nephrologists -including a full diagnostic work-up -is needed in order not to overlook rare forms of rickets, (iv) polar vitamin d metabolites should not be used before the precise type of rickets is known. b. spasojevic-dimitrijeva, m. kostic, a. peco-antic, d. kruscic, d. paripovic, m. stanic university children's hospital, nephrology department, belgrade, serbia t. porowski 1 , w. zoch-zwierz 1 , j. konstantynowicz 2 , k. taranta-janusz 1 1 medical university of bialystok, 1st department of pediatrics, bialystok, poland 2 medical university of bialystok, department of pediatrics and auxology, bialystok, poland there are no published data on calcium oxalate (caox) crystallization and therewith associated kidney stone disease in children. the aim of this study was to determine bonn risk index (bri) in children with urolithiasis in relation to healthy age-and sex-matched controls and to assess possible associations between bri values and the size of renal stones. methods. in this cross-sectional study, we compared bri in 142 caucasian children and adolescents (76 girls, 66 boys) aged 3-18 years (median: 14.3) in whom kidney stones were newly diagnosed and 210 healthy age-and sex-matched controls (105 girls, 105 boys) without urolithiasis. urinary ionized calcium [ca + ] was measured using a selective electrode, while the onset of the spontaneous crystallization was determined using a photometer and titrating with 40 mmol/l ammonium oxalate (ox 2 ). the calculation of bri value was based on [ca 2+ ] to (ox 2 ) ratio. high resolution renal ultrasonography was done to estimate the size of renal stones. results. our results showed that bri values in children with renal stones were greater compared with healthy children without stones. bri was 15-fold greater, bri/kg body weight -10-fold greater, bri/per 1.73 m 2 b. s. -13-fold, whereas bri/bmi was even 23-fold greater in cases with stones than in controls. no significant association was observed between bri and the size of stones. interpretation. children with kidney stones demonstrate increased bone risk index compared with healthy subjects without urolithiasis. an increased bri during growth, although unrelated to renal stone size reflects the risk for crystallization of calcium oxalate and may suggest early metabolic disorders leading to urolithiasis. this simple method appears to be accurate and cost-effective, thus bri may be widely used for discrimination between stone-formers and healthy children. m. dixit, n. dixit florida children's hospital, florida children's kidney center, orlando, united states acute tubulo-interstitial nephritis (atin) is an important cause of acute renal failure resulting from a variety of insults including rare immune complex-mediated tubulo-interstitial injury. drugs including non-steroidal anti-inflammatory drugs (nsaids) are far most frequent cause of atin, but overall as an entity it remains under-diagnosed as symptoms resolve spontaneously if the medication is stopped. we present a 14-year-old male who developed acute renal failure 2 weeks after aortic valve surgery. he was put on aspirin following surgery and took ibuprofen for fever for nearly a week prior to presentation. he then presented to the emergency department feeling quite ill and was found to have bun of 147 mg/dl, creatinine of 15.3 mg/dl and serum potassium of 8.7 meq/l. dialysis therapy was immediately initiated. a kidney biopsy showed inflammatory infiltrate consistent with atin. however, very intense tubular basement membrane (tbm) granular deposits of polyclonal igg and c3 were noted. he needed dialysis for nearly two weeks and was treated successfully with steroids. his renal recovery and disappearance proteinuria took almost a year. in conclusion, we present an unusual case of tbm immune complex-mediated atin due to nsaids with severe but reversible renal failure. the effect of corticosteroid therapy on bone metabolism in nephrotic syndrome background: nephropathic cystinosis is characterised by lysosomal cystine accumulation leading to generalized fanconi syndrome. defective tubular reabsorption of proteins, mainly by the multi ligand receptors megalin and cubilin, is considered to be the cause of proteinuria in cystinosis. whether increased glomerular permeability contributes to proteinuria in cystinosis is investigated in this study by evaluating 1) urinary protein pattern in cystinotic patients and healthy controls 2) expression of megalin, cubilin and their ligands transferrin, albumin, a 1 -microglobulin (a 1 m) and b 2 -microglobulin (b 2 m) in renal tissue. methods: urine of cystinotic patients and controls (n=6), aged 1-16, were immunoblotted using antibodies against megalin, cubilin, transferrin, albumin, a 1 m and b 2 m. additionally, urinary levels of igg, albumin, a 1 m and creatinine were measured. results are expressed as mg/mmol creatinine (median, range). presence of proteins in semithin paraffin sections from cystinotic and control kidneys was evaluated using antibodies mentioned before. results: cystinotic patients had increased urinary excretion of , p 90 <10) and all tested ligands of megalin and cubilin. immuno histochemistry showed comparable expression of megalin, cubilin and their ligands in convoluted proximal tubules (pt), while the ligands in straight pt were only present in cystinotic patients. conclusion: a selective proteinuria with high molecular weight protein excretion such as igg indicates increased permeability of glomerular filtration barrier in cystinosis already at an early age. the presence of the megalin and cubilin ligands in endocytic vesicles suggests functional endocytosis. however, the enhanced staining of the ligands in cystinotic straight pt may be a result of incomplete reabsorption in convoluted pt. background: dent's disease and lowe syndrome are the most frequent x-linked tubulopathies. dent's is characterized by lmw proteinuria, hypercalciuria and nephrocalcinosis. in ca. 60%, this phenotype results from mutations in the clcn5-gene. lowe syndrome (congenital cataract, mental retardation and generalized fanconi syndrome) is due to mutations in the ocrl1 gene. stunted growth is another typical finding in lowe patients. recently, in a subgroup of dent patients ocrl1 gene mutations have been demonstrated (dent-2 disease). aim of the study: comparison of the growth pattern of patients with clcn5 and ocrl1 mutations. patients: boys with proven mutations in clcn5 (n=25, mean age 13.8±9 yrs) were compared with those with dent-2 disease (n=11, 9±4 yrs) and those with ocrl mutations and a lowe phenotype (n=6,14.9±7.7 yrs). comparison of z-scores for height, weightand bmi. results: clcn5 positive boys had a significantly higher height-sds (-0.87±1.4) than ocrl1 positives (dent-2: -2.18±1.1/lowe: -3.46±1.0). there were no significant differences in bmi-sds and weight-sds. the difference between weight and height sds as a parameter for obesity in these small-statured children was higher in lowe than in classical dent patients, with intermediate values being found in dent-2. discussion: although the renal phenotype of dent-2 patients is identical with classical dent, the former are more stunted. therefore, the abnormal growth pattern in dent-2 patients cannot be ascribed to renal dysfunction. taken together with other findings (elevated ck and ldh, mild mental retardation) our findings illustrate that dent-2 is indeed a mild variant of classical lowe syndrome. quantitative ultrasonometry of the calcaneus in children with idiopathic hypercalciuria lesch-nyhan syndrome is a very rare x-linked recessive disorder characterized by mental retardation, spasticity resembling cerebral palsy, choreoathetosis, self-mutilation and hyperuricemia. self-mutilation behavior is a hallmark of the disease. hyperuricemia leads to hyperuricuria and uric acid nephrolithiasis. we report on a 7-year-old boy with lesch-nyhan syndrome with no self-mutilation behavior who was erroneously diagnosed as having athetotic cerebral palsy. besides, he had no renal stones, the only renal abnormality detected were hyperechoic renal medullary pyramids, sonographicaly indistinguishable from medullary nephrocalcinosis. bone disease is frequently observed in children with homozygous beta-thalassemia (thal). we have observed an increased prevalence of renal stones in these patients. in order to understand the cause of this predisposition to renal stone formation we investigated markers of bone metabolism in our thal children. we studied 23 thal children (age range 3-16 years; 8 females and 15 males) with no eviodence of renal stones. thal was diagnosed with haemoglobin hplc study and genetic typing. all received blood transfusion and iron chelants on a regular schedule. serum levels of pth, osteocalcin, telopeptide c-terminale (cross-laps) were determined with eclia technique; serum vitamin d (25ohd3) with elisa technique. serum calcium, phosphate, uric acid, bicarbonate, creatinine, alkalin phosphatase and sodium, calcium, oxalate, citrate and creatinine in 24 hours and fasting urine were determined with common methods. as controls we studied 30 children with comparable age. serum pth and vitamin d were increased in 28% of our patients, serum osteocalcin in 64% and telopeptide c-terminale (cross-laps) in 92%. hypercalciuria was observed in 26%, hyperoxaluria in 16% hypocitraturia in 11%. significant correlation were found between pth and osteocalcin (p<0.01) cau/cru (p<0.05); osteocalcin and cross-laps (p<0.005) and vit d and cau/cru (p<0.05). bone disruption due to bone marrow expansion may produce an increase in vit d and pth production with hypercalciuria producing renal stones. losartan is an angiotensin ii subtype 1 (at1) receptor antagonist used for controlling blood pressure and urinary protein excretion in patients with hypertension and chronic renal disease. there are a few reports about the clinical implications of at-1 receptor antagonism that may interfere with the kidney's defense against an acid load and may thereby exacerbate metabolic acidosis in the literature. the suggested mechanism is that at-1 blockade by losartan exacerbates acidosis by inducing a distal-tubular acidification defect.we observed metabolic acidosis and hyperkalemia in five patients (3 females/2 males, age ranged 5-17 years) whom were given losartan. during the second week of the therapy all patients revealed a metabolic acidosis with (ph; ranged 7,19 to 7,33 and hco3; ranged 11,9 to 17 mmol/l) hiperkalemia (ranged 5,6 to 6,7 mg/dl). the etiologies of chronic renal disease in the patients were focal segmental glomerulosclerosis (fsgs) in one, lupus nephritis in one and three had undergone renal transplantation according to different etiologies. glomerular filtration rate was higher than 60 ml/sec/1,73 m 2 in all patients. immune suppressive regimen of renal transplanted patients was based on tacrolimus in two patients and on cyclosporine a in one. both renal transplanted patients and other two patients with fsgs and lupus nephritis were all receiving steroid, enalapril and mycophenolate mophetil at the same time during the losartan therapy. metabolic acidosis and hyperkalemia were recovered within a week following the exclution of the losartan. in conclusion, we think detailed and controlled studies are necessary to determine the pathogenesis of the metabolic acidosis due to losartan and patients must be followed up very closely for the adverse effects of metabolic acidosis and hyperkalemia during the treatment. objectives of study: bartter's syndrome is a rare renal tubular disorder characterized by hypokalemia and metabolic alkalosis. it is also known to be effectively treated with potassium supplement, potassium sparing diuretics and indometacin. we experienced two sibling cases whose problems were incompletely solved with above mentioned conventional treatment, but rather completely with adjunctive therapy of regular hemodialysis with dialysate of low bicarbonate concentration. case 1: this male patient was diagnosed of bartter's syndrome when he was 3 months old. he was treated with potassium supplement, aldactone and indometacin with marked improvement. but he still had some problems of retarded growth, severe headache and episodes of marked hypokalemia which needed repetitive admission. when he was 10 years old, we put him weekly hemodialysis with dialysate of low bicarbonate concentration (20 meq/l). with hemodialysis, he has been in good condition for 2 years with stable blood ph and serum potassium levels. case 2: this female patient, the elder sister of case 1, was diagnosed of bartter's syndrome at one year of age. with those therapy on conventional medications, she seemed to grow out of failure to thrive, but needed repeated admissions due to episodes of dehydration and hypokalemia. after start of weekly hemodialysis with low bicarbonate dialysate, for one year she has been on good control of blood ph and serum potasium level, and was never admitted with those episodes. conclusion: regular hemodiaysis with dialysate of low bicarbonate concentration can be considered as effective adjunctive therapy in intractable bartter's syndrome. a. deguchtenaere, a. raes, j. dehoorne, c. vande walle, r. mauel, j. vande walle university hospital gent, pediatric uro-nephrologic center, gent, belgiummonosymptomatic enuresis nocturna (mne) may be associated with nocturnal polyuria (np) and low urinary osmolality during the night. besides vasopressin, recent studies have stressed the possible role of renal sodium-handling, hypercalciuria, prostaglandins and/or osmotic excretion.the aim: was to study circadian rhythm of gfr and diuresis in a highly selected group of children with persistant np. methods: population existed of 15 children with mne and np, age 9-14 y, 9 males. controls n=25 children, 9-16 y with mne, but no np. (=b). renal function during 24 h concentration-prophyle, with timed urine samples, and measurement of p and u for na, k, osmol, creatinin. calculation of gfr by creatinine, uosmol, feosmol, diuresis vol (ml/min), fena, fecl, fek, u k /u k +u na %. statistics: paired t-test p<0.05 between d and n, unpaired t-test, between the 2 groups. conclusion: children with nocturnal polyuria have not only lost their circadian rhythm of diuresis and sodium-excretion but also of gfr. another observation for a reanl involvement in mne. sarcoidosis is a systemic disorder of unknown etiology, rare in children, characterized by the presence of noncaseating granuloma in affected organs. we report a 12-year-old boy of french african origin who presented with left hearing loss followed by bilateral deafness within 5 months. a history of bilateral uveitis was secondarily unveiled. mild renal insufficiency (creatinine level 130 μmol/l ; clearance: 68 mmol/min) was diagnosed prior to cochlear implant surgery. a percutaneous renal biopsy evidenced a granulomatous interstitial nephritis with widespread interstitial fibrosis. complementary explorations showed elevated lysozyme activity at 25 μg/l (normal <10) with elevated cd4/cd8 ratio in bronchoalveolar lavage specimen. pulmonary function test was notable for mild diffusion impairment. cerebral mri demonstrated abnormal enhancement involving the periventricular white matter and the intracanalicular portions of both viii cranial nerves. cerebrospinal fluid showed abnormal hyperlymphocytosis (12 lymphocytes per mm3) while protein was normal. three weeks after admission, bilateral uveitis recurred and was cured by local steroid therapy. despite intensive treatment with intravenous prednisone 1 g/1,73 m 2 /j per day, 3 successive days per month associated with oral prednisone during 6 months, glomerular filtration rate did not improve. in conclusion, sarcoidosis may apparently be revealed by acute bilateral deafness, and prompt diagnosis is needed to avoid permanent lesions. 1 gfr cystcin=91.869cystc -1.1227 , 2 formgfr=38*height (cm)/s-creat (mmol/l). d difference between c inulin and tested method. conclusion: none of the tested methods seems to reveal hyperfiltration in type 1 diabetes patients as clearance of inulin. the best correlation was found to clearance of iohexol and second best gfr estimated by 100/cystatin c. creatinine clearance overestimates and formula clearance underestimates gfr in diabetic patients without nephropathy. objective: to study the effects and mechanism of fty720 on the renal interstitial fibrosis in unilateral ureteral obstructic rats. methods: fouty-five males sd rats were randomly divided into sham-operated (sham), unilateral ureteral obstruction (uuo) and uuo treated with fty720 (uuo+fty720) group. 0.5 mg.kg -1 .d -1 of fty720 or vehicle was administrated through daily gavage and begun from two days before the operation till being sacrificed. 24-hour urine protein, blood urea nitrogen and plasma creatinine were determined. the renal tubular interstitial fibrosis lesion and the expression of α-sma,col-i, cd3, ed1 were scored semi-quantitively. results: the amount of 24 hours urine protein was much lower than that in uuo group, (p<0.05). serum creatinine in fty720 treated group were significantly lower than those in uuo group (p<0.05). the scores of renal interstitial fibrosis were lower in fty720 group than that in uuo group. α-sma expression was limited to vessels in sham group, but extended to renal tubule and interstitium in uuo and fty720 treated group, while relatively weaker expression was observed in fty720 group than in uuo group. some collagen expression was found in sham group, which was much enhanced in uuo group and mainly distributed in renal interstitium, the expression in fty720 group was also increased compared to sham group, but much lower than that in uuo group. obvious lymphocyte and macrophage infiltration were found in tubular interstitial area in uuo group but significantly less in fty720 treated group (p<0.01).conclusion: novel immunomodulator fty720 can obviously inhibit renal interstitial lymphocyte and macrophage infiltration, renal tubule cell transdifferentiation, and interstitial fibrosis, thus prevent renal disease progression. background: the mesangial cell, especially as a fundationtal component in normal mature glomeruli, is essential to keep glomerular capillary lumen open and to maintain efficient ultrafiltration. loss of mesangial cells due to pathologic conditions such as glomerulonephritis leads to impaired renal function. the exact developmental origin of mesangial cells is unknown. it has been established that mesenchymal stem cells, which are derived from bone marrow, have a potential to differentiate into different lineages in response to different environments. the purpose of the study is to examined the effect of platelet-derived growth factor (pdgf) in the differentiation of bone marrow-derived cells into mesangail cells. method: isolated bone marrow cells were cultured in the medium containing collagen type i within 24 hours, and then transferred to collagen type i.-coated dishes. the cells attached to collagen type i. in the following 7 days were maintained in the differentiation medium containing 2% horse serum, 200 μmol/l, and 1 μmol/l of pdgf and all-trans retinoic acid. results: after cultivation under the above condition, approximately 10% of cells expressed β actin and desmin, which highly resembled cultured mesangial cells in rat. the induced cultured cells changed into a wide range of shapes from spindle to stellate. the results indicate that bone marrow-derived stem cells could differentiate into mesangial-like cells in vitro. key: cord-010119-t1x9gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober 7‐10, 2017 date: 2017-09-04 journal: transfusion doi: 10.1111/trf.14286 sha: doc_id: 10119 cord_uid: t1x9gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna1 blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks 1, 3, 6, 12 and 24 following index donations from 50 donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna1 samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to 3 months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by 3 months. urine and saliva detection decreased significantly after 2 weeks and was undetectable by 3 months. of donors who were enrolled in the acute pre-seroconversion stage of infection 65% (15/23) developed multiple zikv related symptoms 1 week post index donation, compared to only 30% (7/27) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* 1 , yuelong guo 2 , ritchard g. cable 3 , joseph e. kiss 4 , michael paul busch 5 , grier page 2 , stacy endres-dighe 2 , steve kleinman 6 , simone glynn 7 , alan mast 8 and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) 9 . 1 american red cross, 2 rti international, 3 american red cross blood services, 4 blood systems inc., 5 blood systems research institute, 6 university of british columbia, 7 nih/ nhlbi, 8 blood research institute, 9 nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over 12,600 donors were enrolled from 4 us blood centers for ferritin testing. the study population was enriched for racial minorities [1600 african-american (aa), 1600 asian (as), 1000 hispanic (hisp)] and for "super donors" (1600, who had completed 101 donations in two years without low hemoglobin deferral). the minority donors and the remaining 6800 non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml). results/findings: across all subjects, 19% had ais and 42% had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all 4 groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > 50 years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed %20% decreased risk for ais compared to nhw, while hisp donors had 25% higher risk. daily use of exogenous iron reduced risk for lf and ais by 30 to 40%, respectively, while the estimated benefit from less-than-daily use was lower (5 to 19% protection). regular use of antacids was associated with a 20% or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by %15-20%, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean (6sd) *p < 0.05 compared to batf31/1, 200ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to 25% of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d1 and d14 of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration (62% on d14 vs 100% on d1 of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized (74%) and macrophage-depleted (79%) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than 50% at 2h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at 2h posttransfusion. at 24h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* 1 , amanda l richards 2 and krystalyn e hudson 2 . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod 1 otii 1 mice are predisposed to have autoreactive cd4 1 t cells. study design/method: four cohorts of hodxotii f1 mice (16-48 mice/ cohort) were bled monthly for 15 months to assess for autoab production. peripheral rbcs were stained with anti-complement (c3) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd71 and ter119 to assess for the presence of rbc progenitors. statistical analysis between hod 1 otii 1 autoab 1 vs. hod 1 otii 1 autoabvs. hod -otii 1 was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd4 1 t cells were not deleted in the thymus of hod 1 otii 1 mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod 1 otii 1 . however, as they aged, 15-50% of hod 1 otii 1 were positive for rbc autoab by 6 months. thereafter, $50% of the autoab 1 mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in 3 of the 4 cohorts, 60-100% of autoab 1 mice were female. hod 1 otii 1 autoab 1 mice also had enlarged spleens compared to hod 1 otii 1 autoaband hod -otii 1 mice (0.42g vs. 0.21g and 0.14g, resp., p<0.04). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd71 hi ter119 inter ) were observed in the spleens of hod 1 otii 1 autoab 1 mice but not in hod 1 otii 1 autoaband hod -otii 1 (2.86% vs. 0.06% and 0.05% resp., p<0.03). moreover, autoab and c3 deposition were found (0.1-2% and 3-10%, resp.) on ter119 1 rbcs in all of the hod 1 otii 1 autoab 1 mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b5-a01a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards 1 , christopher a tormey 2 and krystalyn e hudson* 1 . background/case studies: red blood cell (rbc) alloimmunization occurs in up to 10% of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c57bl/6 (b6) mice were treated with pbs, or anti-ly6g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at 18-24 hours post-transfusion. anti-hod alloantibody generation was assessed 14 days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n55), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n55). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls (3/3 experiments, p<0.01). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio1 leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l1), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p<0.05); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b9-a03b cxcr5 1 pd1 1 and ccr7 1 expressions characterize responders to rbc immunization benoît vingert* 1,2,3 , marie tamagne 1,2,3 , sadaf pakdaman 1,2,3 , anoosha habibi 2,3,4 , philippe bierling 1,2,3,4,5 , rachid djoudi 1 and france pirenne 1,2,3,5 . 1 efs ile de france, 2 laboratory of excellence gr-ex, 3 imrb u955 -eq2, 4 ap-hp, 5 universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd4 1 t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd4 1 t cells have a th17 profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd4 1 t cells with a cxcr5 1 pd1 1 phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr5 1 pd1 hi profile, with a differentiated expression of ccr7. ccr7 is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr6 and cxcr3 can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr5 1 pd1 1 lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr5 1 pd1 1 cells were compared in 2 groups of transfused sickle cell patients : alloimmunized (n514) and non-alloimmunized patients (n510). the analysis was also performed in non-transfused healthy controls (n512). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr5 1 pd1 hi subpopulation expression was identical between transfused groups and controls. ccr6 and cxcr3 expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr7 expression was very strong independently of the expression of pd1. in the aim to determine the help of the circulating cxcr5 1 pd1 1 cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for 5 days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr5 1 pd1 1 subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr5 1 pd1 1 cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr7 on circulating cxcr5 1 pd1 1 cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr5 1 pd1 1 profile and the ccr7 1 expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd4 t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* 1 , ashley bennett 1 , kathryn girard-pierce 1 , connie arthur 1 , amanda mener 1 , patty zerra 1 , christopher a tormey 2 , jeanne hendrickson 3 and sean stowell 4 . 1 emory university, 2 yale-new haven hospital, 3 yale university, 4 emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd4 t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd4 t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b6 recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod 1 kel). to examine the role of cd4 t cells, pic/kel primed b6 recipients were cd4 t cell depleted prior to transfusion. in addition, b6 recipients were adoptively transferred with cd4 t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd4 t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < 0.001); pic/kel primed recipients transfused with (hod 1 kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd4 t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < 0.0001) and transfer of cd4 t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < 0.001). conclusion: these results demonstrate that cd4 t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd8 t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within 24 hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd8 t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd8 t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h2kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c57bl/6) mice or oti mice, whose cd8 t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h2kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes 48 hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after 2, 4, 8, 16, and 24 hours and on days 2-5. results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after 8 hours and peaking at 24 hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is 28-35% versus >60% in wt recipients at 24 hours (p<0.05), whereas transfusion of wt platelets into either oti or wt recipients is approximately 60% at 24 hours after transfusion. adoptive transfer of oti cd8 t cells into wt mice recapitulates the effect, with significant mova platelet clearance at 24 hours compared to wt platelet clearance (p<0.05). conclusion: this work extends the ability of cd8 t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv1r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv1r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv1r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c57bl/6 mouse blood and treated with uv1r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv1r treated wbc-rich prp, or uv1r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c57bl/6 donor blood. a second transfusion of untreated wbc-rich c57bl/6 prp was given 2 weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv1r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p<0.01) or necrotic (p<0.05) wbcs, but not those given uv1r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c57bl/6 wbcs were reduced in recipients of either uv1r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv1r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv1r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so 2 ) of venous blood is generally assumed to be around 70-80% as measured from a central venous line. however, a recent investigation of so 2 levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so 2 distribution (mean 45.9%617.5% [yoshida et al. 2017; blood transfusion 15, 172] . the present study was undertaken to determine the distribution of so 2 in lr-rcc produced at a medium-size blood center using a novel non-invasive so 2 probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so 2 levels. study design/method: the so 2 from 977 units of lr-rcc were examined on five consecutive days representing 78% of the collected units during the period at a regional blood center where all the units were processed at room temperature within 8 hours of blood collection. so 2 was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a3u11; pendar technologies, cambridge ma). in addition to so 2 , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n54) from human volunteers were stored in as-3 under normoxic, hyperoxic, or hypoxic conditions for up to 42 days (so 2 ranging from <3 to >95%) prior to uhplc-ms metabolomics analyses in presence of 13 c, 15 n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so 2 carried out non-invasively at a blood center yielded a similar wide distribution as previous study from 497 units of lr-rcc procured and sampled invasively within 24 hours after blood collection [yoshida ibid]. the shape of the so 2 distribution appeared near normal with the mean of 47.0%621.0%, median 45.2%, range < 5% to > 95% and inter-quartile range (iqr) of 31.4%-61.9%. male donors showed higher so 2 compared to female donors (p<0.04). no correlations were observed between so 2 levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so 2 levels ameliorate the energy and oxidative metabolic lesion. lower so 2 levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp 1 ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so 2 levels was observed from lr-rcc manufactured at a blood center using 8-hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a 3-fold increase in the absolute lymphocyte count (ko 7.59 6 4.63 x10 9 /l vs. wt 2.90 6 1.32 x10 9 /l, p 5 0.0303), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko 6.00 6 0.29 fl vs. wt 5.24 6 0.56 fl, p50.0140). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd81foxp31 regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd8 1 foxp3 1 tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd8 1 foxp3 1 tregs derived exosomes and their functions involved in cd8 1 tregs mediated immune-modulation were seldom reported. study design/method: cd8 1 t cells were freshly purified from pbmcs, cultured with anti-cd3/cd28 antibody packaged beads and il-2, and then polarized with tgf-b and rapamycin into cd8 1 foxp3 1 treg cells. the harvest cells were co-cultured with cd3/cd28 beads stimulating cd41cd25effector cells in the transwell plate. the supernatant derived from cd81 tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd63, cd81, tsg101 and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir-155, let-7b, let-7d were measured by qpcr. the precipitated exosomes were further purified by cd63 immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd8 1 treg cells could suppress the proliferation of effector cells with a small decline (p>0.05), which means some non-contact factors involved in the cd8 1 treg mediated immune modulation. a total number of 4.57 6 0.52 3 10 8 /10 6 cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle 50-150nm in diameter (145.16 6.7nm by nta). cd63 and cd81 were expressed on these background/case studies: regulatory t cells (tregs), containing cd4 1 and cd8 1 subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd4 1 foxp3 1 regulatory t cells (ntregs) in inflammation conditions (including instability of foxp3, conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd81 regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd8 1 treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd8 1 regulatory t cells in inflammation and transfusion. study design/method: human cd8 1 tregs were induced with tgf-b1 and rapamycin from cd8 1 t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd8 1 tregs when encountering with inflammation were test by foxp3 expression, th1 and th17 cells conversion in inflammations conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6) on day3, day6 and day9. in vivo, cd8 1 tregs were transfused into cia mice and then their survival in mice and foxp3 express were evaluated to reveal the stability of cd8 1 tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd8 1 tregs when induced factor tgf-b1 and rapamycin were removed by testing the foxp3 expression on day3, day6 and day9. results/finding: ex vivo induced human cd8 1 treg were foxp3 1 (90.40 6 1.40%) and did not secret il17a (both in supernatant and % of cells). foxp3 express in cd8 1 tregs were maintained after induced factor tgf-b1 and rapamycin were removed on day3, day6 and day9. in vitro, foxp3, il2 and ifn-c expression has no significant difference when compared with controlled tregs on day3, day6 and day9 and did not secret il17a when encounter with inflammation conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6). in vivo, cd81 treg cells were transfused into cia mice on the peak of disease onset (35 days after the first collagen immunization, has inflammation condition in vivo) to test cd8 1 tregs survival. cd8 1 tregs were found in cia mice foot (27.4 6 2.03%), blood (4.55 6 1.03%) and spleen cells (1.90 6 0.05%) 72 hours after transfusion and their % of foxp3 1 were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd8 1 tregs are stable in inflammation and transfusion and can maintain foxp3 expression when induced factor were removed, these make cd8 1 treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s6k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd83/cd80/cd86 expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p70s6k and its downstreanm proteins, especially the protein s6, which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s6 related protein translation inhibition. xiaoyun fu* 1,2 , mikayla anderson 1 and james c zimring 1,2 . 1 bloodworksnw research institute, 2 university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in 405 leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day 43 (one day past their expiration). 35 bioactive lipids including 10 common fatty acids, 10 oxylipins, and 15 lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in 405 stored rbc units. for example, arachidonic acid (aa) ranges from 0.5 -10.7 mm, linoleic acid (la) (1.4-28 mm), dihomo-c-linolenic acid (dgla) (0.1-0.8 mm), eicosapentaenoic acid (epa) (0.03-3.1 mm), docosahexaenoic acid (dha) (0.2-3.0 mm), and alpha-linolenic acid (ala) (0.06-2.3 mm). ten oxylipins including hetes, hodes, and dihomes, and 15 lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of 35 analytes quantified, 25 showed a significant difference in concentration among different blood types by one-way anova testing (fdr<0.05). the ab rh1 blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh1). the fold increase of o rh-/o rh1 among pufas ranges from 1.3 to 2.1, suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among 405 stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna-223 was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna-223 targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps (49 6 6 nm) surrounded by a thick fluorescent silica shell (22 6 2 nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r 2 rnaprobe 5 0.96 and r 2 dnaprobe 5 0.98) with mirna-223 concentration, down to a 10-nm limit of detection. hybridization assays in 1% human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in 1% human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. 6.7 6 0.0 a 6.9 6 0.0 6.7 6 0.0 a 6.6 6 0.0 6.7 6 0.0 a 6.9 6 0.0 total atp,lm/ghb 7.6 6 0.3 a 5.2 6 0.3 7.3 6 0.2 a 5.5 6 0.3 7.3 6 0.4 a 4.9 6 0.5 extracellular lactate,mm 5 6 1 a 6 6 1 7 6 0 8 6 0 6 6 0 6 6 1 extracellular glucose,mm 32 6 1 a 50 6 3 2 9 6 1 a 38 6 1 2 5 6 0 a 28 6 1 extracellular na 1 ,mm 142 6 2 143 6 2 138 6 1 a 137 6 1 144 6 1 141 6 1 extracellular k 1 ,mm 1 6 0 a 4 6 0 1 6 0 a 5 6 0 1 6 0 a 4 6 0 a p<0.05, paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso100201600009c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n56) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by 50% (63/12,492) compared to the previous year without pathogen inactivation (42/12, 931, p50.030, chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased (2/15,286 vs 10/ 13,488). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5.0x10 11 ) than unaffected controls (3.5x10 11 ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above 5.0x10 11 . in vitro quality of single dose amotosalen/uva treated platelets in 35% plasma/65% pas-3 after 5 days of storage crystal stanley 1 , marguerite kelher 2 , nero evero 1 , melissa vongoetz 3 , betsy donnelly 3 and anna erickson* 3 . 1 belle bonfils memorial blood center, 2 university of colorado, 3 cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas-3 to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct02298842) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas-3 and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in 35% plasma/65% pas-3, collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, 7.5 6 0.6 310 11 platelets in 602 6 52 ml, were collected on the trima apheresis platform in 35% plasma/65% pas-3. a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n56, were 302 6 26ml (t) and 300 6 27ml (c) with doses of 3.8 6 0.3 3 10 11 (t) and 3.7 6 0.3 3 10 11 (c). all pc were stored under the same conditions and evaluated on day 5 and day 7 for physical/metabolic characteristics. results/finding: on days 5 and 7 all t and c pc had ph228c !6.2. the dose recovery for t was 87%63%. on day 5, t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table 1) . conclusion: trima pc in 35% plasma/65% pas-3 treated with the inter-cept blood system for platelets using the sv set and stored for 5 days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan 1,2 , yang yu 1 , li-ping sun 1 , shu-fang wang 1 , rui wang 1 , lei-ying zhang 1 and deqing wang* 1 . 1 the department of blood transfusion, the pla general hospital, 2 the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: 1 five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d0, d14 and d35. we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in 17% final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. 2 levels of k and lactic acid (la) were tested using automatic biochemical analyzer.3 k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. 4 treated hips-cms with d35 ssrbcs, d35 k and cell culture media for 48h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: 1 d0 ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d14 ssrbcs stop beating, but beating patterns restored at 48h. hips-cms treated with d35 ssrbcs stop beating, and beating patterns did not restored at 48h. 2 levels of k and la in ssrbcs changed most obviously. 3 only d35 k solution made hips-cms stop beating and can restore in 48h; d0 k, d14 k and la solution did not influence the beating pattern in at the end of the treatment for 24h, hips-cms treated with d35 ssrbcs show obvious shrinkage. at the end of the treatments for 48h, cells treated with d35 k and d35 ssrbcs both show obvious shrinkage, the shrinkage in d35 ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. 5 gene expression array results show a total of 140 genes were differentially expressed in d35 ssrbcs group compared with naive group. there was no consistent separation within the d35 k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. 2 in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. 3 further study should be applied to signal pathways on ssrbcs induced cytotoxicity. 4 large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as-3 as compared to sagm. the presence of citrate in as-3 seems to be necessary to prevent hemolysis of thawed cells. during storage in as-3, atp and 2,3-dpg levels rapidly decline. recently developed additive solutions like pag3m and as-7 have shown to better maintain 2,3-dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag3c in which the mannitol of pag3m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at 2-68c in pag3c. study design/method: leukoreduced rcc (n56) in pag3c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at 2-68c. on day 8, rccs were glycerolized using acp215 (haemonetics v r , braintree, ma) to a final concentration of 40% (w/v), frozen and stored for at least two weeks at -808c. after thawing and deglycerolization using acp215, rcc were resuspended in pag3c. during storage at 2-68c, stability (hemolysis), atp and 2,3-dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n58) resuspended in or sagm (n54). results/finding: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels at day 8 as compared to storage in sagm, resp. 9.1 6 7.6 mmol/g hb and 1.9 6 0.7 mmol/g hb. hemolysis during post-thaw storage in pag3c remained below 0.8% for 35 days and was comparable with storage in as-3. in sagm, hemolysis remained below 0.8% for 7 days. during the first 2 weeks of post-thaw storage in pag3c, both atp and 2,3-dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag3c showed significantly higher atp and 2,3-dpg levels compared to as-3 or sagm. while in sagm and as-3, 2,3-dpg levels were undetectable after 7 days post-thaw storage, in pag3c, 2,3-dpg levels only decreased to 6.1 lmol/g hb after 35 days of storage. conclusion: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels. as compared to as-3, post-thaw storage in pag3c showed comparable hemolysis while atp and 2,3-dpg levels were much better maintained. based on a maximum allowed hemolysis of 0.8% and an atp content of >2.7mmol/g hb, thawed rcc can be stored at 2-68c for 35 days in pag3c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold (48c, 4c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only 2 apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n54-5) in 65% iso using a trima or in 65% int using an amicus and stored for 15 days at rt and 4c. samples were tested on day 1 (baseline, bl), 5, 10, and 15 of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at 40% hct. labeled blood was perfused through microfluidic channels (fluxion) coated with 100 ug/ml type-1 collagen at 720s -1 shear rate. images were acquired every 30 sec for 10 min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p<0.05. results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day 5 of storage compared to bl (bl: 11.6 6 1.7%, rt: 4.9 6 1.2%; p<0.005). 4c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, 4c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day 5 (p50.03) and compared to 4c-int by day 10 (p<0.01). conclusion: our work suggests that 4c storage of plts collected with a trima ap system in iso for up to 15 days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at 4c. these results are surprising since both 4c-int and 4c-iso have been shown to express similar levels of cd62p, pac-1, and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at 4c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life 3x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table 1. comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after 2-week storage between unirradiated and irradiated groups (n 5 29) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone (10mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin (250mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th17 cells and increase in cd4 regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th1,th2,tfh and tfr cd4 subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd4 positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* 1 , sarah kloss 1 , sara crew 1 and sandra j nance 2 . 1 american red cross, 2 american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd109 have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to 28 unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only 510k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from 40 plasma and serum clinical specimens. group 1 contained a single hpa alloantibody specificity with or without hla antibodies (n526). group 2 included 5 specimens with hla antibodies alone and group 3 consisted of 9 patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa-1, hpa-2, hpa-3, hpa-4, hpa-5, gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is 100% concordance observed for hpa-1a, hpa-1b and hpa-5b antibodies. the pak-plus assay had difficulty discriminating hpa-5b from hpa-5a antibody when hpa-5a antibody was present (3 false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa-5a when compared to hpa-5b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within 10% of the cut-off for pakplus and <2.0 adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb231 cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac6 specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut1 overexpression on cell motility. results/findings: fut1 overexpression increases both ley expression and cell migration, while fut1 knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut1 overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac6. as tumor promoter, hdac6 becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac6 function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at 48c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n53) and stored at 48c for up to 10 days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and 30 minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd62p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means6sem, and paired student's t-tests were used to determine statistical significance (p<0.05). results/finding: on day 10, p-selectin levels were significantly higher in pre than bl (p50.03). mirasol treatment caused a significant increase in pac-1 expression compared to pre (pre: 10.5 6 3.1%, post: 28.1 6 4.7%; p50.04), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post-30 samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after 10 day storage at 48c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, 48c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day 10 4c-stored pas plts followed by incubation (30 minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* 1 , arthur p. bode 1 , anne s hale 2 , michael stanton 3 , mark johnson 4 and g. michael fitzpatrick 3 . 1 cellphire, 2 bodevet, inc, 3 cellphire, 4 background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to 33, 10, and 3.3% of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control (2-day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the 4 hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to 33% of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of 10% and 33% of the tcpc produced a significant decrease in blood loss. the lcps at 10% and 33% tcpc were as effective in mitigating blood loss as 2-day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to 33% of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of 10% and 33% of the tcpc reduced blood loss. these results suggest a starting dose above 3.3% tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as 10% tcpc had similar efficacy signals as 33% tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso100201300021c. the study on pcr-ssp technique for the genotyping of cd36 329-330del.ac mutation and the genetic polymorphism of cd36 329-330del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd36 (platelet glycoprotein iv, scarb3) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd36 is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd36 deficiency in china. cd36 gene mutation is the main reason that leads to cd36 deficiency. cd36 329-330del.ac (frameshift at aa110) mutation is one of the cd36 mutations that causes cd36 deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in 28 pediatric patients who received both doses of the mmr vaccine at 12 and 18 months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are !90% for all mmr components. results/finding: table 1 shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged 6.8 years (0.5 to 16.5 years). thirteen patients (46%) were chronically transfused at the time of serology. twenty-three patients (82%) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to 6 months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper 1,2 , franklyn cladis 2 , richard saladino 2 , darrell triulzi 3 , barbara a gaines 2 and mark yazer* 1 . 1 university of pittsburgh, 2 children's hospital of pittsburgh of upmc, 3 institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients !3 years old and !15 kg with evidence of hemorrhagic shock were eligible to receive up to 20 cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (<50) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately 11 months, 15 trauma patients received wb: 7 group o and 8 group a recipients, 53% male, median (iqr) age was 11 (4.5-14) and 73% blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of 36 (22-51) and 47% mortality rate. the median (iqr) quantity of wb transfused to group o recipients was 21.9 (14.8-24.3) ml/kg versus 13.4 (9-18) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean 6 standard deviation haptoglobin concentrations for non-group o recipients was 51.3 6 14.4 mg/dl on day 0, 86.3 6 36.8 mg/dl on day 1, and 126.9 6 45.8 mg/dl on day 2; the corresponding haptoglobin concentrations for group o recipients were 51.4 6 38.0 mg/dl, 84.7 6 61.5 mg/dl, and 134.8 6 68.3 mg/dl, respectively (p>0.42 for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n57) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n514). the mean 6 standard deviation platelet volume administered was 112 6 24 cc for whole blood recipients versus 147 6 68 cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to 30 ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) 41 with anti-human igg only, and a 31 positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a 60 minute 378c incubation, followed by 3 automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than 3 days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer 4096 and the breast milk showed anti-d with a titer between 16 and 64. the patient had a consistent plasma anti-d titer of 8. the patient's mother chose to stop breast feeding after 8 weeks, and the patient's hemoglobin was improved at 12 and 16 weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that 30% of c1 scd patients from the west indies and west and central africa are partial c and at (30%) risk for alloimmunization to the c antigen through transfusion of c1 rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce(733g,1006t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce(4-7)-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of 255 patients with genotype/rh phenotype data available, 78 (30.6%) were c antigen positive serologically. the allele frequency of rhce*ce(733g,1006t) was 0.071. in total, 15 (5.9%) patients possessed rhce*ce(733g, 1006t) in the absence of conventional c gene in trans. of the 78 c antigen positive patients, 15 individuals (19.2%) were predicted to be partial c based on four molecular profiles [rhce*ce(733g, 1006t)/rhce*ce:12; rhce*ce(733g, 1006t)/rh*ce:1; rhce*ce(733g, 1006t)/rh*ce(733g):1; rhce*ce(733g, 1006t)/rh*ce(733g, 1006t):1]. in these 15 partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after 60 transfusion exposures (57 c-antigen negative units; mean: 4, range: 0-36), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce(733g, 1006t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* 1 , megan l nguyen 1 , melanie c proctor 1 , david e krysztof 1 , gregory a foster 1 , erin k sash 1 , sandy s dickson 1 , joua yang 1 , jeffrey m linnen 2 , kui gao 3 , jaye p brodsky 4 and susan l stramer 1 . 1 american red cross, 2 grifols diagnostic solutions inc., 3 grifols diagnostic solutions, inc, 4 quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and 4 suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on 6/20/16 (fl, ga, sc, ms, al). following revised guidance on 8/26/16, testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on 12/12/16. travel history questions were discontinued on 1/23/17. confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of 4/8/17, 2,288,855 donations were tested including 393,713 (17%) in 24,611 mps. no reactive donations were identified by mp-nat. of the 1,895,142 id-nat donations, 72 were initial reactive (ir) of which 8 (11%) confirmed positive (cp) by subsequent testing (cp rate of 1:286,107; positive predictive value of 11%; specificity of 99.997%). five (62%) cp donations were id-nat repeat reactive (rr); 3 (38%) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and 3 in fl, 2 of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from 2 to 73 days prior to donation. two donors with a travel risk reported clinical symptoms; 6 cp donors (75%) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than 40 copies (c)/ml to about 8ê 5 c/ml. at the time of writing, the longest period of detection in rbcs was 91 days vs. 17 days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from 1 ir and all rr donors, ranging from 12 to 2000 c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat1 samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of 6 were prepared by diluting nat1 plasma 1:6 and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n5308) were sorted into 4 categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm1/high vl; igm1/low vl. results/finding: of 52,942 donations collected april 3-december 31, 352 were reactive for zikv rna. igm-index donations had higher vls (mean 1.1 x 10 6 vs 8.3 x 10 4 iu/ml) and higher proportions of simulated mp-detectable results (93% vs 23%) than igm1 donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm1 donations increased (table 1) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the 2016 pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r 6800/8800 systems lisa lee pate* 1 , phillip c williamson 2 , michael paul busch 3 , susan rossmann 4 , scott jones 5 , ann butcher 1 , john duncan 1 , jean stanley 1 and susan a galel 1 . 1 roche molecular systems, inc., 2 creative testing solutions, 3 blood systems research institute, 4 gulf coast regional blood center -sugar land, 5 qualtex laboratories background/case studies: in february 2016, the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march 30, 2016 and testing of puerto rico donations began on april 3, 2016. as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august 2016, the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april 3, 2016 -february 28, 2017 using the investigational cobasv r zika for use on the cobas v r 6800/8800 systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a 1:6 dilution to simulate mini-pool testing. results/findings: a total of 1,776,190 blood donations were screened using cobasv r zika. of 56 ir donations, 12 were repeat reactive (rr), 39 non-rr and 5 had no repeat testing. of the 12 rr donations, 7 were positive by altnat; 3 of these were igm positive. all 4 altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the 5 rr donors that were not igm positive on index, 3 enrolled in follow-up and all seroconverted. of 39 non-rr donations, 38 were altnat negative and 1 is pending supplemental testing. 8/38 donors were igm positive on index. 30 donors were igm negative on index; 15/30 enrolled in follow-up; 14 remained igm negative and 1 was 1gm inconclusive. of 5 donations without repeat testing results, 2 met criteria for positive (1 was altnat positive, igm negative and 1 altnat negative, igm positive). 1 donation is pending additional testing. altogether, 22/56 ir donations met the criteria for true positive on the index donation. 9/22 (41%) true positive donations were reactive when retested in a simulated minipool. 16/22 were igm positive. conclusion: 0.001% of the 1,776,190 donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* 1 , marc germain 2 , gilles delage 3 , maria esther lopes 1 and yves gr egoire 3 . 1 hemorio, 2 hemaquebec, 3 h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia (2013) , and in brazil (2015/2016), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since 80% of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january 1 st , 2016 through november, 26 th , 2016, from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: 5 days, with 99% of the values lower than 18 days); 20% of infected donors with symptoms lasting 2 days; 1.2 donation/donor/year for wb and 1.75 for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x (1 -proportion of refused donors) x (1proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain 1:13,598, for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at 4 large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from 6 donations). the residual volume of mp plasma, 0.35 -0.45 ml, is routinely discarded. beginning in april 2016 each blood center saved $67 mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april 3-15) 3 mp6 were combined into pools of 18 donations; thereafter mp6 were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and 95% confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity (95% limit of detection <20 copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first 6 months of samples is complete for 6,292 mp, comprised of 37,752 donations collected from april 3 to october 9, 2016. a total of 77 pools were positive, with 76 detected between april-june 2016. the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over 0.6% of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over 0.4% of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in 2016, zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* 1 , sze sze chua 1 , mars stone 2 , michael paul busch 2 and ai leen ang 1 . 1 health sciences authority, blood services group, 2 blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on 26 august 2016. the numbers rose rapidly to 386 cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since 1 october 2016. zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using 300 blinded frozen samples consisting of 25 replicates of 11 half log dilutions of the who international standard for zikv and 25 replicates of negative controls prepared by bsri. probit analysis was performed to determine the 50% and 95% limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a 14 member blinded zikv reference panel from the usa-fda. results/finding: a total of 63,144 donations were screened from 1 october 2016 to 31 march 2017, with 1 false positive case and 1 zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of 9.54x10 5 copies/ml. zika igm was negative in the index donation sample but present in the 10-day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be 2.1 copies/ml at 50% lod and 10.0 copies/ml at 95% lod. the procleix zikv assay detected rna in 6 out of 9 patient samples and provided 85.7% agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with 1 confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of 1 in 25,888 donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types 1, 2 and 3 express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to 21 reactivity on initial gel testing. if genotyping demonstrated weak d types 1, 2 or 3, the intent was to manage the patient as rhd-positive. if weak d types 1, 2 or 3 were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in 2 to 4 weeks. results/finding: rhdgenotyping was performed on 22 patient samples over 15 months. of these 22 patient samples, 13 (59%) were weak d types 1 or 2. the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type 1 required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types 1 and 2 patients have not received transfusions at this institution since they were genotyped. four of 4 obstetric weak d types 1 and 2 patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this 15 month study period 13 serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using 3 fda approved anti-d reagents. when reactivity with all 3 reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over 80 rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of 8 months we tested 509 rhd-negative blood donors. there were 3 (0.6%) partial-d, 1 weak d (0.2%), and 3 (0.6%) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs5-46_42deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these 8 donors showed that 6 rhd-negative recipients received rbcs from 4 of these donors. five of these recipients underwent antibody screening after an average follow-up period of 5 months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, 5 grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which 63% of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d1" with 31 reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october 2016 to march 2017, we performed routine d typing (neo, immucor) on 1875 obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of 31 using at least 1 antibody. solid phase and manual testing used the series 4 and series 5 reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh1) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all 13 samples. two of 13 (15.4%) were d1 with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs1-29c, rs2301153; ivs3 1 117c, rs28521909; and ivs3 1 124a, rs28562109). two (15.4%) were d1 and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four (30.8%) patients had rhd alleles with known potential to make anti-d (rhd*dol2, rhd*dar1.2, and 2 with weak d type 4.0). one had weak d type 96, which has uncertain susceptibility to alloimmunization and one was weak d type 1, which has not yet been associated with anti-d. interestingly, two (15.4%) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type 2 is a variant of the rhd protein that comprises an amino acid substitution located in the 12th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c.1154 g>c which is the first nucleotide of the exon 9 of the rhd gene and thus could be implicated in exon 9 skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v6 (agilent) and the nextseq500 platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon 9 skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type 2 rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of 10 patients previously characterized by beadchip technology. interestingly, 4 out of 10 carry the c.1154-31c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last 4 patients, one has been previously characterized as rhd weak type 2 carrying the c.1154g>c (p.gly385ala). independently, sanger sequencing on 50 unrelated rhd weak type 2 samples pinpoint to a linkage disequilibrium between c.1154g>c (exac, maf 5 0.001145) and the c.1154-31c>t (exac, maf 5 0.2496). in silico analysis of both mutation located close to the splice acceptor site of the exon 9 does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon 9, mutant rhd c.1154g>c, mutant rhd c.1154-31c>t and double rhd mutants c.1154g>c plus c.1154-31c>t, we showed no influence on skipping of exon 9 due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type 2 between ala385 (transmembrane helix 12) and val183 (transmembrane helix 6) hampering membrane insertion. conclusion: the c.1154-31c>t variation is always associated in cis with the missense mutation c.1154g>c on the allele rhd weak type 2. the c.1154-31c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type 2 red blood cells is due to the substitution of alanine at amino acid position 385 to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has 10 transmembrane (tm) and 2 tilted ureapore a-helices, a long extracellular connector segment, and 2 cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p.280. we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and 2010-2016 aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, 2012) . results/finding: seven snmvs located within 1 amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all 3 at the exofacial ends (p.a93t, p.w240r, p.v333d) are jk-weak; the two jkneg exceptions p.g298e and p.g299e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, 13 snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v10m, p.e44k, p.v76i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the 13 jk-neg variants are within 19 aa (p.270-p.299) of jk a/b at p.280. none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n289s and p.s291p are adjacent to p.288f and p.292l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among 13 jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc14a1 gene, which encodes the urea transporter ut-b1. the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in 1965. in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc14a1 gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within 1 aa from tm a-helix end v76i, a93t, w171r, w240r, g298e, g299e, v333d* cytoplasmic n-terminal v10m, g40s, e44k, l45p in membrane tm and urea-pore a-helices r64w, r64q, g65d, i117t, a183v, l246r, a248t ‡, a270a §, l272f, n289s, s291p, t319m 2/10 * second nucleotide variant in this allele is synonymous (p.p196p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a 5 mbp region in 19q13.11-13.2 with an lod score of 9.6. using deep sequencing, we identified a potential deleterious mutation in the znf850 gene, which deletes 84 bp resulting in loss of an entire zing finger domain. the identical del84-znf850 mutation is present in all affected individuals, and is absent from all controls tested (n>2000). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf850 locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf850del84. none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc14a1gene, is a urea transporter that has been associated with renal function, we found that people with the znf850del84 in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf850, prevalent in southern spain due to a founder mutation, leads to ut-b1 dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-(2-ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis(2-ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at 30 days and 1 year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the 12 pools included 5 group a, 6 group o and 1 group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than -208c within 8 hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day 0 (pool), day 30, and 1 year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day 0, day 30, and 1 year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp 5 2.9 ppm; mehp 5 0.3 ppm; deht 5 0.9 ppm; and meht 5 0.2 ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than 80% of its initial value. plasma stored in deht bags had an average plasticizer content 90% lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at 100 g to separate rbcs from platelet-rich plasma (prp). prp was diluted 3-fold in pipes-saline with 1.4mm pge1 and centrifuged at 1900 g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of 34-40% and 150,000-250,000 platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted 1:1 (spdp50%) with plasma from a patient with type 3 vw disease (t3vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of 1600 s -1 for 180 seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was 20% (spdp/ffp > 0.8). results/finding: six batches of spdp/ffp were evaluated using 17 subjects. there was no statistical difference between the spdp/ffp pairs (p50.7558). the mean ratio of spdp/ffp was 1.21 with a 95% ci of 0.84 -1.57. comparing spdp vs. spdp50%, there was no difference (median ratio 5 1.045, range: 0.95-1.14) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was 20% greater than in samples reconstituted with ffp. the lower limit of the 95 th % ci is a difference of 16%, which is less than the a priori determined margin of noninferiority of 20%. even with 50% dilution with t3vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* 1 , qiyong peter liu 2 , grantham c. peltier 1 , ryan c. carney 2 , ashley s. taylor 1 , colby s. mcintosh 1 , james a. bynum 1 and andrew p cap 1 . 1 u.s. army institute of surgical research, 2 velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: (1) ffp; (2) ffp with 70mm glycine; (3) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; (4) spdp pretreated with glycine-hcl (20mm); and (5) spdp pretreated with glycine-hcl:glycine (20mm:50mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood (40% hct with 200 platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < .05). fibrin polymerization density was slightly diminished in rspdp vs. ffp (0.879 vs. 0.742 o.d., p < .01), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < 0.001). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < .01) and an additional twofold in pretreated spdps vs. rspdp (p < .05). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < 0.01). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples (71.53% surface coverage vs. 30.26-43.87%, p < .05). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* 1 , anita tuip-de boer 1 , ruqayyah almizraq 2 , jason p. acker 3 , philip j. norris 4 , jennifer a muszynski 5 and nicole juffermans 1 . 1 academic medical center, 2 university of alberta, 3 canadian blood services, 4 blood systems research institute, 5 nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from 8 donors (blood type a or b). supernatants were prepared after 4-5 (fresh) and 41-42 days of storage (stored) for measurement of thrombin generation and ev analysis. a549 type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to 25% stretch using a cellstretcher. control cells were not stretched. after 24 hours, il-8 and il-6 production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il-6 and il-8 production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p<0.05) . incubation of stretched cells with stored wbf products resulted in higher il-8 production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by 4 different methods from 5 individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after 4-5 days (fresh) and 41-42 days (expiry). monocytes were co-cultured in media plus 20% rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in 5 replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean 6 sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table 1) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin-8 production was higher after exposure to fresh wbf (248 6 115 % control, p 5 0.02) or wbd at expiry (292 6 111 % control, p 5 0.0005). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* 1 , alessandro tocchio 1 , anita howell 2 , kaushik sridhar 1 , jason p. acker 3 and utkan demirci 1 . 1 stanford university, 2 canadian blood services, centre for innovation, 3 background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of 10 -4 g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at 7, 14, 21, 28, 35 and 42 days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from 24 volunteers with four different age and sex categories (male, 18-40 years; male, >60 years; female, 18-40 years; female, >60 years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young (1.098 g/ml) and older female donors (1.109 g/ml) (p < 0.01). moreover, rbcs from young males (1.096 g/ml) were significantly less dense compared to rbcs profiled from older female donors (1.109 g/ml) (p < 0.05). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il-6 (pg/ml) il-8 (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: 120 icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of 426 rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p<0.001). substantial correlations were also found between orp and free hemoglobin (p<0.05) and orp and free heme (p<0.05). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections 132 6 10 vs 127 6 13 (p<0.05). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than 35 days compared to rbcs stored for 7 days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of 1 to 42 days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to 7 days storage duration -reference group), medium age (at least 1 rbc of 8-35 days storage), and oldest (at least 1 rbc greater than 35 days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for 7 days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every 7 days, and b) a finer partition using cut-points every 3 days. results/finding: 24,726 patients receiving 90,530 rbcs were included in the analysis. exposure to rbcs stored for more than 35 days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for 7 days or less) after adjusting for several fixed and time-dependent potential confounders (hr 5 0.91; 95% ci: 0.72, 1.14; p 5 0.400). exposure to blood stored for at most 8-35 days yielded a similar hazard ratio (hr 5 0.90; 95% ci: 0.73, 1.10; p 5 0.295). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than 35 days compared to exclusive exposure to rbcs stored 7 days or less was not significant (hr 0.90; 95% ci 0.72, 1.14; p 5 0.381). the confidence intervals around the hazard ratios for the other 7-day intervals all include 1. similar findings were obtained with partitioning exposure data into 3 day intervals where exposure to rbcs stored for 40-42 days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for 1-3 days (hr 0.82; 95% ci 0.37, 1.83; p 5 0.635). the confidence intervals around the hazard ratios for the other 3-day intervals all include 1. conclusion: individuals exposed to rbcs stored for more than 35 days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for 7 days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* 1 , cynthia walser 1 , tatsuro yoshida 2 , andrew dunham 2 and pedro cabrales 1 . 1 university of california san diego, 2 new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o 2 ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o 2 carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o 2 saturation <10%) stored rbcs, or anaerobic/hypercapnic (o 2 saturation <10% and pco 2 (@378c) $70mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as-3 after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either 1) conventional; 2) anaerobic; or 3) anaerobic/hypercapnic conditions. rats (150-200g) were hemorrhaged to 50% of blood volume, held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre-hemorrhage with prbc stored for either 1 or 3 weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. 1(11%) neg patient showed short term response and 6(67%) patients showed progressive disease. at the neg group standard eval 1(11%) patient showed response and 3(33%) had progressive disease. 1(11%) neg patient had long term response compared to 11(21%) pos patients. at the pos short term eval 22(42%) patients showed response and 20(38%) patients had progressive disease. at the pos group standard eval, 20(38%) patients showed response and 6(11%) patients had progressive disease. overall, 28(53%) pos patients responded compared to 2(22%) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd38 neutralizing substance could play a role in treatment response. alternatively, reduced cd38 expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a 24-hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april 2017 were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate 24% replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of 0.2m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* 1 , laurie sutor 1,2 , germ an leparc 3 , marjorie doty 3 and william crews 1 . 1 carter bloodcare, 2 ut southwestern medical center, 3 oneblood background/case studies: anti-cd38 drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with 0.2m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a 28 day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and 0.2m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual (18 th edition). each of the 12 plasma aliquots was further separated into 28 aliquots and stored at -208c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. (25) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than 11 with the untreated or dtt-treated cells during the study. conclusion: long term storage of 0.2m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque-10771), pooled, suspended in cryopreservation media (20% dmso; 1:1) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips (378c, 5% co 2 , 1 h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o1) rbcs sensitized with either anti-d (positive control), anti-scianna-2 (sc2) or anti-anwj or lipopolysaccharide stimulated for 2 h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/100 monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed 96.2 6 1% viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il-1b, il-6, il-8, mip-a (p < 0.01), mip-b and gro (p < 0.05) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc2-and anwj-sensitised rbcs resulted in a pi of 9.2 6 2% and 60.2 6 6.4% respectively vs anti-d sensitized rbcs (pi: 72 6 8.7%). a weak (11) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in 41 iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi>5%). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in 5 patients, involving 3 antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all 5 patients had 3-41 positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients 1 and 2 typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c1 by hea precise-type. ega-treated rbcs gave 31 reactions with the same anti-c reagent. patient 1 rbcs gave variable reactivity (vw-11) with bio-rad seraclone and ortho bioclone anti-c. patient 2 rbcs gave 11 reactivity with all 3 anti-c reagents when incubated for the maximum incubation time allowed. patient 3 rbcs were jk(a1) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat1rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients 4 and 5 tested s1 with bio-rad seraclone anti-s (3-41), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat1 rbcs but not all manufacturers include reagent limitations regarding testing of dat1 rbcs. we describe 2 cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and 3 cases with false positive tests with anti-s (n52) and anti-jk a (n51) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with 3-41 positive dat and supports testing to dissociate igg from rbcs strongly dat1 before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/2opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r :5 prob(ab|al-loexp), so that prob(ab) 5 prob(ab|alloexp)*prob(alloexp) and 0 r 1; rewriting prob(alloexp) 5 prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) 5 nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) 5 r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a 12 month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past 12 months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in 2 states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar 3 months before (2/ 2015 -4/2015) and 3 months after (2/2016 -4/2016) was selected; for state b, a similar 4 months before (12/2015 -3/2016) and 4 months after (12/2016 -3/2017) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (<12 months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a 13-and 3-fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from 13 to 567 (state a) and 151 to 1,496 (state b), which annualized, represents a potential gain of 2,216 (state a) and 4,035 (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the 2 states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from 2013 -2015 , there were 181 donors identified who had changed their gender from their birth gender; 121 female donors changed their gender to male and 60 male donors changed their gender to female. there were 7 (6%) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to 10.5 ml/kg or 15% of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the 15% limit. variable volume scales [vvs] can be programmed to vary unit volume (up to 550 ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by 10 ml at ebvs <3.5l in donors !23 yo, but increase by 5-40 ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the 18 mos. before a 6 mo. phased implementation of the vvs, and the subsequent 24 mos. multivariable analysis [mva] by 6-mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods 1 & 2, continued during impl and post-impl periods 1 & 2, returning to the baseline rate in post-impl periods 3 & 4 (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods 3 & 4. the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of 3.8 ml during post-impl periods 1 & 2 from the temporally matched baseline & pre-impl period 1. conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* 1 and yves gr egoire 2 . 1 hema-quebec, 2 h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, 2017. the vaccine is produced with live and attenuated yfv, which can circulate for at least 4 weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a 4 week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate 600 people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received 3,351 blood donors candidates; from those, 2,449 were accepted as a blood donor, after medical interview. the deferral rate was 26.9%. at the same period of the year 2016, there were 1,215 prospective donors, and 883 blood donations. the deferral rate was 27.3%. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, 1,566 additional donors, compared to 2016 same dates. that represents a 177.34% increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from 42.7% in 2016 to 45.8% in 2017. conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of !3.0x10 11 is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: 1,000 apheresis collections from 4 centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of 3.1 x10 11 for single (s), 6.3x1011 11 for double (d), and 9.5x10 11 for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or 100% plasma) assuming i) a minimum dose (allowing for production loss) of 3.5 x10 11 for s and 6.7x10 11 for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as 99%) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc 2rbc plt/p plt plt/rbc/p plt/rbc 2plt 2plt/rbc 2plt/p #donations 47990 63 1775 10832 968 476 67 1150 142 10252 citrate exposure (mls) 41-85 71 138 263 266 300 322 349 478 study design/method: a randomized (2:1), placebo-controlled, single blind, 15 subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into 5 cohorts, receiving increasing doses, ranging from 1/1,000 -1/10 of the lowest effective dose found in the above rabbit model. cohorts 4 and 5 received the 1/10th dose, but cohort 5 received two 1/20th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for 24hrs post infusion and followed for up to 60 days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of 68 aes: 40 were treatment emergent (teae), of which 8 were treatment-related (6 thrombosomes and 2 control). all teaes were mild or moderate in severity. in cohorts 4 and 5, 3/4 thrombosomes subjects had treatment related adverse events. one cohort 4 subject developed an upper respiratory infection and elevated wbcs within 8 hours post infusion, which resolved by 24 hours, and an elevated d-dimer at 24 hours post infusion, which resolved by day 7. this subject also had an elevation of prothrombin fragment 1 1 2 at baseline, which increased post transfusion and peaked at 24 hours with resolution by day 14. one cohort 5 subject developed non-specific t-wave changes at 1 and 2 hours following her 2nd infusion that resolved by day 21 without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort 5 subject developed an igg platelet autoantibody on days 7-21, which was undetectable on days 42-60; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days 7-14, and negative on days 21-60. background/case studies: cryopreservation of platelets (plts) could extend the shelf life from 5-7 days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with 5% dmso and stored at 2808c. after thawing, the unit was reconstituted in thawed ffp spiked with either 500 lm puromycin (pm) or 250 nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after 2, 4 and 24 hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd62p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x-100containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd62p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by 11-fold during 24 hour storage. immunoblot analyses of the plts showed a 2-and 4-fold increase in pm incorporation after 4 and 24 hours of storage, respectively. massspectrometry revealed 23 unique proteins that were synthesized after 4 hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac1, rap1 and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac1, rap1 and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in 2015, the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of 3 days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r 4-496 cooler with 2 units of ffp, 2 units of rbcs, and 1 unit of whole blood. three to 5ml of platelets were collected via syringe from each unit at 0 min (before storage in cooler or refrigerator) and after 0.5, 1, 3, 5, and 6 hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding) were measured by coulter counter, 2 channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p<0.05 deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for 6 hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c49-a03h molecular sieving: beyond genotyping ghazala hashmi 1 , reinhard klemm 2 and michael seul* 1,2 . 1 biomolecular analytics, 2 immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi2005 http://bit.ly/2ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding 401 rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of 30 rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, 4*4*96 samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for 135 sensitized sickle cell anemia ("sca") patients (tb1 in cas-tro2002, http://bit.ly/2oplxhr, excluding le and e(variant) and assuming 1 request per patient), presenting with up to 9 allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only 1 =2 plate holding 4*48 candidate units from actual black donors, followed by profiling of 44 samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for 127 of 135 requests (594.1%), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining 66 pooled samples produced 44 additional assignments to a second set of 135 requests, for a total of 171 assignments from only 92 wells. in another scenario, sieving of a full plate of 4*96 samples, produced $250 assignments for two successive batches of 271 requests from sca patients, a yield exceeding 2.5x. sieving alone typically fills 65-75% of requests of moderate complexity ( 5 ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez 1 , monica kalvelage 2 , ghazala hashmi* 1 and michael seul 1 . 1 biomolecular analytics, 2 lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou2013 http://bit. ly/2ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including 30 at the rhce locus) that encode 401 mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag2" (e.g. e2,c2,e2,c2) to specific combinations of "ag2" (e.g. c2e2k2fya2 and c2e2jsa2) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from 384 (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, 96 pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the 124 c2 samples, 24 that were also v2 and vs2 and, among the e2 samples, 12 that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select 6 specific "ee" pools of which 4 were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b (8 pools), co a|b (8) and others. conclusion: molecularsieving of a single 96-well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c2, e2 and jsa2. these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving 124 132 100 100 360 208 192 40 28 92 52 background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the 36 blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam4 (landsteiner-wiener) and ackr1 (duffy). for longer genes, such as abo of >20 kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of >21 kb each was used for all physically confirmed 48 ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v1.8.3 was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least 4 clades representing clusters of 5 to 11 alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the 4 alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt1 genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors1) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons 266-268, and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors1. a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos1(b3galnt1) cells, and cell-surface expression of fors1 antigen was immunologically monitored with a monoclonal anti-fors1 antibody. results/findings: we found that met69thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon 3 or 4 of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors1 antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met69thr/ ser or exon 3/4 deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt1 genes is reminiscent of common ancestral origin of alpha 1,3-gal(nac) transferase genes. the finding that at can synthesize fors1 implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of 29,308 cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period 7/1/2008 -4/1/2017. abo genotyping targeting specific snps for groups a, a2, b, o1, and o2 and, if needed, gene sequencing was conducted in cases with indeterminate results, and in 4 cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two (0.21%) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in 53% the predicted abo phenotype was a rh neg (table 1a ). the predominant donor race was caucasian (65%). four cbu with abo discrepancy were also evaluated by genotyping (table 1b) . in 3 of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r 6800/8800 systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: 30,695 fresh and 20,029 frozen edta plasma samples from american red cross donors, collected from february 2015-2016, were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r 8800 system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of 50,724 valid results, a total of 3 donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a 65-year old male in indiana, a 21-year old male in california, and a 55-year old female in kentucky. all 3 donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna (1440 iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as 3a, the california donation genotype 3b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was 100% (95% exact ci: 99.993% to 100%). conclusion: based on the 3 confirmed-positive donations of 50,724 tested, the hev prevalence was 0.006% (95% exact ci: 0.001% to 0.017%) with a detection rate of 1:16,667 (95% ci, 1:588-1:100,000). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than 50% of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report 13 months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using 95% confidence intervals. this analysis contains data from 9/1/15-9/30/16. results/findings: among 7,578,462 donations reported (16.2% from firsttime and 83.8% from repeat donors), there were respectively 483, 1489 and 194 cp results for hbv, hcv and hiv with corresponding rates of 6.37,19.63 and 2.56 per 100,000 (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of 23:1, 24:1 and 5.4:1 for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv 8.3 vs 4.2; hcv 23.5 vs 15.2; hiv 3.9 vs 1.0). in general, higher rates for all markers were seen among minority donors, those in the 25-39-year age group (also 18-24 year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when 3-month periods were compared. conclusion: data from 4 major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* 1 , melanie c proctor 2 , deanna self 1 , monique portugal 1 , adrian gurrola 1 , laura tonnetti 2 , sonia bakkour 3 , cheryl lobo 4 , michael paul busch 3 , susan l stramer 2 and jeffrey m linnen 1 . 1 grifols diagnostic solutions inc., 2 american red cross, 3 blood systems research institute, 4 new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to 16 donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening 32,274 unlinked whole blood donations collected from august 25 th 2016 to april 7 th 2017 in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of 16. results/finding: the procleix babesia assay detected all four babesia species with a 95% lod ranging from 7.10-13.51 copies/ml. the preliminary 95% lod in parasites/ml ranged from 0.64-3.61 p/ml for b. microti (n59), from 0.92-1.52 p/ml for b. duncani (n52), and from 0.62-4.95 p/ml for b. divergens (n52). of the 32,274 donations screened, 17 initial reactive and 14 confirmed positive donations were identified for specificity of 99.991% (95%ci: 99.972-99.997%). of the confirmed positive specimens, 8 were reactive by both ifa and pcr, 5 by ifa only and 1 by pcr only. all confirmed positive samples were reactive in lysate pools of 16. donors of reactive donations resided in ct (11), nj (1), nh (1) and me (1) for an overall incidence of 1:2,305, and 1:1,433 in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of 16 thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* 1 , whitney r steele 1 , ed p notari 1 , james haynes 1 , roger y dodd 2 and susan l stramer 1 . 1 american red cross, 2 american red cross (retired) background/case studies: from 2004 -2012 , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from 2008-2015. study design/methods: prevalence was calculated in 2-year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of 18.5, 7.4 and 9.1 days for hbv, hcv and hiv, respectively. linear regressions were calculated with p<0.05 (*) as significant. results/findings: from 1/1/08-12/31/15, there were more than 48 million donations from 13,204,447 donors (51.4% female, 33% first-time (ft), 81.4% caucasian). there were significant decreases in donation prevalence for hbv and hcv (p50.014 and 0.044), but no significant decrease in hiv during the 8 years (see table for f and r 2 values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p50.026 and 0.042). prevalent ft donors were significantly more likely to be male (68.3% -hbv, 59.8% -hcv, 79.7% -hiv; p<0.001). incidence for all agents declined (significant only for hbv; p50.035). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last 2-year period (74 in 2012-2013 vs. 19 in 2014-2015) . hcv incident donors in 2014-2015 were more likely to be male (79.0% vs 46.0% in 2012-2013, p<0.001) and were younger (84.2% vs. 67.6% in 2012-2013 <40 years, p50.011). overall, incident donors were more likely to be caucasian males (p<0.01). rrs for all 3 agents decreased over time with rrs in 2014-2015 of 1 in 1,565,000; 1 in 2,680,000; and 1 in 2,435,000 for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the 8-year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in 2015, mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as-5 rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with 200lm amustaline, and incubated for 18hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the 3hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero76 cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, >6.9 log 10 , or >6.2 log 10 pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was >6.2 log 10 , or >5.5 log 10 pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts-13 inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts13 activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts13 deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts13 activity of <5% and high inhibitor (1.4-8). mean age of cohort 22.8 years (range 17-64). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean 15.2 x 10 9 /l, range 9 -27 x 10 9 /l) and low a-ipc (mean 1.5 x 10 9 /l, range 0.5 -3.6 x 10 9 /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab 375 mg/m 2 (4 patients) and cyclophosphamide 400 mg/m 2 (one patient). tpe continued until platelet count reached 150 x 10 9 /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of 2.4 days [range 1-4 days]) when they achieved a three-fold increase in a-ipc from baseline (mean 11.1 x 10 9 /l, range 2.2 -25.3 x 10 9 /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean 217.6 x 10 9 /l, range 200 -294 x 10 9 /l) and a-ipc (mean 19.4 x 10 9 /l, range 13 -28.5 x 10 9 /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of 11.6 days (range 8-14 days) mean platelet count was 65.4 x 10 9 /l (range 14 176 x 10 9 /l) and mean a-ipc 3.2 x 10 9 /l (range 0.7 -6.6 x 10 9 /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of 20.8 days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts13 inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* 1 , michelle n stram 1 , joan sevcik 2 , alesia kaplan 2,3 and joseph e. kiss 2,3 . 1 department of pathology, university of pittsburgh medical center, 2 blood systems inc., 3 university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within 4-8 hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january 1, 2013 to november 1, 2016 was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts-13 activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version 14 (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the 96 ttp patients identified, 22 were excluded due to missing temporal data for important variables. the majority (85%) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients (20%) had a prior history of ttp and 26% had severe adamts13 deficiency on admission. the median time from tpe request to initiation was 5.6 hours (interquartile range: 4.7-7.2 hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table 1) . treatment was not started within an 8-hour window in 13 patients; the median time to cv access was significantly longer in these patients (5.8 vs 2.47 hours, p<0.001). two of these patients had a prior history of ttp and only four patients had severe adamts-13 deficiency. the majority (more than 70%) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table 1) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus 4-8 hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi-48a transfusion 2017 vol. 57 supplement s3 hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, 2006 through january, 2017, we performed cytapheresis (cy) treatments (txs) for 123 pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. 84 pts (68%) had cml-at and received 319 leukapheresis (lp) txs; 39 pts (32%) had et and received 124 thrombocytapheresis (tc) txs. cml-at pts presented with median wbc 398 x 10 9 /l (range 193-689 x 10 9 /l), of which 63% had blast percent >75% or blast count >100 x 10 9 /l. median age was 42 years (8-79 years); 62% were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. 22% of cml-at pts had no sxs of lks; 40% pts had sxs of either cns or pulm lks (1 sxs), and 38% pts had sxs of both cns and pulm lks (2 sxs). et pts presented with median platelet (plt) count of: 1738 x 10 9 /l (642-3510 x 10 9 /l)and 71% pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was 66 years (31-89 years); 58% pts were male. results/finding: all pts received a course of cy tx with following objectives: 1) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and 2) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) <100 x 10 9 /l for cml-at pts, and plt ct <450 x 10 9 /l for symptomatic et pts and <750 x 10 9 /l for asymptomatic et pts. cml-at pts received median of 3 lp txs (mean 3.9 txs/pt; range 2-8 txs). et pts underwent median of 2 tc txs (mean 3.4 txs/pt; 1-7 txs). outcomes were evaluated by percentage of pts who: 1) reached wbc (or plt ct) tx goal, and 2) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved >50% reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, 76% pts improved, 21% pts stabilized; and 3% pts worsened. in et cohort, 85% improved, 14% stabilized, and 1% were unchanged. for cml-at pts, median final wbc ct 5 96 x 10 9 /l (range 66-307 x 10 9 /l); 94% pts received ind chemo. for et pts, median final plt ct 5 705 x 10 9 /l (263-1087 x 10 9 /l); 95% pts had resolution of thrombotic 49a transfusion 2017 vol. 57 supplement s3 symptoms. 4% of cml-at pts and 0% of et pts expired within 1-4 days after course of cy tx. of 3 expired pts, 2 pts had both blast crisis and sxs of cns/ pulm lks; 1 pt had intracranial hemorrhage or cva; and 2 pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median 2-3 txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used 100% albumin or 80% albumin-20% normal saline (80/20) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered (100% albumin vs 80/20), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where 100% albumin was used versus those that used 80/20. covariates included were fluid types, age and gender. odds ratios (or) and 95% confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < 0.05. results/finding: during the study period, 3650 procedures were documented for 414 subjects (46% female), age range 0-93 years, of which 2,470 (67.7%) received 80/20. the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with 100% albumin had a significantly lower risk of having either event than by using 80/20, [p50.002, or (ci): 0.40(0.22, 0.72)] , and also had a significantly lower risk of causing hypotension [p50.023, or (ci):0.45 (0.22, 0.89)] in addition to a lower risk of causing citrate toxicity [p50.042, or (ci): 0. 24 (0.06, 0.95)]. age had a significant effect on having a hypotensive event [p50.04, or (ci):1.1 (1.0, 1.1)] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a 10 year period and compared it to published literature. study design/method: we conducted a 10-year retrospective study of ta procedures performed and aes were classified according to criteria described in table 1 . during the study period, ta were performed using cobe spectra (software versions 4.7 and 6.1) and since 2013 the spectra optia apheresis system (version 8.0). literature search was conducted for data published on aes associated with ta. four studies from us and 13 non-us studies (canada, europe and japan) were analyzed. trend for ae rates from 2007-16 was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was 6.9% (396 of 5,684 procedures) during 10 year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher (8.5%, p<0.00001) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho 0.7, p50.002) over the 10 years and significant down trend of moderate and severe aes with a spearman rho of -0.64 (p50.04) and -0.83 (p50.003) respectively. there were no fatalities during the study period. majority of aes were grade i (60%) and grade ii (28%): 32/5684 (0.6%) procedures were not completed due to aes. comparison of aes [6.9% (396/5,684)] to both european [11.2% (n513, 12, 256/ 109, 842) ] and other us studies [13.6% (n54, 860/6,324)] showed a statistically significant difference (p<0.0001). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table 1) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether 200 platelet donors with a donation activity of up to 150 platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within 2 hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* 1 , jordy jurgens 1 , jacoline buchner-doeven 1 , joris roelofs 1 , philip spinella 2 , jennifer a muszynski 3 , carel goslings 1 and nicole juffermans 1 . 1 academic medical center, 2 washington university school of medicine, 3 nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells (14 days old) and platelets (5 days old) by washing. plasma was filtered through a 0.22um filter. rats ($350 grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $30% of their estimated blood volume, which was calculated to be 57ml/kg. hemorrhage continued until a mean arterial pressure of 40mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to 4h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of 17ml/kg of blood products in a 1:1:1 ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april 2013, which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and 30-day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january 2008 and july 2015 (n53535). because of missing data on patient characteristics, 257 patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january 2008 to march 2013, n51987) and after (april 2013 to july 2015, n51291) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of 18 baseline variable, generating 969 pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common (8.7% vs 3.7%, p<0.001) and ventilation time was longer (15 h vs 13 h, p50.04) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group (2000 ml vs 2200 ml, p50.009; and 1265 ml vs 1460 ml, p<0.001, respectively). however, 30-day mortality was not statistically different between the groups (1.6% vs 1.4%, p50.82). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april 2014, www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over 12,000 joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table 1 ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy16. length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of 4-factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: 4-factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in 2015. marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh5 rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p5.005) and ptt (p5.05) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p5.03) and plasma (p5.04) after off-label use was significantly greater than on-label use. 20 cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were 5.5 times (p5.0072) more with cell saver or anh, and 5.3 (p5.0130) times more with cpb. post-pcc thromboses were identified in 6 cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every 3 days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after 7 days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least 4 days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to 7 days. the transfusion service medical director reviews the case and gives final approval. we observed only 1 patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight (38) patients were in-patients continuously until delivery. five patients were discharged prior to delivery-1 moved to another state, 1 was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was 17 days (range 0-63). six (6) patients delivered within 3 days of approval. after approval, the mean number of additional specimens per patient was 2.1 (range, 0-9). no patient required transfusion prior to delivery. five patients received transfusion of at least 1 rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only 6 patients delivering within 3 days of approval for extended specimens, 37 patients avoided collection of at least 1 specimen each, and 16 patients avoided at least 4 collections each. since new antibodies are not detectable for at least 10 days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to 7 days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* 1,2 , jan m. binnekade 1 , benjamin nota 2 , pieter r tuinman 3 , kirsten van de groep 4 , olaf l cremer 4 , janneke horn 1 , marcus j schultz 1 , robin van bruggen 2 and nicole p juffermans 1 . 1 academic medical center, 2 sanquin research and landsteiner laboratory, 3 vu university medical center, 4 university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in 2 tertiary icus in the netherlands comparing 30 patients who developed ai during icu stay with 3 control groups: 30 non-anemic patients with sepsis, 30 non-anemic patients without sepsis, and 10 patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron (15.4 vs. 2.9 mmol/l, p<0.001) and transferrin saturation (53 vs. 9 %, p<0.001), and low ferritin (104 vs. 645 mg/l, p<0.001). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells4life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately 2.5 x 10 7 cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to 65%, whilst leaving 25% of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within 30 minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than 1% of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately 65% of the cd341 fraction post separation and freeze thaw (table 1) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table 1) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd451cd611) and early projenitor cells expressing oct4 and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: 1. routine recovery of the wcf at levels higher than current methods, independent of volume. 2. higher percentage recoveries of all cell types tested than can be achieved with existing methods. 3. markedly higher post-thaw recovery of viable nucleated cells than any current methodology. 4. almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd341 target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving >0.5x10 9 lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd341 cell target of 4.0x10 6 /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd341 yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf 1 plerixafor (g1pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< 0.05 considered significant. results/finding: 110 no alc and 159 alc collections occurred among the 50 patients. fenwal amicus was used for 91% of the no alc and 99% of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was 5 hodgkin's and 45 non-hodgkin's lymphoma (no alc); 7 hodgkin's and 43 non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc 49.3, lymph 2.0x10 9 /l) than alc (wbc 39.1, lymph 1.2x10 9 /l). equivalent whole blood (corrected for ac) was processed for no alc (16.4l) and alc (17.1l). for alc group, extra collections beyond cd341 target were: 0 days: 24%, 1 day: 36%, 2 days: 22%, 3 days: 16%, and 5 days: 2%. significantly more patients were mobilized with g1pl in no alc group (n581) than alc group (n560) and 42 collections in alc group had mobilization discontinued after cd341 cell target reached. there was no significant difference in g (13.2x10 9 lymph) compared to g1pl mobilized collections (13.0x10 9 lymph); both were significantly higher than the collections where mobilization had been discontinued (5.9 x10 9 lymph). days to wbc engraftment (13.5 no alc vs 13.0 alc) and platelet engraftment (13.0 no alc vs 12.0 alc) were not significantly different. median number of collections for no alc (2) and alc (3) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the 0.5x10 9 lymph/kg or even the 0.3x10 9 lymph/kg targets. implementation of a lymph target increased patients obtaining 0.5x10 9 lymph/kg from 40% to 54%. only 12% had <0.3x10 9 lymph/ kg. discontinuation of mobilization once cd341 cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* 1,2 and nicolas pineault 2,3 . 1 canadian blood services, 2 university ottawa, 3 canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost 48 hours at room temperature (rt) as long as units are cryopreserved by 48-hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n53) were split with one half processed immediately (baseline 8-12 hours) and the second after 43 hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd451 cells and cd341 cell (n53). primary nsg mice were transplanted with a ucb cell dose that contained a total of 7,500 annexinv neg viable cd341 cells. the latter was done to avoid any bias towards one group or another. short term platelets (190 vs. 140 hplt/ml, p50.06) and leucocytes (1.2% vs. 0.2% hcd451, p<0.02) engraftment at 4-weeks were significantly reduced in stored mice vs. baseline (n53), and similar results were observed long-term at 16-weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated 3 months post-transplant. strikingly, the frequency of human cd451 bm cells was 10-fold greater in baseline vs. stored mice (p<0.01, n52). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured 22-weeks post-transplants were reduced by 30% in unit 1, and by 80% in unit 2. conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($1mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to 97%, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of 1sec). total lymphocyte recovery was 43% and monocyte concentration was reduced 76%. furthermore, in a two-pass process platelets were reduced by 75%. in a 12-fold parallel system we tested rbc separation from plasma and achieved 90% separation at 72ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past 15 years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from 9 clinical protocols from 2008-2016. an infusion reaction was defined as any symptom from the time of nk cell infusion up to 4 hours afterwards. a severe reaction was defined as any symptom with grade 3 or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r 3.3.1. two major endpoints of interest were: 1) infusion reaction with any symptom and 2) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of 127 nk cell infusions. there were 119 (94%) patients with an infusion reaction of any symptom and there were 37 (29%) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median52.55, p50.42) and those with severe reaction (median52.52, p5 0.42). infusion rate (ml/min/kg) was also similar among those with any reaction (median50.03, p50.43) and those with severe reaction (median50.03, p50.15 respectively). incubation of nk cell product overnight in il-2 vs il-15 had similar reaction rates for those with any symptom (88% had reaction with il-2, 86% had reaction with il-15, p50.94) and those with severe reaction (28% had severe reaction with il-2, 24% had severe reaction with il-15, p50.80). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median52.44 x 107) versus those without (median51.92 x 107, p50.02). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade 3 or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan 1 , maryanne c herzig* 1 , barbara a christy 1 , james a. bynum 2 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at -80. mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at 50-60,000 cells/ well and cultured in 96 well plates for 4-48 h in their respective medias. on day 0, mscs were washed, resuspended in pbmc media and incubated with or without 150,000 freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, 0-5 lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by 72 h, with >6 fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was 10.0%. inter-assay variation of msc preps run under identical conditions was 7.5%. inhibition of pbmc proliferation was graded from 0-100% over the range msc concentrations therefore an ec50 of msc cell number resulting in 50% suppression of pbmc could be determined for each msc prep. this ec50 however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within 72 h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute 10% or more of the us blood supply. differences between donors 16-18 years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged 16-49 were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the 2015/16 academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml) were estimated for 16, 17, 18 and 19-49yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of 4265 donors contributed 6219 donations. donors were evenly split by gender, 66% were ft donors, and 87% were 16-18yo. ft and rpt 16-18yo donors had on average lower ferritin values at enrollment (p<.0001), and a greater percentage were iron-depleted than donors 19-49yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged 16-18 have sharply higher risk for iron depletion than donors 19-49yo. odds for lf were 4 to 6 times greater in the younger donors, and for ais were 3-to 4fold higher. preliminary statistical models indicate 16yo donors may have greater risk for lf than 17 or 18yo by 4 to 5 percentage points, controlling for other factors (p5.06). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in 16-18yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on 12/19/ 2016 by a large blood collector. testing was performed on successful 16-18 y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < 20 ng/ml in females (f) and < 30 ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations (12 months for f and 6 months for m) and counseled to take 18-28 mg of elemental iron daily for 60 days. for m and f, a ferritin < 12 ng/ml indicated absent iron stores (ais) and < 26 ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! 20 ng/ml in f and ! 30 ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior 24 months. an appreciable number of donors with no rbc donations in the prior 24 months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* 1 , joan williams 1 , michelle humphries 1 , nancy haubert 1 , ben reynolds 1 , michael phillips 1 , randall spizman 1 , ralph r vassallo 2 , hany kamel 2 , sally caglioti 1 , german leparc 1,3 and phillip c williamson 1 . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. 1 new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. 2 study design/methods: over 28,000 serum samples from donors aged 16, 17 and 18 years were analyzed for ferritin levels using the beckman coulter au680 instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. 3 results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba1c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among 21,007 adolescents (ages 16-19; 61.5% female) who donated blood from 2015 to 2016. study design/method: abnormal risk factor levels were defined as hba1c ! 5.7%, sbp/dbp ! 120/80 mm hg and tc !170mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of 2 or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table 1 shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, 11,283 (53.7%) adolescents had at least one abnormal risk factor (61.8% of males, 48.6% of females). of these, 8,709 adolescents had isolated abnormal risk factors, and 2,574 adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as !13.5 g/dl for men and !12.5 g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from 52 blood services worldwide and complete data were available for 25 blood services. deferral percentages for low hb varied from 0.01% to 8.81% among male donors and 0.03% to 46.73% among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with 53% lower hb deferral rates in men (95% confidence interval [ci] 11% to 75%) and 61% lower rates in women (95%ci 15% to 82%). iron supplementation was associated to 57% lower hb deferral rates among women (95%ci 22% to 76%) but there was no evidence of such an effect among men (p50.680). each one-week increase in minimum donation intervals resulted in 8% lower hb deferral rates among women (95%ci 1% to 14%) but not among men (p50.454). at the 5% level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the 5 previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: 9.15% of all candidates for wb donation were deferred in continental france in 2015. deferral was significantly more frequent in women (11.16%) than in men (7.29%), due to anemia in 24.41% of deferred women and 9.79% of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from 20 to 30 weeks. analysis (table) identified 3 main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the 5 previous years. conclusion: the 3 main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, 2 days stored or 35 days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, 2 ng/kg). blood was sampled every 2 hours up to 8 hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from 1.4e108 (iqr 8.3e107-1.9e108) /ml in the fresh product to 1.7e110 (iqr 7.9e109-2.3e110/ml; p<0.01) in the stored product (p <0.001), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within 6 hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: 200 cases of taco and 405 matched controls were enrolled from 20,845 transfused patients who received 128,263 blood components from may 2015 until july 2016. taco incidence was 1 case per 100 patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation (71% vs. 49%; p < 0.001), experienced longer intensive care (4 vs. 3 days; p50.04) and hospital length of stay following transfusion (10 vs. 7 days; p< 0.001), and had higher mortality (21% vs. 11%; p50.02). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march 2016 fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from 5 to 7 days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july 2008 and to routinely extend ap outdate to day 7 since february 2016. this study reports a 103 month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july 2008-january 2016, ap underwent rt on day 4. day 6 and 7 units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day 8 had a second rt performed. from february 2016-january 2017, ap underwent rt on day 5 with routine outdate extension to 7 days by performing a second rt on day 6 and a third rt on day 7, as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type 1) or repeat rt positive with negative confirmatory culture (type 2). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july 2008, 20,010 ap were entered into inventory. of these, 11,840 (59%) were transfused prior to rt testing. the remaining 8170 (41%) underwent rt on day 4 or day 5. of these 43 (0.5%) were rt positive (29 type 1 fp, returned to inventory; 14 type 2 fp, discarded), leaving a total available inventory of 8156 units tested by rt. of these, 5631 (28% of original inventory) were transfused before the end of day 5 and the remaining 2525 (13% of original inventory) reached a day 5 outdate. a total of 1561 (8% of original inventory) were transfused on day 6 or day 7. of these, 768 underwent a second rt on day 6 (2 rt positives; 1 fp type one and 1 fp type 2) and 233 underwent a third rt on day 7 (no positive results). a total of 964 (5% of original inventory) outdated on day 7. of these, 754 underwent a second rt on day 8 (no positive results). conclusion: to date we have performed 9925 rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted 13% to only 5%. a total of 1522 ap have been tested twice by rt (768 on day 5 and 6; 754 on day 4 and 8) with 2 (0.1%) positive results, both of which were deemed fp by repeat testing or culture. a total of 233 units have been tested 3 times (day 5, day 6 and day 7) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every 24 hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with 8 to 10 million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only 5 cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in 2007. contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between 10-10,000 parasites/ml. each parasite concentration in wb was tested x2. an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at 48c for up to 42 days; platelets were stored at 228c (rt) under agitation for 5 days and plasma was frozen at -208c. aliquots for culture were removed weekly from rbcs, daily from platelets and after 30 days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at 278c for detection of live parasites for up to 16 weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at 48c, rbcs from all units spiked with 10,000 parasites/ml were positive for up to 21 days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with 1000 parasites/ml were positive for up to 7 days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with 10,000 and 1000 parasites/ml were positive up to 5 days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at 48c for up to 3 weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* 1 , marion lanteri 2 and larry corash 1 . 1 cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom (2006 -2015 ), french (2006 -2015 , swiss (2011 -2015 ), and belgium(2009 -2015 hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately 1.35 million dlvbc-screened were issued with a 7 day outdate after release into inventory 3 days after collection, and $2.3 million amotosalen/uva-treated pc were released into inventory on day 1 or 2, with a 5 to 7 day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored >2.83 million conventional, non-dlvbc-screened pc and recorded 58 str and 9 fatalities. concurrently, zero definite and 2 possible str were reported with 607,871 amotosalen/uva-treated pc, significantly fewer than with conventional pc (table 1 ) (20.5 str per million vs. 0.0 per million, p<0.001). one definite, 1 possible, 7 undetermined/indeterminate non-fatal str and 5 contaminated "near miss" pc were reported with 1.35 million dlvbc-screened pc between 2010 and 2015, for a reduced falsenegative rate compared with the prior five years (3.7 str per million vs. 16.3 per million, p <0.05). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october 2016 for all platelets received at our institution. at time of receipt at the blood bank (day 3 post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, 10 ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at 35c for three days. results/finding: a total of 9473/11,066 (85.6%) platelet products were successfully cultured (934/1373 [68.03%] and 1842/1912 [96.3%] in october 2016 and march 2017 respectively). over the 6-month period, two true positive cultures were obtained (incidence of 1 in 4736 platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us16.83 per product tested. the cost per averted case was $us79,707. conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january 2009 and december 2016. the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the 7-year study period, a total of 3280 transfusions reactions were reported, 18 of which were bcptr (0.55% of transfusion reactions). of the 18 bcptr, 15 (83%) were associated with apheresis platelets, 2 (11%) with red blood cells, and 1 (6%) with plasma. recipient diagnoses spanned hematologic/oncology (n512), renal (n53), cardiac (n51), autoimmune (n51), and obstetrics (n51). an organism was identified in both the blood product and recipient in 10 (56%) cases; in 6 (33%) cases an organism was grown in the blood product but not the recipient; and in 2 (11%) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in 5 of the 6 cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever (83%), chills (67%), nausea and vomiting (50%), pain (27%) and dyspnea (22%). blood pressure (bp) decreased in 22%, increased in 17%; 61% of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie 1 , jenna lebedev 1 , linda kapp 1 , xiaohong wang 1 , meghan delaney 2 , lay see er 1 and james c zimring* 3 . 1 bloodworksnw research institute, 2 bloodworks nw, 3 university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg1-igg4), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least 29 natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all 29 known variants. study design/methods: the heavy and light chain variable regions of an anti-k1 monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known 29 igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k11 rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg2, igg3, and igg4 had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table 1). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg1 or for any of the monoclonal ahgs tested. monoclonal anti-igg3 had a blindspot for igg3-04, due to the shorter hinge region on igg3-04. no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* 1 , yves dominique pastore 1 and maryse st-louis 2 . 1 chu sainte-justine, 2 hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since 2008, our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of 2014, 205 scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september 2016, 117 (57%) patients had been transfused and 14 had antibodies with known blood group antigen specificity: anti-c, anti-e (2), anti-hrb, anti-fya, anti-jka, anti-jkb (2), anti-s, anti-m, anti-sc2, anti-leb (2). seventeen patients (8.3%) were either d2 or partial d. rhce results showed that 163 patients expressed a normal c antigen and 32 expressed partial c. as for e antigen, 163 had a normal antigen, 38 bore a partial antigen and 3 were weakly expressed. fy(a2b2) phenotype was found in 182 (89%) patients. a total of 2606 genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos 1 , emilia sippert 1 , mayra dorigan de macedo 1 , sheila fatima perecin menegati 1 and lilian castilho* 1,2 . 1 hemocentro unicamp, 2 university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa-308a, il1b-511t cytokine polymorphisms, rhag 808g>a and hla-drb1*15 alleles may predict a good responder phenotype (sippert et al, transfusion 2017) and that rhag 808a and hla-drb*15 alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included 96 non-alloimmunized patients with scd, homozygous for hbs, receiving a range of 5-289 rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa-308g>a, il1b-511c>t) and the rhag 808g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among 96 non-alloimmunized patients, 21 were homozygous or compound heterozygous for rh variant alleles. from those, 6 had rhag 808a and/or hla-drb*15 alleles and at least one cytokine polymorphism (tnfa-308a or ilb1-511t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other 15 patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and 2 were confirmed by sequencing. the third sample was found to be rhce*cevs.01,rhce*cebi on sequencing (predicted phenotype v1,vs1). the 3 samples were typed as v1 (or ce s ) and vs1 (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[712g]in 2 samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c1 by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the 2 methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising 80% of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies (20% of patients) bound young and old rbcs with no apparent prejudice. band-3 is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band-3 is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band-3 to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band-3 aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from 22 patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band-3 tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing 5 type i and 5 type ii patients, we found that type i is characterized by 5 percollv r fractions (similar to healthy storage-matched controls) but increased band-3 tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by 3-4 percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band-3 tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band-3 suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band 3 suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* 1 , burak bahar 1 , jeanne hendrickson 2 , krystalyn e hudson 3 and christopher a tormey 1 . 1 yale-new haven hospital, 2 yale university, 3 background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam30 abstract algorithm, e value51x10 -6 , word size5 6, gap costs: existence59 exten-sion51). search results were restricted to bacteria and fungi, with a selective threshold of >80% identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from 162 alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b5-0.0017, r 2 50.624 & p50.0197); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of 162 alloimmunized patients reviewed, 105 were culture-positive. of these, 76% of the anti-c/c group (13 of 17 patients) and 16% of the anti-k group (7 of 43 patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from 0 -11.1%. overall, 21.9% (23 of 105 patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of >80% sequence identity. while 27.6% (29 of 105) patients reviewed had positive cultures for klebsiella species, 62.1% of these (18 of 29 patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed 109 tegs performed on 76 patients undergoing cv surgeries at our institution from jan 1 to dec 31, 2016. no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the 56 tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at 30 minutes (min) all within reference range), "hypocoagulable" (r>10 min, k>3 min, a<53 degrees, ma<50 mm) and "hypercoagulable" (r<5 min, k<1 min, a>72 degrees, ma>70 mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of 56 tegs analyzed, 37 patients (66%) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients (8% vs. 32%, p50.02). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused (32% vs. 11%, p50.07). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused (100% versus 33%, p50.06). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from 670 liver transplants, performed from 2011 to 2015 in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, 670 olts were performed. a total of 345 patients was submitted to cs. the median age was 51 years (range 10-78 yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in 31,6% of the patients. the average meld score was 29,6 6 9,4 and it was slightly higher in the cs group (31,3 vs 27,9, p<0,001) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was 8856 6 4503 ml and mean reinfused blood volume was 914 6909 ml. allogeneic blood transfusion was required in 71,8% patients in the cs group, compared to 46,7% patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group (2,4 units vs 3,39 units, p<0,001 background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july 2015-december 2016 was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp (27 and 15 products, respectively) however, obp wastage occurred more frequently in the 18 month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations (23 versus 4 products). this is skewed by one month when 20 products were wasted due to expiration of product on the floor. cooler-related issues (6) and products dwelling too long out of a controlled environment (5) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were 1.7%, 0.3%, and 2.3%, respectively, with a total exsanguination protocol waste rate of 1.33%. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p50.176). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a 17 year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with 5 units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d1) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d(table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* 1 , edward smith 2 , thomas brown 2 , foeks jeremy 2 , metcalf suzanne 2 , james johnson 2 , peter davis 2 , karafa sw badjie 1 and abba zubair 1 . 1 department of laboratory medicine and pathology, transfusion medicine, mayo clinic, 2 department of anesthesia, mayo clinic background/case studies: our institution performs an average of 398 solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a 29 y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused 2 o(1) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused 10 more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the 2 o(1) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in 1% of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a 29 y.o. female patient should not have received o(1) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with 10 admissions during the 5 hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight (107 kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o(1) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a 50 y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o(1) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately 375,000 surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from 6-16%, whereas transfusion rates for vaginal and robotic pfd surgeries range from 0.2-1.6% and 0.3-1.4%, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately 15% of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may 2015 -may 2016 in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p<0.05. results/finding: we identified 66 patients for analysis, of whom 65 (98.5%) had a preoperative t&s ordered. two (3.1%) of these 65 patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine (90.8%) of the 65 patients required a second abo/rh specimen per hospital protocol; 51 (86.4%) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table 1) . no abo/rh discrepancies were identified. one patient received 1 unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution (90.8% vs. 15%, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n527) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of 1.05 g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs-1000i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant 1 removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), 5 units were subjected to the volume reduction while recording the time needed to process all 5 units. this was performed twice for a total of 10 units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of 72% (range 49%-87%). in units between 21 and 42 days (n510), the estimated mean residual k1 was 1.89 meq (range 0.61 to 2.21). in the two mock mtp trials, the time to complete the procedure was approximately 50 minutes and we estimate an additional 5-10 minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal (3) and caesarian (2) births. uc collections were divided into 3 segments to test 3 conditions. segment explants were placed on 0.1% gelatin-coated gridded tissue culture plates (32 explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of 21 days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the 2 remaining tissue segments were soaked in (ab/am) saline solution for 1 hr and 24 hrs at 48c, respectively. tissue segments were frozen in cryo bags with a proprietary 10% dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process 3 or 4 times the total blood volume (bv) of the patient, up to a maximum of 25 liters (l) per procedure, to obtain peripheral blood cd341 stem cells. as a consequence, a patient often would need to spend 6 hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd341 cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our 2016 collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd341 pre-count and cd341 yield, normalized per liter of blood processed, was derived utilizing the patient's cd341 pre-count, the patient's weight in kilograms (kg), and the target cd341 dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd341 stem cells. the initial equation was modified to add an additional 15% to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in 8 patients, representing both allogeneic and autologous donors, the average blood volume processed was 14.8 l. the range was 4.9 l -21.6 l. the target dose was achieved in all patients. our previous practice for these 8 patients would have required, assuming a standard 4 bv procedure, processing an average of up to 28 l per patient, with a range of 20-62 l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd341 yield. the result was a high correlation between these two ratios (r 2 5 0.92), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r 2 5 0.92, confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing 2 hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses (1-2) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd341 mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june 2015 and march 2016. patients (n525) evaluated were diagnosed with malignant lymphoma (n515), multiple myeloma (n59) and primary amyloidosis (n51) and were mobilized according to standard protocols. collection cd341 cellularity target was established ! 2x10e6/kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by >65 years old, previous fludarabine, lenalidomide, or bendamustine treatments or !2 previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd341 count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v4.0. results/finding: the media (range) general collection parameters were: cd341 (day 5) 27.50/ml (4.5-157.5/ml), blood volume processed 23204ml (9718-39618ml) and 4.96 (2-7.30) exchanged volemias. seventeen patients were considered bad mobilizers, 7 needed plerixafor and 5 had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p50.071]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n52), fever (n52) and flu syndrome; all grade 1]. two patients could not undergo hematopoietic stem cell transplantation due low cd341 cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd34) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with 20% fetal bovine serum. bmsc and amsc at passage 2-5 were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses (1.5 3 10 4 /ml, 7 3 10 4 /ml and 1.5 3 10 5 /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par4) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within 30min and 2hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: 334 6 35 seconds, versus low, medium and high doses of amsc (145 6 2, 111 6 6, and 75 6 12 seconds), and bmsc (155 6 2, 90 6 10, 80 6 7.0 seconds), p<0.05), clot formation time (cft, p<0.05) and increased alpha angle (p<0.05) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par4. no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* 1 , neil bagamasbad 2 , reynold dilag 2 , melissa nasser 2 , nicole bauer 2 , jennifer wheeler 3 and mary berg 1 . 1 department of pathology, university of colorado -anschutz medical campus, 2 department of medicine, division of hematology, university of colorado hospital, 3 scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from 120 collection procedures using the mnc protocol and 173 collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including 36 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd34)-positive (cd341) throughput, cd341 collection efficiency (ce%), platelet loss 71a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included 14 and 22 allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd341 throughput was significantly higher in the cmnc group than the mnc group. the cd341 ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded 20 ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient #1, originally typed as an a2, had 1 bone marrow donor and 2 cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient #1 is now typing as type o. patient #2 was originally typed as a2 and received a bone marrow transplant from a type b donor. patient #2 is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient #1 and patient #2 indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with 20% fetal bovine serum under either normoxia (20% o 2 ) or hypoxia (3.5% o 2 ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd90/cd29 and cd45 were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc (1.5 3 10 5 /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by 15%, but depressed the growth of amsc by 30% at day 5 in comparison to normoxia. both bmsc and amsc equally expressed cd90 and cd29 but not cd45 under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: 468 6 64 (control), versus 170 6 13 (bmsc), and 195 6 60 (amsc) seconds) by natem. hypoxia also significantly shortened ct (165 6 20 (bmsc), 169 6 50 (amsc) seconds, p<0.05 as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd341 cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd341 cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd341 cells. study design/method: cryopreserved cd341 cells from 2 healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef1-alpha-yfp lentivirus (2.5% concentration) and media (x-vivo-10, human serum albumin(hsa), 100 ng/ml each of cytokines (scf, tpo and flt3-l) over 2 days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of 5% dmso, 6% pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd34 1 cd38 -cd45ra -cd90 1 cd49f 1 cells) phenotyping and cfu assays were done following first thaw (pt1), post-transduction (ptxn) and second cryopreservation-thaw (pt2). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd34%, cfus were similar before and after pt2. hscs ranged from 824 to 1655 cells/10 6 cd341 cells in the pt2-tr arm compared to a range of 286 to 1416 /10 6 cd341 cells after pt1. viability, % cd341 and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt2 (table) . conclusion: dec of mpb human cd341 cells decreases tnc recovery, but has minimal effects on cd341 cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd34% in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd341 cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd341 cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of 20% hydroxyethyl starch, 18% human serum albumin and 10% dmso at final concentration. pbsc were cryopreserved by direct immersion on -808c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of 03 consecutive days of neutrophil count >0.5 x10 9 /l and platelet count >20 x10 9 /l after 07 days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd341 was below 10 x 10 6 cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd341 on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd341 on the day of the collection versus collected cd341 per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd341 was calculated. final laboratory count of cd341 per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss 23 software. results/findings: among patients collecting hpc for autologous transplantation, 69,23% needed only one day of hpc harvesting, while 25,64% needed two days and 5,13% needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was 49 1-2,91%. after comparing predicted values with cd341 collected in the final product, we found a very strong correlation of 0.873 (p<0.01) for patients and a strong correlation 0.653 for healthy donors (p<0.01). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; !95% cd90, cd105, cd73 and 2% cd14, cd19, cd34, cd45, hla-dr) study design/method: umbilical cord tissue (n510) was washed, blood vessels removed, cut into 0.5-3mm pieces, and washed twice in saline. fresh tissue was immersed in 0.9% saline for same day culture, while frozen tissue was cryopreserved for at least 24 hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a 25cm 2 tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for 10 minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures >80% confluence. all cells were tested on an msc flow panel at passage 2 just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of 8 days (fresh 5 7.8, frozen 5 8.1), and 13 days (total) for the msc's to reach passage 1 (fresh 5 12.6, frozen 13.4). all cells were ready for flow analysis in approximately 3 weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p 5 0.81), or their growth rates (p > 0.05 for all). flow cytometry showed average !95% for positive markers and 2% negative markers. there was no statistical difference between fresh and frozen flow result (p > 0.05). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of 2 up to 11 years (2004 to 2017) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with 10% concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a 378 c water bath and 0.5ml aliquots were diluted at a 1:1 proportion with 5% human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using 7-aad marker through flow cytometric analysis. results/finding: ucb storage period was 7.24 years (mean) and cell recovery was 86.31% (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p 5 0.11). post-thaw cell viability of 63.13% (mean) showed no statistically significant correlation with unit storage period (p 5 0.07). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for 5 years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln 2 vapor in a dmso-based cryoprotectant for 5yrs. (5.49 6 0.431; n54). units were rapidly thawed and rinsed in dpbs, then 25 pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a 5x5 grid pattern in msc-supportive medium and incubated for 7 days, after which the tissue was discarded and media exchanged. cells were isolated on the 14 th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a 100% success rate. cells were positive for the msc markers cd73, cd90, and cd105 (98.8 6 0.7%, 98.7 6 0.6%, and 97.8 6 0.6%, respectively) and negative for the hematopoietic markers cd34/45 (1.1 6 0.7%). passage 1 and passage 2 doubling times were 1.92 6 0.47 days and 2.07 6 0.43 days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical 75a transfusion 2017 vol. 57 supplement s3 research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd34 yield prediction algorithm ines bojanic* 1 , nelly besson 2 , ivana vidovic 1 and branka golubic cepulic 1 . 1 department of transfusion medicine and transplantation biology, university hospital centre zagreb, 2 terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd341cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version 11) in adult and pediatric lvl. a prediction algorithm for cd341cell yield was also tested. study design/method: we evaluated retrospectively 67 lvl performed in 46 adult patients, and 14 lvl in 11 pediatric patients treated in uhc zagreb from march 2016 till september 2016. mobilization regimen combined chemotherapy and filgrastim; 2 poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio 1:24). in patients weighting 25kg (n59), a rbc prime was performed. cd34, lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd341cell count and cd341cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd34 values to real cd34 yield. results are presented as median (iqr). results/finding: in both groups, cd34, ly and mo ces were high. target cd34 dose was successfully reached in 1 procedure in 30 (65,2%)adults and in 9 (81.8%) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in 5 (7.5%) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd341cells and cd341cells collected/ blood volume was observed in both groups (r 2 50.97 and 0.83 in adults and children respectively, p<0.0001) suggesting cd34 yield could be predicted based on precd341cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd34 yield and observed cd34 yield (r 2 50.95 and 0.82 in adults and children respectively, p<0.001) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of 10.1(8.9-12.9)l of blood in 20 adult procedures, and 5.9(3.5-7.8)l in 7 pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd34, ly and mo ce1 were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* 1 , aniko barta 2 , arpad batai 2 , zoltan csukly 2 , zita farkas 2 , laszlo gopcsa 2 , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) 4 times per case weekly at a dose of 1 million cells/kg. clinical response was assessed 28 days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all 12 patients had received 13 cycles of msctreatment (4 dose per cycle). the median age was 47 years old (19-56) with a male/female ratio of 1:2. distribution of the original malignancies (n): acute myeloid leukemia: 6; acute lymphoblastic leukemia: 2; myelofibrosis: 1; myelodysplastic syndrome: 1; multiple myeloma: 1; t-cell lymphoma: 1. nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median 63rd day (7-455). the involved organs were skin (2), gut (4), skin and gut combined (7) and even lung in 3 cases. the median time of msc's first infusion was 274 days after the stem cell transplantation (hsct) and 165 (19-1974) days after the first episode of gvhd. 4 of the 13 cycles of msc-treatment led to complete remission (30.8%) and 7 resulted inpartial remission (53.8%). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with 83% overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is 2 million cd341 cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd341 cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the 2 million cells/kg goal. the ideal minimum cd341 cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from 55 patients to evaluate the predictive value of the cd341 cells/ml level. data was collected over 6 months from every patient who underwent a stem cell collection. four patients were allogenic donors and 51 were autologous donors. the patients' weight, diagnosis, and pre-procedure cd341 cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd341 cells collected were recorded. the collection efficiency and the cd341 cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd341 cells/ml and post-procedure cd341 cells/kg (r50.95). any patient who had a pre-procedure cd341 cells/ml count of 29 or greater had a collection of at least 2 million cells/kg. any patient who had a pre-procedure cd341 cells/ml count of 16 or less collected less than 2 conclusion: the pre-procedure cd341 cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd341 cells/kg level. to confidently know that a patient will be able to produce the desired 2 million cells/kg, a pre-procedure cd341 cells/ml count of at least 29 should be obtained. for any patient with a count below 16, they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between 16 and 29 cd341 cells/ml should be conducted. heidi elmoazzen 1 , antonio giulivi 1 , michael halpenny* 1 , lisa martin 1 , donna perron 1 , chris bredeson 2 , lin yang 1 , locksley mcgann 1 , paul birch 1 and jason p. acker 1 . 1 canadian blood services, 2 ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso (5% final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of 3 phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd34, viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in 5% dmso and 1.7% hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of 12.6 days for anc500 with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current 5% dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using 5% dmso and 1.7% hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from 5% to 40%. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is 0.24 mg/kg, therefore patients weighing >100 kg would require a second vial, thus doubling the drug cost. in 2013 we implemented a policy of capping plerixafor at 24 mg for patients weighing >100 kg. this retrospective study compares the mobilization of patients >100kg who received capped doses (2013) (2014) (2015) (2016) , with historical control patients (2010-2013) who received full or uncapped doses. study design/method: patients weighing >100 kg with crcl >50ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of 47 and 40 consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd341/cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at 24 mg for patients >100 kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd34 in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (<30yrs) have a higher pre-apheresis %cd34 level than any of the other groups, reaching statistical significance when comparing the %cd34 pre-apheresis between the youngest group (<30 yrs) and the oldest group (>540 yrs). hispanic donors show statistically similar %cd34 pre-apheresis levels over all age groups. moreover, the hispanic older age group (>540yrs) had a statistically higher %cd34 pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of 121 sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd34 level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd34 level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last 3 years was analyzed to determine the 95 th percentile, median and 5 th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc (234 x 10 6 cell / ml) and three low wbc (114 x 10 6 cells / ml) concentrations, each at high (505 ml), low (265 ml) and median (355 ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax 2 (pericell protocol, cs.430.1 kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and 7-aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting 7-aad viability of 76 6 8% [range 64-85]% and a hematocrit of 14 6 5% [9-19] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the 6 mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was 97 6 8% [64-105] with a 3 6 3% [-2-11] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of 99 6 4 [92, 104] % and a change in 7-aad viability of 2 6 2 [0, 11] % from the input product. the method was found to have a cv of 2.0%. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating 78a transfusion clinical assessment. in the first phase, 2 cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd34 7-aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to 3 hours post thaw. the second in vivo phase included use of an infusion pump for 10 consecutive autologous patients, with comparison of infusion and transplant outcomes to 18 previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the 2 products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than 20% within 1 hour, while cd341 cell viability remained stable up to 3 hours post thaw. small aggregates appeared after 1 hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were 10.8 6 1.3 and 11.6 6 1.0, respectively (p-value50.075). platelet days to engraftment for pump and drip were 17.9 6 2.2 and 20.2 6 5.0, respectively (p-value50.207). infusion rates were slightly higher for the pump group. for control patients, 2 required transfer of products to syringes due to slow infusion rate and 2 others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the 278 zikv ineligible cbus was: caucasian 52%, asian 9%, black/aa 20%, and multi-race 21%. racial distribution of all clinical cbu donors was caucasian 49%, asian 15%, black/aa 20%, and multi-race 17%, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: 78% of all ineligible cbus and 21% of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete 2,3-diphosphoglycerate (2,3-dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring 2,3-dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a 10 ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n 5 20), which were stored in sagm for 22 days, to act as untreated controls. the remainder of each unit ($270 ml) underwent treatment with the rejuvenation solution (50ml, 60minutes at 37 o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from 39 random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p 1 , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of 22 day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp 215 (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros 60. pc were centrifuged at 1250g in a sorvall rc3c1 centrifuge (sorvall, usa) for 10 min. the combination cryoprotectant dmso1dextran (cryosure dex40, germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at 37 degrees c (barkey plasmatherm) for 10 min. cpc osmolality was measured with an osmomat 030 osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru 169287 u1). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso1dextran (cryosure dex40) , as a cryoprotectant, to obtain a final concentration of 5% dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m3 were instrumental in automating that phase. pc to be frozen had an osmolality of no less than 1500 mosm/l. prp and ppp were frozen at a cooling rate of 1-38c/min and stored at -85 0 in the chest freezer for up to 24 months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru 167874 u1). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to 380 mosm/l. freeze-thaw recovery of platelets was 80% or more of the original population. defrosted pc were stored at 20-24 0 with continuous gentle stirring from a helmer platelet agitator for no longer than 4 hours before transfusion. it took no more than 30 min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over 20 min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day 4 and day 5 evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to 7 days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day 4 for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day 5. a total of 60 lrap units were tested over a 3-month period: 50 were cultured and rapid tested on day 4; 10 were rapid tested on day 5. the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were 59 true negatives (tn) and 1 false positive (fp) on day 1 when tested by bact/alert, with 60 tns on day 4. bactx testing results showed 50 tns on day 4 and 10 tns on day 5. testing using the pgd kit showed 50 tns on day 4; and 8 tns and 2 fps on day 5. fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and 30 minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within 20 minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in 100% plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day 4 and day 5 during the night shift to be accomplished without additional staffing and allows to extend outdate to 7day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for 1 year jos lorinser 1 , pieter f van der meer 1 , hans van der heiden 2 and dirk de korte* 1 . 1 department of product and process development, sanquin blood bank, 2 mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers (140 ml) is unknown. if these products can be stored at -188c it will be feasible to store this product in 3-star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at -188c or <-25 to -358c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from 500 ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <-258c for 3-12 months and controlled thawing, six different sera were used to fill a large number of mini (140 ll) containers, which were refrozen and stored at either -188c or <-258c. during storage at 3 months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <-808c. growth factors tested were pdgf-aa&ab/bb, tgf-ß1/2/3, vegf, 80a transfusion 2017 vol. 57 supplement s3 egf, fgf2. the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß1 were the most abundant growth factors, on average 35, resp. 40 ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average 11 ng/ml. tgf-ß2, egf and vegf were detected at relatively low values, resp. 3 ng/ml, 0.5 ng/ml and 0.3 ng/ml. average levels of fgf2 and tgf-ß3 were close to detection limit (< 0.2 ng/ml). the controls stored at <-808c showed for all growth factors close to 100% of the initial values in samples at t50 (moment of filling mini containers). for serum stored at <-258c for up to 12 months, most factors showed less than 2 % decrease, except for pdgf-aa and tgf-ß2, showing 6% resp. 3% lower values. for serum stored at -188c the values for tgf-ß1, egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß2 showed a decrease of resp. 9, 17 and 3%. conclusion: human serum eye drops can be stored in the new micro dose device at -188c (3-star household freezers) or <-258c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at -188c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond 1 year. ruqayyah almizraq* 1 , heather inglis 2 , phillip norris 2,3 , jennifer a muszynski 4 , nicole juffermans 5 , jelena holovati 1 and jason p. acker 1,6 . 1 university of alberta, 2 blood systems research institute, 3 university of california, san francisco, 4 nationwide children's hospital, 5 academic medical center, 6 canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n58 per method). residual platelets and white blood cells (wbcs) were measured on day 5 using flow cytometer (fc). on storage day 5 and 42, number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day 5, apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p<0.01, wbd p<0.05) and wbf (vs: apheresis p<0.0001, wbd p<0.01) methods. while rcf units yielded the lowest count of platelet-evs (cd41a1) on day 5 and 42, the highest number of platelet-evs were in apheresis (day 5) and in wbd (day 42). similarly, there was significant difference among methods in the number of wbc-evs (cd31, cd141, cd161, cd191, cd66b1) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day 42 vs day 5 in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< 200 nm) was greater than large evs (! 200 nm) in all of the products on day 5 and 42, and the highest level of evs < 200 nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p<0.05). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at 48 centigrade maryanne c herzig* 1 , crystal lafleur 2 , chriselda g fedyk 1 , sherrill j. slichter 3 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research, 3 university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least 2 weeks of storage at 48c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at 48c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a2 (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for 10, 12, 15, or 22 days after collection. units were stored for 12 days without agitation. units stored for 10, 15 or 22 days were agitated during storage with a model 400 hybridization incubator at 48c set for end over end rotation at 2-3 rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at -808c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd40l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai-1) as another fibrinolytic measure; and complement activation markers c3a, c4d, c5a and c5b-9. data was analyzed by one way repeated measure anova. results/finding: only 49 6 12% of the platelets were recovered in units stored for 12 days without agitation. these levels did not meet fda requirements of 5.5 x 10 10 platelets per wb unit. subsequently, wb was agitated and platelet recovery was 71-76%. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t0 (day of collection) and t10, 12, 15, or 22 measurements. significant elevations of pai-1 and scd40l indicate activation of platelets and inhibition of fibrinolysis (p<0.001). activated complement peptides c3a, c5a, and c4d were all elevated over time (p<0.001) while sc5d-9 was not. however, only c3a and c4d levels at t22 were above normal reference ranges at 1.30 and 1.41 times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at 48c for 10-22 days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc5d-9 reported, wb showed elevation of c3a, 5a and c4d and not sc5d-9. complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* 1 and christian todd 2 . 1 cerus corporation, 2 community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly 1.1x10 10 6 5.6x10 9 9.8x10 10 6 5.6x10 10 310 6 330 430 6 440 900 6 260 28000 6 33000 3 6 3 186 31 30 6 22 110 6 97 rcf 1.9x10 10 6 7.4x10 9 4.2x10 10 6 1.1x10 10 13 6 4 316 14 530 6 160 5100 6 2000 3 6 3 96 7 176 7 346 11 apheresis 2.4x10 10 6 2.0x10 10 1.0x10 11 6 6.1x10 10 520 6 320 700 6 310 2200 6 1900 9800 6 4100 14 6 17 7 6 5 466 15 120 6 24 wbd 6.4x10 9 6 3.1x10 9 4.6x10 10 6 1.5x10 10 350 6 140 760 6 360 1000 6 180 4400 6 2400 3 6 2 426 23 57 6 24 120 6 56 platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following 4 collection targets: 4.4x10 11 in 350ml, 6.6x10 11 in 400ml, 6.8x10 11 in 400ml, and 7.0x10 11 in 400ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of 3.0x10 11 or 6.0x10 11 was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: 64% of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was 1.34. conclusion: it is possible to treat 64% of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward 100% while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* 1 , andrey skripchenko 1 , fei xu 1 , ying li 1 , stephen j wagner 2 , pamela h whitley 3 and jaroslav g vostal 1 . 1 fda/cber/ obrr/dbcd/lch, 2 american red cross holland laboratory, 3 american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature (4-6 o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts (11 hrs ct: 1 hr 37 o c [tc]). autologous apheresis plts stored for 7-days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n59) and the same non-labeled plts were also infused into mice (n590). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts <5% were considered background. results/finding: the mean recoveries of infused plts were 51.2 6 16.7% for rt, 37.7 6 12.3% for tc and 23.1 6 8.8% for ct in humans. in mice, mean recoveries of the same plts were 24.9 6 10.3% for rt, 19.1 6 9.8% for ct and 16.2 6 6.9 for ct (mean6sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $74% and ct was $45% of rt. in mice tc was $76% and ct was $64% of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to 100% for rt plts. human tc plts had 26% auc while ct plts had 11% auc compared to rt plts in humans. in comparison, the same tc plts had 39% auc and ct plts had 26% auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are 2.4 and 1.5, respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* 1 and geeta paranjape 1,2 . 1 coastal bend blood center, 2 carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g5 was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g5 with the compomaster net software for data management. implementation was planned for a november 2014 go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g5s and compomaster in june 2014. training and validations were successfully completed and a full launch occurred mid-march 2015. device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december 2014. validation was completed and signed off in march of 2015. manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g5 system. data points were collected from 210 units bi and 302 units ai. results/finding: upon initial implementation, staff training and use, the compomat g5 was found to be easy. plt weight spread was reduced from an average of 22gm to an average of 15 gm. actual plt weights were reduced from an average of 63gm to 59gm, resulting in an average increase in recovered plasma of 3.78ml per unit. plt count on average increased from a count of 1435 to 1506 (10 3 /mm 3 ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by 31.8% after implementation of the compomat g5 and our plt concentrations increased on average by 5%. we were able to consistently produce a smaller volume plt (average 59 gm), which gave us 3.78ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell 1 , angela hill 1 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: 13 abo/rh matched lr sagm rccs were pooled and split to produce 6 large (354 ml) and 6 small (244 ml) rccs. the rccs were stored to 14 d and glycerolized manually by mixing 400 ml of glycerol with the rcc in a 2000 ml freezing bag. units were frozen at -658c for ! 72 h before being removed from frozen storage and thawed in a 378c water bath. 3 large rccs and 3 small rccs were deglycerolized using the organization's current procedure on the cobe 2991 cell processor prior to re-suspension in 0.9% saline, 0.2% dextrose. the remaining rccs were transferred into a 1l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of 75 6 5%, and deglycerolized in a 275 ml centrifuge bowl on the acp-215 with re-suspension in as-3. rbc quality was tested at 24 6 2 h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p50.006, acp215: p50.007) and lower cell recovery (cobe: p50.002, acp215: p<0.001) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe 2991 had higher hemolysis (p<0.001) and supernatant potassium (p50.001) than did small volume rccs. large cobe 2991 rccs had higher hematocrits (p50.033), hemoglobin (p50.006), and recovery (p50.001) than did large acp-215 rccs. however, all cobe 2991 rccs had higher (p<0.001) hemolysis (0.99 6 0.24 %) levels than did acp-215 rccs (0.31 6 0.02 %). cobe 2991 rccs failed to meet regulatory hemolysis standards of 0.8%. conclusion: addition of a 400 ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as-3 and storage for 24 6 2 h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp-215 cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using 0.9% saline and centrifugation and the semi-automated washing method (sam) using the cobe 2991blood cell processor. study design/method: in this study, 20 units of single donor platelets were evaluated (10 washed using the mm and 10 washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas 6000. results/finding: table 1 shows that the average platelet recovery for the sam (92%) was significantly higher compared to the mm (82%). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took 10-15 minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas-3) for input platelet doses of 2.9 to 8.0 3 10 11 platelets in 255 to 420 ml of 47 to 68% plasma and 32-53% pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas-3 containing doses of 6.0 to 12.0 3 10 11 platelets in a volume of 420 to 650 ml. study design/methods: apheresis pcs (amicus v r ) were collected in 35% plasma and 65% pas-3. one study was performed at the nominal dose (9.2 -10.0 x10 11 platelets), volume (558 -629 ml) in 65% pas/35% plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition (9.7 -11.8 x 10 11 platelets in 593 -659 ml) using either single or pooled donations. input pcs (n520) were treated with the intercept ts set by the end of day 1 post collection; the incubation time in the compound adsorption device (cad) container ranged from 4 to 16 hours and the intercept treated pcs were stored in 3 containers (n560). day 5 and 7 post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas-3 treated in the intercept ts set demonstrated acceptable in vitro function (table 1 ). all intercept treated pcs had ph(228c) !6.2. platelet dose and volume recovery post-treatment ranged from 82% to 99% and 88% to 92%, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through 7 days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing 100% plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n56). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n56). numerous in-vitro quality markers (plt concentration, atp, po2, pco2, ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days 1, 3, 5 and 7 for apheresis pcs, and on days 2, 3, 5 and 7 for wb-derived pcs. two flow cytometry assays were used to evaluate cd62p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion 2017 vol. 57 supplement s3 results/finding: platelet recovery was 92 6 5% and 81 6 10% for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells (3% 6 1 (test), vs. 1.7% 6 0.5 (ctl) on day 5) and a higher rate of cd62p expression than control pc units (58% 6 7 (test), vs. 23% 6 6 (ctl)) on day 5). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* 1 , lorraine blagg 2 , christi e marshall 1 , herman woodson 1 , sean erony 1 , krishna patel 1 and eric gehrie 3 . 1 the johns hopkins hospital, 2 johns hopkins hospital transfusion medicine dept, 3 johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. 340 intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day 4. as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day 3. the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of 340 lrap were tested. 335 lraps initially tested negative by bactx, while 5 lraps initially tested positive by bactx. all 5 initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was 98.5%. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only 340 platelet units. the expected rate of bacterial contamination of platelets is less than 1 per 2000 units. the 1.5% initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as-1 and cp2d/as-3 rbc alan d. gray* 1 , matt landrigan 2 , pamela whitley 3 , michael wellington 3 , sherrie sawyer 3 , shalene hanley 3 , emily rondeau 4 , louise herschel 4 , neeta rugg 5 , patricia a.r. brunker 3 , shawnagay nestheide 5 , jose cancelas-perez 5 , larry dumont 6 and zbigniew m. szczepiorkowski 7 . and 2,3-dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for >24 hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood (530-550 ml) was collected and processed at 3 sites into leukocyte-reduced rbc (a total of n563 cpd/ as-1 and n564 cp2d/as-3). 50 ml of rejuvenation solution (citra labs) was added to each rbc on day 35 (d-35), incubated for 60 minutes with agitation at 378c water bath (helmer dh4), washed (haemonetics acp215), and stored in as-3 at 1-6 oc for 7 days (d-36 through d-42). in vitro recovery (%) was calculated and hemolysis, atp, and 2,3-dpg were determined on day 0, d-35, d-35 after rejuvenation and washing (postrjv), d-36, d-38, d-40, and d-42. all units were cultured on d-35 postrjv and on d-42, and then concentrated by centrifugation on d-42. results/finding: in vitro rbc recoveries were 95.7% and 95.5% (as-1 and as-3, respectively) and no bacterial growth was observed. hemolysis on d-42 was maintained <1% in 58/63 (92%) as-1 units and 63/64 (98.4%) as-3 units. all as-1 and as-3 units (100%) had hemolysis <1% following concentration by centrifugation. morphology score was reduced to 77% (as-1) and 74% (as-3) by d-35, restored after rejuvenation (91%, 92%, respectively) and maintained through d-42 (>90%). atp was restored and maintained above fresh levels after rejuvenation. 2,3-dpg was restored above fresh levels and was maintained !80% of fresh levels through d-38. all values were significantly different compared to d-35 except as noted (p<0.001, paired ttest) ( table 1) . conclusion: rejuvenation of stored rbc restores atp and 2,3-dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d-42 when compared to nonrejuvenated rbc on d-35. this study is funded by zimmer biomet. storage >24 hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante 1 , jason p. acker* 1,2 and jelena holovati 1 . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during 42-day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and 2,3-dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome 1 rejuvesol-treated (l1r). the prbcs were incubated for 1 h at 378c with hepes-nacl (sham), liposomes (dopc:chol, 7:3 mol%, 2 mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and 2,3-dpg at day 42 hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control (0.60 6 0.06%): l (0.53 6 0.01%, p50.042), r (0.43 6 0.02%, p50.004), l1r (0.48 6 0.06%, p50.020). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r (0.55 6 0.01, p50.010) and l1r (0.55 6 0.01, p50.010) treatments compared to s (0.53 6 0.01) but not l (0.53 6 0.01, p50.936). rbc rigidity (kei) increased in all treatments compared to sham (1.19 6 0.07): l (1.28 6 0.06, p50.025), r (1.44 6 0.17, p50.010) and r1l (1.44 6 0.06, p50.004). aggregation amplitude was significantly increased by r treatment only (24.07 6 1.67 au vs. 19.12 6 1.38 au, p50.004). atp levels were significantly higher in all treatments compared to sham (1.64 6 0.14 mmol/g hb): l (2.00 6 0.21 mmol/g hb, p50.010), r (4.70 6 1.20 mmol/g hb, p50.004), l1r (5.00 6 1.56 mmol/g hb, p50.004). the levels of 2,3-dpg were no longer detectable in s and l treatments at day 42. the combined treatment was comparable to r (2.38 6 3.26 mmol/g hb vs. 2.62 6 2.20 mmol/g hb, p50.868). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l1r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer 1 , eric ducas 1 , patricia landry 1 , nathalie dussault 1 , jacques bernier 1 , danny brouard* 1 and anne maltais 2 . 1 h ema-qu ebec, 2 institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the 500-ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < 108c) of one to six 500-ml whole blood units (wbu) within 8h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between 18c and 108c for 24h under extreme external conditions (-308c to 408c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned 58c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for 24h at 20-248c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, 500-ml wb bags were filled with 555 ml saline 0.9% at t 5 308c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (-308c) and summer (408c) conditions. shipping boxes were filled with either one or six bags (n5 2). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under 108c in 4.55 6 0.62h and maintain their internal temperature between 18c and 108c for 24h with final values ranging between 6.38c and 9.38c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the 108c threshold value in 2.4 6 0.2h and the bags' internal temperatures were within the acceptable range for 24h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than -138c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* 1 , christopher c. c silliman 2 , beth shaz 1 , marguerite kelher 2 and claudia s. cohn 3 . 1 new york blood center, 2 bonfils blood center, 3 department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either 100% donor plasma (n550) or 65% pas-3 / 35% donor plasma (n550). within 12 hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd40 ligand (scd40l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c (3/50 products) compared to plasma platelets (2/50 products); however, the hla-antibody screen-positive supernatants of pas-86a transfusion 2017 vol. 57 supplement s3 abstract c platelets had fewer hla specificities (2 specificities) compared to those of the plasma platelets (18 specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd40l levels were increased in the supernatant of pas-c compared to plasma platelets (table 1) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd40l and not bioactive lipids. although scd40l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd40l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below 5x10 6 wbc in us and 1x10 6 wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n52) and leukoreduced (lr) rbc units (n52) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of 1000 wbc/ul. the spiked samples of 5, 12.5, 5, 25, 50 and 100 wbc/ul were prepared from the source sample of 1000 wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations (0, 12.5, 5, 25, 50, 100 wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples (0, 5, 25, 50 wbc/ul). 10 tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of 20 samples (plt and rbc) were run on both analyzers, repeated for 5 days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt50.996, slope50.972), (r-rbc50.999, slope50.992). the %diff-plt at 5, 25, 50 wbc/ul were 7.8, 4.7 and 10, respectively. the %diff-rbc at 5, 25, 50 wbc/ul were 10.8, 3.2 and 14.7, respectively. the average total testing time was similar on both instruments; 89 min for the facsvia and 92 min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated 62% (56 of 89 min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of 56 minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* 1 and mickey koh 2 . 1 department of laboratory medicine and pathology, university of minnesota, 2 st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of 62 completed and partially completed surveys were received. results/findings: responses came from 13 countries, but the majority of responses came from the united states (us). of the respondents, 35% reported aprp use in their hospital. aprp was used predominantly for outpatients, though >40% of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by 1-5 mds; however, 3 hospitals had >10 mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures (97%); however, 3 respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the 3 hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, 2017) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n56) under blood bank conditions at day 3 (d3), at day 42 (d42), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from 2.1-8.8% at d3 to 8.3-68.1% at d42. rejuvenation markedly reduced this storage-induced spherocytic shift (1.7-29.3%) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell 1 , angela hill 1 , tracey turner 2 , april xu 2 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units (314 6 15) than in rcf units (275 6 16), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as-3 additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: 12 small rcf (252-263 ml) and 12 large wbf (322-353 ml) rccs were stored for 21 d before being glycerolized and frozen at -658c for !72 h. large rccs whose red cell mass exceeded the capacity of the 275 ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a 378c water bath, deglycerolized and re-suspended in as-3. rccs were stored 14 d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p<0.05) hematocrit, specific gravity, hemoglobin per unit, supernatant k 1 and na 1 concentration, deformability (ei max ), and higher (p<0.001) recovery than did large wbf units. no significant differences in hemolysis, atp, 2,3-dpg, p50, rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table 1) , however 8 of 12 large wbf units had rbc recoveries < 80% due to pre-glycerolization volume reduction, and 2 of the small rcf units had hemoglobin values < 35 g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above 80% and the hemoglobin failure rate would be below 10% of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. 0.03 6 .02 0.33 6 .28 0.83 6 .3 0.32 6 .14 elongation index (30pa) 0.602 6 .008 0.585 6 .017 0.580 6 .017 0.578 6 .017 this study is funded by zimmer biomet. (hasan 1994) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo 2 ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for 42 days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two (52) rbc units (leukocyte-reduced), cpd/as-1 or cp2d/as-3, on day 0, day 42, and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o 2 /g hb) and total releasable oxygen (tro) of the unit (ml o 2 ). orc was determined by assessing the change in % o 2 saturation from 100 mm hg po 2 (e.g., lung) to 40 mm hg po 2 (e.g., venous blood) multiplied by 1.34 ml o 2 /g hb (li 2016). a simulated baseline pretransfusion vo 2 of 115 ml o 2 /min was estimated using the day 0 orc and assuming a 7 g/dl transfusion trigger with a cardiac output of 5 l/min and 5 l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day 42 restored orc and tro to levels greater than day 0 ( table 1) . orc of the rejuvenated unit was 1.5 6 0.2 times and 3.4 6 0.5 times greater than rbc on day 0 and day 42, respectively (p<0.001). vo 2 increased after a simulated single unit transfusion of rbc (day 0, day 42, and pw) by 19.3%, 8.9%, and 28.8% over the pre transfusion vo 2 , respectively (p<0.001). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release 3.3 times the volume of o 2 compared to standard, untreated rbcs stored for 42 days. inferior oxygen delivery to tissues (vo 2 max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for 42 days vs 7 days which seem dependent on genetic variability and storage time (bennett-guerrero 2017). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* 1 , jay srinivasan 1 , gustaaf de ridder 2 , alan d. gray 3 , matt landrigan 4 , keaton charles stoner 5 , angela crabtree 6 , jessica poisson 7 and ian welsby 8 . 1 duke university school of medicine, 2 duke health pathology, 3 citra labs, a zimmer biomet company, 4 zimmer biomet, 5 duke university, 6 department of pathology, durham veterans affairs medical center, 7 duke university hospital, 8 duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted 2,3-diphosphoglycerate (2,3-dpg). the loss of 2,3-dpg increases the oxygen affinity of hemoglobin, resulting in lower p50 (partial pressure of oxygen at 50% hemoglobin saturation). decreased p50 may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and 2,3-dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at 378c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a1, leukoreduced prbc stored in as-1 were obtained from our local blood center. after 3 days of storage, units were divided into 4 separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution (50ml) was added to the cr group, and all groups were then stored for another 12 days at 1-68c. on day 15 of storage, the sr group was incubated for 1 hour at 378c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p50 was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps1) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a 5 0.05. results/finding: significant differences in p50 were noticed between all groups (table 1) . ei, ps1, and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p50) seen over 15 days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p50 that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k 1 , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc (300ml) units stored in as-1, obtained from a regional blood donor center at expiration (42 6 2 days), were passed by gravity through sorbent-devices containing 50 ml of multifunctional polymer bead, at a flow rate of 20 ml/min. supernatants were analyzed for k 1 removal as well as free hb, antibodies and cytokines (27-plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k 1 ] from 54.2 6 5.0 to 1.98 6 1.3 meq/l; equivalent to an 84.6% reduction. free hb was reduced by 96.3% from 2.5 6 1.0 to 0.39 6 0.2 mg/ml. antibodies, specifically igg, iga, and igm decreased from 9.91 6 3.1 to 2.40 6 1.1 mg/ml (77.7%), 0.48 6 0.1 to 0.25 6 0.01 mg/ml (48.9%), and 0.73 6 0.2 to 0.49 6 0.1 mg/ml (31.5%), respectively. inflammatory cytokines were significantly reduced, specifically: ip-10 from 144.27 6 16.2 to 18.43 6 2.7 pg/ml (87.2%), mip-1b from 37.37 6 5.7 to 7.23 6 2.5 pg/ml (80.7%), and pdgf from 1348.3 6 291.9 to 77.91 6 22 pg/ml (94.2%). filtration had no significant impact on cell surface markers of rbc viability (<0.1% decrease) or sensitivity to osmotic changes. values listed represent mean 6 sem (p < 0.01 for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k 1 as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k 1 along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency (1.5%) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in 50 apheresis and 50 whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia 2120 (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on 10000 permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and 2,3-dpg profiles to fresh levels. the objective was to compare 50% hemoglobin-oxygen saturation (p50) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was 97.2 6 2.2%. hemolysis (%) was similar on day 42 before and after dry-air incubation with the rejuvenation solution (0.34 6 0.14% vs 0.35 6 0.14%). percent hemolysis (%) decreased after washing (0.24 6 0.07%) and was maintained below <1% for all units during storage for 24hr (0.51 6 0.19%). average atp and 2,3-dpg were restored above the average fresh values. the morphology score decreased $25% by day 42, which was restored to near fresh values following rejuvenation and washing and storage 24hr (93.7% and 95.1%, respectively). rbc oxygen affinity, as assessed by p50, was restored above fresh values. all values were significantly different compared to day 42 (p<0.001, paired t-test) ( table 1) . conclusion: rbc morphology was restored to near fresh and average atp, 2,3-dpg, and p50 were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and 2,3-dpg were maintained during storage 24hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d9tetrahydrocannabinol (thc) and 11-oh-d9-tetrahydrocannabinol (11-oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and 11-oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and 11-oh-thc to produce samples for lc-ms/ms analysis. lc used a c18 column. post-column detection by ms/ms used positive ion electrospray with q1:q3 ion pairs of m/z 5 605.3:225.3 (internal standard (is), d3-thc), m/ z 5 602.2:225.2 (thc), and m/z 5 618.3:256.1 (11-oh-thc). quantitative results for thc and 11-oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from 0-50 ng/ ml for both thc and 11-oh-thc. limits of quantitation, defined as 5 standard deviations above background, were 0.7 ng/ml for thc and 7 ng/ml for 11-oh-thc. results/finding: a total of 424 donor plasma samples were tested for thc and 11-oh-thc. no samples tested positive for either thc or 11-oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a 50% probability of one or more positives at a prevalence of 0.16% positive samples, and a 95% probability of one or more positives at a prevalence of 0.71% positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than 1% among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than 12 hours for post-exposure detection of thc and/or 11-oh-thc in plasma. conclusion: testing of 424 donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than 1%. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* 1 , ruqayyah almizraq 2 , daniel millar 3 and jason p. acker 4 . 1 university of british columbia, 2 university of alberta, 3 lightintegra technology inc., 4 canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days 7, 21, and 42 of storage. one rcf rcc was tested on days 1, 5, 14, 21, and 43 and six 10 ml aliquots were stored in parallel and tested on days 14, 21, and 43. all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days 14, 21 and 43 (p<0.001) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the 6 aliquots were consistent at each time point but statistically higher than in the original rcc on and after day 21 of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow 100% screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n 5 60) and paired ffp aliquots were stored for 31-33 days at 2-68c and -188c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! 80% levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c1 esterase and alpha 1-proteniase inhibitors). the level of factor xiii in odp was slightly lower, about 70% of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i1ii and ddimer) and complement (c3a and c5a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion 2017 vol. 57 supplement s3 abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to 62% in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to 15 minutes. in addition, donations with collection times between 12 and 15 minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from 12-15 minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n56) were prepared from one 12-15 minutes bc and 60 ml of autologous plasma in a 600 ml pvc-dehp container. as a reference, spc from donations with collection times of <12 minutes were prepared (n55). in addition, pc were prepared from 5 bc, of which at least 4 bc were from 12-15 minutes donations (n55). after pooling of the bc, 300 ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for 8 days at 22 6 28c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean6 sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume (67 6 5 vs. 66 6 16 ml) and platelet content (74 6 11 vs. 71 6 15 x10 9 ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day 8, ph(378c): 6.84 6 0.16 vs 6.83 6 0.17, other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day 6 and/or 8 in 2/5 pc (possibly because sometimes ab0 incompatibility was accepted). on day 8, plt showed low cd62p expression (17.1 6 1.8%) and phosphatidylserine exposure (annexin v binding, 8.9 6 1.9%). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from 12-15 minutes whole blood donations had a normal composition and showed good in vitro quality during 8 day storage. to substantiate that the exclusion of 12-15 minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* 1 , jessie miller 1 , ranee marie wannarka-farlinger 1 , sandra bryant 1 , scott a hammel 1 , sherry stern 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as-3 rbcs, 20 irradiated and 21 non-irradiated, were selected for the study. the units varied in age, ranging from 2 to 42 days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< 0.05) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p50.02). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after 5 days of storage pei lun karen lim* 1 , erma sofia sumardi 1 , isamar eduardo ancheta 1 , susan lim 2 , christina yip 1 , lip kun tan 2 and shir ying lee 3 . 1 national university hospital singapore, 2 national university hospital, 3 national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within 8 hours of processing and stored at -188c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of 24 hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for 5 days and kept at 1 to 68c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than 50 iu/dl. study design/method: randomly selected units of ffp (n510) were measured for fviii concentration based on clotting assay (stav r -deficient 92a transfusion 2017 vol. 57 supplement s3 viii diagnostica stago). fviii levels were measured at five time points: prefreezing, 0, 24, 72 and 120 hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at 30 to 378c for 35 minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration (0 hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at 1 to 68c for 5 days for subsequent testing. results/finding: results obtained were listed in table 1 . units 7 to 9 were not tested for fviii at post thaw-24 hour due to operational issues. the overall fviii concentration decreased at an average of 13% from pre-freezing to post thaw 0 hour. after further storage of tp post thaw-24 hour and -72 hour, residual fviii level remain to be above 50 iu/dl except unit 10 which had a lower initial fviii concentration. at post thaw-120 hour, 7 out of 10 units tested had residual fviii activity within the pre-set standard of 50 iu/ dl. the average decline from 0-hour post-thaw to 24-hour, 72-hour and 120hour post-thaw was 36.5%, 42.7% and 47.9% respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least 50 iu/dl of fviii. typically patients with factor levels below 30 iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi 1 , ayman mohamad sabri 1 , ali abdullah alajeafi 1 , ashwaq hasan alhekri 1 , saleem bin mahfouz 1 , ali hasan alkhodari 1 , rawya saeed shealy 1 , marcus picard-maureau* 2 and hussain bana almalki 1 . 1 king abdulaziz hospital and oncology center, 2 cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in 100% plasma over a 5 day storage period and the new "test" pathogen-reduced, pooled (pools of 5) prp pc in 100% plasma over a 7 day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of 4 leucoreduced test pcs were assessed at day 7 of storage and compared to leucoreduced control pc at day 5 of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day 0; the process was completed by day 1 post-collection. samples were taken daily for quality analysis from test and control pc until day 5 and day 7, respectively. for bacterial spiking, additional pc were spiked with each receiving 4 ml of 1 mcfarland ($ 1.2x10 9 cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was 4.7% 6 2.0, the total average platelet loss at day 7 was 11.2% 62.8. the average platelet loss in the control units at day 5 was 9.5% 61.4. the average ph of the test units at day 7 was 6.64 60.04 and in the same range as the control pc, ph 5 6.89 60.09. glucose concentration in test pc at day 7 (13.8 63.0 mmol/l) was lower than in the day 5 control units (18.32 61.06 mmol/l). lactate levels increased during the course of storage; lactate levels at days 5 and 7 were outside the range of the assay (>15 mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after 7 days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* 1 , jay srinivasan 2 , jessica poisson 3 and ian welsby 4 . 1 duke university, 2 duke university school of medicine, 3 duke university hospital, 4 duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified 256 proteins in cryo; of the 10 most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf (4.44 mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial (0á2 lm) filtration. cryoprecipitate mini-pools (400 6 20 ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di(2-ethylhexyl) phthalate (dehp)] adsorption device and a 0á2 lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table 1) . kit ensured bacterial sterility (table 3 ) and most importantly, final product was free of hbv, hcv and hiv (table 2) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to 4 days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase-1, thereby blocking synthesis of thromboxane a 2 from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n518) were prepared from a nsaid-bc and 60 ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for 8 days at 22 6 28c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n55) were investigated as a reference. values are expressed as mean6sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume (69 6 4 vs. 66 6 16 ml) and plt content (67 6 14 vs. 71 6 15x10 9 ) were similar in both groups. on day 8, both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day 8 was significant higher in a subset of donors who had used ibuprofen (n55). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin (0,0-30, p<0.05), diclofenac (31,1-76) and naproxen (0,0-24, p<0.05), compared to normal controls (76, . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day 1 in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (<24 hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, 2014] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type 2 diabetes (t2d). because of the strong rise of people with t2d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t2d, but accepted as donor. study design/method: twelve whole blood donors with t2d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for 8 days at 22 6 28c and sampled on day 1, 4 or 5 and 8. the diabetic marker hba1c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 3 'good' (ph day8 >6.6) and 3 'poor' (ph day8 <6.3) storing spc were selected and analysed in more detail. results/finding: donors were of age 57 6 10 year and primarily men (75%). donors with t2d had a higher mean bmi (30.3 6 4.6 vs.25.4 6 3.4 kg/m 2 ) and higher hba1c than controls. the spc of both groups had the same volume (70 6 5 vs 726 2 ml) and plt content (71 6 9 vs 73 6 11x10 9 ) but on day 1 glucose concentration was higher in the diabetic group (20.5 6 1.7 vs 18.9 6 1.4 mm, p<0.05). on day 8, the average in vitro quality was comparable in both groups (data not shown). when combining 94a transfusion 2017 vol. 57 supplement s3 the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed (0.14 6 0.04 vs 0.36 6 0.03 mmol/ day/10 11 plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc-1) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day 1 ('poor':2.2 6 0.7 vs 'good':1.1 6 0.2, p<0.01). conclusion: bc from donors with t2d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t2d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of 30 minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of 2017 detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table 1). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to 60 minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* 1,2 , katharine a downes 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts13 are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy 12 month old unvaccinated girl presented with history of diarrhea for 5 days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: 32 x 10 9 /l, platelets: 62 x 10 9 /l, bun: 77mg/dl, creatinine: 2.4mg/dl, lactate dehydrogenase 1940 u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to 44 x 10 9 /l, adamts13 sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation (74 x 10 9 /l and a-ipc of 4.7 x 10 9 /l). two consecutive tpe were completed which resulted in a platelet count decrease to 54 x 10 9 /l and a-ipc of 5.1 x 10 9 /l. a-ipc ratio was 1.1 below the ratio of 3 which has been reported for ttp patients. similarly a-ipc count was not below 5 x 10 9 /l threshold reported in setting of ttp with severe adamts13 deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o157:h7 toxin. testing of c3, c4, factor h, factor h autoantibody, factor i and factor b were normal. adamts13 activity was 93%. patient was treated for the infection and platelet count improved within 10 days to 315 x 10 9 /l, with resolution of her renal failure: bun: 42 mg/dl, creatinine: 0.65 mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts13 testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts13 activity. many patients with severe autoantibody-mediated adamts13 deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts13 activity <5%) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value 2-3 days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column 1), increased peak a-ipc value (results shown as percent increase, column 2), delayed a-ipc peak, and delayed plt recovery (table 1) . moreover, recurrent episodes required more procedures compared to initial presentation (table 1) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect 3 cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february 2016 to march 2017 on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the 2 nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs1 separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version 5.0 after taking consent from the donors. the target collection of each procedure was a dose of 3 x 10 11 platelets in 200-250 ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at 2 consecutive donations within 7 days were considered. data was analyzed by stata 14. within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the 98 donors, 35 repeated the plateletpheresis within a week (group i) and 63 underwent 2 nd plateletpheresis within 8-30 days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the 2 groups (p50.025). though above the eligibility cutoff of 1.5 lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at 2 nd plateletpheresis in group ii donors. there were 49 donors who presented to us for the 3 rd time for plateletpheresis with a mean gap between 1 st and 3 rd plateletpheresis being 46 days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p50.000). plateletpheresis through all the 3 cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type 1 receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type-1 receptor antibody (at1rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at1rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at1r ab are reviewed. results/findings: case 1: the patient is a currently 43-year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age 22, and a second deceased donor transplant due to a rejection of the transplanted kidney at age 38. three years post-transplant, her creatinine (cr) started to rise from 0.7 to 1.35 mg/dl and a biopsy showed banff criteria grade 2 amr, grade 2a t-cell mediated rejection (tcmr) and grade 3 interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at1rab was identified at >40 u/ ml (high: >17 u/ml, intermediate: 12-17 u/ml, negative: <12 u/ml). she received 6 tpe treatments every other day and started losartan. after a course of tpe, at1rab decreased to 32 u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to 1.9 ml/dl. in one month, her at1rab increased again to >40 u/ml, therefore, she received 3 more tpe treatments with a decrease in her at1rab to 16 u/ml. although at1rab level increased slightly to 20 u/ml after 3 months, her cr has been stable at 1.3-1.6 ml/dl. case 2: the patient is a 25-year-old mean 1/-se -54.71/-12.9% * 183.1%1/-12.8%* * p<0.05 96a female with malignant hypertension who received a deceased donor kidney transplant at age 24. her cr started to rise 2 weeks post-transplant from 1.4 to 2.68 mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at1rab level at 18 u/ml. she received 6 tpe procedures every other day and at1rab decreased to 8 u/ml with a decrease of cr to 1.98 mg/dl and improved arteriopathy in histology. because her at1rab level slightly increased to 12 u/ml over the next 2 weeks, she started weekly tpe treatment. after 5 weekly tpe, tpe treatment was stopped because her at1rab level remained relatively unchanged. her cr has been stable at around 1.5 ml/dl to date. conclusion: we present 2 kidney transplant recipients who received tpe treatments for high at1rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at1rab levels; however, weekly tpe had no effect on reducing at1rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a 36 year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < 10%], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a 33 year old man (patient b) with a medical history of hypothyroidism (on synthroid for 2 years), end stage renal disease and non-ischemic cardiomyopathy (ef of 20-25%) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a 1-1.5 plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within 12 hours of the procedure completion. their total t4, t3 and free t4 levels trended to normal or near normal range within 24 hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy 3-4 weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* 1 , laura martínez molina 2 , cristina muniesa montserrat 2 , octavio servitje bedate 2 , silvia cosano navarro 1 and maria isabel gonz alez medina 1 . 1 banc de sang i teixits, 2 dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since 30 years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last 20 years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) 2016, as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category 1. since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of 10 patients diagnosed with ss and compare them in their first evaluation once the 20 th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( 8-methoxypsoralen, 8-mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from 1.5 to 2 total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in 2 where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in 7/10 patients) and with online system (therakos) just in 3. main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is 77'7% (partial remission 66.6% and complete remission 11.1% with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : 3835 u/l) requiring transfusions, mild thrombocytopenia (144 x 10 9 /l), acute kidney injury (bun 175 mg/dl, creatinine 2.51 mg/dl). by the third hospitalization day hgb improved to 10 g/dl, however with worsening thrombocytopenia (16 x 10 9 /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days 6-10). platelet count and a-ipc improved to 52 x 10 9 /l and 6.6 x 10 9 /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count (30 x 10 9 /l) and a-ipc 1.98 x 10 9 /l. these dynamics did not resemble those which had been described for ttp patients with adamts13 deficiency. adamts13 obtained prior to tpe initiation was resulted at this time and was 67%. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc (2.64 x 10 9 /l) that preceded platelet count increase to 80 x 10 9 /l three days later when patient was discharged. other laboratory values at this time were ldh of 635 u/l, hgb: 11.2 g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts13 deficiency eiman hussein* 1 and jun teruya 2 . 1 department of clinical pathology, cairo university, 2 texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts13. since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts13, the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts13. the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january 2008 through march 2017 were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts13 activity of less than 10%. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts13 deficiency. eight patients (25%) were associated with suspected bacterial infection. four of the 8 patients (50%) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in 3 patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than 10 kilograms using a single apheresis procedure. study design/method: in october 2015 and june 2016, two children with possible leukemia were submitted to tl procedure. they were 6 and 9 months old, and weighted 7,0 and 9,1 kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with 285 ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, 64% hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin (750 ml of acd-a and 7,500 units of heparin), at a blood to anticoagulant ratio of 25:1. a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every 30 minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were 120.000 and 150.000/ mm 3 . the formula "collection pump flow 5 0,0003 x inlet flow x preapheresis wbc count" was used with the goal of removing up to 3 x 10 9 leukocytes/ml. a single leukapheresis procedure was performed with 2 total blood volume processed per patient. immediately after the 2-hour procedures, wbc count were 74.000 and 92.000 wbc/mm3, and 12-hour post tl, wbc count were respectively 45.000 and 70.000/mm3. net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing 10 kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since 2006. we report the data from the year 2016. study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of 13,302 apheresis procedures were performed at 37 hospitals. therapeutic plasmapheresis was the most frequent procedure (50.4%) followed by autologous peripheral blood stem cell (pbsc) collection (23.9%), allogeneic pbsc collection (11.0%), donor leukapheresis (4.0%), and therapeutic leukapheresis (3.9%). cobe spectra (37.4%) and amicus (16.8%) were the most widely distributed instruments. centrifugation was the dominant technique (92.2%) for therapeutic plasmapheresis. detailed information was given for 4,199 therapeutic plasmapheresis procedures performed on 786 patients (some items were not completely filled out). spectra optia (42.7%) and cobe spectra (26.6%) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently (47.2%) as the replacement fluid followed by 5% albumin (26.3%), 4% albumin (13.3%), and 5% albumin 1 ffp (11.1%). most of the procedures were performed for 1 plasma volume (72.4%). acd (88.4%) and heparin (11.5%) were used for anticoagulation. central venous catheter (91.9%) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation (24.1%), antibody mediated rejection in renal transplantation (19.9%), thrombotic microangiopathy (11.5%), desensitization for abo compatible renal transplantation (4.7%), neuromyelitis optica spectrum disorders (4.6%), and hyperviscosity in monoclonal gammopathies (4.6%). adverse reactions were observed in 8.5% of the procedures. allergic reaction (55.2%), hypocalcemic symptom (20.4%), and hypotension (6.9%) were frequently reported. therapeutic effect was achieved in 86.5% of the patients. our apheresis registry has been well run for 10 years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride >1000-2000 mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $18 deaths/100,000 cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of 41 pts who were diagnosed with hp from january, 2009 through january, 2017, and referred for immunotherapy evaluation. 27/41 (66%) pts received conventional therapy (ct) and pe (pe group), and 14/41 (34%) pts received ct alone (ct group). mean age was 36 years (range 16-79), and 56% were female. baseline mean triglyceride level (normal <150 mg/dl) for pe group was 6,728 mg/dl (4,652-12,486) versus 3,142 mg/dl (1,697-5,120) for ct group. baseline mean lipase level (normal <393 u/l) for pe group was 1,798 u/l (797-2,745) versus 923 u/l (472-1,796) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving 2-3 medications. 24/27 (89%) of pe group and 11/14 (79%) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. 20/27 (75%) of pe group and 6/14 (43%) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of 2.85 pe treatments (txs) (median of 2, range 1-4 daily txs) using 5% albumin; 7/27 (26%) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were <3000-4000 mg/dl and lipase <950-1375 u/l (2.5-3.5 x upper limit of normal). mean triglyceride levels after 2 pe txs were 1,976 mg/dl (627-3,968) for pe group (mean decrease 72%); mean triglyceride levels after 48 additional hours of ongoing ct were 1,576 mg/dl (487-2,873) for ct group (mean decrease 50%). while the pe group achieved a greater mean decrease in triglyceride levels after 2 pe txs (compared to the ct group after 48 hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p>0.05). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a 47-year-old man with a chronic history of hypertriglyceridemia >1000 mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa 2016 guidelines, in a patient unresponsive to optimal medical management. asfa 2016 guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed 39 tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely 5% albumin for exchange fluid (100% albumin procedures) or partial plasma replacement (2-3 units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while 27 were performed with 100% albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* 1,2 , metha apiwattanakul 3 , sompis santipong 2 , jutaluk jaipian 2 , jettawan siriaksorn 2 and ponlapat rojnuckarin 1 . 1 chulalongkorn university, 2 king chulalongkorn memorial hospital, 3 prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june 2016 through february 2017 were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a 20% solution. before using, it was diluted to a 4% albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of 156 tpes in 38 patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was 3,000 (range 1,750-4,200) ml. although the corrected calcium level was low (<8 mg/dl) in 3.2% (5/156) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in 2 patients. the first patient had 2 events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was 1.9% (3/ 156). in 2014, the incidence of tpe adverse effects was 1.6% (2/125) when commercial albumin was used. the difference was not statistically different (p 5 1.000). median serum albumin levels pre-tpe and post-tpe were 3.6 (1.9-4.4) and 3.9 (2.4-5.0) g/dl. the increase in serum albumin after tpe was statistically significant (p<0.001). eighty-two percent of pre-tpe serum albumin levels were lower than 4.0 g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether 60 outpatients who underwent a total of 100 la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer (75%) and ovarian cancer (20%). based on differences in the study protocols la was performed either one-time (41%), two-times (27%) or three-times (32%), with an interval of at least 2 weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, 3 out of 60 patients (5%) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in 16% of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was 1.4x10 10 wbc consisting of 1.1x10 10 mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was 32% lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg4-related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category 1 indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a 70 year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to 57,000 iu/mls (ref. range < 35) and anti-cyclic citrulline peptide antibody was elevated to 34,339 units (ref. range < 20) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were 4610, 2890 and 1320 mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was 1.74. peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be 8.5 centipoise (cp) at admission (ref. range 1.6 -1.9). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated -1 total plasma volume; replacement fluid -5% albumin and normal saline in a 50:50 ratio; replacement fluid volume: 110% of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to 2.4 cp. serum igg, igm and iga levels decreased to 2040, 1510 and 672 mg/dl respectively. her rf had decreased to 19,900 iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than 3 cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan 2013 -dec 2016). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with 5% human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between 4 to 35 years age (m: f; 1:2) underwent 62 tpe procedures with an average of 5.6 per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three (27%) patients had only visual symptoms, 5 (46%) had both visual as well as muscular symptoms whereas 3 (27%) patients had muscular symptoms only. three (27%) out of the seven tested, were positive for aqp4-igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in 8 patients with grade-1, in 1 patient, and by grade-2, in seven. adverse events were observed in 8% (5/62) of the procedures with allergic reactions to replacement fluid as most common event (n-3) followed by hypotension (n-2). follow up was available in 55% (6/11) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after 3 months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from 6.0 to 6.4 for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version 6.4 as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version 6.0. the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to 1/16/2016, plt collections were performed on nine trima accel machines operating with version 6.0. upgrading and validating all nine machines to version 6.4 occurred from 1/16/2016 to 4/30/ 2016. the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version 6.0 (5/1/2015 to 9/30/2015) was compared to version 6.4 (5/1/2016 to 9/30/ 2016). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version 6.0 and 6.4, adjusting for multiple visits per donor, with significance defined as p-value < 0.05. results/findings: following the upgrade to version 6.4, staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version 6.4 of the trima accel showed a statistically significant increase in possible leukocyte contamination from 3% to 5% of collections as compared with version 6.0. both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version 6.4. conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version 6.4 software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version 6.4 currently does not provide added value over version 6.0 for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in 32 yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is 30 weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of 500 ml 5% albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using 500 ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of 500 ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a 45 years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at 20 years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with 3 acute episodes requiring prolong hospital admission of approximately 2 months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of 4980 mg/dl, lipase 92 u/l, glucose 250 mg/dl, bicarbonate 24 mmol/l, anion gap 12. ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega 3 fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day 3 of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day 3 after admission. results/finding: the patient tg decreased by 52% (2365 mg/dl) with medical therapy, followed by additional 67% (767 mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day 6 after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below 1000 mg/dl at 20 days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from 2009 to 2013, a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected 5,129 cases whose ferritin levels have been monitored more than twice with an interval of detection in 150-160 days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over 50 lg/l. and the upper limit was set to be 244 lg/ l in male and 158 lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: 0 times, 1 to 3 times, 4 to 6 times, 7 to 9 times and more than 10 times. the high frequency (more than 10 times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were 5,129 donors included in the study, of which 4,944 were male (96.4%) and 185 were female (3.6%). the mean ferritin was 82.0 lg/l in male (95% ci: 80.7-83.2 lg/l) and 66.5 lg/l in female (95% ci: 60.9-72.0 lg/l). the result of anova indicates that the group with the highest frequency (more than 10 times) has the significant lowest ferritin level (p<0.05). the average change of ferritin if donation over 10 times would up to 13.4 and 14.1 lg/l in younger and elder 50 y/o male and 18 and 23 lg/l in female. and then for high frequency (half a year more than 10 times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over 10 times in 150$160 days) was reduced from 21.5 lg/l in the first period to 4.1 lg/l in the third period (1 period5150$160 days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* 1 , mary townsend 2 and lizabeth rosenbaum 3 . 1 university of new mexico hospital, 2 blood systems, inc., 3 blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a 22 year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately 10 minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within 24 hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* 1 , william korzun 1 , teresa nadder 1 , susan roseff 2 and elizabeth ripley 1 . 1 virginia commonwealth university, 2 virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for 1% of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a 1-hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of 142 subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from 2015 to 2016 could be a result of a change in the blood drive timing of the training schedule of that location. in 2015, basic trainees at site a were scheduled at day 57 of 70. in january 2016, the blood drive date changed to day 60 of 70. the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from 2015 to 2016 of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in 2016. these observations support the hypothesis that the increase in hemoglobin deferrals in 2016 resulted from the implementation of the male hemoglobin standard change from 12.5 to 13.0 g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional 24% of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* 1 , eva alonso 1 , oscar bascuñana 1 , monica romero 1 , teresa vich 1 , elena castaño 1 , laura carbonell 1 , eva palomas 1 , saray almerge 1 , francesc carpio 1 and xavier curia 2 . 1 banc de sang i teixits, 2 institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january 2015 to december 2016 we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t5 due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t5 and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, 219 donors came to give blood, of these, 15 (7%) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t5 excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t5 but his hemoglobin levels were lower than our selection criteria. of the 204 donors selected for donation 16 (7.8%) had sci lower than t5 and t6. adverse reactions to donation (1.4%) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t5 have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of 2013, automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an 11 month period from january 2013 to november 2013. the same information was assembled for the automated bp process for the 11 month period of january 2014 to november 2014. the automated bp process implemented in mid-december 2013; so the december data for both 2013 and 2014 has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < 0.05. results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p50.006). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < 0.03) with the automated bp while and reactions remained non-significantly lower (p 5 0.086). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below 12.5 g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < 0.05. results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under 12.5. statistically more visits with hgb less than 12.5 g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under 12.5 was 16.5% higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from 1 january to 31 march 2017. after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of 6680 donors who has donated blood in the blood bank's main branch were used as the baseline for this study. 85% of donors (n55678) accepted automatic appointment booking, whereas some donors (n51002) were not comfortable with it. 77% of those who declined still preferred walk-ins (n5771) based on their own time schedule, the rest decided that variable situations (n5112), donation frequency (n569) and choice of preferred donation locations (n550) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was 19%. a comparison was made and found that this study shown a significant increase of acceptance rate by 66%. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has 4 collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. 4) , 3.28 0.4940 4 1 poisson distribution, 2 normal distribution, 3 logistic distribution, 4 lognormal distribution 105a transfusion (p>0.05) in donor and reference populations except in younger (20-44 yrs) male donors (p<0.0021; donor 4.9%, reference 10.0%). mean donor sbp, dbp, and pulse were 125 6 14.7 mmhg, 75.1 6 9.6 mmhg, and 75.9 6 11.2 bpm, respectively. screening blood pressure levels consistent with hypertension (29.4% male; 16.6% female) in the 20-44 year donor group, significantly (p<0.0001) higher than the reference population (11.2% male; 8.7% female). no differences were observed in the 45-64 year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in 20-49 year old females. developing blood donor educational materials gay wehrli* 1 , susan rossmann 2 , louis m. katz 3 and dan a waxman 4 . 1 university of virginia health system, 2 gulf coast regional blood center -sugar land, 3 americas blood centers, 4 indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an 8 th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same 10 multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, 3.5 page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table 1. results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level (8 th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in 2011 for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has 4 major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from 2011 to 2016 after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from 5.6% to 21.4% in 2011 and 2016, respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from 26.8% in 2011 to 57% in 2016. moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to 24 times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately 1.3 million doses of ap transfused within 1.375 billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since 2006. firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than 180 days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than 4 times and had not donated for more than 90 days or less than 4 times with an interval of more than 60 days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than 8 times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by 7.46 times from 5550 to 41420 and the doses of ap increased by 7.41 times from 7363 to 54553 within 10 years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into 5 groups: those who donated ap once, those who donated 2-4 times, 5-9 times, 10-29 times, and those who donated more than 30 times, respectively. it was found that the number of permanent ap donors who donated ap more than 30 times was only 965 (2.1%), but they denoted a total of 76432 doses of ap (29.2%) from 2006 to 2016. conclusion: aps increased at a rapid and steady pace in wuhan blood center from 2006 to 2016, which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at 4 sites on 2 consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer <26 ng/ml and zpp levels >100 umol/mol heme) at 3 hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all 3 hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp 87.8% (r50.937) at first and 86.5% (r50.93) at second donations. at first donation when compared to fs hb, only 10.4% (r50.323) of variation could be explained by variation in fs zpp, 12.3 % (r50.35) by ven zpp and 9.4% (r50.307) by ven fer. at second donation, when compared to fs hb, only 9% (r50.30) of variation could be explained by variation in fs zpp, 14.4% (r50.38) by ven zpp and 20.1% (r50.448) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p<0.001) suggesting strong evidence against correlation. 55% (181) responded to the survey of which 4% (13) reported not feeling well after donation. it should be noted that noted that 1% (3) female study participants reported feeling unwell after the first donation and had ferritin levels below 26ng/ml but the zpp levels were less than 100 umol/mol heme. of the 3% (10) male participants that reported not feeling well none had ferritin levels below 26 ng/ml nor ven or fs zpp levels above 100 umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from 12.5 to 13.0 gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from 59 blood centers over two intervals, july-dec. 2015 and july-dec. 2016 (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male5m, female5f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab 17.0, chicago il). p <.05 was considered significant. results/findings: data were provided by 40 of 59 centers invited, representing 2,420,886 and 2,945,802 wb donations and 272,094 and 319,161 ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from 1.5% to 2.9% in the two intervals among aggregated donation attempts (p<.001), and for m ap from 1.8 to 3.5% (p<.001). the mean "by center" deferral rates (table) were similar to that and significant (p<.001). mean by center hb deferral rates among f donations during the two intervals were 11.6 and 11.9% (p50.241) for wb, 11.8 and 13.0% (p5.041) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only 12 centers could provide specific high vs. low vs. irregular pulse deferrals; 27 provided only a summary (i.e total pulse deferrals), and 1 could provide none. for bp, 8 provided detail (high vs. low), 28 summary and 4 none. p deferrals increased in the successive intervals among f wb donors from a center mean of 0.57 to 1.49% (p5.018) and for m wb donors from 0.78 to 1.16% (p5.006). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba1c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba1c levels among those with rbs >180 mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from 1 st march 2017 to 31 st march 2017. total of 1,861 blood donors were tested for rbs. those with rbs > 180 mg/dl were further tested for hba1c by gold standard hplc method using variant ii biorad. blood donors with >180 mg/dl rbs and hba1c > 6.5% were advised to consult a physician for further evaluation. results/findings: of the 1,861 donors tested, 44 (2.36%) donors showed a rbs of > 180 mg/dl. forty two (95.45%) were males and 2 (4.54%) females with a mean age of 40.55 years (26-56 years). of these, 14 (31.81%) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining 30, 8 (26.66%) of them had a family history of dm. of these 30 donors, 8 donors did not give a consent for testing for hba1c. among the 22 donors tested for hba1c levels, 16 (72.72%) had hba1c > 6.5%. all the 16 donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is 0.87% (16 of 1839 donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* 1 , hwee huang tan 1 and ai leen ang 2 . 1 health sciences authority blood services group, 2 health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from 12.5 to 13.0 g/dl last may 2016. the current minimum acceptable hemoglobin for male donors in singapore is 12.5 g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels (12.5-12.9) and in donors with hemoglobin 13 g/dl and above. study design/method: during a 4 month period, serum ferritin testing was performed on 350 regular male whole blood and 250 regular male apheresis donors who made at least 3 donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin 12.5-12.9) group b (whole blood with hemoglobin !13, group c (apheresis with hemoglobin 12.5-12.9) and group d (apheresis with hemoglobin !13). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below 30 ug/l is considered low and levels below <12 ug/l are considered having absent iron stores. results/findings: 55.1% of donors in the study have ferritin levels below 30 ug/l. there were more donors with low ferritin in group a compared to group b, 80% and 53% respectively (p<0.05). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, 49% and 30% respectively (p50.001876). ferritin results for the 4 groups can be seen in table 1. conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or 54.3% have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after 4 months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin 12.5-12.9 g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to 13.0 g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than 15 % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among 600 students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of 54% male and 46% female students in the age group of 18-28 years. only 65 % of the students have heard about voluntary blood donation and 28 % of the students have given blood once in their lifetime and among them 19 % are blood donors at the moment. 42 % of the participants believed that there is a specific reason why they don't donate blood and 59 % believed that there is a risk involved for the donors, when donating blood. 80 % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. (21); miscellaneous effects were reported in 23 courses. side effects led to interruption of supplementation in 55 instances. ferritin levels (mgt6sd) at entry into the program and at the last visit were 48.9 6 2 and 65.4 6 1.7 mg/l in participants, vs 64.1 6 2.2 and 56.3 6 2.2 mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took !75% of the tablets. ferritin levels<26mg/l were found in 4,8% of participants and 14.7% of controls. deferral for low hemoglobin was below 1% in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only 50% of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under 21cfr630.10 and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § 640.120 to make a collection under this provision if the requirements set forth in § 630.15(a)(2) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in 2001, an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of 50 -75 ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached 547, of whom 365 (67%) are c282y homozygotes. without active recruitment, accrual rate is about 7 per quarter, with 69% of subjects qualifying as allogeneic donors. the mean current age is 59.7 years, 65% male, 96% caucasian. the majority of hh donors (276 of an active cohort of 318) are in the maintenance phase of therapy with an average of 2.6 donations/year and a 4% deferral rate. over the last 5 years, hh donors contributed approximately 8-11% of the hospital's allogeneic blood supply, averaging 475 whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided 30-40% of blood for in vitro research at our institution with an average of 180 wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over 16 years. since 5/23/16, with an increase in male hgb deferral threshold to 13g/dl, there has been only 1 hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm-200 non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm-200 non-invasive occlusion spectroscopy device. over a span of 7 days, 200 eligible blood donors, both male and female, were first screened by the nbm-200 non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the 200 blood donors for the performance of hb measurement on the sysmex hematology analyzer within 1-3 hours of collecting the venous samples. results/finding: the sd of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed 1.1g/dl. the hb measurements obtained from the nbm-200 and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be 0.978 g/dl. the precision of the nbm-200 yielded a co-efficient of variation of .02 g/dl and a standard deviation of .33 g/dl. conclusion: the operators found the nbm-200 easy to install, maintain, and operate with minimal training. the nbm-200 non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder 1 , ravi reddy 1 , dhuly chowdhury 2 , don brambilla 2 and edward l. murphy* 3 . 1 sanbs, 2 rti international, 3 ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in 2014 and followed them for one year. within 56 days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used 4-point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. 2015) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included 2,902 first-time black donors with median age 23 and female predominance (59%). within one year, 1,786 donors (62%) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable (45 strongly agree to 15 strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) 5 1.16, 95% ci 1.06-1.28), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or51.11, 95% ci 1.00-1.23). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or50.83, 95% ci 0.72-0.96) and "i wasn't treated well by the <blood center> staff" (or50.85, 95% ci 0.74-0.97). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or51.19, 95% ci 1.03-1.37). a secondary analysis treating the likert scales as 4-level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard 1 , ramya ghantasala 1 , obhijit d hazarika 1 , nicole leonard 1 , cori a polonski 1 , zachary b wunrow 1 , michelle heleba 2 , jan k carney 1 and mark k fung* 1 . 1 university of vermont larner college of medicine, 2 american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous 16 question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of 292 surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" (27.5 %) and "traveling is a time for me to relax." (30.6 %). of the respondents who travel in the summer, very few reported donating while traveling (3.4 %). summer donation rates between summertime travelers (36.5 %) and non-travelers (36.4 %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website (45.6 %) and phone (28.4%). willingness to use a regional blood donation smartphone app was highest among respondents ages of 18 to 34 (45-55%) and lowest among ages 55 and older (13-15%). of respondents with no prior knowledge of summer seasonal shortages (22 %), 2/3rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv6, and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin-4(il-4) and il7. others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent 2 hard spins at 3800 rpm for 7 minutes with separation after each spin on a compomatev r g5. plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb1 loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers (13m [29%]: 32 [71%] f); median age 42 years (range 21-70) donated a unit (500 ml) blood from which buffy coats (average volume 56 ml) were processed. the buffy coat process was previously validated on 20 wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures 1 and 2. all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by 20% within 12 months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for 34 days. results/findings: there were 158 completed responses, of which 73 (46%) indicated that their hospital had an msbos and 85 (54%) did not. the majority of hospitals without an msbos were academic centers (36/85, 42%) from oceania (26/85, 31%) or europe (23/85, 27%), had between 500-999 beds (30/85, 35%); the majority of these hospitals transfused between 1,001-4,999 rbcs (21/85, 25%) per year. 15/85 (18%) are going to implement an msbos in 2017. of those with an msbos, the majority 23/73 (32%) were from north america. the majority were academic hospitals (39/ 73, 53%) with 500-999 beds (43/73, 59%) that transfused !20,000 rbc units per year (21/73, 29%) offering a wide range of surgical services. on average there were 207 6 577 procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of 30% of the procedures listed, a pre-operative type and screen for 38%, crossmatching rbc units for 28%, and for 4% of procedures a different recommendation was made. most (32/73, 44%) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only 5/73 (7%) of msbos' were created solely by using procedure-specific data, and most (35/73, 48%) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually (30/73, 41%), and the hospital transfusion committee is often (39/73, 53%) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents (30%) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, 23% of respondents felt that it was regularly used by all surgeons and anesthesiologists; 10% felt that it was not used at all at their hospital, 36% did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, 18% of the hospitals currently without one indicated that it would be implemented in 2017 suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a 1490-bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see 93,000 hospital admissions and nearly 300,000 emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in 2015 was approximately $15.8m. in nov. 2015, an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $1.2m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns (2014 and 2015), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee 1. development of evidence based transfusion triggers. 2. education on evidence based transfusion triggers across multiple campuses, specialties and resident programs 3. clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to "1" unit instead of "2" units. 4. updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in 2016, we were still able to reduced blood product expenditures by $933,874 when compared to 2015. conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* 1 , anna w rains 2 and christopher t clark 3 . 1 university of tennessee graduate school of medicine, 2 univeristy of tennessee medical center, 3 univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for 1 week post-delivery, with cost of $1.10 per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in 2016 was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc 3000 system. by using the blood volume values, and assigning a value of 1.5 ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in 2016, there was a total of 3,331 infants delivered at our facility. out of all the deliveries, 487 (14%) infants were transferred to the nicu. of those infants, 27% received at least one red blood cell transfusion and 7% received at least one platelet transfusion. of the 487 infants transferred to the nicu, 98 (20%) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to 1% (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from 1.0% all the way up to 3.9%. in those 98 infants, the birth weight ranged from 400-1650 grams, and the gestational age ranged from 22 weeks to 36 weeks and 4 days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than 2500 grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* 1 , shannon davis 2 , suzan new 2 , vaishali patel 2 and oren guttman 2 . 1 university of texas southwestern medical center, 2 ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable 1:1 communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to 50% of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july 2016; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of 79 randomized controlled trials (8, conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* 1 and stephanie rogers 2 . 1 dignity health st joseph's medical center, 2 dignity health background/case studies: over 12 million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across 39 hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels >5 7.0 g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of 7.0 g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which 4 or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of 7.0 g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to 21 hospitals including 16 cme presentations, online physician and nursing educational videos, communication tools including infographics and "7 is the new 10" buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb >5 7.0 g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy2015 to fytd2017, there was a 26% reduction in prbc units transfused to patients with hgb >5 7.0 g/dl, starting at a baseline of 67% down to 41%. this represents an fy17 annualized savings of $9.732m, from a baseline of 82 units per 1,000 patients days down to an average 71 units and approximately 2,000 fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels >5 7.0 g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm5-0.258) reduced rbc units and two studies decreased the percentage of patients transfused (or5 0.700). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or 50.264) and rbc units transfused (sdm5-0.553). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a-6 method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was 7g/dl/21% or 8g/dl/24%. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately 42% of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was 590 1 24 units/ month. after the first 5 months of buc activation the mean number of units was 439 1 50 units/month a reduction of 151 units/month or 26% of nonsurgical blood use (p50.003 by t-test). non-surgical rbc use now represents approximately 29% of the total rbc use hospital wide a 13% reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital -30 month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital (4001 beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first 30 months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating 115a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance (2q14 vs. 1q17): present and completed consents (66 vs. 94%), present and completed nursing flow sheets (19 vs. 96%), transfusion thresholds supported (73 vs. 100%), discharge instructions provided (17 vs. 86%); (3q15 vs.1q17) vital sign compliance (39% vs.71%). jwp increased from 27 to 225 (04/16-03/17). cost savings were realized by decreased utilization and implementation of bpa. (table 2016 -1q17) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately 30-40 trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about 8 minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only 10 percent of coolers fully used. it also consumed valuable staff time as technologists typically made 20-45 trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november 2016 implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level 1 trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated 6-10 hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains 2-4 units of group o rhd negative, 4 units of o rhd positive, and 4 units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in 2015, the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < 10 k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june 2015-october 2016 were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a 17-month period, 1,270 cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table 01 below. overall, 532 (42%) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." (325), "active bleeding" (303), "platelet count of . . ." (173), and "downtrend" (92), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, 618 (49%) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years 2014-2016. study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by 207 km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of 5,5g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> 9 g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey (12 questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of 32 complete responses (16%). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, 81% with physicians, and of these, 59% reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only 3% responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of 7-8 g/dl (56%), platelet count of 20-50,000 (38%), and inr of greater than 2.0 (69%). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per 1,000 inhabitants may vary 3 folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of 18 key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies (14.4% and 14.0%), for the remaining strategies weights varied between 7.0% and 10.6%. we estimate that 384,704 patients would be eligible for pbm strategies in one year time horizon, resulting in 594 premature death avoided (3.8% reduction) corresponding to a gain of approximately 1,500 life years and a reduction of 3,660 (6.0%) disability adjusted life years (daly) relative to the current clinical practice. a decrease of 233,141 in-hospital days is expected mainly due to a 8.4% reduction in hospital length of stay and a 37.3% reduction in 30-day readmission rate. in this population the overall transfusion rate could decrease to 4.3% from the current 8.7% (51.2% reduction) implying 17,202 blood transfusion avoided and 65,214 red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: 237 adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted <1250ml of rbcs, all cases with 0ml transfused were captured and only 7.8% of the time >1250ml were used. if 1250-2000ml rbcs were predicted to be transfused, >2000ml were used 25% of the time. if predicted usage was >2000ml, 53% of the time it exceeded 2000ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for >1250ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than 2.0 is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a 3 month period (jan to mar 2017). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the 3 month period, there were 274 patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of 494 units of blood were requested. 154 units were crossmatched, of which 138 units were sent to the operating theatre (ot). only 33.3% of blood issued to ot were transfused (n546) while the rest were unutilized. the observed ct ratio was 3.35. conclusion: although only 31% of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of !2.0, with almost 70% of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study 35 patients who received tranexamic acid (txa) (study group) and 31 patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa 1gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < 7.0 g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were 1732 unique alert encounters. of these, 1531 (88.4%) led to a crossmatch being ordered while 201 (11.6%) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds (7.0 g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds (8.0 or 9.0 g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at 4 academic hospitals with data-derived msbos. study design/method: the 4 hospitals were in 2 groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if 5% of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if 5-24% of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if !25% of the patients had been transfused. data were collected at each center over a 1 month period between january to march 2017 and included a maximum of 400 cases per hospital during that one month to ensure equal representation between centers results/finding: between these 4 centers there were a total of 1599 cases analyzed. some of the more frequently performed surgeries included orthopedics (23% of cases), general surgery (16%) and cardiac surgery (11%). there were 1362 t&s ordered for these cases, of which 5 were positive for antibodies on the day of surgery. of all the t&s ordered, 52% were ordered in accord with the msbos recommendation, 26% were ordered when the msbos did not recommend one, and in 0.2% a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio 5 6.12 for prenatal hemoglobin (hgb) 8-8.9). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of 29% among obstetric patients. study design/method: we studied a sample of anemic (hgb<10.0 g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of 301 women were enrolled, with median age 27 (interquartile range 23-32) years, median gravida 2 / para 1 and median gestational age 28 weeks. mean hgb before referral was 7.5 g/dl and most were already taking oral iron therapy. a total of 169 women were hiv positive with mean cd41 lymphocytes counts of 394 cells/ul; 29 (12%) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and 156 (92%) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in 292 (97%) of women. there was concurrent chronic disease (n52), infection (n52), vitamin b12 deficiency (n52) and antenatal hemorrhage (n56); 10 had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a 2 month old boy presented to our institution after a 1 month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige (15270 ku/l; rr: 0-2.9). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp3, while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp31 cd41 lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $1.8%. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of 8.2% over the last 5 years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from 2014 to 2016, with a peak of 11.6% (range 3.2%-11.6%). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for 2016 mtps. results/finding: in 2016, mtp was activated 28 times. in 6 cases the patient did not receive any blood product and in 11 cases plasma was already available at the time of rbc allocation/issue. this left 11 cases to evaluate. the median time to plasma availability was 29 minutes (range 4 minutes -61 minutes). the mean plasma wastage for mtp activations was 32% (range 0-100%). of the 9 cha replies, 3 were using thawed plasma and their wastage was </5 5%. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of 29 minutes. our implementation began with thawing a single unit of plasma for "level 1 neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are 9.5 %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over 40 years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated 5 cases of gtx in pediatric patients with age from 8 months to 16 years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of 56 granulocytes donations from 2015 to 2016 were collected from 43 health donors (21 male/ 22 female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, 5mcg/kg/dose (donors up to 70kg, maximum of 600mcg) and oral dexamethasone (8mg), 12h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with 40gy. results/finding s: granulocytes number increased 5 times from basal levels and the median of cells collected were 4.89 x10 10 (range 1.09-9.85). in general donors reported no significant adverse reactions. from 56 donations, only in 4 was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within 24 hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table 1 . adverse reactions for patients were minor and not frequent (4 in 66 gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* 1 , elena kurnikova 1 and pavel trakhtman 2 . 1 russian institute for paediatric haematology, oncology and immunology, 2 instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period 04/2012-02/2016. donors underwent granulocyte mobilization with g-csf in a dose 3-5 mg/kg s.c. 12-16 hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed 169 granulocytes collections in 153 donors (82 f, 71 m), ages 18 to 58. the mean increment of wbc after stimulation was 22,6x10 9/l (9,2-41,9 x10 9/l; f-23,2 x10 9/l; m-21.9 x10 9/l); in the age groups 18-39 years (n5121) and over 40 years (n532), the mean increment of wbc was respectively 22,2 x10 9/l (9,2-41,9x10 9/l) and 24,02x10 9/l (12,6-35 x10 9/l). the mean volume of treated blood was 566, a50,05) . the mean number of total wbc in the product was 38,43x10 9/l (13.38-77x10 9/ l; f-34,8x10 9/l, m-42,7x10 9/l). only three donors (1,96%) had apheresisrelated adverse effects the analysis included 244 granulocyte transfusions in 35 cases of infectious complications in 32 patients. the mean number of transfusions was 6.9 (1-60). each patient received transfusions from mean 5 donors (1-32); the granulocyte transfusions started at the mean 8 days (2-30) after infection; the mean dose of wbc on the transfusion was 11.6x10 8/kg (2-30.7x10 8/kg). wbc increment was able to estimate after 144 transfusions. wbc increment was recorded after 96 transfusions (66.7%); the mean increment was 1,12x10 9/l (0,01-11,38 x10 9/l). efficacy was determined by the number of patients who survived an infection episode and amounted to 80.6%. in the cases of severe sepsis, the efficacy of transfusions was 76.2%. the frequency of post-transfusion reactions was 22%, (n 5 8): febrile nonhemolytic reactions 8,3% (n 5 3); pulmonary complications 11,1% (n 5 4); the anti-hla antibodies formation 2,7% (n 5 1) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp150 hemolytic disease of the newborn due to anti-rh17 keegan barry-holson* 1 and jennifer andrews 2 . 1 stanford university, dept of pathology, 2 stanford university, dept of pathology & pediatrics background/case studies: anti-rh17 is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh17 has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh17 alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g3p1 > 2 mother with a negative 1 st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the 1 st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at 6 hours of life because he was found to have anemia (hemoglobin 12.0 g/dl), severe hyperbilirubinemia (total bilirubin (t bili) 9.0 mg/dl), reticulocytosis (8%) and a positive direct antiglobulin test (igg 21). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of 16.7mg/dl on day 8 of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin 6.3 g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh17 was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh17 sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d1 c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh17 antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* 1 , cyril jacquot 1 , valli criss 1 , philippe p pary 1 , jay greenberg 1 , naomi lc luban 1 and edward cc wong 2 . 1 children's national medical center, 2 quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as 4-11%. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a 2 month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of 1.6 g/dl/4.9%. wbc counts (19 x 10 9 /l) were mildly elevated and platelet counts (410 x 10 9 /l) were within normal limits. her history was notable for upper respiratory tract infection 6 days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total 7.1 mg/dl, direct 1.4 mg/dl), normal ldh (318 u/l), and undetectable haptoglobin (<7 mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin (3-41 reactivity) with positive autocontrol. dat was 41 positive for anti-igg and negative for c3 despite a positive cold antibody screen. the patient weighed 6.9 kg with an estimated total blood volume of 620 ml. she initially received simple transfusions totaling 20 ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with 463 ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of 40%, utilizing the central venous catheter. no adverse events took place over the course of the 2 hour exchange. her one hour post-exchange hb was 9.5 g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin (21). after initiation of steroid therapy (methylprednisolone, 2 mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of 11 g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels (3168 mg/dl). at a subsequent follow-up visit 3 months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a 24 or 16 gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, 1.431/-0.49 ml/ second) or with a mechanical syringe pump (slowly, 2 ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the 24g catheter, the mean change in hct was -3.531/-0.69% with the one-way valve and 0.221/-0.13% without (p<0.00001). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p<0.0001). during rapid manual transfusion with a 24g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse 4.5ml when using a one-way valve (change in hct versus time: r5-0.75, p<0.0001) compared to a significantly different (p50.0085) slight increase in hemolysis for samples that took less time to transfuse 4.5ml when not using a one-way valve (change in hct versus time: r50.58, p50.23). correlations between time and hemolysis were similar, but insignificant using 24g with washed rbcs and the 16g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in 99.9% of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge3. study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at 24 weeks gestation with passive anti-d and an anti-ge3 titer of 256. she was d-and ge:-2,-3, 4 by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at 37 weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge3. the birth hemoglobin (hgb) was 12.6 g/dl, reticulocyte (retic) was 8.6%, bilirubin (bili) was 2.8 mg/dl; the infant was discharged. on day 7 of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb 7.6 g/dl, retic 2.6%, and bili 6.6 mg/dl. ge3-blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh1. obstetrics had to authorize maternal blood donation due to her hgb of 10.9 g/dl. maternal blood collection and rbc washing was expedited and the infant received 40ml of maternal rbcs within 24 hours, at which time his hgb was 6.1 g/dl. post-transfusion hgb was 10.8 g/dl. one week later, the infant was symptomatic with hgb 7.1 g/dl, retic 1.0%, bili 2.1 mg/dl. a 2 nd aliquot of 60ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb 7.8 g/dl, retic 0.7%, anti-ge3 titer 8, and needed another transfusion. the maternal blood stored for just 3 weeks had hemolyzed necessitating a 2nd maternal donation for baby's 3 rd transfusion. at 6 weeks, the infant's anti-ge3 titer was 2, hgb 9.2 g/dl, retic 1.7%; no transfusion was necessary. at 8 weeks of life, hgb was 10.2 g/dl, retic was 3.3%, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge3 hdfn. molecular analysis revealed that the mother was homozygous ge3-negative ge*01.-03, the father had homozygous wild type ge*01, and the infant was heterozygous ge*01/ge*01.-03. conclusion: the infant had hdfn due to antibodies to the high prevalence ge3 antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge3. hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results (1) supporting a hemoglobin trigger of 7 g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in 2016, 14, 247 rbc orders occurred and the top three patient groups were: 34% in congenital heart disease patients, 25% in hematology/oncology patients and 14% in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was 9.85 g/dl as measured in the 72 hours prior to rbc order placement. in 2016, 3105 ffp orders occurred and the top three patient groups were: 46% in neonates in the nicu, 28% in congenital heart disease patients and 13% in pediatric intensive care patients. average inr of every patient was 2.09 as measured in the 72 hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day 5 bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates (0-4 months of age), infants (>4-12 months of age) and children (>12 months-18 years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after 4 months, pr-plt represented 30% of our platelet inventory (average daily plt inventory: 45 units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among 16 transfused neonates (0-4 m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased 2,3 dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of 60 pediatric patients receiving prbc transfusion over a 12 month period were retrospectively reviewed. a total of 44 patients were identified as receiving allogeneic prbc transfusion. 16 patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was 10.6 g/dl with post-transfusion hgb rising to 14.5 g/dl. the mean prbc volume transfused was 46.3 ml using a dose of 15ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of 10-15 ml/kg in a 2 kg patient, for instance, would translate into 3 full prbc units (about 1000 ml) in an average size adult. the current standard dose of 10-15 ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only 3 case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a 7month-old, previously healthy female infant who presented to the hospital with a 1-week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of 2.7 g/dl and 9.1%, respectively, platelets of 635,000, and a reticulocyte count of 10.3%. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of 4.9 mg/dl with a direct fraction of 0.43 mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens (19 total) were non-detectable. the patient was started on a 5-day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c3. an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her 8-day hospital course, the patient received 2 rbc transfusions on the day of admission and several rbc transfusions thereafter (see table 1 ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day 6. her hemoglobin rose to 8.4 g/dl on hospital day 7 and increased to 9.5 g/dl on hospital day 8. at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next 2 weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to 11.2 g/dl on day 57 after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is 3 or 41. platelet and leukocyte immunohematology, testing and genetics table 1 . of 53 pairs, 7 pairs were complete match (2/2), 26 pairs were partial match (1/2), 20 pairs were complete mismatch (0/2). the matching rate of hla-dpb1 in our study is 13%. conclusion: the matching rate of hla-dpb1 in 10/10 hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb1 in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb1 and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china (81401732) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts13. therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts13 with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a 56 year old male with a 7-year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts13 <5% and inhibitor of 3.6. on day of admission platelet count was 95 x 10 9 /l which decreased within five days to 14 x 10 9 /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were 1 x 10 9 /l and 14 x 10 9 /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to 3.2 x 10 9 /l achieving the ratio of 3 previously shown to be diagnostic of ttp. on day 5 his a-ipc and platelet counts had improved to 7.5 x 10 9 /l and 218 x 10 9 /l respectively. absence of anti-pf4 antibodies ruled out heparin-induced thrombocytopenia at this time. on day 6 he had an unexpected decrease in both a-ipc and platelet count to 4.8 x 10 9 /l and 132 x 10 9 /l respectively, worsening by day 8 to 1.7 x 10 9 /l and 40 x 10 9 /l respectively despite daily tpe. patient received 25 additional tpes that failed to improve a-ipc or platelets which on day 32 were 0.4 x 10 9 /l and 13 x 10 9 /l respectively. a-ipc had remained at this level for 16 days suggesting that the observed decrease was irreversible. adamts13 activity remained <5% low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa-1a, hpa-3a, and hpa-5a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa-1a, hpa-3a, and hpa-5a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p2 and gi9, specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa-1aa, hpa-3ab and hpa-5aa) were collected and reacted with anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex100. the hpa-1a serum was diluted to 10 serial dilutions (from neat to 1/502) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa-1a, hpa-3a, hpa-5a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( .08 vs 37.05), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa-1a, anti-hpa-3a, anti-hpa-5a. furthermore, because the platelet was hpa-5aa, the hpa-5b serum did not react with the coupled beads with mfi was comparable to negative control (286.59 vs 127.25). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa-1a was 1/128 (0.78iu/ml) and 1/64 (1.56iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa-1a, hpa-3a and hpa-5a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in 50% of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from 1 january 2014 to 31 december 2016. clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c3, c4bp, thbd, dgke, cfhr3, cfhr1, cfhr4 and cfhr5) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of 134 patients tested, pathogenic mutations were detected in 13% (18/134) and vus in 35% (47/134). 20% (27/134) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and 9 (33%) had vus. 31% (42/134) of patients had primary ahus; of these, 28% (12/42) had pathogenic mutations and 40% (17/42) had vus. 48% (65/134) of patients had secondary tma; of these, 9% (6/65) had pathogenic mutations and 32% (21/65) had vus. in patients with pathogenic mutations, 39% (7/18) were children, 22.5% (4/18) had a positive family history of ahus and 28% (5/18) had recurrent disease. patients with primary ahus had a significantly lower age at presentation (22 6 18 vs. 33 6 20 yrs; p-value: 0.005) and a higher proportion of pathogenic mutations (28% vs. 9% p-value: 0.009) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl80 and sysmex xn9000 compared with a flow cytometric method farshid ezligini 1 , kjersti roen eriksen 1 , annette vetlesen 1 , thomas larsen titze 1 and geir hetland* 1,2 . 1 oslo university hospital, 2 university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion 2012). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and 33 buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl80 (horiba abx, montpelier, france) and xn9000 sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd41a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x10 9 /l 6 sd were 819 6118, (<) 1106 6137, (<) and 1195 6176 for counting by sysmex toa, pentraxl80, and the gallios flow cytometer, respectively. sysmex count was the very lowest 129a transfusion 2017 vol. 57 supplement s3 abstract (31.4% less than for flow cytometry), but all plt counts were significantly different (p<0.001), although least so (7.4%) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl80 seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing 1 , arishma lata 1 , roland russnak 2 , zachary antovich 2 , heather dunckley 1 and thierry viard* 2 . 1 new zealand blood service, 2 linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of 24 reactions that identify both variants of 12 relevant snps located within hpa genes (hpa-1 through hpa-11, and hpa 15). genomic dna purified from 48 blood samples, previously genotyped for hpa-1,-2,-3,-4,-5 and -15 by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were 100% concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately 90 minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late 2016 and to date has tested 749 dna samples from 400 blood donors (349 donors were tested in duplicate). concordance between the sample replicates was 100%. there were 24 occasions where the assay had to be repeated, giving a repeat rate of 3.2%. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa-3 (4.7%) and hpa-5 (1.2%) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd36 antigen deficiency expression in jiangsu chinese han population qing chen* 1 , jianyu xiao 1 and chengyin huang 2 . 1 jiangsu province blood center, 2 jiangsu province blood center background/case studies: cd36 has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd36 deficiency varies widely among different ethnic populations, with the frequency of 3-11% in asians and 2.4% of african americans, respectively. however, there is little information on the molecular basis of individuals with cd36 deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd36 expression levels and to determine the molecular basis of cd36 deficiency on the platelet surface of the han population in jiangsu region. cd36 expression levels on platelets were detected by flow cytometry among 243 blood donors in jiangsu region. donors without cd36 antigen expression on their platelet surface were further to be determined the expression of cd36 antigen on their peripheral blood monocyte cells. the coding exons of cd36 gene and adjacent introns were amplified and sequenced in cd36 deficient individuals. results/finding: among these 243 blood donors, cd36-deficient and cd36-expression individuals were 2.47% (6/243) and 97.53% (237/243), respectively. the frequencies of type i and type ii cd36 deficiency among the study population were 0.41% (1/243) and 2.06 % (5/243), respectively. among 237 individual with platelet cd36 expression, according to mean fluorescence intensity (mfi) value, 45, 141 and 51 individuals showed low, moderate and high expression levels of cd36, respectively, and their mfis were 1725.9 6 343.6, 3876.1 6 788.5 and 8431.6 6 529.9 (p<0.05), respectively. the type i cd36 deficiency individual were heterozygous for 1200-13a>g and 430-14c>g, respectively. among type ii cd36 deficiency individuals, two harbored a t insertion at position 560 in exon 6 which caused frameshift at codon 187; one has a t>c exchange at position 538 in exon 6 which resulted in a tryptophan to arginine substitution at codon 180; one has a a insertion before the 17th bp of the start codon atg in the promoter region; one were heterozygous for 748 1 2t>c and 1006 1 2t>g, respectively. conclusion: platelet cd36 surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd36 deficiency was higher than that in type i. the study findings indicated that the frequency of cd36 deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd36-deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd36-deficient individuals widely varies among ethnic groups, with 3% to 11% in japanese, 8% in sub-saharan africans, 2.4% in african americans, and 0.3% in caucasians. although some studies of cd36 deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd36 expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from 1282 unrelated platelet-apheresis donors in the eastern china. the expression of cd36 antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd36 and peanti-cd41). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd36-nagtive phenotype. for those donors with cd36-negative platelets, cd36 antigen expression on monocytes was analyzed further to distinguish between cd36 type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china (81570170) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd36 in all 1282 samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd36 phenotypes using the (mean 1 3sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd36 deficiency on platelet, in which one sample was cd36 negative both on platelet and monocyte. the frequency of cd36 type i and type ii deficiency in the eastern chinese donors was 0.08% and 3.3%, respectively. the average mfi of cd36 deficiency samples was significantly lower than cd36 positive samples (15.2 6 7.9 vs 79.8 6 37.8, p< 0.0001). conclusion: the frequency of platelet cd36 deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd36 antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd36-deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd36 antibody. background/case studies: cd36 (gpiv, chromosome 7q11.2) is an 88 kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd36 deficiency (cd36-n) is observed in 3-10% of africans (t1264g) & is classified as either type i (cd36-n plt, cd36-n mono) or type ii (cd36-n plt, cd361 mono). an acquired type ii cd36-n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type 1 cd36-n individuals can develop anti-cd36 alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd36 in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd36 phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd36 staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd36 dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an 80 year-old, group o1 african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin 4.4 g/dl & plt count 5k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, 5-10% blasts & a complex karyotype with del(7)(q22q34) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < 5. hla antibody testing was negative (class i panel reactive antibody (pra)50%). the patient was plt-xm-incompatible with most donors (10/14). a trial of 4 group o, plt-xm-compatible plts was unsuccessful (cci 1). subsequent testing for plt-specific alloantibodies identified anti-cd36. fc-phenotyping showed no cd36 on patient's mono or plt, consistent with type i cd36-n. preliminary dna results show that the patient is heterozygous for t1264g. because cd36-n apheresis plt were unavailable from blood suppliers, the patient's 3 children & grandson were screened as possible donors: all showed normal cd36 expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day 16) demonstrated new class i alloantibodies (pra 5 55%) in response to transfusion (21 apheresis plts, 5 rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd36-n & severe plt refractoriness in the setting of new mds, and 7q-chromosomal abnormalities. the absence of cd36 on plt & mono support congenital type 1 cd36-n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel 1 , kristopher fernandez* 1 , eric senaldi 2 , pascal george 2 , michael seul 1 and ghazala hashmi 1 . 1 biomolecular analytics, 2 central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur1978 http://bit.ly/2q51heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $100 adult donors who had made ! 6 donations in the previous 12 months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for 2, 3, 4, 5, 6, 7, 8, 9, 11, 15 and for hla class 1 (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/2pdplf8) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a *26:82 chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are 16,429 hla alleles documented according to the imgt / hla sequence database in janury2017, and more than 80% of them were identified in the last 10 years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a *26:82 allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn72d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove1 nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a *26:01:01:01, but 1 nucleotide substitution in exon 4, by nt 746 c-a (codon225 acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a 13 year-old pregnant woman typed as ab1, who delivered a baby affected by severe hdfn. the newborn was typed as b1 and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted 41 with the four monoclonal anti-d used (igm clones p3x61 and rum 1 and the blends clones th281ms26 and d1751d415) and were typed as c-c1e-e1. an anti-d was identified in her serum. molecular analysis showed the 410c>t and 455a>c in exon 3, the snp 509t>c changes in exon 4 and the 667t>g nucleotide change in exon 5. the set of snps found is similar to the molecular background of dol3, except for 455a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of 1089 immuno hematological tests (465 abo/d grouping (including 33 newborn samples), 12 extended erythrocytic phenotype, 562 antibody screening, 14 antibody identification, 16 dat) and 20 crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in 99.2% of the abo/d tests (n5265), 99,7% of the antibody screening tests (n5377), 88,8% of the antibody identification tests (n59) and 100% of the dat tests (n510). there were 4 discrepancies (2 abo/d for the same patient, 1 for antibody screening and 1 antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than 10 min). detection threshold of the d antibody was assessed at 2.5 ng/ml (0.0125 ui/ml) whereas the french recommendations are 20 ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso 15189 accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a 31 year old g5p3 presented during her fifth pregnancy with anti-kpb with an initial titer measured of 64. by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at 16 during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams (1.7 moms) peaking at 27 weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at 35 weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was 3.4 mg/dl with 13.6 g/dl hemoglobin. the baby typed as o positive, kp (b1) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at 6 weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by 8 weeks of age. his hemoglobin recovered to 9.0 g/dl with an indirect bilirubin of 1.4 mg/dl at 9 weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c3d-specific dat may be too insensitive to detect low, but significant levels of c3d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a "1111" reaction corresponds to about 500 molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c3d have been published. however, these are mainly designed to quantify the fraction of rbcs with c3d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c3d. study design/method: ten microliters (ul) of 1:80 (after documenting experimentally that this amount ensured maximum binding of anti-c3d) mouse monoclonal anti-human anti-c3d (abcam, clone 7c10) were added to 5 ul of a 2.5% rbc suspension. after incubation for 60 minutes at 4c, samples were washed x3, and 25 ul of 1:10 diluted anti-mouse-f(ab)2-pe (ro480, dako) were added. after incubation at 4c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro480 was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c3d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a1 rbcs stained with 10 levels (2fold dilution, 1:1 -1:512) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c3d, edta-stabilized samples from 4 healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c3d (values ranging from 0 to 3,393 abc) with level of 0-serum dilution (used to sensitize a1 rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r2 5 0.97, p < 0.0001). compared with dc-screening 1, the sensitivity of the flow cytometric assay was superior. it detected c3d sensitization at least 4 dilution steps further. the median normal level of rbc-bound c3d was 11 abc (range 7-20 abc, n54). the assay enabled demonstration of specific c3d-sensitization in the patient; the level of rbc-bound c3d in the sample was significantly elevated (1,907 abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c3d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt1 gene. in humans, the abo and gbgt1 genetic loci are located on chromosome 9q34, and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors1) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt1 genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt1 genes have been constructed of 88 vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt1 genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k1 antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a 14 year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion 2017 vol. 57 supplement s3 the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c1, e-, e1 and k1-. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d1, c-, e-, partial c1, partial e1. the probable rhce genotype, rhce*ce-jal/rhce*ce733g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant woman and her newborn carolina bonet bub* 1 , maria giselda aravechia 1 , thiago costa 1 , marilia sirianni 1 , eduardo bastos 1 , leandro santos 1 , lilian castilho 2 and jos e kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp background/case studies: rhd*weak d type 2 is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion 2007) the c.1154g>c change (p.g385a), which characterizes the rhd*weak d type 2 allele is a splicing variant that induces skipping of the whole rhd exon 9. we report an altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d1 w c-c1e1e1. the samples showed weak hemagglutination reactions (11/21) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon 9 in both dna samples. sequencing showed the c.1154g>c change and the intronic c.1154-8t>a and c.1154-31t>c substitutions, which are associated to the rhd*weak d type 2 allele. conclusion: our results showed that c.1154g>c associated with c.1154-8t>a and c.1154-31t>c variations had probably a functional impact on splicing inducing exclusion of exon 9 in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type 2. background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than 50% of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per 100 transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with 1,057 patients and 3 studies from other regions (brazil, egypt and france) with 641 patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was 0 to 26 years and for the other countries 0 to 20 years. available data from 5 us studies included a total of 91 alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens (18.7%, 16.5%, 15.4%, 7.7%, 7.7% and 6.5% respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per 100 transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of 16.5 % (14.1 to 19.2, 95% ci) vs. 9.4% for non-us studies (7.3-11.8, 95% ci) (p50.0008) and 134a transfusion 2017 vol. 57 supplement s3 more alloantibodies per transfused patient (0.25 vs. 0.096, p50.0001). similarly, the number of alloantibodies per 100 transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort (0á68 vs. 0.33, p50á0005). average number of rbc units transfused per patient in the us was also higher compared to data from france (77 vs. 45, p50.0001). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* 1 , melissa grohotolsky 2 , lisa deblass 1 , bala carver 1 and kip kuttner 2 . 1 health network laboratories, 2 miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: 23 year old white mennonite female g1,p0 presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in 21 panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated 11 reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was 2 at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at 2. although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a 15 year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g1 (r g r) cells. both rhce*c and rhd genes encode ser103 which determines g expression. rare rhd variant antigens lacking ser103 are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e1c1e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r 2 r 2 , r'r, r g r and rr cells. this patient is predicted to be r 2 r 2 (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is 135a transfusion 2017 vol. 57 supplement s3 excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* 1 , christa voliva 1 , kathy fletcher 1 , heather vaught 2 and tracie ingle 1 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology 2011; 27:1-5) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than 7 days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were >21 dat positive, 2 were weakly dat positive and 16 were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman 6.0-8.1ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for 5 minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: 5 bb, 14 bg and 1 bp. the ph of all eluates ranged from 6.9-8.1 with the highest percentage of eluates at a ph of 8.1 (35%). sixteen of the 20 eluates tested yielded the same results in both automation platforms (concordance of 80%). four eluates with different results are summarized in table 1 . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results (3) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in 1900 led to the discovery of human blood groups. in the abo system >200 alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor (38 units over 17 years) who is actually a w . study design/methods: donations were tested with the pk7300 instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns 1,2 and 4 and exons 6 and 7. specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table 1 . tests with anti-a, -a1, -b anti-a,b were negative as were the a2 cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table 2 . the significant changes were found in exons 6 and 7. in exon 6 there was a nucleotide (nt) deletion of 261g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly117ala. mutations in exon 7 included a nt substitution causing a pro156-leu change and a nt deletion 1061c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a2 cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o.01.01/abo*aw.02] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a1 and b cells. this abo discrepancy was caused by the presence of anti-a1 in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a2 cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer >1:1000. (schmidt, nacarrow et al. 1959) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. 136a transfusion 2017 vol. 57 supplement s3 extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to 96 blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to 15 days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: 92 edta blood tubes collected from random blood donors were used to extract dna from 200 microliters of whole blood on day 5, 12 and 15 days post collection. blood samples were stored at 2-8c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a260/a280 using a nanodrop 2000 (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result50) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the 92 samples, 20 samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from 10.8 to 62.6 ng/ul. all readings with the exception of 1 (10.8ng/ ul) had concentrations >5 15ng/ul. interestingly, the one that was <15ng/ ul on day 5, yielded >515ng/ul on day 12 and 15 post collection. over the next 3 months, 67 sets of 92 samples were extracted and tested by hea. eighty-three (1.3%) failed extraction and 82 (1.3%) failed hea. none of the samples that failed extraction were 12 or 15 days post collection; none of those that failed hea were 15 days post collection; 3.7% were >10 <15 days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to 15 days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas 1 , carlos villa 1 , rachel davis-rauser* 1 , helen carpenter 1 and vrunda patel 2 . 1 university of pennsylvania, 2 hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd38 monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in 2015. communications suggest all patients receiving therapy would have a positive antibody screen because cd38 is a common antigen expressed on red blood cells. currently, 154 patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd38 antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for 1 hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at 1:1 dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for 2 negative patients was observed up to a 1:4 dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a 28 y/o g1p0 at approximately 23 weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [1, 2] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m1n-s1s1 phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency >99.9), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [1, 2] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [3] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays 137a transfusion 2017 vol. 57 supplement s3 that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during 3 years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in 67 dna samples from chronically transfused patients with scd, in 65 patients with thalassemia and in 3000 dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in 3 levels: (1) rh and k matching; (2) extended matching and (3) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of 2 donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for 100% of our thalassemic and scd patients at level 1, 90% for scd patients and 70% for patients with thalassemia at level 2 and 30% for patients with scd and 90% for patients with thalassemia at level 3. the patients were transfused with a median of 36.4 rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to 5-10% with c e k matching and <1% with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube (6 drops plasma, 30 minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to 10 minutes and specimen volume to 2 drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of 202 specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of 164 clinically significant antibodies were detected using sprca technique, as well as 9 warm autoantibodies and 97 nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being 100% specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions (30 versus 13), it identified more clinically significant antibodies (129) than liss (93). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd38 on rbcs patricia a arndt* 1 , anthony salazar 2 and regina m. leger 1 . 1 american red cross blood services, 2 long beach memorial medical center background/case studies: monoclonal anti-cd38, e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd38 on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd38 antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or 2aminoethylisothiouronium bromide (aet). chapuy et al described (2015) and validated (2016) 2), and 6% aet (ph 8.0) as per the aabb technical manual, 17th ed. these treated and untreated rbcs were stored in alsevers at 4c and tested on days 1, 2, 4, 5 and 8 by two methods: 1) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from 8 total dara patients were tested with reactivity 5 1-31), and 2) flow cytometry using phycoerythrin (pe)labeled anti-cd38. rbcs were also tested on days 1 and 5 or 8 with a serum containing anti-k by peg iat. results/findings: the 0.2m dtt in ph 8.0 pbs had a final ph of 7.3 and the ph of the commercial 0.2m dtt was 6.5. results are in table 1 ; flow cytometry results from days 2, 4 and 5 (data not shown) were similar to those from days 1 and 8. rbcs treated with 0.2m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd38 by flow cytometry for up to 8 days after treatment. rbcs treated with 0.01m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening (10-30%) of reactivity with pe anti-cd38. background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery 1997 of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week 12 were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon 5 and 10) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr5) in gestational week 25. the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of 1618 pregnant women were included. nip rhd was positive in 987/1618 (61%), negative in 582/1618 (36%) and inconclusive in 49/1618 (3.0%). compared to the postnatal rhd type, 9/987 (0.1%) of nip rhd results were false positive (fp) and 4/582 (0.7%) were false negative. in 5/49 (10%) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n51618) at gestational week 12 was 25.3 (10-and 90-percentiles: 20.0 -32.4). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p50,71). the fraction of ccfdna was calculated for 150 randomly selected nip rhd true positive cases. median ccfdna ratio was 5.47 (the distribution had a highly positive skew, 10-and 90-percentiles: 0.64 -27.2). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r2 50,012; p50.49). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the 25th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an 83 year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of 1, suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only 1 in 20,000 donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with 1 cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, 4 lan-rbc units were transfused over 4 days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day 2, the patient had symptomatic anemia with hemoglobin (hb) 5.3 g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan1 but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of 10.2 g/dl was maintained for 4 days. the antibody screen was negative on day 3 post-transfusion, but strongly panreactive on day 6, with a positive dat (igg 21, c3 11) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from 10.9 g/dl on day 5 to 7.4 g/dl by day 8 with no bleeding identified, and increase in total bilirubin and ldh (peak 2.4 mg/dl and 304 u/l on day 7) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day 8 with good response (hb 8.1 g/dl). the patient remained stable and was discharged to a skilled nursing facility 6 days later. conclusion: transfusion of lan1 rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction 6 days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan1 units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* 1 , andrea gerner 1 , lynne stewart 1 , carol sostok 1 , mollie bell 2 and gregory r halverson 2 . 1 st. elizabeth healthcare, 2 hoxworth blood center background/case studies: anti-f was first described in 1953 by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the 6th antigen assigned to the rh blood group system (isbt rh6). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a 56 year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused 1 unit o-rbcs. two weeks later the patient received an additional o-rbc. within 4 days the hgb had decreased from 8.3 to 7.1 g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused 3 r 1 r 1 (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c1e1f1 phenotype. the rh phenotype and as was repeated on a sample collected 18 days later. the c typing was micro positive, mixed field only after 5 minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post 24 hour hgb increment from the receipt of a standard unit of blood should be near 1 g/dl (or 3% hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a 0.9 g/dl increase, and the second unit was only 0.5 g/dl. the last transfusion of 3 units increased by only 1.2 g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of 3 r 1 r 1 units was nearly complete. in a case from 1989, ohto and kariyone (transf. 1989; vol29, no. 3) reported a 51 cr ásurvival study of f1 rbcs in a patient with anti-f. they showed that the initital survival of f1 cells was fairly normal, however, after 18 days, there was a sudden increase of red cell destruction, and by day 27 all f1 cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd38 drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to 9 days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with 0.2m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table 1 ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to 9 days following the dtt treatment of rrbc. reactions were graded using standard serological grading of 0 (negative) to 41 (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table 1 for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to 9 days. this suggests that dtttreated reagent red cells can be stored for at least 9 days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd38 therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd38 that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd38 is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with 0.2m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract 2-5c, and observed for hemolysis (none was seen) for up to 21 days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted 2-41 with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in 4 patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than 6 months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* 1 , monique scott 2 , garcia curtis 2 , ellice wong 2 , alexa j siddon 1 and christopher a tormey 1 . 1 yale-new haven hospital, 2 va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately 25% to 50% of persons of african descent is characterized by neutrophil count of <1.5x10 9 /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p<0.05. results/finding: subjects who were clinically identified as having probable ben included 7 patients (mean age 48.7; all self-identified as african-american; 6/7 were male) and controls included 50 patients (mean age 68.5; 10 self-identified as african american; (50/50 male). all of the cases (100%) diagnosed with ben had fy(a-b-) phenotype. mean anc (1.95x10 3 /ul) and wbc counts (4.04x10 3 /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p50.0008 and 0.001, respectively) compared with controls (mean anc 5 5.46x10 3 /ul ; mean wbc count 5 8.14x10 3 /ul). there was no significant difference in mean platelet counts (161x10 3 /ul vs 213x10 3 /ul; p50.2301) or mean hemoglobin levels (12.4 g/dl vs 11.7 g/dl; p50.6031) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, 18 subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c3d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih-1000 had 100% concordance for all blood grouping assays. for ahg assays, the ih-1000 detected an anti-jka1e, anti-fya 1 warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih-1000 identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih-1000 with anti-igg,-c3d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih-1000 analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih-1000 is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of 2944 tests were performed on 1,214 adult patient samples and 208 donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in 99.9% of samples tested. there were 4 discrepancies, all antibody screening (2 false positives, 1 failure to detect a very weak prophylactic anti d and 1 positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing 80-100 group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp201 evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* 1 , kathleen bensing 1 , michael schanen 1 , cindy piefer 1 , randall w. velliquette 2 , christine lomas-francis 2 and connie m. westhoff 2 . 1 immunohematology reference laboratory, versiti/bloodcenter of wisconsin, 2 immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new 7/0 7/0 7/0 cecf 1/1 2/0 2/0 rhce*ce or rhce*ce compound heterozygotes ce254g 1 ce733g or ce48c,733g or ces or ceti 6/0 6/0 6/0 ce733g 1 ce48c,712g or 48c,733g 4/0 4/0 4/0 ce733g 1 ces or cemo or ceek or ceek(var) or cern 8/0 8/0 8/0 ce48c,733g 1 ce48c,712g or cemo or ceti 2/0 2/0 2/0 ce48c,712g/ce254g/733g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi 7/0 7/0 7/0 total 106/8 110/4 113/2 142a transfusion 2017 vol. 57 supplement s3 reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd9/4 and rd12/2, and a licensed comparator anti-e (p3gd5121ms63), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n 5 42) or edta blood from donors (n 5 72) and were tested using a manual tube method or on a pk7300 automated platform. a score 6 (11) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table 1) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r 1 r 1 , r 2 r 2 , r 1 r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: 40 were rhce*ce that were in trans to rhce*ce; 16 were various rhce*ce plus rhce*ce48c compound heterozygotes; 31 were rhce*ce or rhce*ce homozygotes; 27 were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for 6 rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with 1 rhce*cear/rhce*ce48c compound heterozygote, and with 1 rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and 1 of 2 rhce*cecf homozygotes were detected using the comparator reagent. rd9/4 and rd12/2 failed with 4 and 1 e variants, respectively (table 1) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd9/4 clone. none of the reagents detected e antigen variant expressed on 1 example of rhce*cehar/rhce*ce. conclusion: rd9/4 and rd12/2 anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the 3 monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of 19 blood group genes associated with the expression of 56 blood group antigens from 17 blood group systems. we used the illumina's hiseq 2000/2500 system to perform next generation sequencing first on sureselect-enriched genes from 16 dna reference samples with average target design coverage of 97.5%, and then on haloplex-enriched genes from 32 dna reference samples with average target design coverage above 97.0%. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for 38 blood group genetic variants in these 19 genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads (80.54%) were mapped to the target regions relative to the sureselect reads (29.23%). the mean sequence coverage depth of the targeted bases was around 200x for sureselect method and 300x for haloplex method. some exons, such as rhd exons 4 and 8, 10, rhce exon 10, ermap exons 5 and 12, cd55 exons 10 and 11, cr1 gene (most exons) and gypb exon 5, are consistently covered with less than 10x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on 38 blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than 90% concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than 90% blood group genetic variants in 19 selected genes. evidence rhce*cehar does not encode for rh34 (hr b ) antigen debra j bailey* 1 , trina horn 2 , paul mansfield 2 , najmi qazi 1 , pamela nickle 2 , jessica keller 2 , margaret a keller 2 and jan r hamilton 1 . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in 1996 and has a phenotype of c2e2c1e1 w f1 w , g2, hr 0 1 w , hr2, hr s 2, rh:33, rh:50 with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh:234 (hr b 2) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c.254c>g and rhd c.1136c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d1c1e2c1e1. her plasma contained an alloanti-s and an antibody that reacted strongly with all random e2k2s2 reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d22 and dc2 red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e2s2k2 red cells homozygous for the rhd*diiia-ce(4-7)-d, rhce*-ce48c,733g,1006t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce48c,733g,1006t/ rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr1 (2 of 2 sources) and hr b 2 (2 of 3 sources) phenotype. conclusion: the rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t haplotype is one of the rh haplotypes expressed by the original hr b 2 individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between 1/1/2006 and 3/ 31/2017. all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately 11 years, 81 patients had htla established at least once by titration studies. serological investigations on a total of 118 samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on 20 samples was successful in rule out in 60% of cases. in an additional 12 patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only 40% of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for 71% of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c.1136c>t (p.thr379met). the dau0 allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion 2016, 56:2520) recently summarized serologic characteristics and associated anti-d alloimmunization for 18 dau family alleles. we investigated two samples with the c.1136c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample 1 was from a 17 yo multiracial female. her rbcs reacted 11 s at immediate spin (is) and 31 in iat with immucor gammaclone and series 4 and 5, and mi1 at is and 41 in iat with ortho bioclone anti-d. rbcs did not react with 2 of 12 (lhm 174/102 & 57/17) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c.1136c>t characteristic of dau. rhd sequencing confirmed c.1136c>t and identified two adjacent changes, c.787g>t and c.788g>t (c.787_788delinstt), in exon 5 encoding p.gly263leu. sample 2 rbcs reacted 1w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series 4 and 5, and quotient albaclone blend and alpha anti-d. papain treated rbcs were 11s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c.1136c>t. sequencing confirmed c.1136c>t and found a new c.761c>t change (p.ser254-leu) in exon 5. the c.761t has not been reported, but c.761g encodes a stop codon (p.ser254stop) in japanese (vox sang 2015, 109:359). conclusion: we report two new alleles: rhd with c.787_788delinstt (p.gly263leu) and rhd with c.761c>t (p.ser254leu), both also carrying the c.1136c>t (p.thr379met) characteristic of the african dau cluster. d antigen associated with p.263leu is a partial d antigen with a novel epitope pattern. the p.254leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by 5/7 commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to 21. the number and diversity of alleles in the dau cluster supports that the c.1136c>t change is a major ancestral african background allele (wagner et al, blood 2002,100:306). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september 2016 to present day we got 130 samples of repeated blood donors who are known to be d negative, c positive and/or e negative from 15 blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon 4, exon 7 and exon 10 in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon 9 to confirm the existence of c.1227g>a and c.1222t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the 130 sample, we identified 71 cases (54.6%) of total rhd deletion, 18 cases (13.8%) of rhd-ce-d hybrid, and 41 cases (31.5%) of rhd variant. 39 of rhd variant were determined to be asian type del with c.1227g>a variation. 2 cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was 10 % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b 3 phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b 3 phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from 30 taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n552) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon 6 and exon 7 of the abo gene were amplified and sequenced. the abo*b3.03 allele was confirmed by pcr-rflp analysis. results/finding: among 52 subjects with b 3 or ab 3 phenotypes, 47 were genotyped as abo*b3.03. the abo*b3.03 group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage (37.81 6 6.62) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other 5 subjects with b 3 or ab 3 subjects, genotyped as abo*b3.06(n51), abo*bw.03(n51), abo*bw.11(n51), abo*bw.12(n51) and abo*bw.29(n51), displayed flow patterns differed from the abo*b3.03 group. the abo*bw.03, abo*bw.11 and abo*b3.06 subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (<10% in abo*bw.03 and abo*bw.11 subjects and <20% in abo*b3.06 subject). both abo*bw.12 and abo*bw.29 displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b3.03 genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout 2016, the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of 398 patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in 12% (n549) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in 22% (n511), 4% (n52), and 74% (n536) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n54), anti-jka (n53), anti-k (n52), anti-jkb (n51), both anti-e and anti-c (n51) (see table 1 ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly 1 in 4 cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr1 alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr1 gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr1 gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study 60 volunteers in the gambela region (nct01282021). the whole ackr1 gene was amplified in one reaction covering 12,125 base pairs (bp). this primary amplicon was re-amplified using nested primers covering 5782 nucleotides. nucleotide sequence was obtained by 14 sequencing reactions and manually annotated using ncbi refseq ng_011626.2. the sequencing covered 1008 bp of both exons, 480 bp of intron, 2101 bp of 5'-flanking region, 947 bp of 5'-utr, 53 bp of 3'-utr and 1092 bp of 3'-flanking region and encompassed all the 470 variations present in dbsnp and nhlbi esp databases. results/finding: among the 60 samples, a total of 15 snps, including one novel snp in 5'-utr were observed. 4 snps occurred in the exons, 5 in 5'and 3'flanking region, 4 in 5'-utr and 2 in the intron. all 60 individuals carried the snp indicative of the common fy:2 phenotype; while 58 individuals were homozygous and 1 was heterozygous for the gata box mutation. no splice site mutation was detected. as 46 individuals were observed as being homozygous or heterozygous for 1 snp, we could unambiguously assign 8 distinct alleles. in the remaining 14 individuals with 2 or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than 5.5 kb of the ackr1 gene and identified at least 8 different alleles. the present study found that the vast majority of alleles (117/120) in the gambela population as defined by 15 snps, were similar to the clinically relevant fy*02n.01 allele, which in turn is defined by only 2 snps at positions c.1-67t>c and c.125g>a. out of the remaining 3 alleles, 2 were similar to fy*02 with the fy(b1) phenotype and 1 was similar to fy*02w.01 with the fy x phenotype. the high frequency of fy*02n.01 (95%) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique (95%-100%). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce(4-7)-d is the most common hybrid and is found in african blacks. it arose by conversion of exons 4-7 of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample 1 (male) and sample 2 (multiracial female), both c1c2e2e1 (presumed r1r1), presented with weaker than expected d typing; 11 is and 31/41 at iat. rhd beadchip identified the common african rhd*diiia-ce(4-7)-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c.733c>g and c.1006g>t (heterozygous) was also detected. as rhce*ce with 733g and 1006t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons 2 and 3 replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce(4-7)-d were found in both samples. sample 3 (scd male), d1c2e2c1e1, by rh beadchip had one conventional rhd and rhd*diii type 8, and rhce*ce733g/ces. as rhd*diiia type 8 has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce(48c) exons 1 and 2 had replaced those exons in the common hybrid rhd*diiia-ce(4-7)-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky926711and ky926710. we report two different and novel complex rh rearrangements: two samples thought to be r1r1 had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia(2-3)-ce. in kind, a sample genotyped as diii type 8 rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce48c(1-2)-diiia(3)-ces(4-7)-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples 2 and 3 have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r1 haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a 81-year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at 81g/l. her pregnancy history was not provided. she had received 5 units of packed red blood cells (rbcs) in the past including 1 unit within the last 3 months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r 1 r 1 , r 2 r 2 , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse 29 polymorphisms which determine 37 antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a1) and lu(b1). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of 36 antigens encoded by the kel gene, is organized into 19 exons. there are approximately 30 kel alleles associated with a kell null phenotype (k 0 ) in which no kell antigens are expressed, and 12 alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a 53 year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons 10, 11, 12,13 and 14 and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k1 kp(a-b1) js(a-b1). kel-cdna sequence analysis was performed and detected a single transcript species with c.578c, c.841c, 1790t, and missing the sequences corresponding to exons 11, 12 and 13. amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after 37c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel*02 allele. this donor was presumed to have a k 0 phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the 11 variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel*02m.12. here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table 1 ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a1 and a2 cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c.261 deleted g, characteristic of o alleles, c.467t, characteristic of a2 and some uncommon o alleles, and c.703a and c.1096a, characteristic of b alleles. genomic sequencing of exons 6 and 7 confirmed the presence of an o allele, abo*o09 261del g, 318t, 467t), and the presence of a b allele (297g, 526g, 657t, 703a, 796a, 803c, and 930a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , diana gazito 2 , afonso cortez 2 , lilian castilho 4 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the 17-bp deletion in smim1 in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs1175550 located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of 400 blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting 21 and in samples with reactivity of 31. dna was isolated from peripheral blood and smim1 was sequenced. results/finding: from 400 donor samples studied, 4 were serologically vel negative by gel-iat but positive by adsorption-elution, 158 presented a 21 reaction and the remained samples showed a reactivity of 31. genotyping results showed that the 4 samples with negative results and 5 of 26 samples that presented 21 reaction were heterozygous for the 17 bp deletion and presented the a allele rs1175550 in homozygous status. from the 21 of 26 remaining samples with reactivity of 21, 19 (90%) had the a allele of rs1175550 and 14 (66.7%) had the a allele of rs6673829. in contrast, in the 16 samples with stronger reactions we found the a allele of rs1175550 in 5 (31.25%) samples and the a allele of rs6673829 in 3 (18.75%) samples. conclusion: the molecular changes rs6673829 and rs1175550 are located in intron 2 distancing 38 nucleotides. this study reinforces the association of the a allele of rs1175550 with reduction of vel expression and suggests the involvement of a new rs6673829 change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as 0 (rh negative), or !31 on the neo or !21 on the echo (rh positive) for both series 4 and series 5 anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series 4 and series 5 anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: 80 patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in 51 of 80 (63.7%) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in 16 of 80 (20%) of samples. 67 of 80 (83.8%) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. 40 of 80 (50%) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining 13 of 80 (17.3%) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but <31 on the galileoneo or positive but <21 on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , rosangela person 2 , lilian castilho 4 , afonso cortez 2 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of 108 samples were included in the study, being 69 previously genotyped as rhd*dar1.2, 37 rhd*dar3.1 and 2 rhd*dau6. the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from 0 -99 corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon 10. rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon 3 was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that 10 of 108 samples (5 dar1.2, 4 dar3.1 and 1 dau6) had 2 rhd genes, were phenotyped as c1e-c1e1 and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these 10 samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in 2 dar1.2 samples showed the rhce*-cear/ce s genotype, in 2 dar3.1 samples the rhce*cevs.02/ce s genotype and in the dau6 sample the rhce*ce s /ce genotype. table 1 describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized 5f9 antibody (hu5f9-g4) that binds human cd47 has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu5f9-g4 (anti-cd47) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a 69 year old female with progressive follicular lymphoma who was enrolled in phase 1b/2 trial of hu5f9 g4 in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a 48 year old male with refractory diffuse large b cell lymphoma enrolled in hu5f9-g4 clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd47 therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing (41 with anti-a, non-reactive with anti-b) and the reverse typing (31 with both a 1 cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase (11 to 41), at liss-37c (11 to 31), at liss-polyspecific ahg (m1), and at peg-anti-igg (m1 to 11). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and 37c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at 37 o c ahg phase. the abo typing of the second case performed after anti-cd47 administration showed a discrepancy between the forward (41 with anti-a) and the reverse (41 with both a 1 and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is (21), at 37c in liss (21), and liss-polyspecific ahg (m1). the dat and autocontrol were negative. his genotype was determined to be a1/o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd47 therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, 37c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd38 interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected 31 reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o.01.01/ o.01.01, consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n523) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt1 and a4galt. papain-treated patient rbcs were used to screen donor plasmas (n578) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with 3 polyclonal anti-a,b and a monoclonal anti-b (clone g1/2) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors1 or nor antigens. the patient was le(a-b1) and thus a secretor. a positive crossmatch was seen with 47% of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type 1 or 2) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type 1 chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $15 years earlier. the reactions are likely due to uptake of recipient-derived ble b (type 1) antigen (isbt no. 007006), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b1). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of 2017. shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive (11) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from 1/29/2017 to 3/31/2017. reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of 19,647 columns run as part of antibody screens, 1,633 (8.3%) columns generated "?" results. assuming 30 seconds of technologist time per "?", we estimate that 13.6 hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table 1) . in 26 cases, all three columns were visually negative but the analyzer reported 11 reactivity with 1 of 3 cells. all cases had mts-gel tm antibody identification panels performed, 25 of 26 also had a mts-gel tm ficin panel. the yield for the 51 panels performed was two routine panels with weak reactivity against hla1 cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; 13 were negative. one patient newly demonstrated anti-jka. fifty percent (13/26) of visually negative but analyzer positive samples were tested with gel card lot number 3, 38% (10/26) with lot 1, and 12% (3/ 26) with lot 2. conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from 28 patients (137 cross-match samples, 301 units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in 16/28 patients. further testing was performed in 9/16. eight were tested for the presence of antibodies at 188c and confirmed in 8/8. rouleaux formation was observed in 5/9 patients, 4/5 had reactivity detectable at 188c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict 378c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* 1 , sandra nance 2 , david moolten 3 and p. dayand borge 3 . (2):47-53). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this 2.5 year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from 150 random allogeneic and 20 autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing 0.6% bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed 50k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of 62 these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* 1 , anna burgos 1 , virginia lew 2 , sunitha vege 1 , susan veneman 3 , christopher j gresens 3 , jonathan hughes 3 and connie m. westhoff 1 . 1 immunohematology and genomics laboratory, new york blood center, 2 blood centers of the pacific, 3 bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons 1-6, and long range sequencing of exon 2-6 (5.4kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least 100 rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m1n-individuals (meyer et al. br j haematol. 2016; 174:624-36) . the st a allele, also described as gyp*401, is a hybrid gypb-gypatranscript with the crossover in intron 3. we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a 24 years old pregnant, african american female g1p0 was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a 1 antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a 3 like phenotype. genetic testing did not support the serological findings of a 3 subgroup and a new abo allele, abo*784c that has never been reported in correlation with an a 3 like subgroup was detected study design/method: the patient rbcs were typed with anti-a 1 (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a 1 antibody work up was performed using three different lots of a 1 cells and three lots of a 2 cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and 48c for 30min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at 35 weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a 3 b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo*784c found in the mother. the previously reported abo*784a allele encoded an aspartic acid to asparagine change at position p.256 consistent with an a weak phenotype. also, at least five other alleles encoding an a 3 phenoytpe consisted of polymorphisms at positions c.745 through c.871, giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a3/ aweak phenotype is encoded by the variant allele abo*784c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated biorobot m48 robot using the magattract dna mini m48 extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of 38.1ng/ml and 1.83 respectively. in the second phase of the study (n 5 39), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was 20-80ng/ll and the recommended purity was an absorbance ratio of 1.63-2.1 (a260/280) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was 100% concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano 1 , izaskun apraiz 1 , maría azcarate 2 , miguel angel vesga 2 , montserrat rubia 2 , mercedes piedrabuena 2 , fernando puente 3 , barbera veldhuisen 4 , ellen van der schoot 4 and m onica l opez* 1 . 1 progenika biopharma, a grifols company, 2 centro vasco de transfusi on y tejidos humanos, 3 banco de sangre y tejidos de arag on, 4 sanquin blood supply research background/case studies: it is well established that weak d 1, 2 and 3 phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type 1, rhd*weak d type 2, rhd*weak d type 3, rhd deletion, rhd*pseudogene and rhd*diiia-ce(3-7)-d and itgb3 gene: hpa1a and hpa1b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa-1 blood group typing. study design/method: a cohort of 1000 previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications 2009/108/ce for a ivd product of list a (!10% clinical samples, >2% neonatal specimens and !2% weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n5160, 16%). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa-1 predicted phenotype were used for comparison. transfusion results/finding: no system failure, 100% call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a 100% concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was 100%. the following id rhd xt predicted phenotype results were obtained: d negative (n5361), no amplification variant (n515), weak d type 1 (n522), weak d type 1 heterozygous (n51), weak d type 2 (n532), weak d type 2 heterozygous (n51), weak d type 3 (n534), weak d type 3 heterozygous (n51), weak d types 1, 2 or 3 not detected (n5533). regarding hpa-1 blood group, the predicted phenotype results obtained by id rhd xt were 100% concordant with bds results: hpa-1a positive (n5157) and hpa-1a negative (n56), hpa-1b positive (n546) and hpa-1b negative (n5117). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types (100% specificity and 100% sensitivity for d antigen, hpa-1a and hpa-1b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with 11 rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value <50.05 (clinical analysis) or <5x10 -8 (gwa) was considered statistically significant. results/finding: of the 2795 cohort patients, 2272 (81.3%) transfused subjects were included with 129 alloimmunized children <18 years (11.0% of 1172) and 224 alloimmunized adults (20.4% of 1100). in multivariable logistic regression models, age (or 4.2, p50.009, for age 501 compared to 0-4), gender (or 1.3, p50.04, for female compared to male), transfusion history (or 3.5, p<0.0001, for 811 transfusions compared to 1-5), site, hemolysis (or 1.3, p50.05, for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or 4.5, p<0.0001) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from 32 newborns less than 2,000 gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on 32 sample pairs. dat test was negative on 30 sample pairs and two were positive. there was 100% concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on 32 placental blood samples and 29 heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < 2,000 g birth weight newborns. o-(7.4%), ab1(6.8%), b-(2.7%), and a-(0.6%). among the tested donors, 89.2% were d positive with r1r being the most common rh phenotype. in the kell blood group system, 4.5% of the donors were k positive, while the k antigen was found to be 99.4%. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a1b1) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a1b1) and m1n-s1s1 at 47% and 22.6% respectively. the le a 1 and le b 1 alleles were seen in 21.7% and 67.3% of donors respectively, while lu b -phenotype was found in 3.3% of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a1b1) and lu(a-b-) were 0.3% , 0.3% and 2.7% respectively, while the m1n-s-s-and m-n1s-s-phenotypes were not found. the frequency of the p1 antigen was found to be at 78.9% similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph 7.2 and the supernatant was discharged. a dilution buffer containing 2% human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with 10ll of washed red cells. the cell suspensions were incubated for 30 min at 378c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional 15 min at 228c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for 515-548nm. events were recorded at a frequency of 1000 cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* 1 , mark yazer 2 , nancy m. dunbar 3 and biomedical excellence for safer transfusion (best) collaborative 1 . 1 university of vermont medical center, 2 university of pittsburgh, 3 dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of 50. study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a 1:50 dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a1 and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a 4-year and 5-year period, respectively. one center provided plasma and wb testing data for a 1-year period. results/findings: in total there were 7106 group a plasma units tested and 654 (9.2%) had a high titer anti-b. the range of high titer group a plasma units between these three centers was 7.4%-12.5%. of the 1778 wb units tested, 388 (21.8%) units had a high titer; 221/1778 (12.4%) of the units had a high titer anti-a, 61/1778 (3.4%) had a high titer anti-b, and 106/1778 (6%) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with 0.2m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd38 on reagent rbcs and render them free from plasma anti-cd38 drug interference. procedures for the preparation of 0.2m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on 0.2m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of 0.2m dtt, was adjusted to ph7.16, ph 7.56, ph 7.96 using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n53) were treated with the 0.2m dtt solutions in parallel by mixing 1:4 ratio of packed rbcs to 0.2m dtt solution followed by incubation at 378c. for up to 45 minutes during treatment, the expression of k antigens was measured every 5 minutes by tube method using 2 different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every 5 minutes for each ph level. the reduction in average scores between different phs were also calculated at every 5 minutes to measure the impact of 0.2m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by !11) after 15 minutes of dtt treatment at ph 7.96; 15 minutes at ph7.56 and 20 minutes at ph7.16. complete loss of k expression was seen after 25 minutes of dtt treatment at ph7.96; 35 minutes at ph7.56 and 35 minutes at ph 7.16. the reactivity patterns of k antigen tested with 2 sources of anti-k correlate with each other. the reductions in average scores were seen between 15 to 30 minutes range of dtt treatment time when ph 7.16 was raised to ph7.56; 15 to 30minutes range when ph7.56 was raised to ph7.96; and 15 to 30 minutes range when ph7.16 was raised to ph7.96. conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to 15 minutes and/or beyond 35 minutes of incubation. the ph of the 0.2m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of 0.2m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n 5 756,221) drawn between july 1, 2013 and june 30, 2014. these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, 11,647 patients were found to possess clinically significant red cell antibodies for an overall incidence of 1.5 percent. the three most commonly encountered antibodies were anti-d (n 5 7639) 63.1%, anti-m (n 5 1288) 0.6%, and anti-e (n 5 1227) with a frequency of 10.1%. a total of 455 (3.9%) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with 182 instances (40.0%) followed by anti-e and anti-c with 79 (17.4%), and anti-c, anti-e with 26 (5.7%). of the multiple antibodies identified, 435 (95.6%) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with 7474 or 61.8% of the total. the west had 2111 (17.4%), the midwest 1538 (12.7%) and the northeast with 979 (8.1%). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of 0.05 and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the 0.05 thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p 5 0.17). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* 1 , lindsy rich 1 , sherry stern 1 , sharon wangen 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous 4 days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was 1 to 2 days. results/finding: of the 30 ntd specimens from the immucor neo, 8 resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all 3 results, it was determined that there was no reactivity and a valid result was present. the other 19 samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the 23 absc specimens that were resulted out as positive on the immucor neo, 11 specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: 3-41 with ms24 (n515), 1-31 s with ms23 (n59), no reaction with ms273, dgc02, p3x255 (n514). 14 samples tested with a polyclonal anti-c showed a 1-31 reactivity. 3 d1c1e1c1e1 cases tested with a polyclonal and monoclonal anti-e (ms16, ms21, ms62, ms63) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c.286g>a mutation in exon 2, predicted to encode the p.gly96ser substitution. for 2 apparent r 1 r 2 donors, a f-negative type allowed the prediction of a rhce*ce286a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce286a allele (c and e in cis) in all samples. 3 d1c1e1c1e1 individuals were reactive 11 s with the original source of anti-rh55, slightly weaker when compared to rhce*ce286a/rhce*ce rbc samples available from our cryobank (21). conclusion: our results confirm that the c.286g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh55). the locr reactivity appears to be rather similar when coded by rhce*ce286a or rhce*ce286a alleles. this was quite an unexpected finding, since the p.gly96ser substitution is close to the critical amino-acid for c/c expression (p.pro103ser). none of our 15 cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce286a was reported to code for a partial c (rh:-26), we consider that rhce*ce286a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly96ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes 1, 2 or 3. the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d 1, 2, or 3 genotypes. study design/methods: between 9/2015 and 2/2017 50 samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types 1, 2, or 3 genotypes, but 156a transfusion 2017 vol. 57 supplement s3 had evidence of rhd genetic sequences in exon 7 and/or intron 4 in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons 1-10 to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients (62%) followed by transfusion patients (28%); 10% had no clinical indication provided. 34 samples (68%) were found to be weak d type 1, 2, or 3 (24, 6, and 4 samples, respectively). 5 samples (10%) appear to be genetically rhd negative. genetic sequencing was performed on 11 samples; 9 had rhd genetic variants that were not weak d types 1, 2, or 3 (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples (4%) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types 1-3. of the 11 samples that had evidence of an rhd gene and did not carry the known weak d types 1-3 polymorphisms, 9 (82%) of were found to have other rhd variants, and 2 (18%) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types 1-3 variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel 2006, curr opin hematol13:476) , that individuals with weak d types 1, 2, and 3 are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report 15 months experience with rhd genotyping on 352 samples referred with discrepant or weak d typing investigated from january 2016 to april 2017. study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for 153 samples (53.3% caucasian, 32.2% african american/african, 6.6% multiracial, 4.6% hispanic, 2% asian, and 1.3% other). results/finding: rhd genotyping identified weak d types 1, 2, and 3 in 155/352 (44%) and alleles known to encode partial d phenotypes in 168/ 352 (47.7%) (table) . uncommon or rare weak d alleles including types 6, 15, 40, 42, 45, 51, 57 (n52), 59, 61, 78, 91 , and 119 were found in 13 (3.7%). the partial d alleles found were diverse, but the largest number included partial rhd*d 4.0 (n562) and *dar ( conclusion: in a multiracial cohort of 352 individuals with weaker than expected d typing 44% were due to weak d types 1, 2, or 3 and would not be considered at risk of clinical significant anti-d, but for 56% there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp242 rhd*07.02 allele causes discrepant genotyping results for rhce small c sabine scholz* 1 , sandra schneider 1 , sabrina k€ onig 1 , susanne helmig 1 and vicky van sandt 2 . 1 inno-train diagnostik gmbh, 2 rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c (307c) and c (307t) is caused by the snp on position 307 on the rhce gene. the rhd*07.02 allele (also known as rhd cat vii type 2) carries the snp 307t>c on the rhd gene and additionally the snp 329t>c. this rhd*07.02 allele has been described to partially express rhc on the d polypeptide (faas, transfusion, 2001) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, 81938) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles (307t>c, 329t>c) confirming a rhd*07.02 and one rhd*01 allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series 4 and 5 anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series 4 and series 5, and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c.463a>g change in exon 3 encoding an amino acid change p.met155val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c.463a>g (p.met155val) change in exon 3. several snps, deletions, and insertions have been reported with changes in exon 3. phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c.463a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* 1 , juan merayo-rodriguez 2 , christopher lough 1 and nancy eckert 3 . 1 lifesouth community blood centers, 2 life south community blood centers, 3 lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is 100% in most populations and greater than 99% in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, 57 year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is 6.4/ 18.8 and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal 510(k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found 251 eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to 7.1/21. the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient 1 was a 29 yo female, c2e2c1e1, whose rbcs reacted 11 by echo and 31 by neo with anti-d4, and '?' with anti-d5. testing with d4 and d5 by tube gave 21 and 11 w on initial spin (is) respectively and 41 by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and 1 w with immucor gammaclone anti-d, and all were 21 at iat. rbcs did not react with two (lhm 174/102 & 57/17) of 12 anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c.780c>a change encoding p.his260gln. patient 2 was a 20 yo pregnant female, c2e2c1e1, whose rbc were 1 w at is and 31 at iat with immucor series 4 and 5 and gammaclone, and moderately reactive, 21 is and 41 iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm 174/102 & 170/45) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon 2 gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon 2 from c.150 to c.203 encoding amino acid changes p.ile60leu and ser68asn. conclusion: we found two previously reported rare alleles: rhd with a c.780c>a (p.his260gln), previously found in france (lefloch et al. genbank ku363612), and rhd*dar with part of exon 2 replaced by rhce, reported in sub-sahara africa (granier et al. transfusion 53:3009) designated rhd*dar(ce2:v505v-s68n) with an allele frequency of 0.002 to 0.016. blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu5f9-g4 is a human monoclonal igg4 antibody recognizing cd47 that is in clinical trials to treat hematologic or solid malignancies. cd47 is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd47 is thought to enhance phagocytosis and promote anti-tumor responses. cd47 is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd47 drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n57) from 2 patients were tested over the course of 1 month treatment. plasma was tested at immediate spin (is) and by iat with r2r2, rr, d--, rh mod and rh null rbcs, as cd47 expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg4) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd47 was observed in plasma as soon as 1 hour post infusion. plasma reacted 31 to 41 at is and 41 with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker (31 and 21) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was 31. in contrast, iat reactivity using gamma-clone anti-igg was only 1 w to 11, and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd47 titer was 1 at is and peg iat with gamma-clone anti-igg, but was ! 256 with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n54) were 31 reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after 4x allo-adsorption with papain treated rr cells, but in some samples low level (micro-11) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu5f9-g4 anti-cd47 therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd47 expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu5f9-g4 is igg4. reactivity was observed in all phases and with all test methods. cd47 is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd47 reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg4, can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd47 on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat1 rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in 1985. in august 2016, she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh1k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was 21 with untreated and 31 with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:-3 etc. had been excluded, k-phenotyping revealed a k 0 -phenotype. a total of 38 silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k 0 -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel*02n.19 with c.2023t encoding p.675ter (reported in an individual from austria in 2007). there are two known k 0 -patients in our country, both homozygous for c.2023t. the daughter was a c.2023t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k 0 -donors are available in our country. with the help of the isbt rare donor working party, a k 0 o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* 1 , kshitij srivastava 2 , houda romdhane 3 , saloua jemni yacoub 4 and willy albert flegel 1 . 1 nih, 2 dtm/cc/nih, 3 regional blood transfusion center sousse, 4 regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, 159a transfusion 2017 vol. 57 supplement s3 since 2009. the tunisian population has the largest known prevalence of weak d type 4.0 alleles, occurring in 1 of 105 rh haplotypes, compared to 1 in 6,060 or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type 4.0 in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of 13,431 random blood donors were serologically screened for the d antigen using 3 routine techniques. samples with weak reactivity were tested with a panel of 6 monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of 67 discrepant samples (0.5%) were observed and expressed the serologic weak d phenotype. among them, 60 carried an allele of the weak d type 4 cluster (89.6%), of which 53 samples (88.3%) showed the weak d type 4.0 allele. only 1 sample each was found for the weak d types 1, 3 and 100 and the dvii, while 3 samples showed the consensus rhd sequence. no mutation in any of the 10 rhd exons was detected in another 3 samples. the molecular analysis of the rhce gene showed that 59 out of 67 samples with serologic weak d phenotype (88.06%) had a variant rhce allele and the most common associations were: weak d type 4.0 linked to rhce*cevs.04.01; weak d type 4.2.2 with cear; and weak d type 4.1 to rhce*cevs.02, while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost 90% of the weak d phenotypes in tunisia were caused by alleles of the weak d type 4 cluster, of which 88% represented the weak d type 4.0 allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type 4.0 in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type 4.0 phenotype. there is a possibility that the rhce*cevs.04.01 allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* 1 , heather simmons 1 , christine lomas-francis 2 , gayane shakarian 2 , sunitha vege 2 , lauren hutelmyer 3 , sandra nance 4 , jessica poisson 1 , nicholas bandarenko 1 and connie m. westhoff 2 . 1 duke university hospital, 2 immunohematology and genomics laboratory, new york blood center, 3 arc pennjersey, 4 american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a1, 2 year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with 0.2m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c2, k2, fy(a2),s2]. reactivity was detected to a titer of 64; it was not removed by prewarm technique or by 4x peg alloadsorption. the adsorbed plasma reacted with 0.2m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k2, k2, js(b2), kp(a2b2) and sc:21,23. her plasma reacted with k o , mcleod, sc:21,22 rbc samples and dtt-treated sc:21 rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused 4 aliquots of crossmatch incompatible kp(b2), s2 rbcs. her post-transfusion dat was 21 with anti-igg, 11 with anti-c3d. the eluate reacted with all rbc samples except 1 kp(b2) sample. she tolerated additional aliquots from 4 phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k2, k1, kp(a2b1), js(a2b1) and sc:1,22, discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c.1481a>t (p.glu494val) (kel*02.10) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a2). sc sequencing found heterozygosity for a 5'-2g>a change (rs12124733, 24 to 30% prevalence) and conventional sc*01, predicting sc:1,22,3. kel and sc results on the mother were kel*02/kel*02.10, heterozygous for the sc change 5'-2g>a, and her rbcs typed k2k1 kp(a2b1), sc31, ula1, consistent with dna predictions. plasma collected 7 months later was nonreactive at rt and in peg iat. her rbcs were dat2 and now typed k1, kp(a2b1), ul(a1) sc11 and sc31,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel*02.10 homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc:21,23 rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b1). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a 55-year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron 5 polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs5-1a)genotype, associated with a jkb null phenotype. anti jk3 was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused 64 old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a1) 41 and jk(b1) 21. genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk3. however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk3 or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms 160a transfusion 2017 vol. 57 supplement s3 (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a1b-); kp(a1b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on 3 separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon 2 revealed a 287g>a mutation, fy01*n.04, known to silence fya. sequencing of kel exons 1-19 exposed a silent polymorphism in exon 8, 846g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy01*n.04 mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly 100% of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, 64 self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have 2 genetic variants not previously reported in those of african descent. only 1 was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient 1. study design/methods: patient 1 was a 7 year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at 3 years of age. anti-b changed from undetectable/weak to strong at the age of 7 years. patient 2 was a 17 month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was 0/11. both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table 1 . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient 1 with 0-11 reactions up to 7 years of age. thereafter, abo typing showed mainly strong anti-b. patient 2 had 0/11 anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for 2 children on long-term tpn. patient 1 had absent/weak anti-b since birth up to 7 years of age, then developed strong anti-b with no change in feeding regiment and medications. patient 2 had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used 28 times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* 1 and elizabeth hart 2 . 1 brigham and women's faulkner hospital, 2 university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title 42, cfr part 493.1271(a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in 2016. a total of 232 samples were evaluated. each abid was subcategorized; (1) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and (2) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of 83 abids were performed on new patient samples. of the new abid samples, 29 (35%) had microscopic absc results. for the previously known antibody patients, there were 35 which accounted for 15% of the total abids performed. when reviewing the total abid workups, a total of 64 (28%) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the 29 new antibody samples were: conclusion: a total of 86% of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: (1) discontinue the use of the microscope, (2) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or (3) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than 99% in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only 10 cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a 56-yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a 59-year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received 10 rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only (1-21), and negative with anti-c3b, c3d reagent. the antibody showed a peak gel-igg iat titer of 32. results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w1), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from 33 to 83%, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody 12 anti-p 1 4 anti-m 2 anti-sd a 6 anti-le b 1 anti-jk a 1 anti-k 1 anti-e 1 anti-c 1 results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of 21% (type 1), 5% (type 2), 9% (type 3), 25% (type 42) and 40% other than 1, 2, 3 or 42. further investigation was conducted to determine the molecular identity of the «others». out of 157 samples, 119 (75%) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon 5 or both exons 4 and 5. a surprising amount of 38 samples were discovered to be normal rhd. conclusion: along with sandler et al. (2015) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types 1, 2, 3 and 42 obtained in serological weak d, 45 years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was 0,6%. no trali happened in the period. prophylaxis were used in 98% of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred 3 times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, 358 rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p <0.05). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) (0.27% vs 0.11%, p <0.001) or aes with a non-allergic type inflammation etiology (0.30% vs 0.14%, p <0.001) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes (0.028% vs 0.024%, p 5 0.614) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for 1, 2, 3, and 4 weeks were 0.25%, 0.32%, 0.39% and 0.41%, respectively (p 5 0.084, logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week (0.25%) and longer than a week (0.35%) (p < 0.05). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than 1 week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd10 codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found 11 patients from 2011-2016, who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these 10 patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these 10 cases, only 2 of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those 10 cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as 1 out of 100 transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was 8.7 g/dl but declined to 7.3 g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days 2 and 3 with poor responses. on day 4, routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from 1.0 mg/dl to 1.8 mg/dl (reference 0.8-1.3 mg/dl), and lactate dehydrogenase was above reportable linearity, >2500 u/l (reference 122-222 u/l). testing revealed additional anti-e, anti-jkb, dat c31, plasma free hb 64.4 mg/dl (reference 1-15.2 mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b1), one of which was also e1. one volume tpe was performed to remove free hb on days 5, 6, and 7 using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at 3.7 mg/dl on day 13, decreased to 2.3 mg/dl before discharge on day results/findings: twenty three cases were identified, of which 20 had medical records available for analysis. ten (50%) patients were male, the mean age was 50.4 years (range 24-76 years), 15 (75%) had an underlying hematologic malignancy or bone marrow disorder, and 3 (15%) had a history of coronary artery disease (cad). the implicated units included 14 (70%) red blood cells and 6 (30%) platelets; 17 (85%) patients received a single unit, and 3 (15%) received two or more within the previous 6 hours; the mean volume transfused was 153.3 ml (range 20-280 ml). the mean time to onset of chest pain was 92.15 minutes (sd 85 minutes), with 90% of patients presenting within 2.5 hours and 100% within 6 hours of starting the transfusion. chest pain was present as the only symptom in 35% of the cases, and for the other cases the accompanying symptoms included dyspnea (30%), fever (25%), back pain (20%), and hypo-and hypertension (10%). a post-transfusion chest x-ray was performed in 65% of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in 70% of cases and showed no findings to suggest acute ischemia. three (15%) patients had a minimal increase in their troponin levels, although 1 had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen (70%) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than 10 minutes in the majority of patients (90%). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a 6 months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts 13. based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, 37 degree, or anti-human globulin phase. check cells were found to be 21. these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o1 to a1) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a 63-year-old o1 man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha-1 antitrypsin deficiency who presented for olt (donor o1). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving 46 units of o1 rbcs and 26 units of o1 plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a1 plasma were transfused to wash out anti-a antibody prior to transfusion of a1 rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for 7 hours post-transplant. the patient's total estimated blood loss was >20l. he received a total of 71 units of rbcs (including 23 a1), 63 units of plasma (including 37 a1), 6 units of cryoprecipitate, and 9 units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o1. on postoperative day (pod) 1, a ts showed predominantly a1 rbcs with trace o1 rbcs, as well as very low anti-a igm and igg titers (table 1) . he received two additional o1 rbc units (1 each on pod 4 and pod 12) with increasing o1 rbcs on ts and rising anti-a titers. his blood type was unequivocally o1 by pod 13. the patient showed recovery of liver synthetic function on pod 1 (factor 5 activity 5 58%) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod 13, the patient had reverted to o1 with recovery of anti-a titers. at 3 months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* 1 , alfred mingo 1 , maria isabel gonzalez 1 , antoni mena 2 and juan pedro benitez 2 . 1 bst, 2 at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h1) and an oncology center (h2). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of 2016 transfusion activity in both h1 and h2 shows 7970 pse and 12572 bca, out of 13163 and 20676 respectively, since the tss deployment in 2015. retrospective analysis and classification of 6700 security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h1, nm related to pse accounted for 42.39% of all, being the mistake in concordance between patient identification and prescription order the most frequent (52.03%). the nm detected in bcas were 12.1% of all and mostly (74.52%) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are 45.51% of all and mostly (36%) the systems detects a not assigned bracelet. for h2, nm related to pse accounted for the 47.49% of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent (65.84%). the nm detected in bcas accounts for 24.48% of all and in 69.08% occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are 28.02% of all and in 65.61% of them the blood components were assigned to another patient. (1, 3, 4, 5, 8, 9, 12, 14, 19, 23, 26, 51, 56, 68) were analyzed via a commercially available elisa. comparison of adequate response to ppv23, defined as ! 2 mcg/ml for >7 serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in 72 patients (alloimmunized, 15); pre-and post-vaccine titers were available for 19 patients (alloimmunized, 6). of the 72 patients, 25 were on chronic transfusions, 24 were on hydroxyurea, 11 were surgical splenectomized, 58 patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv23 in the previous 10 years; 9/44 also reported previous history 13-valent sp conjugate vaccine within the last 5 years. baseline pre-vaccination titers (n572) showed no difference between alloimmunized and non-alloimmunized patients (all p-values >0.13). in the group with pre-and post-vaccination (n519) titers available, 11 out of 13 (85%) non alloimmunized patients had an adequate response versus 4 out of 6 in the alloimmunized group (67%, p 5ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from 2012 to 2016). patient harm events recorded within the veritas system from january to july 2016 were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of 114 tres per month and 1300 per year were found over five years. 81% of tres are associated with pre-bb activities, 10% occur within bb, and 9% are post-bb events. sample collection and handling represent 80% of total tres. most tres (96%) were reported by bb staff, 4% were reported by non-bb staff. patient harm analysis revealed an average of four level 0 (near miss), three level 1 (no known harm), and 0.3 level 2 (patient harm) per month. no deaths related to tre were detected over the seven month january to july 2016 period. patient harm was associated with tres occurring in the bb (17%) and post-bb (83%). these events were reported externally (78%) and by bb staff (22%). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january 1, 2013 and march 28, 2017 was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within 6 hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by 5 and added to the number of individual whole blood plts; apheresis plasma units were multiplied by 2 and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was 31. the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the 4-hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions (18[10 9 /l vs. 14[10 9 /l, p50.029). there were no significant differences in the frequency of effective therapeutic (55% vs. 72%, p50.1) and prophylactic (63% vs. 54%, p50.09) transfusions between the prcs and 25gypcs. we did not find significant differences between prcs and 25gypcs in cci1 after prophylactic (16.0 6 7.1 vs. 19.2 6 8.7) and therapeutic (11.3 6 9.0 vs. 11.8 6 5.8) transfusions, in cc24 after prophylactic (20.0 6 9.2 vs. 22.5 6 12.8) and therapeutic (13.3 6 8.9 vs. 13.9 6 8.) transfusions. there were no significant differences between prcs and 25gypcs also in ma1 after prophylactic (62.2 6 8.5 vs. 60 6 8.5, p50.7) and therapeutic (61.3 6 9.9 vs. 60.9 6 12.7, p50.08) pc transfusions. reduction of the severity of bleeds was obtained in 78 (86%) of the 91 cases after prpc transfusions and in 51 (84%) of 61 cases after 25gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, 3 and 2 cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in 2006. it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in 2014 under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were 593 units administered using the emergency transfusion process in the 3 months before the change was implemented. it was found that 51/593 (9%) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month (2530 components administered), 109/2530 (4%) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: 1) a stable anemic inpatient, 2) a stable anemic inpatient to be discharged, and 3) an asymptomatic post-operative inpatient. results/finding: we identified 67 canadian tm experts: 48 (71.6%) provided a response and most had a primary place of practice in a laboratory setting (38/48; 79.2%). for a stable, non-bleeding, anemic inpatient, 87.5% of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with 31.2% generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period (1-2 hours), a repeat hemoglobin >18 hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse (38.1%) compared to an inpatient potentially symptomatic due to anemia (72.1%). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium-51 ( 51 cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n514) were divided into two 150ml aliquots, which were labeled with selected concentrations of s-nhsbiotin (3 and 30 lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of 38.4 6 1.6%). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately 2 million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that 561nm excitation of phycoerythrin (pe)-sa and high laser power (150mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with 3lg/ml of biotin resulted in $50,000 mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, 95% ci) of 1 in 380,000 (0.0003%). the lld95 for rbc labeled with biotin at 30lg/ ml was $ 1 in 1 million (0.0001%). biotinylation was not associated with increased levels of hemolysis (0.40 6 0.22% before labeling versus 0.34 6 0.12% after labeling; p50.09) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes (150ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer-250 (hboc-201) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: (1) wb, (2) wb110% hboc volume (model of two units in an adult), (3) wb110% fdp, and (4) wb110% hboc110% fdp. samples (5)-(8) simulated autoresuscitation by adding 25% plasmalyte to 1-4. susceptibility to lysis was tested with 75ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of 50%, 60%, 75%, and 100% volume replacement with hboc and/or fdp, with or without prior 25% plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb1hboc, and wb1hboc1fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb1hboc in autodilution simulation (mean lysis 4.79% vs. 16.36%; p<.05). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc (10%) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even 75% hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc-201, there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc1plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in 8/2013 and 2/2015. we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of 81 patients, 24 were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery (43%). pcc was given for warfarin reversal in 31% of cases. a subset of patients received plasma within 2 hours prior to pcc (40%) or 24 hours after (47%). pcc was most frequently ordered in the or/perioperative service (25%). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in 2016, several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; 1) simplify and expedite the process; 2) improve communication and expectations to decrease tat; 3) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood (4 units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of 61 er episodes were received. the average tat from order to delivery at the bedside was reduced by 50% (7.0 minutes compared to 14 minutes previously), while the compliance rate for er orders and physician documentation was 100% (61/61), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february 2016 -february 2017. mt was defined as the transfusion of ! 10 rbc units in a 24-hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at 30 days. results/findings: thirty mts occurred during a one year period. a total of 192,441 blood products were transfused during that time period. gender distribution was 21/30 (70%) males, and the average age of all patients was 68 with a range of 21 to 70 years of age. surgical patients accounted for 26/30 (86.7%) mts, and 4/30 (13.3%) were critical care patients. tumor categories included carcinomas (14/30), sarcomas (13/30), leukemias (2/30) and lymphoma (1/30). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer (6/30) was the most common disease seen followed by sacral chordoma (4/30). mtps were activated in only 8/30 (26.7%) cases. thirty-day survival was seen in 25/30 (83.3%) patients. only 1 of 5 mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage (3/ 5) or perisplenic hematoma (1/5). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a 54-year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a 26-year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a 70-year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* 1,2 , rebecca ross 2 , christopher a tormey 1,2,3 and amit gokhale 1,2 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: daratumumab (dara) is a igg1 monoclonal antibody therapy that specifically targets cd38, a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd38 expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, 54 subjects were identified for analysis. their mean age was 67.8 years, with 29 male and 25 female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of 0% (0/54) prior to administration of dara. of these patients, 22 were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these (0%; 0/22) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the 22 patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of 2015 for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september 1, 2015 through april 7, 2017 were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age 60 was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in 2016, 2,523 unique women receiving a total of 5,889 units of red cells (rc) in 2,906 transfusion episodes were identified. their median age was 45 (range 11 -60). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in 46 (1.6%) and 283 (9.7%). 635 (21.9%) transfusion episodes were associated with the use of 3 units or more rc. as a result, 1385 (47.7%) episodes resulted in a post transfusion hb ! 9g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given 2 (<0.1%) and 116 (4.6%) women respectively. upon discharge, 442 (15.0%), 84 (3.2%) and 1,994 (65.8%) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to 386 (16.0%) women. conclusion: in the present study, it is observed that 49.7% transfusion episodes were given at hb ! 7g/dl. a substantial number of episodes (71.7%) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! 9g/dl). for iron replenishment and bleeding control, up to 16.0% transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes (0.8-1.3x10 10 per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with 25 gy and transfused over 3-4 hours within 24 hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* 1 , lee grabner 1 , brenda herdman 2 , robert fallis 1 , amin kabani 3 and charles musuka 3 . 1 canadian blood services, 2 kenora rainy-river regional laboratory program, 3 diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over 50 years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between 2008 and 2011 before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and 38% were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels (8.7 g/dl; p50.94); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units (9.9 vs 9.8 g/dl; 1.2 vs. 1.1 g/dl; both p50.02). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients (1.3 g/dl vs. 1.0 g/dl; p<0.001). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p50.01). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p50.53 and p50.32, respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than 1% of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered 33 times in total, affecting 15 patients and 21 users. stratified by location, the majority of triggers occurred in the perioperative areas (18 times) and the liver icu (6 times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin 25% iv solution, human albumin 5% iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* 1,2 , mahmut akgul 1,2 , hollie m reeves 1,2 , robert w maitta 1,2 , marcie pokorny 2 , anne capetillo 2 and katharine a downes 1,2 . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a 7-month retrospective study (january-july 2016) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was 200-400 mg/dl with critical value of 50mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were 301 adult (>18 years) orders reviewed by tms out of which 299 were approved. of the 299 approved orders, 136 (45.5%) were in agreement with tms's estimated dose. of 163 (54.5%) orders that were not in agreement with the tms's estimate, 142 (47%) were underestimated and 21 (7%) were overestimated. seventeen of 299 orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of 23.6 mg/dl (range 0.3 to 124 mg/dl) and a median excess of 13.3 mg/dl (range 0.6 to 155 mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was 12 mg/dl above target, which is significantly higher with intervention than without (which could have been 12 mg/dl below the target; p<0.0001). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention (11 vs. 2.7 mg/dl, p50.07). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention (15 vs. -23.5 mg/dl, p<0.0001). seven of 299 (17) 40 (24) orders were for critically low fibrinogen (<50 mg/dl) and 4 of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were 10 and 5 units (59.5% and 25%) i.e. 2 and 1 pools and the most frequent orders in the disagreement group were 10, 1, 5 and 2 units (33%, 20%, 17% and 16%). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was 2.36 and posttransfusion was 1.91. only 22% of patients had their inr corrected to 1.5, while 28% had no change, or had increased inr. (table) . the majority (67%) of patients received 2 units of plasma. the mean plasma dose was 6 ml/kg. there were 4 transfusion reactions reported, 1 non-hemolytic and 3 transfusion associated circulatory overload reactions in which 1 required admission to the icu. two patients experienced bleeding during ir procedures (tips) and 1 developed a hematoma (tunneled central line). the median of inr correction in this study was 1.9 with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is 1.9. randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. 3 of the 111 patients experienced bleeding complications indicating that inr of 1.9 may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately 4% of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up 45 % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population (40%) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately 85% of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july 1, 2014 as follows: 2 units of group a plasma and 3 units of group ab plasma is provided for the massive transfusion protocol (mtp) along with 5 units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed 235 mtps at our institution between july 2014 and march 2017. twenty patients (8.5 %) were transfused with incompatible group a plasma (5 group ab and 15 group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining 15 patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april 2017, our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in 2013, bonfils immunohematology reference lab (irl) sent out approximately 245 special platelets for patients with hla antibodies. by 2016, hla platelet orders increased dramatically and the irl sent out over 650 special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over 10,000 donors in the database with historical hla typing. however, only approximately 3500 of those donors actively donate. in the denver area, one of the most common hla types is a1 a2 b7 b8. only 81 of the 10,000 donors have this type (0.81%). therefore, to fill an hla platelet order request for a common hla type, only 28 donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a9 a11 b17 b35, there is only 1 out of 10,000 donors (0.01%) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a1 for example, all of the a1 positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in 2012, approximately 27% of these special order platelets were pra matched and the remaining 73% were hla matched by donor recruitment. by 2017, approximately 59% of special platelets sent are pra matched. this change resulted in a 2.2 fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains 6 packed red blood cells (prbcs), 4 fresh frozen plasma (ffp) units, and 1 plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august 2013 to march 2017 mtps were activated at ucm, of which 251 orders could be traced to the origin: 118 on inpatient floor (including icus), 58 in the operating rooms, 46 in the emergency department, 25 in labor and delivery, and 4 in other procedure rooms. of the 2207 prbcs that were issued, 1406 were transfused (64% utilized); of the 1446 units of ffp that were issued, 901 were transfused (62% utilized); of the 359 platelet packs that were issued, 246 were transfused (69% utilized); of the 64 units of cryoprecipitate that were issued, 49 were transfused (77% utilized). since march 2016, the time of first product issue after the initiation of an mtp has also been tracked. of the 84 events that fall within this time period, 39 (46%), had the first product issued in 5 minutes or less. another 31 (37%) were issued between 5-10 minutes, resulting in over 80% of patients being issued their first blood product within the first 10 minutes. only 15 of 84 (17%) events had an initial time greater than 10 minutes and none were greater than 21 minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level 1 trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($60-70%). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within 10 minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* 1 , shailesh macwan 1 , arline stein 1 , jane fischman 1 , nancy nikolis 1 , matthew bank 1 , lennart logdberg 1 , alexander indrikovs 2 , sherry shariatmadar 1 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: massive bleeding is generally defined as any patient who requires 1 blood volume replacement within 24 hours and/or receives transfusion of greater than or equal to 4 units in one hour with 177a transfusion 2017 vol. 57 supplement s3 ongoing bleeding. our mtp was officially implemented in 2013 in preparation for an initial verification as a level 1 trauma center by acs. our mtp has the following packages: 1st pack has a ratio of 4:4:1 (rbcs, plasma & platelets) and subsequent packs a ratio of 6:6:1. our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march 2016 to add cryo and pcc at a defined point in the mtp (cryo is included in the 3rd pack and pcc in the 4th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received >20 rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had 8 patients who received >20 rbc in 2015 and 2016. mtp had been activated for all patients and all patients received between 0.5 to 1 unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt >16 seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of 200 mg/dl (table 1) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of >20 rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's 5-day and 114-day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective 10-year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of 63 published cases (31 reports) of ta-pls, 8 (4 reports) were stem cell and 55 (27 reports) were organ transplants. all 8(100%) stem cell transplants and 52 (95%) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the 4 reports of stem cell ta-pls, 3 actively screened for antibodies in the immediate post-transplant period, and of the 27 reports of organ ta-pls, 1 actively screened for antibodies. these screens detected 5 cases of stem cell ta-pls before hemolysis became apparent and 2 cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* 1 , rosario mallari 2 , marc de asis 2 , elaine shu 3 , jonathan hughes 4 and tho pham 1,3 . 1 stanford university, 2 stanford health care, 3 stanford blood center, 4 bloodsource background/case studies: mur antigen is present in 7-10% of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. 41 year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received 1-2 rbc units every 1-2 weeks to maintain a hemoglobin (hb) level of 8 g/dl. the patient remained stable for 5 months when his hb level acutely dropped to 6.6 g/dl. the antibody screen remained negative for an additional 2 months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for 30 of the 33 rbc units he received. 3 units were from an asian donor, and a unit transfused 13 days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of 64,495 donors at a hospital-associated blood center located in a region where asians comprise approximately 30% of the population. results/finding: 6.6% of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for 5245 of 37933 (13.8%) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over 10% of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* 1 , princess maynie 1 , carol chandler 1 , shelia garret 1 and pampee young 2 . 1 vanderbilt, 2 vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [3] [4] [5] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of 26 samples have been analyzed (table) , 17 rh and 9 non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being 2.77 (range 1-7) times higher. the average fold change for rhd/c/e antibody titers were 3.2, whereas the average fold change for non rh titers was 1.03 (range 1-2). the range for anti d titers was particularly variable, 2-7, whereas for c/e, it was 1-3. the overall reproducibility/precision of the automated analyzer was $90%. to correlate the 178a transfusion 2017 vol. 57 supplement s3 increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $3 times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of 250. units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to 33% of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately 65% of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix 0.8% suspension of a1 and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with 15 min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, 32 group a, and 16 group b. results/finding: of the 100 whole blood edta samples tested, 26 (25 group o and 1group b) exceeded a high titer threshold of 250. when the pas samples of these 26 donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of 250 when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of 250, a 96% decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from 2 main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year 2016 was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the 27(59%) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the 2 mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these 3 hospitals had a lower overall rate of wastage including their own donations than the other 13 hospitals that did not collect in-house plt. the other 13 hospitals had wastage rates ranging between 9-54%. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* 1,2 , christopher j gresens 3 , anne capetillo 2 , hollie m reeves 1,2 and katharine a downes 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center, 3 bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of <0.1%. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a 58-year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure (117/65 mm hg to 205/89 mm hg) followed by hematuria (500 ml). chills and rigors resolved; blood pressure stabilized after 15 min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of 256 (igm) and 1,024 (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* 1 , leila patricia de sousa fontenele 1 , isabel nagle reis 1 , carolina bonet bub 1 , araci sakashita 1 , raffael zamper 1 , cristiane nakazawa 1 , tatiane almeida omura paula 1 , patricia silva batista 1 , marcio dias almeida 1 , fernanda loureiro de andrade orsi 2 and jose mauro kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of 671 cases of olt performed between 2011 and 2015 in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of 671 consecutive patients submitted to liver transplantation between 2011 and 2016 were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or 1,726 -95% ci: 1,147-2,597, p:0,009), absence of hcc (or 0,295 -95% ci:0,199-0,437, p:0,0), cirrhosis of any cause (or 4,161 -95% ci 1,816-9,534 -p:0,001), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or 5,236 95%ic 2,212-12,394) and retransplantation due to primary non function of the graft (or 5,791 95%ci 1,33-4,25,206, p: 0,019) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a1/b in group o platelet products. charles k. childers* 1 , mark destree 2 , ashley rose 2 and theresa nester 3,4 . 1 madigan army medical center, 2 bloodworks northwest, 3 bloodworks nw, 4 dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a1 or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a1/b in group o platelet products is presented from a large regional blood center collected over 10-12 months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in 2 ml edta sample tubes. a single 1:150 dilution of plasma was prepared using a hamilton microlab 600 series dilutor using 2235.0 ml saline diluent and 15.0 ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a1 or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at 3175 rpm for 20 seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a1 cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a1 or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of 150 is used, approximately 3% of group o apheresis platelets will have a high titer, most commonly with anti-a1. less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their 5 day outdate. after 10 months of testing pspp units and verifying that the products rarely had a high titer (0.28%), the blood center stopped performing this testing for pspp units. rh1) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to 19% (from 24% on the previous day). his hematocrit did not increase (18%), and over the ensuing 12 hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of 8 in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c3 in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of 24% while reducing the number of a rbcs in his circulation by approximately 70%. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes 7-14 days for antibodies to develop and they are short-lived (3-5 weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the 2016 calendar year from 9 hospitals. an additional hospital* provided data for august-december 2016. rbc transfusions in patients <1 year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/5 70 years was also determined. results/findings: see table 1. the fraction of all transfused rbcs that were oneg ranged from 5-14% (row f). the percentage of oneg rbcs transfused to oneg patients ranged from 37-89% (row g); thus, non-oneg patients received 11-63% of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients (68%-100%; row i). overall use of oneg rbcs could have been reduced by 10%-39% if opos units had been given to all oneg patients >/ 5 70 years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* 1 , nehad mohammed 2 , marwa aly 2 and nashwa fahmy 2 . 1 national blood transfusion services, 2 nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on 206 multitransfused patients who received blood transfusion chronically at our central blood center. they were 129 thalassemia patients (128 bthalassemia patients, one patients with a thalassemia), 10 sickle cell anemia patients and 6 immune hemolytic anemia patients (4 auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). 29 oncology patients, 32 chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: 32 out of 48 (67%) alloimmunized patients and 16 out of48(33%) non alloimunized patients(p<0.001) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june 2016. screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified 136 articles for data abstraction, where 48 articles were transfusion guidelines. there were 12 guidelines (25%) that made a recommendation, 11 for a single unit and 1 for multiple unit transfusion strategy (table 1) . review b identified 3 retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or 9.4, 95% ci 5.02-17.60), although heterogeneity was high (i 2 597%). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december 2016 and february 2017 was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the 3 months, 1723 units of platelets were transfused to 238 recipients. over the 3 months, a median of 4 units was given to each patient with a range of 1 to 69. the overall distribution of products used was 58% plasma, 24% pr, 7% pas f and 11% pas c. thirty percent of patients (n572) received all of their products on a single day. single units were given to 54 patients while 14, 3 and 1 received 2, 3, and 4 units respectively. the distribution by product type was 56% plasma, 25% pr, 13% pas c and 4% pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the 3 month period (p5 1.00). the distribution by service was different for the groups receiving multiple units. for single units the distribution was 44% hematologic malignancy, 22% infusion clinic (nos), 13% solid tumor medicine, 11% surgery, and 9% pediatrics. for those receiving multiple units the distribution was 50% surgery and 16% each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of 0.022. conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the 3 month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* 1,2 , yvemarie b.o. somsen 2 , maike e. van hezel 2 , marleen straat 2 , robin van bruggen 1 and nicole p juffermans 2 . 1 sanquin research and landsteiner laboratory, 2 academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains 220 mg of iron and 25% of the rbcs are cleared by macrophages within 1 hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in 52 icu patients who received one rbc transfusion, different iron parameters were measured before and 24 hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il-6 levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion (4.1 vs. 4.3 mmol/l, p50.69). also, the transfusion had no effect on transferrin saturation (12 vs. 13 %, p50.13), ferritin (531.0 vs. 599.0 mg/l, p50.74) and il-6 levels (35.0 vs. 25.5 pg/ml, p50.09). hepcidin levels increased in these icu patients after rbc transfusion (223 vs 332 ng/ml, p50.01). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (-2.7 vs. 3.7 % change, p50.05). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il-6 or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since 2011 and pathogen reduced platelets have been available since 2016. in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between 2012 and 2016. during this time pas c, pas f and pr went from 13% to 40% of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in 2011 patients received an average of 5.41 units/recipient/month and in 2016 the average was 5.39 units/recipient/month. the intervening data points for 2013, 2014, and 2015 were 5.92, 5.66, and 5.92 respectively. the 5 year average was 5.66. the slope of the graph for all 5 points was y5 -0.004 15.672. the two sample t-test showed that the plt/recipient/month from 2012 to 2016 was not statistically different with a p value of 0.81. conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that 4% to 8% develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab 17 statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least 5 k/ul. the analysis revealed that the mean of 9.35 k/ul (n584) had a 95 percent lower bound confidence interval platelet increment of 7 k/ul (p<50.001) results/findings: 123 (median 4 range [1-43]) hla matched leucoreduced irradiated sda platelets were transfused to 17 (6m/11f) patients, median age 60 years (range 27-83). 15/17 (88%) patients showing broad alloimmunization to hla class i/class ii antigens. 2/17(12%) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority 16/17 (94%) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); 9/11 (81%) female patients had prior exposure via pregnancy and 4/11 (24%) had a history of hsct. 63 (51%) platelets were abo identical-platelet increment median 7 k/ul (range -14 to 61), 53 (43%) were abo compatible -platelet increment median of 2k/ul (range -10 to 46) and 7(6%) were abo incompatible with platelet increments median 8k/ul ( range -10 to 32). platelet counts were performed within 24 hours in 73 (57%) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* 1 , renee leblanc 2 , dongfu xie 2 , alice cabe 1 and yanyun wu 2 . 1 overlake hospital, 2 bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. 1 these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about 350 for 3 years (from 2014 to 2016) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and 68 % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than 0.25% of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a 32-year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of 6.4 g/dl (baseline 9-10 g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was 5.9 g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin (8.8 to 6.2 mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to 6.2 g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october 2015 to march 2017. patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were 139 rbc products transfused to 40 waiha patients. twenty-three (57.5%) patients received at least 1 incompatible unit. the mean age was 51.4 years (range 4-93 yrs) with 50% women. ethnic composition was 55% african-american, 40% caucasian, and 5% patients of mixed/other ethnicity. one hundred fourteen (82%) of these products were released as li products and 25 (18%) were compatible. ninetythree (81.6%) of the li product transfusions had a post-tfn hct change of <3% whereas only 14 (56%) of the compatible product transfusions resulted in a post-tfn hct change of <3% (p50.0092, v 2 (1), exact methods). the mean hct increase in the compatible group was 1.83% per unit vs. a slightly lesser per-unit increase of 1.71% in the li group (p50.82, t-test, 2-tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for 12 units. units that were 31 incompatible had a lower mean post-tfn hct rise compared to all other li units (1.49% vs. 2.15%); however, this difference was not statistically significant (p50.38). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected 3% per unit more frequently than it was for waiha patients who received compatible products (81.6% vs. 56%). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the 31 li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first 4-hours from mt onset) was calculated with 95% and 99.8% control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were 5482 mt cases from 25 hospitals (17 tertiarylevel, 6 smaller/medium sized acute-care and 2 specialist women's). number of mt cases per hospital ranged from 5 to 721. patient median age was 65 years (iqr 49, 76), 62% were male and 73% required admission to intensive care. the most common clinical groups were cardiac surgery (21% cases), trauma (20%) and gastrointestinal hemorrhage (13%); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the 17 tertiary-level hospitals was 21% (range 13% to 33%) and 16/17 (94%) were within the 95% control limit. cb that required !10 rbcs within 24-hours of mt onset occurred in 40% of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals (67% versus 78%; p50.03). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of 5 bcs and 280 ml of platelet additive solution iii (pasiii) were used to produce pcs (n55). pcs were stored on a flatbed agitator (60 cycles/min) in a temperature-controlled cabinet at 22 6 28c for 4-7 days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of 300 mm hg was applied. using clamps, a flow velocity of 90-120 ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted 10-30%, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration (0.8-1.6x10 11 /l) and number (>250x10 9 /unit). simulated transfusion had no effect on the percentages of cd62p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* 1,2 , joan sevcik 2 and joseph e. kiss 1,2 . 1 university of pittsburgh, 2 blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, 2 ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for 3 patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution 1:50 is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient 1 and 2 and 3 (control). results/finding: all 3 patients were ab blood type. patient 1, a 57 year old female with recurrent adamts13 deficient ttp, received 2 courses of tpe (total 12 tpe procedures) for relapse and exacerbation. ten out of 12 procedures were performed with ab and a plasma (average 916 ml of a plasma or 24% of total plasma volume for 10 tpe procedures). patient 2, a 27 year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of 12 tpe procedures. four out of 12 procedures were performed with ab and a plasma (average 1210.5 ml of a plasma or 48% of total plasma volume for 4 tpe procedures). patient 3, a 33 year old female with adamts13 deficient ttp who served as a control, received a total of 10 procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between 3 patients. the trends of hemolysis laboratory data for patient 1 and 2 were comparable with patient 3. all 3 patients had negative dat. only patient 3 received 2 rbc transfusions. all 3 patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to 48% of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd36 negative platelet unit is not available for a patient with anti-cd36 antibodies sameer khatri* 1 , charles harmon 1 , brian r curtis 2 and chisa yamada 1 . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd36) is one of the identified plt surface ags and deficiency is rare, but found in asians (3-11%), sub-saharan africans (7-8%) and also in some people from mediterranean descent. two types of cd36 deficiency have been described. type 1 deficiency is the complete lack of cd36 on both plts and monocyte-macrophages whereas type 2 deficiency lacks cd36 on plts with variable expression (12-99%) on monocytemacrophages. transfusing plts in a patient with cd36 deficiency is challenging given the rarity of cd36 negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd36 negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a 21 year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than 20 units of apheresis plt units over a 2 week period without any significant increase in plt count. cross-match compatible plt unit found in 1 of 32 units and hla matched units were tried without success. at that point, a cd36 ab was identified in the serum and the patient's type 1 cd36 deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was 95% due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd-36 negative (but blood type different and hla 187a transfusion 2017 vol. 57 supplement s3 unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd36 non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every 2 weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd36 antibody positive reactivity in serial dilutions has reduced from 1:32 to 1:2 dilutions and his hla class i pra has decreased to 37%. he is currently receiving 2 apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to 1.0 k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd36 abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct 2015 utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for 2016 to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec 2016, 101,854 blood donations were screened for b microti by immunetics elisa. of those, 267 (0.26%) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between 0.08% and 0.42%. no patient babesia transmission has been reported since implementing this test, but we only had 4 documented babesia ttd cases from 2007-2017. donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of 267 positive test results, 160 lookback investigations were initiated representing 59% of positive donations. lookbacks were only performed when there was a donation within 12 months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to 80% were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented 0.25% loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only 0.25% of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in >90% of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for 53 blood donors, 51 were positive with an average load of 1.49x10 2 copies/ml of plasma, a median value of 80.5 copies/ml of plasma, ranging from 0 to 1.87x10 3 copies/ml. pre-transplant viral loads were similar. for 41 transplant candidates, 40 were positive with an average of 3.70x10 2 copies/ml of plasma, a median value of 88 copies/ml of plasma, ranging from 0 to 1.18x10 5 copies/ml. post-transplant viral loads were remarkably different. for 94 transplant recipients, all were positive with an average of 3.14x10 5 copies/ml of plasma, a median value of 1.25x10 5 copies/ml of plasma, ranging from 0 to 4.6x10 7 copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around 100-200 copies were present in >90% of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least 2 orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt (17) 0.2 post-ivig: all plt (41) 5.5 post-ivig: cd36-negative plt from relative (1) 0.8 post-ivig: single donor apheresis (23) 4.3 post-ivig: cross-match compatible (15) 6.1 post-ivig: flow cross-match compatible plt (2) 12.6 188a transfusion 2017 vol. 57 supplement s3 detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv (1/2/3/4), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows 100% of specificity, with no false positive results on the 40 control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of 1 tcid 50 /ml for denv-1, denv-3 and chikv and of 10 tcid 50 /ml for denv-2, denv-4 and zikv. finally, the first results obtained on 110 denv(1), 69 zikv(1) and 50 chikv(1) clinical samples show 85%, 87% and 96% correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of 4, 8, 16 and 32 were prepared from 192 plasma or 192 serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from 1:128 to 1:1024 (2-fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of 16 or higher and was not eliminated by the addition of a blocking step. pools of 4 or 8 samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of 4 or 8 samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to 8 plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $100 cfu/ml of 12 organisms associated with platelet contamination and incubated at room temperature for 18-24 hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher (1 1 log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc 12924, were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into 4 ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta 3d and virtuo the organism was recovered 100% . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc 12924. however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of 4ml lrap demonstrated 100% recovery when loaded onto the virtuo and 3d ( table 1) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus 1-4, and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in 2014, a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of 150 mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as-5 rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final 200 mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and 3hrs after amustaline addition, respectively, for titration by plaque assay on vero76 cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, >5.1 log 10 or log 10 /ml of rrv was achieved, with >5.5 log 10 or >5.2 log 10 /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; 255-325 ml), large volume (lv; 300-390 ml) and dual storage containers (ds; 300-420 ml) designed to treat platelet doses between 2.9 and 8.0x10 11 . the new triple storage (ts) set was designed to expand the dose range to 12.0x10 11 and the maximum volume to 650 ml, generating either 2 or 3 doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or 100% plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in 47% plasma/53% pas or 100% plasma with a final volume of $650 ml and a dose of 9-12 3 10 11 platelets. these conditions represent inactivation using the lowest amotosalen concentration (135 mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log 10 titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of 6-10 logs in respiratory secretions of mers patients, and with lower genomic titers of 4-5 logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log 10 reduction/ml (log 10 /ml) 47%plasma/ 53% pas e .coli 6.0 <-1.0 >6.0 e. cloacae 6.4 <-1.0 >6.4 k. pneumoniae 6.6 <20.1 >6.5 s. aureus 6.7 <-1.0 >6.7 blue tongue virus 4.9 <-1.0 >4.9 bovine viral diarrhea virus 4.6 <-1.0 >4.6 adenovirus-5 1 3.9 <-0.6 >3.9 100%plasma k. pneumoniae 1 6.5 -0.5 >6.5 s. aureus 1 6.2 <-0.7 >6.2 adenovirus-5 1 4.5 <-0.6 >4.5 1 n53 190a transfusion 2017 vol. 57 supplement s3 abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged 3 times up to 9 days, assessing the infectious titer and genomic titer every 3 rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of !5.8 log infectious titer. no viral replication was observed after 9 days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above 5 logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* 1 , jen-wei chen 1 , chi-ling chen 2 , sheng-nan lu 3 and pei-jer chen 2 . 1 department of research, head office, taiwan blood services foundation, 2 graduate institute of clinical medicine, college of medicine, national taiwan university, 3 division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by 2030, and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since 1992) and 8-sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since 2013) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during 1999-2016 and 2013-2016, respectively. age-standardized prevalence and its 95% confidence interval (95% ci) were calculated with adjustment of who world standard population 2000-2025. for the incidence study, donors who have donated blood two or more times during 2013-2016 and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its 95% confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from 15.2 per 1,000 donors (95% ci: 14.8-15.7) to 4.0 per 1,000 (95% ci: 3.7-4.3) during 1999-2016, and the agestandardized prevalence was also decreased from 27.0 per 1,000 donors (95% ci: 25.6-28.4) to 7.7 per 1,000 (95% ci: 6.9-8.5). the agestandardized prevalence of anti-hcv was generally higher in female donors before 2015, but it was significantly higher in male donors at 2016 (p-value50.03). a total of 1,036 hcv rna positive cases, 1.9% of them were anti-hcv negative, identified from 579,286 first-time donors during 2013-2016, and the crude and age-standardized prevalence of hcv rna was 1.8 per 1,000 (95% ci: 1.7-1.9) and 5.0 per 1,000 (95% ci: 4.3-5.7), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value <0.0001), but no significant difference was found after age standardization (p value50.93). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend<0.0001). in the incidence study, a total of 68 new hcv rna positive cases, 23.5% of them were anti-hcv negative, found from 1,202,165 donors followed for 2,415,668 person-years. the incidence of hcv rna was 2.8 per 100,000 person-years (95% ci: 2.2-3.5), and no significant difference was observed between both genders (p-value50.41) and between age groups (p for trend 0.37). conclusion: the prevalence of hcv infection has been dramatically decreased by 71.5% during 1999-2013. it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* 1 , sahar muhmmad 2 and dalia el dewi 2 . 1 national blood transfusion services, 2 azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is 70-12 days, hiv from 22 to 11 days, and hbv from 25-30 days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from 2012 to 2015, the total number of donor samples to be screened is 178685, the age of the donors ranged from 18 to 50 years, and they were of both sexes (m: f 5 3:1).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v2) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of 75 nat yield donations among 178685 (0.04%) seronegative donors. among these 75 nat yields cases, 53 (0.03%) were reactive for hbv, 20 (0.011%) were reactive for hcv and 2 (0.001%) were reactive for hiv-1. we stratified the age of the donors into 3 groups; group a (18 -28 years), group b (29 -39 years) and group c (40 -50 years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p 5 0.0089; with 95% confidence interval (ci) 5 0.0085 -0.0520 & p 5 0.0247; with 95% ci 5 0.0025 -0.0534 respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p 5 0.0224; with 95% ci 5 0.0032 -0.0413). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p 5 0.0335; with 95% ci 5 0.0015 -0.0352). nat-hcv; did not differ significantly between the three groups (p 5 0.3222; with 95% ci 5-0.0089 to 0.0161 between groups a and b & p 5 0.1340; with 95% ci 5 -0.0055 to 0.0270 between groups a and c & p 5 0.4277; with 95% ci 5 -0.0080 to 0.0215 between groups b and c). nat-hiv; did not also differ significantly between the three groups (p 5 0.3801; with 95% ci 5-0.0077 to 0.0077 between groups a and b & p 5 0.3172; with 95% ci 5 -0.0056 to 0.0077 between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p 5 0.0013; with 95% ci 5 0.0136 to 0.0507). nat hbv was significantly higher in males (p 5 0.002; with 95% ci5 0.0103 -0.0413), but the prevalence of either hcv or hiv did not differ significantly between males and females (p 5 0.3835; with 95% ci 5 -0.0077 -0.0145 & p 5 0.2751; with 95% ci 5 -0.0044 -0.0066; respectively). conclusion: in this study the nat yield of 75 in 178685 assumes more significance when one considers the fact that single donation is used for generating 3 components that can be used by 3 recipients. hence, in effect the nat yield becomes 3 times that is, 225 in 178685. saving 225 recipients from tti out of 178685 (0.13%) is indeed very significant. results/finding: of the 303,569 donors who were tested by our donor center, 1,386 (0.460%) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at 1:128 titer. the screening elia s/co of this donor was 0.2782. both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were 6-10 transfusion transmitted babesia cases per year from 2008-2015 (table 2 ). in the 11 months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed (1 in 303,569 donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of 4 years of additional nat testing at blood bank, dmch, ludhiana from september 2012 to december 2016. results/finding: results 1.73% (2041 of 118021) units were initially nat reactive. these units were further tested, of which 90.98% were discriminated (70 hiv, 1051 hcv, 726 hbv and 10 co-infections). the remaining 6.71% (137) were repeat non-reactive and 1.91% (39) could not be discriminated. overall, nat yield rate was one in 837, whereas virus-specific nat yield rates were one in 59,010 for hiv, one in 1873 for hcv, one in 1639 for hbv and one in 29,505 for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past 4 years has increased the screening sensitivities to check viral load and prevented transmission of 141 probable transfusion transmitted viral infections. assuming 100 % component preparation it saved 423 transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu 1,2 , wei mao 3 , tao he 3 , yashan yang 1,2 , zhan gao 1,2 , chunhong zhang 3 , hongmei liao 3 , jingxing wang 1,2 and miao he* 1,2 . 1 institute of blood transfusion, chinese academy of medical sciences & peking union medical college, 2 sichuan blood safety and blood substitute international science and technology cooperation base, 3 chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from 5,000 voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a 250pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch37 human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as 1e -5 . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: 1.23 gb raw data with 2,450,046 reads were generated in the dna library. meanwhile, 1.98gb raw data with 3,967,242 reads were generated in the rna library. after cleaning the human background, 211 reads from bacteria, 98 reads from viruses, and 341 reads from parasites were identified (table 1) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table 1) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in 2015 most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of 1 february 2016 to 1 february 2017. thrombocyte concentrates are prepared out of 4 single donation units by the buffycoat method. results/finding: over the period 260 platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* 1 , germ an leparc 2 , phillip c williamson 1 , lani palmer 1 , ben reynolds 1 , maria noedel 1 and lindsey houghton 1 . 1 creative testing solutions, 2 oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february 16, 2016 recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march 1, 2016. with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february 16, 2016 and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february 16, 2016, the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within 6 weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in 2015, who reported $212 million new cases worldwide, resulting in >400,000 deaths. malaria prevalence is highest in sub-saharan africa, home to 90% of all infections accounting for 92% of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of 0.2 mm amustaline and 2 mm glutathione (gsh) and a 24h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to 7 days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed 24h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with 5% fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching >1% parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at >7.5 log 10 or >6.0 log 10 /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with 7 day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from 5 to 7 days using an fda cleared rapid test (rt). in february 2016, our hospital based transfusion service implemented the use of rt on day 5, 6 and 7 to routinely extend ap shelf life to 7 days. prior to this, we tested aps by rt on day 4 and transfused day 6 or day 7 units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of 7 day platelets. study design/methods: data were obtained for two 12-month study periods: october 2014-september 2015 (pre-implementation) and february 2016-january 2017 (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table 1 . the number of ap transfusions increased by 7% post-implementation, comparable to a 4% increase in inpatient admissions and an 11% increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different (3.16 pre; 3.12 post, p50.91). the number of rts performed increased by 130%. the percentage of transfused units tested at least once by rt prior to transfusion increased by 21% (p<0.0001). the outdate rate decreased from 5% to 2% (p<0.0001). ad-hoc ordering decreased from 21% to 9% (p<0.0001). conclusion: use of an approved rt for routine ap outdate extension to day 7 was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* 1 , vito scalia 1 , carla osiowy 2 , michael carpenter 2 , anton andonov 2 and margaret fearon 1 . 1 canadian blood services, 2 public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low (6.6 and 4.9 per 100,000 donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in 2011 the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of 6 units. hcv nat was in place since 1999 (using minipools of 24) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march 2016 all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at -20 o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the 5' ntr-e1 and ns5b regions. sanger sequencing of these regions represents approximately 15% of the genome. results/findings: all confirmed positive donations were whole blood donations. there were 42 hbv positive donations. of these, 37 had tested hbv nat positive. genotypes were 8 type a, 6 b, 4 c, 17 d and 2 e. there were 5 samples hbv nat negative but hbsag positive (2 were anti-hbc reactive). of these, 4 could not be sequenced and one was genotype a (also anti-hbc reactive). there were 30 samples considered hcv positive. of these, 17 samples were hcv nat positive. genotypes were 5 type 1a, 3 1b, 3 2c, 2 2b and 4 3a. there were also 13 samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first 8 months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert 3d microbial detection system (bta 3d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert 3d (bta 3d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels (1-20 cfu/ml) of 11 bacterial species commonly associated with platelet contamination, and 20 replicates (10 per instrument) of 4 ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta 3d and the other into virtuo and incubated until signaled positive by the instruments or for up to 7 days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally 98 bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august 2013. this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a 100% voluntary donor program since 2011 and is the only center in the country that has achieved this goal. results/findings: a total of 264,343blood donations were studied from august 2013 to december 2016. in the rbdc, where only voluntary blood donations are recruited, the prevalence was 18 per 100,000 donations for hiv (ic95% 8-34:100,000); 14 per 100,000 for hbv (ic95% 7-29:100,000) and 18 per 100,000 for hcv (ic95% 8-34:100,000). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was 89 per 100,000 donations for hiv (ic95% 77-103:100,000); 70 per 100,000 for hbv (ic95% 59-83:100,000) and 78 per 100,000 for hcv (ic95% 66-91:100,000). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of 1: 66,086 for hbv; 1: 132,172 for hiv and 1: 264,343 for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s201 system. hbv s region was also sequenced. results/finding: 234 obi were found in the 230,000 donations. in the viral loads assay, 43 samples were negative and 104 samples' viral loads were lower 20iu/ml. the mean viral loads was 1.85 6 0.41 (log10) iu/ml in other 87 samples,while the mean viral loads with hbsag1/hbv dna1 samples was 2.38 6 0.83 (log10) iu/ml. 60 samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype (69.0%) and the other was hbv c genotype(31.0%). compared the samples with hbsag1/hbv dna1 ,we found two obi samples carrying with 318t>c mutation, which could cause an amino acid s55f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag1/hbv dna1, and some unique variation was identified in the obi individuals. 195a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat 15 th days, 1month, 3 months& 6 months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may 2015, we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may 2015 to dec 2016 was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s201 platform using pools of 6 serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, 68547 seronegative donations were screened by nat and a total of 20 hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group 31-55 years old showed a large proportion, who accounted for 80% of reported infections. most of the hbv dna cases (about 80.0%) reached senior high school education. the average hbsag dna positive rate was 0.029% (20/ 68547). incidence among apheresis donors in this period for hbsag dna were 2.91/10000. these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf24 with 5 day stability at 228c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control (225 610 ml) and test components (625 ml 625 ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within 8 hr and wbd pf24 within 24 hr. cryo was manufactured according to site sops and frozen at -308c (test 62 6 2 ml, control 22 6 2 ml ). test and control cryos were thawed at 378c, and characterized immediately post thaw (t50), and after 5 d storage at 228c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over 5d at 228c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through 5d storage at 228c. conclusion: pr cryo can be processed from 3 plasma sources, including pf24, and stored at rt for 5 days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf24 with stability over 5 days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , lynne fleischmann 1 , mirjana sarac 3 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics, 3 abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects 6-8 million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over 20 days using a panel of positive and negative samples. sensitivity was evaluated on 407 presumed antibody positive specimens and specificity was evaluated on 7621 random blood donor samples. results/finding: precision was 7% cv or less for positive samples over 20 days. the overall specificity in a blood donor population was 99.99% (7620/ 7621). sensitivity was 100.00% for 407 presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , lynn martin 1 , daniela kaleve 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over 20 days. sensitivity was evaluated using 511 known positive samples, 30 commercially available seroconversion panels, the who standard, 23 hbsag mutants, and 94 hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.98% (7998/8000). sensitivity was 100% for 511 presumed positive samples. sensitivity was 100% for all genotypes. 100% of the mutants were detected vs 83% for the comparator assay. seroconversion detection was equivalent to the comparator assay with 157 reactive samples detected with the alinity s assay and 154 reactive samples detected by the comparator assay. analytical sensitivity ranged from 0.015 to 0.016 iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including 3 hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , kevin callear 1 , susan sullivan 1 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types 1 and 2 (anti-hiv-1/2). blood centers require very high throughput anti-hiv-1/2 assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen was evaluated on the abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv-1, hiv-2 and hiv group o antibodies and hiv-1 p24 antigen. seroconversion sensitivity was evaluated with 41 commercial seroconversion panels. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.96% (8082/8085). sensitivity was 100% for 813 presumed antibody positive samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. also, sensitivity was 100% for 102 antigen positive viral isolate samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 135 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , joyce siregar 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who 1st international standard. seroconversion sensitivity was evaluated using 10 commercial seroconversion panels. results/finding: precision was less than 6% cv for positive samples over 20 days. the blood donor specificity was 99.93% (6946/6951). sensitivity was 100% for 500 samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from 0.57 to 0.62 iu/ml. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 134 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* 1 , anton vanweert 2 , ed bakker 2 , mark paradowski 1 , jane bryant 1 , tuan bui 1 , joyce siregar 1 , george chen 1 , george schlauder 3 and gregg williams 1 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over 20 days using htlv i and htlv ii positive samples. specificity was evaluated using 8,001 blood donor specimens from europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 500 preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot 2.4. results/finding: imprecision was less than 7.0% for positive samples over 20 days. clinical sensitivity was 100.00% (500/500) on preselected htlv i and htlv ii positive samples. the specificity was 99.98% (7,999/8,001) on a blood donor population and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez 1 , michel garcia* 2 , fernando palomino 3 and guillermo orjuela-falla 1 . 1 national blood bank colombian red cross, 2 universidad del rosario, 3 fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, 387 anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than 1.0; abbott architect i2000sr) underwent supplemental testing by immunoblot (either chiron riba hcv 3.0 sia or hcv blot 3.0 test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: 1-4.99, 5-9.99, >10. band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in 57.9% (224/387) of samples, indeterminate in 30.7% (119/387) and were positive in 11.4% (44/ 387). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group 5-9.99 (63.2%) compared with the 1-4.99 (29.9%). in samples with indeterminate results, ns3_2 was the most frequent band detected (52,9%). in contrast, the most frequent band in the group of positive results was core (93,2%). only one sample from the indeterminate group (0.8%) had a strong band intensity (31), compared with 10 samples from the positive group (22.7%). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group (5-9.99) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (>5). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns3_1 and ns3_2 cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* 1 , ivanka mihaljevic 2 , manuela miletic 2 , miljana stojic vidovic 2 , irena jukic 2 , jane bryant 1 , mark paradowski 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 croatian institute of transfusion medicine, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over 20 days using positive samples. specificity was evaluated on samples obtained from 9,101 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 514 preselected positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with 3 confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than 6.0% cv for positive samples over 20 days. clinical sensitivity was 100.00% (514/514) on preselected syphilis positive samples. the specificity was 99.97% (9,063/9,066) for blood donor specimens and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* 1 , ed bakker 2 , anton vanweert 2 , jane bryant 1 , mark paradowski 1 , tuan bui 1 , lynn martin 1 , george chen 1 , gregg williams 1 and george schlauder 3 . 1 abbott laboratories, 2 sanguin diagnostics, 3 background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over 20 days evaluating positive samples. sensitivity was evaluated using 501 preselected positive samples and 30 seroconversion panels. specificity was evaluated on samples obtained from 8,113 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than 7.0% cv for positive samples over 20 days. overall clinical sensitivity was 100% on 501 preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying 5 more bleeds than the comparator assay. the specificity was 99.99% (8,111/8,112) for blood donor specimens and 98. 98% (194/196) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of 2015, was declared as the public health emergency of international concern by who in feb 2016. in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where 2.8% of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, 73.8% of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately 1:5 to 1:6. thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of 1lm and assayed after illumination with visible light from both sides for 5, 15, and 30min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was 4.5 log 50% tissue culture infectious dose (tcid 50 )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for 5min, 15min or 30min and the losses of the infectivity were further demonstrated by 3 repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial 5min of treatment whereby ct-value jumped from 18.25 (control) to 25.50 (mbpt for 5min) (table 1) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* 1 , margaret fearon 2 , susan l stramer 3 , megan l nguyen 3 , france bernier 1 , sheila o'brien 2 , vito scalia 2 , sakina smith 3 , yves gr egoire 1 and boris hogema 4 . 1 h ema-qu ebec, 2 canadian blood services, 3 american red cross, 4 sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in 14,000 canadian blood donors in 2013. in a subset of 4,000 donor samples the seroprevalence was 5.9%. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of 250 iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately 50,000 canadian whole blood donors including 30,000 from canadian blood services (cbs) and 20,000 from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all 199a transfusion 2017 vol. 57 supplement s3 donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test (95% lod 18.6 iu/ml, 95% ci 15.9-25.6) for use on the cobas v r 6800/8800 system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for 6 months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous 6 months are destroyed. recipients will be traced in the event of any products transfused in the previous 6 months. results/finding: as of april 10, 2017, 9 of 39,834 (19,395 cbs, 20,439 hq) tested samples with valid results have been found hev-nat reactive: 8 donors have been confirmed by further testing to date. confirmation is pending in 1 donor. of the 9 donors, 7 were from quebec, and one each from nova scotia and alberta (7 male, 2 female). ages ranged from 21 to 70 years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: 6 ate pork (including 3 who ate pork liver), 4 ate shellfish, 2 ate venison, and 3 drank well water. one donor had no identifiable risk factor. viral loads ranged from 3 to 151 iu/ml, of which 2 were <10, 3 were 10-50, and 3 were >50 iu/ml; 2 were anti-hev igm positive and 4 anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around 1/4400. the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about 300 to 500 million cases and 2 to 3 million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august 2014. both thin and thick glass stained blood smears of 417 blood samples with giemsa was examined using microscope. results/finding: of the 417 donated blood samples, 17 (4.1%) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p>0.05), majority of the blood donors that tested positive belonged to blood group o (64.71%). the prevalence of malaria parasite in the study was 4.1%. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert 3d (bta 3d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life (3, 4 and 5 days after collection). study design/method: pooled lrap were seeded with low levels of 6 organisms commonly associated with platelet contamination at 3, 4 and 5 days post collection. the seeded lrap were inoculated into bpa and bpn bottles on 10 different days (not consecutive) alternating between 2 teams of 2 people each. seeded bottles were loaded into a virtuo and a bta 3d and incubated until declared positive or negative (up to 7 days). additionally, bpa and bpn bottles inoculated with 4 ml of unseeded lrap were tested on the virtuo and the bta 3d (120 and 40 bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of 99.9% for the virtuo and 99.5% for the bta 3d. the virtuo demonstrated an average improved ttd of 3.2 hours, when compared to the bta 3d in the presence of 4 ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within 5 day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began 12 weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for 120 days barring continued zikv testing and nonreactive results. a total of 2,485 donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low (1.0%). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus 17d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn 1 , felicia santa maria 1 , yvette girard 1 , peter bringmann 1 , marion lanteri* 2 and adonis stassinopoulos 2 . 1 microbiology department, cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in 2015. the rapidly increasing number of infections in brazil, with hundreds of fatalities since december 2016, is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the 17d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a 2 weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate 17d-yfv using amotosalen (s-59) and uva light prt of platelet components (pc). study design/method: pc in 65%pas (n53) or 100% plasma (n51) were spiked with high titers of 17d-yfv and treated with s-59/uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero76 cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were 4.71 6 0.7 log 10 pfu/ml for pc in 65% plasma and 5.19 log 10 pfu/ml for pc in 100% plasma while titers in post-prt samples were <-0.7 6 0.0 log 10 pfu/ml for pc in 65% plasma and <-0.7 log 10 pfu/ml for pc in 100% plasma. inactivation to the limit of detection of >5.41 6 0.7 log 10 or inactivation of >4.71 6 0.7 log 10 pfu/ml was achieved for pc in 65% plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately 1000 ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples (1 hiv-1 antibody and 1 hiv-1 p24 antigen, and 1 htlv-i antibody and 1 htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were 0.00 for hcv, hbc, syphilis, cmv igg, and chagas, 0.01 for hiv ag/ab and htlv i/ii, and 0.03 for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were 0.00 % (antibody sample) and 0.36% (antigen sample) for hiv ag/ab combo; 0.90% (htlv i antibody sample) and 0.32% (htlv ii antibody sample); -1.43% for anti-hcv, -2.52% for chagas, -0.71% for hbsag, -0.37% for anti-hbc, -1.62% for syphilis, and -0.59% for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of 1000 ng/ml. robustness of the abbott prism methods to biotin interference c fischer 1 , r schneider 2 , w leonard 2 , m cobb 3 , g schlauder 3 , g williams 3 , m zuske 2 m janulis* 2 . 1 transfusion medicine, abbott diagnostics, wiesbaden, germany, 2 add diagnostics, 3 transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, 3 assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from 10 to 100 colony forming units (cfus)/bag (i.e. 0.03 to 0.3 cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of 10 8 -10 9 cfu/ml over the 5 days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day 2 after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, 24 hours (day 1) after collection a sampling volume of spiked platelets (0.1-1 cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of 25 bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate (7.5%). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by 2030 in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann 1 , frank tolksdorf 2 , wiebke handke 1 , thomas h. m€ uller 1 and axel seltsam* 1 . 1 german red cross blood service nstob, 2 maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from 5 buffy coats using the additive solution ssp1 (macopharma) with a residual plasma content of 35%. for inactivation kinetics, pcs (n53) were spiked with bacteria to a final concentration of approx.10 6 colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately 0.3 cfu/ml. bacteria were allowed to grow for 6 h in the pcs at 22 6 28c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log 10 reduction factors ranged from 6 to 7 for enterobacter cloacae (6.3 6 0.6, pei-b-p-43), pseudomonas fluorescens (7.1 6 0.4, pei-b-p-77), staphylococcus aureus (6.6 6 0.4, pei-b-p-63), and streptococcus bovis (7.0 6 0.3, pei-b-p-61). pcs (n512 for each species) spiked with these different bacteria species were efficiently sterilized (12 out of 12). treated pcs remained sterile during storage for 7 days, while bacteria in non-treated pcs grew to high titers of 10 6 -10 8 cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of 7 days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july 1, 2008 to july 30, 2013 was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version 16 software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than 0.05 were considered significant. results/finding: a total of 173, 207 consecutive blood donors were screened between 2008 and 2013. the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was 5.0%, 1.6%, 1.4% and 0.1% respectively. the hiv-hbv co-infection was higher among blood donors 135(41.79%) followed by hbv-hcv co-infection whish accounts about 103(31.89%). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of 26-35. in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was 746,773.9 ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a 14 month old infant patricia davenport* 1 , geeta paranjape 1 and laurie sutor 1,2 . 1 carter bloodcare, 2 ut southwestern medical center background/case studies: in 2017 at a large pediatric hospital, a 14 month old infant was supported for 31 days by extracorporeal membrane oxygenation (ecmo). over this time 113 blood products were transfused. about 10 days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified 27 donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed 3 years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october 2014 and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an 81 y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of 13 units of red blood cells. approximately 4 weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 20's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of 1:256. the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* 1 , andrea j linscott 2 and donny dumani 3 . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from 2011-2015, 8% of 173 transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a 27-year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day 1 of hospitalization showed no growth after five days. on day 3, the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the 318 ml unit had been aliquoted via sterile connecting device 12 days prior for a pediatric patient. all 26 ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was 37.18c. within 45 minutes of starting the transfusion, the patient's temperature increased to 39.38c and subsequently reached a maximum of 39.58c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at 4-68c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus 3 standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of 6 determinations of a low positive control in 1 run. inter-assay variability was determined by testing at least 3 representative negative production pool samples, at least 1 low positive sample (about 3 s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of 1.00 and were 0.72 and 0.48 respectively. the hbsag assay detection limit was 0.065 iu/ ml for source plasma and 0.120 iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at 15-25 0 c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between 1:10,000 to 1: 1,250,000 depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than 5% for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than 14%. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on 6 donors (3-17 days after the index donation) -3 donors in the follow up study and 3 tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all 6 donors. denv antibodies were negative in 9 donations and equivocal in 1. our initial reactive rate is higher than that reported to date for the procleix zikv tma of 1 per 23, 342 [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since 2014. over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late 2011, for which the program consisted of 2 types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june 2016, bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, 300 blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with 60 ml of plasma, vs. 30 ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, 2017; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation #1: updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation #2: implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over 120, 000 donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on 16-18 year olds and premenopausal women (ages 19-55) donors. on average, 16-18 year olds donate 1.3 times a year and premenopausal women donate 1.49 times a year. if both of these groups were limited to donating once a year, a total of 4,845 donations from 16-18 year olds and 9,272 donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin #17-02, the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for 16-18 year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a 2.5 hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: 97% (101/104) of the simulation group students improved their post-test scores and had an average likert scale rating of 4.1 (very good). 89% (63/71) of hybrid group students improved their post-test scores and had an average likert scale rating of 4.2 (very good). 89% (90/101) of online only students improved their post-test scores and had an average likert scale rating of 3.0 (good). the average changes in scores were statistically significant within all training groups (p value < 0.0001). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p<0.0001) and the hybrid group (p<0.0001). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in 2016. all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july 2016 through january 2017 were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a 7-month period, 504 cases of complex antibody identification workups (65%), transfusion reactions (2%), consultations for blood component utilization (6%), and deviations from standard operating procedures and massive transfusion protocols (27%) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $68,000 of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* 1 , david lancaster 2 , dianne geary 2 , robert scott 1 , anh thu nguyen 1 , adam garcia 2 , raina shankar 1 , leslie buchanan 1 and tho pham 2 . 1 stanford health care, 2 stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to (1) streamline the ordering process to accurately reflect inventory status and transfusion practices and (2) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over 50 product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a 5-month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a 3-month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: (1) the ratio of units transfused per week to the number stocked (t:s), (2) the number of products ordered as stat, and (3) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. 2486 lines of code were written for both programs, including 2 class modules and 34 distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were 1.03, 1.21, and 1.48 before the pilot period compared with 0.88, 1.17, and 1.40 during the pilot period. these differences did not reach statistical significance (p50.28). we also monitored the number of stat ordered products before and during the pilot period, which were 27 and 31 stat units per week, respectively (p50.86). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were 226 and 196 units, respectively (p 5 0.28). an estimated 7 hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to 0.175 fte and $18,200 per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over 360 hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* 1 , yulia lin 2 , troy thompson 1 , allison collins 1 and sheena scheuermann 1 . 1 ontario regional blood coordinating network, 2 sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in 2014 to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that 74 of the province's 158 hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in 2017 indicated that 93% plan to implement or already have implemented the ptqip and 43% of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october 2016 a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in 2017. publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* 1 , rana hajjeh 1 and cees th. smit sibinga 2 . 1 world health organization regional office for the eastern mediterranean, 2 international quality management (iqm) consulting background/case studies: more than 76 million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between 2006 and 2016; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may 2016 in tunisia. results/finding: we found 24 publications on disaster from five countries in the region and 16 publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries (54.5%) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated 10-85% of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. (63, 85, and 108 for 2014-2016) . the number of collections per registered trt donor varied significantly, ranging from 0 to 12 therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from 3.8 to 2.8 between 2014 and 2016. conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from 2014 through 2016. it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than 56 days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of 2010. after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march 2017 to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a 77% response rate (n533). of these, 78.8% have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. 87.9% of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although 93.9% of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only 48% of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate 3% (770 units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs1 to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in 2015, apheresis red cells (arc) represented 4.7 % of total red cell collections at our center. hae mcs1 ln8150 was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs1 operators first and then operators new to apheresis with a training goal of 30% of mobile staff. validation of the 12 alyx began 06/01/16 and took approximately 45 days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs1 machines were removed from service 07/08/16. alyx go-live occurred 07/13/16. additional operator training continued through september 2016. results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs1 and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $19.21 each providing an estimated annual savings of $239,000. conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately 2.5% and decrease our kit costs by 22%. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october 2016-march 2017. background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at 125g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! 130 g/l) and for females (minimum interdonation interval increased from 56 to 84 days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october 2016, changes in rebooking of donation appointments in december 2016 and culminating with eprogesa criteria changes on march 5, 2017. both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by 100. percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from 0.89% in the 3 weeks pre-implementation to 2.16% in the 3 weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were 12.6% in september, 12.0 % in october/november, and 9.9 % from december to march, 2017. conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to 130 g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past 8 years, 59,223 blood products, derived from 10,509 procedures, were distributed to 185 different investigators in over 200 laboratories. whole blood was the most common product (45.2%), followed by unmanipulated mononuclear cell collections (28.6%), and elutriated monocytes or lymphocytes (19.8%). less common requests included platelets (2.5%), plasma (2.5%) and granulocytes (0.8%). adverse donor reactions were infrequent (0.33% of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable 100% collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in 100% plasma. the corresponding pathogen reduction system used for the study has 3 kits with 3 different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than 6.8 x10 11 , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than 1800 x 10 3 /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of 1867 x 10 3 / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of 3.5 x10 11 and platelet concentration of 1167 x10 3 /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of 7.0 x10 11 and 6.8 x10 11 at the volume of 400 mls and platelet yield of 4.2 x10 11 , 4.0 x10 11 , and 3.5 x10 11 at the volume of 300 mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* 1 and steve cihura 2 . 1 bonfils blood center, 2 bbc / bsi background/case studies: in 2012, the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated 3 devices with the following criteria in mind: 1) device disposable costs, 2) reagents/controls/quality control, 3) objective hgb/hct measurement, 4) portability and durability for a mobile environment, 5) ease of use, 6) donor experience, 7) battery life, 8) validation requirements plans, 9) blood center suitability, and 10) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested 50 donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february 2013 with a targeted implementation date of july 2013. after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately 15%. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to 4.59% in 2014 and 4.29% in 2015. during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in 2016, the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may 2016. conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* 1 , ayumi araki 1 , hiromi sanyoshi 1 , hiromi kanai 1 , hiroya kikuchi 2 , katsushi tsukada 2 and kazuhide mure 2 . 1 hokkaido red cross blood center, 2 japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from 354 individuals who donated platelets during the 6-month period between february and august 2015, and data from the 30 donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the 156 men (5.1%) and 22 of the 198 women (11.1%) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the 30 donors in the vvr group was 64.7 6 13.7%, which was significantly higher than the 25.6 6 11.7% in the non-vvr group. at a maximum dbf threshold of 45%, sensitivity for discriminating between vvr and non-vvr donors was 93.3% and accuracy was 94.4%. when 45% dbf was used as the alert level, alerts were issued for 44 donors, including 25 in the vvr group. therefore sensitivity for predicting vvr was 83.3% and specificity was 94.1%. mean time from alert to diagnosis in the vvr group was 4.03 6 4.35 minutes, and accuracy of the alert was 56.8%. some of the vvr could not be predicted even the value of maximum dbf exceeded 45%. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* 1,2 , salam abdus 3 and shabrina shah 3 . 1 inova blood donor services, 2 inova fairfax medical campus, 3 inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of 10 scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every 4 to 8 weeks from december 2013 to march 2017. blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of 10 patients, 3 females and 7 males, who underwent a total of 178 rbcx from october 2013 to march 2017, using an average number of 7 rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for 8 patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the 178 rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of 168 healthy volunteers were collected before blood donation and after blood donation immediately, 1 day, 1 week, 4 weeks, and 12 weeks among men and 16 weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component 3 ( c3) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c3 decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at 12 weeks among men and 16 weeks among women, while c3 significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover 1 week after blood donated and reached their levels before blood donated within 12 weeks among men and 16 weeks among women. conclusion: the biomarkers mutually changed over the course of 12 weeks among men and 16 weeks among women. donating 400 ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low 40% split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate 15 percent and increased concurrent plasma collections by 24 percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than 62 percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below 3 percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october 2015. after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september 2016 for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in 2015 and 2016, a total number 1,097 rns and 965 rns completed the questionnaires, giving a response rate of 78.5% and 67.4% respectively. the overall mean score in 2015 was 6.24 points (range 0 to 8). the mean score in 2016 was 6.57 points (range 2 to 8). the percentage of rns having perfect scores of 8 increased from 8.8% in 2015 to 20.5% in 2016. table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; 264 purposively selected blood prescribing clinicians and nurses from 60 hospitals in 13 countries of the 4 human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at .05 level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r5 .137; p5.03; df5262) and accessibility (r5.184; p5.01; df5262) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f(3,260) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, 7 junior faculty co-investigators from 5 west coast institutions each had 2 months to create a 30 minute powerpoint presentation on a fundamental tm topic, after which 2 other members had 2 months to review and edit. therefore, each member created 1 and reviewed 2 presentations (three total steps). during each step, members wrote 2 multiple-choice questions for those particular topics. in the end, each topic would have 6 quiz questions to assess learning. at completion, 7 evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: 1) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; 2) pre and post-lecture 20 question validated examination (best collaborative) to assess learning; 3) resident in-service examination trends specific to tm. results/finding: six presentations were developed as 6 of the 7 abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables 1 and 2. abo leaders pre-test data could not be obtained for institution b, and 3 trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsõ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all 6 presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a 10 question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the 40 minute vignette performance and the 20 minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean 95 1 4%) when compared to the pre-test scores (mean 67 1 26%) ttest p<0.017. during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and 90% reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than 350 healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between 8.3 to 9.4 (in the range of 4-10). nps varies between 83 and 98. according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased 2011-2016 from 8.4 to 9.0. conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* 1 , anne eder 2 , beth a. dy 1 and mary o'neill 1 . 1 american red cross, 2 georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about 1 in 100,000 apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. 2015, a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to 2,300 hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the 12 months before and after launching the educational outreach. results/finding: the web based course was completed by more than 700 participants; 117 were physicians. based on a review of the evaluations, the course was highly valued with 93% of participants rating it excellent or very good. the blood center physicians gave over 200 presentations to hospital customers. reporting of suspected strs in 2016 increased by 23% compared to the prior year. the increased reporting came from 2 specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required 30-45 minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, 2014.) subscription-based e-learning utilizes 5-7 minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in 2016 by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june 2016, with a new equipment module assigned each month for the following five months. the series concluded in december 2016 with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of 3.5 on a 4-point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with 21 positive and 1 negative comment. level 2: learning the average score of users increased 13% from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of 1.647 and a t-stat value of 5.641. level 4: results while equipment-related errors decreased by 20% after training, there is not enough data to demonstrate a statistical significance. conclusion: our level 1 and 2 evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october 2013, a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n5151) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was 35%. of those, 96% endorse that fbc creates a climate of respect within our transfusion practice, 94% believe it has improved communication between work units, and 98% feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only 56% responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: 41 students of undergraduate semester 3 and 59 students of semester 4 participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with 86% of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, 12/7/16 from 9-11am. there were 17 attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, 4/20/17 from 7-11am. there were 14 attendees, including 2 regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and 11 surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) 50% as patients with fcr 50% may benefit from delaying the procedure for performance in the future. validation process included (1) a deming regression to globally assess the predicted vs. actual results and (2) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| 15%. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table 1 background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during 2 days (18 academic hours) the trainees can attend 6 lectures, discuss the methodical materials, participate in 3 seminars, 2 interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, 2016 with the group capacity 220a transfusion 2017 vol. 57 supplement s3 of up to 35 people the number of medical specialists who have attended the training is nearly 450. results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of 1-100; 1 being least satisfied/comfortable and 100 being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of 35 total technologists, 31 technologists took the pre esd survey and 25 technologists took the one year post esd implementation survey. table 1 shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, 19 (76.0%) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were 14 unplanned sop deviations; in the year after esd implementation there were only 5 deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from 0600 to 1600. the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in 100% plasma is broken up in to two days. on day 0 platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for 12-24 hours. on day 1 products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day 0 and day 1. in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: 13 of 18 employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was 21. for the two month training period the daily average rose to 23. conclusion: our "flip the switch" training plan for implementing prt platelets in 100% plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly 100% of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as 90% of an activity. in 2015 we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of 2015 we performed a supply inventory on all departments to plan future purchases and control residual stocks. in 2016, we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a 2016 cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter 2015 (q4/15) with last quarter 2016 (q4/16). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q4/15 1756 blood bags were used compared to 1998 in q4/16, demonstrating an activity "13.78%. price negotiation resulted in 12.58% readjustment. both indicated an estimated cost "28.10% with a possible impact of over us$ 35,000. we have identified a real cost #2.31% in q4/16, representing an overall #14.89% and us$ 3,716.72 (r$12,235.16) savings. conclusion: economy had deteriorated in our country in 2016 with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih-1000 tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17, statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in 2006. in the 2015-2018 nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on 60% individual and 40% for the other four levels (10% for each). the bonus (%) is calculated based on the iis as follows; category a: 100% (iis >575%), category b: 75% (iis: 50 -<75%), category c: 50% (iis: 25-<50%) and category d: 0% (iis < 25%). on the pilot implementation, the individual scores for 12 staff ranged from 71% to 100%. the iis were 76% to 81%. the number of staff in each bonus categories were 11, 92% (category a) and 1, 8% (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* 1 , deborah r fludd 1 and sandra j nance 2 . background/case studies: rare donors are defined as a blood type occurring in less than 1 in 1000 people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in 2016, there were 65,801 active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the 6398 (9.7% of total active donors) returned contact cards alerting ardp of changes in calendar year 2016, 355 (5.5%) were donors moving from one ardp facility to another, 1369 (21.4%) were donors no longer eligible to donate, and an additional 4324 (68.4%) were address changes. other changes were 115 (1.8%) reactivated donors and 235 (3.5%) donors who we were notified were deceased, or did not want to be listed in the ardp. in 2016, 5390 new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in 2016 was 4709 (355 1 4324), which would be 87.4% of the new donors submitted. conclusion: with nearly a 10% response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in 2016, 4709 donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* 1 , barbara j bachman 2 , mike leamy 2 , susan olson 2 and candace williams 2 . 1 university of florida college of medicine, 2 bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih-1000 (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two (22) runs of one to six (6) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing 153,000 types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. regardless of quality or speed metrics evaluated, the ih-1000 demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < 0.001). ih-1000 process steps and time studies addressed in the table below did not account for the ih-1000 reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih-1000 (77% reduction, a difference of 43 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* 1 , kimberly monnin 1 , barbara j bachman 2 , kyla warren 2 , susan olson 2 and candace williams 2 . 1 clinical pathology labs, 2 bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih-1000 tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of 12 separate test runs of 72 or 144 samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately 211,500 type & screens (t&s). the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih-1000 demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < 0.001). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih-1000 (difference of 120 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih-1000 tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih-1000 and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih-1000 with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17. statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/findings: using automated result verification, only 0.93% out of 6,339 samples evaluated for abo/rh testing would require visual verification, resulting in a 98% reduction in operator touchpoints (p < 0.001) and a labor saving of 444 minutes (7:01 hh:mm) for abo/rh testing. for 8,750 antibody screens, automatic validation of results would result in 99.5% reduction in operator touchpoints (p < 0.001) and a labor savings of 378 minutes (6:18 hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past 2 years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by 1% annually and peaked at 16% in mid 2016. to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an 8week period in late 2016. hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than 10 day shelf life remaining. units with tie tags attached were in hospital inventories for up to 3 months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was 100% of the 56 hospitals surveyed. mean percent o neg rbc gross sales for a 3 month period before, during, and after the survey was 16.6%, 16.0% and 16.5%, respectively. mean percent o neg net sales during the 3-month survey fell to 13.5% compared to an average of 15.4% in the 3 months prior. during the 3-month survey period o neg rbc monthly return rate increased to an average of 28.4% compared to an average of 23.0% in the 3 months prior. for the 3 months after the survey the average o neg rbc return rate further increased to 28.9% while mean percent o neg rbc net sales trended slightly upward to 14.1%. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* 1 , jeremie gachelin 1 , veronique ollivier 2 , thibaut mutin 1 , xavier telot 1 , benoit ho tin noe 2 and sandra sanfilippo 1 . 1 aenitis technologies, 2 hôpital bichat, inserm u1148 background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from 14 donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac1) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than 80 % (p< 0.001) and a purity of platelets close to 91.0 %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over 250.000 blood donations for an area with more than 7 million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in 2005, orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in 2007, when we moved from this equipment to atreus 2c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in 2011 we changed to atreus 3c (terumo bct) and finally in 2013, we moved to reveos system (terumo bct). since the changes in 2007, our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in 2009, bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during 2008 and 2016. conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of 140-160 patients and an average, round-trip travel time of approximately 15-minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about 30% of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november 2015, the hb has significantly improved the turnaround time of rbc issue -from 15-minutes to less than 60-seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately 750 rbc per month out of the window for non-surgical patients. this has been reduced to approximately 300 rbc per month, a 60% average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in 2015, they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the 131 st rescue squadron (131 rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the 131 rqs master sergeant in january 2016. we asked what 131 rqs's order and delivery expectations were. he said sporadic use and the blood order would be 2 rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the 131 rqs needs. staff was trained based on data from january 2016 meeting. we contacted the 131 rqs in september 2016 to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the 131 rqs association with a civilian blood center. based on his field experiences, he changed the blood order from 2 to 6 rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and 131 rqs performed a mock run on october 31, and we felt prepared for any future events. results/finding: on november 11, 2016, the 131 rqs was deployed to a civilian aeromedical evacuation. we anticipated a 6 rbc order. the actual order was 7 rbcs and 4 ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the 131 rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at 4:30am by the 131 rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november 28 th , we established a maximum blood order of 10 rbc and 4 liquid plasma, noting future orders may request fewer products, yet meet the preferred 2 rbc; 1 plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table 1 ). the highest mean fib concentration was 535 mg/donor unit; lowest mean fib concentration was 264 mg/donor unit. all sites had a mean fib concentration at least 100 mg/donor unit above the fda minimum requirement of 150 mg/donor unit. fifteen of 17 blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; 12 manufactured pooled donor cryoprecipitate. most froze plasma in a -188c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than 10 hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of 345 mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every 15 minutes for a 12 hour period or until the temperature exceeded 68c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached >68c in 75 minutes as shown in table 1 . the control thermometer recorded temperatures maintained at 1-68c for the entire 12 hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at 1-68c for more than 45 minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to 27 us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: 100% conventional (c-pc), 100% pr-pc, and mix of 75% c-pc/25% pr-pc. the model predicts a modest ($4%) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january 2011. a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august 2013, inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january 2015. a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from 2010 to 2016 (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in 2016, zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within 4 weeks (sept. phase 1) and nationwide within 12 weeks (nov. phase 2). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools (16-donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab 1 had 86% and lab 2 had 77% of clients requiring universal zkv testing. we evaluated a 12-month test result upload performance period to determine the impact of zkv test implementation. results/finding: during 2016, lab 1 upload time performance ranged from 92% to 94.2% from january to july; upload time performance fell between august through november, returning to 94.2% performance in december. lab 2 upload time performance ranged from 91.4% to 95.3% january to august. performance fell september through december 83.3% -88.5%. lab 1 experienced a low of 75% upload time performance during phase 1 when there was a rapid implementation; 69% clients required zkv nat. improved performance was observed during phase 2, with a 16% increase in zkv clients. for lab 2: phase 1 experienced a modest decline of upload performance ranging from 83.3% to 88.5% with 33.3% of clients implementing zkv nat. performance was 87.1% in phase 2, when an additional 43.3% of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* 1 and colleen a. aronson 2 . 1 advocate lutheran general hospital, 2 acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march 2016 regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in 6 of the 8 hospital transfusion services. the 2 sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the 6 sites which implemented the verax pgd test perform testing on all day 4 and day 5 platelets to be issued for transfusion. this abstract summarizes the data collected for the first 5 weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day 4, day 5, and those that were tested twice. inventory reports were reviewed for platelets issued on day 2 or day 3 that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february (1 week of performing the test), 48.1% of all platelets issued by the 6 participating transfusion services were day 2 or day 3 platelets. in march that number dropped to 29.9%. it is expected that this number will level off at some percentage at or below 29.0% with further data collection. in february 25.9% of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day 2 or day 3) will likely level off at some number at or below 29.9% due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, 5 of the 6 sites performing testing are level 1 trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed 100%. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from 400 ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n57). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd62p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days1, 3, 5 and 7 of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was (86.7 6 1.6)%, relative change rate of hsr was (3.87 6 12.75)%, the residual leukocytes were (0.15 6 0.15)310 6 . the ph, hsr, and the cd62p expression of pooled platelet concentrates before and after filtering were (7.00 6 0.17) vs (7.06 6 0.16), (66.96 6 12.35)% vs (63.22 6 8.26)% and (28.94 6 14.25) % vs (31.60 6 16.77)%. there is significant change for wbc after filtering (p<0.01). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table 1. conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb 18469-2012) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of 500ml and 1000ml. two users then evaluated the ss tubing segment types with 500ml or 1000ml samples for a total of 10 data points. samples were collected into the attached 1ml or 3ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of 10 ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the 5 day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january 2015, a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by 22%. the platelet outdate rates dropped after implementing the platelet inventory tool from 14% (1324 units) to 11% (874 units); a 21% decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of 7 fte to 6.2 fte, lowering fte by 11%. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* 1 , jaclyn mckay 1 , jennifer curnes 1 and rowena punzalan 1,2 . 1 bloodcenter of wisconsin, 2 children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a 3 month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: (1) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, (2) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july 2016 for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within 8-hours after collection. this tight 8hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight 8-hour completion time constraint for cryo production (capacity expansion). in particular, during the 4 th -quarter of 2016, a blood processing region was able to process about 1000 more cryo units/month (an increase of 20%) at a slightly lower collection cost (cost avoidance), resulting in an approximately 40% reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the 2017-the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after 7-day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from 5 to 7 days in the usa using an fda cleared rapid test (rt). in august 2016, our hospital based transfusion service began using a rt on day 6 and 7 to routinely extend ap shelf life to 7 days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of 7-day ap. study design/methods: data were obtained for two study periods: september 2015-february 2016 (pre-implementation) and september 2016-february 2017 (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from 5-day to 7-day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by 3% post-implementation while inpatient admissions and surgical volume increased by 1% and 3%, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by 36% post-implementation and the outdate rate decreased from 29% to 15% (p<0.0001). ad-hoc ordering was not statistically different between study periods (p50.10). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different (2.1 and 1.9, respectively, p50.65). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years (2012) (2013) (2014) (2015) (2016) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was 4892 units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was 8546, 24, and 2 units respectively. the mean number of discarded rbc units of the five years of the study exceeds 50% of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below 1000 mg/dl, and so the rejection threshold for lipaemia is level equal to or more than 1000 mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including 4892 rbcs, 8546 plasma products and 24 apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from 200 mg/dl to 1000 mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* 1,2 , rebecca ross 3 , debra mraz 3 , anne baker 3 , zenna neal 3 , melanie champion 3 and edward l. snyder 2,3 . 1 johns hopkins university school of medicine, 2 yale university, 3 yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first 4 months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, 777 pr-sdp were transfused at our hospital (out of a total of 3286 platelets transfused). after 4 months of scale-up, pr-sdp were approximately 30% of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($12 mg per 100g) to the content in pr-sdp (<1 ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september 2017, the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from 10 donors was collected in k 3 edta tubes. plt concentrations were determined at the qc department using the coulter act 5 diff hematology analyzer (beckman coulter). sample tubes were stored at 20-248c and measured at 24, 48 and 72 hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd (250 ml; n58) or dpd units (500 ml; n54). plt pools were stored at 20-248c under mild agitation for seven days except for dpd, which were split in two 250-ml bags after 18 6 1 h. samples were taken on days 1 and 7. ph, po 2 and pco 2 , hypotonic shock response (hsr), extent of shape change (esc), cd62p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at 24h (247 6 32 pltx10 9 /l), 48h (247 6 27 pltx10 9 /l) and 72h (247 6 32 pltx10 9 /l) postdonation. dpd can be stored in the same collection bag for 24h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits (1141-1526 pltx10 9 / l) before splitting. on day 1, lactate and pco 2 concentrations increased, and po 2 decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march 2017. there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted 12% increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* 1 , colleen vincent 2 and patricia kopko 1 . 1 university of california -san diego, 2 american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only 40-45% of double platelet collections meet requirements for pathogen reduction treatment. 1 blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: 9,500 apheresis platelets annually), which includes two hospitals (750 inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week 0) and the bone marrow transplant (bmt) ward (week 6). an e-mail communication explained the change to all physicians and nurses. in phase 1, we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase 2, we expanded usage to include the inpatient bmt ward. in phase 3, we lifted all restrictions so prp could be used throughout the health system, with the goal to reach 100% prp within 6 months. results/finding: in phase 1 (weeks 1-6), we requested 31 prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of 23 prp weekly (range 9-33), and prp constituted 44% of platelet transfusions in the cancer center. in week 2, excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase 1. in phase 2 (weeks 7-8), we formally expanded issuing of prp to include the inpatient bmt ward and requested 91 prp products weekly. our blood supplier provided an average of 57 prp weekly (range 44-69), and prp constituted 53% of platelet transfusions in the phased-in areas. in phase 3 (weeks 9-10), we began issuing prp throughout the health system. our supplier provided an average of 70 prp weekly (range 61-78), and prp constituted 43% of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises 21 hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of 2 apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, 2 ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a 48 hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table 1 ) resulting in a cost savings of $50k. an additional cost savings of approximately $25k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as 237a transfusion 2017 vol. 57 supplement s3 we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* 1 , james r stubbs 1 , scott a hammel 1 and manish gandhi 2 . 1 mayo clinic, 2 mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing 100% pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of 100 apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of 3.0, 3.1, and 3.3 x10 11 may end up below a 3.0 in the final storage bag and would need a post-processing sample to ensure the product met criteria at !3.0x10 11 platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a 3.4 yield but ended with a yield below 3.0. these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe-2100d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that 99 of 100 results were 0.00 or 0.01x10 3 / mcl with the exception being the wbc failure with a count of 0.29. further monitoring of the wbc counts discovered a result of 0.04 which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of 0.03 will be tested on the adam to confirm a leukoreduced product. we also discovered 2 of 4 (50%) of the 3.4 preprocessing yields products ended with a post processing yield <3.0. we decided to increase the yields requiring post processing samples to include the 3.4. conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of !3.0x10 6 platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from 9/16-12/16 were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need 1 6) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in 2017; the revised prediction method (estimated clinical need 1 11) was then evaluated retrospectively using the same data sources covering 1/17-4/17 and then compared to the prior method. results/finding: the average number of platelets transfused from 9/16-12/ 16 was 18.2 u/d with a standard deviation of 5.3 u/d; the predicted amount was 13.7 u/d. the difference between the predicted amount and the number of units used was -4.5 u/d. 79% days (23d/month) were under-predicted (average: 6 u/d). 17% of days (10) were under-predicted by !10 u (average: 12 u; max: 15 u (4x)). the average number of platelets used from 1/17-4/17 was 17.5 u/d with a standard deviation of 4.4 u/d; the predicted amount was 18.3 u/d. the difference between the predicted and units used was a 10.8 u/d. 38% days (11d/ month) were under-predicted (average: 3.5 u/d). one day (1%) over this period was under-predicted !10 u (11 u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages (17% a 1%), reduced the number of days under-predicted (79% a 38%), and decreased the discrepancy on those under-predicted days (5.9 u a 3.5 u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* 1 , maria noedel 1 , nancy haubert 1 , kenneth hudson 1 , larry morgan 1 , robert shaw 1 , tracy fickett 1 , jamie jue 1 , valerie winkelman 1 , sally caglioti 1 , german leparc 2,3 and phillip c williamson 1 . background/case studies: on 08/26/16, fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within 4 weeks; nationwide in 12 weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on 1 of 2 manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for 44 clients within 4 weeks, and an additional 21 clients within 12 weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the 4-week period but overcome during the 12-week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* 1 , hala samuel boules 1 , fatemah saleh al matroud 1 , rabab hussien ali dashti 1 , hanan alawadhi 2 and reem al radwan 3 . 1 kuwait central blood bank, 2 kuwait central blood bank, 3 kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of 3000 rbcs baby units were randomly chosen to be traced to their final deposition from the year 2012 till 2016. half of them (1500 units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb (60 units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is 100% efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (<5 days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march 2017. results/findings: a total of 339 products were tested. fifteen units (4%) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all 15 by the vendor and none were true positives by re-culture. of the 324 units that were successfully tested, 200 were tested again on day 6 for use on day 7(62%). there were 166 platelets transfused (51%) and 158 expired after day 7 (49%). the cost to test the products including controls was $12,970 and our calculated cost to produce 324 products would be $77,436. if we had needed to import products to meet needs, the cost would be roughly $91,300 without shipping costs which are estimated at $14,815.50. we averaged 40 expired platelet products per month (range 6-67) before verax testing and 26 (range 9-40) after implementation. conclusion: using verax point-of-care testing saved 166 platelet products from discard. the cost savings were $93,145.50 from importing and $64,466 from producing a replacement for those 324 products. the average discard rate per month went from 40 to 26 after verax implementation. extending platelet shelf life to 7 days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december 2016 were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april 2017 were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for 2016 data, 129 units were investigated without the use of secure texting. of these, 118 units were identified as preventable wastage, and 11 wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: 1) product not released after procedure/ or when patient stabilized (42) 2) product returned outside of appropriate temperature range (40) 3) clinician unaware product was assigned (36). thus far in 2017, wastage records have identified 31 units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, 11 responses thanked the transfusion service for the information, and in 8 instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: 99.9% dmso, plas-a, 25% hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) 99.9%dmso: plas-a : 25%hsa51:2:2. plas-a and hsa are kept at room temperature (20-258c, rt) and refrigerated at 48c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point 18.58c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total 14 tests. at least 10 minutes cooling after dmso, before adding the next reagent. see table: (1) after directly adding 99.9% dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. (2) in tube, autologous plasma first, dmso next, powder-like precipitates. (3) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. (4) & (5) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at 48c. (6)&(7) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the 48c group is slightly milder/slower than rt group. so hsa should not be added first. (8)&(9) trace of hsa(<1ml) was mixed into the plas-a bag (500ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below 10 in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5% to 3.2%. with an annual platelet usage of approximately 13,000 units, this reduction equates to approximately 950 units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $450,000 to $650,000, per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* 1 , russell thorsen 1 , rosaline ma 1 , antonio g insigne 1 , amy decourten 1 , florence panganiban 1 , patricia mckean 1 , cyril jacquot 2 , sara bakhtary 1 and ashok nambiar 1 . 1 ucsf health, 2 children's national medical center background/case studies: plasma (ffp, pf24, pf24rt24) stored at 1-6c outdates 24 hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to 5 days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (<4 months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited (24 hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf24 for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk 1 and richard gammon* 2 . 1 oneblood, 2 oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average 5-8 minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average 2-5 minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < 4 hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from 34% to 14%). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp-215 using the low glycerol (40%) freezing method and frozen at -658c within six days of collection. thawing occurred in a 328c water bath in the following order: 7 o positive and 1 o negative on 3 january 2017; 7 o positive and 1 o negative on 7 february 2017; and 8 o negative on 22 february 2017. deglycerolization occurred on site using the acp-215 with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for 13 days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $240-280 and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $400 per unit to produce and distribute. drbcs have a shorter shelf life, 14 days versus the 211 days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp-215's and deglycerolize four units at a time. in january and february 2017, it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of 10 years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c8000 izekial butler* 1 , karen leighton 1 , scott jones 1 and rachel beddard 2 . 1 qualtex laboratories, 2 biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c8000 instrument. precision of the new assay parameters was determined by testing 10 replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from 1 iu/ml to 60 iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of 2.0 to 45.0 iu/ml. stability of samples was determined by testing samples stored at 2-8 0 c and -20 0 c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from 1.2855 to 1.3142. so, precision was acceptable since the %cv for all samples tested was 5%. the mean values for the samples tested in the accuracy study were all 610% of the expected value which was much lower than the acceptance criteria which was 615% of the expected value. the linearity of the assay was acceptable with a r2 ! 99.0%. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to 14 days at 2-8 0 c and up to 1 month at -20 0 c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for 14 days at 2-8 0 c and stored up to one month at -20 0 c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in 2010 to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, 2017) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april 1, 2014 to march 31, 2015. local research ethics board approval was obtained. results/finding: 40 patients received ivig for itp at smh over the study period for a total of 76 unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding (13, 17%), pre-operative or antepartum care (22, 29%), a platelet count of less than 10 and contraindication to corticosteroids (8, 11%). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between 30-50 x 109/l. 6 patients received ivig for a likely diagnosis itp while 245a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. 18/76 (24%) of infusions consisted of 2g/kg over 2 days; the remainder of infusions consisted of 1g/kg. of those who received 2g/kg,3 of patients (17%) had evidence of partial remission after a first 1g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single 1g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our 2016 survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to 32 staff members in early february of 2017. twenty-two technologists responded for a 69% response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, 81% indicated that workspace size was insufficient and 71% that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* 1 , neil bangs 1 and kimberly sanford 2 . 1 vcu health system, 2 virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from 1/1/2015 to 6/30/2015 to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (<4 hours, > 4 hours < 24 hours), and if a second sample was required. results/finding: a total of 3144 t&s orders were initiated from the ed in this time period. 2787 (88.7%) patients were not subsequently transfused any type of blood product within 4 hours of tsd and 2584 (82.2%) patients were not subsequently transfused any type of blood product within 24 hours of tsd. a total of 2119 (67.3%) patients required a second sample. of these patients requiring a second sample, 1960 (92.5%) were not subsequently transfused any type of blood product within 4 hours of tsd and 1886 (89%) were not subsequently transfused any type of blood product within 24 hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within 24 hours of tsd amounted to an estimated $699,706 in unnecessary patient charges and approximately 628.7 nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* 1 , debra berry 1 , yunchuan delores mo 2 and gay wehrli 1 . 1 university of virginia health system, 2 children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* 1 , elizabeth halperin 2 , sharon breining 2 and mona papari 3 . 1 acl laboratories/ advocate hospitals, 2 advocate health care, 3 itxm background/case studies: a large midwest hospital system with 5 level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and 3 months of data were evaluated that contained 29 events. results/finding: there was an equal number of events that were initiated in the ed and the or (12). male patients were involved 69% of the time and 31% of time the patients expired. trauma of some type was the majority of the cause but 13.8% of the cases involved gi bleed and only 6.9% were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average 7.1 with the post hgb average of 9.7. ratios of 1:1 for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of 4:1. it was found that the rbc: plasma was 1.9:1, rbc: plt was 5.9:1 and rbc to cryo was 7.4:1. use of txa was only 24.1% and cacl was utilized in 58.6% of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* 1 , jody a barna 1 , donald e ulinski 1 and nancy m. dunbar 2 . 1 dartmouth hitchcock medical center, 2 dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to 2 weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july 2016, we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior 24 hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august 2015 and november 2015 (pre-implementation) , newly identified clinically significant antibodies were resulted for 56 patients compared to 51 patients between the months of august 2016 and november 2016 (post-implementation). the tat for allergy alert entry for both periods is shown in table 1 . we observed that 57% of allergy comments were performed within 24 hours in the post-implementation period versus only 30% pre-intervention (p50.0067). using the new process, nearly all of the alerts were entered into the emr within 72 hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* 1 , shailesh macwan 1 , nancy nikolis 1 , arline stein 1 , janelle richardson 1 , manju bagu 1 , lennart logdberg 1 , alexander indrikovs 2 , vishesh chhibber 1 and sherry shariatmadar 1 . 1 north shore university hospital, 2 northwell health background/case studies: our institution is a tertiary care facility performing over 1500 cardiovascular surgeries (cvs) in 2016, an increase of 117% after the healthcare system cvs integration in 2015. transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december 2016, the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after 2 reported events in q3 2016 that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: 1. open discussions and collaboration between blood bank and cvs nursing teams 2. mapping the process using flowcharts for additional blood orders from cvs. 3. identify bottlenecks and brainstorm solutions. 4. a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. 5. the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. 6. follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period 12/23/16-4/7/17 the blood bank has received 327 verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* 1 , tracie ingle 1 and heather vaught 2 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter 12-6 and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as 11 or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with 8-11 o, d negative red blood cells over 30 days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series 4, anti-d series 5, a1 cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table 1 summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion 2009; 49:1672 -1677 who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for 75% and 20% of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february 2014-june 2015 as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june 2015-october 2016. results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table 1) . this led to a reduction in observed overall sli (7.2 6 1.8 days vs 6.0 6 1.5 days, p<0.01) and odr (0.9% vs 0.5%). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. (6)) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january 2014 and december 2016 thirty-five (35) notifications were sent to physicians using epic letters and of those, fourteen (14) responded to the epic notification and five (5) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining 21 cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* 1 , denden benabdessadek 2 , annu george 2 and alexandra jimenez 2 . 1 westchester medical center, 2 new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of 941 orders were reviewed. approximately, 30% of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases (89%), but of the issued products, all were returned to the blood bank in 40% of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> 50 type and screens (t&s) per day] blood banks 1 , respectively. our laboratory which serves a large 1278-bed multispecialty academic hospital and receives 275-300 t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of 32.2 6 4.5 and 27.5 6 5.6 minutes 2 , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was 30% longer and had a larger standard deviation (s.d.) than the published trial result of 32.2 6 4.5. transfusion 2017 vol. 57 supplement s3 abstract during phase i visionv r 1 performed 263 panels. during phase ii visionv r 1 performed 351 of the 361 visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* 1 , john roback 2 , ronald arkin 1 , michael bartlett 1 , robert geiger 1 and jaxk reeves 1 . 1 university of georgia, 2 emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february 2016 guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august 2016 guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over 4,000 emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a 10% target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of 317 participants responded to the survey (7.94% response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n-15317-15316 df) of the null hypothesis that the mean50 vs. the alternative that the true mean is> 0. overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on 3 run cycles. ct was comprised of 3 metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of 2 components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table 1 provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc (2:50), xm (32:23), cb (13:18) and dat (10:00). by implementing the future state, an average $1.3 min. lt and vt is saved on each sample loaded for ts equating to a 73% labor reduction over the current state. a 19% improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a 59% lt reduction. on average, a 38 min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $100 type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing 8-column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on 100 patient samples for abo/d typing and antibody screening; of which at least 10 had a positive antibody screen. out of the 10, 5 had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which 17 were d(-) and 25 were d(1). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the 100 patient and 42 donor samples tested (100% concordance). all 10 samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave 100% sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for 5 samples at 3 different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over 3 months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* 1 , craig fletcher 2 and peter millward 1 . 1 beaumont hospital, 2 beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short 5-day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october 2014 and quality data was reviewed from august 2013 to december 2016. the collected data was then analyzed using descriptive statistical methods. results/finding: data from 2016 indicates platelet wastage comprised 1% of total received platelets and 79% of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units 12 months preimplementation of the report was 13 units, compared to 11 units 12 months post-implementation and 5 units 24 months post-implementation. wastage rates improved from 6% (wasted yearly platelets/total received yearly platelet units) in 2014, the year of report implementation, to post-implementation rates of 3% in 2015 and 1% in 2016 (see table) . importantly, this occurred despite a greater than 30% increase in platelet inventory between 2014 and 2016 and resulted in cost savings of over $60,000 in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all 4 participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within 2 years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* 1 , lisa marie button 2 , lori scanlan-hanson 2 , karen koch 2 , janet finley 2 , deepi goyal 2 and camille van buskirk 3 . 1 mayo clinic-rochester, 2 mayo clinic, 3 mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in 2014 to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in 2013 and 2014 were 63/1187 (events/ed transfusions 2013-2014). study design/method: electronic ordering for the ed was implemented march 31 st 2015. any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients <35kg and pediatric patients >35kg. 252a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for 2 years (april 2015 -march 2017), and during that time there was 1 instance of blood being ordered for an unintended patient 0.09% (1/1081). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in 2013, 17/651 (2.61%) units were transfused in the ed but not charted in the patient's medical record. in 2014, 18/536 (3.36%) transfusions were not charted. however, in 2016, the first full year of electronic transfusion order capability, only 4/462 (0.87%) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level 1 trauma center, with over 700 inpatient beds and over 50 operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option 1 (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option 2 (verbal) for verbal orders and coolers set up; and option 3 (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table 1 showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by 68% with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* 1 , steven zibrat 1 , geoffrey wool 2 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in 2015, blood bank accounted for 48% of all rejected clinical laboratory samples, yet comprised only 5% of total laboratory volume; 88% of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was 0.006%. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september 2016. results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of 0.128%, three times the national average of 0.043%. under the new system, rejected blood bank samples decreased from an average of 50% to 28% of all rejected laboratory samples, a 43% decrease. implementation of the new process produced a net savings of $55.8k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. 253a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured 19 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-78 days (mean 19 days, sd 20). in the postimplementation period, we cultured 22 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-12 days (mean 7 days, sd 2; p50.0082). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* 1 , marcus holme 1 , johnathan bakst 1 , gunta musa 1 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an 80 minute turnaround time (tat). in april of 2016, the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased 104%. tat analysis of a representative one week sampling per month showed an increase in outliers from 28 per month to 57 per month. average monthly tys samples performed is 2758. these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was 12/20/ 2016. results/finding: the average number of outliers decreased 61% from 57 per month to 22. further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $134,000 for fiscal year 2017. conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific 7 month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from 0-65 days with a mean of 13.64 days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for 7 months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of 0-7 days with a mean of 2 days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* 1 and randy levine 2 . 1 northwell health, 2 lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, 67 units were transfused on the oncology floor with 38 units (57%) requiring irradiation and only 22 of those 38 units (57%) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, 9 additional irradiated units were issued (31/67; 46%). post-intervention, 29 units were transfused on the oncology floor with 15 units (52%) requiring irradiation and all 15 of those units (100%) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus 23 of the 29 (79%) total units were ordered as irradiated. again, 4 additional irradiated units were issued (27/29;93%) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* 1 , charlotte van dyke 2 , dina garza van hoose 2 and rachel beddard 1 . 1 biobridge global, 2 south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics 8150s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january 1, 2016 to april 19, 2017, 2,097 total collections were flagged for additional qc by our trima accels and haemonetics 8150 instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in 2015, the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: 1) risk assessment, 2) quality control plan and 3) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* 1 , rachel jug 1 , kimberly ingersoll 1 , nicholas bandarenko 2 , nicole guinn 3 and jessica poisson 2 . 1 duke health pathology, 2 duke university hospital, 3 duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced 6:6:1 transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july 2015-december 2016. data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the 6:6:1 ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* 1 , nancy nikolis 1 , linda benison 1 , ruthmire thelusca 1 , renee liberty 1 , sherry shariatmadar 1 , alexander indrikovs 2 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: our blood bank (bb) processes approximately 60,000 specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june 2016, a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed 24/7. the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of 2016. the implementation of this new process has led to a decrease in the number of unacceptable specimens up to 30% quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from 75% to 96%, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* 1,2 , morgan rockwell 2 , joseph hagan 1 , jun teruya 1,2 and shiu-ki hui 2 . 1 texas children's hospital, 2 baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within 24 hours pre and post transfusion were evaluated. patients (0-18 years) receiving prophylactic ptx from july to december 2016 admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < 10ml/kg for patients < 35kg and one apheresis unit (au) for patients >35kg; therefore, patients were separated into 2 groups: < 35 kg and > 35 kg. a significant proportion of orders for both < 35 kg and > 35 kg did not meet patient platelet threshold criteria (p<0.001). conclusion: ptx threshold above ir for both groups were 31 ( 35 kg) and 48% (> 35 kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than 10% of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p>0.05) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to 56 days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding 3 months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only 2.1% of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated 3220 (805x4) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately 68 patients (17x4) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts (97.9%) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, 7 columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by 29% with the communication category average rpn having the greatest reduction of 72%. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* 1 , karen king 1 and joseph sweeney 2 . 1 rhode island hospital, 2 lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level 1 trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was 80 6 32 minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different (30 minutes for the provuev r and 28 for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over 90 minutes are shown in the table. the results show a reduction in tat by 14 minutes with a 20% reduction of tat greater than 90 minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* 1,2 , lorna orengo 3 , monique scott 3 and christopher a tormey 1,2,3 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as 1 in 19,000 blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as 25% prior to interventions, but may potentially be reduced to as little as 1.5%. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of <5%, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding 6 months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged 8.2%. reasons for specimen rejection were divided into 5 groups: 1) hemolysis, 2) blood bank witness collection form errors, 3) quantity not sufficient, abstract 4) duplicate sample, and 5) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table 1) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a 6.8% rejection rate with only 1 rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s 201 system christopher shahan* 1 , christina dejesus 1 , mosi mccall 1 , fallon hampton 1 , tangi herring 1 , judy davis 1 , anjali patel 1 , sonya gomillion 1 and bonnie maltby 2 . 1 qualtex laboratories, 2 qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s 201 system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual 10-12 hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently 42% of whole blood donor testing turnaround time delays are due to issues and failed runs on the s 201 system and we have 3 technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s 201 system. study design/method: the number of technician related failed runs on the s 201 system were tracked from 10/11/2015 thru 12/11/2016. a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the 5 whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s 201 system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s 201 system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s 201 system. after counter measures were implemented, the number of technician related failed runs decreased from 3 to 1.2 failures per week, which was a 58% decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s 201 system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by 58%. patients who were transfused for pre-transfusion hgb >7g/dl with resulting post-transfusion hgb >9g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by 11 volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: 265 patient charts were reviewed. 91 were excluded for bleeding and cardiovascular instability. 106/174 (60.9%) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors (76.19% vs 55.73%, p50.0181) and anemia of chronic disease (76.47% vs 54.1%, p50.006) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb 7.7 g/dl vs 7.3 g/dl, p<0.0001). inappropriately transfused patients also had higher median post-transfusion hemoglobin (9.9 g/dl vs 9.4 g/dl, p<0.0001). moreover, lab evalutions revealed association with lower folate levels (median 8.1 nmol/l vs 15.7 nmol/l, p50.029). 29/106 (27.3%) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. 7/64 providers were responsible for 32.3% of all inappropriate transfusions. 1/68 appropriately-transfused patients experienced an fnhtr. 3 deaths unrelated to transfusion occurred (1 in appropriate, 2 in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* 1 , willem martin smid 2 and ashley john duits 1 . 1 red cross blood bank foundation, 2 sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of 8 worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* 1 , dee dee cassidy 1 , jed b gorlin 1,2 and nancy l van buren 1,2 . 1 hennepin county medical center, 2 innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant 10 or more minutes may be required for transit of units often released in less than 5 minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july 2016. data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january 2015-june 2016, and post change included august 2016-december 2016. july 2016 data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of 99 rbc/month in coolers. post change this dropped to 62 rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of 54 rbc/month were returned (55%). post change, the average rbc/ month returned was 27 (44%), this represents an absolute 50% reduction in number of returned products. each rbc dispensed and returned takes approximately 20 minutes to complete paperwork and transport, therefore this change saved an average of 540 minutes per month. it was also noted that the average rbc/month transfused was 44 for baseline and 57 post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of 15-20 minutes (estimated) was reduced to 3-5 minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over 250 patent-applications have been filed related to "transfusion medicine" and over 400 related to "transfusion alarm", during the last 30 years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an 1/4 inch color cmos sensor, providing effectively 1.0 mp, a 3.6 mm lens, ir-cut, day/night minimum illumination 0.1 lux/f 1 and 808 viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a 24 hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood 260a transfusion 2017 vol. 57 supplement s3 abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h.264, video frame rate (fps) 1-30/s, refresh rate 50 hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/802.11/b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p2p is provided. typical 5v power-supply, sized 165x125x101mm and weighing 370 g. client software is required. the ir range is 10-15 m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of 10 m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a 32 gb sd-card. pan/tilt-horizontal 355 o and pan/tilt-vertical 90 o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo 2 , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than 6 3 %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under 20 $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to 4 participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot (30 cfu), multishot 550 cfu or highdose 10k organism preparations at a low level (< 50 cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a 14 day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at 36 c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < 1 days and the fungal organisms in < 2 days. the overall agreement was 99.8 % in 698 bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* 1 , diane schafer 2 , debra brown 1 , jesse cox 3 , scott koepsell 3 and sara shunkwiler 3 . 1 nebraska medicine, 2 the nebraska medical center, 3 university of nebraska medical center background/case studies: anticipating the implementation of the new (30 th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, 2 nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september 11, 2016), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling 4 in 6 months post implementation compared to 144 in the 6 months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential 2 nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for 2 nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late '90s and in early 2000, intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since 2010 have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january 1, 2005 to june 30, 2016. data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: 57% were female and 7% were less than 34 weeks of gestational age. none had co-existing g6pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn (41%) and rhesus-hdn (59%). antibodies most often implicated in rh-hdn were anti-d (22/57), anti-d and anti-c (22/57) and anti-c (5/57). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was 1 g/kg (range from 0,3 g/kg to 3,8 g/kg). neonates received one to four ivig administrations. table 1 shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, 14 required an et for rh-hdn and 3 for abo-hdn. forty-five (46%) patients needed top-up transfusions during hospitalisation and until three months of age: 8 with abo-hdn and 37 with rh-hdn. the mean number of transfusions was three (range:1 to 7). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over 90% case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a 4-part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include 3 specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than 4 months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, 30 coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate (2 each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and 45 different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than 1 hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on 2 separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to 10 hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, 2 georgia institute of technology ap72 reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with 2 o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add 2 type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to 1a) all adult males (am), 1b) women of non-childbearing age (wncba), and 1c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to 2a) women of childbearing age (wcba) and 2b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed 3%) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap73 saving apheresis platelets through use of verax point of care testing jennifer rhamy* 1 and rebecca wride 2 . 1 st. mary's regional blood donor center, 2 st. mary's regional medical center background/case studies: our rural hospital-based blood center serves 17 hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between 0 and 8 per day in 2017), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson 2 and jun teruya 1,2 . 1 texas children's hospital, 2 baylor college of medicine ap127 vision titers --easier or problematic? (table 1) . results/findings: post intercept, t had volumes of 261-320 ml, with 98 6 4% hemoglobin (hb) recovery. t had 10-fold less extracellular protein than c. after 35 days of storage t had higher atp and na 1 than c while lactate and hemolysis were lower. hct, ph, k 1 and glucose were equivalent between t and c on d35. d35 hemolysis for t was 0.08-0.31%, while for c it was 0.10-0.57%. t and c atp was >2mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table 1) . hematocrit (hct, %) 58.5 6 2.5* 56.3 6 1.7 60.8 6 2.7 61.8 6 2.9 hemoglobin (g/unit) 59 6 7 6 0 6 4 not measured hemolysis (%) 0.02 6 0.01* 0.06 6 0.04 0.16 6 0.07* 0.29 6 0.14 ph (378c) 6.9 6 0.1* 7.3 6 0.1 6.7 6 0.1 6.6 6 0.1 total atp (mmol/g hb) 7.7 6 0.5* 5.0 6 0.4 4.6 6 0.6* 3.9 6 0.5 k1 (mm) 1.5 6 0.3* 5.7 6 2.5 53. total tested total plts issued feb 87 121 64 48 24 185 181 mar 226 516 342 276 133 858 757 totals 352 730 471 375 174 1201 table: 2. resident reports to the intranet "drop box" increased from 53.7% to 69.3% to 100%, each over 2 month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from 17.4 per day or 10,370 dpmo to 6.3 per day or 3,539 dpmo. this is a statistically significant difference since the p-value calculated was 0.002. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately 4,400 specimens per month. since 1998, the requirement of having a second blood type on record was met by:1. utilizing the historical blood type and the current specimen, or 2. having second type performed on same specimen by different technologist, and 3. each type and screen specimen signed by 2 staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards 29 th edition, #5.16.2.2 a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april 2016, the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding:1. there was a minor adjustment period with more phone calls made to blood bank to explain the process. 2. there was minimal impact on turn around times for release of components. 3. aborh retype workload decreased from 1500 to 950 (35% to 20% of t&s volume) per month. 4. unnecessary blood draws minimized, improving patient experience. 5. no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as 15 to 48%. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our 926 bed hospital, a retrospective chart review was performed (07/01/15-12/31/15) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a 11 reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< 2 grade" or "> 2 grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table 1 demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples (48%) were > 2 titer results higher, while the majority was 2 titer results different (52%). the cost analysis is summarized in table 2 . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated 41% decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after 23 samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc.15 samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another 8 samples were measured with fc in ucc maribor. results/finding: 15 samples (6 rbc, 3 plt, 3 ffp-all leucocyte depleted and 3 non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from 44,10 -173,21; plt from 86,60 -100,00; ffp from 86,60 -173,21; and for non-leucocyte depleted ffp from 0,69 -8,85 (table 1) . 8 samples (2 rbc, 2 plt, 2 ffp -all leucocyte depleted and 2 nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from 91, 56; plt from 78, 21, ffp from 66, 21 ; and for non-leucocyte depleted ffp from 11,17 -15,91 (table 2) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than 1x10 9 /unit for leucocyte depleted or 1x10 6 / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january 2017 we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control. key: cord-347710-ff64y6ef authors: wan, qianya; song, dan; li, huangcan; he, ming-liang title: stress proteins: the biological functions in virus infection, present and challenges for target-based antiviral drug development date: 2020-07-13 journal: signal transduct target ther doi: 10.1038/s41392-020-00233-4 sha: doc_id: 347710 cord_uid: ff64y6ef stress proteins (sps) including heat-shock proteins (hsps), rna chaperones, and er associated stress proteins are molecular chaperones essential for cellular homeostasis. the major functions of hsps include chaperoning misfolded or unfolded polypeptides, protecting cells from toxic stress, and presenting immune and inflammatory cytokines. regarded as a double-edged sword, hsps also cooperate with numerous viruses and cancer cells to promote their survival. rna chaperones are a group of heterogeneous nuclear ribonucleoproteins (hnrnps), which are essential factors for manipulating both the functions and metabolisms of pre-mrnas/hnrnas transcribed by rna polymerase ii. hnrnps involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, rnp assembly and stabilization, rna export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. dysregulation of stress proteins is associated with many human diseases including human cancer, cardiovascular diseases, neurodegenerative diseases (e.g., parkinson’s diseases, alzheimer disease), stroke and infectious diseases. in this review, we summarized the biologic function of stress proteins, and current progress on their mechanisms related to virus reproduction and diseases caused by virus infections. as sps also attract a great interest as potential antiviral targets (e.g., covid-19), we also discuss the present progress and challenges in this area of hsp-based drug development, as well as with compounds already under clinical evaluation. stress proteins (sps) are a diverse group of proteins that are synthesized at increased levels when cells are exposed to either intracellular or extracellular stressful stimuli. they exhibit protective effects against stresses. stress proteins include heat shock proteins (hsps), rna chaperone protein (rnps), and proteins mainly function in the endoplasmic reticulum (er): peptidyl-propyl isomerases, protein disulfide isomerases (pdis) and the lectin-binding chaperone system. 1 sps are ubiquitously expressed in all kind of cells, triggering signal cascades for neutralizing and eradicating the stresses occurring both intracellularly (e.g., pathogen invasion) and extracellularly (e.g., starvation, stimulation by cytokines/chemokines or hormones). responses triggered by sps can either activate pathways to promote cell survival or initiate cell death (i.e., apoptosis, necrosis, pyroptosis or autophagic cell death) for eliminating the damaged cells to protect a particular organ/ tissue under given conditions. it is widely noted that the dysregulation of stress proteins is associated with a variety of human diseases, including cardiovascular diseases, neurodegenerative diseases (e.g., parkinson's diseases, alzheimer disease), stroke, human cancers and infectious diseases. in this review, we focus on their functions and update findings involved in infectious diseases, particularly, the diseases caused by viral infections. heat shock proteins in 1962, an italian geneticist ritossa inadvertently elevated the incubation temperature of drosophila larvae and discovered an increased gene transcription of unknown proteins. he nominated these protein as hsps. 2 further studies have revealed a large number of hsps, which form a big family and are ubiquitously expressed in cells. based on the molecular weight, hsps are classified into different families, including hsp100s, hsp90s, hsp70s, hsp60s, hsp40s, and some small hsps . [3] [4] [5] hsps belong to the largest family of chaperones. the hsp expression is rapidly induced when cells meet physiological or environmental attacks such as starvation, high temperature, hypoxia or hyperoxia, pathogen invasion, malnutrition and exposure to chemicals or uv, etc. they form a network to promote or stabilize the correct folding of substrate protein to gain its functional/active conformation (fig. 1 ), although they may not associate with the substrate protein in the final structure. 3, 6 hsps are important factors in regulating cell survival, differentiation and cell death. accumulating evidence shows that some hsps participate in not only innate cellular immunity but also antigen presentation in adaptive immune response. 7, 8 hsps also serve as potential biomarkers for some diseases. it has been shown that the increase of hsp70 in plasma is associated with heart failure, 9 and the elevated hsp27 level in human peripheral blood mononuclear cells is related with coronary artery disease (cad). 10 hsp90s. hsp90, an abundant chaperone in all eukaryotic cells, controls a variety of critical signalling pathways in eukaryotic cells. 5, 6, 11 hsp90 is an atp-dependent chaperone with different isoforms, including 1) hsp90α (hsp90aa1, or hspc1), hsp90α-a2 (hspaa2, or hspc2) and hsp90β (hspab1, or hspc3) locate in cytoplasm. 2) glucose regulated protein grp94 (hspc4 or gp96) locates in the er. 3) trap1 (hspc5) locates in mitochondria. 12 among them, hsp90α and hsp90β account for the greatest proportion in humans. hsp90 contains three regions: an atpasedependent hydrolytic domain in the n-terminal region, a middle linker region, and a dimerization domain in the c-terminal region. 5 like other hsps, hsp90 binds non-native substrate peptide to prevent its aggregation and degradation. when hsp90 binds atp, a transient dimerization of the n-terminal domain allows the substrate peptide binding to hsp90. then the atp hydrolysis and energy release lead to a conformational change of the n-terminal domain that facilitates the correct folding of the substrate petite ( fig. 1) . 5, 6 besides, hsp90 is also involved in telomere maintenance, apoptosis, and cell cycle progression, etc. 6, 13 it is well known that hsp90 not only interacts and contributes to rna polymerase assembly and nuclear import of some (−) ssrna viruses (e.g., pb2 of influenza virus), but plays crucial roles in the folding process of viral capsid proteins and virion assemblies as well. 14 cochaperones of hsp90. cochaperones of hsp90 regulate hsp90 functions at many aspects. cdc37 (also called p50) delivers kinase to hsp90 and inhibits its atpase activity; carboxyl terminus of hsp70-interacting protein (chip) functions as e3 ubiquitin ligase; hsc70/hsp90-organizing protein (hop, also called sti1) inhibits dimerization of the n-terminal domain; and the activator of hsp90 atpase 1 (aha) and p23 participates the maturation of substrate peptides. 11, 15 hsp70s. hsp70 is a subfamily of hsps' superfamily with~70 kda molecular weight. it accounts for the majority of molecular chaperones in cells. 11 the members of the hsp70 family mainly include: (1) hsp72 (hspa1a), hsp70-2 (hspa2), hsp70b' (hspa6) and hsc70 (hspa8) are commonly located in the cytosol; (2) grp75 (hspa9) is located in mitochondria; (3) grp78 (hspa5) is associated with the er. 16 hsp70 consists of two domains: a 44-kda nucleotide-binding domain (nbd) which can be divided into four subdomains (ia, ib, iia, and iib) in the n-terminal region and a 28-kda substrate-binding domain (sbd) composed of c-terminal α-helical (sbdα) and n-terminal β-sheet (sbdβ) subdomains in the c-terminal region. 17, 18 as a critical component of cellular protein surveillance, the atp-dependent molecular chaperone protects cells from damage caused by stress and takes part in a number of folding processes, including folding of newly synthesized polypeptides, recognition and refolding of misfolded or aggregated proteins, solubilization or degradation of proteins, transporting proteins, assembly or disassembly of oligomeric protein complexes, and the regulation of certain natively folded proteins. 5, 13, 19, 20 the functions of hsp70 are not limited to host protein folding. its functions are considerable during viral infection. the members of hsp70s exhibit quite different roles in the course of virus life cycle. for example, hsp72, hsp70b' and hsc70 participate in the hcv viral entry, virion assembly and translation of the viral genome. grp78 in er is associated with the homeostasis of viral proteins and prevents the overload of viral proteins in host cells. in hepatocytes, the elevated grp78 stimulates innate immunity to restrict or eliminate hepatitis b virus (b) replication. 21 grp75 interacts with the ns5a protein of hcv in mitochondria. 22 accumulating evidence shows that hsp70 interacts with viral components of human cytomegalovirus, rabies virus, respiratory syncytial virus, human papillomavirus, herpes simplex virus. chaperone cycle of hsp70. the chaperone cycle is mediated by the n-terminal nbd, which regulates the binding of hsp70 with substrates through switch of two states. the first state is the atpbound state with low affinity for substrate binding, i.e., a high association and dissociation rate of the substrate peptides to the sbd. the second state is atp hydrolysis that switches to the adpbound and nucleotide-free state. at this state, the substrate exchange rates are low while the affinity to substrates is high. the chaperone activity of hsp70 mostly relies on atp hydrolysis. the basal atpase of hsp70 is normally low unless it is stimulated by the substrate peptide itself. it takes 20-30 min for a molecule of atp to be hydrolysed per hsp70 molecule at 30°c. as a result, it is necessary for some cochaperones to encounter with hsp70 atp to induce atp hydrolysis and help the increase of the affinity for substrate peptides. 19, 23 cochaperones of hsp70. the most crucial cochaperones of hsp70 are members of the j-domain proteins (jdps) family and nucleotide exchange factors (nefs) family. previous studies focused on the function of hsp70 machinery and led to the development of a "canonical model" for its mode of action. the model contains two steps. first, the unfolded peptide substrates bind jdps of the hsp40 family; then the substrates are delivered to hsp70 that stimulate the hsp70's atpase activity. simultaneously, jdps prevent the aggregation of unfolded proteins. second, nefs work as substrate release factors that assure the substrate to fold into the correct and active conformation. in this way, the cochaperones strongly facilitate the function of hsp70. therefore, hsp70 generally does not work individually but cooperates with cochaperones. 23, 24 hsp60s. hsp60s are large cylindrical oligomers with two back-toback rings. 19 the non-native proteins of the central cavity in each ring are folded into the native protein through an atp-dependent process. 25, 26 hsp60s are classified into two subfamilies. group i is mainly in prokaryotes, while group ii appears in eukaryotic cytosol and some archaea. 27, 28 the most well-studied one in group i is the groel-groes chaperonin system in the cytosol of bacteria. groel is an about 57 kda protein with two rings arranged back-to-back; and 7 subunits form a tetradecamer structure. groes is the cochaperone of groel. 11 the two rings-tetradecamer structure appears in two fig. 1 the general chaperone cycle of heat shock proteins. initially, unfolded client protein bound to the hsp70-hsp40 chaperones interacts with the hsp90. atp binding to hsp90 induces the client proteins transfer from hsp70 to hsp90. later, the conformation of hsp70-hsp40 chaperones will be released. finally, the hydrolysis of atp induces additional conformation changes leading to the client protein release forms include asymmetric (1 groel: 1 groes) and symmetric (1 groel: 2 groes) complexes, which are described as "bullet" 14, 29, 30 and "american football" 31 shaped respectively. the groel-groes chaperonin undergoes allosteric regulation dependent on atp and which completes the protein folding function. the polypeptide binds to the hydrophobic sites of one of the seven subunits of groel and changes conformation upon atp binging and hydrolysis. with the help of cochaperone groes, the polypeptide finishes its folding process. 32 in contrast to the groel-groes system, the mammalian homolog hsp60/hsp10 system is less studied. hsp60 is thought to be imported into the mitochondria and converted into its mature form with a molecular size of 58 kda. [33] [34] [35] hsp60 exhibits important roles in facilitating protein folding, transportation and proteostasis in mitochondria. 36, 37 group ii chaperonins include the archaeal thermosome and eukaryotic cct (chaperonin-containing tcp1, or called tric), which are oligomers with eight to nine subunits with molecular weight 57-61 kda in each ring. compared to group i, group ii chaperonins show different allosteric movements by atp binding. 11, 19 hsp60 family is known to participate in viral life cycle at various stages from viral attachment to the replication of the viral genome. hsp60s are essential for host cell immunity regulation. some viral proteins require hsp60 for its translocation within host cells. pb2 is a subunit of influenza a viral rna polymerase, which mostly locates in the nucleus but also appears in mitochondria. 38 when the virus infects the host cells, pb2 is responsible for maintaining the function of mitochondria. pb2 interacts with mitochondrial antiviral signaling protein (mavs) to downregulate intracellular immune response by decreasing the level of ifnβ so that the invading virus can easily escape from the defence of host cells. 39 hsp60 takes the role of transporting pb2 from cytosol to mitochondria in the host cells. besides, hsp60 shows great regulatory functions on innate immunity by inducing proinflammatory cytokine release, such as tnf-a, il-6, and il-1b, etc. 40 small heat shock proteins. small heat shock proteins (shsps) are a group of small proteins with a low molecular weight ranging from 15 to 40 kda. 4 there are 10 members in the shsp family and some are ubiquitous including hsp27 (hspb1), hspb5 (αb-crystallin, or αbc), hsp20 (hspb6), and hsp22 (hspb8), while the others are tissue-specific including hspb2 (myotonic dystrophy protein kinase, or mkbp), hspb3, hspb4 (αa-crystallin, or αac), hspb7 (cvhsp), hspb9, and hspb10 (sperm outer dense fiber protein, or odf). 41 shsps can exist as monomers, dimers or even large multimeric complexes in the cells. 42 the structure of shsps is different from the other hsps due to their less conserved sequences. the basic structure of shsps is a conserved α-crystallin domain (acd) flanked by two non-conserved domains including the n-terminal sequence (nts) and the c-terminal sequence (cts). among these domains, acd becomes the characteristic of different shsps. 43, 44 shsps play crucial roles in several physiological processes regarding stress tolerance, apoptosis, aging, and longevity. [45] [46] [47] [48] the phosphorylation together with the n-terminal wdpf motif helps shsps to form homo-or hetero-oligomers. 49, 50 the oligomerization is the hallmark of shsps for supporting their quite different activities. phosphorylation favors small oligomer formation while dephosphorylation provokes a shift toward large oligomer formation. 51 oligomerization dynamics is crucial for chaperone activity because it gives rise to the possibility to format different homo-and hetero-oligomers, each with different binding properties to chaperone a broad range of substrates. 41, 52 for instance, the phosphorylated species are required for actin dynamics. small phosphorylated dimers/tetramers bind f-actin to regulate actin polymerization. 53 among different shsps, hsp27 has been broadly studied. hsp27 exists in all tissues though it is known to mainly express in cardiac, skeletal and smooth muscles. 54 its importance has been demonstrated in cell differentiation, cell survival, cellular innate immunity, viral protein translation, and intracellular virus transport, etc. [55] [56] [57] same as the other shsps, hsp27 shares a highly conserved α-crystallin domain. 41, 58, 59 hsp27 is capable of oligomerization and phosphorylation. there are three serine residues 15, 78, and 82 can be phosphorylated by different kinases including p90rsk, pkg, mapkap kinases, etc. 55 the phosphorylation of hsp27 is a reversible process. the dephosphorylation contributes to the formation of large size oligomers. 60, 61 hsp27 can not only form large homo oligomers up to 800 kda; but it can also cooperate with other shsps (e.g., hsp20) to form heteromeric structures. 56, 58 hsp27 is upregulated and activated upon infections. 62, 63 the elevated hsp27 activity promotes either cytoskeletal stability or cell motility, 64, 65 and prevents apoptosis. 66 hsp27 is required for il-1-induced expression of the pro-inflammatory mediators il-6, il-8, and cyclooxygenase-2. [67] [68] [69] hsp27 is also linked to various signalling pathways involved in the development, differentiation, and cell growth. 70, 71 the long-term and high-level expression of hsp27 stimulated by variety of stresses (such as hbv or ebv infections) enhances carcinogenesis, cell survival, stemness of cancer cells, cancer metastasis, tumour formation and drug resistance. transcriptional regulation of the hsps. heat shock factors (hsfs) display great contributions in regulating the transcriptional activation of hsp genes. in all invertebrate animals, only hsf1 is responsible for the transcriptional activation. in vertebrates, four members of hsf family (hsf1-4) regulate hsp expression. 72 among them, hsf1 is the most critical one. the fibroblasts from hsf1 −/− mice undergo apoptosis upon heat stress because of no hsp transcription. 73 upon stress conditions, the originally monomeric hsf1 in the cytoplasm could trimerize and translocate into the nuclei to promote the hsp expression by binding on the heat shock elements (hse) in the promoter region. 74 protein disulfide isomerase protein disulfide isomerase (pdi) is a multifunctional oxidoreductase and chaperone that catalyses the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (er). during disulfide bond formation, cysteine residues at the cghc active site of pdi accept two electrons from the cysteine residues in polypeptide substrates, leading to the reduction of pdi and oxidation of the substrate. then pdi transfers the electrons to an acceptor to start another cycle of disulfide bond formation. 75 in addition to pdi's catalytic function as a thiol-disulfide isomerase, it also exhibits molecular chaperone properties for glycosylated protein quality control. 76 erp57 (pdia3, grp58) is possibly the most thoroughly studied pdi family member that shares a similar structure consisting of four domains (namely a-b-b'-a') and possesses two localization sequence-an er retention signal (qdel), and a nuclear localization signal (kpkkkkk). unlike other pdi family members that directly bind the substrates for their reductase or isomerase activities, the b domains of erp57 have a high affinity to associate with calreticulin (crt) and calnexin (cnx), which would help to recognize and recruit polypeptide segments of the glycoproteins. 77 if the protein is not correctly folded, udpglucose:glycoprotein glucosyltransferase (uggt) would be recruited to reglycosylate the proteins, allowing them to be recognized and re-associated by erp57/crt/cnx complex. 76, 78, 79 considering the essential roles of pdis in the oxidative folding and chaperone-mediated protein quality control, they are now linked to a growing range of diseases including those are caused by virus infection. proteins that interact non-specifically with rna and resolve the non-functional inhibitory structures are usually referred to as rna chaperones, which have distinct roles without common sequences or motifs. 80, 81 they participate in a large number of cellular processes, including chromatin remodelling, transcription regulation, rnp assembly and stabilization, rna export, histone-like nucleoid structuring, intracellular immunity, and viral rna replication and translation. rna molecules mostly rely on welldefined 3d structures to fulfill their functions. however, the process of rna folding is very complicated. 82 the multitude of possible rna base-pairings together with the high stability of rna duplexes would give rise to a large number of alternative secondary and tertiary structures that are thermodynamically as stable as the functional, native structure. 83 rna chaperones promote rna folding by accelerating the escape from kinetic folding traps and prevent rnas from being trapped in nonfunctional conformations. [84] [85] [86] so far, no protein has been characterized whose primary function is to resolve nonspecifically misfolded rnas in cells. 80, 81 hnrnps are a group of heterogeneous nuclear ribonucleoproteins. they are essential factors for manipulating both the functions and metabolisms of pre-mrnas/hnrnas transcribed by rna polymerase ii. more than 20 hnrnps have been identified to date. hnrnps contain common rna binding motifs like arginine glycine boxes (rgg boxes), rna recognition motifs (rrms), hnrnp k homology (kh)-domains and zinc finger (zf)-domains (khzf domain). 87 well-defined functions of this family include transcription regulation, pre-mrna splicing, 3′-end formation, mrna packaging, rna transport, translational regulation, rna silencing, dna repair, and telomere biogenesis. they also have the ability to shuttle between nucleus and cytoplasm, therefore could transiently help to form rnp complexes in nucleus and also participate in rna metabolism in cytoplasm. 88 a large collection of hnrnps are involved in virus activities, most of which were first identified using viral rna-protein binding assays, followed by functional assays. 89 the importance of stress proteins one of the main functions of stress proteins is to maintain cellular homeostasis. under pressure, stress proteins are hyperactive to release the pressure. hsp27, hsp70 and hsp90 accumulate to a very high level in quite a few types of cancer cells. [90] [91] [92] although the mechanism underlying the increase has not been fully understood, it suggests that the fast increased hsps respond to the folding stresses. with tumor progression, the accumulating oncogenic proteins need powerful protein folding ability. under long-term stresses, hsps participate or promote carcinogenesis, cell survival, anti-apoptosis, angiogenesis, cancer cell stemness, invasion and metastasis. 93 however, hsps and rna chaperones are downregulated in nearly all age-related neurodegenerative diseases including alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, and several polyglutamine diseases such as huntington's disease and different forms of spinocerebellar ataxias 94, 95 (fig. 2) . downregulated rna chaperones lead to disorder of rna metabolism; 94 while the attenuated hsps result in a failure of the protein quality control (pqc) system to adequately handle the folding or timely degradation of proteins in neurologic disease. 95 both mechanisms cause protein aggregates, the hallmark of age-related neurodegenerative diseases. in this review, instead of paying much attention to these topics, we would only focus on the main biological functions and target values of stress proteins in human diseases caused by pathogen infections, particularly by virus infections because the welldeveloped antibiotics have already controlled the bacterial infection very well since 1950s. viruses infection causes a variety of diseases that are highly associated with dysregulation of stress proteins (fig. 2 ), e.g., respiratory symptoms, 96, 97 gastroenteritis, [98] [99] [100] [101] hemorrhagic fevers. 102, 103 critically, it has been demonstrated that neurodegenerative diseases as well as neurobehavioral disorders are the consequences of loss of neurons and axons in the central nervous system with ageing. it is evidenced that these diseases are possibly caused by chronic neuropathic viral infections. 104 stress proteins are involved in many steps of virus infection process, including virus entry, uncoating, replication, gene expression, as well as virus assembly and releasing as steps 1-5 shown (fig. 3 ) some viruses replicate in the nucleus, while stress proteins take part in the virus protein and/or rna nuclear import/export processes as setp a-c shown (fig. 3 ). the summary of the relationship between stress proteins, virus infection as well as host cell response is listed in table 1 . in this section, we would review and present new findings on hsp90 family proteins in the life cycles of different viruses including rna virus, dna virus and retrovirus. in addition, we also discuss their functions in virus-induced cellular response. the function of hsp90 family in rna virus infection virus entry. in the case of rna virus, hsp90 is critical for the entry of enterovirus a71 (ev-a71), 107 japanese encephalitis virus (jev) and dengue virus (denv). 108 ev-a71 entry is significantly blocked when cells are pre-treated with hsp90 inhibitors or hsp90-specific sirnas. 107, 109 since both denv virus and jev belong to the flavivirus, the entry of the these two viruses differently utilize hsp90 with the support of hsp70s in both neuroblastoma and microglial cells. 95, 108 denv depends much more on hsp90 to enter into cells as compared with jev since anti-hsp90 antibodies or hsp90 inhibitors block the majority of denv entry 110 but only a small portion of jev entry. 111 additionally, both hsp90 and hsp70 are associated with membrane lipid rafts in response to denv infection. 110 however, in denv infected liver cells (hepg2), neither hsp90 nor hsp70 works as the receptor to enable denv internalization, 110 indicating alternative entry mechanisms in different cell types. it is likely that the receptor functions of hsp90 and hsp70 are replaced by other unknow molecules. virus replication. hsp90 protein facilitates virus replication in several aspects. first, hsp90 works as a classic chaperone protein to stabilize virus proteins. hsp90 stabilizes paramyxoviruses polymerase and l protein, as well as assists virus replication. inhibition of hsp90 could hamper virus replication and shorten the half-life of l protein in vesicular stomatitis virus (vsv), human parainfluenza viruses-2 (hpiv-2), human parainfluenza viruses-3 (hpiv-3), simian virus 41 (sv41), or respiratory syncytial virus (rsv) infection cells. 112, 113 similarly, hsp90 is shown to maintain the stability of chikungunya virus (chikv) non-structural proteins (nsps) including nsp3 (a protein essential for rna synthesis) and nsp4 (rna-dependent rna polymerase, rdrp); 114 and the protease of hcv nonstructural protein ns3. 115 second, hsp90 modulates virus polymerase activity to enhance virus replication. taking hcv as an example, hsp90 indirectly modulates the hcv polymerase ns5 activity by maintaining the stability of kinase phosphoinositide dependent kinase l (pdk1), an upstream kinase of ns5 phosphorylation kinase prk2. 116 besides, mediated by fkbp8, hsp90 forms a complex with ns5 and directly regulates ns5 activity. 117 hsp90 inhibitors suppress viral replication by disrupting the hsp90-ns5 complex formation. 117 third, hsp90 manipulates the proper location of virus polymerases. during influenza virus infection, hsp90 interacts with the viral rdrp subunits polymerase basic protein-1 (pb1) and -2 (pb2) to form a complex, and then co-translocates into nucleus. 14, 118 in this process, hsp90 maintains the stability of pb1 and pb2. after entry into nucleus, hsp90 dissociates from the hsp90/pb1/pb2 complex and forms a new functional complex with polymerase acidic protein (pa). 119 extending study shows that the state of hsp90 acetylation is strictly regulated by histone deacetylases 6/8 while some viruses (such as influenza, sv40, hbv etc) also enter into nucleus of host cells. they may undergo other steps in their life cycle which are labelled as a-c: virus nucleus import, nucleus export, and virus rna processing. during the process of virus infection, hsp90, hsp70, hsp60, hsp40, hsp27, and pdis participate in the virus entry and uncoating steps. hsp90, hsp70, hsp60, hsp40, hsp27 and rna chaperones take part in the virus replication step. hsp70, hsp40 and rna chaperones are required in virus gene expression step. hsp90, hsp70 and hsp40 assist virus assembly. hsp70 and rna chaperones contribute to the virus release. while hsp90 is also important in virus nucleus import and export. hsp70 plays a role in the virus nucleus import. and rna chaperones play a major role in virus rna processing, including replication, initiation of translation, stabilization and decay 485, 486 (hdac6/8) in the influenza rdrp nuclear import stage. hdac6/8 inhibitors efficiently limit the polymerase nuclear import and suppress virus replication. 120 in the course of influenza infections, hsp90 expression is stimulated through mtor/p70s6k signalling. 121 our recent studies show that hsp90 also exhibits significant importance on ev-a71 replication through pim1 signalling (unpublished). it has been shown that ev-a71 infection elevated both the mrna and protein levels of pim1. 122 the elevated pim promoted ev-a71 replication while knockdown of pim1 decreased ev-a71 replication. knockdown of hsp90β decreases 60% of virus replication 12h post infection (p.i.), while the secreted virions decrease by approximately 80%, indicating the crucial roles of hsp90β in both virus replication and secretion (fig. 4a-c) . other researchers reported that hsp90β is responsible for ev-a71 assembly which may be the reason that hsp90β attenuates the secretion of ev-a71 virions in our study. 107, 109, 122 notably, hsp90β contributes to virus replication more than that of secretion. our data also shows that knockdown or knockout of hsp90β decreased the proteins expression level of ev-a71 structure protein (fig. 4d , e). and hsp90 inhibitors 17-aag, geldanamycin (ga) and ver50588 all dramatically inhibit ev-a71 protein expression (fig. 4f ). among them, ver50588 display the strongest inhibitory effect which has not been reported before. we predicted that hsp90β is a potential target of pim1 (fig. 4g ). to address this hypothesis, we conducted experiments by overexpression pim1 and knockdown of pim1. accordingly, the phosphorylated status of hsp90β is increased and decreased (fig. 4h,i) . more importantly, knockout of hsp90β by crispr/cas9mediated gene editing almost completely abolishes the effects of pim1 signaling on ev-a71 replication (fig. 4j, k) . viral protein maturation, virion assembly, and release. during the viral protein expression and maturation, hsp90 works as a classic chaperone to monitor the proper folding of viral proteins. hsp90 modulates the maturation of hcv nonstructural protein 2/3 (ns2/ 3) kinase. 123 hcv ns2/3 is cleaved into two separate proteins right after translation, a key step of ns2/3 protein maturation. hsp90 strictly regulates the proper folding of newly synthesized ns2/3 protein. 123 hsp90 and its co-chaperone p23 form a complex to assist the proper folding of capsid precursor polyprotein p1 of poliovirus, rhinovirus, and coxsackievirus; 124 while the inhibitor ga reduces the maturation of p1, leading to immature p1 degradation in proteasome. 124 during the virion assembly, hsp90 interacts with capsid vp1 protein of noroviruses and the termini of the murine norovirus 1 genome. 124, 125 this interaction not only stabilizes vp1, but ensures the viral genome to be encapsulated into capsids as well. 124 hsp90 interacts with and stabilizes influenza neuraminidase (na), a major surface glycoprotein involving in virion release. 126 more importantly, it emphasizes the hsp90-na complex formation on promoting cell survival, leading to more virus production. 126 the function of hsp90 family in dna virus infection virus entry. the entry of dna viruses includes steps of crossing over cell membrane and nuclear import. hsp90 is mainly shown the ability to assist the nuclear import of virus. the nuclear transport of many viruses depends on the microtubules (mt) and mt-dependent molecular motor dynein/dynactin complex. 127 virus strictly modulates the status of tubulin acetylation, a critical event for the transportation of viral components. [128] [129] [130] in hsvinfected cells, hsp90 co-localizes with acetylated tubulin and capsid protein vp5. 131 hsp90 inhibitors disrupt its binding to the acetylated tubulin, thereby inhibiting the nuclear transport of hsv capsid protein. 131 during hbv infection, the glucocorticoid receptor shows a strong possibility to enhance the nuclear import of hbv. 132 hsp90 facilitates glucocorticoid receptor redistribution from the cytoplasm to the nucleus. 133 virus replication. during dna retroviral replication, hsp90 mainly contributes to regulating and maintaining the reversetranscriptase (rt) activity. taking hepatitis b virus (hbv) as an example, the reverse transcription is an essential step to generate viral genomic dna in type vii viruses. the beginning of reverse transcription is the recognition and interaction of rt with an rna signal (the packaging signal, ε) on the pre-genomic rna. 134 it was identified that hsp90 is an essential host factor that facilitates duck hepatitis b virus (dhbv) replication by interacting with viral rt. 135 treating with hsp90 inhibitors or monoclonal antibodies (mab) sufficiently block rt-ε binding. 135 hu et al. demonstrated that rt-ε interaction depends on hsp90's atp hydrolysis activity. 136 two independent regions in the terminal protein (tp) and the rt domains of polymerase separately bind with hsp90 at the n-terminal and c-terminal fragments, and both domains are essential for ribonucleoprotein (rnp) and protein priming. 137, 138 although a model is established to show how hsp90 bridges the two separate rt domains of polymerase together to enable the formation of an rnp complex with the hbv rna; 137 there are still some fundamental questions to be addressed. firstly, whether the hsp90 chaperones or hsp70 chaperones are essential for the rt-ε interaction. stahl et al. believed that hsp70 chaperones are much more important for the rt-ε interaction. they proposed that hsp90β another hsp90 family member, is shown a critical regulator in stabilizing and activating rt, allowing its preferential binding to the pregenomic rna during hbv replication. 143 the replication of most dna viruses occurs in the nucleus, where virions form in replication centres. therefore, the proper location of viral proteins is quite important for virus replication. hsp90 is also found to regulate the location of virus dna polymerase in virus-infected cells. after treated with hsp90 inhibitors, hsv polymerases is mislocalized from the nucleus to the cytoplasm and subsequently degraded in a proteasomedependent manner. 144, 145 similar to hsv, the nuclear translocation of dna polymerase of ebv also requires hsp90. 146, 147 during the polymerase transportation, the polymerase catalytic subunit balf5 forms a complex with bmrf1 in the assistance of hsp90β. hsp90 inhibitors effectively block the translocation of viral dna polymerase. 146, 147 virus gene expression. hsp90 is important for virus gene expression both at the transcription and translation levels. the transcription of hsv immediate-early α (ieα) genes is initiated by the transcription factor complex, which is composed of octamerbinding transcription factor 1 (oct-1), host cell factor 1 (hcf-1) and viral protein 16 (vp16). 148, 149 in the complex, vp16 is the major virus-encoded transcriptional activator that controls the efficiency and level of viral gene transcription. in the transcription process, hsp90α is shown to maintain the stability of vp16 by keeping it from degradation in a macroautophagy-mediated manner. 150 similarly, hsp90 also regulates the transcription of human cytomegalovirus (hcmv) immediate-early genes through activating akt and nf-κb signalling pathways, which are critical for major immediate early genes transcription. 151, 152 at the translation level, hsp90 promotes the translation of conserved herpesvirus protein kinases (chpks), including herpes simplex virus type 1 and 2 (hsv-1, hsv-2), varicella-zoster virus (vzv), ebv, kshv. 153 chpks play important roles in multiple processes, including gene expression, 154-156 viral dna replication, [156] [157] [158] capsid nuclear egress, 159, 160 and dna damage responses. 161, 162 the translation of ebv nuclear antigen 1 (ebna1) protein is also manipulated by hsp90. 163, 164 ebna1 is critical for cellular transformation, tumorigenesis, and the maintenance of viral episomes. [165] [166] [167] the ebna1 translation is strictly regulated by hsp90 through the gly-ala repeat domain to keep ebna1 at a relatively low level. 168 it has been demonstrated that hsp90 inhibitors block the translation of ebna1; and mutation of gly-ala repeat domain abrogates the inhibition of ebna1 translation. 163, 164 hsp90 does not interact with ebna1directly, a bridge protein may be involved in this process. inhibiting ebna1 expression strongly suppresses both ebv-induced primary b cell transformation in vitro and lymphoproliferative disease in scid mice in vivo. 163, 164 virus assembly. only a few papers reported the function of hsp90 in dna virus assembly. the activated hsp90 is needed for hbv assembly. 169, 170 hsp90 enhances the affinity of core protein dimers for capsid formation and prevents capsid dissociation. 169 besides, the reactive oxygen species (ros)-promoted hbv capsid assembly also requires an active form of hsp90. 170 virus-induced tumorigenesis. as described before, ebv is the causative regent of several tumors, including burkitt's lymphoma 171 and nasopharyngeal carcinoma (npc). 172 the latent membrane protein 1 (lmp1) is regarded as an oncoprotein that promotes tumor metastasis and invasiveness through inducing the expression of matrix metalloproteinase 9 (mmp9), mimicking the tumor necrosis factor receptor (tnfr) superfamily proteins, and activating the nf-kb, mapk, pi3k/akt and jak/stat signal transduction pathways. 173, 174 hsp90 seems positively promoting cell growth in ebv-positive nasopharyngeal carcinoma cells and ebv-infected t and nk cells. 175, 176 hsp90 inhibitors, at13387 and biib021, potently inhibit cell growth and induce apoptosis by impeding lmp1 function through activating its downstream signaling pathways described above. 175, 176 immunity modulation. we have discussed how hsp90 is hijacked by dna viruses to promote viral replication above. under certain conditions, hsp90 exhibits antiviral activities by promoting cell immunity. in the acute infection stage, hsp90 is induced to express in the cell surface of ebv-infected b cells. it has a strong ability to expand gamma delta t cells (γδ t cells) population. 177 the γδ t cells have potent antiviral ability in the acute phage when a host is infected by hiv, influenza, sendai, coxsackie, vaccinia, vsv, or hsv-1. the γδ t cells work as both early sentinels of the immune system by providing immediate protection and as bridging elements between innate immunity and adaptive immunity. 178 since hsp90 can work as an immune sensor and assist antigen presentation, it may function in the same way in ebv infection. 177, [179] [180] [181] [182] the function of hsp90 family on cell transformation during retrovirus infection several hsps functions as oncoproteins to promote cellular transformation. hsp90 participates in the htlv-1-induced cellular transformation. the tax protein of htlv-1 controls viral replication and induces t lymphocyte transformation. 183 nf-κb pathway is one of the main targets essential for this process; 184 while hsp90 acts as an important partner of tax by binding with and maintaining its stability in nucleus. 153, 185 hsp90 inhibitors (e.g.,17-dmag) or hsp90-depletion by sirnas cause tax degradation in proteasome, inhibition of nf-κb signalling, and activation of the long terminal repeat (ltr) of htlv-1. 153, 185 oral treatment with fig. 4 pim1 signaling promotes ev-a71 replication through hsp90β phosphorylation(unpublished data). a rd cells were treated with scramble/hsp90β sirna for 24h, the effects of hsp90β knockdown were determined by rt-qpcr assay. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. b, c rd cells were treated with scramble /hsp90β sirna for 4h, then infected with ev-a71 at moi of 1 for indicated time. the intracellular (b) and extracellular (c) viral rna levels were detected by rt-qpcr assay. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. d, e knockdown of hsp90β by sirna or knockout by cripsr/cas9 mediated gene editing in rd cells, and then cells were infected with ev-a71 at moi of 1 for indicated time. the protein level of ev-a71 was determined by western blot. f rd cells were treated with hsp90 inhibitors at different concentrations and infected with ev-a71 at moi of 0.01 for 48h. the protein level of ev-a71 was determined by western blot. g pim1protein interaction network was predicted using online genemania program. h pim 1 was overexpressed in 293t cells and cell lysate was collected for immunoprecipitation assay. the phosphorylation status of hsp90 was detected by western blot. i pim1 was overexpressed/ knocked down in 293t cells. and cell lysate was collected for native page analysis. j wt rd cells and hsp90β knockout (hsp90β-ko) cells were pretreated with 2 μm pim1 inhibitor sgi-1776 for 2 h, then the cells were infected cells with ev-a71 at moi of 0.01 for 48h. intracellular viral rna level was determined by rt-qpcr. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. k pim1 was overexpressed in wt/hsp90β -knockout rd cells, and infected with ev-a71 at moi of 1 for 12 h. the protein level of ev-a71 was determined by western blot hsp90 inhibitor 17-dmag significantly suppresses the aggressive infiltration into multiple organs in atl mice. 153, 185 functions of hsp70 family in rna virus infection virus entry. viruses in different families (e.g., picornaviridae, flaviviridae, and reoviridae) take advantage of hsp70 family proteins for their entry into host cells. in the case of picornaviridae, for example, the coxsackievirus a9 (cav-9) uses hsp70 homolog grp78 for its entry. 186 it shows that antibodies against grp78 block 50% of virus binding. integrin αvβ3 is another famous virus receptor. 187 when cells are simultaneously treated with grp78 and integrin αvβ3 antibodies, virus binding is blocked completely. therefore, grp78 functions as a co-receptor of cav-9. besides, grp78 can interact with major histocompatibility complex (mhc) i molecules on the host cell membrane after infection of cav-9. mhc i molecules help virus internalization into mammalian cells. 186 in the course of ev-a71 infection, hsp70 is dramatically upregulated and interacts with ev-a71 on the cell surface. hsp70 antibody significantly inhibits virus binding to the cell surface. 188 besides enteroviruses, many viruses of flaviviridae family also require hsp70 to entry into host cells. by affinity chromatography assay, hsp70 is discovered to form a complex with hsp90 and denv receptor that facilitates viral entry. 110, 189 hsp70 interacts with dnev envelope protein (e protein) and plays a significant role in virus attachment. 190 similarly, antibodies against hsp70 and hsp90 significantly inhibit denv infection. 110, 189 the same mechanism is also observed in jev infection. 191 hsp70 is enriched in the lipid raft and colocalized with the e protein in jev-infected huh7 cells. 192 the depletion of cholesterol disrupts the enrichment and colocalization of the e protein and hsp70 to a raft membrane. eventually, it decreases jev entry without any effects on virus attachment. 192 these results suggest that hsp70 works as a receptor of jev; and lipid rafts serve as an organizing centre to facilitate jev entry. at the late stage of jev entry, hsc70 (isoform d) is upregulated in c6/36 cells upon jev infection. however, it seems that hsc70 is not required for virus attachment to the cell membrane but needed for virus penetration into the host cells. it is suggested that hsc70 holds an intense involvement in clathrinmediated endocytosis at the late stage of viral entry, which helps jev to penetrate into host cells. 193 recently, it is reported that grp78 is also required for jev both in the attachment and entry steps. 194 antibody targeting the n-terminal of grp78 significantly prevents virus attaching to host cells whereas antibody targeting the c-terminus fails to block the attachment. knockdown of grp78 also inhibits jev internalization. the colocalization and interaction between grp78 and jev envelope protein provide solid evidence to show the importance of grp78 in the process of virus attachment and entry. 194 interestingly, grp78 is secreted out of host cells after jev infection, and the secreted grp78 cooperates with jev to promote virus infection. 195 recent studies show that grp78 is a receptor of sars-cov, mers-cov, and sars-cov-2 viruses. 196 zikv infection is positively regulated by hsp70 at multiple stages. 197 hsp70 inhibitors impair virus entry, rna replication, and capsid assembly of different zikv strains in diverse cell lines. 190, 197 rotavirus infection also needs the assistance of membrane-resident hsc70. 198 hsc70-specific monoclonal antibodies inhibit virus internalization and infection without effect on virus attachment. 198 further evidence shows that the whole virus particle and a short domain (or a peptide) in the cterminal region of vp5 is sufficient to bind to hsc70. 199 the atpase domain of hsc70 is proved to be necessary for its interaction with vp5 and induction of virion conformation change for the entry. 200 virus replication. hsp70 family proteins participate viral replication by employing different mechanisms. first, hsp70 family proteins facilitate the formation of virus replication complex and/ or maintain the stability of complex proteins. in some cases, hsp70 family proteins directly interact with viral polymerase to enhance viral replication. for example, during the mumps virus (muv) infection, the expression level of hsp72 is increased. the c-terminal region of hsp72 interacts with the n-terminal region of p protein, which is an essential component of rdrp complex. knockdown of hsp72 results in accumulated ubiquitinated p protein as well as increased cell apoptosis. 201 besides, hsp70 is also reported to regulate l protein, another muv polymerase component. hsp70 cooperates with hsp90 to regulate l protein levels. hsp90 inhibitor, 17-aag, reduces the l protein level through promoting degradation via the c terminus of hsp70interacting protein (chip) -mediated proteasomal pathway. hsp70 inhibitor ver155008 together with 17-aag enhances l protein degradation. therefore, hsp90 and hsp70 together regulate the stability of l protein and ensure the proper virus replication complex (vrc) formation. 202 in the case of canine distemper virus (cdv) infection, the increased hsp70 results in an elevated expression of light nucleocapsid (nc-l) variant, which displays polymerase activity. 203, 204 a more direct evidence is that hsp70 facilitates viral rna production in cell-free transcriptional assays. 204 furthermore, it is demonstrated that hsp70 interacts with and regulates nc polymerase activity dependent on the hsp70 atp activity; because hsp70 antibody significantly inhibits nc polymerase activity and supplementation of purified recombinant hsp70 enhances both the basal and stress-induced nc polymerase activity. 205 the other members of hsp70s also regulate vrc formation. hsp72 physically interacts with several replication proteins of flavivirus including ns5a, ns3, and ns5b (rdrp). for example, hsp72 participates the vrc formation of hcv. 206 downregulating hsp72 leads to a decreased number of vrc in hcv-infected cells, while overexpression of hsp72 raises the number of vrc. 206 hsc70 is associated with vrc by binding on the 3' polyu/uc motif of hcv rna genome. 207 hcv accumulation and virion production are significantly suppressed when cells are treated with hsp70 or hsc70 inhibitors. 208 similar result is reported in the case of rsv infection. ectopic expression of rsv nucleocapsid protein (n protein) and phosphoprotein (p protein) are detected to interact with hsp70 in 293t cells. 209 the n protein is responsible for interacting with the viral rna, and p protein interacts with n protein and with the rdrp l to form the nucleocapsid. in rsvinfected cells, hsp70 redistributes into lipid-raft membranes and colocalizes with virus n protein and lipid raft marker gm1. 210 although hsp70 inhibitors suppress rsv polymerase activity; 210 it only disrupts viral gene expression but do not affect rna polymerization. 211 therefore, more detailed studies are needed to understand the functions of hsp70 in modulating rsv gene expression and replication. in term of ebola virus (ebov) replication, the mechanism of hsp70 involved is much more complicated. by using immunoprecipitation and mass spectrometry assays, it has been identified that the n protein interacts with hsp70, nef, bag2, and the hsp70 co-chaperone dnaja2. 212 the n protein recognizes and binds to the viral rna genome to establish a steady n protein-rna complex structure (rnp). this complex further interacts with viral proteins vp30, vp35 and rdrp l, to finally form vrc. here, hsp70 functions to maintain the stability of n protein and helps to facilitate vrc formation. 212, 213 in addition, hsp70 is also copurified with l polymerase in insect cells. 214 besides, hsc70 interacts with the terminal non-coding regions of the ebov genome. disruption of the interaction by mutating the binding site potently inhibits the minigenome replication of ebov. 215 another mechanism that hsp70 employs to support virus replication is to modulate nuclear import of polymerase or nuclear capsid. some rna viruses also replicate in the nuclear such as cdv and influenza virus. therefore, nuclear transportation becomes a critical step for their replication. upon cdv infection, hsp70 is shown a strong contribution to viral replication by interacting with and promoting the translocation of the nucleocapsid particles from the cytosol to nucleus. 216 similarly, during influenza infection, hsp70 interacts with pb2 or pb1 monomers and pb2/ pb1 heterodimer in hela and hek293t cells, and sequentially translocates into the nucleus with pb2 monomers or pb2/pb1 heterodimers. 217 if hsp70 and pb2/pb1 polymerases are retained in the cytosol, the polymerase activity reduces dramatically. the shuttling of hsp70 between nuclear and cytoplasmic compartments underlies the modulatory effect of hsp70 on influenza virus replication. 217 virus gene expression. besides viral entry and replication, hsp70 also contributes to viral protein translation. positive singlestranded rna viruses (e.g., sars-cov-2, hcv, zika, ev-a71, etc) use the internal ribosome entry site (ires) to initiate the translation of their own proteins but inhibit the host cellular cap-dependent translation through regulation or cleavage of eukaryotic translation initiation /elongation factors (eifs/eefs). in the case of coxsackievirus b3 (cva b3) infection, hsp70 is upregulated to enhance the initiation and elongation of viral translation. 218 in the translation initiation step, hsp70 upregulates ires-acting factor lupus autoantigen protein expression and activates eif4e binding protein 1 (eif4ebp1), a cap-dependent translation suppressor. in the elongation step, hsp70 activates the akt-mammalian target of rapamycin complex 1 (mtorc1) signal cascade, leading to activation of eef2 via kinase p70s6k-and cdc2-mediated phosphorylation and inactivation of eef2 kinase (ef2k). 218 hsc70 enhances the ires activity in ev-a71 infected cells. hsc70 interacts with 2a protease of ev-a71 to enhance eif4g cleavage that impairs host cell cap-dependent translation but enhances viral ires-mediated translation. 219 therefore, hsc70 may serve as an antiviral target against ev-a71 and hcv infections. some viruses do not have ires sequence, and virus replication produces plenty of dsrnas which trigger the activation of protein kinase-rna-activated (pkr)-eif2a signalling cascade that shuts down global translation in cells and releases stresses. 220, 221 to circumvent the pkr-mediated block to viral proliferation, influenza a virus induces the cellular tetratricopeptide repeat (tpr)domains containing jdp protein, p58ipk. influenza virus downregulates pkr in an hsp70-dependent way. [222] [223] [224] [225] [226] in uninfected and unstressed cells, p58ipk activity is clogged with hdj1 by forming a complex. 225, 226 during influenza a infection, the amount of hdj1 in p58ipk-hdj1 complex decreases to an undetectable level. these findings suggest that the activation of p58ipk appears to be a sequel to the hsp70-mediated release of hdj1 from the p58ipk-hdj1 complex, which allows the monomeric p58ipk to inhibit pkr. 225 in the hsp40 chaperone part, we would discuss more details about the regulation of pkr signaling by influenza virus. virion assembly. hsp70 is reported to assist some viruses' assembly. during morphogenesis of the double-stranded rna reovirus, hsp70 contributes to the assembly of trimeric sigma 1 protein, which is responsible for the interaction with host cell receptor. 227 while the n-terminal segment of the sigma 1 protein folds and trimerizes cotranslationally in an hsp70-independent manner, a post-translational fold of the c-terminal globular domain is dependent on hsp70. in this process, hsp70 binds cotranslationally to the region downstream the n-terminal αhelical coiled-coil, which presumably helps to inhibit unwanted interaction and misfolding. trimerization of the c-terminal domain of the sigma 1 protein is coupled to the atp-dependent release of hsp70 from the ribosome. 227 besides, in hela cells infected by poliovirus or coxsackievirus b1, hsp70 is detected to interact with the capsid precursor p1. 228 in the complex, p1 is mainly newly synthesized and has a longer half-life than that of total p1. the hsp70-p1 complex is regarded as an assembly intermediate of picornaviruses. 228 interestingly, some researchers demonstrate that hsp70 inhibits influenza virus replication by blocking nuclear export of viral ribonucleoprotein complex (vrnp), and subsequent viral morphogenesis via disassociating m1 from vrnp. 229, 230 virus release. the evidence of hsps on virus releasing is limited. both hsp70 and hsc70 can interact with ns5a protein; although they play different roles in hcv infection. 231 silencing of hsp70 decreases viral protein expression, but the virus protein level is not affected. 232, 233 instead, interfering hsc70 reduces extracellular virion production. 232, 233 moreover, hsc70 is embedded in the viral capsid. and co-localization between hsc70 and core and e2 structural proteins of hcv has been found in lipid droplets. therefore, hsp70 and hsc70 may regulate hcv infection release at different steps. the function of hsp70 family proteins in dna virus infection virus entry and genome releasing. instead of virus attachment and entry into the host cells, here we talked about virus entry into the cytosol from er. the cytosol entry from er is a key step in sv40 infections. hsc70 is reported to be essential in this step. hsc70 interacts with and is regulated by sgta. 234 further studies show that hsp70 superfamily member hsp105 forms a subunit in the hsc70-sgta complex to facilitate sv40 cytosol entry. 235 in addition, grp78/bip also plays a role in sv40 cytosol entry. grp78 interacts with sv40 capsid protein in a dnajb11-dependent manner to help sv40 disassemble and enter into the cytosol. 236 hsp70 is also needed for the genome release of some dna viruses. such a process is described in adenovirus infection. after the release of the virion from endocytic vesicles into the cytoplasm, hsp70 and hsc70 immediately attach to the hexon protein, one of the major adenovirus coat proteins. 237 hsp70/ hsc70 and its co-chaperone bag3 interact with the penton base protein, the viral capsid constituent responsible for virus internalization. 238, 239 the intact nucleocapsid is transported to nucleus through the typical nls-dependent nuclear import machinery. 240 the nucleocapsid anchors to the nuclear pore through its hexon protein by interacting with components of the pore complex. then viral dna is transferred into the nucleus in a hsp70-dependent manner but leaving hexon outside the nucleus because the purified hexon, instead of viral dna, enter the nucleus in a hsp70-independent manner. 240 a possible explanation is that the intact nucleocapsid is too large to pass through the nuclear pore complex, while the disassembly of nucleocapsid facilitates the entry of viral dna into nucleus. however, more solid evidence is needed for such an explanation. 238 other examples for the contribution of hsc70 in viral genome release in host nucleus are hsv and polyomavirus. in hsv-infected cells, the translocation of hsc70 from the cytosol to nucleus is triggered by the immediate-early viral protein icp0. hsc70 is colocalized with the components of the 26s proteasome and virus ul6 portal protein, which provides the conduit for dna entry and exit from the capsid. ul6 is highly ubiquitinated in the nucleus, indicating that hsc70 may be responsible for the correct folding and degradation of ul6 in the ubiquitin-proteasome pathway, though there was no direct evidence that ul6 is a substrate of hsc70. 241 it is also believed that hsc70 contributes to polyomavirus genome nuclear import through its interaction with all viral capsid proteins vp1, vp2 and vp3 both in vitro and in vivo. hsc70 translocates from the cytosol to the nucleus accompanied by the translocation of capsid proteins upon infection with polyomavirus. 254 gene expression and protein maturation. most viruses manipulate the cellular transcription and translation machinery and shut off host protein synthesis, so that they can take advantage of these machineries and recruit initiation and elongation factors for the expression of viral proteins. some host factors exploited by viruses interact closely with components of hsp70 complex. therefore, the chaperone system is highly important for viral gene expression. several transcription initiation factors are well known to physically interact with the hsp70 co-chaperone bag1 in vitro. bag1 stimulates general transcription activity in an hsp70dependent manner. [242] [243] [244] the stimulation of global transcription is detected in cells upon infections by either human polyomavirus, john cunningham virus (jcv) or hcmv. 245, 246 however, the detailed molecular mechanism of the general transcriptional activation by viruses is to be further investigated. the typical example of hsp70 system in regulating the maturation of viral proteins is shown in hbv large envelope protein (lhbsag). [247] [248] [249] the mature lhbsag has a unique dual transmembrane topology. initially, the c-terminal of lhbsag cotranslationally resides in er, while the n-terminus is resident in the cytosol and later is translocated into er for post-tranreported that the expression of grp78 is stimulatedslation. to ensure the correct topology, hsp70 system strictly regulates the post translocation of n-terminal of lhbsag. during the translation, hsc70 interacts with the n-terminal of lhbsag at amino acids 63 to 107 and suppresses lhbsag translocation into er. 248, 249 hsc70 cochaperones hip and bag1 also regulate the activity of hsc70 in an antagonistic way. 250, 251 overexpressed hip promotes hsc70 activity resulting in more cytosol retention of lhbsag n-terminal; 250 while bag1 overexpression could inhibit hsc70 activity to promote nuclear translocation. 251 in the post-translation process, grp78 binds with lhbsag and hsc70 to facilitate the er translocation of hbv large surface antigen. 247, 248, 252, 253 the function of grp78 is regulated by both positive regulator er-localized dnaj-domain-containing protein 4 (erdj4) and negative regulator bip-associated protein (bap). 253 increased bap destabilizes lhbsag/bip complex. 253 together, hsp70 chaperone system is crucial in modulating the sophisticated topogenesis of hbv envelope protein. 247 virus assembly. a lot of dna viruses normally assemble in the nuclei of infected cells. taking polyomavirus as an example, all capsid proteins are synthesized in the cytosol, whereas subsequent assembly of virions only takes place in the nucleus. during polyomavirus infection, the constitutive form of hsp70 and hsc70 are coimmunoprecipitated with all three viral capsid proteins (vp1, vp2, and vp3). hsp70 is translocated from cytoplasm to the nucleus in the late stage of infection, coincident with localization change of the viral capsid proteins. 254 in vitro studies show that hsp70 functions to keep proper assembly of polyomavirus. 255 the prokaryotic hsp70 chaperone dnak also interacts with recombinant vp1 at the c-terminal domain where it links pentamers in an assembled capsid. 255 when dnak binds to vp1, it inhibits vp1 assembly, which is induced by calcium in vitro. however, combining the hsp70 chaperone system including dnak, dnaj and grpe with vp1 together is sufficient to assemble vp1 into uniform capsids in the presence of atp alone without calcium. 255 the function of hsp70 family in retrovirus infection human t lymphotropic virus type 1 (htlv-1) is a well-investigated example of retrovirus that interacts with hsp70 proteins. during htlv-1 infection, syncytium formation is a key factor for cell-tocell virus transfer. the syncytium formation is subject to close cellto-cell interactions. 256, 257 cell membrane-resident hsc70 is necessary in this process as hsc70-specific monoclonal antibodies eliminates the syncytium formation and htlv-1 infectivity. the same outcome presents when cell is treated with a peptide derived from the htlv-1 glycoprotein gp46, which binds to hsc70. 258, 259 interestingly, although hsc70 enhances the syncytium formation, it has no effect on virus entry. 259 hsp70 also plays an important role in the post-entry steps. during human immunodeficiency virus type 1 (hiv-1) infection, viral protein r (vpr) stimulates interaction between the viral preintegration complex and karyopherin-alpha to facilitate viral nuclear import. hsp70 functionally overlaps with vpr in this process. 260 when vpr is deficient, hsp70 could rescue virus nuclear import by interacting with karyopherin-alpha at the n-terminal that also binds vpr. interestingly, some researchers argue for the antiviral role of hsp70 because hsp70 and vpr share the same substrate. it seems that hsp70 would compete and inhibit vpr function. since hiv-1 needs vpr to manipulate cell cycle and apoptosis, hsp70 neutralizes the function of vpr in hiv-1 infection. 261, 262 recently, hiv is observed to package hsp70 as part of virion core. the virion-incorporated hsp70 atpase activity and correct conformation of hsp70-virion are essential for hiv infection, since inhibition of hsp70 atpase activity interrupts the hsp70-virion core association and diminishes virus infectivity. 263 the effects of hsp70s on host cells upon dna virus infection cellular transformation. dna viruses those do not encode polymerase in their genome are dependent on host dna replication machinery. to replicate in quiescent cells, the virus has to reinitiate cell cycle thereby transforming the host cells. some mechanisms have evolved in enabling viruses to overcome the restriction points of cell cycle. the best-investigated example is sv40. the large and small t antigen (tag) are central for the sv40 transformation ability. at their n terminus, both types of tag contain the j-domain, the signature motif of an hsp70 cochaperone. mutation or deletion of the j-domain disrupts the functional association of tag with hsp70s. the ability of tag to transform mammalian cells is subsequently obliterated. 264 also, tag sequesters the retinoblastoma family proteins (prb, p107, and p130) and liberates members of the e2f family of transcription factors in a hsc70-atp hydrolysis-dependent manner. 265, 266 the free e2f family proteins subsequently trigger the expression of the s-phase genes leading to dna replication. 267 the above observations are interpreted in the following four steps. firstly, the large tag combines with prb-e2f complex. subsequently, the prb-e2f-tag complex associates with hsc70 in the presence of atp as hsc70 atp-bound form presents high substrate association rate to facilitate tag-hsc70 complex formation. the third step is that the j-domain of tag in the tag-hsc70 complex stimulates atp hydrolysis. this process is dependent on the active site of hsc70 and does not occur at the t-antigen binding site. meanwhile, hsc70 transfers to the high-affinity conformation allowing the trap of the substrate protein prb or e2f. in the last step, hsc70 induces the conformation of the substrate protein in the complex which assists the dissociation of prb and e2f. after adp dissociation and rebinding of atp to hsc70, e2f and the prb-tag complex is released from the hsc70-substrate complex. [268] [269] [270] [271] a second strategy of tag to transactivate e2f transcription is independent of the disruption of the prb-e2f complex that also involves the jdomain and hsp70 protein. [271] [272] [273] [274] tag functions like bag1 to assiste the assembly of a transcription initiation complex on the respective promoters in the presence of hsc70. alternatively, tag could induce hsc70 to disassemble an inhibitory silencer complex fig. 5 model for the participation of hsc70 in the tag induced disassembling of prband e2f. firstly, tag combines with prb-e2f complex to facilitate t-antigen-hsc70 complex formation. then tag-hsc70 complex stimulates atp hydrolysis, and hsc70 transfers to the high-affinity conformation allowing the trap of the substrate protein prb or e2f. finally, hsc70 induces the conformation of the substrate protein in the complex which assists the dissociation of prb and e2f to initiate cell cycle reprogram or to assist with remodelling the chromatin (fig. 5) . hpv and adenovirus have similar transforming activities by disrupting prb-e2f complexes. although neither e7 (hpv) nor e1a (adenovirus) protein contains a j-domain, both proteins could transform cells in a way similar to that described for sv40 tag. e7 interacts with tumor suppressor htid-1. 275 the c terminus of e7, which mediates the interaction with htid-1, is essential for the physical disruption of the prb-e2f complex though it is not necessary for direct interaction with prb. 276, 277 these observations suggest that the interaction with htid-1 is involved in the disruption of the prb-e2f complex, providing e7 with the jdomain necessary to recruit hsc70 for the complex dissociation in analogy to the function of sv40 tag. alternatively, the binding of e7 to htid-1 could transform cells through inhibition of the assumed tumor suppressor function of htid-1. e1a directly interacts with hsc70 to disrupt the prb-e2f complex. 278 in conclusion, most double-stranded dna viruses depend on hsp70 chaperones for the reprogramming of the host cells to reenter the cell cycle. cell survival and apoptosis. since hsp70 systems are essential modulators for cell survival under stress conditions, the induction of hsp70 protein facilitates virus infection by keeping the cell alive until mature viruses are ready to leave. this is the main reason why the disruption of apoptotic pathway is a quite common phenomenon in viral infections. [279] [280] [281] in the early stage of viral life cycle, viral reproduction is simply vulnerable to cell death. naturally, viruses are evolved in manipulating cellular apoptosis. in ebv-infected cells, nuclear oncoprotein ebna3a helps hsp70 nuclear translocation and hsp70 chaperone complex formation to immortalize b cells through inhibiting apoptosis. 282 in contrast, apoptosis is beneficial for virus spreading when virions are finally assembled. [283] [284] [285] [286] [287] [288] [289] [290] virions are found in apoptotic bodies and subsequently engulfed by phagocytic cells. it has been suggested that virus can infect neighbour cells without being detected by the host immune system. 291, 292 on the other hand, the decrease of hsp70 mrna level may lead to the timed induction of apoptosis at the late stage of adenovirus infections. innate immunity. hsp70 has been previously described to influence innate immunity and inflammatory responses. 293 it has been believed that hepatocyte is devoid of innate immunities. ma et al. reported that the expression of grp78 is stimulated by hbv replication. the elevated grp78 protein in turn activated innate immune response by induction of ifnβ expression. 294 further studies showed that hsp70 greatly contributes to cellular innate immunity in response to either virus or bacterial infections. in the course of bacterial infections, the elevated intracellular levels of hsp70 protect cells from lps-mediated inflammation. through its interaction with tnf receptor associated factor 6 (traf6), hsp70 can inhibit its ubiquitination and thereby block the activation of transcription factor nf-kb. 295, 296 weiss et al. has presented more details on this mechanism. hsp70 binds with ikk, leading to disturbing the function and stability of nf-κb/ikbα/ikk complex and further impairing ikbα phosphorylation. 297 the disturbance of this complex also affects ikbα proteasomal degradation and nuclear translocation of nf-κb complex. 298 the inhibition of nf-κb signalling has great therapeutic significance because it can prevent massive tissue damage medicated by the excessive inflammation response. a more recent study provides another approach for hsp70 to regulate inflammation. pierre et al. described an nf-κb independent pathway that hsp70 could impact nlrp3/asc inflammasome formation through its association with nlrp3. 299 aside from the anti-inflammation function of intracellular hsp70, the secreted extracellular hsp70 binds to dendritic cells and macrophages before being recognized by its binding elements, most of which are innate immune receptors. 293, 300 toll-like receptors (tlrs) detect virus invasion and immediately trigger intracellular innate antiviral response. 301 they belong to type i integral membrane glycoproteins of il-1 receptor (il-1r) superfamily. 302 it has been suggested that tlr2/tlr4 involved in the initiation of innate immunity by extracellular hsp70. hsp70 utilizes both tlr2 and tlr4 to transduce its proinflammatory signal in a cd14-dependent manner to promote proinflammatory cytokine production via myd88/irak/nf-κb axis signalling cascade. 8, 301 another study clearly demonstrated the function of tlr4 and its direct interaction with hsp70. 303 after ligand binding, tlrs dimerize and undergo a conformational change required for recruiting downstream signaling molecules, including the adaptor molecule myeloid differentiation primary-response protein 88 (myd88), il-1r-associated kinases (iraks), tgfβ-activated kinase (tak1), tak1-binding protein 1 (tab1), tab2 and tnf-receptor-associated factor 6 (traf6). [304] [305] [306] [307] many other innate immunity-related signaling pathways would then be activated, for example, phosphorylation of nf-κb via tak1/ikk activation. mapks p38, jnk, and erk pathway are also activated, then subsequently activate creb and ap-1 transcription factors. both ap-1 and nf-κb activate proinflammatory cytokine expression, including tnfα, il-6, il-1β, and a number of other cytokines and chemokines. 308-311 the effects of hsp60s on host cells upon rna virus infection immunity modulation. few studies show the function of hsp60s in the life cycle of rna virus; however, the function of hsp60 in regulating host immunity has been widely studied. [312] [313] [314] [315] [316] the majority of studies show that hsp60 works as an activator of immune response. in the case of jev infection, hsp60 facilitates virus-induced inflammation by promoting il-1β production via increasing nlrp3 inflammasome activity and nfκb phosphorylation. 317 however, under certain conditions, viruses also utilize hsp60 to evade host cell immune response. the genome-wide rna interference (rnai) screen identifies the interaction of hsp60 with pb2 of influenza virus. 318 hsp60 helps pb2 translocation from the cytosol into mitochondria. 38, 39 then the mitochondrial pb2 interacts with and modulates the activity of mitochondrial antiviral signaling protein (mavs) to suppress interferon β (ifnβ) production. 38 therefore, hsp60 determines the effect of pb2 on both mitochondrial stability and the level of ifn-β production. 38, 39 besides, denv infection also elevates hsp60 expression. silencing of hsp60 results in an increase of ifn-α production and decrease of virus reproduction in macrophages. 319 however, the detailed mechanism remains elusive. apoptosis regulation. viruses use different mechanisms to modulate apoptosis. in the case of hcv infection, ros production is regarded as the major contributor of hcc although it also promotes cell apoptosis. 320 study shows that the n-terminal domain of hcv core protein can induce ros production by interacting with hsp60 and inhibiting the normal function of hsp60 in releasing protein stress. 321, 322 while another rna virus, rotavirus sa11, tries its best to delay the early apoptosis through modulating hsp60 stability. 323 hsp60 helps to translocate nsp4 protein of rotavirus into mitochondria from cytosol and induces apoptosis. 323 to yield more viruses, rotavirus infection increases the hsp60 phosphorylation at tyr 228 by activated src kinase that leads to the ubiquitin-mediated proteasomal degradation. 323 even though virus postpones apoptosis via hsp60, the main function of hsp60 is to refold proteins in mitochondria. 323, 324 the function of hsp60 family in dna virus infection virus replication. in the previous sections, we have discussed that hsp90 is important for rt-ε rna complex formation in hbv infection. however, it has been shown that hsp60 directly regulates rt activity before the rt-ε rna complex formation. 325, 326 hbv replication is markedly suppressed when hsp60 is knocked down by specific sirnas. 325 hsp60 transiently interacts with rt to activate rt in an atp-dependent manner. 326 upon rt activation, hsp60 immediately dissociates from the hsp60/rt complex without being encapsidated into viral nucleocapsid. 326 more detailed research shows that at least one of two rt fragments, residues 1-199 of terminal protein (tp) domain and 680-842 of rnase h (rh), is necessary for hsp60 binding. 327 the tp domain is also responsible for the binding of hsp90 in the rt-ε rna priming step. 138 apoptosis regulation. similar to hcv infection, hbv infection also induces strong apoptosis which is thought mainly contributed by hbx protein. 328 studies show that overexpression of hsp60 facilitates hbx-induced apoptosis. 329, 330 the interaction of hbx and hsp60 has been observed and confirmed by different methods including affinity purification, mass spectrometry and co-immunoprecipitation. 329 hsp60 binds on a small domain (residues 88-117) of hbx. 329 furthermore, hsp60 also forms a complex with hbx and hsp70 in the mitochondria. 330 however, the mechanism of how hsp60 enhances the apoptosis remains unclear during hbv infections. immunity modulation. hsp60 is involved in both the innate and adaptive immune response. 314 here, we focus on how hbv harnesses hsp60 to evade host immune response. numerous studies show that hbv employs active means to escape innate immune response and induce immunosuppression. 331 among these strategies, hbv infection increases the population of cd4 + cd25 + t regulatory cells (tregs) which can produce an amount of il-10 and tgf-β. 332 il-10 is also called cytokine synthesis inhibitory factor (csif) displaying anti-inflammatory properties. 333 it influences both the first and the second line of immune defence. 334 hbv infection increases serum shsp60 level and makes use of hsp60 to activate cd4 + cd25 + regulatory t via tlr2/myd88/il10 signaling. 335 the function of hsp60 family in retrovirus infection although the role of hsp60 in dna/rna virus infection process has been widely studied; only limited knowledge has been obtained in retrovirus infection. hsp60 is encapsidated into hiv particles, 336 but we have no idea about its function. the integrase catalyzes the integration of hiv-1 pro-viral dna in the host genome. 337 a small portion of hsp60 colocalizes and interacts with the viral integrase (in). 338 hsp60-hsp10 complex maintains the integrase at an active form and stimulates its activity in an atp-dependent manner. 338 the integration is a critical step for successful infection of hiv-1. the function of hsp40 family in rna virus infection virus replication. hsp40s regulate rna viral replication by modulating polymerase activity, replication complex and nuclear transportation. in jev-infected cells, hsp40/dnaj homolog hdj2 interacts and colocalizes with ns5 protein, an rdrp essential for viral rna genome replication. 339 overexpressed hdj2 promotes jev replication significantly. however, how hdj2 modulates ns5 activity remains elusive. 339 influenza virus also takes advantage of hsp40 to promote replication by assisting vrc to relocate into nucleus. the replication of influenza virus occurs in the nucleus of host cells; therefore, nuclear trafficking of viral ribonucleoprotein (vrnp) complex is required. the vrnp is composed of viral rna (vrna), polymerase heterotrimer (pa, pb1, pb2) and nucleoprotein (np). 340 np has a key function of interacting with importins through its nuclear localization signals. 341, 342 hsp40s have two strategies to help vrnp transport into nucleus. first, hsp40/ dnajb1 interacts with np at the early stage of infection and ensures efficient association between np and importin alpha. 343 this interaction is mediated by the j domain of hsp40 and the nterminal region of np. 343 another strategy is that hsp40/dnaja1 binds with the pb2 and pa polymerase subunits, then cotranslocates into nucleus with pb1-pa complex. 344 besides, hsp40/dnaja1 enhances viral rna synthesis both in vivo and in vitro. 344 different from dnajb1, dnaja1 mainly depends on its c-terminal substrate-binding domain instead of typical j domain to manage viral rna synthesis. 344 in the replication process, hsp70 is reported to enhance polymerase nuclear translocation. 217 thus it is proposed that dnaja1 cooperates with hsp70 and assist rna polymerase nuclear import. 217, 343 virus gene expression. viral rna replication produces plenty of double-stranded rna (dsrna) molecules. host cell detects these dsrna and activates interferon-induced protein kinase (pkr) to restrict viral replication by phosphorylating eukaryotic initiation factor eif2α and preventing protein synthesis. 220, 221, 345 however, in order to escape from the antiviral response of host cells, the influenza virus smartly blocks the activation of pkr/eif-2α. influenza virus ns1 directly binds the n-terminal of pkr and inhibits pkr activation. 225 besides, np protein also interacts with hsp40, leading to dissociation of hsp40 and p58ipk. 346 it is reported that type iii hsp40/p58ipk is an inhibitor of pkr, [222] [223] [224] 226, 347 while hsp40 is the inhibitor of p58ipk. 225, 348 therefore, the dissociation of hsp40 results in activation of p58ipk. subsequently, p58ipk inhibits the activity of pkr/eif-2α. as a result, influenza virus releases the inhibition of protein synthesis. however, in the late stage of infection, influenza virus m2, hsp40 and p58ipk form a stable complex that would lead to pkr activation, er-stress-induced cell death and virion release. 349 protein maturation. flavivirus genome encodes a large polyprotein which is later cleaved into several mature structures and nonstructure proteins. the mature proteins then form vrcs. at the beginning, hsp40 family protein dnajc14 participates in the vrc formation of flavivirus. during yellow fever virus (yfv) infection, dnajc14 is recruited to non-structural protein clustering sites with ns3 and ns5 to form vrc. 350 however, either knockdown or overexpression of dnajc14 inhibits yfv and hcv replication. 350, 351 later, it has been demonstrated that dnajc14 overexpression affects yfv polyprotein processing and alters vrc assembly. overexpression of dnajc14 alters the cleavage sites of ns3/4a and ns4a/2k and gives rise to abnormal ns3 to ns3-4a ratios, suggesting that the chaperone activity of dnajc14 modulates ns3/ 4a/2k cleavage that ensures appropriate expression level of ns3 and ns4a. the inhibition of vrc formation upon ectopic expressing dnajc14 is caused by chaperone dysregulation. 351, 352 immunity modulation. hsp40s sometimes act to help the virus evade host immunity, while sometimes it exhibits antiviral activity by increasing host immune response under certain conditions. it is reported that the expression of dnajb1/hsp40 and hsp70 is induced by polyi:c stimulation. 353 hsp40 cooperates with hsp70 to suppress the mda5/mavs pathway though interacting with mda5 and inhibiting mda5 multimer formation. 353 however, dnaja3 shows its ability to suppress virus replication. during hfdv infection, vp1 is able to suppress the type i interferon signaling via suppression of phosphorylation, dimerization, and nuclear translocation of irf3. 354 however, dnaja3 induces lysosomal degradation of vp1 protein. therefore, dnaja3 indirectly stimulates the immune response of host cells. 354 the function of hsp40 family in dna virus infection virus replication. evidence suggests that hsp40s regulate the initiation of dna virus replication. in the case of hpv replication, it starts with the recognition of protein e2 on the origin (ori) sequence and recruitments of replication initiator e1, which displays atpase and helicase activities. [355] [356] [357] hsp40 (hdj1 and hdj2) and hsp70 enhance e1 binding on the ori independently and additively. hsp40 directly binds with e1 and remains in the e1ori complex, whereas hsp70 transiently interacts with e1 in an atp-dependent manner. 355 subsequent study reveals an additional role of hdj2 in facilitating e1 helicase function by replacing e2 in the e1/e2/ori complex. 358 the stable association of e2 to ori flanking the e1 binding site may act as a dna clamp to prevent dna unwinding. similarly, hsp40 (htid1) also has similar functions to hdj1 and hdj2 with independent chaperone activity. hsp40 interacts with hsv-1 replication initiator protein and helicase protein ul9, thereby promotes their binding to the replication origin. 359, 360 this provides another example of the involvement of j-proteins in the replication process of eukaryotic dna viruses. except for enhancing hbv replication, a possible negative role of hsp40 has also been reported. the core protein is a key component of viral capsid and essential for virion assembly, while hbx is a multifunctional virulence factor implicated in viral replication and hepatocarcinogenesis in human. a yeast 2-hybrid approach is used to identify interactions between the core protein and two hsp40s, hdj1 and htid1. 361, 362 individual expression of each hsp40 in hepatocytes transfected with a replicationcompetent hbv construct shows an inhibitory effect on both viral replication and capsid formation. further studies reveal that both core and hbx proteins are destabilized by co-expression with hdj1 or htid1; because they are targeted for enhanced proteolytic degradation. except inhibiting hbv replication, hdj1 also facilitates proteasome-mediated degradation of hbx. 361 virus induced cellular transformation. as mentioned above, some viruses can induce cell transformation and tumorigenesis. here, we discuss the role of hsp40 proteins on virus-induced cellular transformation. hsp40 may have a negative role in hbv-induced cell transformation. it has been demonstrated that hbx is the major factor for induction of hepatocyte transformation. [363] [364] [365] [366] nevertheless, overexpression of hsp40s (hdj1 and htid1) significantly enhance the proteasome-mediated degradation of hbx. another study suggests that htid1 interacts with e7 oncoprotein and promotes the cellular transformation by hpv16; 275 because the binding sequence of e7 shares high similarity to the tag oncoproteins of sv40. nuclear entry of viral pre-integration complex. hiv can efficiently infect nondividing cells. 367 this requires active transport of the viral pre-integration complex (pic) into the nucleus without breaking down nuclear membrane. some components of pic implicated in regulating nuclear import include the central dna flap and viral proteins in, ma, and vpr of hiv-1 (or vpx of hiv-2). 288,368-372 hsp40/dnajb6 interacts and enhances the nuclear localization of vpx as well as promotes the nuclear import of viral pic. 373 similarly, dnajb6 also promotes vpr nuclear localization during hiv-1 infection; 374, 375 particularly, the long isoform of dnajb6 is extremely important in this process. 375 the expression level of dnajb6 s/l isoform is regulated by the polyadenylation factor cstf64. high level of cstf64 favors dnajb6-s isoform production, whereas a low level of cstf64 enhances dnajb6-l isoform production. 374 regulation of viral gene expression. a notable example of hsp40 involvement in regulating retrovirus gene expression is its importance in enhancing the gene expression during hiv-1 infection. 376 nef protein, an important viral protein associated with pathogenesis and disease progression, stimulates hsp40 expression by enhancing hsp40 promoter activity via hsf1 transcription factor. [377] [378] [379] hsp40 and nef co-translocate into nucleus where they become a part of cdk9-associated transcription complex to enhance long terminal repeat (ltr) mediated gene expression. 376, 380 the binding of hsf1 on hiv-1 ltr promoter induces viral gene expression directly. 380 interestingly, hsp70 seems to act contrary to hsp40, which also presents in the nef-hsp40 complex. different from hsp40, hsp70 suppresses viral replication and gene expression; while hsp40 rescues the hsp70downreguled viral gene expression. hsp70 inhibits cdk9 phosphorylation, an essential event for high-affinity binding of hiv-1 transactivator of transcription-positive transcription elongation factor b complex for transactivating response rna. 381 it is also reported that some other hsp40 family proteins negatively regulate hiv replication. hsp40a1, b1, b6 and c5 (but not c3) are able to limit hiv-1 production, while they have no effect on viral gene expression upon infection by adenovirus, hsv-1 or vaccinia virus. the conserved dnaj domain is suggested to be responsible for the inhibiting hiv-1 reproduction. the hsp70/ hsp40 complex specifically recognizes and inhibits the rev translation or accelerates its degradation, leading to inhibiting viral gene expression. 382 small heat shock proteins are the most upregulated proteins identified in host cells under stress conditions, for example, when cells are exposed to elevated ros level, abnormally high temperature, or pathogen invasions. 383 in most cases, shsps are responsible for recognizing misfolded proteins and transferring them to other atp-dependent chaperones for proper folding, or proteasomes or autophagosomes for degradation. 49, 384 hsp27 is one of the most ubiquitously expressed shsps with the highest level in skeletal, smooth, and cardiac muscles. 59, 385 like all other shsps, hsp27 shares a highly conserved α-crystallin domain, or socalled c-terminal domain. it contains 6-8 β-strands, forming 2 β-sheets as intermolecular interaction sites. 41, 59 because of the importance of hsp27, in this section, we mainly focus on the function of hsp27 in virus infection. hsp27 has been shown particular importance in viral infections. rajaiya et al. suggested that the association of hsp27 with p38 or nfκb/p65 plays key roles in controlling the expression of proinflammatory mediators in virus-infected cells. 64 fukagawa et al. suggested that the phosphorylated hsp27 is upregulated through the pi3k/akt pathway upon ebv infection. 386 we would discuss the special roles of hsp27 in different viral infections in the following sections. the function of hsp27 in rna virus infection hsp27 is upregulated during ev-a71 infection by proteomics analysis. knockout of hsp27 results in the suppression of virus replication and protein expression level, while their restoration appears after hsp27 is restored. also, hsp27 enhances viral ires activity by promoting 2a pro -mediated eif4g cleavage. 57 interestingly, the nuclear translocation of hsp27 from cytosol is reversely correlated with the relocalization of rna chaperone hnrnpa1 from nucleus to the cytosol for initiating viral protein translation upon ev-a71 infection. however, knockout of hsp27 blocks hnrnpa1 cytosol relocolization, indicating a fundamental role of hsp27 in regulating the import/export of nuclear proteins during virus infection (fig. 6) . 383 hsp27 is rapid upregulated at the early stage (4 hours post infection) of coronavirus infection, 387 suggesting an important role in virus early replication and possibly a good target for treating sars-cov-2 infection. swine fever virus (csfv) is a member of the family flaviviridae. hsp27 is found to bind with ns5a, which is a non-structural protein in response to viral replication and assembly. hsp27 depletion enhances the virus replication while the replication is suppressed by ectopic expression of hsp27 through activating nf-κb signaling in pk-15 cells. 388 porcine epidemic diarrhoea virus (pedv) infection causes high fatality in swine. the hsp27 is obviously upregulated in pedv infected marc-145 cells. 389 however, the virus titer is declined by ectopic expression of hsp27. hsp27 could activate nf-κb signalling and enhance the transcription of ifnβ and downstream interferon stimulated genes (isgs). 389 the function of hsp27 in dna virus infection pro-inflammatory cytokines play crucial roles in early antiviral infections. adenovirus infection causes a wide variety of diseases. 390 the internalization of adenovirus leads to the activation of erk1/2, which could in turn trans-activate nf-κb and ap-1 to induce pro-inflammatory cytokine il-8 expression in different experimental systems. [391] [392] [393] a study shows that downregulation of hsp27 leads to an increased release of il-8 from keratinocytes stimulated with uv or tnfα. the increase of il-8 is suppressed by nf-κb inhibitor and correlated with an enhanced iκb-α degradation and phosphorylated iκb-α accumulation in hsp27-depleted cells. in addition, hsp27 is shown to be associated with the iκb kinase (ikk) complex. because the synthesis of prostanoid pge2 and il-8 is regulated by nf-κb, it could be a likely mechanism of hsp27 in modulating inflammatory cytokine production. 70 further studies also show that hsp27 is of particular importance in the cyclooxygenase-2 and il-6 expression via activating p38 mapk/mk2 signalling and the consequential stabilization of cyclooxygenase-2 and il-6 mrnas. 68 aside from its involvement in pro-inflammatory response regulation, researches have also found the antioxidant role of hsp27 in regulating stress caused by ros. 49 it is commonly agreed that hsp27 regulates enzyme activities by upholding glutathione in reduced form, such as glutathione reductase and glucose-6phosphate dehydrogenase. 394 more recent evidence correlates the level of shsps with the intracellular content of iron, a catalyzer of hydroxyl radical generation. hsp27 also exhibits an important role in oxidized protein degradation machinery. 395, 396 however, there is also controversial evidence indicating the involvement of hsp27 in accumulation of oxidized proteins that benefits herpesvirus replication. experimental models are set up using two distantly related herpesviruses rhesus rhadinovirus (rrv) and hsv-1. they are close relative of kaposi's sarcoma-associated herpesvirus (kshv). the oxidized proteins are accumulated during these viral infections. results show the removal of only a part of oxidized proteins in a proteasome-dependent manner, while some others resisting degradation. 397 oxidized proteins resisting proteolysis become sequestered in foci within the nucleus and coincided with hsp27-enriched foci; although they are not associated with virus-induced chaperone enriched domains (vice). furthermore, the accumulation of oxidized proteins is more pronounced in hsp27-depleted cells. 398 one possible explanation is that hsp27 buffers the toxic effects of those defective proteins undergoing proteolysis through aggregation in the nucleus. the roles of hsp27 are most likely not mutually exclusive during virus infection. hsp27 also contributes to virus replication. porcine circovirus type 2 (pcv2) is a single-stranded dna virus that causes the postweaning multisystemic wasting syndrome (pmws) in pigs. the phosphorylated hsp27 is upregulated in the nucleus in pcv2infected pk-15 cells. hsp27 inhibitors such as sb203580 suppress pcv2 replication. the same result appears upon hsp27 knockdown. in contrast, ectopic expression of hsp27 promotes viral replication. 399 moreover, the phosphorylation of hsp27 is also upregulated in ebv-positive cells, as well as the phosphorylated (activated) akt levels. when ebv-positive cells are treated with pi3k inhibitors, the phosphorylated hsp27 level is decreased, suggesting that the phosphorylation of hsp27 is upregulated through the pi3k/akt signalling pathway upon ebv infection. 386 however, studies also show that hsp27 can both positively and negatively regulate the virus replication depending on the virus/ cell types. tong et al. reported that hsp27 works as an antiviral protein against hbv replication through enhancing ifn production in hepatocytes. the hsp27 expression level is increased in both hbv-infected human liver tissues and hbv-producing hepg2.2.15 cells. 400 the function of pdis on virus entry and uncoating the entry of some viruses into eukaryotic cells is governed by redox-regulated processes. one example is newcastle disease virus (ndv), a bird virus in the family of paramyxoviruses. this negative-sense, single-stranded rna paramyxovirus gains entry to its host cell through large conformational changes in its fusogenic f-protein, which involves thiol/disulfide exchange. 401 overexpression of pdi and erdj5 (a pdi family reductase with an extra j domain) leads to an increase of viral membrane fusion, indicating a route whereby virus can take advantage of the pdi family to gain access to host cells. 402 fig. 6 the controversial function of hsp27 during virus infection. hsp27 would enhance 2a protein-mediated eif4g cleavage, which would in turn promote ires function and viral genome replication. it correlates with hnrnpa1 translocation from host cell nuclei to cytosol, and the consequential virus protein translation. hsp27 is also able to suppress apoptosis via caspase 3 inhibition. on the other hand, hsp27 could help activating ikk complex, modulating nf-κb and ap-1 to induce the expression of pro-inflammatory cytokines reduces β1 and β3 integrins allowing denv entry. 403, 404 also, thiol blockers and pdi inhibitors decrease the entry of rotavirus in ma104 cells, indicating the involvement of thiols for infectivity. 405 the entry of hiv is regulated by pdis. the envelope of hiv becomes unhinged by pdi for entry. ryser et al. firstly reported that cleavage of two disulfide bonds in the gp120 surface component of the hiv-1 envelope is required for virion entry into cd4 + cells. in this process, the pdi on cell surface is responsible for this effect. 406 pdi inhibitors sufficiently prevent the reduction and block the cleavage of surface-bound disulfide conjugate, thereby prevent infection at the level of hiv-1 entry. 407 now, there are numerous studies on how envelope binds on host cells. results from different groups have demonstrated that gp120 moves laterally along the membrane surface until it collides with a patch of pdi in a domain of the membrane that distinguishes from a typical lipid raft. pdi reduces two disulfide bonds in gp120, producing conformation changes that likely stabilize the binding of gp120 to cd4 and expose the v3 loop for subsequent binding to the chemokine coreceptor. following this, gp41 undergoes rearrangement into its fusogenic intermediates and entry occurs. 408, 409 during the entry, galectin-9 binds pdi to regulate the redox environment on the cell surface and enhance hiv entry. 410 taken together, the reports from these three groups have profound implications for our understanding of the hiv virion surface structure and viral entry. the other examples are the nonenveloped polyomavirus (py). py particles contain a layer of coat protein vp1. this single protein, arranged as 72 pentamers, forms the shell surrounding the viral genome. 411 after internalization, py penetrates the er membrane to gain access to the cytosol and then the nucleus for viral genome transcription and replication. pdis isomerize or reduce the virus disulfide bonds to generate a membrane transportcompetent intermediate for host cell membrane penetration. 412 for example, sv40 entry involves caveolar/lipid raft-mediated endocytosis, vesicular transport to er and translocation into the cytosol. erp57 isomerizes the interchain disulfides connecting vp1 for virus uncoating. 413 the further study demonstrates that erp57 and pdi operate in concert with erp29 to unfold the vp1 c-terminal arm. pdi and erp72 reduce py, while erp57 principally isomerizes the virus in vitro. mutagenesis study subsequently identified that the residues c 11 and c 15 of vp1 are important for infection, suggesting a role for these residues during isomerization. 414 pdis regulate viral protein translation a little evidence shows that pdis are involved in viral protein translation. some positive single-stranded rna (+ssrna) virus depends on ires-mediated translation for viral protein synthesis. ev-a71 infection is potently inhibited by an active compound oblongifolin m (om), which is isolated from herb garcinia oblongifolia. further studies show that om suppresses the viral ires-mediated translation of polypeptide via suppressing erp57, and ectopic expression of erp57 increases the ires activity and partially rescues the decreased viral replication caused by om treatment. 415 the detailed mechanism how erp57 downregulates ires activity would be further investigated. several studies have demonstrated the implication of redox balance disruption in the establishment of viral infection and the progression of virus-induced diseases. and accumulated ros in turn may modulate the viral replication and cellular response that also contribute to viral pathogenesis. 416, 417 virus-induced oxidative stress has been reported during hiv, 418 influenza virus, 419 hbv, 420 hcv, 421 encephalomyocarditis virus (emcv), 422 respiratory syncytial virus (rsv), 423 and jev 424 infections. considering its thioredoxin-like sites, erp57 has been thought a main player in the mechanisms of cell protection against oxidative stress, regardless of its subcellular location. redox proteomics analysis of hpv positive tissues shows that the expression level of erp57 and gst is positively correlated with tissue redox status, suggesting its potential association with viral-induced oxidative dna and protein damages. 425 er stress is another consequence caused by virus activities. viral infection would lead to exploitation of the er membrane, accumulation of misfolded proteins, and imbalance of calcium concentration. influenza a virus (iav), hbv, jev, denv, and zika virus all hijack host cell process to enhance viral pathogenesis, such as facilitating viral folding and trafficking, affecting receptor interaction, and modulating host immune responses. [426] [427] [428] therefore, as a major factor in er stress response, erp57 would be critical for viral protein glycosylation and maturation, which may in turn affect virus release and infection (fig. 7) . the life cycle of most rna viruses is completed in the cytoplasm of host cells. to complete the life cycle, virus is often able to induce redistribution of many host cell proteins; particularly, those proteins involved in rna metabolism and functional regulation of viral rnas. hnrnps, such as hnrnp a1, 429 hnrnp c1/c2, 430 hnrnp d 431 and hnrnp i, 432 are mainly resident in the nuclear but quite often shuttle to cytoplasm for stabilizing the viral rna and initiating cap-independent translation. viruses employ various means to redistribute hnrnps. for instance, the ebov inhibits the nuclear import of hnrnp c1/c2. vp24 binds to npi-1 subfamily karyopherin-alpha nuclear import proteins at the c-terminal region (amino acids and prevents their interaction with tyrosine-phosphorylated stat1 (pstat1) and hnrnp c1/c2. the inhibition results in cytoplasm retention of pstat1 and hnrnp c1/c2. 430 435 however, the detailed mechanism of how 2a pro and core protein induce the redistribution remains unknown. virus replication. it has been well documented that hnrnps greatly contribute to the virus replication. for example, hnrnp i/ ptb and hnrnp c participate replication of different viruses, e.g., coronavirus, 436 hcv 437 and poliovirus. 438 the role of hnrnp i/ptbs in viral rna synthesis seems to vary among different viruses. ptbs bind ucuaa pentanucleotide repeats at ires and 3′-utr. ptbs modulate coronavirus rna synthesis. 436 however, the function of hnrnp i/ptb is to some degree complicated in hcv rna replication. it selectively interacts with the 3′ end of the hcv genomic rna. the upstream sl2 and sl3 stem-loop structures are essential for hnrnp-i/ptb i binding whereas the most 3′-terminal stem-loop sl1 is not needed. hnrnp i/ptb i and hnrnp c specifically bind the pyrimidine-rich region of in the 3′ntr of hcv rna genome. 437 possibly, hnrnp i/ptb i is recruited for helping to initiate viral negative-strand (−) rna genome replication, to stabilize rna genome, and/or to regulate the encapsidation of genomic rna. 439, 440 up to date, the detailed function of hnrnp i/ptb i binding on 3′utr has not been elucidated. some studies showed that ptbs have an inhibitory effect on the synthesis of hcv rna genome, 441 while aizaki et al. reported that ptbs are required for efficient hcv rna replication. 442 it has been postulated that they suppress the initiation of viral genome rna replication but enhance ires-mediated translation or facilitate the replication-translation switch. it is shown that hur can compete hnrnp i/ptb i binding on 3′ utr of the viral rna to facilitate la binding to the 3′ utr; while la protein is critical for hcv genome replication. [443] [444] [445] although hnrnp c has many similarities with hnrnp i and both bind with the 3′utr of the hcv rna genome to facilitate viral rna replication; 437 more details demonstrate that only hnrnp c binds the 3′-ends of viral rnas with both negative and positive polarities. 446 besides, hnrnp c also binds at the 5′-end on negative-strand rna of poliovirus to facilitate the synthesis of positive-strand rna; 438 while mir-555 decreases the expression of hnrnp c thereby inhibits poliovirus replication. 447, 448 virus rna splicing. the main function of hnrnps is modulating rna process and metabolism, including splicing, stabilization and transportation. it has been documented that hnrnp k helps the rna splicing of iav. iav is a major human pathogen with a genome comprised of eight negative-stranded rna segments. two viral rna segments, ns1 and m, undergo alternative splicing and yield several proteins including ns1, ns2, m1, and m2 proteins. two of the influenza virus rna segments generate spliced products: ns segment codes for non-structural protein ns1 and nuclear export protein nep/ns2; m segment encodes the matrix protein (m1) and ion channel (m2). ns1-bp properly splices the viral m1 mrna segment to yield the m2 mrna without affecting the splicing of m4 mrna or ns mrna segments. in this process, hnrnp k works as a mediator to bridge the interaction of ns1-bp (binding protein) and m mrna. lack of neither ns1-bp nor hnrnp k ensures the proper splicing of m mrna. 449, 450 further studies show that ns1-bp and hnrnp k bind m mrna downstream of the m2 5′ splice site (5′ ss). ns1-bp binds most proximal to the 5′ss, partially overlapping the u1 snrnp binding site, while hnrnp k binds further downstream and promotes u1 snrnp recruitment. mutation of either or both the hnrnp k and ns1-bp-binding sites results in m segment mis-splicing and attenuated iav replication. 451 virus translation. virus rna translation often harnesses host cellular proteins. most hnrnps positively regulate virus translation. positive-sense single-stranded rna viruses normally contain an ires sequence that drives viral protein translation independence of the cellular cap-dependent translation. hnrnps bind with the ires of different viruses to assist their translation. during hcv virus infection, hnrnp i, hnrnp l, hnrnp d, [452] [453] [454] hnrnp a1 and hnrnp k 455 all participate the translation of hcv viral protein by binding with 5′utr sequence. hnrnp i (ptb3), is one of the polypyrimidine tract-binding proteins (ptbs) that has been reported to bind viral rna recurrently. they could bind the ires of picornaviruses, including cardio-aphthovirus group (including emcv, ev-a71) and entero-rhinovirus group (poliovirus and hrv-2). 89, 456 ptbs also bind hcv utr regions, 441 calicivirus rna 457 and coronavirus rnas. 436 for picornavirus and hcv, it is proposed that ptbs could help ires folding into a translation-competent structure. 458 although unlike canonical transient interactions shared by rna chaperones, it is not clear whether ptbs are eliminated after the ires has folded properly. 459 ptb binds the 5' utr of hcv rna genome to initiate translation. in translation assays, ptb antibody efficiently blocks the ires-mediated translation in vitro. 460 three rrm motifs of ptb monomer directly bind with ires of fmdv to stabilize ires structure and enhance eif4g entry to ires. 459 hnrnp l is capable of binding the 3′ border of the ires. [461] [462] [463] hnrnp l binds with single stranded-hcv rna when preannealing singlestranded rna with mir-122. 463 hnrnp d, also referred to as aurich element rna-binding protein 1 (auf1), shuttles between the cytosol and nucleus. it is found that hnrnp d could also interact with the stem-loop ii of hcv 5′ utr, and its overexpression enhances hcv ires-dependent translation. 452 pim1 inhibitors induce hnrnp d relocalization from nucleus to cytosol so that it binds the ires and inhibit ev-a71 protein translation. 122 similarly, hnrnp k interacts with sl1 located in the 5′ -utr of hcv genome where is bound with mir-122 binding site. 464, 465 mir-122 is required for hcv replication. it binds at a conserved sequence in the 5′ -utr and increases the stability of hcv rna. [466] [467] [468] it is also reported that residues 25-91, a hydrophilic region near the n terminus of hcv core protein, binds to proline-rich domains of hnrnp k and negatively regulates the effect on human thymidine kinase transcription. 469 however, its function on viral replication is not well addressed. while hnrnp a1 binds with both 5′-and 3′-utr of hcv rna, they form a complex with ns5b and septin 6 to assist viral protein translation. the c-terminal of hnrnp a1 and n-terminal of septin-6 are required in the translation process. since hnrnp a1 has many homologous such as hnrnp a/b, hnrnp a2/b1, and hnrnp a2; all of them may substitute for hnrnp a1 in regulating ires-mediated translation. 470 the enteroviruses' ires sequence also interacts with hnrnps. hnrnp a1 specifically binds on the 5′-utr of ev-a71 and enhances ires-dependent translation. 471 apigenin, a dietary flavonoid, interacts with hnrnps and interferes with their rna editing activity. 472 the binding of hnrnp a1 with viral rna is significantly blocked when the cells are infected with ev-a71 upon treating with apigenin, leading to marked suppression of iresmediated translation. it is noted that hnrnp a1 redistribution is not affected in this experiment. recent studies show that hnrnp a1 cytoplasmic translocation is strictly regulated by some signalling or stress proteins, such as mink/p38 mapk pathway 473 and hsp27. 57 p38 inhibitors or hsp27 knockout dramatically block hnrnp a1 translocating from nucleus into cytosol, leading to inhibition of virus replication. 57, 473 except for promoting viral protein translation by binding with the viral ires sequence, hnrnps can directly bind with virus proteins to facilitate virus replication. upon cvb3 infection, 3c pro binds and cleaves hnrnp m to facilitate virus replication. 474 however, hnrnp m does not affect ires activity and rna stability. 475 hnrnp k is required for denv replication. 476 the core protein of dnev interacts with hnrnp k to release the inhibitory effects of hnrnp k on transcriptional activator c/ebpb. 477, 478 other studies demonstrate that hnrnp c1/c2 interact with viral rna and ns1 protein of dnev and facilitate virus reproduction. [479] [480] [481] [482] hnrnp d binds on both 5′-and 3′-utr of enteroviruses and inhibits translation without affecting rna decay. 434 on the other hand, virus also applies strategy to release the inhibitory effects of hnrnp d via protease 3cd cleavage. 434 the cytoplasm translocation of hnrnp d is dependent on the expression of 2a pro and viral rna replication. 433, 434 hnrnp d also inhibits nucleocapsid expression by interacting with an au-rich sequence (nt 41 to 60) in the 3′ utr of niv. 483 virus rna export. hnrnp a2/b1 interacts with ns1 of influenza virus, leading to decrease of the viral ns1 rna/protein levels as well as ns1 rna nuclear export. 484 rna polyadenylation. the gag gene of rous sarcoma virus contains a cis-acting negative regulator of splicing (nrs) element. the general function of nrs is usually manifested by binding serine/arginine-rich (sr) protein hnrnp h and u1/u11 snrnps, resulting in inhibition of splicing by acting as a decoy 5′ splice site. evidenced by in vitro polyadenylation analysis, a new function of nrs element is revealed that it is required for 3′ ltr polyadenylation. in this process, hnrnp h binds on nrs element and promotes polyadenylation; however, mutation of the binding sites of u1 and u11 snrnps to the nrs does not affect polyadenylation. 485, 486 an opposite result shows that polyadenylation stimulatory activity of nrs is dependent of u1 and/or u11 snrnp as well as sr proteins; while hnrnp h seems not participating in the splicing control or rous sarcoma virus polyadenylation. 487 the functions of rna chaperones in regulating viral activities of dna viruses virus replication. the transient lytic dna replication of hcmv relies on the cis-acting element orilyt, six viral-encoded core proteins, the proposed dna replication initiator protein ul84, ie2, irs1 and the gene products from the ul112/113 loci. here it is reported that hnrnp k is sufficient to interact with ul84 protein of hcmv thereby promotes viral replication. the interaction is further enhanced by another two virus proteins ul44 and ie2. 488 gene expression. hnrnps regulate dna virus gene expression at both transcriptional and post-transcription levels either positively or negatively. at the transcription level, hnrnp d0b and hnrnp a/b are capable of binding with cis-acting aagtatgca core element of hpv18 late promoter to suppress the late genes l1 and l2, which are components of virus capsid proteins. 425, 489 hnrnp k binds enhancer ii (enii) to promote hbv replication. ectopic expression of hnrnp k augments hbv replication; while hnrnp k knockdown significantly decreases hbv viral load. 490 further study reveals that apobec3b forms a super complex with hnrnp k that alters the enii binding activity via conformational change, therefore suppresses the s gene promoter activity. 491 in the course of hsv infection, the immediate early protein ie63 (icp27), hnrnp k and casein kinase 2 (ck2) together form a big complex. ck2 is capable of phosphorylating hnrnp k and icp27. the phosphorylated icp27 is responsible for its nucleocytoplasmic translocation and interaction with hnrnp k. up to date, the function of the complex formation is not well elucidated. 492, 493 it may affect icp27 to recruit the cellular rna polymerase ii for the transcription of certain late genes. [494] [495] [496] at the post-transcription level, hnrnps regulate the polyadenylation, splicing, and translation during dna virus infections. hpv genome can be divided into an early region and a late region, followed by the proximal early (pae) and the distal late (pal) polyadenylation signals, respectively. 497, 498 the virion production mainly depends on the differentiation-dependent induction of l1 and l2 late genes. it has been shown that hnrnp h downregulates the late gene l2 at an early stage by interacting with the multiple ggg motifs located 174 nucleotides downstream of the early polyadenylation signal pae. the hnrnp h binding promotes polyadenylation at early polyadenylation signal and inhibits the l2 mrna production, since l2 mrna production need read-through into the late region and polyadenylation of the late transcripts at the pal. 499 while at the late infection stage, hnrnp h is captured by l1 to release the inhibitory effect on l2. 499, 500 this process is called late gene autoregulation which enables rapid viral capsid protein production. 500 another example for hnrnps modulating the polyadenylation is that sm polymerase (pol) mrna of ebv early protein is cleaved and polyadenylated inefficiently. 501 under certain conditions, ebv early protein sm may harness hnrnp a1 and hnrnp c to help the processing of polymerase mrna. viral rna splicing is also modulated by hnrnps. hnrnp a1 negatively regulates the expression of hpv late genes by affecting rna splicing. it directly binds with the late regulatory element (lre) in differentiated hpv16-infected cells. 502 hnrnp a1 binds with splicing silencer element to suppress the use of the 3′ splice site located immediately upstream of aug at late 1 (l1) mrna. hnrnp a1 inhibits the splicing of late mrnas through the splicing silencer sequence and prevents the premature expression of l1 gene. [503] [504] [505] on the other hand, there is another 17 nucleotides resident immediately downstream of the splice site which counteracts the effect of hnrnp a1-binding splicing silencers. 506 hnrnp i also interferes with the splicing inhibitory elements locating at the upstream and downstream of major late 5′ splice site sd3632, thereby activates the late gene expressions. 507 at the translation level, hnrnp i and hnrnp k inhibit the translation of hpv late gene l2 via binding on a specific cis-acting element in the 3′ end of l2 mrna; 508 whereas the inhibition of l2 translation can be disrupted by c-src-mediated phosphorylation of hnrnp k at multiple tyrosine residues. 509 cellular transformation. hnrnps also exhibit some other functions during virus infections. auf1 works as a major component of c promoter binding factor 2 (cbf2) to interact with ebna2 and mediate ebna2 targeting to the latency c promoter (cp) of ebv, thereby inducing b-cell immortalization and viral latency in humans. 510 auf1 also binds with the eber1 noncoding rna of ebv. in ebv-positive cells, eber1 is abundant; therefore it may compete with auf1-interacting targets in the host cells. 511 both ebna2 and eber1 are proposed to facilitate cell transformation. immunity modulation. aside from affecting virus life cycle directly, hnrnps are able to regulate viral activities through modulating immune response. during hsv-1 infection, hnrnp a/b form a complex with viral dna, followed by homodimerization and demethylation. these events result in translocating the complex to cytosol and activating natural immunity through type i interferon signalling. moreover, the complex promotes n6methyladenosine modification and cgas-sting-related mrnas translocation upon infection by dna viruses, further enhancing the immune response for virus elimination. 512 the function of rna chaperones in regulating the viral activities of retroviruses as we have mentioned above, hnrnp a1 can bind to rna/proteins and participate intracellular nucleo-cytoplasmic transportation, 513 as well as alternative splicing of mrna in eukaryotes. during retrovirus infection, hnrnps participate in viral rna transcription, 514 splicing, 515-517 translocation 516 and translation. transcription. in hiv-infected cells, viral nef protein is required for high-level viral replication. 518 it is reported that nef, eed, kinase lck and npkc subfamily (pkcδ/θ) form a complex nakc (nef-associated kinase complex) responsible for promoting viral replication. 519 later on, it is found that hnrnp k is also a partner of the complex. it bridges the interaction of nef, eed and the kinases. the hnrnp k-nucleated complex activates erk1/2 and results in suppressing hiv promoter, enabling suboptimal amounts of tat and transcription factors (e.g., nf-kb) for initiation of transcription. 514 post-transcription. hiv-1 takes advantage of alternative splicing to generate doses of messenger rnas to encode the various viral proteins. it is known that over 40 message rnas are created by alternative splicing from a single pre-mrna. 520,521 alteration of splicing patterns dramatically affects the infectivity and pathogenesis of hiv-1. 521, 522 the splicing of hiv mrna is mainly controlled at the early stages of spliceosome assembly on pre-mrna by a stepwise association of the small nuclear ribonucleoprotein particles (snrnps) u1, u2, and u5·u4/u6. the early steps include the recognition of the 5′ splice site and the branch point sequence by u1 and u2 snrnps, respectively. 523 rs splicing factors, which contain a domain rich in arginines and serines (rs domain), assist these steps. u2af, one of the splicing factors which consists of two subunits u2af65 and u2af35, interacts with the polypyrimidine tract and the 3′ splice site, respectively. 524 then the interaction mediates the association of u2 snrnp with the branch point sequence. [525] [526] [527] sr splicing factors are essential for virtually every step of spliceosome assembly, including early recognition of splice sites, recruitment of basic splicing factors to the pre-mrna, and formation of bridging contacts with other rs domain-containing splicing factors. 528 prior to forming spliceosomes, the pre-mrna is packed with hnrnps. 529 it has been documented that pre-mrnas bind with different subsets of hnrnps, indicating sequence specificity at some degrees. a direct role of hnrnps in constitutive splicing has not been observed; although it seems that the binding of hnrnps exhibits an unspecific nature. it is widely believed that hnrnps employ crucial functions of modelling pre-mrna structure and initiating recognition of splice sites. 530 the cis-acting sequence elements of cellular and viral pre-mrnas undergoing alternative splicing regulate the process either positively or negatively. they bind trans-splicing factor machinery together and form splicing complex. the majority of trans-acting factors are either hnrnps or sr family proteins. these proteins also regulate alternative rna splicing either positively or negatively. sr proteins often regulate splicing in a positive way while some hnrnps mainly do the job in a negative way. 528, 530 alternative incomplete splicing of hiv-1 genomic mrna produces more than 40 unique viral mrna species within an hiv-1-infected cell through highly accurate regulation by cis exon splicing silence elements (ess) and trans hnrnps. the production of tat, rev and vpr proteins is highly controlled because they play key roles in hiv-1 multiplication. ess2 is the first identified ess in the hiv-1 genome that locates downstream of 3′ ss a3 within exon 4 and specifically represses tat mrna splicing. 531 ess2 is mapped to a 10 nt core sequence cuagacuaga. 532 ess3, which locates at exon 7, represses splicing at 3′ ss a7. 533 fine structure mutagenesis indicated that ess3 is bipartite; and each sub-elements [agaucc (ess3a) and uuag (ess3b)] inhibits splicing independently. 534 a third ess (auaguuaguccuagg, essv), which locates downstream of 3' ss a2 in exon 3 within the vif coding sequence, represses splicing at 3′ ss a2. 535 to modulate the expression of tat protein, several hnrnps participate the splicing process by interacting with these ess, intron splicing silencer (iss) elements directly or interfering enhancer splicing element (ese) activity. for example, hnrnp a1 and hnrnp k synergistically bind on ess2 and inhibit the utilization of a3 splicing site. 536, 537 the uag triplets in ess2 is required for hnrnp a1 binding, and several hnrnp a1-binding sites are also found at sls3. the c-terminal gly domain of hnrnp a1 is important for the binding. sr proteins sc35 and srp40, the strong activators of site a3, have similar binding sites to hnrnp a1. hence, they may compete with each other to bind ess2 and ese2. 536 another study shows that hnrnp h displays an inhibitory effect on splicing. hnrnp h binds to ess2 and competes with u2af35 for binding to the exon sequence flanking 3′-splice site a3. this binding results in the inhibition of splicing at the 3′-splice site a3. 538 hnrnp a1 also inhibit tat splicing by binding with iss, which is independent of exon splicing silencer (ess2) and blocks the entry of u2 snrnp but not u2af65. [539] [540] [541] the up1 domain of hnrnp a1 is responsible for the iss binding, while the rgg domain of hnrnp a1 is not needed for the alternative splicing activity. 542, 543 in addition to ess2 and iss elements that are regulated by hnrnp a1, blocking the binding of sr protein sc35 on ese is another strategy for the virus to inhibit the tat expression at the early infection stage. hnrnp a/b proteins bind the ese locating at tat exon 2 to repress splicing, whereas sc35 binds the ese to activate splicing. the binding sites of hnrnp a1 and sc35 are overlapping within the juxtaposed ese/ess9. it seems that hnrnp a1 inhibits the splicing of the upstream intron by binding to the ess and directly masking the sc35 binding site. 544, 545 similarly, hnrnp a1 is also reported to compete with asf/sf2 at ese3/(gaa)3. therefore, the ratio of asf/sf2 to hnrnp a1 determines the utilization of ese3/(gaa)3 for activation or repression at site a7. 515, 541, 546 the expression of other hiv proteins is also regulated by hnrnps via modulating splicing. hnrnp a/b proteins inhibit the splicing at 3′ splice site a2 which is used to generate vpr mrna. hnrnp a/b proteins bind with essv with a consensus sequence pyuag. the splicing at splice site a2 is increased when the three pyuag motifs within essv are mutated, leading to increased vpr mrna levels and reduced skipping of the noncoding exon flanked by a2 and d3. 547 others reported that hnrnp d also binds at essv and functions as an inhibitor of splicing. 548 the binding of hnrnp a/b proteins at essv blocks u2af65 binding to the ppt of the repressed 3′ splice site and inhibits the splicing efficiency of 3′ splice site. 549 interestingly, hnrnp h is found to positively regulate the exon 6d splicing of hiv mrna. it interacts with the sequence cgga and enables u1 snrnp assembly onto exon 6d. 550, 551 further study shows that hnrnp h family proteins function as a splicing enhancer through enhancing the atp-dependent spliceosomal complex formation. 552 rna translocation. after transcription and splicing, lots of spliced and unspliced rna molecules of hiv exist in the nucleus. translocation of rna into the cytoplasm is dependent on the rev protein. on one hand, some hnrnps facilitate rna translocation. in hiv-1 genome, two sequences similar to the hnrnp a2 response element (a2re) function as cis-acting rna trafficking sequence that binds to the trans-acting trafficking factor hnrnp a2. the binding mediates a specific rna trafficking pathway characterized extensively in oligodendrocytes. a2re-1 locates within the major homology region of gag gene; while a2re-2 locates at a region overlapped between vpr and tat coding sequence. hnrnp a2 binds to both a2res in vitro to form a complex, which is necessary for rna transportation in oligodendrocytes in vivo. a2re-mediated rna transport requires both microtubule and hnrnp a2. if gag and vpr rnas containing a2re-1 and a2re-2 respectively are differentially labelled, it is observed that they are co-assembled into the same rna trafficking granules and cotransported to the periphery of the cell. although the tat rna contains a2re-2, it is not transported as efficiently as vpr rna. [553] [554] [555] hnrnp d also has a similar function in assisting rna translocation. 556 on the other hand, hnrnp a2b1, hnrnp c and hnrnp u can retain the hiv-1 genomic rna in nucleus. depletion of hnrnp a2b1 results in cytoplasmic redistribution of the virus rna genome in the absence of rev. 557, 558 an n-terminal fragment of the hnrnp u specifically targets the 3′ long terminal repeat (3′ltr) and blocks the cytoplasmic accumulation of mrnas, thereby affecting hiv gene expression. 559 besides hiv, the regulatory protein orf57 of kaposi's sarcomaassociated herpesvirus (kshv) also interacts with hnrnp k. ck2 can phosphorylate orf57 and promote orf57-hnrnp k interaction. the orf57-hnrnpk-ck2 complex may be important for rna export of kshv since orf57 is responsible for the nuclear export of viral mrnas. 560,561 hnrnp a1 interferes with the binding of rex to rexresponse element (xre). the rex protein of htlv-1 mediates the nuclear export of unspliced and incompletely spliced viral mrnas. this process partly depends on the binding of rex to rex-regulatory sequences xre. 562 viral protein translation. hnrnp a1 is induced to redistribute into the cytoplasm in the late infection stage of hiv and enhances the ires-mediated translation. 563 hnrnp d helps the redistribution of hiv mrna into the cytoplasm, and p45 and p42 isoforms increase viral gag protein synthesis while p40 and p37 suppressed this process. 556 hnrnp i interacts with a novel ires within a latently expressed gene (vcyclin) of kshv and enhances vflip expression. 564 the network of hsps and their co-chaperones is essential for cells to maintain protein homeostasis, including nascent protein folding, protein translocation across membranes, and protein complex formation. many stress stimuli can disrupt protein homeostasis such as thermal stress, nutrient starvation, chemical toxicity, oxidative stress, hypoxia, inflammation, and virus infection. [565] [566] [567] [568] stresses can lead to protein misfolding and aggregation that need to be resolved quickly to prevent cell and tissue damage. 568 hsps and their partners may facilitate protein degradation when cells cannot cope with massive damaged proteins. many lines of evidence suggest hsps as major factors for protein surveillance to protect host cells against virus infections. it was observed that hsp70/hsp90 expression increased dramatically in hsv-infected cells. 569 overexpression of hsp70 inhibits the translocation of viral capsid into nucleoli during flavivirus infections. although the interaction has not yet been investigated in natural infection, the in vitro findings have suggested that hsp70 may act as a negative regulator of viral capsid protein to protect host cells against wnv infection by abolishing cytotoxic effects induced by the viral capsid. 570 studies from ours and other groups show that almost all hsp subfamily members (hsp90s, hsc70, hsp70, grp78, erp57, hsp27) are highly responsible to enterovirus infection and play key roles in all stages of virus life cycle. not surprisingly, demonstrated that all most all hsps (hsp90s, hsp70s, hsp60s, hsp40s and hsp27) participate coronavirus infection, 145, 196, 387, 571 suggesting good target for anti-covid-19 drug development. as discussed in above sections, hsps exhibit immunomodulatory effects on both innate and adaptive immune responses. [572] [573] [574] hsps are capable of regulating not only intracellular innate immunity, but extracellular innate and adaptive immunity as well. hsps released from tumor cells can bind to surface receptors on antigen presenting cells (apcs) and elicit tumor-specific killers by means of antigen crosspresentation. 575, 576 for example, hsp27 positively modulates nf-κb phosphorylation, increases ifnβ transcription and downstream antiviral interferon-induced genes (isgs). pedv infection downregulates hsp27 expression to escape host antiviral surveillance. 389 additionally, extracellular hsps can also regulate cytokine production by dendritic cells (dcs), underscoring a connection between innate and adaptive immune responses modulated by hsps. 577 the purified or recombinant hsp70 can stimulate the production of pro-inflammatory cytokines and c-c chemokines in monocytes, macrophages and dcs, and upregulate mhc and costimulatory molecules in dcs. 578 binding of extracellular hsp72 to human monocytes and dendritic cells can induce the production of the pro-inflammatory cytokines including tnf-α, il-1β, il-6 and il-12 and ifn-γ. 579 in the process of above hspinduced cytokine production, hsps act as internal stimulus of the cd14/tlr (tlr2 and tlr4) complex signal transduction pathways that further activates nf-κb and mapks signalling. 580, 581 although the main functions of hsps are to protect cells upon stresses, they are often hijacked by many viruses to achieve successful infections. recent study has demonstrated that denv ns3 protein acts as a viral suppressor of rna silencing by interacting with hsc70, thereby interrupting host antiviral system by rnai pathway and subsequently enhancing denv replication. 582 ev-a71 takes advantage of hsps (hsp90, hsc70, erp57, and hsp27) to enhance 2a pro -mediated eif4g cleavage that is favour viral protein translation and blocks host protein translation. 57, 219, 415 viruses also initiate er stress in host cells after infection. they have to manipulate upr activation leading to cell survival, rather than inflammation induction, autophagy and apoptosis. denv modulates upr activation in a sequential manner to prolong the viral life cycle by allowing cellular adaptation to cope with the infection-induced er stress. upr is transiently induced by perk pathway resulting in phosphorylation of eif2α and subsequently translational attenuation in the early phase of infection. this transient event allows viral protein synthesis and accumulation that finally trigger upr by ire1-xbp1 (x-box binding protein 1) axis in the mid-phase of infection. this results in the increased expression of grp78 to facilitate protein folding and also the increased expression of gadd34 (growth arrest and dna damage 34), which dephosphorylates eif2ɑ, and thus allowing protein translation to be continued. the increased grp78 also prevents cellular apoptosis-mediated by chop (pro-apoptotic protein during stress persistence). finally, the increased viral proteins transiently trigger atf6 arm of upr to provide the active spliced xbp1 for sustaining upr activation in the late phase. 583 jev infection requires hsp70s in particular stages of its life cycle for survival and establishment of infection, including viral entry, replication, 194 and maturation. 195 cell-surface hsp70 directly interacts with jev envelope protein. 192 antibodies against hsp70 or 90 significantly block virus entry. 111 it is also observed that hsp70 colocalizes and directly interacts with jev replicase complex. in addition, hsp70 also interacts with ns3, ns5 and viral dsrna that stabilizes vrc formation during jev infection. 194, 584 er stress and antiviral in mammalian cells, the er stress is sensed and mediated by three er transmembrane receptors: pancreatic er kinase (pkr)-like er kinase (perk), inositol-requiring enzyme 1 (ire1) and activating transcription factor 6 (atf6). in resting cells, these three sensors are maintained in inactive states via interactions with the er resident chaperone grp78. when unfolded or misfolded proteins accumulate in the er lumen, grp78 dissociates from these three transducers, resulting in activation and initiation of the upr. viruses also take advantage of or revolt er stress response by different means for favour its life cycle at specific stage(s) of infection. the er stress response constitutes a cellular process triggered by a variety of conditions disturbing protein folding in the er. eukaryotic cells have developed an evolutionarily conserved adaptive mechanism, i.e., the er stress and upr, aiming to clear unfolded/misfolded, and excessive amount of protein production; thereby restoring er homeostasis. in cases er stress cannot be resolved, upr would be triggered and lead to cell death. er stress and upr could be observed in a large amount of virus infections, as most of viruses require host er machinery for viral protein synthesis and modification. virus infection usually causes er stress. when large amount of misfolded/unfolded proteins or excessively expressed viral proteins accumulate inside er lumen, er stress response is triggered as indicated by fast elevated expression of er chaperone proteins, including grp78/ bip, grp94, calnexin and calreticulin. it is well documented that er stress response is triggered by a number in some cases, er stress plays a role in virus pathogenesis. for instance, jev, bvdv, tulv, severe acute respiratory syndrome coronavirus (sars-cov) and wnv have all been shown to induce apoptosis through upr. [585] [586] [587] [588] [589] oxidative stress is mediated by er stress during hcv infection. 21 certain viruses modulate er stress response or preferentially activate different pathways of upr. hcv infection activates the atf6 pathway while blocks the ire1 pathway. instead, hbv infection stimulates both atf6 and ire1 signalling but has no effects on perk signalling although both virus infect the same kind of hepatocytes. 590-592 hsv-1 develops a virulence factor enabling dephosphorylation of eif2α, one of the downstream effectors of the perk pathway. 593 some consequences of upr are beneficial for viral life cycle. for example, atf6-induced expression of chaperone proteins may help viral protein folding and prevent protein aggregation. the perk-eif2α-activated atf4 may help re-establish cell metabolism and resume protein translation. the ire1-xbp1 pathway may facilitate virus replication by enhancing er protein-folding ability and er membrane biosynthesis. 594 the activated atf6 promotes the replication of asfv and lymphocytic choromeningitis virus (lcmv). 595,596 denv envelope protein directly interacts with grp78, which provides a scaffold for its association with two other chaperones calnexin and calreticulin. this complex significantly improves the proper folding and stability of denv e protein, thereby leading to increase of virus production. 597 other than denv, grp78 is also demonstrated to promote hcmv, jev and rgnnv infections. 195, 598, 599 the ire1-xbp1 pathway is activated by iav to facilitate its replication. 600 however, other upr outcomes are detrimental for virus replication. the perk-eif2α-mediated global translation attenuation is known as an antiviral response to restrict viral replication, such as infection by denv or wnv. 583, 601 the activated pkr phosphorylates eif2α at the ribosomal interface, which in turn causes a general inhibition of protein synthesis and blockage of vsv replication. 602 the ire1-xbp1(s)-mediated er-associated protein degradation (erad) pathway reduces intracellular hbv particles by degrading its envelope proteins. 603 another approach of er stress contributing to virus pathogenesis goes through modulating host cell immune responses. vsv, hcv and sars-cov are able to inhibit the type i ifn signaling pathway by activating the perk signalling, which leads to the phosphorylation-dependent ubiquitination and subsequent degradation of ifnar1, thereby promoting immune evasion and virus pathogenesis. 604, 605 wnv has also been reported to induce er stress and inhibit type i ifn signaling pathway for escaping from the host immune response. 601 us11 protein of hcmv activates upr to facilitate the degradation of class i major histocompatibility complex (mhc1), leading to immune evasion. 606 moreover, er stress is responsible for viral pathogenesis by interconnecting with the inflammatory responses. for example, hcv induces inflammatory responses by activating ire1, which interacts with traf2 to phosphorylate jnk, leading to activation of inflammation mediators. 607 ns4b and ns5a proteins of hcv activate nf-κb via er stress-elicited calcium depletion and ros production. 604 the x protein (hbx) of hbv induces the expression of cyclo-oxygenase 2 (cos2), a key mediator of inflammation, through perk-eif2α-activated atf4. 608 rna chaperons and human diseases caused by viral infections hnrnps impact mrna metabolism including transcript synthesis, processing and degradation as well as translation. 432 the function of hnrnps is determined or modulated by cellular localization. the mechanisms that regulate the nucleo-cytoplasmic shuttling are, therefore, of extreme importance. most hnrnps possess a conventional nuclear localization signal (nls) and are predominantly present in the nucleus during steady state. they are able to translocate in the cytosol upon post-translational stimulation or by the recruitment of other hnrnps. 609 post-translational modifications like methylation, phosphorylation, ubiquitination and sumoylation are reported to affect biological activity and subcellular localization of hnrnps. 610 as stated in the above sections, virus always employs different strategies to redistribute hnrnps in host cells. 430, 431, 433, 434 how dysregulation of hnrnps causing human diseases has gained an increasing interest. the expression level of hnrnps is altered in many types of diseases, including varieties of human cancers and neurodegenerative diseases (e.g., spinal muscular atrophy (sma), amyotrophic lateral sclerosis (als), alzheimer's disease (ad), and fronto-temporal lobe dementia). in this section, we mainly focus on the relationship between hnrnps and diseases caused by virus infections. enteroviruses are common pathogens that cause human diseases worldwide. although most enteroviral infections are subclinical, they may cause a wide spectrum of diseases including mild upper respiratory illness (common cold), febrile rash (hand, foot, and mouth disease and herpangina), aseptic meningitis, heart failture, pleurodynia, encephalitis, acute flaccid paralysis (paralytic poliomyelitis) and neonatal sepsis-like disease. 293, 611 besides, several studies showed that enterovirus sequences could be detected in neuronal cell bodies of the spinal cord of 60-88% amyotrophic lateral sclerosis (als) patients, suggesting that persistent enterovirus infection may have a relationship with als. 295, 297, 299 enterovirus infections are supposed to cause or increase the risk of als; because the replication and translation of poliovirus, ev-a71, and coxsackievirus redistribute numerous hnrnps (such as hnrnp a1) into the cytoplasm, the same localization during als pathogenesis. for example, hnrnp k, hnrnp a1, hnrnp m and hnrnp d shuttle into cytoplasm to assist virus replication or translation. 300, 455, 472, [612] [613] [614] dislocated hnrnp a1 and loss of splicing function have been regarded as a toxic mechanism in als pathogenesis. 301 hsv infection is one of the most common causes of infectious disease in humans. 302 hsv infection often causes watery blisters in the skin or mucous membranes of the mouth, lips, nose, or genitals. 615 as neurotropic and neuroinvasive viruses, hsv-1 and -2 persist in the infected individuals through hiding in the neuron cells from immune surveillance. when the carrier's immunity compromised, hsv is reactivated that causes new sores. more seriously, accumulating evidence shows that hsv infection is associated with the pathogenesis of alzheimer's disease. 105, 616, 617 during hsv infection, hnrnp k positively promotes hsv replication; 618 while hnrnp a2/b1 negatively regulates hsv replication by triggering immunity response. 512 hbv, hcv, ebv, hpv and hiv are oncogenic viruses that account for over 20% of human cancers. hcv or hbv infection leads to a wide spectrum of liver disease ranging from acute hepatitis (including fulminant hepatic failure) to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (hcc). 619,620 hdv can only infect people who are already infected with hbv. the coinfection of hdv and hbv increases the risk of liver cirrhosis and liver cancer. 621, 622 except for damaging the liver, 10% of hbv infected patients also have other symptoms outside the liver, such as serumsickness-like syndrome, membranous glomerulonephritis, acute necrotizing vasculitis (polyarteritis nodosa) and papular acrodermatitis of childhood (gianotti-crosti syndrome). 623 multiple steps of hcv reproduction need the presence of hnrnps. the rna replication of hcv needs the presence of hnrnp i, hnrnp c; 437, 442, 446 while virus rna translation requires hnrnp a1, hnrnp d, hnrnp i, hnrnp k and hnrnp l. 452, 460, [462] [463] [464] [465] besides, hnrnp a2, hnrnp l, hnrnp u and hnrnp k are highly expressed in hcc tissue. they promote liver cancer progression. 8, 304, 305, 624 ebv has been implicated in several diseases, including infectious mononucleosis, 625 burkitt's lymphoma, 626 hodgkin's lymphoma, 627 nasopharyngeal carcinoma, 628 multiple sclerosis 629 and lymphomatoid granulomatosis. 630 other diseases associated with ebv infections include gianotti-crosti syndrome, erythema multiforme, acute genital ulcers, oral hairy leukoplakia, 631 and disorders related to α-synuclein aggregation (e.g. multiple system atrophy, dementia with lewy bodies, and parkinson's disease). 632 during ebv infection, hnrnp a1 and hnrnp c assist the polyadenylation of ebv rna. hnrnp d takes part in cellular transformation-induced by ebv. 510 hpvs are a group of dna viruses with more than 150 members that infect cutaneous and mucosal epithelia. the acute infection of hpv causes benign cutaneous lesions such as non-genital and genital warts, or flat cervical condylomas. 633 about 15 hpv subtypes that infect genital tract are capable of inducing malignant tumors, most commonly in the cervix. 308,309 these cancer-associated hpvs are grouped into high-risk types, while those not associated with cervical cancer are regarded as low-risk types. 307 most infections by low-risk type hpvs are asymptomatic, except for infection by hpv6 and hpv11 that cause most cases of genital warts (condyloma acuminatum). 309 hpv-related cancers are the result of long-term persistent infection with high-risk types. hpv16 and hpv18 are the most common carcinogenic types which account for a larger proportion of cervical cancers, squamous cell cancers and adenocarcinomas in all regions. 311 hpvs are also implicated in the development of a variable proportion of vulvar, vaginal, penile, and oropharyngeal cancers, anal cancer, 634 head and neck cancers, 635-637 lung cancer and skin cancers. hnrnp a1 is involved in splicing of hpv rna; while hnrnp h plays a role in the polyadenylation of hpv. hnrnp k and hnrnp i are required for viral protein translation. besides acquired immunodeficiency syndrome (aids), hiv infection is also able to cause human cancers because hiv infection impairs immunity system. people with aids are not only more easily infected with bacteria, viruses, fungi, and parasites; 638 but at high risk for developing various viral-induced cancers as well, including burkitt's lymphoma, kaposi's sarcoma, primary central nervous system lymphoma, and cervical cancer. 639 515, [532] [533] [534] [535] [536] [537] [544] [545] [546] [547] [548] [549] [550] [551] [552] [553] [554] [555] [556] [557] [558] [559] 564 potential therapeutic value by targeting stress proteins interest in targeting stress proteins in various diseases is increasing overtime. in this part, we will discuss the potential therapeutic value by targeting stress proteins. first, targeting stress proteins have a wide spectrum of antiviral ability. for example, hsp90 inhibitors, which are originally developed for anticancer, have been demonstrated to possess antiviral activity in cultured cells against poliovirus, rhinovirus, ev-a71, 107,109 coxsackievirus, 124 rsv, 112, 113 vsv, paramyxoviruses (hpiv2, hpiv3, sv5, sv41), influenza virus, 126 chikv, 114 hcv, 115, 124 and hsv, 131, 144, 145, 148, 149 hbv, 135, 169 ebv, 146, 147, 163, 164 hcmv 151, 152 and htlv. 153, 185 depleting hsp60 by sirna functionally suppresses infections by influenza virus, 38,39 denv, 319 hbv, 325, 329, 331 hcv, 321, 322 rotavirus, 323 and hiv. 338 targeting hnrnp a1 is able to inhibit the reproduction of hiv, [539] [540] [541] 563 hpv, 503-505 hcv, 471 ev-a71 472 and htlv-1. 562 inhibition of hnrnp c could be a strategy to combat viral infections by hcv, 438 [461] [462] [463] 475 therefore, stress proteins are particularly attractive as antivirals targets for those lacking therapies and newly emerging viral diseases. second, under disease situations, the cellular need of stress proteins is usually stronger than that in normal conditions which enable specific selectivity of those drugs targeting stress proteins. for example, mutant p53 relies much more on hsp90 function than wild type p53. hsp90 inhibitor gm can easily disturb the association of mutant p53 with hsp90, resulting in mutant p53 degradation while not affecting wild type p53. 310 additionally, stress proteins derived from stressed cells display higher affinity to clients and inhibitors. tumor cell-derived hsp90 exhibits a 100fold higher binding affinity for 17-aag than that from normal cells, since tumor cell-derived hsp90 complexes with activating cochaperones p23 and hop exhibit increased atpase activity and possess higher affinity to hsp90 inhibitors. in contrast, hsp90 in normal cells exists as an uncomplexed species with low atpase activity and low affinity to hsp90 inhibitors. 294 further study demonstrates that the difference is caused by a distinctive portion of hsp90 forming complexes in cancer cells with oncogenic partners, such as bcr-abl-hsp90 complex in mutant b-raf-hsp90 in skmel28 melanoma cells, k562 chronic myeloid leukemia cells, her3-hsp90 and raf1-hsp90 complex in mda-mb-468 breast cancer cells. hsp90 inhibitor pu-h71 selectively binds to specific hsp90-oncoprotein networks in these cancer cells. 570 these characters of cancer cells enable stress proteins to exhibit stronger biological activity and to be discriminated by their inhibition under stress as compared with normal conditions. similarly, inhibitors of stress proteins show high prospect for antiviral. hsp70 inhibitor jc40 inhibits pan-flavivirus (denv2 and denv4) replication in mddcs, with negligible toxicity to host cells. 640 these experiments highlight the feasibility of using hsp inhibitors therapeutically in humans. third, it is not observed drug resistance using hsp inhibitors for antiviral. for instance, hsp90 inhibitors are refractory to develop drug resistance. this is clearly demonstrated in rsv infection. 113 when rsv is repeatedly treated with hsp90 inhibitors, no drug resistance was observed either in extensive passaging of the virus in cultured cells or in mice undergoing long-term treatment with hsp90 inhibitors. similar result is also observed in denv infections. 640 treatment of denv up to 10 passages with hsp70 inhibitor jc40 does not cause any drug resistance. the lack of viral drug resistance to hsp90 and hsp70 inhibitors suggests that such an antiviral approach may be particularly useful for treating chronic viral infections in which drug resistance is most frequently observed. the three features of stress proteins inhibitors make them extremely powerful antiviral agents, suggesting great application potential for treating the diseases caused by virus infections, such as covid-19. challenges and perspectives: target-based drug development targeting hsp90 for antivirus drug development. hsp90 is thought to be the most abundant and evolutionarily conserved heat shock protein. a unique pocket in n-terminal region of hsp90 is required for binding with atp and co-chaperones. 641 hsp90 forms a flexible dimer by interaction of c-terminal domains. the formation and dissociation of compact dimers involving nterminal domains are important for the molecular chaperone activity. 642 the middle domain of hsp90 tends to recruit and facilitate unfolded client proteins to assemble. the c-terminal domain of hsp90 possesses a highly conserved peptide sequence for interacting with co-chaperones. 643 over 20 co-chaperones selectively interact with hsp90 to regulate atpase activity and recruit client proteins to assemble a big complex under certain conditions. [642] [643] [644] [645] it has been shown that more than 300 client proteins require hsp90 and co-chaperones for folding and maturation. 646 the mechanism of selectivity may rely on the direct interaction of co-chaperones with specific clients. therefore, most hsp90 inhibitors achieve their inhibitory effects by suppressing the atpase activity or disrupting the interaction between hsp90 and its co-chaperones. expression of hsp90 and its client proteins are increased during viral infection and in most cancer cells. hsp90 as an effective anticancer drug target has already grabbed attention; and a series of hsp90 inhibitors as potential drugs have been intensively investigated in the laboratories, preclinical and clinical scenarios. the successful use of hsp90 inhibitors in cancer therapy makes it much easier to apply them for treating virus infections. as described above, we have comprehensively summarized the functions of hsp90 and its client proteins in a diversity of virus reproduction processes. the potential of hsp90 inhibitors has been well demonstrated to protect cultured cells against infections by ev-a71, 107,109 poliovirus, rhinovirus, coxsackievirus, 124 paramyxoviruses (hpiv2, hpiv3, sv5, sv41),vsv, rsv, 112,113 influenza virus, 126 chikv, 114 hcv, 115,124 hsv, 131,144,145,148,149 hbv, 135,169 ebv, 146, 147, 163, 164 hcmv, 151, 152 and htlv. 153, 185 notably, administration of hsp90 inhibitors to infected animals exhibits little toxicity while potently suppresses the replication of poliovirus, 107,109 ebv, 163, 164 hbv, 135 chikv 114 and hcv. 647 these experiments highlight the feasibility of using these inhibitors therapeutically in clinic. here we would briefly enumerate the present hsp90 inhibitors and their potential for antiviral therapies. most hsp90 inhibitors hamper hsp90 function by competitively binding to the atp binding site of hsp90, blocking the interaction with co-chaperones, or modulating acetylation. by doing so, we also try to address the possibility of repurposing hsp90 inhibitors as candidate antiviral drugs ( table 2) . inhibitors targeting hsp90 atpase activity. some hsp90 inhibitors competitively bind to the atp pocket in the n-terminus of hsp90, leading to blocking of atp hydrolysis and the closure of nterminus of hsp90 dimer. in addition, another atp binding site has been found in the c-terminus of hsp90. 648 recently, some natural products and their derivatives have been reported to competitively bind to the atp pocket in the c-terminus of hsp90. generally, inhibitors of hsp90 atpase are classified into three types: (i) ansamycins, (ii) non-ansamycins and (iii) those block hsp90 c-terminal atpase activity. ansamycins are antibiotics that possess the benzoquinone as structure core. these antibiotics include geldanamycin (ga), herbimycin a, and the macbecins. they inhibit hsp90 activity and degrade its client proteins. 649 ga competitively binds to the atp pocket in the n-terminus and inhibits the atpase activity. 649 it has shown potent antiviral activity against coronavirus infection in the culture cells, 145 indicating a good potential for treating covid-19. tanespimycin (17-allylamino-17-demethoxygeldanamycin, 17-aag) is an analogue of ga. 650, 651 alvespimycin (17-dimethylaminoethylamino-17demethoxygeldanamycin, 17-dmag) is an analogue of 17-aag, with high solubility in water. in addition, retaspimycin hydrochloride (ipi-504) is a more water-soluble derivative of 17-aag. 652 non-ansamycin inhibitors of hsp90 include luminespib, 653 biib021, 654 ganetespib, 655 onalespib, 656 snx-5422, 657 etc. another type of hsp90 inhibitors binds to the c-terminal atp pocket rather than n-terminal atp pocket. novobiocin blocks the c-terminal atpase activity. 658, 659 in in vitro and in vivo assays, treatment with novobiocin strongly reduces the expression of hsp90-dependent client proteins, such as raf-1 and p60v-src. a natural rotenoid, deguelin, is suggested to bind to the atp pocket in the c-terminus without affecting the atp pocket in the nterminus. 660 treating with deguelin leads to reduced hsp90 clients, such as cdk4, akt and mek1/2. 661 like deguelin, epigallocatechin gallate (ecgc) is reported to bind to the atp pocket in the c-terminus of hsp90 without affecting the nterminal atp pocket. 662 compared with inhibition of the nterminal atp pocket of hsp90, inhibitors targeting the c-terminal atp pocket lead to a stability of hsp70 and a negative instability of client proteins of hsp90. however, the potential of these cterminal inhibitors and their molecular mechanisms remain elusive. the structural features of the c-terminal domain of hsp90 need to be further analysed. such analysis may provide important hints on how to design effective hsp90 inhibitors without hsr induction. inhibitors of hsp90's co-chaperones. as the functions of the hsp90 chaperone machinery are highly dependent on the associated co-chaperones, it is possible to selectively inhibit downstream signaling of hsp90 by disrupting certain protein-protein interactions. inhibitors targeting the interactions may present an alternative approach to prevent the toxicity induced by other hsp90 inhibitors. as a result, great efforts are put into disrupting the interaction between hsp90 and its cochaperones and are rewarded recently in this area. here we briefly present the advance in hsp90-cdc37 complex and hsp90-hop-hsp70 ternary complex. human cdc37 is a well-studied co-chaperone of hsp90. the middle domain and c-terminus of cdc37 are critical for the interaction with hsp90. currently, most of the reported agents for manipulating the hsp90-cdc37 interaction are natural products and their derivatives. these agents include celastrol, aferin a, sulforaphane, kongensin a and platycodin d. they are capable of dissociating the hsp90-cdc37 complex. [663] [664] [665] [666] [667] in addition, a peptide (pep-1, ac-khfgmlrrwdd-nh2) is developed and shows strong inhibitory effects on the formation of hsp90-cdc37 complex. 668 another well-known hsp90/co-chaperone complex is hsp90-hop-hsp70 ternary complex, which facilitates the transfer of unfolded client proteins from hsp70 to hsp90. notably, six active compounds have been identified after screening the ncgc compound library. 669 these compounds possess similar structural cores and have no effect on hsp70 expression. studies on hsp90-co-chaperone inhibitors are limited. great effort should be paid on the efficacy and toxicity for further drug design and clinical studies. hsp90 inhibitors blocking deacetylation of hsp90. the chaperone function of hsp90 is also controlled by posttranslational modification (ptm). the well-studied ptms in hsp90 are acetylation and deacetylation in the m-domain of hsp90. vorinostat (suberoylanilide hydroxamic acid, saha) dissociates her2/erbb2 from hsp90 via acetylation of hsp90 that leads to degradation of her2/erbb2. 670 laq824 induces hsp90 acetylation that reduces hsp90 client protein levels. 671 romidepsin is involved in the dissociation of mutant p53 and raf-1 from hsp90 via acetylation of hsp90. 672 in addition, the nuclear import of iav polymerase needs the deacetylation of hsp90 which is strictly regulated by histone deacetylases 6/8 (hdac6/8). 120 hdac6/8 inhibitors efficiently limit polymerase nuclear import and suppress virus replication. 120 therefore, hsp90 inhibitors that block hsp90 deacetylation would potentially be used to treat influenza a virus infection. their potential usage for combating other viruses (such as sars-cov-2) needs to be extensively explored. novel class of hsp90 inhibitors for hsp90 cleavage. recently, hsp90 cleavage has been observed under various stimuli such as uv irradiation, 673 ascorbate/menadione, 674 hdac inhibitors, 675 proteasome inhibitors 676 etc. therefore, hsp90 cleavage is considered as another mechanism. hsp90 cleavage induced by these inhibitors can be classified into two types: enzymatic cleavage and non-enzymatic cleavage. 674, 676 the enzymatic cleavage produces a 55 kda fragment of hsp90 via caspase 10 activation, while the non-enzymatic cleavage utilizes reactive oxygen species (ros) to chemically degrade hsp90 to an~70 kda fragment. additionally, some substances have been reported to present hsp90 cleavage activity, but the mechanism remains unclear. targeting hsp70s for drug development: one kind of antiviral drugs are currently designed based on different properties of hsps. considering hsp70 and hsc70 are potential targets for hcv, they load the dcs with hsp70 for a prolonged potent antigen-and tumor-specific t cell responses directed against multiple epitopes. 677 hsp70 inhibitors (such as quercetin, ver155008 and jc40) also show great potential for treating flavivirus and enterovirus infections. 202, 208, 640 inhibitors blocking the interaction between grp78 and spike protein of coronavirus would be a useful approach for combating the infection by sars-cov, mers-cov, and sars-cov-2 infections. 196, 387, 571 however, the efficacy and toxicity of these inhibitors are not yet well investigated in clinic studies. targeting hsp60s for drug development: as described above, hsp60s play critical roles in different diseases including autoimmune diseases, human cancers and virus infection-induced diseases. studies show that silencing of hsp60s by sirna significantly decrease influenza virus, 38,39 denv 319 and hbv 325, 331 reproduction by activating immunity response; and is capable of releasing hcv 321,322 , rotavirus, 323 hbv 329 and hiv 338 infectioninduced pathogenesis. therefore, small molecule regents targeting hsp60s are potentially useful as therapeutics in these diseases. 678, 679 currently, the known hsp60 inhibitors are either from natural products or synthetic compounds (table 3) . mechanistically, they can be grouped as two types. type i inhibitors block atp binding and hydrolysis. type ii inhibitors covalently interact with certain cysteine residues of hsp60. however, the detailed binding sites have not been identified. the natural products of hsp60 inhibitors include mizoribine, epolactaene, myrtucommulone a, stephacidin b. mizoribine is an imidazole nucleoside antibiotics isolated from eupenicillium brefeldianum. 680 it directly binds to hsp60 and inhibits the chaperone activity of the hsp60-hsp10 complex. 33 epolactaene is isolated from the fungal strain penicillium sp. bm 1689-p. 681 its derivate tert-butyl ester etb has similar activity as epolactaene. 682 they inhibit hsp60-hsp10's chaperoning activity by covalent and selective bound to hsp60 at residue of cys442, close to the atp binding pocket. 683, 684 interestingly, etb does not inhibit the atpase activity of hsp60, 685 suggesting that the covalent interaction between etb and cys442 may allosterically modulate hsp60-hsp10's chaperoning activity without interfering with its atpase activity. myrtucommulone a (mc) binds to hsp60 and inhibits the refolding activity of the hsp60-hsp10 complex. 686 mc is a non-prenylated acylphloroglucinol with multiple reported bioactivities, including antibacterial, 687,688 antioxidant, 689 antiinflammatory, 690, 691 and anti-tumor properties. 692, 693 in addition to these natural products, several other natural products are also reported to interact with hsp60 without direct proof. stephacidin b is isolated from aspergillus ochraceus wc76466 while avrainvillamide is isolated from aspergillus sp. cnc358. 694, 695 interestingly, dimeric stephacidin b is converted into monomeric avrainvillamide in tissue culture media which implies avrainvillamide is the actual active species. 696 pull down assay only shows hsp60 may be a putative target. 696 further validation studies have yet to be performed. besides the natural products identified above as potential hsp60 inhibitors, quite a few synthetic molecules have also been discovered to be able to modulate hsp60 activity. o-carboranylphenoxyacetanilide is firstly identified as an hif-1α inhibitor. 697 later it is found that o-carboranylphenoxyacetanilide selectively and directly binds to hsp60 and inhibits hsp60-hsp10's atpase activity and refolding activity. 685 hsp60 can interact with hif-1α, suggesting that inhibition of hif-1α activity by carboranylphenoxyacetanilide is possibly through targeting hsp60. 685 the other class of synthetic compounds identified to inhibit hsp60 is gold porphyrins. 698 it directly binds to hsp60 and inhibits the refolding activity of the hsp60-hsp10 complex. although several hsp60 inhibitors potently suppress virus reproduction, a lot of work remains to be done to verify if they can serve as drugs for treating diseases caused by virus infections. targeting hnrnps for antivirus drug development. drugs targeting rna chaperones may have a wide spectrum of antivirus capability. hnrnp a1 participates the reproduction of hiv, [539] [540] [541] 563 hpv, 503-505 hcv, 471 ev-a71 472 and htlv-1. 562 inhibition of hnrnp c suppresses the replication of hcv, 438 514, 536, 537 however, the available regents developed targeting hnrnps as antiviral drugs are limited. to date, only three agents targeting hnrnps have been reported. apigenin is a flavonoid abundant in fruits and vegetables. it targets hnrnp a2 and blocks hnrnp a2 dimerization thereafter affects the alternative splicing of key mrnas. 699 apigenin efficiently inhibits the interaction of hnrnp a1 and a2 with ev-a71 rna thereby inhibits ev-a71 rna translation. 472 quercetin is also a flavonoid abundantly present in plants. it binds to the c-terminal region of hnrnp a1, impairing the ability of hnrnp a1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. 700 in addition, quercetin down-regulates hnrnp a1 expression. 701 the antiviral ability of quercetin has been verified in some viruses such as inhibiting influenza entry, 702 ev-a71, 703 and hcv, 208 jev and denv reproduction. 704 however, in these researches, they explain the antivirus mechanism of quercetin is by inhibiting hsp70 expression 208 or inhibiting virus proteases 703 without mentioning the function of hnrnp a1. another compound vpc-80051 targets rna binding motif of hnrnp a1 and alters hnrnp a1 splicing activity. 705 its antiviral ability remains to be further investigated. targeting er stress pathways for drug development: during the past few years, the enlarging role of er stress-mediated pathways is becoming quite an attractive topic for broad-spectrum antiviral therapy, especially in the field of virus reproduction and pathogenesis, considerable progress has also been made for potential antiviral agents development. among pathways involved in er stress, the three upr pathways are the most thoroughly studied, thus antiviral targets related with these pathways are considered as the first class. extensive investigation for antiviral development has been put int to the perk-eif2α pathway. a small chemical compound named salubrinal, identified by boyce and his colleagues, specifically inhibits the formation of pp1/gadd34 complex as well as suppresses hsv γ134.5-mediated eif2α dephosphorylation. it could also significantly reduce hsv replication. 706 their research suggested that salubrinal was able to attenuate pp1/gadd34-dependent eif2α dephosphorylation, which would in turn inhibit denv replication. 707 a glucose analog 2-deoxy-d-glucose is responsible for the activation and phosphorylation of eif2α, leading to suppression of herpesvirus (kshv) replication. this agent is a potential candidate for anti-herpesvirus, and hopefully is tested in clinical therapies. 708 other than eif2α, tremendous efforts have also been made in developing drugs to target ire1-xbp1 pathway. 3,5-dibromosalicyladehyde is an endoribonuclease that specifically interacts with ire1 and blocks the downstream iav replication. 600 wp1130 is another ire1-xbp1 inhibitor with a broad-spectrum antiviral effects against multiple viruses, namely mnv-1, lcv and so on. 709 er stress-mediated apoptosis signaling is another noteworthy pathway for drug development and selection. rana catesbeiana ribonuclease (rc-rnase) is reported to be efficient in suppression of jev replication via activation of caspase-3, caspase-8 and caspase-9 pathways. 710 dubble-stranded rna activated caspase oligomerizer (draco) is reported to have broad anti-viral effects in clinical therapy. draco selectively activates apoptosis in cells infected with dsdna viruses but has no harmful effect on normal healthy cells. over 15 different kind of viruses can be eradicated by draco, including hiv and dengue virus. 711 drugs targeting other signal transducers are also investigated, for example jnk, jak, bcl-2 and chop. a promising agent vaticanol b can protect host cells from er stress-induced apoptosis. 712, 713 some researchers choose to focus on viral proteins that could trigger er-stress, including non-envelope glycoproteins of sfv, precursor glycoprotein of lcmv, glycoproteins tulv, several nonstructural protein of sars-cov, multiple non-structural proteins of flavivirus family, envelope protein of pedv, e1, e2 and nonstructral protein of hcv, surface protein of mhv, icp0 glycoprotein b of hsv1, structural protein 4 of chikv. viral proteins that are responsible for upr activation and apoptosis induction are given special attentions such as hcv (e1, e2, core), hcmv (pul37x1, pul38) and sv5 v protein. great advances have been made in this area, current progress includes, clemizol for hcv ns4b, vaccine vectors for hsv-1 γ134, and norakin for iav hemagglutinin a. 714, 715 due to their potential side effects on normal cell functions, the usage of inhibitors targeting stress proteins are very cautious in clinic settings; and the gone with wind infection manner of some virus (e.g., sars-cov) limits the opportunity of clinic studies, giving rise to additional challenges for developing stress protein-based drugs against the common and special infectious diseases such as severe acute respiratory symptom (sars) caused by coronavirus. we thank prof. robert weiss at cornell university college of veterinary medicine for critical reading and comments in preparation of this manuscript. the work was partially supported by grants from the science technology and innovation committee of shenzhen municipality [jcyj20170818100531426, jcyj20180507181627057]; grants from national science foundation of china in and out of the er: protein folding, quality control, degradation, and related human diseases a. new puffing pattern induced by temperature shock and dnp in drosophila molecular chaperone functions of heat-shock proteins the growing world of small heat shock proteins: from structure to functions 70 and 90, anti-apoptotic proteins, in clinical cancer therapy molecular chaperones in protein folding and proteostasis the role of heat shock proteins in antigen cross presentation novel signal transduction pathway utilized by extracellular hsp70: role of toll-like receptor (tlr) 2 and tlr4 heat shock protein 70 acts as a potential biomarker for early diagnosis of heart failure hsp27 expression in the human peripheral blood mononuclear cells as an early prognostic biomarker in coronary artery disease patients chaperone machines for protein folding, unfolding and disaggregation the hsp90 family: structure,regulation, function, and implications in health and disease molecular chaperones and protein quality control involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits the hsp90 chaperone machinery heat shock proteins: cellular and molecular mechanisms in the central nervous system structural analysis of substrate binding by the molecular chaperone dnak the human hsp70 family of chaperones: where do we stand? gymnastics of molecular chaperones the kinetic parameters and energy cost of the hsp70 chaperone as a polypeptide unfoldase hepatitis c virus, er stress, and oxidative stress chaperones in hepatitis c virus infection hsp70 chaperone dynamics and molecular mechanism the hsp70 chaperone machinery: j-proteins as drivers of functional specificity two families of chaperonin: physiology and mechanism folding of newly translated proteins in vivo: the role of molecular chaperones mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets group ii chaperonins: new tric(k)s and turns of a protein folding machine structure of holo-chaperonin studied with electron microscopy oligomeric opal0 on top of two layers of cpn60 rings with two stripes each a chaperonin from a thermophilic bacterium, thermus thermophilus. in molecular chaperones review: a structural view of the groe chaperone cycle groel and the groel-groes complex mammalian hsp60 is a major target for an immunosuppressant mizoribine mammalian hsp60 is quickly sorted into the mitochondria under conditions of dehydration mammalian 60-kda stress protein (chaperonin homolog) physicochemical properties of the mammalian molecular chaperone hsp60 down-regulation of hsp60 suppresses the proliferation of glioblastoma cells via the ros/ampk/mtor pathway the pb2 subunit of the influenza virus rna polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon association of the influenza virus rna polymerase subunit pb2 with the host chaperonin cct heat shock protein 60: regulatory role on innate immune cells the small heat shock proteins family: the long forgotten chaperones crystal structure and assembly of a eukaryotic small heat shock protein a first line of stress defense: small heat shock proteins and their function in protein homeostasis the small heat shock protein hsp27: present understanding and future prospects structure and function of small heat shock/α-crystallin proteins: established concepts and emerging ideas mechanism of chaperone function in small heat shock proteins regulation of aging and age-related disease by daf-16 and heatshock factor hsp27 negatively regulates cell death by interacting with cytochrome c some like it hot: the structure and function of small heat-shock proteins small heat shock protein activity is regulated by variable oligomeric substructure small heat shock proteins hsp27 and αb-crystallin: cytoprotective and oncogenic functions heat shock proteins 27 and 70: anti-apoptotic proteins with tumorigenic properties regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27 muscle develops a specific form of small heat shock protein complex composed of mkbp/hspb2 and hspb3 during myogenic differentiation heat shock protein 27 phosphorylation: kinases, phosphatases, functions and pathology heat shock protein 27: its potential role in vascular disease hsp27 responds to and facilitates enterovirus a71 replication by enhancing viral internal ribosome entry site-mediated translation heterooligomeric complexes formed by human small heat shock proteins hspb1 (hsp27) and hspb6 (hsp20) large potentials of small heat shock proteins blocking tumor cell migration and invasion with biphenyl isoxazole derivative kribb3, a synthetic molecule that inhibits hsp27 phosphorylation quantification of hsp27 and hsp70 molecular chaperone activities human adenovirus type 19 infection of corneal cells induces p38 mapk-dependent interleukin-8 expression mapk and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption heat shock protein 27 mediated signaling in viral infection hsp27 expression regulates cck-induced changes of the actin cytoskeleton in cho-cck-a cells increased hsp27 after androgen ablation facilitates androgenindependent progression in prostate cancer via signal transducers and activators of transcription 3-mediated suppression of apoptosis interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of hsp27 heat shock protein 27 functions in inflammatory gene expression and transforming growth factor-beta-activated kinase-1 (tak1)-mediated signaling hsp27 regulates pro-inflammatory mediator release in keratinocytes by modulating nf-κb signaling increased expression of the m (r) 27,000 heat shock protein (hsp27) in in vitro differentiated normal human keratinocytes 28-kda mammalian heat shock protein, a novel substrate of a growth regulatory protease involved in differentiation of human leukemia cells regulation of hsf 1 function in the heat stress response: implications in aging and disease targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis novel aspects of heat shock factors: dna recognition, chromatin modulation and gene expression proteins of the pdi family: unpredicted non-er locations and functions the protein disulfide isomerase family: key players in health and disease erp57 functions as a subunit of specific complexes formed with the er lectins calreticulin and calnexin protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and erp57 rna chaperone stpa loosens interactions of the tertiary structure in the td group i intron in vivo rna chaperones and the rna folding problem probing the folding landscape of the tetrahymena ribozyme: commitment to form the native conformation is late in the folding pathway exposing the kinetic traps in rna folding strategies for rna folding and assembly protein enhancement of hammerhead ribozyme catalysis a. chaperone activity of non-specific rna binding proteins in hammerhead ribozyme catalysis domain analysis of the saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein, nab2p. dissecting the requirements for nab2p-facilitated poly(a) rna export role of rna chaperones in virus replication heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications altered states: selectively drugging the hsp90 cancer chaperone heat shock proteins promote cancer: it's a protection racket heat shock proteins and cancer: intracellular chaperones or extracellular signalling ligands? disruption of rna metabolism in neurological diseases and emerging therapeutic interventions heat shock proteins as potential targets for protective strategies in neurodegeneration severe respiratory viral infections: new evidence and changing paradigms pathogenesis of viral respiratory infection. respiratory disease and infection-a new insight, intechopen lvd viruses causing gastroenteritis: the known, the new and those beyond identification of mw polyomavirus, a novel polyomavirus in human stool discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin a highly prevalent and genetically diversified picornaviridae genus in south asian children immunological features underlying viral hemorrhagic fevers pathogenesis of the viral hemorrhagic fevers the role of viruses in neurodegenerative and neurobehavioral diseases herpes viruses and alzheimer's disease: new evidence in the debate virus infection and human cancer: an overview. recent results cancer res heat shock protein 90: role in enterovirus 71 entry and assembly and potential target for therapy dengue virus entry into liver (hepg2) cells is independent of hsp90 and hsp70 heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells characterization of putative japanese encephalitis virus receptor molecules on microglial cells antiviral activity and rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus chikungunya virus nsp3 & nsp4 interacts with hsp-90 to promote virus replication: hsp-90 inhibitors reduce chikv infection and inflammation in vivo heat-shock protein 90 is essential for stabilization of the hepatitis c virus nonstructural protein ns3 destabilization of pdk1 by hsp90 inactivation suppresses hepatitis c virus replication through inhibition of prk2-mediated viral rna polymerase phosphorylation hepatitis c virus rna replication is regulated by fkbp8 and hsp90 identification of hsp90 as a stimulatory host factor involved in influenza virus rna synthesis hsp90 inhibitors reduce influenza virus replication in cell culture mc1568 inhibits hdac6/8 activity and influenza a virus replication in lung epithelial cells: role of hsp90 acetylation autophagy is involved in regulating influenza a virus rna and protein synthesis associated with both modulation of hsp90 induction and mtor/p70s6k signaling pathway pim1 impacts enterovirus a71 replication and represents a potential target in antiviral therapy host cell factor requirement for hepatitis c virus enzyme maturation evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance molecular chaperone hsp90 is a therapeutic target for noroviruses influenza a virus neuraminidase protein interacts with hsp90, to stabilize itself and enhance cell survival viral transport and the cytoskeleton the hepatitis e virus open reading frame 3 product interacts with microtubules and interferes with their dynamics the ins and outs of tubulin acetylation: more than just a post-translational modification? regulation of microtubule assembly and stability by the transactivator of transcription protein of jembrana disease virus heat-shock protein 90 promotes nuclear transport of herpes simplex virus 1 capsid protein by interacting with acetylated tubulin glucocorticoid stimulates hepatitis b viral gene expression in cultured human hepatoma cells geldanamycin, a heat shock protein 90-binding benzoquinone ansamycin, inhibits steroid-dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus hepatitis b virus replication hsp90 is required for the activity of a hepatitis b virus reverse transcriptase requirement of heat shock protein 90 for human hepatitis b virus reverse transcriptase function in vitro reconstitution of a functional duck hepatitis b virus reverse transcriptase: posttranslational activation by hsp90 hbv polymerase interacts independently with nterminal and c-terminal fragments of hsp90beta chaperone activation of the hepadnaviral reverse transcriptase for template rna binding is established by the hsp70 and stimulated by the hsp90 system efficient hsp90-independent in vitro activation by hsc70 and hsp40 of duck hepatitis b virus reverse transcriptase, an assumed hsp90 client protein hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids hsp90 makes the human hbv pol competent for in vitro priming rather than maintaining the human hbv pol/ pregenomic rna complex expression of stable hepatitis b viral polymerase associated with grp94 in e. coli herpes simplex virus type 1 dna polymerase requires the mammalian chaperone hsp90 for proper localization to the nucleus geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro hsp90 inhibitors: a potential treatment for latent ebv infection? nuclear transport of epstein-barr virus dna polymerase is dependent on the bmrf1 polymerase processivity factor and molecular chaperone hsp90 the herpes simplex virus vp16-induced complex: the makings of a regulatory switch herpes simplex virus disrupts nf-kappab regulation by blocking its recruitment on the ikappabalpha promoter and directing the factor on viral genes heat-shock protein 90α is involved in maintaining the stability of vp16 and vp16-mediated transactivation of α genes from herpes simplex virus-1 geldanamycin, a potent and specific inhibitor of hsp90, inhibits gene expression and replication of human cytomegalovirus curcumin inhibits human cytomegalovirus by downregulating heat shock protein 90 a novel hsp90 inhibitor, 17-dmag, induces tax down-regulation and its oral administration to atl-model mice intervenes against the infiltration property of the atl-like lymphocytes and provides extended survival period processing of the herpes simplex virus regulatory protein alpha 22 mediated by the ul13 protein kinase determines the accumulation of a subset of alpha and gamma mrnas and proteins in infected cells icp22 and the ul13 protein kinase are both required for herpes simplex virus-induced modification of the large subunit of rna polymerase ii epstein-barr virus-encoded protein kinase (bglf4) is involved in production of infectious virus the human cytomegalovirus ul44 protein is a substrate for the ul97 protein kinase distinct and separate roles for herpesvirus-conserved ul97 kinase in cytomegalovirus dna synthesis and encapsidation the human cytomegalovirus ul97 protein kinase, an antiviral drug target, is required at the stage of nuclear egress viral mimicry of cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress conserved herpesvirus kinases target the dna damage response pathway and tip60 histone acetyltransferase to promote virus replication sumo binding by the epstein-barr virus protein kinase bglf4 is crucial for bglf4 function hsp90 inhibitors block outgrowth of ebv-infected malignant cells in vitro and in vivo through an ebna1-dependent mechanism ganetespib, an hsp90 inhibitor, kills epstein-barr virus (ebv)-infected b and t cells and reduces the percentage of ebv-infected cells in the blood ebp2 plays a key role in epstein-barr virus mitotic segregation and is regulated by aurora family kinases epstein-barr virus provides a survival factor to burkitt's lymphomas structure of the p53 binding domain of hausp/usp7 bound to epstein-barr nuclear antigen 1 implications for ebv-mediated immortalization self-inhibition of synthesis and antigen presentation by epstein-barr virus-encoded ebna1 heat shock protein 90 facilitates formation of the hbv capsid via interacting with the hbv core protein dimers reactive oxygen species promote heat shock protein 90-mediated hbv capsid assembly virus particles in cultured lymphoblasts from burkitt's lymphoma epstein-barr virus in the pathogenesis of npc all three domains of the epstein-barr virus-encoded latent membrane protein lmp-1 are required for transformation of rat-1 fibroblasts epstein-barr virus induces invasion and metastasis factors a novel hsp90 inhibitor at13387 induces senescence in ebvpositive nasopharyngeal carcinoma cells and suppresses tumor formation the heat shock protein 90 inhibitor biib021 suppresses the growth of t and natural killer cell lymphomas heat shock protein 90 expression in epstein-barr virus-infected b cells promotes gammadelta t-cell proliferation in vitro tcrgammadelta cells and viruses heat shock protein 90 (hsp90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells efficient cross-presentation by heat shock protein 90-peptide complex-loaded dendritic cells via an endosomal pathway nlr, the nucleotide-binding domain leucine-rich repeat containing gene family the hsp90-sgt1 chaperone complex for nlr immune sensors molecular mechanisms of cellular transformation by htlv-1 tax activation of nf-kappab by htlv-i and implications for cell transformation hsp90 protects the human t-cell leukemia virus type 1 (htlv-1) tax oncoprotein from proteasomal degradation to support nf-κb activation and htlv-1 replication grp78, a coreceptor for coxsackievirus a9, interacts with major histocompatibility complex class i molecules which mediate virus internalization integrin alpha v beta 6 is an rgd-dependent receptor for coxsackievirus a9 heat shock protein 70 as a supplementary receptor facilitates enterovirus 71 infections in vitro surface proteins of c6/36 cells involved in dengue virus 4 binding and entry zika virus dependence on host hsp70 provides a protective strategy against infection and disease heat shock protein 70 on neuro2a cells is a putative receptor for japanese encephalitis virus association of heat-shock protein 70 with lipid rafts is required for japanese encephalitis virus infection in huh7 cells heat shock cognate protein 70 isoform d is required for clathrin-dependent endocytosis of japanese encephalitis virus in c6/36 cells grp78 is an important host factor for japanese encephalitis virus entry and replication in mammalian cells japanese encephalitis virus co-opts the er-stress response protein grp78 for viral infectivity natural products may interfere with sars-cov-2 attachment to the host cell heat shock protein 70 (hsp70) mediates zika virus entry, replication, and egress from host cells heat shock cognate protein 70 is involved in rotavirus cell entry interaction of rotaviruses with hsc70 during cell entry is mediated by vp5 the peptide-binding and atpase domains of recombinant hsc70 are required to interact with rotavirus and reduce its infectivity heat shock protein 70 regulates degradation of the mumps virus phosphoprotein via the ubiquitin-proteasome pathway heat shock protein 90 ensures efficient mumps virus replication by assisting withfang viral polymerase complex formation constitutive overexpression of the major inducible 70 kda heat shock protein mediates large plaque formation by measles virus enhanced production of morbillivirus gene-specific rnas following induction of the cellular stress response in stable persistent infection the highly inducible member of the 70 kda family of heat shock proteins increases canine distemper virus polymerase activity heat shock protein 72 is associated with the hepatitis c virus replicase complex and enhances viral rna replication tylophorine analogs allosterically regulates heat shock cognate protein 70 and inhibits hepatitis c virus replication the heat shock protein inhibitor quercetin attenuates hepatitis c virus production human respiratory syncytial virus n, p and m protein interactions in hek-293t cells evidence for an association between heat shock protein 70 and the respiratory syncytial virus polymerase complex within lipid-raft membranes during virus infection interactome analysis of the human respiratory syncytial virus rna polymerase complex identifies protein chaperones as important cofactors that promote l-protein stability and rna synthesis elucidation of the cellular interactome of ebola virus nucleoprotein and identification of therapeutic targets an rna polymerase ii-driven ebola virus minigenome system as an advanced tool for antiviral drug screening recombinant rna-dependent rna polymerase complex of ebola virus a small stem-loop structure of the ebola virus trailer is essential for replication and interacts with heat-shock protein a8 cellular stress response induces selective intranuclear trafficking and accumulation of morbillivirus major core protein heat shock protein 70 modulates influenza a virus polymerase activity heat shock protein 70 promotes coxsackievirus b3 translation initiation and elongation via akt-mtorc1 pathway depending on activation of p70s6k and cdc2 hsc70 regulates the ires activity and serves as an antiviral target of enterovirus a71 infection the double-stranded rna-dependent protein kinase is also activated by heparin repression of the pkr protein kinase by the hepatitis c virus ns5a protein: a potential mechanism of interferon resistance the 58,000-dalton cellular inhibitor of the interferon-induced double-stranded rna-activated protein kinase (pkr) is a member of the tetratricopeptide repeat family of proteins purification and partial characterization of a cellular inhibitor of the interferon-induced protein kinase of mr 68,000 from influenza virus-infected cells characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsrna-activated protein kinase the molecular chaperone hsp40 regulates the activity of p58ipk, the cellular inhibitor of pkr the cellular inhibitor of the pkr protein kinase, p58(ipk), is an influenza virus-activated co-chaperone that modulates heat shock protein 70 activity c-terminal trimerization, but not n-terminal trimerization, of the reovirus cell attachment protein is a posttranslational and hsp70/atp-dependent process association of heat shock protein 70 with enterovirus capsid precursor p1 in infected human cells heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virus ribonucleoprotein complex heat shock protein 70 inhibits the activity of influenza a virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo polo-like kinase 1 is involved in hepatitis c virus replication by hyperphosphorylating ns5a the ns5a-binding heat shock proteins hsc70 and hsp70 play distinct roles in the hepatitis c viral life cycle structural characterization of the hsp70 interaction domain of the hepatitis c viral protein ns5a sgta-dependent regulation of hsc70 promotes cytosol entry of simian virus 40 from the endoplasmic reticulum a non-enveloped virus hijacks host disaggregation machinery to translocate across the endoplasmic reticulum membrane bip and multiple dnaj molecular chaperones in the endoplasmic reticulum are required for efficient simian virus 40 infection adenovirus capsid proteins interact with hsp70 proteins after penetration in human or rodent cells novel partner proteins of adenovirus penton co-chaperone bag3 and adenovirus penton base protein partnership nuclear import of adenovirus dna in vitro involves the nuclear protein import pathway and hsc70 nuclear sequestration of cellular chaperone and proteasomal machinery during herpes simplex virus type 1 infection transcriptional stimulation by the dna binding protein hap46/bag-1m involves hsp70/hsc70 molecular chaperones transcriptional activation by the human hsp70-associating protein hap50 the hsp70-associating protein hap46 binds to dna and stimulates transcription bag-1, a novel bcl-2-interacting protein, activates expression of human jc virus bag-1m, an isoform of bcl-2-interacting protein bag-1, enhances gene expression driven by cmv promoter chaperone action in the posttranslational topological reorientation of the hepatitis b virus large envelope protein: implications for translocational regulation sequence-specific repression of cotranslational translocation of the hepatitis b virus envelope proteins coincides with binding of heat shock protein hsc70 chaperones involved in hepatitis b virus morphogenesis bag-1m accelerates nucleotide release for human hsc70 and hsp70 and can act concentration-dependent as positive and negative cofactor hsp70 interacting protein hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of bag-1 molecular chaperone grp78/bip interacts with the large surface protein of hepatitis b virus in vitro and in vivo mammalian bip controls posttranslational er translocation of the hepatitis b virus large envelope protein in vivo and in vitro association of hsc70 with polyomavirus capsid proteins chaperone-mediated in vitro assembly of polyomavirus capsids productive infection and cell-free transmission of human t-cell leukemia virus in a nonlymphoid cell line detection of lymphocytes producing a human retrovirus associated with adult t-cell leukemia by syncytia induction assay 71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human t-cell lymphotropic virus type 1 heat shock cognate protein 70 is a cell fusion-enhancing factor but not an entry factor for human t-cell lymphotropic virus type i heat-shock protein 70 can replace viral protein r of hiv-1 during nuclear import of the viral preintegration complex heat-shock proteins reverse the g2 arrest caused by hiv-1 viral protein r heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein r atpgammas disrupts human immunodeficiency virus type 1 virion core integrity the amino-terminal transforming region of simian virus 40 large t and small t antigens functions as a j domain the molecular chaperone activity of simian virus 40 large t antigen is required to disrupt rb-e2f family complexes by an atp-dependent mechanism atp-dependent simian virus 40 tantigen-hsc70 complex formation the regulation of e2f by prb-family proteins mechanism of regulation of hsp70 chaperones by dnaj cochaperones investigation of the interaction between dnak and dnaj by surface plasmon resonance spectroscopy priming polyvalent immunity by dna vaccines expressing chimeric antigens with a stress protein-capturing, viral j-domain loss of p19(arf) eliminates the requirement for the prb-binding motif in simian virus 40 large t antigenmediated transformation novel mechanisms of e2f induction by bk virus large-t antigen: requirement of both the prbbinding and the j domains j domain-independent regulation of the rb family by polyomavirus large t antigen the j domain of simian virus 40 large t antigen is required to functionally inactivate rb family proteins a novel human dnaj protein, htid-1, a homolog of the drosophila tumor suppressor protein tid56, can interact with the human papillomavirus type 16 e7 oncoprotein identification of a novel retinoblastoma gene product binding site on human papillomavirus type 16 e7 protein the human papillomavirus e7 oncoprotein and the cellular transcription factor e2f bind to separate sites on the retinoblastoma tumor suppressor protein differential distribution of the adenovirus e1a proteins and colocalization of e1a with the 70-kilodalton cellular heat shock protein in infected cells to kill or be killed: viral evasion of apoptosis apoptosis in viral pathogenesis viruses and apoptosis epstein-barr virus nuclear antigen (ebna) 3a induces the expression of and interacts with a subset of chaperones and co-chaperones caspase activation is required for permissive replication of aleutian mink disease parvovirus in vitro the mechanisms of direct, virus-induced destruction of neurons the hepatitis c virus core protein interacts with ns5a and activates its caspase-mediated proteolytic cleavage apoptosis in coxsackievirus b3-caused diseases: interaction between the capsid protein vp2 and the proapoptotic protein siva the apoptotic capability of coxsackievirus b3 is influenced by the efficient interaction between the capsid protein vp2 and the proapoptotic host protein siva hiv-1 vpr induces apoptosis through caspase 9 in t cells and peripheral blood mononuclear cells adenovirus encoding hiv-1 vpr activates caspase 9 and induces apoptotic cell death in both p53 positive and negative human tumor cell lines influenza virus ns1 protein induces apoptosis in cultured cells neurovirology and developmental neurobiology programmed cell death in virus infections of the nervous system ts-hsp70 induces protective immunity against trichinella spiralis infection in mouse by activating dendritic cells through tlr2 and tlr4 multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma hsp70 inhibits lipopolysaccharide-induced nf-κb activation by interacting with traf6 and inhibiting its ubiquitination hsp70/dnaja3 chaperone/cochaperone regulates nf-κb activity in immune responses the role of the membrane-initiated heat shock response in cancer enhanced heat shock protein 70 expression alters proteasomal degradation of ikappab kinase in experimental acute respiratory distress syndrome hsp70 is a negative regulator of nlrp3 inflammasome activation functional domains of hsp70 stimulate generation of cytokines and chemokines, maturation of dendritic cells and adjuvanticity hsp70 peptidembearing and peptide-negative preparations act as chaperokines toll-like receptor signalling toll-like receptor 4 (tlr4) is essential for hsp70-like protein 1 (hsp70l1) to activate dendritic cells and induce th1 response toll-like receptor signaling and its inducible proteins inflammation in gastric cancer: interplay of the cox-2/prostaglandin e2 and toll-like receptor/myd88 pathways tak1-ecsit-traf6 complex plays a key role in the tlr4 signal to activate nf-kappab cutting edge: traf6 mediates tlr/il-1r signaling-induced nontranscriptional priming of the nlrp3 inflammasome heme oxygenase-1 regulates dendritic cell function through modulation of p38 mapk-creb/atf1 signaling activated apoptotic cells induce dendritic cell maturation via engagement of toll-like receptor 4 (tlr4), dendritic cell-specific intercellular adhesion molecule 3 (icam-3)-grabbing nonintegrin (dc-sign), and beta2 integrins il-1β induction of muc5ac gene expression is mediated by creb and nf-κb and repressed by dexamethasone induction of proinflammatory response in prostate cancer epithelial cells by activated macrophages autoimmune and inflammatory mechanisms in atherosclerosis heat shock proteins as ligands of toll-like receptors the hsp60 immune system network heat shock protein and innate immunity heat shock proteins: mediators of atherosclerotic development hsp60 critically regulates endogenous il-1β production in activated microglia by stimulating nlrp3 inflammasome pathway genome-wide rnai screen identifies human host factors crucial for influenza virus replication rna interference mediated silencing of hsp60 gene in human monocytic myeloma cell line u937 revealed decreased dengue virus multiplication hepatitis c virus: from oxygen free radicals to hepatocellular carcinoma mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis c virus core protein interaction of hepatitis c virus core protein with hsp60 triggers the production of reactive oxygen species and enhances tnf-alpha-mediated apoptosis tyrosine phosphorylation modulates mitochondrial chaperonin hsp60 and delays rotavirus nsp4-mediated apoptotic signaling in host cells cooperation between heat shock proteins in organizing of proteins spatial structure antisense oligodeoxynucleotides targeted against molecular chaperonin hsp60 block human hepatitis b virus replication human hepatitis b virus polymerase interacts with the molecular chaperonin hsp60 binding site analysis of human hbv pol for molecular chaperonin, hsp60 hepatitis b virus x protein: a multifunctional viral regulator interaction of the hepatitis b virus x protein (hbx) with heat shock protein 60 enhances hbx-mediated apoptosis hbx protein of hepatitis b virus (hbv) can form complex with mitochondrial hsp60 and hsp70 innate immune responses in hepatitis b virus (hbv) infection circulating and liver resident cd4 + cd25 + regulatory t cells actively influence the antiviral immune response and disease progression in patients with hepatitis b interferon effects on interleukin-10 secretion mononuclear cell response to interleukin-10 is normal in multiple sclerosis patients il-10-producing regulatory b cells (b10 cells) in autoimmune disease hepatitis b virus replication could enhance regulatory t cell activity by producing soluble heat shock protein 60 from hepatocytes an hsp60 related protein is associated with purified hiv and siv molecular mechanisms in retrovirus dna integration functional interactions of human immunodeficiency virus type 1 integrase with human and yeast hsp60 dnaj homolog hdj2 facilitates japanese encephalitis virus replication structure of the ribonucleoprotein of influenza virus a classical bipartite nuclear localization signal on thogoto and influenza a virus nucleoproteins an unconventional nls is critical for the nuclear import of the influenza a virus nucleoprotein and ribonucleoprotein human heat shock protein 40 (hsp40/dnajb1) promotes influenza a virus replication by assisting nuclear import of viral ribonucleoproteins dnaja1/hsp40 is co-opted by influenza a virus to enhance its viral rna polymerase activity molecular cloning and characterization of the human doublestranded rna-activated protein kinase induced by interferon influenza a virus nucleoprotein exploits hsp40 to inhibit pkr activation p58ipk, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eif2alpha signaling biochemical and genetic evidence for complex formation between the influenza a virus ns1 protein and the interferon-induced pkr protein kinase interaction of hsp40 with influenza virus m2 protein: implications for pkr signaling pathway flavivirus replication complex assembly revealed by dnajc14 functional mapping identification and characterization of the host protein dnajc14 as a broadly active flavivirus replication modulator chaperone-assisted protein folding is critical for yellow fever virus ns3/4a cleavage and replication dnajb1/hsp40 suppresses melanoma differentiation-associated gene 5-mitochondrial antiviral signaling protein function in conjunction with hsp70 cellular dnaja3, a novel vp1-interacting protein, inhibits footand-mouth disease virus replication by inducing lysosomal degradation of vp1 and attenuating its antagonistic role in the beta interferon signaling pathway human hsp70 and hsp40 chaperone proteins facilitate human papillomavirus-11 e1 protein binding to the origin and stimulate cell-free dna replication targeting the e1 replication protein to the papillomavirus origin of replication by complex formation with the e2 transactivator cell-free replication of the human papillomavirus dna with homologous viral e1 and e2 proteins and human cell extracts chaperone proteins abrogate inhibition of the human papillomavirus (hpv) e1 replicative helicase by the hpv e2 protein the human dnaj protein, htid-1, enhances binding of a multimer of the herpes simplex virus type 1 ul9 protein to oris, an origin of viral dna replication activation of the herpes simplex virus type-1 origin-binding protein (ul9) by heat shock proteins turnover of hepatitis b virus x protein is facilitated by hdj1, a human hsp40/dnaj protein negative regulation of hepatitis b virus replication by cellular hsp40/dnaj proteins through destabilization of viral core and x proteins hepatitis b virus x protein in the pathogenesis of hepatitis b virusinduced hepatocellular carcinoma high-level expression of hepatitis b virus hbx gene and hepatocarcinogenesis in transgenic mice x protein of hepatitis b virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis hepatitis b. x antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma retroviral infection of non-dividing cells: old and new perspectives hiv-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway a nuclear localization signal within hiv-1 matrix protein that governs infection of non-dividing cells hiv-1 genome nuclear import is mediated by a central dna flap functional analysis of the simian immunodeficiency virus vpx protein: identification of packaging determinants and a novel nuclear targeting domain simian immunodeficiency virus vpx is imported into the nucleus via importin alphadependent and -independent pathways hsp40 facilitates nuclear import of the human immunodeficiency virus type 2 vpx-mediated preintegration complex interference of dnajb6/mrj isoform switch by morpholino inhibits replication of hiv-1 and rsv large isoform of mammalian relative of dnaj is a major determinant of human susceptibility to hiv-1 infection heat shock protein 40 is necessary for human immunodeficiency virus-1 nef-mediated enhancement of viral gene expression and replication hiv-1 nef and host cell protein kinases nef triggers a transcriptional program in t cells imitating single-signal t cell activation and inducing hiv virulence mediators the plasma membrane as a combat zone in the hiv battlefield cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways reciprocal regulation of human immunodeficiency virus-1 gene expression and replication by heat shock proteins 40 and 70 novel role of hsp40/dnaj in the regulation of hiv-1 replication heat shock proteins as cellular lifeguards structural and functional diversities between members of the human hspb, hsph, hspa, and dnaj chaperone families muscle develops a dpecific form of small heat shock protein complex composed of mkbp/hspb2 and hspb3 during myogenic differentiation epstein-barr virus upregulates phosphorylated heat shock protein 27 kda in carcinoma cells using the phosphoinositide 3-kinase/akt pathway proteomics analysis of differentially expressed proteins in chicken trachea and kidney after infection with the highly virulent and attenuated coronavirus infectious bronchitis virus in vivo cellular hsp27 interacts with classical swine fever virus ns5a protein and negatively regulates viral replication by the nf-κb signaling pathway down-regulating heat shock protein 27 is involved in porcine epidemic diarrhea virus escaping from host antiviral mechanism the heat-shock response adenovirus type 7 induces interleukin-8 production via activation of extracellular regulated kinase 1/2 nf-κb and the map kinases/ap-1 pathways are both involved in interleukin-6 and interleukin-8 expression in fibroblast-like synoviocytes stimulated by protein i/ii, a modulin from oral streptococci signaling pathways for glycated human serum albumin-induced il-8 and mcp-1 secretion in human rpe cells mammalian small stress proteins protect against oxidative stress through their ability to increase glucose-6-phosphate dehydrogenase activity and by maintaining optimal cellular detoxifying machinery heat shock proteins: essential proteins for apoptosis regulation apoptosis versus cell differentiation: role of heat shock proteins hsp90, hsp70 and hsp27 small heat shock proteins hsp27 (hspb1), αb-crystallin (hspb5) and hsp22 (hspb8) as regulators of cell death modification and reorganization of the cytoprotective cellular chaperone hsp27 during herpes simplex virus type 1 infection heat shock protein 27 is involved in pcv2 infection in pk-15 cells hspb1 is an intracellular antiviral factor against hepatitis b virus role of thiol/disulfide exchange in newcastle disease virus entry overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the newcastle disease virus fusion protein endothelial cell surface expression of protein disulfide isomerase activates beta1 and beta3 integrins and facilitates dengue virus infection protein disulfide isomerase mediates dengue virus entry in association with lipid rafts inhibiting rotavirus infection by membrane-impermeant thiol/disulfide exchange blockers and antibodies against protein disulfide isomerase inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent hiv-1 entry the catalytic activity of protein disulfide isomerase is involved in human immunodeficiency virus envelope-mediated membrane fusion after cd4 cell binding protein-disulfide isomerase-mediated reduction of two disulfide bonds of hiv envelope glycoprotein 120 occurs post-cxcr4 binding and is required for fusion galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance t-cell migration and hiv entry structure of murine polyomavirus complexed with an oligosaccharide receptor fragment how viruses use the endoplasmic reticulum for entry, replication, and assembly simian virus 40 depends on er protein folding and quality control factors for entry into host cells a pdi family network acts distinctly and coordinately with erp29 to facilitate polyomavirus infection oblongifolin m, an active compound isolated from a chinese medical herb garcinia oblongifolia, potently inhibits enterovirus 71 reproduction through downregulation of erp57 cellular self-defense: how cellautonomous immunity protects against pathogens role of free radicals in viral pathogenesis and mutation ex vivo induction of apoptosis in lymphocytes is mediated by oxidative stress: role for lymphocyte loss in hiv infection. free radic role of oxidants in influenza virus-induced gene expression investigation of oxidative stress and antioxidant defense in patients with hepatitis b virus infection and the effect of interferon-alpha plus lamivudine combination therapy on oxidative stress hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production oxidative damage to neurons caused by the induction of microglial nadph oxidase in encephalomyocarditis virus infection cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells japanese encephalitis virus stimulates superoxide dismutase activity in rat glial cultures oxidative stress in hpv-driven viral carcinogenesis: redox proteomics analysis of hpv-16 dysplastic and neoplastic tissues folding and oligomerization of influenza hemagglutinin in the er and the intermediate compartment n-glycosylation of the premembrane protein of japanese encephalitis virus is critical for folding of the envelope protein and assembly of viruslike particles hepatitis c virus glycoprotein folding: disulfide bond formation and association with calnexin an rna chaperone activity of non-specific rna binding proteins in hammerhead ribozyme catalysis the ebola virus vp24 protein prevents hnrnp c1/c2 binding to karyopherin α1 and partially alters its nuclear import hnrnps relocalize to the cytoplasm following infection with vesicular stomatitis virus hnrnp proteins and the biogenesis of mrna direct and indirect effects on viral translation and rna replication are required for auf1 restriction of enterovirus infections in human cells cellular mrna decay protein auf1 negatively regulates enterovirus and human rhinovirus infections heterogeneous nuclear ribonucleoprotein a2 participates in the replication of japanese encephalitis virus through an interaction with viral proteins and rna viral and cellular proteins involved in coronavirus replication hnrnp c and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'ntr of the hcv rna genome mechanistic consequences of hnrnp c binding to both rna termini of poliovirus negative-strand rna intermediates characterization of multimeric complexes formed by the human ptb1 protein on rna heterogeneous nuclear ribonucleoprotein i (hnrnp-i/ptb) selectively binds the conserved 3′ terminus of hepatitis c viral rna role of la autoantigen and polypyrimidine tract-binding protein in hcv replication polypyrimidine-tract-binding protein is a component of the hcv rna replication complex and necessary for rna synthesis hur displaces polypyrimidine tract binding protein to facilitate la binding to the 3′ untranslated region and enhances hepatitis c virus replication the la antigen binds 5′ noncoding region of the hepatitis c virus rna in the context of the initiator aug codon and stimulates internal ribosome entry site-mediated translation hepatitis c virus internal ribosome entry site-mediated translation is stimulated by specific interaction of independent regions of human la autoantigen hur, a protein implicated in oncogene and growth factor mrna decay, binds to the 3′ ends of hepatitis c virus rna of both polarities delayed kinetics of poliovirus rna synthesis in a human cell line with reduced levels of hnrnp c proteins microrna-555 has potent antiviral properties against poliovirus cellular rna binding proteins ns1-bp and hnrnp k regulate influenza a virus rna splicing ns1-binding protein (ns1-bp): a novel human protein that interacts with the influenza a virus nonstructural ns1 protein is relocalized in the nuclei of infected cells co-regulatory activity of hnrnp k and ns1-bp in influenza and human mrna splicing rna-binding protein hnrnp d modulates internal ribosome entry site-dependent translation of hepatitis c virus rna two distinct binding modes of a protein cofactor with its target rna mechanism for nucleic acid chaperone activity of hiv-1 nucleocapsid protein revealed by single molecule stretching heterogeneous nuclear ribonuclear protein k interacts with the enterovirus 71 5′ untranslated region and participates in virus replication the polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation feline calicivirus replication: requirement for polypyrimidine tract-binding protein is temperature-dependent internal initiation of translation from the human rhinovirus-2 internal ribosome entry site requires the binding of unr to two distinct sites on the 5′ untranslated region evidence for an rna chaperone function of polypyrimidine tractbinding protein in picornavirus translation interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis c virus rna genome and its functional requirement in internal initiation of translation hnrnp l is required for the translation mediated by hcv ires heterogeneous nuclear ribonucleoprotein l interacts with the 3 border of the internal ribosomal entry site of hepatitis c virus hnrnp l and nf90 interact with hepatitis c virus 5′-terminal untranslated rna and promote efficient replication a human proteome microarray identifies that the heterogeneous nuclear ribonucleoprotein k (hnrnp k) recognizes the 5′ terminal sequence of the hepatitis c virus rna heterogeneous ribonucleoprotein k (hnrnp k) binds mir-122, a mature liver-specific microrna required for hepatitis c virus replication modulation of hepatitis c virus rna abundance by a liver-specific microrna global epidemiology and genotype distribution of the hepatitis c virus infection position-dependent function for a tandem microrna mir-122-binding site located in the hepatitis c virus rna genome hepatitis c virus core protein interacts with heterogeneous nuclear ribonucleoprotein k an rna-binding protein, hnrnp a1, and a scaffold protein, septin 6, facilitate hepatitis c virus replication hnrnp a1 interacts with the 5' untranslated regions of enterovirus 71 and sindbis virus rna and is required for viral replication apigenin inhibits enterovirus-71 infection by disrupting viral rna association with trans-acting factors the role of misshapen nck-related kinase (mink), a novel ste20 family kinase, in the ires-mediated protein translation of human enterovirus 71 n-terminomics tails identifies host cell substrates of poliovirus and coxsackievirus b3 3c proteinases that modulate virus infection heterogeneous nuclear ribonucleoprotein m facilitates enterovirus infection the heterogeneous nuclear ribonucleoprotein k (hnrnp k) is a host factor required for dengue virus and junín virus multiplication the heterogeneous nuclear ribonucleoprotein k (hnrnp k) interacts with dengue virus core protein identification of heterogeneous nuclear ribonucleoprotein k (hnrnp k) as a repressor of c/ebpmediated gene activation role of human heterogeneous nuclear ribonucleoprotein c1/c2 in dengue virus replication vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein 1 and is important for viral replication and release identification of human hnrnp c1/c2 as a dengue virus ns1-interacting protein heterogeneous nuclear ribonucleoprotein k supports vesicular stomatitis virus replication by regulating cell survival and cellular gene expression downregulation of nipah virus n mrna occurs through interaction between its 3′ untranslated region and hnrnp d hnrnp a2/b1 interacts with influenza a viral protein ns1 and inhibits virus replication potentially through suppressing ns1 rna/ protein levels and ns1 mrna nuclear export the negative regulator of splicing element of rous sarcoma virus promotes polyadenylation a cellular protein, hnrnp h, binds to the negative regulator of splicing element from rous sarcoma virus efficient polyadenylation of rous sarcoma virus rna requires the negative regulator of splicing element analysis of the interactions of viral and cellular factors with human cytomegalovirus lytic origin of replication viral dna replication orientation and hnrnps regulate transcription of the human papillomavirus 18 late promoter host heterogeneous ribonucleoprotein k (hnrnp k) as a potential target to suppress hepatitis b virus replication cytidine deaminase apobec3b interacts with heterogeneous nuclear ribonucleoprotein k and suppresses hepatitis b virus expression the multifunctional herpes simplex virus ie63 protein interacts with heterogeneous ribonucleoprotein k and with casein kinase 2 ck2 protein kinase is stimulated and redistributed by functional herpes simplex virus icp27 protein rna polymerase ii holoenzyme modifications accompany transcription reprogramming in herpes simplex virus type 1-infected cells association of herpes simplex virus type 1 icp8 and icp27 proteins with cellular rna polymerase ii holoenzyme herpes simplex virus 1 icp27 is required for transcription of two viral late (gamma 2) genes in infected cells human papillomaviruses: a compilation and analysis of nucleic acid and amino acid sequences a downstream polyadenylation element in human papillomavirus type 16 l2 encodes multiple ggg motifs and interacts with hnrnp h specific interaction between hnrnp h and hpv16 l1 proteins: implications for late gene auto-regulation enabling rapid viral capsid protein production the epstein-barr virus (ebv) sm protein enhances pre-mrna processing of the ebv dna polymerase transcript the alternative splicing factor hnrnp a1 is up-regulated during virus-infected epithelial cell differentiation and binds the human papillomavirus type 16 late regulatory element a 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hfip1, cstf-64, hnrnp c1/c2, and polypyrimidine tract binding protein identification of an hnrnp a1-dependent splicing silencer in the human papillomavirus type 16 l1 coding region that prevents premature expression of the late l1 gene inhibition of hpv-16 l1 expression from l1 cdnas correlates with the presence of hnrnp a1 binding sites in the l1 coding region identification of a 17-nucleotide splicing enhancer in hpv-16 l1 that counteracts the effect of multiple hnrnp a1-binding splicing silencers polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, sd3632 translational inhibition in vitro of human papillomavirus type 16 l2 mrna mediated through interaction with heterogenous ribonucleoprotein k and poly(rc)-binding proteins 1 and 2 c-src-mediated phosphorylation of hnrnp k drives translational activation of specifically silenced mrnas regulation of the epstein-barr virus c promoter by auf1 and the cyclic amp/protein kinase a signaling pathway auf1/hnrnp d is a novel protein partner of the eber1 noncoding rna of epstein-barr virus nuclear hnrnpa2b1 initiates and amplifies the innate immune response to dna viruses hnrnp a1 selectively interacts through its gly-rich domain with different rna-binding proteins hiv nef enhances tat-mediated viral transcription through a hnrnp-k-nucleated signaling complex cooperative-binding and splicing-repressive properties of hnrnp a1 trafficking of hiv-1 rna is mediated by heterogeneous nuclear ribonucleoprotein a2 expression and impacts on viral assembly actin-associated hnrnp proteins as transacting factors in the control of mrna transport and localization genomic structure of an attenuated quasi species of hiv-1 from a blood transfusion donor and recipients novel (n)pkc kinases phosphorylate nef for increased hiv transcription, replication and perinuclear targeting cloning and functional analysis of multiply spliced mrna species of human immunodeficiency virus type 1 alternative splicing of human immunodeficiency virus type 1 mrna modulates viral protein expression, replication, and infectivity a naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production exon recognition in vertebrate splicing intron recognition comes of age identification, purification, and biochemical characterization of u2 small nuclear ribonucleoprotein auxiliary factor interaction of u2af65 rs region with pre-mrna of branch point and promotion base pairing with u2 snrna a potential role for u2af-sap 155 interactions in recruiting u2 snrnp to the branch site sorting out the complexity of sr protein functions hnrnp complexes: composition, structure, and function alternative pre-mrna splicing: the logic of combinatorial control balanced splicing at the tat-specific hiv-1 3'ss a3 is critical for hiv-1 replication splicing efficiency of human immunodeficiency virus type 1 tat rna is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2 the tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3′ splice site behind the scenes of hiv-1 replication: alternative splicing as the dependency factor on the quiet an exonic splicing silencer downstream of the 3' splice site a2 is required for efficient human immunodeficiency virus type 1 replication biochemical and nmr study on the competition between proteins sc35, srp40, and heterogeneous nuclear ribonucleoprotein a1 at the hiv tat exon 2 splicing site rna biology identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new hiv-1 splicing regulator, protein hnrnp k a second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of tat mrna and binds protein hnrnp h control of hiv-1 env rna splicing and transport: investigating the role of hnrnp a1 in exon splicing silencer (ess3a) function the hnrnp a1 protein regulates hiv-1 tat splicing via a novel intron silencer element hnrnp a1 controls hiv-1 mrna splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure solution structure of the hiv-1 intron splicing silencer and its interactions with the up1 domain of heterogeneous nuclear ribonucleoprotein (hnrnp) a1 a truncated hnrnp a1 isoform, lacking the rgg-box rna binding domain, can efficiently regulate hiv-1 splicing and replication sc35 and heterogeneous nuclear ribonucleoprotein a/b proteins bind to a juxtaposed exonic splicing enhancer/exonic splicing silencer element to regulate hiv-1 tat exon 2 splicing hnrnp a1 recruited to an exon in vivo can function as an exon splicing silencer a janus splicing regulatory element modulates hiv-1 tat and rev mrna production by coordination of hnrnp a1 cooperative binding rna splicing at human immunodeficiency virus type 1 3' splice site a2 is regulated by binding of hnrnp a/b proteins to an exonic splicing silencer element differential hnrnp d isoform incorporation may confer plasticity to the essv-mediated repressive state across hiv-1 exon 3 human immunodeficiency virus type 1 hnrnp a/b-dependent exonic splicing silencer essv antagonizes binding of u2af65 to viral polypyrimidine tracts sr proteins and hnrnp h regulate the splicing of the hiv tev-specific exon 6d the secondary structure of the human immunodeficiency virus type 1 transcript modulates viral splicing and infectivity members of the heterogeneous nuclear ribonucleoprotein h family activate splicing of an hiv-1 splicing substrate by promoting formation of atp-dependent spliceosomal complexes rna trafficking signals in human immunodeficiency virus type 1 trafficking of hiv-1 rna is mediated by heterogeneous nuclear ribonucleoprotein a2 expression and impacts on viral assembly a late role for the association of hnrnp a2 with the hiv-1 hnrnp a2 response elements in genomic rna, gag, and vpr localization differential effects of hnrnp d/auf1 isoforms on hiv-1 gene expression depletion of hnrnp a2/b1 overrides the nuclear retention of the hiv-1 genomic rna mapping of determinants required for the function of the hiv-1 env nuclear retention sequence inhibition of hiv-1 gene expression by a fragment of hnrnp u nuclear mrna export: insights from virology protein kinase ck2 phosphorylation regulates the interaction of kaposi's sarcoma-associated herpesvirus regulatory protein orf57 with its multifunctional partner hnrnp k heterogeneous nuclear ribonucleoprotein a1 interferes with the binding of the human t cell leukemia virus type 1 rex regulatory protein to its response element human immunodeficiency virus type 1 (hiv-1) induces the cytoplasmic retention of heterogeneous nuclear ribonucleoprotein a1 by disrupting nuclear import: implications for hiv-1 gene expression a polypyrimidine tract facilitates the expression of kaposi's sarcoma-associated herpesvirus vflip through an internal ribosome entry site heat shock protein hsp72 plays an essential role in her2-induced mammary tumorigenesis. oncogene the role of heat shock protein 70 in mediating agedependent mortality in sepsis peptides and aptamers targeting hsp70: a novel approach for anticancer chemotherapy activation of hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation monitering heat shock protein 70 and heat shock protein 90 during herpes simplex virus type 1 infection hsp70 functions as a negative regulator of west nile virus capsid protein through direct interaction mitochondrial hsp70, hsp40, and hsp60 bind to the 3′ untranslated region of the murine hepatitis virus genome immunomodulatory activity of extracellular hsp70 mediated via paired receptors siglec-5 and siglec-14 extracellular cell stress proteins as biomarkers of human disease molecular chaperones and protein-folding catalysts as intercellular signaling regulators in immunity and inflammation cross-presentation of the oncofetal tumor antigen 5t4 from irradiated prostate cancer cells-a key role for heat-shock protein 70 and receptor cd91 membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen stress for maintaining memory: hsp70 as a mobile messenger for innate and adaptive immunity novel heat shock protein hsp70l1 activates dendritic cells and acts as a th1 polarizing adjuvant chaperokine activity of heat shock proteins hsp70 as endogenous stimulus of the toll/interleukin-1 receptor signal pathway chlamydial heat shock protein 60 activates macrophages and endothelial cells through toll-like receptor 4 and md2 in a myd88-dependent pathway association of hadha with human rna silencing machinery dengue virus modulates the unfolded protein response in a time-dependent manner heat shock protein 70 is associated with replicase complex of japanese encephalitis virus and positively regulates viral genome replication japanese encephalitis virus activates autophagy through xbp1 and atf6 er stress sensors in neuronal cells bovine viral diarrhea virus infection induces autophagy in mdbk cells the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis the emerging roles of viroporins in er stress response and autophagy induction during virus infection stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy hepatitis c virus suppresses the ire1-xbp1 pathway of the unfolded protein response glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis b virus hepatitis b virus x protein (hbx) activates atf6 and ire1-xbp1 pathways of unfolded protein response herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase perk and mediates eif-2alpha dephosphorylation by the gamma(1)34.5 protein membrane biogenesis and the unfolded protein response the atf6 branch of unfolded protein response and apoptosis are activated to promote african swine fever virus infection role of the host cell's unfolded protein response in arenavirus infection interaction of dengue virus envelope protein with endoplasmic reticulum-resident chaperones facilitates dengue virus production human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone bip/grp78, which is required for virion assembly betanodavirus up-regulates chaperone grp78 via er stress: roles of grp78 in viral replication and host mitochondria-mediated cell death influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (ire1) stress pathway west nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2α kinases perk and pkr activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production hepatitis c virus ns4b induces unfolded protein response and endoplasmic reticulum overload response-dependent nf-κb activation the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor human cytomegalovirus protein us11 provokes an unfolded protein response that may facilitate the degradation of class i major histocompatibility complex products hcv causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response endoplasmic reticulum stress induced by hepatitis b virus x protein enhances cyclo-oxygenase 2 expression via activating transcription factor 4 functional diversity of the hnrnps: past, present and perspectives heterogeneous nuclear ribonucleoproteins (hnrnps) in cellular processes: focus on hnrnp e1's multifunctional regulatory roles acute and chronic disease caused by enteroviruses hnrnp a1 alters the structure of a conserved enterovirus ires domain to stimulate viral translation heterogeneous nuclear ribonuclear protein k interacts with the enterovirus 71 59 untranslated region and participates in virus replication molecular mechanism of action of hnrnp k and rtn3 in the replication of enterovirus 71. chin sherris medical microbiology, 6e anti-herpetic medications and reduced risk of dementia in patients with herpes simplex virus infections-a nationwide, population-based cohort study in taiwan antivirals reduce the formation of key alzheimer's disease molecules in cell cultures acutely infected with herpes simplex virus type 1 the heterogeneous nuclear ribonucleoprotein k is important for herpes simplex virus-1 propagation hepatitis b virus epidemiology and natural history of hcv infection pathobiology of chronic hepatitis virus infection and hepatocellular carcinoma (hcc). tal hepatitis delta virus polyarteritis nodosa and extrahepatic manifestations of hbv infection: the case against autoimmune intervention in pathogenesis autoantibody recognition of an n-terminal epitope of hnrnp l marks the risk for developing hbv-related hepatocellular carcinoma benign lymphadenopathies epstein-barr virus-associated lymphoproliferative disorders: experimental and clinical developments epstein-barr virus-associated hodgkin's lymphoma human papillomavirus and epstein-barr virus in nasopharyngeal carcinoma in a low-incidence population epstein-barr virus infection and multiple sclerosis: a review lymphomatoid granulomatosis: a practical review for pathologists dealing with this rare pulmonary lymphoproliferative process epstein-barr virus and skin manifestations in childhood monoclonal antibodies against epstein-barr virus cross-react with alpha-synuclein in human brain human papillomavirus (hpv) type distribution and serological response to hpv type 6 virus-like particles in patients with genital warts the global health burden of infection-associated cancers in the year 2002 trends in human papillomavirus-associated cancers-united states epidemiology and natural history of human papillomavirus infections in the female genital tract case-control study of human papillomavirus and oropharyngeal cancer review of human immunodeficiency virus type 1-related opportunistic infections in sub-saharan africa the treatment of patients with hiv defining hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection the hsp90 mosaic: a picture emerges the chaperone hsp90: changing partners for demanding clients structure of tpr domain-peptide complexes: critical elements in the assembly of the hsp70-hsp90 multichaperone machine hsp90 and co-chaperones twist the functions of diverse client proteins the 'active life' of hsp90 complexes structure and functional relationships of hsp90 cellular growth kinetics distinguish a cyclophilin inhibitor from an hsp90 inhibitor as a selective inhibitor of hepatitis c virus novobiocin and additional inhibitors of the hsp90 cterminal nucleotide-binding pocket the amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an atp/adp switch domain that regulates hsp90 conformation 17-allylamino-17-demethoxygeldanamycin induces downregulation of critical hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells inhibition of signal transduction by the hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis antitumor efficacy of ipi-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2-positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib nvp-auy922: a novel heat shock protein 90 inhibitor active against xenograft tumor growth, angiogenesis, and metastasis biib021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein hsp90 ganetespib, a unique triazolone-containing hsp90 inhibitor, exhibits potent antitumor activity and a superior safety profile for cancer therapy the heat shock protein 90 inhibitor, at13387, displays a long duration of action in vitro and in vivo in non-small cell lung cancer snx2112, a synthetic heat shock protein 90 inhibitor, has potent antitumor activity against her kinase-dependent cancers novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins the heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized atpbinding domain in the carboxyl terminus of the chaperone design, synthesis, and biological evaluation of novel deguelinbased heat shock protein 90 (hsp90) inhibitors targeting proliferation and angiogenesis structural basis for depletion of heat shock protein 90 client proteins by deguelin epigallocatechin-3-gallate is a novel hsp90 inhibitor novel hsp90 inhibitor platycodin d disrupts hsp90/cdc37 complex and enhances the anticancer effect of mtor inhibitor sulforaphane inhibits pancreatic cancer through disrupting hsp90-p50cdc37 complex and direct interactions with amino acids residues of hsp90 withaferin a targets heat shock protein 90 in pancreatic cancer cells a novel hsp90 inhibitor to disrupt hsp90/cdc37 complex against pancreatic cancer cells natural product kongensin a is a non-canonical hsp90 inhibitor that blocks rip3-dependent necroptosis discovery and identification of cdc37-derived peptides targeting the hsp90-cdc37 protein-protein interaction targeting the hsp90-cdc37-client protein interaction to disrupt hsp90 chaperone machinery activity of suberoylanilide hydroxamic acid against human breast cancer cells with amplification of her-2 chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor laq824 modulation of p53, erbb1, erbb2, and raf-1 expression in lung cancer cells by depsipeptide fr901228 caspase-10-mediated heat shock protein 90β cleavage promotes uvb irradiation-induced cell apoptosis hsp90 cleavage by an oxidative stress leads to its client proteins degradation and cancer cell death suberoylanilide hydroxamic acid induces ros-mediated cleavage of hsp90 in leukemia cells proteasome inhibitor-induced cleavage of hsp90 is mediated by ros generation and caspase 10-activation in human leukemic cells dendritic cells transfected with heat-shock protein 70 messenger rna for patients with hepatitis c virus-related hepatocellular carcinoma: a phase 1 dose escalation clinical trial hsp60 chaperonopathies and chaperonotherapy: targets and agents hsp60 as a drug target studies on bredinin. i. isolation,characterization and biological properties hiroyuki epolactaene, a novel neuritogenic compound in human neuroblastoma cells, produced by a marine fungus structure-activity relationships of epolactaene derivatives: structural requirements for inhibition of hsp60 chaperone activity epolactaene binds human hsp60 cys442 resulting in the inhibition of chaperone activity crystal structure of the human mitochondrial chaperonin symmetrical football complex identification of hsp60 as a primary target of o-carboranylphenoxyacetanilide, an hif-1α inhibitor mitochondrial chaperonin hsp60 is the apoptosis-related target for myrtucommulone isolation and antibacterial activity of acylphloroglucinols from myrtus communis oligomeric acylphloroglucinols from myrtle (myrtus communis) antioxidant activity of oligomeric acylphloroglucinols from myrtus communis l myrtucommulone from myrtus communis, exhibits potent antiinflammatory effectiveness in vivo identification of molecular targets of the oligomeric nonprenylated acylphloroglucinols from and myrtle (myrtus communis) and their implication as anti-inflammatory compounds myrtucommulone from myrtus communis induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9 dual induction of mitochondrial apoptosis and senescence in chronic myelogenous leukemia by myrtucommulone a stephacidin a and b: two structurally novel, selective inhibitors of the testosterone-dependent prostate lncap cells avrainvillamide, a cytotoxic marine natural product, and derivatives thereof evidence for the rapid conversion of stephacidin b into the electrophilic monomer avrainvillamide in cell culture boron-containing phenoxyacetanilide derivatives as hypoxiainducible factor (hif)-1α inhibitors anticancer gold(iii) porphyrins target mitochondrial chaperone hsp60 molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets chemical proteomics identifies heterogeneous nuclear ribonucleoprotein (hnrnp) a1 as the molecular target of quercetin in its anti-cancer effects in pc-3 cells quercetin targets hnrnpa1 to overcome enzalutamide resistance in prostate cancer cells quercetin as an antiviral agent inhibits influenza a virus (iav) entry. viruses inhibition of enterovirus 71 replication and viral 3c protease by quercetin antiviral activity of baicalein and quercetin against the japanese encephalitis virus computer-aided discovery of small molecules targeting the rna splicing activity of hnrnp a1 in castration-resistant prostate cancer a selective inhibitor of eif2alpha dephosphorylation protects cells from er stress dengue virus serotype infection specifies the activation of the unfolded protein response activation of the unfolded protein response by 2-deoxy-dglucose inhibits kaposi's sarcoma-associated herpesvirus replication and gene expression antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response rana catesbeiana ribonuclease inhibits japanese encephalitis virus (jev) replication and enhances apoptosis of jev-infected bhk-21 cells broad-spectrum antiviral therapeutics vaticanol b, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress the hepatitis c virus (hcv) ns4b rna binding inhibitor clemizole is highly synergistic with hcv protease inhibitors spontaneous and engineered compensatory hsv mutants that counteract the host antiviral pkr response open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source [81671995]; the general research grant of hong kong [11100215] ; and strategic funds from city university of hong kong. competing interests: the authors declare no competing interests.commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-350569-dtxtjtfo authors: kasoka, kasoka title: autonomy in hiv testing: a call for a rethink of personal autonomy in the hiv response in sub-saharan africa date: 2020-06-13 journal: med health care philos doi: 10.1007/s11019-020-09959-y sha: doc_id: 350569 cord_uid: dtxtjtfo the author reviews various conceptions of autonomy to show that humans are actually not autonomous, strictly speaking. he argues for a need to rethink the personal autonomy approaches to hiv testing in sub-saharan africa (ssa) countries. hiv/aids has remained a leading cause of disease burden in ssa. it is important to bring this disease burden under control, especially given the availability of current effective antiretroviral regimens in lowand middle-income countries. in most ssa countries the ethic or value of personal autonomy or self-determination is promoted as primary in hiv testing decision-making. ssa policymakers have an ontological and moral duty to adopt hiv testing policies that reflect human and medical realities, relationships, local contexts, and respect human rights for both individuals and others who are affected by hiv in society. without rethinking the value of autonomy in hiv testing decision-making, the article cautions that attainment of the sustainable development goal (sdg) 3 and the unaids fast-track strategy that explicitly call to end the epidemic by 2030 will not be feasible for ssa. the case of covid-19 pandemic has more than ever presented to the world that individual autonomy in society is at best conditional and relational. the coronavirus intrusive measures taken to contain the virus expose the fundamental problem in metaphysics-the issue of what makes a person the same over a period of time-and the moral dilemma posed in bioethics by the fact that an individual who lives in society with others is promoted as a sovereign of her own body, medical choices and life. this dilemma might otherwise be expressed: 'what makes a person the selfsame person today as who he or she was yesterday' (ndete 2016) , and what does autonomy mean to an individual whose exercise of self-governance has inescapable ramifications on the wellbeing and rights of others. thus, should there be a need to rethink the personal autonomy 1 premising of informed consent requirements in hiv testing in most sub-saharan african (ssa) countries, since informed consent requirements in hiv testing are mainly premised on personal autonomy? childress 1979/2000; fraser 2005; united nations 2009; naidoo and vernillo 2012) . or there isn't a need to rethink because humans are, as kantian and related notions of autonomy tell us, normatively and narratively sovereign selves? in this article i seek to show that personal autonomy, strictly speaking, is an illusion, and its primacy in healthcare ethics is morally problematic. in most of ssa countries the hiv prevalence has stabilised at high rates. the ssa region carries a disproportionate hiv burden, accounting for 70% of the worldwide burden of infection (bulstra et al. 2020) . hiv/aids is a leading cause of morbidity and mortality in the region (dwyer-lindgren, et al. 2019 ). 2 this is despite that major scientific breakthroughs (availability of effective hiv therapy) that have shifted the hiv paradigm that was once was considered a death sentence are now mostly available in the region. even with the availability of hiv medication in ssa countries like zambia, aids still remains the most common cause of death. 3 and yet, individual citizens are still celebrated and told through hiv laws or policies that they have a right to refuse hiv testing because they are sovereigns of their own health, life and destiny (castell v. de greef 1994; southern africa litigation centre 2012) . therefore, does this temptation and pursuit to view ourselves as autonomous hiv testing decision-makers blind us to an observable reality that shows that as humans we are interdependent and inextricably social and socialised beings? three decades into the hiv epidemic, hiv still poses a real health challenge from which no country in the world is immune, particularly ssa countries. according to unaids' factsheet, at the end of 2019 37.9 million people worldwide were living with hiv (unaids 2020a, b, c) . the same source indicates that 74.9 million people as of the end of 2018 became infected with hiv since the beginning of the epidemic. of the 74.9 million infected, 32 million have since died from aids-related illnesses. this makes hiv/ aids one of the major causes of morbidity and mortality in the world. hiv/aids is the second leading cause of death in ssa (business insider 2017). given the threat of the epidemic, it is not surprising that all the united nations member states agreed to reach certain targets in response to the disease (suthar et al. 2013 ). these targets were set to be achieved by 2015. some of the targets included the reduction of sexual and parental transmission of hiv by 50%, elimination of vertical hiv transmission, reduction of tb deaths among people living with hiv by 50%, and delivery of art to 15 million people (suthar et al. 2013; udjo and lalthapersad-pillay 2015) . it was noted that the achievement of such goals requires that people test for hiv since it is only through knowledge of one's serostatus that one can be linked to prevention, treatment and care services (suthar et al. 2013) . it is encouraging to note that such efforts have led to significant degrees of progress in response to hiv. overall, hiv infections are approximated to have been reduced by 40% between 2000 and 2013, with 13.6 million people globally reported to have been receiving art as of june 2014 (united nations 2015) . at present, new worldwide efforts are being made to end the epidemic by 2030. on 8 june 2016 united nations general assembly (unga) member states adopted, also in the light of the 2030 agenda for sustainable development and other, a new political declaration that seeks to end aids by 2030 (un general assembly 2016; un news centre 2016). the declaration includes a set of time-bound targets to fast-track response to reach three identified milestones by this year (2020). the 2020 milestones being: reduction of new hiv infections globally to less than 500,000; ensure that 90% of people infected with hiv know their hiv status, 90% of people who know their status are on put on art, and 90% of those on art have suppressed viral load (unaids 2015 (unaids , 2016 un news centre 2016) . 4 according to unaids, scale-up of art has put the reach of the global commitment to end hiv by 2030 on track (unaids 2016) . moreover, kharsany and karim state that the substantial declines in hiv infections are the result of hiv testing scale-up and widespread coverage of art (kharsany and karim 2016, p. 35) . thus, it is apparent that the above unga targets can only be achieved if there is an increased uptake of hiv testing and counselling (htc), and increased access to hiv prevention and care services (suthar et al. 2013, p. 23) . recognising the critical importance of hiv testing, indeed one of unaids' 90-90-90 2020 targets is that 90% of of all persons living hiv will know their hiv status (unaids 2020a, b, c) . as of 2018, 79% of all plhiv knew their hiv status (unaids 2020a, b, c) . the importance of hiv testing uptake cannot be overemphasised. its advantages include: being able to initiate early treatment for those who test positive at the time their immune system is still strong; early diagnosis improves long-term survival rates; knowledge of one's hiv status helps in prevention of hiv transmission to one's sexual partners, foetuses, babies, caregivers and other; knowledge of one's hiv status prevents re-infections, and; a positive test even for those who have been diagnosed later means one can still have access to life-saving treatment (st maarten aids foundation 2003) . put differently, 'an hiv test opens the door to accesing a range of hiv prevention options available depending on a person's hiv status to keep themselves and their loved ones hiv-negative' (unaids n.d.) . hiv testing is indeed critical as an entry point for hiv treatment and care. 5 unaids 4 one of the principles of the fast-track approach also calls for change; that is, among other things, stopping what does not work when it comes to the hiv response 'and scaling-up proven programmes' (unaids 2015, p. 6). 5 'hiv testing services (hts) are the entry point for diagnosis and access to life-saving antiretroviral therapy (art). early diagnosis and initiation of art have been shown to drastically decrease viral load, which reduces individual morbidity and mortality, and limits onward hiv transmission. hts can also offer a pathway for primary prevention interventions, including programs that deliver pre-exposure prophylaxis, voluntary medical male circumcision, and prevention of mother-to-child transmission ' (al., 2019) . and who have confirmed that adherence to an effective art regimen can result in reduction (by 96%) of the risk of transmitting the virus to an uninfected sexual partner, and can cause viral suppression that will lead to a plhiv living a normal lifestyle and longer life expectancy. without art, plhiv develop aids as a result of a compromised immune system, thereby exposing them to development of infections, certain cancers, and other severe clinical manifestations (unaids 2016; un-desa 2017) . in this vein, i am in favour of programmes such as the 'who test and treat' policy in ssa. those countries in ssa which have already adopted and implemented this approach are therefore to be commended. the introduction of 'test and treat' programmes have significantly contributed to an increase in the number of people accessing treatment (skovdal et al. 2016; avert, 2019a, b; unaids, 2020a, b, c) . ssa governments can alocate money, including requesting for hiv programmes funding from international donors, purchase mobile clinics and hire testing community workers to go door-to-door in communities and educate people about hiv prevention and the importance of hiv testing to an individual and common good, and then offer counselling and on-the-spot hiv testing and art initiation whenever individuals consent (egpaf 2017; sulat et al. 2018; silberner 2019) . this should be done in combination with routine hiv testing in healthcare facilities. without further delay, before the preparation of this article, i was aware that issues surrounding personal autonomy in hiv testing are multifaceted -therefore they cannot be all explored in this article. thus, the scope of this article is limited. firstly i have not explored various cultural conceptions of autonomy in this article (for such see the position of, mbiti 1970; gaylin and jennings 2003; woods 2002 woods -2004 traphagan 2013; song 2018) who have suggested that the primacy of the value of autonomy has its roots in western liberalism (particularly american). i have only mentioned in passing the ssa traditional ontological outlook and its potential impact on hiv healthcare ethics. secondly, the scope of the present review does not include a review of issues surrounding autonomy in clinical practice, including whether it is even possible to actualise informed consent requirements in medical practice (for this see, the position of manson and o'neill 2007) . thirdly, i have not explored in detail issues surrounding personal autonomy, hiv stigma and discrimination, and hence, some may argue, the importance of personal autonomy in healthcare practice. i am persuaded that the subject area of stigma versus personal autonomy deserves a critical exploratory article of its own. nonetheless, i have written this article having bourne in mind existing study findings and reports on stigma and discrimination (april 2009; stangl and grossman 2013) , 6 which, by implication, indicate that humans are social beings affected by the social environment. extensive studies that have found that fear for hiv stigma compromise hiv testing uptake and/or art adherence are also arguably an indictment on how respect to personal autonomy in hiv healthcare should be viewed. if indeed humans are self-rulers and rational players, why should social stigma prevent them from making self-rational and moral decisions in directing their own hiv medical therapy for their own good, and direct their lives as they deem fit? why should patients be declared and imputed with personal autonomy and yet at the same time blame the impact of social stigma on preventing potential hiv service-users from seeking medical attention? is this not conceptually and practically conflicting? and lastly, this paper does not explore whether the approach to hiv testing and treatment should be different from how we approach cancer, malaria, tuberculosis, covid-19, etc., even in light of hiv treatment universal health coverage. for my positions on this, i agree with hiv exceptionalism conclusions that criticise the subjectivism tendency to separate hiv response approaches from broader health systems (de cock and johnson 1998; april 2009; oppenheimer and bayer 2009; smith and whiteside 2010; benton 2015) . i agree that hiv exceptionalism was necessary before access to and effective art was made available in ssa. indeed, the hiv exceptionalism force is losing its original power as hiv has become less threatening (smith and whiteside 2010) due to accessible and effective hiv medication-treatment which has now been made available for the majority of affected populations in ssa. 'through the combined efforts of people living with hiv, national public health programmes, global donors and a broad community 6 basing autonomy on guarding against stigma can be problematic. among other things, april (2009) arguing for opt-out hiv testing versus the issue of stigma noted: 'under an opt-in programme, ….[a person] remains oblivious of her infection and avoids any immediate repercussions from her community. yet, this only delays the consequences -she will inevitably progress to aids. in societies with high hiv prevalence, as in much of sub-saharan africa, it is all but certain that her community will find out the cause of her suffering. it will be precisely at the time of her greatest physical ailing and need for emotional support that she will suffer the burden of hiv stigma and discrimination. in the case of an opt-out programme, although her decision to test may not have been borne of her own initiative, her decision not to decline testing will empower her to control the circumstances of her disclosure and formulate a plan for addressing her disease'. of stakeholders, the number of people on antiretroviral therapy (art) rose rapidly across ssa, going from about 100,000 in 2004 to 15.4 million by the end of 2017' (nash et al. 2018, p. 1). 7 hence, almost all countries in ssa have adopted hiv national policies to treat all persons, regardless of cd4 cell count (ssa has an hiv prevalence of 25.7 million people) (nash et al. 2018) . the massive expansion of art treatment in ssa has continued to save millions of lives, hence a need to even advance more appropriate hiv testing ethics. the scope of this article therefore concerns itself with a review whether a person who can test for hiv and have access to art is an autonomous patient who should be encouraged to choose her own medical therepy for her own good. the chief aim of this article is to show by reviewing procedural, substantive, ontological and socio-relational autonomy theories that capacity or achievement of autonomy in human society is an illusion, strictly speaking. thus, premising informed consent requirements of hiv testing testing on personal autonomy is problematic, philosophically and ethically. i have demonstrated that since humans don't exist in a vacuum, but are born into society and live in families and communities with fellow human beings, the promotion of primacy of personal autonomy over the common good 8 is inappropriate. the value of autonomy has been recognised as the core of medical ethics and has been validated by court judgments as a primary good in a free society (faden and beauchamp 1986; planned parenthood v. casey 1992; airedale nhs trust v. bland 1993; c v minister of correctional services 1996; lewanika v. frederick chiluba 1998; huri -laws v. nigeria 2000; diau v botswana building society 2003; southern africa litigation centre 2012) . for example, emphasising its primacy, the south african high court even held that '[i]t is, in principle, wholly irrelevant that [the patient's] attitude is, in the eyes of the entire medical profession, grossly unreasonable, because her rights of bodily integrity and autonomous moral agency entitle her to refuse medical treatment' (castell v. de greef 1994) . compounding this, the worldwide media through television dramas and documentaries foster this perception that individual choices or wishes are/ought to be sovereign in medical decision-making (english et al. 2006) . the individual or health service-user's medical judgment is celebrated as a right to be make one's own decisions for one's own health, body and life (southern africa litigation centre 2012). the individual is held to be a master of her own body and destiny, and is free to resist any violations to her autonomy (diau v. botswana building society 2003) against others of whom she lives with in society. behind the promoted freedom, autonomy, choice, and personal rights now stands a particular vision of what is entailed in being a human being; social relationships and arrangements are only recognised as far as they are able to nurture the atomistic self (gaylin 1996) . and behind this vocabulary of a sovereign human being is the belief that human behaviour is voluntarily chosen, and that other people's conduct can be modified through rational argument (gaylin 1996; gaylin and jennings 2003) . the case of the covid-19 crisis shows otherwise. 9 my article interrogates the personal autonomy arguments and reaches a conclusion that the philosophy surrounding the value is problematic, as well as, it is silent on the ethics of the actual implications of an autonomous decision in hiv testing (selemogo 2010) . this article is, therefore, composed of three themed sections. it begins by giving a brief overview of the origin of the word 'autonomy' and its evolution, and its relationship to informed consent in healthcare ethics. the second section analyses the various conceptions of autonomy. thirdly, an alternative conception of autonomy is reviewed and advanced (a review of whether autonomy is social as opposed to individual). the paper concludes by referencing the implication of a social view of autonomy on hiv testing in ssa where hiv is an epidemic. jones (2020) more 9 in discussing the impact of the coronavirus crisis, tobias jones observes: 'it has been fascinating to see the speed at which other attitudes have changed. the indignation expressed towards people not respecting social distancing (from those who would never normally describe themselves as moralists) has been understandably shrill: here too we've suddenly realised that the wellbeing of the group is endangered by indifferent individuals, and that community -for which we've yearned for so long -means originally simply a pooling of duties…. [t]he philosopher and activist simone weil wrote that "the notion of obligations comes before that of rights. it's a complicated, but convincing case, and it seems to me that in the last month there has been a radical shift in the balance between rights and responsibilities that has changed the timbre of our lives. i've never seen so many news items about applause, or so many social media posts accompanied by clappy emojis. before, "in the absence of adversity", the psychiatrist and philosopher iain mcgilchrist said this month: "we grew flabby, selfish. we manufactured grievances that now can be seen for what they were." now, when people meet their obligations to us we're obliged' (jones 2020) . 7 in east and southern africa, 67% of adults and 62% of children living with hiv are on art (avert 2019a, b) . 8 in this article, i have used the word 'common good' to mean 'the sum of those conditions of social life which allow social groups and their individual members relatively thorough and ready access to their own fulfillment' (velasquez et al. 2014 ). the ' "common good" refers to those facilities-whether material, cultural or institutional-that' individual 'members of a community provide to all members in order to fulfill a relational obligation they all have to care for certain interests that they have in common' (hussain 2018) . and as john finnis held, 'respect for human rights is a requirement of justice and that "the maintenance of human rights is a fundamental component of the common good"' (finnis 1980 , cited in hussain 2018 recently argued that what the coronavirus public health responses worldwide have shown us is that 'wellbeing isn't individual but social', and that humans 'are not actually independent at all'. the origin of autonomy, its relationship with informed consent, and hiv testing in ssa jackson (2013) explains that the word autonomy is from the greek words autos (self) and nomos (rule), which originally was used to refer to cities' independent self-rule. 10 feinberg (1986) , among others, also have noted that the word has a greek origin which comes from the greek for 'self' and 'law', meaning the making of one's own laws. in tracing the roots of personal autonomy, taylor (2005) suggests that individual self-authorship has its roots in the romantic liberalism of john stuart mill (2015) , in which free development of individuality was promoted. feinberg (1986) states that personal autonomy has interrelated meanings, from one's capacity to govern oneself, to the sovereign authority to govern oneself. o'neill (2003) provides that the original view of autonomy in antiquity-meaning self-legislation-never referred to persons, but to property. that is, ancient autonomous citystates made their own laws, colonies being given laws by the colonising parent cities (o'neill 2003, p. 3) . so, unlike its original cities' self-rule application, autonomy has now been extended to indicate the self-governance of rational independent individuals. this extension to an autonomous human has been adopted in medical ethics, and universalised, incuding in ssa. patient autonomy is promoted through informed consent requirements. informed consent has become one of the fundamental principles in medical ethics and law (dhai 2008) . individuals are held to have inviolable bodily and psychological integrity. this body-mind integrity right is violated when a patient is afforded medical intervention: without informing her in a language she understands the nature of the therapy; she has not been told about associated benefits and risks; available options in respect to an intervention have not been disclosed, and; she has not been informed that she has a right to refuse (mcquoid-mason 2008) . unlawful medical interventions that do not respect patient autonomy can constitute assault at law (dove et al. 2017) . in common law, free and informed consent means an inclusive recognition and respect of a: patient's capacity (and competence) to consent; disclosure of information to a patient related to the nature and extent of risks incident to a medical intervention; patient understands risks involved; patient is informed about available medical options, and; patient voluntariliy consents to (or to refuse) a medical intervention (beyleveld and brownsword 2007; mcquoid-mason 2008) . put differently, the legal and ethical elements of informed consent are: capacity; disclosure; understanding, and; voluntariness (dhai 2008) . a healthcare professional who by commission or omission fails to respect these requirements may be held responsible for any injury resulting therefrom. in this vein, '[t]he traditional hippocratic belief that one could do almost anything on a patient as long as the principles of beneficence (best interests) and nonmaleficence (no harm) were upheld has been considerably' stamped by rational and moral individuals whose autonomous actions or choices take precedence (dhai 2008, p. 27) . the principle of informed consent in clinical practice is primarily a negative right of non-interference (schermer 2002; clarke 2009) . '[i]t is closely connected to a particularly western, post-enlightenment idea that an adult person is a bounded individual who is able to live her life freely in accordance with her self-chosen plan, and ideally independently from controlling influences' (dove et al. 2017, p. 150) . its underpinnings can be traced to the influential kantian and millan conceptions of autonomy-or humans as rational self-legislators who are ends in themselves (selemogo 2010) . thus, 'in the domain of western biomedicine, the epitome of personal autonomy is a patient expressing a decision that she has come to autonomously and independently' (dove et al. 2017, p. 150) . to this effect, potential hiv service-users are told that they have the right to 'make choices that meet with their own interests according to their own will…[because humans have] capacity to understand substantial information, form a judgment according to their own values and communicate with the physician freely about their wishes' (song 2018, p. 296) . in this article, i have not questioned the importance of informed consent in healthcare conduct, and in hiv testing in particular. what i question is the premising of informed consent requirements on personal autonomy. i hold that grounding informed consent on autonomy distracts attention from observable human reality-which is that there are various important aspects of and factors in life that obstruct and pose challenges to realisation of personal autonomy in society. thus, instead of promoting personal autonomy, i advance a promotion of greater cooperation between patient, healthcare professional and one'a family and community. beauchamp and childress' definition of an autonomous patient who has freedom from controlling influences is mistaken. different schools of thought have advanced various conceptions of what autonomy means. o'neill (2003) has enumerated such definitions: dignity, integrity, individuality, independence, responsibility and self-knowledge; liberty; self-assertion; knowledge of one's interests; freedom from obligations; absence of external causation; choosing one's own moral position and accepting responsibility for one's own choices; self-mastery, voluntariness; privacy; and choosing freely. in the following review, much reference has therefore been made to killmister's (2013) thoughts on autonomy because the author identifies critical aspects of autonomy that have been overlooked in various theses by celebrated luminaries. killmister's structure and analysis of the subject matter is instructive for the present review. hence, the current analysis will be restricted to a critical review of feminist accounts; kantian accounts of autonomy which have been embraced by a number of feminist thinkers will therefore be reviewed. although feminists reject kantian and rawlsian notions of autonomy due to the former's argument that autonomy is relational, there are still different theses within the feminist school of thought which are arguably similar to kantian conceptions-the only major difference being that such feminist scholars advance a relational account of autonomy due to their rejection of self-sufficiency of human beings. besides marina oshana's socio-relational theory being arguably a more persuasive feminist account of autonomy, other accounts arguably embrace kantian and rawlsian rational choice theories. i commence by considering the reflective or historic endorsement accounts of frankfurt (1988) , dworkin (1988) and christman (2009) . the theses of these great thinkers indicate that an individual's capacity for autonomy lies in her psychological dispositions. nonetheless, christman's reflective endorsement theory has been credited as an account that is placed to address the problem of socialisation-it is argued that his theory overcomes the inadequacies identified in procedural models such as those of dworkin (1988) and frankfurt (killmister 2013) . in fact, christman has argued that gerald dworkin and harry frankfurt's first and second-order desire theories overlook the autonomycompromising nature of manipulation. being autonomous, according to dworkin and frankfurt, is synonymous with a capacity to critically evaluate firstorder values, desires and preferences by referencing them to one's second-order preferences, values, and desires. thus, one's capacity 'to accept or attempt to change these in light of higher-order preferences and values' makes one autonomous (dworkin 1988, p. 20) . 11 it is held that by exercising such a capacity under second-order desires, persons define their nature, give meaning and coherence to their lives, and take responsibility for the kind of person they are (dworkin 1988, p. 20) . under this theory, second-order values ought to take priority over first-order ones; second-order values are considered to be an individual's realm of autonomous decision-making. i wish to argue that dworkin and frankfurt's second-order accounts are philosophically and, consequently, morally problematic. philosophically, this rational thesis of autonomy fails to convincingly account for the problem of socialisation of values, desires and preferences, internalisations of which compromise second-order values. a person's socalled second-order desires can be a product of socialisation. effective manipulation can reach all the way to second-order desires. thus, indeed 'to judge as autonomous individuals who are hypnotized, brain-washed, or otherwise manipulated into developing second-order desires…is to misunderstand the very nature of autonomy' (killmister 2013, p. 98 ). holton has illustrated: 'to see this [philosophical problem of second-order thesis], suppose that i implanted a second order desire in you by hypnosis… then surely you wouldn't have free will if you got your desires to conform to that; but frankfurt's account seems to have the consequence that you would' (holton 2003, p. 2) . 11 'a first order desire is a desire for anything other than a desire; a second order desire is a desire for a desire. so, for instance, you might have a first order desire to smoke a cigarette; and a second order desire that you desire not to smoke a cigarette… thus [another example], i might wish that i wanted to give all money to charity, since i might think that having such a desire would show me to be an excellent person; but i might nonetheless not actually want that desire to be effective… but when a person does want the first order desire to be effective, when they want it to be their will, frankfurt calls this a second order volition' (holton 2003, p. 1) . it can be inferred from this thesis that a person can act autonomously through identification with second-order desires. that is, a person is seen to have autonomy in so far as she has second order volitions and can bring her first order desires into line with second order volitions or desires. thus, according to this account, dogs and children don't have autonomy because they lack second-order desires or volitions (holton 2003) . they don't have autonomous second-order desires which can be used to control first-order desires. first order values can, for example, be values which form the edifice of a given moral system. thus, common good morality can be situated in first-order values (ockham's beard 2010) . frankfurtian and dworkinian accounts possess a great potential to declare someone who is acting on internalised values to be autonomous. such accounts seem to neglect the fact that external conditions (in the form of, e.g., societal rewards and punishments, and limiting human biological factors) have a tendency to make humans conform to certain behaviours (gaylin and jennings 2003) . this can be seen in the present case of coronavirus where, due to fear of receiving fines or fear of being viewed as social deviant, among other reasons, people who would otherwise wish to open their entertainment venues, go to clubs, restaurants, bars and other social facilities, or go on holidays cannot now do so. internalised first-order desires have consequences on second-order desires. internalised ideas, fears, and values which we later mistakenly come to identify as our own have inescapable consequences on our daily choices or preferences (lanier and henry 2010) . 12 a good example of internalised preference can be inferred from the 18-year-old student theorised by benson (1991) . benson explores the case of an 18-year-old whom through internationalisation of the hollywood idea that appearance is a criterion of self-worth ends up regarding beauty and fashion as essential for her self-worth. first and second-order autonomy accounts fail to explain away this lived social reality. although it is possible to hold individuals to have acted autonomously by virtue of fulfilling frankfurtian or dworkinian first and second-order requirements, it can also be argued that morally this thesis of autonomy may be amoral because it has the potential to ignore that well-being can only be achieved when both individuals and others in society act responsibly towards each other. the common good values which sustain and advance the well-being of society can be denigrated through such outlooks, and the consequences of this would be the annihilation of millions of human beings and the end of human civilisation as we know it, as each individual acts according to their own second-order values. what about christman's theory of autonomy which killmister claims is more persuasive? does christman, who criticises frankfurtian theories of autonomy, provide a more realistic perspective? according to christman's view, for an individual to claim autonomy 'necessary endorsement must be directed at the process of desire-formation, rather than the desire itself' (christman 2009; killmister 2013, p. 98 ). christman's theory of autonomy to me is equally an internalist or rational theory of autonomy. stoljar explains: for christman, preferences or desires will be nonautonomous only if they fail either a competence condition or a hypothetical reflection condition. competency corresponds to the capacity of the agent to form effective intentions relative to a desire as well as to reflect critically about the desire. the hypothetical reflection condition employs the notion of non-alienation to characterize authenticity and hence autonomy. (stoljar 2014, p. 235 ). killmister has illustrated and differentiated christman's account from other procedural accounts of autonomy as follows: for instance, we can take an individual who desires to eat a slice of cheesecake. rather than asking, as dworkin and frankfurt would, whether the individual desires to desire to eat a slice of cheesecake, christman asks how she would feel about her desire, were she to be aware of how the desire were formed. if the desire were implanted by a hypnotist, and knowledge of this process caused the individual to feel alienated from the desire, then that desire [according to christman] would not be autonomous. (killmister 2013, p. 98 ). for christman, for an action to be autonomous the individual must personally identify with the desire through a feeling of non-alienation. like frankfurtian and dworkinian visions of autonomy, i wish to argue that christman's endorsement account is unpersuasive. endorsement accounts of autonomy fail to account for internalisation of norms, values which an individual may mistakenly endorse or identify to be her own. indeed, the problem with christman's theory is not the 'inclusion of a historical version of reflective endorse-ment…, but rather that this condition is insufficient to determine the autonomy of an individual' (killmister, 2013 p. 100 ). christman's hypothetical reflection account fails to appreciate that due to deeply ingrained traditional values, imparted through oppressive (and i add non-oppressive) ideological socialisation over many years of one's life, individuals can identify with deformed desires and not feel alienated (stoljar 2014) . effects of ideological socialisation and environmental changes can cause one to treat stereotypes and acquired feelings as natural to oneself and thus formulate one's desires and plans based on such ingrained nonautonomous desires (gaylin and jennings 2003) . recipients of socialised ideologies 'are unlikely to experience alienation from either the 12 'one of the more astonishing features of current political debates on autonomy and coercion is the naïve underlying assumption about rationality and human motivation. psychology -whether based on behaviourism or on its diametric opposite and antagonist, freudianism (or any of the splinter of these two dominant branches) -views few pieces of human conduct as rational choices selected at the moment. rather, modern motivational psychology tends to see most human behaviour as being "conditioned" or "unconsciously determined," a consequence or product of life experiences that tend to make responses to certain stimuli automatic and unchosen…' (gaylin and jennings 2003, pp. 16-17) . norms that they have internalized or the preferences formed on the basis of the norms' (stoljar 2014, p. 235 ). mele's (1995) external historical account is a hypothetical counter-example to procedural accounts (killmister 2013 ). mele dismisses psychological (internalistic) accounts of autonomy (christman 1999) as insufficient. he advances that, if all individual human beings were like athena or a god, procedural accounts of autonomy would indeed capture what amounts to autonomy, and therefore his (mele's) historical account of autonomy would be unnecessary. however, because humans are not athenas, he argues that his alternative account is therefore valid or convincing and necessary (mele 1995) . i actually find mele's account of autonomy more persuasive than frankfurt, dworkin and christman's accounts put together. for mele, for an agent to be autonomous her actions must not be compelled by another: it is a historical property of agents required for responsibility for the possession of a pro-attitude. a necessary condition of an agent s's authentically possessing a pro-attitude p (e.g., a value or preference) that he has over an interval t is that it be false that s's having p over that interval is, as i will say, compelled -where compulsion is not arranged by s. (mele 1995, p. 166 ). the implication of mele's theory is that pro-attitudes can violate an individual's autonomy. to this effect, mele's theory implies that second-order desires (explained above) must not be a product of pro-attitudes or compulsion. he predicates his theory on an understanding that humans are susceptible to acting on socialised values (compelled or instilled pro-attitudes 13 ). according to mele, compelled pro-attitudes, therefore, make individuals non-autonomous (mele 1995) . put differently, an agent's choices are nonautonomous, according to mele's exposition, when: an agent comes to have a pro-attitude because of an external force, rather than via exercise of her skills for critical reflection and evaluative judgment; (ii) the instilled pro-attitude is one she is unable (in the absence of radical counterfactuals) to eradicate or attenuate; (iii) she did not arrange the by-passing herself; and (iv) she does not, nor did she earlier, possess other pro-attitudes that would support her endorsing the instilled pro-attitude. (killmister 2013, p. 101) the implication of this understanding of autonomy is that human motivation is susceptible to socialised pro-attitudes that are difficult to efface. mele's exposition of autonomy makes more sense as it touches on external factors that internalist accounts of autonomy fail to account for. indeed, a fully competent individual who is able or capable of acting rationally upon desires (values which she has critically reflected upon) cannot necessarily by this very token be safely considered autonomous, as the pro-attitudes (which she considers to be her own) could have been socialised earlier in life. however, i do not completely agree with mele's thesis regarding what he then advances as constituting autonomy. this disagreement lies in mele's advancement of an alternative theory of self-control as being a mechanism through which one can achieve autonomy. this theory (capacity for autonomy by virtue of self-control) appears to water down the fact that both inculcated cognitive biases and environmental changes can compose the very mechanics of self-control. 14 mele's self-control theory does not satisfactorily explain how the problem of socialisation of pro-attitudes acquired since childhood can be qualified in the assessment of what amounts to one's autonomous self-control. studies have established that babies do not have competent mental capacities for reflection during early childhood-a period when they acquire pro-attitudes. 15 killmister has argued that there is a problem in mele's theory if it is to be used as a guide for identifying 'autonomy-inhibiting socialization' (killmister 2013, p. 101 ). mele's approach would rule out most early childhood education directed at developing the very necessary mental capacities. killmister postulates that 'if the relevant capacities are inoperative or not yet developed, as they will be in very young children, mele takes them to have been bypassed' (killmister 2013, p. 70) . 13 a pro-attitude is a person's mental attitude (a feeling or opinion about someone or something) directed toward an action under a certain description; pro-attitudes may include moral views, desires, urges, aesthetic principles and economic prejudices (bunnin and yu 2004) . 14 despite an individual 'being perfectly able to appraise critically and act upon her desires and values, the perfectly self-controlled person may nevertheless be acting on desires and values that have been implanted into her by artificial means, ones whose development has bypassed normal reflection and awareness' (christman 1999, p. 96) . 15 'human beings are born biologically premature, so that unlike most mammals, many of our neurological and physical functions and all of our behavioral capacities exist at birth in potential form only. to come to fruition, these potentials must be molded by a social environment' (gaylin and jennings 2003, p. 34) . the conditions which influence our everyday adult conduct are set by our parents and culture early in life through a conflation of coercion, parental encouragement, emotional intimidation, conditioning, and other mechanisms (gaylin and jennings 2003 ). mele has nonetheless acknowledged that bypassing is common in infants but argues that such bypassing is not sufficient for compulsion unless the pro-attitude is also practically non-sheddable. to this, killmister has responded that many people have pro-attitudes (traceable to early childhood socialisation) that they are practically unable to shed. she claims that even our attitudes towards evidence, reason, and reflection are both presumably inculcated and nearly unsheddable: the problem here is that one of the central roles of socialisation is to inculcate the very pro-attitudes that are required for the kind of self-control mele sees as necessary for autonomy. to be self-controlled is to believe and desire on the basis of an assessment of evidence. this will rely upon having the appropriate pro-attitudes towards evidence and reasons, which must at some point have been instilled in the child. (killmister 2013, p. 102) . in our early development each of us is subjected to physical and social forces of which we are largely ignorant, over which we have no control, yet from which we acquire values, beliefs, motivations, and capacities for rational evaluation that subsequently guide our choices and actions. these forces "destroyed" any capacity to become a different sort of person with selfcontrol regarding any unsheddable pro-attitude that we happen to have. consequently, every unsheddable proattitude is compelled, and anyone with firm unshakeable principles of action ends up being inauthentic and non-autonomous. (kapitan 2000, p. 89 ). stoljar rejects procedural approaches to autonomy by replacing them with a substantive account (stoljar 2000 (stoljar , 2014 . she argues that procedural conceptions fail to appreciate that people who have been subjected to false and oppressive internalised norms cannot be autonomous. according to her thesis, oppressive socialisation can lead an individual to internalise false beliefs which the victim may later come to endorse as her own. decisions made on the basis of internalised false beliefs are therefore not autonomous, she declares. stoljar's thesis indicates that mere possession of false/ oppressive beliefs makes an individual non-autonomous. i wish to agree with killmister's observation that stoljar's theory is initially promising as it acknowledges that 'the question for all theories is what kinds of socialisation are incompatible with autonomy' (killmister 2013, p. 104) . i, however, find stoljar's account unpersuasive. what is problematic about stoljar's theory is that it reduces autonomy to an individual who is free from false/ oppressive socialisation (killmister 2013) . such a perspective fails to consider that for an individual to be autonomous she does not only necessarily need to choose her own moral position but also enjoys the absence of any form of external influences on her self-determination capacity. that an autonomous individual is a self-ruler, or a law unto herself, no matter what form of socialisation she underwent, should not be ignored. an autonomous individual freely acts in accordance with a self-chosen plan by virtue of self-law and/ or morality (kant 1785; gaylin and jennings 2003) . what the preceding arguments entail is that even nonoppressive internalised norms can still be non-autonomous, provided they were instilled or regulated by another or something external to the individual claiming autonomy. if to be autonomous is to be self-governing or self-directing or, in other words, to act on motives, values, and reasons that are one's own, then how can we restrict non-autonomous action to only false/oppressive socialisation? moreover, the fact that the person who is supposed to be a law unto herself is at the mercy of false/oppressive socialisation shows a lack of sovereignty, like that of the christian god, hence the illusion of autonomy. the dialogical theses by westlund (2009) and benson (2011) view a person to be a social self. according to these accounts, a person's answerability to others becomes the key condition for autonomy. in other words, autonomy is synonymous with a person's ability to have normative power or authority over one's decisions (mackenzie 2008) . autonomy thus becomes dispositional. what is encouraging about dialogical approaches is that they indicate that human beings are interconnected-in other words, social selves. as will be shown below, however, the thesis of dispositional autonomy is unconvincing because this theory propounds that a capacity for autonomy is premised on an individual's mental capacity. westlund argues that autonomy should not be understood in terms of an individual's psychology or history; rather it is constituted by a particular kind of disposition. her theory suggests that an agent's answerability to others is a key condition for autonomy. she rejects psychological accounts which designate an autonomous agent as one who critically reflects in an appropriate way in evaluating one's preferences, desires and motives. in other words, westlund rejects rational choice accounts of autonomy because such theories merely argue that autonomy is achieved when an individual engages in critical reflection or if an individual acts or chooses to act in accordance with desires which she has self-reflectively endorsed (friedman 2003; christman 2009 ). according to westlund, one's autonomy is instead undermined if one is unable to give reasons in defence for one's actions or choices (westlund 2009; killmister 2013) . so, an individual who readily answers for herself constitutes a self-governor or autonomous person, she suggests. according to benson, an autonomous act is achieved when a selfregarding attitude is expressed in one's willingness to take responsibility for one's actions (benson 2011) . in this case, if an agent claims authority over her own actions, then such an agent can be considered autonomous. these accounts suggest that certain emotional states and attitudes towards oneself are necessary conditions for autonomy. such approaches appear to require that reliance on critical reflection and judgment for autonomy is only possible if a person can endure criticism of her own sense of basic competence and worth. i wish to state that dialogical accounts of autonomy are philosophically and morally problematic. they are philosophically unpersuasive because such approaches ground autonomy on a person's ability to offer reasons for her choices. and these accounts are morally problematic because they are individualistic in outlook-such outlooks appear to promote autonomy on the basis of meeting moral obligations to self. 16 although westlund criticises psychological accounts of autonomy, i wish to argue that dialogical accounts of autonomy are equally psychological or internalist in substance. thus, the criticisms of rational choice accounts of autonomy can to a larger degree equally be applied to dialogical ones; both approaches clearly ignore or brush aside the reality of socialisation and environmental conditioning. kantian psychological or rational conceptions of autonomy often erroneously portray agents as causally and psychologically independent of environmental conditioning. westlund regards someone as non-autonomous if they defer to something or someone rather than providing their own reasons in choice-making; however, she appears to ignore the fact that defending a choice with plausible reasons is not necessarily synonymous with being its author, or authentically identifying or individually valuing a given choice. an agent can defend, for example, oppressive values, not because that is what the agent desires, but because it is required. external circumstances can cause an agent to embrace oppressive values and yet still be in a position to stand behind a given decision as their own. thus, being answerable to one's choices or behaviour does not make one autonomous, but potentially makes one accountable to socialised values which one's external environment through interpersonal and environmental conditions have imposed upon or inculcated into the agent who is claiming autonomy. a person can give a convincing or persuasive reasoned answer to something not because she necessarily has chosen the value and identifies with it, but because of environmental conditioning and limitations. an agent is capable of defending inculcated or internalised oppressive values through adaptive preference formation (elster 2016) . 17 expressed differently, the problem with westlund and benson's conception of autonomy is that individuals can stand behind decisions or actions which are paradigmatically non-autonomous. this is especially true seeing that it is widely accepted that 'we humans are very good at inventing post-facto explanations of our actions, even to ourselves' (moll 2004 , cited in killmister 2013 . killmister (2013) concludes by suggesting that we do not have absolute autonomy, but 'degrees of autonomy'. i hesitantly agree! one agrees in a strict sense that 'degrees of autonomy' can be in the form of restricted autonomies in which individuals have been placed under laws where boundaries have been drawn on how far they can conduct themselves. one may as well call this 'permitted autonomy'. it is within the boundaries of laws (natural or positive), ethics, regulations and conventions where one can agree that 'degrees of autonomy' are apparently found. thus, yes, we humans have no absolute autonomy. for example, the current covid-19 lockdown and social distancing rules indicate that human autonomy is not absolute. being the law unto oneself is limited to what governments proclaim are boundaries within which individuals can be allowed to rule themselves. however, founding autonomy on limited or relative autonomy also crumbles when the whole picture of human motivations, in light of the ever-changing environment, is closely examined. thus, can killmister convincingly enumerate in what circumstances exactly an agent can enjoy this limited autonomy, circumstances which are excluded from the susceptibility to socialisation, internalisation, biological influences or environmental changes? gaylin (1996) has established that human motivation and behaviour is highly susceptible to environmental influences. therefore, he argues that protagonists of autonomy are mistaken in believing that human behaviour is voluntary. gaylin submits that present behaviour is moreover significantly determined by past treatment (gaylin 1996) . he suggests that human behaviour is less rational than we are willing to admit. he advances that our behaviours are influenced by our emotions, which are usually more effective than logical argument, and adds that shame, fear, guilt, greed and pride influence human behaviour. the implication of this analysis is that a combination of societal morality (morality which can be internalised through effective socialisation), the institution of law in society which defines the boundaries of human behaviour, and human natural susceptibility to emotions (as drivers of decisions) makes the argument for capacity for individual autonomy, even in limited circumstances, problematic. discussing the stifling nature of our biological make-up on human decision-making, morison (1984) has observed that besides historical influences human 'agency' is encumbered with genetic or environmental factors which subtly motivate our day-to-day choices. for morison, much of our human behaviour is significantly influenced by events over which we as individual have little or no control. he quotes melvin konner's the tangled wing: biological constraints on the human spirit (1982) 18 work as an extraordinary book which identifies, among others, the roles genes and the environment play towards behaviours and choice: it requires only a little reflection to realise… that our choice is strictly limited to the possibilities displayed before our conscious in real time including those we can call up from memory or create by synthesizing bits and pieces of previous experience… not nearly so clear is the degree to which the final choice is also so conditioned or "shaped" by genetic patterning or by previous experience of which we are no longer conscious. my hunch is that such influences are much more numerous than we imagine and that autonomy in any strict sense is an illusion. (morison 1984, p. 46 ). my review of autonomy so far agrees with feminist relational accounts to the extent that feminists generally highlight 'that agency cannot be separated from interdependence, since large swaths of our lives, especially during the formative years, are spent dependent on others, and those relationships function to inform our preferences, our values, and our understanding of our selves' (wenner 2020) . hence, i have contended above that autonomy cannot be conceptually explained without convincingly explaining away the impact of human social embeddedness and environmental change on human capacity for autonomy. i have thus shown that feminists' relational account analyses are only insufficient to the degree that they theorise that humans can be autonomous by virtue of a psychological state of mind. in the following discourse, i will show that autonomy is a social phenomenon. crittenden (1993) has observed that critics of liberalism, and even some liberal theorists, have criticised the emphasis of autonomy by liberalists. indeed, internalist conceptions of autonomy are not only conceptually unconvincing, but also, since they are associated with self-sufficiency, they exude 'the worst aspects of atomistic individualism, insulation, social isolation, and an indifference to society's values and interests' (crittenden 1993) . the fact that humans are born into society, are interdependent, co-exist, and therefore have to act in solidarity for the common good is almost ignored in rational theories. humans in psychological theories of autonomy become laws and morality unto themselves. crittenden, like gaylin, acknowledges that human beings by nature are interdependent and interconnected. hence, he argues that a conception of autonomy which is premised on self-sufficiency does not represent a fulfilment of autonomy but its abandonment. he submits that autonomy has a social nature and explains that autonomy develops through sociality, and it also requires sociality for its exercise. crittenden quotes anthony arblaster, who has noted that it is not normal or even desirable for a human to be self-sufficient (arblaster 1984) . he also invokes jennifer nedelsky, who submits that 'the perfectly autonomous man is thus the most perfectly isolated' (nedelsky 1989) . for crittenden, autonomy does not entail isolation from the presence of the influence of others or a necessary rejection of societal values, but the very concept of autonomy depends on these very factors. 'the social nature of autonomy appears to undergird a communitarian notion of the self as socially situated' (crittenden 1993, p. 37) . the author argues, among other reasons, that he believes autonomy is social because humans are not born with autonomy, but that autonomy requires psychosocial development. he also argues that 'autonomy is social because we can only be autonomous when we know we are acting autonomously; and we can only know that when we give an account to others of how we arrived at a decision or action' (crittenden 1993, p. 38) . according to crittenden, the only autonomous person is one who is able to remove herself from the social matrix. he argues that since no person is born autonomous; we may 18 konner has demonstrated that man's behaviour is influenced by nature and nurture (konner 1982) . only be autonomous through socially distancing ourselves from the social matrix. he suggests that self-autonomy, provided one was born and lives in society, is impossible: any kind of introspection or reflection must be done in and through language, and the language we use is itself a cultural or social inheritance. we do not create it, and in this sense also autonomy has a social side… to make our actions and judgments intelligible to ourselves, we not only translate them into language, but also form them through language. the only language we have available by and through which to think rationally and self-reflectively, is one based on cultural tradition… there is implicit in every language, as part of that cultural tradition, a system of norms and standards that determine proper use. (crittenden 1993, p. 44 ). i agree with crittenden's theory of social autonomy, even though there are issues which arise from a further reading of his account which i do not necessarily agree with. when crittenden suggests that an autonomous person is one who is able to distance oneself from the social matrix-a matrix which one earlier identified with-this perspective appears to suggest that an individual is capable of extricating herself from the social context, and from her genetic make-up. if crittenden means that an individual can extricate herself from the environment (a reality which has made that person who she is), i argue that doing so is not possible in reality due to biological constraints, interdependence, common human frailty, and socialisation. in other words, because all human beings have internalised pro-attitudes, it is impossible for them to disown or separate themselves from what they are personally disillusioned to believe are their own personal values and aspirations, if they later on choose to live by themselves. that is, even if a person later decides to live alone, like the asocial koala which only has social intercourse with other koalas during the breeding season, that person very likely already has embedded pro-attitudes which they will inadvertently carry with them, unless they were nursed by a koala as a baby and have since lived alone in the jungle. feminist theorist marina oshana grounds autonomy on a socio-relational premise. oshana submits that autonomy is equivalent to self-governance (she states that the synonyms for self-governing are: 'free-standing', to be independent (self-sufficient), to be separate, and to be self-ruling and sovereign) (oshana 1998) . she rejects psychological conceptions of autonomy and advances an externalist account. she invokes both the internalist (psychological) and externalist (socio-relational) perspectives in her conceptualisation of what autonomy is. oshana submits that individual self-governance is in reality social. for oshana, 'autonomy is a condition of persons constituted, in large part, by external, social relations people find themselves in (or in the absence of certain social relations)' (oshana 1998, p. 81) . in this sense oshana's conception is to some extent similar to crittenden's theory. however, unlike crittenden, oshana adds that manipulation, coercion, subjection to the dominant will of others, and also 'the internal phenomena native to the individual such as captivity to desires or physical impulses, psychological neuroses, or weakness of the will' compromise the capacity for autonomy (oshana 1998, p. 83) . oshana criticises dworkinian, frankfurtian and christmanian accounts of autonomy as inadequate because these accounts are internalist. indeed, as shown above, rational accounts of autonomy premise capacity for autonomy on the 'structural and/or historical character of a person's psychological states and dispositions, and on an agent's judgments about them' (oshana 1998, p. 83) . for oshana, an individual does not become non-autonomous merely by being born in an environment where one has no control, but one becomes non-autonomous by virtue of the effects of the conditions of a given environment. she maintains that choice does not guarantee autonomy. this is because a person who may be exercising what appears to be genuine choice may be compelled to do so by others, and endorsing such choices from within does not guarantee autonomous agency (oshana 1998) . in regards to the assumption that critical reflection in a procedurally independent fashion makes one's choice an autonomous one, oshana argues that 'being able to engage in critical reflection, to take stock of oneself and to shape oneself on the basis of this evaluation, does not guarantee that whatever state of affairs ensues from this activity will be one of autonomy' because autonomy does not exclusively depend on circumstances descriptive of an individual's psychology (oshana 1998, p. 89 ). oshana has therefore advanced four conditions necessary for autonomy: critical reflection, procedural independence, access to a range of relevant options, and the condition of social-relational properties. she argues that an individual cannot be autonomous if: (i) one cannot engage in critical reflection or objective appraisal of one's motives or actions, and the environment in which one's putative values develop; (ii) if she is, in fact, influenced or restricted by others in ways that constrain autonomy (such influences can be by way of manipulation or coercion); (iii) that autonomy becomes questionable when a person claims that their decision was autonomous in circumstances where they were lacking in access to an adequate range of options; and, (iv) an individual who is in society (having relations with others) does not limit herself to relations which enable her to pursue her goals in a context of social and psychological security (oshana 1998) . she submits that these four conditions for autonomy are not mutually exclusive; they must all be satisfied for an individual to claim autonomy. for oshana, her account of autonomy has three benefits: it recognises the human condition; it recognises the status of humans as moral agents; and, that a socio-relational account of autonomy 'can easily explain how persons might be selfgoverning even when manifesting external or communal virtues that might appear to reduce autonomy' (oshana 1998, p. 98) . the socio-relational conception of autonomy is more convincing, philosophically and morally. it not only appreciates that humans are unique, but also recognises that humans are only able to exercise their uniqueness or the 'self' 19 in a socialised world encumbered by fear, emotions, the need for social validation, a changing environment, and the need to act responsibly in order to protect and promote the common good for mutual well-being and survival. ravven (2013, pp. 336-411) has shown that mounting data from history of ethics and neuroscience demonstrate that a human being is in reality the 'i that is we'. she states that neurobiological and other evidence have shown that the body-mind boundaries ethics celebrated as inviolable by millan and kantian ontological theses are mistaken. ravven demonstrates that the dimension of the human self extend beyond the scope of the body-mind, and that human investment in others in the world is finally what ethics is all about, and should be acknowledged as such. we are made for togetherness. throughout, her analysis, she convincingly demonstrates how locating the self as distributed beyond our skins and brains can provide a more plausible and appropropriate ethical and moral approach to hiv testing decisionmaking ethics. for ravven, 'the scope of the self as a moral agent, of who is performing a given moral action, can be distributed beyond the individual to groups, and even extend at times to whole contexts'. this indicates that hiv testing decision-making is in reality complex. firstly, by citing the work of clark (2009), ravven has demonstrated that the self as we understand it is joined to society and is one with the world. this is in contrast to individualised informed consent requirements which advance a self self. according to clark (2009), reported by ravven, 'at least some aspects of human cognition… [are] realised by the ongoing work of the body and/or the extraorganismic environment,' so that the 'physical mechanisms of the mind… are not all in the head' or in other central nervous system. ' ravven explains that our attraction to selfhood fails to account for an observable reality that demonstrates that as humans we are not 'locked-in' agents-as beings whose minds and physical abilities are fixed quantities apt (at best) for mere support and scaffolding by…[our] best tools and technologies.' instead, 'our minds and bodies are essentially open to episodes of deep and transformative restructuring in which new equipment (both physical and 'mental') can become quite literally incorporated into the thinking and acting systems that we identify as our own minds and bodies.' she emphasises and advances that humans do not only discover the world within their selves, but also discover themselves as parts of it. this entails that humans ought not to conceive themselves as the whole story of the whole, but as part of the story that makes the whole story complete. this outlook is similar to ssa ontological perspective that situate a human as 'a part of the organic whole' (mbiti 1970; menkiti 1984; diop 1991; tutu 1999; nussbaum 2003; woods 2002 woods -2004 molefe 2019) . therefore, an hiv testing regime that locates hiv as a shared reality will also adopt appropriate ethics and human rights that intergrate this reality. current hiv testing ethics and human rights on decision-making in ssa have not reflected this human reality (eba 2015) . secondly, ravven shows how a person is an 'i that is we' by invoking co-consciousness empirical findings that have demonstrated that 'the self as a fully aware self-aware includes perspectives that were initially 'other': third person perspectives now taken in as one's own have relocated the self outside itself and in another'. she suggests that as humans we create a shared self-a self that is both mine and yours-when we unconsciously integrate our people's values and perspectives with our first person perspectives. she therefore concludes that humans are at best relational selves who discover the self through other's eyes with whom they have a shared reality, and a shared world. ravven indicates that humans share an environment of inescapable embeddedness. thus, ethicists, including hiv policymakers in ssa, should begin to acknowledge and accept that the self cannot exist in a social vacuum. the discovery that the self is not atomistic therefore 'heralds the end of the…cherished illusion… namely the illusory goal of independence, self-sufficiency, and free autonomy' promoted in hiv testing ethics which seek to disintegrate the self from her social embeddedness: at any given time the self is constructed by a relation to another person. 'people have as many selves selves as they have significant others.' these selves emerge from the early relationships with parents, siblings, extended family members, and others who have an impact on one's life, and they profoundly affect motivation and emotions. unconsciously perceived similarity triggers the relational patterns to take hold, without our conscious awareness or even the ability to control the process… the other and the relationships are necessary for the feeling of 'me' of this 'me'… heinz koht [a psychoanalyst]… held that it is through 'expanding its selfexperience to include the whole surround' that an infant comes to be able to (1) feel itself confirmed as a self, (2) pursue its ambitions, and (3) pursues its ideals. (ravven 2013, pp. 369-370) . thirdly, describing findings from neurobiological experiments, ravven reports that research by thomas metzinger and olaf blanke have also demonstrated that the boundaries of the self transcends the boundaries of our bodies and minds: we mistake the feeling of the self as our interior, a soul-thing, a solid bounded essence that is our true self that we alone know and disclose to the world. but that is a false picture. we are more like verbs than nouns; we make parts of the world feel like the self, and we fill our feeling with our engagemnets in the world. (ravven 2013, p. 372) . fourthly, ravven turns to neurochemistry discoveries, like the one by donald w pfaff, that have also illustrated that the self transcends body-mind. and suggests that ethics should be grounded on relational autonomy: pfaff shows that the feeling of the self and where we draw the line between the self and other, self and the world, depend upon particular neurochemicals. these chemicals that produce the self-other divide, he says, are sometimes turned off, and when they are we experience others as if they were ourselves. the neurochemistry that produces the feeling of self as extending into others, he argues, underlies ethics. he says the chemicals that turn off the feeling of self-other boundary operate in fear and in love, in empathy and in aggression, and in other situations. these chemicals create a sense of shared experience and even of merger. pfaff believes that this mechanism produces ethical action by creating empathetic responses to others. (ravven, 2013, p. 373 ). the dominant, individualistic understanding of autonomy that features in' bioethics and medical ethics underpin the idea that individuals are 'in their ideal form, independent, self-interested and rational gain-maximising decision-makers' (dove et al. 2017, p. 150) . my review of autonomy in this article demonstrates that such a view is erroneous as it does not convincingly account for both external and internal (influences of biology on decision-making) environmental factors. this individualistic conception of an autonomous patient is inconsistent with lived human life-it does not capture the breadth of lived human reality that shows the 'i that is we'. conceptions of autonomy, especially in clinical practice, 'should accommodate the fact that people are rarely, if ever, fully independent individuals' (dove et al. 2017, p. 151) , hence, as an example, the challenge posed by hiv stigma to hiv testing uptake. as humans, we are social, cultural and biological beings whose decisions, including in healthcare, are shaped by a conflation of powerful social, cultural and biological forces that often times are beyond us (konner 1982; gaylin and jennings 2003; ravven 2013; sapolsky 2017) . this is the more reason why we need each other as humans: as opposed to atomising ourselves. hiv testing ethics in ssa should be premised on the human reality of a self beyond itself, as this approach captures the physiological, biological, pyschological, sociological and cultural aspects of human condition; unlike the kantian and millan conceptions of personal autonomy which are largely abstract psychological ontologies. instead of advancing abstract ethics of a self within self and unto self, ssa hiv policymakers have also something to learn from ssa traditional and moral analysis that view a person as 'a person through other persons' (mbiti 1970; menkiti 1984 )-an outlook that was developed in the light of ssa's unique experiences and lived reality, besides ontological deliberations. this ssa outlook is consistent with the conclusion of my analysis in this article that suggests that humans are incapable of being autonomous-they are social creatures who constantly, also unconsciously, simulate the values of their socio-cultural environments as their own. drawing ethics that recognise and reflect environmental impacts, including of love, empathy, fear and hate, on individual decision-making, will therefore be a good starting point for reconfiguring individualised hiv testing informed consent requirements in ssa. hiv testing policymakers in africa should cease ignoring the impact of the illusion of personal autonomy on our shared humanity. rather, they should explore their own lived experiences and reality, review studies that indicate that as human we are part of the organic whole, explore potential ethics grounded on the recognition of social attachments as evidenced in sexual and parental love, and even in friendship, resist the illusion of autonomy, and advance realistic ethics where the individualised cold isolated person is restored to who she really is: a unique part of an organic whole. 'parent love and especially motherly love, 'take the blurring of identity between two living beings to new heights,'… [ -] the upshot of all this is that the various mechanisms that induce sexual and parental attachment can be harnessed and be recruited for all kinds of prosocial behaviours and attachments' (ravven 2013, p. 375) , as opposed to surrendering hiv testing ethics to the seduction of autonomy. to this end, i wish to invite ssa hiv policymakers to consider basing hiv testing on altruism, or what ravven (2013) terms, an ethic of: 'shared selves'-'a sefiness that loses the boundaries of the skin and is extended and distributed to others'. a self who is born into society, is interdependent, interrelated, and shares with others shared human frailty and vulnerability. recognising this kind self can lead to an appropriate reconfiguration of hiv testing ethics. hiv testing ethics, in particular informed consent requirements that are now premised on personal autonomy, should reflect a human being who is unique and yet a creature of the inescapable inculcating environment that makes her the 'i that is we'. according to ssa traditional and moral thought, 'i am because we are', and 'we are because i am'. ravven states: mounting data from neurosciences show that evil is rooted in the failure to see others as ourselves. in the case of genocides such as the holocaust, it is a collective failure of society-wide proportions. the self-other boundary beyond the skin is of central importance to rethinking of ethics, to a deeper understanding of both good and evil. (ravven 2013, p. 377) personal autonomy cannot provide a sufficient and persuasive starting point for bioethics (o'neill 2002 (o'neill , 2003 , in particular hiv testing decision-making. it is necessary for ssa countries to reconfigure their healthcare ethics and place them within the overall compass of human condition, and in the light of the setting of ssa's unique socio-economic and cultural background (wood 2002 (wood -2004 ravven 2013; joseph et al. 2018) . it is appropriate to get rid of abstract liberal and libertarian concepts of an autonomous patient and reconsider the observable and lived situation of the relationship between patients and their environments. the emphasis on primacy of patient autonomy distracts attention from important aspects of life and healthcare: 'wellbeing is social'. it is crucial to eradicate the man-made autonomy versus medical intervention crisis and move towards greater cooperation. hiv testing informed consent requirements should be premised on ethics of love, honesty, empathy, sympathy, friendship, togetherness and solidarity. personal autonomy is an illusion. a review of the universalised dominant western liberal view of personal autonomy demonstrates that the general agreement that informed consent in hiv testing is required to respect personal autonomy 'and that autonomy is a basic ethical value, is more apparent than real' (manson and o'neill 2007, p. 17) . personal autonomy, strictly speaking, is an illusion. human lived experience and reality show that our lives and decisions are encumbered by combined forces of socialisation, genetics and changing external environmental factors that make the idea of an ability or capacity for autonomy resemble a fantastic dream which, when we wake up, we realise was just a dream and not something achievable in a real, civilised and striving society. in this vein, universalised ethics which continue to premise informed consent requirements in hiv testing on personal autonomy are inappropriate. human beings are not asocial or atomistic that they can be a law unto themselves; they are individuals who are enmeshed in complex social realities of reciprocity, mutual obligations, and responsibilities for amicable co-existence, well-being and survival. moreover '[h]uman behaviour is less 'voluntary' than libertarians and theorists of autonomy would have" us believe (gaylin and jennings 2003, pp. 7-8) . as indicated in the analysis, "human behaviour is less rational than most of us would like to believe it is', in that 'fear, greed, shame, guilt, and pride fuel the machinery of' our decision-making and choices (gaylin and jennings 2003, pp. 7-8) . it is these aspects which should also be acknowledged and reflected in hiv testing informed consent requirements in ssa. like what has been shown in the covid-19 pandemic response, persons living in ssa countries should be made aware that as social beings and living in complex relations with others in society, they have a duty to test for hiv because hiv is an epidemic that affect the lives of both people living with hiv and others (especially, close family members). the hiv epidemic not only affects the person living with hiv, but causes suffering, and some instances even death, to family members and people in the community. hiv testing, like the involuntary covid-19 lockdowns and social distancing, is a common good. in other words, the effects of aids are often corporately felt within the arena of the 'i that is we'-especially in the ssa setting where relational autonomy is articulated (woods 2002 (woods -2004 . in conclusion, whenever we as humans are faced with the temptation to celebrate our 'individual autonomous right', we should pause, consider, and remember that we are because of the humanity of others: our birth came through others; our name was given to us by others; we have been educated by others; the respect we demand is given by others; the first bath we had was given to us by others when we were born; our last bath will be given by others; our funeral and potentially dignified burial will be organised and conducted by others; and everything we have owned will be inherited by others once we are dead. we are simply not autonomous as individuals; we are but interdependent and interconnected social creatures. therefore, we should act in solidarity towards each other by formulating appropriate hiv testing ethics and encouraging people to test for hiv so those who need it can access appropriate therapy. the covid-19 crisis has taught us that 'wellbeing isn't individual but social' (jones 2020) . this indicates that ssa countries may not be able to achieve the sustainable development goal (sdg) 3 (united nations 2019), together with the unaids' fast-track strategy that explicitly call to end the epidemic by 2030 (unaids 2014), if hiv testing policies in ssa continue to ignore that humans are social beings (gaylin and jennings 2003) and 'wellbeing isn't individual but social' (jones 2020) . it is up to hiv policymakers in these countries to re-examine personal autonomy in hiv testing, and hopefully identify appropriate bioethics and human rights policies which are reflective of natural human condition and are suitable for the ssa socio-cultural setting and hiv epidemic reality. rethinking hiv exceptionalism: the ethics of opt-out hiv testing in sub-saharan africa the rise and decline of western of western liberalism who test & treat policy and change in art uptake principles of biomedical ethics autonomy and opressed socialisation narrative self-understanding and relational autonomy: comments on mackenzie c. poltera, j, 'narrative integration, fragmented selves, and autonomy'. symposia on gender hiv exceptionalism: development through disease in sierra leone mapping and characterising areas with high levels of hiv transmission in sub-saharan africa: a geospatial analysis of national survey data tocno de?id=g9781 40510 6795_chunk _g9781 40510 67951 7_ss1-271#citat ion hiv/aids is no longer the leading cause of death in africa all sa 63 (cc) at 74 (s. afr moral principlism alone is insufficient, and traditional moral theories remain important for practical ethics reviewed work(s): autonomous agents: from self-control to autonomy by alfred r. mele the politics of person: individual autonomy and social-historical selves autonomy and adaptive preferences the social nature of autonomy from exceptionalism to normalisation: a reappraisal of attitudes and practice around hiv testing civilisation or barbarism: an authetntic anthropology beyond individualism: is there a place for relational autonomy in clinical practice and research the theory and practice of autonomy mapping hiv prevalence in sub-saharan hiv-specific legislation in sub-saharan africa: a comprehensive guman rights analysis communities benefit from door-to-door hiv testing sour grapes: studies in the subversion of rationality autonomy and its limits: what place for the public good a history and theory of informed consent harm to self: the moral limits of criminal law the importance of what we care about ethics of hiv testing in general practice without informed consent: a case series worshiping autonomy the perversion of autonomy: coercion and constraints in a liberal society introduction to philosophy: free will ii afr. comm'n on hum. & peoples' rts the common good. the stanford encyclopedia of philosophy medical law: text, cases and materials the penny has dropped that wellbeing isn't individual but social. the guardian groundwork of metaphysic of morals in nous supplement; philosophical perspectives, 14. action and freedom hiv infection and aids in sub-saharan africa: current status, challenges and opportunites autonomy and the problem of socialisation the tangled wing: biological constraints on the human spirit essential criminology relational autonomy, normative authority and perfectionism rethinking informed consent in bioethics african religions and philosophy an introduction to aspects of health law: bioethical principles autonomous agents: from self-control to autonomy person and community in african traditional thought on liberty, utilitarianism, and other essays the biological limits of autonomy ethical and legal issues on hiv testing, policy and the practice of dentistry treating all people living with hiv in sub-saharan africa: a new era calling for new approaches aristotelian reconstruction of the concept of personal identity. sophia reconceiving autonomy: sources, thoughts and possibilities african culture and ubuntu: reflections of a south african values and moral pragmatism the inaugural address: autonomy: the emperor's new clothes the rise and fall of aids exceptionalism personal autonomy and society 833; 112 s.ct. 2791; 120 l the self beyond itself: an alternative history of ethics, the new brain sciences, and the myth of free will autonomy in medical ethics: issues of informed consent evaluating the right to autonomy argument in the debate on coercive antenatal hiv testing in south africa they thought this hiv strategy couldn't work. but it did opportunities and challenges for 'test-andtreat': insights from eastern and southern africa ethical dilemmas concerning autonomy when persons with dementia wish to live at home: a qualitative, hermeneutic study the history of aids exceptionalism autonomy and intervention in medical practice southern africa litigation centre testing: a priority in aids prevention xsom5 2gzau k. accessed global action to reduce hiv stigma and discrimination autonomy and the feminist intuition autonomy and adaptive preference formation the impacts of community-based hiv testing and counselling on testing uptake: a systematic review towards universal voluntary hiv testing and counselling: a systematic review and meta-analysis of community-based approaches kantian personal autonomy rethinking autonomy: a critique of principlism in biomedical ethics no future without forgiveness assessing the achievements of the millenium development goals in southern africa resolution adopted by the general assembly on 8 un general assembly adopts popitical declaration to fast-track progess on ending aids fast-track: ending the aids epidemic by 2030 understanding fast-track: accelerating action to end the aids epidemic by 2030 unaids. 2020. 90-90-90: an ambitious treatment target to help end the aids epidemic unaids. 2020a. global hiv & aids statistics-2019 fact sheet test and treat showing results in uganda and zambia the benefits of knowing your hiv status report of the special rapporteur on the right of everyone to the enjoyment of the highest attainable standard of physical and mental health-a/64/272 united nations united nations division for social policy and development, indigenous peoples united nations department of economic and social affairs (un-desa) the common good non-domination and the limits of relational autonomy rights as slogans: a theory of human rights based on african humanism acknowledgements this paper was prepared thanks to study support from birkbeck, university of london, uk during the author's phd studies. the author is also grateful to prof. matthew weait, deputy vice-chancellor -university of hertfordshire for his supervisory support during research on the subject matter examined in this article. funding none.data availability not applicable.code availability none. conflict of interest the authors declared that they have no conflict of interest. key: cord-354029-mp5r82g4 authors: earp, l. j.; delos, s. e.; park, h. e.; white, j. m. title: the many mechanisms of viral membrane fusion proteins date: 2005 journal: membrane trafficking in viral replication doi: 10.1007/3-540-26764-6_2 sha: doc_id: 354029 cord_uid: mp5r82g4 every enveloped virus fuses its membrane with a host cell membrane, thereby releasing its genome into the cytoplasm and initiating the viral replication cycle. in each case, one or a small set of viral surface transmembrane glycoproteins mediates fusion. viral fusion proteins vary in their mode of activation and in structural class. these features combine to yield many different fusion mechanisms. despite their differences, common principles for how fusion proteins function are emerging: in response to an activating trigger, the metastable fusion protein converts to an extended, in some cases rodlike structure, which inserts into the target membrane via its fusion peptide. a subsequent conformational change causes the fusion protein to fold back upon itself, thereby bringing its fusion peptide and its transmembrane domain—and their attached target and viral membranes—into intimate contact. fusion ensues as the initial lipid stalk progresses through local hemifusion, and then opening and enlargement of a fusion pore. here we review recent advances in our understanding of how fusion proteins are activated, how fusion proteins change conformation during fusion, and what is happening to the lipids during fusion. we also briefly discuss the therapeutic potential of fusion inhibitors in treating viral infections. 1 introduction fusion of enveloped viruses with host cells remains an important topic of research for two major reasons. first, it has recently become clear that fusion is a good target for therapeutic intervention (kilby et al. 1998 ). second, viral fusion reactions continue to serve as models for cellular fusion events. although several viral fusion proteins, such as influenza hemagglutinin (ha) and the human immunodeficiency virus (hiv) envelope glycoprotein (env), have emerged as paradigms, it is important to realize that there are many distinguishing features among viral fusion proteins (table 1 ). viral fusion proteins can be activated for fusion by different mechanisms. they have also been classified according to structural criteria. for some viruses, the viral receptor does not actively participate in fusion, whereas for others, one or more receptors are essential players. the location of the fusion peptide, critical for fusion, can vary. finally, whereas some viruses require a single viral glycoprotein to mediate fusion, others require multiple viral glycoproteins. there are many excellent recent reviews on viral fusion and the glycoproteins that mediate this process (durell et al. 1997; eckert and kim 2001; heinz and allison 2001; skehel and wiley 2000; weissenhorn et al. 1999) . the goal of this review is to give the reader an appreciation for the diversity of viral fusion mechanisms. 2 all fusion proteins exist on virion surfaces in a metastable state in which the fusion peptide, a critical hydrophobic sequence, is hidden or shielded within the glycoprotein oligomer (carr et al. 1997 ; hernandez et al. 1996; rey et al. 1995; skehel and wiley 2000; wilson et al. 1981) . after activation, the fusion peptides are rendered accessible for interaction with a target membrane. a major distinction among viral fusion proteins is the "trigger" for activation. there are two well-recognized mechanisms: (1) exposure to low ph and (2) specific interactions with target cell receptors at neutral ph. a third mechanism involving receptor priming at neutral ph followed by further activation at low ph was recently proposed (mothes et al. 2000) . orthomyxoviruses, togaviruses, flaviviruses, rhabdoviruses, bunyaviruses, arenaviruses, and, apparently, filoviruses require low ph to fuse with target membranes (table 1 ) (doms et al. 1985; gaudin et al. 1999b; stegmann et al. 1987; . these viruses are endocytosed after binding to the target cell surface. the low-ph environment of the endosome activates the viral fusion protein to convert from a metastable state to one that is capable of driving fusion. although the presence of a receptor may modulate the rate or extent of fusion stegmann et al. 1996; white et al. 1982) , receptors are not essential for low-ph-dependent fusion. low-ph-dependent fusion generally occurs within seconds to minutes at 37c but can also occur, albeit more slowly, at t<22c. four main techniques have been used to assess whether a virus requires low ph to fuse. the first technique is testing the effects of agents, such as bafilomycin, that inhibit endosomal acidification. in some studies of this type, fusion has been measured directly by assessing the transfer of fluorescent probes from the virus to the target cell (earp et al. 2003; irurzun et al. 1997; zarkik et al. 1997) . in others, fusion has been inferred by monitoring postfusion events, such as the synthesis of viral dna (mothes et al. 2000) . a second test is to assess whether fusion of bound virions can be induced by briefly warming virus-cell complexes in low-ph medium (mothes et al. 2000; . a third test is to assess whether pretreatment of virions at low ph (in the absence of target membranes) inactivates the virus for fusion. some (bron et al. 1993; corver et al. 2000; di simone and buchmeier 1995; korte et al. 1999; nir et al. 1990; stegmann et al. 1987) , but not all (puri et al. 1988) , viruses that fuse at low ph can be inactivated by this method. viral fusion proteins that are inactivated by low ph undergo irreversible conformational changes. in the case of x:31 ha, this results in insertion of the fusion peptide into the viral membrane (korte et al. 1999; weber et al. 1994) . the fourth test is to assess whether cells expressing the viral fusion protein can fuse. cell-cell fusion can be observed by light or fluorescence microscopy (frey et al. 1995; melikyan et al. 1997b; mothes et al. 2000) , or it can be scored with gene reporter assays that monitor interactions of components from the fusing cells earp et al. 2003; feng et al. 1996; nussbaum et al. 1994) . although cellcell fusion assays are relatively simple to perform, the results do not always correlate with virus-cell fusion or infection (earp et al. 2003; lavillette et al. 1998; schmid et al. 2000) . many enveloped viruses do not require low ph to fuse with target cells. this has generally been established in controlled experiments using the approaches described in sect. 2.1. viruses that can fuse at neutral ph include paramyxoviruses, herpesviruses, coronaviruses, poxviruses, and most retroviruses (table 1) (hernandez et al. 1997; mcclure et al. 1990; stein et al. 1987; taguchi and matsuyama 2002) . the fusion proteins of these viruses are activated via specific interactions with one or more receptors in the target cell membrane (hernandez et al. 1996; hunter 1997; stein et al. 1987) . viruses that can fuse at neutral ph are thought to do so at the plasma membrane. however, they may also be able to fuse with neutral-ph intracellular compartments (e.g., caveosomes) that can be accessed through newly recognized endocytic pathways (pelkmans and helenius 2003; shin and abraham 2001 ) (see also the chapter by sieczkarski and whittaker, this volume). it is important to note, however, that viruses that can fuse at neutral ph may also possess the ability to fuse at low ph (earp et al. 2003; fackler and peterlin 2000) . to date, neutral-ph fusion has been found to display a sharp temperature threshold, with little or no fusion occurring at t<20c. recently, a third model was proposed for the activation of alpharetroviruses. in this model, activation of the alpharetroviral env begins with receptor binding at neutral ph (at t>22c) but is only complete after exposure to low ph (mothes et al. 2000) . the role of low ph in this "two-step" model is derived from two key observations: (1) the continuous presence of endosomal acidification inhibitors prevents production of alpharetroviral reverse transcripts, and (2) cells expressing env and cells expressing the viral receptor only form large syncytia after exposure to low ph (mothes et al. 2000) . our recent work indicates that alpharetrovirus fusion can proceed to the lipid mixing stage at neutral ph (earp et al. 2003) , and that receptor binding and low ph sequentially induce distinct conformational changes in the alpharetrovial env (matsuyama et al. 2004 ). current work is now focused on determining the precise role of low ph in the fusion cascade. all viral fusion proteins contain a relatively large ectodomain, generally a single transmembrane domain, and all contain a cytoplasmic tail. so far, two major groups (class i and class ii) have been defined based on structural criteria lescar et al. 2001) (tables 1 and 2) . class i fusion proteins are synthesized as precursors that are cleaved into two subunits by host cell proteases. in some cases (e.g., influenza ha), the two subunits remain associated through a disulfide bond; in others (e.g., hiv env), the two subunits remain associated through noncovalent interactions. the proteolytic processing event that generates the two subunits is critical, as it creates the metastable state of the fusion protein (chen et al. 1998) . class i fusion proteins exist as relatively long trimeric spikes in both their metastable and activated states. in their metastable states, they project perpendicularly to the viral membrane. the activated forms of the fusion subunits of known class i fusion proteins are highly a-helical , and the final lowestenergy (which we will refer to as "postfusion") forms ( fig. 1) contain "six-helix bundles" (bullough et al. 1994; carr and kim 1993) . all six-helix bundles contain a relatively long (65-115 ) central n-terminal trimeric coiled-coil. some (e.g., hiv env, siv env, and paramyxovirus f) form six-helix bundles that extend to their membrane proximal ends [i.e., three c-terminal helices (fig. 1a , green) pack in the grooves of the central coiled-coil (fig. 1a, blue) ]. others display a mixture of helical and nonhelical segments that pack into the grooves of the central coiledcoil. for example, the ha2 subunit of influenza ha contains a relatively small six-helix bundle (fig. 1 , green/blue) at its membrane distal end, followed by an extended chain ( fig. 1 , yellow) that packs in the groove and extends to the n-terminal (membrane proximal) end of its central coiled-coil (fig. 1b) . because of these variations, the postfusion forms of class i fusion proteins are often referred to as "trimers of hairpins" (eckert and kim 2001) . the general structure of class ii fusion proteins is quite different from that of class i fusion proteins. a well-characterized example is the envelope glycoprotein (e) of tick-borne encephalitis (tbe) virus. during biosynthesis, tbe e and a second viral membrane glycoprotein, the precursor to the membrane protein (prm), form heterodimers. as virions ma[lamb 1993; colman and lawrence 2003] ture, a host cell protease cleaves prm, resulting in reorganization of proteins on the viral surface (allison et al. 1995) . after prm cleavage, the e proteins exist as metastable homodimers. the ectodomains of the dimer are oriented antiparallel to one another. in further contrast to the trimeric class i fusion protein spikes, the ectodomains of the e homodimer lie parallel to the viral membrane and close to the surface. the tbe e protein is composed mostly of b-strand structure rey et al. 1995) . the architecture of the semliki forest virus (sfv) spike, another well-characterized class ii fusion protein, is similar to that of tbe e, but in this case, the metastable oligomer is a heterodimer of two membrane-anchored proteins, e1 and e2, with an associated small protein (e3). in sects. 4.1-4.4, we discuss a few examples of viral fusion proteins that employ different fusion mechanisms in more detail. these will include high-resolution structures are available for both the complete native (metastable) (wilson et al. 1981 ) and activated (bullough et al. 1994; chen et al. 1999 ) forms of the influenza ha. on the viral surface, ha exists as a trimer of heterodimers ( fig. 2a) . each heterodimer consists of ha1, which contains the receptor binding domain (fig. 2 , gray), and ha2, which contains the fusion peptide ( fig. 2 , red) and the transmembrane domain (located at the c-terminus). in the native (neutral ph) structure, the fusion peptide is buried within the ha oligomer. three long helices, one from each monomer, come together to form the triple-stranded coiled-coil of the metastable trimer ( fig. 2a and b, blue and green). low-ph-induced conformational changes within influenza ha. ha1 is depicted in gray. the fusion peptide is red (ha2 residues 1-24). the coiled-coil is blue, with the c-terminal helix colored green. the c-terminal extended region is yellow. the transmembrane domain (not shown) attaches to the c-terminal end, indicated by "c", of ha2. a model for conformational changes: a in the native, metastable, structure of ha, the fusion peptides are buried within the trimer interface. ha1 acts as a clamp to hold ha2 in a metastable state. ha2 is largely shielded by ha1. to illuminate the ha2 core, we have cartooned the portion of ha1 that covers ha2 as a simple (gray) line. b on exposure to low ph, the ha1 headgroups separate, allowing expulsion of the fusion peptide. c a loop-to-helix transition causes the fusion peptide to be repositioned to one end of ha2, where it can bind to the target membrane. d a helix-to-loop transition causes the c-terminal helix and the c-terminal extended region to reverse direction and bind to the grooves of the coiled-coil in an antiparallel orientation on exposure to low ph, ha undergoes dramatic conformational changes. the globular head domains separate, releasing the clamp that holds ha2 in its metastable state (fig. 2b) . as a result, the fusion peptide is exposed (fig. 2c , red) at the top of an extended triple-stranded coiled-coil, in a position where it can interact with the target membrane. a helix-to-loop transition causes a short helix (fig. 2d , green) and the c-terminal extended region (yellow) to flip up and run antiparallel to the central coiled-coil (bullough et al. 1994) . as a result, the fusion peptide and transmembrane domain are brought into close proximity at the same end of the molecule (fig. 2d ). many regions of ha are important for fusion. the fusion peptide is critical for hydrophobic attachment of the virus to the target membrane (sect. 6.1). mutations that prevent (1) globular head domain separation (godley et al. 1992; kemble et al. 1992 ), (2) the "b-loop"-to helix transition (gruenke et al. 2002; qiao et al. 1998 ), or (3) the c-terminal extended region from packing into the grooves of the final coiled-coil (borrego-diaz et al. 2003; park et al. 2003) ablate the ability of ha to reach the lipid mixing stage of fusion. in our model (gruenke et al. 2002) , conversion of ha to a prehairpin intermediate ( fig. 2c ) allows ha to bind to the target membrane. further conversion to the hairpin structure ( fig. 2d ) then drives the formation and opening of a fusion pore. like influenza ha, hiv env is synthesized as a single-chain precursor and cleaved during biosynthesis to yield gp120 and gp41. native (metastable) hiv env is a trimer of the heterodimers of gp120 (the receptor binding subunit) and gp41 (the fusion subunit). env is activated for fusion (at neutral ph) after sequential binding to cd4 and a coreceptor (a chemokine receptor). binding of env to cd4 causes conformational changes in env that permit binding to the coreceptor. after coreceptor binding, additional conformational changes occur in env that lead to fusion (eckert and kim 2001) . crystal structures exist for the core of the gp120 subunit (kwong et al. 1998 ) as well as for the postfusion (fig. 3, step 6) form of gp41 (chan et al. 1997; weissenhorn et al. 1997) . however, there is not yet a crystal structure of the native (metastable) env trimer. therefore, a detailed picture of hiv env activation via receptor interaction is not available. we presume that the first steps of env activation are separation of the globular head domains, expulsion of the fusion peptide, and extension of gp41 into a prehairpin intermediate (fig. 3, step 1). several lines of evidence indicate the existence of the prehairpin intermediate. for example, peptide analogs of the c-terminal helix (fig. 3 , green) strongly inhibit hiv fusion and infection (chan and kim 1998; kilby et al. 1998 ). also, a synthetic peptide corresponding to the c-terminal helix coimmunoprecipitates with hiv env after engagement of receptors (furuta et al. 1998; he et al. 2003 ). the c-terminal helix then packs, in an antiparallel fashion, into the groove of the n-terminal coiled-coil ( fig. 3, step 5). because the c-terminal helices of gp41 extend along the entire length of the n-terminal coiled-coil, this packing would bring the fusion peptide and transmembrane domain very close together. the transition to the six-helix bundle drives membrane merger (melikyan et al. 2000a ). moreover, complete six-helix bundles are needed to form "robust" fusion pores . as mentioned above, hiv studies, primarily using epitope accessibility assays, have indicated that engagement of hiv receptors induces contarget cell receptors are not pictured in this model. on exposure to receptor and coreceptor at t !22c and neutral ph, env undergoes conformational changes that result in exposure of the fusion peptides (step 1), which then insert into the target membrane ( step 2). multiple envs may cluster ( step 3) to form a fusion site. additional conformational changes (steps 4 and 5) lead to the formation of a six-helix bundle, resulting in hemifusion (step 5) (defined as mixing of the outer leaflets of the viral and cellular membranes). eventually a fusion pore forms (step 6) and enlarges (not shown) formational changes in gp120 and gp41 (eckert and kim 2001; xiang et al. 2002) . a remaining issue for all receptor-activated viral fusion proteins is how information is transmitted (after receptor binding) through the receptor binding subunit to the fusion subunit. such transmission is essential to allow rearrangements in the fusion subunit (e.g., six-helix bundle formation) that drive fusion. for hiv, part of the mechanism may involve reduction of one or more disulfide bonds in gp120 (abrahamyan et al. 2003; barbouche et al. 2003; fenouillet et al. 2001; gallina et al. 2002) . in murine retroviral envs, a proline-rich hinge region appears to relay receptor binding information from the n-terminal to the c-terminal region of the receptor binding subunits (su) (barnett and cunningham 2001; lavillette et al. 2001) . because the proline-rich region of su is linked to tm by a disulfide bond (pinter et al. 1997) , this may provide a relay system to trigger conformational changes in the fusion subunit. clearly, the molecular pathways by which receptor-activated fusion proteins change from their metastable to their activated forms need to be defined. in fig. 3 , we show a working model for hiv env-mediated fusion. it is derived in part from studies with influenza ha, and it is similar to other hiv fusion models (eckert and kim 2001) . our hypothesis is that all class i fusion proteins will employ similar mechanisms. we note, however, that even in the case of influenza ha, alternate models are still entertained (see fig. 2 in jahn et al. 2003) . furthermore, others have suggested that different class i fusion proteins may use fundamentally different mechanisms (chen et al. 2001a) . the features that we predict will be common to the fusion mechanisms of all class i fusion proteins (fig. 3) include: (1) conversion from a metastable state to an activated state, (2) exposure and repositioning of the fusion peptide for binding to the target bilayer, (3) recruitment of several activated fusion proteins to a fusion site (blumenthal et al. 1996; danieli et al. 1996; markovic et al. 2001; markovic et al. 1998) , and (4) subsequent conformational changes that result in close apposition of the fusion peptide and the transmembrane domain. the viral fusion proteins that have thus far been discussed in detail contain a receptor binding domain (e.g., the gp120 subunit of hiv env) within the fusion protein spike. in other cases, the receptor binding do-main resides in a separate viral spike. paramyxoviruses have an attachment protein spike and a separate fusion (f) protein spike. most, but not all, paramyxoviruses require both the attachment protein and the f protein for fusion (bagai and lamb 1995; paterson et al. 2000) . in most cases, the attachment protein must come from the same paramyxovirus as the fusion protein (bossart et al. 2002) . in the few cases in which the f protein is sufficient, fusion is enhanced if the attachment protein is also expressed (bagai and lamb 1995) . the need for the attachment protein can be overcome by mutations in the f protein (paterson et al. 2000; seth et al. 2003) or by conducting fusion reactions at t>37c (paterson et al. 2000; wharton et al. 2000) . paramyxovirus fusion proteins thus represent special cases of receptor-activated fusion proteins, in which receptor activation is communicated from one viral spike glycoprotein to another. f proteins are proteolytically cleaved during biosynthesis to generate two disulfide-bonded subunits, f 1 and f 2 (begona ruiz-arguello et al. 2002; gonzalez-reyes et al. 2001; lamb 1993) , found as metastable trimers of dimers (baker et al. 1999) on virions. it has been suggested that binding of the attachment protein to a host cell receptor causes conformational changes in this protein, which in turn cause activating conformational changes in the metastable f protein (lamb 1993; russell et al. 2001; takimoto et al. 2002) . the exact mechanism by which attachment proteins activate f proteins is not known, but several groups have provided evidence for cross talk between attachment and f proteins (bossart et al. 2002; deng et al. 1999; mcginnes et al. 2002; stone-hulslander and morrison 1997; takimoto et al. 2002; yao et al. 1997) . the post-fusion form of the f protein from the paramyxovirus sv5 contains a six-helix bundle (baker et al. 1999 ). similar to hiv env kilby et al. 1998; munoz-barroso et al. 1998 ) and other retroviral fusion proteins (earp et al. 2003; netter 2002) , peptide analogs of the n-and c-terminal helices of paramyxovirus six-helix bundles are potent inhibitors of fusion and infection (bossart et al. 2002; joshi et al. 1998; lambert et al. 1996; young et al. 1999) . as is also the case for hiv env melikyan et al. 2000a ), a recent study showed that conversion of the sv5 f protein to a six-helix bundle drives membrane fusion . issues yet to be addressed for paramyxoviruses are the structure of the complete native (metastable) f trimer and how it is converted to its activated form. the first glimpses at the metastable and postfusion states of the f trimer came from em observations of the respiratory syncytial virus (rsv) f protein. preparations of purified recombinant f protein contained both cone-shaped rods and "lollipop"-shaped structures. on storage, there appeared to be a shift from the cone-shaped to the "lollipop"-shaped structures (calder et al. 2000) . examination of f complexed with specific monoclonal antibodies suggested that the "lollipop" structures contained six-helix bundles composed of n-and c-terminal heptad repeats (calder et al. 2000) . a high-resolution structure of an f protein ectodomain was recently presented (chen et al. 2001a ). the protein used for the analysis contained a mixture of precursor f 0 and proteolytically cleaved f. it also apparently lacked the second heptad repeat, which forms the c-helix in the postfusion form. this trimeric f protein structure is fundamentally different from that of influenza ha; the n-terminal end of its coiled-coil is positioned near the viral membrane end of the molecule (i.e., opposite the orientation of the coiled-coil in the metastable ha trimer). if this f protein structure represents the native metastable f trimer, then it suggests a mechanism of fusion activation for f fundamentally different from that for ha (chen et al. 2001a ). additional work is needed to test this idea. all known class ii fusion proteins are activated by low ph. however, the mechanism by which class ii fusion proteins are initially activated is quite different than the mechanism by which class i fusion proteins are initially activated. for example, the ectodomain of the tbe glycoprotein forms an antiparallel dimer that lies parallel and close to the viral membrane (fig. 4b) . at low ph, the tbe e homodimer converts to an e homotrimer (allison et al. 1995; heinz and allison 2001; stiasny et al. 2001 ). this transformation is thought to occur in two steps: dissociation of the e homodimer, followed by reassociation of e trimers (stiasny et al. 1996) . membrane binding occurs after dimer dissociation and promotes the formation of e homotrimers (stiasny et al. 2002) . homotrimer formation may involve interactions between a-helices in the stem region of the e protein (allison et al. 1999) . the sfv fusion protein also converts from a dimer to a trimer during fusion activation. on native virions, e1 exists as a tight heterodimeric complex with a second membrane protein, e2. on exposure to low ph, e1 dissociates from e2, changes conformation, and forms highly stable e1 homotrimers (ahn et al. 1999; kielian 1995; . during this process, e1 binds hydrophobically through its fusion peptide to target membranes and mediates fu-sion. similar to tbe e, it appears that binding to the target bilayer fosters formation of the activated e1 homotrimer (kielian 1995; kielian et al. 2000) ; the fusion peptide and the transmembrane domain of e1 appear to be important for e1 homotrimer formation (kielian et al. 1996 (kielian et al. , 2000 sjoberg and garoff 2003) . thus both class i and class ii viral fusion proteins appear to function as trimers during fusion. it has been proposed that activated tbe e (helenius 1995) and sfv e1 stand up as trimeric spikes and present their fusion peptides to the target membrane (fig. 4b, right) . this would be analogous to fig. 3, step 1. if this occurs, (kuhm et al. 2002) . note that other possibilities for the dimer to trimer transition exist (for example involving relative movements of domains about hinge regions). for very recent developments regarding the fusion mechanism of class ii fusion proteins, see bressanelli et al. (2004) , gibbons et al. (2004) and modis et al. (2004) then the spike would have to refold to bring the viral and cellular membranes together (e.g., analogous to steps 4 and 5 in fig. 3) . very recent evidence indicates that this is, indeed, the case (bressanelli et al. 2004; gibbons et al. 2004; modis et al. 2004 ). thus far we have focused on the conditions that elicit viral fusion reactions and the conformational changes in viral fusion proteins necessary for fusion. however, it is the viral and cellular bilayer membranes that merge during fusion. lipid bilayers are stable structures that do not fuse spontaneously. fusion proteins have evolved to catalyze the necessary lipid rearrangements. we now review a lipid rearrangement model and focus on the roles of different regions of viral fusion proteins in choreographing the structural changes that the membranes undergo throughout the fusion cascade (fig. 3) . the favored model for the lipid transition state during membrane fusion is the stalk model. in this model, two opposing membranes bend toward each other, creating "dimples" (when viewed from the trans surface) or "nipples" (when viewed from the cis surface) (fig. 3, step 4) . nipples continue to bend until they meet. the two cis leaflets then merge, creating a lipid stalk (see fig. 2 in kozlovsky and kozlov 2002 ) that proceeds to a state of local hemifusion (fig. 3, step 5). in a second step, transient fusion pores form, which give rise to stable pores (fig. 3, step 6). the first direct visualization of a lipid stalk intermediate was achieved by electron diffraction studies of the effect of sequential dehydration on lipid bilayers composed of a lipid that has negative spontaneous curvature (yang and huang 2002). the stalk intermediate was stable at intermediate relative humidities. the results suggested that both the formation of a lipid stalk and its transition to a conformation that can be equated with pore formation require external forces. cellular membranes do not have spontaneous negative curvature and are highly hydrated. membrane curvature can be promoted by introducing defects into the contacting bilayers. thus roles for the fusion protein include pulling the fusing bilayers toward one another (dimpling), dehydrating the membranes, and creating membrane defects that lower the energy barrier for stalk and pore formation. two intermediates in hiv fusion have been trapped: one in which the two membranes are joined by activated envs, but are not yet fused (melikyan et al. 2000a) , and one in which small, "labile" pores have formed that can either expand into stable, "robust" pores or return to the prefusion state . these observations suggest a role for the fusion protein in formation and stabilization of both the fusion stalk and the fusion pore. the mechanism by which a small pore enlarges is not known. however, several possibilities have been proposed. one is that the initial fusion pore is formed by a small number of activated fusion proteins. additional activated fusion proteins then move into the fusion site to buttress and stabilize the pore, thereby allowing it to expand (kozlov and chernomordik 2002) . another possibility is that multiple small fusion pores coalesce to form larger ones. this was supported by em visualization of ha-mediated fusion, in which multiple dimples/nipples were arranged circularly and lipid fragments were seen at the center of a fusion ring (kanaseki et al. 1997 ). as discussed above, roles for the fusion protein in the fusion cascade (fig. 3) include pulling the fusing bilayers toward one another (dimpling) and creating membrane defects that lower the energy barriers for stalk formation and fusion pore opening/enlargement. the fusion peptide and the transmembrane domain must remain stably associated with the target and viral membranes, respectively, for fusion to occur. once the fusion peptide is stably associated with the target bilayer (fig. 3, step 2), we envision that rearrangements in the fusion protein ectodomain that bring the fusion peptide and transmembrane domains close together (fig. 3, step 4) result in dimpling of membranes toward one another. in addition to serving as critical membrane anchors, the fusion peptide and the transmembrane domain likely create membrane defects that facilitate the next stages of fusion. here, we review information about the structure and function of the fusion peptide and the transmembrane domain during fusion. we also review evidence that juxtamembrane sequences, on both sides of the transmembrane domain, participate in fusion. fusion peptides are relatively apolar sequences that interact with membranes and are central to viral fusion reactions (martin and ruysschaert 2000; martin et al. 1999; skehel et al. 2001; white 1990) . they have been fig. 5a , b. characteristics of viral fusion peptides. a selected viral fusion peptide sequences. n-terminal ) and internal fusion peptide sequences are aligned according to their first noncharged residue. b model of ha fusion peptide structure in target membrane at ph 5 (adapted from tamm et al. 2002) . the fusion peptide (red) resides in the target membrane in a kinked structure composed of two a-helices, each penetrating the outer leaflet. the glycine ridge is depicted by a yellow box, the hydrophobic interior face by cyan ovals, and the surface charged residues by blue squares. "c" denotes the direction of the ha2 ectodomain classified as n-terminal or internal depending on their location within the fusion subunit (table 1) . although fusion peptides are highly conserved within each virus family, there is little sequence similarity between fusion peptides of different families (fig. 5a) . generally, however, fusion peptides contain a high percentage of glycines and/or alanines, as well as several critical bulky hydrophobic residues (martin and ruysschaert 2000; martin et al. 1999; skehel et al. 2001; tamm and han 2000; tamm et al. 2002) . a significant body of work has emerged on the structure and function of synthetic fusion peptides (martin and ruysschaert 2000; martin et al. 1999; skehel et al. 2001; tamm and han 2000; tamm et al. 2002) . synthetic fusion peptides are disordered in solution but ordered (a-helix and/or b-sheet) when they associate with membranes. the n-terminal fusion peptides that have been studied insert into membranes at oblique angles and do not penetrate the inner leaflet of the membrane. in general, mutations that abrogate fusion reduce the ability of synthetic fusion peptides to insert at oblique angles and to disrupt membranes (martin et al. 1999) . contradictory conclusions on the precise structure of synthetic fusion peptides in membranes likely stem from the general low solubility of the peptides in aqueous solution and the different experimental methods employed (tamm et al. 2002) . to circumvent solubility problems, a polar sequence was added to the c-terminal end of the influenza ha fusion peptide, rendering it soluble in both aqueous and hydrophobic environments (han et al. 2001) . at ph 5, the ha fusion peptide consists of an n-terminal helix, a kink, and a short c-terminal helix (fig. 5b) . both the n-and c-terminal helices penetrate the outer leaflet of the target bilayer. the kink remains at the phospholipid surface; the interior (lipid-facing surface) of the kink is lined with hydrophobic residues. the conserved glycines form a ridge along the outer face of the n-terminal helix. three charged residues are also found on the outer face (fig. 5b ). an ha in which the conserved glycine at the beginning of the fusion peptide (gly1) has been changed to valine cannot mediate fusion. if gly1 is changed to serine, ha mediates only hemifusion or only forms small nonexpanding fusion pores (qiao et al. 1999; skehel et al. 2001) . interestingly, these mutant fusion peptides have membrane-associated structures and orientations significantly different from those of the wild-type fusion peptide (li et al. 2003) . simulations suggested similar membrane penetrating orientations for the hiv fusion peptide and two fusion-defective mutants (kamath and wong 2002) . in addition to a significant number of apolar residues, many internal fusion peptides contain a conserved proline at or near their centers (fig. 5a) . mutagenesis of this proline in the avian sarcoma/leukosis virus (aslv) enva fusion peptide suggested that it stabilizes a b-turn . this, coupled with the observation that mutating two cysteines that flank the fusion peptide abolishes fusion activity , suggested that the internal enva fusion peptide exists as a looped structure stabilized by a disulfide bond. the ability of the ebola virus fusion protein, which also contains an internal fusion peptide, to support infection was similarly inhibited when its central proline and flanking cysteines were mutated (ito et al. 1999; jeffers et al. 2002) . a similar mutation of a proline within the predicted turn segment of the candidate fusion peptide of vsv g also significantly decreased fusion and abolished infectivity (fredericksen and whitt 1995) . the idea of loop structures for internal fusion peptides is further supported by the known looped structure of the tbe e and sfv ei fusion peptides (rey et al. 1995; lescar et al. 2001) . in some cases, two or more noncontiguous sequence loops may function as a collective fusion peptide (gaudin et al. 1999a; li et al. 1993) . like n-terminal fusion peptides, internal fusion peptides contain a significant number of glycines and hydrophobic residues (fig. 5a ). changing either of two glycines within the sfv e1 fusion peptide to alanines altered the ph threshold for fusion, and changing one of the glycines to aspartic acid abolished fusion (duffus et al. 1995; kielian et al. 1996) . alteration of hydrophobic residues at the beginning, middle, or end of the (internal) aslv enva fusion peptide to charged residues impaired the ability of enva to mediate fusion (hernandez and white 1998) . similarly, a tryptophan and a glycine are critical for ebola gp-mediated infection (ito et al. 1999) . also, a bulky hydrophobic residue is needed at the tip of the tbe e fusion peptide loop (rey et al. 1995) (fig. 4a, red) . collectively, these results suggest that internal fusion peptides function as loops that require a mixture of hydrophobic and flexible residues, similar to those found in n-terminal fusion peptides. fusion peptides appear to act at several steps along the fusion pathway. as demonstrated by mutants in which apolar fusion peptide residues were changed to charged residues (freed et al. 1992; gething et al. 1986; hernandez and white 1998; schoch and blumenthal 1993) , fusion peptides clearly play an important role in anchoring the fusion protein to the target membrane (fig. 3, step 2). the energy provided by inserting the fusion peptides of a single ha trimer into a membrane would be sufficient to initiate stalk formation (gunther-ausborn et al. 2000) . the fusion peptide may also assist in creating the stalk by displacing water from the lipid-water interface, thus decreasing the repulsive force between the two fusing membranes (tamm and han 2000) . fusion peptides may also function in fusion pore opening. in support of this possibility is the observation that an ha mutant in which gly1 was changed to serine mediates extensive lipid, but not content mixing (qiao et al. 1999) . furthermore, defects in syncytium formation and infectivity were observed for hiv env harboring the mutation v2e in its fusion peptide (freed et al. 1992) . biophysical studies comparing a synthetic fusion peptide harboring this mutation with the wild-type peptide suggested a requirement for fusion peptide aggregation in the creation of the hiv fusion pore pereira et al. 1995) . studies with chimeric fusion proteins have suggested that the transmembrane domains of some viral fusion proteins do not require a specific sequence to support fusion (armstrong et al. 2000 and references therein). in contrast, studies with glycosylphosphatidylinositol (gpi)-anchored fusion proteins have demonstrated that there is a strict requirement for a proteinaceous membrane anchor for fusion proteins to efficiently mediate the transition from hemifusion to full fusion (kemble et al. 1994; melikyan et al. 1997a; tong and compans 2000) . there also appears to be a minimum length for the fusion protein transmembrane domain to be able to support this transition (armstrong et al. 2000; west et al. 2001) . therefore, it has been suggested that fusion protein transmembrane domains must span both leaflets of the viral bilayer to mediate fusion pore opening (armstrong et al. 2000) . the transmembrane domains of some fusion proteins appear to have specific amino acid requirements for fusion function. for example, a conserved positively charged residue in the middle of the transmembrane domains of certain retroviral envs appears to be important for the ability to mediate fusion and infection (einfeld and hunter 1994; owens et al. 1994; pietschmann et al. 2000; west et al. 2001) . two glycine residues in the transmembrane domain of vsv-g appear to be important for the transition from hemifusion to full fusion (cleverley and lenard 1998) . studies using a synthetic peptide corresponding to the mutant vsv-g transmembrane domain (dennison et al. 2002) suggested that the vsv-g transmembrane domain lowers the energy barrier for fusion and stabilizes the transient fusion pore, thereby promoting its conversion to a stable fusion pore. two glycines may allow the vsv-g transmembrane domain to adopt alternative conformations under different conditions, and such flexibility may be important for function. the transmembrane domain of ha from the japan (melikyan et al. 2000b ), but not the x:31 (armstrong et al. 2000) , strain of influenza appears to require a glycine near the middle. an ability to adopt alternative conformations was also invoked to explain the requirement for a proline near the middle of the transmembrane domain of the murine leukemia virus (mlv) env glycoprotein (taylor and sanders 1999) . the observations that mutations in fusion peptides or transmembrane domains (armstrong et al. 2000; baker et al. 1999; tamm et al. 2002) can impair the ability to mediate full fusion (fig. 3, step 6) have suggested that both of these apolar domains function in the transition from hemifusion to full fusion. initially, the fusion peptide appears to insert only into the outer leaflet of the target membrane (tamm and han 2000) . it has been proposed that the transmembrane domain and the fusion peptide, which are close to each other after membrane merger, may interact to stabilize the fusion pore (tamm et al. 2002; zhou et al. 1997) . if this is the case, the fusion peptide might span both leaflets of the fused membrane in its final conformation (tamm et al. 2002) . several lines of evidence suggest that ectodomain sequences that lie just before the transmembrane domains of certain viral fusion proteins may be important for fusion. these sequences tend to have a high proportion of tryptophans or other aromatic residues and are predicted to partition into the interfacial regions of membranes (suarez et al. 2000) . indeed, synthetic peptides containing juxtamembrane ectodomain sequences from hiv env and ebola gp partition into the interfacial region of target membranes (saez-cirion et al. 2003; saez-cirion et al. 2002; schibli et al. 2001; suarez et al. 2000) . mutation of three tryptophans within this region of hiv gp41 abrogated infection ), apparently by inhibiting fusion pore enlargement (munoz-barroso et al. 1999) . extending the hiv gp41 c-terminal heptad repeat peptide to include the tryptophan-rich juxtamembrane ectodomain sequence appeared to increase the potency of the peptide as an inhibitor of fusion (kliger et al. 2001) . it was suggested that the extended heptad repeat peptide was more potent because it could bind to two sites on hiv env (the n-terminal coiled-coil and a second, as yet unidentified site) (kliger et al. 2001) . a likely effect of these peptides on late stages of fusion is to prevent formation of a required structure in env that provides additional membrane destabilization. in this manner, partitioning of juxtamembrane sequences into the interfacial region of membranes may promote the transition from a stalk intermediate to a fusion pore (see sect. 5). a specific cytoplasmic tail sequence does not appear to be essential, but it can modulate late stages of fusion. the cytoplasmic tail has been shown to influence the transition from hemifusion to full fusion or fusion pore enlargement (dutch and lamb 2001; kozerski et al. 2000) in some viruses. the mechanism by which cytoplasmic tails may influence these later stages of fusion is not known. some studies using synthetic peptides have suggested a direct interaction between the cytoplasmic tail and the viral membrane (chen et al. 2001b; fujii et al. 1992; gawrisch et al. 1993; haffar et al. 1991; . others have shown that the cytoplasmic tail can influence the structure of the ectodomain of the fusion protein (aguilar et al. 2003; edwards et al. 2002) . the ability of the cytoplasmic tail to affect ectodomain structure is most clearly manifested for those viral fusion proteins that harbor fusion-suppressing sequences. these sequences have been found in the fusion proteins of mlv (ragheb and anderson 1994) , other type c retroviruses (bobkova et al. 2002) , some lentiviruses (kim et al. 2003; luciw et al. 1998) , and a paramyxovirus f protein (tong et al. 2002) . the cytoplasmic tail of mlv env is cleaved during virus budding (schultz and rein 1985) . mlv envs harboring uncleaved cytoplasmic tails do not induce fusion (yang and compans 1996) . although viruses lacking fusion-suppressing sequences display increased cell-cell fusion, they are more susceptible to neutralizing antibodies (januszeski et al. 1997; li et al. 2001; rein et al. 1994; yang and compans 1996) and are impaired in their ability to sustain multiple rounds of infection (cathomen et al. 1998; freed and martin 1996; piller et al. 2000) . acylation of cytoplasmic tails can also affect fusion, apparently at a late stage. for example, a mutant ha from the japan strain of influenza in which three (normally palmitoylated) cysteine residues were mutated appeared to fuse normally when monitored by dye redistribution assays (melikyan et al. 1997b ). however, electrophysiological measurements revealed that fusion pores formed by the mutant ha did not flicker like those formed by wt-ha (melikyan et al. 1997b) . similar mutations in ha from the a/ussr/77 (h1n1) and a/fpv/rostock/34 (h7n1) influenza subtypes were shown, respectively, to inhibit syncytia formation (fischer et al. 1998 ) and the transition from hemifusion to full fusion . palmitoylation of hiv env was also shown to be important for env incorporation into virions and for infectivity (rousso et al. 2000) . thus acylation of cytoplasmic tails appears to have multiple effects on viral fusion reactions, the details of which are not completely understood. lipid rafts are plasma membrane microdomains that are enriched in cholesterol and glycosphingolipids with saturated acyl chains. they are organizational platforms for a variety of cellular functions including sorting of membrane proteins and signaling (brown and london 2000) . although there is growing evidence that certain viruses employ rafts, or raftlike membrane microdomains, during virus assembly (suomalainen 2002) , the question of whether these structures are required at the site of fusion in the target cell is less clear. here, we consider the role of rafts in the fusion of two enveloped viruses, sfv and hiv. it is important to consider whether cholesterol and/or sphingolipids are required for fusion because they are found in lipid rafts, or if they serve some other purpose. for example, cholesterol may interact directly with the fusion protein, thereby facilitating its insertion into the target membrane. alternatively, a need for cholesterol and sphingolipids could reflect an ability of raft structures to concentrate viral receptors. a third possibility is that the cholesterol imparts the membrane fluidity (or other biophysical properties) needed to lower the energy barrier for fusion. sfv requires cholesterol and sphingolipids in the target membrane for fusion. these moieties enable the sfv spike protein to undergo conformational changes and bind to the target membrane (ahn et al. 2002; kielian et al. 2000) . in a recent study, it was shown that after hydrophobic association with target bilayers, the sfv glycoprotein ectodomain associates with membrane structures with properties similar to rafts. however, careful studies using liposomes prepared with specific cholesterol and sphingolipid analogs demonstrated that the cholesterol and sphingolipid requirements in the target membrane did not correlate with their ability to form lipid rafts (ahn et al. 2002) . a related conclusion was drawn based on the fusion activities of both sfv and sindbis virus with liposomes (waarts et al. 2002) . for both viruses, the requirement for cholesterol and sphingolipids in the target membrane appears to be for insertion of the fusion peptide (vashishtha et al. 1998 ). in the case of hiv, several studies have suggested a need for raftlike membrane microdomains for virus entry (kozak et al. 2002; popik et al. 2002) . depleting plasma membrane cholesterol from target cells resulted in reduced levels of virus infectivity or cell-cell fusion. other studies have concluded that rafts are not necessary for hiv entry (percherancier et al. 2003; viard et al. 2002) . in one study, depleting cholesterol from cells that express low levels of virus receptors inhibited hiv env-mediated cell-cell fusion, but depleting cholesterol from cells that express high levels of virus receptors did not (viard et al. 2002) . therefore, it was concluded that rafts per se are not needed for fusion. rather, the presence of raftlike structures in the plasma membrane may concentrate virus receptors. previous work has shown that a critical density of hiv receptors is required for fusion and infection (reeves et al. 2002) . clearly more work is needed to clarify the role of rafts in virus-cell fusion and entry. it has recently become apparent, largely because of the success of t-20 in the inhibition of hiv infection in patients (jiang et al. 2002; kilby et al. 1998) , that fusion is a good target for antiviral intervention. this was originally conceptualized because fusion is an essential early step in the virus infectious cycle, it happens in an exoplasmic space, and strategies can be designed to inhibit fusion without interfering with host cell proteins. some fusion inhibitors function by inhibiting six-helix bundle formation. others function by preventing earlier conformational changes in viral fusion proteins. the peptide t-20 (also known as fuzeon) corresponds to the c-terminal helix of hiv env (fig. 3, green) . t-20 works by preventing six-helix bundle formation. t-20 is a potent inhibitor of infections in tissue culture. peptides corresponding to equivalent regions of other retroviruses as well as several paramyxoviruses function similarly (earp et al. 2003; russell et al. 2001) . notably, all of the viruses that have been shown to be highly susceptible to "c-helix" peptide inhibitors function at neutral ph, at least up to the lipid interacting stage of virus-cell fusion (earp et al. 2003) . peptides corresponding to the n-terminal helices of hiv env and the sv5 f protein also inhibit fusion, although with lower potency (lu et al. 1995; russell et al. 2001) . the mechanism of inhibition by n-terminal peptides is still under consideration ). in the case of the sv5 f, the n-peptide appears to target an earlier intermediate than the c-peptide . other strategies are being considered to stabilize the prehairpin intermediate (fig. 3, step 1) and thereby prevent six-helix bundle formation. one strategy is the development of antibodies that recognize the prehairpin intermediate (golding et al. 2002) . another, exemplified in three studies, is the development of small molecules that prevent six-helix bundle formation (debnath et al. 1999; eckert et al. 1999; ferrer et al. 1999 ). all three studies targeted a hydrophobic pocket in the groove of the central coiled-coil of hiv gp41 that is important for interaction with the c-terminal helix in the post-fusion form. in the first approach, two organic compounds were identified from a screening effort conducted in conjunction with molecular docking, a method to identify small molecules that fit in a target site (debnath et al. 1999) . the second approach replaced three residues of the c-terminal helix that bind to the hydrophobic pocket with organic moieties, generated by combinatorial chemistry (ferrer et al. 1999 ). the third approach used a mirror image phage display library to identify small, d-amino acid containing peptides that bind to the pocket (eckert et al. 1999) . although none of the small molecules identified to date is as potent as t-20, the precedent has been set for attaining this goal. an effort to block the fusion activity of influenza was based on the idea that maintaining ha in its native metastable state should prevent fusion and infection. the first trial targeted a site in x:31 ha that includes part of the fusion peptide. with the use of an antibody-based assay to monitor fusion peptide exposure, a compound, tert-butylhydroquinone (tbhq), that prevents the first stages of the ha conformational change and inhibits infectivity was discovered (bodian et al. 1993) . a follow-up study, targeting a site near the b-loop in ha, yielded additional inhibitors. whereas some functioned like tbhq, a second class was identified that appeared to push ha to an inactive state (hoffman et al. 1997 ). a random screen against an h1 influenza virus identified an inhibitor that appears to function similarly to tbhq. in the latter case, the binding site for the inhibitor was mapped to the vicinity of the fusion peptide (cianci et al. 1999 ). other small molecules that inhibit conformational changes in ha have been identified (staschke et al. 1998 ). to date, none of the ha inhibitors has blocked all ha subtypes and none has an ic 50 value in the submicromolar range. it is not yet clear whether the latter limitation represents a fundamental difficulty in inhibiting viral fusion proteins that function at low ph. in addition to the small molecule approaches described above, antibodies that prevent fusion-inducing changes in viral glycoproteins have been described. the first example was an antibody that prevents low-ph-induced fusion of west nile virus with model liposomes (gollins and porterfield 1986) . recently, a fab fragment that binds to two ha1 monomers was shown to prevent an early conformational change in influenza ha (barbeymartin et al. 2002) , separation of the globular head domains (fig. 2b ). as described above, antibodies have been developed that likely block six-helix bundle formation in the case of hiv env (golding et al. 2002) . the goal of this review was to give the reader an appreciation for the diversity of viral fusion mechanisms while recognizing their common underlying principles. we also summarized what is known about the lipid dynamics and lipid structures involved in fusion, and we also briefly overviewed recent developments in targeting viral fusion as an antiviral strategy. there is clearly much more we need to know about viral fusion proteins, viral fusion reactions, and the design of antifusion agents. we end this review by enumerating some pressing issues and questions that remain about viral fusion. a major goal is to determine highresolution structures for the complete ectodomains of the metastable trimers of class i viral fusion proteins in addition to the influenza ha. structures of a complete paramyxovirus f and a complete retroviral env ectodomain will be highly informative because we currently lack a detailed molecular description of how a receptor activates any viral fusion protein at neutral ph. a second goal will be to further delineate the mechanisms of class ii viral fusion proteins. do the transitions to their recently described low ph forms (bressanelli et al. 2004; gibbons et al. 2004; modis et al. 2004 ) mediate hemifusion or fusion pore opening? what about the mechanisms of the as yet unclassified viral fusion proteins? these include viruses such as rhabdoviruses (e.g., vsv) that need only one protein to promote fusion, as well as more complicated viruses such as herpesviruses and poxviruses that require multiple viral glycoproteins. the ensuing years should also bring a more complete understanding of how viral fusion proteins interact with target membrane bilayers. class ii fusion proteins insert their internal fusion peptides into target membranes as loops (bressanelli et al. 2004; gibbons et al. 2004; modis et al. 2004 ). it has been predicted that the internal fusion proteins of the class i fusion proteins from ebola and avian retroviruses form disulfidebonded loop structures (weisenhorn et al. 1998) , and mutagenesis work has supported this prediction jeffers et al. 2002) . it remains to be seen, from high resolution structural studies, whether all internal fusion peptides, be they from class i, class ii, or other classes of fusion proteins, interact with target bilayers as (disulfide bond) stabilized loops. finally, we expect that there will be major developments in furthering the concept of targeting fusion as a weapon against pathogenic enveloped viruses. particular emphasis will likely be on the development of small molecule inhibitors through the use of combinatorial chemistry in conjunction with high-throughput screens. it will be interesting to learn whether small molecule fusion inhibitors can be identified that block the entry of viruses that fuse in endosomes in response to low ph. this is a challenge for low-ph-activated class i fusion proteins such as influenza ha as well as for all known class ii fusion proteins. stay tuned. there are likely to be exciting develop-ments in our understanding of viral fusion mechanisms as well as in the development of antifusion antivirals in the years ahead. dennison sm, greenfield n, lenard j, and lentz br (2002) receptor binding transforms the surface subunit of the mammalian c-type retrovirus envelope protein from an inhibitor to an activator of fusion effect of proteolytic processing at two distinct sites on shape and aggregation of an anchorless fusion protein of human respiratory syncytial virus and fate of the intervening segment dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cellcell fusion events identification of r-peptides in envelope proteins of c-type retroviruses inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones completion of trimeric hairpin formation of influenza virus hemagglutinin promotes fusion pore opening and enlargement the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex membrane fusion tropism and heterotypic functional activities of the nipah virus and hendra virus envelope glycoproteins structure of a flavivirus envelope glycoprotein in its low-ph-induced membrane fusion conformation membrane fusion of semliki forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein structure and function of sphingolipid-and cholesterol-rich membrane rafts structure of influenza haemagglutinin at the ph of membrane fusion electron microscopy of the human respiratory syncytial virus fusion protein and complexes that it forms with monoclonal antibodies influenza hemagglutinin is spring-loaded by a metastable native conformation a spring-loaded mechanism for the conformational change of influenza hemagglutinin measles viruses with altered envelope protein cytoplasmic tails gain cell fusion competence core structure of gp41 from the hiv envelope glycoprotein hiv entry and its inhibition structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation n-and c-terminal residues combine in the fusion-ph influenza hemagglutinin ha(2) subunit to form an n cap that terminates the triple-stranded coiled coil the structure of the fusion glycoprotein of newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion cellular membrane-binding ability of the c-terminal cytoplasmic domain of human immunodeficiency virus type 1 envelope transmembrane protein gp41 ph-dependent changes in photoaffinity labeling patterns of the h1 influenza virus hemagglutinin by using an inhibitor of viral fusion the transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus g protein the structural biology of type i viral membrane fusion membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system receptor-triggered membrane association of a model retroviral glycoprotein membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers structure-based identification of small molecule antiviral compounds targeted to the gp41 core structure of the human immunodeficiency virus type 1 the central proline of an internal viral fusion peptide serves two important roles critical role for the cysteines flanking the internal fusion peptide of avian sarcoma/leukosis virus envelope glycoprotein mutations in the newcastle disease virus hemagglutinin-neuraminidase protein that interfere with its ability to interact with the homologous f protein in the promotion of fusion try inhibitors from biased combinatorial libraries of non-natural binding elements acylation of the influenza hemagglutinin modulates fusion activity vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity a mutation in the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 dominantly interferes with fusion and infectivity domains of the human immunodeficiency virus type 1 matrix and gp41 cytoplasmic tail required for envelope incorporation into virions temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 a molecular model for membrane fusion based on solution studies of an amphiphilic peptide from hiv gp41 capture of an early fusion-active conformation of hiv-1 gp41 inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent hiv-1 entry mutations in the glycoprotein of viral haemorrhagic septicaemia virus that affect virulence for fish and the ph threshold for membrane fusion rabies virus-induced membrane fusion interaction of peptide fragment 828-848 of the envelope glycoprotein of human immunodeficiency virus type i with lipid bilayers studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus conformational change and protein-protein interactions of the fusion protein of semliki forest virus fusion of rous sarcoma virus with host cells does not require exposure to low ph introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates a new mechanism for the neutralization of enveloped viruses by antiviral antibody cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion new insights into the spring-loaded conformational change of influenza hemagglutinin role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity the cytoplasmic tail of hiv-1 gp160 contains regions that associate with cellular membranes membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin peptides trap the human immunodeficiency virus type 1 envelope glycoprotein fusion intermediate at two sites the machinery for flavivirus fusion with host cell membranes alphavirus and flavivirus glycoproteins: structures and functions virus-cell and cellcell fusion activation of a retroviral membrane fusion protein: soluble receptor-induced liposome binding of the alsv envelope glycoprotein mutational analysis of the candidate internal fusion peptide of the avian leukosis and sarcoma virus subgroup a envelope glycoprotein structure-based identification of an inducer of the low-ph conformational change in the influenza virus hemagglutinin: irreversible inhibition of infectivity viral entry and receptors entry of semliki forest virus into cells: effects of concanamycin a and nigericin on viral membrane fusion and infection mutational analysis of the putative fusion domain of ebola virus glycoprotein membrane fusion functional analysis of the cytoplasmic tail of moloney murine leukemia virus envelope protein covalent modifications of the ebola virus glycoprotein peptide and non-peptide hiv fusion inhibitors a core trimer of the paramyxovirus fusion protein: parallels to influenza virus hemagglutinin and hiv-1 gp41 membrane structure of the human immunodeficiency virus gp41 fusion domain by molecular dynamics simulation structural features of membrane fusion between influenza virus and liposome as revealed by quick-freezing electron microscopy intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion membrane fusion and the alphavirus life cycle mechanisms of mutations inhibiting fusion and infection by semliki forest virus specific roles for lipids in virus fusion and exit. examples from the alphaviruses potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry human t-cell leukemia virus type 1 envelope-mediated syncytium formation can be activated in resistant mammalian cell lines by a carboxy-terminal truncation of the envelope cytoplasmic domain fusion peptides derived from the hiv type 1 glycoprotein 41 associate within phospholipid membranes and inhibit cell-cell fusion. structure-function study mode of action of an antiviral peptide from hiv-1. inhibition at a post-lipid mixing stage a leucine zipper-like sequence from the cytoplasmic tail of the hiv-1 envelope glycoprotein binds and perturbs lipid bilayers conformational intermediates and fusion activity of influenza virus hemagglutinin segregation of cd4 and cxcr4 into distinct lipid microdomains in t lymphocytes suggests a mechanism for membrane destabilization by human immunodeficiency virus modification of the cytoplasmic domain of influenza virus hemagglutinin affects enlargement of the fusion pore the protein coat in membrane fusion: lessons from fission stalk model of membrane fusion: solution of energy crisis structure of dengue virus: implications for flavivirus organization, maturation, and fusion structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody paramyxovirus fusion: a hypothesis for changes peptides from conserved regions of paramyxovirus fusion (f) proteins are potent inhibitors of viral fusion activation of membrane fusion by murine leukemia viruses is controlled in cis or in trans by interactions between the receptor-binding domain and a conserved disulfide loop of the carboxy terminus of the surface glycoprotein a proline-rich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph mutations in the cytoplasmic tail of murine leukemia virus envelope protein suppress fusion inhibition by r peptide mutation analysis of the vesicular stomatitis virus glycoprotein g for membrane fusion domains thermodynamics of fusion peptide-membrane interactions a trimeric structural domain of the hiv-1 transmembrane glycoprotein importance of the intracytoplasmic domain of the simian immunodeficiency virus (siv) envelope glycoprotein for pathogenesis hiv-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation synchronized activation and refolding of influenza hemagglutinin in multimeric fusion machines membrane fusion mediated by baculovirus gp64 involves assembly of stable gp64 trimers into multiprotein aggregates common properties of fusion peptides from diverse systems role of the n-terminal peptides of viral envelope proteins in membrane fusion sequential roles of receptor binding and low ph in forming prehairpin and hairpin conformations of an avian retroviral envelope glycoprotein the ph independence of mammalian retrovirus infection newcastle disease virus hn protein alters the conformation of the f protein at cell surfaces inner but not outer membrane leaflets control the transition from glycosylphosphatidylinositol-anchored influenza hemagglutinin-induced hemifusion to full fusion the role of the cytoplasmic tail region of influenza virus hemagglutinin in formation and growth of fusion pores evidence that the transition of hiv-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion a point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion structure of the dengue virus envelope protein after membrane fusion retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41 role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion fusion of enveloped viruses with cells and liposomes. activity and inactivation fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virusbased assay quantitating cell fusion-dependent reporter gene activation tight binding of influenza virus hemagglutinin to its receptor interferes with fusion pore dilation mutations in the membrane-spanning domain of the human immunodeficiency virus envelope glycoprotein that affect fusion activity leash in the groove mechanism of membrane fusion fusion protein of the paramyxovirus sv5: destabilizing and stabilizing mutants of fusion activation insider information: what viruses tell us about endocytosis hiv-1 entry into tcells is not dependent on cd4 and ccr5 localization to sphingolipid-enriched, detergent-resistant, raft membrane domains liposome destabilization induced by the hiv-1 fusion peptide effect of a single amino acid substitution an evolutionarily conserved positively charged amino acid in the putative membrane-spanning domain of the foamy virus envelope protein controls fusion activity mutational analysis of conserved domains within the cytoplasmic tail of gp41 from human immunodeficiency virus type 1: effects on glycoprotein incorporation and infectivity localization of the labile disulfide bond between su and tm of the murine leukemia virus envelope protein complex to a highly conserved cwlc motif in su that resembles the active-site sequence of thiol-disulfide exchange enzymes human immunodeficiency virus type 1 uses lipid raft-colocalized cd4 and chemokine receptors for productive entry into cd4(+) t cells activation of vesicular stomatitis virus fusion with cells by pretreatment at low ph a specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype specific single or double proline substitutions in the "spring-loaded" coiled-coil region of the influenza hemagglutinin impair or abolish membrane fusion activity ph-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the r peptide and p12e tm in viral entry sensitivity of hiv-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics function of the cytoplasmic domain of a retroviral transmembrane protein: p15e-p2e cleavage activates the membrane fusion capability of the murine leukemia virus env protein the envelope glycoprotein from tick-borne encephalitis virus at 2 resolution palmitoylation of the hiv-1 envelope glycoprotein is critical for viral infectivity membrane fusion machines of paramyxoviruses: capture of intermediates of fusion pre-transmembrane sequence of ebola glycoprotein. interfacial hydrophobicity distribution and interaction with membranes sphingomyelin and cholesterol promote hiv-1 gp41 pretransmembrane sequence surface aggregation and membrane restructuring fatty acids on the a/ussr/77 influenza virus hemagglutinin facilitate the transition from hemifusion to fusion pore formation a conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity the membrane-proximal tryptophanrich region of the hiv glycoprotein, gp41, forms a well-defined helix in dodecylphosphocholine micelles antibodies to cd9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion role of the fusion peptide sequence in initial stages of influenza hemagglutinin-induced cell fusion maturation of murine leukemia virus env proteins in the absence of other viral proteins mutations in the cytoplasmic domain of a paramyxovirus fusion glycoprotein rescue syncytium formation and eliminate the hemagglutinin-neuraminidase protein requirement for membrane fusion caveolae as portals of entry for microbes interactions between the transmembrane segments of the alphavirus e1 and e2 proteins play a role in virus budding and fusion influenza fusion peptides receptor binding and membrane fusion in virus entry: the influenza hemagglutinin inhibition of influenza virus hemagglutinin-mediated membrane fusion by a compound related to podocarpic acid influenza hemagglutinin-mediated membrane fusion: influence of receptor binding on the lag phase preceding fusion effects of low ph on influenza virus. activation and inactivation of the membrane fusion capacity of the hemagglutinin ph-independent hiv entry into cd4-positive t cells via virus envelope fusion to the plasma membrane role of metastability and acidic ph in membrane fusion by tick-borne encephalitis virus structural requirements for low-ph-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus membrane interactions of the tick-borne encephalitis virus fusion protein e at low ph detection of an interaction between the hn and f proteins in newcastle disease virus-infected cells membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion lipid rafts and assembly of enveloped viruses soluble receptor potentiates receptor-independent infection by murine coronavirus role of the hemagglutinin-neuraminidase protein in the mechanism of paramyxovirus-cell membrane fusion viral fusion peptides: a tool set to disrupt and connect biological membranes structure and function of membrane fusion peptides the role of the membrane-spanning domain sequence in glycoprotein-mediated membrane fusion oligomerization, secretion, and biological function of an anchor-free parainfluenza virus type 2 (pi2) fusion protein regulation of fusion activity by the cytoplasmic domain of a paramyxovirus f protein a single point mutation controls the cholesterol dependence of semliki forest virus entry and exit role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells sphingolipid and cholesterol dependence of alphavirus membrane fusion. lack of correlation with lipid raft formation in target liposomes membrane fusion of semliki forest virus involves homotrimers of the fusion protein membrane fusion process of semliki forest virus. i: low ph-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells evidence for h + -induced insertion of influenza hemagglutinin ha2 n-terminal segment into viral membrane crystal structure of the ebola virus membrane fusion subunit, gp2, from the envelope glycoprotein ectodomain structural basis for membrane fusion by enveloped viruses atomic structure of the ectodomain from hiv-1 gp41 mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity temperature dependence of fusion by sendai virus ph-dependent fusion between the semliki forest virus membrane and liposomes fusion of semliki forest virus with the plasma membrane can be induced by low ph membrane fusion activity of influenza virus viral and cellular membrane fusion proteins structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 resolution mutagenic stabilization and/ or disruption of a cd4-bound state reveals distinct conformations of the human immunodeficiency virus type 1 gp120 envelope glycoprotein analysis of the cell fusion activities of chimeric simian immunodeficiency virus-murine leukemia virus envelope proteins: inhibitory effects of the r peptide observation of a membrane fusion intermediate structure association of the parainfluenza virus fusion and hemagglutinin-neuraminidase glycoproteins on cell surfaces interaction of peptides with sequences from the newcastle disease virus fusion protein heptad repeat regions fusion of bovine leukemia virus with target cells monitored by r18 fluorescence and pcr assays proper spacing between heptad repeat b and the transmembrane domain boundary of the paramyxovirus sv5 f protein is critical for biological activity acknowledgements work in the authors laboratory was supported by the nih (ai22470). we thank dr. margaret kielian for helpful comments on the manuscript. abrahamyan lg, markosyan rm, moore jp, cohen fs, and melikyan gb (2003) human immunodeficiency virus type 1 env with an intersubunit disulfide bond engages coreceptors but requires bond reduction after engagement to induce fusion. j virol 77:5829-36 aguilar hc, anderson wf, and cannon pm (2003)